Design, synthesis, and anticonvulsant evaluation of some novel 1, 3 benzothiazol-2-yl hydrazones/acetohydrazones

Article abstract of DOI:10.1007/s00044-011-9768-0

A series of 2-[2-(substituted)hydrazinyl]-1,3-benzothiazole and 2-(1,3-benzothiazol-2-ylsulfanyl)-N′-(substituted)acetohydrazide were designed and synthesized keeping in view the structural requirement of pharmacophore and evaluated for anticonvulsant activity and neurotoxicity. The anticonvulsant activity of the titled compounds was assessed using the 6 Hz psychomotor seizure test. The most active compound of the series was 2-(1,3-benzothiazol-2-ylsulfanyl)-N′-[4-(4-bromophenoxy)benzylidene] acetohydrazide BT 15, which showed 75% protection (3/4, 1.0 h) and 50% protection (2/4, 0.5 h) at a dose of 100 mg/kg in mice. A computational study was carried out for calculation of pharmacophore pattern and prediction of pharmacokinetic properties. Titled compounds have also exhibited good binding properties with epilepsy molecular targets such as GABA (A) alpha-1, glutamate, GABA (A) delta receptors and Na/H exchanger, in Lamarckian genetic algorithmbased flexible docking studies. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9768-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2428–2442
DOI 10.1007/s00044-011-9768-0
ORIGINAL RESEARCH
Design, synthesis, and anticonvulsant evaluation of some novel
1, 3 benzothiazol-2-yl hydrazones/acetohydrazones
•
Praveen Kumar Birendra Shrivastava
•
•
Surendra N. Pandeya Laxmi Tripathi
•
James P. Stables
Received: 13 December 2010 / Accepted: 27 July 2011 / Published online: 10 August 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of 2-[2-(substituted)hydrazinyl]-1,3-
benzothiazole and 2-(1,3-benzothiazol-2-ylsulfanyl)-N0-
(substituted)acetohydrazide were designed and synthesized
keeping in view the structural requirement of pharmaco-
phore and evaluated for anticonvulsant activity and neuro-
toxicity. The anticonvulsant activity of the titled compounds
was assessed using the 6 Hz psychomotor seizure test.
The most active compound of the series was 2-(1,3-ben-
zothiazol-2-ylsulfanyl)-N0-[4-(4-bromophenoxy)benzylidene]
acetohydrazide BT 15, which showed 75% protection (3/4,
1.0 h) and 50% protection (2/4, 0.5 h) at a dose of 100 mg/kg
in mice. A computational study was carried out for calcula-
tion of pharmacophore pattern and prediction of pharmaco-
kinetic properties. Titled compounds have also exhibited
good binding properties with epilepsy molecular targets such
as GABA (A) alpha-1, glutamate, GABA (A) delta receptors
and Na/H exchanger, in Lamarckian genetic algorithm-
based flexible docking studies.
Keywords 2-[2-(substituted)hydrazinyl]-1,3-
benzothiazole ꢀ 2-(1,3-benzothiazol-2-ylsulfanyl)-
N0-(substituted)acetohydrazide ꢀ 6 Hz psychomotor
seizure test ꢀ Neurotoxicity ꢀ Computational study
Introduction
Epilepsy is a common neurological disorder affecting
0.5–1% of the population, i.e., 45–100 million people
worldwide (Bell and Sander, 2002). Current clinically avail-
able drugs produce satisfactory seizure control in 60–70% of
patients (Perucen, 1996). Several new anticonvulsants like
oxacarbazepine, vigabatrin, lamotrigine, gabapentin, topira-
mate, felbamate, rufinamide, and levetiracetam have been put
in clinical practice. Despite familiarity with established anti-
epileptic drugs and the introduction of these new agents in the
past decade, up to one-third of epilepsy patients remain
resistant to optimum drug treatment (Sabers and Gram, 2000).
Moreover, the current drug therapy is associated with adverse
side effects such as drowsiness, ataxia, gastrointestinal dis-
turbance, gingival hyperplasia, hirsutism, and megaloblastic
anemia (Yogeeshwari et al., 2006). These facts triggered
the search for newer more effective and less toxic anti-
convulsants.
P. Kumar (&) ꢀ B. Shrivastava
School of Pharmaceutical Sciences, Jaipur National University,
Jaipur 302025, Rajasthan, India
e-mail: praveensha1977@rediffmail.com
Earlier two-dimensional (2D) modeling on anticonvul-
sants has identified that at least one aryl unit, one or two
electron donor atoms, and/or an NH group in a spatial
arrangement are to be recommended for anticonvulsant
activity (Camerman and Camerman, 1980). A pharmaco-
phore model based on some well-known voltage-gated
sodium channel blockers including phenytoin and lamo-
trigine was also proposed (Unverferth et al., 1998). Liter-
ature review shows that anticonvulsant activity has
been displayed by various hydrazones, acetohydrazones,
S. N. Pandeya
Saroj Institute of Technology and Management,
Lucknow 226 002, Uttar Pradesh, India
L. Tripathi
S D College of Pharmacy & Vocational Studies,
Muzaffarnagar 251001, Uttar Pradesh, India
J. P. Stables
Preclinical Pharmacology Section, Epilepsy Branch, National
Institute of Health, Bethesda, MD 20892-9020, USA
123

Med Chem Res (2012) 21:2428–2442
2429
semicarbazides, and thiosemicarbazides. Aryl acid hydra-
zones, similar to semicarbazones, contains large hydro-
phobic group in close proximity to two electron donor
nitrogen atoms, thus fulfilling the structural requirements
of maximal electroshock (MES) screen (Jones and Wood-
bury, 1982). Various benzothiazole derivatives were syn-
thesized and reported as anticonvulsants (Hays et al., 1994;
Mizoule et al., 1985; Chopade et al., 2000). 1, 3-ben-
zothiazol-2-yl semicarbazones were synthesized and
reported to have significant protection in MES model
(Siddiqui et al., 2007). In fact, these evidences suggest that
2-[substituted]-1,3-benzothiazole moiety 1, possess phar-
macophoric character for anticonvulsant activity. In addi-
tion, the 4-(aryloxy)phenyl semicarbazone 2 were reported
as potential anticonvulsant (Wang et al., 1998).
pharmacophore distance mapping, Log P calculation and
pharmacokinetic parameters. In this study, we have used
AutoDock 4.0 along with its LGA algorithm for automated
flexible ligand docking of compounds with six established
epilepsy molecular targets and docking affinity & count of
probable H-bonds were determined.
Results and discussion
Synthesis and characterization
The reaction sequence leading to the formation of the titled
compounds, viz. 2-{2-[substituted]hydrazinyl}-1,3-benzo-
thiazole BT 1–10 and 2-(1,3-benzothiazol-2-ylsulfanyl)-
N0-(substituted) acetohydrazide BT 11–20 is shown in
Scheme 1. The 4-substituted benzaldehydes 3a–i were
prepared by refluxing various substituted phenols 1a–i with
4-fluoro benzaldehyde 2 in N,N-DMF in the presence of
potassium carbonate. To a suspension of 1, 3-benzothiazol-
2-amine 4 in ethylene glycol, hydrazine hydrate, and conc.
HCl was added with stirring at 5–6°C. This mixture was
refluxed to obtain 2-hydrazinyl-1, 3-benzothiazole 5. A
mixture of 1, 3-benzothiazole-2-thiol 6, chloroethyl acetate
and triethyl amine was refluxed in dry methanol to obtain
ethyl (1, 3-benzothiazol-2-ylsulfanyl) acetate 7. The 7 was
converted to 2-(1, 3-benzothiazol-2-ylsulfanyl) acetohyd-
razide 8 by reacting with hydrazine hydrate in ethanol. The
5 and 8 were condensed with various 4-substituted benz-
aldehyde 3a–i/Isatin 3j to yield the titled compounds BT
1–20. Thin layer chromatography (TLC) was run
throughout the reactions to optimize for purity and com-
pletion. The physical data for the newly synthesized
compounds is presented in Tables 1 and 2.
Continuing our studies on benzfused derivatives that are
attractive candidates as anticonvulsant agents, we designed
a new series of functionalized 2-[2-(substituted)hydrazi-
nyl]-1,3-benzothiazole BT 1–10 and 2-(1,3-benzothiazol-2-
ylsulfanyl)-N0-(substituted)acetohydrazide compounds BT
11–20, exploring 2-amino benzothiazole and 2-mercapto
benzothiazole, respectively, as starting material. The
rational design of 2-[2-(substituted)hydrazinyl]-1,3-benzo-
thiazole BT 1–10, was planned by molecular hybridization
of 2-[substituted]-1,3-benzothiazole 1 and 4-(aryloxy)phenyl
semicarbazones 2. While the rational design of 2-(1,3-ben-
zothiazol-2-ylsulfanyl)-N0-(substituted)acetohydrazide BT
11–20, was planned by molecular hybridization of 1 and 2,
using –S–CH2–CO– group as bridge (Fig. 1). Their chemical
1
structures were characterized using IR, H-NMR, MS, and
elemental analysis techniques. All the synthesized titled
compounds composed of the essential pharmacophoric ele-
ments (Fig. 2) that are necessary for good anticonvulsant
activity (Unverferth et al., 1998). In addition, their anticon-
vulsant activity was evaluated by using 6 Hz psychomotor
seizure test in mice. The rotorod assay was performed in
mice to evaluate the neurotoxicity of the compounds. Com-
putational study was also carried out to highlight the
The structural assignments to new compounds were
1
based on their elemental analysis and spectral (FT-IR, H-
NMR, and mass) data. The formation of 4-substituted
benzaldehydes 3a–i from 4-substituted phenols 1a–i was
S
R
N
Molecular
hybridization using
-S-CH2-C=O group
(1)
Molecular
Hybridization
S
as bridge
O
C
S
H
H2
C
H
N
N
C
O
Ar
S
N
N
C
O
Ar
H
H
N
N
BT 1-10
BT 11-20
O
C
H2N
NHN
C
H
O
Ar
(2)
Fig. 1 Rational concept to new 2-{2-[substituted]hydrazinyl}-1,3-benzothiazole BT 1–10 and 2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(substituted)
acetohydrazide BT 11–20
123

2430
Med Chem Res (2012) 21:2428–2442
Fig. 2 Pharmacophoric pattern
of well-known anticonvulsants
and 2-{2-
[substituted]hydrazinyl}-1,3-
benzothiazole and 2-(1,3-
benzothiazol-2-ylsulfanyl)-N0-
(substituted) acetohydrazide
(BT 2, BT 5, BT 14, BT 15, and
BT 20)
D
N H
D
CH3
A
D
S
H
HD
OH
CH3
A
HA
O
N
N
HD
N
H
A
HA
O
HD H2N
S HA
Albutoin
Carbamazepine
Gabapentin
D
HA
H
N
H
D
O
N
N
O
N
A
H
NH
Cl
N
HD
HD
N
C2H5
O
Cl
H
N
O
N
O
HA
HA
D
CH3
H
H
HD
Lamotrignine
HA
Mephobarbital
Phenytoin
O
O
N
OH
H
H3C
A
N
H
D
O
N
HA
D
HD
HD
N
O
N
A
F
H
S
H
N
H
H
HA
O
H
A
HD
Cl
Progabide
Remacemide
Zonisamide
HD
HD
O
H3C
D
CH3
H
H
H
H
O
N
N
C
N
C
N
A
N
N
N
S
HA
N
HA
S
HA
S
A
A
H
O
O
Cl
H
D
D
HD
Br
NO2
Ralitoline
BT 2
BT 5
HD
HD
HC
O
HC
HN N
O
N
S
N
S
A
A
S
H2C
HN N
S
H2C
HA
HA
O
O
Cl
Cl
D
D
BT 14
BT 15
D
O
HD
S
N
C
NH
H2
S
N
A
HA
NH
O
BT 20
1
confirmed by its IR and H-NMR spectral studies. The IR
spectrum of 4-(4-bromophenoxy) benzaldehyde 3e showed
bands at 1662, 3030, and 1241 cm-1 indicating the
presence of C=Ostr, C–Hstr, and diaryl ether linkage (–O–).
The absence of broad band of phenolic –OH at 2900–
3000 cm-1 confirms the conversion of 4-bromo phenol 1e
123

Med Chem Res (2012) 21:2428–2442
2431
F
K2CO3, DMF, reflux
Ar OH
O
Ar
CHO
CHO
1a-i
2
3a-i
S
S
NH2NH2.H2O
NHNH2
NH2
Ethylene glycol, conc. HCl
N
N
5
4
S
S
S
NH2NH2H2O
Ethanol
ClCH2COOC2H5
SCH2CONHNH2
SH
SCH2COOC2H5
Triethylamine, methanol,
reflux
N
N
N
7
8
6
O
O
S
S
S
Ar
OR
O
NHN
NHN
C
O
Ar
OR
OR
NHNH2
NH
H
N
N
N
CHO
N
H
3a-i
3j
5
BT 10
O
BT 1-9
Ethanol , Reflux
S
S
S
SCH2CONHNH2
SCH2CONHN
SCH2CONHN
C
O
Ar
NH
N
H
N
N
8
O
BT 20
BT 11-19
Scheme 1 Synthesis of 2-{2-[substituted]hydrazinyl}-1,3-benzothiazole BT 1–10 and 2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(substituted)
acetohydrazide BT 11–20
Table 1 Physical data of 2-{2-[substituted]hydrazinyl}-1,3-benzothiazoles BT 1–10
S
S
NHN C
H
O Ar
NHN
NH
N
N
O
BT 10
BT 1-9
Compounds
Ar
Yield (%)
Melting point (°C)
Mol. formulaa (mol. wt.)
Rf
BT 1
BT 2
BT 3
BT 4
BT 5
BT 6
BT 7
BT 8
BT 9
BT 10
a
Phenyl
76
81
78
72
68
74
70
76
67
82
174
172
182
156
240
191
195
198
147
308
C20H15N3OS (345.42)
C20H14N4O3S (390.42)
C21H17N3OS (359.44)
0.58
0.52
0.55
0.50
0.44
0.45
0.51
0.55
0.49
0.57
4-NO2-Phenyl
4-CH3-Phenyl
4-Cl-Phenyl
4-Br-Phenyl
4-F-Phenyl
C20H14ClN3OS (379.86)
C20H14BrN3OS (424.31)
C20H14FN3OS (363.41)
C21H16ClN3OS (393.89)
C24H17N3OS (395.48)
C21H15N3O3S (389.43)
C15H10N4OS (294.33)
3-Methyl-4-chloro phenyl
Naphthalene-2-yl
1,3-benzodioxol-5-yl
–
Solvent used for recrystallization—ethanol:water (9:1)
1
to 4-(4-bromophenoxy) benzaldehyde 3e. In its H-NMR
spectrum a singlet at d 9.81 ppm indicating the presence of
–CHO group, where as an absence of a singlet around d
9.28 ppm for phenolic –OH confirms the conversion of 1e
to 3e. The IR spectrum of the titled compound BT 15
showed N–Hstr at 3178 and 3076 cm-1, C=Ostr at
1664 cm-1, CH=Nstr at 1611 cm-1, and diaryl ether link-
proton resonated as a broad singlet at d 11.15 ppm that
was D2O exchangeable. Aromatic and benzothiazole
protons appeared as a set of signals in the region d 6.88–
7.94 ppm. The S-CH2 protons appeared as singlet at
4.06 ppm. The presence of CH=Nstr at 1611 cm-1 in IR
spectrum and a singlet for imine (CH=N) proton at d
1
8.00 ppm in H-NMR spectrum, confirms the formation
age (–O–) at 1240 cm-1. Its H-NMR spectrum showed a
of BT 15. Further mass spectrum confirmed their purity
and molecular weight.
1
singlet of imine (CH=N) proton at d 8.00 ppm. The NH
123

2432
Med Chem Res (2012) 21:2428–2442
Table 2 Physical data of 2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(substituted) acetohydrazides BT 11–20
S
S
SCH2CONHN
C
H
O Ar
SCH2CONHN
NH
O
N
N
BT 11-19
BT 20
Compounds
Ar
Yield (%)
Melting point (°C)
Mol. formulaa (mol. wt.)
Rf
BT 11
BT 12
BT 13
BT 14
BT 15
BT 16
BT 17
BT 18
BT 19
BT 20
a
Phenyl
76
78
70
64
62
70
77
72
66
80
184
112
178
174
148
162
168
150
130
182
C22H17N3O2S2 (419.52)
C22H16N4O4S2 (464.52)
C23H19N3O2S2 (433.55)
0.54
0.43
0.46
0.41
0.38
0.46
0.43
0.48
0.42
0.47
4-NO2-Phenyl
4-CH3-Phenyl
4-Cl-Phenyl
4-Br-Phenyl
4-F-Phenyl
C22H16ClN3O2S2 (453.96)
C22H16BrN3O2S2 (498.42)
C22H16FN3O2S2 (437.51)
C23H18ClN3O2S2 (467.99)
C26H19N3O2S2 (469.58)
C23H17N3O4S2 (463.53)
C17H12N4O2S2 (368.43)
3-Methyl-4-chloro phenyl
Naphthalene-2-yl
1,3-benzodioxol-5-yl
–
Solvent used for recrystallization—ethanol:water (9:1)
Anticonvulsant and neurotoxicity evaluation
protection. None of the compounds showed neurotoxicity in
the highest administered dose (300 mg/kg).
The newly synthesized 2-{2-[substituted]hydrazinyl}-1,3-
benzothiazoles BT 1–10 and 2-(1,3-benzothiazol-2-ylsul-
fanyl)-N0-(substituted)acetohydrazides BT 11–20 were
subjected to anticonvulsant screening by 6 Hz psychomotor
seizure or minimal clonic seizure test to identify their anti-
convulsant activity at five different time points, i.e., 0.25,
0.5, 1.0, 2.0, and 4.0 h after i.p. administration in mice at a
dose of 100 mg/kg. Neurotoxicity was observed by minimal
motor impairment (MMI) which was measured by the roto-
rod test. The6 Hz psychomotor seizure test is used to assess a
compound’s efficacy against electrically induced seizures
but uses a lower frequency (6 Hz) and longer duration of
stimulation (3 s). It is used to identify clinically useful AEDs
that are ineffective in the standard MES and scMET tests but
still have anticonvulsant activities in vivo. The results of
screening at five different points are summarized in Table 3.
As observed from the results of various tested titled com-
pounds, the most active compound of the series was BT 15,
which showed 75% protection (3/4, 1.0 h) and 50% protec-
tion (2/4, 0.5 h) at a dose of 100 mg/kg in mice. At a dose of
100 mg/kg, compounds BT 14 and BT 20 showed 75%
protection (3/4) at a time point of 1.0 and 0.5 h, respectively.
Compounds BT 2, BT 5, and BT 11 showed 50% protection
at two time point, i.e. (2/4, 0.25 h; 2/4, 1.0 h), (2/4, 0.5 h; 2/
4, 2.0 h), and (2/4, 0.25 h; 2/4, 1.0 h), respectively. At a dose
of 100 mg/kg, compounds BT 1, BT 4, BT 8, and BT 16
showed 50% protection (2/4) at a time point of 2.0, 0.5, 2.0,
and 0.25 h, respectively. BT 6, BT 13, and BT 19 were found
inactive. The other compounds displayed mild to moderate
Computational study
The pharmacophore pattern studies in which distance
between the various groups postulated as essential for
anticonvulsant activity were done on the 3D optimized
structures using ACD/3D viewer version 12.0 and Argus
Lab 4.0 Mark A. Thompson Planaria Software LLC. A
computational study of all compounds was performed for
prediction of ADME properties such as absorption (%
ABS), polar surface area (TPSA), miLog P, number of
rotatable bonds, and violations of Lipinski’s rule of five by
using Molinspiration online property calculation toolkit.
Calculated miLog P for synthesized compounds were then
compared with the experimental Log P data of these com-
pounds. Docking study of titled compounds was performed
with six established epilepsy molecular targets, namely
GABA (A) alpha-1 receptor, GABA (A) delta receptor,
glutamate receptor, Na/H exchanger, Na channel receptor,
and T-type calcium channel receptor by using AutoDock
4.0 along with its LGA algorithm for automated flexible
ligand docking and their docking affinity (Kcal/mol) and
count of probable hydrogen bonds were determined.
Distance mapping
This study involves the correlation of the structural
requirement of well-known and structurally different
123

Med Chem Res (2012) 21:2428–2442
2433
mechanism of action, were selected so as to propose a
generalized pharmacophore model. The pharmacophore
group’s distance estimation was done by molecular
mechanics calculation with the force fields based on both
CHARMm force fields and MM3 parameterization. In this
study, energy minimization was performed on above men-
tioned 10 well-known anticonvulsants and the titled com-
pounds using Argus Lab 4.0. Distance between the various
structural components essential for activity was determined
by ACD/3D viewer. The crucial structural components that
were included in the four point pharmacophore model
(Fig. 3) were the aryl ring center or the lipophilic group (A),
an electron donor atom (D), a hydrogen bond acceptor
(HA), and a hydrogen bond donor (HD). An average dis-
tance range for every point was obtained and compared to
that of 2-{2-[substituted]hydrazinyl}-1,3-benzothiazoles
BT 1–10 and 2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(substi-
tuted)acetohydrazides BT 11–20. Now it may be interesting
to examine whether BT 1–20 reflect the conditions of the
derived pharmacophore model. Our analyses of the distance
relationship showed that the titled compounds BT 1–20 did
fulfill the essential demands of the pharmacophore when
compared to the average distance requirement (Table 4).
Table 3 6 Hz anticonvulsant activity and neurotoxicity of 2-{2-
[substituted]hydrazinyl}-1,3-benzothiazole BT 1–10 and 2-(1,3-ben-
zothiazol-2-ylsulfanyl)-N0-(substituted) acetohydrazide BT 11–20
Compound Test
Dose
(mg/kg)
Time (h) to peak effect (N/F)a
0.25
0.5
1.0
2.0
4.0
BT 1
BT 2
BT 3
BT 4
BT 5
BT 7
BT 8
BT 9
BT 10
BT 11
BT 12
BT 14
BT 15
BT 16
BT 17
BT 18
BT 20
a
6 Hz 100
TOX 100
0/4
0/4
2/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
1/4
0/4
1/4
0/4
2/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
1/4
0/4
1/4
0/4
1/4
0/4
3/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
2/4
0/4
1/4
0/4
3/4
0/4
3/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
2/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
6 Hz 100
TOX 100
Prediction of ADME properties
A computational study for prediction of ADME properties
of titled compounds was performed. Topological polar
surface area (TPSA), i.e., surface belonging to polar atoms,
is a descriptor that was shown to correlate well with pas-
sive molecular transport through membranes and, there-
fore, allows prediction of transport properties of drugs in
the intestine and blood–brain barrier (BBB) crossing (Ertl
et al., 2000). The percentage of absorption (% ABS) was
calculated using TPSA. From all these parameters, it can be
observed that all titled compounds exhibited a great %
ABS ranging from 71.2 to 92.9% (Table 5). Furthermore,
compounds BT 10 and BT 20 did not show any violation in
Lipinski’s parameters, making them potentially promising
agents for epilepsy therapy. Other compounds are showing
only one violation in Lipinski’s parameters.
N/F = number of animals active or toxic over the number tested at
a dose of 100 mg/kg
Log P determination
anticonvulsant compounds with the titled compounds. The
2D modeling on anticonvulsants has identified that at least
one aryl unit, one or two electron donor atoms, and/or an
NH group in a special spatial arrangement is recommended
for anticonvulsant activity. In this study, the 10 well-known
and structurally different compounds with anticonvulsant
activity—albutoin, carbamazepine, gabapentin, lamotri-
gine, mephobarbital, phenytoin, progabide, ralitoline, re-
macemide, and zonisamide (Fig. 2) with different
Titled compounds showed dependence of biological
activity on lipophilic character in a congeneric series. In
particular, for drugs acting on central nervous system to be
potent, they have to cross BBB, thus potency has been
correlated with optimum lipophilicity (Log P) near 2. In
this study, we attempted to correlate the anticonvulsant
activity of congeners with their calculated Log P value.
The experimental Log P values were determined using the
octanol-phosphate buffer method. The data is presented in
123

2434
Med Chem Res (2012) 21:2428–2442
Fig. 3 Four-point 3D
A- Hydrophobic Unit
pharmacophore model for
anticonvulsants derived by
using MM3 and CHARMm
parametrization (Argus Lab 4.0
and ACD/3D viewer)
D- Electron Donar Group
HA- Hydrogen Bond Acceptor Unit
HD- Hydrogen Bond Donar Unit
D
4.30 0.64
C
C
C
C
4.86 1.13
4.37 1.19
A
C
C
6.05 1.87
5.86 2.09
HA
2.54 0.54
HD
Table 4 Distance range between the essential structural elements A, D, and HA–HD
Compounds
A–HA
A–HD
A–D
HA–HD
HD–D
HA–D
Albutoin
5.37
2.72
4.51
2.72
4.03
5.40
Carbamazepine
Gabapentin
Lamotrigine
Mephobarbital
Phenytoin
Progabide
Ralitoline
Remacemide
Zonisamide
Mean SD
BT 2
4.28
4.28
4.25
2.33
5.75
5.67
4.26
4.93
3.83
2.23
3.57
4.50
5.30
7.42
4.54
2.42
4.94
4.25
3.78
5.50
4.81
2.34
4.63
5.23
6.20
4.01
4.35
2.63
3.88
5.17
9.51
9.23
3.79
2.41
6.72
6.79
8.30
5.55
4.56
2.75
2.52
4.85
7.51
8.75
5.38
3.87
3.96
2.43
6.02
6.22
3.01
1.78
3.71
4.31
6.05 1.87
5.60
5.86 2.09
4.69
4.30 0.64
2.92
2.54 0.54
1.41
4.37 1.19
2.68
4.86 1.13
3.07
BT 5
5.47
4.70
2.92
1.42
2.67
3.04
BT 14
9.16
7.95
8.09
1.42
2.24
2.67
BT 15
9.16
7.95
8.09
1.42
2.24
2.67
BT 20
8.45
8.47
7.70
1.42
2.24
3.52
Distances calculated for 3D optimized structures using MM3 and CHARMm parameterization (Argus Lab 4.0 and ACD/3D viewer)
Table 6. As observed some of the experimental values
were in good agreement with the theoretical values. All the
titled compounds showed lipophilic character.
alpha-1 receptor, GABA (A) delta receptor, glutamate
receptor, Na/H exchanger, Na channel receptor, T-type
calcium channel receptor. The description of receptors is
provided in Table 7. The docking affinity (Kcal/mol) and
count of probable hydrogen bonds was evaluated. BT 2
exhibited good binding properties with GABA (A) delta
receptor (affinity value -6.2 kcal/mol and 1 H-bond),
GABA (A) alpha-1 receptor (affinity value -5.8 kcal/mol
and 0 H-bond), glutamate receptor (affinity value -6.4 kcal/
mol and 0 H-bond), and Na/H exchanger (affinity value
Docking study
In this study, we have used AutoDock 4.0 along with its
LGA algorithm for automated flexible ligand docking of
compounds BT 2, BT 5, BT 14, BT 15, and BT 20 with six
established epilepsy molecular targets namely GABA (A)
123

Med Chem Res (2012) 21:2428–2442
2435
Table 5 Pharmacokinetic parameters important for good oral bioavailability of compounds BT 1–20
Compound
% ABS
TPSA (A2)
n-ROTB
MW
MV
n-OHNH
donors
n-ON
acceptors
Lipinski’s
violations
Rule
–
–
–
5
6
5
5
5
5
5
5
5
2
7
8
7
7
7
7
7
7
7
4
\500
–
\5
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
\10
4
B1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
0
BT 1
92.9
77.1
92.9
92.9
92.9
92.9
92.9
92.9
86.5
84.8
87.0
71.2
87.0
87.0
87.0
87.0
87.0
87.0
80.6
78.9
46.51
92.34
46.51
46.51
46.51
46.51
46.51
46.51
64.98
70.14
63.58
109.41
63.58
63.58
63.58
63.58
63.58
63.58
82.05
87.21
345.43
302.05
325.38
318.61
315.59
319.94
306.98
332.15
346.04
325.98
242.20
355.96
379.30
372.53
369.50
373.85
360.90
386.06
399.96
379.89
296.12
BT 2
390.42
359.45
379.87
424.32
363.41
393.89
395.48
389.43
294.33
419.53
464.52
433.55
453.97
498.42
437.52
468.00
469.59
463.54
368.44
7
BT 3
4
BT 4
4
BT 5
4
BT 6
4
BT 7
4
BT 8
4
BT 9
6
BT 10
BT 11
BT 12
BT 13
BT 14
BT 15
BT 16
BT 17
BT 18
BT 19
BT 20
5
5
8
5
5
5
5
5
5
7
6
-5.5 kcal/mol and 0 H-bond). BT 5 exhibited good
binding properties with glutamate receptor (affinity value
-6.3 kcal/mol and 2 H-bonds), Na/H exchanger (affinity
value -5.7 kcal/mol and 2 H-bonds), GABA (A) alpha-1
receptor (affinity value -6.4 kcal/mol and 1 H-bond), and
GABA (A) delta receptor (affinity value -6.4 kcal/mol and
0 H-bond). BT 14 exhibited good binding properties with
glutamate receptor (affinity value -7.7 kcal/mol and 3 H-
bonds), Na/H exchanger (affinity value -5.5 kcal/mol and
1 H-bond), GABA (A) alpha-1 receptor (affinity value
-5.2 kcal/mol and 1 H-bond), and GABA (A) delta
receptor (affinity value -5.9 kcal/mol and 0 H-bond). BT
15 exhibited good binding properties with GABA (A)
alpha-1 receptor (affinity value -6.6 kcal/mol and 2 H-
bonds), glutamate receptor (affinity value -6.6 kcal/mol
and 1 H-bond), GABA (A) delta receptor (affinity value
-6.2 kcal/mol and 0 H-bond), and Na/H exchanger (affinity
value -5.2 kcal/mol and 0 H-bond). BT 20 exhibited good
binding properties with GABA (A) alpha-1 receptor
(affinity value -5.9 kcal/mol and 3 H-bonds), glutamate
receptor (affinity value -6.8 kcal/mol and 2 H-bonds),
GABA (A) delta receptor (affinity value -6.2 kcal/mol and
1 H-bond), and Na/H exchanger (affinity value -5.2 kcal/
mol and 0 H-bond). The above-mentioned compounds did
not show affinity and H-bonding with Na channel and
T-type calcium channel receptors. The docking study
results of BT 2, BT 5, BT 14, BT 15, and BT 20 with these
six receptors are shown in Table 8. The docking images are
shown in Fig. 4. Docking study results shows that the titled
compounds exhibited good binding properties with gluta-
mate, GABA (A) alpha-1, GABA (A) delta receptor, and
Na/H exchanger.
Structure–activity relationship
In this study, various 2-{2-[substituted]hydrazinyl}-1,3-
benzothiazoles BT 1–10 and 2-(1,3-benzothiazol-2-ylsulfa-
nyl)-N0-(substituted)acetohydrazides BT 11–20 were syn-
thesized by using different substituent, in order to investigate
the pharmacophoric substituent, responsible for better
activity. In the synthetic design, the various substituents were
introduced at positions 2 of 1, 3-benzothiazol-2-yl backbone
structure. At position 2, hydrazones/acetohydrazones having
substituent viz. phenyl, 4-nitro phenyl, 4-methyl phenyl, 4-
chloro phenyl, 4-bromo phenyl, 4-flouro phenyl, 4-chloro-3-
methyl phenyl, naphthalene-2-yl, 1, 3-benzodioxol-5-yl at
position 19 and 23, respectively, were introduced. Keeping
in view, the pharmacophoric character of isatin for anti-
convulsant activity, the isatinyl moiety was also attached
to position N-11 and N-15 of 1, 3-benzothiazol-2-yl
123

2436
Med Chem Res (2012) 21:2428–2442
2-yl hydrazones only 4-nitro phenyl and 4-chloro phenyl
substitution at position 19, yields compound with moderate
protection. This observation clearly indicates that ben-
zothiazol-2-yl acetohydrazones are more promising anti-
convulsant agents than 1, 3-benzothiazol-2-yl hydrazones.
Table 6 Log P value for compounds BT 1–20
Compounds
Experimental
Log P
Theoretical
Log P
(miLog Pa)
Rule
–
B5
5.5
5.5
5.9
6.2
6.3
5.7
6.5
6.7
5.4
3.5
5.9
5.8
6.3
6.5
6.7
6.0
6.9
7.1
5.8
3.4
BT 1
BT 2
BT 3
BT 4
BT 5
BT 6
BT 7
BT 8
BT 9
BT 10
BT 11
BT 12
BT 13
BT 14
BT 15
BT 16
BT 17
BT 18
BT 19
BT 20
a
5.2
5.1
5.4
5.6
5.5
5.0
5.5
5.6
5.0
3.2
5.6
5.3
5.7
5.6
5.7
5.5
5.7
5.9
5.5
3.2
Experimental
Chemistry
All the chemicals and solvents, purchased from Merck
(India), Spectrochem (India), Himedia (India) and S. d.
Fine were used without further purification. The progress
of reaction was monitored by TLC, performed on a silica
gel 60 F254 coated aluminum sheet. The melting points
were determined by using Thomas-Hoover melting point
apparatus and are uncorrected. The FT-IR spectra were
recorded on Perkin-Elmer Spectrum BX-II Spectropho-
1
tometer. The H-NMR spectra were recorded on Bruker
300 MHz High Resolution NMR spectrometer using TMS
as an internal standard. Chemical shifts were reported in
ppm (d) and signals were described as singlet (s), doublet
(d), triplet (t), and multiplet (m). All exchangeable protons
were confirmed by addition of D2O. The mass spectra were
recorded on a Waters Micromass ZQ 2000 mass spec-
trometer. Elemental analysis (C, H, N) was undertaken
with Perkin Elmer Model 240C analyzer.
miLog P, logarithm of compound partition coefficient between n-octa-
nol and water calculated as per Molinspiration Online Property Toolkit
Synthesis of 4-substituted benzaldehyde (3a–i)
hydrazones/acetohydrazones. These substituents were cho-
sen based on the literature review and computational study.
The most active compound BT 15 has 4-bromo phenyl at
position 23 of 1, 3-benzothiazol-2-yl acetohydrazones. It has
been observed that at the presence of 4-chloro phenyl and
phenyl at position 23 of 1, 3-benzothiazol-2-yl acetohyd-
razones yields active compounds. The presence of isatinyl at
position N-15 of 1, 3-benzothiazol-2-yl acetohydrazones
also yields the active compound. Among 1, 3-benzothiazol-
A mixture of substituted phenol 1a–i (37.4 mmol),
4-fluorobenzaldehyde 2 (37.4 mmol), and potassium car-
bonate (38.8 mmol) in N,N-dimethylformamide (30 ml)
was refluxed for 16–18 h under nitrogen. After cooling, the
product was extracted from the reaction mixture and
purified by chromatography. (3e) Yield: 72%; m.p.: 47°C;
1
IR (KBr, cm-1) t: 1662 (C=O), 1241 (–O–); H NMR
(CDCl3, 300 MHz) d: 9.81 (s, 1H, –CHO), 6.80–7.82 (a set
Table 7 Description of target receptors used in docking study
Receptor
Description
GABA(A) alpha-1
gi|27808653|sp|P14867.3|GBRA1_HUMAN RecName: Full=Gamma-aminobutyric acid receptor subunit alpha-1;
AltName: Full=GABA(A) receptor subunit alpha-1; Flags: Precursor
GABA(A) delta
gi|59802571|sp|O14764.2|GBRD_HUMAN RecName: Full=Gamma-aminobutyric acid receptor subunit delta;
AltName: Full=GABA(A) receptor subunit delta; Flags: Precursor
Glutamate
gi|4504117|ref|NP_000821.1| glutamate receptor, ionotropic kainate 1 isoform 1 precursor [Homo sapiens]
Na/H exchanger
gi|6919937|sp|Q92581.2|SL9A6_HUMAN RecName: Full=Sodium/hydrogen exchanger 6; AltName:
Full=Na(?)/H(?) exchanger 6; Short = NHE-6; AltName: Full = Solute carrier family 9 member 6
Na channel
gi|126362949|ref|NP_008853.3| sodium channel protein type 3 subunit alpha isoform 1 [Homo sapiens]
T-type calcium
gi|21361077|ref|NP_066919.2| voltage-dependent T-type calcium channel subunit alpha-1I isoform a [Homo sapiens]
123

Med Chem Res (2012) 21:2428–2442
2437
Table 8 Docking study results of compounds BT 2, BT 5, BT 14, BT 15, and BT 20
Ligand Receptor
Affinity
(Kcal/mol)
H-bonds H-binding ligand
Element Atom No. Type
H-binding receptor
Residue Element Atom No. Type
BT 2
GABA(A) alpha-1 -5.8
00
01
00
00
00
00
01
00
02
–
–
09
–
–
–
–
–
452
–
–
GABA (A) delta
Glutamate
-6.2
-6.4
-5.5
0.0
N
–
Donor
Ala324
O
–
Acceptor
–
–
–
Na/H exchanger
Na channel
–
–
–
–
–
–
–
–
–
–
–
–
–
–
T-type calcium
0.0
–
–
–
–
–
–
–
BT 5
GABA(A) alpha-1 -6.4
N
–
09
–
Donor
–
Ser326
–
O
–
349
–
Acceptor
GABA (A) delta
Glutamate
-6.4
-6.3
–
N
N
N
N
–
09
08
09
09
–
Donor
Val541
O
N
O
O
–
952
780
112
117
–
Acceptor
Acceptor His523
Donor
Na/H exchanger
-5.7
02
Donor
Gly254
Acceptor
Donor
Glu255
Acceptor
Na channel
0.0
0.0
00
00
01
00
03
–
–
–
–
–
T-type calcium
–
–
–
–
–
BT 14
GABA(A) alpha-1 -5.2
N
–
6
Acceptor Ser299
O
–
185
–
Both
GABA (A) delta
Glutamate
-5.9
-7.7
–
–
–
–
N
O
N
N
–
6
Acceptor Thr333
Acceptor Thr333
O
O
O
O
–
865
865
865
135
–
Both
30
12
12
–
Both
Donor
Thr333
Both
Na/H exchanger
Na channel
-5.5
0.0
01
00
00
02
Donor
Val256
Acceptor
–
–
–
–
–
T-type calcium
0.0
–
–
–
–
–
BT 15
GABA(A) alpha-1 -6.6
O
O
–
30
30
–
Acceptor Ser297
Acceptor Ser297
O
N
–
168
162
–
Both
Donor
GABA (A) delta
Glutamate
-6.2
-6.6
-5.2
0.0
00
01
00
00
00
03
–
–
–
N
–
12
–
Donor
Leu455
O
–
102
–
Acceptor
Na/H exchanger
Na channel
–
–
–
–
–
–
–
–
–
–
T-type calcium
0.0
–
–
–
–
–
–
–
BT 20
GABA(A) alpha-1 -5.9
N
O
N
N
N
N
–
23
25
06
13
06
06
–
Donor
Ser299
O
O
O
O
N
N
–
185
185
168
446
107
117
–
Both
Both
Both
Acceptor
Donor
Donor
–
Acceptor Ser299
Acceptor Ser297
GABA (A) delta
Glutamate
-6.2
-6.8
01
02
Donor
Ala323
Acceptor Glu456
Acceptor Glu457
Na/H exchanger
Na channel
-5.2
0.0
00
00
00
–
–
–
–
–
–
–
–
–
–
–
T-type calcium
0.0
–
–
–
–
–
Affinity and H-bonds calculations were determined by docking study using AutoDock 4.0 software
of signals, 8H, Ar–H); MS (m/z, %): 276.95 (M??1 for
79Br, 100.00), 278.93 (M??1 for 81Br, 96.70).
and conc. HCl (2 ml) was added at 5–6°C. This mixture
was refluxed for 5–6 h to obtain 2-hydrazinyl-1, 3-benzo-
thiazole 5. Yield: 82%; m.p.: 212–214°C; IR (KBr, cm-1
)
1
Synthesis of 2-hydrazinyl-1, 3-benzothiazole (5)
t: 3345 (NHNH2); H NMR (CDCl3, 300 MHz) d: 11.12
(s, 1H, NH, D2O exchangeable), 7.12–7.96 (m, 4H, Ben-
zothiazole-H), 4.52 (s, 2H, NH2). MS (m/z, %): 166.02
(M??1, 87.43).
To a suspension of 1, 3-benzothiazol-2-amine 4 (0.01 mol)
in ethylene glycol (8 ml), hydrazine hydrate (0.03 mol),
123

2438
Med Chem Res (2012) 21:2428–2442
Fig. 4 Docking images. a BT 2
with GABA (A) delta receptor;
b BT 5 with GABA(A) alpha-1
receptor; c BT 5 with glutamate
receptor, d BT 5 with Na/H
exchanger; e BT 14 with GABA
(A) alpha-1 receptor; f BT 14
with glutamate receptor, g BT
14 with Na/H exchanger; h BT
15 with GABA (A) alpha-1
receptor; i BT 15 with
glutamate receptor; j BT 20
with GABA (A) alpha-1
receptor; k BT 20 with GABA
(A) delta receptor; and l BT 20
with glutamate receptor
Synthesis of ethyl (1, 3-benzothiazol-2-ylsulfanyl)
(s, 2H, S-CH2), 1.34 (t, 3H, OCH2CH3); MS (m/z, %):
254.00 (M??1, 43.26).
acetate (7)
A mixture of 1, 3-benzothiazole-2-thiol 6 (1 mmol), chlo-
roethyl acetate (1.1 mmol) and triethyl amine (1 mmol)
was refluxed in dry methanol for 2–3 h. Ethanol was
removed under reduced pressure and the product was re-
crystallized with ethanol to obtain white crystals of 7.
Yield: 86%; m.p.: 112°C; IR (KBr, cm-1) t: 1672 (C=O),
Synthesis of 2-(1,3-benzothiazol-2-
ylsulfanyl)acetohydrazide (8)
Compound 7 (0.1 mol) and hydrazine hydrate (0.3 mol) in
ethanol (50 ml) were refluxed for 2 h. The excess of sol-
vent was removed under reduced pressure and recrystal-
lized from chloroform-hexane (3:1) to yield white crystals
of compound 8. Yield: 81%; m.p.: 168–170°C; IR
1
721 (C–S–C); H NMR (CDCl3, 300 MHz) d: 7.06–7.92
(m, 4H, Benzothiazole-H), 4.33 (q, 2H, O-CH2), 4.00
123

Med Chem Res (2012) 21:2428–2442
2439
(KBr, cm-1) t: 3335 (NHNH2), 1668 (C=O), 726 (C–S–C);
1H NMR (CDCl3, 300 MHz) d: 11.02 (s, 1H, NH, D2O
exchangeable), 7.02–7.90 (m, 4H, Benzothiazole-H), 4.48
(s, 2H, NH2), 4.06 (s, 2H, S-CH2). MS (m/z, %): 240.02
(M??1, 37.43).
ppm: 11.76 (s, 1H, NH, D2O exchangeable), 7.08–8.21 (a
set of signals, 12H, Ar–H, and Benzothiazole-H), 8.09 (s,
1H, CH=N). MS (m/z, %): 380.06 (M??1 for 35Cl,
100.00), 382.01 (M??1 for 37Cl, 35.13).
2-{2-[4-(4-bromophenoxy)benzylidene]hydrazinyl}-
Synthesis of 2-{2-[substituted]hydrazinyl}-1,3-
1,3-benzothiazole (BT 5)
benzothiazoles (BT 1–10) and 2-(1,3-benzothiazol-2-
ylsulfanyl)-N0-(substituted) acetohydrazides (BT 11–20)
IR (KBr, cm-1) t: 3251, 3068 (NHstr associated), 1606
(CH=Nstr), 1240 (–O–). H NMR (CDCl3, 300 MHz) d in
1
Equimolar quantities (0.01 mol) of 4-substituted benzal-
dehydes 3a–i/isatin 3j and 2-hydrazinyl-1, 3-benzothiazole
5/2-(1, 3-benzothiazol-2-ylsulfanyl) acetohydrazide 8 were
dissolved in warm ethanol containing 0.5 ml of glacial
acetic acid. The reaction mixture was refluxed for 4–6 h
and set aside. The resultant solid was washed with ethanol
and recrystallized from 90% ethanol. The physical data and
elemental analysis data of the titled compounds BT 1–20
are presented in Tables 1 and 2. The spectral data of titled
compounds BT 1–20 are given below.
ppm: 11.79 (s, 1H, NH, D2O exchangeable), 7.01–8.26 (a
set of signals, 12H, Ar–H, and Benzothiazole-H), 8.05 (s,
1H, CH=N). MS (m/z, %): 426.03 (M??1 for 81Br,
100.00), 424.07 (M??1 for 79Br, 95.30).
2-{2-[4-(4-fluorophenoxy)benzylidene]hydrazinyl}-
1,3-benzothiazole (BT 6)
IR (KBr, cm-1) t: 3255, 3066 (NHstr associated), 1602
1
(CH=Nstr), 1241 (–O–). H NMR (CDCl3, 300 MHz) d in
ppm: 11.78 (s, 1H, NH, D2O exchangeable), 7.08–8.21 (a
set of signals, 12H, Ar–H, and Benzothiazole-H), 8.03 (s,
1H, CH=N). MS (m/z, %): 364.02 (M??1, 78.10).
2-[2-(4-phenoxybenzylidene)hydrazinyl]-1,3-
benzothiazole (BT 1)
IR (KBr, cm-1) t: 3251, 3068 (NHstr associated), 1605
(CH=Nstr), 1241 (–O–). H NMR (CDCl3, 300 MHz) d in
1
2-{2-[4-(4-chloro-3-
methylphenoxy)benzylidene]hydrazinyl}-1,3-
benzothiazole (BT 7)
ppm: 11.72 (s, 1H, NH, D2O exchangeable), 7.01–8.28 (a
set of signals, 13H, Ar–H, and Benzothiazole-H), 8.02
(s, 1H, CH=N). MS (m/z, %): 346.07 (M??1, 94.11).
IR (KBr, cm-1) t: 3246, 3061 (NHstr associated), 1609
1
(CH=Nstr), 1240 (–O–). H NMR (CDCl3, 300 MHz) d in
2-{2-[4-(4-nitrophenoxy)benzylidene]hydrazinyl}-1,3-
ppm: 11.72 (s, 1H, NH, D2O exchangeable), 7.09–8.25 (a
set of signals, 11H, Ar–H, and Benzothiazole-H), 8.06 (s,
1H, CH=N), 2.36 (s, 3H, CH3). MS (m/z, %): 394.05
(M??1 for 35Cl, 100.00), 396.10 (M??1 for 37Cl, 36.17).
benzothiazole (BT 2)
IR (KBr, cm-1) t: 3245, 3066 (NHstr associated), 1607
(CH=Nstr), 1523 (N=O), 1240 (–O–). 1H NMR (CDCl3,
300 MHz) d in ppm: 11.76 (s, 1H, NH, D2O exchangeable),
7.04–8.23 (aset ofsignals, 12H, Ar–H, and Benzothiazole-H),
8.09 (s, 1H, CH=N). MS (m/z, %): 391.00 (M??1, 100.00).
2-{2-[4-(naphthalen-2-yloxy)benzylidene]hydrazinyl}-
1,3-benzothiazole (BT 8)
2-{2-[4-(4-methylphenoxy)benzylidene]hydrazinyl}-
IR (KBr, cm-1) t: 3253, 3065 (NHstr associated), 1607
(CH=Nstr), 1240 (–O–), 838, 826 (b-naphthyl). H NMR
1
1,3-benzothiazole (BT 3)
(CDCl3, 300 MHz) d in ppm: 11.74 (s, 1H, NH, D2O
exchangeable), 7.03–8.28 (a set of signals, 15H, Ar–H,
naphthyl-H, and Benzothiazole-H), 8.03 (s, 1H, CH=N).
MS (m/z, %): 396.13 (M??1, 52.13).
IR (KBr, cm-1) t: 3248, 3064 (NHstr associated), 1603
(CH=Nstr), 1239 (–O–). 1H NMR (CDCl3, 300 MHz) d in ppm:
11.71 (s, 1H, NH, D2O exchangeable), 7.01–8.22 (a set of sig-
nals, 12H, Ar–H, and Benzothiazole-H), 8.05 (s, 1H, CH=N),
2.34 (s, 3H, CH3). MS (m/z, %): 360.07 (M??1, 91.71).
2-{2-[4-(1,3-benzodioxol-5-yloxy)
benzylidene]hydrazinyl}-1,3-benzothiazole (BT 9)
2-{2-[4-(4-chlorophenoxy)benzylidene]hydrazinyl}-
1,3-benzothiazole (BT 4)
IR (KBr, cm-1) t: 3251, 3063 (NHstr associated), 1608
(CH=Nstr), 1242 (–O–). 1H NMR (CDCl3, 300 MHz) d
in ppm: 11.78 (s, 1H, NH, D2O exchangeable), 7.11–8.21
(a set of signals, 11H, Ar–H, Benzodioxol-H, and
IR (KBr, cm-1) t: 3254, 3061 (NHstr associated), 1611
1
(CH=Nstr), 1242 (–O–). H NMR (CDCl3, 300 MHz) d in
123

2440
Med Chem Res (2012) 21:2428–2442
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-
bromophenoxy)benzylidene]acetohydrazide (BT 15)
Benzothiazole-H), 8.07 (s, 1H, CH=N). MS (m/z, %):
390.07 (M??1, 91.03).
IR (KBr, cm-1) t: 3178, 3076 (NHstr associated), 1664
(C=Ostr), 1611 (CH=Nstr), 1240 (–O–), 725 (C–S–C). H
3-[2-(1,3-benzothiazol-2-yl)hydrazinylidene]-1,3-
1
dihydro-2H-indol-2-one (BT 10)
NMR (CDCl3, 300 MHz) d in ppm: 11.15 (s, 1H, NH, D2O
exchangeable), 8.00 (s, 1H, CH=N), 6.88–7.94 (a set of
signals, 12H, Ar–H, and Benzothiazole-H), 4.06 (s, 2H, S-
CH2). MS (m/z, %): 499.84 (M??1 for 81Br, 100.00),
497.85 (M??1 for 79Br, 94.12).
IR (KBr, cm-1) t: 3257, 3082 (NHstr associated), 1673
(C=Ostr), 1621 (C=N). H NMR (CDCl3, 300 MHz) d in
ppm: 12.72 (s, 1H, NH of Isatin, D2O exchangeable), 11.68
(s, 1H, NH, D2O exchangeable), 7.20–8.18 (a set of signals,
8H, Isatin-H, and Benzothiazole-H). MS (m/z, %): 295.05
(M??1, 92.11).
1
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-
fluorophenoxy)benzylidene]acetohydrazide (BT 16)
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(4-
phenoxybenzylidene)acetohydrazide (BT 11)
IR (KBr, cm-1) t: 3176, 3077 (NHstr associated), 1668
(C=Ostr), 1607 (CH=Nstr), 1240 (–O–), 725 (C–S–C). H
1
IR (KBr, cm-1) t: 3188, 3079 (NHstr associated), 1661
(C=Ostr), 1607 (CH=Nstr), 1242 (–O–), 723 (C–S–C). H
NMR (CDCl3, 300 MHz) d in ppm: 11.11 (s, 1H, NH, D2O
exchangeable), 8.06 (s, 1H, CH=N), 6.85–7.98 (a set of
signals, 12H, Ar–H, and Benzothiazole-H), 4.03 (s, 2H, S-
CH2). MS (m/z, %): 438.04 (M??1, 76.66).
1
NMR (CDCl3, 300 MHz) d in ppm: 11.09 (s, 1H, NH, D2O
exchangeable), 8.09 (s, 1H, CH=N), 6.80–7.96 (a set of
signals, 13H, Ar–H, and Benzothiazole-H), 4.11 (s, 2H, S-
CH2). MS (m/z, %): 420.10 (M??1, 63.06).
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-chloro-3-
methylphenoxy)benzylidene] acetohydrazide (BT 17)
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-
nitrophenoxy)benzylidene]acetohydrazide (BT 12)
IR (KBr, cm-1) t: 3183, 3078 (NHstr associated), 1672
(C=Ostr), 1607 (CH=Nstr), 1240 (–O–), 721 (C–S–C). H
1
IR (KBr, cm-1) t: 3177, 3075 (NHstr associated), 1666
(C=Ostr), 1604 (CH=Nstr), 1523 (N=O), 1240 (–O–), 728
(C–S–C). 1H NMR (CDCl3, 300 MHz) d in ppm: 11.13 (s,
1H, NH, D2O exchangeable), 8.04 (s, 1H, CH=N), 6.83–
7.91 (a set of signals, 12H, Ar–H, and Benzothiazole-H),
4.10 (s, 2H, S-CH2). MS (m/z, %): 465.05 (M??1, 42.17).
NMR (CDCl3, 300 MHz) d in ppm: 11.18 (s, 1H, NH, D2O
exchangeable), 8.01 (s, 1H, CH=N), 6.82–7.91 (a set of
signals, 11H, Ar–H, and Benzothiazole-H), 4.05 (s, 2H, S-
CH2), 2.37 (s, 3H, CH3). MS (m/z, %): 468.05 (M??1 for
35Cl, 100.00), 470.02 (M??1 for 37Cl, 35.34).
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(naphthalen-2-
yloxy)benzylidene]acetohydrazide (BT 18)
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-
methylphenoxy)benzylidene]acetohydrazide (BT 13)
IR (KBr, cm-1) t: 3212, 3068 (NHstr associated), 1669
(C=Ostr), 1611 (CH=Nstr), 1238 (–O–), 837, 822 (b-naph-
thyl), 727 (C–S–C). 1H NMR (CDCl3, 300 MHz) d in ppm:
11.24 (s, 1H, NH, D2O exchangeable), 8.07 (s, 1H, CH=N),
6.72–7.98 (a set of signals, 15H, Ar–H, naphthyl-H, and
Benzothiazole-H), 4.12 (s, 2H, S-CH2). MS (m/z, %):
470.05 (M??1, 41.92).
IR (KBr, cm-1) t: 3182, 3076 (NHstr associated), 1667
(C=Ostr), 1610 (CH=Nstr), 1240 (–O–), 723 (C–S–C). H
1
NMR (CDCl3, 300 MHz) d in ppm: 11.12 (s, 1H, NH, D2O
exchangeable), 8.07 (s, 1H, CH=N), 6.82–7.94 (a set of sig-
nals, 12H, Ar–H, and Benzothiazole-H), 4.07 (s, 2H, S-CH2),
2.35 (s, 3H, CH3). MS (m/z, %): 434.06 (M??1, 57.78).
N0-[4-(1,3-benzodioxol-5-yloxy)benzylidene]-2-(1,3-
benzothiazol-2-ylsulfanyl)aceto hydrazide (BT 19)
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-[4-(4-
chlorophenoxy)benzylidene]acetohydrazide (BT 14)
IR (KBr, cm-1) t: 3208, 3063 (NHstr associated), 1667
(C=Ostr), 1606 (CH=Nstr), 1240 (–O–), 725 (C–S–C). H
IR (KBr, cm-1) t: 3188, 3079 (NHstr associated), 1666
(C=Ostr), 1612 (CH=Nstr), 1240 (–O–), 723 (C–S–C). H
1
1
NMR (CDCl3, 300 MHz) d in ppm: 11.08 (s, 1H, NH, D2O
exchangeable), 8.02 (s, 1H, CH=N), 6.78–7.91 (a set of
signals, 11H, Ar–H, Benzodioxol-H, and Benzothiazole-
H), 4.09 (s, 2H, S-CH2). MS (m/z, %): 464.02 (M??1,
62.21).
NMR (CDCl3, 300 MHz) d in ppm: 11.18 (s, 1H, NH, D2O
exchangeable), 8.08 (s, 1H, CH=N), 6.85–7.92 (a set of
signals, 12H, Ar–H, and Benzothiazole-H), 4.05 (s, 2H, S-
CH2). MS (m/z, %): 454.02 (M??1 for 35Cl, 77.31), 456.05
(M??1 for 37Cl, 27.05).
123

Med Chem Res (2012) 21:2428–2442
2441
2-(1,3-benzothiazol-2-ylsulfanyl)-N0-(2-oxo-1,2-
dihydro-3H-indol-3-ylidene)acetohydrazide (BT 20)
treated animal falls off this rotating rod three times during
1-min period.
IR (KBr, cm-1) t: 3239, 3080 (NHstr associated), 1677
(C=Ostr), 1621 (C=N), 723 (C–S–C). H NMR (CDCl3,
Computational study
1
300 MHz) d in ppm: 12.77 (s, 1H, NH of Isatin, D2O
exchangeable), 11.11 (s, 1H, NH, D2O exchangeable),
6.89–7.93 (a set of signals, 8H, Isatin-H, and Benzothia-
zole-H), 4.08 (s, 2H, S-CH2). MS (m/z, %): 369.02 (M??1,
56.83).
Distance mapping
The pharmacophore pattern studies in which distance
between the various groups postulated as essential for anti-
convulsant activity were done on the 3D optimized structures
using ACD/3D viewer version 12.01 and Argus Lab 4.0
Mark A. Thompson Planaria Software LLC. In conforma-
tional analysis of the 10 clinically effective, well-known and
structurally different anticonvulsant drugs such as albutoin,
carbamazepine, gabapentin, lamotrigine, mephobarbital,
phenytoin, progabide, ralitoline, remacemide, zonisamide; a
molecular model was suggested on the basis of molecular
dynamics distance estimations (Yogeeswari et al., 2005).
Anticonvulsant and neurotoxicity evaluation
The evaluation of anticonvulsant activity and neurotoxicity
was carried out by the Epilepsy Branch, National Institute
of Neurological Disorder and Stroke, National Institute of
Health, Bethesda, USA following the reported procedures.
Male albino mice (CF-1 strain, 18–25 g) and male
albino rats (Sprague-Dawley, 100–150 g) were used as
experimental animals. The synthesized derivatives were
suspended in 0.5% methyl cellulose and the test compound
is usually manipulated with a mortar pestle to help prepa-
ration of suspension. The titled compounds BT 1–20 were
subjected to anticonvulsant screening by 6 Hz psychomo-
tor seizure test to identify the anticonvulsant activity of the
compounds at five different time points, i.e., 0.25, 0.5, 1.0,
2.0, and 4.0 h after i.p. administration in mice at a dose of
100 mg/kg. Neurotoxicity was observed by MMI which
was measured by the rotorod (neurotoxicity) test.
Calculation of physicochemical parameters
A computational study of titled compounds was performed
for prediction of ADME properties. Polar surface area
(TPSA) (Ertl et al., 2000), miLog P, number of rotatable
bonds, molecular volume, number of hydrogen donor and
acceptor atoms and violations of Lipinski’s rule of five
(Lipinski et al., 2001) were calculated using Molinspiration
online property calculation toolkit (Molinspiration Chem-
informatics, 2010). Absorption (% ABS) was calculated
by: % ABS = 109 - (0.345 9 TPSA) (Zhao et al., 2002).
6 Hz psychomotor seizure test
Log P determination
6 Hz psychomotor seizure test or minimal clonic seizure
(6 Hz) test was used to assess compound’s efficacy against
electrically induced seizures but used a lower frequency
(6 Hz) and longer duration of stimulation (3 s). Test
compounds were pre-administered to mice via i.p. injec-
tion. At varying times, individual mice (four mice per time
point) were challenged with sufficient current delivered
through corneal electrodes to elicit a psychomotor seizure
in 97% animals (32 mA for 3 s). The untreated mice would
display seizure characterized by a minimal clonic phase
followed by stereotyped, automatistic behaviors, described
originally as being similar to the aura of human patients
with partial seizure. Animals not displaying this behavior
are considered to be protected.
The partition coefficient between octanol and phosphate
buffer was determined at room temperature (Farrar et al.,
1993). 10 ml of octanol and 10 ml phosphate buffer were
taken in a glass stoppered graduated tube and 5 mg of
accurately weighed compound was added. The mixture was
then shaken with the help of mechanical shaker for 24 h at
room temperature and then transferred to a separating
funnel and allowed to dynamic equilibrate for 6 h. The
aqueous and octanol phase were separated and filtered
through membrane filter and drug content in aqueous phase
was analyzed by UV spectroscopy. Theoretical miLog P
for synthesized compounds was then compared with the
experimental Log P data.
Neurotoxicity—minimal motor impairment
Docking study
MMI was measured by the rotorod (neurotoxicity) test.
When a mouse is placed on a rod that rotates at a speed of
6 rpm, the animal can maintain its equilibrium for a long
period of time. The compound was considered toxic if the
Compounds BT 2, BT 5, BT 14, BT 15, and BT 20 were
selected as ligands for docking study with six established
epilepsy receptors namely GABA(A) alpha-1, GABA(A)
delta, glutamate, Na/H exchanger, Na channel, and T-type
123

2442
Med Chem Res (2012) 21:2428–2442
Farrar VA, Ciechanowicz MR, Grochowski J, Serda P, Pilati T,
Filippini G, Hinko CN, El-Assadi E, Moore JA, Edafiogho IO,
Andrews CW, Cory M, Nicholson JM, Scott JR (1993) Synthesis
and CLOGP correlation of imidooxy anticonvulsants. J Med
Chem 36:3517–3525
Hays SJ, Rice MJ, Ortwine DF, Johnson G, Schwarz RD, Boyd DK
(1994) Substituted 2-benzothiazolamines as sodium flux inhib-
itors: quantitative structure activity relationships and anticon-
vulsant activity. J Pharm Sci 83:1425
Jones GL, Woodbury DM (1982) Principles of drug action: structure
activity relationships and mechanisms. In: Woodbury DM, Penry
JK, Pippenger CE (eds) Antiepileptic drugs. Raven Press, New
York, pp 83–109
Landmark CJ (2007) Targets for antiepileptic drugs in the synapse.
Med Sci Monit 13:RA1–RA7
calcium channel receptor. The description of receptors used
is shown in Table 7. These receptors are the most impor-
tant molecular targets in the design and discovery of suc-
cessful antiepileptic drugs (Landmark, 2007). In this study,
AutoDock 4.0 with its Lamarckian genetic algorithm
(LGA) was used for automated flexible ligand docking of
BT 2, BT 5, BT 14, BT 15, and BT 20 with above-men-
tioned receptors.
The grid maps were calculated using AutoGrid. In all
dockings, a grid map with 60 9 60 9 60 points, a grid
˚
spacing of 0.375 A (roughly a quarter of the length of a
carbon–carbon single bond) were used, and the mps were
centered on the ligand binding site. For all dockings, 100
independent run with, an initial population of random
individuals with a population size of 150 individuals, a
maximum number of 2.5 9 106 energy evaluations, max-
imum number of generations of 27,000, an elitism value of
1 and a number of active torsion of 9 were used. Auto Dock
4.0 was used to generate both grid and docking parameter
files (i.e., .gpf and.dpf files) and docking affinity (Kcal/
mol) and count of probable H-bonds were determined.
Lipinski CA, Lombardo L, Dominy BW, Feeney PJ (2001) Exper-
imental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv
Drug Deliv Rev 46:3–26
Mizoule J, Meldrum B, Martine M, Croucher M, Ollat C, Uzar A
(1985) 2-amino-6-trifluromethoxy benzothiazole,
a possible
antagonist of excitatory amino acid neurotransmission-1. Anti-
convulsant properties. Neuropharmacology 24:767
Molinspiration Cheminformatics, Bratislava, Slovak Republic,
http://www.molinspiration.com/services/properties.html. Acces-
sed September 16, 2010
Perucen E (1996) The new generation of antiepileptic drugs:
advantages and disadvantages. Br J Clin Pharmacol 42:531–543
Sabers A, Gram L (2000) Newer anticonvulsants: comparative review
of drug interactions and adverse effects. Drugs 60:23–33
Siddiqui N, Rana A, Khan SA, Bhat MA, Haque SE (2007) Synthesis
of benzothiazole semicarbazones as novel anticonvulsants–the
role of hydrophobic domain. Bioorg Med Chem Lett 17(15):
4178–4182
Acknowledgments The authors would like to express their grati-
tude to IIT, Delhi and CDRI, Lucknow for providing the spectral and
elemental data. One of the authors (Praveen Kumar) is thankful to S.
D. College of Pharmacy and Vocational Studies, Muzaffarnagar-
251001, India for providing research facilities. The authors also
appreciate the help and support by Mr. Om Prakash during docking
study.
Unverferth K, Engel J, Hofgen N, Rostock A, Gunther R, Lankau HJ,
Menzer M, Rolfs A, Liebscher J, Muller B, Hofmann HJ (1998)
Synthesis, anticonvulsant activity, and structure-activity rela-
tionships of sodium channel blocking 3-aminopyrroles. J Med
Chem 41:63–73
References
Wang Y, Cai S, Lan N, Keana J, Ilyin V, Weber E (1998) Carbocyclic
and heterocyclic substituted semicarbazones and thiosemicarba-
zones and the use thereof. PCT/US98/08004:1-81
Bell GS, Sander JW (2002) The epidemiology of epilepsy: the size of
the problem. Seizure 11(Suppl A):306–314
Camerman A, Camerman N (1980) Stereochemical similarities in
chemically different antiepileptic drugs. In: Glaser GH, Penry
JK, Woodbury JM (eds) Antiepileptic drugs: mechanism of
action. Raven Press, New York, pp 223–231
Yogeeshwari P, Sriram D, Thirumurgan R, Jit LR, Ragavendran JV,
Kavya R, Rakhra K, Saraswat V (2006) Synthesis of N4-(2, 4-
dimethylphenyl) semicarbazones as 4-aminobutyrate amino-
transferase inhibitors. Acta Pharm 56:259–272
Chopade RS, Bahekar RH, Khedekar PB, Bhusari KP, Rao AR (2000)
Synthesis and anticonvulsant activity of 3-(5-substituted-ben-
Yogeeswari P, Sriram D, Thirumurugan R, Raghavendran JV, Sudhan
K, Pavana RK, Stables JP (2005) Discovery of N-(2, 6-
dimethylphenyl)-substituted semicarbazones as anticonvulsants:
hybrid pharmacophore-based design. J Med Chem 48:6202–
6211
Zhao Y, Abraham MH, Lee J, Hersey A, Luscombe NC, Beck G,
Sherborne B, Cooper I (2002) Rate-limited steps of human oral
absorption and QSAR studies. Pharm Res 19:1446–1457
zothiazol-2-yl)-6-phenyl-[1,
Pharm (Weinheim) 335:881
3]-oxazinane-2-thiones.
Arch
Ertl P, Rohde B, Selzer P (2000) Fast calculation of molecular polar
surface area as a sum of fragment-based contributions and its
application to the prediction of drug transport properties. J Med
Chem 43:3714–3717
123