Synthesis, biological evaluation and mechanism studies of C-23 modified 23-hydroxybetulinic acid derivatives as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2019.111659

A series of C-23 modified 23-hydroxybetulinic acid (HBA) derivatives were synthesized and evaluated for their antiproliferative activity against a panel of cancer cell lines (A2780, A375, B16, MCF-7 and HepG2). The biological screening results showed that most of the derivatives exhibited more potent antiproliferative activity than HBA, and compound 6e exhibited the most potent activity with IC₅₀ values of 2.14 μM, 2.89 μM, and 3.97 μM against A2780, B16, and MCF-7 cells, respectively. Further anticancer mechanism studies revealed that compound 6e induced the generation of intracellular reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) of B16 cells in a dose-dependent manner. Moreover, western blot analysis indicated that compound 6e downregulated the expression of anti-apoptotic protein Bcl-2 and upregulated the expression of proapoptotic protein Bax, activation of caspase 3 to induce cell apoptosis. Meanwhile, compound 6e significantly inhibited the phosphorylation of MEK, ERK, and Akt without affecting the expression of MEK, ERK, and Akt. Furthermore, the in vivo anti-tumor activity of 6e was validated (tumor inhibitory ratio of 68.4% at the dose of 30 mg/kg) in mice with B16 melanoma.

Full text of DOI:10.1016/j.ejmech.2019.111659

Journal Pre-proof
Synthesis, biological evaluation and mechanism studies of C-23 modified 23-
hydroxybetulinic acid derivatives as anticancer agents
Lixue Lu, Hengyuan Zhang, Jie Liu, Yang Liu, Yiwei Wang, Shengtao Xu, Zheying
Zhu, Jinyi Xu
PII:
S0223-5234(19)30803-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111659
EJMECH 111659
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2019
Revised Date: 21 August 2019
Accepted Date: 28 August 2019
Please cite this article as: L. Lu, H. Zhang, J. Liu, Y. Liu, Y. Wang, S. Xu, Z. Zhu, J. Xu, Synthesis,
biological evaluation and mechanism studies of C-23 modified 23-hydroxybetulinic acid derivatives
as anticancer agents, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111659.
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Synthesis, Biological Evaluation and Mechanism Studies of
C-23 Modified 23-Hydroxybetulinic Acid Derivatives as
Anticancer Agents
Lixue Lu a, b, Hengyuan Zhang a, Jie Liu b, , Yang Liu a, Yiwei Wanga, Shengtao Xu a,
Zheying Zhuc, Jinyi Xu a,
a
State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
b Department of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing,
210009, PR China
cDivision of Molecular Therapeutics & Formulation, School of Pharmacy, The University of
Nottingham, University Park Campus, Nottingham NG7 2RD, UK
* Corresponding authors: E-Mail: jinyixu@china.com; cpu-jill@163.com
.
Tel.: +86-25-86185328 (J.Y. Xu)
Abstract
A series of C-23 modified 23-hydroxybetulinic acid (HBA) derivatives were
synthesized and evaluated for their antiproliferative activity against a panel of cancer
cell lines (A2780, A375, B16, MCF-7 and HepG2). The biological screening results
showed that most of the derivatives exhibited more potent antiproliferative activity
than HBA, and compound 6e exhibited the most potent activity with IC50 values of
2.14 µM, 2.89 µM, and 3.97 µM against A2780, B16, and MCF-7 cells, respectively.
Further anticancer mechanism studies revealed that compound 6e induced the
generation of intracellular reactive oxygen species (ROS) and reduction of
mitochondrial membrane potential (MMP) of B16 cells in a dose-dependent manner.

Moreover, western blot analysis indicated that compound 6e downregulated the
expression of anti-apoptotic protein Bcl-2 and upregulated the expression of
proapoptotic protein Bax, activation of caspase 3 to induce cell apoptosis. Meanwhile,
compound 6e significantly inhibited the phosphorylation of MEK, ERK, and Akt
without affecting the expression of MEK, ERK, and Akt. Furthermore, the in vivo
anti-tumor activity of 6e was validated (tumor inhibitory ratio of 68.4% at the dose of
30 mg/kg) in mice with B16 melanoma.
Keywords: 23-hydroxybetulinic acid, structural modification, antiproliferative
activity, melanoma, anticancer mechanism
1. Introduction
Natural products have been one of the most important sources of leads for the
development of drugs for the treatment of cancer [1]. Biologically active polycyclic
triterpenoids (generally triterpenoids with dammarane, lanostane, lupane, oleanane
and ursane skeletons) are one of the most promising groups of naturally occurring
compounds being used as starting molecules in the synthetic transformations [2] for
novel drug discovery. Betulinic acid (BA), one of the well-known and well-studied
pentacyclic lupane-type triterpenes, has been reported to possess a wide spectrum of
biological and pharmacological activities, such as anti-HIV, anti-tumor, and
anti-inflammatory, etc [3].
HBA (1), isolated from the root of Pulsatilla chinensis, is considered as hydroxy
substituted BA and displays similar anti-tumor activities to BA [4]. Furthermore, HBA
exhibited the synergistic cytotoxicity with clinically used drugs such as doxorubicin
(DOX) and paclitaxel in vitro and in vivo [5]. In addition, the HBA derivatives could
inhibit the activity of a series of ATP-Binding Cassette (ABC) transporters which are
known as major factors contributing to multidrug-resistance (MDR) in cancer cells [6,
7]. These results demonstrated that HBA derivatives might be potential therapeutic
leads for further research to develop anticancer agents.

Fig. 1. Chemical structures of betulinic acid, 23-hydroxybetulinic acid and compound 2
Previous studies have revealed that HBA exhibited potent apoptotic activity by
increasing the formation of intracellular ROS and reducing the MMP of murine
melanoma B16 cells [4]. Besides, HBA induced apoptosis of human leukemia HL-60
cells by inhibiting the expression of Bcl-2 and telomerase activity [8]. The results of
subcellular localization and uptake profile studies of fluorescent HBA probe
suggested that mitochondrion was the main site of probe accumulation in B16F10
cells [9]. The mitochondrion-targeted apoptosis in cancer cells along with the low
toxicity in normal cells have made HBA derivatives as new promising
chemotherapeutic candidates for the intervention of cancers.
Previously, our group has reported a series of C-17 modified HBA ester and
amide derivatives [10, 11], we also reported a series of heterocyclic ring-fused HBA
derivatives, including isoxazole, oxadiazole, pyrazole and pyrazine [12-14]. The
promising antiproliferative activity of these derivatives (such as compound 2)
prompted us to further design and synthesize novel HBA derivatives. Our previous
work mainly focused on the chemical modification of A ring and C-28 position of
HBA, less attention was paid to the modification of C-23 position. To improve the
anticancer activity of HBA and further supplement its structure-activity relationships
(SARs), a series of novel C-23 modified HBA derivatives were designed and
synthesized. Herein, we would like to report their synthesis, SARs and anti-tumor
activities. In addition, the underlying cytotoxic mechanisms of representative
compound 6e were also elucidated.
2. Results and discussion

2.1 Chemistry
HBA was isolated from the roots of Pulsatilla chinensis (Bge) Regel [15], and
1
characterized by H NMR, 13C NMR and HR-MS. Derivatives 3a-h, 3j-s were
synthesized by the reaction of HBA with corresponding acid/amide under
condensation condition. Reduction of derivative 3h afforded compound 3i. (Scheme
1).
As outlined in Scheme 2, HBA was protected with BnBr in DMF to yield
28-benzyl-23-hydroxybetulinic ester 4. To synthesize derivative 6a, the C-23
hydroxyl group of compound 4 reacted with 4-(bis(2-chloroethyl)amino)benzoic acid
firstly, and then debenzylation of compound 4 in THF with Pd/C as a catalyst under
atmospheric pressure of hydrogen afforded compound 6a. Intermediates 5d-e were
obtained using the similar synthetic method for the preparation of 6a, the removal of
the protecting group with CF3COOH produced the desired derivatives 6d-e.

H
H
OH
OH
a
H
H
O
O
H
H
HO
HO
HO
RO
HBA
3a-h, 3j-s
H
H
H
H
OH
OH
b
O
O
H
H
HO
RO
HO
RO
3h
3i
O
R =
O
O
O
O
3e
3j
3a
3b
3g
3c
3h
3d
3i
O
H
CH3
H
NO2
H
O
O
O
O
O
O
O
NH2
CF3
3f
NO2
NO2
F
O2N
NO2
O
O
O
F
3n
3o
F
3k
3p
3l
3m
3r
F
F
F3C
Cl
N
O
O
O
O
S
O
3q
3s
N
O
H
N
H
Scheme 1. Synthesis of derivatives 3a-s. Reagents and conditions: (a) Acid/Amide, DCC/EDCI,
DMAP, pyridine, rt, 8-24 h; (b) H2, 10% Pd/C, THF, rt.

H
H
H
OH
OBn
OBn
H
H
H
a
b
O
O
O
H
H
H
HO
HO
HO
HO
HO
RO
HBA
5a-c
4
H
H
OH
OH
H
H
c
d
O
O
H
H
HO
RO
HO
RO
6d-e
6a-c
R =
Cl
O
N
6a
Cl
O
O
6d
6e
6b
6c
NH2
NHBoc
N-Boc
O
O
NH
Scheme 2. Synthesis of 6a, and 6d-e. Reagents and conditions: a) BnBr, K2CO3, DMF, rt; b)
protected amino acid, DCC/EDCI, DMAP, pyridine, rt, 8-24 h; c) H2, 10% Pd/C, THF, rt; d)
CF3COOH, CH2Cl2, 0 °C - rt, 2 h.
2.2 Biological evaluation
2.2.1 In vitro antiproliferative activity
In order to analyze the effect of different C-23 substituted groups on the
antiproliferative activity, twenty-two compounds 3a-s, 6a, and 6d-e with varying
substitution groups were preliminarily screened for their antiproliferative activities in
B16 cells by using MTT assay. The results in Fig. 2 suggested that most of the target
compounds with substitutions at C-23 position exhibited more potent cytotoxicity at
10 µM than HBA.

Fig. 2. Histograms display the inhibitory rates of compounds 3a-s, 6a, and 6d-e against B16 cells.
Based on the above results, we further evaluated the in vitro antiproliferative
activities of representative C-23 modified HBA derivatives on five cancer cell lines
(A2780 human ovarian cancer, A375 human melanoma, B16 mice melanoma, MCF-7
human breast cancer, and HepG2 human liver cancer cells) and one non-cancer cell
lines (L-O2 human normal liver cell line) by MTT assay with doxorubicin as the
positive control. The results summarized in Table 1 are presented as the concentration
of drug inhibiting 50% cell growth (IC50).

Table 1. Antiproliferative activities of C-23 modified HBA derivatives against different tumor
cell lines and human normal liver cell line.
IC50 values (µM)a
Comp.
A2780
A375
B16
MCF-7
HepG2
L-O2
3b
3d
11.31 ± 2.51 23.73 ± 1.79 16.70 ± 1.22 11.40 ± 0.85 20.28 ± 1.52 33.27 ± 1.99
19.94 ± 2.11 15.70 ± 1.08 17.62 ± 1.59 18.80 ± 1.21 12.46 ± 0.73 26.54 ± 1.32
24.19 ± 0.89 20.22 ± 1.45 21.54 ± 1.68 19.18 ± 0.99 21.12 ± 2.58 35.21 ± 1.43
3g
3k
19.78 ± 1.23 23.73 ± 3.01 17.21 ± 1.71 18.75 ± 0.98 16.24 ± 1.06
34.15 ± 1.88
3l
7.26 ± 3.51 13.94 ± 3.51 10.30 ± 3.51 9.52 ± 3.51 10.67 ± 3.51 21.76 ± 3.51
3m
3n
42.97 ± 3.52 43.58 ± 4.31 44.60 ± 3.61 41.84 ± 3.87 44.42 ± 2.65 49.26 ± 3.66
22.61 ± 3.41 20.40 ± 1.54 26.96 ± 3.01 24.84 ± 2.11 18.71 ± 1.31 33.61 ± 1.21
3o
12.23 ± 0.82 13.28 ± 0.76
7.54 ± 0.37
5.61 ± 0.51
13.33 ± 0.98 21.72 ± 1.62
3q
16.18 ± 0.99 26.14 ± 1.72 23.06 ± 1.82 16.92 ± 1.33 22.00 ± 1.98 36.87 ± 3.92
c
-
6a
18.50 ± 1.42 21.50 ± 2.51
8.49 ± 0.66
3.95 ± 0.22
2.89 ± 0.11
26.83 ± 1.97 24.34 ± 2.61
6d
2.85 ± 0.17
2.14 ± 0.08
5.55 ± 0.48
4.84 ± 0.32
3.16 ± 0.29
3.97 ± 0.16
2.71 ± 0.22
3.14 ± 0.22
11.62 ± 0.13
13.39 ± 0.43
6e
HBA
ADM b
79.12 ± 6.50 76.30 ± 5.31 70.33 ± 4.71 74.98 ± 7.38 71.84 ± 5.56 79.15 ± 9.01
0.18 ± 0.02 0.19 ± 0.02 0.21 ± 0.03 0.18 ± 0.001 0.16 ± 0.02 0.14 ± 0.01
a Each data represents mean ± S.D. from three different experiments performed in triplicate.
bADM: Doxorubicin.
cNot determined.
The data in Table 1 indicated that most of the C-23 modified HBA derivatives
exhibited markedly improved antiproliferative activity than HBA, especially against
human ovarian cancer cells A2780 and mice melanoma cells B16. Among which,
compounds 3b, 3d, 3g, 3k-o, and 3q exhibited more potent activity than HBA,
indicating that large steric hindrance substitution at the C-23 position of HBA was
more favorable. Moreover, electron-withdrawing substituents and halogens, especially
fluorine, on benzene rings at the C-23 position were more favored, as exemplified by

compounds 3k, 3l, 3n and 3o.
In order to investigate the effect of hydrophilic amino side chains on
antiproliferative activity, we designed and synthesized compounds 3i, 3r-s, and 6d-e.
In which, compounds 6d-e exhibited significantly improved antiproliferative efficacy
relative to HBA, and were about 3- to 4-fold more potent than compounds 3l and 3o,
which revealed that the introduction of hydrophilic amino side chains was more
beneficial for the activity than the aromatic counterparts. Compound 6e displayed the
most potent activity with IC50 values ranging from 2.14 µM to 4.84 µM against five
cancer cell lines, which is about 30-fold more potent than the parent compound HBA.
Futhermore, IC50 value of compounds 6e for L-O2 cell line is 13.39 µM, which is 4-5
fold less potent than those of in cancer cells. Given the potent antiproliferative activity
and selectivity of compound 6e, it was finally selected as the candidate for further
anticancer mechanism studies.
2.2.2 Compound 6e induced the generation of intracellular ROS.
ROS plays an important role in the induction of physiological and pathological
apoptosis [16], which are mainly produced in mitochondria. To determine the effects
of compound 6e on ROS generation, the fluorescent probe 2′, 7′-dichlorofluorescein
diacetate (DCF-DA) was used to evaluate the intracellular ROS levels. As shown in
Fig. 3, the ratio of DCF-positive cells was increased from 3.65% in cells incubated
with DMSO to 21.2% in cells incubated with 3 µM of 6e. The result demonstrated
that compound 6e significantly enhanced the intracellular ROS levels in a
dose-dependent manner.

Fig. 3. Effect of 6e on ROS generation in B16 cells. A) Generation of ROS was measured using
the ROS-detecting fluorescent dye DCF-DA in combination with FACScan flow cytometry; B)
Histogram displays the DCF-positive cell radio in different concentrations (0, 0.75, 1.5 and 3 µM)
of 6e.
2.2.3 Compound 6e reduced MMP.
Increasing evidence has indicated that mitochondria plays an important role in
regulating cellular functions, which is involved in many pathological processes [17].
Therefore, we further tested the effect of compound 6e on MMP using fluorescent
probe JC-1. As shown in Fig. 4, with the increase of concentrations of 6e from 0 to 3
µM, the green fluorescence intensity (JC-1 monomers, low MMPs) correspondingly
increased from 1.57 to 27.05%, which indicated that compound 6e caused MMP
collapse in a dose-dependent manner.

Fig. 4. Effect of 6e on MMP of B16 cells. A) After incubation with different concentrations (0,
0.75, 1.5 and 3 µM) of 6e in B16 cells for 48 h prior to staining with JC-1 dye, the number of cells
with collapsed MMPs was determined by flow cytometry analysis; B) Histogram displays the
green fluorescence intensity in the absence or presence of 6e.
2.2.4 Compound 6e regulated the expression of cell death-related proteins
2.2.4.1 Effects on mitochondrial apoptosis pathway
To illustrate the potential mechanisms of apoptosis induced by compound 6e, the
expression of several apoptosis-related proteins (Bcl-2, Bax, and Caspase 3) in B16
cells were determined by Western blot. As shown in Fig. 5, the expression level of
caspase 3 in B16 cells was dramatically increased in a dose-dependent manner after
treatment with compound 6e (0.75, 1.5 and 3 µM). Meanwhile, treatments with
compound 6e also significantly upregulated the expression of the pro-apoptotic
protein Bax and downregulated the expression of the anti-apoptotic protein Bcl-2 in a
dose-dependent manner. Collectively, compound 6e triggered mitochondrial apoptosis
pathway and induced cell apoptosis.

Fig. 5. Effect of compound 6e on the expression levels of Bcl-2, Bax, and Caspase 3. A) Western
blot analysis of 6e on the expression of apoptosis-related proteins in B16 cells treated with
different concentrations (0, 0.75, 1.5 and 3 µM) of 6e; B) Histogram displays the density ratios of
Bcl-2, Bax, and Caspase 3 to GADPH: *p <0.05, **p <0.01, ***p <0.001 vs vehicle control.
2.2.4.2 Effects on signaling pathway MEK/ERK, and PI3K/Akt
Ras/Raf/MEK/ERK and PI3K/Akt are two important survival signaling pathways
in malignant melanoma cells [18], so we further tested the effects of compound 6e on
these two signaling pathways by Western blot in B16 cells. As shown in Fig. 6,
compound 6e exhibited significant inhibition on phosphorylation of MEK, ERK and
Akt, while had no obvious effects on the expression levels of MEK, ERK and Akt.
Fig. 6. Effect of compound 6e on the MEK/ERK and PI3K/Akt signaling pathways in B16 cells.
A) Western blot analysis of 6e on the expression of related proteins in B16 cells; B) Quantitative

analysis showing the levels of each protein relative to control GAPDH: *p <0.05, **p <0.01, ***p
<0.001 vs vehicle control.
2.2.5 Compound 6e inhibited the growth of B16 melanoma in mice
Based on the in vitro results and intensive mechanistic studies, we further tested
the anti-tumor activity of compound 6e in vivo by performing an assay in mice with
B16 melanoma. As illustrated in Fig.7, compound 6e significantly and
dose-dependently decreased tumor volume and tumor weight. At the dose of 30
mg/kg, 6e significantly suppressed tumor growth by inhibition ratio of 68.4%, which
was somewhat less effective than that of positive control 5-fluorouracil (5-FU, tumor
inhibition ratio of 70.9%). Meanwhile, 6e did not obviously affect the body weight at
doses of 15 and 30 mg/kg (Fig.7C). Thus, compound 6e is worthy of further
investigation as a potential anti-tumor drug candidate.
Fig. 7. Compound 6e inhibits B16 melanoma growth in vivo. After administering vehicle (group
1), 5-FU (30 mg/kg per 2 days, group 2), 6e (15 mg/kg per day, group 3), 6e (30 mg/kg per day,
group 4) for 3 weeks, the mice were sacrificed, and the tumors were weighed. (A) Images of
tumors from mice at 21 days after initiation of treatment; (B) Tumor volume changes during

treatment; (C) Body weight changes of mice during treatment; (D) Weight of the excised tumors
of each group. **P < 0.01 vs control group.
3. Conclusion
In summary, a series of C-23 modified HBA derivatives were synthesized by
acylation of the unique C-23 hydroxyl group of HBA. The new synthesized
compounds were evaluated for their in vitro antiproliferative activity. The results
showed that most of the C-23 modified HBA derivatives exhibited more potent
anticancer activity than the parent compound HBA, and the most active compound 6e
displayed the IC50 values at 2.14 µM against A2780 cells, 2.89 µM against B16 cells
and 3.97 µM against MCF-7 cells. Further anticancer mechanism studies revealed that
compound 6e induced the generation of intracellular ROS and reduction of MMP in
B16 cells. Moreover, western blot analysis indicated that compound 6e downregulated
the expression of anti-apoptotic protein Bcl-2, upregulated the expression of
pro-apoptotic protein Bax, and activated caspase 3 for causing cell apoptosis.
Meanwhile, compound 6e also significantly inhibited the phosphorylation of MEK,
ERK, and Akt without affecting the expression of MEK, ERK, and Akt. Furthermore,
the in vivo evaluation showed that 6e exhibited remarkable anti-tumor efficacies
against mice bearing B16 melanoma with tumor inhibition rate of 68.4% at the dose
of 30 mg/kg per day (ip). These results provide a further theoretical insight for the
anti-tumor therapeutic application of HBA derivatives. It is expected that the
mechanistic and biological studies described, together with our previous reports about
HBA derivatives, could expedite the development of new natural product-based
therapeutic agents for clinical cancer intervention.
4. Experimental section
4.1. Chemistry
4.1.1 General
Most chemicals and solvents were purchased from commercial sources. Further

purification and drying by standard methods were employed when necessary. Melting
points were determined on an XT-4 micro melting point apparatus and uncorrected. IR
spectra were recorded in CDCl3 or KBr pellets on a Nicolet Impact 410
1
spectrophotometer. H NMR and 13C NMR spectra were recorded with a Bruker
AV-300 spectrometer in the indicated solvents (TMS as internal standard): the values
of the chemical shifts are expressed in d values (ppm) and the coupling constants (J)
in Hz. EI-MS spectra were recorded on an Agilent1100-LC-MSD-Trap/SL
spectrometer and High-resolution mass spectra (HRMS) were recorded using an
Agilent QTOF 6520.
4.1.2 3-Hydroxy-23-acetoxy-lup-20(29)-en-28-oic acid (3a)
To a solution of 1 (100 mg, 0.21 mmol) in anhydrous pyridine (3 mL) was added
DMAP (10 mg, 0.1 mmol) and acetic anhydride (0.02 mL, 0.21 mmol). The mixture
was stirred for 6 h at room temperature and then diluted with ethyl acetate (50 mL),
acidified with 1 N HCl (1 mL). Then the solution was washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether-ethyl acetate 4:1 - 2:1) to obtain
compound 3a as a white solid (237 mg, 68.9%). m.p: 135-138 . IR (film, cm-1) 3462,
1
3171, 2943, 2869, 1719, 1640, 1450, 1385, 1240, 1132, 1043, 982, 878; H NMR
(CDCl3, 400 MHz) δ: 0.76, 0.88, 0.93, 0.97 (each 3H, s, Me-24, -25, -26, -27), 1.74
(3H, s, Me-30), 2.00 (3H, s, 3-OAc), 3.00 (1H, m, H-19), 3.53 (1H, m, H-3), 3.68 (1H,
d, J = 11.4 Hz, H-23a), 3.83 (1H, d, J = 11.4 Hz, H-23b), 4.62 (1H, s, H-29a), 4.77
(1H, s, H-29b) ; ESI-MS m/z: 537.3 [M + Na]+; HRMS (m/z) (ESI): calcd for
C32H50O5Na [M + Na]+: 537.3550; found: 537.3555.
4.1.3 3-Hydroxy-23-O-cyclohexyformyl-lup-20(29)-en-28-oic acid (3b)
To a solution of 1 (100 mg, 0.21 mmol) in anhydrous pyridine (3 mL) was added
DMAP (25 mg, 0.21 mmol), DCC (44 mg, 0.21 mmol) and cyclohexanecarboxylic
acid (27 mg, 0.21 mmol). The mixture was stirred for 8 h at room temperature and
then diluted with ethyl acetate (50 mL), acidified with 1 N HCl (2.5 mL). Then the
solution was washed with water and brine, dried over anhydrous Na2SO4, and

concentrated in vacuo. The residue was purified by chromatography on silica gel
(petroleum ether-ethyl acetate 4:1 - 2:1) to afford compound 3b as a white solid (226
mg, 60.3%). m.p: 131-133 . IR (film, cm-1) 3448, 3152, 2936, 2857, 1707, 1640,
1
1450, 1399, 1385, 1247, 1175, 1133, 1042, 885; H NMR (CDCl3, 300 MHz) δ: 0.67
(3H, s), 0.79 (3H, s), 0.86 (3H, s), 0.89 (3H, s), 1.62 (3H, s), 2.08-2.32 (3H, m), 2.95
(1H, m, H-19), 3.32 (1H, t, J = 8.2 Hz, H-3), 3.74, 4.08 (each 1H, d, J = 11.4 Hz,
H-23), 4.54, 4.67 (each 1H, s, H-29); 13C NMR (CDCl3, 75 MHz) δ: 11.7, 14.5, 16.0,
16.5, 18.1, 19.4, 20.9, 24.7, 25.4, 25.8, 26.2, 29.1, 29.3, 29.7, 30.6, 32.2, 34.0, 37.0,
38.4, 38.7, 40.7, 42.3, 42.4, 43.4, 46.9, 48.1, 49.3, 50.8, 56.4, 66.2, 72.6, 109.6, 150.4,
176.2, 182.0; ESI-MS m/z: 605.4 [M + Na]+, 581.4 [M – H]-, 617.4 [M + Cl]-; HRMS
(m/z) (ESI): calcd for C37H57O5 [M - H]-: 581.4211; found: 581.4218.
4.1.4 3-Hydroxy-23-O-(1-adamantaneformyl)-lup-20(29)-en-28-oic acid (3c)
The titled compound 3c was obtained following the procedure described for 3b.
Yield: 58.1%. White solid. m.p: 143-146 . 1H NMR (CDCl3, 300 MHz) δ: 0.85 (3H,
s), 0.87 (3H, s), 0.97 (6H, s), 1.62 (3H, s), 1.93 (4H, s), 1.94 (2H, s), 2.17-2.30 (2H,
m), 3.00 (1H, m, H-19), 3.39 (1H, d, J = 10.4 Hz, H-23a), 3.62 (1H, t, J = 7.9 Hz,
H-3), 3.69 (1H, d, J = 10.4 Hz, H-23b), 4.62, 4.73 (each 1H, s, H-29); 13C NMR
(CDCl3, 75 MHz) δ: 11.3, 14.7, 16.0, 16.5, 18.4, 19.4, 20.9 , 25.5, 26.8, 27.6, 28.0,
29.7, 30.3, 31.7, 33.6, 34.1, 36.1, 36.3, 36.5, 37.1, 38.0, 38.2, 38.3, 38.5, 40.7, 41.9,
42.5, 46.5, 49.2, 49.8, 50.6, 58.0, 71.5, 109.9, 149.9, 171.9, 180.3; ESI-MS m/z: 669.4
[M + Cl]-.
4.1.5 3-Hydroxy-23-O-benzoyl-lup-20(29)-en-28-oic acid (3d)
The titled compound 3d was obtained following the procedure described for 3b.
Yield: 60.3%. White solid. m.p: 130-132 . IR (film, cm-1) 3435, 3156, 2943, 2870,
1720, 1700, 1640, 1601, 1450, 1399, 1384, 1271, 1176, 1111, 1026, 981, 884, 711; 1H
NMR (CDCl3, 300 MHz) δ: 0.83 (3H, s), 0.90 (3H, s), 0.96 (6H, s), 1.61 (3H, s),
1.92-2.03 (2H, m), 2.16-2.33 (2H, m), 3.02 (1H, m, H-19), 3.52 (1H, t, J = 7.1 Hz,
H-3), 4.01, 4.50 (each 1H, d, J = 11.5 Hz, H-23), 4.62, 4.75 (each 1H, s, H-29), 7.47

(2H, t, J = 7.4 Hz), 7.59 (1H, t, J = 7.4 Hz), 8.06 (2H, d, J = 7.2 Hz); 13C NMR
(CDCl3, 75 MHz) δ: 11.9, 14.5, 16.0, 16.6, 18.2, 19.4, 20.9, 24.7, 25.5, 26.3, 30.6,
32.2, 34.1, 37.1, 38.4, 38.7, 40.7, 42.4, 42.6, 46.9, 48.5, 49.3, 50.9, 56.4, 66.8, 72.4,
109.6, 128.5, 129.6, 130.2, 133.1, 150.4, 166.8, 182.1; ESI-MS m/z: 599.3 [M + Na]+;
HRMS (m/z) (ESI): calcd for C37H52O5Na [M + Na]+: 599.3707; found: 599.3706.
4.1.6 3-Hydroxy-23-O-(4-methoxybenzoyl)-lup-20(29)-en-28-oic acid (3e)
The titled compound 3e was obtained following the procedure described for 3b.
Yield: 56.9%. White solid. m.p: 121-124 . 1H NMR (CDCl3, 300 MHz) δ: 0.81 (3H,
s), 0.88 (3H, s), 0.95 (6H, s), 1.68 (3H, s), 1.88-2.02 (2H, m), 2.13-2.32 (2H, m), 3.00
(1H, m, H-19), 3.88 (3H, s), 3.93 (1H, m, H-3), 3.69 (1H, d, J = 11.6 Hz, H-23a), 4.49
(1H, d, J = 11.6 Hz, H-23b), 4.60, 4.73 (each 1H, s, H-29), 6.94 (2H, d, J = 8.8 Hz),
13
8.00 (2H, d, J = 8.8 Hz); C NMR (CDCl3, 75 MHz) δ: 11.9, 14.5, 16.0, 16.6, 18.2,
19.4, 20.9, 24.8, 25.6, 26.2, 29.6, 30.6, 32.2, 33.8, 34.2, 37.1, 38.4, 38.7, 40.7, 42.4,
42.6, 46.9, 48.5, 49.2, 50.9, 55.5, 56.3, 66.5, 72.4, 109.7, 113.7, 122.2, 131.6, 150.4,
163.5, 166.4, 182.1; ESI-MS m/z: 607.4 [M + H]+.
4.1.7 3-Hydroxy-23-O-(2-nitrobenzoyl)-lup-20(29)-en-28-oic acid (3f)
The titled compound 3f was obtained following the procedure described for 3b.
Yield: 62.5%. White solid. m.p: 136-138 . IR (film, cm-1) 3467, 3127, 2937, 2854,
1726, 1638, 1540, 1450, 1398, 1384, 1292, 1255, 1127, 1075, 967, 884, 791, 735, 699;
1H NMR (CDCl3, 300 MHz) δ: 0.71 (3H, s), 0.79 (3H, s), 0.86 (3H, s), 0.94 (3H, s),
1.61 (3H, s), 2.07-2.24 (2H, m), 2.93 (1H, m, H-19), 3.43 (1H, m, H-3), 3.99 (1H, d, J
= 11.4 Hz, H-23a), 4.32 (1H, d, J = 11.4 Hz, H-23b), 4.61, 4.74 (each 1H, s, H-29),
7.53-7.67 (2H, m), 7.72-7.85 (2H, m); 13C NMR (CDCl3, 75 MHz) δ: 11.7, 14.6, 16.0,
16.6, 18.2, 19.3, 20.9, 24.8, 25.6, 26.6, 29.7, 30.2, 30.6, 32.2, 33.7, 37.2, 38.4, 40.6,
42.4, 42.5, 46.9, 47.5, 49.2, 49.4, 50.3, 56.3, 68.4, 71.8, 109.5, 123.7, 126.9, 130.6,
132.1, 132.5, 150.5, 165.5, 181.6; ESI-MS m/z: 644.3 [M + Na]+; HRMS (m/z) (ESI):
calcd for C37H51NO7Na [M + Na]+: 644.3558; found: 644.3563.
4.1.8 3-Hydroxy-23-O-(3-nitrobenzoyl)-lup-20(29)-en-28-oic acid (3g)

The titled compound 3g was obtained following the procedure described for 3b.
Yield: 60.8%. White solid. m.p: 137-139 . 1H NMR (CDCl3, 300 MHz) δ: 0.84 (3H,
s), 0.91 (3H, s), 0.96 (3H, s), 0.97 (3H, s), 1.69 (3H, s), 2.15-2.33 (2H, m), 3.02 (1H,
m, H-19), 3.50 (1H, m, H-3), 4.05 (1H, d, J = 11.4 Hz, H-23a), 4.58 (1H, d, J = 11.4
Hz, H-23b), 4.61, 4.74 (each 1H, s, H-29), 7.70 (1H, t, J = 8.0 Hz), 8.39 (1H, d, J =
13
7.8 Hz), 8.46 (1H, d, J = 8.2 Hz), 8.86 (1H, s); C NMR (CDCl3, 75 MHz) δ: 11.9,
14.5, 16.0, 16.6, 18.2, 19.3, 20.9, 24.8, 25.4, 25.5, 26.5, 29.4, 30.6, 32.1, 33.6, 34.2,
37.1, 38.4, 38.6, 40.7, 42.3, 42.6, 46.9, 48.6, 49.2, 49.4, 50.9, 56.3, 67.1, 72.2, 109.7,
124.4, 127.5, 129.8, 132.0, 135.3, 148.3, 150.4, 164.6, 181.9; ESI-MS m/z: 622.4 [M
+ H]+.
4.1.9 3-Hydroxy-23-O-(4-nitrobenzoyl)-lup-20(29)-en-28-oic acid (3h)
The titled compound 3h was obtained following the procedure described for 3b.
Yield: 48.9%. White solid. m.p: 135-138 . 1H NMR (CDCl3, 300 MHz) δ: 0.84 (3H,
s), 0.91 (3H, s), 0.95 (3H, s), 0.96 (3H, s), 1.70 (3H, s), 1.89-2.07 (3H, m), 2.16-2.32
(2H, m), 3.03 (1H, m, H-19), 3.52 (1H, t, J = 7.4 Hz, H-3), 4.11, 4.50 (each 1H, d, J =
11.4 Hz, H-23), 4.62, 4.74 (each 1H, s, H-29), 8.21 (2H, d, J = 8.8 Hz), 8.32 (2H, d, J
= 8.8 Hz); 13C NMR (CDCl3, 75 MHz) δ: 11.3, 14.1, 15.5, 16.1, 17.7, 18.8, 20.4, 24.2,
25.0, 26.1, 30.1, 31.6, 32.9, 33.7, 36.6, 37.9, 38.2, 40.2, 41.9, 42.1, 46.4, 48.2, 48.7,
50.5, 55.8, 66.9, 71.8, 109.2, 123.2, 130.1, 135.2, 149.8, 150.1, 164.3, 181.3; ESI-MS
m/z: 620.4 [M – H]-, 656.3 [M + Cl]-.
4.1.10 3-Hydroxy-23-O-(4-aminobenzoyl)-lup-20(29)-en-28-oic acid (3i)
Add 10% Pd/C to a solution of 3h in tetramethylene oxide (15 mL) under H2 at
atmospheric pressure. The mixture was stirred at room temperature for 0.5 h. Then the
solution was filtered and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether-ethyl acetate 2:1) to afford 3i as a
white solid. Yield: 86.5%. m.p: 161-164 . IR (film, cm-1) 3376, 3145, 2941, 2870,
1692, 1624, 1604, 1518, 1459, 1398, 1384, 1310, 1271, 1171, 1109, 1043, 980, 885,
770; 1H NMR (CDCl3, 300 MHz) δ: 0.80 (3H, s), 0.88 (3H, s), 0.94 (3H, s), 0.96 (3H,

s), 1.68 (3H, s), 1.91-2.04 (2H, m), 2.13-2.32 (2H, m), 3.01 (1H, m, H-19), 3.42 (1H, t,
J = 7.3 Hz, H-3), 3.87, 4.48 (each 1H, d, J = 11.5 Hz, H-23), 4.61, 4.74 (each 1H, s,
H-29), 6.65 (2H, d, J = 8.3 Hz), 7.84 (2H, d, J = 8.8 Hz); 13C NMR (CDCl3, 75 MHz)
δ: 11.9, 14.5, 16.0, 16.7, 18.2, 19.4, 20.8, 25.5, 26.0, 29.7, 30.2, 30.6, 31.4, 32.2, 34.1,
37.0, 38.4, 38.7, 40.7, 42.4, 42.6, 46.9, 48.5, 49.2, 50.8, 56.4, 66.3, 72.5, 109.7, 113.9,
119.4, 131.7, 150.4, 151.1, 167.1, 182.2; ESI-MS m/z: 592.4 [M + H]+, 614.4 [M +
Na]+. HRMS (m/z) (ESI): calcd for C37H54NO5 [M + H]+: 592.3997; found: 592.3997.
4.1.11 3-Hydroxy-23-O-(3,5-dinitrobenzoyl)-lup-20(29)-en-28-oic acid (3j)
The titled compound 3j was obtained following the procedure described for 3b.
Yield: 46.6%. White solid. m.p: 145-147 . 1H NMR (CDCl3, 300 MHz) δ: 0.86 (3H,
s), 0.93 (3H, s), 0.98 (6H, s), 1.70 (3H, s), 1.90-2.24 (3H, m), 2.17-2.31 (2H, m), 3.02
(1H, m, H-19), 3.49 (1H, t, J = 7.2 Hz, H-3), 4.12 (1H, d, J = 11.4 Hz, H-23a), 4.61
(1H, s, H-29a), 4.66 (1H, d, J = 11.4 Hz, H-23b), 4.74 (1H, s, H-29b), 9.16 (2H, d, J =
2.0 Hz), 9.26 (1H, s); 13C NMR (CDCl3, 75 MHz) δ: 11.9, 14.5, 16.0, 16.6, 18.3, 19.3,
20.9, 24.8, 25.5, 26.8, 30.6, 32.1, 33.6, 34.3, 37.0, 37.2, 38.4, 40.7, 42.3, 42.6, 46.9,
48.8, 49.2, 50.9, 56.3, 68.0, 72.1, 109.7, 122.4, 129.3, 134.0, 148.8, 150.4, 162.6,
181.7; ESI-MS m/z: 665.3 [M – H]-, 701.3 [M + Cl]-.
4.1.12 3-Hydroxy-23-O-(4-fluorobenzoyl)-lup-20(29)-en-28-oic acid (3k)
The titled compound 3k was obtained following the procedure described for 3b.
Yield: 66.0%. White solid. m.p: 111-114 . IR (film, cm-1) 3448, 3134, 2942, 2870,
1
1718, 1628, 1604, 1508, 1451, 1400, 1384, 1272, 1152, 1115, 1044, 853, 767; H
NMR (CDCl3, 300 MHz) δ: 0.82 (3H, s), 0.89 (3H, s), 0.95 (6H, s), 1.68 (3H, s),
1.93-2.12 (2H, m), 2.14-2.31 (2H, m), 3.03 (1H, m, H-19), 3.47 (1H, t, J = 7.4 Hz,
H-3), 3.99, 4.48 (each 1H, d, J = 11.5 Hz, H-23), 4.61, 4.74 (each 1H, s, H-29), 7.14
13
(2H, m), 8.06 (2H, dd, J = 8.7, 5.6 Hz); C NMR (CDCl3, 75 MHz) δ: 11.8, 14.5,
16.0, 16.6, 18.2, 19.3, 20.9, 25.5, 26.4, 29.6, 30.6, 32.1, 34.2, 37.1, 38.4, 38.7, 40.7,
42.4, 42.6, 46.9, 48.6, 49.2, 51.0, 56.3, 66.8, 72.4, 109.7, 115.5, 115.8, 126.3, 132.0,
132.1, 150.3, 164.3, 165.8; ESI-MS m/z: 593.4 [M – H]-; HRMS (m/z) (ESI): calcd

for C37H52FO5 [M + H]+: 595.3793; found: 595.3797.
4.1.13 3-Hydroxy-23-O-(3-chlorobenzoyl)-lup-20(29)-en-28-oic acid (3l)
The titled compound 3l was obtained following the procedure described for 3b.
Yield: 45.7%. White solid. m.p: 140-142 . 1H NMR (CDCl3, 300 MHz) δ: 0.82 (3H,
s), 0.89 (3H, s), 0.95 (3H, s), 0.98 (3H, s), 1.67 (3H, s), 2.1-2.28 (2H, m), 3.00 (1H, m,
H-19), 3.48 (1H, m, H-3), 4.03 (1H, d, J = 11.4 Hz, H-23a), 4.50 (1H, d, J = 11.4 Hz,
H-23b), 4.59, 4.72 (each 1H, s, H-29), 7.41 (1H, d, J = 7.8 Hz),7.55 (1H, d, J = 8.7
Hz), 7.91 (1H, d, J = 7.8 Hz), 7.98 (1H, s); 13C NMR (CDCl3, 75 MHz) δ: 11.9, 14.5,
16.0, 16.6, 18.2, 19.3, 20.9, 24.8, 25.4, 25.6, 26.5, 29.7, 30.6, 32.1, 33.7, 34.2, 37.1,
38.4, 38.7, 40.6, 42.4, 42.6, 46.9, 48.5, 49.2, 49.4, 50.9, 56.4, 66.8, 72.2, 109.7, 127.7,
129.6, 129.8, 133.1, 150.4, 150.7, 172.5, 182.0; ESI-MS m/z: 609.3 [M – H]-.
4.1.14 3-Hydroxy-23-O-(2-trifluoromethylbenzoyl)-lup-20(29)-en-28-oic acid (3m)
The titled compound 3m was obtained following the procedure described for 3b.
Yield: 56.6%. White solid. m.p: 110-112 . IR (film, cm-1) 3466, 3129, 2939, 2856,
1
1718, 1637, 1450, 1399, 1384, 1314, 1261, 1169, 1139, 1052, 886, 768; H NMR
(CDCl3, 300 MHz) δ: 0.82 (3H, s), 0.89 (3H, s), 0.96 (6H, s), 1.69 (3H, s), 2.18-2.31
(2H, m), 3.01 (1H, m, H-19), 3.52 (1H, m, H-3), 4.14 (1H, d, J = 11.5 Hz, H-23a),
4.43 (1H, d, J = 11.5 Hz, H-23b), 4.61, 4.74 (each 1H, s, H-29), 7.63 (2H, m), 7.78
(1H, m), 7.83 (1H, m); 13C NMR (CDCl3, 75 MHz) δ: 11.7, 14.4, 16.1, 16.6, 18.2,
19.4, 20.8, 24.8, 25.5, 26.4, 29.7, 30.6, 32.2, 33.7, 36.9, 37.1, 38.3, 40.6, 42.4, 46.9,
47.6, 49.2, 49.4, 50.5, 56.3, 68.3, 72.0, 109.6, 126.6, 130.5, 131.3, 131.8, 150.5, 167.2,
181.3; ESI-MS m/z: 667.3 [M + Na]+; HRMS (m/z) (ESI): calcd for C38H50F3O5 [M -
H]-: 643.3616; found: 643.3624.
4.1.15 3-Hydroxy-23-O-(4-trifluoromethylbenzoyl)-lup-20(29)-en-28-oic acid (3n)
The titled compound 3n was obtained following the procedure described for 3b.
1
Yield: 63.5%. White solid. m.p: 112-113 . H NMR (CDCl3, 300 MHz) δ: 0.75 (3H,
s), 0.82 (3H, s), 0.89 (6H, s), 1.61 (3H, s), 1.81-1.98 (3H, m), 2.07-2.24 (2H, m), 2.93
(1H, m, H-19), 3.45 (1H, t, J = 7.7 Hz, H-3), 3.98, 4.42 (each 1H, d, J = 11.5 Hz,

H-23), 4.53, 4.66 (each 1H, s, H-29), 7.65 (2H, d, J = 8.2 Hz), 8.08 (2H, d, J = 8.2
Hz); 13C NMR (CDCl3, 75 MHz) δ: 11.3, 14.0, 15.5, 16.1, 17.7, 18.8, 20.4, 24.1, 25.0,
26.0, , 30.1, 31.6, 32.8, 33.7, 36.6, 37.9, 38.2, 40.2, 41.9, 42.1, 46.4, 48.1, 48.8, 50.5,
55.9, 66.6, 71.8, 109.2, 125.0, 125.1, 129.4, 133.0, 149.8, 165.0, 181.4; ESI-MS m/z:
643.4 [M – H]-, 679.4 [M + Cl]-.
4.1.16 3-Hydroxy-23-O-(2,3,4,5-tetrafluorobenzoyl)-lup-20(29)-en-28-oic acid (3o)
The titled compound 3o was obtained following the procedure described for 3b.
Yield: 60.3%. White solid. m.p: 134-137 . 1H NMR (CDCl3, 300 MHz) δ: 0.74 (3H,
s), 0.81 (3H, s), 0.88 (3H, s), 0.90 (3H, s), 1.62 (3H, s), 1.83-1.97 (3H, m), 2.08-2.24
(2H, m), 2.94 (1H, m, H-19), 3.44 (1H, t, J = 7.7 Hz, H-3), 3.98, 4.42 (each 1H, d, J =
13
11.4 Hz, H-23), 4.54, 4.67 (each 1H, s, H-29), 7.56 (1H, dd, J = 14.1, 7.9 Hz); C
NMR (CDCl3, 75 MHz) δ: 11.1, 13.8, 15.5, 16.0, 17.6, 18.8, 20.4, 24.3, 25.0, 26.2,
29.1, 30.1, 31.6, 33.4, 36.5, 37.9, 38.0, 40.2, 41.7, 41.9, 46.4, 47.4, 48.7, 50.3, 55.8,
67.6, 71.7, 109.1, 112.7, 113.0, 114.5, 149.9, 161.8, 181.2; ESI-MS m/z: 647.3 [M –
H]-.
4.1.17 3-Hydroxy-23-O-(4-benzylsulfonyl)-lup-20(29)-en-28-oic acid (3p)
The titled compound 3p was obtained following the procedure described for 3b.
Yield: 50.9%. White solid. m.p: 165-167 . IR (film, cm-1) 3415, 3128, 2945, 2870,
1686, 1638, 1598, 1500, 1460, 1400, 1385, 1356, 1264, 1189, 1167, 1111, 1045, 960,
834; 1H NMR (CDCl3, 300 MHz) δ: 0.65 (3H, s), 0.81 (3H, s), 0.89 (3H, s), 0.97 (3H,
s), 1.70 (3H, s), 1.97-2.03 (2H, m), 2.17 (1H, m), 2.28 (1H, m), 3.00 (1H, m, H-19),
3.59 (1H, m, H-3), 3.69 (1H, d, J = 9.7 Hz, H-23a), 3.88 (3H, s), 3.96 (1H, d, J = 9.8
Hz, H-23b), 4.61, 4.74 (each 1H, s, H-29), 7.01 (2H, d, J = 8.9 Hz), 7.85 (2H, d, J =
13
8.9 Hz); C NMR (CDCl3, 75 MHz) δ: 11.5, 14.8, 15.9, 16.5, 17.8, 19.3, 20.8, 25.4,
26.6, 29.6, 30.6, 31.1, 33.7, 37.0, 38.2, 38.4, 40.6, 42.4, 42.4, 46.9, 47.0, 49.2, 50.3,
55.7, 56.4, 71.1, 71.5, 109.7, 114.4, 127.4, 130.0, 150.3, 163.7, 182.2; ESI-MS m/z:
665.3 [M + Na]+; HRMS (m/z) (ESI): calcd for C37H53O7S [M - H]-: 641.3517; found:
641.3526.

4.1.18 3-Hydroxy-23-O-(3-picolinoyl)-lup-20(29)-en-28-oic acid (3q)
The titled compound 3q was obtained following the procedure described for 3b.
Yield: 42.1%. White solid. m.p: 154-157 . IR (film, cm-1) 3468, 3127, 2945, 2857,
1
1719, 1638, 1590, 1400, 1385, 1289, 1129, 1042, 880, 740; H NMR (CDCl3, 300
MHz) δ: 0.75 (3H, s), 0.82 (3H, s), 0.86 (3H, s), 0.88 (3H, s), 1.61 (3H, s), 1.82-1.98
(3H, m), 2.10-2.22 (2H, m), 2.94 (1H, m, H-19), 3.45 (1H, t, J = 7.3 Hz, H-3), 4.02,
4.40 (each 1H, d, J = 11.4 Hz, H-23), 4.53, 4.66 (each 1H, s, H-29), 7.36 (1H, dd, J =
7.7, 5.1 Hz), 8.25 (1H, d, J = 7.9 Hz), 8.75 (1H, d, J = 3.7 Hz), 9.16 (1H, s); 13C NMR
(CDCl3, 75 MHz) δ: 11.8, 14.6, 16.0, 16.6, 18.2, 19.3, 20.9, 25.5, 26.6, 30.3, 30.6,
32.2, 34.2, 37.1, 38.4, 38.6, 40.7, 42.4, 42.6, 46.9, 48.6, 49.2, 50.9, 56.3, 67.0, 72.2,
109.6, 123.5, 126.3, 137.3, 150.4, 150.5, 153.3, 165.2, 181.1; ESI-MS m/z: 578.3 [M
+ H]+; HRMS (m/z) (ESI): calcd for C36H50NO5 [M - H]-: 576.3694; found: 576.3702.
4.1.19 3-Hydroxy-23-O-(indole-2-formyl)-lup-20(29)-en-28-oic acid (3r)
The titled compound 3r was obtained following the procedure described for 3b.
Yield: 39.6%. White solid. m.p: 166-168 . 1H NMR (CDCl3, 300 MHz) δ: 0.79 (3H,
s), 0.90 (6H, s), 1.01 (3H, s), 1.63 (3H, s), 2.08-2.27 (2H, m), 2.97 (1H, m, H-19),
3.38 (1H, m, H-3), 4.07, 4.17 (each 1H, d, J = 11.5 Hz, H-23), 4.55, 4.68 (each 1H, s,
H-29), 7.01 (1H, s), 7.10 (1H, m), 7.51 (2H, m); ESI-MS m/z: 614.4 [M – H]-.
4.1.20 3-Hydroxy-23-O-(indole-4-formyl)-lup-20(29)-en-28-oic acid (3s)
The titled compound 3s was obtained following the procedure described for 3b.
1
Yield: 46.7%. White solid. m.p: 161-163 . H NMR (DMSO-d6, 300 MHz) δ: 0.69
(3H, s), 0.83 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 1.64 (3H, s), 2.06 (1H, m), 2.22 (1H,
m), 2.95 (1H, m, H-19), 3.47 (1H, m, H-3), 3.93, 4.17 (each 1H, d, J = 11.0 Hz, H-23),
4.56, 4.69 (each 1H, s, H-29), 6.56 (1H, s), 7.49 (2H, d, J = 2.9 Hz), 7.74 (2H, d, J =
8.4 Hz), 8.26 (1H, s), 11.5 (1H, s), 12.1 (1H, brs); 13C NMR (CDCl3, 75 MHz) δ: 12.7,
14.5, 16.2, 16.7, 18.2, 19.4, 20.9, 25.5, 27.1, 30.5, 30.9, 32.1, 34.1, 37.0, 38.0, 38.8,
40.7, 42.1, 42.4, 47.1, 48.3, 49.0, 50.9, 55.8, 65.8, 70.8, 102.9, 110.1, 111.8, 121.3,
122.2, 122.9, 127.7, 138.9, 150.7, 167.0, 177.7; ESI-MS m/z: 614.4 [M – H]-.

4.1.21 Benzyl 3, 23-dihydroxy-lup-20(29)-en-28-oate (4)
To a solution of 1 (5.00 g, 10.5 mmol) in DMF (60 mL) was successively added
K2CO3 (5.00 g, 36 mmol) and benzyl bromide (1.5 mL, 12.5 mmol). The mixture was
stirred for 12 h at room temperature and then poured into water (15 mL) and extracted
with ethyl acetate (80 mL × 3). The organic layer was washed with water and brine,
dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was
purified by chromatography on silica gel (petroleum ether-ethyl acetate 2:1) to afford
compound 4 as a white solid (5.4 g, 90.3%). 1H NMR (CDCl3, 300 MHz) δ: 0.75 (3H,
s), 0.84 (6H, s), 0.93 (3H, s), 1.67 (3H, s), 2.11-2.22 (1H, m), 2.23-2.29 (1H, m), 3.01
(1H, m, H-19), 3.37 (1H, d, J = 9.9 Hz, H-23a), 3.59 (1H, m, H-3), 3.67 (1H, d, J =
9.8 Hz, H-23b), 4.59, 4.72 (each 1H, s, H-29), 5.05-5.17 (2H, m, -CH2Ar), 7.23-7.42
(5H, m, H-Ar); 13C NMR (CDCl3, 75 MHz) δ: 11.3, 14.7, 15.8, 16.5, 18.4, 19.4, 20.9,
25.5, 26.8, 29.6, 30.6, 32.1, 34.0, 36.9, 37.0, 38.1, 38.4, 40.6, 41.8, 42.4, 46.9, 49.4,
49.9, 50.5, 56.5, 65.7, 71.8, 76.6, 109.6, 128.1, 128.2, 128.5, 136.4, 150.5, 175.8;
ESI-MS m/z: 563.3 [M + H]+, 585.3 [M + Na]+, 601.4 [M + K]+.
4.1.22 3-Hydroxy-23-O-[N,N-bis(2-chloroethyl)aminobenzoyl]-lup-20(29)-en-28-oic
acid (6a)
To a solution of 4 (200 mg, 0.35 mmol) in anhydrous CH2Cl2 (10 mL) was added
DMAP (42 mg, 0.35 mmol), DCC (73 mg, 0.35 mmol) and
4-(bis(2-chloroethyl)amino)benzoic acid (92 mg, 0.35 mmol). The mixture was stirred
for 12 h at room temperature and then diluted with ethyl acetate (50 mL). Then the
solution was washed with 1 N HCl (4 mL), water and brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether-ethyl acetate 8:1 - 5:1) to afford benzyl
product. Added 10% Pd/C to a solution of benzyl product in tetramethylene oxide (15
mL) under H2 at atmospheric pressure. The mixture was stirred at room temperature
for 0.5 h. Then the solution was filtered and concentrated in vacuo. The residue was
purified by chromatography on silica gel (petroleum ether-ethyl acetate 2:1) to afford
1
compound 6a as a white solid (138 mg, 55.3%). m.p: 135-138 . H NMR (CDCl3,

300 MHz) δ: 0.77 (3H, s), 0.80 (3H, s), 0.85 (3H, s), 0.92 (3H, s), 1.67 (3H, s, Me-30),
2.18 (1H, m), 2.27 (1H, m), 3.00 (1H, m, H-19), 3.41 (1H, m, H-3), 3.62-3.68 (4H, m),
3.75-3.83 (4H, m), 3.89 (1H, d, J = 11.4 Hz, H-23a), 4.47 (1H, d, J = 11.4 Hz, H-23b),
4.59 (1H, s, H-29a), 4.72 (1H, s, H-29b), 6.67 (2H, d, J = 9.1 Hz), 7.92 (2H, d, J = 9.1
Hz); 13C NMR (CDCl3, 75 MHz) δ: 11.9, 14.6, 15.8, 16.7, 18.2, 19.4, 20.9, 25.5, 26.1,
29.5, 30.6, 32.1, 34.1, 36.9, 37.1, 38.2, 38.7, 40.1, 40.7, 42.4, 42.7, 46.9, 48.6, 49.4,
50.9, 53.3, 56.5, 65.7, 72.4, 109.6, 110.9, 128.5, 131.8, 149.8, 166.8, 181.2; ESI-MS
m/z: 714.4 [M – H]-.
4.1.23 3-Hydroxy-23-O-(β-aminopropionyl)-lup-20(29)-en-28-oic acid (6d)
To a solution of N-tert-butoxycarbonyl-β-alanine (66 mg, 0.35 mmol) in
anhydrous CH2Cl2 (10 mL) was added DMAP (42 mg, 0.35 mmol), DCC (73 mg,
0.35 mmol). The mixture was stirred for 1 h at room temperature, then added 4 (197
mg, 0.35 mmol), and the mixture was stirred for 12 h at room temperature and then
diluted with ethyl acetate (50 mL). Then the solution was washed with 1 N HCl (4
mL), water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo.
The residue was purified by chromatography on silica gel (petroleum ether-ethyl
acetate 8:1 - 5:1) to afford benzyl product 5b. Added 10% Pd/C to a solution of
benzyl product in tetramethylene oxide (15 mL) under H2 at atmospheric pressure.
The mixture was stirred at room temperature for 0.5 h. Then the solution was filtered
and concentrated in vacuo. The residue was purified by chromatography on silica gel
(petroleum ether-ethyl acetate 2:1) to afford intermediate product 6b. 6b was
dissolved in anhydrous dichloromethane (10 mL), and trifluoroacetic acid (1.5 mL)
was added under ice bath. The mixture was stirred for 3h at room temperature and
then concentrated and chromatographed over silica gel (dichloromethane-methanol
30:1 - 10:1) to give 6d (42 mg, 26.9%) as a white solid. m.p: 153-155 . IR (film, cm-1)
3448, 3139, 2946, 2874, 1759, 1685, 1637, 1450, 1400, 1385, 1190, 1132, 1043, 966,
800, 723; 1H NMR (CDCl3, 300 MHz) δ: 0.87 (6H, s), 0.94 (3H, s), 0.97 (3H, s), 1.69
(3H, s, Me-30), 2.20-2.28 (2H, m), 2.67-2.69 (2H, m), 2.96-3.00 (1H, m, H-19),
3.17-3.19 (2H, m), 3.42 (1H, d, J = 10.3 Hz, H-23a), 3.60-3.65 (m, 1H, H-3), 3.72

(1H, d, J = 10.4 Hz, H-23b), 4.61 (1H, s, H-29a), 4.74(1H, s, H-29b); 13C NMR
(DMSO-d6, 75 MHz) δ: 12.87, 14.84, 16.19, 16.76, 17.93, 19.40, 20.97, 25.62, 27.22,
29.68, 30.58, 32.21, 33.97, 36.21, 36.88, 37.22, 37.85, 38.05, 38.55, 40.65, 42.38,
42.49, 47.11, 48.04, 49.01, 50.44, 55.88, 64.93, 70.81, 110.11, 150.74, 173.29,
177.71;ESI-MS m/z: 544.4 [M + H]+; HRMS (m/z) (ESI): calcd for C33H54NO5 [M +
H]+: 544.3997; found: 544.3998.
4.1.24 3-Hydroxy-23-O-(4-piperidinylformyl)-lup-20(29)-en-28-oic acid (6e)
The titled compound 6e was obtained following the procedure described for 6d.
Yield: 31.5%. White solid. m.p: 158-159 . IR (film, cm-1) 3431, 3134, 2946, 2869,
1
1728, 1637, 1560, 1451, 1399, 1385, 1190, 1107, 1041, 883; H NMR (CDCl3, 300
MHz) δ: 0.75 (3H, s), 0.88 (3H, s), 0.91 (3H, s), 0.95 (3H, s), 1.66 (3H, s), 2.45-2.52
(1H, m), 2.65-2.73 (2H, m), 3.10-3.14 (2H, m), 3.34-3.42 (1H, m, H-3), 3.85 (1H, d, J
= 11.5 Hz, H-23a), 3.94 (1H, s), 4.17(1H, d, J = 11.4 Hz, H-23b), 4.63, 4.76 (each 1H,
s, H-29)；13C NMR (DMSO-d6, 75 MHz) δ: 12.90, 14.86, 16.29, 16.79, 17.96, 19.46,
21.01, 25.62, 27.59, 29.75, 30.74, 32.61, 34.01, 36.89, 37.14, 37.98, 38.43, 38.63,
40.66, 40.96, 42.42, 42.50, 43.93, 47.13, 48.57, 49.14, 50.48, 56.08, 64.85, 70.72,
109.88, 151.12, 176.43, 178.39; ESI-MS m/z: 584.4[M + H]+; HRMS (m/z) (ESI):
calcd for C36H58NO5 [M + H]+: 584.4310; found: 584.4308.
4.2 Biology
4.2.1 Cell lines and cultures
A2780 (human ovarian cancer cell), A375 (human melanoma)，MCF-7 (human
breast cancer) and HepG2 (human liver cancer) were originally obtained from
American Type Culture Collection (ATCC), USA and stock was maintained in
laboratory; B16 (mice melanoma) and L-O2 (human normal liver) were obtained from
Type Culture Collection of Chinese Academy of Sciences Committee. All cell lines
were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (Gibco) and
1% penicillin streptomycin (Gibco) at 37 ℃in the presence of 5% CO2.
4.2.2 Antiproliferative screening using MTT assay

The antiproliferative activity of the compounds was determined using MTT assay.
Cells were seeded in 96-well micro culture plates incubated for 24 h at 37 in CO2
incubator. All of the reported betulinic derivatives were dissolved in DMSO while the
positive control doxorubicin was dissolved in PBS. These tested compounds at
different concentrations were added into wells and cells were treated at 37 for 72 h.
Then MTT (5 mg/mL, in PBS) was added into each well and cultured for another 4 h.
The optical density was detected in a microplate reader at 570 nm. IC50 values were
calculated according to the dose dependent curves.
4.2.3 Flow cytometry
4.2.3.1 Reactive oxygen species (ROS) analysis
B16 cancer cells were plated into 6-well culture plates and treated without or with
compound 6e at indicated concentration for 72 h. DCFH-DA was diluted to 10 µmol/L
with cell medium and incubation with cells for 20 min at 37 °C. Finally, cells were
washed with serum-free medium for three times. Then, the intracellular ROS levels
were detected with flow cytometer (Ex=488 nm; Em=530 nm).
4.2.3.2 Mitochondrial membrane potential (MMP) assay
B16 cells were cultured overnight and incubated in triplicate with the compound
6e (0, 0.75, 1.5, 3.0 µM) for 48 h. The cells were stained with the lipophilic cationic
dye JC-1, according to the manufacturer’s instruction (Keygen, KGA601). The
percentage of cells with healthy or collapsed mitochondrial membrane potentials was
monitored by flow cytometry analysis.
4.2.3.3 Western Blot analysis
B16 cells were incubated in the presence of 6e and collected after 72 h. Cells
were centrifuged and washed two times with ice cold phosphate buffered saline. The
pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30
min, lysates were centrifuged at 13,000 g at 4 °C for 10 min. The protein
concentration in the supernatant was measured with the Braford protein content

detection kit. Equal amounts of protein (70 µg) were electrophoresed using sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8−15% acrylamide
gels) and transferred onto PVDF Hybond-P membrane. Membranes were blocked for
1 h in 5% fat-free milk at room temperature. Membranes were then incubated with
primary antibodies against caspase-3, Bax, Bcl-2, MEK, p-MEK, ERK, p-ERK, Akt,
p-Akt with gentle rotation overnight at 4 °C. Membranes were next incubated with
fluorescent secondary antibodies for 60 min and were determined by densimetric
scanning.
4.2.4 Anti-tumor assay in vivo
A total of 1 × 107 B16 cells were subcutaneously inoculated into the right flank of
ICR mice with body weight of 18 - 22 g according to protocols of tumor transplant
research. After incubation for 1 day, mice were weighted and at random divided into
four groups with eight animals per group. The groups treated with 6e were
administered intraperitoneously 15 mg/kg, 30 mg/kg in a vehicle of 10% DMSO/2%
Tween 80/88% saline, respectively. The positive control group was treated with 5-FU
(30 mg/kg) by intraperitoneous injection. The negative control group received a
vehicle of 10% DMSO/2% Tween 80/88% saline through intraperitoneous injection.
Treatments of 6e was conducted at a frequency of one dose per day for a total 21
consecutive days, while the positive group was treated with 5-FU one dose per two
days. Body weights and tumor volumes were measured every 2 days. After the
treatments, all the mice were sacrificed and weighed. The following formula was used
to determine tumor volumes: tumor volume = L × W2/2, where L is the length and W
is the width. The ratio of inhibition of tumor (%) = (1 − average tumor weight of
treated group/average tumor weight of control group) × 100%. All procedures were
performed following institutional approval in accordance with the NIH Guide for the
Care and Use of Laboratory Animals.
Acknowledgements
This study was supported from the National Natural Science Foundation of China

(No. 81673306) for financial support, and "Double First-Class" University project
CPU2018GY04, CPU2018GY35, China Pharmaceutical University.
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