Design, synthesis, and anticonvulsant activity evaluation of 4-(3-Alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones

Article abstract of DOI:10.1002/ardp.201200201

A series of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones were synthesized using the appropriate synthetic route and evaluated experimentally in the maximal electroshock test; their neurotoxicities were evaluated by the rotarod neurotoxicity test. The structures of these compounds were confirmed by IR, MS, ¹H-NMR, and elementary analysis. All target compounds exhibited anticonvulsant activity to varying degrees in the maximal electroshock test. 4-(3-Benzyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one (4i) was the most promising compound with an ED₅₀ value of 30.5 mg/kg and a protective index (PI) of 18.63, showing a higher safety than the standard carbamazepine (PI = 6.45). In addition, the potency of compound 4i against seizures induced by pentylenetetrazole and 3-mercaptopropionic acid suggested its broad-spectrum activity, and the mechanisms of action including inhibition of voltage-gated ion channels and modulation of GABAergic activity might be involved in its anticonvulsant activity. Copyright

Full text of DOI:10.1002/ardp.201200201

Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
127
Full Paper
Design, Synthesis, and Anticonvulsant Activity Evaluation of
4-(3-Alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones
Bing Shu, Yan Zheng, Shi-Ben Wang, Xian-Qing Deng, and Zhe-Shan Quan
College of Pharmacy, Yanbian University, Jilin, China
A series of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones were synthesized using the appropriate
synthetic route and evaluated experimentally in the maximal electroshock test; their neurotoxicities
were evaluated by the rotarod neurotoxicity test. The structures of these compounds were confirmed
by IR, MS, 1H-NMR, and elementary analysis. All target compounds exhibited anticonvulsant activity
to varying degrees in the maximal electroshock test. 4-(3-Benzyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-
3-one (4i) was the most promising compound with an ED50 value of 30.5 mg/kg and a protective index
(PI) of 18.63, showing a higher safety than the standard carbamazepine (PI ¼ 6.45). In addition, the
potency of compound 4i against seizures induced by pentylenetetrazole and 3-mercaptopropionic
acid suggested its broad-spectrum activity, and the mechanisms of action including inhibition of
voltage-gated ion channels and modulation of GABAergic activity might be involved in its
anticonvulsant activity.
Keywords: Anticonvulsant / Maximal electroshock / Neurotoxicity / Synthesis / Triazolone
Received: May 10, 2012; Revised: September 27, 2012; Accepted: October 9, 2012
DOI 10.1002/ardp.201200201
Introduction
most of the antiepileptic drugs make it difficult to use
rational methodologies in the field of drug discovery.
Fortunately, another design of new antiepileptics is based
on the existence of different pharmacophores that were
established through the analysis of structural characteristics
of clinically effective drugs as well as other antiepileptic
compounds [8–10]. In the literature [11, 12], it is well docu-
mented that one of the important core fragments is defined
by the presence of a hydrophobic unit (A), an electron donor
group (D), and a hydrogen donor/acceptor unit (HAD; Fig. 1).
Much efforts devoted in the recent years based on the phar-
macophore model for the development of novel therapeutics
resulted in the availability of several newer drugs (such as
tiagabine, lamotrigine, pregabalin, stiripentol, zonisamide,
topiramate, levetiracetam) as promising antiepileptics [13].
In the previous work we have reported the synthesis and
anticonvulsant activity evaluation of some compounds con-
taining triazolone. All of them exhibited certain anticonvul-
sant activity [14–17]. A series of 8-alkoxy-4,5-dihydro-[1,2,4]-
triazolo[4,3-a]quinoline-1-ones (I) were found outstanding in
respect of their anticonvulsant activities [17]. As part of our
continuous efforts to find better anticonvulsant agents in this
area, a series of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-
ones were designed and synthesized using the compounds I
Epilepsy is one of the most common neurological disorders,
affecting about 1% of the world’s population. Much efforts
devoted in the recent years to the development of novel
therapeutics resulted in the availability of several newer
drugs as promising anticonvulsants [1, 2]. However, the cur-
rently available anticonvulsants are effective in reducing the
severity and number of seizures in less than 70% of patients.
Moreover, their usage is associated with numerous undesir-
able side effects [3–5]. The currently used antiepileptic drugs
like phenytoin, carbamazepine, ethosuximide, valproic acid,
and barbiturates though widely prescribed produce adverse
effects such as ataxia, hepatotoxicity, gingival hyperplasia,
and megaloblastic anemia [6, 7]. The search for antiepileptic
agents with more selectivity and lower toxicity continues to
be an area of investigation in medicinal chemistry.
The insufficient information on the cellular mechanism of
epilepsy in human and the complex mechanism of action of
Correspondence: Dr. Zhe-Shan Quan, College of Pharmacy, Yanbian
University, No. 977, Park road, Yanji, Jilin 133002, China.
E-mail: zsquan@ybu.edu.cn
Fax: þ86-433-2436020
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Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
Figure 1. Pharmacophoric pattern of well-known antiepileptics and target compounds with their vital structural features.
(Fig. 2) as leading compounds. The target compounds were the
ring-opening product of compounds I. Assigning the fragments
of the target compounds using the above-mentioned model
gave the following results: The phenyl group with the sub-
stituents on it is the A unit, the oxygen atom is the D unit,
and the amide group is the HAD unit (Fig. 1).
neurotoxicity was evaluated using the rotarod test in mice.
For explaining the possible mechanism of action, compound
4i was tested in pentylenetetrazole (PTZ) and 3-mercaptopro-
pionic acid (3-MP) induced seizure tests. In the same con-
dition, the anti-MES activity and the neurotoxicity of the
marketed agent carbamazepine were evaluated in our labora-
tory, taken as comparison.
Their structures were characterized using IR, 1H-NMR, MS,
and elemental analysis techniques. In addition, their anti-
convulsant activity was evaluated using the MES test. Their Results and discussion
Chemistry
The protocol for the synthesis of target compounds 4a–4t is
shown in Scheme 1. N-(3-Hydroxyphenyl)acetamide reacted
with appropriate substituted alkyl halide in acetonitrile gave
compounds 2a–2t. Compounds 2a–2t reacted with appropri-
ate potassium hydroxide solution to obtain compounds 3a–
3t. The target compounds 4a–4t were synthesized through
the reaction of compounds 3a–3t with methyl hydrazinocar-
boxylate and triethyl orthoformate in anhydrous alcohol in
the presence of sodium methylate. All target compounds
Figure 2. The structures of compounds I and target compounds.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
Triazolones as Anticonvulsant Agents
129
Scheme 1. The synthesis route of compounds 4a–4t.
were characterized by elemental analysis, IR, 1H-NMR, and
MS data.
indicated that all compounds in this series were effective in
the i.p. MES screens, indicative of their ability to prevent
seizure spread. Among these compounds, ten compounds 4a–
4c, 4e–4g, 4i, 4k–4l, and 4n showed prominent anticonvul-
sant activities exhibiting protection against MES-induced
seizure at the low dose of 30 mg/kg. Almost all compounds
showed anticonvulsant activity at the dose of 100 mg/kg
except 4h, 4o, 4q, and 4r, which were active at the dose of
200 mg/kg.
Pharmacology
Pharmacological tests of the 4-(3-alkoxy-phenyl)-2,4-dihydro-
[1,2,4]triazol-3-ones (4a–4t) were conducted following the
protocol adopted by the Antiepileptic Drug Development
(ADD) program [18, 19].
The results of the preliminary (phase I) screening of 4a–4t
are summarized in Table 1. The phase I evaluation of 4a–4t
On the basis of the considerable anticonvulsant activity
suggested in phase I testing, compounds 4a–4c, 4e–4g, 4i, 4k–
4l, and 4n were subjected to phase II trials for quantification
of their anticonvulsant activity (indicated by ED50) and neuro-
toxicity (indicated by TD50) in mice. Results of the quantitat-
ive test for the selected compounds, along with the data of
the standard drug carbamazepine, are presented in Table 2.
Among the tested compounds, 4-(3-benzyloxy-phenyl)-2,4-
dihydro-[1,2,4]triazol-3-one (4i) was the most promising com-
pound with relatively high activity and the lowest toxicity. In
the anti-MES potency test, it showed a median effective dose
(ED50) of 30.5 mg/kg, a median toxicity dose (TD50) of
568.1 mg/kg, and a protective index (PI) of 18.6. The high PI
value gave it a bigger safety margin compared to carbamaze-
Table 1. Phase I evaluation of the anticonvulsant activity of target
compounds after intraperitoneal administration in mice.
RO
N
N
NH
O
Compounds
R
MES (mg/kg)a)
100
200
30
4a
4b
4c
4d
4e
4f
4g
4h
4i
–C2H5
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C12H25
3/3b)
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
1/3
3/3
1/3
2/3
3/3
3/3
3/3
3/3
3/3
2/3
3/3
3/3
2/3
0/3
3/3
3/3
3/3
3/3
2/3
3/3
0/3
2/3
0/3
0/3
2/3
3/3
2/5
1/5
3/5
0/5
3/5
3/5
2/5
0/5
4/5
0/5
3/5
2/5
0/5
3/5
0/5
0/5
0/5
0/5
0/5
0/5
Table 2. Phase II quantitative anticonvulsant data in mice (test drug
administered via the intraperitoneal route).
Compounds
ED50a) (MES)
TD50b) (NT)
PIc)
4a
4b
4c
4e
4f
4g
4i
4k
34.1 (29.5–39.3)d)
36.7 (32.1–41.9)
29.5 (24.5–35.3)
28.4 (24.6–32.8)
31.7 (27.5–36.6)
35.3 (30.9–40.4)
30.5 (26.7–34.9)
31.7 (27.4–36.6)
34.1 (29.5–39.3)
35.3 (30.9–40.4)
11.8 (9.7–14.1)
160.6 (120.7–213.8)
59.1 (41.2–78.6)
4.71
1.61
3.21
9.29
8.03
10.38
18.63
9.29
9.99
8.97
6.45
–CH2C6H5
–CH2C6H4(o-F)
–CH2C6H4(m-F)
–CH2C6H4(p-F)
–CH2C6H4(o-Cl)
–CH2C6H4(m-Cl)
–CH2C6H4(p-Cl)
–CH2C6H3(2,4-Cl)
–CH2C6H4(o-Br)
–CH2C6H4(p-Br)
94.7 (82.1–109.2)
264.1 (228.9–304.6)
254.6 (222.7–291.0)
366.6 (320.7–419.7)
568.1 (492.4–624.1)
294.6 (257.7–336.7)
340.8 (294.5–393.2)
316.9 (274.7–365.6)
76.1 (69.1–83.7)
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4l
4n
Carbarmazepine
–CH2C6H4(p-OCH3)
–CH2C6H4(p-CH3)
a)
ED50: median effective dose affording anticonvulsant protec-
tion in 50% of animals, the dose is measured in mg/kg.
a)
b)
Maximal electroshock: doses of 30, 100, and 200 mg/kg were
administrated intraperitoneally in mice. The animals were
examined 0.5 h after administration.
TD50: median toxic dose eliciting minimal neurological
toxicity in 50% of animals, the dose is measured in mg/kg.
c)
PI: protective index (TD50/ED50).
Ninety-five percent confidence intervals given in parentheses.
b)
d)
n1/n2: the animals protected/the animals tested.
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130
B. Shu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
pine, which has a PI value of 6.5. With a TD50 of 59.1 mg/kg,
compound 4b was more toxic than the other compounds.
Compound 4e was the most active compound with respect
to its ED50 of 28.4 mg/kg; however, a relatively high toxicity
(TD50 ¼ 264.1 mg/kg) was also seen.
known to inhibit or attenuate seizures. The findings of the
present study suggest that the newly synthesized compound
4i might inhibit or attenuate PTZ-induced seizures in mice by
enhancing GABAergic neurotransmission.
In the 3-MP induced seizure model, carbamazepine inhib-
ited the clonic seizures, tonic seizures, and death at the rates
of 0, 100, and 100%, respectively. In comparison, compound
4i showed an anticonvulsant effect similar to that of carba-
mazepine in inhibiting the clonic seizures and lethlity, and
inhibited the tonic seizures induced by 3-MP with the inhi-
bition rate of 70% (Table 3). Compound 4i exhibited the
efficacy to inhibit the seizures (tonic seizures) induced by
3-MP though the protection from death is barely satisfactory.
3-MP is a competitive inhibitor of the GABA synthesis enzyme
glutamate decarboxylase (GAD), and it inhibits the synthesis
of GABA resulting in decrease of GABA levels in the brain [23].
Compound 4i showed moderate antagonism to 3-MP induced
seizures suggesting that it might activate GAD or inhibit
aminotransferase (GABA-T) in the brain.
Analyzing the activities of the synthesized compounds 4a–
4t in Tables 1 and 2, the following structure–activity relation-
ships (SAR) were observed. From compounds 4e to 4h, the
length of the alkyl chain appeared to have no direct impact
on the anticonvulsant activity of these derivatives. Among
the benzyloxy-substituted derivatives, the position of the
substituted group on the phenyl ring appeared to greatly
influence the anticonvulsant activity. Comparing the deriva-
tives with different halogen substitutions on the benzene
ring, their activity order was m-F > p-F > o-F, m-Cl > o-Cl, 2,4-
Cl > p-Cl, p-Br > o-Br. Comparing the derivatives with differ-
ent electron-donating substitutions (methyl and methoxy) on
the benzene ring, their activity order was p-CH3 > p-OCH3.
To further investigate the effects of the anticonvulsant
activity in several different models and to speculate about
the possible mechanism of anticonvulsant action, compound
4i was tested against convulsions induced by chemical sub-
stances, including PTZ and 3-MP. Compound 4i was admin-
istered to mice i.p. at a dose of 30 mg/kg, which was higher
than four times its ED50 value and far below its TD50 value.
The reference drug carbamazepine was also administered i.p.
at a dose of 30 mg/kg.
In conclusion, we synthesized a series of 4-(3-alkoxy-phen-
yl)-2,4-dihydro-[1,2,4]triazol-3-ones and tested their anticon-
vulsant activity and neurotoxicity using the MES test and
rotarod tests, respectively. All compounds exhibited anti-
convulsant activity in varying degrees against MES. 4-(3-
Benzyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one (4i) was
the most promising compound with the ED50 value of
30.5 mg/kg and a PI value of 18.63, which showed the higher
safety than the standard carbamazepine (PI ¼ 6.45). The
potency of compound 4i against seizures induced by pentyl-
enetetrazole and 3-mercaptopropionic acid suggested its
broad-spectrum activity, and the mechanisms of action
including inhibition of voltage-gated ion channels and
modulation of GABAergic activity might be involved in its
anticonvulsant activity.
In the sc-PTZ model, carbamazepine inhibited the clonic
seizures, tonic seizures, and death at the rates of 0, 100, and
90%, respectively. While compound 4i inhibited the clonic
seizures, tonic seizures, and lethality at the rates of 0, 40, and
40% induced by sc-PTZ, respectively (Table 3). The data
showed that compound 4i and carbamazepine can protect
the mice from tonic seizures and death, which revealed that
compound 4i possesses activity against sc-PTZ. PTZ has been
reported to produce seizures by inhibiting g-aminobutyric
acid (GABA) neurotransmission [20, 21]. GABA is the main
inhibitory neurotransmitter in the brain, and is widely impli-
cated in epilepsy. Inhibition of GABAergic neurotransmission
or activity has been shown to promote and facilitate seizures
[22], while enhancement of GABAergic neurotransmission is
Experimental
Chemistry
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
Table 3. Effects of compound 4i on chemical-induced seizures in mice (i.p.).
Chemical substances
Pentylenetetrazol
Compound
Doses
(mg/kg)
Test time
(h)
Clonic seizures
(%)
Tonic seizures
(%)
Lethality
(%)
DMSO
Carbamazepine
4i
DMSO
Carbamazepine
4i
–
0.5
0.5
0.5
0.5
0.5
0.5
100
100
100
100
100
100
100
0
60
100
0
100
10
60
100
0
30
30
–
30
30
3-Mercaptopropionic acid
30
10
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Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
Triazolones as Anticonvulsant Agents
131
–
(Bruker, Switzerland), and all chemical shifts were given in ppm
relative to tetramethysilane. Mass spectra were measured on an
HP1100LC (Agilent Technologies, USA). Elemental analyses were
performed on a 204Q CHN (Perkin Elmer, USA). The chemicals
were purchased from Aldrich Chemical Corporation.
IR (KBr) cmꢁ1: 3110 (NH), 1707 (C O); MS-EI m/z 234 (Mþ1).
–
Anal. calcd. for C12H15N3O2: C, 61.79; H, 6.48; N, 18.01. Found:
C, 61.53; H, 6.25; N, 18.19.
4-(3-Pentyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4d
Yield: 30.2%, mp: 108–1108C. 1H-NMR (CDCl3, 300 MHz): d 0.93
(t, 3H, J ¼ 6.97 Hz, –CH3), 1.36–1.50 (m, 4H, –CH2–), 1.76–1.86
(m, 2H, –CH2–), 4.00 (t, 2H, J ¼ 6.51 Hz, –OCH2–), 6.89–7.40
Synthesis of N-(3-alkoxy-phenyl)acetamide 2a–2t
Compound 1 (16.6 mmol) and anhydrous potassium carbonate
(24.9 mmol) were placed into a round-bottomed flask containing
35 mL acetonitrile, and the mixture was stirred at room tempera-
ture for 0.5 h, then alkyl halide (19.9 mmol) was added and
refluxed for 8 h. After the reaction was completed, the solvent
was evaporated under reduced pressure. The residue was washed
with 30 mL water to produce a white solid 2a–2t.
–
(m, 4H, Ar-H), 7.71 (s, 1H, –N CH–), 10.48 (s, 1H, –NHCO–).
–
IR (KBr) cmꢁ1: 3112 (NH), 1707 (C O); MS-EI m/z 248 (Mþ1).
–
–
Anal. calcd. for C13H17N3O2: C,63.14; H, 6.93; N, 16.99. Found:
C, 63.33; H, 6.77; N, 16.76.
4-(3-Hexyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4e
Yield: 37.7%, mp: 120–1228C. 1H-NMR (CDCl3, 300 MHz): d 0.91
(t, 3H, J ¼ 6.83 Hz, –CH3), 1.31–1.36 (m, 4H, –CH2–), 1.42–1.84
(m, 4H, –CH2–), 3.99 (t, 2H, J ¼ 6.50 Hz, –OCH2–), 6.89–7.40
Synthesis of 3-alkoxy-benzenamine 3a–3t
A solution of 50% potassium hydroxide was added into a solution
of compound 2a–2t in 30 mL anhydrous alcohol, then the mix-
ture was stirred and heated at 908C for 18 h. After the solvent was
evaporated under reduced pressure, the residue was dissolved in
dichloromethane (30 mL), washed with water (30 mL ꢀ 3) and
dried over anhydrous MgSO4. Evaporation of the solvents gave a
crude product, which was used without purification in further
syntheses.
–
(m, 4H, Ar-H), 7.70 (s, 1H, –N CH–), 10.18 (s, 1H, –NHCO–).
–
IR (KBr) cmꢁ1: 3109 (NH), 1709 (C O); MS-EI m/z 262 (Mþ1).
–
–
Anal. calcd. for C14H19N3O2: C,64.35; H, 7.33; N, 16.08. Found:
C, 64.13; H, 7.11; N, 16.33.
4-(3-Heptyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4f
Yield: 46.9%, mp: 106–1088C. 1H-NMR (CDCl3, 300 MHz): d 0.89
(t, 3H, J ¼ 6.43 Hz, –CH3), 1.25–1.38 (m, 4H, –CH2–), 1.42–1.48
(m, 2H, –CH2–), 1.75–1.84 (m, 2H, –CH2–), 3.99 (t, 2H, J ¼ 6.48 Hz,
Synthesis of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-
3-one 4a–4t
–
–
–OCH –), 6.89–7.40 (m, 4H, Ar-H), 7.71 (s, 1H, –N CH–), 10.6
2
(s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3108 (NH), 1706 (C O); MS-EI
–
–
m/z 276 (Mþ1). Anal. calcd. for C15H21N3O2: C, 65.43; H, 7.69;
N, 15.26. Found: C, 65.20; H, 7.48; N, 15.06.
Compounds 3a–3t (10 mmol) and methyl hydrazinocarboxylate
(15 mmol) with triethyl orthoformate (15 mmol) were placed
into a round-bottomed flask containing 30 mL anhydrous alco-
hol and refluxed for 24 h, then sodium methylate (15 mmol) was
added and refluxed for 24 h. After the reaction was completed,
the solvent was evaporated under reduced pressure, the residue
was dissolved in dichloromethane (30 mL), washed with water
(10 mL ꢀ 3) and dried over anhydrous MgSO4, which was puri-
fied by silica gel column chromatography with methanol-
dichloromethane (1:80) to a white solid.
4-(3-Octyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4g
Yield: 36.6%, mp: 98–1008C. 1H-NMR (CDCl3, 300 MHz): d 0.88
(t, 3H, J ¼ 6.54 Hz, ꢁCH3), 1.25–1.84 (m, 12H, –CH2–), 3.99
(t, 2H, J ¼ 6.50 Hz, –OCH2–), 6.88–7.40 (m, 4H, Ar-H), 7.71
(s, 1H, –N CH–), 10.57 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3111 (NH),
–
–
–
1707 (C O); MS-EI m/z 276 (Mþ1). Anal. calcd. for C16H23N3O2:
–
C, 66.41; H, 8.01; N, 14.52. Found: C, 66.20; H, 8.28; N, 14.26.
4-(3-Ethoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4a
Yield: 40.2%, mp: 118–1208C. 1H-NMR (CDCl3, 300 MHz): d 1.43
(t, 3H, J ¼ 6.97 Hz, –CH3), 4.07 (q, 2H, J ¼ 6.97 Hz, –OCH2–), 6.88–
4-(3-Dodecyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one
4h
–
7.40 (m, 4H, Ar-H), 7.71 (s, 1H, –N CH–), 10.89 (s, 1H, –NHCO–).
–
Yield: 43.1.2%, mp: 124–1268C. 1H-NMR (CDCl3, 300 MHz): d 0.87
(t, 3H, J ¼ 6.64 Hz, –CH3), 1.25–2.01 (m, 20H, –CH2–), 3.98
(t, 2H, J ¼ 6.51 Hz, –OCH2–), 6.89–7.40 (m, 4H, Ar-H), 7.70
IR (KBr) cmꢁ1: 3109 (NH), 1708 (C O); MS-EI m/z 206 (Mþ1).
–
–
Anal. calcd. for C10H11N3O2: C, 58.53; H, 5.40; N, 20.48. Found:
C, 58.26; H, 5.14; N, 20.24.
(s, 1H, –N CH–), 10.01 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3109 (NH),
–
1707 (C O); MS-EI m/z 346 (Mþ1). Anal. calcd. for C20H31N3O2:
–
C, 69.53; H, 9.04; N, 12.16. Found: C, 69.30; H, 9.28; N, 12.35.
–
–
4-(3-Propoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4b
Yield: 24.9%, mp: 116–1188C. 1H-NMR (CDCl3, 300 MHz): d 1.05
(t, 3H, J ¼ 7.39 Hz, –CH3), 1.77–1.89 (m, 2H, –CH2–), 3.96
(t, 2H, J ¼ 6.51 Hz, –OCH2–), 6.89–7.40 (m, 4H, Ar-H), 7.71
4-(3-Benzyloxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4i
Yield: 49.8%, mp: 152–1548C. 1H-NMR (CDCl3, 300 MHz): d 5.13
(s, 1H, –N CH–), 10.71 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3108 (NH),
–
–
–
(s, 2H, –OCH –), 6.97–7.46 (m, 9H, Ar-H), 7.69 (s, 1H, –N CH–),
2
–
–
9.97 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3109 (NH), 1706 (C O); MS-EI
–
1708 (C O); MS-EI m/z 220 (Mþ1). Anal. calcd. for C11H13N3O2:
–
m/z 268 (Mþ1). Anal. calcd. for C15H13N3O2: C, 67.40; H, 4.90;
N, 15.72. Found: C, 67.18; H, 4.68; N, 15.56.
–
C, 60.26; H, 5.98; N, 19.17. Found: C, 60.06; H, 5.80; N, 19.40.
4-(3-Butoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-one 4c
Yield: 33.8%, mp: 124–1268C. 1H-NMR (CDCl3, 300 MHz): d 0.88
(t, 3H, J ¼ 7.35 Hz, –CH3–), 1.23–1.25 (m, 2H, –CH2–), 1.70–1.79
(m, 2H, –CH2–), 4.01 (t, 2H, J ¼ 6.45 Hz, –OCH2–), 7.01–7.54
4-[3-(2-Fluoro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4j
Yield: 45.2%, mp: 122–1248C. 1H-NMR (CDCl3, 300 MHz): d 5.17
–
–
(m, 4H, Ar-H), 7.67 (s, 1H, –N CH–), 10.23 (s, 1H, –NHCO–).
–
(s, 2H, –OCH –), 6.98–7.50 (m, 8H, Ar-H), 7.71 (s, 1H, –N CH–),
–
2
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B. Shu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
10.04 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3108 (NH), 1706 (C O); MS-EI
–
m/z 346 (Mþ1). Anal. calcd. for C15H12BrN3O2: C, 52.04; H, 3.49;
–
m/z 286 (Mþ1). Anal. calcd. for C15H12FN3O2: C, 63.15; H, 4.24;
N, 12.14. Found: C, 51.89; H, 3.68; N, 12.31.
N, 14.73. Found: C, 63.38; H, 4.48; N, 14.56.
4-[3-(4-Bromo-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4r
4-[3-(3-Fluoro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4k
Yield: 25.7%, mp: 204–2068C. 1H-NMR (CDCl3, 300 MHz): d 5.06
Yield: 38.3%, mp: 108–1108C. 1H-NMR (CDCl3, 300 MHz): d 5.11
(s, 2H, –OCH –), 6.94–7.54 (m, 8H, Ar-H), 7.70 (s, 1H, –N CH–),
–
–
2
–
(s, 2H, –OCH –), 6.96–7.43 (m, 8H, Ar-H), 7.72 (s, 1H, –N CH–),
9.63 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3109 (NH), 1710 (C O); MS-EI
–
–
–
m/z 346 (Mþ1). Anal. calcd. for C15H12BrN3O2: C, 52.04; H, 3.49;
N, 12.14. Found: C, 52.34; H, 3.68; N, 12.02.
2
9.82 (s, 1H, –NH–CO–). IR (KBr) cmꢁ1: 3115 (NH), 1710 (C O); MS-EI
–
–
m/z 286 (Mþ1). Anal. calcd. for C15H12FN3O2: C, 63.15; H, 4.24;
N, 14.73. Found: C, 63.38; H, 4.41; N, 14.58.
4-[3-(4-Methoxy-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4s
4-[3-(4-Fluoro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4l
Yield: 28.3%, mp: 206–2088C. 1H-NMR (CDCl3, 300 MHz): d 3.82
(s, 3H, –OCH3), 5.04 (s, 2H, –OCH2–), 6.92–7.41 (m, 8H, Ar-H), 7.69
Yield: 30.3%, mp: 146–1488C. 1H-NMR (CDCl3, 300 MHz): d 5.07
–
(s, 2H, –OCH –), 6.95–7.44 (m, 8H, Ar-H), 8.46 (s, 1H, –N CH–),
(s, 1H, –N CH–), 9.91 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3112 (NH),
–
–
–
2
10.13 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3113 (NH), 1709 (C O); MS-EI
–
1707 (C O); MS-EI m/z 297 (Mþ1). Anal. calcd. for C16H15N3O3:
–
–
m/z 286 (Mþ1). Anal. calcd. for C15H12FN3O2: C, 63.15; H, 4.24;
N, 14.73. Found: C, 63.31; H, 4.48; N, 14.89.
–
C, 64.64; H, 5.09; N, 14.13. Found: C, 64.41; H, 5.28; N, 14.36.
4-[3-(4-Methyl-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4t
4-[3-(2-Chloro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4m
Yield: 23.5%, mp: 188–1908C. 1H-NMR (CDCl3, 300 MHz): d 2.37
(s, 3H, –CH3), 5.07 (s, 2H, –OCH2–), 6.96–7.42 (m, 8H, Ar-H), 7.69
Yield: 25.8%, mp: 164–1668C. 1H-NMR (CDCl3, 300 MHz): d 5.21
–
(s, 2H, –OCH –), 6.98–7.58 (m, 8H, Ar-H), 7.71 (s, 1H, –N CH–),
(s, 1H, –N CH–), 9.99 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3112 (NH),
–
–
–
2
9.52 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3109 (NH), 1706 (C O); MS-EI
–
1708 (C O); MS-EI m/z 281 (Mþ1). Anal. calcd. for C16H15N3O2:
–
–
m/z 303.5 (Mþ1). Anal. calcd. for C15H12ClN3O2: C, 59.71; H, 4.01;
N, 11.75. Found: C, 59.51; H, 4.39; N, 11.56.
–
C, 68.31; H, 5.37; N, 14.94. Found: C, 68.20; H, 5.21; N, 14.67.
Pharmacology
4-[3-(3-Chloro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4n
The MES 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) [24,
25]. All compounds, which were dissolved in dimethylsulfoxide,
were evaluated for anticonvulsant activities in KunMing mice in
the 18–25 g weight range. In phase I screening, each compound
was administered at the dose levels of 30, 100, and 200 mg/kg for
evaluating preliminarily the anticonvulsant activity. For deter-
mination 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 these data, the respective ED50, TD50 values, and
95% confidence intervals were calculated by probit analysis.
Yield: 27.4%, mp: 144–1468C. 1H-NMR (CDCl3, 300 MHz): d 5.08
–
(s, 2H, –OCH –), 6.96–7.46 (m, 8H, Ar-H), 7.70 (s, 1H, –N CH–),
–
2
10.21 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3114 (NH), 1708 (C O); MS-EI
–
–
m/z 303.5 (Mþ1). Anal. calcd. for C15H12ClN3O2: C, 59.71; H, 4.01;
N, 11.75. Found: C, 59.90; H, 4.31; N, 11.89.
4-[3-(4-Chloro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4o
Yield: 23.2%, mp: 188–1908C. 1H-NMR (CDCl3, 300 MHz): d 5.08
–
(s, 2H, –OCH –), 6.94–7.38 (m, 8H, Ar-H), 7.70 (s, 1H, –N CH–),
–
2
9.49 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3110 (NH), 1706 (C O); MS-EI
–
–
m/z 303.5 (Mþ1). Anal. calcd. for C15H12ClN3O2: C, 59.71; H, 4.01;
N, 11.75. Found: C, 59.49; H, 4.20; N, 11.51.
Maximal electroshock test (MES)
4-[3-(2,4-Dichloro-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4p
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 MES-induced seizures
was defined as the abolition of the hind leg and tonic maximal
extension component of the seizure. The derivatives in MES test
were evaluated 30 min after administration of the compounds.
Yield: 35.2%, mp: 166–1708C. 1H-NMR (CDCl3, 300 MHz): d 5.17
–
(s, 2H, –OCH –), 6.95–7.52 (m, 8H, Ar-H), 7.72 (s, 1H, –N CH–),
–
2
10.72 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3113 (NH), 1708 (C O); MS-EI
–
–
m/z 337 (Mþ1). Anal. calcd. for C15H11Cl2N3O2: C, 53.59; H, 3.30;
N, 12.50. Found: C, 53.41; H, 3.08; N, 12.69.
Neurotoxicity (NT) screening
4-[3-(2-Bromo-benzyloxy)-phenyl]-2,4-dihydro-[1,2,4]-
triazol-3-one 4q
The neurotoxicity of the compounds were measured in mice by
the rotarod test. The mice were trained to stay on an accelerating
rotarod of diameter 3.2 cm that rotates at 10 rpm. Trained
animals were given i.p. injection of the test compounds.
Neurotoxicity was indicated by the inability of the animal to
Yield: 41.6%, mp: 164–1668C. 1H-NMR (CDCl3, 300 MHz): d 5.17
–
(s, 2H, –OCH –), 7.00–7.62 (m, 8H, Ar-H), 7.71 (s, 1H, –N CH–),
–
–
2
10.11 (s, 1H, –NHCO–). IR (KBr) cmꢁ1: 3115 (NH), 1707 (C O); MS-EI
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 127–133
Triazolones as Anticonvulsant Agents
133
maintain equilibrium on the rod for at least 1 min in each of the
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0.5 h. The numbers of clonic seizures (range from exaggerated
twitches of the limbs to violent shaking or vibrating of the
stiffened extremities) and tonic seizures (the extremities pull
towards the body or rigidly push away from it, usually maximal
extension of the hind leg) as well as the number of deaths were
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This work was supported by the National Natural Science Foundation of
China (No. 81160382) and National Science and Technology Major Project
of China (No. 2011ZX09102-003-03).
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The authors have declared no conflict of interest.
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www.archpharm.com