Synthesis of novel ligustrazine derivatives as Na+/H+ exchange inhibitors

Article abstract of DOI:10.1002/cbdv.200900353

A novel series of 3,5,6-trimethylpyrazine-2-methoxy (or methylamino) substituted benzoyl-guanidine derivatives were designed and synthesized as Na⁺/H⁺ exchange (NHE) inhibitors. In this study, compounds with electron-withdrawing substituents on the benzene ring seemed to improve NHE-1 inhibitory activities. Compounds 6d, 6k, and 6l were found to be potent inhibitors of NHE-1 (IC₅₀=3.0±1.6, 3.0±1.4, and 1.6±0.4nmol/l, resp.). Furthermore, they showed a remarkable reduction of infarct size in the rat myocardial infarction model in vivo.

Full text of DOI:10.1002/cbdv.200900353

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Synthesis of Novel Ligustrazine Derivatives as Naþ/Hþ Exchange Inhibitors
by Mei Rena), Jin Donga), Yungen Xu*a), Nan Wenb), Guoqing Gong*b)
a) Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, P. R. China
(phone: þ86-25-83322067; fax: þ86-25-83271487; e-mail: xyg64@126.com)
b) Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, P. R. China
(phone: þ86-25-83604316; fax: þ86-25-83301606; e-mail: gonggq@hotmail.com)
A novel series of 3,5,6-trimethylpyrazine-2-methoxy (or methylamino) substituted benzoyl-
guanidine derivatives were designed and synthesized as Naþ/Hþ exchange (NHE) inhibitors. In this
study, compounds with electron-withdrawing substituents on the benzene ring seemed to improve NHE-1
inhibitory activities. Compounds 6d, 6k, and 6l were found to be potent inhibitors of NHE-1 (IC50 ¼3.0ꢀ
1.6, 3.0ꢀ1.4, and 1.6ꢀ0.4 nmol/l, resp.). Furthermore, they showed a remarkable reduction of infarct
size in the rat myocardial infarction model in vivo.
Introduction. – The Naþ/Hþ exchangers (NHEs) comprise a family of membrane
proteins [1]. To date, nine NHE isoforms (NHE-1–NHE-9) have been identified in
various organs in the human body [2]. NHE-1 is ubiquitously distributed in tissue, and
it is the predominant NHE isoform in the myocardial cell [3][4]. It is rapidly activated
during ischemia by intracellular Hþ accumulation that is exchanged for Naþ. The high
intracellular Naþ level leads to an increase in intracellular Ca2þ via Naþ/Ca2þ exchange
[3–5]. At the cardiac level, this cellular Ca2þ overload is involved in ischemic and
reperfusion injuries such as myocardial stunning and infarction, and tissue necrosis
[5][6].
Although the results of clinical studies of the NHE-1 inhibitors cariporide (Fig.)
and eniporide are quite disappointing, some of these studies indicated that these
NHE-1 inhibitors can indeed provide the protection of the human myocardium from
injury during ischemia and reperfusion in the appropriate setting [7]. These somewhat
contradictory results may be due to the design of their trials or their modest potency
[7][8].
While the improvement of the myocardial protective effects by replacing the
acylguanidine moiety with its bioisostere such as aminoguanidine and 2-aminoimida-
zole was quite limited [9–11], the studies that focused on searching for diverse
templates bearing an acylguanidine moiety have provided promising results [12–15]1).
Because the use of natural product templates is known as a viable source of new drug
candidates [17], in our efforts to discover more potent and safe NHE-1 inhibitors, we
designed our target compounds through combining a natural product possessing good
cardioprotective effects with a benzoylguanidine moiety according to a hybrid ap-
proach.
1
)
A preliminary account of this work has been published: [16].
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Ligustrazine (2,3,5,6-tetramethylpyrazine (TMP); Fig.), a major active component
of the Chinese traditional medicine Chuanxiong (Ligusticum wallichii Franch.), was
selected as the ꢂnatural productꢃ part of the desired molecules. It has been introduced
on the market for the treatment of coronary atherosclerotic disease and ischemic
cerebrovascular disease in China since the 1970s [18]. It also shows good protective
effect on myocardial injury during ischemia and reperfusion [19]. Here, the synthesis
and pharmacological properties of ligustrazine derivatives as NHE-1 inhibitors are
described.
Figure. Structures of ligustrazine and cariporide
Results and Discussion. – Synthesis. As depicted in the Scheme, compound 3 was
synthesized from 2,3,5,6-tetramethylpyrazine hydrochloride (1), which was deproto-
nated with aqueous NaOH to give 2, followed by bromination with N-bromosuccini-
mide (NBS) in CCl4, leading to 3. The reaction temperature is very important in the
bromination reaction. To reduce the yield of polybrominated by-products, the solution
of 2 was stirred at 658, and the product could be used in the next reaction step without
further purification. The intermediates 5a–5n, obtained by the reaction of 3 and ethyl
benzoate or methyl benzoate derivatives 4a–4n, were treated with guanidine in
i
anhydrous PrOH for 0.5–1 h, followed by formation of the hydrochloride salt with
Scheme
a) 1. NaOH, H2O; 2. benzene. b) NBS, CCl4, hn, reflux. c) Na2CO3, acetone or DMF, reflux. d) 1.
i
Guanidine, PrOH, reflux; 2. sat. HCl (g).

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2729
i
HCl gas, to afford our target compounds. In this reaction, using anhydrous PrOH
rather than anhydrous THF as solvent gave better yields.
Biological Activities. The NHE-1 inhibitory activities of 14 target compounds and
cariporide were evaluated by rat platelet swelling assay (PSA). The experimental
procedure was similar to that described in [20][21], with minor modifications. The PSA
results showed that all tested compounds inhibited rat platelet NHE-1 in a
concentration-dependent manner. Compounds 6b–6g, 6k, 6l, and 6n (see Table 1)
are superior to cariporide in NHE inhibition. The IC50 values of compounds 6d, 6k, and
6l were 3.0ꢀ1.6, 3.0ꢀ1.4, and 1.6ꢀ0.4 nm, respectively, rendering them 20, 20, and 40
times more potent than cariporide (65.0ꢀ8.6 nm). However, ligustrazine showed no
effect on NHE-1 under these experimental conditions, suggesting that its protective
effect on myocardial injury during ischemia and reperfusion was not regulated via the
NHE-1 system (Table 2).
Table 1. Chemical Structures and NHE-1 Inhibitory Activities of Compounds 6a–6n
Compound
R
X
Position of
IC50a) [nm]
Cariporide
65.0ꢀ8.6
204ꢀ20
32.8ꢀ6.7
14.7ꢀ3.8
3.0ꢀ1.6
5.7ꢀ2.0
6.6ꢀ2.3
11.7ꢀ2.5
78.0ꢀ5.3
86.9ꢀ7.8
95.7ꢀ5.7
3.0ꢀ1.4
1.6ꢀ0.4
134ꢀ9
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
H
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
NH
4
4
4
4
4
2
2
4
4
4
4
4
2
3
3-MeO
3-Cl
3-Br
3-NO2
H
4-CF3
H
3-Me
3-Cl
3-Br
3-NO2
H
4-Me
54.7ꢀ4.9
a) Drug concentration to achieve half-maximal inhibition of acid-induced swelling in rat platelets.
Starting from 6a, the structure–activity relationship (SAR) was described as
follows. Replacing a benzene H-atom in 6a with a MeO group (i.e., compound 6b)
resulted in a six-times improvement in potency. The 3-Cl, 3-Br, and 3-NO2 analogs (i.e.,

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 2. Cardioprotective Activity of Compounds 6c, 6d, 6e, 6f, 6g, 6k, and 6l against Ischemic-
Reperfusion. Cariporide and the tested compounds were injected intravenously 5 min before LAD (left
anterior descending) occlusion (0.01 mmol/kg).
Dosage [mmol/kg]
CKa) [U/ml]
Infarct sizeb) [%]
Ischemia/reperfusion
Cariporide
–
67.3ꢀ4.8
66.7ꢀ4.3
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
35.6ꢀ3.5**c)
47.5ꢀ6.6**
31.2ꢀ2.9**
42.3ꢀ4.8**
36.5ꢀ4.3**
45.7ꢀ5.2**
32.3ꢀ3.6**
34.4ꢀ5.4**
42.7ꢀ3.2**
52.7ꢀ4.6*
33.3ꢀ2.7**
43.3ꢀ5.2**
39.6ꢀ2.5**
49.8ꢀ3.4**
36.6ꢀ4.1**
38.4ꢀ2.2**
6c
6d
6e
6f
6g
6k
6l
a) Serum CK activity was expressed as U/ml. Values are meansꢀSD, n¼6 or higher. b) Infarct size was
expressed as the ratio of myocardial infarct weight to weight of ventricle at risk. Values are meansꢀSD,
n¼6 or higher. c) *: p<0.05, **: p<0.01 compared with ischemia-reperfusion group.
6c–6e, resp.) showed more significant improvement in potency relative to the starting
compound 6a, which suggested that introducing electron-withdrawing substituents to
benzene ring improve the NHE-1 inhibitory potency. A similar SAR was observed for
the aminobenzene analogs (6j–6l). In addition, although inhibition by compound 6h
was dramatically lower than that of 6a, compound 6d and 6k showed similar potencies,
suggesting that the influence of the atom X (O/N) at the benzene ring on NHE-1
inhibitory activity was limited.
The compounds showing good NHE-1 inhibitory activity were further investigated
for their cardioprotective effects against ischemia-reperfusion injury in Sprague-
Dawley (SD) rat hearts [22]. The infarct size and the creatine kinase (CK) level of
cariporide were 42.7ꢀ3.2 and 35.6ꢀ3.5 U/ml, respectively. Most compounds with a
good NHE-1 inhibitory activity exhibited a good cardioprotective efficacy both in vitro
and in vivo. The infarct size and the CK level of compound 6d, 6k, and 6l were better
than those of cariporide at the same dose. Especially, compound 6d significantly
reduced the infarct size to 33.3ꢀ2.7%.
Conclusions. – A series of novel derivatives of ligustrazine linked with substituted
benzoylguanidine were synthesized and evaluated for their NHE-1 inhibitory activity
and cardioprotective efficacy against ischemia-reperfusion injury. Compound 6d, 6k,
and 6l showed remarkably improved NHE-1 inhibitory activity and reduced the infarct
size and the CK level in the rat model of ischemic heart in vivo. Further
pharmacological studies are in progress.
This project was supported by the National Natural Science Foundation of China (No. 30672512).
The authors are grateful to the Center for Instrumental Analysis at the China Pharmaceutical University
for their contribution to the structural confirmation.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Experimental Part
General. M.p.: RDCSY-I apparatus; uncorrected. IR Spectra: BRUKER Tensor 27 IR spectropho-
tometer, in KBr; in cmꢁ1. 1H- and 13C-NMR Spectra: BRUKER AM-300 spectrometer; d in ppm rel. to
Me4Si, J in Hz. ESI-MS: HP1100 mass spectrometer; in m/z (rel. %). Elemental analyses: Elementar
Vario EL III instrument.
Syntheses. 2,3,5,6-Tetramethylpyrazine (2). A soln. of NaOH (13.9 g, 0.348 mol) in 100 ml of H2O was
poured into a soln. of 2,3,5,6-tetramethylpyrazine hydrochloride (1; 60.0 g, mol) in 250 ml of H2O while
stirring. The white precipitate formed was filtered and washed with H2O until the filtrate was pH-neutral.
The precipitate was dried with benzene through an azeotropic process to give 2 (36.0 g, 76.1%). White
solid. M.p. 83–868.
2-Bromomethyl-3,5,6-trimethylpyrazine (3). N-Bromosuccinimide (NBS; 12.8 g, 0.072 mol) and
benzoyl peroxide (cat. amount) were added to a soln. of 2 (10.0 g, 0.072 mol) in anh. CCl4. The mixture
was stirred at 658 for 10 h under light (60-W tungsten-halogen lamp), then cooled to 58, and filtered to
remove succinimide. The filtrate was concentrated to give a yellow oil, which was purified by vacuum
distillation to give 3 (9.2 g, 58.3%). Colorless oil. B.p. 127–1318/10 mm ([23]: b.p. 99–1008/2 mm).
General Procedure for the Synthesis of Compounds 5a, 5c, and 5e. A mixture of 3 (2 g, 0.01 mol), 4a
(4c or 4e; 0.01 mol), anh. Na2CO3 (0.5 g, 0.0047 mol), and triethyl(benzyl)ammonium chloride (TEBA;
cat. amount) in anh. acetone (50 ml) was heated to reflux for 24 h and then filtered. The filtrate was
adjusted to pH 3 with HCl (g), filtered, and washed with H2O to give a yellowish solid product. A soln.
of this yellowish solid in MeOH was adjusted with MeONa to pH 7, filtered and concentrated to give a
solid.
Ethyl 4-[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoate (5a). Yield: 1.4 g (46.7%). Yellowish solid.
M.p. 62–648. IR (KBr): 3454, 2949, 1713, 1604, 1552, 1510, 1435, 1416, 1359, 1280, 1251, 1172, 1103, 1010,
993, 848, 774, 767. 1H-NMR (500 MHz, CDCl3): 1.37 (t, J ¼ 7.1, Me); 2.51 (s, 2 Me); 2.57 (s, Me); 4.34 (q,
J ¼ 7.1, MeCH2O); 5.20 (s, CH2O); 7.02 (dd, J¼6.9, 2.0, 2 arom. H); 7.99 (dd, J¼2, 6.9, 2 arom. H).
13C-NMR (75 MHz, CDCl3): 20.43; 21.20; 21.52; 51.67; 60.46; 69.81; 114.29 (2 arom. C); 131.36 (2 arom.
C); 144.86; 148.51; 149.78; 151.38; 162.09; 166.56. ESI-MS: 301.1 ([MþH]þ ). Anal. calc. for C17H20N2O3:
C 67.98, H 6.71, N 9.33; found: C 67.66, H 6.85, N 9.34.
Ethyl 3-Chloro-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (5c). Yield: 1.1 g (34.1%). White
solid. M.p. 146–1488. 1H-NMR (300 MHz, CDCl3): 1.37 (t, J ¼ 7.1, Me); 2.51 (s, Me); 2.52 (s, Me); 2.63 (s,
Me); 4.34 (q, J ¼ 7.1, MeCH2O); 5.30 (s, CH2O); 7.16 (d, J ¼ 8.6, 1 arom. H); 7.90 (dd, J¼2.1, 8.6, 1 arom.
H); 8.04 (d, J ¼ 2.1, 1 arom. H).
Ethyl 3-Nitro-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (5e). Yield: 1.1 g (31.2%). Yellow
solid. M.p. 156–1578. 1H-NMR (300 MHz, CDCl3): 1.38 (t, J ¼ 7.1, Me); 2.51 (s, Me); 2.52 (s, Me); 2.62 (s,
Me); 4.37 (q, J ¼ 7.1, MeCH2O); 5.38 (s, CH2O); 7.39 (d, J ¼ 8.9, 1 arom. H); 8.17 (dd, J¼2.1, 8.8, 1 arom.
H); 8.45 (d, J ¼ 2.1, 1 arom. H).
General Procedure for the Synthesis of Compounds 5b and 5d. A mixture of 3 (1.1 g, 0.005 mol), 4b
(or 4d; 0.01 mol), and anh. Na2CO3 (0.53 g, 0.005 mol) in anh. DMF (5 ml) was heated to 1208 while
stirring for 2 h. After cooling, the mixture was poured into ice-H2O (50 ml), while stirring, to yield a solid,
which was then filtered and dried to give the title intermediate.
Ethyl 3-Methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (5b). Yield: 1.1 g (65.2%).
1
White solid. M.p. 98–1018. H-NMR (300 MHz, CDCl3): 1.40 (t, J ¼ 7.1, Me); 2.53 (s, 2 Me); 2.63 (s,
Me); 3.91 (s, MeO); 4.37 (q, J ¼ 7.1, MeCH2O); 5.28 (s, CH2O); 7.08 (d, J ¼ 8.4, 1 arom. H); 7.57 (d, J ¼
2.0, 1 arom. H); 7.66 (dd, J¼2.0, 8.4, 1 arom. H).
Ethyl 3-Bromo-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (5d). Yield: 1.4 g (73.7%). Yellow
1
solid. M.p. 1608. H-NMR (300 MHz, CDCl3): 1.37 (t, J ¼ 7.1, Me); 2.51 (s, Me); 2.52 (s, Me); 2.64 (s,
Me); 4.34 (q, J ¼ 7.1, MeCH2O); 5.30 (s, CH2O); 7.13 (d, J ¼ 8.7, 1 arom. H); 7.95 (dd, J¼2.1, 8.7, 1 arom.
H); 8.21 (d, J ¼ 2.1, 1 arom. H).
General Procedure for the Synthesis of Compounds 5f and 5g. A mixture of 3 (2.6 g, 0.013 mol), 4f
(or 4g; 0.012 mol), and anh. Na2CO3 (1.2 g, 0.011 mol) in anh. DMF (10 ml) was heated to 1208 while
stirring for 2 h. After cooling, the mixture was poured into ice-H2O (50 ml), while stirring, and then
extracted with CHCl3 (3ꢂ20 ml). The combined org. layers were washed with sat. NaCl soln., dried

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
(Na2SO4), filtered, and concentrated. The residue was purified by column chromatography (CC; PE/
AcOEt 4 :1) to afford the title intermediate.
Ethyl 2-[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoate (5f). Yield: 1.8 g (50.0%). White solid.
M.p. 61–648. 1H-NMR (300 MHz, CDCl3): 1.27 (t, J ¼ 7.1, Me); 2.51 (s, 2 Me); 2.63 (s, Me); 4.29 (q, J ¼
7.1, MeCH2O); 5.24 (s, CH2O); 6.98–7.01 (m, 1 arom. H); 7.13–7.16 (m, 1 arom. H); 7.39–7.42 (m, 1
arom. H); 7.76 (dd, J¼1.8, 7.7, 1 arom. H).
Ethyl 4-(Trifluoromethyl)-2-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate (5g). Yield: 3.0 g
(68.7%). Colorless liquid. 1H-NMR (300 MHz, CDCl3): 1.30 (t, J ¼ 7.1, Me); 2.51 (s, 2 Me); 2.64 (s,
Me); 4.33 (q, J ¼ 7.1, MeCH2O); 5.32 (s, CH2O); 7.22 (d, J ¼ 8.0, 1 arom. H); 7.55 (s, 1 arom. H); 7.81 (d,
J ¼ 8.0, 1 arom. H).
General Procedure for the Synthesis of Compounds 5h, 5i, and 5k. A mixture of 3 (1 g, 0.0047 mol),
4h (4i or 4k; 0.0048 mol), and anh. Na2CO3 (0.5 g, 0.0048 mol) in anh. acetone (30 ml) was refluxed for
24 h. Then, the mixture was filtered to remove Na2CO3. After cooling, the precipitate formed from the
filtrate was collected by filtration and dried to give the title intermediate.
Ethyl 4-{[(3,5,6-Trimethylpyrazin-2-yl)methyl]amino}benzoate (5h). Yield: 0.21 g (15.0%). White
1
solid. M.p. 138–1418. H-NMR (300 MHz, CDCl3): 1.36 (t, J ¼ 7.1, Me); 2.53 (s, 2 Me); 2.55 (s, Me);
4.30–4.37 (m, MeCH2O, CH2N); 5.85 (br. s, NH); 6.73 (d, J ¼ 8.7, 2 arom. H); 7.93 (d, J ¼ 8.7, 2 arom. H).
Methyl 3-Methyl-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzoate (5i). Yield: 0.46 g
(32.7%). Yellowish solid. M.p. 168–1708. 1H-NMR (300 MHz, CDCl3): 2.29 (s, Me); 2.54–2.55 (m,
3 Me); 3.86 (s, MeO); 4.38 (s, CH2N); 5.90 (br. s, NH); 6.66 (d, J ¼ 8.4, 1 arom. H); 7.79 (d, J ¼ 1.2, 1
arom. H); 7.88 (dd, J¼1.9, 8.4, 1 arom. H).
Ethyl 3-Bromo-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzoate (5k). Yield: 0.75 g (42.3%).
1
Yellow solid. M.p. 182–1838. H-NMR (500 MHz, CDCl3): 1.38 (t, J ¼ 7.1, Me); 2.53 (s, 2 Me); 2.57 (s,
Me); 4.33 (q, J ¼ 4.3, MeCH2O); 4.39 (s, CH2N); 6.69 (d, J ¼ 8.6, 1 arom. H); 7.92 (dd, J¼2.0, 8.6, 1 arom.
H); 8.16 (d, J ¼ 2.0, 1 arom. H).
General Procedure for the Synthesis of Compounds 5j, 5m, and 5n. A mixture of 3 (2.0 g, 0.009 mol),
4j (4m or 4n; 0.0085 mol), and anh. Na2CO3 (1.2 g, 0.011 mol) in anh. DMF (10 ml) was heated to 1208
while stirring for 2 h. After cooling, the mixture was poured into ice-H2O (50 ml) while stirring to yield a
yellow-brown syrup-like substance, which was extracted with CHCl3 (3ꢂ30 ml). The combined org.
layers were washed with sat. NaCl soln. (3ꢂ90 ml), dried (Na2SO4), filtered, and concentrated. The
residue was purified by CC (PE/AcOEt 4 :1) to give the title intermediate.
Ethyl 3-Chloro-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzoate (5j). Yield: 1.5 g (53.2%).
Yellowish solid. M.p. 155–1588. 1H-NMR (500 MHz, CDCl3): 1.38 (t, J ¼ 7.1, Me); 2.53–2.56 (m, 3 Me);
4.33 (q, J ¼ 7.1, MeCH2O); 4.40 (s, CH2N); 6.61 (s, NH); 6.73 (d, J ¼ 8.5, 1 arom. H); 7.89 (dd, J¼1.8, 8.5,
1 arom. H); 8.00 (d, J ¼ 1.8, 1 arom. H).
Ethyl 2-{[(3,5,6-Trimethylpyrazin-2-yl)methyl]amino}benzoate (5m). Yield: 1.0 g (39.3%). Yellow-
ish solid. M.p. 113–1158. IR (KBr): 3441, 3328, 2978, 2913, 1682, 1608, 1571, 1518, 1477, 1455, 1415, 1371,
1256, 1227, 1183, 1153, 1127, 1094, 1074, 990, 853, 792, 752. 1H-NMR (500 MHz, CDCl3): 1.38 (t, J ¼ 7.1,
Me); 2.51 (s, Me); 2.54 (s, Me); 2.57 (s, Me); 4.35 (q, J ¼ 7.1, MeCH2O); 4.44 (d, J ¼ 4.6, CH2N), 6.62–
6.65 (m, 1 arom. H); 6.82 (d, J ¼ 8.4, 1 arom. H); 7.37–7.40 (m, 1 arom. H); 7.96 (dd, J¼1.7, 8.0, 1 arom.
H); 8.63 (s, NH). 13C-NMR (75 MHz, CDCl3): 14.38; 20.33; 21.41; 21.58; 45.83; 60.21; 111.06; 111.60;
114.90; 131.73; 134.42; 146.47; 147.78; 148.37; 149.62; 150.32; 168.42. ESI-MS: 300.2 ([MþH]þ ). Anal.
calc. for C17H21N3O2: C 68.20, H 7.07, N 14.04; found: C 67.90, H 7.08, N 13.81.
Ethyl 4-Methyl-3-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzoate (5n). Yield: 1.2 g (46.6%).
White solid. M.p. 104–1058. 1H-NMR (500 MHz, CDCl3): 1.39 (t, J ¼ 7.1, Me); 2.45 (s, Me); 2.52 (s, Me);
2.53 (s, Me); 2.55 (s, Me); 4.34–4.38 (m, MeCH2O, CH2N); 5.57 (s, NH); 6.84 (dd, J¼1.5, 7.7, 1 arom. H);
7.16 (dd, J¼1.6, 7.8, 1 arom. H); 7.18–7.21 (m, 1 arom. H).
Synthesis of Ethyl 3-Nitro-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzoate (5l). A soln. of 4l
(0.5 g, 0.0024 mol) in 2 ml of anh. DMF was added to a mixture of NaH (0.05 g. 0.002 mol) suspended in
1 ml of anh. DMF while stirring. Then, the mixture was stirred under N2 for 1 h at r.t., a soln. of 3 (0.5 g,
0.0024 mol) in 2 ml of anh. DMF was added, and the mixture was stirred for 4 h. The yellow precipitate
formed was filtered, 5 ml of AcOEt was added into the filtrate, and the yellow precipitate formed was
filtered. All precipitates were dissolved in CHCl3, filtered, and the filtrate was concentrated to give 5l.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2733
1
Yield: 0.31 g (38.0%). Yellow solid. M.p. 206–2108. H-NMR (500 MHz, CDCl3): 1.41 (t, J ¼ 7.2, Me);
2.70 (s, Me); 2.75 (s, Me); 2.77 (s, Me); 4.38 (q, J ¼ 7.2, MeCH2O); 4.64 (d, J ¼ 2.7, CH2N); 6.98 (d, J ¼
8.9, 1 arom. H); 8.15 (dd, J¼2.0, 8.9, 1 arom. H); 8.95 (d, J ¼ 2.0, 1 arom. H); 9.73 (s, NH).
General Procedure for Synthesis of Compounds 6. Guanidine (2.00 g, 0.0035 mol) and 3 ml of anh.
iPrOH were mixed, and the mixture was then heated to 708 while stirring. A soln. of 5 (0.0017 mol) in 3 ml
i
of anh. PrOH was added, and the mixture was refluxed for 30 min. After cooling, the mixture was
adjusted to pH 3 with HCl (g) in AcOEt, filtered, and concentrated under reduced pressure. The residue
was purified by CC (CHCl3/MeOH 10 :1) to afford a solid product.
N-(Diaminomethylidene)-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydrochloride (6a).
Yield: 0.31 g (51.7%). White solid. M.p. 102–1068. IR (KBr): 3435, 3352, 3129, 2952, 2924, 2853, 1701
(C¼O), 1666 (C¼N), 1606, 1543, 1463 (C¼C), 1261 (CꢁOꢁC), 1174, 991, 809, 762. 1H-NMR
(500 MHz, (D6)DMSO): 2.44 (s, Me); 2.45 (s, Me); 2.49 (s, Me); 5.23 (s, CH2O); 7.10–7.13 (m, 2 arom.
H); 7.88–7.90 (m, 2 arom. H); 12.61 (br. s, NH). ESI-MS: 314.1 ([MþH]þ ). Anal. calc. for C16H19N5O2 ·
HCl·H2O: C 52.25, H 6.03, N 19.04; found: C 51.81, H 6.18, N 18.70.
N-(Diaminomethylidene)-3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydro-
chloride (6b). Yield: 0.37 g (57.7%). White solid. M.p. 1978. IR (KBr): 3329, 3186, 2950, 1695 (C¼O),
1625, 1597, 1521, 1462 (C¼C), 1271, 1218, 1174, 1135, 1027 (CꢁOꢁC), 991, 825, 756. 1H-NMR
(300 MHz, (D6)DMSO): 2.44 (s, Me); 2.46 (s, Me); 2.49 (s, Me); 3.84 (s, MeO); 5.24 (s, CH2O); 7.27 (d,
J ¼ 8.3, 1 arom. H); 7.79–7.80 (m, 2 arom. H); 8.70 (br. s, 4 NH); 12.10 (br. s, NH). 13C-NMR (75 MHz,
CDCl3): 20.08; 20.92; 21.23; 56.01; 70.02; 111.67; 112.78; 122.52; 144.81; 148.32; 148.75; 149.47; 151.20;
152.22; 153.71; 156.27; 167.59. ESI-MS: 344.1 ([MþH]þ ). Anal. calc. for C17H21N5O3 ·HCl·1.5 H2O: C
50.18, H 6.19, N 17.21; found: C 49.80, H 6.22, N 17.38.
3-Chloro-N-(diaminomethylidene)-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydro-
chloride (6c). Yield: 0.25 g (38.4%). White solid. M.p. 162–1648. IR (KBr): 3371, 3130, 2945, 2929,
2872, 1705 (C¼O), 1659 (C¼N), 1599, 1520, 1440 (C¼C), 1267 (CꢁOꢁC), 1175, 989, 813, 755.
1H-NMR (500 MHz, (D6)DMSO): 2.45 (s, Me); 2.47 (s, Me); 2.52 (s, Me); 5.35 (s, CH2O); 7.22 (br. s,
2 NH); 7.40 (d, J ¼ 8.6, 1 arom. H); 7.88–7.91 (m, 2 arom. H); 12.9 (br. s, 3 NH). ESI-MS: 348.1 ([Mþ
H]þ ). Anal. calc. for C16H18ClN5O2 ·HCl·H2O: C 47.77, H 5.26, N 17.41; found: C 47.59, H 5.31, N 17.27.
3-Bromo-N-(diaminomethylidene)-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydro-
chloride (6d). Yield: 0.38 g (52.1%). Yellowish solid. M.p. 175–1768. IR (KBr): 3380, 3189, 2954,
2921, 2877, 2850, 1706 (C¼O), 1657 (C¼N), 1595, 1551, 1494, 1455 (C¼C), 1359, 1265, 1176, 1148, 1043
(CꢁOꢁC), 1043, 990, 811, 786, 755. 1H-NMR (500 MHz, (D6)DMSO): 2.50 (s, 2 Me); 2.59 (s, Me); 5.29
(s, CH2O); 7.20 (d, J ¼ 8.8, 1 arom. H); 7.65 (br. s, 2 NH); 8.06 (dd, J¼2.0, 8.8, 1 arom. H); 8.32 (d, J ¼
2.0, 1 arom. H). ESI-MS: 394.0 ([MþH]þ ). Anal. calc. for C16H18BrN5O2 ·HCl·H2O: C 43.02, H 4.74, N
15.68; found: C 42.87, H 4.82, N 15.57.
N-(Diaminomethylidene)-3-nitro-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydrochlor-
ide (6e). Yield: 0.33 g (48.9%). White solid. M.p. 127–1308. IR (KBr): 3429, 3314, 2923, 2853, 1710
(C¼O), 1681 (C¼N), 1618, 1534, 1462, 1414 (C¼C), 1383 (CꢁNO2), 1274, 1150, 1089 (CꢁOꢁC), 983,
755, 708. 1H-NMR (300 MHz, (D6)DMSO): 2.44 (s, Me); 2.46 (s, Me); 2.49 (s, Me); 5.41 (s, CH2O); 7.55
(d, J ¼ 8.9, 1 arom. H); 7.88 (br. s, 2 NH); 8.27 (dd, J¼2.1, 8.8, 1 arom. H); 8.49 (d, J ¼ 2.1, 1 arom. H).
ESI-MS: 359.1 ([MþH]þ ), 381.1 ([MþNa]þ ). Anal. calc. for C16H18N6O4 ·HCl·0.5 H2O: C 47.59, H
4.99, N 20.81; found: C 47.48, H 5.07, N 20.50.
N-(Diaminomethylidene)-2-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide Hydrochloride (6f).
Yield: 0.26 g (43.4%). Yellowish solid. M.p. 185–1878. IR (KBr): 3356, 3271, 3126, 3028, 2754, 1701
(C¼O), 1687 (C¼N), 1596, 1558, 1458 (C¼C), 1278, 1240, 1211, 1170, 1014 (CꢁOꢁC), 979, 752.
1H-NMR (300 MHz, (D6)DMSO): 2.46 (s, Me); 2.52 (s, Me); 2.60 (s, Me); 5.52 (s, CH2O); 7.08–7.13 (m,
1 arom. H); 7.39 (d, J ¼ 8.4, 1 arom. H); 7.55–7.61 (m, 1 arom. H); 7.70 (dd, J¼1.4, 7.7, 1 arom. H); 8.78
(br. s, 4 NH); 11.66 (br. s, NH). ESI-MS: 314.2 ([MþH]). Anal. calc. for C16H19N5O2 ·HCl·0.5 H2O: C
53.56, H 5.90, N 19.52; found: C 53.27, H 5.97, N 19.49.
N-(Diaminomethylidene)-4-(trifluoromethyl)-2-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzamide
Hydrochloride (6g). Yield: 0.32 g (44.9%). White solid. M.p. 193–1958. IR (KBr): 3356, 3281, 3047, 2927,
1697 (C¼O), 1679 (C¼N), 1622, 1585, 1562, 1500 (C¼C), 1440, 1325 (CꢁF), 1211, 1170, 1132, 979, 719,
696. 1H-NMR (300 MHz, (D6)DMSO): 2.42 (s, Me); 2.44 (s, Me); 2.51 (s, Me); 5.45 (s, CH2O); 7.41 (d,

2734
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
J ¼ 7.9, 1 arom. H); 7.74–7.76 (m, 2 arom. H); 8.24 (br. s, 4 NH); 11.78 (br. s, NH). 1H-NMR (500 MHz,
MeOD): 2.48 (s, Me); 2.50 (s, Me); 2.58 (s, Me); 5.55 (s, CH2O); 7.40 (dd, J¼0.8, 8.0, 1 arom. H); 7.70 (s, 1
arom. H); 7.89 (d, J ¼ 8.0, 1 arom. H). ESI-MS: 382.1 ([MþH]þ ). Anal. calc. for C17H18N5O2 ·HCl: C
48.87, H 4.58, N 16.76; found: C 49.17, H 4.61, N 16.57.
N-(Diaminomethylidene)-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hydrochloride
(6h). Yield: 0.25 g (42.3%). White solid. M.p. 251–2558. IR (KBr): 3361, 3297, 3125, 2987, 2918, 1691
(C¼O), 1605, 1563, 1540, 1482 (C¼C), 1272, 1258, 1191, 1116, 827, 687. 1H-NMR (300 MHz,
(D6)DMSO): 2.43 (s, Me); 2.44 (s, Me); 2.48 (s, Me); 4.43 (d, J ¼ 5.3, CH2N); 6.79 (d, J ¼ 8.9, 2 arom. H);
7.19 (br. s, CNH); 7.91 (d, J ¼ 8.9, 2 arom. H); 8.53 (br. s, 4 NH); 11.39 (br. s, NH). ESI-MS: 313.2 ([Mþ
H]þ ). Anal. calc. for C16H20N6O·HCl: C 55.09, H 6.07, N 24.09; found: C 54.98, H 6.24, N 23.66.
N-(Diaminomethylidene)-3-methyl-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hy-
drochloride (6i). Yield: 0.34 g (54.8%). Yellowish solid. M.p. 2678. IR (KBr): 3368, 3153, 2993, 1689
(C¼O), 1606, 1564, 1450 (C¼C), 1261, 1151, 1113, 990, 810, 760. 1H-NMR (300 MHz, (D6)DMSO): 2.22
(s, Me); 2.43 (s, Me); 2.45 (s, Me); 2.49 (s, Me); 4.50 (s, CH2N); 6.77 (d, J ¼ 8.7, 1 arom. H); 7.81 (s, 1
arom. H); 7.86 (dd, J¼2.1, 8.6, 1 arom. H); 8.50 (br. s, 4 NH). ESI-MS: 327.2 ([MþH]þ ). Anal. calc. for
C17H22N6O·HCl·H2O: C 53.61, H 6.62, N 22.07; found: C 53.77, H 6.58, N 22.29.
3-Chloro-N-(diaminomethylidene)-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hy-
drochloride (6j). Yield: 0.45 g (69.3%). White solid. M.p. 207–2108. IR (KBr): 3356, 3281, 3045, 2926,
1697 (C¼O), 1680, 1622, 1579, 1562, 1500 (C¼C), 1325, 1209, 1170, 1132, 1002, 979, 719, 696. 1H-NMR
(500 MHz, (D6)DMSO): 2.44 (s, Me); 2.47 (s, Me); 2.48 (s, Me); 4.46 (s, CH2N); 6.48 (br. s, NH); 6.84 (d,
J ¼ 8.6, 1 arom. H); 7.65 (br. s, 4 NH); 7.85 (dd, J¼1.8, 8.5, 1 arom. H); 8.02 (d, J ¼ 1.8, 1 arom. H). ESI-
MS: 347.1 ([MþH]þ ). Anal. calc. for C16H19ClN6O·HCl·H2O: C 47.89, H 5.53, N 20.94; found: C 48.02,
H 5.47, N 21.14.
3-Bromo-N-(diaminomethylidene)-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hy-
drochloride (6k). Yield: 0.53 g (72.7%). Yellow solid. M.p. 218–2208. IR (KBr): 3407, 2923, 2770,
1692 (C¼O), 1632, 1596, 1542, 1461 (C¼C), 1267, 1229, 1092, 755, 701. 1H-NMR (300 MHz,
(D6)DMSO): 2.48 (s, Me); 2.52 (s, 2 Me); 4.56 (s, CH2N); 6.94 (d, J ¼ 8.6, 1 arom. H); 7.10 (br. s,
CNH); 8.20 (d, J ¼ 8.3, 1 arom. H); 8.34 (d, J ¼ 1.1, 1 arom. H); 8.55 (br. s, 2 NH); 8.87 (br. s, 2 NH);
11.96 (br. s, NH). ESI-MS: 391.1 ([MþH]þ ). Anal. calc. for C16H19BrN6O·HCl·H2O: C 43.11, H 4.97, N
18.85; found: C 43.46, H 4.82, N 19.10.
N-(Diaminomethylidene)-3-nitro-4-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hydro-
chloride (6l). Yield: 0.23 g (34.2%). Yellow solid. M.p. 207–2108. IR (KBr): 3406, 1697 (C¼O), 1627
(C¼C), 1566, 1421, 1359 (CꢁNO2), 1296, 1186, 1093 (CꢁN), 993, 831, 790. 1H-NMR (500 MHz,
(D6)DMSO): 2.55 (s, Me); 2.56 (s, Me); 2.59 (s, Me); 3.16 (s, CH2N); 7.18 (br. s, CNH); 7.90 (d, J ¼ 8.6, 1
arom. H); 8.21 (d, J ¼ 8.6, 1 arom. H); 8.54 (s, 1 arom. H); 8.79 (br. s, 2 NH); 8.92 (br. s, 2 NH); 12.33 (br.
s, NH). ESI-MS: 358.1 ([MþH]þ ). Anal. calc. for C16H19N7O3 ·HCl·0.5 H2O: C 47.70, H 5.25, N 24.34;
found: C 47.56, H 5.29, N 24.11.
N-(Diaminomethylidene)-2-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hydrochloride
(6m). Yield: 0.17 g (28.7%). White solid. M.p. 247–2508. IR (KBr): 3330, 3227, 2991, 2952, 2920, 2856,
1695 (C¼O), 1672, 1612, 1562, 1515 (C¼C), 1357, 1271, 1161, 1091, 894, 750. 1H-NMR (300 MHz,
(D6)DMSO): 2.40 (s, Me); 2.45 (s, Me); 2.47 (s, Me); 4.43 (s, CH2N); 6.49–6.54 (m, 1 arom. H); 6.76 (d,
J ¼ 8.0, 1 arom. H); 6.90 (br. s, CNH); 7.23 (br. s, 1 arom. H); 7.96 (d, J ¼ 6.8, 1 arom. H); 7.70 (br. s,
2 NH); 9.75 (br. s, 2 NH). ESI-MS: 313.1 ([MþH]þ ). Anal. calc. for C16H20N6O·HCl: C 55.09, H 6.07, N
24.09; found: C 54.88, H 6.12, N 23.87.
N-(Diaminomethylidene)-4-methyl-3-{[(3,5,6-trimethylpyrazin-2-yl)methyl]amino}benzamide Hy-
drochloride (6n). Yield: 0.42 g (67.6%). White solid. M.p. 226–2298. IR (KBr): 3396, 3340, 3140, 2991,
1
2920, 2856, 2810, 1691 (C¼O), 1585, 1504 (C¼C), 1357, 1261, 1145, 1092, 989, 831, 783, 628. H-NMR
(500 MHz, (D6)DMSO): 2.23 (s, Me); 2.44 (s, Me); 2.47 (s, Me); 2.50 (s, Me); 4.41 (d, J ¼ 4.6, CH2N);
6.55 (br. s, CNH); 6.83–6.88 (m, 2 arom. H); 7.11–7.14 (m, 1 arom. H); 8.37 (br. s, 4 NH); 11.80 (br. s,
NH). ESI-MS: 327.1 ([MþH]þ ). Anal. calc. for C17H22N6O·HCl·H2O: C 53.61, H 6.62, N 22.06; found:
C 53.59, H 6.72, N 21.99.
3. Bioassays. Animals. Sprague–Dawley (SD) rats were provided by Shanghai BK Biology Co. The
rats were acclimatized for a week before use. Rodent laboratory chow and tap H2O were provided ad

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2735
libitum, and the animals were maintained under controlled conditions at a temp. of 24ꢀ18 and 50ꢀ10%
humidity, using a 12-h light/12-h dark cycle. All the procedures were in strict accordance with the P. R.
China legislation on the use and care of laboratory animals and with the Guide for the Care and Use of
Laboratory Animals published by the U.S. National Institute of Health.
Rat Platelet Swelling Assay (PSA). Sprague–Dawley rats (380–420 g) were anesthetized with Et2O,
and blood was collected from their eyeholes with 25% (v/v) acid-citrate-dextrose (ACD; sodium citrate,
2.23 g; citric acid, 0.86 g; and glucose, 2.47 g in 100 ml of distilled H2O). Platelet-rich plasma (PRP) was
obtained by centrifugation of whole blood at 1300g/min for 10 min at r.t. The upper two-thirds of the
supernatants were used for the further measurements and stored at r.t. until used. All measurements
were performed within 4–5 h.
All compounds were dissolved in DMSO and diluted with propionate medium (pH 7.4). A soln. of
the tested compound (25 ml) was added to 175 ml of propionate buffer (in mm; sodium propionate,
140 mm; HEPES, 20 qs; glucose, 10 mm; KCl, 5 mm; MgCl2, 1 mm; CaCl2, 1 mm; pH 6.7) contained in a
spectrophotometer cuvette. Then, 50 ml of PRP prewarmed to 378 was added. The suspension was stirred,
and the change in optical density (OD) was recorded each 7.5 s for 2 min at 550 nm (Thermo Multiscan
Spectrum). The decrease of OD corresponded to a monoexponential curve following the equation
OD(t) ¼ODt¼0 e where t is the time (in s) corresponding to the recorded OD, and k is the decrease rate
constant. For each compound, the concentrations were plotted against their corresponding k values. The
maximum platelet swelling was measured in absence of any drug. The minimum swelling was observed in
presence of cariporide (10 mol/l) and is the result of complete NHE-1 inhibition. Sigmoidal curves were
drawn by non-linear regression analysis (Graphpad Prism software). The IC50 value was calculated
according to the regression analysis. Each measurement was performed in triplicate for all molecules.
Cardioprotective Effects in Rat Model of Ischemic Heart. Adult male and female SD rats (280–300 g)
were anesthetized with sodium pentobarbital (60 mg/kg, ip, Shanghai Chemicals Reagent Co., Ltd.,
Shanghai, P. R. China). Coronary artery occlusion was produced by ligating the left anterior descending
(LAD) coronary artery for 1 h. After that, the coronary artery was reperfused by loosing the ligature.
After 2 h of reperfusion, the coronary artery was reoccluded, and 1 ml of a 2% Evans blue was injected
via tail vein. Then, the heart was removed, and blood sample was collected.
The left ventricle of the removed heart was dissected free from other structures and sliced
transversely into 1-mm thick sections. The sections were then incubated in 0.5% triphenyltetrazolium
chloride (Shanghai Chemicals Reagent Co., Ltd., Shanghai, P. R. China) for 10 min at 378 and then fixed
for 1–2 h in a 10% formalin soln. to determine the infarct size. The infarct size was calculated by the
formula as follow:
Myocardial infarct size [%]¼(the weight of undyed myocardium/the weight of left ventricle)ꢂ100
The blood samples were centrifuged at 3000g for 10 min. The supernatant serum was removed and
stored in liquid N2 until the biochemical analysis was performed. Creatine kinase (CK) in serum were
measured by 722 grating photospectrometer (Shanghai Precision & Scientific Instrument Co., LTD.,
Shanghai, P. R. China) using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R.
China).
Target compounds 6c, 6e, 6e, 6d, 6f, 6g, 6k, and 6l, and cariporide were intravenously given 5 min
before reperfusion.
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Received October 23, 2009