Synthesis and studies on the anticonvulsant activity of 5-alkoxy-[1,2,4]triazolo[4,3-a]pyridine derivatives

Article abstract of DOI:10.1055/s-0032-1314821

In this study, a series of new 5-alkoxy-[1,2,4]triazolo[4,3-a]pyridine derivatives was synthesized and their anticonvulsant activity and neurotoxicity was evaluated with the maximal electroshock and rotarod tests, respectively. The most promising compounds, 3p (5-(4-chlorophenoxy)-[1,2,4]triazolo[4,3-a] pyridine) and 3r (5-(4-bromophenoxy)-[1,2,4]triazolo[4,3-a]pyridine), showed a median effective dose of 13.2 and 15.8 mg/kg and had a protective index value of 4.8 and 6.9, respectively. For exploring the putative mechanism of action, compounds 3n, 3p and 3r were tested in chemically induced models. Georg Thieme Verlag KG Stuttgart.New York.

Full text of DOI:10.1055/s-0032-1314821

372 Original Article
Synthesis and Studies on the Anticonvulsant Activity
of 5-alkoxy-[1,2,4]triazolo[4,3-a]pyridine Derivatives
Authors
L.-P. Guan1, R.-P. Zhang2, Y. Sun1, Y. Chang2, X. Sui3
Affiliations
1 School of Food, Drug & Medicine Zhejiang Ocean University, Zhejiang, Zhoushan, China
2 College of Pharmacy, Yanbian University, Yanji, Jili, China
3 Qilu Pharmaceutical L CO.,LTD. Jinan City, China
Key words
Abstract
rophenoxy)-[1,2,4]triazolo[4,3-a]pyridine) and
▶
●
[1,2,4]triazolo[4,3-a]pyri-
dine
3r (5-(4-bromophenoxy)-[1,2,4]triazolo[4,3-a]
pyridine), showed a median effective dose of
13.2 and 15.8mg/kg and had a protective index
value of 4.8 and 6.9, respectively. For exploring
the putative mechanism of action, compounds
3n, 3p and 3r were tested in chemically induced
models.
▼
In this study, a series of new 5-alkoxy-[1,2,4]
triazolo[4,3-a]pyridine derivatives was syn-
thesized and their anticonvulsant activity and
neurotoxicity was evaluated with the maximal
electroshock and rotarod tests, respectively.
The most promising compounds, 3p (5-(4-chlo-
▶
synthesis
anticonvulsant activity
chemically induced model
●
▶
●
▶
●
Introduction
sant activity. A conjugation effect increased the
electron cloud density at the triazole ring because
of the lone-pair electrons of the oxygen atom at
the 5-alkoxy moiety. This in turn increased the
combination ability of compounds 3a–3s to the
receptor. The pharmacology results indicated that
the most promising compounds 3n (5-(2-chlo-
▼
Epilepsy, one of the most common neurologic
diseases, is characterized by epileptic seizures,
which are evoked by unexpected, high-level neu-
ronal discharges in the brain [1]. Anticonvulsant
drugs currently on the market are of unsatisfac-
tory effectiveness in seizure control and cause
adverse reactions such as drowsiness, ataxia, gas-
trointestinal disturbance, hepatotoxicity and
megaloblastic anemia [2–6] and even life-threat-
ening conditions [7]. Therefore, the search for
safer and more effective antiepileptic drugs [2,3]
is of high importance. The development of new
antiepileptic drugs with the desired therapeutic
properties, however, has been a challenge for
medicinal chemists.
rophenoxy)-[1,2,4]triazolo[4,3-a]pyridine),
3p
(5-(4-chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyri-
dine) and 3r (5-(4-bromophenoxy)-[1,2,4]
triazolo[4,3-a]pyridine) showed median effective
doses of 19.0, 13.2 and 15.8 mg/kg- and had pro-
tective index values (PI) of 4.8, 4.8 and 6.9, respec-
tively, which is much better than the PI value of
the reference drug phenobarbital.
The structures of the new derivatives were char-
acterized using IR, 1H NMR, 13C NMR, MS and
elemental analysis techniques. Their anticonvul-
sant activity, reported for the first time, was eval-
uated using the MES test and the neurotoxicity
was evaluated using the rotarod test in mice. In
this contribution, in order to explain the possible
mechanism of action, compounds 3n, 3p and 3r
were tested in the pentylenetetrazol (PTZ), isoni-
azid (ISN), 3-mercaptopropionic acid (3-MP) and
thiosemicarbazide tests. The anti-MES activity
and neurotoxicity of the marketed agent pheno-
barbital was evaluated as a positive control.
received 31.12.2011
accepted 02.05.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1314821
Published online:
July 10, 2012
Arzneimittelforschung 2012;
62: 372–377
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
As part of our program directed towards the
search for central nervous system (CNS) agents
and in order to discover a new anticonvulsant
compound with improved activity, we synthe-
sized compound I (5-phenoxy-[1,2,4]-triazolo
▶
[4,3-a]pyridine) ( Fig. 1), which showed anti-
●
Correspondence
L.-P. Guan
School of Food, Drug &
Medicine
Zhejiang Ocean University
Qixiangtai Road
316004 Zhoushan
China
convulsant activity at an effective dose of 300mg/
kg in the maximal electroshock (MES) test. To
obtain further compounds with better anticon-
vulsant activity, we designed and synthesized a
new series of 5-alkoxy-[1,2,4]triazolo[4,3-a]pyri-
dine derivatives (3a–3s) using compound I as the
lead compound and investigated the contribution
of different alkoxyl groups at position 5 of the
[1,2,4]triazolo[4,3-a]pyridine to the anticonvul-
Tel.: 86/580/2554 536
Fax: 86/580/2554 781
glp730@yahoo.com.cn
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377

Original Article 373
cm-1: 1643 (C=N), 1284, 1061 (C-O-C), 1193 (N-N). MS m/z:
150 (M+1). Anal. calculated for C7H7N3O: C, 56.37; H, 4.73; N,
28.17. Found: C, 56.25; H, 4.65; N, 28.25.
Fig. 1 The structure
of compounds I and
3a–3s.
R
O
N
O
N
N
N
N
N
I
3a–3s
5-Ethoxy-[1,2,4]triazolo[4,3-a]pyridine (3b)
mp 94–96°C, yield=79%. 1H-NMR (CDCl3, 300 MHz) δ 8.88 (1H,
s, triazolo-H), 7.38 (1H, d, J=9.09Hz, 7.22Hz, pyridine-H), 7.30
(1H, dd, J=7.22Hz, 9.09Hz, pyridine-H), 6.02 (1H, d, J=7.20Hz,
7.22Hz, pyridine-H), 4.36 (2H, p, -OCH2), 1.59 (3H, t, -CH3). IR
(KBr) cm-1: 1642 (C=N), 1294, 1060 (C-O-C), 1190 (N-N). MS
m/z: 164 (M+1). Anal. calculated for C8H9N3O: C, 58.88; H, 5.56;
N, 25.75. Found: C, 58.75; H, 5.42; N, 25.60.
Materials and Methods
▼
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) on a
FT-IR1730 apparatus (Bruker), 1H-NMR and 13C-NMR were
measured on an AV-300 spectrometer (Bruker). All chemical
shifts were given in ppm relative to tetramethysilane. Mass
spectra were measured on an HP1100LC spectrometer (Agilent
Technologies). Elemental analysis (CHN) was performed on a
Perkin Elmer 204Q CHN or a Heraeus CHN Rapid Analyzer. The
majority of the chemicals was purchased from Aldrich Chemical
Corporation. All other chemicals were of analytical grade.
5-Isopropoxy-[1,2,4]triazolo[4,3-a]pyridine (3c)
mp 90–92°C, yield=81%. 1H-NMR (CDCl3, 300 MHz) δ 8.86 (1H,
s, triazolo-H), 7.42 (1H, d, J=9.06Hz, 7.29Hz, pyridine -H), 7.32
(1H, dd, J=7.29Hz, 9.06Hz, pyridine-H), 6.06 (1H, d, J=7.26Hz,
7.29Hz, pyridine-H), 4.86 (1H, m, -OCH), 1.54 (3H, s, -CH3), 1.52
(3H, s, -CH3). IR (KBr) cm−1: 1639 (C=N), 1282, 1045 (C-O-C),
1189 (N-N). MS m/z: 178 (M+1). Anal. calculated for C9H11N3O:
C, 61.00; H, 6.26; N, 23.71. Found: C, 59.87; H, 6.38; N, 23.62.
Synthesis of 1-hydrazino-6-chroropyridine (1)
To a solution of hydrazine hydrate (3.04g, 62.8mmol) in 20mL
ethanol, a solution of 2,6-dichloropyridine (1.85g, 12.6mmol) in
30mL ethanol was added dropwise at room temperature. The
mixture was stirred and refluxed for 1h. Then, half of the solvent
was removed under reduced pressure and the solution was
combined with petroleum ether. The precipitate was filtered
and washed with petroleum ether, then kept below 0°C. The
resulting crude compound was purified by recrystallization in
EtOH [8].
5-Butoxy-[1,2,4]triazolo[4,3-a]pyridine (3d)
mp 28–30°C, yield=74%. 1H-NMR (CDCl3, 300 MHz) δ 8.87 (1H,
s, triazolo-H), 7.39 (1H, d, J=9.09Hz, 7.23Hz, pyridine -H), 7.28
(1H, dd, J=7.23Hz, 9.09Hz, pyridine-H), 6.02 (1H, d, J=7.20Hz,
7.23Hz, pyridine-H), 4.28 (2H, t, -OCH2), 1.56–1.96 (4H, m,
-(CH2)2), 1.04 (3H, t, -CH3). IR (KBr) cm−1: 1643 (C=N), 1283,
1053 (C-O-C), 1193 (N-N). MS m/z: 192 (M+1). Anal. calculated
for C10H13N3O: C, 62.81; H, 6.85; N, 21.97. Found: C, 62.69; H,
6.77; N, 21.83.
Synthesis of 5-chloro-[1,2,4]triazolo[4,3-a]pyridine (2)
Compound 1 (4mmol), formic acid (4mmol) and 30mL ethanol
were placed in a 50mL round-bottom flask. The mixture was
stirred for 2h at room temperature to obtain the acid intermedi-
ate. The mixture was then evaporated under reduced pressure
and the acid intermediate was reacted byrefluxing in DMF for
3h. The solution was evaporated to dryness and the oily residue
was filtered on a silica gel chromatographic column (ethyl ace-
tate) to give a light yellow or white solid [8]. Yield=87%. 1H-
NMR (CDCl3, 300MHz ) δ: 8.97 (1H, s, triazolo-H), 7.77 (1H, d,
J=9.24Hz, 7.05Hz, pyridine-H), 7.28 (1H, dd, J=7.05Hz, 9.24Hz,
pyridine-H), 6.93 (1H, d, J=6.99Hz, 7.05Hz, pyridine-H). MS
(M+1): 154.
5-Pentyloxy-[1,2,4]triazolo[4,3-a]pyridine (3e)
Oil, yield=78%. 1H-NMR (CDCl3, 300MHz): δ 8.88 (1H, s, tria-
zolo-H), 7.42 (1H, d, J=9.16Hz, 7.25Hz, pyridine-H), 7.28 (1H,
dd, J=7.25Hz, 9.16Hz, pyridine-H), 6.02 (1H, d, J=7.20Hz,
7.25Hz, pyridine-H), 4.31 (2H, t, -OCH2), 1.43–1.96 (6H, m,
-(CH2)3), 0.98 (3H, t, -CH3). IR (KBr) cm−1: 1641 (C=N), 1281,
1021 (C-O-C), 1189 (N-N). MS m/z: 206 (M+1). Anal. calculated
for C11H15N3O: C, 64.37; H, 7.37; N, 20.47. Found: C, 64.28; H,
7.26; N, 20.39.
5-Hexyloxy-[1,2,4]triazolo[4,3-a]pyridine (3f)
Oil, yield=73%. 1H-NMR (CDCl3, 300 MHz): δ 8.88 (1H, s, tria-
zolo-H), 7.40 (1H, d, J=9.18Hz, 7.21Hz, pyridine-H), 7.36 (1H,
dd, J=7.21Hz, 9.18Hz, pyridine-H), 6.02 (1H, d, J=7.23Hz,
7.21Hz, pyridine-H), 4.28 (2H, t, -OCH2), 1.25–1.99 (8H, m,
-(CH2)4), 0.95 (3H, t, -CH3). IR (KBr) cm−1: 1639 (C=N), 1283,
1054 (C-O-C), 1193 (N-N). MS m/z: 220 (M+1). Anal. calculated
for C12H17N3O: C, 65.73; H, 7.81; N, 19.16. Found: C, 65.62; H,
7.76; N, 19.01.
General protocol for the synthesis of 8-alkoxy-[1,2,4]
triazolo[4,3-a]pyrazine (3a–3s)
Compound 2 (0.5 g, 3.2 mmol) and either alkanol or substituted
phenol (3.2 mmol) were added to a solution of sodium hydrox-
ide (3.2 mmol) or K2CO3 (3.2 mmol) in DMF followed by stirring
and refluxing for 3–5h. After removal of the solvent under
reduced pressure, the solid residue was purified by silica gel col-
umn chromatography with dichloromethane: methanol (20:1).
The yield, melting point and spectral data for each compound
are given below [9,10].
5-Heptyloxy-[1,2,4]triazolo[4,3-a]pyridine (3g)
mp 30–32°C, yield=77%. 1H-NMR (CDCl3, 300 MHz ): δ 8.87 (1H,
s, triazolo-H), 7.39 (1H, d, J=9.19Hz, 7.21Hz, pyridine -H), 7.28
(1H, dd, J=7.21Hz, 9.19Hz, pyridine-H), 6.01 (1H, d, J=7.22Hz,
7.23Hz, pyridine-H), 4.27 (2H, t, -OCH2), 1.34–1.97 (10H, m,
-(CH2)5), 0.91 (3H, t, -CH3). IR (KBr) cm−1: 1642 (C=N), 1282,
1054 (C-O-C), 1191 (N-N). MS m/z: 234 (M+1). Anal. calculated
for C13H19N3O: C, 66.92; H, 8.21; N, 18.01. Found: C, 66.82; H,
8.15; N. 18.16.
5-Methoxy-[1,2,4]triazolo[4,3-a]pyridine (3a)
mp 108–110°C, yield=80%. 1H-NMR (CDCl3, 300 MHz) δ 8.88
(1H, s, triazolo-H), 7.41 (1H, d, J=9.16Hz, 7.24Hz, pyridine-H),
7.29 (1H, dd, J=7.24Hz, 9.16Hz, pyridine-H), 6.04 (1H, d,
J=7.21Hz, 7.24Hz, pyridine-H), 4.13 (3H, s, -OCH3 ). IR (KBr)
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377

374 Original Article
5-Octyloxy-[1,2,4]triazolo[4,3-a]pyridine (3h)
m/z: 246(M+1). Anal. calculated for C12H8ClN3O: C, 58.67; H,
mp 46–48°C, yield=81%. 1H-NMR (CDCl3, 300MHz): δ 8.87 (1H,
s, triazolo-H), 7.39 (1H, d, J=9.09Hz, 7.14Hz, pyridine -H), 7.28
(1H, dd, J=7.14Hz, 9.09Hz, pyridine-H), 6.01 (1H, d, J=7.20Hz,
7.14Hz, pyridine-H), 4.27 (2H, t, -OCH2), 1.30–1.99 (12H, m,
-(CH2)6), 0.90 (3H, t, -CH3). IR (KBr) cm−1: 1639 (C=N), 1281,
1061 (C-O-C), 1193 (N-N). MS m/z: 248 (M+1). Anal. calculated
for C14H21N3O: C, 67.98; H, 8.56; N, 16.99. Found: C, 67.80; H,
8.45; N, 16.78.
3.28; N, 17.10. Found: C, 58.54; H, 3.19; N, 17.01.
5-(3-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3o)
mp 138–140°C, yield=69%. 1H-NMR (CDCl3, 300MHz): δ 9.02
(1H, s, triazolo-H), 7.56 (1H, d, J=9.18Hz, 8.04Hz, pyridine-H),
7.16 (1H, dd, J=8.04Hz, 9.18Hz, pyridine-H), 6.02 (1H, d,
J=7.29Hz, 8.04Hz, pyridine-H), 7.24–7.48 (4H, m, -C6H4). IR
(KBr) cm−1: 1642 (C=N), 1281, 1060 (C-O-C), 1152 (N-N). MS
m/z: 246(M+1). Anal. calculated for C12H8ClN3O: C, 58.67; H,
3.28; N, 17.10. Found: C, 58, 59; H, 3.33; N, 17.20.
5-(2-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyridine (3i)
mp 116–118°C, yield=79%. 1H-NMR (CDCl3, 300MHz): δ 9.10
(1H, s, triazolo-H), 7.60 (1H, d, J=9.15Hz, 6.93Hz, pyridine-H),
7.17 (1H, dd, J=6.93Hz, 9.15Hz, pyridine-H), 5.85 (1H, d,
J=7.35Hz, 6.93Hz, pyridine -H), 7.26–7.39 (4H, m, -C6H4), 2.24
(3H, s, -CH3). IR (KBr) cm−1: 1643 (C=N), 1284, 1044 (C-O-C),
1153 (N-N). MS m/z: 226(M+1). Anal. calculated for C13H11N3O:
C, 69.32; H, 4.92; N, 18.66. Found: C, 69.41; H, 4.81; N, 18.78.
5-(4-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3p)
mp 155–157°C, yield=76%. 1H-NMR (CDCl3, 300MHz): δ 9.03
(1H, s, triazolo-H), 7.53 (1H, d, J=9.18Hz, 7.17Hz, pyridine-H),
7.23 (1H, dd, J=7.17Hz, 9.18Hz, pyridine-H), 5.95 (1H, d,
J=7.29Hz, 7.17Hz, pyridine-H), 7.19–7.50 (4H, m, -C6H4). 13C
NMR (CDCl3 ): δ151.0, 150.6, 146.8, 132.3, 131.9, 130.7, 128.8,
122.0, 121.8, 118.7, 109.5, 92.5. IR (KBr) cm−1: 1641 (C=N),
1284, 1024 (C-O-C), 1153 (N-N). MS m/z: 246(M+1). Anal. calcu-
lated for C12H8ClN3O: C, 58.67; H, 3.28; N, 17.10. Found: C, 58.71;
H 3.20; N 17.19.
5-(3-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyridine (3j)
mp 120–122°C, yield=75%. 1H-NMR (CDCl3, 300MHz): δ 9.04
(1H, s, triazolo-H), 7.57 (1H, d, J=9.09Hz, 7.21Hz, pyridine-H),
7.11 (1H, dd, J=7.21Hz, 9.09Hz, pyridine-H), 6.00 (1H, d,
J=7.35Hz, 7.65Hz, pyridine-H), 7.03–7.41 (4H, m, -C6H4), 2.42
(3H, s, -CH3). IR (KBr) cm−1: 1642 (C=N), 1281, 1054 (C-O-C),
1151 (N-N). MS m/z: 226 (M+1). Anal. calculated for C13H11N3O:
C, 69.32; H, 4.92; N, 18.66. Found: C, 69.23; H, 4.85; N, 18.55.
5-(4-Fluorophenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3q)
mp 168–170°C, yield=60%. 1H-NMR (CDCl3, 300MHz): δ 9.05
(1H, s, triazolo-H), 7.53 (1H, d, J=9.12Hz, 7.47Hz, pyridine-H),
7.21 (1H, dd, J=7.47Hz, 9.12Hz, pyridine-H), 5.89 (1H, d,
J=7.35Hz, 7.47Hz, pyridine-H), 7.17–7.27 (4H, m, -C6H4). IR
(KBr) cm−1: 1643 (C=N), 1294, 1050 (C-O-C), 1152 (N-N). MS
m/z: 230 (M+1). Anal. calculted for C12H8FN3O: C, 62.88; H, 3.52;
N, 18.33. Found: C, 62.79, H 3.46, N, 18.38.
5-(4-Tolyloxy)-[1,2,4]triazolo[4,3-a]pyridine (3k)
mp 124–126°C, yield=83%. 1H-NMR (CDCl3, 300MHz): δ 9.06
(1H, s, triazolo-H), 7.58 (1H, d, J=9.14Hz, 7.41Hz, pyridine-H),
7.33 (1H, dd, J=7.41Hz, 9.14Hz, pyridine-H), 5.98 (1H, d,
J=7.40Hz, 7.41Hz, pyridine-H), 7.18–7.34 (4H, m, -C6H4), 2.39
(3H, s, -CH3). IR (KBr) cm−1: 1642 (C=N), 1281, 1054 (C-O-C),
1153 (N-N). MS m/z: 226(M+1). Anal. calculated for C13H11N3O:
C, 69.32; H, 4.92; N, 18.66. Found: C, 69.25; H, 4.87; N, 18.49.
5-(4-Bromophenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3r)
mp 154–156°C, yield=74%. 1H-NMR (CDCl3, 300MHz): δ 9.03
(1H, s, triazolo-H), 7.52 (1H, d, J=9.30Hz, 7.50Hz, pyridine-H),
7.24 (1H, dd, J=7.50Hz, 9.30Hz, pyridine-H), 5.94 (1H, d,
J=7.20Hz, 7.50Hz, pyridine-H), 7.13–7.66 (4H, m, -C6H4). 13C
NMR (CDCl3 ): δ151.0, 150.6, 146.8, 132.3, 131.9, 130.7, 128.8,
122.0, 121.8, 118.7, 109.5, 92.5. IR (KBr) cm−1: 1642 (C=N),
1282, 1060 (C-O-C), 1151 (N-N). MS m/z: 289 (M+1). Anal. cal-
culated for C12H8BrN3O: C, 49.68; H, 2.78; N, 14.48. Found: C,
49.57; H, 2.65; N, 14.35.
5-(2-Methoxyphenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3l)
mp 118–120°C, yield=85%. 1H-NMR (CDCl3, 300MHz): δ 9.09
(1H, s, triazolo-H), 7.53 (1H, d, J=9.15Hz, 7.50Hz, pyridine-H),
7.23 (1H, dd, J=7.50Hz, 9.15Hz, pyridine-H), 5.83 (1H, d,
J=7.22Hz, 7.50Hz, pyridine -H), 7.06–7.35 (4H, m, -C6H4), 3.77
(3H, s, -OCH3). IR (KBr) cm−1: 1638 (C=N), 1282, 1040 (C-O-C),
1152 (N-N). MS m/z: 242 (M+1). Anal. calculated for C13H11N3O2:
C, 64.72; H, 4.60; N, 17.42. Found: C, 64.83; H, 4.51; N, 17.34.
5-(2,4-Dichlorophenoxy)-[1,2,4]triazolo[4,3-a]pyridine
(3s)
mp 144–146°C, yield=62%. 1H-NMR (CDCl3, 300MHz): δ 9.09
(1H, s, triazolo-H), 7.54 (1H, d, J=9.18Hz, 7.35Hz, pyridine-H),
7.23 (1H, dd, J=7.35Hz, 9.18Hz, pyridine-H), 5.81 (1H, d,
J=7.29Hz, 7.35Hz, pyridine-H), 7.19–7.60 (3H, m, -C6H3). IR
(KBr) cm−1: 1643 (C=N), 1284, 1020 (C-O-C), 1153 (N-N). MS
m/z: 280 (M+1). Anal. calculated for C12H7Cl2N3O: C, 51.45; H,
2.52; N, 15.00. Found: C, 51.34; H, 2.40; N, 15.09.
5-(4-Methoxyphenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3m)
mp 128–130°C, yield=82%. 1H-NMR (CDCl3, 300MHz): δ 9.06
(1H, s, triazolo-H), 7.53 (1H, d, J=9.09Hz, 7.59Hz, pyridine-H),
7.24 (1H, dd, J=7.59Hz, 9.09Hz, pyridine-H), 5.90 (1H, d,
J=7.35Hz, 7.59Hz, pyridine-H), 7.01–7.28 (4H, m, -C6H4), 3.86
(3H, s, -OCH3). IR (KBr) cm−1: 1639 (C=N), 1294, 1043 (C-O-C),
1151 (N-N). MS m/z: 242 (M+1). Anal. calculated for C13H11N3O2:
C, 64.72; H, 4.60; N, 17.42. Found: C, 64.65; H, 4.54; N, 17.35.
Pharmacology
The MES and rotarod tests were carried out according to the
standard described in the Antiepileptic Drug Development Pro-
gram (ADD) of the National Institutes of Health (USA) [11,12]. All
compounds were tested for anticonvulsant activity in KunMing
mice (weight 18–25g) purchased from the Laboratory of Animal
Research, College of Pharmacy, Yanbian University. The tested
compounds were dissolved in DMSO.
5-(2-Chlorophenoxy)-[1,2,4]triazolo[4,3-a]pyridine (3n)
mp 150–152°C, yield=74%. 1H-NMR (CDCl3, 300MHz): δ 9.11
(1H, s, triazolo-H), 7.53 (1H, d, J=9.17Hz, 7.19Hz, pyridine-H),
7.27 (1H, dd, J=7.19Hz, 9.17Hz, pyridine-H), 5.81 (1H, d,
J=7.31Hz, 7.19Hz, pyridine-H), 7.20–7.59 (4H, m, -C6H4). IR
(KBr) cm−1: 1642 (C=N), 1283, 1051 (C-O-C), 1153 (N-N). MS
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377

Original Article 375
The pharmacologic parameters estimated from the phase-I
Table 1 Phase-I evaluation of anticonvulsant activity in mice (i.p.).
screening were then quantified in the phase-II screening for
RO
▶
compounds 3d, 3n–3p and 3r–3s ( Table 2). The anticonvul-
●
sant activity was expressed as the median effective dose (ED50
)
N
and neurotoxicity as the median toxic dose (TD50). For determi-
nation of the ED50 and TD50 values, a dose range of the test com-
pounds was administered intraperitoneally (i.p.) to groups of 10
mice until at least three points with 10–90% seizure protection
or minimal observed neurotoxicity were measured. By plotting
these data, the respective ED50 and TD50 values, 95% confidence
intervals, slope of the regression line, and the standard error of
the slope were calculated by means of a computer program
written at the National Institute of Neurological Disorders and
N
N
Compound
R
Dosage (mg/kg)
MESa
0.5h
4h
I
- C 6H5
300
300
300
100
30
1/3
2/3
2/3
3/3
2/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
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
0/3
3a
3b
3c
3d
3e
3f
n-CH3
n-C2H5
n-CH(CH3)2
n-C4H9
Stroke.
n-C5H11
100
100
100
100
100
100
300
100
100
30
▶
n-C6H13
In the case of chemically induced seizures ( Table 3), mice
●
3g
3h
3i
n-C7H15
were given doses of the compounds that could induce seizures in
at least 97% of the animals. The doses used were: PTZ, 85mg/kg;
ISN, 250mg/kg; 3-MP, 40mg/kg and thiosemicarbazide, 100mg/kg.
The test compounds and standard AEDs were administered i.p.
at 50mg/kg to groups of 10 mice 1h before either i.p. adminis-
tration of ISN and thiosemicarbazide or s.c. injection of PTZ and
3-MP. The mice were placed in individual cages and observed for
30min. The number of clonic seizures, tonic seizures and the
lethality were recorded [13,14]. Thiosemicarbazide was also
administered i.p., and in this case the test compounds were
given to mice i.p. 30min prior to thiosemicarbazide administra-
tion. The mice were placed in individual cages and observed for
2h and the number of clonic seizures, tonic seizures and the
lethality were noted [15].
n-C8H17
2 - C 6H4CH3
3 - C 6H4CH3
4 - C 6H4CH3
2 - C 6H4OCH3
4 - C 6H4OCH3
2 - C 6H4Cl
3 - C 6H4Cl
4 - C 6H4Cl
4 - C 6H4F
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
30
30
100
30
4 - C 6H4Br
2,4-C6H3Cl2
30
a Maximal electroshock test (number of animals protected/number of animals
tested), the number of mice was 3
Results and Discussion
Table 2 Phase-II based, quantitative anticonvulsant data in mice (test drug
administered i.p.).
▼
Synthesis of the new derivatives was carried out starting from
Rotarod Toxicity
▶
●
2,6-dichloropyridine according to Fig. 2. 2,6-Dichloropyridine
b
MES, EDa
TD50
PIc (TD50/ED50
)
Compound
50
reacted with hydrazine hydrate in ethanol to afford compound
1[8]. In a next step, compound 1 reacted with formic acid in
ethanol to obtain compound 2, which in turn reacted with the
appropriate alcohol or substituted phenol to yield the target
compounds 3a–3s [9,10]. All compounds were identified based
on their spectral data. In general, the IR spectra showed the C=N
3d
3n
3o
3p
3r
32.9 (28.0–38.6)d 126.8 (108.1–148.8) 3.9
19.0 (16.2–22.3)
22.8 (19.4–26.8)
13.2 (11.3–15.5)
91.3 (77.8–107.1)
93.0 (79.2–109.2)
63.4 (54.0–74.4)
4.8
4.1
4.8
6.9
2.8
3.2
15.8 (13.5–18.5) 109.5 (93.3–128.5)
3s
27.4 (23.4–32.1)
76.1 (64.9–89.3)
69 (62.8–72.9)
peak at 1638–1643cm−1 and the N-N peak at 1151–1193cm−1
.
Phenobarbital 21.8 (21.8–25.5)
In the nuclear magnetic resonance spectrum (1H-NMR), the sig-
nals of the respective protons of the synthesized compounds
were verified on the basis of their chemical shifts, multiplicities
and coupling constants. The spectra showed the triazolo-H pro-
ton as a singlet at 8.86–9.10ppm.
a ED50-median effective dose required to assure anticonvulsant protection in 50% of
the animals, the number of mice was 10
b T D 50-median toxic dose eliciting minimal neurological toxicity in 50% of the
animals
c PI protective index (TD50/ED50
)
d 95% confidence limits given in parentheses
The anticonvulsant activity and neurotoxicity of the synthesized
compounds 3a–3s were measured according to the procedures
described in the Antiepileptic Drug Development Program
(ADD) of the National Institutes of Health (USA) [11,12]. The
▶
For the phase-I screening ( Table 1), each compound was
●
administered at 30, 100, 300mg/kg for evaluating the anticon-
vulsant activity. The neurotoxicity was measured at 30min and
4h after administration. The anticonvulsant activity was meas-
ured with the MES test for which seizures were elicited in mice
with a 60Hz alternating current of 50 mA. The current was
applied via corneal electrodes for 0.2s. Protection against the
propagation of the MES-induced seizures was defined as the
abolition of the hind leg and tonic maximal extension compo-
nent of the seizure. Neurologic deficits caused by the compounds
were detected with the rotorod ataxia test.
results from the pharmacology tests for all synthesized com-
▶
pounds are shown in Table 1. All compounds showed anticon-
●
vulsant activity. The lead compound I (5-phenoxy-[1,2,4]
triazolo[4,3-a]pyridine) was active in the MES test only at the
high dose of 300mg/kg. 3 of the synthesized compounds (3a, 3b,
and 3k) exhibited an anti-MES effect at 300mg/kg, ten com-
pounds (3c, 3e–3j, 3l, 3m, and 3q) at 100mgkg, and 6 com-
pounds (3d, 3n–3p, 3r, and 3s) at 30mg/kg ( Table 1). However,
▶
●
none of these compounds was active for more than 4h following
administration. The higher activity may be explained by the
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377

376 Original Article
Table 3 E ffect of compounds 3n, 3p and 3r on PTZ-, ISN-, 3-MP-, and thiosemicarbazide-induced convulsion in mice.
Chemical substances
Compound
Doses (mg/kg)
Test time (h)
Clonic seizures (%)
Tonic seizures (%)
Lethality (%)
DMSO
–
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
100
70
100
0
40
0
carbamazepine
50
50
50
50
–
PTZ
3n
40
30
20
30
100
0
0
3p
30
0
3r
10
0
DMSO
100
50
75
0
carbamazepine
50
50
50
50
–
3n
30
50
30
50
100
0
0
ISN
3p
10
0
3r
50
0
DMSO
100
60
100
0
carbamazepine
50
50
50
50
–
3n
60
50
20
0
0
3-MP
3p
30
0
3r
100
100
50
0
DMSO
100
0
100
0
carbamazepine
50
50
50
50
thiosemicarbazide
3n
3p
3r
50
80
60
60
0
70
0
60
0
When assessing the influence of the position of the atomic Cl in
the phenyl ring, it was found that compounds with Cl in position
4 were the most active. Cl in position 2 rendered the compound
more active than Cl in position 3. A double-chlorinated com-
pound (positions 2 and 4) showed the weakest activity. Intro-
N
N
N
N
i
ii
iii
N
N
Cl
N
Cl
N
NHNH2
Cl
Cl
RO
1
2
3a–3s
ducing the Cl atom into the benzyl ring resulted in increased
R:
▶
activity ( Table 2) .
●
3a=n-CH3
3e=n-C5H11 3i=2-C6H5CH3
3f=n-C6H13 3j=3-C6H4CH3
3m=4-C6H4OCH3 3q=4-C6H4F
When comparing the influence of the electron-donor group on
the anticonvulsant activity, it was found that, from the 8 alkyl
chain substituted derivatives 3a–3h, only compound 3d exhib-
ited increased anticonvulsant activity, with an ED50 value of
32.9mg/kg and decreased neurotoxicity (TD50 value of 126.8mg/
3b=n-C2H5
3n=2-C6H4Cl
3r=4-C6H4Br
3c=n-CH(CH3)2 3g=n-C7H15 3k=4-C6H4CH3 3o=3-C6H4Cl
3d=n-C4H9 3h=n-C8H17 3l=2-C6H4OCH3 3p=4-C6H4Cl
3s=2.4-C6H3Cl2
Fig. 2 The synthesis of compounds 3a–3s. Reagents: (i) NH2NH2·H2O/
C2H5OH, reflux; (ii) HCOOH/C2H5OH, reflux; (iii) alkanol/DMSO, NaOH or
K2CO3.
kg). In addition, the results show the superior activity of aryloxy
▶
derivatives in comparison to alkoxy ones ( Table 2) .
●
To further investigate the anticonvulsant activity in different
models, compounds 3n, 3p and 3r were tested against convul-
sions induced by chemical substances, i.e., PTZ, ISN, 3-MP, and
thiosemicarbazide. Compounds 3n, 3p and 3r were adminis-
tered i.p. to mice at 50mg/kg,which corresponded to their
3×ED50, but was far below their TD50. The reference drug car-
bamazepine was administered i.p. at 50mg/kg. Compounds 3n,
3p and 3r inhibited PTZ induced clonic seizures by 60%, 70% and
presence of the triazole ring, which enhances the affinity of the
pyridine ring for the recognition site [16].
In the phase-II pharmacology test, 6 compounds were quantita-
tively evaluated for their anticonvulsant activity (indicated by
▶
ED50) and neurotoxicity (indicated by TD50) ( Table 2) follow-
●
ing i.p. administration. Based on the activities of compounds 3d,
3n–3p and 3r–3s, the following structure activity relationship
(SAR) was gained.
90%, tonic seizures by 70%, 80% and 70% and death by 100%,
▶
100% and 100%, respectively ( Table 3). While carbamazepine
●
Generally, the anticonvulsant activity of an organic compound
may be increased significantly by introducing a halogen atom.
Therefore, some halogen substituted derivatives were designed
and synthesized in this study. Comparison of the activity of the
various halogen substituted derivatives indicates that the halo-
showed inhibition values of 30%, 100% and 100%. Car-
bamazepine inhibited ISN induced clonic seizures, tonic seizures
and death by 50%, 100% and 100%, respectively, while inhibition
values of 70%, 90% and 50% (clonic seizures), of 50%, 70% and
50% (tonic seizures) and 100%, 100% and 100% (death) were
▶
gen atoms contribute to the anticonvulsant activity in the order
measured for compounds 3n, 3p and 3r ( Table 3). PTZ and ISN
●
▶
4-Cl>4-Br>4-F ( Table 2). The ED and PI for phenobarbital
●
were reported to produce seizures by inhibiting aminobutyric
acid (GABA) neurotransmission [17,18]. GABA is the main inhib-
itory neurotransmitter in the brain and has been widely impli-
cated in epilepsy. Inhibition of GABAergic neurotransmission or
activity has been shown to promote and facilitate seizures [19],
whereas enhancement of GABAergic neurotransmission is
known to inhibit or attenuate seizures. The findings of the
present study suggest that the newly synthesized compounds
50
were 21.8mg/kg and 3.2. Remarkably, three compounds, 3n, 3p
and 3r, namely, had lower ED50 (19.0, 13.2 and 15.8mg/kg,
respectively) values than phenobarbital and one of the com-
pounds, 3r, also had a higher PI of 6.9 than the control substance.
Compared with phenobarbital, compound 3r showed a better
anticonvulsant activity and a decreased neurotoxicity, which
indicates a certain safety margin for this compound.
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377

Original Article 377
3n, 3p and 3r may inhibit or attenuate PTZ- and ISN-induced
seizures in mice by enhancing GABAergic neurotransmission.
In the 3-MP-induced seizure model, carbamazepine inhibited
clonic seizures, tonic seizures and death by 40%, 100%, and
100%, respectively, while compounds 3n, 3p and 3r showed
inhibition values of 40%, 70% and 0% (clonic seizures), of 50%,
Conflicts of Interest
▼
We declare that we have no conflict of interest with respect to
this study.
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●
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Conclusion
▼
In conclusion, compounds 3n (5-(2-chlorophenoxy)-[1,2,4]
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highest PI value of the assayed compounds. Compounds 3n, 3p
and 3r showed antagonistic activity against seizures induced by
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▼
This work was supported by the National Natural Science Foun-
dation of China (No. 30960458) and the Natural Science Founda-
tion of Zhejiang Province of China (No. LY12C19005).
Guan L-P et al. [1,2,4]triazolo[4,3-a]pyridine Derivatives… Arzneimittelforschung 2012; 62: 372–377