Synthesis and cytotoxicity of novel ursolic acid derivatives containing an acyl piperazine moiety

Article abstract of DOI:10.1016/j.ejmech.2012.08.048

This study designed and synthesized a series of novel ursolic acid derivatives in an attempt to develop potent antitumor agents. Their structures were confirmed using MS, IR, ¹H NMR and ¹³C NMR. The inhibitory activities of the title compounds against the MGC-803 (gastric cancer cell) and Bcap-37 (breast cancer cell) human cancer cell lines were evaluated using standard MTT assay in vitro. The pharmacological results showed that some of the compounds displayed moderate to high levels of antitumor activities against the tested cancer cell lines and that most exhibited more potent inhibitory activities compared with ursolic acid. The mechanism of compound 4b was preliminarily investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, TUNEL assay and flow cytometry, which revealed that the compound can induce cell apoptosis in MGC-803 cells.

Full text of DOI:10.1016/j.ejmech.2012.08.048

European Journal of Medicinal Chemistry 58 (2012) 128e135
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity of novel ursolic acid derivatives containing an acyl
piperazine moiety
*
*
Ming-Chuan Liu, Sheng-Jie Yang, Lin-Hong Jin, De-Yu Hu, Wei Xue, Bao-An Song , Song Yang
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, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
This study designed and synthesized a series of novel ursolic acid derivatives in an attempt to develop
potent antitumor agents. Their structures were confirmed using MS, IR, 1H NMR and 13C NMR. The
inhibitory activities of the title compounds against the MGC-803 (gastric cancer cell) and Bcap-37 (breast
cancer cell) human cancer cell lines were evaluated using standard MTT assay in vitro. The pharmaco-
logical results showed that some of the compounds displayed moderate to high levels of antitumor
activities against the tested cancer cell lines and that most exhibited more potent inhibitory activities
compared with ursolic acid. The mechanism of compound 4b was preliminarily investigated by acridine
orange/ethidium bromide staining, Hoechst 33258 staining, TUNEL assay and flow cytometry, which
revealed that the compound can induce cell apoptosis in MGC-803 cells.
Received 28 March 2012
Received in revised form
29 August 2012
Accepted 30 August 2012
Available online 6 October 2012
Keywords:
Ursolic acid derivatives
Antitumor
Cytotoxicity
Apoptosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
antitumor activities and bioavailability [16e19]. Research has
shown that keeping a polar substituent at the C-3 position is
Pentacyclic triterpenes exhibit various biological activities [1,2],
such as anti-HIV [3], anti-inflammatory [4], antitumor [5], antiox-
idant [6], antifeedant [7] and antibacterial [8] activities. Ursolic acid
essential for the pharmacological activities of pentacyclic tri-
terpenes [20e22] and that a hydrogen donor group at either C-3 or
C-28 [19], as well as an ester group at C-3 [23], could improve the
(UA; 3
b
-hydoxy-urs-12-en-28-oic acid 1) is a well-known penta-
cytotoxic activity of UA. Liu et al. [24] found a significant
cyclic triterpene that serves as one of the major effective compo-
nents of many traditional Chinese medicines. Muto et al. [9]
reported that UA is one of the most promising tumor-preventive
medications. As an effective natural anticancer drug, UA has also
been reported to show significant cytotoxicity against some tumor
cell lines [5,10e14]. However, the low bioavailability of UA in vivo
restricts its clinical application [15]. Chemical modifications in UA
have been widely investigated in recent years to improve its
improvement in cell proliferation inhibition when acyl groups were
introduced at the C-28 position. Piperazine-based drug discovery
has attracted considerable attention in recent years. Studies have
shown that the incorporation of a piperazine moiety could occa-
sionally provide unexpected improvements in the bioactivity of
compounds [25e27].
In order to search for ursolic derivatives with high antitumor
bioactivities, the present study introduced an acyl piperazine
moiety to the C-28 position of UA. A series of UA derivatives
containing an acyl piperazine moiety were designed and synthe-
sized. Their antiproliferative activities in vitro against the MGC-
803 and Bcap-37 cancer cell lines as well as the NIH3T3 (mouse
fibroblast cell) normal cell line were evaluated. Results showed
that the target compounds can inhibit proliferation of the two
tumor cell lines at moderate to high rates. Preliminary investiga-
tion of the mode of action of compound 4b found that it can
induce cell apoptosis in MGC-823 cells with a higher apoptosis
ratio compared with the positive controls UA and Hydrox-
ycamptothecin (HCPT) [28,29].
Abbreviation: ADM, adriamycin; AO/EB, acridine orange/ethidium bromide; 13C
NMR, 13C nuclear magnetic resonance; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; ESI-MS, electrospray ionization mass spectrometry; HCPT, 10-
hydroxyl camptothecine; 1H NMR, proton nuclear magnetic resonance; IR, infra-
red; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL,
terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; UA, ursolic
acid.
* Corresponding authors. Ctr for R&D of Fine Chemicals, Guizhou University,
Huaxi St., Guiyang 550025, PR China. Tel.: þ86 851 829 2171; fax: þ86 851 829 2170.
E-mail addresses: songbaoan22@yahoo.com (B.-A. Song), jhzx.msm@gmail.com
(S. Yang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.048

M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
129
2. Results and discussion
corresponding related inhibitory ratios were 92.1% and 85.3% for
compound 4c, 93.0% and 69.1% for 4d as well as 98.9% and 94.8% for
4k. The IC50 values of UA against the MGC-803 and Bcap-37 cell
2.1. Chemistry
lines were 24.32 and 28.69
mM, respectively (Table 1). The IC50
UA was used as the leading compound, and structure modifi-
cations were performed at the C-28 position. The synthetic path-
ways are shown in Scheme 1.
values against these two cell lines ranged from 2.5 to 14.01
most of the title compounds, among which compounds 4b, 4c, 4d
and 4k were found to have the corresponding values of 2.50 and
m
M for
UA (compound 1) was treated with ethylene dibromide in DMF
for 24 h to obtain compound 2, which was introduced with piper-
azine in DMF in the presence of K2CO3 at 80 ꢀC, and then reacted
with aromatic, heterocyclic or aliphatic amines to obtain
compounds 4ae4w. The target compounds were purified by flash
chromatography with petroleum ether/ethyl acetate or chloroform/
methanol as the eluent. The structures of title compounds were
confirmed by IR, ESI-MS, 1H NMR and 13C NMR.
9.24
5.34
m
M, 4.18 and 4.32
mM, 4.66 and 7.26 mM as well as 3.59 and
mM, respectively. These data indicate that the incorporation of
an acyl piperazine moiety at C-28 while retaining the polar group at
C-3 significantly improved the antitumor bioactivities of the
compounds.
Table 1 also shows that acyl substitutions affect the antitumor
bioactivities. Among the acids tested, substituted benzoic acids
(compounds 4be4l) and alkyl carboxylic acids (compounds 4ue
4w) exhibited higher and moderate activities, respectively,
whereas substituted pyridine carboxylic acids (compounds 4pe4s)
demonstrated weak antitumor activities. Meanwhile, halogen or
methyl at the ortho position of the acyl phenyl ring favored the
antitumor bioactivities. On the other hand, the introduction of
a nitro group to the phenyl ring significantly reduced the activities
of compounds 4me4o against the tumor cell lines.
2.2. Evaluation of antitumor activities
In vitro inhibitory activities against the MGC-803 and Bcap-37
cell lines were evaluated for all the synthesized compounds, with
UA and adriamycin (ADM) used as positive controls [30e32]. All the
tested compounds were dissolved in DMSO and subsequently
diluted in culture medium before treatment of cultured cells. The
cells treated with 0.1% DMSO served as control. The inhibitory rates
The antiproliferative bioactivities of the title compounds against
the NIH3T3 cell line were also evaluated. Most of the title
compounds showed stronger antiproliferative activities against the
tumor cell lines than the NIH3T3 cell line (Table 1).
of cell viability with 10 mM concentration of each compound for
72 h and the IC50 values of some compounds are given in Table 1.
As shown in Table 1, most of the title compounds containing an
acyl piperazine moiety at C-28 displayed higher inhibitory activity
than UA against the MGC-803 and Bcap-37 cancer cell lines. The
inhibitory ratios of UA against the MGC-803 and Bcap-37 cell lines
2.3. Preliminary investigation of the apoptosis-inducing effect of
title compound 4b
at 10
mM were 28.9% and 11.9%, respectively, whereas those of
The ability of UA and its derivatives to potentially induce
compound 4b were 95.6% and 76.0%, respectively. The
apoptosis in certain cancer cell lines has been previously reported
Scheme 1. Synthetic route to UA derivatives containing acyl piperazine moieties at C-28. Reagents and conditions: (a) Br(CH2)2Br, K2CO3, DMF, r.t.; (b) piperidine, K2CO3, DMF,
80 ꢀC; (c) EDCI, RCOOH, DCM, r.t.

130
M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
Table 1
Green live MGC-803 cells with normal morphology were seen in
the negative control group. Green yellow or orange dots were
detected in the HCPT after 48 h. However, cells under UA treatment
changed only minimally and pycnosis could only be seen after 48 h.
Cells treated with compound 4b displayed yellow dots as well as
cell budding in MGC-803 cells at 24 h and stained green 48 h after
treatment. Moreover, the emerging orange dots at 48 h also showed
typical late apoptotic cells.
In conclusion, the cells presented with an apoptotic
morphology. The nearly complete absence of red cells in
compounds 4b and UA indicated that it was associated with very
low cytotoxicity. These findings show that compounds 4b and UA
could induce apoptosis with low cytotoxicity.
Effect of UA derivatives against cell viability of different cell lines.
Compound Inhibition rates against
different cells (%)a
IC50 (m
M)b
MGC-803 Bcap-37
NIH3T3
MGC-803
Bcap-37
1
2
3
28.9 ꢂ 4.9 11.9 ꢂ 2.7 14.0 ꢂ 8.3
39.4 ꢂ 4.7 23.1 ꢂ 5.2 11.0 ꢂ 5.5
86.6 ꢂ 3.6 84.4 ꢂ 3.0 60.1 ꢂ 5.7
21.4 ꢂ 3.4 23.9 ꢂ 2.2 19.1 ꢂ 5.8
95.6 ꢂ 1.0 76.0 ꢂ 1.5 32.1 ꢂ 4.8
92.1 ꢂ 0.2 85.3 ꢂ 3.1 24.4 ꢂ 6.8
93.0 ꢂ 1.5 69.1 ꢂ 1.6 29.8 ꢂ 6.9
24.32 ꢂ 0.57 28.69 ꢂ 0.35
12.43 ꢂ 0.98 NTc
7.25 ꢂ 0.41
NT
7.50 ꢂ 0.31
NT
9.24 ꢂ 0.53
4.32 ꢂ 0.42
7.26 ꢂ 0.46
10.00 ꢂ 0.99
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
2.50 ꢂ 0.25
4.18 ꢂ 0.33
4.66 ꢂ 0.12
68.6 ꢂ 3.7 52.0 ꢂ 4.9
2.9 ꢂ 13.1 8.59 ꢂ 0.53
32.1 ꢂ 5.2 60.5 ꢂ 7.3 18.1 ꢂ 6.8
71.8 ꢂ 5.3 83.7 ꢂ 0.6 71.3 ꢂ 5.9
45.3 ꢂ 6.2 32.1 ꢂ 6.3 33.5 ꢂ 5.1
71.2 ꢂ 6.8 38.6 ꢂ 2.3 16.2 ꢂ 8.4
12.45 ꢂ 0.62 8.31 ꢂ 0.44
7.29 ꢂ 0.68
5.12 ꢂ 0.40
NT
>20d
2.3.1.2. Hoechst 33258 staining. Hoechst 33258 is a membrane-
permeable blue fluorescent dye that stains the cell nucleus. Live
cells with uniformly light blue nuclei were observed under fluo-
rescence microscope after treatment with Hoechst 33258, whereas
apoptotic cells had bright blue nuclei because of karyopyknosis and
chromatin condensation; the nuclei of dead cells could not be
8.72 ꢂ 0.49
10.87 ꢂ 0.42
11.76 ꢂ 1.00
5.34 ꢂ 0.41
11.13 ꢂ 0.55
NT
72.3 ꢂ 4.6 45.5 ꢂ 1.8
5.5 ꢂ 12.4 5.02 ꢂ 0.52
98.9 ꢂ 0.6 94.8 ꢂ 1.0 60.3 ꢂ 6.5
24.5 ꢂ 5.7 51.8 ꢂ 2.2 73.1 ꢂ 2.2
24.3 ꢂ 8.2 19.1 ꢂ 5.6 65.4 ꢂ 3.8
57.3 ꢂ 2.0 33.2 ꢂ 5.1 77.7 ꢂ 6.4
24.7 ꢂ 7.4 10.7 ꢂ 6.6 37.2 ꢂ 1.2
3.59 ꢂ 0.25
NT
NT
9.41 ꢂ 0.78
NT
NT
NT
32.1 ꢂ 7.0
8.3 ꢂ 7.0 55.4 ꢂ 6.1
13.26 ꢂ 0.70 NT
stained. MGC-803 cells treated with compound 4b at 5
mM from 12
22.0 ꢂ 5.7 24.5 ꢂ 9.6 52.3 ꢂ 1.8
24.1 ꢂ 1.1 19.0 ꢂ 3.8 44.3 ꢂ 4.7
NT
NT
NT
NT
to 48 h were stained with Hoechst 33258. HCPT and UA were used
as positive controls at 10
Fig. 2.
mM for 48 h. The results are shown in
4s
4t
34.3 ꢂ 4.1 29.0 ꢂ 6.3
4.3 ꢂ 14.1 14.01 ꢂ 1.10 NT
51.1 ꢂ 1.9 54.4 ꢂ 3.2 39.0 ꢂ 8.3
23.4 ꢂ 2.0 14.7 ꢂ 5.4 35.0 ꢂ 7.1
44.2 ꢂ 1.1 12.5 ꢂ 3.8 12.0 ꢂ 9.2
80.8 ꢂ 4.7 68.6 ꢂ 3.4 54.5 ꢂ 9.3
12.59 ꢂ 0.20 9.22 ꢂ 0.40
Fig. 2 shows that cells treated with the negative control DMSO
were normally blue (in the web version). On the other hand, the cell
nuclei of HCPT (positive control)-treated cells appeared to be
compact and condensed. UA had no obvious morphological
changes, but most cell nuclei appeared to be highly condensed
(brightly stained). For 4b treatment, the cells exhibited strong blue
fluorescence and revealed typical apoptotic morphology after 12,
24 and 48 h. These findings demonstrate that compounds 4b and
UA induced apoptosis against MGC-803 cell lines, consistent with
the results for AO/EB double staining.
4u
4v
4w
HCPTe
ADMf
NT
>20
NT
NT
6.89 ꢂ 0.85
4.16 ꢂ 0.29
29.1 ꢂ 2.6 28.1 ꢂ 1.0
9.3 ꢂ 4.2
>20
>20
92.1 ꢂ 1.3 92.1 ꢂ 1.1 99.2 ꢂ 0.1
0.7 ꢂ 0.2
1.28 ꢂ 0.28
a
Inhibitory percentages of cells treated with 10
mM concentration of each
compound for 72 h.
b
Agent concentration (mM) that inhibited cell growth by 50% at 72 h after
treatment.
c
d
e
f
Not tested.
Inhibition (50%) was higher than 20
Hydroxycamptothecin, positive control.
Adriamycin, positive control.
m
M.
2.3.2. TUNEL assay
TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick
end labeling) is a popular method for identifying apoptotic cells in
situ by detecting DNA fragmentation. Due to degradation of DNA
caused by the activation of Ca/Mg-dependent endonucleases in
apoptotic cells, DNA cleavage occurred and led to strand breakage
within the DNA. These strand breaks of cleaved DNA could be
identified by terminal deoxynucleotidyl transferase that catalyzed
the addition of biotin-dUTP. The biotin-labeled cleavage sites were
then detected by reaction with streptavidineHRP and visualized by
diaminobenzidine, as indicated by a brown color (in the web
version). The results are illustrated in Fig. 3.
[16e18,33]. In the present study, compound 4b was selected and its
mechanism of growth inhibition of MGC-803 cells was evaluated.
2.3.1. Fluorescence staining
Changes in the morphological character of MGC-803 cells were
investigated using acridine orange (AO)/ethidium bromide (EB) and
Hoechst 33258 staining under fluorescence microscopy to deter-
mine whether the growth inhibitory activity of the selected
compound was related to the induction of apoptosis.
2.3.1.1. AO/EB staining. AO is a vital dye that can stain nuclear DNA
across an intact cell membrane, whereas EB can only stains cells
that had lost their membrane integrity. Thus, after simultaneous
treatment with AO and EB, live cells will be uniformly stained as
green (in the web version) and early apoptotic cells will be densely
stained as green yellow or show green yellow fragments (in the web
version), whereas late apoptotic cells will be densely stained as
orange or display orange fragments and necrotic cells will be
stained as orange with no condensed chromatin. The cytotoxicity of
Fig. 3 shows that cells in the negative group (DMSO treatment)
did not appear as brown precipitates (Fig. 3, 1). The cells treated
with compound 4b at different concentrations (Fig. 3, 4e6), UA
(Fig. 3, 3) and HCPT (positive control; Fig. 3, 2) appeared as brown
precipitate. Therefore, we further concluded that compound 4b
induced apoptosis against MGC-803. The results were identical
with the previous experiment.
2.3.3. Flow cytometry
compound 4b at the concentration of 5
from 12 to 48 h was detected by AO/EB staining, with HCPT and UA
m
M against MGC-803 cells
The apoptosis ratios induced by compound 4b in tumor cells
were quantitatively assessed by flow cytometry. In the early stages
of apoptosis, phosphatidylserine was translocated from within the
cell membrane to its outer part. Annexin V, a calcium-dependent
phospholipid-binding protein associated with a high affinity for
phosphatidylserine, was used to detect early apoptotic cells. Pro-
pidine iodide (PI) is a red fluorescent dye that stains cells that had
lost their membrane integrity. Cells stained with Annexin V-FITC
and PI were classified as necrotic cells (B1; Annexinꢁ/PIþ), late
used as positive controls at 10
The results are given in Fig. 1.
mM against MGC-803 cells for 48 h.
Fig.1 shows that the cells treated with 4b from 12 to 48 h and UA
for 48 h had changed in MGC-803 cells. The nuclei stained as yellow
green or orange, and the morphology showed pycnosis, membrane
blebbing and cell budding. These phenomena are associated with
cell apoptosis.

M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
131
Fig. 1. AO/EB staining of compound 4b in MGC-803 cells. (2,3) Respectively HCPT and UA (10
mM each) as positive for 48 h (4e6) treatment with compound 4b (5
m
M) for
respectively 12 h, 24 h and 48 h.
apoptotic cells (B2; Annexinþ/PIþ), intact cells (B3; Annexinꢁ/PIꢁ)
or early apoptotic cells (B4; Annexinþ/PIꢁ). The results are given in
Fig. 4.
Fig. 5 shows that compound 4b (10 mM) could induce apoptosis
in MGC-803 cells. Apoptosis ratios (including the early and late
3. Conclusions
UA is an important natural product with antitumor bioactivity.
In this study, a series of UA derivatives containing an acyl pipera-
zine moiety were designed and synthesized, and their cell growth
inhibition activities against the MGC-803 and Bcap-37 cell lines
were evaluated using MTT assay. The corresponding IC50 values of
compounds 4b, 4c, 4d and 4k against MGC-803 and Bcap-37 were
apoptosis ratios) for compound 4b were obtained after 36 h of
treatment at a concentration of 10 mM, with the highest apoptosis
ratio being 33.35%. Furthermore, as shown in Fig. 5, the apoptosis of
MGC-803 cells treated with compound 4b increased gradually in
a time-dependent manner; meanwhile, the apoptosis ratios of
compound 4b measured at different time points (23.57%, 28.01%
and 33.35% at 12, 24 and 36 h, respectively) were higher than those
of HCPT (17.37%, 18.66% and 24.95%, respectively).
2.50 ꢂ 0.25 and 9.24 ꢂ 0.53
4.66 ꢂ 0.12 and 7.26 ꢂ 0.46
M, respectively. Incorporation of an acyl piperazine
m
M, 4.18 ꢂ 0.33 and 4.32 ꢂ 0.42
mM,
m
M as well as 3.59 ꢂ 0.25 and
5.34 ꢂ 0.41
m
moiety at C-28 while retaining the polar group at C-3 significantly
improved the antitumor bioactivities of the compounds. Moreover,
Fig. 2. Hoechst 33258 staining of compound 4b in MGC-803 cells. (2,3) Respectively HCPT and UA (10 mM each) as positive for 48 h (4e6) treatment with compound 4b (5 mM) for
respectively 12 h, 24 h and 48 h.

132
M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
Fig. 3. TUNEL assay of compound 4b in MGC-803 cells for 24 h (2,3). Respectively HCPT and UA (10
M (5) and 10 M (6) for 24 h.
mM each) as positive for 24 h (4e6) treatment with compound 4b at 2.5 mM (4),
5
m
m
most of the title compounds showed lower antiproliferative effects
against NIH3T3 cells than the tumor cells. The apoptosis-inducing
activity of compound 4b in MGC-803 cells was investigated by
AO/EB staining, Hoechst 33258 staining, TUNEL assay and flow
cytometry. This compound clearly demonstrated cell apoptosis-
inducing effects, with its inducing ratio being higher than the
ratios observed for the positive control HCPT and UA. Further
studies of the specific mechanisms of these compounds in human
malignant tumors are currently underway.
Compound 2 (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 ethyl acetate (3 ꢃ 20 mL). The organic layer was
washed with saturated sodium chloride, dried over Na2SO4 and
purified via silica gel column chromatography with chloroform/
methanol (10:1, v/v) to obtain compound 3.
Compound 3 (1 mmol) and amine compounds (1.2 mmol) and
HATU (1.2 mmol) or EDCI (1.2 mmol) and HOBt (1.2 mmol) were
added to DCM (10 mL) containing Et3N (0.5 mmol); the mixture was
then stirred at room temperature for 6e12 h. After the reaction was
completed, the mixture was poured onto 100 mL of distilled water
and partitioned withethyl acetate(3ꢃ 50mL). The target compounds
were purified on a flash column with chloroform/methanol (20:1, v/
v) to yield compounds 4ae4w. The structures were confirmed by IR,
ESI-MS, 1H NMR and 13C NMR (see Supporting information).
4. Experimental
4.1. General
UA with more than 90% purity was purchased from Hainan
Super Biotech Co., Ltd. Reagents of analytical grade were obtained
from Yuda Chemistry Co., Ltd., and used without further purifica-
tion unless otherwise noted. Silica gel (200e300 mesh) used in
column chromatography was provided by Tsingtao Marine Chem-
istry Co., Ltd. IR spectra were recorded on a Bruker VECTOR22
spectrophotometer in KBr disks. ESI-MS spectra were determined
with Agilent 1100 IC/MDS Trap XCT and are reported as m/z. 1H
NMR and 13C NMR were performed on a JEOL-ECX500 spectrom-
eter at 22 ꢀC, with TMS as the internal standard.
4.2.1.1. (2-(Piperazin-1-yl)ethyl) 3-hydroxy-urs-12-en-28-oate (3).
According to the general procedure, compound 2 was treated with
piperazine and then purified on silica gel column using chloroform/
methanol (10:1, v/v) to obtain compound 3, Rf [petroleum ether/
ethyl acetate (3:1, v/v)] ¼ 0.31.
Yield: 55.5%; colorless oil; ESI-MS: 569[M þ H]þ; IR (KBr, cmꢁ1
)
v: 3269, 3163, 2941, 2868, 1730, 1463, 1377, 1220, 1029; 1H NMR
(CDCl3, 500 MHz): 5.23 (1H, s, H-12), 4.14 (2H, t, J ¼ 5 Hz, CH2), 3.20
(1H, dd, J ¼ 10 Hz, 10 Hz, H-3), 2.89 (4H, t, J ¼ 5 Hz, H in piperazine),
2.65 (2H, t, J ¼ 5 Hz, CH2), 2.48 (4H, brs, H in piperazine), 2.22 (1H, d,
J ¼ 10 Hz, H-18), 1.07 (CH3, s, H-27), 0.98 (CH3, s, H-25), 0.94 (CH3, s,
H-26), 0.93 (CH3, s, H-24), 0.91 (CH3, s, H-23), 0.86 (CH3, d, J ¼ 10 Hz,
H-30), 0.77 (CH3, d, J ¼ 10 Hz, H-29); 13C NMR (CDCl3, 125 MHz):
177.4 (C-28), 138.1 (C-13), 125.6 (C-12), 78.9 (C-3), 61.8 (CH2), 57.2
(CH2), 55.2 (C-5), 54.7 (CH2), 52.9 (C-18), 48.0 (C-17), 47.6 (C-9), 46.1
(CH2), 42.1 (C-14), 39.6 (C-8), 39.1 (C-19), 38.9 (C-1), 38.8 (C-4), 38.6
(C-20), 37.0 (C-10), 36.7 (C-22), 33.1 (C-7), 30.7 (C-21), 28.2 (C-23),
28.0 (C-15), 27.3 (C-2), 24.2 (C-16), 23.6 (C-11), 23.3 (C-27), 21.2 (C-
30), 18.3 (C-6), 17.2 (C-26), 17.0 (C-29), 15.7 (C-25), 15.5 (C-24).
4.2. Synthesis
4.2.1. General procedure for compounds 2, 3 and 4ae4w
UA (1 mmol) and K2CO3 (2 mmol) were added to DMF (15 mL)
and stirred at room temperature for 30 min, after which bro-
moalkane or dibromoalkane (4 mmol) was added and KI
(0.5 mmol) was dripped into the mixture. After being stirred for
another 12 h, the reaction mixture was poured onto 100 mL of
distilled water and partitioned with ethyl acetate (3 ꢃ 20 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 compound 2.

M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
133
Fig. 4. Apoptosis ratio detection of compound 4b by Annexin V/PI assay. (1e3) Cells were treated with HCPT at 10
mM for 12 h, 24 h, and 36 h, respectively. (4e6) Cells were treated
with compound 4b at 10 M for 12 h, 24 h, and 36 h, respectively. (7) Cells were treated with 0.1% DMSO for 36 h.
m
4.2.1.2. (2-(4-(2,6-Dichlorobenzoyl)piperazin-1-yl)ethyl)3-hydroxy-
urs-12-en-28-oate (4b). According to the general procedure,
compound 3 was treated with aromatic amines and then purified
on silica gel column using chloroform/methanol (10:1, v/v) to
obtain compound 4b, Rf [chloroform/methanol (5:1, v/v)] ¼ 0.22.
Yield: 51.5%; yellow colorless oil; ESI-MS: 741[M þ H]þ; IR (KBr,
cmꢁ1) v: 3444, 2924, 2868, 1718, 1637, 1429, 1139, 794, 754; 1H NMR
(CDCl3, 500 MHz): 7.34e7.28 (3H, m, PhH), 5.22 (1H, s, H-12), 4.12 (2H,
t, J ¼ 7.5 Hz, CH2), 3.84 (2H, m, H in piperazine), 3.23 (2H, t, J ¼ 7.5 Hz, H
in piperazine), 3.16 (1H, d, J ¼ 5 Hz, H-3), 2.65 (2H, t, J ¼ 5 Hz, CH2), 2.61
(2H, m, H in piperazine), 2.49 (2H, brm, H in piperazine), 2.21 (1H, d,
J ¼ 10 Hz, H-18), 1.07 (CH3, s, H-27), 0.98 (CH3, s, H-25), 0.94 (CH3, s, H-
26), 0.93 (CH3, s, H-24), 0.90 (CH3, s, H-23), 0.86 (CH3, d, J ¼ 5 Hz, H-30),
0.77 (CH3, d, J ¼ 15 Hz, H-29); 13C NMR (CDCl3,125 MHz): 177.4 (C-28),
161.9 (C),159.7 (C),157.7 (C),138.1 (C-13),131.9 (CH2),130.8 (CH2),125.6
(C-12),124.3 (C),114.5 (CH2), 79.0 (C-3), 61.7 (CH2), 56.5 (CH2), 55.2 (C-
5), 53.4 (CH2), 52.9 (C-18), 52.8 (CH2), 48.1 (C-17), 47.5 (C-9), 46.6 (CH2),
42.1 (C-14), 41.8 (CH2), 39.6 (C-8), 39.1 (C-19), 38.9 (C-1), 38.8 (C-4),
38.6 (C-20), 37.0 (C-10), 36.8 (C-22), 33.1 (C-7), 30.7 (C-21), 28.2 (C-23),
28.0 (C-15), 27.2 (C-2), 24.3 (C-16), 23.6 (C-11), 23.3 (C-27), 21.2 (C-30),
18.3 (C-6), 17.2 (C-26), 17.0 (C-29), 15.7 (C-25), 15.5 (C-24).
Fig. 5. Apoptosis ratios of MGC-803 cells treated with compound 4b (10
(10 M) as assessed by flow cytometry at 12, 24 and 36 h. The apoptosis ratio includes
the early (B4) and late (B2) apoptosis ratios.
mM) and HCPT
m

134
M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
4.3. Cell lines and culture
were washed with PBS and fixed in 4% paraformaldehyde for
40 min. After washing once with PBS, cells were permeabilized
with Immunol staining wash buffer (Beyotime) for 2 min on ice.
Cells were washed once with PBS again and incubated in 0.3%
H2O2 in methanol at room temperature for 20 min to inactivate
the endogenous peroxidases, after which the cells were washed
The MGC-803, Bcap-37 and NIH3T3 cell lines used in this study
were all obtained from the Institute of Biochemistry and Cell
Biology, China Academy of Sciences. MGC-803 is stomach cancer,
Bcap-37 is breast cancer and NIH3T3 is normal mouse fibroblast.
The MGC-803 and Bcap-37 cell lines were maintained in RPMI 1640
medium, whereas the NIH3T3 cell line was maintained in DMEM
medium; all were supplemented with 10% heat-inactivated fetal
bovine serum in a humidified atmosphere of 5% CO2 at 37 ꢀC.
thrice with PBS. Thereafter, the cells were incubated with 2
of terminal deoxynucleotidyl transferase enzymes and 48 L of
biotin-dUTP per specimen for 60 min at 37 ꢀC. After termination
for 10 min, cells were incubated with streptavidineHRP (50
per specimen) conjugate diluted at 1:50 in diluent of
streptavidineHRP for 30 min. After washing thrice with PBS,
cells were incubated with diaminobenzidine solution (200
mL
m
mL
a
4.4. MTT assay against cancer cell viability
mL
per specimen) for 10 min. This was again followed by washing
with PBS two times, and the result was imaged under an XDS-
1B inverted biological microscope (Chongqing Photoelectric
Device Co.).
All tested compounds were dissolved in DMSO and subse-
quently diluted in culture medium before treatment of cultured
cells. Tested cells were plated in 96-well plates at a density of
2 ꢃ 103 cells/well per 100
mL of the proper culture medium and
treated with the compounds at 1e20
mM for 72 h. In parallel, the
cells treated with 0.1% DMSO served as negative control and
those treated with ADM as positive control. Finally, 100 L of MTT
m
4.8. Flow cytometry
was added, and the cells were incubated for 4 h. The MTT
formazan formed by metabolically viable cells was dissolved in
Prepared MGC-803 cells (1 ꢃ 106 cells/mL) were washed twice
100
mL of SDS for 12 h. Absorbance, which is directly proportional
with cold PBS and then resuspended gently in 500 mL of binding
to the number of living cells in culture, was then measured at
595 nm with a microplate reader (Model 680; BIO-RAD). The
percentage of cell viability inhibition was calculated using the
following formula:
buffer. Thereafter, cells were stained in 5
shaken well. Finally, the cells were mixed with 5
for 20 min in the dark and subsequently analyzed using FACSCali-
bur (Becton Dickinson).
m
L of Annexin V-FITC and
mL of PI, incubated
ðControl abs ꢁ Blank absÞ ꢁ ðTest abs ꢁ Blank absÞ
ðControl abs ꢁ Blank absÞ
% viability inhibition ¼
ꢃ 100
4.5. AO/EB staining
4.9. Statistical analysis
Cells were seeded at a concentration of 5 ꢃ 104 cell/mL in
a volume of 0.6 mL on a sterile cover slip in six-well tissue culture
plates. Following incubation, the medium was removed and
replaced with fresh medium plus 10% fetal bovine serum and
All statistical analysis was performed with SPSS Version 10. Data
was analyzed by one-way ANOVA. Mean separations were per-
formed using the least significant difference method. Each experi-
ment was replicated thrice, and all experiments yielded similar
results. Measurements from all the replicates were combined, and
treatment effects were analyzed.
supplemented with compounds (10
period, the cover slip with monolayer cells was inverted on a glass
slide with 20 L of AO/EB stain (100 g/mL). Fluorescence was read
mM). After the treatment
m
m
on an IX71SIF-3 fluorescence microscope (OLYMPUS Co., Japan).
Authors contributions
4.6. Hoechst 333258 staining
Ming-Chuan Liu synthesized the compounds and carried out
most of the bioassay experiments. Sheng-Jie Yang did part of the
bioassay experiments. Lin-Hong Jin took part in the compound
structural elucidation and bioassay experiments. De-Yu Hu carried
out some structure elucidation experiments. Wei Xue assisted in
structural elucidation experiments. Prof. Bao-An Song and Song
Yang are the co-corresponding authors for this work.
Cells grown on a sterile cover slip in six-well tissue culture
plates were treated with compounds for a certain range of time. The
culture medium containing compounds was removed, and the cells
were fixed in 4% paraformaldehyde for 10 min. After being washed
twice with PBS, the cells were stained with 0.5 mL of Hoechst
33258 (Beyotime) for 5 min and then again washed twice with PBS.
The stained nuclei were observed under an IX71SIF-3 fluorescence
microscope using 350 nm excitation and 460 nm emission.
Conflicts of interest
None.
4.7. TUNEL assay
TUNEL assays were performed with a colorimetric TUNEL
apoptosis assay kit (Beyotime) according to the manufacturer’s
instructions. Cells grown in six-well culture clusters were
treated as in the mitochondrial depolarization assay. In short,
MGC-803 or Bcap-37 cells grown in six-well tissue culture plates
Acknowledgments
This work was supported by the National Key Program for Basic
Research (Grant No. 2010CB 126105), the National Basic Research
Preliminary Program of China (Grant No. 2010CB134504) and the

M.-C. Liu et al. / European Journal of Medicinal Chemistry 58 (2012) 128e135
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