Ligustrazine derivatives. Part 8: Design, synthesis, and preliminary biological evaluation of novel ligustrazinyl amides as cardiovascular agents

Article abstract of DOI:10.2174/157340641130900042

A series of novel Ligustrazinyl amides was designed, synthesized and evaluated for their protective effect onthe injured vascular endothelial cells. The preliminary results demonstrated that some compounds possessed more potentactivities than that of Ligustrazine in stimulating replication of the injured human umbilical vascular endothelial cells(HUVECs) that is damaged by hydrogen peroxide. Among the active compounds, compounds 8i, 8t and 8u exhibited thehighest potency with low EC50 values of 0.037, 0.070 and 0.055 mM, respectively. Structure-activity relationships werebriefly discussed.

Full text of DOI:10.2174/157340641130900042

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Medicinal Chemistry, 2014, 10, 81-89
81
Ligustrazine Derivatives. Part 8: Design, Synthesis, and Preliminary
Biological Evaluation of Novel Ligustrazinyl Amides as Cardiovascular
Agents
Zhenyu Lia, Fang Yua, Lei Cuib, Wenmin Chena, Shouxun Wangb, Peng Zhana, Yuemao Shena and
Xinyong Liua*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharma-
b
ceutical Sciences, Shandong University, No.44 Wenhuaxi Road, Jinan 250012, China; Department of Biological
Chemistry and Molecular Biology, School of Medicine, Weifang Medical University, No.7166, Baotongxi Road, Weifang
261053, China
Abstract: A series of novel Ligustrazinyl amides was designed, synthesized and evaluated for their protective effect on
the injured vascular endothelial cells. The preliminary results demonstrated that some compounds possessed more potent
activities than that of Ligustrazine in stimulating replication of the injured human umbilical vascular endothelial cells
(HUVECs) that is damaged by hydrogen peroxide. Among the active compounds, compounds 8i, 8t and 8u exhibited the
highest potency with low EC50 values of 0.037, 0.070 and 0.055 mM, respectively. Structure-activity relationships were
briefly discussed.
Keywords: Ligustrazine, pyrazine amides, synthesis, cardiovascular disease.
In our previous work, we had designed and synthesized
several series of Ligustrazine derivatives, such as Ligustraz-
inyl esters [9], Ligustrazinyl alkyl/acylpiperazines [10, 11]
and Ligustrazinyl acylguanidines [12]. From biological
evaluation, several promising lead compounds were discov-
ered and exhibited good drugability. Among them, one of the
representative Ligustrazinyl esters is the ferulic acid ester
(Fig. 2), which has potent activity in animal model for anti-
cerebrocardiac vascular ischemia, but is prone to hydrolyze
in gastrointestinal tract, subjecting to fast metabolic fate with
short half-life.
INTRODUCTION
Pyrazine derivatives [1] have become of particular inter-
est to chemists and biologists, because of their broad spec-
trum of biological activities and potential pharmacological
applications [2]. For example, Pyrazinamide (PZA) is widely
used in chemotherapy for tuberculosis. Amiloride, 3,5-
diamino-6-chloropyrazinoyl-guanidine, is a potent Na+/H+
exchanger (NHE) inhibitor, and Ligustrazine (tetramethyl-
pyrazine, TMP) is one of the calcium antagonists, both are
clinically used for the treatment of cerebrocardiac vascular
disease (CVD) (See Fig. 1) [3-7].
In continuation of our research, we decided to undertake
a study of the Ligustrazinyl amide series (Fig. 2), because
amides relatively have metabolic stability when compared to
Ligustrazinyl esters. In the designed Ligustrazinyl amides,
carbolic acid moieties were selectively used the active com-
pounds in cardiovascular biology, such as nicotinic acid,
salicylic acid, fumalic acid, cinnamic acid, gallic acid, salvi-
anic acid, mandelic acid etc., in order to acquire the pharma-
cologically combinational effects based on the hybridization
and prodrug principles [13, 14]. Herein, we report the prepa-
ration of the novel Ligustrazinyl amides, as well as their bio-
logical evaluation for protective effects on the injured vascu-
lar endothelial cells.
Ligustrazine is an active component of Chinese tradi-
tional medicine herb Chuanxiong (Ligusticum wallichii
Franchat), which is currently widely used in clinic for the
treatment of coronary atherosclerotic cardiovascular disease
and ischemic cerebrocardiac vascular disease [8]. However,
pharmacokinetics studies found that Ligustrazine presented
low bioavailability with a short half-life in vivo (T1/2=2.89h),
so accumulated toxicity often appeared in the patients for
keeping an effective plasma concentration by the frequent
administration [9]. Therefore, it is necessary to develop new
generation of the Ligustrazine’s cardiovascular drugs by
molecular modification.
MATERIALS AND METHODS
Chemistry
*Address correspondence to this author at the Department of Medicinal
Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, No.44 Wenhuaxi
Road, Jinan 250012, China; Tel: +86 0531 88380270;
Infrared spectra were measured using a nicolet nexus 470
FT-IR spectrometer using smear KBr crystal or KBr plate.
Fax: +86 0531 88382731; E-mail: xinyongl@sdu.edu.cn
1875-6638/14 $58.00+.00
© 2014 Bentham Science Publishers

82 Medicinal Chemistry, 2014, Vol. 10, No. 1
Li et al.
Fig. (1). Some pyrazine derivatives used in clinic.
Fig. (2). Design strategy of Ligustrazinyl amide derivatives.
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 electrospray ionization
mass spectrometer as the value m/z. Thin-layer chromatogra-
phy (TLC) was self-made silica gel (GF254) sheets. Flash
chromatography was performed using 200-300 mesh silica
gel. The yields were calculated by the last step reaction.
2H, CH2), 2.58 (s, 3H, CH3), 2.54-2.53 (m, 6H, CH3 x 2);
13C-NMR (150 MHz, CDC13, ꢀ ppm): 168.32, 168.21
(C=O), 149.78, 147.85, 147.34, 144.81 (pyrazine-C), 134.46,
134,12, 128.64, 128.24, 127.17, 127.08 (benzene-C), 42.28
(CH2), 21.33, 21.21, 20.08 (CH3); ESI-MS: 282.2 (M+H)+.
A solution of compound 6 (28.1 g, 100 mmol) and 50%
hydrazine hydrate (10 mL, 100 mmol) in ethanol (100 mL)
was refluxed for 2.5 h, and filtered. The filtrate was adjusted
to pH 2-3 with concentrated hydrochloride and filtered. The
solvent was evaporated off under reduced pressure, and then
40% sodium hydroxide (60 mL) was added. The organic
layer was separated to give the product 7 as yellow oil (8.2 g,
Preparation of (3,5,6-Trimethylpyrazin-2-yl)methanol (4)
Compound 4 was prepared according to our previous re-
ported method by using one-pot reaction [10, 15]. The crude
product was purified by recrystallization with n-hexane to
obtain yellow needles, mp: 88-89 °C, with total yield of
64%.
1
54.2%). H-NMR (600 MHz, CDCl3, ꢀ ppm): 8.70 (s, 2H,
NH2), 4.53 (d, 2H, CH2), 2.56 (s, 3H, CH3), 2.53-2.52 (m,
6H, CH3 x 2); 13C-NMR (150 MHz, CDC13, ꢀ ppm): 149.77,
147.90, 147.75, 144.63 (pyrazine-C), 41.72 (CH2), 21.66,
21.42, 20.04 (CH3); ESI-MS: 152.3 (M+H)+.
Synthesis of 2-Chloromethyl-3,5,6-trimethylpyrazine
Hydrochloride (5)
General Procedure for the Preparation of Ligustrazinyl
Amides (8a-8l, Method A of Scheme 1), Example of N-
((3,5,6-trimethylpyrazin-2-yl)methyl)benzamide (8a)
To a solution of (3,5,6-trimethylpyrazin-2-yl)methanol
(4) (15.50 g, 102 mmol) in anhydrous CH2Cl2 (300 mL),
thionyl chloride (7.41 mL, 102 mmol) was added drop by
drop at 0 °C. After finishing dropping, the mixture was
stirred at room temperature for 2.5 h. The solvent was evapo-
rated in vacuo and the product 2-chloromethyl-3,5,6-
trimethylpyrazine hydrochloride (5) was obtained as yellow
solid (21.11 g, 100%), mp: 102-105 °C [11].
A solution of 2-aminemethyl-3,5,6-trimethylpyrazine (7)
(0.755 g, 0.005 mol) and pyridine (0.4 g, 0.0055 mol) in
chloroform (30 mL), benzoyl chloride (0.0775 g, 0.0055
mol) was added drop by drop at 0 °C. After 2 h of stirring at
room temperature, the mixture was washed with water (3 x
30 mL). The organic phase was dried over anhydrous sodium
sulfate and the solvent evaporated in vacuo. The residue was
purified by flash column chromatography and then recrystal-
lized from ethanol to afford 8a as white crystals (0.66 g,
52%), mp: 115-116 °C; IR (KBr, cm-1): 3279.86 (NH),
1652.95 (C=O), 1530.02, 1486.59, 1446.19, 1416.15 (C=N,
Preparation of 2-aminemethyl-3,5,6-trimethylpyrazine (7)
To a solution of 2-chloromethyl-3,5,6-trimethylpyrazine
(5) (20.7 g, 100 mmol) in DMF (100 mL), there was added a
solution of phthalimide potassium salt (18.5 g, 150 mmol) in
60 mL of ethanol. After 1.5 h of stirring at 80 °C, the solids
were filtered off. The filtrate was evaporated under reduced
pressure to obtain the crude product, which was purified by
recrystallization from ethanol affording the compound 6 as
colorless crystals (20.8 g, 74.3%), mp: 154-156 °C. 1H-NMR
(600 MHz, CDCl3, ꢀ ppm): 7.91-7.84 (m, 4H, Ar-H), 4.92 (d,
1
C=C); H-NMR (600 MHz, CDCl3, ꢀ ppm): 7.92 (s, 1H,
NH), 7.90 (d, 2H, Ar-H, J = 7.2 Hz), 7.54 (dd, 1H, Ar-H, J1
= 6.9 Hz, J2 = 1.8 Hz), 7.49 (dd, 2H, Ar-H, J1 = 7.0 Hz, J2 =
7.1 Hz), 4.69 (d, 2H, CH2, J = 4.1 Hz), 2.58 (s, 3H, CH3),
2.54 (s, 3H, CH3), 2.53 (s, 3H, CH3); 13C-NMR (150 MHz,

Ligustrazine Derivatives. Part 8: Design, Synthesis, and Preliminary Biological Evaluation
Medicinal Chemistry, 2014, Vol. 10, No. 1 83
CDC13, ꢀ ppm): 167.33 (C=O), 149.73, 147.92, 147.78,
144.85 (pyrazine-C), 134.46, 131.53, 128.62, 127.06 (ben-
zene-C), 41.43 (CH2) 21.56, 21.45, 20.06 (CH3); ESI-MS:
256.0 (M+H)+.
(E)-3-(4-chlorophenyl)-N-((3,5,6-trimethylpyrazin-2-yl)me-
thyl)acrylamide (8f)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 50%; white crystal; mp: 169-171 °C; IR (KBr,
cm-1): 3264.29 (NH), 1619.16, 1543.28, 1493.85, 1442.58
(C=N, C=C), 1651.74 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ
ppm): 7.32 (s, 1H, NH), 7.65 (d, H, C=CH, J = 15.60 Hz),
6.55 (d, 1H, C=CH, J = 15.60 Hz), 7.48 (d, 2H, Ar-H, J =
8.45 Hz), 7.35 (d, 2H, Ar-H, J = 8.44 Hz), 4.61 (s, 2H, CH2);
2.53-2.51(m, 9H, CH3 x 3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 165.55 (C=O), 133.36, 135.50 (C=CH), 41.26 (CH2),
20.08, 21.41, 21.45 (CH3); ESI-MS: 316.3 (M+H)+.
2-phenyl-N-((3,5,6-trimethylpyrazin-2-yl)methyl)acetamide
(8b)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 53%; yellow crystal; mp: 125-126 °C; IR (KBr,
cm-1): 3377.86 (NH), 1507.64, 1496.82, 1454.64 (C=N,
C=C), 1662.40 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.17 (s, H, NH), 7.40-7.33 (m, 5H, Ar-H), 4.43 (d, 2H, CH2,
J = 4.1 Hz), 2.46 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.34 (s,
3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ ppm): 171.13
(C=O), 144.57, 147.50, 147.73, 149.51 (pyrazine-C), 127.31,
128.97, 129.70, 134.91 (benzene-C), 41.02, 43.84 (CH2),
19.91, 21.21, 21.34 (CH3); ESI-MS: 270.5 (M+H)+.
(E)-3-(4-fluorophenyl)-N-((3,5,6-trimethylpyrazin-2-yl)me-
thyl)acrylamide (8g)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 52%; white crystal; mp: 160-161 °C; IR (KBr,
cm-1): 3266.11 (NH), 1618.59, 1540.61, 1510.52, 1444.70
(C=N, C=C), 1652.47 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ
ppm): 7.31 (s, 1H, NH), 7.65 (d, H, C=CH, J = 15.60 Hz),
6.51 (d, H, C=CH, J = 15.61 Hz), 7.54 (m, 2H, Ar-H), 7.07
(m, 2H, Ar-H), 4.62 (s, 2H, CH2), 2.54(s, 3H, CH3), 2.54(s,
3H, CH3), 2.51(s, 3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 165.71 (C=O), 139.90, 131.08 (C=CH), 41.22 (CH2),
20.09, 21.38, 21.45 (CH3), C: 163.53 (1JC-F = 248.55 Hz), C:
120.27 (4JC-F = 1.5 Hz), CH: 115.85 (2JC-F = 21.5 Hz), CH:
129.64 (3JC-F = 8.6 Hz); ESI-MS: 300.6 (M+H)+.
3-chloro-N-((3,5,6-trimethylpyrazin-2-yl)methyl)benzamide
(8c)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 45%; white crystal; mp: 110-111 °C; IR (KBr,
cm-1): 3406.14 (NH), 1523.23, 1565.89, 1523.23 (C=N,
1
C=C), 1671.43 (C=O); H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.98 (s, H, NH), 7.89 (t, H, Ar-H), 7.76 (dd, 1H, Ar-H, J1 =
1.11 Hz, J2 = 7.73 Hz), 7.50 (m, 1H, Ar-H ), 7.40 (m, 1H,
Ar-H), 4.67 (d, 2H, CH2, J = 4.51 Hz), 2.56 (s, 3H, CH3),
2.54 (s, 3H, CH3), 2.53 (s, 3H, CH3); 13C-NMR (150 MHz,
CDC13, ꢀ ppm): 165.99 (C=O), 144.61, 147.84, 148.00,
149.96 (pyrazine-C), 125.09, 127.55, 129.93, 133.14,
134.79, 136.15 (benzene-C), 41.41 (CH2), 20.00, 21.40,
21.49 (CH3); ESI-MS: 290.5 (M+H)+.
(E)-3-(4-bromophenyl)-N-((3,5,6-trimethylpyrazin-2-yl)me-
thyl)acrylamide (8h)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 45%; white crystal; mp: 165-166 °C; IR (KBr,
cm-1): 3261.65 (NH), 1651.52, 1617.67, 1543.39, 1488.76
(C=N, C=C), 1757.39 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ
ppm): 7.79 (d, H, C=CH, J = 15.92), 7.63 (d, H, C=CH, J =
15.59 Hz), 7.52 (d, 2H, Ar-H, J = 8.36 Hz), 7.40 (d, 2H, Ar-
H, J = 8.36 Hz), 7.34 (s, H, NH), 4.62 (d, 2H, CH2, J = 4.0
Hz), 2.63 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.53 (s, 3H, CH3);
13C-NMR (150 MHz, CDC13, ꢀ ppm): 165.54 (C=O),
117.28, 123.81 (C=CH), 41.23 (CH2), 20.07, 21.33, 21.42
(CH3); ESI-MS: 362.4 (M+H)+.
N-((3,5,6-trimethylpyrazin-2-yl)methyl)nicotinamide (8d)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 40%; white crystal; mp: 125-129 °C; IR (KBr,
cm-1): 3275.19 (NH), 1591.62, 1548.10, 1438.75 (C=N,
1
C=C), 1642.60 (C=O), H-NMR (600 MHz, CDCl3, ꢀ ppm):
8.23 (s, H, NH), 7.45 (m, H, Ar-H), 8.25 (dt, 1H, Ar-H, J1 =
7.86 Hz, J2 = 1.98 Hz), 8.75 (dd, 1H, Ar-H, J1 = 4.81 Hz, J2 =
1.63 Hz), 9.12 (m, 1H, Ar-H), 4.68 (d, 2H, CH2, J = 4.08
Hz), 2.58 (s, 3H, CH3), 2.54-2.53 (m, 6H, CH3 x 2); 13C-
NMR (150 MHz, CDC13, ꢀ ppm): 165.36 (C=O), 123.60,
130.08, 135.27, 144.28, 147.71 (pyridine-C), 41.31 (CH2),
20.00, 21.46, 21.54 (CH3); ESI-MS: 257.6 (M+H)+.
N-((3,5,6-trimethylpyrazin-2-yl)methyl)isonicotinamide (8i)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 38%; yellow crystal; mp: 119-120 °C; IR (KBr,
cm-1): 3275.81 (NH), 1591.52, 1547.44, 1478.53 (C=N,
2-(((3,5,6-trimethylpyrazin-2-yl)methyl)carbamoyl)phenyl
acetate (8e)
1
C=C), 1642.22 (C=O), H-NMR (600 MHz, CDCl3, ꢀ ppm):
8.06 (s, H, NH), 7.45 (m, H, Ar-H), 8.25 (dt, 1H, Ar-H, J1 =
7.86 Hz, J2 = 1.98 Hz), 8.75 (dd, 1H, Ar-H, J1 = 4.81 Hz, J2 =
1.63 Hz), 9.12 (m, 1H, Ar-H), 4.68 (d, 2H, CH2, J = 3.75
Hz), 2.54 (s, 9H, 3 ꢀ CH3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 165.34 (C=O), 123.59, 130.13, 135,26, 144.26, 147.73
(pyridine-C), 41.30 (CH2), 19.99, 21.45, 21.51 (CH3); ESI-
MS: 257.4 (M+H)+.
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 46%; yellow crystal; mp: 113-114 °C; IR (KBr,
cm-1): 3443.58 (NH), 1523.88, 1606.85, 1479.13 (C=N,
C=C), 1758.85 (C=O), 1653.75 (C=O); 1H-NMR (600 MHz,
CDCl3, ꢀ ppm): 8.11 (s, H, NH), 7.93 (dd, H, Ar-H, J1 = 1.67
Hz, J2 = 7.7 6Hz), 7.51 (dt, 1H, Ar-H, J1 = 1.68 Hz, J2 = 7.66
Hz), 7.35 (dt, 1H, Ar-H, J1 = 1.06 Hz, J2 = 7.60 Hz), 7.15
(dd, 1H, Ar-H, J1 = 0.98 Hz, J2 = 8.12 Hz ), 4.66 (s, 2H,
CH2), 2.53 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.52 (s, 3H,
CH3), 2.31 (s, 3H, COCH3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 165.34, 169.09 (C=O), 41.78 (CH2), 20.09, 21.23,
21.37, 21.44 (CH3); ESI-MS: 314.4 (M+H)+.
(E)-3-(4-methoxyphenyl)-N-((3,5,6-trimethylpyrazin-2-yl)
methyl)acrylamide (8j)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 48%; white crystal; mp: 185-187 °C; IR (KBr,
cm-1): 3285.87 (NH), 1615.61, 1603.50, 1576.22, 1567.12

84 Medicinal Chemistry, 2014, Vol. 10, No. 1
Li et al.
(C=N, C=C), 1655.75 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ
ppm): 7.65 (d, H, C=CH, J = 15.58 Hz), 7.51 (d, 2H, Ar-H, J
= 8.68 Hz), 6.91 (d, 2H, Ar-H, J = 8.69 Hz), 6.46 (d, H,
C=CH, J = 15.60 Hz), 4.63(s, H, CH2), 3.85 (s, 3H, OCH3),
2.55 (s, 3H, CH3), 2.54 (s, 3H, CH3), 2.53 (s, 3H, CH3); 13C-
NMR (150 MHz, CDC13, ꢀ ppm): 166.24 (C=O), 129.95,
127.56 (C=CH), 41.19 (CH2), 55.36 (OCH3), 20.10, 21.32,
21.42 (CH3); ESI-MS: 312.6 (M+H)+.
CH3); 13C-NMR (150 MHz, CDC13, ꢀ ppm): 166.34 (C=O),
124.56, 132.56 (C=CH), 41.34 (CH2), 55.82, 56.13 (OCH3),
20.13, 21.18, 21.43 (CH3); ESI-MS: 342.5 (M+H)+.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-((3,5,6-trimethylpyr-
azin-2-yl)methyl)acrylamide (8n)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 35%; white crystal; mp: 204-206 °C; IR (KBr,
cm-1): 3324.79 (OH), 2996.19 (NH), 1599.66, 1583.17,
1583.17, 1559.39, 1510.43 (C=N, C=C), 1650.75 (C=O); 1H-
NMR (600 MHz, CDCl3, ꢀ ppm): 7.22 (s, H, NH), 7.05 (s,
H, Ar-H), 7.12 (d, H, Ar-H, J = 8.05 Hz), 6.93 (d, H, Ar-H, J
= 8.14 Hz), 6.44 (d, H, C=CH, J = 15.53 Hz), 7.63 (d, H,
C=CH, J = 15.52 Hz), 4.63 (d, 2H, CH2, J = 3.51 Hz), 3.95
(s, 3H, OCH3), 2.55 (s, 3H CH3), 2.52 (s, 3H, CH3), 2.49 (s,
3H, CH3), 5.97 (s, H, OH); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 166.72 (C=O), 127.43, 141.14 (C=CH), 41.30 (CH2),
55.97 (OCH3), 20.10, 21.42, 21.44 (CH3); ESI-MS: 328.6
(M+H)+.
(E)-3-p-tolyl-N-((3,5,6-trimethylpyrazin-2-yl)methyl)acryl-
amide (8k)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 46%; white crystal; mp: 152-153 °C; IR (KBr,
cm-1): 3237.75 (NH), 1622.78, 1549.82, 1444.59 (C=N,
C=C), 1667.90 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.66 (d, H, C=CH, J = 15.60 Hz), 7.46 (d, 2H, Ar-H, J =
8.02 Hz), 7.20 (d, 2H, Ar-H, J = 7.94 Hz), 6.54 (d, H,
C=CH, J = 15.60 Hz), 4.63(d, 2H, CH2, J = 4.25 Hz), 2.54
(s, 3H, CH3), 2.40 (s, 3H, CH3), 2.38 (s, 3H, CH3), 2.19 (s,
3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ ppm): 166.07
(C=O), 132.13, 139.97 (C=CH), 41.25 (CH2), 20.10, 21.37,
21.42, 21.43 (CH3); ESI-MS: 296.6.
2-hydroxy-N-((3,5,6-trimethylpyrazin-2-yl)methyl)benzam-
ide (8o)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 41%; white crystal; mp: 152-153 °C; IR (KBr,
cm-1): 3381.89 (NH), 1595.08, 1523.00, 1487.51 (C=N,
C=C), 1636.94 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
12.49 (s, H, OH), 8.26 (s, H, NH), 7.57 (d, H, Ar-H, J = 2.2
Hz), 7.43 (t, H, Ar-H, J = 7.8 Hz), 7.01 (d, H, Ar-H, J=7.8
Hz), 6.62 (t, H, Ar-H, J = 7.8 Hz), 4.64 (s, 2H, CH2), 2.56
(m, 9H, 3 ꢀ CH3); ESI-MS: 342.4 (M+H)+.
(E)-3-(4-hydroxyphenyl)-N-((3,5,6-trimethylpyrazin-2-yl)
methyl)acrylamide (8l)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 30%; white crystal; mp: 210-212 °C; IR (KBr,
cm-1): 3113.90 (NH), 3394.06 (OH), 1602.85, 1580.64,
1
1515.34, 1440.15 (C=N, C=C), 1650.06 (C=O); H-NMR
(600 MHz, CDCl3, ꢀ ppm): 7.19 (s, H, NH), 6.40 (d, H,
C=CH, J = 15.59 Hz), 6.86 (d, 2H, Ar-H, J = 8.52 Hz), 7.45
(d, 2H, Ar-H, J = 8.44 Hz), 7.62 (d, H, C=CH, J = 15.58
Hz), 4.63 (d, 2H, CH2, J = 4.19 Hz), 2.55 (s, 3H, CH3), 2.54
(s, 3H, CH3), 2.53 (s, 3H, CH3); 13C-NMR (150 MHz,
CDC13, ꢀ ppm): 166.28 (C=O), 118.13, 127.64 (C=CH),
41.26 (CH2), 20.11, 21.35, 21.43 (CH3); ESI-MS: 298.6
(M+H)+.
3,4,5-triacetoxy-N-((3,5,6-trimethylpyrazin-2-yl)methyl)be-
nzamide (8p)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 53%; white crystal; mp: 179-180 °C; IR (KBr,
cm-1): 3278.44 (NH), 1667.94, 1541.30, 1491.03 (C=N,
C=C), 1777.84 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.90 (s, H, NH), 7.66 (s, 2H, Ar-H), 4.65 (d, 2H, CH2, J = 4.2
Hz), 2.54 (m, 9H, 3 ꢀ OCH3), 2.32 (m, 9H, 3ꢀCH3); 13C-
NMR (150 MHz, CDC13, ꢀ ppm): 167.67, 166.62, 165.03
(C=O), 41.38 (CH2), 20.01, 20.23, 20.67 (CH3), 21.49
(OCOCH3); ESI-MS: 430.6 (M+H)+.
General Procedure for the Preparation of Ligustrazinyl
Amides (8m-8u, Method B in Scheme 1). Example of (E)-
3-(2,5-dimethoxyphenyl)-N-((3,5,6-trimethylpyr-azin-2-
yl) methyl) Acrylamide (8m)
A mixture of 2,5-dimethoxylcinnamic acid (1.04 g, 0.005
mol), HOBt (0.73 g, 0.0055 mol) and DCC (1.12 g, 0.0055
mol) in anhydrous THF (20 mL) was stirred at 0 °C for 5 h,
and then the solids were filtered off. A solution of 2-
aminemethyl-3,5,6-trimethylpyrazine (7) (0.755 g, 0.005
mol) in anhydrous THF (15 mL) was then added to the fil-
trate. After the mixture was stirred at room temperature
overnight, the solvent was evaporated in vacuo and the resi-
due was poured into ethyl acetate and washed with saturated
sodium bicarbonate, 50% citric acid and brine. The organic
layer was dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness. The crude product was purified by
recrystallisation from 90-95% ethanol. yield 38%; white
crystal; mp: 138-139 °C; IR (KBr, cm-1): 3265.84 (NH),
1608.61, 1539.66, 1500.09 (C=N, C=C), 1645.42 (C=O); 1H-
NMR (600 MHz, CDCl3, ꢀ ppm): 7.93 (d, H, HC=C, J =
15.81 Hz), 7.24 (s, H, NH), 7.06 (s, H, Ar-H), 6.89 (d, 2H,
Ar-H, J = 8.95 Hz), 6.66 (d, H, C=CH, J = 8.89 Hz), 4.63(s,
2H, CH2), 3.84 (s, 3H, CH3), 3.81 (s, 3H, CH3), 2.59 (s, 3H,
4-(((3,5,6-trimethylpyrazin-2-yl)methyl)carbamoyl)phenyl
acetate (8q)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 52%; white crystal; mp: 127-128 °C; IR (KBr,
cm-1): 3284.37 (NH), 1632.71, 1541.40, 1541.40, 1501.51
(C=N, C=C), 1752.65 (C=O), 1632.71 (C=O); 1H-NMR (600
MHz, CDCl3, ꢀ ppm): 7.93 (s, H, NH), 7.92 (s, 2H, Ar-H),
7.22 (m, 2H, Ar-H), 4.67 (d, 2H, CH2, J = 3.6 Hz), 2.34 (s,
3H, OCOCH3), 2.53 (m, 9H, 3 ꢀ CH3); 13C-NMR (150 MHz,
CDC13, ꢀ ppm): 166.50 (C=O), 41.36 (CH2), 20.03, 21.18,
21.44 (CH3), 21.49 (OCOCH3); ESI-MS: 314.5 (M+H)+.
2-oxo-1-phenyl-2-((3,5,6-trimethylpyrazin-2-yl)methylam-
ino)ethyl acetate (8r)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 54%; white crystal; mp: 112-114 °C; IR (KBr,
cm-1): 3366.26 (NH), 1694.91, 1685.48, 1548.19 (C=N,
C=C), 1717.79 (C=O), 1735.81 (C=O); 1H-NMR (600 MHz,

Ligustrazine Derivatives. Part 8: Design, Synthesis, and Preliminary Biological Evaluation
Medicinal Chemistry, 2014, Vol. 10, No. 1 85
CDCl3, ꢀ ppm): 7.93 (s, 1H, NH), 7.50 (m, 5H, Ar-H), 6.20
(s, H, CH-O), 4.50 (m, 2H, CH2), 2.51 (m, 9H, 3 ꢁ CH3),
2.26 (s, 3H, COCH3); 13C-NMR (150 MHz, CDC13, ꢀ ppm):
169.24, 168.38 (C=O), 40.61 (CH2), 67.09 (OCOCH3),
19.93, 21.04, 21.40 (CH3); ESI-MS: 328.6 (M+H)+
(t, 2H, Ar-H , J = 3.9 Hz), 7.51 (d, 2H, Ar-H, J = 8.4 Hz),
4.55 (d, 2H, CH2, J = 4.2 Hz), 2.48 (s, 3H, CH3), 2.45 (s, 3H,
CH3), 2.43 (s, 3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 172.24 (C=O), 40.02 (CH2), 67.09 (OCOCH3), 19.92,
21.33, 21.37 (CH3); ESI-MS: 286.5 (M+H)+; C16H19N3O2.
2-hydroxy-2-phenyl-N-((3,5,6-trimethylpyrazin-2-yl)meth-
yl)acetamide (8s)
Biological Evaluation for Protective Effects on HUVECs
Damaged by Hydrogen Peroxide [11, 16, 17]
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 48%; white crystal; mp: 107-108 °C; IR (KBr,
cm-1): 3256.31 (NH), 1521.74, 1443.77, 1412.98, (C=N,
C=C), 1653.10 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.63 (s, 1H, NH), 5.17 (s, 1H, OH), 7.35 (m, H, Ar-H), 7.40
(t, 2H, Ar-H, J = 3.9 Hz), 7.51 (d, 2H, Ar-H, J = 8.4 Hz),
4.55 (d, 2H, CH2, J = 4.2 Hz), 2.48 (s, 3H, CH3), 2.45 (s, 3H,
CH3), 2.43 (s, 3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ
ppm): 172.24 (C=O), 40.02 (CH2), 67.09 (OCOCH3), 19.92,
21.33, 21.37 (CH3); ESI-MS: 286.5 (M+H)+.
The human umbilical vascular endothelial cells were
plated and grown for 24 h in cultured medium, then were
switched to fresh medium in the presence of 0.025, 0.050,
0.100, 0.200 mM Ligustrazine and its derivatives. After 0.5 h
incubation, 150 ꢂM of hydrogen peroxide was added and
the cells were incubated for an additional 12 h. The results
were expressed as the values of absorbance at 570 nm.
The proliferation rates (P%) of damaged cells were calcu-
lated by [OD570(Compd)-OD570(H2O2)]/[OD570(Control) -OD570
ꢀꢁ100%, which was then used to obtain EC50 values
(H2O2)
(see Table 1), according to the equation: -pEC50 = log Cmax
-
N-((3,5,6-trimethylpyrazin-2-yl)methyl)cinnamamide (8t)
log2 (ꢀP -0.75 +0.25Pmax + 0.25Pmin), where Cmax, maximum
concentration; ꢀP, sum of proliferation rates; Pmax, maxi-
mum value of proliferation rate; and Pmin, minimum value
of proliferation rate.
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 56%; yellow crystal; mp: 142-144 °C; IR (KBr,
cm-1): 3308.13 (NH), 1618.19, 1540.95, 1445.92 (C=N,
C=C), 1652.30 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.32 (s, 1H, NH), 7.72 (d, 1H, C=CH, J = 15.6 Hz), 7.48 (m,
5H, Ar-H), 6.51 (d, 1H, C=CH, J = 15.4 Hz), 4.63 (d, 2H,
CH2, J = 4.1 Hz), 2.55 (s, 3H, CH3), 2.53 (s, 3H, CH3), 2.53
(s, 3H, CH3); 13C-NMR (150 MHz, CDC13, ꢀ ppm): 165.88
(C=O), 134.87, 129.7 (C=CH), 41.28 (CH2), 20.11, 21.44,
21.47 (CH3); ESI-MS: 282.5 (M+H)+.
RESULTS AND DISCUSSION
Synthesis of the Compounds 8a-8u
The important intermediate (3,5,6-trimethylpyrazin-2-
yl)methanol (3) was prepared by the Boekelheide reaction
[18] starting from Ligustrazine (1) and directly hydrolyzed
with sodium hydroxide water solution to obtain hydroxyl
Ligustrazine (HTMP, 4). This process was used one-pot re-
action according to our previous publication with 64% of the
total yield (see Scheme 1) [9]. The 2-chloromethyl-3,5,6-
trimethylpyrazine hydrochloride (5) was synthesized by the
chlorination of 4 with SOCl2 in anhydrous CH2Cl2 [11]. 2-
aminemethyl-3,5,6-trimethylpyrazine (7) was synthesized by
typical Gabriel reaction through hydraziniolysis of the key
2-hydroxy-2-phenyl-N-((3,5,6-trimethylpyrazin-2-yl)me-
thyl)acetamide (8u)
Flash column chromatography: ethyl acetate/cyclohexane
= 4:1; yield 48%; white crystal; mp: 107-108 °C; IR (KBr,
cm-1): 3256.31 (NH), 1521.74, 1443.77, 1412.98, (C=N,
C=C), 1653.10 (C=O); 1H-NMR (600 MHz, CDCl3, ꢀ ppm):
7.63 (s, 1H, NH), 5.17 (s, 1H, OH), 7.35 (m, H, Ar-H), 7.40
N
N
N
N
iii
i
ii
O
OH
N
O
N
N
N
O
2
1
3
4
O
N
N
N
N
N
N
HCl
iv
vi
v
N
Cl
NH2
O
6
5
7
N
vii
H
N
R
A) or B)
8a-u
N
O
Scheme 1. Reagents: (i) 30% H2O2, AcOH; (ii) Ac2O; (iii) 20% NaOH; (iv) SOCl2, anhydrous CH2Cl2; (v) phthalimide, DMF; (vi) 50%
hydrazine hydrate, ethanol; (vii) A) RCOCl, Py or B) HOBt, DCC, anhydrous THF.

86 Medicinal Chemistry, 2014, Vol. 10, No. 1
Li et al.
Table 1. The Structures of the Synthesized Compounds
Compound
Structure
Compound
8b
Structure
N
N
N
N
H
N
H
N
8a
O
O
N
N
N
H
N
H
N
8c
8e
8g
8i
8d
8f
8h
8j
8l
N
Cl
N
O
O
N
N
N
Cl
Br
H
H
N
N
N
O
OCOCH3
O
O
N
N
N
N
F
H
N
H
N
O
N
N
OCH3
OH
N
N
N
H
N
H
N
O
O
N
CH3
N
N
H
H
N
8k
N
N
O
O
OCH3
N
OH
N
H
H
8m
8o
8n
8p
N
N
N
OCH3
N
O
O
OCH3
OCOCH3
N
N
N
OCOCH3
H
N
H
N
N
OCOCH3
O
OH
O
O
N
N
OCOCH3
N
H
H
N
8q
8s
8r
8t
N
N
O
O
N
N
N
OH
OCOCH3
H
H
N
N
N
O
N
OH
H
N
8u
N
O

Ligustrazine Derivatives. Part 8: Design, Synthesis, and Preliminary Biological Evaluation
Medicinal Chemistry, 2014, Vol. 10, No. 1 87
intermediate of N-2-((3,5,6-trimethylpyrazin-2-yl)methyl)
phthalimide (6) [19]. Ligustrazinyl amides 8 were prepared
from 7 in the following two methods. In method (A), the
carboxylic acid was firstly transformed to acyl chloride, and
then reacted with compound 7 in the presence of pyridine
base. In method (B), the carboxylic acid and HOBt were
firstly transformed to active ester in the presence of DCC,
and then reacted with compound 7, obtaining the target com-
pounds 8. All the newly synthesized compounds (Table 1)
were confirmed by IR, 1H-NMR and MS spectrometries.
damaged by hydrogen peroxide. TMP was used as the con-
trol drug. The viability of normal and injured HUVECs was
assessed by MTT assay [20]. The maximum percentage of
stimulating proliferation (Pmax%) corresponding to the con-
centrations of the compounds, and the proliferation effective
concentration (EC50) were listed in (Table 2).
The results showed that some of the newly synthesized
compounds significantly increased the viability of the dam-
aged HUVECs (with low EC50 values) in comparison with
the positive stimulator of TMP (EC50 = 0.600 mM). Among
the active compounds, 8i containing isonicotinoyl group was
the most active one with the EC50 value at 0.037mM, which
is 16 times higher than the reference drug TMP. The excel-
lent potency of 8i might be caused by the isonicotinoyl
Biological Evaluation and Discussion
All newly synthesized Ligustrazinyl amide derivatives
were evaluated for protecting effects on the HUVECs that is
Table 2. The EC50 for Protecting Damaged HUVECs of the Novel Ligustrazine Derivatives
N
H
N
R
N
O
No.
RCO-
Method
P
max%/C(mM)a
EC50(mM)b
8a
8b
8c
Benzoyl
Phenylacetyl
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
36.30/0.025
11.46/0.1
0.194 0.011
0.335 0.034
0.094 0.012
0.108 0.014
8.914 0.912
0.370 0.024
23.873 1.452
16.217 1.875
0.037 0.004
0.109 0.017
2.520 0.321
2.229 0.213
2.618 0.324
2.170 0.223
4.004 0.342
1.592 0.146
0.273 0.012
4.265 0.567
3.767 0.456
0.070 0.008
0.055 0.004
0.600 0.073
3-Chlorobenzoyl
Nicotinoyl
97.45/0.05
82.17/0.2
8d
8e
Acetylsalicyloyl
4-Chlorocinnamoyl
4-Fluorocinnamoyl
4-Bromocinnamoyl
Isonicotinoyl
-84.08/0.1
12.74/0.025
-103.82/0.05
-113.38/0.05
98.73/0.1
8f
8g
8h
8i
8j
4-Methoxylcinnamoyl
4-Methylcinnamoyl
4-Hydroxylcinnamoyl
2,5-Dimethylcinnamoyl
Ferulolyl
84.71/0.05
31.85/0.1
8k
8l
29.30/0.05
-51.81/0.025
-38.55/0.2
-36.14/0.05
-1.2/0.025
53.50/0.1
8m
8n
8o
8p
8q
8r
Salicyloyl
3,4,5-Triacetylgalloyl
Cinnamoyl
4-Acetoxybenzoyl
4-Hydroxylbenzoyl
2-acetoxy-2-phenylacetoyl
Mandeloyl
-58.60/0.025
36.14/0.025
87.95/0.2
8s
8t
8u
106.02/0.2
TMP
10/0.05
aResults were expressed as maximum values of stimulating HUVCEs proliferation (Pmax%) at the corresponding concentration (C/mM). Pmax(%)=ODmax-ODa/ ODb-ODa.
bThe activity of compounds was expressed as EC50. The EC50 is defined as the effective concentration of compounds stimulating the damaged endothelial cells to proliferate by 50%.
Values represent means SD of three individual experiments.

88 Medicinal Chemistry, 2014, Vol. 10, No. 1
Li et al.
group, as the counterpart 8a bearing benzoyl group showed
less potency with the EC50 value at 0.194 mM. The analog
8d (EC50 = 0.108 mM) containing nicotinoyl group showed
less potent than 8i, which revealed the significance of the
position of the N atom.
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The authors confirm that this article content has no
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ACKNOWLEDGEMENTS
The financial supports of this work by National Special
Key Projects of New Drugs R&D (2009ZX09103-117), and
Shandong Natural Science Foundation, Shandong Science
and Technology Bureau Foundation (2006GG2202063) are
gratefully acknowledged.

Ligustrazine Derivatives. Part 8: Design, Synthesis, and Preliminary Biological Evaluation
Medicinal Chemistry, 2014, Vol. 10, No. 1 89
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Received: December 31, 2012
Revised: September 03, 2013
Accepted: September 03, 2013