Novel NO-releasing scopoletin derivatives induce cell death via mitochondrial apoptosis pathway and cell cycle arrest

Article abstract of DOI:10.1016/j.ejmech.2020.112386

A series of phenylsulfonyfuroxan-based NO-releasing scopoletin derivatives were designed and synthesized in the study. All target compounds showed significantly improved antiproliferative activity against four cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A459) and lower cytotoxicity toward normal liver LO2 cells. Derivative 47 concentration-dependently inhibited the colony formation of MDA-MB-231 cells. NO-releasing assessment indicated that the intracellular NO level was almost positively correlated with the antiproliferative ability. Compound 47, which released the highest amounts of NO, showed the best potency (IC₅₀ = 1.23 μM) against MDA-MB-231 cells. Mechanism research revealed for the first time that 47 blocked the proliferation of MDA-MB-231 cells by activating mitochondrial apoptosis pathway and arresting cell cycle at G2/M phase. Taken together, as a novel scopoletin derivative, 47 exhibited excellent inhibitory effects against malignant cancer cells and lower toxicity on normal cells. Thus, an in-depth evaluation of 47 to explore its complete therapeutic potential for cancer treatment is warranted.

Full text of DOI:10.1016/j.ejmech.2020.112386

Journal Pre-proof
Novel NO-releasing scopoletin derivatives induce cell death via mitochondrial
apoptosis pathway and cell cycle arrest
Zhixian Shi, Na Li, Cheng Chen, Yujin Wang, Zhichao Lei, Li Chen, Jianbo Sun
PII:
S0223-5234(20)30356-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112386
EJMECH 112386
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 March 2020
Revised Date: 23 April 2020
Accepted Date: 23 April 2020
Please cite this article as: Z. Shi, N. Li, C. Chen, Y. Wang, Z. Lei, L. Chen, J. Sun, Novel NO-releasing
scopoletin derivatives induce cell death via mitochondrial apoptosis pathway and cell cycle arrest,
European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112386.
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Novel NO-releasing scopoletin derivatives induce cell death via
mitochondrial apoptosis pathway and cell cycle arrest
1
1
Zhixian Shi , Na Li , Cheng Chen, Yujin Wang, Zhichao Lei, Li Chen*, and Jianbo Sun**
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People's Republic of China
*
Corresponding author: Tel: +86 83271447
E-mail address: chenli627@cpu.edu.cn (Li Chen)
*
* Corresponding author: Tel: +86 83271415
sjbcpu@gmail.com (Jianbo Sun)
1
These authors contributed equally.

Novel NO-releasing scopoletin derivatives induce cell death via
mitochondrial apoptosis pathway and cell cycle arrest
1
1
Zhixian Shi , Na Li , Cheng Chen, Yujin Wang, Zhichao Lei, Li Chen*, and Jianbo
Sun**
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School
of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
2
10009, People's Republic of China
*
Corresponding author: Tel: +86 83271447
E-mail address: chenli627@cpu.edu.cn (Li Chen); sjbcpu@gmail.com (Jianbo Sun)
1
These authors contributed equally.

Abstract
A series of phenylsulfonyfuroxan-based NO-releasing scopoletin derivatives were
designed and synthesized in the study. All target compounds showed significantly
improved antiproliferative activity against four cancer cell lines (MDA-MB-231,
MCF-7, HepG2 and A459) and lower cytotoxicity toward normal liver LO2 cells.
Derivative 47 concentration-dependently inhibited the colony formation of
MDA-MB-231 cells. NO-releasing assessment indicated that the intracellular NO
level was almost positively correlated with the antiproliferative ability. Compound 47,
which released the highest amounts of NO, showed the best potency (IC = 1.23 μM)
5
0
against MDA-MB-231 cells. Mechanism research revealed for the first time that 47
blocked the proliferation of MDA-MB-231 cells by activating mitochondrial
apoptosis pathway and arresting cell cycle at G2/M phase. Taken together, as a novel
scopoletin derivative, 47 exhibited excellent inhibitory effects against malignant
cancer cells and lower toxicity on normal cells. Thus, an in-depth evaluation of 47 to
explore its complete therapeutic potential for cancer treatment is warranted.
Key words: Scopoletin; Nitric oxide; Mitochondrial apoptosis pathway; Cell cycle
arrest.

1
. Introduction
Scopoletin (7-hydroxy-6-methoxy-2H-chromen-2-one) (Fig. 1), existing in diverse
medicinal plants (Scopolia japonica Maxim., Atropa belladonna Linn., Saposhnikovia
divaricata (Turcz.) Schischk., etc.) [1], possesses multiple pharmacological activities,
including antitumor [2], antioxidant [3], and anti-inflammatory [4-5]. Of these, the
favorable potency of scopoletin against various tumor cells has attracted many
researchers’ attention [6-9]. Further mechanism investigation demonstrates that
scopoletin inhibits the multiplication of prostate cancer cells via inducing cell
apoptosis and cell cycle arrest [10]. Similar results have also been reported in
cholangiocarcinoma cells and cervical cancer cells [11-12]. Although scopoletin has
no obvious toxicity on the viability of normal cells, the moderate or low anticancer
activity hampers its clinical application. Therefore, considerable scopoletin
derivatives have been designed and synthesized to enhance its antitumor activity
[
13-15]. Accordingly, for the past few years, our research group has been committed
to designing scopoletin derivatives, and demonstrated that several compounds (such
as compounds 1-4, Fig. 1) exhibited superior antitumor activity compared to
scopoletin in vitro [16-18]. However, the antitumor ability of these derivatives has not
yet achieved desired effect, so further improvement is needed.
Fig. 1. Scopoletin and representative scopoletin derivatives

Hybridization strategy, chemically incorporating two or more pharmacophores
with similar bioactivities to acquire a new molecular entity, has been widely and
successfully applied in the discovery of anticancer drugs, antiviral drugs, etc. [19-21].
Nitric oxide (NO), an intracellular messenger molecule in cellular activities, has been
proved to affect the progress of cancer cells in vitro and in vivo [22]. In cells, high
levels of intracellular NO are involved in the up-regulation of signals such as p53,
PARP, and DNA-PK, which can restrain DNA repair mechanisms [23]. Then, this
process contributes to proliferation inhibition of cancer cells. Thus, many chemical
NO donors have been developed to provide exogenous NO [24]. Among them,
furoxan is an important NO donor and can generate high levels of intracellular NO.
Particularly, as one of furoxan, phenylsulfonylfuroxan (Fig. 2) has been successfully
applied for the design of anticancer hybrids [25-34]. For example, the furoxan-based
NO-releasing hybrids 5-12 (Fig. 2) both exhibited improved antitumor activities in
vitro compared with parent compounds. Hence, it is an effective way to enhance the
antiproliferative activity of parent compounds by introducing phenylsulfonylfuroxan
into their molecular structures while synthesizing NO-releasing hybrids.

Fig. 2. Phenylsulfonylfuroxan and phenylsulfonylfuroxan-based hybrids
Thus, we attempted here to design novel NO-releasing scopoletin derivatives by
introducing phenylsulfonylfuroxan at 7-position in scopoletin to enhance its antitumor
effect (Fig. 3). In addition, given that introducing an acetylamino or amino group at
3
-position of scopoletin is favorable for cytotoxic activity [14, 16-17], we also
designed and synthesized a series of 3-acetylamino scopoletin/phenylsulfonylfuroxan
and 3-amino scopoletin/phenylsulfonylfuroxan hybrids (Fig. 3). Their
antiproliferative activities against cancer cells and cytotoxicities toward normal cells
were measured. Further, the intracellular NO generated by target compounds was
measured and the effect on antiproliferative activity was evaluated. Moreover, we
elucidated the mitochondrial apoptotic pathway by apoptosis, mitochondrial
membrane potential depolarization, ROS generation, and mitochondrial apoptosis
protein determination. Simultaneously, cell cycle arrest was analyzed to explore
further potential mechanism of these derivatives.

Fig. 3. The design of novel scopoletin/phenylsulfonyfuroxan hybrids
. Results and discussion
.1. Chemistry
The synthesis route of target compounds (36-50) is shown in Scheme 1. 3,
-diphenylsulfonyl-furoxan (15) was obtained from benzenethiol (12) as described in
2
2
4
the literature [35]. The starting material 12 was converted to 2-(phenylthio)acetic acid
13) via treatment with 2-chloroacetic acid, sodium hydroxide (NaOH), and sodium
carbonate (Na CO ). Following this, 13 was treated with hydrogen peroxide (H O ) to
(
2
3
2
2
generate (phenylsulfonyl)methanoic acid (14), which was then mixed with fuming
nitric acid to provide intermediate 15. Further, scopoletin and 20a were synthesized
from 2,4,5-trimethoxybenzaldehyde (16) according to the method reported by our
group [16-18]. Compound 20b was obtained from intermediate 19 in the presence of
hydrochloric acid [36]. Subsequently, intermediate scopoletin, 20a, and 20b were
separately treated with different halo alcohols to yield compounds 21-35. Finally,
2
1-35 were treated separately with 15 to provide target compounds 36-50 in the
presence of 8-diazabicyclo [5.4.0] undec-7-ene (DBU) [25].

Scheme 1. Reagents and conditions: (a) ClCH COOH, Na CO , NaOH, EtOH, reflux,
2
2
3
4
h; (b) H O , AcOH, rt, 3h; (c) fuming HNO , reflux, 4h; (d) AlCl , CTAB, dry DCM,
2 2 3 3
reflux, 4h; (e) malonic acid, aniline, pyridine, rt, 24h; (f) glycine, Ac O, NaOAc,
2
reflux, 4h; (g) pyridine, reflux, 6h; (h) K CO , MeOH, reflux, 1h; (i) EtOH, HCl,
2
3
8
0°C, 1h; (j) BrCH (CH ) OH, DMF, 60°C, 8h; (k) 15, DBU, DCM, -15°C, 4h;
2 2 n
2
.2. Antiproliferative activity in vitro
Firstly, we measured the proliferation inhibition rates of scopoletin, 20a, 20b,
and 36-50 in MCF-7 cells via the MTT assay at a concentration of 10 μM. All target
compounds (36-50) showed favorable inhibitory effect on MCF-7 cell proliferation
(inhibition rates: 48.63%-91.55%) (Table 1). However, the proliferation inhibition
rates of 20a and 20b (7.95%, 5.21%, 7.13% respectively) were not significantly
different, suggesting that the introduction of either acetylamino or amino at C-3
position without other modifications failed to boost up the antiproliferative activity.

Table 1. The proliferation inhibition rates of the tested compounds in MCF-7 cells
a
a
Cpd.
IR (%)
89.19
88.57
84.37
87.06
84.80
83.72
88.51
80.41
73.64
Cpd.
45
IR (%)
48.63
88.55
91.55
89.02
88.78
84.37
36
37
38
39
40
41
42
43
44
46
47
48
49
50
Scopoletin 7.95
20a
20b
5.21
7.13
a
IR, inhibition rate, MCF-7 cells were treated with the tested compounds (10 μM) for 48 h (mean
SD, n = 3)
±
Next, the target compounds 36-50 were tested in the following cancer cell lines:
MDA-MB-231, MCF-7, HepG2, and A549. Doxorubicin was selected as positive
control. As shown in Table 2, compared with scopoletin, 36-50 showed potential
anticancer potency in all studied cancer cells. Further, compounds 36-50 showed
higher activities in MDA-MB-231, MCF-7, and HepG2 than in A549, indicating the
selectivity of these scopoletin derivatives against different tumor cells. Subsequently,
3
6-50 were also studied in human normal liver cell line LO-2 for evaluating their
cytotoxicity towards normal cells. All scopoletin derivatives exhibited reduced
cytotoxicity toward LO-2 cell (IC = 7.82-17.33 μM) compared with cancer cell lines.
5
0

Among all derivatives, compound 47 was the most active derivative with an IC of
5
0
1
.23 μM in MDA-MB-231, slightly higher than that of doxorubicin (IC = 0.55 μM)
50
(Table 2). However, compound 47 showed approximately 15-fold less toxic than
doxorubicin in LO-2 cell (IC = 0.59 µM).
5
0
With an amino group in C-3 position, compound 50 did not show favorable
antiproliferative activities in all cell lines except HepG2 due to six carbons in the
linker. Similar results were also observed in derivatives 36-45, which shared the same
substituent group at C-3 position. Compounds 40 and 45 showed inferior antitumor
effects also due to their six carbon length linkers. Although such scopoletin
derivatives with acetamido or amino group attached (compounds 36-40 or 41-45 or
4
6-50) have no significant difference in anticancer activity, scopoletin derivatives still
show a certain pattern structure-activity relationship. According to biological studies
conducted in 3-acetylamino scopoletin/phenylsulfonylfuroxan hybrids (compounds
4
1-45), the introduction of acetamido is generally not conducive to anticancer efficacy,
but compound 42 showed potential antitumor activity in MCF-7 and HepG2 cells.
The activity difference between scopoletin/phenylsulfonylfuroxan and 3-amino
scopoletin/phenylsulfonylfuroxan (compounds 36-39 and 46-49) is the smallest;
compounds 37 and 47 have the highest activity among all derivatives. Unfortunately,
compound 37 was more active than 47 in LO-2. Overall, the biological data shown in
Table 2 revealed that these modifications to scopoletin resulted in increased
anticancer activity, and the most active compound (47) was selected for further study.
Table 2 The IC values of 36-50 against cancer cell lines and human liver LO2 cells
5
0

a
IC (μM)
5
0
Compounds
MDA-MB-231
2.86 ± 0.11
1.85 ± 0.08
2.79 ± 0.14
3.17 ± 0.16
3.35 ± 0.06
4.99 ± 0.25
3.81 ± 0.20
3.15 ± 0.14
4.34 ± 0.27
7.89 ± 0.16
2.46 ± 0.18
1.23 ± 0.10
1.93 ± 0.09
2.40 ± 0.13
2.93 ± 0.14
>25
MCF-7
HepG2
A549
LO2
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.54 ± 0.11
2.27 ± 0.12
3.04 ± 0.06
3.85 ± 0.13
4.48 ± 0.32
6.89 ± 0.33
2.40 ± 0.18
3.44 ± 0.13
6.27 ± 0.21
11.30 ± 0.07
2.77 ± 0.24
1.91 ± 0.14
2.28 ± 0.15
2.62 ± 0.08
3.14 ± 0.17
>25
3.61 ± 0.16
2.90 ± 0.19
3.25 ± 0.15
3.42 ± 0.21
3.79 ± 0.17
4.83 ± 0.18
2.82 ± 0.03
3.29 ± 0.07
5.42 ± 0.11
6.59 ± 0.11
3.06 ± 0.09
2.81 ± 0.13
2.64 ± 0.12
3.46 ± 0.16
3.06 ± 0.11
>25
7.24 ± 0.37
5.45 ± 0.14
6.14 ± 0.30
10.62 ± 0.09
9.43 ± 0.15
8.79 ± 0.17
9.81 ± 0.13
7.19 ± 0.28
11.36 ± 0.30
14.95 ± 0.24
5.91 ± 0.13
4.18 ± 0.15
5.03 ± 0.21
6.88 ± 0.27
8.06 ± 0.22
>25
10.94 ± 0.52
7.82 ± 0.23
9.33 ± 0.16
8.86 ± 0.53
12.42 ± 0.22
10.25 ± 0.12
8.36 ± 0.28
9.19 ± 0.04
13.76 ± 0.16
17.33 ± 0.21
9.82 ± 0.34
9.16 ± 0.38
8.63 ± 0.42
12.25 ± 0.41
10.26 ± 0.24
>25
Scopoletin
b
Doxorubicin
0.55 ± 0.01
0.60 ± 0.04
0.62 ± 0.15
0.96 ± 0.06
0.59 ± 0.09
a
MTT assay, cells were treated with tested compounds for 48 h (mean ± SD, n = 3)
Positive control
b
2
.3. Effect of compound 47 on colony formation
Plate clone formation assay was performed to evaluate the effect of 47 on cellular
proliferative ability based on the colony forming efficiency. As presented in Fig. 4, at
.25 μM, compound 47 slightly suppressed colony formation in MDA-MB-231 cell.
0
Compared with control, the cells treated with 0.5 or 1 μM 47 displayed much smaller
and fewer colonies, particularly those exposed to the higher drug concentration. The

absorbance after staining with crystal violet tested at 590 nm also decreased in
dose-dependent manner. These results confirmed that 47 suppressed colony formation
at nanomolar concentrations, and demonstrated the antiproliferative activity of 47
against MDA-MB-231 cell.
Fig. 4. Compound 47 inhibited the colony formation of MDA-MB-231 cells. (A)
Cells were incubated with DMSO or 47 (0.25, 0.5, and 1 μM）for 24 h and then
cultured in a fresh medium for another 10 days, followed by staining with crystal
violet. (B) The absorbance of crystal after staining at 590 nm (mean ± SD, n = 3). **P
<
0.01, *P < 0.05
2
.4. Determination of intracellular NO
Cellular imaging, using the fluorescent probe DAF-FM DA, was conducted to
determine whether 47 can release NO in MDA-MB-231 cells. As presented in Fig. 5A,
after incubated with 47 (1 and 10 μM ) for 2.5 h, the cells showed an increase of
DAF-FM fluorescence intensity in a concentration-dependent manner. In contrast, no
green fluorescence was observed in cells exposed to scopoletin. This result manifested
that 47 can release NO in cells and implicated that all derivatives led to NO release in

MDA-MB-231cells.
Next, we used the total nitric oxide assay kit to quantify the levels of intracellular
NO generated by all derivatives in MDA-MB-231 cell. As shown in Fig. 5B,
intracellular NO levels after treatment with each synthesized hybrid for 2.5 h were
different (approximately 3.99-10.33 μM). Treatment with compounds 46-50 resulted
in higher levels of intracellular NO and showed higher anticancer potency among the
target compounds. We speculate that the introduction of amino group increased the
molecule polarity for improving lipo-hydro partition coefficient, which enable more
drugs enter the cells and was beneficial to NO release. Derivative 47 with the highest
anticancer potency showed the highest released levels of NO in MDA-MB-231 cell.
The NO released by compound 37 was similar to that of 47, indicating the potency of
strong anticancer activities. Derivatives 36 and 38-45 showed moderate NO release,
which indicated relatively poor potency. Therefore, the abilities of NO release for
compounds 36-50 were generally positive consistent with their antitumor activities.
Furthermore, to investigate the relationship between NO release and
antiproliferative activity of 47, cells were pretreated with indicated concentrations of
hemoglobin (NO scavenger). As presented in Fig. 5C, the proliferation inhibition rate
decreased from 91.22% to 39.47% with an increasing concentration of hemoglobin
(2.5-40 μM). The result indicated that the enhanced cytotoxicity of 47 towards
MDA-MB-231 cell was partly due to the release of intracellular NO.

Fig. 5. Intracellular NO generated by the studied derivatives in MDA-MB-231 cells.
A) Cells were pretreated with DAF-FM DA and then incubated with scopoletin (10
(
μM) or 47 (1 and 10 μM) for 2.5 h, and then observed using a laser scanning confocal
microscope (Olympus, Japan). (B) After cellular treatment with tested compounds (25
μM)) for 2.5 h, levels of intracellular NO were quantified using the total nitric oxide
assay kit (mean ± SD, n = 3). (C) Cells were pretreated with hemoglobin (2.5, 5, 10,
2
0, and 40 μM) for 2 h and then incubated with 47 (4 μM) for another 48 h. The
inhibition rates were measured by the MTT assay (mean ± SD, n = 3)
2
.5. Effect of an NO donor on the antiproliferative activity of 47
To further explore the effect of NO on the antiproliferative activity, compound 47
was chemically split into two pharmacophores (compounds 51 and 52), and their
antiproliferative activities against MDA-MB-231 cell were measured by the MTT

assay (Fig. 6). The results showed that 47 (IC = 1.23 μM) was at least 5-fold and
5
0
8
-fold more active than 51 (IC = 7.25 μM) and 52 (IC > 25 μM), respectively.
50 50
Furthermore, 47 was significantly more active (by approximately 5-fold) when
compared with the equimolar combination of 51 and 52 (1:1, IC50 = 6.36 μM),
indicating the benefits of our derivative. These results indicated that the introduction
of NO donor moieties was beneficial to anticancer potency, in line with the
presumption of the NO-based hybrid design strategy.
Fig. 6. Antiproliferative abilities of 51 and 52 and their equimolar mixture against
MDA-MB-231 cells
2
.6. Compound 47 induced MDA-MB-231 cell death via apoptosis
Apoptosis and necrocytosis are considered as two recognized pathways for
anticancer agents due to their effects on cell survival and virulence [37]. To research
the underlying mechanism of 47 on the proliferation inhibition of MDA-MB-231 cells,
necrostatin-1 (necroptosis inhibitor) or Z-VAD-FMK (apoptosis inhibitor) was added
prior to drug treatment. As presented in Fig. 7A, treatment with necrostatin-1 (10 μM)
or Z-VAD-FMK (20 μM) showed no effect on cell viability in MDA-MB-231 cell.
However, compared with cells treated with necrostatin-1, the inhibition rate of

MDA-MB-231 cell pretreated with Z-VAD-FMK decreased more significantly (Fig.
B). The result suggested that derivative 47 induced the death of MDA-MB-231 cells
7
via apoptosis pathway.
Fig. 7. (A) MDA-MB-231 cells were separately treated with necrostain-1 (10 μM)
and Z-VAD-FMK (20 μM) for 48 h, and the cell viability was measured by MTT
method. (B) Cells were pretreated with necrostatin-1 or Z-VAD-FMK for 1 h and then
incubated with 47 (1.5, 2, and 3 μM) for another 48 h, and the inhibition rates were
determined by MTT method (mean ± SD, n = 3). **P < 0.01, *P < 0.05
2
.7. Effect of 47 on cell apoptosis
To confirm that compound 47 induced apoptosis of MDA-MB-231 cells, the
Hoechst 33342 staining assay was performed (Fig. 8A). Compared with control cells,
MDA-MB-231 treated with 47 for 24 h exhibited morphological apoptosis as judged
from the bright blue stained apoptotic cells; the density of cells decreased
dose-dependently, which was in agreement with the inhibitory effect of 47. To further

confirm apoptosis induction by 47, the cells were stained with Annexin V and PI and
tested by flow cytometry. As observed in Fig. 8B and 8C, the proportion of total
apoptotic (Q2 + Q3) cells increased from 9.9% to 34.0% in cells treated with
compound 47 for 24 h; the early apoptotic cells (Q2) and late apoptotic cells (Q3)
showed a similar increasing tendency. Based on these results, it was determined that
compound 47 could induce the apoptosis of MDA-MB-231 cell.
Fig. 8. Compound 47 induced the apoptosis of MDA-MB-231 cells. (A) Cells were
treated with DMSO or 47 (0.5, 1, and 1.5 μM) for 24 h, and stained with Hoechst
3
3342. Cells were then observed under a fluorescent inverted microscope. (B) After

treatment with DMSO or 47 (1, 1.5, 2, and 3 μM) for 24 h, cells were collected and
sequentially stained with Annexin V and Propidium Iodide (PI), followed by flow
cytometric analysis. (C) The percentages of early, late, and total apoptotic cells were
calculated (mean ± SD, n = 3). **P < 0.01, *P < 0.05 vs. the negative control group.
2
.8. Effect of 47 on the mitochondrial membrane potential
Mitochondrial, death receptor, and endoplasmic reticulum stress-induced
pathways are the three major apoptotic pathways [38]. The mitochondrion is a crucial
cell organelle that regulates cellular survival and apoptosis, and many anticancer
agents exhibit their pharmacological effects by acting on it. The mitochondrial
function depends on the stabilization of the mitochondrial membrane structure [39],
and scopoletin derivatives reported by our group can cause mitochondrial dysfunction
[
40]. Thus, we performed the JC-1 staining assay to estimate whether 47 had any
effect on the mitochondrial membrane potential. Compared with the control group, the
treatment group showed an increase in the proportion of cells with depolarized
mitochondria (Q3) from 5.4% to 31.5% and a decrease in the proportion of
hyperpolarized mitochondria (Q2) from 5.4% to 31.5% (Fig. 9). This result indicated
that compound 47 induced the mitochondrial membrane potential depolarization in a
concentration-dependent manner and disrupted mitochondrial function in
MDA-MB-231 cells.

Fig. 9. Compound 47 affected the mitochondrial membrane potential of
MDA-MB-231 cells. (A) Cells were treated with 47 (1, 1.5, and 2 μM) and DMSO
(control) for 18 h, harvested, and then stained with JC-1, followed by flow cytometric
analysis. (B) The percentage of cells with green fluorescence or red fluorescence was
determined (mean ± SD, n = 3), **P < 0.01, *P < 0.05 vs. the negative control group.
2
.9. Effect of 47 on reactive oxygen species (ROS) generation
Since the primary sources of ROS derived from mitochondria, the high level of
intracellular ROS lead to the mitochondrial dysfunction and then activate apoptosis
pathway [41]. To investigate whether 47 could promote the accumulation of ROS in
MDA-MB-231 cell, the relative levels of ROS were monitored by DCF-DA staining
assay and flow cytometric analysis. As presented in Fig. 10, the relative levels of ROS
increased from 1.31 to 2.16 with the increase of the drug concentration, which showed
significant difference compared with the levels observed in the control group.
Pretreatment with NAC (N-acetyl-L-cysteine, ROS scavenger) before treatment with

4
7 (2 μM) resulted in a marked reduction in intracellular ROS levels; the relative
levels of intracellular ROS decreased to 1.09, with no significant difference compared
with control (Fig. 10). This result demonstrated that 47 was involved in ROS-induced
apoptosis of MDA-MB-231 cell, which was associated with the mitochondrial
apoptotic pathway.
Fig. 10. Compound 47 induced ROS generation in MDA-MB-231 cells. (A) Cells
were incubated with 47 (1, 1.5, and 2 μM) and DMSO (control) for 24 h, and then
collected and stained with 2',7'-Dichlorofluorescin diacetate (DCFH-DA), followed
by flow cytometric analysis. (D) The relative levels of fluorescent DCF were
measured and plotted (mean ± SD, n = 3). **P < 0.01, *P < 0.05
2
.10. Compound 47 regulated the levels of mitochondrial apoptosis-related proteins
The abovementioned experiments indicated that compound 47 induced cell death
through the mitochondrial apoptotic pathway. The mitochondrial apoptotic pathway is
mainly regulated by the Bcl family (e.g., Bcl-2, Bax, and Bad). Down-regulating the

level of Bcl-2 (an anti-apoptotic protein) and up-regulating the level of Bax and Bad
(
pro-apoptotic proteins) can increase the permeability of mitochondrial membrane
42]. Further, the increase in the permeability of the mitochondrial membrane
[
promotes caspase-9 to form cleaved-caspase-9. Similarly, cleaved-caspase-3 is
derived from caspase-3, which acts an executioner in the apoptotic signaling pathway.
The cleaved-caspase-3 further cleaves PARP and eliminates PARP’s ability to repair
DNA, which is followed by apoptosis [42]. Therefore, we used western blot to
thoroughly understand the effect of 47 on mitochondrial apoptosis-related proteins.
Data from western blot showed that incubation of 47 reduced the expression of Bcl-2
in MDA-MB-231 cell, and the relative levels of Bax and Bad increased with the drug
concentration (Fig. 11). This result indicated mitochondrial dysfunction, which was in
accordance with 47-induced mitochondrial depolarization concluded above. Bedsides,
the relative levels of cleaved-caspase-3, cleaved-caspase-9, and cleaved-PARP
increased in a concentration-dependent manner (Fig. 11). Taking into account the
induced-apoptosis, mitochondrial depolarization, and ROS generation effects verified
for our derivative, the mitochondrial apoptotic pathway was found to be responsible
for the antiproliferative ability of 47.

Fig. 11. Compound 47 affected the expressions of mitochondrial apoptosis-related
proteins in MDA-MB-231 cells. (A) Cells were incubated with DMSO or 47 (1.5, 3,
and 6 μM ） for 8 h, and levels of Bcl-2, Bax, Bad, cleaved-caspase-9,
cleaved-caspased-3, and cleaved-PARP were determined by western blotting. (B)
Relative levels of mitochondrial apoptosis-related proteins were determined (mean ±
SD, n = 3). **P < 0.01, *P < 0.05 vs. the negative control group
2
.11. Compound 47 blocked the Cell Cycle in the G2/M Phase
Cell cycle is the basis of cell life activity and controls the entry of stationary phase
cells into the proliferative phase [43]. Previous study showed that scopoletin could
induce cell cycle at G2/M phase in HeLa cells [12]. To explore whether compound 47
had similar pharmacology function, PI staining assay and flow cytometry were
performed. As shown in Fig. 12, compared with the control group, the treatment
group showed a G2/M phase fraction of 24.58%, 30.11%, and 40.29% in
MDA-MB-231 cells incubated with 1, 1.5, and 2 μM of 47, respectively. However,
the percentage of cells in G0/G1 and S phases decreased from 45.71% to 38.05% and
3
6.25% to 20.82%, respectively. This result proved that compound 47 could arrest cell
cycle at G2/M phase in MDA-MB-231 cells, which was another important mechanism

for the antitumor effect of 47.
Fig. 12. Compound 47 blocked the cell cycle in the G2/M phase. (A) MDA-MB-231
cells were treated with 47 (1, 1.5, and 2 μM) or DMSO (control) for 24 h, and then
collected, stained with PI, and analyzed by flow cytometric analysis. (B) The
percentage of cells in each cell phase was determined (mean ± SD, n = 3). **P<0.01,
*
P<0.05 vs. the negative control group
3
. Conclusions
A number of scopoletin derivatives were synthesized by introducing
phenylsulfonyfuroxan moieties. All derivatives exhibited enhanced antiproliferative
activities against cancer cell lines and reduced cytotoxicity toward normal cells.
Derivative 47 was significantly more active than scopoletin and slightly weaker than
doxorubicin against MDA-MB-231 cell. Analysis of NO release by our derivatives in
MDA-MB-231 cell revealed that high anticancer potency of derivatives was

positively associated with their abilities to release intracellular NO. The mechanism
research revealed that 47 induced mitochondrial depolarization and promoted
intracellular ROS generation, and then regulated the expressions of mitochondrial
apoptosis-related proteins (Bcl-2, Bax, Bad, Cleaved-caspase-3, Cleaved-caspase-9,
Cleaved-PARP), finally activated the apoptosis. In addition, 47 arrested
MDA-MB-231 cell cycle at the G2/M Phase. These derivatives blocked the growth of
breast cancer MDA-MB-231 cell through mitochondrial apoptosis pathway and cell
cycle arrest. Overall, compound 47 was a potential anticancer candidate and deserved
further optimization.
4
. Experimental protocol
4
.1. Chemical Analysis
Reagents and solvents were commercially available. Each reaction was monitored
by silica gel GF-254 plate at 254 nm or 365 nm ultraviolet light. Products were
purified by recrystallization or silica gel column chromatography (200-300 mesh,
Qingdao city, China). The structures of target compounds were confirmed by
1
13
characterization with nuclear magnetic resonance ( H NMR and C NMR) and high
resolution mass spectrometer (HRMS). NMR spectra were recorded on the Bruker
AVANCE instrument (300 MHz) at 25°C using TMS as the internal standard in CDCl3
or DMSO-d . HRMS was run on an Agilent Q-TOF mass spectrometer. The purity of
6
scopoletin derivatives 36-50, found in all cases as > 95%, was determined by
SHIMADZU Prominence-i LC-2030C system equipped with a SHIMADZU
Shim-pack GIST C18 4.6×250 mm (5 μm) column (column temperature equal 25ºC)

and a variable wavelength UVvis detector (detection at λmax = 339 nm); the flow rates
were 0.8 mL/min with injection volumes of 10 μL in acetonitrile mixtures and the
mobile phase: 30:70 H O/CH CN. Intermediates 15, scopoletin, 20a and 20b were
2
3
obtained according to previous reported procedures [16-18, 33, 36].
4
.1.1. General procedure for synthesis of 36-50
Respectively mixing intermediate scopoletin, 20a, and 20b (0.5 mmol) with
indicated halo alcohol (0.6 mmol) in DMF (10 ml), K CO (0.6 mmol) was added.
2
3
The mixture was stirred at 60°C for 8 h. The mixture was cooled to room temperature,
0% NaOH aqueous solution was added into the mixture. Intermediate 21-35 were
1
obtained by suction filtering, water washing, recrystallization with ethanol, and drying.
Compounds 21-35 (0.3 mmol) separately reacted with 15 (0.45 mmol) in CH Cl (10
2
2
ml) using DBU (1, 0.36 mmol) as a trigger. The mixture was stirred at -15°C for 4 h.
Then, it was diluted with CH Cl (20 ml), washed with 5% HCl, water, and dried over
2
2
anhydrous Na SO , and then concentrated. The crude products were purified by silica
2
4
gel column chromatography using CH Cl /MeOH (V/V, 150:1) to provide target
2
2
compounds 36-50.
4
4
.1.1.1.
-(2-((6-methoxy-2-oxo-2H-chromen-7-yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadi
1
azole 2-oxide (36). White powder, 58.9% yield. Mp 203.8–205.4°C; H NMR (300
MHz, DMSO-d ) δ : 7.97–8.00 (2H, m), 8.00 (1H, d, J = 9.3 Hz), 7.77–7.82 (1H, m),
6
H
7
.59–7.64 (2H, m), 7.29 (1H, s), 7.18 (1H, s), 6.36 (1H, d, J = 9.3 Hz), 4.83–4.85 (2H,
1
3
m), 4.48–4.51 (2H, m), 3.79 (3H, s) C NMR (75 MHz, DMSO-d ) δ : 160.98,
6
C

1
2
59.42, 151.43, 149.64, 146.31, 144.72, 137.60, 136.44, 130.29 (C × 2), 128.64 (C ×
), 113.50, 112.18, 111.07, 109.78, 101.65, 70.14, 67.21, 56.38. HRMS (POSI)
+
calculated for C H N O S [M+H] 461.06493, found 461.06527. Purity: 96.121%
2
0
17
2
9
(by HPLC).
4
4
.1.1.2.
-(3-((6-methoxy-2-oxo-2H-chromen-7-yl)oxy)propoxy)-3-(phenylsulfonyl)-1,2,5-oxad
1
iazole 2-oxide (37). White powder, 45.1% yield. Mp 196.8–198.4°C; H NMR (300
MHz, DMSO-d ) δ : 8.04 (1H, d, J = 9.6 Hz), 7.98–8.05 (2H, m), 7.84–7.90 (1H, m),
6
H
7
.69–7.75 (2H, m), 7.29 (1H, s), 7.12 (1H, s), 6.35 (1H, d, J = 9.6 Hz), 4.60 (2H, t, J
1
3
=
6.3 Hz), 4.23 (2H, t, J = 6.3 Hz), 3.81 (3H, s), 2.28–2.36 (2H, m). C NMR
(
75MHz, DMSO-d ) δ : 161.01, 159.29, 151.88, 149.75, 146.37, 144.76, 137.63,
6 C
1
6
4
4
4
36.51, 130.40 (C × 2), 128.81 (C × 2), 113.29, 111.87, 111.01, 109.60, 101.30, 68.70,
+
5.50, 56.38, 28.05. HRMS (POSI) calculated for C H N NaO S [M+Na]
2
1
18
2
9
97.06252, found 497.06244. Purity: 97.729% (by HPLC).
.1.1.3
-(4-((6-methoxy-2-oxo-2H-chromen-7-yl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadi
1
azole 2-oxide (38). White powder, 25.9% yield. Mp 211.4–213.7°C; H NMR (300
MHz, CDCl ) δ : 8.01–8.04 (2H, m), 7.71–7.76 (1H, m), 7.61 (1H, d, J = 9.6 Hz),
3
H
7
.56–7.61 (2H, m), 6.87 (1H, s), 6.86 (1H, s), 6.30 (1H, d, J = 9.6 Hz), 4.67 (2H, t, J
1
3
=
6.0 Hz), 4.26 (2H, t, J = 6.0 Hz), 3.87 (3H, s), 2.41–2.49 (4H, m). C NMR (75
MHz, DMSO-d ) δ : 161.05, 159.32, 152.32, 149.86, 146.39, 144.80, 137.66, 136.57,
6
C
1
30.45 (C × 2), 128.77 (C × 2), 113.04, 111.58, 110.90, 109.45, 101.11, 71.82, 69.06,

+
5
5
4
4
6.33, 28.33, 28.00. HRMS (POSI) calculated for C H N NaO S [M+Na]
22 20 2 9
11.46603, found 511.46607. Purity: 98.326% (by HPLC).
.1.1.4.
-((5-((6-methoxy-2-oxo-2H-chromen-7-yl)oxy)pentyl)oxy)-3-(phenylsulfonyl)-1,2,5-o
1
xadiazole 2-oxide (39). White powder, 37.1% yield. Mp 207.2–209.6°C; H NMR
(
300 MHz, DMSO-d ) δ : 8.03–8.06 (2H, m), 7.98 (1H, d, J = 9.6 Hz), 7.87–7.93 (1H,
6 H
m), 7.73–7.78 (2H, m), 7.27 (1H, s), 7.12 (1H, s), 6.32 (1H, d, J = 9.6 Hz), 4.44 (2H, t,
J = 6.3 Hz), 4.13 (2H, t, J = 6.3 Hz), 3.82 (3H, s), 1.80–1.91 (4H, m), 1.51–1.61(2H,
1
3
m). C NMR (75 MHz, DMSO-d ) δ : 161.06, 159.34, 152.34, 149.88, 146.42,
6
C
1
1
44.81, 137.69, 136.58, 130.47 (C × 2), 128.78 (C × 2), 113.06, 111.60, 110.92,
09.49, 101.14, 71.84, 69.08, 56.36, 28.34, 28.02, 22.29. HRMS (POSI) calculated
+
for C H N NaO S [M+Na] 525.09382, found 525.09375. Purity: 97.983% (by
2
3
22
2
9
HPLC).
4
4
.1.1.5.
-((6-((6-methoxy-2-oxo-2H-chromen-7-yl)oxy)hexyl)oxy)-3-(phenylsulfonyl)-1,2,5-ox
1
adiazole 2-oxide (40). White powder, 46.5% yield. Mp 201.9–203.6°C; H NMR (300
MHz, DMSO-d ) δ : 8.02–8.05 (2H, m), 7.97 (1H, d, J = 9.3 Hz), 7.89–7.93 (1H, m),
6
H
7
.74–7.79 (2H, m), 7.27 (1H, s), 7.08 (1H, s), 6.31 (1H, d, J = 9.3 Hz), 4.42 (1H, t, J
6.3 Hz), 4.11 (1H, t, J = 6.3 Hz), 3.83 (3H, s), 1.76–1.85 (4H, m), 1.40–1.55 (4H,
=
1
3
m). C NMR (75 MHz, DMSO-d ) δ : 161.03, 159.32, 152.33, 149.85, 146.39,
6
C
1
44.77, 137.68, 136.56, 130.45 (C × 2), 128.75 (C × 2), 113.02, 111.55, 110.88,
09.48, 101.06, 71.83, 69.04, 56.36, 28.69, 28.20, 25.38, 25.12. HRMS (POSI)
1

+
calculated for C H N NaO S [M+Na] 539.10947, found 539.10953. Purity:
2
4
24
2
9
9
4
4
9.526% (by HPLC).
.1.1.6.
-(2-((3-acetamido-6-methoxy-2-oxo-2H-chromen-7-yl)oxy)ethoxy)-3-(phenylsulfonyl
1
)
-1,2,5-oxadiazole 2-oxide (41). White powder, 41.3% yield. Mp 205.4–207.0°C; H
NMR (300 MHz, DMSO-d ) δ : 9.67 (1H, s), 8.63 (1H, s), 7.97–8.00 (2H, m), 7.78–
6
H
7
3
1
1
.83 (1H, m), 7.59–7.65 (2H, m), 7.33 (1H, s), 7.17 (1H, s), 4.84 (2H, s), 4.48 (2H, s),
13
.78 (1H, s), 2.19 (3H, s). C NMR (75 MHz, DMSO-d ) δ : 170.43, 159.44, 158.32,
6
C
49.61, 146.84, 145.25, 137.60, 136.48, 130.31 (C × 2), 128.65 (C × 2), 125.33,
22.85, 112.77, 110.08, 109.57, 101.55, 70.23, 68.94, 56.43, 24.34. HRMS (POSI)
+
calculated for C H N O S [M+NH ] 535.11294, found 535.11298. Purity:
2
2
20
4
10
4
9
4
4
9.649% (by HPLC).
.1.1.7.
-(3-((3-acetamido-6-methoxy-2-oxo-2H-chromen-7-yl)oxy)propoxy)-3-(phenylsulfon
1
yl)-1,2,5-oxadiazole 2-oxide (42). White powder, 46.7% yield. Mp 196.4–198.1°C; H
NMR (300 MHz, DMSO-d ) δ : 9.66 (1H, s), 8.61 (1H, s), 8.01–8.04 (2H, m), 7.84–
6
H
7
.89 (1H, m), 7.69–7.74 (2H, m), 7.32 (1H, s), 7.11 (1H, s), 4.59 (2H, t, J = 6.0 Hz),
1
3
4
.20 (2H, t, J = 6.0 Hz), 3.80 (3H, s), 2.29–2.32 (2H, m), 2.18 (3H, s). C NMR
(
300MHz, DMSO-d ) δ : 170.34, 159.33, 158.34, 150.51, 146.89, 145.46, 137.67,
6 C
1
1
36.57, 130.46 (C × 2), 128.75 (C × 2), 125.51, 122.49, 112.03, 110.88, 109.26,
00.88, 71.83, 68.96, 56.40, 28.75, 24.31. HRMS (POSI) calculated for
+
C H N O S [M+H] 532.10204, found 532.10229. Purity: 98.898% (by HPLC).
23
22
3
10