Synthesis and α-glucosidase inhibitory activity of oleanolic acid derivatives

Article abstract of DOI:10.1080/10286020903405456

Glucosidations of oleanolic acid (1) and its dihydroxy-olide derivatives (2) were carried out to provide eight glycosides. All synthesized compounds were evaluated by in vitro α-glucosidase inhibitory activity assay. 3-Acetyl dihydroxy-olide oleanolic der

Full text of DOI:10.1080/10286020903405456

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Synthesis and α-glucosidase inhibitory
activity of oleanolic acid derivatives
Shan Qian a , Jiao Hai Li a , Yu Wei Zhang b , Xin Chen b & Yong Wu
a
a Key Laboratory of Drug Targeting , West China School of
Pharmacy, Sichuan University , Chengdu, China
b Di Ao Pharmaceutical Group , Chengdu, China
Published online: 01 Feb 2010.
To cite this article: Shan Qian , Jiao Hai Li , Yu Wei Zhang , Xin Chen & Yong Wu (2010) Synthesis
and α-glucosidase inhibitory activity of oleanolic acid derivatives, Journal of Asian Natural Products
Research, 12:1, 20-29, DOI: 10.1080/10286020903405456
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Journal of Asian Natural Products Research
Vol. 12, No. 1, January 2010, 20–29
ORIGINAL ARTICLE
Synthesis and a-glucosidase inhibitory activity
of oleanolic acid derivatives
Shan Qiana1, Jiao Hai Lia2, Yu Wei Zhangb3, Xin Chenb4 and Yong Wua*
aKey Laboratory of Drug Targeting, West China School of Pharmacy,
Sichuan University, Chengdu, China; Di Ao Pharmaceutical Group,
Chengdu, China
b
(Received 16 July 2009; final version received 9 October 2009)
Glucosidations of oleanolic acid (1) and its dihydroxy-olide derivatives (2) were
carried out to provide eight glycosides. All synthesized compounds were evaluated by
in vitro a-glucosidase inhibitory activity assay. 3-Acetyl dihydroxy-olide oleanolic
derivative 15 showed the most potent inhibitory effect. Structure–activity relationships
within these compounds are discussed.
Keywords: oleanolic acid; glycosides; a-glucosidase; inhibition ratio
1. Introduction
new OA derivatives in order to find more
potent therapeutic agents with better
bioavailability [6,7].
Pentacyclic triterpenoids are widely dis-
tributed in plants, and they have attracted
much attention due to their broad biologi-
cal activities. The most well-known
member of this family of compounds is
probably oleanolic acid (OA, 1; Figure 1),
which has been in active clinical use as an
anti-hepatitis drug in China for over 20
years, and possesses some attractive
biological activities including protection
of the liver against toxic injury [1], anti-
inflammation [2], anti-HIV [3], anti-tumor
[4], anti-hyperglycemia [5], etc. However,
it is well known that pentacyclic triter-
penes including OA have very poor H2O
solubility and thus have poor bioavail-
ability. Given the significant biological
importance and potentially new clinical
utilities of OA as a promising modulator of
glycogen metabolism, it is highly desirable
to synthesize and biologically evaluate
a-Glucosidase is a membrane-bound
enzyme at the epithelium of the small
intestine, which hydrolyses the cleavage of
glucose from disaccharides and oligo-
saccharides. The inhibitors of this enzyme
delay carbohydrate digestion, prolong the
overall carbohydrate digestion time, and
thus cause a reduction in the rate of
glucose absorption and lower the post-
prandial rise in blood glucose. Therefore,
inhibition of a-glucosidase is considered
important in managing non-insulin-
dependent diabetes [8].
Previously, Ali et al. [9] reported the
synthesis and biological evaluation of
some oleanolic derivatives as a-glucosi-
dase inhibitors. Among the tested samples,
dihydroxy-olide derivatives (2; Figure 1)
were found to be the most active inhibitor
*Corresponding author. Email: wyong@scu.edu.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020903405456
http://www.informaworld.com

Journal of Asian Natural Products Research
21
(1 ! 2)-a-L-arabinopyranosyl moiety, as
glycosyl donors. So far, no a-glucosidase
inhibitory activity evaluation toward OA
glycoside bearing this disaccharide has
been reported. As described by Cheng et al.
[12], selective shelter of hydroxyl groups
at C-3 and C-4 of the arabinose residue on
8 was successfully carried out using 2,2-
dimethoxypropane. Coupling of 8 and 4a
promoted by TMSOTf gave glycoside 9.
Et3N was added after glycosylation to
neutralize the Lewis acid, and an
additional step of deprotection was per-
formed to provide intermediate 10, fol-
lowed by removal of the benzoyl group in
NaOMe–MeOH to afford 11. Finally,
removal of the benzyl group through
catalytic hydrogenation afforded 12
(Scheme 2).
Figure 1. Structures of compounds 1 and 2.
of a-glucosidase. On the other hand, the
activity of saponins, which are glycosides
of terpenoid, is well known, and strongly
depends on the linkage between the
glucose units and on their configurations
[10]. Here, we report, for the first time, the
synthesis of glycosides of 2 by conjugating
2 with sugar moieties for investigating
whether the influence of the carbohydrate
residue is comparable. As a result of our
interest in chemical studies on the
glycosidic constituents of oleanolic
derivatives, we synthesized a series of
OA glycosides by introducing different
sugar moieties in C-3, and their inhibitor
activities against a-glucosidase were eval-
uated in vitro to study their structure–
activity relationships.
Treatment of 1 with ozone produced
2
dihydroxy-olide derivatives
[9]
1
(Scheme 3). As shown in the H NMR
spectrum, the formation of 2 could be
explained by the resonances of an olide-
type bridge between C-13 and C-28.
Subsequently, we planned to introduce
sugar moieties in C-3 and C-12 of 2 by
glycosylation with trichloroacetimidates
(4a) under promotion with TMSOTf.
Initially, we failed to obtain the target
product 13. Instead of 13, trichloroacetimi-
dates 4a were unexpectedly transferred to
2,3,4,6-benzoyl-glucopyranose under effec-
tiveness of Lewis acid. We were faced with
the problem of glycosylation of hindered
C-3 and C-12 positions on 2. We have
probed different glycosylation methods in
the experiment. AgOTf, Ag2CO3, etc. have
been employed to promote the glycosyla-
tion. Finally, hydroxyls of 2 were glycosy-
lated successfully under promotion with
TMSOTf. In a typical experiment, 2 and
excess of trichloroacetimidates (4a) were
dissolvedinabsoluteCH2Cl2 inthepresence
2. Results and discussion
Five 3-glycosides of 1 bearing different
natural monosaccharides and disacchar-
ides were easily prepared in four steps
from OA (1), as described in the literature
[11]. First, 1 was reacted with BnBr and
K2CO3 in dry DMF, which gave benzyl
oleanate (3) in a high yield. Esterification
of 1 was followed by glycosylation with
trichloroacetimidates (4) under promotion
with trimethylsilyl trifluoromethanesulfi-
nate (TMSOTf). Consequently, removal of
benzoyl on 5 through ester exchange in
NaOMe–MeOH afforded glycosides 6.
The benzyl moiety was then removed
under catalytic hydrogenolysis to give free
carboxylic acids 7 (Scheme 1).
˚
of 4 A molecular sieves. The vigorously
In order to investigate whether the
influence of the carbohydrate residue of
oleanane saponins is comparable, we used
the disaccharides, b-D-glucopyranosyl-
stirred mixture was treated at 2408C with
TMSOTf added dropwise. After standing
overnight, thereactionwascompleteandthe
product was obtained in a satisfactory yield.

22
S. Qian et al.
Scheme 1. The synthetic route of 7a–7e. Reagents and conditions: (a) BnBr, K2CO3, DMF, 508C,
˚
97%; (b) TMSOTf, 4 A MS, CH2Cl2, 2408C; (c) NaOMe, CH2Cl2–MeOH, r.t., 75–80% for two
steps; (d) H2, Pd/C (10%), MeOH, r.t., 90%.
Removal of benzoyl on 13 through ester
exchange in NaOMe–MeOH afforded
target glycoside 14. Compound 2 containing
two hydroxyl functions introduced acetyl
in C-3 selectively with acetic anhydride in
pyridine to obtain 15 [9]. Compound 15 was
glycosylated with trichloroacetimidates 4a
under promotion with TMSOTf to get 16.
All the benzoyl groups on the sugar parts
were removed by NaOMe–MeOH, while
the acetyl linkage was not affected under the
condition. Compound 17 was hydrolyzed
under the effectiveness of NaOH to afford
18. The configuration of the glucosidic bond
wasdeterminedfromthe1HNMR spectra of
the products. The stereochemistry of the
glycosides was assigned on the basis of
coupling constants. The coupling constants
(6.0–8.0 Hz) indicated the b-glucosidic
bond junction between pyranose 4a and A
ring of 2. The hydroxyl on C-12 of 2 was
converted into ketonic functions 19 by
treating with Jones reagent [9]. The
derivatization of 2 into derivatives 13–19
is summarized in Scheme 3, and the
glycosides 13, 14, 16–18 were the new
compounds.
All the derivatives of OA were
screened for their in vitro a-glucosidase
inhibitory activity for the determination of
inhibition ratio (%). The inhibition ratio
of the compounds is reported in Table 1.
The known active compound against
a-glucosidase, voglibose, was included
in the assays as controls and reference
points. Its inhibition ratio is 92.63%
(1 mg/ml).
of H-10 proton (J1 ,2 ) in products 14 and 18
0
0

Journal of Asian Natural Products Research
23

24
S. Qian et al.
Scheme 3. The synthetic route of 13–19. Reagents and conditions: (a) O3, CH2Cl2, 2708C, 62%;
˚
(b) 4a, TMSOTf, CH2Cl2, 4 A MS, 2408C, 57%; (c) NaOMe, CH2Cl2–MeOH, r.t., 85%; (d) Ac2O,
˚
pyridine, 08C, 64%; (e) 4a, TMSOTf, CH2Cl2, 4 A MS, 2408C; (f) NaOMe, CH2Cl2–MeOH, r.t.,
41% for two steps; (g) NaOH, THF–MeOH, 67%; (h) 3 mol/l Jones reagent, 08C, 92%.
OA (1) exhibited low a-glucosidase
inhibitory activity with an inhibition ratio of
2.92%. Benzyl oleanolate (3) was fourfold
more active than the parent acid 1.
Introduction of the sugar moiety at the C-3
position of OA (1) and benzyl oleanolate (3)
resulted in an increase in inhibitory activity
of the parent triterpenes. On the other hand,
the corresponding glycosides of 1 and
glycosides of 3 exhibited similar inhibitory
activity. 3-(b-D-Glucopyranosyl-(1 ! 2)-
a-L-arabinopyranosyl) benzyl oleanolate
(11) showed the highest activity of OA
glycosides, with an inhibition ratio of
15.77%.
Inthederivativesofdihydroxy-olide(2),
almost all compounds exhibited a-glucosi-
dase inhibitory activity. Compound 2
showed higher activity than the parent
acid 1, with an inhibition ratio of 11.92%.
The corresponding acetate 15 showed the
highest activity, with an inhibition ratio of
17.50%. The results indicated that acety-
lation of the hydroxyl group of 2 resulted
in an increase in activity. Incorporation of
the sugar moiety at the C-3 and C-12
positions of 2 and at the C-12 position
of acetate 15 resulted in a slight increase
in a-glucosidase inhibitory potency.
The ketonic derivative 19 showed less

Journal of Asian Natural Products Research
25
(J) are expressed in Hz. Mass spectra were
recorded on an Agilent 1946B ESI-MS
instrument.
Table 1. a-Glucosidase inhibitory activity
(in vitro) of the derivatives of OA.
Inhibition
(%)
Inhibition
(%)
Compound
Compound
1
2
3
2.92
11.92
10.82
13.33
8.97
10.48
15.11
15.18
11.35
15.49
9.04
7d
7e
11
12
13
14
15
16
17
18
19
14.71
14.38
15.77
12.32
13.92
16.8
3.2 General procedures for the
synthetic compounds
3.2.1 Oleanolic acid
3-O-b-D-glucopyranoside (7a)
6a
6b
6c
6d
6e
7a
7b
7c
According to the reported method [11], 3-
O-b-D-glucopyranoside benzyl oleanolate
(6a) was obtained as a white amorphous
solid from starting material 1, yield 72%
17.5
15.83
17.17
15.79
14.07
20
for three steps. Mp 1528C; ½aꢀD ¼ þ17.1
(c ¼ 0.5, methanol); IR (KBr) nmax: 3418,
Notes: The concentrations of all the determined
compounds were 5 mg/ml. The standard drug,
voglibose, showed an inhibition ratio of 92.63%
(1 mg/ml).
2945, 1727, 1668, 1459, 1366, 1161,
1078 cm21 1H NMR (CDCl3): d 7.38
;
(s, 5H), 5.30 (s, 1H), 5.10 (d, J ¼ 12.4 Hz,
1H), 5.04 (d, J ¼ 12.4 Hz, 1H), 4.33 (d,
J ¼ 6.8 Hz, 1H), 3.80 (s, 2H), 3.48–3.64
(m, 5H), 3.10–3.40 (m, 3H), 2.90
(d, J ¼ 12.8 Hz, 1H), 1.13 (s, 3H), 0.99
(s, 3H), 0.89–0.91 (s each, 3H each, 9H),
0.80 (s, 3H), 0.64 (s, 3H); ESI-MS m/z:
709 [MþH]þ. Compound 7a was obtained
as a white amorphous solid from 6a
activity than the parent compound 15,
indicating a preference for hydrophilic
groups over hydrophobic groups at C-12.
Further biological evaluation of these
oleanolic derivatives and extensive lead
optimizations are ongoing in our labora-
tory, and more results will be reported in
due time.
according to the reported method [11],
20
yield 90%. Mp 240–2418C; ½aꢀD
¼
þ21.3 (c ¼ 0.5, methanol). IR (KBr)
max: 3422, 2945, 2653, 1694, 1647,
3. Experimental
n
3.1 General experimental procedures
1462, 1387, 1364, 1077, 1022 cm21; H
NMR (DMSO-d6): d 12.00 (br, 1H), 5.19
(s, 1H), 4.90 (m, 3H), 4.38 (t, J ¼ 5.4 Hz,
1H), 4.13 (d, J ¼ 7.6 Hz, 2H), 3.64 (dd,
J ¼ 11.2 Hz, 4.6, 1H), 3.06–3.36 (m, 5H),
2.72 (d, J ¼ 10.0 Hz, 1H), 1.09 (s, 3H),
0.98 (s, 3H), 0.89–0.91 (s each, 3H each,
9H), 0.75 (s, 3H), 0.70 (s, 3H); ESI-MS
m/z: 620 [MþH]þ.
1
TLC was performed using precoated silica
gel GF254 (0.2 mm; Chemical Industry
Institute, Yantai, China), and column
chromatography was performed using
silica gel (100–200 mesh; Haiyang
Chemical Co., Ltd, Qingdao, China).
Melting points were observed in an open
capillary tube and are uncorrected. Optical
rotations were determined on a Perkin-
Elmer model 241 polarimeter. IR spectra
were obtained on a Perkin-Elmer 983.
Elemental analyses were performed by
Atlantic Microlab (Atlanta, GA, USA).
NMR spectra were taken on a Varian
INOVA 400 and on a Bruker AC-E200.
Chemical shifts (d) are expressed in ppm,
tetramethylsilane (TMS) used as the
internal reference, and coupling constants
Compounds 7b–7e were prepared
according to the same procedure described
for 7a.
3.2.1.1 Oleanolic acid 3-O-b-D-galacto-
pyranoside (7b). Mp 235–2368C;
½aꢀD ¼ þ19.0 (c ¼ 0.5, methanol); 1H
20
NMR (DMSO-d6): d 12.00 (s, 1H), 5.15 (s,
1H), 4.75–4.29 (br, 2H), 4.10 (d,

26
S. Qian et al.
J ¼ 6.4 Hz, 1H), 3.60–3.20 (m, 8H), 3.03
(d, J ¼ 7.6 Hz, 1H), 2.73 (d, J ¼ 12.8 Hz,
1H), 1.09 (s, 3H), 0.98 (s, 3H), 0.89–0.91
(s each, 3H each, 9H), 0.75 (s, 3H), 0.70
(s, 3H); ESI-MS m/z: 619 [MþH]þ.
3.2.2 Benzyl oleanolate 3-O-3,4-O-
isopropylidene-a-L-arabinopyranoside (8)
According to the reported method [12], 8
was obtained as a white foam from 6c,
20
yield 85%. ½aꢀD ¼ þ13.8 (c ¼ 0.5,
1
methanol); H NMR (400 MHz, CDCl3):
d 7.34 (s, 5H), 5.29 (t, J ¼ 3.6 Hz, 1H),
5.09 (d, J ¼ 12.8 Hz, 1H), 5.04 (d,
J ¼ 12.4 Hz, 1H), 4.20 (m, 3H), 4.05 (dd,
J ¼ 7.8, 6.0 Hz, 1H), 3.75 (dd, J ¼ 14.0,
3.4 Hz, 1H), 3.63 (t, J ¼ 7.8 Hz, 1H), 3.12
(dd, J ¼ 11.6, 4.6 Hz, 1H), 2.90 (dd,
J ¼ 13.8, 3.3 Hz, 1H), 1.54, 1.38, 1.12,
0.98, 0.86, 0.85, 0.84, 0.81, 0.60 (s each,
3H each); ESI-MS m/z: 719 [MþH]þ.
The spectral data of 8 are similar to those
reported in the literature [12].
3.2.1.2 Oleanolic acid 3-O-b-D-arabino-
pyranoside (7c). Mp 236–2388C;
½aꢀD ¼ þ18.7 (c ¼ 0.5, methanol); 1H
20
NMR (DMSO-d6): d 12.0 (br, 1H), 5.19 (s,
1H), 4.90 (br, 1H), 4.18 (d, J ¼ 7.6 Hz,
1H), 3.63 (dd, J ¼ 11.2, 5.4 Hz, 1H),
2.91–3.08 (m, 5H), 2.60 (dd, J ¼ 13.6,
4.0 Hz, 1H), 1.09 (s, 3H), 0.98 (s, 3H),
0.89–0.91 (s each, 3H each, 9H), 0.75 (s,
3H), 0.70 (s, 3H); ESI-MS m/z: 590
[MþH]þ.
3.2.3 Benzyl oleanolate 3-O-2,3,4,6-
tetra-O-benzoyl-b-D-glucopyranosyl-
(1 ! 2)-3,4-O-isopropylidene-a-L-
arabinopyranoside (9)
3.2.1.3 Oleanolic acid 3-O-b-D-gluco-
pyranoside (7d). Mp 238–2398C;
½aꢀD ¼ þ18.6 (c ¼ 0.5, methanol); 1H
20
Compound 9 was prepared from 8 accord-
ing to the same procedure described for 7a,
NMR (DMSO-d6): d 5.19 (s, 1H), 4.62 (d,
J ¼ 6.4 Hz, 1H), 4.20 (dd, J ¼ 10.8,
4.6 Hz, 1H), 4.15 (m, 2H), 2.89 (m, 1H),
3.54 (m, 1H), 3.21 (d, J ¼ 10.0 Hz, 1H),
2.98 (dd, J ¼ 11.2, 3.8 Hz, 1H), 1.09
(s, 3H), 0.98 (s, 3H), 0.89–0.91 (s each,
3H each, 9H), 0.75 (s, 3H), 0.70 (s, 3H);
ESI-MS m/z: 589 [MþH]þ.
20
yield 75%. ½aꢀD ¼ þ18.2 (c ¼ 0.5,
1
methanol); H NMR (400 MHz, CDCl3):
d 8.03–7.28 (m, 25H), 5.80 (t, J ¼ 9.6 Hz,
1H), 5.72 (t, J ¼ 9.6 Hz, 1H), 5.50 (dd,
J ¼ 9.6, 8.2 Hz, 2H), 5.42 (d, J ¼ 8.0 Hz,
1H), 5.38 (t, J ¼ 3.4 Hz, 1H), 5.10 (d,
J ¼ 12.8 Hz, 1H), 5.04 (d, J ¼ 12.4 Hz,
1H), 4.58 (dd, J ¼ 12.4, 3.0 Hz, 1H), 4.48
(dd, J ¼ 12.4, 4.2 Hz, 1H), 4.36 (d,
J ¼ 6.8 Hz, 1H), 4.12 (m, 2H), 3.80 (m,
3H), 3.68 (dd, J ¼ 12.8, 4.8 Hz, 1H), 3.00
(dd, J ¼ 11.2, 4.8 Hz, 1H), 2.90 (dd,
J ¼ 13.6, 4.0 Hz, 1H), 1.50, 1.24, 1.10,
0.95, 0.92, 0.90, 0.79, 0.72, 0.58 (s each,
3H each); ESI-MS m/z: 1298 [MþH]þ.
The spectral data of 9 are similar to those
reported in the literature [12].
3.2.1.4 Oleanolic acid 3-O-b-D-gluco-
pyranoside (7e). Mp 220–2218C;
½aꢀD ¼ þ14.9 (c ¼ 0.6, methanol); 1H
20
NMR (DMSO-d6): d 5.18 (s, 1H), 5.10
(d, J ¼ 4.0 Hz, 1H), 5.05 (d, J ¼ 4.4 Hz,
1H), 4.80 (d, J ¼ 4.8 Hz, 1H), 4.64–4.68
(m, 2H), 4.53 (d, J ¼ 4.4 Hz, 1H), 4.46
(t, J ¼ 6.0 Hz, 1H), 4.18–4.22 (m, 2H),
3.72 (dd, J ¼ 11.2, 5.0 Hz, 1H), 3.40–3.60
(m, 4H), 3.14 (m, 1H), 2.78 (m, 1H), 1.09
(s, 3H), 0.98 (s, 3H), 0.89–0.91 (s each,
3H each, 9H), 0.75 (s, 3H), 0.70 (s, 3H);
ESI-MS m/z: 781 [MþH]þ.
3.2.4 Oleanolic acid 3-O-b-D-
glucopyranosyl-(1 ! 2)-a-L-
arabinopyranoside (12)
The spectral data of 7a–7e are similar
to those reported in the literature [11].
Compound 12 was prepared from 9
according to the reported method [12],

Journal of Asian Natural Products Research
27
20
yield 74% for three steps. Mp 220–2218C;
20
solid. Mp 141–1438C; ½aꢀD ¼ þ13.4
(c ¼ 0.6, CH2Cl2).
½aꢀD ¼ þ16.0 (c ¼ 0.5, methanol); IR
(KBr) nmax: 3421, 2942, 1693, 1643,
;
To a solution of 13 (0.1 g, 0.06 mmol)
in dry CH2Cl2–MeOH (1:1, 15 ml), a
newly prepared NaOMe in MeOH solution
(30%, 65 ml) was added. The mixture was
stirred at room temperature for 4 h and
neutralized with acetic acid to pH 7.
The solution was concentrated and the
resulting residue was subjected to silica gel
column chromatography (CH2Cl2 –
MeOH–H2O ¼ 5:1:0.05) to give 14
(40.6 mg, 85%) as a white amorphous
1460, 1386, 1079 cm21
(400 MHz, DMSO-d6):
1H NMR
d 5.34 (d,
J ¼ 4.0 Hz, 1H), 5.15 (s, 1H), 4.98 (d,
J ¼ 2.0 Hz, 1H), 4.92 (d, J ¼ 4.0 Hz, 2H),
4.63 (d, J ¼ 4.0 Hz, 1H), 4.40 (d,
J ¼ 4.8 Hz, 1H), 4.35 (d, J ¼ 7.6 Hz,
1H), 4.21 (t, J ¼ 5.0 Hz, 1H), 3.56–3.68
(m, 5H), 3.44–3.51 (m, 1H), 2.94–3.17
(m, 5H), 2.75 (dd, J ¼ 9.2, 4.4 Hz, 1H),
1.09 (s, 3H), 0.98 (s, 3H), 0.82–0.88
(s each, 3H each, 9H), 0.74 (s, 3H), 0.72
(s, 3H); ESI-MS m/z: 751 [MþH]þ.
The spectral data of 12 are similar to
those reported in the literature [12].
20
solid. Mp 182–1848C; ½aꢀD ¼ þ27.9
(c ¼ 0.5, methanol); IR (KBr) nmax: 3418,
2936, 2873, 1727, 1645, 1459, 1384, 1076,
1026cm21; 1H NMR(DMSO-d6):d5.38(d,
J ¼ 8.0 Hz, 1H), 5.21 (d, J ¼ 3.6 Hz, 1H),
5.11 (d, J ¼ 6.4 Hz, 1H), 4.96 (dd, J ¼ 10.4,
5.0 Hz, 2H), 4.83–4.91 (m, 4H), 4.80 (s,
2H), 4.69 (dd, J ¼ 7.6, 4.8 Hz, 2H), 4.40
(dd, J ¼ 10.8, 5.6 Hz, 2H), 4.32
(t, J ¼ 5.6 Hz, 1H), 4.15 (d, J ¼ 7.6 Hz,
1H), 0.96, 0.93, 0.90, 0.84, 0.81, 0.74, 0.66
(s each, 3H each); elemental analyses found:
C, 63.22%; H, 8.53%; O, 28.26%; calcd for
C42H68O14: C, 63.30%; H, 8.60%; O,
28.11%; ESI-MS m/z: 798 [MþH]þ.
3.2.5 12-Dihydroxy-olide-olean (2)
According to the reported method [9], 2
was obtained in a pure form on chromato-
20
graphy, yield 62%. Mp .2508C; ½aꢀD
¼
þ19.5 (c ¼ 0.5, methanol); IR (KBr) nmax
:
3516, 2948, 2871, 1740, 1705, 1463, 1393,
1256 cm21; H NMR (400 MHz, CDCl3):
1
d 3.90 (s, 1H), 3.22 (dd, J ¼ 11.2, 4.8 Hz,
1H), 1.29, 1.13, 0.98, 0.89, 0.97, 0.86, 0.76
(s each, 3H each); elemental analyses
found: C, 76.25%; H, 10.21%; O, 13.58%;
calcd for C30H48O4: C, 76.23%; H, 10.24%;
O, 13.54%; ESI-MS m/z: 473 [MþH]þ.
The spectral data of 2 are similar to those
reported in the literature [9].
3.2.7 3-Acetyl dihydroxy-olide oleanolic
derivative (15)
According to the reported method [9], 15
was obtained (131.6 mg, 64%) as a white
amorphous solid on chromatography.
20
Mp .2508C; ½aꢀD ¼ þ18.1 (c ¼ 0.5,
3.2.6 3,12-b-D-Glucopyranosyl-12-
dihydroxy-olide-olean (14)
1
methanol); H NMR (CDCl3): d 4.50 (dd,
J ¼ 10.4, 6.4 Hz, 1H), 3.90 (s, 1H), 2.05 (s,
3H), 1.30 (s, 3H), 1.15 (s, 3H), 0.99 (s,
3H), 0.90–0.92 (s each, 3H each, 6H),
0.88 (s, 3H), 0.87 (s, 3H); ESI-MS m/z: 516
[MþH]þ. The spectral data of 15
are similar to those reported in the
literature [9].
Compound 2 (0.2 g, 0.4 mmol), trichloro-
acetimidates 4a (1.7 g, 2.0 mmol), and
˚
powdered 4 A molecular sieves (4.0 g)
were stirred for 30 min at 2408C in dry
CH2Cl2 (20 ml). A solution of TMSOTf
(23 ml) in dry CH2Cl2 was added dropwise
at 2408C. The mixture was stirred for 24 h
followed by addition of Et3N (0.1 ml) and
filtration. The mixture was concentrated
and purified through silica gel column
chromatography (CH2Cl2–MeOH) to give
13 (0.4 g, 57%) as a white amorphous
3.2.8 Dihydroxy-olide oleanolic-12-
glycosides (16)
Compound 16 was prepared from 15
according to the same procedure described

28
S. Qian et al.
20
for 13. ½aꢀD ¼ þ11.9 (c ¼ 0.5, metha-
nol); 1H NMR (400 MHz, CDCl3): d 7.83–
8.06 (m, 8H), 7.28–7.58 (m, 12H), 6.01
(m, 2H), 5.75 (m, 2H), 4.58 (dd, J ¼ 12.0,
2.8 Hz, 1H), 4.49 (m, 2H), 4.30 (m, 1H),
2.75 (d, J ¼ 13.6 Hz, 1H), 2.58 (d,
J ¼ 4.4 Hz, 1H), 2.03 (s, 3H), 0.89 (s,
3H), 0.86 (s, 3H), 0.79–0.81 (s each, 3H
each, 9H), 0.65 (s, 3H), 0.62 (s, 3H);
elemental analyses found: C, 72.54%; H,
7.03%; O, 20.44%; calcd for C66H76O14:
C, 72.51%; H, 7.01%; O, 20.49%; ESI-MS
m/z: 1094 [MþH]þ.
elemental analyses found: C, 68.14%; H,
9.23%; O, 22.63%; calcd for C36H58O9: C,
68.11%;H, 9.21%;O,22.68%;ESI-MSm/z:
635 [MþH]þ.
3.2.9 3-Acetyl dihydroxy-olide
oleanolic-12-oxo-derivative (19)
According to the reported method [9], 19
was obtained as a white solid after
concentration and repeated column
chromatography, yield 92%. Mp .2508C;
½aꢀD ¼ þ16.1 (c ¼ 0.5, methanol); 1H
20
Compound 17 was prepared from 16
according to the same procedure described
for 14, yield 41% for two steps.
NMR (CDCl3): d 4.50 (dd, J ¼ 11.2,
4.8 Hz, 1H), 2.71 (t, J ¼ 14.2 Hz, 1H),
2.54 (t, J ¼ 8.2 Hz, 1H), 2.39 (d,
J ¼ 2.8 Hz, 1H), 2.37 (d, J ¼ 3.2 Hz, 1H),
1.32 (s, 1H), 0.96–0.98 (s each, 3H each,
9H), 0.94 (s, 3H), 0.87 (s, 3H), 0.86 (s, 3H);
ESI-MS m/z: 514 [MþH]þ. The spectral
data of 19 are similar to those reported in
the literature [9].
20
½aꢀD ¼ þ12.3 (c ¼ 0.5, methanol); IR
(KBr) nmax: 3429, 2945, 2874, 1736,
1468, 1369, 1248, 1369, 1247, 1073,
1
1033 cm21; H NMR (400 MHz, CDCl3):
d 5.68 (d, J ¼ 7.6 Hz, 1H), 4.50 (dd,
J ¼ 11.2, 4.8 Hz, 1H), 3.80 (m, 2H), 3.40–
3.60 (m, 5H), 2.05 (s, 3H), 0.80–1.00 (s
each, 21H); elemental analyses found: C,
67.40%; H, 8.98%; O, 23.62%; calcd for
C38H60O10: C, 67.43%; H, 8.93%; O,
23.64%; ESI-MS m/z: 678 [MþH]þ.
3.3 Enzymatic activity assays
The inhibitory activity of all samples
against a-glucosidase (Sigma G-0660),
supplied by Sigma-Aldrich Co. Ltd (St
Louis, MO, USA), was measured spectro-
photometrically at pH 6.8 and at 378C
using 0.2 units/ml enzyme in 0.67 mM
sodium phosphate buffer. Voglibose
(1 mg/ml) was used as a positive control.
The reaction system was stirred at 378C
for 10 min, and 0.1 mol/l maltose was
added. After 10 min, a reagent (200 ml)
for detecting glucose, supplied by
Jiancheng Co. Ltd (Nanjing, China), was
added and the OD value in absorption at
490 nm was monitored continuously with
the spectrophotometer. The results are
shown in Table 1.
To
a solution of 17 (90.0 mg,
0.14mmol) in THF–MeOH (1.5:1, 15ml),
NaOH (4mol/l, 0.72ml) was added. The
mixture was stirred at room temperature for
5 h and neutralized with dilute hydrochloric
acid to pH 7. The solution was extracted
with CH2Cl2. The CH2Cl2 layer was
separated and washed with water, dried
over sodium sulfate, filtered and concen-
trated to a yellow solid, and the resulting
residue was subjected to silica gel column
chromatography (CH2Cl2–MeOH) to give
18 (60.0 mg, 67%) as a white amorphous
20
solid. ½aꢀD ¼ þ21.6 (c ¼ 0.5, methanol);
IR (KBr) nmax: 3422, 2931, 2868, 1727,
1692, 1467, 1367, 1074, 1034cm21 1H
;
NMR (DMSO-d6): d 5.37 (d, J ¼ 8.4 Hz,
1H), 5.29 (d, J ¼ 6.0 Hz, 1H), 5.05 (d,
J ¼ 4.8 Hz, 1H), 4.98 (d, J ¼ 4.8 Hz, 1H),
4.43(t, J ¼ 5.6 Hz, 1H), 4.32(d, J ¼ 5.2 Hz,
1H), 1.24 (s, 3H), 0.86–0.94 (s each, 3H
each, 12H), 0.79 (s, 3H), 0.68 (s, 3H);
Notes
1. Email: qians33@163.com
2. Email: tgxx903@163.com
3. Email: xinchanel@126.com
4. Email: wu.y903@163.com

Journal of Asian Natural Products Research
29
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