1,2,3,4,6-Pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-glucopyranose (PGG) analogs: Design, synthesis, anti-tumor and anti-oxidant activities

Article abstract of DOI:10.1016/j.carres.2016.04.021

1,2,3,4,6-Pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-glucopyranose (PGG) 12 has been reported for its antioxidant activities, where the free OH groups in PGG seem to be critical for activities. To explore PGG-based compounds as chemotherapeutic agents and to analyze the contribution of specific OH groups in PGG for anti-cancer activities, we designed and synthesized a series of 27 benzoic and cinnamic acid analogs of PGG. These analogs were tested for cytotoxicities against two human lung (A549 and H1299) and two human colon (HCT116 and HT29) cancer cell lines. Compound 12 (PGG) had highest cytotoxicities against HCT116 and A549 cells with IC₅₀ of 1.61 μM and 3.02 μM, respectively. In contrast, the compound 16 (1,2,3,4,6-pentakis[-O-(4-hydroxy-3-methoxybenzoyl)]-α,β-D-glucopyranose, PVG) was most effective at killing HT29 and H1299 cells with IC₅₀ of 1.76 μM and 3.65 μM, respectively, indicating the mutual contribution of m-methoxy and p-hydroxy groups to the observed cytotoxicities. Moreover, cinnamic acid analogs were less active than the benzoic acid analogs evidenced by higher IC₅₀ values. Furthermore, in cinnamic acid analogs the hydrogenation of double bond to saturated 2-C side chain enhance the cytotoxicities in all four cell lines. Compounds also possess good anti-oxidant and reducing activities. Compound 12 and 26 show the highest antioxidant and reducing activities.

Full text of DOI:10.1016/j.carres.2016.04.021

Carbohydrate Research 430 (2016) 72–81
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
1,2,3,4,6-Pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-glucopyranose
(PGG) analogs: design, synthesis, anti-tumor and anti-oxidant
activities
Qurat-ul-ain Shaikh a,1, Meiting Yang a,1, Khadim Hussain Memon b, Mehreen Lateef c,
Du Na a, Shengbiao Wan a, Deslandes Eric d, Lijuan Zhang a,*, Tao Jiang a,**
a Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, China
b Department of Zoology, Shah Abdul Latif University, Pakistan
c Multidisciplinary laboratory, Medical and Dental College, Bahria University Karachi, Pakistan
d European Institute for Marines Studies, University of Western Brittany, France
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 10 February 2016
Received in revised form 14 April 2016
Accepted 21 April 2016
Available online 28 April 2016
1,2,3,4,6-Pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-glucopyranose (PGG) 12 has been reported for its
antioxidant activities, where the free OH groups in PGG seem to be critical for activities. To explore PGG-
based compounds as chemotherapeutic agents and to analyze the contribution of specific OH groups in
PGG for anti-cancer activities, we designed and synthesized a series of 27 benzoic and cinnamic acid
analogs of PGG. These analogs were tested for cytotoxicities against two human lung (A549 and H1299)
and two human colon (HCT116 and HT29) cancer cell lines. Compound 12 (PGG) had highest cytotox-
icities against HCT116 and A549 cells with IC50 of 1.61 ꢀM and 3.02 ꢀM, respectively. In contrast, the
compound 16 (1,2,3,4,6-pentakis[-O-(4-hydroxy-3-methoxybenzoyl)]-α,β-D-glucopyranose, PVG) was most
effective at killing HT29 and H1299 cells with IC50 of 1.76 ꢀM and 3.65 ꢀM, respectively, indicating the
mutual contribution of m-methoxy and p-hydroxy groups to the observed cytotoxicities. Moreover, cin-
namic acid analogs were less active than the benzoic acid analogs evidenced by higher IC50 values.
Furthermore, in cinnamic acid analogs the hydrogenation of double bond to saturated 2-C side chain enhance
the cytotoxicities in all four cell lines. Compounds also possess good anti-oxidant and reducing activi-
ties. Compound 12 and 26 show the highest antioxidant and reducing activities.
Keywords:
Pentagalloylglucose (PGG)
Pentavanilloylglucose (PVG)
Anti-tumor
Inhibition
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Gallo-tannins are widely distributed in foods4 and belong to
the class of hydrolysable tannins, which can be cleaved hydrolyti-
Tannins are the most common water-soluble polyphenolic sec-
ondary metabolites of higher plants with molecular mass ranging
from 300 to 3000 D. Their well-known human health benefits are
explained by the incomparable abilities of tannin polyphenol groups
in quenching reactive oxygen species (ROS) in human body, such
as superoxide radical, singlet oxygen, peroxylradical, nitric oxide,
hydroxyl radical, nitrogen dioxide and peroxynitrite, and by their
strong chelating property towards oxidative metal ions, which also
leads to the reduction of ROS.1–3
cally by acids, bases or hydrolytic enzyme (tannases) into
carbohydrate (majorly D-glucose) and gallic acid (GA). Although
Gallic acid at 200 μM (Fig. 1c) induces apoptosis of lung cancer
A549 cells,5 the Gallo-tannins are more potent cancer cell-killing
compounds and have been shown to have a variety of biological
activities such as anti-inflammatory, anti-diabetic, anti-cancer,
and anti-viral activities.6–9 1, 2, 3, 4, 6-penta-O-galloyl- β -D-
glucose (β-PGG, Fig. 1), is a core compound in the biosynthetic
pathway of Gallo-tannins. PGG is one of the most potent antioxi-
dant in tannins when tested in several model systems.10 PGG’s
anti-tumor effects both in vitro or in vivo have been reported such
as in prostate cancer,11,12 lung cancer,13 sarcoma,14 breast cancer,
leukemia, melanoma, and liver cancer. PGG also inhibits the
growth of angiogenesis-dependent Lewis lung cancer (LLC) allograft.13
However, the structure activity relationships (SAR), especially the
contributions of specific OH-groups in PGG, have not been system-
atically studied.
*
Corresponding author. School of Medicine and Pharmacy, Ocean University of
China, 5 Yushan Road, Qingdao 266003, China. Tel.: +86 532 82031615; fax: +86 532
82031615.
E-mail address: lijuanzhang@ouc.edu.cn (L. Zhang).
** Corresponding author. School of Medicine and Pharmacy, Ocean University of
China, 5 Yushan Road, Qingdao 266003, China. Tel.: +86 532 82032712; fax: +86 532
82033054.
In addition to hydroxybenzoic acid (HBAs) polyphenols,
hydroxycinnamic acid (HCAs) polyphenols are also abundantly
E-mail address: jiangtao@ouc.edu.cn (T. Jiang).
Co-first authors.
1
http://dx.doi.org/10.1016/j.carres.2016.04.021
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
73
Fig. 1. Common structure of PGG and natural phenolic acids.
present in nature15–17 and serve as an important part of human and
2. Results and discussions
animal nutrition. These polyphenols also have anti-allergic, anti-
microbial, immunomodulatory, antitumor, and cardiovascular disease
modification properties.1 Chlorogenic acid (b), ferulic acid (d), and
caffeic acid (e) (Fig. 1) are the most studied analogs in the HCA poly-
phenol family.
2.1. Chemistry
The PGG and its analogous were synthesized by using conven-
tional synthetic method of Steglich esterification.9 The same methods
were also worked well for the synthesis of sterically hindered and
cinnamic acid derivative of PGG analogs.
To our knowledge, the anti-cancer activities of HCA-glucose
analogs have not been studied. In the present study, we used
caffeic acid (e), salicylic acid (g), vanillic acid (f) and
other aromatic acids to synthesize the analogs of PCG
(pentacaffeoylglucose), PSG (pentasalicylloylglucose), and PVG
(pentavanilloylglucose), respectively, in addition to the OH-
protected penta-phenolic acid glucose esters. We found that
p-hydroxy group on aromatic rings was the major contributor to
the observed cytotoxicities of the PGG analogs. Moreover, cinna-
mic acid analogs were less active than the benzoic acid analogs
evidenced by higher IC50 values. Furthermore, when the double
bond in the cinnamic acid analogs was reduced by H2/Pd, the 2-C
side chain-containing analog had greatly increased cytotoxicities
in all four-cell lines tested.
The methoxy analogous (1–6 and 17–21) were synthesized by
coupling of D-glucose and respective methoxy phenylic acids via
single step reaction with yields of 35%–65%, (Scheme 1). The alpha/
beta anomeric isomers for some compounds (Fig. 2) were separated
by column chromatography. Common and odorless solvents (DCM/
hexane/petroleum ether/EtoAc) in different ratios were used for
column chromatography rather than bed ordered solvent.18
The critical step was to remove the protecting methoxy groups
in order to obtain the hydroxylated PGG analogs. We were unable
to get targeted compounds by following previously reported Lewis
acid (BBr3, AlCl3 and AlCl3/EtSH) methods,19–21 and many side prod-
ucts were observed on TLC. The expected compounds could not be
Scheme 1. Synthesis of compound 19 and route for compounds 1–6 and 17–21.

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Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
Fig. 2. Structures of methoxylated analogs of PGG.
obtained even by using reverse phase chromatography (data not
shown). To overcome the de-protection problem, benzyl group was
used as protecting group that could be easily removed by H2/Pd/C
method.22,23
The targeted compound 16 was synthesized by using steglich es-
terification method. Firstly, the suspension of benzyl protected
vanillic acid and D-glucose in the presence of DCC and DMAP in DCM
was refluxed to obtained the 11, which gave compound 16
(pentavanilloylglucose, PVG) after debenzylation over H2/Pd/C and
crystallization (Scheme 2).
Compounds 7–11 and 22–23 (Fig. 3) were synthesized by ester
coupling of D-glucopyranose with its corresponding benzyl-
protected benzoic and cinnamic acid, respectively, by using Scheme 2
method. Compounds 12–16 (Scheme 2) were synthesized by the
debenzylation of compounds 7–11 over H2/Pd/C.
The debenzylation of compounds 22 and 23 with H2/Pd/C method
gave rise to compounds 26 and 27, respectively (Scheme 3), because
H2 hydrogenated the olifenic bond of cinnamic acid to single bond.
It was difficult to synthesize compounds 24 and 25. Using re-
ported debenzylation methods of AlCl3/EtSH (hard/soft acid/base)
Scheme 2. Synthetic rout for compound 16 and 12–15.

Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
75
compounds 22 and 23 with TiCl4 in dry DCM for 10 to 15 minutes
at −15 °C converted the benzyl group to hydroxyl group and up to
10 hydroxyl groups were de-protected in a single step without af-
fecting the double bonds. The products were easily recovered by
solvent extraction after quenching with dilute acidic solution
(Scheme 3). After evaporating the solvent, the residues were crys-
tallized with acetone and water. Compounds 24 and 25 were
obtained in 70–80% yields by using above methods.
2.2. Biological assay
2.2.1. Tumor cell inhibition assay
All synthesized compounds were screened for cytotoxicities against
four cancer cell lines, which include two human lung cancer (A549
and H1299) and two human colon cancer (HCT116 and HT29) cells.
Primary screening of compounds 1–6, 8–11, 17–23 with methylated
and protected hydroxyl groups was performed at concentration of
100 μM. At this concentration, compounds 1–6, 8–11, 17–23 showed
almost no cytotoxicities in all tested cancer cell lines. In contrast, at
the same concentration the hydroxylated compounds 12–16 and 24–
27 showed considerable cytotoxicities. These results indicated that
hydroxyl groups in PGG analogs were critical for their cytotoxicities.
Subsequently, their IC50 in four cancer cell lines were further ex-
plored by using Doxorubicin as a positive control. Table 1 summarizes
the IC50 values of the selected compounds. Compound 12 (1,2,3,4,
Fig. 3. Structure of benzyloxylated cinnamic acid analogs of PGG.
or AlCl3 in dry dichloromethane19–21 only generated unpredictable
products, but not the desired compounds 24 and 25. We then tried
the condition of TiCl4 in dry DCM,24,25 which can debenzylate the
saturated ethers in the presence of olefinic bond. Treating
Scheme 3. Synthesis of compounds 24–27.
Table 1
Antitumor and antioxidant activities of hydroxylated compounds 12–16 and 24–27
Compounds
IC50(μM)
Antioxidant
activity
IC50 (μM)
Reducing
activity
IC50 (μM)
HCT116
HT29
A549
H1299
Doxorubicin
12
13
14
0.20 0.09
1.61 0.36
7.46 1.94
10.27 1.77
>100
0.35 0.07
4.46 1.16
19.96 4.15
13.37 1.31
>100
0.32 0.06
3.02 0.50
7.89 1.63
15.25 4.02
>100
6.70 0.59
3.84 0.58
20.56 4.29
12.89 2.56
>100
—
—
20.5 0.19
42.5 0.43
43.4 0.09
>500
45.6 0.15
59.2 0.87
78.4 0.87
>500
15
16
24
25
26
2.02 0.28
>100
15.18 2.26
44.71 3.69
6.03 0.55
—
1.76 0.23
>100
12.78 1.16
29.37 4.19
4.55 0.88
—
4.16 1.01
>100
16.56 3.85
78.74 4.95
7.44 0.48
—
3.65 0.10
>100
20.53 1.62
49.89 1.01
6.31 1.45
—
>500
>500
67.8 0.23
>500
36.2 0.59
98.5 0.56
30.4 0.24
59.6 0.29
59.2 0.08
32.9 0.56
65.9 0.39
12.6 0.36
67.2 0.56
15.1 0.14
29.5 0.25
44.2 0.09
27
Gallic acid
Caffeic acid
BHA
—
—
—
—
—
—
—
—

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Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
Fig. 4. Structures of PGG analogs with highest cytotoxicities against cancer cells.
6-pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-glucopyranose (PGG)
was more active than other compounds against HCT116 and A549
cells with IC50 1.61 ꢀM and 3.02 ꢀM, respectively. Interestingly, the
compound 16 (1,2,3,4,6-pentakis[-O-(4-hydroxy-3-methoxybenzoyl)]-
α,β-D-glucopyranose, PVG) was most effective at killing HT29 and
H1299 cells with IC50 1.76 ꢀM and 3.65 ꢀM, respectively. Com-
pounds 12, 16, and 27 (Fig. 4) were more effective than positive control
doxorubicine (IC50 = 6.70 ꢀM) in killing H1299 cells and have IC50
3.84 ꢀM, 3.65 ꢀM and 6.31 ꢀM, respectively, where as in other three
cancer cell lines doxorubicine was slightly more active than these
compounds.
The cytotoxicity studies indicated that the substitutions on ar-
omatic ring and the double bond-containing side chain had dominant
effect on cytotoxicities of the analogs. The co-planner orientation
of OH groups on the ring allowed the formation of highly stabiliz-
ing intramolecular (O)H. . .O interactions, which might play a role
in the bioactivity via localized stability effect.26 Hence, the number
of OH groups and their orientation on aromatic ring had draw-
string effect on cytotoxicity.
dihydroxy > p-hydroxymethoxy > p-hydroxy.27 However, in our cy-
totoxicity studies in four cancer cell lines, compound 16 with
p-hydroxymethoxy substitution appeared to be more toxic than
dihydroxy/p-hydroxy substituted compounds 13/14, which indi-
cated that antioxidant effects of the PGG analogs might not be
equivalent to their cytotoxicities and different molecular targets in
cells might be involved for these activities. Therefore, compound 16
might not be a good candidate for been developed as an antioxidant,
but might be an interesting PGG-based cytotoxic compound.
Compound 15 (pentasalicylloylglucose, PSG) had very low cy-
totoxicities. The reason might be the intramolecular hydrogen bond
between OH and CO of carbonyl group (Fig. 5), which was consis-
tent with previous reports28,29 in that o-hydroxybenzoic acid has
H-bond both in water and in gas phase.
Cinnamic acid analogs (24, 25) had relative low cytotoxicities
except for compound 27 (Fig. 4) where the double bond of the cin-
namic acid analogs was reduced to the 2-C side chain by H2/Pd/C,
Increasing OH substitution enhanced the cytotoxicities in the
benzoic acid analogs in general. The cytotoxicities of compound 13
(PPG) against HCT116 and A549 cells (IC50 of 7.46 ꢀM and 7.89 ꢀM)
were higher than that of compound 14 (penta-(4-hydroxybenzoyl)-
glucose, PHG) (IC50 of 10.27 ꢀM and 15.25 ꢀM). These results resembled
the antioxidant studies conducted by Fausta et al.27 in that increas-
ing hydroxy/methoxy ratio in the aromatic ring with at least one OH
at p-position increases the antioxidant activities of PGG analogs.
However, the compound 14 (PHG) appeared to be more toxic than
13 (PPG) in HT29 and H1299 cells, which indicated that the cyto-
toxicity and antioxidant activity might not affect the same set of target
molecules. Therefore, different expression of protein targets in each
cell lines might explain the cell-type specific effects of the analogs.
Furthermore, Relation between antioxidant activity and substitu-
tions on aromatic ring has been reported as p-hydroxydimethoxy >
Fig. 5. H-bond in salicylic acid and possible H-bond in compound 15.

Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
77
which made the compound 27 almost as toxic as compounds 12 and
13 in HT29 and HCT116/A549 cells with IC50 of 4.55 ꢀM, 6.03 ꢀM
and 7.44 ꢀM, respectively.
[K3Fe(CN)6]. The results are given in Table 1. Almost the similar ac-
tivity results are obtained as for DPPH scavenging activity.
3. Conclusion
2.2.2. Anti-oxidant and reducing activity
Synthesized polyphenols were also screened for their anti-
oxidant potentials using different assays including 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activity as well as iron
reduction capability.
We are reporting the synthesis of the benzoic and cinnamic acid
analogs of pentagalloylglucose (PGG) with different substitutions
and numbers of hydroxyl groups on aromatic ring. Anti-tumor anal-
ysis of synthesized compounds was explored against four cell lines,
which include the two human lung cancer (A549 and H1299) and
two human colon cancer (HCT116 and HT29). A competent new
compound 16 (PVG) showed highest activity with IC50 values of
1.76 ꢀM and 3.65 ꢀM, respectively, against HT29 and H1299 cancer
cell lines representing an interesting drug candidate for further
studies. In addition, most of the newly synthesized compounds
showed potent cancer cell-killing properties. Compounds also possess
good anti-oxidant and reducing activities. Compound 12 and 26 show
the highest antioxidant and reducing activities.
2.2.2.1. DPPH radical scavenging activity. DPPH radical scavenging
assay is most widely used method to evaluate the anti-oxidant po-
tentials in relatively short time as compare with other methods.
DPPH is a stable free radical, it accepts an electron or hydrogen radical
to become a stable diamagnetic molecule.30
Reduction capability of DPPH is measured by fall in its absor-
bance at 517 nm, which is induced by antioxidants. The absorbance
of DPPH is decreased due to a reaction with antioxidant mol-
ecules which scavenge the radical by hydrogen donation. Completion
of scavenging reaction is visualized by discoloration from purple
to yellow.31
4. Experimental
Many in vitro studies have defined the antioxidant potential of
the polyphenols as direct radical scavengers.32 The antioxidant ac-
tivity of polyphenolic acids is reasonably related to their structure,
namely, the substitution (hydroxyl or/and methoxy) on the aro-
matic ring and their side chain. In the present work, we try to focus
on the effect of different acids analogs and sugar on the antioxi-
dant activity.
Compounds with hydroxyl functional groups were selected for
antioxidant and reducing activity due to their hydrogen donation
capability. Antioxidant and reducing activities of compounds 12–
16 and 24–27 are given in Table 1. Compounds 12 (PGG) and 26 show
the highest potency for DPPH scavenging activity with IC50 20.5 μM
and 12.6 μM, respectively, both compounds are more active then
positive control BHA which has IC50 of 44.2 μM. Unexpectedly the
compound 16 with p-hydroxy, m-methoxy substitution found to be
inactive against DPPH scavenging, though in free state, the
m-methoxy substitution considered to have positive effect on the
stability of p-hydroxy free radical.33 This activity of compound 16
is evident in our statement that the synthesized analogs are not
equivalent in their antioxidant and cytotoxicity activities and dif-
ferent molecular targets are involved for these activities. Compound
13 show more antioxidant activity than compound 14, which is ev-
idence that the cytotoxicity and antioxidant activity not affect the
same set of target molecule.
4.1. General
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) pur-
chased from MeiGao Ltd. (Qingdao, China). TLC (thin layer
chromatography) 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) and
MestReNova 500 spectrometer with tetramethylsilane (Me4Si) as the
internal standard, and chemical shifts were recorded as ä values.
Mass spectra were recorded on a Q-TOF Global mass spectrometer.
4.2. Chemistry
The synthesis of methoxylated analogs of PGG is given in Sup-
plementary material.
4.2.1. Synthesis of benzyl protected carboxylic acids
The benzyl protected acids were synthesized from their respec-
tive hydroxy acids by the reported methods.9,34,35
In addition to hydroxyl substitution, acid side chain also has the
considerable effect on antioxidant activity. In free state, cinnamic
acid are more efficient then their benzoic acid counterparts due to
delocalized stabilization of free radical. In our work we found the
cinnamic acid analogs less active then their benzoic acid analogs
Table 1. Esterification of acids with glucose reduces their antioxi-
dant potency, through compound 25 was found to be
(IC50 = 65.9 0.39) less potent then free caffeic acid (IC50 = 29.5 0.25).
Saturation of side chain in cinnamic acid analogs increases activi-
ty up to many folds. Compound 26, with saturated 2-C side chain
analog of caffeic acid show the highest activity among all com-
pounds with (IC50 = 12.6 0.36), the results can be determined by
the conformational arrangement of sp3 carbons of side chains can
affect the drug and target interactions. Tetrahedral carbons in com-
pounds 26 and 27 might have the out of plane arrangement of
aromatic rings, which leads to better interaction between com-
pound and target.
4.2.1.1. General procedure. A suspension of D-glucose (0.03 g,
0.16 mmol), 4-(benzyloxy)-3-methoxybenzoic acid (0.34 g,
1.30 mmol), dicyclohexylcarbodiimide (DCC) (0.28 g, 1.32 mmol) and
N,N-(di-methylamino) pyridine (DMAP) (0.16 g, 1.36 mmol) in freshly
distilled dichloromethane (DCM) was refluxed for 16–18 hrs (moni-
tored by TLC). After cooling reaction mixture at room temperature,
urea by-product was removed by gravity filtration and the filtrate
was evaporated. Compound 11 was purified by column chroma-
tography eluting with a mixture of dichloromethane, hexane, and
ethyl acetate. The product was collected, evaporated and precipi-
tated using toluene and cyclohexane.
4.2.1.2. 1,2,3,4,6-Pentakis[-O-(3,4,5-tribenzyloxybenzoyl)]-α,β-D-
glucopyranoside (7). White solid, mp 162–164 °C; Rf (94% toluene/
6% ethyl acetate) 0.55, IR (KBr) 1724, 1589, 1499, literature9 Rf (94%
toluene/6% ethyl acetate) 0.54 and 0.55 for separate anomers.
4.2.1.3. 1,2,3,4,6-Pentakis[-O-(3,4-dibenzyloxybenzoyl)]-α,β-D-
glucopyranoside (8). Dirty white (50%), Rf (DCM:Hex:EtoAc, 8:6:1)
0.36; IR (KBr) 1727, 1598, 1520, 1H NMR (600 MHz CDCl3) δ 7.78
(dd J = 1.65 & 8.25 Hz, 0.5H, ArH α-isomer), 7.70 (d J = 2.2 Hz, 0.5H,
2.2.2.2. Iron reduction capability. Polyphenols are also good reduc-
ing agents. The synthesized compounds were also checked for their
reducing activity against iron using potassium ferricyanide

78
Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
ArH α-isomer), 7.64–7.59 (m 4H, ArH α/β-isomer), 7.55 (dd J = 2.2
& 8.80 Hz, 0.5H, ArH α-isomer), 7.52–7.27 (m 85H, which include,
8H, ArH α/β-isomer, 25H, BnAr, α-isomer, 50H, BnAr, β-isomer), 7.18–
7.15 (m 2H, ArH α/β-isomer), 6.95 (d J = 8.80 Hz, 0.5H, ArH α-isomer),
6.87–6.84 (m 2H, ArH α/β-isomer), 6.82–6.80 (m 1.5H, ArH α/β-
isomer), 6.78 (br 2d J = 8.80 & 8.25 Hz, 1.5H, ArH α/β-isomer), 6.75
(d J = 3.85 Hz, 0.5H, α-isomer), 6.23 (t J = 9.9 Hz, 0.5H, α-isomer),
6.218 (d, J = 8.25 Hz, 1H, β-isomer), 5.95 (t, J = 9.91 Hz, 1H, β-isomer),
5.76–5.69 (m, 1.5H, α/β-isomer), 5.57 (dd J = 3.85 & 10.45 Hz, 0.5H,
α-isomer), 5.20–5.00 (m, 30H, Bn-CH2, α/β-isomer), 4.92 (dd J = 11.55
& 25.29 Hz, 1H, β-isomer) 4.66–4.60 (m 1.5H, α/β-isomer), 4.50–
4.47 (m 0.5H, α-isomer), 4.35–4.29 (m 2.5H, α/β-isomer).
149.4, 149.1, 149.0, 148.9, 148.8, 148.8, 144.6, 140.1, 140.0, 137.8,
136.3, 136.2, 136.1, 136.0, 128.9, 128.7, 128.6, 128.1, 128.0, 127.9,
127.2, 127.1, 127.0, 125.2, 124.2, 123.9, 123.8, 123.7, 123.6, 122.3,
122.2, 121.5, 121.4, 121.3, 121.0, 120.9, 112.8, 112.7, 112.6, 112.5,
112.4, 112.3, 112.3, 112.2, 112.2, 92.6, 92.1, 73.2, 72.6, 70.8, 70.6,
70.6, 70.3, 69.1, 68.7, 55.9, 55.9, 55.9, 55.8, 55.6.
4.2.1.7. 1,2,3,4,6-Pentakis[-O-(3,4-dibenzyloxycinnamoyl)]-α,β-D-
glucopyranoside (22). White solid (52%), mp 85–87 °C; Rf
(DCM:Hex:EtoAc, 8:6:1) 0.32; MASS (ESI+) m/z 1892 [M+], 1893
[M + H+]; 1H NMR (500 MHz CDCl3) δ 7.70 (d, J = 15.59 trans, 1H, ole-
finic H), 7.64 (d, J = 15.59 trans, 1H, olefinic H), 7.59–7.27 (m, 109H
(8H olefinic H, 100H BnArH, α/β-isomer), 7.10–6.81 (m, 30H ArH,
α/β-isomer), 6.60 (d, J = 3.50 Hz, 1H, H-1 α-isomer), 6.42 (d, J = 16.0
trans, 1H, olefinic H), 6.29–6.11 (m, 9H olefinic H), 6.04 (d, J = 8.25 Hz,
1H, H-1 β-isomer), 5.93 (t, J = 10.0 Hz, 1H), 5.65 (t, J = 10.0 Hz, 1H),
5.52–5.44 (m, 3H, sugarH α/β-isomer), 5.40 (dd J = 3.50 & 10.50 Hz,
1H), 5.21–5.07 (m, 41H BnCH2, α/β-isomer), 4.41–4.31 (m, 5H sugar
H α/β-isomer), 4.14–4.10 (m, 1H). 1313C NMR (126 MHz, CDCl3) δ
166.6, 166.4, 166.1, 165.9, 165.5, 165.0, 164.9, 151.5, 151.4, 151.3,
151.2, 151.0, 149.1, 148.9, 146.8, 146.4, 146.0, 145.3, 136.8, 136.7,
136.6, 128.5, 127.9, 127.4, 127.3, 127.2, 127.1, 123.6, 123.5, 123.3,
123.1, 115.3, 115.2, 114.6, 114.4, 114.2, 114.1, 113.6, 113.5, 113.4,
92.1, 89.3, 71.3, 71.2, 70.8, 70.3, 69.9, 69.8, 69.6, 68.6.
4.2.1.4. 1,2,3,4,6-Pentakis[-O-(4-benzyloxybenzoyl)]-α,β-D-
glucopyranoside (9). White solid (40%), mp 147–149 °C; Rf
1
(DCM:Hex:EtoAc, 8:6:1) 0.42; IR (KBr) 1738, 1610, 1515; H NMR
(600 MHz CDCl3) δ 8.12 (d, J = 8.80 Hz, 0.6H (2H), ArH α-isomer),
7.99–7.96 (m, 4.6H (6H), ArH α/β-isomer), 7.89 (d, J = 9.35 Hz, 0.57H
(2H), ArH α-isomer), 7.87–7.81 (m 8H (10H), ArH α/β-isomer), 7.43–
7.31 (m 32.9H (6.5H-α-isomer, 25H-β-isomer) BnAr), 7.08 (d
J = 8.80 Hz, 0.56H (2H), ArH α-isomer), 6.96–6.94 (m 4.8H (6H), ArH
α/β-isomer), 6.90–6.84 (m, 8.8H (12H), ArH α/β-isomer), 6.77 (d,
J = 3.30 Hz, 0.27H (1H), H-1 α-isomer), 6.24–6.20 (t, J = 11.0 & 8.25 Hz,
1.26H (2H include H-1 α-isomer), 5.95 (t, J = 9.90 Hz, 1H, β-isomer),
5.78–5.72 (m 2.34H (3H), sugar H α/β-isomer), 5.60 (dd J = 3.85 &
10.45 Hz, 0.27H (1H), α-isomer),5.17 (s, 0.57H (2H), BnCH2, α-isomer),
5.11–5.03 (m, 12.0H (18H), BnCH2, α/β-isomer), 4.61 (dd J = 2.75 &
12.10 Hz, 1H, β-isomer), 4.57–4.51 (m 0.56H (2H), α-isomer), 4.45
(dd, J = 4.95 & 12.6 Hz, 1H, β-isomer), 4.41 (m, 0.26H (1H), α-isomer),
4.34–4.31 (m, 1H, β-isomer). MASS (ESI+) m/z 1231.0, 1232.6 [M + H+].
4.2.1.8. 1,2,3,4,6-Pentakis[-O-(4-benzyloxycinnamoyl)]-α,β-D-
glucopyranoside (23). White solid (43.6%), mp 142–144 °C; Rf
(DCM:Hex:EtoAc, 8:6:1) 0.30; IR (KBr) 3033, 1720, 1601, 1509, 1249;
1H NMR (600 MHz CDCl3) δ 7.78 (d, J = 15.95 trans, 1H, olefinic H),
7.72 (d, J = 15.95 trans, 1H, olefinic H), 7.66–7.54 (m, 8H, olefinic H,
α/β-isomer), 7.46–7.31 (m, 50H, BnArH, α/β-isomer), 7.02–6.88 (m,
20H, ArH, α/β-isomer), 6.62 (d, J = 3.30 Hz, 1H, H-1, α-isomer), 6.49
(d, J = 15.95 trans, 1H, olefinic H), 6.35 (d, J = 15.95 trans, 2H, ole-
finic H), 6.27–6.19 (m, 7H, olefinic H, α/β-isomer), 6.06 (d, J = 8.25 Hz,
0.5H (1H) H-1, β-isomer), 5.95 (t, J = 9.90 Hz, 1H), 5.68 (t, J = 9.35,
9.90 Hz, 0.5H (1H) sugarH), 5.54–5.48 (m, 3H, α/β-isomer), 5.42 (dd,
J = 3.85 & 9.90 Hz, 1H), 5.12–5.05 (m, 20H BnCH2, α/β-isomer), 4.40–
4.35 (m, 5H), 4.16 (m, 0.5H (1H), β-isomer). 13C NMR (126 MHz,
CDCl3) δ 166.7, 166.6, 166.3, 166.1, 166.0, 165.9, 165.5, 165.0, 164.9,
160.9, 160.8, 160.7, 160.6, 160.4, 146.8, 146.5, 146.1, 145.9, 145.8,
145.7, 145.1, 145.0, 144.9, 136.4, 136.3, 130.2, 130.1, 129.9, 129.8,
128.9, 128.6, 128.5, 128.1, 128.0, 127.4, 127.3, 127.2, 127.1, 127.0,
126.9, 125.2, 115.2, 115.1, 115.0, 114.9, 114.3, 114.2, 114.1, 114.0,
113.9, 92.1, 89.3, 72.9, 72.3, 70.2, 70.1, 70.0, 69.9, 69.7, 69.6, 68.6,
68.5, 62.3, 62.2.
4.2.1.5. 1,2,3,4,6-Pentakis[-O-(2-benzyloxybenzoyl)]-α,β-D-
glucopyranoside (10). White solid (69.8%), mp 70–72 °C; Rf
(DCM:Hex:EtoAc, 8:12:1) 0.20;, IR (KBr) 2924, 1738, 1627, 1598; 1H
NMR (600 MHz CDCl3) δ 7.92–7.88 (m, 4H, ArH α/β-isomer), 7.76–
7.68 (m, 4H, ArH, α/β-isomer), 7.62 (dd, J = 1.65 & 7.70 Hz, 1H, ArH)
7.47–7.40 (m, 5H ArH α/β-isomer), 7.38–7.11 (m, 40H (15H-α-
isomer, 25H-β-isomer) BnAr), 6.98–6.89 (m, 6H, ArH α/β-isomer),
6.85–6.80 (m, 3H, ArH α/β-isomer), 6.77–6.68 (m, 9H (1H, H-1
α-isomer, 8H ArH)), 6.64 (t, J = 8.25 & 7.15 Hz, 1H), 6.36 (t, J = 9.90 Hz,
1H), 6.17 (d, J = 8.25 Hz, 1H, H-1, β-isomer), 5.97 (t, J = 9.90 & 9.35 Hz,
1H), 5.88 (t, J = 9.90 Hz, 1H), 5.83 (m, 1H,), 5.65 (dd J = 3.30 & 10.45 Hz,
1H,), 5.30–4.90 (m, 16H (20H α/β-isomer), CH2Bn), 4.48–4.43 (m,
3H, α/β-isomer), 4.36 (m, 2H), 4.03 (m, 1H). 13C NMR (126 MHz,
CDCl3) δ 165.4, 164.8, 164.7, 164.6, 164.5, 164.2, 164.1, 164.0, 162.8,
159.1, 158.6, 158.5, 158.3, 158.2, 158.1, 157.9, 137.0, 136.9, 136.8,
8, 136.7, 136.6, 136.5, 134.3, 134.1, 133.7, 133.6, 133.5, 133.4, 133.3,
132.7, 132.4, 132.2, 132.1, 132.0, 131.8, 131.7, 131.6, 128.6, 128.5–
128.4, 128.3, 127.8, 127.7, 127.6, 127.4, 127.3, 127.2, 127.1, 126.8–
126.7, 126.6, 126.4, 126.3, 120.5, 120.5, 120.4, 120.3, 119.7, 119.6,
119.5–119.4, 118.9, 118.2, 113.9, 113.8, 113.7, 113.6, 113.5, 113.4,
113.3, 92.2, 89.9, 73.1, 72.6, 70.5, 70.4, 70.3, 70.3, 70.2, 70.1, 70.0,
69.9, 69.9, 69.9, 68.7, 68.5, 62.5, 62.2.
4.2.2. Synthesis of hydroxylated analogs of PGG
4.2.2.1. General procedure. A suspension of (60 mg, 0.043 mmol) of
11 and (12.0 mg 0.091 mmol) of palladium (10 wt. % on activated
carbon) in dry THF was stirred at 40 °C under a hydrogen gas at-
mosphere for 20 h. The reaction mixture was cooled and filtered
through Celite filter-aid, and the filtrate was evaporated. The residue
was crystallized from acetone and/or water to obtained required
product 16.
4.2.1.6. 1,2,3,4,6-Pentakis[-O-(4-benzyloxy,3-methoxybenzoyl)]-α,β-D-
glucopyranoside (11). White solid (52.1%), mp 80–82 °C; Rf
4.2.2.2. 1,2,3,4,6-Pentakis[-O-(3,4,5-trihydroxybenzoyl)]-α,β-D-
glucopyranoside (12). White solid (60%), mp 134–137 °C; Rf
(MeOH:H2O, 9.5:0.5) 0.83; IR (KBr) 3367, 1706, 1616, 1542; MASS
(ESI−) m/z 939.2 [M-H+], HRMS (−) calcd for (C41 H31 O26) 939.1098,
found 939.1123; 1H NMR (600 MHz CD3COCD3) δ 8.12 (br s, 30H,
OH α/β-isomer), 7.15 (s 2H, ArH), 7.06 (s 2H, ArH), 7.04 (s 2H, ArH),
6.98 (s 2H, ArH), 6.95 (s 2H, ArH), 6.92 (s 2H, ArH), 6.88–6.87 (3s
6H, ArH), 6.83 (s 2H, ArH), 6.61 (d J = 3.3 Hz, 1H, H-1 α-isomer), 6.21
(d J = 8.0 Hz, 1H, H-1 β-isomer), 6.05 (t J = 8.25 Hz, 1H), 5.89 (t
J = 8.0 Hz, 1H), 5.67 (t J = 8.0 Hz, 1H), 5.55 (m, 1H), 5.36 (dd J = 3.0
& 9.0 Hz, 1H), 4.58 (m, 1H), 4.44–4.38 (m, 4H), 4.29–4.26 (m, 2H).9
+
(DCM:Hex:EtoAc, 8:3:1) 0.16; HRMS calcd for (C81 H123 O17
)
1381.8786, found 1381.8799; 1H NMR (500 MHz, CDCl3) δ 7.81 (dd,
J = 8.4, 1.8 Hz, 1H), 7.57–7.25 (m, 95H), 7.17–7.04 (m, 14H), 6.69 (d,
J = 3.6 Hz, 1H), 6.44 (d, J = 8.1 Hz, 1H), 6.17 (t, J = 9.5 Hz, 1H), 6.12
(t, J = 10.0 Hz, 1H), 5.79 (t, J = 10.0 Hz, 1H), 5.66 (dt, J = 17.9, 8.9 Hz,
3H), 5.55 (d, J = 7.9 Hz, 1H), 5.23 (s, 2H), 5.16–5.07 (m, 14H), 4.75–
4.70 (m, 2H), 4.55 (t, J = 11.6 Hz, 2H), 4.36–4.31 (m, 2H), 3.86 (s, 3H),
3.82 (s, 3H), 3.78 (s, 3H), 3.74–3.72 (m, 9H), 3.56 (s, 3H). 13C NMR
(151 MHz, CDCl3) δ 165.8, 165.7, 165.6, 165.3, 164.9, 164.8, 164.2,
164.0, 162.7, 162.2, 152.8, 152.7, 152.5, 152.4, 152.3, 152.2, 152.1,

Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
79
4.2.2.3. 1,2,3,4,6-Pentakis[-O-(3,4-dihydroxybenzoyl)]-α,β-D-
glucopyranoside (13). White solid (55%), mp 172–176 °C; Rf
(DCM:MeOH, 8:2) 0.65; IR (KBr) 3374, 1708, 1606, 1523; MASS (ESI−)
m/z 859.3 [M-H+], HRMS (−) calcd for (C41 H32 O21) 883.1328, found
1H), 2.76–2.62 (m, 20H, CH2, α/β-isomer), 2.54–2.38 (m, 16H (20H),
CH2, α/β-isomer). 13C NMR (126 MHz, CD3COCD3) δ 171.9, 171.8, 171.7,
171.5, 171.4, 171.3, 171.2, 170.7, 164.8, 144.9, 144.8, 143.4, 143.2,
143.1, 132.5, 132.4, 132.3, 132.2, 132.1, 132.0, 131.9, 119.6, 119.5,
119.4, 119.3, 115.5, 115.4, 115.3, 115.2, 115.1, 89.7, 88.6, 71.3, 69.7,
69.6, 69.5, 69.4, 68.8, 67.6, 67.0, 62.1, 61.5, 61.2, 35.7, 35.6, 35.5, 35.4,
35.3, 35.2, 35.1, 33.5, 31.9, 29.9, 29.8, 29.7, 29.6, 29.4, 26.1, 25.8, 25.5,
25.3, 25.2, 24.7, 24.6.
1
883.1329; H NMR (500 MHz CD3COCD3) δ 8.81–8.30 (m, 15H, OH
α/β-isomer), 7.63–7.27 (m 15H, ArH, α/β-isomer), 6.97 (d, J = 8.25 Hz,
0.5H, ArH α-isomer), 6.85–8.71 (m 7.5H, which include 7H, ArH α/β-
isomer & 0.5H, H-1 α-isomer), 6.31 (d, J = 8.50 Hz, 1H, H-1, β-isomer),
6.21 (t, J = 9.50 Hz, 0.6H, α-isomer), 6.01 (t, J = 10.0 Hz, 1H, β-isomer),
5.79 (t, J = 10.0 Hz, 0.6H, α-isomer), 5.69–5.61(m, 2H, α/β-isomer),
5.51 (dd J = 4.0 & 10.5 Hz, 0.6H, α-isomer), 4.68 (br d, J = 11.5 Hz,
0.6H, α-isomer), 4.56–4.51 (m, 2.6H, α/β-isomer), 4.42–4.38 (m, 1.6H,
α/β-isomer).9
4.2.2.8. 1,2,3,4,6-Pentakis-[-O-{3-(4-hydroxyphenyl)-propanoyl}]-α,β-D-
glucopyranoside (27). White solid, (62.1%), mp 140–143 °C; Rf
(DCM:MeOH, 18:2) 0.30; MASS (ESI−) m/z 919.3 [M-H+], HRMS (−)
calcd for (C51 H51 O16) 919.3172, found 919.3183; 1H NMR (500 MHz
CD3COCD3) δ 8.13–8.07 (m, 8H (10H), OH, α/β-isomer) 7.04–6.91 (m,
15H (20H), ArH, α/β-isomer), 6.71–6.66 (m, 15H (20H), ArH, α/β-
isomer), 6.19 (d, J = 3.85 Hz, 1H, H-1, α-isomer), 5.84 (d, J = 8.25 Hz,
0.5H (1H) H-1, β-isomer), 5.44 (t, J = 9.90 Hz, 1H), 5.39 (t, J = 9.36,
9.89 Hz, 0.5H (1H) sugarH), 5.13 (m, 2H), 5.04 (d of t, J = 9.90 & 9.37,
1H), 4.97 (dd, J = 3.85 & 10.45 Hz, 1H), 4.11 (dd, J = 4.95 & 12.10 Hz,
0.5H (1H) β-isomer), 4.06–4.00 (m, 2H), 3.98 (dd, J = 2.20 & 12.65 Hz,
0.5H (1H) β-isomer), 3.90–3.87 (m, 2H), 2.77–2.60 (m, 15H (20H),
CH2, α/β-isomer), 2.53–2.31 (m, 15H (20H), CH2, α/β-isomer). 13C
NMR (151 MHz, CD3COCD3) ä 172.6, 172.7, 172.4, 172.3, 172.1, 172.0,
171.7, 171.5, 171.4, 156.8, 156.7, 156.6, 132.3, 132.2, 132.1, 132.0,
131.9, 131.8, 130.2, 130.1, 130.0, 129.9, 116.2, 116.1, 116.0, 92.3, 89.5,
73.2, 73.0, 71.1, 70.6, 70.4, 70.3, 68.9, 68.5, 62.4, 62.1, 36.6, 36.5, 36.4,
36.336.2, 36.1, 34.4, 30.2, 30.0, 29.9, 29.8, 29.7, 29.5, 29.4.
4.2.2.4. 1,2,3,4,6-Pentakis[-O-(4-hydroxybenzoyl)]-α,β-D-glucopyranoside
(14). White solid (35%), mp 117–120 °C; Rf (DCM:MeOH, 9:1) 0.60;
IR (KBr) 3327, 1723, 1626, 1577; MASS (ESI−) m/z 779.1 [M-H+], HRMS
(−) calcd for (C41 H32 O16) 803.1583, found 803.1398; 1H NMR
(500 MHz, acetone) δ 6.77–6.74 (m, 1H), 6.60–6.40 (m, 7H), 6.14 (d,
J = 8.7 Hz, 14H), 5.67 (m, 2H), 5.57 (d, J = 8.6 Hz, 2H), 5.51 (d, J = 8.7 Hz,
15H), 5.47 (d, J = 7.6 Hz, 2H), 5.07 (d, J = 8.4 Hz, 1H), 4.99–4.94 (m,
1H), 4.77 (t, J = 9.0 Hz 2H), 4.57–4.52 (m, 1H), 4.47 (t, J = 10.0 Hz 1H),
4.41 (t, J = 8.5 Hz 1H), 4.32–4.28 (m, 1H), 4.13 (br s, 1H), 3.32 (s, 3H),
3.17–3.11 (m, 1H).
4.2.2.5. 1,2,3,4,6-Pentakis[-O-(2-hydroxybenzoyl)]-α,β-D-glucopyranoside
(15). Dirty white solid (65.0%), mp 85–90 °C; Rf (DCM:Hex:EtoAc,
4:3:2) 0.27; MASS (ESI−) m/z 779.1 [M-H+], 780.1, HRMS (−) calcd
for (C41 H31 O16) 779.1607, found 779.1618; 1H NMR (500 MHz,
acetone) δ 10.56–10.08 (m, 10H, hydroxyl OH), 8.02–7.85 (m, 8H),
7.74–7.65 (m, 1H), 7.58–7.43 (m, 9H), 7.05–6.79 (m, 18H), 6.73 (d,
J = 6.7 Hz 1H), 6.55 (t, J = 10.0 Hz 1H), 6.47 (t, J = 9.0 Hz 1H), 6.37 (t,
J = 9.5 Hz 1H), 6.24–6.04 (m, 3H), 5.95 (t, J = 9.5 Hz 1H), 5.85 (br s,
1H), 5.57 (dd, J = 3.5, 10.0 Hz 1H), 4.99 (m, 1H), 4.94 (m, 1H), 4.85
(m, 2H).
4.2.2.9. 1,2,3,4,6-Pentakis[-O-(3,4-dihydroxycinnamoyl)]-α,β-D-
glucopyranoside (24). To a solution of compound 22 (200 mg,
0.1 mmol) in dry DCM with constant stirring, TiCl4 (13.2 ml,
0.3 mmol) in dry DCM was added at −10 °C under nitrogen atmo-
sphere, reaction mixture was stirred for 15–20 minutes. After the
completion of the reaction, the mixture was quenched with 2 N HCl
and extracted with ethyl acetate (100 ml), dried, and concen-
trated. The product was obtained after crystallization in acetone and
water.
Yellow solid (80%), mp 140–143 °C; Rf (DCM:MeOH, 15:6) 0.51;
IR (KBr) 3406, 1707, 1601, 1513; MASS (ESI−) m/z 989.2 [M-H+], HRMS
(−) calcd for (C51 H41 O21) 989.2135, found 989.2147; 1H NMR
(600 MHz, CD3COCD3) δ 8.33 (s, 15H), 7.57–7.48 (m, 11H), 6.83–
6.76 (m, 16H), 6.59–6.51 (m, 5H), 6.29–6.21 (m, 6H), 6.18–6.14 (m,
5H), 5.89 (t, J = 9.6 Hz, 1H), 5.73 (t, J = 9.5 Hz, 1H), 5.50–5.39 (m, 3H),
5.32 (dd, J = 10.2, 3.6 Hz, 1H), 4.52–4.47 (m, 1H), 4.38–4.24 (m, 5H).
13C NMR (151 MHz, CD3COCD3) δ 166.1, 165.8, 165.7, 165.4, 165.3,
164.9, 164.8, 164.6, 148.5, 148.4, 148.2, 148.1, 148.0, 147.2, 146.7,
146.5, 146.2, 145.5, 145.4, 145.3, 128.9, 128.8, 128.7, 128.6, 128.4,
128.3, 127.2, 127.0, 126.7, 126.6, 126.5, 126.4, 126.3, 126.0, 125.9,
125.8, 125.7, 122.4, 122.3, 122.2, 122.1, 122.0, 121.8, 121.7, 115.6,
115.5, 114.6, 114.55, 114.4, 114.2, 113.7, 113.6, 113.5, 113.4, 113.3,
113.2, 113.1, 113.0, 89.0, 83.7, 79.0, 74.0, 73.9, 72.8, 72.3, 70.3, 69.9,
69.7, 62.0.
4.2.2.6. 1,2,3,4,6-Pentakis[-O-(4-hydroxy, 3-methoxy benzoyl)]-α,β-
D-glucopyranoside (16). White solid (77.5%), mp 116–118 °C; Rf
(DCM:MeOH, 9:1) 0.68; IR (KBr) 3421, 2923, 1719, 1598, 1514; MASS
(ESI−) m/z 929.24 [M-H+], HRMS (−) calcd for (C46 H41 O21) 929.2135,
1
found 929.2155; H NMR (500 MHz, d2o) δ 8.19 (d, J = 7.7 Hz, 1H),
8.05 (s, 1H), 7.90 (d, J = 10.0 Hz, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.63–
7.40 (m, 12H), 7.11 (d, J = 7.5 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.98–
6.83 (m, 7H), 6.78 (d, J = 8.3 Hz, 1H), 6.54 (d, J = 8.3 Hz, 1H, H-1
β-isomer), 6.43–6.39 (t, J = 9.5 Hz, 1H), 6.29–6.25 (t, J = 9.5 Hz, 1H),
6.06–5.94 (m, 3H), 5.79 (dd, J = 10.2, 4.3 Hz, 1H), 5.60 (dd, J = 17.7,
6.3 Hz, 1H), 4.61–4.50 (m, 3H), 4.40–4.37 (m, 1H), 4.19–4.16 (m, 1H),
4.03 (s, 3H), 3.97–3.85 (m, 15H), 3.81–3.72 (m, 6H), 3.66 (m, 3H).
13C NMR (151 MHz, CD3COCD3) δ 166.2, 166.1, 166.0, 165.9, 165.8,
165.7, 164.8, 153.1, 153.0, 152.8, 152.7, 152.6,152.5, 152.4, 152.3,
148.2, 148.1, 148.0, 125.4, 125.3, 125.2, 125.1, 125.0, 124.9, 124.8,
122.2, 121.6, 121.5, 121.4, 121.3, 120.8, 115.8, 115.7, 115.6, 115.5,
113.6, 113.5, 113.4, 100.7, 93.6, 73.9, 73.6, 72.1, 71.6, 70.2, 63.4, 56.3,
56.2, 56.2, 54.9.
4.2.2.10. 1,2,3,4,6-Pentakis[-O-(4-hydroxycinnamoyl)]-α,β-D-
glucopyranoside (25). To a solution of compound 23 (100 mg,
0.1 mmol) in dry DCM with constant stirring, TiCl4 (13.2 ml,
0.3 mmol) in dry DCM was added at −10 °C under nitrogen atmo-
sphere, reaction mixture was stirred for 15–20 minutes. After the
completion of the reaction, the mixture was quenched with 2N HCl
and extracted with ethyl acetate (100 ml), dried, and concen-
trated. The product was obtained after crystallization in acetone and
water.
Light orange solid, (89.3%), mp 132–135 °C; Rf (DCM:MeOH, 18:2)
0.40; IR (KBr) 3399, 1701, 1601, 1513, 1163;MASS (ESI−) m/z 909.24
[M-H+], HRMS (−) calcd for (C51 H41 O16) 909.2389, found 909.2388;
1H NMR (600 MHz CD3COCD3) δ 8.98 (br s, 8H (10H), OH, α/β-
4.2.2.7. 1,2,3,4,6-Pentakis-[-O-{3-(3,4-dihydroxyphenyl) propanoyl}]-
α,β-D-glucopyranoside (26). Off-white solid (75.0%), mp 170–
172 °C; (decompose color change to brown), Rf (DCM:MeOH, 8:3)
0.88; IR (KBr) 3406, 1734, 1606, 1521; HRMS (−) calcd for (C51 H51
O21) 999.2917, found 999.2933; 1H NMR (500 MHz CD3COCD3) δ 8.98
(br s, 17H (20H), OH, α/β-isomer), 6.72–6.63 (m, 16H ArH, α/β-
isomer), 6.57–6.44 (m, 9H ArH, α/β-isomer), 6.23 (d, J = 4.0 Hz, 1H,
H-1, α-isomer), 5.86 (d, J = 8.25 Hz, 0.5H (1H) H-1, β-isomer), 5.57
(t, J = 12.5 Hz, 1H), 5.48 (t, J = 10.0 Hz, 1H), 5.16 (t, J = 10.0 Hz, 1H),
5.06 (m, 2H), 4.80 (dd, J = 3.50 & 10.5 Hz, 1H), 4.23 (m, 1H), 4.17 (dd,
J = 5.0 & 12.0 Hz, 1H), 4.10–4.03 (m, 3H), 3.67 (dd, J = 4.5 & 8.50 Hz,

80
Q. Shaikh et al. / Carbohydrate Research 430 (2016) 72–81
isomer), 7.82 (d, J = 15.95 trans, 1H, olefinic H), 7.72 (d, J = 15.95 trans,
1H, olefinic H), 7.67–7.46 (m, 22H (include (7H olefinic H, & 15H
ArH), 6.95 (d, J = 8.80 Hz, 2H, ArH), 6.89–6.81 (m, 15H (18H), ArH,
α/β-isomer), 6.64–6.61 (2d, J = 15.95 Hz & 3.85 Hz, 2H, H-1, α-isomer
& olefinic H), 6.38–6.23 (m, 9H, olefinic H), 6.21 (d, J = 7.70 Hz, 1H,
H-1, β-isomer), 5.96 (t, J = 10.1 Hz, 1H, sugarH), 5.79 (t, J = 9.35 Hz,
1H, sugarH), 5.54 (t, J = 9.67 Hz, 1H, sugarH), 5.49–5.43 (m, 2H), 5.38
DPPH scavenging effect % = A c−A s A c×100,
(
)
where Ac is the absorbance of control (DMSO treated) and As the
absorbance of sample.
4.4.2. Iron reducing activity
The reducing power of compounds was determined according
to the method previously described by Oyaizu.37 Different concen-
trations of (10–1000 μM) in 1 ml of distilled water were mixed with
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricya-
nide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50 °C
for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added
to the mixture, which was then centrifuged at 3000 rpm for 10 min.
The upper layer of the solution (2.5 ml) was mixed with distilled
water (2.5 ml) and FeCl3 (0.5 ml. 0.1%) and the absorbance was mea-
sured at 700 nm. Increased absorbance of the reaction mixture
indicated increased reducing power. Ascorbic acid was used as the
standard. Phosphate buffer (pH 6.6) was used as blank solution. The
absorbance of the final reaction mixture of two parallel experi-
ments was taken and is expressed as mean standard deviation.
(dd, J = 9.90 Hz & 6.60 Hz, 1H), 4.54 (m, 1H), 4.41–4.34 (m, 5H). 13
C
NMR (151 MHz, CD3COCD3) δ 166.3, 166.2, 165.9, 165.7, 165.6, 165.5,
165.4, 164.9, 164.7, 160.3, 160.2, 160.1, 160.0, 159.9, 159.8, 146.9,
146.4, 146.2, 146.1, 146.0, 145.9, 145.3, 145.2, 130.7–130.3 (m) 130.2,
129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 126.2, 126.1,
126.0, 125.9, 125.9, 125.9, 125.86, 125.8, 116.0, 115.9, 115.8, 115.2,
114.1, 113.4, 113.2, 113.1, 89.1, 89.0, 72.7, 70.3, 70.0, 69.7, 68.7, 62.2.
4.3. Biological methods
4.3.1. Anti-tumor activity
4.3.1.1. Materials and instruments. 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 and
fetal bovine serum (FBS) were from Gibco; RPMI-1640 medium, pen-
icillin, and streptomycin were from Hyclone; trypsin was from
Cellgro; Resazurin was from Sigma-Aldrich; DMSO was from Solarbio.
Spectramax M5 plate reader was from Molecular Devices.
OLYMPUSCKX41 inverted microscope and cell counting chamber
were purchased from Shanghai Qiujing biochemical reagents In-
strument Co., Ltd. Cell culture dishes and 96-well standard plates
were from Corning; and the 0.22 μm millipore filter papers were
from Whatman.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by grants from the Key International
Cooperation Project of CMST (Grant No. 2013DFG32160) for T. Jiang,
National Natural Science Foundation of China (NSFC) (No. 91129706)
for L. Zhang and the NSFC–Shandong Joint Fund (U1406402).
4.3.1.2. Cell culture. HCT116 and HT29 cells were maintained in
McCoy’s 5A media supplemented with 5% FBS, 100 U/ml of peni-
cillin, and 0.1 mg/ml of streptomycin at 37 °C with 5% CO2 and 95%
air in the incubator. A549 and H1299 cells were maintained in RPMI-
1640 media supplemented with 5% FBS, 100 U/ml of penicillin, and
0.1 mg/ml of streptomycin at 37 °C with 5% CO2 and 95% air. Cells
were kept sub-confluent and media were changed every other day.
All cells used were between 3 and 30 passages. DMSO was used as
the vehicle to dissolve the compounds, and the final concentra-
tion of DMSO in all experiments was 0.1%.
Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2016.04.021.
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