Ligustrazine derivatives. Part 6: Design, synthesis and evaluation of novel ligustrazinyl acylguanidine derivatives as potential cardiovascular agents

Article abstract of DOI:10.2174/157340612802084243

A series of novel Ligustrazinyl acylguanidines was designed, synthesized and evaluated for their protective effect on injured vascular endothelial cell (ECV-304). The preliminary results demonstrated that some compounds possessed more potent activities than that of Ligustrazine in stimulating replication of the injured endothelial cell. Among the active compounds, compounds 8b, 8f and 8l displayed remarkable antioxidative activity with low EC50 values of 0.097, 0.059 and 0.094 mM, respectively. Structure-activity relationships were briefly discussed.

Full text of DOI:10.2174/157340612802084243

928
Medicinal Chemistry, 2012, 8, 928-933
Ligustrazine Derivatives. Part 6: Design, Synthesis and Evaluation of
Novel Ligustrazinyl Acylguanidine Derivatives as Potential Cardiovascular
Agents
Zhenyu Li,1 Fang Yu,1 Lei Cui,2 Peng Zhan,1 Shouxun Wang,2 Yuemao Shen1 and Xinyong Liu 1,*
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology, Ministry of Education, School of Pharma-
ceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, China
2Department 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 acylguanidines was designed, synthesized and evaluated for their protective ef-
fect on injured vascular endothelial cell (ECV-304). The preliminary results demonstrated that some compounds pos-
sessed more potent activities than that of Ligustrazine in stimulating replication of the injured endothelial cell. Among the
active compounds, compounds 8b, 8f and 8l displayed remarkable antioxidative activity with low EC50 values of 0.097,
0.059 and 0.094 mM, respectively. Structure-activity relationships were briefly discussed.
Keywords: Ligustrazine, Acylguanidines, Synthesis, Cardiovascular disease.
INTRODUCTION
to develop new generations of Ligustrazine-based cardio-
vascular drugs through molecular modification.
Cardiovascular diseases (CVDs) seriously threaten peo-
ples’ health with 21% proportion of the death worldwide.
Endothelial cells play an important role in maintaining the
function of normal vessel and organ [1]. Much evidence
proves that endothelial cell damage as an initial event causes
the alteration of endothelial permeability barrier and vascular
tone, eventually development of atherosclerosis and other
CVDs. It is also demonstrated that oxidative stress, as a ma-
jor cardiovascular risk factor, significantly contributes to
endothelial injury during atherogenesis. Therefore, the pro-
tection of endothelial cells against oxidative stress is a cru-
cial strategy in clinical CVDs therapy [1-3].
In our previous work, we have designed and synthesized
a series of novel Ligustrazine derivatives by incorporation of
Ligustrazine with pharmacophores or drug-like groups from
active cardiovascular agents [1-3, 7]. Through biological
activity studies, several Ligustrazine derivatives were ad-
vanced into promising drug candidates that are now under
preclinical evaluation.
Recently, acylguanidine derivatives have become of par-
ticular interest to medicinal chemists because of their broad
spectrum of cardiovascular activities and potential pharma-
cological applications. For examples, some acylguanidine
derivatives was reported to be active inhibitors of ADP-
induced platelet aggregation [8], potent FXa inhibitors as
anticoagulant agents [9], and Na+/H+ exchanger (NHE) in-
hibitor, e.g. amiloride, clinically for treatment of hyperten-
sion [10-11]. Inspired by structural characteristics of Ligus-
trazine and acylguanidine derivatives, we integrated Ligus-
trazine ring and acylguanidine fragment into the one mole-
cule according to the principles of hybridization and bioisos-
teric replacement in medicinal chemistry, constructing a se-
ries of novel Ligustrazinyl acylguanidine derivatives (Fig.
1). Herein, we reported the preparation of the novel Ligus-
Ligustrazine (Lig; 2,3,5,6-tetramethylpyrazine, TMP,
Fig. 1), a major active component of the Chinese traditional
medicine herb Chuanxiong (Ligusticum wallichii Franchat),
possesses variety of cardiovascular activities, acting as plate-
let aggregation inhibitor, vasodilator, free radical scavenger,
calcium channel antagonist, anti-thrombosis and anti-
hypertension agent, and endothelial injury protector, which
is now widely used in clinical for treatment of coronary athe-
rosclerotic cardiovascular disease and ischemic cardiovascu-
lar disease [4-5]. However, pharmacokinetics studies showed
that Ligustrazine was rapidly absorbed when taken orally,
but quickly excreted in the urine, displaying a low bioavail-
ability [2-3, 6]. As a result, accumulated toxicity often oc-
curred in the patients by the frequent administration for
keeping an plasma concentration. Therefore, it is necessary
H3C
N
CH3
H3C
H3C
N
N
CH3
CH3
H
H
N
N
H3C
N
R
O
NH
Ligustrazine
*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; Fax: +86 0531
88382731; E-mail: xinyongl@sdu.edu.cn
Ligustrazinyl acylguanidines
Fig. (1). Ligustrazine and Newly Designed Ligustrazinyl Acylgua-
nidine Derivatives.
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

Ligustrazine Derivatives
Medicinal Chemistry, 2012, Vol. 8, No. 5 929
N
N
N
N
iv
i
ii
iii
O
OH
N
O
N
N
N
O
1
3
4
2
NH
R
H2N
N
N
N
5
N
N
N
H
v
7
H
H
OH
N
N
N
N
N
R
vi
O
O
O
NH
6
8a-m
Scheme 1. Reagents: (i) 30% H2O2, AcOH; (ii) Ac2O; (iii) 20% NaOH; (iv) KMnO4/H2O, Na2SO3; (v) CDI/DMF, Room Temperature; (vi)
5h, Room Temperature.
SO3H
SO2H
NH
C
S
ii
i
NH
H2N
C
H2N
H2N
C
NH2
9
R1
NH
iii
R1
.H2CO3
9
H2N
N
NH2
H
NH
NH
NH
iv
v
R
CH3
.H2SO3
CH3NH2
H2N
H2N
H2N
N
NH
NH
H
9
7
NH2
iv
.H2SO3
R2
9
R2
Scheme 2. Reagents: (i) 30% H2O2; (ii) peracetic acid or H+/ H2O2; (iii) K2CO3, EtOH; (iv) EtOH; (v) NaH.
(6), generated in situ from compound 5. The substituents 7
were prepared by the reaction of fatty amine and arylamine
with thiourea trioxide (9), which was synthesized through
two steps oxidation of thiourea (Scheme 2). The synthesized
compounds 8a-m were characterized by physicochemical
and the MS, IR and NMR spectral data were found in
agreement with the assigned molecular structures (see Ex-
perimental part).
trazinyl derivatives, as well as their biological evaluation for
protective effects on injured ECV-304 cells.
RESULTS AND DISCUSSION
Synthesis of Compound 8a-m
The important intermediate (3,5,6-trimethylpyrazin-2-
yl)methyl acetate (3) was prepared by the Boekelheide reac-
tion starting from Ligustrazine (1) and directly hydrolyzed
with sodium hydroxide water solution to obtain hydroxyl
Ligustrazine (HTMP, 4) [1]. This process was used one-pot
reaction according to our previous publication with 64% of
the total yield (Scheme 1). The 3,5,6-trimethylpyrazine-2-
carboxylic acid (5) was synthesized by the oxidation of 4
with potassium permanganate in the water. The target com-
pounds 2-acylguanidino-3,5,6-trimethylpyrazine (8) were
readily prepared in moderate yields by the reaction of vari-
ous of substituted guanidines (7) with the key intermediate
(1H-imidazol-1-yl)(3,5,6-trimethylpyrazin-2-yl)methanone
Biological Evaluation
Setting Ligustrazine as the positive control drug, all the
synthesized compounds were tested for their protective ef-
fects on the human umbilical vascular endothelial cells
(ECV-304 cells) damaged by hydrogen peroxide [2-3]. The
viability of normal and injured ECV-304 cells was assessed
by MTT assay. The proliferation rate (P%) and 50% effec-
tive concentration for protecting damaged ECV-304 cells of
newly synthesized compounds were shown in Table 1.

930 Medicinal Chemistry, 2012, Vol. 8, No. 5
Li et al.
Table 1. The Proliferation Rate (%) and EC50 Values for Protecting Damaged ECV-304 Cells of the Ligustrazinyl Acylguanidine
Derivatives
N
H
H
N
N
N
R
O
NH
Proliferation Rate (%)
No.
R
EC50(mM) a
50μM
100μM
200μM
400μM
8a
8b
8c
H
Methyl
1.93
70.57
2.47
10.54
47.17
6.39
14.24
15.01
11.13
58.25
31.54
100.84
-25.98
28.18
-8.87
32.34
4.29
0.769
0.097
0.866
0.147
>100
0.059
-
4-Methylphenyl
4-Methoxylphenyl
4-Fluorobenzyl
3-Chlorobenzyl
4-Methoxybenzyl
2-Fluorobenzyl
2-Chlorobenzyl
Benzyl
26.60
102.92
32.73
155.95
-66.19
29.44
8.66
8d
8e
0.84
36.12
31.34
76.62
-65.98
27.97
-12.99
28.14
25.34
57.70
-61.51
14.64
29.14
40.29
-19.79
25.68
-45.15
25.95
1.75
8f
8g
8h
8i
>100
>100
0.564
0.391
0.094
-
8j
31.54
30.99
93.76
-44.99
26.23
42.91
49.90
-5.85
8k
8l
2,4-Dichlorobenzyl
3,4-Dichlorobenzyl
3,5,6-Trimethylpyrazine-2-yl
-
4.09
8m
TMP
-103.34
11.03
-24.96
43.08
0.549
aConcentration of compound required to achieve 50% protection of ECV-304 cell from H2O2 induced cytotoxicity, as determined by the MTT method.
In general, from the obtained data, it was observed that
some of the Ligustrazinyl acylguanidine derivatives pro-
tected the proliferation of injured ECV-304 cells with effects
comparable to or better than TMP (EC50= 0.549 mM).
Among all the Ligustrazinyl acylguanidine derivatives, 8b,
8f and 8l displayed remarkable antioxidative activity (EC50=
0.097, 0.059 and 0.094 mM, respectively). Particularly, the
compound 8f presented almost 10 times higher potency than
TMP, and exhibited the greatest proliferation rate (P% =
155.95%) at 0.040 mM. Some other compounds 8d and 8k
also exhibited reasonable antioxidant activities, similar to
that of TMP.
position of the chlorine can largely affect the activities of the
compounds. The active sequence of the substituents at the
benzyl moiety was as follows: 3-Cl>3,4-2Cl>3,5-2Cl>>2-Cl.
Interestingly, between the compounds 8b and 8g, the ac-
tivity order was 8b >> 8g, which might reflect the impor-
tance of carbon numbers between the phenyl group and gua-
nidino group. No carbon between the phenyl group and gua-
nidino group seems more favorable than one carbon. In addi-
tion, it also can be seen that the proliferation rates presented
in the Table 1 were increased with the increasing of the con-
centration of the compounds. However, this rule is not suit-
able for compound 8b.
The structure-activity relationships (SARs) analysis
showed that the nature of the substituents greatly influenced
the antioxidative activity of these compounds. When sub-
stituents were fatty group (8a and 8b), methyl group was
more active than hydrogen. In phenyl case, the activity order
was 8d > 8c, illustrating that methoxy group was more
needed for keeping high protective effect than methyl group.
CONCLUSION
In conclusion, a series of novel Ligustrazinyl acylgua-
nidine derivatives were designed, synthesized and biologi-
cally evaluated for their protective effect on injured vascular
endothelial cell (ECV-304). The results showed that some of
the compounds displayed high activities, demonstrating that
the Ligustrazinyl acylguanidines are potent cardiovascular
agents. Among all the active compounds, compound 8f dis-
played an excellent antioxidant activity with low EC50 value
of 0.059 mM, which about 10 times higher than that of
Ligustrazine, presenting a most promising lead for further
investigation.
In the benzyl series, the active sequence of derivatives
was 8f > 8l > 8k > 8j >>8e, 8h, 8i, 8g, which reflect the sig-
nificance of substituents at the benzyl moiety. Chloro-
substitution at the phenyl group seems more active than
other substituents. Furthermore, when compared chloro-
substituted compounds 8f, 8i, 8k and 8l, we can see that the

Ligustrazine Derivatives
Medicinal Chemistry, 2012, Vol. 8, No. 5 931
EXPERIMENTAL SECTION
Chemistry
To a mixture of 0.26 mL concentrated sulfuric acid and
6.5 mL 30% hydrogen peroxide, 5.4g thiourea dioxide was
added one spoon by one spoon at 55 ºC for 80 min. After
finishing adding, the mixture was stirred for 15 min to
generate a large number of colorless granular crystals. Then
placed the mixture in the refrigerator overnight, and filtered
to yield colorless crystalline thiourea trioxide monohydrate
4.5g. Yield: 83.3%.
The melting points of the compounds were metered on a
micromelting point apparatus. Infrared spectra were recorded
with a Nexus 470 FT-IR Spectrometer. 1HNMR spectra were
determined by a Bruker Avance (600 MHz) NMR-
spectrometer in the indicated solvents. Chemical shifts are
expressed in d units and TMS as internal reference. Mass
spectra were recorded with an LC Autosampler Device:
Standard G1313A instrument. TLC was performed on silica
gel GF254 for TLC (Merck) and spots were visualized by
iodine vapors or by irradiation with UV light (254 nm).
Flash column chromatography was performed on column
packed with silica gel 60 (230–400 mesh). Solvents were
reagent grade and, when necessary, were purified and dried
by standard methods. Concentration of the reaction solutions
involved the use of rotary evaporator at reduced pressure.
The yields were calculated by the last step reaction.
General Procedure for the Preparation of Substituted
Phenylguanidine
To a solution of substituted phenylamine 0.005 mol and
anhydrous potassium carbonate (0.76 g, 0.0055 mol) in 8 mL
ethanol-H2O (ethanol : H2O = 1:1) at 35-40 ºC water bath,
thiourea trioxide (0.67 gꢀ0.0055 mol) was added one spoon
by on spoon. After finishing adding, the mixture was stirred
for 4h at at 35 ºC. Then placed the mixture for 24h at room
temperature and the product was filtered off, washing with
acetone. The crude product was recrystallized from water to
get white solid. Yield: 45-48%.
Preparation of (3,5,6-Trimethylpyrazin-2-yl)Methanol
(4)
General Procedure for the Preparation of Substituted
Benzylguanidine and Adipic Guanidine
Compound 4 was prepared according to our previous
reported method by using one-port reaction [1]. The crude
product was purified by recrystallization with n-hexane
to obtain yellow needles. Yield: 60%. M.p. 88-89 ºC (lit. 88–
89 ºC) [1].
A solution of substituted benzylamine (methylguanidine
was prepared by 25% methylamine) 0.01 mol in 15 mL
ethanol at 35-40 ºC water bath, thiourea trioxide (1.33g,
0.011mol) was added to the solution. After finishing adding,
the mixture was stirred for 4h at at 35 ºC. Then placed the
mixture in the refrigerator overnight and the product was
filtered off, washing with ethanol. The crude product was
recrystallized from water to get white solid. Yield: 16-50%.
Preparation of 3,5,6-trimethylpyrazine-2-carboxylic Acid
(5)
To a solution of (3,5,6-Trimethylpyrazin-2-yl)methanol
(4) (10.268 g, 0.068 mol) in 60 ml water, potassium perman-
ganate (KMnO4) aqueous solution (14.4 g KMnO4 : 240 mL
water) was added dropwise at room temperature for about 40
min. After finishing dropping, the mixture was stirred at
room temperature for 30 min. Then saturated aqueous
solution of sodium bisulfite was added to neutralize the re-
manent KMnO4, filtration, and filter was washed with 400
mL water (90 ºC). The filtrate and washing liquor were
merged, cooling to 0-5 ºC, and regulated pH to 2.0 with
concentrated hydrochloric acid. Extraction was performed
with ethyl acetate (100 mL ꢀ 5), and the organic phase was
dried with anhydrous sodium sulfate. The solvent was re-
moved by distillation under vacuum, and the residue was
recrystallized with methyl ethyl ketone to yield light yellow
solid 5.8g. Yield: 52.0%. M.p. 161-164 ºC.
General Procedure for the Preparation of Compounds
8a-8m
To a mixture of NaH (0.2 g, 60%, 0.005 mol) and various
substituted guanidine 0.005 mol in 10 ml dry DMF-dioxane
(DMF-dioxane = 1:1), heated to 50 - 60 ºC for 20 min under
the protection of nitrogen. Then sodium salt was filtered off
to obtain the substituted guanidine. 3,5,6-Trimethylpyrazine-
2-carboxylic acid (5) (0.83 g, 0.005 mol) and carbonyl-
diimidazole (CDI, 0.89 g, 0.0055 mol) was added to 10 ml
dry DMF-dioxane (DMF-dioxane = 1:1), the mixture stirred
for 2 h. Two kinds of reaction solution were mixed and
stirred at room temperature for 5h. The solvent was removed
by distillation under vacuum to get the crude product. The
final product was purified by flash column chromatography
[dichloromethane/methanol = 3:1 (8a); 5:1 (8b); 20:1 (8c-
8m)] and recrystallized from methanol (8a) or water (8b-
8m).
Preparation of Amidine Agent Thiourea Trioxide (9)
A solution of thiourea 10g in 100 mL water, cooling to
about 8 ºC in ice-water bath, 30% hydrogen peroxide was
added dropwise to control the reaction temperature of about
15 °C. When the pH value descended to 3-5, another 10 g of
thiourea was added and 30% hydrogen peroxide was added
dropwise (66g was added in all). After finishing dropping,
the mixture was stirred at room temperature for 3 h. Abun-
dant precipitation seperated out (pH = 2-3), cooling to about
5 ºC, filtration, and washed with ethanol to get thiourea
dioxide. The product was dried at 50 ºC under vacuum.
Yield: 57.3%.
N-carbamimidoyl-3,5,6-trimethylpyrazine-2-carboxamide
(8a)
Light yellow crystal. Yield: 38.9%. M.p. 220-223°C. IR
(KBr, cm-1): 3350.61 (NH), 1588.00, 1450.21, 1410.35
(C=N, C=C), 1671.04 (C=O); 1H-NMR (DMSO-d6, ppm) ꢁ:
9.31 (s, 1H, NH), 8.55 (br, 2H, NH2), 2.62 (s, 3H, CH3),
2.57 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS: m/z 208.3
[M+H]. C9H13N5O (207.23).

932 Medicinal Chemistry, 2012, Vol. 8, No. 5
Li et al.
3,5,6-Trimethyl-N-(N-methylcarbamimidoyl)pyrazine-2-
carboxamide (8b)
(s, 2H, CH2), 2.37-2.49 (m, 9H, 3ꢁCH3); ESI-MS: m/z 316.3
[M+H]. C16H18FN5O (315.35).
N-(N-(2-chlorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8i)
White solid. Yield: 14.5%. M.p. 86-88°C. IR (KBr,
cm-1): 3356.64 (NH), 1574.28, 1431.23, 1405,76
(C=N,C=C), 1672.55 (C=O); H-NMR (DMSO-d6, ppm) ꢀ:
9.32 (s, 1H, NH), 8.43 (s, 1H, NH), 3.63 (s, 3H, NCH3), 2.59
(s, 3H, CH3), 2.51 (s, 3H, CH3), 2.48 (s, 3H, CH3); ESI-MS:
m/z 222.3 [M+H]. C10H15N5O (221.26).
1
White solid. Yield: 28.9%. M.p. 97-100°C. IR (KBr,
cm-1): 3367.70, 3245.99, 2923.85 (NH), 1600.34, 1514.26,
1474.32, 1425.18 (C=N,C=C), 1628.03 (C=O); H-NMR
(DMSO-d6, ppm) ꢀ: 9.27 (s, 1H, NH), 7.39 (s, 1H, NH),
7.02-7.35 (m, 4H, Ar-H), 4.45 (s, 2H, CH2), 2.42-2.51 (m,
9H, 3ꢁCH3); ESI-MS: m/z 332.4 [M+H]. C16H18ClN5O
(331.80).
1
3,5,6-Trimethyl-N-(N-p-tolylcarbamimidoyl)pyrazine-2-
carboxamide (8c)
Yellow solid. Yield: 24.0%. M.p. 168-171°C. IR (KBr,
cm-1): 3367.38 (NH), 1656.42, 1577.45, 1478.53 (C=N,
C=C), 1686.37 (C=O); 1H-NMR (DMSO-d6, ppm) ꢀ: 9.35 (s,
1H, NH), 7.40 ( s, 1H, NH), 7.30-7.37 (m, 4H, Ar-H), 4.34
(s, 2H, CH2), 2.52 (s, 3H, Ph-CH3), 2.37-2.50 (m, 9H,
3ꢁCH3); ESI-MS: m/z 298.5 [M+H]. C16H19N5O (297.35).
N-(N-benzylcarbamimidoyl)-3,5,6-trimethylpyrazine-2-
carboxamide (8j)
White solid. Yield: 27.7%. M.p. 146-149°C. IR (KBr,
cm-1): 3452.31, 3394.06, 3087.78 (NH), 1590.42, 1422.05
(C=N, C=C), 1669.39 (C=O); 1H-NMR (DMSO-d6, ppm) ꢀ:
9.36 (s, 1H, NH), 7.30-7.37 (m, 6H, Ar-H and NH), 4.42 (s,
2H, CH2), 2.39-2.51 (m, 9H, 3ꢁCH3); ESI-MS: m/z 298.3
[M+H]. C16H19N5O (297.35).
N-(N-(4-methoxyphenyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8d)
White solid. Yield: 29.3%. M.p. 160-164°C. IR (KBr,
cm-1): 3350.65 (NH), 1592.31, 1514.23, 1427.14 (C=N,
C=C), 1632.49 (C=O); 1H-NMR (DMSO-d6, ppm) ꢀ: 9.29 (s,
1H, NH), 7.38 ( s, 1H, NH), 7.28-7.38 (m, 4H, Ar-H), 4.30
(s, 1H, NH), 2.92 (s, 3H, O-CH3), 2.32-2.49 (m, 9H,
3ꢁCH3); ESI-MS: m/z 314.3 [M+H]. C16H19N5O2 (313.35).
N-(N-(2,4-dichlorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8k)
White solid. Yield: 64.3%. M.p. 117-120°C. IR (KBr,
cm-1): 3360.46 (NH), 1589.60, 1434.81 (C=N, C=C),
1
1621.47 (C=O); H-NMR (DMSO-d6, ppm) ꢀ: 9.30 (s, 1H,
NH), 7.39 (s ,1H, NH), 6.99-7.38 (m, 3H, Ar-H), 4.42 (s,
2H, CH2), 2.38-2.45 (m, 9H, 3ꢁCH3); ESI-MS: m/z 366.2
[M], 367.1 [M+H]. C16H17Cl2N5O (366.25).
N-(N-(4-fluorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8e)
Lightyellow solid. Yield: 32.1%. M.p. 93-94°C. IR (KBr,
cm-1): 3331.56 (NH), 1516.13, 1423.95, 1338.51
N-(N-(3,4-dichlorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8l)
1
(C=N,C=C), 1596.98 (C=O); H-NMR (DMSO-d6, ppm) ꢀ:
9.34 (s, 1H, NH), 7.14-7.39 (m, 5H, Ar-H and NH), 4.28 (s,
2H, CH2), 2.38-2.44 (m, 9H, 3ꢁCH3), 2.14 (s, 1H, NH); ESI-
MS: m/z 314.3 [M-H]. C16H18FN5O (315.35).
White solid. Yield: 59.5%. M.p. 98-101°C. IR (KBr,
cm-1): 3322.90 (NH), 1589.88, 1467.43, 1425.01
(C=N,C=C), 1635.78 (C=O); H-NMR (DMSO-d6, ppm) ꢀ:
1
9.23 (s, 1H, NH), 7.49 (s, 1H, NH), 7.18-7.40 (m, 3H, Ar-
H), 4.41 (s, 2H, CH2), 2.39-2.50 (m, 9H, 3ꢁCH3); ESI-MS:
m/z 366.2 [M], 367.4 [M+H]. C16H17Cl2N5O (366.25).
N-(N-(3-chlorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8f)
White solid. Yield: 26.3%. M.p. 95-100°C. IR (KBr,
cm-1): 6293.04 (NH), 1597.71, 1514.69, 1422.68
N,N'-(iminomethylene)bis(3,5,6-trimethylpyrazine-2-
carboxamide) (8m)
1
(C=N,C=C), 1743.37 (C=O); H-NMR (DMSO-d6, ppm) ꢀ:
9.35 (s, 1H, NH), 7.43 ( s, 1H, NH), 6.92-7.35 (m, 4H, Ar-
H), 4.43 (s, 2H, CH2), 2.42-2.52 (m, 9H, 3ꢁCH3); ESI-MS:
m/z 332.1 [M+H]. C16H18ClN5O (331.80).
White solid. Yield: 38.1%. M.p. 233-234°C. IR (KBr,
cm-1): 3393.97 (NH), 1598.32, 1577.66, 1439.46, 1409.51
(C=N,C=C), 1701.75 (C=O); H-NMR (DMSO-d6, ppm) ꢀ:
9.36 (s, 1H, NH), 9.30 (s ,1H, NH), 2.34-2.55 (m, 18H,
6ꢁCH3); ESI-MS: m/z 357.2 [M+H]. C17H21N7O2 (355.39).
1
N-(N-(4-methoxybenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8g)
Protective Effect on Damaged ECV-304 Cells Assay [2,
12]
White solid. Yield: 33.3%. M.p. 84-86°C. IR (KBr,
cm-1): 3319.22 (NH), 1513.55, 1422.22, 1399.13
(C=N,C=C), 1590.66 (C=O); 1H-NMR (DMSO-d6, ppm) ꢀ:
9.25 (s, 1H, NH), 6.88-7.29 (m, 5H, Ar-H and NH), 4.33 (s,
2H, CH2), 2.93 (s, 3H, O-CH3), 2.39-2.50 (m, 9H, 3ꢁCH3);
ESI-MS: m/z 328.5 [M+H]. C17H21N5O2 (327.38).
ECV-304 cells were seeded in a 24-well plate at a density
of 6ꢁ103 /well and allowed to grow to the desired conflu-
ence. The cells were pretreated with various concentrations
of Ligustrazinyl derivatives for 24 h, and then exposed to
150 μM H2O2 for another 12 h. Control cells were incubated
with a media containing an equivalent solvent amount with-
out the test materials. The plate was incubated at 37 °C in a
humidified 5% CO2 atmosphere. 12 hours later, 0.01 mL
MTT solution (5 mg/mL) was added to each well and then
incubated for 4 h. Ligustrazine derivatives were dissolved in
dimethyl sulfoxide (DMSO) and added into the wells (the
N-(N-(2-fluorobenzyl)carbamimidoyl)-3,5,6-
trimethylpyrazine-2-carboxamide (8h)
White solid. Yield: 37.5%. M.p. 141-142°C. IR (KBr,
cm-1): 3372.21 (NH), 1601.72, 1490.80, 1424.43 (C=N,
C=C), 1629.53 (C=O); 1H-NMR (DMSO-d6, ppm) ꢀ: 9.36 (s,
1H, NH), 7.48 (s, 1H, NH), 6.98-7.30 (m, 4H, Ar-H), 4.32

Ligustrazine Derivatives
Medicinal Chemistry, 2012, Vol. 8, No. 5 933
injured vascular endothelial cell damaged by hydrogen peroxide.
Bioorg. Med. Chem. Lett., 2003, 13(13), 2123-6.
Cheng, X.C.; Liu, X.Y.; Xu, W.F.; Guo, X.L.; Ou, Y. Design,
synthesis, and biological activities of novel Ligustrazine deriva-
tives. Bioorg. Med. Chem., 2007, 15(10), 3315-20.
Cheng, X.C.; Liu, X.Y.; Xu, W.F.; Guo, X.L.; Zhang, N.; Song,
Y.N. Ligustrazine derivatives. Part 3: Design, synthesis and evalua-
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diac vascular agents. Bioorg. Med. Chem., 2009, 17(8), 3018-24.
Ren, M.; Dong, J.; Xu, Y.; Wen, N.; Gong, G. Synthesis of novel
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1065.
final concentration of Ligustrazinyl derivatives was to 400,
200, 100, 50 μM, and the DMSO content should never ex-
ceed 0.05%) and were incubated with cells for 24 h before
the addition of H2O2. The supernatant was removed carefully
by pipetting from wells without disturbing the attached cells
and formazan crystals were solubilized by adding 200 μL of
DMSO to each well and shaken for 15 min. The absorbance
at 570 nm was measured with a microplate reader, using
wells without cells as control. The proliferation rates of
damaged ECV-304 cells were calculated by [OD570 (Com-
[2]
[3]
[4]
[5]
pound)-OD570
(H2O2)]/[OD570
(Control)-
OD570
(H2O2)]ꢀ100%, which was then used to obtain EC50 values,
according to the equation: -pEC50 = logCmax- 2ꢀ(ꢁP- 0.75 +
0.25Pmax + 0.25Pmin), where Cmax = maximum concentration,
ꢁP = sum of proliferation rates, Pmax = maximum value of
proliferation rate and Pmin = minimum value of proliferation
rate.
[6]
Ju, X.D.; Deng, M.; Ao, Y.F.; Yu, C.L.; Wang, J.Q.; Yu, J.K.; Cui,
G.Q.; Hu, Y.L. The protective effect of tetramethylpyrazine on car-
tilage explants and chondrocytes. J. Ethnopharmacol, 2010,
132(2), 414-20.
[7]
[8]
[9]
Liu, X.Y.; Cheng, X.C.; Xu, W.F.; Guo, X.L.; Ou, Y. Design,
synthesis, and biological activities of novel Ligustrazine deriva-
tives. Bioorg. Med. Chem., 2007, 15(10), 3315-3320.
Thomas, E.W.; Nishizawa, E.E.; Zimmermann, D.C.; Williams,
D.J. Synthesis of acylguanidine analogues: inhibitors of ADP-
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O'Connor, S.P.; Atwal, K.; Li, C.; Liu, E.C.; Seiler, S.M.; Shi, M.;
Shi, Y.; Stein, P.D.; Wang, Y. Synthesis and evaluation of acyl-
guanidine FXa inhibitors. Bioorg Med Chem Lett, 2008, 18(16),
4696-9.
CONFLICT OF INTEREST
The author(s) declare that they have no competing inter-
ests.
ACKNOWLEDGMENTS
The financial supports of this work by National Special
Key Projects of New Drugs R&D (2009ZX09103-117),
Shandong Natural Science Foundation, Shandong Science
and Technology Bureau Foundation (2006GG2202063) are
gratefully acknowledged.
[10]
[11]
Zerbini, G.; Maestroni, A.; Mangili, R.; Pozza, G. Amiloride-
insensitive Na+-H+ exchange: a candidate mediator of erythrocyte
Na+-Li+ countertransport. J. Am. Soc. Nephrol., 1998, 9(12), 2203-
11.
Pedersen, S.F.; King, S.A.; Nygaard, E.B.; Rigor, R.R.; Cala, P.M.
NHE1 inhibition by amiloride- and benzoylguanidine-type com-
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Received: August 25, 2011
Revised: April 12, 2012
Accepted: April 14, 2012