Nitric oxide-releasing derivatives of brefeldin A as potent and highly selective anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.05.018

A series of NO-donating mono- or diester derivatives of brefeldin A were designed, synthesized and biologically evaluated. Some derivatives exhibited potent antiproliferative activity with low IC₅₀ values. The most potent NO-donating hybrid 13b exhibited stronger cytotoxicity against human prostate cancer PC-3?cells, human colon carcinoma HT-29?cells and human liver cancer HepG-2?cells than BFA with IC₅₀ values of 25?nM, 160?nM and 180?nM, respectively. More importantly, compound 13b showed good selectivity between human normal and tumor liver cells with selectivity index of 33. Additionally, 13b released higher levels of NO in HepG-2?cells than L-02?cells. Further mechanism concerning cellular apoptosis showed that 13b induced apoptosis and S phase cell cycle arrest in HepG-2?cells. Incubation with 13b increased the number of HepG-2?cells with collapsed mitochondrial membrane at low concentrations in dose-dependent manner. In addition, by using the Human Apoptosis Protein Array kit, several apoptosis-related proteins, including HO-1, HO-2 and survivin, were found to be markedly downregulated by 13b in HepG-2?cells. Furthermore, in western blot assay, 13b increased the expression of Bax, Cyt c and caspase 3, and reduced the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG-2?cells.

Full text of DOI:10.1016/j.ejmech.2017.05.018

European Journal of Medicinal Chemistry 136 (2017) 131e143
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Nitric oxide-releasing derivatives of brefeldin A as potent and highly
selective anticancer agents
,
,
, **
Kangtao Tian a 1, Fanxing Xu b 1, Xiang Gao a, Tong Han a, Jia Li a, Huaqi Pan c
,
Linghe Zang d, Dahong Li a ***, Zhanlin Li a, Takahiro Uchita e, Ming Gao e, Huiming Hua a
,
, *
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
b Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
c Shenyang Institute of Applied Ecology, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang 110016, PR China
d School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
e School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien, Nishinomiya 663-8179, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of NO-donating mono- or diester derivatives of brefeldin A were designed, synthesized and
biologically evaluated. Some derivatives exhibited potent antiproliferative activity with low IC50 values.
The most potent NO-donating hybrid 13b exhibited stronger cytotoxicity against human prostate cancer
PC-3 cells, human colon carcinoma HT-29 cells and human liver cancer HepG-2 cells than BFA with IC50
values of 25 nM, 160 nM and 180 nM, respectively. More importantly, compound 13b showed good
selectivity between human normal and tumor liver cells with selectivity index of 33. Additionally, 13b
released higher levels of NO in HepG-2 cells than L-02 cells. Further mechanism concerning cellular
apoptosis showed that 13b induced apoptosis and S phase cell cycle arrest in HepG-2 cells. Incubation
with 13b increased the number of HepG-2 cells with collapsed mitochondrial membrane at low con-
centrations in dose-dependent manner. In addition, by using the Human Apoptosis Protein Array kit,
several apoptosis-related proteins, including HO-1, HO-2 and survivin, were found to be markedly
downregulated by 13b in HepG-2 cells. Furthermore, in western blot assay, 13b increased the expression
of Bax, Cyt c and caspase 3, and reduced the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG-
2 cells.
Received 17 February 2017
Received in revised form
1 May 2017
Accepted 4 May 2017
Available online 5 May 2017
Keywords:
Brefeldin A
Antitumor
Nitric oxide
Apoptosis
© 2017 Published by Elsevier Masson SAS.
1. Introduction
mechanism was widely investigated, that BFA could induce
apoptosis in numerous human cancer cell lines [7,8] and disrupt the
Brefeldin A (BFA) (1, Fig. 1) was first isolated from Penicillium
decumbens by Singleton et al. [1]. It is a macrolide antibiotic and
secondary metabolite of Ascomycetes species. Most importantly,
BFA exhibited a variety of biological activities including antitumor
[2], antifungal [3], antiviral [4], antimitotic [5] and so on. The potent
antiproliferative activity of BFA was tested by the National Cancer
Institute's 60 cancer cell line assay (NCI-60) with the mean graph
midpoint (MGM) GI50 value of 40 nM [6]. Furthermore, the
cis Golgi apparatus [9]. Treatment of eukaryotic cells with BFA could
induce the breakdown of vesicle-mediated protein transport,
which resulted in the Golgi complex redistributing into the endo-
plasmic reticulum (ER) [10]. Moreover, it has been reported that
BFA would cause apoptotic cell death in ovarian carcinoma cell lines
by activating the mitochondrial pathway and the caspase-8- and
Bid-dependent pathways [11]. In addition, BFA also effectively
inhibited clonogenic activity and the migration and matrix
metalloproteinases-9 activity of MDA-MB-231 cells. Western blot
analysis indicated that BFA could mediate the down-regulation of
breast cancer stem cells marker CD 44 and anti-apoptotic proteins
Bcl-2 and Mcl-1, as well as the reversal of epithelial-mesenchymal
transition [12]. These biological results and mechanism investiga-
tion prompted BFA to be a potential therapeutic lead for future
investigation [13,14].
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: panhq@iae.ac.cn (H. Pan), lidahong0203@163.com (D. Li),
huimhua@163.com (H. Hua).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.05.018
0223-5234/© 2017 Published by Elsevier Masson SAS.

132
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
2. Results and discussion
2.1. Chemistry
The derivatives 11aꢀf, 12aꢀf and 13aꢀf were synthesized from
thiophenol and BFA. The BFA was isolated from the fermentation
liquor and mycelium of Eupenicillium brefeldianum, and character-
ized by 1H NMR, 13C NMR, high-resolution mass spectra, X-ray
crystal structure analysis and optical rotation [24]. Thiophenol 2
was treated with chloroacetic acid in sodium hydroxide yielding
the (phenylthio)acetic acid 3. A one-pot reaction of (phenylthio)
acetic acid by oxidization with 30% H2O2 produced compound 4,
which was further reacted with fuming HNO3 at 90 ꢁC to yield 5.
Reacting 5 with ethanediol or propanediol in the presence of 30%
NaOH in THF afforded 6aꢀb, which were further treated with an-
hydride, triethylamine (Et3N) and 4-dimethylaminopyridine
(DMAP) to obtain intermediates 7aꢀf (Scheme 1). In order to get
the ester derivatives at 4-position, the 4,7-OH groups of BFA were
protected with tert-butyldimethylsilyl (TBS) groups using tert-
butyldimethylsilyltriflate (TBSOTf) and 2,6-lutidine in dichloro-
methane (DCM). Then the TBS group on 4-OH of compound 8 was
removed by tert-butylammonium fluoride (TBAF) in THF to obtain
the intermediate 9. Reacting compound 9 with intermediates 7aꢀf
in the presence of DMAP and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI) afforded the intermediates
10aꢀf. The derivatives 11aꢀf were got from 10aꢀf by the reaction
with TBAF in THF (Scheme 2). Target compounds 12aꢀf and 13aꢀf
were prepared from the combination of 7aꢀf and BFA under the
condition of DMAP and EDCI in DCM, followed by column chro-
matography on silica gel using petroleum ether-ethyl acetate (V:V
2:1) (Scheme 3). Intermediate 7a was further treated with
TMSCHN2 to offer compound 14. 15 was prepared by the reaction of
BFA with succinic anhydride in 2,6-lutidine (Scheme 4).
Fig. 1. Chemical structure and key numbering of brefeldin A.
Although the cytostatic and apoptotic effects of BFA have been
well studied, it is unsuitable to subject BFA to direct clinical
development mainly because of its undesirable drug-like proper-
ties [15,16], including low selectivity between tumor and normal
cells. In this study, the antiproliferative activity, especially the
selectivity between cancerous and normal cells was improved,
which was probably contributed to the introducing of nitric oxide
(NO) donor moieties.
NO is known as a notable mediator in biological system and a
wide range of physiological processes [17,18]. Active macrophages
produce NO, which induce cytostasis and generate cytotoxity on
tumor cells, and the mechanism seems to be the inhibition of
ribonucleotide reductase [19]. Recently, NO donor hybrid com-
pounds have become a focus in the field of oncotherapy since NO
influences the apoptosis process of tumor cells [18]. It is reported
that OA/furoxan hybrid compounds exhibited remarkably
improved cytotoxicity because of the cytotoxic effects of NO
released from the furoxan moieties [20]. In addition, nonsteroidal
anti-inflammatory drugs/nitrate hybrids have been identified as
the prototypical chemopreventive agents against many forms of
cancers [21]. We have also synthesized a variety of furoxan-based
NO donating compounds, such as evodiamine/furoxan [22] and
spirolactone-type diterpenoid/furoxan hybrids [23] with potent
antitumor activities and/or selectivity between cancerous and
normal cells. Therefore, generation of furoxan-based hybrids is a
promising strategy to develop potential antitumor drug candidates.
On the basis of above, the present article outlined the synthesis
of 18 mono- or diester derivatives of BFA, which were obtained by
introducing furoxan-type NO donors into the two hydroxyl (at C4
and C7 position) groups. The cytotoxicity, NO releasing ability and
cellular mechanism of these derivatives were investigated. The
research herein aimed to obtain a befitting dual BFA/NO releasing
agent that still maintained the antiproliferative activity and showed
selectivity between cancerous and normal cells.
2.2. Pharmacology
2.2.1. The antiproliferative activities of BFA derivatives
Applying
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay, the anti-
proliferative activities of BFA and the derivatives against one
normal (L-02) and three tumor (PC-3, HT-29 and HepG-2) cell lines
were evaluated and the results were summarized in Table 1. 5-FU
was served as positive control. Comparing the antiproliferative
activities of NO-donating BFA derivatives (11aꢀf, 12aꢀf and 13aꢀf)
with those of NO donors 7aꢀf and BFA derivative 15, it could be
found that the hybrids showed better antiproliferative profiles. It
Scheme 1. Synthesis of NO donors 7aꢀf. Reagents and conditions: (a) ClCH2COOH, NaOH (aq), 140 ꢁC, 2 h; (b) 30% H2O2, AcOH, rt, 3 h; (c) fuming HNO3, AcOH, 90 ꢁC, 4 h; (d) (1)
HOCH2CH2OH, THF, 30% NaOH, 0 ꢁC, 4e8 h; (2) HOCH2CH2CH2OH, THF, 30% NaOH, 0 ꢁC, 72 h; (e) (1) succinic anhydride, Et3N, DMAP, DCM, rt, 2e3 h; (2) glutaric anhydride, Et3N,
DMAP, DCM, rt, 6e24 h; (3) phthalic anhydride, Et3N, DMAP, DCM, rt, 12e15 h.

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
133
Scheme 2. Synthesis of BFA derivatives 11aꢀf. Reagents and conditions: (a) TBSOTf, 2,6-lutidine, DCM, 0e25 ꢁC, 3 h; (b) TBAF, THF, 0e25 ꢁC, 24 h; (c) 7a¡f, EDCI, DMAP, DCM, rt,
1e2 h; (d) TBAF, THF, 0e25 ꢁC, 20 h.
Scheme 3. Synthesis of BFA derivatives 12aꢀf and 13aꢀf. Reagents and conditions: (a) 7a¡f, EDCI, DMAP, DCM, rt, 4e72 h.
was obvious that most of diester derivatives of BFA (except 13f)
exhibited more potent cytotoxic activities against three human
cancer cell lines than the monoester derivatives. Moreover, the IC50
values of BFA derivatives against three cancer cell lines were lower

134
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
Scheme 4. Synthesis of NO donor and BFA derivatives 14 and 15. Reagents and conditions: (a) TMSCHN2, CH3OH, 0 ꢁC, 10 min; (b) succinic anhydride, 2,6-lutidine, 110 ꢁC, 12 h.
Table 1
Table 2
Antiproliferative activity (IC50
,
mM) of BFA derivatives and NO donors against three
NO releasing ability of the BFA derivatives (m
M/L) in vitro.a
cancer and one normal cell lines.
Compound
HepG-2
L-02
Compounds
IC50
PC-3
(m
M)a
SIc
11b
12b
13a
13b
13d
13e
68.53 2.84
65.19 3.08
98.37 1.54
98.47 3.25
92.30 3.62
89.28 2.51
13.90 1.04
14.57 0.63
19.54 1.23
22.36 0.98
23.42 0.75
20.60 1.47
HT-29
HepG-2
L-02
BFA
11a
12a
13a
11b
12b
13b
11e
12e
13e
11c
12c
13c
11d
12d
13d
11f
12f
13f
15
0.068 0.003
0.12 0.006
0.14 0.004
0.056 0.002
0.046 0.004
0.059 0.002
0.025 0.001
0.11 0.005
0.064 0.003
0.13 0.007
0.25 0.009
0.73 0.05
0.12 0.008
0.095 0.006
0.16 0.01
0.083 0.008
0.15 0.01
0.095 0.004
1.3 0.09
0.16 0.007
0.23 0.008
0.46 0.02
0.22 0.01
0.28 0.02
0.18 0.01
0.16 0.01
0.4 0.02
0.28 0.01
0.23 0.01
0.79 0.03
0.91 0.04
1.2 0.05
5.7 0.4
1.6 0.09
0.62 0.03
0.86 0.05
0.3 0.02
4.4 0.3
6.3 0.2
>10
0.35 0.01
0.51 0.01
0.91 0.02
0.51 0.02
0.41 0.02
0.82 0.03
0.18 0.01
0.82 0.04
0.54 0.04
0.55 0.03
0.73 0.03
1.3 0.08
0.84 0.04
1.8 0.09
0.75 0.06
0.38 0.02
0.62 0.04
0.62 0.04
4.0 0.3
1.6 0.1
>10
4.0 0.2
>10
6.4 0.3
>10
>10
29.1 0.9
<0.0004
5.0 0.4
5.2 0.2
5.9 0.3
5.1 0.2
5.1 0.2
6.0 0.1
5.7 0.4
5.9 0.4
6.2 0.3
6.9 0.5
7.8 0.4
7.2 0.4
6.7 0.2
6.7 0.3
5.1 0.4
5.9 0.4
5.4 0.2
9.8 0.5
9.5 0.4
36 0.9
NCd
10
6
12
12
6
33
7
11
11
9
6
9
4
9
13
10
9
2
6
a
Results are expressed as mean SD of three independent experiments.
100 mM of 11b, 12b, 13a, 13b, 13d or 13e, respectively. The levels of
NO generated in the cell lysates were determined and presented as
those of nitrite. As shown in Table 2, the tested compounds could
produce various levels of nitrite at the time point of 1 h in human
liver cancer HepG-2 cells with the concentrations ranging from
65.19 mM to 98.47 mM, among which, the most active compound
13b released the highest concentration of nitrite. As expected, the
treatment with selected compounds resulted in less nitrite in hu-
man normal liver L-02 cells which was consistent with our previous
study [22]. It was also observed that diester derivatives 13a, 13b,
13d and 13e released higher levels of NO and exhibited stronger
inhibitory effects than monoester derivatives 11b and 12b against
HepG-2 cells, indicating that the amounts of NO released by these
compounds were positively correlated with their antiproliferative
activity against HepG-2 cells.
1.6 0.07
7a
3.8 0.3
NC
8
NC
5
NC
NC
NC
7b
5.1 0.3
>10
>10
>10
>10
>10
4.9 0.3
32
37 0.8
29
35
1
7e
9.9 0.4
7c
>10
2
1
7d
3.3 0.2
7f
4.9 0.3
40 0.8
5-FU
24.3 0.4
NTb
a
2.2.3. Apoptosis and cell cycle effects of 13b on HepG-2 cells
IC50: concentration of the tested compound that inhibits 50% of cell growth.
Not tested.
Selectivity index expressed as the ratio of IC50(L-02)/IC50(HepG-2) and rounded to
b
c
Cell cycle arrest is an important sign for the inhibition of pro-
liferation and the series of events take place in cells, leading to cell
division and duplication (replication). Some NO-donating hybrids
were reported to exhibit cell cycle arrest properties [25]. Based on
the antiproliferative data, the most potent compound 13b was
selected for further mechanistic study and the effects of 13b on
HepG-2 cell cycle were examined. HepG-2 cells were treated with
the nearest integer.
d
NC: not calculated. Results are expressed as the means SD of three indepen-
dent experiments.
than those against human normal liver cell line L-02. Among these
hybrids, compound 13b exhibited the most potent activities against
PC-3, HT-29 and HepG-2 cells with IC50 values of 25 nM, 162 nM
and 178 nM, respectively and also exhibited 15-fold less inhibitory
activity against human normal liver L-02 cells than BFA. The
selectivity indexes (SI) between HepG-2 and L-02 cells were
calculated and compound 13b showed the highest SI of above 30
which deserved further investigation.
different concentrations of 13b (0, 0.0625
mM, 0.125 mM and
0.25 M) for 48 h and stained with propidium iodide (PI), followed
m
by flow cytometry analysis. As shown in Fig. 2A, compared with the
control cells treated with 0.1% DMSO, when HepG-2 cells were
treated with increasing concentrations of 13b, the percentage of
cells in the S phase increased from 43.88% to 48.20%, 51.22% and
72.16%, respectively. The results indicated that compound 13b
suppressed HepG-2 cell proliferation through inducing cell cycle
arrest at S phase.
2.2.2. The ability of NO-releasing
The NO-releasing ability of some synthesized derivatives was
investigated by Griess assay. HepG-2 and L-02 cells were exposed to
Cancer cells arrested at S phase are connected with high
sensitivity to apoptotic triggers [26]. Changes of the morphological

Fig. 2. (A) Effects of 13b on HepG-2 cell cycle. Cells were treated with the indicated concentrations of 13b for 48 h and stained with PI, followed by flow cytometry analysis. (B) Effects of 13b on cell cycle of hemoglobin pretreated HepG-
2 cells. HepG-2 cells were pretreated with 10 M of hemoglobin for 1 h and then treated with different concentrations of 13b for 48 h.
m

136
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
characters in HepG-2 cells were studied by Hoechst 33258 staining
under fluorescence microscopy to estimate whether the growth
inhibitory activities of 13b were related to the inducing of
apoptosis. As shown in Fig. 3, the vehicle-treated HepG-2 cells (0.1%
DMSO) displayed weak homogeneous blue in the nuclei while the
2.2.4. Mitochondrial membrane potential assay
The mitochondrion is an organelle that plays an essential role in
bio-energetic function. Its dysfunction, including the loss of mito-
chondrial membrane potential, is a key event that takes place
during drug-induced apoptosis [29]. JC-1 is a commercially avail-
able florescent probe. In healthy cells, JC-1 aggregates in the
mitochondria and spontaneously forms complexes which fluoresce
red. In necrotic or apoptosis cells, JC-1 exists in the monomeric
form and stains the cytosol green. HepG-2 cells were incubated
13b (0.0625
mM, 0.125 mM and 0.25 mM) treated HepG-2 cells
exhibited bright chromatin condensation and nuclear fragmenta-
tion, a hallmark of apoptosis. These data indicated that the treat-
ment of 13b induced HepG-2 cells apoptosis.
Apoptosis is a process of programmed cell death and cancer cells
usually have an abnormal ability of proliferation mainly due to the
lack of apoptosis. Thus, activation of apoptosis could reduce the
accumulation of cancer cells. Furthermore, high levels of NO could
act as a tumor cell apoptosis inducer [27,28]. So, we investigated
whether the antiproliferative activity of 13b against HepG-2 cells
was attributed to apoptosis. HepG-2 cells were treated with vari-
with different concentrations (0.0625 mM, 0.125 mM and 0.25 mM)
of hybrid 13b for 48 h prior to staining with JC-1. As depicted in
Fig. 5, the percentage of apoptotic cells increased in a dose-
dependent fashion (16.58%, 35.45% and 60.14%, respectively).
These results showed that incubation with 13b increased the
number of HepG-2 cells with collapsed mitochondrial membrane at
low concentrations and in dose-dependent manner. So compound
13b would induce apoptosis in HepG-2 cells via mitochondria-
related pathways.
able concentrations of hybrid 13b (0.0625
mM, 0.125 mM and
0.25 M). The cells were harvested and stained with 7-
m
aminoactinomycin D (7-AAD) and annexin-V, then the percentage
of apoptotic cells was determined by flow cytometry analysis.
Significantly, it was found that hybrid 13b induced apoptosis of
HepG-2 cells in a dose-dependent manner (Fig. 4A). After 48 h
treatment, the observed apoptotic cells were 14.86%, 34.65% and
55.24% (early and late apoptosis) at the indicated concentrations,
indicating the remarkable apoptosis inducing effects of hybrid 13b,
while the vehicle-treated HepG-2 cells displayed 6.54% of apoptotic
cells.
2.2.5. The effects on apoptosis-related proteins of 13b-treated
HepG-2 cells
Heme oxygenase (HO) is a cytoprotective enzyme that can be
overexpressed in some pathological conditions, including certain
cancers [30]. Recent work reported significant tumor regression
mediated by the downregulation of both HO-1 and HO-2 systems
[31]. Survivin is a member of the inhibitor in apoptosis family that
has been found to abundantly express in fetal tissues and the most
common human tumors, and is considered absent in normal tissues
in initial studies [32]. At the same time, survivin, a member of the
inhibitor of apoptosis protein family that inhibits caspases and
blocks cell death, is highly expressed in most cancers and associ-
ated with poor clinical outcome [33]. In order to identify the
contribution of cellular proteins involved in apoptosis, we detected
the relative expression of 35 key apoptosis associated proteins in
HepG-2 cells by using the Human Apoptosis Protein Array kit
To verify the contribution of NO to apoptosis and cell cycle ar-
rest, NO scavenger hemoglobin was explored in the tests of
apoptosis and cell cycle arrest. HepG-2 cells were pretreated with
10
mM of hemoglobin for 1 h and then treated with 13b (0,
0.0625
m
M, 0.125 M and 0.25 M) for 48 h. The apoptotic and cell
m
m
cycle effects were reduced (Figs. 2B and 4B). These results sug-
gested that NO produced by 13b would contribute to the apoptosis
and cell cycle arrest of HepG-2 cells.
Fig. 3. Hoechst staining of 13b treated HepG-2 cells. Cells were treated with the indicated concentrations of 13b or vehicle (control) for 48 h, stained with Hoechst 33258 and
examined with a fluorescent microscope.

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
137
Fig. 4. Flow cytometry analysis of 13b induced HepG-2 cell apoptosis. (A) Cells were incubated with the indicated concentrations of 13b for 48 h, and the apoptotic cells were
determined by flow cytometry after staining with 7-aminoactinomycin D (7-AAD) and annexin-V. (B) Effects of 13b on apoptosis of hemoglobin pretreated HepG-2 cells. HepG-
2 cells were pretreated with 10
mM of hemoglobin for 1 h and then treated with different concentrations of 13b for 48 h. The apoptotic cells were determined by flow cytometry
after staining with PI and annexin-V.
Fig. 5. The effects of 13b on mitochondrial membrane potential (MMP) of HepG-2 cells.
[34,35]. Cell lysates were collected after the treatment of 13b for the
analysis of apoptosis-related proteins. The images of the most
significantly changed proteins in the Human Apoptosis Protein
Array were showed in Fig. 6. It is notable that 13b could

138
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
Fig. 6. The effects of 13b on the expression of apoptosis-related proteins of HepG-
2 cells. Cells were treated with 13b or control for 24 h and the total cell lysates were
collected for the analysis by Human Apoptosis Protein Array kit.
downregulate the expression of HO-1, HO-2 and survivin. Besides,
the whole photographic films of the Human Apoptosis Protein
Array assay were shown in supplementary materials. NO is known
to induce apoptosis by activating the intrinsic mitochondrion-
mediated pathway, which results from a variety of key events,
including the loss of mitochondrial membrane potential, the
regulation of both pro- and anti-apoptotic Bcl-2 family proteins, the
release of cytochrome c (Cyt c) into the cytoplasm, and finally the
activation of caspase 9 and caspase 3. We therefore conducted
western blot analysis of some apoptosis related proteins. And the
results revealed that 13b could dramatically increase the relative
expression of pro-apoptotic Bax, total Cyt c and caspase 3, but
reduced the expression of anti-apoptotic Bcl-2, Bcl-xl and pro-
caspase 3 levels in HepG-2 cells (Fig. 7).
3. Conclusions
In summary, a novel series of BFA derivatives capable of
releasing NO were designed and synthesized for the treatment of
cancer by the multifunctional drug approach. All the synthesized
compounds were evaluated for their antiproliferative effects
against PC-3, HT-29, HepG-2 and L-02 cell lines and the NO-
releasing ability was also measured. Compound 13b exhibited the
strongest cytotoxicity against three selected tumor cell lines with
IC50 values of 25 nM, 160 nM and 180 nM, respectively. Compound
13b also displayed 15-fold less cytotoxicity than BFA against L-
02 cells. So NO-donor/BFA hybrid 13b was highly selective between
tumor and normal liver cells and NO donating groups might be
effective to increase antiproliferative selectivity. Additionally, 13b
released higher levels of NO than other tested derivatives in HepG-
2 cells. And all the selected derivatives released lower levels of NO
in normal L-02 liver cells than tumorous HepG-2 cells. Further
mechanistic studies revealed that 13b induced apoptosis and S
phase arrest in HepG-2 cells. Incubation with 13b increased the
number of cells with collapsed mitochondrial membrane potentials
at low concentrations in dose-dependent manner. So the mito-
chondrial pathway would be involved in BFA derivatives-mediated
apoptosis. Results of the Human Apoptosis Protein Array assay
disclosed that the apoptosis related proteins survivin, HO-1 and
HO-2 were downregulated. These proteins played a critical role in
the apoptotic process of HepG-2 cells induced by compound 13b. In
addition, western blot analysis revealed that treatment with 13b
increased the expression of Bax, Cyt c and caspase 3 and reduced
the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG-2 cells.
Taken together, compound 13b, a potent compound with NO
releasing ability, was discovered and deserved further investigation
as a potential chemotherapeutic agent for colon and liver cancer.
Fig. 7. Western blot of apoptosis-related proteins in HepG-2 cells after exposure to
different concentrations (0, 0.0625 M, 0.125 M and 0.25 M) of 13b for 48 h.
m
m
m
sources. Further purification by standard methods were employed
when necessary. Melting points were determined on an XT-4 micro
melting point apparatus and uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker ARX-400 NMR spectrometer in
the indicated solvents (TMS as internal standard): the values of the
chemical shifts were expressed in
d values (ppm) and the coupling
constants (J) in Hz. Mass spectra were obtained on Agilent 1100 Ion
trap mass spectrometer. HR-MS were carried out on Agilent Q-TOF
B.05.01 (B5125.2).
4.2. General procedure to synthesize compounds 11aꢀf, 12aꢀf and
13aꢀf
6aꢀb was obtained from thiophenol (2) in a four-step sequence
according to the literature [26,27]. Then 6aꢀb (0.5 mmol) in 10 mL
of anhydrous DCM was mixed with corresponding anhydride
(1 mmol), Et3N (2.5 mmol) and catalytic amount of DMAP by stir-
ring at room temperature for 2e24 h. The mixture was dissolved in
15 mL of H2O and extracted with DCM (15 mL ꢂ 3). The organic
layer was combined, washed with water and saturated NaCl solu-
tion sequentially, dried over anhydrous Na2SO4, and concentrated
in vacuo. The crude product was purified by column chromatog-
raphy (petroleum ether-ethyl acetate 1:1) to afford compounds
4. Experimental
4.1. General methods
Most chemicals and solvents were purchased from commercial

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
139
7aꢀf.
9 was obtained from 1 in a two-step sequence according to the
(1H, dd, J ¼ 15.0, 9.6 Hz), 5.00 (1H, m), 4.72 (1H, m), 4.37e4.60 (8H,
t, J ¼ 4.4 Hz), 2.55e2.76 (8H, m), 0.72e2.54 (15H, m); 13C NMR
literature [6]. To a solution of 9 (32 mg, 0.08 mmol) in 2.5 mL of
anhydrous DCM, 7aꢀf (0.08 mmol), EDCI (29 mg, 0.15 mmol) and
catalytic amount of DMAP were added. The reaction solution was
stirred for 1e2 h at room temperature. The mixture was poured
into 1 mL of H2O, and extracted with DCM (5 mL ꢂ 3). The organic
layer was combined, washed with saturated NaCl solution, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residues
were purified by chromatography on silica gel (petroleum ether-
ethyl acetate 5:1) to afford compounds 10aꢀf. Then 10aꢀf
(0.06 mmol) in 2 mL of THF was mixed with TBAF (0.19 mL,
0.19 mmol) by stirring at room temperature for 20 h. The mixture
was dissolved in 1 mL of H2O and extracted with ethyl acetate
(5 mL ꢂ 3). The organic layer was combined, washed with water
and saturated NaCl solution sequentially, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residues were purified by
column chromatography (petroleum ether-ethyl acetate 2:1) to
yield compounds 11aꢀf.
(400 MHz, DMSO-d6): d 171.87,171.83,171.39,170.93,164.87,158.68,
158.68, 148.02, 137.22, 137.20, 136.11, 136.09, 135.76, 130.66, 129.97,
129.97, 129.97, 129.97, 128.33, 128.33, 128.33, 128.33, 117.24, 110.46,
110.44, 76.09, 75.06, 71.25, 69.21, 69.21, 61.59, 61.46, 49.14, 49.14,
42.43, 40.06, 37.38, 33.38, 31.29, 28.78, 28.57, 28.47, 26.26, 20.56;
HRMS (ESI) m/z calcd for C44H48N4O20S2 [MþNa]þ 1039.2196, found
1039.2197.
4.2.4. 4-(2-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)-5-oxopentanoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11b)
Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.72e8.01
(5H, m), 7.24 (1H, dd, J ¼ 15.8, 3.2 Hz), 5.70 (1H, m), 5.58 (1H, dd,
J ¼ 15.9, 1.7 Hz), 5.19e5.23 (2H, m), 4.72 (1H, m), 4.41e4.62 (4H, t,
J ¼ 3.9 Hz), 4.54 (1H, d, J ¼ 3.4 Hz), 4.04 (1H, m), 2.50 (4H, m),
0.73e2.40 (17H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.64,
To a solution of 7aꢀf (0.5 mmol) in 10 mL of anhydrous DCM,
BFA (140 mg, 0.5 mmol), EDCI (191.7 mg, 1 mmol) and catalytic
amount of DMAP were added. The reaction solution was stirred for
4e24 h at room temperature and quenched with 10 mL of H2O,
extracted with DCM (10 mL ꢂ 3), dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (petroleum ether/ethyl acetate 2:1) to
afford the title compounds 12aꢀf and 13aꢀf.
171.99, 165.29, 159.07, 149.03, 137.60, 137.08, 136.48, 130.34, 130.34,
130.13, 128.69, 128.69, 117.27, 110.84, 76.44, 71.62, 70.73, 69.72,
61.67, 49.48, 43.32, 43.20, 40.68, 33.76, 32.78, 32.73, 31.75, 26.74,
20.96, 20.00; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ
685.2038, found 685.2046.
4.2.5. 4-(2-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)-5-oxopentanoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12b)
4.2.1. 4-(2-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)-4-oxobutanoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(11a)
Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.73e7.99
(5H, m), 7.32 (1H, dd, J ¼ 15.6, 3.0 Hz), 5.70 (2H, m), 5.19 (1H, d,
J ¼ 5.6 Hz), 5.14 (1H, dd, J ¼ 15.1, 9.6 Hz), 5.00 (1H, m), 4.70 (1H, m),
4.39e4.60 (4H, t, J ¼ 4.2 Hz), 4.00 (1H, m), 2.32e2.37 (4H, m),
White solid, 21% yield: mp. 148e151 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
7.72e8.09 (5H, m), 7.23 (1H, dt, J ¼ 15.8, 3.1 Hz), 5.71
0.73e2.46 (17H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.30,
(1H, m), 5.62 (1H, dt, J ¼ 15.7, 1.6 Hz), 5.22 (2H, m), 4.72 (1H, m),
172.09, 165.60, 158.69, 154.03, 137.20, 136.27, 136.14, 130.12, 129.98,
129.98, 128.31, 128.31, 116.47, 110.46, 74.99, 74.00, 70.89, 69.33,
61.25, 51.88, 42.69, 38.16, 38.07, 33.43, 32.71, 32.38, 31.37, 26.38,
20.69, 19.79; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ
685.2038, found 685.2039.
4.36e4.60 (5H, m), 4.06 (1H, m), 2.59e2.73(4H, m), 0.72e1.99 (15H,
m); 13C NMR (400 MHz, DMSO-d6):
d 171.86, 171.00, 164.98, 158.69,
148.43, 136.70, 136.17, 131.59, 130.16, 130.00, 129.81, 128.74, 128.36,
117.00, 110.48, 76.38, 71.26, 70.35, 69.23, 61.70, 54.92, 49.03, 42.93,
42.84, 33.38, 31.38, 28.51, 27.22, 26.39, 20.61; HRMS (ESI) m/z calcd
for C30H36N2O12S [MþNa]þ 671.1881, found 671.1881.
4.2.6. 4,4'-((((5,5'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo-
4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(5-oxopentanoyl))bis(oxy))
bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide) (13b)
4.2.2. 4-(2-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)-4-oxobutanoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12a)
Yellow oil, 13% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.72e8.02
Yellow oil, 7% yield: 1H NMR (400 MHz, DMSO-d6):
d
7.74e8.01
(10H, m), 7.23 (1H, dd, J ¼ 15.7, 3.3 Hz), 5.73 (1H, m), 5.60 (1H, dd,
J ¼ 15.8,1.7 Hz), 5.28 (1H, m), 5.16 (1H, dd, J ¼ 15.1, 9.8 Hz), 5.01 (1H,
m), 4.73 (1H, m), 4.43e4.62 (8H, m), 2.39e2.63 (8H, m), 0.73e2.34
(5H, m), 7.32 (1H, dd, J ¼ 15.5, 3.0 Hz), 5.70e5.75 (2H, m), 5.21 (1H,
d, J ¼ 5.7 Hz), 5.16 (1H, dd, J ¼ 15.3, 9.7 Hz), 4.98 (1H, m), 4.70 (1H,
m), 4.39e4.59 (4H, t, J ¼ 4.2 Hz), 4.00 (1H, m), 2.54e2.60 (4H, m),
(19H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.83, 172.29, 172.05,
0.73e2.46 (15H, m); 13C NMR (400 MHz, DMSO-d6):
d
171.86,
171.56, 164.86, 158.69, 158.69, 148.34, 137.21, 136.13, 136.13, 136.10,
135.78, 130.68, 129.97, 129.97, 129.97, 129.97, 128.31, 128.31, 128.31,
128.31, 117.13, 110.46, 110.46, 75.76, 74.74, 71.26, 69.33, 69.33, 61.28,
61.24, 51.28, 42.43, 42.40, 37.43, 33.40, 32.71, 32.66, 32.40, 32.28,
31.27, 26.26, 20.57, 19.82, 19.77; HRMS (ESI) m/z calcd for
171.42, 165.60, 158.68, 154.03, 137.21, 136.14, 130.10, 129.99, 129.99,
128.34, 128.34, 116.46, 110.47, 75.29, 73.99, 70.89, 69.23, 61.47,
51.89, 42.67, 40.05, 38.17, 33.43, 31.38, 28.81, 28.58, 26.37, 20.69,
20.69; HRMS (ESI) m/z calcd for C30H36N2O12S [MþNa]þ 671.1881,
found 671.1864.
C
46H52N4O20S2 [MþNa]þ 1067.2509, found 1067.2510.
4.2.3. 4,4'-((((4,4'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo-
4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(4-oxobutanoyl))bis(oxy))
bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide) (13a)
4.2.7. 4-(2-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)carbonyl)benzoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11e)
Yellow oil, 5% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.51e7.95
White solid, 11% yield: mp. 60e65 ꢁC; 1H NMR (400 MHz,
(9H, m), 7.26 (1H, dd, J ¼ 15.9, 3.4 Hz), 5.71 (1H, m), 5.55 (1H, dd,
J ¼ 15.8,1.7 Hz), 5.42 (1H, dq, J ¼ 10.6,1.78 Hz), 5.24 (1H, dd, J ¼ 15.1,
9.76 Hz), 4.59e4.73 (5H, m), 4.55 (1H, d, J ¼ 3.6 Hz), 4.08 (1H, m),
DMSO-d6):
d
7.72e8.02 (10H, m), 7.22 (1H, dd, J ¼ 15.8, 3.1 Hz), 5.75
(1H, m), 5.64 (1H, dd, J ¼ 15.7, 1.9 Hz), 5.27 (1H, d, J ¼ 9.6 Hz), 5.18

140
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
0.76e2.56 (15H, m); 13C NMR (400 MHz, DMSO-d6):
d
167.16,
128.73,128.73,116.83,110.91, 75.64, 74.37, 71.27, 68.55, 60.71, 52.28,
43.05, 38.56, 38.45, 33.81, 31.77, 30.17, 29.25, 29.00, 27.75, 21.07;
HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ 685.2038, found
685.2035.
165.73, 165.16, 159.06, 148.26, 137.56, 137.06, 136.32, 132.51, 132.27,
132.03, 130.96, 130.39, 130.12,130.12,128.66,128.66,128.66,128.66,
117.61, 110.76, 78.08, 71.69, 70.78, 69.36, 63.05, 49.70, 43.36, 43.16,
40.73, 33.80, 31.78, 26.65, 20.91; HRMS (ESI) m/z calcd for
C
34H36N2O12S [MþNa]þ 719.1881, found 719.1862.
4.2.12. 4,4'-((((4,4'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo-
4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(4-oxobutanoyl))bis(oxy))
bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide) (13c)
4.2.8. 4-(2-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)carbonyl)benzoyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12e)
Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.71e8.01
Yellow oil, 6% yield: 1H NMR (400 MHz, DMSO-d6):
d
7.53e7.95
(10H, m), 7.22 (1H, dd, J ¼ 15.9, 3.3 Hz), 5.74 (1H, m), 5.63 (1H, dd,
J ¼ 15.8, 1.7 Hz), 5.25 (1H, m), 5.18 (1H, dd, J ¼ 15.0, 9.7 Hz), 4.94
(1H, m), 4.71 (1H, m), 4.15e4.45 (8H, t, J ¼ 6.1 Hz), 2.52e2.70 (8H,
(9H, m), 7.33 (1H, dd, J ¼ 15.7, 3.04 Hz), 5.72 (2H, m), 5.22 (1H, d,
J ¼ 5.7 Hz), 5.19 (1H, m), 5.13 (1H, dd, J ¼ 15.2, 9.58 Hz), 4.64e4.71
(5H, m), 4.03 (1H, m), 0.73e2.26 (15H, m); 13C NMR (400 MHz,
m), 0.72e2.52 (19H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.36,
DMSO-d6):
d
166.77, 166.10, 165.57, 158.71, 153.94, 137.17, 136.08,
172.36, 171.83, 171.40, 165.26, 159.15, 159.15, 148.45, 137.53, 137.50,
136.50, 136.50, 136.16, 131.03, 130.37, 130.37, 130.37, 130.37, 128.72,
128.72, 128.72, 128.72, 117.60, 110.88, 110.88, 76.43, 75.42, 71.62,
68.62, 68.56, 60.90, 60.73, 49.57, 42.80, 40.42, 37.78, 33.76, 31.67,
29.22, 28.98, 28.90, 28.90, 27.75, 27.75, 26.64, 20.93; HRMS (ESI) m/
z calcd for C46H52N4O20S2 [MþNa]þ 1067.2509, found 1067.2456.
136.00, 131.74, 131.69, 131.43, 131.39, 130.25, 129.81, 129.81, 128.87,
128.70, 128.19, 128.19, 116.50, 110.45, 76.65, 73.97, 70.86, 69.09,
62.59, 51.87, 42.69, 40.04, 38.05, 33.41, 31.33, 26.34, 20.66; HRMS
(ESI) m/z calcd for
719.1884.
C
34H36N2O12
S
[MþNa]þ 719.1881, found
4.2.9. 4,4'-((((2,2'-((((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo-
4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(carbonyl))bis(benzoyl))
bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide) (13e)
4.2.13. 4-(3-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)-5-oxopentanoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11d)
White solid, 23% yield: mp. 55e60 ꢁC; 1H NMR (400 MHz,
White solid, 27% yield: mp. 50e54 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
d
7.70e8.00 (5H, m), 7.22 (1H, dd, J ¼ 15.8, 3.3 Hz), 5.68
DMSO-d6):
d
7.51e7.92 (18H, m), 7.25 (1H, dd, J ¼ 15.8, 3.5 Hz), 5.75
(1H, m), 5.55 (1H, dd, J ¼ 15.8, 1.6 Hz), 5.19 (2H, m), 4.69 (1H, m),
4.52 (1H, d, J ¼ 3.7 Hz), 4.13e4.44 (4H, t, J ¼ 6.1 Hz), 4.03 (1H, m),
2.32e2.45 (4H, m), 0.68e2.10 (19H, m); 13C NMR (400 MHz, DMSO-
(1H, m), 5.58 (1H, dd, J ¼ 15.8, 1.6 Hz), 5.51 (1H, m), 5.24 (1H, m),
5.16 (1H, dd, J ¼ 15.0, 9.8 Hz), 4.71 (1H, m), 4.63 (8H, m), 0.73e2.32
(15H, m); 13C NMR (400 MHz, DMSO-d6):
d
167.05, 167.02, 166.54,
d6): d 172.78, 171.95, 165.29, 159.16, 149.05, 137.56, 137.09, 136.51,
165.79, 165.10, 159.06, 159.02, 147.84, 137.54, 137.54, 136.36, 136.29,
135.90, 132.45, 132.32, 132.20, 132.01, 131.92, 131.81, 131.54, 131.26,
131.06, 130.17 130.17, 130.09, 130.09, 129.37, 129.33, 129.29, 129.15,
128.62, 128.62, 128.56, 128.56, 117.96, 110.82, 110.74, 77.83, 76.91,
71.68, 69.41, 69.31, 62.99, 62.92, 49.73, 42.99, 40.43, 37.77, 33.81,
31.67, 26.49, 20.87; HRMS (ESI) m/z calcd for C52H48N4O20S2
[MþNa]þ 1135.2196, found 1135.2176.
130.38, 130.38, 130.14, 128.72, 128.72, 117.25, 110.88, 76.42, 71.62,
70.73, 68.62, 60.62, 49.50, 43.32, 43.20, 40.69, 33.76, 32.83, 32.79,
31.75, 27.76, 26.74, 20.97, 20.28; HRMS (ESI) m/z calcd for
C
32H40N2O12S [MþNa]þ 699.2194, found 699.2196.
4.2.14. 4-(3-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)-5-oxopentanoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12d)
4.2.10. 4-(3-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)-4-oxobutanoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11c)
Yellow oil, 11% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.73e8.02
(5H, m), 7.33 (1H, dd, J ¼ 15.5, 3.0 Hz), 5.72 (2H, m), 5.20 (1H, d,
J ¼ 5.7 Hz), 5.16 (1H, dd, J ¼ 15.1, 9.7 Hz), 5.00 (1H, m), 4.71 (1H, m),
4.14e4.45 (4H, t, J ¼ 6.1 Hz), 4.00 (1H, m), 2.28e2.36 (4H, m),
Yellow oil, 5% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.72e8.02
(5H, m), 7.23 (1H, dd, J ¼ 15.8, 3.1 Hz), 5.71 (1H, m), 5.61 (1H, dd,
J ¼ 15.8, 1.6 Hz), 5.18e5.24 (2H, m), 4.71 (1H, m), 4.57 (1H, s),
4.15e4.45 (4H, t, J ¼ 6.1 Hz), 4.03 (1H, m), 2.59e2.73 (4H, m),
0.73e2.46 (19H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.82,
172.44, 165.99, 159.16, 154.43, 137.55, 136.68, 136.53, 130.51,130.40,
130.40, 128.71, 128.71, 116.84, 110.88, 75.40, 74.38, 71.28, 68.63,
60.59, 52.31, 43.06, 40.44, 38.57, 33.81, 33.16, 32.82, 31.77, 27.75,
0.72e2.48 (17H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.38,
171.45, 165.33, 159.14, 148.79, 137.54, 137.09, 136.51, 130.38, 130.38,
130.15, 128.72, 128.72, 117.35, 110.89, 76.72, 71.60, 70.72, 68.62,
60.89, 49.43, 43.31, 43.20, 40.63, 33.75, 31.75, 28.93, 28.93, 27.76,
26.76, 20.96; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ
685.2038, found 685.2039.
26.77, 21.07, 20.26; HRMS (ESI) m/z calcd for
C32H40N2O12S
[MþNa]þ 699.2194, found 699.2193.
4.2.15. 4,4'-((((5,5'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo-
4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(5-oxopentanoyl))bis(oxy))
bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5-
oxadiazole 2-oxide) (13d)
4.2.11. 4-(3-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)-4-oxobutanoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12c)
Yellow oil, 19% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.70e7.99
(10H, m), 7.21 (1H, dd, J ¼ 15.8, 3.3 Hz), 5.72 (1H, m), 5.56 (1H, dd,
J ¼ 15.9, 1.8 Hz), 5.26 (1H, m), 5.15 (1H, dd, J ¼ 15.2, 9.8 Hz), 4.99
(1H, m), 4.69 (1H, m), 4.10e4.44 (8H, m), 2.30e2.48 (8H, m),
Yellow oil, 8% yield: 1H NMR (400 MHz, CDCl3):
d 7.63e8.10 (5H,
m), 7.34 (1H, dd, J ¼ 15.7, 3.1 Hz), 5.91 (1H, dd, J ¼ 15.6, 1.9 Hz), 5.71
(1H, m), 5.21 (1H, dd, J ¼ 15.2, 9.7 Hz), 5.08 (1H, m), 4.86 (1H, m),
4.30e4.51 (4H, t, J ¼ 6.2 Hz), 4.12 (1H, m), 2.59e2.64 (4H, m),
0.70e2.65 (23H, m); 13C NMR (400 MHz, DMSO-d6):
d 172.79,
172.76, 172.37, 171.92, 165.21, 159.15, 159.15, 148.68, 137.56, 137.56,
136.49, 136.49, 136.13, 131.07, 130.36, 130.36, 130.36, 130.36, 128.70,
128.70, 128.70, 128.70, 117.52, 110.86, 110.86, 76.15, 75.13, 71.62,
0.71e2.38 (17H, m); 13C NMR (400 MHz, CDCl3):
d 172.37 171.88,
165.98, 159.16, 154.42, 137.51, 136.68, 136.53, 130.46, 130.38, 138.38,

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
141
68.60, 68.60, 60.60, 60.57, 55.27, 49.60, 42.82, 37.80, 33.77, 33.10,
32.81, 32.81, 32.70, 31.67, 27.75, 27.75, 26.63, 20.93, 20.21, 20.21;
HRMS (ESI) m/z calcd for C48H56N4O20S2 [MþNa]þ 1095.2822,
found 1095.2771.
DMSO and 5-FU was used as the positive reference. The IC50 values
were calculated according to the inhibition ratios.
4.4. Griess assay
4.2.16. 4-(3-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6-
methyl-4-oxo-4,6, 7,8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-1-yl)oxy)carbonyl)benzoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11f)
NO-releasing data were acquired for tested compounds using
the Griess reaction in HepG-2 and L-02 cells according to the
manufacturer's instructions (Beyotime, China). Briefly, cells were
treated with 100 mM of each compound for 60 min. Subsequently,
Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6):
d
7.70e8.02
the cells were harvested and their cell lysates were prepared and
then mixed with Griess reagent for 10 min at 37 ꢁC, followed by
measurement at 550 nm on a microplate reader. The cells treated
with 0.4% of DMSO in medium were used as negative controls for
the background levels of nitrite production, while sodium nitrite at
different concentrations was prepared as the positive control for
the establishment of a standard curve [22].
(9H, m), 7.27 (1H, dd, J ¼ 15.8, 3.44 Hz), 5.71 (1H, m), 5.66 (1H, dd,
J ¼ 15.8, 1.7 Hz), 5.38 (1H, m), 5.25 (1H, dd, J ¼ 15.2, 9.8 Hz), 4.74
(1H, m), 4.55 (1H, d, J ¼ 3.5 Hz), 4.37e4.49 (4H, t, J ¼ 6.2 Hz), 4.09
(1H, m), 0.75e2.57 (17H, m); 13C NMR (400 MHz, DMSO-d6):
d
167.48,165.72,165.24,159.20,148.26,137.51,137.06,136.51,132.57,
131.97, 130.58, 130.36, 130.36, 130.20, 129.32, 129.18, 129.04, 128.73,
128.73, 117.75, 110.92, 78.02, 71.71, 70.77, 68.54, 62.09, 49.71, 43.41,
43.16, 40.76, 38.46, 33.80, 28.74, 27.58, 20.93; HRMS (ESI) m/z calcd
for C35H38N2O12S [MþNa]þ 733.2038, found 733.2048.
4.5. Cell cycle assay
Cell cycle effects were tested by flow cytometry with PI (KGA511,
KeyGEN Biotech, Nanjing, China). HepG-2 cells were plated in 6-
well plates and incubated at 37 ꢁC for 24 h. Cells were then incu-
bated with 13 b at certain concentrations for 48 h. Besides, HepG-
4.2.17. 4-(3-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6-
methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta
[f][1]oxacyclotridecin-13-yl)oxy)carbonyl)benzoyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12f)
2 cells were pretreated with 10
treated with different concentrations (0, 0.0625
0.25 M) of 13b for 48 h. Then, cells were centrifuged and fixed in
70% ethanol at 4 ꢁC overnight and resuspended in PBS containing
100 L RNase A and 400 L PI. Cellular DNA content, for cell cycle
mM of hemoglobin for 1 h and then
Yellow oil, 9% yield: 1H NMR (400 MHz, DMSO-d6):
d
7.66e8.02
m
M, 0.125 M and
m
(9H, m), 7.33 (1H, dd, J ¼ 15.6, 3.0 Hz), 5.71 (2H, m), 5.23 (1H, d,
J ¼ 5.7 Hz), 5.14 (2H, m), 4.70 (1H, m), 4.38e4.50 (4H, t, J ¼ 6.2 Hz),
4.03 (1H, m), 0.73e2.46 (17H, m); 13C NMR (400 MHz, DMSO-d6):
m
m
m
d
167.41, 166.57, 165.97, 159.20, 154.36, 137.50, 136.52, 136.52,
distribution analysis, was measured using a flow cytometer (FACS
Calibur BectoneDickinson) [37].
132.20, 132.08, 131.90, 131.67, 130.64, 130.36, 130.36,129.17, 129.06,
128.75, 128.75, 116.90, 110.94, 77.03, 74.37, 71.26, 68.50, 61.93,
52.37, 43.09, 40.44, 38.55, 33.81, 31.74, 27.63, 26.73, 21.06; HRMS
(ESI) m/z calcd for
733.2045.
4.6. Cell apoptosis assay
C
35H38N2O12
S
[MþNa]þ 733.2038, found
Apoptosis was analyzed using Annexin-V and 7-
aminoactinomycin D (7-AAD) double staining by flow cytometry
according to the manufacturer's instructions (KGA1024, KeyGEN
Biotech, Nanjing, China) in order to detect apoptotic cells [38]. The
HepG-2 cells were seeded in 6-well plates to grow overnight, and
then treated with or without 13 b at indicated concentrations for
48 h. Cells were then washed twice in PBS and resuspended in
Annexin V binding buffer. Annexin V-FITC was then added and the
mixture was incubated for 15 min under dark conditions at 25 ꢁC. 7-
AAD was added just prior to acquisition. The percentage of cells
positive for 7-AAD and/or Annexin V-FITC was reported inside the
4.2.18. 4,4'-((((2,2'-((((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-
oxo-4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1]
oxacyclotridecine-1,13-diyl)bis(oxy))bis(carbonyl))bis(benzoyl))
bis(oxy))bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-
1,2,5-oxadiazole2-oxide) (13f)
Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6):
d 7.69e7.99
(18H, m), 7.28 (1H, dd, J ¼ 15.8, 3.6 Hz), 5.77 (1H, m), 5.69 (1H, dd,
J ¼ 15.8, 1.7 Hz), 5.50 (1H, m), 5.21 (2H, m), 4.74 (1H, m), 4.34e4.44
(8H, m), 0.74e2.32 (19H, m); 13C NMR (400 MHz, DMSO-d6):
d
167.36, 167.25, 166.63, 165.77, 165.16, 159.16, 159.14, 147.91, 137.50,
quadrants. In addition, HepG-2 cells were pretreated with 10
hemoglobin for 1 h and then treated with different concentrations
(0, 0.0625 M, 0.125 M and 0.25 M) of 13b for 48 h. The per-
centages of apoptotic HepG-2 cells were determined by PI and
annexin-V staining and flow cytometry.
mM of
137.49, 136.48, 136.48, 135.95, 132.49, 132.34, 132.09, 131.97, 131.97,
131.91, 131.79, 131.26, 130.66, 130.33, 130.33, 130.33, 130.33, 129.34,
129.20, 129.11, 129.04, 128.70, 128.70, 128.70, 128.70, 118.06, 110.89,
110.89, 76.90, 71.71, 71.52, 68.53, 67.78, 62.11, 61.87, 49.83, 43.01,
40.44, 40.44, 38.45, 30.17, 28.74, 27.58, 23.62, 22.77; HRMS (ESI) m/z
calcd for C54H52N4O20S2 [MþNa]þ 1163.2509, found1163.2464.
m
m
m
4.7. Mitochondrial membrane potential assay
4.3. MTT assay
Briefly, HepG-2 cells were incubated with 13b or vehicle for
48 h, and then washed with PBS and stained with JC-1 dye under
dark conditions according to the manufacturer's instruction
(KGA601, KeyGEN Biotech, Nanjing, China). The percentage of cells
with healthy or collapsed mitochondrial membrane potentials was
monitored by flow cytometry analysis [39].
The antiproliferative activities were determined by the MTT
method as previously described [36]. The assay was performed in
96-well plates. Cells were added to each well and incubated for
24 h at 37 ꢁC in a humidified atmosphere of 5% CO2. Then cells were
incubated in the presence or absence of test compounds. After 72 h,
20
cultured medium, which was incubated for another 4 h. Then,
DMSO (150 L) was added to each well and the plates were shaken
for 10 min at room temperature. After 10 min, the OD of each well
was measured on a Microplate Reader (BIO-RAD) at the wavelength
of 570 nm. In these experiments, the negative reference was 0.1%
m
L of MTT solution (5 mg/mL) per well was added to each
4.8. Human Apoptosis Protein Array assay
m
The Human Apoptosis Protein Array (Proteome Profiler™
Ary009; R&D Systems) was used to analyze the relative expression
levels of 35 apoptosis-related proteins according to manufacturer
instructions in HepG-2 cells. Proteins were extracted from cells

142
K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
[12] C.N. Tseng, Y.R. Hong, H.W. Chang, T.J. Yu, T.W. Hung, M.F. Hou, S.S. Yuan,
C.L. Cho, C.T. Liu, C.C. Chiu, C.J. Huang, Brefeldin reduces anchorage-
treated for 24 h with compound 13b which were incubated with
the array membrane overnight at 4 ꢁC, followed by incubation with
a biotinylated detection antibody cocktail at room temperature for
1 h. A digital imaging system (Bio Pioneer Tech) was used to detect
the chemiluminescent signals which were further analyzed using
the Image J program [40].
A
independent survival, cancer stem cell potential and migration of MDA-MB-
231 human breast cancer cells, Molecules 19 (2014) 17464e17477.
[13] R.G. Shao, T. Shimizu, Y. Pommier, Brefeldin A is a potent inducer of apoptosis
in human cancer cells independently of p53, Exp. Cell. Res. 227 (1996)
190e196.
[14] H. Nojiri, H. Manya, H. Isono, H. Yamana, S. Nojima, Induction of terminal
differentiation and apoptosis in human colonic carcinoma cells by brefeldin A,
a drug affecting ganglioside biosynthesis, FEBS Lett. 453 (1999) 140e144.
[15] S. Kikuchi, K. Shinpo, S. Tsuji, I. Yabe, M. Niino, K. Tashiro, Brefeldin A-induced
neurotoxicity in cultured spinal cord neurons, J. Neurosci. Res. 71 (2003)
591e599.
4.9. Western blot assay
HepG2 cells were extracted with a cell lysis buffer (Beyotime).
Then, cells were centrifuged and washed twice. The pellet was then
resuspended in lysis buffer. After the cells were lysed for 20 min on
ice, lysates were centrifuged for 15 min 4 ꢁC [25,41]. The total
protein concentration was measured and adjusted to equal con-
centrations across different samples. The protein was separated on
a 12% sodium dodecyl sulfateꢀpolyacrylamide gel electrophoresis
gel and transferred onto a polyvinylidene difluoride (PVDF) mem-
brane (Millipore). The PVDF membranes were incubated with the
indicated primary antibodies overnight at 4 ꢁC and then incubated
with the secondary antibodies conjugated to horseradish peroxi-
dase. The proteins were visualized by a Keygen ECL system (Key-
GEN Biotech, Nanjing, China) and scanned with a Clinx ChemiScope
chemiluminescence imaging system. The relative optical densities
of the specific proteins were determined with a ChemiScope
analysis program.
[16] L.R. Phillips, J.G. Supko, L. Malspeis, Analysis of brefeldin A in plasma by gas
chromatography with electron capture detection, Anal. Biochem. 211 (1993)
16e22.
[17] C. Szabo, Gasotransmitters in cancer: from pathophysiology to experimental
therapy, Nat. Rev. Drug Discov. 15 (2016) 185e203.
[18] A.W. Carpenter, M.H. Schoenfisch, Nitric oxide release: part II. Therapeutic
applications, Chem. Soc. Rev. 41 (2012) 3742e3752.
[19] S. Pervin, R. Singh, G. Chaudhuri, Nitric oxide-induced cytostasis and cell cycle
arrest of a human breast cancer cell line (MDA-MB-231): potential role of
cyclin D1, Proc. Natl. Acad. Sci. U. S. A 98 (2001) 3583e3588.
[20] L. Chen, Y. Zhang, X. Kong, E. Lan, Z. Huang, S. Peng, D.L. Kaufman, J. Tian,
Design, synthesis, and antihepatocellular carcinoma activity of nitric oxide
releasing derivatives of oleanolic acid, J. Med. Chem. 51 (2008) 4834e4838.
[21] B. Rigas, K. Kashfi, Nitric-oxide-donating NSAIDs as agents for cancer pre-
vention, Trends Mol. Med. 10 (2004) 324e330.
[22] N. Zhao, K.T. Tian, K.G. Cheng, T. Han, X. Hu, D.H. Li, Z.L. Li, H.M. Hua, Anti-
proliferative activity and apoptosis inducing effects of nitric oxide donating
derivatives of evodiamine, Bioorg. Med. Chem. 24 (2016) 2971e2978.
[23] D.H. Li, T. Han, K.T. Tian, S. Tang, S.T. Xu, X. Hu, L. Wang, Z.L. Li, H.M. Hua,
J.Y. Xu, Novel nitric oxide-releasing spirolactone-type diterpenoid derivatives
with in vitro synergistic anticancer activity as apoptosis inducer, Bioorg. Med.
Chem. Lett. 26 (2016) 4191e4196.
Acknowledgments
[24] H.Y. Li, R. Huang, C. Qing, T. Sha, L.F. Li, Z.W. Zhao, Brefeldin A, acytotoxin from
an endophytic fungal strain of Eupenicillium brefeldianum isolated from Ari-
saemae rubescens, Nat. Prod. Res. Dev. 20 (2008) 24e27.
This work was financially supported by the National Natural
Science Foundation of China (41576136).
[25] J. Fu, L. Liu, Z. Huang, Y. Lai, H. Ji, S. Peng, J. Tian, Y. Zhang, Hybrid molecule
from O2-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid:
a
gluta-
thione S-transferase
p-activated nitric oxide prodrug with selective anti-
human hepatocellular carcinoma activity and improved stability, J. Med.
Chem. 56 (2013) 4641e4655.
Appendix A. Supplementary data
[26] Y. Ai, Y. Hu, F. Kang, Y. Lai, Y. Jia, Z. Huang, S. Peng, H. Ji, J. Tian, Y. Zhang,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.018.
Synthesis and biological evaluation of novel olean-28,13b-lactams as potential
antiprostate cancer agents, J. Med. Chem. 58 (2015) 4506e4520.
[27] C. Han, Z. Huang, C. Zheng, L. Wan, L. Zhang, S. Peng, K. Ding, H. Ji, J. Tian,
Y. Zhang, Novel hybrids of (phenylsulfonyl)furoxan and anilinopyrimidine as
potent and selective epidermal growth factor receptor inhibitors for inter-
vention of non-small-cell lung cancer, J. Med. Chem. 56 (2013) 4738e4748.
[28] D. Fukumura, S. Kashiwagi, R.K. Jain, The role of nitric oxide in tumour pro-
gression, Nat. Rev. Cancer 6 (2006) 521e534.
References
[1] V.L. Singleton, N. Bohonos, A.J. Ullstrup, Decumbin, a new compound from a
species of Penicillium, Nature 181 (1958) 1072e1073.
[2] B.M. Fox, J.A. Vroman, P.E. Fanwick, M. Cushman, Preparation and evaluation
of sulfide derivatives of the antibiotic brefeldin A as potential prodrug can-
didates with enhanced aqueous solubilities, J. Med. Chem. 44 (2001)
3915e3924.
[3] A. Argade, R.D. Haugwitz, R. Devraj, M. Cushman, Highly efficient diaster-
eoselective Michael addition of various thiols to (þ)-brefeldin A, J. Org. Chem.
63 (1998) 273e278.
[4] A.B. Argade, R. Devraj, J.A. Vroman, R.D. Haugwitz, M. Hollingshead,
M. Cushman, Design and synthesis of brefeldin A sulfide derivatives as pro-
drug candidates with enhanced aqueous solubilities, J. Med. Chem. 41 (1998)
3337e3346.
[5] A. Takatsuki, I. Yamaguchi, G. Tamura, T. Misato, K. Arima, Correlation be-
tween the anti-animal and anti-plant-virus activities of several antibiotics.
(Studies on antiviral and antitumor antibiotics. XIX), J. Antibiot. 22 (1969)
442e445.
[6] N.O. Anadu, V.J. Davisson, M. Cushman, Synthesis and anticancer activity of
brefeldin A ester derivatives, J. Med. Chem. 49 (2006) 3897e3905.
[7] J.L. Moon, S.Y. Kim, S.W. Shin, J.W. Park, Regulation of brefeldin A-induced ER
stress and apoptosis by mitochondrial NADPþ-dependent isocitrate dehy-
drogenase, Biochem. Biophys. Res. Commun. 417 (2012) 760e764.
[8] D.E. Larsson, M. Wickstrom, S. Hassan, K. Oberg, D. Granberg, The cytotoxic
agents NSC-95397, brefeldin A, bortezomib and sanguinarine induce
apoptosis in neuroendocrine tumors in vitro, Anticancer Res. 30 (2010)
149e156.
[29] R. Horbay, R. Bilyy, Mitochondrial dynamics during cell cycling, Apoptosis 21
(2016) 1327e1335.
ꢀ
[30] L. Salerno, V. Pittala, G. Romeo, M.N. Modica, Novel imidazole derivatives as
heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) inhibitors and their
cytotoxic activity in human-derived cancer cell lines, Eur. J. Med. Chem. 96
(2015) 162e172.
[31] L. Vanella, C. Di Giacomo, R. Acquaviva, I. Barbagallo, G. Li Volti, V. Cardile,
N.G. Abraham, V. Sorrenti, Effects of ellagicacid on angiogenic factors in
prostate cancer cells, Cancers 5 (2013) 726e738.
[32] G. Ambrosini, C. Adida, D.C. Altieri, A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma, Nat. Med. 3 (1997) 917e921.
[33] P.K. Jaiswal, A. Goel, R.D. Mittal, Survivin: a molecular biomarker in cancer,
Indian J. Med. Res. 141 (2015) 389e397.
[34] G.M. Nitulescu, C. Draghici, O.T. Olaru, L. Matei, A. Ioana, L.D. Dragu, C. Bleotu,
Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell
lines, Bioorg. Med. Chem. 23 (2015) 5799e5808.
[35] S.Y. Wu, A.T. Wu, S.H. Liu, Microrna-17-5p regulated apoptosis-related protein
expression and radiosensitivity in oral squamous cell carcinoma caused by
betel nut chewing, Oncotarget 7 (2016) 51482e51493.
[36] C.M. Sai, D.H. Li, C.M. Xue, K.B. Wang, P. Hu, Y.H. Pei, J. Bai, Y.K. Jing, Z.L. Li,
H.M. Hua, Two pairs of enantiomeric alkaloid dimers from Macleaya cordata,
Org. Lett. 17 (2005) 4102e4105.
[37] Q.Y. Liu, W. Li, L. Sheng, C.Y. Zou, H.X. Sun, C.F. Zhang, Y. Liu, J.Y. Shi, E.L. Ma,
L. Yuan, Design, synthesis and biological evaluation of novel asperphenamate
derivatives, Eur. J. Med. Chem. 110 (2016) 76e86.
[38] H.Y. Zhang, Y.W. Wang, P.Q. Zhu, J. Liu, S.T. Xu, H.Q. Yao, J.Y. Jiang, W.C. Ye,
X.M. Wu, J.Y. Xu, Design, synthesis and antitumor activity of triterpenoid
pyrazine derivatives from 23-hydroxybetulinic acid, Eur. J. Med. Chem. 97
(2015) 235e244.
[39] S.T. Xu, S.S. Luo, H. Yao, H. Cai, X.M. Miao, F. Wu, D.H. Yang, X.M. Wu, W.J. Xie,
H.Q. Yao, Z.S. Chen, J.Y. Xu, Design and synthesis of imidazo[2,1-b]thiazole
linked triazole conjugates: microtubule-destabilizing agents, J. Med. Chem.
59 (2016) 5022e5034.
[9] A. Dinter, E.G. Berger, Golgi-disturbing agents, Histochem. Cell Biol. 109
(1998) 571e590.
[10] J. Lippincott-Schwartz, L.C. Yuan, J.S. Bonifacino, R.D. Klausner, Rapid redis-
tribution of Golgi proteins into the ER in cells treated with brefeldin A: evi-
dence for membrane cycling from Golgi to ER, Cell 56 (1989) 801e813.
[11] S.A. Lee, Y.J. Kim, C.S. Lee, Brefeldin A induces apoptosis by activating the
mitochondrial and death receptor pathways and inhibits focal adhesion
kinase-mediated cell invasion, Basic Clin. Pharmacol. Toxicol. 113 (2013)
329e338.

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143
143
[40] G.M. Nitulescu, C. Draghici, O.T. Olaru, L. Matei, A. Ioana, L.D. Dragu, C. Bleotu,
Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell
lines, Bioorg. Med. Chem. 23 (2015) 5799e5808.
H. Yao, W. Xie, Z.S. Chen, J. Xu, A novel potent anticancer compound opti-
mized from a natural oridonin scaffold induces apoptosis and cell cycle arrest
through the mitochondrial pathway, J. Med. Chem. 60 (2017) 1449e1468.
[41] S. Xu, H. Yao, S. Luo, Y.K. Zhang, D.H. Yang, D. Li, G. Wang, M. Hu, Y. Qiu, X. Wu,