Synthesis of oleanolic acid saponins mimicking components of Chinese folk medicine Di Wu

Article abstract of DOI:10.1016/j.carres.2009.04.005

3,28-Di-O-rhamnosylated oleanolic acid saponins, mimicking components of Chinese folk medicine Di Wu, have been designed and synthesized. One-pot glycosylation and 'inverse procedure' technologies have been applied thus significantly simplifying the prepa

Full text of DOI:10.1016/j.carres.2009.04.005

Carbohydrate Research 344 (2009) 1153–1158
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of oleanolic acid saponins mimicking components of Chinese
folk medicine Di Wu
c
Xing Huang a,b, Shuihong Cheng a,b, Yuguo Du a,b, , Feihong Bing
*
a Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b College of Chemistry and Chemical Engineering, Graduate University of Chinese Academy of Sciences, Beijing 100049, China
c Hubei College of Traditional Chinese Medicine, Hubei, China
a r t i c l e i n f o
a b s t r a c t
Article history:
3,28-Di-O-rhamnosylated oleanolic acid saponins, mimicking components of Chinese folk medicine Di
Wu, have been designed and synthesized. One-pot glycosylation and ‘inverse procedure’ technologies
have been applied thus significantly simplifying the preparation of desired saponins. The cytotoxic
Received 14 February 2009
Received in revised form 25 March 2009
Accepted 7 April 2009
activity of compounds 3-O-[
rhamnopyranosyl-(1?4)-b- -glucopyranosyl-(1?6)-b-
anosyl]oleanolic acid 28-O-[ -rhamnopyranosyl- (1?4)-b-
syl] ester (4), 3-O-[ -rhamnopyranosyl]oleanolic acid 28-O-[
3-O-[ -rhamnopyranosyl]oleanolic acid 28-O-[6-O-( -rhamnopyranosyl)hexyl] ester (6) was prelim-
a
-
L
-rhamnopyranosyl-(1?2)-b-
-glucopyranosyl] ester (3), 3-O-[
-glucopyranosyl-(1?6)-b-
-rhamnopyranosyl] ester (5), and
D
-xylopyranosyl]oleanolic acid 28-O-[
-rhamnopyr-
-glucopyrano-
a-L-
Available online 14 April 2009
D
D
a-L
D
a
-
L
D
Keywords:
Saponin
Apoptosis
Natural product
Chinese folk medicine
Cytotoxicity
a
-
L
a-L
a
-
L
a-L
inarily evaluated against HL-60 human promyelocytic leukemia cells. The natural saponin 3 and designed
saponin 4 exhibited comparable moderate cytotoxic activity under our testing conditions.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Literature searching suggested that a-L-rhamnopyranosyl moi-
ety of some natural soponins may play a crucial role in triggering
cell death by apoptosis.12 Several hypotheses have been proposed
for the mechanism of their activity.13–15 However, the related
SAR study is sporadic and unsystematic due to the difficulty in
obtaining adequate analogical glycosides from nature. We have re-
cently synthesized compound 3 which inhibited ConA-induced
lymphocyte proliferation.8 However, the structural complexity,
especially having 20-OH branched sugar chain on C-3 of oleanolic
acid, has hindered the large-scale preparation of this type of bides-
mosidic triterpene saponins.6,16–23 To simplify the structure and
Saponins, notably steroid or triterpene plant glycosides, have
been used worldwide in the treatments of diseases and health care
practices by taking advantages of their generally non-ionic surfac-
tant and membrane-disrupting properties.1–5 The diversity of struc-
tures, the challenges of isolation and purification, their wide
abundance in the plant kingdom and their broad spectrum of bio-
logical and pharmacological activities have driven chemists to the
research field of saponins.6 It is surprising that more than half of
the triterpene saponins are glycosides of oleanolic acid or its deriv-
atives, with one sugar chain attached through an ether linkage at C-
3 and another through an ester linkage at C-28.7 In our ongoing
project of Di Wu,8 a Chinese folk medicine from dry rhizome of
Anemone flaccida Fr. Schmidt, we proved that the major
investigate the role of L-rhamnopyranosyl residues in oleanane-
type saponins, especially in Di Wu saponins, we here report the de-
sign, synthesis, and bioactivity of some structural analogues.
components (Scheme 1) of Di Wu are 3-O-[
(1?2)-b- -glucopyranosyl]oleanolic acid 28-O-[
anosyl-(1?4)-b- -glucopyranosyl-(1?6)-b- -glucopyranosyl] ester
(1), 3-O-[ -rhamnopyranosyl-(1?2)- -arabinopyranosyl] ole-
anolic acid 28-O-[ -rhamnopyranosyl-(1?4)-b- -glucopyranosyl-
(1?6)-b- -glucopyranosyl] ester (2), and 3-O-[ -rhamnopyrano-
syl-(1?2)-b- -xylopyranosyl]oleanolic acid 28-O-[ -rhamnopyr-
-glucopyranosyl] ester
a
-
L
-rhamnopyranosyl-
2. Results and discussion
D
a-L-rhamnopyr-
D
D
The target molecules are shown in Scheme 2. To prepare
compound 4, trisaccharide 10 was first synthesized via one-pot
a
-
L
a-L
a-L
D
glycosylation technology (Scheme 3). Thus, 2,3,4-tri-O-benzoyl-
a
-
D
a-L
L
-rhamnopyranosyl trichloroacetimidate (8, 1.4 equiv)24 was
D
a-L
slowly added into ethyl 2,3-di-O-acetyl-6-O-benzoyl-1-thio-b-
D
-
anosyl-(1?4)-b-
D-glucopyranosyl-(1?6)-b-D
glucopyranoside25 (7, 1.2 equiv) in dry CH2Cl2 in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.17 equiv) at
ꢀ70 °C. The reaction mixture was warmed up to rt and stirred at
this condition for another 30 min, then acceptor 4-methoxyphenyl
(3).9–11
* Corresponding author. Tel.: +86 10 62849126; fax: +86 10 62923563.
E-mail address: duyuguo@rcees.ac.cn (Y. Du).
2,3,4-tri-O-acetyl-b-D
-glucopyranoside (9, 1.0 equiv)8,26 and
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.04.005

1154
X. Huang et al. / Carbohydrate Research 344 (2009) 1153–1158
excellent yield. Selective removal of the acetate and benzoate pro-
tecting groups in 14 with catalytic amount of NaOMe (CH2Cl2–
MeOH, 2:1, v/v), taking advantage of a kinetic steric effect of the
neighboring quaternary center on C-28,16 furnished the target 3-
O
R2
R3
O-
syl-(1?4)-b-
70% yield from 12.
a
-
L
-rhamnopyranosyl oleanolic acid 28-O-
a-L-rhamnopyrano-
O
R1
O
O
OH
D
-glucopyranosyl-(1?6)-b- -glucopyranoside (4) in
D
O
HO
O
O
O
Similarly (Scheme 5), condensation of 13 with 8 as described in
the preparation of compound 14 gave derivative 15, which was
subjected to deacylation with NaOMe in CH2Cl2–MeOH to give de-
HO
OH
Me
HO
O
HO
O
OH
HO
HO
OH
1 = OH, R2 = H, R3 = CH2OH
1 = H, R2 = OH, R3 = H
O
sired saponin 3-O-
a-L-rhamnopyranosyl oleanolic acid 28-O-a-L-
1
R
R
Me
HO
rhamnopyranoside (5) in excellent overall yield.
2
OH
3 R1 = OH, R2 = H, R3 = H
OH
In the preparation of compound 6 (Scheme 6), 3-O-acetyl
oleanolic acid 1630 was treated with oxalyl chloride in dry CH2Cl2,
the resulting acid chloride was then immediately reacted with
1,6-hexanediol in the presence of triethylamine affording 3-O-
acetyl oleanolic acid 28-O-(6-hydroxyhexyl) ester (17). Removal
of acetyl group with NaOMe in CH2Cl2/MeOH (?18), followed by
glycosylation with 8 using the same procedure applied in the syn-
thesis of 13, gave diglycosylated compound 19 in 90% yield. Global
debenzoylation of 19 with catalytic amount of NaOMe (CH2Cl2–
MeOH, 2:1, v/v) in the presence of the C-28 ester glycosidic linkage
gave the target compound 6 in 60% yield from 16.
Scheme 1. Natural saponins isolated from Di Wu.
O
O
O
OH
O
The cytotoxic activities of compounds 3–6, and oleanolic acid
were evaluated31,32 against HL-60 human promyelocytic leukemia
cells (Table 1). Saponins 3 and 4 exhibited comparable moderate
O
Me
HO
O
O
HO
OH
HO
O
OH
HO
OH
HO
HO
cytotoxic activity with IC50 values at 2.7 and 3.0 lg/mL, respec-
O
Me
tively. Compared to 4, structural analogues 5 and 6 showed only
weak activities under the same testing conditions, suggesting that
sugar moieties (or may be polyhydroxyl linkers) are important be-
HO
4
OH
tween a-L-rhamnopyranosyl residue and C-28 carboxyl group. Ole-
anolic acid itself was not cytotoxic under our assay conditions. The
current results should be valuable for the related molecule design,
synthesis, and bioactivity screening.33,34
O
O
O
O
OH
Me
HO
OH
Me
HO
3. Experimental
O
5
HO
OH
3.1. General methods
Optical rotations were determined at 25 °C with a Perkin–El-
mer Model 241-Mc automatic polarimeter. 1H NMR, 13C NMR,
and 1H–1H, 1H–13C COSY spectra were recorded with a Bruker
ARX 400 spectrometer for solutions in CDCl3, MeOD, or
DMSO-d6. Chemical shifts are given in ppm downfield from
internal Me4Si. Mass spectra were measured using a MALDI-
O
O
O
Me
HO
O
O
HO
Me
HO
O
OH
TOF-MS with
a-cyano-4-hydroxycinnamic acid as matrix. Thin-
layer chromatography (TLC) was performed on silica gel HF254
with detection by charring with 30% (v/v) H2SO4 in MeOH, or
by UV detector. Column chromatography was conducted by elu-
tion of a column of silica gel (100 mesh) with EtOAc–petroleum
ether (60–90 °C) as the eluent, or a column of Biogel P2 with
water as the eluent. Solutions were concentrated at <50 °C un-
der reduced pressure.
HO
6
OH
Scheme 2. Designed saponins mimicking natural Di Wu saponins.
N-iodosuccinimide (NIS) in dry CH2Cl2, respectively, were added at
ꢀ15 °C, obtaining 4-methoxyphenyl 2,3,4-tri-O-benzoyl-
rhamnopyranosyl-(1?4)-2,3-di-O-acetyl-6-O-benzoyl-b- -glu-
copyranosyl-(1?6)-2,3,4-tri-O-acetyl-b- -glucopyranoside (10) in
a-L-
D
D
3.2. 4-Methoxyphenyl 2,3,4-tri-O -benzoyl-
syl-(1?4)-2,3-di-O-acetyl-6-O -benzoyl-b- -glucopyranosyl-(1?
6)-2,3,4-tri-O -acetyl-b- -glucopyranoside (10)
a-L-rhamnopyrano-
a total yield of 53%. Treatment of 10 with ceric ammonium nitrate
(CAN) in CH3CN–H2O (4:1, v/v), followed by trichloroacetimidation
(Cl3CCN, DBU, CH2Cl2),27 furnished the trisaccharide donor 11 in
80% yield. Coupling of oleanolic trityl ester 12 with donor 8 was
smoothly completed within 30 min in the presence of a catalytic
amount of TMSOTf at ꢀ35 °C, obtaining key intermediate 13 in
92% yield (Scheme 4). Sugar-ester formation between trityl-ester
13 and trisaccharide donor 11 was achieved using a so-called ‘In-
verse Procedure’28,29 in dry dichloromethane under the promotion
of TMSOTf leading to the fully protected saponin derivative 14 in
D
D
A suspension of 7 (495 mg, 1.2 mmol), 8 (870 mg, 1.4 mmol),
and 4 Å MS in dry CH2Cl2 (15 mL) was stirred at rt for 0.5 h, then
cooled to ꢀ70 °C, and a solution of TMSOTf (30
lL, 0.17 mmol) in
CH2Cl2 (0.2 mL) was added under N2 protection. The reaction mix-
ture was allowed to stir at these conditions for about 3 h, at the end
of which time, TLC (petroleum ether–EtOAc 3:1) indicated a com-
plete consumption of 8. To the above mixture was added

X. Huang et al. / Carbohydrate Research 344 (2009) 1153–1158
1155
OC(NH)CCl3
Me
BzO
O
BzO
OBz
BzO
AcO
8
O
O
O
BzO
a
O
Me
BzO
O
AcO
HO
O
AcO
R2
SEt
AcO
AcO
AcO
BzO
b
OBz
AcO
R1
7
HO
1
= H, R2 = OMP
O
10
11
R
AcO
c
OMP
1
2
AcO
R = OC(NH)CCl3, R = H
AcO
9
Scheme 3. One-pot synthesis of trisaccharide 10. Reagents and conditions: (a) TMSOTf, CH2Cl2, ꢀ70 °C, 3 h; (b) NIS, CH2Cl2, ꢀ15 °C, 2 h, 53% overall yield; (c) CAN, CH3CN–
H2O (4:1, v/v); then Cl3CCN, DBU, CH2Cl2, rt, 80%.
charide 10 as a foamy solid (640 mg, 53% overall yield), which pre-
sented the same physical data as that reported before {½a D25
ꢁ
+75
(c 2, CHCl3), lit.8
½
a 2D5
+74 (c 2.1, CHCl3)}.
ꢁ
OTr
+
8
3.3. 3-O-[2,3,4-Tri-O-benzoyl-a-L-rhamnopyranosyl]oleanolic
O
acid 28-O-trityl ester (13)
HO
12
To a mixture of compounds 8 (220 mg, 0.35 mmol) and 12
(200 mg, 0.29 mmol) in anhyd CH2Cl2 (5 mL) was added TMSOTf
a
(7
l
L, 0.04 mmol) under an N2 atmosphere at ꢀ35 °C. The mixture
was stirred under these conditions for 30 min, at the end of which
time TLC (3:1 petroleum ether–EtOAc) indicated that all starting
materials were consumed. The reaction mixture was neutralized
with Et3N and concentrated. Column chromatography (4:1 petro-
leum ether–EtOAc) of the residue gave 13 as a foamy solid
OTr
O
O
(305 mg, 92%): ½a D25
ꢁ
+45 (c 1, CHCl3); 1H NMR (400 MHz, CDCl3):
Me
BzO
O
d 0.35, 0.82, 0.88, 0.90, 0.91, 1.11, 1.29, 1.31 (8s, 8 ꢂ 3H, 8CH3),
0.78–1.79 (m, 22H), 2.83 (dd, 1H, J 4.0, 13.3 Hz, H-18 of oleanolic
acid), 3.25 (dd, 1H, J 4.6, 11.5 Hz, H-3 of oleanolic acid), 4.28–
4.31 (m, 1H, H-5), 5.07 (d, 1H, J 2.0 Hz, H-1), 5.24 (br s, 1H, H-12
of oleanolic acid), 5.62–5.69 (m, 2H, H-2, H-4), 5.82 (dd, 1H, J 3.2,
BzO
13
OBz
11
10.1 Hz, H-3), 7.20–8.09 (m, 30H, 6Ph). Anal. Calcd for C76H84O10
C, 78.86; H, 7.31; Found: C, 79.14; H, 7.22.
:
b
O
3.4. 3-O-[2,3,4-Tri-O-benzoyl-
acid 28-O-[2,3,4-tri-O-benzoyl-
2,3-di-O-acetyl-6-O-benzoyl-b-
tri-O-acetyl-b- -glucopyranosyl] ester (14)
a
a
-
L
-rhamnopyranosyl]oleanolic
-rhamnopyranosyl-(1?4)-
-glucopyranosyl-(1?6)-2,3,4-
-L
O
OR1
O
D
O
D
O
Me
R2O
O
R2O
O
O
OR1
1
R O
R2O
OR1
To a solution of compound 13 (243 mg, 0.21 mmol) in dry
dichloromethane (5 mL) was added TMSOTf (20 L, 0.11 mmol)
OR2
R1O
l
O
Me
under an N2 atmosphere at 0 °C. The mixture was stirred at this
condition for 1 h, then cooled to ꢀ35 °C, and trisaccharide 11
(306 mg, 0.24 mmol) in anhyd CH2Cl2 (3 mL) was added dropwise.
The reaction mixture was stirred under these conditions for further
30 min, quenched by Et3N, and concentrated. The residue was puri-
fied by silica gel column chromatography (7:2, petroleum ether–
EtOAc) to give compound 14 as a foamy solid (381 mg, 90%):
R2O
R2O
c
OR2
R
R
1 = Ac, R2 = Bz
1 = R2 = H
14
4
Scheme 4. Synthesis of saponin 4. Reagents and conditions: (a) TMSOTf, CH2Cl2,
ꢀ35 °C, 92%; (b) TMSOTf, CH2Cl2, 0 °C, 1 h, then 11, ꢀ35 °C, 30 min, 90%; (c) NaOMe,
CH2Cl2–MeOH, 2:1, v/v, 85%.
½
a 2D5 +98 (c 1.1, CHCl3); 1H NMR (400 MHz, CDCl3): 0.76, 0.85,
ꢁ
0.89, 0.95, 0.99, 1.12, 1.17, 1.29, 1.56 (9s, 9 ꢂ 3H, 9CH3), 0.76–
1.79 (m, 22H), 1.99, 2.00, 2.02, 2.08, 2.11 (5s, 5 ꢂ 3H, 5Ac), 2.80
(dd, 1H, J 4.2, 10.1 Hz, H-18 of oleanolic acid), 3.22 (dd, 1H, J 7.3,
11.6 Hz, H-3 of oleanolic acid), 3.60 (dd, 1H, J 5.6, 11.5 Hz, H-6aGlc),
3.79–3.89 (m, 3H), 4.05 (t, 1H, J 9.3 Hz, H-5Glc), 4.14–4.16 (m, 1H,
H-5Glc), 4.30–4.33 (m, 1H, H-5Rha), 4.60–4.64 (m, 2H), 4.86–4.99
(m, 3H), 5.07 (d, 1H, J 2.1 Hz, H-1Rha), 5.13–5.33 (m, 5H), 5.51 (br
s, 1H, H-12 of oleanolic acid), 5.55–5.80 (m, 6H), 7.23–8.09 (m,
35H, 7Ph). 13C NMR (100 MHz, CDCl3): 175.9, 170.8, 170.7, 170.5,
compound 9 (412 mg, 1.0 mmol) in dry CH2Cl2 (2.5 mL) at ꢀ15 °C,
followed by adding NIS (450 mg, 2.0 mmol), and a further portion
of TMSOTf (10 lL, 0.055 mmol) in CH2Cl2. The reaction mixture
was stirred at these conditions for 2 h, then quenched by Et3N, di-
luted with CH2Cl2, washed with satd aq NaHCO3 and water. The or-
ganic layer was combined, dried, and concentrated. Purification by
column chromatography (1:1 petroleum ether–EtOAc) gave trisac-

1156
X. Huang et al. / Carbohydrate Research 344 (2009) 1153–1158
O
O
O
O
OR
a
Me
RO
OR
13
Me
RO
O
8
+
RO
OR
15 R = Bz
5 R = H
b
Scheme 5. Synthesis of saponin 5. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 1 h, then 8, ꢀ35 °C, 30 min, 93%; (b) NaOMe, CH2Cl2–MeOH, 2:1, v/v, 92%.
32.3, 31.1, 29.9, 28.9, 28.4, 26.2, 26.0, 24.1, 23.5, 21.7, 21.3, 21.2,
18.9, 18.1, 18.0, 17.6, 17.2, 16.0. Anal. Calcd for C113H126O33: C,
67.45; H, 6.31; Found: C, 67.21; H, 6.27.
OH
3.5. 3-O-[a-L-Rhamnopyranosyl]oleanolic acid 28-O-[a-L-
rhamnopyranosyl- (1?4)-b-D-glucopyranosyl-(1?6)-b-D-
O
AcO
16
glucopyranosyl] ester (4)
a
To a solution of 14 (202 mg, 0.1 mmol) in dry CH2Cl2–MeOH
(1:2, v/v, 15 mL) was added 1.0 M NaOMe in MeOH at 0 °C. The
mixture was stirred at rt until all starting materials were con-
sumed, and then neutralized with Dowex H+ resin. The mixture
was filtered and filtrate was concentrated under diminished pres-
sure. The resulting residue was purified by Biogel P2 column chro-
matography using H2O as eluent to afford, after freeze drying,
O
OH
O
RO
compound 4 as an amorphous solid (91 mg, 85%): ½a D25
ꢁ
+7.8 (c 1,
17
18
R = Ac
R = H
b
MeOH); 1H NMR (400 MHz, MeOD): 0.81 (s, 6H, 2CH3), 0.92-0.97
(m, 12H, 4CH3), 1.17 (s, 3H, CH3), 1.24 (d, 3H, J 6.2 Hz), 1.27 (d,
3H, J 6.4 Hz), 0.81ꢀ1.71 m, 22H), 2.86 (dd, 1H, H-18 of oleanolic
acid), 3.09 (dd, 1H, J 6.9, 11.1 Hz, H-3 of oleanolic acid), 3.23–
3.84 (m, 19H), 3.96–3.98 (m, 1H), 4.10 (d, 1H, J 10.8 Hz), 4.74 (d,
1H, J 7.8 Hz), 4.41 (d, 1H, J 7.8 Hz), 5.27 (br s, 1H, H-12 of oleanolic
acid), 5.35 (d, 1H, J 8.0 Hz). 13C NMR (100 MHz, MeOD): 171.2,
144.1, 122.4, 103.0 (C-1), 102.9 (C-1), 101.5 (C-1), 94.3 (C-1),
89.0, 78.7, 77.4, 77.2, 76.0, 75.9, 74.5, 73.3, 73.0, 72.9, 71.8, 71.7,
71.6, 71.4, 79.1, 69.8, 69.1, 68.6, 61.2, 55.4, 46.4, 42.1, 41.7, 39.9,
39.2, 38.9, 37.1, 34.1, 33.1, 32.8, 32.4, 30.8, 28.0, 25.6, 23.4, 18.7,
8
c
O
O
O
Me
RO
O
O
RO
Me
RO
O
OR
19
6
R = Bz
R = H
d
RO
OR
17.1, 17.1, 17.0, 16.3, 15.3. MALDITOF-MS: calcd for C54H88O21
1072.58 [M]+; found, 1095.6 [M+Na]+. Anal. Calcd for C54H88O21
C, 60.43; H, 8.26; Found: C, 60.67; H, 8.32.
:
:
Scheme 6. Synthesis of saponin 6. Reagents and conditions: (a) oxalyl chloride,
CH2Cl2; 1,6-hexanediol, triethylamine, 82%; (b) NaOMe, MeOH, 91%; (c) TMSOTf,
CH2Cl2, ꢀ35 °C, 90%; (d) NaOMe, CH2Cl2–MeOH, 2:1, v/v, 89%.
3.6. 3-O-[2,3,4-Tri-O -benzoyl-
acid 28-O-[2,3,4-tri-O-benzoyl-
a
-
L
-Rhamnopyranosyl]oleanolic
a-L-rhamnopyranosyl] ester (15)
Table 1
Cytotoxicity of compounds 3–6 and oleanolic acid against HL-60 Cellsa
Coupling of compounds 8 (248 mg, 0.4 mmol) and 13 (417 mg,
0.36 mmol) using the same procedure as described in the prepara-
tion of 14 from 11 and 13 obtained compound 15 as a foamy solid
(460 mg, 93%): ½a2D5 +43 (c 1, CHCl3); 1H NMR (400 MHz, CDCl3):
0.79, 0.87, 0.88, 0.90, 1.00, 1.01, 1.14 (7s, 7 ꢂ 3H), 1.26 (d, 3H, J
6.2 Hz, H-6)), 1.30 (d, 3H, J 6.2 Hz, H-6)), 0.73–1.86 (m, 22H),
2.97 (dd, 1H, J 3.6, 13.4 Hz, H-18 of oleanolic acid), 3.17 (dd, 1H, J
6.9, 10.8 Hz, H-3 of oleanolic acid), 4.19–4.23 (m, 2H, 2H-5), 5.02
(d, 1H, J 1.9 Hz, H-1), 5.36 (br s, 1H, H-12 of oleanolic acid),
5.57–5.79 (m, 6H), 6.22 (d, 1H, J 1.8 Hz, H-1), 7.19–8.04 (m, 30H,
6Ph). 13C NMR (100 MHz, CDCl3): 175.4, 166.5, 166.3, 166.2 (2C),
166.1, 165.8, 144.5, 134.2, 134.1, 134.0, 133.8 (2C), 133.6, 131.5,
130.6, 130.5, 130.3, 130.2, 130.0, 129.9, 129.8 (2C), 129.7, 129.4,
129.2 (2C), 129.1, 129.0, 128.9, 128.8, 124.0, 100.3 (C-1), 90.9 (C-
1), 73.4, 72.6, 71.8, 71.1, 70.8, 70.5, 70.3, 69.8, 69.7, 67.4, 66.1,
48.1, 48.0, 46.1, 42.6, 42.5, 40.1, 39.6, 39.1, 37.4, 34.3, 33.7, 33.4,
33.2, 31.4, 31.2, 30.3, 29.0, 28.2, 26.5, 26.0, 24.1, 23.7, 21.6, 19.8,
Entry
Compound
IC50 (lg/mL)
1
2
3
4
5
3
4
5
6
2.7
3.0
20.1
14.5
>30
Oleanolic acid
a
The cells were continuously treated with each sample for 72 h, and the cell
growth was evaluated using MTT reduction assay. Data are mean values of three
experiments performed in triplicate.
170.0, 169.5, 166.5, 166.3, 166.3, 166.2, 166.0, 166.0, 142.0, 134.0,
134.1, 133.8, 133.7, 133.6, 130.5, 130.3, 130.3, 130.1, 130.0, 129.9,
129.8, 129.8, 129.7, 129.1, 129.0, 129.0, 128.8, 124.0, 100.1 (C-1),
99.8 (C-1), 99.7 (C-1), 92.1 (C-1), 89.5, 74.8, 74.5, 73.6, 73.5, 72.6,
72.5, 72.0, 71.8, 71.7, 70.8, 70.6, 70.0, 69.4, 68.7, 68.2, 67.4, 56.0,
54.4, 48.2, 47.3, 46.4, 42.4, 41.7, 39.9, 39.6, 39.0, 37.4, 34.3, 33.5,

X. Huang et al. / Carbohydrate Research 344 (2009) 1153–1158
1157
18.9, 18.4, 18.3, 18.2, 17.8, 17.2, 16.0, 14.3. Anal. Calcd for
C84H92O17: C, 73.45; H, 6.75; Found: C, 73.72; H, 6.68.
tion mixture was stirred under these conditions for 30 min, at
the end of which time TLC (petroleum ether–EtOAc 5:2) indicated
that all starting materials were consumed. The reaction mixture
was neutralized with Et3N and concentrated. Column chromatog-
raphy of the residue gave 19 as a white foam (477 mg, 90%): ½aD25
+89 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): 0.79, 0.88, 0.92,
0.96, 1.00, 1.08, 1.16 (7s, 7 ꢂ 3H, 7CH3), 1.34, 1.39 (2d, 2 ꢂ 3H, J
6.3 Hz, 2H-6Rha), 0.79–1.82 (m, 30H), 2.90 (dd, 1H, H-18 of olean-
olic acid), 3.25 (dd, 1H, H-3 of oleanolic acid), 3.55–3.58 (m, 1H,
CH2OH), 3.81-3.84 (m, 1H, CH2OH), 4.08 (m, 2H, CH2OC@O), 4.20
(m, 1H, H-5Rha), 4.31-4.33 (m, 1H, H-5Rha), 5.03 (d, 1H, J 2.4 Hz,
H-1Rha), 5.09 (d, 1H, J 2.1 Hz, H-1Rha), 5.32 (br s, 1H, H-12 of olean-
olic acid), 5.66-5.88 (m, 6H), 7.28–8.14 (m, 30H, 6Ph). 13C NMR
(100 MHz, CDCl3): 178.4, 166.5, 166.3, 166.2 (2C), 166.1, 166.0,
144.5, 134.0 (2C), 133.9, 133.7 (2C), 130.5, 130.3 (2C), 130.2,
130.1, 130.0, 129.9, 129.8 (2C), 129.4, 129.2, 129.0, 128.9, 124.5,
100.3 (C-1), 98.2 (C-1), 90.0, 73.4, 72.5 (2C), 71.8, 71.6, 70.8, 70.7,
68.9, 67.4, 67.2, 64.8, 56.0, 48.2, 47.3, 46.5, 42.3, 41.9, 40.0, 39.6,
39.0, 37.4, 34.5, 33.7, 33.3, 33.1, 31.3, 30.0, 29.2, 29.0, 28.2, 26.6,
26.4, 26.0, 24.2, 24.1, 23.6, 19.8, 18.9, 18.3, 18.1, 17.7, 17.2, 16.0.
Anal. Calcd for C90H104O18: C, 73.35; H, 7.11; Found: C, 73.06; H,
7.18.
3.7. 3-O-[a-L-Rhamnopyranosyl]oleanolic acid 28-O-[a-L-rhamno-
pyranosyl] ester (5)
Compound 15 (215 mg, 0.16 mmol) was treated with NaOMe,
as described in the preparation of 4, affording compound 5
(108 mg, 92%) as a foamy solid: 1H NMR (400 MHz, MeOD): ½a2D5
+11 (c 1.5, H2O); d 0.79–1.91 (m, 49H), 2.90 (dd, 1H, H-18 of olean-
olic acid), 3.09 (dd, 1H, H-3 of oleanolic acid), 3.35 (t, 1H, J 9.5 Hz),
3.43 (t, 1H, J 9.5 Hz), 3.61–3.72 (m, 4H), 3.75 (br s, 1H), 3.82 (br s,
1H), 4.72 (d, 1H, J 2.1 Hz, H-1), 5.31 (br s, 1H, H-12 of oleanolic
acid), 5.92 (d, 1H, J 2.3 Hz, H-1). 13C NMR (100 MHz, DMSO-d6):
175.3, 143.6, 122.7, 103.1 (C-1), 93.6 (C-1), 87.8, 72.4, 71.8, 71.4,
71.1, 71.0, 70.9, 69.9, 68.8, 55.2, 47.3, 46.9, 45.7, 41.6, 41.5, 38.4,
36.7, 33.5, 33.1, 32.8, 32.5, 30.8, 28.3, 27.35, 25.98, 26.0, 25.3,
23.7, 23.4, 22.8, 18.2, 18.1, 17.3, 16.8, 15.5. MALDITOF-MS: calcd
for C42H68O11: 748.5 [M]+; found, 771.88 [M+Na]+. Anal. Calcd for
C42H68O11: C, 67.35; H, 9.15; Found: C, 67.60; H, 9.08.
3.8. 3-O-Acetyl oleanolic acid 28-O-(6-hydroxyhexyl) ester (17)
To a stirred solution of 16 (500 mg, 1.0 mmol) and N,N-dimeth-
3.11. 3-O-[a-L-Rhamnopyranosyl]oleanolic acid 28-O-[6-O-(a-L-
ylformamide (125
oxalyl chloride (430
l
L, 1.50 mmol) in dry CH2Cl2 (5 mL) was added
L, 5.01 mmol) at room temperature under
rhamnopyrano syl)hexyl] ester (6)
l
nitrogen atmosphere. The reaction mixture was stirred at this con-
dition for 1 h, then the organic layer was transferred into a solution
of 1,6-hexanediol (354 mg, 3.00 mmol), triethylamine (0.85 mL,
6.00 mmol), and 4-methylaminopyridine (15 mg) in dry CH2Cl2
(20 mL) at rt under N2 protection. The mixture was stirred for
6 h, then poured into cold water, and the organic phase was
washed with NaHCO3 aqueous solution, dried over Na2SO4, and
concentrated. The crude product was purified by flash column
chromatography (petroleum ether–EtOAc 4:1) to give 17
(491 mg, 82%) as an amorphous solid: ½a2D5 +65 (c 0.5, CHCl3); 1H
NMR (400 MHz, CDCl3): 0.74, 0.86, 0.87, 0.90, 0.93, 0.93, 1.13 (7s,
7 ꢂ 3H, 7CH3), 0.74–1.87 (m, 30H), 2.05 (s, 3H, Ac), 2.87 (dd, 1H,
J 4.0, 13.8 Hz, H-18 of oleanolic acid), 4.49 (t, 1H, J 8.1 Hz, H-3 of
oleanolic acid), 3.63-3.67 (m, 2H, CH2OH), 3.99–4.03 (m, 2H,
CH2OC@O), 5.28 (br s, 1H, H-12 of oleanolic acid). Anal. Calcd for
C38H62O5: C, 76.21; H, 10.43; Found: C, 75.96; H, 10.40.
To a solution of 19 (148 mg, 0.1 mmol) in dry CH2Cl2/MeOH
(1:2, v/v, 9 mL) was added 1 M NaOMe (12 L, 0.012 mmol) in
l
MeOH. The mixture was stirred at rt until all starting materials
were consumed, and then neutralized with Dowex-50 (H+) ion
exchange resin. Filtration, concentration of the filtrate, and puri-
fication of the resulting residue by LH-20 column chromatogra-
phy using H2O as eluent afforded compound 6 (76 mg, 89%) as
a white powder: ½a2D5 +6 (c 1.2, water); 1H NMR (400 MHz,
MeOD): 0.77, 0.80, 0.92, 0.95, 0.96, 0.97, 1.18 (7s, 7 ꢂ 3H,
7CH3), 1.24, 1.27 (2d, 2 ꢂ 3H, J 6.3 Hz, H-6Rha), 0.77–2.02 (m,
30H), 2.91 (dd, 1H, H-18 of oleanolic acid), 3.10 (dd, 1H, J 4.6,
11.5 Hz, H-3 of oleanolic acid), 3.31–3.41 (m, 3H), 3.56–3.72
(m, 5H), 3.79–3.82 (m, 1H), 3.83–3.85 (m, 1H), 4.03 (t, 2H, J
6.2 Hz), 4.66 (d, 1H, J 1.6 Hz, H-1Rha), 4.74 (d, 1H, J 1.4 Hz,
H-1Rha), 5.27 (br s, 1H, H-12 of oleanolic acid). 13C NMR
(100 MHz, MeOD): 178.7, 144.3, 123.0, 103.6 (C-1), 100.8 (C-1),
89.5, 73.3, 73.2, 71.7, 71.5, 69.1, 68.9, 67.6, 64.7, 56.8, 47.6,
42.1, 42.0, 39.9, 39.2, 38.8, 37.1, 34.0, 33.1, 32.9, 32.7, 30.8,
29.9, 28.9, 28.0, 26.3, 26.1, 25.8, 25.6, 23.8, 23.2, 18.6, 17.3,
3.9. Oleanolic acid 28-O-(6-hydroxyhexyl) ester (18)
1 M NaOMe was added into a solution of compound 17 (443 mg,
0.74 mmol) in MeOH/CH2Cl2 (3 mL/7 mL). The reaction mixture
was stirred under reflux for 72 h at pH 9.5, then neutralized with
Dowex-50 (H+) ion exchange resin. The mixture was filtered, the
filtrate concentrated, and the resulting residue was purified by sil-
ica gel column chromatography (petroleum ether–EtOAc 2:1) to af-
ford compound 18 as a white foam (375 mg, 91%): ½a2D5 +72 (c 1,
CHCl3); 1H NMR (400 MHz, CDCl3): 0.73, 0.77, 0.90, 0.90, 0.92,
0.98, 1.13 (7s, 7 ꢂ 3H, 7CH3), 0.73–2.01 (m, 30H), 2.86 (dd, 1H, J
4.4, 13.9 Hz, H-18 of oleanolic acid), 3.21 (dd, 1H, J 4.3, 10.8 Hz,
H-3 of oleanolic acid), 3.63–3.67 (m, 2H, CH2OH), 3.98–4.03 (m,
2H, CH2OC@O), 5.27 (br s, 1H, H-12 of oleanolic acid). Anal. Calcd
for C36H60O4: C, 77.65; H, 10.86; Found: C, 77.91; H, 10.78.
17.1, 17.0, 16.2, 15.2. MALDITOF-MS: calcd for C48H80O12
848.56 [M]+; found, 871.72 [M+Na]+. Anal. Calcd for C48H80O12
C, 67.89; H, 9.50; Found: C, 67.63; H, 9.44.
:
:
3.12. Cell culture assay
HL-60 cells were maintained in the RPMI 1640 medium con-
taining 10% fetal bovin serum supplemented with -glutamine,
100 units/mL penicillin, and 100 g/mL streptomycin. The leuke-
L
l
mia cells were washed and suspended in the above medium to
1 ꢂ 105 cells/mL, and 100
lL of this cell suspension was placed
in each well of
a
96-well plate. The cells were incubated
L/mL of
DMSO–H2O (1:20, v/v) solution containing the sample was
added to give the final concentration of 0.1–30 L/mL, while
L/mL of DMSO–H2O (1:20, v/v) was added into control
in 5% CO2/air for 24 h at 37 °C. After incubation, 3
l
3.10. 3-O-[2,3,4-Tri-O-benzoyl-
a-
L-Rhamnopyranosyl]oleanolic
l
acid 28-O-[6-O -(2,3,4-tri-O -benzoyl-
a
-
L
-rhamnopyranosyl)-
3 l
hexyl] ester (19)
wells. The cells were further incubated for 72 h in the presence
of each agent, and then cell growth was evaluated using
To a solution of compounds 8 (480 mg, 0.77 mmol) and 18
modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(200 mg, 0.36 mmol) in anhyd CH2Cl2 (5 mL) was added TMSOTf
bromide (MTT) reduction assay, and the IC50 values were calcu-
(16
l
L, 0.09 mmol) under an N2 atmosphere at ꢀ35 °C. The reac-
lated accordingly.31,32

1158
X. Huang et al. / Carbohydrate Research 344 (2009) 1153–1158
12. Chang, L. C.; Tsai, T. R.; Wang, J. J.; Lin, C. N.; Kuo, K. W. Biochem. Biophys. Res.
Acknowledgment
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This work was supported in partial by NNSF of China (Projects
20621703, 20872172, and 20732001).
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Supplementary data
19. Deng, S.; Yu, B.; Hui, Y. Tetrahedron Lett. 1998, 39, 6511–6514.
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