Synthesis and studies on anticonvulsant activity of 8-alkoxy-[1,2,4] triazolo[4,3-a]pyrazine

Article abstract of DOI:10.14233/ajchem.2013.13703

In this study, a series of new 8-alkoxy-[1,2,4]triazolo[4,3-a]pyrazine derivatives were synthesized and their anticonvulsant activity and neurotoxicity were evaluated by the maximal electroshock test and the rotarod test, respectively. The most promising compounds 3d (8-butoxy-[1,2,4]triazolo[4,3-a] pyrazine) and 3f (8-hexyloxy-[1,2,4]triazolo[4,3-a]pyrazine) showed a median effective dose of 44 and 35.3 mg/kg and had protective index value of 3.2 and 4.8, respectively. To explain the possible mechanism of anticonvulsant activity, all compounds were tested in chemical induced model in anti pentylenetetrazol test.

Full text of DOI:10.14233/ajchem.2013.13703

Asian Journal of Chemistry; Vol. 25, No. 7 (2013), 3660-3664
http://dx.doi.org/10.14233/ajchem.2013.13703
Synthesis and Studies on Anticonvulsant Activity of 8-Alkoxy-[1,2,4]triazolo[4,3-a]pyrazine
1,*
2
1
LI-PING GUAN , RUI-PENG ZHANG , YUE CHANG1,2 and XI-XI GAN
1School of Food, Drug & Medicine, Zhejiang Ocean University, Zhoushan 316004, P.R. China
2College of Pharmacy, Yanbian University, Yanji 133000, , Jili Province, P.R. China
*Corresponding author: Tel: +86 580 2554536; E-mail: glp730@yahoo.com.cn
(Received: 9 March 2012;
Accepted: 7 January 2013)
AJC-12669
In this study, a series of new 8-alkoxy-[1,2,4]triazolo[4,3-a]pyrazine derivatives were synthesized and their anticonvulsant activity and
neurotoxicity were evaluated by the maximal electroshock test and the rotarod test, respectively. The most promising compounds 3d
(8-butoxy-[1,2,4]triazolo[4,3-a]pyrazine) and 3f (8-hexyloxy-[1,2,4]triazolo[4,3-a]pyrazine) showed a median effective dose of 44 and
35.3 mg/kg and had protective index value of 3.2 and 4.8, respectively. To explain the possible mechanism of anticonvulsant activity, all
compounds were tested in chemical induced model in anti pentylenetetrazol test.
Key Words: [1,2,4]Triazolo[4,3-a]pyrazine, Anticonvulsant activity, Maximal electroshock test, Pentylenetetrazol.
but its activity was higher than that of flunitrazepam. With the
introduction of a triazole ring to the first and second position
of flunitrazepam, the physical space occupied by this imidazo-
benzodiazepine is similar to that found for flunitrazepam, but
it has a significantly higher affinity for the recognition site
(Fig. 1).
INTRODUCTION
Epilepsy, one of the most common neurologic diseases,
is characterized by epileptic seizures, which are evoked by
unexpected high-level neuronal discharges in the brain1.Anti-
convulsant drugs currently on the market are of unsatisfactory
effectiveness in seizure control and cause adverse reactions
such as drowsiness, ataxia, gastrointestinal disturbance, hepato-
toxicity and megaloblastic anemia2-4 and even life-threatening
conditions5. Therefore the search for safer and more effective
antiepileptic drugs is necessary and the development of these
drugs has been important and challenging for medicinal chemists
to develop new antiepileptic drugs with desirable therapeutic
properties.
RO
S
N
RO
S
N
H
O
N
N
pre.1
pre. 1'
RO
In the previous study, we reported the synthesis and anti-
convulsant activities of 7-alkoxyl-4H-[1,2,4]triazolo[4,3-d]-
benzo[b]-[1,4]thiazines (pre. 1')6 and 6-alkoxy-[1,2,4]triazolo-
[4,3-b]pyridazine (pre. 2')7. From compounds pre. 1 to
compounds pre. 1' and from compounds pre. 2 to compounds
pre. 2', in which we incorporated triazole ring at the first and
second position for compounds pre. 1 and at the second and
third position for compounds pre. 2 (Fig. 1). The pharma-
cology test showed the anticonvulsant activity increase when
incorporated triazole ring. It may have higher affinity for the
receptor and enhance anticonvulsant activity, after the triazole
ring was introduced and there were some similar design
reports8. In addition, Chau et al.9, carried out a similar docking
experiment with the imidazobenzodiazepine and alprazolam.
Alprazolam is an agonist benzodiazepine, like flunitrazepam,
RO
Cl
N
N
N
N
N
N
pre. 2
pre. 2'
Cl
F
O2N
N
N
N
O
N
N
N
Flunitrazepam
Alprazolam
Fig. 1. Structrue of previous studied compounds, 1, 2, flunitrazepam and
alprazolam

Vol. 25, No. 7 (2013)
Synthesis and Studies on Anticonvulsant Activity of 8-Alkoxy-[1,2,4]triazolo[4,3-a]pyrazine 3661
In an attempt to discover a novel anticonvulsant compound
with better activity, keeping the above-mentioned in view,
compound I (8-phenoxy-[1,2,4]triazolo[4,3-a]pyrazine) was
designed and synthesized as the leading compound to investi-
gate the contribution of different alkoxyl groups at position 8
of the 1,2,4-triazolo[4,3-a]pyrazine to obtain a series of
8-alkoxy-[1,2,4]triazolo[4,3-a]pyrazine derivatives (Fig. 2).
3.2 mmol) and appropriate alkanol or substituted phenol (3.2
mmol) were added to a solution of sodium hydroxide (3.2
mmol) or K2CO3 (3.2 mmol) in DMF with stirring and refluxing
for 3-5 h.After the solvent was removed under reduced pressure,
the solid residue was purified by silica gel chromatography
with dichloromethane:methanol (20:1). The yield, melting
point, spectral data of each compound is given below7.
8-Methoxy-[1,2,4]triazolo[4,3-a]pyrazine (3a): Yield:
1
Their structures were characterized using IR, H NMR and
1
MS. Their anticonvulsant activity was evaluated using the
maximal electroshock (MES) test and reported for the first
time. Their neurotoxicity was evaluated using the rotarod test
in mice. In this contribution, to explain the possible mechanism
of action, compound 3a-s was tested in the pentylenetetrazol
(PTZ) test. Under identical conditions, the anti pentylenete-
trazol activity of the marketed agent carbamazepine was
evaluated as a comparison.
83 %; m.p: 152-154 ºC. H NMR (CDCl3): δ 8.90 (1H, s,
triazolo-H), 7.75 (1H, d, J = 4.68 Hz, pyrazine-H), 7.42 (1H,
d, J = 4.68 Hz, pyrazine-H), 4.21 (3H, s, -OCH3). IR (KBr,
νmax, cm-1): 1623 (C=N), 1258, 1031 (C-O-C), 1128 (N-N).
MS m/z: 151(M + 1).
8-Ethoxy-[1,2,4]triazolo[4,3-a]pyrazine (3b):Yield: 78
%; m.p. 134-136 ºC. 1H NMR (CDCl3): δ 8.87 (1H, s, triazolo-
H), 7.73 (1H, d, J = 4.64 Hz, pyrazine-H), 7.40 (1H, d, J =
4.64 Hz, pyrazine-H), 4.66 (2H, p, -OCH2), 1.55 (3H, t, -CH3).
IR (KBr, νmax, cm-1): 1623 (C=N), 1254, 1030 (C-O-C), 1127
(N-N). MS m/z: 165 (M + 1).
O
N
N
O
N
N
R
8-Isopropoxy-[1,2,4]triazolo[4,3-a]pyrazine (3c):Yield:
1
75 %; m.p. 116-118 ºC. H NMR (CDCl3): δ 8.85 (1H, s,
N
N
N
N
triazolo-H), 7.70 (1H, d, J = 4.74 Hz, pyrazine-H), 7.40 (1H,
d, J = 4.74 Hz, pyrazine-H), 5.59 (1H, m, -OCH), 1.50 (3H, s,
-CH3), 1.52 (3H, s, -CH3). IR (KBr, νmax, cm-1): 1623 (C=N),
1252, 1034 (C-O-C), 1126 (N-N). MS m/z: 179 (M + 1).
8-Butoxy-[1,2,4]triazolo[4,3-a]pyrazine (3d):Yield: 72
%; m.p. 104-106 ºC. 1H NMR (CDCl3): δ 8.88 (1H, s, triazolo-
H), 7.72 (1H, d, J = 4.71 Hz, pyrazine-H), 7.40 (1H, d, J =
4.71 Hz, pyrazine-H), 4.58 (2H, t, -OCH2), 1.51-1.94 (4H, m,
-(CH2)2), 1.00 (3H, t, -CH3). IR (KBr, νmax, cm-1): 1622 (C=N),
1253, 1033 (C-O-C), 1126 (N-N). MS m/z: 193 (M + 1).
8-Pentyloxy-[1,2,4]triazolo[4,3-a]pyrazine (3e):Yield:
71 %; m.p. 58-60 ºC. 1H NMR (CDCl3): δ 8.84 (1H, s, triazolo-
H), 7.70 (1H, d, J = 4.74 Hz, pyrazine-H), 7.39 (1H, d, J =
4.74 Hz, pyrazine-H), 4.57 (2H, t, -OCH2), 1.11-1.72 (6H, m,
-(CH2)3), 0.95 (3H, t, -CH3). IR (KBr, νmax, cm-1): 1622 (C=N),
1251, 1034 (C-O-C), 1127 (N-N). MS m/z: 207(M + 1).
8-Hexyloxy-[1,2,4]triazolo[4,3-a]pyrazine(3f): Yield:
I
3a-s
Fig. 2. Structure of compounds I and 3a-s
EXPERIMENTAL
Melting points were determined in open capillary tubes
and were uncorrected. IR spectra were recorded (in KBr) on a
FT-IR1730 (Bruker, Switzerland), 1H NMR was measured on
an AV-300 (Bruker, Switzerland) and all chemical shifts were
given in ppm relative to tetramethysilane. Mass spectra were
measured on an HP1100LC (Agilent Technologies, USA). The
major chemicals were purchased from Alderich Chemical
Corporation. All other chemicals were the analytical grade.
Synthesis of 1-hydrazino-4-chroropyrazine (1): To a
solution of hydrazine hydrate (3.04 g, 62.8 mmol) in 20 mL
of ethanol, the solution of 2,3-dichloropyrazine (2.50 g, 12.6
mmol) in 30 mL ethanol was added dropwise at room tempe-
rature. The mixture was stirred and refluxed for 1 h, then, the
half of the solvent was removed under reduced pressure and
the solution was poured into petroleum ether. The precipitate
was filtered and washed with petroleum ether, then kept below
0 ºC. The resulting crude intermediates were used for the next
step10.
1
69 %; m.p. 102-104 ºC. H NMR (CDCl3): δ 8.93 (1H, s,
triazolo-H), 7.81 (1H, d, J = 4.71 Hz, pyrazine-H), 7.40 (1H,
d, J = 4.71 Hz, pyridazine-H), 4.57 (2H, t, -OCH2), 1.32-1.94
(8H, m, -(CH2)4), 0.90 (3H, t, -CH3). IR (KBr, νmax, cm-1): 1624
(C=N), 1252, 1034 (C-O-C), 1127 (N-N). MS m/z: 221
(M + 1).
8-Heptyloxy-[1,2,4]triazolo[4,3-a]pyrazine (3g):Yield:
1
76 %; m.p. 110-112 ºC. H NMR (CDCl3): δ 8.85 (1H, s,
Synthesis of 8-chloro-[1,2,4]triazolo[4,3-a]pyrazine
(2): Compound 1 (4 mmol), formic acid (4 mmol) and ethanol
30 mL was placed in a 50 mL round-bottomed flask. The
mixture was stirred for 2 h at room temperature to obtain the
acid intermediate. The mixture was evaporated under reduced
pressure. The acid intermediate was reacted in refluxing DMF
for 3 h. The solution was evaporated to dryness and the oily
residue was filtered on a silica gel chromatographic column
(ethyl acetate) to give a light yellow or white solid.Yield 47.6 %.
1H NMR (DMSO) δ: 7.83 (1H, d, J = 4.64 Hz, pyrazine-H),
8.70 (1H, d, J = 4.64 Hz, pyrazine-H), 9.61 (1H, s, triazolo-H).
MS (M + 1): 155.
triazolo-H), 7.71 (1H, d, J = 4.74 Hz, pyrazine-H), 7.39 (1H,
d, J = 4.74 Hz, pyrazine-H), 4.57 (2H, t, -OCH2), 1.30-1.97
(10H, m, -(CH2)5), 0.89 (3H, t, -CH3). IR (KBr, νmax, cm-1):
1622 (C=N), 1251, 1036 (C-O-C), 1127 (N-N). MS m/z: 235
(M + 1).
8-Octyloxy-[1,2,4]triazolo[4,3-a]pyrazine (3h): Yield:
1
80 %; m.p. 112-114 ºC. H NMR (CDCl3): δ 8.86 (1H, s,
triazolo-H), 8.11 (1H, d, J = 4.76 Hz, pyrazine-H), 7.07 (1H,
d, J = 4.76 Hz, pyrazine-H), 4.57 (2H, t, -OCH2), 1.29-1.97
(12H, m, -(CH2)6), 0.88 (3H, t, -CH3). IR (KBr, νmax, cm-1):
1623 (C=N), 1251, 1034 (C-O-C), 1124 (N-N). MS m/z: 249
(M + 1).
General procedure for the synthesis of 8-alkoxy-
[1,2,4]triazolo[4,3-a]pyrazine (3a-s): Compound 2 (0.5 g,

3662 Guan et al.
Asian J. Chem.
8-(2-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyrazine (3i):
-C6H4). IR (KBr, νmax, cm-1): 1622 (C=N), 1250, 1032
(C-O-C), 1127 (N-N). MS m/z: 290 (M + 1).
8-(2,4-Dichlorophenoxy)-[1,2,4]triazolo[4,3-a]-
Yield: 81 %; m.p. 136-138 ºC. 1H NMR (CDCl3): δ 8.96 (1H,
s, triazolo-H), 7.77 (1H, d, J = 4.77 Hz, pyrazine -H), 7.36
(1H, d, J = 4.77 Hz, pyrazine-H), 7.20-7.32 (4H, m, -C6H4),
2.24 (3H, s, -CH3). IR (KBr, νmax, cm-1): 1623 (C=N), 1254,
1034 (C-O-C), 1126 (N-N). MS m/z: 227 (M + 1).
1
pyrazine (3s): Yield: 59 %; m.p. 255-257 ºC. H NMR
(CDCl3): δ 8.97 (1H, s, triazolo-H), 7.83 (1H, d, J = 4.68 Hz,
pyrazine-H), 7.55 (1H, d, J = 4.68 Hz, pyrazine-H), 7.28-7.39
(3H, m, -C6H3). IR (KBr, νmax, cm-1): 1623 (C=N), 1254, 1030
(C-O-C), 1125 (N-N). MS m/z: 281 (M + 1).
8-(3-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyrazine (3j):
Yield: 83 %; m.p. 142-144 ºC. 1H NMR (CDCl3): δ 8.97 (1H,
s, triazolo-H), 7.82 (1H, d, J = 4.70 Hz, pyrazine-H), 7.38
(1H, d, J = 4.70 Hz, pyrazine-H), 7.09-7.34 (4H, m, -C6H4),
2.42 (3H, s, -CH3). IR (KBr, νmax, cm-1): 1622 (C=N), 1251,
1032 (C-O-C), 1126 (N-N). MS m/z: 227(M + 1).
Pharmacology: The maximal electroshock test and
rotarod test were carried out according to procedures described
in the Antiepileptic Drug Development Program (ADD) of
the National Institutes of Health (USA)11,12. All compounds,
which were dissolved in polyethylene glycol-400, were evalu-
ated for anticonvulsant activities in KunMing mice in the 18-
25 g weight range. Groups of 10 mice were given a range of
intraperitoneal doses of the test drug until at least three points
were established in the range of 10-90 % seizure protection or
minimal observed neurotoxicity. From the plots of these data,
the respective ED50 and TD50 values, 95 % confidence intervals,
slopes of the regression line and the standard error of the slopes
were calculated by computer program written by the National
Institute of Neurological Disorders and Stroke.
8-(4-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyrazine(3k):
Yield: 82 %; m.p. 224-226 ºC. 1H NMR (CDCl3): δ 8.94 (1H,
s, triazolo-H), 7.78 (1H, d, J = 4.71 Hz, pyrazine-H), 7.37
(1H, d, J = 4.71 Hz, pyrazine-H), 7.18-7.30 (4H, m, -C6H4),
2.40 (3H, s, -CH3). IR (KBr, νmax, cm-1): 1622 (C=N), 1251,
1032 (C-O-C), 1127 (N-N). MS m/z: 227(M + 1).
8-(2-Methoxyphenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3l):Yield: 77 %; m.p. 174-176 ºC. 1H NMR (CDCl3): δ 8.03
(1H, s, triazolo-H), 7.87 (1H, d, J = 4.70 Hz, pyrazine-H),
7.35 (1H, d, J = 4.70 Hz, pyrazine-H), 7.02-7.30 (4H, m,
-C6H4), 3.75 (3H, s, -OCH3). IR (KBr, νmax, cm-1): 1623 (C=N),
1252, 1030 (C-O-C), 1126 (N-N). MS m/z: 243 (M + 1).
8-(4-Methoxyphenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3m):Yield: 74 %; m.p. 170-172 ºC. 1H NMR (CDCl3): δ 8.96
(1H, s, triazolo-H), 7.80 (1H, d, J = 4.60 Hz, pyrazine-H),
7.38 (1H, d, J = 4.60 Hz, pyrazine-H), 6.98-7.27 (4H, m,
-C6H4), 3.85 (3H, s, -OCH3). IR (KBr, νmax, cm-1): 1623 (C=N),
1254, 1033 (C-O-C), 1127 (N-N). MS m/z : 243(M + 1).
8-(2-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3n): Yield: 61 %; m.p. 172-174 ºC. 1H NMR (CDCl3): δ 9.00
(1H, s, triazolo-H), 7.86 (1H, d, J = 4.71 Hz, pyrazine-H),
7.35 (1H, d, J = 4.71 Hz, pyrazine-H), 7.25-7.54 (4H, m,
-C6H4). IR (KBr, νmax, cm-1): 1622 (C=N), 1253, 1033
(C-O-C), 1126 (N-N). MS m/z: 247(M + 1).
Maximal electroshock test: Seizures were elicited with
a 60 Hz alternating current of 50 mA intensity in mice. The
current was applied via corneal electrodes for 0.2 s. Protection
against the spread of maximal electroshock-induced seizures
was defined as the abolition of the hind leg and tonic maximal
extension component of the seizure. The derivatives in maximal
electroshock test were evaluated 15 min after administration
of the compounds.
Rotarod test: After 15 min administration of the comp-
ounds (with different doses), the animals were tested on a 2.5
cm diameter knurled plastic rod rotating at 6 rpm for 1 min.
Neurotoxicity was indicated by the inability of an animal to
maintain equilibrium in each of three trials13.
Pentylenetetrazole-induced seizures: The doses used
were: pentylenetetrazole, 85 mg/kg. The test compounds and
standard AEDs were administered i.p. in a volume of 100 mg/
kg to groups of 10 mice 0.5 h before s.c. injection of pentyl-
enetetrazole. The mice were placed in individual cages and
observed for 0.5 h, the number of clonic seizures, tonic seizures
and the lethality were recorded14.
8-(3-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3o): Yield: 65 %; m.p. 194-196 ºC. 1H NMR (CDCl3): δ 8.97
(1H, s, triazolo-H), 7.83 (1H, d, J = 4.70 Hz, pyrazine-H),
7.38 (1H, d, J = 4.70 Hz, pyrazine-H), 7.23-7.45 (4H, m,
-C6H4). IR (KBr, νmax, cm-1): 1622 (C=N), 1251, 1030
(C-O-C), 1127 (N-N). MS m/z : 247 (M + 1).
8-(4-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3p): Yield: 70 %; m.p. 250-252 ºC. 1H NMR (CDCl3): δ 9.47
(1H, s, triazolo-H), 8.32 (1H, d, J = 4.74 Hz, pyrazine-H),
7.48 (1H, d, J = 4.74 Hz, pyrazine-H), 7.34-7.57 (4H, m,
-C6H4). IR (KBr, νmax, cm-1): 1621 (C=N), 1250, 1031
(C-O-C), 1126 (N-N). MS m/z : 247 (M + 1).
RESULTS AND DISCUSSION
Target compounds 3a-s was prepared by three-step
synthesis.As illustrated in Scheme-I, in the first step, the starting
material 2,3-dichloropyrazine reacted with hydrazine hydrate
in ethanol to afford10 compound 1. The second step, compound
1 reacted further with formic acid in ethanol to obtain compound
2. Finally, compound 2 reacted with appropriate alcohol and
substituted phenol to produce the target compounds 3a-s7. All
the compounds were identified by spectral data. In general,
IR spectra showed the C=N peak at 1624-1621 cm-1 and the
N-N peak at 1128-1124 cm-1. In the nuclear magnetic resonance
spectra (1H NMR) the signals of the respective protons of the
synthesized compounds were verified on the basis of their chemi-
cal shifts, multiplicities and coupling constants. The spectra
showed the triazolo-H proton as a singlet at 8.03-9.47 ppm.
8-(4-Fluorophenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
(3q): Yield: 58 %; m.p. 253-255 ºC. 1H NMR (CDCl3): δ 9.46
(1H, s, triazolo-H), 8.31 (1H, d, J = 4.74 Hz, pyrazine-H),
7.42 (1H, d, J = 4.74 Hz, pyrazine-H), 7.32-7.40 (4H, m,
-C6H4). IR (KBr, νmax, cm-1): 1622 (C=N), 1252, 1033
(C-O-C), 1127 (N-N). MS m/z: 231 (M + 1).
8-(4-Bromophenoxy)-[1,2,4]triazolo[4,3-a]pyrazine
1
(3r): Yield: 72.1 %; m.p. 251-253 ºC. H NMR (CDCl3): δ
8.98 (1H, s, triazolo-H), 8.13 (1H, d, J = 4.72 Hz, pyrazine-
H), 7.37 (1H, d, J = 4.72 Hz, pyrazine-H), 7.21-7.62 (4H, m,

Vol. 25, No. 7 (2013)
Synthesis and Studies on Anticonvulsant Activity of 8-Alkoxy-[1,2,4]triazolo[4,3-a]pyrazine 3663
N
N
N
N
N
N
Cl
Cl
O
N
Cl
i
ii
Cl iii
N
R
N
N
N
N
NHNH2
3a-s
2
1
R
3a = -CH3
3b = -C2H5
3e = -C5H11
3f = -C6H13
3g = -C7H15
3h = -C8H17
3i = 2-C6H4CH3
3j = 3-C6H4CH3
3m = 4-C6H4OCH3
3n = 2-C6H4Cl
3o = 3-C6H4Cl
3p = 4-C6H4Cl
3q = 4-C6H4F
3r = 4-C6H4Br
3s = 2,4-C6H3Cl2
–
3c = -CH(CH3)2
3d = -C4H9
3k = 4-C6H4CH3
3l = 2-C6H4OCH3
Reagents:(i) NH2NH2·H2O/C2H5OH, reflux; (ii) HCOOH/C2H5OH, reflux; (iii) alkanol or phenol, DMSO, NaOH or K2CO3.
Scheme-I: Synthesis route of compounds 3a-s
TABLE-1
PRIMARY EVALUATION OF COMPOUNDS I
Pharmacology: The anticonvulsant activity and neuroto-
xicity of the synthesized compounds 3a-s were carried out
AND 3a-s IN ANTICONVULSANT ACTIVITY
according to procedures described in the Antiepileptic Drug
(TEST DRUG ADMINISTERED i.p.)
O
Development Program (ADD) of the National Institutes of
Health (USA)15,16
N
O
N
R
.
N
The initial evaluation of the anticonvulsant activity of
synthesized compounds 3a-s (Table-1) indicated that thirteen
compounds displayed differently anticonvulsant activity, while
compounds 3d, 3f possessed anticonvulsant activity against
MES-induced seizure at 30 mg/kg, compound 3g were active
at 100 mg/kg. The rotarod toxicity test result indicated that all
compounds did not show toxicity at 100 mg/kg.
N
N
N
N
N
I
3a-s
MESa
Rotarodb
0.5 h
Dosage
(mg/kg)
Comp.
R
0.5 h
1/3
0/3
0/3
0/3
1/3
1/3
1/3
1/3
1/3
0/3
0/3
1/3
0/3
1/3
1/3
1/3
1/3
1/3
1/3
1/3
4 h
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
4 h
0/3c
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
-C6H5
-CH3
-C2H5
300
300
300
300
30
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
I
3a
3b
3c
3d
3e
3f
In the phase-II pharmacology test, compounds 3d and 3f
were quantitatively evaluated for anticonvulsant activity
(indicated by ED50) and neurotoxicity (indicated by TD50)
(Table-2) by intraperitoneal (i.p.) administration. The most
promising compounds 3d and 3f showed a median effective
dose of 44.0 and 35.3 mg/kg and had protective index value
of 3.2 and 4.8, respectively, in which had better than the leading
compound I (the dose of 300 mg/ kg).
-CH(CH3)2
-C4H9
-C5H11
-C6H13
-C7H15
-C8H17
300
30
100
300
300
300
300
300
300
300
300
300
300
300
300
3g
3h
3i
2-C6H4CH3
3-C6H4CH3
4-C6H4CH3
3j
3k
3l
To further investigate the effect of anticonvulsant activity
in chemical induced model, all compounds were tested against
convulsions induced by chemical substances pentylenetetrazol.
Compounds 3a-s were administered (i.p.) into mice at 100
mg/kg. The reference drug carbamazepine was administered
(i.p.) at 50 mg/kg.
2-C6H4OCH3
4-C6H4OCH3
2-C6H4Cl
3m
3n
3o
3p
3q
3r
3s
3-C6H4Cl
4-C6H4Cl
4-C6H4F
As shown in Table-3, all compounds completely or partly
inhibited the clonic seizures induced by sc-pentylenetetrazol,
but the reference drug carbamazepine did not show any
inhibition activity. Whereas compounds 3d, 3i, 3j and 3o
inhibited the tonic seizures and death induced by pentylene-
tetrazol at values of 100 and 100 %, respectively, in which
was similarly to the reference drug carbamazepine. Pentylenete-
trazol have been reported to produce seizures by inhibiting
4-C6H4Br
2,4-C6H3Cl2
aMaximal electroshock seizure test (number of animals
b
protected/number of animals tested). Rotorod toxicity (number of
animals exhibiting toxicity/number of animals tested). cCompounds
are metabolized/excreted at 4 h.
aminobutyric acid (GABA) neurotransmission17,18. GABA is
the main inhibitory neurotransmitter in the brain and is widely
TABLE-2
PHASE-II QUANTITATIVE ANTICONVULSANT DATA IN MICE
(TEST DRUG ADMINISTERED VIA THE INTRAPERITONEAL ROUTE)
a
PIc (TD50/ED50)
b
Compound
MES, ED50
TD50
44.0 (36.7-52.8) d
35.3 (28.3-44.1)
21.8 (21.8–25.5)
141.4 (117.8-169.7)
169.7 (141.4-203.7)
69 (62.8-72.9)
3.2
4.8
3.2
3d
3f
Phenobarbital
aED50: Median effective dose affording anticonvulsant protection in 50 % of animals, the dose is measured in mg/kg. bTD50: Median toxic dose
eliciting minimal neurological toxicity in 50 % of animals, the dose is measured in mg/kg. cPI: Protective index (TD50/ED50). d95 % confidence
intervals given in parentheses.

3664 Guan et al.
Asian J. Chem.
TABLE-3
EFFECT OF COMPOUNDS I AND 2a-s ON
PENTYLENETETRAZOL (PTZ) CONVULSION IN MICE
electroshock test, sc-PTZ. The only two compounds had better
anticonvulsant activity. But all compounds showed antagonistic
activity against seizures induced by pentylenetetrazol. These
experiments suggested that compound may activate GAD or
inhibit GABA-T, thereby enhancing GABAergic neurotrans-
mission.
Test
time seizures seizures
Clonic
Tonic
Dosage
(mg/kg)
Lethality
(%)
Compound
(h)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
(%)
0
(%)
100
100
100
60
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
–
100
100
60
0
I
0
3a
ACKNOWLEDGEMENTS
0
3b
0
3c
This work was supported by the National Natural Science
Foundation of China (No. 30960458).
0
0
0
3d
0
30
0
3e
60
30
30
0
100
60
0
3f
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0
3g
60
0
3h
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0
0
3i
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3j
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3k
3l
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3m
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Conclusion
In the present study, we synthesized a series of 8-alkoxy-
[1,2,4]triazolo[4,3-a]pyrazine derivatives and tested their
anticonvulsant activity and neurotoxicity using the maximal