Anomeric selectivity and influenza A virus inhibition study on methoxylated analogues of Pentagalloylglucose

Article abstract of DOI:10.1016/j.carres.2014.10.011

Anomeric selectivity in galloylation of d-glucose and d-mannose with carboxylic acid was explored under steglich conditions. Base catalyst 4-dimethylaminopyridine favored the formation of alpha-anomers, while adding an acid and carbodiimide favored the formation of beta-anomers. Steric hindrance between α,β-unsaturated acid and C-2 OH stereochemistry (adjacent carbon to anomeric) influenced anomeric selectivity for both d-glucose and d-mannose. The influenza A virus inhibition activities of the synthesized compounds were evaluated in Madin-Darby canine kidney cell line using the cytopathic effect inhibition assay. All the synthetic methoxylated analogues showed more considerable activity against influenza A virus than their corresponding acids, which indicated the sugar core as key functionality for anti-viral activity. The activities of trimethoxy-cinnamic acid Pentagalloylglucose analogues, 3α, 3β, 4α, and 4β (IC_50, 109.1 μM, 134.4 μM, 119.5 μM, 111.1 μM, respectively) were better than those of trimethoxy-benzoic acid Pentagalloylglucose analogues, 1-αβ and 2α, 2β (IC_50, 209.8 μM, 132.9 μM, 161.2 μM, respectively), which suggested that the double bond in cinnamic acid Pentagalloylglucose analogues makes the major contribution for influenza A virus inhibitory activity. Notably, several anomeric mixtures showed better activities than pure alpha or beta anomer and were almost two times more effective than Ribavirin, a clinically used anti-viral drug.

Full text of DOI:10.1016/j.carres.2014.10.011

Carbohydrate Research 402 (2015) 152–157
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Anomeric selectivity and influenza A virus inhibition study
on methoxylated analogues of Pentagalloylglucose
⇑
Shaikh Qurat-ul-ain, Wei Wang, Meiting Yang, Na Du, Shengbiao Wan, Lijuan Zhang, Tao Jiang
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2014
Received in revised form 14 October 2014
Accepted 18 October 2014
Available online 28 October 2014
Anomeric selectivity in galloylation of D-glucose and D-mannose with carboxylic acid was explored under
steglich conditions. Base catalyst 4-dimethylaminopyridine favored the formation of alpha-anomers,
while adding an acid and carbodiimide favored the formation of beta-anomers. Steric hindrance between
a
,b-unsaturated acid and C-2 OH stereochemistry (adjacent carbon to anomeric) influenced anomeric
selectivity for both -glucose and -mannose. The influenza A virus inhibition activities of the synthe-
D
D
sized compounds were evaluated in Madin–Darby canine kidney cell line using the cytopathic effect inhi-
bition assay. All the synthetic methoxylated analogues showed more considerable activity against
influenza A virus than their corresponding acids, which indicated the sugar core as key functionality
Keywords:
Steglich esterification
Anomeric selectivity
Galloylation
for anti-viral activity. The activities of trimethoxy-cinnamic acid Pentagalloylglucose analogues, 3
, and 4b (IC50, 109.1 M, 134.4 M, 119.5 M, 111.1 M, respectively) were better than those of tri-
methoxy-benzoic acid Pentagalloylglucose analogues, 1- b and 2 , 2b (IC50, 209.8 M, 132.9 M,
161.2 M, respectively), which suggested that the double bond in cinnamic acid Pentagalloylglucose ana-
a, 3b,
4
a
l
l
l
l
Influenza A virus inhibition
a
a
l
l
l
logues makes the major contribution for influenza A virus inhibitory activity. Notably, several anomeric
mixtures showed better activities than pure alpha or beta anomer and were almost two times more effec-
tive than Ribavirin, a clinically used anti-viral drug.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
group at p-position.12 Therefore, the methoxy/hydroxyl/cinnamic
acid derivatives of PGG could be interesting antiviral agents for
Tannins are the most common water-soluble polyphenolic sec-
ondary metabolites of higher plants with molar masses in the
range of 300–3000D.1 According to recently proposed biosynthetic
mechanism of natural polyphenols,2,3 the polyphenols derived/
formed through shikimate/phenylpropanoid pathway are identi-
fied as secondary metabolites.
drug designing and screening.
Penta-O-galloyl-b- -glucose (PGG) and tannic acid (Fig. 1) are
D
two backbone units of poly/oligomericgallo-ellagitannins with
various biological activities.6,13,14 The gallo-ellagitannins with the
b-D-glucose core are the major isomers in nature because of the
b-glucogallin (Fig 2) formation at the earlier stage of Gallo-tannins
Gallo- and ellagi-tannins (hydrolyzable tannins) are natural
biosynthesis.15,16
esters of
D
-glucose, which show various biological activities, such
In pure forms, various bioactivities have been reported for both
anomers of hydrolyzable tannin. For example, b-PGG inhibits DNA
replication synthesis in PCa cells (S-phase arrest) and G1 arrest
with cyclin D1 down-regulation.17 It also can induce the growth
inhibition in MDA-MB-231 and BT474 Cells through the down-
as anticancer, anti-angiogenesis, P-gp inhibition, anti-oxidant,
anti-inflammatory, anti-ulcer, and so on.4–6 In addition, penta-O-
galloyl-b-D-glucose (PGG), gallic acid, methoxygallic acid, and their
esters also have antiviral activities against a variety of viruses, such
as influenza A, rabies, HIV, hepatitis B, ADV, HSV, and so on.7–10
Acid esters have been reported as potent antiviral agents than their
corresponding acids. For example, the antiviral activity of gallic
acid esters is higher than gallic acid.11 Curcumin (Fig. 1) with
methoxy group at p-position of cinnamic acid moiety has a stron-
ger inhibitory effect of influenza A virus production than the OH
regulating Skp2 protein.18
a-PGG is an IR (insulin receptor) signal-
ing activator, which induces apoptosis in RKO cells through p53,
Bax, and caspase 3.19 Arbutin (Fig. 1) is a glycosylated hydroqui-
none and is a simple gallotannin, both
a- and b-anomers of arbutin
possess anti-oxidant and depigmentation properties in pure forms.
a
-Arbutin is more stable and ten times more active than b-anomer
inhibition of a tyrosinase from B16 mouse melanoma.20
Since the anomeric isomers of gallotannins and ellagitannins
glycosides co-exist, few studies have been done on comparative
⇑
Corresponding author. Tel.: +86 532 82032712; fax: +86 532 82033054.
E-mail address: jiangtao@ouc.edu.cn (T. Jiang).
http://dx.doi.org/10.1016/j.carres.2014.10.011
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

S. Qurat-ul-ain et al. / Carbohydrate Research 402 (2015) 152–157
153
OH
OH
HO
OH
OH
HO
OH
O
O
O
O
OH
O
HO
O
O
OH
O
O
O
O
OH
O
H3CO
OCH3
O
(D)
curcumin
(A)
PGG
β−
OH
OH
OH
HO
OH
HO
OH
OH
OH
O
OH
O
OH
HO
O
OH
OH
OH
HO
O
OH
OH
O
HO
OH
O
O
O
O
OH
O
O
HO
HO
O
O
O
O
O
OH
OH
O
O
O
(B)
β−arbutin
O
OH
(C)
tannic acid
OH
OH
OH
OH
O
O
O
HO
HO
HO
OH
OH
HO
Figure 1. Major components of gallo-ellagitannins (hydrolyzable tannins).
OH
O
OH
gallate 1-beta-
OH
glucosyltransferase
OH
OH
O
O
+ UDP-glucose
- UDP
HO
HO
HO
OH
O
OH
OH
β−glucogalin
Gallic acid
Figure 2. Basic unit of gallo-ellagitannins (hydrolyzable tannins) biosynthesis pathway.
effect of pure or/and mixed anomers. Recently, stereoselective syn-
thesis of galloyl esters of glucose has been reported with pure ano-
meric isomer of D-glucose as starting material. The stereoselective
synthesis of pure anomers of galloyl esters has also reported using
acyl halides and glucose coupling reaction in the presence of
TMEDA as base catalyst in DCM medium.21,22 In the present work,
we tried to make effort on the stereochemical study for the synthe-
sis of pure anomeric galloyl esters by using the steglich esterifica-
tion method and exploring their anti-viral bioactivity in pure and
different ratio mixtures.
synthesized with benzoic and cinnamic acid under adjusted condi-
tions. The required products were obtained by using previously
reported methods23 in yields of 35–50% (Scheme 1). The alpha
and beta isomers were purified by using a normal silica gel column
chromatography with a solvent combination of dichloromethane,
hexane, and ethylacetate (V/V/V, 8:3:2) as eluent. These optimized
reaction conditions were used for the aroylation of sugars with all
kinds of carboxylic acids, even with sterically hindered carboxylic
acids.
The anomeric selectivity of the reaction depends on rates of ano-
merization against the rate of esterification in a given medium.22 In
a given reaction medium, fast anomerization relative to esterifica-
tion predominantly favors b-isomer, while slow anomerization rel-
2. Results and discussion
ative to esterification predominantly favors
a
-isomer.24 Based on
2.1. Synthesis and stereochemical studies
the mentioned kinetics of the mechanism, we analyzed stereo-
chemical results after using the different reagents (DMAP as cata-
lyst, DCC carbodiimide as coupling agent and benzoic acid) with
various concentrations in the reaction medium (DCM) for the syn-
thesis of compound 1. With the same ratios, different reagents
resulted in different anomeric selectivities. The difference in ano-
meric selectivity could be caused by steric interference and ano-
meric neighboring stereochemical center. Stoichiometric molar
The aroylation of sugars is a well-studied PGG synthesis reac-
tion.21,22 However, the anomeric selectivity of glucosyl ester reac-
tions needs to be optimized to get the desired products with
common and easy methods. In this paper, the effect of different
stoichiometric amounts of reagents and their structural influence
on the anomeric selectivity were tested. Penta-O-galloyl
D-glucose
(PGG) and penta-O-galloyl- -mannose (PGM) analogues were
D

154
S. Qurat-ul-ain et al. / Carbohydrate Research 402 (2015) 152–157
OH
OGMe
O
O
HO
HO
MeGO
MeGO
i
OH
GMeOH
OGMe
O
OH
OGMe
O
or
GMe =
MeO
OMe
MeO
OMe
OMe
OMe
1-αβ
3-αβ
Scheme 1. Esterification of monosaccharide with gallic acid derivatives. Reagents and conditions: (i) DCC, DMAP, DCM, 16–18 h reflux.
Table 2
ratio of each reagent affected the anomeric selectivity; the results
are given in Table 1. Poor anomeric selectivity was observed,
approximately equal amounts of alpha and beta isomers were
obtained when large amounts of reagents were used Table 1 (entry
1). Decreased amount of base (DMAP) equivalent equaled one, it
Effect of anomeric neighboring stereo center and olefinic bond on selectivitya
Entry
Sugar
ACID
a:b
HO
O
gave a slight increased ratio of
could be read as the anomerization rate (
a
-anomer (Table 1, entry 2); the ratio
) versus the esterification
R1
=
1
2
R1
R1
53.0:47.0
52.2:47.8
D
-Glucose
a
O
O
rate (b). Therefore, the decrease in DMAP molar ratio also decreased
the anomerization rate. Evidenced by previous findings, the exces-
sive DMAP increased the anomerization rate and dominantly
favored the beta anomers.22,25 b-Anomer was obtained dominantly
(Table 1 entry 3) when five equivalents of the 3,4,5-trimethoxy ben-
zoic acid were used to esterify the 5-hydroxyl groups (1 equiv: for 1
OH). In contrast to the effect caused by the DMAP, lower yields and
increased anomerization rate were observed with reduced concen-
tration of 3,4,5-trimethoxy benzoic acid. The anomerization under
proposed conditions favors the b-anomer. Stoichiometric amount
of carbodiimide also favors b-anomer with significantly decreased
yield. The presence of excess amounts (unreacted) of DMAP in the
reaction mixture could be a reason to the increase of rate of
anomerization for both acid and carbodiimide which resulted in
the formation of b-anomer as a major product.
O
O
D-Mannose
HO
O
3
4
R
R
43.4: 56.6
64.9:35.1
D-Glucose
R =
O
O
D-Mannose
a
DCC (N,N0-dicyclohexylcarbodiimide), DMAP.
hand, both sugar cores showed different ratios of anomers when
coupled with trimethoxycinnamic acid (Table 2 entries 3 and 4).
b-Anomer was dominant with glucose while mannose gives
DCC highly activates the acids by increasing electrophilicity at
carbonyl center. Acids and coupling reagents show similar effects
on selectivity because of the dominant effect of DMAP in the med-
ium. The excessive amount of DMAP increases the anomerization
a
-anomer as the major isomer. The effect influenced by C-2 center
in hexoses could be elucidated in such a point, that, the equatorial
OH in -glucose and the axial OH in -mannose cause steric hin-
drance with double bond in cinnamic acid analogues, which
resisted the and b-attacks for both sugars, respectively.
The olefinic bond also influenced the anomeric selectivity for
products. The adjacent equatorial OH of -glucose shows steric
hindrance for -attack with trimethoxycinnamic acid, as a result,
b-isomer dominantly formed (Table 2 entries 1 & 3). Similarly,
the axial OH at the adjacent carbon of -mannose has steric
hindrance for b-attack with trimethoxybenzoic acid, thus the
-isomer formed as a major product (Table 2 entries 2 & 4). The
D
D
rate for
a-anomer. Thus, DMAP and acid with specific equivalent
ratios can increase the selectivity for
respectively.
a and b-anomers,
a
To explore the effects of neighboring carbon stereochemistry
and ,b-unsaturated acid on anomeric selectivity, -mannose
methyl-PGG analogues were prepared in the same way. Table 2
summarizes the obtained selectivity results for -mannose and
-glucose sugars. The similar anomeric selectivity ratios were
D
a
D
a
D
D
D
observed for the both mannose and glucose sugars when coupled
with trimethoxybenzoic acid. (Table 2 entries 1 & 2). Thus, the
axial/equatorial configuration at the adjacent carbon of anomeric
center shows no influence on the anomeric selectivity. On other
a
observed results for both C-2 and double bond influence evidently
confirm the steric hindrance as the major influencing element for
selectivity.
2.2. Biological studies
Table 1
The
a- and b-anomers in pure and mixture (different ratios)
Anomeric selectivity for compound 1 under different reaction conditions
forms were screened for anti-influenza A virus activity in MDCK
cells using the CPE inhibition assay along with three positive con-
trols. Firstly, trimethoxy-benzoic acid and trimethoxy-cinnamic
acid (entries 13 and 14 in Table 3) showed no anti-viral activity.
In contrast, the synthetic methoxylated analogues of PGG showed
more considerable activity against influenza A virus than their
corresponding acids. The results indicates that, the sugar core is
critical for their anti-viral activity. Subsequently, entries 1–7 in
Entry ACID
(equiv)
DMAP
(BASE)
(equiv) (equiv)
DCC
a
:b
Yield
(%)
(coupling reagent)
1
2
3
8
8
8
1
8
9
9
9
53.0:47.0 37
65.6:34.4 50
33.7:66.3 25
5 (1 for each
OH)
8
4
8
1
34.2:65.8 12

S. Qurat-ul-ain et al. / Carbohydrate Research 402 (2015) 152–157
155
Table 3
(2-
glucopyranos (3-
cinnamoyl)]- ,b-
using tri-methoxy benzoic or tri-methoxy cinnamic acids with
either -mannose or -glucose. Their - and b-isomers were sepa-
a
b), 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]-
b), and 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxy-
-mannopyranos (4- b), were synthesized by
a,b-D-
Influenza A virus inhibition activity of PGG analoguesb
a
D
a
a
a
a
Entry Compound IC50
(lM)
Entry Compound
IC50 (lM)
1
1-
b = 53/47)
2-
2-b
3-
3-b
a
b (
a
/
209.8 20.8
8
3-
a
b (
a
/b = 63/37) 66.5 4.9
D
D
a
2
3
4
5
a
132.9 13.6
161.2 5.6
109.1 7.3
9
10
11
3-
3-
4-
a
a
a
b (
b (
b (
a
a
a
/b = 80/20) 66.3 5.1
/b = 20/80) 79.2 1.5
/b = 63/37) 49.9 3.8
rated using normal silica gel column chromatography with an elu-
ent of dichloromethane, hexane, and ethylacetate (V/V/V, 8:3:2).
a
Effects of each reagent on the
esterification coupling were investigated. Among the synthesized
compounds, four pure - and b-isomers exhibited comparable
anti-influenza A virus inhibition activity as that of an anti-viral drug,
ribavirin. However, the mixed anomers 3- b (1,2,3,4,6-Pentakis[-O-
(3,4,5-trimethoxycinnamoyl)]- ,b- -glucopyranos) and 4-
(1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]- ,b- -manno-
pyranos) showed significant anti-influenza A virus activity with
IC50 value of 66.5 M, 66.3 M, 79.2 M, and 49.9 M, respectively.
These IC50 values were only half of that of Ribavirin and pure or b
anomers. In addition, the in vitro tests showed that the synthetic
compounds had no cytotoxicity at concentrations up to 100 M in
a/b-anomeric selectivity in steglich
134.4 10.5 12
Ribavirin (positive 113.1 2.7
control)
a
6
7
4-
a
119.5 11.8 13
3,4,5-Trimethoxy
benzoic acid
P500
4-b
111.1 9.6
14
3,4,5-Trimethoxy
cinnamic acid
P500
a
a
D
ab
a
IC50 value was calculated from three independent experiments performed in
triplicate.
a
D
b
The sugar core of 1 and 3 is
mannose.
D-glucose and the sugar core of 2 and 4 is D-
l
l
l
l
a
l
Table 3, the anti-viral activity of trimethoxy-cinnamic acid PGG
analogues, 3b, and 4b (with the IC50 of 109.1 M,
134.4 M, 119.5 M, 111.1 M, respectively) is better than that
of trimethoxy-benzoic acid PGG analogues, 1- b and 2 , 2b (with
the IC50 of 209.8 M, 132.9 M, 161.2 M respectively). These
four cancer cell lines, including two lung cancer (A549 and
H1299) and two colon cancer (HCT116 and HT29) cell lines. All
the results suggested that the anomer mixtures were potential
candidates for anti-viral drug development.
3a
,
4a
l
l
l
l
a
a
l
l
l
results suggests the cinnamic acid double bond in PGG analogues
makes the major contribution for anti-viral activity and it could
be a major pharmacophore for influenza A virus inhibition.
4. Experimental section
4.1. Chemistry
Interestingly, the synthetic
(with ratio 63/37, entry 8 in Table 3), was much more effective
against influenza A virus inhibition with IC50 of 66.5 M than the
pure - or b-anomer compounds, 3- and 3-b (entries 4 and 5 in
Table 3) with IC50 of 109.1 M and 134.4 M, respectively. Hence,
in order to confirm the anomer mixture is more active than the
pure anomers, we mixed the pure anomeric compounds, 3- and
3-b, 4- and 4-b, in different ratios to obtain 3- b ( /b = 80/20),
3- b ( /b = 20/80) and 4- b ( /b = 63/37) (entries 9–11 in Table 3).
The results indicates that the mixed anomers 3- b (1,2,3,4,6-Pen-
takis[-O-(3,4,5-trimethoxycinnamoyl)]- ,b- -glucopyranos) and
4- (1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]- ,b-
mannopyranos) show significant anti-influenza A virus activity
with IC50 value of 66.5 M, 66.3 M, 79.2 M and 49.9 M (Table 3,
entries 9–11) respectively than pure or b anomers. In addition,
the mixed anomers 3- b and 4- b are almost two times more
effective than Ribavirin (an anti-viral drug) which has IC50 value
of 113.1 M (Table 3, entry 13).
We next examined the in vitro cytotoxicity of the synthetic
a- and b-anomer mixture, 3-ab
l
4.1.1. General
a
a
All starting materials and solvents were obtained from com-
mercial sources and used without further purification. Column
chromatography was performed using silica gel (200–300 mesh)
purchased from MeiGao Ltd (Qingdao, China). TLC (Thin layer chro-
matography) was performed on pre coated silica-gel 60 F254
plates purchased from Merck Ltd (Darmstadt, Germany). Melting
points were determined on a Mitamura-Riken micro-hot stage
and were uncorrected. 1H NMR and 13C NMR spectra were taken
on a Jeol JNM-ECP 600 spectrometer (Jeol Ltd, Tokyo, Japan) with
tetramethylsilane (Me4Si) as the internal standard, and chemical
shifts were recorded as d values. Mass spectra were recorded on
a Q-TOF Global mass spectrometer.
l
l
a
a
a
a
a
a
a a
a
a
D
ab
a
D-
l
l
l
l
a
a
a
4.1.2. General procedure for synthesis of galloyl esters
l
A suspension of D-glucose 0.02 g (0.11 mmol), 3,4,5-trimethoxy
benzoic acid 0.18 g (0.88 mmol), (DCC)/(EDC) 0.20 g (0.99 mmol),
and N,N-(di-methylamino) pyridine (DMAP) 0.10 g (0.88 mmol)
in 15 mL of freshly distilled dichloromethane (DCM) was refluxed
for 16–18 h (monitored by TLC). After cooling reaction mixture at
room temperature, urea by-product was filtered out by gravity fil-
compound
1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]-
a,b-D
-glucopyranos and 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxy-
cinnamoyl)]-a,b-D-mannopyranos) and their a or b pure anomers
in four cancer cell lines including two lung cancer (A549 and
H1299) and two colon cancer (HCT116 and HT29) cells, in an effort
to quantitatively evaluate safety profile. Primary screening of com-
tration. The filtrate was evaporated. The
ab isomer in the residue
was purified by column chromatography, where the column was
eluting with 4:3:1 (v/v/v) mixture of dichloromethane, hexane,
and ethyl acetate or a mixture of alpha beta isomers (whose ratio
was calculated by 1H NMR) was obtained by eluting the column
with 8:3:2 (v/v/v) of the same solvents. The product fractions were
collected separately, evaporated, and precipitated from toluene
and cyclohexane.
pounds, 3-
with methyl protected groups of hydroxyl groups was performed
at concentration of 100 M. At this concentration, all the tested
a, 3-b, 4-a, 4-b, and the anomer mixture 3-ab and 4-ab,
l
compounds show no cytotoxicity in these four cell lines. As
expected, the introduction of tri-methoxy-substituted group to
cinnamic acid of compounds 3 and 4 has decrease the compound
cytotoxicity, which clearly suggested that these compounds should
be safer anti-viral agents.
4.1.2.1. 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxybenzoyl)]-
a,b-D-
glucopyranose (1). ,b-Anomer): White solid (37%), Rf
(a
(DCM/Hex/EtoAc, 8:3:2) 0.30, mp 89–95 °C. IR (KBr) 2939, 1724,
1587, 1124, MASS (ESI+) m/z 1173.8 [M+Na+], HRMS calcd for
(C56 H62 O26+NH4)+ 1168.3868, found 1168.3856. 1H NMR
(600 MHz CDCl3) d 7.41 (s 2H, ArH), 7.31 (s 2H, ArH), 7.29 (2s
4H, ArH), 7.15–7.14 (br s, 8H, ArH), 7.12 (s 2H, ArH), 7.09 (s 2H,
3. Conclusion
In present work, methoxy PGG analogues, 1,2,3,4,6-Pentakis
[-O-(3,4,5-trimethoxybenzoyl)]-
6-Pentakis[-O-(3,4,5-trimethoxylbenzoyl)]-
a
,b-
D
-glucopyranos (1-
ab), 1,2,3,4,
a
,b- -mannopyranos)
D

156
S. Qurat-ul-ain et al. / Carbohydrate Research 402 (2015) 152–157
ArH), 6.78 (d J = 3.3 Hz, 1H, H-1
a-isomer), 6.27 (t J = 10.4 Hz, 1H,
(2s 4H, ArH), 6.65 (s 2H, ArH), 6.56–6.53 (d J = 15.95 trans, 1H, ole-
H-3
a
-isomer), 6.20 (d J = 8.25 Hz, 1H, H-1 b-isomer), 6.02 (t
finic H), 6.49–6.46 (d J = 15.95 trans, 1H, olefinic H), 6.40 (d
J = 2.2 Hz, 1H, H-1), 6.37–6.34 (d J = 15.95 trans, 1H, olefinic H),
6.32–6.30 (d J = 15.95 trans, 1H, olefinic H), 6.28–6.25 (d J = 15.95
trans, 1H, olefinic H), 5.81–5.79 (dd J = 3.3 & 9.9 Hz, 1H, H-3),
5.76 (t J = 9.35 Hz, 1H, H-4), 5.62–5.61 (m, 1H, H-2), 4.48–4.37
(m, 3 h, H-6b, H-5, & H-6a), 3.94–3.83 (7s 33H, OCH3), 3.787–
3.782 (2s 12H, OCH3). 13C NMR (126 MHz, CDCl3) d 166.49,
165.91, 165.79, 165.65, 164.14, 153.48, 153.47, 153.37, 153.32,
147.51, 146.83, 146.67, 146.46, 145.71, 140.66, 140.50, 140.37,
140.21, 129.55, 129.34, 129.25, 129.21, 116.39, 115.79, 115.74,
115.60, 115.35, 105.50, 105.41, 105.39, 105.38, 105.26, 90.78,
70.65, 68.95, 68.83, 66.61, 63.00, 62.99, 62.94, 61.03, 61.00,
60.97, 60.96, 60.93, 56.22, 56.15, 56.09, 56.07, 56.02, 50.87.
(b-Anomer): Off-white solid (37.1%), Rf (DCM/Hex/EtoAc, 8:3:2)
J = 9.90 Hz, 1H, H-3 b-isomer), 5.81–5.70 (m 3H, H-4
H-2,H-4 b-isomer), 5.57 (dd J = 3.3 & 9.9 Hz, 1H, H-2
a-isomer,
a-isomer),
4.79–4.75 (m 2H, H-6b
1H, H-5 -isomer), 4.45–4.37 (m 3H, H-6a
isomer), 3.95–3.82 (m 84H, OCH3), 3.72 (s 6H, OCH3). 13C NMR
(126 MHz, CDCl3) 165.64, 165.48, 165.01, 164.91, 164.87,
164.18, 153.21, 152.96, 152.89, 152.87, 152.82, 143.05, 142.84,
142.80, 142.68, 142.42, 124.45, 123.50, 123.47, 123.41, 123.16,
109.99, 107.44, 107.37, 107.15, 107.10, 107.05, 106.99, 92.90,
90.34, 73.26, 72.97, 71.16, 70.78, 70.60, 70.56, 69.70, 69.23,
63.16, 61.01, 60.94, 60.89, 60.86, 60.83, 56.39, 56.19, 56.16,
56.10, 55.94.
a
-isomer, H-6b b-isomer), 4.66–4.63 (m
a
a
-isomer, H-5,H-6a b-
d
4.1.2.2. 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxybenzoyl)]-
a
,b-
D
-
0.20, mp 117–120 °C. [
a
]
ꢀ0.0965 (CH2Cl2) IR (KBr) 2915, 1716,
D
mannopyranose (2).
The data for compound 2 are the same
1502, 1124, HRMS calcd for (C66 H72 O26+Na)+ 1303.4204, found
1303.4165. MASS (ESI+) m/z 1303.9, 11304 [M+Na+], 1H NMR
(600 MHz CDCl3) d 7.72–7.69 (d J = 15.95 trans, 1H, olefinic H),
7.65–7.59 (3d J = 15.83 & 15.95 trans, 3H, olefinic H), 7.56–7.53
(d J = 15.95 trans, 1H, olefinic H), 6.81 (s 2H, ArH), 6.70 (2s 4H,
ArH), 6.67 (s 2H, ArH), 6.65 (s, 2H, ArH), 6.39–6.23 (4d J = 15.95
trans, 5H, olefinic H), 6.21 (s 1H, H-1) 5.88 (d J = 2.75 Hz, 1H, H-
2), 5.70 (t J = 9.9 Hz, 1H, H-4), 5.53 (dd J = 3.3 & 9.9 Hz, 1H, H-3),
4.48 (br d J = 4.4 Hz, 2H, H-5 H-6b), 417–4.13 (m 1H H-6a), 3.90
(s 3H, OCH3), 3.88 (s 6H, OCH3) 3.6–3.85 (2s 9H, OCH3) 3.84–3.83
(2s 9H, OCH3) 3.81 (s 6H, OCH3) 3.78–3.77 (2s 12H, OCH3). 13C
NMR (126 MHz, CDCl3) d 166.43, 166.32, 165.69, 165.29, 164.67,
153.52, 153.37, 153.36, 147.17, 146.79, 146.78, 146.44, 145.68,
140.73, 140.57, 140.51, 140.27, 129.66, 129.32, 129.29, 129.28,
129.26, 116.41, 115.72, 115.60, 115.40, 115.37, 105.53, 105.49,
105.48, 105.40, 89.37, 70.06, 69.99, 69.56, 68.42, 61.99, 61.00,
60.94, 60.93, 56.24, 56.14, 56.13, 56.11.
as previously reported.21
4.1.2.3. 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]-
-glucopyranose (3). -Anomer): Off-white solid (31.4%), Rf
(DCM/Hex/EtoAc, 8:3:2) 0.25, mp 118–120 °C. [ 0.129 (CH2Cl2)
a,b-
D
(a
a
]
D
IR (KBr) 2933, 1716, 1502, 1120, MASS (ESI+) m/z 1304.0
[M+Na+], HRMS calcd for (C66 H72 O26+NH4)+ 1298.4650, found
1298.4633. 1H NMR (600 MHz CDCl3) d 7.73–7.70 (d J = 15.95 trans,
1H, olefinic H), 7.64–7.54 (m, 4H, olefinic H),6.82 (s 2H, ArH), 6.76
(s 2H, ArH), 6.69–6.66 (3s 6H, ArH), 6.64 (d J = 3.85 Hz, 1H, H-1),
6.53- 6.50 (d J = 15.40 trans, 1H, olefinic H) 6.42–6.39 (d J = 15.95
trans, 1H, olefinic H), 6.31–6.24 (3d J = 15.95, 15.40. 15.40 trans,
3H, olefinic H), 5.98 (t J = 9.9 Hz, 1H, H-3), 5.55 (t J = 9.9 Hz, 1H,
H-4), 5.47 (dd J = 3.3 & 6.6 Hz, 1H, H-2), 4.42–4.38 (m, 2H, H-5-
6b), 4.13 (dd J = 7.1 & 14.3 Hz, 1H, H-6a), 3.93 (s 6H, OCH3), 3.91
(s 3H, OCH3) 3.88–3.86 (3s 12H, OCH3) 3.83 (br s 18H, OCH3)
3.80 (s 6H, OCH3). 13C NMR (126 MHz, CDCl3) d 166.46, 166.34,
165.71, 165.30, 164.68, 153.50, 153.35, 153.33, 147.19, 146.83,
146.80, 146.47, 145.70, 140.63, 140.47, 140.41, 140.17, 129.66,
129.31, 129.28, 129.27, 129.26, 116.39, 115.69, 115.58, 115.37,
115.35, 110.09, 109.99, 105.44, 105.40, 105.31, 89.34, 70.04,
69.95, 69.52, 68.34, 61.95, 61.02, 60.96, 60.95, 56.22, 56.12,
56.10, 56.09, 50.85.
4.2. Biological methods
4.2.1. Materials and instruments
Madin–Darby canine kidney (MDCK) cells, Human colorectal
cancer cell lines HCT116 and HT29 and human lung cancer cell
lines A549 and H1299 were obtained from the Type Culture
Collection of the Chinese Academy of Sciences Shanghai, China),
McCoy’s 5A medium was from Gibco; RPMI-1640 medium was
from Hyclone; Fetal Bovine Serum (FBS) was from Gibco; penicillin
and streptomycin were from Hyclone; trypsin was from Cellgro;
Resazurin was from Sigma; DMSO was from Solarbio. Spectramax
M5 plate reader was from Molecular Devices). OLYMPUSCKX41
inverted microscope and cell counting chamber were from
Shanghai Qiujing biochemical reagents Instrument Co., Ltd. Cell
culture dishes and 96-well standard plates were from Corning
(b-Anomer): Light yellow solid (40%), Rf (DCM/Hex/EtoAc,
8:3:2) 0.13, mp 121–123 °C. [
a
]
D
ꢀ0.010 (CH2Cl2) IR (KBr) 2915,
1718, 1504, 1124, MASS (ESI+) m/z 1304.0 [M+Na+], HRMS calcd
for (C66 H72 O26+NH4)+ 1298.4635, found 1298.4635. 1H NMR
(600 MHz CDCl3) d 7.63–7.61 (d J = 15.95 trans, 1H, olefinic H),
7.58–7.55 (d J = 15.74 trans, 1H, olefinic H), 7.54–7.51 (d J = 15.95
trans, 1H, olefinic H) 7.53–7.51 (d J = 15.95 trans, 1H, olefinic H),
7.50–7.47 (d J = 15.95 trans, 1H, olefinic H), 6.70–6.68 (2s 4H,
ArH), 6.64–6.61(3s 6H, ArH), 6.36–6.20 (5d J = 15.95 trans, 5H, ole-
finic H) 6.05 (d J = 8.25, 1H, H-1), 5.67 (t J = 9.4 Hz, 1H, H-3), 5.52–
5.45 (m, 2H, H-2 & H-4), 4.40–4.35 (m, 2H H-5 & H-6b), 4.14–4.11
(m, 1H, H-6a), 3.81–3.77 (2m, 45H, OCH3). 13C NMR (126 MHz,
CDCl3) d 166.67, 166.20, 165.69, 165.59, 165.10, 151.60, 151.41,
151.35, 151.15, 149.23, 149.15, 149.14, 149.12, 147.32, 146.59,
146.41, 146.33, 145.46, 127.30, 127.01, 126.94, 123.34, 123.16,
122.87, 115.05, 114.14, 114.09, 113.99, 113.94, 110.94, 110.88,
110.84, 109.64, 109.59, 109.56, 92.13, 73.04, 72.47, 70.30, 68.51,
62.28, 55.96, 55.93, 55.86, 55.84.
USA; and the 0.22 lm millipore filter papers were from Whatman.
Ribavirin injection (50 mg/mL) was obtained from LuKang Cisen
(China).
4.2.2. Antiviral activity
The antiviral activity against H1N1 was evaluated by the CPE
inhibition assay as described previously.26,27 Confluent MDCK cells
with the influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) were
firstly incubated at 37 °C for 1 h. After removing the virus inocu-
lum, cells were maintained in infecting media (RPMI 1640, 4
mL of trypsin) containing different concentrations of test com-
pounds at 37 °C. After 48 h, cells were fixed with 100 L of 4%
formaldehyde for 20 min at room temperature. After removal of
the formaldehyde, the cells were stained with 0.1% crystal violet
for 30 min. The plates were washed and dried, and the intensity
of crystal violet staining for each well was measured in a micro-
plate reader (Bio-Rad, USA) at 570 nm. The virus CPE inhibition
lg/
4.1.2.4. 1,2,3,4,6-Pentakis[-O-(3,4,5-trimethoxycinnamoyl)]-
-mannopyranose (4). -Anomer): Off-white solid (20.0%),
Rf (DCM/Hex/EtoAc, 8:3:2) 0.25, mp 106–109 °C. [
a,b-
D
(a
l
a]
ꢀ0.0079
D
(CH2Cl2) IR (KBr) 2917, 1714, 1502, 1122, HRMS calcd for (C66
H72 O26+NH4)+ 1298.4650, found 1298.4639. MASS (ESI+) m/z
1303.9 [M+Na+], 1H NMR (600 MHz CDCl3)
d
7.75–7.52 (5d
J = 15.95 trans, 5H, olefinic H), 6.81–6.80 (2s, 4H, ArH), 6.69–6.68

S. Qurat-ul-ain et al. / Carbohydrate Research 402 (2015) 152–157
157
(%) was calculated by the equation: Virus inhibition (%) = [(Asample
570 ꢀ Avirus 570)/(Amock570 ꢀ Avirus 570)] ꢁ 100; where, Amock 570 was
the absorbance without virus infection, Asample 570 was absorbance
with virus infection and drug treatment, and Avirus 570 was absor-
bance with virus infection but without drugs. The concentration
required for a test compound to reduce the CPE of the virus by
50% (IC50) was determined. Ribavirin was used as the positive
control.
W. Wang and Chinese Government scholarship for foreign nationality
students, awarded by Chinese Scholarship Council (CSC).
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This research was supported by Key International S&T
Cooperation Projects (2013DFG32160) of Ministry of Science and
Technology of the People’s Republic of China for T. Jiang, National
Natural Sciences Foundation of China (NSFC No. 81302811) for