Design, synthesis and biological evaluation of novel α-hederagenin derivatives with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2017.09.016

In an attempt to arrive at a more potent cytotoxic agent than the parent compound α-hederagenin (H), 24 α-hederagenin derivatives (5–8, 11–24, 27–28, 31–32, and 35–36) were synthesized in a concise and efficient strategy and screened for in vitro cytotoxicity against the human cancer cell lines MKN45 and KB. Among these compounds, the polyamine derivative 15 exhibited more potency than the parent compound with IC₅₀ values in the range of 4.22 μM–8.05 μM. Compound 15 increased Bax/bcl-2 ratio that disrupted the mitochondrial potential and induced apoptosis. Therefore, the present studies highlight the importance of polyamine derivatives of α-hederagenin in the discovery and development of novel anticancer agents.

Full text of DOI:10.1016/j.ejmech.2017.09.016

Accepted Manuscript
Design, synthesis and biological evaluation of novel α-hederagenin derivatives with
anticancer activity
Xian-xuan Liu, Yan-ting Yang, Xiao Wang, Kai-yi Wang, Jia-qi Liu, Lei Lei, Xiao-min
Luo, Rong Zhai, Feng-hua Fu, Hong-bo Wang, Yi Bi
PII:
S0223-5234(17)30698-0
10.1016/j.ejmech.2017.09.016
EJMECH 9731
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2017
Revised Date: 8 September 2017
Accepted Date: 10 September 2017
Please cite this article as: X.-x. Liu, Y.-t. Yang, X. Wang, K.-y. Wang, J.-q. Liu, L. Lei, X.-m. Luo, R.
Zhai, F.-h. Fu, H.-b. Wang, Y. Bi, Design, synthesis and biological evaluation of novel α-hederagenin
derivatives with anticancer activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.09.016.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel α-hederagenin
derivatives with anticancer activity
Xian-xuan Liu1,#, Yan-ting Yang1,#, Xiao Wang1, Kai-yi Wang1, Jia-qi Liu1, Lei Lei1,2, Xiao-min
Luo3, Rong Zhai1, Feng-hua Fu1, Hong-bo Wang1,*, Yi Bi1,3,
*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation
Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing, 100050, China
3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
* Correspondence: hongbowangyt@gmail.com (H. W.), beeyee_413@163.com (Y. B.)
# These authors contributed equally to this work
Graphical abstract

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel α-hederagenin
derivatives with anticancer activity
Xian-xuan Liu1,#, Yan-ting Yang1,#, Xiao Wang1, Kai-yi Wang1, Jia-qi Liu1, Lei Lei1,2, Xiao-min
Luo3, Rong Zhai1, Feng-hua Fu1, Hong-bo Wang1,*, Yi Bi1,3,
*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation
Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing, 100050, China
3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
* Correspondence: hongbowangyt@gmail.com (H. W.), beeyee_413@163.com (Y. B.)
# These authors contributed equally to this work
Abstract
In an attempt to arrive at a more potent cytotoxic agent than the parent compound α-hederagenin (H), 24 α-hederagenin
derivatives (5–8, 11–24, 27–28, 31–32, and 35–36) were synthesized in a concise and efficient strategy and screened for in vitro
cytotoxicity against the human cancer cell lines MKN45 and KB. Among these compounds, the polyamine derivative 15
exhibited more potency than the parent compound with IC50 values in the range of 4.22 µM–8.05 µM. Compound 15 increased
Bax/bcl-2 ratio that disrupted the mitochondrial potential and induced apoptosis. Therefore, the present studies highlight the
importance of polyamine derivatives of α-hederagenin in the discovery and development of novel anticancer agents.
Keywords: α-hederagenin derivatives, synthesis, apoptosis, cancer
1. Introduction
Cancer, a notably complex, widespread and lethal disease, responsible for nearly 7.6 million deaths per year, is one of the
major diseases that threatens human health [1,2]. Several lines of evidence support the view that chemotherapy is one of the most
significant treatment approaches in cancer management, but severe side effects are critical obstacles to cancer therapy in the
clinic [3–5]. Consequently, the search for drugs that can kill tumor cells without serious side effects is of vital importance in the
study and development of new antitumor drugs.
Several natural products and their derivatives have shown potent antitumor activity, such as Ursolic acid (UA) [6] (Fig. 1),
and there continues to be significant research into novel antitumor agents from natural products. Pentacyclic triterpenoids,
including oleanolic, lupine and friedelane groups are widely distributed in many medicinal plants. A large number of studies have
shown that most of the pentacyclic triterpenoids have a wide range of pharmacological effects, biological activity and low normal
tissue toxicity in the cancer setting [7–9]. For example, Konopleva et al. [10] found that 2-cyano-3,12-dioxooleana-
1,9-dien-28-oic acid (CDDO) (Fig. 1) blocked the growth and size of transplanted MCF7/HER2 tumors in female nude mice

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through reducing cyclin D1 mRNA and protein expression. In addition, CDDO showed therapeutic potential in advanced breast
cancer. Furthermore, methyl-25-hydroxy-3-oxoolean-12-en-28-oate (AMR-Me) (Fig. 1) inhibited the incidence and burden of
mammary tumors induced by 7,12-dimethylbenzanthracene (DMBA) in female Sprague-Dawley rats through antiproliferative
and proapoptotic effects [11–13]. Therefore, the antitumor activity and mechanisms of these natural products are becoming
increasingly important.
D
COOH
A
28
COOH
3
HO
A
C
HO
23
OH
H
UA
B
O
HO
H
COOH
CH2
COOCH3
CN
O
O
AMR-Me
CDDO
Fig. 1. Chemical structure of α-Hederagenin, UA, CDDO, AMR-Me.
α-Hederagenin (H) (Fig. 1) derived from Hedera nepalensis var.sinensis is a pentacyclic triterpene and a derivative of
oleanolic acid. Studies have shown that α-Hederagenin has clear anti-cancer effects [14, 15]. Zhou et al. [16] found that H
induced apoptosis in human LoVo colon cells through a mitochondrial pathway. Diego Rodrigue-hernandez et al. [17] found that
H derivatives showed cytotoxic activity against several tumor cell lines derived from skin, ovary, colon and breast carcinomas.
To generate novel antitumor agents, we designed and synthesized a series of α-Hederagenin derivatives and biologically
evaluated the antitumor activity of these compounds.

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Scheme 1. Synthesis of H derivatives. (1) BrBn, DMF, K2CO3, 50 ; (2) TBSCl, DMAP, CH2Cl2, r.t; (3) PCC, CH2Cl2, r.t; (4) S, H2NCH2CH2NH2, morpholine,
reflux; (5)/(8) HCl, acetone, r.t; (6)/(9) DMAP, CH2Cl2, anhydrides, r.t; (7)/(10) DMAP, EDCI (diamine), CH2Cl2, r.t; (11) NH2OH·HCl, pyridine, 80°C.
Scheme 2. Synthesis of nitrate side chains. (1) ClCOOCH2CH3, ice-bath, NaOH; (2) HNO3, ice-bath, acetic anhydride; (3) ethanol amine, ethanol; (4)
1,2-Dibromoethane, THF; (5) AgNO3, CH3CN.

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COOBn
COOBn
N
N
O
O
O
27 n=4
28 n=5
31 n=4
ONO2
ONO2
O
O
n
n
32 n=5
(6)
(8)
COOBn
COOBn
N
N
O
O
O
25 n=4
26 n=5
29 n=4
30 n=5
Br
Br
O
O
n
n
(5)
(7)
COOBn
COOBn
COOBn
COOBn
(1)
N
N
(3)
N
N
O
O
OH
O
R3 OH
10-11
OH
O
R3 OH
6-7
9
5
R3
=
R4
=
(4)
(2)
O
O
ONO2
O
O
17,21
18,22
O
O
COOBn
COOBn
N
N
O
ONO2
O
O
ONO2
O
R3 R4
17-20
O
R3 R4
21-24
O
O
19,23
20,24
O
O
O
ONO2
O
COOBn
COOBn
COOBn
(9)
(10)
O
O
Br
O2NO
HO
O
n
O
n
O
O
33 n=4
n=5
35 n=4
36
n=5
Br
ONO2
OH
O
O
n
n
34
1
Scheme 3. Synthesis of the nitrated derivatives of H. (1)/(3) DMAP, CH2Cl2, anhydrides, r.t; (2)/(4) DMAP, EDCI, CH2Cl2, a/b, r.t; (5)/(7)/(9) DMAP, EDCI,
CH2Cl2, bromopentanoic acid, r.t; (6)/(8)/(10) AgNO3, CH3CN, 70°C.
2. Results and discussion
The structure of H consists of a 30-carbon skeleton which has four available sites for chemical modification at C-3, C-23,
C-28 and the A-ring position (Fig. 1). Based on previous research, three target compounds were selected as lead compounds
following structural modification of the A-ring. We obtained the lead compound 9 by oxidizing 3-OH and protecting 28-COOH
[18]. Many derivatives with good biological activity have previously been obtained by splicing a heterocycle on the A-ring of
pentacyclic triterpene [19,20]. Therefore, we designed the lead compound 5 by splicing a pyrazine ring on the A-ring of H. In
addition, we introduced the oxime group into the C-3 position of H to generate the lead compound 16 [18]. After that, several

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derivatives were obtained by structural modification of the C-23 position using the splicing principle, and their biological
activities were evaluated.
Table 1
In vitro cytotoxicities of α-hederagenin (H) and its derivatives against MKN45 and KB at 10 µM concentration. Cell viability was calculated from the results,
and data are presented as the mean ± SD of three independent experiments.
Compound
Cell viability
(10 µM)
MKN45
77% ± 10%
70% ± 8%
84% ± 14%
86% ± 5%
40% ± 4%
92% ± 6%
99% ± 2%
93% ± 10%
7% ± 0%
64% ± 9%
100% ± 8%
93% ± 8%
78% ± 6%
89% ± 12%
100% ± 8%
93% ± 8%
78% ± 6%
KB
75% ± 14%
98% ± 34%
81% ± 12%
57% ± 9%
51% ± 11%
51% ± 7%
50% ± 8%
62% ± 13%
26% ± 2%
76% ± 17%
53% ± 16%
76% ± 26%
86% ± 9%
67% ± 6%
69% ± 9%
50% ± 5%
49% ± 3%
5
6
7
8
11
12
13
14
15
16
17
18
19
20
21
22
23

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84% ± 14%
54% ± 4%
75% ± 1%
89% ± 9%
84% ± 12%
97% ± 3%
85% ± 2%
75% ± 3%
23% ± 2%
55% ± 12%
60% ± 6%
50% ± 4%
59% ± 12%
63% ± 7%
58% ± 7%
61% ± 29%
81% ± 4%
15% ± 0%
24
27
28
31
32
35
36
H
Paclitaxel (100 nM)
2.1. Cell growth inhibition
The anti-proliferative activity of all the synthesized compounds at 10 µM was determined by an MTT assay in two cancer
cell lines: MKN45 (gastric cancer) and KB (oral epidermoid carcinoma). The results are summarized in Table 1. H showed 19%–
25% growth inhibition of the tested cancer cell lines, and many of its derivatives displayed different growth inhibition effects.
Compound 15 exhibited an inhibitory effect against all cell lines (74%–93%) and the greatest growth inhibitory effect of all
compounds tested.
In this study, 24 α-hederagenin derivatives have been synthesized and biologically evaluated as novel antitumor agents. Within
this series of compounds, polyamine compound 15 (cell viability: MKN45: 7%±0%; KB: 26%±2%) was more potent than its
parent compound H. The structure activity relationship (SAR) analysis showed that polyamine compound 15 that possessed three
nitrogen atoms at C-23 exhibited more potent antitumor activity than other compounds that possess zero to two nitrogen atoms at
C-23. Previous and present studies are summarized, it is found that α-hederagenin derivatives containing more nitrogen atoms
generally have good pharmacological activity [17, 21]. Our laboratory is currently designing and synthesizing new α-hederagenin
derivatives using existing results and previous literature [21,22] to improve pharmacological activity. Results from these studies

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will be reported in due course.
We further extended our characterization of the antiproliferative activity of 15 in seven cancer cell lines: KB, KBV
(vincristine resistant oral epidermoid carcinoma cell), Hela (Hela Cells), A549 (human alveolar adenocarcinoma cell), MKN45,
BGC-823 (human gastric cancer cell) and AGS (human gastric adenocarcinoma cell). The IC50 (50% growth inhibition) is shown
in Table 2. Compound 15 displayed robust anti-proliferative activity in all the tested cell lines with IC50 values of 4.22 µM–8.05
µM. Interestingly, compound 15 also demonstrated the maximum growth inhibition in KBV cells that was more than the effect
observed for the parent molecule H. KBV cells are resistant to paclitaxel because of overexpression of the drug efflux pump
p-glycoprotein and the increased effect on growth inhibition by 15 may suggest that its structure is not a target for this pump. We
therefore investigated if this antiproliferative activity was the result of apoptotic mechanisms.
Table 2
The IC50 values of compound 15 against a panel of human cancer cell lines.
Cancer cell lines
IC50 (µM)
15
H
KB
4.75 ± 0.05
8.05 ± 0.08
4.22 ± 0.15
6.30 ± 0.19
7.0 ± 0.45
5.15 ± 0.38
4.40 ± 1.45
54.43 ± 2.79
KBV
>100
Hela
53.96 ± 2.44
>100
A549
MKN45
BGC-823
AGS
53.69 ± 0.56
52.07 ± 3.49
36.14 ± 3.74
2.2. Compound 15 induced apoptosis in MKN45 cells
Since deregulated cell cycle control and the inhibition of apoptosis are two of the hallmarks of cancer, the effect of
compound 15 on the cell cycle and apoptosis was explored using flow cytometry. The results showed that compound 15 at a
concentration of 10 µM had no obvious effect on cell cycle distribution. However, after 48 h of treatment 15 induced apoptosis
(Fig. 2A, B), that was indicated by the ratio of cells in the sub-G0/G1 phase. Consistent with this, western blotting showed that
compound 15 increased the abundance of cleaved poly-ADP ribose polymerase (PARP), another biomarker of apoptosis (Fig.
2C).

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2.3. Compound 15 triggers apoptosis via disruption of the mitochondrial membrane potential.
The reduction in mitochondrial membrane potential induced by anticancer drugs is considered as one of the major pathways
that triggers apoptosis, and the BCL2 family of proteins, such as BCL2, apoptosis regulator (BCL2) and BCL2 associated X,
apoptosis regulator (BAX), play pivotal roles [23]. Our results showed that treatment with compound 15 decreased the expression
of BCL2, one of the main mitochondrial-associated antiapoptotic proteins in a dose-dependent manner (Fig. 3C, D).
Downregulation of antiapoptotic proteins changes the balance of mitochondrial proteins, activates the mitochondrial permeability
transition pores (PTP) and induces the loss of mitochondrial membrane potential (∆ψmt) [24]. Treatment with compound 15 in
MKN45 cells induced significant loss of mitochondrial membrane potential, which may further trigger the apoptotic machinery
by activating proapoptotic factors such as BAX (Fig. 3C, D). During apoptosis, BAX creates pores in the mitochondrial outer
membrane that result in an increase in mitochondrial permeability [25]. Compound 15 significantly increased the expression of
the proapoptotic protein BAX, and dramatically elevated the ratio of Bax/Bcl-2 (Fig. 3C, D). Therefore, our results suggest that
compound 15 induces apoptosis via the intrinsic mitochondrial pathway (Fig. 3).
Fig. 2. Apoptotic profile of compound 15 in MKN45 cells. A) and B) DNA cell cycle analyses of MKN45 cells exposed to
compound 15. MKN45 cells were treated with indicated concentrations of compound 15 for 48 h and stained with propidium
iodide (PI, 50 µg/mL) to determine DNA fluorescence and cell cycle phase distribution as described in materials and methods.
Data were analyzed for the proportions of different cell cycle phases. The fraction of cells from apoptotic, G1, S and G2 phases
were analyzed from FL2-A vs. cell counts and are shown in percent. Data are representative of three experiments. C) Effect of
compound 15 on the expression of key apoptotic proteins. MKN45 cells (2×105) were treated with 5 µM or 10 µM of compound
15 for 48 h. Protein lysates were prepared and electrophoresis as described in materials and methods. β-actin was used as an
internal control. Specific antibodies were used for detection of the indicated proteins in the lysates. Data are representative of
three experiments.

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Fig. 3. Compound 15 induced loss of mitochondrial membrane potential (∆ψmt) in MKN45 cells. A) and B) Cells were incubated
with the indicated doses of compound 15 for 24 h. Thereafter, cells were stained with JC-1and analyzed by flow cytometer. Data
are representative of three experiments. C) and D) Influence of compound 15 on the Bax/bcl-2 ratio in MKN45 cells. The relative
density of each band was measured as arbitrary units by ImageJ software.
3. Conclusion
The analogues of α-hederagenin described herein induced a loss of proliferative capacity in several diverse cancer cell
lines. The present study demonstrates that introduction of a polyamine substituent at C-23 positions of α-hederagenin generated
compounds that displayed promising antiproliferative activity. Therefore, modification of C-23, by the introduction of side chains
containing multiple N atoms, may be beneficial for enhancing activity. We report here for the first time that a derivative of H,
compound 15, induced apoptosis via a mitochondrial pathway in MKN45 cells. In summary, the present studies highlight the
importance of polyamine derivatives of α-hederagenin in the discovery and development of novel anticancer agents. Further
research on compound 15 is currently underway and the results will be submitted for publication in due course.
4. Experimental section
4.1. Chemistry
The chemicals and reagents were analytically pure or dried with standard methods when necessary. The progress of all
reactions were monitored by TLC on silica gel HSGF254 using a 10% ethanol sulfate solution for detection of the spots. All the
compounds synthesized were purified by column chromatography on silica gel. All NMR spectra (1H and 13C NMR) were
routinely measured using Bruker av400 or Bruker av300 instruments using CDCl3 as solvent with TMS as internal standard.
Chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hertz. High resolution mass spectra (HRMS)
were recorded on Agilent QTOF 6520 spectrometer. Melting points of compounds were recorded on Micro melting point
apparatus XT3A.

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4.1.1. Benzyl 3-oxo-23-t-butyldimethylsilyloxy-olean-12-en-28-oate (3)
To a solution of 3β, 23-dihydroxy-olean-12-en-28-oic acid (0.47 g, 1.00 mmol) in DMF (15 mL), potassium carbonate (0.30
g, 2.10 mmol) and benzyl bromide (0.15 mL, 1.25 mmol) were added, and the mixture was stirred for 8 h at 50 . The reaction
mixture was then diluted with ethyl acetate (25 mL), washed with water and brine successively, dried over anhydrous sodium
sulfate, concentrated and purified by column chromatography (8:1 petroleum ether:ethyl acetate). Benzyl (3β, 4α)-3,
23-dihydroxy-olean-12-en-28-oate (1) was obtained as a white solid (0.47 g, 83% yield).
A solution of 1 (0.46 g, 0.81 mmol) in dichloromethane (20 mL) was added DMAP (0.12 g, 1.000 mmol) and
tert-butyldimethylsilyl chloride (0.36 g, 2.400 mmol), and the reaction mixture was stirred at room temperature for 6 h. The
solvent was removed under reduced pressure, ethyl acetate was (20 mL) added. Then diluent was washed with 10% HCl to
acidity, water and brine successively, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography
(20:1 petroleum ether:ethyl acetate). Benzyl 3(β)-hydroxy-23-t-butyldimethylsilyloxy-olean-12-en-28-oate (2) was obtained as a
white solid (0.38 g, 70% yield).
Pyridinium chlorochromate (0.30 g, 1.300 mmol) was added to a solution of 2 (0.38 g, 0.560 mmol) in dichloromethane (15
mL). The mixture was stirred for 8 h at room temperature. The solvent was removed under reduced pressure, ethyl acetate was
(20 mL) added, then washed with water and brine to neutral, dried over anhydrous sodium sulfate, concentrated and purified by
column chromatography (25:1 petroleum ether:ethyl acetate). Benzyl 3-oxo-23-t-butyldimethylsilyloxy-olean-12-en-28-oate (3)
was obtained as a white solid (0.32 g, 84% yield).
4.1.2. Benzyl 23-t-butyldimethylsilyloxy-olean[3,2-b]pyrazine-12-en-28-oate (4)
Sulphur (0.32 g, 10.00 mmol) and ethylene diamine (0.28 g, 4.50 mmol) were added to a solution of 3 (0.50 g, 0.74 mmol) in
dry morpholine (25 mL), and the mixture was refluxed for 8 h. The reaction mixture was then diluted with ethyl acetate (30 mL),
washed with water and brine successively, dried over anhydrous sodium sulfate, concentrated and purified by column
chromatography (20:1 petroleum ether:ethyl acetate) to yield a white solid (0.36 g, 68% yield) .
4.1.3. General procedure for the synthesis of 5 and 9
To a solution of 3 or 4 (0.450 mmol) in acetone (20 mL), 10% hydrochloric acid solution (2 mL) was added, and the mixture
was stirred for 4 h at room temperature. The reaction mixture was then diluted with ethyl acetate (20 mL), washed with water and
brine to neutral, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (6:1-8:1 petroleum
ether:ethyl acetate) to yield the white solids 9 or 5.
4.1.3.1. Benzyl 23-hydroxy-olean[3,2-b]pyrazine-12-en-28-oate (5). Yield: 89% (after chromatograph with petroleum ether/ethyl

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acetate, 6:1) as a white solid. mp 128-131 . Rf = 0.67 (chloroform/methanol 30:1 v/v); [α]20D = 23.38 (c =0.08 , CH3OH/CHCl3
3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.70 (3H, s, -CH3), 0.91 (3H, s, -CH3), 0.92 (3H, s, -CH3), 0.94 (3H, s, -CH3), 1.18 (3H, s,
-CH3), 1.32 (3H, s, -CH3), 2.52 (1H, d, J = 16.6 Hz, H-1A), 2.94 (1H, dd, J = 3.3, 13.56 Hz, H-18), 3.03 (1H, d, J = 16.6 Hz,
H-1B), 3.47 (1H, d, J = 10.48 Hz, H-23A), 3.80 (1H, d, J = 10.56 Hz, H-23B), 5.07 (1H, d, CH2Ar), 5.09 (1H, d, CH2Ar), 5.37
(1H, t, H-12), 7.30-7.37 (5H, m, H-Ar), 8.35, 8.38 (each 1 H, ds, J = 2.3 Hz, H-pyrazine); 13C NMR (100 MHz, CDCl3) δ 16.05,
16.56, 19.36, 20.07, 23.01, 23.29, 23.56, 25.68, 27.51, 29.65, 30.57, 31.84, 32.27, 33.05, 33.81, 36.42, 39.13, 41.46, 41.87, 43.28,
45.63, 45.74, 46.73, 47.01, 47.53, 65.91, 70.55, 122.22, 127.74, 127.90, 127.97, 128.38, 128.46, 136.33, 141.58, 141.87, 143.60,
151.96, 158.00. ESI-HRMS m/z calcd. for C39H52N2O3 [M + H]+ 597.4018, found: 597.4077.
4.1.3.2. Benzyl 3-oxo-23-hydroxy-olean-12-en-28-oate (9). Yield: 74% (after chromatograph with petroleum ether/ethyl acetate,
6:1) as a white solid. mp 178-182 . Rf = 0.38 (petroleum ether/ethyl acetate 4:1 v/v); [α]20D = 98.82 (c = 0.34, CH3OH/CHCl3
3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.66 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.92 (3H, s, -CH3), 1.01 (3H, s, -CH3), 1.10 (3H, s,
-CH3), 1.13 (3H, s, -CH3), 2.28 (1H, m, H-2A), 2.36(1H, br s, HO-23), 2.61(1H, m, H-2B), 2.94 (1H, dd, J = 3.9, 13.9 Hz, H-18),
3.42 (1H, d, J = 11.3 Hz, H-23A), 3.64 (1H, d, J = 11.2 Hz, H-23B), 5.07 (1H, d, CH2Ar), 5.08 (1H, d, CH2Ar), 5.30 (1H, t,
H-12), 7.30-7.35 (5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 12.77, 15.20, 16.80, 17.00, 19.20, 23.10, 23.50, 23.60, 25.90,
27.60, 30.70, 32.10, 32.30, 33.10, 33.90, 35.20, 36.60, 38.80, 39.30, 41.50, 41.90, 45.90, 46.76, 46.80, 49.20, 52.40, 65.90, 67.00,
73.31, 122.10, 127.90, 128.00, 128.40, 136.40, 143.90, 177.40, 219.00.
4.1.4. General procedure for the synthesis of 6 and 10
DMAP (0.08 mmol) and butanedioic anhydride (0.15 mmol) were added to a solution of 5 or 9 (0.10 mmol) in dry
dichloromethane (8 mL), and the mixture was stirred for 8-15 h at room temperature. The reaction mixture was then diluted with
dichloromethane (10 mL), washed with 5% HCl, water, and brine successively, dried over anhydrous sodium sulfate,
concentrated and purified by column chromatography to provide the compound 6 or 10.
4.1.4.1. 4-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-oxo-butyric acid (6). Yield: 75% (after chromatograph with
chloroform/methanol, 40:1-30:1) as a white solid. mp 158-161 ; Rf = 0.53 (chloroform/methanol 25:1 v/v); [α]20D = 30.00 (c =
0.30, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.69 (3H, s, -CH3), 0.87 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.93 (3H,
s, -CH3), 1.18 (3H, s, -CH3), 1.27 (3H, s, -CH3), 2.34-2.45 (4H, m), 2.54-2.58 (1H, m, H-1A), 2.91-2.92 (1H, m, H-1B), 2.94 (1H,
dd, J = 3.3, 13.56 Hz, H-18), 4.30 (1H, d, J = 10.53 Hz, H-23A), 4.40 (1H, d, J = 10.53 Hz, H-23B), 5.07 (1H, d, CH2Ar), 5.09
(1H, d, CH2Ar), 5.37 (1H, t, H-12), 7.31-7.37 (5H, m, H-Ar), 8.35, 8.38 (each 1 H, ds, J = 2.3 Hz, H-pyrazinem); 13C NMR (100
MHz, CDCl3) δ 15.89, 16.62, 19.81, 20.10, 23.08, 23.36, 23.61, 23.78, 23.9, 25.67, 27.59, 28.49, 28.96, 30.71, 31.81, 32.32,
32.56, 33.09, 33.87, 36.44, 39.16, 41.55, 41.90, 43.36, 45.60, 45.79, 46.69, 46.81, 65.98, 70.65, 122.22, 127.50, 127.96, 128.03,
128.44, 136.33, 141.87, 142.97, 143.71, 151.41, 171.58, 174.88, 177.45. ESI-HRMS m/z calcd. for C43H56N2O6 [M + H]+:
697.4138, found: 697.4208.

ACCEPTED MANUSCRIPT
4.1.4.2. 4-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-4-oxo-butyric acid (10). Yield: 70% (after chromatograph with
chloroform/methanol, 50:1-30:1) as a white solid. mp 310-313 ; Rf = 0.66 (chloroform/methanol 15:1 v/v); [α]20D = 27.68 (c =
0.40, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.65 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.92 (3H, s, -CH3), 1.01 (3H,
s, -CH3), 1.03 (3H, s, -CH3), 1.13 (3H, s, -CH3), 2.33-2.39 (1H, m, H-2A), 2.47-2.51 (1H, m, H-2B), 2.59-2.67 (4H, m), 2.92 (1H,
dd, J = 3.9, 13.9 Hz, H-18), 4.13 (2H, s, H-23), 5.08 (1H, d, CH2Ar), 5.10 (1H, d, CH2Ar), 5.31 (1H, t, H-12), 7.31-7.36 (5H, m,
H-Ar); 13C-NMR (100 MHz, CDCl3) δ 14.88, 16.83, 17.50, 19.38, 20.45, 22.97, 23.2, 23.39, 23.57, 23.89, 25.62, 27.48, 28.77,
28.83, 30.64, 31.90, 32.24, 33.04, 33.78, 34.80, 36.25, 37.57, 39.15, 41.42, 41.75, 45.74, 46.51, 46.70, 48.14, 50.23, 65.92, 67.65,
122.10, 127.50, 127.89, 127.96, 128.37, 136.31, 143.78, 171.63, 177.14, 177.39. ESI-HRMS m/z calcd. for C41H56O7 [M + H]+:
661.4026, found: 661.4101.
4.1.5. General procedure for the synthesis of 7 and 11
DMAP (0.08 mmol) and phthalic anhydride (0.15 mmol) were added to a solution of 5 or 9 (0.10 mmol) in dry
dichloromethane (8 mL), and the mixture was stirred for 8-15 h at room temperature. The reaction mixture was then diluted with
dichloromethane (10 mL), washed with 5% HCl, water, and brine successively, dried over anhydrous sodium sulfate,
concentrated and purified by column chromatography to provide the compound 7 or 11.
4.1.5.1. 2-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-oxo-benzoic acid (7). Yield: 55% (after chromatograph with
chloroform/methanol, 40:1-30:1) as a white solid. mp 138-141 ; Rf = 0.43 (chloroform/methanol 25:1 v/v); [α]20D = 48.58 (c
= 0.26, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.69 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.92 (3H, s, -CH3), 0.93
(3H, s, -CH3), 1.13 (3H, s, -CH3), 1.30 (3H, s, -CH3), 2.53-2.57 (1H, m, H-1A), 2.94 (1H, dd, J = 3.3, 13.56 Hz, H-18),
2.94-2.98 (1H, m, H-1B), 4.50-4.56 (2H, m, H-23), 5.07 (1H, d, CH2Ar), 5.09 (1H, d, CH2Ar), 5.36 (1H, t, H-12), 7.18(1 H, m,
H-Ar), 7.31-7.37 (5H, m, H-Ar), 7.35-7.38(2 H, m, H-Ar), 7.68(1 H, m, H-Ar), 8.19 (1 H, s, H-pyrazinem), 8.44 (1 H, s,
H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.83, 16.60, 19.78, 19.94, 23.05, 23.11, 23.29, 23.56, 25.65, 27.55, 29.67,
30.67, 31.77, 32.29, 33.06, 33.85, 36.51, 39.14, 41.52, 41.88, 43.22, 45.56, 45.6, 45.79, 46.77, 47.15, 65.93, 71.81, 76.60,
76.90, 77.30, 77.52, 77.60, 78.41, 122.20, 127.91, 127.97, 128.40, 129.53, 130.73, 136.36, 141.29, 142.59, 143.66, 152.04,
156.19, 177.40. ESI-HRMS m/z calcd. for C47H56N2O6 [M + H]+: 745.4138, found: 745.4199.
4.1.5.2. 2-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-oxo-benzoic acid (11). Yield: 56% (after chromatograph with
chloroform/methanol, 50:1-30:1) as a white solid. mp 280-283 ; Rf = 0.43 (chloroform/methanol 30:1 v/v); [α]20 = 7.00 (c =
D
0.10, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.64 (3H, s, -CH3), 0.88 (3H, s, -CH3), 0.91 (3H, s, -CH3), 1.03 (3H,
s, -CH3), 1.06 (3H, s, -CH3), 1.07 (3H, s, -CH3), 2.44-2.52 (2H, m, H-1A), 2.89 (1H, dd, J = 3.9, 13.6 Hz, H-18), 4.30 (1H, d, J =
10.72 Hz, H-23A), 4.32 (1H, d, J = 11.30 Hz, H-23B), 5.07 (1H, d, CH2Ar), 5.10 (1H, d, CH2Ar), 5.29 (1H, t, H-12), 7.32-7.33
(5H, m, H-Ar), 7.53-7.56 (2H, m, H-Ar), 7.68 (1H, t, J = 3.52 Hz, H-Ar), 7.84 (1H, t, J = 5 Hz, H-Ar); 13C NMR (100 MHz,

ACCEPTED MANUSCRIPT
CDCl3) δ 14.93, 16.85, 17.59, 19.43, 22.98, 23.39, 23.56, 25.55, 27.48, 29.66, 30.64, 31.90, 32.24, 33.04, 33.81, 34.80, 36.28,
37.51, 39.15, 41.44, 41.77, 45.73, 46.47, 46.72, 48.06, 50.28, 65.92, 68.65, 76.68, 76.99, 77.31, 122.10, 127.89, 128.39, 128.81,
129.70, 131.02, 131.75, 132.45, 136.34, 143.76, 167.60, 170.32, 177.39, 215.26. ESI-HRMS m/z calcd. for C45H56O7 [M + Na]+:
731.4026, found: 731.3939.
4.1.6. 4-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-4-oxo-butyryl-p-phenylenediamine (12)
EDCI (0.01 g, 0.05 mmol) and DMAP (0.01 g, 0.06 mmol) were added to a solution of 10 (0.02 g, 0.03 mmol) in dry
dichloromethane (5 mL), and the mixture was instilled relaxedly in p-phenylenediamine (0.01 g, 0.09 mmol) solution. The
mixture was stirred for 3 h at room temperature. The reaction mixture was then washed with 5% HCl, water, and brine
successively, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (200:1
chloroform:methanol) to provide a white solid (0.02 g, 70% yield) mp 340-343 ; Rf = 0.47 (chloroform/methanol 20:1 v/v);
[α]20D = 16.25 (c = 0.08, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.65 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.92 (3H,
s, -CH3), 1.01 (3H, s, -CH3), 1.12 (3H, s, -CH3), 1.25 (3H, s, -CH3), 2.34-2.37 (1H, m, H-2A), 2.49-2.51 (1H, m, H-2B),
2.62-2.71 (4H, m), 2.93 (1H, dd, J = 3.9, 13.9 Hz, H-18), 4.09 (2H, s, H-23), 5.08 (1H, d, CH2Ar), 5.10 (1H, d, CH2Ar), 5.31 (1H,
t, H-12), 6.61 (2H, d, J = 8.6, H-Ar), 7.28 (2H, d, H-Ar), 7.31-7.37 (5H, m, H-Ar), 7.53(1H, s, -NH-); 13C NMR (100 MHz,
CDCl3) δ 14.96, 16.83, 17.50, 19.43, 22.98, 23.39, 23.57, 25.72, 27.47, 28.87, 29.33, 29.66, 30.12, 30.64, 31.85, 31.90, 32.27,
32.7, 33.04, 33.78, 34.80, 36.25, 37.57, 39.14, 41.41, 41.77, 45.72, 46.45, 46.71, 48.19, 50.30, 65.90, 67.62, 76.68, 115.38,
121.71, 122.01, 127.90, 127.96, 128.39, 136.35, 142.78, 143.84, 169.20, 172.63, 177.42, 214.90. ESI-HRMS m/z calcd. for
C47H62N2O6 [M + H]+: 751.4648, found: 751.4701.
4.1.7. General procedure for the synthesis of 8, 13-14
EDCI (0.05 mmol) and DMAP (0.06 mmol) were added to a solution of 6, 10-11 (0.03 mmol) in dry dichloromethane (5 mL),
and the mixture was instilled relaxedly in o-phenylenediamine (0.09 mmol) solution. The mixture was stirred for 3 h at room
temperature. The reaction mixture was then washed with 5% HCl, water, and brine successively, dried over anhydrous sodium
sulfate, concentrated and purified by column chromatography to provide the compounds 8, 13-14.
4.1.7.1. 4-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-oxo-butyryl-o-phenylenediamine (8). Yield: 60% (after
chromatograph with chloroform/methanol, 200:1-100:1) as a white solid. mp 180-183 ; Rf = 0.45 (chloroform/methanol 25:1
1
v/v); [α]20D = 43.87 (c = 0.38, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.68 (3H, s, -CH3), 0.86 (3H, s, -CH3),
0.91 (3H, s, -CH3), 0.93 (3H, s, -CH3), 1.14 (3H, s, -CH3), 1.27 (3H, s, -CH3), 2.41-2.56 (4H, m), 2.42-2.44 (1H, m, H-1A),
2.89-2.92 (1H, m, H-1B), 2.94 (1H, dd, J = 3.3, 13.56 Hz, H-18), 4.30 (1H, s ), 5.07 (1H, d, J = 12.57 Hz, CH2Ar), 5.09 (1H, d,
J = 12.57 Hz, CH2Ar), 5.35 (1H, t, H-12), 6.39-6.41 (1H, d, J = 7.8 Hz, H-Ar), 6.71-6.73 (1H, d, J = 7.72 Hz, H-Ar),
7.02-7.06 (2H, m, H-Ar), 7.31-7.37 (5H, m, H-Ar), 7.5 (1H, d, -NH-), 8.35, 8.38 (each 1 H, ds, J = 2.3 Hz, H-pyrazinem); 13
C
NMR (100 MHz, CDCl3) δ 15.65, 16.54, 19.77, 22.99, 23.22, 23.55, 23.46, 25.64, 27.48, 29.24, 30.64, 31.71, 31.76, 32.27,

ACCEPTED MANUSCRIPT
33.05, 33.80, 36.48, 39.06, 41.42, 41.78, 43.09, 45.51, 45.72, 46.05, 46.70, 47.54, 65.88, 71.03, 76.68, 76.99, 77.31, 106.38,
109.57, 110.90, 122.14, 127.86, 127.96, 128.36, 129.55, 136.31, 138.81, 141.74, 142.30, 143.60, 152.32, 155.94, 169.24,
172.70, 177.40. ESI-HRMS m/z calcd. for C49H62N4O5 [M + H]+: 787.472, found: 787.4783.
4.1.7.2. 4-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-4-oxo-butyryl-o-phenylenedia-mine (13). Yield: 68% (after
chromatograph with chloroform/methanol, 200:1-100:1) as a white solid. mp 340-343 ; Rf = 0.56 (chloroform/methanol 25:1
1
v/v); [α]20D = 27.50 (c = 0.12, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.65 (3H, s, -CH3), 0.90 (3H, s, -CH3),
0.92 (3H, s, -CH3), 1.00 (3H, s, -CH3), 1.12 (3H, s, -CH3), 1.25 (3H, s, -CH3), 2.31-2.32 (1H, m, H-2A), 2.48-2.52 (1H, m,
H-2B), 2.55-2.60 (2H, m), 2.65-2.69 (2H, m), 2.93 (1H, dd, J = 4.2, 14.0 Hz, H-18), 4.10 (2H, s, H-23), 5.07 (1H, d, CH2Ar),
5.10 (1H, d, CH2Ar), 5.30 (1H, t, H-12), 6.40-6.42 (1H, d, J = 7.6 Hz, H-Ar), 6.71-6.73 (1H, d, J = 7.76 Hz, H-Ar), 7.02-7.06
(1H, t, J = 8 Hz, H-Ar), 7.14 (1H, s, H-Ar), 7.31-7.37 (5H, m, H-Ar), 7.66 (1H, s, -NH-); 13C NMR (100 MHz, CDCl3) δ 14.95,
16.82, 17.44, 19.43, 22.98, 23.39, 23.57, 25.73, 27.47, 28.87, 29.33, 29.57, 29.66, 30.65, 31.85, 32.18, 32.26, 32.27, 33.04,
33.78, 34.86, 36.28, 37.55, 39.14, 41.42, 41.77, 45.74, 46.45, 46.71, 48.16, 50.33, 65.90, 67.59, 106.49, 109.75, 111.00, 122.01,
127.90, 127.96, 128.39, 129.58, 136.35, 142.78, 143.84, 169.48, 172.58, 177.40. ESI-HRMS m/z calcd. for C47H62N2O6 [M +
H]+: 751.4648, found: 751.4668.
4.1.7.3. 2-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-oxo-benzoy-o-phenylenedia-mine (14). Yield: 65% (after chromatograph
with chloroform/methanol, 250:1-100:1) as a white solid. mp 340-343 ; Rf = 0.56 (chloroform/methanol 20:1 v/v); [α]20D = 3.75
(c = 0.08, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.65 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.93 (3H, s, -CH3), 1.02
(3H, s, -CH3), 1.03 (3H, s, -CH3), 1.14 (3H, s, -CH3), 2.45-2.48 (2H, m, H-2), 2.89 (1H, dd, J = 3.9, 13.6 Hz, H-18), 4.24 (1H, d,
J = 10.95 Hz, H-23A), 4.32 (1H, d, J = 10.95 Hz, H-23B), 5.07 (1H, d, J = 12.57 Hz, CH2Ar), 5.10 (1H, d, J = 12.57 Hz,
CH2Ar), 5.31 (1H, t, H-12), 6.62 (1H, d, J = 7.6 Hz, H-Ar), 7.08 (1H, d, J = 7.76 Hz, H-Ar), 7.10 (1H, t, J = 8 Hz, H-Ar), 7.14
(1H, s, H-Ar), 7.31-7.37 (5H, m, H-Ar), 7.43-7.46 (1H, m, H-Ar), 7.49-7.51 (2H, m, H-Ar), 7.55-7.63 (1H, m, H-Ar), 7.78 (1H, d,
-NH-); 13C NMR (100 MHz, CDCl3) δ 15.04, 16.80, 17.59, 19.43, 22.98, 23.39, 23.56, 25.55, 27.48, 29.57, 29.66, 30.64, 31.90,
32.18, 32.24, 32.26, 32.27, 33.04, 33.81, 34.80, 36.28, 37.51, 39.15, 41.44, 41.77, 45.73, 46.47, 46.72, 48.06, 50.28, 65.92, 68.40,
71.03, 76.68, 76.99, 77.31, 106.51, 109.76, 111.02, 122.03, 127.91, 128.39, 128.81, 129.70, 131.02, 131.75, 132.45, 136.36,
143.89, 167.03, 177.40. ESI-HRMS m/z calcd. for C51H62N2O6 [M + H]+: 799.4608, found: 799.4687.
4.1.8. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide 4-(benzyl 3-oxo-olean-12-en-28- oate-23-oxy)-4-oxo-benzoate (15)
To a solution of EDCI (0.01 g, 0.05 mmol) and DMAP (0.01 g, 0.06 mmol) in dry dichloromethane (5 mL), compound 11
(0.02 g, 0.03 mmol) was added. The mixture was stirred for 5 h at room temperature, then washed with 5% HCl, water, and brine
successively, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (50:1
chloroform:methanol) to provide the compound 15. White solid (0.02 g, 61% yield) mp 100-103
. Rf = 0.48
1
(chloroform/methanol 15:1 v/v); [α]20 = 16.14 (c = 0.14, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.66 (3H, s,
D
-CH3), 0.89 (3H, s, -CH3), 0.92 (3H, s, -CH3), 1.06 (3H, s, -CH3), 1.09 (3H, s, -CH3), 1.25 (3H, s, -CH3), 2.50-2.53 (2H, m), 2.92

ACCEPTED MANUSCRIPT
(1H, dd, J = 3.9, 13.6 Hz, H-18), 4.30 (1H, d, J = 10.72 Hz, H-23A), 4.32 (1H, d, J = 11.30 Hz, H-23B), 5.06 (1H, d, J = 12.76
Hz, CH2Ar), 5.10 (1H, d, J = 12.76 Hz, CH2Ar), 5.30 (1H, t, H-12), 7.31-7.36 (5H, m, H-Ar), 7.48(1H, t, J = 7.32 Hz, H-Ar),
7.61-7.63 (2H, m, H-Ar), 7.89-7.91 (1H, m, H-Ar), 9.12 (1H, m, -NH-); 13C NMR (100 MHz, CDCl3) δ 14.10, 14.60, 14.92,
16.88, 17.49, 19.54, 22.99, 29.54, 23.46, 23.57, 25.61, 27.56, 29.67, 30.66, 31.97, 32.11, 32.25, 32.28, 32.56, 33.04, 33.05, 34.75,
35.42, 36.39, 37.94, 39.19, 41.49, 41.83, 44.57, 45.80, 46.69, 46.73, 48.66, 50.41, 65.93, 68.48, 71.13, 76.54, 76.86, 77.21,
122.07, 126.21, 127.92, 128.00, 128.40, 129.27, 130.09, 131.02, 133.16, 136.36, 143.83, 167.60, 177.39. ESI-HRMS m/z calcd.
for C53H73N3O7 [M + H]+: 864.5435, found: 864.5501.
4.1.9. Benzyl 3-hydroxyimino-23-hydroxy-olean-12-en-28-oate (16)
To a solution of 9 (0.06 g, 0.10 mmol) in pyridine (6 mL), hydroxylamine hydrochloride (0.06 g, 0.80 mmol) was added, and
the mixture was stirred for 5 h at 80 . The reaction mixture was then diluted with ethyl acetate (20 mL), then diluent was
washed with 10% HCl to acidity, water and brine successively, dried over anhydrous sodium sulfate, concentrated and purified
by column chromatography (6:1 petroleum ether:ethyl acetate) to provide the compound 16. White solid (0.05 g, 81% yield) mp
1
228-232 ; Rf = 0.65 (chloroform/methanol 30:1 v/v); [α]20 = 17.7 (c = 0.1, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz,
D
CDCl3) δ 0.70 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.91 (3H, s, -CH3), 1.03 (3H, s, -CH3), 1.05 (3H, s, -CH3), 1.11 (3H, s, -CH3),
2.91 (1H, dd, J = 3.92, 14.24 Hz, H-18), 3.10-3.13 (1H, m, H-2B), 3.51 (1H, d, J = 11.28 Hz, H-23A), 3.62 (1H, d, J = 11.36 Hz,
H-23B), 5.05 (1H, d, CH2Ar), 5.10 (1H, d, CH2Ar), 5.29 (1H, t, H-12), 7.30-7.37 (5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ
15.11, 16.93, 17.31, 18.46, 18.82, 22.98, 23.42, 23.61, 25.82, 27.53, 29.68, 30.67, 32.18, 32.30, 33.07, 33.80, 36.78, 37.84, 39.24,
41.36, 41.76, 44.78, 45.76, 46.70, 46.93, 49.81, 65.92, 67.60, 69.53, 122.21, 127.90, 127.97, 128.39, 136.36, 143.80, 166.65,
177.47. ESI-HRMS m/z calcd. for C37H53NO4 [M + H]+: 576.3975, found: 576.4058.
4.1.10. 4-[(2-nitroxy)ethyl] phenol (a)
A solution of 2-(4-Hydroxyphenyl)ethanol (1.38 g, 10.00 mmol) in sodium hydroxide solution (10 ml, 1 M) was cooled to
-5 . Then ethyl chlorocarbonate (1.31 g, 12.00 mmol) was added slowly and stirred for 5 h at room temperature. After the pH of
mixture was adjusted to 2 with 10% HCl, the solution was extracted by ethyl acetate and the organic layer was washed with brine,
dried over anhydrous sodium sulfate and the solvent was filtered and evaporated in vacuo. The oily residue was purified by
column chromatography (50:1 petroleum ether:ethyl acetate) to provide a1 as a yellow oil (1.80 g, 92% yield).
A solution of a1 (1.60 g, 10.00 mmol) and acetic anhydride (1 ml) in dichloromethane was cooled to 0 , nitrosonitric acid
(1 ml) was added slowly and stirred for 4 h at room temperature. The solution was washed with water followed by brine, then
dried over anhydrous sodium sulfate, filtered and evaporated in vacuo. Purification by column chromatography (60:1 petroleum
ether:ethyl acetate) gave a2 as a yellow oil (2.17 g, 90% yield).
A solution of a2 (2.00 g, 10.00 mmol) and cholamine (1 ml) in 95% ethanol (25 ml) was stirred for 6 h at room temperature.
The mixture was neutralized by 10% HCl, and was added ethyl acetate. The organic layer was washed with water and brine,

ACCEPTED MANUSCRIPT
dried over anhydrous sodium sulfate, filtered and evaporated. Purification by column chromatography (50:1 petroleum
ether:ethyl acetate) gave a as a yellow oil (1.45 g, 86% yield).
4.1.11. 4-[O-(2-nitro)ethyl] phenylethanol (b)
To a solution of 2-(4-Hydroxyphenyl) ethanol (1.38 g, 10.00 mmol) and potassium carbonate (2.96 g, 20.00 mmol) in THF
(15 ml) was added 1,2-dibromoethane (15.00 mmol) slowly. The resulting mixture was stirred for 5 h at room temperature, then
washed with water and brine, dried over anhydrous sodium sulfate, filtered and evaporated. Purification by column
chromatography (30:1 petroleum ether:acetone) gave b1 as a yellow solid (2.17 g, 89% yield).
A solution of b1 (2.27 g, 10.00 mmol), silver nitrate (3.25 g, 20.00 mmol) in acetonitrile (12 ml) was stirred for 6 h at 70
under protection from light. The resulting mixture was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, filtered and evaporated. Purification by column chromatography (50:1 petroleum ether:acetone) gave
b as a yellow solid (1.93 g, 85% yield).
4.1.12. General procedure for the synthesis of 17-20 and 21-24
To a solution of the appropriate nitrate (a or b) (0.09 mmol), DMAP (0.06 mmol) and EDCI (0.05 mmol) in dry
dichloromethane (8 ml), compound 6-7 or 10-11 (0.03 mmol) was added. The mixture was stirred for 6 h at room temperature.
The organic solution was washed with 5% HCl, water and brine successively, dried over anhydrous sodium sulfate, concentrated
and purified by silica gel column chromatography (500:1 chloroform:methanol) to provide the compounds 21-24 and 17-20.
4.1.12.1. Phenyl [4-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-4-oxo]-4-[(2-nitroxy)ethyl] butyrate (17). Yield: 81% (after
chromatograph with chloroform/methanol, 500:1) as a white solid. mp 220-223 ; Rf = 0.45 (petroleum ether/ethyl acetate 4:1
1
v/v); [α]20 = 1.67 (c = 0.06, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.65 (3H, s, -CH3), 0.90 (3H, s, -CH3),
D
0.92 (3H, s, -CH3), 1.01 (3H, s, -CH3), 1.02 (3H, s, -CH3), 1.08 (3H, s, -CH3), 2.33-2.37 (1H, m, H-2A), 2.549-2.51 (1H, m,
H-2B), 2.69-2.71 (2H, m, -COCH2-), 2.81-2.85 (2H, m, -COCH2-), 2.92 (1H, dd, J = 3.9, 13.9 Hz, H-18), 3.0 (2H, t, J = 7.04 Hz,
-OCH2-), 4.13 (2H, s, H-23), 4.6 (2H, t, J =7.04 Hz, CH2Ar), 5.06 (1H, d, J = 12.52 Hz, CH2Ar), 5.11 (1H, d, J = 12.52 Hz
CH2Ar), 5.31 (1H, t, H-12), 7.05 (2H, d, J = 8.4 Hz, H-Ar), 7.21 (2H, d, J = 8.4 Hz, H-Ar), 7.31-7.35 (5H, m, H-Ar); 13C NMR
(100 MHz, CDCl3) δ 14.88, 16.82, 17.48, 19.40, 22.96, 23.38, 23.55, 25.61, 26.23, 27.47, 27.55, 29.02, 29.19, 30.63, 31.86,
32.24, 32.66, 33.04, 33.79, 34.82, 36.27, 37.58, 39.14, 41.40, 41.75, 45.77, 46.48, 46.70, 48.12, 50.25, 65.90, 67.69, 73.08, 76.68,
76.99, 77.20, 77.31, 121.8, 122.01, 127.89, 127.96, 128.37, 12978, 136.33, 143.78, 170.67, 171.63, 177.33, 214.36. ESI-HRMS
m/z calcd. for C49H63NO10 [M + H]+: 826.4452, found: 826.4525.
4.1.12.2. Phenethyl [4-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-4-oxo]-4-[O-(2-nitro)ethyl] butyrate (18). Yield: 77% (after
chromatograph with chloroform/methanol, 500:1) as a white solid. mp 210-213 ; Rf = 0.56 (petroleum ether/ethyl acetate 4:1

ACCEPTED MANUSCRIPT
v/v); [α]20D = 2.94 (c = 0.18, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.66 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.92
(3H, s, -CH3), 1.00 (3H, s, -CH3), 1.01 (3H, s, -CH3), 1.14 (3H, s, -CH3), 2.38-2.40 (1H, m, H-2A), 2.49-2.51 (1H, m, H-2B),
2.60-2.63 (2H, m, -COCH2-), 2.68-2.71 (2H, m, -COCH2-), 2.86-2.88 (2H, m, -OCH2-), 2.94 (1H, dd, J = 3.9, 13.9 Hz, H-18),
3.0 (2H, t, J = 7.04 Hz, -OCH2-), 4.13 (2H, s, H-23), 4.21-4.23 (2H, m, -OCH2-), 4.80 (2H, t, J = 4.56 Hz, CH2Ar), 5.06 (1H, d,
J = 12.52 Hz, CH2Ar), 5.11 (1H, d, J = 12.52 Hz CH2Ar), 5.31 (1H, t, H-12), 6.82(2H, d, J = 8.6 Hz), 7.12 (2H, d, J = 8.52 Hz),
7.31-7.37 (5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 14.89, 16.85, 17.52, 19.45, 23.00, 23.43, 23.58, 25.70, 27.56, 28.83,
29.02, 29.25, 29.67, 30.66, 31.90, 32.28, 33.05, 33.81, 34.12, 34.90, 36.29, 37.54, 38.33, 39.18, 41.45, 41.80, 45.78, 46.46, 46.72,
48.09, 50.21, 64.04, 65.30, 65.92, 67.75, 70.99, 76.24, 76.88, 77.20, 77.46, 114.61, 122.10, 127.91, 127.98, 128.39, 136.36,
143.80, 156.67, 171.82, 177.36. ESI-HRMS m/z calcd. for C51H67NO11 [M + H]+: 870.4714, found: 870.4786.
4.1.12.3. Phenyl [2-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-oxo]-4-[(2-nitroxy)ethyl] benzoate (19). Yield: 81% (after
chromatograph with chloroform/methanol, 500:1) as a white solid. mp 200-203 ; Rf = 0.48 (petroleum ether/ethyl acetate 4:1
v/v); [α]20D = 1.70 (c = 0.10, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.64 (3H, s, -CH3), 0.88 (3H, s, -CH3), 0.91
(3H, s, -CH3), 1.03 (3H, s, -CH3), 1.06 (3H, s, -CH3), 1.26 (3H, s, -CH3), 2.47-2.52 (2H, m, H-2), 2.89 (1H, dd, J = 3.9, 13.6 Hz,
H-18), 3.04 (2H, t, J = 7.04 Hz, -COCH2-), 4.30 (1H, d, J = 10.72 Hz, H-23A), 4.32 (1H, d, J = 10.40 Hz, H-23B), 4.6 (2H, t, J
= 7.04 Hz, CH2Ar), 5.06 (1H, d, J = 12.52 Hz, CH2Ar), 5.11 (1H, d, J = 12.52 Hz CH2Ar), 5.29 (1H, t, H-12), 7.25-7.27 (4H, m,
H-Ar), 7.31-7.36 (5H, m, H-Ar), 7.53-7.57 (2H, m, H-Ar), 7.71-7.73 (1H, m, H-Ar), 7.83-7.85 (1H, m, H-Ar); 13C NMR (100
MHz, CDCl3) δ 14.95, 16.87, 17.53, 19.49, 22.98, 23.44, 23.60, 25.57, 27.47, 28.23, 28.67, 29.71, 29.88, 30.69, 31.95, 32.28,
32.77, 33.10, 33.84, 34.80, 36.34, 37.74, 39.15, 41.46, 41.77, 45.82, 46.61, 46.73, 48.30, 50.52, 65.96, 68.30, 70.10, 70.67, 71.24,
71.66, 73.19, 73.33, 121.94, 122.10, 127.95, 127.21, 128.02, 128.43, 128.97, 129.22, 129.93, 131.42, 131.53, 132.45, 136.34,
143.76, 177.20. ESI-HRMS m/z calcd. for C53H63NO10 [M + Na]+: 896.4452, found: 896.4345.
4.1.12.4. Phenethyl [2-(benzyl 3-oxo-olean-12-en-28-oate-23-oxy)-oxo]-4-[O-(2-nitro)ethyl] benzoate (20). Yield: 78% (after
chromatograph with chloroform/methanol, 500:1) as a white solid. mp 200-203 ; Rf = 0.43 (petroleum ether/ethyl acetate 4:1
v/v); [α]20D = 2.50 (c = 0.12, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.64 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.91
(3H, s, -CH3), 1.03 (3H, s, -CH3), 1.06 (3H, s, -CH3), 1.25 (3H, s, -CH3), 2.48-2.52 (2H, m, H-2), 2.89 (1H, dd, J = 3.9, 13.6 Hz,
H-18), 3.00 (2H, t, J = 7.04 Hz, -COCH2-), 4.19-4.22 (2H, m, H-23), 4.20-4.24 (2H, m, -COCH2-), 4.45-4.48 (2H, m, -COCH2-),
4.80-4.83 (2H, m, CH2Ar), 5.06 (1H, d, J = 12.52 Hz, CH2Ar), 5.11 (1H, d, J = 12.52 Hz CH2Ar), 5.30 (1H, t, H-12), 6.8 (2H, d,
J = 8.56 Hz, H-Ar), 7.19 (2H, d, J = 8.56 Hz), 7.24-7.28 (4H, m, H-Ar), 7.31-7.36 (5H, m, H-Ar), 7.50-7.53 (2H, m, H-Ar),
7.61-7.66 (2H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 14.90, 16.86, 17.58, 19.49, 22.99, 23.42, 23.58, 25.57, 27.56, 28,11,
28.56, 29.68, 29.88, 30.67, 31.67, 31.95, 32.26, 33.06, 33.84, 34.83, 36.30, 37.57, 39.17, 41.47, 41.81, 45.77, 46.50, 46.73, 48.11,
50.29, 64.04, 65.94, 66.28, 68.42, 70.20, 70.34, 71.20, 71.86, 73.21, 73.33, 114.65, 122.14, 127.93, 128.00, 128.41, 128.68,
130.12, 130.82, 130.96, 131.73, 136.36, 143.76, 156.68, 167.05, 177.37. ESI-HRMS m/z calcd. for C55H67NO11 [M + H]+:
918.4714, found: 918.4775.

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4.1.12.5. Phenyl [4-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-oxo]-4-[(2-nitroxy)ethyl] butyrate (21). Yield: 81%
(after chromatograph with chloroform/methanol, 500:1) as a white solid. mp 220-223 ; Rf = 0.62 (chloroform/methanol
1
150:1 v/v); [α]20D = 14.44 (c = 0.18, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.60 (3H, s, -CH3), 0.80 (3H, s,
-CH3), 0.83 (3H, s, -CH3), 0.86 (3H, s, -CH3), 1.00 (3H, s, -CH3), 1.20 (3H, s, -CH3), 2.40-2.43 (2H, m, H-2), 2.53-2.58 (1H, m,
H-1A), 2.59-2.62 (2H, m, -COCH2-), 2.90-2.93 (1H, m, H-1B), 2.92-2.95 (1H, m, H-18), 2.94-2.98 (2H, m, -COCH2-),
4.25-2.67 (2H, m, H-23), 4.55 (2H, t, J = 2.3 Hz, -OCH2-), 5.00 (1H, d, J = 12.57 Hz, CH2Ar), 5.05 (1H, d, J = 12.54 Hz,
CH2Ar), 5.29 (1H, t, H-12), 7.05 (2H, d, J = 8.49 Hz, H-Ar), 7.21 (2H, d, J = 8.49 Hz, H-Ar), 7.31-7.37 (5H, m, H-Ar), 8.35,
8.38 (each 1 H, ds, J = 2.3 Hz, H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.76, 16.61, 19.83, 23.05, 23.31, 23.61, 25.67,
26.23, 27.56, 27.67, 28.43, 28.96, 29.09, 29.68, 30.71, 31.75, 32.32, 32.72, 33.13, 33.87, 36.55, 39.15, 41.49, 41.84, 43.23,
45.62, 45.84, 46.07, 46.77, 47.48, 65.97, 68.45, 68.99, 70,24, 71.03, 76.68, 76.99, 77.20, 77.31, 121.86, 122.22, 127.95,
128.02, 128.44, 129.84, 136.38, 142.53, 143.69, 149.63, 152.09, 171.42. ESI-HRMS m/z calcd. for C51H63N3O9 [M + H]+:
862.4564, found: 862.4645.
4.1.12.6. Phenethyl [4-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-4-oxo]-4-[O-(2-nitro)ethyl] butyrate (22). Yield: 75%
(after chromatograph with chloroform/methanol, 500:1) as a white solid. mp 223-227 ; Rf = 0.52 (chloroform/methanol 150:1
v/v); [α]20D = 11.69 (c = 0.16, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.70 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.92
(3H, s, -CH3), 0.95 (3H, s, -CH3), 1.19 (3H, s, -CH3), 1.29 (3H, s, -CH3), 2.39-2.42 (2H, m, H-2), 2.54-2.58 (1H, m, H-1A), 2.85
(2H, t, J = 7.08 Hz, -COCH2-), 2.92-2.95 (1H, m, H-1B), 2.95-2.99 (1H, m, H-18), 4.17-4.20 (2H, m, H-23), 4.24-4.33 (4H, m,
-OCH2-), 4.80-4.84 (2H, m, -OCH2-), 5.09 (1H, d, J = 12.57 Hz, CH2Ar), 5.10 (1H, d, J = 12.54 Hz, CH2Ar), 5.39 (1H, t, H-12),
6.86 (2H, d, J = 8.52 Hz, H-Ar), 7.28 (2H, d, J = 8.52 Hz, H-Ar), 7.33-7.37 (5H, m, H-Ar), 8.31, 8.44 (each 1 H, ds, J = 2.3 Hz,
H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.75, 16.63, 19.82, 23.08, 23.34, 23.60, 25.76, 27.61, 28.86, 28.93, 29.20, 29.78,
30.71, 31.81, 32.34, 33.09, 33.88, 34.13, 36.57, 38.66, 39.18, 41.53, 41.89, 43.22, 45.64, 45.82, 46.06, 46.80, 47.20, 47.66, 47.53,
64.07, 65.28, 65.97, 69.36, 71.02, 76.24, 76.88, 77.20, 77.46, 114.64, 122.27, 127.95, 128.02, 128.44, 130.01, 136.38, 142.46,
143.70, 152.22, 156.4, 171.87, 177.20. ESI-HRMS m/z calcd. for C53H67N3O10 [M + H]+: 906.4826, found: 906.4904.
4.1.12.7. Phenyl [2-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-oxo]-4-[(2-nitroxy)ethyl] benzoate (23). Yield: 73%
(after chromatograph with chloroform/methanol, 500:1) as a white solid. mp 196-200 ; Rf = 0.60 (chloroform/methanol 80:1
v/v); [α]20D = 1.25 (c = 0.08, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.70 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.92
(3H, s, -CH3), 0.95 (3H, s, -CH3), 1.19 (3H, s, -CH3), 1.29 (3H, s, -CH3), 2.39-2.42 (2H, m, H-2), 2.54-2.58 (1H, m, H-1A), 2.85
(2H, t, J = 7.08 Hz, -COCH2-), 2.92-2.95 (1H, m, H-1B), 2.95-2.99 (1H, m, H-18), 4.17-4.20 (2H, m, H-23), 4.24-4.33 (4H, m,
-OCH2-), 4.80-4.84 (2H, m, -OCH2-), 5.09 (1H, d, J = 12.57 Hz, CH2Ar), 5.10 (1H, d, J = 12.54 Hz, CH2Ar), 5.39 (1H, t, H-12),
6.86 (2H, d, J = 8.52 Hz, H-Ar), 7.28 (2H, d, J = 8.52 Hz, H-Ar), 7.33-7.37 (5H, m, H-Ar), 8.31, 8.44 (each 1 H, ds, J = 2.3 Hz,
H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.67, 16.57, 19.77, 19.83, 23.00, 23.27, 23.56, 25.59, 27.50, 29.67, 29.88, 30.67,
31.73, 32.26, 32.73, 33.06, 33.56, 33.81, 36.55, 39.09, 41.47, 41.81, 41.99, 43.41, 45.61, 45.81, 46.12, 46.73, 47.62, 65.92, 71.77,
76.24, 76.88, 77.20, 77.46, 121.87, 122.14, 127.91, 127.98, 128.40, 128.50, 128.94, 129.90, 130.01, 131.14, 131.20, 132.18,
133.80, 136.34, 141.66, 142.50, 143.68, 149.87, 152.16, 156.08. ESI-HRMS m/z calcd. for C55H63N3O9 [M + H]+: 910.4578,

ACCEPTED MANUSCRIPT
found: 910.4649.
4.1.12.8. Phenethyl [2-(benzyl olean[3,2-b]pyrazine-12-en-28-oate-23-oxy)-oxo]-4-[O-(2-nitro)ethyl] benzoate (24). Yield: 71%
(after chromatograph with chloroform/methanol, 500:1) as a white solid. mp 198-203 ; Rf = 0.59 (chloroform/methanol 150:1
v/v); [α]20D = 9.81 (c = 0.16, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.69 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.93
(3H, s, -CH3), 1.14 (3H, s, -CH3), 1.25 (3H, s, -CH3), 1.33 (3H, s, -CH3), 2.50 (1H, d, J = 16.5 Hz, H-1A), 2.92 (1H, dd, J = 3.3,
13.56 Hz, H-18), 2.97-2.99 (1H, m, H-1B), 3.0-3.2 (2H, m, H-2), 4.21-4.25 (2H, m, H-23), 4.37-4.41 (2H, m, -OCH2-), 4.45-4.49
(2H, m, -OCH2-), 4.80-4.83 (2H, m, -OCH2-), 5.09 (1H, d, J = 12.57 Hz, CH2Ar), 5.10 (1H, d, J = 12.54 Hz, CH2Ar), 5.36 (1H, t,
J = 3.2 Hz, H-12), 6.86 (2H, d, J = 8.6 Hz, H-Ar), 7.28 (2H, d, J = 8.4 Hz, H-Ar), 7.18-7.34 (7H, m, H-Ar), 7.45 (1H, td, J =
7.52, J = 1.16 Hz, H-Ar), 7.53 (1H, dd, J = 7.56, J = 0.96 Hz, H-Ar), 8.26 (1H, d, J = 2.36 Hz, H-pyrazinem)), 8.41 (1H, d, J =
1.92 Hz, H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.69, 16.60, 19.81, 19.86, 23.03, 23.30, 23.56, 25.66, 27.57, 29.68,
29.87, 30.68, 31.79, 32.28, 33.06, 33.83, 34.21, 36.57, 39.13, 41.50, 41.87, 43.35, 45.62, 45.80, 46.12, 46.76, 47.60, 64.02, 65.94,
66.14, 71.00, 71.68, 76.56, 76.89, 77.20, 114.63, 122.21, 127.92, 127.99, 128.23, 128.41, 128.71, 130.06, 130.70, 131.12, 131.44,
132.32, 136.35, 141.62, 142.46, 143.64, 152.23, 156.16, 156.68, 166.83, 167.34, 177.40. ESI-HRMS m/z calcd. for C57H63N3O10
[M + H]+: 954.4840, found: 954.4914.
4.1.13. General procedure for the synthesis of 27-28 and 31-32
To a solution of bromoacetic acid, 5-bromopentanoic acid or 6-bromohexanoic acid (0.06 mmol), triethylamine (2 drops),
DMAP (0.05 mmol) and EDCI (0.05 mmol) in dry dichloromethane (6 ml), compound 9 or 5 (0.04 mmol) was added. The
mixture was stirred for 5 h at room temperature, then washed with 5% HCl, water and brine successively, dried over anhydrous
sodium sulfate, concentrated and purified by silica gel column chromatography (16:1 petroleum ether:ethyl acetate) to provide
the compounds 25-26 and 29-30.
A solution of 25 (or 26, 29, 30) (0.03 mmol), silver nitrate (0.07 mmol) in dry acetonitrile (8 ml) was stirred for 8 h at 75
under the conditions of protection from light. The mixture was washed with 10% HCl, water and brine successively, dried over
anhydrous sodium sulfate, concentrated and purified by column chromatography to provide the compounds 27-28 and 31-32.
4.1.13.1. Benzyl 3-oxo-23-(5-nitrooxy-valeryl)-olean-12-en-28-oate (27). Yield: 70% (after chromatograph with petroleum
ether/ethyl acetate, 12:1) as a white solid. mp 92-95 ; Rf = 0.63 (petroleum ether/ethyl acetate 4:1 v/v); [α]20D = 11.35 (c = 0.20,
1
CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.67 (3H, s, -CH3), 0.91 (3H, s, -CH3), 0.94 (3H, s, -CH3), 1.02 (3H, s,
-CH3), 1.05 (3H, s, -CH3), 1.13 (3H, s, -CH3), 2.37 (2H, t, J = 6.75 Hz, -COCH2-), 2.43-2.60 (2H, m, H-2), 2.94 (1H, dd, J = 3.9,
13.9 Hz, H-18), 4.10 (2H, s, H-23), 4.45 (2H, d, J = 5.88 Hz, -OCH2-), 5.01 (1H, d, J = 12.57 Hz, CH2Ar), 5.05 (1H, d, J =
12.54 Hz, CH2Ar), 5.25 (1H, t, H-12), 7.31-7.37 (5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 14.85, 16.82, 17.48, 19.39, 21.13,
22.93, 23.38, 23.55, 25.54, 26.10, 27.49, 29.63, 30.63, 31.98, 32.22, 33.02, 33.45, 33.76, 34.79, 36.24, 37.67, 39.14, 41.40, 41.72,
45.70, 46.62, 46.67, 48.30, 50.23, 65.89, 67.33, 72.59, 122.10, 127.89, 127.96, 128.36, 136.31, 143.70, 177.39, 177.32, 214.30.

ACCEPTED MANUSCRIPT
ESI-HRMS m/z calcd. for C42H59NO8 [M + H]+: 706.4227, found: 706.4303.
4.1.13.2. Benzyl 3-oxo-23-(6-nitrooxy-hexanoyl)-olean-12-en-28-oate (28). Yield: 68% (after chromatograph with petroleum
ether/ethyl acetate, 8:1) as a white solid. mp 92-95 ; Rf = 0.67 (petroleum ether/ethyl acetate 4:1 v/v); [α]20D = 10.21 (c = 0.14,
1
CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.67 (3H, s, -CH3), 0.91 (3H, s, -CH3), 0.94 (3H, s, -CH3), 1.02 (3H, s,
-CH3), 1.05 (3H, s, -CH3), 1.13 (3H, s, -CH3), 2.37 (2H, t, J = 6.75 Hz, -COCH2-), 2.43-2.60 (2H, m, H-2), 2.94 (1H, dd, J = 3.9,
13.9 Hz, H-18), 4.10 (2H, s, H-23), 4.45 (2H, d, J = 5.88 Hz, -OCH2-), 5.01 (1H, d, J = 12.57 Hz, CH2Ar), 5.05 (1H, d, J =
12.54 Hz, CH2Ar), 5.25 (1H, t, H-12), 7.31-7.37 (5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 14.91, 16.88, 17.56, 19.46, 21.57,
22.88, 23.00, 23.46, 23.61, 24.45, 25.19, 25.65, 26.47, 27.58, 30.70, 32.05, 32.29, 33.07, 33.83, 33.93, 34.88, 36.31, 37.75, 39.21,
41.49, 41.80, 45.79, 46.69, 46.75, 48.33, 50.29, 65.96, 67.36, 72.95, 122.19, 127.96, 128.03, 128.43, 136.40, 143.79, 177.20,
214.30. ESI-HRMS m/z calcd. for C43H61NO8 [M + H]+: 720.4397, found: 720.4479.
4.1.13.3. Benzyl 23-(5-nitrooxy-valeryl)-olean[3,2-b]pyrazine-12-en-28-oate (31). Yield: 71% (after chromatograph with
petroleum ether/ethyl acetate, 8:1) as a white solid. mp 75-78 ; Rf = 0.45 (petroleum ether/ethyl acetate 4:1 v/v); [α]20D = 20.64
(c = 0.14, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.59 (3H, s, -CH3), 0.78 (3H, s, -CH3), 0.81 (3H, s, -CH3), 0.83
(3H, s, -CH3), 1.07 (3H, s, -CH3), 1.15 (3H, s, -CH3), 2.05 (2H, t, J = 8.04 Hz, -COCH2-), 2.41 (1H, d, J = 16.53 Hz, H-1A), 2.90
(1H, dd, J = 3.3, 13.56 Hz, H-18), 2.95 (1H, d, J = 16.53 Hz, H-1B), 4.20 (2H, s, H-23), 4.24 (2H, m, -OCH2-), 4.97 (1H, d, J =
12.54 Hz, CH2Ar), 5.03 (1H, d, J = 12.54 Hz, CH2Ar), 5.28 (1H, t, H-12), 7.31-7.37 (5H, m, H-Ar), 8.21, 8.35 (each 1 H, ds, J =
2.3 Hz, H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.74, 16.62, 19.82, 21.11, 23.06, 23.33, 23.60, 25.60, 25.98, 27.56, 29.12,
29.66, 30.72, 30.89, 31.90, 32.32, 33.09, 33.87, 36.54, 39.16, 41.53, 41.86, 43.23, 45.78, 45.84, 46.79, 47.53, 65.97, 70.71, 71.86,
71.97, 72.48, 122.26, 127.96, 128.03, 128.44, 136.38, 141.25, 142.59, 143.63, 152.02, 172.30, 177.30. ESI-HRMS m/z calcd. for
C44H59N3O7 [M + H]+: 742.4353, found: 742.4417.
4.1.13.4. Benzyl 23-(6-nitrooxy-hexanoyl)-olean[3,2-b]pyrazine-12-en-28-oate (32). Yield: 75% (after chromatograph with
petroleum ether/ethyl acetate, 8:1) as a white solid. mp 75-78 ; Rf = 0.54 (petroleum ether/ethyl acetate 4:1 v/v); [α]20D = 25.45
(c = 0.22, CH3OH/CHCl3 3:2 v/v); 1H NMR (400 MHz, CDCl3) δ 0.70 (3H, s, -CH3), 0.89 (3H, s, -CH3), 0.93 (3H, s, -CH3), 0.95
(3H, s, -CH3), 1.18 (3H, s, -CH3), 1.29 (3H, s, -CH3), 2.05 (2H, t, J = 8.04 Hz, -COCH2-), 2.45 (1H, d, J = 16.53 Hz, H-1A), 2.90
(1H, dd, J = 3.3, 13.56 Hz, H-18), 3.0 (1H, d, J = 16.53 Hz, H-1B), 4.29 (2H, s, H-23), 4.38 (2H, t, J = 6.6 Hz, -OCH2-), 5.0 (1H,
d, J = 12.54 Hz, CH2Ar), 5.1 (1H, d, J = 12.54 Hz, CH2Ar), 5.39 (1H, t, H-12), 7.31-7.37 (5H, m, H-Ar), 8.21, 8.35 (each 1 H, ds,
J = 2.3 Hz, H-pyrazinem); 13C NMR (100 MHz, CDCl3) δ 15.70, 16.62, 19.81, 23.06, 23.34, 23.61, 24.37, 25.63, 26.37, 27.58,
29,22, 29.68, 30.72, 30.89, 31.91, 32.32, 33.08, 33.88, 36.55, 39.17, 41.53, 41.86, 43.23, 45.85, 45.84, 46.27, 46.79, 47.69, 65.97,
70.61, 71.66, 71.84, 72.84, 122.28, 127.96, 128.03, 128.44, 136.38, 141.45, 142.50, 143.65, 152.15, 173.50, 177.42. ESI-HRMS
m/z calcd. for C45H61N3O7 [M + H]+: 756.4510, found: 756.4579.

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4.1.14. General procedure for the synthesis of 35 and 36
To a solution of bromoacetic acid, 5-bromopentanoic acid or 6-bromohexanoic acid (0.12 mmol), triethylamine (2 drops),
DMAP (0.05 mmol) and EDCI (0.08 mmol) in dry dichloromethane (6 ml), compound 1 (0.04 mmol) was added. The mixture
was stirred for 5 h at room temperature, then washed with 5% HCl, water and brine successively, dried over anhydrous sodium
sulfate, concentrated and purified by silica gel column chromatography (8:1 petroleum ether:ethyl acetate) to provide the
compounds 33 or 34.
A solution of 33 or 34 (0.03 mmol), silver nitrate (0.07 mmol) in dry acetonitrile (8 ml) was stirred for 8 h at 75 under the
conditions of protection from light. The mixture was washed with 10% HCl, water and brine successively, dried over anhydrous
sodium sulfate, concentrated and purified by column chromatography (12:1 petroleum ether:ethyl acetate) to provide the
compounds 35 or 36.
4.1.14.1. Benzyl 3-(5-nitrooxy-valeryl)-23-(5-nitrooxy-valeryl)-olean-12-en-28-oate (35). Yield: 61% (after chromatograph with
petroleum ether/ethyl acetate, 6:1) as a white solid. mp 135-138 ; Rf = 0.66 (chloroform/methanol 150:1 v/v); [α]20D = 19.23 (c
1
= 0.22, CH3OH/CHCl3 3:2 v/v); H NMR (400 MHz, CDCl3) δ 0.60 (3H, s, -CH3), 0.83 (3H, s, -CH3), 0.90 (3H, s, -CH3), 0.92
(3H, s, -CH3), 0.94 (3H, s, -CH3), 1.11 (3H, s, -CH3), 2.30 (2H, t, J = 7.73 Hz, H-2), 2.31-2.43 (4H, m, -COCH2-), 2.91 (1H, dd,
J = 4.0, 13.92 Hz, H-18), 3.72 (1H, d, J = 11.64 Hz, H-23A), 3.64 (1H, d, J = 11.6 Hz, H-23B), 4.44-4.47 (4H, m, -OCH2-),
4.75-4.80 (1H, m, H-3), 5.05 (1H, d, J = 12.52 Hz, CH2Ar), 5.11 (1H, d, J = 12.56 Hz, CH2Ar), 5.30 (1H, t, H-12), 7.31-7.37
(5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 13.01, 15.76, 16.81, 17.89, 21.11, 21.14, 22.95, 23.33, 23.59, 25.18, 25.65, 26.17,
26.22, 27.53, 29.65, 30.65, 32.31, 33.03, 33.55, 33.74, 33.80, 36.71, 37.76, 39.24, 40.67, 41.35, 41.58, 45.79, 46.69, 47.72, 47.90,
49.20, 65.09, 65.87, 72.67, 74.36, 122.25, 127.87, 127.95, 128.36, 136.40, 143.66, 172.26, 172.50, 177.34. ESI-HRMS m/z calcd.
for C47H68N2O12 [M + Na]+: 875.4786, found: 875.4638.
4.1.14.2. Benzyl 3-(6-nitrooxy-hexanoyl)-23-(6-nitrooxy-hexanoyl)-olean-12-en-28-oate (36). Yield: 65% (after chromatograph
with petroleum ether/ethyl acetate, 6:1) as a white solid. mp 135-138 ; Rf = 0.66 (chloroform/methanol 150:1 v/v); [α]20
=
D
11.83 (c = 0.18, CH3OH/CHCl3 3:2 v/v); 1H NMR (CDCl3, 400 MHz) δ 0.60 (3H, s, -CH3), 0.83 (3H, s, -CH3), 0.90 (3H, s, -CH3),
0.92 (3H, s, -CH3), 0.94 (3H, s, -CH3), 1.11 (3H, s, -CH3), 2.30 (2H, t, J = 7.73 Hz, H-2), 2.35 (4H, t, J = 7.2 Hz, -COCH2-), 2.90
(1H, dd, J = 4.0, 13.92 Hz, H-18), 3.70 (1H, d, J = 11.56 Hz, H-23A), 3.85 (1H, d, J = 11.6 Hz, H-23B), 4.45 (4H, m, -OCH2-),
4.75-4.80 (1H, m, H-3), 5.05 (1H, d, J = 12.56 Hz, CH2Ar), 5.11 (1H, d, J = 12.56 Hz, CH2Ar), 5.30 (1H, t, H-12), 7.31-7.37
(5H, m, H-Ar); 13C NMR (100 MHz, CDCl3) δ 13.12, 15.73, 16.82, 17.88, 21.12, 21.14, 22.94, 22.96, 23.33, 23.59, 25.11, 25.19,
25.68, 26.45, 26.48, 27.55, 29.95, 30.65, 32.30, 33.02, 33.80, 34.00, 34.20, 36.69, 37.80, 39.24, 40.61, 41.36, 41.58, 45.79, 46.68,
47.74, 47.86, 65.04, 65.87, 72.96, 74.24, 122.28, 127.86, 127.95, 128.36, 136.4, 143.64, 172.63, 172.88, 177.33. ESI-HRMS m/z
calcd. for C49H72N2O12 [M + Na]+: 903.5099, found: 903.4993.
4.2. Biology

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4.2.1. Cell culture and treatment
The cancer cell lines KB, KBV, Hela, A549, MKN45, BGC-823 and AGS were obtained from the Cell Resource Center, the
Shanghai Institute of Life Sciences (Shanghai, China). All cells were cultured in DMEM (Hyclone) with 10% heat-inactivated
fetal calf serum (Life Technology), and 100 U/ml penicillin and l00 g/ml streptomycin, in an atmosphere of 5% CO2 at 37°C.
Cells in logarithmic growth phase were used for further experiments.
4.2.2. MTT assay
The cell viability was measured using an MTT assay according to our previously published protocol. Briefly, the cells were
seeded into 96-well plates and treated with the tested articles at the desired concentration for 72 h. MTT solution was then added
into the wells and incubated, and then DMSO was added and the optical density was measured at 570 nm, in which the cell
survival rate were calculated.
4.2.3. Flow cytometric analysis
The cell cycle analysis was measured using the flow cytometry according to our previously published protocol. Briefly,
MKN45 cells (2×105 cells/well) were cultured in 6-well plates and exposed to the indicated concentrations of compound for 24
and 48 h. The cells were harvested and fixed, and then stained by propidium iodide solution. Fluorescence emitted from the
propidium iodide-DNA complex was quantitated after excitation of the fluorescent dye by FACScan cytometry.
4.2.4. Western blotting assay
The cells were collected and prepared for the lysates after treatment, and then the cell lysates were performed to detect the
target protein as previously described. Briefly, total cellular proteins were electrophoresed, and were then transferred to a PVDF
membrane (Millipore, USA). Subsequently, the membranes were blocked and then incubated overnight at 4˚C with primry
antibodies to Bax (2772, USA), BCL2 (2876, USA), PARP (46D11, USA) and β-actin (C4,USA). The membranes were washed
thrice and incubated with secondary antibodies to goat anti-mouse IgG conjugated to horseradish peroxidase, then visualized
with an enhanced chemiluminescence detection kit (GE, USA).
4.2.5. Mitochondrial membrane potential
MKN45 cells (2×105 cells/well) were cultured in 6-well plates and exposed to the indicated concentrations of compound for
24 h. After that, the cells were staining with JC-1 solution for 20 minutes and then detected with flow cytometry, in which the
changes in mitochondrial membrane potential due to mitochondrial damage was calculated.
Acknowledgements

ACCEPTED MANUSCRIPT
These work supported by the State Key Laboratory of Drug Research (SIMM1705KF-07), Taishan Scholar Project, Graduate
Innovation Foundation of Yantai University, GIFYTU.
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Design, synthesis and biological evaluation of novel α-hederagenin
derivatives with anticancer activity
Xian-xuan Liu1,#, Yan-ting Yang1,#, Xiao Wang1, Kai-yi Wang1, Jia-qi Liu1, Lei Lei1,2,Xiao-min
Luo3, Rong Zhai1, Feng-hua Fu1, Hong-bo Wang1,*, Yi Bi1,3,
*
1School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation
Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, China
2State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing, 100050, China
3State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
*Correspondence: hongbowangyt@gmail.com (H.W.),beeyee_413@163.com (Y. B.)
# These authors contributed equally to this work
Highlights：


Synthesis of 24 novel α-hederagenin derivatives.
α-Hederagenin derivative 15 was active against human cancer lines, such as Hela (IC50=4.22
± 0.15 μM) and KBV (IC50=8.05 ± 0.08 μM).

It was the first time to discovered that the active molecule induces apotosis through the
mitochondrial pathway.