Design, synthesis and biological evaluation of novel trimethylpyrazine-2- carbonyloxy-cinnamic acids as potent cardiovascular agents

Article abstract of DOI:10.1039/c4md00022f

A series of novel trimethylpyrazine-2-carbonyloxy-cinnamic acids and esters were designed, synthesized and evaluated for their inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation in vitro and also assayed for their protective effect against hydrogen peroxide (H ₂O₂)-induced oxidative damage on Ea.hy926 cells. The results showed that many compounds exhibited high activity in one or both of the assays, of which, compound F′10 displayed the highest protective effect on the proliferation of the damaged Ea.hy926 cells (EC₅₀ = 1.7 μM), presenting almost 40 times higher potency than that of lipoic acid, and compound F3 was the most active anti-platelet aggregation agent with IC ₅₀ = 9.6 μM, comparable to that of clopidogrel. The structure-activity relationships of these compounds were also discussed.

Full text of DOI:10.1039/c4md00022f

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Design, synthesis and biological evaluation of novel
trimethylpyrazine-2-carbonyloxy-cinnamic acids
as potent cardiovascular agents†
Cite this: Med. Chem. Commun., 2014,
5, 711
Hongfei Chen,a Guoning Li,a Peng Zhan,a Hong Li,b Shouxun Wangb
a
*
and Xinyong Liu
A series of novel trimethylpyrazine-2-carbonyloxy-cinnamic acids and esters were designed, synthesized
and evaluated for their inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation in
vitro and also assayed for their protective effect against hydrogen peroxide (H2O2)-induced oxidative
damage on Ea.hy926 cells. The results showed that many compounds exhibited high activity in one or
both of the assays, of which, compound F010 displayed the highest protective effect on the proliferation
of the damaged Ea.hy926 cells (EC50 ¼ 1.7 mM), presenting almost 40 times higher potency than that of
lipoic acid, and compound F3 was the most active anti-platelet aggregation agent with IC50 ¼ 9.6 mM,
comparable to that of clopidogrel. The structure–activity relationships of these compounds were also
discussed.
Received 19th January 2014
Accepted 10th March 2014
DOI: 10.1039/c4md00022f
www.rsc.org/medchemcomm
Under normal conditions, the endothelium prevents plate-
lets from interacting with the vessel wall by synthesizing platelet
Introduction
inhibitors, such as prostaglandin I2 and NO, and shielding the
subendothelial adhesive proteins from the platelets' contact.9
When the endothelial cell lining is injured, platelets rapidly
adhere to the exposed subendothelial matrix and adhesive
proteins such as von Willebrand factor, collagen and brin-
ogen.10 Subsequent platelet activation results in the recruitment
of additional platelets and then leads to aggregation to form a
stable platelet plug.11,12
Ligustrazine (tetramethylpyrazine, TMP) is one of the alka-
loids isolated from the Chinese herb, Chuanxiong. Ligustrazine
has been routinely used to treat patients with coronary vascular
diseases. It is reported that ligustrazine showed signicant
inhibition on shear-induced platelet aggregation and calcium
channels in the platelet membrane.13,14 In recent studies, lig-
ustrazine is found to increase the production of NO in human
umbilical vein endothelial cells (HUVECs) and had an immu-
nomodulatory effect on HUVECs stimulated with TNF-a by
downregulating the expression of 1 intercellular adhesion
molecule and 60 heat shock proteins.15 These results suggested
that ligustrazine protects the endothelium via inhibition of
immunological reactions.16 Ligustrazine could also attenuate
oxidative damage and apoptosis and ameliorate the down-
regulation of superoxide dismutase (SOD) activity.17,18
Cardiovascular diseases (CVDs) such as ischemic heart disease
and stroke are the leading causes of death in the world.1
Advances in pathophysiologic research have revealed that CVDs
are caused by pathological states such as redox imbalance
(oxidative stress), endothelial dysfunction, endothelial inam-
mation, and excessive vascular remodeling.2
Although the mechanisms underlying endothelial dysfunc-
tion are complicated and multifactorial, much evidence indi-
cated that abnormally high levels of reactive oxygen species
(ROS) may have a vital role in this phenomenon.3
ROS include hydrogen peroxide (H2O2), peroxynitrite
(ONOOꢀ), superoxide anions (O2cꢀ), hydroxyl radicals (HOc) and
nitric oxide (NOc), which are created as part of normal cellular
metabolism and defense systems.4 Low levels of ROS are crucial
for the redox homeostasis and various cellular signaling path-
ways and regulate fundamental cell activities including cellular
growth, differentiation, proliferation and apoptosis.5–7 At high
concentrations, ROS leads to oxidative stress, which causes cell
injury and death and contributes to the pathogenesis of
atherosclerosis.8
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology
(Educational Ministry of China), School of Pharmaceutical Sciences, Shandong
University, No. 44 Wenhuaxi Road, Jinan 250012, China. E-mail: xinyongl@sdu.
edu.cn
bDepartment of Biological Chemistry and Molecular Biology, School of Medicine,
Weifang Medical University, No. 7166 Baotongxi Road, Weifang 261053, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00022f
Cinnamic acid and some of its derivatives (e.g. ferulic acid)
(Fig. 1) are components of plants such as soybeans, peanuts and
coffee, and they have been reported to display anti-oxidative
properties. It is believed that cinnamic acids are potential
scavengers of free radicals (such as O2cꢀ) and other oxidative
species, which is one of the mechanisms for antioxidant
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potential of oilseeds.19,20 Ozagrel (Fig. 1), a derivative of cin-
namic acid, is reported to be a highly selective thromboxane
(TXA2) synthase inhibitor. It is clinically used for the treatment
of acute cerebral thrombosis and prevention of postoperative
cerebral ischaemia, by precluding the TXA2 induced platelet
aggregation.21,22
In our previous work, it was found that some of ligustrazine
metabolites, such as 3,5,6-trimethylpyrazine-2-carboxylic acid,
displayed moderate activity on lowering the level of serum
cholesterol and low density lipoprotein in vivo (Fig. 1).
Inspired by the structural characteristics and the drug-like
properties of cinnamic acid and in continuation of our work, we
conjugated the ligustrazinyloxy group with a cinnamic acid
group to obtain a novel series of trimethylpyrazine-2-carbon-
yloxy-cinnamic acids (see Fig. 2), in the hope of getting novel
ligustrazine derivatives with high anti-oxidative activity or anti-
platelet aggregation activity. It is a further objective of this
paper to understand the structure–activity relationships and to
afford information through diversied molecular modication
of ligustrazine.
Scheme 1 Reagents and conditions: (i) KMnO4, H2O, 35 ꢁC, 24 h, (ii)
oxalyl chloride, anhydrous DCM, ice bath, 10 min, (iii) Et3N, anhydrous
DCM, r.t., 1 h, and (iv) EtOH, SOCl2, reflux, 12 h.
Scheme 2 Preparation of the hydroxyl cinnamic acids. Reagents and
conditions: (i) malonic acid, pyridine, cat. piperidine, 120 ꢁC, 1.5 h.
Table 1 Structures of the synthesized compounds
Results and discussion
Compound Structure
Compound Structure
Chemistry
A convenient synthetic route to title compounds is shown in
Scheme 1. In this approach 3,5,6-trimethylpyrazine-2-carboxylic
acid (2), previously synthesized by oxidation of ligustrazine (1)
with potassium permanganate, was reacted with oxalyl chloride
in dichloromethane in an ice bath for 10 min, to obtain 3,5,6-
trimethylpyrazine-2-carbonyl chloride (3), which was used for
the next step directly. Compounds F(1–10) were synthesized
from 3 by reaction with cinnamic acids, and followed by ester-
ication to afford the corresponding trimethylpyrazine-2-car-
bonyloxy-cinnamic acid esters F0(1–10). Cinnamic acids with
different substitutents at the phenyl group were prepared
according to the Knoevenagel condensation,23 depicted in
F1
F2
F01
F02
F3
F4
F5
F03
F04
F05
F6
F7
F06
F07
Fig. 1 Structure of cinnamic acid, ferulic acid, ozagrel and 3,5,6-tri-
methylpyrazine-2-carboxylic acid.
F8
F9
F08
F09
F10
F010
Fig. 2 Design of trimethylpyrazine-2-carbonyloxy-cinnamic acids.
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Scheme 2. All the newly synthesized compounds (Table 1) were trimethylpyrazine-2-carbonyloxy position at the phenyl group of
1
structurally conrmed by IR, H-NMR and MS.
cinnamic acids for antiplatelet activity was observed as the
following: ortho-position > meta-position > para-position. In the
ortho-substituted series F(3, 6–8), compounds containing a
methoxyl or chloro group at the phenyl ring of cinnamic acids
F(6–8) displayed reduced anti-platelet activities (IC50 ¼ 57.2,
133.8, and 49.0 mM, respectively) compared to monosubstituent
compound F3. The structure–activity relationship (SAR) analysis
provided important information for further molecular
optimization.
It is noteworthy that the activity of some compounds is not
concentration dependent, such as for F1, F01 and F2, whose AIR
value decreased with higher concentrations. Since the platelet
aggregation is a rather complicated process, it is not clear which
factor affected the aggregation in the assay. A possible reason is
that high concentrations of those compounds activated the
platelets and induced the aggregation.
The possible anti-platelet aggregation mechanisms of the
synthesized ligustrazine derivatives are (1) that they are prob-
ably TXA2 synthase inhibitors, considering that they contain a
similar cinnamic acid moiety with ozagrel and (2) that the lig-
ustrazine moiety played a role in inhibiting calcium channels in
the platelet membrane. Further molecular biological study is
underway to conrm the mechanism of these compounds.
Protective effect on damaged Ea.hy926 cells. Setting lipoic
acid and butylated hydroxyanisole (BHA) as the positive
Biological results
Anti-platelet activity. The newly synthesized compounds,
ligustrazine and some cinnamic acids/ethyl esters were tested
for their inhibition against rabbit platelet aggregation in vitro;24
ozagrel and clopidogrel were used as control drugs. The results,
expressed as the aggregation inhibition rate (AIR) and IC50, are
summarized in Table 2.
As shown in Table 2, 4-OH-cinnamic acid, 4-OH-ethyl cin-
namate, 2-OH-cinnamic acid and ethyl ferulate displayed
moderate anti-platelet aggregation activity (IC50 ¼ 74.8, 130.4,
115.8 and 193.2 mM, respectively), while ligustrazine possessed
weaker anti-platelet aggregation activity (IC50 ¼ 265.3 mM). The
corresponding ligustrazine–cinnamic acids/ethyl esters (F1, F01,
F3 and F04) exhibited enhanced activity (IC50 ¼ 37.8, 24.4, 9.6
and 58.4 mM, respectively), indicating that the conjugation is an
effective approach to obtain more potent compounds.
Among all the synthesized compounds, F01, F2, F3, F5, F07
and F09 were the most potent platelet aggregation inhibitors
with IC50 values 24.4, 26.4, 9.6, 41.8, 28.2 and 37.5 mM,
respectively, much more active than that of ozagrel (IC50 ¼ 144.1
mM), but slightly lower than that of clopidogrel (IC50 ¼ 7.6 mM).
In the case of compounds derived from monosubstituted
cinnamic acid (F1, F2 and F3),
a
clear order of
Table 2 The IC50 for inhibition of platelet aggregation
a
Compound
AIR 100 mM
AIR 50 mM
AIR 25 mM
AIR 12.5 mM
IC50 (mM)
F1
25.3%
20.5%
8.2%
41.7%
32.2%
28.0%
59.6%
53.8%
39.0%
46.2%
49.9%
59.8%
32.2%
37.0%
44.4%
34.1%
18.7%
48.8%
72.9%
51.2%
55.9%
44.5%
37.9%
25.0%
92.3%
53.2%
25.5%
30.5%
17.9%
15.3%
53.3%
39.1%
38.5%
62.2%
54.8%
16.6%
22.8%
37.2%
53.5%
20.6%
38.1%
36.9%
35.1%
32.6%
46.5%
73.9%
17.4%
47.2%
25.0%
36.4%
4.8%
64.3%
68.0%
73.4%
25.1%
32.9%
11.8%
60.6%
31.0%
21.6%
22.3%
30.0%
26.3%
16.5%
84.2%
43.4%
70.5%
12.0%
44.0%
32.1%
22.0%
6.4%
37.8 ꢂ 9.3
24.4 ꢂ 8.5
26.4 ꢂ 3.9
35.8 ꢂ 15.2
9.6 ꢂ 5.2
F01
F2
F02
69.4%
17.2%
42.4%
73.2%
64.4%
75.2%
39.7%
74.5%
61.7%
40.9%
47.0%
59.8%
60.7%
52.1%
59.5%
53.6%
58.4%
36.8%
94.5%
54.5%
37.9%
42.9%
26.5%
19.6%
F3
F03
110.2 ꢂ 17.2
44.6 ꢂ 7.3
58.4 ꢂ 5.8
41.8 ꢂ 4.2
143.0 ꢂ 13.7
57.2 ꢂ 6.4
67.1 ꢂ 4.1
133.8 ꢂ 19.1
28.2 ꢂ 4.2
49.0 ꢂ 3.9
195.8 ꢂ 30.4
82.3 ꢂ 5.5
37.5 ꢂ 4.5
84.6 ꢂ 6.2
77.4 ꢂ 5.8
144.1 ꢂ 12.1
7.6 ꢂ 2.3
F4
F04
F5
F05
F6
F06
F7
F07
F8
F08
F9
F09
F10
F010
Ozagrel
Clopidogrel
4-OH-cinnamic acid
4-OH-ethyl cinnamate
2-OH-cinnamic acid
Ethyl ferulate
Ligustrazine
77.6%
29.9%
16.4%
21.4%
11.5%
8.6%
86.9%
17.2%
5.8%
10.8%
4.0%
74.8 ꢂ 3.7
130.4 ꢂ 8.9
115.8 ꢂ 5.5
193.2 ꢂ 11.2
265.3 ꢂ 14.5
3.4%
a
IC50: concentration of compound required to achieve 50% inhibition on aggregation of rabbit platelets.
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Table 3 The EC50 values for protection on damaged Ea.hy926 cells and P% at different concentrations of the compounds
a
Compound
P% (100 mM)
P% (50 mM)
P% (25 mM)
P% (12.5 mM)
EC50 (mM)
F1
93.2%
96.1%
103.0%
47.0%
80.1%
40.9%
55.4%
71.6%
216.3%
117.0%
52.2%
11.0%
18.0%
39.9%
79.2%
46.2%
163.0%
ꢀ47.0%
601.4%
375.3%
58.1%
43.4%
81.9%
69.1%
155.6%
146.9%
40.9%
74.1%
62.6%
75.3%
39.5%
64.9%
36.9%
49.1%
69.7%
159.6%
89.0%
23.7%
33.7%
31.6%
20.7%
62.9%
27.8%
96.6%
ꢀ13.6%
284.8%
210.8%
48.7%
28.0%
62.9%
32.0%
149.5%
134.8%
22.1%
46.6%
58.8%
64.3%
34.2%
61.8%
17.8%
40.3%
53.3%
128.1%
60.1%
26.0%
62.5%
ꢀ52.1%
16.9%
45.0%
23.4%
24.0%
20.9%
271.1%
207.0%
ꢀ12.3%
22.6%
36.8%
24.9%
108.5%
88.4%
13.6%
34.2%
42.7%
61.6%
27.9%
45.7%
9.6%
38.7%
52.4%
41.3%
26.4%
6.9%
64.9%
39.7%
ꢀ57.6%
21.7%
15.9%
5.7%
96.6%
92.8%
75.7%
30.5%
17.2%
34.9%
4.9%
38.0 ꢂ 2.2
34.9 ꢂ 3.7
24.0 ꢂ 2.1
92.6 ꢂ 2.8
36.7 ꢂ 3.5
108.4 ꢂ 4.1
69.4 ꢂ 4.9
38.6 ꢂ 1.7
8.8 ꢂ 1.1
F01
F2
F02
F3
F03
F4
F04
F5
F05
29.9 ꢂ 1.4
95.2 ꢂ 4.2
36.1 ꢂ 1.6
115.3 ꢂ 4.9
137.7 ꢂ 5.2
48.6 ꢂ 2.2
104.6 ꢂ 4.4
34.7 ꢂ 1.5
21.4 ꢂ 1.5
2.2 ꢂ 1.1
F6
F06
F7
F07
F8
F08
F9
F09
F10
F010
1.7 ꢂ 0.9
Lipoic acid
BHA
3-OH-4-OCH3-cinnamic acid
3,5-diOCH3-4-OH-ethyl cinnamate
Caffeic acid
68.0 ꢂ 5.9
111.4 ꢂ 5.2
29.3 ꢂ 2.3
67.2 ꢂ 2.1
12.6 ꢂ 1.8
11.6 ꢂ 1.0
83.4 ꢂ 4.1
16.7%
80.4%
11.4%
Ethyl caffeate
Ligustrazine
a
EC50: concentration of compound required to achieve 50% protection of Ea.hy926 cells from H2O2 induced cytotoxicity, as determined by the MTT
method.
control drugs, all the synthesized compounds, ligustrazine the para position of the cinnamic acids, F10 and F010 (EC50
¼
and some cinnamic acids/ethyl esters, were also tested for 2.2 and 1.7 mM, respectively) with the hydroxyl group at the 3
their protective effect on the Ea.hy926 cells damaged by position of the benzene ring showed better protective effects
hydrogen peroxide.25,26 Table 3 revealed the proliferation rate than compounds F1 and F01 (EC50 ¼ 38.0 and 34.9 mM), which
(P%) at different concentrations and 50% effective concen- may be attributed to the anti-oxidative effect of the hydroxyl
tration for protecting damaged Ea.hy926 cells of newly group. From the proliferation rate presented in Table 3, it also
synthesized compounds.
can be found that the potencies of compounds F10 and F010
As shown in Table 3, 3-OH-4-OCH3-cinnamic acid, 3,5- were concentration dependent, reaching the maximum value
diOCH3-4-OH-ethyl cinnamate, caffeic acid (F10) and ethyl caf- (P% ¼ 601.4%, 375.3%, respectively) at 100 mM, whereas
feate (F010) displayed moderate to high anti-platelet aggregation compounds F4 and F04 containing a methoxyl group at the 3
activity (IC50 ¼ 29.3, 67.2, 12.6 and 11.6 mM, respectively), while position of the phenyl group did not give desirable potencies.
ligustrazine possessed mild anti-platelet aggregation activity Among compounds F(2, 5) and F0(2, 5) substituted with tri-
(IC50 ¼ 83.4 mM). The corresponding ligustrazine–cinnamic methylpyrazine-2-carbonyloxy groups at the meta position of
acids/ethyl esters (F5, F09, F10 and F010) exhibited enhanced the cinnamic acid, F5 and F05 with the methoxyl group at the 5
activity (IC50 ¼ 8.8, 21.4, 2.2 and 1.7 mM, respectively).
position of the phenyl ring showed enhanced protective
8.8 and 29.9 mM, respectively) than
From the obtained results, it was observed that some of the effects (EC50
¼
trimethylpyrazine-2-carbonyloxy-cinnamic acids/ethyl esters compounds F2 and F02 (EC50
protected the proliferation of injured Ea.hy926 cells with respectively).
¼
24.0 and 92.6 mM,
commensurable or higher potency compared to control drugs
As listed in the table, the activity of most compounds' is
lipoic acid or BHA (EC50 ¼ 68.0 mM, 111.4 mM, respectively). concentration dependent, except for F06, F7 and F09, whose P%
Among all the trimethylpyrazine-2-carbonyloxy-cinnamic acid value decreased with higher concentration. The possible reason
and esters, F2, F5, F05, F09, F10, and F010 displayed remarkable is that high concentrations of these compounds exhibited
anti-oxidative activity (EC50 ¼ 24.0, 8.8, 29.9, 21.4, 2.2, and toxicity rather than protection to the cells.
1.7 mM, respectively). Particularly, the compound F010 pre-
sented almost 40 times higher potency than lipoic acid.
Among compounds F(1, 4, 9, 10) and F0(1, 4, 9, 10) effect of both ligustrazine and cinnamic acids/ethyl esters, the
substituted with trimethylpyrazine-2-carbonyloxy groups at conjugation of which led to a synergic effect.
The mechanism of the synthesized compounds' anti-oxida-
tive activity may be attributed to the free radical-scavenging
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Cinnamic acid (2 mmol) was added into the dichloro-
methane solution of 3,5,6-trimethylpyrazine-2-carbonyl chlo-
ride (2 mmol) obtained in the above step at room temperature;
triethylamine (4 mmol) was added to the mixture which was
then stirred at 25 ꢁC for 1 h (checked by TLC). The solvent was
removed and the residue was dissolved in ethyl acetate
(100 mL), washed with water and brine, successively. The
organic layer was dried with Na2SO4, ltered and concentrated
under reduced pressure to afford the crude product. The nal
product was puried by ash column chromatography and
recrystallization from ethyl acetate.
(E)-3-(4-((3,5,6-Trimethylpyrazine-2-carbonyl)oxy)phenyl)-
acrylic acid (F1). White needle crystals, yield: 63%, mp: 212–214
ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 12.46 (s, 1H, COOH),
7.82 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.66 (d, 1H, Ar-CH]C, J ¼ 16.0
Hz), 7.36 (d, 2H, Ar-H, J ¼ 8.4 Hz), 6.58 (d, 1H, C]CH–C]O, J ¼
16.0 Hz), 2.72 (s, 3H, CH3), 2.57 (s, 3H, CH3), 2.55 (s, 3H, CH3).
IR (KBr, cmꢀ1): 3400 (OH), 2925 (CH3), 1743, 1693 (C]O), 1628
(C]C), 1599, 1583, 1506 (C]N, C]C), 1164 (C–O). ESI-MS:
313.3 (M + H)+, calcd for C17H16N2O4 312.32.
Conclusion
In conclusion, a series of novel trimethylpyrazine-2-carbon-
yloxy-cinnamic acids were designed, synthesized and biologi-
cally evaluated for their inhibitory activities against the platelet
aggregation, and their protective effect on the damaged
Ea.hy926 cell proliferation. The results showed that some
compounds exhibited high activity in one or both of the assays,
of which compound F010 displayed the highest protective effect
on the proliferation of the damaged Ea.hy926 cells (EC50
¼
1.7 mM), and compound F3 was the most active anti-platelet
aggregation agent with IC50 ¼ 9.6 mM, demonstrating the
potential values in further development of cardiovascular
agents. The structure–activity relationship was discussed in
detail. In addition, the molecular research of these novel
compounds is underway. Further studies are ongoing in our
laboratories and will be reported in due course.
Experimental
(E)-3-(3-((3,5,6-Trimethylpyrazine-2-carbonyl)oxy)phenyl)-
acrylic acid (F2). Yellow powder, yield: 54%, mp: 208–210 ꢁC.
1H-NMR (400 MHz, CDCl3, d ppm): 12.48 (s, 1H, COOH), 7.68–
7.60 (m, 3H, Ar-H, Ar-CH]C), 7.54 (t, 1H, Ar-H, J ¼ 7.8 Hz), 7.36
(dd, 1H, Ar-H, J1 ¼ 1.5 Hz, J2 ¼ 7.9 Hz), 6.64 (d, 1H, C]CH–C]
O, J ¼ 16.0 Hz), 2.73 (s, 3H, CH3), 2.57 (s, 3H, CH3), 2.55 (s, 3H,
CH3). IR (KBr, cmꢀ1): 3450 (OH), 2966, 2852 (CH3), 1736, 1692
(C]O), 1631 (C]C), 1580, 1539 (C]N, C]C), 1172 (C–O). ESI-
MS: 313.4 (M + H)+, calcd for C17H16N2O4 312.32.
The melting points of the compounds were metered on a
micromelting point apparatus. Infrared spectra were recorded
with a Nexus 470 FT-IR Spectrometer. 1H NMR spectra were
determined using a Bruker Avance (400 MHz) NMR-spectrometer
in the indicated solvents. Chemical shis are expressed in d units
and TMS as the internal reference. Mass spectra were recorded
with an LC Autosampler Device: Standard G1313A instrument.
TLC was performed on silica gel GF254 for TLC (Merck) and spots
were visualized by iodine vapors or by irradiation with UV light
(254 nm). Flash column chromatography was performed on a
column packed with silica gel 60 (230–400 mesh). Solvents were
of reagent grade and, when necessary, were puried and dried by
standard methods. The concentration of the reaction solutions
involved the use of a rotary evaporator at reduced pressure. The
yields were calculated using the last step reaction.
(E)-3-(2-((3,5,6-Trimethylpyrazine-2-carbonyl)oxy)phenyl)-
acrylic acid (F3). Light yellow needle crystals, yield: 57%, mp:
1
204–206 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.48 (s, 1H,
COOH), 7.94 (d, 1H, Ar-H, J ¼ 7.8 Hz), 7.67 (d, 1H, Ar-CH]C, J ¼
15.6 Hz), 7.56–7.53 (m, 1H, Ar-H), 7.42–7.38 (m, 2H, Ar-H), 6.75
(d, 1H, C]CH–C]O, J ¼ 15.6 Hz), 2.71 (s, 3H, CH3), 2.58 (s, 3H,
CH3), 2.50 (s, 3H, CH3). IR (KBr, cmꢀ1): 3450 (OH), 2980, 2929
(CH3), 1733, 1692 (C]O), 1627 (C]C), 1577, 1543 (C]N, C]C),
1166 (C–O). ESI-MS: 313.4 (M + H)+, calcd for C17H16N2O4
General procedure for the preparation of the compounds F(1–10)
To an aqueous solution of 2,3,5,6-tetramethylpyrazine (0.1 mol) 312.32.
was added potassium permanganate (0.4 mol). The reaction
mixture was stirred at 35 C for 24 h to give the intermediate phenyl) acrylic acid (F4). White needle crystals, yield: 61%, mp:
3,5,6-trimethylpyrazine-2-carboxylic acid. Oxalyl chloride 214–218 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.44 (s, 1H,
(E)-3-(3-Methoxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
ꢁ
1
(2 mmol) was added to the dichloromethane (10 mL, anhy- COOH), 7.63 (d, 1H, Ar-CH]C, J ¼ 15.6 Hz), 7.56 (s, 1H, Ar-H),
drous) solution of 3,5,6-trimethylpyrazine-2-carboxylic acid 7.34 (d, 1H, Ar-H, J ¼ 7.8 Hz), 7.30 (d, 1H, Ar-H, J ¼ 7.8 Hz), 6.65
(2 mmol) in an ice bath. When the addition was completed with (d, 1H, C]CH–C]O, J ¼ 16.2 Hz), 3.85 (s, 3H, OCH3), 2.70 (s,
further stirring for 10 min, the solvent and redundant oxalyl 3H, CH3), 2.56 (s, 3H, CH3), 2.51 (s, 3H, CH3). 13C-NMR (400
chloride were removed under reduced pressure to obtain 3,5,6- MHz, CDCl3, d ppm): 119.94, 121.87, 124.21, 133.62, 137.93,
trimethylpyrazine-2-carbonyl chloride.
141.17, 143.73, 147.73, 150.10, 151.48, 155.96, 165.11 (–COO–),
Cinnamic acids with different substitutents at the phenyl 168.11 (–COO–). 13C-NMR (400 MHz, CDCl3, d ppm): 21.51
group were prepared according to the Knoevenagel condensa- (–CH3), 22.38 (–CH3), 22.61 (–CH3), 56.19 (–OCH3), 112.25,
tion. To the solution of hydroxybenzaldehyde (2.0 mmol) in 119.94, 121.87, 123.62, 133.62, 137.93, 141.17, 143.73, 150.10,
pyridine were added malonic acid (2.4 mmol) and piperidine. 151.48, 151.80, 155.96, 163.69 (–COO–), 168.11 (–COO–). IR
The mixture was reuxed at 120 ꢁC for 1.5 h. Upon completion (KBr, cmꢀ1): 3422 (OH), 2924, 2852 (CH3), 1745, 1696 (C]O),
of the reaction, the mixture was diluted with ethyl acetate 1630 (C]C), 1591 (C]N, C]C), 1152 (C–O). ESI-MS: 343.5 (M +
(100 mL) and washed with 0.1 N HCl solution. The organic layer H)+, calcd for C18H18N2O5 342.35. HRMS: m/z calcd for
was dried with Na2SO4, ltered and concentrated under reduced
pressure to afford cinnamic acids.
C
18H18N2O5 [M + H]+ 343.1290, found 343.1288.
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6.69 (d, 1H, C]CH–C]O, J ¼ 15.6 Hz), 5.35 (s, 2H, OH), 2.71 (s,
3H, CH3), 2.56–2.50 (m, 6H, CH3 ꢃ 2). 13C-NMR (400 MHz,
CDCl3, d ppm): 21.47 (–CH3), 22.32 (–CH3), 22.67 (–CH3), 117.20,
119.56, 123.29, 128.20, 133.60, 138.31, 139.11, 143.74, 149.64,
149.91, 151.47, 155.64, 162.75 (–COO–), 172.47 (–COO–). IR
(KBr, cmꢀ1): 3470 (OH), 2966, 2852 (CH3), 1736, 1692 (C]O),
1631 (C]C), 1580, 1539 (C]N, C]C), 1172 (C–O). ESI-MS:
329.5 (M + H)+, calcd for C17H16N2O5 328.11. HRMS: m/z calcd
for C17H16N2O6 [M + H]+ 329.1137, found 329.1132.
(E)-3-(4-Methoxy-3-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
phenyl)acrylic acid (F5). White needle crystals, yield: 64%, mp:
1
202–204 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.36 (s, 1H,
COOH), 7.68–7.61 (m, 3H, Ar-H, Ar-CH]C), 7.24 (d, 1H, Ar-H, J
¼ 8.4 Hz), 6.48 (d, 1H, C]CH–C]O, J ¼ 16.2 Hz), 3.84 (s, 3H,
OCH3), 2.71 (s, 3H, CH3), 2.57–2.49 (m, 6H, CH3 ꢃ 2). IR (KBr,
cmꢀ1): 3488 (OH), 2957, 2927, 2848 (CH3), 1753, 1720 (C]O),
1636 (C]C), 1610 (C]N, C]C), 1161 (C–O). ESI-MS: 343.5 (M +
H)+, calcd for C18H18N2O5 342.35.
(E)-3-(5-Chloro-2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
phenyl)acrylic acid (F6). White needle crystals, yield: 89%, mp:
General procedure for the preparation of the compounds F0(1–10)
1
229–233 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.79 (s, 1H,
To an icy solution of the ligustrazinacyl–cinnamic acids
(10 mmol) in ethanol (30 mL) was added thionyl chloride
(0.5 mL) dropwise. The reaction mixture was reuxed for 24 h
to give the corresponding crude ethyl cinnamate. Upon
completion of the reaction, the solvent was removed and the
residue was diluted with ethyl acetate, and washed with water
and brine, successively. The organic layer was dried with
Na2SO4, ltered and concentrated under reduced pressure to
afford the crude product. The nal product was puried by
ash column chromatography and recrystallization from
n-hexane.
(E)-4-(3-Ethoxy-3-oxoprop-1-en-1-yl)phenyl-3,5,6-trimethylpyr-
azine-2-carboxylate (F01). White needle crystals, yield: 81%, mp:
77–80 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 7.86 (d, 2H, Ar-H, J
¼ 8.6 Hz), 7.72 (d, 1H, Ar-CH]C, J ¼ 16.0 Hz), 7.37 (d, 2H, Ar-H, J
¼ 8.6 Hz), 6.68 (d, 1H, C]CH–C]O, J ¼ 16.0 Hz), 4.24 (q, 2H,
OCH2, J ¼ 7.0 Hz), 2.72 (s, 3H, CH3), 2.57 (s, 3H, CH3), 2.55 (s,
3H, CH3), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr, cmꢀ1): 3063 (C]
C–H), 2988, 2929 (CH3), 1747, 1712 (C]O), 1642 (C]C), 1601,
1541, 1509 (C]N, C]C), 1169 (C–O). ESI-MS: 341.4 (M + H)+,
calcd for C19H20N2O4 340.37.
(E)-3-(3-Ethoxy-3-oxoprop-1-en-1-yl)phenyl-3,5,6-trimethylpyr-
azine-2-carboxylate (F02). White needle crystals, yield: 81%, mp:
87–91 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 7.72–7.66 (m, 3H,
Ar–H, Ar-CH]C), 7.55 (t, 1H, Ar-H, J ¼ 7.9 Hz), 7.37 (dd, 1H, Ar-
H, J1 ¼ 1.6 Hz, J2 ¼ 7.9 Hz), 6.74 (d, 1H, C]CH–C]O, J ¼ 16.0
Hz), 4.23 (q, 2H, OCH2, J ¼ 7.1 Hz), 2.73 (s, 3H, CH3), 2.57 (s, 3H,
CH3), 2.55 (s, 3H, CH3), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr,
cmꢀ1): 3061 (C]C–H), 2977, 2927 (CH3), 1731, 1716 (C]O),
1637 (C]C), 1604, 1583, 1541 (C]N, C]C), 1169 (C–O). ESI-MS:
341.4 (M + H)+, calcd for C19H20N2O4 340.37.
(E)-2-(3-Ethoxy-3-oxoprop-1-en-1-yl)phenyl-3,5,6-trimethylpyr-
azine-2-carboxylate (F03). White needle crystals, yield: 81%, mp:
77–79 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 7.97 (d, 1H, Ar-H, J
¼ 7.8 Hz), 7.75 (d, 1H, Ar-CH]C, J ¼ 16.2 Hz), 7.61–7.54 (m, 1H,
Ar-H), 7.45–7.38 (m, 2H, Ar-H), 6.93 (d, 1H, C]CH–C]O, J ¼
16.1 Hz), 4.23 (q, 2H, OCH2, J ¼ 7.2 Hz), 2.73 (s, 3H, CH3), 2.57–
2.49 (m, 6H, CH3 ꢃ 2), 1.30 (t, 3H, CH3, J ¼ 7.2 Hz). 13C-NMR
(400 MHz, CDCl3, d ppm): 14.54 (–CH3), 21.57 (–CH3), 22.39
(–CH3), 22.46 (–CH3), 60.25 (–OCH2–), 119.84, 121.38, 123.87,
127.22, 129.37, 131.96, 137.98, 140.42, 149.59, 150.18, 151.88,
156.25, 164.12 (–COO–), 166.52 (–COO–). IR (KBr, cmꢀ1): 3080
(C]C–H), 2988, 2957 (CH3), 1745, 1705 (C]O), 1629 (C]C),
1601, 1577, 1541 (C]N, C]C), 1151 (C–O). ESI-MS: 341.4 (M +
H)+, calcd for C19H20N2O4 340.37. HRMS: m/z calcd for
COOH), 8.06 (d, 1H, Ar-CH]C, J ¼ 2 Hz), 7.63–7.58 (m, 2H, Ar-
H), 7.50 (d, 1H, Ar-H, J ¼ 8.7 Hz), 6.88 (d, 1H, C]CH–C]O, J ¼
16.0 Hz), 2.72 (s, 3H, CH3), 2.62–2.48 (m, 6H, CH3 ꢃ 2). IR (KBr,
cmꢀ1): 3429 (OH), 2960, 2925, 2854 (CH3), 1739, 1696 (C]O),
1629 (C]C), 1571, 1541 (C]N, C]C), 1159 (C–O). ESI-MS:
347.3 (M + H)+, calcd for C17H15ClN2O4 346.76.
(E)-3-(4-Methoxy-2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
phenyl) acrylic acid (F7). White needle crystals, yield: 84%, mp:
1
210–212 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.32 (s, 1H,
COOH), 7.88 (d, 1H, Ar-H, J ¼ 8.8 Hz), 7.59 (s, 1H, Ar-CH]C, J ¼
16.0 Hz), 7.04 (d, 1H, Ar-H, J ¼ 2.5 Hz), 6.99 (dd, 1H, Ar-H, J1 ¼
2.6 Hz, J2 ¼ 8.8 Hz), 6.61 (d, 1H, C]CH–C]O, J ¼ 16.0 Hz), 3.83
(s, 3H, OCH3), 2.72 (s, 3H, CH3), 2.58 (s, 3H, CH3), 2.51 (s, 3H,
CH3). IR (KBr, cmꢀ1): 3478 (OH), 2956, 2927 (CH3), 1761, 1720
(C]O), 1636 (C]C), 1610 (C]N, C]C), 1161 (C–O). ESI-MS:
343.4 (M + H)+, calcd for C18H18N2O5 342.35.
(E)-3-(5-Methoxy-2-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
phenyl)acrylic acid (F8). White powder, yield: 81%, mp: 213–215
ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 12.48 (s, 1H, COOH),
7.61 (d, 1H, Ar-CH]C, J ¼ 16.2 Hz), 7.44 (s, 1H, Ar-H), 7.33 (d,
1H, Ar-H, J ¼ 9 Hz), 7.10 (m, 1H, Ar-H), 6.80 (d, 1H, C]CH–C]
O, J ¼ 16.2 Hz), 3.84 (s, 3H, OCH3), 2.70 (s, 3H, CH3), 2.57–2.44
(m, 6H, CH3 ꢃ 2). IR (KBr, cmꢀ1): 3442 (OH), 2977, 2924, 2836
(CH3), 1732, 1693 (C]O), 1632 (C]C), 1582, 1541 (C]N, C]C),
1161 (C–O). ESI-MS: 343.4 (M + H)+, calcd for C18H18N2O5
342.35.
(E)-3-(3,5-Dimethoxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)-
oxy)phenyl)acrylic acid (F9). White needle crystals, yield: 82%,
1
ꢁ
mp: 228–232 C. H-NMR (400 MHz, CDCl3, d ppm): 12.44 (s,
1H, COOH), 7.62 (d, 1H, Ar-CH]C, J ¼ 16.0 Hz), 7.19 (s, 2H, Ar-
H), 6.70 (d, 1H, C]CH–C]O, J ¼ 16.0 Hz), 3.83 (s, 6H, OCH3 ꢃ
2), 2.70 (s, 3H, CH3), 2.56–2.50 (m, 6H, CH3 ꢃ 2). 13C-NMR (400
MHz, CDCl3, d ppm): 21.53 (–CH3), 22.38 (–CH3), 22.51 (–CH3),
56.75 (–OCH3 ꢃ 2), 105.74 (Ar-carbon ꢃ 2), 120.44, 129.65,
133.49, 138.04, 144.15, 150.17, 151.33, 152.36 (Ar-carbon ꢃ 2),
155.92, 163.41 (–COO–), 168.07 (–COO–). IR (KBr, cmꢀ1): 3429
(OH), 2941, 2847 (CH3), 1755, 1706 (C]O), 1641 (C]C), 1595,
1541 (C]N, C]C), 1167 (C–O). ESI-MS: 373.3 (M + H)+, calcd for
C
19H20N2O6 372.37. HRMS: m/z calcd for C19H20N2O6 [M + H]+
373.1396, found 373.1394; [M + Na]+ 395.1213, found 395.1214.
(E)-3-(3-Hydroxy-4-((3,5,6-trimethylpyrazine-2-carbonyl)oxy)-
phenyl)acrylic acid (F10). White needle crystals, yield: 84%, mp:
1
220–222 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): 12.45 (s, 1H,
COOH), 7.62 (d, 1H, Ar-CH]C, J ¼ 16.2 Hz), 7.19 (s, 2H, Ar-H),
716 | Med. Chem. Commun., 2014, 5, 711–718
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C19H20N2O4 [M + H]+ 341.1501, found 341.1496; [M + Na]+ (C]C–H), 2981, 2963 (CH3), 1740, 1704 (C]O), 1635 (C]C),
363.1317, found 363.1315.
1581, 1543 (C]N, C]C), 1157 (C–O). ESI-MS: 371.4 (M + H)+,
(E)-4-(3-Ethoxy-3-oxoprop-1-en-1-yl)-2-methoxyphenyl-3,5,6- calcd for C20H22N2O5 370.40.
trimethylpyrazine-2-carboxylate (F04). White needle crystals,
(E)-4-(3-Ethoxy-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl-
yield: 81%, mp: 107–110 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 3,5,6-trimethylpyrazine-2-carboxylate (F09). White needle crys-
7.70 (d, 1H, Ar-CH]C, J ¼ 16.0 Hz), 7.61 (s, 1H, Ar-H), 7.38 (d, tals, yield: 75%, mp: 83–85 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm):
1H, Ar-H, J ¼ 8.2 Hz), 7.31 (d, 1H, Ar-H, J ¼ 8.2 Hz), 6.77 (d, 1H, 7.68 (d, 1H, Ar-CH]C, J ¼ 16.0 Hz), 7.23 (s, 2H, Ar-H), 6.82 (d, 1H,
C]CH–C]O, J ¼ 16.0 Hz), 4.23 (q, 2H, OCH2, J ¼ 7.1 Hz), 3.85 C]CH–C]O, J ¼ 16.0 Hz), 4.23 (q, 2H, OCH2, J ¼ 7.1 Hz), 3.85 (s,
(s, 3H, OCH3), 2.70 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.51 (s, 3H, 6H, OCH3 ꢃ 2), 2.70 (s, 3H, CH3), 2.56–2.50 (m, 6H, CH3 ꢃ 2),
CH3), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). 13C-NMR (400 MHz, CDCl3, d 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). 13C-NMR (400 MHz, CDCl3, d ppm):
ppm): 14.65 (–CH3), 21.49 (–CH3), 22.35 (–CH3), 22.55 (–CH3), 14.64 (–CH3), 21.50 (–CH3), 22.35 (–CH3), 22.51 (–CH3), 56.75
56.59 (–OCH3), 60.50 (–OCH2–), 112.60, 119.15, 122.17, 123.62, (–OCH3 ꢃ 2), 60.51 (–OCH2–), 105.89 (Ar-carbon ꢃ 2), 119.31,
133.88, 137.99, 141.39, 144.16, 150.06, 151.52, 151.72, 155.89, 129.84, 133.26, 138.03, 144.61, 150.14, 151.34, 152.38 (Ar-carbon
163.67 (–COO–), 166.66 (–COO–). IR (KBr, cmꢀ1): 3064 (C]C– ꢃ 2), 155.89, 163.39 (–COO–), 166.68 (–COO–). IR (KBr, cmꢀ1):
H), 2983, 2928 (CH3), 1737, 1703 (C]O), 1676 (C]C), 1601, 3075 (C]C–H), 2977, 2925 (CH3), 1691 (C]O), 1633 (C]C), 1600,
1510 (C]N, C]C), 1158 (C–O). ESI-MS: 371.4 (M + H)+, calcd for 1515 (C]N, C]C), 1156 (C–O). ESI-MS: 401.4 (M + H)+, calcd for
C
20H22N2O5 370.40. HRMS: m/z calcd for C20H22N2O5 [M + H]+ C21H24N2O6 400.43. HRMS: m/z calcd for C21H24N2O6 [M + H]+
371.1608, found 371.1601; [M + Na]+ 393.1422, found 393.1421. 401.1706, found 401.1707; [M + Na]+ 423.1521, found 423.1527.
(E)-5-(3-Ethoxy-3-oxoprop-1-en-1-yl)-2-methoxyphenyl-3,5,6- (E)-4-(3-Ethoxy-3-oxoprop-1-en-1-yl)-2-hydroxyphenyl-3,5,6-
trimethylpyrazine-2-carboxylate (F05). White needle crystals, trimethylpyrazine-2-carboxylate (F010). White needle crystals,
1
1
ꢁ
yield: 54%, mp: 119–122 ꢁC. H-NMR (400 MHz, CDCl3, d ppm): yield: 64%, mp: 90–92 C. H-NMR (400 MHz, CDCl3, d ppm):
7.74–7.61 (m, 3H, Ar-H, Ar-CH]C), 7.24 (d, 1H, Ar-H, J ¼ 8.6 7.62 (d, 1H, Ar-CH]C, J ¼ 16.2 Hz), 7.19 (s, 2H, Ar-H), 6.69 (d,
Hz), 6.59 (d, 1H, C]CH–C]O, J ¼ 16.0 Hz), 4.23 (q, 2H, OCH2, J 1H, C]CH–C]O, J ¼ 15.6 Hz), 5.35 (s, 2H, OH), 4.23 (q, 2H,
¼ 7.0 Hz), 3.84 (s, 3H, OCH3), 2.71 (s, 3H, CH3), 2.57–2.50 (m, OCH2, J ¼ 7.1 Hz), 2.71 (s, 3H, CH3), 2.56–2.50 (m, 6H, CH3 ꢃ 2),
6H, CH3 ꢃ 2), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr, cmꢀ1): 3064 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). 13C-NMR (400 MHz, CDCl3, d ppm):
(C]C–H), 2976, 2930 (CH3), 1741, 1705 (C]O), 1636 (C]C), 14.32 (–CH3), 21.10 (–CH3), 22.17 (–CH3), 22.45 (–CH3), 60.46
1611, 1512 (C]N, C]C), 1155 (C–O). ESI-MS: 371.5 (M + H)+, (–OCH2–), 116.56, 118.05, 122.46, 127.22, 133.79, 138.47, 139.82,
calcd for C20H22N2O5 370.40.
143.65, 149.74, 150.26, 152.89, 156.26, 163.14 (–COO–), 167.02
(E)-4-Chloro-2-(3-ethoxy-3-oxoprop-1-en-1-yl)phenyl-3,5,6-tri- (–COO–). IR (KBr, cmꢀ1): 3384.13 (OH), 3064 (C]C–H), 2976,
methylpyrazine-2-carboxylate (F06). White needle crystals, yield: 2930 (CH3), 1741, 1705 (C]O), 1636 (C]C), 1611, 1512 (C]N,
75%, mp: 96–99 ꢁC. 1H-NMR (400 MHz, CDCl3, d ppm): 8.09 (d, C]C), 1155 (C–O). ESI-MS: 357.4 (M + H)+, calcd for C19H20N2O6
1H, Ar-CH]C, J ¼ 2.5 Hz), 7.70–7.60 (m, 2H, Ar-H), 7.52 (d, 1H, 356.14. HRMS: m/z calcd for C19H20N2O6 [M + H]+ 357.1449,
Ar-H, J ¼ 8.7 Hz), 7.04 (d, 1H, C]CH–C]O, J ¼ 16.2 Hz), 4.18 (q, found 357.1445; [M + Na]+ 379.1267, found 379.1264.
2H, OCH2, J ¼ 7.1 Hz), 2.71 (s, 3H, CH3), 2.60–2.50 (m, 6H, CH3
ꢃ 2), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr, cmꢀ1): 3088 (C]C–
Anti-platelet activity assay27
H), 2994, 2981 (CH3), 1735, 1717 (C]O), 1634 (C]C), 1570,
1542 (C]N, C]C), 1180 (C–O). ESI-MS: 375.4 (M + H)+, calcd for Rabbit blood was obtained using a cardiac puncture and
C
19H19ClN2O4 374.82.
transferred to a test tube containing 3.8% sodium citrate
(E)-2-(3-Ethoxy-3-oxoprop-1-en-1-yl)-5-methoxyphenyl-3,5,6- aqueous solution. Platelet-rich plasma (PRP) was obtained
trimethylpyrazine-2-carboxylate (F07). White needle crystals, following blood sample centrifugation at 1000 rpm for 5 min.
1
ꢁ
yield: 80%, mp: 87–89 C. H-NMR (400 MHz, CDCl3, d ppm): The PRP samples were again centrifuged at 3000 rpm for 15 min
7.70 (d, 1H, Ar-CH]C, J ¼ 16.2 Hz), 7.61 (s, 1H, Ar-H), 7.38 (d, to obtain platelet-poor plasma (PPP), which was used as a
1H, Ar-H, J ¼ 7.8 Hz), 7.31 (d, 1H, Ar-H, J ¼ 8.4 Hz), 6.77 (d, 1H, reference solution in aggregation assays. The platelets of the
C]CH–C]O, J ¼ 15.6 Hz), 4.23 (q, 2H, OCH2, J ¼ 7.1 Hz), 3.86 precipitate were adjusted to the suitable number (3 ꢃ 1011 Lꢀ1
)
(s, 3H, OCH3), 2.70 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.51 (s, 3H, for the aggregation assay. All platelet preparations were con-
CH3), 1.30 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr, cmꢀ1): 3064 (C]C– ducted at room temperature. 90 mL PRP and 5 mL sample solu-
H), 2976, 2930 (CH3), 1741, 1705 (C]O), 1636 (C]C), 1611, tion (nal concentration: 400, 200, 100, and 50 mM) was added
1512 (C]N, C]C), 1155 (C–O). ESI-MS: 371.5 (M + H)+, calcd for into the microplate and incubated on 37 ꢁC for 5 min. The
C
20H22N2O5 370.40.
microplate was put on an aggregometer and vibrated for 10 min,
(E)-2-(3-Ethoxy-3-oxoprop-1-en-1-yl)-4-methoxyphenyl-3,5,6- then monitored by measuring 570 nm transmission (A0). The
trimethylpyrazine-2-carboxylate (F08). White needle crystals, monitoring took place every 30 s. Aer that 5 mL ADP (work
1
ꢁ
yield: 80%, mp: 78–80 C. H-NMR (400 MHz, CDCl3, d ppm): concentration: 5 mM) was added and 570 nm transmission was
7.69 (d, 1H, Ar-CH]C, J ¼ 16.1 Hz), 7.48 (s, 1H, Ar-H), 7.35 (d, monitored every 30 s until it became stable (A). The aggregation
1H, Ar-H, J ¼ 8.9 Hz), 7.11 (dd, 1H, Ar-H, J1 ¼ 3.0 Hz, J2 ¼ 8.9 rate (AR) ¼ (Abs PRP ꢀ Abs sample)/(Abs PRP ꢀ Abs PPP). The
Hz), 6.97 (d, 1H, C]CH–C]O, J ¼ 16.1 Hz), 4.18 (q, 2H, OCH2, J aggregation inhibition rate (AIR) ¼ [1 ꢀ (ARsample/ARcontrol)] ꢃ
¼ 7.1 Hz), 3.84 (s, 3H, OCH3), 2.70 (s, 3H, CH3), 2.59–2.50 (m, 100%. Analysis of the derived data using a curve tting soware
6H, CH3 ꢃ 2), 1.33 (t, 3H, CH3, J ¼ 7.1 Hz). IR (KBr, cmꢀ1): 3064 from OriginLab gave the IC50 values.
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Protective effect on damaged Ea.hy926 cells25,28
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Ea.hy926 cells were seeded in a 96-well plate at a density of 6 ꢃ
103 per well and allowed to grow to the desired conuence. The
cells were pretreated with various concentrations of ligustrazine
derivatives for 24 h, and then exposed to 150 mM H2O2 for
another 12 h. Control cells were incubated with a media con-
taining an equivalent solvent amount without the test materials.
The plate was incubated at 37 ꢁC in a humidied 5% CO2
atmosphere. 12 hours later, 0.01 mL 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) solution (5 mg mLꢀ1 in
DMSO) was added to each well and then incubated for 4 h.
Ligustrazine derivatives were dissolved in dimethyl sulfoxide
(DMSO) and added into the wells (the nal concentration of
ligustrazine derivatives was to 100, 50, 25, and 12.5 mM, and the
DMSO content should never exceed 0.05%) and were incubated
with cells for 24 h before the addition of H2O2. The supernatant
was removed carefully by pipetting from wells without disturb-
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adding 200 mL of DMSO to each well and shaken for 15 min. The
absorbance at 570 nm was measured with a microplate reader,
using wells without cells as the control. The proliferation rates of
damaged Ea.hy926 cells were calculated by [OD570 (compound)
ꢀ OD570 (H2O2)]/[OD570 (control) ꢀ OD570 (H2O2)] ꢃ 100%,
which was then used to obtain EC50 values.
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Acknowledgements
The nancial supports of this work by the National Key Projects
of New Drugs R&D (2009ZX09103-117), Shandong Natural
Science Foundation, Science and Technology Development
Foundation of Shandong Province (2006GG2202063) are grate-
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718 | Med. Chem. Commun., 2014, 5, 711–718
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