Glycoside modification of oleanolic acid derivatives as a novel class of anti-osteoclast formation agents

Article abstract of DOI:10.1016/j.carres.2009.01.019

Oleanolic acid, a natural product, possesses an anti-osteoclast formation activity. Targeting at discovery of novel and potent anti-bone resorption agents, 22 glycosides of oleanolic acid derivatives (including d-galactopyranosides, d-glucopyranosides, d-xylopyranoses, d-arabopyranoses and d-glycuronic acids) were synthesized at phase-transfer-catalyzed conditions (K₂CO₃, Bu₄NBr, CH₂Cl₂-H₂O) and their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α, 25-dihydroxy vitamin D₃ was evaluated in a co-culture assay system. The structure-activity relationships of these compounds were also discussed.

Full text of DOI:10.1016/j.carres.2009.01.019

Carbohydrate Research 344 (2009) 599–605
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Glycoside modification of oleanolic acid derivatives as a novel class
of anti-osteoclast formation agents
*
Jun-Feng Li, Song-Jie Chen, Yu Zhao, Jian-Xin Li
Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Oleanolic acid, a natural product, possesses an anti-osteoclast formation activity. Targeting at discovery
Received 5 December 2008
Received in revised form 14 January 2009
Accepted 18 January 2009
Available online 23 January 2009
of novel and potent anti-bone resorption agents, 22 glycosides of oleanolic acid derivatives (including
galactopyranosides, -glucopyranosides, -xylopyranoses, -arabopyranoses and -glycuronic acids)
were synthesized at phase-transfer-catalyzed conditions (K2CO3, Bu4NBr, CH2Cl2–H2O) and their inhibi-
tory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1 , 25-dihydroxy
D-
D
D
D
D
a
vitamin D3 was evaluated in a co-culture assay system. The structure–activity relationships of these com-
pounds were also discussed.
Keywords:
Oleanolic acid
Glycoside derivatives
Osteoclast formation
Inhibitory activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
that heterocyclic system such as indole, phenylhydrazone moieties
produce effective anti-osteoporosis activity.18,19 Those reports, to-
Osteoporosis is widely recognized as a major public health
problem, particularly among the postmenopausal women. Cur-
rently, it is estimated that over 200 million people worldwide have
osteoporosis1 and osteoporosis related fractures accounted for
more disability-adjusted life years (DALYs) lost than common can-
cers except for lung cancer in Europe.2 It is a common skeletal dis-
ease characterized by gradual loss of bone mass and disrupted
bone architecture as a result of an imbalance between the bone
resorption activity of osteoclasts and the bone formation activity
of osteoblasts.3 In a research project focused on finding new anti-
osteoporosis agents, we discovered that the extracts from the root
of Achyranthes bidentata Blume (Amaranthaceae) showed the anti-
osteoporosis activity in vitro and in vivo and further research re-
vealed that oleanolic acid (OA, 1) and its glycosides were the active
components of the extracts responsible for the anti-osteoporosis
activity.4,5 OA, a natural product, and its derivatives possess a vari-
ety of biological activities, such as anti-inflammatory,6,7 hepato-
protective effects,8,9 anti-HIV,10,11 antitumour,12,13 inhibitory
effect of glycogen phosphorylase14 and suppressive activity of ni-
tric oxide production.15,16 However, the effect of oleanolic acid
and its derivatives on anti-osteoclast formation had not been stud-
ied sufficiently (Fig. 1).
gether with our previous results of oleanolic acid as a potential
anti-osteoporosis lead compound inspired us to incorporate indole,
phenylhydrazone and saccharide segments in the syntheses of ole-
anolic acid derivatives to improve the inhibitory activity on osteo-
clast formation. In the present study, as a part of our continuing
efforts targeting at discovery of more potent anti-osteoclast forma-
tion molecules, we report here the synthesis, activity evaluation of
22 glycosides of oleanolic acid derivatives.
2. Results and discussion
2.1. Chemistry
The preparation of compounds 2–4 is outlined in Scheme 1. 3-
Keto oleanolic acid (2) was synthesized by Jones oxidation of ole-
anolic acid.20 A mixture of 3-keto oleanolic acid (2) and phen-
12
13
17
COOH
C
H
Triterpenoid glycosides are a large family of natural products
with a great variety of structures and bio-effects, especially some
of them show prominent activities.17 Several papers also reported
28
2
3
A
H
HO
1
* Corresponding author. Tel.:/fax: +86 25 83686419.
E-mail address: lijxnju@nju.edu.cn (J.-X. Li).
Figure 1. Chemical structure of oleanolic acid.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.01.019

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J.-F. Li et al. / Carbohydrate Research 344 (2009) 599–605
COOH
COOH
H
H
a
H
H
HO
O
1
2
b
c
COOH
COOH
H
H
H
H
H
O2N
N N
N
H
NO2
4
3
Scheme 1. Reagents and conditions: (a) Jones oxidation;20 (b) phenylhydrazine, AcOH, reflux;21 (c) 2,4-dinitrophenylhydrazine, AcOH, reflux.
ylhydrazine in the presence of acetic acid was refluxed under N2
for 1–2 h to provide indole target compound 3.21 A mixture of 3-
keto oleanolic acid (2) and o,p-dinitrophenylhydrazine in the pres-
ence of acetic acid was refluxed under N2 for 1–2 h to afford phen-
ylhydrazone 4.
tion of
reported when deprotection of an acetyl group at COOH of fully acet-
ylated -glucuronic acid in the presence of iodine followed by treat-
D-glucuronic acid was achieved by a one-pot method. Tosin
D
ment with methanol, the reaction gave the methyl ester, not the
desired carboxylic acid.26 However, this result gave us a clue to pro-
The optimized synthesis route of compound 5 is outlined in
Scheme 2. Oleanolic acid was acetylated with 10 equiv of acetic
anhydride in anhydrous pyridine to give 3-acetoxy-oleanolic acid
(6). Oxidation of compound 6 with 30% H2O2 in formic acid at
50 °C overnight afforded 3-acetoxy-12-oxo-oleanolic acid (7).
Dihydrooleanolic acid (5) was produced by Wolff–Kishner reduc-
tion of 7 in diethylene glycol solution. Although the synthesis of
dihydrooleanolic acid (5) had been reported,22,23 it is worth to indi-
cate that in the present synthetic process: (i) Unprotection of the
C-17 carboxyl group of 2 did not impact the yield of dihydroolean-
olic acid 5. (ii) Oxidation of 6 to 12-oxo compound 7 could be
achieved by one step with 30% H2O2 in acidic medium (formic
acid), which was superior to that with 3-chloroperbenzoic acid
and then reflux with ethereal boron trifluoride (two steps, in acetic
acid).
tection of COOH. As expected, the acetylation of D-glucuronic acid in
the presence of iodine in acetic anhydride followed by treatment
with methanol in one pot gave target product, 1,2,3,4-tetra-O-acet-
yl-b-D-glycuronic acid-6-methyl ester. Thereafter, bromide 12 was
synthesized with the same protocol as 8–11 (Fig. 2).
In recent years, a phase-transfer-catalyzed method has been ap-
plied for glycosidation due to its mild conditions and high
yields.27,28 The present glycosidations of the oleanolic acid deriva-
tives were achieved by the reaction of acceptors (1–5) with donors
(8–12) under the optimized phase-transfer-catalyzed conditions
(K2CO3, Bu4NBr, CH2Cl2–H2O, reflux). Subsequent removal of the
protecting groups (benzoyl) using sodium methoxide (cat.) in
MeOH gave the corresponding glycosides a–d. Removal of the
glycuronic acid protecting groups was slightly modified. The re-
lated compounds were treated with sodium methoxide in MeOH
and then water to yield compounds 1e and 5e, respectively
(Scheme 3).
D-Sugar bromides 8–11 (Fig. 2) were prepared through benzoyla-
tion (BzCl in pyridine) and bromination (HBr in HOAc).24,25 Protec-
COOH
COOH
H
H
a
H
H
HO
AcO
1
6
b
O
COOH
H
COOH
H
c
H
H
HO
AcO
5
7
Scheme 2. Reagents and conditions: (a) Ac2O, Py, DMAP, rt; (b) formic acid, 30% H2O2, CHCl3, 0–50 °C; (c) KOH, diethylene glycol, NH2NH2ꢀH2O, reflux, 160–210 °C.

J.-F. Li et al. / Carbohydrate Research 344 (2009) 599–605
601
As presented in Table 1, all of the tested compounds remained
inhibitory effect on OCLs formation. Fortunately, except for hetero-
cyclic derivative (compound 3), the skeleton modifications of OA
(compounds 2, 4 and 5) displayed a more potent activity than that
OA did, especially compound 4 showed much stronger activity
compared with OA.
OBz
OBz
O
BzO
BzO
BzO
O
BzO
BzO
O
BzO
BzO
BzO
BzO
Br
Br
Br
9
10
8
A lot of papers have reported that glycosidation could improve
BzO
the bioactivity of parent molecules.31–33 As expected, introducing
MeOOC
AcO
O
O
BzO
monosaccharide moieties including
D-glucose, D-galactose, D-xy-
Br
AcO
lose and -arabinose at C28-position of parent compounds 1–4
D
AcO
OBz
Br
greatly enhanced the inhibitory activity compared with their par-
ent compounds, especially indole 3 and phenylhydrazone 4 series
(3a–d, 4a–d) exhibited quite strong activity, almost 100% inhibi-
11
12
Figure 2. Five sugar donors (8–12).
tion on OCLs formation at 20 lM. Unfortunately, dihydrooleanolic
glycosides (5a, 5c–e) showed a quite weaker activity compared
with compound 5. Together with our previous results,4 it could
be concluded that double bond at 12 and 13 position in C-ring is
a key for the potent anti-OCLs formation activity of glycosides,
any changes at the double bond would negatively impact the activ-
For the stereochemistry of the glycosidic bonds, bromides 8–9
were configuration which were identical with the reported
data.24,29 Bromides 10 and 12 were elucidated to be
configura-
a
a
tion (H-1, 6.66–6.85 ppm, d, J = 3.7–3.9 Hz), while 11 was b config-
uration (H-1, 6.93 ppm, d, J = 3.2 Hz). To target glycosides, all of the
glycosidic bonds were b configuration (a–c and e series: H-10, 5.34–
ity. Furthermore, the activity of the compounds at 2 lM was as-
sayed. As can be seen in Table 1, most of the compounds
displayed a weak activity. Meaningfully, the xylose and glycuronic
acid glycosides improved the activity except for 1c. However, for
phenylhydrazone 4, adding a glycone section at the C-28 position
suppressed the inhibitory activity (about 4a, 99.0%; 4b, 91.3%; 4c,
88.3%; 4d, 59.4%). Encouragingly, compounds 1e, 1f, 2c, 3a, 5c, 5e
displayed a quite strong activity compared with parent compounds
6.35 ppm, d, J = 6.4–8.5 Hz;
J = 6.1–7.9 Hz).
d
series, H-10, 5.41–6.28 ppm, d,
2.2. Biological activity
Since potentiated bone resorption by osteoclasts is one of the
major causes of osteoporosis, inhibitors of osteoclasts may there-
fore present useful agents to prevent the excessive bone resorption
associated with osteoporosis. Thus, an inhibitory activity of all
compounds on TRAP-positive osteoclast-like multinucleated cells
(OCLs) formation was assayed. The OCLs formation was induced
even at 2
coside showed the strongest activity (about 16.0%). For the cyto-
toxicity of the tested compounds by
recovery test,34 no
lM, among those compounds, 1e, a glucuronic acid gly-
a
significant toxicity for all of the tested compounds was observed.
From the above data, it is quite clear that any changes in double
bond at C-ring of OA gave a negative impact on the anti-OCLs for-
mation activity. The most glycosidations are of quite benefit to the
improvement of the activity, especially, xylose and glycuronic acid
glycosides. A phenylhydrazone moiety at A-ring of OA greatly im-
proved the activity, while its glycosides did not bring any benefit
on the activity enhancement.
In conclusion, we have synthesized a series of glycosides of ole-
anolic acid derivatives, and assayed their anti-OCLs formation
activity. 12-Keto oleanolic acid (7) was afforded by one step with
30% H2O2 in acidic medium instead of the two steps method. Pro-
by 1a, 25-dihydroxy vitamin D3 [1a, 25(OH)2D3] in a co-culture
system with mouse bone marrow cells and osteoblast-like cells.30
12
13
COOH
H
O
a
+
Br
H
COR
H
8-12
tection of D-glucuronic acid was achieved by a one pot reaction.
Twenty two novel oleanolic acid type glycosides were synthesized
at phase-transfer-catalyzed conditions (K2CO3, Bu4NBr, CH2Cl2–
H2O). The preliminary biological evaluation results showed that
compound 1e, 2c and 4 displayed quite strong inhibitory activity
12(13)
1-4:
a-e
5: 13-H
R
1
2
3
4
5
even at 2 lM. Our results revealed that the glycosides of OA deriv-
CH2OH
O
HO
HO
O
atives represent an interesting class of compounds for further
pharmacological studies and preclinical developments. Further
syntheses of glycosides, detailed studies on compound 1e and 2c
in vivo are in progress in our lab.
1a
2a
3a
3b
4a
5a
5b
(Gal)
OH
CH2OH
O
O
O
(Glc)
(Xyl)
(Ara)
1b
2b
4b
HO
HO
OH
3. Experimental
O
O
1c
2c
2d
3c
3d
4c
5c
5d
HO
HO
OH
3.1. General methods
HO
OH
O
1d
4d
HO
All the solvents used were commercially available. Reactions
were monitored by thin-layer chromatography (TLC) on silica gel
plates, visualizing with ultraviolet light or iodine spray. NMR spec-
tra (300 MHz 1H and 75 MHz 13C) were taken in CDCl3, C5D5N-d5 or
MeOD solution on Bruker Avance II-300 spectrometer. Melting
points were determined using X4 apparatus and uncorrected.
ESI-MS spectra were obtained on a Finnigan TSQ-7000 mass
spectrometer.
COOH
O
O
(GA)
1e
5e
HO
HO
OH
Scheme 3. Reagents and conditions: (a) (i) CH2Cl2/H2O (10:1), K2CO3, Bu4NBr,
reflux; (ii) MeONa (cat.), MeOH, rt or MeONa (cat.), MeOH, H2O, rt.

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J.-F. Li et al. / Carbohydrate Research 344 (2009) 599–605
Table 1
Effects of tested compounds on 1
a
, 25(OH)2D3 induced TRAP(+)-OCLs formation
OCLs (%)
Compound
Compound
OCLs (%)
20
l
M
2
l
V
20
lM
2 lM
Control
1
100.0 9.7#
20.3 5.9**
100.0 6.2#
89.2 10.2**
98.2 5.2
Normal
3c
3d
4
4a
4b
4c
4d
5
5a
5b
5c
5d
5e
20.0 5.0
0**
20.0 8.6
61.3 13.7**
96.7 10.9*
29.7 4.7**
99.0 6.7
0**
**
0
1a
1b
1c
1d
1e
2
2a
2b
2c
2d
3
1.3 0.2**
54.7 2.9**
5.7 0.5**
19.3 1.1**
1.4 3.4**
0**
98.7 5.2
39.8 8.7**
0**
95.5 5.0*
99.3 5.2
0**
91.3 12.2**
88.3 8.2**
59.4 18.3**
66.3 3.5**
87.2 4 .6**
85.7 4.5**
56.5 3.0**
90.9 4.8**
54.6 2.9**
16.0 0.8**
79.2 12.0**
78.1 9.3**
64.4 12.8**
30.2 10.7**
98.4 7.4
0**
0**
28.1 7.0**
77.1 4.1**
0**
0**
0**
9.5 5.0
60.3 3.2**
55.5 2.9**
41.9 2.2**
**
51.1 6.2**
94.6 9.6**
54.4 15.6**
98.5 6.9
3a
3b
0**
0**
Control: cultured with 1a
, 25(OH)2D3 (10ꢂ8 M). Normal: cultured without any additions. Samples: cultured with 25(OH)2D3 (10ꢂ8 M) and each compound . Each value was
expressed as mean SD, n = 4. The data of control group was pegged as 100%, while other data were calculated relative to it. Data were analyzed by student’s t test. Significant
**
differences in TRAP (+)-OCLs compared with control group, *p < 0.05, p < 0.01, #p < 0.01 versus normal group.
3.2. 3-Keto oleanolic acid (2) and [3,2-b] indole oleanolic acid (3)
(100 mL). The mixture was refluxed at 160 °C for 3 h. After removal
of water and hydrazine hydrate, the mixture was then stirred at
210 °C for 6 h. After cooling room temperature, the reaction mix-
ture was neutralized with dilute aqueous HCl (pH <7). The mixture
was poured into water and the resulting suspension was filtered.
Crude product was purified by recrystallization to provide com-
pound 5 (6.62 g, 72%). White solid; 1H NMR (CDCl3): d 3.23 (dd,
1H, J 9.4, 6.3 Hz), 2.90 (dd, 1H, J 13.0, 3.4 Hz); 13C NMR (CDCl3): d
174.5; MS (ESI) m/z: 457.41 [MꢂH]ꢂ.
3-Keto oleanolic acid (2) and [3,2-b] indole acid (3) were pre-
pared following the Gribble’s method20,21 and the 1H NMR, 13C
NMR and MS data were identical with the reported ones.
3.3. 3-[2,4-Dinitrophenylhydrazone] oleanolic acid (4)
To 3-keto oleanolic acid (2) (8.67 g, 19.10 mmol) stirring in ace-
tic acid (80 mL) was added 2,4-dinitrophenylhydrazine (3.97 g,
20 mmol). The mixture was refluxed for 1 h. The reaction mixture
was pipetted into distilled water and then extracted with Et2O
(4 ꢁ 80 mL). After removing Et2O, the residue was purified by col-
umn chromatography (silica gel; EtOAc/PE) to provide phenylhyd-
razone 4 (yield 65%). Orange solid; mp 272 °C; 1H NMR (CDCl3): d
11.19 (s, 1H), 9.15 (s, 1H), 8.32 (d, 1H, J 9 Hz), 7.87 (d, 1H, J 9 Hz),
5.33 (s, 1H), 2.86 (d, 1H, J 12 Hz); 13C NMR (CDCl3): d 184.7, 166.9,
145.5, 143.6, 137.4, 129.9, 128.9, 123.6, 122.3, 116.4; ESI-MS (neg-
ative) m/z: 633.23 [MꢂH]ꢂ.
3.5. General procedure for preparation of glycosides of
oleanolic acid derivatives a–d
To bromine glycoside (0.52 mmol) and oleanolic acid derivative
(0.40 mmol) stirring in 22 mL CH2Cl2/H2O (10:1) was added K2CO3
(138 mg) and Bu4NBr (51.52 mg). The mixture was refluxed under
nitrogen atmosphere. After completion (TLC) the reaction mixture
was diluted with CHCl3 and washed with water, then brine and
dried. The crude was purified by recrystallization or column
chromatography.
3.4. Preparation of 12,13-dihydrooleanolic acid (5)
To the above compound (0.24 mmol) stirring in methanol was
added sodium methoxide (cat.). The mixture was stirred at room
temperature. After completion (TLC) the reaction mixture was neu-
tralized with HCl (1 M). Water was added and the resulting sus-
pension was filtered. Crude product was purified by
recrystallization or column chromatography. Compounds a–d
were deprotected following this protocol.
3.4.1. 3-O-Acetyl oleanolic acid (6)
To OA (1) (456 mg, 1 mmol) stirring in pyridine (19 mL) in an
ice bath was added acetic anhydride (9.45 mL). DMAP (122 mg,
1 mmol) was added and the mixture was stirred at room temper-
ature for 5 h. The mixture was poured into ice water and was then
extracted with CH2Cl2. The organic layer was washed with dilute
aqueous HCl, water and brine, respectively. After removing the
solution, the residue was purified by recrystallization to provide
compound 6 (4.93 g, yield 99%). 1H NMR (CDCl3): d 5.32 (s, 1H),
4.48 (t, 1H, J 7.9 Hz), 2.80 (dd, 1H, J 13.3, 3.5 Hz), 2.04 (s, 3H).
3.5.1. Oleanolic acid 28-O-[b-D-galactopyranosyl] ester (1a)
White solid; yield 91.0%; mp 243–245 °C; 1H NMR (CDCl3): d
5.38 (d, 1H, J 8.2 Hz, Gal-1-H), 5.26 (s, 1H), 3.91 (s, 1H), 3.72–
3.70 (m, 2H), 3.66 (d, 1H, J 8.2 Hz), 3.61 (dd, 1H, J 9.2, 5.9 Hz),
3.54 (dd, 1H, J 9.7, 2.7 Hz), 3.16 (dd, 1H, J 10.6, 4.0 Hz), 2.88 (dd,
1H, J 13.7, 3.0 Hz); 13C NMR (CDCl3): d 177.3, 141.2, 122.3, 96.9;
MS (ESI) m/z: 641.2 [M+Na]+. Anal. Calcd for C36H58O8: C, 69.87;
H, 9.45. Found: C, 69.57; H, 9.37.
3.4.2. 3-O-Acetyl-12-oxo oleanolic acid (7)
To compound 6 (2.50 g, 5 mmol) in CHCl3 (19 mL) was added
H2O2 (10 mL, 30%) and formic acid (2.25 mL). The mixture was stir-
red at 50 °C for 24 h. After removal of the solvent, crude product
was purified by recrystallization to give compound 7 (1.82 g,
71%).1H NMR (CDCl3): d 4.47 (dd, 1H, J 10.5, 4.8 Hz), 2.71 (t, 1H, J
14.3 Hz), 2.54 (t, 1H, J 8.3 Hz), 2.37 (dd, 1H, J 14.6, 3.0 Hz).
3.5.2. Oleanolic acid 28-O-[b-D-glucopyranosyl] ester (1b)
White solid; yield 87.6%; mp 239–241 °C; 1H NMR (MeOD): d
5.57 (d, 1H, J 7.8 Hz, Glc-1-H), 5.26 (s, 1H), 3.85 (d, 1H, J 11.9 Hz),
3.69 (d, 1H, J 12.1 Hz), 3.46–3.33 (m, 4H), 3.12 (dd, 1H, J 11.1,
5.7 Hz), 2.87 (d, 1H, J 13.7 Hz); 13C NMR (MeOD): d 185.2, 145.8,
122.7, 95.6; MS (ESI) m/z: 641.2 [M+Na]+. Anal. Calcd for
C36H58O8: C, 69.87; H, 9.45. Found: C, 69.62; H, 9.40.
3.4.3. 12,13-Dihydrooleanolic acid (5)
To compound
7 (10.28 g, 20 mmol) in diethylene glycol
(200 mL) was added KOH (26 g, 0.46 mol) and hydrazine hydrate

J.-F. Li et al. / Carbohydrate Research 344 (2009) 599–605
603
3.5.3. Oleanolic acid 28-O-[b-
D
-xylopyranosyl] ester (1c)
3.73 (d, 1H, J 11.7 Hz), 3.62 (d, 1H, J 12.3 Hz), 3.43–3.32 (4H, m);
13C NMR (CDCl3): d 176.7, 143.8, 140.9, 136.1, 128.5, 124.5,
121.2, 119.0, 118.1, 110.4, 106.7, 94.5, 76.7, 72.1, 69.5, 65.6; ESI-
MS m/z: 712.27 [M+Na]+. Anal. Calcd for C42H59NO7: C, 73.12; H,
8.62; N, 2.03. Found: C, 72.85; H, 8.60; N, 2.04.
White solid; yield 95.0%; mp >280 °C; 1H NMR (MeOD): d 5.34
(d, 1H, J 6.4 Hz, Xyl-1-H), 5.25 (s, 1H), 3.90–3.84 (m, 2H), 3.54–
3.46 (m, 2H), 3.39 (dd, 1H, J 8.1, 3.2 Hz), 3.14 (dd, 1H, J 11.8,
4.4 Hz), 2.87 (dd, 1H, J 13.9, 3.4 Hz); 13C NMR (C5D5N-d5): d
178.1, 145.8, 122.4, 97.9; MS (ESI) m/z: 611.17 [M+Na]+. Anal. Calcd
for C35H56O7: C, 71.39; H, 9.59. Found: C, 71.14; H, 9.63.
3.5.11. [3,2-b] Indole oleanolic acid 28-O-[b-D-xylopyranosyl]
ester (3c)
3.5.4. Oleanolic acid 28-O-[b-
D
-arabopyranosyl] ester (1d)
Orange solid; yield 69%; mp 196 °C; 1H NMR (CDCl3): d 7.76 (s,
1H), 7.43–7.05 (m, 4H), 5.55 (d, 1H, J 6.0 Hz, Xyl-1-H), 5.43 (s, 1H),
3.95–3.85 (m, 2H), 3.58–3.46 (m, 2H), 3.41 (dd, 1H, J 8.6, 2.3 Hz);
13C NMR (CDCl3): d 176.4, 143.0, 140.9, 136.2, 128.7, 123.2,
121.0, 119.0, 118.1, 110.4, 106.9, 94.4, 76.7, 72.1, 69.5, 65.6; ESI-
MS m/z: 682.11 [M+Na]+. Anal. Calcd for C41H57NO6: C, 74.62; H,
8.71; N, 2.12. Found: C, 74.90; H, 8.75; N, 2.13.
White solid; yield 78.2%; mp 260–262 °C; 1H NMR (C5D5N-d5): d
6.21 (d, 1H, J 7.8 Hz, Aar-1-H), 5.25 (s, 1H), 4.63 (dd, 1H, J 7.6,
6.3 Hz), 4.35–4.27 (m, 2H), 4.24–3.83 (m, 2H), 3.14 (dd, 1H, J
10.3, 4.7 Hz), 2.81 (d, 1H, J 10.4 Hz); 13C NMR (C5D5N-d5): d
178.1, 145.8, 121.9, 97.9; MS (ESI) m/z: 611.17 [M+Na]+. Anal. Calcd
for C35H56O7: C, 71.39; H, 9.59. Found: C, 71.10; H, 9.53.
3.5.5. 3-Keto oleanolic acid 28-O-[b-
D
-galactopyranosyl] ester
3.5.12. [3,2-b] Indole oleanolic acid 28-O-[b-D-arabopyranosyl]
(2a)
ester (3d)
White solid; yield 69%; mp 210 °C; 1H NMR (CDCl3): d 5.41 (d,
1H, J 8.1 Hz, Gal-1-H), 5.30 (t, 1H, J 3.0 Hz), 3.90 (s, 1H), 3.75–
3.70 (m, 2H), 3.64 (d, 1H, J 8.3 Hz), 3.60 (dd, 1H, J 9.0, 6.0), 3.52
(dd, 1H, J 9.3, 2.9); 13C NMR (CDCl3): d 218.2, 176.5, 143.6, 122.3,
94.5, 74.1, 70.4, 68.3; ESI-MS m/z: 639.15 [M+Na]+. Anal. Calcd
for C36H56O8: C, 70.10; H, 9.15. Found: C, 69.85; H, 9.11.
Orange solid; yield 68%; mp 193 °C; 1H NMR (CDCl3): d 7.76 (s,
1H), 7.42–7.03 (m, 4H), 5.49 (d, 1H, J 7.9 Hz, Ara-1-H), 5.41 (s, 1H),
4.01 (dd, 1H, J 7.1, 6.0 Hz), 3.95–3.83 (m, 2H), 3.69–3.60 (m, 2H);
13C NMR (CDCl3): d 176.4, 143.0, 140.9, 136.2, 128.3, 123.2,
121.1, 119.0, 118.1, 110.4, 107.0, 94.4, 73.3; ESI-MS m/z: 682.19
[M+Na]+. Anal. Calcd for C41H57NO6: C, 74.62; H, 8.71; N, 2.12.
Found: C, 74.37; H, 8.68; N, 2.14.
3.5.6. 3-Keto oleanolic acid 28-O-[b-D-glucopyranosyl] ester
(2b)
3.5.13. 3-[2,4-Dinitrophenylhydrazone] oleanolic acid 28-O-[b-
White solid; yield 71%; mp 192 °C; 1H NMR (C5D5N-d5): d 6.35
(d, 1H, J 8.1 Hz, Glc-1-H), 5.46 (s, 1H), 4.42 (d, 1H, J 11.9 Hz), 4.21
(d, 1H, J 12.1 Hz), 4.01–3.95 (m, 4H); 13C NMR (C5D5N-d5): d
217.8, 178.0, 145.7, 124.2, 97.3, 80.9; ESI-MS m/z: 639.17
[M+Na]+. Anal. Calcd for C36H56O8: C, 70.10; H, 9.15. Found: C,
70.39; H, 9.18.
D
-galactopyrano-syl] ester (4a)
Orange solid; yield 71%; mp 215 °C; 1H NMR (C5D5N-d5): d
11.20 (s, 1H), 9.14 (s, 1H), 8.42 (d, 1H, J 9.0 Hz), 8.05–8.02 (m,
1H), 6.29 (d, 1H, J 8.2 Hz, Gal-1-H), 5.47 (t, 1H, J 3 Hz), 4.70–4.61
(m, 2H), 4.49 (dd, 1H, J 12.9, 5.9 Hz), 4.26 (dd, 1H, J 13.0, 6.1 Hz),
4.22–4.16 (m, 2H); 13C NMR (C5D5N-d5): d 178.1, 169.3, 147.3,
145.7, 139.2, 131.7, 131.0, 124.2, 118.1, 97.8, 79.3, 77.3, 73.0,
71.6; ESI-MS m/z: 798.21 [M+H]+. Anal. Calcd for C42H60N4O11: C,
63.30; H, 7.59; N, 7.03. Found: C, 63.03; H, 7.52; N, 7.06.
3.5.7. 3-Keto oleanolic acid 28-O-[b-D-xylopyranosyl] ester (2c)
White solid; yield 73%; mp 252 °C; 1H NMR (C5D5N-d5): d 6.25
(d, 1H, J 7.9 Hz, Xyl-1-H), 5.47 (s, 1H), 4.42 (d, 1H, J 11.3 Hz),
4.30–4.18 (m, 3H), 3.90–3.80 (m, 1H), 2.47 (dd, 1H,
J
3.4,
3.5.14. 3-[2,4-Dinitrophenylhydrazone] oleanolic acid 28-O-[b-
13.9 Hz); 13C NMR (C5D5N-d5): d 217.7, 178.2, 145.7, 122.3, 97.8,
79.9, 75.26, 72.4, 69.3; ESI-MS m/z: 609.27 [M+Na]+. Anal. Calcd
for C35H54O7: C, 71.64; H, 9.28. Found: C, 71.39; H, 9.22.
D
-glucopyranosyl] ester (4b)
Orange solid; yield 70%; mp 210 °C; 1H NMR (C5D5N-d5): d
11.16 (s, 1H), 9.12 (s, 1H), 8.28 (d, 1H, J 9.0 Hz), 7.96–7.91 (m,
1H), 5.50 (d, 1H, J 8.2 Hz, Glc-1-H), 5.32 (s, 1H), 4.45 (d, 1H, J
11.9 Hz), 4.26 (d, 1H, J 12.1 Hz), 4.01–3.85 (m, 4H); 13C NMR
(C5D5N-d5): d 176.7, 167.1, 145.6, 143.8, 137.6, 130.2, 129.0,
123.7, 122.4, 116.6, 93.9; ESI-MS m/z: 798.18 [M+H]+. Anal. Calcd
for C42H60N4O11: C, 63.30; H, 7.59; N, 7.03. Found: C, 63.01; H,
7.52; N, 7.05.
3.5.8. 3-Keto oleanolic acid 28-O-[b-D-arabopyranosyl] ester
(2d)
White solid; yield 70%; mp 240 °C; 1H NMR (C5D5N-d5): d 5.41
(d, 1H, J 7.5 Hz, Ara-1-H), 5.32 (s, 1H), 4.34–4.29 (m, 2H), 4.23
(dd, 1H, J 7.6, 3.4 Hz), 3.85 (dd, 1H, J 10.5, 1.6 Hz), 2.86 (dd, 1H, J
5.1, 13.9 Hz); 13C NMR (C5D5N-d5): d 218.4, 176.3, 143.2, 122.3,
94.3, 73.1, 70.4, 67.8, 66.0; ESI-MS m/z: 587.19 [M+H]+. Anal. Calcd
for C35H54O7: C, 71.64; H, 9.28. Found: C, 71.85; H, 9.32.
3.5.15. 3-[2,4-Dinitrophenylhydrazone] oleanolic acid 28-O-[b-
D
-xylopyranosyl] ester (4c)
Orange solid; yield 71%; mp 171 °C; 1H NMR (C5D5N-d5): d
3.5.9. [3,2-b] Indole oleanolic acid 28-O-[b-
ester (3a)
D
-galactopyranosyl]
11.17 (s, 1H), 9.13 (s, 1H), 8.42 (d, 1H, J 9.0 Hz), 8.03–7.99 (m,
1H), 6.22 (d, 1H, J 7.9 Hz, Xyl-1-H), 5.45 (s, 1H), 4.32 (d, 1H, J
11.5 Hz), 4.15–4.01 (m, 3H), 3.81–3.71 (m, 1H); 13C NMR (C5D5N-
d5): d 178.2, 169.2, 147.3, 145.7, 139.2, 131.7, 124.3, 118.1, 97.3;
ESI-MS m/z: 767.11 [M+H]+. Anal. Calcd for C41H58N4O10: C,
64.21; H, 7.62; N, 7.31. Found: C, 63.96; H, 7.57; N, 7.35.
Orange solid; yield 67%; mp 229 °C; 1H NMR (CDCl3): d 7.74 (s,
1H), 7.43–7.03 (m, 4H), 5.49 (d, 1H, J 7.9 Hz, Gal-1-H), 5.42 (s, 1H),
3.98 (s, 1H), 3.78–3.72 (m, 2H), 3.69 (d, 1H, J 8.1 Hz), 3.63 (dd, 1H, J
9.3, 6.0 Hz), 3.56 (dd, 1H, J 9.2, 2.8 Hz); 13C NMR (CDCl3): d 177.7,
143.9, 141.9, 136.3, 128.8, 124.5, 121.2, 119.0, 118.1, 110.4,
106.7, 95.6; ESI-MS m/z: 712.35 [M+Na]+. Anal. Calcd for
C42H59NO7: C, 73.12; H, 8.62; N, 2.03. Found: C, 72.90; H, 8.59;
N, 2.05.
3.5.16. 3-[2,4-Dinitrophenylhydrazone] oleanolic acid 28-O-[b-
D
-arabopyranosyl] ester (4d)
Orange solid; yield 72%; mp 178 °C; 1H NMR (C5D5N-d5): d
11.16 (s, 1H), 9.12 (s, 1H), 8.29 (d, 1H, J 9.0 Hz), 7.87–7.84 (m,
1H), 6.25 (d, 1H, J 7.8 Hz, Ara-1-H), 5.32 (s, 1H), 4.59 (dd, 1H, J
7.4, 6.2 Hz), 4.34–4.29 (m, 2H), 4.23 (dd, 1H, J 7.6, 3.4 Hz), 3.85
(dd, 1H, J 10.5, 1.6 Hz); 13C NMR (C5D5N-d5): d 178.2, 169.2,
147.3, 145.7, 139.2, 131.7, 131.0, 122.5, 118.1, 97.8; ESI-MS (nega-
3.5.10. [3,2-b] Indole oleanolic acid 28-O-[b-D-glucopyranosyl]
ester (3b)
Orange solid; yield 68%; mp 212 °C; 1H NMR (CDCl3): d 7.73 (s,
1H), 7.42–7.02 (m, 4H), 5.49 (d, 1H, J 8.1 Hz, Glc-1-H), 5.40 (s, 1H),

604
J.-F. Li et al. / Carbohydrate Research 344 (2009) 599–605
tive) m/z: 767.12 [M+H]+. Anal. Calcd for C41H58N4O10: C, 64.21; H,
3.6.2. 12,13-Dihydrooleanolic acid 28-O-[b-D-glycuronic acid]
7.62; N, 7.31. Found: C, 62.99; H, 7.58; N, 7.34.
ester (5e)
White solid; yield 71.3%; mp 200–202 °C; 1H NMR (CDCl3): d
5.80 (d, 1H, J 7.4 Hz, GA-1-H), 3.49 (d, 1H, J 5.8 Hz), 3.22–3.16
(m, 3H), 3.10 (m, 1H); 13C NMR (C5D5N-d5): d 178.2, 175.3, 98.4;
MS (ESI) m/z: 633.23 [MꢂH]ꢂ. Anal. Calcd for C36H58O9: C, 68.11;
H, 9.21. Found: C, 68.31; H, 9.28.
3.5.17. 12,13-Dihydrooleanolic acid 28-O-[b-D-
galactopyranosyl] ester (5a)
White solid; yield 92.9%; mp 259–261 °C; 1H NMR (C5D5N-d5): d
6.30 (d, 1H, J 8.5 Hz, Gal-1-H), 4.61–4.55 (m, 2H), 4.42 (dd, 1H, J
13.0, 6.4 Hz), 4.28 (dd, 1H, J 13.0, 6.6 Hz), 4.16–4.10 (m, 2H),
3.33–3.27 (m, 1H); 13C NMR (C5D5N-d5): d 181.3, 98.2; MS (ESI)
m/z: 643.16 [M+Na]+. Anal. Calcd for C36H60O8: C, 69.64; H, 9.74.
Found: C, 69.93; H, 9.78.
3.7. Formation of OCLs
A
co-culture assay using mouse bone marrow cells and
osteoblast-like cells was used for the evaluation of the effects
of the compounds on OCLs formation.27 Briefly, osteoblast-like
cells were prepared from calvaria of 2-day-old ICR mice. Mouse
bone marrow cells were obtained from tibiae of 6-week-old
male ICR mouse. Osteoblast-like cells (1 ꢁ 104/well) and bone
marrow cells (1 ꢁ 105/well) were co-cultured for 6 days with
3.5.18. 12,13-Dihydrooleanolic acid 28-O-[b-D-glucopyranosyl]
ester (5b)
White solid; yield 90.2%; mp 249–251 °C; 1H NMR (MeOD): d
5.51 (d, 1H, J 8.1 Hz, Glc-1-H), 3.83 (d, 1H, J 12.1 Hz), 3.71 (dd,
1H, J 12.3, 2.3 Hz), 3.43–3.36 (m, 4H), 3.14 (dd, 1H, J 11.3,
5.5 Hz); 13C NMR (MeOD): d 185.2, 95.6; MS (ESI) m/z: 643.16
[M+Na]+. Anal. Calcd for C36H60O8: C, 69.64; H, 9.74. Found: C,
69.39; H, 9.69.
1a,
25(OH)2D3 (10ꢂ8 M) and each compound. The medium
was replaced every 2 days. After 6 days, adherent cells on the
well surface were fixed with 10% formalin-phosphate buffered
saline (PBS, pH 7.2) for 10 min and dehydrated with ethanol–
acetone (1:1, v/v) for 1 min. The cells were stained by tar-
trate-resistant acid phosphatase (TRAP) for 12 min at room tem-
perature and TRAP-positive cells containing three or more
nuclei were counted. The percentages of TRAP(+)-OCLs cultured
3.5.19. 12,13-Dihydrooleanolic acid 28-O-[b-D-xylopyranosyl]
ester (5c)
White solid; yield 91.8%; mp 149–152 °C; 1H NMR (C5D5N-d5): d
6.21 (d, 1H, J 7.5 Hz, Xyl-1-H), 4.24 (d, 1H, J 11.7 Hz), 4.10–3.90 (m,
3H), 3.72–3.69 (m, 1H), 3.30 (m, 1H); 13C NMR (C5D5N-d5): d 179.2,
97.8; MS (ESI) m/z: 613.22 [M+Na]+. Anal. Calcd for C35H58O7: C,
71.15; H, 9.89. Found: C, 71.46; H, 9.92.
with compounds were compared to that only with
25(OH)2D3 group.
1a,
All procedures involving animals were approved by the Jiangsu
Animal Care and Use Committee, China and followed the national
and institutional rules considering animal experiments. Mice were
housed in a climate-controlled room, 12 h light/dark photoperiod,
water and food ad libitium.
3.5.20. 12,13-Dihydrooleanolic acid 28-O-[b-D-arabopyranosyl]
ester (5d)
White solid; yield 82.5%; mp 238–240 °C; 1H NMR (C5D5N-d5): d
6.28 (d, 1H, J 6.1 Hz, Ara-1-H), 4.52 (dd, 1H, J 7.3, 6.5 Hz), 4.33–4.28
(m, 2H), 4.24 (dd, 1H, J 7.7, 3.1 Hz), 3.83 (dd, 1H, J 10.9, 1.3 Hz),
3.30 (t, 1H, J 7.8 Hz); 13C NMR (C5D5N-d5): d 179.2, 97.8; MS (ESI)
m/z: 613.22 [M+Na]+. Anal. Calcd for C35H58O7: C, 71.15; H, 9.89.
Found: C, 70.87; H, 9.84.
Acknowledgements
This work was supported by National Natural Science Fund for
Creative Research Groups of China (20821063), 973 Program
(2007CB714504) and Specialized Research Fund for the Doctoral
Program of Higher Education (20050284027).
3.6. General procedure for preparation of oleanolic acid
glycoside derivatives 1e and 5e
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5.73 (d, 1H, J 6.8 Hz, GA-1-H), 5.27 (s, 1H), 3.59 (d, 1H, J 6.2 Hz),
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