Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine

Article abstract of DOI:10.1016/j.bmc.2016.05.001

The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releasing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based derivatives 13a-c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity between tumor and normal liver cells and was further investigated for its apoptotic properties on human hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent pathways.

Full text of DOI:10.1016/j.bmc.2016.05.001

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiproliferative activity and apoptosis inducing effects of nitric
oxide donating derivatives of evodiamine
a
Nan Zhao a,y, Kang-tao Tian a,y, Ke-guang Cheng b, Tong Han a, Xu Hu a, Da-hong Li a,b, , Zhan-lin Li ,
⇑
Hui-ming 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, China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road,
Guilin 541004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releas-
ing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity
was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human
liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based
derivatives 13a–c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity
between tumor and normal liver cells and was further investigated for its apoptotic properties on human
hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest
at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent
pathways.
Received 27 February 2016
Revised 30 April 2016
Accepted 2 May 2016
Available online xxxx
Keywords:
Nitric oxide
Evodiamine
Antiproliferative activity
Apoptosis
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
3,4,10,13-position modified evodiamine,27 carboxyl derivatives at
position 7 targeting topoisomerase I and sirtuins,28 a diverse
Natural products or their derivatives take an active role in the
development of new therapeutic drugs.1 Evodiamine (1, Scheme 1),
a quinazolinocarboline alkaloid isolated from the fruits of Euodia
rutaecarpa, possesses many biological effects, such as antitu-
mor,2–5 anti-inflammation,6–8 antiobesity,9–11 and so on.12–14 Par-
ticularly, numerous studies have comprehensively demonstrated
that 1 exhibited considerable cytotoxicity on a wide variety of
human cancer cell lines,15–17 and apoptosis inducing ability to sup-
press the proliferation of tumor cells by various mechanisms,18–20
library containing 11 evodiamine-inspired novel scaffolds and
their derivatives as multitargeting antitumor agents,29 hybrid
molecules of 3-amino-10-hydroxylevodiamine and SAHA as triple
inhibitors of topoisomerase I/II and HDAC,30 and so on. Because
of its broad-spectrum and multitargeting antitumor profile, evodi-
amine represented a good lead; more work of structure modifica-
tion was still in urgent need to be taken out.
Nitric oxide (NO) is a small and reactive molecule, which has
various physiological and biological properties.31 High concentra-
tion of NO has shown great potential in inhibiting carcinogenesis
and tumor growth by inducing tumor cell apoptosis, inhibiting
tumor metastasis, and so on.32,33 Unfortunately, the delivery of
gaseous NO to tumor directly is not really effective due to its short
half-life and chemical instability.34,35 NO donors, capable of pro-
ducing a sustained release with a wide range of half-time lives,
and a predictable estimated dose had become useful tools to study
the biological properties of NO in cells and in vivo models of car-
cinogenesis. Recently, great deals of NO donor hybrids spring up,
which primarily served as anticancer drugs.36–38 We were very
interested in what aspects of druggability these hybrids would
performance.
such as, PI3K/Akt/caspase, Fas-L/NF-j
B signaling pathways,21 cas-
pase-dependent and -independent pathways,22,23 and MTDH-
dependent signaling pathway.24 Though there were a great deal
of reports which clarified the antiproliferation and apoptosis func-
tions of evodiamine, it was unpractical to develop it directly as
clinic agents owing to its moderate anticancer activity.25 Besides,
hepatotoxicity caused by the plant E. rutaecarpa had not received
serious attention providing new challenges.26 Some promising
derivatives of
1
had already been reported,27–30 including
⇑
Corresponding author. Tel.: +86 24 23986465.
E-mail addresses: lidahong0203@163.com (D.-h. Li), huimhua@163.com
Inspired by the above reasons, a novel series of evodiamine
derivatives bearing NO-donating groups (organic nitrate or furoxan)
(H.-m. Hua).
y
Co-first authors.
http://dx.doi.org/10.1016/j.bmc.2016.05.001
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
cycle arrest, and mitochondrial membrane potential were also
disclosed.
a
HO
Br
Br
ONO2
n
n
n =
n =
2a
2b
3
6
3
3a
3
6
2
4
3b
2. Results and discussion
2.1. Chemistry
c
b
SH
SCH2COOH
SO2CH2COOH
6
5
The procedures for the synthesis of NO-releasing evodiamine
derivatives were illustrated in Scheme 1. The corresponding bro-
mohydrin 2 was treated with fuming HNO3 and concentrated
H2SO4, which afforded nitrate 3. The reaction of thiophenol 4 with
chloroacetic acid in the presence of sodium hydroxide solution
yielded the thiophenylacetic acid 5, which was further oxidated
with 30% H2O2 and AcOH, generating the oxidation product 6. 6
was treated with fuming HNO3 at 90 °C, which lead to 7. 3,4-Diben-
zenesulfonyl furoxan 7 was treated with ethanediol in the pres-
ence of 30% NaOH in THF to offer 8. Treatment of 8 with
triethylamine, succinic anhydride, and 4-dimethylaminopyridine
PhO2S
O(CH2)2OH
PhO2S
SO2Ph
d
e
N
N
N
N
O
O
O
O
7
8
PhO2S
O(CH2)2OCO(CH2)2COOH
g
f
N
N
O
O
9
PhO2S
O(CH2)2OCO(CH2)2COOCH3
(DMAP) produced the intermediate 9.
9 was reacted with
TMSCHN2 to get 10. For 11a–b, the reaction of 1 with 3a or 3b,
in the presence of NaH, was carried out in DMF. 1 was treated with
bromohydrin in the presence of NaH and DMF to offer 12a–c, and
then 7 was added in the presence of DBU in CH2Cl2 to give evodi-
amine–furoxan hybrids 13a–c. Intermediate 11 was reacted with 9
to afford another series of evodiamine–furoxan hybrids 14 with
long linkage.
N
N
O
O
10
N
O
N
O
h
N
H
H3C
N
n
N
N
O2NO
H3C
n =
11a
3
6
11b
11
1
2.2. NO releasing ability
In order to investigate whether the evodiamine derivatives
including NO donor possessed the ability to release NO and if there
were any differences of NO releasing in tumor and normal cells,
Griess assay was carried out in BGC-823, Bel-7402 and L-02 cell
lines. As shown in Table 1, all the derivatives released more than
N
O
N
O
i
N
N
H
n
N
N
n =
HO
N
H3C
H3C
12
12a
12b
12c
2
3
6
75
lM/L of NO at the time point of 1 h in BGC-823 and Bel-7402
1
cells, and less than 28.12
lM/L of NO in L-02 cells. This would be
caused of special chemical environment (such as low pH) in tumor
cells. In further investigation, control release of NO might be a good
topic. Almost all target compounds released a little more NO in
BGC-823 cells than Bel-7402 cells (except 11b). Of all the deriva-
O
N
n
N
j
PhO2S
O
H3C
n =
2
3
6
13a
13b
13c
N
N
O
O
tives, 11b released the highest amount of NO of 104.18
Bel-7402 cells at the time point of 1 h, while in L-02 cells the least
NO of 6.59 M/L was produced by 14a.
lM/L in
13
l
N
O
N
O
N
O
n
2.3. Antiproliferative activity
N
N
k
n
O
N
H3C
HO
O
PhO2S
H3C
O
n =
2
3
The antiproliferative activity of target compounds was pre-
formed on BGC-823, A549, Bel-7402, and L-02 cell lines by the
standard MTT method. The results were summarized in Table 2.
The nitrate derivatives 11a and 11b only exhibited moderate activ-
12a
12b
n
=
2
3
O
N
N
14a
14b
O
12
O
14
Scheme 1. Synthetic routine of NO-releasing evodiamine derivatives 11, 13 and 14.
Reagents and conditions: (a) fuming HNO3, H2SO4, 0 °C, 3 h; (b) ClCH2COOH, NaOH
(aq), 140 °C, 2 h; (c) 30% H2O2, AcOH, rt, 3 h; (d) fuming HNO3, 90 °C, 4 h; (e)
HOCH2CH2OH, THF, 30% NaOH, 0 °C, 4–8 h; (f) triethylamine, succinic anhydride,
DMAP, rt, 1 h; (g) TMSCHN2, MeOH, 0 °C, 10 min; (h) 3, NaH, DMF, rt, 1.5 h; (i) HO
(CH2)nBr, NaH, DMF, rt, 3 h; (j) 7, DBU, CH2Cl2, ꢀ15 °C, 3 h; (k) 9, EDCI, DMAP, rt,
12 h.
ity against Bel-7402 cells with IC50 values of 12.82 and 28.79 lM,
respectively. And shorter linkage (11a) was favorable. As for fur-
oxan-based derivatives 13a–c, 14a and 14b, they showed very
promising activity against BGC-823 cells with IC50 values ranging
from 0.02 to 0.08 lM. These results revealed that the synthetic
NO donating derivatives of 1 were very sensitive to BGC-823 cells.
13a was the most potent one against A549 and Bel-7402 cells with
IC50 values of 0.23 and 0.55 lM, and other compounds were
weaker than parent compound 1. In L-02 cells, significant differ-
ences were observed that 13a still exhibited the strongest cytotox-
were designed and synthesized at N-13. The antiproliferative
activity against human hepatoma cells (Bel-7402), human non-
small-cell lung cancer cells (A549), human gastric carcinoma cells
(BGC-823) and human normal liver cells (L-02) was evaluated. The
NO releasing property was also measured. Typical selected com-
pound 13c was further investigated for its apoptotic properties
on human hepatocarcinoma Bel-7402 cells, in order to gain a better
understanding of the mode of action. The effects of apoptosis, cell
icity with IC50 value of only 0.02
antiproliferative activity (>100 M) was exhibited by 13c. 14a with
a linker of 2 carbons showed IC50 values of 0.02, 3.04 and 1.57
against three tumor cell lines, respectively, which was stronger
than 14b of 3 carbons with IC50 values of 0.06, 5.32 and 4.65 M.
l
M
and no obvious
l
lM
l
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N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
control group displayed 9.21%, indicating the remarkable effect of
compound 13c to induce apoptosis in a concentration-dependent
manner.
NO releasing ability (l
M/L) of target derivativesa
Compd
BGC-823
Bel-7402
L-02
11a
11b
13a
13b
13c
14a
14b
83.79 3.58
99.96 1.90
78.75 2.86
83.64 2.77
87.35 2.57
93.49 3.32
97.57 2.85
78.49 1.82
104.18 5.30
77.46 2.21
83.10 1.10
82.17 1.83
88.71 2.72
97.05 2.02
19.37 0.98
16.39 0.80
18.50 1.85
16.37 1.20
25.98 2.14
6.59 0.41
17.98 1.57
2.6. Effect of mitochondrial depolarization
Apoptosis played an important role in cancer, since its induc-
tion in tumor cells was essential for successful treatment and mito-
chondria was essential in the propagation of apoptosis. On the
other hand, NO was known to induce apoptosis by activating the
intrinsic mitochondria-related pathway, which mainly resulted in
a
Results are expressed as mean SD of three independent experiments.
the loss of mitochondrial membrane potential (Dw
m).40,42 We
This trend reversed in L-02 cells. For 13a–c, the linkage of 2 car-
bons (13a) was more beneficial to antiproliferative activity than
3 carbons (13b). 13c with the linkage of 6 carbons showed IC50 val-
ues between those of 13a and 13b against tumor cells, and good
selectivity between tumor and normal liver cells. These results
confirmed with previous reports39,40 that some NO donating
derivatives showed less cytotoxicity to normal cells. So it was
selected for further mechanism study to search for a potential safer
chemotherapy agent.
therefore studied the effects of 13c on mitochondria-mediated
apoptosis in Bel-7402 cells. After being exposed to different con-
centrations of 13c (0, 1, 2, and 3 lM) for 72 h, Dwm values were
determined by flow cytometry analysis (Fig. 3) using the lipophilic
mitochondrial probe JC-1. 4.26%, 13.50%, 26.57%, and 44.37% apop-
totic cells were observed, respectively. These results demonstrated
that incubation with 13c increased the number of cells with col-
lapsed mitochondrial membrane potentials at low concentrations
and in dose-dependent manner.
2.4. Effect of cell cycle
2.7. Effect of apoptosis-related proteins
Cell cycle arrest was an important sign for inhibition of prolifer-
ation and the series of events that took place in a cell leading to its
division and duplication (replication). Some NO donating hybrids
exhibited cell cycle arrest properties.40 To determine whether the
suppression of cell growth by 13c was caused by cell-cycle effect,
the DNA content of cell nuclei was detected in Bel-7402 cells by
flow cytometry. As shown in Figure 1, the cells in G1, S and G2
phase of control group accounted for 43.13%, 33.70% and 23.17%,
respectively. After cells were treated with compound 13c at con-
During the process of apoptosis, apoptotic signals could result
in the release of cytochrome C from mitochondria to cytoplasm,
a down-regulation of Bcl2, and an up-regulation of Bax. Caspases
also played an important role in the apoptotic signaling network,
and apoptotic pathways depended on activation of caspases for
the final execution of apoptosis. Therefore, the effect of apopto-
sis-related proteins by 13c was carried out by western blotting
analysis. It was found that 13c could up-regulate proapoptotic
caspase 3, caspase 8, caspase 9, Bax, FAS and cytochrome-C, and
down-regulate anti-apoptotic Bcl-2 expression (Fig. 4).
centrations of 1, 2, and 3 lM for 24 h, the ratio of G1 phase were
almost not changed. The cells of S phase increased to 39.34%,
47.32% and 51.51%, respectively, confirming that Bel-7402 cells
were arrested at S phase.
3. Conclusion
In summary, several NO-donor/evodiamine hybrids were
designed and synthesized. Of which, most compounds showed sig-
nificant antitumor activity against three selected human tumor cell
lines (BGC-823, A549 and Bel-7402). The selectivity of antiprolifer-
ative activity could be observed since some derivatives showed no
obvious cytotoxicity against normal human liver L-02 cells. The
NO-releasing ability of all the synthetic NO-donor/evodiamine
hybrids was measured by Griess assay. Interestingly, all the target
compounds released much higher amount of NO in Bel-7402 cells
than in L-02 cells. High NO releasing ability should, at least to some
extent, contribute to the strong cytotoxicity. 13c with IC50 values
2.5. Induction of apoptosis
Apoptosis was a process of programmed cell death and cancer
cells usually had an abnormal ability of proliferation mainly due
to the defective apoptosis. Thus, activation of apoptosis could
reduce accumulation of cancer cells. High levels of NO could act
as a tumor cell apoptosis inducer,36,41 in order to examine the
influence of 13c on apoptosis, an annexin V-FITC/propidium iodide
(PI) binding assay was carried out. The Bel-7402 cells were treated
with variable concentrations of 13c (1, 2, and 3 lM). The percent-
age of apoptotic cells was shown in Figure 2. After 72 h treatment,
the observed apoptotic cells were 19.63%, 36.38% and 52.57% (early
and late stage apoptosis) at the indicated concentrations, and the
of 0.07, 2.31 and 2.10
lines, respectively, and almost no antiproliferative activity
(IC50 >100 M) against L-02 cells, was further investigated for its
lM against BGC-823, A549 and Bel-7402 cell
l
apoptotic properties on human hepatocarcinoma Bel-7402 cells
in order to gain a better understanding of the mode of action.
The results revealed that 13c caused cell-cycle arrest of S phase
and induced apoptosis in Bel-7402 cells through mitochondria-
related caspase-dependent pathways. These stimulated our great
interest in the possibility to develop new antitumor agents from
NO-donor/evodiamine hybrids.
Table 2
The antiproliferative activity (IC50a lM) of NO-releasing evodiamine derivativesb
Compd
BGC-823
A549
Bel-7402
L-02
1
9
10
11a
11b
13a
13b
13c
14a
14b
1.16 0.22
17.96 0.58
21.37 0.83
>100
0.98 0.28
27.85 0.28
24.73 0.28
>100
1.23 0.21
21.45 1.03
27.86 0.76
12.82 0.07
28.79 1.09
0.55 0.14
6.21 0.17
2.10 0.15
1.54 0.07
4.65 0.29
0.34 0.11
38.73 1.55
43.27 1.48
>100
>100
>100
>100
0.02 0.01
0.08 0.02
0.07 0.01
0.02 0.01
0.06 0.01
0.23 0.03
5.85 1.01
2.31 0.21
3.04 0.10
5.32 1.02
0.02 0.01
2.35 0.12
>100
17.10 0.58
6.38 0.72
4. Experimental
4.1. Chemistry
a
NMR spectra were recorded with a Bruker ARX-400 NMR
spectrometer in the indicated solvents (TMS): the values of the
IC50: concentration that inhibits 50% of cell growth.
Results are expressed as the mean SD of three independent experiments.
b
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N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. The influence of Bel-7402 cell cycle by compound 13c. Bel-7402 cells were incubated with the indicated concentrations of 13c for 24 h before staining with PI.
Cellular DNA content for cell-cycle distribution analysis was measured by flow cytometry. The diagrams show the distribution of cells according to their DNA content; the
inserts give the percentages of cells in various cell-cycle phases.
chemical shifts are expressed in d values (ppm) and the coupling
constants (J) in Hz. Melting points were taken on an XT-4 micro
melting point apparatus and uncorrected. Mass spectra were
obtained using Agilent 1100 Ion trap mass spectrometer. HR-MS
were carried out with Agilent Q-TOF B.05.01 (B5125.2). All
commercially available solvents and reagents were used without
further purification.
trated in vacuo to give the crude product 3a. 3b was obtained
according to similar procedures. No further purification was
needed for the next step.
Compound 1 (91 mg, 0.3 mmol) was mixed with 3a or 3b
(0.4 mmol), and NaH (10 mg, 0.36 mmol) in 5 mL of anhydrous
DMF and stirred at room temperature for 1.5 h. The mixture was
poured into 5 mL of H2O, and extracted with ethyl acetate (EA,
5 mL ꢁ 3). The organic layer was combined, washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo.
The crude product was purified by column chromatography
(petroleum ether, PE/acetone 2:1 v/v) to give 11a or 11b.
4.2. Synthesis of 10
Compound 9 (13 mg, 0.34 mmol) was dissolved in 6 mL anhy-
drous MeOH at °C. TMSCHN2 (about 0.6 mL) was added dropwise
until no air bubble was produced anymore. The reaction mixture
was concentrated in vacuo and subjected to silica gel column chro-
matography (PE/EA 5:1 v/v) to give 10 (13 mg).
4.3.1. Compound 11a
Yellow oil, 85% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.22–
7.68 (8H, m, H-1–4, 9–12), 6.00 (1H, br s, H-13b), 4.96 (1H, m, H-
15a), 4.59–4.49 (3H, m, H-7b, 15b, 17a), 4.36 (1H, m, H-17b),
3.17 (1H, m, H-7a), 3.01 (1H, m, H-8b), 2.88 (1H, m, H-8a), 2.38
(3H, s, –NCH3), 2.29–2.41 (2H, m, H2-16); 13C NMR (100 MHz,
CDCl3), d (ppm): 164.53, 150.88, 137.28, 133.12, 129.01, 128.20,
125.88, 124.69, 124.27, 123.37, 123.17, 120.05, 119.29, 113.99,
109.32, 70.36, 68.14, 50.83, 40.28, 36.57, 27.49, 20.37; ESI-MS
m/z 407.2 [M+H]+; HR-MS (ESI, M+Na) m/z: calcd for C22H22N4
NaO4: 429.1533, found 429.1529.
Compound 10, colorless oil, 98% yield: 1H NMR (400 MHz,
DMSO-d6), d (ppm): 7.73–8.02 (5H, m, H-Ph), 4.39–4.61 (4H, m,
–OCH2CH2O–), 3.57 (3H, s, O-CH3), 2.57–2.61 (4H, m, –CO-CH2CH2
-CO–); 13C NMR (100 MHz, DMSO-d6), d (ppm): 172.35, 171.84,
158.70, 137.20, 136.16, 129.99, 129.99, 128.34, 128.34, 110.48,
69.27, 61.53, 51.47, 28.52, 28.33; ESI-MS m/z 423.0 [M+Na]+.
4.3. General procedure to synthesize 11
Concentrated H2SO4 (1 mL) was added into the fuming HNO3
(0.84 mL) at 0 °C and stirred for 10 min. Dichloromethane (DCM,
10 mL) and 1-bromo-3-propanol (0.9 mL, 10 mmol) were put in
dropwise. The reaction mixture was stirred at room temperature
for another 3 h, then poured into 10 mL of H2O, and extracted with
DCM (10 mL ꢁ 3). The organic layer was combined, washed with
saturated NaCl solution, dried over anhydrous Na2SO4, and concen-
4.3.2. Compound 11b
Yellow oil, 87% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 6.05–
7.71 (8H, m, H-1–4, 9–12), 6.05 (1H, br s, H-13b), 4.24–5.21 (6H, m,
H2-7, H2-15, H2-20), 2.97–3.51 (4H, m, H2-8, H2-16), 2.40 (3H, s,
–NCH3), 1.75–1.98 (6H, m, H2-17, H2-18, H2-19); 13C NMR
(100 MHz, CDCl3),
d (ppm): 164.65, 150.98, 137.22, 133.03,
129.11, 128.36, 125.82, 124.38, 124.24, 123.14, 122.74, 119.70,
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N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 2. Apoptosis inducing effect of 13c in Bel-7402 cells. Bel-7402 cells were incubated with the indicated concentrations of 13c for 72 h before staining with annexin
V-FITC and PI, followed by flow cytometric analysis. The inserts give the percentages of cells in each quadrant.
119.17, 113.26, 109.79, 73.16, 68.18, 43.81, 39.41, 36.57, 30.07,
27.74, 26.82, 25.60, 20.47; ESI-MS m/z 449.2 [M+H]+; HR-MS (ESI,
M+K) m/z: calcd for C25H28KN4O4: 487.1742, found 487.1753.
(1H, m, H-15a), 4.90–4.93 (1H, m, H-15b), 4.63–4.77 (3H, m, H-7b,
H2-16), 3.30 (1H, m, H-7a), 3.04 (1H, m, H-8b), 2.92 (1H, m, H-8a),
2.42 (3H, s, –NCH3); 13C NMR (100 MHz, CDCl3), d (ppm): 164.55,
158.65, 150.92, 137.92, 137.12, 135.59, 132.94, 129.73 (ꢁ2),
129.21, 128.33 (ꢁ2), 127.64, 126.35, 124.67, 123.47, 123.31,
120.35, 119.37, 114.08, 110.52, 109.60, 69.77, 68.10, 60.52, 42.28,
39.15, 36.74, 20.41; ESI-MS m/z 572.2 [M+H]+; HR-MS (ESI, M+H)
m/z: calcd for C29H26N5O6S: 572.1598, found 572.1576.
4.4. General procedure to synthesize 13
Compound 1 (91 mg, 0.3 mmol) was mixed with NaH (10 mg,
0.36 mmol), bromohydrin (0.6 mmol) in 5 mL of anhydrous DMF
and stirred at room temperature for 3 h. The mixture was poured
into 10 mL of H2O, and extracted with EA (10 mL ꢁ 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 12a–c were got. 7 was obtained from thiophe-
nol (4) in a three-step sequence according to the literature.36–38 12
(0.3 mmol) was mixed with 7 (219.6 mg, 0.6 mmol), and DBU
(58.5 mL, 0.4 mmol) in 5 mL of DCM and stirred at -15 °C for 3 h.
The reaction mixture was then poured into 10 mL of H2O, and
extracted with DCM (10 mL ꢁ 3). The organic layer was combined,
washed with water and brine, sequentially, dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatography (PE/EA 5:1–2:1 v/v) to give 13.
4.4.2. Compound 13b
Yellow oil, 80% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.16–
8.14 (13H, m, H-1–4, 9–12, –Ph), 5.98 (1H, br s, H-13b), 4.42–4.91
(6H, m, H2-7, H2-15, H2-17), 3.21 (1H, m, H-8b), 3.03 (1H, m, H-8a),
2.92–3.03 (2H, m, H2-16), 2.40 (3H, s, –NCH3). 13C NMR (100 MHz,
CDCl3), d (ppm): 164.74, 159.04, 151.07, 138.17, 137.36, 135.75,
133.01, 129.77 (ꢁ2), 129.07, 128.68 (ꢁ2), 128.64, 125.88, 124.22,
123.11, 122.76, 119.76, 119.15, 113.27, 110.63, 109.87, 68.68,
67.88, 60.53, 40.72, 39.48, 30.27, 28.95, 20.51; ESI-MS m/z 586.2
[M+H]+; HR-MS (ESI, M+H) m/z: calcd for C30H28N5O6S: 586.1755,
found 586.1722.
4.4.3. Compound 13c
4.4.1. Compound 13a
Yellow oil, 78% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.16–
8.15 (13H, m, H-1–4, 9–12, –Ph), 5.98 (1H, br s, H-13b), 4.16–4.99
(4H, m, H2-15, H2-20), 3.16–3.38 (2H, m, H2-7), 2.89–3.05 (2H, m,
Yellow oil, 76% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.23–
8.15 (13H, m, H-1–4, 9–12, –Ph), 6.33 (1H, br s, H-13b), 5.19–5.27
Please cite this article in press as: Zhao, N.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.001

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N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 3. Effect of 13c on the mitochondrial membrane potentials in Bel-7402 cells. Bel-7402 cells were incubated with the indicated concentrations of 13c for 72 h prior to
staining with JC-1. The inserts give the percentages of cells in each quadrant.
H2-8), 2.41 (3H, s, –NCH3), 1.55–1.87 (8H, m, H2-16, H2-17, H2-18,
H2-19); 13C NMR (100 MHz, CDCl3), d (ppm): 163.53, 158.83,
150.73, 137.20, 136.79, 136.04, 132.87, 129.95 (ꢁ2), 128.23 (ꢁ2),
128.09, 127.57, 125.48, 125.26, 123.59, 122.16, 119.10, 118.74,
112.15, 110.41, 110.06, 71.36, 69.81, 66.88, 48.60, 43.25, 29.42,
29.08, 27.68, 25.89, 24.70, 19.86; ESI-MS m/z 650.3 [M+Na]+;
HR-MS (ESI, M+H) m/z: calcd for C 33H34N5O6S: 628.2224, found
628.2180.
4.5. General procedure to synthesize 14
To a solution of 9 (61 mg, 0.16 mmol) in 10 mL of anhydrous
DCM, 12 (60 mg, 0.17 mmol), EDCI (93 mg, 0.6 mmol), and cat-
alytic amount of DMAP were added. The reaction solution was stir-
red for 12 h at room temperature. The mixture was poured into
10 mL of H2O, and extracted with DCM (10 mL ꢁ 3). The organic
layer was combined, washed with saturated NaCl solution, dried
Figure 4. Effect of 13c on the expression of apoptosis-related proteins in Bel-7402
cells. Bel-7402 cells were incubated with different doses of 13c for 72 h. The target
proteins in the membranes were probed with monoclonal antibodies.
over anhydrous Na2SO4, and concentrated in vacuo. The crude
product was purified by column chromatography (PE/EA 2:1 v/v).
Please cite this article in press as: Zhao, N.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.001

N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.5.1. Compound 14a
4.9. Analysis of cellular apoptosis
Yellow oil, 7% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.18–
8.14 (13H, m, H-1–4, 9–12, –Ph), 6.01 (1H, br s, H-13b), 4.43–4.94
(8H, m, H2-15, H2-16, H2-21, H2-22), 3.01–3.24 (2H, m, H2-7), 2.50–
2.92 (6H, m, H2-8, H2-18, H2-19), 2.39 (3H, s, –NCH3); 13C NMR
Apoptosis was analyzed using Annexin-V and PI double staining
by flow cytometry according to the manufacturer’s instructions
(KGA1024, KeyGEN Biotech, Nanjing, China) in order to detect
apoptotic cells.48,49 The Bel-7402 cells were seeded in 6-well plates
to grow overnight, and then treated with or without 13c at indi-
cated concentrations for 72 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. PI was added just prior to acquisition.
The percentage of cells positive for PI and/or Annexin V-FITC was
reported inside the quadrants.
(100 MHz, CDCl3),
d (ppm): 171.70, 171.66, 163.43, 158.67,
148.41, 139.77, 137.22, 137.17, 136.11, 132.94, 129.96, 129.96,
128.32, 128.32, 128.06, 127.58, 125.47, 125.37, 122.37, 119.42,
118.77, 112.83, 110.46, 110.19, 69.21, 67.08, 63.00, 61.48, 46.50,
42.05, 40.06, 36.03, 28.44, 28.28, 19.84; ESI-MS m/z 738.2
[M+Na]+; HR-MS (ESI, M+Na) m/z: calcd for C35H33N5NaO10S:
738.1840, found 738.1850.
4.5.2. Compound 14b
Yellow oil, 5% yield: 1H NMR (400 MHz, CDCl3), d (ppm): 7.39–
8.36 (13H, m, H-1–4, 9–12, –Ph), 5.98 (1H, br s, H-3), 4.50–4.91
(10H, m, H2-7, H2-15, H2-17, H2-22, H2-23), 2.90–3.30 (8H, m,
H2-8, H2-16, H2-19, H2-20), 2.39 (3H, s, –NCH3). 13C NMR
4.10. Mitochondrial membrane potential assay
Briefly, Bel-7402 cells were incubated with the 13c or vehicle
for 72 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.48,50
(100 MHz, CDCl3),
d (ppm): 171.86, 171.81, 163.54, 158.29,
148.44, 139.74, 136.74, 136.14, 131.74, 131.61, 129.98, 128.32,
127.60, 127.60, 126.14, 125.47, 125.47, 123.35, 121.69, 120.16,
118.77, 118.60, 118.48, 113.53, 111.48, 110.88, 69.27, 67.41,
61.57, 42.12, 40.06, 38.08, 29.79, 29.03, 28.44, 18.57. ESI-MS
m/z 752.2 [M+Na]+; HR-MS (ESI, M+Na) m/z: calcd for
4.11. Western blot analysis
C36H35N5NaO10S: 752.1997, found 752.2105.
Bel-7402 cells were incubated with different doses of 13c for
72 h. The cells were harvested and lysed using lysis buffer, and
the solution was centrifuged. Then the protein concentrations were
determined, and individual cell lysates (50 mg per lane) were sep-
arated by sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (10% gel, SDS–PAGE) and transferred onto nitrocellulose
membranes. After being blocked with 5% fat-free milk, the target
proteins in the membranes were probed with monoclonal anti-
4.6. MTT assay
Cytotoxicity of all the tested compounds against Bel-7402,
A549, BGC-823 and L-02 cells was determined by MTT assay.43,44
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 atmo-
sphere of 5% CO2. Then cells were incubated in the presence or
absence of test compounds. After 72 h, 20
(5 mg/mL) per well was added to each cultured medium, which
was incubated for another 4 h. Then, DMSO (150 L) was added
l
L of MTT solution
Bax (KGA714, KeyGEN Biotech, Nanjing, China), anti-Bcl
(KGA715), anti-caspase 3 (KGA717), anti-caspase 9 (KGA720),
anti-cyto (KGA723) and anti-b-actin antibodies (KGA731),
2
l
C
to each well and the plates were shaken for 10 min at room tem-
perature. 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 agent was 0.1% DMSO;
evodiamine and 5-Fu were used as the positive reference.
respectively. The bound antibodies were detected by horseradish
peroxidase (HRP) conjugated second antibodies and visualized
using an enhanced chemiluminescent reagent.47,51
Acknowledgments
4.7. Griess assay
This work was financially supported by the National Natural
Science Foundation of China (31570350, 21502121), China
Postdoctoral Science Foundation (2015M570258), General
Scientific Research Projects of Department of Education in Liaoning
Province (L2014382), Key Laboratory for the Chemistry and Molec-
ular Engineering of Medicinal Resources (Guangxi Normal Univer-
sity), Ministry of Education of China (CMEMR2015-B07) and Career
Development Support Plan for Young and Middle-aged Teachers in
Shenyang Pharmaceutical University.
NO-release data were acquired for test compounds using the
Griess reaction in Bel-7402 and L-02 cells according to the manu-
facturer’s instructions (S0024, Beyotime, China). Briefly, cells were
treated with 100 lM of each compound for 150 min. Subsequently,
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 540 nm by a microplate reader. The cells treated
with 0.4% 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.37,45
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.05.001.
4.8. Cell cycle study
References and notes
Cell cycle effect was assessed by flow cytometry with PI
(KGA511, KeyGEN Biotech, Nanjing, China). Bel-7402 cells were
plated in 6-well plates and incubated at 37 °C for 24 h. Cells were
then incubated with 13c at a certain concentration. After 48 h, cells
were centrifuged and fixed in 70% ethanol at 4 °C overnight and
1. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311.
2. Zhao, N.; Li, Z. L.; Li, D. H.; Sun, Y. T.; Shan, D. T.; Bai, J.; Pei, Y. H.; Jing, Y. K.; Hua,
H. M. Phytochemistry 2015, 109, 133.
3. Sachita, K.; Kim, Y.; Yu, H. J.; Cho, S. D.; Lee, J. S. Phytother. Res. 2015, 29, 1145.
4. Han, S.; Woo, J. K.; Jung, Y.; Jeong, D.; Kang, M.; Yoo, Y. J.; Lee, H.; Oh, S. H.; Ryu,
J. H.; Kim, W. Y. Biochem. Biophys. Res. Commun. 2016, 469, 1153.
5. Zhong, Z. F.; Tan, W.; Wang, S. P.; Qiang, W. A.; Wang, Y. T. Sci. Rep. 2015, 5,
16415.
resuspended in PBS containing 100 lL RNase A and 400 lL PI.
Cellular DNA content, for cell cycle distribution analysis, was mea-
sured using a flow cytometer (FACS Calibur Becton–Dickinson).46,47
Please cite this article in press as: Zhao, N.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.001

8
N. Zhao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
6. Liu, Y. N.; Pan, S. L.; Liao, C. H.; Huang, D. Y.; Guh, J. H.; Peng, C. Y.; Chang, Y. L.;
Jarho, E.; Zuco, V.; Zunino, F.; Martinet, N.; Dapiaggi, F.; Pieraccini, S.; Sironi,
M.; Dalla Via, L.; Gia, O. M.; Passarella, D. Bioorg. Med. Chem. 2013, 21, 6920.
29. Wang, S.; Fang, K.; Dong, G.; Chen, S.; Liu, N.; Miao, Z.; Yao, J.; Li, J.; Zhang, W.;
Sheng, C. J. Med. Chem. 2015, 58, 6678.
30. He, S.; Dong, G.; Wang, Z.; Chen, W.; Huang, Y.; Li, Z.; Jiang, Y.; Liu, N.; Yao, J.;
Miao, Z.; Zhang, W.; Sheng, C. ACS Med. Chem. Lett. 2015, 6, 239.
31. Mocellin, S. Curr. Cancer Drug Targets 2009, 9, 214.
Teng, C. M. Shock 2009, 32, 263.
7. Ko, H. C.; Wang, Y. H.; Liou, K. T.; Chen, C. M.; Chen, C. H.; Wang, W. Y.; Chang,
S.; Hou, Y. C.; Chen, K. T.; Chen, C. F.; Shen, Y. C. Eur. J. Pharmacol. 2007, 555,
211.
8. Chiou, W. F.; Sung, Y. J.; Liao, J. F.; Shum, A. Y.; Chen, C. F. J. Nat. Prod. 1997, 60,
708.
9. Wang, T.; Kusudo, T.; Takeuchi, T.; Yamashita, Y.; Kontani, Y.; Okamatsu, Y.;
Saito, M.; Mori, N.; Yamashita, H. PLoS One 2013, 8, e83264.
10. Bak, E. J.; Park, H. G.; Kim, J. M.; Kim, J. M.; Yoo, Y. J.; Cha, J. H. Int. J. Obes. 2010,
34, 250.
32. Carpenter, A. W.; Schoenfisch, M. H. Chem. Soc. Rev. 2012, 41, 3742.
33. Pervin, S.; Singh, R.; Chaudhuri, G. Nitric Oxide 2008, 19, 103.
34. Gladwin, M. T.; Lancaster, J. R.; Freeman, B. A.; Schechter, A. N. Nat. Med. 2003,
9, 496.
11. Hu, Y.; Fahmy, H.; Zjawiony, J. K.; Davies, G. E. Fitoterapia 2010, 81, 259.
12. Ge, X.; Chen, S.; Liu, M.; Liang, T.; Liu, C. Int. J. Mol. Sci. 2015, 16, 28180.
13. Lv, Q.; Xue, Y.; Li, G.; Zou, L.; Zhang, X.; Ying, M.; Wang, S.; Guo, L.; Gao, Y.; Li,
G.; Xu, H.; Liu, S.; Xie, J.; Liang, S. Int. Immunopharmacol. 2015, 28, 1044.
14. Yu, H.; Jin, H.; Gong, W.; Wang, Z.; Liang, H. Molecules 2013, 18, 1826.
15. Peng, X.; Zhang, Q.; Zeng, Y.; Li, J.; Wang, L.; Ai, P. Cancer Chemother. Pharmacol.
2015, 76, 1173.
16. Huang, J.; Chen, Z. H.; Ren, C. M.; Wang, D. X.; Yuan, S. X.; Wu, Q. X.; Chen, Q. Z.;
Zeng, Y. H.; Shao, Y.; Li, Y.; Wu, K.; Yu, Y.; Sun, W. J.; He, B. C. Oncol. Rep. 2015,
34, 3203.
35. Song, Q.; Tan, S.; Zhuang, X.; Guo, Y.; Zhao, Y.; Wu, T.; Ye, Q.; Si, L.; Zhang, Z.
Mol. Pharm. 2014, 11, 4118.
36. Han, C.; Huang, Z. J.; Zheng, C.; Wan, L. D.; Zhang, L. W.; Peng, S. X.; Ding, K.; Ji,
H. B.; Tian, J. D.; Zhang, Y. H. J. Med. Chem. 2013, 56, 4738.
37. Ai, Y.; Kang, F.; Huang, Z.; Xue, X.; Lai, Y.; Peng, S.; Tian, J.; Zhang, Y. J. Med.
Chem. 2015, 58, 2452.
38. Liu, M. M.; Chen, X. Y.; Huang, Y. Q.; Feng, P.; Guo, Y. L.; Yang, G.; Chen, Y. J.
Med. Chem. 2014, 57, 9343.
39. Fang, L.; Appenroth, D.; Decker, M.; Kiehntopf, M.; Lupp, A.; Peng, S.; Fleck, C.;
Zhang, Y.; Lehmann, J. J. Med. Chem. 2008, 51, 7666.
17. Wen, Z.; Feng, S.; Wei, L.; Wang, Z.; Hong, D.; Wang, Q. Int. J. Mol. Med. 2015, 36,
1657.
40. Fu, J.; Liu, L.; Huang, Z.; Lai, Y.; Ji, H.; Peng, S.; Tian, J.; Zhang, Y. J. Med. Chem.
2013, 56, 4641.
18. Fei, X. F.; Wang, B. X.; Li, T. J.; Tashiro, S.; Minami, M.; Xing, D. J.; Ikejima, T.
Cancer Sci. 2003, 94, 92.
19. Liao, C. H.; Pan, S. L.; Guh, J. H.; Chang, Y. L.; Pai, H. C.; Lin, C. H.; Teng, C. M.
Carcinogenesis 2005, 26, 968.
41. Fukumura, D.; Kashiwagi, S.; Jain, R. K. Nat. Rev. Cancer 2006, 6, 521.
42. Corbiere, C.; Liagre, B.; Terro, F.; Beneytout, J. L. Cell Res. 2004, 14, 188.
43. Sai, C. M.; Li, D. H.; Xue, C. M.; Wang, K. B.; Hu, P.; Pei, Y. H.; Bai, J.; Jing, Y. K.; Li,
Z. L.; Hua, H. M. Org. Lett. 2015, 17, 4102.
20. Zhao, L. C.; Li, J.; Liao, K.; Luo, N.; Shi, Q. Q.; Feng, Z. Q.; Chen, D. L. Int. J. Mol. Sci.
2015, 16, 27411.
44. Bai, Y.; Li, D.; Zhou, T.; Qin, N.; Li, Z.; Yu, Z.; Hua, H. J. Funct. Foods 2016, 20, 453.
45. Li, D.; Wang, L.; Cai, H.; Zhang, Y.; Xu, J. Molecules 2012, 17, 7556.
46. Shen, T.; Li, W.; Wang, Y. Y.; Zhong, Q. Q.; Wang, S. Q.; Wang, X. N.; Ren, D. M.;
Lou, H. X. Arch. Pharm. Res. 2014, 37, 412.
21. Wang, C.; Li, S.; Wang, M. W. Toxicol. In Vitro 2010, 24, 898.
22. Chen, M. C.; Yu, C. H.; Wang, S. W.; Pu, H. F.; Kan, S. F.; Lin, L. C.; Chi, C. W.; Ho,
L. L.; Lee, C. H.; Wang, P. S. J. Cell Biochem. 2010, 110, 1495.
23. Lee, T. J.; Kim, E. J.; Kim, S.; Jung, E. M.; Park, J. W.; Jeong, S. H.; Park, S. E.; Yoo,
Y. H.; Kwon, T. K. Mol. Cancer Ther. 2006, 5, 2398.
47. Li, D.; Xu, S.; Cai, H.; Pei, L.; Wang, L.; Wu, X.; Yao, H.; Jiang, J.; Sun, Y.; Xu, J.
ChemMedChem 2013, 8, 812.
_
48. Lepiarczyk, M.; Kałuza, Z.; Bielawska, A.; Czarnomysy, R.; Gornowicz, A.;
24. Zou, Y.; Qin, X.; Xiong, H.; Zhu, F.; Chen, T.; Wu, H. Tumour Biol. 2015, 36, 5187.
25. Song, S.; Chen, Z.; Li, S.; Huang, Y.; Wan, Y.; Song, H. Molecules 2013, 18, 15750.
26. Cai, Q.; Wei, J.; Zhao, W.; Shi, S.; Zhang, Y.; Wei, R.; Zhang, Y.; Li, W.; Wang, Q.
Molecules 2014, 19, 21168.
Bielawski, K. Arch. Pharm. Res. 2015, 38, 628.
49. Yan, X.; Yu, Y.; Ji, P.; He, H.; Qiao, C. Eur. J. Med. Chem. 2015, 102, 180.
50. Qian, W.; Salamoun, J.; Wang, J.; Roginskaya, V.; Van Houten, B.; Wipf, P.
Bioorg. Med. Chem. Lett. 2015, 25, 856.
27. Dong, G.; Wang, S.; Miao, Z.; Yao, J.; Zhang, Y.; Guo, Z.; Zhang, W.; Sheng, C. J.
Med. Chem. 2012, 55, 7593.
51. Li, D.; Xu, S.; Cai, H.; Pei, L.; Zhang, H.; Wang, L.; Yao, H.; Wu, X.; Jiang, J.; Sun,
Y.; Xu, J. Eur. J. Med. Chem. 2013, 64, 215.
28. Christodoulou, M. S.; Sacchetti, A.; Ronchetti, V.; Caufin, S.; Silvani, A.; Lesma,
G.; Fontana, G.; Minicone, F.; Riva, B.; Ventura, M.; Lahtela-Kakkonen, M.;
Please cite this article in press as: Zhao, N.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.001