Synthesis, anticonvulsant activity and 3D-QSAR study of some prop-2-eneamido and 1-acetyl-pyrazolin derivatives of aminobenzothiazole

Article abstract of DOI:10.1016/j.ejmech.2009.09.037

A series of 6-substituted-[3-substituted-prop-2-eneamido]benzothiazole 9-32 and 6-substituted-2-[(1-acetyl-5-substituted)-2-pyrazolin-3-yl]aminobenzothi azole 33-56 were synthesized using appropriate synthetic route and evaluated experimentally against ma

Full text of DOI:10.1016/j.ejmech.2009.09.037

European Journal of Medicinal Chemistry 45 (2010) 149–159
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anticonvulsant activity and 3D-QSAR study of some
prop-2-eneamido and 1-acetyl-pyrazolin derivatives
of aminobenzothiazole
*
Nikhil D. Amnerkar, Kishore P. Bhusari
Department of Medicinal Chemistry, Sharad Pawar College of Pharmacy, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 441 110, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 6-substituted-[3-substituted-prop-2-eneamido]benzothiazole 9–32 and 6-substituted-2-
Received 24 May 2009
Received in revised form
11 September 2009
Accepted 22 September 2009
Available online 30 September 2009
[(1-acetyl-5-substituted)-2-pyrazolin-3-yl]aminobenzothiazole 33–56 were synthesized using appropriate
synthetic route and evaluated experimentally against maximal electroshock test. Selected compounds were
evaluated for neurotoxicity, hepatotoxicity and behavioral study. The most active compound, 6-methyl-2-
[(1-acetyl-5-(4-chlorophenyl))-2-pyrazolin-3-yl]aminobenzothiazole 52 exhibited an ED50 of 25.49
mmol/
kg, TD50 of 123.87 mol/kg and high protective index (PI) of 4.86 compared to standard drug phenytoin. The
m
3D-QSAR analysis was carried out by PHASE program and a statistically reliable model with good predictive
power (r2 ¼ 0.9220, q2 ¼ 0.8144) was achieved. The 3D-QSAR plots illustrated insights into the structure
activity relationship of these compounds which may aids in the design of potent aminobenzothiazole
derivatives as anticonvulsant agents.
Keywords:
Aminobenzothiazole
Prop-2-eneamide
Pyrazoline
Anticonvulsant activity
3D-QSAR
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
effective in reducing seizure, whilst their therapeutic efficacy is
overcome by some undesirable side effects such as headache,
Epilepsy is a chronic neurological disorder characterized by the
periodic and unpredictable occurrence of seizures that affects the
people of all ages [1,2]. Being one of the world’s oldest recognized
disorders, it is surrounded by fear, discrimination, social and
frightening manifestation [3]. A global campaign against epilepsy
conducted by World Health Organization (WHO) in partnership
with International Bureau for Epilepsy (IBE) and International
League Against Epilepsy (ILAE) suggests that around 1% of world
population at any time (about 50 million people worldwide) is
afflicted with this neurological disorder. Every year about 2.4
million new cases are added to these figures [4,5]. Currently
available antiepileptic drugs (AEDs) provide adequate seizure
control in many patients, still about 28–30% of patients are esti-
mated to be poorly treated [6,7]. Much efforts devoted in the recent
years for the development of novel therapeutics resulted in the
availability of several newer drugs (such as pregabalin, stiripentol,
zonisamide, tiagabine, lamotrigine, levetiracetam, topiramate) as
promising anticonvulsants [8–10]. These drugs have proven to be
nausea, hepatotoxicity, anorexia, ataxia, drowsiness, gastrointes-
tinal disturbances and hirsutism [11,12]. These observations affirm
the further scope and need for the development of newer agents.
Benzothiazole is among the usually occurred heterocyclic nuclei in
many marine as well as natural plant products possessing the wide
range of biological applications [13–17]. Recently, 2-amino-
benzothiazoles emerged as new classes of anticonvulsant agents and
one of its derivatives, riluzole is clinically available drug as anticon-
vulsant [18–23]. Moreover, pyrazoline ring is also an important
building block inmedicinalchemistryled tothe discoveryof a number
of derivatives endowed with anticonvulsant, anti-inflammatory,
antitubercular, antitumour and antidiabetic effects [24–28]. N–CO
moietyiswell recognized as pharmacophoric requirements insomeof
the well-known anticonvulsant agents with varied mechanism of
action [6,29]. Previously, we have reported some [1,3]-oxazinane-2-
thiones [18] and thioquinazoline [30] derivatives of 2-amino-
benzothiazoles as moderately active anticonvulsant agents. These
findings supported the significance of pyrazoline ring for anticon-
vulsant activity of 2-aminobenzothiazoles. In view of these and as
a part of our enduring studies in the area of anticonvulsant agents it
was thought of interest to combine the both pharmacophoric groups
(pyrazoline ring and N–CO moiety). We have synthesized a series of
6-substituted-[3-substituted-prop-2-eneamido]benzothiazole 9–32
* Corresponding author. Tel.: þ91 7104 236352; fax: þ91 7104 235084.
E-mail addresses: nikhilamnerkar@gmail.com (N.D. Amnerkar), kishore.
bhusari@gmail.com (K.P. Bhusari).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.037

150
N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
and 6-substituted-2-[(1-acetyl-5-substituted)-2-pyrazolin-3-yl]ami-
nobenzothiazole 33–56, and evaluated for their potential as
anticonvulsant agents.
4. Results and discussion
The present study describes the synthesis and anticonvulsant
evaluation of some prop-2-eneamido and 1-acetyl-pyrazolin
derivatives of aminobenzothiazole and development of 3D-QSARs
using the generated data. A series of 48 new aminobenzothiazole
derivatives (compounds 9–32 and 33–56) were synthesized in
satisfactory yields (52–80%) as illustrated in Scheme 1 and their
structures were characterized by spectral data. The 6-substituted-
[3-substituted-prop-2-eneamido]benzothiazole derivatives
(compounds 9–32) showed N–H and C]O stretching bands in the
region of 3360–3298 and 1730–1690 cmꢀ1, respectively in their IR
spectrum. While in their 1H NMR spectra for these compounds
The synthesized compounds and their anticonvulsant activity
data were used to generate 3D-QSAR models in order to gain
insights into the structural and molecular properties of these
compounds using the PHASE [31,32] program (Schro¨dinger). In
general, 3D-QSAR is a useful tool in molecular design and medicinal
chemistry which allows the prediction of activity of structurally
diverse compounds and also may assists in identifying new mole-
cules with improved activity.
2. Chemistry
exhibited two doublets for C–H in the regions of
(CO–CH) and 7.14–7.42 ppm (CH–Ar) in addition to the singlet in
the region 12.42–13.65 ppm (N–H). The IR spectrum of the
d 6.48–6.89 ppm
d
The synthesis of 6-substituted-[3-substituted-prop-2-eneamido]-
benzothiazoles (compounds 9–32) and 6-substituted-2-[(1-acetyl-
5-substituted)-2-pyrazolin-3-yl]aminobenzothiazoles (compounds
33–56) was accomplished as portrayed in Scheme 1. The
compounds 5–8 were obtained by acetylation of compounds 1–4
with acetyl chloride following the methodology as described by A.
Flohr et al. [33]. The aminobenzothiazole-chalcones (compounds
9–32) were prepared by Claisen–Schmidt condensation of
compounds 5–8 with appropriate aromatic aldehydes in ethanol
and 10% NaOH at room temperature. Further, cyclization of chal-
cones (compounds 9–32) in ethanolic hydrazine hydrate and glacial
acetic acid yielded 6-substituted-2-[(1-acetyl-5-substituted)-2-
pyrazolin-3-yl]aminobenzothiazole (compounds 33–56). The
structures of all the synthesized compounds were confirmed by
spectral data (FTIR, 1H NMR and EI-MS) and elemental analyses.
d
6-substituted-2-[(1-acetyl-5-substituted)-2-pyrazolin-3-yl]amino-
benzothiazoles (compounds 33–56) showed the stretching bands
in the region of 1510–1470 cmꢀ1 (N–N) and 1125–1085 cmꢀ1 (C–N),
respectively. In their 1H NMR spectrum these derivatives exhibited
the characteristic signals of HA, HB, Hx of pyrazoline ring as doublet
of doublet in the regions d 2.95–3.14, 3.32–3.95 and 5.06–5.85 ppm,
respectively. However, all the other protons were resolved in
appropriate regions confirming the assigned structures.
The result of anticonvulsant activity of the compounds 9–32 and
33–56 against MES induced seizures is depicted in Table 1. All of the
compounds were active in the MES test which is an indicative of
their ability to prevent seizure spread. Selected compounds 40–42,
44, 46, 50–54 and 56 showed similar results when tested against
scPTZ. In the neurological testing, none of these compounds
demonstrated any sign of neurotoxicity. These compounds exhibi-
ted neurotoxicity (TD50) at the maximum dose administered only
after 0.5 h post-administration and the neurological deficit was
found to be less compared to the standard drug, phenytoin. The
protective index i.e. TD50/ED50 showed significant results (Table 1).
3. Pharmacology
All the synthesized compounds 9–32 and 33–56 were evaluated
for their anticonvulsant activity against Maximal Electroshock
(MES) induced seizures while the selected compounds were eval-
uated against subcutaneous pentylenetetrazole (scPTZ) induced
seizure model in mice [18]. The minimal motor impairment was
measured by the rotorod (neurotoxicity) test [20]. The test
compounds were administered intraperitonealy at various dose
The most active compound 52 exhibited ED50 of 25.49
a TD50 of 123.87 mol/kg and high protective index (PI) of 4.86
compared to phenytoin. Compound 13 with the ED50 of
781.33 mol/kg and TD50 898.07 mol/kg was the least effective
mmol/kg and
m
m
m
and more toxic compound. The statistical analyses were carried out
using one way ANOVA (Dunnet’s test) at 95% confidence interval
and all the activity data on comparison with vehicle control reaches
the statistical significance (p < 0.05).
levels ranging from 20–1000 mmol/kg body weight and the median
effective dose (ED50), median toxic dose (TD50) and protection index
(PI) values were determined. Selected compounds (40, 44, 51–54
and 56) with significant anticonvulsant activity were evaluated for
the behavioral study using an actophotometer-scoring technique
[34] and CNS study by determining the immobility time using Por-
solt’s swimpool test [35] with the standard drug carbamazepine, at
The behavioral activity of selected compounds 40, 44, 51–54 and
56 is summarized in Table 2. Compounds 44 and 52 along with
phenytoin showed significant decrease in the locomotor activity,
while the compound 56 did not showed any behavioral despair
effect when compared with the control at p < 0.05 (Student’s t test).
The effects of selected compounds 40, 44, 51–54 and 56 on
duration of immobility time is presented as mean ꢁ SEM using
Porsolt’s swimpool test (Table 3). No significant CNS depression
was exhibited by compounds 44, 52 and 53, whilst the other
compounds showed increase in immobility time compared with
control at p < 0.05 and emerged as CNS depressants.
the dose level of 100 mmol/kg. Compounds 52, 53 and 56 were tested
for hepatotoxic side effects using liver function tests [36].
O
N
S
N
CH3
a
b
NH2
N
H
S
R1
R1
5-8
1-4
Selected compounds 52, 53 and 56 were analyzed for their
hepatotoxic side effects using liver function tests. The compounds
were administered chronically to rats for 15 days at a dose of
R2
O
100 mmol/kg. The enzyme serum glutamate oxaloacetate trans-
N
CH3
O
aminase (SGOT) and serum glutamate pyruvate transaminase
(SGPT) levels were estimated. All the tested compounds were safe
since the serum level of these enzymes did not show any significant
variation compared to the control (Table 4).
N
N
N
S
c
N
H
N
H
R2
S
R1
R1
33-56
9-32
The anticonvulsant activity (ED50) generated in the present
studies for the series of 6-substituted-[3-substituted-prop-2-enea-
mido]benzothiazoles (compounds 9–32) and 6-substituted-2-
Scheme 1. General scheme for the synthesis of compounds 9–32 and 33–56. Reagents
and conditions: (a) CHCl3, CH3COCl, stir, 3 h, 0–5 ꢂC; (b) dry EtOH, Ar–CHO, NaOH 10%,
stir, 10–12 h, r.t.; (c) dry EtOH, NH2NH2.H2O 99%, CH3COOH, reflux, 6–8 h.

Table 1
Anticonvulsant activity and neurotoxicity of compounds (N ¼ 6)
PId
QSAR sete
pED50
f
Compd
R1
R2
Intraperitoneal administration to mice
MESa
scPTZb
NTc
Actual
Predg
9
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
646
315
367
209
517
249
495
259
781
382
435
226
425
161
204
79.47
322
106
320
127
544
212
259
93.29
485
216
259
132
382
162
372
178
597
274
315
145
380
106
152
25.46
271
50.21
271
73.91
505
160
209
37.32
13.78
NDh
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
81.52
ND
107.35
ND
132.48
ND
ND
ND
97.32
ND
ND
ND
137.65
ND
ND
ND
ND
ND
ND
ND
149.08
ND
111.57
ND
832
498
402
364
925
513
556
382
898
538
540
369
570
245
576
345
413
210
470
276
658
387
465
187
633
357
787
328
602
210
793
256
834
355
466
246
534
265
408
123
387
157
321
147
603
243
468
174
110
1.28
1.57
1.10
1.73
1.78
2.06
1.12
1.47
1.15
1.41
1.24
1.63
1.34
1.52
2.82
4.35
1.28
1.97
1.46
2.16
1.20
1.82
1.79
2.01
1.30
1.65
3.03
2.48
1.57
1.29
2.13
1.43
1.39
1.29
1.47
1.69
1.40
2.49
2.67
4.86
1.42
3.14
1.18
1.99
1.19
1.51
2.24
4.68
8.01
Training
Training
Test
Training
Training
Test
3.19
3.50
3.44
3.68
3.29
3.60
3.31
3.59
3.11
3.42
3.36
3.65
3.37
3.79
3.69
4.10
3.49
3.97
3.51
3.89
3.26
3.67
3.59
4.03
3.31
3.67
3.59
3.88
3.42
3.79
3.43
3.75
3.22
3.56
3.50
3.84
3.42
3.97
3.82
4.59
3.57
4.30
3.57
4.13
3.30
3.79
3.68
4.43
ꢀ
3.21
3.61
3.33
3.77
3.20
3.72
3.21
3.60
3.05
3.39
3.29
3.54
3.48
3.89
3.73
3.94
3.52
4.16
3.66
3.94
3.38
3.72
3.69
3.89
3.36
3.75
3.47
3.76
3.34
3.88
3.30
3.65
3.15
3.52
3.42
3.71
3.58
4.00
3.78
4.22
3.68
4.14
3.60
4.01
3.35
3.84
3.60
4.35
ꢀ
33
10
34
11
35
12
36
13
37
14
38
15
39
16
40
17
41
18
42
19
43
20
44
21
45
22
46
23
47
24
48
25
49
26
50
27
51
28
52
29
53
30
54
31
55
32
56
Phenytoin
4-Cl-C6H5
4-Cl-C6H5
4-OH-C6H5
4-OH-C6H5
3-OH, 4-OMe-C6H5
3-OH, 4-OMe-C6H5
C4H3O
Test
Training
Training
Training
Test
Test
Training
Test
C4H3O
4-OMe-C6H5
4-OMe-C6H5
C6H5
C6H5
4-Cl-C6H5
4-Cl-C6H5
4-OH-C6H5
4-OH-C6H5
3-OH, 4-OMe-C6H5
3-OH, 4-OMe-C6H5
C4H3O
Test
Training
Training
Test
Test
Test
Training
Training
Training
Test
Training
Training
Training
Training
Training
Training
Training
Training
Test
C4H3O
4-OMe-C6H5
4-OMe-C6H5
C6H5
C6H5
4-Cl-C6H5
4-Cl-C6H5
4-OH-C6H5
4-OH-C6H5
3-OH, 4-OMe-C6H5
3-OH, 4-OMe-C6H5
C4H3O
C4H3O
Training
Training
Test
4-OMe-C6H5
4-OMe-C6H5
C6H5
Test
C6H5
Training
Training
Test
Test
Training
Test
Training
Test
Training
Test
Training
ꢀ
4-Cl-C6H5
4-Cl-C6H5
4-OH-C6H5
4-OH-C6H5
3-OH, 4-OMe-C6H5
3-OH, 4-OMe-C6H5
C4H3O
C4H3O
4-OMe-C6H5
4-OMe-C6H5
27.46
ND
54.31
ND
75.64
ND
ND
ND
39.44
ꢀ
a
All compounds were administered by ip injection at doses of 20–1000
m
mol/kg and values were determined at t ¼ 0.5 h. MES indicates maximum electroshock test and the
data are represented in terms of ED50
.
b
Subcutaneous pentylenetetrazole test.
c
d
e
f
Neurotoxicity screening using rotorod test and values are expressed in terms of TD50
Protection index (PI ¼ TD50/ED50).
.
It represents the training set (29 compound) and test set (19 compound).
Biological activity expressed as pED50 ¼ ꢀlog(ED50 ꢃ 10ꢀ6)] and used for 3D-QSAR studies.
g
h
Predicted activity from PHASE 3D-QSAR program.
ND: activity not determined.

152
N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
Table 2
Table 4
Behavioral activity of the selected compounds (N ¼ 6).
SGOT and SGPT estimation of selected compounds in serum level.
Compd
Activity score using actophotometer
% Change in
Compd
SGOTa (units/ml)
SGPTb (units/ml)
locomotor activityb
Control
Post-treatmenta
Control
52
53
92.2 ꢁ 2.78
93.3 ꢁ 3.13
89.6 ꢁ 1.17
95.4 ꢁ 1.74
101.5 ꢁ 5.13
35.4 ꢁ 2.68
33.1 ꢁ 1.62
30.2 ꢁ 2.51
38.4 ꢁ 1.83
41.3 ꢁ 2.18
(24 h before) ‘X’
(0.5 h after) ‘Y’
40
44
51
52
53
54
56
120 ꢁ 2.7
142 ꢁ 3.4
188 ꢁ 4.2
160 ꢁ 1.3
148 ꢁ 0.9
122 ꢁ 3.2
144 ꢁ 1.6
167 ꢁ 2.2
82 ꢁ 1.6
91 ꢁ 2.1
31.66 (Y)
35.91 (Y)
23.93 (Y)
35.62 (Y)
20.94 (Y)
29.50 (Y)
2.08 (Y)
56
phenytoin
143 ꢁ 1.1
103 ꢁ 1.4
117 ꢁ 0.8
86 ꢁ 1.7
a
Each value represents the mean ꢁ SEM of six rats, not significant from the
control value at p < 0.05 (Student’s t test).
b
Control group (six rats) were treated with 0.5% methyl cellulose for 15 days.
141 ꢁ 2.5 NS
98 ꢁ 1.3
Phenytoin
41.31 (Y)
a
Each value represents the mean ꢁ SEM significantly different from the control at
p < 0.05, NS denotes not significant at p < 0.05 (Student’s t test), locomotor activity
score was measured for 10 min.
indicates greater degree of confidence means F is significant at the
95% level. The low standard deviation (SD) and root-mean-squared
error (RMSE) contributes significantly to the model. Fig.1 shows the
graph of actual versus predicted activities for training and test set
molecules using this model.
b
% Change is calculated as 100ꢀ[(Yx100)/X].
The common pharmacophore generated from the best PHASE
hypothesis with aligned compound 52 is shown in Fig. 2. It showed
pharmacophoric features as red sphere for hydrogen-bond acceptor
(A) with the arrows pointing in the direction of lone pair, blue
sphere for positively charged group (P) and orange torus for
aromatic rings (R). In addition, the 3D-QSAR results are also visu-
alized as 3D plots of crucial pharmacophore regions in which the
blue cubes indicate increase and red cubes (regions) indicate
decrease in activity for specific feature in the ligand regions. Fig. 3
a–e shows the 3D-QSAR plots with specific features relating to
most active conformer overlaid with the best-generated pharma-
cophore hypothesis (compound 52) and their relation with the
biological activity of molecules.
In the electron withdrawing (includes hydrogen-bond acceptor)
plot (Fig. 3a), the blue regions at 3-nitrogen and 2-NH position of
benzothiazole ring, in the vicinity of N-CO moiety and 40 position of
5-phenyl ring favors the activity (compounds 34, 40, 46 and 52),
while electron withdrawing unfavorable red region surrounding 6
position of benzothiazole ring reduces the activity (compounds 39
and 43). In the hydrogen-bond donor plots, the blue regions at 6-
and 2-NH position of benzothiazole ring show the significance of
proton in the hydrogen bond formation with the receptor surface
(compounds 53, 54 and 56). The red regions in the vicinity of 30 and
40 position of 5-phenyl ring suggested that the substructure frag-
ments with hydrogen-bond donor in this area may reduce the
activity (compounds 36, 42 and 48, Fig. 3b). In the negative ionic
plots (Fig. 3c), blue regions at 40 position of 5-phenyl ring suggests
that increased activity may be anticipated by moderate negatively
charged substituents at 40 position (compounds 34 and 52),
whereas red region surrounding the 6 position of benzothiazole
ring disfavors the activity (compounds 40 and 46). In addition, the
[(1-acetyl-5-substituted)-2-pyrazolin-3-yl]aminobenzothiazoles
(compounds 33–56) was employed for the generation of 3D-QSAR
models with the aim that these models could provide useful phar-
macophoric information for the future efforts in the development of
more potent molecules in these series of chemical classes. The
statistically significant 3D-QSAR models were generated using 29
molecules as the training set and was validated using test set (19
compounds) (Table 1) employing PHASE, the pharmacophore
modeling tool [37]. Using PHASE, one can exhaustively identify the
spatial arrangements of functional groups that are common and
essential for the biological activity of the ligands under investigation
[38,39].
In general, the total of 101 five point hypotheses were obtained
upon the completion of scoring process and the top four high
ranking pharmacophore hypotheses were analyzed. The statistical
and partial least square (PLS) analyses results for four of the top
ranking pharmacophores (model 1–4) are summarized in Table 5.
The model 1 was consisting of two hydrogen-bond acceptor, one
positive group and two aromatic rings. Model 2 was composed of
two hydrogen-bond acceptor and three aromatic rings. The model 3
consisted of three hydrogen-bond acceptors and two aromatic
rings while model 4 possessed one hydrogen-bond acceptor, one
positive group and three aromatic rings. The model 1 exhibited
comparatively better PLS statistical qualities and excellent predic-
tion of the external test set molecules, therefore was considered for
explaining the SAR of the molecules under investigations. Model 1
showed conventional correlation coefficient (r2) of 0.9220 with four
components, test set prediction (q2) of 0.8144 and Pearson-R (rp) of
0.9114. The high value of variance ratio (F) observed for this model
indicates its statistical robustness which is further supported by
significance level of variance ratio (P). The value of P < 0.05
Table 3
Effect of selected compounds on duration of immobility time in Porsolt’s swimpool
test.
Table 5
Summary of PHASE 3D-QSAR statistical results.
Compd
Mean average immobility time (s)a
Pharmacophore
Model 1
AAPRR
Model 2
AARRR
Model 3
AAARR
Model 4
APRRR
Control (24 before)
Post-treatment (0.5 h after)
Hypotheses
40
44
51
52
53
54
56
92 ꢁ 1.2
82 ꢁ 2.9
104 ꢁ 1.8
87 ꢁ 1.5
128 ꢁ 2.4
113 ꢁ 3.1
97 ꢁ 2.2
78 ꢁ 0.9
134 ꢁ 2.4
88 ꢁ 1.7 NS
151 ꢁ 3.6
91 ꢁ 0.8 NS
136 ꢁ 1.6
177 ꢁ 3.2
163 ꢁ 2.4
145 ꢁ 1.4
PLS statistics of QSAR model
r2
0.9220
0.0585
82.4
0.9128
0.0682
76.2
0.9011
0.0843
61.3
0.9052
0.0781
64.5
SD
F
P
1.51eꢀ13
4
4.22eꢀ12
4
3.43eꢀ11
4
6.46eꢀ12
4
Number of PLS factor
External test set prediction
q2
rp
0.8144
0.9114
0.1385
0.8112
0.9105
0.1346
0.8021
0.9068
0.1312
0.8078
0.9024
0.1363
Carbamazepine
a
Each value represents the mean ꢁ SEM of six rats significantly different from the
control at p < 0.05, NS denotes not significant at p < 0.05 (Student’s t test).
RMSE

N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
153
4.5
4.25
4
b
a
4.5
4.25
4
3.75
3.5
3.25
3
3.75
3.5
3.25
3
Test set
Training set
3
3.25
3.5
3.75
4
4.25
4.5
4.75
3
3.25
3.5
3.75
4
4.25
4.5
Actual activity
Actual activity
Fig. 1. Plots of predicted versus actual activity values for the training set (a) and test set (b) molecules based on PHASE 3D-QSAR model.
positive ionic favorable blue region (Fig. 3d) observed at 6 position
of benzothiazole and near tertiary nitrogen of pyrazoline ring
defines that an increase in positive group may result in enhanced
activity (compound 33). These regions include partial positive
charges associated with hydrogen atoms bound to carbon, which
can be correlated with lipophilic interactions. The red region was
observed at 40 position of 5-phenyl ring which results in reduced
activity (compounds 33 and 38). The hydrophobic (nonpolar) plot
indicates desired/undesired hydrophobic regions around two
phenyl rings (Fig. 3e). The blue region observed in the vicinity of
NCOCH3 group and 6 position of benzothiazole ring signifies that
substitution with hydrophobic group may result in enhanced
activity (compounds 35 and 53); whereas a slight variation in the
phenyl ring results in decreased activity (compound 55). The
disfavored red region in the vicinity of 40 position of 5-phenyl ring
suggests that hydrophobic group in this area resulted in decreased
activity (compounds 53 and 56).
were synthesized successfully in convenient steps in 52–80% yields.
All compounds were tested for anticonvulsant activity to determine
ED50 values and the activity data was supported by CNS depressant
and hepatotoxicity studies. Although the most active in derivatives
in these series (compound 52) showed relatively less activity than
the phenytoin which can be overruled as the activities of compound
52 was statistically significant and can be used as a lead for further
development of more potent anticonvulsant agents. In view of this
and our ongoing efforts to develop anticonvulsant agents, it was
thought of interest to study the SAR quantitatively using these
series of molecules although the substituents are not so diverse but
they are varying significantly in the 3D space. The statistically
significant 3D-QSAR model with r2 of 0.9220 and q2 of 0.8144 was
developed using PHASE and analyzed in order to understand the
trends of these molecules for their anticonvulsant properties. The
influence of electron withdrawing, hydrogen-bond donor, negative/
positive ionic and hydrophobic groups was analyzed and discussed
using the 3D-QSAR PHASE hypotheses. We believe that the derived
3D-QSAR as well as clues for possible structural modifications will
be of interest and significance for the strategic design of more
potent molecules in the benzothiazoles as anticonvulsant agents.
Additionally, the compounds 9–32 displayed weaker anticon-
vulsant activity which might be due to the lack of positive group
(tertiary ring nitrogen) which is a part of best pharmacophore model
and variation in the position of N-CO moiety. From these findings,
one can assumed 1-acetyl-pyrazolin ring as a crucial pharmaco-
phoric group for the anticonvulsant activity of benzothiazoles.
6. Experimental protocols
5. Conclusions
6.1. Instrumentation and chemicals
In the present studies, a series of 6-substituted-[3-substituted-
prop-2-eneamido]benzothiazole 9–32 and 6-substituted-2-[(1-
acetyl-5-substituted)-2-pyrazolin-3-yl]aminobenzothiazole 33–56
All the chemicals and solvents employed in the synthesis were
supplied by Merck (Germany), Fluka (Germany) and SD Fine chem-
icals (India) and used without purification. Melting point were
determined on a digital melting point apparatus Electrothermal 1A
9200 (U.K.) and are uncorrected. All the reactions were monitored by
TLC performed on 2.0–6.0 cm aluminium sheets precoated with silica
gel 60 (HF-254, E. Merck, India). The IR spectra were recorded on
a Shimadzu FTIR 8400S spectrophotometer (Kyto, Japan) using KBr
optics. 1H NMR spectra were recorded in CDCl3 on a Varian Mercury
YH-300 MHz spectrophotometer (Palo Alto, CA, USA) and chemical
shifts (d) are given inppm relativetoTMS. Massspectrawere recorded
at 70 eV on Jeol D-300 spectrometer (Tokyo, Japan). Elemental anal-
yses were carried out using FLASH EA 1112 CHN analyzer (Thermo
Finnigan, Italy) and found within ꢁ0.4% of theoretical values.
6.2. Syntheses
Fig. 2. Common pharmacophore generated from the best PHASE hypothesis. Phar-
macophore features are red sphere for hydrogen-bond acceptors (A) with the arrows
pointing in the direction of lone pair, blue sphere for positively charged group (P) and
orange torus for aromatic rings (R). Compound 52 aligned to the pharmacophore for
which blue indicates nitrogen, yellow refers to sulphur, green for chlorine, gray indi-
cates carbon and white refers to hydrogen.
6.2.1. General synthesis of 6-substituted-[3-substituted-prop-2-enea-
mido]-benzothiazole (9–32)
To
a
solution of 6-substituted-2-acetamidobenzothiazole
(0.01 mol) in dry ethanol (50 ml), appropriate aldehydes (0.01 mol)

154
N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
Fig. 3. (a–e) PHASE 3D plots of crucial pharmacophore region based on model 1 displayed with compound 52. Positive coefficient favored areas (contributing for increase in
activity) are represented by blue cubes. Negative coefficient favored areas (contributing for decrease in activity) are represented by red cubes. (3a) Hydrogen-bond acceptor; (3b)
Hydrogen-bond donor; (3c) Negative-ionic groups; (3d) Positive-ionic groups; (3e) Hydrophobic region.
were added in the presence of NaOH solution (5 ml, 10%). The
reaction mixture was stirred at room temperature for 10–12 h. The
excess solvent was distilled off in vacuum. The remaining mixture
was poured onto ice–water, filtered and washed with water. The
residue was dried and purified by recrystallization from ethanol
(70%) to obtain 6-substituted-[3-substituted-prop-2-eneamido]-
benzothiazoles 9–32.
0.58 (ethyl acetate); IR (KBr, cmꢀ1): 3620 (OH), 3330 (NH), 1700
(C]O), 1530 (C]N); 1H NMR (CDCl3):
3.83 (s, 3H, OCH3), 6.88 (d,
J ¼ 7.9 Hz, 1H, CO–CH), 7.35 (d, J ¼ 6.5 Hz, 1H, CH–Ar), 7.62–8.14 (m,
7H, Ar–H), 9.46 (s, 1H, OH), 13.58 (s, 1H, NH); EI-MS: m/z [M þ H]þ
327; Anal. Calcd for C17H14N2O3S: C, 62.56; H, 4.32; N, 8.58; S, 9.82.
Found: C, 62.55; H, 4.33; N, 8.57; S, 9.84.
d
6.2.1.5. 3-(Furan-2-yl)-prop-2-eneamidobenzothiazole (13). Reddish-
brown crystals; Yield: 59%; mp 186–188 ꢂC; Rf 0.51 (ethyl acetate);
IR (KBr, cmꢀ1): 3350 (NH), 1710 (C]O), 1540 (C]N); 1H NMR
6.2.1.1. 3-Phenyl-prop-2-eneamidobenzothiazole (9). White crys-
tals; Yield: 79%; mp 149–151 ꢂC; Rf 0.78 (ethyl acetate); IR (KBr,
cmꢀ1): 3320 (NH), 1710 (C]O), 1540 (C]N); 1H NMR (CDCl3):
(CDCl3):
d
6.87 (d, J ¼ 8.1 Hz, 1H, CO–CH), 7.38 (d, J ¼ 6.9 Hz, 1H,
d
6.85 (d, J ¼ 7.6 Hz, 1H, CO–CH), 7.42 (d, J ¼ 6.8 Hz, 1H, CH–Ar),
CH–Ar), 7.74–8.21 (m, 7H, Ar–H), 13.61 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 271; Anal. Calcd for C14H10N2O2S: C, 62.21; H, 3.73; N,
10.36; S, 11.86. Found: C, 62.22; H, 3.72; N, 10.37; S, 11.84.
7.58–8.14 (m, 9H, Ar–H), 13.58 (s, 1H, NH); EI-MS: m/z [M þ H]þ
281; Anal. Calcd for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99; S, 11.44.
Found: C, 68.53; H, 4.33; N, 9.98; S, 11.46.
6.2.1.6. 3-(4-Methoxyphenyl)-prop-2-eneamidobenzothiazole (14).
White crystals; Yield: 52%; mp 243–245 ꢂC; Rf 0.74 (ethyl acetate);
IR (KBr, cmꢀ1): 3340 (NH), 1690 (C]O), 1550 (C]N); 1H NMR
6.2.1.2. 3-(4-Chlorophenyl)-prop-2-eneamidobenzothiazole (10). White
crystals; Yield: 73%; mp 162–164 ꢂC; Rf 0.62 (ethyl acetate); IR (KBr,
cmꢀ1): 3330 (NH), 1720 (C]O), 1550 (C]N); 1H NMR (CDCl3):
(CDCl3):
d
3.79 (s, 3H, OCH3), 6.87 (d, J ¼ 8.3 Hz, 1H, CO–CH), 7.37 (d,
d
6.86 (d, J ¼ 7.8 Hz, 1H, CO–CH), 7.43 (d, J ¼ 6.8 Hz, 1H, CH–Ar),
J ¼ 7.2 Hz,1H, CH–Ar), 7.53–8.18 (m, 8H, Ar–H),13.63 (s,1H, NH); EI-
MS: m/z [M þ H]þ 280; Anal. Calcd for C16H11N2OS: C, 68.80; H,
3.97; N, 10.03; S, 11.48. Found: C, 68.81; H, 3.97; N, 10.05; S, 11.46.
7.62–8.22 (m, 8H, Ar–H), 13.62 (s, 1H, NH); EI-MS: m/z [M þ H]þ
315; Anal. Calcd for C16H11ClN2OS: C, 61.05; H, 3.52; N, 8.90; S,10.19.
Found: C, 61.06; H, 3.53; N, 8.89; S, 10.20.
6.2.1.7. 6-Chloro-[3-phenyl-prop-2-eneamido]benzothiazole (15). Pale
yellow crystals; Yield: 65%; mp 166–168 ꢂC; Rf 0.64 (ethyl acetate);
IR (KBr, cmꢀ1): 3360 (NH), 1710 (C]O), 1530 (C]N); 1H NMR
6.2.1.3. 3-(4-Hydroxyphenyl)-prop-2-eneamidobenzothiazole (11). Pale
white crystals; Yield: 59%; mp 261–262 ꢂC; Rf 0.61 (ethyl acetate); IR
(KBr, cmꢀ1): 3485 (OH), 3350 (NH), 1710 (C]O), 1560 (C]N); 1H
(CDCl3):
d
6.89 (d, J ¼ 7.8 Hz, 1H, CO–CH), 7.37 (d, J ¼ 6.9 Hz, 1H, CH–
NMR (CDCl3):
d
6.88 (d, J ¼ 8.3 Hz,1H, CO–CH), 7.39 (d, J ¼ 6.6 Hz,1H,
Ar), 7.56–8.13 (m, 8H, Ar–H), 13.62 (s, 1H, NH); EI-MS: m/z [M þ H]þ
316; Anal. Calcd for C16H11ClN2OS: C, 61.05; H, 3.52; N, 8.90; S,10.19.
Found: C, 61.07; H, 3.52; N, 8.89; S, 10.20.
CH–Ar), 7.61–8.09 (m, 8H, Ar–H), 9.44 (s, 1H, OH), 13.64 (s, 1H, NH);
EI-MS: m/z [M þ H]þ 297; Anal. Calcd for C16H12N2O2S: C, 64.85; H,
4.09; N, 9.45; S, 10.82. Found: C, 64.84; H, 4.10; N, 9.44; S, 10.83.
6.2.1.8. 6-Chloro-[3-(4-chlorophenyl)-prop-2-eneamido]benzothia-
zole (16). White-creamish crystals; Yield: 61%; mp 188–190 ꢂC; Rf
0.62 (ethyl acetate); IR (KBr, cmꢀ1): 3350 (NH), 1700 (C]O), 1540
6.2.1.4. 3-(3-Hydroxy-4-methoxyphenyl)-prop-2-eneamidobenzo-
thiazole (12). Muddy white crystals; Yield: 64%; mp 218–220 ꢂC; Rf

N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
155
(C]N); 1H NMR (CDCl3):
(d, J ¼ 7.2 Hz, 1H, CH–Ar), 7.57–8.18 (m, 7H, Ar–H), 13.63 (s, 1H, NH);
EI-MS: m/z [M þ H]þ 350; Anal. Calcd for C16H10Cl2N2OS: C, 55.03;
H, 2.89; N, 8.02; S, 9.18. Found: C, 55.05; H, 2.88; N, 8.03; S, 9.17.
d
6.87 (d, J ¼ 8.1 Hz, 1H, CO–CH), 7.35
6.2.1.16. 6-Nitro-[3-(3-hydroxy-4-methoxyphenyl)-prop-2-eneamido]-
benzothiazole (24). Yellow solid; Yield: 71%; mp 186–188 ꢂC; Rf 0.68
(ethyl acetate); IR (KBr, cmꢀ1): 3540 (OH), 3345 (NH), 1723 (C]O),
1568 (C]N); 1H NMR (CDCl3):
d 3.88 (s, 3H, OCH3), 6.58
(d, J ¼ 7.9 Hz, 1H, CO–CH), 7.22 (d, J ¼ 6.7 Hz, 1H, CH–Ar), 7.66–8.12
(m, 7H, Ar–H), 9.87 (s, 1H, OH), 12.74 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 373; Anal. Calcd for C17H13N3O5S: C, 54.98; H, 3.53; N,
11.31; S, 8.64. Found: C, 54.97; H, 3.53; N, 11.30; S, 8.64.
6.2.1.9. 6-Chloro-[3-(4-hydroxyphenyl)-prop-2-eneamido]benzothia-
zole (17). Pale brown crystals; Yield: 69%; mp 262–264 ꢂC; Rf 0.57
(ethyl acetate); IR (KBr, cmꢀ1): 3555 (OH), 3340 (NH), 1700 (C]O),
1545 (C]N); 1H NMR (CDCl3):
d
6.88 (d, J ¼ 8.5 Hz,1H, CO–CH), 7.34
(d, J ¼ 7.3 Hz, 1H, CH–Ar), 7.49–8.13 (m, 7H, Ar–H), 9.44 (s, 1H, OH),
13.62 (s, 1H, NH); EI-MS: m/z [M þ H]þ 331; Anal. Calcd for
C16H11ClN2O2S: C, 58.09; H, 3.35; N, 8.47; S, 9.69. Found: C, 58.08; H,
3.36; N, 8.48; S, 9.68.
6.2.1.17. 6-Nitro-[3-(furan-2-yl)-prop-2-eneamido]benzothiazole
(25). Yellow solid; Yield: 52%; mp 220–222 ꢂC; Rf 0.53 (ethyl
acetate); IR (KBr, cmꢀ1): 3298 (NH), 1708 (C]O), 1547 (C]N); 1H
NMR (CDCl3):
d
6.83 (d, J ¼ 8.1 Hz, 1H, CO–CH), 7.36 (d, J ¼ 7.2 Hz,
1H, CH–Ar), 7.72–8.38 (m, 6H, Ar–H), 13.08 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 317; Anal. Calcd for C14H9N3O4S: C, 53.33; H, 2.88; N,
13.33; S, 10.17. Found: C, 53.31; H, 2.87; N, 13.35; S, 10.19.
6.2.1.10. 6-Chloro-[3-(3-hydroxy-4-methoxyphenyl)-prop-2-eneami-
do]benzothiazole (18). Muddy crystalline solid; Yield: 58%; mp
217–219 ꢂC; Rf 0.54 (ethyl acetate); IR (KBr, cmꢀ1): 3520 (OH),
3340 (NH), 1710 (C]O), 1530 (C]N); 1H NMR (CDCl3):
d
3.83
6.2.1.18. 6-Nitro-[3-(4-methoxyphenyl)-prop-2-eneamido]benzo-
thiazole (26). Yellow solid; Yield: 64%; mp 192–194 ꢂC; Rf 0.66 (ethyl
acetate); IR (KBr, cmꢀ1): 3322 (NH), 1714 (C]O), 1556 (C]N); 1H
(s, 3H, OCH3), 6.86 (d, J ¼ 8.3 Hz, 1H, CO–CH), 7.38 (d, J ¼ 7.1 Hz,
1H, CH–Ar), 7.53–8.18 (m, 6H, Ar–H), 9.46 (s, 1H, OH), 13.65 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 362; Anal. Calcd for C17H13ClN2O3S:
C, 56.59; H, 3.63; N, 7.76; S, 8.89. Found: C, 56.58; H, 3.62; N, 7.75;
S, 8.90.
NMR (CDCl3):
d
3.83 (s, 3H, OCH3), 6.64 (d, J ¼ 8.1 Hz,1H, CO–CH), 7.14
(d, J ¼ 6.9 Hz,1H, CH–Ar), 7.58–8.04 (m, 7H, Ar–H), 12.42 (s, 1H, NH);
EI-MS: m/z [M þ H]þ 357; Anal. Calcd for C17H13N3O4S: C, 57.46; H,
3.69; N, 11.82; S, 9.02. Found: C, 57.44; H, 3.68; N, 11.83; S, 9.03.
6.2.1.11. 6-Chloro-[3-(furan-2-yl)-prop-2-eneamido]benzothiazole
(19). Yellowish-brown solid; Yield: 67%; mp 238–240 ꢂC; Rf 0.48
(ethyl acetate); IR (KBr, cmꢀ1): 3350 (NH), 1730 (C]O), 1560
6.2.1.19. 6-Methyl-[3-phenyl-prop-2-eneamido]benzothiazole
(27). Pale brown crystals; Yield: 73%; mp 177–179 ꢂC; Rf 0.55 (ethyl
acetate); IR (KBr, cmꢀ1): 3318 (NH), 1694 (C]O), 1572 (C]N); 1H
(C]N); 1H NMR (CDCl3):
d
6.78 (d, J ¼ 8.2 Hz, 1H, CO–CH), 7.42
(d, J ¼ 6.8 Hz,1H, CH–Ar), 7.68–8.23 (m, 6H, Ar–H),13.13 (s,1H, NH);
EI-MS: m/z [M þ H]þ 306; Anal. Calcd for C14H9ClN2O2S: C, 55.18; H,
2.98; N, 9.19; S, 10.52. Found: C, 55.20; H, 2.98; N, 9.18; S, 10.51.
NMR (CDCl3):
d
2.48 (s, 3H, Ar–CH3), 6.48 (d, J ¼ 8.1 Hz, 1H, CO–CH),
7.23 (d, J ¼ 6.5 Hz, 1H, CH–Ar), 7.61–8.35 (m, 8H, Ar–H), 12.73 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 296; Anal. Calcd for C17H14N2OS: C, 69.36;
H, 4.79; N, 9.52; S, 10.89. Found: C, 69.37; H, 4.77; N, 9.53; S, 10.88.
6.2.1.12. 6-Chloro-[3-(4-methoxyphenyl)-prop-2-eneamido]benzo-
thiazole (20). Pale yellow crystals; Yield: 62%; mp 243–245 ꢂC; Rf
0.62 (ethyl acetate); IR (KBr, cmꢀ1): 3350 (NH), 1690 (C]O), 1540
6.2.1.20. 6-Methyl-[3-(4-chlorophenyl)-prop-2-eneamido]benzothia-
zole (28). Pale orange solid; Yield: 78%; mp 188–190 ꢂC; Rf 0.63
(ethyl acetate); IR (KBr, cmꢀ1): 3343 (NH), 1708 (C]O),1547 (C]N);
(C]N); 1H NMR (CDCl3):
d
3.81 (s, 3H, OCH3), 6.87 (d, J ¼ 7.8 Hz, 1H,
CO–CH), 7.37 (d, J ¼ 6.7 Hz, 1H, CH–Ar), 7.51–8.16 (m, 7H, Ar–H),
13.64 (s, 1H, NH); EI-MS: m/z [M þ H]þ 346; Anal. Calcd for
C17H13ClN2O2S: C, 59.21; H, 3.80; N, 8.12; S, 9.30. Found: C, 59.22; H,
3.81; N, 8.11; S, 9.31.
1H NMR (CDCl3):
d
2.39 (s, 3H, Ar–CH3), 6.57 (d, J ¼ 8.4 Hz, 1H, CO–
CH), 7.38 (d, J ¼ 6.8 Hz, 1H, CH–Ar), 7.52–8.18 (m, 7H, Ar–H), 13.14 (s,
1H, NH); EI-MS: m/z [M þ H]þ 330; Anal. Calcd for C17H13ClN2OS: C,
62.10; H, 3.98; N, 8.52;S, 9.75. Found: C, 62.12; H, 3.97; N, 8.51; S, 9.76.
6.2.1.13. 6-Nitro-[3-phenyl-prop-2-eneamido]benzothiazole
(21). Yellow solid; Yield: 52%; mp 263–265 ꢂC; Rf 0.48 (ethyl
acetate); IR (KBr, cmꢀ1): 3307 (NH), 1695 (C]O), 1569 (C]N); 1H
6.2.1.21. 6-Methyl-[3-(4-hydroxyphenyl)-prop-2-eneamido]benzo-
thiazole (29). Pale white crystals; Yield: 56%; mp 221–223 ꢂC; Rf
0.57 (ethyl acetate); IR (KBr, cmꢀ1): 3545 (OH), 3304 (NH), 1693
NMR (CDCl3):
d
6.75 (d, J ¼ 8.2 Hz, 1H, CO–CH), 7.34 (d, J ¼ 6.8 Hz,
(C]O), 1564 (C]N); 1H NMR (CDCl3):
d 2.44 (s, 3H, Ar–CH3), 6.73
1H, CH–Ar), 7.65–8.14 (m, 8H, Ar–H), 12.86 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 327; Anal. Calcd for C16H11N3O3S: C, 59.07; H, 3.41; N,
12.92; S, 9.86. Found: C, 59.08; H, 3.40; N, 12.93; S, 9.87.
(d, J ¼ 8.1 Hz, 1H, CO–CH), 7.31 (d, J ¼ 6.4 Hz, 1H, CH–Ar), 7.63–8.37
(m, 7H, Ar–H), 9.36 (s, 1H, OH), 12.52 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 312; Anal. Calcd for C17H14N2O2S: C, 65.79; H, 4.55; N,
9.03; S, 10.33. Found: C, 65.81; H, 4.54; N, 9.02; S, 10.34.
6.2.1.14. 6-Nitro-[3-(4-chlorophenyl)-prop-2-eneamido]benzothia-
zole (22). Yellow solid; Yield: 63%; mp 241–243 ꢂC; Rf 0.51 (ethyl
acetate); IR (KBr, cmꢀ1): 3324 (NH), 1704 (C]O), 1553 (C]N); 1H
6.2.1.22. 6-Methyl-[3-(3-hydroxy-4-methoxyphenyl)-prop-2-enea-
mido]benzothiazole (30). Pale brown solid; Yield: 67%; mp
198–200 ꢂC; Rf 0.64 (ethyl acetate); IR (KBr, cmꢀ1): 3580 (OH), 3352
NMR (CDCl3):
d
6.62 (d, J ¼ 7.9 Hz, 1H, CO–CH), 7.21 (d, J ¼ 6.7 Hz,
1H, CH–Ar), 7.72–8.27 (m, 7H, Ar–H), 12.72 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 361; Anal. Calcd for C16H10ClN3O3S: C, 53.41; H, 2.80; N,
11.68; S, 8.91. Found: C, 53.43; H, 2.81; N, 11.67; S, 8.90.
(NH), 1718 (C]O), 1573 (C]N); 1H NMR (CDCl3):
d 2.36 (s, 3H,
Ar–CH3), 3.82 (s, 3H, Ar–OCH3), 6.53 (d, J ¼ 7.7 Hz, 1H, CO–CH), 7.17
(d, J ¼ 6.4 Hz, 1H, CH–Ar), 7.62–8.27 (m, 6H, Ar–H), 9.67 (s, 1H, OH),
13.06 (s, 1H, NH); EI-MS: m/z [M þ H]þ 342; Anal. Calcd for
C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23; S, 9.42. Found: C, 63.53; H,
4.73; N, 8.24; S, 9.41.
6.2.1.15. 6-Nitro-[3-(4-hydroxyphenyl)-prop-2-eneamido]benzothia-
zole (23). Yellow solid; Yield: 58%; mp 207–209 ꢂC; Rf 0.62 (ethyl
acetate); IR (KBr, cmꢀ1): 3610 (OH), 3308 (NH), 1723 (C]O), 1542
(C]N); 1H NMR (CDCl3):
d
6.48 (d, J ¼ 8.3 Hz, 1H, CO–CH), 7.36 (d,
6.2.1.23. 6-Methyl-[3-(furan-2-yl)-prop-2-eneamido]benzothiazole
(31). Pale reddish-brown solid; Yield: 56%; mp 236–238 ꢂC; Rf 0.67
(ethyl acetate); IR (KBr, cmꢀ1): 3324 (NH), 1704 (C]O), 1552
J ¼ 7.1 Hz, 1H, CH–Ar), 7.68–8.12 (m, 7H, Ar–H), 9.71 (s, 1H, OH),
12.88 (s, 1H, NH); EI-MS: m/z [M þ H]þ 343; Anal. Calcd for
C16H11N3O4S: C, 56.30; H, 3.25; N, 12.31; S, 9.39. Found: C, 56.32; H,
3.26; N, 12.30; S, 9.38.
(C]N); 1H NMR (CDCl3):
1H, CO–CH), 7.26 (d, J ¼ 6.7 Hz, 1H, CH–Ar), 7.78–8.32 (m, 6H,
d
2.43 (s, 3H, Ar–CH3), 6.57 (d, J ¼ 7.5 Hz,

156
N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
Ar–H), 12.56 (s, 1H, NH); EI-MS: m/z [M þ H]þ 286; Anal. Calcd for
C15H12N2O2S: C, 63.36; H, 4.25; N, 9.85; S, 11.28. Found: C, 63.37; H,
4.24; N, 9.85; S, 11.26.
m/z [M þ H]þ 384; Anal. Calcd for C19H18N4O3S: C, 59.67; H, 4.74; N,
14.65; S, 8.38. Found: C, 59.61; H, 4.78; N, 14.62; S, 8.34.
6.2.2.5. 2-[(1-Acetyl-5-(furan-2-yl))-2-pyrazolin-3-yl]aminobenzo-
thiazole (37). Pale brown crystals; Yield: 78%; mp 244–246 ꢂC; Rf
0.57 (ethyl acetate); IR (KBr, cmꢀ1): 3290 (NH), 1710 (C]O), 1564
6.2.1.24. 6-Methyl-[3-(4-methoxyphenyl)-prop-2-eneamido]benzo-
thiazole (32). Pale yellow crystalline solid; Yield: 52%; mp 216–
218 ꢂC; Rf 0.61 (ethyl acetate); IR (KBr, cmꢀ1): 3298 (NH), 1715
(C]N), 1480 (N–N), 1125 (C–N); 1H NMR (CDCl3):
d 2.21 (s, 3H, CO–
(C]O),1568 (C]N); 1H NMR (CDCl3):
d
2.53 (s, 3H, Ar–CH3), 3.76 (s,
CH3), 2.98 (dd, 1H, HA, JAB ¼ 17.92 Hz, JAX ¼ 6.33 Hz), 3.57 (dd, 1H,
HB, JAB ¼ 16.68 Hz, JBX ¼ 10.24 Hz), 5.72 (dd, 1H, HX, JAX ¼ 6.75 Hz,
JBX ¼ 9.68 Hz), 7.64–8.09 (m, 7H, Ar–H), 9.10 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 328; Anal. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N,
17.17; S, 9.82. Found: C, 58.81; H, 4.28; N, 17.25; S, 9.84.
3H, Ar–OCH3), 6.64 (d, J ¼ 8.2 Hz, 1H, CO–CH), 7.26 (d, J ¼ 7.3 Hz, 1H,
CH–Ar), 7.58–8.13 (m, 7H, Ar–H), 12.86 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 326; Anal. Calcd for C18H16N2O2S: C, 66.64; H, 4.97; N,
8.64; S, 9.88. Found: C, 66.65; H, 4.96; N, 8.65; S, 9.86.
6.2.2. General synthesis of 6-substituted-2-[(1-acetyl-5-
6.2.2.6. 2-[(1-Acetyl-5-(4-methoxyphenyl))-2-pyrazolin-3-yl]amino-
benzothiazole (38). White crystals; Yield: 57%; mp 211–213 ꢂC; Rf
0.38 (ethyl acetate); IR (KBr, cmꢀ1): 3340 (NH), 1690 (C]O), 1570
substituted)-2-pyrazolin-3-yl]aminobenzothiazole (33–56)
To a solution of appropriate 6-substituted-[3-substituted-prop-
2-eneamido]-benzothiazoles 9–32 (0.01 mol) in dry ethanol
(40 ml), hydrazine hydrate (99%, 0.02 mol) and few drops of glacial
acetic acid were added. The reaction mixture was refluxed for 6–
8 h. The excess solvent was distilled off in vacuum and the
remaining mixture was poured onto ice–water. The residue was
filtered, washed with water dried and recrystallized from ethanol
(70%) to obtain 6-substituted-2-[(1-acetyl-5-substituted)-2-pyr-
azolin-3-yl]aminobenzothiazole 33–56.
(C]N), 1510 (N–N), 1095 (C–N); 1H NMR (CDCl3):
d 2.14 (s, 3H, CO–
CH3), 3.14 (dd, 1H, HA, JAB ¼ 16.85 Hz, JAX ¼ 6.43 Hz), 3.74 (s, 3H,
OCH3), 3.92 (dd, 1H, HB, JAB ¼ 17.46 Hz, JBX ¼ 9.51 Hz), 5.85 (dd, 1H,
HX, JAX ¼ 7.12 Hz, JBX ¼ 11.56 Hz), 7.76–7.96 (m, 8H, Ar–H), 8.72 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 368; Anal. Calcd for C19H18N4O2S: C, 62.28;
H, 4.95; N, 15.29; S, 8.75. Found: C, 62.22; H, 4.99; N, 15.34; S, 8.74.
6.2.2.7. 6-Chloro-2-[(1-acetyl-5-phenyl)-2-pyrazolin-3-yl]amino-
benzothiazole (39). Pale yellow solid; Yield: 63%; mp 242–244 ꢂC;
Rf 0.52 (ethyl acetate); IR (KBr, cmꢀ1): 3315 (NH), 1710 (C]O), 1545
6.2.2.1. 2-[(1-Acetyl-5-phenyl)-2-pyrazolin-3-yl]aminobenzothiazole
(33). White crystals; Yield: 73%; mp 182–184 ꢂC; Rf 0.57 (ethyl
acetate); IR (KBr, cmꢀ1): 3356 (NH), 1716 (C]O), 1552 (C]N), 1485
(C]N), 1490 (N–N), 1110 (C–N); 1H NMR (CDCl3):
d 2.11 (s, 3H, CO–
CH3), 3.14 (dd,1H, HA, JAB ¼ 17.02 Hz, JAX ¼ 6.32 Hz), 3.79 (dd,1H, HB,
JAB ¼ 16.96 Hz, JBX ¼ 10.53 Hz), 5.08 (dd, 1H, HX, JAX ¼ 6.44 Hz,
JBX ¼ 11.31 Hz), 7.45–7.88 (m, 8H, Ar–H), 8.65 (s, 1H, NH); EI-MS: m/
z [M þ H]þ 371; Anal. Calcd for C18H15ClN4OS: C, 58.30; H, 4.08; N,
15.11; S, 8.65. Found: C, 58.22; H, 4.14; N, 15.04; S, 8.69.
(N–N), 1120 (C–N); 1H NMR (CDCl3):
d 2.08 (s, 3H, CO–CH3), 3.12
(dd, 1H, HA, JAB ¼ 16.84 Hz, JAX ¼ 6.42 Hz), 3.38 (dd, 1H, HB,
JAB ¼ 16.84 Hz, JBX ¼ 10.23 Hz), 5.42 (dd, 1H, HX, JAX ¼ 7.86 Hz,
JBX ¼ 11.4 Hz), 7.56–7.86 (m, 9H, Ar–H), 8.80 (s, 1H, NH); EI-MS: m/z
[M þ H]þ 317; Anal. Calcd for C18H16N4OS: C, 64.26; H, 4.79; N,
16.65; S, 9.53. Found: C, 64.22; H, 4.81; N, 16.62; S, 9.55.
6.2.2.8. 6-Chloro-2-[(1-acetyl-5-(4-chlorophenyl))-2-pyrazolin-3-yl]-
aminobenzothiazole (40). White crystals; Yield: 73%; mp
287–289 ꢂC; Rf 0.43 (ethyl acetate); IR (KBr, cmꢀ1): 3367 (NH), 1685
(C]O), 1560 (C]N), 1510 (N–N), 1090 (C–N); 1H NMR (CDCl3):
6.2.2.2. 2-[(1-Acetyl-5-(4-chlorophenyl))-2-pyrazolin-3-yl]amino-
benzothiazole (34). White crystals; Yield: 59%; mp 196–198 ꢂC; Rf
0.68 (ethyl acetate); IR (KBr, cmꢀ1): 3342 (NH), 1705 (C]O),
d
2.20 (s, 3H, CO–CH3), 2.96 (dd, 1H, HA, JAB ¼ 16.24 Hz, JAX ¼ 7.4 Hz),
1562 (C]N), 1497 (N–N), 1110 (C–N); 1H NMR (CDCl3):
d
2.15 (s,
3.82 (dd, 1H, HB, JAB ¼ 16.45 Hz, JBX ¼ 9.63 Hz), 5.17 (dd, 1H, HX,
JAX ¼ 7.43 Hz, JBX ¼ 10.23 Hz), 7.78–8.30 (m, 7H, Ar–H), 8.87 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 407; Anal. Calcd for C18H14Cl2N4OS: C,
53.34; H, 3.48; N, 13.82; S, 7.91. Found: C, 53.26; H, 3.43; N, 13.88; S,
7.94.
3H, CO–CH3), 3.08 (dd, 1H, HA, JAB ¼ 16.26 Hz, JAX ¼ 6.12 Hz), 3.95
(dd, 1H, HB, JAB ¼ 16.34 Hz, JBX ¼ 9.37 Hz), 5.06 (dd, 1H, HX,
JAX ¼ 6.4 Hz, JBX ¼ 10.59 Hz), 7.76–8.10 (m, 8H, Ar–H), 8.56 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 372; Anal. Calcd for C18H15ClN4OS: C,
58.30; H, 4.08; N, 15.11; S, 8.65. Found: C, 58.33; H, 4.11; N,
15.08; S, 8.63.
6.2.2.9. 6-Chloro-2-[(1-acetyl-5-(4-hydroxyphenyl))-2-pyrazolin-3-
yl]aminobenzothiazole (41). Pale brown crystals; Yield: 57%; mp
224–226 ꢂC; Rf 0.53 (ethyl acetate); IR (KBr, cmꢀ1): 3540 (OH), 3290
(NH), 1710 (C]O), 1560 (C]N), 1490 (N–N), 1110 (C–N); 1H NMR
6.2.2.3. 2-[(1-Acetyl-5-(4-hydroxyphenyl))-2-pyrazolin-3-yl]amino-
benzothiazole (35). White crystals; Yield: 63%; mp 219–221 ꢂC; Rf
0.54 (ethyl acetate); IR (KBr, cmꢀ1): 3540 (OH), 3364 (NH), 1695
(C]O), 1570 (C]N), 1497 (N–N), 1088 (C–N); 1H NMR (CDCl3):
(CDCl3):
d
2.22 (s, 3H, CO–CH3), 3.09 (dd, 1H, HA, JAB ¼ 17.86 Hz,
JAX ¼ 6.32 Hz), 3.54 (dd, 1H, HB, JAB ¼ 17.21 Hz, JBX ¼ 9.65 Hz), 5.28
(dd, 1H, HX, JAX ¼ 6.88 Hz, JBX ¼ 10.18 Hz), 7.40–7.82 (m, 7H, Ar–H),
8.51 (s, 1H, NH), 10.11 (s, 1H, OH); EI-MS: m/z [M þ H]þ 388; Anal.
Calcd for C18H15ClN4O2S: C, 55.88; H, 3.91; N, 14.48; S, 8.29. Found:
C, 55.76; H, 3.88; N, 14.52; S, 8.34.
d
2.10 (s, 3H, CO–CH3), 2.95 (dd, 1H, HA, JAB ¼ 16.67 Hz, JAX ¼ 7.9 Hz),
3.36 (dd, 1H, HB, JAB ¼ 16.85 Hz, JBX ¼ 11.16 Hz), 5.54 (dd, 1H, HX,
JAX ¼ 7.24 Hz, JBX ¼ 11.07 Hz), 7.46–7.92 (m, 8H, Ar–H), 8.76 (s, 1H,
NH), 9.87 (s, 1H, OH); EI-MS: m/z [M þ H]þ 354; Anal. Calcd for
C18H16N4O2S: C, 61.35; H, 4.58; N, 15.90; S, 9.10. Found: C, 61.32; H,
4.54; N, 15.93; S, 9.11.
6.2.2.10. 6-Chloro-2-[(1-acetyl-5-(3-hydroxy4-methoxyphenyl))-
2-pyrazolin-3-yl]-aminobenzothiazole (42). Pale brown crystals;
Yield: 65%; mp 212–214 ꢂC; Rf 0.71 (ethyl acetate); IR (KBr, cmꢀ1):
3630 (OH), 3385 (NH), 1680 (C]O), 1575 (C]N), 1495 (N–N), 1120
6.2.2.4. 2-[(1-Acetyl-5-(3-hydroxy-4-methoxyphenyl))-2-pyrazolin-
3-yl]aminobenzothiazole (36). Brown crystals; Yield: 72%; mp
197–199 ꢂC; Rf 0.34 (ethyl acetate); IR (KBr, cmꢀ1): 3620 (OH), 3323
(NH), 1668 (C]O), 1552 (C]N), 1492 (N–N), 1105 (C–N); 1H NMR
(C–N); 1H NMR (CDCl3):
d 2.15 (s, 3H, CO–CH3), 3.12 (dd, 1H, HA,
JAB ¼ 16.65 Hz, JAX ¼ 7.4 Hz), 3.37 (dd, 1H, HB, JAB ¼ 16.82 Hz,
JBX ¼ 11.17 Hz), 3.80 (s, 3H, OCH3), 5.53 (dd, 1H, HX, JAX ¼ 7.24 Hz,
JBX ¼ 11.16 Hz), 7.63–8.12 (m, 6H, Ar–H), 8.76 (s, 1H, NH), 10.18 (s, 1H,
OH); EI-MS: m/z[M þ H]þ 418;Anal. CalcdforC19H17ClN4O3S:C,54.74;
H, 4.11; N, 13.44; S, 7.69. Found: C, 54.67; H, 4.07; N, 13.42; S, 7.75.
(CDCl3):
d
2.15 (s, 3H, CO–CH3), 3.05 (dd, 1H, HA, JAB ¼ 17.22 Hz,
JAX ¼ 6.53 Hz), 3.48 (dd, 1H, HB, JAB ¼ 17.76 Hz, JBX ¼ 10.34 Hz), 3.84
(s, 3H, Ar–OCH3), 5.54 (dd, 1H, HX, JAX ¼ 6.98 Hz, JBX ¼ 11.12 Hz),
7.82–8.23 (m, 8H, Ar–H), 8.85 (s, 1H, NH), 9.46 (s, 1H, OH); EI-MS:

N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
157
6.2.2.11. 6-Chloro-2-[(1-acetyl-5-(furan-2-yl))-2-pyrazolin-3-yl]a-
minobenzothiazole (43). Pale brown solid; Yield: 61%; mp 227–
229 ꢂC; Rf 0.56 (ethyl acetate); IR (KBr, cmꢀ1): 3340 (NH), 1670
(C]O), 1550 (C]N), 1482 (N–N), 1090 (C–N); 1H NMR (CDCl3):
6.2.2.17. 6-Nitro-2-[(1-acetyl-5-(furan-2-yl))-2-pyrazolin-3-yl]amino-
benzothiazole (49). Yellow solid; Yield: 57%; mp 281–283 ꢂC; Rf
0.43 (ethyl acetate); IR (KBr, cmꢀ1): 3368 (NH), 1710 (C]O), 1562
(C]N), 1496 (N–N), 1112 (C–N); 1H NMR (CDCl3):
d 2.16 (s, 3H,
d
2.07 (s, 3H, CO–CH3), 3.11 (dd, 1H, HA, JAB ¼ 16.05 Hz,
CO–CH3), 3.14 (dd, 1H, HA, JAB ¼ 17.64 Hz, JAX ¼ 6.53 Hz), 3.82 (dd,
1H, HB, JAB ¼ 17.32 Hz, JBX ¼ 10.16 Hz), 5.12 (dd, 1H, HX,
JAX ¼ 6.87 Hz, JBX ¼ 10.18 Hz), 7.48–7.92 (m, 6H, Ar–H), 8.55 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 373; Anal. Calcd for C16H13N5O4S: C,
51.75; H, 3.53; N, 18.86; S, 8.63. Found: C, 51.64; H, 3.56; N, 18.92;
S, 8.60.
JAX ¼ 7.28 Hz), 3.82 (dd, 1H, HB, JAB ¼ 16.17 Hz, JBX ¼ 11.19 Hz), 5.24
(dd, 1H, HX, JAX ¼ 7.54 Hz, JBX ¼ 11.24 Hz), 7.46–7.98 (m, 6H, Ar–H),
9.11 (s, 1H, NH); EI-MS: m/z [M þ H]þ 362; Anal. Calcd for
C16H13ClN4O2S: C, 53.26; H, 3.63; N, 15.53; S, 8.89. Found: C, 53.18;
H, 3.54; N, 15.60; S, 8.93.
6.2.2.12. 6-Chloro-2-[(1-acetyl-5-(4-methoxyphenyl))-2-pyrazolin-3-
yl]aminobenzothiazole (44). Pale white crystals; Yield: 72%; mp
192–194 ꢂC; Rf 0.66 (ethyl acetate); IR (KBr, cmꢀ1): 3323 (NH), 1710
(C]O), 1565 (C]N), 1510 (N–N), 1085 (C–N); 1H NMR (CDCl3):
6.2.2.18. 6-Nitro-2-[(1-acetyl-5-(4-methoxyphenyl))-2-pyrazolin-3-yl]-
aminobenzothiazole (50). Yellow solid; Yield: 76%; mp 244–246 ꢂC;
Rf 0.61 (ethyl acetate); IR (KBr, cmꢀ1): 3355 (NH), 1690 (C]O), 1546
(C]N), 1510 (N–N), 1105 (C–N); 1H NMR (CDCl3):
d 2.08 (s, 3H, CO–
d
2.24 (s, 3H, CO–CH3), 3.12 (dd, 1H, HA, JAB ¼ 17.42 Hz,
CH3), 2.97 (dd, 1H, HA, JAB ¼ 16.62 Hz, JAX ¼ 7.8 Hz), 3.32 (dd, 1H, HB,
JAB ¼ 16.85 Hz, JBX ¼ 11.17 Hz), 3.70 (s, 1H, OCH3), 5.51 (dd, 1H, HX,
JAX ¼ 7.27 Hz, JBX ¼ 11.09 Hz), 7.52–7.97 (m, 7H, Ar–H), 9.10 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 413; Anal. Calcd for C19H17N5O4S: C,
55.47; H, 4.16; N, 17.02; S, 7.79. Found: C, 55.41; H, 4.13; N, 17.05; S,
7.81.
JAX ¼ 6.44 Hz), 3.71 (dd, 1H, HB, JAB ¼ 17.56 Hz, JBX ¼ 11.5 Hz), 3.88 (s,
3H, OCH3), 5.11 (dd, 1H, HX, JAX ¼ 7.92 Hz, JBX ¼ 9.45 Hz), 7.67–8.21
(m, 7H, Ar–H), 9.05 (s, 1H, NH); EI-MS: m/z [M þ H]þ 402; Anal.
Calcd for C19H17ClN4O2S: C, 56.93; H, 4.27; N, 13.98; S, 8.00. Found:
C, 56.81; H, 4.31; N, 13.92; S, 7.98.
6.2.2.13. 6-Nitro-2-[(1-acetyl-5-phenyl)-2-pyrazolin-3-yl]amino-
benzothiazole (45). Yellow solid; Yield: 63%; mp 210–212 ꢂC; Rf
0.45 (ethyl acetate); IR (KBr, cmꢀ1): 3293 (NH), 1670 (C]O), 1560
6.2.2.19. 6-Methyl-2-[(1-acetyl-5-phenyl)-2-pyrazolin-3-yl]amino-
benzothiazole (51). White crystals; Yield: 61%; mp 204–206 ꢂC; Rf
0.68 (ethyl acetate); IR (KBr, cmꢀ1): 3371 (NH), 1723 (C]O), 1557
(C]N), 1490 (N–N), 1110 (C–N); 1H NMR (CDCl3):
d
2.08 (s, 3H,
(C]N), 1490 (N–N), 1092 (C–N); 1H NMR (CDCl3):
d 2.24 (s, 3H, CO–
CO–CH3), 3.06 (dd, 1H, HA, JAB ¼ 17.54 Hz, JAX ¼ 6.23 Hz), 3.42 (dd,
1H, HB, JAB ¼ 17.94 Hz, JBX ¼ 10.02 Hz), 5.84 (dd, 1H, HX,
JAX ¼ 7.32 Hz, JBX ¼ 11.09 Hz), 7.68–8.12 (m, 8H, Ar–H), 8.77 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 383; Anal. Calcd for C18H15N5O3S: C,
56.68; H, 3.96; N, 18.36; S, 8.41. Found: C, 56.54; H, 3.91; N, 18.44;
S, 8.45.
CH3), 2.45 (s, 3H, Ar–CH3), 3.14 (dd, 1H, HA, JAB ¼ 17.23 Hz,
JAX ¼ 7.52 Hz), 3.87 (dd, 1H, HB, JAB ¼ 17.92 Hz, JBX ¼ 10.22 Hz), 5.43
(dd, 1H, HX, JAX ¼ 6.53 Hz, JBX ¼ 10.54 Hz), 7.74–8.27 (m, 8H, Ar–H),
8.72 (s, 1H, NH); EI-MS: m/z [M þ H]þ 352; Anal. Calcd for
C19H18N4OS: C, 65.12; H, 5.18; N, 15.99; S, 9.15. Found: C, 65.03; H,
5.12; N, 16.05; S, 9.20.
6.2.2.14. 6-Nitro-2-[(1-acetyl-5-(4-chlorophenyl))-2-pyrazolin-3-yl]a-
minobenzothiazole (46). Yellow solid; Yield: 56%; mp 238–240 ꢂC;
Rf 0.63 (ethyl acetate); IR (KBr, cmꢀ1): 3377 (NH), 1715 (C]O),
6.2.2.20. 6-Methyl-2-[(1-acetyl-5-(4-chlorophenyl))-2-pyrazolin-3-yl]-
aminobenzothiazole (52). Pale white crystals; Yield: 73%; mp 187–
189 ꢂC; Rf 0.51 (ethyl acetate); IR (KBr, cmꢀ1): 3292 (NH), 1705
(C]O), 1564 (C]N), 1485 (N–N), 1115 (C–N); 1H NMR (CDCl3):
1580 (C]N), 1495 (N–N), 1095 (C–N); 1H NMR (CDCl3):
d 2.21 (s,
3H, CO–CH3), 3.08 (dd, 1H, HA, JAB ¼ 17.22 Hz, JAX ¼ 7.65 Hz), 3.81
(dd, 1H, HB, JAB ¼ 16.24 Hz, JBX ¼ 9.82 Hz), 5.08 (dd, 1H, HX,
JAX ¼ 7.15 Hz, JBX ¼ 10.85 Hz), 7.46–8.02 (m, 7H, Ar–H), 9.15 (s, 1H,
NH); EI-MS: m/z [M þ H]þ 417; Anal. Calcd for C18H14ClN5O3S: C,
51.99; H, 3.39; N, 16.84; S, 7.71. Found: C, 51.87; H, 3.45; N, 16.79;
S, 7.73.
d 2.17 (s, 3H, CO–CH3), 2.38 (s, 3H, Ar–CH3), 3.12 (dd, 1H, HA,
JAB ¼ 17.84 Hz, JAX ¼ 6.54 Hz), 3.64 (dd, 1H, HB, JAB ¼ 16.98 Hz,
JBX ¼ 10.76 Hz), 5.12 (dd, 1H, HX, JAX ¼ 7.23 Hz, JBX ¼ 9.87 Hz), 7.56–
8.12 (m, 7H, Ar–H), 9.14 (s, 1H, NH); EI-MS: m/z [M þ H]þ 386; Anal.
Calcd for C19H17ClN4OS: C, 59.29; H, 4.45; N,14.56; S, 8.33. Found: C,
59.23; H, 4.47; N, 14.59; S, 8.37.
6.2.2.15. 6-Nitro-2-[(1-acetyl-5-(4-hydroxyphenyl))-2-pyrazolin-3-yl]-
aminobenzothiazole (47). Yellow solid; Yield: 62%; mp 285–287 ꢂC;
Rf 0.43 (ethyl acetate); IR (KBr, cmꢀ1): 3615 (OH), 3290 (NH), 1690
(C]O), 1565 (C]N), 1480 (N–N), 1115 (C–N); 1H NMR (CDCl3):
6.2.2.21. 6-Methyl-2-[(1-acetyl-5-(4-hydroxyphenyl))-2-pyrazolin-3-
yl]aminobenzothiazole (53). Pale yellow solid; Yield: 65%; mp
232–234 ꢂC; Rf 0.46 (ethyl acetate); IR (KBr, cmꢀ1): 3564 (OH),
3368 (NH), 1723 (C]O), 1548 (C]N), 1497 (N–N), 1084 (C–N); 1H
d
2.14 (s, 3H, CO–CH3), 3.15 (dd, 1H, HA, JAB ¼ 17.98 Hz,
NMR (CDCl3): d 2.25 (s, 3H, CO–CH3), 2.46 (s, 3H, Ar–CH3), 3.09
JAX ¼ 6.38 Hz), 3.44 (dd, 1H, HB, JAB ¼ 17.46 Hz, JBX ¼ 9.64 Hz), 5.31
(dd, 1H, HX, JAX ¼ 7.12 Hz, JBX ¼ 11.41 Hz), 7.58–7.96 (m, 7H, Ar–H),
8.66 (s, 1H, NH), 9.53 (s, 1H, OH); EI-MS: m/z [M þ H]þ 399; Anal.
Calcd for C18H15N5O4S: C, 54.40; H, 3.80; N, 17.62; S, 8.07. Found: C,
54.32; H, 3.74; N, 17.68; S, 8.11.
(dd, 1H, HA, JAB ¼ 17.34 Hz, JAX ¼ 6.86 Hz), 3.82 (dd, 1H, HB,
JAB ¼ 17.58 Hz, JBX ¼ 9.86 Hz), 5.64 (dd, 1H, HX, JAX ¼ 6.24 Hz,
JBX ¼ 9.21 Hz), 7.66–8.30 (m, 7H, Ar–H), 8.86 (s, 1H, NH), 9.57 (s,
1H, OH); EI-MS: m/z [M þ H]þ 368; Anal. Calcd for C19H18N4O2S:
C, 62.28; H, 4.95; N, 15.29; S, 8.75. Found: C, 62.17; H, 4.91; N,
15.34; S, 8.78.
6.2.2.16. 6-Nitro-2-[(1-acetyl-5-(3-hydroxy-4-methoxyphenyl))-2-
pyrazolin-3-yl]-aminobenzothiazole (48). Yellow solid; Yield: 72%;
mp 274–276 ꢂC; Rf 0.56 (ethyl acetate); IR (KBr, cmꢀ1): 3585 (OH),
3344 (NH), 1724 (C]O), 1578 (C]N), 1482 (N–N), 1090 (C–N); 1H
6.2.2.22. 6-Methyl-2-[(1-acetyl-5-(3-hydroxy-4-methoxyphenyl))-2-
pyrazolin-3-yl]-aminobenzothiazole (54). Pale white crystals; Yield:
72%; mp 251–253 ꢂC; Rf 0.52 (ethyl acetate); IR (KBr, cmꢀ1): 3624
(OH), 3292 (NH), 1684 (C]O), 1576 (C]N), 1482 (N–N), 1124 (C–N);
NMR (CDCl3):
d
2.23 (s, 3H, CO–CH3), 3.11 (dd,1H, HA, JAB ¼ 17.98 Hz,
JAX ¼ 6.24 Hz), 3.52 (dd, 1H, HB, JAB ¼ 17.07 Hz, JBX ¼ 9.54 Hz), 3.72
(s, 3H, OCH3), 5.13 (dd, 1H, HX, JAX ¼ 6.76 Hz, JBX ¼ 9.09 Hz), 7.65–
8.20 (m, 6H, Ar–H), 9.11 (s, 1H, NH), 10.18 (s, 1H, OH); EI-MS: m/z
[M þ H]þ 429; Anal. Calcd for C19H17N5O5S: C, 53.39; H, 4.01; N,
16.38; S, 7.50. Found: C, 53.29; H, 4.06; N, 16.34; S, 7.54.
1H NMR (CDCl3):
d 2.12 (s, 3H, CO–CH3), 2.54 (s, 3H, Ar–CH3), 2.96
(dd, 1H, HA, JAB ¼ 17.85 Hz, JAX ¼ 6.75 Hz), 3.42 (dd, 1H, HB,
JAB ¼ 16.52 Hz, JBX ¼ 9.19 Hz), 3.84 (s, 3H, Ar–OCH3), 5.74 (dd,1H, HX,
JAX ¼ 7.81 Hz, JBX ¼ 9.16 Hz), 7.40–7.85 (m, 6H, Ar–H), 9.11 (s, 1H,
NH), 9.86 (s, 1H, OH); EI-MS: m/z [M þ H]þ 398; Anal. Calcd for

158
N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
C20H20N4O3S: C, 60.59; H, 5.08; N, 14.13; S, 8.09. Found: C, 60.51; H,
5.05; N, 14.16; S, 8.12.
results are represented as TD50 which are further used in the
calculation of PI (TD50/ED50) (Table 1).
6.2.2.23. 6-Methyl-2-[(1-acetyl-5-(furan-2-yl))-2-pyrazolin-3-yl]a-
minobenzothiazole (55). Pale yellow crystals; Yield: 56%; mp 208–
210 ꢂC; Rf 0.63 (ethyl acetate); IR (KBr, cmꢀ1): 3355 (NH), 1710
(C]O), 1545 (C]N), 1470 (N–N), 1090 (C–N); 1H NMR (CDCl3):
6.3.4. Behavioral activity
The activity was measured as digital score using actophotometer
[34] with the ip administration of drug (100 mmol/kg) to mice. The
mice were placed in the box and the behavior was noted for 10 min.
Further, the animals were treated with the drug and after 0.5 h of
drug administration the animal were re-tested. The activity score
was noted and based on these results, % change was calculated and
represented in Table 2.
d
2.18 (s, 3H, CO–CH3), 2.47 (s, 3H, Ar–CH3), 3.09 (dd, 1H, HA,
JAB ¼ 17.32 Hz, JAX ¼ 6.24 Hz), 3.52 (dd, 1H, HB, JAB ¼ 17.87 Hz,
JBX ¼ 9.53 Hz), 5.76 (dd, 1H, HX, JAX ¼ 7.63 Hz, JBX ¼ 10.84 Hz), 7.52–
7.96 (m, 6H, Ar–H), 8.76 (s, 1H, NH); EI-MS: m/z [M þ H]þ 342; Anal.
Calcd for C17H16N4O2S: C, 59.98; H, 4.74; N, 16.46; S, 9.42. Found: C,
59.91; H, 4.72; N, 16.53; S, 9.46.
6.3.5. Porsolt’s swimpool test
The CNS depressant study was carried out using Porsolt’s
swimpool test [35]. The rats were placed in chamber containing
water and subjected to a 15 min swimming session 24 h before the
test. The rats were administered intraperitoneally at dose of
6.2.2.24. 6-Methyl-2-[(1-acetyl-5-(4-methoxyphenyl))-2-pyrazolin-3-
yl]aminobenzothiazole. (56)White crystals; Yield: 71%; mp
235–237 ꢂC; Rf 0.54 (ethyl acetate); IR (KBr, cmꢀ1): 3365 (NH), 1686
(C]O),1552 (C]N),1486 (N–N),1110 (C–N); 1H NMR (CDCl3):
d
2.14
100 mmol/kg and 0.5 h before the test. The duration of immobility
(s, 3H, CO–CH3), 2.54 (s, 3H, Ar–CH3), 3.08 (dd,1H, HA, JAB ¼ 17.12 Hz,
JAX ¼ 6.86 Hz), 3.56 (dd, 1H, HB, JAB ¼ 16.54 Hz, JBX ¼ 10.26 Hz), 3.77
(s, 3H, Ar–OCH3), 5.84 (dd, 1H, HX, JAX ¼ 7.35 Hz, JBX ¼ 11.41 Hz),
7.63–8.16 (m, 7H, Ar–H), 8.87 (s, 1H, NH); EI-MS: m/z [M þ H]þ 382;
Anal. Calcd for C20H20N4O2S: C, 63.14; H, 5.30; N, 14.73; S, 8.43
Found: C, 63.08; H, 5.32; N, 14.69; S, 8.48.
was recorded during the 5 min test period (Table 3).
6.3.6. Liver function test
The rats were divided into groups of six, and the control group
received a basal diet and vehicle. The other groups were adminis-
tered the test drug in a dose of 100 mmol/kg/day (in methyl cellu-
lose) for 15 days. After the stipulated period, each animal was
anesthetized and the blood was collected from the liver to assess
the serum level of SGOT and SGPT (Table 4) according to the
reported procedure [27].
6.3. Anticonvulsant activity
The anticonvulsant activity was carried out on albino mice (20–
25 g) of either sex or male albino rats (100–150 g) as experimental
animals. The animals were housed under standard conditions and
allowed free access to standard pellet diet and water (ab libitum).
The test compounds and standard drug were suspended in 0.5%
methyl cellulose/water mixture.
6.4. 3D-QSAR analysis
6.4.1. Data set and biological activity
A set of 48 molecules (Table 1) was used for the generation of
3D-QSAR model. The data set was divided into a training set (29
molecules) and test set (19 molecules) based on the Tanimoto
similarity coefficient [40]. All the biological activity data were
converted to negative logarithmic molar concentration of ED50
values [pED50 ¼ ꢀlog(ED50 ꢃ 10ꢀ6)] and used for 3D-QSAR studies.
6.3.1. Maximum electroshock (MES) test
The test compounds were administered intraperitoneally (ip)
at a dose of 20–1000 mmol/kg body weight. Maximum electro-
shock seizure were elicited using electroconvulsometer with
a 60 cycle altering current of 50 mA for 0.25 s via ear clip
electrode as per the reported procedure and effect was assessed
at 0.5 h after administration by recording the tonic extension of
the hind limbs at different doses. The reduction in time or
absence of hind limb tonic extension of seizure is defined as
protection. Median effective dose (ED50) was calculated at the
doses of test drugs until atleast three points were established in
the range of 10–90% seizure protection or minimal observed
neurotoxicity (Table 1).
6.4.2. Molecular modeling and PHASE methodology
The 3D-QSAR studies were carried out using PHASE version
3.0.110 implemented in the Maestro 8.5.111 modeling package
(Schro¨dinger, Inc., LLC, New York, USA) installed on a Pentium IV
3.06 GHz PC with Linux OS (RedHat Enterprise WS 4.0). The
structures of the molecules were built using structure drawing tool
which was cleaned and minimized using LigPrep program to attach
hydrogens and generate stereoisomers at neutral pH 7 using
‘ionizer’ subprogram. Conformer generation was carried out with
Macromodel ligand torsion search using MMFs force field with
implicit GB/SA distance dependent dielectric solvent model at
cutoff root mean square deviation (RMSD) of 1 Å. The active and
inactive threshold of 3.8 and 3.3, respectively, were applied to the
training set. The pharmacophore feature sites for the molecules
were assigned using a terminal box size of 1 Å and applying a set of
features defined in PHASE as hydrogen-bond acceptor (A),
hydrogen-bond donor (D), hydrophobic (H), negative ionic (N),
positive ionic (P) and aromatic (R) with the requirement that all
seven active matches. The common pharmacophore was generated
by considering five sites that matches seven active ligands.
Hypotheses were generated by a systematic variation of number of
sites (nsites) and the number of matching active compounds (nact).
With nact ¼ nactꢀtot initially (nactꢀtot is the total number of active
compounds in the training set), nsites was varied from 7 to 3 until
atleast one hypotheses was found and scored successfully. If this
failed, then nact was decreased by one and the nsites cycle was
6.3.2. Subcutaneous pentylenetetrazole (scPTZ) seizure threshold
test
Pentylenetetrazole dissolved in 0.9% sodium chloride solution
was administered in the posterior midline of the mice and the onset
and severity of convulsion was noted for the control group. The test
group was administered with the selected compounds 0.5 h prior
to the administration of PTZ and the anticonvulsant activity was
detected in terms of ED50 and is presented in Table 1.
6.3.3. Neurotoxicity screening
The neurotoxicity of all the test compounds was evaluated using
rotorod test. Mice were trained to balance on the rotating rod
(3.2 cm diameter) that rotates at 6 rpm. Trained animals were
treated with test compounds at a dose of 20–1000 mmol/kg
administered intraperitoneally. Neurotoxicity was determined by
the inability of the animal to remain on the rod for 1 min. The

N.D. Amnerkar, K.P. Bhusari / European Journal of Medicinal Chemistry 45 (2010) 149–159
159
Health Organization, Geneva, 2009.http://www.who.int/entity/mediacentre/
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site, vector, volume, selectivity, number of matches and energy
terms. The hypotheses that emerged from this process subse-
quently scored with respect to the three inactives, again with
default parameters. Atom based 3D-QSAR models were generated
for the hypotheses using the 29 member training set with four (4)
PLS factor and a grid spacing of 1 Å.
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Acknowledgements
We are grateful to Dr. P. Trivedi and C. Kartikeyan, Computa-
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Bhoomendra Bhongade, International Medical University, Kuala
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