Synthesis and evaluation of the anticonvulsant activity of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives

Article abstract of DOI:10.3109/14756366.2013.776555

Two series of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4] thiazepine derivatives (6a-q and 7a-q) were synthesized and evaluated for their anticonvulsant activity using the maximal electroshock (MES) method. All of the compounds prepared were e

Full text of DOI:10.3109/14756366.2013.776555

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(2): 272–280
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.776555
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ORIGINAL ARTICLE
Synthesis and evaluation of the anticonvulsant activity of
8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine
derivatives
Xian-Qing Deng1,2, Ming-Xia Song1,2, Shi-Ben Wang1, and Zhe-Shan Quan1
2
1College of Pharmacy, Yanbian University, Yanji, Jilin, China and Medical College, Jinggangshan University, Ji’an, Jiangxi, China
Abstract
Keywords
Two series of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives (6a–q
and 7a–q) were synthesized and evaluated for their anticonvulsant activity using the maximal
electroshock (MES) method. All of the compounds prepared were effective in the MES screens.
Among which, compound 7j was considered as the most promising one with an ED50 value
of 26.3 mg/kg and a superior protective index value of 12.6. The potency of compound 7j
against seizures induced by pentylenetetrazole, 3-mercaptopropionic acid and bicuculline
suggested that two different mechanisms of action might potentially be involved in its
anticonvulsant activity, including the inhibition of voltage-gated ion channels and the
modulation of GABAergic activity. A computational study was also conducted to predict the
pharmacokinetic properties of the compounds prepared, with the results supporting the use
of these compounds as a group of promising antiepileptic agents.
Anticonvulsant, benzothiazepines, maximal
electroshock, neurotoxicity, triazole
History
Received 8 January 2013
Revised 30 January 2013
Accepted 30 January 2013
Published online 11 March 2013
Introduction
of the AEDs makes it difficult to apply rational methodologies in
the field of AED discovery. Fortunately, a design method for the
development of new antiepileptic agents based on the existence
of different pharmacophores has been established through the
analysis of different structural characteristics of clinically effect-
ive drugs as well as other antiepileptic compounds10–13. Several
key pharmacophoric elements essential for good anticonvulsant
activity have been identified and well documented in the
literature13–15, including a hydrophobic unit (A), an electron
donor group (D) and a hydrogen donor/acceptor unit (HAD)
(Figure 1). With the recent development of novel therapeutics
based on the pharmacophore model, several new drugs (including
tiagabine, lamotrigine, pregabalin, stiripentol, zonisamide, topir-
amate and levetiracetam) have already reached the market as
promising antiepileptic agents16.
Epilepsy is one of the most common neurological disorders, and
is characterized by excessive temporary neuronal discharge
resulting in recurrent unprovoked seizures1,2. It has been reported
that about 1% of the world’s population (about 50 million people
worldwide) are suffering with this neurological disorder at any
one time2. In recent years, significant efforts have been invested in
the development of novel therapeutics, resulting in the emergence
of several novel drugs as promising anticonvulsant agents3,4. The
anticonvulsants currently available for the treatment of this
disorder, however, can only reduce the severity and number
of seizures in less than 70% of patients. Moreover, their usage is
often associated with undesirable and numerous side effects5–7
.
The current antiepileptic drugs (AEDs) used widely, such as
phenytoin, carbamazepine, ethosuximide, valproic acid and
barbiturates, produce many adverse side effects, including
ataxia, hepatotoxicity, gingival hyperplasia and megaloblastic
anaemia8,9. High levels of toxicity and intolerance, and a lack
of efficacy also represent further limitations of the current
AEDs. With all of this in mind, there is an urgent need for the
development of novel AEDs with higher levels of potency and
lower levels of toxicity.
The 1,2,4-triazole nucleus has been incorporated into a
wide variety of therapeutically important agents, such as
antimicrobial17–19, antiviral agents20, anti-HIV21,22 and anti-
inflammatory23. Moreover, the chemistry of 1,2,4-triazoles and
their fused heterocyclic derivatives has also received considerable
attention owing to their synthetic and biological importance.
In our previous work, we reported the synthesis and bioactivity
evaluation of several series of compounds with a triazole ring.
All of them exhibited a certain degree of anticonvulsant
The lack of information pertaining to the cellular mechanism
of epilepsy in humans together with the complex mechanisms
activity24–30
. Among which, 7-alkoxy-4H-[1,2,4]triazolo[4,3-
d]benzo[b][1,4]thiazines (I) were found to exhibit the most
remarkable levels of anticonvulsant activities29. Herein, as part
of our ongoing research towards the development of novel
anticonvulsant agents, we described the design and synthesis
of a series of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-
d][1,4]thiazepines (6a–q) using compounds I (Figure 2, I) as
the lead compound. Compounds 6a–q (Figure 2, II) were
Address for correspondence: Zhe-Shan Quan, College of Pharmacy,
Yanbian University, Yanji, Jilin 133002, China. Tel: +86-433-24360200.
Fax: + 86-433-2436020. E-mail: zsquan@ybu.edu.cn

DOI: 10.3109/14756366.2013.776555
Activity of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives 273
NH2
D
D
A
A
O
D
O
H3C
H3C
OH
A
N
CH2
A
A
HAD
N
O
D
N
NH
O
A
HN
A
HN
HAD
HAD
O
NH2
S
Cl
F
Carbamazepine
Progabide
Albution
Phenytoin
HAD
D
O
A
H
O
S
NH
HAD
NH2
O
N
O
NH2
F
HAD
HAD
H2N
O
O
O
A
HN
N
N
N
N
A
D
O
O
O
O
D
A
F
D
Rufinamide
Phenobarbital
Febamate
Zonisamide
A
A
A
D
D
S
S
RO
RO
A
D
O
CH3
O
Cl
D
N
N
N
N
N
A
N
N
N
H
N
N
O
H
O
H
HAD
Mephenytoin
HAD
HAD
Target compounds I and II
HAD
Nordazepam
Figure 1. The vital structural features included in well-known antiepileptic agents and target compounds: hydrophobic unit (A), electron donor group
(D), HAD unit.
Figure 2. Structures of lead compounds I and
target compounds II (6a–q), III (7a–q).
the ring-expanded analogues of compounds I. Furthermore, Experimental protocols
our previous investigations suggested that the triazolone ring
was relevant to the observed levels of activity. So, a series of
Chemistry
8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepin- Melting points were determined in open capillary tubes and were
1(2H)-ones (7a–q) were also been synthesized (Figure 2, III) uncorrected. IR spectra were recorded (in KBr) on IRPrestige-21.
with the aim of identifying promising compounds and establish- 1H-NMR and 13C-NMR spectra were measured on an AV-300
¨
ing some structure–activity relationships (SARs). As shown (Bruker, Fallanden, Switzerland), and all chemical shifts were
in Figure 1, all four of the essential pharmacophoric elements given in ppm relative to tetramethysilane. Mass spectra were
mentioned above were successfully incorporated into the com- measured on an HP1100LC (Agilent Technologies, Santa Clara,
pounds synthesized.
CA). Elemental analyses were performed on a 204Q CHN (Perkin
The newly synthesized compounds were characterized by IR, Elmer, Cambridge, MA). Microanalyses of C, N and H were
1H and 13C NMR, mass spectral and elemental analyses. Their performed using a Heraeus CHN Rapid Analyzer (Perkin Elmer,
anticonvulsant activities and neurotoxicities were evaluated using Waltham, MA). The major chemicals were purchased from
the maximal electroshock (MES)-induced seizure model and the Aldrich Chemical Corporation (St. Louis, MO).
rotarod assay in mice, respectively. To effectively elucidate the
possible mechanism of action of these compounds, the most
Synthesis of 2-amino-5-methoxybenzenethiol (1)
active compound 7j was tested in pentylenetetrazole (PTZ),
3-mercaptopropionic acid (3-MP) and bicuculline (BIC)-induced
A
mixture of 6-methoxybenzo[d]thiazol-2-amine (6.00 g,
seizure tests. Furthermore, a computational study was conducted 33 mmol) and 50% KOH solution (40 mL) was refluxed for
for predicting the ADME properties, toxicity risks and druglike- 16 h. After cooling, the solution was diluted with water (50 mL)
ness of all the synthesized compounds.
and filtered. The filtrated was neutralized with acetic acid to

274 X.-Q. Deng et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 272–280
pH 6. The precipitate formed was filtered and washed with water. General procedure for the synthesis of 8-alkoxy-4,5-
The crude product quickly dried in drying oven was subjected into dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine (6a–q)
the next reaction immediately. Yield 5.12 g (81%); m.p. 74–76 ꢀC;
A solution of 5a–q (3 mmol) in appropriate n-butanol and formyl
1H NMR (CDCl3, 300 MHz) ꢀ 3.77 (s, 3 H, OCH3), 6.43 (dd, 1H,
hydrazine (6 mmol) was refluxed for 2–3 d (TLC monitoring), the
J ¼ 2.6, 8.5 Hz, Ar-H), 6.57 (d, 1H, J ¼ 8.5 Hz, Ar-H), 6.66 (d,
solvent was evaporated to dryness under reduced pressure, and
1H, J ¼ 2.6 Hz, Ar-H).
the residue poured by water (50 mL) was extracted twice with
dichloromethane (60 mL). The dichloromethane layer was
Synthesis of 8-methoxy-2,3-dihydro-1,5-benzothiazepin-
washed three times with saturated aqueous NaCl (60 mL ꢂ 3)
4(5H)-one (2)
and dried over anhydrous MgSO4. After removing the solvents,
products were purified by silica gel column chromatography
A mixture of freshly prepared 6-methoxybenzo[d]thiazol-2-amine
(1; 3.1 g, 20 mmol), acrylic acid (1.6 mL, 24 mmol) and toluene
with CH2Cl2–CH3OH (70:1). Characterization for 6a: m.p. 126–
(40 mL) was heated to reflux for 6 h. After cooling, the crystal
1
128 ꢀC, yield 56%. H-NMR (CDCl3, 300 MHz) ꢀ 1.05 (t, 3H,
formed (black green) was filtered off, and washed with
J ¼ 7.4 Hz, CH3), 1.76–1.88 (m, 2H, CH2CH3), 3.15 (t, 2H,
J ¼ 6.9 Hz, CNCH2), 3.43 (t, 2H, J ¼ 6.9 Hz, SCH2), 3.96
(t, J ¼ 6.5 Hz, 2H, OCH2), 6.97–7.29 (m, 3H, Ar-H), 8.28
(s, 1H, triazole-H). IR (KBr) cmꢁ1: 1601 1501 (N¼C). MS m/z
262 (M þ H). Anal Calcd for C13H15N3OS (%): C, 59.74; H, 5.79;
N, 16.08. Found (%): C, 59.61; H, 5.88; N, 16.25. The
characterizations of the other compounds (6b–q) are available
in Supplementary material.
methanol. Yield 1.9 g (45%); m.p. 208–210 ꢀC; 1H-NMR
(CDCl3, 300 MHz), ꢀ 2.40 (t, 2H, J ¼ 6.8 Hz, COCH2), 3.35
(t, 2H, J ¼ 6.8 Hz, SCH2), 3.74 (s, 3H, OCH3), 6.93–7.09 (m, 3H,
Ar-H), 9.54 (s, 1H, CONH). IR (KBr) cmꢁ1: 3219 (N-H), 1707
(C¼O). MS m/z 210 (M þ H).
Synthesis of 8-hydroxy-2,3-dihydro-1,5-benzothiazepin-
4(5H)-one (3)
8-Methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (2; 2.09 g,
10 mmol) was dissolved in 60 mL dichloromethane. BBr3
(2.78 mL, 30 mmol) was added dropwise to the solution and
the mixture was stirred at room temperature. After 4 h, 20 mL ice
water was added slowly into the mixture and allowed to stir for
half an hour. After removing the dichloromethane under
reduced pressure, the resulting white precipitate was obtained
by filtration. Yield 1.85 g (95%); m.p. 247–249 ꢀC; 1H-NMR
(CDCl3, 300 MHz), ꢀ 2.37 (t, 2H, J ¼ 6.5 Hz, COCH2), 3.31
(t, 2H, J ¼ 6.5 Hz, SCH2), 6.72–6.93 (m, 3H, Ar-H), 9.44 (s, 1H,
Ar-OH). 9.59 (s, 1H, CONH). MS m/z 196 (M þ H).
General procedure for the synthesis of 8-alkoxy-4,5-
dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepin-1(2H)-
one (7a–q)
A solution of 5a–q (3 mmol) in appropriate n-butanol and
methyl hydrazinecarboxylate (6 mmol) was refluxed for 2–3 d
(TLC monitoring), the solvent was evaporated to dryness under
reduced pressure, and the residue poured by water (50 mL)
was extracted twice with dichloromethane (60 mL). The dichlor-
omethane layer was washed three times with saturated aqueous
NaCl (60 mL ꢂ 3) and dried over anhydrous MgSO4. After
removing the solvents, products were purified by silica gel
column chromatography with CH2Cl2–CH3OH (70:1).
General procedure for the synthesis of 8-alkoxy-2,3-dihydro-1,5-
benzothiazepin-4(5H)-one (4a–q)
1
Characterization for 7a: m.p. 136–138 ꢀC, yield 48%. H-NMR
(CDCl3, 300 MHz) ꢀ 1.03 (t, 3H, J ¼ 7.4 Hz, CH3), 1.79–1.84
(m, 2H, CH2CH3), 2.86 (t, 2H, J ¼ 6.8 Hz, CNCH2), 3.27 (t, 2H,
J ¼ 6.8 Hz, SCH2), 3.96 (t, J ¼ 6.4 Hz, 2H, OCH2), 6.99–7.55
(m, 3H, Ar-H), 10.16 (s, 1H, CONH). IR (KBr) cmꢁ1: 3291
(N-H), 1697 (C¼O), 1601 (N¼C). MS m/z 278 (M þ H).
Anal Calcd for C13H15N3O2S (%): C, 56.30; H, 5.45; N, 15.15.
Found (%): C, 56.02; H, 5.69; N, 15.37. The characteriza-
tions of the other compounds (7b–q) seen in Supplementary
material.
K2CO3 (1.24 g, 10 mmol) and 8-hydroxy-2,3-dihydro-1,5-ben-
zothiazepin-4(5H)-one (3; 5 mmol) were dissolved in acetonitrile
(60 mL) and refluxed for 30 min, then the appropriate alkyl
bromide or benzyl chloride (6 mmol) was added to the mixture.
The reaction mixture was heated at reflux temperature for 4–24 h.
After removing the most solvent, 100 mL of water was poured
into the flask and the precipitate formed was filtered. Yield
79–88%. Characterization for 4a: Yield 81%; m.p. 156–158 ꢀC;
1H-NMR (CDCl3, 300 MHz), ꢀ 1.05 (t, 3H, J ¼ 7.4 Hz, CH3),
1.76–1.88 (m, 2H, CH2CH3), 2.62 (t, 2H, J ¼ 6.9 Hz, COCH2),
3.44 (t, 2H, J ¼ 6.9 Hz, SCH2), 3.93 (t, 2H, J ¼ 6.5 Hz, OCH2),
6.89 (dd, 1H, J1 ¼ 2.8 Hz, J2 ¼ 8.7 Hz, Ar-H), 7.04 (d, 1H,
J ¼ 8.7 Hz, Ar-H), 7.15 (d, 1H, J ¼ 2.8 Hz, Ar-H), 8.03 (s, 1H,
CONH). MS m/z 238 (M þ H).
Pharmacology
MES seizure test
The MES test was carried out according to the methods
described in the ADD of the National Institutes of Health
(USA)31,32. 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 a. After 0.5 and 4.0 h of drug
administration, the activities were evaluated in MES test. In
phase-I screening, each compound was administered at the dose
levels of 30, 100 and 300 mg/kg for evaluating the preliminary
anticonvulsant activity. For determination of the median effect-
ive dose (ED50) 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 neurotoxicity, NT). From these data, the respect-
ive ED50, TD50 values, and 95% confidence intervals were
calculated by probit analysis.
General procedure for the synthesis of 8-alkoxy-2,3-dihydro-1,5-
benzothiazepin-4(5H)-thione (5a–q)
A mixture of crude 8-alkoxy-2,3-dihydro-1,5-benzothiazepin-
4(5H)-one (4a–q; 4 mmol), Lawesson’s reagent (0.97 g,
2.4 mmol) and toluene (40 mL) was heated to reflux for 2–4 h.
The excess solvent was removed under reduced pressure, and the
residue was purified by silica gel column chromatography with
CH2Cl2–ether (2:1) to
a
white solid. Yield 65–74%.
1
Characterization for 5a: Yield 69%; m.p. 134-136 ꢀC; H-NMR
(CDCl3, 300 MHz), ꢀ 1.05 (t, 3H, J ¼ 7.4 Hz, CH3), 1.77–1.89
(m, 2H, CH2CH3), 3.08 (t, 2H, J ¼ 6.6 Hz, COCH2), 3.55 (t, 2H,
J ¼ 6.6 Hz, SCH2), 3.94 (t, 2H, J ¼ 6.5 Hz, OCH2), 6.90 (dd, 1H,
J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz, Ar-H), 7.08 (d, 1H, J ¼ 8.7 Hz, Ar-H), 7.15
(d, 1H, J ¼ 2.7 Hz, Ar-H), 9.86 (s, 1H, CONH). MS m/z
254 (M þ H).

DOI: 10.3109/14756366.2013.776555
Activity of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives 275
OH
S
H CO
H CO
H CO
S
N
3
3
KOH/H O
SH
NH
3
2
O
NH
2
N
H
2
O
1
2
S
S
S
HO
RO
RO
BBr
RX/K CO
3
LR
2
3
N
H
N
H
N
O
O
S
H
3
4a-4q
5a-5q
S
N
S
RO
RO
S
RO
CHONHNH
NH NHCO CH
2
2
2
3
N
N
H
N
N
S
N
N
O
H
6a-6q
5a-5q
7a-7q
R= a: C3H7;
f: C8H17
b: C4H9;
g: CH2C6H5
c: C5H11
h: CH2C6H5(o-F);
;
d: C6H13
i: CH2C6H5(m-F)
;
e: C7H15
j: CH2C6H5(p-F);
;
;
k: CH2C6H5(o-Cl); l: CH2C6H5(m-Cl); m: CH2C6H5(p-Cl); n: CH2C6H5(2,4-2Cl); o: CH2C6H5(o-Br)
p: CH2C6H5(p-Br); q: CH2C6H5(p-CH3);
Scheme 1. Synthetic route to the target compounds (6a–q and 7a–q).
NT screening
Results and discussion
The NT of the compounds was measured in mice by the Chemistry
rotarod test31,32. The mice were trained to stay on an accelerating
All of the target compounds were synthesized according to the
route depicted in Scheme 1, with 6-methoxybenzo[d]thiazol-2-
amine being used as the starting material. Briefly, the starting
material was hydrolyzed in a 50% aqueous KOH solution to afford
2-amino-5-methoxybenzenethiol (1). It is noteworthy that com-
pound 1 was unstable and was therefore immediately progressed
into the subsequent reaction39. Compound 1 was treated with
acrylic acid in toluene for 6 h to provide 8-methoxy-2,3-dihydro-
rotarod of diameter 3.2 cm that rotates at 10 rpm. Trained
animals were given an intraperitoneal injection of the test
compounds. NT was indicated by the inability of the animal to
maintain equilibrium on the rod for at least 1 min in each of the
trials.
Chemical induced seizure tests
In chemically induced seizures (Table 4, Supplementary mater- 1,5-benzothiazepin-4(5H)-one (2). The structure of compound 2
1
ial), mice were given doses of convulsants that could induce was confirmed by its H NMR spectrum, which clearly revealed
seizures at least 97% of animals. The doses used were: PTZ, the resonance signals of the amide (9.54 ppm) and methylene
85 mg/kg; 3-MP acid, 40 mg/kg and BIC, 5.4 mg/kg. The test groups (2.40 and 3.35 ppm), in accordance with the proposed
compounds and standard AEDs were administered as an structure. Unfortunately, however, the yield for this reaction was
intraperitoneal (i.p.) injection in a volume of 30 mg/kg to poor (the best is 45% in our lab), because of the occurrence of
groups of ten mice 30 min before injection of PTZ (subcutane- several competing side-reactions (Figure 3). Compound 2 was
ously), 3-MP acid (i.p.) and BIC (subcutaneously). The mice then demethyled with boron tribromide to give 8-hydroxy-2,3-
were placed in individual cages and observed for 30 min, the dihydro-1,5-benzothiazepin-4(5H)-one (3), which was subse-
number of clonic seizures, tonic seizures and the lethality were quently alkylated with a variety of different alkylating agents to
recorded33,34
.
afford the corresponding 8-alkoxy-2,3-dihydro-1,5-benzothiaze-
pin-4(5H)-one (4a–q). Compounds 4a–q were then treated with
Lawesson’s reagent to provide the key 8-alkoxy-2,3-dihydro-1,5-
benzothiazepin-4(5H)-thione intermediates (5a–q). Finally, the
target compounds 6a–q and 7a–q were obtained following the
Computational study
Prediction of ADME properties
A computational study of titled compounds was performed for treatment of the key intermediates 5a–q with formyl hydrazine
the prediction of ADME properties. Polar surface area (PSA), and methyl hydrazinecarboxylate, respectively.
1
miLog P, number of rotatable bonds, molecular volume, number
Their chemical structures were characterized by IR, H and
of hydrogen donor and acceptor atoms and violations of 13C NMR, mass spectral and elemental analyses. The detailed
Lipinski’s rule of five35 were calculated using Molinspiration physical and analytical data are listed in experimental protocols.
online property calculation toolkit36. Absorption (%ABS) was
calculated by: % ABS ¼ 109 – (0.345 ꢂ PSA)37.
Biological evaluation
Toxicity risks prediction and drug-score
The toxicity prediction and drug-score of the compounds were The choice of an appropriate animal model for the initial
calculated using Osiris property calculator38.
compound screening process represents a very important step in
Phase I evaluation of anticonvulsant activity

276 X.-Q. Deng et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 272–280
HO
O
O
OH
OH
S
N
H CO
H CO
H CO
3
SH
NH
3
S
3
O
O
NH
2
O
HO
O
OH
Figure 3. The side-products formed during the synthesis of compound 2.
AED discovery. There are currently three models available for Structure activity relationships
the in vivo evaluation of the anticonvulsant activity of a drug
In the present work, we synthesized two series of compounds
including 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]
thiazepines (6a–q) and 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]tria-
zolo[4,3-d][1,4]thiazepin-1(2H)-ones (7a–q). Analyzing the
activities of the synthesized compounds, the following SARs
were obtained. Among compounds 6a–q, compounds 6a–f
possessed different alkoxyl group attached to the core
candidate, including the MES, subcutaneous PTZ (scPTZ) and
kindling models, which are routinely used by most AED
discovery programs. Among these tests, the MES and scPTZ
seizure models represent the two animal seizure models most
widely used in the search for new AEDs40,41. In the present study,
the MES seizure model was selected to screen the anticonvulsant
activities of target compounds.
In the preliminary screening process (MES and rotarod
tests), each compound was administered (i.p.) at three different
dose levels, including 30, 100 and 300 mg/kg. Anticonvulsant
and neurotoxic effects were assessed at 0.5 and 4 h intervals
after administration in mice. The results obtained are listed in
Table 1.
As shown in Table 1, all the compounds synthesized were
active when administered at the dose of 300 mg/kg in the MES
test. At the dose of 100 mg/kg, most of the compounds, with the
exception of compounds 6h, 6i, 6n, 6p, 6q and 7n, showed some
degree of protection at time period 0.5 h. At the dose of 30 mg/kg,
compounds 6j, 6m, 7b, 7f and 7i exhibited protection in only 1/3
of the mice, whereas compounds 6d, 6g, 7c–g and 7j exhibited
protection in 2/3 at time period 0.5 h. None of the compounds
showed protection at the 4 h period, indicating that they all had a
rapid onset and short duration of anticonvulsant activity.
In the NT screen, all of the synthesized compounds exhibited
neurotoxic effects at the high dose of 300 mg/kg at 0.5 h. With
the exception of compounds 6g, 6m, 6o, 7b–e, 7k, 7o and 7q,
the majority of the compounds did not show any NT at the
dose of 100 mg/kg at 0.5 h. At the 4 h period, no NT could be
detected.
triazolobenzodiazepine fragment, ranging from
a propoxy
group to an octyloxy 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 (n-hexane chain) being the most active.
From compound 6d–f, although the alkyl chain length increased
further, the anticonvulsant activity decreased, suggesting that
the n-hexane chain was optimal. Compound 6d was the most
active compound with an ED50 value of 23.7 mg/kg, indicating
the optimal partition coefficient associated with the easiest
crossing of the biological membranes in the congeners.
Compound 6g was substituted with a benzyloxy group at the
8-position of the triazolobenzodiazepine core and F, Cl, Br and
CH3 groups were subsequently added on to the benzene ring of
the benzyloxy group of 6g at different positions providing
compounds 6h–q. Unfortunately, the introduction of different
halogen or methyl groups to the phenyl ring led to a reduction
in the anticonvulsant activity in comparison with derivative 6g.
For the three F-substituted derivatives (6h–j), a potency order
of p-F4m-F ¼o-F was observed. Of the four Cl-substituted
derivatives (6k–n), a potency order of p-Cl4o-Cl4m-Cl42,4-
Cl2 was observed. Between the two Br-substituted derivatives
(6o, 6p), the potency order was o-Br4p-Br. The SARs of
compounds 7a–q were on the whole similar to those of 6a–q.
Phase II evaluation of anticonvulsant activity
On the basis of the results recorded in the phase I test, compounds For the alkoxyl group substituted compounds (7a–f), the trend
6d, 6g, 7c–g and 7j were subjected to phase II trials for in activity was the same as that observed in compounds 6a–q,
quantification of their anticonvulsant activities (indicated by with compound 7d (with an n-hexane alkyl chain) being
ED50) and neurotoxicities (indicated by TD50) in mice. The results identified as the most active compound in the series. For
of the quantitative tests for the selected compounds, together with compounds 7g–q, substituted with a benzyloxy groups that were
the corresponding data for the currently marketed AEDs, either halogenated or non-halogenated on their phenyl ring, the
including carbamazepine, phenobarbital and valproate, are p-F substituted compound 7j was found to be the most active
shown in Table 2. Compound 6g was found to be the most with an ED50 value of 26.3 mg/kg, and also exhibited a higher
active of this group of compounds with an ED50 value of 20.7 mg/ level of activity than the non-substituted compound 7g
kg. Unfortunately, compound 6g also showed a high level of NT (ED50 ¼ 36.7 mg/kg). The other compounds (7h–i and 7k–q)
with a TD50 value of 163.7 mg/kg, which consequently resulted in all led to a reduction in anticonvulsant activity when compared
a protective index (PI) value of 7.9. Considering all these factors to derivative 7g. The influence of the position of the halogens
(i.e. efficacy, toxicity, and safety), compound 7j was considered on the phenyl ring was also similar to that of series 6a–q. For
to be the most promising compound in this study owing to the three F-substituted derivatives (7h–j), the potency order
its favorable efficacy and low NT (ED50 ¼ 26.3 mg/kg and was p-F4m-F4o-F. For the four Cl-substituted derivatives
TD50 ¼ 331.3 mg/kg, resulting in a PI value of 12.6). It is (7k–n), the potency order was p-Cl ¼ o-Cl4m-Cl42,4-Cl2. The
noteworthy that all of the selected compounds showed a higher potency order of the two Br-substituted derivatives (6o, 6p) was
PI than the standard drugs, except the compound 7c (PI value o-Br4p-Br. Comparing the compounds 6a–q and 7a–q, no
of 4.3).
significant difference was found with regard of anticonvultant

DOI: 10.3109/14756366.2013.776555
Activity of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives 277
Table 1. Phase-I preliminary evaluation of anticonvulsant activity in mice (i.p.)
MES*(mg/kg)
0.5 h
Toxicity (mg/kg)
4 h
0.5 h
100
4 h
Compounds
R
30
100
300
30
100
300
30
300
30
100
300
6a
6b
6c
6d
6e
6f
6g
6h
6i
C3H7
C4H9
C5H11
C6H13
C7H15
C8H17
0/3
0/3
0/3
2/3
0/3
0/3
2/3
0/3
0/3
1/3
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
1/3
2/3
3/3
2/3
2/3
2/3
0/3
1/3
2/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
1/3
1/3
2/3
3/3
1/3
1/3
3/3
0/3
0/3
3/3
2/3
1/3
3/3
0/3
2/3
0/3
0/3
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
1/3
2/3
0/3
2/3
1/3
1/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
1/3
3/3
2/3
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
3/3
2/3
3/3
2/3
2/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
–y
–
–
–
–
–
0/3
–
–
–
–
–
0/3
–
0/3
–
0/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
1/3
0/3
0/3
0/3
1/3
1/3
1/3
1/3
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/3
0/3
1/3
0/3
1/3
1/3
1/3
2/3
2/3
1/3
1/3
3/3
2/3
3/3
3/3
2/3
2/3
3/3
0/3
3/3
1/3
2/3
2/3
3/3
3/3
3/3
2/3
1/3
2/3
1/3
2/3
2/3
3/3
2/3
2/3
1/3
2/3
2/3
2/3
–
–
–
–
–
–
0/3
–
–
–
–
–
0/3
–
0/3
–
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
CH2C6H5
CH2C6H5(o-F)
CH2C6H5(m-F)
CH2C6H5(p-F)
CH2C6H5(o-Cl)
CH2C6H5(m-Cl)
CH2C6H5(p-Cl)
CH2C6H5(2,4-2Cl)
CH2C6H5(o-Br)
CH2C6H5(p-Br)
CH2C6H5(p-CH3)
C3H7
C4H9
C5H11
C6H13
C7H15
C8H17
CH2C6H5
CH2C6H5(o-F)
CH2C6H5(m-F)
CH2C6H5(p-F)
CH2C6H5(o-Cl)
CH2C6H5(m-Cl)
CH2C6H5(p-Cl)
CH2C6H5(2,4-2Cl)
CH2C6H5(o-Br)
CH2C6H5(p-Br)
CH2C6H5(p-CH3)
6j
6k
6l
6m
6n
6o
6p
6q
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
–
–
–
–
0/3
0/3
0/3
0/3
–
–
–
–
–
0/3
–
–
–
0/3
–
0/3
0/3
0/3
0/3
–
–
–
–
–
0/3
–
–
–
0/3
–
7m
7n
7o
7p
7q
0/3
0/3
All positive reaction numbers are in bold italic.
*MES test (number of animals protected/number of animals tested), the number of mice is three.
yNot tested.
Table 2. Phase-II quantitative anticonvulsant evaluation in Mice (i.p.).
Compounds
R
ED50 (mg/kg) (MES)
TD50 (mg/kg) (NT)
PI*
6d
6g
7c
7d
7e
7f
7g
C6H13
CH2C6H5
C5H11
C6H13
C7H15
C8H17
CH2C6H5
CH2C6H5(p-F)
23.7 (20.5–27.3)y
20.7 (14.8–29.0)
36.6 (31.0–43.3)
21.1 (17.8–24.9)
29.5 (25.8–33.7)
51.7 (40.9–65.3)
36.7 (25.7–52.4)
26.3 (22.2–31.1)
9.8 (8.9–10.8)
236.6 (176.2–317.8)
163.7 (138.4–193.6)
158.3 (113.1–221.6)
148.0 (105.7–207.2)
193.7 (142.2–263.9)
464.6 (356.9–604.8)
221.2 (169.9–288.0)
331.3 (246.7–444.9)
44.0 (40.2–48.1)
10.0
7.9
4.3
7.0
6.6
8.9
6.0
12.6
4.5
3.2
1.6
7j
Carbamazepine
Phenobarbital
Valproate
–
–
–
21.8 (21.8–25.5)
272 (247–338)
69 (62.8–72.9)
426 (369–450)
*PI ¼ TD50/ED50
.
yThe 95% confidence limits.

278 X.-Q. Deng et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 272–280
Table 3. Effects of compound 7j on chemical-induced seizures in mice (i.p.).
Doses
(mg/kg)
Test time
(h)
Clonic seizures
(%)
Tonic seizures
(%)
Lethality
(%)
Chemical substances
Pentylenetetrazol
Compound
DMSO
Carbamazepine
7j
DMSO
Carbamazepine
7j
DMSO
Carbamazepine
7j
–
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
100
100
90
100
100
100
100
100
90
100
0
100
10
10
100
10
70
100
60
50
50
50
–
50
50
–
20
100
20
20
100
0
3-MP
acid
BIC
50
50
10
activity and toxicity. The additional C¼O of compounds 7a–q seizures and death at rates of 0%, 100% and 40%, respectively.
seems not to influence the anticonvulsant activity.
Compound 7j showed inhibition of clonic and tonic seizures
and death at rates of 10%, 90% and 50%, respectively (Table 3).
BIC is a competitive antagonist of GABAA receptor, which
produces convulsions through its interference of the GABAA
receptor46. As compound 7j inhibited the seizures induced
by BIC, it likely exerts anticonvulsant activity, at least partially,
through GABAA-mediated mechanisms.
Chemical-induced seizure tests
In this study, the majority of synthesized compounds were highly
potent in the MES test. As the MES test is known to be sensitive
to sodium channel inhibitors (e.g. phenytoin, carbamazepine),
the tested compounds may inhibit voltage-gated ion channels
(particularly sodium channels). To further investigate the effects
of the anticonvulsant activity in several different models and Computational study
speculate about the possible mechanism action of anticonvulsant,
compound 7j was tested against convulsions induced by chemical
Prediction of ADME properties
substances, including PTZ, 3-MP and BIC. The reasons for the A computational study was conducted to predict the ADME
chose of compound 7j are its higher PI value than others and its properties of all of the synthesized compounds (Table 4,
good profile in toxicity and druglikeness prediction. Compound 7j Supplementary material). Two significant barriers exist in the
was administered as an intraperitoneal injection to mice at a dose human body, with one of these being the intestinal epithelium,
of 50 mg/kg, which was higher than its ED50 value and far below which modulate drug absorption following oral administration.
its TD50 value. The reference drug carbamazepine was also With the exception of medications interrupting status epilepticus,
administered at the dose of 50 mg/kg.
most of the AEDs are administered orally, and newly synthesized
In the sc-PTZ model, carbamazepine inhibited the clonic antiepileptic substances should therefore be efficiently absorbed
seizures, tonic seizures and death at the rates of 0%, 100% and in the digestive system. It has been demonstrated experimentally
90%, respectively. While compound 7j inhibited clonic seizures, that the intestinal absorption of drugs is significantly correlated
tonic seizures and lethality at the rates of 10%, 80% and 90% with their PSA. The PSA of a molecule effectively represent the
induced by sc-PTZ, respectively (Table 3). The data showed that portion of its surface belonging to polar atoms, such as oxygen,
compound 7j and carbamazepine can protect the mice from nitrogen and attached hydrogen atoms, and is a descriptor related
tonic seizures and death overwhelmingly, which revealed that to the passive molecular transport of a molecule through
compound 7j possessed excellent activity in sc-PTZ model. PTZ membranes. With this in mind, PSA could be used to predict
has been reported to produce seizures by inhibiting g-aminobu- the transport properties of drugs in the intestines47. Palm et al.48
tyric acid (GABA) neurotransmission42,43. GABA is the main have proven, based on Caco-2 cell studies, that drugs with a PSA
inhibitory neurotransmitter in the brain, and is widely implicated value below 60 were completely absorbed in the intestine. For the
in epilepsy. Inhibition of GABAergic neurotransmission or target compounds 6a–q and 7a–q, the PSA values were ranged
2
activity has been shown to promote and facilitate seizures44, from 39.9 to 59.9 A and the level of intestinal absorption
˚
while enhancement of GABAergic neurotransmission is known to (%ABS) according to the algorithm described by Zhao et al.37 was
inhibit or attenuate seizures. As such, our newly synthesized in the range of 88.3–95.2%. These findings indicated that these
compound 7j might inhibit or attenuate PTZ-induced seizures in compounds (6a–q and 7a–q) would possess good transport
mice by enhancing GABAergic neurotransmission.
In the 3-MP induced seizure model, carbamazepine inhibited
properties in the intestines.
The blood-brain barrier (BBB) represents the second of the two
the clonic seizures, tonic seizures and death at the rates of 0%, significant barriers in the human body, and is responsible for
80% and 90%, respectively. In comparison, compounds 7j showed regulating the influx of different chemical compounds into the
an anticonvulsant effect similar to that of carbamazepine in brain. The permeability (or lack thereof) of the BBB affects drug
inhibiting the clonic and tonic seizures; however, its effect on efficiency in the central nervous system. Chemical substances may
death rates induced by 3-MP was less, with the inhibition rate be transported to the brain by means of passive and facilitated
of 30% (Table 3). 3-MP is competitive inhibitor of the GABA diffusion processes or active transport49. According to Kaliszan &
synthesis enzyme glutamate decarboxylase (GAD), and it inhibits Markuszewski50, the lipophilicity (LogP) and molecular weight
the synthesis of GABA resulting in decrease of GABA levels (MW) ofa compound are the main factors affecting the delivery ofa
in the brain45. Compound 7j showed antagonism to 3-MP induced drug across the BBB. Lipophilicity affects the absorption of a drug,
seizures suggesting that it might activate GAD or inhibit hydrophobic drug-receptor interactions, drug metabolism and drug
aminotransferase (GABA-T) in the brain.
toxicity. It has been reported that a LogP value of 2.0 represent the
In the BIC induced seizure model, both carbamazepine and 7j optimum value for drugs acting on the central nervous system51.
inhibited tonic seizures and death, but did not inhibit clonic Drugs possessing this value will be inhibited to a lesser extent in
seizures. Carbamazepine showed inhibition of clonic and tonic their movement through the aqueous and lipophilic phases of living

DOI: 10.3109/14756366.2013.776555
Activity of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiazepine derivatives 279
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Conclusion
Based on our previous work, we synthesized two novel series
of 8-alkoxy-4,5-dihydrobenzo[b][1,2,4]triazolo[4,3-d][1,4]thiaze-
pine derivatives (6a–q and 7a–q) and evaluated their anticonvul-
sant activities. The compounds tested in the MES screens were
effective at various doses, with the most promising compound
being identified as 8-(4-fluorobenzyloxy)-4,5-dihydroben-
zo[b][1,2,4]triazolo[4,3-d][1,4]thiazepin-1(2H)-one (7j), which
showed an ED50 value of 26.3 mg/kg and a superior PI value of
12.6 when compared to standard drugs in the MES test in mice.
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Declaration of Interest
We declare no conflict of interest.
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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