Synthesis and biological activity of trans-tiliroside derivatives as potent anti-diabetic agents

Article abstract of DOI:10.3390/molecules15129174

A set of novel trans-tiliroside derivatives were synthesized. The structures of the derivatives were identified by their IR, 1H-NMR, and MS spectra analysis. Their anti-diabetic activities were evaluated on the insulin resistant (IR) HepG2 cell model. As a result, compounds 7a, 7c, 7h, and trans-tiliroside exhibited significant glucose consumption-enhancing effects in IR-HepG2 cells compared with the positive control (metformin). This research provides useful clues for further design and discovery of anti-diabetic agents.

Full text of DOI:10.3390/molecules15129174

Molecules 2010, 15, 9174-9183; doi:10.3390/molecules15129174
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Activity of trans-Tiliroside Derivatives
as Potent Anti-Diabetic Agents
Yujin Zhu 1,2, Yanjun Zhang 3, Yi Liu 1, Hongwan Chu 1 and Hongquan Duan 1,*
1
School of Pharmaceutical Sciences, Research Center of Basic Medical Sciences, Tianjin Medical
University, Tianjin 300070, China; E-Mails: wjyxyzyj@yahoo.com.cn (Y.J.Z.);
aliu_yi@yahoo.com.cn (Y.L.); chw40542135@yahoo.com.cn (H.W.C.)
2
Chinese People’s Armed Police Forces, Tianjin Provincial Corps Hospital, Tianjin 300162, China
3
Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;
E-Mail: zyjsunye@163.com (Y.J.Z.)
 Author to whom correspondence should be addressed; E-Mail: duanhq@tijmu.edu.cn;
Tel.:+86-022-23542018; Fax: +86-022-23542018.
Received: 29 October 2010; in revised form: 7 December 2010 / Accepted: 8 December 2010 /
Published: 10 December 2010
Abstract: A set of novel trans-tiliroside derivatives were synthesized. The structures of the
1
derivatives were identified by their IR, H-NMR, and MS spectra analysis. Their
anti-diabetic activities were evaluated on the insulin resistant (IR) HepG2 cell model. As a
result, compounds 7a, 7c, 7h, and trans-tiliroside exhibited significant glucose
consumption-enhancing effects in IR-HepG2 cells compared with the positive control
(metformin). This research provides useful clues for further design and discovery of
anti-diabetic agents.
Keywords: trans-tiliroside; derivative synthesis; Novozyme435; anti-diabetic activity
1. Introduction
The complex metabolic syndrome, diabetes mellitus, is a major worldwide human health concern
that is estimated to affect 300 million people by the year 2025 [1]. Most of the patients who have
diabetes have non-insulin dependent diabetes mellitus (NIDDM). Resistance to the biological actions

Molecules 2010, 15
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of insulin in the liver and peripheral tissues, together with pancreatic cell defects, is a major feature of
the pathophysiology of human NIDDM [2-4].
In a previous study of traditional Chinese herbal medicines, we were particularly interested in the
extraction and separation of anti-hyperglycemic constituents from Potentilla chinensis Ser. (Rosaceae)
which has been reported having anti-hyperglycemic activity in the clinic [5]. Further photochemical
and bioactive analysis studies afforded an anti-diabetic compound, kaempferol-3-O-β-D-(6-O-trans-
p-cinnamoyl)glucopyranoside
(trans-tiliroside,
Figure
1),
which
revealed
significant
anti-hyperglycemic effects when compared with phenethyldiguanide in alloxan mice [6]. As a part of
trans-tiliroside, kaempferol-3-O-β-D-glucopyranose (4) and related analogues revealed weak
anti-diabetes activity [7,8], so the cinnamoyl part of trans-tiliroside is presumed to be the the critical
factor for increasing the activity. This paper deals with the synthesis of a series of novel trans-tiliroside
derivatives by replacing the cinnamoyl ring, as well as their biological activities.
Figure 1. The structure of trans-tiliroside.
HO
HO
O
O
OH
O
O
OH
O
O
OH
OH
OH
2. Results and Discussion
2.1. Synthesis
In the current work, a set of novel trans-tiliroside derivatives were synthesized, among which
compounds 5, and 7c-7h have not been reported before. All relevant reactions are depicted in Scheme
1. 6-(Acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (1) was prepared by treating
anhydrous D-glucose with perchloric acid in acetic anhydride (yield 100%). Treatment of 1 with
bromine and red phosphorus produced 2, which was treated with kaempferol in DMSO in the presence
of excess anhydrous potassium carbonate at room temperature to yield compound 3. Compound 4 was
prepared by treating 3 with sodium methoxide in methanol. Shaking compound 4 with dibenzyl
malonate at 45 °C for 120 hours led to 5 in 30% yield. Catalytic hydrogenation of 5 on Pd/C in THF
solution afforded pure 6 in quantitative yield after catalyst filtration and solvent evaporation at room
temperature. Compound 6 was used as a key intermediate for the synthesis of derivatives 7a-7h, which
were obtained by the Knoevenagel-Doebner reaction of 6 with a series of aldehydes in the presence of
pyridine [9-15]. The derivatives were characterized by IR, 1H-NMR, and MS spectroscopy.

Molecules 2010, 15
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Scheme 1. Synthesis of compounds 7a-7h.
HO
AcO
AcO
HO
HO
O
O
OH
AcO
OAc
O
OH
O
O
OAc
c
O
O
OH
b
a
O
OAc
OH
+
O
OAc
OH
HO
Br
OAc
OH
OH
OAc
OAc
OH
OAc
OAc
OAc
3
1
2
HO
HO
O
OH
O
O
OH
O
O
O
e
HO
O
O
O
d
O
O
OH
+
O
O
O
O
OH
O
OH
OH
HO
OH
HO
OH
5
4
HO
HO
O
OH
O
OH
O
f
g
R
O
HO
O
+
O
O
O
OH
O
O
O
O
OH
R
H
O
OH
O
OH
HO
HO
OH
OH
6
7a-h
Cl
H3CO
HO
Cl
Cl
S
F3C
NC
HO
H3C
7g
7h
7a
7b
7c
7d
7e
7f
Reagents and conditions: (a) acetic anhydride, D-glucose, 70% perchloric acid, 2 h, 100%; (b)
acetic anhydride, red phosphorus and bromine, 2 h, 56%; (c) dimethyl sulfoxide and potassium
carbonate, 12h; (d) methanol and sodium methoxide, 2 h, 22%; (e) Novozyme 435, pyridine and
acetone, 45 C, 5 d, shaken, 30%; (f) tetrahydrofuran and hydrogen, room temperature, 3 d, 90%;
(g) pyridine and piperidine, 80%, 2 h.
2.2. Biological Activity
In this research, an insulin resistant (IR) HepG2 cell model [16-18] was used for appraising the
anti-hyperglycemic effectws of the synthesized trans-tiliroside derivatives. As a result, compounds 7a,
7c, 7h, and trans-tiliroside itself all exhibited significant glucose consumption-enhancing effects in
IR-HepG2 cells compared with a positive control (metformin; Table 1). From the in vitro EC50 values
we presume that meta and para-substitution in the benzene ring of cinnamoyl with electron
withdrawing groups such as cyano-group and chloride group in compounds 7c and 7h resulted in
enhanced anti-diabetic activity.
So far, many anti-diabetic flavonoids have been reported [19-22], such as myricetin with
insulinomimetic effects [23], quercetin with antidiabetic effects in STZ diabetic rats [24], and
kaempferol-3,7-O-(α)-dirhamnoside with hypoglycemic and antioxidant effects [7,8]. However, the
abovementioned flavonoids had weak activity as revealed by their high doses and comparison with
market drugs. trans-Tiliroside is a known natural compound, and its anti-oxidant [25], tyrosinase
inhibitory [26], anti-complement activity [27], acetylcholinesterase inhibition [28], and anti-obesity
effects [29] have been reported. For the first time, we synthesized a series of trans-tiliroside derivatives,
which revealed significant anti-diabetic activities compared with the market drug of Metformin. The

Molecules 2010, 15
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results suggest that trans-tiliroside derivatives can be considered promising candidates in the
development of new antidiabetic lead compound. Further biological evaluations are in progress.
Table 1. Effects on glucose consumption of tiliroside derivatives in IR HepG2 cells.
Compound
EC50 (µM)
0.109
>10
7a
7b
0.048
>10
7c
7d
>10
7e
>10
7f
7g
0.813
0.011
0.155
0.270
7h
trans-tiliroside
metformin
3. Experimental
3.1. General
All reactions were monitored by TLC on silica gel GF-254 plates purchased from Qingdao Haiyang
1
Corporation. H-NMR and 13C-NMR spectra were taken on a Bruker AV400 MHz. DMSO-d6 or
CD3OD were used as the solvent. Chemical shifts are reported in parts per million shift (δ value) from
Me4Si (δ 0 ppm for 1H). Melting points were measured with an X4 melting apparatus. IR spectra were
recorded on a Nicolet 380 spectrometer (KBr). HRMS was taken on a Varian 7.0T HRFTICR-MS.
3.2. Materials
2-(Acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl-triacetate (2). To a mixture of acetic
anhydride (100 mL) and D-glucose (0.5 g, 2.78 mmol), 70% perchloric acid (0.6 mL) was added
dropwise. The mixture was then stirred at 35-40 °C on a water bath until the solution became clear.
After that, anhydrous D-glucose (24.5 g, 0.136 mol) was added over a period of 0.5 h and the reaction
continued for 2 h. The mixture was then cooled to 20-25 °C. Red phosphorus (7.5 g) and bromine
(14.5 mL) was added to the reaction mixture. After 0.5 h, H2O (9 mL) was added and the reaction
continued for 2 h. When the reaction ended, chloroform (75 mL) was added to the mixture and the
resulting reaction mixture was filtered. The filtrate was then poured into ice water, extracted with
chloroform (40 mL), washed with cold water and saturated sodium carbonate, dried with anhydrous
magnesium sulfate, filtered and evaporated. The resulting solid was finally re-crystallized with ether on
an ice bath and afforded product as a white solid (31 g, 56% yield). m.p. 88-89 °C.
5,7-Dihydroxy-2-(4-hydroxyphenyl)-3-(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl-
oxy)-4H-chromen-4-one (4). Kaempferol (650 mg, 2.27 mmol) and tetraacetyl-α-1-bromoglucose
(2.7 g, 6.55 mmol) were dissolved in dimethyl sulfoxide (50 mL) and then stirred overnight in the

Molecules 2010, 15
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presence of potassium carbonate. The resulting mixture was then adjusted to an acidic pH by adding a
few drops of formic acid. The precipitate formed in the acidic solution was separated by centrifugation,
washed and concentrated. Anhydrous MeOH (100 mL) was added to the precipitate, the solution was
adjusted to pH = 8 with Sodium methoxide and kept at room temperature for 2 h, followed by
neutralization and filtration. The filtrate was subjected to silica gel FC (AcOEt/MeOH/AcOH, 5:1:0.5).
The product was obtained as a yellow solid (240 mg, 22% yield). m.p. 163-164 °C. 1H-NMR (400 MHz,
DMSO), : 8.04 (2H, d, J = 8.8 Hz, 2’,6’-H), 6.88 (2H, d, J = 8.8 Hz, 3’,5’-H), 6.44 (1H, br s, 8-H), 6.21
(1H, br s, 6-H), 5.45 (1H, d, J = 7.4 Hz, Gal H-1), 4.01-4.21 (2H, m, 6’’-H), 3.50-3.20 (4H, m, 2’’, 3’’, 4’’,
5’’-H). ESI-MS: m/z [M+Na]+, calcd. For C21H20O11Na: 471.1; found: 471.1.
Benzyl(6-(5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4a,8a-dihydro-4H-chromen-3-yloxy)-3,4,5-tri-
hydroxytetrahydro-2H-pyran-2-yl)methyl malonate (5). To a solution of the flavonol glycoside 4
(125 mg, 0.28 mmol) in anhydrous Me2CO were added 10% pyridine (3 mL), dibenzyl malonate
(15 equiv) and Novozyme 435 (600 mg) were added, and the suspension was shaken at 45 °C and
250 rpm for 5 days. After filtration of the enzyme and evaporation of the solvent, the residue was
repeatedly washed with hexane five times and then purified by column chromatography using
AcOEt/MeOH (3:1) as eluent. The product was obtained as a yellow solid (50 mg, 30% yield). m.p.
214-215 C. 1H-NMR (400 MHz, CD3OD), : 7.95 (2H, d, J = 9.0 Hz, 2’,6’-H), 7.21-7.31 (5H, m, -ph),
6.80 (2H, d, J = 9.0 Hz, 3’,5’-H), 6.30 (1H, d, J = 1.8 Hz, 8-H), 6.13 (1H, d, J = 1.8 Hz, 6-H), 5.12 (1H,
d, J = 7.2 Hz, 1’’-H), 4.96-5.12 (2H, m, ph-CH2-O), 4.05-4.24 (2H, m, 6’’-H), 3.39 (3H, m, 2’’,3’’,4’’-H),
3.27 (1H, m, 5’’-H), 3.27 (2H, s, -CO-CH2-CO-). ESI-MS: m/z [M-H]- , calcd. For C31H27O14: 623.1;
found: 623.1.
3-((6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2H
-pyran-2-yl)methoxy)-3-oxopropanoic acid (6). A solution of the 3-flavone glycoside benzyl malonate
5 (200 mg, 0.32 mmol) in anhydrous THF (5 mL) was stirred with a catalytic amount of Pd/C (5%) for
3 days under a H2 atmosphere. The catalyst was filtered and solvent was removed under vacuum at
room temperature to afford the malonyl glycoside in quantitative yield as a yellow solid (150 mg, 90%
yield). m.p. 178-179 C. 1H-NMR (400 MHz, CD3OD), : 7.96 (2H, d, J = 8.0 Hz, 2’,6’-H), 6.87 (2H, d,
J = 8.0 Hz, 3’,5’-H), 6.43 (1H, br s, 8-H), 6.20 (1H, br s, 6-H), 5.34 (1H, d, J = 6.8 Hz, 1’’-H), 3.99-4.18
(2H, m, 6’’-H), 3.23 (3H, m, 2’’,3’’,4’’-H), 3.20 (1H, m, 5’’-H), 3.07 (2H, s, -CO-CH2-CO-). ESI-MS: m/z
[M-H]- , calcd. For C24H21O14: 533.1; found: 533.1.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2H
-pyran-2-yl)methyl3-(4-hydroxy-3-methoxyphenyl)acrylate (7a). Crude 6 (24.6 mg, 0.046 mmol) was
dissolved in anhydrous pyridine (3 mL) containing 4-hydroxy-3-methoxybenzaldehyde (60 µL,
3 equiv.) and piperidine (20 µL). After the addition of molecular sieves, the mixture was heated at
60 °C for 2.5 h. Usual workup and purification by FC (AcOEt/MeOH/AcOH, 10:1:0.5) gave 7a as a
yellow power (23 mg, 80% yield). m.p. 203-204 C. IR(KBr), max cm-1 3392, 1649, 1597, 1512.
1H-NMR (400 MHz , DMSO-d6), : 7.99 (2H, d, J = 8.8 Hz, 2’,6’-H), 7.39 (1H, d, J = 15.8 Hz, 3’’’-H),
7.22 (1H, s, 5’’’-H), 6.94 (1H, d, J = 7.8 Hz, 9’’’-H), 6.80 (1H, d, J = 7.8 Hz, 8’’’-H), 6.85 (2H, d,

Molecules 2010, 15
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J = 8.8 Hz, 3’,5’-H), 6.27 (1H, br s, 8-H), 6.06 (1H, br s, 6-H), 6.28 (1H, d, J = 15.8 Hz, 2’’’-H), 5.42
(1H, d, J = 7.2 Hz, 1’’-H), 4.21-4.31 (2H, m, 6’’-H), 3.47 (3H, m, 2’’, 3’’, 4’’-H), 3.34 (1H, m, 5’’-H). HR
ESI-MS: m/z [M+Na]+ , calcd. For C31H28O14Na: 647.1377; found: 647.1362. Compounds 7b-7h were
synthesized in the same manner.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-tri-hydroxytetrahydro-
H-pyran-2-yl)methyl cinnamate (7b). Evaporation of the solvent gave 7b as a yellow power (79%); m.p.
231-232 C. IR (KBr) max cm-1: 3374, 1654, 1606, 1503. 1H-NMR (400 MHz, CD3OD), : 8.00 (2H, d,
J = 8.8 Hz, 2’,6’-H), 7.38-7.48 (5H, m, 5’’’~9’’’-H), 7.42 (1H, d, J = 16.0 Hz, 3’’’-H), 6.82 (2H, d,
J = 8.8 Hz, 3’,5’-H), 6.29 (1H, d, J = 2.0 Hz, 8-H), 6.25 (1H, d, J = 16.0 Hz, 2’’’-H), 6.11 (1H, d,
J = 2.0 Hz, 6-H), 5.25 (1H, d, J = 7.2 Hz, 1’’-H), 4.21-4.30 (2H, m, 6’’-H), 3.45 (3H, m, 2’’,3’’,4’’-H),
3.35 (1H, m, 5’’-H). HR ESI-MS: m/z [M+Na]+ , calcd. For C30H26O12Na: 601.1322; found: 601.1319.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2
H-pyran-2-yl)methyl 3-(4-cyanophenyl)acrylate (7c). Evaporation of the solvent gave 7c as a yellow
1
power (75%); m.p. 217-218 C. IR (KBr), maxcm-1 3407, 1643, 1601, 1507. H-NMR (400 MHz,
DMSO-d6), : 7.96 (2H, d, J = 8.2 Hz, 2’,6’-H), 7.84 (2H, d, J = 8.0 Hz, 5’’’, 9’’’-H), 7.71 (2H, d,
J = 8.0 Hz, 6’’’, 8’’’-H), 7.44 (1H, d, J = 16.0 Hz, 3’’’-H), 6.84 (2H, d, J = 8.2 Hz, 3’,5’-H), 6.51 (1H, d,
J = 16.0 Hz, 2’’’-H), 6.23 (1H, br s, 8-H), 5.98 (1H, br s, 6-H), 5.44 (1H, d, J = 7.5 Hz, 1’’-H), 4.21-4.30
(2H, m, 6’’-H), 3.47 (3H, m, 2’’, 3’’, 4’’-H), 3.35 (1H, m, 5’’-H). HR ESI-MS: m/z [M+Na]+ , calcd. For
C31H25NO12Na: 626.1274; found: 626.1257.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2
H-pyran-2-yl)methyl3-(4-(trifl uoromethyl) phenyl) acrylate (7d). Evaporation of the solvent gave 7d
1
as a yellow power (81%); m.p. 189-190 C. IR (KBr), max cm-1 3412, 1647, 1611, 1509. H-NMR
(400 MHz, DMSO-d6), : 7.98 (2H, d, J = 8.8 Hz, 2’, 6’-H), 7.74 (4H, 6’’’, 8’’’, 5’’’, 9’’’-H), 7.49 (1H, d,
J = 16.0 Hz, 3’’’-H), 6.85 (2H, d, J = 8.8 Hz, 3’, 5’-H), 6.50 (1H, d, J = 16.0 Hz, 2’’’-H), 6.29 (1H, br s,
8-H), 6.04 (1H, br s, 6-H), 5.44 (1H, d, J = 7.5 Hz, 1’’-H), 4.13-4.31 (2H, m, 6’’-H), 3.34 (3H, m, 2’’, 3’’,
4’’-H), 3.25 (1H, m, 5’’-H). HR ESI-MS: m/z [M+Na]+ , calcd. For C31H25F3O12Na: 669.1196; found:
669.1189.
(E)-((3S,4R,6R)-6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxyte
trahydro-2H-pyran-2-yl)methyl 3-(thiophen-2-yl) acrylate (7e). Evaporation of the solvent gave 7e as a
1
yellow power (72%); m.p. 170-171 C. IR (KBr), max cm-1 3375, 1654, 1609, 1505. H-NMR
(400 MHz, DMSO-d6), : 7.97 (2H, d, J = 9.0 Hz, 2’, 6’-H), 7.71 (1H, d, J = 5.4 Hz, 8’’’-H), 7.58 (1H, d,
J = 15.6 Hz, 3’’’-H), 7.40 (1H, d, J = 3.6 Hz, 6’’’-H), 7.13 (1H, dd, J = 4.8 Hz, 7’’’-H), 6.84 (2H, d,
J = 9.0 Hz, 3’, 5’-H), 6.31 (1H, d, J = 1.8 Hz, 8-H), 6.09 (1H, d, J = 1.8 Hz, 6-H), 6.01 (1H, d,
J = 15.6 Hz, 2’’’-H), 5.40 (1H, d, J = 7.2 Hz, 1’’-H), 4.08-4.26 (2H, m, 6’’-H), 3.23 (3H, m, 2’’, 3’’, 4’’-H),
3.18 (1H, m, 5’’-H). HR ESI-MS: m/z [M+Na]+ , calcd. For C28H24SO12Na: 607.0886; found:607.0879.

Molecules 2010, 15
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(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2H
-pyran-2-yl)methyl3-(3-chlo ro -4-hydroxyphenyl) acrylate (7f). Evaporation of the solvent gave 7f as a
1
yellow power (76%); m.p. 200-201 C. IR (KBr), max cm-1 3415, 1650, 1601,1505. H-NMR
(400 MHz, DMSO-d6), : 7.98 (2H, d, J = 8.3 Hz, 2’,6’-H), 7.57 (1H, br s, 5’’’-H), 7.34 (1H, d,
J = 15.8 Hz, 3’’’-H), 7.28 (1H, br d, J = 8.4 Hz, 9’’’-H), 6.90 (1H, d, J = 8.4 Hz, 8’’’-H), 6.84 (2H, d,
J = 8.3 Hz, 3’,5’-H), 6.37 (1H, br s, 8-H), 6.16 (1H, d, J = 15.8 Hz, 2’’’-H), 6.09 (1H, br s, 6-H), 5.41
(1H, d, J = 8.7 Hz, 1’’-H), 4.21-4.30 (2H, m, 6’’-H), 3.45 (3H, m, 2’’,3’’,4’’-H), 3.35 (1H, m, 5’’-H). HR
ESI-MS: m/z [M+Na]+ , calcd. For C30H25Cl O13Na: 651.0881; found: 651.0880.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2H
-pyran-2-yl)methyl 3-p-tolylacrylate (7g). Evaporation of the solvent gave 7g as a yellow power(83%);
1
m.p. 219-220 C. IR (KBr), max cm-1 3419, 1650, 1605, 1509. H-NMR (400 MHz, CD3OD), : 7.99
(2H, d, J = 8.3 Hz, 2’, 6’-H), 7.44 (1H, d, J = 16.2 Hz, 3’’’-H), 7.34 (2H, d, J = 7.8 Hz, 5’’’, 9’’’-H), 7.21
(2H, d, J = 7.8 Hz, 6’’’, 8’’’-H), 6.82 (2H, d, J = 8.3 Hz, 3’, 5’-H), 6.26 (1H, br s, 8-H), 6.21 (1H, d,
J = 16.2 Hz, 2’’’-H), 6.12 (1H, br s, 6-H), 5.20 (1H, d, J = 7.2 Hz, 1’’-H), 4.21-4.30 (2H, m, 6’’-H), 3.50
(3H, m, 2’’, 3’’, 4’’-H), 3.36 (1H, m, 5’’-H), 2.38 (3H, s, -CH3). HR ESI-MS: m/z [M+Na]+ , calcd. For
C31H28O12Na: 615.1478; found: 615.1467.
(E)-(6-(5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yloxy)-3,4,5-trihydroxytetrahydro-2
H-pyran-2-yl)methyl 3-(3,4-dichlorophenyl)acrylate (7h). Evaporation of the solvent gave 7h as a
1
yellow power (70%); m.p. 183-184 C. IR (KBr), max cm-1 3387, 1646, 1606, 1509. H-NMR
(400 MHz, DMSO-d6), : 7.95 (2H, d, J = 9.0 Hz, 2’, 6’-H), 7.68 (1H, s, 5’’’-H), 7.66 (1H, d,
J = 15.6 Hz, 3’’’-H), 7.61 (1H, d, J = 8.4 Hz, 9’’’-H), 7.42 (1H, d, J = 8.4 Hz, 8’’’-H), 6.83 (2H, d,
J = 9.0 Hz, 3’, 5’-H), 6.36 (1H, br s, 8-H), 6.32 (1H, d, J = 15.6 Hz, 2’’’-H), 6.16 (1H, br s, 6-H), 5.41
(1H, d, J = 7.2 Hz, 1’’-H), 4.09-4.28 (2H, m, 6’’-H), 3.36 (3H, m, 2’’, 3’’, 4’’-H), 3.21 (1H, m, 5’’-H). HR
ESI-MS: m/z [M+Na]+ , calcd. For C30H24Cl2O12Na: 669.0543; found: 669.0542.
3.3. Biological Activity
Cell culture and insulin resistant HepG2 model: HepG2 cells were cultured in high-glucose DMEM
supplemented with 10% FBS. After confluence, cells were cultured in 96-well cluster plates in
high-glucose DMEM supplemented with 10% FBS for 24 h, and then the cells were treated with
10-7 mol/L insulin for 36 h in serum-free and phenol red-free high-glucose DMEM. After 36 h high
concentration insulin stimulated, the cells were washed with pH = 4 high-glucose DMEM four times
and PBS two times, then serum-free and phenol red-free high-glucose DMEM was added in with
compounds in different concentrations and incubated for 24 h. After 24 h, the glucose content in the
culture medium was measured by a glucose assay kit to study the effect on glucose consumption of
insulin resistance HepG2. The enhancement ratio of glucose consumption (GC) was calculated as
follows: GC % = (drug group of GC – model group of GC) / model group of GC × 100.

Molecules 2010, 15
9181
4. Conclusions
In this research, eight novel trans-tiliroside derivatives were synthesized and characterized by IR,
1H-NMR, and MS analyses. Preliminary bioassay data indicates that the target compounds 7a, 7c, 7h,
and trans-tiliroside showed anti-hyperglycemic activities compared to the reference compound
metformin. Further biological evaluations and the mechanism of the active compounds are in progress.
Acknowledgments
This work was supported by the National Nature Science Foundation of China (NO.30772635) and
supported by science foundation of ministry of education of china.
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Sample Availability: Samples of compounds 7a-7h are available from the authors.
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