Synthesis and cytotoxicity assay of four ganglioside GM3 analogues Dedicated to Professor Max Malacria on the occasion of his 65th birthday.

Article abstract of DOI:10.1016/j.ejmech.2014.01.054

A concise and efficient synthetic route for preparation of four ganglioside GM3 analogues was described. The key step is a highly regioselective and stereoselective α-sialylation from a suitably protected glycoside acceptor with a sialyl xanthate to provide the sialo-oligosaccharide in good yield. The cytotoxic properties of the synthetic gangliosides were evaluated against normal human keratinocytes and human HCT116 and K562 cancer cells. Two of them exhibited good antiproliferative activity and displayed a better cytotoxicity against cancer cell than HaCaT normal cell.

Full text of DOI:10.1016/j.ejmech.2014.01.054

European Journal of Medicinal Chemistry 75 (2014) 247e257
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity assay of four ganglioside GM3 analogues
,
Huanhuan Qu a b, Jian-Miao Liu c, Joanna Wdzieczak-Bakala c, Dan Lu a, Xianran He d,
,d,e,
Wenji Sun e, Matthieu Sollogoub a, Yongmin Zhang a
*
a Sorbonne Universités, UPMC Univ Paris 06, LabEx Michem, CNRS, UMR 8232, IPCM, F-75005 Paris, France
b Glycochemistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Pudong,
Shanghai 201203, China
c Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, avenue de la Terrasse, F-91198 Gif sur Yvette, France
d Institute for Interdisciplinary Research, Jianghan University, Wuhan Economic and Technological Development Zone, Wuhan 430056, China
e Biomedicine Key Laboratory of Shaanxi Province, Northwest University, Xi’an 710069, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A concise and efficient synthetic route for preparation of four ganglioside GM3 analogues was described.
Received 18 October 2013
Received in revised form
21 January 2014
Accepted 24 January 2014
Available online 28 January 2014
The key step is a highly regioselective and stereoselective
a-sialylation from a suitably protected
glycoside acceptor with a sialyl xanthate to provide the sialo-oligosaccharide in good yield. The cytotoxic
properties of the synthetic gangliosides were evaluated against normal human keratinocytes and human
HCT116 and K562 cancer cells. Two of them exhibited good antiproliferative activity and displayed a
better cytotoxicity against cancer cell than HaCaT normal cell.
Dedicated to Professor Max Malacria on the
occasion of his 65th birthday.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Synthesis
Sialylation
Cytotoxicity
Cancer
Ganglioside GM3
Sialic acid
1. Introduction
Our previous researches cooperated with Hakomori’s group
indicate that clustered GSLs associated with c-Src, small G-protein
Glycosphingolipids (GSLs) are components of all animal cell
membranes and are involved in many cellular functions including
proliferation, adhesion, motility, and differentiation [1]. Ganglio-
(Rho A), and focal adhesion kinase (FAK) are involved in GSL-
dependent cell adhesion coupled with activation of these signal
transducers [4]. A typical example is GM3 ganglioside clustering at
the cell surface of mouse melanoma B16, forming a “glycosignaling
domain” (GSD) separable from cholesterol- and caveolin-enriched
membrane fractions derived from caveolae [5]. GM3 in B16 cells
is also recognized as a melanoma-associated antigen, and may have
a role in initiating adhesion of melanoma to endothelial cells, the
first step in metastasis [6]. Antigenicity, mediation of adhesion, and
initiation of signaling through GM3 ganglioside at the B16 cell
surface are thought to be maintained by GM3 clustering in GSD. If
GM3 clustering in GSD is inhibited, antigenicity, adhesion, and
signaling through GM3 could be blocked. This concept suggests
that compounds having structural features analogous to those of
GM3, may destroy or reduce clustering of GM3 in GSD, and inhibit
GM3-dependent adhesion and signaling. So the purpose of this
work was to search for ganglioside GM3 analogues which could
disrupt the structure and function of GSD in cancer cells.
side GM3 (NeuAca3Galb4Glcb1Cer), the first and simplest member
in the metabolic series of a GSLs family containing sialic acids (N-
acetyl- and N-glycolyl-neuraminic acids and their O-acyl de-
rivatives), is known as one of the most abundant tumor-associated
carbohydrate antigens on several types of tumors [2]. Glyco-
sphingolipid structures, and their changes associated with biolog-
ical functions, have been the central focus of our studies, since
structural change is the starting point for understanding biological
significance, and enzymatic/genetic mechanisms [3].
* Corresponding author. Université Pierre et Marie Curie-Paris 6, Institut Parisien
de Chimie Moléculaire, CNRS UMR 8232, 4 place Jussieu, 75005 Paris, France.
E-mail address: yongmin.zhang@upmc.fr (Y. Zhang).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.054

248
H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
Studies utilizing synthetic molecules that resemble natural GSLs
respectively a monosaccharide from natural ganglioside GM3. But
we failed in the preparation of (2 / 4) sialosides (NeuAc 4Glc).
Next, we tried the sialylations of glucose at position C-3, but the
yields were less than 6%. Fortunately, the yield of -sialylation of
glucose at position C-6 was high. In this paper, we describe in
complete detail the total synthesis of our target GSLs.
provide important information that is not available from studies
utilizing molecules from natural sources. Recently, the develop-
ment of synthetic gangliosides has attracted much attention. Gly-
cosylations to form anomeric linkages of sialic acid (sialylations) are
often complicated by the intrinsic structural features of sialic acid,
resulting in poor yields and/or stereoselectivities [7]. Although
notable progress has been made in this research field in the last
decade, good yields along with complete control of stereo-
selectivities for the synthesis of sialosides are still a goal to be
reached.
Herein we would like to report the preparation of four GM3
analogues 1e4 (Fig. 1) based on a facile protocol that can provide a
series of gangliosides. As a part of a parallel investigation, in vitro
cytotoxicity of these compounds was determined against the hu-
man keratinocyte and human HCT116 and K562 cancer cells. We
believe this work will facilitate construction of various GM3 ana-
logues with analogous or even improved biological properties
compared to those of the natural GSLs, and provide meaningful and
useful references for exploring new carbohydrate anticancer
agents.
a
a
a
As illustrated in Table 1, we compared three sialic acid donors 5
[9], 5a [10] and 5b [10] to optimize the a-sialylation of galactoside
acceptors with the sialic acid donors [11e13]. Considering the
synthetic strategy after the sialylation, we gave priority to use
previously reported diol 6a [14] as the acceptor. Unfortunately, we
didn’t obtain our desired product (entry 1e4). Along with the re-
sults reported in Ref. [15], using acetyl groups as the protecting
groups of acceptor was inappropriate (entry 5), the yield of sialy-
lation was low. Entry 6e9, galactoside acceptor 6 containing benzyl
as protective groups was used. The reaction in the presence of
benzenesulfenyl chloride (PhSCl), Silver trifluoromethanesulfonate
(AgOTf) and di-tert-butylpyridine (DTBP) as promoters in a 2:1
mixture of MeCN/CH2Cl2 at low temperature (entry 6) afforded a-
sialoside in significantly higher yield than the other three entries.
So for the sialylation of glucoside, we directly selected this Marti-
chonok and Whitesides’ method [9]. Similarly, considering the
synthetic strategy, the previously reported 2-(trimethylsilyl)ethyl
glycoside 7 [16] was our first choice to be used as the glucoside
acceptor. Not only because it was convenient to prepare 7, but also
because using acetyl groups as the protecting groups simplified the
synthesis after the sialylation. The sialylation of glucoside acceptor
2. Results and discussion
Sialylation is the key step of the total synthesis of our target
glycosides. In spite of extensive efforts and notable progress, the
chemical synthesis of sialosides in high yield with complete ster-
eoselectivity remains a significant challenge [7]. The presence of a
destabilizing electron-withdrawing carboxylic group along with a
tertiary anomeric center and the lack of a participating auxiliary
often drive glycosylation reactions toward competitive elimination
7 with sialic acid donors 5 afforded a-sialoside in 82% yield. This
yield is regarded as satisfactory, so there is no need to try other
protecting groups. Based on these observations, for synthesis of our
target gangliosides, compounds 5, 6 [17], 7 and 8 [18] were chosen
as building blocks (Fig. 2).
reactions resulting in poor stereoselectivity (b-anomer) and in the
formation of a 2,3-dehydro derivative [8]. To overcome these
problems, different approaches have emerged. A variety of leaving
groups and activation conditions has been developed. The original
Sialylation of acceptor 6 occurred at the more reactive 3-OH
position and gave rise predominantly to the
proton signal H-3eq for sialic acid residue appeared at
-product
¼ 2.41 ppm). Catalytic hydrogenolysis of 9 provided
the disaccharide 10. Acetylation of 10 for protection of the
a
-product 9 since the
d
¼ 2.53 ppm
purpose of our study was to synthesize
a(2 / 3) sialosides (Neu-
(b
d
Ac 3Gal) and (2 / 4) sialosides (NeuAca
a
a
4Glc), which removed
Fig. 1. Structures of ganglioside GM3 and the target GM3 analogues.

H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
249
Table 1
Optimization of the a-sialylation.
Entry
Acceptor [equiv]
Donor [equiv]
Promoter [equiv]
Conditions
Solvent
Product
Yield [%]d
Reference
b
b
1
2
3
4
5
6
7
8
9
6a (1)
6a (1.5)
6a (3)
6a (1)
6b (Npa)
6 (1)
6 (1.5)
6 (3)
6 (1)
5 (1.5)
5a (1)
5b (1)
5b (1)
5a (Np)
5 (1.5)
5a (1)
5b (1)
5b (1)
PhSCl/AgOTf/DTBP (2/2/2.1)
NIS/TfOH (1.5/0.4)
Ag2CO3 (3.5)
Hg(CN)2/HgBr2 (1.4/0.5)
NIS/TfOH (Np)
PhSCl/AgOTf/DTBP (2/2/2.1)
NIS/TfOH (1.5/0.4)
Ag2CO3 (3.5)
ꢁ68 ꢀC/3h
MeCN/CH2Cl2
MeCN
CH2Cl2
CH2Cl2
MeCN
MeCN/CH2Cl2
MeCN
CH2Cl2
9a
9a
9a
9a
9bc
9
9
9
9
Nde
Nd
Nd
Nd
26c
75
39
5
[9]
ꢁ45 ꢀC/3h
[11]
[12]
[13]
[15]
[9]
[11]
[12]
[13]
ꢁ45 ꢀC / r.t./2 days
r.t./2 days
ꢁ35 ꢀC/2 days
ꢁ68 ꢀC/3h
ꢁ45 ꢀC/3h
ꢁ45 ꢀC / r.t./2 days
r.t./2 days
Hg(CN)2/HgBr2 (1.4/0.5)
CH2Cl2
8
a
Not reported.
Solvent ratio of 2:1.
This experiment was described in Ref. [15].
Based on the stating material (acceptor or donor) present in the smallest amount.
Not detected.
b
c
d
e
remaining hydroxyl groups yielded the octaacetylated derivative 11
quantitatively. Acid catalyzed cleavage of the 2-(trimethylsilyl)ethyl
glycoside 11 was performed in CH2Cl2 using trifluoroacetic acid to
reaction with NaOMe/MeOH. After adding several drops of water,
compound 17 was obtained quantitatively. Then azide 17 was
reduced with propanedithiol/triethylamine [20] to afford the new
ganglioside 2 in 93% yield.
As shown in Schemes 3 and 4, the synthetic route of ganglio-
sides 3 and 4 is similar to that described above.
In vitro cytotoxicity of the synthetic gangliosides 1e4 was
determined against the human keratinocyte and human HCT116
and K562 cancer cells. The IC50 (mM) values obtained with tested
cell lines are summarized in Table 2. Results demonstrate that
compound 3 and 4 have a similar antiproliferative activity and
display a better cytotoxicity against cancer cell than HaCaT normal
cell. Compound 2 had no effect on cell proliferation as compared to
compound 1. The effect of selected compounds 3 and 4 on the cell
afford hemiacetal 12 as a mixture of
was then treated with trichloroacetonitrile in the presence of 1,8-
diazabicyclo[5,4,0]undec-7-ene (DBU) to give the tri-
chloroacetimidate 13 in 88% yield for two steps (Scheme 1). The 1H
NMR spectrum showed that -trichloroacetimidate is the pre-
dominant product (
a/b isomers. This hemiacetal
a
a
/
b
¼ 94/6) of this reaction on the basis of the
H-10 and H-20 coupling constant (J1 ,2 ¼ 3.9 Hz) of the galactose
residue. This is because an axial trichloroacetimidate is the ther-
modynamically more stable isomer [19].
0
0
Condensation of trichloroacetimidate 13 with azidosphingosine
derivative 8 was performed using BF3∙Et2O as promoter to provide
the desired glycolipid 14 in 83% yield, as shown in Scheme 2. The
b
morphology was then investigated at 100 mM. As shown in Fig. 3,
configuration of the newly introduced glycosidic linkage was
after 3 days of treatment, changes can be clearly observed.
confirmed from the 1H NMR spectrum (J1 ,2 ¼ 8.0 Hz). The azide
group of 14 was reduced by triphenylphosphine in a mixture of
toluene and water at 45 ꢀC for 12 h to give an amino derivative 15 in
76% yield. Then 15 was condensed with stearic acid in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in dry
CH2Cl2 to give the glycosyl ceramide 16. The acyl groups of com-
pound 16 on hydroxyls were subsequently removed by reaction
with NaOMe/MeOH. After adding several drops of water, ganglio-
side 1 was obtained in 81% yield for two steps. On the other hand,
all the acyl groups of compound 14 on hydroxyls were removed by
0
0
3. Conclusions
In this study, we accomplished the synthesis of four GM3 ana-
logues from simple and commercially available substrates and re-
agents. The key step is a highly regioselective and stereoselective a-
sialylation from a suitably protected glycoside acceptor with a sialyl
xanthate to provide the sialo-oligosaccharide in good yield. Two
glucose-containing GM3 analogues, in which sialic acids are linked
by (a2 / 6)-glycosidic linkage to glucosides, exhibited good
Fig. 2. Key building blocks for synthesis of the target gangliosides.

250
H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
Scheme 1. Reagents and conditions: (a) MeCN, CH2Cl2, 4 A powdered molecular sieves, 1 h, AgOTf, DTBP, ꢁ68 ꢀC, PhSCl, 3 h, 75%; (b) Pd/C, MeOH, H2, 40 ꢀC, 5 h, 98%; (c) Pyr., Ac2O,
r.t., 18 h, quant.; (d) CF3COOH, CH2Cl2, 0 ꢀC, 1 h, r.t., 1 h; (e) CCl3CN, DBU, CH2Cl2, ꢁ5 ꢀC, 3 h, 88% (two steps from 11).
ꢀ
antiproliferative activity and displayed a better cytotoxicity against
cancer cell than HaCaT normal cell, indicating that a ( 2 / 6)-
glycosidic linkage is more important than a ( 2 / 3) one in GM3
GM3 analogues, it is logical that these compounds demonstrated a
similar biological activity. Further study is needed to understand
the mechanism.
a
a
molecule for such a biological activity. Based on this observation,
we are planning to design and synthesize galactose-containing
4. Experimental section
GM3 analogues having a structure of NeuAca2 / 6Gal to analyze
their in vitro cytotoxicity in future. The cytotoxicity of these de-
rivatives is possibly caused by their inhibition on activity of various
growth factor receptor (GFR)-associated tyrosine kinases, as it was
reported that exogenous addition of GM3 inhibited BHK cell growth
induced by fibroblast growth factor [21] and the phosphorylation of
platelet-derived GFR [22] and epidermal GFR (EGFR) [23]. Being
4.1. General chemical methods
All chemicals were purchased as reagent grade and used
without further purification. PhSCl was prepared as previously re-
ported [9]. CH2Cl2 was freshly distilled from P2O5. All reactions
were carried out under anhydrous conditions with freshly distilled
Scheme 2. Reagents and conditions: (a) BF3.Et2O, CH2Cl2, 4 A powdered molecular sieves, ꢁ15 ꢀC, 2.5 h, 83%; (b) PPh3, toluene, H2O, 45 ꢀC, 12 h, 76%; (c) Stearic acid, EDC, CH2Cl2, r.t.,
ꢀ
20 h; (d) NaOMe, MeOH, r.t., 14 h; H2O, 0 ꢀC, 1 h, 81% (two steps from 15); (e) NaOMe, MeOH, r.t., 14 h; H2O, 0 ꢀC, 1 h, 98%; (f) HS(CH2)3SH, Et3N, MeOH, r.t., 4 days, 93%.

H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
251
Scheme 3. Reagents and conditions: (a) MeCN, CH2Cl2, 4 A powdered molecular sieves, 1 h, AgOTf, DTBP, ꢁ68 ꢀC, PhSCl, 3 h, 82%; (b) CF3COOH, CH2Cl2, 0 ꢀC, 1 h, r.t., 1 h; (c) CCl3CN,
DBU, CH2Cl2, ꢁ5 ꢀC, 3 h, 85% (two steps from 18).
ꢀ
solvents, unless otherwise noted. Reactions were monitored by
thin-layer chromatography (TLC) on a precoated plate of silica gel
60 F254 (Merck) and detection by staining with sulfuric acid or
acidic ceric ammonium molybdate. Solvents were evaporated un-
der reduced pressure and below 40 ꢀC (bath). Flash column chro-
matography was performed on silica gel 60 (230e400 mesh,
Merck). Proton nuclear magnetic resonance (1H NMR) and Carbon-
13 nuclear magnetic resonance (13C NMR) spectra were recorded at
400 (100.6) and 300 (75.5) MHz with Bruker AVANCE DRX 400 and
300 spectrometers. The chemical shifts were referenced to the
were recorded with a Bruker micrOTOF spectrometer in electro-
spray ionization (ESI) mode, using Tuning-Mix as reference. Optical
rotations were measured at 589 nm (Na line) at 20 ꢀC with a Per-
kineElmer Model 343 digital polarimeter, using a 10 cm, 1 mL cell.
4.2. 2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
(2 / 3)-2,6-di-O-benzyl-b-D-galactopyranoside (9)
4.2.1. Method A (5 þ 6)
solvent peak,
d
¼ 7.26 ppm (1H) and
d
¼ 77.16 ppm (13C) for CDCl3,
A mixture of compound 5 (1.06 g, 1.78 mmol) and 6 (548.0 mg,
¼ 3.31 ppm (1H) and
d
¼ 49.00 ppm (13C) for CD3OD,
¼ 2.50 ppm
d
ꢀ
d
1.19 mmol), 4 A powdered molecular sieves (2.50 g), dry CH3CN
(1H) and
d
¼ 39.52 ppm (13C) for DMSO, at 25 ꢀC, and coupling
(20 mL), and dry CH2Cl2 (10 mL) was stirred under nitrogen for 1 h.
AgOTf (611.5 mg, 2.38 mmol) and DTBP (0.56 mL, 2.50 mmol) were
constants were given in Hz. High-resolution mass spectra (HRMS)
Scheme 4. Reagents and conditions: (a) BF3.Et2O, CH2Cl2, 4 A powdered molecular sieves, ꢁ15 ꢀC, 2.5 h, 77%; (b) PPh3, toluene, H2O, 45 ꢀC, 12 h, 82%; (c) Stearic acid, EDC, CH2Cl2, r.t.,
ꢀ
20 h; (d) NaOMe, MeOH, r.t., 14 h; H2O, 0 ꢀC, 1 h, 79% (two steps from 22); (e) NaOMe, MeOH, r.t., 14 h; H2O, 0 ꢀC, 1 h; (f) HS(CH2)3SH, Et3N, MeOH, r.t., 4 days, 90% (two steps from
21).

252
H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
Table 2
chromatographed (Cy-EtOAc 1:3) to give 9 as a white amorphous
solid (833.6 mg, 75%).
Cytotoxicity (IC50a) of synthetic compounds against different human cell lines.
4.2.2. Method B (5a þ 6)
To a mixture of compound 5a (39.1 mg, 0.07 mmol) and 6
ꢀ
(50.5 mg, 0.11 mmol), 4 A powdered molecular sieves (150 mg) was
added MeCN (10 mL), and the mixture was stirred at room tem-
perature for 1 h. A solution of N-iodosuccinimide (24.7 mg,
0.11 mmol) in MeCN (0.3 mL) was added under nitrogen, and the
temperature was lowered to ꢁ45 ꢀC. Trifluoromethanesulfonic acid
(4 mL, 0.05 mmol) was added, and the mixture was stirred for 3 h
at ꢁ45 ꢀC. iPr2NH (50
mL) was added, and the stirring was continued
for 5 min. The mixture was filtered (Celite), washed (saturated
aqueous NaHCO3 and water), dried (MgSO4), and concentrated. The
residue was chromatographed (Cy-EtOAc 1:3) to give 9 as a white
amorphous solid (25.5 mg, 39%).
4.2.3. Method C (5b þ 6)
To a stirred mixture of 6 (262.6 mg, 0.57 mmol), Ag2CO3
(183.4 mg, 0.67 mmol), and 482.1 mg of drierite was added CH2Cl2
(20 mL), and the mixture was stirred at room temperature for 1 h,
then the mixture was cooled to ꢁ45 ꢀC. A solution of 5b (96.9 mg,
0.19 mmol) in 2 mL of CH2Cl2 was added dropwise. After stirring
at ꢁ45 ꢀC for 20 h, then at room temperature for 1 day, the mixture
was filtered. The filtrate was washed with water, dried (MgSO4),
and concentrated. The residue was chromatographed (Cy-EtOAc
1:3) to give 9 as a white amorphous solid (9.1 mg, 5%).
Human cell lines
Compounds
1
2
3
4
HaCaT (human HaCaT keratinocyte line)
ndb ndb ndb ndb
HCT116 (human colorectal carcinoma HCT116 cells) 540 ndb 480 430
K562 (human leukemia K562 cells)
ndb ndb 270 320
a
IC50(mM): A sample’s concentration which produces a 50% reduction cell growth.
Each drug concentration was tested in triplicate.
b
nd: Not-detected.
4.2.4. Method D (5b þ 6)
added, and the mixture was cooled to ꢁ68 ꢀC and kept protected
from light. PhSCl (0.28 mL, 2.42 mmol) in dry CH2Cl2 (1 mL) was
added by running the solution down the cold wall of the reaction
flask, and the stirring was continued for 3 h at ꢁ68 ꢀC. The mixture
was diluted with a suspension of silica gel (5 g) in EtOAc (30 mL),
filtered (Celite), washed (saturated aqueous NaHCO3 and water),
To a stirred mixture of 6 (180.7 mg, 0.39 mmol), HgBr2
ꢀ
(194.6 mg, 0.54 mmol), Hg(CN)2 (48.0 mg, 0.19 mmol), and 4 A
powdered molecular sieves (300 mg) in dry CH2Cl2 (15 mL) was
added 5b (200.2 mg, 0.39 mmol) in CH2Cl2 (2 mL). The mixture was
stirred at room temperature for 2 days and then diluted with EtOAc
(20 mL), and the solution was washed with 30% KI aqueous solution
(20 mL ꢂ 3), dried over MgSO4, and concentrated. The residual
dried
(MgSO4),
and
concentrated.
The
residue
was
Fig. 3. The effect of selected compounds 3 and 4 on the cell morphology.

H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
253
syrup was chromatographed (Cy-EtOAc 1:3) to give 9 as a white
2.4 Hz, 1H, H-7), 5.16 (d, J ¼ 10.0 Hz, 1H, NH), 4.99 (dd, J ¼ 9.9,
8.2 Hz, 1H, H-20), 4.89 (d, J ¼ 2.6 Hz, 1H, H-40), 4.84 (dd, J ¼ 10.7,
4.5 Hz, 1H, H-4), 4.56 (d, J ¼ 8.0 Hz, 1H, H-10), 4.51 (dd, J ¼ 10.1,
3.3 Hz, 1H, H-30), 4.34 (dd, J ¼ 12.4, 2.5 Hz, 1H, Ha-9), 4.08e3.91 (m,
5H, H2-60, H-5, Hb-9, OCHaCH2Si), 3.86e3.79 (m, 4H, H-50, COOCH3),
3.63 (dd, J ¼ 10.8, 2.6 Hz, 1H, H-6), 3.57 (dt, J ¼ 9.8, 5.1 Hz, 1H,
OCHbCH2Si), 2.56 (dd, J ¼ 12.7, 4.6 Hz, 1H, H-3eq), 2.18 (s, 3H, OAc),
2.16 (s, 3H, OAc), 2.06 (d, J ¼ 1.4 Hz, 3H, OAc), 2.05 (s, 3H, OAc), 2.03
(s, 3H, OAc), 2.01 (s, 3H, OAc),1.98 (s, 3H, OAc), 1.83 (s, 3H, NAc),1.69
(t, J ¼ 12.4 Hz, 1H, H-3ax), 1.03e0.85 (m, 2H, OCH2CH2Si), ꢁ0.01 (s,
amorphous solid (29.1 mg, 8%). Rf
¼
0.38 (Cy-EtOAc 1:5).
20
[
a]
¼ ꢁ12.0 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3):
d 7.43e
D
7.25 (m,10H, AreH), 5.46e5.35 (m,1H, H-8), 5.30 (dd, J ¼ 8.1, 2.1 Hz,
1H, H-7), 5.19 (d, J ¼ 9.6 Hz, 1H, NH), 4.92e4.80 (m, 2H, H-4,
OCH2Ph), 4.71 (d, J ¼ 11.8 Hz, 1H, OCH2Ph), 4.58 (s, 2H, OCH2Ph),
4.43 (d, J ¼ 7.7 Hz,1H, H-10), 4.31 (dd, J ¼ 12.5, 2.6 Hz,1H, Ha-9), 4.14
(dd, J ¼ 9.6, 3.3 Hz, 1H, H-30), 4.11e3.94 (m, 4H, H-5, H-6,
OCHaCH2Si, Hb-9), 3.83e3.70 (m, 6H, H-40, H2-60, COOCH3), 3.67e
3.59 (m, 2H, H-50, OCHbCH2Si), 3.51 (dd, J ¼ 9.5, 7.8 Hz, 1H, H-20),
2.53 (dd, J ¼ 13.0, 4.7 Hz, 1H, H-3eq), 2.09 (s, 3H, OAc), 2.07e2.03
(m, 1H, H-3ax), 2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.95 (s, 3H, OAc),
1.86 (s, 3H, NAc), 1.02 (dd, J ¼ 9.3, 8.0 Hz, 2H, OCH2CH2Si), 0.01 (s,
9H, Si(CH3)3). 13C NMR (100 MHz, CDCl3):
d 170.96 (C]O), 170.66
(C]O), 170.59 (C]O), 170.46 (C]O), 170.45 (2 ꢂ C]O), 169.74
(2 ꢂ C]O), 168.11 (C]O), 100.59 (C-10), 96.89 (C-2), 72.14 (C-6),
71.69 (C-30), 70.54 (C-50), 69.96 (C-20), 69.45 (C-4), 68.00 (C-8),
67.82 (C-40), 67.46 (OCH2CH2Si), 67.16 (C-7), 62.51 (C-9), 62.15 (C-
60), 53.23 (COOCH3), 49.21 (C-5), 37.64 (C-3), 23.25 (CH3, NAc), 21.55
(CH3, OAc), 21.15 (CH3, OAc), 20.86 (3 ꢂ CH3, OAc), 20.82 (CH3, OAc),
20.75 (CH3, OAc), 18.05 (OCH2CH2Si), ꢁ1.31 (3C, Si(CH3)3). ESI-
HRMS (m/z) calcd for C37H57NO21SiNa [M þ Na]þ: 902.3085,
found: 902.3117.
9H, Si(CH3)3). 13C NMR (100 MHz, CDCl3):
d 171.01 (C]O), 170.71
(C]O), 170.39 (C]O), 170.21 (C]O), 170.11 (C]O), 168.79 (C]O),
139.31 (C aromatic), 138.37 (C aromatic), 128.47 (2C, CH aromatic),
128.22 (2C, CH aromatic), 127.84 (2C, CH aromatic), 127.74 (C, CH
aromatic), 127.69 (2C, CH aromatic), 127.41 (C, CH aromatic), 103.31
(C-10), 98.12 (C-2), 77.85 (C-20), 75.85 (C-30), 74.95 (OCH2Ph), 73.64
(OCH2Ph), 72.86, 72.79 (C-50, C-6), 69.47 (C-60), 69.20 (C-4), 68.93
(C-8), 68.37 (C-40), 67.48 (OCH2CH2Si), 67.40 (C-7), 62.46 (C-9),
53.13 (COOCH3), 49.41 (C-5), 37.01 (C-3), 23.28 (CH3, NAc), 21.26
(CH3, OAc), 20.94 (CH3, OAc), 20.89 (CH3, OAc), 20.76 (CH3, OAc),
18.59 (OCH2CH2Si), ꢁ1.31 (3C, Si(CH3)3). ESI-HRMS (m/z) calcd for
4.5. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 3)-2,4,6-tri-O-
acetyl- -galactopyranosyl trichloroacetimidate (13)
D-
a-D
b-D
C
45H63NO18SiNa [M þ Na]þ: 956.3707, found: 956.3714.
To a solution of compound 11 (151.8 mg, 0.173 mmol) in 2 mL of
CH2Cl2 was added 4 mL of trifluoroacetic acid dropwise at 0 ꢀC. The
mixture was stirred at 0 ꢀC for 1 h and then at room temperature for
1 h. Then the mixture was diluted with CH2Cl2 and washed with
saturated aqueous NaHCO3 and then with brine, dried over MgSO4.
After concentration, the residue was purified by flash column
chromatography (CH2Cl2eMeOH 25:1). The crude intermediate 12
4.3. 2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero- -galacto-2-nonulopyranosylonate)-
(2 / 3)- -galactopyranoside (10)
D
a-D
b-D
A solution of 9 (524.2 mg, 0.561 mmol) in methanol (15 mL) was
treated with Pd/C (10%, 200 mg) under H2 (160 kPa) for 5 h at 40 ꢀC,
then filtered and evaporated. The residue was purified by flash
column chromatography (CH2Cl2eMeOH 15:1). Compound 10 was
obtained in
a/b
isomers, Rf ¼ 0.25 (CH2Cl2eMeOH 25:1), were
dissolved in 4 mL of dry CH2Cl2, 0.4 mL of trichloroacetonitrile was
obtained (414.6 mg, 98%) as a white amorphous solid. Rf ¼ 0.39
added to the solution and then 34 mL of DBU was added dropwise
20
(CH2Cl2eMeOH 15:1). [
a]
¼ ꢁ10.2 (c 1.0 in CHCl3). 1H NMR
at ꢁ5 ꢀC under nitrogen. The mixture was stirred at ꢁ5 ꢀC for 3 h.
After concentration, the residue was purified by flash column
chromatography (CH2Cl2eMeOH 25:1) to afford compound 13 as
D
(400 MHz, CDCl3): d 5.48e5.36 (m, 2H, H-8, NH), 5.31e5.25 (m, 1H,
H-7), 4.99e4.87 (m, 1H, H-4), 4.39 (d, J ¼ 7.7 Hz, 1H, H-10), 4.29 (dd,
J ¼ 12.4, 2.4 Hz,1H, Ha-9), 3.99 (ddd, J ¼ 13.6, 9.9, 4.6 Hz, 5H, H-6, H-
30, Hb-9, H-5, OCHaCH2Si), 3.84 (t, J ¼ 5.7 Hz, 2H, H2-60), 3.80 (s, 3H,
COOCH3), 3.73 (s, 1H, H-40), 3.68e3.58 (m, 2H, H-20, OCHbCH2Si),
3.52 (t, J ¼ 5.5 Hz, 1H, H-50), 2.83 (s, 1H, OH), 2.74e2.64 (m, 3H, OH,
H-3eq, OH), 2.10 (d, J ¼ 2.3 Hz, 6H, 2 ꢂ OAc), 2.07e2.04 (m, 1H, H-
3ax), 2.01 (s, 6H, 2 ꢂ OAc), 1.86 (s, 3H, NAc), 1.11e0.94 (m, 2H,
OCH2CH2Si), ꢁ0.01 (s, 9H, Si(CH3)3). 13C NMR (100 MHz, CDCl3):
white foam (140.7 mg, 88% for two steps). Rf ¼ 0.31 (CH2Cl2eMeOH
20
25:1). [
a]
¼ þ7.9 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3):
d 8.64
D
(d, J ¼ 9.8 Hz, 1H, C]NH), 6.49 (d, J ¼ 3.9 Hz, 1H, H-10), 5.52 (dd,
J ¼ 5.6, 3.1 Hz, 1H, H-8), 5.39e5.20 (m, 3H, H-7, NH, H-20), 4.96 (d,
J ¼ 3.5 Hz, 1H, H-40), 4.86 (dd, J ¼ 4.5, 1.6 Hz, 1H, H-4), 4.71 (dd,
J ¼ 10.1, 3.5 Hz, 1H, H-30), 4.39 (dd, J ¼ 12.4, 2.4 Hz, 1H, Ha-9), 4.09e
3.89 (m, 5H, H2-60, H-50, H-5, Hb-9), 3.83 (s, 3H, COOCH3), 3.64 (dd,
J ¼ 10.7, 2.7 Hz, 1H, H-6), 2.57 (dd, J ¼ 12.6, 4.6 Hz, 1H, H-3eq), 2.15
(s, 3H, OAc), 2.13 (d, J ¼ 5.7 Hz, 3H, OAc), 2.09 (s, 3H, OAc), 2.03 (s,
3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc),1.97 (s, 3H, OAc),1.81 (s,
3H, NAc), 1.69 (t, J ¼ 12.4 Hz, 1H, H-3ax). 13C NMR (100 MHz, CDCl3):
d
170.99 (C]O), 170.83 (C]O), 170.48 (C]O), 170.30 (C]O), 170.15
(C]O), 168.36 (C]O), 102.64 (C-10), 97.84 (C-2), 76.90 (C-30), 73.73
(C-50), 72.76 (C-6), 69.37 (C-20), 68.72 (C-4), 68.62 (C-40), 68.38 (C-
8), 67.26 (OCH2CH2Si), 67.16 (C-7), 62.63 (C-9), 62.19 (C-60), 53.34
(COOCH3), 49.53 (C-5), 37.49 (C-3), 23.21 (CH3, NAc), 21.29 (CH3,
OAc), 20.91 (CH3, OAc), 20.85 (CH3, OAc), 20.84 (CH3, OAc), 18.28
(OCH2CH2Si), ꢁ1.32 (3C, Si(CH3)3). ESI-HRMS (m/z) calcd for
d
170.89 (C]O), 170.67 (2 ꢂ C]O), 170.45 (C]O), 170.42 (C]O),
170.30 (C]O), 169.72 (C]O), 169.45 (C]O), 168.00 (C]O), 161.17
(C]NH), 96.68 (C-2), 94.11 (C-10), 90.72 (CCl3), 72.30 (C-6), 71.67
(C-50), 71.17 (C-30), 69.40 (C-4), 68.80 (C-20), 68.31 (C-8), 67.46 (C-
40), 67.29 (C-7), 62.62 (C-9), 61.64 (C-60), 53.26 (COOCH3), 49.09 (C-
5), 37.56 (C-3), 23.19 (CH3, NAc), 21.53 (CH3, OAc), 20.83 (4 ꢂ CH3,
OAc), 20.76 (CH3, OAc), 20.73 (CH3, OAc). ESI-HRMS (m/z) calcd for
C
31H51NO18SiNa [M þ Na]þ: 776.2768, found: 776.2804.
4.4. 2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero- -galacto-2-nonulopyranosylonate)-
(2 / 3)-2,4,6-tri-O-acetyl- -galactopyranoside (11)
D
a-D
b-D
C
34H45Cl3N2O21Na [M þ Na]þ: 945.1473, found: 945.1437.
A solution of 5 (350.2 mg, 0.465 mmol) in 8 mL of pyridine and
4 mL of acetic anhydride was stirred at room temperature for 8 h
and then concentrated, co-evaporated with toluene. The resulting
residue was purified by flash column chromatography (Cy-EtOAc
4.6. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 3)-(2,4,6-tri-
O-acetyl- -galactopyranosyl)-(1 / 1)-(2S,3R,4E)-2-azido-3-O-
benzoyl-4-octadecene-l,3-diol (14)
D-
a-D
b-D
1:5). Compound 7 was obtained (408.8 mg, quantitative) as a white
20
amorphous solid. Rf ¼ 0.34 (EtOAc). [
a]
¼ ꢁ9.2 (c 1.0 in CHCl3). 1H
A solution of 13 (138.1 mg, 0.149 mmol) and 3-O-benzoyl-azi-
dosphingosine 6 (115.6 mg, 0.269 mmol) in 5 mL of dry CH2Cl2 was
D
NMR (400 MHz, CDCl3):
d
5.60e5.49 (m, 1H, H-8), 5.35 (dd, J ¼ 9.1,

254
H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
4), 67.19 (C-7), 62.48 (C-9), 62.02 (C-60), 53.69 (C-200), 53.22
(COOCH3), 49.19 (C-5), 37.62 (C-3), 32.50 (CH2, C-600), 31.99, 29.75,
29.73, 29.67, 29.52, 29.42, 29.31, 29.00, 22.76 (11 ꢂ CH2), 23.22
(CH3, NAc), 21.53 (CH3, OAc), 21.05 (CH3, OAc), 20.84 (3 ꢂ CH3, OAc),
20.78 (CH3, OAc), 20.69 (CH3, OAc), 14.19 (CH3). ESI-HRMS (m/z)
calcd for C57H85N2O23 [M þ H]þ: 1165.5538, found: 1165.5532.
ꢀ
stirred with 4 A powdered molecular sieves (300 mg) under nitro-
gen. The mixture was cooled to ꢁ15 ꢀC, and BF3∙Et2O (94
mL,
0.74 mmol) was added dropwise, stirred for 2.5 h at ꢁ15 ꢀC and then
filtered through Celite. The filtrate was washed with saturated
aqueous NaHCO3 and then with water, dried over MgSO4 and
concentrated. The residue was applied to a flash chromatography
eluted with CH2Cl2eMeOH 25:1 to give the product 14 (147.3 mg,
83%) as an amorphous solid. Rf ¼ 0.38 (CH2Cl2eMeOH 25:1).
4.8. O-(5-Acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulo
20
[a]
¼ ꢁ14.5 (c 1.0 in CHCl3). 1H NMR (400 MHz, CDCl3):
d
8.11e7.97
pyranosylonic acid)-(2 / 3)-( -galactopyranosyl)-(1/1)-
b-D
D
(m, 2H, AreH), 7.62e7.38 (m, 3H, AreH), 5.92 (dd, J ¼ 14.2, 7.3 Hz,1H,
H-500), 5.65e5.45 (m, 3H, H-300, H-400, H-8), 5.38 (dd, J ¼ 9.0, 2.7 Hz,
1H, H-7), 5.16 (d, J ¼ 10.2 Hz,1H, NH), 5.05 (dd, J ¼ 10.1, 8.0 Hz,1H, H-
20), 4.88 (ddd, J ¼ 12.1, 9.1, 3.6 Hz, 2H, H-4, H-40), 4.64 (d, J ¼ 8.0 Hz,
1H, H-10), 4.55 (dd, J ¼ 10.1, 3.4 Hz,1H, H-30), 4.35 (dd, J ¼ 12.4, 2.7 Hz,
1H, Ha-9), 4.09e3.93 (m, 5H, H-5, H2-60, H-200, Hb-9), 3.89e3.79 (m,
5H, Ha-100, H-50, COOCH3), 3.71e3.60 (m, 2H, Hb-100, H-6), 2.57 (dd,
J ¼ 12.7, 4.6 Hz,1H, H-3eq), 2.26e2.20 (m, 3H, OAc), 2.16 (d, J ¼ 2.2 Hz,
3H, OAc), 2.08 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.05 (m, 2H, H2-600), 2.02
(s, 3H, OAc), 1.99 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.84 (s, 3H, NAc), 1.70
(td, J ¼ 12.5, 4.1 Hz, 1H, H-3ax), 1.36 (m, 2H, H2-700), 1.24 (s, 20H,
10 ꢂ CH2), 0.86 (t, J ¼ 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3):
(2S,3R,4E)-2-octadecanamido-4-octadecene-l,3-diol (1)
The mixture of 15 (19.9 mg, 0.017 mmol), stearic acid (16.8 mg,
0.059 mmol), EDC (16 mL, 0.090 mmol) in 4 mL of CH2Cl2 was stirred
at room temperature for 20 h. Then the mixture was washed with
water, dried over MgSO4 and concentrated. The resulting residue
was purified by flash column chromatography (Cy-EtOAc 1:3) to
afford crude intermediate 16 as an amorphous solid. Rf ¼ 0.23 (Cy-
EtOAc 1:3). A solution of this crude product in 5 mL of NaOMe/
MeOH (0.04 M) was stirred at room temperature for 14 h. A few
drops of water were added at 0 ꢀC. After stirring at room tempera-
ture for 1 h, the mixture was neutralized by Amberlite IR 120/Hþ ion
exchange resin. After filtration and concentration, the residue ob-
tained was flash-chromatographed, eluting with CHCl3eMeOH 3:1
to afford 1 (14.0 mg, 81% for two steps) as a white amorphous solid.
Rf ¼ 0.35 (EtOAc-iPrOH-H2O 3:2:1). The NMR spectral data were in
good agreement with those reported in literature [24]. ESI-HRMS
(m/z) calcd for C53H97N2O16 [M ꢁ H]ꢁ: 1017.6833, found: 1017.6865.
d
170.99 (C]O), 170.72 (C]O), 170.59 (C]O), 170.48 (C]O), 170.43
(C]O), 170.41 (C]O), 169.75 (C]O), 169.74 (C]O), 168.11 (C]O),
165.21 (PhC ¼ O), 138.88 (C-500), 133.25 (C, CH aromatic), 130.15 (C
aromatic),129.87 (2C, CH aromatic),128.54 (2C, CH aromatic),122.92
(C-400), 100.71 (C-10), 96.91 (C-2), 74.92 (C-300), 72.21 (C-6), 71.50 (C-
30), 70.88 (C-50), 69.51 (C-40), 69.43 (C-20), 68.04 (2C, C-8, C-100), 67.71
(C-4), 67.19 (C-7), 63.80 (C-200), 62.40 (C-9), 62.08 (C-60), 53.27
(COOCH3), 49.26 (C-5), 37.62 (C-3), 32.48 (CH2, C-600), 32.02, 29.78,
29.75, 29.69, 29.52, 29.45, 29.28, 28.80, 22.78 (11 ꢂ CH2), 23.27 (CH3,
NAc), 21.57 (CH3, OAc), 21.08 (CH3, OAc), 20.87 (3 ꢂ CH3, OAc), 20.81
(CH3, OAc), 20.71 (CH3, OAc), 14.21 (CH3). ESI-HRMS (m/z) calcd for
4.9. O-(5-Acetamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulo
pyranosylonic acid)-(2 / 3)-( -galactopyranosyl)-(1 / 1)-
b-D
(2S,3R,4E)-2-azido-4-octadecene-l,3-diol (17)
C
57H82N4O23Na [M þ Na]þ: 1213.5262, found: 1213.5272.
A solution of compound 14 (66.9 mg, 0.056 mmol) in 5 mL of
NaOMe/MeOH (0.04 M) was stirred at room temperature for 14 h. A
few drops of water were added at 0 ꢀC. After stirring at room
temperature for 1 h, the mixture was neutralized by Amberlite IR
120/Hþ ion exchange resin. After filtration and concentration, the
residue obtained was flash-chromatographed, eluting with CHCl3e
4.7. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 3)-(2,4,6-tri-
O-acetyl- -galactopyranosyl)-(1 / 1)-(2S,3R,4E)-2-amino-3-O-
D-
a-D
b-D
benzoyl-4-octadecene-l,3-diol (15)
MeOH 3:1 to afford 17 (42.5 mg, 98%) as an amorphous solid.
20
To a solution of compound 14 (58.3 mg, 0.049 mmol) in 5 mL of
toluene and 0.2 mL of water was added 32.1 mg of triphenyl-
phosphine. The mixture was stirred at 45 ꢀC for 12 h. After con-
centration, the residue obtained was flash-chromatographed,
Rf ¼ 0.33 (EtOAc-iPrOH-H2O 3:2:1). [
a]
¼ ꢁ5.5 (c 1.0 in CHCl3e
D
MeOH 1:1). 1H NMR (400 MHz, CD3OD):
d
5.76 (m, 1H, H-500), 5.58e
5.44 (m, 1H, H-400), 4.30 (d, J ¼ 7.8 Hz, 1H, H-10), 4.22e4.16 (m, 1H,
H-300), 4.03 (dd, J ¼ 9.6, 3.1 Hz, 1H, H-30), 3.95e3.91 (m, 1H, H-40),
3.88 (dd, J ¼ 10.1, 2.9 Hz, 1H, Ha-100), 3.86e3.83 (m, 1H, H-8), 3.82e
3.78 (m, 1H, Ha-9), 3.72 (ddt, J ¼ 8.1, 6.7, 4.2 Hz, 5H, Ha-60, H-4, H-5,
Hb-100, Hb-60), 3.67e3.59 (m, 4H, H-200, Hb-9, H-6, NH), 3.59e3.55
(m,1H, H-20), 3.51 (dd, J ¼ 10.6, 8.9 Hz, 2H, H-7, H-50), 2.89e2.81 (m,
1H, H-3eq), 2.07 (dd, J ¼ 13.3, 6.5 Hz, 2H, H2-600), 2.01 (s, 3H, NAc),
1.80e1.72 (m, 1H, H-3ax), 1.40 (m, 2H, H2-700), 1.29 (s, 20H,
10 ꢂ CH2), 0.90 (t, J ¼ 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CD3OD):
eluting with CH2Cl2eMeOH 20:1 to afford 15 (43.4 mg, 76%) as an
20
amorphous solid. Rf ¼ 0.32 (CH2Cl2eMeOH 20:1). [
a
]
¼ þ1.0 (c 1.0
D
in CHCl3). 1H NMR (400 MHz, CDCl3):
d
8.13e7.72 (m, 2H, AreH),
7.57e7.34 (m, 3H, AreH), 5.93e5.68 (m, 1H, H-500), 5.52 (ddd,
J ¼ 12.0, 7.3, 2.0 Hz, 2H, H-400, H-8), 5.44e5.30 (m, 2H, H-300, H-7),
5.26e5.21 (m, 1H, NH), 5.05e4.93 (m, 1H, H-20), 4.87 (dt, J ¼ 12.1,
4.6 Hz, 2H, H-40, H-4), 4.59 (d, J ¼ 7.9 Hz, 1H, H-10), 4.57e4.50 (m,
1H, H-30), 4.34 (dd, J ¼ 12.3, 2.6 Hz, 1H, Ha-9), 4.06e3.91 (m, 4H, H-
5, H2-60, Hb-9), 3.87e3.76 (m, 5H, H-50, COOCH3, Ha-100), 3.76e3.69
(m, 1H, Hb-100), 3.63 (dt, J ¼ 10.5, 3.3 Hz, 1H, H-6), 3.30 (dd, J ¼ 10.1,
6.5 Hz, 1H, H-200), 3.09 (s, 2H, NH2), 2.56 (dd, J ¼ 12.7, 4.6 Hz, 1H, H-
3eq), 2.19 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.05 (s, 3H,
OAc), 2.02 (d, J ¼ 5.3 Hz, 2H, H2-600), 2.00 (s, 3H, OAc), 1.97 (d,
J ¼ 7.5 Hz, 6H, 2 ꢂ OAc), 1.83 (s, 3H, NAc), 1.68 (t, J ¼ 12.4 Hz, 1H, H-
3ax), 1.39e1.31 (m, 2H, H2-700), 1.24e1.17 (m, 20H, 10 ꢂ CH2), 0.85 (t,
d
175.43 (C]O), 135.77 (C-500), 129.63 (C-400), 104.96 (C-10), 77.83 (C-
30), 76.61 (C-50), 74.96 (C-6), 73.48 (C-300), 72.84 (C-8), 70.66 (C-20),
69.98 (C-100), 69.94 (C-7), 69.28 (C-4), 68.98 (C-40), 67.32 (C-200),
64.44 (C-9), 62.66 (C-60), 53.92 (C-5), 42.03 (C-3), 33.40 (CH2, C-600),
33.05, 30.77, 30.73, 30.59, 30.45, 30.25, 30.19, 23.71 (11 ꢂ CH2),
22.62 (CH3, NAc), 14.42 (CH3). ESI-HRMS (m/z) calcd for
C
35H61N4O15 [M ꢁ H]ꢁ: 777.4139, found: 777.4109.
J ¼ 6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3):
d
170.96 (C]O),
4.10. O-(5-Acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-nonulo
pyranosylonic acid)-(2 / 3)-( -galactopyranosyl)-(1 / 1)-
(2S,3R,4E)-2-amino-4-octadecene-l,3-diol (2)
170.66 (C]O), 170.59 (C]O), 170.50 (C]O), 170.39 (2 ꢂ C]O),
169.85 (C]O), 169.74 (C]O), 168.06 (C]O), 165.39 (PhC ¼ O),
137.78 (C-500), 133.03 (C, CH aromatic), 130.55 (C aromatic), 129.70
(2C, CH aromatic), 128.45 (2C, CH aromatic), 124.43 (C-400), 100.88
(C-10), 96.88 (C-2), 76.26 (C-300), 72.16 (C-6), 71.45 (C-30), 70.70
(C-50), 70.66 (C-100), 69.82 (C-20), 69.43 (C-40), 67.99 (C-8), 67.71 (C-
b-D
To a solution of 17 (27.8 mg, 0.036 mmol) in dry MeOH (3 mL)
were added under nitrogen propane-1,3-dithiol (0.3 mL) and trie-
thylamine (0.3 mL), and the mixture was stirred at room

H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
255
temperature for 4 days. A white precipitate was formed. After
filtration, and washing with MeOH, the filtrate was concentrated.
The residue obtained was flash-chromatographed, eluting with
mixture was stirred at 0 ꢀC for 1 h and then at room temperature for
1 h. Then the mixture was diluted with CH2Cl2 and washed with
saturated aqueous NaHCO3 and then with brine, dried over MgSO4.
After concentration, the residue was purified by flash column
chromatography (Cy-EtOAc 1:3). The crude intermediate 19 ob-
CHCl3eMeOH 3:1 to afford 2 (25.2 mg, 93%) as a white amorphous
20
solid. Rf ¼ 0.28 (EtOAc-iPrOH-H2O 3:2:1). [
a]
¼ ꢁ2.3 (c 1.0 in
D
CHCl3eMeOH 1:1). 1H NMR (400 MHz, CD3OD):
d
5.93e5.81 (m, 1H,
tained in
a
/
b
isomers, Rf ¼ 0.33 (EtOAc), were dissolved in 12 mL of
H-500), 5.54e5.44 (m, 1H, H-400), 4.35 (d, J ¼ 7.8 Hz, 1H, H-10), 4.33e
4.29 (m, 1H, H-300), 4.04 (dd, J ¼ 9.7, 3.1 Hz, 1H, H-30), 3.99e3.93 (m,
2H, Ha-100, H-40), 3.91 (d, J ¼ 2.7 Hz, 1H, Hb-100), 3.87e3.69 (m, 6H,
H-8, Ha-9, Ha-60, H-4, H-5, Hb-60), 3.64e3.48 (m, 5H, Hb-9, H-6, H-
20, H-50, H-7), 3.42e3.37 (m, 1H, H-20), 3.35 (s, 1H, NH), 2.87 (dd,
J ¼ 12.5, 3.9 Hz, 1H, H-3eq), 2.10 (dd, J ¼ 13.8, 6.7 Hz, 2H, H2-600),
2.02 (d, J ¼ 1.7 Hz, 3H, NAc),1.70 (d, J ¼ 11.3 Hz, 1H, H-3ax), 1.42 (dd,
J ¼ 13.9, 6.7 Hz, 2H, H2-700),1.29 (s, 20H,10 ꢂ CH2), 0.90 (t, J ¼ 6.9 Hz,
dry CH2Cl2, 1.2 mL of trichloroacetonitrile was added to the solution
and then 93
m
L of DBU was added dropwise at ꢁ5 ꢀC under nitro-
gen. The mixture was stirred at ꢁ5 ꢀC for 3 h. After concentration,
the residue was purified by flash column chromatography (Cy-
EtOAc 1:4) to afford compound 20 as white foam (322.0 mg, 85% for
20
two steps). Rf ¼ 0.43 (EtOAc). [
a]
¼ þ3.8 (c 1.0 in CHCl3). 1H NMR
D
(300 MHz, CDCl3):
d
8.62 (s, 1H, NH), 6.53 (d, J ¼ 3.6 Hz, 1H, H-10),
5.51 (t, J ¼ 9.8 Hz, 1H, H-30), 5.39 (m, 1H, H-8), 5.30e5.27 (m, 2H, H-
50, H-7), 5.09 (dd, J ¼ 10.2, 3.6 Hz, 1H, H-20), 4.86e4.80 (m, 1H, H-4),
4.23 (d, J ¼ 11.3 Hz,1H, Ha-9), 4.03 (m, 5H, H-40, Hb-9, H-5, Ha-60, H-
6), 3.78 (s, 3H, COOCH3), 3.40 (d, J ¼ 11.1 Hz, 1H, Hb-60), 2.59 (dd,
J ¼ 12.7, 4.4 Hz, 1H, H-3eq), 2.13 (s, 3H, OAc), 2.12 (s, 3H, OAc), 2.07
(s, 3H, OAc), 2.01 (s, 9H, 3 ꢂ OAc), 1.98 (s, 3H, OAc), 1.92 (m, 1H, H-
3H, CH3). 13C NMR (100 MHz, CD3OD):
d 175.58 (C]O), 174.78 (C]
O), 136.74 (C-500), 128.29 (C-400), 104.21 (C-10), 101.00 (C-2), 77.60 (C-
30), 76.95 (C-50), 74.99 (C-6), 72.92 (C-8), 70.86 (C-300), 70.75 (C-20),
70.01 (C-7), 69.20 (C-4), 68.94 (C-40), 66.84 (C-100), 64.56 (C-9),
62.76 (C-60), 56.79 (C-200), 53.99 (C-5), 42.19 (C-3), 33.37 (CH2, C-600),
33.05, 30.77, 30.73, 30.72, 30.62, 30.45, 30.40, 30.16, 23.71
(11 ꢂ CH2), 22.60 (CH3, NAc), 14.42 (CH3). ESI-HRMS (m/z) calcd for
3ax), 1.85 (s, 3H, NAc). 13C NMR (75 MHz, CDCl3):
d 171.16 (C]O),
170.72 (C]O), 170.32 (2 ꢂ C]O), 170.19 (C]O), 170.12 (C]O),
170.00 (C]O), 169.12 (C]O), 167.70 (C]O), 161.03 (C]NH), 98.37
(C-2), 93.26 (C-10), 90.84 (CCl3), 72.41, 70.66, 70.38, 69.92, 69.09,
68.03, 67.54, 67.25 (C-6, C-40, C-30, C-20, C-4, C-8, C-50, C-7), 62.48
(C-9), 61.96 (C-60), 53.03 (COOCH3), 49.43 (C-5), 37.79 (C-3), 23.31
(CH3, NAc), 21.24 (CH3, OAc), 20.97 (CH3, OAc), 20.93 (CH3, OAc),
20.85 (CH3, OAc), 20.73 (2 ꢂ CH3, OAc), 20.57 (CH3, OAc). ESI-HRMS
(m/z) calcd for C34H45Cl3N2O21Na [M þ Na]þ: 945.1473, found:
945.1431.
C
35H63N2O15 [M ꢁ H]ꢁ: 751.4234, found: 751.4265.
4.11. 2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero- -galacto-2-nonulopyranosylonate)-
(2/6)-2,3,4-tri-O-acetyl- -glucopyranoside (18)
D
a-D
b-D
A mixture of compound 5 (740.0 mg, 1.24 mmol) and 7 (336 mg,
ꢀ
0.83 mmol), 4 A powdered molecular sieves (2.0 g), dry CH3CN
(20 mL), and dry CH2Cl2 (10 mL) was stirred under nitrogen for 1 h.
AgOTf (427 mg, 1.66 mmol) and DTBP (0.39 mL, 1.74 mmol) were
added, and the mixture was cooled to ꢁ68 ꢀC and kept protected
from light. PhSCl (0.20 mL, 1.74 mmol) in dry CH2Cl2 (1 mL) was
added by running the solution down the cold wall of the reaction
flask, and the stirring was continued for 3 h at ꢁ68 ꢀC. The mixture
was diluted with a suspension of silica gel (5 g) in EtOAc (30 mL),
filtered (Celite), washed (saturated aqueous NaHCO3 and water),
dried (MgSO4), and concentrated. The residue was chromato-
4.13. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 6)-(2,3,4-tri-
O-acetyl- -glucopyranosyl)-(1 / 1)-(2S,3R,4E)-2-azido-3-O-
benzoyl-4-octadecene-l,3-diol (21)
D-
a-D
b-D
A solution of 20 (114.6 mg, 0.124 mmol) and 3-O-benzoyl-azi-
dosphingosine 6 (90.2 mg, 0.21 mmol) in 6 mL of dry CH2Cl2 was
ꢀ
stirred with 4 A powdered molecular sieves (500 mg) under ni-
graphed (Cy-EtOAc 1:3) to give 18 as a white amorphous solid
trogen. The mixture was cooled to ꢁ15 ꢀC, and BF3∙Et2O (78
mL,
20
(598.9 mg, 82%). Rf ¼ 0.55 (Cy-EtOAc 1:3, 2 times). [
a
]
¼ ꢁ0.6 (c
0.62 mmol) was added dropwise, stirred for 2.5 h at ꢁ15 ꢀC and
then filtered through Celite. The filtrate was washed with saturated
aqueous NaHCO3 and then with water, dried over MgSO4 and
concentrated. The residue was applied to a flash chromatography
D
1.0 in CHCl3). 1H NMR (300 MHz, CDCl3):
d
5.37e5.26 (m, 2H, H-8,
H-7), 5.18e5.06 (m, 3H, H-50, H-30, NH), 4.98e4.79 (m, 2H, H-20, H-
4), 4.44 (d, J ¼ 8.0 Hz, 1H, H-10), 4.27 (dd, J ¼ 12.4, 2.5 Hz, 1H, Ha-9),
4.13e3.86 (m, 5H, Hb-9, H-5, H-6, OCHaCH2Si, Ha-60), 3.78 (s, 3H,
COOCH3), 3.63e3.48 (m, 3H, H-40, Hb-60, OCHbCH2Si), 2.61 (dd,
J ¼ 12.8, 4.6 Hz, 1H, H-3eq), 2.13 (d, J ¼ 2.8 Hz, 6H, 2 ꢂ OAc), 2.05 (s,
3H, OAc), 2.02 (d, J ¼ 3.0 Hz, 9H, 3 ꢂ OAc), 1.99 (s, 3H, OAc), 1.96e
1.88 (m, 1H, H-3ax), 1.86 (s, 3H, NAc), 0.98e0.80 (m, 2H,
OCH2CH2Si), ꢁ0.01 (s, 9H, Si(CH3)3). 13C NMR (100 MHz, CDCl3):
eluted with Cy-EtOAc 1:2 to give the product 21 (113.7 mg, 77%) as
20
an amorphous solid. Rf ¼ 0.48 (EtOAc). [
a]
¼ ꢁ39.1 (c 1.0 in CHCl3).
D
1H NMR (400 MHz, CDCl3):
d
8.07e8.01 (m, 2H, AreH), 7.59e7.53
(m, 1H, AreH), 7.48e7.41 (m, 2H, AreH), 5.95e5.87 (m, 1H, H-500),
5.59e5.49 (m, 2H, H-300, H-400), 5.36e5.27 (m, 2H, H-8, H-7), 5.16
(ddd, J ¼ 19.1, 8.1, 5.2 Hz, 3H, H-50, H-30, NH), 5.03e4.95 (m, 1H, H-
20), 4.85 (ddd, J ¼ 12.3, 9.7, 4.7 Hz,1H, H-4), 4.47 (d, J ¼ 7.9 Hz,1H, H-
10), 4.26 (dd, J ¼ 12.4, 2.6 Hz, 1H, Ha-9), 4.02 (ddd, J ¼ 11.2, 9.7,
3.2 Hz, 3H, Hb-9, H-5, H-6), 3.96e3.85 (m, 3H, H-200, Ha-60, Ha-100),
3.75 (s, 3H, COOCH3), 3.62 (dd, J ¼ 5.7, 2.5 Hz, 1H, H-40), 3.57e3.50
(m, 2H, Hb-100, Hb-60), 2.59 (dd, J ¼ 12.8, 4.6 Hz, 1H, H-3eq), 2.12 (d,
J ¼ 1.5 Hz, 6H, 2 ꢂ OAc), 2.08 (s, 3H, OAc), 2.06 (d, J ¼ 2.1 Hz, 2H, H2-
600), 2.05 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.00 (s, 3H,
OAc), 1.96e1.90 (m, 1H, H-3ax), 1.86 (s, 3H, NAc), 1.37 (d, J ¼ 6.9 Hz,
2H, H2-700), 1.24 (s, 20H, 10 ꢂ CH2), 0.87 (t, J ¼ 6.9 Hz, 3H, CH3). 13C
d
171.18 (C]O), 170.75 (C]O), 170.64 (C]O), 170.33 (C]O), 170.16
(2 ꢂ C]O), 169.45 (C]O), 169.19 (C]O), 167.65 (C]O), 100.38 (C-
10), 98.60 (C-2), 73.54 (C-30), 72.44 (C-6), 72.38 (C-40), 71.58 (C-20),
69.17 (C-4), 68.45 (C-50), 68.17 (C-8), 67.47 (OCH2CH2Si), 67.29 (C-7),
62.89 (C-60), 62.49 (C-9), 52.82 (COOCH3), 49.47 (C-5), 37.76 (C-3),
23.34 (CH3, NAc), 21.31 (CH3, OAc), 21.00 (CH3, OAc), 20.98 (CH3,
OAc), 20.89 (2 ꢂ CH3, OAc), 20.83 (CH3, OAc), 20.81 (CH3, OAc),17.97
(OCH2CH2Si), ꢁ1.29 (3C, Si(CH3)3). ESI-HRMS (m/z) calcd for
C
37H57NO21SiNa [M þ Na]þ: 902.3085, found: 902.3124.
NMR (100 MHz, CDCl3):
d 171.12 (C]O), 170.71 (C]O), 170.55 (C]
4.12. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 6)-2,3,4-tri-O-
acetyl- -glucopyranosyl trichloroacetimidate (20)
D
-
O), 170.32 (C]O), 170.17 (C]O), 170.11 (C]O), 169.40 (C]O),
169.13 (C]O),167.75 (C]O),165.21 (PhC ¼ O),139.22 (C-500),133.33
(C, CH aromatic), 130.08 (C aromatic), 129.89 (2C, CH aromatic),
128.59 (2C, CH aromatic), 122.73 (C-400), 100.91 (C-10), 98.61 (C-2),
75.02 (C-300), 73.26 (C-30), 72.67 (C-40), 72.52 (C-6), 71.28 (C-20),
69.12 (C-4), 68.41 (C-50), 68.35 (C-100), 68.17 (C-8), 67.35 (C-7), 63.71
a-D
b-D
To a solution of compound 18 (361 mg, 0.41 mmol) in 3 mL of
CH2Cl2 was added 6 mL of trifluoroacetic acid dropwise at 0 ꢀC. The

256
H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
(C-200), 62.91 (C-60), 62.55 (C-9), 52.85 (COOCH3), 49.52 (C-5), 37.78
(C-3), 32.53 (CH2, C-600), 32.05, 29.81, 29.78, 29.73, 29.54, 29.48,
29.31, 28.90, 22.81 (11 ꢂ CH2), 23.32 (CH3, NAc), 21.26 (CH3, OAc),
20.96 (CH3, OAc), 20.95 (CH3, OAc), 20.85 (CH3, OAc), 20.79 (CH3,
OAc), 20.78 (CH3, OAc), 20.76 (CH3, OAc),14.24 (CH3). ESI-HRMS (m/
were in good agreement with those reported in literature [25]. ESI-
HRMS (m/z) calcd for C53H97N2O16 [M ꢁ H]ꢁ: 1017.6844, found:
1017.6864.
4.16. O-(5-Acetamido-3,5-dideoxy-
D
-glycero-a-D-galacto-2-
z) calcd for
C
57H82N4O23Na [M
þ
Na]þ: 1213.5262, found:
nonulopyranosylonic acid)-(2 / 6)-(b-D-glucopyranosyl)-(1 / 1)-
1213.5274.
(2S,3R,4E)-2-amino-4-octadecene-l,3-diol (4)
4.14. O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero- -galacto-2-nonulopyranosylonate)-(2 / 6)-(2,3,4-tri-
O-acetyl- -glucopyranosyl)-(1 / 1)-(2S,3R,4E)-2-amino-3-O-
benzoyl-4-octadecene-l,3-diol (22)
D
-
A solution of compound 21 (29.8 mg, 0.025 mmol) in 5 mL of
NaOMe/MeOH (0.04 M) was stirred at room temperature for 14 h. A
few drops of water were added at 0 ꢀC. After stirring at room
temperature for 1 h, the mixture was neutralized by Amberlite IR
120/Hþ ion exchange resin. After filtration and concentration, the
residue was dried in vacuo to afford crude intermediate 24, Rf ¼ 0.32
(EtOAc-iPrOH-H2O 3:2:1). To a solution of this crude product in dry
MeOH (3 mL) were added under nitrogen propane-1,3-dithiol
(0.3 mL) and triethylamine (0.3 mL), and the mixture was stirred
at room temperature for 4 days. A white precipitate was formed.
After filtration, and washing with MeOH, the filtrate was concen-
trated. The residue obtained was flash-chromatographed, eluting
with CHCl3eMeOH 3:1 to afford 4 (16.9 mg, 90% for two steps) as a
a-D
b-D
To a solution of compound 21 (51.2 mg, 0.043 mmol) in 5 mL of
toluene and 0.2 mL of water was added 28.2 mg of triphenyl-
phosphine. The mixture was stirred at 45 ꢀC for 12 h. After con-
centration, the residue obtained was flash-chromatographed,
eluting with CH2Cl2eMeOH 30:1 to afford 22 (41.1 mg, 82%) as an
20
amorphous solid. Rf ¼ 0.24 (CH2Cl2eMeOH 30:1). [
a
]
¼ þ0.4 (c 1.0
D
in CHCl3). 1H NMR (400 MHz, CDCl3):
d
8.07e7.96 (m, 2H, AreH),
7.59e7.41 (m, 3H, AreH), 5.93e5.84 (m, 1H, H-500), 5.51 (dd, J ¼ 15.4,
7.7 Hz, 1H, H-400), 5.40e5.28 (m, 4H, H-8, H-300, NH, H-7), 5.18e5.12
(m, 2H, H-50, H-30), 5.00e4.94 (m,1H, H-20), 4.94e4.87 (m,1H, H-4),
4.46 (d, J ¼ 8.0 Hz, 1H, H-10), 4.27 (dd, J ¼ 12.4, 2.5 Hz, 1H, Ha-9),
4.03 (td, J ¼ 11.7, 7.4 Hz, 3H, Hb-9, H-6, H-5), 3.90 (dd, J ¼ 11.3,
4.5 Hz, 1H, Ha-60), 3.82e3.78 (m, 1H, Ha-100), 3.75 (s, 3H, COOCH3),
3.61 (dd, J ¼ 9.8, 4.4 Hz, 2H, H-40, Hb-100), 3.50 (dd, J ¼ 11.3, 2.2 Hz,
1H, Hb-60), 3.28 (dd, J ¼ 11.0, 6.4 Hz, 1H, H-200), 2.58 (dd, J ¼ 13.1,
4.8 Hz, 1H, H-3eq), 2.13 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.10e2.06 (m,
2H, H2-600), 2.05 (s, 6H, 2 ꢂ OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc),
2.00 (s, 3H, OAc), 1.94 (dd, J ¼ 9.3, 3.5 Hz, 1H, H-3ax), 1.86 (s, 3H,
NAc), 1.40e1.33 (m, 2H, H2-700), 1.26e1.21 (m, 20H, 10 ꢂ CH2), 0.87
white amorphous solid. Rf
¼
0.28 (EtOAc-iPrOH-H2O 3:2:1).
20
[a]
d6):
¼ þ1.4 (c 1.0 in CHCl3eMeOH 1:1). 1H NMR (400 MHz, DMSO-
D
d
8.41 (d, J ¼ 50.0 Hz, 1H, NH), 5.74e5.60 (m, 1H, H-500), 5.45
(dd, J ¼ 15.4, 6.0 Hz, 1H, H-400), 4.17 (d, J ¼ 7.4 Hz, 2H, H-300, H-10),
3.83 (d, J ¼ 9.1 Hz,1H, Ha-100), 3.69 (dt, J ¼ 10.5, 9.1 Hz, 3H, Ha-9, Hb-
100, Hb-9), 3.57 (d, J ¼ 9.2 Hz, 3H, Ha-60, H-4, H-40), 3.39e3.30 (m,
3H, H-30, H-5, Hb-60), 3.22 (d, J ¼ 8.6 Hz, 2H, H-8, H-50), 3.17e3.08
(m, 2H, H-6, H-200), 3.02 (dd, J ¼ 16.4, 8.5 Hz, 2H, H-20, H-7), 2.63 (d,
J ¼ 7.4 Hz, 1H, H-3eq), 2.07e1.92 (m, 2H, H2-600), 1.89 (s, 3H, NAc),
1.34 (m, 3H, H-3ax, H2-700), 1.24 (s, 20H,10 ꢂ CH2), 0.85 (t, J ¼ 6.8 Hz,
3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 172.45 (C]O), 170.72
(t, J ¼ 6.9 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3):
d
171.09 (C]O),
(C]O), 132.91 (C-500), 128.58 (C-400), 103.24 (C-10), 100.08 (C-2),
76.31 (C-6), 74.88 (C-8), 73.14 (C-7), 72.74 (C-30), 71.35 (C-4), 70.15
(C-20), 69.58 (C-300), 69.05 (C-50), 67.67 (C-100), 67.05 (C-40), 63.33 (C-
9), 63.28 (C-60), 55.72 (C-200), 53.06 (C-5), 41.65 (C-3), 31.67 (CH2, C-
600), 31.31, 29.08, 29.03, 28.96, 28.72, 28.66, 22.11 (11 ꢂ CH2), 22.50
(CH3, NAc), 13.96 (CH3). ESI-HRMS (m/z) calcd for C35H63N2O15
[M ꢁ H]ꢁ: 751.4234, found: 751.4263.
170.73 (C]O), 170.55 (C]O), 170.40 (C]O), 170.17 (C]O), 170.07
(C]O), 169.46 (C]O), 169.20 (C]O), 167.82 (C]O), 165.43
(PhC ¼ O), 138.05 (C-500), 133.16 (C, CH aromatic), 130.53 (C aro-
matic), 129.71 (2C, CH aromatic), 128.56 (2C, CH aromatic), 124.31
(C-400),101.32 (C-10), 98.50 (C-2), 76.43 (C-300), 73.31 (C-30), 72.67 (C-
40), 72.28 (C-6), 71.92 (C-100), 71.52 (C-20), 69.18 (C-4), 68.50 (C-50),
68.30 (C-8), 67.39 (C-7), 62.78 (C-60), 62.50 (C-9), 53.83 (C-200),
52.90 (COOCH3), 49.63 (C-5), 37.60 (C-3), 32.57 (CH2, C-600), 32.06,
29.79, 29.74, 29.58, 29.49, 29.36, 29.09, 22.83 (11 ꢂ CH2), 23.34
(CH3, NAc), 21.25 (CH3, OAc), 21.02 (CH3, OAc), 21.01 (CH3, OAc),
20.87 (CH3, OAc), 20.83 (CH3, OAc), 20.80 (CH3, OAc), 20.77 (CH3,
OAc), 14.25 (CH3). ESI-HRMS (m/z) calcd for C57H85N2O23 [M þ H]þ:
1165.5538, found: 1165.5527.
4.17. Cell culture
Human leukemia K562 and colorectal carcinoma HCT116 cells
were obtained from the American Type Culture Collection (Rock-
ville, MD, USA) and were grown in RPMI 1640 containing 10% fetal
calf serum (FCS) and 1% glutamine. Human HaCaT keratinocyte line
was purchased from Cell Lines Service GmbH (CLS, Eppelheim,
Germany) and cultured in DMEM medium (high glucose) supple-
4.15. O-(5-Acetamido-3,5-dideoxy-
D-glycero-a-D-galacto-2-
nonulopyranosylonic acid)-(2 / 6)-(
(2S,3R,4E)-2-octadecanamido-4-octadecene-l,3-diol (3)
b
-
D
-glucopyranosyl)-(1/1)-
mented with 2 mM L-glutamine and 10% FCS. All cell lines were
maintained at 37 ꢀC in a humidified atmosphere containing 5% CO2.
The mixture of 22 (16.3 mg, 0.014 mmol), stearic acid (13.9 mg,
5. Cytotoxicity assay
0.049 mmol), EDC (13 mL, 0.075 mmol) in 4 mL of CH2Cl2 was stirred
at room temperature for 20 h. Then the mixture was washed with
water, dried over MgSO4 and concentrated. The resulting residue
was purified by flash column chromatography (Cy-EtOAc 1:3) to
afford crude intermediate 23 as an amorphous solid. Rf ¼ 0.41 (Cy-
EtOAc 1:3). A solution of this crude product in 5 mL of NaOMe/
MeOH (0.04 M) was stirred at room temperature for 14 h. A few
drops of water were added at 0 ꢀC. After stirring at room temper-
ature for 1 h, the mixture was neutralized by Amberlite IR 120/Hþ
ion exchange resin. After filtration and concentration, the residue
obtained was flash-chromatographed, eluting with CHCl3eMeOH
3:1 to afford 3 (11.3 mg, 79% for two steps) as a white amorphous
solid. Rf ¼ 0.35 (EtOAc-iPrOH-H2O 3:2:1). The NMR spectral data
In order to evaluate the cytotoxicity of the studied compounds,
we used three different human cell lines: 2 types of cancer cell and
a normal cell line. Stock solution of each compound was prepared
in DMSO at a concentration of 0.02 M and then diluted to their final
concentration in triplicate with medium before use. 0.1% DMSO has
been used as the vehicle control for all the experiments. Cell
viability was assessed using the Promega CellTiter-Blue reagent
(Promega, WI, USA) according to the manufacturer’s instructions.
Briefly, the cells were seeded in 96-well plates (5000 cell/well)
containing 50
were supplemented with 50
in DMSO (less than 0.1% in each preparation). After 72 h of
mL of growth medium. After 24 h of culture, the cells
mL of the studied compound dissolved

H. Qu et al. / European Journal of Medicinal Chemistry 75 (2014) 247e257
[10] A. Marra, P. Sinaÿ, Carbohydrate Research 187 (1989) 35e42.
257
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rescence was recorded (560 nm Ex/590 nm Em) using a Victor
microtiter plate fluorimeter (PerkineElmer, USA). The IC50 value
corresponds to the concentration of the compounds that caused a
decrease of 50% in fluorescence of drug-treated cells relative to
control cells. After treatment, cells were analyzed by inverted light
microscope (TE 2000E, Nikon, Champigny-sur-Marne, France).
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Acknowledgments
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We thank the China Scholarship Council for a Ph.D. fellowship to
Huanhuan Qu. Financial supports from the Centre National de la
Recherche Scientifique (CNRS) and the Université Pierre et Marie
Curie (LIA Programme) are gratefully acknowledged.
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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