Synthesis and vasorelaxant and platelet antiaggregatory activities of a new series of 6-Halo-3-phenylcoumarins

Article abstract of DOI:10.3390/molecules15010270

A series of 6-halo-3-hydroxyphenylcoumarins (resveratrol-coumarins hybrid derivatives) was synthesized in good yields by a Perkin reaction followed by hydrolysis. The new compounds were evaluated for their vasorelaxant activity in intact rat aorta rings pre-contracted with phenylephrine (PE), as well as for their inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. These compounds concentration-dependently relaxed vascular smooth muscle and some of them showed a platelet antiaggregatory activity that was up to thirty times higher than that shown by trans-resveratrol and some other previously synthesized derivatives.

Full text of DOI:10.3390/molecules15010270

Molecules 2010, 15, 270-279; doi:10.3390/molecules15010270
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Vasorelaxant and Platelet Antiaggregatory
Activities of a New Series of 6-Halo-3-phenylcoumarins †
Elías Quezada 1, Giovanna Delogu 2,*, Carmen Picciau 2, Lourdes Santana 3, Gianni Podda 2,
Fernanda Borges 1, Verónica García-Morales 4, Dolores Viña 4 and Francisco Orallo 4
1
Chemistry Department, Faculty of Sciences, University Porto, 4169-007 Porto, Portugal;
E-Mails: elias.quezada@usc.es (E.Q.); mfernandamborges@gmail.com (F.B.)
2
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Cagliari, Via Ospedale 72,
09124 Cagliari, Italy; E-Mails: carmenpicciau@alice.it (C.P); gpodda@unica.it (G.P.)
3
Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela,
15782 Santiago de Compostela, Spain; E-Mail: lourdes.santana@usc.es (L.S.)
4
Department of Pharmacology, Faculty of Pharmacy, University of Santiago de Compostela, 15782
Santiago de Compostela, Spain; E-Mails: mdolores.vina@usc.es (D.V.); francisco.orallo@usc.es
(F.O.); veronica.garcia9@rai.usc.es (V.G.M.)
† In memory of Prof. Francisco Orallo.
* Author to whom correspondence should be addressed; E-Mail: delogug@unica.it;
Tel.: +39-0706758566; Fax: +39-0706758553.
Received: 4 December 2009; in revised form: 21 December 2009 / Accepted: 23 December 2009 /
Published: 12 January 2010
Abstract: A series of 6-halo-3-hydroxyphenylcoumarins (resveratrol-coumarins hybrid
derivatives) was synthesized in good yields by a Perkin reaction followed by hydrolysis.
The new compounds were evaluated for their vasorelaxant activity in intact rat aorta rings
pre-contracted with phenylephrine (PE), as well as for their inhibitory effects on platelet
aggregation induced by thrombin in washed human platelets. These compounds
concentration-dependently relaxed vascular smooth muscle and some of them showed a
platelet antiaggregatory activity that was up to thirty times higher than that shown by
trans-resveratrol and some other previously synthesized derivatives.
Keywords: resveratrol; coumarin; vasorelaxant; platelet antiaggregatory activity

Molecules 2010, 15
271
1. Introduction
Coumarins (or benzopyrones) are a large family of compounds of natural and synthetic origin that
show numerous biological activities, including cardiovascular properties [1]. For instance,
Carbochromen (3-diethylaminoethyl-7-ethoxycarbonylmethoxy-4-methylcoumarin (Figure 1) is a
potent specific coronary vasodilator that has been used for many years in the treatment of angina
pectoris [2,3]. Futhermore, warfarin [3-(2-acetyl-1-phenylethyl)-4-hydroxycoumarin (Figure 1) is a
coumarin with potent anticoagulant activity and a good pharmacokinetic profile [4].
Figure 1. Chemical structures of trans-resveratrol, carbochromen, and warfarin.
Et
N
OH
O
Et
O
Et
O
O
O
O
O
O
Warfarin
Carbochromen
OH
HO
trans-Resveratrol
OH
trans-Resveratrol (t-RESV; 3,4',5-trihydroxy-trans-stilbene; Figure 1) is a natural phenolic
component of Vitis vinifera L. (Vitaceae). It is abundant in the skin of the grapes and it is present in
higher concentrations in red than in white wines. trans-Resveratrol has shown a number of biological
activities, including protection against coronary heart disease, as a result of different effects:
significant antioxidant activity, modulation of lipoprotein metabolism, and vasodilatatory and platelet
antiaggregatory properties [5–8].
Because of their similar characteristics, it was of interest to design and synthesize hybrids that
incorporate the nucleus of the coumarins and resveratrol molecules. In previous work, our research
group had reported the vasorelaxant and platelet antiaggregatory activities of a series of coumarin-
resveratrol hybrids (3-arylcoumarins), bearing hydroxy or methoxy groups on the coumarin and/or on
the 3-phenyl ring. 6-Hydroxy-3-(3’,5’-dihydroxyphenyl)coumarin showed vasorelaxant and platelet
antiaggregatory activity higher than that of trans-resveratrol [9].
Based on this, and with the aim of improving the vasorelaxant and platelet antiaggregatory
activities of resveratrol-coumarin hybrids and establishing a relationship between the structure and
activity for this type of compounds we have studied the effects of substitution on the 3-arylcoumarin
moiety with groups showing different steric and electronics effects. In this paper we report the
synthesis and evaluation of a new series of 3-arylcoumarins in which the hydroxy group in the 6
position has been changed for a halogen group, and different positions of hydroxyl group in 3-phenyl
ring were explored.

Molecules 2010, 15
272
2. Results and Discussion
The 3-phenylcoumarins 6-11 [10] were prepared from the conveniently substituted phenylacetic
acids 1-3, the appropriate salicylaldehyde 4, 5 and dicyclohexylcarbodiimide (DCC) by a Perkin
reaction [11–13] in dimethylsulfoxide (DMSO). These reactions gave 46%, 40%, 27%, 29%, 31% and
33% yield, respectively. Hydrolysis of the methoxy groups [14] by treatment with HI in acetic
acid/acetic anhydride gave the hydroxy derivatives 12, 13, 14, 15, 16 and 17 [15–16], in 28%, 70%,
60%, 73%, 94% and 57% yield, respectively (Scheme 1).
Scheme 1. General synthetic route to obtain compounds 6-17.
R2
O
R1
R3
O
OH
R1
X
a
X
H
+
OH
R2
O
O
R3
6 X = Cl; R1 = MeO; R2 = R3 = H
7 X = Cl; R2 = MeO; R1 = R3 = H
8 X = Cl; R3 = MeO; R1 = R2 = H
9 X = Br; R1 = MeO; R2 = R3 = H
10 X = Br; R2 = MeO; R1 = R3 = H
11 X = Br; R3 = MeO; R1 = R2 = H
1 R1 = MeO; R2 = R3 = H
2 R2 = MeO; R1 = R3 = H
3 R3 = MeO; R1 = R2 = H
4 X = Cl
5 X = Br
b
R2
R1
R3
X
O
O
12 X = Cl; R1 = OH; R2 = R3 = H
13 X = Cl; R2 = OH; R1 = R3 = H
14 X = Cl; R3 = OH; R1 = R2 = H
15 X = Br; R1 = OH; R2 = R3 = H
16 X = Br; R2 = OH; R1 = R3 = H
17 X = Br; R3 = OH; R1 = R2 = H
Reagents and conditions: (a) DCC, DMSO, 110 °C, 12h; (b) HI/AcOH/Ac2O, 0 °C to rt, 3 h.
The vasorelaxant effects of compounds 12-17 were studied on pre-contracted rat aortic rings with
endothelium [6]. The cumulative addition of t-RESV or the new compounds (1-100 μM) caused a
concentration-dependent relaxation of the contractions induced by phenylephrine (PE, 1 μM) in intact
rat aortic rings. The corresponding IC50 values are shown in Table 1.

Molecules 2010, 15
273
Table 1. Vasorelaxant activity (IC50 in μM) of tested compounds.
Compound
PE (1 μM)
36.63 ± 2.46*
57.63 ± 3.87*
**
12
13
14
15
16
48.79 ± 3.27*
46.67 ± 3.13*
**
17
t-RESV
3.12 ± 0.26
*
P < 0.01 versus the corresponding IC50 values of t-RESV; ** Inactive at 100 μM (highest
concentration tested). At higher concentrations compounds precipitate.
All evaluated compounds resulted less efficient than t-RESV in relaxing the contractions induced
by PE. Furthermore, substitution with a p-hydroxy group in the 3-phenyl ring (compounds 14 and 17)
leads to inactive compounds. Platelet aggregation studies were also performed. Compounds 12, 13 and
17 inhibited platelet aggregation more effectively than t-RESV when thrombin (0.25 U/mL) was used
as the stimulating agent (Table 2).
Table 2. Antiplatelet activity (IC50 in μM) for tested compounds.
Compound
Thrombin (0.25 U/mL)
12
13
14
91.36 ± 6.13*
6.41 ± 2.15*
**
15
**
16
17
t-RESV
**
20.1 ± 1.35*
195.50 ± 13.82
*
P < 0.01 versus the corresponding IC50 values of t-RESV; ** Inactive at 100 μM (highest
concentration tested). At higher concentrations compounds precipitate.
These results indicate that the variation of the position of the hydroxyl group on the 3-phenyl and
the substitution with different halogen groups in the 6 position of the coumarin ring, in this type of
molecules (resveratrol-coumarins hybrid), can give derivatives with a significantly higher
pharmacological potency than t-RESV. This is the case for compound 13, which has a platelet
antiaggregatory activity that is more than 32 times higher than that of t-RESV.
3. Experimental
3.1. General
Melting points were determined in a Reichert Kofler thermopan or in capillary tubes in a Buchi 510
apparatus, and are uncorrected. Infrared (IR) spectra were recorded in a Perkin-Elmer 1640FT
spectrometer (KBr disks, ν in cm-1).13C and 1H spectra of samples approximately 10% solutions in
chloroform-d, were recorder at room temperature in 5 mm o.d. tubes. TMS was used as internal

Molecules 2010, 15
274
standard, chemical shifts are expressed in ppm (δ), J in Hz. NMR spectra were recorded with a Bruker
AMX 500 (1H-, 500 MHz; 13C-, 125 MHz) instrument. ). Mass spectra were obtained using a
Hewlett-Packard 5988A spectrometer (70 eV). Silica gel (35-60 mesh) was used for flash
chromatography (FC). Analytical TLC was performed on plates precoated with silica gel (Merck 60
F254, 0.25 mm). Elemental analyses were performed with a Perkin-Elmer 240B microanalyser.
Phenylacetic acids 1-3, salicylaldehydes 4, 5, hydriodic acid (HI), acetic acid (AcOH), acetic
anhydride (Ac2O), dicyclohexylcarbodiimide (DCC) and dimethylsulfoxide (DMSO) were
commercially available (Aldrich).
3.2. Chemistry
The studied compounds are all known and were prepared by a traditional Perkin reaction carried out
in refluxing dimethylsulfoxide (DMSO) between conveniently substituted phenylacetic acids 1-3 and
the corresponding salicylaldehyde 4, 5, using dicyclohexylcarbodiimide (DCC) as dehydrating agent
(Scheme 1) [11–13]. Hydrolysis of methoxy groups [14], by treatment with HI in acetic acid/acetic
anhydride gave the desired hydroxyl derivatives 12-17 [15–16].
6-Chloro-3-(2’-methoxy)phenylcoumarin (6). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 177–179 ºC; IR (KBr): 2920, 1705, 1610, 1302, 1125, 780 cm-1; H-NMR
(CDCl3) δ (ppm): 3.83 (s, 3H, CH3O), 7.00 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 7.22 (d,
13
J = 8.2 Hz, 1H), 7.41 (m, 3H), 7.60 (m, 1H), 7.66 (s, 1H); C-NMR (CDCl3) δ (ppm): 55.7, 111.3,
116.7, 118.2, 120.6, 121.0, 126.9, 130.0, 130.4, 130.7, 131.0, 133.8, 140.2, 152.2, 156.9, 157.1; MS
m/z (%): 288 ([M+2]+, 33), 286 (M+, 100), 269(16), 251 (8), 240 (5), 193 (21), 152 (16), 118 (14);
Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87. Experimental: C, 67.25; H, 3.99.
6-Chloro-3-(3’-methoxy)phenylcoumarin (7). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 144–146 ºC; IR (KBr): 2925, 1700, 1615, 1300, 1115, 775 cm-1; H-NMR
(CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.96 (dd, J = 2.5; 8.2 Hz, 1H), 7.25 (m, 2H), 7.35 (m, 2H), 7.48
(m, 2H), 7.72 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.32, 114.2, 114.8, 117.8, 120.6, 120.8, 127.0,
129.2, 129.5, 129.7, 131.3, 135.5, 138.5, 151.8, 159.5, 159.8; MS m/z (%): 288 ([M+2]+, 29), 286 (M+,
100), 258(30), 215 (20), 201 (5), 152 (7); Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87.
Experimental: C, 67.20; H, 3.95.
6-Chloro-3-(4’-methoxy)phenylcoumarin (8). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 194–196 ºC; IR (KBr): 2918, 1710, 1614, 1300, 1117, 779 cm-1; H-NMR
(CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.99 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.48 (m, 2H),
13
7.66 (d, J = 8.9 Hz, 2H), 7.71 (s, 1H); C-NMR (CDCl3) δ (ppm): 55.4, 113.9 (2C), 117.7, 118.3,
120.3, 121.1, 126.8, 129.8 (2C), 130.8, 136.9, 141.1, 151.9, 156.3, 158.2; MS m/z (%): 288 ([M+2]+,
37), 286 (M+, 100), 258(13), 243 (16), 180 (3), 152 (5); Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87.
Experimental: C, 67.10; H, 3.99.

Molecules 2010, 15
275
6-Bromo-3-(2’-methoxy)phenylcoumarin (9). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 183–185 ºC; IR (KBr): 2909, 1709, 1615, 1305, 1120, 783 cm-1; H-NMR
(CDCl3) δ (ppm): 3.82 (s, 3H, CH3O), 6.99 (d, J = 7.5 Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H), 7.24 (d,
13
J = 8.4 Hz, 1H), 7.36 (m, 2H), 7.60 (m, 2H), 7.66 (s, 1H); C-NMR (CDCl3) δ (ppm): 55.7, 111.3,
116.7, 118.2, 120.6, 121.0, 123.5, 127.6, 130.0, 130.5, 130.7, 133.8, 140.2, 152.5, 157.1, 159.6; MS
m/z (%): 332 ([M+2]+, 100), 330 (M+, 97), 315(23), 237 (32), 152 (15), 118 (16); Anal. Calcd for
C16H11BrO3: C, 58.03; H, 3.35. Experimental: C, 58.25; H, 3.49.
6-Bromo-3-(3’-methoxy)phenylcoumarin (10). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 148-150 ºC; IR (KBr): 2898, 1700, 1620, 1300, 1110, 789 cm-1; H-NMR
(CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.96 (dd, J = 2.3; 8.2 Hz, 1H), 7.20 (m, 2H), 7.36 (m, 2H), 7.60
13
(d, J = 8.3 Hz, 1H), 7.75 (s, 1H), 7.85 (s, 1H); C-NMR (CDCl3) δ (ppm): 55.2, 114.1, 114.6, 116.9,
118.0, 120.7, 121.0, 129.0, 129.4, 130.0, 133.9, 135.3, 138.3, 152.1, 159.3, 159.6; MS m/z (%): 332
([M+2]+, 100), 330 (M+, 28), 302(34), 195 (10), 180 (10), 152 (17); Anal. Calcd for C16H11BrO3: C,
58.03; H, 3.35. Experimental: C, 58.15; H, 3.50.
6-Bromo-3-(4’-methoxy)phenylcoumarin (11). Purified by chromatography using 9:1 hexane/ethyl
1
acetate as eluent; Mp: 205–206 ºC; IR (KBr): 2900, 1712, 1612, 1298, 1110, 781 cm-1; H-NMR
(CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.97 (d, J = 8.8 Hz, 2H), 7.23 (m, 1H), 7.57 (dd, J = 2.2, 8.7 Hz,
13
1H), 7.63 (m, 3H), 7.67 (s, 1H); C-NMR (CDCl3) δ (ppm): 55.3, 113.9 (2C), 114.1, 116.9, 118.1,
121.4, 126.5, 128.9, 129.8 (2C), 129.9, 133.6, 136.8, 152.0, 160.4; MS m/z (%): 332 ([M+2]+, 100),
330 (M+, 13), 302(12), 288 (40), 259 (18), 195 (10), 180 (17), 152 (14); Anal. Calcd for C16H11BrO3:
C, 58.03; H, 3.35. Experimental: C, 58.15; H, 3.42.
6-Chloro-3-(2’-hydroxy)phenylcoumarin (12). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent. Mp: 225–227 ºC. IR (KBr): 3500, 2920, 1705, 1613, 1300, 1120, 780 cm-1. 1H NMR
(DMSO-d6) δ (ppm): 6.86 (m, 2H), 7.23 (m, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.78 (dd, J = 2.3; 8.8 Hz,
1H), 7.97 (s, 1H), 8.00 (s, 1H), 9.64 (bs, 1H, 1OH). 13C NMR (DMSO-d6) δ (ppm): 115.7, 116.0,
118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 140.7, 152.1, 155.1. MS m/z (%):
274 ([M+2]+, 23), 272 (M+, 100), 244(35), 180 (19), 152 (20), 118 (10). Anal. Calcd for C15H9ClO3: C,
66.07; H, 3.33. Experimental: C, 66.25; H, 3.49.
6-Chloro-3-(3’-hydroxy)phenylcoumarin (13). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent; Mp: 221–223 ºC; IR (KBr): 3502, 2925, 1701, 1614, 1310, 1110, 785 cm-1; 1H-NMR
(DMSO-d6) δ (ppm): 6.82 (dd, J = 2.2; 8.0 Hz, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.44 (d, J = 8.8 Hz,
13
1H), 7.61 (dd, J = 2.5; 8.8 Hz, 1H), 7.86 (d, J = 2.5 Hz, 1H), 8.14 (s, 1H), 9.60 (bs, 1H, 1OH); C-
NMR (DMSO-d6) δ (ppm): 115.5, 115.9, 117.8, 119.2, 120.9, 127.6, 128.0, 128.2, 129.3, 131.1, 135.5,
139.1, 151.5, 157.1, 159.2; MS m/z (%): 274 ([M+2]+, 21), 272 (M+, 100), 244(48), 180 (14), 152 (23),
118 (10); Anal. Calcd for C15H9ClO3: C, 66.07; H, 3.33. Experimental: C, 66.30; H, 3.45.
6-Chloro-3-(4’-hydroxy)phenylcoumarin (14). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent; Mp: 241–243 ºC; IR (KBr): 3500, 2918, 1708, 1610, 1296, 1119, 783 cm-1; 1H-NMR

Molecules 2010, 15
276
(DMSO-d6) δ (ppm): 6.84 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.58 (m, 3H), 7.84 (d,
13
J = 2.5 Hz, 1H), 8.09 (s, 1H), 9.81 (bs, 1H, 1OH); C-NMR (DMSO-d6) δ (ppm): 115.1 (2C), 117.7,
121.1, 124.8, 127.2, 127.8, 128.1, 129.9 (2C), 130.6, 137.1, 151.2, 158.2, 159.5; MS m/z (%): 274
([M+2]+, 20), 272 (M+, 100), 244(74), 180 (15), 152 (40), 118 (5); Anal. Calcd for C15H9ClO3: C,
66.07; H, 3.33. Experimental: C, 66.22; H, 3.47.
6-Bromo-3-(2’-hydroxy)phenylcoumarin (15). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent; Mp: 230–232 ºC; IR (KBr): 3510, 2918, 1709, 1620, 1310, 1120, 780 cm-1; 1H-NMR
(DMSO-d6) δ (ppm): 6.87 (m, 2H), 7.23 (m, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.74 (dd, J = 2.3; 8.8 Hz,
1H), 7.99 (s, 1H), 8.02 (s, 1H), 9.64 (bs, 1H, 1OH); 13C-NMR (DMSO-d6) δ (ppm): 115.7, 116.0,
118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 152.1, 155.1, 158.8; MS m/z (%):
318 ([M+2]+, 47), 316 (M+, 71), 300(8), 288 (40), 180 (54), 152 (100); Anal. Calcd for C15H9BrO3: C,
56.81; H, 2.86. Experimental: C, 56.95; H, 3.00.
6-Bromo-3-(3’-hydroxy)phenylcoumarin (16). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent; Mp: 219–221 ºC; IR (KBr): 3505, 2919, 1709, 1622, 1312, 1123, 780 cm-1; 1H-NMR
(DMSO-d6) δ (ppm): 6.82 (dd, J = 2.0; 7.7 Hz, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.38 (d, J = 8.8 Hz,
13
1H), 7.73 (dd, J = 2.3; 8.8 Hz, 1H), 8.00 (d, J = 2.3 Hz, 1H), 8.14 (s, 1H), 9.59 (bs, 1H, 1OH); C-
NMR (DMSO-d6) δ (ppm): 115.5, 115.9, 116.1, 118.1, 119.2, 121.4, 128.0, 129.3, 130.6, 133.9, 135.5,
139.0, 151.9, 157.1, 159.1; MS m/z (%): 318 ([M+2]+, 46), 316 (M+, 79), 288 (46), 180 (19), 152
(100); Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Experimental: C, 56.98; H, 3.01.
6-Bromo-3-(4’-hydroxy)phenylcoumarin (17). Purified by chromatography using 8:2 hexane/ethyl
acetate as eluent; Mp: 226–228 ºC; IR (KBr): 3520, 2939, 1710, 1622, 1302, 1133, 784 cm-1; 1H-NMR
(DMSO-d6) δ (ppm): 6.84 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 9.1 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.66
13
(m, 1H), 7.99 (s, 1H), 8.09 (s, 1H), 9.81 (bs, 1H, 1OH); C-NMR (DMSO-d6) δ (ppm): 115.1 (2C),
116.0, 118.0, 121.6, 124.8, 127.7, 129.9 (2C), 130.2, 133.3, 137.0, 151.6, 158.2, 159.4; MS m/z (%):
318 ([M+2]+, 31), 317 ([M+1]+, 100), 316 (M+, 40), 288 (39), 180 (14), 152 (46); Anal. Calcd for
C15H9BrO3: C, 56.81; H, 2.86. Experimental: C, 56.87; H, 3.0.
3.3. Vasorelaxant activity
Vascular rings were prepared from aortae of male or female Wistar rats weighing 230–270 g. After
an equilibration period of at least 1 h, isometric contractions induced by PE (1 μM) were obtained.
When contraction of the tissue in response to the corresponding vasoconstrictor agent had stabilized
(after about 20 min), cumulatively increasing concentrations of the compounds were added to the bath
at 15–20 min intervals (the time needed to obtain steady-state relaxation). Control tissues were
subjected to the same procedure simultaneously, but in this case omitting the compounds and adding
the vehicle [appropriate dilutions of dimethylsulfoxide (DMSO)]. Results shown in the tables are
expressed as means ± SEM from five experiments. Means were compared by one-way analysis of
variance (ANOVA) followed by Dunnett’s post hoc test. The inhibitory effects of the tested
compounds in rat aorta and human platelet are expressed as IC50 (concentrations that produce a 50%

Molecules 2010, 15
277
inhibition) estimated by least-squares linear regression using the program Origin 5.0, with
X = log molar concentration of the tested compounds and Y = % of pharmacological response.
3.4. Antiplatelet activity
Preparation of washed platelets. Washed human platelets were prepared from blood anticoagulated
with citrate-phosphate-dextrose, which was obtained from Centro de Transfusion de Galicia (Santiago
de Compostela, Spain). Bags containing buffy coat from individual donors were diluted with the same
volume of washing buffer (NaCl, 120 mM; KCl, 5 mM; trisodium citrate, 12 mM; glucose, 10 mM;
sucrose, 12.5 mM; pH 6) and centrifuged at 400 g for 9 min. The upper layer containing platelet
(platelet-rich plasma) was removed and centrifuged at 400 g for 9 min. The resulting platelet pellet
was recovered, resuspended with washing buffer, and centrifuged again at 1,000 g for 15 min. Finally,
the platelet pellet from this step was resuspended in a modified Tyrode-HEPES buffer (HEPES,
10 mM; NaCl, 140 mM; KCl, 3 mM; MgCl2, 0.5 mM; NaHCO3, 5 mM; glucose, 10 mM; pH 7.4) to
afford a cell density of 2.5–3.5 × 108 platelet/mL. The calcium concentration in the extracellular
medium was 2 mM.
Platelet aggregation studies. Platelet aggregation was measured using a dual channel aggregometer
(Chrono-log, Havertown, PA, USA). Each tested compound, dissolved in DMSO, was incubated with
washed platelet at 37 °C for 5 min. Stimulus (thrombin) was then added to induce platelet aggregation
and the light transmission was monitored over 5 min period. Platelet aggregation is measured as the
maximum change in light transmission during this period. The 100% aggregation value was obtained
when vehicle (DMSO) was added instead of the compounds. The final DMSO concentration was
below 1% (v/v) in all cases.
4. Conclusions
In conclusion, some of the new synthesized molecules have been characterized as agents with
remarkable human platelet antiaggregatory activity and significant vasorelaxant effects in intact rat
aorta. Further experiments are in progress aimed at providing new data to clarify the precise
mechanism by which coumarin-resveratrol hybrid derivatives produce their characteristic vasorelaxant
and platelet antiaggregatory effects.
Acknowledgements
We thank Progetto di Ricerca Scientifica 2007-Università di Cagliari and Fondazione del Banco di
Sardegna, Spanish Ministerio de Sanidad y Consumo (FISS PI061537, PI061457), Xunta de Galicia
(Spain; INCITE08PXIB203022PR) and Spanish Ministerio de Ciencia e Innovación (FISS
PS09/00618). D. Vina acknowledges sponsorships for a tenure-track research position at the
University of Santiago de Compostela from the Isidro Parga Pondal Programme of the “Dirección
Xeral de Investigación e Desenvolvemento, Xunta de Galicia”. E. Quezada thanks for a postdoctoral
grant from FCT (Portugal). Verónica García-Morales thanks for a predoctoral grant (FPU, AP2008-

Molecules 2010, 15
278
02609, Spanish Ministerio de Ciencia e Innovación). C. Picciau thanks for a predoctoral grant Master
& Back-Regione Sardegna.
References and Notes
1. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Simple coumarins and analogues in
medicinal chemistry: Occurrence, synthesis and biological activity. Curr. Med. Chem. 2005, 12,
887–916.
2. Fiedler, V.B.; Scholtholt, J. Effects of carbocromene on myocardial oxygen consumption in
isolated dog hearts. J. Pharmacol. Exp. Ther. 1981, 217, 306–313.
3. Opherk, D.; Schuler, G.; Waas, W.; Dietz, R.; Kubler, W. Intravenous carbochromen: A potent
and effective drug for estimation of coronary dilatory capacity. Eur. Heart J. 1990, 11, 342–347.
4. Cao, Y.G.; Liu, X.Q.; Chen, Y.C.; Hao, K.; Wang, G.J. Warfarin maintenance dose adjustment
with indirect pharmacodynamic model in rats. Eur. J. Pharm. Scien. 2007, 30, 175–180.
5. Bradamante, S.; Barenghi, L.; Villa, A. Cardiovascular protective effects of resveratrol.
Cardiovasc. Drug Rev. 2004, 22, 169–188.
6. Orallo, F.; Alvarez, E.; Camina, M.; Leiro, J.M.; Gomez, E.; Fernandez, P. The possible
implication of trans-Resveratrol in the cardioprotective effects of long-term moderate wine
consumption. Mol. Pharmacol. 2002, 61, 294–302.
7. Wu, J.M.; Wang, Z.R.; Hsieh, T.C.; Bruder, J.L.; Zou, J.G.; Huang, Y.Z. Mechanism of
cardioprotection by resveratrol, a phenolic antioxidant present in red wine. Int. J. Mol. Med. 2001,
8, 3–17.
8. Hao, H.D.; He, L.R. J. Mechanisms of cardiovascular protection by resveratrol. Med. Food 2004,
7, 290–298.
9. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.; Cano, E.;
Orallo, F. Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-
resveratrol hybrids. Bioorg. Med. Chem. Lett. 2006, 16, 257–261.
10. Oda, N.; Yoshida, Y.; Nagai, S.; Ueda, T.; Sakakibara, J. Synthesis of coumarins by Nucleophilic
Denitrocyclization Reaction. Chem. Pharm. Bull. 1987, 35, 1796–1802.
11. Hans, N.; Singhi, M.; Sharma, V.; Grover, S.K. A novel one-step synthesis of 3-phenyl-, 4-
methyl-3-phenyl- and 3-phenyl-4-styrylcoumarins using DCC-DMSO. Indian J. Chem. 1996,
35B, 1159–1162.
12. Perkin, W.H. On the artificial production of coumarin and formation of its homologues. J. Chem.
Soc. 1868, 21, 53–63.
13. Perkin, W.H. On the formation of coumarin and of cinnamic and of other analogous acids from
the aromatic aldehydes. 1877, 31, 388–427.
14. Begala, M.; Delogu, G.; Maccioni, E.; Podda, G.; Tocco, G.; Quezada, E.; Uriarte, E.; Fedrigo,
M.A.; Favretto, D.; Traldi, P. Electrospray ionisation tandem mass spectrometry in the
characterisation of isomeric benzofurocoumarin. Rapid Commun. Mass Spectrom. 2001, 15,
1000–1010.
15. Walter, R.; Zimmer, H.; Purcell, T.C. Synthesis and cyclization reactions of 3-(2-
hydroxybenzylidene)-2(3H)-coumaranones. J. Org. Chem. 1966, 31, 3854–3857.

Molecules 2010, 15
279
16. Mimai, M.; Suzuki, Y.; Hamma, N.; Murayama, E.; Aono, S. Coumarin derivatives. JP Pat.
49054371, 1974.
Sample Availability: Contact the authors.
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).