Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2015.04.006

Structural modification was performed at the C-28 position of betulonic acid (BetA). Twenty-five BetA derivatives were synthesized, and evaluated for their antitumor activities against MGC-803, PC3, Bcap-37, A375, and MCF-7 human cancer cell lines by MTT assay. Among the derivatives, most of the derivatives had significant antiproliferative ability (IC₅₀ < 19 μM). Compound 3k, the most active compound, showed IC₅₀ values of 3.6, 5.6, 4.2, 7.8, and 5.2 μM on the five cancer cell lines respectively, and was selected to investigate cell apoptosis by subsequent florescence staining and flow cytometry analysis. The results revealed that compound 3k could induce apoptosis in MGC-803 cell lines, and the apoptosis ratios reached 28.33% after 36 h of treatment at 10 μM. In addition, the study of cancer cell apoptotic signaling pathway indicated that the apoptosis of MGC-803 cells induced by compound 3k could be through the mitochondrial intrinsic pathway.

Full text of DOI:10.1016/j.ejmech.2015.04.006

Accepted Manuscript
Synthesis and biological evaluation of betulonic acid derivatives as antitumor agents
Sheng-Jie Yang, Ming-Chuan Liu, Qi Zhao, De-Yu Hu, Wei Xue, Song Yang
PII:
S0223-5234(15)00255-X
10.1016/j.ejmech.2015.04.006
EJMECH 7821
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 August 2014
Revised Date: 1 April 2015
Accepted Date: 1 April 2015
Please cite this article as: S.-J. Yang, M.-C. Liu, Q. Zhao, D.-Y. Hu, W. Xue, S. Yang, Synthesis and
biological evaluation of betulonic acid derivatives as antitumor agents, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.006.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Twenty-five betulonic acid derivatives were designed and synthesized and found to possess good
antiproliferative activities against five cell lines, among which compound 3k was found to induce
cell apoptosis on MGC-803 cells through the mitochondrial intrinsic pathway.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of betulonic acid derivatives as
antitumor agents
Sheng-Jie Yanga,b,c¶, Ming-Chuan Liua,b¶, Qi Zhaoa, De-Yu Hua, Wei Xuea, and Song Yanga*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, P.R. China
b Research Institute, Sinphar Tian-Li Pharmaceutical Co., Ltd, Hangzhou, 311100, P.R. China
c Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co.,
Ltd, Taizhou, 225321, P.R. China
¶Both authors contributed equally to this work.
*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang,
China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170.
E-mail address: jhzx.msm@gmail.com
Conflicts of interest: None.
Abbreviation list:
ADM: adriamycin
AO/EB: acridine orange/ethidium bromide
BetA: betulonic acid
13C NMR: 13C Nuclear Magnetic Resonance
DMF: N, N-dimethylformamide
DMSO: dimethyl sulfoxide
EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
HCPT: 10-hydroxyl camptothecine
HOBt: 1-hydroxybenzotriazole
1H NMR: Proton Nuclear Magnetic Resonance
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IR: Infra-red
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
TUNEL: terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
Abstract: Structural modification was performed at the C-28 position of betulonic acid (BetA).
Twenty-five BetA derivatives were synthesized, and evaluated for their antitumor activities against
MGC-803, PC3, Bcap-37, A375, and MCF-7 human cancer cell lines by MTT assay. Among the
derivatives, most of the derivatives had significant antiproliferative ability (IC50 < 19 ꢀM).
Compound 3k, the most active compound, showed IC50 values of 3.6, 5.6, 4.2, 7.8, and 5.2 ꢀM on
the five cancer cell lines respectively, and was selected to investigate cell apoptosis by subsequent
florescence staining and flow cytometry analysis. The results revealed that compound 3k could
induce apoptosis in MGC-803 cell lines, and the apoptosis ratios reached 28.33% after 36 h of
treatment at 10 ꢀM. In addition, the study of cancer cell apoptotic signaling pathway indicated that
the apoptosis of MGC-803 cells induced by compound 3k could be through the mitochondrial
intrinsic pathway.
Keywords: betulonic acid derivatives; synthesis; antitumor; apoptosis; mitochondrial pathway
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1. Introduction
Cancer is the major cause of human deaths worldwide because of its high incidence and mortality
[1,2]. In order to improve the survival and quality of life of cancer patients, the need of new
therapies or therapeutic combinations is very urgent all over the world [3]. Natural products have
played an important role in the antitumor drug development [4,5], and about 80% of clinically
antitumor agents originate from natural products [6-9]. In recent years, interest in synthetic
transformations of natural products for the purpose of preclinical development is a major objective
of antitumor research programs [10]. Pentacylic triterpenes are one of the most abundant natural
compounds found in plant kingdom [11-13].
Betulonic acid (BetA, 3-oxo-20(29)-lupen-28-oic acid), is the one of the major effective
components of many traditional Chinese medicine [14], and it can be derived rather easily from
betulin by oxidation with chromium trioxide [15], and betulin forms up to 30% of the dry weight
of the extractive (the bark of birch trees) [16]. BetA and its derivatives have been found to possess
several medicinal properties, such as anti-inflammatory [17,18], anti-melanoma [19] and anti-viral
[20,21] activity. Furthermore, BetA was also found to be as an antitumor agent. Some studies have
shown that BetA could inhibit the growth of various types of human tumor cell lines, including
SGC-7901, HepG-2, LNCaP, DU-145 and PC3 tumor cell lines [22,23]. The antitumor activity of
BetA has attracted the attention of the pioneers who aim to develop novel antitumor agents. The
structure-activity relationships (SAR) of other pentacyclic triterpene acids such as oleanolic acid
(OA), ursolic acids (UA), and GA (glycyrrhetinic acid) had been established [24-26]. Interestingly,
modification of C-28 carboxylic acid group of UA into amides and amines analogous led to a rise
in activity [27]. The amides of BetA with esters of amino acids and with aliphatic and heterocyclic
amines were synthesized, and were revealed valuable biological activity [20,28]. BetA and other
pentacyclic triterpenes shared similar observations of SAR [29]. In the previous work, our group
have synthesized novel nitrogen-containing betulinic acid derivatives. And the pharmacological
evaluations revealed the target compounds had potent antitumor activity compared with betulinic
acid in addition to improved aqueous solubility [30].
In view of the mentioned facts above and an attempt to discover more potent and selective
antitumor agents based on BetA scaffold, a series of new BetA derivatives were also synthesized.
The new products were evaluated their antitumor activities against MGC-803, PC3, Bcap-37,
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A375, and MCF-7 human cancer cell lines. Furthermore, the possible mechanism of MGC-803
cell growth inhibition by compound 3k was also investigated in the present study.
2. Results
2.1. Chemistry
The BetA was derived from betulin by oxidation with Jones regent. The syntheses of BetA
derivatives are summarized in Schemes 1-2. The structure modification of BetA used as the
leading compound was done at the position C-28. BetA was reacted with 1,2-dibromoethane,
1,3-dibromopropane, or 1,4-dibromobutane in the presence of K2CO3 in DMF at room temperature
to give the compounds 2a-2c in high yield. And then, the compounds 2a, 2b, or 2c were reacted
with corresponding amines to yield the nitrogen-containing derivatives 3a-3l, respectively
(Scheme 1). Compound 2a was treated with piperazine in DMF in the presence of K2CO3 at 80 oC,
and reacted with aromatic or aliphatic carboxylic acids in the presence EDCI and HOBT to afford
compounds 5a-5i (Scheme 2). All the compounds were fully characterized by various
spectroscopic methods.
Scheme 1
Scheme 2
2.2. Biology
The in vitro antitumor activity of BetA derivatives on five different human cancer cell lines:
gastric carcinoma cell MGC-803, prostate carcinoma cell PC3, breast carcinoma cell Bcap-37,
malignant melanoma cell A375, and breast carcinoma cell MCF-7 cell lines were studied by MTT
assay. HCPT (hydroxycamptothecine) and ADM (Adriamycin) were used as the positive controls.
The negative control group was treated with culture medium containing 0.1% DMSO. All the
compounds including BetA derivatives, ADM, and HCPT were dissolved in DMSO. Each
experiment was repeated at least three times. The BetA derivatives showed dose dependent
antitumor activity against the investigated cell lines. If IC50 value could not be reached at the
highest concentration, then > 20 was given. The IC50 values are summarized in Table 1.
Table 1
As shown in table 1, when the 28-COOH was only substituted with a dibromoalkane (compounds
2a-2c), the effect was significantly reduced, compared with the parent BetA. The previous study in
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UA or BA derivatives showed that significant improvement of cell growth inhibition was not
observed when the 28-COOH was just acetylated with a fatty alkyl group [30,31]. Similar results
were seen in this study.
Interestingly, the most active compounds (compound 3a-3l) with selected amino groups
introduced were found to be two to eight times more active than BetA, IC50 values between 3 and
18 ꢀM. And the activity of the compounds were increased as the length of the carbon chain was
increased (4 > 3 > 2). The compound 3k, one of the most active compounds, had the IC50 values of
3.6, 5.6, 4.2, 7.8, and 5.2 ꢀM on the five cancer cell lines, respectively.
Among the derivatives substituted with acids, substituted benzoic acids (compounds 5a-5g)
exhibited high and moderate activities, respectively, whereas substituted alkyl carboxylic acids
(compounds 5h-5i) demonstrated weak antitumor activity. Compounds 5h-5i did not show
significant antitumor activity as reported in the literature. One reason for low activity could be due
the long fatty alkyl group. The results were similar with other pentacylic triterpenes [31].
In addition, selectivity of the active compounds (3i, 3j, 3k, 5a, 5e, and 5f) was also assessed on
normal cell line NIH3T3. The selectivity index was calculated by IC50 value in normal cells
divided by IC50 value in cancer cells, and the results are summarized in Table 2. It was observed
that these compounds were 2 to 9 times more selective towards cancer cells than normal cells.
That is to say that these compounds did not show significant cytotoxic effect on normal cells.
Table 2
2.3. Preliminary investigation of the apoptosis-inducing effect of title compound 3k
To determine whether the growth inhibitory activity of the selected compound (compound 3k)
were related to the induction of apoptosis, the morphological character changes of MGC-803 cells
were investigated using the AO/EB, Hoechst 33258 staining under fluorescence microscopy.
The morphologic changes in the cell after treatment with compound 3k were assessed by
fluorescene microscopy after staining with AO/EB. AO is a vital dye and will stain both live and
dead cells, but EB will stain only cells that have lost membrane integrity. The stained cells
revealed four different types under a fluorescence microscope: living cells (normal green nucleus),
early apoptotic (bright green nucleus with condensed or fragmented chromatin), late apoptotic
(orange-stained nuclei with chromatin condensation or fragmentation), and necrotic cells
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(uniformly orange-stained cell nuclei) [32]. With HCPT as positive control at 10 ꢀM for 48 h, the
compound 3k at 5 ꢀM against MGC-803 cells from 12 to 48 h was detected via AO/EB staining.
As can be seen in Figure 1A, the cells treated with compound 3k from 12 to 48 h had changed.
Yellow and orange dots in MGC-803 cells showed early and late apoptotic cells, and the
appearance of little red cells indicated that compound 3k was low cytotoxicity. Therefore, it can be
concluded that compound 3k could induce apoptosis without any significant cytotoxicity.
Hoechst 33258 staining was also used to investigate the apoptosis induction on cells. Live cells
with uniformly light blue nuclei were treated with Hoechst 33258 and observed under a
fluorescence microscope [33]. Hoechst 33258 staining showed apoptosis in all four types of cells,
which was characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and
apoptosis body [34]. With HCPT as positive control at 10 ꢀM for 48 h, the compound 3k at 5 ꢀM
against MGC-803 cells from 12 to 48 h was detected via Hoechst 33258 staining. As can be seen
in Figure 1B, the cells of the negative group (DMSO) were normal blue. However, the cells of
HCPT group appeared compact condensed, and crescent-shaped. The cells exhibited strong blue
fluorescence, revealing the typical apoptosis characteristics. The cells treated with compound 3k
from 12 to 48 h had changed. The cell nuclei appeared to be highly condensed and
crescent-shaped; indicating that compound 3k could induce apoptosis against MGC-803 cell lines.
These results were identical with the previous AO/EB double staining.
In addition, TUNEL is a popular method for identifying apoptotic cells by detection of DNA
fragmentation [33]. The cells were observed that where brown precipitate was the result of
positive apoptosis. With HCPT as positive control at 10 ꢀM for 48 h, the compound 3k at 5 ꢀM
against MGC-803 cells from 12 to 48 h was detected via TUNEL assay. As can be seen in Figure
1C, the cells of the negative group (DMSO) did not appear as brown precipitates, whereas the
other groups, namely, HCPT, appeared as brown precipitates. The cells treated with compound 3k
from 12 to 48 h had changed. Therefore, it can be further concluded that compound 3k induced
apoptosis against MGC-803 cells. The results were identical with the previous experiment.
Figure 1
The apoptosis ratios induced by compound 3k in tumor cells were quantitatively assessed by FCM.
In early apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from
the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular
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environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a
high affinity for PS, and is used to detect early apoptotic cells. PI (Propidine Iodide) was a red
fluorescent dye and stained cells that had lost membrane integrity. So, the different periods of
apoptotic cells could be distinguished when Annexin V matched with PI: the upper left quadrant
contains the necrotic cells (Annexin-/PI+); the upper right quadrant contains late apoptotic cells
(Annexin-/PI+); the lower left quadrant contains the intact cells (Annexin-/PI+); the lower right
quadrant contains the early apoptotic cells (Annexin-/PI+) [35]. As shown in Figure 2, with HCPT
as positive control, compound 3k (10 ꢀM) could induce apoptosis of MGC-803 cells, and highest
apoptosis ratios, 28.33% for compound 3k, were obtained after 36 h of treatment at a
concentration of 10 ꢀM. Furthermore, as shown in Figure 3 the apoptosis of MGC-803 cells which
treated with compound 3k increased gradually in a time-dependent manner.
Figure 2
Figure 3
2.4. The study of cancer cell apoptotic signaling pathway
p53 can initiate apoptosis, the programmed cell death, if DNA damage proves to be irreparable,
and it is able to reduce bcl-2 and promote Bax expression [36]. Thus, cytochrome c, which is a
component of the electron transport chain in mitochondria, will be released [37]. This release of
cytochrome c in turn activates caspase 9, a cysteine protease. And then, caspase 9 can activate
caspase 3 to induce cell apoptosis. As shown in Figure 4A, the expression of p53 and Bax, was
significantly promoted [38]. At the same time, the treatment of compound 3k also resulted in a
significant activation of caspase 3/9 (Figure 4B). These findings indicated that compound 3k
could induce apoptosis of MGC-803 cells through the mitochondrial intrinsic pathway, involving
suppression of the expressions of p53, Bax, caspase 9 and caspase 3.
Figure 4
3. Conclusions
In conclusion, we have described the synthesis of BetA derivatives and their in vitro antitumor
activity on MGC-803, PC3, Bcap-37, A375, and MCF-7 human cell lines. The pharmacological
results showed that most of the compounds displayed moderate to high levels of antitumor activity,
against the five cancer cell lines and that most exhibited more potent inhibitory activity compared
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with the parent BetA. Meanwhile, the results also indicated that (1) Significant improvement of
the cell growth inhibition was achieved when amino groups were introduced at the C-28 position.
(2) Substituted benzoic acids (compounds 5a-5g) displayed moderate to high inhibitory activity,
whereas substituted alkyl carboxylic acids (compounds 5h-5i) demonstrated weak antitumor
activity. (3) When the 28-COOH was only substituted with a dibromoalkane (compounds 2a-2c),
the effect was significantly reduced.
Moreover, the compound 3k, one of the most active compounds, had the IC50 values of 3.6, 5.6,
4.2, 7.8, and 5.2 ꢀM on the five cancer cell lines respectively, and it was selected to analyze the
mechanism by subsequent florescence staining and flow cytometry analysis. The preliminary
mechanism indicated that compound 3k could induce apoptosis in MGC-803 cell lines, and the
apoptosis ratio reached 28.33% after 36 h of treatment at 10 ꢀM, higher than the ratios observed
for the positive control HCPT. Anything else, compound 3k can induce apoptosis of MGC-803
cells through the mitochondrial intrinsic pathway, involving suppression of the expressions of p53,
Bax, caspase 9 and caspase 3. These suggest that BetA derivatives could possess a strong potential
for development as a preventive and therapeutic agent against human carcinomas. Further study is
needed to investigate the active BetA derivatives and the underlying mechanism. And this study
also provides a very powerful incentive for further research on the chemical modification and
structure-activity relationships of BetA and other triterpenoid acids.
4. Experimental
4.1. General
Betulin with more than 98% purity was purchased from Zhejiang Tiancao Biotech Co., Ltd.
Reagents of analytical grade were obtained from Yuda Chemistry Co., Ltd., and used without
further purification unless otherwise noted. Infrared spectra were recorded on a Bruker
VECTOR22 spectrometer in KBr disks. 1H-NMR and 13C-NMR were recorded using a
JEOL-ECX500 spectrometer at 22 °C, with tetramethylsilane as the internal standard and CDCl3
as the solvent. Column chromatography was performed using silica gel (200–300 meshes)
(Qingdao Marine Chemistry Co., Qingdao, China)
4.2. Synthesis
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4.2.1. General procedure for compounds (3a-3l).
BetA (1 mmol) and K2CO3 (2 mmol) were added to DMF (25 mL) and stirred at room temperature
for 10 min, after which 1,2-dibromoethane, 1,3-dibromopropane, or 1,4-dibromobutane (4 mmol)
was added. After being stirred for another 24 h, the reaction mixture was poured onto 100 mL of
distilled water and partitioned with DCM (3×25 mL). The organic layer was washed with
saturated sodium chloride, dried over Na2SO4 and purified via silica gel column chromatography
with petroleum ether/ethyl acetate to obtain compounds 2a-2c. The compounds 2a, 2b or 2c (1
mmol) and K2CO3 (2 mmol) were assed to DMF (10 mL) and stirred at room temperature for 10
min, after saturated nitrogen heterocyclic rings (2 mmol) was added. After being stirred for 24 h,
the reaction mixture was poured into the 100 mL distilled water and partitioned with DCM (3×30
mL). The organic layer was washed with saturated sodium chloride, dried over Na2SO4, and
purified via silica gel column chromatography with CHCl3/MeOH (10:1, v/v) to obtain
compounds 3a-3l.
4.2.1.1. (4-(piperidin-1-yl)butyl) 3-oxo-20(29)-lupen-28-oate (3k)
According to the general procedure, compound 2a was treated with 1,4-dibromobutane, and then
purified on silica gel column using petroleum ether/ethyl acetate (15:1, v/v) to get compound 3k,
Rf [petroleum ether/ ethyl acetate (3:1, v/v)] = 0.24.
1
Yield: 72.8%; pale yellow oil; IR (KBr) : νmax 3441, 2938, 2877, 1689, 1640, 880 cm-1; H NMR
(CDCl3, 500 MHz) δ: 4.67 (1H, s, Hb-29), 4.54 (1H, s, Ha-29), 4.04 (2H, m, CH2), 2.94 (3H, m,
H-2, 19), 2.44 (4H, m, CH2), 2.38 (2H, m, CH2), 1.63 (3H, s, CH3), 1.58 (2H, m, CH2), 1.41 (4H,
m, CH2), 1.01 (3H, s, CH3), 0.96 (3H, s, CH3), 0.91 (3H, s, CH3), 0.89 (3H, s, CH3), 0.86 (3H, s,
CH3); 13C NMR (CDCl3, 125 MHz) δ: 218.2 (C-3), 176.1 (C-28), 150.5 (C-20), 109.7 (C-29), 63.7
(CH2), 58.6 (CH2), 56.5 (C-17), 54.9 (C-5), 54.3 (CH2), 49.9 (C-9), 49.4 (C-18), 47.4 (C-4), 47.0
(C-19), 42.5 (C-14), 40.6 (C-8), 39.7 (C-1), 38.4 (C-13), 37.0 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7
(C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.8 (C-23), 26.7 (CH2), 25.6 (C-12), 25.4 (CH2),
24.1 (CH2), 22.9 (CH2), 21.5 (C-11), 21.1 (C-24), 19.7 (C-6), 19.4 (C-30), 16.0 (C-26), 15.9
(C-25), 14.7 (C-27).
4.2.2. General procedure for compounds (5a-5i).
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Compound 2a (1 mmol) and K2CO3 (2 mmol) were added to DMF (15 mL) and stirred at room
temperature for 10 min, and then piperazine (5 mmol) was dripped into the mixture, which was
stirred at 80 °C for 5 h. After cooling to room temperature, the reaction mixture was poured onto
100 mL of distilled water and partitioned with DCM (3×20 mL). The organic layer was washed
with saturated sodium chloride, dried over Na2SO4 and purified via silica gel column
chromatography with CHCl3/MeOH (10:1, v/v) to obtain compound 4. Compound 4 (1 mmol),
amine compounds (1.2 mmol), EDCI (1.2 mmol) and HOBt (1.2 mmol) were added to DCM (25
mL) containing Et3N (0.5 mmol); the mixture was then stirred at room temperature for 6-12 h.
After the reaction was completed, the mixture was poured onto 100 mL of distilled water and
partitioned with ethyl acetate (3×50 mL). The target compounds were purified on a flash column
with chloroform/methanol (20:1, v/v) to yield compounds 5a-5i.
4.2.2.1. (2-(4-benzoylpiperazin-1-yl)ethyl) 3-oxo-20(29)-lupen-28-oate (5a)
According to the general procedure, compound 4 was treated with aromatic acids and then purified
on silica gel column using chloroform/methanol (10:1, v/v) to obtain compound 5a, Rf
[chloroform/methanol (5:1, v/v)] = 0.28.
Yield: 71.1%; colorless oil; IR (KBr): νmax 3444, 2931, 2869, 1713, 1644, 881, 755, 701 cm-1; 1H
NMR (CDCl3, 500 MHz) δ: 7.37 (5H, s, PhH), 4.69 (1H, s, Hb-29), 4.57 (1H, s, Ha-29), 4.19 (2H,
t, J = 5 Hz, CH2), 3.43 (2H, brs, H in piperazine), 2.97 (3H, m, H-2, 19), 2.64 (2H, t, J = 6.5 Hz,
CH2), 2.46 (2H, m, H in piperazine), 2.38 (2H, m, H in piperazine), 1.67 (3H, s, CH3), 1.04 (3H, s,
CH3), 0.99 (3H, s, CH3), 0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.89 (3H, s, CH3); 13C NMR
(CDCl3, 125 MHz) δ: 218.2 (C-3), 175.9 (C-28), 170.4 (C=O), 150.5 (C-20), 135.8 (C), 129.8
(CH), 128.6 (CH), 127.1 (CH), 109.8 (C-29), 60.8 (CH2), 56.8 (CH2), 55.6 (C-17), 55.0 (C-5),
53.7 (CH2), 53.1 (CH2), 49.9 (C-9), 49.3 (C-18), 47.8 (CH2), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14),
42.2 (CH2), 40.7 (C-8), 39.7 (C-1), 38.4 (C-13), 36.9 (C-22), 36.9 (C-10), 34.2 (C-2), 33.7 (C-7),
32.1 (C-16), 30.7 (C-21), 29.7 (C-15), 26.6 (C-23), 25.5 (C-12), 21.5 (C-11), 21.1 (C-24), 19.7
(C-6), 19.4 (C-30), 16.0 (C-26), 15.9 (C-25), 14.7 (C-27).
4.3. Cell lines and culture
MGC-803, PC3, Bcap-37, A375, and MCF-7 cell lines were obtained from the Institute of
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Biochemistry and Cell Biology, China Academy of Science. MGC-803 is gastric cancer cell line,
PC3 is prostate cancer cell line, A375 is malignant melanoma cell line, Bcap-37, and MCF-7 are
breast cancer cell lines. The entire cancer cell lines were maintained in the RPMI 1640 medium.
They were supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified
atmosphere of 5% CO2 at 37 °C. All cell lines were maintained at 37 °C in a humidified 5%
carbon dioxide and 95% air incubator.
4.4. MTT assays
All compounds were dissolved in DMSO and subsequently diluted in the culture medium before
treatment of the cultured cells. When the cells were 80-90% confluent, they were harvested by
treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in
supplemented growth medium. Cells (2×103/well) were seeded in 96-well plates overnight. The
tested compounds at different concentrations were added into wells and cells were treated at 37 oC
for 72 h. In parallel, the cells treated with 0.1% DMSO served as negative control and ADM
(Adriamycin) as positive control. Then MTT (5 mg/ml, in PBS) was added into each well and
cultured for another 4 h. The MTT-formazan formed by metabolically viable cells was dissolved in
100 ꢀL of SDS for 12 h. The absorbance was then measured at 595 nm with a microplate reader,
which is directly proportional to the number of living cells in culture. The percentage cytotoxicity
was calculated using the formula. IC50 values were calculated according to the dose-dependent
curves [39].
4.5. AO/EB staining
The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates.
Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS
and then supplemented with compounds. After the treatment period, cells were washed with
1×PBS once, and the cover slip with monolayer cells was inverted on the glass slide with 20 ꢀL of
AO/EB stain (100 ꢀg/mL) [40]. The fluorescence was read using an IX71SIF-3 fluorescence
microscope.
4.6. Hoechst 33258 staining
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The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates.
Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS
and then supplemented with compounds (5 ꢀmol/L). After the treatment period, cells were washed
with 1×PBS twice, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were
washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining
solution for 5 min. Then the wells were washed twice, (3min/once) with 1×PBS after staining.
Later, coverslips having cells were covered the glass slide, detected by IX71SIF-3 fluorescence
microscope at 350 nm excitation and 460 nm emissions [41].
4.7. TUNEL assay
The cells (5×104 cell/mL in a volume of 0.6 mL) were seeded in 6-well tissue culture plates.
Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS
and then supplemented with compounds. TUNEL assays were performed using a colorimetric
TUNEL apoptosis assay kit according to the manufacturer’s instructions. (1) After the treatment
period, cells were washed with 1×PBS and fixed in 4% paraformaldehyde for 40 min. The cells
were washed once with PBS, and were consequently permeabilized with immunol staining wash
buffer for 2 min on ice. (2) The cells were rewashed once with PBS, and were consequently
incubated in 0.3% H2O2 in methanol at room temperature for 20 min to inactivate the endogenous
peroxidases, after which the cells were washed thrice with PBS. (3) The cells were incubated with
2 ꢀL of TdT-enzyme and 48 ꢀL of Biotin-dUTP per specimen for 60 min at 37 °C. The cells were
terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 ꢀL per
specimen) conjugate diluted at 1:50 in sample diluent for 30 min. (4) The cells were washed three
times with PBS, and were consequently incubated with diaminobenzidine solution (200 ꢀL per
specimen) for 10 min. At last, the cells were rewashed twice with PBS, and were consequently
imaged under an XDS-1B inverted biological microscope [42].
4.8. Flow cytometry analysis
Prepared MGC-803 cells (1×106/mL) were washed twice with cold PBS and then re-suspended
gently in 500 ꢀL binding buffer. Thereafter, cells were stained in 5 ꢀL Annexin V-FITC and
shaked well. Finally, 5 ꢀL PI was added to these cells and incubated for 20 min in a dark place,
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analyzed by FACS Calibur, Becton Dickinson.
4.9. Western blotting analysis
Cells were collected after treatment with compound 3k at 2.5, 5, and 10 ꢀM for 12 h, respectively.
Western blotting analysis was performed as previously described [43], using the following
antibodies at dilutions of 1: 500 to 1:1000: anti-p53, anti-Bax, and anti-β actin.
5.0. Caspase 3 enzyme assay
Cells were collected after treatment with compound 3k at 2.5, 5, and 10 ꢀM for 12 h, respectively.
Prepared MGC-803 cells (1×106/mL, 5ml) were washed twice with cold PBS. Then, 100 ꢀL of
lysis buffer was added to the cells for 25 min on ice and centrifuged at 18000 g for 15 min. 80 ꢀL
of reaction buffer and 10 ꢀL of Ac-DEVED-pNA were added to 10 ꢀL of supernatant liquid. After
incubating at 37 oC for 2-3 h in darkness, the absorbance was measured at 405 nm, with the lysis
buffer and reaction buffer as control.
5.1. Caspase 9 enzyme assay
Cells were collected after treatment with compound 3k at 2.5, 5, and 10 ꢀM for 12 h, respectively.
Prepared MGC-803 cells (1×106/mL, 5ml) were washed twice with cold PBS. Then, 100 ꢀL of lysis
buffer was added to the cells for 25 min on ice and centrifuged at 16000 g for 15 min. 80 ꢀL of
reaction buffer and 10 ꢀL of Ac-LEHD-pNA were added to 10 ꢀL of supernatant liquid. After
incubating at 37 oC for 2-3 h in darkness, the absorbance was measured at 405 nm, with the lysis
buffer and reaction buffer as control.
5.2. Statistical Analysis
All statistical analyses were performed using SPSS 10.0, and the data were analyzed using
one-way ANOVA. The mean separations were performed using the least significant difference
method. Each experiment was performed in triplicate, and all experiments were run thrice and
yielded similar results. Measurements from all the replicates were combined, and the treatment
effects were analyzed.
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Acknowledgments
The authors wish to thank the National Nature Science Foundation of China (Grant Nos.
21172048, 21372052), Key Technologies R&D Program (No. 2011BAE06B02), the Research
Project of Chinese Ministry of Education (No. 213033A), and Guizhou Key S&T Project (Nos.
[2012]6012, [2011]3016, [2010]3052) for the financial support.
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Table 1 Antitumor activity in a panel of various human cancer cell lines in vitro
Figure 1. Apoptosis induction studies of compound 3k. (A) AO\EB staining. (B) Hoechst 33258
staining. (C) TUNEL assay.
Figure 2. The apoptosis ratios, including the early (B4) and late apoptosis ratios (B2).
Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. A: negative control; B, C, and D:
cells treated with HCPT (10 ꢀM) as positive control; E, F, and G: cells treated with compound 3k
(10 ꢀM).
Figure 4. Expression levels of caspases, p53, and Bax. A: western blot analysis of p53 and Bax in
MGC-803 cells; B: activation of caspase 3/9 in MGC-803 cells.
Scheme 1. Regents and conditions: (i) BrCH2CH2Br, BrCH2CH2CH2Br, or BrCH2(CH2)2CH2Br,
K2CO3, DMF, r.t.; (ii) amine, K2CO3, DMF, r.t.
Scheme 2. Reagents and conditions: (i) piperazine, K2CO3, DMF, 80 °C; (ii) EDCI, HOBT,
aromatic carboxylic acids, DCM, r.t..
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Table 1 Antitumor activity in a panel of various human cancer cell lines in vitro
IC50 in ꢀM for cancer cell lines
Compound
MGC-803
17.7 ± 0.3
> 20
PC3
Bcap-37
25.7 ± 0.4
> 20
A375
MCF-7
1
13.9 ± 0.7
> 20
28.9 ± 0.7
> 20
18.2 ± 0.3
> 20
2a
2b
2c
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
3a
3b
3c
11.7 ± 1.1
10.8 ± 0.1
12.5 ± 1.5
9.4 ± 0.3
6.8 ± 0.4
7.4 ± 0.1
6.3 ± 0.3
5.6 ± 0.3
3.8 ± 0.1
4.0 ± 0.1
3.6 ± 0.1
5.6 ± 0.3
5.3 ± 0.3
4.9 ± 0.2
5.3 ± 0.2
13.4 ± 0.1
4.5 ± 0.3
4.4 ± 0.2
16.6 ± 0.4
> 20
10.1 ± 0.7
12.3 ± 0.3
18.8 ± 0.2
11.3 ± 0.7
7.0 ± 0.6
7.6 ± 0.8
5.7 ± 0.3
6.6 ± 0.8
4.4 ± 0.8
6.7 ± 0.4
5.6 ± 0.3
7.2 ± 0.7
4.1 ± 0.6
6.8 ± 0.5
9.3 ± 0.2
11.0 ± 0.6
8.7 ± 0.8
4.6 ± 0.7
14.1 ± 0.5
> 20
15.6 ± 0.8
10.2 ± 0.9
16.9 ± 1.3
8.8 ± 0.4
6.1 ± 0.3
9.2 ± 0.4
9.5 ± 0.4
7.2 ± 0.8
5.2 ± 0.2
4.7 ± 0.8
4.2 ± 0.1
5.2 ± 0.5
7.4 ± 0.4
6.4 ± 0.1
7.5 ± 0.4
9.4 ± 0.5
6.4 ± 0.4
3.9 ± 0.8
> 20
14.3 ± 1.4
9.7 ± 0.3
17.1 ± 0.1
10.5 ± 0.6
9.3 ± 0.2
8.3 ± 0.4
9.5 ± 0.6
8.6 ± 0.4
9.2 ± 0.7
8.5 ± 0.7
7.8 ± 0.4
6.8 ± 0.5
6.3 ± 0.5
7.6 ± 0.3
8.4 ± 0.2
9.3 ± 0.4
4.5 ± 0.9
7.2 ± 0.4
17.4 ± 0.1
> 20
12.8 ± 0.3
9.4 ± 0.4
16.0 ± 0.2
10.1 ± 0.7
7.3 ± 0.6
8.4 ± 0.3
6.6 ± 0.4
9.3 ± 0.2
7.9 ± 0.5
5.4 ± 0.2
5.2 ± 0.5
8.5 ± 0.6
4.7 ± 0.4
5.4 ± 0.2
8.1 ± 0.1
7.3 ± 0.4
5.4 ± 1.0
3.7 ± 0.7
10.8 ± 1.2
17.6 ± 0.1
15.4 ± 0.7
48.2 ± 0.4
1.35 ± 0.1
3d
3e
3f
3g
3h
3i
3j
3k
3l
5a
5b
5c
5d
5e
5f
5g
5h
5i
> 20
13.4 ± 0.9
29.1 ± 2.6
0.7 ± 0.2
> 20
18.7 ± 1.2
28.1 ± 1.0
1.2 ± 0.2
> 20
HCPT
ADM
34.5 ± 1.5
0.6 ± 0.1
27.8 ± 1.2
1.0 ± 0.6
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Table 2 Selectivity index of the compounds towards tumor cells
Compound
MGC-803
6.15
PC3
5.32
4.28
4.75
9.85
2.43
4.02
Bcap-37
4.50
A375
2.54
3.38
3.41
6.41
4.69
2.57
MCF-7
2.96
5.31
5.11
3i
3j
3k
5a
5e
5f
7.18
6.11
7.38
6.33
7.62
5.46
8.60
3.91
5.00
4.69
3.30
4.20
4.74
20

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Figure 1. Apoptosis induction studies of compound 3k. (A) AO\EB staining. (B) Hoechst 33258
staining. (C) TUNEL assay.
21

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Figure 2. The apoptosis ratios, including the early (B4) and late apoptosis ratios (B2).
22

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Fi
Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. A: negative control; B, C, and D:
cells treated with HCPT (10 ꢀM) as positive control; E, F, and G: cells treated with compound 3k
(10 ꢀM).
23

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Figure 4. Expression levels of caspases, p53, and Bax. A: western blot analysis of p53 and Bax in
MGC-803 cells; B: activation of caspase 3/9 in MGC-803 cells.
24

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Scheme 1. Regents and conditions: (i) BrCH2CH2Br, BrCH2CH2CH2Br, or BrCH2(CH2)2CH2Br,
K2CO3, DMF, r.t.; (ii) amine, K2CO3, DMF, r.t.
25

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Scheme 2. Reagents and conditions: (i) piperazine, K2CO3, DMF, 80 °C; (ii) EDCI, HOBT,
aromatic carboxylic acids, DCM, r.t..
26

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Highlights
1. Two series of betulonic acid derivatives were designed and synthesized.
2. The new compounds showed good antiproliferative activities against tumor cells.
3. Further mechanism study indicates that the new compounds can induce cell apoptosis through
the mitochondrial intrinsic pathway.

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Supporting Information
Synthesis and biological evaluation of betulonic acid derivatives as
antitumor agents
Sheng-Jie Yanga,b,c¶, Ming-Chuan Liua,b¶, Qi Zhaoa, De-Yu Hua, Wei Xuea, and Song Yanga*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, P.R. China
b Research Institute, Sinphar Tian-Li Pharmaceutical Co., Ltd, Hangzhou, 311100, P.R. China
c Research Institute of Traditional Chinese Medicine, Yangtze River Pharmaceutical Group Co.,
Ltd, Taizhou, 225321, P.R. China
¶Both authors contributed equally to this work.
*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang,
China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170.
E-mail address: jhzx.msm@gmail.com
Data of other compounds
o
Compound (1): Yield: 81.8%; white powder; mp 291-293 C; IR (KBr, cm-1): νmax 3440, 2945,
1
1689, 1680; H NMR (CDCl3, 500 MHz) δ: 4.71 (1H, s, H-29), 4.58 (1H, s, H-29), 3.01 (3H, m,
H-2, 19), 1.71 (3H, s, CH3), 1.10 (3H, s, CH3), 0.99 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s,
CH3); 13C NMR (CDCl3, 125 MHz) δ: 219.5 (C-3), 181.8 (C-28), 150.4 (C-20), 109.7 (C-29), 55.9
(C-17), 54.8 (C-5), 49.8 (C-9), 49.5 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.6 (C-8), 39.7
(C-1), 38.6 (C-13), 37.2 (C-22), 36.8 (C-10), 34.2 (C-2), 33.7 (C-7), 32.2 (C-16), 30.7 (C-21),
29.7 (C-15), 26.8 (C-23), 25.6 (C-12), 21.5 (C-11),21.3 (C-24), 19.5 (C-6), 19.4 (C-30), 16.1
(C-26), 15.8 (C-25), 14.5 (C-27). The above data were identical to the literature data [1].
Compound (2a): Yield: 76.9%; colorless oil; IR (KBr, cm-1): νmax 3444, 2955, 2870, 1640, 1428,
1
881; H NMR (CDCl3, 500 MHz) δ: 4.72 (1H, s, Hb-29), 4.60 (1H, s, Ha-29),4.24 (2H, t, J = 7.5
1