Synthesis and anticonvulsant activity of some new thiazolo[3,2-a][1,3] diazepine, benzo[d]thiazolo[5,2-a][12,6]diazepine and benzo[d]oxazolo[5,2-a][12, 6]diazepine analogues

Article abstract of DOI:10.1016/j.ejmech.2011.09.021

A new series of 6,7-dihydro-thiazolo[3,2-a][1,3]diazepines (9-12), benzo[d]thiazolo[5,2-a][12,6]diazepines (19-21) and benzo[d]oxazolo[5,2-a][12,6] diazepine (24) analogues were synthesized and evaluated for their anticonvulsant activity. Compounds (E)-2-bromo-6,7-dihydro-thiazolo[3,2-a][1,3]diazepine-8(5H) -thione (12), 3-chloro-benzo[d]thiazolo[5,2-a][12,6]diazepin-10-one (20), and 4-chloro-benzo[d]oxazolo[5,2-a][12,6] diazepin-10-one (24) showed 100% protection against PTZ- and bicuculline-induced seizures; 70%, 33%, 70% protection against MES-induced tonic extension; and 70%, 66%, 100% protection against picrotoxin-induced convulsions, respectively. Compounds 12, 20, and 24 proved to act as GABA_A receptor agonists, with ED₅₀ values of 252, 380, 251 mg/kg; TD₅₀ values of 398, 417, 355 mg/kg; PI values of 1.58, 1.09, 1.41; LD₅₀ values of 380, 617, 537 mg/kg and TI values of 1.51, 1.62, 2.14, respectively.

Full text of DOI:10.1016/j.ejmech.2011.09.021

European Journal of Medicinal Chemistry 46 (2011) 5567e5572
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of some new thiazolo[3,2-a][1,3]diazepine,
benzo[d]thiazolo[5,2-a][12,6]diazepine and benzo[d]oxazolo[5,2-a][12,6]
diazepine analogues
,
b
,
,
,
Hussein I. El-Subbagha , Ghada S. Hassan c, Adel S. El-Azabb d, Alaa A.-M. Abdel-Azizb c, Adnan A. Kadib,
*
Abdulrahman M. Al-Obaidb, Othman A. Al-Shabanahe, Mohamed M. Sayed-Ahmede
a Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, Cairo 12311, Egypt
b Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
c Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
d Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
e Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 6,7-dihydro-thiazolo[3,2-a][1,3]diazepines (9e12), benzo[d]thiazolo[5,2-a][12,6]dia-
zepines (19e21) and benzo[d]oxazolo[5,2-a][12,6]diazepine (24) analogues were synthesized and eval-
uated for their anticonvulsant activity. Compounds (E)-2-bromo-6,7-dihydro-thiazolo[3,2-a][1,3]
diazepine-8(5H)-thione (12), 3-chloro-benzo[d]thiazolo[5,2-a][12,6]diazepin-10-one (20), and 4-chloro-
benzo[d]oxazolo[5,2-a][12,6] diazepin-10-one (24) showed 100% protection against PTZ- and
bicuculline-induced seizures; 70%, 33%, 70% protection against MES-induced tonic extension; and 70%,
66%, 100% protection against picrotoxin-induced convulsions, respectively. Compounds 12, 20, and 24
proved to act as GABAA receptor agonists, with ED50 values of 252, 380, 251 mg/kg; TD50 values of 398,
417, 355 mg/kg; PI values of 1.58, 1.09, 1.41; LD50 values of 380, 617, 537 mg/kg and TI values of 1.51, 1.62,
2.14, respectively.
Received 3 August 2011
Received in revised form
13 September 2011
Accepted 15 September 2011
Available online 22 September 2011
Keywords:
Synthesis
Thiazolo[3,2-a][1,3]diazepine
Benzo[d]thiazolo[5,2-a][12,6]diazepine
Benzo[d] oxazolo[5,2-a][12,6]diazepine
Anticonvulsant activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
derivatives such as imidazo[2,1-b]thiazepine (1, Chart 1), exhibited
affinity for BzR. Their profile of action was characterized as partially
GABA (
g
-aminobutyric acid) is the principal inhibitory trans-
agonistic at the BzRs on the basis of the GABA shift [8].
mitter of many CNS pathways regulating numerous neurological
functions including convulsions, anxiety and sleep activity. GABA
acts on the GABAA chloride ion channel [1,2]. Molecular biology
studies have demonstrated that several different receptor subunits
combine to form the GABAA receptor complex with a number of
receptor subtypes such as benzodiazepine receptor (BzR) [3e6].
Ligands acting at the BzR allosteric binding site of the GABAA
receptor modulate the action of GABA on chloride ion flux. They
show a wide variety of pharmacological actions ranging from full
agonistic effects such as sedative/hypnotic, anxiolytic and anti-
convulsant activities; to inverse agonistic effects such as anxio-
genic, and proconvulsant activities [7]. Several classes of chemical
compounds such as benzodiazepines, steroids and barbiturates
bind to the GABAA chloride ion channel complex. The azepine
Thiazolo-[3,2-a][1,3]diazepine, and their analogues represent
a new class of ultra-short acting hypnotics [9]. The ultra-short
acting activity of ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a]
[1,3]diazepin-3-carboxylate (HIE-124, 2, Chart 1), overcomes many
of the disadvantages and problems that usually associate the use of
conventional intravenous anaesthetic agents. Compound
2
undergoes liver esterases enzymatic hydrolysis producing the free
acid 3 (Chart 1), which is rapidly cleared out of the brain, termi-
nating the hypnotic activity [9e14].
In the present study, a new series of 6,7-dihydro-thiazolo[3,2-a]
[1,3]diazepine (9e12) was synthesized as bioisosteres of the imi-
dazo[2,1-b]thiazepine nucleus; and evaluated for their anticon-
vulsant activity. The 3-ethyl carboxylate moiety of 2 was eliminated
as an attempt to slow down its rapid metabolic clearance out of the
brain (compounds 9e12). Meanwhile, lipid solubility of the thia-
zolo[3,2-a][1,3]diazepine nucleus was increased either by the
introduction of bromine atom (compounds 10 and 12) or the
thiation of the 8-oxo function (compounds 11 and 12). The thiazole
* Corresponding author. Tel.: þ20 2 26186100; fax: þ20 2 26186111.
E-mail address: subbagh@yahoo.com (H.I. El-Subbagh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.021

5568
H.I. El-Subbagh et al. / European Journal of Medicinal Chemistry 46 (2011) 5567e5572
moiety of 2 was replaced by benzo-thiazole or benzo-oxazole
heterocycles, producing benzo[d]thiazolo[5,2-a][12,6]diazepine
(19e21) and the benzo[d]oxazolo[5,2-a][12,6] diazepine analogue
24, to evaluate the effect of such isosteric replacement on the
anticonvulsant activity.
Cl
Cl
N
N
K2CO3, Toluene
R
R
+
N
NH2
S
Cl
S
H
O
O
16
13
, R = H
, R = Cl
, R = H
, R = Cl
6
17
14
18, R = CH3
15, R = CH3
Piperidine,
Toluene
2. Chemistry
The reaction of 2-aminothiazole (4) or 2-amino-5-bromo-thia-
zole (5) with 4-chlorobutyryl chloride (6) in toluene at room
temperature afforded 4-chloro-N-(thiazol-2-yl)-butanamide (7) or
N-(5-bromothiazol-2-yl)-4-chlorobutanamide (8) which were
cyclized upon reflux with piperidine/toluene to give (E)-6,7-
dihydro-thiazolo[3,2-a][1,3]diazepin-8(5H)-one (9) or (E)-2-bro-
mo-6,7-dihydro-thiazolo[3,2-a][1,3]diazepin-8(5H)-one (10). Com
pound 9 and 10 were thiated by refluxing with Lawsson reagent in
dioxane to afford (E)-6,7-dihydro-thiazolo[3,2-a][1,3]diazepine-
8(5H)-thione (11) and (E)-2-bromo–6,7-dihydro-thiazolo[3,2-a]
[1,3]diazepine-8(5H)-thione (12), (Scheme 1).
N
R
N
S
O
19
20
, R = H
, R = Cl
21, R = CH3
Scheme 2. Synthesis of the target compounds 19e21.
administration of test compounds, taking zero time as 100%, while
hypnotic effect of test compounds was performed using Writing
Reflex method.
2-Amino-6-substituted-benzo[d]thiazoles
(13e15)
were
allowed toreact with 4-chlorobutyrylchloride (6) in tolueneat room
temperature afforded 4-chloro-N-(6-substituted-benzo[d]thiazol-
2-yl)butanamides (16e18) in good yields. Compounds 16e18 were
cyclized upon refluxing in piperidine/toluene to give 3-substituted-
benzo [d]thiazolo[5,2-a][12,6]diazepin-10-one (19e21), Scheme 2.
Meanwhile, 2-amino-5-chloro-benzo[d]oxazole (22) was allowed to
react with 4-chlorobutyryl chloride (6) in toluene at room temper-
ature afforded 4-chloro-N-(5-chloro-benzo[d]oxazol-2-yl) butana-
mide (23) in reasonable yield. Compound 23 was cyclized upon
refluxing in piperidine/toluene to give 4-chloro-benzo[d]oxazolo
[5,2-a][12,6]diazepin-10-one (24), Scheme 3.
4. Results
Compounds 9, 10, 11, 12, 19, 20, and 24 were submitted for
anticonvulsant activity evaluation. Results showed that compounds
9 and 11 caused 33% and 50% mortality within 7e11 min before
even PTZ injection was administered, while compounds 10 showed
marginal anticonvulsant activity. Compounds 12, 20, and 24
showed noticeable sedative effect in addition to 100% protection
against PTZ-induced seizures (Table 1).
Compounds 12, 20, and 24 produced 70%, 33% and 70% protec-
tion against MES-induced tonic extension seizure, respectively.
These finding suggested that those compounds have the ability to
prevent the spread of seizure discharge throughout neuronal
tissues and to raise seizure threshold. Compounds 9, 10, 11 and 19
proved inactive towards MES-induced seizure. Picrotoxin (Pic.) is
a chloride channel blocker producing hyperpolarization and clonic
convulsions. Compounds 12, 20, and 24 produced 70%, 66% and
100% protection against Pic.-induced convulsions, respectively,
suggesting that those compounds may act as GABAA receptor
agonist by increasing chloride influx via brain chloride channels.
Bicuculline (Bic.) is a GABAA receptor blocker when given to mice, it
induces clonic convulsions. All tested compounds produced 100%
protection against Bic.-induced convulsions suggesting that those
compounds may act directly as GABAA receptor agonist or indirectly
by increasing GABA synthesis or its release as a brain inhibitory
neurotransmitter (Table 2).
3. Pharmacology
Pentylenetetrazole (PTZ) seizure threshold test is well-known to
evaluate the potential anticonvulsant activity of compounds under
investigation [15]. PTZ was used at dose level of 83 mg/kg, s.c.; this
dose is the minimum dose that induces 100% clonic convulsions.
Test compounds were used at different dose levels i.p. Sodium
valproate was used as positive control at dose level of 200 mg/kg
(1.38 mmol/kg, s.c.) 30 min after the administration of test
compounds. At 15 and 25 min after i.p. administration of each of the
test compounds at three dose levels, the inability of mice to climb
up backwards in a glass tube of 25 cm length and 3 cm inner
diameter within 30 s was recorded and taken as a measure of
neurological deficits [16]. Normal mice climb up in 5e10 s. Motor
activity was measured using activity metre at zero and 30 min after
Cl
Cl
Cl
N
N
Cl
Cl
K2CO3, Toluene
Cl
+
R
NH2
R
Cl
N
H
N
N
S
S
K2CO3, Toluene
O
+
O
NH2
N
H
Cl
O
O
7,
R = H
4,
R = H
6
O
O
8, R = Br
5, R = Br
6
22
23
Piperidine,
Toluene
Piperidine,
Toluene
N
Cl
N
Lawsson Reagent
R
N
R
N
S
S
N
S
O
N
O
O
9, R = H
10,
11, R = H
12,
R = Br
R = Br
24
Scheme 1. Synthesis of the target compounds 9e12.
Scheme 3. Synthesis of the target compound 24.

H.I. El-Subbagh et al. / European Journal of Medicinal Chemistry 46 (2011) 5567e5572
5569
Table 1
Anticonvulsant activity of the new synthesized compounds against PTZ-induced convulsions.
Compd
Dose (mmol/kg)
% Protectionb
% Chimneyc
% Motor activityd
% Hypnotice
% Mortality
CLogP
9a
1.5
0.0
nt
nt
0.0
nt
nt
80
40
10
100
30
10
100
25
0.0
0.0
nt
nt
100
25
0.0
100
50
17
100
0.0
nt
nt
0.0
nt
nt
33
nt
nt
1.19
2.05
1.62
2.48
1.93
2.64
2.21
2.76
0.75
0.375
1.5
0.75
0.375
1.5
0.75
0.375
3.0
1.5
0.75
1.5
0.75
0.375
3.0
10
11a
12
19
20
24
30
12
13
15
11
26
30
92
84
52
0.0
nt
30
0.0
0.0
0.0
0.0
0.0
40
14
0.0
0.0
nt
0.0
0.0
0.0
50
10
0.0
0.0
nt
0.0
0.0
0.0
0.0
0.0
100
100
50
nt
0.0
nt
70
0.0
0.0
100
50
25
100
67
nt
nt
nt
67
33
20
80
50
33
nt
20
0.0
0.0
17
0.0
0.0
nt
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.5
0.75
3.0
1.5
0.75
1.38
33
100
Sod. valproate
a
Compound 9 and 12 showed 33 and 50% mortality, respectively before PTZ injection.
Compounds showed anticonvulsant activity against PTZ-induced convulsions.
Chimney test at 15 and 25 min after I.P. administration of test compounds.
Motor activity was measured using activity metre at zero and 30 min after administration of test compounds, taking zero time as 100%.
Hypnotic effect of tested compounds was performed using Writing Reflex.
b
c
d
e
A dose-response curve for the remarkably active anticonvulsant
acting hypnotic compound (E)-ethyl 8-oxo-5,6,7,8-tetrahydro-
thiazolo[3,2-a][1,3]diazepine-3-carboxylate (HIE-124, 2) [9].
Removal of the 3-ethyl carboxylate function of 2, produced
agents 12, 20, and 24 were performed along with the median
effective (ED50), median sedative (TD50), protective index (PI), the
acute toxicity determination (LD50) and therapeutic index [17].
Results are shown in Table 2. Compounds 12, 20, and 24 showed
ED50 values of 252, 380, 251 mg/kg; TD50 values of 398, 417,
355 mg/kg; PI (95% CL) values of 1.58, 1.09, 1.41; LD50 (95% CL)
values of 380, 617, 537 mg/kg and TI (95% CL) values of 1.51, 1.62,
2.14, respectively.
compounds
(E)-6,7-dihydro-thiazolo[3,2-a][1,3]diazepin-8(5H)-
one (9) and (E)-6,7-dihydro-thiazolo[3,2-a][1,3]diazepine-8(5H)-
thione (11) with total loss of hypnotic activity. Introduction of
bromine atom to position 2- of 9 (CLogP 1.19) produced (E)-2-
bromo-6,7-dihydro-thiazolo[3,2-a][1,3]diazepin-8(5H)-one
(10,
CLogP 2.05) with the rise of a new type of CNS activity which is the
anticonvulsant potency (30% protection against PTZ-induced
convulsion). It seemed that lipophilicity plays a role in enhancing
such activity as evidenced by the increase of anticonvulsant
potency to 100% protection upon thiation of the 8-ketonic function
of 10 to produce (E)-2-bromo-6,7-dihydro-thiazolo[3,2-a][1,3]
5. Discussion
Correlation of the obtained anticonvulsant results and structure
variation necessitate the comparison with the parent ultra-short
Table 2
Dose-response curve, ED50, TD50, PI, LD50 and TI for compounds 12, 20 and 24.
Compda
Dose
Dose
% PTZ
% Chimb
% MESc
% Picd
% Bice
ED50
TD50
PIg
LD50
TIh
f
f
(mmol/kg)
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
12
3.0
1.5
0.75
0.37
3.0
505
252
126
63
759
380
190
95
710
355
178
89
100
100
50
25
100
50
100
25
0.0
0.0
100
25
0.0
0.0
100
50
70
40
10
0.0
33
0.0
0.0
0.0
70
10
0.0
0.0
70
66
20
0.0
66
100
50
10
0.0
100
66
10
10
100
50
20
252
(195e324)
398
(309e513)
1.58
380
(302e478)
1.51
20
24
380
(295e490)
417
(324e537)
1.09
1.41
617
(479e794)
1.62
2.14
1.5
20
0.75
0.37
3.0
1.5
0.75
0.37
25
0.0
0.0
100
40
10
0.0
0.0
100
67
33
0.0
251
(195e324)
355
(275e457)
537
(427e661)
17
0.0
0.0
a
Each dose of selected compounds was tested using 10 animals and the percentage of animals protected was recorded and the anticonvulsant activity was calculated.
Chim: Chimney test, was applied 15, and 25 min after the administration of each compound.
MES: Maximal electroshock test, was applied 30 min after the administration of each compound.
Pic:Picrotoxin (3.15 mg/kg, sc) was given 30 min after the administration of each compound.
Bic: Bicuculline (1 mg/kg, sc) was given 30 min after the administration of each compound.
b
c
d
e
f
ED50 and TD50: Median effective and median sedative doses, respectively.
g
h
PI: Protective index ¼ TD50/ED50
.
TI: Therapeutic index ¼ LD50/ED50
.

5570
H.I. El-Subbagh et al. / European Journal of Medicinal Chemistry 46 (2011) 5567e5572
diazepine-8(5H)-thione (12, CLogP 2.48). The same analogy was
noticed upon comparing (E)-7,8-dihydro-benzo[d]thiazolo[3,2-a]
[1,3]diazepin-9(6H)-ones (19, CLogP 1.93, 70% protection against
PTZ-induced convulsions) and 3-Chloro-benzo [d]thiazolo[5,2-a]
[12,6]diazepin-10-one (20, CLogP 2.64, 100% protection). Isosteric
replacement of the thiazole moiety with oxazole produced 4-
chloro-benzo[d]oxazolo[5,2-a][12,6]diazepin-10-one (24, CLogP
2.21, 100% protection) Structure activity relationship (SAR) recom-
mendations withdrawn from those findings revealed that (i) 3-
ethyl carboxylate function connected to tetrahydro-thiazolo[3,2-
a][1,3]diazepine as in 2 is essential for hypnotic activity, (ii) the
removal of 3-ethyl carboxylate function of 2 will yield compounds
with anticonvulsant activity, (iii) lipophilicity manipulate the
magnitude of anticonvulsant potency of such chemical synthons.
EtOOC
HOOC
O
H3CO
N
N
N
S
S
N
N
N
S
O
O
1
3
HIE-124, 2
Chart 1. Literature cited active azepine analogues 1, 2 and the inactive metabolic
product 3.
spectra obtained at 500 MHz. Bruker instrument, using TMS as
internal standard. Thin layer and flash chromatography were per-
formed using E. Merck Silica gel (230e400 mesh). Preparative thin
layer chromatography was performed on Harrison model 7924 A
chromatotron using Analtech silica gel GF rotors. Compounds 16
and 19 were previously reported [18]. Male Swiss albino mice
(SWR) weighing 25e30 g, were obtained from the Animal Care
Center, College of Pharmacy, King Saud University, Riyadh, Saudi
Arabia and were housed in metabolic cages under controlled
environmental conditions (25 ꢁC and a 12 h light/dark cycle).
Animals had free access to pulverized standard rat pellet food and
tap water unless otherwise indicated. The protocol of this study has
been approved by Research Ethics Committee of College of Phar-
macy, King Saud University (KSU), Riyadh, Kingdom of Saudi Arabia
(KSA). Test compounds were freshly dissolved in 99% DMSO. In
preliminary screening, all tested compounds were used at dose
level of 3.0, 1.5, 0.75 and 0.375 mmol/kg, i.p. Pentylenetetrazole
(PTZ) was freshly dissolved in 0.9% NaCl and used at dose level of
83 mg/kg, s.c. Picrotoxin (Pic) was freshly dissolved in 1 ml 0.1 N
warmed HCl and the final volume was made up with 0.9% NaCl. It
has been used at dose level of 3.15 mg/kg s.c. and was given 30 min
after the administration of test compounds. Bicuculline (Bic) was
freshly dissolved in 1 ml 0.1 N warmed HCl and the final volume
was made up with 0.9% NaCl. It has been used at dose level of 1 mg/
kg sc and was given 30 min after the test compounds. Sodium
valproate was freshly dissolved in 0.9% NaCl and used at dose level
of 200 mg/kg s.c. 30 min after the administration of test
compounds.
6. Conclusion
In the present study, a new series of compounds belong to 6,7-
dihydro-thiazolo[3,2-a][1,3]diazepine,
benzo[d]thiazolo[5,2-a]
[12,6]diazepine and benzo[d] oxazolo[5,2-a][12,6]diazepine nuclei
were synthesized. The synthesized compounds were subjected to
anticonvulsant activity evaluation. Four animal models were
adopted, namely: pentylenetetrazole (PTZ), maximal electroshock
(MES), picrotoxin (Pic), and bicuculline (Bic) induced convulsions.
Compounds
(E)-2-bromo-6,7-dihydro-thiazolo[3,2-a][1,3]dia-
zepine-8(5H)-thione (12), 3-chloro-benzo[d]thiazolo[5,2-a][12,6]
diazepin-10-one (20), and 4-chloro-benzo[d]oxazolo[5,2-a][12,6]
diazepin-10-one (24) showed 100% protection against PTZ-induced
seizures (Fig. 1). Compounds 12, 20, and 24 produced 70%, 33% and
70% protection against MES-induced tonic extension seizure, and
70%, 66% and 100% protection against picrotoxin-induced convul-
sions, respectively. All of the tested compounds produced 100%
protection against bicuculline-induced convulsions. Those
compounds proved to possess the ability to prevent the spread of
seizure discharge throughout neuronal tissues and to raise seizure
threshold. Those compounds may act as GABAA receptor agonist by
increasing chloride influx via brain chloride channels or act directly
as GABAA receptor agonist or indirectly by increasing GABA
synthesis or its release as a brain inhibitory neurotransmitter.
7.1. Chemistry
7. Experimental protocols
7.1.1. 4-Chloro-N-(thiazol-2-yl)-butanamide 7
Unless otherwise specified all chemicals were of commercial
grade, used without further purification and were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Solvents used for work ups
were dried over MgSO4, filtered and removed on a rotary evapo-
rator. Elemental analyses were performed at College of Pharmacy,
King Saud University-Central Laboratory. All of the new compounds
were analyzed for C, H and N and agreed with the proposed
structures within ꢀ0.4% of the theoretical values. 1H and 13C NMR
A
mixture of 2-aminothiazole (4, 4.0 g, 0.04 mol),
4-chlorobutyryl chloride (6, 11.3 g, 0.08 mol), and potassium
carbonate (1.4 g, 0.01 mol) in toluene (100 ml) was stirred at room
temperature for 24 h. The toluene was then evaporated under
reduced pressure. The residue was then quenched with water,
stirred, and filtered. The solid obtained was washed, dried and
recrystallized from water to give the required product 7; 92% yield,
mp 125e127 ꢁC, 1H NMR (CDCl3):
d 12.92 (s, 1H, NH), 7.54 (d, 1H,
J ¼ 3.5 Hz, thiazole-H), 7.04 (d, 1H, J ¼ 3.5 Hz, thiazole-H), 3.71 (t,
2H, J ¼ 6.0 Hz, CH2), 2.81 (t, 2H, J ¼ 6.0 Hz, CH2), 2.31e2.26 (m, 2H,
Cl
CH2). 13C NMR:
d 27.3, 32.8, 44.4, 113.6, 136.2, 160.1, 170.0. MS: m/z
N
N
N
Cl
113 (100%), M ¼ 204 (20%), M þ 2 ¼ 206 (5%). (C7H9ClN2OS) C, H, N.
Br
N
N
N
S
S
S
O
O
O
7.1.2. N-(5-bromo-thiazol-2-yl)-4-chloro-butanamide 8
24
12
20
A mixture of 2-amino-5-bromothiazole hydrobromide (5, 1.3 g,
0.005 mol), 4-chlorobutyryl chloride (6, 1.1 ml, 0.01 mol), potas-
sium carbonate (1.4 g, 0.01 mol) in toluene (50 ml) was stirred at
room temperature for 24 h. The solvent was then evaporated under
reduced pressure. The residue obtained was dissolved in chloro-
form and neutralized with ammonia solution. The organic layer
was separated, dried over sodium sulphate and then evaporated to
obtain the crude product which was recrystallized from toluene to
Dose
% Anticonvulsant Activity
Compd
CLogP
mmol/kg
PTZ
MES
Pic
Bic
12
20
24
3.0
3.0
3.0
100
100
100
70
33
70
70
66
100
100
100
100
2.48
2.64
2.21
Fig. 1. Structures of the most active anticonvulsant agents.

H.I. El-Subbagh et al. / European Journal of Medicinal Chemistry 46 (2011) 5567e5572
5571
obtain the pure compound 8; 94% yield, mp 153e155 ꢁC 1H NMR
(CDCl3): 12.37 (s, 1H, NH), 7.47 (s, 1H, thiazole-H), 3.65 (t, 2H,
J ¼ 6.5 Hz, CH2), 2.60 (t, 2H, J ¼ 6.5 Hz, CH2), 2.03e2.05 (m, 2H, CH2).
reduced pressure. The residue was then quenched with water,
stirred, and filtered. The obtained solid was dissolved in chloroform
and neutralized with ammonia solution. The organic layer was
separated, dried over sodium sulphate and then evaporated to
obtain the crude product which was used in the next reaction
without further treatment [18].
d
13C NMR:
d 27.8, 32.4, 45.1, 101.9, 138.9, 158.8, 171.2. MS: m/z 179
(100%), Mþ ¼ 283 (30%), M þ 2 ¼ 285 (8%). (C7H8BrClN2OS) C, H, N.
7.1.3. (E)-6,7-dihydro-thiazolo [3,2-a][1,3]diazepin-8(5H)-one 9
A mixture of 4-chloro-N-(thiazol-2-yl)butanamide (7, 0.82 g,
0.004 mol) and piperidine (0.8 ml, 0.008 mol) in toluene (50 ml)
was heated under reflux for 3 h. The reaction mixture was cooled,
poured into water and stirred. Toluene was separated dried and
evaporated to give a crude product which was purified by repeated
silica gel and neutral alumina column chromatography eluting with
CHCl3/hexane (90:10 v/v); 56% yield, mp 75e77 ꢁC 1H NMR (CDCl3):
7.1.8. 4-Chloro-N-(6-chloro-benzo[d]thiazol-2-yl)-butanamide 17
A mixture of 2-amino-6-chloro-benzo[d]thiazole (14, 7.4 g,
0.04 mol) 4-chlorobutyryl chloride (6, 11.3 g, 0.08 mol), and
potassium carbonate (7.0 g, 0.05 mol) in toluene (100 ml) was
stirred at room temperature for 24 h then continued as mentioned
under 16. The crude product was recrystallized from toluene to
obtain the pure compound 17; 89% yield, mp 135e137 ꢁC 1H NMR
d
7.35 (d, 1H, J ¼ 3.5 Hz, thiazole-H), 6.91 (d, 1H, J ¼ 7.5 Hz, thiazole-
(CDCl3):
AreH), 7.13e7.14 (m, 1H, AreH), 3.45e3.46 (m, 2H, CH2), 2.50e2.52
(m, 2H, CH2), 1.98e1.99 (m, 2H, CH2). 13C NMR:
27.4, 32.5, 44.1,
d 11.83 (s, 1H, NH), 7.55 (s, 1H, AreH), 7.42e7.43 (m, 1H,
H), 4.05 (t, 2H, J ¼ 7.5 Hz, CH2), 2.58 (t, 2H, J ¼ 7.5 Hz, CH2),
2.18e2.12 (m, 2H, CH2). 13C NMR:
d
18.0, 31.6, 47.8,113.6,137.4,157.7,
d
173.3. Ms: m/z 113 (100%), Mþ ¼ 168 (42%), M þ 2 ¼ 170 (2%).
120.6, 121.5, 126.3, 128.5, 133.3, 147.4, 158.5, 171.4. MS: m/z 184
(C7H8N2OS) C, H, N.
(100%), Mþ ¼ 289 (20%), M þ 2 ¼ 290 (2%). (C11H10Cl2N2OS) C, H, N.
7.1.4. (E)-2-bromo-6,7-dihydro-thiazolo [3,2-a][1,3]diazepin-
8(5H)-one 10
7.1.9. 4-Chloro-N-(6-methyl-benzo[d]thiazol-2-yl)-butanamide 18
A mixture of 2-amino-6-methyl-benzo[d]thiazole (15, 6.6 g,
0.04 mol), 4-chlorobutyryl chloride (6, 11.3 g, 0.08 mol), and
potassium carbonate (7.0 g, 0.05 mol) in toluene (100 ml) was
stirred at room temperature for 24 h then continued as mentioned
under 16. The crude product was used in the next reaction without
further treatment.
A mixture of N-(5-bromo-thiazol-2-yl)-4-chloro-butanamide (8,
1.14 g, 0.004 mol) and piperidine (0.8 ml, 0.008 mol) in toluene
(50 ml) was heated under reflux for 3 h. The reaction mixture was
cooled, poured into water and stirred. Toluene was separated dried
and evaporated to give a crude product which was purified by
repeated silica gel and neutral alumina column chromatography
eluting with CHCl3/hexane (90:10 v/v); 45% yield, mp 118-20 ꢁC. 1H
7.1.10. 3-Substituted-benzo[d]thiazolo [5,2-a][12,6]diazepin-10-
ones 19e21
NMR (CDCl3):
d
7.41 (s, 1H, thiazole-H), 4.16 (t, 2H, J ¼ 7.5 Hz, CH2),
2.40 (t, 2H, J ¼ 7.5 Hz, CH2), 2.24 (t, 2H, J ¼ 7.5 Hz, CH2). 13C NMR:
A mixture of the butanamide analogues 16e18 (0.004 mol) and
piperidine (0.8 ml, 0.008 mol) in toluene (50 ml) was heated under
reflux for 3 h. The reaction mixture was cooled, poured into water
and stirred. Toluene was separated dried and evaporated to give
a crude product which was purified by repeated silica gel and
neutral alumina column chromatography eluting with CHCl3/
hexane (90:10 v/v). 19: 61% yield, mp 188ꢂ90 ꢁC 1H NMR (CDCl3):
d
18.2, 31.5, 47.3, 103.7, 138.2, 157.7, 173.6. MS: m/z 179 (100%),
Mþ ¼ 247 (65%), M þ 2 ¼ 249 (60%). (C7H7BrN2OS) C, H, N.
7.1.5. (E)-6,7-dihydro-thiazolo [3,2-a][1,3]diazepine-8(5H)-thione
11
A mixture of (E)-6,7-dihydrothiazolo[3,2-a][1,3]diazepin-8(5H)-
one (9, 0.67 g, 0.004 mol) and excess lawsson reagent was refluxed
in dioxin (40 ml) for 3 h. The reaction mixture was cooled, poured
into water and stirred. The solid was filtered and purified by silica
gel column chromatography eluting with CHCl3/hexane (95:5 v/v);
d
7.73e7.77 (m, 2H, AreH), 7.35e7.38 (m, 1H, AreH), 7.19e7.27 (m,
1H, AreH), 4.19e4.12 (m, 2H, CH2), 2.65e2.68 (m, 2H, CH2),
2.19e2.28 (m, 2H, CH2). 13C NMR:
d 18.1, 32.0, 48.2, 121.3, 121.4,
124.0, 126.1, 132.4, 148.6, 157.0, 174.3. MS: m/z 150 (100%), Mþ ¼ 218
(15%), M þ 2 ¼ 220 (1%). (C11H10N2OS) C, H, N [18];. 20: 48% yield,
65% yield, mp 70e71 ꢁC 1H NMR (CDCl3):
d
7.60 (d, 1H, J ¼ 3.5 Hz,
thiazole-H), 7.06 (d, 1H, J ¼ 3.5 Hz, thiazole-H), 4.60 (t, 2H,
mp 251e53 ꢁC 1H NMR (CDCl3):
d 7.73e7.78 (m, 2H, AreH),
J ¼ 7.5 Hz, CH2), 3.28 (t, 2H, J ¼ 7.5 Hz, CH2), 2.31e2.25 (m, 2H, CH2).
7.38e7.40 (m, 1H, AreH), 4.26 (t, 2H, J ¼ 7.5 Hz, CH2), 2.76 (t, 2H,
13C NMR:
d
20.3, 46.6, 56.7, 114.1, 137.2, 158.6, 199.7. Ms: m/z 113
J ¼ 7.5 Hz, CH2), 2.29e2.35 (m, 2H, CH2). 13C NMR:
d 18.1, 31.9, 48.2,
(100%), Mþ ¼ 184 (15%). (C7H8N2S2) C, H, N.
121.0, 122.1, 126.8, 129.4, 133.7, 147.3, 157.3, 174.4. MS: m/z 184
(100%), Mþ ¼ 252 (20%), M þ 2 ¼ 254 (6%). (C11H9ClN2OS) C, H, N.
7.1.6. (E)-2-bromo-6,7-dihydro-thiazolo [3,2-a][1,3]diazepine-
8(5H)-thione 12
21: 39% yield, mp 218ꢂ20 ꢁC 1H NMR (CDCl3):
d 7.73e7.75 (d, 1H,
J ¼ 8.0 Hz, AreH), 7.62 (s, 1H, AreH), 7.26e7.27 (d, 1H, J ¼ 8.0 Hz,
A mixture of (E)-2-bromo-6,7-dihydro-thiazolo [3,2-a][1,3]dia-
zepin-8(5H)-one (10, 1.0 g, 0.004 mol) and excess lawsson reagent
was refluxed in dioxin (40 ml) for 3 h. The reaction mixture was
cooled, poured into water and stirred. The solid was filtered and
purified by silica gel column chromatography eluting with CHCl3/
hexane (95:5 v/v); 52% yield, mp 175ꢂ77 ꢁC 1H NMR (DMSO-d6):
AreH), 4.28 (t, 2H, J ¼ 7.5 Hz, CH2), 2.75 (t, 2H, J ¼ 7.5 Hz, CH2), 2.49
(s, 3H, AreCH3), 2.29e2.32 (m, 2H, CH2). 13C NMR:
d 18.1, 21.5, 32.0,
48.2,120.9, 121.2,127.6,132.5, 134.0,146.6,156.4,174.2. MS: m/z 164
(100%), Mþ ¼ 232 (38%), M þ 2 ¼ 234 (1%). (C12H12N2OS) C, H, N.
7.1.11. 4-Chloro-benzo[d]oxazolo [5,2-a][12,6]diazepin-10-one 24
A mixture of 2-amino-5-chloro-benzo[d]oxazole (22, 6.7 g,
0.04 mol), 4-chloro-butyryl chloride (6, 11.3 g, 0.08 mol), and
potassium carbonate (7.0 g, 0.05 mol) in toluene (100 ml) was
stirred at room temperature for 24 h then continued as mentioned
under 16. The crude product of 4-chloro-N-(5-chloro-benzo[d]
oxazol-2-yl)butanamide (23) was used without further treatment.
A mixture of 23 (1.1 g, 0.004 mol) and piperidine (0.8 ml, 0.008 mol)
in toluene (50 ml) was heated under reflux for 3 h. The reaction
mixture was cooled, poured into water and stirred. Toluene was
separated dried and evaporated to give a crude product which was
d
7.80 (s, 1H, thiazole-H), 4.47 (t, 2H, J ¼ 7.5 Hz, CH2), 3.21 (t, 2H,
J ¼ 7.5 Hz, CH2), 2.21 (m, 2H, J ¼ 7.5 Hz, CH2). 13C NMR:
d 200.5,
157.5, 138.4, 104.0, 56.2, 45.9, 19.8. Ms: m/z 179 (100%), Mþ ¼ 263
(42%), M þ 2 ¼ 265 (38%). (C7H7BrN2S2) C, H, N.
7.1.7. N-(benzo[d]thiazol-2-yl)-4-chloro-butanamide 16
A mixture of 2-amino-benzo[d]thiazole (13, 6.0 g, 0.04 mol),
4-chlorobutyryl chloride (6, 11.3 g, 0.08 mol) and potassium
carbonate (7.0 g, 0.05 mol) in toluene (100 ml) was stirred at room
temperature for 24 h. The toluene was then evaporated under

5572
H.I. El-Subbagh et al. / European Journal of Medicinal Chemistry 46 (2011) 5567e5572
purified by repeated silica gel and neutral alumina column chro-
matography eluting with CHCl3/hexane (90:10 v/v). 54% yield, mp
mice. 30 min later, animals were injected s.c. with Pic (3.15 mg/kg)
and observed for 30 min and the percentage of anticonvulsion of
tested compounds was calculated [15].
230e2 ꢁC 1H NMR (CDCl3):
d
7.50e7.51 (d, 1H, J ¼ 1.5 Hz, AreH),
7.35e7.36 (d, 1H, J ¼ 8.5 Hz, AreH), 7.14e7.16 (dd, 1H, J ¼ 1.5, 8.5 Hz,
AreH), 4.06 (t, 2H, J ¼ 7.5 Hz, CH2), 2.63 (t, 2H, J ¼ 7.5 Hz, CH2),
7.2.5. Effect of tested compounds against bicuculline-induced
convulsion
2.18e2.24 (m, 2H, CH2). 13C NMR:
d 18.3, 32.2, 47.7, 111.1, 118.9,
124.0, 130.2, 141.6, 147.3, 155.9, 172.9. MS: Mþ ¼ 236 (16%),
M þ 2 ¼ 238 (6%). (C11H9ClN2O2) C, H, N.
A 3.0 mmol/kg of each compound which produced 100%
protection against PTZ-induced seizures were injected i.p. in 6
mice. 30 min later, animals were injected s.c. with Bic (1 mg/kg) and
observed for 30 min and the percentage of anticonvulsion of tested
compounds was calculated [15].
7.2. Anticonvulsant screening
7.2.1. Pentylenetetrazole (PTZ)-induced convulsion
Pentylenetetrazole (PTZ) seizure threshold test is one of well-
known chemical tests used to evaluate the potential anticonvul-
sant activity of the tested compounds [15]. Each compound was
tested in 6 mice and was given i.p. at four dose levels of 3.0, 1.5, 0.75
and 0.375 mmol/kg 30 min later, animals were received PTZ (83 mg/
kg, s.c.) and observed for 30 min. A single 5 s episode of clonic spasms
was taken as a threshold seizure. Compounds produced 100%
protection against PTZ-induced seizures was further tested against
maximal electroshock seizure (MES), picrotoxin and bicuculline-
induced clonic convulsions, to explore the involvement of GABA-
ergic receptors in their anticonvulsant activity.
7.3. Determination of LD50 and dose-response curve
Several doses of compounds 12, 20 and 24 which produced
70e100% protection against MES, Pic and Bic-induced convulsions
were used to construct a dose-response curve. Also, LD50 for
compounds 12, 20 and 24 was determined using the Spear-
maneKarber method in which the doses were selected at equal
logarithmic intervals [17].
Acknowledgement
7.2.2. Minimal neurotoxicity (Chimney test)
The authors would like to acknowledge the financial support of
King Abdulaziz City for Science and Technology, grant number
ARP-27-43.
At 15 and 25 min after i.p. administration of each compound at
four dose levels of 3.0, 1.5, 0.75 and 0.375 mmol/kg, the inability of
mice to climb up backwards in a glass tube of 25 cm length and
3 cm inner diameter within 30 s was recorded and taken as
a measure of neurological deficits [16]. Normal mice climb up in
5e10 s.
References
[1] F.E. Bloom, in: J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon,
A.G. Gilman (Eds.), The pharmacological basis of therapeutics, McGraw-Hill,
NewYork, 1996, pp. 267e293.
[2] G.B. Smith, R.W. Olsen, Trends Pharmacol. Sci 16 (1995) 162e168.
[3] W. Sieghart, Pharmacol. Rev. 47 (1995) 181e234.
7.2.3. Effect of tested compounds against the maximal electroshock
(MES)-induced convulsion
The maximal electroshock seizure (MES) is one of the electrical
tests used to evaluate anticonvulsant activity [15]. In this test, MES
that induced 100% maximal seizures was found to be 50 mA
alternating current of 100 Hz frequency for 0.2 s, using ECT UNIT
(model number 7801, UGO Basile, Varese, Italy). A 3.0 mmol/kg of
test compounds which produced 100% protection against PTZ-
induced seizures were injected i.p. 30 min later, mice were
restrained by hand and subjected to electric shock through their
ears, and released immediately following electrical stimulation, to
permit observation of the maximal seizure. The maximal seizure
typically consists of a short period of initial tonic flexion and
a prolonged period of tonic extension (especially the hind limb).
Protection was defined as complete absence of hind limb tonic
extension.
[4] H.H. Yeh, E.V. Grigorenko, J. Neurosci. Res. 41 (1995) 567e571.
[5] E.A. Barnard, P. Skolnick, R.W. Olsen, H. Mohler, W. Sieghart, G. Biggio,
C. Braestrup, A.N. Bateson, S.Z. Langner, Pharmacol. Rev. 50 (1998) 291e313.
[6] R.M. McKernan, P.J. Whiting, Trends Neurosci. 19 (1996) 139e143.
[7] C. Bellantuono, V. Reggi, G. Tognoni, S. Garattini, Drugs 19 (1980) 195e219.
[8] K.K. Kononowicz, J.K. Wojciechowska, B. Michalak, E. Pvkala, B. Schumacher,
C.E. Müller, Eur. J. Med. Chem. 39 (2004) 205e218.
[9] J. Lehmann, H. I. El-Subbagh, H. A. El-Kashef, DE 103 20 732 A1, (2004).
[10] H.I. El-Subbagh, H.A. El-Kashef, A.A. Kadi, A.A.-M. Abdel-Aziz, G.S. Hassan,
J. Tettey, J. Lehmann, Bioorg. Med. Chem. Let. 18 (2008) 72e77.
[11] A.A. Kadi, H.A. El-Kashef, A.A.-M. Abdel-Aziz, G.S. Hassan, J. Tettey, H.M. Grant,
J. Lehmann, H.I. El-Subbagh, Arch. Pharm. Chem. Life Sci. 341 (2008) 81e89.
[12] E.A. Abourashed, M.M. Hefnawyand, H.I. El-Subbagh, J. Planar Chromatog.
(JPC) 22 (2009) 183e186.
[13] M. Hefnawy, M. Al-Omar, S. Julkhuf, S. Attia, E. Abourashed, H. El-Subbagh,
Analytica Chim. Acta 673 (2010) 194e199.
[14] A. Kadi, M. Hefnawy, A. Al-Majed, S. Alonezi, Y. Asiri, S. Attia, E. Abourashed,
H. El-Subbagh, Analyst 136 (2011) 591e597.
[15] H.S. White, J.H. Woodhead, M.R. Franklin, E.A. Swinyard, H.H. Wolf, in:
R.H. Levy, R.H. Mattson, B.S. Meldrum (Eds.), Antiepileptic drugs, fourth ed.
Raven, New York, 1995, pp. 99e121.
[16] J.-R. Boissier, J. Tardy, J.-C. Diverres, Med. Exp. 3 (1960) 81e84 Basel.
[17] R. Ulrich, J. Miller, Percept Psychophys. 66 (2004) 517e533.
[18] G. Tsatsas, E. Costakis, Chem. Commun. 19 (1967) 991.
7.2.4. Effect of tested compounds against picrotoxin-induced
convulsion
A 3.0 mmol/kg of each compound which produced 100%
protection against PTZ-induced seizures were injected i.p. in 6