Synthesis and anticonvulsant activity evaluation of 7-alkoxy-2, 4-dihydro-1H-benzo[b][1,2,4]triazolo[4,3-d][1,4]thiazin-1-ones in various murine experimental seizure models

Article abstract of DOI:10.1007/s00044-013-0778-y

A novel series of 7-alkoxy-2,4-dihydro-1H-benzo [b][1,2,4]triazolo[4,3-d] [1,4]-thiazin-1-ones have been synthesized and tested for their anticonvulsant activity using the maximal electroshock (MES) method. The majority of the compounds prepared were effe

Full text of DOI:10.1007/s00044-013-0778-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1829–1838
DOI 10.1007/s00044-013-0778-y
ORIGINAL RESEARCH
Synthesis and anticonvulsant activity evaluation of 7-alkoxy-2,
4-dihydro-1H-benzo[b][1,2,4]triazolo[4,3-d][1,4]thiazin-1-ones
in various murine experimental seizure models
•
Xu Cao Shi-Ben Wang Xian-Qing Deng
•
•
•
Da-Chuan Liu Zhe-Shan Quan
Received: 9 June 2013 / Accepted: 30 August 2013 / Published online: 21 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A novel series of 7-alkoxy-2,4-dihydro-1H-benzo
[b][1,2,4]triazolo[4,3-d][1,4]-thiazin-1-ones have been syn-
thesized and tested for their anticonvulsant activity using the
maximal electroshock (MES) method. The majority of the
compounds prepared were effective in the MES screens at a
dose level of 100 mg/kg. Of the compounds tested, the most
promising was 7-[(4-fluorobenzyl)oxy]-2,4-dihydro-1H-
[1,2,4]-triazolo[4,3-d][1,4]-benzothiazin-1-one (6m), which
showed an ED50 value of 9.2 mg/kg in the MES test in mice.
Furthermore, the compound exhibited a PI value of 15.4 which
was superior to the standard drug carbamazepine (PI value of
6.4). As well as demonstrating the anti-MES efficacy of
compound 6m, its potency against seizures induced by
pentylenetetrazole, 3-mercaptopropionic acid, and bicuculline
were also established, with the results suggesting that several
different mechanisms of action might be involved in its anti-
convulsant activity, including the inhibition of voltage-gated
ion channels and the modulation of GABAergic activity.
Introduction
Epilepsy is one of the most common neurological disorders,
characterized by excessive temporary neuronal discharge
resulting in recurrent unprovoked seizures (Strine et al.,
2005; Mc Namara et al., 2006). There have been reports
suggesting that around 1 % of the world’s population at any
time (about 50 million people worldwide) is afflicted with
this neurological disorder. In the recent years, extensive
efforts have been devoted to the development of novel
therapeutics, and these efforts have resulted in the develop-
ment of several new drug candidates as promising anticon-
vulsants (Stefan and Feuerstein, 2007; Donner and Snead,
2006). The currently available anticonvulsants, however, are
effective only in reducing the severity and number of sei-
zures in less than 70 % of patients. Furthermore, their usage
is often associated with numerous undesirable side effects
(Bootsma et al., 2009; Kennedy and Lhatoo, 2008; Penovich
and James Willmore, 2009). Although many of the antiepi-
leptic drugs (AEDs) currently available, including phenyt-
oin, carbamazepine, ethosuximide, valproic acid and the
barbiturates, are widely prescribed, they can induce a num-
ber of adverse side effects, including ataxia, hepatotoxicity,
gingival hyperplasia, and megaloblastic anemia (Bialer
et al., 2007; Bialer and Yagen, 2007). With this in mind, it is
clear that a number of the current AEDs are limited by their
toxicity, intolerance, and lack of efficacy. There is, therefore,
an urgent need for the development of novel AEDs with
greater levels of efficacy and lower levels of toxicity.
Keywords Synthesis Á Anticonvulsant Á
Triazolothiazinone Á Maximal electroshock Á
Neurotoxicity
X. Cao Á S.-B. Wang Á X.-Q. Deng Á D.-C. Liu Á
Z.-S. Quan (&)
College of Pharmacy, Yanbian University, No. 977, Park Road,
Yanji 133002, Jilin, China
e-mail: zsquan@ybu.edu.cn
In our previous work, we synthesized a series of 6-alkoxy-
3,4-dihydro-2(1H)-quinolinone derivatives (I) with anticon-
vulsant activities. Of these compounds, 6-hexyloxy-3,4-dihy-
dro-2(1H)-quinolinone showed the strongest activity, with an
ED50 value of 24.0 mg/kg in the maximal electroshock test
(MES) and a PI value of 2.4 (Quanet al., 2005). In a subsequent
X.-Q. Deng
Medical College, Jinggangshan University,
Ji’an 343009, Jiangxi, China
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Med Chem Res (2014) 23:1829–1838
1
study, a second series of compounds II was designed and
synthesized according to a strategy involving the incorporation
of a triazolone moiety into the initial series of compounds I (Jin
et al., 2006). In our laboratory, we have reported the synthesis
and anticonvulsant activity evaluation of some compounds
containing triazolone, the majority of them exhibited potent
anticonvulsant activity (Shu et al., 2013; Zheng et al., 2013).
This new design was based on the research reported by Gitto,
et al. (2003), in which the introduction of a triazolone to the
precursor compounds III provided a remarkable increase in
their anticonvulsant activity. The pharmacological results
achieved with series II were also in agreement with those
reported by Gitto et al. Following the incorporation of the
triazolone moiety into series I, the anticonvulsant activity of
compounds II were markedly increased. Consequently, in the
current study, a series of 7-alkoxy-2,4-dihydro-1H-benzo[b]
[1,2,4]triazolo[4,3-d][1,4]thiazin-1-one derivatives were
designed and synthesized according to a strategy involving the
introduction of a triazolone moiety to compound series IV, with
the latter having already been reported as a potent anticonvul-
sant in our previous research (Zhang et al., 2010), in which,
7-(pentyloxy)-2H-benzo[b][1,4]thiazin-3(4H)-one (IV-e) was
the most active compound from series IV (leading structures)
with an ED50 value of 34.1 mg/kg, TD50 value of 305.5 mg/kg
and PI of 8.9 (Fig. 1).
IRPrestige-21. H NMR and 13C NMR spectra were mea-
sured on an AV-300(Bruker, Switzerland), and all chemical
shifts were given in ppm relative to tetramethysilane. Mass
spectra were measured on an HP1100LC (Agilent Tech-
nologies, USA). The major chemicals were purchased from
Aldrich Chemical Corporation.
Chemistry
Synthesis of 2-amino-5-methoxy-thiophenol (1)
2-Amino-6-methoxybenzothiazole (36.0 g; 206.4 mmol)
was suspended in 50 % KOH (180 g KOH dissolved in
180 ml water). The suspension was heated to reflux for
20 h. Upon cooling to room temperature, concentrated
hydrochloric acid was added to adjust the pH to 6 to give a
large number of pale yellow solid. Filter cake was obtained
by vacuum-filtration and it was dried rapidly.
Synthesis of 7-methoxy-2H-benzo[b][1,4]thiazin-3(4H)-
one (2)
Compound 1 (24.54 g; 158 mmol), sodium bicarbonate
(53.20 g; 633 mol), and benzyltriethylammonium chloride
(TEBA) (25.18 g; 110 mmol) were placed into a round-bot-
tomed flask containing 500 ml ofacetonitrile. Themixture was
put into an ice–salt bath to keep the temperature at 0–5 °C.
2-Chloroacetyl-chloride (23.26 g; 21 mmol) was added into
the mixture at a moderate speed and kept for 40 min.
Replacing the ice–salt bath with an oil bath, the mixture was
refluxed for 2 h, then the solvent was removed under reduced
pressure. The resultant residue was purified by recrystalliza-
tion with ethanol–water (1:1) to obtain pure compound 2.
The structures of target compounds were characterized using
IR, 1H NMR, MS, and 13C NMR techniques. The anticonvul-
sant activities of the newly synthesized compounds were
evaluated using MES-induced seizure in mice as an experi-
mental epilepsy model. The rotarod assay was performed in
mice to evaluate their neurotoxicity. To elucidate the possible
mechanism of action, the most active compound 6m was tested
in pentylenetetrazole (PTZ), 3-mercaptopropionic acid (3-MP),
and bicuculline (BIC)-induced seizure tests.
Synthesis of 7-hydroxy-2H-benzo[b][1,4]thiazin-
Materials and methods
3(4H)-one (3)
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) on
To a stirred solution of boron tribromide (9 mmol) in
anhydrous CH2Cl2, a CH2Cl2 solution of compound 2
Fig. 1 The structures of
compound series I, II, III, and
IV, and the target compounds
O
R
O
RO
H
H
3CO
NH
N
H
N
O
N
NH
3CO
O
II
I
R
III
R
O
S
N
R
O
S
N
H
N
NH
O
O
a- u
IV
6
6
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7-Ethoxy-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]ben-
(0.6 g; 3 mmol) was added drop-wise and the reaction
continued for 1 h at 0 °C and an additional 2 h at 20 °C.
Following the addition of ice-cold water, the organic layer
was separated and the aqueous layer extracted with ethyl
acetate. The combined organic layers were dried over with
anhydrous magnesium sulfate and evaporated under
reduced pressure to give a brown solid.
zothiazin-1-one (6a) White solid; mp. 184–186 °C. IR
1
(KBr) cm-1: 3189 (NH), 1708 (C=O). H NMR (CDCl3,
300 MHz): d 1.42 (t, 3H, J = 6.0 Hz, –CH3), 3.83 (s, 2H,
–SCH2–), 4.03 (q, 2H, J = 7.0 Hz, –OCH2–), 6.85–8.27
(m, 3H, Ar–H), 9.91 (s, 1H, –NHCO–). 13C–NMR (CDCl3,
300 MHz): d 14.71, 24.12 (ethyoxy-C), 63.95 (–SCH2–),
113.79, 114.25, 120.40, 124.39, 125.54, 141.49 (Ar–C),
152.99 (triazolone, C=N), 156.86 (triazolone, C=O). MS-
EI m/z 250 (M?1).
Synthesis of 7-alkoxy-2H-benzo[b][1,4]thiazin-3
(4H)-ones (4a–4t)
7-Propoxy-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]ben-
K2CO3 (0.42 g; 3 mmol), acetonitrile (40 ml), and
7-hydroxy-2H-benzo[b][1,4] thiazin-3(4H)-one 3 (0.53 g;
3 mmol) were mixed in a 100 ml round-bottomed flask
equipped with reflux condenser. After refluxing the mixture
30 min, alkyl bromide or benzyl chloride derivative
(4 mmol) was added drop-wise into the mixture. The
mixture was heated under reflux for 4–24 h, cooled down
and then poured into 100 ml of water. Aqueous layer was
extracted with dichloromethane (30 ml 9 3). The com-
bined layers of dichloromethane were dried by anhydrous
MgSO4. Evaporation of the solvent gave a crude product,
which was purified by silica gel column chromatography
with petroleum ether: ethyl acetate (3:2) to a light-yellow
solid.
zothiazin-1-one (6b) White solid; mp. 160–162 °C. IR
1
(KBr) cm-1: 3178 (NH), 1704 (C=O). H NMR (CDCl3,
300 MHz): d 1.04 (t, 3H, J = 7.5 Hz, –CH3), 1.76–1.87
(m, 2H, –CH2–), 3.83 (s, 2H, –SCH2–), 3.92 (t, 2H, J = 6.0
Hz, –OCH2–), 6.86–8.27 (m, 3H, Ar–H), 9.85 (s, 1H,
–NHCO–). 13C NMR (CDCl3, 300 MHz): d 10.47, 22.47,
24.13 (propoxy-C), 69.96 (–SCH2–), 113.79, 114.28,
120.40, 124.38, 125.54, 141.19 (Ar–C), 153.33 (triazolone,
C=N), 157.04 (triazolone, C=O). MS-EI m/z 264 (M?1).
7-Butoxy-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]ben-
zothiazin-1-one (6c) White solid; mp. 154–156 °C. IR
1
(KBr) cm-1: 3190 (NH), 1704 (C=O). H NMR (CDCl3,
300 MHz): d 0.98 (t, 3H, J = 6.0 Hz, –CH3), 1.43–1.55
(m, 2H, –CH2–), 1.72–1.81 (m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.96 (t, 2H, J = 6.0 Hz, –OCH2–), 6.85–8.27
(m, 3H, Ar–H), 10.16 (s, 1H, –NHCO–). 13C NMR
(CDCl3, 300 MHz): d 13.83, 19.18, 24.11, 31.16 (butoxy-
C), 68.16 (–SCH2–), 113.79, 114.26, 120.40, 124.39,
125.51, 141.33 (Ar–C), 153.34 (triazolone, C=N), 157.06
(triazolone, C=O). MS-EI m/z 278 (M?1).
Synthesis of 7-alkoxy-2H-benzo[b][1,4]thiazin-3(4H)-
thiones (5a–5t)
To a toluene solution in a three-necked round-bottomed
flask in an oil bath, Lawesson’s reagent (0.6 eq) and 4a–4t
was added, and the solution was refluxed for 3–6 h under
nitrogen. After removing the solvent under reduced pres-
sure, the residue was dissolved in dichloromethane (30 ml),
washed with water (30 ml 9 3) and dried over anhydrous
MgSO4. Evaporation of the solvents gave a crude product,
which was purified by silica gel column chromatography
with dichloromethane to a light-yellow solid.
7-(Pentyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6d) White solid; mp. 120–122 °C.
IR (KBr) cm-1: 3186 (NH), 1701 (C=O). 1H NMR (CDCl3,
300 MHz): d 0.94 (t, 3H, J = 7.0 Hz, –CH3), 1.35–1.47
(m, 4H, –CH2–), 1.74–1.83 (m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.95 (t, 2H, J = 6.5 Hz, –OCH2–), 6.85–8.26
(m, 3H, Ar–H), 9.95 (s, 1H, –NHCO–). 13C NMR (CDCl3,
300 MHz): d 14.03, 22.43, 24.09, 28.13, 28.82 (pentyloxy-
C), 68.45 (–SCH2–), 113.73, 114.23, 120.39, 124.41,
125.53, 141.11 (Ar–C), 153.50 (triazolone, C=N), 157.01
(triazolone, C=O). MS-EI m/z 292 (M?1).
Synthesis of target compounds (6a–6t)
A solution of 5a–5t (3 mmol) in appropriate n-butanol and
methyl hydrazinecarboxylate (9 mmol) was refluxed for
2–3 days (TLC monitoring), the solvent was evaporated to
dryness under reduced pressure, and the residue was
extracted twice with dichloromethane (60 ml). The
dichloromethane layer was washed three times with satu-
rated aqueous NaCl (60 ml 9 3) and dried over anhydrous
MgSO4. After removing the solvents, products were puri-
fied by silica gel column chromatography with CH2Cl2–
CH3OH (70:1). The yield, melting point and spectral data
of each compound were given below.
7-(Hexyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6e) White solid; mp. 118–120 °C. IR
1
(KBr) cm-1: 3185 (NH), 1702 (C=O). H NMR (CDCl3,
300 MHz): d 0.91 (t, 3H, J = 6.5 Hz, –CH3), 1.33–1.47
(m, 6H, –CH2–), 1.73–1.82 (m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.95 (t, 2H, J = 6.5 Hz, –OCH2–), 6.84–8.26
(m, 3H, Ar–H), 10.23 (s, 1H, –NHCO–). 13C NMR
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Med Chem Res (2014) 23:1829–1838
(CDCl3, 300 MHz): d 14.04, 22.60, 24.12, 25.66, 29.09,
31.54 (hexyloxy-C), 68.48 (–SCH2–), 113.78, 114.25,
120.39, 124.39, 125.52, 141.18 (Ar–C), 153.32 (triazolone,
C=N), 157.04 (triazolone, C=O). MS-EI m/z 306 (M?1).
120.40, 124.40, 125.50, 141.18 (Ar–C), 153.43 (triazolone,
C=N), 157.05 (triazolone, C=O). MS-EI m/z 418 (M?1).
7-(Benzyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6j) White solid; mp. 178–180 °C. IR
1
7-(Heptyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6f) White solid; mp. 110–112 °C.
IR (KBr) cm-1: 3180 (NH), 1704 (C=O). 1H NMR (CDCl3,
300 MHz): d 0.90 (t, 3H, J = 6.6 Hz, –CH3), 1.31–1.47
(m, 8H, –CH2–), 1.73–1.82 (m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.95 (t, 2H, J = 6.5 Hz, –OCH2–), 6.85–8.26
(m, 3H, Ar–H), 9.76 (s, 1H, –NHCO–). 13C NMR (CDCl3,
300 MHz): d 14.09, 22.61, 24.11, 25.94, 29.02, 29.13,
31.76 (heptyloxy-C), 68.47 (–SCH2–), 113.77, 114.24,
120.39, 124.39, 125.53, 141.13 (Ar–C), 153.35 (triazolone,
C=N), 157.03 (triazolone, C=O). MS-EI m/z 320 (M?1).
(KBr) cm-1: 3183 (NH), 1721 (C=O). H NMR (CDCl3,
300 MHz): d 4.02 (s, 2H, –SCH2–), 5.12 (s, 2H, –OCH2–),
7.14–8.16 (m, 8H, Ar–H), 12.00 (s, 1H, –NHCO–). 13C
NMR (DMSO-d6, CDCl3, 300 MHz): d 23.90 (–OCH2–),
70.15 (–SCH2–), 113.88, 114.52, 120.05, 124.58, 126.36,
127.65, 127.65, 128.14, 128.64, 128.64, 136.63, 140.37
(Ar–C), 152.8 (triazolone, C=N), 156.13 (triazolone, C=O).
MS-EI m/z 312 (M?1).
7-[(2-Fluorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6k) White solid; mp.
1
196–198 °C. IR (KBr) cm-1: 3188 (NH), 1720 (C=O). H
7-(Octyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
NMR (CDCl3, 300 MHz): d 3.84 (s, 2H, –SCH2–), 5.14 (s,
2H, –OCH2–), 6.96–8.29 (m, 7H, Ar–H), 9.47 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
28.60 (–OCH2–), 68.98 (–SCH2–), 118.56, 119.28, 120.12,
120.40, 124.83, 128.32, 128.51, 129.32, 129.47, 131.33,
135.18, 145.15 (Ar–C), 157.54 (triazolone, C=N), 160.68
(triazolone, C=O). MS-EI m/z 330 (M?1).
benzothiazin-1-one (6g) White solid; mp. 94–96 °C. IR
1
(KBr) cm-1: 3181 (NH), 1709 (C=O). H NMR (CDCl3,
300 MHz): d 0.89 (t, 3H, J = 6.0 Hz, –CH3), 1.29–1.44
(m, 10H, –CH2–), 1.73–1.82(m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.94 (t, 2H, J = 7.5 Hz, –OCH2–), 6.84–8.26 (m,
3H, Ar–H), 10.53 (s, 1H, –NHCO–). 13C NMR (DMSO-d6,
300 MHz): d 14.41, 22.54, 23.51, 25.91, 29.13, 31.70, 37.42,
51.76 (octyloxy-C), 68.35 (–SCH2–), 113.91, 114.20,
120.06, 124.85, 125.10, 141.05 (Ar–C), 152.75 (triazolone,
C=N), 156.58 (triazolone, C=O). MS-EI m/z 334 (M?1).
7-[(3-Fluorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazol-
o[4,3-d][1,4]benzothiazin-1-one (6l) White solid; mp.
1
132–134 °C. IR (KBr) cm-1: 3178 (NH), 1718 (C=O). H
NMR (CDCl3, 300 MHz): d 3.83 (s, 2H, –SCH2–), 5.07 (s,
2H, –OCH2–), 6.92–8.29 (m, 7H, Ar–H), 9.19 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.82 (–OCH2–), 69.24 (–SCH2–), 113.86, 114.07, 114.55,
114.70, 114.98, 120.07, 123.19, 124.68, 126.51, 130.47,
139.54, 140.41 (Ar–C), 155.86 (triazolone, C=N), 159.38
(triazolone, C=O). MS-EI m/z 330 (M?1).
7-(Dodecyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6h) White solid; mp. 92–94 °C. IR
1
(KBr) cm-1: 3179 (NH), 1711 (C=O). H NMR (CDCl3,
300 MHz): d 0.88 (t, 3H, J = 6.0 Hz, –CH3), 1.26–1.45
(m, 18H, –CH2–), 1.70–1.83 (m, 2H. –CH2–), 3.83 (s, 2H,
–SCH2–), 3.95 (t, 2H, J = 6.0 Hz, –OCH2–), 6.83–8.24
(m, 3H, Ar–H), 10.52 (s, 1H, –NHCO–). 13C NMR
(CDCl3, 300 MHz): d 14.13, 22.69, 24.09, 25.98, 29.12,
29.36, 29.49, 29.60, 29.64, 29.66, 31.92, 50.75 (dodecyl-
oxy-C), 68.47 (–SCH2–), 113.75, 114.23, 120.36, 124.41,
125.48, 141.16 (Ar–C), 153.14 (triazolone, C=N), 157.04
(triazolone, C=O). MS-EI m/z 390 (M?1).
7-[(4-Fluorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6m) White solid; mp.
1
202–204 °C. IR (KBr) cm-1: 3184 (NH), 1715 (C=O). H
NMR (CDCl3, 300 MHz): d 3.84 (s, 2H, –SCH2–), 5.03 (s,
2H, –OCH2–), 6.93–8.29 (m, 7H, Ar–H), 9.42 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.86 (–OCH2–), 69.41 (–SCH2–), 113.83, 114.51, 115.32,
115.60, 120.05, 124.61, 129.66, 129.77, 132.63, 132.67,
139.06, 140.39 (Ar–C), 152.82 (triazolone, C=N), 155.98
(triazolone, C=O). MS-EI m/z 330 (M?1).
7-(Tetradecyloxy)-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-1-one (6i) White solid; mp. 112–114 °C. IR
1
(KBr) cm-1: 3177 (NH), 1709 (C=O). H NMR (CDCl3,
300 MHz): d 0.88 (t, 3H, J = 6.2 Hz, –CH3), 1.26–1.47
(m, 22H, –CH2–), 1.73–1.82 (m, 2H, –CH2–), 3.83 (s, 2H,
–SCH2–), 3.95 (t, 2H, J = 6.0 Hz, –OCH2–), 6.85–8.27
(m, 3H, Ar–H), 10.19 (s, 1H, –NHCO–). 13C NMR
(CDCl3, 300 MHz): d 14.14, 22.71, 24.10, 25.98, 29.13,
29.38, 29.48, 29.50, 29.58, 29.61, 29.67, 29.78, 31.94,
50.74 (tetradecyloxy-C), 68.47 (–SCH2–), 113.76, 114.25,
7-[(2-Chlorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazol-
o[4,3-d][1,4]benzothiazin-1-one (6n) White solid; mp.
1
212–214 °C. IR (KBr) cm-1: 3179 (NH), 1701 (C=O); H
NMR (DMSO-d6, 300 MHz): d 4.04 (s, 2H, –SCH2–), 5.17
(s, 2H, –OCH2–), 7.19–8.18 (m, 7H, Ar–H), 12.00 (s, 1H,
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1833
–NHCO–). 13C NMR (DMSO-d6, 300 MHz): d 23.50
(–OCH2–), 67.81 (–SCH2–), 114.25, 114.66, 120.13,
125.05, 126.66, 127.87, 129.91, 130.50, 130.78, 133.18,
134.43, 141.06 (Ar–C), 152.79 (triazolone, C=N), 156.05
(triazolone, C=O). MS-EI m/z 346 (M?1).
7-{[3-(Trifluoromethyl)benzyl]oxy}-2,4-dihydro-1H-[1,2,4]
triazolo[4,3-d][1,4]benzothiazin-1-one (6s) White solid;
mp. 176–178 °C. IR (KBr) cm-1: 3189 (NH), 1710 (C=O).
1H NMR (CDCl3, 300 MHz): d 3.84 (s, 2H, –SCH2–), 5.11
(s, 2H, –OCH2–), 6.92–8.30 (m, 7H, Ar–H), 9.44 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.91 (–OCH2–), 69.30 (–SCH2–), 113.81, 114.58, 120.10,
122.36, 124.05, 124.68, 126.60, 129.34, 130.20, 130.63,
131.06, 137.89, 140.31 (Ar–C), 152.85 (triazolone, C=N),
155.82 (triazolone, C=O). MS-EI m/z 380 (M?1).
7-[(3-Chlorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6o) White solid; mp.
1
194–196 °C. IR (KBr) cm-1: 3182 (NH), 1721 (C=O). H
NMR (CDCl3, 300 MHz): d 3.84 (s, 2H, –SCH2–), 5.04 (s,
2H, –OCH2–), 6.92–8.29 (m, 7H, Ar–H), 9.18 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.85 (–OCH2–), 69.18 (–SCH2–), 113.85, 114.54, 120.06,
124.69, 125.84, 126.53, 127.40, 128.09, 130.22, 134.02,
139.06, 140.38 (Ar–C), 152.80 (triazolone, C=N), 155.82
(triazolone, C=O). MS-EI m/z 346 (M?1).
2-{[(1-Oxo-2,4-dihydro-1H-[1,2,4]triazolo[4,3-d][1,4]
benzothiazin-7-yl)oxy]methyl}benzonitrile (6t) White solid;
mp. 212–214 °C. IR (KBr) cm-1: 3188 (NH), 1721 (C=O).
1H NMR (CDCl3, 300 MHz): d 3.85 (s, 2H, –SCH2–), 5.27
(s, 2H, –OCH2–), 6.96–8.32 (m, 7H, Ar–H), 9.25 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, 300 MHz): d 23.48
(–OCH2–), 68.46 (–SCH2–), 111.89, 114.33, 114.73,
117.65 (–CN), 120.13, 125.08, 125.82, 129.71, 130.24,
133.84, 133.94, 140.09, 141.05 (Ar–C), 152.74 (triazolone,
C=N), 155.90 (triazolone, C=O). MS-EI m/z 337 (M?1).
7-[(4-Chlorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6p) White solid; mp.
1
198–200 °C. IR (KBr) cm-1: 3179 (NH), 1705 (C=O). H
NMR (CDCl3, 300 MHz): d 3.83 (s, 2H, –SCH2–), 5.04 (s,
2H, –OCH2–), 6.91–8.29 (m, 7H, Ar–H), 9.45 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.95 (–OCH2–), 69.35 (–SCH2–), 113.84, 114.54, 120.08,
124.56, 126.45, 128.68, 128.68, 129.01, 129.01, 133.52,
135.23, 140.30 (Ar–C), 152.87 (triazolone, C=N), 155.89
(triazolone, C=O). MS-EI m/z 346 (M?1).
Biological assay
Anticonvulsant activity
All the target compounds were screened for their anticon-
vulsant activity using the MES test, which is the most
frequently used method for evaluating the anticonvulsant
activity of compounds. Neurotoxicity was assessed using
the rotarod test. The MES and rotarod tests were carried
out according to the anticonvulsant drug development
(ADD) program protocol. All of compounds, which were
dissolved in DMSO, were evaluated for their anticonvul-
sant activity and neurotoxicity using KunMing mice in the
18–22 g weight range. KunMing mice were purchased
from the Laboratory of Animal Research of the College of
Pharmacy at Yanbian University. Several compounds
possessed good activities when they were quantified for
their anticonvulsant activity and neurotoxicity, and com-
pound 6m, which was the most potent, was tested in the
PTZ, 3-MP, and BIC-induced seizure tests.
7-[(2,6-Dichlorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6q) White solid; mp.
1
220–222 °C. IR (KBr) cm-1: 3184 (NH), 1709 (C=O). H
NMR (CDCl3, 300 MHz): d 3.85 (s, 2H, –SCH2–), 5.29 (s,
2H, –OCH2–), 7.00–8.23 (m, 6H, Ar–H), 9.21 (s, 1H,
–NHCO–). 13C NMR (DMSO-d6, CDCl3, 300 MHz): d
23.89 (–OCH2–), 69.60 (–SCH2–), 113.89, 114.56, 120.10,
124.73, 126.72, 128.70, 128.70, 131.18, 131.67, 136.74,
136.74, 140.39 (Ar–C), 152.82 (triazolone, C=N), 156.22
(triazolone, C=O). MS-EI m/z 381 (M?1).
7-[(4-Methylbenzyl)oxy]-2,4-dihydro-1H-[1,2,4]triazolo
[4,3-d][1,4]benzothiazin-1-one (6r) White solid; mp.
1
164–166 °C. IR (KBr) cm-1: 3187 (NH), 1717 (C=O). H
Maximal electroshock seizure (MES) test
NMR (CDCl3, 300 MHz): d 2.37 (s, 3H, –CH3), 3.83 (s,
2H, –SCH2–), 5.03 (s, 2H, –OCH2–), 6.92–8.27 (m, 7H,
Ar–H), 9.66 (s, 1H, –NHCO–). 13C NMR (DMSO-d6,
CDCl3, 300 MHz): d 21.26 (–CH3), 23.80 (–OCH2–),
70.03 (–SCH2–), 113.95, 114.52, 120.03, 124.62, 126.32,
127.91, 127.91, 129.30, 129.30, 133.74, 137.58, 140.49
(Ar–C), 152.78 (triazolone, C=N), 156.17 (triazolone,
C=O). MS-EI m/z 326 (M?1).
The MES test was carried out by the methods described in the
ADD of the National Institutes of Health (USA) (Krall et al.,
1978; Poter et al., 1984). 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 absence of tonic extension of the hind leg. After 0.5 h of
123

1834
Med Chem Res (2014) 23:1829–1838
drug administration, the activities were evaluated in MES
test. In phase-I screening, each compound was administered
at the dose levels of 100, 30, 15, and 7.5 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 5
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
neurotoxicity. From these data, the respective ED50, TD50
values, and 95 % confidence intervals were calculated by
probit analysis.
Results and discussion
Chemistry
6-Methoxybenzo[d]thiazol-2-amine was used as a starting
material for the preparation of all compounds described in
the current paper. All of the compounds were prepared as
outlined in Scheme 1. 6-Methoxybenzo[d]thiazol-2-amine
was hydrolyzed in a 50 % KOH solution to give 2-amino-
5-methoxybenzenethiol (1), which was treated with chlo-
roacetyl chloride to give compound 2 in accordance with a
procedure reported in the literature (Shridhar et al., 1984).
Compound 2 was successfully demethylated by treatment
with boron tribromide to give the corresponding phenolic
species 7-hydroxy-2H-benzo[b][1,4]thiazin-3(4H)-one (3).
This material was then alkylated with a variety of different
alkylating agents to give the 7-alkoxy-2H-benzo[b][1,4]
thiazin-3(4H)-ones (4a–t). These compounds 4a–t were
then treated with Lawesson’s reagent to give the corre-
sponding intermediate 7-alkoxy-2H-benzo[b][1,4]thiazin-
3(4H)-thiones (5a–t) (Zhang et al., 2010), which were
converted to the target compounds 6a–t by treatment with
methyl hydrazinecarboxylate in refluxing n-butanol. Their
chemical structures were characterized using IR, 1H NMR,
MS, and 13C NMR techniques. A detailed overview of their
physical and analytical data has been provided in Synthesis
of target compounds (6a–6t).
Neurotoxicity screening (NT)
Theneurotoxicityofthecompoundswasmeasuredinmiceby
the rotarod test. The mice were trained to stay on an accel-
erating rotarod of diameter an inch that rotates at 10 rpm.
Trained animals were given i.p. injection of the test com-
pounds. 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 (Krall et al., 1978; Poter et al., 1984).
Chemical induced seizsure tests
In chemically induced seizures (see Table 3), mice were
given doses of convulsant drugs that could induce seizures
at least 97 % of animals. The doses used were: PTZ,
85 mg/kg; 3-mercaptopropionic acid, 40 mg/kg; and BIC,
5.4 mg/kg. The test compounds and standard AEDs were
administered i. p. in a volume of 30 mg/kg to groups of ten
mice 30 min before injection of PTZ (s. c.), 3-MP (i. p.)
and BIC (s. c.). The mice were placed in individual cages
and observed for 30 min, the number of clonic seizures,
tonic seizures, and the lethality were recorded (Arnoldi
Pharmacology
Phase I evaluation of the anticonvulsant activity
The choice of an appropriate animal model for the
initial compound screening process represents a very
important step in antiepileptic drug discovery. There are
currently three models available for the in vivo
¨
et al., 1990; Loscher and Schmidt, 1988).
Scheme 1 The synthesis of the
O
O
S
O
SH
S
N
a
b
target compounds 6a–6t a 50 %
KOH, reflux, 20 h. b NaHCO3,
TEBA, ClCOCH2Cl, CH3CN,
reflux, 2 h. c BBr3, CH2Cl2,
0 °C, 3 h. d K2CO3, alkyl
bromide or benzyl chloride
derivative, CH3CN, reflux,
4–24 h e Lawesson’s reagent,
toluene, reflux, 3–6 h.
NH2
N
H
O
NH2
1
2
HO
S
RO
RO
S
c
e
d
N
H
O
N
H
O
N
f NH2NHCO2CH3, n-Butanol,
reflux, 2–3d
3
4a-4t
S
RO
S
f
N
N
H
S
NH
O
6a-6t
5a-5t
123

Med Chem Res (2014) 23:1829–1838
1835
evaluation of a drug candidate’s anticonvulsant activity,
namely the MES, subcutaneous pentylenetetrazole
(scPTZ), and kindling models, which are routinely used
by most AED discovery programs. Of these tests, the
MES and scPTZ seizure models represent the two ani-
mal seizure models most widely used in the search for
new AEDs (White, 2003; Levy et al., 1995). The MES
test is believed to provide an effective prediction of
drugs that will be effective against generalized seizures
of the tonic–clonic (grand mal) type, whereas the scPTZ
test is used to find drugs that are effective against the
generalized seizures of the petit mal (absence) type. In
the current study, the MES seizure model was used for
screening the anticonvulsant activity of target com-
pounds. The calculated Log P (Clog P) values were
calculated using the ChemOffice 12.0 software (Cam-
bridgeSoft, USA).
During the preliminary screening process, compounds
were administered as an intraperitoneal (i.p.) injection at
dose levels of 100, 30, 15, and 7.5 mg/kg. Their anticon-
vulsant effects were assessed at 0.5 h intervals following
administration in mice. The following observations were
obtained and listed in Table 1.
In the MES test, most of the compounds were active at a
dose of 100 mg/kg, indicating their potent anticonvulsant
properties. Of the compounds tested, compounds 6a–6e,
6j–6m, 6o, and 6r exhibited absolute protection, whereas
compounds 6f, 6p, 6q, and 6s showed partial protection (1/
3 or 2/3) at a dose of 100 mg/kg. At a dose of 30 mg/kg,
compounds 6b–6f, 6j–6m, and 6o–6r showed different
degrees of protection against the MES-induced seizures. At
a dose of 15 mg/kg, compounds 6b–6f, 6j–6m, and 6r still
exhibited activity, with compounds 6c–6e in particular
providing complete protection for all of the mice tested
Table 1 Phase-I preliminary evaluation of the anticonvulsant activity in mice (i.p.)
RO
S
N
N
NH
O
Compound
R
MESa mg/kg
100
CLog Pb
30
15
7.5
c
6a
6b
6c
6d
6e
6f
–C2H5
3/3
3/3
3/3
3/3
3/3
2/3
0/3
0/3
0/3
3/3
3/3
3/3
3/3
0/3
3/3
2/3
1/3
3/3
1/3
0/3
0/5
5/5
5/5
5/5
5/5
2/5
–
–
–
1.44
1.97
2.50
3.03
3.56
4.09
4.62
6.73
7.79
2.68
2.83
2.83
2.83
3.40
3.40
3.40
4.11
3.18
3.57
2.26
n –C3H7
3/5
5/5
5/5
5/5
1/5
–
2/5
3/5
3/5
3/5
0/5
–
n –C4H9
n –C5H11
n –C6H13
n –C7H15
6g
6h
6i
n –C8H17
n –C12H25
–
–
–
n –C14H29
–
–
–
6j
–CH2C6H5
4/5
3/5
2/5
5/5
–
3/5
1/5
1/5
3/5
–
3/5
0/5
0/5
2/5
–
6k
6l
–CH2C6H4(o-F)
–CH2C6H4(m-F)
–CH2C6H4(p-F)
–CH2C6H4(o-Cl)
–CH2C6H4(m-Cl)
–CH2C6H4(p-Cl)
–CH2C6H3(2,6-Cl2)
–CH2C6H4(p-CH3)
–CH2C6H4(m-CF3)
–CH2C6H4(o-CN)
6m
6n
6o
6p
6q
6r
6s
6t
2/5
2/5
1/5
5/5
0/5
–
0/5
0/5
0/5
3/5
–
–
–
–
0/5
–
–
–
a
Maximal electroshock seizure test (number of animals protected/number of animals tested)
CLog P was calculated using software ChemOffice 12.0
Not tested
b
c
123

1836
Med Chem Res (2014) 23:1829–1838
Structure–Activity Relationships
(5/5). Six compounds (6b–6e, 6j, and 6m) exhibited
moderate protection at the lower dose of 7.5 mg/kg.
In the current study, we have synthesized a series of 7-alk-
oxy-2,4-dihydro-1H-benzo[b][1,2,4]triazolo[4,3-d][1,4]thi-
azin-1-one derivatives (6a–6t). Following an analysis of the
activities of the synthesized compounds, the following
structure–activity relationships (SARs) were obtained. Of all
of the compounds tested, compounds 6a–6i possessed dif-
ferent alkoxy groups attached to the core triazolobenzothi-
azine fragment ranging from an ethoxy group to a
tetradecyloxy group. The length of the alkyl chain appeared
to have an impact on the anticonvulsant activity of the
derivatives. For example, as the alkyl chain length increased
from 6a to 6d, the anticonvulsant activity gradually
increased with compound 6d (with the n-pentane chain)
being the most active compound. From 6d to 6i, although the
alkyl chain length increased further, the anticonvulsant
activity decreased and ultimately disappeared. Compound
6d was the most active compound with an ED50 value of
7.1 mg/kg, indicating the optimal partition coefficient
associated with the easiest crossing of the biological mem-
branes in the congeners. Compound 6j was substituted with a
benzyloxy group at the 7-position of the triazolobenzothi-
azine core and F, Cl, Br, CH3, CF3, and CN groups were
subsequently added on to the benzyloxy of 6j in different
position providing compounds 6k–6t. Unfortunately, the
introduction of the halogens and other groups to the phenyl
ring led to a reduction in the anticonvulsant activity relative
to 6j, with the exception of compound 6m (p-F substituent).
Compounds 6g, 6h, and 6i did not show activity in the MES
test, and their Clog P values were in the range of 4.62–7.79,
and therefore considered too high for the absorption and
distribution of drugs that act on the central nervous system
(Lien et al., 1979). These results provided an indication of
the importance of the lipophilicity as well as electronic
Phase II evaluation of the anticonvulsant activity
Based on the considerable anticonvulsant activity demon-
strated during the phase I testing, compounds 6b–6e, 6j,
and 6m were subjected to phase II 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 collected for the current antiepileptic drug carbam-
azepine, are shown in Table 2. The six compounds tested
gave ED50 values in the range of 7.1–12.2 mg/kg and TD50
values in the range of 141.4–81.8 mg/kg, leading to pro-
tective index values in the range of 7.0–15.4. All the
compounds tested showed a higher protective index (PI)
than carbamazepine, which gave a PI value of 6.4. Of the
six compounds tested, compound 6d was the most active
possessing strong anti-MES activity with an ED50 value of
7.1 mg/kg. Unfortunately, however, it also showed a high
level of neurotoxicity with a TD50 value of 81.8 mg/kg that
consequently resulted in a PI value of 11.5. With all these
factors in mind (i.e., efficacy, toxicity, and safety), com-
pound 6m was considered as the most promising compound
in this study because of its efficacy and low neurotoxicity
(ED50 = 9.2 mg/kg and TD50 = 141.4 mg/kg, resulting in
a PI value of 15.4). Compound IV-e was the most active
compound from series IV (leading structures) with an ED50
value of 34.1 mg/kg, TD50 value of 305.5 mg/kg and PI of
8.9. As shown in Table 2, all of the tested compounds
(6b–6e, 6j, and 6m) showed better levels of anticonvul-
sant activity than compound IV-e, and compounds
6c–6e and 6m also showed a higher safety margin than
compound IV-e.
Table 2 Phase-II quantitative anticonvulsant evaluation in Mice (i.p.)
Compound
R
EDa50 (mg/kg)
TDb50 (mg/kg)
PIc (TD50/ED50
)
6b
n –C3H7
n –C4H9
n –C5H11
n –C6H13
–CH2C6H5
–CH2C6H4(p-F)
–
12.2 (4.1–36.1)d
7.9 (6.9–9.1)
84.9 (64.9–110.9)
117.8 (103.1–134.7)
81.8 (62.2–106.9)
89.6 (50.6–158.8)
94.7 (71.1–126.0)
141.4 (123.7–161.6)
305.5 (217.3–29.6)
76.1 (55.8–103.7)
7.0
14.9
11.5
11.3
7.8
6c
6d
7.1 (6.2–8.2)
6e
7.9 (6.8–9.2)
6j
12.2 (4.1–36.1)
9.2 (7.0–12.2)
34.08 (23.9–48.6)
11.8 (8.5–16.4)
6m
15.4
8.9
Compound IVe
Carbamazepine
–
6.4
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
b
c
d
PI protective index (TD50/ED50
)
95 % confidence intervals given in parentheses
123

Med Chem Res (2014) 23:1829–1838
1837
Table 3 Effects of compound 6m on chemically-induced seizures in mice (i.p.)
Chemical substances
Pentylenetetrazole
Compound
Doses (mg/kg) Test time (h) Clonic seizures (%) Tonic seizures (%) Lethality (%)
DMSO
–
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
100
100
100
100
100
100
100
100
90
100
0
100
10
Carbamazepine 30
6m
3-Mercaptopropionic acid DMSO
30
–
10
100
0
80
100
0
Carbamazepine 30
6m
30
–
0
60
Bicuculline
DMSO
100
0
100
60
Carbamazepine 30
6m 30
10
30
properties of the substituents on the activity of these
compounds.
rates of 0, 100, and 100 %, respectively. By comparison,
compounds 6m showed an anticonvulsant effect similar to
that of carbamazepine in inhibiting the clonic and tonic
seizures. While its protection for the death induced by
3-MP cannot compare with carbamazepine, with inhibition
of death induced by 3-MP at rate of 40 % (Table 3). 3-MP
is competitive inhibitor of the GABA synthesis enzyme
glutamate decarboxylase (GAD), and it inhibits the syn-
thesis of GABA resulting in decrease of GABA levels in
the brain (Loscher, 1979). Compound 6m exhibited the
efficacy to inhibit the tonic seizures induced by 3-MP
though the protection for death is barely satisfactory, which
suggesting that the increase of GABA levels might be
involved in its anticonvulsant mechanisms.
Speculation of mechanism
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., phenyt-
oin, carbamazepine), which suggested that the tested
compounds may inhibit voltage-gated ion channels (par-
ticularly sodium channels). To further investigate the
effects of the anticonvulsant activity in several different
models and speculate about the possible mechanism action
of anticonvulsant, compound 6m was tested against con-
vulsions induced by chemical substances, including PTZ,
3-MP, and BIC. Compound 6m 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 BIC-induced seizure model, both carbamazepine
and 6m 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 6m showed inhibition of
clonic and tonic seizures and death at rates of 10, 90, and
70 %, respectively (Table 3). BIC is a competitive antag-
onist of the GABAA receptor (Macdonald and Barker,
1978). As compound 6m inhibited the seizures induced by
BIC, it likely exerts anticonvulsant activity, at least par-
tially, through GABAA-mediated mechanisms.
In the sc-PTZ model, carbamazepine inhibited the clonic
seizures, tonic seizures and death at the rates of 0, 100, and
90 %, respectively. Compound 6m inhibited the clonic
seizures, tonic seizures, and lethality at the rates of 0, 90, and
20 % induced by sc-PTZ, respectively (Table 3). From these
data we can see that compound 6m can inhibit the tonic
seizures induced by PTZ. PTZ has been reported to produce
seizures by inhibiting c-aminobutyric acid (GABA) neuro-
transmission (Okada et al., 1989; Olsen 1981). GABA is the
main inhibitory neurotransmitter in the brain, and is widely
implicated in epilepsy. Inhibition of GABAergic neuro-
transmission or activity has been shown to promote and
facilitate seizures (Gale, 1992), while GABAergic
enhancement is known to inhibit or attenuate seizures. The
inhibition of compound 6m upon the PTZ-induced tonic
seizures might be related to the enhancing of GABAergic
neurotransmission or activity.
Conclusion
In summary, the current study has demonstrated that a
series of 7-alkoxy-2,4-dihydro-1H-benzo[b][1,2,4]triazol-
o[4,3-d][1,4]thiazin-1-one derivatives 6a–t were effective
in the MES screens. The most promising of these com-
pounds was 7-[(4-fluorobenzyl)oxy]-2,4-dihydro-1H-[1,2,4]
triazolo[4,3-d][1,4]benzothiazin-1-one (6m), which showed
an ED50 value of 9.2 mg/kg and a PI value of 15.4. These
values were superior to those provided by carbamazepine
(ED50 and PI values of 11.8 and 6.4, respectively) in the
In the 3-MP induced seizure model, carbamazepine
inhibited the clonic seizures, tonic seizures and death at
123

1838
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PTZ, 3-MP, and BIC were also established, with the results
suggesting that several different mechanisms of action
might be involved in its anticonvulsant activity, including
the inhibition of voltage-gated ion channels and the mod-
ulation of GABAergic activity.
¨
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Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (No. 81160382) and National
Science and Technology Major Project of China (No. 2011ZX09102-
003-03).
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York, pp 501–526
Conflict of interest We declare that we have no conflict of interest
with respect to this study.
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