Synthesis and anticonvulsant activity evaluation of 6-substituted-[1,2,4] triazolo[3,4-a](tetrazolo[5,1-a])phthalazine derivatives

Article abstract of DOI:10.3109/14756366.2012.684052

With the aim of finding new anticonvulsant drugs, new 6-substituted-[1,2,4] triazolo[3,4-a] (tetrazolo[5,1-a]) phthalazine derivatives (1-34) have been designed and synthesized. All the compounds were evaluated for their anticonvulsant activities using the maximal electroshock test (MES). Most of the synthesized compounds exhibited potent anticonvulsant activities in the MES. The most promising compound 14 showed significant anticonvulsant activity in MES test with ED50 value of 9.3 mg/kg. It displayed a wide margin of safety with protective index much higher than the standard drug Carbamazepine. And the potency of compound 14 against seizures induced by Pentylenetetrazole, Isoniazid, Thiosemicarbazide and 3-Mercaptopropionic acid in the chemical-induced seizure tests suggested that compound 14 displayed wide spectrum of activity in several models.

Full text of DOI:10.3109/14756366.2012.684052

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(4): 792–800
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.684052
RESEARCH ARTICLE
Synthesis and anticonvulsant activity evaluation of
6-substituted-[1,2,4]triazolo[3,4-a](tetrazolo[5,1-a])
phthalazine derivatives
Ming Bian1,2, Xian-Qing Deng1, Guo-Hua Gong1,2, Cheng-Xi Wei2 and Zhe-Shan Quan1
1College of Pharmacy, Yanbian University, Yanji City, Jilin, China and 2Institute of Pharmaceutical Chemistry and
Pharmacology, Inner Mongolia University for Nationalities, Inner Mongolia Autonomous Region, Tongliao, China
Abstract
With the aim of finding new anticonvulsant drugs, new 6-substituted-[1,2,4]triazolo[3,4-a] (tetrazolo[5,1-a])
phthalazine derivatives (1–34) have been designed and synthesized. All the compounds were evaluated for their
anticonvulsant activities using the maximal electroshock test (MES). Most of the synthesized compounds exhibited
potent anticonvulsant activities in the MES. The most promising compound 14 showed significant anticonvulsant
activity in MES test with ED50 value of 9.3 mg/kg. It displayed a wide margin of safety with protective index much
higher than the standard drug Carbamazepine. And the potency of compound 14 against seizures induced by
Pentylenetetrazole, Isoniazid, Thiosemicarbazide and 3-Mercaptopropionic acid in the chemical-induced seizure
tests suggested that compound 14 displayed wide spectrum of activity in several models.
Keywords: Triazole, Tetrazole, Phthalazine, Anticonvulsant, Maximal electroshock
Introduction
seizure tests (Figure 1)6–12. From the currently used AEDs,
Epilepsy, a ubiquitous disease characterized by recur-
rent seizures, afflicts more than 60 million people world-
wide according to epidemiological studies1. For epilepsy
treatment, nearly 95% of clinically available drugs were
approved before 1985 and they could provide satisfac-
tory seizure control for 60–70% of patients. ese drugs,
however, also cause notable adverse side effects such as
drowsiness, ataxia, gastrointestinal disturbance, hepa-
totoxicity and megaloblastic anemia2–4, and even life
threatening conditions5. Research to find more effective
and safer antiepileptic drugs is, therefore, imperative and
challenging in medicinal chemistry.
We previously described the synthesis and anticon-
vulsant activity evaluation of some compounds contain-
ing triazole, which exhibited potent activity in maximal
electroshock test (MES) and in several chemical mod-
els such as Pentylenetetrazole- and Isoniazid-induced
the major characteristics important in newly synthesized
compounds are the inclusion of a hydrophobic unit (R), an
electron donor group (D), a hydrogen donor/acceptor unit
(HAD). With respect to the compounds mentioned above,
obviously the R is the phenyl group and the substituents
on it, the D is the oxygen atom and the HAD is the triazole.
As part of our continuous efforts to find better anticon-
vulsant agents in this area, a series of 6-substituted-[1,2,4]
triazolo[3,4-a](tetrazolo[5,1-a])phthalazine
derivatives
(1–34) were designed and synthesized in this study, which
also possessed the three parts mentioned above.
All the 34 new compounds were synthesized and
evaluated as anticonvulsant agents in experimental
epilepsy models. e rotarod assay was performed in
mice to evaluate the neurotoxicity of the compounds.
e anti-MES activity and the neurotoxicity of the
marketed agent carbamazepine were evaluated in our
Address for Correspondence: Zhe-Shan Quan, College of Pharmacy, Yanbian University, No. 977, GongYuan Road, Yanji City, Jilin 133000,
China. Tel: +86–433–2436020. E-mail: zsquan@ybu.edu.cn; Cheng-Xi Wei, No. 996 Xila Mulun Street, Tongliao, Inner Mongolia Autonomous
Region, 028007, China. E-mail: weichengxi1224@yahoo.com.cn
(Received 09 February 2012; revised 31 March 2012; accepted 02 April 2012)
792

Triazolophthalazines as anticonvulsants 793
Figure 1. Structures of some compounds in containing triazole and the sketch map. (R) hydrophobic unit; (D) electron donor group; (HAD)
hydrogen donor/acceptor unit.
laboratory under the same conditions for the purpose of
comparison.
petroleum ether, then kept below 0°C. e compound 37
obtained was pure enough for the following step.
Synthesis of 6-chloro-[1,2,4]triazolo[3,4-a]phthalazine (38)
In a round-bottomed flask, compound 37 (2.2 g, 11.3
mmol) was dissolved in Triethoxymethane (60 mL), and
the solution was refluxed for 2 h, then the solvent was
removed under reduced pressure. e residue was puri-
fied by silica gel chromatography using dichloromethane.
Experimental protocols
Melting points were determined in open capillary tubes
and were uncorrected. IR spectra were recorded (in KBr)
on IR Prestige-21. 1H NMR spectra were measured on an
AV-300 (Bruker, Switzerland), and all chemical shifts were
given in parts per million relative to tetramethysilane.
Mass spectra were measured on a HP1100LC (Agilent
Technologies, USA). Elemental analyses were performed
on a 204Q CHN (Perkin Elmer, USA). e chemicals were
purchased from Aldrich Chemical Corporation.
The general procedure for the synthesis of compounds 1–25
Compound 38 (0.65 g, 3.2 mmol), K2CO3 (0.44 g, 3.2 mmol),
benzyltriethylammonium chloride (TEBA) (0.23 g, 1 mmol)
and corresponding alcohol (0.70 mmol) were suspended
in acetonitrile (50 mL) reacted with stirring and refluxing
for about 3 h. After the acetonitrile was removed under
reduced pressure, the solid residue was purified by silica
gel chromatography with ethyl acetate: petroleum ether
(2:3) to obtain compounds 1–25. e yield, melting point
and spectral data of each compound were given below.
Chemistry
Synthesis of 1,4-dichlorophthalazine (36)
Compound 35 (8.5 g, 52.5 mmol) was dissolved in phos-
phorus oxychloride (45 mL) and stirred under reflux for
4 h. en the solvent was removed under vacuum. e
residue was dissolved in dichloromethane (200 mL) and
stirred rapidly, and the solution was neutralized by the
addition of solid and aqueous sodium hydrogen carbon-
ate (cautiously). When effervescence had ceased, the
organic layer was separated and the aqueous layer was
extracted with dichloromethane (200 mL twice). e
combined organic layers were dried over MgSO4, filtered
and evaporated. e residue was purified by silica gel
column chromatography with ethyl acetate: petroleum
ether (1:8) and gained 9.0 g.
6-(Benzyloxy)-[1,2,4]triazolo[3,4-a]phthalazine (1)
1
Yield: 87%, m.p. 170–172°C. H-NMR (CDCl3): δ 8.93 (s,
1H, 3-H), 8.90 (d, 1H, J = 9.0 Hz, 10-H), 8.27 (d, 1H, J = 9.0
Hz, 7-H), 8.00 (t, 1H, J = 9.0 Hz, 9-H), 7.85 (t, 1H, J = 9.0
Hz, 8-H), 7.55 (d, 2H, J = 9.0 Hz, 2′6′-H), 7.49-7.37 (m, 3H,
3′, 4′, 5′-H), 5.57 (s, 2H, CH2). IR (KBr) cm−1: 1532 (C=N).
MS (APCI) m/z 277 (M+1). Anal. Calc. for C16H12N4O: C,
69.55; H, 4.38; N, 20.28. Found: C, 69.32; H, 4.41; N, 20.33.
6-(2-Fluorobenzyloxy)-[1,2,4]triazolo[3,4-a]
Synthesis of 1-hydrazine-4-chrorophthalazin (37)
phthalazine (2)
A solution of compound 36 (5 g, 25.1 mmol) in THF
(60 mL) was added dropwise to a solution of hydrazine
hydrate (6.28 g, 125.6 mmol) in THF (10 mL) at room
temperature. e mixture was stirred and heated at
60°C for 1 h, then half of the solvent was removed under
reduced pressure and the solution was poured into petro-
leum ether. e precipitate was filtered and washed with
1
Yield: 89%, m.p. 177–179°C. H-NMR (CDCl3): δ 8.87 (s,
1H, 3-H), 8.62 (d, 1H, J = 9.0 Hz, 10-H), 8.17 (d, 1H, J =
9.0 Hz, 7-H), 7.89 (t, 1H, J = 9.0 Hz, 9-H), 7.78 (t, 1H, J =
9.0 Hz, 8-H), 7.58 (t, 1H, J = 9.0 Hz, 5′-H), 7.37-7.42 (m,
1H, 4′-H), 7.25-7.16 (m, 2H, 3′-H), 5.60 (s, 2H, CH2). IR
(KBr) cm−1: 1519 (C=N). MS (APCI) m/z 295 (M+1). Anal.
© 2013 Informa UK, Ltd.

794 M. Bian et al.
Calc. for C16H11FN4O: C, 65.30; H, 3.77; N, 19.04. Found: C,
65.28; H, 3.82; N, 19.22.
C16H11ClN4O: C, 61.84; H, 3.57; N, 18.03. Found: C, 61.77;
H, 3.49; N, 18.07.
6-(2-Bromobenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (8)
Yield: 78%, m.p. 177–179°C. H-NMR (CDCl3): δ 8.90 (s,
1
1H, 3-H), 8.55 (d, 1H, J = 9.0 Hz, 10-H), 8.17 (d, 1H, J = 9.0
Hz, 7-H), 7.88 (t, 1H, J = 9.0 Hz, 9-H), 7.74 (t, 1H, J = 9.0
Hz, 8-H), 7.64-7.59 (m, 1H, 3′ and 6′-H), 7.38 (d, 1H, J =
9.0 Hz, 4′-H), 7.28-7.23 (m, 1H, 5′-H), 5.56 (s, 2H, CH2). IR
(KBr) cm−1: 1509 (C=N). MS (APCI) m/z 355 (M+1). Anal.
Calc. for C16H11BrN4O: C, 54.10; H, 3.12; N, 15.77. Found:
C, 54.01; H, 3.23; N, 15.81.
6-(3-Bromobenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (9)
1
Yield: 84%, m.p. 186–187°C. H-NMR (CDCl3): δ 8.89 (s,
1H, 3-H), 8.78 (d, 1H, J = 9.0 Hz, 10-H), 8.22 (d, 1H, J = 9.0
Hz, 7-H), 7.98 (t, 1H, J = 9.0 Hz, 9-H), 7.82 (t, 1H, J = 9.0
Hz, 8-H), 7.56-7.51 (m, 2H, 2′ and 4′-H), 7.18-7.12 (m, 2H,
5′ and 6′-H), 5.53 (s, 2H, CH2). IR (KBr) cm−1: 1525 (C=N).
MS (APCI) m/z 355 (M+1). Anal. Calc. for C16H11BrN4O: C,
54.10; H, 3.12; N, 15.77. Found: C, 54.04; H, 3.21; N, 15.79.
6-(2-Methoxybenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (10)
Yield: 78%, m.p. 187–189°C. 1H-NMR (CDCl3): δ 8.87 (s, 1H,
3-H), 8.59 (d, 1H, J = 9.0 Hz, 10-H), 8.19 (d, 1H, J = 9.0 Hz,
7-H), 7.87 (t, 1H, J = 9.0 Hz, 9-H), 7.76 (t, 1H, J = 9.0 Hz, 8-H),
7.53 (d, 1H, J = 9.0 Hz, 6′-H), 7.37 (t, 1H, J = 9.0 Hz, 5′-H), 7.05
(t, 1H, J = 9.0 Hz, 4′-H), 6.97 (d, 1H, J = 9.0 Hz, 3′-H), 5.56 (s,
2H, CH2), 3.88 (s, 3H, -OCH3). IR (KBr) cm−1: 1512 (C=N).
MS (APCI) m/z 307 (M+1). Anal. Calc. for C17H14N4O2: C,
66.66; H, 4.61; N, 18.29. Found: C, 66.71; H, 4.69; N, 18.21.
6-(2-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (5)
Yield: 91%, m.p. 166–167°C. H-NMR (CDCl3): δ 8.89 (s,
1
1H, 3-H), 8.72 (d, 1H, J = 9.0 Hz, 10-H), 8.24 (d, 1H, J = 9.0
Hz, 7-H), 7.95 (t, 1H, J = 9.0 Hz, 9-H), 7.80 (t, 1H, J = 9.0
Hz, 8-H), 7.62 (t, 1H, J = 9.0 Hz, 5′-H), 7.50-7.47 (m, 1H,
4′-H), 7.39-7.33 (m, 2H, 3′ and 6′-H), 5.66 (s, 2H, CH2). IR
(KBr) cm−1: 1532 (C=N). MS (APCI) m/z 311 (M+1). Anal.
Calc. for C16H11ClN4O: C, 61.84; H, 3.57; N, 18.03. Found:
C, 61.81; H, 3.65; N, 18.11.
6-(3-Methoxybenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (11)
6-(3-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (6)
1
Yield: 80%, m.p. 156–158°C. H-NMR (CDCl3): δ 8.86 (s,
1H, 3-H), 8.63 (d, 1H, J = 9.0 Hz, 10-H), 8.22 (d, 1H, J = 9.0
Hz, 7-H), 7.96 (t, 1H, J = 9.0 Hz, 9-H), 7.80 (t, 1H, J = 9.0
Hz, 8-H), 7.37 (t, 1H, J = 9.0 Hz, 5′-H), 7.13-7.7.08 (m, 2H,
2′ and 6′-H), 6.95 (d, 1H, 4′-H), 5.56 (s, 2H, CH2), 3.90 (s,
3H, -OCH3). IR (KBr) cm−1: 1541 (C=N). MS (APCI) m/z
307 (M+1). Anal. Calc. for C17H14N4O2: C, 66.66; H, 4.61; N,
18.29. Found: C, 66.72; H, 4.65; N, 18.22.
Yield: 90%, m.p. 176–178°C. 1H-NMR (CDCl3): δ 8.85
(s, 1H, 3-H), 8.58 (d, 1H, J = 9.0 Hz, 10-H), 8.18 (d, 1H,
J = 9.0 Hz, 7-H), 7.89 (t, 1H, J = 9.0 Hz, 9-H), 7.77 (t, 1H,
J = 9.0 Hz, 8-H), 7.53 (s, 1H, 2′-H), 7.44-7.29 (m, 2H, 4′,5′
and 6′-H), 5.49 (s, 2H, CH2). IR (KBr) cm−1: 1511 (C=N).
MS (APCI) m/z 311 (M+1). Anal. Calc. for C16H11ClN4O: C,
61.84; H, 3.57; N, 18.03. Found: C, 61.90; H, 3.67; N, 18.04.
6-(4-Methoxybenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (12)
6-(4-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (7)
Yield: 89%, m.p. 210–212°C. H-NMR (CDCl3): δ 8.88 (s,
Yield: 88%, m.p. 198–200°C. 1H-NMR (CDCl3): δ 8.86
(d, 1H, J = 9.0 Hz, 10-H), 8.58 (s, 1H, 3-H), 8.17 (d, 1H,
J = 9.0 Hz, 7-H), 7.90 (t, 1H, J = 9.0 Hz, 9-H), 7.74 (t, 1H,
J = 9.0 Hz, 8-H), 7.47 (d, 2H, J = 9.0 Hz, 2′ and 6′-H), 6.96
(d, 2H, J = 9.0 Hz, 3′ and 5′-H), 5.46 (s, 2H, CH2), 3.85 (s,
3H, -OCH3). IR (KBr) cm−1: 1519 (C=N). MS (APCI) m/z
1
1H, 3-H), 8.78 (d, 1H, J = 9.0 Hz, 10-H), 8.22 (d, 1H, J =
9.0 Hz, 7-H), 7.98 (t, 1H, J = 9.0 Hz, 9-H), 7.82 (t, 1H, J =
9.0 Hz, 8-H), 7.49 (d, 2H, J = 9.0 Hz, 2′ and 6′-H), 7.42 (d,
2H, J = 9.0 Hz, 3′ or 5′-H), 5.52 (s, 2H, CH2). IR (KBr) cm−1:
1524 (C=N). MS (APCI) m/z 311 (M+1). Anal. Calc. for
Journal of Enzyme Inhibition and Medicinal Chemistry

Triazolophthalazines as anticonvulsants 795
307 (M+1). Anal. Calc. for C17H14N4O2: C, 66.66; H, 4.61; N,
18.29. Found: C, 66.71; H, 4.59; N, 18.19.
C15H16N4O: C, 67.15; H, 6.01; N, 20.88. Found: C, 67.21;
H, 6.19; N, 20.77.
6-(2-(Trifluoromethyl)benzyloxy)-[1,2,4]triazolo[3,4-a]
6-(1-Phenylethoxy)-[1,2,4]triazolo[3,4-a]phthalazine
(18)
phthalazine (13)
Yield: 89%, m.p. 188–190°C. H-NMR (CDCl3): δ 8.88 (s,
1
Yield: 71%, m.p. 68–70°C. 1H-NMR (CDCl3): δ 8.56 (s, 1H,
3-H), 8.19 (d, 1H, J = 8.4 Hz, 10-H), 7.91 (d, 1H, J = 8.4 Hz,
7-H), 7.50-7.44 (m, 2H, 8 and 9-H), 7.42 (d, 2H, J = 7.8 Hz,
2′ and 6′-H), 7.13 (t, 2H, J = 7.8 Hz, 3′ and 5′-H), 7.08 (d,
J = 7.8 Hz, 4′-H), 6.01 (q, 1H, J = 6.6 Hz, -OCH), 1.62 (d, 3H,
J = 6.6 Hz, -CH3). IR (KBr) cm−1: 1513 (C=N). MS (APCI)
m/z 269 (M+1). Anal. Calc. for C17H14N4O: C, 70.33; H,
4.86; N, 19.30. Found: C, 70.37; H, 4.91; N, 19.29.
1H, 3-H), 8.71 (d, 1H, J = 9.0 Hz, 10-H), 8.24 (d, 1H, J = 9.0
Hz, 7-H), 7.94 (t, 1H, J = 9.0 Hz, 9-H), 7.81 (t, 1H, J = 9.0
Hz, 8-H), 7.62 (t, 1H, J = 9.0 Hz, 5′-H), 7.51-7.49 (m, 1H,
4′-H), 7.40-7.33 (m, 2H, 3′ and 6′-H), 5.64 (s, 2H, CH2). IR
(KBr) cm−1: 1518 (C=N). MS (APCI) m/z 345 (M+1). Anal.
Calc. for C17H11F3N4O: C, 59.31; H, 3.22; N, 16.27. Found:
C, 59.29; H, 3.19; N, 16.32.
6-(1-(4-Fluorophenyl)ethoxy)-[1,2,4]triazolo[3,4-a]
phthalazine (19)
6-(3-(Trifluoromethyl)benzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (14)
Yield: 71%, m.p. 68–70°C. 1H-NMR (CDCl3): δ 8.78 (s, 1H,
3-H), 8.52 (d, 1H, J = 8.4 Hz, 10-H), 8.22 (d, 1H, J = 8.4
Hz, 7-H), 7.87 (t, 1H, J = 8.6 Hz, 9-H), 7.77 (t, 1H, J = 8.6
Hz, 8-H), 7.53-7.49 (m, 2H, 3′ and 5′-H), 7.07-7.09 (t, 2H,
J = 7.8 Hz, 2′ and 6′-H), 6.24 (q, 1H, J = 6.5 Hz, -OCH),
1.62 (d, 3H, J = 6.5 Hz, -CH3). IR (KBr) cm−1: 1509 (C=N).
MS (APCI) m/z 309 (M+1). Anal. Calc. for C17H13FN4O: C,
66.23; H, 4.25; N, 18.17. Found: C, 66.12; H, 4.34; N, 18.21.
Yield: 87%, m.p. 164–166°C. 1H-NMR (CDCl3): δ 8.86 (d,
1H, J = 9.0 Hz, 10-H), 8.62 (s, 1H, 3-H), 8.19 (d, 1H, J =
9.0 Hz, 7-H), 7.95 (t, 1H, J = 9.0 Hz, 9-H), 7.81 (s, 1H,
2′-H), 7.79 (t, 1H, J = 9.0 Hz, 8-H), 7.76 (d, 1H, J = 9.0 Hz,
4′-H), 7.68 (d, 1H, J = 9.0 Hz, 6′-H), 7.58 (d, 1H, J = 9.0
Hz, 5′-H), 5.59 (s, 2H, CH2). IR (KBr) cm−1: 1517 (C=N).
MS (APCI) m/z 345 (M+1). Anal. Calc. for C17H11F3N4O:
C, 59.31; H, 3.22; N, 16.27. Found: C, 59.35; H, 3.23; N,
16.38.
6-(1-(4-Cholorophenyl)ethoxy)-[1,2,4]triazolo[3,4-a]
phthalazine (20)
6-(4-(Trifluoromethyl)benzyloxy)-[1,2,4]triazolo[3,4-a]
1
Yield: 78%, m.p. 130–132°C. H-NMR (CDCl3): δ 8.77 (s,
phthalazine (15)
1H, 3-H), 8.52 (d, 1H, J = 8.4 Hz, 10-H), 8.21 (d, 1H, J =
8.4 Hz, 7-H), 7.85 (t, 1H, J = 8.6 Hz, 9-H), 7.78 (t, 1H, J =
8.6 Hz, 8-H), 7.45 (d, 2H, J = 7.8 Hz, 3′ and 5′-H), 7.34 (d,
2H, J = 7.8 Hz, 2′ and 6′-H), 6.24 (q, 1H, J = 6.6 Hz, -OCH),
1.62 (d, 3H, J = 6.6 Hz, -CH3). IR (KBr) cm−1: 1521 (C=N).
MS (APCI) m/z 325 (M+1). Anal. Calc. for C17H13ClN4O: C,
62.87; H, 4.03; N, 17.25. Found: C, 62.79; H, 4.12; N, 17.33.
1
Yield: 87%, m.p. 210–212°C. H-NMR (CDCl3): δ 8.89
(s, 1H, 3-H), 8.79 (d, 1H, J = 9.0 Hz, 10-H), 8.23 (d, 1H,
J = 9.0 Hz, 7-H), 7.99 (t, 1H, J = 9.0 Hz, 9-H), 7.83 (t, 1H,
J = 9.0 Hz, 8-H), 7.50 (d, 2H, J = 9.0 Hz, 2′ and 6′-H), 7.45
(d, 2H, J = 9.0 Hz, 3′ or 5′-H), 5.54 (s, 2H, CH2). IR (KBr)
cm−1: 1503 (C=N). MS (APCI) m/z 345 (M+1). Anal.
Calc. for C17H11F3N4O: C, 59.31; H, 3.22; N, 16.27. Found:
C, 59.33; H, 3.29; N, 16.34.
6-(1-(4-Methoxyphenyl)ethoxy)-[1,2,4]triazolo[3,4-a]
phthalazine (21)
Yield: 86%, m.p. 84–86°C. 1H-NMR (CDCl3): δ 8.78 (s, 1H,
3-H), 8.54 (d, 1H, J = 8.6 Hz, 10-H), 8.24 (d, 1H, J = 8.4 Hz,
7-H), 7.90 (t, 1H, J = 8.6 Hz, 9-H), 7.75 (t, 1H, J = 8.4 Hz,
8-H), 7.45 (d, 2H, J = 7.8 Hz, 3′ and 5′-H), 6.90 (d, 2H, J =
7.8 Hz, 2′ and 6′-H), 6.22 (q, 1H, J = 6.6 Hz, -OCH), 3.80 (s,
3H, -OCH3), 1.79 (d, 3H, J = 6.6 Hz, -CH3). IR (KBr) cm−1:
1512 (C=N). MS (APCI) m/z 321 (M+1). Anal. Calc. for
C18H16N4O2: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.54;
H, 5.23; N, 17.33
6-(4-Methylbenzyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine (16)
1
Yield: 88%, m.p. 196–198°C. H-NMR (CDCl3): δ 8.87 (s,
1H, 3-H), 8.72 (d, 1H, J = 9.0 Hz, 10-H), 8.21 (d, 1H, J = 9.0
Hz, 7-H), 7.94 (t, 1H, J = 9.0 Hz, 9-H), 7.78 (t, 1H, J = 9.0
Hz, 8-H), 7.43 (d, 2H, J = 9.0 Hz, 2′ and 6′-H), 7.25 (d, 2H,
J = 9.0 Hz, 3′ or 5′-H), 5.50 (s, 2H, CH2), 2.45 (s, 3H, CH3).
IR (KBr) cm−1: 1507 (C=N). MS (APCI) m/z 291 (M+1).
Anal. Calc. for C17H14N4O: C, 70.33; H, 4.86; N, 19.30.
Found: C, 70.31; H, 4.96; N, 19.32.
6-(1-4-Tolylethoxy)-[1,2,4]triazolo[3,4-a]phthalazine
(22)
6-(Cyclohexyloxy)-[1,2,4]triazolo[3,4-a]phthalazine
(17)
Yield: 81%, m.p. 68–70°C. 1H-NMR (CDCl3): δ 8.76 (s, 1H,
3-H), 8.52 (d, 1H, J = 8.6 Hz, 10-H), 8.23 (d, 1H, J = 8.4
Hz, 7-H), 7.83 (t, 1H, J = 8.6 Hz, 9-H), 7.76 (t, 1H, J = 8.4
Hz, 8-H), 7.40 (d, 2H, J = 7.8 Hz, 3′ and 5′-H), 7.18 (d, 2H,
J = 7.8 Hz, 2′ and 6′-H), 6.22 (q, 1H, J = 6.6 Hz, -OCH),
2.33 (s, 3H, -CH3), 1.77 (d, 3H, J = 6.6 Hz, -CH3). IR (KBr)
cm−1: 1509 (C=N). MS (APCI) m/z 305 (M+1). Anal. Calc.
1
Yield: 67%, m.p. 139–142°C. H-NMR (CDCl3): δ 8.95
(s, 1H, 3-H), 8.43 (d, 1H, J = 9.0 Hz, 10-H), 8.18 (d, 1H,
J = 9.0 Hz, 7-H), 7.90 (t, 1H, J = 9.0 Hz, 9-H), 7.73 (t,
1H, J = 9.0 Hz, 8-H), 5.24-5.16 (m, 1H, CH), 2.36-2.09
(m, 2H, CH2), 1.88-1.25 (m, 8H, (CH2)4). IR (KBr) cm−1:
1509 (C=N). MS (APCI) m/z 269 (M+1). Anal. Calc. for
© 2013 Informa UK, Ltd.

796 M. Bian et al.
for C18H16N4O: C, 71.04; H, 5.30; N, 18.41. Found: C, 71.11;
H, 5.26; N, 18.45.
6-(Benzyloxy)tetrazolo[5,1-a]phthalazine (26)
Yield: 89%, m.p. 165–167°C; H-NMR (CDCl3, 300 MHz)
1
δ: 8.61 (d, 1H, J = 8.5 Hz, 9-H), 8.33 (d, 1H, J = 8.2 Hz, 6-H),
8.01 (t, 1H, J = 4.2 Hz, 8-H), 7.94 (t, 1H, J = 4.0 Hz, 7-H),
7.58 (d, 2H, J = 7.9 Hz, 3′ and 5′-H), 7.28-7.48 (m, 3H, 2′,
4′ and 6′-H), 5.68 (s, 2H, CH2). IR (KBr) cm−1: 1509 (C=N).
MS (APCI) m/z: 278 (M+1); Anal. Calcd for C15H11N5O: C,
64.97; H, 4.00; N, 25.26. Found: C, 64.78; H, 4.13; N, 25.12.
6-(Benzhydryloxy)-[1,2,4]triazolo[3,4-a]phthalazine
(23)
Yield: 84%, m.p. 188–190°C. 1H-NMR (CDCl3): δ 8.76 (s, 1H,
3-H), 8.58 (d, 1H, J = 7.2 Hz, 10-H), 8.36 (d, 1H, J = 7.2 Hz,
7-H), 7.90 (t, 1H, J = 7.2 Hz, 9-H), 7.80 (t, 1H, J = 7.2 Hz, 8-H),
7.51 (d, 4H, J= 8.4 Hz, (2′and 6′-H)2), 7.41-7.30 (m, 6H, (3′, 4′
and 5′-H)2), 7.24 (s, 1H, -OCH). IR (KBr) cm−1: 1516 (C=N).
MS (APCI) m/z 353 (M+1). Anal. Calc. for C22H16N4O: C,
74.98; H, 4.58; N, 15.90. Found: C, 75.01; H, 4.62; N, 15.78.
6-(4-Fluorobenzyloxy)tetrazolo[5,1-a]phthalazine (27)
Yield: 86%, m.p. 164–166°C; 1H-NMR (CDCl3, 300 MHz) δ:
8.64 (d, 1H, J = 8.0 Hz, 9-H), 8.33 (d, 1H, J = 8.0 Hz, 6-H),
8.05 (t, 1H, J = 4.2 Hz, 8-H), 7.96 (t, 1H, J = 4.0 Hz, 7-H),
7.57 (d, 2H, J = 7.9 Hz, 3′ and 5′-H), 7.13 (d, 2H, J = 7.9 Hz,
2′ and 6′-H), 5.66 (s, 2H, CH2). IR (KBr) cm−1: 1523 (C=N).
MS (APCI) m/z: 296 (M+1); Anal. Calcd for C15H10FN5O:
C, 61.02; H, 3.41; N, 23.72. Found: C, 60.87; H, 3.53; N,
23.68.
6-(Bis(4-fluorophenyl)methoxy)-[1,2,4]triazolo[3,4-a]
phthalazine (24)
Yield: 82%, m.p. 180–182°C. 1H-NMR (CDCl3): δ 8.77 (s, 1H,
3-H), 8.60 (d, 1H, J = 8.0, 10-H), 8.32 (d, 1H, J = 7.8 Hz, 7-H),
7.94 (t, 1H, J = 8.0 Hz, 9-H), 7.82 (t, 1H, J = 7.8 Hz, 8-H), 7.48-
7.44 (m, 4H, (3′ and 5′-H)2),7.22 (s, 1H, -OCH), 7.06 (t, 4H,
J = 8.2 Hz, (2′ and 6′-H)2). IR (KBr) cm−1: 1524 (C=N). MS
(APCI) m/z 389 (M+1). Anal. Calc. for C22H14N4O: C, 68.04;
H, 3.63; N, 14.43. Found: C, 68.23; H, 3.51; N, 14.39.
6-(2-Chlorobenzyloxy)tetrazolo[5,1-a]phthalazine (28)
1
Yield: 89%, m.p. 162–164°C; H-NMR (CDCl3, 300 MHz)
δ: 8.61 (d, 1H, J = 8.0 Hz, 9-H), 8.34 (d, 1H, J = 8.0 Hz, 6-H),
8.03 (t, 1H, J = 4.2 Hz, 8-H), 7.97 (t, 1H, J = 7.6 Hz, 7-H),
7.69-7.34 (m, 4H, 3′, 4′, 5′, and 6′-H), 5.77 (s, 2H, CH2). IR
(KBr) cm−1: 1527 (C=N). MS (APCI) m/z: 312 (M+1); Anal.
Calcd for C15H10ClN5O: C, 57.79; H, 3.23; N, 22.47. Found:
C, 57.81; H, 3.39; N, 22.23.
6-((4-Chlorophenyl)(phenyl)methoxy)-[1,2,4]
triazolo[3,4-a]phthalazine (25)
1
Yield: 69%, m.p. 182–185°C. H-NMR (CDCl3): δ 8.76 (s,
1H, 3-H), 8.54 (d, 1H, J = 7.8 Hz, 10-H), 8.32 (d, 1H, J = 7.6
Hz, 7-H), 7.87 (t, 1H, J = 7.8 Hz, 9-H), 7.80 (t, 1H, J = 7.6
Hz, 8-H), 7.52-7.33 (m, 9H, Ar-H), 7.20 (s, 1H, -OCH). IR
(KBr) cm−1: 1532 (C=N). MS (APCI) m/z 387 (M+1). Anal.
Calc. for C22H15ClN4O: C, 68.31; H, 3.91; N, 14.48. Found:
C, 68.23; H, 3.78; N, 14.61.
6-(4-Chlorobenzyloxy)tetrazolo[5,1-a]phthalazine (29)
1
Yield: 89%, m.p. 190–192°C; H-NMR (CDCl3, 300 MHz)
δ: 8.65 (d, 1H, J = 8.0 Hz, 9-H), 8.33 (d, 1H, J = 8.0 Hz,
6-H), 8.06 (t, 1H, J = 4.2 Hz, 8-H), 7.94 (t, 1H, J = 7.6 Hz,
7-H), 7.57 (d, 2H, J = 7.9 Hz, 3′ and 5′-H), 7.42 (d, 2H,
J = 7.9 Hz, 2′ and 6′-H), 5.66 (s, 2H, CH2). IR (KBr) cm−1:
1517 (C=N). MS (APCI) m/z: 312 (M+1); Anal. Calcd for
C15H10ClN5O: C, 57.79; H, 3.23; N, 22.47. Found: C, 57.58;
H, 3.42; N, 22.39.
Synthesis of 6-chlorotetrazolo[5,1-a]phthalazine (39)
Compound 37 (3 g, 15.4 mmol) was dissolved in 50 ml of
30% H2SO4, then a solution of NaNO2 (1.06 g, 15.4 mmol)
in 15 ml H2O was added dropwise to the mixture under
ice-bath, be sure that the reaction temperature was below
5°C. en the mixture was stirred at room temperature for
2 h (TLC monitoring). e mixture was extracted twice
with dichloromethane (60 ml). e dichloromethane
layer was washed three times with saturated aqueous
NaCl (60 × 3) and dried over anhydrous MgSO4. After
removing the solvents, 2.15 g light yellow product was
gained, yield 68%.
6-(3-Bromobenzyloxy)tetrazolo[5,1-a]phthalazine (30)
1
Yield: 81%, m.p. 180–183°C; H-NMR (CDCl3, 300 MHz)
δ: 8.67 (d, 1H, J = 8.20 Hz, 9-H), 8.35 (d, 1H, J = 8.20 Hz,
6-H), 8.07 (t, 1H, J = 4.2 Hz, 8-H), 7.97 (t, 1H, J = 7.6 Hz,
7-H), 7.73-7.33 (m, 4H, Ar-H), 5.66 (s, 2H, CH2). IR (KBr)
cm−1: 1519. MS (APCI) m/z: 356 (M+1); Anal. Calcd for
C15H10BrN5O: C, 50.58; H, 2.83; N, 19.66. Found: C, 50.62;
H, 2.91; N, 19.57.
The general procedure for the synthesis of compounds 26–34
Compound 38 (0.65 g, 3.2 mmol), K2CO3 (0.44 g, 3.2
mmol), benzyltriethylammonium chloride (TEBA)
(0.23g, 1 mmol) and corresponding alcohol (0.70 mmol)
were suspended in acetonitrile (50 mL), and reacted with
stirring and refluxing for about 3 h. After the acetonitrile
was removed under reduced pressure, the solid residue
was purified by silica gel chromatography with ethyl
acetate: petroleum ether (2:3) to obtain compounds
26–34. e yield, melting point and spectral data of each
compound were given below.
6-(3-(Trifluoromethyl)benzyloxy)-tetrazolo[5,1-a]
phthalazine (31)
Yield: 80%, m.p. 155–157°C; H-NMR (CDCl3, 300 MHz)
1
δ: 8.87 (d, 1H, J = 9.00 Hz, 10-H), 8.35 (d, 1H, J = 9.00 Hz,
7-H), 8.05 (t, 1H, J = 9.0 Hz, 9-H), 7.98 (s, 1H, 2′-H), 7.79 (t,
1H, J = 9.0 Hz, 8-H), 7.76 (d, 1H, J = 9.0 Hz, 4′-H), 7.68 (d,
1H, J = 9.0 Hz, 6′-H), 7.58 (d, 1H, J = 9.0 Hz, 5′-H), 5.59 (s,
2H, CH2). IR (KBr) cm−1: 1527. MS (APCI) m/z: 346 (M+1);
Anal. Calcd for C16H10F3N5O: C, 55.66; H, 2.92; N, 20.28.
Found: C, 55.87; H, 2.91; N, 20.34.
Journal of Enzyme Inhibition and Medicinal Chemistry

Triazolophthalazines as anticonvulsants 797
were calculated by means of a computer program writ-
ten at National Institute of Neurological Disorders and
6-(4-(Methyl)benzyloxy)-tetrazolo[5,1-a]phthalazine
(32)
Stroke13,14
.
1
Yield: 85%, m.p. 153–155°C; H-NMR (CDCl3, 300 MHz)
δ: 8.61 (d, 1H, J = 7.90 Hz, 9-H), 8.31 (d, 1H, J = 8.20 Hz,
6-H), 8.02 (t, 1H, J = 8.2 Hz, 8-H), 7.90 (t, 1H, J = 8.2 Hz,
7-H), 7.46 (d, 2H, J = 8.0 Hz, 3′ and 5′-H), 7.25 (d, 1H, J =
8.0 Hz, 2′ and 6′-H), 5.63 (s, 2H, CH2), 2.39 (s, 3H, CH3). IR
(KBr) cm−1: 1523. MS (APCI) m/z: 292 (M+1); Anal. Calcd
for C16H13N5O: C, 65.97; H, 4.50; N, 24.04. Found: C, 65.78;
H, 4.65; N, 24.09.
Neurotoxicity (NT) screening
e neurotoxicity of the compounds was measured in
Kunming mice by the rotarod test for three times. e
mice were trained to stay on an accelerating rotarod
of diameter 3.2 cm that rotates at 10 rpm. Trained ani-
mals were given i.p. injection of the test compounds.
Neurotoxicity was indicated by the inability of the animal
to maintain equilibrium on the rod for at least 1 min in
6-(1-(4-Fluorophenyl)ethoxy)tetrazolo[5,1-a]
phthalazine (33)
each of the trials15,16
.
Yield: 82%, m.p. 113–115°C; 1H-NMR (CDCl3, 300 MHz)
δ: 8.55 (d, 1H, J = 7.90 Hz, 9-H), 8.37 (d, 1H, J = 7.90 Hz,
6-H), 8.01-7.95 (m, 2H, Ar-H), 7.61-7.56 (m, 2H, Ar-H),
7.10-7.04 (m, 2H, Ar-H), 6.45 (q, 2H, J = 6.5 Hz CH2),
1.89 (d, 3H, J = 6.5 Hz, CH3). IR (KBr) cm−1: 1509. MS
(APCI) m/z: 310 (M+1); Anal. Calcd for C16H12FN5O:
C, 62.13; H, 3.91; N, 22.64. Found: C, 62.21; H, 4.02; N,
22.56.
Sc-PTZ-induced seizures
At 0.5 h after the administration of the test compound, 85
mg/kg PTZ dissolved in saline was administered sc. e
animals (10 KunMing mice in each group) were placed
in individual cages and observed for 0.5 h. e numbers
of clonic seizure (range from exaggerated twitches of
the limbs to violent shaking or vibrating of the stiffened
extremities) and tonic seizure (the extremities pull
towards the body or rigidly push away from it, usually
maximal extension of the hind leg) as well as the number
6-(Bis(4-fluorophenyl)methoxy)tetrazolo[5,1-a]
phthalazine (34)
Yield: 89%, m.p. 204–206°C; H-NMR (CDCl3, 300 MHz)
of death were noted15,16
.
1
δ: 8.65 (d, 1H, J = 7.90 Hz, 9-H), 8.47 (d, 1H, J = 7.90 Hz,
6-H), 8.07 (t, 1H, J = 7.20 Hz, 8-H), 7.99 (t, 1H, J = 7.20 Hz,
7-H), 7.54-7.49 (m, 4H, Ar-H), 7.44 (s, 1H, CH), 7.12-7.06
(m, 4H, Ar-H). IR (KBr) cm−1: 1544. MS (APCI) m/z: 390
(M+1); Anal. Calcd for C21H13F2N5O: C, 64.78; H, 3.37; N,
17.99. Found: C, 64.86; H, 3.45; N, 18.12.
Isoniazid-induced seizures test
At 0.5 h after the administration of the test compound,
the animals (10 KunMing mice in each group) were given
ISO by ip at a dose of 250 mg/kg at which 100% of the ani-
mals showed convulsive reactions. e mice were placed
in individual cages and observed for 1 h. e numbers of
clonic and tonic seizures as well as the number of death
were noted17.
Biological assays
Anticonvulsant effects in the maximal electroshock seizure
(MES) test
3-MP induced seizures test
e MES test and rotarod test were carried out accord-
ing to procedures described in the Antiepileptic Drug
Development Program (ADD) of the National Institutes
of Health (USA) (15, 16). Seizures were elicited with a 60
Hz alternating current of 50 mA intensity in KunMing
mice. e current was applied via corneal electrodes
for 0.2 s. Protection against the spread of MES-induced
seizures was defined as the abolition of the tonic maxi-
mal extension of the hind leg. At 0.5 h after the adminis-
tration of the compounds, the activities were evaluated
in MES test. In phase-I screening, each compound was
administered at the dose levels of 30 and 100 mg/kg for
evaluating the preliminary anticonvulsant activity. For
determination of the median effective dose (ED50) and
the median toxic dose (TD50), the phase-II screening
was prepared. Groups of 10 mice were given a range
of intraperitoneal doses of the tested compound until
at least three points were established in the range of
10–90% seizure protection or minimal observed neuro-
toxicity. From the plot of this data, the respective ED50
and TD50 values, 95% confidence intervals, slope of
the regression line, and the standard error of the slope
At 0.5 h after the administration of the test compound,
60 mg/kg of 3-MP in saline solution was injected by sc to
mice (10 KunMing mice in each group). e mice were
placed in individual cages and observed for 0.5 h. e
numbers of clonic and tonic seizures as well as the num
ber of death were noted18.
Thiosemicarbazide-induced seizures test
At 0.5 h after the administration of the test compound,
the animals (10 KunMing mice in each group) were given
an i.p. dose of TSC (50 mg/kg). e mice were placed
in individual cages and observed for 2.5 h. e number
of clonic seizures, tonic seizures and the lethality were
recorded19.
Results and discussion
Chemistry
Allthetargetcompounds1–34weresynthesizedaccording
to Scheme 1. Compound 36, a key substrate for the whole
reaction, was prepared by an established procedure20.
e starting material 2,3-dihydrophthalazine-1,4-dione
© 2013 Informa UK, Ltd.

798 M. Bian et al.
Scheme 1. Synthesis route of Compounds 1–34.
(35) reacted with the refluxing phosphorus oxychloride
to yield compound 36. Compound 36 reacted further
with hydrazine hydrate in tetrahydrofuran to afford
19 and 24 were subjected to phase II trials for quanti-
fication of their anticonvulsant activity (indicated by
ED50) and neurotoxicity (indicated by TD50) in mice.
Results of the quantitative test for selected compounds,
along with the data on the current antiepileptic drug,
were shown in Table 2. Among the tested compounds,
6-(3-(Trifluoromethyl)benzyloxy)-[1,2,4]triazolo[3,4-
a] phthalazine (14) was the most active and promising
compound in this work. It possessed strong anti-MES
activity with ED50 of 9.3 mg/kg, which was much better
than currently used antiepileptic drug Carbamazepine.
e neurotoxicity caused by it was minimal and was
markedly lower than the drug compared. It showed a
protective index of 91.7, which was many folds higher
than the current antiepileptic drug having the PI value
of 6.9. Furthermore, compounds 4, 13, 15 and 24 also
showed higher protective index than Carbamazepine.
1-hydrazine-4-chrorophthalazin
(37).
Cyclization
of compound 37 with triethyl orthoformate yielded
compound 38. e target compounds 1–25 were gained
from compound 38 by reacting with corresponding
alcohol in acetonitrile in the presence of K2CO3. And via
the diazotization of compound 37 with NaNO2 at 5°C in
30% H2SO4, 6-chloro-tetrazolo[5,1-a] phthalazine (39)
was obtained21. e target compounds 26–34 were gained
from compound 39 by reacting with corresponding
alcohol in acetonitrile in the presence of K2CO3.
Biological evaluation
Phase I evaluation of anticonvulsant activity
As we know, there are two models in vivo, the maxi-
mal electroshock seizure (MES), the subcutaneous
Pentylenetetrazole (sc-PTZ) models represent the two
animal seizure models most widely used in the search
for new AEDs22,23. In the present study, we used the MES
seizure model for screening the anticonvulsant activity of
target compounds. Compounds 1–25 were prepared to
evaluate the influence of the triazole ring and 26–34 were
prepared to evaluate the influence of the tetrazole ring on
anticonvulsant activity. eir preliminary anticonvulsant
activities were obtained and listed in Table 1. After i.p.
injection in mice using doses of 30 and 100 mg/kg, the
following observations can be made. At the dose of 100
mg/kg, compounds 4, 6, 8, 9, 13, 14, 15, 16, 18, 20–22,
24, 25 and 34 showed complete protection. Compounds
4, 13, 14, 15, 19 and 24 exhibited complete protection at
30 mg/kg.
Structure activity relationships discussions
Analysing the activities of the synthesized compounds,
the following structure activity relationships (SAR)
were obtained. We can divide all the compounds into
two groups, in the first group compounds (1–25) with
triazole ring compounds: among compounds 1–16, R1
was H and R2 was substituted part, effects of different
substitutions at benzyl were observed. e bioevaluation
led to an understanding of the importance of the position
of the substituted group at the phenyl. For example, the
activity order of the F position on the phenyl ring was
p-F > m-F > o-F; the activity order of the Cl position
on the phenyl ring was m-Cl > o-Cl > p-Cl; the activity
order of the -OCH3 position was o-OCH3 > m-OCH3 >
p-OCH3; and the activity order of the –CF3 position was
m-CF3 > p-CF3 > o-CF3. Among compounds 18–22:
R1 was CH3 and R2 was substituted part, effects of
different substitutions at benzyl were observed. Among
the five para-position compounds (18–22), the similar
Phase II evaluation of anticonvulsant activity
On the basis of the considerable anticonvulsant activity
suggested in phase I testing, compounds 4, 13, 14, 15,
Journal of Enzyme Inhibition and Medicinal Chemistry

Triazolophthalazines as anticonvulsants 799
Table 1. Phase I evaluation of anticonvulsant activity in
mice (i.p.).
Table 3. Effects of compound 13 on chemical-induced seizures in
mice.
Chemical
Doses Test Clonic
Tonic
sub-
(mg/ time seizures seizures Lethality
stances Compound
kg)
—
30
30
—
30
30
—
30
30
—
30
30
(h)
0.5
0.5
0.5
1
(%)
100
100
20
(%)
60
0
(%)
60
0
Sc-PTZ DMSO
CBZ
14
0
0
ISO
DMSO
CBZ
14
100
50
100
0
60
0
1
1
50
0
0
3-MP
TSC
DMSO
CBZ
14
0.5
0.5
0.5
2.5
2.5
2.5
100
100
40
100
0
100
0
No. R1 R2
100 30 No. R1
0/3 0/3 18 CH3
0/3 1/3 19 CH3
2/3 1/3 20 CH3
3/3 3/3 21 CH3
1/3 0/3 22 CH3
3/3 2/3 23 C6H5
R2
100 30
1
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3/3 1/3
0
0
a
o-F
p-F
p-Cl
—
3/3
DMSO
CBZ
14
100
100
40
100
0
100
0
3
4
m-F
p-F
3/3 1/5
p-OCH3 3/3 1/3
p -CH3 3/3 1/3
0
20
5
6
7
8
o-Cl
m-Cl
p-Cl
o-Br
m-Br
H
1/6
—
models and speculate about the possible mechanism of
anticonvulsant action, compound 14 was tested against
convulsions induced by chemical substances, including
PTZ, ISO, 3-MP, and TSC. Compound 14 was adminis-
tered to mice at 30 mg/kg i.p., which was higher than its
ED50 value and far below its TD50 value. e reference drug
Carbamazepine was also administered at 30 mg/kg i.p..
In the sc-PTZ model, Carbamazepine inhibited the
clonic seizures, tonic seizures and death at the rates of
0%, 100% and 100%, respectively. While compound 14
inhibited the clonic seizures, tonic seizures and lethality
at the rates of 80%, 100% and 100% induced by sc-PTZ
(Table 3), respectively, which revealed that compound 14
possessed excellent activity against sc-PTZ. Compound
14, exhibiting high anticonvulsant activity in the MES
and sc-PTZ models which are most widely used in the
search for new AEDs, suggested that it really possesses
a good anticonvulsant profile. In the ISO model, both
Carbamazepine and compound 14 inhibited the clonic
seizures, tonic seizures and death induced by ISO at
the rates of 50%, 100% and 100%, respectively (Table 3).
PTZ and ISO have been reported to produce seizures by
inhibiting γ-aminobutyric acid (GABA) neurotransmis-
sion24,25. GABA is the main inhibitory neurotransmitter in
the brain, and is widely implicated in epilepsy. Inhibition
of GABAergic neurotransmission or activity has been
shown to promote and facilitate seizures26, while
enhancement of GABAergic neurotransmission is known
to inhibit or attenuate seizures. e findings of the pres-
ent study suggest that the newly synthesized compound
14 might inhibit or attenuate PTZ-induced seizures and
ISO-induced seizures in mice by enhancing GABAergic
neurotransmission.
0/3
—
24 C6H4(p-F) p-F
3/3 3/3
3/3 1/3
3/3 1/3 25 C6H4(p-Cl) p-Cl
9
3/3 0/3 26
H
H
H
H
H
H
H
H
0/3
0/3
0/3
0/3
0/3
—
—
—
—
—
—
—
—
10
11
12
13
14
15
16
17b
o-OCH3 2/3 0/3 27
m-OCH3 1/3 0/3 28
p-OCH3 0/3
p-F
o-Cl
p-Cl
m-Br
—
29
o-CF3
m-CF3
p-CF3
p-CH3
3/3 3/3 30
3/3 3/3 31
3/3 3/3 32
m-CF3 0/3
p-CH3
p-F
0/3
0/3
3/3 0/3 33 CH3
2/3 0/3 34 C6H4(p-F) p-F
3/3 0/3
aNot tested.
bFor compound 17, the cyclohexane group was on the O atom.
Table 2. Quantitative anticonvulsant data in mice (i.p.).
ED50 (mg/kg)
Compound
(MES)
TD50 (mg/kg) PI (TD50/ED50)
4
13
14
15
19
24
CBZa
12.5 (9.5–18.4) 560.3 (354–726)
16.1 (10.2–24.5) 448.7 (256–667)
9.3 (6.7–14.2) 852.8 (645–1032)
15.4 (10.2–23.8) 663.2 (502–872)
19.8 (14.4–30.2) 67.5 (45.5–86.5)
14.6 (10.1–21.3) 267.4 (205–312)
44.8
27.9
91.7
43.1
3.4
18.3
6.4
11.8 (7.2–15.6)
76.1 (54.4–99.5)
aCBZ: carbamazepine
anticonvulsant activities were observed. Among
compound 23–25: R1 was a substituted phenyl and R2
were H, p-F and p-Cl respectively, the activity order of
three compounds was p-F > p-Cl > H. We also can see
that the other nine compounds (26–34) exhibited lower
anticonvulsant activity with tetrazole ring structure,
In the 3-MP induced seizure model, carbamazepine
inhibited the clonic seizures, tonic seizures and death at the
rates of 0%, 100% and 100%, respectively. In comparison,
compound 14 showed the anticonvulsant effect higher
than Carbamazepine in inhibiting the clonic seizures
induced by 3-MP with the inhibition rate of 60% (Table
3). In the TSC-induced seizure model, the anticonvulsant
Chemicals induced seizures
In this study, the majority of synthesized compounds
were highly potent in the MES test, and the MES test is
known to be sensitive to sodium channel inhibitors (e.g.
phenytoin, carbamazepine). To further investigate the
effects of the anticonvulsant activity in several different
© 2013 Informa UK, Ltd.

800 M. Bian et al.
7. Guo LJ, Wei CX, Jia JH, Zhao LM, Quan ZS. Design and synthesis
of 5-alkoxy-[1,2,4]triazolo[4,3-a]quinoline derivatives with
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effect was similar to that of the 3-MP induced seizure
model. Compared with the control group, Carbamazepine
showed inhibition of clonic and tonic seizures and death at
the rates of 0%, 100% and 100%, respectively. Compound
14 showed inhibition of clonic seizure, tonic seizures
and death at the rates of 60%, 100% and 80% induced by
TSC (Table 3), respectively. 3-MP and TSC are competi-
tive inhibitors of the GABA synthesis enzyme glutamate
decarboxylase (GAD), and they inhibit the synthesis of
GABA resulting in decrease of GABA levels in the brain27.
Compound 14 showed moderate antagonism to both
3-MP induced seizures and TSC-induced seizures.
9. Deng XQ, Wei CX, Li FN, Sun ZG, Quan ZS. Design and synthesis
of 10-alkoxy-5, 6-dihydro-triazolo[4,3-d]benzo[f][1,4]oxazepine
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In the present study we described the syntheses and
anticonvulsant activity evaluation of 6-substituted-[1,2,4]
triazolo[3,4-a](tetrazolo[5,1-a])phthalazine (1–34). Most
of the synthesized compounds exhibited potent anticon-
vulsant activities in the maximal electroshock test (MES).
e most promising compound 14 showed significant
anticonvulsant activity in MES test with ED50 value of 9.3
mg/kg. It displayed a wide margin of safety with protective
index much higher than the standard drug Carbamazepine.
And the potency of compound 14 against seizures induced
by Pentylenetetrazole, Isoniazid, iosemicarbazide and
3-Mercaptopropionic acid in the chemical-induced sei-
zure tests, suggested that compound 14 displayed wide
spectrum activity in several models.
Declaration of interest
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is work was supported by the National Natural Science
Foundation of China (No. 81160382).
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