Synthesis and anticonvulsant activity evaluation of 7-alkoxy[1,2,4] triazolo[3,4-b]benzothiazol-3(2H)-ones

Article abstract of DOI:10.1002/ardp.201300277

A new series of 7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-ones were synthesized and evaluated for their anticonvulsant activities. Among these compounds, 7-propoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4c) and 7-butoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4d) showed the highest activity against maximal electroshock (MES)-induced tonic extension [effective dose (ED)₅₀: 11.4 and 13.6 mg/kg, respectively]. It is worth mentioning that compound 4d showed especially low neurotoxicity, which led to a high protective index (PI >51). The orally anticonvulsant activity data of compound 4d further confirmed its efficacy, in an MES test, and its high safety with a PI value of 50.2. In addition, the potency of compound 4h against seizures induced by pentylenetetrazole, 3-mercaptopropionic acid, and bicuculline in the chemical-induced seizure tests suggested that compound 4d may exert its anticonvulsant activity through affecting the GABAergic system.

Full text of DOI:10.1002/ardp.201300277

268
Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Full Paper
Synthesis and Anticonvulsant Activity Evaluation of
7-Alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-ones
Da-Chuan Liu1, Xian-Qing Deng1,2, Shi-Ben Wang1, and Zhe-Shan Quan1
1
College of Pharmacy, Yanbian University, Yanji, Jilin, China
Medical College, Jinggangshan University, Ji’an, Jiangxi, China
2
A new series of 7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-ones were synthesized and evaluated for
their anticonvulsant activities. Among these compounds, 7-propoxy[1,2,4]triazolo[3,4-b]benzothiazol-3
(2H)-one (4c) and 7-butoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4d) showed the highest activity
against maximal electroshock (MES)-induced tonic extension [effective dose (ED)50: 11.4 and 13.6 mg/kg,
respectively]. It is worth mentioning that compound 4d showed especially low neurotoxicity, which led
to a high protective index (PI >51). The orally anticonvulsant activity data of compound 4d further
confirmed its efficacy, in an MES test, and its high safety with a PI value of 50.2. In addition, the potency
of compound 4h against seizures induced by pentylenetetrazole, 3-mercaptopropionic acid, and
bicuculline in the chemical-induced seizure tests suggested that compound 4d may exert its
anticonvulsant activity through affecting the GABAergic system.
Keywords: Anticonvulsant activity / Benzothiazole / Maximal electroshock / Pentylenetetrazole / Triazolone
Received: July 22, 2013; Revised: September 29, 2013; Accepted: October 1, 2013
DOI 10.1002/ardp.201300277
Introduction
ED50 of 8.0 mg/kg and a PI [toxic dose (TD)50/ED50] of 15.0 in the
maximal electroshock test (MES). In the current study, a series of
7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-ones (II) were
designed and synthesized, which replaced the triazole in
compounds I by triazolone. The design is based on a hypothesis
that the carbonyl group in the triazolone may increase
anticonvulsant activity of the aim compounds. This hypothesis
has already been confirmed in our previous work [9, 10]. The
structures of target compounds were characterized using IR, 1H
NMR, MS, and elemental analysis techniques. The anticonvul-
sant activities of the newly synthesized compounds were
evaluated using MES-induced seizures in mice as an experi-
mental epilepsy model. The rotarod assay was performed in
mice to evaluate the neurotoxicity of these compounds. To
elucidate the possible mechanism of action, the most active
compound, 4d, was tested in pentylenetetrazole (PTZ), 3-
mercaptopropionic acid (3-MP), and bicuculline (BIC)-induced
seizure tests. Orally anticonvulsant activity against MES-
induced seizures and orally neurotoxicity in mice were also
evaluated for compound 4d.
Epilepsy is a syndrome of different cerebral disorders of the
central nervous system (CNS), characterized by paroxysmal,
excessive, and hypersynchronous discharges of large numbers
of neurons [1]. In recent years, extensive efforts have been
devoted to the development of novel therapeutics, and these
efforts have resulted in the development of several new
promising anticonvulsant drug candidates [2, 3]. Currently
however, available anticonvulsants are only effective in
reducing the severity and number of seizures in <70% of
patients. Furthermore, their usage is often associated with
numerous undesirable side effects [4–6] and even life-
threatening conditions [7]. Research to find more effective
and safer antiepileptic drugs is therefore imperative, yet
challenging in medicinal chemistry.
In our previous work, we reported the synthesis and
anticonvulsant activity of 7-alkoxy-triazolo[3,4-b]benzo[d]thia-
zoles (Fig. 1, I) [8]. Among these compounds, 7-octyloxy-triazolo-
[3,4-b]benzo[d]thiazole was the most active compound with an
Chemistry
Correspondence: Dr. Zhe-Shan Quan, College of Pharmacy, Yanbian
University, Yanji, Jilin 133002, China.
E-mail: zsquan@ybu.edu.cn
Based on previous studies in our laboratory, we designed and
prepared 7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
Fax: þ86 433 2436020
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Triazolones as Anticonvulsant Agents
269
Table 1. Preliminary screening of anticonvulsant activity in mice
(i.p.).
MESa)
TOXb)
(mg/kg)
(mg/kg)
Compound
R
30 100 200
100
C log pc)
4a
4b
4c
4d
4e
4f
4g
4h
4i
–CH3
–C2H5
–C3H7
–C4H9
–C5H11
–C6H13
–C7H15
–C8H17
–C12H25
–
0/3 0/3
0/3
1/3
2/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
1/3
1/3
1/3
0/3
2/3
1/3
0/3
0/3
0/3
0/3
0/3
1.4
1.9
2.4
3.0
Figure 1. Structure of compounds I and II.
0/3 1/3 2/3
3/3 3/3
3/3 3/3
1/3 2/3 3/3
0/3 1/3 2/3
0/3 1/3 3/3
–
–
–
–
–
3.5
derivatives (4a–u). Target compounds 4a–u were synthesized
according to Scheme 1. The starting material, 6-methoxy-1,3-
benzothiazol-2-amine, was reacted with hydrobromic acid
(48% water solutions) to obtain compound 1 [11], which was
reacted further with appropriate alkanol and substituted
phenol in acetone to obtain derivatives 2a–u [12]. Compounds
2a–u were treated with hydrazine hydrate in the presence
of sulfuric acid (98% water solutions) to produce derivatives
3a–u [8]. Finally, compounds 3a–u were converted to the
target compounds 4a–u by mixing with urea in the molten
state [13]. Their chemical structures were characterized using
IR, 1H NMR, MS, and elemental analysis techniques. A detailed
overview of their physical and analytical data has been
provided in the Experimental part.
4.01
4.54
5.07
7.19
8.24
3.13
3.28
3.28
3.28
3.85
3.85
3.85
3.63
4.02
2.71
4.56
0/3 1/3
0/3 0/3
0/3 0/3
4j
4k
4l
–C14H29
–C6H5
0/3 1/3 2/3
2/3 3/3
0/3 1/3 2/3
0/3 1/3 1/3
0/3 1/3 2/3
0/3 2/3 3/3
–
–
–
–
–
–C6H5(o-F)
–C6H5(m-F)
–C6H5(p-F)
–C6H5(o-Cl)
–C6H5(m-Cl)
–C6H5(p-Cl)
–C6H5(p-CH3)
–C6H5(m-CF3)
–C6H5(o-CN)
–C6H5(2,6-2Cl)
–
4m
4n
4o
4p
4q
4r
4s
4t
0/3 0/3
0/3 0/3
0/3 0/3
0/3 0/3
0/3 0/3
4u
a)
Maximal electroshock seizure test (number of animals
protected/number of animals tested).
Results and discussion
b)
Rotarod toxicity (number of animals exhibiting toxicity/
number of animals tested).
Lipid–water partition coefficient, calculated by ChemDraw
Ultra 8.0.
c)
Preliminary screening of the anticonvulsant activity
During the preliminary screening process, compounds were
administered by intraperitoneal (i.p.) injection at dosages of
200, 100, and 30 mg/kg. Their anticonvulsant effects and
neurovirulence were assessed at 0.5-h intervals following
administration in mice. The following observations were
obtained and are listed in Table 1.
From Table 1, we can see that 4b–g and 4k–p displayed
noticeable anticonvulsant activity at a dosage of 100 mg/kg. Of
these compounds, compounds 4c and 4d exhibited absolute
protection at a dosage of 30 mg/kg, whereas compounds 4e
and 4l showed partial protection (1/3 or 2/3) at this dosage.
Some of these compounds displayed a little neurovirulence at
a dosage of 100 mg/kg, but not so strong.
Quantitative evaluation of the anticonvulsant activity
Based on the considerable anticonvulsant activity demon-
strated during the preliminary screening test, compounds
4c–e and 4l were subjected to quantitative evaluation trials
for quantification of their anticonvulsant activity (indicated
by ED50) and neurotoxicity (indicated by TD50) in mice. Results
of the quantitative testing of selected compounds, together
with data of the antiepileptic drug carbamazepine (measured
in the same condition), are shown in Table 2.
The four compounds tested gave ED50 values in the range of
11.4–73.7 mg/kg. Compound 4c is 11.4 mg/kg (45.78 mmol/kg);
4d is 13.6 mg/kg (51.71 mmol/kg); 4e is 73.3 mg/kg
(264.62 mmol/kg); and 4l is 26.4 mg/kg (83.8 mmol/kg), and
TD50 values in the range of 96.0 to more than 700 mg/kg,
leading to PI values in the range of 4.2 to more than 51.
Among the compounds tested, compound 4c showed equal
MES activity (ED50 ¼ 11.4 mg/kg, 45.78 mmol/kg) and PI (8.5) to
carbamazepine (ED50 ¼ 11.8 mg/kg, 49.94 mmol/kg, PI ¼ 6.4).
Compound 4d had a comparable MES activity (ED50 ¼ 13.6
mg/kg, 51.71 mmol/kg) and a much lower neurotoxicity
Scheme 1. Synthetic route of the target compounds (4a–u).
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

270
D.-C. Liu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Table 2. Quantitative anticonvulsant data in mice (i.p.).
a)
b)
Compound
R
MES, ED50 (mg/kg)
TOX, TD50 (mg/kg)
PIc)
8.5
>51
4.2
4c
4d
4e
4l
–C3H7
–C4H9
–C5H11
–C6H5(o-F)
–
11.4 (9.85–13.11)d)
13.6 (11.81–15.72)
73.3 (64.14–83.81)
26.4 (22.89–30.47)
11.8 (8.5–16.4)
96.0 (71.93–129.65)
>700
311.1 (238.13–406.53)
126.8 (109.87–146.23)
76.1 (55.8–103.7)
4.8
6.4
Carbamazepine
a)
ED50: median effective dose affording anticonvulsant protection in 50% of animals; the dose is measured in mg/kg.
TD50: median toxic dose eliciting minimal neurological toxicity in 50% of animals; the dose is measured in mg/kg.
PI: protective index (TD50/ED50).
b)
c)
d)
95% confidence intervals given in parentheses.
(TD50 > 700 mg/kg) compared to carbamazepine, resulting in
a PI of more than 51.
promising compound with an ED50 of 26.4 mg/kg, TD50 of
126.8 mg/kg, and PI of 4.8. Comparing the derivatives with
different F-substitution positions on the benzyl ring, their
activity order was 2-F > 3-F > 4-F. The activity order of the Cl-
substituted derivatives was 3-Cl > 2-Cl > 4-Cl > 2.4-2Cl. Some
derivatives 4r–t, which have other substituent groups
containing 4-CH3, 3-CF3, and 2-CN, were also designed and
prepared. However none of these exhibited any activity,
independent of the dosage administered.
As a result of the quantitative evaluation, compound 4d
showed strong anticonvulsant activity and the best PI value in
MES test, so it was then selected for further investigations
against seizures induced by PTZ, 3-MP, and BIC to prove its
anticonvulsant activity and speculate about the possible
mechanism of anticonvulsant action. We used 10 mice per
group in our experiment. Compound 4d was administered to
mice at 30 mg/kg i.p., which was higher than its ED50 value
and far below its TD50 value. The reference drug carbamaze-
pine 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 90%, respectively. Compound 4d inhibited the clonic
seizures, tonic seizures, and lethality at the rates of 10%, 70%,
and 70% induced by sc-PTZ, respectively (Table 3). From these
data, we can see that compound 4d can inhibit the tonic
seizures and lethality induced by PTZ. PTZ has been reported
to produce seizures by inhibiting g-aminobutyric acid (GABA)
neurotransmission [15, 16]. GABA is the main inhibitory
Structure–activity relationships
Following activity analysis of the synthesized compounds, the
following structure–activity relationships (SARs) were
obtained. For the 12 alkyl chain-substituted derivatives 4a–
j, the length of the alkyl chain appeared to have a direct
impact on the anticonvulsant activity of the derivatives. For
example, as the alkyl chain length increased from 4a to 4c, the
anticonvulsant activity gradually increased, with compound
4c (with the n-propane chain) being the most active
compound. From 4c to 4k, although the alkyl chain length
increased further, the anticonvulsant activity decreased and
ultimately disappeared. This SARs might be associated with
the lipid–water partition coefficients of the compounds,
which affects drug absorption, hydrophobic drug–receptor
interactions, metabolism of molecules, and especially the
ability to cross the blood–brain barrier (BBB). All these factors
will have an effect on drug efficiency in the CNS. The optimum
log p-value of drugs acting on the CNS is around 2.0 [14], since
at this point their movement through the aqueous and
lipophilic phases of living tissues will be inhibited to a lesser
extent. From the calculated log p values (C log p), it can be
observed that compounds 4a–j exhibited C log p values
ranging from 1.4 to 7.2 (Table 1), and the C log p of the most
active compound, 4c, was 2.4, which is quite close to the
optimum log p-value of 2.0. To some compounds, which have
large substituent group (like 4i, 4j, 4s, etc.), their low activity
may be related to their large substituent.
Table 3. Effects of compound 4d on sc pentylenetetrazol-induced
Compound 4k was substituted with a benzyloxy group at
the 6-position of the triazolobenzothiazole core and F, Cl, CH3,
CF3, and CN groups were subsequently added onto the
benzyloxy group of 4k in different positions, providing
compounds 4l–u. The anticonvulsant activities of these
compounds were weaker than for the compounds mentioned
above. Only one compound (4l) showed an MES activity below
30 mg/kg, whereas the others showed weak activity at 100 or
200 mg/kg. Among 4k–u, compound 4l was the most
seizures in micea) (i.h.).
Test
Clonic
Tonic
Doses time seizures seizures Lethality
Compound
(mg/kg)
(h)
(%)
(%)
(%)
DMSO
Carbamazepine
4d
0.5
0.5
0.5
100
100
90
100
0
30
100
10
30
30
30
a)
The number of animals tested is 10.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Triazolones as Anticonvulsant Agents
271
neurotransmitter in the brain, and is widely implicated in
epilepsy. Inhibition of GABAergic neurotransmission or
activity has been shown to promote and facilitate seizures
[17]. While GABAergic enhancement is known to inhibit or
attenuate seizures. The inhibition of compound 4d upon the
PTZ-induced tonic seizures might be related to the enhancing
of GABAergic neurotransmission or activity.
antagonist of the GABAA receptor [19]. As compound 4d
inhibited the tonic seizures induced by BIC, it likely exerts
anticonvulsant activity, at least partially, through GABAA-
mediated mechanisms.
Pharmacological evaluation of compound 4d
administered orally to mice
In the 3-MP-induced seizure model, carbamazepine inhib-
ited the clonic seizures, tonic seizures, and death at rates of
0%, 100%, and 100%, respectively. By comparison, compound
4d showed an anticonvulsant effect similar to that of
carbamazepine in inhibiting the clonic and tonic seizures.
But its protection for the death induced by 3-MP cannot
compare with carbamazepine, with inhibition of death
induced by 3-MP at rate of 30% (Table 4). 3-MP is competitive
inhibitor of the GABA synthesis enzyme glutamate decarbox-
ylase (GAD), and it inhibits the synthesis of GABA resulting in
decrease of GABA levels in the brain [18]. Compound 4d
exhibited the efficacy to inhibit the tonic seizures induced by
3-MP, though the protection for death is less satisfactory than
carbamazepine, suggesting that it might activate GAD or
inhibit GABA transaminase (GABA-T) in the brain.
To obtain the oral time to peak effect (TPE) of compound 4d,
we conducted a time-course test, in which compound 4d
reached the TPE at 1.5 h after administering oral medications
(p.o.; Fig. 2). We next evaluated the orally anticonvulsant
activity of compound 4d against MES-induced seizures and
oral neurotoxicity in mice (Table 6) with carbamazepin as the
reference. We also got the oral LD50 data of 4d (Table 6). The
data in Table 6 clearly indicate a decrease in the anticonvul-
sant potency and neurotoxicity of 4d in p.o. compared to
intraperitoneal (i.p.) administration. Additionally, the oral
MES activity was lower than that of carbamazepin. Neverthe-
less, the PI value was still relatively high (PI ¼ 50.2), which
gives compound 4d a broader safety margin than that of
carbamazepin (PI ¼ 50.2 vs. 11.1).
In the BIC-induced seizure model, both carbamazepine and Conclusion
4d inhibited tonic seizures and death, but did not inhibit
clonic seizures. Carbamazepine showed inhibition of clonic
and tonic seizures and death at rates of 0%, 100%, and 40%,
respectively. Compound 4d showed inhibition of tonic
seizures at rate of 80%, but no protection for the clonic
and seizures and death (Table 5). BIC is a competitive
Among the series of 7-alkoxy[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-ones, compounds 4c and 4d were the most active in this
study with high activity in the MES assay (ED50 ¼ 11.4,
13.6 mg/kg, respectively). Compound 4d showed a special low
neurotoxicity, much lower than compound 4c. It also led to
the conclusion that 4d has a high PI (>51). The PI has also
higher safety than marketed drug carbamazepine (PI ¼ 6.4).
The orally anticonvulsant activity data of compound 4d also
showed that 4d has a high safety with a PI value of 50.2. The
potency of compound 4d against seizures induced by
pentylenetetrazole, 3-MP, and BIC in the chemical-induced
seizure test suggested that compound 4d displayed wide
Table 4. Effects of compound 4d on 3-mercaptopropionic acid-
induced seizures in micea) (i.h.).
Test
Clonic
Tonic
Doses time seizures seizures Lethality
Compound
(mg/kg)
(h)
(%)
(%)
(%)
DMSO
Carbamazepine
4d
0.5
0.5
0.5
100
100
100
100
0
20
100
0
70
30
30
a)
The number of animals tested is 10.
Table 5. Effects of compound 4d on bicuculline-induced seizures
in micea) (i.h.).
Test
Clonic
Tonic
Doses time seizures seizures Lethality
Compound
(mg/kg)
(h)
(%)
(%)
(%)
DMSO
Carbamazepine
4d
0.5
0.5
0.5
100
80
100
100
0
20
100
60
100
30
30
Figure 2. Time-course of compound 4d (50 mg/kg) in the maximal
electroshock seizure test (the number of animals at each point was
8; o.p.).
a)
The number of animals tested is 10.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

272
D.-C. Liu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Table 6. Pharmacological evaluation of compound 4d and carbamazepin administered orally to mice.
Compound
R
MES, ED50 (mg/kg)
TOX, TD50 (mg/kg)
PI
4d
–C4H9
–
34.1 (29.54–39.32)
18.3 (16.04–20.95)
1710.4 (1587.24–1843.09)
203.7 (178.19–232.82)
50.2
11.1
Carbamazepine
spectrum activity in several models. Though the specific
mechanism is still unclear, the results suggest that GABA-
mediated mechanisms might be involved in its anticonvul-
sant activity, such as enhancing of GABAergic neurotransmis-
sion or activity, activating GAD or inhibiting GABA-T, and
mediating GABAA-receptors.
the mixture and heated at 140°C for 5 h. After cooling to the room
temperature, the mixture was added into 50 mL of ice water. The
precipitate formed was filtered and washed with water to obtain a
light green needle-like solid.
General procedure for the synthesis of 7-alkoxy[1,2,4]
triazolo[3,4-b]benzothiazol-3(2H)-ones (4a–u)
A mixture of compounds 3a–u (10 mmol) and diaminomethanal
(80 mmol) was heated to be fused for 4 h. Then the mixture
was poured into hot water with stirring for 0.5 h. The remaining
precipitate was filtered to obtain a crude product, which
was purified by silica gel column chromatography with
CH2Cl2–CH3OH (50:1) to a white solid.
Experimental
Chemistry
General methods
Melting points were determined in open capillary tubes and are
uncorrected. IR spectra were recorded (in KBr) on a FT-IR1730
(Perkin-Elmer, USA). 1H NMR spectra were measured on a
BRUKER-300 (Bruker Bioscience, Billerica, MA, USA), and all
chemical shifts were given in ppm relative to tetramethylsilane.
Mass spectra were measured on an HP1100LC (Hewlett-Packard,
PaloAlto, CA, USA). Microanalyses of C, N, and H were performed
using a Heraeus CHN Rapid Analyzer (Heraeus GmbH, Hanau,
Germany). The major chemicals were purchased from Aldrich
Chemical Corporation. All other chemicals were of analytical
grade.
The yield, melting point, analytical data, and spectral data of
each compound are given below.
7-Methoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4a)
M.p. 266–268°C; yield: 68.6%; 1H NMR (300 MHz, CDCl3), d 3.79
(t, J ¼ 7.1 Hz, 3H, –O–CH3), 6.95 (dd, J ¼ 8.8, 2.6 Hz, 1H, Ar–H), 7.58
(d, J ¼ 2.6 Hz, 1H, Ar–H), 7.73 (d, J ¼ 8.8 Hz, 1H, Ar–H), 12.15 (s, 1H,
–NH). 13C NMR (300 MHz, CDCl3), d 63.8, 109.4, 113.5, 114.5, 124.5,
129.9, 156.8, 157.3, 158.2. IR (KBr, cmꢀ1): 3156 (NH), 1699 (C O).
–
–
MS m/z 222 (Mþ1). Anal. calcd. for C9H7N3O2S: C, 48.86; H, 3.19; N,
18.99. Found: C, 49.65; H, 3.30; N, 18.86.
Synthesis of 6-hydroxy-2-aminobenzothiazole (1)
A mixture of 6-methoxy-2,3-dihydrobenzo[d]thiazol-2-amine (10 g,
55.56 mmol) and 40 mL of hydrobromic acid (48% water solutions)
was refluxed for 20 h. The mixture was allowed to cool to room
temperature and neutralized with NaOH solution to pH 7–8.
The precipitate formed was filtered and washed with water. The
filtrate was stirred with 100 mL of hot water for 0.5 h, and the
remaining precipitate was filtered to yield a brown solid.
7-Ethoxyl[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4b)
M.p. 249–251°C; yield: 55.8%; 1H NMR (300 MHz, CDCl3), d 1.44
(t, J ¼ 6.7 Hz, 3H, –CH3), 4.06 (d, J ¼ 6.7 Hz, 2H, –OCH2–), 6.95
(d, J ¼ 8.8, 2.6 Hz, 1H, Ar–H), 7.06 (d, J ¼ 2.6 Hz, 1H, Ar–H), 7.89
(d, J ¼ 8.8 Hz, 1H, Ar–H), 9.65 (s, 1H, –NH). 13C NMR (300 MHz,
CDCl3), d 14.2, 66.5, 109.4, 113.5, 114.5, 124.6, 129.8, 156.9, 157.1,
–
Anal. calcd. for C10H9N3O2S: C, 51.05; H, 3.86; N, 17.86. Found:
C, 51.18; H, 3.77; N, 17.96.
157.6. IR (KBr, cmꢀ1): 3155 (NH), 1698 (C O). MS m/z 236 (Mþ1).
–
General procedure for the synthesis of 6-alkoxy-2-
aminobenzothiazoles (2a–u) [8]
A mixture of compound 1 (2 g, 12 mmol), potassium carbonate
(2 g, 14.4 mmol), appropriate alkyl bromide or benzyl chloride
derivatives (1.32 mmol), and a catalytic amount of benzyltriethyl-
amine chloride (TEBA) in 50 mL of acetonitrile was heated under
reflux for 24–48 h. After removing the solvent under reduced
pressure, 80 mL of hot water was poured into the flask, and the
mixture was stirred for 0.5 h to eliminate excess potassium
carbonate. The remaining precipitate was filtered to yield a Russet
solid, which was used without further purification.
7-Propoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4c)
M.p. 178–180°C; yield: 46.3%; 1H NMR (300 MHz, CDCl3), d 1.06
(t, J ¼ 7.4 Hz, 3H, –CH3), 1.66–1.89 (m, 2H, –CH2–), 3.95
(t, J ¼ 6.5 Hz, 2H, –OCH2–), 6.96 (dd, J ¼ 8.8, 2.4 Hz, 1H, Ar–H),
7.07 (d, J ¼ 2.4 Hz, 1H, Ar–H), 7.90 (d, J ¼ 8.8 Hz, 1H, Ar–H), 9.88
(s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 10.5, 22.5, 70.3, 109.5,
113.5, 114.5, 124.6, 130.0, 148.3, 151.2, 157.3. IR (KBr, cmꢀ1): 3154
–
(NH), 1696 (C O). MS m/z 250 (Mþ1). Anal. calcd. for C11H11N3O2S:
–
C, 53.00; H, 4.45; N, 16.86. Found: C, 51.99; H, 4.67; N, 17.02.
General procedure for the synthesis of 6-alkoxy-2-
hydrazinobenzothiazoles (3a–u) [8]
A mixture of compounds 2a–u (20 mmol) and 0.6 mL of 98%
H2SO4 solution (water solutions) in 20 mL of glycol was refluxed
for 0.5 h at 80°C. Then 10 mL of hydrazine hydrate was added into
7-Butoxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one (4d)
M.p. 162–164°C; yield: 36.5%; 1H NMR (300 MHz, CDCl3), d 0.99
(t, J ¼ 7.2 Hz, 3H, –CH3), 1.39–1.62 (m, 2H, –CH2–), 1.67–1.90
(m, 2H, –CH2–), 3.99 (t, J ¼ 6.3 Hz, 2H, –OCH2–), 6.95 (dd, J ¼ 8.8,
2.6 Hz, 1H, Ar–H), 7.06 (d, J ¼ 2.6 Hz, 1H, Ar–H), 7.89 (d, J ¼ 8.8 Hz,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Triazolones as Anticonvulsant Agents
273
1H, Ar–H), 10.15 (s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 13.8,
(d, J ¼ 2.9 Hz, 1H, Ar–H), 7.89 (d, J ¼ 8.8 Hz, 1H, Ar–H), 9.70 (s, 1H,
–NH). 13C NMR (300 MHz, CDCl3), d 14.1, 22.5, 27.1, 29.1, 29.1,
29.3, 29.3, 29.4, 29.4, 29.8, 31.8, 68.6, 109.4, 113.5, 114.5, 124.6,
–
MS m/z 376 (Mþ1). Anal. calcd. for C20H29N3O2S: C, 63.97; H, 7.78;
N, 11.19. Found: C, 64.18; H, 7.59; N, 11.31.
19.2, 31.2, 68.5, 109.5, 113.2, 114.5, 124.6, 129.9, 148.3, 151.2,
157.3. IR (KBr, cmꢀ1): 3152 (NH), 1697 (C O). MS m/z 264 (Mþ1).
–
–
Anal. calcd. for C12H13N3O2S: C, 54.74; H, 4.98; N, 15.96. Found: C,
54.83; H, 4.79; N, 16.01.
129.9, 156.9, 157.3, 157.6. IR (KBr, cmꢀ1): 3142 (NH), 1695 (C O).
–
7-Pentyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
7-Tetradecyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-
(4e)
M.p. 150–152°C; yield: 46.3%; 1H NMR (300 MHz, CDCl3), d 0.96
(t, J ¼ 6.6 Hz, 3H, –CH3), 1.43–1.82 (m, 6H, –(CH2)3–), 3.98
(d, J ¼ 6.2 Hz, 2H, –OCH2–), 6.95 (dd, J ¼ 8.8, 2.6 Hz, 1H, Ar–H),
7.07 (d, J ¼ 2.6 Hz, 1H, Ar–H), 7.90 (d, J ¼ 8.8 Hz, 1H, Ar–H), 9.53
(s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 14.0, 22.4, 28.1, 28.9,
68.8, 109.4, 113.5, 114.5, 124.6, 130.0, 148.3, 151.2, 157.3. IR (KBr,
one (4j)
M.p. 211–213°C; yield: 70.2%; 1H NMR (300 MHz, CDCl3), d 0.88
(t, J ¼ 6.1 Hz, 3H, –CH3), 1.26 (s, 22H, –(CH2)11–), 1.76–1.87 (m, 2H,
–CH2–), 3.98 (t, J ¼ 6.1 Hz, 2H, –OCH2–), 6.95 (dd, J ¼ 8.6, 2.3 Hz, 1H,
Ar–H), 7.06 (d, J ¼ 2.3 Hz, 1H, Ar–H), 7.90 (d, J ¼ 8.6 Hz, 1H, Ar–H),
10.05 (s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 14.1, 22.5, 27.1,
29.1, 29.1, 29.2, 29.3, 29.3, 29.3, 29.4, 29.4, 29.8, 31.8, 68.5,
109.2, 113.8, 114.5, 124.8, 129.7, 157.0, 157.4, 157.9. IR (KBr,
cmꢀ1): 3151 (NH), 1696 (C O). MS m/z 278 (Mþ1). Anal. calcd. for
–
–
C13H15N3O2S: C, 56.30; H, 5.45; N, 15.15. Found: C, 56.39; H, 5.38;
cmꢀ1): 3140 (NH), 1690 (C O). MS m/z 404 (Mþ1). Anal. calcd. for
–
–
N, 15.29.
C22H33N3O2S: C, 65.47; H, 8.24; N, 10.41. Found: C, 65.32; H, 8.09;
N, 10.56.
7-Hexyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
(4f)
M.p. 139–141°C; yield: 45.2%; 1H NMR (300 MHz, CDCl3), d 0.91
(t, J ¼ 7.3 Hz, 3H, –CH3), 1.33–1.82 (m, 8H, –(CH2)4–), 3.97
(t, J ¼ 6.1 Hz, 2H, –OCH2–), 6.94 (dd, J ¼ 8.8, 2.4 Hz, 1H, Ar–H),
7.04 (d, J ¼ 8.8, 2.4 Hz, 1H, Ar–H), 7.89 (d, J ¼ 8.8 Hz, 1H, Ar–H),
10.46 (s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 14.0, 19.9, 26.9,
28.9, 31.3, 68.5, 109.4, 113.5, 114.5, 124.6, 129.9, 156.9, 157.4,
7-Benzyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
(4k)
M.p. 231–233°C; yield: 47.2%; 1H NMR (300 MHz, DMSO), d 5.02
(s, 2H, –OCH2–), 6.93–7.75 (m, 8H, Ar–H), 11.33 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 70.4, 113.6, 115.5, 118.5, 127.6,
128.1, 128.6, 128.7, 136.8, 144.6, 153.3, 154.9, 156.5. IR
157.6. IR (KBr, cmꢀ1): 3149 (NH), 1697 (C O). MS m/z 292 (Mþ1).
(KBr, cmꢀ1): 3165 (NH), 1720 (C O). MS m/z 298 (Mþ1). Anal.
–
–
–
–
Anal. calcd. for C14H17N3O2S: C, 57.71; H, 5.88; N, 14.42. Found: C,
57.97; H, 5.66; N, 14.62.
calcd. for C15H11N3O2S: C, 60.59; H, 3.73; N, 14.13. Found: C, 60.82;
H, 3.60; N, 14.37.
7-Heptyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
7-(2-Fluorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
(4g)
3(2H)-one (4l)
M.p. 158–160°C; yield: 43.3%; 1H NMR (300 MHz, CDCl3), d 0.90
(t, J ¼ 6.6 Hz, 3H, –CH3), 1.30–1.86 (m, 10H, –(CH2)5–), 3.98
(t, J ¼ 6.5 Hz, 2H, –OCH2–), 6.95 (dd, J ¼ 8.8, 2.2 Hz, 1H, Ar–H),
7.07 (d, J ¼ 2.2 Hz, 1H, Ar–H), 7.90 (d, J ¼ 8.8 Hz, 1H, Ar–H), 9.60
(s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 14.1, 22.6, 26.0, 28.7,
29.0, 31.3, 68.5, 109.4, 113.6, 114.1, 124.5, 129.9, 156.9, 157.3,
M.p. 164–166°C; yield: 45.8%; 1H NMR (300 MHz, CDCl3), d 5.17
(s, 2H, –OCH2–), 7.03–7.93 (m, 7H, Ar–H), 10.06 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 61.3, 114.6, 114.9, 114.2, 115.1,
115.2, 115.3, 116.3, 124.3, 124.6, 124.4, 128.0, 129.8, 154.5, 160.5.
IR (KBr, cmꢀ1): 3167 (NH), 1721 (C O). MS m/z 316 (Mþ1). Anal.
–
–
calcd. for C15H10FN3O2S: C, 57.14; H, 3.20; N, 13.33. Found: C,
57.32; H, 3.39; N, 13.51.
157.7. IR (KBr, cmꢀ1): 3148 (NH), 1694 (C O). MS m/z 306 (Mþ1).
–
–
Anal. calcd. for C15H19N3O2S: C, 58.99; H, 6.27; N, 13.76. Found: C,
59.10; H, 6.39; N, 13.92.
7-(3-Fluorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-one (4m)
M.p. 238–240°C; yield: 49.9%; 1H NMR (300 MHz, DMSO), d 5.02
(s, 2H, –OCH2–), 6.92–7.77 (m, 7H, Ar–H), 11.69 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 61.7, 114.8, 115.1, 115.4, 115.8,
115.0, 115.1, 116.3, 122.7, 125.3, 130.3, 132.4, 139.7, 154.5, 163.1.
7-Octyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
(4h)
M.p. 152–154°C; yield: 52.2%; 1H NMR (300 MHz, CDCl3), d 0.89
(s, J ¼ 6.8 Hz, 3H, –CH3), 1.37–1.68 (m, 10H, –(CH2)5–), 1.75–1.84
(m, 2H, –CH2–), 3.96 (t, J ¼ 6.6 Hz, 2H, –OCH2–), 7.00 (dd, J ¼ 8.7,
3.1 Hz, 1H, Ar–H), 7.09 (d, J ¼ 3.1 Hz, 1H, Ar–H), 7.89 (d, J ¼ 8.7 Hz,
1H, Ar–H), 10.29 (s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 14.1,
22.5, 27.1, 29.0, 29.1, 29.5, 31.8, 68.5, 109.4, 113.5, 114.5, 124.5,
IR (KBr, cmꢀ1): 3168 (NH), 1722 (C O). MS m/z 316 (Mþ1). Anal.
–
–
calcd. for C15H10FN3O2S: C, 57.14; H, 3.20; N, 13.33. Found: C,
57.29; H, 3.36; N, 13.45.
129.8, 156.9, 157.4, 157.5. IR (KBr, cmꢀ1): 3146 (NH), 1693 (C O).
–
–
MS m/z 320 (Mþ1). Anal. calcd. for C16H21N3O2S: C, 60.16; H, 6.63;
N, 13.16. Found: C, 60.39; H, 6.87; N, 13.02.
7-(4-Fluorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-one (4n)
M.p. 230–232°C; yield: 42.6%; 1H NMR (300 MHz, CDCl3), d 5.03
(s, 2H, –OCH2–), 6.90–7.40 (m, 7H, Ar–H), 9.31 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 61.7, 113.8, 115.4, 115.6, 118.4,
116.5, 122.6, 126.8, 127.9, 129.3, 132.5, 144.5, 153.2, 156.2, 162.2.
7-Dodecyloxy[1,2,4]triazolo[3,4-b]benzothiazol-3(2H)-one
(4i)
M.p. 134–136°C; yield: 45.2%; 1H NMR (300 MHz, CDCl3), d 0.88
(t, J ¼ 6.4 Hz, 3H, –CH3), 1.27–1.82 (m, 20H, –(CH2)10–), 3.98
(t, J ¼ 6.4 Hz, 2H, –OCH2–), 6.95 (dd, J ¼ 8.8, 2.9 Hz, 1H, Ar–H), 7.07
IR (KBr, cmꢀ1): 3166 (NH), 1721 (C O). MS m/z 316 (Mþ1). Anal.
–
–
calcd. for C15H10FN3O2S: C, 57.14; H, 3.20; N, 13.33. Found: C,
56.93; H, 3.03; N, 13.21.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

274
D.-C. Liu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
7-(2-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
7-(2,6-Dichlorobenzyloxy)-[1,2,4]triazolo[3,4-b]-
3(2H)-one (4o)
benzothiazol-3(2H)-one (4u)
M.p. 207–209°C; yield: 52.3%; 1H NMR (300 MHz, CDCl3), d 5.20
(s, 2H, –OCH2–), 7.04–7.94 (m, 7H, Ar–H), 9.83 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 64.3, 113.6, 114.9, 114.2, 115.1,
115.2, 115.3, 116.3, 124.0, 124.1, 124.4, 128.2, 129.9, 154.5, 170.3.
M.p. 230–232°C; yield: 56.3%; 1H NMR (300 MHz, DMSO), d 5.18
(s, 2H, –OCH2–), 6.90–7.82 (m, 6H, Ar–H), 11.67 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 61.5, 111.6, 112.4, 122.5, 126.1,
128.3, 128.9, 130.2, 131.7, 133.8, 136.8, 144.6, 153.4, 155.0, 156.6.
IR (KBr, cmꢀ1): 3172 (NH), 1718 (C O). MS m/z 333 (Mþ1). Anal.
IR (KBr, cmꢀ1): 3176 (NH), 1722 (C O). MS m/z 367 (Mþ1). Anal.
–
–
–
–
calcd. for C15H10ClN3O2S: C, 54.30; H, 3.04; N, 12.67. Found: C,
54.52; H, 2.89; N, 12.85.
calcd. for C15H9Cl2N3O2S: C, 49.19; H, 2.48; N, 11.47. Found: C,
48.83; H, 2.33; N, 11.56.
7-(3-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
Pharmacology
The MES test, chemical-induced epileptic test, and rotarod test
were carried out according to the procedures described in
Anticonvulsant Screening Program with some modification [20, 21].
In the MES test and rotarod test, the anticonvulsant effects and the
neurovirulence of the compounds were assessed at 0.5-h intervals
following administration in mice. And in preliminary neurotoxicity
screening, compounds only were administered by intraperitoneal
(i.p.) injection at dosages of 100mg/kg to avoid wasting animals.
Electro-convulsions were produced by an electric stimulation
generator (JTC-1, ChengDu, China). All compounds, which were
dissolved in dimethylsulfoxide (DMSO), were evaluated for
anticonvulsant activities with KunMing mice in the 18–22g weight
range purchased from the Laboratory of Animal Research, College
of Pharmacy, Yanbian University.
3(2H)-one (4p)
M.p. 180–182°C; yield: 60.5%; 1H NMR (300 MHz, DMSO), d 5.01
(s, 2H, –OCH2–), 6.93–7.76 (m, 7H, Ar–H), 11.68 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 69.7, 113.6, 114.2, 114.4, 114.8,
115.0, 115.1, 116.5, 122.6, 125.3, 130.5, 132.2, 139.8, 154.5, 171.2.
IR (KBr, cmꢀ1): 3174 (NH), 1717 (C O). MS m/z 333 (Mþ1). Anal.
–
–
calcd. for C15H10ClN3O2S: C, 54.30; H, 3.04; N, 12.67. Found: C,
54.44; H, 2.97; N, 12.91.
7-(4-Chlorobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-one (4q)
M.p. 227–229°C; yield: 42.8%; 1H NMR (300 MHz, DMSO), d 4.97
(s, 2H, –OCH2–), 6.94–7.76 (m, 7H, Ar–H), 11.79 (s, 1H, –NH).
13C NMR (300 MHz, CDCl3), d 69.7, 113.5, 115.4, 115.6, 118.4,
116.5, 122.4, 126.7, 127.9, 129.3, 131.4, 144.4, 153.1, 156.3, 171.8.
The maximal electroshock (MES) test
IR (KBr, cmꢀ1): 3175 (NH), 1716 (C O). MS m/z 333 (Mþ1). Anal.
–
–
Seizures were elicited with a 60 Hz alternating current of 50 mA
intensity in mice. The current was applied via ear-clip electrodes
for 0.2 s. Protection against the spread of MES-induced seizures
was defined as the absence of tonic extension of the hind leg. At
30 min after the administration of the compounds, the activities
were evaluated in MES test. In preliminary screening, each
compound was administered at the dose levels of 200, 100, and
30 mg/kg for evaluating the preliminary anticonvulsant activity.
For determination of the median ED50 and the median TD50, the
quantitative evaluation 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 neurotoxicity. From these data, the
respective ED50, TD50 values, and 95% confidence intervals were
calculated by probit analysis.
calcd. for C15H10ClN3O2S: C, 54.30; H, 3.04; N, 12.67. Found: C,
54.47; H, 2.79; N, 12.82.
7-(4-Methylbenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-one (4r)
M.p. 188–190°C; yield: 43.3%; 1H NMR (300 MHz, DMSO), d 2.27
(s, 3H, –CH3), 4.97 (s, 2H, –OCH2–), 6.91–7.76 (m, 7H, Ar–H), 11.40
(s, 1H, –NH). 13C NMR (300 MHz, CDCl3), d 22.5, 70.5, 113.6, 115.4,
115.5, 116.0, 118.5, 127.7, 129.6, 128.7, 133.8, 137.8, 144.6, 153.4,
155.0, 156.6. IR (KBr, cmꢀ1): 3169 (NH), 1724 (C O). MS m/z 312
–
–
(Mþ1). Anal. calcd. for C16H13N3O2S: C, 61.72; H, 4.21; N, 13.50.
Found: C, 61.55; H, 4.03; N, 13.32.
7-(3-Trifluoromethylbenzyloxy)-[1,2,4]triazolo[3,4-b]-
benzothiazol-3(2H)-one (4s)
M.p. 168–170°C; yield: 45.2%; 1H NMR (300 MHz, DMSO), d 5.08
(s, 2H, –OCH2–), 6.93–7.79 (m, 7H, Ar–H), 11.67 (s, 1H, –NH). 13C
NMR (300 MHz, CDCl3), d 70.5, 113.6, 115.4, 124.9, 125.6, 126.0,
128.3, 128.7, 131.3, 133.7, 133.3, 136.2, 141.6, 153.5, 154.7, 156.7.
Pentylenetetrazol-induced seizure test [22]
At 30 min after the administration of the test compound,
100 mg/kg of PTZ dissolved in saline was administered sc. The
animals were placed in individual cages and observed for 30 min.
The number of clonic and tonic seizures as well as the number of
deaths was noted.
IR (KBr, cmꢀ1): 3167 (NH), 1719 (C O). MS m/z 366 (Mþ1). Anal.
–
–
calcd. for C16H10F3N3O2S: C, 52.60; H, 2.76; N, 11.50. Found: C,
52.73; H, 2.89; N, 11.62.
3-Mercaptopropionic acid-induced seizures [23]
At 30 min after the administration of the test compound, 40 mg/
kg of 3-MP dissolved in saline solution was injected sc. The
animals were placed in individual cages and observed for 30 min.
The number of clonic and tonic seizures as well as the number of
deaths was noted.
7-(2-Cyanobenzyloxy)-[1,2,4]triazolo[3,4-b]benzothiazol-
3(2H)-one (4t)
M.p. 239–241°C; yield: 52.3%; 1H NMR (300 MHz, DMSO), d 5.27
(s, 2H, –OCH2–), 7.16–7.96 (m, 7H, Ar–H), 11.79 (s, 1H, –NH). 13C
NMR (300 MHz, CDCl3), d 70.5, 113.6, 115.4, 115.5, 116.0, 128.5,
128.7, 129.6, 128.7, 133.3, 133.7, 136.2, 144.5, 153.4, 155.2, 156.3.
IR (KBr, cmꢀ1): 3170 (NH), 1720 (C O). MS m/z 323 (Mþ1). Anal.
–
Bicuculline-induced seizures test [24]
At 30 min after the administration of the test compound,
5.4 mg/kg of BIC (within 15–45 min after preparation due to
–
calcd. for C16H10N4O2S: C, 59.62; H, 3.13; N, 17.38. Found: C, 59.85;
H, 3.40; N, 17.21.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 268–275
Triazolones as Anticonvulsant Agents
275
instability) was injected s.c. The animals were placed in individual
cages and observed for 30 min. The number of clonic and tonic
seizures as well as the number of deaths was noted.
[5] G. M. Kennedy, S. D. Lhatoo, CNS Drugs 2008, 22, 739–760.
[6] P. E. Penovich, L. James Willmore, Epilepsia 2009, 50, 37–41.
[7] Y. A. Al-Soud, N. A. Al-Masoudi, Ael.-R. Ferwanah, Bioorg. Med.
Chem. 2003, 11, 1701–1708.
Rotarod test [25]
[8] X. Q. Deng, M. X. Song, C. X. Wei, F. N. Li, Z. S. Quan, Med.
Chem. 2010, 6 (5), 313–320.
The neurotoxicity of the compounds was measured in mice by the
rotarod test. The mice were trained to stay on an accelerating
rotarod of diameter an inch 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
maintain equilibrium on the rod for at least 1 min in each of the
trials.
[9] L. J. Cui, Z. F. Xie, H. R. Piao, G. Li, K. Y. Chai, Z. S. Quan, Biol.
Pharm. Bull. 2005, 28, 1216–1220.
[10] H. G. Jin, X. Y. Sun, K. Y. Chai, H. R. Piao, Z. S. Quan, Bioorg.
Med. Chem. 2006, 14, 6868–6873.
[11] T. D Gross, A. R. Prasad, J. R. Citineni, PCT Int. Appl. 2011026939,
2011.
[12] Y. L. Chen, H. M. Hung, C. M. Lu, K. C. Li, C. C. Tzeng, Bioorg.
Med. Chem. 2004, 12, 6539–6546.
Pharmacological evaluation of compound 4d administered
orally to mice
[13] M. V. Deshmukh, Indian J. Chem. (B) 1998, 9, 921–924.
The time-course effect of compound 4d in the MES test was
determined. A suspension of compound 4d was given in mice by
oral gavages (50 mg/kg in a dose volume of 0.5% methylcellulose).
The mice were divided into seven groups (n ¼ 8). Subsequently,
the animals were subjected to the MES test at various times: 0.5, 1,
1.5, 2, 2.5, and 3 h. The TPE was 1.5 h after the p.o. injection. Then,
compound 4d was evaluated for its orally anticonvulsant activity
against MES-induced seizures and oral neurotoxicity at its TPE.
This test involved the same procedures for determining ED50 and
TD50 as used in the MES test and the Tox test screening, except
that the test drug was administered orally to mice.
[14] E. J. Lien, R. C. Liao, H. G. Shinouda, J. Pharm. Sci. 1979, 68,
463–465.
[15] R. Okada, N. Negishi, H. Nagaya, Brain Res. 1989, 480, 383–
387.
[16] R. W. Olsen, J. Neurochem. 1981, 37, 1–13.
[17] K. Gale, Epilepsia 1992, 33, S3–S12.
[18] W. Loscher, Biochem. Pharmacol. 1979, 28, 1397–1407.
[19] R. L. Macdonald, J. L. Barker, Neurology 1978, 28, 325–
330.
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).
[20] R. L. Krall, J. K. Penry, B. G. White, H. J. Kupferberg,
E. A. Swinyard, Epilepsia 1978, 19, 409–428.
[21] R. J. Porter, J. J. Cereghino, G. D. Gladding, B. J. Hessie,
H. J. Kupferberg, B. Scoville, B. G. White, Cleve. Clin. Q 1984, 51,
293–305.
The authors have declared no conflict of interest.
[22] M. Geurts, J. H. Poupaert, G. K. Scriba, D. M. Lambert, J. Med.
Chem. 1998, 41, 24–30.
References
[23] A. Arnoldi, A. Bonsignori, P. Melloni, L. Merlini, M. L. Quadri,
A. C. Rossi, M. Valsecchi, J. Med. Chem. 1990, 33 (10), 2865–
2869.
[1] M. Gasior, R. B. Carter, S. R. Goldberg, J. M. Witkin, J.
Pharmacol. Exp. Ther. 1997, 282, 543–553.
[2] H. Stefan, T. Feuerstein, Pharmacol. Ther. 2007, 113, 165–183.
[3] E. J. Donner, O. C. Snead, NeuroRX 2006, 3, 170–180.
[24] R. Bernasconi, M. Klein, P. Martin, P. Christen, T. Hafner,
C. Portet, M. Schmutz, J. Neural Transm. 1988, 72, 213–
233.
[4] H. P. Bootsma, L. Ricker, Y. A. Hekster, J. Hulsman,
D. Lambrechts, M. Majoie, A. Schellekens, D. M. Krom,
A. P. Aldenkamp, Seizure 2009, 18 (5), 327–331.
[25] X. Y. Sun, Y. Z. Jin, F. N. Li, G. Li, K. Y. Chai, Z. S. Quan, Arch.
Pharm. Res. 2006, 29, 1080–1085.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com