Design and synthesis of coumarin-3-acylamino derivatives to scavenge radicals and to protect DNA

Article abstract of DOI:10.1016/j.ejmech.2014.07.009

In this study, a series of coumarin-3-acylamino derivatives containing phenethylamine moiety or tyramine moiety were synthesized and their antioxidant activities were evaluated by Cu²⁺/glutathione(GSH)-, OH- and 2,2′-azobis(2-amidinopropane hyd

Full text of DOI:10.1016/j.ejmech.2014.07.009

Accepted Manuscript
Design and Synthesis of coumarin-3-acylamino derivatives to scavenge radicals and
to protect DNA
Yang Yang, Qing-Wen Liu, Ye Shi, Zhi-Guang Song, Ying-Hua Jin, Zai-Qun Liu
PII:
S0223-5234(14)00615-1
10.1016/j.ejmech.2014.07.009
EJMECH 7129
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 November 2013
Revised Date: 27 June 2014
Accepted Date: 3 July 2014
Please cite this article as: Y. Yang, Q.-W. Liu, Y. Shi, Z.-G. Song , Y.-H. Jin, Z.-Q. Liu, Design and
Synthesis of coumarin-3-acylamino derivatives to scavenge radicals and to protect DNA, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.07.009.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Capacities of coumarin-3-acylamino derivatives to scavenge ABTS+· radical and to protect DNA
against AAPH-induced oxidation.

ACCEPTED MANUSCRIPT
ꢀ Several coumarin-3-acylamino derivatives were synthesized for the first time.
ꢀ Antioxidant abilities of coumarin-3-acylamino derivatives were evaluated.
ꢀ Hydroxyl and ortho-methoxy group can enhance the antioxidant abilities remarkably.
ꢀ
Structure-activity relationships give interesting results.

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Running title: Antioxidant property of coumarin-3-acylamino derivatives
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Design and Synthesis of coumarin-3-acylamino
derivatives to scavenge radicals and to protect DNA
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b
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Yang Yang a, Qing-Wen Liu a, Ye Shi a, Zhi-Guang Song a, , Ying-Hua Jin ,
Zai-Qun Liu a
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a Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China
b Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University,
Changchun 130012, China
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Corresponding author: Zhi-Guang Song
Department of Organic Chemistry, College of Chemistry
Jilin University
No. 2519 Jiefang Road
Changchun 130021
China
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Email: szg@jlu.edu.cn
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Abstract
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In this study, a series of coumarin-3-acylamino derivatives containing phenethylamine moiety or
tyramine moiety were synthesized and their antioxidant activities were evaluated by
Cu2+/glutathione(GSH)-, ꢀOH- and 2,2′-azobis(2-amidinopropane hydrochloride)(AAPH)-induced
oxidation of DNA. It was found that both hydroxyl and ortho-methoxy groups at A ring, hydroxyl
group at B ring and peptide bond can enhance the abilities of coumarin-3-acylamino derivatives to
protect DNA against ꢀOH- and AAPH-induced oxidation. Moreover, these coumarin-3-acylamino
derivatives were employed to scavengeand 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic
radical (ABTS+•). We found that tyramine moiety, hydroxyl and ortho-methoxy are the key groups to
enhance the activities of antioxidants to quench ABTS+•. Therefore, tyramine linked with coumarin-3-
carboxyl acid which containing hydroxyl and ortho-methoxy exhibited powerful antioxidant abilities.
Keywords coumarin-3-acylamino, coumarin, free radical, oxidation, antioxidant, DNA.
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1. Introduction
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Numerously clinic and biochemical evidence has demonstrated that the pathogenesis of many fatal
diseases [1] is related to the free radical-initiated oxidation in vivo. The damages to proteins, lipids, cell
membranes and DNA [2,3] caused by the free radicals from the metabolism [4] or the environment [5]
can lead to aging, cancer, atherosclerosis, and some other serious diseases [6]. The supplementation of
antioxidants is beneficial for avoiding oxidative damages [7], maintaining fitness [8] and curing
diseases [9]. Therefore, lots of research efforts have been put into the development of novel
antioxidants and evaluation of their activities in various experimental systems.
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Over the past few decades, coumarin and its derivatives have attracted considerable attention as
valuable building blocks due to their various biological activities, such as anticancer [10,11],
antioxidant [12,13], anti-inflammatory [14], antimicrobial [15], antiviral [16], and enzymatic inhibitory
[17]. They also are key components of flavoring agent for a diverse set of food, such as soft drinks,
yogurt, muffins etc. [18]. However, it is susceptible that the side effect of coumarin is to induce
hepatotoxicity for a particular group of the population [19]. In clinical trials in the U.S. [20] and Ireland
[21], some of the patients receiving coumarin developed signs of drug-induced liver toxicity. Therefore,
the coumarin derivatives with stronger antioxidant activities, which can be used to treat patient at lower
dose, need to be designed and synthesized to alleviate potential side effects. Our group has recently
reported on the antioxidant activity of courmarin derivatives xanthotoxol, methyl-substituted
xanthotoxol and 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety [22]. In an effort
to further enhance antioxidant activities, 3-carboxylic acid and phenethylamine moieties were
introduced into the coumarin nucleus in this study. Herein, a series of coumarin-3-acylamino
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derivatives containing phenethylamine moiety or tyramine moiety were synthesized (Scheme 1) and
their antioxidant activities were evaluated.
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Scheme 1 is here
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2. Experiment
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2.1. Materials and instrumentation
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Diammonium salt of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), was purchased
from Fluka Chemie GmbH, Buchs, Switzerland. AAPH, the naked DNA sodium salt, and GSH were
purchased from ACROS ORGANICS, Geel, Belgium. Other agents were analytical grade, and
purchased from Beijing Chemical Reagent Co., China. Flash column chromatography was performed
on a silica gel (300–400 mesh) and thin layer chromatography (TLC) inspections were carried out on a
silica gel GF254 plates. The structures of compound 2a-c and 3a-3f were identified by 1H and 13C
NMR (Bruker–400 MHz NMR spectrometer) and high solution mass spectra equipped with ESI as the
ionization mode (Agilent1290-micrOTOF Q II). The synthetic compounds were analyzed by high
performance liquid chromatography, and the purities of these compounds were larger than 98.0%.
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2.2. Synthesis of title compound 2a-c and 3a-3f
2.2.1. General procedure A for the synthesis of coumarin-3-carboxyl acid derivatives
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In a round-bottomed flask the selected salicylaldehyde (1mmol) and Meldrum’s acid (1.2 mmol)
in water (2 ml) were heated at reflux under stirring for 10 h; then, the reaction mixture was cooled and
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filtered on Büchner funnel. The products were purified by recrystallization from methanol [23].
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2.2.1.1. Coumarin-3-carboxyl acid (2a) The reaction was conducted according to the general procedure
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A and the product 2a was obtained as a white solid (0.181 g, 95% yield). m. p. 145-146°C. H NMR
(400 MHz, DMSO-d6): δ (ppm) 8.71(s, 1H, 4-CH), 7.88(d, J = 4Hz, 1H, 5-CH), 7.71(t, J = 8Hz, 1H,
7-CH), 7.41(d, J = 8Hz, 1H, 8-CH), 7.38(t, J = 7.2Hz, 1H, 6-CH). 13C NMR (100 MHz, DMSO-d6): δ
(ppm) 164.42(-COOH), 157.16(2-CO), 154.91(8-CH=COC-2), 148.82(4-CH), 134.71(7-CH),
130.63(5-CH), 125.25(4-CH), 118.76(4-CH-C=CH-5), 118.43(3-CH), 116.57(8-CH). HRMS (ESI)
m/z calad for C10H7O4 [M+H+]: 191.0339, found 191.0387.
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2.2.1.2. 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (2b) The reaction was conducted according
to the general procedure A and the product 2b was obtained as a white solid ( 0.185g, 90% yield ). m. p.
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189–191°C. H NMR (400 MHz, DMSO-d6): δ (ppm) 12.84(s, 1H, –COOH), 11.09(s, 1H, –OH),
8.69(s, 1H, 4-CH), 7.75(d, J = 8Hz, 1H, 5-CH), 6.85(d, J = 8Hz, 1H, 6-CH), 6.74(s, 1H, 8-CH). 13C
NMR (100 MHz, DMSO-d6): δ (ppm) 164.68(-COOH), 164.39(7-COH), 158.07(2-CO),
157.45(8-CH=COC-2),
149.88(4-CH),
132.48(5-CH),
114.47(3-C),
112.90(6-CH),
111.09(4-CH-C=CH-5), 102.26(8-CH). HRMS (ESI) m/z calad for C10H7O5 [M+H+]: 207.0288, found
207.0332.
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2.2.1.3. 7-hydroxy-6-methoxy-2-oxo-2H-chromene-3-carboxylic acid (2c) The reaction was conducted
according to the general procedure A and the product 2c was obtained as a yellow solid (0.176g, 75%
yield). m. p. 199–201°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 12.81(s, 1H, –COOH), 10.95(s, 1H,
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–OH), 8.67(s, 1H, 4-CH), 7.44(s, 1H, 5-CH), 6.82(s, 1H, 8-CH), 3.83(s, 1H, -OCH3). C NMR (100
MHz, DMSO-d6): δ (ppm) 164.71(-COOH), 158.68(2-CO), 154.76(8-CH=COC-2), 152.03(6-C-OCH3),
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149.80(7-COH), 146.26(4-CH), 112.74(5-CH), 111.23(3-C), 110.43(4-CH-C=CH-5), 102.81(8-CH),
56.52(-OCH3). HRMS (ESI) m/z calad for C11H9O6 [M+H+]: 237.0394, found 237.0443.
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2.2.2. General procedure B for the synthesis of coumarin-3-acylamino derivative
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To a solution of coumarin-3-carboxyl acid derivatives 2 (1mM) in dry N,N-dimethylformamide
(DMF) was added dicyclohexylcarbodiimide (DCC, 1.1mM) as the dehydrant and
N,N-dimethylaminopyridine (DMAP, 5mg) as the catalyst. The mixture was added
N,N-diisopropylethylamine (DIPEA, 0.5mM) and phenethylamine or tyramine(1.1mM) after stirred for
10 min, then heated at 50°C for 8h. After the reaction mixture was cooled, the precipitates were
filtrated, the solution was extracted by CH3COOC2H5 and water, combined organic layers and dried
over Na2SO4 and evaporated to afford crude compound 3. The crude product 3 was further purified by
flash column chromatography with corresponding eluent.
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2.2.2.1. 2-oxo-N-phenethyl-2H-chromene-3-carboxamide (3a) The reaction was conducted according to
the general procedure B, the product was further purified by flash column chromatography with
chloroform and ethyl acetate (10:1, v:v) as eluent. Compound 3a was obtained as a white solid (0.237g,
81% yield). m. p. 175–177°C. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.91(s, 1H, –CONH), 8.88(s, 1H,
4-CH), 7.70-7.64(m, 2H, 5-CH, 7-CH), 7.38-7.26(m, 7H, 6-CH, 8-CH, 5H of B ring), 3.73(s, 2H,
-NHCH2CH2), 2.95(s, 2H, -NHCH2CH2). 13C NMR (100 MHz, CDCl3): δ (ppm) 156.22(-CONH),
156.13(2-CO), 149.16(8-CH=COC-2), 143.01(1-C of B ring), 133.56(4-CH), 128.76(7-CH),
124.57(6-CH), 123.56(2-CH of B ring), 123.40(3-CH of B ring), 121.32(4-CH of B ring),
120.03(5-CH),
113.42(4-CH-C=CH-5),
113.24(3-C),
111.38(8-CH),
36.13(-NHCH2CH2),
30.43(-NHCH2CH2). HRMS (ESI) m/z calad for C18H16NO3 [M+H+]: 294.1125, found 294.1211.
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2.2.2.2. N-(4-hydroxyphenethyl)-2-oxo-2H-chromene-3-carboxamide (3b) The reaction was conducted
according to the general procedure B, the product was further purified by flash column chromatography
with chloroform and ethyl acetate (5:1, v:v) as eluent. Compound 3b was obtained as a white solid
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(0.24g, 78% yield). m. p. 189–190°C. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.90(s, 2H, –CONH,
4-CH), 7.68(s, 2H, 5-CH, 7-CH), 7.39(s, 2H, 6-CH, 8-CH), 7.12(d, J = 4Hz, 2H, 2-CH of B ring),
6.80(d, J = 4Hz, 2H, 3-CH of B ring), 5.47(s, 1H, 4-COH of B ring), 3.68(d, J = 4Hz, 2H,
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-NHCH2CH2), 2.87(s, 2H, -NHCH2CH2). C NMR (100 MHz, DMSO-d6): δ (ppm) 161.39(-CONH),
160.88(2-CO), 156.21(4-COH of B ring), 154.33(8-CH=COC-2), 147.96(1-C of B ring), 134.55(4-CH),
130.75(7-CH), 130.02(2-CH of
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ring), 129.63(6-CH), 125.61(5-CH), 119.31(3-C),
ring), 41.52(-NHCH2CH2),
118.95(4-CH-C=CH-5), 116.60(8-CH), 115.68(3-CH of
B
34.64(-NHCH2CH2). HRMS (ESI) m/z calad for C18H16NO4 [M+H+]: 310.1074, found 310.1137.
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2.2.2.3. 7-hydroxy-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (3c) The reaction was conducted
according to the general procedure B, the product was further purified by flash column chromatography
with chloroform and ethyl acetate (5:1, v:v) as eluent. Compound 3c was obtained as a yellow white
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solid (0.21g, 68% yield). m. p. 201–202°C. H NMR (400 MHz, DMSO-d6): δ (ppm) 11.05(s, 1H,
-OH), 8.79(s, 1H, –CONH), 8.70(s, 1H, 4-CH), 7.82(d, J = 6Hz, 1H, 5-CH), 7.31-7.22(m, 5H, 5H of B
ring), 6.89(d, J = 8Hz, 1H, 6-CH), 6.80(s, 1H, 8-CH), 3.56(d, J = 4Hz, 2H, -NHCH2CH2), 2.84(s, 2H,
-NHCH2CH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 164.11(-CONH), 161.93(2-CO),
161.49(7-COH), 156.73(8-CH=COC-2), 148.53(1-C of B ring), 139.71(4-CH), 132.46(5-CH),
129.13(2-CH of B ring), 128.87(3-CH of B ring), 126.68(4-CH of B ring), 114.81(6-CH), 114.01(3-C),
111.60(4-CH-C=CH-5), 102.26(8-CH), 41.08(-NHCH2CH2), 35.56(-NHCH2CH2). HRMS (ESI) m/z
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calad for C18H16NO4 [M+H+]: 310.1074, found 310.1132.
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2.2.2.4. 7-hydroxy-N-(4-hydroxyphenethyl)-2-oxo-2H-chromene-3-carboxamide (3d) The reaction was
conducted according to the general procedure B, the product was further purified by flash column
chromatography with chloroform and methanol (20:1, v:v) as eluent. Compound 3d was obtained as a
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yellow solid (0.23g, 73% yield). m. p. 210–212°C. H NMR (400 MHz, DMSO-d6): δ (ppm) 10.91(s,
1H, 7-COH), 9.19(s, 1H, 4-COH of B ring), 8.79(s, 1H, –CONH), 8,67(s, 1H, 4-CH), 7.82(s, 1H,
5-CH), 7.05(s, 2H, 2-CH of B ring), 6.89(s, 1H, 6-CH), 6.80(s, 1H, 8-CH), 6.70(s, 2H, 3-CH of B ring),
3.50(s, 2H, -NHCH2CH2), 2.72(s, 2H, -NHCH2CH2). 13C NMR (100 MHz, DMSO-d6): δ (ppm)
163.62(-CONH), 161.38(2-CO), 161.01(7-COH), 156.22(8-CH=COC-2), 155.68(4-COH of B ring),
147.99(4-CH), 131.95(5-CH), 129.51(2-CH of B ring), 129.19(1-CH of B ring), 115.17(3-C),
114.32(3-CH of B ring), 113.51(6-CH), 111.09(4-CH-C=CH-5), 101.75(8-CH), 40.91(-NHCH2CH2),
34.21(-NHCH2CH2). HRMS (ESI) m/z calad for C18H16NO5 [M+H+]: 326.1023, found 326.1094.
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2.2.2.5. 7-hydroxy-6-methoxy-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (3e) The reaction was
conducted according to the general procedure B, the product was further purified by flash column
chromatography with chloroform and methanol (20:1, v:v) as eluent. Compound 3e was obtained as a
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yellow solid (0.28g, 82% yield). m. p. 199–201°C. H NMR (400 MHz, DMSO-d6): δ (ppm) 10.87(s,
1H, OH), 8.79(s, 1H, -CONH), 8.76(t, J = 4Hz, 1H, 4-CH), 7.52(s, 1H, 5-CH), 7.33-7.20(m, 5H, 5H of
B ring), 6.87(s, 1H, 8-CH), 3.85(s, 3H, -OCH3), 3.57(dd, J = 6.4Hz, J = 6.8Hz, 2H, -NHCH2CH2),
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2.85(t, J = 4Hz, 2H, -NHCH2CH2). C NMR (100 MHz, DMSO-d6): δ (ppm) 162.03(-CONH),
161.69(2-CO), 154.39(7-COH), 151.26(8-CH=COC-2), 148.42(6-C-OCH3), 146.49(1-CH of B ring),
139.73(4-CH), 129.14(2-CH of B ring), 128.88(3-CH of B ring), 126.68(4-CH of B ring), 113.94(3-C),
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111.22(5-CH), 110.98(4-CH-C=CH-5), 102.76(8-CH), 56.58(-OCH3), 41.07(-NHCH2CH2),
35.59(-NHCH2CH2). HRMS (ESI) m/z calad for C19H18NO5 [M+H+]:340.1179, found 340.1245.
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2.2.2.6. 7-hydroxy-N-(4-hydroxyphenethyl)-6-methoxy-2-oxo-2H-chromene-3-carboxamide (3f) The
reaction was conducted according to the general procedure B, the product was further purified by flash
column chromatography with chloroform and methanol (10:1, v:v) as eluent. Compound 3f was
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obtained as a yellow solid (0.23g, 65% yield). m. p. 217–219°C. H NMR (400 MHz, DMSO-d6): δ
(ppm) 10.87(s, 1H, 7-COH), 9.18(s, 1H, 4-COH of B ring), 8.78(s, 1H, -CONH), 8.71(s, 1H, 4-CH),
7.51(s, 1H, 5-CH), 7.04(d, J=7.2Hz, 2H, 2-CH of B ring), 6.86(s, 1H, 8-CH), 6.69(d, J=7.6Hz, 2H,
3-CH of B ring), 3.84(s, 1H, -OCH3), 3.49(d, J=5.6Hz, 2H, -NHCH2CH2), 2.71(s, 2H, -NHCH2CH2).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 161.47(-CONH), 161.20(2-CO), 155.68(4-COH of B ring),
153.91(7-COH), 150.75(8-CH=COC-2), 147.90(6-C-OCH3), 145,99(4-CH), 129.51(2-CH of B ring),
129.20(1-CH of B ring), 115.16(3-CH of B ring), 113.42(3-C), 110.68(5-CH), 110.46(4-CH-C=CH-5),
102.24(8-CH), 56.06(-OCH3), 40.89(-NHCH2CH2), 34.24(-NHCH2CH2). HRMS (ESI) m/z calad for
C19H18NO6 [M+H+]: 356.1129, found 356.1202.
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2.3. Cu2+/GSH-induced oxidation of DNA test
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Cu2+/GSH-induced oxidation of DNA was carried out following a previous report [24] with a
slight modification. DNA , CuSO4, and GSH were dissolved in phosphate buffered solution (PBS1: 6.1
mM, Na2HPO4, 3.9 mM, NaH2PO4), and coumarin-3-acylamino derivatives were dissolved in dimethyl
sulfoxide (DMSO). Then, 2.0 mg/mL DNA , 5.0 mM Cu2+, 3.0 mM GSH, and 150 µM
coumarin-3-acylamino derivatives were mixed to form a solution. 2.0 mL of the solution was transfered
into individual. The test tubes were incubated at 37°C to initiate the oxidation of DNA , and three of
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them were taken out at every 30 min and cooled immediately. PBS1 solution of EDTA (1.0 mL, 30.0
mM) was added to chelate Cu2+, followed by adding 1.0 mL of thiobarbituric acid (TBA) solution (1.00
g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS1) and 1.0 mL of 3.0% trichloroacetic acid
aqueous solution. The test tubes were heated in boiling water for 30 min and cooled to room
temperature; 1.5 mL of n-butanol was added and shaken vigorously to extract thiobarbituric acid
reactive substance (TBARS) whose absorbance was measured at 535 nm.
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2.4. ·OH-induced oxidation of DNA test
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·OH was generated by mixing H2O2 with tetra-chlorohydroquinone (TCHQ, dissolved in DMSO
as the stock solution) as described in literature [25]. DNA and H2O2 were dissolved in phosphate
buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 10.0 mM EDTA), DNA (2.0 mg/mL),
4.0 mM TCHQ, 2.0 mM H2O2, and 150 µM coumarin-3-acylamino derivatives (dissolved in DMSO as
the stock solution) were mixed to form a solution. 2.0 mL of the solution was transfered into individual
tubes. The test tubes were incubated at 37°C for 30 min and cooled immediately. The following
operation was the same as in Cu/GSH-induced oxidation of DNA except EDTA was not added. The
absorbance in the control experiment and in the presence of coumarin-3-acylamino derivatives was
assigned as A0 and Adetect, respectively. The effects of coumarin-3-acylamino derivatives
on ·OH-induced oxidation of DNA were expressed by Adetect/A0 × 100.
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2.5. AAPH-induced oxidation of DNA test
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The experiment of AAPH-induced oxidation of DNA was performed as described in the literature
[26]. DNA and AAPH were dissolved in phosphate buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9
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mM NaH2PO4, 10.0 mM EDTA) with the final concentrations at 2.0 mg/mL and 4.0 mM, respectively.
Various concentrations coumarin-3-acylamino derivatives 2a-c and 3a-f in DMSO were added to the
above mixture. The solution was poured into test tubes as 2.0 ml aliquots. The test tubes were
incubated in a water bath at 37°C to initiate the oxidation of DNA . Three tubes were taken out at every
120 min and cooled immediately. The following operation was the same as in ·OH-induced oxidation
of DNA except the heating period was 15 min after TBA and trichloroacetic acid were added. The
absorbance of TBARS was plotted versus the incubation period.
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2.6. Scavenging ABTS+•
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ABTS+• was produced from 2.0 mL of a mixture containing 4.0 mM ABTS aqueous solution and
1.41 mM K2S2O8 after kept for 16 h and diluted by 100 mL of ethanol. The absorbance of ABTS+•
solution was around 1.00 at 734 nm ( εABTS+• =1.6×104 M−1cm−1 ) [27]. The DMSO solutions of
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coumarin-3-acylamino derivatives were added to 1.9 mL of ABTS+• with the final concentrations of
coumarin-3-acylamino derivatives being 20 µ µM. The
M and concentration of ABTS+• being 64.88
decreases of the absorbance of this radical were recorded at 25°C with a certain time interval.
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2.7. Statistical analysis
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All the data were the average value from at least three independent measurements with the
experimental error within 10%. The equations were analyzed by one-way ANOVA in Origin 8
professional software, and p < 0.001 indicated a significant difference.
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3. Results and discussion
3.1. Chemistry
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Coumarin-3-carboxylic acids are traditionally prepared via Knoevenagel condensation of
ortho-hydroxyaryl aldehydes with malonic acid, malonic ester, or cyanoacetic ester [28]. In this paper,
a simple, efficient and environmentally friendly procedure was used for the reaction of Meldrum’s acid
with salicylaldehydes as described in the literature [23]. Coumarin-3-carboxylic acids 2a-c were
synthesized with high yields (75–95%) and excellent selectivities (95–98%) by carrying out the
reaction in water at reflux for 10 h, without any catalyst. Then, the reaction mixture was cooled and
filtered on Büchner funnel. The products were purified by recrystallization from methanol. Finally,
coumarin-3-acylamino derivatives 3a-f were obtained by DCC condensation in the presence of
coumarin-3-carboxylic acids and phenethylamine or tyramine following the literature [29]. Further
purification by silica chromatography was performed when necessary.
226
227
3.2. Antioxidant profiling
3.2.1. Effects of compounds 2a-c and 3a-f on AAPH-induced oxidation of DNA
228
229
230
231
232
233
234
235
A C-centered radical can be generated at a stable rate from the decomposition of AAPH at 37°C,
and combined with oxygen to form peroxyl radical (ROO·) rapidly. The peroxyl radical (ROO·) is able
to abstract H atom from the C-4′ atom of DNA, resulting in strand breaks and further generating more
than 20 carbonyl species called thiobarbituric acid reactive substance (TBARS) which has a feature
absorbance at 535 nm (λmax) [26]. Hence, the in-vitro assays of the inhibitory activities of synthesized
compounds on DNA oxidation can be realized by monitoring the absorbance change of TBARS when
the compounds are added in DNA solution. As predicted, the addition of various concentrations of
compounds 2a-c and 3a-f can significantly change the absorbance of TBARS as shown in Fig. 1.
12

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236
Figure 1 is here
237
238
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245
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249
250
251
252
253
254
The line for 0 µM (blank experiment) in Fig. 1 shows a continuous increase of the absorbance in
the blank experiment indicating that more TBARS were formed in AAPH-induced oxidation of DNA
with the increase of incubation period of time. Same trend of the absorbance change was observed with
the addition of various concentrations of compounds 2a and 3a. By comparing compounds 2a and 3a
with compounds 2b and 3b respectively, it can be found that the hydroxyl group attaching to A ring
enhanced their antioxident ability to protect DNA against AAPH-induced oxidation. In particular, as
shown in Fig. 1, the addition of compounds 3c and 3d retarded the amount of TBARS formed in
AAPH-induced oxidation of DNA, but they cannot generate tinh even at high concentration. Moreover,
the addition of various concentrations of compounds 2b, 2c, 3b, 3d, and 3f can efficiently stop the
increase of the absorbance of TBARS at early stage of the incubation, but the absorbance increased in
the same trend as the blank experiment afterwards. This period was designated as the inhibition period
(tinh), implying their ability to inhibit the oxidation of DNA, and was prolonged with the increase of the
concentrations of these five compounds. As shown in Fig. 1, the tinh can be measured from the cross
point of two tangents being related to the inhibition and oxidation periods. The relationship between tinh
and the concentration of compounds is illustrated in Fig. 2. The quantitative equations between tinh and
the concentration of compounds 2b, 2c, 3b, 3d, and 3f were listed in Table 1. The Eq. (1) was used to
evaluate the antioxidant efficacy through the linear correlation between tinh and the concentration of the
antioxidant [30].
255
256
tinh = (n/ Ri)[antioxidant]
(1)
Figure 2 is here
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265
From the dynamic point of view, the n refers to the stoichiometric factor, expressing the number
of the radical propagation terminated by one molecule of the antioxidant, and Ri is the initiation rate of
the radical-induced reaction. This equation has been employed in the study of the protection of DNA
against AAPH-induced oxidation by homoisoflavonoids [31,32]. Ri is equal to the generation rate (Rg)
from the decomposition of AAPH (Rg = (1.4 ± 0.2) × 10–6 [AAPH]s–1 [30] ). This is because both
sodium salt of DNA and AAPH are dissolved in the same water phase, and radicals generated from
AAPH attack DNA in the water phase. Thus, the n values of 2b, 2c, 3b, 3d, and 3f are the products of
coefficients in the corresponding equation multiplied by Ri = Rg = 1. 4 × 10–6 × 40 mM/s= 3.36
µM/min and listed in Table 1 as well.
266
Table 1 is here
267
268
269
270
271
272
273
274
275
276
277
From Table 1, it can be seen that the order of the inhibitory effect of assayed compounds 2b, 2c,
3b, 3d, and 3f on the oxidation of DNA is 3f > 2c > 3e > 3b > 2b. Obviously, the n value of compound
2c (9.81) is much higher than that of 2b (2.75), which indicates that the ortho-methoxy group on A ring
could enhance the antioxidant activity to inhibit the oxidation of DNA. The structural difference
between 3f and 3e is the substitute of R3 on B ring. With hydroxyl group on B ring, 3f shows a higher
protective activity (n = 24.96) than that of 3e (n = 5.41), indicating the hydroxyl group on B ring can
enhance the antioxidant effectiveness sharply. Thus, it can be concluded based on Table 1 that hydroxyl
group and ortho-methoxy can enhance the antioxidant effectiveness of coumarin-3-carboxyl acid
derivatives remarkably. This finding is in agreement with our previous result that compound FRTA can
efficiently terminate 3.01 radical-propagation in AAPH-induced oxidation of DNA [29], but the n value
of compound 3f are much higher than that of compound FRTA, indicating that coumarin-3-carboxyl
14

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279
acid used to replace ferulic acid can significantly increase the conjugation system of compound which
can enhance the antioxidant activity to inhibit the oxidation of DNA.
280
3.2.2. Effects of compounds 2a-c and 3a-f on Cu2+/GSH-induced oxidation of DNA
281
282
283
284
285
286
The destroy of DNA caused by the intracellular GSH and Cu(II) may be carcinogenesis because of
the formation of GSH radical (GSꢀ) in the presence of Cu(II). The decomposition of DNA produces
carbonyl species, which can be detected after reacting with TBA to form colorful TBARS [33]. As
shown in Fig. 3, the absorbance line of the blank experiment locates above all the other lines and
increases with the reaction period, indicating that the amount of TBARS increases in the mixture of
DNA , Cu2+ and GSH.
287
Figure 3 is here
288
289
290
291
292
293
294
295
296
297
Obviously, without any other substitutes, coumarin-3-carboxyl acid (2a) shows the lowest
absorbance and the highest antioxidant activity among these derivatives. The absorbance of compounds
2b, 2c and 3a are stronger than that of 2a demonstrating that hydroxyl groups or ortho-methoxy group
at A ring and even peptide bond attenuated their antioxidant capabilities. On the other hand, the
additions of compounds 3b-f slightly decrease the absorbance than that of the blank experiment,
demonstrating that the compounds 3b-f exhibit relatively weak ability to inhibit GSꢀ-induced
degradation of DNA. It can be concluded that hydroxyl group at A ring or hydroxyl group at B ring can
greatly reduce their antioxidant capabilities to inhibit GSꢀ-induced degradation of DNA. This was
because phenolic hydroxyl group can reduce Cu(II) to form Cu(I), and Cu(I) can cause the
decomposition of DNA [34].
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298
3.2.3. Promoting ꢀOH-induced oxidation of DNA
299
300
301
302
303
304
305
306
307
308
309
310
ꢀOH can be readily generated by the reaction between tetra-chlorohydroquinone (TCHQ) and
H2O2, and attack deoxyribose in DNA to produce malondialdehyde [35]. To reveal the effects of
compounds on ꢀOH-induced oxidation of DNA, the absorbances of the solutions with the presence of
compounds 2a-c and 3a-f were compared with that of blank experiment (normalized as 100 %), shown
in Fig. 4. The percentage of TBARS in the presence of 3e (73.6%) is lower than that of 3c (75.3%), and
the result is similar to the comparison between 3f (68.1%) and 3d (71.5%), implying that the
ortho-methoxy group can enhance the antioxidant effect of compounds in this case. In the same way,
the percentage of TBARS in the presence of 3d (71.5%) is lower than that of 3c (75.3%), and 3f
(68.1%) < 3e (73.6%), demonstrating that the hydroxyl group enhances the antioxidant effect at B ring.
It is worthy to note that the percentage of TBARS in the presence 3a-f is lower than that of 2a-c,
indicating that the peptide bond can significantly enhance the antioxidant effectiveness of
coumarin-3-carboxyl acid derivatives.
311
312
Figure 4 is here
3.2.4. Rate constant of coumarin-3-acylamino derivatives to scavenge ABTS+•
313
314
315
316
317
Free radical-scavenging capability is the key property for a compound as an antioxidant [36].
ABTS+• is usually used to test the ability of an antioxidant to trap radicals [37,38]. As shown in Fig. 5,
in the presence of coumarin-3-acylamino derivatives the concentrations of ABTS+• decreases with the
increase of reaction period, demonstrating that coumarin-3-acylamino derivatives can efficiently
quench ABTS+•.
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318
Figure 5 is here
319
320
321
322
The measurement of the rate constants (k) of an antioxidant to trap ABTS+• can reveal the
radical-scavenging kinetics. Thus, we statistically elucidated the data from Fig. 5 and found that the
correlation between the concentrations of ABTS+• and reaction period (t) fits equation (2). The
relationship between the reaction rate (r = −d[radical]/ dt)and the reaction period (t) can be identified
323
324
325
326
clearly by using equation (3) which is the differential style of equation (2). The reaction rate at t = 0
( r0 ) can be calculated by equation (3) when t is assigned to be 0. According to the concept of reaction
rate, the reaction rate at t = 0( r0 ) correlates with the concentrations of radical and antioxidant at the
same time as shown in equation (4), in which
Ae−(t/a) Be−(t/b)
(A / a)e−(t/a)
k is the rate constant.
327
328
329
[radical]
=
+
+
C
(2)
(3)
(4)
−
d[radical]/ dt
=
r
=
+
(B / b)e−(t/b)
r
= k[radical]0[antioxidant]0
0
330
331
332
333
334
335
336
337
The rate constant (
and antioxidant, and r0 are known. This method has been applied to calculate the rate constant (
dihydropyrimidine and oxadiazole derivatives in trapping ABTS+• in the litherature [39]. By employing
the same approach, the values of synthesized coumarin-3-acylamino derivatives were calculated and
summarized in Table 2. It shows that the
value of 2a (4.27 nM-1s-1) is higher than that of 3a (1.47
nM-1s-1), and 2c has a greater
value than 3e, indicating that acylamino group can not enhance the
ability of 2a and 2c to quench ABTS+•, even though the value of 3c (2.77 nM-1s-1) was slightly higher
k
) can be calculated based on equation (4), where the concentrations of radical
k
) of
k
k
k
k
than that of 2b (2.03 nM-1s-1). Moreover, the
k
value of compounds 3b, 3d and 3f were much higher
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340
341
342
than that of 3a, 3c and 3d, respectively, demonstrating that hydroxyl group at B ring can significantly
increase the radical-scavenging ability. It was worthy to note that the k value of compounds 2c, 3e and
3f was much higher than that of other compounds, indicating that the ortho-methoxyl group greatly
enhanced the capability of compounds to scavenge ABTS+•. This result demonstrates that
ortho-methoxyl group at A ring and hydroxyl group at B ring play the major role in trapping ABTS+•.
343
344
Table 2 is here
4. Conclusion
345
346
347
348
349
350
351
352
353
354
355
In this study, we developed a series of coumarin-3-acylamino derivatives and investigated their
antioxidant activities. We found that both hydroxyl and ortho-methoxy groups at A ring, hydroxyl
group at B ring increase the protective abilities of coumarin-3-acylamino derivatives to protect DNA
against AAPH-induced oxidation, while decrease the protective abilities in Cu2+/GSH-induced
oxidation system. The ortho-methoxyl group at A ring and the hydroxyl group at B ring possess high
ability to inhibit •OH-induced oxidation of DNA. Furthermore, they play the major role in trapping
ABTS+•. We believe that the information obtained in this work is useful for understanding the
relationship between molecular structure with antioxidant capability of coumarin compounds and the
further progress of development of antioxidant can be achieved by a rational design of molecular
architecture.
Acknowledgments
356
357
This work was supported by grants from the National Nature Science Foundation of China
(Projects NSFC31240078).
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5. References and Notes
[1] M.S. Cooke, M.D. Evans, M. Dizdaroglu, J. Lunec, FASEB J. 17 (2003) 1195–1214.
[2] K. Sugamura, J.F. Keaney, Jr., Free Radic. Biol. Med. 51 (2011) 978–992.
[3] C.G. Fraga, P.I. Oteiza, Free Radic. Biol. Med. 51 (2011) 813–823.
[4] J. van Horssen, M.E. Witte, G. Schreibelt, H.E. de Vries, Biochim. Biophys. Acta 1812 (2011)
141–150.
[5] S. Mena, A. Ortega, J.M. Estrela, Mutat. Res. 674 (2009) 36–44.
[6] Z.-Q. Liu, Chem. Rev. 110 (2010) 5675–5691.
[7] M.E. Lönn, J.M. Dennis, R. Stocker, Free Radical Biol. Med. 53 (2012) 863–884.
[8] C. Riganti, E. Gazzano, M. Polimeni, E. Aldieri, D. Ghigo, Free Radical Biol. Med. 53 (2012)
421–436.
[9] I. Rahman, Biochim. Biophys. Acta 1822 (2012) 714–728.
[10] F. Belluti, G. Fontana, L.D. Bo, N. Carenini, C. Giommarelli, F. Zunino, Bioorg. Med. Chem.
18 (2010) 3543–3550.
[11] M.E. Riveiro, A. Moglioni, R. Vazquez, N. Gomez, G. Facorro, L. Piehl, E.R. de Celis, C.
Shayo, C. Davio, Bioorg. Med. Chem. 16 (2008) 2665–2675.
[12] M. Roussaki, C.A. Kontogiorgis, D. Hadjipavlou-Litina, S. Hamilakis, A. Detsi, Bioorg. Med.
Chem. Lett. 20 (2010) 3889–3892.
[13] T. Neichi, Y. Koshihara, S. Murota, Biochim. Biophys. Acta. 753 (1983) 130–132.
[14] K.C. Fylaktakidou, D.J. Hadjipavlou-Litina, K.E. Litinas, D.N. Nicolaides, Curr. Pharm.
Des.10 (2004) 3813–3833.
[15] F. Chimenti, B. Bizzarri, A. Bolasco, D. Secci, P. Chimenti, A. Granese, S. Carradori, D.
20

ACCEPTED MANUSCRIPT
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
Rivanera, A. Zicari, M.M. Scaltrito, F. Sisto, Bioorg. Med. Chem. Lett. 20 (2010) 4922–4926.
[16] I. Kostova, Curr. HIV Res. 4 (2006) 347–363.
[17] A. Chilin, R. Battistutta, A. Bortolato, G. Cozza, S. Zanatta, G. Poletto, M. Mazzorana, G.
Zagotto, E. Uriarte, A. Guiotto, F. Meggio, S. Moro, J. Med. Chem. 51 (2008) 752–759.
[18] S.-Y. Peng, L. Wang, H.-B. Guo, S.-F. Sun, J. Wang, Org. Biomol. Chem. 10 (2012)
2537–2541.
[19] Y.-H. Wang, B. Avula, N. P. Dhammika Nanayakkara, J.-P. Zhao, I.A. Khan, J. Agric. Food
Chem. 61 (2013) 4470−4476.
[20] C.L. Loprinzi, J.W. Kugler, J.A. Sloan, T.W. Rooke, S.K. Quella, P. Novotny, R.B. Mowat,
J.C. Michalak, P.J. Stella, R. Levitt, L.K. Tschetter, H. Windschitl, N. Engl. J. Med. 340 (1999)
346−350.
[21] D. Cox, R. O’ Kennedy, R.D. Thornes, Hum. Toxicol. 8 (1989) 501−506.
[22] C. Xiao, Z.-G. Song, Z.-Q. Liu, Eur. J. Med. Chem. 45 (2010) 2559−2566.
[23] R. Maggi, F. Bigi, S. Carloni, A. Mazzacani, G. Sartori, Green Chemistry, 3 (2001) 173–174.
[24] C.J. Reed, K.T. Douglas, Biochem. J. 275 (1991) 601–608.
[25] B.Z. Zhu, N. Kitrossky, M. Chevion, Biochem. Biophys. Res. Commun. 270 (2000) 942–946.
[26] P. Zhang, S.T. Omaye, Food Chem. Toxicol. 39 (2001) 239-246
[27] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radical Biol.
Med. 26 (1999) 1231–1237.
[28] A. Song, X.-B. Wang, K.S. Lam, Tet. Lett. 44 (2003) 1755–1758.
[29] Y. Yang, Z.-G. Song, Z.-Q. Liu, Free Radical Res. 45 (2011) 445–453.
[30] L. Zennaro, M. Rossetto, P. Vanzani, V.D. Marco, M. Scarpa, L. Battistin, A. Rigo, Arch.
21

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404
405
406
407
408
409
410
411
412
413
414
Biochem. Biophys. 462 (2007) 38–46.
[31] Y.-F. Li, Z.-Q. Liu, X.-Y. Luo, J. Agric, Food Chem. 58 (2010) 4126–4131.
[32] G.-X. Li, Z.-Q. Liu, J. Agric. Food Chem. 57 (2009) 3943–3948.
[33] A. Jain, N.K. Alvi, J.H. Parish, S.M. Hadi, Mutat. Res. 357 (1996) 83–88.
[34] G.-L. Xi, Z.-Q. Liu, Eur. J. Med. Chem, 68 (2013) 385–393.
[35] M.F. Janicek, W.A. Haseltine, W.D. Henner, Nucleic Acids Res. 13 (1985) 9011–9029.
[36] B. Halliwell, Trends Pharmacol. Sci. 32 (2011) 125–130.
[37] J.L. Munoz-Munoz, F. Garcia-Molina, R. Varon, J. Tudela, F. García-Cánovas, J.N.
Rodriguez-Lopez, J. Agric. Food Chem. 58 (2010) 2062–2070.
[38] S.-K. Yim, S.-J. Yun, C.-H. Yun, J. Biochem. Mol. Biol. 37 (2004) 629–633.
[39] R. Wang, Z.-Q. Liu, J. Org. Chem. 77 (2012) 3952–3958.
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6. LEGENDS OF FIGURES
Figure 1. The variation of the absorbance of TBARS in the mixture of 2.0 mg/mL DNA , 40 mM
AAPH, and various concentrations of coumarin-3-acylamino derivatives at 37°C.
Figure 2. The linear relationship between the concentrations of coumarin-3-acylamino derivatives and
inhibition period (tinh) in protecting DNA against AAPH-induced oxidation.
Figure 3. The increase of the absorbance at 535 nm in the absence and presence of 150 µM
coumarin-3-acylamino derivatives in the mixture of 2.0 mg/mL DNA , 5.0 mM Cu2+ and 3.0
mM GSH.
Figure 4. The percentages of TBARS in the mixture of 2.0 mg/mL DNA, 4.0 mM H2O2, 2.0 mM
TCHQ, and 150 µM coumarin-3-acylamino derivatives at 37°C for 30 min.
Figure 5. The decrease of the absorbance of ABTS+• (734 nm) in the presence of 20 µM
coumarin-3-acylamino derivatives and 64.88 µM ABTS+• at 25°C for 30 min.
Scheme 1. Synthesis routines of coumarin-3-acylamino derivatives.
23

ACCEPTED MANUSCRIPT
429
430
Table 1. The equations of tinh ~ [ coumarin-3-acylamino derivatives ], and n of coumarin-3-acylamino
derivatives in protecting DNA against AAPH-induced oxidation.
tinh (min) = (n / R )a[antioxidant(ꢀM)]+ constantb
Antioxidant
n
i
tinh (min) = 0.82[2b]+ 94.59
tinh (min) = 2.92[2c]− 41.79
tinh (min) =1.28[3b]+102.41
tinh (min) =1.61[3e]−3.76
tinh (min) = 7.43[3f ]+ 78.84
2b
2c
3b
3e
3f
2.75
9.81
4.30
5.41
24.96
a
431
432
Ri = Rg = 1.4×10-6 [AAPH] s-1 = 3.36 µM min−1 when [AAPH] = 40 mM in protecting DNA, thus, n
= coefficient × 3.36 µM min−1.
433
b The constant was generated from the linear regression analysis
.
24

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Table 2. Equation of [ ABTS+• ] ~ t and its differential style (
−
d[ABTS+• ]/d
t
~t), reaction rate at
t
= 0 (r0), and rate constant (
k )
a
434
Compound
2a
Equation of
−
d[ABTS+• ]/dt ~ t
r0
(
µ
Ms-1)
k
(nM-1 s-1)
Equation of [ ABTS+• ] ~
t
t
t
d[ABTS+• ]
t
t
−
−
−
[ABTS+• ] = 4.69e− + 4.18e
+55.74
0.94
7.28
5.54
2.63
4.27
2.03
0.94
7.28
−
= 4.97e
+ 0.57e
dt
t
t
d[ABTS+• ]
t
t
−
−
−
[ABTS+• ] = 2,36e− +15.43e
+ 46.80
0.95
106.90
2b
2c
3a
3b
3c
3d
3e
3f
0.95
106.90
−
= 2.49e
+ 0.14e
dt
t
t
d[ABTS+• ]
t
t
−
−
−
[ABTS+• ] =16.49e− +14.83e 3.51 +33.43
3.51
210.64
1.91
162.33
1.47
0.08
−
= 206.42e 0.08 + 4.22e
dt
t
t
d[ABTS+• ]
t
t
−
−
−
[ABTS+• ] = 68.58e−
+1.84e 1.11 −5.96
273.64
1.11
273.64
−
−
−
−
= 0.25e
+1.66e
dt
t
t
d[ABTS+• ]
t
t
−
−
[ABTS+• ] =19.43e−9.33 +19.47e− + 24.37
0.62
= 2.08e 9.33 + 31.22e
33.30
3.60
25.66
2.77
0.62
dt
t
t
d[ABTS+• ]
t
t
−
−
−
[ABTS+• ] =11.37e−
+1.49e
+52.02
25.19
0.47
25.19
0.47
= 0.45e
+3.15e
dt
t
t
d[ABTS+• ]
t
t
−
= 2.83e
+16.97e−
[ABTS+• ] =19.72e− +14.09e−0.83 +31.22
6.97
0.83
19.80
140.74
214.27
15.26
108.46
165.13
6.97
dt
t
t
d[ABTS+• ]
t
t
−
−
−
−
[ABTS+• ] =16.63e− +15.57e
+ 32.33
0.12
6.27
0.12
6.27
−
−
=138.26e
+ 2.48e
dt
t
t
d[ABTS+• ]
t
t
−
[ABTS+• ] = 27.34e−0.13 +19.96e− +16.91
= 210.11e 0.13 + 4.16e
4.80
4.80
dt
435
a The concentrations of compounds 2a-c and 3a-f were 20 µM and concentration of ABTS+• was 64.88 µM.
25

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2a
3b
3a
0
µ
µ
µ
µ
M
M
M
M
0
µ
µ
µ
µ
M
M
M
M
0
µM
µM
µM
µM
µM
1.2
0.9
0.6
17
50
83
167
50
75
125
150
100
200
250
300
µ
M
µ
M
0
250
500
750
0
0
0
250
500
750
750
750
0
250
500
750
Incubation period (min)
3d
0
50
100
150
200
3c
0
100
150
200
250
2b
0
83
100
134
167
µ
M
M
M
M
M
µM
µM
µM
µM
µM
µM
µM
µM
µM
µM
1.2
0.9
0.6
µ
µ
µ
µ
0
250
500
750
0
250
500
750
250
500
Incubation period(min)
2c
0
3e
0
80
3f
µM
µM
µM
µM
µM
µM
µM
µM
µM
µM
0
µ
M
1.2
0.9
0.6
0.3
67
83
125
166
13
20
27
33
µ
µ
µ
µ
M
M
M
M
133
213
267
tinh
0
250
500
750
0
300
600
250
500
Incubation period (min)
436
437
Figure 1
26

ACCEPTED MANUSCRIPT
450
300
150
2c
3f
3e
3b
2b
0
100
200
300
Concentration (µM)
438
439
Figure 2
27

ACCEPTED MANUSCRIPT
Blank
3d
3e
3f
3c
3b
3a
2b
2c
0.64
0.48
0.32
0.16
2a
0
60
120
180
Reaction period (min)
440
441
Figure 3
442
28