Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid hybrids

Article abstract of DOI:10.1016/j.ejmech.2015.01.040

A series of hybrids of scopoletin and substituted cinnamic acid were designed, synthesized and evaluated in vitro and in vivo against five human tumor cell lines [MCF-7, MDA-MB-231, A549, HCT-116, and HeLa] with doxorubicin as the positive control. Compounds 17a, 17b, 17c and 17g exhibited potent cytotoxic activity. Especially, compound 17b displayed broad spectrum activity with IC₅₀ values ranging from 0.249 μ1/4M to 0.684 μ1/4M. Moreover, in a preliminary pharmacological study, 17b not only remarkably induced cellular apoptosis, but also clearly induced A549 cells cycle arrest at S phase. In vivo study showed that 17b significantly suppressed tumor growth in a dose-dependent manner without causing the loss of the mean body weight of mice, which was superior to doxorubicin. These preliminary results indicate that 17b is an optimal anti-cancer leading compound and merit further structural modification.

Full text of DOI:10.1016/j.ejmech.2015.01.040

Accepted Manuscript
Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid hybrids
Linhu Li, Peng Zhao, Jinglin Hu, Jinhong Liu, Yan Liu, Zhiqiang Wang, Yufeng Xia,
Yue Dai, Li Chen
PII:
S0223-5234(15)00060-4
10.1016/j.ejmech.2015.01.040
EJMECH 7656
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 September 2014
Revised Date: 19 January 2015
Accepted Date: 20 January 2015
Please cite this article as: L. Li, P. Zhao, J. Hu, J. Liu, Y. Liu, Z. Wang, Y. Xia, Y. Dai, L. Chen,
Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid hybrids, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.01.040.
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Graphic abstract
Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid
hybrids
Linhu Lia, †, Peng Zhaob, †, Jinglin Hua, Jinhong Liua, Yan Liua, Zhiqiang Wanga,
Yufeng Xia c, *, Yue Dai b, Li Chena, *
A series of novel scopoletin-cinnamic acid hybrids were synthesized as antitumor agents. The
most potent compound 17b showed potent activity in vivo and vitro by causing cell cycle arrest
and induced apoptosis in A549 cell lines.
*Correspondence: Li Chen: E-mail chenliduo12@gmail.com Phone/Fax, +86-25-83202334
Yufeng Xia: E-mail yfxiacpu@126.com

ACCEPTED MANUSCRIPT
Synthesis, in vitro and in vivo antitumor activity of scopoletin-cinnamic acid
hybrids
Linhu Lia, †, Peng Zhaob, †, Jinglin Hua, Jinhong Liua, Yan Liua, Zhiqiang Wanga,
Yufeng Xia c, *, Yue Dai b, Li Chena, *
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, People’s Republic of China.
b Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China.
c Department of Chinese Materia Medica Analysis, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, People’s Republic of China.
† These two authors contributed equally to this work
*
Corresponding authors: E-mailꢀchenliduo12@gmail.com; Tel/Fax: +86-25-83202334 (L. Chen).
E-mailꢀyfxiacpu@126.com (Y.F. Xia).
Abstract
A series of hybrids of scopoletin and substituted cinnamic acid were designed, synthesized and
evaluated in vitro and in vivo against five human tumor cell lines [MCF-7, MDA-MB-231, A549,
HCT-116, and HeLa] with doxorubicin as the positive control. Compounds 17a, 17b, 17c and 17g
exhibited potent cytotoxic activity. Especially, compound 17b displayed broad spectrum activity
with IC50 values ranging from 0.249µM to 0.684 µM. Moreover, in a preliminary pharmacological
study, 17b not only remarkably induced cellular apoptosis, but also clearly induced A549 cells
cycle arrest at S phase. In vivo study showed that 17b significantly suppressed tumor growth in a
dose-dependent manner without causing the loss of the mean body weight of mice, which was
superior to doxorubicin. These preliminary results indicate that 17b is an optimal anti-cancer
leading compound and merit further structural modification.
Keywords: scopoletin, cinnamic acid, antitumor activity
1. Introduction
Coumarins widely present in higher plants such as Rutaceae, Apiaceae, Leguminosae,
Thymelaeaceae, as well as occur as animal and microbial metabolite, playing an important role in
the agricultural and pharmaceutical industries [1]. Most of them and their derivatives are found to
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possess versatile biological activities, including anticancer [2-7], enzyme inhibition [8], antioxidant
[9, 10], anti-inflammatory [11], and anti-HIV [12].
Fig. 1
Scopoletin (6-methoxy-7-hydroxy-coumarin) is a phenolic coumarin (Fig. 1) and has been
isolated from many medicinal plants, such as Erycibe obtusifolia, Aster tataricus, and Foeniculum
vulgare, which is commonly used in traditional Chinese medicine (TCM) for treating various
rheumatoid diseases with a long history [13]. Reports have indicated that scopoletin exhibits
significant anti-tumor [14-16] and anti-angiogenesis activity [17]. These studies demonstrated that
scopoletin could induce cell cycle arrest and increase apoptosis in human prostate tumor cells and
human leukemia cell line via activation of caspase-3 and blocking angiogenesis by inhibiting the
endothelial cell growth. While its antitumor activity is not potent enough to be used directly in
clinic. Therefore, scopoletin is considered to be an ideal lead compound for anti-tumor agents.
Many derivatives have been designed and synthesized to increase its anti-tumor activity [18-21].
Cinnamic acid (Fig. 1) is a naturally occurring aromatic fatty acid composed of a phenyl ring
substituted with an acrylic acid group, commonly in the trans-geometry and with low toxicity in
human exposure [22]. Cinnamic acid and hydroxyl cinnamic acid derivatives, such as caffeic acid,
sinapinic acid and ferulic acid (Fig. 1), were found in coffee, apples, citric fruits, vegetable oils,
propolis and wine [23]. In recent years, trans-cinnamic acid derivatives have attracted many
attentions due to their anticancer, antioxidant, and antimicrobial properties [24]. Mechanism study
indicated that cinnamic acid could induce apoptotic cell death and cytoskeleton disruption in
human melanoma cells in 2013 [25]. P. De et al. provides a comprehensive review concerning
cinnamic acid derivatives in anticancer research [26]. In this review, we found that many natural
and synthetic compounds would have an increasing anticancer activity when cinnamoylresidues
were introduced in the form of amides or esters. In addition, cinnamic acid possessing α,
β-unsaturated ketones carbonyl moiety are often applied in the design of antitumor drugs [27, 28].
These findings prompted us to design and synthesize the hybrids of cinnamic acid and scopoletin
as anti-tumor agents with high potency and low toxicity.
Herein, a series of scopoletin-cinnamic hybrids (Fig. 2) were synthesized and screened for
cytotoxicity against five human tumor cell line using doxorubicin as a potent antitumor reference.
Then, the most potent cytotoxic compounds 17a, 17b, 17c and 17g were assayed for their
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anti-proliferative activity indicated by IC50 values. 17b was selected for further pharmacological
studies in A549 human lung cancer cell line to more fully elucidate the compound’ mechanisms of
action and anti-tumor activity in mice with lung cancer.
Fig. 2
2. Results and discussion
2.1 Chemistry
The synthetic route of target compounds 7a-f is outlined in Scheme 1. The synthesis of
scopoletin 6 was reported previously [18, 19]. Compound 4 was synthesized in a one-step reaction
from commercially 2, 4, 5-trimethoxybenzaldehyde 3 by treatment with aluminium ( ) chloride in
dry dichloromethane and followed acid hydrolysis. Compound 4 was treated with malonic acid in
pyridine at room temperature (rt) using phenylamine as catalysts affording scopoletin-3-carboxylic
acid 5, which was then decarboxylated by refluxing in pyridine/ethylene glycol mixture to give
scopoletin 6. Cinnamic acid analogs 1a-f were converted to acid chlorides 2a-f in the present of
thionyl chloride using DMF as catalyst. Finally, intermediates 2a-f were treated with scopoletin in
pyridine to obtain target hybrids 7a-f.
Scheme 1.
In the synthesis of 7g and 7h, a benzyl protected group was used as shown in Scheme 2. to
avoid side reactions of the phenolic hydroxyl group of ferulic acid 1h[29]. Benzyl
O-benzylferulate 9 was obtained through the alkylation of 1h with benzylbromide and potassium
carbonate in DMF. The protected O-benzylferulic acid 1g was afforded by the hydrolysis of ester 9
with potassium hydroxide in methanol-water (9:1). The combination of 2g with scopoletin was the
same as that of 7a-f. Removal of the benzyl group was achieved by hydrogenation in chloroform
at room temperature using Pd/C. But the double bond was reduced simultaneously and 7h was
obtained unexpectedly.
Scheme 2.
The synthetic pathway of target compounds 17a-g as shown in Scheme 3. was also started from
4, which was condensed with glycine in acetic anhydride using sodium acetate as base. Burk and
Allen’s one-pot protocol was employed to convert the acetamide 12 to Boc-protected
3-amino-scopoletin 14 [30]. The key intermediate 15 was afforded after removing the Boc-group
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using 15% TFA/CHCl3. The next step involved double acylation of hydroxyl group and amine
group to afford 16a-g without purification, which were selectively hydrolyzed by K2CO3 in
methanol to give the target compounds 17a-g.
Scheme 3.
The chemical structures of all the new compounds were established on the basis of analytical
and spectral data. The mass spectra showed molecular ion peaks [M+H]+ for 7a-h and [M-H]+ for
17a-g. In the IR spectra, the target compounds exhibited the characteristic peak of cinnamic acid
double bond at 1680-1620 cm-1. The two absorption bands in the region 1750-1650 cm-1 revealed
1
the presence of C=O from coumarin and cinnamic acid. In H-NMR spectra, aromatic protons
resonated at δ 6.4 – 7.9 ppm for 7a-h, and δ 6.8 – 8.9 ppm for 17a-g. The presence of -CONH-
group and Ar-OH group of 17a-g was confirmed by two proton singlet between δ 9.6 – 9.9 ppm
and δ 10.0 – 10.7 ppm respectively. The conjugated double bond of cinnamic acid showed two
doublet around δ 7.8 and 6.6 ppm, and the E-isomeric form was confirmed and characterized on
the basis of coupling constant values (J = 13.8 - 17.1 Hz). Cis coupling (J = 9.5 - 9.6 Hz) was
observed between H3 and H4 of 7a-h coumarin skeleton. The coupling was disappeared in
1H-NMR spectra of 17a-g due to the substitution of H3 with -NH-, and the chemical shift of H4
moved to δ 8.7-8.9.
2.2 Pharmacology
2.2.1. In vitro cytotoxic activity of the compounds
All target compounds 7a-h and 17a-g were preliminarily evaluated in vitro for their cytotoxicity
to human breast cancer cells MCF-7 and MDA-MB-231, lung cancer cell line A549, colorectal
cancer cell line HCT-116, cervical cancer cells HeLa, and human umbilical vein endothelial cell
(HUVEC) respectively by using the standard MTT method adopting doxorubicin as a positive
control. All data were given in Table 1. The results showed that several compounds exhibited
strong growth inhibitory activities against the tested cells, such as 17a, 17b, 17c and 17g, which
were comparable to doxorubicin.
Table 1.
Compounds 17a, 17b, 17c and 17g were next assayed for their anti-proliferative activity against
A549, MDA-MB-231 and HCT-116 indicated by IC50 values, which were summarized in Table 2.
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It was found that these compounds displayed significant enhanced anti-proliferative activity
compared to scopoletin and some were even more potent than doxorubicin. Especially, compound
17b displayed the strongest anti-proliferative activity against most human cancer lines with IC50
values ranging from 0.249~0.684 µM. Thus, compound 17b was regarded as the promising
compound and selected for further study.
Table 2.
2.2.2 Effects of 17b on the cell cycle and apoptosis in A549 cells
To understand the mechanism of 17b induced proliferation inhibition, we examined the effect of
17b on cell cycle progression and apoptosis by flow cytometric analysis. As shown in Fig. 3A, 3C,
the cells treated with 17b significantly increased population in S phase. For example, the cells in
the S phase increased by 9.2%, 19.6% and 19.3% at 0.1 ꢀM, 1 ꢀM and 10 ꢀM of 17b, repectively.
At the same time, the percentage of cells in G0/G1 phase was notably reduced, compared with the
control groups, the percentage of cells in G2/M phase was not significantly different. However,
after treatment of A549 cells with 17b at 0.1 ꢀM, 1 ꢀM and 10 ꢀM for 24 h, the apoptotic cell rate
was 2.7%, 3.7% and 22.3%, respectively, as shown in Fig. 3B, 3D. These results suggested that
growth inhibition of the A549 cells proliferation by 17b may be related to induction of S phase
arrest, and induction of apoptosis.
Fig. 3
2.2.3 In vivo antitumor activity of 17b
Given the potent anti-tumor activities of 17b in vitro, we next evaluated whether 17b could
inhibit tumor growth on the tumor-bearing mice. 17b was delivered intraperitoneally every day for
14 days with doses of 2.5, 5 and 10 mg/kg. Positive control doxorubicin and negative control
vehicle were delivered in the same way. As shown in Fig. 4, compound 17b significantly
suppressed tumor growth. And 17b dose-dependently suppressed tumor growth by 18.1, 33.6 and
49.5% at increasing doses of 2.5, 5 and 10 mg/kg, respectively. During the course of the
anti-tumor evaluation, the mean body weight variation of mice was as same as the negative control.
On the other hand, doxorubicin caused 52.5% tumor growth regression, but significantly
decreased the mean body weight of mice at dose of 2.5 mg/kg, indicating 17b was safer. Therefore,
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these results further supported that 17b has an effective therapy for lung cancer.
Fig. 4
3. Conclusions
Starting from the commercially available 2, 4, 5-trimethoxybenzaldehyde, a practical synthesis
of a series of novel scopoletin-cinnamic acid hybrids as well as the biological evaluation have
been accomplished. The present study demonstrated that 3-cinnamoylaminoscopoletin hybrids 17a,
17b, 17c and 17g could markedly inhibit the proliferation of cancer cell lines (MCF-7,
MDA-MB-231, A549, HCT-116, and HeLa). Among them, compound 17b displayed a stronger
anti-proliferative activity than doxorubicin in vitro with IC50 values of 0.249 µM on A549 cells,
0.231 µM on MDA-MB-231 cells, respectively. Gratifyingly, in vivo study showed that 17b
significantly dose-dependently suppressed tumor growth without causing the decreasing of the
mean body weight of mice which was superior to doxorubicin. Further mechanism study
suggested that compound 17b arrested the cell cycle at the S phase and induced apoptosis. These
results showed that compound 17b is an effective therapy agent for lung cancer. Given the
anti-tumor potential observed for the hybrids of cinnamic-scopoletin derivatives in this study, a
more comprehensive SAR (structure-activity relationship) exploration would be warranted. In
addition, the determination of the PK (pharmacokinetic)/PD (pharmacodynamic) properties of
compound 17b and the further mechanistic studies of its anti-tumor effect are currently under way
in our laboratories.
4. Experimental section
4.1. Chemistry
Unless noted, all solvents and reagents were freshly distilled or purified according to standard
procedures. Melting points were taken on X-4 micro melting point apparatus without correction.
Mass spectra were recorded on electrospray ionization (ESI) techniques. High resolution mass
spectra (HRMS) were measured on an Agilent Q-TOF 6520. Compounds were visualized under
1
UV lamp (254 nm and 365nm). H-NMR and 13C-NMR spectra were obtained on a Bruker
AV-300 MHz NMR spectrometer at ambient temperature. H-NMR spectra were reported in ppm
on the δ scale and referenced to the internal tetramethylsilane. The data were presented as follows:
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chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br =
broad, app = apparent), coupling constant (s) in Hertz (Hz), and integration. Chemical shifts (δ)
were recorded relative to residual DMSO-d6 (δ = 2.50 in 1H-NMR and δ = 35.2 in 13C-NMR). The
reactions were monitored by thin lay chromatography (TLC) on pre-coated silica GF 254 plates.
The purities of all new compounds were more than 96%, which was confirmed by HPLC.
4.2. General experimental procedure for the synthesis of compounds 7a-g
1.2 equiv. of cinnamic acid derivatives 1a-h was dissolved in 5 equiv. of SOCl2 and a catalytic
amount of DMF was added. The reaction mixture was refluxed for 4 h and then solvent was
evaporated under vacuum to get the product 2a-h in the form of solid residue in quantitative yield.
The solid residue was directly added partially to an ice-cold stirred solution of 1 equiv of
scopoletin in dry pyridine (200 mg/mL). After the addition, the mixture was warmed to room
temperature and stirred for 2 h. Triple water was added to the reaction mixture. The solid particles
was filtered off and recrystallized from ethanol to give 7a-g.
4.2.1. 6-Methoxy-2-oxo-2H-chromen-7-yl cinnamate (7a)
Light yellow powder. Yield 69.8%, mp 173-175℃; ESI-MS (m/z): 323 [M+H]+; IR (KBr, cm-1):
3128 (CꢁC—H), 1736, 1717(CꢁO)ꢂ1635(CꢁC); 1H-NMR (CDCl3, 300 MHz) δ: 3.89 (3H, s,
OCH3), 6.42 (1H, d, J = 9.60 Hz, H3), 6.96 (1H, d, J = 16.2 Hz, CH), 7.00 (1H, s, Ar-H8), 7.18
(1H, s, Ar-H5), 7.26-7.62 (5H, m, Ar-H), 7.68 (1H, d, J = 9.60 Hz, H4), 7.90 (1H, d, J = 16.2 Hz,
Ar-H). 13C NMR (DMSO-d6, 75 MHz) δ: 164.4, 160.3, 148.4, 148.1, 147.6, 144.2, 142.6, 134.1,
131.5, 129.4, 129.2, 117.4, 116.8, 116.4, 112.0, 111.0, 56.8. HRMS (ESI) calcd. for C19H15O5
[M+H]+, 323.0914; found, 323.0917.
4.2.2. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(4-chlorophenyl)acrylate (7b)
Light yellow powder. Yield 59.3%, mp 173-175℃; ESI-MS (m/z): 357 [M+H]+; IR (KBr, cm-1):
3127 (CꢁC—H), 1739, 1719 (CꢁO)ꢂ1638(CꢁC); 1H NMR (CDCl3, 300 MHz) δ: 3.89 (3H, s,
OCH3), 6.43 (1H, d, J = 9.60 Hz, H3), 6.64 (1H, d, J = 15.9 Hz, CH), 7.00 (1H, s, Ar-H8), 7.17
(1H, s, Ar-H5), 7.40 (2H, dd, J = 8.46 Hz, J = 1.77 Hz, Ar-H), 7.53 (2H, dd, J = 8.46 Hz, J = 1.77
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Hz, Ar-H), 7.68 (1H, d, J = 9.60 Hz, H4 ), 7.85 (1H, d, J = 15.9 Hz, CH). 13C NMR (DMSO-d6, 75
MHz) δ: 164.2, 160.3, 148.4, 146.2, 144.2, 142.6, 133.1, 130.9, 129.5, 117.6, 117.4, 112.0, 111.0,
56.8. HRMS (ESI) calcd. for C19H14ClO5 [M+H]+, 357.0524; found, 357.0530.
4.2.3. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(4-fluorophenyl)acrylate (7c)
White powder. Yield 59.3%. mp > 250℃; ESI-MS (m/z): 341 [M+H]+; IR (KBr, cm-1): 3127 (C
1
ꢁC—H), 1727, 1714 (CꢁO), 1630 (CꢁC); H NMR (CDCl3, 300 MHz ) δ: 3.89 ( 3H, s,
OCH3 ) , 6.43 (1H, d, J = 9.54 Hz, H3), 6.61 (1H, d, J = 16.05 Hz, CH ),7.00 (1H, s, Ar-H8 ) ,7.17
(1H, s, Ar-H5 ), 7.10 (2H, dd, J = 8.61 Hz, J = 2.47 Hz, Ar-H), 7.60 (2H, dd, J = 8.61 Hz , J =
2.47 Hz, Ar-H), 7.68 (1H, d, J = 9.54 Hz, H4), 7.85 (1H, d, J = 16.05 Hz, CH). HRMS (ESI) calcd.
for C22H13O4 [M+H]+, 341.0808; found, 323.0818.
4.2.4. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(4-methoxyphenyl)acrylate (7d)
Light yellow powder. Yield 68.1%, mp 190-192℃; ESI-MS (m/z): 353 [M+H]+; IR (KBr, cm-1):
3122 (CꢁC—H), 1729, 1710 (CꢁO)ꢂ1629(CꢁC); 1H NMR (CDCl3, 300 MHz) δ: 3.88 (3H, s,
OCH3), 3.89 (3H, s, OCH3), 6.42 (1H, d, J = 9.51 Hz, H3), 6.54 (1H, d, J = 13.9 Hz, CH), 6.99
(1H, s, Ar-H8), 7.17 (1H, s, Ar-H5), 7.46 (2H, dd, J = 8.47Hz, J = 1.76 Hz), 7.66 (2H, dd, J = 8.47
Hz, J = 1.76 Hz), 7.68 (1H, d, J = 9.51 Hz, H4), 7.85 (1H, d, J = 13.9 Hz); 13C NMR (DMSO-d6,
75 MHz) δ: 164.6, 162.1, 160.4, 148.5, 148.1, 147.4, 144.2, 142.8, 131.1, 126.8, 117.3, 116.3,
114.9, 113.9, 112.1, 111.0, 56.78, 55.86. HRMS (ESI) calcd. for C20H17O6 [M+H]+, 353.1020;
found, 353.1021.
4.2.5. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(3,4,5-trimethoxyphenyl)acrylate (7e)
Light yellow powder. Yield 56.9%, mp > 250℃; ESI-MS (m/z): 413 [M+H]+; IR (KBr, cm-1):
3108 (CꢁC—H), 1742, 1722 (CꢁO)ꢂ1630(CꢁC); 1H NMR (CDCl3, 300 MHz) δ: 3.89 (3H, s,
OCH3 ), 3.91 ( 9H, m, 3 × OCH3), 6.43 (1H, d, J = 9.54 Hz, H3), 6.59 (1H, d, J = 15.9 Hz, CH),
6.60 (2H, s, Ar-H), 7.00 (1H, s, Ar-H), 7.18 ( 1H, s, Ar-H), 7.10 ( 2H, dd, J = 8.61 Hz , J = 2.47
Hz), 7.68 (1H, d, J = 9.54 Hz, H4), 7.85 (1H, d, J = 15.9 Hz, CH). HRMS (ESI) calcd. for
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C22H21O8 [M+H]+, 413.1231; found, 413.1231.
4.2.6. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(4-cyanophenyl)acrylate (7f)
Yellow powder. Yield 71.4%, mp > 250℃; ESI-MS (m/z): 348 [M+H]+; IR (KBr, cm-1): 3125 (C
1
ꢁC—H), 1724, 1684 (CꢁO)ꢂ1635(CꢁC), 2227 (CꢃN); H NMR (CDCl3, 300 MHz) δ: 3.89
(3H, s, OCH3 ), 6.43 (1H, d, J = 9.58 Hz, H3), 6.61 (1H, d, J = 17.05 Hz, CH),7.00 (1H, s, Ar-H8 ),
7.17 (1H, s, Ar-H5), 7.15 (2H, dd, J = 8.81 Hz, J = 2.49 Hz), 7.80 (2H, dd, J = 8.81 Hz, J = 2.49
Hz), 7.68 (1H, d, J = 9.58 Hz, H4), 7.85 (1H, d, J = 17.05 Hz, CH). HRMS (ESI) calcd. for
C20H17O6K [M+K]+, 386.0431; found, 386.0431.
4.2.7. (E)-6-Methoxy-2-oxo-2H-chromen-7-yl 3-(4-(benzyloxy)-3-methoxyphenyl) acrylate (7g)
Light yellow powder. Yield 51.6%; mp 201-203℃; ESI-MS (m/z): 459 [M+H]+; IR (KBr, cm-1):
3380ꢂ3124 (CꢁC—H), 1757, 1715 (CꢁO)ꢂ1605(CꢁC); 1H NMR (CDCl3, 300 MHz) δ: 3.88
(3H, s, OCH3), 3.94 (3H, s, OCH3), 5.22 (2H, s, CH2), 6.43 (1H, d, J = 9.54 Hz, H3), 6.59 (1H, d,
J = 15.9 Hz, CH), 6.99 (1H, s, Ar-H), 7.10 (1H, d, J = 2.54 Hz, Ar-H), 7.13 (1H, d, J = 9.54 Hz,
Ar-H), 7.17 (1H, s, Ar-H), 7.10 (2H, dd, J = 8.61 Hz, J = 2.47 Hz), 7.68 (1H, d, J = 9.54 Hz, H4),
7.85 (1H, d, J = 15.9 Hz, CH). HRMS (ESI) calcd. for C27H23O7 [M+H]+, 459.1438; found,
459.1439.
4.2.8. 6-methoxy-2-oxo-2H-chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl) propanoate (7h)
A solution of 7g (155 mg, 0.34 mmol) in chloroform (50mL) was hydrogenated over 10% Pd/C
was removed by filtration through Celite. The filtrate was evaporated and the residue was
crystallized from chloroform-ethyl acetate to give 7h (110mg, 92%). Light yellow powder. Yield
51.6%; mp 201-203℃; ESI-MS(m/z): 371 [M+H]+; IR (KBr, cm-1): 3376 (OH), 3125 (CꢁC—H),
1757, 1714 (CꢁO)ꢂ1604(CꢁC); 1H NMR (CDCl3, 300 MHz) δ: 2.88 (2H, t, CH2 ), 3.02 (2H, t,
CH2), 3.82 (3H, s, OCH3), 3.89 (3H, s, OCH3), 5.51 (1H, s, OH), 6.43 (1H, d, J = 9.54 Hz, H3),
6.78 (1H, d, J = 5.7 Hz, Ar-H), 6.89 (1H, d, J = 5.7 Hz, Ar-H ), 6.95 (1H, s, Ar-H), 7.03 (1H, s,
Ar-H), 7.64 (1H, d, J = 9.54 Hz, H4). HRMS (ESI) calcd. for C20H19O7 [M+H]+, 371.1125; found,
371.1128.
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4.3. Experimental procedure for the synthesis 12
A mixture of 4 (8.41g, 50.0mmol), glycine (4.50g,60mmol), anhydrous sodium acetate (16.4g,
200.0mmol) and acetic anhydride (25.0mL) was heated at 120-125ꢄ for 3.5h. The reaction
solution was cooled to room temperature. The yellow solid was filtered out and taken up with
20ml of ice-cold water. The mass was broken up with a spatula and the solid was suction filtered
and washed 3-4 times with cold water. The resulting solid was dried under vacuum for overnight
to afford key intermediate 12 as a light yellow solid (7.25g, 49.8%); mp 245-247℃; ESI-MS (m/z):
207.26 [M+H] +; 1H NMR (CDCl, 300 MHz) δ: 2.06 (3H, s, CH3), 2.18 (3H, s, CH3), 3.81 (3H, s,
OCH3), 6.84 (1H, s, H5), 6.93 (1H, s, H8), 7.99 (1H, s, H4), 8.58 (1H, s, NH). 13C NMR
(DMSO-d6, 75MHz) δ: 171.8, 170.1, 158.7, 147.3, 145.8, 143.0, 122.7, 117.1, 112.23, 110.7, 106.5,
57.8, 24.3, 21.9.
4.4. Experimental procedure for the synthesis of key intermediate 15
To a solution of intermediate 12 (14.33g, 49.2mmol) and DMAP (1.30g, 10.0mmol) in THF
(200mL), Di-tert-butyl dicarbonate was added and the mixture was magnetically stirred at room
temperature. After 30min, hydrazine hydrate (8.0mL, 257mmol) and methanol (200mL) were
added to the mixture. The system was stirred at room temperature for 4 hours and then poured into
dichloromethane (300mL) which was washed with 1M aqueous HCl solution, 1 M aqueous CuSO4
solution, saturated aqueous NaHCO3 solution and brine, respectively. The resulting organic layer
was dried over anhydrous sodium sulfate, concentrated under reduced pressure to yield crude
product of intermediate 14 which used without purification. A solution of 15% TFA/chloroform
(400mL) was added to the residue. The mixture was stirred at room temperature for 4h followed
by concentrated under reduced pressure. The residue was diluted with water (50mL) and the
insoluble substance was filtered out. To make the filtrate basic, sodium bicarbonate was added
followed by stirring for 1hour. The precipitate came out which was filtered as yellow solid and
further washed with water. The crude product was purified by recrystallization from ethanol to
afford 15 (3.26, combined yield of one-pot reaction 32.0%). mp = 251-253℃; ESI-MS (m/z): 206
[M-H]-; IR (KBr, cm-1): 3368 (OH), 3409 (NH), 3317 (OH), 3127 (CꢁC—H), 1712 (CꢁO),
10 / 16

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1
1635 (CꢁC); H NMR (CDCl3, 300 MHz) δ: 3.92 (3H, s, OCH3), 6.64 (1H, s, H8),6.69 (1H, s,
H5), 6.89 (1H, s, H4)
4.5. General experimental procedure for the synthesis of compounds 17a-g
To an ice-cold stirred solution of 1 equiv. of compound 15 in dry pyridine (200 mg/mL), 2a-g
was directly added partially. After the addition, the mixture was warmed to room temperature and
stirred for 2 h. Triple water was added to the reaction mixture. The solid particles was filtered and
recrystallized from ethanol to give 16a-g. To a suspension of 16a-g in methanol (200 mg/6 mL),
1.5 equiv. of K2CO3 was added and the mixture was refluxed for 1 h. The reaction mixture was
evaporated to dryness. The residue was dissolved in water (10 mL/100 mg) and insoluble
substance was filtered off. After acidification of filtrate with 1N hydrochloric acid, the product
was filtered off and recrystallized from ethanol to give target compound 17a-g.
4.5.1. N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)cinnamamide (17a)
Yellow powder. Yield 94.5%, mp > 250℃; ESI-MS (m/z): 336 [M-H]-; IR (KBr, cm-1): 3368
(OH), 3302(N—H), 3120, 3024(CꢁC—H), 1707, 1667(CꢁO)ꢂ1624(CꢁC); 1H NMR
(DMSO-d6, 300 MHz) δ: 3.83 (3H, s, OCH3), 6.81 (1H, s, H8), 7.27 (1H, d, J = 13.9 Hz, CH),
7.34-7.45 (3H, m, Ar-H), 7.50 (1H, s, H5 ), 7.60-7.62 (2H, m, Ar-H), 7.64 (1H, d, J = 13.9 Hz,
CH), 8.72 (1H, s, H4), 9.7 (1H, s, NH), 10.15 (1H, s, OH). HRMS (ESI) calcd. for C19H16NO5
[M+H]+, 338.1023; found, 338.1025.
4.5.2. (E)-3-(4-Chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)acrylamide (17b)
Yellow powder. Yield 81.4%, mp > 250℃; ESI-MS(m/z): 370 [M-H]-; IR (KBr, cm-1): 3523
1
(OH), 33342(N—H), 3120(CꢁC—H), 1708, 1677(CꢁO)ꢂ1622(CꢁC). H NMR (DMSO-d6,
300 MHz) δ: 3.83 (3H, s, OCH3), 6.81 (1H, s, H8), 7.28 (1H, d, J = 13.8 Hz, CH), 7.35 (2H, d, J =
8.1 Hz, Ar-H), 7.51 (1H, s, H5), 7.53 (2H, d, J = 8.1 Hz, Ar-H), 7.64 (1H, d, J = 13.8 Hz, CH),
8.73 (1H, s, H4), 9.76 (1H, s, NH), 10.10 (1H, s, OH ). HRMS (ESI) calcd. for C19H15ClNO5
[M+H]+, 372.0633; found, 372.0632.
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4.5.3. (E)-3-(4-Fluorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (17c)
Yellow powder. Yield 75.2%, mp > 250℃; ESI-MS (m/z): 354[M-H]-; IR (KBr, cm-1): 3368
(OH), 3331(N—H), 3144(CꢁC—H), 1710, 1673(CꢁO)ꢂ1623(CꢁC); 1H NMR (DMSO-d6, 300
MHz) δ: 3.83 (3H, s, OCH3), 6.81 (1H, s, H8), 7.23 (1H, d, J = 13.8 Hz, CH), 7.32 (2H, d, J = 8.1
Hz, Ar-H), 7.49 (1H, s, H5), 7.54 (2H, d, J = 8.1 Hz, Ar-H), 7.67 (1H, d, J = 13.8 Hz, CH), 8.86
(1H, s, H4), 9.84 (1H, s, NH), 10.17 (1H, s, OH). HRMS (ESI) calcd. for C19H15FNO5 [M+H]+,
356.0929; found, 356.0927.
4.5.4. (E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(4-(trifluoromethyl)phenyl)acryl-
amide (17d).
Yellow powder. Yield 65.2%, mp > 250℃; ESI-MS (m/z): 404 [M-H]-; IR (KBr, cm-1): 3514
(OH), 3327(N—H), 3113, 3024(CꢁC—H), 1703, 1675(CꢁO)ꢂ1635(CꢁC); 1H NMR
(DMSO-d6, 300 MHz) δ: 3.84 (3H, s, OCH3) , 6.81 (1H, s, H8), 7.28 (1H, d, J = 13.8 Hz, CH),
7.41 (2H, d, J = 8.1 Hz, Ar-H), 7.48 (1H, s, H5), 7.62 (2H, d, J = 8.1 Hz, Ar-H ), 7.67 (1H, d, J =
13.8 Hz, CH), 8.74 (1H, s, H4), 9.88 (1H, s, NH), 10.10 (1H, s, OH). HRMS (ESI) calcd. for
C20H15F3NO5 [M+H]+, 406.0897; found, 356.0896.
4.5.5. (E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(3,4,5-trimethoxyphenyl)acrylami
-de (17e)
Light yellow powder. Yield 85.8%, mp > 250℃; ESI-MS (m/z): 426 [M-H]-, IR (KBr, cm-1):
3375 (OH), 3142(N—H), 3100(CꢁC—H), 1704, 1664(CꢁO)ꢂ1624(CꢁC); 1H NMR (DMSO-d6,
300 MHz) δ: 3.70 (3H, s, OCH3), 3.83 (9H, s, OCH3), 6.81 (1H, s, H8), 6.99 (1H, s, H5), 7.28 (1H,
d, J = 13.8 Hz, CH), 7.31 (2H, d, J = 8.1 Hz, Ar-H), 7.48 (2H, d, J = 8.1 Hz, Ar-H), 7.53 (1H, d, J
= 13.8 Hz, CH), 8.73 (1H, s, H4), 9.62 (1H, s, NH), 10.08 (1H, s, OH); 13C NMR (DMSO-d6, 75
MHz) δ: 165.1, 158.4, 153.5, 149.7, 146.2, 145.6, 141.1, 130.8, 125.5, 122.0, 121.8, 111.4, 109.8,
105.7, 103.0, 60.5, 56.5, 56.7. HRMS (ESI) calcd. for C22H22NO8 [M+H]+, 428.1340; found,
428.1347.
4.5.6. (E)-3-(4-Cyanophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)acrylamide (17f)
12 / 16

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Light yellow powder. Yield 87.3%, mp > 250℃; ESI-MS (m/z): 361 [M-H]-; IR (KBr, cm-1):
1
3322(N—H), 3120(CꢁC—H), 1704, 1673(CꢁO)ꢂ1667, 1623(CꢁC), 2226 (CN); H NMR
(DMSO-d6, 300 MHz) δ: 3.84 (3H, s, OCH3), 6.81 (1H, s, H8), 7.28 (1H, d, J = 13.8 Hz, CH),
7.45 (2H, d, J = 8.1 Hz, Ar-H), 7.48 (1H, s, H5), 7.82 (2H, d, J = 8.1 Hz, Ar-H), 7.92 (1H, d, J =
13.8 Hz, CH), 8.74 (1H, s, H4), 9.80 (1H, s, NH), 10.10 (1H, s, OH). HRMS (ESI) calcd. for
C20H15N2O5 [M+H]+, 363.0975; found, 363.0978.
4.5.7.(E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(4-methoxyphenyl)acrylamide
(17g)
Yellow powder. Yield 73.6%, mp 196-198℃; ESI-MS (m/z): 366 [M-H]-; IR (KBr, cm-1): 3407
(OH), 3331(N—H), 3123(CꢁC—H), 1704, 1665(CꢁO)ꢂ1625(CꢁC); 1H NMR (DMSO-d6, 300
MHz) δ: 3.81 (3H, s, OCH3), 3.85 (3H, m, OCH3), 6.81 (1H, s, H8), 7.18 (1H, d, J = 13.8 Hz, CH),
7.02 (1H, d, J = 8.1 Hz, Ar-H), 7.28 (1H, s, H5 ), 7.50 ( 2H, d, J = 8.1 Hz, Ar-H), 7.59 (1H, d, J =
13.8 Hz, CH), 8.72 (1H, s, H4), 9.64 (1H, s, NH), 10.07 (1H, s, OH). HRMS (ESI) calcd. for
C20H18NO6 [M+H]+, 368.1129; found, 368.1126.
4.6. Cytotoxicity assay
Cytotoxicity was assessed using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium
bromide (MTT) assay. Cells were planted on 96-well plates with 5000 per well and incubated
overnight and then exposed to drug at different concentrations for 72 h. Then, the MTT solution (5
mg/mL) was added (20 ꢀL/well), and cells were further incubated for 4 h, and the absorbance was
recorded at 570 nm (EL800, BIO-TEK Instruments Inc.). 50% cell viability inhibition (IC50) was
calculated by the logit method. Each concentration was repeated at least three times.
4.7. Flow cytometry cell cycle analysis
Cells were planted at 3 × 105 per well in 6-well plate and incubated overnight and then
exposed to drug at different concentrations. Then cells were harvested and washed in cold PBS,
and fixed in cold 70% alcohol at least 24 h. After that, DNA was treated with RNase A solution
13 / 16

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and stained with propidium iodide (50 ꢀg/mL) at room temperature before analysis. Flow
cytometry analysis was done by FACSCalibur flow cytometry (Becton–Dickinson).
4.8. Quantification of apoptosis
Cells were planted at 3×105 per well in 6-well plate and incubated overnight and then exposed
to drug at different concentrations. Then cells were harvested and washed in cold PBS. and stained
with the Annexin V/PI Cell Apoptosis Detection Kit according to the manufacturer’s instructions.
Flow cytometry analysis was done by FACSCalibur flow cytometry (Becton–Dickinson).
4.9. In vivo therapeutic activity
Female C57BL/6 mice with body weight of 18–22 g were transplanted with lung lewis cancer
cells in oxter. The xenograft model was generated by subcutaneous injection of 3 × 106 lung lewis
cancer cells into the right frank of mouse. The experiment was performed after the tumor cells
were inoculated for 7 to 21 days. The mice were divided randomly into five groups (with 10 mice
/ group): saline control group, 17b 2.5 mg/kg group, 17b 5 mg/kg group, 17b 10 mg/kg group and
doxorubicin 2.5 mg/kg group. All groups were administered intraperitoneally every day. And body
weights were recorded every day from the first treatment. 14 days later, all mice were killed and
the tumor was segregated, weighed and stored at -80°C.
Acknowledgments
This work was supported by the Fundamental Research Funds for the Central
Universities (JKZ2011016) and Qing Lan Project of Jiangsu Province.
References
[1] R.D.H. Murray, J. Mendez, S.A. Brown, John Wiley & Son Ltd, New York, 1982, P. 21.
[2] M.E. Riveiro, A. Moglioni, R. Vazquez, N. Gomez, G. Facorro, L. Piehl, E.R. de Celis, C.
Shayo and C. Davio, Bioorg. Med. Chem. 16 (2008) 2665-2675.
[3] M.E. Riveiro, R. Vazquez, A. Moglioni, N. Gomez,A. Baldi, C. Davio, C. Shayo, Biochem.
Pharmacol. 75 (2008) 725-736.
[4] R. Vázquez, M.E. Riveiro, M. Vermeulen, E. Alonso, C. Mondillo, G. Facorro, L. Piehl, N. Gó
14 / 16

ACCEPTED MANUSCRIPT
mez, A. Moglioni, N. Fernández, A. Baldi, C. Shayo, C. Davio, Bioorg. Med. Chem. 20 (2012)
5537-554.
[5] Rajesh NG, Sharad GJ. J Exp Clin Med, 4(2012) 165-169.
[6] S. Serra, A.Chicca, G. Delogu, S. Vázquez-Rodríguez, L. Santana, E. Uriarte, L. Casu, J.
Gertsch, Bioorg. Med. Chem. Lett. 22 (2012) 5791-5794.
[7] A.A. AI-Amiery, R.I.H. AI-Bayati, K.Y. Saour, M.F. Radi, Res. Chem. Intermed. 38 (2012)
559-569.
[8] A. Maresca, C. Temperini, L. Pochet, B. Masereel, A. Scozzafava, C.T. Supuran, J. Med. Chem.
53 (2010) 335-344.
[9] A. Goel, A.K. Prasad, V.S. Parmar, B. Ghosh, N. Saini, FEBS. Lett. 581 (2007) 2447-2454.
[10] A.Goel, A.K. P, V.S. Parmar, B. Ghosh, N. Saini, Chem-Biol. Interact. 179 (2009) 363-374.
[11] E.Y. Bissonnette, G.M. Tremblay, V. Turmel, B. Pirotte, M. Reboud-Ravaux, Int.
Immunopharmacol. 9 (2009) 49-54.
[12] Y. Kashman, K.R. Gustafson, R.W. Fuller, J.H. Cardellina, J.B. McMahon, M.J. Currens, R.W.
Buckheit, S.H. Hughes, G.M. Cragg, M.R. Boyd, J. Med. Chem. 35 (1992) 2735-2743.
[13] R. Pan, X.H. Gao, D. Lu, X.X. Xu, Y.F. Xia, Y. Dai, Int. Immunopharmacol. 11 (2011)
2007-2016.
[14] X.L. Liu, L. Zhang, X.L. Fu, K. Chen, B.C. Qian, Acta. Pharmacol. Sin. 22 (2001) 929-933.
[15] E.K. Kim, K.B. Kwon, B.C. Shin, E.A. Seo, Y.R. Lee, J.S. Kim, J.W. Park, B.H. Park, D.G.
Ryu, Life. Sci. 77 (2005) 824-836.
[16] Y. Li, Y. Dai, M. Liu, R. Pan, Y.B. Luo, Y.F Xia, X.F. Xia, Drug. Dev. Res. 70 (2009)
378-385.
[17] R. Pan, Y. Dai, X.H. Gao, D. Lu, Y.F. Xia, Vasc.Pharmacol. 54 (2011) 18-28.
[18] J.P. Zhou, L. Wang, L.J. Wei, Y. Zheng, H.B. Zhang, Y.B. Wang, P. Cao, A. Niu, J. Wang and
Y. Dai, Lett. Drug. Des. Discov. 9 (2012) 397-401.
[19] W.K. Liu, J. Hua, J.P. Zhou, H.B. Zhang, H.Y. Zhu, Y.H. Cheng, R. Gust, Bioorg. Med. Chem.
Lett. 22 (2012) 5008-5012.
[20] S.S. Bhattacharyya, S.K. Mandal, R. Biswas, S. Paul, S. Pathak, N. Boujedaini, P. Belon, A.R.
Khuda-Bukhsh, Exp. Biol. Med. 233 (2008) 1591-1601.
[21] S.S. Bhattacharyya, S. Paul, S.K. Mandal, A. Banerjee, N. Boujedaini, A.R. Khuda-Bukhsh,
15 / 16

ACCEPTED MANUSCRIPT
Eur. J. Pharmacol. 614 (2009) 128-136.
[22] L. Liu, W.R. Hudqins, S. Shack, M.Q. Yin, D. Samid, Int. J. Cancer. 62 (1995) 345-350.
[23] J.A. Hoskins, J. Appl. Toxicol. 4 (1984) 283-292.
[24] G.C. Yen, Y.L. Chen, F.M. Sun, Y.L. Chiang, S.H. Lu, C.J. Weng, Eur. J. Pharm. Sci. 44 (2011)
281-287.
[25] E.L. de Oliveira Niero, G.M. Machado-Santelli, J. Exp. Clin. Cancer. Res. 32 (2013) 31.
[26] P. De, M. Baltas, F. Bedos-Belval, Curr. Med. Chem. 18 (2011) 1672-1703.
[27] W.F. Fong, X.L. Shen, C. Globisch, M. Wiese, G.Y. Chen, G.Y. Zhu, Z.L. Yu, A.K.W. Tse, Y.J.
Hu, Bioorg. Med. Chem. 16 (2008) 3694-3703.
[28] T. Zhou, Q. Shi, K.F. Bastow, K.H. Lee, J. Med. Chem. 53 (2010) 8700-8708.
[29] A. Leschot, R.A. Tapia, J. Eyzaguirre. Synthetic. Commun. 32 (2002) 3219-3223.
[30] M.J. Burk, J.G. Allen, J. Org. Chem. 62 (1997) 7054-7057.
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Captions:
Table 1. Growth inhibition (%) obtained from the single dose (10-5 M, 72 h) test
Table 2. IC50 values of compounds 17a, 17b, 17c and 17g as determind based on
MTT assay
Fig. 1 Structures of some natural and synthetic cinnamic and scopoletin derivatives
Fig. 2 Design of scopoletin-substituted cinnamic acid hybrids
Fig. 3 17b induces S phase cell cycle arrest and apoptosis in A549 cancer cells. A and
C, Effects of 17b on cell cycle were investigated using PI (Propidium Iodide) staining,
Cells were treated with (0, 0.01, 0.1, 1 µM) 17b for 24 h, collected and stained with
PI; B and D, Effects of 17b on cell apoptosis were investigated using PI and Annexin
V-FITC staining, A549 cells were treated with (0, 0.01, 0.1, 1 µM, A-D) 17b for 24 h,
collected and stained with PI and Annexin V-FITC in darkness for 15 min and
analyzed by flow cytometry.
Fig.4 Anti-tumor activity of 17b in mice, LLC tumor-bearing mice were
intraperitoneally administered for 14 days with normal saline, 17b (10, 5, 2.5 mg/kg),
DOX (2.5 mg/kg). (A) the tumor mode; (B) the tumor weight; (C) body weight. Data
represent means ± SEM from three independent experiments performed induplicate. *
P < 0.05, ** P < 0.01.
Scheme 1. The synthesis of substituted cinnamoyl-7-O-scopoletin analogs 7a-f.
Reaction conditions and reagents: (i) AlCl3, CH2Cl2, concentrated HCl; (ii) malonic
acid, pyridine, phenylamine; (iii) pyridine, reflux; (iv) thionyl chloride, DMF.(v)
Pyridine, ice bath to rt.
Scheme 2. The synthesis of 7g and 7h. Reaction conditions and reagents: (i) PhCH2Br,
K2CO3, DMF; (ii) KOH, MeOH-H2O (9: 1); (iii) Benzene, SOCl2, reflux; (iv)
pyridine, scopoletin; (v) CHCl3, H2, 10% Pd-C, rt, 10 h.
Scheme 3. The synthesis of substituted cinnamoyl-3-NH2-scopoletin analogs 17a-g.
Reaction conditions and reagents: (i) AlCl3, CH2Cl2; (ii) glycine, Ac2O, AcONa,
reflux; (iii) Boc2O, THF, DMAP, reflux; (iv) Hydrazine hydrate, MeOH, rt, 4h; (v)
trifluoroacetic acid; (vi) pyridine; (vii) MeOH, K2CO3, concentrated HCl.

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Table 1. Growth inhibition (%) obtained from the single dose (10-5 M, 72 h) test
Growth Inhibition (%)
Compounds
MCF-7
MDA-MB-231
A549
HCT-116
Hela
Scopoletin
2.63
3.65
8.47
10.1
4.23
19.43
2.73
1.36
12.2
0.98
19.35
22.39
3.89
7a
22.83
1.77
20.31
19.28
14.26
11.81
1.39
21.15
17.99
13.6
7b
21.42
17.91
20.59
10.39
6.16
7c
10.51
18.02
9.91
13.22
0.93
7f
18.89
4.36
7e
7g
2.36
8.55
9.73
16.13
31.6
15.37
19.48
58.32
86.15
67.12
19.03
9.45
7h
13.71
6.06
20.79
44.52
70.85
69.17
24.56
12.94
13.12
61.44
89.34
0.53
7d
65.72
87.92
85.60
26.42
4.87
60.34
64.34
54.24
16.97
7.11
17a
70.11
58.40
12.63
18.34
27.31
44.81
84.90
17b
17c
17d
17e
36.92
88.61
92.03
14.08
75.67
79.01
27.1
17f
60.92
82.01
17g
Doxorubicin

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Table 2. IC50 values of compounds 17a, 17b, 17c and 17g as determind based on MTT assay
IC50 (µM
)
Compounds
A549
>30
MDA-MB-231
>30
HCT-116
>30
Scopoletin
17a
2.178
0.249
0.612
0.649
1.280
0.369
1.152
0.684
5.543
0.505
0.231
17b
2.346
17c
17g
0.578
Doxorubicin
1.546
0.477

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O
HO
O
O
Scopoletin
HO
HO
O
O
OH
O
OH
O
HO
OH
O
HO
OH
O
O
Cinnamic acid
Caffeic acid
Ferulic acid
Sinapic acid
O
O
AcHN
O
O
O
H
N
H
N
O
O
R
O
O
R
O
Scopoletin derivatives
Fig. 1 Structures of some natural and synthetic cinnamic and scopoletin derivatives

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O
O
R1
O
O
O
O
HO
O
O
R2
R3
Scopoletin
R1
R2
R3
O
H
N
O
OH
R
O
HO
O
O
Cinnamic acid
a. R1 = H, R2 = H, R3 = H
b. R1 = H, R2 = Cl, R3 = H
c. R1 = H, R2 = F, R3 = H
d. R1 = H, R2 = OMe, R3 = H
e. R1 = OMe, R2 = OMe, R3 = OMe
f. R1 = H, R2 = CN, R3 = H
Fig. 2 Design of scopoletin-substituted cinnamic acid hybrids

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Fig. 3 17b induces S phase cell cycle arrest and apoptosis in A549 cancer cells. A and C, Effects of 17b on cell
cycle were investigated using PI (Propidium Iodide) staining, Cells were treated with (0, 0.01, 0.1, 1 µM) 17b for
24 h, collected and stained with PI; B and D, Effects of 17b on cell apoptosis were investigated using PI and
Annexin V-FITC staining, A549 cells were treated with (0, 0.01, 0.1, 1 µM, A-D) 17b for 24 h, collected and
stained with PI and Annexin V-FITC in darkness for 15 min and analyzed by flow cytometry.

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Fig.4 Anti-tumor activity of 17b in mice, LLC tumor-bearing mice were intraperitoneally administered for 14 days
with normal saline, 17b (10, 5, 2.5 mg/kg), DOX (2.5 mg/kg). (A) the tumor mode; (B) the tumor weight; (C) body
weight. Data represent means ± SEM from three independent experiments performed induplicate. * P < 0.05, ** P
< 0.01.

ACCEPTED MANUSCRIPT
O
O
R3
R3
R2
a. R1 = H, R2 = H, R3 = H
b. R1 = H, R2 = Cl, R3 = H
c. R1 = H, R2 = F, R3 = H
d. R1 = H, R2 = OMe, R3 = H
e. R1 = OMe, R2 = OMe, R3 = OMe
f. R1 = H, R2 = CN, R3 = H
Cl
OH
iv
R2
R1
R1
1a-f
2a-f
CHO
OH
H3CO
H3CO
CHO
H3CO
HO
COOH
O
H3CO
HO
ii
iii
i
OCH3
O
3
5
4
OCH3
O
OCH3
OH
2,
v
O
O
R3
R1
O
O
O
7a-f
6
R2
Scheme 1. The synthesis of substituted cinnamoyl-7-O-scopoletin analogs 7a-f. Reaction conditions and reagents:
(i) AlCl3, CH2Cl2, concentrated HCl; (ii) malonic acid, pyridine, phenylamine; (iii) pyridine, reflux; (iv) thionyl
chloride, DMF.(v) Pyridine, ice bath to rt.

ACCEPTED MANUSCRIPT
O
O
O
O
OH
OBn
OH
Cl
O
O
O
O
i
iii
ii
HO
BnO
BnO
BnO
1h
2g
9
1g
OCH3
O
OCH3
O
iv
v
O
O
O
O
OMe
OBn
OMe
OH
O
O
7h
7g
Scheme 2. The synthesis of 7g and 7h. Reaction conditions and reagents: (i) PhCH2Br, K2CO3, DMF; (ii) KOH,
MeOH-H2O (9: 1); (iii) Benzene, SOCl2, reflux; (iv) pyridine, scopoletin; (v) CHCl3, H2, 10% Pd-C, rt, 10 h.

ACCEPTED MANUSCRIPT
CHO
CHO
OCH3
AcHN
OCH3
OAc
OH
iii
i
ii
H3CO
O
O
H3CO
OCH3
12
OH
3
4
Boc
Boc
AcN
OCH3
OAc
H2N
O
OCH3
OH
iv
HN
OCH3
OH
v
vi
O
O
O
O
O
13
15
14
R3
R1
R2
R2
R1
R3
O
vii
O
O
O
O
O
O
R2
R1
N
HN
O
H
O
O
OH
17a-g
16a-g
R3
a. R1 = H, R2 = H, R3 = H
b. R1 = H, R2 = Cl, R3 = H
c. R1 = H, R2 = F, R3 = H
d. R1 = H, R2 = CF3, R3 = H
e. R1 = OMe, R2 = OMe, R3 = OMe
f. R1 = H, R2 = CN, R3 = H
g. R1 = H, R2 = OMe, R3 = H
Scheme 3. The synthesis of substituted cinnamoyl-3-NH2-scopoletin analogs 17a-g. Reaction conditions and
reagents: (i) AlCl3, CH2Cl2; (ii) glycine, Ac2O, AcONa, reflux; (iii) Boc2O, THF, DMAP, reflux; (iv) Hydrazine
hydrate, MeOH, rt, 4h; (v) trifluoroacetic acid; (vi) pyridine; (vii) MeOH, K2CO3, concentrated HCl.

ACCEPTED MANUSCRIPT
Highlights:
ꢀ
ꢀ
ꢀ
A series of novel scopoletin-cinnamic acid hybrids have been synthesized.
Most of the hybrids showed improved antitumor activity than parent compound.
The most potent compound 17b was able to cause S cell cycle arrest and cell
apoptosis at A549 cells.
ꢀ
Compound 17b showed superior activity to that of the positive doxorubicin in
vivo.

ACCEPTED MANUSCRIPT
Compound 7a 1H-NMR
Compound 7a IR (KBr, cm-1)