Synthesis of scutellarein derivatives with antiproliferative activity and selectivity through the intrinsic pathway

Article abstract of DOI:10.1016/j.ejmech.2018.09.047

To explore antitumor agents with potent efficacy and low toxicity, scutellarein derivatives with benzoic acid mustard (10a?c, 11a?c and 13a?c) were designed and synthesized. The antiproliferative activities of the target derivatives against A549, MCF-7 and Bel-7402 cancer cell lines were tested. Compound 10a showed the strongest potency with an IC₅₀ value of 1.50 μM against MCF-7 cell line, and displayed low toxicity against human liver L-O2 normal cells (IC₅₀ > 50 μM), showing specificity between normal and malignant cells. The mechanism studies revealed that 10a could induce apoptosis in MCF-7 cells, arrest MCF-7 cell cycle at the G1 phase and cause mitochondrial dysfunction in a concentration-dependant manner. Furthermore, the enhanced expression of the pro-apoptotic proteins caspase-9, caspase-3, Bax and cytochrome c, and the reduced expression of the anti-apoptotic protein Bcl-2 confirmed that 10a induced the intrinsic apoptosis pathway in MCF-7 cells. The potent antiproliferative activity and good selectivity guaranteed 10a a lead compound for the further development into anticancer therapeutics, especially for breast cancer.

Full text of DOI:10.1016/j.ejmech.2018.09.047

Accepted Manuscript
Synthesis of scutellarein derivatives with antiproliferative activity and selectivity
through the intrinsic pathway
Tong Han, Yan Wang, Mingying Wang, Xu Li, Keguang Cheng, Xiang Gao, Zhanlin
Li, Jiao Bai, Huiming Hua, Dahong Li
PII:
S0223-5234(18)30819-5
10.1016/j.ejmech.2018.09.047
EJMECH 10758
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2018
Revised Date: 21 August 2018
Accepted Date: 14 September 2018
Please cite this article as: T. Han, Y. Wang, M. Wang, X. Li, K. Cheng, X. Gao, Z. Li, J. Bai, H. Hua,
D. Li, Synthesis of scutellarein derivatives with antiproliferative activity and selectivity through the
intrinsic pathway, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.047.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis
of
scutellarein
derivatives
with
antiproliferative activity and selectivity through the
intrinsic pathway
a
b
a
c
d
a
Tong Han , Yan Wang , Mingying Wang , Xu Li , Keguang Cheng , Xiang Gao ,
a
a
a
a,∗
Zhanlin Li , Jiao Bai , Huiming Hua , Dahong Li
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
Yantai Valiant Pharmaceutical Co., Ltd., 60 Taiyuan Road, Dajijia Industrial Park, YEDA Yantai
2
64006, P. R. China
c
Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Kangyuan Road, Lianyungang 222001, PR China
d
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and
School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Raod, Guilin 541004,
PR China
∗
Corresponding author. E-mail addresses: lidahong0203@163.com (D. Li).

ACCEPTED MANUSCRIPT
ABSTRACT
To explore antitumor agents with potent efficacy and low toxicity, scutellarein
derivatives with benzoic acid mustard (10a−c, 11a−c and 13a−c) were designed and
synthesized. The antiproliferative activities of the target derivatives against A549,
MCF-7 and Bel-7402 cancer cell lines were tested. Compound 10a showed the
strongest potency with an IC50 value of 1.50 µM against MCF-7 cell line, and
displayed low toxicity against human liver L-O2 normal cells (IC50 > 50 µM),
showing specificity between normal and malignant cells. The mechanism studies
revealed that 10a could induce apoptosis in MCF-7 cells, arrest MCF-7 cell cycle at
the G1 phase and cause mitochondrial dysfunction in a concentration-dependant
manner. Furthermore, the enhanced expression of the pro-apoptotic proteins caspase-9,
caspase-3, Bax and cytochrome c, and the reduced expression of the anti-apoptotic
protein Bcl-2 confirmed that 10a induced the intrinsic apoptosis pathway in MCF-7
cells. The potent antiproliferative activity and good selectivity guaranteed 10a a lead
compound for the further development into anticancer theraputics, especially for
breast cancer.
Keywords: scutellarein, benzoic acid mustard, antiproliferative activity, selectivity

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1
. Introduction
Cancer is the second cause of mortality worldwide and continuing to be a serious
health problem in developing countries [1,2]. According to the data of China, the
incidence and mortality of cancer have been increasing. This situation will become
more severe with the rapid population aging process in the future [3]. Current studies
show that chemotherapy is still the most efficacious cancer treatment approach.
However, side effects seriously impede its clinical application [4,5]. Therefore, new
drugs which can cause tumor cell death without obvious side effects are of great
importances for cancer chemotherapy.
At present, many antitumor compounds have the potentials to alkylate RNA,
DNA and several proteins [6]. Among which, classic DNA alkylating agents nitrogen
mustards with the advantages of strong potency and broad spectrum have been
extensively used [7]. However, the lack of specific affinity for tumor cells necessitates
large doses to achieve certain local concentrations, leading to side effects and
systemic toxicity [8]. Structural modifications of nitrogen mustards have been done
during the last decades in order to overcome these deficiencies. In this regard,
changing the combined parts is one of the most effective methods, such as benzene
derivatives [9,10], polyamine [11], distamycin [12], steroids [13,14], diterpenoids
[
15,16], macrolide [17] and so forth. Some achieved good results, especially the
hybrids of nitrogen mustards and nature products, such as oridonin (A) [15],
glutathione (B) [18], enmein (C) [16] and brefeldin A (D) [17], which exhibited
significant antitumor activities and low toxicity (Figure 1). This fact prompts us to
explore new carriers from other natural skeletons to obtain nitrogen mustard
derivatives with improved selectivity and low toxicity.
Flavonoids, one of the most common classes of naturally occurring phenolic

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phytochemicals, have drawn tremendous attention because of their various benficial
biological activities and low toxicity [19]. Scutellarin, the main effective flavonoid
component of Erigeron breviscapus (vant.) Hand-Mazz extract breviscapine [20], has
the potency to treat cardiovascular diseases [21−24]. It could also induce apoptosis
[
25], suppress the migration and invasion [26,27] and sensitize cancer cells to
chemotherapy [28,29]. Scutellarein is one of the main metabolites of scutellarin in
vivo after breviscapine administration and possesses some similar therapeutic effects
as scutellarin [30]. Some recent researches have focused on the anticancer activity of
scutellarein [31−38]. Scutellarein was also found to show non-toxic under the long
administration duration and/or high dose [39]. These results suggest that scutellarein
would be a promising carrier of nitrogen mustards for the discovery of new anticancer
agents.
Some nature products and benzoic acid mustard hybrids showed significant
antiproliferative activities and benzoic acid mustard was reported to show low toxicity
[
40,41]. Therefore, in this study, scutellarein and its two analogues were selected to
combine with benzoic acid mustard. The target hybrids were evaluated against human
tumor A549, Bel-7402 and MCF-7 cells and L-O2 normal hepetic cells for the
antiproliferative activities. Further mechanism study concerning apoptosis related
properties were also investigated.
2
. Result and Discussion
2
.1. Chemistry
The synthetic methods of target derivatives 10a−c, 11a−c and 13a−c are shown in
Scheme 1. According to the literature [15], 2 was reacted with ethylene oxide in acetic
solution to give 3. Benzoic acid mustard 4 was obtained by the sequences of reaction

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of 3 and phosphorus oxychloride, and hydrolysis by using concentrated hydrochloric
acid. Treatment of scutellarin 1 with 6 N HCl in ethanol under reflux, scutellarein 5
was synthesized. 6 was got from 2 by the treatment with dichlorodiphenylmethane
[
42]. 7 was synthesized by the reaction of 5 and 1,2-dibromoethane.
Compounds 8a−c and 12a−c were synthesized in good yields by the reaction of
intermediate 6 or 7 with corresponding dibromoalkane in anhydrous acetone, and then
converted to 9a−c and 13a−c by the reaction with 4 and K CO in anhydrous DMF.
2
3
1
0a−c was obtained by removing the diphenylmethylene ketal of 9a−c with HOAc
solution. The reaction of 10a−c with Me SO in anhydrous acetone provided 11a−c.
2
4
1
13
NMR ( H and C) and HRMS (high resolution mass spectrum) were used to confirm
the structures.
2
2
.2. Biological evaluation
.2.1 Antiproliferative activity
The antiproliferative activities of 10a−c, 11a−c and 13a−c against three human
cancer cell lines MCF-7 (breast cancer), A549 (lung cancer) and Bel-7402
hepatocellular carcinoma), and normal cells L-O2 (liver) were tested and compared
with scutellarein (2), benzoic acid mustard (4), chlorambucil and positive control
-FU (5-fluorouracil) in each panel (Table 1). Some derivatives showed
(
5
antiproliferative activities against tumor cells to some extent and low toxicity against
L-O2 nomal cells with IC values greater than 50 µM, exhibiting selectivity. Among
50
them, 10a−c exhibited stronger antiproliferative activities against MCF-7 and
Bel-7402 cells with IC values between 1.50 and 9.07 µM. 10b and 10c displayed
5
0
moderate activities against A549 cell line with IC values of 21.47 and 23.51 µM,
5
0
respectively. While, 11a−c, 13b and 13c showed weak antiproliferative activities
against Bel-7402 cell line with IC50 values ranging from 25.69 to 45.72 µM. The

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results that hybrids 10a−c showed more potent antiproliferative activities than 11a−c
and 13a−c indicated the unsubstituted 6-OH and 7-OH were beneficial to the
antiproliferative activities. The length of the linkers does not seem to regularly affect
activities. In particular, 10a−c also exhibited more potent inhibitory activities with
IC values of 1.50−4.78 µM than benzoic acid mustard (IC > 50 µM), scutellarein
5
0
50
(
IC > 50 µM), chlorambucil (IC > 50 µM) and 5-FU (IC 27.89 µM) against
50 50 50
MCF-7 cells. Especially, 10a showed the smallest IC50 value of 1.50 µM.
Consequently, 10a was selected for further investigation of the possible cellular
mechanisms in MCF-7 cell line.
2
.2.2 Cell cycle effect
To determine the effects of 10a on the cell cycle arrest, MCF-7 cells were treated
with certain concentrations of 10a (0.75, 1.5 and 3 µM) for 48 h and stained with PI
propidium iodide). The DNA content of cell nuclei was analyzed by flow cytometry
(
with non-treated cells as control. G1 phase cells increased from 41.49% (control
group) to 44.16%, 50.99% and 59.49% (Figure 2) in a concentration-dependent
manner. The results revealed that 10a caused G1 phase arrest of MCF-7 cells in a
concentration-dependent manner.
2
.2.3 Cell apoptosis effect
Annexin V-FITC/PI binding assay was carried out to evaluate the apoptosis
inducing effects of 10a. MCF-7 cells were treated with the same concentrations of
0a as the cell cycle test for 48 h. Then, the annexin-V and PI were used to dyeing
1
cells, and the percentages of apoptotic cells were determined by flow cytometry. The
result were given in Figure 3, the apoptosis of MCF-7 cells treated with 10a increased
from 15.99% to 29.31% and 65.21% at the concentrations of 0.75, 1.5 and 3 µM,
respectively, compared to 6.09% in the control group. The results proved that 10a

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caused remarkable apoptosis of MCF-7 cells in a concentration-dependent manner.
2
.2.4 Mitochondria membrane potential analysis
Mitochondria plays a key role in the induction of apoptosis. The dissipation of
mitochondria membrane potential is an significant early event in apoptosis, and then
some pro-apoptotic factors such as cytochrome c, Bax were released [42,43]. Thus,
the effects of 10a on mitochondrial membrane potentials in MCF-7 cell line were
investigated. MCF-7 cells were treated with the same concentrations of 10a as the cell
cycle test for 48 h, and stained with the dye JC-1, the changes of mitochondrial
membrane potentials were monitored by flow cytometry. As shown in Figure 4, 10a
induced a concentration-dependent increase in depolarized cell population from
1
.61% of control to 19.61%, 43.48% and 64.80%, respectively. This result illustrated
that compound 10a could induced MCF-7 cell apoptosis through mitochondrial
intrinsic) pathways.
(
2
.2.5 Effects on apoptosis-related proteins
The mitochondria-dependent apoptotic pathway is regulated by the Bcl-2 family
of pro- and anti-apoptotic proteins such as Bax (pro-apoptotic protein) and Bcl-2
anti-apoptotic protein), which could induce cytochrome c released into the cytosol,
(
resulting in the activation of the caspase-9 and -3, finally triggering the execution of
apoptosis [44,45]. Therefore, to determine compound 10a involved in the
mitochondria-dependent apoptosis pathway in MCF-7 cells, we examined the
expression of Bcl-2 and Bax, cyto-c, caspase-9 and -3 activities by Western blot
analysis. As shown in Figure 5, in comparison with the control cells, 10a could
up-regulation the level of pro-apoptotic protein Bax and down-regulation the level of
anti-apoptotic protein Bcl-2 in a dose-dependent fashion. Moreover, the expression of
cytochrome c remarkably increased with the elevation of 10a concentration, this is

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probably due to the release from mitochondria (Figure 5). Then, the caspase-9 was
activated, which further increased the level of caspase-3 compared to the control in
MCF-7 cells. These results indicated that 10a could induced cell apoptosis via
increased the Bax/Bcl-2 ratio, leading to the release of cyto-c, caspase-9 and -3
activation, which triggered the execution of apoptosis.
3
. Conclusion
In summary, a series of hybrids of scutellarein and benzoic acid mustard were
synthesized and evaluated. Some of them showed strong antiproliferative activities
against MCF-7 and Bel-7402 cancer cell lines and weak activities against L-O2
human normal liver cells. Among them, compound 10a exhibited the strongest
antiproliferative potency against MCF-7 cell line with an IC value of 1.50 µM. 10a
5
0
also displayed no obvious inhibitory effects against L-O2 cells. In addition,
compound 10a was found to induce apoptosis, G1 phase arrest, and collapse of
mitochondrial membrane in a concentration-dependent manner in MCF-7 cells.
Therefore, mitochondria-dependent apoptotic pathway would be involved in 10a
mediated apoptosis. Western blot assays indicated that 10a induced the expression of
Bax, cyto-c, caspase-9 and -3, while suppressed the expression of Bcl-2. In conclusion,
compound 10a could be considered as a promising anticancer agent and further
evaluation of 10a is on-going.
4
. Experimental
4
.1. Chemistry
Chemicals and solvents were purchased from commercial sources. Further
1
13
purification by standard methods were employed when necessary. H NMR and C

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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
δ values (ppm) and the coupling constants (J) in Hz. HRESIMS data
were obtained using Bruker micro-TOFQ-Q mass spectrometer.
4
.1.2 General procedures to synthesize 10a−c, 11a−c and 13a−c
To a solution of 6 (450 mg, 1 mmol) in 20 mL of anhydrous acetone,
corresponding dibromoalkane (3 mmol) and K CO (417 mg, 3 mmol) were added.
2
3
Followed by refluxing for 12 h and vacuum filtration. This procedure yielded
concentrated filter liquor, from which a yellow solid 8a−c was obtained. 8a−c (0.3
mmol) in 5 mL of DMF was mixed with benzoic acid mustard 4 (0.35 mmol) and
K CO (0.6 mmol) by stirring at room temperature for 24 h. The mixture was poured
2
3
into 20 mL of H O and extracted with ethyl acetate (15 mL × 3). The organic layers
2
were combined, washed with water and saturated NaCl solution, dried over anhydrous
Na SO , and concentrated in vacuo, sequentially. The crude material 9a−c was
2
4
obtained. Without further purification, 9a−c was dissolved in 10 mL of HOAc/H O
2
(
4:1) solution, then refluxed under N atmosphere for 1.5 h. After being cooled down
2
to the room temperature, the mixture was poured into 50 mL water, extracted with
ethyl acetate (20 mL × 3), then the organic layer was washed with saturated NaCl
solution, dried over Na SO , filtered and concentrated. The residues were purified by
2
4
column chromatography (dichloromethane/methanol 20:1) to yield 10a−c.
K CO (0.4 mmol) and Me SO (0.4 mmol) were added to a solution of 10a−c
2
3
2
4
(0.09 mmol) in anhydrous acetone (20 mL). The solution was refluxed for 8 h and
filtered in vacuum. The filter liquor was concentrated to obtain a yellow solid. The
residues were purified by column chromatography (petroleum ether/ethyl acetate 4:1)
to yield 11a−c. 13a−c were prepared by a similar procedure as 10a−c.

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4
.1.1.1 Compound 10a. 3-(4-(5,6,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenoxy)
propyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 85 mg; yield: 41%;
+
1
HRMS (ESI) m/z calcd for C H Cl NO [M + H] 588.1147, found 588.1164; H
2
9
27
2
8
NMR (DMSO-d , 400 MHz) δ (ppm): 12.35 (s, 1H, 5-OH), 10.48 (s, 1H, 7-OH), 8.81
6
(
s, 1H, 6-OH), 8.12 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.80 (d, 2H, J = 8.9 Hz, Ar-H), 7.14
d, 2H, J = 8.9 Hz, Ar-H), 6.83 (d, 3H, J = 8.9 Hz, H-3′,5′, H-8), 6.27 (s, 1H, H-3),
(
4
.37 (t, 2H, J = 6.2 Hz, -CH -), 4.24 (t, 2H, J = 6.2 Hz, -CH -), 3.76−3.81 (m, 8H,
2 2
1
3
-
CH -), 2.18 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.59,
2 2 6
1
1
6
4
4
66.10, 164.28, 163.54, 161.99, 153.53, 150.77, 145.95, 131.74, 131.63, 131.63,
28.96, 128.96, 123.56, 117.64, 115.42, 115.42, 111.62, 111.62, 103.80, 103.50, 99.15,
5.36, 61.25, 52.17, 52.17, 41.31, 41.31, 28.66.
.1.1.2 Compound 10b, 4-(4-(5,6,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenoxy)butyl
-(bis(2-chloroethyl)amino)benzoate. Yellow power, 46 mg; yield: 25%; HRMS (ESI)
+
1
m/z calcd for C H Cl NO [M + H] 602.1304, found 602.1339; H NMR
3
0
29
2
8
(
DMSO-d , 400 MHz) δ (ppm): 12.76 (s, 1H, 5-OH), 10.38 (s, 1H, 7-OH), 8.82 (s, 1H,
6
6
-OH), 8.01 (d, 2H, J = 8.8 Hz, H-2′,6′), 7.77 (d, 2H, J = 9.0 Hz, Ar-H), 7.09 (d, 2H, J
9.0 Hz, Ar-H), 6.82 (s, 1H, H-8), 6.79 (d, 2H, J = 8.8 Hz, H-3′,5′), 6.60 (s, 1H, H-3),
.27 (s, 2H, -CH -), 4.15 (s, 2H, -CH -), 3.74−3.79 (m, 8H, -CH -), 1.87 (s, 4H,
=
4
2
2
2
1
3
-
CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.50, 166.10, 163.51, 161.98,
2 6
1
53.84, 150.70, 150.16, 147.49, 131.54, 131.54, 131.44, 128.64, 128.64, 123.46,
117.76, 115.47, 115.47, 111.58, 111.58, 104.52, 103.33, 94.37, 67.94, 64.00, 52.15,
5
2.15, 41.28, 41.28, 25.82, 25.52.
4
.1.1.3 Compound 10c. 5-(4-(5,6,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenoxy)
pentyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 61 mg; yield: 33%;
+
1
HRMS (ESI) m/z calcd for C H Cl NO [M + H] 616.1460, found 616.1496; H
3
1
31
2
8

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NMR (DMSO-d , 400 MHz) δ (ppm): 12.76 (s, 1H, 5-OH), 10.34 (s, 1H, 7-OH), 8.91
6
(
s, 1H, 6-OH), 8.00 (d, 2H, J = 8.8 Hz, H-2′,6′), 7.78 (d, 2H, J = 9.0 Hz, Ar-H), 7.09
d, 2H, J = 9.0 Hz, Ar-H), 6.82 (t, 3H, J = 8.8 Hz, H-3′,5′, H-8), 6.59 (s, 1H, H-3),
(
4
.23 (t, 2H, J = 6.1 Hz, -CH -), 4.09 (t, 2H, J = 6.3 Hz, -CH -), 3.75−3.80 (m, 8H,
2 2
1
3
-
CH -), 1.83−1.74 (m, 4H, -CH -), 1.56 (m, 2H, -CH -). C NMR (DMSO-d , 100
2 2 2 6
MHz) δ (ppm): 182.50, 166.14, 163.53, 162.07, 153.85, 150.71, 150.16, 147.49,
31.53, 131.53, 131.43, 128.64, 128.64, 123.42, 117.84, 115.45, 115.45, 111.61,
11.61, 104.51, 103.32, 94.37, 68.19, 64.13, 52.18, 52.18, 41.31, 41.31, 28.62, 28.49,
1
1
2
2.59.
4
.1.1.4 Compound 11a. 3-(4-(5-hydroxy-6,7-dimethoxy-4-oxo-4H-chromen-2-yl)
phenoxy)propyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 37 mg; yield:
+
6
0%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 616.1460, found 616.1452;
3
1
31
2
8
1
H NMR (DMSO-d , 400 MHz) δ (ppm): 12.73 (s, 1H, 5-OH), 8.02 (d, 2H, J = 8.9 Hz,
6
H-2′,6′), 7.80 (d, 2H, J = 8.9 Hz, Ar-H), 7.16 (d, 2H, J = 8.9 Hz, Ar-H), 6.91 (s, 1H,
H-8), 6.82 (d, 2H, J = 8.9 Hz, H-3′,5′), 6.59 (s, 1H, H-3), 4.37 (t, 2H, J = 6.0 Hz,
-
CH -), 4.24 (t, 2H, J = 6.2 Hz, -CH -), 3.91 (s, 1H, -OCH ), 3.83 (s, 1H, -OCH ),
2 2 3 3
1
3
3
.73−3.80 (m, 8H, -CH -), 2.18 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ
2 2 6
(ppm): 182.65, 166.11, 163.95, 162.18, 158.88, 153.14, 150.78, 149.23, 131.63,
1
1
4
31.63, 128.95, 128.77, 128.77, 123.35, 117.63, 115.66, 115.66, 111.62, 111.62,
04.39, 103.82, 96.48, 65.43, 62.59, 61.59, 56.97, 53.30, 53.30, 41.33, 41.33, 28.63.
.1.1.5 Compound 11b. 4-(4-(5-hydroxy-6,7-dimethoxy-4-oxo-4H-chromen-2-yl)
phenoxy)butyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 21 mg; yield:
+
4
3%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 630.1617, found 630.1673;
3
2
33
2
8
1
H NMR (DMSO-d , 400 MHz) δ (ppm): 12.74 (s, 1H, 5-OH), 8.02 (d, 2H, J = 8.6 Hz,
6
H-2′,6′), 7.77 (d, 2H, J = 8.9 Hz, Ar-H), 7.14 (d, 2H, J = 8.9 Hz, Ar-H), 6.91 (s, 1H,

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H-8), 6.80 (d, 2H, J = 9.0 Hz, H-3′,5′), 6.58 (s, 1H, H-3), 4.27 (s, 2H, -CH -), 4.15 (s,
2
2
1
1
1
6
4
H, -CH -), 3.91 (s, 1H, -OCH ), 3.83 (s, 1H, -OCH ), 3.74−3.78 (m, 8H, -CH -),
2 3 3 2
1
3
.87 (s, 4H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.65, 166.10, 163.95,
2
6
62.27, 158.86, 157.13, 150.70, 149.22, 131.98, 131.53, 131.53, 128.72, 128.72,
23.20, 117.77, 115.64, 115.64, 111.57, 111.57, 104.39, 103.76, 96.47, 67.99, 63.98,
1.57, 56.96, 52.15, 52.15, 41.27, 41.27, 25.78, 25.52.
.1.1.6 Compound 11c. 5-(4-(5-hydroxy-6,7-dimethoxy-4-oxo-4H-chromen-2-yl)
phenoxy)pentyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 34 mg; yield:
+
5
3%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 644.1773, found 644.1635;
3
3
35
2
8
1
H NMR (DMSO-d , 400 MHz) δ (ppm): 12.75 (s, 1H, 5-OH), 8.05 (d, 2H, J = 8.9 Hz,
6
H-2′,6′), 7.78 (d, 2H, J = 8.9 Hz, Ar-H), 7.14 (d, 2H, J = 8.9 Hz, Ar-H), 6.92 (s, 1H,
H-8), 6.81 (d, 2H, J = 8.9 Hz, H-3′,5′), 6.59 (s, 1H, H-3), 4.24 (s, 2H, -CH -), 4.11 (s,
2
2
1
H, -CH -), 3.92 (s, 1H, -OCH ), 3.83 (s, 1H, -OCH ), 3.75−3.80 (m, 8H, -CH -),
2 3 3 2
1
3
.72−1.85 (m, 4H, -CH -), 1.56 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ
2
2
6
(ppm): 182.66, 166.13, 164.01, 162.38, 158.88, 157.14, 150.71, 149.24, 131.53,
1
1
2
4
31.53, 128.74, 128.74, 123.17, 117.84, 115.65, 115.65, 111.61, 111.61, 104.39,
03.76, 96.49, 68.27, 64.14, 61.59, 56.97, 52.17, 52.17, 41.31, 41.31, 28.61, 28.50,
2.60.
.1.1.7 Compound 13a. 3-(4-(10-hydroxy-9-oxo-2,3-dihydro-9H-[1,4]dioxino[2,3-g]
chromen-7-yl)phenoxy)propyl-4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 13
+
mg; yield: 36%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 614.1304, found
3
1
29
2
8
1
1
6
14.1364; H NMR (DMSO-d , 400 MHz) δ (ppm): H NMR (DMSO-d , 400 MHz)
6
6
δ (ppm): 12.32 (s, 1H, 5-OH), 8.00 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.79 (d, 2H, J = 8.9
Hz, Ar-H), 7.14 (d, 2H, J = 8.9 Hz, Ar-H), 6.94 (s, 1H, H-8), 6.80 (d, 2H, J = 8.9 Hz,
H-3′,5′), 6.33 (s, 1H, H-3), 4.42−4.30 (m, 6H, -CH -), 4.23 (m, 2H, -CH -), 3.76−3.80
2
2

ACCEPTED MANUSCRIPT
1
3
(
m, 8H, -CH -), 2.19 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm):
2 2 6
1
1
1
4
82.48, 166.10, 163.63, 162.16, 153.49, 150.77, 150.42, 145.05, 131.73, 131.63,
31.63, 128.69, 128.69, 123.19, 117.65, 115.60, 115.60, 111.48, 111.48, 105.26,
04.19, 99.52, 65.63, 65.42, 64.29, 61.25, 52.24, 52.24, 41.31, 41.31, 28.64.
.1.1.8 Compound 13b. 4-(4-(10-hydroxy-9-oxo-2,3-dihydro-9H-[1,4]dioxino[2,3-g]
chromen-7-yl)phenoxy)butyl 4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 21
+
mg; yield: 44%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 628.1460, found
3
2
31
2
8
1
6
28.1462; H NMR (DMSO-d , 400 MHz) δ (ppm): 12.32 (s, 1H, 5-OH), 7.99 (d, 2H,
6
J = 8.9 Hz, H-2′,6′), 7.77 (d, 2H, J = 8.9 Hz, Ar-H), 7.13 (d, 2H, J = 8.9 Hz, Ar-H),
.93 (s, 1H, H-8), 6.79 (d, 2H, J = 8.9 Hz, H-3′,5′), 6.33 (s, 1H, H-3), 4.42 (s, 2H,
CH -), 4.38 (s, 2H, -CH -), 4.28 (s, 2H, -CH -), 4.15 (s, 2H, -CH -), 3.74−3.78 (m,
6
-
2
2
2
2
1
3
8
1
1
6
4
H, -CH -), 1.87 (s, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.48,
2 2 6
66.10, 163.67, 162.27, 153.50, 150.70, 150.42, 149.68, 131.73, 131.53, 131.53,
28.66, 128.66, 123.04, 117.78, 115.60, 115.60, 111.58, 111.58, 105.27, 104.13, 99.52,
7.99, 65.64, 64.30, 64.00, 52.16, 52.16, 41.27, 41.27, 25.81, 25.52.
.1.1.9 Compound 13c. 5-(4-(10-hydroxy-9-oxo-2,3-dihydro-9H-[1,4]dioxino[2,3-g]
chromen-7-yl)phenoxy)pentyl 4-(bis(2-chloroethyl)amino)benzoate. Yellow power, 34
+
mg; yield: 53%; HRMS (ESI) m/z calcd for C H Cl NO [M + H] 642.1617, found
3
3
33
2
8
1
6
42.1695; H NMR (DMSO-d , 400 MHz) δ (ppm): 12.32 (s, 1H, 5-OH), 7.98 (d, 2H,
6
J = 8.9 Hz, H-2′,6′), 7.78 (d, 2H, J = 9.0 Hz, Ar-H), 7.11 (d, 2H, J = 9.0 Hz, Ar-H),
6
.92 (s, 1H, H-8), 6.81 (d, 2H, J = 8.9 Hz, H-3′,5′), 6.32 (s, 1H, H-3), 4.42 (d, 2H, J =
.5 Hz, -CH -), 4.38 (d, 2H, J = 4.7 Hz, -CH -), 4.23 (t, 2H, J = 6.2 Hz, -CH -), 4.08
4
2
2
2
(
t, 2H, J = 6.3 Hz, -CH -), 3.80−3.75 (m, 8H, -CH -), 1.82−1.73 (m, 4H, -CH -), 1.55
2 2 2
1
3
(
m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.45, 166.12, 163.65,
2 6
1
62.33, 153.48, 150.68, 150.41, 149.65, 131.73, 131.53, 131.53, 128.64, 128.64,

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1
6
4
22.97 117.83, 115.53, 115.53, 111.58, 111.58, 105.24, 104.08, 99.50, 68.22, 65.62,
4.28, 64.13, 52.17, 52.17, 41.29, 41.29, 28.63, 28.51, 22.60.
.2 MTT assay
The cell viability were assessed by the MTT method. Three cancer cell in
o
logarithmic growth were seeded to each well and incubated for 24 h at 37 C in a
humidified atmosphere of 5% CO . Then target hybrids, scutellarein, benzoic acid
2
mustard and 5-FU were added. 20 µL fresh MTT solution (5 mg/mL) per well was
added to each cultured medium after 72 h, and incubated for another 4 h. The cells
were dissolved with DMSO (150 µL) and shaken for 10 min at room temperature.
After that, the OD of each single well was measured on a Microplate Reader
(
BIO-RAD) and the IC values were calculated [46].
50
4
.3 Cell cycle analysis
MCF-7 cells in logarithmic growth were plated in 6-well plates and incubated at
o
3
7 C for 24 h, then cells were incubated with 10a at different concentrations (0, 0.75,
1
.5 and 3 µM). After 48 h, cells were collected, centrifuged and fixed with 70%
o
ethanol at 4 C overnight. The fixed cells were washed with ice-cold PBS and treated
with 100 µL RNase A at 37 °C for 30 min, after that stained with 400 µL PI in the
dark at 4 °C for 30 min. The cellular DNA content for cycle distribution analysis was
performed using a fow cytometer (FACS Calibur Becton–Dickinson) [47].
4
.4 Cell apoptosis assay
MCF-7 cells in logarithmic growth were plated in 6-well plates and incubated at
o
3
7 C for 24 h, then cells were incubated with 10a at different concentrations (0, 0.75,
1
.5 and 3 µM). After 48 h, cells were collected and washed with PBS, then 500 µL
binding buffer was added. Finally, double stained Annexin V-FITC/PI (KGA1024,
KeyGEN Biotech, Nanjing, China) were added and incubated at room temperature for

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1
5 min in the dark. The cells were detected to analyze apoptosis by fow cytometry
[
48].
4
.5 Mitochondrial membrane potential assay
Briefly, MCF-7 cells in logarithmic growth were plated in 6-well plates and
incubated with 10a at different concentrations (0, 0.75, 1.5 and 3 µM) for 48 h. Cells
were collected and washed with PBS. JC-1 dye was used to stained cells under dark
conditions according to the manufacturer’s instructions (KGA601, KeyGEN Biotech,
Nanjing, China). The percentage of cells with collapsed mitochondrial membrane
potentials was monitored by flow cytometry [49].
4
.6 Western blot assay
MCF-7 cells in logarithmic growth were plated in 6-well plates and incubated
with 10a at different concentrations (0, 0.75, 1.5 and 3 µM) for 48 h, the cells were
harvested. Then the cells were lysed using lysis buffer, and the solution was
o
centrifuged at 14000 g for 10 min at 4 C. Thereafter, the protein concentrations were
determined with bicinchoninic acid protein assay kit (Beyotime Co, Shanghai, China),
and individual cell lysates (25 µg) were separated by SDS-PAGE on a 12%
polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes
were blocked with 5% fat-free milk, the membranes were incubated with monoclonal
antibodies against Bcl-2, Bax, cyto-c, caspase-9, -3 and GAPDH (KeyGEN Biotech,
o
Nanjing, China) at 4 C overnight, respectively. Then, the bound antibodies washed
with TBST, and incubated with appropriate second antibodies and followed by
enhanced chemiluminescence detection [50].
Acknowledgment
This work was financially supported by the Key Laboratory for the Chemistry and

ACCEPTED MANUSCRIPT
Molecular Engineering of Medicinal Resources (Guangxi Normal University), the
Ministry of Education of China (CMEMR2015-B07), Natural Science Foundation of
Liaoning Province (20170540858), General Scientific Research Projects of
Department of Education in Liaoning Province (2017LQN05), Key Laboratory of
Quality Control of TCM of Liaoning Province (17-137-1-00), and Career
Development Support Plan for Young and Middle-aged Teachers in Shenyang
Pharmaceutical University.
References
[
1] C. Allemani, H.K. Weir, H. Carreira, R. Harewood, D. Spika, X.S. Wang, F.
Bannon, J.V. Ahn, C.J. Johnson, A. Bonaventure, R. Marcos-Gragera, C. Stiller, G.
Azevedo e Silva, W.Q. Chen, O.J. Ogunbiyi, B. Rachet, M.J. Soeberg, H. You, T.
Matsuda, M. Bielska-Lasota, H. Storm, T.C. Tucker, M.P. Coleman, CONCORD
Working Group, Global surveillance of cancer survival 1995-2009: analysis of
individual data for 25,676,887 patients from 279 population-based registries in 67
countries (CONCORD-2), Lancet 385 (2015) 977−1010.
[
2] A.B. Ryerson, C.R. Eheman, S.F. Altekruse, J.W. Ward, A. Jemal, R.L. Sherman,
S.J. Henley, D. Holtzman, A. Lake, A.M. Noone, R.N. Anderson, J. Ma, K.N. Ly, K.A.
Cronin, L. Penberthy, B.A. Kohler, Annual report to the nation on the status of cancer,
1
975−2012, featuring the increasing incidence of liver cancer, Cancer 122 (2016)
312−1337.
1
[
3] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J.
He, Cancer statistics in China, 2015, CA-Cancer J. Clin. 66 (2016) 115−132.
4] W. Derek, J.J. Turchi, Chemotherapy induced DNA damage response, Cancer Biol.
Ther. 14 (2013) 379−389.
[

ACCEPTED MANUSCRIPT
[
5] K. Nurgali, R.T. Jagoe, R. Abalo, Editorial: adverse effects of cancer
chemotherapy: Anything new to improve tolerance and reduce sequelae? Front.
Pharmacol. 9 (2018) 245.
[
6] P. Saha, C. Debnath, G. Bérubé, Steroid-linked nitrogen mustards as potential
anticancer therapeutics: A review, J. Steroid Biochem. Mol. Biol. 137 (2013)
71−300.
7] L.F. Povirk, D.E. Shuker, DNA damage and mutagenesis induced by nitrogen
mustards, Mutat. Res. 318 (1994) 205−226.
8] B.J. Sanderson, A.J Shield, Mutagenic damage to mammalian cells by therapeutic
alkylating agents, Mutat. Res. 355 (1996) 41−57.
9] S. Li, X. Wang, Y. He, M. Zhao, Y. Chen, J. Xu, M. Feng, J. Chang, H. Ning, C. Qi,
2
[
[
[
Design and synthesis of novel quinazoline nitrogen mustard derivatives as potential
therapeutic agents for cancer, Eur. J. Med. Chem. 67 (2013) 293−301.
[
10] B. Marvania, R. Kakadiya, W. Christian, T.L. Chen, M.H. Wu, S. Suman, K. Tala,
T.C. Lee, A. Shah, T.L. Su, The synthesis and biological evaluation of new
DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates
containing a urea linker, Eur. J. Med. Chem. 83 (2014) 695−708.
[
11] P.M. Cullis, L. Merson-Davies, R. Weaver, Conjugation of a polyamine to the
bifunctional alkylating agent chlorambucil does not alter the preferred cross-linking
site in duplex DNA, J. Am. Chem. Soc. 117 (1995) 8033−8034.
[
12] P.G. Baraldi, R. Romagnoli, A.E. Guadix, M.J. de las Infantas Pineda, M.A. Gallo,
A. Espinosa, A. Martinez, J.P. Bingham, J.A. Hartley, Design, synthesis, and
biological activity of hybrid compounds between uramustine and DNA minor groove
binder distamycin A, J. Med. Chem. 45 (2002) 3630−3638.
[
13] D. Bastien, R. Hanna, V. Leblanc, É. Asselin, G. Bérubé, Synthesis and

ACCEPTED MANUSCRIPT
preliminary in vitro biological evaluation of 7α-testosterone-chlorambucil hybrid
designed for the treatment of prostate cancer, Eur. J. Med. Chem. 64 (2013) 442−447.
[
14] P.C. Acharya, R. Bansal. Synthesis of androstene oxime-nitrogen mustard
bioconjugates as potent antineoplastic agents, Steroids 123 (2017) 73−83.
15] S. Xu, L. Pei, C. Wang, Y.K. Zhang, D. Li, H. Yao, X. Wu, Z.S. Chen, Y. Sun, J.
[
Xu, Novel hybrids of natural oridonin-bearing nitrogen mustards as potential
anticancer drug candidates, ACS Med. Chem. Lett. 5 (2014) 797−802.
[
16] X. Gao, J. Li, M. Wang, S. Xu, W. Liu, L. Zang, Z. Li, H. Hua, J. Xu, D. Li,
Novel enmein-type diterpenoid hybrids coupled with nitrogen mustards: Synthesis of
promising candidates for anticancer therapeutics, Eur. J. Med. Chem. 146 (2018)
5
88−598.
[
17] T. Han, K. Tian, H. Pan, Y. Liu, F. Xu, Z. Li, T. Uchita, M. Gao, H. Hua, D. Li,
Novel hybrids of brefeldin A and nitrogen mustards with improved antiproliferative
selectivity: Design, synthesis and antitumor biological evaluation, Eur. J. Med. Chem.
1
50 (2018) 53−63.
[
18] Y.Z. Xu, X.Y. Gu, S.J. Peng, J.G. Fang, Y.M. Zhang, D.J. Huang, J.J. Chen, K.
Gao, Design, synthesis and biological evaluation of novel sesquiterpene mustards as
potential anticancer agents, Eur. J. Med. Chem. 94 (2015) 284−297.
[
19] M. Singh, M. Kaur, O. Silakari, Flavones: An important scaffold for medicinal
chemistry, Eur. J. Med. Chem. 84 (2014) 206−239.
20] Z.H. Shi, N.G. Li, Z.J. Wang, Y.P. Tang, Z.X. Dong, W. Zhang, P.X. Zhang, T. Gu,
[
W.Y. Wu, J.P. Yang, J.A. Duan, Synthesis and biological evaluation of methylated
scutellarein analogs based on metabolic mechanism of scutellarin in vivo, Eur. J. Med.
Chem. 106 (2015) 95−105.
[
21] X. Chen, X. Shi, X. Zhang, H. Lei, S. Long, H. Su, Z. Pei, R. Huang, Scutellarin

ACCEPTED MANUSCRIPT
attenuates hypertension-induced expression of brain Toll-like receptor 4/nuclear factor
kappa B, Mediators Inflamm. 2013 (2013) 432623.
[
22] Z. Pan, W. Zhao, X. Zhang, B. Wang, J. Wang, X. Sun, X. Liu, S. Feng, B. Yang,
Y. Lu, Scutellarin alleviates interstitial fibrosis and cardiac dysfunction of infarct rats
by inhibiting TGFβ1 expression and activation of p38-MAPK and ERK1/2, Br. J.
Pharmacol. 162 (2011) 688−700.
[
23] H. Zhou, X. Chen, L. Chen, X. Zhou, G. Zheng, H. Zhang, W. Huang, J. Cai,
Anti-fibrosis effect of scutellarin via inhibition of endothelial-mesenchymal transition
on isoprenaline-induced myocardial fibrosis in rats, Molecules 19 (2014)
1
5611−15623.
[
24] Z. Wang, J. Yu, J. Wu, F. Qi, H. Wang, Z. Wang, Z. Xu, Scutellarin protects
cardiomyocyte ischemia-reperfusion injury by reducing apoptosis and oxidative stress,
Life Sci. 157 (2016) 200–207.
[
25] Y. Feng, S. Zhang, J. Tu, Z. Cao, Y. Pan, B. Shang, R. Liu, M. Bao, P. Guo, Q.
Zhou, Novel function of scutellarin in inhibiting cell proliferation and inducing cell
apoptosis of human Burkitt lymphoma Namalwa cells, Leuk. Lymphoma 53 (2012)
2
456–2464.
26] H. Li, H. Fan, Z. Wang, J. Zheng, W. Cao, Potentiation of scutellarin on human
tongue carcinoma xenograft by low-intensity ultrasound, Plos One 8 (2013) e59473.
27] Y. Ke, T. Bao, X. Wu, H. Tang, Y. Wang, J. Ge, B. Fu, X. Meng, L. Chen, C.
[
[
Zhang, Y. Tan, H. Chen, Z. Guo, F. Ni, X. Lei, Z. Shi, D. Wei, L. Wang, Scutellarin
suppresses migration and invasion of human hepatocellular carcinoma by inhibiting
the STAT3/Girdin/Akt activity, Biochem. Biophys. Res. Commun. 483 (2017)
5
09–515.
[
28] J.Y. Chan, B.K. Tan, S.C. Lee, Scutellarin sensitizes drug-evoked colon cancer

ACCEPTED MANUSCRIPT
cell apoptosis through enhanced caspase-6 activation, Anticancer Res. 29 (2009)
3
043–3047.
[
29] C. Gao, Y. Zhou, Z. Jiang, Y. Zhao, D. Zhang, X. Cong, R. Cao, H. Li, W. Tian,
Cytotoxic and chemosensitization effects of scutellarin from traditional Chinese herb
Scutellaria altissima L. in human prostate cancer cells, Oncol. Rep. 38 (2017)
1
491–1499.
[
30] Z. Sang, X. Qiang, Y. Li, R. Xu, Z. Cao, Q. Song, T. Wang, X. Zhang, H. Liu, Z.
Tan, Y. Deng, Design, synthesis and evaluation of scutellarein-O-
acetamidoalkylbenzylamines as potential multifunctional agents for the treatment of
Alzheimer's disease, Eur. J. Med. Chem. 135 (2017) 307–323.
[
31] H. Xu, S. Zhang, Scutellarin-induced apoptosis in HepG2 hepatocellular
carcinoma cells via a STAT3 pathway, Phytother. Res. 27 (2013) 1524–1528.
32] N. Yang, Y. Zhao, Z. Wang, Y. Liu, Y. Zhang, Scutellarin suppresses growth and
[
causes apoptosis of human colorectal cancer cells by regulating the p53 pathway, Mol.
Med. Rep. 15 (2017) 929–935.
[
33] P. Thirusangu, V. Vigneshwaran, B.R. Vijay Avin, H. Rakesh, H.M. Vikas, B.T.
Prabhakar, Scutellarein antagonizes the tumorigenesis by modulating cytokine VEGF
mediated neoangiogenesis and DFF-40 actuated nucleosomal degradation, Biochem.
Biophys. Res. Commun. 484 (2017) 85.
[
34] L. You, H. Zhu, C. Wang, F. Wang, Y. Li, Y. Li Y. Wang, B. He, Scutellarin
inhibits Hela cell growth and glycolysis by inhibiting the activity of pyruvate kinase
M2, Bioorg. Med. Chem. Lett. 27 (2017) 5404–5408.
[
35] D. Goh, Y.H. And, E.S. Ong, Inhibitory effects of a chemically standardized
extract from Scutellaria barbata in Human colon cancer cell lines, LoVo, J. Agric.
Food Chem. 53 (2005) 8197–8204.

ACCEPTED MANUSCRIPT
[
36] X. Shi, G. Chen, X. Liu, Y. Qiu, S. Yang, Y. Zhang, X. Fang, C. Zhang, X. Liu,
Scutellarein inhibits cancer cell metastasis in vitro and attenuates the development of
fibrosarcoma in vivo, Int. J. Mol. Med. 35 (2015) 31–38.
[
37] M. Feng,·Y. Song, J. Wu, X. Chen, Y. Zhang, Study on antitumour activity of
scutellarin and its metabolite scutellarein by combining activity screening, target
tissue distribution and pharmacokinetics, Chromatographia 80 (2017) 427–435.
[
38] F. Guo, F. Yang, Y. Zhu. Scutellarein from Scutellaria barbata induces apoptosis
of human colon cancer HCT116 cells through the ROS-mediated
mitochondria-dependent pathway, Nat. Prod. Res. (2018) https://doi.org/10.1080/14
7
86419. 2018.1440230.
39] X. Li, L. Wang, Y. Li, L. Bai, M. Xue, Acute and subacute toxicological
evaluation of scutellarin in rodents, Regul. Toxicol. Pharmacol. 60 (2011) 106–111..
40] Q.Z. Zheng, F. Zhang, K. Cheng, Y. Yang, Y. Chen, Y. Qian, H.J. Zhang, H.Q. Li,
[
[
C.F. Zhou, S.Q. An, Q.C. Jiao, H.L. Zhu, Synthesis, biological evaluation and
molecular docking studies of amide-coupled benzoic nitrogen mustard derivatives as
potential antitumor agents, Bioorg. Med. Chem. 18 (2010) 880–886.
[
41] Z.H. Shi, N.G. Li, Q.P. Shi, W. Zhang, Z.X. Dong, Y.P. Tang, P.X. Zhang, T. Gu,
W.Y. Wu, F. Fang, X. Xue, H.M. Li, J.P. Yang, J.A. Duan, Synthesis of scutellarein
derivatives to increase biological activity and water solubility, Bioorg. Med. Chem. 23
(2015) 6875–6884.
[
42] T.S. Reddy, S.H. Privér, N. Mirzadeh, S.K. Bhargava, Synthesis of gold(I)
phosphine complexes containing the 2-BrC F PPh ligand: Evaluation of anticancer
6
4
2
activity in 2D and 3D spheroidal models of HeLa cancer cells, Eur. J. Med. Chem.
45 (2018) 291–301.
43] X. Hu, R. Jiao, H. Li, X. Wang, H. Lyu, X. Gao, F. Xu, Z. Li, H. Hua, D. Li,
1
[

ACCEPTED MANUSCRIPT
Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their
apoptosis inducing properties, Eur. J. Med. Chem. 151 (2018) 376–388.
[
44] J.F.R. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon
with wide-ranging implications in tissue kinetics, Br. J. Canc. 26 (1972) 239–257.
45] G.M. Cohen, Caspases: the executioners of apoptosis, Biochem. J. 326 (1997)
–16.
46] J. Chen, T. Wang, S. Xu, P. Zhang, A. Lin, L. Wu, H. Yao, W. Xie, Z. Zhu, J. Xu,
[
1
[
Discovery of novel antitumor nitric oxide-donating β-elemene hybrids through
inhibiting the PI3K/Akt pathway, Eur. J. Med. Chem. 28 (2017) 414–423.
[
47] T. Han, J. Li, J. Xue, H. Li, F. Xu, K. Cheng, D. Li, Z. Li, M. Gao, H. Hua,
Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological
evaluation, Eur. J. Med. Chem. 135 (2017) 270–281.
[
48] K. Tian, F. Xu, X. Gao, T. Han, J. Li, H. Pan, L. Zang, D. Li, Z. Li, T. Uchita, M.
Gao, H. Hua, Nitric oxide-releasing derivatives of brefeldin A as potent and highly
selective anticancer agents, Eur. J. Med. Chem. 136 (2017) 131–143.
[
49] J. Chen, T. Wang, S. Xu, A. Lin, H. Yao, W. Xie, Z. Zhu, J. Xu, Design, synthesis
and biological evaluation of novel nitric oxide-donating protoberberine derivatives as
antitumor agents, Eur. J. Med. Chem. 132 (2017) 173–183.
[
50] S. Xu, H. Yao, L. Pei, M. Hu, D. Li, Y. Qiu, G. Wang, L. Wu, H. Yao, Z. Zhu, J.
Xu, Design, synthesis, and biological evaluation of NAD(P)H: Quinone
oxidoreductase (NQO1)-targeted oridonin prodrugs possessing indolequinone moiety
for hypoxia-selective activation, Eur. J. Med. Chem. 132 (2017) 310–321.

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Table 1 Antiproliferative activities of target hybrids against different cell lines.
a
Compound
IC (µM)
50
A549
> 50
MCF-7
1.50 ± 0.06
3.55 ± 0.19
4.78 ± 0.31
> 50
Bel-7402
7.96 ± 0.45
9.07 ± 0.34
4.59 ± 0.23
27.83 ± 1.02
25.69 ± 1.33
32.46 ± 2.30
>50
L-O2
> 50
1
0a
1
0b
21.47 ± 1.26
23.51 ± 1.42
> 50
> 50
1
0c
1a
> 50
1
> 50
1
1b
47.28 ± 2.87
> 50
> 50
> 50
1
1c
3a
> 50
> 50
1
44.52 ± 1.90
> 50
45.04 ± 2.47
> 50
> 50
1
3b
3c
37.64 ± 2.26
45.72 ± 1.78
23.94 ± 1.34
> 50
> 50
1
> 50
34.70 ± 1.88
> 50
> 50
4
> 50
32.86 ± 2.39
> 50
scutellarein
chlorambucil
> 50
> 50
> 50
> 50
> 50
> 50
b
5
-FU
2.05 ± 0.11
27.89 ± 1.66
18.65 ± 1.31
NT
a
IC : concentrations inhibit 50% of cell growth measured by the MTT assay. The values are
expressed as average ± standard deviations of three independent experiments. NT: not tested.
5
0
b

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1
. Legends of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Scheme 1
Fig. 1. The chemical structures of some reported natural product/benzoic acid mustard hybrids.
Fig. 2. Cell cycle analysis of 10a in MCF-7 cells by flow cytometry.
Fig. 3. Flow cytometry analysis of apoptosis induced by 10a in MCF-7 cells.
Fig. 4. 10a induced mitochondrial depolarization in MCF-7 cells.
Fig. 5. 10a affected the expressions of the mitochondria pathway related proteins in MCF-7 cells.
Con is short for control.
Scheme 1. Synthesis of benzoic acid mustard/scutellarein hybrids 10a−c, 11a−c and 13a−c.
o
Reagents and conditions: (a) ethylene oxide, H O, CH COOH, rt, 24 h; (b) POCl , 50 C, 0.5 h; (c)
2
3
3
o
1
0% HCl, 12 h; (d) HCl, EtOH, N , reflux, 36 h; (e) Ph CCl , Ph O, 175 C, 1.5 h; (f) 1,2-
2 2 2 2
o
dibromoethane, K CO , DMF, 85 C, 3 h; (g) corresponding dibromoalkane, K CO , anhydrous
2
3
2
3
acetone, reflux, 6-8 h; (h) 4, K CO , anhydrous DMF, rt, 12 h; (i) HAc : H O (4:1), reflux, 1.5 h;
2
3
2
(
j) Me SO , K CO , anhydrous acetone, reflux, 8 h; (k) corresponding dibromoalkane, K CO ,
2
4
2
3
2
3
anhydrous acetone, reflux, 6-8 h; (l) 4, K CO , anhydrous DMF, rt, 12 h.
2
3

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2
. Graphics for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Scheme 1
Figure 1

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Figure 2

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Figure 3

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Figure 4