Novel benzothiazole hydrazine carboxamide hybrid scaffolds as potential in vitro GABA AT enzyme inhibitors: Synthesis, molecular docking and antiepileptic evaluation

Article abstract of DOI:10.1016/j.bmcl.2019.05.007

In the present study, a series of newer benzothiazole derivatives containing thiazolidin-4-one (5a-g) and azetidin-2-one (6a-g), were synthesized by the cyclization of benzothiazolyl arylidene hydrazine carboxamide derivatives with thioglycolic acid and chloroacetyl chloride, respectively. Results of in vivo anticonvulsant screening revealed that compounds having 2,4-dicholoro (5c and 6c) and 4-nitro substituent (5g) at the phenyl ring have promising anticonvulsant activities without any neurotoxicity. Selected compounds were also evaluated for their in vitro GABA AT inhibition. The results indicated that compound 5c (IC₅₀ 15.26 μM) exhibited excellent activity as compared to the standard drug vigabatrin (IC₅₀ 39.72 μM) suggesting the potential of these benzothiazole analogues as new anticonvulsant agents.

Full text of DOI:10.1016/j.bmcl.2019.05.007

Accepted Manuscript
Novel benzothiazole hydrazine carboxamide hybrid scaffolds as potential in vi-
tro GABA AT enzyme inhibitors: Synthesis, molecular docking and antiepi-
leptic evaluation
Sadaf Jamal Gilani, Mohd. Zaheen Hassan, Syed Sarim Imam, Chandra Kala,
Surya Prakash Dixit
PII:
S0960-894X(19)30293-8
https://doi.org/10.1016/j.bmcl.2019.05.007
BMCL 26427
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 January 2019
2 May 2019
5 May 2019
Please cite this article as: Jamal Gilani, S., Zaheen Hassan, Mohd., Sarim Imam, S., Kala, C., Prakash Dixit, S.,
Novel benzothiazole hydrazine carboxamide hybrid scaffolds as potential in vitro GABA AT enzyme inhibitors:
Synthesis, molecular docking and antiepileptic evaluation, Bioorganic & Medicinal Chemistry Letters (2019), doi:
https://doi.org/10.1016/j.bmcl.2019.05.007
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Novel benzothiazole hydrazine carboxamide hybrid scaffolds as potential in vitro GABA AT
enzyme inhibitors: Synthesis, molecular docking and antiepileptic evaluation
a*
b
c
c
Sadaf Jamal Gilani , Mohd. Zaheen Hassan , Syed Sarim Imam , Chandra Kala , Surya
Prakash Dixitd
aCollege of Pharmacy, Jouf University, Aljouf, Sakaka, KSA.
bCollege of Pharmacy, King Khalid University, Abha, KSA.
cGlocal School of Pharmacy, Glocal University, Saharanpur, UP, India
dKIET School of Pharmacy, Ghaziabad, UP, India.
Corresponding Author:
Dr. Sadaf Jamal Gilani
Assistant Professor
Department of Pharmaceutical Chemistry
College of Pharmacy, Jouf University
Aljouf, Sakaka, KSA
E-mail: gilanisadaf@gmail.com
sjjilani@ju.edu.sa
Tel.: +966 507686858
1

Abstract:
In the present study, a series of newer benzothiazole derivatives containing thiazolidin-4-one (5a-g)
and azetidin-2-one (6a-g), were synthesized by the cyclization of benzothiazolyl arylidene
hydrazine carboxamide derivatives with thioglycolic acid and chloroacetyl chloride, respectively.
Results of in vivo anticonvulsant screening revealed that compounds having 2,4-dicholoro (5c and
6
c) and 4-nitro substituent (5g) at the phenyl ring have promising anticonvulsant activities without
any neurotoxicity. Selected compounds were also evaluated for their in vitro GABA AT inhibition.
The results indicated that compound 5c (IC 15.26ꢀμM) exhibited excellent activity as compared to
5
0
the standard drug vigabatrin (IC50 39.72 μM) suggesting the potential of these benzothiazole
analogues as new anticonvulsant agents.
Keywords: Benzothiazole, Antiepileptic, γ-aminobutyric acid aminotransferase (GABA AT),
Molecular docking.
2

Epilepsy is characterized by recurrent abnormal electrical activity in the brain which affects
majority of people all over the world 1,2. Although, several newer classes of anticonvulsant drugs
3
,4
have emerged in the previous 20 years, but complete treatment of epilepsy is still a long way .
Most of these anti-epileptic drugs (AEDs) are ineffective against the 30% refractory epileptic
patients and possess severe side effects viz., motor impairment, continuous pain in head,
biliousness, loss of appetite, unsteady movements and staggering gait, liver damage, weariness,
5
digestive disorder and gingival enlargement etc . The restriction with the available conventional
drugs leads to the evolution of novel drugs for convulsion 6.
Previously we have reported various heterocyclic compounds of nitrogen containing
moieties with potential pharmacological activities. The promising results of these studies prompted
us to further extend our synthesis of heterocyclic compounds and evaluation against the epileptic
7
-9
stimuli . Encouraged by the antiepileptic profile of riluzole which is a benzothiazolamine
derivative, recently research on benzothiazole as anticonvulsants has gained great momentum.
Many of the reported benzothiazole analogues exhibited promising protective activity comparable
to standard AEDs against mice 10-12. Furthermore, thiazolidin-4-one and azetidin-2-one are also
important class of heterocyclic compounds possessing diverse pharmacological properties including
anticonvulsant activity. In our search for new compounds with anticonvulsant activity, the present
series has been designed as a hybrid molecule containing benzothiazole and thiazolidin-4-
one/azetidin-2-one connected through amide linkage. Previously, it has been observed that
molecular hybridization of two lipophilic moieties connected through hydrogen bonding domain
13
14
(HBD) viz amide linkage had promising anticonvulsant effects as it fulfilled the pharmacophoric
requirements of anticonvulsant putative receptors 15,16. In perspective of the above and in
continuation of our interest in the benzothiazole derivatives, the present manuscript gives an
account of the synthesis and anticonvulsant activity of some new hybrid analogues containing
benzothiazole and thiazolidin-4-one/azetidin-2-one ring systems connected through amide linkage
(Scheme 1).
O
O
O
O
Cl
Cl
S
N
N
O
S
N
S
N
N
N
N
N
N
H
H
H
O
Lipophilic
Domain
Hydrogen Bonding
Domain
Benzothiazole
Amide replaced by urea
N
N
NH
NH
S
NH
N
Cl
S
NH
N
Cl
O
O
O
O
R
R
S
5(a-g)
6(a-g)
Cl
3

Scheme 1. 6-Chlorobenzothiazole derivatives containing thiazolidinone and azetidinone analogues
1
4,15
connected through urea linkage.
Titled compounds benzothiazolyl derivatives 5(a-g) and 6(a-g) were synthesized by
following the reactions as given in Scheme 2. Intermediates arylidene hydrazine carboxamide 4(a-
g)17 were prepared by reacting chlorobenzothiazol hydrazine carboxamide (3) with substituted
benzaldehyde in alcohol. The product undergoes cyclization by reacting with thioglycolic acid to
1
8
19
form thiazolidine-4-one derivatives 5(a-g) . Formation of the azetidin-2-one derivatives 6(a-g)
was also accomplished in one step by adding chloroacetyl chloride and triethylamine to arylidene
hydrazine carboxamide 4(a–g) in dioxane. The formation of compound was ascertained by by IR,
1H NMR, and LRMS13,20
.
N
S
N
S
N
NaOCN
GAA
NH NH .H O, NaOH
2 2 2
NH2
NH
O
NH
O
EtOH
NH2
S
3
NH
NH2
Cl
Cl
Cl
1
2
GAA R-CHO
HO
SH
N
S
N
O
NH
NH
NH
N
Cl
O
S
NH
N
R
H
Cl
O
R
O
4
(a-g)
S
5(a-g)
Cl
Cl
O
N
S
NH
O
NH
N
Cl
O
R
(a-g)
6
Cl
Scheme 2. Protocol for the synthesis of titled compounds 5&6(a-g)
All the pharmacological screenings were carried out using adult male albino mice having
weight of 20±2 g. The present study was carried out by following the protocols accepted by the
Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) of
KIET School of Pharmacy, Ghaziabad (UP) (Registration number & date of registration:
1
099/c/07/CPCSEA, dated 27.07.2009). Animals were housed in groups of 4-5 in an atmospheric
temperature (25±2ºC) and were permitted free access to food and water. Animals were not disturbed
unnecessarily and treated gently. Previously sterilized instruments were used for drug preparation
and animal injection. The anticonvulsant screening of the synthesized compounds was assessed by
4

following the standard protocols of Antiepileptic Drug Development (ADD) program by NINDS
National Institute of Neurological Disorders and Stroke), US21-24. At two pretreatment time
intervals (0.5 h and 4 h), all the synthesized compounds were administered intraperitoneally into
(
-1
mice at doses of 30, 100 and 300 mgkg . Table 1 shows the results of the in vivo anticonvulsant
activity of the synthesized moieties.
Table 1: Anticonvulsant profile and rotarod toxicity of the synthesized compounds 5 & 6(a-g)
i.p. injection in micea
Compound
R
MES
.5h
scPTZ
0.5h
Neurotoxicity C Log Pc
0
4h
4h
0.5h
4h
Ph
3.639
4.352
5.065
4.088
2.988
3.558
3.382
3.448
4.161
4.874
3.897
2.797
3.367
3.191
2.08*
2.38*
2.76*
0.39*
5
a
300
100
30
300
300
300
-b
-
2
-ClPh
5
b
100
100
300
300
-
300
300
-
-
2
,4-Cl Ph
2
5
c
100
100
-
-
2
-CH Ph
3
5
d
300
300
300
30
-
-
300
300
2
-CH COOPh
3
5
e
-
-
-
-
4
-CH OPh
3
5
f
-
-
-
-
4
-NO Ph
2
5
6
g
a
100
300
300
100
300
300
300
100
100
-
-
Ph
300
300
30
-
-
-
-
2
-ClPh
6
b
-
-
-
-
2
,4-Cl Ph
2
6
c
100
100
-
300
-
-
300
-
2
-CH Ph
3
6
d
300
300
300
-
-
-
-
-
-
2
-CH COOPh
3
6
e
4
-CH OPh
3
6
f
-
-
4
-NO Ph
2
6
g
100
30
100
30
300
-
300
-
-
-
Phenytoin
Carbamazepine
Valproic acid
Ethosuximide
-
-
-
-
100
100
30
-
100
100
300
300
100
100
300
-
-
-
-
-
-
-
-
-
a
-1
The data indicate the minimum dose (mgkg ) whereby bioactivity was demonstrated in half or
b
c
more of the mice (six in each group). A dash indicates the absence of activity. CLogP was
calculated using software ACD/log P; *Reported Clog P
5

Substitution with different electronic properties are attempted on the phenyl ring attached
with the thiazolidin-4-ones and azetidin-2-ones to study their effects on anticonvulsant activity.
Among the thiazolidin-4-ones (5a-g), all compounds showed some extent of protection in maximal
electroshock screening (MES), which confirmed their therapeutic efficacy against convulsion at a
deferent dose level. All the compounds showed quick onset and longer duration of protection except
compound 5f. Compounds 5c (2,4-Cl Ph) and 5g (4-NO Ph) showed protection of hind limb
2
2
-1
extension at a lower dose of 30 mgkg after 0.5 h exhibiting their promising nature having quick
-1
onset. Compound 5b (2-ClPh) revealed average protection at a dose of 100 mgkg whereas, rest of
-1
the compounds were least effective at the optimum dose of 300 mgkg . In subcutaneous
pentylenetetrazole (scPTZ) test, compounds 5c (2,4-Cl Ph) and 5g (4-NO Ph) elevated seizure
2
2
-1
threshold at a dose of 100 mgkg whereas 5a (Ph) and 5b (2-ClPh) exhibited protection at a
-1
maximum dose of 300 mgkg . Thus, compounds 5c and 5g showed protections against both MES
and scPTZ tests indicating their broad spectrum of anticonvulsant activity.
Azetidin-2-one derivatives (6a-g) also showed promising results against the maximal
electroshock screening by preventing the hind limb tonic extension at different dose of 30-300
-1
-1
mgkg . Only compound 6c (2,4-Cl Ph) showed protection at a lower dose of 30 mgkg . In the
2
scPTZ test, only compounds 6c (2,4-Cl Ph) and 6g (4-NO Ph) effectively increases the convulsive
2
2
-1
threshold at a dose of 100 mgkg . The anticonvulsant efficacy of 5c, 5g and 6c were comparable to
-1
-
the standard drug carbamazepine in MES test effective at dose of 30 mgkg (after 0.5 h), 100 mgkg
1(
after 4.0 h) and scPTZ test at a dose of 100 mgkg (after 0.5 and 4.0 h). In the rotarod
-1
neurotoxicity test, all the compound except 5d (2-CH Ph) and 6d (2-CH Ph) were devoid of any
3
3
-1
sign of motor impairment at a higher dose of 300 mgkg .
Agents effective against the scPTZ models are known to act through γ-aminobutyric acid
GABA) modulatory mechanisms 25,26. Therefore, some selected compounds 5c, 5g and 6c effective
(
against the scPTZ were also evaluated for their whole brain GABA estimation to further get insights
of their mechanism of seizure protective actions. Compared to the control animals (p < 0.01), whole
brain GABA estimations revealed (Fig.1) that oral administration of compound 5c for one week
elevated the brain GABA level remarkably (by 1.5 fold). Thus, the anticonvulsant actions of these
benzothiazolyl derivatives might be through increased GABA neurotransmission.
6

Fig. 1. Liver enzyme levels and GABA levels of compounds 5c, 5g and 6c treated mice.Each value
represents the mean±SEM of six mice, significantly different from the control at *p < 0.05; **p <
0
.01 (Student’s t test).
In order to reveal more about GABA modulatory action of benzothiazolyl derivatives, in-
2
7
vitro enzymatic assay was performed. These newly synthesized compounds were also evaluated in
vitro at γ-aminobutyric acid aminotransferase (GABA AT) activity to determine the IC value.
5
0
GABA AT is a pyridoxal 5′-phosphate-dependent enzyme responsible for the breakdown of
inhibitory neurotransmitter GABA. All the three analogues exhibited encouraging inhibitory results
in the micromolar range. The median inhibitor potency (IC ) of most promising compound 5c was
5
0
found to be 15.26 µM as compared to the standard drug vigabatrin showing median inhibitory
concentration 39.72 μM (Table 2). These results suggested that the increased concentration of
GABA might be through the inhibition of GABA AT enzyme. Moreover, the results of in vitro
inhibitions are consistent with the in vivo seizure protective efficacies of the benzothiazolyl
analogues.
Table 2. In silico and in vitro GABA AT enzyme inhibition
Compound
Docking
score
-6.40
-2.30
-2.58
Predicted inhibition
constant (Ki)
90.10µM
Experimental inhibition constant
(IC50, µM)
5
5
6
c
g
c
15.26±0.48
58.13±0.65
51.36±0.45
75.23mM
79.16mM
Vigabatrin
-2.803
82.76mM
39.72 ±0.47
7

An attempt has also been made to confirm the in vitro GABA AT inhibition of
benzothiazole derivatives through in silico molecular docking studies on GABA AT enzyme. The
X-ray crystal structures of GABA AT was retrieved form the Research Collaboratory for Structural
Bioinformatics (RCSB) Protein Data Bank (PDB) having PDB ID 1OHV and 96% resemblance
with the human brain enzyme. Results of molecular docking studies showed that compounds 5c, 5g
and 6c had binding free energies from -2.30 to 6.40 kcal/mol. Compound 5c showed minimum
docked energy of -6.40 among the three mentioned analogues. Fig. 2A-B depict the docked
conformations of the ligands 5c bound to the active site of GABA AT. The oxo group of
thiazolidinone seems to have an crucial role in strong hydrogen bonding with the active site residue
Lys329. The residue Lys 329 is in close proximity and is probably the general base catalyst for the
proton transfer reaction and drug vigabatrin inhibit this enzyme by covalent bond formation
with Lys-329. Lipophilicity also appears to play crucial role in 5c inhibitory activity, as 2,4-
dichlorophenyl and thiazolidinone rings oriented to the more lipophilic area of GABA-AT binding
site and formed π-π interactions with Ile72 and Phe189, respectively. The results of binding studies
revealed that compound 5c binds strongly in a mode very similar to standard drug vigabatrin (Table
3
).
Fig 2A. Compound 5c at the active site of GABA AT enzyme
8

Fig 2B. 2D binding modes of compound 5c
Table 3. Interactions of compound 5c at the active site of GABA AT
Hydrogen bonds
Halogen-bond
SER 74
Polar
SER137
Hydrophobic
ILE72
Other
CYS135
SER328
LYS329
GLN71
PHE189
As major problem associated with the clinically available antiepileptic drugs are their liver
toxicity7.8 therefore, the most promising compounds 5c, 5g and 6c were also analyzed for their
hepatotoxic profile. Repeated dose of the test compounds was administered orally in a dose of
3
0mg/kg daily for one months. At the end of the study, the levels of serum SGOT, SGPT and
alkaline phosphatase were examined and any significant alterations in these enzymes were
considered as liver toxicity. As shown in Fig.1, compound 5c significantly decreased the levels of
all enzymes (P < 0.01) as compared to the control group. However, compounds 5g and 6c
moderately increased the levels of SGPT and ALP enzymes (P < 0.05) and decreased the levels of
SGOT (P < 0.01). The liver histopathological studies of compound 5c treated animals showed no
remarkable diseased conditions as compared to control group. Section of liver showed normal
hepatocytes with clearing of cytoplasm (Fig. 3A-B). Thus, compound 5c was not only effective
against the MES and scPTZ test but also a safer agent devoid of any sign of neurotoxicity and liver
toxicity.
9

Fig. 3. A: Section of control liver showed normal hepatic parenchyma. PT=Portal Triad
CV=Central Vein (100x); B: Section of compound 5c treated liver showed normal hepatocytes with
clearing of cytoplasm. PT=Portal Triad CV=Central Vein (100x)
In conclusion, various benzothiazole analogues containing thiazolidin-4-one and azetidin-2-
one connected through hydrogen bonding domain linkage were synthesised and examined for their
antiepileptic activity. The results of antiepileptic screen exhibited that all the synthesized
benzothiazole derivatives 5(a-g) and 6(a-g) exhibit good to moderate anticonvulsant activity in
mice. Three compounds 5c, 5g and 6c showed promising anticonvulsant activity. The promising
results could be due to the presence of electron withdrawing substituents viz. 2,4-Cl (5c) and 4-
2
NO (5g) groups linked to the benzene moiety at second position of the thiazolidin-4-one ring, and
2
2
,4-C H Cl (6c) attached to the β-lactam ring. The promising results of in vitro and in silico studies
6 3 2
clearly indicates that, the increased GABA concentration in brain is due to inhibition of GABA AT
enzyme. Moreover, no overt toxicity was observed for the synthesized compounds in the various
toxicity studies. Therefore, hybridization of the benzothiazole with thiazolidin-4-one/azetidin-2-
one moieties provided a new opportunity for possible modification of anticonvulsant
pharmacophoric requirements and future exploitation.
Acknowledgements
The authors are thankful to the Head of the Department, Pharmaceutical Chemistry for providing
laboratory and animal facilities.
Conflict of interest : None
References and notes
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1
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1
0

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4
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1
1
1
1. Murtuja, S.; Shaquiquzzaman, M.; Amir M. Lett. Drug Des. Dis. 2018, 15, 398-405.
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9
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2
-yl)hydrazinecarboxamide 4(a-g): Equimolar methanolic solution of N-(6-
chlorobenzothiazol-2-yl)hydrazine carboxamide (0.01 mol) and substituted
benzaldehyde (0.01 mol) were taken. To this solution, 1-2 drops of glacial acetic acid
was included. For 5-6 hr this solution was then refluxed on a water bath. After
completion of the reaction, the solution was kept aside to cool. After cooling, the
solution was poured on the crushed ice and recrystallized from methanol.
1
8. General method for the synthesis of 1-(6-chlorobenzothiazol-2-yl)-3-(4-oxo-2-
substituted phenylthiazolidin-3-yl)urea 5(a–g) : The reaction mixture of 2-substituted
benzylidene-N-(6-chlorobenzothiazol-2-yl) hydrazinecarboxamide (0.01 mol) and
thioglycollic acid (0.01 mol) was boiled on an oil bath for 7hr at 120-125 ºC with
precaution. To the cooled solution sodium bicarbonate (10%) was added. Finally, the
product was filtered, dried and recrystallized from methanol–dioxane in the ratio of 4:1.
1
1

1
9. General method for the synthesis of 1-(3-Chloro-2-oxo-4-substituted phenylazetidin-1-
yl)-3-(6-chlorobenzothiazol-2yl)urea 6(a–g): Stirred mixture of chloroacetyl chloride
(0.012 mol) and triethyl amine (0.012 mol) in dioxane (10 mL) maintained at 0–5 ºC, a
solution of 2-substituted benzylidene-N-(6-chlorobenzothiazol-2-yl) hydrazine-
carboxamide (0.01 mol) in dioxane (15 mL) was added. The combination was constantly
stirred for six hour and kept aside for two days at 37 ºC. Finally, it was treated with cold
water, washed and filtered. The solution was recrysrtallized from methanol to get the
pure titled compound.
-1
2
0. Analytical data of the selected compound: Compound 5c: IR (KBr, ν cm ): 3210 (N-H),
3
1
114 (C-H aromatic), 1734 (C=O thiazolidinone), 1663 (C=O), 1538 (C-C aromatic),
433 (C-N benzothiazole), 836 (C-Cl), 683 (C-S-C thiazolidinone), 614 (C-S-C
1
benzothiazole); H NMR (300 MHZ, DMSO-d ): δ 11.82 (s,1H,CONH), 8.00
6
1
3
(
s,1H,NH), 7.68-7.71 (m, 6H, Ar-H), 2.47 (s, 2H, CH thiazolidinone); C NMR (75
2
MHz, DMSO-d ) δ: 174.4, 168.7, 153.7,151.2,135.5, 134.2, 132.3, 131.4,130.4, 129.7,
6
+
1
4
26.9, 125.9, 121.3,118.4,100.7, 58.8, 35.3 (CH thiazolidinone); MS: m/z: 477[M +2],
2
+
+
75[M ], 473[M -2]
2
2
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2
7. Bansal, S.K.; Sinha, B. N.; Khosa, R. L. Med. Chem. Res. 2013, 22. 134–146.
8. Amir, M.; Ali,I., Zaheen, H.M. Med. Chem. 2013, 9, 571.
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Legends for Figures
Scheme 1 : 6-Chlorobenzothiazole derivatives containing thiazolidinone and azetidinone
analogues connected through urea linkage
Scheme 2 : Protocol for the synthesis of titled compounds 5&6 (a-g)
Figure 1 : Fig. 1. Liver enzyme levels and GABA levels of compounds 5c, 5g and 6c treated
mice. Each value represents the mean±SEM of six mice, significantly different from
the control at *p < 0.05; **p < 0.01 (Student’s t test).
Figure 2A-B: Compound 5c at the active site of GABA AT enzyme
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D binding modes of compound 5c
Figure 3:
A: Section of control liver showed normal hepatic parenchyma. PT=Portal Triad
CV=Central Vein (100x); B: Section of compound 5c treated liver showed normal
hepatocytes with clearing of cytoplasm. PT=Portal Triad CV=Central Vein (100x)
Highlights

Compounds 5c, 5g & 6c have optimum in-vivo antiepileptic activities without neurotoxicity.

GABA modulatory effects-5c, 5g & 6c showed encouraging inhibitory results against GABA
AT.
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
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Docking studies-5c binds strongly in a mode similar to standard drug vigabatrin.
Results of in vitro inhibitions are consistent with in vivo seizure protection.
Toxicity studies-No overt toxicity was observed for synthesized compounds.
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Graphical abstract
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