Ligustrazine derivatives. Part 5: Design, synthesis and biological evaluation of novel ligustrazinyloxy-cinnamic acid derivatives as potent cardiovascular agents

Article abstract of DOI:10.1016/j.ejmech.2011.09.030

A series of novel ligustrazinyloxy-cinnamic acid derivatives 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 ECV-304 cells. Some compounds exhibited high activity in one or both of the assays, of which, compound 2e displayed the highest protective effect on the proliferation of the damaged ECV-304 cells (EC ₅₀ = 0.020 mM), and compound 2f was the most active anti-platelet aggregation agent (EC₅₀ = 0.054 mM). Structure-activity relationships were briefly discussed.

Full text of DOI:10.1016/j.ejmech.2011.09.030

European Journal of Medicinal Chemistry 46 (2011) 5609e5615
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Ligustrazine derivatives. Part 5: Design, synthesis and biological evaluation of
novel ligustrazinyloxy-cinnamic acid derivatives as potent cardiovascular agents
*
Hongfei Chen, Guoning Li, Peng Zhan, Xinyong Liu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44, Wenhuaxi Road, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel ligustrazinyloxy-cinnamic acid derivatives 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 ECV-304
cells. Some compounds exhibited high activity in one or both of the assays, of which, compound 2e dis-
played the highest protective effect on the proliferation of the damaged ECV-304 cells (EC50 ¼ 0.020 mM),
and compound 2f was the most active anti-platelet aggregation agent (EC50 ¼ 0.054 mM). Structuree
activity relationships were briefly discussed.
Received 30 May 2011
Received in revised form
17 September 2011
Accepted 20 September 2011
Available online 29 September 2011
Keywords:
Ligustrazine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cinnamic acid
Synthesis
Cardiovascular disease
Platelet aggregation
Oxidative damage
1. Introduction
which is clinically used for the treatment of acute cerebral throm-
bosis and prevention of postoperative cerebral ischemia [10e13].
Cardiovascular diseases (CVDs) such as ischemic heart disease
and stroke are the leading cause of death in the world. According to
the World Health Organization (WHO), CVDs led to about 32% of all
deaths in women and 27% in men in 2004, far outstripping deaths
due to malaria, HIV/AIDS and tuberculosis [1].
CVDs are triggered by various risk factors, among which athe-
rothrombosis, characterized by disruption of atherosclerotic lesion
with superimposed thrombus formation, is thought to be the major
pathogeny [2,3]. Much evidence shows that endothelial cell
damage and platelet aggregation plays a crucial role in the devel-
opment of atherosclerosis. Impaired endothelial cells lose their
ability of maintaining vascular homeostasis, and produce more
vasoconstrictors, cytokines and cell adhesion molecules, leading to
abnormal platelet activation. After being activated, the platelets
immediately adhere to the injury surface of artery and form
a platelet-rich thrombus over the disruption site, being the initial
events of atherothrombosis [4e6].
Ligustrazine (tetramethyl pyrazine, TMP, Fig.1), a major effective
component of Chinese traditional herbal medicine Chuanxiong
(Ligusticum wallichii Franch), has been reported to be a multi-
functional drug in treatment of CVDs, with pharmacological
activities such as anti-platelet aggregation, anti-hypertension,
vasodilation, free radical scavenge effect and protection against
vascular endothelial cell injury [14e19]. Nowadays ligustrazine is
widely used in China for CVDs therapies.
Based on the structural characteristics of ozagrel and related
research works, and continued to study the molecular modification
of ligustrazine, we replaced imidazole moiety of the ozagrel with
ligustrazinyl group according to the isosterism principle of medicinal
chemistry, constructing a series of novel ligustrazinyloxy-cinnamic
acid derivatives through an ether bond, in order to find more
potent agents for the treatment of CVDs. In this paper, we describe
the synthesis, biological evaluation of anti-platelet aggregation and
protective effect on the injured endothelial cells, as well as the
structureeactivity relationship (SAR) of these novel compounds.
Although remarkable therapeutic advances in the treatment of
atherothrombosis-related CVDs have been made, the main
approaches are still anti-platelet and anti-thrombotic therapies
[7e9]. Ozagrel (Fig. 1) is a potent platelet aggregation inhibitor
2. Results and discussion
2.1. Chemistry
The target compounds were synthesized by two methods. In
method 1 as shown in Scheme 1, esterification of various cinnamic
* Corresponding author. Tel.: þ86 0531 88380270; fax: þ86 0531 88382731.
E-mail addresses: xinyongl@sdu.edu.cn, xinyongllab@163.com (X. Liu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.030

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H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
Fig. 1. Ozagrel, ligustrazine and designed compounds.
acids bearing phenolic hydroxyl group was carried out, producing
the corresponding ethyl cinnamates. The final ligustrazinyloxy-
cinnamic acids 2(aef) were prepared by alkylation of ethyl cinna-
mates at the phenolic hydroxyl group with 2-chloromethyl-3,5,6-
trimethylpyrazine, a key intermediate synthesized from tetra-
methyl pyrazine according to our previous publication [15], and
followed by hydrolysis of corresponding ligustrazinyloxy-cinnamic
acid esters 1(aef) in 20% (W/V) NaOH aqueous solution.
In method 2 as shown in Scheme 2, various substituted
hydroxybenzaldehydes underwent alkylation reaction with the
intermediate 2-chloromethyl- 3,5,6-trimethylpyrazine, to afford
the ligustrazinyloxyl benzaldehydes (1). Compounds (1) were
reacted with malonic acid catalyzed by piperidine in pyridine to
afford ligustrazinyloxy-cinnamic acids 2(gej), which were then
esterified to produce the compounds 1(gej). All the newly
synthesized compounds (Table 1) were structurally confirmed by
IR, 1H NMR and MS.
Scheme 2. Reagents and conditions: (i) K2CO3, TBAB, DMF, 60 ꢀC, 6 h, (ii) malonic acid,
pyridine, cat. piperidine, 120 ꢀC, 1.5 h, (iii) EtOH, SOCl2, reflux, 12 h.
phenyl ring of cinnamic acids (2b, 2c and 2d) showed better anti-
platelet potencies than that of no other substituent compound 2a.
Among the activecompounds, 2c (IC50 ¼ 0.157 mM) with a methoxyl
substituent neighbor to ligustrazinyloxy group showed slightly
improved potency compared with 2b and 2d (IC50 ¼ 0.176 and
0.182 mM, respectively). Compound 2j bearing 3,5-dibromo groups
displayed a reduced anti-platelet activity (IC50 ¼ 1.287 mM). The
results demonstrated that methoxy or hydroxyl is a favorable group
in this series for keeping high anti-platelet activity.
In the ortho-substituted ligustrazinyloxy series 2fei, the
compound 2f (IC50 ¼ 0.054 mM) possessed the highest potency of
all compounds. 2g (IC50 ¼ 0.522 mM) with additive methoxyl group
at the ortho-position of ligustrazinyloxy presented decreased
potency. The introduction of halogens into 2f led to compounds 2h
(chloro) and 2i (dibromo) with significantly decreased potencies
(IC50 ¼ 1.051 and 1.310 mM, respectively), suggesting that electron-
withdrawing groups might be harmful for the anti-platelet activity.
The structureeactivity relationship (SAR) analysis above provided
important information for further molecular modification.
2.2. Anti-platelet aggregation activity
These ligustrazinyloxy-cinnamic acid derivatives were tested for
their inhibition on rabbit platelet aggregation in vitro, and ozagrel
was used as control drug. The results, expressed as aggregation
inhibition rate (AIR) and IC50, were summarized in Table 2.
As can be seen from Table 2, most of the tested compounds
displayed high potencies in anti-platelet aggregation activities.
Among which, compounds 2f, 2c and 2e were the most potent
platelet aggregation inhibitors with IC50 values 0.054, 0.157 and
0.161 mM, respectively, more active than that of ozagrel
(IC50 ¼ 0.360 mM). Some other compounds, 1c, 1d, 1e and 2g, also
exhibited reasonable anti-platelet potencies (IC50 < 1.000 mM).
It was noticed that all the ligustrazinoxy-cinnamic acids
inhibited platelet aggregation with IC50 values lower than corre-
sponding ligustrazinoxy-cinnamate esters, demonstrating that the
carboxyl group is indispensable for maintaining the anti-platelet
activity.
2.3. Protective effect on damaged ECV-304 cells
Setting ligustrazine as the positive control drug, all the
synthesized compounds were also tested for their protective effect
on the ECV-304 cells damaged by hydrogen peroxide [15,20].
Table 3 revealed the proliferation rate (P%) at different concentra-
tion and 50% effective concentration for protecting damaged ECV-
304 cells of newly synthesized compounds.
From the results obtained, it was observed that some of the
ligustrazinyloxy-cinnamic acid derivatives protected the prolifera-
tion of injured ECV-304 cells with effects comparable to or better
than TMP (EC50 0.600 mM). Among all the ligustrazine-cinnamic
acid derivatives, 2a, 2b and 2e displayed remarkable anti-
oxidation activity (EC50 ¼ 0.086, 0.046 and 0.020 mM, respectively).
Particularly, the compound 2e presented almost 30 times higher
potency than TMP, and exhibited the greatest proliferation rate (P
% ¼ 472.99%) at 0.050 mM.
In the case of compounds derived from monosubstituted cin-
namic acid (2a, 2e and 2f), a clear order of ligustrazinyloxy position
at the phenyl group of cinnamic acids for anti-platelet activity was
observed as the following: ortho-position > meta-position > para-
position. In the para-substituted ligustrazinyloxy series 2(aed, j),
compounds containing hydroxyl, methoxyl and dimethoxyl at
Among compounds 1(aed, j) and 2(aed, j) substituted with
ligustrazinyloxy groups at para-position of the cinnamic acid, 1b
and 2b (EC50 ¼ 0.139 and 0.046 mM, respectively) with hydroxyl
group at the 3 position of benzene ring showed better protective
effects than compounds 1a and 2a (EC50 ¼ 0.206 and 0.086 mM),
confirming the necessity of phenolic hydroxyl group in anti-
oxidative effect. From the proliferation rate presented in the
Table 3, it also can be found that the potency of compound
2b was concentration dependent, reaching to the maximum value
(P% ¼ 170.13%) at 100 mM. Whereas compounds 1c and 2c con-
taining a methoxyl at the 3 position of phenyl group did not give
desirable potencies, instead remarkably decreased potencies with
Scheme 1. Reagents and conditions: (i) EtOH, SOCl2, reflux 12 h, (ii) K2CO3, TBAB, DMF,
60 ꢀC, 6 h, (iii) 20% NaOH, 5 h.

H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
5611
Table 1
The structures of the synthesized compounds.
Compound
Structure
Compound
Structure
1a
2a
1b
1c
2b
2c
1d
1e
2d
2e
1f
2f
1g
1h
2g
2h
1i
1j
2i
2j

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H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
Table 2
3. Conclusion
Platelet aggregation inhibition of the ligustrazinyloxy-cinnamic acid derivatives.
a
In conclusion, a series of novel ligustrazinyloxy-cinnamic acid
derivatives were designed, synthesized and biologically evaluated
for their inhibitory activities against the platelet aggregation,
and their protective effect on the damaged endothelial cell prolif-
eration. The results showed that some of the compounds displayed
high activities in both assays, demonstrating the potential values in
further development of cardiovascular agents. Structure activity
relationship was discussed in detail.
Compound
AIR
(400
AIR
(200
AIR
(100
AIR
(50
IC50 (mM)
m
M)
m
M)
m
M)
m
M)
(n ¼ 5)
1a
2a
1b
2b
1c
22.71%
27.56%
41.75%
87.00%
49.45%
73.20%
48.15%
64.30%
43.04%
60.78%
48.12%
76.77%
28.08%
47.56%
18.66%
67.03%
16.08%
37.38%
20.72%
55.40%
55.31%
15.47%
28.97%
25.71%
60.00%
40.19%
51.85%
38.45%
47.31%
34.81%
57.21%
33.42%
70.23%
12.54%
28.41%
14.47%
45.58%
15.80%
33.15%
16.31%
38.62%
45.39%
11.89%
13.43%
23.01%
11.80%
36.89%
46.99%
35.03%
40.95%
29.42%
46.57%
30.08%
62.29%
10.54%
26.07%
11.27%
38.35%
13.47%
20.00%
10.74%
62.75%
27.26%
9.80%
>2
18.78%
13.89%
3.46%
1.402
1.380
0.176
0.449
0.157
0.631
0.182
0.628
0.161
0.502
0.054
>2
0.522
>2
1.051
>2
1.310
>2
1.287
0.360
26.17%
17.47%
31.03%
32.97%
14.54%
30.03%
16.43%
46.11%
6.97%
8.97%
7.66%
24.66%
9.80%
18.08%
6.77%
2c
1d
2d
1e
2e
1f
4. Experimental section
2f
4.1. Chemistry
1g
2g
1h
2h
1i
2i
1j
2j
Ozagrel
The melting points of the compounds were metered on a micro-
melting point apparatus. Infrared spectra were recorded with
a Nexus 470 FT-IR Spectrometer. 1HNMR spectra were determined
by a Bruker Avance (600 MHz) NMR-spectrometer in the indicated
solvents. Chemicalshifts are expressed in d units and TMS as 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 columnpacked with silica gel 60
(230e400 mesh). Solvents were reagent grade and, when necessary,
were purified and dried by standard methods. Concentration of the
reaction solutions involved the use of rotary evaporator at reduced
pressure. The yields were calculated by the last step reaction.
5.30%
23.30%
a
IC50: concentration of compound required to achieve 50% inhibition on aggre-
gation of rabbit platelet.
dimethoxyl at 3,5 position of the compounds 1d and 2d
(EC50 ¼ 2.308 and 3.532 mM, respectively).
Among compounds 1(fei) and 2(fei) substituted with lig-
ustrazinyloxy groups at ortho-position of the cinnamic acid, 1g and
2g with methoxyl group at the 5 position of the phenyl ring showed
diminished protective effects (EC50 ¼ 3.039 and 3.592 mM,
respectively) than compounds 1f and 2f (EC50 ¼ 1.543 and
0.499 mM for each). However, those compounds with chloro at the
same position (1h and 2h) almost lost their effect. Interestingly,
compounds 1i and 2i containing dibromo groups possessed
equivalent or slightly higher proliferous effects (EC50 ¼ 1.593 and
0.155 mM, respectively) than 1f and 2f.
4.1.1. General procedure for the preparation of (E)-ethyl (3-
(substituted-2-yl) methoxy) phenyl acrylate (1aef, method 1 as
shown in Scheme 1)
To an icy solution of hydroxy cinnamic acid (10 mmol) in
ethanol (30 mL) was added thionyl chloride (0.5 mL) dropwise. The
reaction mixture was refluxed for 24 h to give the corresponding
crude ethyl cinnamate.
Potassium carbonate (2 mmol) and catalytic amount of tetra-n-
butylammonium bromide (TBAB) were added to the dimethyl
formamide (10 mL) solution of ethyl cinnamate (2 mmol) obtained
in the above step at room temperature. When the addition was
finished with further stirring for 10 min, 2 mmol of 2-
chloromethyl-3,5,6-trimethylpyrazine hydrochloride in 10 mL
dimethyl formamide was added to the mixture which was then
stirred at 60 ꢀC for 6 h (checked by TLC). The mixture was diluted
with ethyl acetate (100 ml), washed with water and brine,
successively. The organic layer was dried with Na2SO4, filtered and
concentrated under reduced pressure to afford crude product. The
final product was purified by flash column chromatography and
recrystallization from n-hexane.
Table 3
The EC50 for protecting damaged ECV-304 cells of the ligustrazinyloxy-cinnamic acid
derivatives.
Compound
Proliferation rate (%)
100 50
EC50
(mM)
m
M
mM
25
m
M
12.5 mM
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
ꢂ7.56
46.65
37.32
ꢂ6.36
90.36
10.32
ꢂ10.75
2.01
ꢂ1.79%
ꢂ55.02
ꢂ18.73
15.26
0.206
0.086
0.139
0.046
0.109
0.295
2.308
3.532
4.333
0.020
1.543
0.499
3.039
3.592
16.909
9.305
1.593
0.155
1.971
3.283
0.600
ꢂ8.45
170.13
70.21
156.71
50.92
131.49
43.52
29.51
ꢂ19.61
ꢂ179.50
ꢂ132.00
ꢂ507.11
174.92
ꢂ396.43
ꢂ34.59
ꢂ332.04
ꢂ271.88
ꢂ306.94
ꢂ217.78
ꢂ307.33
ꢂ24.79
ꢂ289.69
ꢂ85.84
42.75
ꢂ17.82
ꢂ94.15
ꢂ95.93
277.66
472.99
327.07
236.84
16.58
ꢂ22.36
ꢂ81.89
ꢂ33.84
ꢂ180.41
378.38
ꢂ109.02
369.92
58.66
ꢂ12.53
ꢂ31.08
ꢂ74.64
ꢂ1.52
4.1.1.1. (E)-ethyl 3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)
acrylate (1a). White needle crystals, yield: 63%, mp: 78e82 ꢀC. 1H
306.50
ꢂ1.240602
183.46
ꢂ92.45
ꢂ164.01
ꢂ0.46
NMR (600 MHz, CDCl3,
d
ppm): 7.63 (d, 1H, J ¼ 15.6 Hz, AreCH]C);
7.48e7.46 (m, 2H, AreH, AreH); 7.02e7.00 (m, 2H, AreH, AreH);
6.32e6.29 (m, 1H, C]CHeC]O); 5.18 (s, 2H, CH2eOAr); 4.25 (dd,
2H, J ¼ 14.4 Hz, J ¼ 7.2 Hz, CH2CH3); 2.60 (m, 2H, CH3); 2.54e2.53 (m,
6H, CH3ꢁ2); 1.34e1.32 (t, 3H, J ¼ 7.2 Hz, CH3eCH2). IR (KBr, cmꢂ1):
3384.13 (OH), 3047.89 (C]CeH), 2997.27, 2975.72 (CH3), 1702.00
(C]O), 1631.61 (C]C), 1603.68, 1513.76 (C]N, C]C), 1245.07
(CeO). ESI-MS: 325.6 (M ꢂ H)þ, calcd. for C19H22N2O3 326.16.
2f
1g
2g
1h
2h
1i
2i
1j
2j
TMP
50.28
ꢂ0.71
ꢂ186.46
ꢂ153.47
73.91
48.55
66.06
ꢂ94.44
ꢂ43.98
ꢂ6.88
ꢂ98.33
ꢂ25.70
ꢂ22.29
ꢂ130.32
ꢂ83.83
10.40
4.21
57.03
ꢂ113.07
ꢂ103.54
22.08
12.71
4.1.1.2. (E)-ethyl
methoxy)phenyl)acrylate (1b). Yellow powders, yield: 54%, mp:
142e146 ꢀC. 1H NMR (CDCl3,
ppm): 9.40 (s, 1H, OH); 7.60e7.58 (m,
3-(3-hydroxy-4-((3,5,6-trimethylpyrazin-2-yl)
EC50: concentration of compound required to achieve 50% protection of MT-4 cell
from H2O2 induced cytotoxicity, as determined by the MTT method.
d

H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
5613
1H, CH]C); 7.16e7.15 (m, 1H, AreH); 7.10e7.08 (m, 1H, AreH);
7.01e6.99 (m, 1H, AreH); 6.32e6.30 (m, 1H, CH]C); 5.20 (s, 2H,
CH2); 4.25 (dd, 2H, J ¼ 14.4 Hz, J ¼ 7.2 Hz, CH2CH3); 2.57 (m, 3H,
CH3); 2.52 (m, 3H, CH3); 2.49e2.48 (m, 3H, CH3); 1.34e1.32 (t, 3H,
J ¼ 7.2 Hz, CH3eCH2). IR (KBr, cmꢂ1): 3455.65 (OH), 3061.76 (C]
CeH), 2982.25 (CH3), 1707.41 (C]O), 1633.29 (C]C), 1611.70,
1579.70, 1508.78 (C]N, C]C), 1267.26 (CeO). ESI-MS: 343.5
(M þ H)þ, calcd. for C19H22N2O4 342.16.
4.1.2.1. (E)-3-(4-((3,5,6-Trimethylpyrazin-2-yl)methoxy)phenyl)
acrylic acid (2a). White needle crystals, yield: 84% mp: 142e145 ꢀC.
1H NMR (DMSO,
ppm): 7.73e7.70 (m, 1H, AreCH]C); 7.51e7.49
d
(m, 2H, AreH, AreH); 7.03e7.01 (m, 2H, AreH, AreH); 6.31 (d, 1H,
J ¼ 15.6 Hz, C]CHeC]O); 5.20 (s, 2H, CH2eOAr); 2.62 (m, 3H,
CH3); 2.57e2.55 (m, 6H, CH3ꢁ2). 13C NMR (CDCl3,
d ppm): 168.23
(eCOOH), 160.39 (eOeAr), 151.26, 149.45, 149.07, 145.76 (pyrazine-
C), 144.03, 130.35, 127.70, 117.21, 115.54 (eCH]CHeAr), 69.71
(pyrazine eCH2eOe), 21.44, 21.37, 20.34 (eCH3). IR (KBr, cmꢂ1):
3450.50 (OH), 2922.88, 2580.38 (CH3), 1692.22 (C]O), 1625.74 (C]
C), 1602.48, 1575.69, 1510.37 (C]N, C]C), 1252.83 (CeO). ESI-MS:
299.6 (M þ H)þ, calcd. for C17H18N2O3 298.13.
4.1.1.3. (E)-ethyl
methoxy)phenyl) acrylate (1c). Light yellow needle crystals, yield:
57%, mp: 104e108 ꢀC. 1H NMR (CDCl3,
ppm): 7.61 (d, 1H,
3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)
d
J ¼ 15.6 Hz, eCH]), 7.27 (s, 3H, 3AreH), 6.31 (d, 1H, J ¼ 15.6 Hz, ]
CHe), 5.23 (s, 2H, eCH2eOe), 4.25 (m, 2H, eOeCH2e), 3.87 (s, 3H,
eOCH3), 2.61 (s, 3H, eCH3), 2.51 (s, 6H, 2-CH3),1.33 (t, 3H, J ¼ 7.2 Hz,
eCH3). IR(KBr, cmꢂ1): 3000.62 (C]CeH), 2932.66 (CH3), 2831.29
(eOCH2eH), 1702.93 (C]O), 1636.93 (C]C), 1597.21, 1508.84 (C]
N, C]C), 1258.74 (CeO). ESI-MS: 357.4 (M þ H)þ, calcd. for
C20H24N2O4 356.17.
4.1.2.2. (E)-3-(3-hydroxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)
phenyl) acrylic acid (2b). White powders, yield: 81% mp:
100e110 ꢀC. 1H NMR (DMSO,
d ppm): 12.23 (m, 1H, COOH); 9.31 (s,
1H, OH); 7.46e7.43 (m, 1H, CH]C); 7.08 (m, 3H, AreH); 6.26e6.24
(m, 1H, CH]C); 5.20 (s, 2H, CH2); 3.35 (m, 3H, CH3); 2.49e2.45 (m,
6H, CH3). 13C NMR (CDCl3,
d ppm): 169.22 (eCOOH), 151.03, 150.91
(eOeAr), 149.70,149.26,148.28,145.54 (pyrazine-C),140.78,128.87,
119.78, 116.01, 114.41, 112.40 (eCH]CHeAr), 70.27 (pyrazine
eCH2eOe), 21.25, 20.93, 20.19 (eCH3). IR (KBr, cmꢂ1): 3378.20
(OH), 3080.37 (C]CeH), 2923.47 (CH3), 1689.40 (C]O), 1634.68
(C]C), 1600.77, 1582.44, 1511.85 (C]N, C]C), 1270.59 (CeO). ESI-
MS: 315.2 (M þ H)þ, calcd. for C17H18N2O4 314.13.
4.1.1.4. (E)-ethyl 3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)
methoxy)phenyl) acrylate (1d). White needle crystals, yield: 61%,
mp: 90e94 ꢀC. 1H NMR (CDCl3,
d
ppm): 7.60 (d, 1H, J ¼ 15.6 Hz,
AreCH]C); 6.73 (s, 2H, AreH, AreH); 6.36 (d, 1H, J ¼ 15.6 Hz, C]
CHeC]O); 5.12 (s, 2H, CH2eOAr); 4.27 (dd, 2H, J ¼ 14.4 Hz,
J ¼ 7.2 Hz, CH2CH3); 3.84 (s, 6H, OCH3ꢁ2); 2.70 (m, 3H, CH3); 2.50
(m, 3H, CH3); 2.48 (m, 3H, CH3); 1.36e1.33 (t, 3H, J ¼ 7.2 Hz,
CH3eCH2). IR (KBr, cmꢂ1): 2944.14 (CH3), 2841.04 (OCH2eH),
1708.76 (C]O), 1640.52 (C]C), 1584.88, 1501.43 (C]N, C]C),
1239.51 (CeO). ESI-MS: 387.4 (M þ H)þ, calcd. for C21H26N2O5
386.18.
4.1.2.3. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)
phenyl)acrylic acid (2c). White needle crystals, yield: 82%, mp:
156e160 ꢀC. 1H NMR (DMSO,
d ppm): 12.20 (s, 1H, eCOOH); 7.52 (s,
1H, AreH); 7.35 (d, 1H, AreH); 7.20 (d, 1H, AreH); 7.13 (d, 1H,
J ¼ 15.6 Hz, eCH]); 6.45 (d, 1H, J ¼ 15.6 Hz, ]CHe); 4.99 (s, 2H,
eCH2eOe); 3.79 (s, 3H, eOCH3); 2.50 (s, 3H, eCH3); 2.45 (s, 6H, 2-
4.1.1.5. (E)-ethyl 3-(3-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)
CH3). 13C NMR (CDCl3,
d ppm): 167.79 (eCOOH), 151.02, 149.57
acrylate (1e). White needle crystals, yield: 64%, mp: 76e80 ꢀC. 1H
(eOeAr), 149.46, 149.23,148.20, 145.09 (pyrazine-C),143.93,127.60,
122.31, 116.95, 113.25, 110.59 (eCH]CHeAr), 69.94 (pyrazine
eCH2eOe), 55.58 (eOeCH3), 21.16, 20.86, 20.03 (eCH3). IR (KBr,
cmꢂ1): 3445.65 (OH), 2946.49, 2922.86 (CH3), 2830.29 (eOCH2eH),
1701.17 (C]O), 1635.99 (C]C), 1596.38, 1514.15 (C]N, C]C),
1260.89 (CeO). ESI-MS: 329.7 (M þ H)þ, calcd. for C18H20N2O4
328.14.
NMR (CDCl3,
d
ppm): 7.64 (d, 1H, J ¼ 15.6 Hz, AreCH]C); 7.31e7.27
(m, 1H, AreH); 7.21e7.20 (m, 1H, AreH); 7.13e7.11 (m, 1H, AreH);
7.04e7.02 (m, 1H, AreH); 6.42 (d, 1H, J ¼ 15.6 Hz, C]CHeC]O);
5.18 (s, 2H, CH2-OAr); 4.29e4.25 (m, 2H, CH2CH3); 2.59 (m, 3H,
CH3); 2.53e2.52 (m, 6H, CH3ꢁ2); 1.35e1.32 (t, 3H, J ¼ 7.2 Hz,
CH3eCH2). IR (KBr, cmꢂ1): 3060.61 (C]CeH), 2981.20, 2953.95
(CH3), 1711.29 (C]O), 1638.35 (C]C), 1508.03 (C]N, C]C), 1231.67
(CeO). ESI-MS: 327.5 (M þ H)þ, calcd. for C19H22N2O3 326.16.
4.1.2.4. (E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)
methoxy)phenyl)acrylic acid (2d). White needle crystals, yield: 87%,
4.1.1.6. (E)-ethyl 3-(2-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)
mp: 146e150 ꢀC. 1H NMR (DMSO,
d
ppm): 7.67 (d, 1H, J ¼ 15.6 Hz,
acrylate (1f). White needle crystals, yield: 58%, mp: 66e70 ꢀC. 1H
AeCH]C); 6.76e6.75 (m, 2H, AreH, AreH); 6.36 (d, 1H, J ¼ 15.6 Hz,
C]CHeC]O); 5.27e5.26 (s, 2H, CH2eOAr); 3.91e3.90 (s, 6H,
OCH3ꢁ2); 3.08 (m, 3H, CH3); 2.89 (m, 3H, CH3); 2.67 (m, 3H, CH3).
IR (KBr, cmꢂ1): 3322.56 (OH), 2946.68 (CH3), 2848.64 (eOCH2eH),
2572.68, 2421.50, 1702.40 (C]O), 1633.65 (C]C), 1585.67, 1503.29
(C]N, C]C), 1279.43 (CeO). ESI-MS: 359.4 (M þ H)þ, calcd. for
C19H22N2O5 358.15.
NMR (CDCl3,
d
ppm): 7.98 (d, 1H, J ¼ 16.2 Hz, AreCH]C); 7.53e7.53
(m, 1H, AreH); 7.35e7.33 (m, 1H, AreH); 7.11e7.09 (m, 1H, AreH);
6.99e6.96 (m, 1H, AreH); 6.52 (d, 1H, J ¼ 16.2 Hz, C]CHeC]O);
5.23 (s, 2H, CH2eOAr); 4.23 (dd, 2H, J ¼ 14.4 Hz, J ¼ 7.2 Hz, CH2CH3);
2.59 (m, 3H, CH3); 2.54e2.53 (m, 6H, CH3ꢁ2); 1.32e1.30 (t, 3H,
J ¼ 7.2 Hz, CH3eCH2). IR(KBr, cmꢂ1): 3076.51 (C]CeH), 2981.33,
2926.84 (CH3), 1717.04 (C]O), 1624.77 (C]CC]C), 1597.84 (C]N,
C]C), 1245.09 (CeO). ESI-MS: 327.4 (M þ H)þ, calcd. for
C19H22N2O3 326.16.
4.1.2.5. (E)-3-(3-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)
acrylic acid (2e). White needle crystals, yield: 89%, mp: 86e90 ꢀC.
1H NMR (DMSO,
d
ppm): 7.72 (d, 1H, J ¼ 15.6 Hz, AreCH]C);
4.1.2. General procedure for the preparation of (E)-(3-(substituted-
2-yl) methoxy) phenyl acrylic acid (2aef, method 1 of Scheme 1)
An aqueous solution of sodium hydroxide (4 mmol) was added
7.33e7.31 (m, 1H, AreH); 7.23 (m, 1H, AreH); 7.15e7.14 (m, 1H,
AreH); 7.06e7.04 (m, 1H, AreH); 6.44 (d, 1H, J ¼ 15.6 Hz, C]
CHeC]O); 5.18 (s, 2H, CH2eOAr); 2.61 (m, 3H, CH3); 2.54e2.53 (m,
to
a
solution of (E)-ethyl-(3-(substituted-2-yl)methoxy)phenyl
6H, CH3ꢁ2). 13C NMR (CDCl3,
d ppm): 167.66 (eCOOH), 158.78
acrylate (1ae6, 2 mmol) in ethanol. The mixture was stirred for
24 h at room temperature. Upon completion of the reaction, pH was
adjusted to 4 with hydrochloric acid, and then the solution was
extracted with ethyl acetate (3 ꢁ 30 mL). The organic layer was
dried over anhydrous Na2SO4. The drying agent was filtered off and
the product was obtained by removing the solvent in vacuo.
(eOeAr), 151.10, 149.48, 148.33, 145.31 (pyrazine-C), 144.17, 135.77,
130.04, 128.70, 121.38, 119.77, 113.66 (eCH]CHeAr), 69.47 (pyr-
azine eCH2eOe), 21.29, 20.99, 20.21 (eCH3). IR (KBr, cmꢂ1):
3380.50 (OH), 2924.37 (CH3), 2595.22, 1697.92 (C]O), 1632.19 (C]
C), 1581.24 (C]N, C]C), 1259.30(CeO). ESI-MS: 299.4 (M þ H)þ,
calcd. for C17H18N2O3 298.13.

5614
H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
4.1.2.6. (E)-3-(2-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)
1245.02 (CeO). ESI-MS: 457.3; 459.3 (M þ H)þ, calcd. for
acrylic acid (2f). White needle crystals, yield: 84%, mp: 144e148 ꢀC.
C17H20Br2N2O3 454.95.
1H NMR (DMSO,
d
ppm): 8.06 (d, 1H, J ¼ 15.6 Hz, AeCH]C);
7.55e7.54 (m, 1H, AreH); 7.37e7.34 (m, 1H, AreH); 7.12e7.10 (m,
1H, AreH); 7.01e6.99 (m, 1H, AreH); 6.51 (d, 1H, J ¼ 15.6 Hz, C]
CHeC]O); 5.24e5.19 (s, 2H, CH2eOAr); 2.61e2.60 (m, 3H, CH3);
2.57e2.55 (m, 6H, CH3ꢁ2). IR (KBr, cmꢂ1): 3069.66 (C]CeH),
2956.75 (CH3), 1715.90 (C]O), 1629.09 (C]C), 1601.93 (C]N, C]
4.1.3.4. (E)-3-(3,5-dibromo-4-((3,5,6-trimethylpyrazin-2-yl)
methoxy)phenyl)acrylic acid (2j). White needle crystals, yield:
81%, mp: 204e208 ꢀC. 1H NMR (DMSO,
d ppm): 8.06 (m, 2H,
AreCH]C); 7.53 (m, 1H, AreH); 6.65e6.63 (m, 1H, C]CHeC]O);
5.17 (s, 2H, CH2eOAr); 2.63 (m, 3H, CH3); 2.51 (m, 3H, CH3); 2.46
(m, 3H, CH3). IR (KBr, cmꢂ1): 3361.44 (OH), 3080.79 (C]CeH),
2974.02, 2925.69 (CH3), 1696.21 (C]O), 1640.74 (C]C), 1536.17
(C]N, C]C), 1260.73 (CeO). ESI-MS: 455 (M þ H)þ, calcd. for
C17H20Br2N2O3 454.95.
C), 1250.05 (CeO). 13C NMR (CDCl3,
d ppm): 167.88 (eCOOH), 156.76
(eOeAr), 151.14, 149.15, 148.45, 145.07 (pyrazine-C), 138.53, 131.69,
128.70, 122.86, 121.22, 119.61, 113.14 (eCH]CHeAr), 69.74 (pyr-
azine eCH2eOe), 21.27, 20.98, 20.11 (eCH3). ESI-MS: 299.5
(M þ H)þ, calcd. for C17H18N2O3 298.13.
4.1.4. General procedure for the preparation of (E)-ethyl (3-
(substituted-2-yl) methoxy) phenyl acrylate (1gej, method 2 of
Scheme 2)
To an ice cooled solution of (E)-(3-(substituted-2-yl)methoxy)
phenylacrylic acid (10 mmol) in ethanol (30 mL) was added thionyl
chloride (0.5 mL) dropwise. The reaction mixture was refluxed for
24 h to give the corresponding crude ethyl cinnamate. The corre-
sponding ethyl acrylates (1ge10) were obtained by removing the
solvent in vacuo.
4.1.3. General procedure for the preparation of (E)-(3-(substituted-
2-yl) methoxy) phenyl acrylic acid (2gej, method 2 of Scheme 2)
Potassium carbonate (2 mmol) and catalytic amount of TBAB
were added to the dimethyl formamide (10 mL) solution of
hydroxybenzaldehyde (2 mmol) at room temperature. When the
addition was finished with further stirring for 10 min, 2 mmol of 2-
chloromethyl-3,5,6-trimethylpyrazine hydrochloride in 10 mL
dimethyl formamide was added to the mixture which was then
stirred at 60 ꢀC for 6 h, catalyzing by tetra-n-butylammonium
bromide. The mixture was diluted with ethyl acetate (100 ml),
washed with water and brine, successively. The organic layer was
dried with Na2SO4, filtered and concentrated under reduced pres-
sure to afford ligustrazinyloxyl phenyl formaldehydes (1). The
compound (1) was purified by flash column chromatography and
recrystallization from n-hexane. To the solution of compound (1) in
pyridine was added malonic acid (2.4 mmol) and piperidine. The
mixture was refluxed at 120 ꢀC for 1.5 h. The mixture was diluted
with ethyl acetate (100 ml) and washed with 0.1 N HCl solution.
The organic layer was dried with Na2SO4, filtered and concentrated
under reduced pressure to afford crude product. The final product
was purified by flash column chromatography and recrystallization
from n-hexane.
4.1.4.1. (E)-ethyl
methoxy)phenyl) acrylate (1g). White needle crystals, yield: 54%,
mp: 78e82 ꢀC. 1H NMR (CDCl3,
ppm): 7.86 (d, 1H, J ¼ 15.6 Hz,
3-(3-methoxy-2-((3,5,6-trimethylpyrazin-2-yl)
d
AreCH]C); 7.11e7.06 (m, 2H, AreH, AreH); 6.96e6.95 (m, 1H,
AreH); 6.34 (d, 1H, J ¼ 15.6 Hz, C]CHeC]O); 5.15 (s, 2H,
CH2eOAr); 4.23 (dd, 2H, J ¼ 13.8 Hz, J ¼ 7.2 Hz, CH2CH3); 3.90e3.89
(s, 3H, OCH3); 2.63 (m, 3H, CH3); 2.50e2.48 (m, 6H, CH3ꢁ2);
1.35e1.31 (t, 3H, J ¼ 7.2 Hz, CH3eCH2). IR (KBr, cmꢂ1): 3434.88 (OH),
3074.79 (C]CeH), 2976.91, 2944.73 (CH3), 2839.67 (OCH2eH),
1702.07 (C]O), 1630.41 (C]C), 1582.66 (C]N, C]C), 1252.83
(CeO). ESI-MS: 357.3 (M þ H)þ, calcd. for C20H24N2O4 356.17.
4.1.4.2. (E)-ethyl 3-(5-chloro-2-((3,5,6-trimethylpyrazin-2-yl) methoxy)
phenyl) acrylate (1h). White needle crystals, yield: 80%, mp:
4.1.3.1. (E)-3-(3-methoxy-2-((3,5,6-trimethylpyrazin-2-yl)methoxy)
phenyl)acrylic acid (2g). White needle crystals, yield: 81%, mp:
92e96 ꢀC. 1H NMR (CDCl3,
d
ppm): 7.88 (d, 1H, J ¼ 16.2 Hz, AreCH]
C); 7.48e7.47 (m, 1H, AreH); 7.28e7.27 (m, 1H, AreH); 7.06e7.04 (m,
1H, AreH); 6.48 (d, 1H, J ¼ 16.2 Hz, C]CHeC]O); 5.21 (s, 2H,
CH2eOAr); 4.26 (dd, 2H, J ¼ 14.4 Hz, J ¼ 7.2 Hz, CH2CH3); 2.58 (m, 3H,
CH3); 2.53 (m, 6H, CH3ꢁ2); 1.32e1.39 (t, 3H, J ¼ 7.2 Hz, CH3eCH2). IR
(KBr, cmꢂ1): 3070.47 (C]CeH), 2991.11, 2922.42 (CH3), 1707.92 (C]
O), 1626.97 (C]C), 1595.31 (C]N, C]C), 1252.83 (CeO). ESI-MS:
361.5; 363.5 (M þ H)þ, calcd. for C19H21N2O3 360.12.
156e160 ꢀC. 1H NMR (DMSO,
d
ppm): 7.90 (d, 1H, J ¼ 16.2 Hz,
AreCH]C); 7.12e7.07 (m, 2H, AreH, AreH); 6.99e6.97 (m, 1H,
AreH); 6.29 (d, J ¼ 16.2 Hz, C]CH); 5.20 (s, 2H, CH2); 3.92 (s, 3H,
OCH3); 2.65 (m, 3H, CH3); 2.52 (m, 3H, CH3); 2.49 (m, 3H, CH3). IR
(KBr, cmꢂ1): 3432.50 (OH), 2938.55 (CH3), 2836.14, 2556.36,
1698.35 (C]O), 1630.43 (C]C), 1577.54 (C]N, C]C), 1267.37
(CeO). ESI-MS: 329.6 (M þ H)þ, calcd. for C18H20N2O4 328.14.
4.1.4.3. (E)-ethyl
methoxy)phenyl) acrylate (1i). White needle crystals, yield: 80%,
mp: 184e188 ꢀC. 1H NMR (CDCl3,
ppm): 7.74e7.73 (m, 1H,
3-(3,5-dibromo-2-((3,5,6-trimethylpyrazin-2-yl)
4.1.3.2. (E)-3-(5-chloro-2-((3,5,6-trimethylpyrazin-2-yl)methoxy)
phenyl)acrylic acid (2h). White needle crystals, yield: 81%, mp:
d
204e208 ꢀC. 1H NMR (DMSO,
d
ppm): 7.95 (d, 1H, J ¼ 15.6 Hz,
AreCH]C); 7.67e7.66 (m, 1H, AreH); 7.57e7.56 (m, 1H, AreH);
6.29e6.26 (m, 1H, C]CHeC]O); 5.10 (s, 2H, CH2eOAr);
4.36e4.35 (m, 2H, CH2CH3); 2.66e2.65 (m, 3H, CH3); 2.54e2.53
(m, 3H, CH3); 2.48e2.47 (m, 3H, CH3); 1.22e1.20 (m, 3H,
CH3eCH2). IR (KBr, cmꢂ1): 3080.34 (C]CeH), 2972.23, 2933.22
(CH3), 1715.97 (C]O), 1621.88 (C]C), 1544.73 (C]N, C]C),
1252.83 (CeO). ESI-MS: 483.2; 485.3; 487.3 (M þ H)þ, calcd. for
C19H20Br2N2O3 481.98.
AreCH]C); 7.49 (m, 1H, AreH); 7.29e7.26 (m, 2H, AreH, AreH);
6.47 (d, 1H, J ¼ 15.6 Hz, C]CHeC]O); 5.23e5.17 (s, 2H, CH2eOAr);
2.54e2.53 (m, 9H, CH3ꢁ3). IR (KBr, cmꢂ1): 3390.15 (OH), 3088.37
(C]CeH), 2926.37 (CH3), 2486.65, 1702.78 (C]O), 1634.35 (C]C),
1593.67, 1571.09 (C]N, C]C), 1266.88 (CeO). ESI-MS: 333.6; 335.6
(M þ H)þ, calcd. for C17H17N2O3 332.09.
4.1.3.3. (E)-3-(3,5-dibromo-2-((3,5,6-trimethylpyrazin-2-yl)
methoxy)phenyl)acrylic acid (2i). White needle crystals, yield: 81%,
4.1.4.4. (E)-ethyl
methoxy)phenyl) acrylate (1j). White needle crystals, yield: 80%,
mp: 116e120 ꢀC. 1H NMR (CDCl3,
ppm): 7.67 (m, 1H, AreCH]C);
7.63 (m, 1H, AreH); 7.51e7.49 (m, 1H, AreH); 6.38e6.35 (m, 1H, C]
CHeC]O); 5.21 (s, 2H, CH2eOAr); 4.26 (dd, 2H, J ¼ 13.8 Hz,
J ¼ 7.2 Hz, CH2CH3); 2.74 (m, 3H, CH3); 2.54 (m, 3H, CH3); 2.49 (m,
3H, CH3); 1.37e1.32 (m, 3H, CH2CH3). IR (KBr, cmꢂ1): 3073.96 (C]
3-(3,5-dibromo-4-((3,5,6-trimethylpyrazin-2-yl)
mp: 216e220 ꢀC. 1H NMR (DMSO,
d ppm): 8.03e7.96 (m, 1H,
AreCH]C); 7.52e7.42 (m, 1H, AreH); 7.39e7.35 (m, 1H, AreH);
6.47e6.42 (m, 1H, C]CHeC]O); 5.09 (s, 2H, CH2eOAr); 2.56e2.51
(m, 3H, CH3); 2.51e2.45 (m, 3H, CH3); 2.43e2.36 (m, 3H, CH3). IR
(KBr, cmꢂ1): 3423.31 (OH), 3045.10 (C]CeH), 2960.59, 2923.12
(CH3), 1693.92 (C]O), 1626.41 (C]C), 1543.71 (C]N, C]C),
d

H. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 5609e5615
5615
CeH), 2981.49, 2937.36 (CH3), 1714.45 (C]O), 1639.10 (C]C),
1535.52 (C]N, C]C), 1252.83 (CeO). ESI-MS: 483.2; 485.3; 487.3
(M þ H)þ, calcd. for C19H20Br2N2O3 481.98.
Foundation of Shandong Province (2006GG2202063) are gratefully
acknowledged.
Supplementary data
4.2. Anti-platelet activity [21]
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.05.030. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
Rabbit blood was obtained using the cardiac puncture and
transferred to a test tube containing 3.8% sodium citrate aqueous
solution. Platelet-rich plasma (PRP) was obtained following blood
sample centrifugation at 1000 rpm for 5 min. The PRP samples
were again centrifuged at 3000 rpm for 15 min to obtain platelet-
poor plasma (PPP), which was used as a reference solution in
aggregation assays. The platelets of the precipitate were adjusted to
the proper number (3 ꢁ1011/L) for the aggregation assay. All
platelet preparations were conducted at room temperature. The
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90
100, 50
m
L PRP and 5
mL sample solution (final concentration: 400, 200,
m
M) were added into the microplate and incubated on 37 ꢀC
for 5 min. The microplate was put on the aggregometer and
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mission (A0). The monitoring took place every 30 s. After that 5
mL
ADP (work concentration: 5 M) was added and 570 nm trans-
m
mission was monitored every 30 s until it became stable (A). The
aggregation rate (AR) ¼ (Abs PRP ꢂAbs sample)/(Abs PRP ꢂAbs
PPP). The aggregation inhibition rate (AIR) ¼ [1 ꢂ (ARsample/
ARcontorl)] ꢁ 100%. The IC50 was obtained by linear regression
method.
4.3. Protective effect on damaged ECV-304 cells [20,22]
ECV-304 cells were seeded in a 24-well plate at a density of
6 ꢁ 103/well and allowed to grow to the desired confluence. 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 containing an
equivalent solvent amount without the test materials. The plate
was incubated at 37 ꢀC in a humidified 5% CO2 atmosphere. After
12 h, 0.01 mL MTT solution (5 mg/mL) 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 final
concentration of ligustrazine derivatives was to 400, 200, 100,
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lized by 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 control. The proliferation rates of
damaged ECV-304 cells were calculated by [OD570 (Compd) ꢂ
OD570 (H2O2)]/[OD570 (Control)- OD570 (H2O2)] ꢁ 100%, which
was then used to obtain EC50 values, according to the equation:
-pEC50 ¼ logCmax
-
2 ꢁ (SP ꢂ 0.75 þ 0.25Pmax þ 0.25Pmin), where
Cmax ¼ maximum concentration, SP ¼ sum of proliferation rates,
Pmax ¼ maximum value of proliferation rate and Pmin ¼ minimum
value of proliferation rate.
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The financial supports of this work by National Key Projects
of New Drugs R&D (2009ZX09103-117), Shandong Natural
Science Foundation, Science and Technology Development