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

DOI:

10.1016/j.ejmech.2015.01.040

Reference:

EJMECH 7656

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphic abstract

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

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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

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most potent compound 17b showed potent activity in vivo and vitro by causing cell cycle arrest

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and induced apoptosis in A549 cell lines.

*Correspondence: Li Chen: E-mail [email protected] Phone/Fax, +86-25-83202334

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Yufeng Xia: E-mail [email protected]

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

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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.

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Department of Chinese Materia Medica Analysis, China Pharmaceutical University, 24 Tong Jia Xiang,

† These two authors contributed equally to this work

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Nanjing 210009, People’s Republic of China.

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*Corresponding authors: E-mail [email protected]; Tel/Fax: +86-25-83202334 (L. Chen). E-mail [email protected] (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,

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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

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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

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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 1 / 16

ACCEPTED MANUSCRIPT 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

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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

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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

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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

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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

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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

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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 2 / 16

ACCEPTED MANUSCRIPT 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.

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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

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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 (Ⅲ) chloride in

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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

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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

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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

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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 3 / 16

ACCEPTED MANUSCRIPT 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.

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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

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the presence of C=O from coumarin and cinnamic acid. In 1H-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-

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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 H-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.

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2.2 Pharmacology

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2.2.1. In vitro cytotoxic activity of the compounds

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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. 4 / 16

ACCEPTED MANUSCRIPT 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

Table 2.

2.2.2 Effects of 17b on the cell cycle and apoptosis in A549 cells

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compound and selected for further study.

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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,

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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 µM, 1 µM and 10 µM 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 µM, 1 µM and 10 µM for 24 h, the apoptotic cell rate

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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.

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Fig. 3

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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, 5 / 16

ACCEPTED MANUSCRIPT these results further supported that 17b has an effective therapy for lung cancer. Fig. 4

3. Conclusions

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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,

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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,

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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

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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

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compound 17b and the further mechanistic studies of its anti-tumor effect are currently under way

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in our laboratories.

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 UV lamp (254 nm and 365nm). 1H-NMR and

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C-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: 6 / 16

ACCEPTED MANUSCRIPT 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.

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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

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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.

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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.

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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 (C C—H), 1736, 1717(C O) 1635(C C); 1H-NMR (CDCl3, 300 MHz) δ: 3.89 (3H, s,

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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,

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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 (C C—H), 1739, 1719 (C O) 1638(C C); 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 7 / 16

ACCEPTED MANUSCRIPT 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,

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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 C—H), 1727, 1714 (C O), 1630 (C C); 1H NMR (CDCl3, 300 MHz ) δ: 3.89 ( 3H, s,

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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 =

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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):

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3122 (C C—H), 1729, 1710 (C O) 1629(C C); 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

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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,

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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 (C C—H), 1742, 1722 (C O) 1630(C C); 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 8 / 16

ACCEPTED MANUSCRIPT 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

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C—H), 1724, 1684 (C O) 1635(C C), 2227 (C N); 1H 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

C20H17O6K [M+K]+, 386.0431; found, 386.0431.

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Hz), 7.68 (1H, d, J = 9.58 Hz, H4), 7.85 (1H, d, J = 17.05 Hz, CH). HRMS (ESI) calcd. for

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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): 3380 3124 (C C—H), 1757, 1715 (C O) 1605(C C); 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,

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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,

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459.1439.

4.2.8. 6-methoxy-2-oxo-2H-chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl) propanoate (7h)

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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 (C C—H), 1757, 1714 (C O) 1604(C C); 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. 9 / 16

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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 120-125

for 3.5h. The reaction

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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):

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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).

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C NMR

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(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

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(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

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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

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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 (C C—H), 1712 (C O), 10 / 16

ACCEPTED MANUSCRIPT 1635 (C C); 1H 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

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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),

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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

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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 C — H), 1707, 1667(C

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(OH), 3302(N — H), 3120, 3024(C

O)

1624(C

C); 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,

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CH), 8.72 (1H, s, H4), 9.7 (1H, s, NH), 10.15 (1H, s, OH). HRMS (ESI) calcd. for C19H16NO5

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[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 (OH), 33342(N—H), 3120(C C—H), 1708, 1677(C O) 1622(C C). 1H 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.

11 / 16

ACCEPTED MANUSCRIPT 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(C C—H), 1710, 1673(C O) 1623(C C); 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

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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.

(E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(4-(trifluoromethyl)phenyl)acryl-

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4.5.4.

amide (17d).

(OH), 3327(N — H), 3113, 3024(C

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Yellow powder. Yield 65.2%, mp > 250℃; ESI-MS (m/z): 404 [M-H]-; IR (KBr, cm-1): 3514 C — H), 1703, 1675(C

O)

1635(C

C); 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

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C20H15F3NO5 [M+H]+, 406.0897; found, 356.0896.

-de (17e)

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4.5.5. (E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(3,4,5-trimethoxyphenyl)acrylami

Light yellow powder. Yield 85.8%, mp > 250℃; ESI-MS (m/z): 426 [M-H]-, IR (KBr, cm-1):

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3375 (OH), 3142(N—H), 3100(C C—H), 1704, 1664(C O) 1624(C C); 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

ACCEPTED MANUSCRIPT Light yellow powder. Yield 87.3%, mp > 250℃; ESI-MS (m/z): 361 [M-H]-; IR (KBr, cm-1): 3322(N—H), 3120(C C—H), 1704, 1673(C O) 1667, 1623(C C), 2226 (CN); 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), 7.45 (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 =

C20H15N2O5 [M+H]+, 363.0975; found, 363.0978.

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13.8 Hz, CH), 8.74 (1H, s, H4), 9.80 (1H, s, NH), 10.10 (1H, s, OH). HRMS (ESI) calcd. for

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4.5.7.(E)-N-(7-Hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl)-3-(4-methoxyphenyl)acrylamide (17g)

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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(C C—H), 1704, 1665(C O) 1625(C C); 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

4.6. Cytotoxicity assay

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C20H18NO6 [M+H]+, 368.1129; found, 368.1126.

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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

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overnight and then exposed to drug at different concentrations for 72 h. Then, the MTT solution (5 mg/mL) was added (20 µL/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

ACCEPTED MANUSCRIPT and stained with propidium iodide (50 µg/mL) at room temperature before analysis. Flow cytometry analysis was done by FACSCalibur flow cytometry (Becton–Dickinson).

4.8. Quantification of apoptosis

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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.

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Flow cytometry analysis was done by FACSCalibur flow cytometry (Becton–Dickinson).

4.9. In vivo therapeutic activity

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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

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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

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the tumor was segregated, weighed and stored at -80°C.

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central

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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.

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[8] A. Maresca, C. Temperini, L. Pochet, B. Masereel, A. Scozzafava, C.T. Supuran, J. Med. Chem.

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53 (2010) 335-344.

[9] A. Goel, A.K. Prasad, V.S. Parmar, B. Ghosh, N. Saini, FEBS. Lett. 581 (2007) 2447-2454.

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[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.

2007-2016.

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[13] R. Pan, X.H. Gao, D. Lu, X.X. Xu, Y.F. Xia, Y. Dai, Int. Immunopharmacol. 11 (2011)

[14] X.L. Liu, L. Zhang, X.L. Fu, K. Chen, B.C. Qian, Acta. Pharmacol. Sin. 22 (2001) 929-933.

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[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.

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[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)

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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.

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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.

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[29] A. Leschot, R.A. Tapia, J. Eyzaguirre. Synthetic. Commun. 32 (2002) 3219-3223.

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[30] M.J. Burk, J.G. Allen, J. Org. Chem. 62 (1997) 7054-7057.

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ACCEPTED MANUSCRIPT

Captions:

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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.

ACCEPTED MANUSCRIPT Table 1. Growth inhibition (%) obtained from the single dose (10-5 M, 72 h) test Growth Inhibition (%) Compounds MDA-MB-231

A549

HCT-116

Hela

Scopoletin

2.63

8.47

4.23

1.36

0.98

7a

3.65

10.1

19.43

7b

22.83

20.31

2.73

7c

1.77

19.28

21.42

7f

10.51

14.26

17.91

7e

18.02

11.81

20.59

18.89

0.93

7g

9.91

1.39

10.39

4.36

2.36

7h

8.55

9.73

6.16

16.13

15.37

7d

13.71

20.79

0.53

31.6

19.48

17a

6.06

44.52

65.72

60.34

58.32

17b

70.11

70.85

87.92

64.34

86.15

17c

58.40

69.17

85.60

54.24

67.12

17d

12.63

24.56

26.42

16.97

19.03

17e

18.34

12.94

4.87

7.11

9.45

17f

27.31

13.12

36.92

14.08

27.1

44.81

61.44

88.61

75.67

60.92

84.90

89.34

92.03

79.01

82.01

12.2

19.35

21.15

22.39

17.99

3.89

13.6

13.22

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Doxorubicin

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17g

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MCF-7

ACCEPTED MANUSCRIPT Table 2. IC50 values of compounds 17a, 17b, 17c and 17g as determind based on MTT assay

IC50 (µM ) Compounds MDA-MB-231

HCT-116

Scopoletin

>30

>30

>30

17a

2.178

0.369

1.152

17b

0.249

0.231

0.684

17c

0.612

2.346

5.543

17g

0.649

Doxorubicin

1.280

SC 0.578

0.505

1.546

0.477

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A549

ACCEPTED MANUSCRIPT O HO

O

O

Scopoletin

HO HO

OH

O

O

HO

OH

O

Cinnamic acid

O

Caff eic acid

Ferulic acid

AcHN

H N

O O

R

O

O O

O

O

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Scopoletin derivatives

O

H N

O

SC

O

OH

Sinapic acid

O O

HO

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OH

O

R

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Fig. 1 Structures of some natural and synthetic cinnamic and scopoletin derivatives

ACCEPTED MANUSCRIPT O O HO

R1 O

O

O

R2

R1 R2

O H N

HO

Cinnamic acid

O

O

R3 O

RI PT

O

OH

R

O

O

R3

Scopoletin

O

a. R1 = H, R 2 = H, R 3 = H b. R1 = H, R 2 = Cl, R 3 = H c. R 1 = H, R 2 = F, R 3 = H d. R1 = H, R 2 = OMe, R 3 = H e. R1 = OMe, R 2 = OMe, R3 = OMe f. R 1 = H, R 2 = CN, R 3 = H

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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

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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

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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

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< 0.01.

ACCEPTED MANUSCRIPT

O OH

R3

iv

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, R 2 = CN, R3 = H

Cl

R2

R2

R1

R1

2a-f

1a-f H 3CO

CHO

H 3CO

i

H 3CO

OCH3

CHO

HO

3

ii

H3CO HO

OH

OCH3

O

OH

O

O

O

O

O O

6 7a-f

R3

SC

O

2, v

iii

5

4

OCH3

COOH

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O R3

R1

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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

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chloride, DMF.(v) Pyridine, ice bath to rt.

ACCEPTED MANUSCRIPT O

O OH

OBn O

i

HO

OH O

ii

BnO

1h

O OMe

O

2g OCH 3

v

O

O

BnO 1g

OCH3 O

Cl O

iii

BnO 9

iv

O

OBn 7g

O

O

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O

O

OMe

O 7h

OH

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Scheme 2. The synthesis of 7g and 7h. Reaction conditions and reagents: (i) PhCH2Br, K2CO3, DMF; (ii) KOH,

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TE D

M AN U

MeOH-H2O (9: 1); (iii) Benzene, SOCl2, reflux; (iv) pyridine, scopoletin; (v) CHCl3, H2, 10% Pd-C, rt, 10 h.

ACCEPTED MANUSCRIPT CHO

CHO

OCH 3

OH

i

H 3CO

AcHN

OCH3

O

H 3CO

OCH3 Boc

iii

OAc

O 12

OH 4

3

Boc OCH 3 O

iv

HN

OAc

OCH 3

O

O 14

13

v

H 2N O

OH

R3 R2 R2

R1 O

O

O

OH

vii O

R3

O

R2

N H

R1 R3

HN

17a-g

O

O

M AN U

16a-g

SC

O

O

vi

15

R1

O

OCH 3

RI PT

AcN O

ii

a. R1 = H, R2 = H, R3 = H b. R1 = H, R2 = Cl, R3 = H c. R1 = H, R 2 = F, R3 = H d. R1 = H, R2 = CF 3, R3 = H

O OH

e. R1 = OMe, R2 = OMe, R 3 = OMe f. R 1 = H, R 2 = CN, R3 = H g. R1 = H, R2 = OMe, R 3 = 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

AC C

EP

TE D

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

RI PT




apoptosis at A549 cells.

Compound 17b showed superior activity to that of the positive doxorubicin in

EP

TE D

M AN U

SC

vivo.

AC C




M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7a 1H-NMR

Compound 7a IR (KBr, cm-1)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7a ESI-HRMS (m/z)

Compound 7a 13C-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7b ESI-HRMS (m/z)

Compound 7b IR (KBr, cm-1)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7b 1H-NMR

Compound 7b 13C-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7d 1H-NMR

Compound 7d 13C-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7d IR (KBr, cm-1)

Compound 7d ESI-HRMS (m/z)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 7e 1H-NMR

Compound 7e IR (KBr, cm-1)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

EP

TE D

Compound 7e ESI-HRMS (m/z)

O

O

AC C

O

H N

HO

O

O

O

Compound 17e 1H-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 17e IR (KBr, cm-1)

Compound 17e 13C-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 17e ESI-HRMS (m/z)

Compound 17d 1H-NMR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 17d IR (KBr, cm-1)

Compound 17d ESI-HRMS (m/z)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 17g 1H-NMR

Compound 17g IR (KBr, cm-1)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Compound 17g ESI-HRMS (m/z)