Synthesis and evaluation of immunostimulant plasmalogen lysophosphatidylethanolamine and analogues for natural killer T cells

Article abstract of DOI:10.1016/j.bmc.2014.04.012

Plasmalogen lysophosphatidylethanolamine (pLPE) had been identified as a self antigen for natural killer T cells (NKT cells). It is very important in the development, maturation and activation of NKT cells in thymus. Besides, pLPE is a novel type of antigen for NKT cells. To evaluate the structure-activity relationship (SAR) of this new antigen, pLPE and its analogues referred to different aliphatic chains and linkages at the sn-1 position of the glycerol backbone were synthesized, and the biological activities of these analogues was characterized. It is discovered that the linkages between phosphate and lipid moiety are not important for the antigens' activities. The pLPE analogues 1, 3, 4, 7 and 9, which have additional double bonds on lipid parts, were identified as new NKT agonists. Moreover, the analogues 4, 7 and 9 were discovered as potent Th2 activators for NKT cells.

Full text of DOI:10.1016/j.bmc.2014.04.012

Bioorganic & Medicinal Chemistry 22 (2014) 2966–2973
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of immunostimulant plasmalogen
lysophosphatidylethanolamine and analogues for natural killer
T cells
Guanghui Ni , Zhiyuan Li , Kangjiang Liang a,b, Ting Wu a,b, Gennaro De Libero , Chengfeng Xia
a,b
a
c
a,
⇑
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
University of the Chinese Academy of Sciences, Beijing 100049, China
Experimental Immunology, Department of Biomedicine, University Hospital Basel, 4031 Basel, Switzerland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 February 2014
Revised 3 April 2014
Accepted 4 April 2014
Available online 13 April 2014
Plasmalogen lysophosphatidylethanolamine (pLPE) had been identified as a self antigen for natural killer
T cells (NKT cells). It is very important in the development, maturation and activation of NKT cells in thy-
mus. Besides, pLPE is a novel type of antigen for NKT cells. To evaluate the structure–activity relationship
(SAR) of this new antigen, pLPE and its analogues referred to different aliphatic chains and linkages at the
sn-1 position of the glycerol backbone were synthesized, and the biological activities of these analogues
was characterized. It is discovered that the linkages between phosphate and lipid moiety are not impor-
tant for the antigens’ activities. The pLPE analogues 1, 3, 4, 7 and 9, which have additional double bonds
on lipid parts, were identified as new NKT agonists. Moreover, the analogues 4, 7 and 9 were discovered
as potent Th2 activators for NKT cells.
Keywords:
NKT cells
Antigen,
lysophospholipids
SAR
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
to produce Th1 and Th2 cytokines. However, the efficacy of
a
-GalCer has been largely limited because of the reciprocal inhibi-
1
1,12
Natural killer T cells (NKT cells) are specific subpopulations of T
tion exhibited by Th1 and Th2 cytokines.
Therefore, com-
lymphocytes, which express both conserved semi-invariant T cell
receptors (TCR) and natural killer cell (NK) receptors.1–3 The NKT
cells regulate a broad range of immune responses, and have been
recognized as potentially significant factor in the diagnosis and
treatment of many human diseases.4 The NKT cells can be stimu-
lated by the lipid antigens which are presented by major histocom-
patibility complex class-I-like protein CD1d through TCR. After
being activated, NKT cells secrete both T helper 1 (Th1) and Th2
pounds that can increase the selectivity toward either Th1 or Th2
cytokines responses may be more advantageous. Since the discov-
ery of
onstrate their structure–activity relationships (SAR).
these analogues showed Th1- or Th2-biased cytokine production
as compared to -GalCer. OCH, of which the sphingosine chain is
substantially shorter than that of -GalCer (Fig. 1), stimulate NKT
cells to produce many more Th2 cytokines than Th1 cytokines.
a-GalCer, numerous analogues have been developed to dem-
1
3–22
Some of
a
a
23
types of cytokines. Th1 type cytokines such as interferon (IFN)-
c
On the contrast, C20:2 which contains two double bonds in the
acyl chain can activate NKT cells to preferentially produce Th1
cytokines and induced cellular proliferation.
produce the proinflammatory response, and mediate protective
immune functions like tumor rejection and microbial infection;
whereas Th2 type cytokines such as IL-4 and IL-10, mediate regu-
2
4
Recognition of self antigens is very important for the develop-
ment, maturation and activation of NKT cells in thymus. Facciotti
et al. extracted lipids from mouse thymus and fractionated them
5
–8
latory immune functions to ameliorate autoimmune diseases.
Until now, several lipid antigens involved in the activation of
NKT cells have been identified (Fig. 1). Alpha-galactosylceramide
2
5,26
base on their polarities,
and then evaluated the activity on
(
a
-GalCer) is the first and most potent agonist of NKT cells, which
stimulating NKT cells in vitro. The active lipid fractions were
identified to be 1-O-1 -(Z)-hexadecenyl-2-hydroxy-sn-glycero-3-
phosphoethanolamine (plasmalogen C16-lysophosphatidyletha-
nolamine, pLPE) (Fig. 1), which is a metabolic product of the
peroxisomal enzyme glyceronephosphate O-acyltransferase
0
was derived by medicinal chemistry from agelsaphin, a glycolipid
found in the marine sponge Agelas mauritianus.
sented by CD1d will activate NKT cells by means of TCR recognition
9,10
a-GalCer pre-
(
GNPAT). The mice deficient in the enzyme GNPAT which lack of
⇑
Corresponding author. Tel.: +86 871 65223354.
E-mail address: xiachengfeng@mail.kib.ac.cn (C. Xia).
pLPE had fewer total NKT cells and significant alteration of the
http://dx.doi.org/10.1016/j.bmc.2014.04.012
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
2967
HO OH
HO OH
removal of the silyl protecting group on the primary hydroxyl with
hydrogen fluoride–pyridine complex, provided the corresponding
O-alkynyl ether 12a–c. To prepare vinyl ether linked pLPE and ana-
logue 1, the alkynes 12a and 12b were converted to cis-enol ethers
O
O
5 11
C H
O
C
24
H
49
O
9
HO
HO
NH OH
NH OH
HO
HO
O
O
C
14
H
29
14 29
C H
OH
OH
1
3a and 13b by partial hydrogenation with Lindlar catalyst in hex-
C20:2
α -GalCer
ane/EtOAc (1:1) in the presence of quinoline. Finally, the reaction
of alcohols 13a and 13b with phosphorus oxychloride in the pres-
ence of triethylamine and pyridine in dichloromethane, followed
by trapping of N-(monomethoxytrityl)-ethanolamine, and subse-
quently desilyation in the presence of hydrogen fluoride–pyridine
to afford pLPE and analogue 1. The preparation of alkynyl ether
linked pLPE analogues 2–4 were achieved by directly phosphoryla-
tion of alcohols 12a–c with phosphorus oxychloride and trapping
with N-(monomethoxytrityl)-ethanolamine, and finally removal
of the TBS protecting group by hydrogen fluoride–pyridine.
The analogues 5–9 were synthesized as shown in Scheme 2. For
the alkyl ether linked analogues 5–7, the 2,3-O-isopropylidene-
sn-glycerol was treated with an excess of n-alkyl bromide under
phase-transfer conditions (50% aqueous NaOH, tetrabutylammo-
nium bromide) at 80 °C, and the isopropylidene protective group
was then removed yielding 14a–14c. The diols (14a–14c) were
protected with TBS groups and then selectively deprotected at
the primary position in the presence of hydrogen fluoride–pyridine
to produce 15a–15c. The reaction of alcohol 15a–15c with phos-
phorus oxychloride in the presence of triethylamine and pyridine
in dichloromethane, followed by trapping of N-(monomethoxytri-
tyl)-ethanolamine, and subsequently desilyation in the presence
of hydrogen fluoride–pyridine to afford analogues 5–7. On another
hand, the ester linked analogues 8 and 9 were generated by firstly
acylated with acyl chloride (for 14d) or acid (for 14e) and then fol-
lowing the same procedures as which for analogues 5–7.
HO OH
O
O
C
22
H
45
OH
HO
NH OH
O
P
O
O
C14H29
HO
H
2
N
O
O
OH
5 11
C H
OH
OCH
Figure 1. Structures of
pLPE
-GalCer, OCH, C20:2 and pLPE.
a
thymic maturation of NKT cells. Thus, pLPE is a self-antigen that
not only stimulates the activation of NKT cells but also is required
for the generation of a full NKT cell repertoire in vivo.
Unlike
a-GalCer and its analogues, which are glyco-
sphingolipids, pLPE is a mono-alkyl glycerophosphate. To the best
of our knowledge, few analogues of pLPE have been synthesized to
explore its structure–activity relationship (SAR) on NKT cells.
Herein, we report the synthesis of pLPE and its analogues 1–9
(
Fig. 2) and evaluated their associated stimulatory activity on
NKT cells. To explore the most efficient linkage at the sn-1 position
of the glycerol backbone, we synthesized the analogues which con-
tain different aliphatic chains at the sn-1 position. 1 contains a
vinyl ether bond linking the sn-1 aliphatic chain to the glycerol
backbone, while an alkynyl ether linkage contained in 2, 3 and 4;
as well as saturated ether linkage contained in 5–7; and an ester
linkage contained in 8 and 9. Moreover, the aliphatic chains of 1,
3
, 4, 7 and 9 were designed to contain one or more carbon–carbon
double bonds, since compound C20:2 has two additional double
bonds and showed excellent Th1 selectivity. The biological evalua-
tion of these analogues would explore the SAR of linkages, and
length and unsaturated bonds of lipid parts.
2
.2. Immunological assay
The biological activity of pLPE analogues as NKT agonists was
preliminarily assayed in vitro by measuring the production of
Interleukin-2 (IL-2) of mouse NKT cell hybridoma 2H4. The ana-
logues were presented to stimulate 2H4 cell directly by surface-
bound CD1d protein in the plate-bound CD1d presenting assay.
As depicted in Figure 3, analogues 1, 3, 4, 7 and 9 induced obvious
IL-2 production. The results illustrated that they had the ability to
activate NKT cells directly and thus caused the release of IL-2
cytokines.
2
2
. Results and discussion
.1. Synthesis
The pLPE and all the analogues were initiated with commer-
cially available 2,3-O-isopropylidene-sn-glycerol. The methodolo-
gies to prepare pLPE and analogues 1–4 are outlined in
Scheme 1. Treatment of 2,3-O-isopropylidene-sn-glycerol with
potassium hydride and trichloroethylene in THF followed by
deacetonation with p-toluenesulfonic acid in MeOH gave 10 in
As a functional assay, we further cultured whole spleen cells in
the presence of indicated immunostimulant (1, 3, 4, 7 and 9) or
vehicle and determined the production of Th1 and Th2 prototypic
cytokines, Interferon-
2 h of culture. We found analogues 4, 7 and 9 induced a
significantly greater secretion of IL-4 than pLPE, whereas all ana-
c (IFN-c) and Interleukin- 4 (IL-4) by ELISA at
9
6% yield. The two hydroxyl groups were protected by tert-butyldi-
methylsilanyl (TBS) groups to yield 11. Reaction of dichloroethenyl
1 with n-butyllithium and aliphatic iodide, and then selectively
7
1
logues elicited slightly less IFN-
c than pLPE (Fig. 4A). When
OH
P
O
OH
OH
O
O
O
O
O
O
O
C
5
H
11
O
O
O
P
O
O
C
14
H
29
O
O
P
O
O
8
5 11
C H
8
H
2
N
H
2
N
H
H
2
N
N
OH
1
O
OH
2
O
OH
3
OH
OH
OH
P
O
8
C
8
H
17
P
O
OC
8
H
17
P
O
OC16H
33
H
2
N
H
2
N
2
O
OH
4
O
OH
5
O
OH
6
O
O
O
OH
P
OH
OH
8 17
C H
O
P
O
O
C
15
H
31
O
P
O
7
8 17
C H
12
H
2
N
H
2
N
H
2
N
O
OH
O
OH
O
OH
7
8
9
Figure 2. Structures of pLPE analogues.

2
968
G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
Cl
Cl
OH
O
b)
R
c)
O
a)
HO
d)
O
CHCl
TBSO
O
OTBS
CHCl
O
O
OH
1
0
1
1
OH
P
e)
O
O
R
HO
R
HO
O
2
H N
O
OH
OTBS
2a, R = C14
2b, R = C
2c, R = C
OTBS
13a, R = C14H29
11 13b, R = C H
1
1
1
H
29
pLPE, R = C14
H
29
8
H
16
C
5
H
8
16
C H
5
11
1
, R =
C H
8
16
5 11
C H
8
H
16
8 17
C H
OH
P
O
2
3
4
, R = C14
H
29
16
16
e)
O
O
O
R
2
H N
, R = C
, R = C
8
H
C H
5 11
OH
8
H
8 17
C H
Scheme 1. aReagents and conditions: (a) (i) KH (30%), 0 °C, trichloroethylene, ꢀ42 °Cꢁrt, THF, (ii) TsOH, MeOH, 96% for two steps; (b) TBSCl, imidazole, DMAP, DMF/THF(3:1),
9
5%; (c) (i) n-BuLi(2.5 M in hexane, THF, ꢀ78 °C ꢁ ꢀ42 °C, RI, HMPA, ꢀ42 °C ꢁ rt, (ii) HFꢂpyridine, pyridine, THF, 0 °C ꢁ rt 36–52% for two steps; (d) H
2 4
, Pd/BaSO ,quinoline,
O, 0 °C ꢁ rt, (ii) HFꢂpyridine, pyridine, THF, 47–61% for two steps.
hexane/EtOAc (1:1), quantitative yield; (e) (i) POCl
3
, TEA, pyridine, MMTrNHCH
2
CH
2
OH, H
2
OH
a)
b)
O
HO
OR
17
HO
OR
17
To more meaningfully address the biological activity of the ana-
logues and physiological consequences, we resorted to in vivo
study. We administered 1 g of the analogues intraperitoneally
and measured the levels of IFN- and IL-4 in serum at 2 and
4 h. Similarly to splenocyte cultures, all compounds induced obvi-
O
OH
OTBS
1
1
4a, R = C H
4b, R = C16H33
15a, R = C H
15b, R = C16H33
8
8
l
c
1
4c, R = C12H24
C H
15c, R = C12H24
8 17
C H
8
17
2
c)
ous production of IL-4 upon in vivo administration as detected in
the serum of treated mice 2 h later, and analogues 4, 7 and 9
5
6
, R = C H
, R = C16H33
8
17
OH
O
O
P
OR
induced
Fig. 5), while IFN-
ground levels at 2 and 24 h in any treatment (Fig. 5).
a
significantly greater secretion of IL-4 than pLPE
2
H N
7
, R = C12
H
24
C
8
H
17
b)
O
OH
(
c
induction in vivo was weak barely over back-
O
O
OH
d) or e)
O
HO
O
R
R
HO
O
R
O
2
.3. Discussion
OH
OTBS
1
4d, R = C 5H
1
31
14
15d
,
R = C15
H
31
1
4e, R = C
7
H
C
8
H
17
In this study, we designed and synthesized a series of mono-
1
5e, R = C
7
H
14
8 17
C H
O
aliphatic chain modified pLPE analogues, and evaluated their
biological activity. According to the reactivity of mouse NKT cell
hybridomas to a series of pLPE analogues presented by CD1d pro-
tein, in which assay the affects from the antigen presenting cells
were excluded, we found analogues 1, 3, 4, 7, and 9 could also
stimulate NKT cells activation significantly while the activity of
OH
O
c)
8
, R = C15H31
O
P
O
O
2
H N
OH
9, R = C
7
H
14
C
8 17
H
Scheme 2. aReagents and conditions: (a) (i) RBr, NaOH(50%, aq), 80 °C, (ii) AcOH,
50%, aq), 50 °C, 90–98% for two steps; (b) (i) TBSCl, imidazole, DMAP, DMF/
THF(3:1), (ii) HFꢂpyridine, pyridine, THF, 0 °C ꢁ rt; 71–78% for two steps; (c) (i)
POCl , TEA, pyridine, MMTrNHCH CH OH, H
2% for two steps; (d) for 14d (i) C15 31COCl, TEA, DMAP, DCM, (ii) AcOH, (50%, aq),
0 °C, quantative yield for two steps; (e) for 14e (i) C 17CH@CHC 14CO H, DCC,
(
2, 5, 6, and 8 were weak. There is a 16-carbon chain with a vinyl
3
2
2
2
O, (ii) HFꢂpyridine, pyridine, THF, 54–
6
5
H
ether bond linking to the sn-1 position of the glycerol of pLPE. 2,
6 and 8 had the same 16-carbon chain as pLPE but with different
linkage at the sn-1 position: an alkynyl ether linkage, a saturated
ether linkage, or an ester linkage respectively. It indicated that only
the change of the vinyl ether bond in pLPE would impair its activ-
ity. Meanwhile, the activity of 5 which had an 8-carbon chain with
a saturated ether linkage at the sn-1 position like 6 also became
weaker. Interestingly, we found that analogues 3, 4, 7 and 9 which
did not contain a vinyl ether bond could still stimulate NKT cells
obviously. It is notably that the aliphatic chains of 3, 4, 7 and 9 con-
tained one or more carbon–carbon double bonds. It suggested that
carbon–carbon double bonds in the aliphatic chains made a great
improvement on the immunostimulatory activity in comparison
with 2, 6 and 8 which did not contain a vinyl ether bond. Unlike
8
H
7
H
2
DMAP, DCM, (ii) AcOH, (50%, aq), 50 °C, 98% for two steps.
5
4
3
2
0
0
0
0
*
*
*
*
*
*
h
1
2
3
4
5
6
7
8
9
ve
pLPE
3, 4, 7 and 9, we noticed the activity of 1, which contained car-
Figure 3. Reactivity of mouse NKT cell hybridoma to a series of pLPE analogues.
ELISA of IL-2 released by 2H4 cells stimulated with plate-bound mCD1d loaded with
equal concentrations of various compounds (100 ng/mL). Data are expressed as
mean ± SD from three independent experiments. Statistical significance of the
difference in secretion levels was determined by Student’s t test. P <0.05, compared
with vehicle.
bon–carbon double bonds in the aliphatic chains with a vinyl ether
bond linkage at the sn-1 position, was not higher than that of pLPE.
It indicated that carbon–carbon double bonds in the aliphatic
chains could not enhance the activity when there was a vinyl ether
bond linkage at the sn-1 position of the glycerol. In other words,
pLPE analogues could activate NKT cells obviously if they con-
tained a saturated aliphatic chain with a vinyl ether bond linkage
at the sn-1 position, or an unsaturated aliphatic chain with an
alkynyl ether or saturated ether or ester linkage, which was in
agreement with a report LPC that contained an unsaturated
aliphatic chain with an ester linkage could also stimulate NKT cells
⁄
expressed as the IL-4/IFN-c ratio (Fig. 4B), all compounds induced a
Th2 biased cytokine response, and especially compound 4, 7 and 9
induced more Th2-biased immunity than pLPE. The data strongly
suggest that 4, 7 and 9 were recognized by NKT cells stimulating
their activation in a way that seems to be more biased toward Th2.

G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
2969
A 20
B
60
Th1
Th2
5
4
3
2
1
*
*
1
5
0
5
*
*
4
0
0
*
*
1
2
h
1
3
4
7
9
h
1
3
4
7
9
ve
ve
LPE
p
pLPE
h
1
3
4
7
9
ve
PE
pL
5
Figure 4. Effect of pLPE analogues on Th1/Th2 cytokine production by mouse splenocytes. Splenocytes (5 ꢃ 10 ) from C57BL/6 mice were cultured for72 h with 100 ng/mL of
pLPE analogues or DMSO vehicle. Cytokines in the supernatants were measured by ELISA. (A) Induction of IFN-
control. Assays were performed in triplicate, and data are given as mean ± SD. P <0.05, compared with pLPE.
c
and IL-4. (B) Ratio of IL-4 over IFN-
c
, normalized DMSO
⁄
1
1
1
1
1
8
6
4
2
0
8
6
4
2
0
key structure on NKT cell antigen can result in large affinity differ-
ences among binding partners and have a surprising degree of
influence on cytokine-inducing selectivity.
veh
pLPE
1
3
4
7
9
3. Conclusion
In summary, we described total synthesis of pLPE, an endoge-
nous antigen for NKT cells, and its analogues. We identified pLPE
analogues 1, 3, 4, 7 and 9 as new NKT agonists and 4, 7 and 9
become more potent Th2 activators for NKT cells which have supe-
rior properties for the treatment of autoimmune and inflammatory
diseases. It will provide a new strategy and theoretical basis for the
designation and clinical application of NKT cell antigens.
2
h
24h
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
*
*
*
4
. Experimental
4.1. General
2
h
24h
All the reagents were obtained from commercial suppliers and
used without further purification unless stated otherwise. THF
Figure 5. In vivo cytokine induction in mice. Amounts of 1
pLPE were i.p. injected into C57BL/6 mice, and IFN-
measured after 2 h and 24 h. Data are given as mean ± SD. P <0.05, compared with
l
g of the analogues and
(
tetrahydrofuran) was distilled from sodium/benzophenone, DCM
(dichloromethane) was distilled from CaH , acetone was distilled
from anhydrous CaSO , pyridine was dried by boiling with CaH
2
c
and IL-4 in serum were
⁄
2
pLPE.
4
prior to distillation and stored over KOH, DMF(N,N-Dimethylform-
amide) was stored over 4 Å molecular sieves, TEA (triethylamine)
was distilled from Ca(OH) and stored over KOH. All the reactions
2
activation.27 Besides, the observation of immunostimulatory activ-
ity of 7 and 9 will benefit the further SAR study on pLPE, because it
was much more economic and easier to synthesize saturated ether
or ester linkage than a vinyl ether bond linkage.
Although the modification of the mono-aliphatic chain of pLPE
by changing the vinyl ether bond linkage did not elicit greater
IL-2 release of NKT cell hybridoma, we found 4, 7 and 9 polarized
were performed in oven-dried or flame-dried apparatus under
argon atmosphere. Reactions were monitored by thin-layer chro-
matography (TLC), performed on 0.2 mm silica gel GF254 plates.
Visualization on TLC was achieved using UV light, iodine vapor
and/or phosphomolybdic acid reagent. Chromatographic purifica-
tion of products was accomplished using forced-flow chromatogra-
phy on 300–400 mesh silica gel. NMR spectra were recorded on
Bruker 400, Bruker 500, Bruker 600 or Avance 600. High-resolution
mass spectra (HRMS) were performed on Waters AutoSpec Premier
P776 or Agilent G6230.
the release of cytokines more obviously. As we know,
a-GalCer
induces both Th1 and Th2 cytokines, and the two types of cyto-
kines inhibit each other. There were no clinical responses in a
phase I study of
the effects of Th1 cytokines were hindered by Th2 cytokines. As
a result, the efficacy of -GalCer has been limited. Therefore, the
a-GalCer in patients with solid tumors because
1
2
0
0
a
4.1.1. 1-O-(1 ,2 -Dichloroethenyl)-sn-glycerol (10)
selectivity toward either Th1 or Th2 cytokines responses is more
critical. The five immunostimulants (1, 3, 4, 7 and 9) were further
evaluated for their in vitro and in vivo immune-modulating effects.
We found they all induced a Th2-biased cytokine response, as
deduced from the high level of IL-4 in the splenocyte cultures
To a suspension of KH (8.1 g, 30%, 60.7 mmol) in THF (250 mL)
was added 2,3-O-isopropylidene-sn-glycerol (5.0 mL, 40.5 mmol)
dropwise at 0 °C and stirred for 1 h. The reaction mixture was
cooled to ꢀ42 °C and trichloroethylene (4.0 mL, 44.6 mmol) was
added dropwise. The resulting mixture was warmed to room tem-
and in vivo assays compared with the very weak IFN-
c
induction.
4
perature and stirred for 1 h. Saturated NH Cl solution was added to
Especially, compounds 4, 7 and 9, induced more Th2-biased immu-
nity than pLPE. Thus, 4, 7 and 9 have an efficacy as potent Th2
response inducer but do not activate Th1 cytokines, so avoiding
quench the reaction and the solution was extracted with EtOAc.
The combined organic layers were washed with brine, dried over
Na
(100 mL). After stirring overnight, the mixture was neutralized by
NH OH, concentrated and purified by column chromatography
(petroleum ether/acetone 3:1) to give 7.23 g (96%) diol 10 as light
2 4
SO , concentrated and treated with TsOH (500 mg) in methanol
one main problem in the therapeutical application of
a-GalCer,
the simultaneous and potent induction of contradictory responses.
These results demonstrate that even relatively minor changes in
4

2
970
G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
yellow oil: 1H NMR (400 MHz, CDCl
.15–3.95 (m, 3H), 3.80 (dd, J = 11.6, 3.2 Hz, 1H), 3.70 (dd,
) d 5.58 (d, J = 8.7 Hz, 1H),
3
3
CDCl ) d 5.48–5.14 (m, 4H), 4.05–3.99 (m, 1H), 3.99–3.94 (m,
4
1H), 3.90 (dd, J = 10.2, 6.3 Hz, 1H), 3.70–3.61 (m, 1H), 3.61–3.48
(m, 1H), 2.76 (t, J = 7.0 Hz, 2H), 2.13–1.87 (m, 6H), 1.46–1.19 (m,
1
3
J = 11.6, 5.2 Hz, 1H), 2.87 (s, 2H); C NMR (100 MHz, CDCl
43.59, 98.72, 72.83, 70.07, 63.32; HRMS (EI+) calcd for C
3
) d
Cl
1
3
1
H
5 8
O
3
2
18H), 0.94–0.81 (m, 12H), 0.13–0.08 (m, 6H); C NMR (150 MHz,
CDCl ) d 130.34, 130.28, 128.11, 128.07, 89.67, 78.72, 70.45,
3.78, 31.67, 29.82, 29.77, 29.62, 29.49, 29.45, 29.32, 29.00,
+
[
M] 185.9850, found 185.9855.
3
6
0
0
4
.1.2. 2,3-O-bis-tert-Butyldimethylsilanyl-1-O-(1 ,2 -
27.37, 27.34, 26.24, 26.21, 25.92, 25.89, 25.77, 22.72, 17.24,
dichloroethenyl)-sn-glycerol (11)
14.22, ꢀ4.54, ꢀ4.77; HRMS (EI+) calcd for C29
H
54
O
3
Si [M]+
To a solution of diol 10 (207.1 mg, 1.1 mmol) in DMF (9 mL) and
478.3842, found 478.3864.
THF (3 mL), was added imidazole (238.3 mg, 3.5 mmol) and DMAP
0
0
(
(
24.7 mg, 0.22 mmol), followed by tert-butyldimethylsilyl chloride
497.4 mg, 3.3 mmol). The resulting reaction mixture was stirred
4.1.3.3.
ynyl-sn-glycerol (12c).
2-O-tert-Butyldimethylsilanyl-1-O-11 -(Z)-icosen-1 -
1
3
36% yield; H NMR (400 MHz, CDCl )
overnight. The reaction mixture was diluted with Et
washed with saturated NH Cl, saturated NaHCO , brine, and then
dried over Na SO . The solvents were removed under reduced
pressure and the crude product was purified by flash chromatogra-
phy on silica gel (petroleum ether). The desired bis-TBS (431.6 mg,
2
O and was
d 5.49–5.16 (m, 2H), 4.05–4.00 (m, 1H), 3.97 (dd, J = 10.2, 5.2 Hz,
1H), 3.94–3.84 (m, 1H), 3.68–3.61 (m, 1H), 3.61–3.51 (m, 1H),
2.09 (t, J = 7.0 Hz, 2H), 2.05–1.90 (m, 4H), 1.86 (dd, J = 7.7, 5.2 Hz,
1H), 1.47–1.37 (m, 2H), 1.38–1.13 (m, 22H), 0.91–0.87 (m, 12H),
0.20–0.08 (m, 6H); C NMR (150 MHz, CDCl ) d 130.09, 130.00,
3
87.07, 78.73, 70.47, 63.81, 37.54, 32.06, 29.93, 29.79, 29.68,
29.64, 29.47, 29.33, 29.02, 27.36, 26.26, 26.00, 25.90, 22.83,
4
3
2
4
1
3
9
5
3
5%) was obtained as colorless oil: 1H NMR (400 MHz, CDCl
.44 (s, 1H), 4.08 (dd, J = 9.9, 4.1 Hz, 1H), 4.02–3.83 (m, 2H),
3
) d
1
3
.77–3.50 (m, 2H), 0.97–0.86 (m, 18H), 0.19–0.02 (m, 12H);
) d 144.15, 96.95, 72.94, 71.75, 64.37,
6.04, 25.90, 18.44, 18.24, ꢀ4.56, ꢀ4.67, ꢀ5.26, ꢀ5.30; HRMS
C
18.22, 17.26, 14.26, ꢀ4.53, ꢀ4.76; HRMS (EI+) calcd for C29
56 3
H O Si
+
NMR (100 MHz, CDCl
2
3
[M] 480.3999, found 480.4008.
+
(
EI+) calcd for C17
H
36
O
3
Si
Cl
2 2
[M] 414.1580, found 414.1588.
4.1.4. General procedure for the synthesis of compounds 13a
and 13b
4
1
.1.3. General procedure for the synthesis of compounds
2a–12c
To a solution of dichloroethenyl (11, 1 equiv) in THF was added
To a solution of the O-alkynyl ether (12a and 12b) in hexane/
EtOAc (1:1) were added Pd/BaSO and quinoline. The resulting sus-
pension was degassed by H and then a H balloon was applied.
4
2
2
n-BuLi (2.5 M in hexane, 2 equiv) dropwise at ꢀ78 °C. After 1 h, the
reaction mixture was warmed to ꢀ42 °C. RI (1.1 equiv) in HMPA
was added. The solution was warmed to room temperature and
After stirring overnight, the mixture was filtered through a plug
of Celite, washed with EtOAc and concentrated. The residue was
purified by flash chromatography on silica gel (petroleum ether/
EtOAc 100:1) to affored the desired cis-enol ether (13a and 13b)
as colorless oil.
stirred for 24 h. Saturated NH
ture was extracted with Et O three times, the combined organic
layers were washed with water and brine, dried over Na SO
4
Cl solution was added and the mix-
2
2
4
,
0
filtered and concentrated. The residue was purified by flash chro-
matography on silica gel (petroleum ether) to affored the desired
O-alkynyl ether as colorless oil. A stirring solution of the bis-TBS-
protected alcohol prepared above in THF at 0 °C was treated with
HF-pyridine in pyridine/THF (prepared by mixing 1 volume of
HF-pyridine complex with 4 volume of pyridine and diluted with
4.1.4.1. 2-O-tert-Butyldimethylsilanyl-1-O-1 -(Z)-hexadecenyl-
1
2-hydroxy-sn-glycerol (13a).
(400 MHz, CDCl
Quantative yield;
H NMR
3
) d 5.90 (d, J = 6.2 Hz, 1H), 4.33 (dd, J = 13.6,
7.2 Hz, 1H), 3.95–3.88 (m, 1H), 3.77–3.62 (m, 3H), 3.62–3.52 (m,
1H), 2.12–2.00 (m, 2H), 1.91 (dd, J = 7.4, 5.4 Hz, 1H), 1.50–1.14
13
(m, 24H), 0.93–0.88 (m, 12H), 0.20–0.09 (m, 6H);
(100 MHz, CDCl ) d 145.02, 107.48, 73.39, 71.50, 64.33, 32.08,
29.94, 29.86, 29.82, 29.69, 29.52, 29.50, 25.93, 24.17, 22.85,
C NMR
4
volume of THF) dropwise via addition funnel. The reaction was
3
allowed to slowly warm to rt and stirred for 1 h. The reaction mix-
ture was cooled to 0 °C and quenched by the dropwise addition of
52 3
O
Si [M]+
18.23, 14.27, ꢀ4.48, ꢀ4.74; HRMS (EI+) calcd for C25
H
saturated NaHCO
3
and stirring continued until no gas evolution
428.3686, found 428.3703.
was observed. The solution was transferred into a separatory fun-
nel and the two layers were allowed to separate. The organic layer
was collected and the aqueous layer extracted with EtOAc three
times, the combined organic layers were washed with water and
0
0
0
4.1.4.2. 2-O-tert-Butyldimethylsilanyl-1-O-1 ,11 ,14 -(Z,Z,Z)-ico-
1
satrienyl-sn-glycerol (13b).
Quantative yield;
(400 MHz, CDCl ) d 5.90 (d, J = 6.2 Hz, 1H), 5.63–5.12 (m, 4H),
H
NMR
3
brine, dried over Na
was purified by flash chromatography on silica gel (petroleum
ether/EtOAc 100:1) to affored the desired primary alcohol
2
SO
4
, filtered and concentrated. The residue
4.46–4.20 (m, 1H), 4.13–3.98 (m, 1H), 3.95–3.83 (m, 1H), 3.71–
3.67 (m, 1H), 3.66–3.62 (m, 1H), 3.60–3.53 (m, 1H), 2.77 (t,
J = 6.4 Hz, 2H), 2.15–1.98 (m, 6H), 1.38–1.23 (m, 18H), 0.91–0.89
1
3
(12a–c) as colorless oil.
(m, 12H), 0.17–0.09 (m, 6H);
3
C NMR (150 MHz, CDCl ) d
1
45.03, 130.34, 128.10, 128.08, 107.42, 79.40, 73.38, 71.48, 70.26,
0
4
.1.3.1.
hydroxy-sn-glycerol (12a).
CDCl ) d 4.03 (dd, J = 10.8, 4.7 Hz, 1H), 3.97 (dd, J = 10.2, 5.2 Hz,
2-O-tert-Butyldimethylsilanyl-1-O-1 -hexadecynyl-2-
64.31, 63.65, 31.68, 29.92, 29.84, 29.69, 29.65, 29.48, 27.40,
27.35, 26.25, 25.92, 25.88, 25.78, 24.16, 22.72, 19.31, 14.22,
44% yield; 1H NMR (400 MHz,
+
3
ꢀ3.89, ꢀ4.48; HRMS (EI+) calcd for C29
H
56
O
3
Si [M] 480.3999,
1
3
H), 3.90 (dd, J = 10.2, 6.4 Hz, 1H), 3.65 (dd, J = 11.3, 3.7 Hz, 1H),
.60–3.47 (m, 1H), 2.08 (t, J = 6.9 Hz, 2H), 1.92 (br s, 1H), 1.42
found 480.3987.
(
dd, J = 14.2, 7.1 Hz, 2H), 1.37–1.12 (m, 22H), 0.93–0.84 (m, 12H),
4.1.5. General procedure for the synthesis of pLPE, 1–4
To a solution of the POCl (1.05 equiv) in DCM was added TEA
(20 equiv) and pyridine (1 equiv) dropwise at 0 °C. After 0.5 h, alco-
hol (12a–12c, 13a–13b, 1 equiv) in DCM was added dropwise.
After 1 h, N-(monomethoxytrityl)-ethanolamine(2.5 equiv) in
DCM was added dropwise and the mixture was stirred 1 h before
1
3
0
7
2
.18–0.07 (m, 6H); C NMR (100 MHz, CDCl
3
) d 89.63, 78.68,
3
0.41, 63.76, 37.53, 32.07, 29.84, 29.81, 29.77, 29.75, 29.51,
9.36, 29.01, 26.24, 25.88, 22.84, 18.21, 17.24, 14.27, ꢀ4.55,
+
ꢀ
4.78; HRMS (EI+) calcd for C25
50
H O
3
Si [M] 426.3529, found
4
26.3528.
2
H O (0.5 mL) was added. After another 1 h stirring, the mixture
was treated with HF-pyridine (2 equiv) and stirred overnight. The
resulting mixture was dissolved with DCM and washed with
0
0
4
.1.3.2. 2-O-tert-Butyldimethylsilanyl-1-O-11 , 14 -(Z,Z)-icosadi-
0
52% yield; 1H NMR (600 MHz,
en-1 -ynyl-sn-glycerol (12b).

G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
2971
H
2
O. The organic layer was concentrated, and the residue was puri-
fied by flash chromatography on silica gel (CHCl /MeOH/H
0:10:1) to affored the compounds (pLPE, 1–5) as white solid.
(15.2 mmol) and Bu
4
NBr (245.0 mg, 0.76 mmol). After disappear-
3
2
O
ance of the starting materials (2 h), the reaction mixture was
cooled and extracted with CH Cl . The organic solution was
washed with water, brine, dried over Na SO , concentrated and
7
2
2
2
4
0
4
.1.5.1. 1-O-1 -(Z)-Hexadecenyl-2-hydroxy-sn-glycero-3-phos-
purified by column chromatography (petroleum ether, then 20:1
petroleum ether/ethyl acetate) to give the 1-O-n-alkyl-2,3-O-iso-
propylidene-sn-glycerol. The 1-O-n-alkyl-2,3-O-isopropylidene-
sn-glycerol was dissolved in 80% aqueous AcOH (1 mmol/5 mL).
The mixture was stirred overnight at room temperature and then
evaporated to dryness in vacuo. The residue was purified by col-
umn chromatography (3:1 petroleum ether/acetone) to afford the
1-O-n-alkyl-sn-glycerol.
1
phoethanolamine (pLPE).
CDCl /MeOD) d 8.31 (br s, 2H), 5.93 (d, J = 6.1 Hz, 1H), 4.37–4.30
m, 1H), 4.26–4.06 (m, 2H), 4.06–3.91 (m, 2H), 3.90–3.80 (m, 1H),
61% yield; H NMR (500 MHz,
3
(
3
2
1
2
1
.79–3.65 (m, 2H), 3.29–3.15 (m, 2H), 2.09–1.86 (m, 2H), 1.25 (s,
4H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl
3
/MeOD) d
44.80, 107.76, 72.43, 69.55, 67.48, 62.19, 45.94, 32.02, 29.91,
9.85, 29.82, 29.81, 29.76, 29.63, 29.49, 29.46, 24.05, 22.77,
4.18, 8.60; P NMR (162 MHz, CDCl /MeOD) d 0.61; HRMS (EI+)
3
3
1
+
4.1.6.1. 1-O-n-Octyl-sn-glycerol (14a)29
calcd for C21
H44NO
6
P [M] 437.2906, found 437.2915.
.
White solid;
) d 4.01–3.80 (m,
1
7
61.2 mg; 98% yield; H NMR (400 MHz, CDCl
3
0
0
0
4
3
CDCl
.1.5.2. 1-O-1 ,11 ,14 -(Z,Z,Z)-icosatrienyl-2-hydroxy-sn-glycero-
1H), 3.80–3.56 (m, 2H), 3.56–3.37 (m, 4H), 2.97 (d, J = 4.8 Hz,
1H), 2.63 (t, J = 5.8 Hz, 1H), 1.55 (dd, J = 14.0, 6.9 Hz, 2H), 1.51–
1.13 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H).
-phosphoethanolamine (1).
) d 6.10–5.76 (m, 1H), 5.57–5.16 (m, 4H), 4.32 (dd, J = 13.2,
.4 Hz, 1H), 4.24–3.59 (m, 5H), 3.53–3.05 (m, 4H), 2.77 (t,
J = 6.4 Hz, 2H), 2.30–2.03 (m, 6H), 1.68–1.06 (m, 18H), 0.89 (t,
47% yield; 1H NMR (400 MHz,
3
6
4.1.6.2. 1-O-n-Hexadecyl-sn-glycerol (14b)30.
White solid;
) d 3.85 (d, J = 4.5 Hz,
1
3
1
J = 6.8 Hz, 3H);
3
C NMR (150 MHz, CDCl ) d 145.02, 130.34,
1.17 g; 98% yield; H NMR (400 MHz, CDCl
3
1
3
2
30.28, 128.11, 128.08, 109.57, 71.97, 69.66, 62.45, 45.98, 40.64,
2.06, 31.67, 29.89, 29.85, 29.79, 29.58, 29.50, 29.47, 27.42,
1H), 3.71 (dd, J = 11.4, 3.5 Hz, 1H), 3.64 (dd, J = 11.4, 5.2 Hz, 1H),
3.58–3.35 (m, 4H), 2.68 (s, 1H), 2.26 (s, 1H), 1.62–1.52 (m, 2H),
1.25 (s, 26H), 0.87 (t, J = 6.7 Hz, 3H).
3
1
7.35, 26.23, 25.78, 24.15, 22.84, 22.73, 14.24, 8.76; P NMR
+
(
162 MHz, CDCl
3 6
) d ꢀ0.24; HRMS (EI+) calcd for C25H48NO P [M]
4
89.3219, found 489.3229.
4.1.6.3. 1-O-cis-Docos-13-enyl-sn-glycerol (14c).
Colorless
1
oil; 1.37 g; 90% yield; H NMR (400 MHz, CDCl
3
) d 5.57–5.06 (m,
0
4
.1.5.3. 1-O-1 -Hexadecynyl-2-hydroxy-sn-glycero-3-phospho-
2H), 3.85 (d, J = 3.9 Hz, 1H), 3.71 (dd, J = 11.3, 3.5 Hz, 1H), 3.64
(dd, J = 11.4, 5.3 Hz, 1H), 3.56–3.41 (m, 4H), 2.75 (s, 1H), 2.36 (s,
1H), 2.12–1.90 (m, 4H), 1.66–1.49 (m, 2H), 1.34–1.22 (m, 30H),
1
ethanolamine (2).
MeOD) d 4.25–3.92 (m, 6H), 3.91–3.83 (m, 1H), 3.29–3.11 (m,
3
63% yield; H NMR (600 MHz, CDCl /
1
3
2H), 2.07 (t, J = 7.2 Hz, 2H), 1.49–1.38 (m, 2H), 1.29–1.20 (m,
2H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR(150 MHz, CDCl
2 /MeOD) d
89.49, 78.32, 68.53, 67.11, 62.54, 40.56, 37.56, 34.27, 32.10,
2
0.87 (t, J = 6.8 Hz, 3H);
3
C NMR (100 MHz, CDCl ) d 130.03,
3
130.01, 72.56, 71.98, 70.66, 64.35, 32.03, 29.90, 29.83, 29.78,
29.74, 29.72, 29.70, 29.65, 29.59, 29.45, 27.33, 26.20, 22.80,
3
1
+
9.95, 29.93, 29.87, 29.56, 25.09, 22.86, 17.35, 14.27, 0.14;
/MeOD) d 0.41; HRMS (EI+) calcd for C21
P [M] 435.2750, found 435.2739.
P
14.23; HRMS (EI+) calcd for C25
398.3751.
50
H O
3
[M] 398.3760, found
NMR (162 MHz, CDCl
NO
3
H
42-
+
6
4
.1.7. 1-O-Hexadecanoyl-sn-glycerol (14d)31
To an ice-cold solution of 2,3-O-isopropylidene-sn-glycerol (1 g,
0
0
0
4
.1.5.4. 2-Hydroxy-1-O-11 ,14 -(Z,Z)-icosadien-1 -ynyl-sn-glyce-
1
ro-3-phosphoethanolamine (3).
600 MHz, CDCl /MeOD) d 5.39–5.16 (m, 4H), 4.11–3.72 (m, 7H),
.18–2.96 (s, 2H), 2.68 (t, J = 6.8 Hz, 1H), 2.30–2.13 (m, 2H),
.11–1.76 (m, 4H), 1.64–1.47 (m, 2H), 1.36–1.17 (m, 18H), 0.79
52% yield;
H
NMR
6 mmol) and DMAP(17.0 mg, 0.15 mmol) in DCM (10 mL) and trieth-
ylamine (3 mL) was added palmitoyl chloride (2.5 g, 9.0 mmol) in
DCM (3 mL) dropwise. The reaction mixture was warmed to room
temperature and stirred overnight. The mixture was filtered, and
(
3
2
3
1
3
(
t, J = 6.0 Hz, 3H); C NMR (150 MHz, CDCl
3
/MeOD) d 130.16,
30.09, 127.94, 127.89, 89.34, 72.86, 71.47, 64.79, 61.83, 37.20,
4.05, 31.86, 31.48, 29.64, 29.47, 29.30, 29.16, 28.91, 27.19,
the filtrate was washed with 1 N HCl, saturated NaHCO
3
, brine, and
1
3
2
then dried over Na SO . The solvents were removed under reduced
2
4
pressure and the crude product dissolved in 80% aqueous AcOH
(27 mL). The mixture was stirred overnight at room temperature
and then evaporated to dryness in vacuo. The residue was purified
by column chromatography (3:1 petroleum ether/acetone) to afford
3
1
7.15, 25.57, 24.82, 22.52, 17.03, 13.96, ꢀ0.16;
P NMR
(
[
162 MHz, CDCl
M] 487.3063, found 487.3068.
3
/MeOD) d 0.55; HRMS (EI+) calcd for C25H46NO P
6
+
2
.51 g 1-O-hexadecanoyl-sn-glycerol as a white solid (quantitative
0
0
1
4
.1.5.5.
1-O-11 -(Z)-icosen-1 -ynyl-2-hydroxy-sn-glycero-3-
3
yield): H NMR (400 MHz, CDCl ) d 4.26–4.02 (m, 2H), 4.00–3.86
phosphoethanolamine (4).
CDCl /MeOD) d 8.26 (br s, 2H), 5.56–5.06 (m, 2H), 4.68–4.32 (m,
H), 4.31–3.88 (m, 4H), 3.88–3.56 (m, 2H), 3.17–2.82 (m, 2H),
.30 (d, J = 7.2 Hz, 1H), 2.10–1.86 (m, 6H), 1.66–1.05 (m, 24H),
.88 (t, J = 6.6 Hz, 3H);
49% yield; 1H NMR (400 MHz,
(m, 1H), 3.69 (dd, J = 11.5, 3.5 Hz, 1H), 3.59 (dd, J = 11.4, 5.9 Hz,
1H), 2.91 (br s, 1H), 2.57 (br s, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.70–
1.53 (m, 2H), 1.24 (s, 24H), 0.87 (t, J = 6.4 Hz, 3H).
3
1
2
0
1
3
2
13
4.1.8. 1-O-cis-Octadec-9-enoyl-sn-glycerol (14e)32
3
C NMR (150 MHz, CDCl /MeOD) d
29.99, 129.87, 89.35, 72.02, 68.71, 64.85, 62.12, 40.32, 34.13,
2.65, 31.94, 29.83, 29.80, 29.75, 29.70, 29.59, 29.56, 29.53,
9.35, 29.24, 29.01, 27.24, 24.91, 22.72, 17.13, 14.12, 8.53, ꢀ0.02;
DCC (1.18 g, 5.7 mmol) and DMAP (213.1 mg, 1.9 mmol) were
added to a solution of 2,3-O-isopropylidene-sn-glycerol (0.50 g,
3.8 mmol) and oleic acid(1.8 mL, 5.7 mmol) in 50 mL of DCM. After
the mixture was stirred at room temperature overnight, the sol-
vent was removed under vacuum. The crude product was dissolved
in 80% aqueous AcOH (17 mL) and stirred overnight. The mixture
was concentrated in vacuo and the residue was purified by column
chromatography (3:1 petroleum ether/acetone) to afford 1.33 g
3
1
P NMR (162 MHz, CDCl
3
/MeOD) d ꢀ0.61; HRMS (EI+) calcd for
+
C
25
H48NO
6
P [M] 489.3219, found 489.3221.
4
1
.1.6. General procedure for the synthesis of compounds
4a–14c
,3-O-Isopropylidene-sn-glycerol (500.0 mg, 3.8 mmol) was
added to a 50% aqueous solution of NaOH (8 mL). The reaction mix-
ture was heated at 80 °C, before the addition of the n-alkyl bromide
2
8
2
(98%) 1-O-cis-octadec-9-enoyl-sn-glycerol as a white solid: 1
NMR (400 MHz, CDCl ) d 5.57–5.12 (m, 2H), 4.39–4.02 (m, 2H),
3.99–3.87 (m, 1H), 3.70 (dd, J = 11.4, 3.8 Hz, 1H), 3.60 (dd,
H
3

2
972
G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
J = 11.4, 5.8 Hz, 1H), 2.57 (br s, 1H), 2.35 (t, J = 7.6 Hz, 2H), 2.14 (br
s, 1H), 2.01 (dd, J = 14.3, 8.5 Hz, 4H), 1.66–1.60 (m, 2H), 1.40–1.23
25.05, 22.84, 18.21, 14.26, ꢀ4.50, ꢀ4.68; HRMS (EI+) calcd for C25
+
52
H O
4
Si [M] 444.3635, found 444.3633.
(
m, 20H), 0.88 (t, J = 6.7 Hz, 3H).
4
.1.9.5. 2-O-tert-Butyldimethylsilanyl-1-O-cis-octadec-9-enoyl-
1
4
1
.1.9. General procedure for the synthesis of compounds 15a–
5e
To a solution of diol (14a–14e, 1 equiv) in DMF and THF, was
sn-glycerol (15e).
3
71% yield; H NMR (400 MHz, CDCl ) d
5.48–5.18 (m, 2H), 4.16–4.02 (m, 2H), 3.99–3.86 (m, 1H), 3.68–
3.48 (m, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.10–1.82 (m, 4H), 1.67–1.58
(m, 2H), 1.38–1.23 (m, 20H), 0.95–0.83 (m, 12H), 0.11 (d,
added imidazole (10 equiv) and DMAP (0.2 equiv), followed by
tert-butyldimethylsilyl chloride (3 equiv). The resulting reaction
mixture was stirred overnight. The reaction mixture was diluted
1
3
J = 1.9 Hz, 6H);
3
C NMR (100 MHz, CDCl ) d 173.88, 130.17,
129.89, 70.74, 64.95, 64.05, 34.36, 32.06, 29.92, 29.84, 29.67,
29.47, 29.31, 29.27, 29.24, 27.37, 27.32, 25.88, 25.04, 22.83,
with Et
2
O and was washed with saturated NH
4
Cl, saturated
3
2
SO . The solvents were
4
18.21, 14.26, ꢀ4.50, ꢀ4.68; HRMS (EI+) calcd for C27
54 4
H O
Si [M]+
NaHCO , brine, and then dried over Na
removed under reduced pressure and the crude product was puri-
fied by flash chromatography on silica gel (petroleum ether) to
afford the desired 2, 3-O-bis-tert-butyldimethylsilanyl-sn-glycerol
as a colorless oil. A stirring solution of the bis-TBS-protected
prepared above in THF at 0 °C was treated with HF-pyridine in
pyridine/THF (prepared by mixing 1 volume of HF-pyridine com-
plex with 4 volume of pyridine and diluted with 4 volume of THF)
dropwise via addition funnel. The reaction was allowed to slowly
warm to rt and stirred for 1 h. The reaction mixture was cooled to
470.3791, found 470.3788.
4.1.10. General procedure for the synthesis of compounds 5–9
To a solution of the POCl (1.05 equiv) in DCM was added TEA
3
(20 equiv) and pyridine (1 equiv) dropwise at 0 °C. After 0.5 h,
alcohol (15a–15e, 1 equiv) in DCM was added dropwise. After
1 h, N-(monomethoxytrityl)-ethanolamine (2.5 equiv) in DCM
2
was added dropwise and the mixture was stirred 1 h before H O
(0.5 mL) was added. After another 1 h stirring, the mixture was
treated with HF-pyridine (2 equiv) and stirred overnight. The
resulting mixture was dissolved with DCM and washed with
0
°C and quenched by the dropwise addition of saturated NaHCO
3
and stirring continued until no gas evolution was observed. The
solution was transferred into a separatory funnel and the two layers
were allowed to separate. The organic layer was collected and the
aqueous layer extracted with EtOAc three times, the combined
organic layers were washed with water and brine, dried over Na2-
H
2
O. The organic layer was concentrated, and the residue was puri-
fied by flash chromatography on silica gel (CHCl /MeOH/H
70:10:1) to afford the compounds (5–9) as white solid.
3
2
O
SO
4
, filtered and concentrated. The residue was purified by flash
4.1.10.1. 1-O-n-Octyl-2-hydroxy-sn-glycero-3-phosphoethanol-
1
chromatography on silica gel (petroleum ether/EtOAc 100:1) to aff-
ored the desired primary alcohol(15a–e) as colorless oil.
amine (5).
3.91–3.79 (m, 2H), 3.79–3.65 (m, 2H), 3.66–3.55 (m, 1H), 3.28–
.15 (m, 4H), 2.97–2.82 (m, 2H), 1.31 (dd, J = 13.8, 6.9 Hz, 2H),
3
62% yield; H NMR (400 MHz, CDCl /MeOD) d
3
1
3
4
.1.9.1. 2-O-tert-Butyldimethylsilanyl-1-O-n-octyl-sn-glycerol
1.13–0.94 (m, 10H), 0.63 (t, J = 6.8 Hz, 3H); C NMR (100 MHz,
CDCl /MeOD) d 71.49, 71.19, 69.48, 67.27, 61.35, 40.21, 31.50,
29.21, 29.11, 28.92, 25.68, 22.28, 13.52; P NMR (162 MHz, CDCl
1
(
15a).
78% yield; H NMR (400 MHz, CDCl
3
) d 3.93–3.82 (m,
3
3
1
1
2
0
7
2
H), 3.70–3.60 (m, 1H), 3.60–3.53 (m, 1H), 3.49–3.35 (m, 4H),
.23–1.97 (m, 1H), 1.56–1.48 (m, 2H), 1.37–1.25 (m, 10H), 0.90–
3
+
/
MeOD) d 0.70; HRMS (ESI+) calcd for C13
H
31NO
6
P ([M+H] )
.84 (m, 12H), 0.10 (s, 6H); 13C NMR (150 MHz, CDCl
328.1889, found 328.1891.
3
) d 72.92,
1.95, 71.29, 65.28, 31.96, 29.80, 29.57, 29.39, 26.25, 26.02,
5.95, 22.80, 18.27, 14.25, ꢀ4.45, ꢀ4.72; HRMS (EI+) calcd for C17
4.1.10.2. 1-O-n-Hexadecyl-2-hydroxy-sn-glycero-3-phosphoeth-
anolamine (6). 57% yield; H NMR (600 MHz, CDCl /MeOD) d
.98–3.85 (m, 2H), 3.85–3.72 (m, 2H), 3.70–3.65 (m, 1H), 3.30–
3.28 (m, 2H), 3.20–3.15 (m, 2H), 2.98–2.89 (m, 2H), 1.52–1.24 (m,
+
H
38
O
3
Si [M] 318.2590, found 318.2592.
3
3
4
.1.9.2. 2-O-tert-Butyldimethylsilanyl-1-O-n-hexadecyl-sn-glyc-
1
13
erol (15b).
75% yield; H NMR (400 MHz, CDCl
3
) d 3.95–3.82
2H), 1.23–1.02 (m, 26H), 0.70 (t, J = 7.0 Hz, 3H);
(150 MHz, CDCl /MeOD) d 71.65, 71.29, 69.61, 67.44, 61.39, 40.28,
31.74, 29.50, 29.46, 29.44, 29.38, 29.34, 29.17, 25.84, 22.48,
C NMR
(
m, 1H), 3.71–3.52 (m, 2H), 3.49–3.27 (m, 4H), 2.13 (dd, J = 7.4,
3
5
0
7
2
.3 Hz, 1H), 1.55–1.48 (m, 2H), 1.25 (s, 26H), 0.91–0.85 (m, 12H),
.10 (d, J = 0.9 Hz, 6H); C NMR (100 MHz, CDCl
1.31, 65.28, 32.08, 29.85, 29.81, 29.76, 29.62, 29.51, 26.26,
1
3
31
3
) d 72.93, 71.96,
13.77; P NMR (162 MHz, CDCl
3
/MeOD) d 4.77; HRMS (ESI+) calcd
+
for C21H46NNaO P ([M+Na] ) 462.2960, found 462.2966.
6
5.95, 22.84, 18.27, 14.27, ꢀ4.44, ꢀ4.72; HRMS (EI+) calcd for C25
+
H
54
O
3
Si [M] 430.3842, found 430.3855.
4.1.10.3. 1-O-cis-Docos-13-enyl-2-hydroxy-sn-glycero-3-phos-
1
phoethanolamine (7).
3
59% yield; H NMR (400 MHz, CDCl /
4
.1.9.3.
2-O-tert-Butyldimethylsilanyl-1-O-cis-docos-13-enyl-
72% yield; 1H NMR (400 MHz, CDCl
) d
.49–5.18 (m, 2H), 3.93–3.82 (m, 1H), 3.73–3.51 (m, 2H), 3.48–
MeOD) d 5.28–5.07 (m, 2H), 3.98–3.84 (m, 2H), 3.82–3.70 (m,
2H), 3.70–3.63 (m, 1H), 3.27 (t, J = 6.8 Hz, 4H), 3.19–3.12 (m, 2H),
1.97–1.70 (m, 4H), 1.47–1.26 (m, 2H), 1.08 (s, 30H), 0.70 (t,
sn-glycerol (15c).
3
5
3
6
1
3
.27 (m, 4H), 2.14 (dd, J = 7.3, 5.3 Hz, 1H), 2.01 (dd, J = 11.6,
.0 Hz, 4H), 1.54 (dd, J = 13.7, 6.8 Hz, 2H), 1.26 (s, 30H), 0.92–0.80
3
J = 6.0 Hz, 3H); C NMR (100 MHz, CDCl /MeOD) d 129.67, 71.65,
71.30, 69.51, 67.44, 39.46, 31.70, 29.55, 29.46, 29.37, 29.30,
1
3
31
(
7
2
m, 12H), 0.10 (s, 6H); C NMR (100 MHz, CDCl
3
) d 130.05, 72.93,
1.95, 71.31, 65.27, 32.06, 29.93, 29.81, 29.74, 29.68, 29.63, 29.48,
7.36, 26.26, 25.95, 22.83, 18.26, 14.26, ꢀ4.45, ꢀ4.72; HRMS (EI+)
29.11, 26.97, 25.83, 22.45, 13.74; P NMR (162 MHz, CDCl
3
/MeOD)
+
d 0.96; HRMS (ESI+) calcd for C27H56NNaO P ([M+Na] ) 544.3743,
6
found 544.3747.
+
64 3
calcd for C31H O Si [M] 512.4625, found 512.4631.
4
.1.10.4. 1-O-Hexadecanoyl-2-hydroxy-sn-glycero-3-phospho-
1
4
.1.9.4.
glycerol (15d).
.01 (m, 2H), 3.99–3.87 (m, 1H), 3.68–3.45 (m, 2H), 2.31 (t,
J = 7.5 Hz, 2H), 1.96 (dd, J = 7.2, 5.8 Hz, 1H), 1.70–1.59 (m, 2H),
2-O-tert-Butyldimethylsilanyl-1-O-hexadecanoyl-sn-
ethanolamine (8).
3
62%; H NMR (400 MHz, CDCl /MeOD) d
1
76% yield; H NMR (400 MHz, CDCl
3
) d 4.17–
3.93–3.86 (m, 2H), 3.86–3.75 (m, 2H), 3.76–3.67 (m, 1H), 3.66–
3.54 (m, 2H), 2.98–2.92 (m, 1H), 2.89–2.82 (m, 1H), 2.18 (t,
J = 7.6 Hz, 2H), 1.54–1.30 (m, 2H), 1.23–0.97 (m, 24H), 0.72 (t,
4
1
3
1
.42–1.12 (m, 24H), 0.98–0.78 (m, 12H), 0.11 (d, J = 1.9 Hz, 6H);
J = 6.8 Hz, 3H); C NMR (150 MHz, CDCl
3
/MeOD) d 174.28, 64.72,
1
3
3
C NMR (100 MHz, CDCl ) d 173.91, 70.74, 64.95, 64.06, 34.37,
61.42, 57.60, 53.27, 41.54, 33.98, 31.80, 29.57, 29.53, 29.50,
29.37, 29.23, 29.17, 29.04, 24.74, 22.55, 13.88;
31
3
2.07, 29.84, 29.80, 29.75, 29.60, 29.51, 29.41, 29.30, 25.87,
P NMR

G. Ni et al. / Bioorg. Med. Chem. 22 (2014) 2966–2973
2973
(
(
162 MHz, CDCl
[MꢀH] ) 452.2777, found 452.2785.
3
/MeOD) d 0.81; HRMS (ESIꢀ) calcd for C21
7
H43NO P
Technology Professionals Introduction Program(2010CI117), the
National Natural Science Foundation of China (21272242), and
National Basic Research Program of China (2011CB915500).
ꢀ
4
.1.10.5.
phosphoethanolamine (9).
CDCl ) d 8.34 (brs, 2H), 5.46–5.33 (m, 2H), 4.26–4.03 (m, 4H),
.02–3.93 (m, 2H), 3.88–3.80 (m, 1H), 3.40–3.11 (m, 2H), 2.93–2.60
m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.17–1.85 (m, 4H), 1.73–1.49 (m,
1-O-cis-Octadec-9-enoyl-2-hydroxy-sn-glycero-3-
54% yield; 1H NMR (400 MHz,
References and notes
3
1
2
3
.
.
.
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4
(
1
3
2
H), 1.43–1.16 (m, 20H), 0.88 (t, J = 5.3 Hz, 3H);
) d 173.98, 130.15, 129.83, 68.99, 67.65, 64.90,
1.96, 34.24, 32.06, 31.67, 29.92, 29.69, 29.47, 29.37, 27.37, 25.78,
C
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6
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(
100 MHz, CDCl
3
6
2
3
1
5.04, 22.83, 14.26; P NMR (162 MHz, CDCl
3
) d 0.54; HRMS
P ([M+Na] ) 502.2910, found 502.2906.
+
7
8
.
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Godfrey, D. I.; Pellicci, D. G.; Patel, O.; Kjer-Nielsen, L.; McCluskey, J.; Rossjohn,
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(
ESI+) calcd for C23
H
46NNaO
7
4
4
.2. Methods for measurement of biological activity
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1
1
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The 2H4 mouse NKT hybridoma was kindly provided by
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Blomberg, B. M. E.; Scheper, R. J.; van der Vliet, H. J. J.; van den Eertwegh, A. J.
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2
mM
L-glutamine, and 50
l
M 2-mercaptoethanol.
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2
1
5. Toba, T.; Murata, K.; Futamura, J.; Nakanishi, K.; Takahashi, B.; Takemoto, N.;
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4
.2.2. Plate-bound CD1d presenting assay
The stimulation of mouse NKT cell hybridomas in a cell-free
1
1
1
1
6. Tashiro, T.; Shigeura, T.; Watarai, H.; Taniguchi, M.; Mori, K. Bioorg. Med. Chem.
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antigen presentation assay was performed as described by pub-
lished protocol , with some modification. 1 lg mCD1d protein
was coated in 96-well flat-bottom plate at 4 °C overnight. The
plates were washed with PBS and blocked by PBS and 10% FCS
for 1 h. After washing, the pLPE and anologs at indicated concen-
tration were added to each well and incubated for 24 h at 37 °C.
3
3
4
After washing, 2H4 hybridoma cells (5 ꢃ 10 ) were added to each
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2
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2
2
2
2. Tashiro, T. Biosci. Biotechnol. Biochem. 2012, 76, 1055.
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4
.2.3. Mouse primary splenocytes assay
5
Splenocytes (5 ꢃ 10 ) from C57BL/6 mice were cultured for 72 h
with 100 ng/mL of pLPE and analogues or DMSO vehicle in 96-well
U-bottom plates containing RPMI1640 supplemented with 10%
FCS,
in humidified 5% CO
supernatants were collected and IFN-
2
5. Facciotti, F.; Ramanjaneyulu, G. S.; Lepore, M.; Sansano, S.; Cavallari, M.;
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L-glutamine, 2-mercaptoethanol, penicillin and streptomycin
2
2
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2
at 37 °C. For cytokine determination, cell-free
c and IL-4 levels were mea-
sured by ELISA.
2
2
4
.2.4. In vivo assays
g of the pLPE and analogues in 200
intraperitoneally to C57BL/6 mice. Sera were collected at 2 time
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l
lL of PBS were injected
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We are owe to NIH Tetramer Facility for providing mCD1d. We
gratefully acknowledge financial support from Yunnan High-End