Design and synthesis of novel aspirin-caffeic acid ester hybrids for cardioprotection with reduced risk of hemorrhagic stroke

Article abstract of DOI:10.14233/ajchem.2015.17896

A series of novel aspirin-caffeic acid ester hybrids for cardioprotection with reduced risk of hemorrhagic stroke were designed and synthesized by coupling aspirin and caffeic acid esters inspired by NCX-4016. The synthesized compounds were evaluated for

Full text of DOI:10.14233/ajchem.2015.17896

Asian Journal of Chemistry; Vol. 27, No. 4 (2015), 1342-1346
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.17896
Design and Synthesis of Novel Aspirin-Caffeic Acid Ester Hybrids for
Cardioprotection with Reduced Risk of Hemorrhagic Stroke
1,2
1,*
1,*
1
1
ZHI-HAO SHI , NIAN-GUANG LI , YU-PING TANG , QIAN-PING SHI , WEI ZHANG ,
1
1
1
1
PENG-XUAN ZHANG , ZE-XI DONG , WEI LI and JIN-AO DUAN
1National and Local Collaborative Engeering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine,
Nanjing University of Chinese Medicine, Nanjing, P.R. China
2Department of Organic Chemistry, China Pharmaceutical University, Nanjing, P.R. China
*Corresponding authors: Tel/Fax: +86 25 85811916; E-mail: linianguang@njutcm.edu.cn; yupingtang@njutcm.edu.cn
Received: 26 April 2014;
Accepted: 15 July 2014;
Published online: 4 February 2015;
AJC-16788
A series of novel aspirin-caffeic acid ester hybrids for cardioprotection with reduced risk of hemorrhagic stroke were designed and
synthesized by coupling aspirin and caffeic acid esters inspired by NCX-4016. The synthesized compounds were evaluated for their in
vitro antiplatelet aggregations induced by ADP and antioxidant activity of the caffeic acid esters which could be released from the hybrids
by esterases in vivo like NCX 4016 were determined by DPPH assay. The results showed that compound 3d exhibited potent antiplatelet
activity than aspirin and its intermediate caffeic acid isopropyl ester (2d) showed good antioxidant activity and thus could be considered
to be novel potent cardioprotectic drug candidates with reduced risk of hemorrhagic stroke.
Keywords: Hemorrhagic stroke, Aspirin, Caffeic acid, Synthesis.
occur7. A promising alternative solution to minimize the side
effects of aspirin on gastric mucosa without compromising its
INTRODUCTION
Aspirin, which has been known as an antipyretic and
antithrombotic efficacy is a newly designed category of NO-
analgesic for 100 years, has undergone a resurgence of popu-
releasing aspirin. NCX-4016 molecule (Fig. 1) is actually
larity since it became appreciated in the 1980s as the most
consisted of an aspirin group, a spacer joint by an ester linkage
cost-effective agent for the secondary prevention of coronary
and an NO-releasing group8. When administered in vivo,
artery disease1. Aspirin is also the basic antiplatelet agent for
esterases separate the aspirin moiety from the benzene spacer-
all kinds of acute disease that may cause platelet-dependent
NO complex, leading to the release of NO from this complex.
thrombotic vessel occlusion2. It is recommended as the first-
Interestingly, NO possesses some properties of prostaglandins
line antiplatelet drug. Aspirin imparts its primary antithro-
within the gastric mucosa, therefore leading to cytoprotection,
mbotic effects through the inhibition of PGH-synthase/COX
possibly through the increase of mucosal blood flow and
by the irreversible acetylation of a specific serine moiety3.
mucous fluid secretion by the gastric epithelial cells.
Unfortunately, its long-term administration is accom-
panied by an increased risk of gastrointestinal ulceration
with consequent gastrointestinal bleeding4. This side effect is
O
believed to result from the blockade of COX-1. COX-1 is
constitutively expressed and has clear physiological functions
in many tissues including the gastric mucosa, where it generates
cytoprotective meditors such as prostacyclin5. So new COX-2
selective inhibitors have been synthesized6, the consequence
of this effort has been the development of compounds such as
rofecoxib and celecoxib that are now widely prescribed.
However the initial expectation that such new generation of
NSAIDs would lack gastrointestinal side effects has been
challenged by reports showing that these side effects also
ONO2
O
O
O
Fig. 1. Structure of NCX 4016
Recently, a meta-analysis included 287 randomized trials
that together enrolled 212,000 high-risk patients by the Anti-
thrombotic Trialists' Collaboration1 indicated that antiplatelet

Vol. 27, No. 4 (2015)
Design and Synthesis of Novel Aspirin-Caffeic Acid Ester Hybrids for Cardioprotection 1343
therapy by aspirin reduced the risk of serious vascular events
by 25 % and the risk of non-fatal myocardial infarction (MI)
alone by 34 %. However, there was a proportional increase in
fatal or nonfatal hemorrhagic stroke of 22 %.
mmYantai silica gel plates (RSGF 254) using UV light as the
visualizing agent. Chromatography was performed on Qingdao
silica gel (160-200 mesh) using petroleum ether (60-90) and
1
ethyl acetate as the eluating solvent. H NMR spectra were
Atherosclerosis played a vital role in stroke, among major
risk factors for the development of atherosclerosis are increased
levels of low-density lipoprotein (LDL), oxidative modification
of low-density lipoprotein and an impairment of endothelial
derived relaxing factor (EDRF, nitric oxide, NO)-mediated
obtained using a BrukerAV-300 (300 MHz). Chemical shifts were
recorded in ppm downfield from tetramethylsilane. ESI-MS
spectra were recorded on a Waters Synapt HDMS spectrometer.
General procedures for the esterification of caffeic acid
through microwave irradiation: To a stirring mixture of
caffeic acid (940 mg, 5 mmol) in alcohol (10 mL) was added
the concentrated sulfuric acid (0.067 mL, 1.25 mmol) and the
reaction mixture was refluxed for 3-6 min in a sealed reaction
vessel of Discover (CEM, USA) under microwave irradiation,
where the power was set at 200 W, the temperature was set at
some centigrade which was 20 above the boiling point of the
alcohol and the PSI was set at 180. After cooling to 25 °C,
ethyl acetate was added and washed with water and brine.
The ethyl acetate layer was dried over MgSO4, filtered and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography eluted with petroleum
ether-EtOAc (v:v = 8:1) to afford caffeic acid esters.
bioactions9,10
.
The accumulation of low-density lipoprotein and reactive
oxygen species (ROS) in the sub-endothelial space induces a
high degree of low-density lipoprotein oxidation. According
to the oxidative hypothesis of atherosclerosis, this is an early
event in a complex process that leads to the formation of foam
cells that constitute a fatty streak, a forerunner to the develop-
ment of mature atherosclerotic plaques11. An increasing
number of studies seem to indicate that the administration of
exogenous antioxidants might decrease the impact of athero-
sclerosis in animals and humans through the regulation and
protection of several aspects of endothelial function12-14
.
On these bases and inspired by the invention of NCX-
4016, here we designed and synthesized a novel series of
hybrid structures in which some selected antioxidants were
linked to aspirin moieties through appropriate spacers. As
antioxidants, we chose caffeic acid (3,4-dihydroxycinnamic
acid), which is one of the most common phenolic acids and
occurs in fruits15, grains16 and dietary supplements17 for human
consumption as simple ester with quinic acid or saccharides.
The intake of caffeic acid from foods, mainly from tomatoes
and potatoes, was estimated to be about 0.2 mg/kg body weight
per day. It has been reported that caffeic acid possesses several
pharmacological properties like antioxidant18, free radical
scavenging19 and lipoxygenase inhibitor20. However, caffeic
acid has only limited solubility in hydrophobic media, which
reduces its antioxidant effects in inhibiting autooxidation of
fats and oils21. The strategy of esterification of hydrophilic
caffeic acid with lipophilic molecules, such as aliphatic alcohols,
could be employed to alter its solubility in a hydrophobic
medium. So in order to improve the solubility of the target
compounds in ester, we synthesized a series of caffeic acid
esters before they are combined with aspirin. As a result, the
aspirin-caffeic acid ester hybrids 3a-h were synthesized and
their activities were evaluated.
(E)-Methyl-3-(3,4-dihydroxyphenyl)acrylate (2a)22:
Yield 94 %; IR (KBr, νmax, cm-1): 3473, 3098, 1676, 1443,
1
1279, 583; H NMR (DMSO-d6, 300 MHz) δ: 3.69 (s, 3H,
OCH3), 6.27 (d, J = 15.9 Hz, 1H, C=CH), 6.76 (d, J = 8.1 Hz,
1H, Ar-H), 6.98-7.05 (m, 2H, 2Ar-H), 7.49 (d, J = 15.9 Hz,
1H, CH=C), 9.13 (s, 1H, OH), 9.57 (s, 1H, OH); ESI-MS: m/z
195 [M + H]+.
(E)-Ethyl-3-(3,4-dihydroxyphenyl)acrylate (2b)22:Yield
93%; IR (KBr, νmax, cm-1): 3453, 3088, 1855, 1668, 1607, 1531;
1H NMR (DMSO-d6, 300 MHz) δ: 1.26 (s, 3H, CH3), 4.15 (q,
J = 7.1 Hz, 2H, COOCH2), 6.25 (d, J = 15.9 Hz, 1H, C=CH),
6.76 (d, J = 8.2 Hz, 1H, Ar-H), 6.98-7.04 (m, 2H, 2Ar-H),
7.47 (d, J = 15.9 Hz, 1H, CH=C), 9.31 (s, 2H, 2OH); ESI-MS:
m/z 209 [M + H]+.
(E)-Propyl-3-(3,4-dihydroxyphenyl)acrylate (2c)22:
Yield 93 %; IR (KBr, νmax, cm-1): 3457, 2968, 1666, 1607,
1530, 1446; 1H NMR (DMSO-d6, 300 MHz) δ: 0.92 (t, J = 7.4
Hz, 3H, CH3), 1.64 (q, J = 7.1 Hz, 2H, CH2), 4.01 (t, J = 6.6
Hz, 2H, COOCH2), 6.26 (d, J = 15.9 Hz, 1H, C=CH), 6.76 (d,
J = 8 Hz, 1H, Ar-H), 6.99-7.04 (m, 2H, Ar-H), 7.47 (d, J =
15.9 Hz, 1H, CH=C), 9.11 (s, 1H, OH), 9.56 (s, 1H, OH);
ESI-MS: m/z 223 [M + H]+.
(E)-Isopropyl-3-(3,4-dihydroxyphenyl)acrylate (2d)23:
Yield 92 %; IR (KBr, νmax, cm-1): 3464, 3312, 2976, 1682,
EXPERIMENTAL
1
1599, 1530, 1445; H NMR (DMSO-d6, 300 MHz) δ: 1.23
Reagents and solvents were purchased from commercial
sources and used without further purification unless otherwise
specified. Air- and moisture-sensitive liquids and solutions
were transferred via syringe or stainless steel cannula. Organic
solutions were concentrated by rotary evaporation below 45 °C
at approximately 20 mm Hg. All non-aqueous reactions were
carried out under anhydrous conditions using flame-dried
glassware within an argon atmosphere in dry, freshly distilled
solvents, unless otherwise noted.Yields refer to chromatogra-
phically and spectroscopically (1H NMR) homogeneous
materials, unless otherwise stated. Reactions were monitored
by thin-layer chromatography (TLC) carried out on 0.15-0.20
(m, 6H, 2CH3), 4.99 (m, 1H, COOCH), 6.23 (d, J = 15.9 Hz,
1H, C=CH), 6.76 (d, J = 8 Hz, 1H, Ar-H), 6.98-7.04 (m, 2H,
Ar-H), 7.45 (d, J = 15.9 Hz, 1H, CH=C), 9.11 (s, 1H, OH),
9.56 (s, 1H, OH); ESI-MS: m/z 223 [M + H]+.
(E)-Butyl-3-(3,4-dihydroxyphenyl)acrylate (2e)22:Yield
93 %; IR (KBr, νmax, cm-1): 3484, 3340, 2953, 1601, 1533; 1H
NMR (DMSO-d6, 300 MHz) δ: 0.91 (t, J = 7.3 Hz, 3H, CH3),
1.36 (m, 2H, CH2), 1.59 (m, 2H, CH2), 4.11 (t, J = 6.6 Hz, 2H,
COOCH2), 6.26 (d, J = 15.9 Hz, 1H, C=CH), 6.76 (d, J = 8
Hz, 1H, Ar-H), 6.99-7.04 (m, 2H, Ar-H), 7.46 (d, J = 15.9 Hz,
1H, CH=C), 9.13 (s, 1H, OH), 9.59 (s, 1H, OH); ESI-MS: m/z
237 [M + H]+.

1344 Shi et al.
Asian J. Chem.
(E)-Isobutyl-3-(3,4-dihydroxyphenyl)acrylate (2f)23:
11H, Ar-H), 7.66 (d, J = 15.9 Hz, 1H, CH=C), 8.08 (m, 2H,
OH); ESI-MS: m/z 547 [M + H]+.
Yield 91 %; IR (KBr, νmax, cm-1): 3724, 3087, 1666, 1606, 1530,
1445; 1H NMR (DMSO-d6, 300 MHz) δ: 0.92 (d, J = 6.8 Hz,
6H, 2CH3), 1.93 (m, 1H, CH), 3.90 (d, J = 6.6 Hz, 2H, COOCH2),
6.27 (d, J = 15.9 Hz, 1H, C=CH), 6.76 (d, J = 8 Hz, 1H, Ar-H),
7-7.05 (m, 2H,Ar-H), 7.48 (d, J = 15.9 Hz, 1H, CH=C), 9.12 (s,
1H, OH), 9.58 (s, 1H, OH); ESI-MS: m/z 237 [M + H]+.
(E)-Pentyl-3-(3,4-dihydroxyphenyl)acrylate (2g)24:
Yield 91 %; IR (KBr, νmax, cm-1): 3342, 2861, 1684, 1599,
1532, 1445; 1H NMR (DMSO-d6, 300 MHz) δ: 0.88 (m, t, J =
7.1 Hz, 3H, CH3), 1.33 (m, 4H, 2CH2), 1.61 (m, 2H, CH2),
4.10 (t, J = 6.6 Hz, 2H, COOCH2), 6.25 (d, J = 15.9 Hz, 1H,
C=CH), 6.75 (d, J = 8 Hz, 1H, Ar-H), 6.99-7.04 (m, 2H, Ar-
H), 7.46 (d, J = 15.9 Hz, 1H, CH=C), 9.11 (s, 1H, OH), 9.58
(s, 1H, OH); ESI-MS: m/z 251 [M + H]+.
(E)-Butyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3e): Yield 61 %; IR (KBr, νmax, cm-1): 3492, 3064,
2958, 2357, 1755, 1700; 1H NMR (CDCl3, 300 MHz) δ: 0.97
(t, J = 7.3 Hz, 3H, CH3), 1.44 (m, 2H, CH2), 1.70 (m, 2H,
CH2), 2.28 (s, 6H, COCH3), 4.22 (t, J = 6.6 Hz, 2H, COOCH2),
6.43 (d, J = 15.9 Hz, 1H, C=CH), 7.09-7.60 (m, 11H, Ar-H),
7.67 (d, J = 15.9 Hz, 1H, CH=C), 8.07 (m, 2H, OH); ESI-MS:
m/z 561 [M + H]+.
(E)-Isobutyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3f): Yield 65 %; IR (KBr, νmax, cm-1): 3493, 3424,
3075, 2962, 2610, 2357; 1H NMR (CDCl3, 300 MHz) δ: 0.98
(d, J = 6.6 Hz, 6H, 2CH3), 2.04 (m, 1H, CH), 2.28 (s, 6H,
COCH3), 4.01 (d, J = 6.8 Hz, 2H, COOCH2), 6.46 (d, J = 15.9
Hz, 1H, C=CH), 7.08-7.60 (m, 11H, Ar-H), 7.68 (d, J = 15.9
Hz, 1H, CH=C), 8.05 (m, 2H, OH); ESI-MS: m/z 561 [M + H]+.
(E)-Pentyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3g): Yield 70 %; IR (KBr, νmax, cm-1): 2952, 2357,
1757, 1607, 1492, 1371; 1H NMR (CDCl3, 300 MHz) δ: 0.93
(t, J = 7 Hz, 3H, CH3), 1.37-1.41 (m, 4H, 2CH2), 1.72 (m, 2H,
CH2), 2.29 (s, 6H, 2COCH3), 4.21 (t, J = 6.7 Hz, 2H,
COOCH2), 6.44 (d, J = 15.9 Hz, 1H, C=CH), 7.09-7.60 (m,
11H, Ar-H), 7.68 (d, J = 15.9 Hz, 1H, CH=C), 8.09 (m, 2H,
OH); ESI-MS: m/z 575 [M + H]+.
(E)-Isopentyl-3-(3,4-dihydroxyphenyl)acrylate (2h)23:
Yield 90 %; IR (KBr, νmax, cm-1): 3483, 2955, 1684, 1601,
1532, 1447; 1H NMR (DMSO-d6, 300 MHz) δ: 0.90 (m, 6H,
2CH3), 1.52 (m, 2H, CH2), 1.68 (m, 1H, CH), 4.14 (t, J = 6.8
Hz, 2H, COOCH2), 6.25 (d, J = 15.9 Hz, 1H, C=CH), 6.75 (d,
J = 8 Hz, 1H, Ar-H), 6.99-7.04 (m, 2H, Ar-H), 7.46 (d, J =
15.9 Hz, 1H, CH=C), 9.12 (s, 1H, OH), 9.58 (s, 1H, OH);
ESI-MS: m/z 251 [M + H]+.
General procedures for the condensation of compound
2 with aspirin: To a stirring solution of aspirin (540 mg,
3 mmol), N,N-dicyclohexylcarbodiimide (DCC) (618 mg,
3 mmol) and N,N-(dimethylamino)pyridine (DMAP) (73.2
mg, 0.6 mmol) in dry CH2Cl2 (50 mL) at room temperature
was added compound 2 (1 mmol), then the reaction mixture
was stirred at room temperature for 4-10 h, at last the reaction
mixture was filtered. Removal of the solvent under vacuo
followed by silica gel column chromatographic purification
of the residue using petroleum ether-EtOAc (v:v = 8:1)
afforded the compounds (yields 57-73 %).
(E)-Isopentyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3h): Yield 66 %; IR (KBr, νmax, cm-1): 3525, 2959,
1
2357, 1757, 1639, 1606, 1496, 1371; H NMR (CDCl3, 300
MHz) δ: 0.97 (m, 6H, 2CH3), 1.60 (m, 2H, CH2), 1.76 (m, 1H,
CH), 2.28 (s, 6H, 2COCH3), 4.25 (d, J = 6.8 Hz, 2H, COOCH2),
6.44 (d, J = 15.9 Hz, 1H, C=CH), 7.09-7.58 (m, 11H, Ar-H),
7.67 (d, J = 15.9 Hz, 1H, CH=C), 8.08 (m, 2H, OH); ESI-MS:
m/z 575 [M + H]+.
Determination of antiplatelet aggregations activity:
New Zealand male Rabbits were housed in a conventional
animal facility and were allowed to acclimate for 7 days under
normal conditions (21-24 °C temperature and 60-70 % relative
humidity) before experimentation. All the protocols for the
animal study were approved by the Internal Animal Ethics
Committee of SK Chemical and Ethics Committee of Animal
Service Center at Nanjing University of Chinese Medicine.
Blood was withdrawn from carotid artery of New Zealand
rabbits, which was local anaesthetized by pentobarbital sodium
(30 mg/kg) and it was drawn into a plastic syringe containing
3.8 % trisodium citrate solution (1:9 citrate/blood, v/v). Platelet
rich plasma (PRP) was prepared by centrifugation at room
temperature for 10 min at 800 rpm. Platelet poor plasma (PPP)
was obtained from the precipitated fraction of platelet rich
plasma by centrifugation at room temperature for 10 min at
3000 rpm. Platelet rich plasma (280 µL) plus different samples
10 µL were incubated at 37 °C for 5 min in the aggregometer.
After incubation, the tested tube was turned into the test channel
and platelet aggregation was induced by the addition of ADP
(Final concentration is 5 µM). The aggregation inhibition rate
(AIR) was calculated with the following equation. X(%) =
(A-B)/A × 100, where X was platelet aggregation inhibition,
A was maximal aggregation of the control, B was maximal
aggregation of administration group. The IC50 value was calcu-
lated by the least-squares method25.
(E)-Methyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3a): Yield 73 %; IR (KBr, νmax, cm-1): 3433, 3322,
2959, 2850, 2354, 1756; 1H NMR (CDCl3, 300 MHz) δ: 2.39
(d, 6H, 2COCH3), 3.93 (s, 3H, COOCH3), 6.55 (d, J = 15.9
Hz, 1H, C=CH), 7.20-7.68 (m, 11H, Ar-H), 7.80 (d, J = 15.9
Hz, 1H, CH=C), 8.18 (m, 2H, 2OH); ESI-MS: m/z 519 [M + H]+.
(E)-Ethyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3b): Yield 70 %; IR (KBr, νmax, cm-1): 3547, 3328,
1
1756, 1634; H NMR (CDCl3, 300 MHz) δ: 1.33 (t, J = 7.1
Hz, 3H, CH3), 2.28 (s, 6H, 2COCH3), 4.27 (q, J = 7.1 Hz, 2H,
COOCH2), 6.43 (d, J = 15.9 Hz, 1H, C=CH), 7.08-7.58 (m,
11H, Ar-H), 7.68 (d, J = 15.9 Hz, 1H, CH=C), 8.08 (m, 2H,
OH); ESI-MS: m/z 533 [M + H]+.
(E)-Propyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3c): Yield 70 %; IR (KBr, νmax, cm-1): 2965, 2357,
1757, 1605, 1495; 1H NMR (CDCl3, 300 MHz) δ: 0.99 (t, J =
7.5 Hz, 3H, CH3), 1.74 (m, 2H, CH2), 2.28 (s, 6H, 2COCH3),
4.16 (t, J = 6.8 Hz, 2H, COOCH2), 6.44 (d, J = 15.9 Hz, 1H,
C=CH), 7.08-7.58 (m, 11H, Ar-H), 7.68 (d, J = 15.9 Hz, 1H,
CH=C), 8.04 (m, 2H, OH); ESI-MS: m/z 547 [M + H]+.
(E)-Isopropyl-3-[3,4-bis(2-acetoxybenzoyloxy)phenyl]-
acrylate (3d): Yield 57 %; IR (KBr, νmax, cm-1): 3510, 3326,
3074, 2982, 2933; 1H NMR (CDCl3, 300 MHz) δ: 1.31 (d, J =
6.2 Hz, 6H, 2CH3), 2.28 (s, 6H, 2COCH3), 5.14 (m, 1H,
COOCH), 6.42 (d, J = 15.9 Hz, 1H, C=CH), 7.08-7.58 (m,

Vol. 27, No. 4 (2015)
Design and Synthesis of Novel Aspirin-Caffeic Acid Ester Hybrids for Cardioprotection 1345
Determination of antioxidant activity: DPPH radical
scavenging activity was determined using the method of Wang
et al.26 with minor modifications. The solution of the sample
(10 µL) in ethanol was added to 90 µL of a 0.1 mM DPPH
radical in ethanol in a 96-well plate. The sample solution refers
to the tested compounds and the reference antioxidants at
various concentrations, as well as ethanol as a control. The
solutions of the tested compounds had concentrations ranging
from 3 to 1000 µg/mL, whereas the concentrations of the
solutions of the reference compounds varied from 0.1 to 1000
µg/mL. The reaction leading to the scavenging of DPPH radical
was complete within 10 min at 25 °C. The absorbance of the
mixture was then measured at 517 nm using a microplate
reader. The reduction of DPPH radical was expressed as
percentage: Scavenged DPPH (%) = (1-Atest/Acontrol) × 100,
where Atest is the absorbance of a sample at a given concen-
tration after 10 min reaction time and Acontrol is the absorbance
recorded for 10 µL ethanol. The IC50 value is defined as the
concentration of sample that causes 50 % loss of the DPPH
radical.
In recent years, microwave-assisted reactions have received
a great deal of attention, because reactions under microwave
irradiation were in general not only faster compared with the
conventional heating reactions, but also potentially more
efficient, cleaner and safer30. Further improvements have also
been reported which can offer enhanced reaction rates, higher
yields and greater selectivity to the targeted product under
milder reaction conditions31. So a highly efficient synthesis of
alkyl caffeates under microwave irradiation was adopted and
implemented in this paper, the time of these reactions ranged
from 3 to 6 min, which was much shorter than the traditional
synthetic methods22 and the alkyl caffeates (2a-h) were
obtained in higher yields.
Then compound 2a-h was condensed with aspirin,
respectively in CH2Cl2 in the presence of N,N-dicyclohexyl-
carbodiimide (DCC) and a catalytic amount of N,N-(dimethyl-
amino)pyridine (DMAP) to afford the corresponding target
compounds 3a-h.
Biological activity: The synthesized compounds were
evaluated for their antiplatelet aggregations induced by ADP
in vitro, aspirin was taken as a positive control drug in the
assays25. Firstly, the results reported in Table-1 showed that
for derivatives with same atom numbers, the antiplatelet
aggregation activity for caffeic acid esters with branched
carbon chains was much stronger than those with linear carbon
chains, for instance, the IC50 of compound 3e was 29.2 µM,
while the IC50 of compound 3f was 18.3 µM. Secondly, all the
target compounds exhibited potent antiplatelet aggregations
activity than aspirin, the most potent compound was compound
3d, which showed about 22 times more potent than aspirin
(The IC50 for these two compounds were 4.3 mmol/L and 90
mmol/L, respectively).
RESULTS AND DISCUSSION
Compounds 3a-h were prepared as shown in Scheme-I.
The intermediate compound 2a-h could be synthesized through
the method where the acid directly refluxed with alcohols in
the presence of various catalysts such as conc. sulfuric acid,
hydrogen chloride, boron trifluoride, aluminum chloride,
trifluoroacetic anhydride, polyphosphate ester, neodymium
oxide, dicyclohexylcarbodiimide, graphite bisulfate, etc27-29
.
The disadvantages of using these catalysts, such as long reac-
tion time, low yield, expensive reagents and tedious operation
were difficult to avoid. For instance, in 2002, Kadota's group22
described a classical esterification of caffeic acid in the
presence of methanol and p-TsOH to afford methyl caffeate
and the same procedure was used in the presence of ethanol to
afford ethyl caffeate, however, the yields of methyl caffeate
and ethyl caffeate were only 40 and 17 %, respectively.
Furthermore, such as in the case of NCX-4016, when
administered in vivo, esterases separated the aspirin moiety
from the benzene spacer-NO complex, leading to the release
of NO from this complex8. So in order to pre-evaluate the
antioxidant activity of the target hybrids in vivo, all the
TABLE-1
in vitro ANTIPLATELET AGGREGATIONS (IC50 In µM) INDUCED BY ADP OF TARGET COMPOUNDS
AND ANTIOXIDANT ACTIVITY OF INTERMEDIATE CAFFEIC ACID ESTERS IN DPPH ASSAY (IC50 In µM)
Compdounds
Antiplatelet
29.1
Compounds
Antiplatelet
29.2
Compounds
DPPH
7.16
7.67
25.28
6.98
9.3
Compounds
DPPH
33.78
25.48
14.12
7.4
3a
3b
3c
3d
Aspirin
3e
3f
3g
3h
2a
2b
2c
2d
2e
2f
2g
2h
31.7
33.7
4.3
90.0
18.3
46.6
40.4
Caffeic acid
O
OR
OH
O
OR
O
O
OH
O
O
HO
HO
HO
HO
AcO
OAc
DCC, DMAP
ROH, H2SO4
microwave irradiation
O
O
2a-h
3a-h
1
OAc
R= CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, (CH2)3CH3, CH CH(CH3)2, (CH2)4CH , CH CH CH(CH3)2
2
3
2
2
Scheme-I: Synthetic route of aspirin-caffeic acid ester hybrids (3a-h)

1346 Shi et al.
Asian J. Chem.
intermediate caffeic acid esters were evaluated by DPPH assay
through the measurement of the hydrogen-donating ability
to covert stable DPPH free radical to 1,1-diphenyl-2-picryl-
hydrazine26. The reaction was accompanied by a change in
color from deep-violet to light-yellow and was monitored
spectrophotometrically, caffeic acid served as reference anti-
oxidant in this assay. Table-1 showed that the intermediate
caffeic acid esters (2a) (IC50 = 7.16 µM) compound 2b (IC50 =
7.67 µM) compound 2d (IC50 = 6.98 µM) and compound 2h
(IC50 = 7.4 µM) exhibited good antioxidant activity in DPPH
assay, which were more potent than the reference antioxidant
caffeic acid (IC50 = 9.3 µM).
Because oxidative damage played a vital role in stroke,
so a kind of compound with both antiplatelet aggregation and
antioxidant activity would be very important for cardiopro-
tection. In this paper, we found that compound 3d not only
exhibited potent antiplatelet activity than aspirin, but also its
intermediate caffeic acid isopropyl ester (2d) showed good
antioxidant activity and thus compound 3d could be considered
to be novel potent cardioprotectic drug candidates with reduced
risk of hemorrhagic stroke.
1-13), Construction Project for Jiangsu Engineering Center
of Innovative Drug from Blood-conditioning TCM Formulae
and Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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In conclusion, we have obtained a new series of aspirin-
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confirmed that they were actually pharmacodynamic hybrids
possessing antiplatelet aggregations induced by ADP. In
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ACKNOWLEDGEMENTS
This work was supported by the Program for New Century
Excellent Talents by the Ministry of Education (NCET-12-
0741), Six Talents Project Funded by Jiangsu Province (2013-
YY-010), 333 High-level Talents Training Project Funded by
Jiangsu Province, National Natural Science Foundation of
China (No. 21302225, 81274058), Technology Innovation
Venture Fund by Nanjing University of Chinese Medicine
(CX201301), Jiangsu Collaborative Innovation Center of
Chinese Medicinal Resources Industrialization (ZDXMHT-
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