Ligustrazine derivatives. Part 3: Design, synthesis and evaluation of novel acylpiperazinyl derivatives as potential cerebrocardiac vascular agents

Article abstract of DOI:10.1016/j.bmc.2009.03.012

A series of novel acylpiperazinyl Ligustrazine derivatives was designed, synthesized, and their protective effects on damaged ECV-304 cells and antiplatelet aggregation activities were evaluated. The results showed that compound E33 displayed most potential protective effects on the ECV-304 cells damaged by hydrogen peroxide, and compound E1 was found to be the most active antiplatelet aggregation agent. Structure-activity relationships were briefly discussed.

Full text of DOI:10.1016/j.bmc.2009.03.012

Bioorganic & Medicinal Chemistry 17 (2009) 3018–3024
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ligustrazine derivatives. Part 3: Design, synthesis and evaluation of novel
acylpiperazinyl derivatives as potential cerebrocardiac vascular agents q
a
b
b
b
Xian-Chao Cheng a, Xin-Yong Liu a, , Wen-Fang Xu , Xiu-Li Guo , Ning Zhang , Yu-Ning Song
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, China
b Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, 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 acylpiperazinyl Ligustrazine derivatives was designed, synthesized, and their protective
effects on damaged ECV-304 cells and antiplatelet aggregation activities were evaluated. The results
showed that compound E33 displayed most potential protective effects on the ECV-304 cells damaged
by hydrogen peroxide, and compound E1 was found to be the most active antiplatelet aggregation agent.
Structure–activity relationships were briefly discussed.
Received 13 January 2009
Revised 7 March 2009
Accepted 10 March 2009
Available online 13 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ligustrazine
Ligustrazine derivatives
Synthesis
Cerebro-cardiac vascular activity
1. Introduction
nant of its pharmacodynamics, while the substituted groups might
primarily govern its pharmacokinetics and toxicity.8
Substituted pyrazine derivatives play a very important role in
pharmaceutical research for the treatment of cardiovascular disor-
ders. Ligustrazine (Lig; tetramethylpyrazine, TMP; see Fig. 1) is a
cardiovascular drug containing a pyrazine ring, which is widely
used in China for the treatment of coronary atherosclerotic cardio-
vascular disease and ischemic cerebrocardiac vascular disease.2
Ligustrazine has been reported to inhibit the platelet aggrega-
tion,3 to cause negative chronotropic and inotropic responses on
isolated atria,4 to inhibit vasoconstriction in isolated vascular
strips,5 and to act as a vasodilator, a free radical scavenger, anti-
thrombosis and anti-hypertention agent.2 More recently, it has
been found to be more effective in protection against vascular
endothelial cell injury.6
However, pharmacokinetics studies found that Ligustrazine
presented low bioavailability and to be metabolized fast in vivo
with short half-life of T1/2 = 2.89 h, so accumulated toxicity often
appeared in the patients for keeping an effective plasma concentra-
tion by the frequent administration.7 Therefore, it is necessary to
develop new generation of the cerebrocardiac vascular drugs from
molecular modification of Ligustrazine.
According to the principles of hypridization and bioisosteric
replacement in medicinal chemistry, some drug-like groups and
pharmacophores can be introduced to the methyl position of
Ligustrazine, for acquiring the pharmacologically additive or syner-
getic effects to improve pharmacokinetic properties.
Calcium channel antagonists, such as Cinnarizine and Flunari-
zine (see Fig. 1) are very important cerebrocardiac vascular drugs
currently used in the clinic. A piperazine moiety as a linker is the
common characters existed in their molecular structures, which
is considered as the functional group for keeping the drugs’ poten-
tial.9 Based on the structures of the calcium antagonists above,
Ligustrazine were modified by combination with a piperazine
and some pharmacophores or drug-like groups, such as acetylsali-
cyloyl, nicotinoyl, cinnamoyl, furoyl and salicyloyl to form the new
integrated structural pattern (see Fig. 1). Some substituted benzoyl
groups were also introduced in order to further expand our explo-
ration and better understand the structure–activity relationships
of Ligustrazine derivatives.
2. Chemistry
Structure–activity relationship studies indicated that pyrazine
ring in the molecule of Ligustrazine might largely be the determi-
In the synthesis, 2-hydroxymethyl-3,5,6-trimethylpyrazine (B)
was prepared by the Boekelheide reaction starting from Ligustr-
azine trihydrate (A), but it was used one-pot reaction according
to our previous publication with 64% of the total yield (mp 88–
89 °C).10 The important intermediate of 2-chloromethyl-3,5,6-
q
See Ref. 1.
* Corresponding author. Tel.: +86 531 8380270; fax: +86 531 8382731.
E-mail address: xinyongl@sdu.edu.cn (X.-Y. Liu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.012

X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
3019
R
N
N
N
N
N
N
N
N
R
X
R
R=H
R=F
Cinnarizine
Flunarizine
Ligustrazine
(Lig; Tetramethylpyrazine,TMP)
Ligustrazine derivatives
Figure 1. Structures of Ligustrazine, Cinnarizine, Flunarizine and newly synthesized acylpiperazinyl Ligustrazine derivatives.
N
N
N
N
N
c
a
b
3H2O
HCl
OH
Cl
N
N
R
Method 1
N
A
N
B
N
C
X
E 1-32
e
d
Method 3
f
Method 2
N
N
N
NH
N
N
N
N
D
E33
O
OH
Scheme 1. Reagents and conditions: (a) Boekelheide reaction; (b) SOCl2/anhydrous CH2Cl2; (c) various mono N-substituted acylpiperazines, toluene, NaI, Et3N/reflux for 10 h;
(d) piperazine; (e) RCOCl, anhydrous CH2Cl2/reflux for 10 h; (f) RX = acetylsalicyloyl, NH3ÁH2O.
trimethylpyrazine hydrochloride (C) was synthesized by the chlo-
rination of B with SOCl2 in anhydrous CH2Cl2 (see Scheme 1).11
Acylpiperazinyl Ligustrazine derivatives (E1–33) were synthe-
sized by the following three methods. In method 1,2-chloro-
methyl-3,5,6-trimethylpyrazine hydrochloride (C) was directly
reacted with the mono-substituted N-acylpiperazines, which were
prepared in our lab, to afford acylpiperazinyl Ligustrazine deriva-
tives (E10–22, 24, 26–30, see Method 1 of Scheme 1).12,13 In Meth-
od 2, 2-chloromethyl-3,5,6-trimethylpyrazine hydrochloride (C)
alkylated with anhydrous piperazine in chloroform to produce
the intermediate 2-(1-piperazinmethyl)-3,5,6-trimethylpyrazine
(D), which was reacted with various acyl chloride to give the cor-
responding acylpiperazinyl Ligustrazine derivatives (E1–9, 23, 25,
31, 32, see Method 2 of Scheme 1).13 In method 3, E33 was ob-
tained by hydrolysis of E1 in ammonium hydroxide medium (see
Method 3 of Scheme 1). The chemical structures of these com-
pounds were confirmed by IR, 1H NMR and ESI-MS.
taining salicyloyl group was the most active one with the EC50
value at 6.13 M. The excellent potency of E33 might be caused
by the hydroxyl group at phenyl moiety, as the acetylated coun-
terpart E1 showed less potency with the EC50 value at
159.24 lM.
l
Introduction of iso-nicotinoyl group (E3) favored the protective
effects on ECV-304 cells, however, replacement of this group by
nicotinoyl group produced E2 with tenfold less potency.
Furoyl group, galloyl group, and 2-(4-chlorophenoxy)-2-
methyl-propanoyl group are confirmed as pharmacophores of
cerebrocardiac vascular profiles. Introduction of furoyl group
(E10) and galloyl group (E11) produced protective effect on ECV-
304 cells. However, introduction of 2-(4-chlorophenoxy)-2-
methyl-propanoyl pharmacophore (E4) did not show protective ef-
fect with EC50 value not determined.
Among compounds E12, E24 and E26, the activity order was
E26 > E24 > E12, which might reflect the importance of carbon
numbers between the phenyl group and carbonyl group. Two car-
bons between the phenyl group and carbonyl group seem most
favorable. In addition, the compounds containing cinnamoyl or sul-
fonyl group exhibited less potency as compared with Ligustrazine.
And the benzoyl series compounds did not present satisfactory po-
tency with the exception of E20.
Ligustrazine has been reported to inhibit the platelet aggrega-
tion,3 so these Ligustrazine derivatives were further tested for anti-
platelet aggregation induced by adenosine diphosphate (ADP).15
The preliminary antiplatelet aggregation results obtained from
the experiments showed that Ligustrazine derivatives bearing
pharmacophores or drug-like groups, such as E1 (acetylsalicyloyl,
3.02 3.78%), E2 (nicotinoyl, 5.08 1.45%), E3 (iso-nicotinoyl,
6.07 6.48%), E6 ((E)-3,4-dimethoxylcinnamoyl, 6.13 7.23%), E8
(acetylferuloyl, 5.84 4.21%) exhibited the high potent activities
3. Biological evaluation and discussion
Endothelial cells play a critical physiological role in maintaining
normal vessel and organ function. Much evidence showed that vas-
cular endothelial cells damage that causes the alteration of endo-
thelial permeability barrier and vascular tone is
a major
promoter of atherogenesis, thrombosis and consequently cardio-
vascular events. Oxidative stress is a cardiovascular risk factor
and contributes significantly to endothelial injury during athero-
genesis. Therefore, the protection of endothelial cells against dam-
age caused by oxidative stress is a very important therapeutic
strategy.14
The newly synthesized Ligustrazine derivatives were assayed
for the protective effects on the human umbilical vascular endo-
thelial cells (ECV-304 cells) damaged by hydrogen peroxide. The
results (Table 1) showed that Ligustrazine and most of its deriva-
tives presented protective effects on the damaged ECV-304 cells
and some of the Ligustrazine derivatives were more active (with
in antiplatelet aggregation at 200 lM, and with lower values than
that of Ligustrazine (7.86 4.17%). Compound E1 (acetylsalicyloyl,
3.02%) was found to be the most active antiplatelet aggregation
agent (Table 1).
Deacetylation of E1 produced the compound E33 (salicyloyl,
16.71 2.13%), which caused a decreased antiplatelet aggregation
activity. Most of the substituted benzoyl derivatives showed higher
antiplatelet aggregation activities than the non-substituted ben-
lower EC50 values) than Ligustrazine (EC50 value at 154.35
The derivatives E3, E10, E11, E20, E30 and E33 exhibited high
potency with EC50 values below 100 M, among which E33 con-
lM).
l

3020
X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
Table 1
The structures, EC50 for protecting damaged ECV-304 cells and platelet aggregation rate (A%) of Liqustrazine derivatives E1–33
N
N
N
R
N
X
No.
X
RX-
Method
EC50
(lM)
A% (200 l
M)a
E1
E2
E3
E4
E5
E6
E7
E8
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
SO2
SO2
SO2
CO
CO
CO
CO
Acetylsalicyloyl
Nicotinoyl
iso-Nicotinoyl
2-(4-Chlorophenoxy)-2-methyl-propanoyl
(E)-2,5-Dimethoxylcinnamoyl
(E)-3,4-Dimethoxylcinnamoyl
(E)-2,3-Dimethoxylcinnamoyl
Acetylferuloyl
(E)-3-Nitrocinnamoyl
Furoyl
3,4,5-Trimethoxylbenzoyl
Benzoyl
2-Methoxylbenzoyl
4-Methoxylbenzoyl
3,4-Dimethoxylbenzoyl
2-Chlorobenzoyl
3-Chlorobenzoyl
4-Chlorobenzoyl
2,4-Dichlorobenzoyl
2-Iodobenzoyl
4-Iodobenzoyl
4-Nitrobenzoyl
3,5-Dinitrobenzoyl
Phenylacetyl
4-Nitrophenylacetyl
b-Phenylpropionoyl
Benzenesulfonyl
4-Methylbenzenesulfonyl
Methanesulfonyl
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
1
1
1
1
1
2
2
3
159.24
220.02
29.73
3.02 3.78
5.08 1.45
6.07 6.48
13.68 5.17
9.78 3.51
6.13 7.23
8.78 1.97
5.84 4.21
13.07 5.54
21.76 5.87
14.26 4.43
28.46 8.57
24.27 2.76
26.06 5.58
29.06 4.67
27.24 4.97
24.74 6.43
25.62 2.09
16.08 6.83
25.45 5.54
25.17 8.79
28.64 7.24
15.67 2.98
25.59 1.85
18.24 3.16
13.29 4.95
24.49 7.04
25.13 5.83
33.51 3.95
15.25 6.89
24.89 4.29
26.11 5.07
16.71 2.13
7.86 4.17
ND
376.28
282.47
340.48
ND
421.50
37.57
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
E31
E32
E33
Lig
88.73
240.57
248.56
210.47
169.95
161.14
121.46
208.03
248.56
67.80
125.33
461.88
178.81
188.61
376.93
150.00
480.30
ND
162.22
76.59
147.52
304.37
6.13
Exthoxycarbonyl
Chloroacetyl
Phenylethyldione
Salicyloyl
154.35
ND: not determined.
a
The A% value for blank is 0.73 2.93, and for ADP is 39.77 3.69.
zoyl derivatives (E12), but with some exceptions of 3,4-dimethoxy,
4-nitryl substituents. Among the compounds E12, E24 and E26,
there were increasing antiplatelet aggregation activities with the
increasing length of side chain at the phenyl moiety, which might
reflect the importance of alkyl chain length of the aralkyl carbox-
ylic acid. All features described above should be considered in
the design of novel Ligustrazine derivatives.
ter; J values are in hertz. 13C NMR spectra were also recorded on
the Bruker Avance spectrometer. Mass spectra were recorded on
an electro-spray ionization mass spectrometer as the value m/z.
Thin-layer chromatography (TLC) was performed on E. Merck Sil-
ica Gel 60-F-254 plates. Flash chromatography was performed
using 300 mesh silica gel. The yields were calculated by the last
step reaction.
5.1.1. (3,5,6-Trimethylpyrazin-2-yl)methanol (B)
4. Conclusion
2,3,5,6-Tetramethylpyrazine trihydrate (A) (30.40 g, 160 mmol)
was heated with 30% hydrogen peroxide (18 ml, 160 mmol) in gla-
cial acetic acid (40 ml) for 4 h at 70 °C. Further 30% hydrogen per-
oxide (18 ml, 160 mmol) was then added and the heating was
continued for 4 h. The solution was made alkaline with 50% sodium
hydroxide and extracted with chloroform. The combined extracts
were dried and evaporated in vacuo; tetramethylpyrazine mono-
N-oxide was obtained. To this, acetic anhydride (15.1 ml,
160 mmol) was added and the mixture was refluxed for 3 h, check-
ing for product formation via TLC. The excess of acetic anhydride
was evaporated and (3,5,6-trimethylpyrazin-2-yl)methyl acetate
was obtained, which was directly saponified with 20% NaOH
(155 ml), and extracted with chloroform. The combined extracts
were dried and the solvent removed. The residual oil was recrystal-
lized from n-hexane; (3,5,6-trimethylpyrazin-2-yl)methanol (B)
was obtained as yellow needles (15.50 g, 64%); mp 88–89 °C
(lit.10 88–89 °C).
In conclusion, a series of novel acylpiperazinyl Ligustrazine
derivatives was designed and synthesized. Activity assay identified
compound E1 as the most active antiplatelet aggregation agent,
and compound E33 with most potent protective effect on the dam-
aged ECV-304 cells. Structure–activity relationships were briefly
discussed. Further bioassay of these compounds on cerebrocardiac
vascular activity on animal models is underway.
5. Experimental
5.1. Synthetic methods and spectroscopic details
Infrared spectra were measured using a nicolet nexus 470 FT-
IR spectrometer using smear KBr crystal or KBr plate. 1H NMR
spectra were recorded on a Bruker Avance (600 MHz) spectrome-

X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
3021
5.1.2. 2-Chloromethyl-3,5,6-trimethylpyrazine hydrochloride
(C)
cmÀ1): 2913.72 (CH), 1624.65 (C@O), 1604.84 (C@C), 1578.23
(C@N); 1H NMR (CDCl3, d): 7.41 (m, 5H, Ar-H), 2.56–3.66 (m,
10H, CH2), 2.59 (s, 3H, CH3), 2.49 (s, 3H, CH3), 2.48 (s, 3H, CH3);
ESI-MS: 325.5 (M+1).
Thionyl chloride (7.41 ml, 102 mmol) was added dropwise to
(3,5,6-trimethylpyrazin-2-yl)methanol (B) (15.50 g, 102 mmol) in
anhydrousCH2Cl2 (300 ml) at0 °C. Themixturewasallowedtostand
for 2.5 h, checking for product formation via TLC. The solvent was
evaporated in vacuo and the crude product 2-chloromethyl-3,5,6-
trimethylpyrazine hydrochloride (C) was obtained as a yellow solid
(21.11 g, 100%); mp 102–105 °C. Compound C was basified and then
purified to give 2-chloromethyl-3,5,6-trimethylpyrazine as oil for
spectral confirmation. IR (KBr, cmÀ1): 2993 (CH), 2952 (CH), 2923
(CH), 2856 (CH), 1548 (C@N); 1H NMR (CDCl3, d): 4.68 (s, 2H, CH2),
2.63 (s, 3H, CH3), 2.54 (s, 3H, CH3), 2.52 (s, 3H, CH3); 13C NMR (CDCl3,
d): 151.6 (C@N), 149.3 (C@N), 149.0 (C@N), 146.3 (C@N), 44.8 (CH2),
21.6, 21.4 and 20.5 (3 Â CH3); ESI-MS: 171 (M+1).
5.1.4.4. 2-[4-(2-Methoxylbenzoyl)-1-piperazinmethyl]-3,5,6-tri-
methylpyrazine (E13). Flash column chromatography: chloro-
form–acetone = 8:1; yield 44%; white crystal; mp 104–105 °C; IR
(KBr, cmÀ1): 2917.00 (CH), 1622.38 (C@O), 1600.03 (C@C),
1253.43 (C–O), 1023.52 (C–O); 1H NMR (CDCl3, d): 7.33 (t, 1H,
Ar-H, J = 7.53 Hz), 7.23 (d, 1H, Ar-H, J = 7.31 Hz), 6.98 (t, 1H, Ar-H,
J = 7.46 Hz), 6.90 (d, 1H, Ar-H, J = 8.32 Hz), 3.80 (3H, OCH3), 2.40–
3.70 (m, 10H, CH2), 2.58 (s, 3H, CH3), 2.49 (s, 3H, CH3), 2.47 (s,
3H, CH3); ESI-MS: 355.4 (M+1).
5.1.4.5. 2-[4-(4-Methoxylbenzoyl)-1-piperazinmethyl]-3,5,6-tri-
methylpyrazine (E14). Flash column chromatography: chloro-
form–acetone = 8:1; yield 45%; yellow crystal; mp 82–85 °C; IR
(KBr, cmÀ1): 2921.16 (CH), 1621.72 (C@O), 1573.75 (C@C),
1514.22 (C@N), 1245.92 (C–O), 1033.00 (C–O); 1H NMR (CDCl3,
d): 7.37 (d, 2H, Ar-H, J = 8.68 Hz), 6.90 (d, 2H, Ar-H, J = 8.67 Hz),
3.82 (3H, OCH3), 3.60–3.70 (m, 10H, CH2), 2.57 (s, 3H, CH3), 2.49
(s, 3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 355.4 (M+1).
5.1.3. 2,3,5-Trimethyl-6-(piperazin-1-ylmethyl)pyrazine (D)
2-Chloromethyl-3,5,6-trimethylpyrazine hydrochloride (C)
(20.7 g, 100 mmol) in chloroform (100 ml) was added dropwise
into anhydrous piperazine (49.88 g, 580 mmol) in chloroform
(300 ml) at 0 °C. The solution was left at room temperature for
5 h, checking for product formation via TLC. The mixture solution
was washed with aqueous ammonia (4 M), and the organic layers
were dried. The solvent was evaporated in vacuo and the crude
product was recrystallized from n-hexane to give 2,3,5-trimethyl-
6-(piperazin-1-ylmethyl)pyrazine (D) as white crystals (11 g,
50%); mp 94 °C. IR (KBr, cmÀ1): 3443, 3272 (NH), 2943 (CH),
1546 (C@N); 1H NMR (CDCl3, d): 2.57–3.60 (m, 10H, CH2), 2.49
(s, 3H, CH3), 2.48 (s, 3H, CH3), 2.47 (s, 3H, CH3), 1.90 (s, 1H, NH);
13C NMR (CDCl3, d): 153.6 (C@N), 150.4 (C@N), 148.7 (C@N),
147.2 (C@N), 56.6, 54.4, 54.1, 45.2 and 44.4 (5 Â CH2), 21.4, 21.0
and 20.1 (3 Â CH3); ESI-MS: 221 (M+1).
5.1.4.6. 2-[4-(3,4-Dimethoxylbenzoyl)-1-piperazinmethyl]-3,5,
6-trimethylpyrazine (E15). Flash column chromatography: chlo-
roform–acetone = 8:1; yield 43%; yellow crystal; mp 110–112 °C;
IR (KBr, cmÀ1): 2916.64 (CH), 1629.45 (C@O), 1583.80 (C@C),
1518.01 (C@N), 1266.25 (C–O), 1026.80 (C–O); 1H NMR (CDCl3,
d): 6.97 (t, 2H, Ar-H, J = 7.56 Hz), 6.85 (d, 1H, Ar-H, J = 8.12 Hz),
3.89 (6H, OCH3), 3.60–3.70 (10H, CH2), 2.57(s, 3H, CH3), 2.49 (s,
3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: 385.3 (M+1).
5.1.4. General procedure for the preparation of 2-(4-substitu-
ted-1-piperazinmethyl)-3,5,6-trimethylpyrazine (E10–22, E24,
E26–30, Method 1 of Scheme 1)
5.1.4.7. 2-[4-(2-Chlorobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E16). Flash column chromatography: chloro-
form–acetone = 3:1; yield 50%; yellow crystal; mp 148 °C; IR
(KBr, cmÀ1): 2953.76 (CH), 1632.68 (C@O), 1591.87 (C@C); 1H
NMR (CDCl3, d): 7.35 (m, 4H, Ar-H), 2.30–3.83 (m, 10H, CH2),
2.58 (s, 3H, CH3), 2.49 (s, 3H, CH3), 2.47 (s, 3H, CH3); ESI-MS:
359.4 (M+1).
2-Chloromethyl-3,5,6-trimethylpyrazine hydrochloride (C)
(2.07 g, 10 mmol) and various mono N-substituted acylpiperazines
(10 mmol) were dissolved in toluene (70 ml). Triethylamine
(3.46 ml, 25 mmol) and NaI (catalytic quantity) were added to
the solution. The mixture solution was refluxed for 10 h until the
reaction was complete (monitored by TLC). After cooling, the mix-
ture was filtered and the filtrate was evaporated in vacuo. The final
product was purified by flash column chromatography and recrys-
tallization from n-hexane.
5.1.4.8. 2-[4-(3-Chlorobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E17). Flash column chromatography: chloro-
form–acetone = 3:1; yield 41%; yellow crystal; mp 136 °C; IR
(KBr, cmÀ1): 2920.53 (CH), 1629.36 (C@O), 1594.43 (C@C),
1563.11(C@N); 1H NMR (CDCl3, d): 7.39 (m, 2H, Ar-H), 7.33 (t,
1H, Ar-H), 7.27 (t, 1 H, Ar-H), 2.40–3.66 (m, 10H, CH2), 2.60 (s,
3H, CH3), 2.49 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: 359.4
(M+1).
5.1.4.1. 2-(4-Furoyl-1-piperazinmethyl)-3,5,6-trimethylpyrazine
(E10). Flash column chromatography: chloroform–acetone = 3:1;
yield 51%; yellow crystal; mp 100–102 °C; IR (KBr, cmÀ1): 3098.74
(@CH), 2948.27 (CH), 1621.05 (C@O), 1581.79 (C@C), 1292.18 (C–
O), 1023.80 (C–O); 1H NMR (CDCl3, d): 7.46 (1H, furan-2-H), 6.97
(1H, furan-4-H, J = 3.32 Hz), 6.47 (1H, furan-3-H, J2–3 = 1.57 Hz, J3–
4 = 3.13 Hz), 2.50–3.78 (m, 10H, CH2), 2.59 (s, 3H, CH3), 2.56 (s, 3H,
CH3), 2.49 (s, 3H, CH3); ESI-MS: 315.3 (M+1).
5.1.4.9. 2-[4-(4-Chlorobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E18). Flash column chromatography: chloro-
form–acetone = 4:1; yield 58%; white crystal; mp 102–104 °C; IR
(KBr, cmÀ1): 2935.07 (CH), 1614.37 (C@O), 1566.14 (C@C); 1H
NMR (CDCl3, d): 7.39 (d, 2H, Ar-H, J = 8.35 Hz), 7.36 (d, 2H, Ar-H,
J = 8.44 Hz), 3.40–3.77 (m, 10H, CH2), 2.51 (s, 3H, CH3), 2.50 (s,
3H, CH3), 2.49 (s, 3H, CH3); ESI-MS: 359.3 (M+1).
5.1.4.2. 2-[4-(3,4,5-Trimethoxylbenzoyl)-1-piperazinmethyl]-3,
5,6-trimethylpyrazine (E11). Flash column chromatography:
chloroform–acetone = 3:1; yield 44%; yellow oil; IR (KBr, cmÀ1):
2939.22 (CH), 1636.18 (C@O), 1583.82 (C@C), 1505.51 (C@N),
1231.28 (C–O), 1127.39 (C–O); 1H NMR (CDCl3, d): 6.62 (s, 2H,
Ar-H), 3.85–3.87 (9H, OCH3), 2.56–3.66 (m, 10H, CH2), 2.59 (s,
3H, CH3), 2.50 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: 415.3 (M+1).
5.1.4.10. 2-[4-(2,4-Dichlorobenzoyl)-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (E19). Flash column chromatography: chloro-
form–acetone = 2:1; yield 51%; yellow crystal; mp 142–144 °C; IR
(KBr, cmÀ1): 2916.04 (CH), 1631.66 (C@O), 1587.77 (C@C),
1553.47 (C@N); 1H NMR (CDCl3, d): 7.43 (d, 1H, Ar-H,
J = 8.00 Hz), 7.31 (t, 1H, Ar-H, J = 8.11 Hz), 7.23 (t, 1H, Ar-H,
J = 5.30 Hz), 2.50–3.83 (m, 10H, CH2), 2.58 (s, 3H, CH3), 2.49 (s,
3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: 393.2 (M+1).
5.1.4.3.
2-(4-Benzoyl-1-piperazinmethyl)-3,5,6-trimethylpyr-
azine (E12). Flash column chromatography: chloroform–ace-
tone = 10:1; yield 61%; yellow crystal; mp 138–140 °C; IR (KBr,

3022
X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
5.1.4.11. 2-[4-(2-Iodobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E20). Flash column chromatography: chloro-
form–acetone = 5:1; yield 42%; white crystal; mp 163–166 °C; IR
(KBr, cmÀ1): 2949.93 (CH), 1628.84 (C@O), 1583.72 (C@C); 1H
NMR (CDCl3, d): 7.82 (d, 1H, Ar-H, J = 7.92 Hz), 7.38 (t, 1H, Ar-H,
J = 7.38 Hz), 7.19 (d, 1H, Ar-H, J = 6.44 Hz), 7.06 (t, 1H, Ar-H,
J = 7.04 Hz), 2.30–3.81 (m, 10H, CH2), 2.57 (s, 3H, CH3), 2.48 (s,
3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 451.3 (M+1).
1161.38 (SO2), 1132.50 (SO2); 1H NMR (CDCl3, d): 3.75 (2H, CH2),
3.31–3.32 (m, 8H, CH2), 2.79 (3H, SO2CH3), 2.57 (s, 3H, CH3), 2.54
(s, 3H, CH3), 2.49 (s, 3H, CH3); ESI-MS: 299.4 (M+1).
5.1.4.19. 2-(4-Exthoxycarbonyl-1-piperazinmethyl)-3,5,6-trim-
ethylpyrazine (E30). Flash column chromatography: chloro-
form–acetone = 4:1; yield 58%; yellow crystal; mp 94–96 °C; IR
(KBr, cmÀ1): 2929.29 (CH), 1702.38 (C@O), 1236.10 (C–O),
1129.85 (C–O); 1H NMR (CDCl3, d): 3.44–4.13 (m, 10H, CH2), 2.56
(s, 3H, CH3), 2.48 (s, 3H, CH3), 2.47 (s, 3H, CH3), 2.44 (2H, CH2),
1.24 (t, 3H, CH3, J = 7.12 Hz); ESI-MS: 293.4 (M+1).
5.1.4.12. 2-[4-(4-Iodobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E21). Flash column chromatography: chloro-
form–acetone = 5:1; yield 52%; white crystal; mp 166–168 °C; IR
(KBr, cmÀ1): 2938.22 (CH), 1619.75 (C@O), 1586.17 (C@C); 1H
NMR (CDCl3, d): 7.75 (d, 2H, Ar-H, J = 8.26 Hz), 7.14 (d, 2H, Ar-H,
J = 8.25 Hz), 2.57–3.65 (m, 10H, CH2), 2.50 (s, 3H, CH3), 2.48 (s,
3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 451.3 (M+1).
5.1.5. General procedure for the preparation of 2-(4-substitu-
ted-1-piperazinmethyl)-3,5,6-trimethylpyrazine (E1–9, E23,
E25, E31 and E32, Method 2 of Scheme 1)
To a mixture of 2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyra-
zine (D) (2.2 g, 10 mmol) and Na2CO3 (3.18 g, 30 mmol) in anhy-
drous CH2Cl2 (100 ml), was added dropwise various acyl chloride
(10 mmol) in anhydrous CH2Cl2 (100 ml) at room temperature.
The mixture was refluxed for 10 h (checked by TLC), and the sol-
vent was evaporated in vacuo. The final product was purified by
flash column chromatography and recrystallization from n-hexane.
E1–4, E11–15, E29, E31, E37 and E38 are obtained.
5.1.4.13. 2-[4-(4-Nitrobenzoyl)-1-piperazinmethyl]-3,5,6-trim-
ethylpyrazine (E22). Flash column chromatography: chloro-
form–acetone = 8:1; yield 63%; yellow crystal; mp 151–153 °C; IR
(KBr, cmÀ1): 2919.51 (CH), 1638.20 (C@O), 1600.48 (C@C),
1521.12 (NO2), 1350.51 (NO2); 1H NMR (CDCl3, d): 8.34 (d, 2H,
Ar-H, J = 8.36 Hz), 7.63 (d, 2H, Ar-H, J = 8.50 Hz), 3.40–3.87 (m,
10H, CH2), 2.65 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.54 (s, 3H, CH3);
ESI-MS: 370.4 (M+1).
5.1.5.1. 2-(4-Acetylsalicyloyl-1-piperazinmethyl)-3,5,6-trimeth-
ylpyrazine (E1). Flash column chromatography: chloroform–ace-
tone = 3:1; yield 48%; yellow oil; IR (KBr, cmÀ1): 2920.52 (CH),
1766.95 (C@O), 1640.86 (C@O), 1606.93 (C@N), 1195.56 (C–O);
1H NMR (CDCl3, d): 7.29 (m, 1H, Ar-H), 7.19 (dd, 1H, Ar-H), 7.14
(t, 1H, Ar-H, J = 7.42 Hz), 7.05 (d, 1H, Ar-H, J = 8.12 Hz), 3.18–3.66
(m, 10H, CH2), 2.47 (s, 3H, CH3), 2.37 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 2.16 (t, 3H, COCH3, J = 4.98 Hz); ESI-MS: 383.3 (M+1).
5.1.4.14. 2-(4-Phenylacetyl-1-piperazinmethyl)-3,5,6-trimethyl-
pyrazine (E24). Flash column chromatography: chloroform–ace-
tone = 8:1; yield 39%; yellow crystal; mp 67–69 °C; IR (KBr,
cmÀ1): 2955.83 (CH), 1636.90 (C@O), 1602.07 (C@C); 1H NMR
(CDCl3, d): 7.30 (m, 5H, Ar-H), 3.77 (2H, Ar-CH2), 2.30–3.69 (m,
10H, CH2), 2.53 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.50 (s, 3H, CH3);
ESI-MS: 339.4 (M+1).
5.1.5.2. 2-(4-Nicotinoyl-1-piperazinmethyl)-3,5,6-trimethylpyr-
azine (E2). Flash column chromatography: chloroform–ace-
tone = 1:1; yield 44%; white crystal; mp 120–121 °C; IR (KBr,
cmÀ1): 2916.66 (CH), 1630.39 (C@O), 1590.11 (C@C), 1567.99
(C@N); 1H NMR (CDCl3, d): 8.65 (m, 2H, Ar-H), 7.73 (m, 1H, Ar-
H), 7.34 (m, 1H, Ar-H), 3.42–3.77 (m, 10H, CH2), 2.57 (s, 3H, CH3),
2.49 (s, 3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 326.5 (M+1).
5.1.4.15. 2-[4-(b-Phenylpropionoyl)-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (E26). Flash column chromatography: chloro-
form–acetone = 8:1; yellow crystal; yield 47%; mp 74–76 °C; IR
(KBr, cmÀ1): 2935.95 (CH), 1630.00 (C@O); 1H NMR (CDCl3, d):
7.27 (t, 2H, Ar-H, J = 5.15 Hz), 7.20 (d, 3H, Ar-H, J = 7.57 Hz),
2.90–3.59 (m, 8H, CH2), 2.60 (2H, CH2), 2.56 (s, 3H, CH3), 2.49 (s,
3H, CH3), 2.48 (s, 3H, CH3), 2.44 (t, 2H, J = 4.73 Hz), 2.36 (t, 2H,
J = 4.58 Hz); ESI-MS: 353.3 (M+1).
5.1.5.3. 2-(4-iso-Nicotinoyl-1-piperazinmethyl)-3,5,6-trimethyl-
pyrazine (E3). Flash column chromatography: chloroform–ace-
tone = 1:1; yield 42%; yellow crystal; mp 156 °C; IR (KBr, cmÀ1):
2939.60 (CH), 1629.98 (C@O), 1599.69 (C@C), 1551.20 (C@N); 1H
NMR (CDCl3, d): 8.68 (q, 2H, Ar-H, J = 1.58 Hz), 7.27 (q, 2H, Ar-H,
J = 1.58 Hz), 2.43–3.78 (m, 10H, CH2), 2.57 (s, 3H, CH3), 2.49 (s,
3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 326.5 (M+1).
5.1.4.16. 2-(4-Benzenesulfonyl-1-piperazinmethyl)-3,5,6-trim-
ethylpyrazine (E27). Flash column chromatography: chloro-
form–acetone = 1:1; yield 48%; yellow crystal; mp 118–120 °C; IR
(KBr, cmÀ1): 2919.28 (CH), 1353.05 (SO2), 1331.33 (SO2), 1181.05
(SO2), 1174.51 (SO2); 1H NMR (CDCl3, d): 7.75 (d, 2H, Ar-H,
J = 7.30 Hz), 7.59 (t, 1H, Ar-H, J = 7.31 Hz), 7.52 (t, 2H, Ar-H,
J = 7.56 Hz), 3.60 (2H, CH2), 2.60–3.05 (m, 8H, CH2), 2.53 (s, 3H,
CH3), 2.47 (s, 3H, CH3), 2.46 (s, 3H, CH3); ESI-MS: 361.4 (M+1).
5.1.5.4. 2-[4-[2-(4-Chlorophenoxy)-2-methyl-]propanoyl-1-pip-
erazinmethyl]-3,5,6-trimethylpyrazine (E4). Flash column chro-
matography: chloroform–acetone = 5:1; yield 49%; white crystal;
mp 100–101 °C; IR (KBr, cmÀ1): 2937.20 (CH), 1629.32 (C@O),
1596.54 (C@C), 1578.56 (C@N), 1242.16 (C–O), 1002.05 (C–O); 1H
NMR (CDCl3, d): 7.19 (m, 2H, Ar-H), 6.75 (m, 2H, Ar-H), 3.80 (s,
2H, CH2), 3.63 (s, 2H, CH2), 3.50 (s, 2H, CH2), 2.51 (s, 3H, CH3),
2.48 (s, 3H, CH3), 2.46 (s, 3H, CH3), 2.41 (s, 2H, CH2), 2.19 (s, 2H,
CH2), 1.62 (s, 6H, C(CH3)2); ESI-MS: 417.6 (M+1).
5.1.4.17. 2-[4-(4-Methylbenzenesulfonyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (E28). Flash column chromatography:
chloroform–acetone = 1:1; yield 46%; white crystal; mp 119–
120 °C; IR (KBr, cmÀ1): 2920.51 (CH), 1595.88 (C@C), 1662.32
(C@N), 1350.98 (SO2), 1329.04 (SO2), 1168.24 (SO2),
1130.92(SO2); 1H NMR (CDCl3, d): 7.67 (d, 2H, Ar-H, J = 8.17 Hz),
7.37 (d, 2H, Ar-H, J = 8.03 Hz), 3.65 (2H, CH2), 2.63–3.07 (m, 8H,
CH2), 2.63 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.49
(3H, Ar-CH3); ESI-MS: 375.3 (M+1).
5.1.5.5. 2-[4-[(E)-2,5-Dimethoxylcinnamoyl]-1-piperazinmeth-
yl]-3,5,6-trimethylpyrazine (E5). Flash column chromatography:
chloroform–acetone = 1:1; yield 56%; yellow oil; IR (KBr, cmÀ1):
2919.80 (CH), 1645.01 (C@O), 1604.46 (C@C), 1496.96 (C@N),
1222.62 (C–O), 1044.67 (C–O), 985.00 (@CH); 1H NMR (CDCl3, d):
7.86 (d, 1H, @CH, J = 15.57 Hz), 7.02 (d, 1H, Ar-H, J = 2.74 Hz),
6.95 (d, 1H, @CH, J = 15.60 Hz), 6.85 (m, 2H, Ar-H), 3.83 (s, 3H,
5.1.4.18. 2-(4-Methanesulfonyl-1-piperazinmethyl)-3,5,6-trim-
ethylpyrazine (E29). Flash column chromatography: chloro-
form–acetone = 3:1; yield 41%; white crystal; mp 116–117 °C; IR
(KBr, cmÀ1): 2947.79 (CH), 1641.98 (C@C), 1324.32 (SO2),

X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
3023
OCH3), 3.81 (s, 3H, OCH3), 3.60–3.80 (m, 10H, CH2), 2.54 (s, 3H,
CH3), 2.49 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: 411.6 (M+1).
cmÀ1): 2941.71 (CH), 1668.60 (C@O), 1654.54 (C@N); 1H NMR
(CDCl3, d): 4.06 (s, 2H, ClCH2), 3.51–3.67 (m, 10H, CH2), 2.59 (s,
3H, CH3), 2.51 (s, 3H, CH3), 2.49 (s, 3H, CH3); ESI-MS: 297.5 (M+1).
5.1.5.6. 2-[4-[(E)-3,4-Dimethoxylcinnamoyl]-1-piperazinmeth-
yl]-3,5,6-trimethylpyrazine (E6). Flash column chromatography:
chloroform–acetone = 2:1; yield 52%; yellow oil; IR (KBr, cmÀ1):
2935.64 (CH), 1645.14 (C@O), 1597.84 (C@C), 1513.44 (C@N),
1262.19 (C–O), 1024.92 (C–O), 999.80 (@CH); 1H NMR (CDCl3, d):
7.59 (d, 1H, @CH, J = 15.34 Hz), 7.09 (dd, 1H, Ar-H, J = 8.29 Hz),
7.02 (d, 1H, Ar-H, J = 1.57 Hz), 6.84 (d, 1H, Ar-H, J = 8.29 Hz), 6.70
(d, 1H, @CH, J = 15.34 Hz), 3.90 (6H, OCH3), 2.52–3.65 (m, 10H,
CH2), 2.57 (s, 3H, CH3), 2.53 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-
MS: 411.5 (M+1).
5.1.5.13. 2-(4-Phenylethyldione-1-piperazinmethyl)-3,5,6-trim-
ethylpyrazine (E32). Flash column chromatography: chloroform–
acetone = 1:1; yield 47%; yellow oil; IR (KBr, cmÀ1): 2920.22 (CH),
1680.18 (C@O), 1645.37 (C@O), 1596.29 (C@C), 1579.49 (C@N); 1H
NMR (CDCl3, d): 7.93 (d, 2H, Ar-H, J = 7.49 Hz), 7.63 (t, 1H, Ar-H,
J = 7.42 Hz), 7.50 (t, 2H, Ar-H, J = 7.80 Hz), 2.40–3.80 (m, 10H,
CH2), 2.54 (s, 3H, CH3), 2.47 (s, 3H, CH3), 2.45 (s, 3H, CH3); ESI-
MS: 353.5 (M+1).
5.1.6. Procedure for the preparation of 2-(4-salicyloyl-1-
piperazinmethyl)-3,5,6-trimethylpyrazine (E33, Method 3 of
Scheme 1)
5.1.5.7. 2-[4-[(E)-2,3-Dimethoxylcinnamoyl]-1-piperazinmeth-
yl]-3,5,6-trimethylpyrazine (E7). Flash column chromatography:
chloroform–acetone = 2:1; yield 44%; yellow oil; IR (KBr, cmÀ1):
2936.85 (CH), 1647.52 (C@O), 1606.19 (C@C), 1578.12 (C@N),
1268.27 (C–O), 1071.58 (C–O), 1002.77 (@CH); 1H NMR (CDCl3,
d): 7.87 (d, 1H, @CH, J = 15.64 Hz), 7.10 (d, 1H, Ar-H, J = 7.82 Hz),
7.03 (t, 1H, Ar-H, J = 8.00 Hz), 6.95 (d, 1H, @CH, J = 15.64 Hz),
6.90 (d, 1H, Ar-H, J = 8.06 Hz), 3.87 (s, 3H, OCH3), 3.84 (3H,
OCH3), 2.54–3.70 (m, 10H, CH2), 2.60 (s, 3H, CH3), 2.54 (s, 3H,
CH3), 2.49 (s, 3H, CH3); ESI-MS: 411.5 (M+1).
A mixture of E1 (10 mmol) and ammonia (25–28%, 50 mmol)
was stired at room temperature for 5 h and checked for product
formation via TLC. When the reaction finished, the mixture was ex-
tracted by chloroform and the extraction was evaporated in vacuo.
The final product was purified by flash column chromatography
and E33 was obtained. Flash column chromatography: chloro-
form–acetone = 1:1; yield 51%; yellow oil; IR (KBr, cmÀ1):
3065.70 (OH), 2919.83 (CH), 1614.15 (C@O); 1H NMR (CDCl3, d):
9.68 (s, 1H, OH), 7.26 (m, 1H, Ar-H), 7.20 (d, 1H, Ar-H,
J = 7.72 Hz), 6.96 (m, 1H, Ar-H), 6.82 (t, 1H, Ar-H, J = 7.33 Hz),
2.58–3.70 (m, 10H, CH2), 2.56 (s, 3H, CH3), 2.48 (s, 3H, CH3), 2.46
(s, 3H, CH3); ESI-MS: 341.4 (M+1).
5.1.5.8. 2-(4-Acetylferuloyl-1-piperazinmethyl)-3,5,6-trimethyl-
pyrazine (E8). Flash column chromatography: chloroform–ace-
tonechloroform–acetone = 2:1; yield 42%; yellow oil; IR (KBr,
cmÀ1): 2920.41 (CH), 1765.60 (C@O), 1648.46 (C@O), 1603.67
(C@C), 1509.17 (C@N), 1260.13 (C–O), 1196.81 (C–O), 1001.28
(@CH); 1H NMR (CDCl3, d): 7.61 (d, 1H, @CH, J = 15.38 Hz), 7.12
(d, 1H, Ar-H, J = 9.79 Hz), 7.07 (s, 1H, Ar-H), 7.03 (d, 1H, Ar-H,
J = 8.14 Hz), 6.79 (d, 1H, @CH, J = 15.38 Hz), 3.86 (3H, s, OCH3),
2.50–3.80 (m, 10H, CH2), 2.58 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.50
(s, 3H, CH3), 2.32 (s, 3H, COCH3); ESI-MS: 439.6 (M+1).
5.2. Biological evaluation
5.2.1. Protective effects on ECV-304 cells damaged by hydrogen
peroxide
The ECV-304 cells were plated and grown for 24 h in cultured
medium, then were switched to fresh medium in the presence of
12.5, 25, 50, 100, 200
lM Ligustrazine and its derivatives. After
5.1.5.9. 2-[4-[(E)-3-Nitrocinnamoyl]-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (E9). Flash column chromatography: chloro-
form–acetone = 1:2; yield 62%; yellow oil; IR (KBr, cmÀ1):
2920.27 (CH), 1651.78 (C@O), 1610.95 (C@C), 1529.96 (NO2),
1351.87 (NO2), 996.53 (@CH); 1H NMR (CDCl3, d): 8.38 (s, 1H, Ar-
H), 8.19 (d, 1H, Ar-H, J = 6.88 Hz), 7.78 (d, 1H, Ar-H, J = 7.70 Hz),
7.69 (d, 1H, @CH, J = 15.46 Hz), 7.56 (t, 1H, Ar-H, J = 7.93 Hz),
6.99 (d, 1H, @CH, J = 15.46 Hz), 2.59–3.71 (m, 10H, CH2), 2.56 (s,
3H, CH3), 2.50 (s, 3H, CH3), 2.49 (s, 3H, CH3); ESI-MS: 396.4 (M+1).
0.5 h incubation, 150
cells were incubated for an additional 6 h. The results were ex-
pressed as the values of absorbance at 570 nm. The date are mean-
lM hydrogen peroxide was added and the
s
SEM (n = 6). The proliferation rates of damaged cells were
calculated by [OD570 (Compd)-OD570 (H2O2)]/[OD570 (Control)-
OD570 (H2O2)] Â 100%, which was then used to obtain EC50 values
(see Table 1), according to the equation: ÀpEC50 = logCmax
À
P
2Â( P À 0.75 + 0.25Pmax + 0.25Pmin), where Cmax = maximum
P
concentration,
P = sum of proliferation rates, Pmax = maximum
value of proliferation rate and Pmin = minimum value of prolifera-
5.1.5.10. 2-[4-(3,5-Dinitrobenzoyl)-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (E23). Flash column chromatography: chloro-
form–acetone = 5:1; yield 45%; yellow crystal; mp 198–199 °C; IR
(KBr, cmÀ1): 2951.71 (CH), 1639.02 (C@O), 1591.30 (C@C), 1542.23
(NO2), 1344.54 (NO2); 1H NMR (CDCl3, d): 9.09 (s, 1H, Ar-H), 8.59
(d, 2H, Ar-H, J = 1.68 Hz), 2.66–3.84 (m, 10H, CH2), 2.53 (s, 3H,
CH3), 2.50 (s, 3H, CH3), 2.49 (s, 3H, CH3); ESI-MS: 415.5 (M+1).
tion rate.
5.2.2. Antiplatelet aggregation
Citric acid trisodium (3.8%, 0.3 ml), rabbit blood (2.7 ml), ADP
and Ligustrazine derivatives (200 lM) were added to cuvette in se-
quence, another two cuvette using as blank and ADP control. The
blood samples were centrifugated (270 g/5 min) and the super
stratum was extracted as platelet-abundant plasma. The remnant
blood samples were centrifugated again (1000g/10 min) and the
super stratum was extracted as platelet penurious plasma (PPP),
which was measured by aggregometer. The results were listed in
Table 1 and expressed as the mean value of the platelet aggrega-
tion rate (A%, n = 6).
5.1.5.11. 2-[4-(4-Nitrophenylacetyl)-1-piperazinmethyl]-3,5,
6-trimethylpyrazine (E25). Flash column chromatography: chlo-
roform–acetone = 5:1; yield 41%; yellow crystal; mp 50 °C; IR
(KBr, cmÀ1): 2920.09 (CH), 1647.97 (C@O), 1604.78 (C@N),
1519.28 (NO2), 1345.87 (NO2); 1H NMR (CDCl3, d): 8.20 (d, 2H,
Ar-H, J = 8.52 Hz), 7.42 (d, 2H, Ar-H, J = 8.45 Hz), 3.46–3.82 (m,
10H, CH2), 2.57 (s, 3H, CH3), 2.50 (s, 3H, CH3), 2.48 (s, 3H, CH3),
2.42 (s, 2H, Ar-CH2); ESI-MS: 384.4 (M+1).
Acknowledgments
The financial supports of this work by Shandong Natural Sci-
ence Foundation, Shandong Science and Technology Bureau Foun-
dation (2006GG2202063) and Jinan Excellent Young Scientists
Program (2003–12) are gratefully acknowledged.
5.1.5.12. 2-(4-Chloroacetyl-1-piperazinmethyl)-3,5,6-trimethyl-
pyrazine (E31). Flash column chromatography: chloroform–ace-
tone = 3:1; yield 34%; yellow crystal; mp 76–77 °C; IR (KBr,

3024
X.-C. Cheng et al. / Bioorg. Med. Chem. 17 (2009) 3018–3024
8. Jiang, G. H.; Wang, S. Z. Chinese Academy of Medical Sciences & Peking Union
References and notes
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