Design of benzothiazole-1,3,4-thiadiazole conjugates: Synthesis and anticonvulsant evaluation

Article abstract of DOI:10.1002/ardp.201300083

Various 2-[(6-substituted-1,3-benzothiazol-2-yl)amino]-N-[5-substituted- phenyl-1,3,4-thiadiazol-2-yl]acetamides were synthesized with a prospective exploration of "lead hopping", using pharmacophoric elements for in vivo anticonvulsant activity. This yielded three potent candidates (5i, 5t, and 5u) in the preliminary screening employing the maximal electroshock seizure (MES) and the subcutaneous pentylenetetrazole (scPTZ) test, showing minimal neurotoxicity. Their quantitative study indicated an increase of nearly 2-10 times for the MES test and 7- to 67-fold for the scPTZ test in the protective index, the keystone in drug discovery for anticonvulsant activity. Various 2-[(6-substituted-1,3-benzothiazol-2-yl)amino]-N-[5-substituted-phenyl-1,3, 4-thiadiazol-2-yl]acetamides were synthesized with a prospective exploration of "lead hopping", using pharmacophoric elements for in vivo anticonvulsant activity. Three potent candidates (5i, 5t, and 5u) with minimal neurotoxicity were identified in the MES and scPTZ tests.

Full text of DOI:10.1002/ardp.201300083

Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
819
Full Paper
Design of Benzothiazole-1,3,4-thiadiazole Conjugates:
Synthesis and Anticonvulsant Evaluation
Nadeem Siddiqui, Priya Ahuja, Sachin Malik, and Satish K. Arya
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Jamia Hamdard, Hamdard Nagar, New Delhi,
India
Various 2-[(6-substituted-1,3-benzothiazol-2-yl)amino]-N-[5-substituted-phenyl-1,3,4-thiadiazol-2-yl]acet-
amides were synthesized with a prospective exploration of “lead hopping”, using pharmacophoric
elements for in vivo anticonvulsant activity. This yielded three potent candidates (5i, 5t, and 5u) in the
preliminary screening employing the maximal electroshock seizure (MES) and the subcutaneous
pentylenetetrazole (scPTZ) test, showing minimal neurotoxicity. Their quantitative study indicated an
increase of nearly 2–10 times for the MES test and 7- to 67-fold for the scPTZ test in the protective index,
the keystone in drug discovery for anticonvulsant activity.
Keywords: Anticonvulsants / Benzothiazole / Neurotoxicity / Phenytoin / Thiadiazole
Received: March 7, 2013; Revised: July 22, 2013; Accepted: July 26, 2013
DOI 10.1002/ardp.201300083
Introduction
patients. Approximately 25–30% of patients continue to suffer
from seizures despite state-of-the-art treatment [5].
Epilepsy is one of the common chronic diseases affecting 0.5–
1% of the population worldwide. It ranks as the third most
frequent neurological disorder, after cerebrovascular disease
and dementia [1]. According to the International League
against Epilepsy and the International Bureau of Epilepsy,
epilepsy is the disorder of the brain characterized by an
enduring predisposition to generate epileptic seizures and by
the neurobiologic, cognitive, psychological, and social con-
sequences of the condition [2]. Current antiepileptic drugs are
effective in controlling seizures in about 70% of the patients,
but their use is often limited by side effects [3]. About one-third
of the patients do not respond well to the currently available
treatments, even if multiple drugs with complementary
activities are used [4].
Over the last decades, the optimum therapy for seizures
manifests complete control of seizures, absence of bother-
some side effects, and an emphasis on maximizing quality of
life. In spite of the concerted attempts by the physicians to
utilize the available pharmacotherapies to their full advan-
tage, the prospect of freedom from seizures and adverse
effects remains elusive for a considerable number of epileptic
One example of a rationally designed anticonvulsant agent
having an aminoacetamide linkage is the phase III drug
brivaracetam, chemically 2-(2-oxo-4-propyl-pyrrolidin-1-yl)-
butamide. It is an agonist of the synaptic vesicle protein
SV2A and an alkylation hybrid of valproic acid and
levetiracetam, an anticonvulsant drug made by UCB Pharma
(Fig. 1). Moreover, the presynaptic N-methyl-D-aspartate
(NMDA) glutamate receptor antagonist rilutek (riluzole), a
2-aminobenzothiazole analog generated by Rhone-Poulenc
Rover, Inc. Collegeville (PA, USA) and marketed by Sanofi-
Aventis (chemically, 6-(trifluoromethoxy)benzo[d]thiazol-2-
amine), contributed to the designing of the synthesized
compounds. The Food and Drug Administration (FDA) granted
brivaracetam orphan drug designation for the treatment of
symptomatic myoclonus in November 2005. The European
Medicines Agency (EMA) granted orphan drug designation to
brivaracetam for the treatment of progressive myoclonic
epilepsies in August 2005. In December 2010, a phase III trial
was initiated for partial-onset seizures [6]. A chemical drawing
of the new targeted compounds, as rationally designed
through brivaracetam along with riluzole, is represented in
Fig. 1.
For over a century, despite the refined and efficient
methods accrued for the synthesis of thiadiazole, a five-
membered aromatic system having three heteroatoms at
symmetrical positions has been studied extensively, owing to
Correspondence: Nadeem Siddiqui, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Jamia Hamdard, Hamdard Nagar 110062,
New Delhi, India.
E-mail: nadeems005@gmail.com
Fax: þ91 11 26059688 ext: 5307
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N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Figure 1. Rationally designed template for the targeted compounds as anticonvulsant agents.
its interesting pharmacological activities [7–12]. Based on our
prior research on benzothiazole derivatives as anticonvulsant
nuclei [13], it was found that these potential agents lacked the
derivatization of a lead to yield compounds with enhanced
anticonvulsant activity.
pharmacophoric element of a hydrophobic domain. To gain Results and discussion
more insight, the benzothiazole and thiadiazole moieties
coalesced through an aminoacetamide linkage were envi-
sioned as postulates with a view to produce promising
anticonvulsant agents.
Chemistry
A pragmatic approach to synthesize a series of 2-(1,3-
benzothiazol-2-ylamino-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acet-
amides 5a–x is delineated here. Initially, substituted
benzaldehyde reacts with thiosemicarbazide to yield substi-
tuted (2E)-2-benzylidenehydrazinecarbothioamides 1a–f by
extrusion of a water molecule. The Schiff bases undergo
intramolecular oxidative cyclization by the coordination of
ferric chloride into 5-phenyl-1,3,4-thiadiazole-2-amines 2a–f.
However, mild citric acid in sodium citrate treatment was
required to facilitate the cyclization. Further, chloroacetyl
chloride selectively condenses with the amine group in the
Hence, the following approaches were utilized for the
design of these derivatives: (a) hybridization of the thiadiazole
and benzothiazole bioactive nuclei to elucidate their
synergistic activity [14], (b) the synthesis of molecular scaffolds
possessing essential pharmacophoric elements that are
present in well-established anticonvulsant drugs such as
phenytoin, carbamazepine, gabapentin, zonisamide, etc. [15],
(c) a conceivable strategic approach for the discovery of
efficacious drugs devoid of toxicity alerts, and (d) the
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
821
and 1440 cmꢀ1, indicating the presence of imine C N and
–
str
–
presence of dimethyl formamide to afford 2-chloro-N-(5-
phenyl-substituted-1,3,4-thiadiazol-2-yl)acetamides 3a–f (path
1 in Scheme 1).
A well-established literary procedure incorporating aniline
derivatives and potassium thiocyanate via chemoselective
bromination and base in cold glacial acetic acid produced
–
–
the characteristic alkene CH
peak, respectively, formed
bend
via the removal of a water molecule through the reaction of
the primary substrates. The absence of the exocyclic carbonyl
function stretching peak derived from the aryl aldehyde at
1656 cmꢀ1 and a broad band at 3338 cmꢀ1 for –NHNH2
further supported their formation. The strong absorption
peaks at 1510 and 696 cmꢀ1 for compounds 2a–f, attributable
to the heteroaromatic ring vibrations and the C–S–Cstr band,
provided an affirmation for the cyclization of benzylidene
hydrazine carbothioamide into the five-membered thiadia-
annulated 1,3-benzothiazol-2-amines 4a–e (path
2
in
Scheme 1) [16].
Gratifyingly, cyclocondensation of acetamides with benzo-
thiazol-2-amines furnished the quantitative formation of the
conjugate addition products 5a–x, using absolute ethanol as
solvent (path 3 in Scheme 1).
zole ring. The C O peak at 1650 cmꢀ1 for the amide bond
–
–
str
The chemical identification studies (1H NMR and IR) of 1a–f
substantiated their formation from aryl aldehyde and
thiosemicarbazide. In the IR spectra of the compounds
1a–f, it was possible to observe the absorption peaks at 1640
was assigned to the acetamide formation in compounds 3a–f.
In path 2, the synthesis of compounds 4a–e from
–
substituted aniline indicated an IR peak of C N
at
–
str
1565 cmꢀ1 and a characteristic amidine (N–C N) peak at
–
–
1686 cmꢀ1, evidently from the formation of the aromatic
benzothiazole ring. The compounds 5a–x in path 3 gave a
strong CH2–N linkage peak at 2830 cmꢀ1, attributable to the
hybrid formation via the amine residue in the benzothiazole
ring with the acetamide residue of the thiadiazole ring in the
final compounds.
1
–
In the H NMR spectra of 1a, a singlet of the imine (CH N)
–
proton resonated at 8.25 ppm, unlike the aldehydic singlet
resonating at 9.08 ppm in the substrate. Two D2O-exchange-
able singlets derived from thiosemicarbazide appeared at
–
–
9.26 (–NH ) and 10.80 ppm ( N–NH–CS). The link NH –C S is
–
–
2
2
considerably more polar and stronger than the fused
thiadiazole ring C–S–C–NH2; consequently, the peak is not
intense and is deshielded to 4.80 ppm in compounds 2a–f. The
1H NMR spectra of 3a–f exhibited the signals belonging to an
amide group and a methylene proton at 11.54 and 4.01 ppm,
afforded by the acetamide linkage in compounds 4a–e. The
characteristic peak intensity ranging from 4.16 to 4.43 ppm of
–CH2–CO–N– affirmed the formation of the title compounds
5a–x.
Pharmacology
The newly synthesized compounds 2-[(6-substituted-1,3-ben-
zothiazol-2-yl)amino]-N-[5-(4-substituted-phenyl)-1,3,4-thiadia-
zol-2-yl]acetamide 5a–x were subjected to anticonvulsant
screening based on the NIH Anticonvulsant Drug Develop-
ment (ADD) Program protocol (Table 1) [17]. The preliminary
activity was established through electrical and chemical
induction of convulsions in the maximal electroshock seizure
(MES) and subcutaneous pentylenetetrazole (scPTZ) tests,
respectively [18, 19]. The neurotoxicity of the active
compounds was determined using the minimal motor
impairment-Rotarod test [20]. The intraperitoneal injections
of the synthesized compounds were administered to the mice
at doses of 30, 100, and 300 mg/kg. Phenytoin and carbamaze-
pine were selected as the standard drugs for this purpose [21].
Scheme 1. Synthetic process for the preparation of compounds
5a–x.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
The evaluation was carried out at two time intervals of 0.5 and
4.0 h, respectively.
The synthesized compounds 5b, 5d, 5j, 5l, 5p, 5v, and 5w
had shown activity at a dose of 100 mg/kg at both the reported
intervals, except for 5b, 5j, 5p, and 5v, which exhibited
activity at 300 mg/kg after 4.0 h of duration. The compounds
5a, 5c, 5e, 5h, 5m, 5o, 5q, and 5s showed nearly 50%
protection at the maximum dose of 300 mg/kg at 0.5 and 4.0 h
of duration, with the exception of 5e and 5m, which exhibited
25% protection at the 0.5-h and no protection at the 4.0-h
interval, indicating rapid absorption but cessation of activity
at longer duration.
The compounds exhibiting a better anticonvulsant profile
against electroshock-induced convulsions were further evalu-
ated for chemically induced convulsions. In the scPTZ screen,
the compounds 5i, 5n, 5r, 5t, and 5u showed anti-scPTZ
activity at the minimum dose of 100 mg/kg at the two chosen
time intervals, except for 5n and 5r, which showed protection
at a higher dose at 4.0 h duration, indicating quick onset and
prolonged protection against absence seizures, but at a higher
dose. Interestingly, compound 5i exhibited 100% protection at
both the selected time intervals, making it a good anticon-
vulsant lead compared to carbamazepine. Compound 5g
The MES screen for the evaluation of generalized tonic–
clonic seizures identified some clinical drug candidates that
prevented seizure spread (Table 2). In this test, compounds 5f,
5g, 5i, 5k, 5n, 5r, 5t, 5u, and 5x were found to be active at
30 mg/kg at 0.5 h of duration. Interestingly, 5g, 5i, 5n, 5t, and
5u continued to protect against the seizures at a dose of
30 mg/kg at 4.0 h, indicating rapid onset and longer duration
of action at a lower dose, equal to the effects of the standard
drug phenytoin. Compounds 5i and 5t showed 100%
protection, whereas 75% protection was reported for com-
pounds 5g, 5n, and 5u. Compounds 5f, 5k, 5r, and 5x were
also active at 4.0 h, but at a higher dose of 100 mg/kg,
suggesting rapid onset and shorter duration of action at a
lower dose. The percentage of protection depicted for
compounds 5f, 5k, and 5x was less than or equal to 50% at
both the administered doses, thereby rendering them less
efficient as compared to the standard drug carbamazepine.
Compound 5r had an activity nearly equal to that of
carbamazepine.
Table 1. Physicochemical constants of the synthesized compounds 5a–x and Lipinski evaluation of active compounds.
Lipinski rule (for active compounds)c)
Compd.
R
R0
Molecular formulaa) % Yield M.p. (°C)b)
MW
MR
No. of atom tPSA Log P HBD/HBA
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
–H
–H
–H
–H
–H
–H
–Br
–Cl
–F
–CH3
–H
–Br
–Cl
–F
–CH3
–H
–Br
–Cl
–F
–CH3
–H
–Br
–Cl
–F
C17H13N5OS2
C17H12BrN5OS2
C17H12ClN5OS2
C17H12FN5OS2
C18H15N5OS2
71.2
74.6
73.9
80.4
76.8
70.9
75.3
72.7
78.4
78.1
79.6
83.1
78.6
81.1
80.5
76.2
77.4
76.5
78.7
79
165.5
162.6
163.4
160.8
149.2
167.4
159.8
186.8
156.2
150.6
190.2
175.5
188.1
177.2
160.1
230.4
155.2
180.2
172.6
168.4
184.7
169.3
174.3
166.7
367.45
446.35
401.89
385.44
381.48
412.45
491.34 117.41
446.89
430.44 109.72
426.47
383.45
462.34
417.89
401.44 105.05
397.47
397.47
476.37
431.92 114.75
415.46
411.5
436.34 111.37
480.79
480.79
–NO2
–NO2
–NO2
–NO2
–NO2
–OH
–OH
–OH
–OH
–OH
–OCH3
–OCH3
–OCH3
–OCH3
C17H12N6O3S2
C17H11BrN6O3S2
C17H11ClN6O3S2
C17H11FN6O3S2
C18H14N6O3S2
C17H13N5O2S2
C17H12BrN5O2S2
C17H12ClN5O2S2
C17H12FN5O2S2
C18H15N5O2S2
C18H15N5O2S2
C18H14BrN5O2S2
C18H14ClN5O2S2
C18H14FN5O2S2
C19H17N5O2S2
C17H11Cl2N5OS2
C17H11BrClN5OS2
C17H11BrClN5OS2
C17H11Br2N5OS2
40
40
130.02
130.02
4.90
4.26
2/9
2/9
39
42
98.44
87.44
3.82
4.86
3/7
2/7
5t
–OCH3 –CH3
114.68
45
38
87.44
78.21
4.64
4.86
2/7
2/6
5u
5v
5w
5x
–Cl
–Cl
–Br
–Br
–Cl
–Br
–Cl
–Br
77
78.5
71.2
74.3
525.24
MW, molecular weight; MR, molar refractivity; tPSA, total polar surface area; HBD/HBA, hydrogen bond donor/acceptor.
a)
Solvent used for crystallization of compounds: ethanol.
Melting point of compounds at their decomposition.
Data evaluation based on [32].
b)
c)
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Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
823
Table 2. Preliminary evaluation of synthesized compounds 5a–x
for anticonvulsant activity using maximal electroshock test.
The outcomes of the preliminary screening encouraged
further studies of the active compounds for quantitative
screening [22]. In the quantitative study, MES attainments
depicted compound 5i as having a protective index much
higher than the standard drugs phenytoin, carbamazepine,
and riluzole [23]. However, compounds 5t and 5u showed a
much wider protective window than the standard drugs.
Likewise the scPTZ quantitative study indicated a remarkable
increase in the protective indices for compounds 5i and 5t,
whereas a comparable value was found for 5u (Table 4).
A instill into the Antiepileptic Drug Development (ADD)
Program disposition compounds 5i, 5t, and 5u were also
speculated for the determination of their anticonvulsant
activity in the psychomotor 6-Hz seizure test [24–26]. The 6-Hz
screen has been validated recently as a model of therapy-
resistant epilepsy. It has not been widely used because it lacks
clinical validity, since the hydantoins such as phenytoin failed
to show protective activity. However, the clinically effective
antiepileptic drug levetiracetam, which is not active in the
conventional MES and scPTZ tests, does display protective
activity in the 6-Hz model. This suggested that the 6-Hz model
may be capable of identifying antiseizure agents with a novel
spectrum of activity and unknown mechanism of anticonvul-
sant action. The results upon intraperitoneal administration
in mice are summarized in Table 5 and show 5i as the most
active compound, with maximal (100%) protection at the time
points 0.5, 1, and 2 h. The other molecules were less active and
protected up to 75% of the mice. Interestingly, the quantita-
tive studies for 2-[(6-fluoro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-
nitrophenyl)-1,3,4-thiadiazol-2-yl]acetamide (5i) identified the
compound as the most promising candidate, having an ED50
comparable to that of levetiracetam [25] used as model
anticonvulsant (Table 6).
MES screen
Dosage (mg/kg)a)
Protectionb)
Compd. no.
0.5 h
4 h
0.5h
4h
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
300
100
300
100
300
30
30
300
30
100
30
100
300
30
300
100
300
30
300
30
300
300
300
100
–
2/4
2/4
2/4
2/4
1/4
2/4
3/4
2/4
4/4
2/4
1/4
2/4
1/4
3/4
2/4
2/4
2/4
4/4
2/4
4/4
3/4
1/4
2/4
1/4
4/4
4/4
2/4
2/4
2/4
1/4
0/4
1/4
3/4
2/4
4/4
1/4
1/4
3/4
0/4
3/4
2/4
1/4
2/4
3/4
2/4
4/4
3/4
1/4
1/4
1/4
4/4
4/4
100
30
300
30
100
100
300
–
30
300
300
300
100
300
30
5s
5t
5u
5v
5w
5x
PHYc)
CBZc)
30
30
100
100
30
30
30
300
100
100
30
100
(–) Indicates devoid of activity at the maximum dose administered
(300 mg/kg).
a)
All the compounds were administered intraperitoneally.
Number of animals protected/number of animals tested, the
b)
number of mice is four.
c)
Data taken from [21] and dose in mg/kg.
To infer the effects of the synthesized compounds on the
gamma amino butyric acid (GABA) receptor, the highly active
compounds 5i, 5t, and 5u were selected and subjected to
neurochemical investigation, to estimate the level of GABA in
the different regions of the rat brain [27]. The statistical data
(Table 7) showed that the concentration of GABA increased
significantly in the olfactory lobe and midbrain of the rat
brain after administration of the compounds 5i and 5u
(p < 0.01) compared to 5t (p < 0.05). Non-significant results
were obtained for 5t and 5u in the medulla oblongata and the
cerebellum. In the midbrain area, the cerebellum, the
olfactory lobe, and the medulla oblongata, compound 5i
significantly elevated the GABA concentration with respect to
the control.
showed anticonvulsant activity at a maximum dose of
300 mg/kg at both durations of time.
Determination of the minimal motor impairment was
performed using the Rotarod test with the compounds
exhibiting protection against MES- and scPTZ-induced con-
vulsions. Compounds 5i, 5t, and 5u were found to be devoid of
any neurotoxicity, even at the maximum dose, thereby
rendering them much safer than the standard drug
phenytoin. Compounds 5g and 5n gave equivalent neurotox-
icity profiles compared to carbamazepine at 0.5 h, but the
percentage of toxicity increased after 4.0 h of duration from
25% to 75%, suggesting it to be more toxic than carbamaze-
pine after prolonged absorption. Compound 5r, although
having a minimal motor impairment dose equivalent to that
of the standard drug phenytoin, was found to be toxic in
nearly 100% of the mice after 4.0 h of duration (Table 3).
Hepatotoxicity and total protein imbalance are the major
limitations of the currently marketed antiepileptic drugs.
Hence, the results regarding toxicity were established
through the concentrations of alkaline phosphatase, serum
glutamate oxaloacetate transaminase (SGOT), serum
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Table 3. Preliminary evaluation of selected compounds for anticonvulsant activity using subcutaneous pentylenetetrazole test and
neurotoxicity.
scPTZ screen
Dosage (mg/kg)a)
Minimal motor impairment test
Dosage (mg/kg)a)
Protectionb)
Toxicityc)
Compd. no.
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
5g
5i
5n
5r
300
100
100
100
100
100
x
300
100
300
300
100
100
x
3/4
4/4
2/4
2/4
4/4
3/4
x
2/4
4/4
2/4
2/4
3/4
3/4
x
100
300
100
100
300
300
100
100
300
300
300
100
300
300
100
300
1/4
0/4
1/4
3/4
0/4
1/4
1/4
1/4
3/4
0/4
3/4
4/4
1/4
1/4
0/4
0/4
5t
5u
PHYd)
CBZd)
100
300
4/4
4/4
a)
All the compounds were administered intraperitoneally.
b)
c)
d)
Number of animals protected/number of animals tested; the number of mice is four.
Number of animals toxic/number of animals tested; the number of mice is four.
Data taken from [21] and dose in mg/kg. (x) Indicates not tested.
glutamate pyruvate transaminase (SGPT), and total body
protein determined in serum, and the values were repre-
sented as mean ꢁ SEM [28–30]. Compounds 5i and 5u showed
moderate but significant changes in the levels of the
mentioned liver enzymes as compared to the control
(p < 0.01), except for 5u, which exhibited no significant
difference regarding SGPT. Compound 5t was also found to
alter the SGOT level, although the change was not very
significant (p < 0.05). 5i showed a significant change in the
total protein level (p < 0.01) compared to the other selected
compounds with p < 0.05 significance. All the other values
were not significant, indicating the nontoxic nature of the
tested compounds. Thus, considering the biochemical
parameters estimated to establish the liver function test
with selected compounds, it was clearly indicated that none of
the compounds showed any malfunctioning or toxicity of the
liver as compared to the control (Table 8).
Structure–activity relationship
Dimmock et al. [15] proposed a pharmacophore model for
anticonvulsants that consisted of (a) a hydrophobic domain,
(b) a hydrogen bonding domain (HBD), (c) a distal hydrophobic
domain, and (d) an electron donor moiety, based on which the
compounds were synthesized (Fig. 1). The procedure, as
depicted in Scheme 1, utilizes constrained pharmacophore
characteristics of thiadiazole chemistry, i.e., a “hydrogen
binding domain” and a “two-electron donor system” [31]. With
the identification of suitable reaction conditions, a survey of
the various substituents was executed. The insights into the
structure–activity relationship (SAR) of our active compounds
Table 4. Quantification studies of the selected compounds after intraperitoneal administration in mice.
a)
ED50
PIc)
b)
Compd. no.
MES
scPTZ
TD50
MES
scPTZ
5i
5t
5u
6.5
7.4
7.8
9.2
8.8
8.5
8.5
10.8
30
127.0
109.5
75.0
65.6
71.6
38.6
19.54
14.80
9.62
7.13
8.14
4.54
14.94
10.14
2.5
<0.22
<0.72
<1.93
PHYd)
CBZd)
RILZd)
>300
>100
>20
All the administered doses are in mg/kg (n ¼ 6).
a)
ED50 – median effective dose eliciting anticonvulsant protection in 50% animals.
TD50 – median toxic dose eliciting minimal neurological toxicity in 50% animals.
b)
c)
d)
PI (protective index) ¼ TD50/ED50
.
Data taken from [23].
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
825
Table 5. Anticonvulsant activity – psychomotor seizure test
(6-Hz, current 32 mA).
nuclei improved the activity (5n, 5r). This can be attributed to
the fact that substituents such as –OH and –OCH3 (5n, 5r, and
5t) on the distal aryl ring, which are capable of forming
intermolecular hydrogen bonds with the receptor sites,
favored by electronegative substituents or a lipophilic alkyl
chain (5t), possibly increase the substituent effect of the
synthesized compounds. The introduction of hydrophobic
substituents in the para position of the distal aryl and the 6th
position of the benzothiazole ring is proposed to be a
significant factor for the difference in anticonvulsant
potential of our hybrid compounds. Unsubstituted derivatives
of the phenyl ring attached to the thiadiazole nuclei resulted
in a substantial loss of activity.
Intraperitoneal injection into micea)
Compd. no.
0.25 h
0.5 h
1 h
2 h
4 h
5i
5t
5u
1
0
0
4
2
0
4
3
2
4
3
2
3
2
1
a)
Dose of 100 mg/kg was administered. The data indicate the
number of mice that were protected as four.
Table 6. Quantification data – psychomotor seizure test (6 Hz,
current 32 mA) after intraperitoneal injection into mice.
The corporate compound inventory entailed zero Lipinski
violations, a polar surface area ꢂ120 and no more than 10
rotatable bonds [32]. The refined hits of the MES, scPTZ, and
neurotoxicity analysis (5i, 5t, and 5u) subjected to quantita-
tive screening rendered a comparable increment of nearly 2–
10 times for MES and 7- to 67-fold for scPTZ (Table 4) in the
protective index, the mainstay in the drug discovery.
Furthermore, these achievements were corroborated with
the aid of the psychomotor seizure test (Tables 5 and 6), liver
enzyme estimation, total protein estimation of the body, and
determination of the GABA concentration in the rat brain. The
results confirm levels of significance of ꢃꢃp < 0.01 and
ꢃp < 0.05 (Tables 7 and 8).
b)
c)
TPEa)
(h)
ED50
TD50
(mg/kg) (TD50/ED50)
PId)
Compd. no.
(mg/kg)
5i
2
1
25.9 (19.4–26.4)
19.4 (9.90–36.0)
ND
>500
ND
>26
Levetiracetame)
ND, not determined.
a)
Time to peak effect.
ED50 – median effective dose required to assure anticonvulsant
b)
protection in 50% animals.
c)
TD50 – median toxic dose eliciting minimal neurological
toxicity in 50% animals.
d)
PI – protective index (TD50/ED50).
Data taken from Ref. [25].
e)
Conclusion
(5g, 5i, 5n, 5r, 5t, and 5u) envisaged the thiadiazole–
benzothiazole hybrid pharmacophore as a lead towards
efficacious anticonvulsant drugs. While the parent unsub-
stituted hybrid showed moderate activity, substitution of
electron-donating groups in compounds 5g, 5i, and 5t or an
electron-withdrawing group in 5u afforded stronger drugs.
Furthermore, the combination of an electron-donating
substitution on the distal aryl ring attached to the thiadiazole
nuclei and an electron-withdrawing substitution on the 6th
position of the hydrophobic domain of the benzothiazole
In conclusion, the unprecedented study describes a facile
synthesis of new promising anticonvulsant thiadiazole
derivatives and also probed the importance of joining this
nucleus to the benzothiazole hydrophobic domain through
aminoacetamide linkage, rendering new effective hybrid
molecules. The biological evaluation of the synthesized
compounds displayed two most active candidates, 5i and
5t, likewise consistent with the toxicity studies. Stimulated by
the outcomes of this viable schematic strategy, experiments
Table 7. GABA estimation of the selected compounds.
GABA conc. (g/100 mg tissue) ꢁ SEM
Compd. no.a)
Olfactory lobe
Mid brain
Medulla oblongata
Cerebellum
Control
5i
5t
5u
12.74 ꢁ 0.812
16.02 ꢁ 0.221ꢃꢃ
14.55 ꢁ 0.019ꢃ
15.65 ꢁ 0.112ꢃꢃ
44.35 ꢁ 1.044
60.65 ꢁ 1.881ꢃꢃ
51.47 ꢁ 1.108ꢃ
58.02 ꢁ 1.652ꢃꢃ
38.57 ꢁ 0.665
40.54 ꢁ 0.377ꢃ
39.65 ꢁ 0.115ns
40.01 ꢁ 0.169ns
23.72 ꢁ 1.404
30.25 ꢁ 1.226ꢃꢃ
25.62 ꢁ 1.005ns
27.69 ꢁ 1.011ns
ns ¼ not significant. The mean level was calculated using ANOVA followed by Dunnett’s multiple comparison test (n ¼ 6).
a)
The compounds were tested at a dose of 100 mg/kg intraperitoneally.
ꢃp < 0.05.
ꢃꢃp < 0.01.
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826
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Table 8. Liver enzyme and total protein estimation of the selected compounds.
Compd. no.a)
Alkaline phosphatase ꢁ SEM
SGOT ꢁ SEM
SGPT ꢁ SEM
Total protein (g/100 mL) ꢁ SEM
Control
5i
5t
5u
15.51 ꢁ 0.421
11.76 ꢁ 0.201ꢃꢃ
14.80 ꢁ 0.200ns
11.20 ꢁ 0.450ꢃꢃ
151.06 ꢁ 0.958
125.23 ꢁ 0.855ꢃꢃ
147.46 ꢁ 0.455ꢃ
123.67 ꢁ 0.560ꢃꢃ
25.18 ꢁ 0.663
20.09 ꢁ 0.990ꢃꢃ
22.37 ꢁ 0.011ꢃ
24.18 ꢁ 0.020ns
1.93 ꢁ 0.015
2.28 ꢁ 0.025ꢃꢃ
2.03 ꢁ 0.018ꢃ
2.04 ꢁ 0.033ꢃ
ns ¼ not significant. The mean level of SGOT/SGPT ꢁ SEM was calculated using ANOVA followed by Dunnett’s multiple comparison test
(n ¼ 6).
a)
A dose of 25 mg/kg/day of all the compounds was administered by oral route.
ꢃp < 0.05.
ꢃꢃp < 0.01.
for elucidating further benefits of these compounds are
underway and will be reported in due course.
was filtered off, washed with distilled water, and allowed to dry.
The final crystallization was effected by ethanol to afford 2a–f.
Synthesis of the 2-chloro-N-(5-phenyl-substituted-1,3,4-
thiadiazol-2-yl)acetamides (3a–f)
Experimental
The amines 2a–f (1 mol) were reacted with chloroacetylchloride
(2 mol) in N,N-dimethylformamide at room temperature for 2 h to
give compounds 3a–f.
Chemistry
All reagents were used as purchased from commercial suppliers
without further purification. Thin-layer chromatography (TLC)
was performed on silica gel 60 F254 TLC aluminum sheets (Merck
KGaA, Darmstadt, Germany) using toluene/ethyl acetate/formic
acid (5:4:1) as eluent. Ashless Whatmann No. 1 filter paper was
used for vacuum filtration. Melting points were determined by
using open capillary tubes in a Hicon melting point apparatus
(Hicon, India) and are uncorrected. Elemental data of C, H, and N
were within ꢁ0.4% and 0.3%, respectively, of the theoretical value
as determined by Vario EL III CHNS Elementar (Analysensysteme
GmbH, Hanau, Germany). The infrared spectra of the compounds
were recorded on a Bio-Rad FTS-135 spectrophotometer using KBr
pellets. 1H NMR and 13C NMR spectra of DMSO-d6/CDCl3 solutions
were recorded at 400 and 100 MHz, respectively, with a Bruker
400 Ultra shield TM NMR spectrometer (400 MHz; Bruker
Bioscience, USA), using TMS [(CH3)4Si] as internal standard.
Splitting patterns are designated as follows: s, singlet; d, doublet;
t, triplet; m, multiplet. Chemical shifts (d) values are given in parts
per million (ppm). J values are given in Hz. The mass spectra were
recorded on a Waters Micro-mass ZQ 2000 spectrophotometer
(Jamia Hamdard, New Delhi, India).
Synthesis of the 6-substituted-1,3-benzothiazol-2-amines
(4a–e)
Aryl amines (0.01 mol) and potassium thiocyanate (0.01 mol) in
glacial acetic acid (10%) were cooled and stirred. During stirring,
cold bromine (0.01 mol, 3 mL in 10 mL) was added dropwise.
Stirring was continued for an additional 3 h. The separated
hydrochloride salt was filtered off, washed with acetic acid,
dissolved in hot water, neutralized with aqueous ammonia
solution (25%), and filtered off. Finally, the products 4a–e were
washed with cold water, dried, and recrystallized from benzene.
Synthesis of the 2-(6-substituted-1,3-benzothiazol-2-
ylamino-N-(5-phenyl-substituted-1,3,4-thiadiazol-2-yl)-
acetamides (5a–x)
2-Chloro-N-(5-phenyl-substituted-1,3,4-thiadiazol-2-yl)acetamides
3a–f (0.01 mol) and 1,3-benzothiazol-2-amines 4a–e (0.01 mol)
were dissolved in ethanol and stirred for 4 h and filtered. The
filtrate so collected was left for crystallization overnight, to yield
the target products 5a–x.
Synthesis of the substituted (2E)-2-benzylidenehydrazine-
carbothioamides (1a–f)
Aryl aldehydes (0.05 mol) in warm alcohol (300 mL) and a solution
of thiosemicarbazide (0.05 mol) in 300 mL hot water were mixed
slowly with continuous stirring. The product (1a–f), which was
separated, was filtered off after cooling.
2-(1,3-Benzothiazol-2-ylamino)-N-(5-phenyl-1,3,4-thiadia-
zol-2-yl)acetamide (5a): Light-brown shiny crystalline pow-
der; solubility: chloroform, methanol. Elemental analysis (%):
Found C, 55.65; H, 3.52; N, 19.01; calcd. C, 55.57; H, 3.57; N, 19.06.
IR (KBr) n (cmꢀ1) 3418.51 (NH str), 3072.08 (C–H str aromatic),
–
–
–
1704.16 (C C str), 1624.60 (C O str), 1599.54 (C N str), 689.74
–
–
–
(C–S–C str); 1H NMR d (ppm) (400 MHz, CDCl3): 11.76 (s, 1H,
NH–CO), 8.68 (s, 1H, NH), 8.10 (d, 2H, 20,60HPh, J ¼ 7.2 Hz), 8.05 (m,
Synthesis of the 5-phenyl substituted-1,3,4-thiadiazol-2-
amines (2a–f)
3H, 30,40,50HPh), 8.02 (d, 1H, 400HBT, J ¼ 7.9 Hz), 7.97 (d, 1H, 700HBT
,
Compounds 1a–f (0.03 mol) were suspended in 300 mL of distilled
water and, after adding a mixture of citric acid (0.06 mol) and
sodium citrate (0.03 mol), were heated in the presence of ferric
chloride solution (0.15 mol) and stirred for 45 min. After cooling,
the solution mixture was neutralized by aqueous ammonia (10%),
rendering the solution to precipitate. The precipitate so obtained
J ¼ 7.9 Hz), 7.56 (m, 1H, 500HBT), 7.47 (m, 1H, 600HBT), 4.02 (s, 2H,
CH2); 13C NMR d (ppm) (DMSO-d6, TMS): 168.2 (NH–CO), 165.4 (C-2),
160.8 (C-200), 155.5 (C-5), 148.7 (C-800), 132.2 (C-10), 130.6 (C-900), 128.5
(C-400), 127.3 (C-700), 124.9 (C-500), 124.8 (C-600), 120.7 (C-20 and C-60),
120.5 (C-30 and C-50), 118.5 (C-40), 40.5 (CH2–NH); MS (70 eV):
m/z ¼ 368.31 (Mþ1).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
827
2-[(6-Bromo-1,3-benzothiazol-2-yl)amino]-N-(5-phenyl-
(C-700), 120.9 (C-500), 120.5 (C-20 and C-60), 120.1 (C-30 and C-50), 118.3
(C-40), 40.6 (CH2–NH), 22.4 (–CH3); MS (70 eV): m/z ¼ 382.11
(Mþ1).
1,3,4-thiadiazol-2-yl)acetamide (5b): Yellowish-brown shiny
crystalline powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 45.79; H, 2.69; N, 15.65; calcd. C, 45.75; H,
2.71; N, 15.69. IR (KBr) n (cmꢀ1) 3401.53 (NH str), 3084.70 (C–H str
2-(1,3-Benzothiazol-2-ylamino)-N-[5-(4-nitrophenyl)-1,3,4-
–
–
–
aromatic), 1699.22 (C C str), 1652.86 (C O str), 1607.14 (C N str),
–
–
–
thiadiazol-2-yl]acetamide (5f): Dark-brown crystalline pow-
der; solubility: chloroform, methanol. Elemental analysis (%):
Found C, 49.55; H, 2.90; N, 20.32; calcd. C, 49.51; H, 2.93; N, 20.38.
IR (KBr) n (cmꢀ1) 3416.06 (NH str), 3078.83 (C–H str aromatic),
1
883.78 (C–Br str), 692.45 (C–S–C str); H NMR d (ppm) (400 MHz,
CDCl3): 11.96 (s, 1H, NH–CO), 8.35 (s, 1H, NH), 8.04 (d, 2H, 20,60HPh
,
J ¼ 7.0 Hz), 7.94 (m, 3H, 30,40,50HPh), 7.92 (s, 1H, 700HBT), 7.20 (d, 1H,
500HBT, J ¼ 7.8 Hz), 6.89 (d, 1H, 400HBT, J ¼ 8.0 Hz), 4.12 (s, 2H, CH2);
13C NMR d (ppm) (DMSO-d6, TMS): 169.3 (NH–CO), 165.6 (C-2), 161.4
(C-200), 155.8 (C-5), 148.2 (C-800), 132.5 (C-10), 130.1 (C-900), 126.3
(C-600), 125.5 (C-400), 124.7 (C-700), 122.9 (C-500), 120.5 (C-20 and C-60),
120.4 (C-30 and C-50), 118.8 (C-40), 40.8 (CH2–NH); MS (70 eV):
m/z ¼ 447.38 (Mþ1), 448.19 (Mþ2).
–
–
–
1709.02 (C C str), 1647.26 (C O str), 1624.74 (C N str), 1596.39
–
–
–
1
–
(N O), 692.75 (C–S–C str); H NMR d (ppm) (400 MHz, CDCl ): 12.60
–
3
(s, 1H, NH–CO), 9.54 (s, 1H, NH), 8.90 (d, 2H, 30,50HPh, J ¼ 7.5 Hz),
8.50 (d, 2H, 20,60HPh, J ¼ 7.2 Hz), 7.88 (d, 1H, 400HBT, J ¼ 7.9 Hz), 7.54
(d, 1H, 700HBT, J ¼ 7.9 Hz), 7.25 (m, 1H, 500HBT), 7.13 (m, 1H, 600HBT),
4.13 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6, TMS): 168.3 (NH–CO),
165.6 (C-2), 160.5 (C-200), 155.2 (C-5), 146.9 (C-800), 132.5 (C-10), 130.8
(C-900), 130.1 (C-40), 128.3 (C-400), 127.9 (C-700), 125.7 (C-30 and C-50),
124.8 (C-500), 124.7 (C-600), 124.4 (C-20 and C-60), 40.7 (CH2–NH); MS
(70 eV): m/z ¼ 413.36 (Mþ1).
2-[(6-Chloro-1,3-benzothiazol-2-yl)amino]-N-(5-phenyl-
1,3,4-thiadiazol-2-yl)acetamide (5c): Light-yellow shiny crys-
talline powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 50.88; H, 2.98; N, 17.38; calcd. C, 50.81;
H, 3.01; N, 17.43. IR (KBr) n (cmꢀ1) 3416.11 (NH str), 3083.75 (C–H
2-[(6-Bromo-1,3-benzothiazol-2-yl)amino]-N-[5-(4-nitro-
–
–
–
–
str aromatic), 1703.05 (C C str), 1657.39 (C O str), 1609.13 (C N
–
–
phenyl)-1,3,4-thiadiazol-2-yl]acetamide (5g): Creamy-white
shiny needle-shaped crystals; solubility: chloroform, ethanol,
methanol. Elemental analysis (%): Found C, 41.37; H, 2.19; N,
17.16; calcd. C, 41.56; H, 2.26; N, 17.10. IR (KBr) n (cmꢀ1): 3472.20
–
str), 865.95 (C–Cl str), 691.02 (C–S–C str); 1H NMR d (ppm)
(400 MHz, CDCl3): 12.08 (s, 1H, NH–CO), 9.22 (s, 1H, NH), 8.07 (d,
2H, 20,60HPh, J ¼ 6.9 Hz), 8.03 (m, 3H, 30,40,50HPh), 7.98 (s, 1H,
700HBT), 7.25 (d, 1H, 500HBT, J ¼ 7.7 Hz), 7.01 (d, 1H, 400HBT, J ¼ 8.0 Hz),
4.34 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6, TMS): 168.8 (NH–CO),
165.2 (C-2), 161.8 (C-200), 155.6 (C-5), 148.9 (C-800), 132.7 (C-10), 129.8
(C-900), 128.4 (C-400), 125.7 (C-700), 124.8 (C-500), 124.1 (C-600), 120.6 (C-20
and C-60), 120.3 (C-30 and C-50), 118.9 (C-40), 40.7 (CH2–NH); MS
(70 eV): m/z ¼ 402.74 (Mþ1), 403.63 (Mþ2).
(NH str), 3190.65 (C–H str aromatic), 1598.70 (C C str), 1585.20
–
–
–
–
(C O str), 1552.42 (C N str), 1509.99 (N O), 879.38 (C–Br str),
–
–
–
663.39 (C–S–C str); 1H NMR d (ppm) (400 MHz, CDCl3): 12.67 (s, 1H,
NH–CO), 9.58 (s, 1H, NH), 8.83 (d, 2H, 30,50HPh, J ¼ 7.4 Hz), 8.41 (d,
2H, 20,60HPh, J ¼ 6.8 Hz), 7.80 (s, 1H, 700HBT), 7.09 (d, 1H, 500HBT
,
J ¼ 7.6 Hz), 6.97 (d, 1H, 400HBT, J ¼ 7.9 Hz), 4.16 (s, 2H, CH2); 13C NMR
d (ppm) (DMSO-d6, TMS): 168.7 (NH–CO), 165.3 (C-2), 160.8 (C-200),
156.2 (C-5), 146.7 (C-800), 132.6 (C-10), 130.4 (C-900), 130.2 (C-40), 126.4
(C-600), 125.5 (C-30 and C-50), 125.1 (C-400), 124.8 (C-700), 124.3 (C-20 and
C-60), 122.8 (C-500), 40.7 (CH2–NH); MS (70 eV): m/z ¼ 492.22 (Mþ1),
493.45 (Mþ2).
2-[(6-Fluoro-1,3-benzothiazol-2-yl)amino]-N-(5-phenyl-
1,3,4-thiadiazol-2-yl)acetamide (5d): Yellow crystalline pow-
der; solubility: chloroform, methanol. Elemental analysis (%):
Found C, 53.03; H, 3.09; N, 18.11; calcd. C, 52.97; H, 3.14; N, 18.17.
IR (KBr) n (cmꢀ1) 3441.52 (NH str), 3091.20 (C–H str aromatic),
–
–
–
1708.25 (C C str), 1646.28 (C O str), 1608.89 (C N str), 874.81
–
–
–
2-[(6-Chloro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-nitro-
(C–F str), 693.23 (C–S–C str); 1H NMR d (ppm) (400 MHz, CDCl3):
12.01 (s, 1H, NH–CO), 9.10 (s, 1H, NH), 8.06 (d, 2H, 20,60HPh
,
phenyl)-1,3,4-thiadiazol-2-yl]acetamide (5h): Light-yellow
shiny crystalline powder; solubility: chloroform, methanol.
Elemental analysis (%): Found C, 45.74; H, 2.46; N, 18.78; calcd.
C, 45.69; H, 2.48; N, 18.81. IR (KBr) n (cmꢀ1): 3456.75 (NH str),
J ¼ 7.3 Hz), 8.04 (s, 1H, 700HBT), 7.99 (m, 3H, 30,40,50HPh), 7.98 (d, 1H,
500HBT, J ¼ 7.7 Hz), 7.56 (d, 1H, 400HBT, J ¼ 7.9 Hz), 4.18 (s, 2H, CH2);
13C NMR d (ppm) (DMSO-d6, TMS): 168.6 (NH–CO), 165.4 (C-2), 160.9
(C-200), 156.1 (C-5), 155.4 (C-600), 148.1 (C-800), 132.9 (C-10), 129.3
(C-900), 120.8 (C-500), 120.5 (C-20 and C-60), 120.2 (C-30 and C-50), 118.7
(C-40), 118.2 (C-400), 114.5 (C-700), 40.8 (CH2–NH); MS (70 eV):
m/z ¼ 386.35 (Mþ1).
–
–
3090.30 (C–H str aromatic), 1707.62 (C C str), 1633.41 (C O str),
–
–
–
–
1534.11 (C N str), 1445.36 (N O), 859.19 (C–Cl str), 689.43 (C–S–C
–
–
str); 1H NMR d (ppm) (400 MHz, CDCl3): 12.68 (s, 1H, NH–CO), 9.62
(s, 1H, NH), 8.80 (d, 2H, 30,50HPh, J ¼ 7.2 Hz), 8.60 (d, 2H, 20,60HPh
,
J ¼ 7.0 Hz), 7.67 (s, 1H, 700HBT), 7.42 (d, 1H, 500HBT, J ¼ 7.7 Hz), 7.08
(d, 1H, 400HBT, J ¼ 8.1 Hz), 4.19 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-
d6, TMS): 168.5 (NH–CO), 165.2 (C-2), 160.9 (C-200), 156.1 (C-5), 148.3
(C-800), 132.4 (C-10), 130.4 (C-40), 128.6 (C-900), 128.4 (C-400), 126.5
(C-700), 125.4 (C-30 and C-50), 124.9 (C-20 and C-60), 124.7 (C-500), 124.3
(C-600), 40.9 (CH2–NH); MS (70 eV): m/z ¼ 447.56 (Mþ1), 448.40
(Mþ2).
2-[(6-Methyl-1,3-benzothiazol-2-yl)amino]-N-(5-phenyl-
1,3,4-thiadiazol-2-yl)acetamide (5e): Brown powder; solubil-
ity: chloroform, methanol. Elemental analysis (%): Found C, 56.69;
H, 3.92; N, 18.31; calcd. C, 56.67; H, 3.96; N, 18.36. IR (KBr) n (cmꢀ1
)
–
–
3400.93 (NH str), 3073.76 (C–H str aromatic), 1701.67 (C C str),
1
–
–
1653.77 (C O str), 1604.28 (C N str), 685.41 (C–S–C str); H NMR d
–
–
(ppm) (400 MHz, CDCl3): 11.82 (s, 1H, NH–CO), 8.87 (s, 1H, NH),
8.03 (d, 2H, 20,60HPh, J ¼ 7.0 Hz), 7.97 (m, 3H, 30,40,50HPh), 6.89 (d,
1H, 400HBT, J ¼ 7.8 Hz), 6.55 (d, 1H, 500HBT, J ¼ 8.0 Hz), 6.47 (m, 1H,
700HBT), 4.28 (s, 2H, CH2), 2.27 (s, 3H, CH3); 13C NMR d (ppm) (DMSO-
d6, TMS): 167.9 (NH–CO), 165.5 (C-2), 160.7 (C-200), 155.8 (C-5), 148.2
(C-800), 131.9 (C-10), 130.5 (C-900), 130.2 (C-600), 125.4 (C-400), 124.7
2-[(6-Fluoro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-nitro-
phenyl)-1,3,4-thiadiazol-2-yl]acetamide (5i): Light-yellow
needle shaped crystal; solubility: ethanol, chloroform. Elemental
analysis (%): Found C, 47.28; H, 2.52; N, 19.57; calcd. C, 47.44; H,
2.58; N, 19.52. IR (KBr) n (cmꢀ1) 3386.39 (NH str), 3071.08 (C–H str
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

828
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
–
–
–
–
aromatic), 1707.66 (C C str), 1640.16 (C O str), 1606.41 (C N str),
2-[(6-Chloro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-hydrox-
–
–
1576.52 (N O), 852.38 (C–F str), 689.43 (C–S–C str); 1H NMR d
–
–
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide (5m): Light-brown
crystalline powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 48.92; H, 2.84; N, 16.72; calcd. C, 48.86; H,
2.89; N, 16.76. IR (KBr) n (cmꢀ1) 3450.04 (NH str), 3394.14 (OH str),
(ppm) (400 MHz, CDCl3): 12.66 (s, 1H, NH–CO), 9.58 (s, 1H, NH),
8.80 (d, 2H, 30,50HPh, J ¼ 7.0 Hz), 8.41 (d, 2H, 20,60HPh, J ¼ 7.3 Hz),
8.06 (s, 1H, 700HBT), 7.99 (d, 1H, 500HBT, J ¼ 7.8 Hz), 7.62 (d, 1H,
400HBT, J ¼ 8.2 Hz), 4.18 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6,
TMS): 167.8 (NH–CO), 164.9 (C-2), 161.2 (C-200), 156.2 (C-5), 155.5 (C-
600), 147.9 (C-800), 132.7 (C-10), 130.4 (C-40), 128.7 (C-900), 126.3 (C-30
and C-50), 124.2 (C-20 and C-60), 120.8 (C-500), 118.1 (C-400), 114.6
(C-700), 41.2 (CH2–NH)); MS (70 eV): m/z ¼ 431.34 (Mþ1).
–
–
3080.77 (C–H str aromatic), 1693.13 (C C str), 1633.47 (C O str),
–
–
1
–
1609.31 (C N str), 837.90 (C–Cl str), 709.62 (C–S–C str); H NMR d
–
(ppm) (400 MHz, CDCl3): 12.79 (s, 1H, NH–CO), 12.14 (s, 1H, OH),
9.81 (s, 1H, NH), 8.02 (d, 2H, 20,60HPh, J ¼ 7.0 Hz), 7.93 (s, 1H, 700HBT),
7.68 (d, 2H, 30,50HPh, J ¼ 7.4 Hz), 7.62 (d, 1H, 500HBT, J ¼ 7.8 Hz), 7.44
(d, 1H, 400HBT, J ¼ 8.1 Hz), 4.22 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-
d6, TMS): 168.3 (NH–CO), 165.1 (C-2), 161.4 (C-200), 155.5 (C-5), 148.6
(C-800), 138.3 (C-40), 132.2 (C-10), 129.2 (C-900), 127.8 (C-400), 127.1 (C-30
and C-50), 124.8 (C-700), 124.5 (C-500), 124.3 (C-600), 123.6 (C-20 and
C-60), 40.8 (CH2–NH); MS (70 eV): m/z ¼ 418.71 (Mþ1), 419.54 (Mþ2).
2-[(6-Methyl-1,3-benzothiazol-2-yl)amino]-N-[5-(4-nitro-
phenyl)-1,3,4-thiadiazol-2-yl]acetamide (5j): Light-brown
shiny crystalline powder; solubility: chloroform, methanol.
Elemental analysis (%): Found C, 50.75; H, 3.27; N, 19.67; calcd.
C, 50.69; H, 3.31; N, 19.71. IR (KBr) n (cmꢀ1) 3401.16 (NH str),
–
–
–
–
3081.33 (C–H str aromatic), 1696.17 (C C str), 1652.02 (C O str),
2-[(6-Fluoro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-hydrox-
1620.91 (C N str), 1584.77 (N O), 694.98 (C–S–C str); 1H NMR d
–
–
–
–
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide (5n): White crys-
talline powder; solubility: DMF. Elemental analysis: Found C,
50.70; H, 2.95; N, 17.54; calcd. C, 50.86; H, 3.01; N, 17.45. IR (KBr) n
(cmꢀ1) 3390.24 (NH str), 2924.52 (OH str), 2853.17 (C–H str
(ppm) (400 MHz, CDCl3): 12.64 (s, 1H, NH–CO), 9.57 (s, 1H, NH),
8.90 (d, 2H, 30,50HPh, J ¼ 7.3 Hz), 8.30 (d, 2H, 20,60HPh, J ¼ 7.0 Hz),
6.81 (d, 1H, 400HBT, J ¼ 7.9 Hz), 6.57 (d, 1H, 500HBT, J ¼ 7.6 Hz), 6.50
(m, 1H, 700HBT), 4.16 (s, 2H, CH2), 2.19 (s, 3H, CH3); 13C NMR d (ppm)
(DMSO-d6, TMS): 168.3 (NH–CO), 164.8 (C-2), 160.5 (C-200), 155.7
(C-5), 148.1 (C-800), 131.8 (C-10), 130.5 (C-40), 130.3 (C-900), 130.1 (C-600),
126.0 (C-30 and C-50), 125.4 (C-400), 124.9 (C-700), 124.8 (C-20 and C-60),
120.6 (C-500), 40.6 (CH2–NH), 22.6 (–CH3); MS (70 eV): m/z ¼ 427.30
(Mþ1).
–
–
–
aromatic), 1692.23 (C C str), 1639.20 (C O str), 1608.34 (C N str),
–
–
–
838.88 (C–F str), 708.71 (C–S–C str); 1H NMR d (ppm) (400 MHz,
DMSO-d6): 12.77 (s, 1H, NH–CO), 12.12 (s, 1H, OH), 9.70 (s, 1H, NH),
8.04 (s, 1H, 700HBT), 8.01 (d, 2H, 20,60HPh, J ¼ 6.8 Hz), 7.91 (d, 1H,
500HBT, J ¼ 7.8 Hz), 7.74 (d, 1H, 400HBT, J ¼ 8.0 Hz), 7.71 (d, 2H,
30,50HPh, J ¼ 7.5 Hz), 4.25 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6,
TMS): 168.3 (NH–CO), 165.5 (C-2), 161.5 (C-200), 155.8 (C-5), 155.3 (C-
600), 148.4 (C-800), 137.9 (C-40), 132.4 (C-10), 129.3 (C-900), 127.3 (C-30
and C-50), 123.1 (C-20 and C-60), 120.4 (C-500), 118.2 (C-400), 114.6
(C-700), 40.8 (CH2–NH)); MS (70 eV): m/z ¼ 402.50 (Mþ1).
2-(1,3-Benzothiazol-2-ylamino)-N-[5-(4-hydroxyphenyl)-
1,3,4-thiadiazol-2-yl]acetamide (5k): Cream crystalline pow-
der; solubility: chloroform, methanol. Elemental analysis (%):
Found C, 53.30; H, 3.39; N, 18.22; calcd. C, 53.25; H, 3.42; N, 18.26.
IR (KBr) n (cmꢀ1): 3446.27 (NH str), 3263.11 (OH str), 3080.00 (C–H
N-[5-(4-Hydroxyphenyl)-1,3,4-thiadiazol-2-yl]-2-[(6-meth-
–
–
–
–
–
–
str aromatic), 1636.24 (C C str), 1530.12 (C O str), 1510.29 (C N
yl-1,3-benzothiazol-2-yl)amino]acetamide (5o): Creamish-
white powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 54.45; H, 3.75; N, 17.58; calcd. C, 54.39;
H, 3.80; N, 17.62. IR (KBr) n (cmꢀ1) 3395.04 (NH str), 3052.72 (OH
str), 700.08 (C–S–C str); 1H NMR d (ppm) (400 MHz, CDCl3): 12.72 (s,
1H, NH–CO), 12.60 (s, 1H, OH), 9.73 (s, 1H, NH), 7.99 (d, 1H, 400HBT
J ¼ 7.8 Hz), 7.97 (d, 2H, 20,60HPh, J ¼ 6.8 Hz), 7.84 (d, 1H, 700HBT
,
,
J ¼ 7.9 Hz), 7.58 (m, 1H, 500HBT), 7.54 (d, 2H, 30,50HPh, J ¼ 7.2 Hz),
7.49 (m, 1H, 600HBT), 4.18 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6,
TMS): 168.1 (NH–CO), 165.3 (C-2), 160.6 (C-200), 155.4 (C-5), 148.6
(C-800), 136.8 (C-40), 132.2 (C-10), 130.7 (C-900), 128.4 (C-400), 127.9
(C-700), 127.5 (C-30 and C-50), 124.5 (C-500), 124.2 (C-600), 123.6 (C-20 and
C-60), 40.5 (CH2–NH); MS (70 eV): m/z ¼ 384.22 (Mþ1).
str), 2920.61 (C–H str aromatic), 1693.11 (C C str), 1636.03 (C O
–
–
–
–
str), 1608.34 (C N str), 705.58 (C–S–C str); 1H NMR d (ppm)
–
–
(400 MHz, CDCl3): 12.76 (s, 1H, NH–CO), 12.01 (s, 1H, OH), 9.68 (s,
1H, NH), 7.98 (d, 2H, 20,60HPh, J ¼ 7.0 Hz), 7.66 (d, 2H, 30,50HPh
J ¼ 7.4 Hz), 6.79 (d, 1H, 400HBT, J ¼ 7.9 Hz), 6.56 (d, 1H, 500HBT
,
,
J ¼ 7.5 Hz), 6.48 (m, 1H, 700HBT), 4.23 (s, 2H, CH2), 2.27 (s, 3H, CH3);
13C NMR d (ppm) (DMSO-d6, TMS): 168.4 (NH–CO), 165.4 (C-2), 161.2
(C-200), 155.4 (C-5), 148.3 (C-800), 137.9 (C-40), 130.8 (C-10), 130.4 (C-900),
130.1 (C-600), 127.2 (C-30 and C-50), 125.1 (C-400), 124.8 (C-700), 123.6
(C-20 and C-60), 120.2 (C-500), 40.8 (CH2–NH), 22.5 (–CH3); MS (70 eV):
m/z ¼ 398.09 (Mþ1).
2-[(6-Bromo-1,3-benzothiazol-2-yl)amino]-N-[5-(4-hydrox-
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide (5l): Light-yellow
shiny crystalline powder; solubility: chloroform, methanol.
Elemental analysis (%): Found C, 44.22; H, 2.59; N, 15.09; calcd.
C, 44.16; H, 2.62; N, 15.15. IR (KBr) n (cmꢀ1): 3451.94 (NH str),
–
2-(1,3-Benzothiazol-2-ylamino)-N-[5-(4-methoxyphenyl)-
3269.75 (OH str), 3086.55 (C–H str aromatic), 1631.49 (C C str),
–
1,3,4-thiadiazol-2-yl]acetamide (5p): Yellowish-brown shiny
crystalline powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 54.44; H, 3.76; N, 17.59; calcd. C, 54.39; H,
3.80; N, 17.62. IR (KBr) n (cmꢀ1) 3399.03 (NH str), 3053.28 (C–H str
–
–
–
–
1527.32 (C O str), 1443.40 (C N str), 862.01 (C–Br str), 695.28
(C–S–C str); 1H NMR d (ppm) (400 MHz, CDCl3): 12.78 (s, 1H,
NH–CO), 12.12 (s, 1H, OH), 9.76 (s, 1H, NH), 8.01 (d, 2H, 20,60HPh
,
J ¼ 7.1 Hz), 7.89 (s, 1H, 700HBT), 7.59 (d, 2H, 30,50HPh, J ¼ 7.5 Hz), 7.15
(d, 1H, 500HBT, J ¼ 7.7 Hz), 6.91 (d, 1H, 400HBT, J ¼ 8.0 Hz), 4.23 (s, 2H,
CH2); 13C NMR d (ppm) (DMSO-d6, TMS): 168.2 (NH–CO), 165.6 (C-2),
160.9 (C-200), 155.8 (C-5), 148.1 (C-800), 137.1 (C-40), 132.5 (C-10), 130.2
(C-900), 127.5 (C-30 and C-50), 126.3 (C-600), 125.3 (C-400), 124.7 (C-700),
123.3 (C-20 and C-60), 122.8 (C-500), 40.7 (CH2–NH); MS (70 eV):
m/z ¼ 463.12 (Mþ1), 464.00 (Mþ2).
–
–
–
aromatic), 1698.51 (C C str), 1642.36 (C O str), 1605.79 (C N str),
–
–
–
1268.27 (O–CH3), 681.02 (C–S–C str); 1H NMR d (ppm) (400 MHz,
CDCl3): 12.66 (s, 1H, NH–CO), 9.54 (s, 1H, NH), 7.96 (d, 1H, 400HBT
J ¼ 8.0 Hz), 7.86 (d, 2H, 20,60HPh, J ¼ 6.7 Hz), 7.75 (d, 1H, 700HBT
,
,
J ¼ 7.7 Hz), 7.59 (m, 1H, 500HBT), 7.58 (d, 2H, 30,50HPh, J ¼ 7.2 Hz),
7.51 (m, 1H, 600HBT), 4.28 (s, 2H, CH2), 4.19 (s, 3H, OCH3); 13C NMR d
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
829
(ppm) (DMSO-d6, TMS): 168.3 (NH–CO), 165.3 (C-2), 160.5 (C-200),
158.5 (C-40), 155.6 (C-5), 148.1 (C-800), 132.2 (C-10), 130.2 (C-900), 128.7
(C-400), 127.4 (C-700), 124.9 (C-500), 124.4 (C-600), 123.7 (C-20 and C-60),
116.6 (C-30 and C-50), 45.6 (O–CH3), 40.9 (CH2–NH); MS (70 eV):
m/z ¼ 398.14 (Mþ1).
nol. Elemental analysis: Found C, 55.50; H, 4.22; N, 17.09; calcd. C,
55.46; H, 4.16; N, 17.02. IR (KBr) n (cmꢀ1) 3408.02 (NH str), 3082.79
–
–
(C–H str aromatic), 1701.08 (C C str), 1684.96 (C O str), 1617.18
–
–
(C N str), 1274.54 (O–CH ), 697.32 (C–S–C str); 1H NMR d (ppm)
–
–
3
(400 MHz, CDCl3): 12.70 (s, 1H, NH–CO), 9.70 (s, 1H, NH), 7.96 (d,
2H, 20,60HPh, J ¼ 6.7 Hz), 7.68 (d, 2H, 30,50HPh, J ¼ 7.0 Hz), 6.82 (d,
1H, 400HBT, J ¼ 8.1 Hz), 6.60 (d, 1H, 500HBT, J ¼ 7.9 Hz), 6.44 (m, 1H,
2-[(6-Bromo-1,3-benzothiazol-2-yl)amino]-N-[5-(4-methox-
700HBT), 4.21 (s, 2H, CH2), 3.58 (s, 3H, OCH3), 2.27 (s, 3H, CH3); 13
C
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide (5q): Yellowish-
white shiny powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 45.42; H, 2.92; N, 14.67; calcd. C, 45.38; H,
2.96; N, 14.70. IR (KBr) n (cmꢀ1): 3450.70 (NH str), 3080.37 (C–H str
NMR d (ppm) (DMSO-d6, TMS): 168.3 (NH–CO), 165.5 (C-2), 160.3 (C-
200), 158.3 (C-40), 155.6 (C-5), 148.2 (C-800), 131.1 (C-10), 130.8 (C-900),
130.4 (C-600), 125.4 (C-400), 124.6 (C-700), 123.6 (C-20 and C-60), 120.5 (C-
500), 116.4 (C-30 and C-50), 45.6 (O–CH3), 40.8 (CH2–NH), 22.6 (–CH3);
MS (70 eV): m/z ¼ 412.31 (Mþ1).
–
–
–
aromatic), 1693.01 (C C str), 1633.84 (C O str), 1609.23 (C N str),
–
–
–
1
1275.66 (O–CH3), 837.59 (C–Br str), 709.86 (C–S–C str); H NMR d
(ppm) (400 MHz, CDCl3): 12.68 (s, 1H, NH–CO), 9.57 (s, 1H, NH),
7.98 (d, 2H, 20,60HPh, J ¼ 6.8 Hz), 7.88 (s, 1H, 700HBT), 7.59 (d, 2H,
30,50HPh, J ¼ 7.1 Hz), 7.25 (d, 1H, 500HBT, J ¼ 7.7 Hz), 6.99 (d, 1H,
400HBT, J ¼ 8.0 Hz), 4.32 (s, 2H, CH2), 4.20 (s, 3H, OCH3); 13C NMR d
(ppm) (DMSO-d6, TMS): 168.7 (NH–CO), 165.2 (C-2), 160.9 (C-200),
158.2 (C-40), 155.5 (C-5), 148.3 (C-800), 132.3 (C-10), 130.4 (C-900), 126.2
(C-600), 125.7 (C-400), 124.7 (C-700), 123.1 (C-20 and C-60), 122.6 (C-500),
116.2 (C-30 and C-50), 45.4 (O–CH3), 40.8 (CH2–NH); MS (70 eV):
m/z ¼ 477.06 (Mþ1), 478.02 (Mþ2).
2-(6-Chlorobenzo[d]thiazol-2-ylamino-N-(5-(4-chloro-
phenyl)-1,3,4-thiadiazol-2-yl)acetamide (5u): Creamy-white
shiny crystals; solubility: chloroform, ethanol. Elemental analy-
sis: Found C, 46.71; H, 2.46; N, 15.99; calcd. C, 46.79; H, 2.54; N,
16.05. IR (KBr) n (cmꢀ1) 3418.54 (NH str), 3072.84 (C–H str
–
–
–
aromatic), 1704.67 (C C str), 1624.09 (C O str), 1599.56 (C N str),
–
–
–
1
836.98 (C–Cl str), 689.71 (C–S–C str); H NMR d (ppm) (400 MHz,
DMSO-d6): 12.68 (s, 1H, NH–CO), 9.58 (s, 1H, NH), 7.91 (d, 2H,
30,50HPh, J ¼ 7.4 Hz), 7.88 (s, 1H, 700HBT), 7.82 (d, 2H, 20,60HPh
,
,
J ¼ 7.0 Hz), 7.32 (d, 1H, 500HBT, J ¼ 7.8 Hz), 7.04 (d, 1H, 400HBT
2-[(6-Chloro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-methox-
J ¼ 8.0 Hz), 4.43 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6, TMS):
168.4 (NH–CO), 165.4 (C-2), 160.3 (C-200), 155.5 (C-5), 148.2 (C-800),
134.3 (C-40), 132.7 (C-10), 129.1 (C-900), 128.6 (C-30 and C-50), 127.1
(C-400), 125.3 (C-700), 124.7 (C-500), 124.4 (C-600), 124.2 (C-20 and C-60),
40.8 (CH2–NH); MS (70 eV): m/z ¼ 437.35 (Mþ1), 438.54 (Mþ2),
440.85 (Mþ4).
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide
(5r): Creamy
white amorphous flakes; solubility: DMF. Elemental analysis:
Found C, 49.88; H, 3.22; N, 16.29; calcd. C, 50.05; H, 3.27; N, 16.21.
IR (KBr) n (cmꢀ1) 3456.78 (NH str), 3081.69 (C–H str aromatic),
–
–
–
1693.19 (C C str), 1634.38 (C O str), 1609.31 (C N str), 1275.68
–
–
–
(O–CH3), 837.92 (C–Cl str), 700.03 (C–S–C str); 1H NMR d (ppm)
(400 MHz, DMSO-d6): 12.71 (s, 1H, NH–CO), 9.61 (s, 1H, NH), 7.91
(s, 1H, 700HBT), 7.86 (d, 2H, 20,60HPh, J ¼ 7.0 Hz), 7.71 (d, 2H, 30,50HPh
,
,
2-(6-Bromobenzo[d]thiazol-2-ylamino)-N-(5-(4-chloro-
J ¼ 7.5 Hz), 7.30 (d, 1H, 500HBT, J ¼ 7.6 Hz), 7.01 (d, 1H, 400HBT
phenyl)-1,3,4-thiadiazol-2-yl)acetamide (5v): Light-brown
crystals; solubility: chloroform, ethanol. Elemental analysis:
Found C, 42.53; H, 2.40; N, 14.52; calcd. C, 42.47; H, 2.31; N,
14.57. IR (KBr) n (cmꢀ1) 3415.49 (NH str), 3070.44 (C–H str
J ¼ 8.0 Hz), 4.39 (s, 2H, CH2), 4.26 (s, 3H, OCH3); 13C NMR d (ppm)
(DMSO-d6, TMS): 168.5 (NH–CO), 165.4 (C-2), 160.3 (C-200), 158.3
(C-40), 155.5 (C-5), 148.2 (C-800), 132.7 (C-10), 129.1 (C-900), 127.1 (C-400),
125.3 (C-700), 124.7 (C-500), 124.4 (C-600), 123.2 (C-20 and C-60), 116.6
(C-30 and C-50), 45.6 (O–CH3), 40.8 (CH2–NH)); MS (70 eV):
m/z ¼ 433.10 (Mþ1), 434.40 (Mþ2).
–
–
–
aromatic), 1703.59 (C C str), 1622.00 (C O str), 1600.03 (C N str),
–
–
–
1
838.94 (C–Br str), 832.09 (C–Cl str), 686.57 (C–S–C str); H NMR d
(ppm) (400 MHz, DMSO-d6): 12.62 (s, 1H, NH–CO), 9.55 (s, 1H, NH),
7.89 (d, 2H, 30,50HPh, J ¼ 7.5 Hz), 7.86 (s, 1H, 700HBT), 7.79 (d, 2H,
20,60HPh, J ¼ 7.0 Hz), 7.14 (d, 1H, 500HBT, J ¼ 7.7 Hz), 7.01 (d, 1H,
400HBT, J ¼ 8.0 Hz), 4.39 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6,
TMS): 168.8 (NH–CO), 162.9 (C-2), 160.8 (C-200), 154.8 (C-5), 147.6 (C-
800), 138.4 (C-40), 132.9 (C-10), 130.5 (C-900), 127.8 (C-30 and C-50), 126.1
(C-600), 125.5 (C-400), 124.6 (C-700), 123.9 (C-20 and C-60), 122.3 (C-500),
39.9 (CH2–NH); MS (70 eV): m/z ¼ 481.51 (Mþ1), 482.19 (Mþ2),
484.86 (Mþ4).
2-[(6-Fluoro-1,3-benzothiazol-2-yl)amino]-N-[5-(4-methox-
yphenyl)-1,3,4-thiadiazol-2-yl]acetamide (5s): Yellow shiny
crystalline powder; solubility: chloroform, methanol. Elemental
analysis (%): Found C, 52.10; H, 3.36; N, 16.82; calcd. C, 52.04; H,
3.40; N, 16.86. IR (KBr) n (cmꢀ1) 3392.13 (NH str), 3055.60 (C–H str
–
–
–
aromatic), 1693.14 (C C str), 1639.22 (C O str), 1608.73 (C N str),
–
–
–
1275.86 (O–CH3), 837.19 (C–F str), 708.70 (C–S–C str); 1H NMR d
(ppm) (400 MHz, CDCl3): 12.70 (s, 1H, NH–CO), 9.59 (s, 1H, NH),
8.08 (s, 1H, 700HBT), 8.01 (d, 1H, 500HBT, J ¼ 7.9 Hz), 7.89 (d, 1H,
400HBT, J ¼ 8.0 Hz), 7.84 (d, 2H, 20,60HPh, J ¼ 7.2 Hz), 7.69 (d, 2H,
30,50HPh, J ¼ 7.5 Hz), 4.38 (s, 2H, CH2), 4.24 (s, 3H, OCH3); 13C NMR d
(ppm) (DMSO-d6, TMS): 168.4 (NH–CO), 165.3 (C-2), 160.8 (C-200),
158.3 (C-40), 155.5 (C-5), 155.1 (C-600), 148.1 (C-800), 132.4 (C-10), 129.3
(C-900), 123.7 (C-20 and C-60), 120.4 (C-500), 118.4 (C-400), 116.3 (C-30
and C-50), 114.5 (C-700), 45.5 (O–CH3), 40.9 (CH2–NH); MS (70 eV):
m/z ¼ 416.18 (Mþ1).
N-(5-(4-Bromophenyl)-1,3,4-thiadiazol-2-yl)-2-(6-
chlorobenzo[d]thiazol-2-ylamino)acetamide (5w): Creamy-
yellow shiny crystals; solubility: chloroform, methanol. Elemen-
tal analysis: Found C, 42.50; H, 2.39; N, 14.49; calcd. C, 42.47; H,
2.31; N, 14.57. IR (KBr) n (cmꢀ1) 3417.42 (NH str), 3070.90 (C–H str
–
–
–
aromatic), 1702.55 (C C str), 1625.67 (C O str), 1596.24 (C N str),
–
–
–
1
836.74 (C–Br str), 833.01 (C–Cl str), 687.05 (C–S–C str); H NMR d
(ppm) (400 MHz, DMSO-d6): 12.63 (s, 1H, NH–CO), 9.56 (s, 1H, NH),
7.90 (s, 1H, 700HBT), 7.84 (d, 2H, 30,50HPh, J ¼ 7.5 Hz), 7.66 (d, 2H,
20,60HPh, J ¼ 7.2 Hz), 7.30 (d, 1H, 500HBT, J ¼ 7.8 Hz), 7.04 (d, 1H,
400HBT, J ¼ 7.9 Hz), 4.31 (s, 2H, CH2); 13C NMR d (ppm) (DMSO-d6,
TMS): 167.8 (NH–CO), 163.4 (C-2), 161.2 (C-200), 155.0 (C-5), 148.3
N-[5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl]-2-[(6-meth-
yl-1,3-benzothiazol-2-yl)amino]acetamide (5t): Yellowish-
brown shiny crystalline powder; solubility: chloroform, metha-
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N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
(C-800), 137.7 (C-40), 132.5 (C-10), 131.4 (C-900), 126.8 (C-30 and C-50),
126.5 (C-600), 125.7 (C-400), 124.9 (C-700), 123.5 (C-20 and C-60), 122.8
(C-500), 40.4 (CH2–NH); MS (70 eV): m/z ¼ 481.54 (Mþ1), 482.27
(Mþ2), 484.91 (Mþ4).
scPTZ test
In the scPTZ test [19], a dose of pentylenetetrazol of 70 mg/kg was
utilized. This produced clonic seizures in >95% of the animals,
lasting for a period of at least 5 s. The test compounds were
administered at three graded doses, viz., 30, 100, and 300 mg/kg
intraperitoneally. At the anticipated time, the convulsant was
administered as 0.5% solution, subcutaneously, in the posterior
midline. Animals were observed over a 30-min period. Absence of
clonic spasms in half or more of the animals in the observed time
period indicates the ability of a compound to abolish the effect of
pentylenetetrazol at the seizure threshold.
2-(6-Bromobenzo[d]thiazol-2-ylamino)-N-(5-(4-bromo-
phenyl)-1,3,4-thiadiazol-2-yl)acetamide (5x): Dark yellow-
ish crystals; solubility: chloroform, ethanol. Elemental analysis:
Found C, 38.79; H, 2.06; N, 13.39; calcd. C, 38.87; H, 2.11; N, 13.33.
IR (KBr) n (cmꢀ1) 3416.07 (NH str), 3073.99 (C–H str aromatic),
–
–
–
1701.65 (C C str), 1623.75 (C O str), 1597.98 (C N str), 837.06
–
–
–
(C–Br str), 688.25 (C–S–C str); 1H NMR d (ppm) (400 MHz, DMSO-d6):
12.58 (s, 1H, NH–CO), 9.55 (s, 1H, NH), 7.85 (s, 1H, 700HBT), 7.81 (d,
2H, 20,60HPh, J ¼ 6.7 Hz), 7.64 (d, 2H, 30,50HPh, J ¼ 7.0 Hz), 7.17 (d,
1H, 500HBT, J ¼ 7.7 Hz), 7.00 (d, 1H, 400HBT, J ¼ 8.0 Hz), 4.24 (s, 2H,
CH2); 13C NMR d (ppm) (DMSO-d6, TMS): 168.9 (NH–CO), 163.1 (C-2),
160.5 (C-200), 155.2 (C-5), 147.8 (C-800), 137.5 (C-40), 132.5 (C-10), 130.4
(C-900), 126.7 (C-30 and C-50), 126.0 (C-600), 125.4 (C-400), 124.8 (C-700),
123.5 (C-20 and C-60), 122.2 (C-500), 40.1 (CH2–NH); MS (70 eV):
m/z ¼ 526.09 (Mþ1), 527.00 (Mþ2), 529.79 (Mþ4).
Minimal motor impairment test (Rotarod test)
The minimal motor impairment is measured in mice by the
Rotarod test [20]. The mice were trained to stay on an accelerating
Rotarod of 3.2 cm in diameter, rotating at 10 rpm. The animals
were injected intraperitoneally with the test compounds at a dose
of 25 mg/kg. Neurotoxicity was indicated by the inability of the
animal to maintain equilibration on the rod for at least 1 min in
each of the three trials. The dose at which 50% of the animals were
unable to balance themselves and fell off the rotating rod was
determined.
Pharmacology
The investigations were conducted on Swiss male albino mice
(20 ꢁ 2 g) and adult Wistar rats (180 ꢁ 10 g). The animals
were housed in wire-mesh cages under laboratory conditions
(26 ꢁ 2°C, 12 h/12 h light/dark). They were allowed to acclimatize,
with free access to food and water, for a 24-h period before testing.
All the experimental protocols were carried out with the
permission from the Institutional Animal Ethics Committee
(IAEC). Animals were obtained from the Central Animal House
Facility (173/CPCSEA, 28 January, 2000), Hamdard University,
New Delhi.
Quantitative anticonvulsant study
The quantification study of the selected compounds for the
determination of the median effective dose (ED50), the median
neurotoxic dose (TD50), and the protective index (PI) was done
using the literary procedure. For the determination of ED50 and
TD50 values, groups of six mice were given
a range of
intraperitoneal doses of the test drug, until at least three points
were established in the range of 10–90% seizure protection or
minimal observed neurotoxicity. From the plots of these data, the
respective ED50 and TD50 values, 95% confidence intervals, slope
of the regression line, and the standard error of the slope were
calculated by means of a computer program [22].
Preliminary anticonvulsant screening
The synthesized compounds were assessed for anticonvulsant
activity using the MES test and the scPTZ test. The electro-
convulsometer (Ugo Basile ECT unit, pulse generator 57800;
Comerio VA, Italy) with an ear clip electrode was used for the
assessment of the anticonvulsant potential against electroshock-
induced seizures. Neurotoxicity was ascertained by the minimal
motor impairment test using the Rotarod test (Ugo Basile Srl
motor function unit, 47600–Mouse Rota-Rod; Comerio VA, Italy).
Four animals (Swiss albino mice) were selected for each treatment
and standard group. Phenytoin and carbamazepine were used as
standards.
Psychomotor seizure test
The psychomotor seizure analysis was executed according to the
protocol first described by Brown et al. [24] and more recently by
Barton et al. [25] and Kaminski et al. [26]. The kernels employed for
this alternate electroshock paradigm included a constant-current
corneal stimulation (0.2 ms rectangular pulse width) at a low
frequency (6 Hz) for a longer duration (3 s) at a dose of 100 mg/kg
intraperitoneally in a group of four mice. The mice were
manually restrained and instantly released into the observation
cage following the stimulation. The stunned posture colligating
rearing, forelimb automatic movements, and clonus, twitching of
the vibrissae and Straub tail conduced to the seizure. The
duration of seizure in untreated animals varied from 60 to 120 s,
following which the animals resumed their normal exploratory
behavior. Protection is manifested by attainment of exploratory
behavior within 10 s from the stimulation.
MES test
In the MES test [18], mice were pre-screened for 24 h before
delivering the maximal electroshock (50 mA, 60 Hz, 0.2 s dura-
tion) by means of corneal electrodes. A drop of 0.9% sodium
chloride was instilled in each eye prior to the application of the
electrodes, in order to prevent death of the animal. The test
compounds were suspended in a 0.5% methyl cellulose/water
mixture or in polyethylene glycol (PEG). The test solutions of each
compound at three doses (30, 100, and 300 mg/kg body mass)
were administered intraperitoneally and the anticonvulsant
activity was assessed after 0.5- and 4.0-h intervals, respectively.
Abolition of the hind limb tonic extensor component of
the seizure in half or more of the animals was defined as
protection.
GABA estimation
The most active compounds found after initial anticonvulsant
screening were subjected to neurochemical estimation of the
GABA levels in adult Wistar rat brain, to assess the effects of the
synthesized compounds on the GABA levels in various regions of
the rat brain. Adult Wistar rats weighing 180 ꢁ 10 g were used in
this study. The animals were divided into groups of six. The
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Arch. Pharm. Chem. Life Sci. 2013, 346, 819–831
Benzothiazole-1,3,4-thiadiazole Anticonvulsant Conjugates
831
control group was treated only with the vehicle (30% v/v PEG-400).
The animals were sacrificed after 2 h of intraperitoneal drug
administration by decapitation, and the brain regions, i.e.,
midbrain, olfactory lobe, cerebellum, and medulla oblongata,
were transferred to separate vessel containing 6–8 mL of ice-cold
80% ethanol and processed further according to the reported
procedure [27].
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Liver function test
The animals were divided into groups of six, and the control
group received a basal diet and vehicle (0.5% methylcellulose).
The active drugs were administered orally to each animal at a
dose of 25 mg/kg/day (in 0.5% methylcellulose) for 2 weeks. After
the stipulated period, each animal was anesthetized by anesthetic
ether and blood was collected by cardiac puncture to assess the
biochemical parameters such as SGOT, SGPT, alkaline phosphate,
and total protein, according to the 2,4-dinitrophenyl hydrazine
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All the statistical analyses were performed using the software
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The authors extend their gratitude to Jamia Hamdard, New Delhi,
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