Structure-activity relationships of 3-O-β-chacotriosyl ursolic acid derivatives as novel H5N1 entry inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.029

A series of methyl ursolate 3-O-β-chacotrioside analogs have been designed, synthesized and evaluated as H5N1 entry inhibitors based on a small molecule inhibitor saponin 3 previously discovered by us. Detailed structureeactivity relationships (SARs) studies on the aglycone of compound 3 indicated that both the type of pentacyclic triterpene and the subtle modification of ursolic acid as an aglycon had key influences on the antiviral activity. These results suggested that either the introduction of a disubstituted amide structure at the 17-COOH of ursolic acid or alteration of the C-3 configuration of ursolic acid from 3β-to 3α-forms was helpful to significantly improve the selective index while keeping their antiviral activities.

Full text of DOI:10.1016/j.ejmech.2015.02.029

European Journal of Medicinal Chemistry 93 (2015) 431e442
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships of 3-O-
b-chacotriosyl ursolic acid
derivatives as novel H5N1 entry inhibitors
,
*
,
,
Gaopeng Song a 1, Xintian Shen b 1, Sumei Li c, Yibin Li a, Yunpeng Liu d, Yushan Zheng a,
Ruheng Lin a, Jihong Fan a, Hanming Ye a, Shuwen Liu b
,
*
a College of Resources and Environment, South China Agricultural University, Guangzhou, 510642, China
b School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China
c Department of Human Anatomy, School of Medicine, Jinan University, Guangzhou, 510632, China
d Center for Diagnostics and Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA, 30303, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of methyl ursolate 3-O-b-chacotrioside analogs have been designed, synthesized and evaluated
Received 5 October 2014
Received in revised form
22 January 2015
Accepted 7 February 2015
Available online 20 February 2015
as H5N1 entry inhibitors based on a small molecule inhibitor saponin 3 previously discovered by us.
Detailed structureeactivity relationships (SARs) studies on the aglycone of compound 3 indicated that
both the type of pentacyclic triterpene and the subtle modification of ursolic acid as an aglycon had key
influences on the antiviral activity. These results suggested that either the introduction of a disubstituted
amide structure at the 17-COOH of ursolic acid or alteration of the C-3 configuration of ursolic acid from
3
b
-to 3
a-forms was helpful to significantly improve the selective index while keeping their antiviral
Keywords:
3-O-b-chacotriosyl saponins
H5N1 entry inhibitors
activities.
© 2015 Elsevier Masson SAS. All rights reserved.
Structureeactivity relationships
1. Introduction
can prevent H5N1 virus from binding to and entering the host cell,
which may achieve the inhibition of H5N1 viral infection [6].
In our previous study [7], we have discovered three small
molecule H5N1 viral entry inhibitors 1e3 (Fig.1A) using an efficient
H5N1 avian influenza A virus is the cause of significant
morbidity among humans, posing a serious threat to public health
due to its transmission from animals to human being [1,2]. The two
common classes of antiviral drugs used to treat influenza are
neuraminidase inhibitors, like zanamivir and oseltamivir, and in-
hibitors of the viral M2 protein, such as amantadine and rimanta-
dine [3]. It is known that ion channel inhibitors have given rise to
the rapid emergence of drug-resistant viral strains which cause CNS
side effects [4], and therefore are not recommended for general use.
However, resistance of H5N1 to an NA inhibitor oseltamivir has also
been recently observed [5]. Therefore, there is an urgent demand
for new classes of agents with new mechanism of action to combat
avian H5N1 variants.
HIV-based pseudotyping system to screen
a saponin library
generated from semisynthesis. These compounds show good
inhibitory activity against H5N1 entry with the IC50 values being
6.00e9.25
chacotriosyl
anosyl-(1 / 4)]-
m
M. Structurally, these compounds bear the same
-rhamnopyranosyl- (1 2)-[ -rhamnopyr-
-glucopyranosyl) residue that is known as
b-
(a
-L
/
a-L
b-D
chacotriose. A preliminary structureeactivity relationship (SAR)
study [7,8] has indicated that both the chacotriosyl residue and the
aglycone moiety of compounds 1e3 are important to the antiviral
activity, though several modifications are tolerated at these posi-
tions. The 3-O-
methylating one or two sugar hydroxyls of the
moiety and altering the -chacotriosyl moiety of compound 1 into
-rhamnopyranosyl-(1 / 4)- -glucopyranosyl moiety or
rhamnopyranosyl-(1 / 2)- -glucopyranosyl moiety resulted in
b-chacotriosyl residue is essential for activity as
The most critical and first step of virual infection influenza is
viral entry that is mediated by the interaction of viral envelope
protein hemagglutinin (HA), the receptor of which is sialic acid
sugars on the host cell surface. Therefore, potent entry inhibitors
b-chacotriosyl
b
a-
L
b-
D
a-L-
b-D
the loss of activity. In addition, replacement of the aglycone moiety
of compound 1 with dihydrochlorogenin, dehydroisoandrosterone
or stigmasterine led to the loss of activity except in the case of
methyl ursolate (i.e., compound 3), suggesting that the subtle
modifications of aglycone may be tolerated without losing antiviral
* Corresponding authors.
E-mail addresses: vinsin1021@126.com (G. Song), liusw@smu.edu.cn (S. Liu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.02.029
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

432
G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
Fig. 1. A. Saponin inhibitors for H5N1 viral entry. B. Saponins designed for SARs studies.
activity. Notably, change of the aglycone moiety of compounds 1
and 2 into pentacyclic triterpene (to afford compound 3) led to
slightly stronger inhibition against HA (QH) [7], suggesting that
replacement of the aglycone moiety of compound 1 with penta-
cyclic triterpene can enhance the activity.
for multiple pharmacological effects, such as anti-inflammatory
[12], antiviral [13,14], and also readily available in large amounts
from the extract of liquorice roots. Thus, methyl oleanolate, methyl
glycyrrhetinate and methyl betulinate were selected as the agly-
cone substitutes and the resulting three saponins 4e6 were derived
(Fig. 1B).
In order to study the influence of esterification of the 17-COOH
group of ursolic acid on the inhibitory activity, compounds 7e11
(Fig. 1B) were synthesized. Compounds 12e14 (Fig. 1B), bioisosteric
surrogates of ursolic acid ester, were synthesized to study the ef-
fects of acylation at the17-COOH position of ursolic acid with alkyl
amines on the activity. To understand the influence of ursane tri-
terpenoids with modified rings A and C on the antiviral activity, we
synthesized compounds 15 and 16 (Fig. 1B). Compound 15 bears an
It has been reported that the defense activities of pentacyclic
triterpenes stem from their ability to prevent various pathogen and
herbivore infections in the host [9]. Certain pentacyclic triterpe-
noids with conserved structural features displayed in vitro anti-
influenza virus activity that is comparable to or higher than that
of oseltamivir [10,11]. Based on the above results, we chose com-
pound 3 as the lead compound and designed analogs 4e16 with the
aim to investigate the SARs of pentacyclic triterpenes with different
aglycones. Pentacyclic triterpenes can be classified into three major
types based on structural features: (a) oleanane type triterpenes,
(b) ursane type triterpenes, and (c) lupane type triterpenes. The
most well-known member of each category is oleanolic acid (OA),
ursolic acid (UA) and betulinic acid (BA), respectively. We first
investigated the effect of different types of pentacyclic triterpene as
the aglycone residue on the inhibitory activity. In addition, glycyr-
rhyzinic acid (GL), a member of oleanane type triterpenes, is known
a-OH at the C-3 position. Thus, comparison with compound 3 with
a
b
-OH at the same position provides a means to judge the effects of
the C-3 configuration on the activity. Compound 16 was derived
from the incorporation of a carbonyl group at the C-11 position of
compound 3 to determine whether the introduction of an addi-
tional hydrogen bond acceptor was beneficial to the activity.

G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
433
2. Results and discussion
2.2. Inhibition of the infection of H5N1 pseudovirus
Previously, we have found that methyl ursolate 3-O-
2.1. Chemistry
b
-chaco-
trioside (3) could inhibit the highly pathogenic H5N1 avian influ-
enza A virus by blocking viral entry [7]. The inhibitory activity of
compound 3 is specific to hemagglutinin, since it has no inhibitory
activity toward VSV-G pseudovirus [7].
As depicted in Scheme 1, oxidation of methyl ursolate 17 [7] ob-
tained by treating ursolic acid with iodomethane in the presence of
pyridinium chlorochromate (PCC) gave the 3-oxo compound 18.
Meerwein-Ponndorf reduction of compound 18 afforded 3
a
-ursolic
Due to the safety concerns in studying viral H5N1 pathogens,
the single-cycle pseudovirus was used instead of live H5N1 avian
influenza virus to evaluate the inhibitory activities of compounds
4e16 against H5N1 entry. Compounds 3e16 and 41 were evaluated
for the inhibitory activity against the entry of H5N1 influenza virus
based on an efficient HIV-based pseudotyping system with lucif-
erase report element established by us [18]. This pseudovirus was
enveloped with influenza glycoprotein HA and NA, and packed with
HIV-backbone [18].
acid methyl ester 19 as the major product (70%), along with com-
pound 17 (18%) as a minor product. The coupling constant of H-3 in
the 1H NMR of 19 [
d
3.42 (1H, t, J ¼ 1.7 Hz)] compared with that of 17
3.23 (1H, dd, J ¼ 11.4, 4.7 Hz)] indicated that H-3 was orientated
in 19 and orientated in 17, respectively. Treatment of ursolic acid
[d
a
b
with allyl bromide in the presence of K2CO3 in DMF provided allyl
ursolate 20 [15] in good yield. The 3-hydroxy-11-oxo methyl ursolate
derivative 21 [16] was prepared following literature procedures.
Compound 4 was prepared following our previous procedure
[7]. Compounds 5e16 were synthesized via a route similar to that of
compound 3 [7,8]. As shown in Scheme 2, glycosylation of the in-
termediates 19e21, methyl glycyrrhetinate 22 [12] and methyl
The obtained IC50 values were summarized in Table 1 and
indicate that all the compounds showed inhibitory activity against
H5N1 pseudovirus with
a
potency ranging from moderate
M, which is comparable to the
(IC50 > 10 M) to potent (IC50 < 4
m
m
betulinate 23 [17], with 2,3,4,6-tetra-O-benzoyl-
trichloroacetimidate 24 [7], respectively, under the action of
TMSOTf gave the 3-O- -glucopyranosides 25e29, respectively.
Removal of the benzoyl groups with NaOMe in MeOH afforded
30e34 and selective protection of the 3,6-OHs of the -glucopyr-
anosyl residues with 1-(benzoyloxy)-benzotriazole (1-BBTZ) in the
presence of Et3N afforded intermediates 35e39. Subsequent
glycosylation of the 2, 4-OHs in 35e39 with 2,3,4-tri-O-acetyl-L-
rhamnopyranosyl trichloroacetimidate 40 [7] under the “inverse
addition conditions” with BF3$Et2O as the promoter, followed by
deprotection of the acyl groups with MeONa, afforded target sa-
ponins 5, 6, 15, 16 and the important intermediate 41, respectively.
The target saponins 7e11 and 12e14 were prepared as depicted
in Scheme 3. Intermediate 41 was subjected to PdCl2 to provide the
target compound 7. Treatment of 7 with different halohydrocarbon
in the presence of K2CO3 in DMF yielded the target compounds
8e11. Protection of all the OHs in 7 by using Ac2O in the presence of
DMAP produced compound 42. The key intermediate 42 was
treated with oxalyl chloride to give 28-acyl chloride, which was
then condensed with appropriate amines and finally deprotected
with MeONa to yield the corresponding target saponins 12e14.
D
-glucopyranosyl
IC50 of a positive compound CL-385319 [18e20]). Notably, the
active compounds in series 8e10 and 13e15 display a comparable
IC50 value with compound 3, whereas lower cytotoxicity against
Madin Darby Canine Kidney (MDCK) cells than compound 3.
Representative compounds 3, 13 and 15 had no effect on VSV-G
enveloped pseudovirus (Fig. 2A), which was similar with both
compound 3 and HA targeted compound CL-385319 [18e20]. These
results demonstrate that these compounds did not inhibit HIV-
backbone and luciferase reporting activity. In addition, com-
b
b
pounds 3, 13 and 15 at 20 mg/mL did not inhibit neuraminidase (NA)
(Fig. 2B), a glycoprotein enveloped in the pseudovirus system.
These results suggested that compounds 4e15 might interfere with
the entry of influenza virus by targeting hemagglutinin, the only
other glycoprotein enveloped in the pseudovirus system.
2.3. SAR analysis of compounds 3e16
It can be seen from the IC50 values that compound 3 and its
derivatives 4e16 could inhibit infection by H5N1 influenza A virus
in MDCK cells in varying extents (IC50 values 0.98e22.50
mM).
Among them, compound 13 showed the most potent inhibition
Scheme 1. Reagents and conditions: (a) CH3I, K2CO3, DMF; (b) PCC, CH2Cl2; (c) Al(O-i-Pr)3, i-PrOH, reflux; (d) AllylBr, K2CO3, DMF.

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G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2; (b) MeONa, CH3OH; (c) 1-BBTZ, Et3N, CH2Cl2; (d) (i) BF3$Et2O, CH2Cl2, (ii) MeONa, CH3OH.
Scheme 3. Reagents and conditions: (a) PdCl2, CH3OHeCH2Cl2; (b) ReBr, K2CO3, DMF; (c)Ac2O, DMAP, pyridine; (d) (i) (COCl)2, CH2Cl2 (ii) Et3N, CH2Cl2; (iii) CH3ONa, CH3OH.

G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
435
Table 1
length of carbon side chain from one to five had little effect on the
antiviral activity but introduction of bulky groups or unsaturated
hydrocarbon should be avoided.
ICa values (
m
M) of lead compounds 3e16, 41 and CL-385319 screened in MDCK
50
cells infected with H5N1 pseudovirus and CCb cytotoxicity values (
m
M) of lead
50
compounds 3e17 and 41 against MDCK cells (data derived from the mean of three
The introduction of an amine structure at the 17-COOH position
was done to determine whether a disubstituted amide structure
was beneficial to inhibition. As a result, compounds 13 and 14 had
similar activity as compounds 3 and 8, while compound 12 has
lower activity than compound 13, possibly due to the polar NH
group at the 17-COOH position. This was further supported by the
fact that keeping a polar group at the 17-COOH position was not
essential for inhibition activity, which should be esterified or
amidated. It was interesting to note that there was an excellent
change in the IC50 of cytotoxicity against MDCK cells in vitro be-
tween compound 3 and 14. Compound 14, amidated from com-
pound 3, had an improved selective index (12.8e50), indicating
that amidation at the 17-COOH position could significantly improve
the safety of these compounds while keeping their antiviral
activity.
independent assays).
Compound
ICa
CCb
SIc
50
50
3
4
5
6
7
8
9
10
11
12
13
14
15
16
41
1.02 0.13
3.74 0.14
4.93 0.52
2.08 0.29
10.09 0.23
1.16 0.05
2.30 0.16
3.72 0.68
4.91 0.26
22.50 0.12
0.98 0.07
1.41 0.07
1.33 0.15
5.68 0.11
33.95 0.75
4.45 1.25
13.02 0.88
16.85 0.51
17.12 0.36
33.90 0.80
26.15 0.80
19.05 0.18
19.22 0.18
20.10 0.85
24.25 0.37
>500
48.64 0.35
47.04 0.81
>200
38.37 0.75
21.83 0.68
1480 10
12.8
4.5
3.5
16.3
2.58
16.4
8.4
5.4
4.9
>15
49.6
50.0
>162.6
6.76
0.64
332.3
Of the two 3-hydroxyl group isomers, the 3
a-form 15 had a
CL-385319
similar inhibition as the 3 -form 3, but 3 -form 15 exhibited
b
a
ICa50 values (
mM) of lead compounds 3e16 and 41 screened in MDCK cells infected
significantly decreased cytotoxic activity against MDCK cells in vitro
compared to compound 3. These results confirmed that the
configuration at C-3 is an important factor for the selection index,
rather than for the antiviral activity. As far as selective index is
concerned, compound 15 is a more promising lead than compound
3. Lastly, introduction of an additional oxo group to the 11-position
did not improve the inhibition (16 vs. 3).
with H5N1 pseudovirus (data derived from the mean of three independent assays).
CCb50 cytotoxicity values (
M) of lead compounds 3e16 and 41 against MDCK cells
(data derived from the mean of three independent assays).
The selection index (SIc) was CCb50/ICa
m
.
50
with an IC50 of 0.98 mM and a selection index (SI, CC50/IC50) of 49.6,
respectively. Thus, compound 13 is exploitable as a lead compound.
Pentacyclic triterpene as aglycone residue had significant in-
fluence on the inhibitory activity. The activity in the order of
compounds 3 > 6 > 4 > 5 suggests that ursane type triterpenes are
superior to the other kinds of triterpenes as aglycone residue. It was
found that esterification of the 17-COOH group of ursolic acid with
saturated alkane groups enhanced the inhibitory activity
(8e11 > 7). We supposed that the hydroxyl group of 17-COOH did
not contribute to the interaction of the aglycone residue with the
receptor. When 17-COOH was substituted by a fatty alkyl group, the
length of the carbon chain from one to five led to slightly reduced
activity. However, introduction of the allyl group to the 17-COOH
position (to give compound 41), resulted in a loss in the inhibi-
tory activity. In addition, compound 3 showed slightly higher ac-
tivity than compound 11, suggesting that the introduction of a
bulky group could increase the steric hindrance and decrease
binding. Taken together these results suggested that when the 17-
COOH was esterified, introduction of short straight saturated alkyl
groups was helpful to enhance the inhibitory activity, whereas the
3. Conclusion
Based on our previously discovered small molecule inhibitor 3,
intensive SARs studies on the aglycone of compound 3 were con-
ducted. The results showed that both the type of pentacyclic tri-
terpene and the modification of ursolic acid as aglycon had
influence on the antiviral activity. The above results indicated that
when the 17-COOH of ursolic acid was esterified or amidated,
introduction of short straight saturated alkyl groups or a disubsti-
tuted amide structure was helpful in enhancing inhibitory activity,
but introduction of bulky groups or unsaturated hydrocarbons
should be avoided. The configuration at C-3 was found to be
important for the selection index. Compounds with the 3
kept antiviral activity and greatly decreased cytotoxicity against
MDCK cells compared to compounds with 3 -form. The SARs
a-form
b
described herein may serve as the basis for the rational design of
more potent ursolic acid derivatives as novel H5N1 entry inhibitors.
Fig. 2. A. Representative compounds 3, 13 and 15 did not inhibit VSVG pseudovirus. B. Representative compounds 3, 13 and 15 did not inhibit neuraminidase activity.

436
G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
4. Experimental protocols
51.4, 49.0, 48.1, 47.4, 42.1, 39.7, 38.9, 37.3, 36.7, 33.1, 32.8, 30.7, 28.3,
25.2, 24.2, 23.7, 23.2, 22.3, 21.2, 18.3, 17.0, 16.9, 15.2; HRESIMS calcd
for C31H50O3Na 493.3652; found 493.3659.
4.1. General methods
Solvents were purified in a conventional manner. 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). 1H NMR and 13C
NMR spectra were taken on a JEOL JNM-ECP 600 spectrometer with
tetramethylsilane as an internal standard, and chemical shifts are
recorded in ppm values. Mass spectra were recorded on a Q-TOF
Global mass spectrometer.
4.5. 3-Hydroxy-urs-12-en-28-oic acid allyl ester (20)
To a solution of UA (1.00 g, 2.19 mmol) in dry DMF (20 mL) was
added potassium carbonate (0.60 g, 4.38 mmol). After 4 h of stirring
at room temperature, allyl bromide (0.48 mL, 6.57 mmol) was
added and the mixture was stirred for an additional 8 h. The sol-
vents were evaporated and the crude residue was dissolved in a
mixture of CH2Cl2 (100 mL) and hydrochloric acid (30 mL, 1.0 M).
The aqueous layer was extracted with CH2Cl2 (3 ꢀ 50 mL), the
combined organic layers were washed with brine (100 mL), dried
(Na2SO4), filtered and the solvent was evaporated. Recrystallization
from EtOH provided compound 20 (0.99 g, 91%) as a colorless solid;
4.2. 3- Hydroxy-urs-12-en-28-oic acid methyl ester (17)
To a solution of UA (1.00 g, 2.19 mmol) in dry DMF (20 mL) was
added potassium carbonate (0.60 g, 4.38 mmol). After 20 min of
stirring at room temperature, iodomethane (0.41 mL, 6.57 mmol)
was added and the mixture was stirred for an additional 5 h. The
solvents were evaporated and the crude residue was dissolved in a
mixture of CH2Cl2 (100 mL) and hydrochloric acid (30 mL, 1.0 M).
The aqueous layer was extracted with CH2Cl2 (3 ꢀ 50 mL). The
combined organic layer was washed with brine (100 mL), dried
(Na2SO4) and concentrated under reduced pressure. Recrystalliza-
tion from EtOH provided the known compound 17 (0.98 g, 95%) as a
1H NMR (CDCl3):
d
5.86e5.91 (m, 1H, CH¼), 5.32 (dd, 1H, J ¼ 17.0,
1.7 Hz, CH2]CH-1), 5.23 (t, 1H, J ¼ 3.3 Hz, H-12), 5.21 (dd, 1H,
J ¼ 10.5, 1.3 Hz, CH2]CH-2), 4.51 (d, 2H, J ¼ 5.6 Hz, OCH2eCH¼),
3.22 (dd,1H, J ¼ 11.2, 4.7 Hz, H-3), 2.26 (d,1H, J ¼ 11.3 Hz, H-18),1.99
(td, 1H, J ¼ 17.8, 4.3 Hz), 1.08, 1.00, 0.91, 0.79, 0.64 (each s, each 3H,
CH3), 0.94 (d, 3H, J ¼ 6.3 Hz, CH3), 0.86 (d, 3H, J ¼ 6.2 Hz, CH3).
4.6. 3-Hydroxy-11-oxo-urs-12-en-28-oic acid methyl ester (21)
Compound 21 was prepared following the same procedure as
colorless solid; 1H NMR (CDCl3):
d
5.26 (t, 1H, J ¼ 3.6 Hz, H-12), 3.62
(s, 3H, OCH3), 3.23 (dd, 1H, J ¼ 11.4, 4.7 Hz, H-3), 2.24 (d, 1H,
J ¼ 11.5 Hz, H-18), 1.09, 1.00, 0.93, 0.80, 0.76 (each s, each 3H, CH3),
0.95 (d, 3H, J ¼ 6.2 Hz, CH3), 0.88 (d, 3H, J ¼ 6.6 Hz, CH3).
described by C.M Ma [16]. 1H NMR (CDCl3):
d 5.58 (brs, 1H, H-12),
3.56 (s, 3H, OCH3), 3.18 (dd, 1H, J ¼ 10.5, 5.7 Hz, H-3), 2.75 (dt, 1H,
J ¼ 14.0, 3.5 Hz, H-1a), 2.36 (d, 1H, J ¼ 11.0 Hz, H-18), 2.25 (s, 1H, H-
9), 1.22, 1.08, 0.95, 0.86, 0.75 (each s, each 3H, CH3), 0.93 (d, 3H,
J ¼ 6.3 Hz, CH3), 0.82 (d, 3H, J ¼ 6.2 Hz, CH3).
4.3. 3-Oxo-urs-12-en-28-oic acid methyl ester (18)
To a solution of compound 17 (2.50 g, 5.31 mmol) in CH2Cl2
(80 mL) was added PCC (1.72 g, 7.97 mmol). After being stirred at
room temperature for 8 h, the mixture was concentrated and par-
titioned with H2O and CH2Cl2. The CH2Cl2 layer was concentrated
and purified by chromatography on a silica gel column eluted with
n-hexane-acetone (15:1) to give 18 (2.15 g, 86%) as a colorless solid;
4.7. General procedure for the preparation of 25e29
To a solution of compounds 19e23 (1 eq), 2,3,4,6-tetra-O-ben-
zoyl- -glucopyranosyl trichloroacetimidate 24 (1.3 eq) and 4 Å
D
molecular sieves in dry CH2Cl2 was added TMSOTf (0.1 eq) at 0 ꢁC
under argon. The reaction mixture was stirred for 1 h and warmed
to room temperature for 1 h. The reaction was quenched by Et3N
and concentrated. The residue was purified by silica gel column
chromatography (petroleum ether-EtOAc, 6:1) to afford com-
pounds 25e29, respectively.
Rf 0.53 (1:8, EtOAc-petroleum ether); 1H NMR (CDCl3):
d 5.29 (t-
like, 1H, H-12), 3.63 (s, 3H, OCH3), 2.53e2.59 (m, 1H, H-2a),
2.37e2.41 (m, 1H, H-2b), 2.26 (d, 1H, J ¼ 11.5 Hz, H-18), 1.10 (s, 6H,
2 ꢀ CH3), 1.05 (s, 6H, 2 ꢀ CH3), 0.96 (d, 3H, J ¼ 6.2 Hz, CH3), 0.88 (d,
3H, J ¼ 6.3 Hz), 0.81 (s, 3H, CH3); 13C NMR (CDCl3):
d 217.8 (C-3),
178.0 (C-28), 138.3 (C-13), 125.3 (C-12), 55.3, 53.0, 51.5, 48.1, 47.4,
46.8, 42.1, 39.5, 39.3, 39.1, 38.9, 36.7, 36.6, 34.2, 32.5, 30.7, 28.0, 26.6,
24.2, 23.5, 23.4, 21.5, 21.2, 19.6, 17.0, 16.9, 15.2; HRESIMS calcd for
4.7.1. 3
en-28-oic acid methyl ester (25)
Compound 25 was synthesized as a white solid in 93% yield; Rf
0.42 (1:4, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.29e8.03
a-O-(2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl)-urs-12-
C
31H48O3Na 491.3496; found 491.3498.
d
(m, 20H, AreH), 5.94 (t, 1H, J ¼ 9.7 Hz, H-30), 5.66 (t, 1H, J ¼ 9.6 Hz,
H-40), 5.12 (t, 1H, J ¼ 3.7 Hz, H-12), 4.79 (d, 1H, J ¼ 7.9 Hz, H-10), 4.66
(dd, 1H, J ¼ 11.9, 3.5 Hz, H-60-1), 4.51 (dd, 1H, J ¼ 11.9, 5.5 Hz, H-60-
2), 4.08e4.12 (m, 1H, H-50), 3.59 (s, 3H, OCH3), 3.35 (t-like, 1H,
J ¼ 1.6 Hz, H-3), 2.21 (d, 1H, J ¼ 11.2 Hz, H-18), 1.07, 0.94, 0.78, 0.76,
0.65 (each s, each 3H, CH3), 0.96 (d, 3H, J ¼ 6.2 Hz, CH3), 0.95 (d, 3H,
4.4. 3 -Hydroxyurs-12-en-28-oic acid methyl ester (19)
a
A mixture of compound 18 (2.10 g, 4.63 mmol), freshly prepared
Al (O-i-Pr)3 (18.9 g, 92.57 mmol), catalytic amount of AlCl3 (61.7 mg,
0.46 mmol) and dry i-PrOH (50 mL) was refluxed for 4 h. After the
mixture was cooled to room temperature, 1 M HCl (50 mL) was
added. The mixture was extracted with EtOAc (3 ꢀ 100 mL). The
combined extract was washed with saturated NaHCO3 (3 ꢀ 100 mL)
and brine (3 ꢀ 100 mL), dried over Na2SO4, filtered, and concen-
trated to afford a yellow solid, which was purified by flash chro-
matography (petroleumether-EtOAc, 6:1) to give 19 as a white solid
(1.49 g, 70%), together with 17 as a minor product. Rf 0.48 (1:4,
J ¼ 6.6 Hz, CH3); 13C NMR (CDCl3):
d 178.1 (C-28), 166.1, 165.8, 165.3,
165.0, 138.2 (C-13), 133.4, 133.2, 133.1, 133.0, 129.8, 129.7 (three),
129.6, 128.9 (two), 128.4, 128.3, 128.2 (two), 125.5 (C-12), 99.0 (C-
10), 82.9, 73.0, 71.9, 71.8, 70.4, 63.1, 52.9, 51.4, 49.0, 48.1, 47.0, 41.9,
39.5, 39.0, 38.9, 36.7 (two), 36.5, 33.0, 32.7, 30.6, 28.6, 28.0, 24.2,
23.6, 22.9, 22.1, 21.4, 21.2, 17.9, 17.3, 16.8, 15.3; HRESIMS calcd for
C65H76O12Na 1071.5229; found 1071.5233.
EtOAc-petroleum ether); 1H NMR (CDCl3):
d
5.26 (t, 1H, J ¼ 3.6 Hz,
H-12), 3.62 (s, 3H, OCH3), 3.42 (t, 1H, J ¼ 1.7 Hz, H-3), 2.24 (d, 1H,
J ¼ 11.3 Hz, H-18), 1.10, 0.96, 0.94, 0.85, 0.76 (each s, each 3H, CH3),
0.95 (d, 3H, J ¼ 6.4 Hz, CH3), 0.87 (d, 3H, J ¼ 6.5 Hz, CH3); 13C NMR
4.7.2. 3
en-28-oic acid allyl ester (26)
Similarly, 26 was prepared as a white solid in 92% yield; Rf 0.57
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.29e8.06 (m,
b-O-(2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl)-urs-12-
(CDCl3):
d
178.1 (C-28), 138.1 (C-13), 125.6 (C-12), 76.1 (C-3), 52.9,
d

G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
437
20H, AreH), 5.93 (t, 1H, J ¼ 9.7 Hz, H-30), 5.83e5.92 (m, 1H, CH ¼ ),
5.60 (t, 1H, J ¼ 9.7 Hz, H-20), 5.58 (dd, 1H, J ¼ 9.7, 7.9 Hz, H-20),
5.29e5.32 (m, 1H, CH2]CH-1), 5.27 (t, 1H, J ¼ 3.5 Hz, H-12),
5.19e5.23 (m, 1H, CH2]CH-2), 4.86 (d, 1H, J ¼ 7.9 Hz, H-10),
4.50e4.59 (m, 4H, H-60, OCH2), 4.15e4.18 (m,1H, H-50), 3.08 (dd,1H,
J ¼ 11.9, 4.8 Hz, H-3), 2.27 (d, 1H, J ¼ 12.3 Hz, H-18), 1.06, 0.85, 0.71,
0.70, 0.63 (each s, each 3H, CH3), 0.97 (d, 3H, J ¼ 6.2 Hz, CH3), 0.92
(m, 2H), 1.80e1.85 (m, 1H), 0.93, 0.87, 0.76, 0.67, 0.62 (each s, each
3H, CH3); 13C NMR (CDCl3):
176.7 (C-28), 166.1, 165.9, 165.3, 165.0,
d
150.7 (C-20), 133.4, 133.2, 133.1, 129.9, 129.8, 129.7, 128.4 (two),
128.3 (two), 109.5 (C-29), 103.2 (C-10), 90.7, 73.0, 72.1, 72.0, 70.3,
63.5, 56.6, 55.6, 51.3, 50.5, 49.4, 47.0, 42.3, 40.7, 38.8, 38.6, 38.3, 37.0,
36.8, 34.3, 34.2, 32.1, 30.6, 29.6, 27.5, 26.0, 25.6, 20.9, 19.5, 18.1, 16.0
(two), 15.9, 14.6; HRESIMS calcd for C65H76O12Na 1071.5229; found
1071.5238.
(d, 3H, J ¼ 6.6 Hz, CH3); 13C NMR (CDCl3):
d 177.7 (C-28), 166.6,
166.5, 165.9, 165.6, 138.6 (C-13), 134.1, 133.8, 133.7, 133.2, 130.7,
130.5, 130.4 (two), 130.3 (two), 130.0, 129.5, 129.4, 129.3, 129.0,
128.9 (two), 126.2 (C-12), 118.4, 103.9 (C-10), 91.2, 79.7, 73.6, 72.7,
72.6, 71.0, 65.4, 64.1, 56.1, 55.8, 53.5, 48.7, 48.2, 42.6, 40.1, 39.7, 39.3,
39.1, 37.3, 37.1, 33.6, 31.3, 28.7, 28.5, 28.2, 26.4, 24.8, 24.1, 23.8, 21.8,
18.6, 17.6, 16.8, 15.9; HRESIMS calcd for C67H78O16Na 1097.5385;
found 1097.5383.
4.8. General procedure for the preparation of 30e34
Compounds 25e29 was dissolved in CH2Cl2 and CH3OH
(V:V ¼ 1:1) and then NaOMe was added until pH ¼ 10, respectively.
After stirred at r.t. for 8 h, the solution was neutralized with Dowex
50 ꢀ 8 (Hþ) resin until pH ¼ 7, filtered and concentrated. Then the
residue was purified by silica gel column chromatography
(CH2Cl2eMeOH, 10:1) to give compounds 30e34, respectively.
4.7.3. 3
12-en-28-oic acid methyl ester (27)
Similarly, 27 was prepared as a white solid in 90% yield; Rf 0.52
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.29e8.03 (m,
b-O-(2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl)-oxo-urs-
4.8.1. 3
ester (30)
Compound 30 was synthesized as a white solid in 94% yield; Rf
0.35 (8:1, CHCl3eMeOH); 1H NMR (CDCl3):
a-O-(b-g-Glucopyranosyl)-urs-12-en-28-oic acid methyl
d
20H, AreH), 5.92 (t,1H, J ¼ 9.6 Hz, H-30), 5.62 (brs,1H, H-12), 5.59 (t,
1H, J ¼ 9.7 Hz, H-40), 5.57 (dd, 1H, J ¼ 9.7, 7.9 Hz, H-20), 4.87 (d, 1H,
J ¼ 8.0 Hz, H-10), 4.65 (dd 1H, J ¼ 11.8, 4.2 Hz, H-6-10), 4.50 (dd, 1H,
J ¼ 11.8, 7.0 Hz, H-6-20), 4.15e4.18 (m, 1H, H-50), 3.61 (s, 3H, OCH3),
3.13 (dd, 1H, J ¼ 11.3, 5.0 Hz, H-3), 2.73 (dt, 1H, J ¼ 13.7, 3.3 Hz, H-
1a), 2.43 (d, 1H, J ¼ 11.2 Hz, H-18), 2.31 (s, 1H, H-9), 1.27, 1.08, 0.87,
0.71, 0.68 (each s, each 3H, CH3), 0.99 (d, 3H, J ¼ 6.4 Hz, CH3), 0.91
d
5.26 (t, 1H, J ¼ 3.5 Hz,
H-12), 4.29 (d, 1H, J ¼ 7.8 Hz, H-10), 3.88 (dd, 1H, J ¼ 12.0, 3.1 Hz, H-
60-1), 3.83 (dd, 1H, J ¼ 11.8, 3.5 Hz, H-60-2), 3.64 (t, 1H, J ¼ 9.2 Hz, H-
40), 3.62 (s, 3H, OCH3), 3.58 (t-like, 1H, J ¼ 9.2, 8.9 Hz, H-30), 3.41(t-
like, 1H, J ¼ 8.8, 8.2 Hz, H-20), 3.36 (t, 1H, J ¼ 1.7 Hz, H-3), 3.29e3.32
(m, 1H, H-50), 2.24 (d, 1H, J ¼ 11.2 Hz, H-18), 1.11, 0.95, 0.94, 0.86,
0.75 (each s, each 3H, CH3), 0.96 (d, 3H, J ¼ 6.3 Hz, CH3), 0.88 (d, 3H,
(d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CDCl3):
d 199.6 (C-11), 177.2 (C-
28), 166.1, 165.9, 165.3, 164.9, 162.6 (C-13), 133.4, 133.3, 133.2, 133.1,
133.0, 130.6 (C-12), 129.8, 129.7, 129.6, 128.5, 128.4, 128.3 (two),
103.1 (C-10), 90.5, 73.0, 72.1, 72.0, 70.3, 63.4, 61.4, 55.2, 52.7, 51.9,
47.7, 44.6, 43.7, 39.0, 38.6, 36.8, 36.0, 32.9, 30.3, 28.3, 27.6, 25.8, 23.9,
21.0, 18.9, 17.2, 17.1, 16.2, 16.1; HRESIMS calcd for C65H74O13Na
1085.5022; found 1085.5030.
J ¼ 6.4 Hz, CH3); 13C NMR (CDCl3):
d 178.1 (C-28), 138.1 (C-13), 125.6
(C-12), 100.4 (C-10), 82.4, 76.4, 75.2, 73.6, 70.0, 62.0, 52.9, 51.5, 49.8,
48.1, 47.2, 42.0, 39.7, 39.1, 38.9, 36.9 (two), 36.7, 33.5, 32.7, 30.7, 28.9,
28.0, 24.2, 23.9, 23.2, 22.7, 21.2, 18.1, 17.1, 16.9, 15.4; HRESIMS calcd
for C37H60O8Na 655.4180; found 655.4184.
4.8.2. 3b-O-(b-D-Glucopyranosyl)-urs-12-en-28-oic acid allyl ester
4.7.4. Methyl 3
11-oxo-olean-12 -en-30-oate (28)
Similarly, 28 was prepared as a white solid in 88% yield; Rf 0.50
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.34e8.01 (m,
b
-O-(2,3,4,6-Tetra-O-benzoyl-
b
-D-glucopyranosyl)-
(31)
Similarly, 31 was prepared as a white solid in 93% yield; Rf 0.33
(8:1, CHCl3eMeOH); 1H NMR (CD3OD):
d
5.90e5.96 (m, 1H, CH]
d
CH2), 5.33 (dd, 1H, J ¼ 17.2, 1.6 Hz, CH]CH2-1), 5.15 (t, 1H,
J ¼ 3.4 Hz, H-12), 5.22e5.23 (m, 1H, CH]CH2-2), 4.50 (d, 2H,
J ¼ 5.6 Hz, OCH2eCH¼), 4.34 (d, 1H, J ¼ 7.8 Hz, H-10), 3.85 (dd, 1H,
J ¼ 11.9, 2.2 Hz, H-60-1), 3.67 (dd, 1H, J ¼ 11.9, 5.5 Hz, H-60-2),
3.28e3.36 (m, 2H, H-30, H-40), 3.24e3.27 (m, 1H, H-50), 3.28e3.36
(m, 2H, H-3, H-20), 2.25 (dd,1H, J ¼ 11.2, 4.1 Hz, H-18),1.13,1.07, 0.97,
0.86, 0.79 (each s, each 3H, CH3), 0.98 (d, 3H, J ¼ 6.3 Hz, CH3), 0.90
20H, AreH), 5.70 (brs,1H, H-12), 5.63 (t,1H, J ¼ 9.7 Hz, H-30), 5.51 (t,
1H, J ¼ 9.7 Hz, H-40), 4.62 (d, 1H, J ¼ 8.2 Hz, H-10), 4.61 (dd, 1H,
J ¼ 11.9, 3.2 Hz, H-60-1), 4.46 (dd, 1H, J ¼ 11.9, 7.1 Hz, H-60-2),
4.01e4.08 (m, 1H, H-20), 3.87e3.91 (m, 1H, H-50), 3.72 (s, 3H, OCH3),
3.24 (dd, 1H, J ¼ 11.7, 4.7 Hz, H-3), 2.77 (dt, 1H, J ¼ 13.7, 3.5 Hz, H-1),
2.54 (d,1H, J ¼ 3.0 Hz, H-9), 2.29 (s,1H), 2.11 (dd,1H, J ¼ 13.6, 3.6 Hz,
H-18), 2.08 (dd, 1H, J ¼ 13.4, 4.6 Hz, H-15), 2.01e2.03 (m, 1H, H-21),
1.95e1.97 (m, 1H, H-19), 1.90e1.93 (m, 1H, H-2), 1.37, 1.18, 1.15, 1.14,
(d, 3H, J ¼ 6.3 Hz, CH3); 13C NMR (CD3OD):
d 177.5 (C-28), 138.1 (C-
13),132.3,125.7 (C-12),117.0,105.3 (C-10), 89.4, 76.9, 76.2, 74.3, 70.2,
64.7, 61.4, 55.6, 53.0, 41.8, 39.5, 39.0, 38.8, 36.6, 36.3, 32.9, 30.2, 27.7,
27.2, 25.5, 22.9, 22.6, 20.1, 17.9, 16.2, 15.7; HRESIMS calcd for
1.05, 0.87, 0.83 (each s, each 3H, CH3); 13C NMR (CDCl3):
d 199.9 (C-
11), 177.0 (C-30), 169.1 (C-13), 166.5, 166.0, 165.5, 133.4, 133.3, 133.2,
129.9, 129.8, 129.7, 129.6, 129.2, 128.9 (C-12), 128.5 (two), 128.4
(two), 104.9 (C-10), 90.4, 75.0, 73.3, 72.0, 69.9, 63.5, 61.8, 55.3, 51.8,
48.4, 45.4, 44.4, 43.2, 41.2, 39.3, 39.0, 37.8, 36.8, 32.7, 31.8, 31.2, 28.5,
28.4, 28.2, 26.5, 26.4, 25.9, 23.4, 18.7, 17.4, 16.7, 16.3; HRESIMS calcd
for C65H74O13Na 1085.5022; found 1085.5023.
C39H62O8Na 681.4337; found 681.4345.
4.8.3. 3 -O-( -D-Glucopyranosyl)-oxo-urs-12-en-28-oic acid
methyl ester (32)
b
b
Similarly, 32 was prepared as a white solid in 90% yield; Rf 0.30
(8:1, CHCl3eMeOH); 1H NMR (CDCl3):
d 5.59 (s, 1H, H-12), 4.35 (d,
4.7.5. Methyl betulinate 3
glucopyranoside (29)
b
-O-2,3,4,6-Tetra-O-benzoyl-
b
-D-
1H, J ¼ 7.8 Hz, H-10), 3.78e3.83 (m 2H, H-60), 3.56e3.60 (m, 1H, H-
50), 3.62 (s, 3H, OCH3), 3.51 (t, 1H, J ¼ 9.0 Hz, H-40), 3.41 (t-like, 1H,
J ¼ 8.2 Hz, H-30), 3.27 (d, 1H, J ¼ 9.3 Hz, H-3), 3.16 (t, 1H, J ¼ 8.2 Hz,
H-20), 2.77 (d, 1H, J ¼ 13.1 Hz, H-1a), 2.42 (d, 1H, J ¼ 11.2 Hz, H-18),
2.30 (s, 1H, H-9), 1.30, 1.12, 1.02, 0.90, 0.84 (each s, each 3H, CH3),
0.98 (d, 3H, J ¼ 6.4 Hz, CH3), 0.88 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR
Similarly, 29 was prepared as a white solid in 93% yield; Rf 0.40
(1:4, EtOAc-petroleum ether); 1H NMR (CDCl3):
d
7.29e8.04 (m,
20H, AreH), 5.92 (t, 1H, J ¼ 9.6 Hz, H-30), 5.60 (t, 1H, J ¼ 9.7 Hz, H-
40), 5.57 (dd, 1H, J ¼ 9.6, 8.0 Hz, H-20), 4.87 (d, 1H, J ¼ 7.9 Hz, H-10),
4.77 (d, 1H, J ¼ 2.2 Hz, H-29-1), 4.65e4.66 (m, 1H, H-29-2), 4.61 (dd,
1H, J ¼ 11.9, 3.4 Hz, H-60-1), 4.55 (dd, 1H, J ¼ 11.9, 6.8 Hz, H-60-2),
4.13e4.18 (m, 1H, H-50), 3.67 (s, 3H, OCH3), 3.08 (dd, 1H, J ¼ 11.8,
4.5 Hz, H-3), 2.99e3.03 (m, 1H, H-19), 2.17e2.24 (m, 2H), 1.87e1.94
(CDCl3): d 199.7 (C-11), 177.1 (C-30), 162.7 (C-13), 130.7 (C-12), 105.2
(C-10), 89.7, 76.4, 75.3, 73.9, 69.5, 61.7, 61.4, 55.3, 52.7, 51.8, 47.1,
44.6, 43.7, 39.5, 39.2, 38.7, 38.5, 36.9, 36.0, 33.0, 30.3, 28.4, 28.0,
26.1, 24.0, 21.0 (two), 18.9, 17.3, 17.2, 16.7, 16.3; HRESIMS calcd for

438
G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
C37H58O9Na 669.3973; found 669.3976.
1H, J ¼ 11.8, 4.4 Hz, H-3), 2.28 (d, 1H, J ¼ 11.1 Hz, H-18); 1.08, 1.00,
0.89, 0.81, 0.74 (each s, each 3H, CH3), 0.98 (d, 3H, J ¼ 6.3 Hz, CH3),
4.8.4. Methyl 3
b
-O-(
b-D-glucopyranosyl)-11-oxo-olean-12-en-30-
0.93 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CDCl3):
d 177.2 (C-28), 167.8,
oate (33)
166.6, 144.0, 138.1 (C-13), 133.5, 133.1, 132.6, 130.1, 130.0, 129.8,
128.5, 128.4, 128.3, 125.6 (C-12), 117.8, 104.8 (C-10), 90.1, 78.8, 74.2,
72.7, 70.2, 64.8, 63.9, 55.5, 52.9, 48.1, 47.6, 42.1, 39.6 39.1, 38.9, 38.8,
38.5, 36.7, 36.6, 33.1, 30.6, 28.2, 28.0, 25.9, 24.2, 23.5, 23.3, 21.2, 18.1,
17.0, 16.1, 15.4; HRESIMS calcd for C53H70O10Na 889.4867; found
889.4861.
Similarly, 33 was prepared as a white solid in 89% yield; Rf 0.27
(10:1, CHCl3eMeOH); 1H NMR (CDCl3):
d
5.66 (s, 1H, H-12), 4.37 (d,
1H, J ¼ 7.5 Hz, H-10), 3.82e3.89 (m, 2H, H-60), 3.69 (s, 3H, OCH3),
3.62 (t, 1H, J ¼ 9.2 Hz, H-30), 3.60 (t, 1H, J ¼ 9.1 Hz, H-40), 3.44 (t-like,
1H, J ¼ 9.2, 8.0 Hz, H-20), 3.29e3.30 (m, 1H, H-50), 3.20 (t-like, 1H,
J ¼ 9.8, 7.8 Hz, H-3), 2.78 (d, 1H, J ¼ 15.2 Hz, H-1), 2.34 (s, 1H, H-9),
2.06e2.09 (m, 1H, H-18), 2.00e2.03 (m, 1H, H-15), 1.36, 1.15, 1.14,
1.12,1.05, 0.86, 0.81 (each s, each 3H, CH3); 13C NMR (CDCl3):
d
200.1
4.9.3. 3
en-28-oic acid methyl ester (37)
Similarly, 37 was prepared as a white solid in 72% yield; Rf 0.36
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.45e8.09 (m,
b-O-(3,6-Di-O-benzoyl-b-D-glucopyranosyl)-oxo-urs-12-
(C-11), 176.7 (C-30), 169.2 (C-13), 128.5 (C-12), 104.9 (C-10), 89.5,
76.3, 75.2, 74.0, 61.8, 60.4, 55.2, 51.8, 48.4, 45.4, 44.1, 43.2, 41.1, 39.5,
39.2, 37.7, 36.8, 32.8, 31.8, 31.1, 28.5, 28.3, 28.1, 26.5, 26.1, 23.4, 18.7,
17.4, 16.7, 16.4; HRESIMS calcd for C37H58O9Na 669.3973; found
669.3982.
d
10H, AreH), 5.63 (s, 1H, H-12), 5.22 (t, 1H, J ¼ 9.2 Hz, H-30), 4.71 (dd,
1H, J ¼ 11.6, 1.3 Hz, H-6-10), 4.63 (dd, 1H, J ¼ 11.6, 5.6 Hz, H-6-20),
4.50 (d, 1H, J ¼ 7.8 Hz, H-10), 3.74e3.78 (m, 3H, H-30, H-40, H-50),
3.62 (s, 3H, OCH3), 3.27 (brs, 1H, H-20), 3.19 (dd, 1H, J ¼ 11.8, 4.6 Hz,
H-3), 2.74 (dt, 1H, J ¼ 13.6, 3.4 Hz, H-1a), 2.45 (d, 1H, J ¼ 11.6 Hz, H-
18), 2.23 (s, 1H, H-9),1.30,1.21,1.01, 0.91, 0.84 (each s, each 3H, CH3),
0.99 (d, 3H, J ¼ 6.4 Hz, CH3), 0.92 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR
4.8.5. Methyl betulinate 3
Similarly, 34 was prepared as a white solid in 95% yield; Rf 0.31
(10:1, CHCl3eMeOH); 1H NMR (CDCl3):
4.76 (brs,1H, H-29-1), 4.63
b-O-b-D-glucopyranoside (34)
d
(brs, 1H, H-29-2), 4.34 (d, 1H, J ¼ 7.3 Hz, H-10), 3.78e3.85 (m, 2H, H-
60), 3.62 (t,1H, J ¼ 8.8 Hz), 3.55e3.58 (m, 1H), 3.43 (t,1H, J ¼ 6.8 Hz),
3.28e3.29 (m, 1H, H-50), 3.66 (s, 3H, OCH3), 3.10 (dd, 1H, J ¼ 10.3,
4.1 Hz, H-3), 2.99e3.03 (m, 1H, H-19), 2.19e2.25 (m, 2H), 1.89e1.90
(m, 2H), 1.81e1.84 (m, 1H), 1.17, 0.97, 0.96, 0.92, 0.82, 0.79 (each s,
(CDCl3): d 199.6 (C-11), 177.2 (C-28), 167.8 (C-13), 166.8, 162.7, 133.5,
133.2, 130.7 (C-12), 130.0, 129.8, 129.7, 129.4, 128.5 (two), 104.8 (C-
10), 90.0, 78.6, 74.2, 72.8, 70.0, 63.9, 61.4, 55.3, 52.7, 51.8, 47.7, 44.6,
43.7, 39.3, 39.1, 38.6 (two), 36.8, 36.0, 33.0, 30.3, 28.4, 28.2, 25.8,
23.9, 21.0 (two), 18.9, 17.3, 17.1, 16.6, 16.1; HRESIMS calcd for
each 3H, CH3); 13C NMR (CDCl3):
d 176.6 (C-28), 150.4 (C-20), 109.6
(C-29), 105.1 (C-10), 90.2, 76.2, 75.2, 73.8, 69.5, 61.5, 56.5, 55.7, 51.2,
50.5, 49.5, 47.0, 42.4, 40.7, 39.2, 38.8, 38.3, 37.0, 36.9, 34.4, 32.2,
30.6, 29.7, 27.9, 26.3, 25.5, 20.9, 19.4, 18.2, 16.5, 16.2, 16.0, 14.7;
HRESIMS calcd for C37H60O8Na 655.4180; found 655.4188.
C51H66O11Na 877.4497; found 877.4506.
4.9.4. Methyl 3
oxo-olean-12-en-30-oate (38)
Similarly, 38 was prepared as a white solid in 70% yield; Rf 0.35
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.45e8.07 (m,
b- O-(3,6-Di-O-benzoyl-b-D-glucopyranosyl)-11-
4.9. General procedure for the preparation of 35e39
d
To a mixture of 30e34 (1 eq) and 1-BBTZ (3 eq) in dried CH2Cl2
(100 mL) was added Et3N (4 eq). After stirred at r.t. for 24 h, the
mixture was concentrated and purified by silica gel column chro-
matography (EtOAc/petroleum ether/CH2Cl2, 1:8:2) to provide
35e39 as white solids, respectively.
10H, AreH), 5.70 (s, 1H, H-12), 5.24 (t, 1H, J ¼ 8.1 Hz, H-30), 4.72 (dd,
1H, J ¼ 11.6, 1.1 Hz, H-6-10), 4.62 (dd, 1H, J ¼ 11.8, 5.7 Hz, H-6-20),
4.52 (d, 1H, J ¼ 7.8 Hz, H-10), 3.74e3.78 (m, 3H, H-20, H-40, H-50),
3.70 (s, 3H, OCH3), 3.22 (dd, 1H, J ¼ 11.8, 4.6 Hz, H-3), 2.77 (dt, 1H,
J ¼ 13.3, 2.9 Hz, H-1), 2.29 (s, 3H, H-9), 2.09e2.11 (m, 1H, H-18),
2.02e2.04 (m, 1H, H-15), 1.36, 1.18, 1.14, 1.13, 1.03, 0.86, 0.82 (each s,
4.9.1. 3
oic acid methyl ester (35)
Compound 35 was synthesized as a white solid in 74% yield; Rf
0.32 (1:4, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.46e8.11
a-O-(3,6-Di-O-benzoyl-
b
-D-glucopyranosyl)-urs-12-en-28-
each 3H, CH3); 13C NMR (CDCl3):
d 199.9 (C-11), 176.9 (C-30), 169.1
(C-13), 167.7, 133.5, 133.3, 130.0, 129.8, 128.5 (C-12), 128.4, 104.8 (C-
10), 90.0, 78.5, 74.2, 72.8, 70.0, 63.9, 61.8, 55.3, 51.8, 48.4, 45.3, 44.1,
43.3, 41.2, 39.2, 39.0, 37.7, 36.9, 32.7, 31.8, 31.1, 28.5, 28.4, 28.2, 26.5,
26.4, 25.9, 23.4, 18.7, 17.3, 16.7, 16.3; HRESIMS calcd for C51H66O11Na
877.4497; found 877.4501.
d
(m,10H, AreH), 5.23e5.26 (m, 2H, H-12, H-30), 4.71 (dd,1H, J ¼ 11.9,
5.3 Hz, H-60-1), 4.66 (dd, 1H, J ¼ 11.9, 2.6 Hz, H-60-2), 4.45 (d, 1H,
J ¼ 7.7 Hz, H-10), 3.74e3.80 (m, 2H, H-40, H-20), 3.69e3.72 (m, 1H, H-
50), 3.61 (s, 3H, OCH3), 3.41 (t, 1H, J ¼ 1.1 Hz, H-3), 2.22 (d, 1H,
J ¼ 11.2 Hz, H-18); 1.07, 0.94, 0.93, 0.81, 0.74 (each s, each 3H, CH3),
0.95 (d, 3H, J ¼ 6.3 Hz, CH3), 0.85 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR
4.9.5. Methyl betulinate 3b-O-3,6-Di-O-benzoyl-b-D-
glucopyranoside (39)
(CDCl3):
d
178.1 (C-28), 167.7, 166.8, 138.1 (C-13), 133.5, 133.2, 130.0,
Similarly, 39 was prepared as a white solid in 71% yield; Rf 0.38
129.9, 129.8, 129.7, 129.5, 128.5, 128.4 (two), 128.3, 126.3, 125.5 (C-
12), 100.4 (C-10), 82.3, 78.6, 74.3, 72.2, 69.9, 63.7, 52.9, 51.4, 49.9,
48.1, 47.4, 42.0, 39.7, 39.1, 38.9, 36.9, 36.8, 36.7, 33.7, 32.8, 30.7, 28.5,
28.0, 24.2, 23.8, 23.2, 22.4, 21.2, 18.1, 17.0, 16.9, 15.4; HRESIMS calcd
for C51H68O10Na 863.4705; found 863.4702.
(1:3, EtOAc-petroleum ether); 1H NMR (CDCl3):
d
8.08 (td, 4H,
J ¼ 8.2, 1.2 Hz, AreH), 7.59 (t, 2H, J ¼ 7.8 Hz, AreH), 7.46 (td, 4H,
J ¼ 8.3, 1.4 Hz, AreH), 5.23 (t, 1H, J ¼ 8.8 Hz, H-30), 4.78 (d, 1H,
J ¼ 1.7 Hz, H-29-1), 4.70 (dd, 1H, J ¼ 11.7, 2.2 Hz, H-60-1), 4.61e4.65
(m, 1H, H-60-2), 4.65 (s, 1H, H-29-2), 4.50 (d, 1H, J ¼ 7.8 Hz, H-10),
3.73e3.77 (m, 3H, H-20, H-40, H-50), 3.68 (s, 3H, OCH3), 3.14 (dd, 1H,
J ¼ 11.9, 4.5 Hz, H-3), 3.00e3.04 (m, 1H, H-19), 2.24e2.26 (m, 1H),
2.18e2.23 (m, 1H), 1.74, 0.98, 0.96, 0.91, 0.79, 0.79 (each s, each 3H,
4.9.2. 3
oic acid allyl ester (36)
Similarly, 36 was prepared as a white solid in 71% yield; Rf 0.31
(1:4, EtOAc-petroleum ether); 1H NMR (CDCl3):
7.44e8.08 (m,
b-O-(3,6-Di-O-benzoyl-b-D-glucopyranosyl)-urs-12-en-28-
CH3); 13C NMR (CDCl3):
d 176.7 (C-28), 167.8, 166.6, 150.6 (C-20),
d
133.5,133.1,130.0 (two),129.8 (two),129.4,128.4 (two),128.3 (two),
109.5 (C-29), 104.8 (C-10), 90.2, 78.7, 74.2, 72.8, 70.1, 64.0, 56.6, 55.7,
51.3, 49.5, 46.9, 42.4, 40.7, 39.0, 38.6, 38.3, 37.0, 36.8, 34.3, 32.2, 30.7,
29.7, 28.0, 26.0, 25.5, 20.9, 19.5, 18.1, 16.4, 16.1, 15.9, 14.7; HRESIMS
calcd for C51H68O10Na 863.4705; found 863.4713.
10H, AreH), 5.87e5.94 (m, 1H, CH]CH2), 5.32 (dd, 1H, J ¼ 17.2,
1.6 Hz, CH]CH2-1), 5.28 (t, 1H, J ¼ 3.2 Hz, H-12), 5.21e5.24 (m, 2H,
CH]CH2-2, H-30), 4.68 (d, 2H, J ¼ 3.6 Hz, OCH2eCH¼), 4.49e4.52
(m, 3H, H-6΄ ꢀ 2, H-10), 3.73e3.79 (m, 3H, H-50, H-40, H-20), 3.15 (dd,

G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
439
4.10. General procedure for the preparation of 5, 6, 15, 16, 41
(m, 1H, CH]CH2), 5.37 (d, 1H, J ¼ 1.3 Hz, Rha-H-1), 5.32e5.35 (m,
1H, CH]CH2-1), 5.25 (t, 1H, J ¼ 3.3 Hz, H-12), 5.21e5.23 (m, 1H,
CH]CH2-2), 4.86 (s, 1H, Rha-H-1), 4.50 (d, 2H, J ¼ 5.2 Hz,
OCH2eCH¼), 4.44 (d, 1H, J ¼ 7.7 Hz, H-10), 3.98e4.00 (m, 2H),
3.91e3.93 (m, 1H), 3.85 (dd, 1H, J ¼ 3.1, 1.8 Hz, Rha-H-2), 3.81 (dd,
1H, J ¼ 12.1, 1.7 Hz, H-60-1), 3.76 (dd, 1H, J ¼ 9.5, 3.4 Hz, Rha-H-3),
3.67 (dd, 1H, J ¼ 12.1, 4.1 Hz, H-60-2), 3.64 (dd, 1H, J ¼ 9.4, 3.3 Hz,
Rha-H-3), 3.60 (t, 1H, J ¼ 8.7 Hz), 3.55 (t, 1H, J ¼ 9.3 Hz), 3.46 (t, 1H,
J ¼ 8.2 Hz), 3.38e3.44 (m, 2H), 3.33e3.34 (m, 1H), 3.19 (dd, 1H,
J ¼ 11.6, 4.1 Hz, H-3), 2.25 (d, 1H, J ¼ 11.2 Hz, H-18), 1.27 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6), 1.22 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.13, 1.07, 0.97,
0.87, 0.79 (each s, each 3H, CH3), 0.98 (d, 3H, J ¼ 6.4 Hz, CH3), 0.90
To a mixture of 35e39 (1 eq) and 4 Å molecular sieves in dried
CH2Cl2 (40 mL) at ꢂ40 ꢁC under argon was added BF3$Et2O (1.5 eq),
followed by a solution of imidate 40 (5 eq) in CH2Cl2 (10 mL). After
stirring at - 40 ꢁC for 2 h and then at r.t. for 1 h, the reaction was
quenched with Et3N. The solid was filtered, and the filtrate was
concentrated under vacuum to give yellow oil. The oil was sub-
jected to column chromatography on silica gel (EtOAc-petroleum
ether, 1:4) to give the desired crude trisaccharide. The above
trisaccharide mixture was dissolved in CH2Cl2 and CH3OH
(V:V ¼ 1:1) and then NaOMe was added until pH ¼ 10. After stirred
at r.t. for 24 h, the solution was neutralized with Dowex 50 ꢀ 8 (Hþ)
resin until pH ¼ 7, filtered and concentrated. Then the residue was
purified by silica gel column chromatography (CH2Cl2eMeOH, 6:1)
to afford compounds 5, 6, 15, 16 and 41, respectively.
(d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 175.9 (C-28), 136.6 (C-
13), 130.9, 124.2 (C-12), 115.4, 102.5 (C-10), 100.2 (Rha-C-1), 99.2
(Rha-C-1), 87.4, 75.2, 71.1, 70.7, 69.5, 67.9, 67.2, 63.2, 59.1, 54.4, 51.5,
40.3, 38.0, 37.5, 35.1, 34.9, 31.4, 28.8, 26.2, 25.6, 22.4, 21.2, 16.4, 15.0,
14.7, 13.3; HRESIMS calcd for
973.5493.
C51H82O16Na 973.5495; found
4.10.1. Methyl 3
glucopyranosyl]-11- oxo-olean-12-en-30-oate (5)
Compound 5 was synthesized as a white solid in 51% yield for
two steps; Rf 0.35 (4:1, CHCl3eMeOH); 1H NMR (CD3OD):
5.58 (s,
b- O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
d
4.10.4. 3a-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
1H, H-12), 5.38 (d, 1H, J ¼ 1.3 Hz, Rha-H-1), 4.60 (brs, 1H, Rha-H-1),
4.44 (d, 1H, J ¼ 7.7 Hz, H-10), 3.97e4.00 (m, 2H), 3.91e3.93 (m, 1H),
3.85 (dd, 1H, J ¼ 3.2, 1.7 Hz, Rha-H-2), 3.81 (dd, 1H, J ¼ 12.0, 1.5 Hz,
H-60-1), 3.76 (dd, 1H, J ¼ 9.5, 3.3 Hz, Rha-H-3), 3.67 (dd, 1H, J ¼ 12.1,
4.1 Hz, H-60-2), 3.63 (dd, 1H, J ¼ 9.4, 3.2 Hz, Rha-H-3), 3.61 (s, 3H,
OCH3), 3.60 (t, 1H, J ¼ 8.9 Hz), 3.55 (t, 1H, J ¼ 8.9 Hz), 3.38e3.48 (m,
2H), 3.21 (dd, 1H, J ¼ 11.8, 4.3 Hz, H-3), 3.09e3.13 (m, 1H, H-50), 2.72
(dt, 1H, J ¼ 13.5, 2.8 Hz, H-1), 2.46 (s, 1H, H-19), 1.28 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6), 1.25 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.43, 1.16, 1.15,
1.14, 1.08, 0.90, 0.83 (each s, each 3H, CH3), 0.99 (d, 3H, J ¼ 6.4 Hz,
glucopyranosyl]-12-en-28-oic acid methyl ester (15)
Similarly, 15 was prepared as a white solid in 53% yield for two
steps; Rf 0.49 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d 5.28 (d, 1H,
J ¼ 1.3 Hz, Rha-H-1), 5.22 (t, 1H, J ¼ 3.2 Hz, H-12), 4.86 (d, 1H,
J ¼ 1.3 Hz, Rha-H-1), 4.39 (d, 1H, J ¼ 7.7 Hz, H-10), 4.02 (dd, 1H,
J ¼ 3.3, 1.7 Hz, Rha-H-2), 3.88e3.95 (m, 2H), 3.85 (dd, 1H, J ¼ 3.2,
1.8 Hz, Rha-H-2), 3.82 (dd, 1H, J ¼ 11.9, 1.9 Hz, H-60-1), 3.76 (dd, 1H,
J ¼ 9.5, 3.4 Hz, Rha-H-3), 3.67 (dd, 1H, J ¼ 12.0, 4.5 Hz, H-60-2), 3.64
(dd, 1H, J ¼ 9.4, 3.3 Hz, Rha-H-3), 3.61 (s, 3H, OCH3), 3.58 (t, 1H,
J ¼ 8.7 Hz), 3.54 (t, 1H, J ¼ 9.3 Hz), 3.49 (t, 1H, J ¼ 0.9 Hz, H-3), 3.42
(t,1H, J ¼ 9 0.5 Hz), 3.34e3.39 (m, 2H), 3.28e3.31 (m,1H, H-50), 2.22
(d, 1H, J ¼ 11.2 Hz, H-18), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.25 (d,
3H, J ¼ 6.2 Hz, Rha-H-6), 1.18, 0.98, 0.97, 0.88, 0.76 (each s, each 3H,
CH3), 0.91 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 201.2 (C-11),
171.2 (C-30), 171.1 (C-13), 121.5 (C-12), 104.1 (C-10), 101.7 (Rha-C-1),
100.6 (Rha-C-1), 88.1, 79.1, 78.1, 77.9, 76.8, 75.1, 72.6, 72.3, 71.0, 70.7,
70.6, 69.4, 68.7, 61.8, 60.6, 55.3, 50.9, 48.5, 45.3, 43.9, 43.2, 41.0,
39.2, 39.1, 37.6, 36.7, 32.4, 31.5, 30.6, 27.7, 27.1, 27.0, 26.1, 25.9, 25.8,
22.4, 17.9, 17.0, 16.6, 16.5, 15.7, 15.6; HRESIMS calcd for C49H78O17Na
961.5131; found 961.5138.
CH3), 0.99 (d, 3H, J ¼ 6.4 Hz, CH3), 0.91 (d, 3H, J ¼ 6.4 Hz, CH3); 13
C
NMR (CD3OD):
d 178.6 (C-28), 138.1 (C-13), 125.7 (C-12), 101.7 (C-
10), 100.6 (Rha-C-1), 99.6 (Rha-C-1), 81.2, 79.3, 78.2, 76.2, 75.1, 72.5,
72.3, 71.0, 70.8, 70.7, 69.4, 68.8, 60.8, 53.0, 50.7, 50.0, 46.5, 41.9, 39.5,
39.0, 36.7, 36.5, 33.1, 32.6, 30.3, 27.8, 27.7, 23.9, 22.9, 20.6, 20.1, 17.9,
17.3, 16.5, 16.4, 16.3, 14.8; HRESIMS calcd for C49H80O16Na 947.5339;
found 947.5346.
4.10.2. Methyl betulinate 3
-D- glucopyranoside (6)
Similarly, 6 was prepared as a white solid in 55% yield for two
steps; Rf 0.38 (4:1, CHCl3eMeOH); 1H NMR (CD3OD):
5.37 (d, 1H,
b-O-2,4-Di-O-(a-L-rhamnopyranosyl)-
b
d
J ¼ 1.2 Hz, Rha-H-1), 4.86 (s, 1H, Rha-H-1), 4.73 (s, 1H, H-29-1), 4.64
(s, 1H, H-29-2), 4.43 (d, 1H, J ¼ 7.7 Hz, H-10), 3.96e4.00 (m, 2H),
3.90e3.94 (m, 1H), 3.84 (dd, 1H, J ¼ 3.2, 1.8 Hz, Rha-H-2), 3.80 (d,
1H, J ¼ 11.2 Hz, H-60-1), 3.76 (dd, 1H, J ¼ 9.5, 3.0 Hz, Rha-H-3),
3.66e3.68 (m, 2H), 3.67 (s, 3H, OCH3), 3.63 (dd, 1H, J ¼ 9.4,
2.9 Hz, Rha-H-3), 3.59 (t, 1H, J ¼ 8.3 Hz), 3.55 (t, 1H, J ¼ 9.2 Hz),
3.39e3.47 (m, 3H), 3.16 (dd, 1H, J ¼ 11.2, 3.5 Hz, H-3), 2.98e3.03 (m,
1H, H-19), 2.21e2.24 (m, 2H), 1.95e1.97 (m, 1H), 1.85e1.90 (m, 2H),
1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.22 (d, 3H, J ¼ 6.2 Hz, Rha-H-6),
1.71, 1.04, 1.01, 0.95, 0.88, 0.85 (each s, each 3H, CH3), 0.98 (d, 3H,
J ¼ 6.4 Hz, CH3), 0.90 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
4.10.5. 3b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranosyl]-oxo-urs-12-en-28-oic acid methyl ester (16)
Similarly, 16 was prepared as a white solid in 52% yield for two
steps; Rf 0.46 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d 5.54 (s, 1H,
H-12), 5.37 (d, 1H, J ¼ 1.3 Hz, Rha-H-1), 4.86 (d, 1H, J ¼ 1.3 Hz, Rha-
H-1), 4.44 (d, 1H, J ¼ 7.8 Hz, H-10), 3.97e3.99 (m, 2H), 3.89e3.93
(m, 1H), 3.84 (dd, 1H, J ¼ 3.2, 1.9 Hz, Rha-H-2), 3.80 (dd, 1H,
J ¼ 11.8, 1.6 Hz, H-6-10), 3.75 (dd, 1H, J ¼ 9.6, 3.4 Hz, Rha-H-3), 3.67
(dd, 1H, J ¼ 11.9, 4.1 Hz, H-6-20), 3.63 (dd, 1H, J ¼ 9.6, 3.3 Hz, Rha-
H-3), 3.62 (s, 3H, OCH3), 3.59 (t, 3H, J ¼ 8.7 Hz), 3.55 (t, 3H,
J ¼ 8.8 Hz), 3.46 (t, 3H, J ¼ 8.2 Hz), 3.38e3.44 (m, 2H), 3.20 (dd, 1H,
J ¼ 11.8, 4.3 Hz, H-3), 2.70 (dt, 1H, J ¼ 13.5, 2.9 Hz, H-1a), 2.43 (d,
1H, J ¼ 11.7 Hz, H-18), 2.41 (s, 1H, H-9), 1.36, 1.14, 1.07, 0.93, 0.88
(each s, each 3H, CH3), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.23 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6), 1.00 (d, 3H, J ¼ 6.3 Hz, CH3), 0.90 (d, 3H,
d
176.8 (C-28), 150.4 (C-20), 108.9 (C-29), 104.1 (C-10), 101.7 (Rha-C-
1), 100.6 (Rha-C-1), 89.0, 79.1, 78.1, 77.8, 76.8, 75.0, 72.5, 72.3, 71.0,
70.7, 70.5, 69.4, 68.6, 60.6,56.5, 56.0, 50.6, 50.4, 49.3, 42.1, 40.5, 38.9
(two), 38.3, 36.7, 36.5, 34.2, 31.7, 30.2, 29.4, 27.0, 26.0, 25.5, 20.7,
18.1, 17.9, 16.6, 16.5, 15.6, 15.5, 15.2, 13.7; HRESIMS calcd for
J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 199.3 (C-11), 175.9 (C-28),
C
49H80O16Na 947.5339; found 947.5338.
163.0 (C-13), 128.6 (C-12), 102.3 (C-10), 99.9 (Rha-C-1), 98.9 (Rha-
C-1), 87.0, 77.5, 76.3, 75.2, 73.5, 71.0, 70.7, 69.5, 69.2, 69.1, 67.8,
67.1, 59.9, 59.0, 53.8, 51.5, 49.5, 43.1, 42.2, 37.6, 37.5, 37.1, 36.7, 35.2,
34.2, 31.2, 28.3, 26.5, 25.5, 24.2, 22.1, 18.4, 18.3, 16.6, 15.4, 15.1, 15.0,
14.6, 14.3, 14.0; HRESIMS calcd for C49H78O17Na 961.5131; found
961.5138.
4.10.3. 3
glucopyranosyl]-12-en-28-oic acid allyl ester (41)
Similarly, 41 was prepared as a white solid in 54% yield for two
steps; Rf 0.48 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
5.90e5.97
b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
d

440
G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
4.11. 3
12-en-28-oic acid (7)
b
-O-[2,4-Di-O-(
a
-
L
-rhamnopyranosyl)-
b
-
D
-glucopyranosyl]-
5.28 (t, 1H, J ¼ 3.4 Hz, H-12), 4.85 (brs, 1H, Rha-H-1), 4.45 (d, 1H,
J ¼ 7.9 Hz, H-10), 3.98e4.00 (m, 2H), 3.96 (t, 2H, J ¼ 6.4 Hz,
OCH2CH2), 3.90e3.93 (m,1H), 3.85 (dd, 1H, J ¼ 3.2,1.8 Hz, Rha-H-2),
3.82 (d, 1H, J ¼ 11.0 Hz, H-6-10), 3.76 (dd, 1H, J ¼ 9.5, 3.3 Hz, Rha-H-
3), 3.67 (dd, 1H, J ¼ 12.0, 3.9 Hz, H-6-20), 3.63 (dd, 1H, J ¼ 9.4, 3.1 Hz,
Rha-H-3), 3.60 (t, 1H, J ¼ 8.6 Hz), 3.55 (t, 1H, J ¼ 8.9 Hz), 3.44 (t, 1H,
J ¼ 8.5 Hz), 3.38e3.43 (m, 2H), 3.19 (dd, 1H, J ¼ 11.6, 4.0 Hz, H-3),
2.25 (d, 1H, J ¼ 11.2 Hz, H-18), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.23
(d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.13, 1.07, 0.97, 0.88, 0.79 (each s, each
3H, CH3), 0.98 (d, 3H, J ¼ 6.2 Hz, CH3), 0.96 (t, 3H, J ¼ 6.2 Hz,
Compound 41 (475 mg, 0.50 mmol) was dissolved in 1:1 MeOH/
CH2Cl2 (20 mL), PdCl2 (22.6 mg, 0.25 mmol) was then added. The
mixture was vigorously stirred at r.t. for 36 h and then filtered over
a celite pad and concentrated under vacuum. The mixture was
subjected to column chromatography on silica gel (1:5,
MeOHeCH2Cl2) to give compound 7 (415 mg, 91%) as a colorless
solid; Rf 0.42 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d 5.38 (d, 1H,
J ¼ 1.2 Hz, Rha-H-1), 5.24 (t, 1H, J ¼ 3.3 Hz, H-12), 4.86 (d, 1H,
J ¼ 1.2 Hz, Rha-H-1), 4.45 (d,1H, J ¼ 7.7 Hz, H-10), 3.97e4.01 (m, 2H),
3.91e3.94 (m, 1H), 3.85 (dd, 1H, J ¼ 3.1, 1.8 Hz, Rha-H-2), 3.81 (dd,
1H, J ¼ 12.1, 1.7 Hz, H-60-1), 3.76 (dd, 1H, J ¼ 9.5, 3.4 Hz, Rha-H-3),
3.67 (dd, 1H, J ¼ 12.1, 4.1 Hz, H-60-2), 3.64 (dd, 1H, J ¼ 9.4, 3.3 Hz,
Rha-H-3), 3.60 (t, 1H, J ¼ 8.7 Hz), 3.55 (t, 1H, J ¼ 9.3 Hz), 3.46 (t, 1H,
J ¼ 8.1 Hz), 3.38e3.44 (m, 2H), 3.33e3.34 (m, 1H), 3.19 (dd, 1H,
J ¼ 11.7, 4.1 Hz, H-3), 2.21 (d, 1H, J ¼ 11.3 Hz, H-18), 2.05 (dd, 1H,
J ¼ 13.4, 4.2 Hz), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.23 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6),1.13,1.07, 0.97, 0.87, 0.86 (each s, each 3H, CH3),
0.98 (d, 3H, J ¼ 6.0 Hz, CH3), 0.90 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR
CH2CH3), 0.90 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 178.1 (C-
28), 138.2 (C-13), 125.7 (C-12), 104.0 (C-10), 101.7 (Rha-C-1), 100.7
(Rha-C-1), 89.0, 79.1, 77.8, 76.8, 75.0, 72.5, 72.3, 71.0, 70.7, 70.6, 69.4,
68.6, 65.8, 60.6, 55.9, 53.0, 41.8, 39.5, 39.0, 38.8, 36.6, 36.4, 32.9,
30.3, 27.7, 21.1, 25.8, 23.9, 23.0, 22.7, 21.6, 20.1, 17.9, 16.6, 16.5 (two),
16.2, 15.8, 14.8, 9.6; HRESIMS calcd for C51H84O16Na 975.5652;
found 975.5656.
4.12.3. 3b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranosyl]-12-en-28-oic acid n-pentyl ester (10)
Similarly, 10 was prepared as a white solid in 94% yield; Rf 0.49
(CD3OD):
d
180.2 (C-28), 138.2 (C-13), 125.5 (C-12), 104.1 (C-10),
(3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d
5.37 (d, 1H, J ¼ 1.4 Hz,
101.6 (Rha-C-1), 100.6 (Rha-C-1), 89.0, 79.1, 77.8, 76.8, 75.0, 72.5,
72.3, 71.0, 70.7, 70.6, 69.4, 68.5, 60.6, 55.9, 53.0, 41.8, 39.4, 39.0,
38.9, 36.7, 36.4, 32.9, 30.4, 27.8, 27.1, 25.8, 23.0, 22.7, 20.2, 17.9, 16.6,
16.5,16.5,16.3,15.8,14.8; HRESIMS calcd for C48H78NaO16 933.5182;
found 933.5184.
Rha-H-1), 5.24 (t, 1H, J ¼ 3.4 Hz, H-12), 4.86 (d, 1H, J ¼ 1.5 Hz, Rha-
H-1), 4.45 (d, 1H, J ¼ 7.7 Hz, H-10), 3.96e4.03 (m, 4H), 3.90e3.94
(m, 1H), 3.85 (dd, 1H, J ¼ 3.2, 1.8 Hz, Rha-H-2), 3.81 (dd, 1H,
J ¼ 12.1, 1.9 Hz, H-60-1), 3.76 (dd, 1H, J ¼ 9.5, 3.4 Hz, Rha-H-3), 3.67
(dd, 1H, J ¼ 12.1, 4.1 Hz, H-60-2), 3.63 (dd, 1H, J ¼ 9.5, 3.3 Hz, Rha-
H-3), 3.60 (t, 1H, J ¼ 8.6 Hz), 3.55 (t, 1H, J ¼ 8.9 Hz), 3.46 (t, 1H,
J ¼ 8.2 Hz), 3.39e3.44 (m, 2H), 3.32e3.24 (m, 1H), 3.18 (dd, 1H,
J ¼ 11.6, 4.2 Hz, H-3), 2.24 (d, 1H, J ¼ 11.2 Hz, H-18), 2.07 (td, 1H,
J ¼ 13.6, 4.6 Hz), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.22 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6), 1.13, 1.07, 0.96, 0.88, 0.79 (each s, each 3H,
CH3), 0.98 (d, 3H, J ¼ 6.4 Hz, CH3), 0.94 (d, 3H, J ¼ 7.0 Hz, CH2CH3),
4.12. General procedure for the preparation of 8e11
A mixture of compound 41 (1 mmol) and K2CO3 (2 mmol) in
DMF (10 mL) was stirred at room temperature for 4 h. The bro-
moalkane (4 mmol) was then dripped slowly into the mixture. After
being stirred for another 12 h, the reaction mixture was poured into
the 40 mL distilled water and partitioned with butyl alcohol
(3 ꢀ 80 mL). The organic layer was washed with saturated sodium
chloride, dried over Na2SO4, and purified via silica gel column
chromatography (MeOH/CH2Cl2, 1:8) to provide 8e11 as white
solids, respectively.
0.90 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 177.8 (C-28),
138.1 (C-13), 125.7 (C-12), 104.2 (C-10), 101.8 (Rha-C-1), 100.6 (Rha-
C-1), 88.9, 79.1, 77.8, 76.9, 76.8, 75.0, 72.5, 72.3, 71.0, 70.7, 70.6,
69.4, 68.6, 60.6, 55.9, 53.0, 41.9, 39.6, 39.0, 38.8, 36.6, 36.4, 33.0,
32.1, 31.9, 30.3, 27.6, 27.1, 25.8, 23.9, 23.2, 23.1, 23.0, 22.5, 20.1, 17.9,
16.7, 16.6, 16.5, 16.2, 15.8, 14.8; HRESIMS calcd for C53H88O16Na
1003.5965; found 1003.5967.
4.12.1. 3
glucopyranosyl]-12-en-28-oic acid ethyl ester (8)
Compound 8 was synthesized as a white solid in 96% yield; Rf
0.48 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
5.38 (d, 1H, J ¼ 1.3 Hz,
b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
4.12.4. 3b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranosyl]-12-en-28-oic acid cyclopentyl ester (11)
d
Rha-H-1), 5.25 (t, 1H, J ¼ 3.4 Hz, H-12), 4.86 (d, 1H, J ¼ 1.3 Hz, Rha-
H-1), 4.45 (d, 1H, J ¼ 7.7 Hz, H-10), 4.06 (q, 2H, J ¼ 7.0 Hz, OCH2CH3),
3.97e4.01 (m, 2H), 3.90e3.95 (m, 1H), 3.85 (dd, 1H, J ¼ 3.2, 1.8 Hz,
Rha-H-2), 3.79e3.83 (m, 2H), 3.76 (dd, 1H, J ¼ 9.5, 3.4 Hz, Rha-H-3),
3.68 (dd, 1H, J ¼ 12.0, 4.1 Hz, H-60-1), 3.64 (dd, 1H, J ¼ 9.4, 3.2 Hz,
Rha-H-3), 3.59 (t, 1H, J ¼ 8.7 Hz), 3.55 (t, 1H, J ¼ 8.9 Hz), 3.46 (t, 1H,
J ¼ 8.1 Hz), 3.39e3.44 (m, 2H), 3.19 (dd, 1H, J ¼ 11.8, 4.2 Hz, H-3),
2.24 (d, 1H, J ¼ 11.2 Hz, H-18), 1.28 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.26
(d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.13, 0.98, 0.97, 0.88, 0.74 (each s, each
3H, CH3), 1.23 (d, 3H, J ¼ 6.4 Hz, CH3), 0.91 (d, 3H, J ¼ 6.4 Hz, CH3);
Similarly, 11 was prepared as a white solid in 92% yield; Rf 0.49
(3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d
5.38 (d, 1H, J ¼ 1.5 Hz,
Rha-H-1), 5.24 (t, 1H, J ¼ 3.4 Hz, H-12), 4.86 (d, 1H, J ¼ 1.6 Hz, Rha-
H-1), 4.45 (d, 1H, J ¼ 7.7 Hz, H-10), 3.96e3.99 (m, 1H), 3.98 (dd, 1H,
J ¼ 3.4, 1.8 Hz, Rha-H-2), 3.90e3.92 (m, 1H), 3.85 (dd, 1H, J ¼ 3.2,
1.8 Hz, Rha-H-2), 3.81 (dd, 1H, J ¼ 12.1, 1.9 Hz, H-60-1), 3.76 (dd, 1H,
J ¼ 9.5, 3.4 Hz, Rha-H-3), 3.67 (dd, 1H, J ¼ 12.1, 4.1 Hz, H-60-2), 3.64
(dd, 1H, J ¼ 9.5, 3.3 Hz, Rha-H-3), 3.60 (t, 1H, J ¼ 8.6 Hz), 3.55 (t,
1H, J ¼ 8.9 Hz), 3.46 (t, 1H, J ¼ 8.1 Hz), 3.38e3.44 (m, 2H),
3.32e3.24 (m, 1H), 3.19 (dd, 1H, J ¼ 11.9, 4.2 Hz, H-3), 2.20 (d, 1H,
J ¼ 11.2 Hz, H-18), 2.07 (td, 1H, J ¼ 13.6, 4.6 Hz), 1.28 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6), 1.23 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.13, 1.07,
0.98, 0.88, 0.82 (each s, each 3H, CH3), 0.97 (d, 3H, J ¼ 6.4 Hz, CH3),
0.90 (d, 3H, J ¼ 7.0 Hz, CH2CH3), 0.90 (d, 3H, J ¼ 6.4 Hz, CH3); 13C
13C NMR (CD3OD):
d 177.9 (C-28), 138.0 (C-13), 125.7 (C-12), 104.1
(C-10), 101.6 (Rha-C-1), 100.5 (Rha-C-1), 88.8, 79.1, 77.8, 76.8, 75.0,
72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6, 60.6, 60.5, 55.9, 53.0, 41.8, 39.5,
39.0, 38.8, 36.5, 36.4, 32.9, 30.3, 27.7, 27.1, 25.8, 23.9, 23.0, 22.7, 20.1,
17.9, 16.7, 16.6, 16.5, 16.2, 15.8, 14.8, 13.1; HRESIMS calcd for
NMR (CD3OD):
d 178.0 (C-28), 138.3 (C-13), 125.7 (C-12), 103.9 (C-
C
50H82O16Na 961.5495; found 961.5496.
10), 101.6 (Rha-C-1), 100.6 (Rha-C-1), 89.0, 79.1, 77.8, 76.7, 76.8,
75.0, 72.5, 72.3, 71.0, 70.7, 70.6, 69.3, 68.6, 64.2, 60.6, 55.9, 53.0,
41.8, 39.5, 39.0, 38.8 (two), 36.6, 36.4, 32.9, 30.3, 28.1, 28.0, 27.7,
27.1, 25.8, 23.9, 22.9, 22.7, 21.9, 20.1, 17.9, 16.6, 16.5 (two), 16.2, 15.8,
14.7, 13.0; HRESIMS calcd for C53H86O16Na 1001.5808; found
1001.5810.
4.12.2. 3 -O-[2,4-Di-O-( -rhamnopyranosyl)-b-D-
glucopyranosyl]-12-en-28-oic acid n-propyl ester (9)
b
a-L
Similarly, 9 was prepared as a white solid in 92% yield; Rf 0.49
(3:1, CHCl3eMeOH); 1H NMR (CD3OD):
d 5.38 (brs, 1H, Rha-H-1),

G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
441
4.13. 3
b
-O-[2,4-Di-O-(2,3,4-tri-O-acetyl-
a
-
L
-rhamnopyranosyl)-
b
-
(two), 38.8, 38.7, 37.1, 36.4, 32.6, 30.5, 29.3, 27.5, 27.2, 25.8, 23.9,
23.0, 22.8, 20.2, 17.9, 16.6, 16.5, 16.4, 16.1, 15.8, 14.7, 14.0; HRESIMS
calcd for C49H81O15NNa 946.5498; found 946.5505.
(3,6-two-O-acetyl)-D-glucopyranosyl] -12-en-28-oic acid (42)
Compound 7 (455 mg, 0.50 mmol) was dissolved in pyridine
(10 mL), Ac2O (0.76 mL, 8.0 mmol) and DMAP (30.5 mg, 0.25 mmol)
were then added, and the mixture was vigorously stirred at r.t. for
12 h. The mixture was concentrated and dissolved in CH2Cl2
(100 mL), then washed with 1 M HCl (3 ꢀ 50 mL), saturated NaHCO3
(3 ꢀ 50 mL) and brine (2 ꢀ 50 mL), dried over Na2SO4, filtered, and
concentrated to afford a yellow solid, which was purified by flash
chromatography [petroleumether-EtOAc (2:1)] to afford 42 as a
white solid (0.58 g, 94%); Rf 0.36 (1:2, EtOAc-petroleum ether); 1H
4.14.2. N-{3b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranosyl]-urs-12-en-28-oyl}- dimethylamine (13)
Similarly, 13 was prepared as a white solid in 94% yield for three
steps; Rf 0.45 (3:1, CHCl3eMeOH); 1H NMR (DMSO-d6):
d 5.18 (s,
1H, Rha-H-1), 5.05 (brs, 1H, H-12), 4.67 (s, 1H, Rha-H-1), 4.26 (d, 1H,
J ¼ 7.7 Hz, H-10), 3.77e3.81 (m, 3H), 3.60 (dd,1H, J ¼ 3.1, 1.7 Hz, Rha-
H-2), 3.56 (d, 1H, J ¼ 11.1 Hz, H-6-20), 3.47 (dd, 1H, J ¼ 9.5, 3.1 Hz,
Rha-H-3), 3.40e3.42 (m, 2H), 3.36e3.37 (m, 2H), 3.27 (t, 1H,
J ¼ 7.7 Hz), 3.18 (t, 1H, J ¼ 9.3 Hz), 3.17 (t, 1H, J ¼ 9.4 Hz), 3.13e3.15
(m, 1H), 3.02 (dd, 1H, J ¼ 10.8, 2.9 Hz, H-3), 2.87 (s, 6H, 2 ꢀ CH3),
2.36 (d, 1H, J ¼ 10.5 Hz, H-18), 1.09 (d, 3H, J ¼ 6.1 Hz, Rha-H-6), 1.05
(d, 3H, J ¼ 6.1 Hz, Rha-H-6), 1.00, 0.92, 0.83, 0.72, 0.62 (each s, each
3H, CH3), 0.88 (d, 3H, J ¼ 6.0 Hz, CH3), 0.80 (d, 3H, J ¼ 6.0 Hz, CH3);
NMR (CDCl3):
d
5.23e5.26 (m, 3H), 5.18 (dd,1H, J ¼ 10.1, 3.1 Hz, Rha-
H-3), 5.11 (dd, 1H, J ¼ 3.2, 17 Hz, Rha-H-2), 5.01e5.06 (m, 4H), 4.81
(d, 1H, J ¼ 1.2 Hz, Rha-H-1), 4.53 (d, 1H, J ¼ 7.3 Hz, H-10), 4.49 (d, 1H,
J ¼ 12.1 Hz, H-60-1), 4.22e4.28 (m, 2H), 3.85e3.88 (m, 1H), 3.77 (t,
1H, J ¼ 9.3 Hz), 3.68 (t, 1H, J ¼ 8.3 Hz), 3.60e3.62 (m, 1H), 3.16 (dd,
1H, J ¼ 11.5, 4.1 Hz, H-3), 2.55 (d, 1H, J ¼ 12.4 Hz, H-18), 2.14, 2.13,
2.12, 2.11, 2.05, 2.02, 1.99, 1.98 (each s, each 3H, each CH3CO),1.18 (d,
3H, J ¼ 6.2 Hz, Rha-H-6), 1.16 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.09, 1.03,
0.92, 0.81, 0.77 (each s, each 3H, CH3), 0.95 (d, 3H, J ¼ 6.0 Hz, CH3),
13C NMR (CD3OD):
d 176.1 (C-28), 128.8 (C-13), 128.6 (C-12), 104.1
(C-10), 101.7 (Rha-C-1), 100.6 (Rha-C-1), 89.0, 79.1, 77.8, 76.8, 75.1,
72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6, 60.6, 56.0, 48.7, 47.5, 39.3, 38.9
(two), 38.0, 36.4, 33.6, 32.8, 30.2, 27.8, 27.1, 25.8, 23.0, 20.2 17.9,16.6,
16.5, 16.1, 15.8, 14.8; HRESIMS calcd for C50H83O15NNa 960.5655;
found 960.5664.
0.86 (d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CDCl3):
d 182.8 (C-28), 170.5,
170.2, 170.1 (two), 170.0 (three), 169.6, 137.8 (C-13), 125.8 (C-12),
103.6 (C-10), 99.4 (Rha-C-1), 97.0 (Rha-C-1), 90.0, 77.9, 75.7, 75.4,
72.1, 71.1, 70.6, 69.9, 69.7, 68.6, 68.5, 67.9, 66.7, 62.1, 55.9, 52.6, 47.9,
47.6, 42.0, 39.5, 39.0, 38.8, 36.7, 32.9, 30.6, 28.0, 27.8, 26.0, 24.1, 23.6,
23.3, 21.4, 21.2, 20.9 (two), 20.8 (two), 20.7 (two), 18.2, 17.2, 17.1,
17.0, 16.9, 16.2, 15.6; HRESIMS calcd for C64H94O24Na 1269.6027;
found 1269.6031.
4.14.3. N-{3
glucopyranosyl]-urs-12-en-28-oyl}- diethylamine (14)
Similarly, 14 was prepared as a white solid in 93% yield for three
steps; Rf 0.46 (3:1, CHCl3eMeOH); 1H NMR (CD3OD):
5.37 (d, 1H,
b-O-[2,4-Di-O-(a-L-rhamnopyranosyl)-b-D-
d
J ¼ 1.3 Hz, Rha-H-1), 5.21 (t, 1H, J ¼ 3.1 Hz, H-12), 4.86 (d, 1H,
J ¼ 1.2 Hz, Rha-H-1), 4.44 (d,1H, J ¼ 7.8 Hz, H-10), 3.98e4.01 (m, 2H),
3.91e3.93 (m, 1H), 3.85 (dd, 1H, J ¼ 3.1, 1.8 Hz, Rha-H-2), 3.81 (dd,
1H, J ¼ 12.0, 1.7 Hz, H-6-10), 3.76 (dd, 1H, J ¼ 9.6, 3.4 Hz, Rha-H-3),
3.67 (dd, 1H, J ¼ 12.1, 4.1 Hz, H-6-20), 3.64 (dd, 1H, J ¼ 9.4, 3.3 Hz,
Rha-H-3), 3.60 (t, 1H, J ¼ 8.7 Hz), 3.55 (t, 1H, J ¼ 9.3 Hz), 3.46 (t, 1H,
J ¼ 8.5 Hz), 3.36e3.44 (m, 3H), 3.29e3.31 (m, 4H, NCH2), 3.18 (dd,
1H, J ¼ 11.6, 4.1 Hz, H-3), 2.41 (d, 1H, J ¼ 11.0 Hz, H-18), 1.28 (d, 3H,
J ¼ 6.2 Hz, Rha-H-6),1.22 (d, 3H, J ¼ 6.2 Hz, Rha-H-6),1.13,1.06, 0.97,
0.87, 0.81 (each s, each 3H, CH3), 0.98 (d, 3H, J ¼ 6.4 Hz, CH3), 0.91
4.14. General procedure for the preparation of 12e14
To a solution of compound 42 (1.5 mmol) in 20 mL dried CH2Cl2
was added oxalyl chloride (1 mL). The mixture was stirred at room
temperature for 36 h under argon and then concentrated to dryness
under reduced pressure. Hexane (3 ꢀ 10 mL) was added to the
residue, then concentrated to dryness. To a dried CH2Cl2 (10 mL)
solution of methylamine hydrochloride, dimethylamine hydro-
chloride or diaethylamine (2.0 mmol) was added to the above acid
chloride in the presence of triethylamine (4.0 mmol). The reaction
mixture was stirred at room temperature for 5 h under argon and
then concentrated. The obtained residue was dissolved in 2:1
MeOH/CH2Cl2 (15 mL) and then NaOMe was added until pH ¼ 10.
After stirred at r.t. for 12 h, the solution was neutralized with
Dowex 50 ꢀ 8 (Hþ) resin until pH ¼ 7, filtered and concentrated.
Then the residue was purified by silica gel column chromatography
(CH2Cl2eMeOH, 6:1) to give compounds 12e14.
(d, 3H, J ¼ 6.4 Hz, CH3); 13C NMR (CD3OD):
d 176.1 (C-28), 128.9 (C-
13),128.6 (C-12),104.1 (C-10),101.7 (Rha-C-1),100.6 (Rha-C-1), 89.0,
79.1, 77.9, 76.7, 75.0, 72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6, 60.6, 56.0,
52.7, 41.8, 39.4, 38.8 (two), 36.5, 32.6, 30.3, 29.3, 27.5, 27.1, 25.8,
23.0, 22.8, 20.3, 17.9, 16.6 (two), 16.5, 16.4, 16.1, 15.8, 14.8; HRESIMS
calcd for C52H87O15NNa 988.5968; found 988.5976.
4.15. Measurement of the inhibitory activity against
H5N1pseudovirus
4.14.1. N-{3
b
-O-[2,4-Di-O-(
a
-
L
-rhamnopyranosyl)-
b
-
D
-
MDCK cells and 293T cells were obtained from the American
Type Culture Collection (ATCC). Cells were grown in Dulbecco's
Modified Eagle Medium (DMEM, Gibco) containing glutamine,
supplemented with 10% fetal calf serum (FCS). The H5N1 pseudo-
viruses were prepared by transfecting HA plasmid from the H5
subtype strain A/Thailand/Kan353/2004 (H5N1) strain and the NA
plasmid from the N1 subtype strain A/Thailand/Kan353/2004.
Briefly, 293T cells (70e80% confluent) were co-transfected with
glucopyranosyl]-urs-12-en-28-oyl}- methylamine (12)
Compound 12 was synthesized as a white solid in 92% yield for
three steps; Rf 0.30 (4:1, CHCl3eMeOH); 1H NMR (CD3OD):
d 5.36
(d, 1H, J ¼ 1.2 Hz, Rha-H-1), 5.32 (t, 1H, J ¼ 3.4 Hz, H-12), 4.84 (d, 1H,
J ¼ 1.2 Hz, Rha-H-1), 4.42 (d,1H, J ¼ 7.7 Hz, H-10), 3.95e3.98 (m, 2H),
3.88e3.92 (m, 1H), 3.84 (dd, 1H, J ¼ 3.1, 1.8 Hz, Rha-H-2), 3.79 (dd,
1H, J ¼ 12.1, 1.7 Hz, H-6-20), 3.75 (dd, 1H, J ¼ 9.5, 3.4 Hz, Rha-H-3),
3.62e3.67 (m, 2H), 3.58 (t, 1H, J ¼ 8.7 Hz), 3.53 (t, 1H, J ¼ 9.3 Hz),
3.43 (t, 1H, J ¼ 8.5 Hz), 3.36e3.40 (m, 2H), 3.30e3.32 (m, 1H), 3.16
(dd, 1H, J ¼ 11.5, 4.1 Hz, H-3), 2.64 (s, 3H, NHCH3), 2.09 (d, 1H,
J ¼ 11.0 Hz, H-18), 1.27 (d, 3H, J ¼ 6.0 Hz, CH3), 1.26 (d, 3H, J ¼ 6.2 Hz,
Rha-H-6), 1.20 (d, 3H, J ¼ 6.2 Hz, Rha-H-6), 1.10, 1.04, 0.96, 0.95,
0.84, 0.76 (each s, each 3H, each CH3), 0.89 (d, 3H, J ¼ 6.2 Hz, CH3);
1 mg HA plasmid, 1 mg NA plasmid and 3 mg HIV backbone plasmid
(pNL4-3.luc.R_E_) into six-well plate with polyethylenimine (PEI)
[18]. Forty-eight hours after transfection, the culture supernatants
were harvested and centrifuged at 2000 g for 10 min. Aliquots were
stored at ꢂ80 ꢁC. For measuring the inhibitory activities of test
compounds, MDCK cells (104/well) were seeded in 96-well plates
and grown overnight. Tested compounds at indicated concentra-
tions were incubated with pseudotyped particles for 30 min at
37 ꢁC. Subsequently, the virus-compound mixture was transferred
13C NMR (CD3OD):
d 179.6 (C-28), 138.6 (C-13), 125.8 (C-12), 104.0
(C-10), 101.5 (Rha-C-1), 100.6 (Rha-C-1), 89.1, 78.8, 78.0, 76.6, 75.0,
72.5, 72.3, 71.0, 70.7, 70.6, 69.2, 68.7, 63.8, 60.6, 55.8, 52.7, 41.8, 39.4

442
G. Song et al. / European Journal of Medicinal Chemistry 93 (2015) 431e442
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The neuraminidase activity was measured by a fluorescence
based assay using a Neuraminidase Inhibitors Screen Kit by
following the manufacturer's instruction (Beyotime Institute of
Biotechnology, China). Briefly, 10
aminidase was added to 70 L of detection buffer, followed by
adding 10 L of a test compound and 10 L of neuraminidase
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m
m
m
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Acknowledgments
This project was supported by the National Science Foundation
for Young Scientists of China (No. 21202047), Foundation for
Distinguished Young Talents in Higher Education of Guangdong,
China (No. LYM10037), The PhD Start-up Fund of Natural Science
Foundation of Guangdong Province, China (S2012040007711) and
Specialized Research Fund for the Doctoral Program of Higher Ed-
ucation (New Teachers), China (No. 20114404120016).
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