Synthesis, biological evaluation and structure-activity relationship studies of hederacolchiside E and its derivatives as potential anti-Alzheimer agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.040

Inspired by the previously reported neuroprotective activity of hederacolchiside E (1), we synthesized hederacolchiside E for the first time along with eleven of its derivatives. The neuroprotective effects of these compounds were further evaluated agains

Full text of DOI:10.1016/j.ejmech.2017.11.040

Accepted Manuscript
Synthesis, biological evaluation and structure-activity relationship studies of
hederacolchiside E and its derivatives as potential anti-Alzheimer agents
Hui-ning Li, Yang Liu, Zuo-peng Zhang, Zhi-peng Wang, Jing-zheng Hao, Feng-ran
Li, Zhan-fang Fan, Li-bo Zou, Mao-sheng Cheng
PII:
S0223-5234(17)30943-1
10.1016/j.ejmech.2017.11.040
EJMECH 9915
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 August 2017
Revised Date: 16 November 2017
Accepted Date: 17 November 2017
Please cite this article as: H.-n. Li, Y. Liu, Z.-p. Zhang, Z.-p. Wang, J.-z. Hao, F.-r. Li, Z.-f. Fan, L.-
b. Zou, M.-s. Cheng, Synthesis, biological evaluation and structure-activity relationship studies of
hederacolchiside E and its derivatives as potential anti-Alzheimer agents, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.040.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation and structure-activity relationship studies of
hederacolchiside E and its derivatives as potential anti-Alzheimer agents
Hui-ning Lia, Yang Liu*,a, Zuo-peng Zhanga, Zhi-peng Wanga, Jing-zheng Haoa, Feng-ran Lia,
Zhan-fang Fana, Li-bo Zoub, Mao-sheng Cheng*,a
a Key Laboratory of Structure-Based Drugs Design and Discovery (Ministry of Education), School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016 China
E-mail addresses: y.liu@syphu.edu.cn (Y. Liu); mscheng@syphu.edu.cn (M. Cheng)
b
School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang
110016, China
Abstract
Inspired by the previously reported neuroprotective activity of hederacolchiside E (1), we
synthesized hederacolchiside E for the first time along with eleven of its derivatives. The
neuroprotective effects of these compounds were further evaluated against H2O2- and Aβ1-42-induced
injury using cell-based assays. The derivatives showed obvious differences in activity due to structural
variations, and two of them exhibited better neuroprotective effects than 1 in the Aβ1-42-induced injury
model. Compound 7 was the most active derivative and had a relatively simple chemical structure.
Moreover, 1 and 7 can significantly reduce the release of lactate dehydrogenase (LDH), level of
intracellular reactive oxygen species (ROS) and extent of malondialdehyde (MDA) increase resulting
from Aβ1-42 treatment, which demonstrated that these kinds of compounds show neuroprotective effects
in Alzheimer’s disease (AD) models via modulating oxidative stress. Compound 7 could be used as
promising lead for the development of a new type of neuroprotective agent against AD.
Keywords:
Hederacolchiside E; Alzheimer’s disease; Neuroprotective effects; Antioxidation;
Structure-activity relationship.
1. Introduction
Alzheimer’s disease (AD), characterized by progressive loss of memory and cognitive function,
dementia and eventually death, is the most common neurodegenerative disorder of this era that
substantially lowers the life quality of patients [1]. According to Alzheimer’s Disease International
(ADI) [2], there were 46.8 million people worldwide living with dementia in 2015; a number that is
estimated to reach 131.5 million by 2050. However, existing drug treatments are insufficient in
preventing or reversing its inexorable neurodegenerative progress [3] and show only marginal
symptomatic improvement in moderately to severely affected patients [4]. Although the mechanism of
this dementia has not been thoroughly elucidated, the abnormal accumulation of β-amyloid (Aβ) in the
hippocampal and cortical regions is hypothesized to be a major pathological hallmark of AD [5-7]. In
addition, some studies have reported evidence that Aβ-induced oxidative stress and mitochondrial
dysfunctions also play roles in this kind of neuronal cytotoxicity [8,9]. Therefore, searching for
antioxidants or free radical scavengers is one strategy for the treatment of AD.
For centuries, natural products and natural product-related structures have played a significant role
in the drug discovery and development process. Triterpenoid saponins are a large family of natural
products with diverse structures and bioactivities [10]. Many of them have been shown to cause

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improvements against neurotic atrophy and memory defect in dementia research [11]. For example,
uncarinic acid C, extracted from U. rhynchophylla [12], could effectively inhibit Aβ aggregation, and a
large number of oleanane triterpenes from the root of Bupleurum chinese [13] and the leaves of
Eleutherococcus senticosus [14] also showed potent neuroprotective activities. However, the isolation
of pure saponins from crude plant extracts, especially in suitable amounts, is relatively tedious and
difficult because of the microheterogeneity of these complex structures [15]. Chemical synthesis would
therefore provide us a feasible route to homogeneous saponins, as well as their derivatives, in sufficient
amounts for elucidating the structure activity relationships (SARs), the biological mechanisms and for
potential commercial applications.
Hederacolchiside
3-O-(α- -rhamnopyranosyl-(1→2)-[β-
α- -rhamnopyranosyl-(1→4)-β-
E
(1),
namely
L
D
-glucopyranosyl-(1→4)]-α-
L
-arabinopyranosyl) oleanolic acid
L
D
-glucopyranosyl-(1→6)-β-
D
-glucopyranosyl ester, is a bidesmosidic
oleanane saponin first isolated in 1970 from the leaves of Hedera colchica (Figure 1) [16]. Previously,
Han’s group reported that hederacolchiside E, the main bioactive component in the root extract of
Pulsatilla koreana, had prominent cognition-enhancing abilities and neuroprotective effects in
preliminary vitro and vivo activity-guided fractionation studies [17]. In the subsequent study,
researchers found that the oleanolic-glycoside saponins enriched fraction SK-PC-B70M, which
contained hederacolchiside E as the most active ingredient, could attenuate AD-like pathology in the
brain of Tg2576 mice [18]. The antioxidative activity of hederacolchiside E demonstrated in previous
reports may play an important role in the mechanism of its neuroprotective effect against Aβ [19].
Because of the unique biological activity in combination with isolation and purification difficulties,
herein we report the chemical synthesis and biological evaluation of hederacolchiside E and its
derivatives. Since the α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl moiety at 3-OH usually
appears in oleanane-type triterpenoid saponins, we replaced the glucopyranosyl on the 4-OH of the
arabinose by other monosaccharide moieties; thus, compounds 2-6 were designed. To elucidate the
contribution of each sugar moiety in the biological activity, we removed one, two or three sugar
moieties to give compounds 7-12 (Figure 2). The SAR study of these natural and non-natural saponins
against H2O2-induced oxidative damage, as well as Aβ-mediated neuronal cytotoxicity, is discussed.
Further investigation of compounds 1 and 7 on related neuroprotective indicators demonstrated that the
neuroprotective activities of this kind of compound are highly correlated with their antioxidative
activities.
Figure 1. The structure of hederacolchiside E (1)
2. Chemistry
The synthesis of the naturally occurring saponin hederacolchiside E and its glycosylate-replaced
and deglycosylated derivatives is described herein. Intermediates 18-23 were synthesized by routes
analogous to the efficient synthetic route to 9 reported previously by our group (Scheme 1) [20].

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Deprotection of the benzoyl groups of 13 generated intermediate 14. The 3- and 4-OH groups of 14
were selectively protected as an isopropylidene acetal, and the 2-OH group was then glycosylated with
a peracetylated rhamnopyranosyl donor to afford compound 16. The isopropylidene group was
1
hydrolyzed using p-TsOH, producing 18 with an abnormal C4 conformation of the arabinopyranosyl
residue (3-O-α-Ara H-1 J1-2 = 3.0) [21]. The conformation changing did not influence the subsequent
selective glycosylation of 4-OH of the arabinopyranosyl residue by different trichloroacetimidate
donors [22] under the promotion of trimethylsilyl triflate (TMSOTf) to provide intermediates 18-23.
As shown in Scheme 2, trichloroacetimidate donors linked with the carboxyl group of C-28 were
synthesized in a straightforward manner from known 1-thio-β-D-glucopyranoside 24. Regioselective
silylation of the 6-OH of 24 by tert-butyldimethylsilyl chloride (TBSCl) in pyridine was followed by
acetylation with acetic anhydride in one pot to afford 25 in excellent yield. The Lewis acid BF3·Et2O
was used to remove the TBS group from 25 to efficiently generate 26 with no observable acetyl
migration [23]. Glycosylation of 26 with perbenzoylated glucopyranosyl trichloroacetimidate afforded
27. Hydrolyzation of the thioglycoside using the efficient green reagent trichloroisocyanuric acid
(TCCA)
[24]
followed
by
trichloroacetimidation
with
CCl3CN
catalyzed
with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) furnished the disaccharide building block 28. To prepared
trisaccharide donor 36, the 4- and 6-OH groups of 24 were first protected using benzaldehyde dimethyl
acetal to provide 29. Acetylation of the two remaining hydroxyl groups followed by removal of the
benzylidene group by p-TsOH afforded 31. Regioselective benzoylation of 31 on the 6-OH with
benzoyl chloride in pyridine at 0 °C generated 32 in 85% yield. Coupling 32 with a peracetylated
rhamnopyranosyl donor provided 33, which was followed by trichloroacetimidation to generate 34.
Glycosylation of 26 and 34 using catalytic amounts of TMSOTf successfully gave the trisaccharide 35.
The final trisaccharide donor 36 was prepared via hydrolysis and trichloroacetimidation of 35 in the
same manner as above.
With key trisaccharide trichloroacetimidate donor 36 in hand, we started to formally assemble
target saponins 1-8. As shown in Scheme 3, ready removal of the benzyl group through catalytic
hydrogenation provided intermediates 37-42. Esterification of 37-42 with trichloroacetimidate donor 36
promoted by TMSOTf in dry CH2Cl2 under an atmosphere of N2, followed by global deprotection of
the benzoyl and acetyl groups by 1 M NaOMe in the presence of the C-28 ester glycosidic linkage,
afforded target compound 1 and its derivatives 2-6. The conformation of the arabinopyranosyl moiety
4
returned to the normal C1 form (3-O-α-Ara H-1 J1-2 = 6.0) [20,21,25] after deprotection due to the
attenuated steric hindrance of the rhamnopyranosyl moiety and led to the correct final products.
The preparation of target compounds 7 and 8 were similar to the synthetic route described above
(Scheme 3). Catalytic hydrogenation of 13 afforded intermediate 49, which was converted to
compound 7 by esterification and global deprotection via the general procedure. Deprotecting the
carboxyl group of compound 15 followed by esterification with 36 furnished compound 52.
Deprotection of the isopropylidene by p-TsOH and benzoyl and acetyl groups by NaOMe afforded
compound 8. Changing substituent groups on the carboxyl group of 37 led to compounds 9-12. Direct
removal of the benzoyl and acetyl groups of 37 furnished compound 9. Coupling 37 with
perbenzoylated glucopyranosyl trichloroacetimidate (or 28, 34) followed by global deprotection of the
benzoyl and acetyl groups to provide compounds 10-12 (Scheme 4).

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Figure 2. Chemical structures of target compounds 1-12
Scheme 1. Synthesis of intermediates 18-23. Reagents and conditions: (a) NaOMe, MeOH-CH2Cl2 2:1,
v/v, rt, 10 min; (b) Me2C(OMe)2, p-TsOH, CH2Cl2, rt, 5 h; (c) peracetylated rhamnopyranosyl
trichloroacetimidate, TMSOTf, CH2Cl2, 4 Å MS, 0 °C, 30 min; (d) p-TsOH, MeOH-CH2Cl2 2:1, v/v, rt,
2 h; (e) perbenzoylated sugar trichloroacetimidates, TMSOTf, CH2Cl2, 4 Å MS, 0 °C, 30 min.

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Scheme 2. Synthesis of intermediates 28, 34 and 36. Reagents and conditions: (a) TBSCl, DMAP,
pyridine, rt, 24 h; (b) Ac2O, pyridine, 0 °C to rt, 5 h; (c) BF3·Et2O, CH2Cl2, rt, 1 h; (d) sugar
trichloroacetimidates, TMSOTf, CH2Cl2, 4 Å MS, 0 °C, 30 min; (e) TCCA, acetone-H2O 9:1, v/v,
0 °C to rt, 1 h; (f) CCl3CN, DBU, CH2Cl2, rt, 1 h; (g) PhCH(OMe)2, p-TsOH, CH3CN, 50 °C, 1 h;
(h) p-TsOH, MeOH-CH2Cl2 2:1, v/v, rt, 2 h; (i) BzCl, pyridine, 0 °C to rt, 6 h.
Scheme 3. Synthesis of target compounds 1-8. Reagents and conditions: (a) 10% Pd-C, H2, EtOAc,
reflux, 2.5 h; (b) TMSOTf, CH2Cl2, 4 Å MS, N2, 0 °C, 30 min; (c) NaOMe, MeOH-CH2Cl2 2:1, v/v, rt,
5 h; (d) p-TsOH, MeOH-CH2Cl2 2:1, v/v, 40 °C.

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Scheme 4. Synthesis of target compound 9-12. Reagents and conditions: (a) NaOMe, MeOH-CH2Cl2
2:1, v/v, rt, 5 h; (b) sugar trichloroacetimidates, TMSOTf, CH2Cl2, 4 Å MS, N2, 0 °C, 30 min.
3. Results and discussion
3.1. Neuroprotective effects on H2O2-induced apoptosis in PC12 cells
It has been demonstrated that neurodegenerative disorders are a class of disease that induce
cellular injury by oxidative stress. H2O2 can generate exogenous free radicals and simulate damage
caused by oxidative stress in nerve cells [26]. High concentrations of H2O2 can help us investigate
neuroprotective activities of compounds in an economical way. The neuroprotective effects of
compounds 1-12 on the H2O2-induced decrease in cell viability in rat pheochromocytoma (PC12) cells
were assessed in preliminary screenings using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, and the results are expressed as a percentage of untreated control. As shown in
Figure 3, the cell viabilities decreased to approximately 58% when treated with H2O2 alone. Natural
saponin 1 exhibited a significant and dose-dependent effect on protecting PC12 cells from H2O2
damage, and derivatives 2-6, which kept the six-sugar skeleton, also showed dose-dependent activities.
The antioxidant activities of these compounds were equivalent to Edaravone, the positive control.
Compound 7 displayed a moderate activity and compound 8 showed potent neuroprotective effects at 1
and 10 µM, however as the concentration of the compounds increased to 50 µM, some toxicity
appeared. Compound 9, with no substituent on carboxyl group of C-28, is another natural product,
hederacolchiside A1, and it showed potent cytotoxicity against PC12 cells which is consistent with
previous reports [27]. Derivatives 10 and 11 showed cytotoxicity at high concentration; however,
compound 12 showed moderate neuroprotective effects in this model test.

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Figure 3. The neuroprotective effects of compounds 1−12 on the H
2
O2-induced decrease in cell
viability in PC12 cells. The cell viability in control was taken as 100%, and the average value of cell
viability under H2O2 (200 µM) exposure was 57.9 ± 2.9%. Data are expressed as mean ± the standard
deviation (SD), n = 3. Edaravone (Eda) was used as positive control. ###p < 0.001 vs the control group.
*p < 0.05, **p < 0.01, ***p < 0.001 vs the H2O2-treated group.
3.2. Neuroprotective effects on Aβ1-42-induced apoptosis in PC12 cells
To further examine the neuroprotective effects of these compounds, a model of Aβ1-42-induced
damage was chosen. Aβ is widely used to generate AD-like models to analyze the protective effects
and mechanisms of action of new pharmacotherapies for AD. H2O2-mediated toxicity to neurons in this
system occurs through covalent cross-linking of Aβ [28]. The results showed that the neuroprotective
effects of these compounds against Aβ1-42-induced damage had obvious structure-based activity
differences that were not exactly in line with H2O2-induced damage (Table 1). Based on the data, the
preliminary SARs were analyzed and were found to be the following:
a) The glycosyl residue on 4-OH of arabinose is crucial for the activity since the six-sugar
skeleton compounds 1-6 showed different effects against Aβ1-42-induced damage. Natural product 1
showed remarkable activity at 50 and 100 µM. However, when the glucopyranosyl moiety was
substituted with a rhamnopyranosyl moiety, compound 3 showed more potent anti-Aβ1-42 activity than
1. Compounds 2, 4, 5 and 6 that had alternative sugar substituents showed diminished neuroprotective
activity in this model test.
b) For the substituent group on C-3 position, the situation was mixed. Compound 7, bearing only
an arabinopyranosyl moiety on C-3, displayed more potent activity against Aβ1-42-induced damage than
compound
α- -rhamnopyranosyl-(1→2)-α-
feeble activity at 50 µM.
1
did
at
50
and
100
µM.
Compound
8,
with
the
L
L-arabinopyranosyl moiety, was less potent than 1 and showed only
c) The activities of compounds 10-12, which were designed by changing the substituent group on
C-28, were consistent in both H2O2- and Aβ1-42-induced models. Only substitution by one or two
glucopyranosyl moieties (10, and 11) lead to cytotoxicity, and substitution with glucopyranosyl and
rhamnopyranosyl (12) afforded comparable neuroprotective activity to that of 1. The results indicated
that the presence of the rhamnopyranosyl residue at the C-28 position is of vital importance for the
neuroprotective activity.

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Table 1. The neuroprotective effects of compounds 1-8, 10-12 on the Aβ1-42-induced decrease in cell
viability in PC12 cells.
Cell Viability (% of Control)
Compd.
10 µM
50 µM
100 µM
1
57.0 ± 2.5 65.8 ± 1.6b 70.4 ± 3.0b
c
2
58.3 ± 2.2
59.7 ± 2.6
–
3
61.4 ± 2.1a 68.4 ± 3.7b 74.6 ± 2.1b
4
52.6 ± 1.2
52.7 ± 1.2
58.5 ± 1.1
58.6 ± 0.5
55.7 ± 2.1
57.9 ± 1.5
–
–
–
5
6
7
55.9 ± 2.7 69.9 ± 3.3b 79.1 ± 4.2b
8
54.3 ± 1.0 60.3 ± 2.4a
–
–
–
–
10
11
14.5 ± 0.3
29.5 ± 0.4
10.3 ± 1.0
19.8 ± 2.6
12
model
59.3 ± 0.7a 63.3 ± 2.0b
55.3 ± 1.5
The cell viability in control was taken as 100%, and the average value of cell viability under Aβ1-42 (40
µM) exposure was 55.3 ± 1.5%. Data are expressed as mean ± SD, n = 3.
ap < 0.05 vs the Aβ1-42-treated group.
bp < 0.01 vs the Aβ1-42-treated group.
cThe – means not test.
3.3. Effects on Aβ1-42-induced lactate dehydrogenase (LDH) leakage in PC12 cells
LDH is a soluble enzyme present in most eukaryotic cells, it released from damaged cells into the
culture medium because of damage to the plasma membrane [29]. Measuring the activity of LDH can
quantitatively assess cytotoxicity. As shown in Figure 4, when PC12 cells were exposed to 40 µM
Aβ1-42 for 24 h, the LDH leakage was substantially increase to 153% compared with the control group
(100%). Pretreatment of the cells with various concentrations (10, 50, and 100 µM) of compounds 1
and 7 led to significantly decreases in LDH. The LDH leakage percentages of samples treated with
compound 1 were 147.4%, 138.5%, and 126.7%, respectively, and similar levels were found with
compound 7 (143.7%, 134.4% and 126.3%, respectively). The results indicated that the two
compounds can prevent Aβ1-42-induced damage by reducing the permeability of the plasma
membrane.

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Figure 4. The effects of compounds 1 (a) and 7 (b) at different concentrations on the LDH leakage in
Aβ1-42-treated PC12 cells. PC12 cells were treated with 40 µM Aβ1-42 for 24 h in the presence or
absence of compounds (10, 50, 100 µM). The results are expressed as mean ± SD, n = 3.##p < 0.01 as
*
compared with control group. p < 0.05 vs the Aβ1-42-treated group. **p < 0.01 vs the Aβ1-42-treated
group.
3.4. Effects on Aβ1-42-induced intracellular reactive oxygen species (ROS) in PC12 cells
Overproduction of ROS can lead to severe impairment of cellular functions. Once ROS cannot be
cleared by the antioxidant system, oxidative stress may occur. For instance, it can peroxidize membrane
lipids, oxidize proteins and attack mitochondrial DNA [30]. The effect of hederacolchiside E and
compound 7 on the intracellular ROS level of PC12 cells was measured using a ROS-sensitive
fluorescent probe (DCFH-DA). DCFH-DA can passively enter the cell and react with ROS to produce
highly fluorescent dichlorofluorescein (DCF), which is readily detected by flow cytometry. As shown
in Figure 5, after treatment with 40 µM Aβ1-42 for 24 h, the intracellular ROS level of PC12 cells
substantially increased to 51.4% relative to the control value (1.12%). When the cells were
preconditioned with 10, 50, and 100 µM of 1 or 7, the Aβ1-42-induced ROS levels were reduced in a
dose-dependent manner. The experiments demonstrated that compound 7 significantly reduced ROS
levels to 45.0%, 30.7%, and 20.6%, respectively, and showed better ROS-lowering activity than 1
(43.0%, 35.0%, and 29.2%, respectively) in high-dose groups.
Figure 5. The effects of compounds 1 and 7 on Aβ1-42-induced ROS level in PC12 cells as measured by
flow cytometry. The figure shows representative flow cytometric histograms. (a) Control; (b) 40 µM
Aβ1-42 alone; (c) 10 µM compound 1+40 µM Aβ1-42; (d) 50 µM compound 1+40 µM Aβ1-42; (e) 100 µM

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compound 1+40 µM Aβ1-42; (f) 10 µM compound 7+40 µM Aβ1-42; (g) 50 µM compound 7+40 µM
Aβ1-42; (h) 100 µM compound 7+40 µM Aβ1-42
.
3.5. Effects on Aβ1-42-induced malondialdehyde (MDA) in PC12 cells
Lipid peroxidation is one of the earliest recognized effects of oxygen toxicity in the cell
membranes that can lead to various degradation products such as MDA. This effect has been studied
widely as an indicator of lipid peroxidation and a marker of oxidative stress [31]. The in vitro MDA
expression levels in samples treated with compounds 1 and 7 are displayed in Figure 6. The results
showed that these two compounds significantly decreased the MDA levels in a dose-dependent manner,
and compound 7 possessed more potent activity (2.0, 1.6 and 1.2 nmol/mL) than 1 (2.5, 1.9 and 1.3
nmol/mL) at all test concentrations.
Figure 6. The effects of compounds 1 (a) and 7 (b) at different concentrations on the MDA levels in
Aβ1-42-treated PC12 cells. PC12 cells were treated with 40 µM of Aβ1-42 for 24 h in the presence or
absence of compounds (10, 50, 100 µM). The results are expressed as mean ± SD, n = 3. ##p < 0.01 as
compared with control group. **p < 0.01 vs the Aβ1-42-treated group.
4. Conclusions
Natural saponin hederacolchiside E was first synthesized in a concise and practical way, and its
analogs 2-12 were designed and synthesized based on the structure-guided SAR. The neuroprotective
effects of these compounds against H2O2- and Aβ1-42-induced injury in PC12 cells and the SAR were
evaluated and discussed. The twelve compounds showed activity difference in the test and some of
them possessed potent neuroprotective effects. Among them, compound 7 showed the best activity
against Aβ1-42-induced cell damage, which was noticeably superior to the natural compound 1. The in
vitro detection of related indicators in terms of neuroprotection showed that natural saponin 1 and
compound
7 could significantly decrease the levels of LDH, ROS and MDA in a
concentration-depended manner, indicating that the anti-Aβ activities of these compounds were highly
related to their antioxidant activities. In particular, compound 7, which exhibited a relatively simple
chemical structure and potent neuroprotection, could be used as a promising lead for the development
of a new series of neuroprotective agents against AD. The pharmacokinetics of these kinds of saponins
require further study.
5. Experimental
5.1. Chemistry
All the commercial reagents were used without further purification unless otherwise specified.

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Solvents were dried and redistilled prior to use in the usual manner. Analytical thin-layer
chromatography (TLC) was performed with silica gel HF254. Preparative column chromatography was
performed with silica gel H (200-300 mesh). Melting points were measured with a BÜCHI Melting
1
Point B-540. Optical rotations were recorded on an Anton Paar MCP 200 digital polarimeter. H and
13C NMR spectra were recorded on a Bruker ARX 600 MHz spectrometer, using Me4Si as the internal
standard if not otherwise mentioned. J values are given in hertz. HR-MS spectra were obtained on a
Bruker micro-TOF-Q spectrometer.
5.1.1. Benzyl Oleanolate 3-O-2,3,4-tri-O-acetyl-α-
(17)
L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside
A solution of compound 16 (5.03 g, 5.08 mmol) in CH2Cl2 – MeOH (1:2, v/v, 50 mL) was added
p-TsOH (2.62 g, 15.24 mmol), and stirred at rt for 4 h. When TLC (2:1, petroleum ether–EtOAc)
showed the deprotection was complete, Et3N (2 mL) was added to quench the reaction and the mixture
was concentrated and purified by a silica gel column chromatography (1.2:1, petroleum ether–EtOAc)
to give compound 17 (4.20 g, 87%) as a white amorphous solid. Rf = 0.36 (1:1, petroleum ether –
EtOAc); Mp 141 – 143 °C; [α]2D0 +9.62 (c 2.08, CH3OH); 1H NMR (600 MHz, CDCl3) δ 7.36 – 7.28 (m,
Ⅰ
5H, Ar–H), 5.30 – 5.24 (m, 3H), 5.06 (m, 4H), 4.70 (d, J = 3.0 Hz, 1H, H-1 ), 4.03 – 3.98 (m, 1H),
3.90 (dt, J = 7.9, 4.0 Hz, 1H), 3.88 – 3.85 (m, 1H), 3.85 – 3.83 (m, 1H), 3.74 (dd, J = 11.7, 8.3 Hz, 1H),
3.59 (dd, J = 11.7, 4.3 Hz, 1H), 3.11 (dd, J = 11.8, 4.4 Hz, 1H), 2.90 (dd, J = 13.7, 4.0 Hz, 1H), 2.14,
Ⅱ
2.03, 1.98 (s each, 3H each, 3 × CH3CO), 1.87 – 1.20 (m, 18H), 1.18 (d, J = 6.3 Hz, 3H, H-6 ), 1.10,
0.96, 0.91, 0.89, 0.87, 0.79, 0.59 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, CDCl3) δ 177.55,
170.19, 170.13, 170.10, 143.83 , 136.54, 128.53, 128.51, 128.11, 128.03, 122.55, 102.02, 98.22, 90.40,
76.00, 71.00, 70.60, 69.83, 69.02, 67.09, 66.06, 65.43, 61.07, 55.60, 47.73, 46.85, 46.01, 41.79, 41.49,
39.41, 39.21, 38.64, 36.82, 33.98, 33.22, 32.77, 32.48, 30.82, 28.22, 27.73, 25.97, 25.85, 23.77, 23.52,
23.16, 21.01, 20.91, 20.83, 18.35, 17.45, 16.97, 16.50, 15.44; HRMS(ESI): calcd. for [M+Na]+
C54H78O14Na: 973.5284, found 973.5291.
5.1.2. General procedure for the preparation of intermediates 18-23
A mixture of 17 (0.70 g, 0.74 mmol), trichloroacetimidate donors (0.89 mmol) and 4 Å molecular
sieves in dry CH2Cl2 (50 mL) wad stirred at 0 °C for 20 min. A solution of TMSOTf (0.22 mL, 0.34
mmol/mL) in dry CH2Cl2 was injected to the reaction mixture. The mixture was stirred under these
conditions for 30 min, then quenched with Et3N (0.1 mL). The mixture was the diluted with CH2Cl2
and filtered. The filtrate was concentrated and purified by a silica gel column chromatography (2:1
1
petroleum ether – EtOAc) to afford 18-23. The H-NMR and 13C-NMR data of intermediates 19-23
was shown in supplementary data.
5.1.3.
Benzyl
Oleanolate
3-O-2,3,4-tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→2)-[2,3,4,6-tetra-O-benzoyl-β-
D-glucopyranosyl-(1
→4)]-α-L-arabinopyranoside (18)
Following the general procedure mentioned above to get compound 18 (0.97g, 86%) as a white
foam. Rf = 0.32 (2:1, petroleum ether – EtOAc); Mp 119 – 121 °C; [α]2D0 –92.04 (c 2.26, CH3OH); 1H
NMR (600 MHz, CDCl3) δ 8.03 – 7.27 (m, 25H, Ar–H), 5.92 (t, J = 9.7 Hz, 1H), 5.69 (t, J = 9.7 Hz,
1H), 5.56 (dd, J = 9.8, 7.9 Hz, 1H), 5.30 – 5.25 (m, 2H), 5.23 (dd, J = 3.4, 1.6 Hz, 1H), 5.11 – 5.00 (m,
Ⅱ
4H, PhCH2), 4.97 (d, J = 0.9 Hz, 1H, H-1 ), 4.68 (dd, J = 12.1, 3.2 Hz, 1H), 4.56 (d, J = 3.6 Hz, 1H,
Ⅰ
H-1 ), 4.50 (dd, J = 12.1, 5.0 Hz, 1H), 4.21 (ddd, J = 9.6, 4.7, 3.5 Hz, 1H), 4.06 – 4.02 (m, 1H), 4.00
(dd, J = 10.0, 6.4 Hz, 1H), 3.96 (dt, J = 7.2, 3.5 Hz, 1H), 3.80 – 3.76 (m, 1H), 3.74 (dd, J = 5.6, 3.8 Hz,
1H), 3.63 (dd, J = 11.9, 3.5 Hz, 1H), 3.03 (dd, J = 11.8, 4.4 Hz, 1H), 2.90 (dd, J = 13.5, 3.9 Hz, 1H,

ACCEPTED MANUSCRIPT
Ⅱ
H-18), 2.13, 2.04, 1.98 (s each, 3H each, 3 × CH3CO), 1.16 (d, J = 6.2 Hz, 3H, H-6 ), 1.10, 0.93, 0.92,
0.89, 0.84, 0.74, 0.58 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, CDCl3) δ 177.57, 170.12,
166.20, 165.90, 165.44, 165.28, 143.79, 136.56, 133.56, 133.36, 133.32, 129.98, 129.90, 129.74,
129.27, 128.93, 128.54, 128.51, 128.42, 128.12, 128.04, 122.65, 102.40, 101.90, 97.89, 90.24, 75.95,
75.92, 72.92, 72.43, 72.21, 71.08, 70.41, 69.83, 69.70, 69.11, 66.83, 66.07, 65.46, 63.12, 60.42, 55.72,
47.74, 46.87, 46.02, 42.12, 41.78, 41.50, 39.42, 39.17, 38.70, 36.80, 33.99, 33.24, 32.78, 32.51, 30.84,
30.27, 29.83, 29.26, 28.22, 27.73, 25.99, 25.85, 23.78, 23.53, 23.49, 23.22, 23.18, 21.03, 20.93, 20.86,
18.35, 17.42, 16.98, 16.49, 15.44, 14.22; HRMS(ESI): calcd. for [M+Na]+ C88H104O23Na: 1551.6861,
found 1551.6864.
5.1.4.
Phenyl
D-glucopyranosyl-(1→6)-2,3,4-tri-O-acetyl-1-thio-β-D-glucopyranoside
2,3,4,6-tetra-O-benzoyl-β-
(27)
A
mixture of compound 26 (0.30 g, 0.75 mmol), perbenzoylated glucopyranosyl
trichloroacetimidate (0.67 g, 0.90 mmol) and 4 Å molecular sieves in dry CH2Cl2 (30 mL) was stirred
for 20 min at 0 °C. A solution of TMSOTf (0.22 mL, 0.34 mmol/mL) in dry CH2Cl2 was injected to the
reaction mixture. The mixture was stirred under these conditions for 30 min, then quenched with Et3N
(0.1 mL). The mixture was the diluted with CH2Cl2 and filtered. The residue was purified by a silica
gel column chromatography (2.5:1 petroleum ether – EtOAc) to give compound 27 (0.61 g, 83%) as a
white foam. Rf = 0.27 (2:1, petroleum ether – EtOAc); Mp 100 – 103 °C; [α]2D0 –1.06 (c 1.89, CH3OH);
1H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 7.2 Hz, 2H), 7.89 (d, J = 7.2 Hz,
2H), 7.83 (d, J = 7.2 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H),
7.46 – 7.42 (m, 5H), 7.38 – 7.35 (m, 5H), 7.33 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.8 Hz, 2H), 5.83 (t, J =
9.6 Hz, 1H), 5.63 (t, J = 9.7 Hz, 1H), 5.49 (dd, J = 9.7, 7.9 Hz, 1H), 5.13 (t, J = 9.3 Hz, 1H), 4.92 (d, J
= 7.9 Hz, 1H), 4.85 (t, J = 9.7 Hz, 1H), 4.78 (t, J = 9.8 Hz, 1H), 4.66 – 4.59 (m, 2H), 4.46 (dd, J = 12.1,
5.4 Hz, 1H), 4.08 – 4.03 (m, 1H), 3.83 (dd, J = 11.8, 2.1 Hz, 1H), 3.80 – 3.75 (m, 1H), 3.69 (ddd, J =
9.6, 7.2, 2.1 Hz, 1H), 2.05 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 170.05,
169.57, 169.24, 166.11, 165.78, 165.23, 165.16, 133.48, 133.22, 132.91, 131.64, 129.84, 129.84,
129.80, 129.77, 129.58, 129.19, 128.87, 128.82, 128.52, 128.48, 128.45, 128.34, 128.32, 100.86, 85.41,
77.93, 73.83, 72.92, 72.39, 71.80, 70.00, 69.70, 68.84, 68.02, 63.02, 20.74, 20.57, 20.50; HRMS(ESI):
calcd. for [M+Na]+ C52H48O17SNa: 999.2504, found 999.2484.
5.1.5.
2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-acetyl-β-D-glucopyranosyl
trichloroacetimidate (28)
A solution of compound 27 (0.54 g, 0.55 mmol) in acetone-H2O (9:1, 50 mL) was added TCCA
(0.13 g, 0.55 mmol) slowly at rt. The solution was stirred for 1 h and then acetone was removed in
vacuum. The reaction mixture was dissolved with CH2Cl2 and successively washed with saturated
NaHCO3 and followed by brine, then dried over Na2SO4. Rf = 0.35 (1:1, petroleum ether – EtOAc). A
mixture of crude solid, CCl3CN (0.22 mL, 2.20 mmol) and DBU (0.01 mL, 0.07 mmol) in CH2Cl2 (30
mL) was strried at rt for 1 h. After completion of the reaction based on TLC, the solvent was removed
in vacuum and the resulting residue was purified by a silica gel column chromatography (2:1 petroleum
ether–EtOAc) to give compound 28 (0.47 g, 83% over two steps) as a white foam. Rf = 0.25 (2:1,
petroleum ether – EtOAc); Mp 107 – 109 °C; [α]2D0 +35.54 (c 1.21, CH3OH); 1H NMR (600 MHz,
CDCl3) δ 8.43 (s, 1H), 8.02 (d, J = 7.1 Hz, 2H), 7.95 (d, J = 7.2 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 7.81
(d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.42 (dd, J = 15.0, 7.5 Hz, 4H), 7.37
(t, J = 7.8Hz, 1H), 7.33 (t, J = 7.7 Hz, 2H), 7.27 (t, J = 7.7, 2H), 6.39 (d, J = 3.6 Hz, 1H), 5.87 (t, J =

ACCEPTED MANUSCRIPT
9.6 Hz, 1H), 5.65 (t, J = 9.7 Hz, 1H), 5.51 – 5.46 (m, 2H), 4.96 (t, J = 4.2, 1H), 4.94 – 4.91 (m, 2H),
4.63 (dd, J = 12.1, 3.2 Hz, 1H), 4.49 (dd, J = 12.1, 5.2 Hz, 1H), 4.19 – 4.12 (m, 2H), 3.96 (dd, J = 11.6,
2.0 Hz, 1H), 3.70 (dd, J = 11.6, 6.1 Hz, 1H), 1.97 (s, 6H), 1.96 (s, 3H); 13C NMR (150 MHz, CDCl3) δ
169.88, 169.74, 169.53, 166.12, 165.78, 165.20, 165.07, 160.61, 133.44, 133.21, 133.15, 133.13,
129.93, 129.85, 129.76, 129.63, 129.30, 128.83, 128.81, 128.44, 128.41, 128.31, 128.29, 100.78, 92.62,
90.63, 72.87, 72.34, 71.65, 71.38, 69.78, 69.72, 68.49, 67.40, 63.06, 20.64, 20.52, 20.41; HRMS(ESI):
calcd. for [M+Na]+ C48H44O18NCl3Na: 1050.1516, found 1050.1492.
5.1.6.
2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2,3-di-O-acetyl-6-O-benzoyl-β-D-
glucopyranosyl trichloroacetimidate (34)
Crude 34 was prepared from 33 (1.51 g, 2.06 mmol) by the same procedure as for 28, and purified
by a silica gel column chromatography (3:1 petroleum ether–EtOAc) to give compound 34 (1.32 g, 81%
over two steps) as a white foam. Rf = 0.41 (1:1, petroleum ether – EtOAc); Mp 82 – 84 °C; [α]D20
+23.78 (c 2.08, CH3OH); 1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H, NH), 8.04 (d, J = 7.4 Hz, 2H, Ph),
Ⅴ
7.58 (t, J = 7.4 Hz, 1H, Ph), 7.45 (t, J = 7.7 Hz, 2H, Ph), 6.51 (d, J = 3.7 Hz, 1H, H-1 ), 5.62 (t, J = 9.7
Hz, 1H), 5.26 (dd, J = 10.1, 3.2 Hz, 1H), 5.19 – 5.15 (m, 1H), 5.09 – 5.00 (m, 2H), 4.92 (d, J = 1.5 Hz,
Ⅵ
1H, H-1 ), 4.80 (dd, J = 12.5, 1.4 Hz, 1H), 4.54 (dd, J = 12.5, 3.7 Hz, 1H), 4.28 (d, J = 8.9 Hz, 1H),
3.99 (t, J = 9.7 Hz, 1H), 3.92 (dq, J = 12.4, 6.1 Hz, 1H), 2.09, 2.08, 2.05, 2.01, 1.99 (s each, 3H each, 5
Ⅵ
× CH3CO), 1.18 (d, J = 6.2 Hz, 3H, H-6 ). 13C NMR (150 MHz, CDCl3) δ 170.17, 170.06, 170.02,
169.83, 165.90, 161.02, 133.28,129.94, 129.82, 128.51, 99.58, 93.11, 90.87, 76.76, 71.31, 70.90, 70.78,
70.35, 70.15, 68.66, 67.99, 62.02, 21.22, 20.95, 20.91, 20.83, 20.58, 17.39; HRMS(ESI): calcd. for
[M+Na]+ C31H36O16NCl3Na: 806.0992, 808.0962, found 806.0954, 808.0943.
5.1.7.
Phenyl
2,3,4-tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→4)-2,3-di-O-acetyl-6-O-benzoyl-β-
-glucopyranoside (35)
D-glucopyranosyl-(1→
6)-2,3,4-tri-O-acetyl-1-thio-β-
D
Crude 35 was prepared from 34 (1.34 g, 1.71 mmol) with 26 by the same procedure as for 27, and
purified by a silica gel column chromatography (3:2 petroleum ether – EtOAc) to give compound 35
(1.52 g, 87%) as a white foam. Rf = 0.23 (1.5:1, petroleum ether – EtOAc); Mp 102 – 104 °C; [α]D20
–
44.34 (c 1.06, CH3OH); 1H NMR (600 MHz, CDCl3) δ 8.03 (dd, J = 8.2, 1.1 Hz, 2H, Ph), 7.56 (t, J =
7.4 Hz, 1H, Ph), 7.47 – 7.40 (m, 4H, Ph), 7.34 – 7.28 (m, 3H, Ph), 5.24 – 5.16 (m, 3H), 5.13 (dd, J =
3.1, 2.1 Hz, 1H), 5.04 (t, J = 10.0 Hz, 1H), 4.94 – 4.90 (m, 1H), 4.89 (d, J = 1.8 Hz, 1H), 4.88 – 4.79
(m, 3H), 4.69 (d, J = 10.1 Hz, 1H), 4.58 (d, J = 7.9 Hz, 1H), 4.45 (dd, J = 12.3, 4.3 Hz, 1H), 3.91 (t, J =
9.3 Hz, 1H), 3.88 – 3.82 (m, 2H), 3.74 – 3.69 (m, 1H), 3.66 (ddd, J = 9.7, 4.1, 1.9 Hz, 1H), 3.61 (dd, J
= 11.3, 7.3 Hz, 1H), 2.07, 2.06, 2.06, 2.03, 1.97, 1.96, 1.95, 1.95 (s each, 3H each, 8 × CH3CO), 1.16 (d,
J = 6.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 170.30, 170.23, 170.14, 170.04, 169.98, 169.66,
169.40, , 165.91, 133.28, 132.27, 132.24, 129.94, 129.83, 129.31, 128.57, 128.30, 100.52, 99.21, 85.71,
77.47, 74.11, 74.03, 73.02, 72.04, 70.78, 70.15, 70.06, 68.82, 68.65, 68.36, 67.93, 62.32, 21.17, 20.94,
20.91, 20.87, 20.83, 20.79, 20.71, 20.65, 17.35; HRMS(ESI): calcd. for [M+Na]+ C47H56O23S1Na:
1043.2825, found 1043.2833.
5.1.8.
2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→4)-2,3-di-O-acetyl-6-O-benzoyl-β-
-glucopyranosyl trichloroacetimidate (36)
D-glucopyranosyl-(1
→6)-2,3,4-tri-O-acetyl-β-
D
Crude 36 was prepared from 35 (1.52 g, 1.50 mmol) by the same procedure as for 34, and purified
by a silica gel column chromatography (1.5:1 petroleum ether – EtOAc) to give compound 36 (1.33 g,
83%) as a white foam. Rf = 0.47 (1:1, petroleum ether – EtOAc); Mp 100 – 102 ºC; [α]2D0 +15.43 (c

ACCEPTED MANUSCRIPT
1.88, CH3OH); 1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H, NH), 8.03 (d, J = 7.9 Hz, 2H, Ph), 7.56 (t, J
Ⅳ
= 7.2 Hz, 1H, Ph), 7.45 (t, J = 7.6 Hz, 2H, Ph), 6.50 (d, J = 3.6 Hz, 1H, H-1 ), 5.51 (t, J = 9.8 Hz, 1H),
5.22 – 5.18 (m, 2H), 5.11 (s, 1H), 5.06 – 4.99 (m, 3H), 4.87 – 4.83 (m, 2H), 4.77 (d, J = 11.3 Hz, 1H),
4.58 (d, J = 7.9 Hz, 1H), 4.47 (dd, J = 12.3, 4.1 Hz, 1H), 4.15 – 4.11 (m, 1H), 3.91 (t, J = 9.6 Hz, 2H),
3.87 – 3.82 (m, 1H), 3.73 (d, J = 9.4 Hz, 1H), 3.54 (dd, J = 11.2, 5.2 Hz, 1H), 2.05, 2.05, 2.04, 2.02,
Ⅵ
2.00, 1.99, 1.99, 1.96 (s each, 3H each, 8 × CH3CO), 1.14 (d, J = 6.2 Hz, 3H, H-6 ); 13C NMR (150
MHz, CDCl3) δ 170.27, 170.14, 170.12, 170.03, 170.00, 169.90, 169.48, 160.85, 133.26, 129.93,
129.85, 128.53, 100.38, 99.14, 92.89, 90.83, 76.85, 74.01, 73.01, 71.83, 70.78, 70.11, 70.07, 69.84,
68.63, 68.24, 67.87, 67.22, 62.45, 21.13, 20.90, 20.80, 20.68, 20.55, 17.33; HRMS(ESI): calcd. for
[M+Na]+ C43H52Cl3NO24Na: 1094.1837, found 1094.1832.
5.1.9. General procedure for the preparation of intermediates 37-42
A sustention of 18 (or 19-23) (0.50 mmol) and 10% Pd-C (0.2 g) in EtOAc (50 mL) was refluxed
and bubbled up with H2 (70 mL/min) for 3.5 h. After completion of the reaction based on TLC, Pd-C
was removed through filtration and the filtrate was concentrated to dryness and purified by a silica gel
column chromatography (1.5:1 petroleum ether – EtOAc) to afford 37-42. The 1H-NMR and 13C-NMR
data of intermediates 38-42 was shown in supplementary data.
5.1.10.
Oleanolic
acid
3-O-2,3,4-tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→2)-[2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1
→4)]-α-L-arabinopyranoside (37)
Following the general procedure mentioned above to get compound 37 (0.65g, 91%) as a white
foam. Rf = 0.19 (2:1, petroleum ether – EtOAc); Mp 175 – 178 °C; [α]2D0 +27.27 (c 1.98, CH3OH);1H
NMR (600 MHz, CDCl3) δ 8.03 – 7.28 (m, 20H, Ar–H), 5.91 (t, J = 9.7 Hz, 1H), 5.69 (t, J = 9.7 Hz,
1H), 5.56 (dd, J = 9.7, 8.0 Hz, 1H), 5.30 – 5.25 (m, 2H), 5.23 (dd, J = 3.3, 1.5 Hz, 1H), 5.04 (dd, J =
Ⅱ
Ⅰ
19.7, 9.0 Hz, 2H), 4.94 (br s, 1H, H-1 ), 4.68 (dd, J = 12.1, 3.1 Hz, 1H), 4.59 (d, J = 3.1 Hz, 1H, H-1 ),
4.50 (dd, J = 12.1, 4.9 Hz, 1H), 4.23 – 4.18 (m, 1H), 4.05 – 3.95 (m, 3H), 3.82 – 3.77 (m, 1H), 3.77 –
3.73 (m, 1H), 3.64 (dd, J = 11.5, 3.3 Hz, 1H), 3.04 (dd, J = 11.6, 4.4 Hz, 1H), 2.81 (dd, J = 13.6, 3.8
Ⅱ
Hz, 1H), 2.13, 2.03, 1.98 (s each, 3H each, 3 × CH3CO), 1.16 (d, J = 6.2 Hz, 3H, H-6 ), 1.12, 0.93,
0.92, 0.90, 0.87, 0.74, 0.74 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, CDCl3) δ 183.11, 170.12,
166.21, 165.90, 165.44, 165.29, 143.66, 133.56, 133.34, 133.22, 129.99, 129.91, 129.75, 129.31,
128.99, 128.95, 128.94, 128.54, 128.52, 128.42, 122.75, 102.28, 101.86, 97.99, 90.23, 76.07, 75.75,
72.95, 72.42, 72.20, 71.08, 70.15, 69.85, 69.71, 69.12, 66.89, 63.13, 60.14, 55.71, 47.78, 46.67, 46.05,
41.76, 41.11, 39.41, 39.20, 38.69, 36.86, 33.96, 33.20, 32.74, 32.52, 30.80, 29.84, 28.22, 27.80, 26.01,
25.84, 23.71, 23.54, 23.13, 21.03, 20.93, 20.87, 18.34, 17.43, 17.09, 16.46, 15.46; HRMS(ESI): calcd.
for [M+Na]+ C81H98O23Na: 1461.6391, found 1461.6398.
5.1.11. General procedure for the preparation of compounds 1-6
A mixture of compounds 37 (or 38-42) (0.45 mmol), 36 (0.58 g, 0.54 mmol) and 4 Å molecular
sieves in dry CH2Cl2 (30 mL) were stirred under an atmosphere of N2 for 30 min at 0 °C. A solution of
TMSOTf (0.13 mL, 0.34 mmol/mL) in dry CH2Cl2 was injected. The mixture was stirred under these
conditions for 30 min, then quenched with Et3N (0.1 mL), and concentrated to dryness to afford crude
intermediates 42-48. The resulting residue was dissolved in dry MeOH-CH2Cl2 (2:1, v/v, 30 mL), to
which a freshly prepared solution of NaOMe in MeOH solution (1.0 mol/L, 0.45 mL) was added. The
solution was stirred at rt for 5 h and then neutralized with Dowex H+ resin to pH 7 and filtered. The
filtrate was concentrated and purified on a Sephadex LH-20 column using MeOH as the eluent to
afford compound 1-6.

ACCEPTED MANUSCRIPT
5.1.12.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D
-glucopyranosyl-(1→4)]-α- -arabinopyranosyl]oleanolic acid
L
28-O-[α-
L-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] ester (1)
Following the general procedure mentioned above to get compound 1 (0.52 g, 84% over two steps)
as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 226 – 228 °C; [α]2D0 –21.74 (c 1.15,
Ⅳ
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.40 – 6.45 (m, 16H. OH), 6.27 (d, J = 8.1 Hz, 1H, H-1 ),
Ⅱ
Ⅵ
6.20 (br s, 1H, H-1 ), 5.93 (d, J = 9.0 Hz, 1H, OH), 5.88 (br s, 1H, H-1 ), 5.42 (br s, 1H, H-12), 5.16
Ⅲ
Ⅴ
Ⅰ
(d, J = 7.8 Hz, 1H, H-1 ), 4.99 (d-like, J = 6.0 Hz, 1H, H-1 ), 4.78 (d, J = 6.0 Hz, 1H, H-1 ), 4.75 (s,
1H), 4.71 – 4.62 (m, 4H), 4.57 (dd, J = 9.1, 2.5 Hz, 1H), 4.58 – 4.52 (m, 2H), 4.47 – 4.38 (m, 3H), 4.38
– 4.25 (m, 8H), 4.25 – 4.20 (m, 3H), 4.19 – 4.09 (m, 4H), 4.06 (t, J = 8.4 Hz, 1H), 3.96 (t, J = 8.4 Hz,
1H), 3.93 – 3.90 (m, 1H), 3.82 (d, J = 10.8 Hz, 1H), 3.68 (d, J = 9.4 Hz, 1H), 3.26 – 3.16 (m, 2H), 2.32
Ⅵ
(td, J = 13.6, 2.6 Hz, 1H), 2.11 – 2.00 (m, 2H), 1.99 – 1.74 (m, 7H), 1.72 (d, J = 6.1 Hz, 3H, H-6 ),
Ⅱ
1.66 (d, J = 6.1 Hz, 3H, H-6 ), 1.25, 1.18, 1.13, 1.11, 0.90, 0.90, 0.89 (s each, 3H each, 7 × CH3); 13C
Ⅲ
NMR (150 MHz, C5D5N) δ 176.53 (C-28), 144.13 (C-13), 122.88 (C-12), 106.42 (C-1 ), 104.95 (C-1
Ⅰ
Ⅴ
Ⅵ
Ⅱ
Ⅳ
), 104.90 (C-1 ), 102.76 (C-1 ), 101.80 (C-1 ), 95.67 (C-1 ), 88.69, 79.67, 78.81, 78.76, 78.56, 78.23,
78.08, 77.19, 76.52, 76.35, 75.50, 75.37, 74.09, 74.02, 73.90, 72.79, 72.60, 72.51, 72.32, 71.27, 70.87,
70.32, 69.84, 69.22, 64.53, 62.55, 61.29, 56.00, 48.07, 47.05, 46.24, 42.16, 41.68, 39.90, 39.50, 38.93,
37.03, 34.01, 33.14, 32.56, 30.77, 28.29, 28.07, 26.63, 26.07, 23.81, 23.70, 23.38, 18.67, 18.55, 17.51,
17.04, 15.68; HRMS(ESI): calcd. for [M+Na]+ C65H106O30Na: 1389.6661, found 1389.6677.
5.1.13.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[α-
L
-arabinopyranosyl-(1→4)]-α-
L
-arabinopyranosyl]oleanolic
-glucopyranosyl] ester (2)
acid 28-O-[α-
L
-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D
Following the general procedure mentioned above to get compound 2 (0.49 g, 82% over two steps)
as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 230 – 233 °C; [α]2D0 –51.49 (c 1.34,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.09 (s, 1H, OH), 7.54 – 7.52 (m, 2H, OH), 7.44 (d, J = 4.3
Hz, 1H, OH), 7.36 (d, J = 4.8 Hz, 1H, OH), 6.86 (d, J = 3.0 Hz, 2H, OH), 6.83 (d, J = 4.4 Hz, 1H, OH),
6.81 – 6.77 (m, 1H, OH), 6.76 (d, J = 4.1 Hz, 1H, OH), 6.71 (d, J = 4.5 Hz, 1H, OH), 6.67 (d, J = 3.6
Ⅳ
Ⅱ
Hz, 1H, OH), 6.54 – 6.51 (m, 3H, OH), 6.27 (d, J = 8.1 Hz, 1H, H-1 ), 6.16 (br s, 1H, H-1 ), 5.90 (br s,
Ⅵ
Ⅴ
1H, H-1 ), 5.73 (d, J = 8.8 Hz, 1H, OH), 5.41 (s, 1H, H-12), 5.04 – 5.00 (m, 2H, H-1 ), 4.93 (d, J = 7.4
Ⅲ
Ⅰ
Hz, 1H, H-1 ), 4.81 (d, J = 5.8 Hz, 1H, H-1 ), 4.75 (br s, 1H), 4.71 – 4.69 (m, 2H), 4.67 – 4.63 (m,
2H), 4.60 – 4.57 (m, 1H), 4.53 – 4.43 (m, 4H), 4.40 – 4.31 (m, 4H), 4.31 – 4.25 (m, 4H), 4.25 – 4.20
(m, 2H), 4.20 – 4.14 (m, 2H), 4.14 – 4.08 (m, 3H), 3.99 – 3.95 (m, 8.4 Hz, 1H), 3.87 (d, J = 11.0 Hz,
Ⅵ
1H), 3.72 – 3.67 (m, 2H), 3.25 – 3.18 (m, 2H), 2.32 (t, J = 12.6 Hz, 1H), 1.73 (d, J = 6.1 Hz, 3H, H-6 ),
Ⅱ
1.65 (d, J = 6.0 Hz, 3H, H-6 ), 1.25, 1.19, 1.13, 1.10, 0.90, 0.90, 0.88 (s each, 3H each, 7 × CH3); 13C
Ⅲ
NMR (150 MHz, C5D5N) δ 176.53 (C-28), 144.08 (C-13), 122.88 (C-12), 106.91 (C-1 ), 104.93 (C-1
Ⅰ
Ⅴ
Ⅵ
Ⅱ
Ⅳ
), 104.88 (C-1 ), 102.74 (C-1 ), 101.74 (C-1 ), 95.66 (C-1 ), 88.70, 79.13, 78.74, 78.18, 78.06, 77.17,
76.50, 76.33, 75.36, 74.78, 74.05, 74.01, 73.95, 73.88, 72.78, 72.58, 72.50, 72.31, 70.82, 70.31, 69.82,
69.79, 69.18, 67.47, 64.53, 61.25, 55.99, 49.68, 48.06, 47.03, 46.21, 42.13, 41.66, 39.88, 39.50, 38.92,
37.02, 33.99, 33.14, 32.53, 30.76, 29.98, 28.27, 28.06, 26.63, 26.06, 23.79, 23.68, 23.36, 18.65, 18.55,
17.50, 17.02, 15.67; HRMS(ESI): calcd. for [M+Na]+ C64H104O29Na: 1359.6555, found 1359.6563.
5.1.14.
3-O-[α-
acid 28-O-[α-
Following the general procedure mentioned above to get compound 3 (0.51 g, 84% over for two
L
-rhamnopyranosyl-(1→2)-[α-
L
-rhamnopyranosyl-(1→4)]-α-
L
-arabinopyranosyl]oleanolic
L
-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-
D-glucopyranosyl] ester (3)

ACCEPTED MANUSCRIPT
steps) as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 211 – 214 °C; [α]2D0 –36.76 (c 1.36,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 7.54 – 7.36 (m, 4H, OH), 6.89 – 6.29 (m, 12H, OH), 6.27 (d, J
Ⅳ
Ⅱ
Ⅵ
Ⅲ
= 8.1 Hz, 1H, H-1 ), 6.03 (br s, 1H, H-1 ), 5.90 (br s, 1H, H-1 ), 5.77 (br s, 1H, H-1 ), 5.41 (s, 1H,
Ⅴ
Ⅰ
H-12), 5.03 – 5.00 (m, 2H, H-1 ), 4.92 (d, J = 4.3 Hz, 1H, H-1 ), 4.76 (s, 1H), 4.71 – 4.69 (m, 2H),
4.65 (d, J = 4.3 Hz, 1H), 4.63 – 4.56 (m, 3H), 4.55 – 4.50 (m, 2H), 4.48 – 4.45 (m, 2H), 4.43 – 4.37 (m,
3H), 4.37 – 4.32 (m, 4H), 4.31 – 4.20 (m, 3H), 4.20 – 4.05 (m, 5H), 3.97 (t, J = 6.9 Hz, 1H), 3.89 (d, J
= 10.1 Hz, 1H), 3.68 (d, J = 9.4 Hz, 1H), 3.62 (d, J = 4.2 Hz, 1H), 3.20 (t, J = 16.7 Hz, 2H), 2.32 (t, J =
Ⅵ
Ⅱ
12.5 Hz, 1H), 1.73 (d, J = 6.1 Hz, 3H, H-6 ), 1.69 (d, J = 6.0 Hz, 3H, H-6 ), 1.64 (d, J = 6.1 Hz, 3H),
1.26, 1.17, 1.11, 1.09, 0.90, 0.90, 0.87 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ
Ⅰ
Ⅴ
Ⅵ
176.52 (C-28), 144.09 (C-13), 122.85 (C-12), 104.85 (C-1 ), 104.81 (C-1 ), 102.73 (C-1 ), 102.52
Ⅲ
Ⅱ
Ⅳ
(C-1 ), 101.98 (C-1 ), 95.63 (C-1 ), 89.01, 78.72, 78.17, 78.04, 77.16, 76.49, 76.32, 75.34, 74.94,
74.02, 73.86, 72.76, 72.57, 72.29, 72.16, 72.01, 70.80, 70.30, 70.27, 70.12, 69.16, 62.90, 61.24, 55.88,
49.68, 48.04, 47.03, 46.21, 42.13, 41.65, 39.87, 39.48, 38.86, 37.00, 33.98, 33.14, 32.52, 30.75, 29.97,
28.26, 28.03, 26.50, 26.05, 23.78, 23.68, 23.36, 18.71, 18.56, 18.54, 17.47, 16.90, 15.62; HRMS(ESI):
calcd. for [M+Na]+ C65H106O29Na: 1373.6712, found 1373.6672.
5.1.15.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D
-galactopyranosyl-(1→4)]-α-
L
-arabinopyranosyl]oleanolic
-glucopyranosyl] ester (4)
acid 28-O-[α-
L
-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D
Following the general procedure mentioned above to get compound 4 (0.52 g, 84% over two steps)
as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 228 – 231 °C; [α]2D0 –43.52 (c 2.16,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.18 (d, J = 3.0 Hz, 1H, OH), 7.55 – 7.32 (m, 4H, OH), 6.88 –
Ⅳ
Ⅱ
6.66 (m, 8H, OH), 6.54 – 6.51 (m, 3H, OH), 6.27 (d, J = 8.1 Hz, 1H, H-1 ), 6.17 (br s, 1H, H-1 ), 5.90
Ⅵ
Ⅲ
(br s, 1H, H-1 ), 5.81 (d, J = 8.9 Hz, 1H, OH), 5.42 (s, 1H, H-12), 5.08 (d-like, J = 8.9 Hz, 1H, H-1 ),
Ⅴ
Ⅰ
5.03 – 5.01 (m, 2H, H-1 ), 4.78 (d, J = 5.9 Hz, 1H, H-1 ), 4.74 (s, 1H), 4.71 – 4.69 (m, 2H), 4.68 –
4.61 (m, 2H), 4.61 – 4.52 (m, 3H), 4.51 – 4.48 (m, 2H), 4.47 – 4.43 (m, 3H), 4.39 – 4.31 (m, 4H), 4.30
– 4.28 (m, 2H), 4.26 – 4.18 (m, 3H), 4.18 – 4.10 (m, 5H), 4.07 (t, J = 5.8 Hz, 1H), 3.97 (td, J = 8.4, 4.6
Hz, 1H), 3.83 (d, J = 11.1 Hz, 1H), 3.68 (d, J = 9.5 Hz, 1H), 3.25 – 3.16 (m, 2H), 2.32 (t, J = 11.7 Hz,
Ⅵ
Ⅱ
1H), 1.73 (d, J = 6.2 Hz, 3H, H-6 ), 1.65 (d, J = 6.1 Hz, 3H, H-6 ), 1.25, 1.18, 1.14, 1.11, 0.90, 0.90,
0.89 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ 176.53 (C-28), 144.11 (C-13), 122.89
Ⅲ
Ⅰ
Ⅴ
Ⅵ
Ⅱ
Ⅳ
(C-12), 107.06 (C-1 ), 104.92 (C-1 , C-1 ), 102.77 (C-1 ), 101.74 (C-1 ), 95.68 (C-1 ), 88.68, 79.56,
78.78, 78.21, 78.09, 77.24, 77.20, 76.53, 76.36, 75.45, 75.38, 74.09, 74.04, 73.90, 73.05, 72.80, 72.61,
72.52, 72.33, 70.87, 70.36, 70.33, 69.81, 69.23, 64.62, 62.36, 61.28, 56.00, 49.69, 48.06, 47.05, 46.23,
42.15, 41.68, 39.89, 39.50, 38.92, 37.03, 34.01, 33.15, 32.56, 30.77, 30.00, 28.29, 28.07, 26.64, 26.07,
23.81, 23.70, 23.38, 18.67, 18.56, 17.52, 17.04, 15.68; HRMS(ESI): calcd. for [M+Na]+ C65H106O30Na:
1389.6661, found 1389.6717.
5.1.16.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D
-xylopyranosyl-(1→4)]-α- -arabinopyranosyl]oleanolic acid
L
28-O-[α-
L-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] ester (5)
Following the general procedure mentioned above to get compound 5 (0.51 g, 84% over two steps)
as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 228 – 232 °C; [α]2D0 –33.55 (c 1.52,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.34 (d, J = 3.8 Hz, 1H, OH), 7.54 – 7.34 (m, 5H, OH), 7.20
Ⅳ
Ⅱ
(d, J = 4.5 Hz, 1H, OH), 6.85 – 6.54 (m, 8H, OH), 6.27 (d, J = 8.1 Hz, 1H, H-1 ), 6.19 (br s, 1H, H-1 ),
Ⅵ
Ⅲ
5.89 (br s, 1H, H-1 ), 5.80 (d, J = 9.0 Hz, 1H, OH), 5.41 (s, 1H, H-12), 5.03 – 4.99 (m, 3H, H-1 , H-1
Ⅴ
Ⅰ
), 4.81 (d, J = 5.8 Hz, 1H, H-1 ), 4.76 (s, 1H), 4.72 – 4.68 (m, 2H), 4.68 – 4.62 (m, 2H), 4.61 – 4.56

ACCEPTED MANUSCRIPT
(m, 1H), 4.54 – 4.51 (m, 1H), 4.50 – 4.44 (m, 2H), 4.38 – 4.31 (m, 6H), 4.30 – 4.28 (m, 1H), 4.25 –
4.20 (m, 3H), 4.20 – 4.15 (m, 2H), 4.15 – 4.08 (m, 4H), 4.03 (td, J = 8.4, 4.1 Hz, 1H), 3.97 (td, J = 8.1,
4.4 Hz, 1H), 3.89 (d, J = 10.7 Hz, 1H), 3.69 – 3.63 (m, 2H), 3.21 (ddd, J = 16.2, 12.3, 3.3 Hz, 2H), 2.32
Ⅵ
Ⅱ
(t, J = 11.5 Hz, 1H), 1.73 (d, J = 6.2 Hz, 3H, H-6 ), 1.66 (d, J = 6.1 Hz, 3H, H-6 ), 1.25, 1.18, 1.12,
1.10, 0.90, 0.89, 0.88 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ 176.56 (C-28),
Ⅲ
Ⅰ
Ⅴ
Ⅵ
Ⅱ
144.13 (C-13), 122.92 (C-12), 106.96 (C-1 ), 104.94 (C-1 , C-1 ), 102.80 (C-1 ), 101.84 (C-1 ),
Ⅳ
95.70 (C-1 ), 88.73, 79.33, 78.80, 78.66, 78.23, 78.11, 77.23, 76.55, 76.37, 75.41, 75.24, 74.11, 74.06,
73.93, 72.83, 72.64, 72.55, 72.37, 71.01, 70.89, 70.36, 69.90, 69.25, 67.47, 64.55, 61.31, 56.02, 49.72,
48.09, 47.08, 46.25, 42.18, 41.71, 39.92, 39.53, 38.96, 37.05, 34.03, 33.18, 32.58, 30.80, 28.32, 28.11,
26.64, 26.10, 23.83, 23.72, 23.41, 18.70, 18.59, 17.54, 17.07, 15.69; HRMS(ESI): calcd. for [M+Na]+
C64H104O29Na: 1359.6555, found 1359.6449.
5.1.17.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D
-ribofuranosyl-(1→4)]-α- -arabinopyranosyl]oleanolic acid
L
28-O-[α-
L-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] ester (6)
Following the general procedure mentioned above to get compound 6 (0.47 g, 79% over two steps)
as a white foam. Rf = 0.24 (4:1:5, n-BuOH-AcOH-H2O); Mp 220 – 223 °C; [α]2D0 –72.32 (c 1.12,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 7.53 – 7.30 (m, 4H, OH), 7.00 – 6.47 (m, 11H), 6.27 (d, J =
Ⅳ
Ⅱ
Ⅵ
7.9 Hz, 1H, H-1 ), 6.17 (s, 1H, OH), 6.09 (s, 1H, H-1 ), 5.90 (s, 1H, H-1 ), 5.65 (d, J = 2.4 Hz, 1H,
Ⅲ
Ⅴ
Ⅰ
H-1 ), 5.41 (s, 1H, H-12), 5.01 (d, J = 7.6 Hz, 2H, H-1 ), 4.87 (d, J = 3.4 Hz, 1H, H-1 ), 4.74 (s, 1H),
4.71 – 4.68 (m, 2H), 4.67 – 4.60 (m, 2H), 4.59 (d, J = 9.1 Hz, 1H), 4.54 – 4.51 (m, 1H), 4.50 – 4.43 (m,
3H), 4.41 – 4.28 (m, 7H), 4.27 – 4.06 (m, 11H), 3.99 – 3.95 (m, 1H), 3.88 (d, J = 10.2 Hz, 1H), 3.68 (d,
J = 8.6 Hz, 1H), 3.22 (ddd, J = 15.3, 11.5, 1.9 Hz, 2H), 2.32 (t, J = 13.7 Hz, 1H), 1.73 (d, J = 5.6 Hz,
Ⅵ
Ⅱ
3H, H-6 ), 1.67 (d, J = 5.5 Hz, 3H, H-6 ), 1.25, 1.19, 1.11, 1.11, 0.90, 0.90, 0.89 (s each, 3H each, 7 ×
Ⅰ
CH3); 13C NMR (150 MHz, C5D5N) δ 176.53 (C-28), 144.11 (C-13), 122.87 (C-12), 104.91 (C-1 , C-1
Ⅴ
Ⅲ
Ⅵ
Ⅱ
Ⅳ
), 103.57 (C-1 ), 102.77 (C-1 ), 101.97 (C-1 ), 95.67 (C-1 ), 88.85, 78.77, 78.19, 78.08, 77.20, 77.02,
76.52, 76.36, 75.38, 74.04, 73.90, 72.88, 72.81, 72.61, 72.55, 72.52, 72.30, 70.87, 70.33, 70.08, 70.03,
69.22, 65.33, 61.28, 55.95, 48.07, 47.05, 46.23, 42.15, 41.68, 39.89, 39.51, 38.92, 37.03, 34.00, 33.15,
32.55, 30.77, 30.00, 28.29, 28.07, 26.60, 26.07, 23.80, 23.69, 23.38, 18.68, 18.57, 17.50, 16.96, 15.66;
HRMS(ESI): calcd. for [M+Na]+ C64H104O29Na: 1359.6555, found 1359.6423.
5.1.18.
3-O-α-
L
-arabinopyranosyl-oleanolic
-glucopyranosyl] ester (7)
acid
28-O-[α-
L
-rhamnopyranosyl-(1→4)-β- -glucopyranosyl-(1→6)-β-D
D
Compound 7 was synthesized from 49 (0.41 g, 0.45 mmol) by the same procedure as for
compound 1-6 and purified on a Sephadex LH-20 column using MeOH as the eluent. (0.42 g, 89%
over two steps), white foam; Rf = 0.36 (4:1:5, n-BuOH-AcOH-H2O); Mp 186 – 189 °C; [α]2D0 –27.50 (c
1
1.12, CH3OH); H NMR (600 MHz, C5D5N) δ 7.51 (d, J = 5.6 Hz, 1H, OH), 7.49 (d, J = 4.5 Hz, 1H,
OH), 7.39 (d, J = 4.5 Hz, 1H, OH), 7.33 (d, J = 5.0 Hz, 1H, OH), 6.97 (d, J = 4.9 Hz, 1H, OH), 6.82 (d,
J = 4.3 Hz, 1H, OH), 6.79 (d, J = 5.0 Hz, 1H, OH), 6.74 (dd, J = 7.4, 5.3 Hz, 1H, OH), 6.66 (d, J = 4.6
Hz, 1H, OH), 6.58 (d, J = 5.7 Hz, 1H, OH), 6.50 (d, J = 6.4 Hz, 1H, OH), 6.31 (d, J = 4.0 Hz, 1H, OH),
Ⅳ
Ⅵ
Ⅴ
6.27 (d, J = 8.1 Hz, 1H, H-1 ), 5.89 (s, 1H, H-1 ), 5.42 (s, 1H, H-12), 5.00 (d, J = 5.4 Hz, 1H, H-1 ),
Ⅰ
4.78 (d, J = 7.1 Hz, 1H, H-1 ), 4.70 – 4.68 (m, 2H), 4.60 – 4.55 (m, 1H), 4.47 – 4.44 (m, 2H), 4.38 –
4.31 (m, 5H), 4.26 – 4.20 (m, 2H), 4.19 – 4.14 (m, 3H), 4.13 – 4.09 (m, 2H), 3.96 (td, J = 8.5, 4.5 Hz,
1H), 3.85 (d, J = 10.9 Hz, 1H), 3.68 (d, J = 9.5 Hz, 1H), 3.35 (dd, J = 11.7, 4.2 Hz, 1H), 3.20 (dd, J =
Ⅵ
13.6, 3.8 Hz, 1H), 1.72 (d, J = 6.1 Hz, 3H, H-6 ), 1.28, 1.25, 1.11, 0.98, 0.90, 0.90, 0.90 (s each, 3H
each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ 176.52 (C-28), 144.12 (C-13), 122.90 (C-12), 107.47

ACCEPTED MANUSCRIPT
Ⅰ
Ⅴ
Ⅵ
Ⅳ
(C-1 ), 104.87 (C-1 ), 102.76 (C-1 ), 95.65 (C-1 ), 88.70, 78.75, 78.33, 78.05, 77.16, 76.53, 75.35,
74.65, 74.01, 73.88, 72.93, 72.77, 72.57, 70.92, 70.32, 69.52, 69.25, 66.74, 61.33, 55.91, 49.68, 48.10,
47.06, 46.25, 42.15, 41.70, 39.93, 39.57, 38.84, 37.06, 34.02, 33.14, 32.56, 30.77, 28.30, 28.25, 26.65,
26.08, 23.83, 23.72, 23.38, 18.52, 17.53, 16.94, 15.63; HRMS(ESI): calcd. for [M+H]+ C53H87O21:
1059.5734, found 1059.5745.
5.1.19.
Oleanolic
acid
3-O-2,3,4-tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→2)-3,4-O-isopropylidene-α-
L
-arabinopyranoside (51)
A suspension of 16 (0.60 g, 0.61 mmol) and 10% Pd-C (0.18 g) in EtOAc (30 mL) was refluxed
and bubbled up with H2 (70 mL/min) for 1.5 h. Pd-C was removed through filtration and the filtrate
was concentrated to dryness and purified by a silica gel column chromatography (2:1 petroleum ether –
EtOAc) to afford 51 (0.47 g, 87%) as a white foam. Rf = 0.34 (2:1, petroleum ether – EtOAc); Mp 144
– 148 °C; [α]2D0 –24.75 (c 1.01, CH3OH); H NMR (600 MHz, CDCl3) δ 7.27 (s, 1H), 5.36 – 5.31 (m,
1
3H), 5.29 – 5.27 (m, 2H), 5.05 (t, J = 9.9 Hz, 1H), 4.36 (d, J = 7.5 Hz, 1H), 4.22 – 4.18 (m, 2H), 4.17 –
4.14 (m, 1H), 4.11 (dd, J = 10.4, 2.7 Hz, 1H), 3.78 – 3.73 (m, 2H), 3.10 (dd, J = 11.8, 4.3 Hz, 1H), 2.82
(dd, J = 13.7, 3.9 Hz, 1H), 2.15 (s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.93 – 1.54 (m, 14H), 1.53 (s, 3H),
1.33 (s, 3H), 1.20 (d, J = 6.2 Hz, 3H), 1.13 (s, 3H), 1.06 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H),
0.82 (s, 3H), 0.74 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 183.11, 170.17, 169.99, 169.94, 143.55,
122.66, 110.38, 103.14, 95.25, 89.05, 79.11, 75.20, 73.35, 71.28, 69.63, 69.09, 66.23, 62.61, 55.88,
47.67, 46.55, 45.88, 41.67, 41.09, 39.33, 39.08, 38.74, 36.77, 33.83, 33.07, 32.66, 32.46, 30.68, 28.05,
27.78, 27.64, 26.10, 25.92, 23.58, 23.42, 22.95, 20.97, 20.81, 20.72, 18.23, 17.36, 16.92, 16.35, 15.40;
HRMS(ESI): calcd. for [M+Na]+ C50H76O14Na: 923.5127, found 923.5116.
5.1.20.
3-O-[2,3,4-tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→2)-3,4-O-isopropylidene-α-
L
-arabinopyranoside]ol
acid
eanolic
28-O-2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranosyl-(1→4)-2,3-di-O-acetyl-6-O-benzoyl-β-
-glucopyranosyl ester (52)
D-glucopyranos
yl-(1→6)-2,3,4-tri-O-acetyl-β-
D
A mixture of compounds 51 (0.47 g, 0.53 mmol), 36 (0.68 g, 0.64 mmol) and 4 Å molecular
sieves in dry CH2Cl2 (30 mL) were stirred under an atmosphere of N2 for 30 min at 0 °C. A solution of
TMSOTf (0.16 mL, 0.34 mmol/mL) in dry CH2Cl2 was injected. The mixture was stirred under these
conditions for 30 min, then quenched with Et3N (0.1 mL), and concentrated to dryness to afford crude
intermediates 52. The residue was purified by a silica gel column chromatography (5:2 petroleum ether
– EtOAc) to give compound 52 (0.83 g, 87%) as a white foam. Rf = 0.3 (3:2, petroleum ether – EtOAc);
Mp 154 – 156 °C; [α]2D0 –21.67 (c 2.18, CH3OH); 1H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 7.2 Hz,
2H), 7.58 (t, J = 7.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 2H), 5.54 (d, J = 8.3 Hz, 1H), 5.36 – 5.34 (m, 2H),
5.31 (dd, J = 3.5, 1.7 Hz, 1H), 5.30 – 5.29 (m, 1H), 5.22 – 5.17 (m, 3H), 5.13 – 5.09 (m, 2H), 5.05 (t, J
= 8.1 Hz, 1H), 5.02 (t, J = 8.0 Hz, 1H), 4.95 (t, J = 9.7 Hz, 1H), 4.87 – 4.82 (m, 2H), 4.77 (d, J = 10.6
Hz, 1H), 4.55 (d, J = 7.9 Hz, 1H), 4.48 (dd, J = 12.3, 4.3 Hz, 1H), 4.36 (d, J = 7.5 Hz, 1H), 4.22 – 4.18
(m, 2H), 4.18 – 4.14 (m, 1H), 4.14 – 4.10 (m, 1H), 3.90 (t, J = 9.3 Hz, 1H), 3.87 – 3.82 (m, 2H), 3.79 –
3.73 (m, 3H), 3.69 (ddd, J = 9.7, 4.0, 1.9 Hz, 1H), 3.56 (dd, J = 11.5, 5.6 Hz, 1H), 3.09 (dd, J = 11.7,
4.3 Hz, 1H), 2.78 (dd, J = 13.5, 3.8 Hz, 1H), 2.15 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.06 (s, 3H), 2.03
(s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.97 (s, 6H), 1.96 (s, 3H), 1.92 – 1.56 (m, 11H), 1.53 (s,
3H), 1.51 – 1.35 (m, 4H), 1.33 (s, 3H), 1.32 – 1.24 (m, 5H), 1.20 (d, J = 6.3 Hz, 3H), 1.15 (d, J = 6.2
Hz, 3H), 1.11 (s, 3H), 1.05 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H), 0.73 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 175.27, 170.16, 170.13, 170.07, 169.99, 169.92, 169.85, 169.38, 168.90,

ACCEPTED MANUSCRIPT
165.79, 142.86, 133.13, 132.33, 130.91, 129.81, 129.77, 128.85, 128.44, 122.85, 110.36, 103.18,
100.28, 99.08, 95.15, 91.47, 88.99, 79.15, 75.02, 73.99, 73.83, 73.39, 72.91, 72.87, 71.80, 71.27, 70.70,
69.99, 69.60, 69.07, 68.80, 68.52, 67.76, 67.60, 66.19, 65.56, 62.66, 62.36, 55.84, 53.43, 47.62, 46.72,
45.81, 41.72, 41.06, 39.30, 39.07, 38.75, 36.76, 33.72, 32.95, 31.69, 30.56, 29.70, 28.01, 27.85, 27.78,
25.57, 23.49, 23.41, 22.83, 21.03, 20.98, 20.79, 20.72, 20.69, 20.65, 20.59, 20.54, 19.19, 18.19, 17.35,
17.21, 16.99, 16.40, 15.47, 13.73; HRMS(ESI): calcd. for [M+H]+ C91H127O37: 1811.8051, found
1811.8015.
5.1.21.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-α-
L
-arabinopyranosyl]oleanolic
-glucopyranosyl] ester (8)
acid
28-O-[α-
L
-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl-(1→6)-β-D
P-TsOH (0.15 g, 0.90 mmol) was added to a solution of 52 (0.81 g, 0.45 mmol) in
MeOH-CH2Cl2 (2:1, v/v, 30 mL). The mixture was stirred at 40 °C for 6 h followed by addition of
NaOMe in MeOH solution (1.0 mol/L, 1.35 mL). The mixture was stirred at rt for 5 h and then
neutralized with Dowex H+ resin to pH 7 and filtered. The filtrate was concentrated and purified on a
Sephadex LH-20 column using MeOH as the eluent to afford compound 8 (0.43 g, 79% over two steps)
as a white foam. Rf = 0.34 (4:1:5, n-BuOH-AcOH-H2O); Mp 168 – 172 °C; [α]2D0 –21.37 (c 1.17,
Ⅳ
Ⅱ
CH3OH); 1H NMR (600 MHz, C5D5N) δ 6.28 (d, J = 8.2 Hz, 1H, H-1 ), 6.20 (s, 1H, H-1 ), 5.91 (s, 1H,
Ⅵ
Ⅴ
Ⅰ
H-1 ), 5.42 (s, 1H, H-12), 5.04 – 5.01 (m, 3H, H-1 ), 4.91 (d, J = 5.4 Hz, 1H, H-1 ), 4.79 (d, J = 1.8
Hz, 1H), 4.73 – 4.65 (m, 3H), 4.65 – 4.57 (m, 3H), 4.47 (t, J = 9.3 Hz, 1H), 4.40 – 4.27 (m, 7H), 4.26 –
4.10 (m, 6H), 3.97 (t, J = 8.4 Hz, 1H), 3.85 (d, J = 9.9 Hz, 1H), 3.68 (d, J = 9.4 Hz, 1H), 3.26 (dd, J =
11.6, 4.1 Hz, 1H), 3.19 (dd, J = 13.9, 3.8 Hz, 1H), 2.33 (t, J = 12.6 Hz, 1H), 1.73 (d, J = 6.2 Hz, 3H,
Ⅵ
Ⅱ
H-6 ), 1.65 (d, J = 6.1 Hz, 3H, H-6 ), 1.26, 1.18, 1.11, 1.10, 0.91, 0.90, 0.89 (s each, 3H each, 7 ×
Ⅴ
CH3); 13C NMR (150 MHz, C5D5N) δ 176.53 (C-28), 144.12 (C-13), 122.85 (C-12), 104.83 (C-1 ),
Ⅰ
Ⅵ
Ⅱ
Ⅳ
104.76 (C-1 ), 102.73 (C-1 ), 101.73 (C-1 ), 95.65 (C-1 ), 88.78, 78.72, 78.30, 78.05, 77.14, 76.51,
75.94, 75.33, 74.05, 73.98, 73.87, 73.68, 72.74, 72.57, 72.55, 72.36, 70.89, 70.30, 69.89, 69.23, 68.59,
64.58, 61.31, 55.93, 48.07, 47.06, 46.24, 42.16, 41.68, 39.90, 39.49, 38.93, 37.04, 34.01, 33.13, 32.55,
30.76, 28.27, 28.11, 26.51, 26.06, 23.80, 23.70, 23.38, 18.56, 18.51, 17.51, 16.98, 15.64; HRMS(ESI):
calcd. for [M+Na]+ C59H96O25Na: 1227.6133, found 1227.6101.
5.1.22. General procedure for the preparation of compounds 10-12
A mixture of compounds 37 (0.6 g, 0.41 mmol), trichloroacetimidate donors (perbenzoylated
glucosyl trichloroacetimidate, 28 or 34), (0.49 mmol) and 4 Å molecular sieves in dry CH2Cl2 (30 mL)
were stirred under an atmosphere of N2 for 30 min at 0 °C. A solution of TMSOTf (0.12 mL, 0.34
mmol/mL) in dry CH2Cl2 was injected. The mixture was stirred under these conditions for 30 min, then
quenched with Et3N (0.1 mL), and concentrated to dryness to afford crude intermediates 53-55. The
resulting residue was dissolved in dry MeOH-CH2Cl2 (2:1, v/v, 30 mL), and a newly prepared solution
of NaOMe in MeOH (1.0 mol/L, 0.41 mL) was added. The solution was stirred at rt for 5 h and then
neutralized with Dowex H+ resin to pH 7 and filtered. The filtrate was concentrated and purified on a
Sephadex LH-20 column using MeOH as the eluent to afford compound 10-12.
5.1.23.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
-glucopyranosyl] ester (10)
D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl]oleanolic acid
28-O-β-
D
Following the general procedure mentioned above to get compound 10 (0.38 g, 88% over two
steps) as a white foam. Rf = 0.36 (4:1:5, n-BuOH-AcOH-H2O); Mp 217 – 220 °C; [α]2D0 –5.16 (c 1.55,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.50 (s, 1H, OH), 7.57 – 7.29 (m, 5H, OH), 6.84 – 6.48 (m,
Ⅳ
Ⅱ
5H, OH), 6.37 (d, J = 8.2 Hz, 1H, H-1 ), 6.23 (s, 1H, H-1 ), 5.99 (d, J = 9.0 Hz, 1H, OH), 5.45 (s, 1H,

ACCEPTED MANUSCRIPT
Ⅲ
Ⅰ
H-12), 5.18 (d, J = 7.8 Hz, 1H, H-1 ), 4.80 – 4.72 (m, 2H, H-1 ), 4.70 – 4.61 (m, 2H), 4.58 – 4.48 (m,
3H), 4.46 – 4.41 (m, 4H), 4.37 – 4.19 (m, 7H), 4.08 – 4.06 (m, 2H), 3.96 – 3.89 (m, 1H), 3.82 (d, J =
Ⅱ
11.1 Hz, 1H), 3.26 – 3.14 (m, 2H), 2.37 (t, J = 12.8 Hz, 1H), 1.67 (d, J = 6.1 Hz, 3H, H-6 ), 1.27, 1.19,
1.13, 1.12, 0.93, 0.90, 0.87 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ 176.45 (C-28),
Ⅲ
Ⅰ
Ⅱ
Ⅳ
144.12 (C-13), 122.88 (C-12), 106.47 (C-1 ), 104.98 (C-1 ), 101.82 (C-1 ), 95.78 (C-1 ), 88.64,
79.78, 79.40, 78.95, 78.84, 78.58, 76.36, 75.51, 74.18, 74.09, 72.52, 72.33, 71.23, 71.06, 69.84, 64.61,
62.52, 62.17, 55.97, 48.05, 47.01, 46.19, 42.15, 41.75, 39.90, 39.49, 38.91, 37.01, 34.00, 33.16, 32.55,
30.79, 28.25, 28.06, 26.64, 26.09, 23.81, 23.67, 23.42, 18.69, 18.53, 17.48, 17.04, 15.65; HRMS(ESI):
calcd. for [M+H]+ C53H87O21: 1059.5734, found 1059.5739.
5.1.24.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl]oleanolic acid
28-O-[β-
D
-glucopyranosyl-(1→6)-β- -glucopyranosyl] ester (11)
D
Following the general procedure mentioned above to get compound 11 (0.43 g, 86% over two
steps) as a white foam. Rf = 0.32 (4:1:5, n-BuOH-AcOH-H2O); Mp 215 – 217 °C; [α]2D0 –16.67 (c 1.50,
CH3OH); 1H NMR (600 MHz, C5D5N) δ 8.48 (s, 1H, OH), 7.56 – 7.29 (m, 5H, OH), 6.83 – 6.45 (m,
Ⅳ
Ⅱ
4H, OH), 6.30 (d, J = 8.1 Hz, 1H, H-1 ), 6.22 (s, 1H, H-1 ), 5.98 (d, J = 8.0 Hz, 1H, OH), 5.43 (s, 1H,
Ⅲ
Ⅴ
Ⅰ
H-12), 5.18 (d, J = 7.9 Hz, 1H, H-1 ), 5.06 (d, J = 7.8 Hz, 1H, H-1 ), 4.78 (d, J = 5.9 Hz, 1H, H-1 ),
4.76 – 4.74 (m, 2H), 4.70 – 4.60 (m, 2H), 4.57 – 4.47 (m, 3H), 4.45 – 4.37 (m, 5H), 4.34 (t, J = 9.5 Hz,
1H), 4.32 – 4.20 (m, 7H), 4.18 – 4.13 (m, 2H), 4.09 – 4.02 (m, 2H), 3.94 – 3.90 (m, 2H), 3.83 (d, J =
Ⅱ
11.0 Hz, 1H), 3.27 – 3.17 (m, 2H), 2.33 (t, J = 11.9 Hz, 1H), 1.67 (d, J = 6.1 Hz, 3H, H-6 ), 1.26, 1.18,
1.13, 1.12, 0.90, 0.89, 0.89 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ 176.55 (C-28),
Ⅲ
Ⅴ
Ⅰ
Ⅱ
144.15 (C-13), 122.87 (C-12), 106.44 (C-1 ), 105.35 (C-1 ), 104.96 (C-1 ), 101.83 (C-1 ), 95.71 (C-1
Ⅳ
), 88.67, 79.72, 78.84, 78.79, 78.58, 78.52, 78.42, 78.02, 76.37, 75.51, 75.19, 74.09, 73.92, 72.52,
72.33, 71.48, 71.24, 70.90, 69.85, 69.38, 64.54, 62.60, 62.52, 55.98, 48.06, 47.05, 46.24, 42.16, 41.69,
39.89, 39.49, 38.90, 37.02, 33.98, 33.14, 32.54, 30.78, 30.00, 28.28, 28.06, 26.63, 26.08, 23.81, 23.68,
23.39, 18.69, 18.54, 17.51, 17.04, 15.70; HRMS(ESI): calcd. for [M+H]+ C59H97O26: 1221.6263, found
1221.6290.
5.1.25.
3-O-[α-
L
-rhamnopyranosyl-(1→2)-[β-
D-glucopyranosyl-(1→4)]-α-
L-arabinopyranosyl]oleanolic acid
28-O-[α-
L-rhamnopyranosyl-(1→4)-β-
D-glucopyranosyl] ester (12)
Following the general procedure mentioned above to get compound 12 (0.42 g, 86% over two
steps) as a white foam. Rf = 0.34 (4:1:5, n-BuOH-AcOH-H2O); Mp 162 – 165 °C; [α]2D0 –37.50 (c 1.44,
Ⅳ
Ⅱ
CH3OH); 1H NMR (600 MHz, C5D5N) δ 6.28 (d, J = 8.2 Hz, 1H, H-1 ), 6.22 (s, 1H, H-1 ), 5.95 (s,
Ⅵ
Ⅲ
1H, H-1 ), 5.43 (s, 1H, H-12), 5.18 (d, J = 7.8 Hz, 1H, H-1 ), 5.02 – 4.97 (m, 2H), 4.79 (d, J = 6.0 Hz,
Ⅰ
1H, H-1 ), 4.77 (s, 1H), 4.73 – 4.64 (m, 4H), 4.60 – 4.52 (m, 5H), 4.44 – 4.33 (m, 5H), 4.32 – 4.20 (m,
7H), 4.20 – 4.09 (m, 3H), 4.07 (t, J = 8.5 Hz, 1H), 3.95 – 3.91 (m, 1H), 3.84 – 3.83 (m, 1H), 3.21 (t, J =
Ⅵ
Ⅱ
12.5 Hz, 2H), 2.32 (t, J = 12.5 Hz, 1H), 1.72 (d, J = 6.2 Hz, 3H, H-6 ), 1.67 (d, J = 6.1 Hz, 3H, H-6 ),
1.26, 1.19, 1.13, 1.09, 0.93, 0.90, 0.86 (s each, 3H each, 7 × CH3); 13C NMR (150 MHz, C5D5N) δ
Ⅲ
Ⅰ
Ⅵ
176.40 (C-28), 144.05 (C-13), 122.91 (C-12), 106.29 (C-1 ), 104.86 (C-1 ), 102.68 (C-1 ), 101.77
Ⅱ
Ⅳ
(C-1 ), 95.41 (C-1 ), 88.69, 79.45, 78.71, 78.49, 77.89, 77.08, 76.41, 75.46, 74.23, 74.03, 73.94 73.89,
73.83, 72.73, 72.66, 72.54, 72.47, 72.26, 71.31, 70.41, 69.83, 65.64, 64.39, 62.55, 61.02, 55.98, 48.05,
47.01, 46.18, 42.15, 41.74, 39.90, 39.48, 38.93, 37.02, 34.00, 33.12, 32.54, 30.76, 28.24, 28.09, 26.59,
26.07, 23.79, 23.64, 23.37, 18.61, 18.52, 17.44, 17.00, 15.62; HRMS(ESI): calcd. for [M+H]+
C59H97O25: 1205.6313, found 1205.6294.

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5.2. Biology
5.2.1. Cell culture and treatments
PC12 cells were purchased from Jiangsu Keygen Biotech Company, China, grown in Roswell
Park Memorial Institute-1640 (Gibico), supplemented with 15% fetal bovine serum and 1% antibiotic
mixture comprising penicilline and streptomycin (100 U/mL of penicillin and 100 µg/mL of
streptomycin), in a humidified atmosphere at 37 °C with 5% CO2. Cells were sub-cultured every 2 days
and in culture for 8 days before being used for experiments.
5.2.2. Cell viability assay
Cells were trypsinized with 0.25% trypsin, counted, and seeded in 96-well culture plates (1 × 104
cells/well). After 12 h of incubation, the cells were pretreated with compounds in different
concentrations for 12 h before incubation in medium containing H2O2 (200 µM) or Aβ1-42 (40 µM).
After 12 h of treatment, 20 µL of MTT (5 mg/mL) was added. After incubation for 4 h the supernatant
was discarded, and DMSO (150 µL/well) was added. The 96-well plate was vibrated on a
microvibrator for 10 min, and the optical density at 490 nm was measured using a microplate reader.
5.2.3. LDH release assay
The amount of LDH release was determined by using an assay kit according to the manufacturer’s
protocol. At the end of drug treatment, the supernatant and cell lysates were transferred to 96-well
plates and incubated with 1mg/ml NADH in pyruvate substrate solution for 15 min at 37 °C. After
additional incubation for 15 min with 2,4-dinitrophenylhydrazine at 37 °C, the reaction was stopped by
adding 0.4 mol/L NaOH. The changes in absorbance were determined at 450 nm by using a microplate
reader. LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the
supernatant + LDH in the cell lysate), according to the equation: %LDH released = (LDH activity in
the medium/total LDH activity) × 100%.
5.2.4. Measurement of intracellular ROS level
ROS level was measured by using DCFH-DA method [29]. After the treatment, PC12 cells were
washed with PBS and incubated with DCFH-DA at a final concentration of 10 µmol/L for 20 min at 37
oC in darkness. Then the cells were washed with PBS three times. The fluorescence intensity was
measured by using a flow cytometer (BD FACSCallibur 488/530 nm).
5.2.5. Measurement of MDA level
The MDA content was determined using the 2-thiobarbituric acid (TBA) method [32]. After the
treatment, two volumes of 2-thiobarbituric acid reagent (0.375% TBA, 15% trichloroacetic acid, and
0.1 mM EDTA) were added to the cell samples and boiled at 95 °C for 40 min. After cooling and
centrifugation at 3000 × g for 10 min, the absorbance of each supernatant was measured at 532 nm by
using a microplate reader.
5.2.6. Statistical analysis
All samples were cultured in triplicate. The data are expressed as the means ± SD of three assays.
Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunnett’s test. P
< 0.05 was considered significant.
Acknowledgments
This project was financially supported by National Natural Science Foundation of China (No.
81473087 and 81273358). Dr. Y. Liu wishes to express his thanks for the support by Career
Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical
University (ZQN2016005).

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Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version.
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homogenates, Food Chem. 107 (2008) 739-744.

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Highlights
‹
‹
Natural saponin hederacolchiside E and eleven of its derivatives were synthesized for the
first time.
This series of compounds showed obvious differences in activity in AD-like models due
to structural variations
.
‹ Compound 7 with a relatively simple chemical structure showed the best activity against
Aβ1-42-induced cell damage.
‹ Hederacolchiside E and compound 7 could significantly reduce the levels of LDH, ROS
and MDA resulting from Aβ1-42 treatment in a concentration-depended manner.