Synthesis and anti-nociceptive and anti-inflammatory effects of gaultherin and its analogs

Article abstract of DOI:10.1080/10286020.2011.596830

The synthesis of gaultherin (1) and its analogs was carried out to provide 11 glycosides under phase-transfer catalytic conditions. The activities of all synthesized compounds were evaluated by nitric oxide production inhibitory assay in vitro. Methyl 2-O-(4-O-β- d-galactopyranosyl)- β- d-glucopyranosylbenzoate (5f) showed significantly anti-nociceptive and anti-inflammatory effects by the evaluation in vivo. Structure-activity relationships within these compounds were discussed.

Full text of DOI:10.1080/10286020.2011.596830

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Synthesis and anti-nociceptive and anti-
inflammatory effects of gaultherin and
its analogs
Chao Wang a , Tian-Tai Zhang a , Guan-Hua Du a & Dong-Ming Zhang
a
a State Key Laboratory of Bioactive Substances and Functions of
Natural Medicines , Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College , Beijing,
100050, China
Published online: 10 Aug 2011.
To cite this article: Chao Wang , Tian-Tai Zhang , Guan-Hua Du & Dong-Ming Zhang (2011) Synthesis
and anti-nociceptive and anti-inflammatory effects of gaultherin and its analogs, Journal of Asian
Natural Products Research, 13:9, 817-825, DOI: 10.1080/10286020.2011.596830
To link to this article: http://dx.doi.org/10.1080/10286020.2011.596830
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Journal of Asian Natural Products Research
Vol. 13, No. 9, September 2011, 817–825
Synthesis and anti-nociceptive and anti-inflammatory effects of
gaultherin and its analogs
Chao Wang, Tian-Tai Zhang, Guan-Hua Du and Dong-Ming Zhang*
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China
(Received 25 April 2011; final version received 9 June 2011)
The synthesis of gaultherin (1) and its analogs was carried out to provide 11 glycosides
under phase-transfer catalytic conditions. The activities of all synthesized compounds
were evaluated by nitric oxide production inhibitory assay in vitro. Methyl 2-O-(4-O-
b-D-galactopyranosyl)-b-D-glucopyranosylbenzoate (5f) showed significantly anti-
nociceptive and anti-inflammatory effects by the evaluation in vivo. Structure–activity
relationships within these compounds were discussed.
Keywords: gaultherin; analogs; phase-transfer catalytic method; anti-nociceptive;
anti-inflammatory
1. Introduction
with prolonged effects and reduced
adverse effects [1]. Pharmacokinetic
study showed that the release of salicylic
acid would be slowed down in patients
who had taken gaultherin [1]. Given the
significant biological superiority, it was
highly desirable to synthesize and biologi-
cally evaluate gaultherin and its analogs,
which could contribute to the structure–
activity relationships study and the new
drug discovery.
Gaultherin (1; Figure 1) is a glycoside of
methyl salicylate with a disaccharide,
which was obtained from the plant of
Gaultheria yunnanensis Rehd [1]. It is
well known that drugs containing salicylic
acid are key members of nonsteroidal anti-
inflammatory drugs, clinically used as
antipyretic, anti-inflammatory, antirheu-
matic, antithrombotic, and antitumor
agents [2]. However, various side effects
come out in some of the patients who had
taken these drugs, such as inciting the
gastrointestinal tract, preventing grume,
and injuring liver, kidney and central
nervous system. The most concerned
adverse effect is inciting the gastrointes-
tinal tract with common symptoms of
indigestion, stomach ulcers, bleeding, and
perforation [3]. Recently, it was found that
gaultherin from the natural product had the
activities of antipyretic, anti-inflamma-
tory, antirheumatic, and antithrombotic,
Previously, Robertson reported the
synthesis of monotropitoside (gaultherin)
with dry active silver oxide and ammonia
as catalyst, respectively [4]. Zang et al.
reported the synthesis and bioactivity of
salicylic acid saccharide carboxylate as
inducer in plants [5]. Here, we reported the
synthesis of gaultherin by phase-transfer
catalytic method catalyzed by tetrabutyl
ammonium bromide (TEAB) without the
limitation of anhydrous condition, which
was facile for the industrial production.
*Corresponding author. Email: zhangdm@imm.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.596830
http://www.informaworld.com

818
C. Wang et al.
(Scheme 1). The structures of 1 and 12
O
HO
were determined by the spectral data.
To investigate the influence of differ-
ent sugar moieties and to compare the
anti-inflammatory and analgesic effects of
the glycosides, we used a series of
monosaccharides and disaccharides such
as D-glucose, D-galactose, D-mannose,
D-xylose, L-rhamnose, maltose, and lac-
tose as glycosyl donors. So far, few anti-
inflammatory and analgesic activities of
methyl salicylate glycosides bearing these
sugar moieties have been reported
[5,12,13]. According to the same pro-
cedure described for 5a, compounds 5b–
5g were prepared (Scheme 2). We also
used salicylic acid as the receptor to afford
saccharide carboxylate 13a–13b, accord-
ing to the literature [5,13]. The configur-
ation of the glucosidic bond was
HO
O
O
OCH3
HO
HO
O
O
HO
HO
Figure 1. The structure of gaultherin (1).
To study the structure–activity relation-
ship of glycosides of salicylate, we
synthesized a series of analogs by
introducing different sugar moieties in 2-
OH of salicylate by phase-transfer cataly-
tic method, and their anti-nociceptive and
anti-inflammatory effects were evaluated
in vitro and in vivo.
2. Results and discussion
After analyzing the structure of
gaultherin, we designed the method and
prepared the target compound (1) suc-
cessfully in 11 steps from methyl
salicylate, D-glucose, and D-xylose.
First, 4a was afforded when methyl
salicylate reacted with acetobromoglu-
cose (3a) under promotion with phase-
transfer catalyst TEAB [6–9]. Conse-
quently, the removal of the acetyl of 4a
through ester exchange in NaOMe–
MeOH afforded glycoside 5a [10].
Treatment of 5a with protective groups
such as benzaldehyde and acetic anhy-
dride produced the key intermediate 8
[10,11]. The glucopyranosyl of 8 had two
hydroxyls (4-OH and 6-OH) with differ-
ent activations. It is well known that the
reaction activity of 6-OH was better than
4-OH, and thus we supposed to get 10
when 8 was reacted with equal trichlor-
oacetimidate (9) under promotion with
boron trifluoride ether solution. When the
reaction was brought into practice, the
target compound 10 was obtained as
supposed along with the by-product 11,
which was the glycoside of methyl
salicylate bearing trisaccharide [10].
Finally, removal of acetyl groups by
NaOMe–MeOH afforded 1 and 12
1
determined from the H NMR spectra of
the products. The stereochemistry of the
glycosides was assigned on the basis of
coupling constants (Table 1).
Gaultherin and all the analogs were
screened for their nitric oxide (NO)
production
inhibitory
effects
at
1 £ 1026 M in vitro. The inhibition ratio
of the compounds is listed in Table 2.
Gaultherin (1), 5f, 12, and 13b exhibited
remarkable NO production inhibitory
activities with an average inhibition ratio
of 40.2%. The results indicated that the
activities of glycosides with disaccharides
were better than those with monosacchar-
ides. Based on the evaluation in vitro and
the benefit of industrial production, we
prepared the glycoside with disaccharide
(5f) to evaluate the activity in vivo. The
anti-inflammatory effect of 5f was eval-
uated by croton oil-induced ear edema in
mice (Table 3). The anti-nociceptive effect
of 5f by acetic acid-induced writhing
response in mice was observed (Table 4).
The known drug aspirin was included in
the assay as a positive control. As a result,
the anti-inflammatory effect of 5f
(400 mg/kg) was similar to aspirin
(200 mg/kg), and the anti-nociceptive

Journal of Asian Natural Products Research
819
OR1
O
OCH3
a
OAc
O
OCH3
O
HO
R1O
R2O
O
R2O
O
AcO
AcO
2
AcO
4a R1 = Ac, R2 = Ac
5a R1 = H, R2 = H
Br
b
c
3a
6 R1 = PhCH, R2 = H
7 R1 = PhCH, R2 = Ac
8 R1 = H, R2 = Ac
d
e
O
AcO
AcO
O
CCl3
AcO
f
HN
9
O
AcO
AcO
O
AcO
O
O
OCH3
O
AcO
HO
AcO
AcO
O
OCH3
AcO
O
O
O
AcO
AcO
O
O
O
AcO
AcO
AcO
AcO
10
11
g
g
O
HO
HO
O
HO
HO
O
O
OCH3
HO
HO
O
O
OCH3
HO
O
O
O
O
HO
HO
O
HO
HO
O
HO
HO
HO
1
12
Scheme 1. The synthetic route of 4a–12. Reagents and conditions: (a) TEAB, NaOH, CH2Cl2,
408C, 4 h, 66%; (b) NaOMe, MeOH, r.t., 3 h, 45%; (c) ZnCl2, PhCHO, 12 h, r.t., 49%; (d) pyridine,
˚
Ac2O, 10 h, r.t., 75%; (e) 80% HOAc, 808C, 3 h, 79%; (f) BF3–Et2O, CH2Cl2, 4 A MS, N2, r.t., 20 h;
(g) NaOMe, MeOH, r.t., 3 h, 45%.
1
effect of 5f (800 mg/kg) was similar to
aspirin (200 mg/kg).
as KBr pellets. H and 13C NMR spectra
were run on INOVA-500 spectrometer with
solvent peaks as references. ESIMS were
obtained on an Agilent 1100 series
LC/MSD Trap SL mass spectrometer.
Column chromatography was carried out
on silica gel (100–200, 200–300 mesh,
Qingdao Marine Chemistry Company,
Qingdao, China). Silica gel GF254 (Qing-
dao Marine Chemistry Company) was used
to make silica gel plates.
3. Experimental
3.1 General experimental procedures
The optical rotations were determined on a
Perkin-Elmer digital polarimeter. UV spec-
tra were taken on a Shimadzu UV-300
spectrophotometer. IR spectra were
recorded on a Nicolet 5700 spectrometer

820
C. Wang et al.
O
OCH3
O
OCH3
R–Br
3a–3g
HO
R = P1-P7
RO
a
2
4a–4g R = P1-P7
5a–5g R = S1-S7
b
R–Br
COOR
COOH
3e–3f R = P5-P6
OH
OH
a
13a R = P5
13b R = P6
OH
OH
HO
OH
O
HO
O
HO
O
O
HO
S2 = HO
S1 =
S4 =
S3 =
HO
HO
HO
HO
OH
OH
OH
OH
HO
OH
OH
O
OH
O
HO
S5 =
S6 =
HO
O
O
HO
S7 =
HO
HO
O
O
OH
O
HO
HO
HO
HO
HO
HO
OAc
OAc
AcO
OAc
AcO
O
O
O
O
AcO
AcO
AcO
AcO
P1 =
P2 =AcO
P3 =
P4 =
AcO
AcO
AcO
OAc
AcO
OAc
AcO
AcO
OAc
AcO
AcO
O
O
AcO
AcO
O
O
P5 =
AcO
AcO
P6 =
P7 =
O
O
OAc
O
AcO
AcO
AcO
AcO
Scheme 2. The synthetic route of 5a–5 g and 13a–13b. Reagents and conditions: (a) TEAB,
NaOH, CH2Cl2, 408C, 4 h, 66%; (b) NaOMe, MeOH, r.t., 3 h, 45%.
3.2 General procedures for the target
compounds
glucopyranosylbenzoate (4a) was obtained
as a white amorphous powder from starting
material D-glucose and methyl salicylate,
yielding 66% for two steps. Compound 5a
was obtained as a white amorphous powder
from 4a after the removal of acetyl groups
3.2.1 Methyl 2-O-b-D-
glucopyranosylbenzoate (5a)
According to the reported method [6–9],
methyl 2-O-(2,3,4,6-tetra-O-acetyl)-b-D-
Table 1. Configuration of the glucosidic bond of compounds.
0
J(1 ,2 )(Hz)
0
0 0
J(1 ,2 ) (Hz)
Compounds
Configuration
Compounds
Configuration
1
7.6
7.5
6.5
8.0
1.5
–
b
b
b
b
a
a
5e
5f
5 g
13a
13b
7.0
8.0
7.5
6.5
8.0
b
b
b
b
b
12
5a
5b
5c
5d

Journal of Asian Natural Products Research
821
(MeOH) lmax: 230, 286 nm; IR (KBr)
max: 3469, 3453, 2944, 1685, 1605, 1491,
1454, 1432, 1316, 1238, 1086, 753 cm21
Table 2. Inhibitory effects of NO production
of compoundsa.
n
;
Groups
(x¯ ^ s (n ¼ 3))
Inhibition (%)
1H-NMR (DMSO-d6, 500 MHz) dH: 7.26
(1H, br.d, J ¼ 8.5 Hz, H-3), 7.50 (1H, m,
H-4), 7.07 (1H, t, J ¼ 8.0 Hz, H-5), 7.62
(1H, dd, J ¼ 8.0, 1.5 Hz, H-6), 3.79 (3H, s,
H-8), 4.84 (1H, d, J ¼ 8.0 Hz, H-10),
3.0 , 4.0 (H-20, 30, 40, 50, 60a, 60b); ESI-
MS m/z: 337 [M þ Na]þ.
Normal
Control
1
12
5a
5b
5c
5d
5e
5f
5 g
13a
13b
0.306 ^ 0.062
3.235 ^ 0.062
0.306 ^ 0.007
0.307 ^ 0.058
3.190 ^ 0.040
2.956 ^ 0.090
0.334 ^ 0.014
0.334 ^ 0.009
0.340 ^ 0.007
3.287 ^ 0.068
3.188 ^ 0.108
0.356 ^ 0.028
0.300 ^ 0.027
–
–
39.8
39.4
1.5
9.5
24.8
24.70
*
21.5
38.5
1.6
13.0
43.0
**
*
**
*
*
*
3.2.2.2 Methyl 2-O-a-D-mannopyrano-
sylbenzoate (5c). [a ]2D0 þ 52.6 (c ¼ 0.10,
MeOH); UV (MeOH) lmax: 212, 229,
287 nm; IR (KBr) nmax: 3405, 2949, 1718,
1601, 1489, 1455, 1435, 1304, 1255, 1087,
**
Notes: a Results are expressed as means ^ SD.
p , 0.05, p , 0.01, significantly different from
control by Student’s t-test.
*
**
1
757 cm21; H-NMR (CD3OD, 500 MHz)
dH: 7.37 (1H, d, J ¼ 8.5 Hz, H-3), 7.43
(1H, m, H-4), 7.03 (1H, t, J ¼ 8.5 Hz, H-
5), 7.68 (1H, dd, J ¼ 8.5, 1.5 Hz, H-6),
3.82 (3H, s, H-8), 5.48 (1H, d, J ¼ 1.5 Hz,
H-10), 3.0 , 4.0 (H-20, 30, 40, 50, 60a, 60b);
ESI-MS m/z: 337 [M þ Na]þ.
according to the reported method [10],
yielding 45%. m.p. 126.1–127.88C; [a]D20
228.7 (c ¼ 0.09, MeOH); UV (MeOH)
l
max: 234, 286 nm; IR (KBr) nmax: 3426,
2906, 1723, 1602, 1491, 1450, 1437, 1364,
1264, 1081, 747 cm21; 1H-NMR (DMSO-
d6, 500 MHz) dH: 7.26 (1H, d, J ¼ 8.5 Hz,
H-3), 7.51 (1H, m, H-4), 7.08 (1H, t,
J ¼ 8.5 Hz, H-5), 7.62 (1H, dd, J ¼ 8.5,
1.5 Hz, H-6), 3.80 (3H, s, H-8), 4.89 (1H, d,
J ¼ 6.5 Hz, H-10), 3.0 , 5.0 (H-20, 30, 40, 50,
60a, 60b); ESI-MS m/z: 337 [M þ Na]þ.
3.2.2.3 Methyl 2-O-a-L-rhamnopyrano-
sylbenzoate (5d). [a]2D0 278.9 (c ¼ 0.10,
MeOH);UV (MeOH)lmax: 233, 287 nm; IR
(KBr) nmax: 3360, 2940, 1713, 1601, 1490,
1455, 1435, 1304, 1254, 1087, 756 cm21
;
1H-NMR (DMSO-d6, 500 MHz) dH: 7.30
(1H, d, J ¼ 8.5 Hz, H-3), 7.52 (1H, m, H-4),
7.10 (1H, m, H-5), 7.77 (1H, dd, J ¼ 7.5,
1.5 Hz, H-6), 3.90 (3H, s, H-8), 5.51 (1H, s,
H-10), 1.24 (3H, d, J ¼ 6.5 Hz, H-60),
3.0 , 4.5 (H-20, 30, 40, 50); ESI-MS m/z:
298 [M þ Na]þ.
3.2.2 Compounds 5b–5 g were prepared
according to the same procedure
described for 5a
3.2.2.1 Methyl 2-O-b-D-galactopyrano-
sylbenzoate (5b). m.p. 171.3–172.98C;
[a ]2D0 222.0 (c ¼ 0.10, MeOH); UV
Table 3. Anti-inflammatory effect of 5f by croton oil-induced ear edema in micea.
Groups
Dose (mg/kg)
n
Swelling value (mg)
Inhibition (%)
–
45
***
Control
Aspirinb
5f
0
200
400
10
10
10
19.3 ^ 1.3
11.6 ^ 1.6
12.8 ^ 1.8
39
***
Notes: a Results are expressed as means ^ SD.
b Positive control substance.
p , 0.001, significantly different from control by Student’s t-test.
***

822
C. Wang et al.
Table 4. Anti-nociceptive effect of 5f by acetic acid-induced writhing response in micea.
Groups
Dose (mg/kg)
n
Writhing response (times)
Inhibition (%)
–
Control
5f
5f
0
200
400
800
200
10
10
10
10
10
29.04 ^ 3.05
16.72 ^ 4.45
15.73 ^ 3.56
9.83 ^ 2.60
7.67 ^ 2.12
45.84
49.36
69.62
77.07
*
**
***
***
5f
Aspirinb
Notes: a Results are expressed as means ^ SD.
b Positive control substance.
p , 0.05, p , 0.01,
* **
p , 0.001, significantly different from control by Student’s t-test.
***
3.2.2.4 Methyl 2-O-b-D-xylopyranosyl-
benzoate (5e). [a]2D0 287.6 (c ¼ 0.10,
MeOH); UV (MeOH) lmax: 209, 230,
286 nm; IR (KBr) nmax: 3393, 2894, 1713,
1601, 1490, 1452, 1435, 1262, 1088,
3.2.2.6 Methyl 2-O-(4-O-b-D-gluco-
pyranosyl)-b-D-glucopyranosylbenzoate
(5 g). m.p. 178.0–180.08C; [a ]2D0 þ 25.4
(c ¼ 0.10, MeOH); UV (MeOH) lmax
:
231, 287 nm; IR (KBr) nmax: 3491, 2929,
2911, 2868, 1677, 1603, 1491, 1460, 1438,
;
1322, 1248, 1068, 762 cm21 1H-NMR
1048, 761 cm21
;
1H-NMR (CD3OD,
500 MHz) dH: 7.24 (1H, d, J ¼ 8.0 Hz,
H-3), 7.52 (1H, dt, J ¼ 8.0, 1.5 Hz, H-4),
7.07 (1H, t, J ¼ 8.0 Hz, H-5), 7.73 (1H,
dd, J ¼ 8.0, 1.5 Hz, H-6), 3.83 (3H, s, H-
8), 4.90 (1H, d, J ¼ 7.0 Hz, H-10),
3.0 , 4.0 (H-20, 30, 40, 50a, 50b); ESI-MS
m/z: 307 [M þ Na]þ.
(DMSO-d6, 500 MHz) dH: 7.27 (1H, d,
J ¼ 8.0 Hz, H-3), 7.52 (1H, m, H-4), 7.10
(1H, d, J ¼ 8.0 Hz, H-5), 7.64 (1H, dd,
J ¼ 8.0, 1.5 Hz, H-6), 3.80 (3H, s, H-8),
4.98 (1H, d, J ¼ 7.5 Hz, H-10), 3.0 , 6.0
(H-20, 30, 40, 50, 60a, 60b, 100, 200, 300, 400,þ500,
600a, 600b); ESI-MS m/z: 499 [M þ Na] .
3.2.2.5 Methyl 2-O-(4-O-b-D-galacto-
pyranosyl)-b-D-glucopyranosylbenzoate
(5f). m.p. 177.1–178.68C; [a]2D0 271.2
(c ¼ 0.10, MeOH); UV (MeOH) lmax:
209, 230, 286 nm; IR (KBr) nmax: 3382,
3.2.2.7 1-(2,3,4-Tri-O-acetyl)-b-D-xylo-
pyranosyl-2-hydroxybenzoate
(13a).
m.p. 140.1–144.58C; [a ]2D0 281.7
(c ¼ 0.15, CHCl3); UV (CHCl3) lmax:
249, 313 nm; IR (KBr) nmax: 2948, 1754,
2921, 2882, 1717, 1602, 1491, 1458,
1319, 1250, 1077, 1049, 761 cm21 1H-
;
1687, 1614, 1483, 1369, 1245, 1221, 1063,
763 cm21
;
1H-NMR
(DMSO-d6,
NMR (DMSO-d6, 500 MHz) dH: 7.26
(1H, d, J ¼ 8.0 Hz, H-3), 7.52 (1H, m,
H-4), 7.09 (1H, d, J ¼ 7.5 Hz, H-5), 7.62
(1H, dd, J ¼ 7.5, 2.0 Hz, H-6), 3.79 (3H,
s, H-8), 5.00 (1H, d, J ¼ 8.0 Hz, H-10),
4.51 (1H, d, J ¼ 7.0 Hz, H-100), 3.3 , 5.0
(H-20, 30, 40, 50, 60a, 60b, 200, 300, 400, 500, 600a,
600b). 13C-NMR (DMSO-d6, 125 MHz) dC:
121.7 (C-1), 156.0 (C-2),116.4 (C-3),
133.3 (C-4), 121.4 (C-5), 130.3 (C-6),
166.4 (C-7), 103.8 (C-10), 100.4 (C-100),
80.0, 75.5, 75.0, 74.7, 73.3, 73.1, 70.6,
68.2, 60.4, 60.1 (C-20, 30, 40, 50, 60, 200, 300,
400, 500, 600), 52.0 (C-8); ESI-MS m/z: 499
[M þ Na]þ.
500 MHz) dH: 7.00 (1H, d, J ¼ 8.5 Hz,
H-3), 7.54 (1H, m, H-4), 6.96 (1H, m, H-
5), 7.71 (1H, dd, J ¼ 8.0, 1.5 Hz, H-6),
10.19 (1H, s, -OH), 6.09 (1H, d,
J ¼ 6.5 Hz, H-10), 3.6 ,5.5 (4H, H-20, 30,
40, 50), 2.04 (3H, s, -OAc), 2.03 (3H, s, -
OAc), 2.02 (3H, s, -OAc); ESI-MS m/z:
419 [M þ Na]þ.
3.2.2.8 1-[2,3,6-Tri-O-acetyl-4-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl)]-b-D-glucopyranosyl-2-hydroxy-
benzoate (13b). [a]2D0 226.1 (c ¼ 0.10,

Journal of Asian Natural Products Research
823
CHCl3); UV (CHCl3) lmax: 244, 313 nm; IR
(KBr) nmax: 2965, 1754, 1696, 1486, 1370,
1043, 762 cm21
;
1H-NMR (CD3OD,
400 MHz) dH: 7.40 (1H, d, J ¼ 8.0 Hz, H-
3), 7.51 (1H, m, H-4), 7.07 (1H, d,
J ¼ 7.6 Hz, H-5), 7.70 (1H, dd, J ¼ 7.6,
1.6 Hz, H-6), 3.83 (3H, s, H-8), 4.81 (1H, d,
J ¼ 7.6 Hz, H-10), 4.27 (1H, d, J ¼ 7.6 Hz,
H-100), 3.0 , 4.1 (H-20, 30, 40, 50, 60a, 60b, 200,
300, 400, 500a, 500b); 13C-NMR (CD3OD,
100 MHz) dC: 122.3 (C-1), 158.6 (C-2),
119.2 (C-3), 135.4 (C-4), 123.7 (C-5), 132.0
(C-6), 168.5 (C-7), 52.8 (C-8), 103.9 (C-10),
105.5 (C-100), 77.7, 77.6, 77.4, 75.0, 74.9,
71.3, 71.2, 69.9, 66.9 (C-20, 30, 40, 50, 60, 200,
300, 400, 500); ESI-MS m/z: 469 [M þ Na]þ.
1
1229, 1074, 762 cm21; H-NMR (DMSO-
d6, 500 MHz) dH: 7.00 (1H, d, J ¼ 8.5 Hz,
H-3), 7.52 (1H, m, H-4), 6.94 (1H, t,
J ¼ 8.0 Hz, H-5), 7.61 (1H, d, J ¼ 8.0 Hz,
H-6), 10.18 (1H, s, -OH), 6.12 (1H, d,
J ¼ 8.0 Hz, H-10), 3.0 , 5.5 (H-20, 30, 40, 50,
60a, 60b, 100, 200, 300, 400, 500, 600a, 600b), 2.15
(3H, s, -OAc), 2.06 (3H, s, -OAc), 2.00 (9H,
s, -OAc £ 3), 1.95 (3H, s, -OAc), 1.89 (3H,
s, -OAc); ESI-MS m/z: 779 [M þ Na]þ.
3.2.3 Methyl 2-O-(2,3-di-O-acetyl-4,6-
O-benzylidene)-b-D-glucopyranosyl
benzoate (7)
3.2.5 Methyl 2-O-[4-(O-b-D-
xylopyranosyl)-6-(O-b-D-xylopyranosyl)]-
b-D-glucopyranosylbenzoate (12)
When selective shelter of hydroxyl groups
at C-2, C-3, C-4, and C-6 of the glucose
residue on 5a was successfully carried out
using benzaldehyde and acetic anhydride,
compound 7 was afforded, yielding 37%
for two steps. m.p. 180.0–185.08C; [a]D20
2153.6 (c ¼ 0.10, CHCl3); UV (CHCl3)
Compound 12 was prepared according to
the same procedure described for 1.
m.p. 130.0–132.08C; [a ]2D0 267.5
(c ¼ 0.10, MeOH); UV (MeOH) lmax:
233, 285 nm; IR (KBr) nmax: 3392, 2892,
1708, 1602, 1491, 1451, 1310, 1267, 1075,
1043, 763 cm21
lmax: 242, 282 nm; IR (KBr) nmax: 2949,
;
1H-NMR (CD3OD,
2882, 1752, 1732, 1715, 1604, 1490, 1455,
1373, 1307, 1251, 1232, 1096, 1082, 1062,
400 MHz) dH: 7.29 (1H, d, J ¼ 8.0 Hz, H-
3), 7.45 (1H, m, H-4), 7.04 (1H, d,
J ¼ 8.0 Hz, H-5), 7.67 (1H, dd, J ¼ 8.0,
1.6 Hz, H-6), 3.79 (3H, s, H-8), 4.86 (1H, d,
J ¼ 7.5 Hz, H-10), 4.43 (1H, d, J ¼ 8.0 Hz,
H-100), 4.27 (1H, d, J ¼ 7.2 Hz, H-1000),
3.0 , 4.2 (H-20, 30, 40, 50, 60a, 60b, 200, 300, 400,
500a, 500b, 2000, 3000, 4000, 5000a, 5000b); 13C-NMR
(CD3OD, 100 MHz) dC: 122.5 (C-1), 158.5
(C-2), 119.1 (C-3), 135.2 (C-4), 123.8 (C-
5), 132.1 (C-6), 168.5 (C-7), 52.9 (C-8),
103.7 (C-10), 105.4 (C-100), 105.2 (C-1000),
79.9, 77.9, 77.8, 75.6, 75.5, 74.9, 74.8, 74.7,
71.2, 71.1, 68.6, 67.1, 67.0 (C-20, 30, 40, 50,
60, 200, 300, 400, 500, 2000, 3000, 4000, 5000); ESI-MS
m/z: 601 [M þ Na]þ.
1
1031, 757, 700 cm21; H-NMR (CDCl3,
400 MHz) dH: 7.12 (2H, m, H-3, 4), 7.45
(1H, m, H-5), 7.77 (1H, d, J ¼ 7.6 Hz,
H-6), 3.86 (3H, s, H-8), 7.36 , 7.49 (5H,
m, H-20, 30, 40, 50, 60), 5.54 (1H, s, H-70),
5.21 (1H, d, J ¼ 6.8 Hz, H-100), 5.38 (2H,
m, H-200, 300), 4.43 (1H, dd, J ¼ 10.4,
4.4 Hz, H-600a), 3.71 , 3.83 (3H, m, H-400,
500, 600b), 2.07 (6H, s, -OAc £ 2); ESI-MS
m/z: 425 [M þ K]þ.
3.2.4 Gaultherin (1)
The removal of the protective group on 7 in
80% HOAc at 808C for 3 h afforded 8,
yielding 79% [11]. When xylose as a donor
attached to the C-6 of the glucopyranosyl of
8 under promotion with BF3–Et2O, 1 was
afforded. m.p. 104.0–105.08C; [a]2D0 286.0
(c ¼ 0.10, MeOH); UV (MeOH) lmax: 231,
287 nm; IR (KBr) nmax: 3391, 2891, 1708,
1601, 1491, 1451, 1437, 1309, 1266, 1074,
3.3 Anti-nociceptive and anti-
inflammatory assay
3.3.1 Acetic acid-induced writhing test
A slight modification of the acetic acid-
induced writhing method was used [14].

824
C. Wang et al.
One hour after administering 5f (200–
800 mg/kg), aspirin (200 mg/kg), and the
vehicle orally to groups of 10 mice, 0.6%
aqueous solution of acetic acid was
administered intraperitoneally to each
mouse at a dose of 10 ml/kg body weight.
Each animal was placed in a transparent
observation cage 5 min after the acetic acid
injection, and the number of writhes per
mouse was counted over a 10 min period.
Writhing was defined as a contraction of
the abdominal muscles together with a
stretching of the hind limbs.
medium was used as the blank in all
experiments [14,16].
3.3.4 Statistical analysis
All values were expressed as ^SD. The
Student’s t-test for unpaired observations
between normal or control and tested
samples was carried out to identify
statistical differences; p values less than
0.05 were considered as significantly
different.
3.3.2 Croton oil-induced ear edema test
Acknowledgements
Aspirin (200 mg/kg), 5f (400 mg/kg), and
the vehicle were administered orally for
3 days before the topical application of
1 mg croton oil dissolved in 50 ml acetone
(20 ml/ear) to the right ear of mice. The
activity was evaluated using the following
process: the animals were sacrificed by
deep ether anesthesia 3 h after the topical
treatment. Eight millimeter diameter disks
were removed from each ear and weighed
on a balance [14,15].
The authors are grateful to the Department of
Instrumental Analysis in the Institute of
Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College
for all spectroscopic analysis. We also thank the
Department of Pharmacology of the Institute of
Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College
for bioactivity tests. This work was financially
supported by the National Science and
Technology Major Project (2009ZX09102-
034).
References
3.3.3 NO assay
[1] Y. Ding, G.H. Du, J.B. Li, and B. Zhang,
Chinese patent CN 1727354A (2006).
[2] Y.J. Gao, Pract. Pharm. Clin. Reme. 12,
122 (2009).
[3] Y.L. Xu, Med. Inf. 22, 2762 (2009).
[4] A. Robertson and R.B. Waters, J. Chem.
Soc. 1881 (1931).
[5] H.J. Zang, Z.M. Li, C.C. Ni, Z. Shen,
Z.J. Fan, and X.F. Liu, Chem. J. Chin.
Univ. 10, 1877 (2006).
[6] D. Dess, H.P. Kleine, D.V. Weinberg,
R.J. Kaufman, R.S. Sidhu, S.D. Dess,
H.P. Kleine, D.V. Weinberg, R.J. Kauf-
man, and R.S. Sidhu, Synthesis 883
(1981).
[7] W. Li, H.M. Liu, and Q.D. You, J. Chin.
Pharm. Univ. 35, 397 (2004).
[8] N.A. Bessie, Mbadugha, and F.M. Menger,
Org. Lett. 5, 4041 (2003).
[9] S. Xu, X.L. Ling, and H.M. Liu,
Chem. Res. Appl. 18, 870 (2006).
[10] N.L. Son and G.H. Jacquelyn, J. Org.
Chem. 70, 8772 (2005).
Murine macrophage RAW264.7 cells were
cultured at 378C in RPMI1640, containing
10% fetal bovine serum, in a humidified
atmosphere containing 5% CO2. The cells
were pretreated with all the synthetic
compounds (1 £ 1026 M) 1 h before being
treated with 0.5 mg/ml LPS for 24 h. The
nitrite concentration in the culture medium
was measured as an indicator of NO
production based on the Griess reaction.
A total of 70 ml of each supernatant was
mixed with the same volume of Griess
reagent (1% sulfanilamide in 5% phos-
phoric acid and 0.1% naphthylethylene-
diamine dihydrochloride in water), and the
resulting mixture was then incubated at
room temperature for 10 min. The absor-
bance at 540 nm was then measured using
a microplate reader. Freshly cultured

Journal of Asian Natural Products Research
825
[11] M.S. Cai and Z.J. Li, Carbohydrate
Chemistry (Chemical Industry Press,
Beijing, 2006), p. 51.
B.K. Kim, B.O. Lim, D.K. Choi, and W.S.
Choi, J. Ethnopharmacol. 112, 49 (2007).
[15] Y.C. Chen, C.Y. Cai, B. Ji, F.F. Gao,
Y.M. Zhang, J.Z. Wang, Z.Q. Huang, and
G.G. Shi, J. Shantou Univ. Med. Coll. 23,
7 (2010).
[16] K.M. Shin, I.T. Kim, Y.M. Park, J. Ha,
J.W. Choi, H.J. Park, Y.S. Lee, and K.T.
Lee, Biochem. Pharmacol. 68, 2327
(2004).
[12] X.F. Wu, W. Liu, M. Lu, and J.W. Chen,
Chin. J. Synth. Chem. 13, 461 (2005).
[13] G.Z. Sun and J.Y. Liu, J. Tianjin Med.
Univ. 9, 19 (2003).
[14] M.H. Lee, J.M. Lee, S.H. Jun, S.H. Lee,
N.W. Kim, J.H. Lee, N.Y. Ko, S.H. Mun,