Synthesis and cytotoxicity of oleanolic acid trisaccharide saponins

Article abstract of DOI:10.1016/j.carres.2017.02.010

An array of oleanolic acid-type saponins based on β-hederin has been synthesized in a linear or one-pot manner. The cell viability assays indicate that synthetic saponins show antiproliferation activities in three cancer cell lines with IC₅₀ values of 2.4–15.1?μM and hederacolchiside A₁ being the most potent. The results demonstrate that the type of terminal monosaccharides and linkage position have apparent effects on cytotoxicities and selectivities of these saponins against cancer cell lines tested. This study is helpful for future development of more potent anticancer leads.

Full text of DOI:10.1016/j.carres.2017.02.010

Carbohydrate Research 442 (2017) 9e16
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and cytotoxicity of oleanolic acid trisaccharide saponins
,
b
,
1
,
,
c
,
, **
Liming Wang a
2, Zengshang Wang a 1, Sheng Su a, Ying Xing c, Yali Li c
,
Ming Li b ***, Jiangyun Liu a *, Shilin Yang a
,
,
a College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
b Key Lab of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
c Department of the VIP Internal Medicine, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, China
a r t i c l e i n f o
a b s t r a c t
Article history:
An array of oleanolic acid-type saponins based on b-hederin has been synthesized in a linear or one-pot
Received 2 February 2017
Received in revised form
27 February 2017
Accepted 27 February 2017
Available online 28 February 2017
manner. The cell viability assays indicate that synthetic saponins show antiproliferation activities in three
cancer cell lines with IC50 values of 2.4e15.1 M and hederacolchiside A1 being the most potent. The
results demonstrate that the type of terminal monosaccharides and linkage position have apparent ef-
fects on cytotoxicities and selectivities of these saponins against cancer cell lines tested. This study is
helpful for future development of more potent anticancer leads.
m
© 2017 Published by Elsevier Ltd.
Keywords:
Triterpenoid saponin
b
-hederin
Glycosylation
Antiproliferation activity
1. Introduction
over the past few decades.
Studies on structure-activity relationship of bioactive natural
Saponins are a vast group of naturally occurring plant glyco-
sides, characterized by their strong foam-forming properties in
aqueous solution. Saponins show diverse biological effects
including anti-tumor [1,2], anti-inflamatory, anti-diabetic and anti-
fungal functions [3]. They are also the key ingredients of traditional
Chinese medicines and other folk medicines worldwide. Among
products are crucial steps for discovering new lead compounds. In
recent years, we have been devoted to discovering pharmacologi-
cally anticancer agents. A diverse range of oleanane-type saponins
has been identified from various traditional Chinese medicines,
including Pulsatilla chinensis [16,17] and Patrinia scabiosaefolia
Fisch. [18,19] Previous studies on synthesis [20,21] and isolation
them, oleanane-type saponins such as
b
-hederin, hederacolchiside
[7e9] have revealed that
potential antitumor agents. Herein we report synthesis and cyto-
toxiticity evaluation of a family of saponins based on -hederin
(1e11, Fig. 1), which can help future development of more prom-
ising anticancer agents.
b-hederin and hederacolchiside A1 are
A1 (HA1) from Ivy [4], and other memebers from Pulsatilla chinensis
[5] and Anemone flaccida [6] have drawn considerable attention due
to their strong anti-tumor activities. Because of micro-
heterogenecity and limited amounts of saponins from natural
sources, natural saponins can't meet the current needs for biolog-
ical evaluation. Thus, total synthesis of complex saponins has
beenan active field [7e15], and has witnessed a significant progress
b
2. Results and discussion
2.1. Chemistry
* Corresponding author.
** Corresponding author.
*** Corresponding author.
Based on the structures of
classified into two types. The first class constitutes
b
-hederin, saponins 1e11 can be
-hederin (1)
b
E-mail addresses: lylemai@sina.cn (Y. Li), lmsnouc@ouc.edu.cn (M. Li),
liujiangyun@suda.edu.cn (J. Liu).
and its advanced analogues (2e7) featuring the different mono-
saccharide at 4-OH of the arabinosyl unit. The second class is
composed of saponins 8e11 with sugar chain extension at 3-OH of
1
These authors contributed equally to this work.
Current address: Central Institute for Engineering, Electronics and Analytics,
2
the rhanmosyl moiety of
b-hederin. The syntheses of saponins 1e4
Analytics (ZEA-3), Jülich Research Center, Wilhelm Johnen Straße, Jülich 52425,
Germany.
have been accomplished using a linear strategy by Cheng's group
http://dx.doi.org/10.1016/j.carres.2017.02.010
0008-6215/© 2017 Published by Elsevier Ltd.

10
L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
Fig. 1. Structures of saponins 1e11.
[7,8]. Following that protocol, our synthesis commenced with the
preparation of -hederin (Scheme 1). Glycosyl tri-
chloroacetimidates (13a e13f, Fig. 2) were prepared as efficient
sugar donors according to the previously reported procedures
[22e24]. Briefly, glycosylation of benzyl oleanolate 12 [7] with
perbenzoylated arabinosyl trichloroacetimidate 13a under catalytic
monosaccharide 14 in excellent yield [25]. Debenzoylation of 14
using methanolic NaOCH3 in the presence of CH2Cl2 followed by
isopropylidene formation with 2,2-dimethoxypropane mediated by
p-toluenesulfonic acid hydrate (p-TsOH$H2O) proceeded smoothly
to furnish saponin 15 with 2-OH of arabinosyl moiety free and
ready for subsequent glycosylation. Coupling of compound 15 with
13b was carried out to generate 16 under catalytic amount of
b
trimethylsilyl
trifluoromethanesulfonate
(TMSOTf)
gave
Scheme 1. Synthesis of saponins 1e7. Reagents and conditions: (a) TMSOTf, CH2Cl2, 4 Å MS, 0 ꢀC; (b) i) NaOCH3, CH2Cl2-MeOH, r. t; ii) Me2C(OMe)2, p-TsOH$H2O, acetone, 0 ꢀC to rt;
(c) BF3. Et2O, CH2Cl2, 4 Å MS, ꢁ78 ꢀC; (d) p-TsOH$H2O, CH2Cl2-MeOH, rt; (e) TMSOTf, CH2Cl2, 4 Å MS, ꢁ30 ꢀC, 75%, 72%, 69%, 72%, 70% and 65% for 18a-18f, respectively; (f) i) 10% Pd-
C, H2, CH2Cl2-MeOH; ii) NaOCH3, CH2Cl2-MeOH, rt, 86%, 80.4%, 82%, 78%, 80%, 76% and 77.5% for 1e7.

L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
11
Fig. 2. Structures of glycosyl trichloroacetimidates 13ae13f used in this work.
BF3,Et2O. It should be noted that the reaction had to be conducted
at ꢁ78 ꢀC to prevent the cleavage of isopropylidene group. 16 was
treated with methanol/p-TsOH$H2O to give 17 in 94% yield. Ben-
HL-7702 as reference. The results are listed in Table 1. By compar-
isons of their IC50 values with that of -hederin (1), most of com-
pounds tested showed good antiproliferation effects against 3
cancer lines (2.4e15.1 M), with HA1 (4) being the most potent (IC50
2.4e4.5 M). On the other hand, they all possessed comparable
toxicities against normal HL-7702 cell either, but with relative
lower activity (higher than 9.2 M) by comparisons with -hederin
(4.9 M). These results were in agreement with previous reports
[7,9,13]. As for the same terminal monosaccharide, compounds of
type 1 usually showed stronger activities than that of type 2, such
as comparisons between compounds 4 and 10. However, com-
pounds 3 (possessed little cytotoxity) and 9 with terminal rham-
nosyl moieties did not follow the same trend. As for saponins of the
same type such as 2e7, different compounds showed different
cytotoxicities and selectivities on the cell lines tested with similar
trend. In general, as for SMMC-7721 cell, different terminal
monosaccharides affect antiproliferation activities in following or-
b
ꢀ
zoates of 17 were cleaved under Zemplen conditions to afford the
m
desired
b-hederin (1).
m
It has been observed that arabinopyranosyl ring in 17 changes
from 4C1 to 1C4 conformation leaving 4-OH in equatorial orientation
[8]. This arrangement makes 4-OH sterically less hindered and
more accessible than vicinal 3-OH in axial orientation in the ara-
binosyl unit, thus offering opportunities for regioselective glyco-
sylation at 4-OH. With this assumption in mind, diol 17 was
subjected to TMSOTf-catalyzed glycosylation with glycosyl tri-
chloroacetimidates 13a-13f. The reactions proceeded uneventfully
at ꢁ30 ꢀC to regioselectively afford the expected triglycosides
18ae18f, respectively, in satisfactory yields. Finally, benzyl group
and benzoyl groups of 18ae18f were sequentially removed by
hydrogenolysis and transesterification to give the corresponding
saponins 2e7.
m
b
m
der: Glc (4) > Ara (2) > Xyl (5) > Man (7), b-hederin > Gal (6) [ Rha
One-pot strategy has proven to be a highly efficient approach to
synthesis of complex triterpenoid saponins [10,11,26,27]. Encour-
aged by these advances, we employed this strategy to synthesize
saponins 8e11, as shown in Scheme 2. TMSOTf-catalyzed coupling
of 2-methyl-5-tert-butylphenyl-rhamnosyl thioglycoside 19 [10,28]
with glycosyl trichloroacetimidate 13a was conducted at ꢁ78 ꢀC to
provide the desired disaccharyl thioglycoside intermediate 20a.
When the reaction went to completion, the mixture was warmed
to ꢁ10 ꢀC followed by sequential addition of glycosyl acceptor 17, N-
iodosuccinimide (NIS), and another portion of TMSOTf (0.5 equiv).
The reaction was stirred for another hour, thus providing protected
saponin 18a in 75% yield in a one-pot manner after work-up.
Analogously, replacement of 13a with 13be13d led to trisaccha-
ride saponins 21ae21d via disaccharide intermediates 20be20d.
Isopropylidene moiety of 21ae21d was removed with p-TsOH-
catalyzed transacetalization in CH3OH/CH2Cl2 to afford 22ae22d.
Similar to 15, the arabinopyranosyl unit in these saponins also as-
sumes a 1C4 chair conformation. Finally, benzyl group and benzoyl
groups of 22ae22d were removed to afford saponins 8e11. Their
structures were determined by extensive NMR experiments and by
comparing spectroscopic data with those of analogues in the
literature [11,29,30].
(3). These results suggested that many saponins within the same
herbal extract (such as Pulsatilla chinensis) played certain roles and
contributed together to the overall anti-tumor effect, while being
less toxic than the single one [5]. It was well recognized that hae-
molytic activity, cytotoxicity and membrane cell permeabilization
were prevailed in such kinds of saponins [9,13], and glycosylations
played important role in their bioactivities and selectivities
[12e15]. The follow-up studies, such as anti-tumor effect in vivo,
mechanisms of action and pharmacokinetics, should be conducted
on these derivatives.
3. Conclusions
In summary, an array of triterpenoid saponins based on
b-
hederin has been synthesized using either a linear or a one-pot
glycosylation strategy. The anticancer activities of the synthesized
saponins were evaluated in vitro against four cell lines. The results
demonstrate that the nature of the terminal monosaccharide and
its position of its glycosidic bond exert a certain influence on the
antiproliferation activities and selectivities of these compounds,
thus providing information for future development of better anti-
cancer agents.
2.2. Cell viability assay
4. Experimental section
The antiproliferation effects of compounds 1e11 were assessed
using MTT assay as reported [5,21]. Three human cancer cell lines,
SMMC-7721, Bel-7402, and A-549 were tested with normal cell line
4.1. Chemicals and general methods
Commercial reagents were used without further purification

12
L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
Scheme 2. Synthesis of saponins 8e11. Reagents and conditions: (a) i) 0.2 equiv TMSOTf, CH2Cl2, 4 Å MS, ꢁ78 ꢀC; (b) 2.0 eq NIS, 0.5 eq TMSOTf, CH2Cl2,4 Å MS, ꢁ10 ꢀC, 66%, 62%,
60.3%, and 64.5% for 21a-21d, respectively, over two steps; (c) TsOH, CH2Cl2/MeOH; (d) i) 10% Pd-C, H2, CH2Cl2-MeOH; ii) NaOCH3, CH2Cl2 - MeOH, rt, 77%, 74%, 75%, and 78% for
8e11, respectively, over two steps.
Table 1
were prepared according to literature methods [7,8,22e25].
In vitro cytotoxicity of compound 1e11 against 3 cancer cell lines and normal cell line
HL-7702.
4.2.1. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-a-L-
arabinopyranoside (14)
Compound
IC50 (m
M)a
A suspension of 12 (1212 mg, 2.23 mmol), 13a (1553 mg,
2.56 mmol), and powdered 4A molecular sieves (2.30 g) were
stirred for 30 min at 0 ꢀC in dry CH2Cl2 (50 mL) and then cooled
SMMC-7721
Bel-7402
A-549
HL-7702
1
2
3
4
5
6
7
8
7.49 0.22
3.47 0.11
15.13 0.56
3.07 0.09
4.84 0.18
9.55 0.25
7.63 0.31
4.77 0.17
9.57 0.33
4.39 0.15
8.71 0.29
10.95 0.39
9.63 0.28
>100
7.65 0.31
4.21 0.11
41.00 1.9
2.43 0.07
8.86 0.31
9.82 0.39
13.61 0.38
4.60 0.13
5.60 0.17
3.78 0.15
5.54 0.23
4.87 0.17
10.85 0.41
37.51 1.77
13.95 0.59
15.00 0.65
10.80 0.50
12.85 0.53
9.20 0.38
11.65 0.51
11.70 0.55
9.27 0.39
to ꢁ30 ꢀC. TMSOTf (2.7
mL, 0.015 mmol) was added and the mixture
was stirred at ꢁ30 ꢀC for 3 h before the reaction was quenched by
Et3N (0.3 mL). The suspension was then filtered, concentrated and
purified by a silica gel column (10:1, petroleum ether - EtOAc) to
afford 14 (1950 mg, 89%). Rf ¼ 0.45 (5:1, petroleum ether - EtOAc.
4.57 0.19
8.48 0.32
10.66 0.39
10.79 0.31
8.80 0.36
8.40 0.27
4.98 0.22
8.58 0.25
[
a
]20D þ107.6 (c 1.58, CHCl3); 1H NMR (600 MHz, CDCl3)
d:
9
10
11
8.09e7.26 (m, 20H, aromatic H), 5.76 (dd, J 8.8, 6.6, 1H, H-20), 5.67
(m, 1H, H-40), 5.58 (dd, J 8.8, 3.5, 1H, H-30), 5.30 (t, J 3 .0, 1H, H-12),
5.06 (dd, J 19.9, 12.6, 2H, PhCH2), 4.79 (d, J 6.6, 1H, H-10), 4.33 (dd, J
12.9, 3.8,1H, H-50-1), 3.87 (m, 1H, H-50-2), 3.16 (dd, J 11.0, 4.9,1H, H-
3), 2.9 (dd, J 10.1, 3.2, 1H, H-18), 1.11, 0.92, 0.90, 0.86, 0.79, 0.66, 0.58
(s each, 3H each, 7 ꢂ CH3).
a
Data represent mean values
standard deviation for three independent
experiments.
unless specialized. Solvents were dried and redistilled prior to use
in the usual way. Thin layer chromatography (TLC) were performed
on precoated silica gel plates 60 F254 (Qingdao Haiyang Chemical
Co., LTD), silica gel (200e300 mesh, Qingdao Haiyang Chemical Co.,
4.2.2. Benzyl oleanolate 3-O-3, 4 -O-isopropylidene-a-L-
arabinopyranoside (15)
To a solution of 14 (1927 mg, 1.95 mmol) in dry CH2Cl2/MeOH
(1:2, 60 mL) was added NaOCH3 in MeOH solution (0.5 mol/L,
3.57 mL). The mixture was stirred at r.t. for 2 h and neutralized with
Dowcx Hþ resin to pH 7 and then filtered. The filtrate was
concentrated and the resulting residue was purified by a silica gel
China) and Cosmosil C18 reversed-phase silica gel (40e60
mm,
Japan) were used for column chromatography. The spray reagent
was the ethanolic H2SO4 (10%). Optical rotations were determined
with a PerkineElmer PL341 polarimeter. Mass spectra were per-
formed with an 4500 QTRAP (AB SCIEX, Concord, Canada) or a
TripleTOF 5600 (AB Sciex, Foster City, CA) mass spectrometry sys-
tem in positive electrospray ionization (ESI) mode. 1H and 13C NMR
spectra were taken on a Jeol JNM-ECP 600 spectrometer.
column (EtOAc) to gave Benzyl oleanolate 3-O-
anoside (1230 mg, 93%) as a white amorphous solid; To a solution of
Benzyl oleanolate 3-O- -L-arabinopyranoside (1000 mg,
a-L-arabinopyr-
a
1.47 mmol) in dry acetone(30 mL) stirred at 0 ꢀC was added
Me2C(OMe)2 (0.5 mL, 4.16 mmol) and TsOH$H2O (40 mg,
0.21 mmol).The mixture was stirred for 5 h at r.t before the reaction
was quenched by Et3N (0.5 mL). The solvent was evaporated in
vacuum and purified through a silica gel column(6:1, petroleum
4.2. Synthesis
Benzyl oleanolate 12 and glycosyl trichloroacetimidates 13aef

L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
13
ether -EtOAc) to afford 15 (845 mg, 80%) as a white powder.
Rf ¼ 0.50(petroleum ether -EtOAc, 4:1); [ ]20D þ45.3 (c 1.55,
CHCl3); 1H NMR (600 MHz, CDCl3)
7.38e7.35 (m, 5H, aromatic H),
3H,H-600),1.13, 1.11, 0.93, 0.93, 0.90, 0.63(s each, 3H each, 7 ꢂ CH3).
a
d
4.2.6. Benzyl oleanolate 3 - O - 2,3,4- tri- O- benzoyl- a-Le
5.28 (t, J 3.0, 1H, H-12), 5.08 (dd, J 18.7,12.6, 2H, PhCH2), 4.22e4.17
(m, 3H, H-10, H-40, H-50-1), 4.06 (dd, J 7.8, 6.1, 1H, H-30), 3.73 (dd, J
13.9, 3.5, 1H, H-50-2), 3.63 (dd, J 7 .8, 7.8, 1H, H-20), 3 .12 (dd, J 11.5,
4.6,1H, H-3), 2.89 (dd, J 13.8, 3.3,1H, H-18), 2.30 (m,1H),1.54,1.36 (s
each, 3H each, Oe(CH3)2CeO), 1.12, 0.98, 0.92, 0 .89, 0.88, 0.80, 0.59
(s each, 3H each, 7 ꢂ CH3).
rhamnopyranosyl -(1 / 2) - [2,3,4,6-tetra-O-benzoyl-b-
Deglucopyranosyl-(1 / 4)]-a-Learabinopyranoside (18c)
Compound 18c was prepared in 69% yield from 17 (200 mg,
0.176 mmol) and 13c (156 mg, 0.211 mmol), purified by silica gel
chromatography using petroleum ether -EtOAc (3:1) as the eluant.
Rf ¼ 0.36 (3:1, petroleum ether - EtOAc). [
a
]20D þ55.3 (c 0.52,
CHCl3). 1H NMR (600 MHz, CDCl3)
d
8.08e7.23 (m, 40H, aromatic H),
4.2.3. Benzyl oleanolate 3- O ꢁ2,3,4-tri-O-benzoyl-
a
-L-
5.94 (t, J 9.7 Hz,1H, H-3000), 5.79 (dd, J 10.2, 3.4 Hz,1H, H-300), 5.71 (t, J
9.7 Hz, 1H), 5.65e5.57 (m, 3H), 5.29 (s, 1H, H-12), 5.20 (s, 1H, H-100),
5.13 (d, J 7.8 Hz, 1H, H-1000), 5.07 (dd, J 33.5, 12.6 Hz, 2H, PhCH2), 4.71
(dd, J 12.1, 3.2 Hz, 1H), 4.65 (d, J 3.8 Hz, 1H, H-10), 4.52 (dd, J 12.0,
4.9 Hz, 1H), 4.37e4.28 (m, 1H), 4.28e4.21 (m, 1H), 4.08 (dd, J 12.1,
7.6 Hz, 2H), 3.90 (s, 1H), 3.86e3.81 (m, 1H), 3.69 (d, J 8.7 Hz, 1H),
3.10 (dd, J 11.6, 4.6 Hz, 1H, H-3), 2.91 (dd, J 15.0, 3.8 Hz, 1H, H-18),
1.29 (d, J 6.3 Hz, 3H, H-600), 1.11, 1.03, 0.92, 0.90, 0.86, 0.81, 0.60 (s
each, 3H each, 7 ꢂ CH3).
rhamnopyranosyl-(1 / 2)- -L earabinopyranoside (17)
a
A solution of compound 16 (470 mg, 0.40 mmol) and p-
TsOH$H2O (75 mg, 0.39 mmol) in CH2Cl2/MeOH (1:2, 30 ml) was
stirred at r. t. . When TLC (petroleum ether - EtOAc, 2:1) showed
that deprotection had completed, the reaction was quenched by
Et3N (0.3 mL).Then the solvent was evaporated in vacuum and
purified by a silica gel column (3:1, petroleum ether -EtOAc) to
afford 17 (427 mg, 94%) as a white powder. Rf ¼ 0.27 (2:1, petroleum
ether -EtOAc); [
CDCl3)
a
]20D þ80.0 (c 2.50, CHCl3); 1H NMR (600 MHz,
d
8.12e7.23 (m, 20H, aromatic H)), 5.84 (dd, J 10.2, 3.2,1H, H-
4.2.7. Benzyl oleanolate 3 - O - 2,3,4- tri- O- benzoyl-
a-Le
300), 5.65 (m,2H, H-200, H-400), 5.37 (s, 1H, H-100), 5.30 (t, J 3.0, 1H, H-
12), 5.07 (dd, J 18.7,12.6, 2H, PhCH2), 4.79 (d, J 1.3,1 H, H-10), 4.34 (m,
1H, H-500), 4.12e3.96 (m, 3H, H-20,H-40,OH), 3.82 (m, 1H, H-50-1),
3.67 (m, 1H, H-50-2), 3.45 (d, J 7.9, 1H, H-30), 3.18 (dd, J 11.0, 3.2, 1H,
H-3), 2.90 (m, 1H, H-18), 2.52 (br s, 1H, OH), 1.34 (d, J 6.1, 3H, H-600),
1.12, 1.05, 0.92, 0.89, 0.88, 0.84, 0.60 (s each, 3H each, 7 ꢂ CH3).
rhamnopyranosyl -(1 / 2) - [2,3,4,6-tetra-O-benzoyl-b
xylopyranosyl-(1 / 4)]-a-Learabinopyranoside (18d)
-D-
Compound 18d was prepared in 72% yield from 17 (200 mg,
0.176 mmol) and 13d (128 mg, 0.211 mmol), purified by silica gel
chromatography using petroleum ether - EtOAc (4:1) as the eluant.
Rf ¼ 0.39 (3:1, petroleum ether - EtOAc). 1H NMR (600 MHz, CDCl3)
d
8.08e7.27 (m, 35H, aromatic H), 5.86e5.80 (m, 2H), 5.69 (dd, J 3.0,
4.2.4. Benzyl oleanolate 3 - O - 2,3,4- tri- O- benzoyl-
rhamnopyranosyl -(1 / 2) - [2,3,4,6-tetra-O-benzoyl-a-
Learabinopyranosyl-(1 / 4)]-a-Learabinopyranoside(18a)
A suspension of 17 (200 mg, 0.176 mmol), 13a (128 mg,
0.211 mmol, 1.2eq) and powdered 4A molecular sieves (300 mg) in
dry CH2Cl2 (30 mL) was stirred at r.t. for 30 min and then cooled
a
-Le
1.5 Hz, 1H), 5.64 (t, J 10.0 Hz, 1H), 5.43 (dd, J 7.2, 5.2 Hz, 1H), 5.35 (s,
1H), 5.33 (d, J 4.5 Hz, 1H, H-100), 5.30 (t, J 3.1 Hz, 1H, H-12), 5.12 (s,
1H), 5.10 (d, J 7.9 Hz, 1H, H-1000), 5.05 (d, J 12.6 Hz, 1H, PhCH2), 4.67
(d, J 4.4 Hz, 1H, H-10), 4.54 (dd, J 12.3, 4.2 Hz, 1H), 4.37 (dd, J 9.8,
6.2 Hz, 1H), 4.12 (dd, J 11.9, 6.4 Hz, 1H), 4.06 (d, J 3.0 Hz, 1H), 3.97 (s,
1H), 3.92e3.88 (m, 1H), 3.81 (dd, J 12.3, 6.6 Hz, 1H), 3.68 (dd, J 11.9,
2.7 Hz, 1H), 3.17 (dd, J 11.8, 4.3 Hz, 1H), 3.05 (d, J 9.9 Hz, 1H), 2.92
(dd, J 13.6, 3.6 Hz, 1H), 1.28 (d, J 6.3 Hz, 3H, H-600), 1.13, 1.10, 0.93,
0.90, 0.89, 0.62(s each, 3H each, 7 ꢂ CH3).
to ꢁ30 ꢀC. TMSOTf (30
mL, 0.155 mmol) was added and the mixture
was stirred at ꢁ30 ꢀC for 3 h before the reaction was quenched by
Et3N (0.1 mL). The suspension was then filtered and the filtrate was
concentrated and purified by a silica gel column (3:1, petroleum
ether - EtOAc) to afford 18a (209 mg, 75%) as a white powder.
Rf ¼ 0.38 (3:1, petroleum ether - EtOAc). 1H NMR (600 MHz, CDCl3)
4.2.8. Benzyl oleanolate 3 - O - 2,3,4- tri- O- benzoyl-
a-Le
rhamnopyranosyl -(1 / 2) - [2,3,4,6-tetra-O-benzoyl-
b
-D-
d
8.09e7.26 (m, 35H, aromatic H),5.83e5.77 (m, 2H), 5.72 (dd, J 5.9,
xylopyranosyl-(1 / 4)]-a-Learabinopyranoside (18e)
3.6 Hz, 1H), 5.64 (m, 3H), 5.30e5.27 (m, 1H, H-12), 5.26 (s, 1H,H-100),
5.10 (d, J 12.5 Hz, 1H,H-1000), 5.05 (d, J 7.8 Hz, 1H, PhCH2), 5.04 (d, J
6.1 Hz,1H, PhCH2), 4.70 (d, J 4.0 Hz,1H, H-10), 4.38 (dd, J 12.9, 4.0 Hz,
1H), 4.33 (dd, J 9.9, 6.3 Hz, 1H), 4.11 (m, 2H), 4.03e3.98 (m, 1H), 3.95
(d, J 4.2 Hz, 1H), 3.89 (dd, J 5.8, 4.1 Hz, 1H), 3.74 (dd, J 11.4, 2.9 Hz,
1H), 3.14 (dd, J 11.9, 4.4 Hz,1H), 3.07 (d, J 9.5 Hz,1H), 2.90 (dd, J 13.5,
4.3 Hz, 1H, H-18), 1.27 (d, J 6.3 Hz, 3H, H-600), 1.11, 1.06, 0.92, 0.89,
0.88, 0.85, 0.61(s each, 3H each, 7 ꢂ CH3).
Compound 18e was prepared in 70% yield from 17 (200 mg,
0.176 mmol) and 13e (156 mg, 0.211 mmol), purified by silica gel
chromatography using petroleum ether -EtOAc (4:1) as the eluant.
Rf ¼ 0.35 (3:1, petroleum ether -EtOAc). 1H NMR (600 MHz, CDCl3)
d
8.14e7.22(m, 40H, aromatic H) , 6.05 (d, J 3.4 Hz, 1H), 5.89 (dd, J
10.3, 8.0 Hz,1H), 5.81 (dd, J 10.2, 3.4 Hz,1H), 5.67e5.61 (m, 3H), 5.31
(t, J 3.4 Hz, 1H, H-12), 5.19 (s, 1H, H-100), 5.14e5.09 (m, 2H, H-1000,
PhCH2), 5.05 (d, J 12.5 Hz, 1H, PhCH2), 5.05 (d, J 12.5 Hz, 1H),
4.73e4.70 (m, 1H), 4.70 (d, J 4.0 Hz, 1H, H-10), 4.52e4.40 (m, 2H),
4.38e4.27 (m, 1H), 4.16 (dd, J 11.7, 7.3 Hz, 1H), 4.10 (dt, J 7.0, 3.4 Hz,
1H), 3.90 (d, J 23.3 Hz, 1H), 3.89e3.84 (m, 1H), 3.81e3.69 (m, 1H),
3.14 (dd, J 11.8, 4.5 Hz, 1H, H-3), 2.92 (dd, J 13.6, 4.2 Hz, 1H, H-18),
1.31 (d, J 6.2 Hz, 3H, H-600), 1.13, 1.06, 0.93, 0.90, 0.89, 0.84, 0.62(s
each, 3H each, 7 ꢂ CH3).
4.2.5. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-
Lerhamnopyranosyl-(1 / 2)-[2,3,4,6- tetra -O-benzoyl-
Lerhamnopyranosyl-(1 / 4)]- -Learabinopyranoside (18b)
a-
a-
a
Compounds 18b-f were synthesized following the procedure for
synthesis of 18a. 18b was prepared in 72% yield from 17 (200 mg,
0.176 mmol) and 13b (131 mg, 0.211 mmol), purified by silica gel
chromatography using petroleum ether - EtOAc (5:1) as the eluant.
Rf ¼ 0.62 (3:1, petroleum ether - EtOAc). 1H NMR (600 MHz, CDCl3)
4.2.9. Benzyl oleanolate 3 - O - 2,3,4- tri- O- benzoyl-
a-Le
rhamnopyranosyl -(1 / 2) - [2,3,4,6-tetra-O-benzoyl-
b
-D-
d
8.11e7.23 (m, 35H,aromatic H),5.88 (ddd, J 10.2, 3.4, 1.4 Hz, 2H),
xylopyranosyl-(1 / 4)]-a-Learabinopyranoside (18f)
5.78 (ddd, J 16.5, 3.3,1.7 Hz, 2H), 5.70 (t, J 10.0 Hz, 2H), 5.49 (s, 1H,H-
1000), 5.30 (s, 2H,H-12,H-100), 5.08 (dd, J 30.5,12.6 Hz, 2H,PhCH2), 4.79
(d, J 3.8 Hz, 1H,H-10), 4.41 (dq, J 12.8, 6.3 Hz, 2H), 4.17 (ddd, J 9.9, 9.4,
5.5 Hz, 3H), 4.07 (dd, J 5.7, 4.0 Hz, 1H), 3.72 (dd, J 11.6, 2.9 Hz, 1H),
3.61 (d, J 11.0 Hz, 1H), 3.22 (dd, J 11.6, 4.6 Hz, 1H,H-3), 2.92 (dd, J
13.9, 3.9 Hz, 1H,H-18), 1.42 (d, J 6.2 Hz, 3H,H-6000), 1.39 (d, J 6.2 Hz,
Compound 18f was prepared in 65% yield from 17 (200 mg,
0.176 mmol) and 13c (156 mg, 0.211 mmol), purified by silica gel
chromatography using petroleum ether -EtOAc (3:1) as the eluant.
Rf ¼ 0.38 (3:1, petroleum ether - EtOAc). 1H NMR (600 MHz, CDCl3)
d
8.14e7.21 (m, 40H, aromatic H) , 6.11 (t, J 10.2 Hz, 1H), 6.00 (dd, J
10.2, 3.3 Hz, 1H), 5.80 (dd, J 10.2, 3.4 Hz, 1H), 5.77 (dd, J 3.2, 1.8 Hz,

14
L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
1H), 5.69e5.64 (m, 2H), 5.38 (d, J 1.5 Hz, 1H, H-100), 5.31 (t, J 3.7 Hz,
1H,H-12), 5.16 (s, 1H, H-1000), 5.08 (dd, J 29.9, 12.6 Hz, 2H,PhCH2),
4.94e4.87 (m, 2H), 4.77 (dd, J 12.3, 2.3 Hz, 1H, H-10), 4.53 (dd, J 12.2,
4.5 Hz, 1H), 4.34e4.26 (m, 2H), 4.25e4.19 (m, 1H), 4.14e4.04 (m,
1H), 3.81e3.71 (m, 2H), 3.17 (dd, J 11.5, 4.7 Hz, 1H, H-3), 2.92 (dd, J
13.7, 4.2 Hz, 1H, H-18), 1.35 (d, J 6.3 Hz, 3H, H-600), 1.13, 1.01, 0.94,
0.93, 0.90, 0.88, 0.63(s each, 3H each, 7 ꢂ CH3).
10.1, 5.9 Hz, 1H), 3.74e3.63 (m, 2H), 3.06 (dd, J 11.5, 4.3 Hz, 1H, H-3),
2.91 (dd, J 13.1, 2.9 Hz, 1H, H-18), 2.10, 1.76(s each, 3H each, Ac ꢂ 2),
1.52, 1.32 (s each, 3H each, Oe(CH3)2CeO), 1.13 (d, J 6.2 Hz, 3H, H-
600), 1.10, 1.00, 0.92, 0.90, 0.88, 0.81, 0.61(s each, 3H each, 7 ꢂ CH3).
4.2.13. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoy-
Dexylopyranosyl-(1 / 3)-2,4-di-O-acetyl - -L-rhamnopyranosyl-
(1 / 2)-3,4-di-O-benzoyl- -Learabinopyranoside (21d)
b-
a
4.2.10. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-
arabinopyranosyl-(1 / 3)-2,4-di-O- acetyl- -L-rhamnopyranosyl-
(1 / 2)-3,4-di-O-benzoyl- -D- -Learabinopyranoside (21a)
a-L-
a
a
Compound 21d was prepared in 64.5% yield from 19 (50 mg,
0.122 mmol), 13d (148 mg, 0.244 mmol), and 15 (88 mg,
0.122 mmol), purified by silica gel chromatography using petro-
leum ether- EtOAc (4:1) as the eluant. 1H NMR (600 MHz, CDCl3)
b
a
Rhamnosyl thioglycoside 19 [10,11] (50 mg, 0.122 mmol) was
condensed with corresponding compound 13a (148 mg,
0.244 mmol, 2.0 eq) with the use of catalytic amount of TMSOTf (0.2
eq) in CH2Cl2 (10 mL) at ꢁ78 ꢀC for 30 min, providing the desired
disaccharide Mbp thioglycoside donor 20a. Then, the reaction
mixture was warmed to ꢁ10 ꢀC, the 15 (88 mg, 0.122 mmol), N-
iodosuccinimide (NIS, 50 mg, 0.11 mmol), and TMSOTf (0.5 eq) were
sequentially added. After the resulting mixture was stirred for 1 h,
the reaction was quenched with Et3N (0.10 ml) followed by puri-
fication with silica gel column (petroleum ether - EtOAc, 3:1) to
afford the fully protected saponin 21a (122 mg, 66%). 1H NMR
d
8.04e7.29 (m, 20H, aromatic H), 5.73 (t, J 7.8 Hz, 1H, H-30), 5.36
(dd, J 3.4, 1.7 Hz, 1H, H-200), 5.32 (s, 1H, H-100), 5.30e5.25 (m, 3H, H-
12, H-20, H-40), 5.13e5.04 (m, 3H, PhCH2, H-400), 4.87 (d, J 5.1 Hz, 1H,
H-10), 4.37 (dd, J 12.4, 4.1 Hz, 1H), 4.31 (d, J 7.5 Hz, 1H, H-1000), 4.22
(dd, J 10.3, 3.1 Hz, 1H), 4.20e4.11 (m, 3H), 4.02 (dd, J 8.9, 6.2 Hz, 1H),
3.73 (dd, J 9.6, 8.2 Hz, 2H), 3.65 (dd, J 12.9, 6.1 Hz, 1H), 3.06 (dd, J
11.5, 4.3 Hz, 1H, H-3), 2.91 (dd, J 13.1, 2.9 Hz, 1H, H-18), 2.10, 1.76(s
each, 3H each, Ac ꢂ 2), 1.52, 1.32(s each, 3H each, Oe(CH3)2CeO),
1.13 (d, J 6.2 Hz, 3H, H-600), 1.10, 1.00, 0.92, 0.90, 0.88, 0.81, 0.61 (s
each, 3H each, 7 ꢂ CH3).
(600 MHz, CDCl3) d 8.03e7.35 (m, 20H, aromatic H), 5.65 (d, J 5.4 Hz,
1H), 5.61 (dd, J 7.8, 3.4 Hz, 1H), 5.57e5.52 (m, 1H), 5.37e5.35 (m,
1H), 5.32 (s,1H, H-100), 5.29 (t, J 3.3 Hz,1H, H-12), 5.11 (s,1H), 5.10 (d,
J 3.0 Hz, 1H, PhCH2), 5.07 (d, J 12.6 Hz, 1H, PhCH2), 4.82 (d, J 5.2 Hz,
1H, H-10), 4.31 (d, J 7.5 Hz, 1H, H-1000), 4.24 (dd, J 10.0, 3.4 Hz, 1H),
4.22e4.11 (m, 5H), 4.01 (dq, J 12.6, 6.2 Hz, 1H), 3.82 (dd, J 12.5,
2.7 Hz, 1H), 3.75e3.69 (m, 2H), 3.06 (dd, J 11.5, 4.3 Hz, 1H, H-3), 2.91
(dd, J 13.1, 2.9 Hz, 1H, H-18), 2.10, 1.76(s each, 3H each, Ac ꢂ 2), 1.52,
1.32(s each, 3H each, Oe(CH3)2CeO),1.13 (d, J 6.2 Hz, 3H, H-600),1.10,
1.00, 0.92, 0.90, 0.88, 0.81, 0.6(s each, 3H each, 7 ꢂ CH3).
4.2.14. Oleanolic acid 3- O- a-L-rhamnopyranosyl-(1 / 2)-a-L-
arabinopyranoside (1)
To a solution of 17 (170 mg, 0.150 mmol) in CH2Cl2/MeOH (1:1,
30 mL), 10% Pd-C (110 mg) was added and stired under H2 over-
night, then filtered and concentrated when the reaction was
completed as monitored by TLC (petroleum ether e EtOAc,1:1). The
residue was dissolved in CH2Cl2 eCH3OH (1:2, 30 mL), NaOCH3
(150 mg) was then added and stirred at room temperature for 4 h,
neutralized with Dowex ion-exchange resin (Hþ), and then filtered
and concentrated. The residue was purified through silica gel
4.2.11. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoyl-
rhamnopyranosyl-(1 / 3)-2,4-di-O- acetyl- -L-rhamnopyranosyl-
(1 / 2)-3,4-di-O-benzoyl- -D- -Learabinopyranoside (21b)
a-L-
a
b
a
chromatography (CHCl3-MeOH, 6:1) to afford 1 (
86% yield). Rf ¼ 0.32 (6:1, CHCl3-MeOH). [ ]20D þ4.7 (c 0.60,
CH3OH). 1H NMR (600 MHz, pyridine-d5) 6.12 (s,1H, H-100), 5.43 (s,
b-hederin, 95 mg,
Compounds 21b-d were synthesized following the procedure
for synthesis of 21a. 21b was prepared in 62% yield from 19 (50 mg,
0.122 mmol), 13b (151 mg, 0.244 mmol), and 15 (88 mg,
0.122 mmol), purified by silica gel chromatography using petro-
leum ether - EtOAc (4:1) as the eluant. 1H NMR (600 MHz, CDCl3)
a
d
1H, H-12), 4.86 (d, J 5.4 Hz, 1H, H-10), 4.72 (dd, J 3.4, 1.6 Hz, 1H), 4.60
(dd, J 9.3, 3.4 Hz, 1H), 4.58e4.50 (m, 2H), 4.32e4.21 (m, 4H), 3.80
(dd, J 11.5, 2.2 Hz, 1H), 3.26 (dd, J 13.9, 4.4 Hz, 1H, H-3), 3.21 (dd, J
11.7, 4.3 Hz, 1H, H-18), 1.59 (d, J 6.2 Hz, 3H, H-600), 1.26, 1.14, 1.03,
0.97, 0.95, 0.93, 0.80(s each, 3H each, 7 ꢂ CH3); 13C NMR (150 MHz,
d
8.11e7.26(m, 20H, aromatic H), 5.69 (dd, J 10.1, 3.1 Hz, 1H),
5.67e5.63 (m, 1H), 5.43(dd, J 2.8, 1.9 Hz, 1H, H-2000), 5.41 (s, 1H), 5.34
(s, 1H, H-100), 5.28 (t, J 3.3 Hz, 1H, H-12), 5.21 (t, J 9.9 Hz, 1H), 5.15 (d,
J 1.2 Hz, 1H), 5.07 (q, J 12.5 Hz, 2H, PhCH2), 4.33 (d, J 5.7 Hz, 1H, H-
1000), 4.31 (d, J 3.4 Hz, 1H, H-10), 4.26 (dq, J 12.5, 6.2 Hz, 1H), 4.19 (dt, J
5.7, 2.9 Hz, 1H), 4.17e4.14 (m, 2H), 4.13e4.07 (m, 2H), 3.78e3.70 (m,
2H), 3.07 (dd, J 11.7, 4.4 Hz, 1H, H-3), 2.90 (dd, J 13.7, 4.0 Hz, 1H, H-
18), 2.30, 2.20(s each, 3H each, Ac ꢂ 2), 1.55, 1.32 (s each, 3H each,
Oe(CH3)2CeO), 1.31 (d, J 6.6 Hz, 3H, H-6000), 1.23 (d, J 6.2 Hz, 3H, H-
600), 1.10, 1.00, 0.92,0.89, 0.88, 0.84, 0.59 (s each, 3H each, 7 ꢂ CH3).
pyridine-d5)
d
180.2(C-28), 144.8(C-13), 122.5(C-12), 104.9(C-10),
101.7(C-100), 88.7(C-3), 75.9, 74.0, 73.9, 72.6, 72.4, 69.9, 68.8, 64.8,
55.9, 48.0, 46.7, 46.5, 42.1, 42.0, 39.7, 39.5, 38.8, 37.0, 34.2, 33.3, 33.1,
31.0, 28.3, 28.1, 26.5, 26.2, 23.8, 23.7, 18.6, 18.5, 17.4, 17.0, 15.5. ESI
-MS (m/z): 787.5 ([MþNa]þ).
4.2.15. Oleanolic acid 3-O-
a
-L-rhamnopyranosyl-(1 / 2)-[
a-
Learabinopyranosyl-(1 / 4)] -a- Learabinopyranoside (2)
4.2.12. Benzyl oleanolate 3-O-2,3,4-tri-O-benzoy-
glucopyranosyl-(1 / 3)-2,4-di-O-acetyl - -L-rhamnopyranosyl-
(1 / 2)-3,4-di-O-benzoyl- -Learabinopyranoside (21c)
b
-D-
Compounds 2e11 were synthesized following the procedure for
synthesis of 1. 2 was prepared in 80.4% yield from 18a (200 mg,
0.127 mmol), purified by silica gel chromatography using CHCl3 -
a
a
Compound 21c was prepared in 60.3% yield from 19 (50 mg,
0.122 mmol), 13c (180 mg, 0.244 mmol), and 15 (88 mg,
0.122 mmol), purified by silica gel chromatography using petro-
leum ether - EtOAc (3:1) as the eluant. 1H NMR (600 MHz, CDCl3)
CH3OH (V/V ¼ 3:1) as the eluant. [
a
]20D þ1.6 (c 0.22, CH3OH). 1H
NMR (600 MHz, pyridine- d5): d
6.12 (br s, 1H, H-100), 5.42 (br s, 1H,
H-12), 4.88 (d, 1H, J 7.8, H-10), 4.76 (d, 1H, J 6.0, H-1000), 1.60 (d, 3H, J
6.2, H-600), 1.25, 1.15, 1.07, 0.97, 0.94, 0.92, 0.79 (each s, each 3H,
d
8.04e7.24 (m, 25H, aromatic H), 5.90 (t, J 9.7 Hz, 1H, H-3000), 5.67 (t,
7 ꢂ CH3). 13C NMR (150 MHz, pyridine-d5):
d 180.2 (C-28), 144.8 (C-
J 9.7 Hz, 1H, H-4000), 5.49 (dd, J 9.8, 7.8 Hz, 1H, H-2000), 5.39e5.33 (m,
1H, H-200), 5.30 (d, J 1.3 Hz, 2H, H-100,H-12), 5.09 (q, J 12.5 Hz, 2H,
PhCH2), 5.02 (t, J 9.8 Hz, 1H, H-400), 4.86 (d, J 7.8 Hz, 1H, H-1000),
4.57e4.48 (m, 2H, H-6000), 4.18 (s, 1H), 4.16 (d, J 4.2 Hz, 1H, H-10),
4.15e4.13 (m, 1H), 4.12e4.07 (m, 2H), 4.07e4.02 (m, 1H), 3.94 (dd, J
13), 122.6 (C-12), 107.0 (C-1000), 105.0 (C-10), 101.8 (C-100), 88.8 (C-3),
79.3, 76.4, 74.9, 74.1, 72.9, 72.6, 72.4, 69.9, 67.5, 64.6, 56.0, 48.1, 46.7,
46.5, 42.2, 42.0, 39.8, 39.5, 38.9, 37.1, 34.3, 33.3, 33.2, 31.0, 28.4, 28.1,
26.7, 26.2, 23.8, 23.7, 18.7, 18.6, 17.4, 17.1, 15.6. ESI-MS (m/z): 889.5
([MþNa]þ).

L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
15
4.2.16. Oleanolic acid 3-O-
rhamnopyranosyl-(1 / 4)]-
Compound 3 was prepared in 82% yield from 18b (200 mg,
0.125 mmol), purified by silica gel chromatography using CHCl3
CH3OH (3:1) as the eluant. [
]20D -14.8 (c 0.21, CH3OH). 1H NMR
(600 MHz, pyridine- d5)
a
-L-rhamnopyranosyl-(1 / 2)-[
- Learabinopyranoside (3)
a
-L-
2.2, H-1000), 5.43 (m, 1H, H-12), 4.92 (d, 1H, J 6.3, H-10), 1.64 (d, 3H, J
6.2, H-600), 1.28, 1.09, 1.00, 0.98, 0.97, 0.94, 0.80 (each s, 3H, 7 ꢂ CH3).
a
13C NMR (150 MHz, pyridine- d5):
d 180.2 (C-28), 144.9 (C-13), 122.7
-
(C-12), 104.5 (C-1000), 101.9 (C-10), 100.3 (C-100), 89.2 (C-3), 76.1, 74.1,
73.1, 72.6, 72.4, 71.3, 69.9, 64.6, 62.6, 56.0, 48.1, 46.7, 46.5, 42.2, 42.0,
39.8, 39.5, 38.9, 37.1, 34.3, 33.3, 33.2, 31.0, 28.4, 28.1, 26.6, 26.2, 23.8,
a
d
5.97 (br s, 1H, H-100), 5.72 (br s, 1H, H-1000),
5.43 (br s, 1H, H-12), 4.87 (d, 1H, J 6.0, H-10), 1.64 (d, 3H, J 5.8, H-600),
23.7,18.7,18.6,17.4,17.0,15.6. ESI-MS (m/z): 919.5 ([MþNa]þ), HRMS
þ
1.58 (d, 3H, J 5.8, H-6000), 1.26, 1.13, 1.02, 0.97, 0.95, 0.92, 0.78 (each s,
(m/z): calcd for [C47H76NaO16
919.5029.
]
([MþNa]þ) 919.5026, found:
each 3H, 7 ꢂ CH3); 13C NMR (150 MHz, pyridine-d5):
d
180.3 (C-28),
144.8 (C-13), 122.5 (C-12), 104.8 (C-10), 102.6 (C-1000), 102.0 (C-100),
89.0 (C-3), 76.4, 75.0, 74.1, 72.6, 72.4, 72.2, 72.1, 70.3, 70.2, 63.0, 55.9,
39.8, 39.5, 38.9, 37.0, 34.3, 33.3, 33.2, 31.0, 28.4, 28.1, 26.6, 26.2, 23.8,
23.7, 18.7, 18.6, 17.4, 16.9, 15.5. ESI-MS (m/z): 903.5 ([MþNa]þ).
4.2.21. Oleanolic acid 3-O-[
rhamnopyranosyl- (1 / 2)] -
Compound 8 was prepared in 77% yield from 21a (200 mg,
a
-Learabinopyranosyl -(1 / 3) -
a-L-
a-Learabinopyranoside (8)
0.127 mmol), purified by silica gel charomatography using CHCl3
-
4.2.17. Oleanolic acid 3-O-
a
-L-rhamnopyranosyl-(1 / 2)-[
b
-
CH3OH (3:1) as the eluant. [
a
]20D þ2.9 (c 0.24, CH3OH). 1H NMR
Deglucopyranosyl -(1 / 4)] -
a-L earabinopyranoside (4)
(600 MHz, pyridine-d5): d
6.25 (br s, 1H, H-100), 5.44 (m, 1H, H-12),
Compound 4 was prepared in 78% yield from 18c (200 mg,
0.117 mmol) by purification by silica gel chromatography using
5.28 (d, 1H, J 7.4, H-1000), 4.84 (d, 1H, J 6.0, H-10), 1.51 (d, 3H, J 6.0, H-
600), 1.30, 1.29, 1.14, 0.98, 0.97, 0.92, 0.81 (each s, 3H, 7 ꢂ CH3). 13C
CHCl3 - CH3OH (3:1) as the eluant. [
NMR (600 MHz, pyridine- d5):
a
]20D -2.4 (c 0.14, CH3OH). 1H
NMR (150 MHz, pyridine-d5): d 180.4 (C-28), 145.0 (C-13), 122.7 (C-
d
6.13 (br s, 1H, H-100), 5.43 (m, 1H, H-
12), 107.5 (C-1000), 105.5 (C-10), 101.6 (C-100), 88.8 (C-3), 82.9, 75.4,
74.6, 73.2, 73.1, 72.0, 69.7, 69.6, 67.2, 56.0, 48.1, 46.7, 42.0, 39.8, 39.6,
37.0, 33.2, 31.0, 28.2, 26.2, 24.0, 18.6, 17.4, 17.2, 15.5. ESI-MS (m/z):
889.5 ([MþNa]þ).
12), 5.09 (d, 1H, J 7.8, H-1000), 4.74 (d, 1H, J 6.0, H-10), 1.64 (d, 3H, J 6.2,
H-600), 1.28, 1.17, 1.10, 1.00, 0.97, 0.94, 0.82 (each s, 3H, 7 ꢂ CH3). 13C
NMR (150 MHz, pyridine-d5): d 180.2 (C-28), 144.8 (C-13), 122.6 (C-
12), 106.4 (C-1000), 105.0 (C-10), 101.8 (C-100), 88.7 (C-3), 79.7, 78.8,
78.6, 76.4, 75.5, 74.1, 74.1, 72.5, 72.4, 71.3, 69.9, 64.6, 62.6, 56.0, 48.1,
46.7, 46.5, 42.2, 42.0, 39.8, 39.5, 38.9, 37.1, 34.3, 33.3, 33.2, 31.0, 28.4,
28.1, 26.6, 26.2, 23.8, 23.7, 18.6, 18.4, 17.3, 17.0, 15.5. ESI-MS (m/z):
919.5 ([MþNa]þ).
4.2.22. Oleanolic acid 3-O-[
rhamnopyranosyl- (1 / 2)] -
Compound 9 was prepared in 74% yield from 21b (200 mg,
0.127 mmol), purified by silica gel charomatography using CHCl3
CH3OH (3:1) as the eluant. [
]20D -14.2 (c 0.17, CH3OH).1H NMR
(600 MHz, pyridine-d5):
a
-L-rhamnopyranosyl-(1 / 3)-
a-L-
a-Learabinopyranoside (9)
-
a
4.2.18. Oleanolic acid 3-O-
Dexylopyranosyl-(1 / 4)]-
Compound 5 was prepared in 80% yield from 18d (200 mg,
0.127 mmol), purified by silica gel chromatography using CHCl3
CH3OH (3:1) as the eluant. [
]20D -6.4 (c 0.21, CH3OH). 1H NMR
(600 MHz, pyridine-d5):
6.14 (br s, 1H, H-100), 5.42 (m, 1H, H-12),
a
-L-rhamnopyranosyl-(1 / 2)-[
b
-
d
6.23 (br s, 1H, H-100), 6.04 (br s,1H, H-1000),
a- Learabinopyranoside (5)
5.43 (br s, 1H, H-12), 4.87 (d, 1H, J 6.3, H-10), 1.60 (d, 3H, J 6.2, H-600),
1.58 (d, 3H, J 6.2, H-6000), 1.27, 1.26, 1.09, 0.97, 0.95, 0.92, 0.79 (each s,
-
3H, 7 ꢂ CH3). 13C NMR (150 MHz, pyridine- d5):
d 180.4 (C-28),145.0
a
(C-13), 122.7 (C-12), 107.7 (C-1000), 105.5 (C-10), 101.7 (C-100), 88.9 (C-
3), 83.2, 78.7, 75.8, 75.5, 73.2, 72.3, 71.3, 69.9, 69.6, 67.7, 56.2, 48.2,
46.9, 42.2, 39.9, 39.7, 37.1, 33.5, 33.3, 31.5, 28.4, 26.4, 24.0, 18.6, 17.6,
17.4, 15.7. ESI-MS (m/z): 903.5 ([MþNa]þ).
d
4.95 (d, 1H, J 7.8, H-1000), 4.76 (d, 1H, J 6.0, H-10), 1.60 (d, 3H, J 6.2, H-
600), 1.25, 1.15, 1.07, 0.97, 0.94, 0.92, 0.79 (each s, each 3H, 7 ꢂ CH3).
13C NMR (150 MHz, pyridine- d5):
d 180.2 (C-28), 144.8 (C-13), 122.6
(C-12), 106.9 (C-1000), 105.0 (C-10), 101.8 (C-100), 88.7(C-3), 79.3, 76.4,
74.9, 74.1, 72.9, 72.6, 72.4, 69.9, 67.5, 67.4, 55.9, 48.1, 46.7, 46.5, 42.2,
42.0, 39.8, 39.5, 38.9, 37.1, 34.3, 33.3, 33.2, 31.0, 28.4, 28.1, 26.6, 26.2,
23.8, 23.7, 18.7, 18.5, 17.3, 17.0, 15.6. ESI-MS (m/z): 889.5 ([MþNa]þ).
4.2.23. Oleanolic acid 3-O-[
rhamnopyranosyl- (1 / 2)] -
Compound 10 was prepared in 75% yield from 21c (200 mg,
0.127 mmol), purified by silica gel chromatography using CHCl3
CH3OH (3:1) as the eluant. [
]20D -7.8 (c 0.26, CH3OH). 1H NMR
(600 MHz, pyridine- d5):
6.19 (br s, 1H, H-100), 5.49 (d, 1H, J 7.8, H-
b
-Deglucopyranosyl-(1 / 3)-
a-L-
a- Learabinopyranoside (10)
-
a
4.2.19. Oleanolic acid 3-O-
Degalactopyranosyl -(1 / 4)] -
Compound 6 was prepared in 76% yield from 18e (200 mg,
0.117 mmol), purified by silica gel chromatography using CHCl3
CH3OH (V/V ¼ 3:1) as the eluant. [
]20D -3.2 (c 0.20, CH3OH). 1H
NMR (600 MHz, pyridine- d5):
6.10 (br s, 1H, H-100), 5.43 (br s, 1H,
a-L-rhamnopyranosyl-(1 / 2)-[
b-
d
a-Learabinopyranoside (6)
1000), 5.48 (br s,1H, H-12), 4.86 (d, 1H, J 6.0, H-10), 1.58 (d, 3H, J 6.0, H-
600), 1.35, 1.33, 1.15, 1.03, 0.99, 0.98, 0.85 (each s, 3H, 7 ꢂ CH3). 13C
-
NMR (150 MHz, pyridine- d5): d 180.0 (C-28), 144.6 (C-13), 122.4 (C-
a
12), 105.0 (C-10), 104.1 (C-1000), 101.5 (C-100), 88.6 (C-3), 79.6, 75.5,
74.6, 74.0, 72.6, 72.2, 71.9, 69.9, 69.8, 69.7, 55.9, 47.9, 46.5, 42.0, 41.9,
39.5, 39.3, 36.8, 33.1, 33.0, 30.8, 28.1, 26.0, 23.6, 18.5, 17.1, 17.0, 15.4.
ESI-MS (m/z): 919.5 ([MþNa]þ).
d
H-12), 5.00 (d, 1H, J 7.8, H-1000), 4.69 (d, 1H, J 6.0, H-10), 1.76 (d, 3H, J
6.2, H-600), 1.28, 1.17, 1.10, 1.00, 0.97, 0.94, 0.82 (each s, 3H, 7 ꢂ CH3).
13C NMR (150 MHz, pyridine- d5):
d 180.1 (C-28), 144.7 (C-13), 122.5
(C-12), 106.3 (C-1000), 104.9 (C-10), 101.7 (C-100), 88.6 (C-3), 79.6, 78.7,
78.5, 76.3, 75.5, 74.0, 72.4, 72.3, 71.2, 69.9, 64.6, 62.6, 56.0, 48.1, 46.7,
46.5, 42.2, 42.0, 39.8, 39.5, 38.9, 37.1, 34.3, 33.3, 33.2, 31.0, 28.3, 28.0,
4.2.24. Oleanolic acid 3-O-[
rhamnopyranosyl- (1 / 2)]-
Compound 11 was prepared in 78% yield from 21d (200 mg,
0.127 mmol), purified by silica gel chromatography using CHCl3
CH3OH (3:1) as the eluant.[
]20D -5.1 (c 0.24, CH3OH). 1H NMR
(600 MHz, pyridine-d5):
6.30 (br s, 1H, H-100), 5.49 (m, 1H, H-12),
b
-D-xylopyranosyl -(1 / 3) -
a-L-
a- Learabinopyranoside (11)
26.5, 26.1, 23.7, 23.6, 18.7, 18.5, 17.2, 17.0, 15.4. ESI-MS (m/z): 919.5
-
þ
([MþNa]þ). HRMS (m/z): calcd for [C47H76NaO16
]
([MþNa]þ)
a
919.5026, found: 919.5030.
d
5.39 (d, 1H, J 7.4, H-1000), 4.89 (d, 1H, J 6.0, H-10), 1.57 (d, 3H, J 6.0, H-
600), 1.34, 1.33, 1.16, 1.03, 0.99, 0.98, 0.85 (each s, 3H, 7 ꢂ CH3). 13C
4.2.20. Oleanolic acid 3-O-
Demannopyranosyl -(1 / 4)] -
a
-L-rhamnopyranosyl-(1 / 2)-[
-Learabinopyranoside (7)
b-
a
NMR (150 MHz, pyridine- d5): d 180.4 (C-28), 145.0 (C-13), 122.7 (C-
Compound 7 was prepared in 77.5% yield from 18f (200 mg,
0.117 mmol) by purification by silica gel chromatography using
12), 107.7 (C-1000), 105.7 (C-10), 101.7 (C-100), 88.9 (C-3), 83.2, 78.7,
75.8, 75.5, 73.2, 72.3, 71.3, 69.9, 69.6, 67.7, 56.2, 48.2, 46.9, 42.2,
39.9, 39.7, 37.2, 33.5, 33.3, 31.5, 28.4, 26.4, 24.0, 18.7, 17.6, 17.4, 15.7.
ESI-MS (m/z): 889.5 ([MþNa]þ).
CHCl3 - CH3OH (3:1) as the eluant. [
a
]20D -4.1 (c 0.18, CH3OH). 1H
NMR (600 MHz, pyridine- d5)
d
5.92 (br s, 1H, H-100), 5.61 (d, 1H, J

16
L. Wang et al. / Carbohydrate Research 442 (2017) 9e16
4.3. Cell viability assays
References
[1] S. Man, W. Gao, Y. Zhang, L. Huang, C. Liu, Fitoterapia 81 (2010) 703e714.
4.3.1. Cell culture
[2] I. Podolak, A. Galanty, D. Sobolewska, Phytochem. Rev. 9 (2010) 425e474.
[3] S.G. Sparg, M.E. Light, J. van Staden, J. Ethnopharmacol. 94 (2004) 219e243.
[4] C. Barthomeuf, E. Debiton, V. Mshvildadze, E. Kemertelidze, G. Balansard,
Planta Med. 68 (2002) 672e675.
[5] Q.-M. Xu, Z. Shu, W.-J. He, L.-Y. Chen, S.-L. Yang, G. Yang, X.-R. Li, Phytome-
dicine 19 (2012) 293e300.
[6] J.-C. Lu, B.-B. Xu, S. Gao, L. Fan, H.-F. Zhang, R.-X. Liu, H. Kodama, Fitoterapia 80
(2009) 345e348.
[7] M.-S. Cheng, M.-C. Yan, Y. Liu, L.-G. Zheng, J. Liu, Carbohydr. Res. 341 (2006)
60e67.
The human cancer cell lines SMMC-7721, Bel-7402 and A-549,
and normal cell line HL-7702 were purchased from Shanghai In-
stitutes for Biological Sciences and maintained in a humidified at-
mosphere at 37 ꢀC in 5% CO2. The cells were grown in RPMI-1640
(GIBCO) media containing 10% (for cancer cell lines) or 20% (for
HL-7702) heat-inactivated fetal bovine serum (FBS).
4.3.2. Cell viability assay
[8] M.-C. Yan, Y. Liu, W.-X. Lu, H. Wang, Y. Sha, M.-S. Cheng, Carbohydr. Res. 343
(2008) 780e784.
The effect of compound 1e11 pre-treatment on cell viability was
assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay as reported [5,21]. Briefly, cells
were seeded in 96-well microtiter plates at a density of 1 ꢂ 104 cells
per well. After 24 h incubation, cells were treated with various
ꢀ
[9] M. Chwalek, N. Lalun, H. Bobichon, K. Ple, L. Voutquenne-Nazabadioko, Bio-
chim. Biophys. Acta 1760 (2006) 1418e1427.
[10] Q. Liu, P. Wang, L. Zhang, T. Guo, G. Lv, Y. Li, Carbohydr. Res. 344 (2009)
1276e1281.
[11] Q. Liu, H. Liu, L. Zhang, G. Guo, P. Wang, M. Geng, Y. Li, Eur. J. Med. Chem. 64
(2013) 1e15.
[12] T. Guo, S. Wu, S. Guo, L. Bai, Q. Liu, N. Bai, Pharm. Med. Chem. 348 (2015)
615e628.
[13] C. Gauthier, J. Legault, K. Girard-Lalancette, V. Mshvildadze, A. Pichette, Bio-
org. Med. Chem. 17 (2009) 2002e2008.
[14] A.M. Daines, B.W. Greatrex, C.M. Hayman, S.M. Hook, W.T. Mcburney, T. Rades,
I.M. Sims, Bioorg. Med. Chem. 17 (2009) 5207e5218.
[15] J. Xu, X. Nie, Y. Hong, Y. Jiang, G. Wu, X. Yin, X. Wang, Carbohydr. Res. 424
(2006) 42e53.
[16] Z. Shu, Z. Chen, X.-J. Ding, B.-Q. Lu, C.-J. Ji, Q. Xu, X.-J. Ding, Heterocycles 83
(2011) 2365e2372.
[17] Z. Shu, Z. Chen, Y.-L. Liu, W.-F. Zhu, Y.-L. Feng, Q.-M. Xu, S.-L. Yang, Nat. Prod.
Res. 27 (2013) 2196e2201.
concentrations (generally 1.25e40
solved in DMSO), cultured for 48 h. At the end of the treatment
period, 20 L of the MTT (5 mg/mL) reagent was added to each well.
After 1 h incubation at 37 ꢀC for 1 h. Supernatants were removed
from the wells, and the MTT dye was solubilized in 200 L/well
mM) of compound 1e11 (dis-
m
m
DMSO. Absorbance was measured at 540 nm with an ELISA plate
reader. Each condition was performed with 6 replicates and all
assays were performed in triplicate. The 50% inhibitory concen-
tration (IC50) values were then determined for each compound
from a plot of log (drug concentration) versus percentage of loss of
viability.
[18] L. Gao, L. Zhang, N. Li, J.-Y. Liu, P.-L. Cai, S.-L. Yang, Carbohydr. Res. 346 (2011)
2881e2885.
[19] L. Gao, L. Zhang, L. Wang, J.-Y. Liu, P.-L. Cai, S.-L. Yang, J. Asian Nat. Prod. Res.
14 (2012) 333e341.
[20] Z. Chen, H.-Q. Duan, M.-L. Wang, L. Han, Y.-L. Liu, Y.-M. Zhu, S.-L. Yang, Bioorg.
Med. Chem. Lett. 25 (2015) 2550e2554.
Conflict of interests
[21] Y.-Y. Fang, Z.-H. Yang, H. Ouyang, R.-K. Wang, J. Li, H.-S. Huang, Y. Jin, Y.-
L. Feng, S.-L. Yang, Bioorg. Med. Chem. Lett. 26 (2016) 4576e4579.
[22] R.R. Schmidt, Angew. Chem. Int. Ed. Engl. 25 (1986) 212e235.
[23] S.-J. Deng, B. Yu, J.-M. Xie, Y.-Z. Hui, J. Org. Chem. 64 (1999) 7265e7266.
[24] B. Yu, J.-M. Xie, S.-J. Deng, Y.-Z. Hui, J. Am. Chem. Soc. 121 (1999)
12196e12197.
[25] B. Yu, H. Yu, Y.-Z. Hui, X.-W. Han, Synlett 6 (1996) 753e755.
[26] B. Yu, Y.-C. Zhang, P.-P. Tang, Eur. J. Org. Chem. 2007 (2001) 5145e5161.
[27] B. Yu, J.-S. Sun, X.-Y. Yang, Acc. Chem. Res. 45 (2012) 1227e1236.
[28] M. Collot, J. Savreux, J.M. Mallet, Tetrahedron 64 (2008) 1523e1535.
[29] K. Nakayama, H. Fujino, R. Kasai, O. Tanaka, J. Zhou, Chem. Pharm. Bull. 34
(1986) 2209e2213.
The authors have declared no conflict of financial interest.
Acknowledgements
This work was financially supported by National Natural Science
Foundation of China (Nos. U1603124 and 81460634), and Jiangsu
Provincial Science and Technology Commission (BE2012649).
Appendix A. Supplementary data
[30] Q. Zheng, K. Koike, L.-K. Han, H. Okuda, T. Nikaido, J. Nat. Prod. 67 (2004)
604e613.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.carres.2017.02.010.