Design, synthesis, and biological activities of novel Ligustrazine derivatives

Article abstract of DOI:10.1016/j.bmc.2007.03.033

A series of novel Ligustrazine derivatives was designed, synthesized, and assayed for their protective effects on damaged ECV-304 cells and antiplatelet aggregation activities. The results showed that most Ligustrazine derivatives exhibited lower EC₅₀ values for protective effects on the ECV-304 cells damaged by hydrogen peroxide in comparison with Ligustrazine. And some Ligustrazine derivatives presented better antiplatelet aggregation activities than Ligustrazine. The derivatives containing the bisphenylmethyl pharmacophore (7a-c) exhibited highest potency. Compound 7a displayed most potential protective effects on the ECV-304 cells damaged by hydrogen peroxide, and compound 7c was found to be the most active antiplatelet aggregation agent. Structure-activity relationships were briefly discussed.

Full text of DOI:10.1016/j.bmc.2007.03.033

Bioorganic & Medicinal Chemistry 15 (2007) 3315–3320
Design, synthesis, and biological activities of novel
Ligustrazine derivatives
Xian-Chao Cheng,a Xin-Yong Liu,a,* Wen-Fang Xu,a Xiu-Li Guob and Yang Oub
aInstitute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road,
Jinan 250012, China
bInstitute of Pharmacology, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, China
Received 4 February 2007; revised 8 March 2007; accepted 9 March 2007
Available online 15 March 2007
Abstract—A series of novel Ligustrazine derivatives was designed, synthesized, and assayed for their protective effects on damaged
ECV-304 cells and antiplatelet aggregation activities. The results showed that most Ligustrazine derivatives exhibited lower EC50
values for protective effects on the ECV-304 cells damaged by hydrogen peroxide in comparison with Ligustrazine. And some
Ligustrazine derivatives presented better antiplatelet aggregation activities than Ligustrazine. The derivatives containing the
bisphenylmethyl pharmacophore (7a–c) exhibited highest potency. Compound 7a displayed most potential protective effects on
the ECV-304 cells damaged by hydrogen peroxide, and compound 7c was found to be the most active antiplatelet aggregation agent.
Structure–activity relationships were briefly discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
for keeping an effective plasma concentration by the
frequent administration.6 Therefore, it is necessary to
develop new generation of the cerebrocardiac vascular
drugs from molecular modification of Ligustrazine.
Ligustrazine (Lig; tetramethylpyrazine, TMP; see Fig. 1)
is one major efficient component from Chinese tradi-
tional medicine herb Chuanxiong(Ligusticum wallichii
Franchat), which is currently widely used in China as
a new kind of calcium channel antagonist for the treat-
ment of coronary atherosclerotic cardiovascular disease
and ischemic cerebrocardiac vascular disease.1 Ligustr-
azine has been reported to inhibit the platelet aggrega-
tion,2 to cause negative chronotropic and inotropic
responses on isolated atria,3 to inhibit vasoconstriction
in isolated vascular strips,4 and to act as a vasodilator,
a free-radical scavenger, and anti-thrombosis and anti-
hypertension agent.1 More recently, it has been found
to be more effective in protection against injured vascu-
lar endothelial cell.5
Structure–activity relationship studies indicated that
pyrazine ring in the molecule of Ligustrazine might lar-
gely be the determinant of its pharmacodynamics, while
the substituted groups might primarily govern its phar-
macokinetics and toxicity.7 So some drug-like groups
and pharmacophores can be introduced to the methyl
position of Ligustrazine, for acquiring the pharmacolog-
ically additive or synergetic effects to improve pharma-
cokinetic properties.
Calcium channel antagonists such as Cinnarizine, Flu-
narizine, and Lomerizine (see Fig. 1) are very important
cerebrocardiac vascular drugs currently used in the
clinic. As common characters in their molecular struc-
tures, piperazine ring acts as a linker to be considered
as the functional group for keeping the drugs’
potential.8
However, pharmacokinetics studies found that Ligustr-
azine presented low bioavailability and to be metabo-
lized fast in vivo with short half-life of T1/2 = 2.89 h,
so accumulated toxicity often appeared in the patients
According to the principles of hybridization and bioisos-
teric replacement in medicinal chemistry, we designed a
series of novel Ligustrazine derivatives by combination
with a piperazine and some pharmacophores or drug-
like groups, such as substituted benzyl, cinnamyl,
Keywords: Ligustrazine; Ligustrazine derivatives; Synthesis; Cerebro-
cardiac vascular activity.
*
Corresponding author. Tel.: +86 531 88380270; fax: +86 531
88382731; e-mail: xinyongl@sdu.edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.033

3316
X.-C. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 3315–3320
N
N
F
O
O
R
N
N
N
N
N
N
N
N
R
O
Novel designed Ligustrazine
derivatives 7a-u
R =H
R =F
Cinnarizine
Flunarizine
Lomerizine
Ligustrazine
F
R
Figure 1. Structures of Ligustrazine, Lomerizine, Cinnarizine, Flunarizine, and newly synthesized Ligustrazine derivatives 7a–u.
bisphenylmethyl, ligustrazinyl to form the new inte-
grated structural pattern (see Fig. 1). Some other groups
such as phenyl, b-phenylethyl were also introduced in
order to further expand our exploration and better
understand the structure–activity relationships of
Ligustrazine derivatives.
3. Biological evaluation and discussion
Endothelial cells play a critical physiological role in
maintaining normal vessel and organ function. Much
evidence showed that vascular endothelial cell damage
that causes the alteration of endothelial permeability
barrier and vascular tone is a major promoter of athero-
genesis, thrombosis, and consequently cardiovascular
events. Oxidative stress is a cardiovascular risk factor
and contributes significantly to endothelial injury during
atherogenesis. Therefore, the protection of endothelial
cells against damage caused by oxidative stress is a very
important therapeutic strategy.10
2. Chemistry
In the synthesis, (3,5,6-trimethylpyrazin-2-yl)methanol
(4) was prepared by the Boekelheide reaction starting
from Ligustrazine trihydrate (1), but one-pot reaction
was used according to our previous publication.9 The
important intermediate 2-chloromethyl-3,5,6-trimethyl-
pyrazine hydrochloride (5) was synthesized by the chlo-
rination of 4 with SOCl2 in anhydrous CH2Cl2 (see
Scheme 1). Ligustrazine derivatives (7a–u) were synthe-
sized by the following two methods. In Method 1, 2-
chloromethyl-3,5,6-trimethylpyrazine hydrochloride (5)
was directly reacted with the mono-substituted N-alkyl-
piperazines to afford alkylpiperazinyl Ligustrazine
derivatives (7a–d and 7t, see Method 1 of Scheme 1).
In Method 2, anhydrous piperazine was alkylated with
compound 5 in chloroform to produce the intermediate
2,3,5-trimethyl-6-(piperazin-1-ylmethyl)pyrazine (6), which
was reacted with various substituted benzyl halide to
give the corresponding alkylpiperazinyl Ligustrazine
derivatives (7e–s and 7u, see Method 2 of Scheme 1).
The chemical structures of the newly synthesized com-
The newly synthesized Ligustrazine derivatives were as-
sayed for the protective effects on the human umbilical
vascular endothelial cells (ECV-304 cells) damaged by
hydrogen peroxide.11 The results (Table 1) showed that
Ligustrazine and its derivatives presented protective ef-
fects on the damaged ECV-304 cells and most of the
Ligustrazine derivatives were more active (with lower
EC50 values) than Ligustrazine (EC50 value at 452 lM).
The derivatives containing bisphenylmethyl substituents
7a–c exhibited high potency with EC50 values below
300 lM, among which 7a was the most active one with
the EC50 value at 218 lM. As expected, the introduction
of bisphenylmethyl group, which is confirmed active in
cerebrocardiac vascular profiles of calcium channel
antagonists such as Cinnarizine, Flunarizine, and
Lomerizine, produced the remarkable combinational
effects. However, this principle did not work in 7d
(EC50 value at 428 lM), because introduction of
cinnamyl pharmacophore only moderately favored the
protective effects as compared with Ligustrazine.
1
pounds were confirmed by IR, H NMR, and ESI-MS.
N
N
N
N
N
N
i
ii
Cl
N
iii
3H2O
In the benzyl series, non-substituted compound at the
phenyl moiety (7f) exhibited highest potency and its
EC50 values were revealed at 246 lM. 4-Nitro-substitu-
tion at the phenyl moiety (7s) moderately impaired the
activity (EC50 value at 299 lM) and other substituted
compound seriously impaired the activity. 3-Nitril sub-
stitution (7q) showed highest EC50 value (1114 lM), al-
most completely losing the activity.
O
O
O
2
1
3
N
N
N
iv
v
HCl
OH
N
N
Method 1
N
4
N
5
N
R
7a-u
vii
vi
Method 2
The 2,20-piperazinyl-diligustrazine (7e) showed a low
EC50 value at 311 lM, reflecting the combinational ef-
fects of two ligustrazinyl groups. This compound may
act as a promising prodrug with improved pharmacoki-
netics properties.
N
NH
N
6
Scheme 1. Reagents and conditions: (i) 30% H2O2/AcOH, 70 ꢁC; (ii)
Ac2O/reflux for 2 h; (iii) 20% NaOH; (iv) SOCl2/anhydrous CH2Cl2;
(v) various N-mono-substituted piperazines, toluene, NaI, Et3N/reflux
for 10 h; (vi) piperazine; (vii) RX, toluene, NaI, Et3N/reflux for 10 h.
Among compounds 7f, 7t, and 7u, the activity order was
7f > 7u > 7t, which might reflect the importance of

X.-C. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 3315–3320
3317
Table 1. The structures, EC50 for protecting damaged ECV-304 cells, and platelet aggregation rate (A%) of Ligustrazine derivatives 6 and 7a–u
N
N
N
N
R
Compound
R
Method
EC50 (lM)
A%a (200 lM)
6
H
2
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
481
218
223
253
428
311
246
328
375
431
405
503
339
424
784
845
637
1114
417
299
665
542
452
11.36 6.52
9.88 4.26
6.32 8.22
5.19 3.52
8.36 7.11
19.36 5.84
9.06 1.08
17.69 2.64
13.56 4.07
15.32 3.30
9.67 3.02
10.56 7.06
12.31 3.22
5.70 3.56
7.68 4.66
13.26 8.75
12.16 3.45
14.19 5.22
11.37 3.56
14.22 1.05
21.43 2.63
9.77 5.39
7.86 4.17
7a
7b
7c
7d
7e
7f
Bisphenylmethyl
Bis(4-Fluorophenyl)methyl
(4-Chlorophenyl)(phenyl)methyl
(E)-Cinnamyl
Ligustrazinyl
Benzyl
7g
7h
7i
4-Methylbenzyl
4-Ethylbenzyl
4-tert-Butylbenzyl
2-Chlorobenzyl
3-Chlorobenzyl
4-Chlorobenzyl
2,4-Dichlorobenzyl
2-Bromobenzyl
4-Bromobenzyl
2-Nitrilbenzyl
3-Nitrilbenzyl
4-Nitrilbenzyl
4-Nitrobenzyl
Phenyl
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
Lig
b-Phenylethyl
a The A% value for blank is 0.73 2.93, and for ADP is 39.77 3.69.
carbon numbers between the phenyl group and pipera-
zine ring. One carbon between the phenyl group and
piperazine ring seems more favorable than other carbon
numbers. This assumption can be further confirmed by
the EC50 values of compounds 7a–c.
agent, presenting the pharmacologically additive or syn-
ergetic effects of Ligustrazine with pharmacophores.
In the benzyl-substituted series, 2-substituted or unsub-
stituted derivatives presented higher antiplatelet aggre-
gation activities than 4-substituted derivatives, but
with some exceptions of 2-, 3- or 4-nitril derivatives,
which showed almost no changes in platelet aggregation
rates. 2-Halogen substitution might favor the antiplate-
let aggregation activities, while alkyl, nitril or nitro-sub-
stitution impaired antiplatelet aggregation activities.
Benzyl or b-phenylethyl derivatives showed higher anti-
platelet aggregation activities than the phenyl derivative,
which might reflect the importance of methylene be-
tween the phenyl and piperazine ring.
The activity comparison between Ligustrazine and com-
pound 6 indicated that introduction of a piperazine ring
into Ligustrazine did not favor the protective effect of
Ligustrazine. However, further N-alkylation at pipera-
zine with R group produced compounds 7a–u, some of
which favored the activity of compound 6. The pharma-
cophore-based compounds, such as 7a–c showed lower
EC50 values as compared with compound 6. The struc-
ture–activity relationship studies above encouraged us
to connect compound 6 with another series of pharma-
cophores containing acyl groups, such as acetylsalicy-
loyl, nicotinoyl, and gallic acyl groups, which would
be reported later.
4. Conclusions
In conclusion, a series of novel Ligustrazine derivatives
was designed and synthesized. Most Ligustrazine deriva-
tives exhibited lower EC50 values for protective effects on
the ECV-304 cells damaged by hydrogen peroxide in com-
parison with Ligustrazine. And some Ligustrazine deriv-
atives presented better antiplatelet aggregation activities
than Ligustrazine. The derivatives containing the bisphe-
nylmethyl pharmacophore (7a–c) exhibited highest po-
tency. Compound 7a displayed most potential
protective effects on the ECV-304 cells damaged by
hydrogen peroxide, and compound 7c was found to be
the most active antiplatelet aggregation agent. Struc-
ture–activity relationships were briefly discussed. Further
bioassay of these compounds on cerebrocardiac vascular
activity on animal models is underway.
Ligustrazine has been reported to inhibit the platelet
aggregation,2 so these Ligustrazine derivatives were fur-
ther tested for antiplatelet aggregation induced by
ADP.12 The preliminary antiplatelet aggregation results
(Table 1) obtained from the experiments showed that
Ligustrazine derivatives bearing pharmacophores or
drug-like groups, such as 7b (bis(4-fluorophenyl)methyl,
6.32 18.22%), 7c ((4-chlorophenyl)(phenyl)methyl,
5.19 3.52%), 7m (2,4-dichlorobenzyl, 5.70 3.56%),
7n (2-bromobenzyl, 7.68 4.66%), exhibited the high
potent activities in antiplatelet aggregation at the con-
centration of 200 lM, and with lower values than that
of Ligustrazine (7.86 4.17%). Compound 7c was
found to be the most active antiplatelet aggregation

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X.-C. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 3315–3320
5. Experimental
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
(6) as white crystals (11 g, 50%); mp 94 ꢁC. IR (KBr,
cmꢀ1): 3443, 3272 (NH), 2943 (CH), 1546 (C@N).
NMR: dH (600 MHz, CDCl3) 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); dC (150 MHz, CDCl3)
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· CH2), 21.4,
21.0, and 20.1 (3· CH3). ESI-MS: 221 (M+1).
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) spectrometer; J values are in Hz. 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 silica gel 60-F-254 plates. Flash chromatogra-
phy was performed using 300 mesh silica gel. The yields
were calculated by the last step reaction.
5.1.4. General procedure for the preparation of 2-(4-substi-
tuted-1-piperazinmethyl)-3,5,6-trimethylpyrazine (7a–d and
7t, Method 1 of Scheme 1). 2-Chloromethyl-3,5,6-trimeth-
ylpyrazine hydrochloride (5) (2.07 g, 10 mmol) and
mono-substituted N-alkylpiperazines (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 mixture was filtered and the filtrate
was evaporated in vacuo. The final product was purified
by flash column chromatography and recrystallization
from n-hexane.
5.1.1. (3,5,6-Trimethylpyrazin-2-yl)methanol (4). 2,3,5,6-
Tetramethylpyrazine trihydrate (1) (30.40 g, 160 mmol)
was heated with 30% hydrogen peroxide (18 ml,
160 mmol) in glacial acetic acid (40 ml) for 4 h at
70 ꢁC. Further 30% hydrogen peroxide (18 ml,
160 mmol) was then added and the heating was contin-
ued 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 (2) was obtained. To
this, acetic anhydride (15.1 ml, 160 mmol) was added
and the mixture was refluxed for 3 h, checking for prod-
uct formation via TLC. The excess of acetic anhydride
was evaporated and (3,5,6-trimethylpyrazin-2-yl)methyl
acetate (3) was obtained, which was directly saponified
with 20% NaOH (155 ml) and extracted with chloro-
form. The combined extracts were dried and the solvent
removed. The residual oil was recrystallized from n-hex-
ane; (3,5,6-trimethylpyrazin-2-yl)methanol (4) was ob-
tained as yellow needles (15.50 g, 64%); mp 88–89 ꢁC.
5.1.4.1. 2-(4-Biphenylmethyl-1-piperazinmethyl)-3,5,6-
trimethylpyrazine (7a). Flash column chromatography:
ethyl acetate/cyclohexane = 1:3; yield 56%; white crystal;
mp 121–122 ꢁC; IR (KBr, cmꢀ1): 2809.86 (CH), 1598.06
1
(C@C), 1585.41(C@N); H NMR (CDCl3, d ppm): 7.41
(d, 4H, Ar–H, J = 7.75 Hz), 7.26 (t, 4H, Ar–H,
J = 7.70 Hz), 7.16 (t, 2H, Ar–H,J = 7.46 Hz), 4.22 (1H,
CH), 2.30–3.62 (m, 10H, CH2), 2.56 (s, 3H, CH3), 2.52
(s, 3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 387.5 (M+1).
5.1.2. 2-Chloromethyl-3,5,6-trimethylpyrazine hydrochlo-
ride (5). Thionyl chloride (7.41 ml, 102 mmol) was added
dropwise to (3,5,6-trimethylpyrazin-2-yl)methanol (4)
(15.50 g, 102 mmol) in anhydrous CH2Cl2 (300 ml) at
0 ꢁC. The mixture was allowed to stand for 2.5 h, check-
ing for product formation via TLC. The solvent was
evaporated in vacuo and the crude product 2-chloro-
methyl-3,5,6-trimethylpyrazine hydrochloride (5) was
obtained as a yellow solid (21.11 g, 100%); mp 102–
105 ꢁC. Compound 5 was basified and then purified to
give 2-chloromethyl-3,5,6-trimethylpyrazine as oil for
spectral confirmation. IR (KBr, cmꢀ1): 2993 (CH),
2952 (CH), 2923 (CH), 2856 (CH), 1548 (C@N).
NMR: dH (600 MHz, CDCl3) 4.68 (s, 2H, CH2), 2.63
(s, 3H, CH3), 2.54 (s, 3H, CH3), 2.52 (s, 3H, CH3); dC
(150 MHz, CDCl3) 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· CH3). ESI-MS: 171 (M+1).
5.1.4.2.
2-[4-(4,40-Difluro)biphenylmethyl-1-piper-
azinmethyl]-3,5,6- trimethyl pyrazine (7b). Flash column
chromatography: ethyl acetate/cyclohexane = 1:1; yield
56%; white crystal; mp 138–140 ꢁC; IR(KBr, cmꢀ1):
2954.35 (CH), 1603.08 (C@C), 1505.49 (C@N); 1H
NMR (CDCl3, d ppm): 7.34 (q, 4H, Ar–H, J = 7.04 Hz),
6.97 (t, 4H, Ar–H, J = 8.65 Hz), 4.21 (s, 1H, CH), 3.61–
3.62 (m, 10H, CH2), 2.57 (s, 3H, CH3), 2.51 (s, 3H,
CH3), 2.47 (s, 3H, CH3); ESI-MS: 423.6 (M+1).
5.1.4.3.
2-[4-(4-Chlorophenyl)phenylmethyl-1-piper-
azinmethyl]-3,5,6-trimethyl pyrazine (7c). Flash column
chromatography: ethyl acetate/cyclohexane = 1:2; yield
45%; white crystal; mp 112–114 ꢁC; IR(KBr, cmꢀ1):
2950.77 (CH), 1599.71 (C@C), 1546.64 (C@N); 1H
NMR (CDCl3, d ppm): 7.35 (q, 4H, Ar–H), 7.27 (t,
2H, Ar–H), 7.24 (t, 2H, Ar –H), 7.18 (t, 1H, Ar–H,
J = 7.27 Hz), 4.20 (1H, CH), 2.30–3.62 (m, 10H, CH2),
2.56 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.47 (s, 3H,
CH3); ESI-MS: 421.5 (M+1).
5.1.3. 2,3,5-Trimethyl-6-(piperazin-1-ylmethyl)pyrazine
(6). 2-Chloromethyl-3,5,6-trimethylpyrazine hydrochlo-
ride (5) (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 prod-
uct formation via TLC. The mixture solution was
5.1.4.4. 2-[4-[(E)-Cinnamyl]-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (7d). Flash column chromatography:
ethyl acetate/cyclohexane = 1:1; yield 43%; yellow crys-
tal; mp 74–75 ꢁC; IR (KBr, cmꢀ1): 2935.55 (CH),
1
1596.95 (C@N), 977.98 (@CH); H NMR (CDCl3, d,

X.-C. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 3315–3320
3319
ppm): 7.36 (d, 2H, Ar–H, J = 7.49 Hz), 7.29 (q, 2H, Ar–
H), 7.22 (t, 1H, Ar–H, J = 7.33 Hz), 6.51 (d, 1H, @CH,
J = 15.85 Hz), 6.27 (m, 1H, =CH), 2.50–3.63 (m, 12H,
CH2), 2.57(s, 3H, CH3), 2.56 (s, 3H, CH3), 2.48 (s, 3H,
CH3); ESI-MS: 337.5 (M+1).
5.1.5.5. 2-[4-(4-tert-Butylbenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7i). Flash column chromatogra-
phy: ethyl acetate; yield 47%; yellow oil; IR (KBr,
cmꢀ1): 2960.15 (CH), 1656.00 (C@C), 1513.44 (C@N);
1H NMR (CDCl3, d ppm): 7.32 (t, 2H, Ar–H,
J = 6.61 Hz), 7.24 (d, 2H, Ar–H, J = 8.20 Hz), 3.48–
3.62 (m, 12H, CH2), 2.57 (s, 3H, CH3), 2.54 (s, 3H,
CH3), 2.47 (s, 3H, CH3), 1.32 (s, 9H, C(CH3)3); ESI-
MS: 367.4 (M+1).
5.1.4.5. 2-(4-Phenyl-1-piperazinmethyl)-3,5,6-trimeth-
ylpyrazine (7t). Flash column chromatography: ethyl
acetate; yield 40%; white crystal; mp 82–84 ꢁC; IR
1
(KBr, cmꢀ1): 2957.59 (CH), 1603.01 (C@C); H NMR
(CDCl3, d ppm): 7.26 (m, 2H, Ar–H), 6.92 (d, 2H, Ar–
H, J = 7.99 Hz), 6.85 (t, 1H, Ar–H, J = 7.28 Hz), 2.67–
3.70 (m, 10H, CH2), 2.62 (s, 3H, CH3), 2.56 (s, 3H,
CH3), 2.51 (s, 3H, CH3); ESI-MS: 297.6 (M+1).
5.1.5.6.
2-[4-(2-Chlorobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7j). Flash column chromatogra-
phy: ethyl acetate; yield 41%; white crystal; mp 60 ꢁC; IR
(KBr, cmꢀ1): 2935.80 (CH), 1594.16 (C@C), 1571.32
1
(C@N); H NMR (CDCl3, d ppm): 7.49 (s, 1H, Ar–H),
5.1.5. General procedure for the preparation of 2-(4-substi-
tuted-1-piperazinmethyl)-3,5,6-trimethylpyrazine (7e–s and
7u, Method 2 of Scheme 1). To a mixture of 2,3,5-
trimethyl-6-(piperazin-1-ylmethyl)pyrazine (6) (2.2 g,
10 mmol) and Na2CO3 (3.18 g, 30 mmol) in anhydrous
CH2Cl2 (100 ml) was added dropwise various substituted
benzyl halide (10 mmol) in anhydrous CH2Cl2 (100 ml)
at room temperature. The mixture was refluxed for
10 h (checked by TLC), and the solvent was evaporated
in vacuo. The final product was purified by flash
column chromatography and recrystallization from
n-hexane.
7.35 (d, 1H, Ar–H, J = 7.83 Hz), 7.24 (t, 1H, Ar–H,
J = 7.42 Hz), 7.19 (t, 1H, Ar–H, J = 7.39 Hz), 2.50–
3.64 (m, 12H, CH2), 2.59 (s, 3H, CH3), 2.55 (s, 3H,
CH3), 2.49 (s, 3H, CH3); ESI-MS: 345.4 (M+1).
5.1.5.7.
2-[4-(3-Chlorobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7k). Flash column chromato-
graphy: ethyl acetate; yield 31%; yellow oil; IR (KBr,
cmꢀ1): 2936.45 (CH), 1596.95 (C@C), 1574.90 (C@N);
1H NMR (CDCl3, d ppm): 7.32 (s, 1H, Ar–H), 7.20
(m, 3H, Ar–H), 3.45–3.62 (m, 12H, CH2), 2.57 (s, 3H,
CH3), 2.53 (s, 3H, CH3), 2.46 (s, 3H, CH3); ESI-MS:
345.4 (M+1).
5.1.5.1. 2-[4-[(3,5,6-Trimethyl)-2-pyrazinmethyl]-1-
piperazinmethyl]-3,5,6-trimethylpyrazine (7e). Flash col-
umn chromatography: ethyl acetate/cyclohexane = 1:1;
yield 59%; yellow crystal; mp 162–164 ꢁC; IR (KBr,
5.1.5.8.
2-[4-(4-Chlorobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7l). Flash column chromatogra-
phy: ethyl acetate; yield 44%; yellow crystal; mp 68 ꢁC;
IR (KBr, cmꢀ1): 2946.03 (CH), 1596.71 (C@C),
1575.99 (C@N); 1H NMR (CDCl3, d ppm): 7.35 (d,
2H, Ar–H, J = 8.24 Hz), 7.29 (d, 2H, Ar–H,
J = 8.21 Hz), 2.30–3.52 (m, 12H, CH2), 2.47 (s, 3H,
CH3), 2.39 (s, 3H, CH3), 2.38 (s, 3H, CH3); ESI-MS:
345.4 (M+1).
1
cmꢀ1): 2937.71 (CH); H NMR (CDCl3, d ppm): 3.65
(m, 12H, CH2), 2.58 (s, 3H, CH3), 2.53 (s, 3H, CH3),
2.48 (s, 3H, CH3); ESI-MS: 355.6 (M+1).
5.1.5.2. 2-(4-Benzyl-1-piperazinmethyl)-3,5,6-trimeth-
ylpyrazine (7f). Flash column chromatography: ethyl
acetate; yield 44%; white crystal; mp 66 ꢁC; IR (KBr,
1
cmꢀ1): 2936.68 (CH); H NMR (CDCl3, d ppm): 7.30
5.1.5.9. 2-[4-(2,4-Dichlorobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7m). Flash column chromato-
graphy: ethyl acetate; yield 34%; white crystal; mp 62–
64 ꢁC; IR (KBr, cmꢀ1): 2943.87 (CH), 1587.77 (C@C),
1561.51 (C@N); 1H NMR (CDCl3, d ppm): 7.63 (s,
1H, Ar–H), 7.53 (q, 2H, Ar–H), 7.40 (t, 1H, Ar–H,
J = 7.69 Hz), 2.40–3.62 (m, 12H, CH2), 2.56 (s, 3H,
CH3), 2.52 (s, 3H, CH3), 2.47 (s, 3H, CH3); ESI-MS:
379.5 (M), 381.4 (M+2).
(m, 4H, Ar–H), 7.24 (m, 1H, Ar–H), 3.49–3.61 (m,
12H, CH2), 2.56 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.47
(s, 3H, CH3); ESI-MS: 311.5 (M+1).
5.1.5.3. 2-[4-(4-Methylbenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7g). Flash column chromatog-
raphy: ethyl acetate; yield 42%; white crystal; mp
66 ꢁC; IR (KBr, cmꢀ1): 2929.10 (CH), 1515.58 (C@N);
1H NMR (CDCl3, d ppm): 7.26 (m, 1H, Ar–H), 7.20
(d, 1H, Ar–H, J = 7.57 Hz), 7.14 (q, 2H, Ar–H), 3.44–
3.62 (m, 12H, CH2), 2.57 (s, 3H, CH3), 2.56 (s, 3H,
CH3), 2.47 (s, 3H, CH3), 2.35 (s, 3H, Ar–CH3); ESI-
MS: 325.6 (M+1).
5.1.5.10. 2-[4-(2-Bromobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7n). Flash column chromato-
graphy: ethyl acetate; yield 33%; white crystal; mp
90 ꢁC; IR (KBr, cmꢀ1): 2928.22 (CH), 761.48 (cuH);
1H NMR (CDCl3, d ppm): 7.51 (d, 1H, Ar–H,
J = 7.88 Hz), 7.47 (d, 1H, Ar–H, J = 7.54 Hz), 7.26 (m,
1H, Ar–H), 7.08 (t, 1H, Ar–H, J = 7.89 Hz), 3.58–3.62
(m, 12H, CH2), 2.57 (s, 3H, CH3), 2.53 (s, 3H, CH3),
2.47 (s, 3H, CH3); ESI-MS: 389.3 (M+1).
5.1.5.4. 2-[4-(4-Ethylbenzyl)-1-piperazinmethyl]-3,5,6-
trimethylpyrazine (7h). Flash column chromatography:
ethyl acetate; yield 32%; yellow oil; IR (KBr, cmꢀ1):
2932.21 (CH), 1512.87 (C@N); 1H NMR (CDCl3, d
ppm): 7.27 (t, 1H, Ar–H, J = 5.92 Hz), 7.21 (d, 1H,
Ar–H, J = 7.88 Hz), 7.18 (t, 1H, Ar–H), 7.11 (q, 1H,
Ar–H), 2.58–4.60 (m, 14H, CH2), 2.52 (s, 3H, CH3),
2.49 (s, 3H, CH3), 2.46 (s, 3H, CH3), 1.22 (3H,
CH2CH3); ESI-MS: 339.5 (M+1).
5.1.5.11. 2-[4-(4-Bromobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7o). Flash column chromato-
graphy: ethyl acetate; yield 44%; yellow crystal; mp
64–66 ꢁC; IR (KBr, cmꢀ1): 2925.61 (CH), 1592.58

3320
X.-C. Cheng et al. / Bioorg. Med. Chem. 15 (2007) 3315–3320
1
(C@C); H NMR (CDCl3, d ppm): 7.42 (d, 2H, Ar–H,
J = 7.37 Hz), 7.19 (d, 2H, Ar–H, J = 7.81 Hz), 2.40–
3.61 (m, 12H, CH2), 2.56 (s, 3H, CH3), 2.51 (s, 3H,
CH3), 2.47 (s, 3H, CH3); ESI-MS: 391 (M+2).
incubation, 150 lM hydrogen peroxide was added and
the cells were incubated for an additional 6 h. The
results were expressed as the values of absorbance at
570 nm. The data are means SEM (n = 6). The prolif-
eration rates of damaged cells were calculated by
[OD570 (Compd) ꢀ OD570 (H2O2)]/[OD570 (Con-
trol) ꢀ OD570 (H2O2)] · 100%, which was then used
5.1.5.12.
2-[4-(2-Nitrilbenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7p). Flash column chromato-
graphy: ethyl acetate; yield 45%; white crystal; mp
106–108 ꢁC; IR (KBr, cmꢀ1): 2931.17 (CH), 2219.88
(C„N), 1652.92 (C@C), 1599.30 (C@N); 1H NMR
(CDCl3, d ppm): 7.62 (d, 1H, Ar–H, J = 7.69 Hz), 7.54
(m, 2H, Ar–H), 7.33 (m, 1H, Ar–H), 2.50–3.69 (m,
12H, CH2), 2.56 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.47
(s, 3H, CH3); ESI-MS: 336.6 (M+1).
to obtain EC50 values (see Table 1), according to the
P
equation: ꢀpEC50 = log Cmax ꢀ log2 · ( P ꢀ 0.75 +
0.25Pmax + 0.25Pmin), where Cmax, maximum concen-
P
tration; P, sum of proliferation rates; Pmax, maximum
value of proliferation rate; and Pmin, minimum value of
proliferation rate.
5.2.2. Antiplatelet aggregation. Citric acid trisodium
(3.8%, 0.3 ml), rabbit blood (2.7 ml), ADP, and Ligustr-
azine derivatives (200 lM) were added to cuvette in se-
quence, another two cuvette using as blank and ADP
control. The blood samples were centrifuged (270 g/
5 min) and the superstratum was extracted as platelet-
abundant plasma. The remnant blood samples were cen-
trifuged again (1000 g/10 min) and the superstratum was
extracted as platelet penurious plasma (PPP), which was
measured by aggregometer. The results are listed in Ta-
ble 1 and expressed as the mean value of the platelet
aggregation rate (A%, n = 6).
5.1.5.13.
2-[4-(3-Nitrilbenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7q). Flash column chromatogra-
phy: ethyl acetate; yield 43%; yellow crystal; mp
116–118 ꢁC; IR (KBr, cmꢀ1): 2925.96 (CH), 2229.33
1
(C„N); H NMR (CDCl3, d ppm): 7.64 (s, 1H, Ar–H),
7.54 (q, 2H, Ar–H), 7.40 (t, 1H, Ar–H, J = 7.70 Hz),
2.40–3.62 (m, 12H, CH2), 2.56 (s, 3H, CH3), 2.52 (s,
3H, CH3), 2.47 (s, 3H, CH3); ESI-MS: 336.6 (M+1).
5.1.5.14.
2-[4-(4-Nitrilbenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7r). Flash column chromatogra-
phy: ethyl acetate; yield 34%; white crystal; mp 94–
96 ꢁC; IR (KBr, cmꢀ1): 2953.65 (CH), 2227.05 (C„N),
1
1607.08 (C@N), 1503.74 (C@C); H NMR (CDCl3, d
Acknowledgments
ppm): 7.59 (d, 2H, Ar–H, J = 8.15 Hz), 7.44 (d, 2H,
Ar–H, J = 8.05 Hz), 2.40–3.63 (m, 12H, CH2), 2.57 (s,
3H, CH3), 2.53 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-
MS: 336.6 (M+1).
The financial supports of this work by Shandong Natu-
ral Science Foundation, Shandong Science & Technol-
ogy Bureau Foundation, and Jinan Excellent Young
Scientists Program
acknowledged.
(2003-12)
are
gratefully
5.1.5.15.
2-[4-(4-Nitrobenzyl)-1-piperazinmethyl]-
3,5,6-trimethylpyrazine (7s). Flash column chromatogra-
phy: ethyl acetate; yield 39%; white crystal; mp 82–
83 ꢁC; IR (KBr, cmꢀ1): 2929.48 (CH), 1605.75 (C@C),
1522.47 (NO2), 1343.16 (NO2); 1H NMR (CDCl3, d
ppm): 8.17 (d, 2H, Ar–H, J = 8.49 Hz), 7.51 (d, 2H, Ar–
H, J = 7.62 Hz), 3.61–3.62 (m, 12H, CH2), 2.58 (s, 3H,
CH3), 2.49 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS:
356.5 (M+1).
References and notes
1. Xu, H.; Shi, D. Z.; Guan, C. Y. Chin. J. Integr. Tradit.
West. Med. 2003, 23(5), 376.
2. Liu, S. Y.; Sylvester, D. M. Thromb. Res. 1994, 75(1),
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3. Zou, L. Y.; Hao, X. M.; Zhang, G. Q.; Zhang, M.; Guo, J.
H.; Liu, T. F. Can. J. Physiol. Pharmacol. 2001, 79(7), 621.
4. Kwan, C. Y.; Daniel, E. E.; Chen, M. C. J. Cardiovasc.
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5. Yan, F.; Luo, R. J. Chin. Med. Mater. 2002, 25(2), 143.
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21(1), 58.
7. Jiang, G.H.; Wang, S.Z. Chinese Academy of Medical
Sciences & Peking Union Medical College Doctorial
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5.1.5.16. 2-(4-b-Phenylethyl-1-piperazinmethyl)-3,5,6-
trimethylpyrazine (7u). Flash column chromatography:
chloroform/acetone, 1:1; yield 40%; white crystal; mp
108 ꢁC; IR (KBr, cmꢀ1): 2929.26 (CH), 1601.92 (C@C);
1H NMR (CDCl3, d ppm): 7.30 (d, 2H, Ar–H,
J = 7.93 Hz), 7.21 (d, 3H, Ar–H, J = 7.29 Hz), 3.60–3.65
(m, 14H, CH2), 2.58 (s, 3H, CH3), 2.53 (s, 3H, CH3),
2.48 (s, 3H, CH3); ESI-MS: 325.6 (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 25, 50, 100, 200,
400 lM Ligustrazine and its derivatives. After 0.5-h