Synthesis and anti-angiogenetic activity evaluation of N-(3-aryl acryloyl)aminosaccharide derivatives

Article abstract of DOI:10.1016/j.carres.2013.08.022

In order to find novel potent inhibitors for signal pathways of FGF/FGFR, nineteen N-(3-aryl acryloyl)aminosaccharide derivatives were designed and synthesized based on the binding sites of FGF and oligosaccharides of heparin. Their structures were confirmed by IR, ¹H NMR, ¹³C NMR, MS and elemental analysis. The nineteen target compounds were evaluated for biological activity against HUVEC cell. In vitro assays showed that compound 10s (IC₅₀ = 5.3 μM) exhibited comparable inhibitory effects on endothelial cell growth with topotecan (IC₅₀ = 2.7 μM). Compound 10s (10 μg/egg) also showed obvious anti-angiogenetic activity in the in vivo chicken chorio allantoic membrane (CAM) assay, and the potency was similar to topotecan (10 μg/egg).

Full text of DOI:10.1016/j.carres.2013.08.022

Carbohydrate Research 381 (2013) 83–92
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and anti-angiogenetic activity evaluation
of N-(3-aryl acryloyl)aminosaccharide derivatives
Kun Liu a, Shuowei Yao b, Yinghan Lou b, Yungen Xu a,b,
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2013
Received in revised form 17 August 2013
Accepted 21 August 2013
Available online 29 August 2013
In order to find novel potent inhibitors for signal pathways of FGF/FGFR, nineteen N-(3-aryl acry-
loyl)aminosaccharide derivatives were designed and synthesized based on the binding sites of FGF and
oligosaccharides of heparin. Their structures were confirmed by IR, 1H NMR, 13C NMR, MS and elemental
analysis. The nineteen target compounds were evaluated for biological activity against HUVEC cell. In
vitro assays showed that compound 10s (IC50 = 5.3
thelial cell growth with topotecan (IC50 = 2.7 M). Compound 10s (10
angiogenetic activity in the in vivo chicken chorio allantoic membrane (CAM) assay, and the potency was
similar to topotecan (10 g/egg).
lM) exhibited comparable inhibitory effects on endo-
l
lg/egg) also showed obvious anti-
Keywords:
Tumor angiogenesis inhibitors
Fibroblast growth factor
Heparin mimic
l
Ó 2013 Elsevier Ltd. All rights reserved.
Aminosaccharide
1. Introduction
could regulate FGF signal pathway and have little influence on
the thrombin system in the meantime.8,9 These results suggest that
shorter chain oligosaccharides may avoid the inhibition of the
thrombin system while retaining the action of FGF signal pathway.
Mono-saccharide-based glycoconjugates such as compound 1
(Fig. 1) exhibit potent bioactivities in both a FGF-2 binding assay
and an endothelial cell survival assay.10 Further more, Rawe et al.
found per-O-acetylated glucosamine derivatives such as com-
pound 2 possessing significantly improved inhibitory potency
compared to their corresponding polyhydroxylated deriva-
tives.11,12 Our group also designed and synthesized a series of N-
heteroaroyl aminosaccharide derivatives, and found compound 3
could inhibit heparin- and FGF-2-dependent BaF3 cell
proliferation.13
Cinnamic acid derivatives, such as ferulic acid and sodium caff-
eate, are known to possess notable inhibitory effect on endothelial
cell growth due to their tumor angiogenetic inhibitory activity.
Here, we designed and synthesized a series of N-(3-aryl acry-
loyl)aminosaccharide derivatives by combining aminosaccharide
with cinnamic acid, and evaluated their inhibitory effects on HU-
VEC cell growth through the MTT assay and their anti-angiogenetic
activities through the in vivo CAM assay.
Tumor angiogenesis inhibitors can inhibit the formation of new
blood vessels and block tumor growth, metastasis and relapse.1,2
They showed great advantages when compared to traditional
anti-tumor drugs. Fibroblast growth factors (FGFs) are an impor-
tant class of angiogenic factors. Studies have shown that the FGF
signaling pathway dysregulation is closely related to tumor blood
vessel growth. This signaling pathway particularly plays an impor-
tant role in Angiogenic Rescue.3 For example, animal experiment
results showed that the anti-VEGFR-2 antibody therapy can induce
the release of FGF-2.4 Therefore, the FGF signal pathway is a poten-
tial target for cancer therapy.
Heparin can regulate FGF signal pathway. Heparin/HS is re-
quired to form a ternary complex with FGF and FGFR, which stabi-
lize and activate FGF signaling for a long enough period of time to
elicit a specific response.5 Heparin and its analogs have been found
as FGF signal pathway regulating agents. For example, PI-88,6
a
mixture of highly sulfated monophosphorylated mannose oligo-
saccharides, has entered phase III clinical trial as an anti-tumor
agent. However, bleeding complications of these heparin analogs
limit their clinical application due to the inhibition of thrombin
system.7 The further studies showed that different signal pathways
require different heparin sugar chain lengths. The pentose se-
quence is required for anticoagulant effect. Researchers have found
that disaccharide, trisaccharide, or tetrasaccharide heparanoids
2. Results and discussion
2.1. Synthesis
2,3,4,6-Tetra-O-acetyl-1-deoxy-b-
D
-galactopyranosamine (G1-
⇑
Corresponding author. Tel.: +86 25 86185390.
E-mail addresses: xyg@cpu.edu.cn, xu_yungen@hotmail.com (Y. Xu).
NH2),14,15 1,3,4,6-tetra-O-acetyl-2-deoxy-b-
D-galactopyranosamine
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.08.022

84
K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
OAc
OAc
O
OH
O
O
H
N
H
N
S
S
AcO
AcO
AcO
AcO
HO
HO
OAc
S
OAc
OH
HN
O
O
CH3
O
1
2
3
Figure 1. The structures of compound 1, 2, and 3.
(G2-NH2), and16 methyl 6-amino-6-deoxy-
(G3-NH2)17–21 were synthesized according to the reported refer-
a
-D
-galactopyranoside
imide hydrochloride/1-hydroxybenzotriazole (EDCI/HOBt) as con-
densing agent (Scheme 2). In order to avoid undesirable
deacetylation, the amount of triethylamine as acid-binding agent
should be limited to between 1 and 2 equiv. Compound 10q was
synthesized through acetylation of the compound 10o (Scheme 3).
ences. 1,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-
pyranosyl)-2-amino-2-deoxy-b- -glucopyranose
D
-galacto-
D
p-
toluenesulfonate (G4-NH2ꢀTsOH) was synthesized from lactose (4)
via five steps (Scheme 1).22–25
Treatment of hexa-O-acetyl-D-lactal (5) with cerium(IV) ammo-
2.2. Biological evaluation and discussion
nium nitrate and sodium azide gave the azidonitrates 6a, 6b, and
6c (Scheme 1).22,23 Compound 6c could be easily removed from
the mixture of 6a, 6b, and 6c by column chromatography. How-
ever, 6a and 6b were difficult to be separated under the same con-
dition because of their similar polarity. When the mixture of 6a
and 6b (2:1 according to 1H NMR) reacted with LiBr, the com-
pounds 6a and 6c were easily converted into bromination product
7, and the compound 6b remained unchanged. G4-NH2ꢀTsOH could
be obtained from compound 7 by reaction with Hg(OAc)2, followed
by hydrogenation in the presence of 10% Pd–C and p-toluenesul-
fonic acid.
2.2.1. In vitro MTT assay against HUVEC cell growth
The results of the in vitro MTT assay showed that majority of
target compounds exhibited better inhibitory effect on HUVEC cell
growth than positive compound 2 except 10d and 10k which pro-
moted HUVEC cell proliferation. The IC50 value of compounds 10a,
10j, 10n, and 10o could not be measured due to their poor solubil-
ity in DMSO and medium (Table 1).
Among the active compounds, compounds 10l, 10m, and 10s
showed good inhibitory effects on HUVEC cell growth. Especially
compound 10s (IC50 = 5.3
lM) showed comparable inhibitory
Target compounds 10a–10f, 10r, and 10s were obtained
through the reaction of G1-NH2 or G4-NH2 with the corresponding
acyl chloride derived from diverse cinnamic acids. Compounds
10g–10p were obtained by reacting G2-NH2 or G3-NH2 with diverse
cinnamic acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
activity against HUVEC cell growth with topotecan (IC50 = 2.7
l
M).
The structure–activity relationship showed that electron-with-
drawing group substituted cinnamic acids were more potent than
electron-donating group substituted ones when the saccharide
portion of conjugates was G1-NH2, the order being 3-methoxy-4-
OH
OAc
OH
O
HO
HO
OAc
O
AcO
AcO
O
(1)
O
O
OH
O
HO
AcO
OH
OAc
OH
OAc
4
5
(2)
OAc
OAc
OAc
O
OAc
OAc
O
AcO
AcO
OAc
O
AcO
AcO
AcO
AcO
O
N
3O
O
+
O
+
O
O
ONO2
AcO
AcO
AcO
OAc
OAc
OAc
N3
N3
ONO2
ONO2
6b
6a
6c
(3)
OAc
O
OAc
O
AcO
AcO
6b
+
O
AcO
OAc
(4)
N3
Br
7
OAc
O
OAc
O
OAc
OAc
O
AcO
AcO
AcO
AcO
(5)
O
O
O
OAc
OAc
AcO
AcO
OAc
OAc
.
NH2
N3
TsOH
8
G4-NH2.TsOH
Scheme 1. Synthesis of compound G4-NH2ꢀTsOH. Reagents and conditions: (1) Ac2O, HBr/HAc, rt, 10 h; CuSO4ꢀ5H2O/NaOAc/HAc, 20 °C, 2 h; (2) NaN3, CAN, CH3CN, ꢁ15 °C, N2,
16 h; (3) LiBr, CH3CN, rt, 6 h; (4) Hg(OAc)2/HAc, rt, 17 h; (5) H2, p-TsOH, THF, 10% Pd–C, rt, 6 h.

K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
R1
85
O
R2
G
G
G1-NH2 or G4-NH2
N
H
G-NH2 = G1-NH2
(1)SOCl2
R1
O
or
G4-NH2
R3
R2
R3
(2)Na2CO3, CH2Cl2 /H2O
OH
10a 10f, 10r, 10s
~
R1
O
9
G2-NH2 or G3-NH2
R2
R3
G-NH2 = G2-NH2
N
H
or
G3-NH2
Et3N, EDCI / HOBt
10g~10p
OAc
NH2
AcO
AcO
OAc
O
OAc
AcO
AcO
AcO
AcO
OAc
O
HO
HO
O
O
O
O
NH2
OAc
OAc
AcO
OAc
OAc
NH2
NH2
OH
OCH3
G1-NH2
G2-NH2
G3-NH2
G4-NH2
10a
G-NH G -NH
=
1
R1 = H
R2 = OCH3 R3 = OCH3
10b R1 = OCH3 R2 = OCH3 R3 = OCH3 G-NH = G1-NH
10c R1 = H
R2 = OCH3 R3 = OAc
G-NH = G1-NH
10d
G-NH G -NH
R1 = H
R2 = OAc
R2 = H
R3 = OAc
R3 = H
=
10e R1 = Cl
10f R1 = Cl
G-NH = G11-NH
R2 = H
R3 = Cl
G-NH = G1-NH
10g
G-NH G -NH
=
R1 = H
R2 = H
R3 = OCH3
10h R1 = H
R2 = OCH3 R3 = OCH3 G-NH = G22-NH
10i R1 = OCH3 R2 = OCH3 R3 = OCH3 G-NH = G2-NH
10j
G-NH G -NH
R1 = H
10k R1
10l R1 = Cl
R2 = OCH3 R3 = OAc
=
=
H
R2 = OAc
R2 = H
R2 = H
R3 = OAc
R3 = H
R3 = Cl
G-NH = G22-NH
G-NH = G2-NH
10m
G-NH G -NH
=
R1 = Cl
10n R1 = H
10o R1 = H
R2 = OCH3 R3 = OCH3 G-NH = G23-NH
R2 = OCH3 R3 = OAc
G-NH = G3-NH
10p
10r
G-NH G -NH
=
R1 = Cl
1 = H
R2 = H
R3 = H
R
R2= OCH3 R3 = OCH3 G-NH = G43-NH
10s R1 = Cl
R2= H
R3 = Cl
G-NH = G4-NH
Scheme 2. The structures and synthetic routes of the target compounds 10a–10p, 10r, and 10s.
O
O
OCH3
OAc
OCH3
OAc
NH
O
Ac2O, Pyridine
Et3N, 4-DMAP
NH
O
HO
HO
AcO
AcO
OH
OAc
OCH3
OCH3
10o
10q
Scheme 3. The synthetic route of the target compound 10q.
acetoxy > 2,4-dichloro > 2-chloro ꢂ 2,3,4-trimethoxy. By compar-
ing compound 10m versus 10i, 10p versus 10n, and 10s versus
10r, coincident conclusion could be obtained that electron-with-
drawing group substituted cinnamic acids is beneficial to improve
the expected anti-angiogenetic activity when the saccharide por-
tion was substituted with G2-NH2, G3-NH2, or G4-NH2.
On the other hand, electron-withdrawing group substituted cin-
namic acids conjugated with different aminosaccharides possessed
different levels of inhibitory activities on HUVEC cell growth. Com-
pound 10p was about two times weaker than 10e and 10l. The rea-
son may be the multihydroxy increasing the compounds polarity,
which lead to cell membrane impermeability in the in vitro assay
although they can be transported into cells through active trans-
Table 1
Inhibitory effects of N-(3-aryl acryloyl)aminosaccharide derivatives on endothelial
cell proliferation
Compd
IC50 (lM)
Compd
IC50 (lM)
2
660
2.7
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
>1000a
Topotecan
10a
10b
10c
10d
10e
10f
10g
10h
10i
nob
>1000a
475.6
114.3
nob
93.1
89
>1000a
>1000a
290.1
186.3
281.2
5.3
145.71
132.4
1013.9
356.1
312.2
portation. The activity of 10s (IC50 = 5.3 lM) was 15–30 times
more potent than 10f and 10m although they contained the same
3-(2,4-dichlorophenyl) acryloyl group. Thus, we may infer that
disaccharide conjugates possessed much stronger proliferation
inhibition activities than monosaccharides. It is possible that the
a
>1000: Compounds in setting concentrations appeared to different levels of
turbidity, and their IC50 value could not be measured.
b
Promote proliferation.

86
K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
space structure of the disaccharides is better for combination with
bFGF than monosacchrides.
2.2.2. In vivo CAM assay
Effects of compounds 10m and 10s on angiogenesis of CAM are
shown in Figure 2. The anti-angiogenetic activities of compounds
10m and 10s were semiquantitatively analyzed using Graph Pad
Prism 5.0. (shown in Figure 3). The result showed that both 10m
and 10s (10 lg, p < 0.05) could inhibit the angiogenesis of CAM.
The anti-angiogenetic activity of compound 10s was comparable
with topotecan in the in vivo CAM assay at the same dose
(10 lg/egg).
3. Conclusions
Nineteen novel N-(3-aryl acryloyl)aminosaccharide derivatives
were synthesized and evaluated for their anti-angiogenetic activity
on HUVEC cells through the MTT assay. The results of bioassay and
SAR study showed that disaccharide structure and electron-with-
drawing group substituted cinnamic acids are important to im-
prove the expected anti-angiogenetic activity. This conclusion
was also corroborated by the results of further CAM study. It
pointed out the direction for further modification. What’s more,
we obtained compound 10s which had the potential to be devel-
oped as novel tumor angiogenesis inhibitor candidate. According
to the above conclusion, further investigations will explore the cell
biological studies to determine their action mechanism. Since the
activity observed is possible in general non-specific inhibition,
investigations are also underway to demonstrate whether the
aminosaccharide derivatives have the specific modulation of bFGF.
Figure 3. The anti-angiogenetic activity of compounds 10m and 10s.
NMR spectra were recorded on a Brucker AV-300 or AV-500 NMR
spectrometer using tetramethylsilane (TMS) as an internal stan-
dard and chemical shifts were given in d ppm with TMS. Mass
spectra were recorded on an Agilent 1100 series LC/MSD Tarp
(SL). The Elemental analysis data were obtained using an Elemen-
tar Vario EL III instrument. All solvents were purchased from com-
mercial sources and used as received unless otherwise stated.
4.1.1. Hexa-O-acetyl-lactal (5)
To a stirring suspension of lactose (5.0 g, 14.6 mmol) in acetic
anhydride (13.4 g, 0.13 mol) was added slowly 31% HBr/HAc
(5.0 g, 3.5 mL) at room temperature. After stirring for 24 h at room
temperature CuSO4ꢀ5H2O (0.91 g, 40 mmol) in 50 mL water and
NaOAc (27.4 g, 0.33 mol) in 75 mL acetic acid were added, and
the reaction mixture was stirred violently for 2 h keeping temper-
ature at about 20 °C. The mixture was filtered, and the filter cake
was washed with ethyl acetate (250 mL), water (250 mL). The or-
ganic layer was washed with saturated aqueous NaHCO3
(150 mL) twice and brine (150 mL) twice, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was subjected to flash silica gel column chromatography
using petroleum ether–acetic ether (2:1, v/v) to give compound 5
as a clean oil (3.40 g, 41.5%); 1H NMR (300 MHz, CDCl3) d (ppm):
6.42 (1H, d, J = 6.2 Hz, –CH@), 5.41 (1H, dd, J = 3.9 Hz, J = 3.9 Hz,
GlcAH-3), 5.37 (1H, d, J = 2.7 Hz, GalBH-4), 5.20 (1H, m, GalBH-2),
5.01 (1H, dd, J = 3.3 Hz, J = 10.5 Hz, GalBH-3), 4.84 (1H, dd,
J = 3.6 Hz, J = 5.9 Hz, –CH@), 4.67 (1H, d, J = 7.8 Hz, GalBH-1), 4.44
(1H, d, J = 9.6 Hz, GlcAH-6a), 4.23–4.09 (4H, m, GlcAH-4, GlcAH-
6b, GalBH-6a, GalBH-6b), 4.00 (1H, m, GlcAH-5), 3.98 (1H, dd,
J = 6.9 Hz, J = 6.9 Hz, GalBH-5), 2.16, 2.12, 2.09, 2.07, 2.06, 1.98
(each s, each 3H, 6 ꢃ OAc).
4. Experimental
4.1. General
Melting points were determined on a RDCSY-I capillary appara-
tus and were uncorrected. The IR spectra (in KBr pellets) were re-
corded on a Nicolet Impact 410 spectrophotometer. The 1H and 13
C
4.1.2. 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-azido-2-deoxy-a/b-D-glucopyranosyl/
mannopyranosyl nitrate (6)
NaN3 (1.18 g, 18.2 mmol) and ceric ammonium nitrate (16.6 g,
30.3 mmol) were added into 100 mL reaction flask under N2 pro-
tection at temperature of ꢁ15 °C. Then 5 (6.80 g, 12.1 mmol) in
68 mL anhydrous acetonitrile was added slowly into reaction flask
and the reaction mixture was stirred violently for 16 h. Cold
diethyl ether (80 mL) and cold water (80 mL) were added into
the reaction solution. The aqueous layer was extracted with cold
diethyl ether (60 mL). The combined organic layer was washed
with water (80 mL) twice, dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was chro-
Figure 2. Effects of 10m and 10s on angiogenesis of CAM.

K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
87
matographed on a silica gel column by use of petroleum ether–ace-
tic ether (2:1, v/v) to give the compound as follows:
(1) white solid, 4.0 g, Rf = 0.75 (petroleum/ether–acetic
ether = 2/1).
C@O), 1436, 1372, 1219, 1073, 1046, 896; MS(ESI(+)70 V, m/z):
679.1 [M+NH4]+.
4.1.5. 1,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-amino-2-deoxy-b-D-glucopyranose p-
4.1.2.1. Compound 6a: 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-
toluenesulfonate (G5-NH2ꢀTsOH)
acetyl-b-
syl nitrate.
J = 8.6 Hz, GlcAH-1).
D
-galactopyranosyl)-2-azido-2-deoxy-b-
D
-glucopyrano-
Compound 8 (1.3 g, 1.97 mmol) was dissolved in THF (50 mL).
Then p-TsOHꢀH2O (0.37 g, 1.97 mmol) and 10% Pd–C (0.13 g) were
added into above solution in turn. The reaction mixture was stirred
at room temperature for 6 h. The catalyst was filtered and the fil-
trate was evaporated under reduced pressure. The residue was
chromatographed on a silica gel column by use of dichloromethane
methanol (30:1, v/v) to give G5-NH2ꢀTsOH as a yellow oil (1.0 g,
61.3%). 1H NMR (500 MHz, CDCl3) d (ppm): 8.5 (3H, br s, OH,
NH2), 7.71 (2H, d, J = 7.3 Hz, ArH), 7.12 (2H, d, J = 6.5 Hz, ArH),
5.98 (1H, d, J = 8.7 Hz, GlcNAH-1), 5.52 (1H, dd, J = 9.2 Hz, GlcNA-
H-3), 5.31 (1H, d, J = 3.5 Hz, GalBH-4), 5.04 (1H, dd, J = 7.9 Hz,
J = 10.3 Hz, GalBH-2), 4.92 (1H, dd, J = 3.5 Hz, J = 10.4 Hz, GalBH-
3), 4.48 (1H, d, J = 7.9 Hz, GalBH-1), 4.43 (1H, d, J = 10.7 Hz, GlcNA
H-6a), 4.12–4.05 (3H, m, GalA H-6b, GalB H-6a, GalB H-6b), 3.84–
3.79 (3H, m, GlcNA H-2, GalAH-4, GalB H-5), 3.35 (1H, m, GlcNA
H-5), 2.36 (3H, s, CH3), 2.17, 2.14, 2.10, 2.04, 2.01, 1.96, 1.94 (each
3H, each s, 7 ꢃ OAc); MS(ESI(+)70 V, m/z): 636.2 [M+H]+;
1H NMR (500 MHz, CDCl3) d (ppm): 5.56 (2H, d,
4.1.2.2. Compound 6b: 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-
acetyl-b- -galactopyranosyl)-2-azido-2-deoxy- -mannopyr-
1H NMR (500 MHz, CDCl3) d (ppm): 6.13 (1H,
D
a-D
anosyl nitrate.
d, J = 3.4 Hz, GlcAH-1).
(2) white solid, 1.80 g, Rf = 0.60 (petroleum/ether–acetic
ether = 2/1).
4.1.2.3. Compound 6c: 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-
acetyl-b-
osyl nitrate.
D
-galactopyranosyl)-2-azido-2-deoxy-
a-D-glucopyran-
1H NMR (500 MHz, CDCl3) d (ppm): 6.27 (1H, d,
J = 4.2 Hz, GlcNAH-1), 5.39–5.36 (2H, m, GlcNAH-3, GalBH-4), 5.11
(1H, dd, J = 8.0 Hz, J = 10.3 Hz, GalBH-2), 4.95 (dd, 1H, J = 3.4 Hz,
J = 10.4 Hz, GalBH-3), 4.48 (1H, d, J = 7.9 Hz, GalBH-1), 4.43 (1H,
dd, J = 1.8 Hz, J = 12.3 Hz, GlcNA H-6a), 4.19–4.15 (2H, m, GalBH-
6a, GalBH-6b), 4.10–4.06 (2H, m, GalB H-5, GlcNA H-6b), 3.89–
3.80 (2H, m, GlcNA H-4, GalAH-5), 3.70 (1H, dd, J = 4.2 Hz,
J = 10.8 Hz, GlcNA H-2), 2.17, 2.14, 2.11, 2.06, 2.04, 1.96 (each 3H,
each s, 6 ꢃ OAc).
4.1.6. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-D-galactopyranosyl)-
3-(3,4-dimethoxyphenyl)-2-propenamide (10aꢀ0.5H2O)
3,4-dimethoxycinnamic acid (0.36 g, 1.73 mmol) was sus-
pended in anhydrous CHCl3 (2 mL). SOCl2 (1 mL) was slowly added
to this mixture while cooling in an ice bath, and the resulting solu-
tion was stirred at room temperature for 1 h. After the completion
of the reaction, the solvent was removed under reduced pressure
to give acyl chloride as yellow solid which was used without fur-
ther purification. The new prepared acyl chloride was dissolved
in CH2Cl2 (4 mL) and the solution was slowly added into a biphasic
mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous CH2Cl2 (7 mL)
and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The reaction mixture
was stirred at room temperature for 12 h. Then the organic layer
was removed and the aqueous layer was extracted with CH2Cl2
(10 mL ꢃ 2). The combined organic layer was washed with satu-
rated aqueous NaHCO3 (10 mL ꢃ 2) and brine (10 mL ꢃ 2), dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was chromatographed on a silica gel column
by use of petroleum ether–acetic ether (2:1, v/v) to give compound
4.1.3. 1-Bromo-3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-2-azido-2-deoxy- -glucopyranoside (7)
D-
a-D
The mixture of 6a and 6b (0.5 g, 0.75 mmol) was dissolved in
anhydrous acetonitrile (5 mL). Then LiBr (0.5 g) and molecular
sieve (1.0 g) were added into above solution. The reaction mixture
was stirred at room temperature for 6 h. The filtrate was evapo-
rated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether
(2:1, v/v) to give compound 7 as a white solid (0.26 g, 51.0%), mp
154–155 °C (lit.26 mp 156–157 °C); 1H NMR (500 MHz, CDCl3) d
(ppm): 6.37 (1H, d, J = 3.9 Hz, GlcNAH-1), 5.49 (1H, d, J = 9.6 Hz,
J = 9.6 Hz, GlcNAH-3), 5.37 (1H, d, J = 3.4 Hz, GalBH-4), 5.13 (1H,
m, GalBH-2), 4.96 (1H, dd, J = 3.4 Hz, J = 10.4 Hz, GalBH-3), 4.52–
4.48 (2H, m, GlcNAH-6a, GalBH-1), 4.23–4.17 (3H, m, GlcNA H-6b,
GalBH-6a, GalBH-6b), 4.10–4.07 (1H, m, GalB H-5), 3.91–3.86 (2H,
m, GlcNA H-4, GalAH-5), 3.68 (1H, dd, J = 10.2 Hz, J = 3.9 Hz, GlcNA
H-2), 2.17, 2.14, 2.13, 2.07, 2.06, 1.97 (each 3H, each s, 6 ꢃ OAc);
MS(ESI(+)70 V, m/z): 699.1 [M+NH4]+.
10a as a white solid (0.45 g, 58.4%), mp 98–102 °C; ½a D15:8
ꢁ9.88 (c
ꢄ
0.2450, CHCl3); 1H NMR (300 MHz, CDCl3) d (ppm): 7.60 (1H, d,
J = 15.6 Hz, –CH@), 7.15 (1H, d, J = 8.4 Hz, ArH), 7.02 (1H, s, ArH),
6.87 (1H, d, J = 8.4 Hz, ArH), 6.40 (1H, d, J = 9.3 Hz, NH), 6.21 (1H,
d, J = 15.6 Hz, –CH@), 5.47 (1H, s, H-4), 5.37 (1H, t, J = 9.0 Hz, H-
1), 5.23–5.19 (2H, m, H-2, H-3), 4.19–4.10 (3H, m, H-5, H-6a, H-
6b), 3.92, 3.88 (each 3H, each s, 2 ꢃ OCH3), 2.17, 2.07, 2.04, 1.95
(each 3H, each s, 4 ꢃ OAc); 13C NMR (300 MHz, CDCl3) d (ppm):
171.57, 170.34, 169.98, 169.71 (4C, ester C@O), 166.02 (1C, amide
C@O), 151.20, 149.32, 127.29, 122.56, 111.19, 109.84 (6C, ArC),
143.08, 117.19 (2C, CH@CH), 78.84, 72.35, 70.91, 68.52, 67.28,
61.15 (6C, carbohydrate ring carbons), 55.98 (2C, 2 ꢃ OCH3),
20.77, 20.63, 20.56, 20.51 (4C, 4 ꢃ CH3); IR (cmꢁ1): 3466 (NH),
2938 (CH), 1751 (ester, C@O), 1677 (amide, C@O), 1630 (C@C),
1599, 1516, 1466, 1370, 1226, 1083, 1052, 909, 847; MS(E-
SI(+)70 V, m/z): 538.0 [M+H]+; Anal. Calcd for C25H31NO12ꢀ0.5H2O:
C, 54.94, H, 5.90, N, 2.56. Found: C, 54.92, H, 6.34, N, 2.64.
4.1.4. 1,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-2-azido-2-deoxy-b- -glucopyranose (8)
Compound (0.3 g, 0.44 mmol) and Hg(OAc)2 (0.28 g,
D-
D
7
0.88 mmol) were dissolved in acetic acid (5 mL). The mixture
was stirred at room temperature for 17 h. After CH2Cl2 (50 mL)
was added, the mixture was washed with KBr (40 mL) three times
and water (50 mL) twice, dried over anhydrous Na2SO4 overnight.
The filtrate was evaporated under reduced pressure. The residue
was chromatographed on a silica gel column by use of petroleum
ether–acetic ether (2:1, v/v) to give 8 as a white solid (0.26 g,
89.7%), mp 72–75 °C; 1H NMR (500 MHz, CDCl3) d (ppm): 5.52
(1H, d, J = 8.5 Hz, GlcNAH-1), 5.38–5.36 (1H, m, GalBH-4), 5.13–
5.07 (2H, m, GlcNAH-3, GalBH-2), 4.96 (1H, dd, J = 3.4 Hz,
J = 10.4 Hz, GalBH-3), 4.50–4.43 (2H, m, GlcNAH-6a, GalBH-1),
4.19–4.06 (3H, m, GlcNA H-6b, GalBH-6a, GalBH-6b), 3.89–3.88
(1H, m, GalB H-5), 3.77–3.73 (2H, m, GlcNA H-4, GalAH-5), 3.58
(1H, d, J = 10.2 Hz, GlcNA H-2), 2.17, 2.16, 2.13, 2.12, 2.08, 2.03,
1.96 (each 3H, each s, 7 ꢃ OAc); IR (cmꢁ1): 2117 (N3), 1753 (ester,
4.1.7. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-D-gluctopyranosyl)-3-
(2,3,4-trimethoxyphenyl)-2-propenamide (10b)
3,4,5-Trimethoxycinnamic acid (0.41 g, 1.73 mmol) was sus-
pended in anhydrous CHCl3 (2 mL). SOCl2 (1 mL) was slowly added
to this mixture while cooling in an ice bath, and the resulting solu-

88
K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
tion was stirred at room temperature for 1 h. After the completion
of the reaction, the solvent was removed under reduced pressure
to give acyl chloride as yellow solid which was used without fur-
ther purification. The new prepared acyl chloride was dissolved
in CH2Cl2 (4 mL) and the solution was slowly added into a biphasic
mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous CH2Cl2 (7 mL)
and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The reaction mixture
was stirred at room temperature for 12 h. Then the organic layer
was removed and the aqueous layer was extracted with CH2Cl2
(10 mL ꢃ 2). The combined organic layer was washed with satu-
rated aqueous NaHCO3 (10 mL ꢃ 2) and brine (10 mL ꢃ 2), dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was chromatographed on a silica gel column
by use of petroleum ether–acetic ether (3:2, v/v) to give compound
4.1.9. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-D-galactopyranosyl)-
3-(3,4-diacetoxyphenyl)-2-propenamide (10d)
Caffeic acid (1.5 g, 8.33 mmol) was dissolved in water (11 mL)
containing NaOH (1.15 g, 28.75 mmol) in an ice bath. Then Ac2O
(2.27 mL, 20.82 mmol)was added dropwise and the mixture was
stirred for 1.5 h at room temperature. After the reaction completed,
the pH was adjusted to 2 with 10% H2SO4, and the mixture was
stirred for 1 h. The mixture was filtered, and the filter cake was
washed three times with water and dried under infrared lamp to
give (E)-3-(3,4-diacetoxyphenyl)acrylic acid as gray white solid
(2.01 g, 91.36%). (E)-3-(3,4-Diacetoxyphenyl)acrylic acid (0.46 g,
1.73 mmol) was suspended in anhydrous CHCl3 (2 mL). SOCl2
(1 mL) was added dropwise in an ice bath and the mixture was
heated to reflux for 1 h. The solvent was removed under reduced
pressure to give acyl chloride as yellow oil which was used without
further purification. The new prepared acyl chloride was dissolved
in CH2Cl2 (4 mL) and the solution was slowly added into a biphasic
mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous CH2Cl2 (7 mL)
and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The reaction mixture
was stirred at room temperature for 24 h. Then the organic layer
was removed and the aqueous layer was extracted with CH2Cl2
(10 mL ꢃ 2). The combined organic layer was washed with satu-
rated aqueous NaHCO3 (10 mL ꢃ 2) and brine (10 mL ꢃ 2), dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was chromatographed on a silica gel column
by use of petroleum ether–acetic ether (3:2, v/v) to give compound
10b as a white solid (0.25 g, 30.5%), mp 91–94 °C; ½a D19:9
ꢁ1.24 (c
ꢄ
0.225, CHCl3); 1H NMR (300 MHz, CDCl3) d (ppm): 7.80 (1H, d,
J = 15.9 Hz, –CH@), 7.21 (1H, d, J = 8.7 Hz, ArH), 6.69 (1H, d,
J = 8.7 Hz, ArH), 6.40–6.34 (2H, overlapping, –CH@, NH), 5.47 (1H,
s, H-4), 5.39 (1H, s, H-1), 5.23–5.19 (2H, m, H-2, H-3), 4.16–4.10
(3H, m, H-5, H-6a, H-6b), 3.91, 3.87, 3.82 (each 3H, each s,
3 ꢃ OCH3), 2.31 (3H, s, OAc), 2.05, 2.04, 2.00, 1.98 (each 3H, each
s, 4 ꢃ OAc); IR (cmꢁ1): 3349 (NH), 2941 (CH), 1751 (ester, C@O),
1684 (amide, C@O), 1626 (C@C), 1594, 1536, 1497, 1228, 1096,
1046, 909, 801; MS(ESI(+)70 V, m/z): 568.0 [M+H]+; Anal. Calcd
for C26H33NO13: C, 55.02, H, 5.86, N, 2.47. Found: C, 54.70, H,
5.92, N, 2.18.
10d as a white solid (0.48 g, 56.5%), mp 103–105 °C; ½a D16:5
ꢁ7.1 (c
ꢄ
4.1.8. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-
D
-galactopyranosyl)-
0.2, CHCl3); 1H NMR (300 MHz, CDCl3) d (ppm): 7.60 (1H, d,
J = 15.6 Hz, –CH@), 7.40–7.21 (3H, m, ArH), 6.44 (1H, d, J = 8.7 Hz,
NH), 6.27 (1H, d, J = 15.6 Hz, –CH@), 5.47 (1H, d, J = 1.5 Hz, H-4),
5.35 (1H, t, J = 9.0 Hz, H-1), 5.18 (2H, m, J = 9.6 Hz, H-2, H-3),
4.16–4.10 (3H, m, H-5, H-6a, H-6b), 2.38 (3H, s, COCH3), 2.32
(3H, s, COCH3), 2.17, 2.06, 2.05, 2.01 (each 3H, each s, 4 ꢃ OAc);
IR (cmꢁ1): 3359 (NH), 3063, 2940 (CH), 1751 (ester, C@O), 1687
(amide, C@O), 1635 (C@C), 1537, 1506, 1221, 1112, 1050, 837;
3-(3-methoxy-4-acetoxyphenyl)-2-propenamide (10c)
Ferulic acid(1.5 g, 7.73 mmol) was dissolved in water (7.7 mL)
containing NaOH (0.8 g, 20.6 mmol) in an ice bath. Then Ac2O
was added dropwise and the mixture was stirred for 1.5 h at room
temperature. After the reaction completed, the pH was adjusted to
2 with 10% H2SO4, and the mixture was stirred for 1 h. The mixture
was filtered, and the filter cake was washed three times with water
and dried under infrared lamp to give (E)-3-(4-acetoxy-3-
methoxyphenyl)acrylic acid as gray white solid (1.74 g, 95.35%).
MS(ESI(+)70 V, m/z): 594.0 [M+H]+; Anal. Calcd for C27H31NO14
C, 54.64, H, 5.26, N, 2.36. Found: C, 54.62, H, 5.22, N, 2.17.
:
(E)-3-(4-Acetoxy-3-methoxyphenyl)acrylic
acid
(0.34 g,
1.44 mmol) was suspended in anhydrous CH2Cl2 (10 mL). Oxalyl
chloride (0.47 mL, 5.5 mmol) was added dropwise into the solu-
tion. Then a drop of DMF was added and the solution was stirred
for 24 h at room temperature. The solvent was removed under re-
duced pressure to give acyl chloride as yellow solid which was
used without further purification. The new prepared acyl chloride
was dissolved in CH2Cl2 (4 mL) and the solution was slowly added
into a biphasic mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous
CH2Cl2 (7 mL) and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The
reaction mixture was stirred at room temperature for 24 h. Then
the organic layer was removed and the aqueous layer was ex-
tracted with CH2Cl2 (10 mL ꢃ 2). The combined organic layer was
washed with saturated aqueous NaHCO3 (10 mL ꢃ 2) and brine
(10 mL ꢃ 2), dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether (3:2,
v/v) to give compound 10c as a white solid (0.44 g, 54.3%), mp
4.1.10. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-D-galactopyranosyl)-
3-(2-chlorophenyl)-2-propenamide (10e)
2-Chlorocinnamic acid (0.32 g, 1.73 mmol) was suspended in
SOCl2 (1 mL) and the mixture was heated to reflux for 7 h. After
the completion of the reaction, the solvent was removed under re-
duced pressure to give acyl chloride as yellow oil which was used
without further purification. The new prepared acyl chloride was
dissolved in CH2Cl2 (4 mL) and the solution was slowly added into
a biphasic mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous CH2-
Cl2 (7 mL) and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The reac-
tion mixture was stirred at room temperature for 12 h. Then the
organic layer was removed and the aqueous layer was extracted
with CH2Cl2 (10 mL ꢃ 2). The combined organic layer was washed
with saturated aqueous NaHCO3 (10 mL ꢃ 2) and brine
(10 mL ꢃ 2), dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether (2:1,
v/v) to give compound 10e as a white solid (0.33 g, 44.6%). mp 77–
117–120 °C; ½a 2D4
ꢄ
ꢁ6.53 (c 0.095, CHCl3); 1H NMR (300 MHz, CDCl3)
d (ppm): 7.61 (1H, d, J = 15.6 Hz, –CH@), 7.12–7.03 (3H, m, ArH),
6.39 (1H, d, J = 8.7 Hz, NH), 6.27 (1H, d, J = 15.6 Hz, –CH@), 5.47
(1H, d, J = 1.5 Hz, H-4), 5.37 (1H, t, J = 9.0 Hz, H-1), 5.19–5.17 (2H,
m, J = 9.6 Hz, H-2, H-3), 4.15–4.08 (3H, m, H-5, H-6a, H-6b), 3.87
(3H, s, OCH3), 2.32 (3H, s, COCH3), 2.16, 2.06, 2.04, 2.01 (each 3H,
each s, 4 ꢃ OAc); IR (cmꢁ1): 3357 (NH), 2942 (CH), 1751 (ester,
C@O), 1686 (amide, C@O), 1633 (C@C), 1600, 1535, 1512, 1224,
1158, 1123, 1051, 834; MS(ESI(+)70 V, m/z): 566.0 [M+H]+;-
81 °C; ½a 1D5:9
ꢄ
ꢁ9.27 (c 0.300, CHCl3); 1H NMR (300 MHz, CDCl3) d
(ppm): 7.59 (1H, d, J = 15.6 Hz, –CH@), 7.49 (1H, s, ArH), 7.39–
7.29 (3H, m, ArH), 6.49 (1H, d, J = 9 Hz, NH), 6.34 (1H, d,
J = 15.6 Hz, –CH@), 5.47 (1H, s, H-4), 5.37 (1H, t, J = 9.0 Hz, H-1),
5.19–5.17 (2H, m, H-2, H-3), 4.16–4.07 (3H, m, H-5, H-6a, H-6b),
2.16, 2.06, 2.04, 2.01 (each 3H, each s, 4 ꢃ OAc);IR (cmꢁ1): 3355
(NH), 3066, 2965 (CH), 1751 (ester, C@O), 1688 (amide, C@O),
1636 (C@C), 1594, 1538, 1370, 1225, 1123, 1083, 1052, 790; MS(E-
SI(+)70 V, m/z): 512.0 [M+H]+; Anal. Calcd for C23H26ClNO10: C,
53.96, H, 5.12, N, 2.74. Found: C, 53.66, H, 5.44, N, 2.43.
MS(ESI(ꢁ)70 V, m/z): 563.9 [MꢁH]ꢁ; Anal. Calcd for C26H31NO13
:
C, 55.22, H, 5.53, N, 2.48. Found: C, 55.52, H, 5.93, N, 2.17.

K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
89
4.1.11. N-(2,3,4,6-Tetra-O-acetyl-1-deoxy-b-
3-(2,4-dichlorophenyl)-2-propenamide (10f)
D
-galactopyranosyl)-
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), 3,4-dimethoxycinnamic acid (0.33 g, 1.56 mmol), and
EDCI (0.30 g, 1.56 mmol) were added successively, and the reaction
mixture was stirred at room temperature for 36 h. Then the mix-
ture was washed with water (20 mL ꢃ 2), saturated aqueous
NaHCO3 (20 mL ꢃ 2), and brine (20 mL ꢃ 2), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column by use of
petroleum ether–acetic ether (1:1, v/v) to give compound 10h as
2,4-Dichlorocinnamic acid (0.38 g, 1.73 mmol) was suspended
in SOCl2 (1 mL) and the mixture was heated to reflux for 2 h. After
the completion of the reaction, the solvent was removed under re-
duced pressure to give acyl chloride as white oil which was used
without further purification. The new prepared acyl chloride was
dissolved in CH2Cl2 (4 mL) and the solution was slowly added into
a biphasic mixture of G1-NH2 (0.5 g, 1.44 mmol) in anhydrous CH2-
Cl2 (7 mL) and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL). The reac-
tion mixture was stirred at room temperature for 12 h. Then the
organic layer was removed and the aqueous layer was extracted
with CH2Cl2 (10 mL ꢃ 2). The combined organic layer was washed
with saturated aqueous NaHCO3 (10 mL ꢃ 2) and brine
(10 mL ꢃ 2), dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether (3:2,
v/v) to give compound 10f as a white solid (0.52 g, 66.3%), mp 95–
a white solid (0.44 g, 63.0%), mp 105–108 °C; ½a D24
ꢄ
+23.2 (c 0.075,
d (ppm): 7.55 (1H, d,
CHCl3); 1H NMR (500 MHz, CDCl3)
J = 15.5 Hz, –CH@), 7.07 (1H, d, J = 8.2 Hz, ArH), 7.01 (1H, d,
J = 1.88 Hz, ArH), 6.85 (1H, d, J = 8.3 Hz, ArH), 6.17 (1H, d,
J = 15.5 Hz, –CH@), 5.76 (1H, d, J = 8.8 Hz, NH), 5.45 (1H, d,
J = 9.5 Hz, H-1), 5.41 (1H, d, J = 2.5 Hz, H-4), 5.15 (1H, dd,
J = 3.2 Hz, J = 11.3 Hz, H-3), 4.63 (1H, dd, J = 9.5 Hz, J = 10.5 Hz, H-
2), 4.22–4.06 (3H, m, H-5, H-6a, H-6b), 3.91 (3H, s, OCH3), 3.90
(3H, s, OCH3), 2.19, 2.11, 2.05, 2.00 (each 3H, each s, 4 ꢃ OAc); IR
(cmꢁ1): 3457 (NH), 2965, 2939 (CH), 1750 (ester, C@O), 1662
(amide, C@O), 1626 (C@C), 1516, 1423, 1370, 1264, 1220, 1074,
1040, 846, 810; MS(ESI(+)70 V, m/z): 538.1 [M+H]+; Anal. Calcd
for C25H31NO12: C, 55.86, H, 5.81, N, 2.61. Found: C, 55.81, H,
5.87, N, 2.44.
97 °C; ½a 1D6:6
ꢄ
ꢁ3.06 (c 0.085, CHCl3); 1H NMR (300 MHz, CDCl3) d
(ppm): 7.96 (1H, d, J = 15.6 Hz, –CH@), 7.52–7.26 (3H, m, ArH),
6.50 (1H, d, J = 8.4 Hz, NH), 6.32 (1H, d, J = 15.6 Hz, –CH@), 5.47
(1H, d, J = 1.5 Hz, H-4), 5.37 (1H, t, J = 9.0 Hz, H-1), 5.19–5.17 (2H,
m, J = 9.6 Hz, H-2, H-3), 4.12–4.10 (3H, m, H-5, H-6a, H-6b), 2.16,
2.07, 2.05, 2.01 (each 3H, each s, 4 ꢃ OAc); 13C NMR (300 MHz,
CDCl3) d (ppm): 171.60, 170.34, 169.97, 169.69 (4C, ester C@O),
165.07 (1C, amide C@O), 136.32, 135.59, 131.28, 130.64, 128.40,
127.55 (6C, ArC), 137.85, 122.68 (2C, CH@CH), 78.90, 72.48,
70.82, 68.53, 67.26, 61.17 (6C, carbohydrate ring carbons), 20.77,
20.64, 20.56, 20.50 (4C, 4 ꢃ CH3); IR (cmꢁ1): 3369 (NH), 2961,
2935 (CH), 1751 (ester, C@O), 1688 (amide, C@O), 1632 (C@C),
1584, 1537, 1370, 1225, 1123, 1084, 1051, 789; MS(ESI(+)70 V,
m/z): 546.0 [M+H]+; Anal. Calcd for C23H25Cl2NO10: C, 50.56, H,
4.61, N, 2.56. Found: C, 50.27 H, 4.61, N, 2.48.
4.1.14. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-
3-(2,3,4-trimethoxyphenyl)-2-propenamide (10i)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), 2,3,4-trimethoxycinnamic acid (0.37 g, 1.56 mmol),
and EDCI (0.30 g, 1.56 mmol) were added successively, and the
reaction mixture was stirred at room temperature for 36 h. Then
the mixture was washed with water (20 mL ꢃ 2), saturated
aqueous NaHCO3 (20 mL ꢃ 2), and brine (20 mL ꢃ 2), dried over
anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was chromatographed on a silica gel column
by use of petroleum ether–acetic ether (2:1, v/v) to give compound
4.1.12. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-
3-(4-methoxyphenyl)-2-propenamide (10g)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), 4-Methoxycinnamic acid (0.28 g, 1.56 mmol), and
EDCI (0.30 g, 1.56 mmol) were added successively, and the reaction
mixture was stirred at room temperature for 36 h. Then the mix-
ture was washed with water (20 mL ꢃ 2), saturated aqueous
NaHCO3 (20 mL ꢃ 2), and brine (20 mL ꢃ 2), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column by use of
petroleum ether–acetic ether (1:1, v/v) to give compound 10g as
10i as a white solid (0.38 g, 51.6%), mp 92–94 °C; ½a D24
ꢄ
+21.3 (c 0.28,
CHCl3); 1H NMR (500 MHz, CDCl3) d (ppm): 7.75 (1H, d, J = 15.8 Hz,
–CH@), 7.20 (1H, d, J = 8.8 Hz, ArH), 6.67 (1H, d, J = 8.8 Hz, ArH),
6.32 (1H, d, J = 15.7 Hz, –CH@), 5.77 (1H, d, J = 8.8 Hz, NH), 5.51
(1H, d, J = 9.6 Hz, H-1), 5.41 (1H, d, J = 2.5 Hz, H-4), 5.15 (1H, dd,
J = 3.2 Hz, J = 11.3 Hz, H-3), 4.64 (1H, dd, J = 9.4 Hz, J = 10.5 Hz,
H-2), 4.20–4.07 (3H, m, H-5, H-6a, H-6b), 3.90, 3.89, 3.86 (each
3H, each s, 3 ꢃ OCH3), 2.19, 2.11, 2.05, 2.00 (each 3H, each s,
4 ꢃ OAc); IR (cmꢁ1): 3471 (NH), 2942 (CH), 1751 (ester, C@O),
1660 (amide, C@O), 1624 (C@C), 1548, 1437, 1370, 1220, 1096,
1041, 800; MS(ESI(+)70 V, m/z): 568.2 [M+H]+; MS(ESI(ꢁ)70 V,
m/z): 566.1 [MꢁH]ꢁ; Anal. Calcd for C26H33NO13: C, 55.02, H,
5.86, N, 2.47. Found: C, 54.71, H, 6.02, N, 2.24.
a white solid (0.32 g, 48.6%), mp181–183 °C; ½a D21
ꢄ
+25.68 (c 0.19,
CHCl3); 1H NMR (300 MHz, CDCl3)
d (ppm): 7.57 (1H, d,
J = 15.6 Hz, –CH@), 7.44 (2H, d, J = 8.7 Hz, ArH), 6.89 (2H, d,
J = 8.7 Hz, ArH), 6.18 (1H, d, J = 15.3 Hz, –CH@), 5.78 (1H, d,
J = 8.7 Hz, NH), 5.57 (1H, d, J = 9.6 Hz, H-1), 5.41 (1H, d, J = 2.7 Hz,
H-4), 5.17 (1H, dd, J = 3.3 Hz, J = 11.3 Hz, H-3), 4.63 (1H, dd,
J = 9.3 Hz, J = 10.5 Hz, H-2), 4.23–4.04 (3H, m, H-5, H-6a, H-6b),
3.83 (3H, s, OCH3), 2.19, 2.11, 2.05, 2.00 (each 3H, each s, 4 ꢃ OAc);
IR (cmꢁ1): 3329 (NH), 2974, 2941 (CH), 1745 (ester, C@O), 1659
(amide, C@O), 1630 (C@C), 1538, 1451, 1370, 1221, 1080, 1041;
4.1.15. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-
3-(3-methoxy-4-acetoxyphenyl)-2-propenamide (10j)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.175 g,
MS(ESI(ꢁ)70 V, m/z): 506.0 [MꢁH]ꢁ; Anal. Calcd for C24H29NO11
:
1.3 mmol),
(E)-3-(4-acetoxy-3-methoxyphenyl)acrylic
acid
C, 56.80, H, 5.76, N, 2.76. Found: C, 56.95, H, 5.85, N, 2.75.
(0.31 g, 1.3 mmol), and EDCI (0.25 g, 1.3 mmol) were added succes-
sively, and the reaction mixture was stirred at room temperature
for 36 h. Then the mixture was washed with water (20 mL ꢃ 2),
saturated aqueous NaHCO3 (20 mL ꢃ 2), and brine (20 mL ꢃ 2),
dried over anhydrous Na2SO4, filtered, and concentrated under re-
duced pressure. The residue was chromatographed on a silica gel
column by use of dichloromethane–methanol (100:1–30:1, v/v)
4.1.13. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-
3-(3,4-dimethoxyphenyl)-2-propenamide (10h)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred

90
K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
to give compound 10j as a white solid (0.17 g, 23.3%), mp 183–
93.08, 71.99, 70.43, 66.41, 61.28, 50.06 (6C, carbohydrate ring car-
bons), 20.82, 20.61, 20.60, 20.58 (4C, 4 ꢃ CH3), 15.60 (1C, CH3); IR
(cmꢁ1): 3375 (NH), 3073, 2970 (CH), 1751 (ester, C@O), 1666
(amide, C@O), 1631 (C@C), 1547, 1431, 1369, 1221, 1077, 1041,
788, 745; MS(ESI(ꢁ)70 V, m/z): 509.9 [MꢁH]ꢁ; Anal. Calcd for C23-
H26ClNO10: C, 53.96, H, 5.12, N, 2.74. Found: C, 53.91, H, 5.42, N,
2.44.
185 °C; ½a 2D4
ꢄ
+20.63 (c 0.095, CHCl3); 1H NMR (300 MHz, CDCl3) d
(ppm): 7.56 (1H, d, J = 15.6 Hz, –CH@), 7.06 (3H, m, ArH), 6.24
(1H, d, J = 15.6 Hz, –CH@), 5.77 (1H, d, J = 8.7 Hz, NH), 5.56 (1H,
d, J = 9.6 Hz, H-1), 5.41 (1H, d, J = 3 Hz, H-4), 5.15 (1H, dd,
J = 3.3 Hz, J = 11.3 Hz, H-3), 4.63 (1H, dd, J = 9.6 Hz, J = 10.5 Hz, H-
2), 4.17–4.06 (3H, m, H-5, H-6a, H-6b), 3.86 (3H, s, OCH3), 2.32
(3H, s, COCH3), 2.19, 2.11, 2.06, 2.00 (each 3H, each s, 4 ꢃ OAc);
IR (cmꢁ1): 3337 (NH), 2950 (CH), 1745 (ester, C@O), 1663 (amide,
C@O), 1630 (C@C), 1601, 1520, 1221, 1126, 1038, 860; MS(E-
SI(+)70 V, m/z): 567.0 [M+H]+; MS(ESI(ꢁ)70 V, m/z): 564.0
[MꢁH]ꢁ; Anal. Calcd for C26H31NO13: C, 55.22, H, 5.53, N, 2.48.
Found: C, 55.32, H, 5.46, N, 2.47.
4.1.18. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-
galactopyranosyl)-3-(3,4-dichlorophenyl)-2-propenamide
(10m)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), 2,4-dichlorocinnamic acid (0.34 g, 1.56 mmol), and
EDCI (0.30 g, 1.56 mmol) were added successively, and the reaction
mixture was stirred at room temperature for 36 h. Then the mix-
ture was washed with water (20 mL ꢃ 2), saturated aqueous
NaHCO3 (20 mL ꢃ 2), and brine (20 mL ꢃ 2), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was chromatographed on a silica gel column by use of
petroleum ether–acetic ether (1:1, v/v) to give compound 10m as
4.1.16. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-
galactopyranosyl)-3-(3,4-diacetoxyphenyl)-2-propenamide
(10k)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), (E)-3-(3,4-diacetoxyphenyl)acrylic acid (0.41 g,
1.56 mmol), and EDCI (0.30 g, 1.56 mmol) were added successively,
and the reaction mixture was stirred at room temperature for 36 h.
Then the mixture was washed with water (20 mL ꢃ 2), saturated
aqueous NaHCO3 (20 mL ꢃ 2) and brine (20 mL ꢃ 2), dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was chromatographed on a silica gel column by
use of petroleum ether–acetic ether (1:1, v/v) to give compound
a white solid (0.38 g, 53.5%), mp 153–156 °C; ½a D24
ꢄ
+16.22 (c
0.185, CHCl3); 1H NMR (300 MHz, CDCl3) d (ppm): 7.91 (1H, d,
J = 15.6 Hz, –CH@), 7.50 (1H, d, J = 8.4 Hz, ArH), 7.43 (1H, s, ArH),
7.24 (1H, d, J = 8.7 Hz, ArH), 6.132 (1H, d, J = 15.6 Hz, –CH@), 5.87
(1H, d, J = 9.3 Hz, NH), 5.81 (1H, d, J = 8.7 Hz, H-1), 5.41 (1H, d,
J = 2.4 Hz, H-4), 5.19 (1H, dd, J = 3.3 Hz, J = 11.4 Hz, H-3), 4.61
(1H, dd, J = 9.3 Hz, J = 10.5 Hz, H-2), 4.23–4.07 (3H, m, H-5, H-6a,
H-6b), 2.20, 2.12, 2.06, 2.01 (each 3H, each s, 4 ꢃ OAc); IR
(cmꢁ1): 3342 (NH), 3069, 2973 (CH), 1748 (ester, C@O), 1665
(amide, C@O), 1629 (C@C), 1532, 1471, 1373, 1223, 1081, 1044;
MS(ESI(ꢁ)70 V, m/z): 543.9 [MꢁH]ꢁ; Anal. Calcd for C23H25Cl2-
NO10: C, 50.56, H, 4.61, N, 2.56. Found: C, 50.40, H, 4.64, N, 2.15.
10k as a white solid (0.34 g, 46.6%), mp 115–119 °C; ½a D19:8
ꢄ
+24.10 (c 0.195, CHCl3); 1H NMR (300 MHz, CDCl3) d (ppm): 7.55
(1H, d, J = 15.6 Hz, –CH@), 7.37–7.19 (3H, m, ArH), 6.21 (1H, d,
J = 15.6 Hz, –CH@), 5.76 (1H, d, J = 8.7 Hz, NH), 5.59 (1H, d,
J = 9.6 Hz, H-1), 5.41 (1H, d, J = 3 Hz, H-4), 5.13 (1H, dd, J = 3.3 Hz,
J = 11.4 Hz, H-3), 4.63 (1H, dd, J = 9.6 Hz, J = 10.5 Hz, H-2), 4.19–
4.05 (3H, m, H-5, H-6a, H-6b), 3.49 (3H, s, OCH3), 2.31 (3H, s,
COCH3), 2.19, 2.11, 2.06, 2.00 (each 3H, each s, 4 ꢃ OAc); IR
(cmꢁ1): 3385 (NH), 2939 (CH), 1751 (ester, C@O), 1668 (amide,
C@O), 1632 (C@C), 1544, 1506, 1429, 1219, 1074, 1041; MS(E-
SI(+)70 V, m/z): 594.1 [M+H]+; Anal. Calcd for C27H31NO14.0ꢀ5H2O:
C, 53.82, H, 5.35, N, 2.32. Found: C, 53.91, H, 5.26, N, 1.90.
4.1.19. Methyl 6-[3-(3,4-dimethoxyphenyl)-propenamido]-6-
deoxy-a-D-galactopyranoside (10n)
3,4-Dimethoxycinnamic acid (0.46 g, 2.2 mmol), HOBt (0.3 g,
2.2 mmol) and EDCI (0.42 g, 2.2 mmol) were dissolved in anhy-
drous DMF(25 mL). G3-NH2 (0.42 g, 2.2 mmol) dissolved in
DMF(3 mL) was slowly added into this solution and the reaction
mixture was stirred at room temperature for 24 h. Then the mix-
ture was concentrated under reduced pressure. The residue was
chromatographed on a silica gel column by use of dichlorometh-
ane–methanol (20:1, v/v) to give compound 10n as a white solid
4.1.17. N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-D-galactopyranosyl)-
3-(2-chlorophenyl)-2-propenamide (10l)
G2-NH2ꢀHCl (0.5 g, 1.3 mmol) was suspended in anhydrous CH2-
Cl2 (20 mL). Et3N (0.17 mL, 1.30 mmol) was added to this mixture
while cooling in an ice bath, and the resulting solution was stirred
at room temperature for 1 h. To this solution, HOBt (0.21 g,
1.56 mmol), 2-Chlorocinnamic acid (0.29 g, 1.56 mmol), and EDCI
(0.30 g, 1.56 mmol) were added successively, and the reaction mix-
ture was stirred at room temperature for 36 h. Then the mixture
was washed with water (20 mL ꢃ 2), saturated aqueous NaHCO3
(20 mL ꢃ 2), and brine (20 mL ꢃ 2), dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column by use of petroleum
ether–acetic ether (1:1, v/v) to give compound 10l as a white solid
(0.42 g, 50.0%), mp 113–114 °C; ½a D20:8
ꢄ
+86.2 (c 0.0650, CH3OH);
1H NMR (500 MHz, DMSO-d6) d (ppm): 8.01 (1H, t, J = 5.9 Hz,
CONH), 7.36 (1H, d, J = 15.7, –CH@), 7.15 (1H, d, J = 1.9 Hz, ArH),
7.11 (1H, dd, J = 2.0 Hz, J = 8.4 Hz, ArH), 6.98 (1H, d, J = 8.4 Hz,
ArH), 6.56 (1H, d, J = 15.7 Hz, –CH@), 4.56 (1H, d, J = 3.6 Hz, H-1),
4.55–4.47 (3H, m, 3 ꢃ OH), 3.79 (3H, s, OCH3), 3.78 (3H, s, OCH3),
3.68–3.64 (2H, m, H-5, H-2), 3.59–3.39 (3H, m, H-3, H-4, H-6a),
3.24 (3H, s, OCH3), 3.17 (1H, m, H-6b); IR (cmꢁ1): 3397 (br, NH,
CH), 2936, 2838 (CH), 1657, 1614 (amide, C@O), 1599, 1515,
1264, 1141, 1079, 1023, 847; MS(ESI(+)70 V, m/z): 384.2 [M+H]+;
Anal. Calcd for C18H25NO8.H2O: C, 53.86, H, 6.78, N, 3.49. Found:
C, 53.70, H, 6.74, N, 3.65.
(0.36 g, 54.5%), mp 100–102 °C; ½a D24
ꢄ
+18.4 (c 0.075, CHCl3); 1H
NMR (500 MHz, CDCl3) d (ppm): 7.54 (1H, d, J = 15.6 Hz, –CH@),
7.47 (1H, s, ArH), 7.35–7.27 (3H, m, ArH), 6.32 (1H, d, J = 15.6 Hz,
–CH@), 5.81 (2H, d, J = 8.8 Hz, overlapping, NH, H-1), 5.42 (1H, d,
J = 2.7 Hz, H-4), 5.21 (1H, dd, J = 3.1 Hz, J = 11.2 Hz, H-3), 4.60
(1H, dd, J = 9.3 Hz, J = 10.5 Hz, H-2), 4.22–4.08 (3H, m, H-5, H-6a,
H-6b), 2.20, 2.11, 2.05, 2.00 (each 3H, each s, 4 ꢃ OAc); 13C
NMR(300 MHz, CDCl3) d (ppm): 170.75, 170.32, 170.10, 169.50
(4C, ester C@O), 165.48 (1C, amide C@O), 136.19, 134.90, 130.08,
129.91, 127.55, 126.20 (4C, ArC), 140.80, 120.97 (2C, CH@CH),
4.1.20. Methyl 6-[3-(3-methoxy-4-acetoxyphenyl)-
propenamido]-6-deoxy-a-D-galacto-pyranoside (10o)
3-Methoxy-4-acetoxycinnamic acid (1.25 g, 5.3 mmol), HOBt
(0.72 g, 5.3 mmol) and EDCI (1.02 g, 5.3 mmol) were dissolved in
anhydrous DMF(50 mL). G3-NH2 (1.02 g, 5.3 mmol) dissolved in
DMF(6 mL) was slowly added into this solution and the reaction
mixture was stirred at room temperature for 24 h. Then the mix-

K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
91
ture was concentrated under reduced pressure. The residue was
chromatographed on a silica gel column by use of dichlorometh-
ane–methanol (20:1, v/v) to give compound 10o as a white solid
2 ꢃ OCH3), 20.72, 20.65, 20.54, 20.54 (4C, 4 ꢃ CH3);IR
(cmꢁ1):3409 (NH), 2948, 2842 (CH), 1762 (ester, C@O), 1661,
1630 (amide, C@O), 1517, 1227, 1200, 1046, 837; MS(ESI(+)70 V,
m/z): 538.2 [M+H]+; Anal. Calcd for C25H31NO12: C, 55.86, H, 5.81,
N, 2.61. Found: C, 55.54, H, 6.17, N, 2.47.
(1.26 g, 57.8%), mp 119–124 °C; ½a D22:1
ꢄ
+120.25 (c 0.0800, CH3OH);
1H NMR (500 MHz, DMSO-d6) d (ppm): 8.11 (1H, t, J = 5.8 Hz,
CONH), 7.42 (1H, d, J = 15.8 Hz, –CH@), 7.31 (1H, d, J = 1.8 Hz,
ArH), 7.15 (1H, dd, J = 1.8 Hz, J = 8.3H, ArH), 7.11 (1H, d,
J = 8.1 Hz, ArH), 6.68 (1H, d, J = 15.8 Hz, –CH@), 4.55 (1H, d,
J = 3.6 Hz, H-1), 4.56–4.48 (3H, m, 3 ꢃ OH), 3.82 (3H, s, OCH3),
3.68–3.65 (2H, m, H-5, H-2), 3.59–3.52 (3H, m, H-3, H-4, H-6a),
3.24 (3H, s, OCH3), 3.20 (1H, m, H-6b), 2.27 (3H, s, OAc); 13C
NMR(300 MHz, CDCl3) d (ppm): 168.35 (1C, ester C@O), 165.24
(1C, amide C@O), 151.04, 140.15, 133.88, 123.20, 122.38, 111.60
(6C, ArC), 140.15, 120.00 (2C, CH@CH), 100.11 (1C, C-1), 69.40,
69.33, 68.64, 68.26 (4C, C-2, C-3, C-4, C-5), 40.37 (C-6), 55.78,
54.45 (2C, 2 ꢃ OCH3), 20.32 (1C, CH3); IR (cmꢁ1):3422 (br, NH,
OH), 2939, 2841 (CH), 1762 (ester, C@O), 1659, 1620 (amide,
C@O), 1549, 1263, 1199, 1155, 1124, 1033, 788; MS(ESI(ꢁ)70 V,
m/z): 410.1 [MꢁH]ꢁ; Anal. Calcd for C19H25NO9: C, 55.47, H, 6.13,
N, 3.40. Found: C, 55.28, H, 6.45, N, 3.29.
4.1.23. N-[1,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-2-amino-2-deoxy-b- -glucopyranosyl]-3-
(3,4-dimethoxyphenyl)-2-propenamide (10r)
D-
D
3,4-dimethoxycinnamic acid (0.13 g, 0.62 mmol) was sus-
pended in anhydrous CHCl3 (1 mL). SOCl2 (0.5 mL) was slowly
added to this mixture while cooling in an ice bath, and the result-
ing solution was stirred at room temperature for 1 h. After the
completion of the reaction, the solvent was removed under re-
duced pressure to give acyl chloride as yellow solid which was
used without further purification. The new prepared acyl chloride
was dissolved in CH2Cl2 (4 mL) and the solution was slowly added
into a biphasic mixture of G4-NH2ꢀTsOH (0.5 g, 0.62 mmol) in anhy-
drous CH2Cl2 (7 mL) and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL).
The reaction mixture was stirred at room temperature for 12 h.
Then the organic layer was removed and the aqueous layer was ex-
tracted with CH2Cl2 (10 mL ꢃ 2). The combined organic layer was
washed with saturated aqueous NaHCO3 (10 mL ꢃ 2) and brine
(10 mL ꢃ 2), dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether (1:1,
v/v) to give compound 10r as a white solid (0.36 g, 58.4%), mp 127–
4.1.21. Methyl 6-[3-(2-chloro)-propenamido]-6-deoxy-
a-D-
galactopyranoside (10p)
2-Chlorocinnamic acid (0.4 g, 2.2 mmol), HOBt (0.3 g, 2.2 mmol)
and EDCI (0.42 g, 2.2 mmol) were dissolved in anhydrous
DMF(25 mL). G3-NH2 (0.42 g, 2.2 mmol) dissolved in DMF(3 mL)
was slowly added into this solution and the reaction mixture
was stirred at room temperature for 24 h. Then the mixture was
concentrated under reduced pressure. The residue was chromato-
graphed on a silica gel column by use of dichloromethane–metha-
nol (20:1, v/v) to give compound 10p as a white solid (0.42 g,
130 °C; ½a 2D3
ꢄ
+26.6 (c 0.1150, CHCl3); 1H NMR (500 MHz, CDCl3) d
(ppm): 7.55 (1H, d, J = 15.6 Hz, –CH@), 7.07 (1H, dd, J = 8.3 Hz,
J = 1.8 Hz, ArH), 7.02 (1H, d, J = 1.8 Hz, ArH), 6.84 (1H, d,
J = 8.3 Hz, ArH), 6.25 (1H, d, J = 15.6 Hz, –CH@), 6.04 (1H, d,
J = 9.8 Hz, NH), 5.71 (1H, d, J = 7.9 Hz, GlcNAH-1), 5.37 (1H, d,
J = 3.4 Hz, GalBH-4), 5.18–5.12 (2H, m, GlcNA H-3, GalBH-2), 4.99
(1H, dd, J = 3.4 Hz, J = 10.4 Hz, GalBH-3), 4.53 (1H, d, J = 7.9 Hz, GalB-
H-1), 4.49–4.43 (2H, m, GlcNA H-2, GlcNA H-6a), 4.19–4.08 (3H, m,
GalA H-6b, GalB H-6a, GalB H-6b), 3.92–3.89 (2H, m, GalAH-5, GalB
H-5), 3.88 (6H, s, 2 ꢃ OCH3), 3.83–3.80 (1H, m, GlcNA H-4), 2.15,
2.13, 2.10, 2.06, 2.04, 1.97, 1.91 (each 3H, each s, 7 ꢃ OAc); 13C
NMR(500 MHz, CDCl3) d (ppm): 170.67, 170.31, 170.26, 170.04,
169.96, 169.41, 169.37 (7C, ester C@O), 166.07 (1C, amide C@O),
150.85, 149.14, 127.41, 122.26, 111.07, 109.78 (6C, ArC), 141.98,
117.49 (2C, CH@CH), 100.82, 92.55, 76.75, 74.88, 73.55, 72.20,
70.79, 69.02, 66.63, 62.08, 60.80, 51.93 (12C, carbohydrate ring
carbons), 55.89, 55.83 (2C, 2 ꢃ OCH3), 20.84, 20.74, 20.57, 20.54,
20.53, 20.48, 20.41 (7C, 7 ꢃ CH3); IR (cmꢁ1): 3481 (NH), 2960
(CH), 1752 (ester, C@O), 1664 (amide, C@O), 1628 (C@C), 1516,
1424, 1372, 1223, 1064, 897, 846; MS(ESI(+)70 V, m/z): 826.1
[M+H]+; MS(ESI(ꢁ)70 V, m/z): 824.3 [MꢁH]ꢁ; Anal. Calcd for
57.8%), mp 105–107 °C; ½a D21:0
ꢄ
+100.5 (c 0.1100, CH3OH); 1H NMR
(500 MHz, DMSO-d6) d (ppm): 8.14 (1H, t, J = 5.8 Hz, CONH), 7.62
(1H, s, ArH), 7.52 (1H, m, ArH), 7.42 (3H, m, ArH, –CH@), 6.75
(1H, d, J = 15.8 Hz, –CH@), 4.55 (1H, d, J = 3.6 Hz, H-1), 4.56–4.48
(3H, m, 3 ꢃ OH), 3.68–3.64 (2H, m, H-5, H-2), 3.59–3.41 (3H, m,
H-3, H-4, H-6a), 3.24 (3H, s, OCH3), 3.17 (1H, m, H-6b); IR
(cmꢁ1):3416 (br, NH, OH), 2935, 2838 (CH), 1660, 1621 (amide,
C@O), 1565, 1228, 1193, 1146, 1127, 1044, 785; MS(ESI(+)70 V,
m/z): 358.2 [M+H]+; MS(ESI(ꢁ)70 V, m/z: 356.0 [MꢁH]ꢁ; Anal.
Calcd for C16H20ClNO6ꢀ0.75H2O: C, 51.76, H, 5.84, N, 3.77. Found:
C, 51.70, H, 5.61, N, 4.26.
4.1.22. Methyl 2,3,4-tri-O-acetyl-6-[3-(3-meoxy-4-
acetoxyphenyl)-propenamido]- 6-deoxy-a-D-galactopyranoside
(10q)
In a 100 mL round bottom flask, were placed 10p (0.3 g,
0.73 mmol), 4-DMAP (0.05 g, 0.37 mmol), Et3N (0.05 mL,
0.37 mmol), Ac2O (0.69 mL, 7.3 mmol), and Pyridine (43 mL). The
mixture was stirred for 12 h at room temperature. The solvent
was removed under reduced pressure and the residue was chro-
matographed on a silica gel column by use of petroleum ether–ace-
tic ether (1:1, v/v) to give compound 10q as a white solid (0.29 g,
C37H47NO20: C, 53.82, H, 5.74, N, 1.70. Found: C, 53.80, H, 5.73, N,
1.48.
4.1.24. N-[1,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-2-amino-2-deoxy-b-D-glucopyranosyl]-3-
74.4%), mp 224–226 °C; ½a D21:9
ꢄ
+73.6 (c 0.1250, CHCl3); 1H NMR
(2,4-dichlorophenyl)-2-propenamide (10s)
(500 MHz, CDCl3) d (ppm): 7.58 (1H, d, J = 15.6 Hz, –CH@), 7.11–
7.02 (3H, m, ArH), 6.31 (1H, d, J = 15.6 Hz, –CH@), 5.97 (1H, t,
CONH), 5.44 (1H, d, J = 3.3 Hz, H-4), 5.36 (1H, dd, J = 10.8 Hz,
J = 3.4 Hz, H-3), 5.17 (1H, dd, J = 10.8 Hz, J = 3.6 Hz, H-2), 4.99
(1H, d, J = 3.6 Hz, H-1), 4.14–4.10 (1H, m, H-5), 3.86 (3H, s,
OCH3), 3.62–3.56 (1H, m, H-6a), 3.40 (3H, s, OCH3), 3.37–3.31
(1H, m, H-6b), 2.31, 2.19, 2.09, 2.00 (each 3H, each s, 4 ꢃ OAc);
2,4-Dichlorocinnamic acid (0.14 g, 0.62 mmol) was suspended
in SOCl2 (0.5 mL) and the mixture was heated to reflux for 2 h.
After the completion of the reaction, the solvent was removed un-
der reduced pressure to give acyl chloride as white solid which was
used without further purification. The new prepared acyl chloride
was dissolved in CH2Cl2 (4 mL) and the solution was slowly added
into a biphasic mixture of G4-NH2.TsOH (0.5 g, 0.62 mmol) in anhy-
drous CH2Cl2 (7 mL) and Na2CO3 (0.15 g, 1.4 mmol) in H2O (7 mL).
The reaction mixture was stirred at room temperature for 12 h.
Then the organic layer was removed and the aqueous layer was ex-
tracted with CH2Cl2 (10 mL ꢃ 2). The combined organic layer was
washed with saturated aqueous NaHCO3 (10 mL ꢃ 2) and brine
13C NMR(300 MHz, CDCl3)
d (ppm): 170.88, 170.36, 169.71,
168.69 (4C, ester C@O), 165.70 (1C, amide C@O), 151.31, 141.06,
133.61, 123.11, 120.76, 111.34 (6C, ArC), 140.94, 120.32 (2C,
CH@CH), 97.20 (1C, C-1), 69.26 (1C, C-4), 68.29 (1C, C-2), 67.58
(1C, C-3), 66.58 (1C, C-5), 38.91 (1C, C-6), 55.89, 55.51 (2C,

92
K. Liu et al. / Carbohydrate Research 381 (2013) 83–92
(10 mL ꢃ 2), dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was chromatographed
on a silica gel column by use of petroleum ether–acetic ether (1:1,
v/v) to give compound 10s as a white solid (0.19 g, 36.5%), mp 109–
The eggs were returned back to the incubator. On day-10, the eggs
were divided into following groups: model group, DMSO group and
topotecan group of (10
group for each candidate compound (10, 3, 1
l
g/egg), and the high, medium, low dose
g/egg). Each group
l
110 °C; ½a 2D3
ꢄ
+14.2 (c 0.1200, CHCl3); 1H NMR (300 MHz, CDCl3) d
has three eggs. After adding drugs, the eggs were returned back
to the incubator. On day-13, the transparent tape was removed
and fixative containing equal methanol and acetone was dropped
on the observation window. Then the CAMs were excised, fixed,
and photographed.
(ppm): 7.92 (1H, d, J = 15.7 Hz, –CH@), 7.51 (1H, d, J = 8.5 Hz,
ArH), 7.41 (1H, d, J = 2.1 Hz, ArH), 7.23 (1H, dd, J = 2.1 Hz,
J = 8.4 Hz, ArH), 6.41 (1H, d, J = 15.7 Hz, –CH@), 6.30 (1H, d,
J = 9.7 Hz, NH), 5.73 (1H, d, J = 7.5 Hz, GlcNAH-1), 5.37 (1H, d,
J = 3.4 Hz, GalBH-4), 5.18–5.11 (2H, m, GlcNA H-3, GalBH-2), 4.99
(1H, dd, J = 3.4 Hz, J = 10.5 Hz, GalBH-3), 4.52 (1H, d, J = 7.8 Hz, GalB-
H-1), 4.49–4.44 (2H, m, GlcNA H-2, GlcNA H-6a), 4.21–4.11 (3H, m,
GalA H-6b, GalB H-6a, GalB H-6b), 3.94–3.84 (3H, m, GalAH-5, GalA-
H-4, GalB H-5), 2.15, 2.14, 2.10, 2.09, 2.08, 2.01, 1.99 (each 3H, each
s, 7 ꢃ OAc); 13C NMR(500 MHz, CDCl3) d (ppm): 170.51, 170.30,
170.25, 170.02, 169.96, 169.56, 169.33 (7C, ester C@O), 165.10
(1C, amide C@O), 135.89, 135.40, 131.44, 129.90, 128.27, 127.38
(6C, ArC), 136.69, 123.05 (2C, CH@CH), 100.67, 92.41, 74.40,
73.48, 71.89, 70.85, 70.72, 69.01, 66.65, 62.20, 60.82, 51.73 (12C,
carbohydrate ring carbons), 20.87, 20.75, 20.62, 20.54, 20.43 (7C,
7 ꢃ CH3); IR (cmꢁ1): 3463 (NH), 2938 (CH), 1751 (ester, C@O),
1668 (amide, C@O), 1630 (C@C), 1583, 1548, 1433, 1371, 1225,
1067, 898, 863; MS(ESI(ꢁ)70 V, m/z): 833.9 [MꢁH]ꢁ; Anal. Calcd
for C35H41Cl2NO18: C, 50.37, H, 4.95, N, 1.68. Found: C, 50.58, H,
4.95, N, 1.65.
Acknowledgements
The Project was supported by the Natural Science Foundation of
Jiangsu province, China(Grant No.BK2010436), and the Specialized
Research Fund for the Doctoral Program of Higher Education of
China(Grant No. 20110096110001).
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4.2.2. In vivo CAM assay
Fertilized chicken eggs (Guangda Co. Ltd., Zhejiang, China) were
positioned in a horizontal position and incubated at 37 °C under
60% relative humidity in an egg incubator equipped with a turner
which automatically turned eggs 6 times/day. On day 6, a rectangle
window (1.0 cm ꢃ 1.0 cm) was cut in the shell as a portal of access
for the CAM. Seal the pseudo air chamber with transparent tape.