Synthesis and antitumor activities of naturally occurring oleanolic acid triterpenoid saponins and their derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.04.016

Twenty-six naturally occurring oleanolic acid saponins and their derivatives, 16 of which were synthesized in this study, were preliminarily evaluated against human cancer cells. From SAR studies, the presence of α-l-rhamnosyl residue at the terminal of both C-3 and C-28 position for oleanolic acid bidesmosides was important to enhance cytotoxicity, and introducing more sugar residues at C₃-OH of compound 12 with C-28 carboxylic acid is a favorable modification to ameliorate the anticancer activity. Furthermore, α-l-rhamnosyl moiety linked to C₂-OH of the first monosaccharide (α-l-alabinose, β-d-xylose, β-d-galactose or β-d-glucose) in C₃-OH of oleanolic acid was helpful to improve the cytotoxicity. According to the predicted log P values, lipophilicity of the synthesized saponins was not an important factor for cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2013.04.016

European Journal of Medicinal Chemistry 64 (2013) 1e15
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activities of naturally occurring oleanolic acid
triterpenoid saponins and their derivatives
Qingchao Liu a, Hongchun Liu d, Lei Zhang c, Tiantian Guo c, Peng Wang c, Meiyu Geng d,
,
Yingxia Li b
*
a Department of Pharmaceutical Engineering, Northwest University, Xi’an 710069, China
b School of Pharmacy, Fudan University, Shanghai 201203, China
c School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
d Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-six naturally occurring oleanolic acid saponins and their derivatives, 16 of which were synthe-
sized in this study, were preliminarily evaluated against human cancer cells. From SAR studies, the
Received 4 March 2013
Received in revised form
4 April 2013
Accepted 6 April 2013
Available online 15 April 2013
presence of
a-L-rhamnosyl residue at the terminal of both C-3 and C-28 position for oleanolic acid
bidesmosides was important to enhance cytotoxicity, and introducing more sugar residues at C3eOH of
compound 12 with C-28 carboxylic acid is a favorable modification to ameliorate the anticancer activity.
Furthermore,
a
-
L-rhamnosyl moiety linked to C2eOH of the first monosaccharide (
a-L-alabinose, b-D-
xylose, -galactose or b-D
According to the predicted log P values, lipophilicity of the synthesized saponins was not an important
factor for cytotoxicity.
b
-
D
-glucose) in C3eOH of oleanolic acid was helpful to improve the cytotoxicity.
Keywords:
Oleanolic acid triterpenoid saponins
Cytotoxic activity
Glycosylation
Log P
Ó 2013 Elsevier Masson SAS. All rights reserved.
IC50 value
1. Introduction
As we all known, the most predominant member of this family
of triterpenoids is probably oleanolic acid [11,12], which has been
Triterpenoid saponins, glycosylated plant secondary metabolites
[1], which exist widely in many significant foods, forage crops and
herbal medicinal plants with high contents [2], act as natural
chemical barriers to fungal attack because of their remarkable
antifungal properties [3]. Apart from the natural protective activity,
many of them are also discovered as resources for drugs at the
folkloric usage, such as the saponin extracts from ginseng, licorice,
senega roots and ivy leaves [4,5], or food crops such as oats and
legumes [6]. However, the occurrence of triterpenoid saponins
with prominent structural microheterogeneity makes separation of
the homogeneous component, especially in a suitable amount,
comparatively difficult, which results in the hysteretic development
on elucidating the structureeactivity relationships and the bio-
logical mechanisms of action of triterpenoid saponins components
[7,8]. Therefore, chemical synthesis would provide a feasible access
to these biologically triterpenoid components for further under-
standing and application of this type of natural products [7,9,10].
clinically used as a hepatoprotective/antihepatitis drug in China for
several decades. However, its low water solubility results in its
unfavorably absolute oral bioavailability [13,14]. Therefore, com-
pounds derived from chemical modification of oleanolic acid with
prodrug strategy, or isolation of oleanolic acid saponins from nat-
ural resources for improving pharmacokinetics was researched
[15,16]. In previous communications [17e20], we reported that the
naturally occurring oleanolic acid saponins (1e10) with significant
biological activities were synthesized via concise and efficient
strategies. With the continuous interest in the oleanolic acid sa-
ponins on the biological activity and in order to search for potential
new antitumor agents, we decided to investigate the effect of
modification of sugar moiety of oleanolic acid saponins on the tu-
mor cell growth inhibitory activity. Herein, we disclose the efficient
synthesis of some natural (12, 14e16) [21e25] (Fig. 1) and non-
natural sugar-modified oleanolic acid saponins (11, 13, 17e26)
(Fig. 2). Furthermore, structureeactivity relationships analysis of
this series of natural and non-natural oleanolic acid saponins
against tumor cells including human promyelotic leukemia cancer
(HL-60), non-small-cell lung cancer (A549) and human melanoma
cancer (A375) is discussed. These results obtained have provided
* Corresponding author. Tel./fax: þ86 21 51980127.
E-mail address: liyx417@fudan.edu.cn (Y. Li).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.016

2
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
O
O
R1
O
OH
O
O
O
R2
O
HO
HO
O
O
HO
O
OH
O
O
O
HO
HO
O
HO
O
HO
HO
HO
HO
HO
R1O
OH
O
R2O
OH
OH
HO
5 R1 = OH, R2 = H
6 R1 = H, R2 = OH
HO
O
R2 = H
HO
HO
1
R1
=
HO
HO
O
HO
O
O
HO
2
3
R1
R1
=
=
R
2 = H
HO
R1
OH
OH
O
HO
HO
HO
HO
R2
HO
O
O
O
O
R2 = H
O
HO
HO
HO
HO
HO
7 R1 = OH, R2 = H
8 R1 = H, R2 = OH
HO
HO
O
4
R
1 = H R2
=
HO
HO
OH
O
O
O
HO
HO
HO
O
OH
OH
OH
O
O
HO
O
O
HO
HO
O
O
O
O
HO
O
HO
O
HO
HO
HO
HO
HO
HO
HO
HO
O
HO
O
O
O
O
O
OH
OH
HO
HO
HO
HO
HO
10
9
Fig. 1. Chemical structures of previous synthesized natural oleanolic acid saponins 1e10.
valuable clues to the understanding of the cytotoxic profile for this
type of oleanolic acid saponins.
followed by deprotection of 2,3-isopropylidene acetal with p-tolue-
nesulfonic acid in CH3OHeCH2Cl2 afforded compound 33e in 92%
yield for 2 steps. The resulting compound was benzoylated using
BzCl in pyridine, and then regioselectively removal of acetyl group
with 1% AcCl in CH3OH afforded compound 33g. Reaction of 33g and
33h in dichloromethane under the promotion of TMSOTf at ꢁ78 ꢀC
gave disaccharide 33i, which was converted to hemiacetal 33j by
treatment with NBS in acetone and water. Finally, compound 33j was
treated with trichloroacetonitrile in the presence of DBU affording
the disaccharide trichloroacetimidate donor 33.
According to our previous strategy [16], the synthesis of target
compounds 14 and 15 was carried out employing the “one-pot
sequential glycosylation” with the combined use of an odorless
2-methyl-5-tert-butylphenyl (Mbp) thioglycoside and tri-
chloroacetimidate donors. Glycosylation of Mbp thioglycoside 34
[18] and imidate 32 [17] (or 33) was completed within 45 min with
the use of catalytic amount of TMSOTf at ꢁ78 ꢀC, affording the
desired disaccharide (or trisaccharide) Mbp thioglycoside donor.
Without purification, the reaction mixture was warmed to ꢁ10 ꢀC,
and then saponin acceptor 29 was added, followed by addition of
NIS and TMSOTf, providing the desired saponin 35 (or 36). The
isopropylidene acetal of saponin 35 (or 36) was hydrolyzed using p-
TsOH in CH2Cl2 and CH3OH, affording the intermediate 37 (or 38)
with the abnormal 1C4 conformation of arabinopyranosyl residue.
After removal of the 28-O-benzyl group in 37 (or 38) by hydro-
genolysis in the presence of 10% PdeC and acetyl groups with
NaOMe in MeOH and CH2Cl2, the expected target compound 14 (or
15) was provided (the arabinopyranosyl moiety returned to the
normal 4C1 conformation), whose analytical data are identical in all
respects to those reported in the literature.
2. Results and discussion
2.1. Chemistry
We have completed the efficient synthesis of compound 4
(flaccidoside II) previously by one-pot sequential glycosylation
employing two glycosyl trichloroacetimidate donors under the
promotion of TMSOTf, followed by removal of the benzoyl groups in
the presence of NaOMe in MeOHeCH2Cl2 [19]. For the synthesis of
target compound 11, the intermediate 28 that used for synthesis of
compound 4 was removed the protection of the benzoyl groups,
affording the desired product 11 in 87% yield (Scheme 1).
The preparation of target compounds 12e15 was carried out as
shown in Schemes 2e4. Glycosylation between known acceptor 29
[18,21] and donor 30a [26,27] (or 30b) [28] was performed through
thioglycoside activation by N-iodosuccinimide (NIS) under the
promotion of AgOTf to provide the desired product 31a (or 31b).
The isopropylidene acetal was hydrolyzed employing p-TsOH in
CH2Cl2eCH3OH followed by 10% PdeC promoted hydrogenolysis of
the benzyl group (or deprotection of acetyl groups with NaOMe),
affording the target saponins 12 and 13.
Disaccharide trichloroacetimidate donor 33 was prepared in a
straightforward manner from known p-tolyl
b-D-xylopyranoside
(33a) [17]. Protection of the C4eOH with a 2-methoxypropane group
and 2,3-isopropylidene formation using 2,2-dimethoxypropane
and trifluoroacetic acid in N,N-dimethylformamide (DMF) simulta-
neously followed by removal of 2-methoxypropane group selectively
with p-toluenesulfonic acid in CH3OH at 0 ꢀC gave compound 33c in
88% yield [29]. Acetylation of compound 33c with Ac2O in pyridine
The synthesis of target compounds 16e18 was done by the
similar route as that for compounds 14e15 described. As depicted

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
3
O
O
OH
OH
O
O
O
O
O
HO
HO
O
HO
O
O
O
OH
HO
13
HO
O
HO
HO
HO
OH
HO
OH
11
O
OH
O
HO
O
OH
O
HO
HO
O
HO
O
OH
O
O
HO
20
HO
R1
OH
OH
O
O
R2
HO
O
R1
OH
OR5
O
O
R2
R3O
O
O
HO
R4O
R3O
OH
O
21 R1 = OH, R2 = R3 = R5 = H, R4
22 R1 = OH, R2 = R4 = R5 = H, R3
=
=
HO
HO
12 R1 = OH, R2 = H, R3 = H
HO
OH
O
14 R1 = OH, R2 = H, R3
=
HO
HO
O
HO
HO
HO
OH
O
O
O
15 R1 = OH, R2 = H, R3
16 R1 = H, R2 = OH, R3
=
=
HO
HO
O
HO
23 R1, R5 = PhCHO, R2 = H, R3 = R4
=
HO
HO
HO
HO
HO
HO
O
OH
HO
HO
O
24 R1 = R3 = R5 =H, R2 = OH, R4
=
HO
HO
HO
HO
HO
O
HO
HO
HO
O
O
17 R1 = H, R2 = OH, R3
18 R1 = H, R2 = OH, R3
=
HO
HO
OH
HO
HO
O
25 R1 = R4 = R5 = H, R2 = OH, R3
26 R1 = R5 = H, R2 = OH, R3 = R4
=
O
=
HO
OH
HO
OH
O
19
R
1 = H, R2 = OH, R3 = H
=
OH
Fig. 2. Chemical structures of synthesized natural and non-natural oleanolic acid saponins 11e26.
in Scheme 5, with the glycosyl trichloroacetimidate donor 32 (or
39e40) [17,30], 2-methyl-5-tert-butylphenyl (Mbp) thioglycoside
34 and acceptor 41 in hand, their coupling reaction was performed
under the promotion of TMSOTf and NIS to obtain 42 (or 43e44),
which was subjected to the benzyl cleavage with 10% PdeC and
acetyl deprotection with NaOMe in CH2Cl2eMeOH to provide the
target compound 16 (or 17e18) efficiently.
donor 40 was achieved in the presence of TMSOTf to afford the
desired saponin 45 in 91% yield, which was subjected to benzyl
cleavage with 10% PdeC, followed by removal of acetyl groups with
NaOMe in CH2Cl2eMeOH, providing the target saponin 19.
As depicted in Scheme 7, the synthesis of target saponin 20 was
commenced with known p-tolyl 3,4-O-isopropylidene-1-thio-a-L-
arabinopyranoside 40, which was coupled with donor 46 [31] un-
der the promotion of TMSOTf in CH2Cl2 at low temperature
(ꢁ78 ꢀC) to provide the desired disaccharide 47, followed by
The preparation of target compound 19 was performed as out-
lined in Scheme 6. Glycosylation between acceptor 41 [20] and
Scheme 1. Synthesis of non-natural oleanolic acid saponin 11.

4
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
STol
O
OBn
O
AcO
LevO
O
(1) p-TsOH, CH2Cl2-CH3OH
30a OAc
12
13
O
O
O
(2)10%Pd-C, H2; NaOMe
53% for 3 steps
O
AgOTf, NIS,
CH Cl , 76A%cO
O
O
31a
LevO
OAc
OBn
O
O
O
AgOTf, NIS,
CH Cl , 43%
O
O
(1) p-TsOH, CH Cl -CH OH
OBn
2
2
3
O
O
HO
29
O
STol
O
O
(2)10%Pd-C, H2
65% for 2 steps
O
O
BnO
BnO
BnO
BnO
OBn
30b OBn
31b
Scheme 2. Synthesis of oleanolic acid saponins 12 and 13.
glycosylation with the acceptor 48 [32,33] in the presence of NIS
and AgOTf stereoselectively affording the fully protected interme-
diate 49 in 35% yield. The diisopropylidene was achieved by
treatment of compound 49 with p-TsOH in CH2Cl2eMeOH solution
to obtain 50. Subsequent deprotection of the benzyl ether with 10%
PdeC and the benzoyl ester with NaOMe in CH2Cl2eMeOH was
achieved to afford the target compound 20.
The synthesis of non-natural saponins 21e26 was achieved in a
straight-forward manner. As shown in Scheme 8, the oleanolic acid
saponin acceptor 51 (or 53) was condensed with the known donor
40 in the presence of TMSOTf to obtain the intermediates 52aec (or
54aec). Subsequent removal of benzyl and acetyl groups provided
the desired saponins 21e23 (or 24e26).
by saponin 11 (IC50 4.7e10.3
show any cytotoxic activity (IC50 > 20
A375 cell lines. These results proved that the presence of
rhamnosyl residue at the terminal of both C-3 and C-28 position
was critical to enhance cytotoxicity.
In the second group, most of compounds except 20, 22e23 and
25e26 exerted better anticancer activity. When compared com-
pounds 6, 16e17 with 1e3, the influence of free C-28 carboxylic
acid on enhancing the cytotoxicity was clear. As for compound 12,
m
M). In contrast, saponins 1e3 did not
m
M) against HL-60, A549 and
a-L-
we anticipated that changing the spatial configuration of L-alabi-
nosyl moiety at the C-3 position of oleanolic acid should improve
anticancer activity. Actually, the changed compound 20 exhibited
no cytotoxicity against all tested cell lines (IC50 > 20
mM). However,
when -rhamnosyl residue of compound 12 was converted to b-L-
a-
L
rhamnosyl residue, the derived compound 13 exerted good cyto-
2.2. Cytotoxic activity
toxicity, especially enhanced the cytotoxicity about 2-fold against
HL-60 cell line (IC50 4.2
6.1 M), 9 (IC50 3.5 M), 10 (IC50 3.1
(IC50 4.2 M), were 1.5- to 3-fold more potent against HL-60 cell
line comparing with compound 12 (IC50 9.2 M), which suggested
that introducing more sugar residues at C3eOH of compound 12 is a
favorable modification to ameliorating the anticancer activity.
m
M). Meanwhile, compounds 5 (IC50
To examine the potential ability of the natural and non-natural
oleanolic acid saponins 1e26, the in vitro cytotoxic activity against
human promyelotic leukemia cancer (HL-60), non-small-cell lung
cancer (A549) and human melanoma cancer (A375) was assessed
using methyl-thiazol-tetrazolium (MTT) reduction test and sulfo-
rhodamine B (SRB) staining methods for protein. The cytotoxicity
results presented in Table 1 are shown as the IC50 values. They are
subdivided into two groups: Oleanolic acid bidesmosides (1e4, 11),
and Oleanolic acid monodesmosides (5e10, 12e26). In the first
group, saponin 4 exhibited the most cytotoxic activity against the
m
m
m
M), 14 (IC50 3.4 M), and 15
m
m
m
Replacement of
glucose at the C3eOH of 12 afforded compound 19, 21 or 24, which
has no significant increase in cytotoxicity. However, adding
rhamnosyl moiety at the C2eOH and C3eOH (or C3eOH solely) of
-galactose or -glucose respectively, obtained compounds
a-L-alabinose by b-D-xylose, b-D-galactose or b-D-
a-L-
b-D
b-D
three tumor cells (with IC50 ranging from 3.2 to 10.5 mM) followed
Scheme 3. Synthesis of disaccharide donor 33.

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
5
Scheme 4. Synthesis of target compounds 14e15.
22e23 or 25e26, which did not exhibit desirable antitumor activ-
3. Conclusions
ity. These results demonstrated that the type of the first mono-
saccharide linked to C3eOH of oleanolic acid has no obvious
A series of natural and non-natural sugar-modified oleanolic
acid saponins was synthesized in a concise and practical way and
their cytotoxicity was evaluated in vitro. In terms of structureeac-
tivity relationships, we can conclude that: (i) with regard to ole-
influence on improving the cytotoxicity, and
linked to C2eOH with free C3eOH of the first monosaccharide (
alabinose by -xylose, -galactose or -glucose) favored the
cytotoxic activity.
a-L-rhamnose moiety
a-L-
b-
D
b
-D
b-D
anolic acid bidesmosides, the presence of a-L-rhamnosyl residue at
The logarithm of a partition coefficient (log P) is a parameter
which reflects a drug equilibrium partition ratio between polar
(water) and non-polar (octanol) phases, and it has been proved to
determine the absorption, the distribution, the biological avail-
ability, and pharmacological activity of drugs. In this study, we have
employed the ACD lab program to predict the values of log P for
each oleanolic acid saponin 1e26. The results are arranged in
Table 2, comparing the log P values of all synthesized compounds,
we found that the order of antitumor activity was not consistent
with the order of lipophilicity, which indicated that lipophilicity of
the compounds had no important effect on cytotoxicity.
the terminal of both C-3 and C-28 position was critical to enhance
cytotoxicity; (ii) for oleanolic acid monodesmosides, free C-28
carboxylic acid favors the cytotoxicity; (iii) changing the spatial
configuration of L-alabinosyl moiety at the C-3 position of oleanolic
acid disfavor anticancer activity; (iv) introducing more sugar resi-
dues at C3eOH of compound 12 is a favorable modification to
ameliorate the anticancer activity; (v) the type of the first mono-
saccharide linked to C3eOH of oleanolic acid has no obvious in-
fluence on improving the cytotoxicity; (vi)
linked to C2eOH with free C3eOH of the first monosaccharide (
alabinose by -xylose, -galactose or -glucose) was helpful
a-L-rhamnose moiety
a-L-
b-
D
b
-D
b-D
Scheme 5. Synthesis of target compounds 16e18.

6
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
O
CCl3
NH
OBz
O
O
BzO
BzO
40
OBn
O
O
BzO
BzO
O
OBn
O
O
BzO
BzO
O
TMSOTf, CH2Cl2
O
HO
BzO
BzO
41
45
OBz
O
10%Pd-C, H2
NaOMe, CH2Cl2-CH3OH
OH
O
O
HO
HO
O
O
HO
19
HO
OH
Scheme 6. Synthesis of target compound 19.
to improve the cytotoxic activity. Meanwhile, lipophilicity of the
compounds was not an important factor for cytotoxicity.
(MeOHeCHCl3eH2O, 1:25:0.1) to give 11 (40 mg, 87%) as a white
amorphous solid. ½a D25
ꢂ
ꢁ13.3 (c 0.25, CH3OH); IR (KBr) nmax 3499,
2938, 1731, 1645, 1453, 1073 cmꢁ1
;
1H NMR (500 MHz, C5D5N)
d:
6.23 (d, J ¼ 8.1 Hz,1H, H-100), 5.85 (br s, 1H, H-1000), 5.39 (t, J ¼ 3.6 Hz,
1H, H-12), 4.98 (d, J ¼ 7.8 Hz, 1H, H-10), 4.96 (qd, J ¼ 9.1, 6.0 Hz, 1H,
H-5000), 4.82 (d, J ¼ 7.5 Hz, 1H, H-10000), 4.68 (dd, J ¼ 3.2, 1.7 Hz, 1H, H-
2000), 4.65 (m, 1H, H-40), 4.54 (dd, J ¼ 9.3, 3.2 Hz, 1H, H-3000), 4.41 (t,
J ¼ 9.6 Hz, 1H, H-300), 4.31e4.37 (m, H-30000, H-4000, H-50000-1, H-60-1),
4.09e4.23 (m, 8H, H-200, H-30, H-400, H-40000, H-50, H-500, H-600-1, H-60-
2), 4.02 (t-like, J ¼ 8.8, 7.9 Hz, 1H, H-20000), 3.93 (t-like, J ¼ 8.7, 8.3 Hz,
1H, H-20), 3.78 (t, J ¼ 11.0 Hz, 1H, H-50000-2), 3.64 (m, 1H, H-600-2),
3.33 (dd, J ¼ 11.7, 4.3 Hz, 1H, H-3), 3.15 (dd, J ¼ 13.7, 3.7 Hz, 1H, H-
18), 1.69 (d, J ¼ 6.0 Hz, 3H, H-6000), 1.29, 1.24, 1.09, 0.99, 0.89, 0.89,
4. Experimental section
4.1. Chemistry
Thin-layer chromatography (TLC) was performed on precoated
E. Merck Silica Gel 60 F254 plates. Flash column chromatography
was performed on silica gel (200e300 mesh, Qingdao, China).
Optical rotations were determined with a PerkineElmer Model 241
MC polarimeter. 1H NMR and 13C NMR spectra were taken on a JEOL
JNM-ECP 600 spectrometer with tetramethylsilane as the internal
standard, and chemical shifts are recorded in d values. Mass spectra
were recorded on a Q-TOF Global mass spectrometer.
Commercial reagents were all analytically or chemically pure and
used without further purification unless specified. All anhydrous
solvents were dried and redistilled prior to use in the usual way.
0.89 (s each, 3H each, CH3 ꢃ7). 13C NMR (125 MHz, C5D5N)
d: 176.5
(C-28), 144.0 (C-13), 122.9 (C-12), 107.6 (C-10000), 104.9 (C-100), 102.8
(C-1000), 95.6 (C-10), 88.7 (C-3), 78.8, 78.6, 78.4, 78.1, 77.2, 76.6, 75.5,
75.4, 74.0, 73.9, 72.8, 72.6, 71.3, 71.0, 70.3, 69.3, 67.1, 62.0, 56.0, 49.7,
48.2, 47.1, 46.3, 42.2, 41.8, 40.0, 39.6, 38.9, 37.1, 34.1, 33.2, 32.6, 30.8,
28.4, 28.3, 26.8, 26.1, 23.9, 23.8, 23.4, 18.5, 17.6, 17.0, 15.7. HR-MS
(ESI): m/z calcd for C53H86O21Na [M þ Na]þ 1081.5554, found
1081.5579.
4.1.1. 28-O-a-L
-Rhamnopyranosyl-(1 / 4)-O- -glucopyranosyl-
b-D
(1 / 6)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)
To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH
(1:2, 12 mL) was added a newly prepared NaOMe in MeOH solution
(1.0 mol/L, 0.20 mL). The mixture was stirred at room temperature
for 5 h and neutralized with Dowex Hþ resin to pH 7 and
then filtered. The filtrate was concentrated and the resulting
residue was subjected to a silica gel column chromatography
4.1.2. General procedure for synthesizing compounds 31ae31b
ꢀ
A mixture of compound 29 (0.139 mmol), powdered 4 A mo-
lecular sieves and compound 30a or 30b (0.209 mmol) in dry
CH2Cl2 (5 mL) was stirred at room temperature for 30 min then
cooled to 0 ꢀC. NIS (0.221 mmol) and AgOTf (0.016 mmol) was
Scheme 7. Synthesis of target compound 20.

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
7
Scheme 8. Synthesis of target compounds 21e26.
added, and the reaction mixture was stirred for 30 min and then
warmed to room temperature. The product was detected on TLC
(2:1, petroleum ethereEtOAc). After completion of the reaction, the
reaction mixture was quenched by addition of Et3N, and then
filtered. The filtrate was concentrated and purified by a silica gel
column chromatography to afford the products.
3H, H-600), 1.12, 1.04, 0.92, 0.89, 0.88, 0.81, 0.60 (s each, 3H each,
CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
d 206.2, 177.5 (C-28), 171.4,
170.2, 143.7 (C-13), 136.4, 128.4, 128.0, 127.9, 122.5 (C-12), 110.4
((CH3)2C), 103.1 (C-10), 95.2 (C-100), 89.0 (C-3), 79.1, 75.1, 73.3, 70.9,
70.5, 69.7, 69.6, 69.3, 66.2, 65.9, 62.6, 55.8, 47.6, 46.7, 45.9, 41.7, 41.4,
39.3, 39.0, 37.8, 37.7, 37.6, 36.7, 33.9, 33.1, 32.7, 32.4, 30.9, 30.7, 29.7,
28.0, 27.8, 27.6, 26.1, 25.9, 23.7, 23.4, 23.0, 21.0, 20.8, 18.2, 17.3, 16.9,
16.4, 15.4, 15.3. HR-MS (ESI): m/z calcd for C60H86O15Na [M þ Na]þ
1069.5859, found 1069.5883.
4.1.2.1. Benzyl oleanolate 2,4-di-O-acetyl-3-O-levulinoyl-
a
-
L
-rham-
-arabinopyranoside
þ21.3 (c 0.8, CHCl3); IR (KBr) vmax 2929,
1H NMR (CDCl3,
nopyranosyl-(1 / 2)-3,4-O-isopropylid-ene-a-L
(31a). Yield: 76%; ½a D25
ꢂ
1731, 1447, 1356, 1123, 1023, 707, 673 cmꢁ1
;
4.1.2.2. Benzyl oleanolate 2,3,4-tri-O-benzyl-
b
-
L
-rhamnopyranosyl-
-arabin-opyranoside (31b).
þ5.92 (c 1.18, CHCl3); IR (KBr) vmax 2939, 1733,
600 MHz):
d
7.07e7.35 (m, 5H, PheH), 5.34 (s, 1H, H-100), 5.29e5.32
(1
/
Yield: 43%; ½a 2D3
2)-3,4-O-isopropylidene-a-L
(m, 3H, H-30, H-40, H-12), 5.03e5.11 (m, 3H, H-200, PhCH2), 4.34
(d, J ¼ 7.7 Hz,1H, H-10), 4.10e4.21 (m, 4H, H-300, H-400, H-500, H-50-1),
3.73e3.78 (m, 2H, H-20, H-50-2), 3.08 (dd, J ¼ 11.8, 4.4 Hz, 1H, H-3),
2.90 (dd, J ¼ 13.9, 4.0 Hz, 1H, H-18), 2.72e2.76 (m, 1H, LeveCHH),
2.61e2.66 (m, 1H, LeveCHH), 2.50e2.55 (m, 1H, LeveCHH), 2.39e
2.44 (m,1H, LeveCHH), 2.16, 2.15, 2.05 (s each, 3H each, Ac ꢃ2, Leve
CH3), 1.53, 1.33 (s each, 3H each, Oe(CH3)2CeO), 1.20 (d, J ¼ 6.2 Hz,
ꢂ
1454, 1368, 1116, 1023, 738, 685 cmꢁ1; 1H NMR (CDCl3, 600 MHz):
d
7.27e7.34 (m, 20H, PheH), 5.33 (d, J ¼ 1.1 Hz, 1H, H-100), 5.29
(t, J ¼ 3.6 Hz, 1H, H-12), 5.11 (d, J ¼ 12.7 Hz, 1H, PhCHH), 5.05
(d, J ¼ 12.7 Hz, 1H, PhCHH), 5.01 (d, J ¼ 15.4 Hz, 1H, PhCHH), 4.95
(d, J ¼ 13.5 Hz, 1H, PhCHH), 4.76 (d, J ¼ 12.7 Hz, 1H, PhCHH), 4.67
(d, J ¼ 11.0 Hz, 1H, PhCHH), 4.64 (d, J ¼ 10.4 Hz, 1H, PhCHH), 4.56

8
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
Table 1
4.1.2.3. 1-(4-Tolyl)thio-2,3-O-isopropylidene-4-O-(1-methoxy-1-
methylethyl)- -xylopyranoside (33b). Compound 33a (1.60 g,
6.24 mmol) was dissolved in DMF (2 mL) and heated to 40 ꢀC.
Trifluoroacetic acid (100 L, 1% TFA in DMF) was added followed by
In vitro cytotoxicity of oleanolic acid bidesmosides (1e4, 11), and oleanolic acid
monodesmosides (5e10, 12e26) against cancer cell lines.
b-D
Groupa
Compound
IC50 (m
mol/L)b
m
2-methoxypropene (2 mL, 21.22 mmol). The reaction mixture was
stirred for 12 h at this temperature. TLC (10:1 petroleum ethere
EtOAc) indicated that the reaction was complete. The reaction was
HL-60
A549
A375
I
1
2
3
4
11
5
6
7
8
>20
>20
>20
>20
>20
>20
>20
>20
>20
quenched with Et3N (20
mL), and the mixture was diluted with
3.2 ꢄ 1.0
5.7 ꢄ 1.3
6.1 ꢄ 2.3
4.6 ꢄ 2.9
10.1 ꢄ 3.7
12.5 ꢄ 2.6
3.5 ꢄ 1.3
3.1 ꢄ 1.8
9.2 ꢄ 2.9
4.2 ꢄ 1.5
3.4 ꢄ 1.2
4.2 ꢄ 0.3
4.5 ꢄ 1.5
2.5 ꢄ 0.9
9.3 ꢄ 0.8
3.6 ꢄ 0.1
>20
10.5 ꢄ 2.3
11.9 ꢄ 4.1
16.7 ꢄ 1.5
14.7 ꢄ 1.6
17.9 ꢄ 4.2
15.3 ꢄ 3.2
7.9 ꢄ 1.3
6.2 ꢄ 1.1
15.8 ꢄ 4.3
17.5 ꢄ 3.4
15.4 ꢄ 1.3
>20
11.5 ꢄ 3.1
7.8 ꢄ 4.4
16.2 ꢄ 3.4
10.7 ꢄ 4.2
>20
7.3 ꢄ 1.4
13.5 ꢄ 1.7
12.1 ꢄ 1.1
5.5 ꢄ 1.7
9.7 ꢄ 2.3
10.3 ꢄ 3.1
5.9 ꢄ 0.9
3.5 ꢄ 0.4
8.4 ꢄ 1.8
7.7 ꢄ 3.8
8.9 ꢄ 2.4
22.1 ꢄ 6.1
6.3 ꢄ 2.9
6.1 ꢄ 1.3
8.4 ꢄ 3.3
5.0 ꢄ 1.8
>20
CH2Cl2 and sequentially washed with saturated NaHCO3, saturated
NaCl, dried over Na2SO4, filtered and concentrated. The residue was
purified by column chromatography (10:1 petroleum ethereEtOAc)
affording compound 33b (1.50 g, 65%) as a white solid; Rf ¼ 0.35
II
(petroleum ethereEtOAc, 10:1); 1H NMR (CDCl3, 600 MHz):
d 7.47
9
(d, J ¼ 8.2 Hz, 2H, PheH), 7.13 (d, J ¼ 7.8 Hz, 2H, PheH), 4.69 (d,
J ¼ 9.1 Hz, 1H, H-1), 4.09 (dd, J ¼ 11.5, 5.0 Hz, 1H, H-5-1), 4.00 (ddd,
J ¼ 9.6, 9.2, 5.0 Hz, 1H, H-4), 3.53 (t, J ¼ 9.1 Hz, 1H, H-3), 3.22 (s, 3H,
OCH3), 3.18e3.21 (m, 2H, H-2, H-5-2), 2.35 (s, 3H, SPhCH3), 1.46,
1.41, 1.37, 1.33 (s each, 3H each, CH3 ꢃ4); HRESIMS: calcd for
[M þ Naþ] C19H28O5SNa 391.1550, found 391.1576.
10
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
ADMc
HCPTd
4.1.2.4. 1-(4-Tolyl)thio-2,3-O-isopropylidene-b-D-xylopyranoside
(33c). Compound 33b (1.40 g, 3.80 mmol) was dissolved in MeOH
(15 mL) and cooled to 0 ꢀC. A solution of p-toluenesulfonic acid in
2.5 ꢄ 1.1
12.4 ꢄ 5.5
15.2 ꢄ 0.7
>20
5.1 ꢄ 1.2
15.4 ꢄ 0.9
>20
>20
MeOH (1 mg/mL, 200 mL) was added. The reaction mixture was
>20
stirred and monitored by. TLC (6:1 petroleum ethereEtOAc) When
the starting material was disappeared, the reaction was quenched
with strongly basic ion exchange resin (OHꢁ form). The solution
was filtrated, and the filtrate was concentrated. The residue was
purified by column chromatography (6:1 petroleum ethereEtOAc)
affording compound 33c as a white solid (1.00 g, 88%): Rf ¼ 0.31
12.9 ꢄ 2.7
23.5 ꢄ 2.7
11.6 ꢄ 1.8
>20
>20
>20
>20
>20
>20
0.90 ꢄ 0.3
0.56 ꢄ 0.2
0.75 ꢄ 0.1
0.25 ꢄ 0.1
0.30 ꢄ 0.1
0.15 ꢄ 0.1
a
(I) oleanolic acid bidesmosides; (II) oleanolic acid monodesmosides.
Data represent mean values ꢄ standard deviation for three independent ex-
b
(petroleum ethereEtOAc, 6:1); 1H NMR (CDCl3, 600 MHz):
d 7.47
periments made in triplicate.
c
(d, J ¼ 7.8 Hz, 2H, PheH), 7.13 (d, J ¼ 7.8 Hz, 2H, PheH), 4.73
(d, J ¼ 9.6 Hz, 1H, H-1), 4.12 (dd, J ¼ 11.5, 5.0 Hz, 1H, H-5-1), 3.94e
3.99 (m, 1H, H-4), 3.52 (t, J ¼ 9.2 Hz, 1H, H-3), 3.20e3.24 (m, 2H,
H-2, H-5-2), 2.35 (s, 3H, SPhCH3), 1.49, 1.44 (s each, 3H each,
CH3 ꢃ2); ESI-MS (m/z): 297.1 (M þ Hþ); 615.2 (2M þ Naþ);
HRESIMS: calcd for [M þ Naþ] C15H20O4SNa 319.0975, found
319.0997.
Adriamycin, positive control.
d
Hydroxycamptothecin, positive control.
(d, J ¼ 12.7 Hz, 1H, PhCHH), 4.31 (d, J ¼ 7.2 Hz, 1H, H-10), 4.21
(dd, J ¼ 10.4, 4.4 Hz, 1H, H-50-1), 3.99e4.02 (m, 2H, H-40, H-400), 3.95
(dq, J ¼ 9.1, 6.6 Hz, 1H, H-500), 3.88 (dd, J ¼ 9.4, 2.8 Hz, 1H, H-50-2),
3.69e3.72 (m, 2H, H-20, H-300), 3.61 (dd, J ¼ 9.6, 3.8 Hz, 1H, H-30),
3.01 (dd, J ¼ 12.1, 4.9 Hz, 1H, H-3), 2.91 (dd, J ¼ 13.7, 3.8 Hz, 1H, H-
18), 1.48, 1.33 (s each, 3H each, Oe(CH3)2CeO), 1.28 (d, J ¼ 6.6 Hz,
3H, H-600), 1.11, 0.92, 0.91, 0.89, 0.88, 0.81, 0.61 (s each, 3H each,
4.1.2.5. 1-(4-Tolyl)thio-4-O-acetyl-
solution of compound 33c (140 mg, 0.472 mmol) in pyridinee
CH2Cl2 (2 mL, 1:1), Ac2O (120 L) and DMAP (5 mg) was added.
b-D-xylopyranoside (33e). To a
m
CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
d 177.7 (C-28), 143.9 (C-13),
The mixture was stirred for 2 h. After completion of the reaction
(monitored by TLC), the reaction mixture was quenched with
CH3OH, concentrated under reduced pressure. The residue was
diluted with EtOAc and sequentially washed with saturated 1 mol/
L HCl, saturated NaHCO3, saturated NaCl, dried over Na2SO4,
filtered and concentrated. The crude product 33d was dissolved in
CH3OHeCH2Cl2 (5 mL, 1:1), and then p-toluenesulfonic acid
(24 mg) was added. The mixture was stirred for 2 h. The reaction
mixture was concentrated and purified by column chromatog-
raphy (petroleum ethereEtOAc, 2:1) to afford 33e as a white solid
(130 mg, 92% for two steps); Rf ¼ 0.31 (petroleum ethereEtOAc,
138.9, 138.6, 136.6, 128.6, 128.4, 128.3, 128.2, 122.8 (C-12), 110.4
((CH3)2C), 103.3 (C-10), 96.7 (C-100), 88.9 (C-3), 80.1, 80.0, 79.3, 75.3,
74.9, 73.5, 72.3, 68.7, 68.5, 66.1, 55.9, 47.8, 46.9, 46.2, 41.7, 41.5, 40.0,
39.3, 38.2, 36.9, 33.6, 33.3, 30.9, 28.0, 26.3, 23.9, 23.6, 18.1, 17.1, 15.9,
15.6; HRMALDIMS: calcd for [M þ Naþ] C72H94O11Na 1157.6686,
found 1157.6688.
Table 2
Lipophilicity (log P) of oleanolic acid saponins 1e26.
Compound
Log Pa
Compound
Log Pa
2:1); ½a 2D3
ꢂ
ꢁ54.2 (c 1.70, CHCl3); IR (KBr) nmax 3403, 3337, 2972,
1H
7.35 (d, J ¼ 8.2 Hz, 2H, PheH), 7.15
1
2
3
4
5
6
7
8
6.26
4.06
3.86
6.11
7.49
7.49
6.80
6.80
7.42
4.78
5.83
9.96
9.96
14
15
16
17
18
19
20
21
22
23
24
25
26
10.02
7.38
10.02
7.42
10.51
9.96
2913, 2860, 1713, 1487, 1242, 1070, 950, 824, 804, 592 cmꢁ1
;
NMR (DMSO-d6, 600 MHz):
d
(d, J ¼ 8.2 Hz, 2H, PheH), 5.51 (d, J ¼ 5.9 Hz, 1H, C3eOH), 5.41
(d, J ¼ 5.5 Hz, 1H, C2eOH), 4.61 (d, J ¼ 9.2 Hz, 1H, H-1), 4.50 (ddd,
J ¼ 10.1, 5.5 Hz, 1H, H-4), 3.84 (dd, J ¼ 11.0, 5.0 Hz, 1H, H-5-1), 3.43
(ddd, J ¼ 9.2, 5.0 Hz, 1H, H-2), 3.26 (dd, J ¼ 11.0, 10.1 Hz, 1H, H-5-
2), 3.09 (ddd, J ¼ 9.2, 5.9 Hz, 1H, H-3), 2.28 (s, 3H, SPhCH3), 2.01
9.96
10.06
9.87
10.34
10.06
9.87
9
10
11
12
13
(s, 3H, AceCH3); 13C NMR (DMSO-d6, 150 MHz):
d 170.7, 137.5,
133.2, 130.1, 129.6, 88.2 (C-1), 74.5, 72.5, 71.6, 65.7, 21.2, 21.0; ESI-
MS (m/z): 321.1 (M þ Naþ); ESIHRMS: m/z calcd for C14H18O5NaS
[M þ Naþ] 321.0773; found: 321.0780.
10.34
a
Predicted octanol/water partition coefficient.

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
9
4.1.2.6. 1-(4-Tolyl)thio-2,3-di-O-benzoyl-4-O-acetyl-
side (33f). To a solution of 33e (100 mg, 0.335 mmol) and
b
-
D
-xylopyrano-
1065.6 (M þ Naþ); ESIHRMS: m/z calcd for C60H50O15NaS [M þ Naþ]
1065.2768; found: 1065.2798.
4-dimethylamino-pyridine (5 mg, 0.034 mmol) in pyridine (5 mL)
and CH2Cl2 (10 mL) was added benzoyl chloride (156
mL,
4.1.2.9. 2,3,4,6-Tetra-O-benzoyl-
b-D-glucopyranosyle(1 / 4)-2,3-di-
1.01 mmol) dropwise at 0 ꢀC. The reaction mixture was stirred for
3 h, and then quenched with CH3OH. The solvents were evapo-
rated in vacuo. The resulting residue was diluted with EtOAc and
sequentially washed with 1 N HCl, saturated NaHCO3, and satu-
rated NaCl, dried over Na2SO4, filtered and concentrated. The
residue was purified by column chromatography (petroleum
ethereEtOAc, 6:1) to afford 33f as a white solid (163 mg, 94%);
O-benzoyl- -xylopyranoside trichloroacetimidate (33). To a solu-
b-D
tion of compound 33i (150 mg, 0.144 mmol) in 10 mL of acetone-
H2O (9/1), NBS (67 mg, 0.374 mmol) were added at ꢁ20 ꢀC. The
mixture was stirred for 5 min, quenched with satd aq NaHCO3. The
reaction mixture was concentrated, and the residue was diluted
with EtOAc, washed with satd aq NaHCO3, brine, dried over Na2SO4,
and concentrated. The residue was purified by silica gel column
chromatography (3:1 to 1:1, petroleum ethereEtOAc) to give 33j
(115 mg, 85%) as a white solid. A solution of 33j (100 mg,
0.107 mmol), CNCCl3 (0.08 mL, 0.859 mmol) and DBU (0.02 mL,
0.054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at room
temperature, then the solvent was evaporated in vacuo to give a
residue, which was purified by silica-gel flash column chromatog-
raphy (petroleum ethereEtOAc, 3:1) to afford 33 (97 mg, 83%) as a
white solid; Rf ¼ 0.36 (petroleum ethereEtOAc, 1:1); 1H NMR
Rf ¼ 0.41 (petroleum ethereEtOAc, 5:1); ½a D23
þ21.2 (c 1.30,
ꢂ
CHCl3); IR (KBr) nmax 2946, 2873, 1739, 1229, 1123, 1056, 804,
705 cmꢁ1; 1H NMR (CDCl3, 600 MHz):
d 7.11e8.02 (m, 14H, PheH),
5.56 (t, J ¼ 7.1 Hz, 1H, H-3), 5.36 (t, J ¼ 7.1 Hz, 1H, H-2), 5.12 (td,
J ¼ 7.7, 4.4 Hz, 1H, H-4), 5.06 (d, J ¼ 7.1 Hz, 1H, H-1), 4.48 (dd,
J ¼ 12.1, 4.4 Hz, 1H, H-5-1), 3.64 (dd, J ¼ 12.1, 7.1 Hz, 1H, H-5-2),
2.34 (s, 3H, SPhCH3), 2.01 (s, 3H, AceCH3); 13C NMR (CDCl3,
150 MHz):
d 170.1, 165.5, 165.2, 138.8, 133.7, 133.6, 130.1, 130.0,
128.9, 128.7, 128.6, 86.8 (C-1), 71.6, 70.2, 68.4, 64.4, 21.4, 21.0; ESI-
MS (m/z): 529.1 (M þ Naþ); ESIHRMS: m/z calcd for C28H26O7NaS
[M þ Naþ] 529.1297; found: 529.1302.
(CDCl3): d 8.52 (s, 1H, N-H), 7.21e8.18 (m, 30H, PheH), 6.57 (d,
J ¼ 3.7 Hz, 1H, H-1), 5.98 (t, J ¼ 9.6 Hz, 1H, H-3), 5.83 (t, J ¼ 9.6 Hz,
1H, H-30), 5.52 (t, J ¼ 9.6 Hz,1H, H-40), 5.45 (dd, J ¼ 9.6, 7.7 Hz,1H, H-
20), 5.35 (dd, J ¼ 10.1, 3.7 Hz, 1H, H-2), 4.97 (d, J ¼ 8.2 Hz, 1H, H-10),
4.26 (m, 1H, H-4), 4.09 (m, 2H, H-60-1, H-60-2), 3.97 (m, 1H, H-50),
3.89 (dd, J ¼ 11.5, 5.5 Hz, 1H, H-5-1), 3.77 (t, J ¼ 11.0 Hz, 1H, H-5-2);
ESI-MS: m/z 1080.3 (M þ Hþ); HRESIMS: calcd for C55H45O16NCl3
[M þ Hþ]: m/z 1080.1804; found: m/z 1080.1849.
4.1.2.7. 1-(4-Tolyl)thio-2,3-di-O-benzoyl-4-
To a solution of 33f (120 mg, 0.237 mmol) in CH3OH (10 mL) was
added acetyl chloride (120
L) dropwise at 0 ꢀC. The reaction
b-D-xylopyranoside (33g).
m
mixture was stirred for 5 h, and then quenched with Et3N. The
solvent was evaporated in vacuo. The resulting residue was purified
by column chromatography (petroleum ethereEtOAc, 4:1) to afford
33g as a white solid (100 mg, 90%); Rf ¼ 0.32 (petroleum ethere
4.1.3. General procedure for synthesizing compounds 35e36
ꢀ
A solution of Mbp thioglycoside 34 (40 mg, 0.112 mmol) and 4 A
EtOAc, 3:1); ½a 2D3
ꢂ
þ47.1 (c 0.88, CHCl3); IR (KBr) nmax 3456, 3059,
MS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature
under argon for 30 min, and then cooled to ꢁ78 ꢀC. At this tem-
perature, a solution of TMSOTf (0.2 equiv.) in dry CH2Cl2 was
2846, 1726, 1487, 1269, 1063, 804, 705 cmꢁ1 1H NMR (CDCl3,
;
600 MHz):
d
7.12e8.04 (m, 14H, PheH), 5.40 (t-like, J ¼ 7.8, 7.3 Hz,
1H, H-3), 5.32 (t-like, J ¼ 7.8, 7.3 Hz, 1H, H-2), 5.01 (d, J ¼ 7.3 Hz, 1H,
H-1), 4.42 (dd, J ¼ 11.9, 4.6 Hz, 1H, H-5-1), 3.98 (td, J ¼ 7.7, 4.6 Hz,
1H, H-4), 3.57 (dd, J ¼ 11.9, 7.8 Hz, 1H, H-5-2), 2.34 (s, 3H, SPhCH3);
injected and after 10 min a trichloroacetimidate 32 or 33
(2.1 equiv.) in dry CH2Cl2 was added. The mixture was stirred for
additional 30 min and then warmed up to ꢁ10 ꢀC. To the above
mixture was added a solution of saponin acceptor 29 (79 mg,
0.112 mmol, 1.0 equiv.) in CH2Cl2 (2 mL) followed by NIS (50 mg,
0.112 mmol, 2.0 equiv.). After being stirred for 1 h, the reaction
mixture was quenched with Et3N, and then filtered through a pad
of Celite. The filtrate was concentrated. The residue was purified
silica gel column chromatography (2.5:1, petroleum ethereEtOAc)
to give the fully protected saponin. The amounts of the reactants
and the yields of the saponin products were calculated based on
saponin accepter 29.
13C NMR (CDCl3, 150 MHz):
d 167.2, 165.3, 138.7, 133.9, 133.7, 133.5,
130.3, 129.0, 128.7, 87.1 (C-1), 76.3, 70.3, 68.6, 67.8, 21.4; ESI-MS
(m/z): 487.1 (M þ Naþ); ESIHRMS: m/z calcd for C26H24O6NaS
[M þ Naþ] 487.1191; found: 487.1174.
4.1.2.8. 2,3,4,6-Tetra-O-benzoyl-
b-D-glucopyranosyle(1 / 4)-1-(4-
tolyl)thio-2,3-di-O-benzoyl- -xylopyranoside (33i). A mixture of
b-D
ꢀ
compound 33g (100 mg, 0.22 mmol), powdered 4 A molecular
sieves and compound 33h (239 mg, 0.32 mmol) in dry CH2Cl2
(3 mL) was stirred at r.t. for 30 min then cooled to ꢁ78 ꢀC. TMSOTf
(10
m
L, 0.02 mmol) was added dropwise and the reaction mixture
4.1.3.1. Benzyl oleanolate 2,3,4-tri-O-benzoyl-
(1 / 3)-2,4-di-O-acetyl- -rhamnopyranosyl-(1 / 2)-3,4-O-iso-
propylidene-
b-D-xylopyranosyl-
was stirred for 30 min and then warmed to r.t. The product was
detected on TLC (2:1, petroleum ethereEtOAc). After completion of
the reaction, the reaction mixture was quenched with Et3N
(0.05 mL) and filtered. The filtrate was concentrated and purified by
a silica gel column chromatography (3:1, petroleum ethereEtOAc)
a-L
a-L-arabinopyranoside (35). Yield: 73% for two steps;
½
a 2D2
ꢂ
þ3.68 (c 2.88, CHCl3); Mp: 137e139 ꢀC; IR (KBr) nmax 2939,
1719, 1448, 1255, 1096, 705 cmꢁ1 1H NMR (CDCl3, 600 MHz):
;
d
7.25e8.03 (m, 20H, PheH), 5.72 (t-like, J ¼ 7.3, 6.9 Hz, 1H, H-30),
to afford 33i (204 mg, 91%); ½a D23
ꢂ
ꢁ11.8 (c 0.92, CHCl3); IR (KBr) nmax
5.35 (dd, J ¼ 3.2, 1.9 Hz, 1H, H-200), 5.31 (d, J ¼ 1.9 Hz, 1H, H-100),
5.26e5.29 (m, 3H, H-12, H-20, H-40), 5.11 (d, J ¼ 12.9 Hz,1H, PhCHH),
5.08 (d, J ¼ 12.7 Hz, 1H, PhCHH), 5.07 (t, J ¼ 10.1 Hz, 1H, H-400), 4.86
(d, J ¼ 5.0 Hz, 1H, H-10), 4.36 (dd, J ¼ 12.4, 4.1 Hz, 1H, H-50-1), 4.31
(d, J ¼ 7.3 Hz, 1H, H-1000), 4.21 (dd, J ¼ 10.1, 3.7 Hz, 1H, H-300), 4.15e
4.19 (m, 2H, H-4000, H-5000-1), 4.10e4.14 (m, 2H, H-3000, H-5000-2), 4.01
(dq, J ¼ 9.2, 6.0 Hz, 1H, H-500), 3.72 (m, 1H, H-2000), 3.65 (dd, J ¼ 12.4,
6.4 Hz, 1H, H-50-2), 3.05 (dd, J ¼ 11.9, 4.6 Hz, 1H, H-3), 2.91 (dd,
J ¼ 13.4, 4.1 Hz, 1H, H-18), 2.09, 2.04 (s each, 3H each, Ac ꢃ2), 1.57,
1.31 (s each, 3H each, Oe(CH3)2eO), 1.12 (d, J ¼ 6.4 Hz, 3H, H-600),
3456, 3059, 2846, 1726, 1487, 1269, 1063, 804, 705 cmꢁ1
;
1H NMR
(CDCl3, 600 MHz):
d
7.08e8.03 (m, 34H, PheH), 5.81 (t, J ¼ 9.6 Hz,
1H, H-30), 5.68 (t, J ¼ 7.4 Hz, 1H, H-3), 5.44 (dd, J ¼ 9.7, 7.8 Hz, 1H, H-
20), 5.36 (t, J ¼ 9.6 Hz, 1H, H-40), 5.31 (t-like, J ¼ 8.7, 7.0 Hz, 1H, H-4),
5.30 (d, J ¼ 7.3 Hz, 1H, H-1), 4.99 (t, J ¼ 7.8, 6.4 Hz, 1H, H-2), 4.97 (d,
J ¼ 7.8 Hz, 1H, H-10), 4.25 (dd, J ¼ 12.4, 4.6 Hz, 1H, H-60-1), 4.17 (dd,
J ¼ 11.9, 3.2 Hz, 1H, H-5-1), 4.03 (m, 1H, H-50), 3.98 (dd, J ¼ 11.9,
5.5 Hz, 1H, H-5-2), 3.45 (dd, J ¼ 12.4, 7.7 Hz, 1H, H-60-2), 2.31 (s, 3H,
SPhCH3); 13C NMR (CDCl3, 150 MHz):
d 171.4, 166.1, 165.9, 165.3,
165.1, 165.0, 138.5, 133.6, 133.4, 133.2, 130.0, 129.9, 129.8, 129.6,
128.6, 128.5, 128.4, 101.9 (C-1), 86.8 (C-10), 73.0, 72.8, 72.1, 70.5,
69.6, 65.2, 63.1, 60.6, 29.9, 22.9, 21.4, 21.3, 14.4; ESI-MS (m/z):
1.09, 0.99, 0.92, 0.89, 0.88, 0.80, 0.60 (s each, 3H each, CH3 ꢃ7); 13
C
NMR (CDCl3, 150 MHz):
d 177.5 (C-28), 170.5, 169.7, 165.6, 165.5,
143.7 (C-13), 136.5, 133.5, 130.0, 128.5, 122.6 (C-12), 110.4 ((CH3)2C),

10
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
103.5 (C-1000), 101.6 (C-10), 94.9 (C-100), 89.1 (C-3), 76.1, 75.8, 73.4,
71.7, 70.5, 66.0, 62.8, 61.3, 60.9, 55.9, 47.7, 46.8, 46.3, 41.8, 39.4, 36.8,
30.8, 28.2, 27.8, 26.2, 23.7, 20.6, 17.5, 16.5, 15.5, 13.8; HRMALDIMS:
20.6, 18.3, 17.5, 16.9, 16.5, 15.4; HRMALDIMS: calcd for [M þ Naþ]
C
78H96O20Na: 1375.6371; found: m/z 1375.6387.
calcd for [M
1415.6700.
þ
Naþ] C81H100O20Na: 1415.6721; found: m/z
4.1.4.2. Benzyl oleanolate 2,3,4,6-tetra-O-benzoyl-
b-D-glucopyr-
anosyl-(1 / 4)-2,3-di-O-benzoyl- -xylopy-ranosyl-(1 / 3)-2,4-
b-D
di-O-acetyl-a-L-rhamnopyranosyl-(1 / 2)-a-L-arabinopyranoside
4.1.3.2. Benzyl oleanolate 2,3,4,6-tetra-O-benzoyl-
b
-
D
-glucopyr-
(38). Yield: 90%; ½a D22
ꢂ
ꢁ0.6ꢀ (c 0.85, CHCl3); IR (KBr) nmax 3451,
anosyl-(1 / 4)-2,3-di-O-benzoyl- -xylopyr-anosyl-(1 / 3)-2,4-
b
-
D
2922, 1735, 1447, 1256, 1093, 1062, 1023, 711 cmꢁ1; 1H NMR (CDCl3,
di-O-acetyl-
L
a
-
L
-rhamnopyranosyl-(1 / 2)-3,4-O-isopropylidene-
a
-
600 MHz):
d
7.26e7.94 (m, 35H, PheH), 5.82 (t, J ¼ 9.9 Hz, 1H, H-
-arabinopyranoside (36). Yield: 73% for two steps; ½a D22
ꢂ
þ12.0ꢀ (c
30000), 5.61 (t-like, J ¼ 7.7, 7.1 Hz, 1H, H-3000), 5.47 (dd, J ¼ 9.9, 7.7 Hz,
1H, H-20000), 5.41 (t, J ¼ 9.4 Hz, 1H, H-40000), 5.28 (t-like, J ¼ 3.8, 3.3 Hz,
1H, H-12), 5.18 (dd, J ¼ 3.4, 1.1 Hz, 1H, H-200), 5.14 (dd, J ¼ 7.7, 5.5 Hz,
1H, H-2000), 5.10 (d, J ¼ 12.6 Hz, 1H, PhCHH), 5.06 (d, J ¼ 12.7 Hz, 1H,
PhCHH), 5.01 (t, J ¼ 9.9 Hz, 1H, H-400), 4.97 (d, J ¼ 7.7 Hz, 1H, H-10000),
4.93 (d, J ¼ 1.7 Hz, 1H, H-100), 4.74 (d, J ¼ 5.5 Hz, 1H, H-1000), 4.70 (d,
J ¼ 2.8 Hz, 1H, H-10), 4.22 (dd, J ¼ 11.6, 1.6 Hz, 1H, H-60000-1), 4.03e
1.10, CHCl3); IR (KBr) nmax 2934, 1731, 1455, 1369, 1260, 1097, 1058,
1027, 707 cmꢁ1 1H NMR (CDCl3, 600 MHz):
; d 7.27e7.98 (m, 35H,
PheH), 5.82 (t, J ¼ 9.9 Hz, 1H, H-30000), 5.63 (t, J ¼ 7.1 Hz, 1H, H-30),
5.47 (dd, J ¼ 9.9, 7.7 Hz, 1H, H-20000), 5.41 (t, J ¼ 9.9 Hz, 1H, H-40000),
5.29 (t, J ¼ 3.6 Hz, 1H, H-12), 5.27 (dd, J ¼ 3.3, 1.7 Hz, 1H, H-200), 5.25
(d, J ¼ 1.7 Hz, 1H, H-100), 5.13 (dd, J ¼ 7.1, 5.5 Hz, 1H, H-20), 5.11
(d, J ¼ 12.1 Hz, 1H, PhCHH), 5.07 (d, J ¼ 12.1 Hz, 1H, PhCHH), 5.02
(t, J ¼ 9.9 Hz, 1H, H-400), 4.97 (d, J ¼ 7.7 Hz, 1H, H-10000), 4.67
(d, J ¼ 5.5 Hz, 1H, H-10), 4.27 (d, J ¼ 7.7 Hz, 1H, H-1000), 4.20 (dd,
J ¼ 12.1, 2.8 Hz, 1H, H-5000-1), 4.17 (m, 1H, H-4000), 4.09e4.13 (m, 2H,
H-3000, H-60000-1), 4.08 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-600), 4.02e4.06 (m,
2H, H-40, H-50000), 3.96e4.01 (m, 3H, H-50-1, H-500, H-60000-2), 3.68e
3.72 (m, 2H, H-2000, H-50-2), 3.36 (dd, J ¼ 12.7, 7.1 Hz, 1H, H-5000-2),
3.02 (dd, J ¼ 12.1, 4.4 Hz, 1H, H-3), 2.89 (dd, J ¼ 13.7, 4.4 Hz, 1H, H-
18), 2.04, 2.01 (s each, 3H each, Ac ꢃ2), 1.49, 1.29 (s each, 3H each,
Oe(CH3)2eO), 1.10 (d, J ¼ 6.0 Hz, 3H, H-600), 1.13, 0.92, 0.90, 0.87,
0.86, 0.75, 0.60 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3,
4.08 (m, 2H, H-4000, H-50000), 3.96e4.02 (m, 3H, H-300, H-5000-1, H-60000
-
2), 3.82e3.86 (m, 3H, H-20, H-30, H-40), 3.79 (dq, J ¼ 9.9, 6.6 Hz, 1H,
H-500), 3.70 (dd, J ¼ 11.5, 8.8 Hz,1H, H-50-1), 3.70 (dd, J ¼ 11.5, 4.9 Hz,
1H, H-50-2), 3.40 (dd, J ¼ 12.1, 7.7 Hz, 1H, H-5000-2), 3.08 (dd, J ¼ 11.5,
4.4 Hz, 1H, H-3), 2.90 (dd, J ¼ 13.7, 3.8 Hz, 1H, H-18), 2.04, 2.01 (s
each, 3H each, Ac ꢃ2), 1.08 (d, J ¼ 6.1 Hz, 3H, H-600), 1.10, 0.91, 0.90,
0.89, 0.87, 0.76, 0.59 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3,
150 MHz):
d 177.4 (C-28), 170.4, 169.6, 165.9, 165.8, 165.7, 165.1,
165.0, 164.8, 143.7 (C-13), 136.4, 133.4, 133.2, 133.0, 129.7, 129.6,
129.5, 129.4, 129.2, 128.9, 128.5, 128.3, 127.9, 127.8, 122.4 (C-12),
101.6 (C-10), 101.5 (C-10000), 101.4 (C-1000), 98.0 (C-100), 90.4 (C-3), 75.7,
75.3, 72.9, 72.3, 71.8, 71.6, 70.7, 70.5, 70.0, 69.4, 67.1, 65.9, 62.9, 61.9,
60.1, 55.4, 47.6, 46.7, 45.9, 41.7, 41.3, 39.2, 39.0, 38.4, 36.7, 33.8, 33.1,
32.6, 30.7, 29.7, 28.1, 27.6, 25.8, 25.6, 23.6, 23.4, 23.0, 20.9, 20.4, 18.2,
150 MHz):
d 177.7 (C-28), 170.6, 166.1, 165.9, 165.5, 165.3, 165.1,
143.9 (C-13), 136.6, 133.6, 133.5, 133.4, 133.3, 129.9, 129.8, 129.7,
128.6, 128.5, 128.2, 123.2 (C-12), 110.5 ((CH3)2C), 103.5 (C-1000), 102.0
(C-10), 101.8 (C-10000), 95.0 (C-100), 89.1 (C-3), 79.0, 73.1, 72.1, 71.1,
69.4, 66.6, 66.1, 63.1, 60.6, 56.0, 47.8, 47.0, 46.1, 42.0, 41.6, 39.3, 38.9,
36.9, 35.6, 33.3, 32.8, 32.5, 30.9, 29.9, 28.2, 27.9, 26.3, 26.1, 23.9,
23.3, 21.3, 20.6, 18.4, 17.6, 17.1, 16.6, 15.6, 14.4; HRMALDIMS: calcd
for [M þ Naþ] C108H122O28Na: 1889.8059; found: m/z 1889.8015.
17.3, 16.8, 16.4, 15.3; HRMALDIMS: calcd for [M
105H118O28Na: 1849.7706; found: m/z 1849.7702.
þ
Naþ]
C
4.1.5. General procedure for synthesizing compounds 42e44
ꢀ
A solution of Mbp thioglycoside 34 (40 mg, 0.112 mmol) and 4 A
MS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under
argon for 30 min, and then cooled to ꢁ78 ꢀC. At this temperature, a
solution of TMSOTf (0.2 equiv.) in dry CH2Cl2 was injected and after
10 min a trichloroacetimidate 32, 39 or 40 (2.1 equiv.) in dry CH2Cl2
was added. The mixture was stirred for additional 30 min and then
warmed up to ꢁ10 ꢀC. To the above mixture was added a solution of
saponin acceptor 41 (99 mg, 0.112 mmol, 1.0 equiv.) in CH2Cl2 (2 mL)
followed by NIS (50 mg, 0.112 mmol, 2.0 equiv.). After being stirred
for 1 h, the reaction mixture was quenched with Et3N, and then
filtered through a pad of Celite. The filtrate was concentrated. The
residue was purified silica gel column chromatography (2.5:1, pe-
troleum ethereEtOAc) to give the fully protected saponin. The
amounts of the reactants and the yields of the saponin products were
calculated based on saponin accepter 41.
4.1.4. General procedure for synthesizing compounds 37e38
To a solution of compound 35 or 36 (0.054 mmol) in CH2Cl2e
MeOH (V:V/1:2, 5 mL) was added p-TsOH (0.054 mmol) was stirred
at room temperature. When TLC (3:2, petroleum ethereEtOAc)
showed that deprotection had completed, Et3N (0.1 mL) was added
and the mixture was concentrated and purified through a silica gel
column chromatography (petroleum ethereEtOAc), affording the
product 37 or 38.
4.1.4.1. Benzyl oleanolate 2,3,4-tri-O-benzoyl-b-D-xylopyranosyl-
(1 / 3)-2,4-di-O-acetyl-a-L-rhamnopyranosyl-(1 / 2)-a-L-arabi-
nopyranoside (37). Yield: 95%; ½a D23
ꢂ
ꢁ17.6ꢀ (c 3.65, CHCl3); Mp: 141e
143 ꢀC; IR (KBr) nmax 2945, 1731, 1451, 1373,1260,1097, 707 cmꢁ1; 1H
NMR (CDCl3, 600 MHz):
d 7.27e8.04 (m, 20H, PheH), 5.70 (t-like,
J ¼ 7.1, 6.6 Hz,1H, H-3000), 5.27e5.30 (m, 4H, H-12, H-300, H-2000, H-4000),
5.10 (d, J ¼ 12.1 Hz,1H, PhCHH), 5.08 (s,1H, H-200), 5.06 (d, J ¼ 12.7 Hz,
1H, PhCHH), 5.01 (s, 1H, H-100), 4.97 (d, J ¼ 5.0 Hz, 1H, H-1000), 4.74 (br
s, 1H, H-10), 4.35 (dd, J ¼ 12.7, 3.8 Hz, 1H, H-5000-1), 4.10 (dd, J ¼ 9.9,
3.8 Hz, 1H, H-50-1), 3.88e3.92 (m, 2H, H-30, H-40), 3.83e3.87 (m, 2H,
H-20, H-500), 3.71e3.75 (m, 2H, H-400, H-50-2), 3.61 (dd, J ¼ 11.5, 4.4 Hz,
1H, H-5000-2), 3.10 (dd, J ¼ 11.5, 4.4 Hz, 1H, H-3), 2.91 (dd, J ¼ 13.7,
3.8 Hz, 1H, H-18), 2.09, 1.80 (s each, 3H each, Ac ꢃ2), 1.12 (d,
J ¼ 6.6 Hz, 3H, H-600), 1.11, 0.94, 0.92, 0.89, 0.88, 0.78, 0.60 (s each, 3H
4.1.5.1. Benzyl oleanolate
anosyl-(1 / 3)-2,4-di-O-acetyl-
di-O-benzoyl-
-xylopyranoside (42). Yield: 67%; Mp: 140e142 ꢀC;
a 2D5
ꢁ28.1 (c 2.04, CHCl3); IR (KBr) nmax 2945, 1731, 1603, 1451,
1256, 1093, 711 cmꢁ1 1H NMR (CDCl3, 600 MHz):
7.27e8.10 (m,
3
b
-O-2,3,4-tri-O-benzoyl-
-rhamno-pyranosyl-(1 / 2)-3,4-
b-D-xylopyr-
a-L
b-D
½
ꢂ
;
d
30H, PheH), 5.59 (t, J ¼ 7.1, 6.6 Hz, 2H, H-30, H-3000), 5.29 (t,
J ¼ 3.8 Hz, 1H, H-12), 5.20e5.22 (m, 2H, H-200, H-2000), 5.14e5.17 (m,
2H, H-40, H-4000), 5.10 (d, J ¼ 12.7 Hz, 1H, PhCHH), 5.05 (s, 1H, H-100),
5.04 (d, J ¼ 12.7 Hz, 1H, PhCHH), 5.03 (m, 1H, H-400), 4.78 (d,
J ¼ 4.9 Hz, 1H, H-10), 4.60 (d, J ¼ 5.0 Hz, 1H, H-1000), 4.37 (dd, J ¼ 12.1,
3.9 Hz, 1H, H-50-1), 4.11 (dd, J ¼ 12.1, 3.8 Hz, 1H, H-5000-1), 4.01e4.04
(m, 2H, H-300, H-500), 3.97 (t, J ¼ 5.5 Hz, 1H, H-20), 3.62 (dd, J ¼ 12.1,
6.6 Hz, 1H, H-50-2), 3.33 (dd, J ¼ 12.1, 6.6 Hz, 1H, H-5000-2), 3.15 (dd,
J ¼ 11.5, 4.4 Hz, 1H, H-3), 2.91 (dd, J ¼ 13.2, 4.4 Hz, 1H, H-18), 1.92,
1.77 (s each, 3H each, Ac ꢃ2), 1.13 (d, J ¼ 6.6 Hz, 3H, H-600), 1.12, 0.95,
each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
d 177.5 (C-28), 170.5,
169.8, 165.6, 165.5, 165.0, 143.8 (C-13), 136.5, 133.5, 133.4, 130.0, 129.9,
128.5, 128.4, 128.1, 122.5 (C-12), 101.7 (C-10), 101.3 (C-1000), 98.0 (C-100),
90.5 (C-3), 75.8, 75.6, 71.9, 71.8, 70.2, 70.1, 69.9, 68.9, 67.3, 66.0, 64.9,
61.1, 60.4, 55.5, 47.7, 46.8, 46.0, 41.8, 41.5, 39.4, 39.2, 38.6, 36.8, 33.9,
33.2, 32.7, 32.5, 30.8, 29.8, 28.2, 27.7, 25.9, 25.8, 23.7, 23.5, 23.1, 21.1,

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
11
0.92, 0.90, 0.89, 0.74, 0.60 (s each, 3H each, CH3 ꢃ7); 13C NMR
CH2Cl2 (5 mL) was stirred at room temperature for 30 min then
cooled to 0 ꢀC. TMSOTf (10
L, 0.02 mmol) was added dropwise and
(CDCl3, 150 MHz):
d
177.6 (C-28), 169.9, 169.5, 165.8, 165.6, 165.5,
m
165.4, 165.2, 143.8 (C-13), 136.5, 133.5, 133.4, 129.9, 129.8, 128.6,
128.5,128.4, 122.6 (C-12), 103.1 (C-10), 101.1 (C-1000), 97.6 (C-100), 89.4
(C-3), 75.9, 74.1, 71.4, 71.2, 70.3, 69.5, 69.1, 67.2, 66.0, 60.9, 55.7, 47.7,
46.8, 46.0, 41.8, 41.5, 39.4, 39.2, 38.8, 36.8, 33.9, 33.2, 32.8, 32.5,
30.8, 28.1, 27.7, 26.0, 25.9, 23.7, 23.5, 23.1, 20.9, 20.6, 18.3, 17.5, 16.9,
16.4, 15.5; HRMALDIMS: calcd for [M þ Naþ] C92H104O22Na:
1583.6952; found: m/z 1583.6912.
the reaction mixture was stirred for 30 min and then warmed to
room temperature. The product was detected on TLC (2:1, petro-
leum ethereEtOAc). After completion of the reaction, the reaction
mixture was quenched with Et3N (0.05 mL) and filtered. The filtrate
was concentrated and purified by a silica gel column chromatog-
raphy (3:1, petroleum ethereEtOAc) to afford 45 (171 mg, 91%);
½
a 2D0
ꢂ
þ63.2 (c 0.52, CHCl3); 1H NMR (CDCl3, 600 MHz):
d 7.23e8.05
(m, 30H, PheH), 5.82 (dd, J ¼ 10.1, 3.2 Hz, 1H, H-300), 5.71 (t-like,
J ¼ 7.3, 6.9 Hz, 1H, H-30), 5.60 (t-like, J ¼ 10.1, 9.6 Hz, 1H, H-400), 5.56
(dd, J ¼ 3.2, 1.4 Hz, 1H, H-200), 5.34 (d, J ¼ 1.8 Hz, 1H, H-100), 5.30
(t-like, J ¼ 3.7, 3.2 Hz, 1H, H-12), 5.21e5.24 (m, 1H, H-40), 5.09
(dd, J ¼ 34.4, 12.8 Hz, 2H, CH2ePh), 4.87 (d, J ¼ 5.5 Hz, 1H, H-10),
4.48e4.52 (m, 1H, H-500), 4.40 (dd, J ¼ 12.4, 4.6 Hz, 1H, H-50-1), 4.12
(dd, J ¼ 6.9, 6.0 Hz, 1H, H-20), 3.66 (dd, J ¼ 12.4, 7.4 Hz, 1H, H-50-2),
3.25 (dd, J ¼ 11.9, 4.6 Hz, 1H, H-3), 2.91 (dd, J ¼ 14.2, 4.6 Hz, 1H, H-
18), 1.99 (dt, J ¼ 13.2, 4.1 Hz, 1H, H-16), 1.31 (d, J ¼ 5.9 Hz, 3H, H-600),
1.13, 1.12, 0.92, 0.89, 0.89, 0.83, 0.62 (s each, 3H each, CH3 ꢃ7); 13C
4.1.5.2. Benzyl oleanolate 3
b
-O-2,3,4,6,20,30,40,60-octa-O-benzoyl-
3)-2,4-di-O-acetyl- -rhamnopyranosyl-
-xylopyranoside (43). Yield: 66%; Mp:
þ26.2 (c 1.92, CHCl3); IR (KBr) nmax 2945, 1731,
7.27e
b-D-
cellobicopyranosyl-(1
/
a-L
(1 / 2)-3,4-di-O-benzoyl-b-D
144e146 ꢀC; ½a 2D4
ꢂ
1599,1451,1264,1085, 707 cmꢁ1; 1H NMR (CDCl3, 600 MHz):
d
8.07 (m, 50H, PheH), 5.65 (t, J ¼ 9.4 Hz,1H, H-3000), 5.51e5.54 (m, 2H,
H-2000, H-30), 5.40 (t-like, J ¼ 9.9, 3.3 Hz,1H, H-30000), 5.27e5.31 (m, 2H,
H-12, H-300), 5.25 (dd, J ¼ 9.6, 7.8 Hz, 1H, H-20000), 5.15 (m, 1H, H-40),
5.10 (d, J ¼ 12.1 Hz, 1H, PhCHH), 5.06 (d, J ¼ 12.6 Hz, 1H, PhCHH),
4.92e4.95 (m, 3H, H-100, H-200, H-400), 4.74 (d, J ¼ 7.7 Hz, 1H, H-1000),
4.66 (d, J ¼ 4.9 Hz, 1H, H-10), 4.34 (dd, J ¼ 11.6, 3.8 Hz, 1H, H-50-1),
4.28 (dd, J ¼ 12.6, 2.7 Hz, 1H, H-6000-1), 4.22 (t, J ¼ 9.9 Hz, 1H, H-4000),
4.05 (d, J ¼ 7.8 Hz,1H, H-10000), 4.03 (t, J ¼ 9.6 Hz,1H, H-40000), 3.99 (dd,
J ¼ 12.1, 2.8 Hz, 1H, H-60000-1), 3.89e3.92 (m, 1H, H-500), 3.85e3.87 (m,
1H, H-5000), 3.84 (t, J ¼ 6.6 Hz, 1H, H-20), 3.67 (m, 1H, H-50000), 3.57 (dd,
J ¼ 12.1, 5.5 Hz, 1H, H-60000-2), 3.51 (dd, J ¼ 12.1, 5.6 Hz, 1H, H-50-2),
3.12 (dd, J ¼ 11.5, 4.4 Hz,1H, H-3), 2.91 (dd, J ¼ 13.7, 3.8 Hz, 1H, H-18),
2.71 (t, J ¼ 10.5 Hz, 1H, H-6000-2), 1.74, 1.74 (s each, 3H each, Ac ꢃ2),
1.04 (d, J ¼ 6.1 Hz, 3H, H-600), 1.12, 0.92, 0.90, 0.89, 0.86, 0.67, 0.59 (s
NMR (CDCl3, 150 MHz):
d 177.5 (C-28), 165.9, 165.8, 165.2, 143.8
(C-13), 136.6, 133.4, 130.0, 128.4, 122.6 (C-12), 103.5 (C-10), 97.7
(C-100), 89.6 (C-3), 74.2, 72.5, 71.9, 70.6, 66.0, 61.3, 55.8, 47.8, 46.8,
46.0, 39.4, 39.3, 36.9, 33.2, 30.8, 28.2, 25.9, 23.8, 17.5, 16.6, 15.5;
ESI-MS (m/z): 1345.6 (M þ Hþ); HRMALDIMS: calcd for [M þ Naþ]
C83H92O16Na: 1367.6309; found: m/z 1367.6278.
4.1.7. 2,3,4-Tri-O-benzoyl-
tolyl)thio-3,4-O-isopropylidene-
a
-
L
-rhamnopyranosyl-(1 / 2)-1-(4-
-arabino-pyranoside (47)
a-L
ꢀ
A mixture of compound 46 (100 mg, 0.34 mmol), powdered 4 A
molecular sieves and compound 39 (314 mg, 0.51 mmol) in dry
CH2Cl2 (5 mL) was stirred at room temperature for 30 min then
each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
¼ 177.5 (C-28),
d
169.8, 169.7, 165.7, 165.6, 165.4, 165.3, 165.1, 143.8 (C-13), 136.5, 133.3,
129.8, 129.7, 128.5, 128.3, 128.1, 122.6 (C-12), 103.4 (C-10), 100.9 (C-
1000), 100.5 (C-10000), 97.9 (C-100), 88.9 (C-3), 76.4, 75.4, 73.0, 72.4, 72.3,
71.9, 69.7, 69.6, 66.9, 66.6, 66.0, 62.7, 61.7, 55.7, 47.7, 46.8, 45.9, 41.8,
41.5, 39.4, 39.2, 38.8, 36.8, 33.2, 32.8, 30.8, 28.0, 26.0, 23.7, 23.1, 20.7,
20.1, 17.4, 16.9, 16.2, 15.5; HRMALDIMS: calcd for [M þ Naþ]
cooled to ꢁ78 ꢀC. TMSOTf (10
mL, 0.02 mmol) was added dropwise
and the reaction mixture was stirred for 30 min and then warmed
to room temperature. The product was detected on TLC (2:1, pe-
troleum ethereEtOAc). After completion of the reaction, the reac-
tion mixture was quenched with Et3N (0.05 mL) and filtered. The
filtrate was concentrated and purified by a silica gel column chro-
matography (5:1, petroleum ethereEtOAc) to afford 47 (221 mg,
C127H132O32Na: 2191.8557; found: m/z 2191.8594.
4.1.5.3. Benzyl oleanolate 3
anosyl-(1 / 3)-2,4-di-O-acetyl-
di-O-benzoyl-
-xylopyranoside (44). Yield: 70%; Mp: 136e138 ꢀC;
a 2D4
þ36.9 (c 2.10, CHCl3); IR (KBr) nmax 2945, 1727, 1603, 1451, 1264,
1093, 707 cmꢁ1 1H NMR (CDCl3, 600 MHz):
7.28e8.12 (m, 30H,
b-O-2,3,4-tri-O-benzoyl-
a-L
-rhamnopyr-
86%); ½a 2D3
þ77.9 (c 0.95, CHCl3); IR (KBr) vmax 3364, 3231, 2979,
ꢂ
a-
L
-rha-mnopyranosyl-(1 / 2)-3,4-
1726, 1448, 1255, 1109, 831, 705 cmꢁ1 1H NMR (CDCl3, 600 MHz):
;
b-
D
d
7.13e8.09 (m, 19H, PheH), 5.81 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-30), 5.74
½
ꢂ
(dd, J ¼ 3.3, 1.6 Hz, 1H, H-20), 5.66 (t-like, J ¼ 9.9, 9.8 Hz, 1H, H-40),
5.49 (d, J ¼ 1.7 Hz, 1H, H-10), 4.82 (d, J ¼ 8.3 Hz, 1H, H-1), 4.55 (dq,
J ¼ 9.9, 6.1 Hz, 1H, H-50), 4.32 (m, 1H, H-4), 4.29 (t, J ¼ 6.1 Hz, 1H, H-
3), 4.20 (dd, J ¼ 13.2, 3.8 Hz, 1H, H-5-1), 3.98 (dd, J ¼ 8.3, 6.1 Hz, 1H,
H-2), 3.80 (dd, J ¼ 12.7, 3.8 Hz, 1H, H-5-2), 2.33 (s, 3H, SToleCH3),
1.52, 1.35 (s each, 3H each, Oe(CH3)2CeO), 1.32 (d, J ¼ 6.6 Hz, 3H, H-
;
d
PheH), 5.57e5.62 (m, 3H, H-30, H-3000, H-400), 5.38 (t-like, J ¼ 2.3,
1.9 Hz, 1H, H-2000), 5.29 (t, J ¼ 3.7 Hz, 1H, H-12), 5.24 (br s, 1H, H-200),
5.13e5.19 (m, 2H, H-40, H-400), 5.09 (d, J ¼ 12.4 Hz, 1H, PhCHH), 5.08
(s, 1H, H-100), 5.06 (d, J ¼ 12.9 Hz, 1H, PhCHH), 4.89 (s, 1H, H-1000),
4.81 (d, J ¼ 4.6 Hz, 1H, H-10), 4.48 (dd, J ¼ 12.4, 4.1 Hz, 1H, H-50-1),
4.12e4.15 (m, 3H, H-300, H-500, H-5000), 4.00 (t-like, J ¼ 6.0, 5.0 Hz, 1H,
H-20), 3.63 (dd, J ¼ 11.9, 6.4 Hz, 1H, H-50-2), 3.18 (dd, J ¼ 11.9, 4.6 Hz,
1H, H-3), 2.90 (dd, J ¼ 13.8, 4.6 Hz, 1H, H-18), 2.22, 2.10 (s each, 3H
each, Ac ꢃ2), 1.23 (d, J ¼ 5.9 Hz, 3H, H-600), 1.12, 0.98, 0.92, 0.89, 0.89,
0.77, 0.60 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
60); 13C NMR (CDCl3, 150 MHz):
d 166.0, 165.8, 165.7, 138.1, 133.7,
133.5, 133.3, 132.8, 130.2, 130.0, 129.9, 129.8, 129.7, 129.6, 128.8,
128.7, 128.6, 128.5, 110.7 (C-1), 96.8 (C-10), 86.9 (C-30), 78.5, 76.0,
72.8, 71.9, 70.9, 70.3, 67.7, 65.1, 28.0, 26.3, 21.4, 17.7; HRMALDIMS:
calcd for [M þ Naþ] C42H42O11SNa: m/z 777.2359; found: m/z
777.2340.
d
¼ 177.6 (C-28), 170.4, 170.0, 165.8, 165.9, 165.8, 165.4, 143.8 (C-13),
136.5, 133.7, 133.4, 133.1, 129.9, 128.8, 128.5, 128.1, 122.6 (C-12), 102.9
(C-10), 98.7 (C-1000), 97.6 (C-100), 89.5 (C-3), 74.7, 73.8, 72.5, 71.6, 71.0,
69.5, 67.5, 67.2, 66.0, 60.8, 55.7, 47.7, 46.8, 46.0, 41.8, 41.5, 39.4, 39.3,
38.8, 36.8, 33.9, 33.2, 32.7, 30.8, 28.1, 27.7, 26.1, 26.0, 23.7, 23.1, 21.0,
20.9, 18.3,17.7,17.4,16.9,16.5,15.5; HRMALDIMS: calcd for [M þ Naþ]
4.1.8. Benzyl oleanolate 2,3,4-tri-O-benzoyl-
a-L-rhamnopyranosyl-
(1 / 2)-3,4-O-isopropylidene- -arabinopyranoside (49)
b-L
ꢀ
A mixture of compound 48 (80 mg, 0.15 mmol), powdered 4 A
molecular sieves and compound 47 (166 mg, 0.22 mmol) in dry
CH2Cl2 (5 mL) was stirred at room temperature for 30 min then
cooled to 0 ꢀC. NIS (54 mg, 0.23 mmol) and AgOTf (5 mg,
0.02 mmol) was added, the reaction mixture was stirred for 30 min
and then warmed to room temperature. The product was detected
on TLC (3:1, petroleum ethereEtOAc). After completion of the re-
action, the reaction mixture was quenched with Et3N (0.20 mL) and
filtered. The filtrate was diluted with CH2Cl2, and then washed with
C
93H106O22Na: 1597.7089; found: m/z 1597.7068.
4.1.6. Benzyl oleanolate 3
b
-O-[3-O-(2,3,4-tri-O-benzoyl-
a-L-
rhamnopyranosyl)-3,4-di-O-benzoyl-
A mixture of compound 41 (120 mg, 0.14 mmol), powdered 4 A
molecular sieves and compound 40 (104 mg, 0.17 mmol) in dry
b-D
-xylo-pyranoside] (45)
ꢀ

12
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
saturated Na2S2O3 and brine, respectively. The organic layer was
dried over anhydrous Na2SO4, and then concentrated in vacuo. The
resulting residue was purified by a silica gel column chromatog-
raphy (3:1, petroleum ethereEtOAc) to afford 49 (60 mg, 35%);
600 MHz):
d
7.23e8.07 (m, 25H, PheH), 5.90 (dd, J ¼ 10.6, 3.7 Hz,
1H, H-300), 5.85 (d, J ¼ 1.8 Hz, 1H, H-30), 5.81 (m, 1H, H-40), 5.65
(t, J ¼ 10.1 Hz, 1H, H-400), 5.55 (s, 1H, PhCHO), 5.30 (t, J ¼ 3.6 Hz, 1H,
H-12), 5.09 (dd, J ¼ 40.8, 12.8 Hz, 2H, CH2ePh), 4.61e4.64 (m, 1H,
H-500), 4.52 (d, J ¼ 7.3 Hz, 1H, H-10), 4.32 (dd, J ¼ 10.2, 1.8 Hz, 1H,
H-60-1), 4.18 (d, J ¼ 2.8 Hz, 1H, H-100), 4.08e4.10 (m, 2H, H-50, H-60-
2), 3.96 (m, 2H, H-20, H-200), 3.20 (dd, J ¼ 11.9, 4.1 Hz, 1H, H-3), 2.91
(dd, J ¼ 13.3, 3.7 Hz, 1H, H-18), 1.1.36 (d, J ¼ 6.4 Hz, 3H, H-600), 1.14,
0.97, 0.93, 0.92, 0.90, 0.89, 0.63 (s each, 3H each, CH3 ꢃ7); 13C NMR
½
a 2D3
1096, 705 cmꢁ1
ꢂ
þ131.7 (c 0.50, CHCl3); IR (KBr) vmax 2919, 1726, 1441, 1255,
;
1H NMR (CDCl3, 600 MHz):
d
7.10e8.10 (m, 20H,
PheH), 5.84 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-300), 5.77 (dd, J ¼ 3.3, 1.7 Hz,
1H, H-200), 5.64 (t, J ¼ 9.9 Hz,1H, H-400), 5.34 (d, J ¼ 1.7 Hz,1H, H-100),
5.29 (t-like, J ¼ 3.8, 3.3 Hz, 1H, H-12), 5.11 (d, J ¼ 12.7 Hz, 1H,
PhCHH), 5.10 (d, J ¼ 3.3 Hz, 1H, H-10), 5.06 (d, J ¼ 12.7 Hz, 1H,
PhCHH), 4.44 (dd, J ¼ 7.7, 6.0 Hz, 1H, H-30), 4.27e4.30 (m, 2H, H-40,
H-500), 4.11 (m, 1H, H-50-1), 3.97 (d, J ¼ 12.7 Hz, 1H, H-50-2), 3.90
(dd, J ¼ 7.7, 3.3 Hz, 1H, H-20), 3.29 (dd, J ¼ 11.5, 3.8 Hz, 1H, H-3),
2.90 (dd, J ¼ 14.3, 3.8 Hz, 1H, H-18), 1.55, 1.36 (s each, 3H each,
Oe(CH3)2CeO), 1.35 (d, J ¼ 6.4 Hz, 3H, H-600), 1.13, 1.05, 0.95, 0.92,
0.92, 0.90, 0.62 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3,
(CDCl3, 150 MHz):
d 177.5 (C-28), 165.9, 165.6, 143.8 (C-13), 137.5,
136.5, 133.0, 128.3, 128.0, 122.7 (C-12), 104.0 (C-10), 101.3 (PhC(O)),
97.2 (C-1000), 89.9 (C-3), 72.1, 70.7, 70.1, 66.5, 56.1, 47.8, 46.9, 46.0,
41.5, 39.5, 39.3, 33.2, 30.8, 28.3, 26.0, 23.8, 17.6, 17.0, 16.9;
HRMALDIMS: calcd for [M þ Naþ] C77H90O15Na: 1277.6202; found:
m/z 1277.6172.
150 MHz):
d
177.7 (C-28), 165.8, 143.9 (C-13), 136.6, 133.7, 133.5,
4.1.10.2. Benzyl
oleanolate
3b-O-[3-O-(2,3,4-tri-O-benzoyl-a-L-
130.0, 129.5, 128.6, 128.5, 128.3, 128.2, 122.6 (C-12), 109.2, 99.3
(C-100), 93.5 (C-10), 88.8 (C-3), 81.1 (C-300), 71.0, 70.4, 70.2, 69.9, 69.3,
67.2, 66.1, 62.7, 55.7, 47.8, 46.9, 41.9, 41.7, 39.5, 38.9, 38.1, 37.8, 33.3,
33.1, 32.8, 32.7, 31.5, 31.3, 31.2, 30.0, 29.9, 28.7, 28.0, 23.8, 23.7, 23.5,
23.3, 23.2, 18.0, 17.9, 16.5, 15.5, 14.2, 11.6; HRMALDIMS: calcd for
[M þ Naþ] C72H88O14Na: m/z 1199.6078; found: m/z 1199.6066.
rhamnopyranosyl)-4,6-di-O-benzylidene-b-D-galactopyranoside]
(52b). Yield: 29%; ½a D20
ꢂ
þ80.8 (c 0.55, CHCl3); 1H NMR (CDCl3,
600 MHz):
d
7.04e8.11 (m, 25H, PheH), 5.96 (dd, J ¼ 10.5, 3.2 Hz,
1H, H-300), 5.83 (t, J ¼ 10.1 Hz, 1H, H-400), 5.76 (m, 1H, H-200), 5.58 (t,
J ¼ 10.1 Hz, 1H, H-30), 5.53 (s, 1H, H-100), 5.38 (s, 1H, PhCHO), 5.30 (br
s, 1H, H-12), 5.10 (dd, J ¼ 34.4, 12.5 Hz, 2H, CH2ePh), 4.91 (m, 1H, H-
500), 4.66 (d, J ¼ 7.3 Hz, 1H, H-10), 4.55 (m, 1H, H-40), 4.43 (t,
J ¼ 8.7 Hz, 1H, H-20), 4.33 (dd, J ¼ 11.5, 5.0 Hz, 1H, H-60-1), 4.11 (dd,
J ¼ 9.2, 6.8 Hz, 1H, H-60-2), 3.86 (m, 1H, H-50), 3.28 (dd, J ¼ 11.4,
4.4 Hz, 1H, H-3), 2.92 (dd, J ¼ 13.7, 3.2 Hz, 1H, H-18), 1.40 (d,
J ¼ 6.0 Hz, 3H, H-600), 1.14, 0.93, 0.93, 0.92, 0.92, 0.90, 0.63 (s each,
4.1.9. Benzyl oleanolate 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-
(1 / 2)- -arabinopyranoside (50)
b-L
A mixture of compound 49 (100 mg, 0.09 mmol) and p-TsOH
(15 mg, 0.09 mmol) in CH2Cl2eMeOH (V:V/1:2, 6 mL) was stirred at
r.t. When TLC (3:2, petroleum ethereEtOAc) showed that depro-
tection had completed, Et3N (0.1 mL) was added and the mixture
was concentrated and purified through a silica gel column chro-
matography (2:1, petroleum ethereEtOAc) to afford 50 (81 mg,
3H each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
d 177.5 (C-28), 165.8,
165.5, 163.4, 143.8 (C-13), 137.8, 136.5, 133.3, 129.8, 128.3, 122.7 (C-
12), 105.3 (C-10), 101.2 (PhC(O)), 99.9 (C-1000), 89.9 (C-3), 81.8, 70.9,
70.3, 70.1, 66.0, 60.5, 55.8, 47.8, 46.8, 46.0, 41.8, 39.2, 36.9, 33.2, 30.8,
28.3, 26.0, 23.8, 19.2, 17.8, 16.8, 15.4; HRMALDIMS: calcd for
[M þ Naþ] C77H90O15Na: 1277.6173; found: m/z 1277.6172.
84%) as a white solid; ½a D23
ꢂ
þ156.2 (c 0.40, CHCl3); IR (KBr) vmax
2938, 1727, 1455, 1260, 1108, 1066, 707 cmꢁ1
;
1H NMR (CDCl3,
600 MHz):
d
7.27e8.07 (m, 20H, PheH), 5.80 (dd, J ¼ 9.9, 3.3 Hz, 1H,
H-300), 5.75 (dd, J ¼ 2.8, 1.6 Hz, 1H, H-200), 5.66 (t, J ¼ 9.9 Hz, 1H,
H-400), 5.28 (br s, 2H, H-100, H-12), 5.22 (d, J ¼ 3.8 Hz, 1H, H-10), 5.12
(d, J ¼ 12.7 Hz, 1H, PhCHH), 5.05 (d, J ¼ 12.7 Hz, 1H, PhCHH), 4.38
(dq, J ¼ 9.9, 6.0 Hz, 1H, H-500), 4.20 (d, J ¼ 8.8 Hz, 1H, H-30), 4.09
(m, 1H, H-40), 4.02 (dq, J ¼ 9.3, 3.3 Hz, 1H, H-20), 3.91 (d, J ¼ 12.6 Hz,
1H, H-50-1), 3.75 (d, J ¼ 12.1 Hz, 1H, H-50-2), 3.31 (dd, J ¼ 11.6,
3.9 Hz, 1H, H-3), 2.89 (dd, J ¼ 13.7, 4.4 Hz, 1H, H-18), 1.35 (d,
J ¼ 6.0 Hz, 3H, H-600), 1.13, 1.07, 0.97, 0.91, 0.89, 0.87, 0.61 (s each, 3H
4.1.10.3. Benzyl oleanolate 3b-O-[2,3-di-O-(2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl)-4,6-di-O-benzyl-idene-b-D-galactopyranoside]
(52c). Yield: 7%; ½a D20
ꢂ
þ64.1 (c 0.65, CHCl3); 1H NMR (CDCl3,
600 MHz):
d
7.15e8.11 (m, 40H, PheH), 5.90 (dd, J ¼ 10.0, 3.6 Hz,1H,
H-3000), 5.83 (dd, J ¼ 10.1, 3.7 Hz, 1H, H-300), 5.77 (m, 1H, H-40), 5.70
(m, 1H, H-4000), 5.62 (t, J ¼ 10.1 Hz, 1H, H-400), 5.56 (s, 1H, PhCHO),
5.32 (d, J ¼ 1.9 Hz, 1H, H-1000), 5.30 (t, J ¼ 3.6 Hz, 1H, H-12), 5.08 (dd,
J ¼ 31.1, 12.4 Hz, 2H, CH2ePh), 4.48e4.50 (m, 1H, H-500), 4.44e4.47
(m, 1H, H-5000), 4.40 (d, J ¼ 7.3 Hz, 1H, H-10), 4.35 (d, J ¼ 3.2 Hz, 1H,
H-100), 4.33 (dd, J ¼ 11.5, 1.6 Hz, 1H, H-60-1), 4.08e4.12 (m, 4H, H-20,
H-30, H-50, H-60-2), 3.75 (dd, J ¼ 7.0, 3.7 Hz, 1H, H-200), 3.17 (dd,
J ¼ 11.9, 4.6 Hz, 1H, H-3), 2.91 (dd, J ¼ 12.8, 4.1 Hz, 1H, H-18), 1.44 (d,
J ¼ 5.0 Hz, 3H, H-6000), 1.34 (d, J ¼ 5.9 Hz, 3H, H-600), 1.12, 1.05, 0.91,
0.89, 0.86, 0.85, 0.60 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3,
each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
d 177.7 (C-28), 171.4,
165.8, 144.0 (C-13), 136.7, 133.7, 133.6, 133.3, 130.1, 129.9, 128.8,
128.6, 128.5, 128.2, 122.6 (C-12), 99.3 (C-100), 93.5 (C-10), 88.7 (C-3),
81.2 (C-300), 71.9, 70.8, 70.2, 69.9, 68.5, 67.2, 66.1, 62.7, 60.6, 55.7,
47.8, 46.9, 46.1, 41.9, 41.6, 39.5, 38.9, 38.3, 37.1, 34.1, 33.3, 32.9, 32.5,
30.9, 28.7, 27.8, 26.1, 23.8, 23.6, 23.2, 21.3, 21.1, 18.5, 17.9, 17.1, 16.9,
15.5, 14.4; ESI-MS: m/z 1159.7 (M þ Naþ).
150 MHz):
d 177.5 (C-28), 165.5, 165.2, 164.9, 143.8 (C-13), 137.6,
136.5, 129.8, 128.5, 128.3, 128.0, 122.7, 104.3 (C-10), 101.2 (C-100),
100.2 (PhC(O)), 97.7 (C-1000), 89.6 (C-3), 85.8, 72.5, 71.6, 70.9, 66.0,
56.3, 47.9, 46.9, 46.0, 41.5, 39.5, 39.3, 33.2, 30.8, 28.3, 26.0, 23.8,17.5,
17.1, 15.6; HRMALDIMS: calcd for [M þ Naþ] C104H112O22Na:
1735.7533; found: m/z 1735.7538.
4.1.10. General procedure for synthesizing compounds 52aec and
54aec
Compound 51 or 53 (0.13 mmol), trichloroacetimidate 40
ꢀ
(0.13 mmol) and powdered 4 A molecular sieves (0.10 g) were
stirred for 40 min at room temperature in dry CH2Cl2 (2 mL).
TMSOTf (0.002 mL, 0.013 mmol) was added dropwise. The mixture
was stirred for 10 min followed by addition of Et3N and filtration.
The filtrate was concentrated and purified by a silica gel column
chromatography (5:1, petroleum ethereEtOAc) to afford the
saponin products 52aec and 54aec.
4.1.10.4. Benzyl
oleanolate
3b-O-[2-O-(2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl)-4,6-di-O-benzylidene-b-D-glucopyranoside]
(54a). Yield: 45%; ½a D20
ꢂ
þ49.5 (c 0.50, CHCl3); 1H NMR (CDCl3,
600 MHz):
d
7.29e8.09 (m, 25H, PheH), 5.91 (dd, J ¼ 10.1, 3.2 Hz,
1H, H-300), 5.83 (dd, J ¼ 5.0, 1.8 Hz, 1H, H-200), 5.76 (br s, 1H, H-100),
5.69 (t, J ¼ 10.1 Hz, 1H, H-400), 5.53 (s, 1H, PhCH(O)), 5.30 (t,
J ¼ 3.6 Hz, 1H, H-12), 5.09 (dd, J ¼ 37.6, 12.4 Hz, 2H, CH2ePh), 4.67
(d, J ¼ 7.3 Hz, 1H, H-10), 4.52e4.57 (m, 1H, H-500), 4.35 (dd, J ¼ 10.5,
5.5 Hz, 1H, H-60-1), 4.07 (m, 1H, H-30), 3.83e3.88 (m, 1H, H-20), 3.78
4.1.10.1. Benzyl
oleanolate
3b-O-[2-O-(2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl)-4,6-di-O-benzylidene-b-D-galactopyranoside]
(52a). Yield: 51%; ½a D20
ꢂ
þ70.8 (c 0.62, CHCl3); 1H NMR (CDCl3,

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
13
(t, J ¼ 10.1 Hz, 1H, H-60-2), 3.56 (t, J ¼ 9.6 Hz, 1H, H-40), 3.45 (dt,
J ¼ 10.1, 5.0 Hz, 1H, H-50), 3.25 (dd, J ¼ 11.9, 4.6 Hz, 1H, H-3), 2.91
(dd, J ¼ 13.1, 3.2 Hz, 1H, H-18), 1.35 (d, J ¼ 6.0 Hz, 3H, H-600), 1.14,
0.94, 0.93, 0.92, 0.91, 0.89, 0.63 (s each, 3H each, CH3 ꢃ7); 13C NMR
was purified by column chromatography on silica gel (3:1, CHCl3e
MeOH) to give the products 14e26.
4.1.11.1. Oleanolate 3b-O-b-D-xylopyranosyl-(1 / 3)-a-L-rhamno-
pyranosyl-(1 / 2)-a-L-arabinopyranoside (14). Yield: 74% for two
(CDCl3, 150 MHz):
d
177.6 (C-28), 165.9, 165.7, 143.8 (C-13), 137.1,
136.5, 133.4, 129.8, 128.4, 122.6 (C-12), 104.7 (C-10), 101.9 (PhC(O)),
97.7 (C-1000), 89.9 (C-3), 80.8, 75.1, 72.5, 70.7, 69.5, 67.2, 66.1, 65.8,
56.0, 47.8, 46.8, 46.0, 41.8, 41.5, 39.4, 38.5, 34.1, 33.2, 30.8, 28.1, 26.0,
25.9, 23.8, 22.9, 21.6, 19.2, 17.6, 16.6, 15.5; HRMALDIMS: calcd for
[M þ Naþ] C77H90O15Na: 1277.6210; found: m/z 1277.6172.
steps; ½a 2D5
ꢂ
ꢁ4.30 (c 0.80, CH3OH); Mp: 219e221 ꢀC; IR (KBr) nmax
3397, 2941, 1688, 1455, 1385, 1046 cmꢁ1
;
1H NMR (C5D5N,
600 MHz):
d
6.30 (br s, 1H, H-100), 5.49 (t, J ¼ 3.6 Hz, 1H, H-12), 5.39
(d, J0¼ 7.4 Hz, 1H, H-1000), 4.95 (br s, 1H, H-200), 4.89 (d, J ¼ 5.9 Hz, 1H,
H-1 ), 4.77 (dd, J ¼ 9.6, 3.2 Hz,1H, H-300), 4.67 (dq, J ¼ 9.6, 5.9 Hz,1H,
H-500), 4.62 (t-like, J ¼ 6.9, 6.4 Hz, 1H, H-20), 4.52 (t-like, J ¼ 9.7,
9.1 Hz, 1H, H-400), 4.32e4.35 (m, 2H, H-40, H-5000-1), 4.26e4.30 (m,
2H, H-30, H-50-1), 4.23 (m, 1H, H-4000), 4.19 (t-like, J ¼ 8.7, 8.3 Hz, 1H,
H-3000), 4.11 (t, J ¼ 8.2 Hz, 1H, H-2000), 3.84 (d, J ¼ 10.9 Hz, 1H, H-50-2),
3.72 (t, J ¼ 11.0 Hz, 1H, H-5000-2), 3.30e3.33 (m, 3H, H-3, H-18), 1.57
(d, J ¼ 6.0 Hz, 3H, H-600), 1.34, 1.33, 1.16, 1.03, 1.00, 0.98, 0.85 (s each,
4.1.10.5. Benzyl
oleanolate
3b-O-[3-O-(2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl)-4,6-di-O-benzylidene-b-D-glucopyranoside]
(54b). Yield: 35%; ½a D20
ꢂ
þ35.9 (c 1.04, CHCl3); 1H NMR (CDCl3,
600 MHz):
d
7.20e8.08 (m, 25H, PheH), 5.83 (dd, J ¼ 10.1, 3.7 Hz,
1H, H-300), 5.72 (dd, J ¼ 3.7, 1.9 Hz, 1H, H-200), 5.63 (s, 1H, PhCH(O)),
5.57 (t-like, J ¼ 10.1, 9.6 Hz 1H, H-400), 5.53 (d, J ¼ 1.4 Hz, 1H, H-100),
5.29 (t, J ¼ 3.7 Hz, 1H, H-12), 5.08 (dd, J ¼ 31.1, 12.8 Hz, 2H, CH2ePh),
4.53 (dt, J ¼ 12.4, 6.4 Hz, 1H, H-500), 4.47 (d, J ¼ 7.8 Hz,1H, H-10), 4.35
(dd, J ¼ 10.6, 5.0 Hz, 1H, H-60-1), 4.01 (t-like, J ¼ 9.2, 9.1 Hz, 1H,
H-30), 3.86 (t, J ¼ 9.6 Hz, 1H, H-60-2), 3.74e3.77 (m, 1H, H-20), 3.72
(t-like, J ¼ 9.7, 9.1 Hz, 1H, H-40), 3.49 (dt, J ¼ 9.6, 5.0 Hz, 1H, H-50),
3.20 (dd, J ¼ 11.5, 4.6 Hz,1H, H-3), 2.9 (dd, J ¼ 13.7, 4.1 Hz,1H, H-18),
2.00 (dt, J ¼ 13.7, 4.1 Hz, 1H, H-16), 1.00 (d, J ¼ 6.0 Hz, 3H, H-600), 1.11,
0.99, 0.91, 0.89, 0.89, 0.83, 0.60 (s each, 3H each, CH3 ꢃ7); 13C NMR
3H each, CH3 ꢃ7); 13C NMR (C5D5N, 150 MHz):
d 180.8 (C-28), 145.3
(C-13), 122.5 (C-12), 107.9 (C-1000), 105.8 (C-10), 101.9 (C-100), 89.3
(C-3), 83.4 (C-300), 78.9, 76.1, 75.9, 73.5, 72.5, 71.6, 70.2, 67.9, 66.1,
56.5, 48.6, 47.2, 47.0, 42.7, 42.5, 40.3, 40.1, 39.4, 37.6, 34.8, 33.8, 33.7,
31.5, 30.5, 28.8, 28.7, 27.2, 26.7, 24.3, 18.9, 17.9, 17.7, 16.1; ESI-HRMS:
m/z calcd for C46H73O15 [M ꢁ Hþ]: 865.4949; found: 865.4961.
4.1.11.2. Oleanolate 3
b
-O-
b
-
D
-glucopyranosyl-(1 / 4)-
-arabinopyrano-
ꢁ17.9 (c 0.80, CH3OH); IR
b-D-xylopyr-
anosyl-(1 / 3)-a-L-rhamnopyranosyl-(1 / 2)-a-L
(CDCl3, 150 MHz):
d
177.5 (C-28), 165.7, 143.8 (C-13), 137.3, 136.5,
side (15). Yield: 75% for two steps; ½a D25
ꢂ
133.5, 128.5, 128.2, 122.6 (C-12), 105.7 (C-10), 101.8 (PhC(O)), 97.7
(C-1000), 90.3 (C-3), 78.2, 71.5, 70.9, 70.2, 68.5, 66.9, 66.0, 55.6, 47.7,
46.8, 46.3, 41.8, 39.4, 36.8, 33.2, 30.8, 28.4, 27.6, 25.9, 23.8, 23.7,
23.5, 17.7, 17.1, 16.8, 15.4; HRMALDIMS: calcd for [M þ Naþ]
(KBr) nmax 3412, 2945, 1693, 1499, 1071 cmꢁ1 1H NMR (C5D5N,
;
500 MHz):
d
6.19 (s, 1H, H-100), 5.49 (t, J ¼ 3.6 Hz, 1H, H-12), 5.23
(d, J0¼0 7.0 Hz,1H, H-1000), 5.01 (d, J ¼ 7.9 Hz,1H, H-10000), 4.87 (br s,1H,
H-2 ), 4.85 (d, J ¼ 5.0 Hz, 1H, H-10), 4.67 (dd, J ¼ 9.5, 3.0 Hz, 1H,
H-300), 4.60 (dq, J ¼ 9.3, 5.5 Hz, 1H, H-500), 4.53e4.56 (m, 2H, H-400,
H-60000-1), 4.47 (t, J ¼ 9.5 Hz, 1H, H-3000), 4.37 (dd, J ¼ 12.5, 2.7 Hz, 1H,
H-60000-2), 4.24e4.33 (m, 5H, H-30, H-40, H-50-1, H-4000, H-30000), 4.18
(t, J ¼ 9.6 Hz, 1H, H-40000), 3.97e4.06 (m, 4H, H-2000, H-20000, H-5000-1,
H-50000), 3.82 (dd, J ¼ 12.1, 2.3 Hz, 1H, H-5000-2), 3.63 (m, 1H, H-50-2),
3.28e3.31 (m, 2H, H-3, H-18), 1.55 (d, J ¼ 6.0 Hz, 3H, H-600), 1.29,
1.26, 1.10, 0.99, 0.96, 0.94, 0.82 (s each, 3H each, CH3 ꢃ7); 13C NMR
C
77H90O15Na: 1277.6201; found: m/z 1277.6172.
4.1.10.6. Benzyl oleanolate 3
b
-O-[2,3-di-O-(2,3,4-tri-O-benzoyl-a-L-
rhamnopyranosyl)-4,6-di-O-benzylid-ene-b-D-glucopyranoside]
(54c). Yield: 20%; ½a D20
ꢂ
þ37.9 (c 0.64, CHCl3); 1H NMR (CDCl3,
600 MHz):
d
7.16e7.97 (m, 40H, PheH), 5.86 (dd, J ¼ 10.5, 3.7 Hz,
1H, H-300), 5.79 (dd, J ¼ 10.1, 3.2 Hz, 1H, H-3000), 5.75 (dd, J ¼ 3.2,
1.3 Hz, 1H, H-200), 5.68 (t, J ¼ 10.1 Hz, 1H, H-400), 5.63 (dd, J ¼ 3.2,
1.4 Hz, 1H, H-2000), 5.62 (s, 1H, H-1000), 5.58 (s, 1H, H-100), 5.50 (t,
J ¼ 10.1 Hz, 1H, H-4000), 5.37 (s, 1H, PhCH(O)), 5.30 (t, J ¼ 3.2 Hz, 1H,
H-12), 5.09 (dd, J ¼ 34.3, 12.4 Hz, 2H, CH2ePh), 4.82 (d, J ¼ 6.4 Hz,
1H, H-10), 4.64e4.69 (dt, J ¼ 12.4, 6.0 Hz, 1H, H-500), 4.49 (dt,
J ¼ 12.4, 5.9 Hz, 1H, H-5000), 4.40 (dd, J ¼ 10.5, 5.0 Hz, 1H, H-600-1),
4.24 (dd, J ¼ 9.2, 7.3 Hz, 1H, H-30), 4.10 (t-like, J ¼ 7.3, 6.9 Hz, 1H,
H-200), 3.96 (t-like, J ¼ 9.5, 9.2 Hz, 1H, H-40), 3.82e3.88 (m, 1H,
H-60-2), 3.64 (td, J ¼ 9.6, 4.6 Hz, 1H, H-50), 3.32 (dd, J ¼ 11.5, 4.1 Hz,
1H, H-3), 2.91 (dd, J ¼ 13.7, 4.6 Hz, 1H, H-18), 1.37 (d, J ¼ 6.0 Hz,
3H, H-600), 0.88 (d, J ¼ 5.9 Hz, 3H, H-6000), 1.18, 1.14, 0.97, 0.92, 0.90,
0.89, 0.63 (s each, 3H each, CH3 ꢃ7); 13C NMR (CDCl3, 150 MHz):
(C5D5N, 125 MHz):
d 180.2 (C-28), 145.0 (C-13), 122.7 (C-12), 106.8
(C-1000), 104.9 (C-10), 103.5 (C-10000), 101.4 (C-100), 88.7 (C-3), 82.9
(C-300), 78.7, 78.1, 77.7, 76.1, 75.5, 75.2, 74.2, 73.9, 72.7, 71.7, 71.6,
69.6, 64.7, 64.3, 62.6, 56.6, 48.0, 46.6, 46.4, 42.1, 41.9, 39.7, 39.5, 36.9,
34.2, 33.2, 33.1, 32.0, 30.9, 30.7, 29.8, 29.7, 29.5, 28.2, 28.1, 26.5, 26.1,
23.7, 23.6, 22.8, 18.4, 18.3, 17.3, 17.0, 15.4, 14.1; ESI-HRMS: m/z calcd
for C52H83O20 [M ꢁ Hþ]: 1027.5478; found: 1027.5460.
4.1.11.3. Oleanolic acid 3
nopyranosyl-(1 / 2)- -xylopyranoside (16). Yield: 78% for2 steps;
a 2D5
ꢁ3.82(c0.50, CH3OH);IR (KBr)nmax 3412,2938,1696,1536,1455,
1385, 1046 cmꢁ1; 1H NMR (C5D5N, 600 MHz): 6.62 (br s, 1H, H-100),
b-O-b-D-xylopyranosyl-(1 / 3)-a-L-rham-
b-D
½
ꢂ
d
d
177.6 (C-28), 165.4, 165.3, 163.6, 143.8 (C-13), 137.2, 136.5, 132.9,
5.50(br s,1H, H-12), 5.44 (d, J ¼ 7.8 Hz,1H, H-1000), 5.06 (br s,1H, H-200),
4.88 (d, J ¼ 7.8 Hz, 1H, H-10), 4.83e4.87 (m, 2H, H-20, H-500), 4.58 (t,
J ¼ 9.1 Hz,1H, H-30), 4.36e4.39 (m, 2H, H-40, H-4000), 4.30 (t, J ¼ 8.8 Hz,
1H, H-400), 4.21e4.27 (m, 5H, H-3000, H-400, H-50-1, H-5000-1, H-5000-2),
4.14 (dd, J ¼ 8.7, 7.3 Hz,1H, H-2000), 3.74e3.79(m, 2H, H-3, H-50-2), 3.39
(dd, J ¼ 11.5, 4.1 Hz, 1H, H-18), 1.69 (d, J ¼ 5.9 Hz, 3H, H-600),1.43,1.35,
1.26,1.05, 0.99, 0.99, 0.88 (s each, 3H each, CH3 ꢃ7); 13C NMR (C5D5N,
129.7, 128.4, 122.6 (C-12), 103.5 (C-10), 102.2 (C-100), 97.4 (PhC(O)),
97.1 (C-1000), 91.9 (C-3), 89.1, 78.7, 77.5, 71.8, 70.0, 68.9, 67.1, 66.0,
55.9, 47.8, 46.9, 41.8, 39.2, 36.9, 33.2, 30.8, 28.3, 26.1, 25.8, 23.7,
17.5, 17.1, 15.5; HRMALDIMS: calcd for [M þ Naþ] C104H112O22Na:
1735.7564; found: m/z 1735.7538.
4.1.11. General procedure for synthesizing compounds 14e26
150 MHz):
d
¼ 176.9 (C-28), 145.8 (C-13), 123.8 (C-12), 108.3 (C-1000),
To a solution of 37, 38, 42e45, 50, 52aec, or 54aec (50 mg) in
CH2Cl2eMeOH (V:V/1:1, 8 mL) was added 10% PdeC (30 mg) and
AcOH (2 drops) under 1 atm of H2 for 4 h. The reaction mixture was
then filtered and the filtrate was concentrated to dryness to give a
white solid. The solid was dissolved in MeOHeCH2Cl2 (V:V/2:1,
8 mL), and then NaOMe (40 mg) was added. After stirring at room
temperature for 8 h, the solution was neutralized with ion-
exchange resin (Hþ), then filtered and concentrated. The residue
106.9 (C-10), 102.4 (C-100), 89.4 (C-3), 83.8 (C-300), 80.6, 79.4, 78.1, 76.5,
73.7, 72.7, 72.4, 72.0, 70.5, 68.3, 67.9, 57.0, 48.9, 43.0, 40.5, 37.9, 35.1,
34.2, 31.8, 29.2, 29.0, 27.7, 27.0, 24.6, 19.5, 18.1, 16.4; ESI-HRMS: m/z
calcd for C46H73O15 [M ꢁ Hþ]: 865.4949; found: 865.4980.
4.1.11.4. Oleanolic acid 3
b
-O-
b
-
D
-glucopyranosyl-(1 / 4)-
-xylopyr-
ꢁ5.86 (c 0.50, CH3OH); IR
b-D-glu-
copyranosyl (1 / 3)-a-L-rhamnopyranosyl-(1 / 2)-b-D
anoside (17). Yield: 78% for 2 steps; ½a D25
ꢂ

14
Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
(KBr) nmax 3397, 2945, 1692, 1459, 1389, 1042 cmꢁ1
;
1H NMR
3), 77.3, 73.9, 72.5, 72.2, 71.4, 70.3, 64.8, 64.4, 57.2, 47.8, 47.4, 43.0,
42.9, 40.7, 39.8, 39.5, 38.3, 35.1, 34.2, 34.0, 33.7, 31.8, 29.4, 29.0, 26.6,
24.7, 24.2, 24.1, 22.8, 19.6, 18.2, 17.9, 17.4, 16.1; ESI-HRMS: m/z calcd
for [M ꢁ Hþ] C41H65O11: 733.4527; found: 733.4543.
(C5D5N, 600 MHz):
d
6.51 (br s, 1H, H-100), 5.49 (d, J ¼ 7.3 Hz, 1H, H-
1000), 5.47 (br s, 1H, H-12), 5.05 (br s, 1H, H-200), 4.85 (dd, J ¼ 9.6,
3.5 Hz, 1H, H-300), 4.82 (d, J ¼ 7.3 Hz, 1H, H-10), 4.79 (dq, J ¼ 9.3,
5.5 Hz, 1H, H-500), 4.54e4.58 (m, 3H, H-20, H-400, H-6000-1), 4.42 (dd,
J ¼ 12.1, 5.2 Hz, 1H, H-60000-1), 4.33e4.37 (m, 2H, H-4000, H-6000-2),
4.1.11.8. Oleanolic acid 3-O-
a
-
L
-rhamnopyranosyl-(1 / 2)-
b
-
D
-gal-
4.26e4.30 (m, 2H, H-30, H-40), 4.13e4.22 (m, 5H, H-3000, H-30000
,
actopyranoside (21). Yield: 56% for two steps; ½a D20
ꢂ
ꢁ11.6 (c 0.64,
H-40000, H-50-1, H-60000-2), 4.10 (t, J ¼ 8.3 Hz, 1H, H-2000), 4.07 (t,
J ¼ 8.3 Hz, 1H, H-20000), 4.10 (m, 1H, H-50000), 3.95 (m, 1H, H-5000), 3.72
(m, 1H, H-50-2), 3.32 (m, 2H, H-3, H-18), 1.67 (d, J ¼ 5.9 Hz, 3H,
H-600), 1.39, 1.32, 1.22, 1.03, 0.98, 0.98, 0.86 (s each, 3H each,
CH3OH); 1H NMR (CD3OD, 600 MHz):
d
5.24 (t, J ¼ 3.7 Hz, 1H, H-12),
5.05 (d, J ¼ 1.9 Hz, 1H, H-100), 4.31 (d, J ¼ 7.8 Hz, 1H, H-10), 3.96 (dd,
J ¼ 3.8, 1.4 Hz, 1H, H-200), 3.92 (d, J ¼ 3.2 Hz, 1H, H-40), 3.75e3.79 (m,
2H, H-300, H-500), 3.69e3.73 (m, 2H, H-60-1, H-60-2), 3.65 (dd, J ¼ 9.7,
7.8 Hz, 1H, H-20), 3.51 (dd, J ¼ 9.6, 3.2 Hz, 1H, H-30), 3.49 (t-like,
J ¼ 6.4, 5.9 Hz, 1H, H-50), 3.39 (t-like, J ¼ 9.7, 9.1 Hz, 1H, H-400), 3.18
(dd, J ¼ 11.5, 4.1 Hz, 1H, H-3), 2.86 (dd, J ¼ 13.3, 3.2 Hz, 1H, H-18),
2.01 (dt, J ¼ 14.2, 3.7 Hz,1H, H-16),1.24 (d, J ¼ 6.4 Hz, 3H, H-600),1.16,
1.06, 0.95, 0.94, 0.91, 0.84, 0.81 (s each, 3H each, CH3 ꢃ 7); 13C NMR
CH3 ꢃ7); 13C NMR (C5D5N, 150 MHz):
d 178.9 (C-28), 145.5 (C-13),
122.9 (C-12), 107.0 (C-1000), 106.7 (C-10), 105.5 (C-10000), 102.1 (C-100),
89.0 (C-3), 83.9 (C-300), 81.6, 79.9, 79.0, 78.8, 77.3, 75.9, 75.3, 73.5,
72.0, 67.5, 62.9, 62.3, 56.6, 48.6, 47.2, 47.0, 42.7, 42.5, 40.3, 39.4, 37.6,
33.8, 31.5, 28.9, 28.7, 27.2, 26.7, 24.3, 19.2, 19.0, 17.9, 17.8, 16.1; ESI-
HRMS: m/z calcd for C53H85O21 [M ꢁ Hþ]: 1057.5583; found:
1057.5569.
(CD3OD, 150 MHz):
d
144.0 (C-13), 122.3 (C-12), 104.8 (C-10), 100.7
(C-100), 88.9 (C-3), 75.0, 74.9, 72.7, 70.7, 69.7, 61.1, 60.9, 56.1, 41.6,
39.3, 36.6, 32.7, 32.3, 30.3, 27.2, 25.1, 23.2, 22.7, 18.0, 16.7, 15.9, 14.7;
ESI-HRMS: m/z calcd for [M þ H]þ C42H69O12: 765.4789; found:
765.4783.
4.1.11.5. Oleanolic acid
3
b-O-
a-L-rhamnopyranosyl-(1 / 3)-a-L-
rhamnopyranosyl-(1 / 2)-b-D-xylopyranoside (18). Yield: 73% for 2
a 25
steps; ½ ꢂD ꢁ11.1 (c 0.65, CH3OH); IR (KBr) nmax 3350, 2922, 1595,
1544,1424,1042, 704 cmꢁ1; 1H NMR (C5D5N, 600 MHz):
d
6.60 (br s,
4.1.11.9. Oleanolic acid 3-O-
a
-
L
-rhamnopyranosyl-(1 / 3)-
b
-
D
-gal-
1H, H-100), 6.11 (br s, 1H, H-1000), 5.47 (br s, 1H, H-12), 4.99 (br s, 1H,
H-200), 4.86 (d, J ¼ 6.8 Hz, 1H, H-10), 4.78e4.84 (m, 3H, H-2000, H-300,
H-5000), 4.74 (dq, J ¼ 9.1, 6.4 Hz,1H, H-500), 4.67 (dd, J ¼ 9.1, 3.2 Hz,1H,
actopyranoside (22). Yield: 58% for two steps; ½a D20
ꢂ
þ5.43 (c 0.56,
CH3OH); 1H NMR (CD3OD, 600 MHz):
d
5.37 (s, 1H, H-100), 5.27 (t,
J ¼ 3.6 Hz, 1H, H-12), 4.40 (d, J ¼ 7.7 Hz, 1H, H-10), 3.98e4.03 (m, 2H,
H-200, H-500), 3.80 (d, J ¼ 3.3 Hz, 1H, H-40), 3.78 (dd, J ¼ 9.9, 3.3 Hz,
1H, H-30), 3.74 (dd, J ¼ 5.5, 2.8 Hz, 1H, H-300), 3.70 (t-like, J ¼ 8.8,
7.7 Hz, 1H, H-20), 3.60e3.63 (m, 2H, H-60-1, H-60-2), 3.50 (t-like,
J ¼ 6.6, 6.1 Hz, 1H, H-50), 3.41 (t-like, J ¼ 9.9, 9.3 Hz, 1H, H-400), 3.20
(dd, J ¼ 12.1, 4.4 Hz, 1H, H-3), 2.87 (dd, J ¼ 13.5, 3.2 Hz, 1H, H-18),
1.24 (d, J ¼ 6.6 Hz, 3H, H-600), 1.19, 1.08, 0.97, 0.97, 0.94, 0.89, 0.84 (s
H-50-1), 4.49 (t, J ¼ 9.6 Hz, 1H, H-40), 4.30e4.36 (m, 3H, H-30, H-3000
,
H-400), 4.27 (dd, J ¼ 8.7, 7.2 Hz, 1H, H-20), 4.19 (m, 1H, H-40), 3.74 (t,
J ¼ 11.0 Hz, 1H, H-50-2), 3.30e3.36 (m, 2H, H-3, H-18), 1.69 (d,
J ¼ 6.4 Hz, 3H, H-6000), 1.62 (d, J ¼ 6.4 Hz, 3H, H-600), 1.38, 1.32, 1.23,
1.02, 0.97, 0.97, 0.85 (s each, 3H each, CH3 ꢃ7); 13C NMR (C5D5N,
150 MHz):
d
176.4 (C-28), 143.5 (C-13), 123.7 (C-12), 106.5 (C-10),
104.7 (C-1000), 101.9 (C-100), 89.0 (C-3), 80.1 (C-300), 80.0 (C-3000), 78.7,
74.7, 73.1, 72.8, 72.6, 72.4, 70.0, 68.5, 56.6, 49.3, 42.5, 40.1, 39.9, 37.4,
33.7, 31.3, 30.3, 28.7, 26.5, 24.2, 19.0, 17.6, 15.9; ESI-HRMS: m/z calcd
for C47H75O15 [M ꢁ Hþ]: 879.5106; found: 879.5103.
each, 3H each, CH3 ꢃ7); 13C NMR (CD3OD, 150 MHz):
d 143.9 (C-13),
122.3 (C-12), 105.9 (C-10), 102.6 (C-100), 89.5 (C-3), 80.5, 74.8, 72.8,
71.4, 70.8, 68.8, 60.8, 55.8, 41.4, 39.3, 38.3, 36.6, 32.3, 27.3, 25.7, 25.1,
23.2, 22.7, 16.7, 16.4, 15.7, 14.6; ESI-HRMS: m/z calcd for [M þ Na]þ
C42H68O12Na: 787.4608; found: 787.4595.
4.1.11.6. Oleanolic acid 3-O-
a
-
L
-rhamnopyranosyl-(1
/
2)-
b-D-
xylopyranoside (19). Yield: 65% for 2 steps; ½a D20
ꢂ
þ5.96 (c 0.62,
4.1.11.10. Oleanolic acid 3-O-2,3-di-O-(a-L-rhamnopyranosyl)-4,6-di-
CH3OH); 1H NMR (CD3OD, 600 MHz):
d
5.35 (s, 1H, H-100), 5.27 (br s,
O-benzylidene-b-D-galactopyranoside (23). Yield: 51% for two steps;
1H, H-12), 4.41 (d, J ¼ 6.1 Hz, 1H, H-10), 3.95e3.98 (m, 2H, H-200, H-
5000), 3.87 (dd, J ¼ 11.6, 5.5 Hz, 1H, H-50-1), 3.25 (dd, J ¼ 9.9, 3.3 Hz,
1H, H-300), 3.48e3.51 (m, 1H, H-40), 3.39e3.46 (m, 3H, H-20, H-30, H-
50-2), 3.20 (t, J ¼ 9.9 Hz, 1H, H-400), 3.14 (dd, J ¼ 12.1, 4.4 Hz,1H, H-3),
2.88 (dd, J ¼ 13.7, 4.4 Hz, 1H, H-18), 1.25 (d, J ¼ 6.0 Hz, 3H, H-600),
1.19, 1.07, 0.97, 0.97, 0.94, 0.89, 0.85 (s each, 3H each, CH3 ꢃ7); 13C
½
a 2D0
ꢂ
ꢁ2.72 (c 0.75, CH3OH); 1H NMR (CD3OD, 600 MHz):
d 5.56 (br
s, 1H, PhCHO), 5.24 (t, J ¼ 3.8 Hz, 1H, H-12), 5.13 (d, J ¼ 1.1 Hz, 1H, H-
100), 4.90 (s, 1H, H-1000), 4.53 (d, J ¼ 7.7 Hz, 1H, H-10), 4.38 (d,
J ¼ 3.3 Hz, 1H, H-40), 3.97 (dq, J ¼ 9.4, 6.1 Hz, 1H, H-500), 3.91 (dd,
J ¼ 3.3, 1.2 Hz, 1H, H-2000), 3.88 (br s, 1H, H-200), 3.83e3.86 (m, 2H, H-
20, H-5000), 3.77 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-30), 3.68 (dd, J ¼ 9.4, 3.3 Hz,
1H, H-300), 3.66 (dd, J ¼ 9.4, 3.3 Hz, 1H, H-3000), 3.54 (br s, 1H, H-60-2),
3.39 (t, J ¼ 9.9 Hz, 1H, H-4000), 3.36 (t, J ¼ 9.3 Hz, 1H, H-400), 3.20 (dd,
J ¼ 12.1, 4.4 Hz, 1H, H-3), 2.86 (dd, J ¼ 13.7, 3.8 Hz, 1H, H-18), 1.21 (d,
J ¼ 6.0 Hz, 3H, H-600), 1.19 (d, J ¼ 6.1 Hz, 3H, H-6000), 1.17, 1.08, 0.96,
0.94, 0.91, 0.89, 0.82 (s each, 3H each, CH3 ꢃ7); 13C NMR (CD3OD,
NMR (CD3OD, 150 MHz):
d
143.9 (C-13), 122.3 (C-12), 104.9 (C-10),
100.6 (C-100), 88.8 (C-3), 77.5, 77.3, 72.7, 70.8, 70.3, 68.7, 55.9, 41.6,
40.9, 39.3, 38.9, 36.6, 32.7, 32.3, 30.3, 27.2, 25.1, 22.7, 18.0, 16.7, 15.8,
14.7; ESI-HRMS: m/z calcd for [M þ Na]þ C41H66O11Na: 757.4503;
found: 757.4509.
150 MHz):
d 182.6 (C-28), 162.7, 145.4 (C-13), 139.7, 130.0, 129.2,
4.1.11.7. Oleanolic acid
3
b
-O-
a
-
L
-rhamnopyranosyl-(1 / 2)-
b
-
L
-
127.6, 123.7 (C-12), 106.0 (C-10), 104.9 (C-1000), 102.7 (C-100), 102.4
(PhCHO), 90.8 (C-3), 84.4 (C-300), 77.6 (C-3000), 75.6, 74.0, 73.7, 72.5,
72.4, 71.9, 70.7, 70.5, 67.7, 57.6, 43.1, 42.9, 40.7, 40.5, 40.3, 38.1, 35.1,
34.2, 33.8, 31.8, 29.0, 28.6, 27.4, 26.6, 24.7, 24.2, 19.5, 18.3, 18.2, 18.0,
arabinopyranoside (20). Yield: 74% for 2 steps; ½a D20
þ78.3 (c 0.84,
ꢂ
CH3OH); IR (KBr) vmax 3420, 2941, 1688, 1455, 1139, 1066 cmꢁ1; 1H
NMR (CD3OD, 600 MHz):
d
5.24 (t, J ¼ 3.3 Hz, 1H, H-12), 5.05 (d,
J ¼ 3.3 Hz, 1H, H-10), 4.95 (d, J ¼ 1.1 Hz, 1H, H-100), 3.95 (dd, J ¼ 3.3,
1.6 Hz, 1H, H-200), 3.91 (dd, J ¼ 9.9, 3.3 Hz, 1H, H-30), 3.87 (m, 2H, H-
20, H-40), 3.85 (dd, J ¼ 9.8, 3.3 Hz, 1H, H-50-1), 3.75 (qd, J ¼ 9.9,
6.0 Hz, 1H, H-500), 3.66 (dd, J ¼ 9.4, 3.3 Hz, 1H, H-300), 3.57 (dd,
J ¼ 12.1, 1.6 Hz, 1H, H-50-2), 3.40 (t-like, J ¼ 9.9, 9.3 Hz, 1H, H-400),
3.20 (dd, J ¼ 12.1, 4.4 Hz, 1H, H-3), 2.84 (dd, J ¼ 12.1, 3.8 Hz, 1H, H-
18), 1.26 (d, J ¼ 6.0 Hz, 3H, H-600), 1.16, 1.03, 0.96, 0.94, 0.91, 0.85,
17.2, 16.2; HRMALDIMS: calcd for [M
1021.5507; found: m/z 1021.5495.
þ
Naþ] C55H82O16Na:
4.1.11.11. Oleanolic acid 3-O-
a
-
L
-rhamnopyranosyl-(1 / 2)-
b
-
D
-glu-
copyranoside (24). Yield: 55% for two steps; ½a D20
ꢂ
þ5.95 (c 1.00,
CH3OH); 1H NMR (CD3OD, 600 MHz):
d
5.37 (s, 1H, H-100), 5.24 (t,
J ¼ 3.6 Hz, 1H, H-12), 4.41 (d, J ¼ 7.7 Hz, 1H, H-10), 3.98 (td, J ¼ 9.4,
6.1 Hz, 1H, H-500), 3.95 (dd, J ¼ 3.4, 1.6 Hz, 1H, H-200), 3.83 (dd,
J ¼ 11.5, 1.6 Hz, 1H, H-60-1), 3.74 (dd, J ¼ 9.3, 3.3 Hz, 1H, H-300), 3.66
0.82 (s each, 3H each, CH3 ꢃ7); 13C NMR (CD3OD, 150 MHz):
d 182.1
(C-28), 145.4 (C-13), 123.7 (C-12), 103.8 (C-100), 96.0 (C-10), 83.3 (C-

Q. Liu et al. / European Journal of Medicinal Chemistry 64 (2013) 1e15
15
(dd, J ¼ 12.1, 5.5 Hz, 1H, H-60-2), 3.46 (t, J ¼ 8.8 Hz, 1H, H-30), 3.41 (t-
Acknowledgments
like, J ¼ 8.8, 7.7 Hz, 1H, H-20), 3.38 (t-like, J ¼ 9.8, 9.4 Hz, 1H, H-400),
3.29 (t, J ¼ 8.8 Hz, 1H, H-1-40), 3.21e3.24 (m, 1H, H-50), 3.18 (dd,
J ¼ 12.1, 4.4 Hz, 1H, H-3), 2.86 (dd, J ¼ 14.3, 3.8 Hz, 1H, H-18), 1.21 (d,
J ¼ 6.6 Hz, 3H, H-600), 1.16, 1.05, 0.95, 0.94, 0.91, 0.86, 0.82 (s each, 3H
This project was financially supported by the National Natural
Science Foundation of China (No. 30701046) and China Post-
doctoral Science Foundation funded project (No. 2012M512023).
each, CH3 ꢃ7); 13C NMR (CD3OD, 150 MHz):
d 143.9 (C-13), 122.3 (C-
12), 104.3 (C-10), 100.5 (C-100), 88.9 (C-3), 78.2, 77.6, 76.3, 70.8, 70.7,
68.7, 61.5, 56.0, 41.6, 39.3, 38.9, 36.6, 32.3, 30.3, 27.5, 25.9, 25.1, 22.7,
Appendix A. Supplementary data
16.7, 16.4, 15.9, 14.7; ESI-HRMS: m/z calcd for [M
þ
Na]þ
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.016.
C42H68O12Na: 787.4608; found: 787.4584.
4.1.11.12. Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 / 3)-b-D-
glucopyranoside (25). Yield: 54% for two steps; ½a D20
þ2.90 (c 0.65,
ꢂ
References
CH3OH); 1H NMR (CD3OD, 600 MHz):
d 5.27 (br s, 1H, H-12), 5.17 (s,
1H, H-100), 4.35 (d, J ¼ 8.2 Hz, 1H, H-10), 4.01 (dq, J ¼ 9.4, 6.6 Hz, 1H,
H-500), 3.97 (br s, 1H, H-200), 3.86 (dd, J ¼ 10.9, 3.5 Hz, 1H, H-60-1),
3.71 (ddd, J ¼ 9.4, 5.0, 3.3 Hz, 1H, H-50), 3.49 (t, J ¼ 8.8 Hz, 1H, H-30),
3.42 (t-like, J ¼ 9.9, 9.3 Hz, 1H, H-400), 3.36 (t-like, J ¼ 9.4, 9.3 Hz, 1H,
H-20), 3.28e3.32 (m, 2H, H-40, H-60-2), 3.21 (dd, J ¼ 11.6, 4.4 Hz, 1H,
H-3), 2.89 (dd, J ¼ 12.1, 3.3 Hz, 1H, H-18), 1.27 (d, J ¼ 6.1 Hz, 3H, H-
600), 1.19, 1.08, 0.98, 0.97, 0.93, 0.87, 0.84 (s each, 3H each, CH3 ꢃ7);
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13C NMR (CD3OD, 150 MHz):
d 143.9 (C-13), 122.3 (C-12), 105.2 (C-
10), 101.6 (C-100), 89.6 (C-3), 83.7, 74.8, 72.9, 71.1, 70.9, 69.0, 68.8,
61.4, 55.7, 41.6, 39.3, 38.8, 38.5, 36.6, 32.3, 30.3, 27.3, 25.1, 23.2, 22.7,
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C42H69O12Na: 765.4789; found: 765.4806.
þ
H]þ
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glucopyranoside (26). Yield: 57% for two steps; ½a D20
ꢁ1.49 (c 0.75,
ꢂ
CH3OH); 1H NMR (CD3OD, 600 MHz):
d 5.24 (br s, 1H, H-12), 5.13 (s,
1H, H-100), 4.92 (s, 1H, H-1000), 4.48 (d, J ¼ 7.1 Hz, 1H, H-10), 3.99 (br s,
1H, H-200), 3.90e3.96 (m, 3H, H-2000, H-50, H-5000), 3.84e3.86 (m, 2H,
H-3000, H-40), 3.64e3.71 (m, 3H, H-400, H-50, H-60-1), 3.58 (t-like,
J ¼ 8.8, 8.3 Hz, 1H, H-300), 3.42e3.49 (m, 3H, H-20, H-4000, H-60-2),
3.39 (t-like, J ¼ 9.9, 9.4 Hz,1H, H-30), 3.22 (dd, J ¼ 12.1, 4.4 Hz,1H, H-
3), 2.87 (dd, J ¼ 13.2, 3.3 Hz, 1H, H-18), 1.27 (d, J ¼ 6.6 Hz, 3H, H-600),
1.22 (d, J ¼ 6.1 Hz, 3H, H-6000), 1.16, 1.05, 0.95, 0.94, 0.91, 0.87, 0.82 (s
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d 144.2 (C-13),
122.2 (C-12), 104.0 (C-10), 102.2 (C-1000), 100.7 (C-100), 88.8 (C-3),
86.9, 77.4,76.1, 72.2, 71.9, 70.8, 70.5, 69.8, 68.9, 63.0, 61.3, 56.0, 41.6,
39.3, 39.0, 36.6, 32.5, 32.3, 30.3, 27.2, 25.1, 23.2, 22.7, 16.7, 16.5, 15.8,
14.7; ESI-HRMS: m/z calcd for [M þ Na]þ C48H78O16Na: 933.5188;
found: 933.5175.
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4.2. Cytotoxic assay
The cytotoxicity of all synthesized oleanolic acid saponins was
examined using a panel of human tumor cell lines, including one
human promyelotic leukemia cell line (HL-60), one human non-
small-cell lung cancer cell line (A549) and one human melanoma
cancer cell lines (A375). Cells were seeded into 96-well plates and
treated in triplicate with gradient concentrations of tested com-
pounds at 37 ꢀC for 72 h. Cytotoxicity to HL-60 cells was assessed by
MTT assay, and cytotoxicity to A549 and A375 was assessed by SRB
assay as previous described [34,35]. The cytotoxicity of tested
compounds was expressed as an IC50, determined by the Logit
method from at least three independent experiments.
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