Synthesis of glucoconjugates of oleanolic acid as inhibitors of glycogen phosphorylase

Article abstract of DOI:10.1016/j.carres.2009.02.012

Synthesis and biological evaluation of glucoconjugates of oleanolic acid, linked by either a triazole moiety or an ester function, as novel inhibitors of glycogen phosphorylase have been described. Several triterpene-glycoside conjugates exhibited moderate-to-good inhibitory activity against rabbit muscle GPa. Compound 12 showed the best inhibition with an IC₅₀ value of 1.14 μM. Structure-activity relationship (SAR) analysis of these inhibitors is also discussed. Possible binding modes of compound 12 were explored by molecular docking simulations.

Full text of DOI:10.1016/j.carres.2009.02.012

Carbohydrate Research 344 (2009) 841–850
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of glucoconjugates of oleanolic acid as inhibitors
of glycogen phosphorylase
a,
Keguang Cheng a,b, Jun Liu c, Xiaofeng Liu d, Honglin Li d,e, Hongbin Sun b, , Juan Xie
*
*
a PPSM, ENS Cachan, CNRS, UniverSud, 61 av President Wilson, F-94230 CACHAN, France
b Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
c Jiangsu Center for Drug Screening, China Pharmaceutical University, 1 Shennonglu, Nanjing 210038, China
d Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
e School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and biological evaluation of glucoconjugates of oleanolic acid, linked by either a triazole moiety
or an ester function, as novel inhibitors of glycogen phosphorylase have been described. Several triter-
pene–glycoside conjugates exhibited moderate-to-good inhibitory activity against rabbit muscle GPa.
Compound 12 showed the best inhibition with an IC50 value of 1.14 lM. Structure–activity relationship
(SAR) analysis of these inhibitors is also discussed. Possible binding modes of compound 12 were
Received 31 December 2008
Received in revised form 7 February 2009
Accepted 11 February 2009
Available online 14 February 2009
explored by molecular docking simulations.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Glycogen phosphorylase
Oleanolic acid
Glucoconjugate
Inhibitor
Diabetes
Click chemistry
1. Introduction
toxic injury, anti-inflammation, anti-HIV, anti-tumor, anti-oxida-
tion, anti-hyperglycemia, and cardiovascular activities.7 Given the
Type 2 diabetes is associated with disorders in glucose metabo-
lism by the liver and periphery, and an ideal antidiabetic agent
should be capable of lowering blood glucose in both fed and fasted
states. Inhibition of glycogen phosphorylases (GP) has been re-
garded as a therapeutic strategy for blood glucose control in diabe-
tes, and various studies have shown the efficacy of GP inhibitors in
lowering blood glucose in animal models of diabetes and in clinical
trials.1 Several structural classes of GP inhibitors have been re-
ported, and at least six potential regulatory binding sites have been
identified in GP.2 A series of glucose analogues have developed as
GP inhibitors that bind at the catalytic site of the enzyme.1c,e,3,4
On the other hand, we have recently reported that oleanolic acid
(1, OA) and related pentacyclic triterpenes represent a new class
of GP inhibitors,5,6 and the results of X-ray crystallographic studies
disclose the molecular basis of the pentacyclic triterpenes binding
to GP at the allosteric site.5
biological importance and clinical significance of OA, lead modifica-
tion based on OA is highly desirable for the study of structure–activ-
ity relationships and drug development. Considering that OA might
bind to GP at the allosteric site,5 and glucose analogue inhibitors
bind to GP at the catalytic site, it would be interesting to design
glucoconjugates of OA in order to find more potent GP inhibitors.
Recent investigation on the click chemistry,8 the Cu(I)-catalyzed
Huisgen [2+3] dipolar cycloaddition reaction between an organic
azide and an alkyne, has attracted much attention in drug design.
This transformation is especially useful for drug discovery since it
is a reliable conjugating reaction. However, to the best of our knowl-
edge, the application of this approach to pentacyclic triterpenes has
not been explored. Herein, we report the synthesis of glucoconju-
gates of oleanolic acid by conjugating together OA and a glycoside
through a triazole or ester linkage. Biological evaluation of the
synthesized glucoconjugates as new GP inhibitors is also presented.
Moreover, the possible binding mode of the most active compound
12 was investigated by molecular docking simulation.
Oleanolic acid (1, OA), which has been in active clinical use as an
anti-hepatitis drug in China for over 20 years, possesses some attrac-
tive biological activities including protection of the liver against
2. Results and discussion
* Corresponding authors. Tel.: +33 1 47405586; fax: +33 1 47402454 (J.X.);
tel.: +86 25 85327950; fax: +86 25 83271335 (H.B.S.)
E-mail addresses: hbsun2000@yahoo.com (H. Sun), joanne.xie@ens-cachan.fr
(J. Xie).
The syntheses are summarized in Schemes 1–4. To realize click
chemistry between oleanolic acid and glycoside derivatives, azide
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.02.012

842
K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
30
19
29
21
20
H
12
H
18
11
22
25
13
26
14
O
17
a
CO2H
1
4
N3
28
2
3
9
6
16
O
10
8
15
27
7
HO
5
HO
2
1
24
23
Ref. 6
b
H
H
c
H
CO2Bn
CO2Bn
O
O
O
HO
4
5
HO
3
Scheme 1. Reagents and conditions: (a) K2CO3, Br(CH2)6N3, DMF, rt, 92%. (b) K2CO3, propargyl bromide, DMF, rt, 97%; and (c) NaH, propargyl bromide, THF, 0 °C to reflux, 88%.
OH
O
HO2C
BnO
BnO
(88%) and 25 (94%), respectively. Treatment of 4 with 2-chloroethyl
chloroformate led to the carbonate 27 (92%) that was then treated
with 7 to afford glucoconjugate 28 (91%). Hydrogenolysis of 22, 25,
and 28 with Pd/C (10%) in THF–MeOH at room temperature gave
the target compounds 23 (85%), 26 (86%), and 29 (81%), respectively.
The above-synthesized derivatives were evaluated in an
enzyme inhibition assay against rabbit muscle glycogen phosphor-
ylase a (RMGPa) (Table 1), an enzyme that shares considerable
sequence similarity with human liver GPa. As described previ-
ously,13 the activity of rabbit muscle GPa was measured through
detecting the release of phosphate from glucose-1-phosphate in
the direction of glycogen synthesis.
It is interesting to notice that glucosides modified at 6-position
(compounds 8 and 9) also exhibit GP inhibition activity, since only
anomerically modified glucose derivatives have been reported as
GP inhibitors.14 Compared to oleanolic acid 1, modification at
either 3- or 28-position with an alkyl group diminished the inhib-
itory activity (1 vs 3, 5, and 21). Conjugation of oleanolic acid with
glucosides exhibited moderate to good inhibitory activity against
rabbit muscle GPa. SAR analysis shows that the distance between
the triterpene moiety and sugar moiety is important: a shorter lin-
ker led to a better GPa inhibitory activity (e.g., 12 vs 18, 20, 23, and
26). The different potency between compounds 17 vs 18, 19 vs 20,
22 vs 23, and 25 vs 26 indicated that with a long carbon-chain lin-
ker, benzyl-protected sugar derivatives displayed stronger affinity
toward GPa. With a shorter linker, hydrophilic conjugates showed
better inhibitory activity (11 vs 12, 14 vs 15, and 28 vs 29). Pres-
ence of an ester function at the C-6 position of the sugar is also
beneficial (17 vs 19, 8 vs 9). The 3-hydroxyl and 28-carboxyl
groups of triterpene moiety seem to contribute to the GPa enzyme
inhibitory activity (12 vs 14 and 15), although it is less predictable.
Nevertheless, modification at the 28-carboxyl group led to more
potent inhibitors (12 and 17) than those modified at the 3-position
(15 and 29). The presence of an azido group was not favorable to
inhibition (e.g., 2 and 10). The acetyl group in the sugar moiety
could decrease the potency (15 vs 16). Within this series of com-
pounds, triterpene–glucoside conjugate 12 is the most potent GP
a
BnO
BnO
O
BnO
BnO
7
8
OMe
OMe
6
O
O
O
b
BnO
BnO
BnO
OMe
c
O
O
6
BnO
9
BnO
BnO
OMe
Scheme 2. Reagents and conditions: (a) TEMPO, KBr, NaOCl, NaHCO3, acetone, rt,
92%; (b) K2CO3, propargyl bromide, DMF, rt, 97%; and (c) NaH, propargyl bromide,
THF, 0 °C to rt, 86%.
or alkyne functions were first introduced into the 3- and 28-posi-
tion of oleanolic acid 1 (Scheme 1). Esterification of 1 with 1-azi-
do-6-bromohexane or propargyl bromide gave, respectively,
azide 2 (92%) and alkyne 3 (97%). Protection of oleanolic acid with
benzyl chloride, followed by an etherification with propargyl
bromide, afforded the alkyne 5 (88%).
Two alkyne-functionalized glycoside derivatives 8 and 9 have
been prepared as shown in Scheme 2. Oxidation of the primary
hydroxyl group of methyl glucoside 6 by TEMPO–KBr–NaOCl–NaH-
CO3 in acetone afforded carboxylic acid 7 in 92% yield.9 Esterification
of 7 with propargyl bromide afforded alkyne 8 (97%). Etherification
of 6 with propargyl bromide afforded alkyne 910 (86%).
Click chemistry was handled as shown in Scheme 3. The corre-
sponding azide and alkyne were dissolved in CH2Cl2–H2O, followed
by the addition of catalytic sodium ascorbate and CuSO4ꢀ5H2O.
Reaction of the known azide 1011 with alkyne 3 afforded triazole
11 in 93% yield. Similarly, the oleanolate–glycoside conjugates
13, 17, and 19 have been prepared in more than 83% yield. Cleav-
age of acetyl groups in 11 and 13 with MeONa–MeOH gave tria-
zoles 12 (43%) and 14 (78%), respectively. The poor yield of 12
can be explained by a partial removal of the acetyl groups. Hydrog-
enolysis of 13, 14, 17, and 19 over Pd/C (10%) in THF–MeOH at
room temperature gave the corresponding triterpene derivatives
15 (81%), 16 (85%), 18 (76%), and 20 (38%), respectively.
inhibitor with an IC50 value of 1.14
than that of the parent oleanolic acid (IC50 = 14
l
M. This value is much lower
l
M).5,6
To explore the corresponding binding site and mode of the glu-
coconjugates of oleanolic acid for GP inhibition, the most potent
compound 12 was selected for subsequent molecular docking sim-
ulation. Both the dimer interface site and the catalytic site are bur-
ied deeply in their respective binding cavities and are extremely
spatially constrained, indicating a low compatibility between the
Ester-linked oleanolate–glycoside conjugates have also been
prepared (Scheme 4). Esterification of 1 with 1,6-dibromohexane
afforded bromide 21 (52%). Further esterification of 21 with sugar
carboxylic acid 7 or 2412 afforded protected glucoconjugates 22

K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
843
N
N
N3
O
H
N
a
AcO
AcO
O
H
+ 3
OMe
O
RO
RO
O
AcO
10
OR
HO
OMe
11 R = Ac
12 R = H
b
CO2R2
N
N
N
O
a
10 + 5
R1O
R1O
O
13 R1= CH3CO R2 = Bn
b
R1= H
R2= Bn
R2= H
14
R1O
c
c
15 R1= H
OMe
16 R1= CH3CO R2= H
H
a
2 + 8
O
N
N
O
O
17 R= Bn
18
O
RO
RO
N
O
HO
c
R= H
RO
OMe
H
a
2 + 9
O
N
N
O
O
N
O
HO
RO
RO
19 R= Bn
20
c
R= H
RO
OMe
Scheme 3. Reagents and conditions: (a) CuSO4, sodium ascorbate, CH2Cl2–H2O, rt, 83–93%; (b) MeONa, MeOH, rt, for 12: 43%; for 14: 78%; and (c) H2, Pd/C (10%), THF–MeOH,
rt, 38–85%.
highly extended and flexible glucoconjugates of oleanolic acid and
these two binding sites. This assumption was proved by molecular
docking simulations during which no rational binding position of
compound 12 was observed for either site. Compound 12 was pre-
dicted to bind at the T-state allosteric site exclusively as shown in
Figure 1a due to the steric and volume incompatibility with the
catalytic site, and no reasonable binding attitudes against this site
were observed from the docking results (data not shown). The top-
ranked binding position of 12 at the T-state allosteric site of GP was
determined as a preferred docking mode with the calculated Glide
GScore of ꢁ10.086 (as shown in Fig. 1), which is superior to the
docking result against the inhibitor site with GScore value of
ꢁ7.31. The predicted binding position of the oleanolic acid moiety
of compound 12 occupied the same location as the T-state alloste-
ric site of asiatic acid5 in the crystal structure (see Fig. 1c). How-
ever, the newly attached sugar moiety shielded the carboxyl
group of oleanolic acid from forming the salt bridge with Arg310
(A), and consequently reversed the orientation of the oleanolic acid
as well as propelled the sugar moiety to extend more deeply into
the alloteric dimer interface. As a result, a new hydrogen-bonding
network formed between the sugar hydroxyls and the carboxyl of
Asp227 (A) plus the guanidino of Arg193 (A), respectively, enhanc-
ing their contribution to the stability of the complex (Fig. 1b). This
preferred binding pose was stabilized by extra hydrogen-bonding
networks involving the 3-hydroxyl of the triterpene moiety and
the guandino groups of Arg242 (A) and Arg309 (A) plus the
carboxyl of Asp306 (A). These hydrogen-bonding networks were
critical for maintaining the good activity and highlighted the con-
tribution of unshielded hydroxyls in the sugar moiety (12 vs 11).
Other interactions contributing to the high affinity of 12 included
hydrophobic contacts between pentacyclic triterpenes and Tyr75
(A), Val45 (B), and Phe196 (A).
In summary, a series of glucoconjugates of oleanolic acid have
been synthesized using click chemistry and esterification methods.
The results of GP inhibition assay and SAR analysis indicate a clear
preference for hydrophobic groups with a long chain linker and
hydrophilic groups with a short chain linker. Further research
and drug development on pentacyclic triterpenes as promising
modulators of glycogen metabolism are ongoing in our laborato-
ries, and the results will be reported in due course.
3. Experimental
3.1. Chemistry
All commercially available solvents and reagents were used
without further purification. Melting points were measured on a
RY-1 melting point apparatus. Column chromatography was
carried out on E. Merck Silica Gel 60 (230–400 mesh) or on silica
gel (200–300 mesh, Qindao Ocean Chemical Company, China). IR
spectra were recorded on a Shimadzu FTIR-8400S spectrometer.
1H and 13C NMR spectra were measured on Bruker AV-300 or
AGH-250 spectrometers. Chemical shifts are reported as values
from an internal tetramethylsilane standard. Mass spectral data

844
K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
H
H
a
O
O
a
O
O
Br
1
+
Br(CH2)6Br
O
7
O
O
RO
RO
HO
HO
21
RO
22
R= Bn
OMe
b
23 R= H
OR
O
RO
RO
OBn
O
a
21
RO
BnO
BnO
O
H
25
R= Bn
b
O
O
BnO
24
26 R= H
CO2H
O
HO
H
CO2R
O
H
O
O
O
CO2Bn
a
7
c
O
RO
RO
4
O
O
Cl
28 R= Bn
29
O
O
b
27
R= H
RO
OMe
Scheme 4. Reagents and conditions: (a) K2CO3, DMF, rt or 40 °C, 52–94%; (b) H2, Pd/C (10%), THF–MeOH, rt, 81–86%; and (c) ClCH2CH2OCOCl, K2CO3, DMAP, CH2Cl2, 35 °C,
92%.
Table 1
IC50 values (
3.1.2. General procedure for the O-alkylation of oleanolic acid
or glucoside
l
M) for RMGPa inhibition assay results
a
a
A solution of alcohol (0.22 mmol) in anhyd THF (2 mL) was
added dropwise to a suspension of NaH (60%, 0.56 mmol) in anhyd
THF (2 mL) at 0 °C. After 30 min of further stirring at 0 °C, alkyl bro-
mide (0.48 mmol) was added dropwise, and the mixture was stir-
red at rt for 12 h. MeOH (0.1 mL) was added at 0 °C. After stirring
for 15 min, the mixture was concentrated under reduced pressure.
The residue was diluted in EtOAc, washed successively with water
and brine, dried (MgSO4), filtered, and concentrated. The residue
was purified by column chromatography.
Compd
GPa IC50
Compd
GPa IC50
1b
2
14
19
136.5
na
50.4
na
41.6
na
26.5
466.9
na
66.2
na
574.7
na
62.6
83.1
nac
405
461
32.7
na
20
22
23
25
3
4b
21
6
26
7
8
9
10
24
116
12
17
18
na
5
37.2
289.3
na
13
14
15
na
16
3.1.3. General procedure for the click reaction
na
27
To a solution of alkyne (0.51 mmol) and azide (0.51 mmol) in
CH2Cl2 (2 mL) and H2O (2 mL) were added CuSO4ꢀ5H2O (0.2 mmol)
and sodium ascorbate (0.4 mmol). The resulting solution was stirred
at rt for 8 h. The reaction mixture was extracted with CH2Cl2 (3ꢂ).
The combined organic layer was dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromatography.
1.14
11.6
na
28
29
Caffeine
a
Values are means of three experiments.
See Ref.6.
na = no activity.
b
c
were obtained on an Agilent 1100 LC/DAD/MSD spectrometer or
Q-Tof Micro MS/MS spectrometer. Elementary analysis was mea-
sured on a Vario EL III instrument (Elementar, Germany). Optical
rotations were measured using a Perkin–Elmer 141 polarimeter
or Jasco P-2000 polarimeter.
3.1.4. General procedure for the deacetylation
To a solution or suspension of the acetate (0.2 mmol) in dry
MeOH (10 mL) was added a solution of MeONa (0.2 M in MeOH,
40 mL) with stirring at rt for 24 h. The mixture was concentrated
in vacuo, and the residue was taken up in EtOAc (50 mL), washed
successively with 1 N HCl (3 ꢂ 25 mL), water (3 ꢂ 15 mL) and brine
(3 ꢂ 15 mL), dried (Na2SO4), filtered, and concentrated. The residue
was purified by column chromatography.
3.1.1. General procedure for the esterification of oleanolic acid
or uronic acid
To a solution of the carboxylic acid (3.4 mmol) in DMF (8 mL)
were added alkyl bromide (3.8 mmol) and K2CO3 (6.8 mmol). The
reaction mixture was stirred at rt for 6–18 h, then concentrated.
The residue was diluted with EtOAc (50 mL), and the extract was
washed successively with 1 N HCl, water, satd NaHCO3, water
and brine, dried (MgSO4), filtered, and concentrated. The residue
was purified by column chromatography.
3.1.5. General procedure for the debenzylation
To a solution of benzyl ether or ester (0.117 mol) in THF
(1.5 mL) and MeOH (1.5 mL) was added 10% Pd/C (0.09 g), with
stirring under hydrogen atmosphere at rt for 12 h. The catalyst
was filtered off, and the filtrate was concentrated to give a crude
product that was purified by flash column chromatography.

K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
845
Figure 1. Stereoview of the binding mode of compound 12 at the dimer allosteric site of RMGP (a) and enlarged view of binding site (b) with residues around the docked
position within 4.0 Å (labeled by capital letter A or B to indicate which chain they belong to). The comparison of docking modes of 12 and oleanolic acid, and asiatic acid
crystallized conformation of 2QN1 is shown in c. The RMGP structure is presented in the cartoon with green and cyan indicating each monomer, respectively. The residues of
the binding site are shown in line format, and compound 12, oleanolic acid and asiatic acid are shown as stick structures. The coloring rules for 12 are as follows: carbon
atoms, magenta; oxygen atoms, red; nitrogen atoms, blue; respectively. The carbon atoms of oleanolic acid and asiatic acid are colored in cyan and green, respectively. All
hydrogen atoms are omitted for clarity. The hydrogen bonds in b are represented by red-dashed lines.
3.1.6. 6-Azidohexyl 3b-hydroxyolean-12-en-28-oate (2)
J 2.4 Hz, CH), 2.91 (dd, 1H, J18,19a 4.1, J18,19b 13.9 Hz, H-18), 3.01
(dd, 1H, J2a,3 4.3, J2b,3 11.7 Hz, H-3), 4.14 (dd, 1H, J 2.4, 16.0 Hz,
CH–O), 4.23 (dd, 1H, J 2.4, 15.9 Hz, CH-O), 5.06 (d, 1H, J 12.5 Hz,
CHPh), 5.08 (d, 1H, J 12.5 Hz, CHPh), 5.29 (t, 1H, J 3.5 Hz, H-12),
7.31–7.35 (m, 5H, Ph-H). 13C NMR (75 MHz, CDCl3): d 15.3, 16.4,
17.0, 18.4, 22.6, 23.2, 23.5, 23.7, 25.9, 27.8, 28.2, 30.7, 32.5, 32.9,
33.1, 34.0, 37.1, 38.5, 38.6, 39.5, 41.6, 41.9, 46.1, 46.9, 47.8, 56.0,
56.5, 66.0, 73.3, 73.4, 81.1, 86.1, 122.7, 127.9, 128.0, 128.4, 136.6,
143.8, 177.4. ESIMS m/z: [M+Na]+ calcd for C40H56NaO3: 607.4;
found: 607.3.
This compound was prepared from 1 (1.55 g, 3.4 mmol) and 1-
azido-6-bromohexane (0.78 g, 3.8 mmol) according to the general
procedure 3.1.1. The residue was purified by column chromatogra-
phy (1:6 EtOAc–petroleum ether). Yield: 1.82 g, 92%; white solid,
mp 73–75 °C; Rf 0.5 (2:7 EtOAc–petroleum ether). [a] +53.9 (c
D
0.55, CH2Cl2). IR (KBr, cmꢁ1): 3514, 3402, 2942, 2863, 2095,
1721, 770, 759. 1H NMR (300 MHz, CDCl3): d 0.73, 0.78, 0.90,
0.91, 0.92, 0.99, 1.13 (7s, each 3H, 7 ꢂ CH3), 0.73–1.97 (m, 30H),
2.88 (dd, 1H, J18,19a 3.9, J18,19b 13.5 Hz, H-18), 3.20 (t, 1H, J 5.7 Hz,
H-3), 3.27 (t, 2H, J 6.8 Hz, CH2N3), 4.01 (t, 2H, J 6.4 Hz, CO2CH2),
5.27 (br s, 1H, H-12). 13C NMR (75 MHz, CDCl3): d 15.3, 15.6,
17.1, 18.3, 23.1, 23.4, 23.6, 25.7, 25.9, 26.4, 27.2, 27.7, 28.1, 28.5,
28.8, 30.7, 32.5, 32.8, 33.1, 33.9, 37.1, 38.5, 38.8, 39.4, 41.4, 41.7,
45.9, 46.7, 47.6, 51.4, 55.3, 64.0, 79.0, 122.4, 143.9, 177.7. ESIMS
m/z: [M+Na]+ calcd for C36H59N3NaO3: 604.4; found: 604.5.
3.1.9. Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranosiduronic acid
(7)
KBr (0.028 g, 0.24 mmol), NaHCO3 (5%, 10 mL), 2,2,6,6-tetra-
methyl-1-piperidinyloxy (TEMPO, 0.374 g, 2.4 mmol) and NaOCl
(5%, 1.8 mL) were added at 0 °C to a solution of 6 (1.0 g, 2.2 mmol)
in acetone (15 mL). After the mixture had been stirred for 1 h at
0 °C, additional NaOCl (5%, 4.9 mL) was added, and the mixture
was stirred further at rt for 20 h. The solution was concentrated
and diluted in EtOAc (30 mL), washed with 10% HCl and brine,
dried (MgSO4), filtered, and concentrated to give a crude yellow
oil. Purification by flash column chromatography (1:1 EtOAc–
petroleum ether) gave 7 (1.0 g, 92%) as a yellow oil. Rf 0.7 (7:1
CH2Cl2–MeOH). IR (KBr, cmꢁ1): 3063, 3031, 2932, 2874, 1748,
1731, 1089, 1071, 1050, 1029, 770, 737, 698. 1H NMR (300 MHz,
CDCl3): d 3.48 (s, 3H, OCH3), 3.62 (dd, 1H, J1,2 3.5, J2,3 9.7 Hz, H-
2), 3.74 (dd, 1H, J 8.8, 9.9 Hz, H-4), 4.06 (t, 1H, J 9.4 Hz, H-3),
4.28 (d, 1H, J 9.9 Hz, H-5), 4.66–5.03 (m, 7H, H-1, 3 ꢂ PhCH2),
7.27–7.40 (m, 15H, Ph-H). 13C NMR (75 MHz, CDCl3): d 55.8, 69.4,
73.6, 75.3, 75.9, 79.1, 79.3, 81.4, 98.6, 127.7, 127.93, 127.96,
128.1, 128.11, 128.18, 128.4, 128.42, 128.5, 137.4, 137.8, 138.4,
173.0. ESIMS m/z: [M+Na]+ calcd for C28H30NaO7: 501.2; found:
501.1.
3.1.7. Propargyl 3b-hydroxyolean-12-en-28-oate (3)
This compound was prepared from 1 (1 g, 2.2 mmol) and prop-
argyl bromide (0.27 mL, 2.4 mmol) according to the general proce-
dure 3.1.1. The residue was purified by column chromatography
(1:6 EtOAc–petroleum ether). Yield: 1.05 g, 97%; white solid, mp
121–122 °C; Rf 0.33 (1:4 EtOAc–petroleum ether). [
a]
+67.9 (c
D
0.50, CH2Cl2). IR (KBr, cmꢁ1): 3308, 2945, 2866, 1731, 1157,
1032, 739. 1H NMR (300 MHz, CDCl3): d 0.74, 0.77, 0.92, 0.98,
1.13 (5s, each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3), 0.71–2.04 (m,
22H), 2.41 (t, 1H, J 2.6 Hz, CH), 2.87 (dd, 1H, J18,19a 4.1, J18,19b
9.5 Hz, H-18), 3.21 (dd, 1H, J2a,3 5.1, J2b,3 10.7 Hz, H-3), 4.56 (dd,
1H, J 2.6, 15.4 Hz, CO2CH2), 4.68 (dd, 1H, J 2.6, 15.4 Hz, CO2CH2),
5.30 (t, 1H, J 3.5 Hz, H-12). 13C NMR (75 MHz, CDCl3): d 15.3,
15.6, 17.1, 18.3, 23.0, 23.4, 23.6, 25.8, 27.2, 27.7, 28.1, 30.7, 32.2,
32.8, 33.1, 33.8, 37.0, 38.5, 38.8, 39.4, 41.3, 41.7, 45.9, 46.8, 47.6,
51.6, 55.2, 74.4, 78.1, 79.0, 122.63, 143.4, 176.8. ESIMS m/z:
[M+Na]+ calcd for C33H50NaO3: 517.4; found: 517.3.
3.1.10. Propargyl (methyl 2,3,4-tri-O-benzyl-a-D-
glucopyranosid)uronate (8)
3.1.8. Benzyl 3-O-propargyl-3b-hydroxyolean-12-en-28-oate (5)
This compound was prepared from 4 (1.0 g, 1.8 mmol) and
propargyl bromide (0.8 mL, 7.2 mmol) according to the general
procedure 3.1.2. The residue was purified by column chromatogra-
phy (1:40 EtOAc–petroleum ether). Yield: 0.94 g, 88%; white solid,
This compound was prepared from 7 (0.28 g, 0.59 mmol) and
propargyl bromide (0.072 mL, 0.64 mmol) according to the general
procedure 3.1.1. The residue was purified by column chromatogra-
phy (1:7 EtOAc –petroleum ether). Yield: 0.29 g, 97%, colorless oil;
Rf 0.48 (1:4 EtOAc–petroleum ether). [a]D +34.1 (c 0.71, CH2Cl2). IR
(KBr, cmꢁ1): 3281, 2917, 1752, 1609, 1509, 1452, 1359, 1180, 1091,
1071, 1044, 697. 1H NMR (250 MHz, CDCl3): d 2.37 (t, 1H, J 2.3 Hz,
CH), 3.33 (s, 3H, OCH3), 3.50 (dd, 1H, J1,2 3.6, J2,3 9.6 Hz, H-2), 3.67
(t, 1H, J 9.5 Hz, H-4), 3.92 (t, 1H, J 9.4 Hz, H-3), 4.18 (d, 1H, J
mp 153–155 °C; Rf 0.51 (1:6 EtOAc–petroleum ether). [a]D +71.0 (c
0.48, CH2Cl2). IR (KBr, cmꢁ1): 3308, 2945, 2865, 1725, 1076, 758,
696, 666. 1H NMR (300 MHz, CDCl3): d 0.61, 0.78, 0.89, 0.90, 0.92,
0.99, 1.13 (7s, each 3H, 7 ꢂ CH3), 0.61–1.85 (m, 22H), 2.36 (t, 1H,

846
K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
10.0 Hz, H-5), 4.53 (d, 1H, J1,2 3.3 Hz, H-1), 4.54–4.91 (m, 8H,
CO2CH2 and 3 ꢂ PhCH2), 7.16–7.28 (m, 15H, Ph-H). 13C NMR (75
MHz, CDCl3): d 52.8, 55.7, 70.1, 73.5, 75.1, 75.5, 75.8, 76.8, 79.2,
79.4, 81.3, 98.7, 127.6, 127.7, 127.8, 127.92, 127.97, 128.1,
128.25, 128.3, 128.4, 137.8, 137.9, 138.5, 168.8. ESIMS m/z:
[M+NH4]+ calcd for C31H36NO7: 534.2; found: 534.3; [M+Na]+ calcd
for C31H32NaO7: 539.2; found: 539.2.
1H), 4.68–4.77 (m, 2H), 5.14 (d, 1H, Jgem 12.7 Hz, CO2CH), 5.18 (d,
1H, Jgem 12.7 Hz, CO2CH), 5.27 (s, 1H, H-12), 7.74 (s, 1H, CH@N).
13C NMR (75 MHz, CDCl3): d 15.4, 15.6, 16.9, 18.4, 23.1, 23.4,
23.7, 25.8, 27.2, 27.7, 28.2, 29.7, 30.7, 32.4, 32.8, 33.0, 33.9, 37.1,
38.6, 38.8, 39.4, 41.4, 41.8, 45.9, 46.8, 47.7, 55.3, 55.7, 57.6, 77.2,
79.0, 122.6, 143.6. ESIMS m/z: [M+K]+ calcd for C40H63KN3O8:
752.4; found: 752.4. [M+H]+ calcd for C40H64N3O8: 714.5; found:
714.5.
3.1.11. Methyl 2,3,4-tri-O-benzyl-6-O-propargyl-a-D-
glucopyranoside (9)
3.1.14. Benzyl 3-O-{[(methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-glu-
This compound was prepared from 6 (0.1 g, 0.22 mmol) and
propargyl bromide (0.052 mL, 0.48 mmol) according to the general
procedure 3.1.2. The residue was purified by column chromatogra-
phy (1:6 EtOAc–petroleum ether). Yield: 93 mg, 86%, colorless oil;
Rf 0.4 (1:2 EtOAc–petroleum ether,). IR (KBr, cmꢁ1): 3292, 2917,
2872, 1728, 1510, 1453, 1359, 1159, 1134, 1094, 1070, 1049,
1030, 740, 698. 1H NMR (300 MHz MHz, CDCl3): d 2.43 (t, 1H, J
2.4 Hz, CH), 3.44 (s, 3H, OCH3), 3.62 (dd, 1H, J1,2 3.7, J2,3 9.6 Hz,
H-2), 3.65 (t, 1H, J 10.0 Hz, H-4), 3.70 (dd, 1H, J5,6 2.0, Jgem
copyranosid-6-yl)-1H-1,2,3-triazol-4-yl]methyl} 3b-hydroxyolean-
12-en-28-oate (13)
This compound was prepared from 5 (0.4 g, 0.68 mmol) and 10
(0.239 g, 0.69 mmol) according to the general procedure 3.1.3. The
residue was purified by column chromatography (1:1 EtOAc–
petroleum ether). Yield: 0.53 g, 83%, white solid, mp 107–108 °C.
Rf 0.18 (1:2 EtOAc–petroleum ether). [a] +102.5 (c 0.11, CHCl3).
D
IR (KBr, cmꢁ1): 2947, 2866, 1748, 1368, 1247, 1221, 1046, 760.
1H NMR (250 MHz, CDCl3): d 0.42, 0.57, 0.72, 0.74, 0.93 (5s, each
3H, 5 ꢂ CH3), 0.70 (s, 6H, 2 ꢂ CH3), 0.42–1.66 (m, 22H), 1.83,
1.89, 1.92 (3s, each 3H, 3 ꢂ COCH3), 2.76–2.81 (m, 2H, H-3, H-
18), 2.94 (s, 3H, OCH3), 3.96–4.04 (m, 1H, H-5Glc), 4.17 (dd, 1H,
J5,6 8.6, Jgem 14.1 Hz, H-6Glc), 4.35–4.44 (2H, m, H-60Glc, OCH),
4.57–4.73 (m, 4H, H-1Glc, H-2Glc, H-4Glc, OCH), 4.88 (d, 1H, Jgem
12.5 Hz, PhCH), 4.90 (d, 1H, Jgem 12.5 Hz, PhCH), 5.12 (t, 1H, J
4.0 Hz, H-12), 5.30 (t, 1H, J 9.6 Hz, H-3Glc), 7.13–7.17 (m, 5H, Ph-
H), 7.46 (s, 1H, CH@N). 13C NMR (75 MHz, CDCl3): d 15.3, 16.4,
16.8, 18.2, 20.6, 20.61, 20.63, 22.64, 23.0, 23.4, 23.6, 25.8, 27.6,
28.1, 30.7, 32.3, 32.7, 33.1, 33.8, 36.9, 38.2, 38.6, 39.3, 41.4, 41.6,
45.8, 46.7, 47.6, 50.7, 55.4, 55.6, 63.0, 65.9, 67.9, 69.7, 70.1, 70.7,
86.6, 96.6, 122.5, 123.7, 127.8, 127.9, 128.3, 136.4, 143.6, 146.6,
169.8, 169.82, 170.1, 177.4. ESIMS m/z: [M+Na]+ calcd for
0
10.0 Hz, H-6), 3.80–3.85 (m, 1H, H-5), 3.91 (dd, 1H, J5,6 3.5, Jgem
10.1 Hz, H-60), 4.05 (t, 1H, J 9.2 Hz, H-3), 4.18 (dd, 1H, J 2.4,
16.0 Hz, OCHC„C), 4.27 (dd, 1H, J 2.6, 15.8 Hz, OCHC„C), 4.67
(d, 1H, J1,2 3.7 Hz, H-1), 4.70–5.07 (m, 6H, 3 ꢂ PhCH2), 7.30–7.47
(m, 15H, Ph-H). 13C NMR (75 MHz, CDCl3): d 55.1, 58.5, 68.0,
69.8, 73.3, 74.8, 74.9, 75.6, 79.4, 79.7, 82.0, 98.2, 127.5, 127.6,
127.82, 127.83, 128.1, 128.3, 128.4, 138.1, 138.4, 138.8. ESIMS m/
z: [M+Na]+ calcd for C31H34NaO6: 525.2; found: 525.2.
3.1.12. [1-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-
glucopyranosid-6-yl)-1H-1,2,3-triazol-4-yl]methyl 3b-
hydroxyolean-12-en-28-oate (11)
This compound was prepared from 3 (0.25 g, 0.51 mmol) and 10
(0.18 g, 0.51 mmol) according to the general procedure 3.1.3. The
residue was purified by column chromatography (3:2 EtOAc–
petroleum ether). Yield: 0.39 g, 93%, white solid, mp 138–139 °C.
C53H75N3NaO11
:
952.5; found: 952.5. [M+H]+ calcd for
C53H76N3O11: 930.5; found: 930.5. Anal. Calcd for C53H75N3O11: C,
68.44; H, 8.13; N, 4.52. Found: C, 68.16; H, 8.15; N, 4.40.
Rf 0.13 (1:2 EtOAc–petroleum ether). [a] +95.2 (c 0.11, CHCl3).
D
IR (KBr, cmꢁ1): 2946, 2866, 1750, 1246, 1221, 1046, 769. 1H NMR
(250 MHz, CDCl3): d 0.54, 0.71, 0.83, 0.91, 1.04 (5s, each 3H,
5 ꢂ CH3), 0.81 (s, 6H, 2 ꢂ CH3), 0.54–1.82 (m, 22H), 1.94, 2.00,
2.03 (3s, each 3H, 3 ꢂ CH3CO), 2.75 (m, 1H, H-18), 3.03 (s, 3H,
OCH3), 3.11–3.17 (m, 1H, H-3), 4.03–4.14 (m, 1H, H-5Glc), 4.20–
3.1.15. Benzyl 3-O-{[1-(methyl 6-deoxy-a-D-glucopyranosid-6-yl)-
1H-1,2,3-triazol-4-yl]methyl} 3b-hydroxyolean-12-en-28-oate (14)
This compound was prepared from 13 (0.2 g, 0.22 mmol)
according to the general procedure 3.1.4. The residue was purified
by column chromatography (EtOAc). Yield: 0.134 g, 78%, white so-
4.29 (m, 1H, H-6Glc), 4.51 (dd, 1H, J5,6 1.9, Jgem 14.1 Hz, H-60Glc),
lid, mp 116–118 °C, Rf 0.43 (7:1 CH2Cl2–MeOH). [a]D +85.5 (c 0.11,
0
4.74–4.80 (m, 2H, H-2Glc,4Glc), 4.83 (d, J1,2 3.5 Hz, H-1Glc), 5.10 (s,
2H, CO2CH2), 5.21 (br s, 1H, H-12), 5.41 (t, 1H, J 9.6 Hz, H-3Glc),
7.64 (s, 1H, CH@N). 13C NMR (75 MHz, CDCl3): d 15.3, 15.5, 16.8,
18.3, 20.5, 20.6, 22.9, 23.3, 23.5, 25.8, 27.2, 27.6, 28.1, 30.6, 32.3,
32.7, 33.0, 33.8, 37.0, 38.4, 38.7, 39.3, 41.3, 41.7, 45.8, 46.6, 47.5,
50.7, 55.2, 55.4, 57.4, 67.8, 69.6, 70.0, 70.7, 78.9, 96.6, 122.5,
125.4, 143.2, 143.5, 169.78, 169.81, 170.0, 177.5. ESIMS m/z:
[M+Na]+ calcd for C46H69N3NaO11:862.5; found: 862.3. [M+H]+
CHCl3). IR (KBr, cmꢁ1): 3419, 2943, 2865, 1725, 1156, 1097, 1052,
1011, 751, 696. 1H NMR (300 MHz, CDCl3 + D2O): d 0.60, 0.74, 0.87,
0.91, 1.11 (5s, each 3H, 5 ꢂ CH3), 0.89 (s, 6H, 2 ꢂ CH3), 0.60–1.97
(m, 22H), 2.88–2.96 (m, 2H, H-18, H-3), 3.14 (s, 3H, OCH3), 3.18
(m, 1H, H-2Glc), 3.43 (dd, 1H, J 3.0, 9.6 Hz, H-5Glc), 3.72 (t, 1H, J
9.2 Hz, H-4Glc), 3.86 (t, 1H, J 7.3 Hz, H-3Glc), 4.46–4.57 (m, 2H, H-
6
Glc), 4.66 (d, 1H, J1,2 3.2 Hz, H-1Glc), 4.76 (d, 2H, J 12.9 Hz, OCH2),
5.05 (d, 1H, Jgem 12.4 Hz, PhCH), 5.07 (d, 1H, Jgem 12.4 Hz, PhCH),
5.29 (br s, 1H, H-12), 7.28–7.33 (m, 5H, Ph-H), 7.68 (s, 1H, CH@N).
13C NMR (75 MHz, CDCl3): d 15.3, 16.5, 17.0, 18.3, 22.8, 23.1, 23.5,
23.7, 25.9, 27.7, 28.2, 30.7, 32.4, 32.8, 33.1, 33.9, 37.0, 38.4, 38.8,
39.4, 41.5, 41.8, 46.0, 46.8, 47.7, 51.0, 55.5, 55.8, 63.2, 65.9, 70.2,
71.0, 72.1, 74.1, 87.0, 99.7, 122.6, 124.2, 127.9, 128.0, 128.4,
136.5, 143.7, 146.3, 177.4. ESIMS m/z: [M+Na]+ calcd for C47H69Na-
N3O8: 826.5; found: 826.5. [M+H]+ calcd for C47H70N3O8: 804.5;
found: 804.5. HRESIMS: [M+H]+ calcd for C47H70N3O8: 804.5157;
Found: 804.5183.
calcd for C46H70N3O11: 840.5; found: 840.4. Anal. Calcd for
C46H69N3O11: C, 65.77; H, 8.28; N, 5.00. Found: C, 65.21; H, 8.30;
N, 4.89.
3.1.13. 3-O-[1-(Methyl 6-deoxy-a-D-glucopyranosid-6-yl)-1H-
1,2,3-triazol-4-yl]methyl 3b-hydroxyolean-12-en-28-oate (12)
This compound was prepared from 11 (0.18 g, 0.2 mmol)
according to the general procedure 3.1.4. The residue was purified
by column chromatography (8:1 EtOAc–petroleum ether). Yield:
66 mg, 43%, white solid, mp 142–143 °C. Rf 0.15 (EtOAc). [a]
D
+56.8 (c 0.17, MeOH). IR (KBr, cmꢁ1): 3416, 2944, 2873, 1727,
1157, 1049, 1009. 1H NMR (300 MHz, CDCl3 + D2O): d 0.63, 0.78,
0.98, 1.12, 1.24 (5s, each 3H, 5 ꢂ CH3), 0.89 (s, 6H, 2 ꢂ CH3),
0.63–1.87 (m, 22H), 2.83 (d, 1H, J 11.0 Hz, H-18), 3.10–3.16 (m,
2H), 3.19 (3H, s, OCH3), 3.41 (1H, dd, J1,2 2.9, J2,3 9.4 Hz, H-2Glc),
3.73 (t, 1H, J 9.0 Hz, H-4Glc), 3.88 (t, 1H, J 7.0 Hz, H-3Glc), 4.55 (m,
3.1.16. 3-O-{[1-(Methyl 6-deoxy-a-D-glucopyranosid-6-yl)-1H-1,
2,3-triazol-4-yl]methyl} 3b-hydroxyolean-12-en-28-oic acid (15)
This compound was prepared from 14 (0.094 g, 0.117 mol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (EtOAc). Yield: 67 mg, 81%, white so-
lid, mp 249–251 °C, Rf 0.17 (15:1 CH2Cl2–MeOH). [
a
]
D
+147 (c

K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
847
0.06, MeOH). IR (KBr, cmꢁ1): 3416, 2942, 2874, 1710, 1464, 1386,
169.7, 177.6. ESIMS m/z: [M+Na]+ calcd for C67H91NaN3O10
:
1120.7; found: 1120.6. [M+H]+ calcd for C67H92N3O10: 1098.7;
found: 1098.6. Anal. Calcd for C67H91N3O10ꢀ0.5H2O: C, 72.66; H,
8.37; N, 3.79. Found: C, 72.66; H, 8.42; N, 3.68.
1147, 1099, 1051, 1035, 1011. 1H NMR (300 MHz, pyridine-d5): d
0.83, 0.86, 0.96, 1.00, 1.01, 1.02, 1.27 (7s, each 3H, 7 ꢂ CH3),
0.83–2.14 (m, 22H), 3.05 (dd, 1H, J 3.5, 11.0 Hz, H-18), 3.24 (s,
3H, OCH3), 3.31 (dd, 1H, J 3.3, 13.3 Hz, H-3), 3.82 (t, 1H, J 9.3 Hz,
H-4Glc), 4.03 (dd, 1H, J1,2 3.6, J2,3 9.5 Hz, H-2Glc), 4.44–4.50 (m, 2H,
H-6Glc), 4.79 (d, 1H, Jgem 12.0 Hz, OCH), 4.85 (q, 1H, J 8.1 Hz, H-
3.1.19. 6-[4-(Methyl a-D-glucopyranosiduronylmethyl)-1H-
1,2,3-triazol-1-yl]hexyl 3b-hydroxyolean-12-en-28-oate (18)
This compound was prepared from 17 (0.18 g, 0.164 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (10:1 CH2Cl2–MeOH). Yield: 0.103 g,
76%, white solid, mp 98–100 °C. Rf 0.16 (20:1 CH2Cl2–MeOH),
5
1
Glc), 4.99 (d, 1H, Jgem 12.2 Hz, OCH), 5.06 (d, 1H, J1,2 3.6 Hz, H-
Glc), 5.30 (m, 1H, H-3Glc), 5.49 (br s, 1H, H-12), 8.23 (s, 1H, CH@N).
13C NMR (75 MHz, pyridine-d5): d 15.5, 16.9, 17.5, 18.7, 23.0, 23.8,
26.2, 28.4, 31.0, 33.3, 34.4, 37.3, 38.6, 39.0, 39.9, 42.1, 42.3, 46.6,
46.8, 48.1, 52.1, 55.2, 56.1, 63.3, 71.9, 73.0, 73.7, 75.2, 86.2,
101.6, 122.6, 124.7, 144.9, 146.3, 180.1. Negative-mode ESIMS m/
z: [MꢁH]+ calcd for C40H62N3O8: 712.5; found: 712.6. Positive-
mode ESIMS m/z: [M+H]+ calcd for C40H64N3O8: 714.5; found:
714.4 [M+H]+. Anal. Calcd for C40H63N3O8ꢀ0.5H2O: C, 66.45; H,
8.92; N, 5.81. Found: C, 66.41; H, 9.02; N, 5.79.
[a
]
D
ꢁ9.3 (c 0.08, MeOH). IR (KBr, cmꢁ1): 3419, 2941, 2863, 1747,
1726, 1177, 1048. 1H NMR (300 MHz, CDCl3 + D2O): d 0.71, 0.77,
0.92, 0.98, 1.13 (5s, each 3H, 5 ꢂ CH3), 0.89 (s, 6H, 2 ꢂ CH3),
0.71–1.92 (m, 30H), 2.83–2.86 (m, 1H, H-18), 3.20 (dd, 1H, J 5.3,
10.4 Hz, H-3), 3.46 (s, 3H, OCH3), 3.62 (dd, 1H, J1,2 3.8, J2,3 9.3 Hz,
H-2Glc), 3.68 (t, 1H, J 9.1 Hz), 3.79 (t, 1H, J 8.9 Hz), 3.99 (t, 2H, J
6.4 Hz, CO2CH2), 4.20 (d, 1H, J 9.5 Hz), 4.34 (t, 2H, J 7.2 Hz), 4.76
(br s, 1H), 4.87 (d, 1H, J1,2 3.6 Hz, H-1Glc), 5.27 (br s, 1H, H-12),
5.54 (d, 1H, J 13.2 Hz), 7.62 (1H, s, CH@N). 13C NMR (75 MHz,
CDCl3): d 15.3, 15.6, 17.1, 18.4, 23.1, 23.5, 23.6, 25.5, 25.9, 26.1,
27.2, 27.7, 28.1, 28.4, 30.1, 30.7, 32.6, 32.8, 33.0, 33.9, 37.1, 38.5,
38.8, 39.4, 41.4, 41.8, 46.0, 46.8, 47.7, 50.5, 55.3, 56.0, 58.4, 63.9,
71.1, 71.7, 71.8, 73.6, 79.0, 99.8, 122.4, 123.0, 143.8, 169.8, 177.7.
ESIMS m/z: [M+Na]+ calcd for C46H73NaN3O10: 850.5; found:
850.5. [M+H]+ calcd for C46H74N3O10: 828.5; found: 828.5. Anal.
Calcd for C46H73N3O10: C, 66.72; H, 8.89; N, 5.07. Found: C,
66.54; H, 9.08; N, 4.91.
3.1.17. 3-O-{[1-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-glucopy-
ranosid-6-yl)-1H-1,2,3-triazol-4-yl]methyl} 3b-hydroxyolean-
12-en-28-oic acid (16)
This compound was prepared from 13 (0.1 g, 0.108 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (2:1 EtOAc–petroleum ether). Yield:
0.077 g, 85%, white solid, mp 262–263 °C, Rf 0.61 (3:1 EtOAc–
petroleum ether). [
a]
+237.8 (c 0.06, MeOH). IR (KBr, cmꢁ1):
D
3438, 2945, 2873, 1755, 1463, 1368, 1223, 1075, 1046, 602. 1H
NMR (300 MHz, CDCl3): d 0.74, 0.75, 0.88, 0.90, 0.91, 0.92, 1.11
(7s, each 3H, 7 ꢂ CH3), 0.74–1.87 (m, 22H), 2.00, 2.06, 2.10 (3s,
each 3H, 3 ꢂ COCH3), 2.81 (dd, 1H, J 3.5, 13.0 Hz, H-18), 2.98 (dd,
1H, J 3.8, 11.3 Hz, H-3), 3.12 (s, 3H, OCH3), 4.18 (t, 1H, J 8.4 Hz,
H-4Glc), 4.34 (dd, 1H, J5,6 8.6, Jgem 14.0 Hz, H-6Glc), 4.54–4.61 (m,
2H, H-60Glc, H-1Glc), 4.75–4.91 (m, 4H, OCH2, H-2Glc, H-5Glc), 5.27
(br s, 1H, H-12), 5.47 (t, 1H, J 9.7 Hz, H-3Glc), 7.63 (s, 1H, CH@N).
13C NMR (75 MHz, CDCl3): d 15.3, 16.4, 17.2, 18.3, 20.6, 22.8,
23.0, 23.5, 23.6, 25.9, 27.7, 28.2, 30.7, 32.5, 32.7, 33.0, 33.9, 37.1,
38.4, 38.8, 39.4, 41.1, 41.7, 45.9, 46.7, 47.7, 50.8, 55.5, 55.8, 63.2,
68.0, 69.8, 70.2, 70.9, 86.7, 96.7, 122.7, 123.7, 143.6, 146.7, 169.8,
3.1.20. 6-[4-(Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranosid-6-
yloxy)-methyl-1H-1,2,3-triazol-1-yl]hexyl 3b-hydroxyolean-12-
en-28-oate (19)
This compound was prepared from 2 (0.108 g, 0.19 mmol) and 9
(0.093 g, 0.19 mmol) according to the general procedure 3.1.3. The
residue was purified by column chromatography (2:1 EtOAc–
petroleum ether). Yield: 0.175 g, 88%, white solid, mp 72–74 °C,
Rf 0.23 (1:1 EtOAc–petroleum ether), [a] +59.2 (c 0.05, CHCl3).
D
IR (KBr, cmꢁ1): 3498, 2941, 2864, 1721, 1454, 1047, 767, 698. 1H
NMR (250 MHz, CDCl3): d 0.65, 0.70, 0.85, 0.91, 1.07 (5s, each 3H,
5 ꢂ CH3), 0.82 (s, 6H, 2 ꢂ CH3), 0.65–1.96 (m, 30H), 2.76–2.80 (m,
1H, H-18), 3.09–3.16 (m, 1H, H-3), 3.29 (s, 3H, OCH3), 3.46 (dd,
1H, J1,2 3.5, J2,3 9.8 Hz, H-2Glc), 3.53–3.74 (m, 4H, H-4Glc, H-5Glc, H-
170.1, 182.5. ESIMS m/z: [M+Na]+ calcd for C46H69NaN3O11
:
862.5; found: 862.5. [M+H]+ calcd for C46H70N3O11: 840.5; found:
840.5. Anal. Calcd for C46H69N3O11ꢀ0.5H2O: C, 65.07; H, 8.31; N,
4.95. Found: C, 64.93; H, 8.17; N, 5.09.
6
Glc), 3.87–3.94 (m, 3H, H-3Glc, CH2N), 4.16 (t, 2H, J 7.3 Hz, CO2CH2),
3.1.18. 6-[4-(Methyl 2,3,4-tri-O-benzyl-
nylmethyl)-1H-1,2,3-triazol-1-yl]hexyl 3b-hydroxyolean-12-en-
28-oate (17)
This compound was prepared from 2 (0.162 g, 0.28 mmol) and 8
(0.144 g, 0.28 mmol) according to the general procedure 3.1.3. The
residue was purified by column chromatography (3:2 EtOAc–
petroleum ether). Yield: 0.278 g, 88%, white solid, mp 73–74 °C,
a
-
D
-glucopyranosiduro-
4.54 (d, 1H, J1,2 3.5 Hz, H-1Glc), 4.41–4.92 (m, 8H, 3 ꢂ PhCH2,
NCCH2O), 5.20 (br s, 1H, H-12), 7.12–7.25 (m, 15H, Ph-H), 7.37 (s,
1H, CH@N). 13C NMR (75 MHz, CDCl3): d 14.1, 15.3, 15.5, 17.0,
18.3, 21.0, 23.0, 23.4, 23.6, 23.8, 25.4, 25.5, 25.8, 26.1, 27.1, 27.6,
28.1, 28.3, 30.1, 30.6, 32.4, 32.7, 33.0, 33.8, 37.0, 38.4, 38.7, 39.3,
41.3, 41.7, 45.8, 46.6, 47.6, 50.1, 53.4, 55.1, 55.2, 60.3, 63.8, 65.0,
67.9, 68.8, 70.0, 73.3, 74.9, 75.7, 78.9, 79.8, 82.0, 98.2, 122.1,
122.3, 127.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.31, 128.33,
128.37, 138.1, 138.2, 138.7, 143.8, 144.9, 177.6. ESIMS m/z:
[M+Na]+ calcd for C67H93NaN3O9: 1106.7; found: 1106.6. [M+H]+
calcd for C67H94N3O9: 1084.7; found: 1084.7. Anal. Calcd for
C67H93N3O9ꢀ0.5H2O: C, 73.59; H, 8.66; N, 3.84. Found: C, 73.27;
H, 8.64; N, 3.66.
Rf 0.15 (1:2 EtOAc–petroleum ether). [a] +23.1 (c 0.07, CHCl3).
D
IR (KBr, cmꢁ1): 3027, 2942, 2864, 1747, 1721, 1180, 1092, 1071,
1047, 754, 698, 666. 1H NMR (250 MHz, CDCl3): d 0.65, 0.70,
0.85, 0.91, 1.06 (5s, each 3H, 5 ꢂ CH3), 0.82 (s, 6H, 2 ꢂ CH3),
0.65–1.97 (m, 30H), 2.89 (m, 1H, H-18), 3.26 (m, 1H, H-3), 3.31
(s, 3H, OCH3), 3.49 (dd, 1H, J1,2 3.5, J2,3 9.5 Hz, H-2Glc), 3.68 (t, 1H,
J 9.5 Hz, H-4Glc), 3.90 (t, 1H, J 9.1 Hz, H-3Glc), 3.91 (t, 2H, J 6.4 Hz,
CH2N), 4.03–4.10 (m, 1H, H-5Glc), 4.11 (t, 2H, J 9.9 Hz, CO2CH2),
4.52 (d, 1H, J1,2 3.5 Hz, H-1Glc), 4.34–4.89 (m, 6H, 3 ꢂ PhCH2),
5.18–5.22 (m, 3H, NCCH2O, H-12), 7.19–7.28 (m, 15H, Ph-H), 7.32
(s, 1H, CH@N). 13C NMR (75 MHz, CDCl3): d 15.3, 15.5, 17.0, 18.3,
23.0, 23.4, 23.6, 25.4, 25.8, 26.1, 27.2, 27.6, 28.1, 28.4, 30.0, 30.7,
32.5, 32.7, 33.0, 33.8, 37.0, 38.4, 38.7, 39.3, 41.3, 41.7, 45.8, 46.6,
47.6, 50.1, 55.2, 55.7, 58.7, 63.8, 70.2, 73.6, 74.8, 75.8, 78.9, 79.3,
79.6, 81.3, 98.8, 122.3, 123.7, 127.5, 127.6, 127.7, 127.9, 128.0,
128.1, 128.2, 128.3, 128.5, 137.9, 138.0, 138.5, 142.1, 143.8,
3.1.21. 6-[4-(Methyl a-D-glucopyranosid-6-yloxy)methyl-1H-
1,2,3-triazol-1-yl]hexyl 3b-hydroxyolean-12-en-28-oate (20)
This compound was prepared from 19 (0.045 g, 0.042 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (10:1 CH2Cl2–MeOH). Yield: 13 mg,
38%, colorless oil, Rf 0.1 (21:1 CH2Cl2–MeOH). 1H NMR (300 MHz,
CDCl3): d 0.71, 0.77, 0.88, 0.89, 0.91, 0.98, 1.12 (7s, each 3H,
7 ꢂ CH3), 0.71–2.04 (m, 30H), 2.85 (dd, 1H, J 3.8, 13.3 Hz, H-18),
3.21 (dd, 1H, J 5.0, 10.4 Hz, H-3), 3.41 (s, 3H, OCH3), 3.54–3.91

848
K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
(m, 6H, H-2Glc, H-3Glc, H-4Glc, H-5Glc, H-6Glc), 3.99 (t, 2H, J 6.5 Hz,
NCH2), 4.33 (t, 2H, J 7.2 Hz, CO2CH2), 4.67 (d, 1H, J 12.8 Hz, NCCHO),
4.75 (d, 1H, J 12.5 Hz, NCCHO), 4.77 (d, 1H, J1,2 3.8 Hz, H-1Glc), 5.26
(t, 1H, J 3.3 Hz, H-12), 7.51 (s, 1H, CH@N). 13C NMR (75 MHz,
CDCl3): d 15.4, 15.6, 17.1 18.4, 23.1, 23.5, 23.7, 25.6, 25.9, 26.2,
27.3, 27.7, 28.2, 28.5, 29.7, 30.2, 30.7, 32.6, 32.9, 33.1, 34.0, 37.1,
38.6, 38.8, 39.5, 41.5, 41.8, 46.0, 46.8, 47.7, 50.4, 55.3, 55.4, 63.9,
64.8, 69.8, 70.4, 70.6, 72.4, 74.7, 77.2, 79.1, 99.6, 122.0, 122.4,
143.9, 177.8. ESIMS m/z: [M+Na]+ calcd for C46H75NaN3O9: 836.5;
found: 836.5. [M+H]+ calcd for C46H76N3O9: 814.6; found: 814.5.
Anal. Calcd for C46H75N3O9: C, 67.87; H, 9.29; N, 5.16. Found: C,
67.33; H, 9.14; N, 5.06.
1H, H-12). 13C NMR (75 MHz, CDCl3): d 15.3, 15.6, 17.0, 18.3,
23.0, 23.4, 23.6, 25.3, 25.6, 25.9, 27.2, 27.7, 28.1, 28.4, 28.5, 29.7,
30.7, 32.5, 32.8, 33.1, 33.9, 37.0, 38.5, 38.7, 39.4, 41.3, 41.7, 45.9,
46.7, 47.6, 55.2, 55.9, 64.0, 65.8, 70.4, 71.7, 73.8, 79.0, 99.6,
122.4, 130.6, 143.8, 170.2, 177.8. ESIMS m/z: [M+Cl]+ calcd for
C43H70ClO10: 781.5; found: 781.5. Anal. Calcd for C43H70O10ꢀH2O:
C, 67.51; H, 9.49. Found: C, 67.77; H, 9.81.
3.1.25. 6-[2-(Methyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)
ethanoyl]hexyl 3b-hydroxyolean-12-en-28-oate (25)
This compound was prepared from 21 (0.12 g, 0.19 mmol) and 24
(0.11 g, 0.19 mmol) according to the general procedure 3.1.1. The
residue was purified by column chromatography (1:6 EtOAc–petro-
leum ether). Yield: 0.20 g, 94%, white solid, mp 53–55 °C, Rf 0.21
(1:6 EtOA –petroleum ether). IR (KBr, cmꢁ1): 3509, 2942, 2865,
1727, 1457, 1090, 737. 1H NMR (300 MHz, CDCl3): d 0.73, 0.77, 0.92,
0.98, 1.13 (5s, each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3), 0.73–1.97
(m, 30H), 2.69–2.78 (m, 2H, CH2CO2), 2.87 (dd, 1H, J 3.8, 13.9 Hz,
3.1.22. 6-Bromohexyl 3b-hydroxyolean-12-en-28-oate (21)
This compound was prepared from 1 (1.87 g, 4.1 mmol) and
1,6-dibromohexane (0.63 mL, 4.1 mmol) according to the general
procedure 3.1.1. The residue was purified by column chromatogra-
phy (1:6 EtOAc–petroleum ether). Yield: 1.3 g, 52%, white solid, mp
71–73 °C, Rf 0.7 (1:3 EtOAc–petroleum ether). IR (KBr, cmꢁ1): 3515,
2931, 2864, 1722, 1463, 1386, 1364, 1261, 1176, 1161, 1031, 1001,
763, 667. 1H NMR (300 MHz, CDCl3): d 0.73, 0.78, 0.92, 0.98, 1.13
(5s, each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3), 0.73–2.04 (m, 30H),
2.85 (dd, 1H, J 3.2, 13.5 Hz, H-18), 3.20 (dd, 1H, J 4.0, 10.1 Hz, H-
3), 3.41 (t, 2H, J 6.7 Hz, BrCH2), 4.02 (t, 2H, J 6.3 Hz, CO2CH2),
5.28 (s, 1H, H-12). 13C NMR (75 MHz, CDCl3): d 15.3, 15.6, 17.1,
18.4, 23.1, 23.5, 23.6, 25.3, 25.9, 27.3, 27.75, 27.82, 28.2, 28.5,
30.7, 32.6, 32.7, 32.9, 33.1, 33.4, 34.0, 37.1, 38.6, 38.8, 39.5, 41.5,
41.8, 46.0, 46.8, 47.7, 55.4, 64.0, 77.2, 79.1, 122.4, 143.9, 177.7.
ESIMS m/z: [M+Na]+ calcd for C36H59BrNaO3: 641.4; found: 641.4.
H-18), 3.17 (dd, 1H, J 5.0, 10.8 Hz, H-3), 3.62–3.81 (m, 6H, H-2Glc
,
H-3Glc, H-4Glc, H-5Glc, H-6Glc), 4.00–4.03 (m, 4H, 2 ꢂ CO2CH2), 4.44–
4.93 (m, 9H, H-1Glc, 4xPhCH2), 5.28 (br s, 1H, H-12), 7.11–7.32 (m,
20H, Ph-H). 13C NMR (75 MHz, CDCl3): d 15.25, 15.32, 15.6, 17.1,
18.4, 23.1, 23.5, 23.6, 25.6, 25.8, 25.9, 27.3, 27.7, 28.1, 28.5, 30.7,
32.5, 32.8, 33.1, 33.9, 37.1, 38.5, 38.8, 39.4, 41.4, 41.8, 46.0, 46.7,
47.7, 55.3, 64.1, 64.6, 68.9, 71.5, 72.3, 73.2, 73.5, 75.0, 75.4, 79.0,
79.3, 82.2, 122.4, 127.6, 127.7, 127.8, 127.83, 127.9, 128.3, 128.4,
138.0, 138.1, 138.2, 138.6, 143.9,171.2, 177.7. ESIMS m/z: [M+K]+
calcd for C72H96KO10:1159.7; found: 1159.9.
3.1.26. 6-[2-(Methyl a-D-glucopyranosyl) ethanoyl]hexyl
3b-hydroxyolean-12-en-28-oate (26)
3.1.23. 6-(Methyl 2,3,4-tri-O-benzyl-
a-
D-glucopyranosiduronyl)
hexyl 3b-hydroxyolean-12-en-28-oate (22)
This compound was prepared from 25 (0.14 g, 0.12 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (15:1 CH2Cl2–MeOH). Yield: 0.082 g,
This compound was prepared from 21 (0.26 g, 0.4 mmol) and 7
(0.2 g, 0.4 mmol) according to the general procedure 3.1.1. The res-
idue was purified by column chromatography (1:6 EtOAc–petro-
leum ether). Yield: 0.38 g, 88%, white solid, mp 59–61 °C, Rf 0.27
(1:6 EtOAc–petroleum ether). IR (KBr, cmꢁ1): 3531, 2940, 2864,
1747, 1720, 1455, 1369, 1259, 1194, 1071, 1046, 734, 697, 463.
1H NMR (300 MHz, CDCl3): d 0.72, 0.77, 0.92, 0.97, 1.13 (5s, each
3H, 5 ꢂ CH3), 0.89 (s, 6H, 2 ꢂ CH3), 0.72–1.97 (m, 30H), 2.85 (dd,
1H, J 4.4, 13.5 Hz, H-18), 3.20 (dd, 1H, J 4.8, 10.7 Hz, H-3), 3.41 (s,
3H, OCH3), 3.58 (dd, 1H, J1,2 3.5, J2,3 10.0 Hz, H-2Glc), 3.73 (t, 1H, J
9.5 Hz, H-4Glc), 3.95–4.14 (m, 5H, 2 ꢂ CO2CH2, H-3Glc), 4.19 (d, 1H,
J4,5 10.1 Hz, H-5Glc), 4.55–4.97 (m, 7H, 3 ꢂ PhCH2, H-1Glc), 5.27 (t,
1H, J 3.5 Hz, H-12), 7.24–7.34 (m, 15H, Ph-H). 13C NMR (75 MHz,
CDCl3): d 15.3, 15.7, 17.1, 18.4, 23.1, 23.4, 23.6, 25.5, 25.7, 25.9,
27.2, 27.7, 28.1, 28.4, 28.5, 30.7, 32.5, 32.8, 33.1, 33.9, 37.1, 38.5,
38.8, 39.4, 41.4, 41.7, 45.9, 46.7, 47.7, 55.3, 55.6, 64.0, 65.6, 70.3,
73.6, 75.0, 75.8, 79.0, 79.5, 79.7, 81.4, 98.8, 122.4, 127.7, 127.9,
128.0, 128.1, 128.3, 128.4, 128.5, 138.0, 138.1, 138.6, 143.8,
169.8, 177.7. ESIMS m/z: [M+K]+ calcd for C64H88KO10: 1055.6;
found: 1055.8.
86%, white solid, mp 89–91 °C, Rf 0.09 (20:1 CH2Cl2–MeOH). [a]
D
+72.8 (c 0.10, MeOH). IR (KBr, cmꢁ1): 3402, 2940, 2863, 1722,
1635, 1386, 1176, 1067, 1033. 1H NMR (300 MHz, CDCl3): d 0.72,
0.77, 0.92, 0.97, 1.13 (5s, each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3),
0.60–2.00 (m, 30H), 2.67–2.72 (m, 2H, CH2CO2), 2.86 (d, 1H, J
10.3 Hz, H-18), 3.23 (t, 1H, J 7.1 Hz, H-3), 3.50–3.81 (m, 6H, H-
2
Glc, H-3Glc, H-4Glc, H-5Glc, H-6Glc), 3.94–4.07 (m, 4H, 2xCO2CH2),
4.51 (br s, 1H, H-1Glc), 5.27 (br s, 1H, H-12). 13C NMR (75 MHz,
CDCl3): d 15.3, 15.7, 17.1, 18.4, 23.1, 23.5, 23.7, 25.4, 25.8, 25.9,
27.1, 27.7, 28.2, 28.5, 28.6, 29.7, 30.7, 31.9, 32.6, 32.8, 33.1, 33.9,
37.1, 38.5, 38.8, 39.4, 41.4, 41.7, 45.9, 46.8, 47.6, 55.2, 61.6, 64.1,
65.1, 72.9, 73.2, 77.2, 79.0, 122.4, 143.9, 172.2, 177.9. ESIMS m/z:
[M+HCOO]+ calcd for C45H73O12: 805.5; found: 805.5. Anal. Calcd
for C44H72O10ꢀH2O: C, 67.84; H, 9.57. Found: C, 67.19; H, 9.76.
3.1.27. Benzyl 3-O-(2-chloroethoxycarbonyl)-3b-hydroxyolean-
12-en-28-oate (27)
To a solution of benzyl oleanolate 4 (2.6 g, 4.8 mmol) in anhyd
CH2Cl2 (100 mL) were added K2CO3 (13 g, 0.095 mol), DMAP
(0.06 g, 0.48 mmol), and then 2-chloroethyl chloroformate
(9.8 mL, 0.095 mol) in three portions during 9 h. After stirring at
35 °C for 12 h, the reaction mixture was filtered, and the filtrate
was diluted with water (200 mL), and extracted with CH2Cl2
(3 ꢂ 100 mL). The combined organic layers were washed with
water and brine, dried (Na2SO4), filtered, and concentrated. Purifi-
cation by flash column chromatography (1:60 EtOAc–petroleum
ether) afforded 27 (2.87 g, 92%) as a white solid. mp 61–63 °C, Rf
0.61 (1:8 EtOAc–petroleum ether). IR (KBr, cmꢁ1): 2947, 2878,
1743, 1460, 1391, 1157, 1253, 1011, 966, 739, 457. 1H NMR
(300 MHz, CDCl3): d 0.60, 0.87, 0.92, 1.01, 1.12 (5s, each 3H,
5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3), 0.60–1.99 (m, 22H), 2.91 (dd, 1H,
J 3.9, 13.7 Hz, H-18), 3.70 (t, 2H, J 5.8 Hz, CH2Cl), 4.32–4.43 (m,
3.1.24. 6-(Methyl a-D-glucopyranosiduronyl)hexyl 3b-
hydroxyolean-12-en-28-oate (23)
This compound was prepared from 22 (0.24 g, 0.24 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (20:1 CH2Cl2–MeOH). Yield: 0.15 g,
85%, white solid, mp 84–86 °C, Rf 0.23 (20:1 CH2Cl2–MeOH), [a]
D
+73.1 (c 0.06, MeOH). IR (KBr, cmꢁ1): 3431, 2940, 1732, 1048. 1H
NMR1H NMR (300 MHz, CDCl3): d 0.72, 0.78, 0.92, 0.98, 1.13 (5s,
each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3), 0.72–1.99 (m, 30H), 2.85
(dd, 1H, J 3.6, 13.7 Hz, H-18), 3.21 (dd, 1H, J 4.8, 10.2 Hz, H-3),
3.50 (s, 3H, OCH3), 3.68–3.82 (m, 3H, H-2Glc, H-3Glc, H-4Glc), 4.01
(t, 2H, J 6.1 Hz, CO2CH2), 4.12 (d, 1H, J4,5 9.4 Hz, H-5Glc), 4.22 (2H,
t, J 6.8 Hz, CH2OCO), 4.85 (d, 1H, J1,2 3.6 Hz, H-1Glc), 5.27 (br s,

K. Cheng et al. / Carbohydrate Research 344 (2009) 841–850
849
3H, H-3, CO2CH2), 5.06 (d, 1H, Jgem 12.6 Hz, PhCH), 5.08 (d, 1H, Jgem
12.6 Hz, PhCH), 5.28 (t, 1H, J 3.3 Hz, H-12). 13C NMR (75 MHz,
CDCl3): d 15.3, 16.6, 16.9, 18.2, 23.1, 23.4, 23.5, 23.6, 25.8, 27.6,
28.0, 30.7, 32.4, 32.7, 33.1, 33.9, 36.9, 38.0, 38.1, 39.3, 41.0, 41.2,
41.4, 41.7, 45.9, 46.8, 47.6, 55.4, 65.9, 66.8, 67.5, 77.2, 86.0,
122.4, 127.9, 128.0, 128.4, 136.5, 143.7, 154.4, 154.9, 177.4. ESIMS
m/z: [M+K]+ calcd for C40H57ClKO5: 691.4; found: 691.5. [M+Na]+
calcd for C40H57ClNaO5: 675.4; found: 675.6.
molybdate and 0.38 mg/mL Malachite Green, reactions were run
at 22 °C for 25 min, and then the phosphate absorbance was mea-
sured at 655 nm. The IC50 values were estimated by fitting the inhi-
bition data to a dose-dependent curve using a logistic derivative
equation.
3.3. Molecular docking simulation
To further identify the putative binding site and corresponding
binding poses of the compounds, molecular docking simulation
was performed against the GP protein. Glide 4.015 was used. The
high-resolution crystal structure of GP complexed with asiatic acid
at the allosteric site was received from the Protein Data Bank16
(PDB code: 2QN15), and the corresponding dimer was manually
generated as the receptor according to the symmetry information
provided in the PDB file. All the co-crystallized ligands and waters
were removed. Preparation of the target protein with the Protein
Preparation Wizard script in Maestro17 involved the addition of
hydrogen atoms to the macromolecule and optimization of the
states for hydroxyl, Asn, Gln, and His residues. Three-dimensional
affinity grids were positioned around the allosteric site, inhibitor
site, catalytic site, and dimer interface site, and calculated with
Receptor Grid Generation module of Glide, respectively. The start-
ing 3D conformations of compound 12 and olealonic acid were pre-
pared with LigPrep.18 The extra precision (XP) mode of Glide was
applied in molecular docking, and positions with a Coulomb–van
der Waals score greater than 0 kcal/mol were rejected. All the
docking positions were evaluated with Glide GScore. The best-
ranked position from each of the binding sites was determined
by selecting the one with the lowest calculated GScore values.
3.1.28. Benzyl 3-O-[2-O-(methyl 2,3,4-tri-O-benzyl-a-D-glucopy-
ranosiduronyl ethoxycarbonyl]-3b-hydroxyolean-12-en-28-
oate (28)
This compound was prepared from 27 (0.13 g, 0.2 mmol) and 7
(0.19 g, 0.4 mmol) according to the general procedure 3.1.1. The
residue was purified by column chromatography (1:7 EtOAc–
petroleum ether). Yield: 0.20 g, 91%, white solid, mp 62–63 °C, Rf
0.33 (1:5 EtOAc–petroleum ether). IR (KBr, cmꢁ1): 2947, 1745,
1454, 1365, 1256, 754, 698. 1H NMR (300 MHz, CDCl3): d 0.60,
0.80, 0.88, 0.92, 1.12 (5s, each 3H, 5 ꢂ CH3), 0.90 (s, 6H, 2 ꢂ CH3),
0.60–1.97 (m, 22H), 2.89–2.92 (m, 1H, H-18), 3.40 (s, 3H, OCH3),
3.57 (dd, 1H, J1,2 3.5, J2,3 9.6 Hz, H-2Glc), 3.74 (t, 1H, J 9.5 Hz, H-
4
Glc), 3.99 (t, 1H, J 9.3 Hz, H-3Glc), 4.19–4.38 (m, 6H, H-3, H-5Glc
,
CO2CH2CH2), 4.59–4.97 (m, 7H, H-1Glc, 3 ꢂ PhCH2), 5.06 (d, 1H, Jgem
12.6 Hz, PhCH), 5.08 (d, 1H, Jgem 12.5 Hz, PhCH), 5.29 (br s, 1H, H-
12), 7.22–7.34 (m, 20H, Ph-H). 13C NMR (75 MHz, CDCl3): d 15.3,
16.6, 16.9, 18.2, 23.1, 23.4, 23.6, 25.8, 27.6, 28.0, 30.7, 32.4, 32.7,
33.1, 33.9, 36.9, 37.9, 38.1, 39.3, 41.4, 41.7, 45.9, 46.8, 47.6, 55.3,
55.7, 63.1, 64.8, 65.9, 70.2, 73.6, 75.1, 75.8, 77.2, 79.4, 79.6, 81.4,
85.9, 98.8, 122.4, 127.6, 127.7, 127.8, 127.9, 127.99, 128.0, 128.1,
128.3, 128.37, 128.40, 128.5, 138.0, 138.6, 143.7, 155.0, 169.5,
177.4. ESIMS m/z: [M+K]+ calcd for C68H86KO12: 1133.6; found:
1133.3.
Acknowledgments
3.1.29. 3-O-[2-O-(Methyl a-D-glucopyranosiduronyl)
K.C. thanks the French Embassy in China for a co-tutor Doctorate
fellowship. This work is supported by a grant from Region Ile-de-
France. H.S. thanks financial support by National Natural Science
Foundation of China (Grants 30672523 and 90713037), research
grants from Chinese ministry of Education (Grants 706030 and
20050316008) and program for New Century Excellent Talents in
University (NCET-05-0495).
ethoxycarbonyl]-3b-hydroxy olean-12-en-28-oic acid (29)
This compound was prepared from 28 (0.12 g, 0.11 mmol)
according to the general procedure 3.1.5. The residue was purified
by column chromatography (23:1 CH2Cl2–MeOH). Yield: 0.065 g,
81%, white solid, mp 204–206 °C. Rf 0.14 (20:1 CH2Cl2–MeOH).
[
a
]
+91.1 (c 0.07, MeOH). IR (KBr, cmꢁ1): 3412, 2942, 1744,
D
1692, 1273, 1051, 1033, 1014. 1H NMR (300 MHz, CDCl3): d 0.76,
0.87, 0.93, 0.97, 1.15 (5s, each 3H, 5 ꢂ CH3), 0.96 (s, 6H, 2 ꢂ CH3),
0.76–2.04 (m, 22H), 2.84–2.88 (m, 1H, H-18), 3.49 (s, 3H, OCH3),
3.67 (m, 1H, H-2Glc), 3.75–3.83 (m, 2H, H-3Glc, H-4Glc), 4.19 (d, 1H,
J 9.3 Hz, H-5Glc), 4.34–4.47 (m, 5H, H-3, CO2CH2CH2), 4.86 (br s,
1H, H-1Glc), 5.30 (br s, 1H, H-12). 13C NMR (75 MHz, CDCl3): d
15.4 16.6, 17.5, 18.2, 22.8, 23.4, 23.5, 23.6, 26.0, 27.7, 28.0, 29.7,
30.7, 32.5, 32.6, 33.1, 33.8, 37.0, 38.0, 39.3, 40.9, 41.5, 45.8, 46.5,
47.5, 55.3, 55.9, 63.5, 65.0, 70.8, 71.6, 73.5, 85.9, 99.8, 122.6,
143.7, 155.1, 169.8, 184.0. Negative-mode ESIMS m/z: [MꢁH]+
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.02.012.
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