Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as glycogen synthase kinase-3β inhibitors with anti-depressant activity

Article abstract of DOI:10.1016/j.bmcl.2016.06.084

A series of benzimidazole based thiadiazole and carbohydrazide conjugates have been synthesized and evaluated for inhibition of glycogen synthase kinase-3β and anti-depressant effect. Compounds 4f, 4j, 5b, 5g and 5i were found to be the most potent inhibitors of GSK-3β in vitro amongst the twenty-five benzimidazole based thiadiazole and carbohydrazide conjugates synthesized. Compound 5i was also found to exhibit significant antidepressant activity in vivo at 50?mg/kg, when compared to fluoxetine, a known antidepressant drug. The molecular docking studies revealed multiple hydrogen bond interactions by the synthesized compounds with various amino acid residues, viz, ASP-133, LYS-183, PRO-136, VAL-135, TYR-134, or LYS-60 at the GSK-3β receptor site.

Full text of DOI:10.1016/j.bmcl.2016.06.084

Accepted Manuscript
Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as
Glycogen Synthase Kinase-3β inhibitors with anti-depressant activity
Imran Khan, Mushtaq A Tantray, Hinna Hamid, MohammadSarwarAlam, Abul
Kalam, Abhijeet Dhulap
PII:
S0960-894X(16)30695-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.084
BMCL 24037
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 January 2016
23 April 2016
29 June 2016
Please cite this article as: Khan, I., Tantray, M.A., Hamid, H., Alam, M.S., Kalam, A., Dhulap, A., Synthesis of
benzimidazole based thiadiazole and carbohydrazide conjugates as Glycogen Synthase Kinase-3β inhibitors with
anti-depressant activity, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.06.084
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Synthesis of benzimidazole based thiadiazole and carbohydrazide
conjugates as Glycogen Synthase Kinase-3β inhibitors with anti-
depressant activity
Imran Khana, Mushtaq A Tantraya, Hinna Hamida*, Mohammad Sarwar Alama, Abul
Kalamb, Abhijeet Dhulapc
aDepartment of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University),
New Delhi 110 062, India.
bDepartment of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), New Delhi 110 062, India..
cCSIR Unit for Research and Development of Information Products, Pune 411038.
Correspondence to Dr. Hinna Hamid, Hamdard University, New Delhi 110 062, Email
ID. hhamid@jamiahamdard.ac.in. Phone: +91 11 26059688 (5559).
Abstract
A series of benzimidazole based thiadiazole and carbohydrazide conjugates have been
synthesized and evaluated for inhibition of glycogen synthase kinase-3β and anti-
depressant effect. Compounds 4f, 4j, 5b, 5g and 5i were found to be the most potent
inhibitors of GSK-3β in vitro amongst the twenty-five benzimidazole based thiadiazole
and carbohydrazide conjugates synthesized. Compound 5i was also found to exhibit
significant antidepressant activity in vivo at 50 mg/Kg, when compared to fluoxetine, a
known antidepressant drug. The molecular docking studies revealed multiple hydrogen
bond interactions by the synthesized compounds with various amino acid residues, viz,
ASP-133, LYS-183, PRO-136, VAL-135, TYR-134, or LYS-60 at the GSK-3β receptor
site.
Key words: Benzimidazole; thiadiazole; molecular docking; glycogen synthase kinase;
carbohydrazide.

Glycogen synthase kinase 3 (GSK-3) a serine/threonine kinase of the CMGC
kinase group (cyclin dependent kinases, mitogen activated protein kinases, glycogen
synthase kinase and cell kinase-2), has been originally described as a key regulator of
glycogen synthesis and glucose metabolism.1 Glycogen synthase kinase 3β (GSK-3β)
acts as a downstream regulatory switch that determines the output of numerous signaling
pathways initiated by diverse stimuli.2 GSK-3β targets a wide range of substrates, namely
metabolic proteins (e.g. cyclin D1, APP (amyloid precursor protein) and presenilin),
structural proteins (e.g. tau and other microtubule associated proteins) and transcription
factors (e.g. NF-κB, p53 and Notch).2 This plethora of biological substrates places GSK-3
as an important feature in cell cycle and development, bioenergetics and apoptosis,
unveiling its role as a master regulator of key physiological processes.3 Consequently the
abnormal regulation of GSK-3β results in the development of psychiatric disorders like
bipolar disorder, mood disorders, depression and schizophrenia4-6, besides other non-
psychiatric diseases such as cancer, aging immune disorders and metabolic disorders.7, 8
Depression is a common, debilitating, life-threatning illness with a significant
incidence in the population. GSK-3β has been implicated in the mechanism of action of
the mood stabilizers lithium and valproate9, 10, largely used in the treatment of bipolar
disorder.11 Deficient serotonergic neurotransmission observed in depression12,
considering that serotonergic activity contributes to the inhibitory control of GSK-3β in
mammalian brain in vivo.13, 14 Serotonin (5-HT) itself, as well as fluoxetine (a selective
serotonin re-uptake inhibitor) and 5-HT1A agonists, augment serine 9 phosphorylation
with the consequent inhibition of GSK-3β.13 In addition, classical antidepressants, as well
as atypical antipsychotics involve inhibition of GSK3β, either directly or following its
phosphorylation on Ser9 by AKT in their mechanism of action.15, 16 There have been
several reports demonstrating that pharmacologic inhibition of GSK-3 activity produces
antidepressant-like effects in rodents.17-20
Benzimidazole constitutes an indispensible construction motif for the
development of compounds of medicinal interest due to its diverse pharmaceutical
applications and its derivatives have also been identified as strong GSK-3β inhibitors.21-23
Suitable structural combinations of benzimidazole core with various pharmacophores in a
single molecular framework have resulted in improved GSK-3β inhibitory potential and

alleviation of associated disorders.22 The thiadiazole as well as its bioisosteric oxadiazole
heterocyclic frameworks have been regarded as the privileged scaffolds for GSK-3
inhibitory activity.24, 25 Also, the carbohydrazide motif possesses favourable H-bond
donors as well as acceptors for it to interact strongly with the GSK-3β target site and
hence could give higher GSK-3β inhibition (and consequently better antidepressant
activity). Keeping this in view, we attempted conjugation of benzimidazole with 2-
(alkyl/aryl)amino-1,3,4-thiadiazoles and aryl/heteroaryl carbohydrazides under a single
construct through methylene linkage. A library of twenty-five benzimidazole based 1,3,4-
thiadiazoles and carbohydrazide conjugates (4a-m and 5a-l) has been synthesized as
shown in Table S.1 (Supplementary data). Various substitutions have been made to
explore their effect on the GSK-3β inhibition and generate the structure-activity
relationship. Among these four compounds (4a, 4k, 4l and 5a), which are already known
26
in the literature and structurally similar to the benzimidazole based 1,3,4-thiadiazoles
and carbohydrazides conjugates were also taken up for synthesis and evaluated for GSK-
3β inhibition and anti-depressant activity.
The designed inhibitors were synthesized using the methods in Scheme 1. The
key intermediate used for the synthesis of both series of the final compounds was 2-(2-
methyl-1H-benzimidazol-1-yl)acetohydrazide (2)27, which in turn was prepared by the
reaction of 2-methyl-1H-benzimidazole (1) with ethyl bromoacetate in the presence of
anhydrous potassium carbonate as a base, followed by the reaction with hydrazine
hydrate. The benzimidazole thiosemicarbazides (3) were prepared by condensing
compound 2 with appropriate isothiocyanates. Cyclization of compounds 3 in Conc.
H2SO4 under room temperature gave the benzimidazole-1,3,4-thiadiazoles 4a-m.28
Further, another series of benzimidazole carbohydrazides (5a-l) was synthesized by using
EDC coupling.17

O
N
N
N
N
Br
NH2NH2.H2O
Abs Alcohol
N
O
H
O
N
NH2
K2CO3
N
H
Dry Acetone
O
O
2
1
R2 COOH
EDC.HCl
HOBT
R1 NCS
Abs Alcohol
N
O
H
N
N
N
R2
N
N
H
H
N
O
NH
R1
5a-l
N
O
S
H
3
Conc.H2SO4
N
N
N
N
S
R1
N
H
4a-m
Scheme 1: Synthesis of benzimidazole based 1,3,4-thiadiazoles and carbohydrazide
conjugates.
The attachment of the benzimidazole ring with the 1,3,4-thiadiazole nucleus (4a-
m) was confirmed by the appearance of methylene protons at δ 5.65-6.06 as singlet’s in
the 1HNMR spectra. Further structural confirmation of the final compounds was provided
by the presence of singlet’s for amino protons attached to the 1,3,4-thiadiazole ring at δ
9.71-11.10 (4a-m). The formation of benzimidazole carbohydrazide compounds (5a-l)
was confirmed by the apperance of singlet of the methylene proton attached to aromatic
ring at δ 3.38-3.55 and 4.86-5.04 for methylene protons attached to benzimidazole rings.
1
The structure of all the compounds was further confirmed by the IR, H-NMR, 13C-NMR
spectra and ESI-MS mass spectral analysis.
All the synthesized compounds were evaluated for GSK-3β inhibitory activity in a
non-radioactive assay using Kinase-Glo method and the results are shown in Table 1.
Staurosporine, a well known kinase inhibitor was used as the reference compound. The

test compounds were first screened at a primary concentration of 10 µM. The compounds
showing considerable inhibition at 10 µM were next tested over a wide range of
concentrations and IC50 values were determined from the dose response curves.29 The
results of kinase inhibitory assays demonstrated that compounds 4f, 4j, 5b, 5g and 5i
were the more potent amongst benzimidazole based 1,3,4-thiadiazoles/carbohydrazides
with IC50 value of 76, 107, 92, 80 and 72 nM respectively.
Table 1: GSK-3β inhibitory activity of benzimidazole based 1,3,4-thiadiazoles and
carbohydrazides.
N
N
N
N
O
R2
H
N
N
N
N
H
S
O
R1
N
H
4a-m
5a-l
GSK-3β
GSK-3β IC50
R1
Compound
R2
Compound
IC50 (nM)
(nM)
Phenyl
4a
4b
157
145
4-Chloro Benzyl
2-Methyl Benzyl
5a
131
4-Chloro
Phenyl
5b
92
2-Chloro
Phenyl
4-Methoxy
Benzyl
4c
4d
4e
4f
122
134
173
76
5c
5d
5e
5f
379
205
148
124
80
4-Flouro
Phenyl
4-Nitro Benzyl
3-Indole
4-Bromo
Phenyl
2-Flouro
Phenyl
2-Pyrrole
4-Methoxy
Phenyl
4-
4g
151
5g
Phenoxyphenyl-

4-oxobutanoyl
Benzyl
2-Methoxy
Phenyl
4h
4i
184
293
5h
105
4-Ethoxy
Phenyl
3-indole
5i
72
glyoxalyl
4-Nitro
2-acetyl-Indole
2-Naphthyl
5j
135
157
4j
107
Phenyl
Cyclohexyl
Butyl
4k
406
5k
4-
4l
582
190
Methoxyphenyl-
4-oxobutanoyl
5l
-
561
54
2-Bromo
Phenyl
4m
Staurosporine
Evaluation of antidepressant activity involves rodent behavioural tests and two
behavioural models i.e., forced swim test (FST) and tail suspension test (TST) are widely
used. Both models are based on the application of stress to the animal and measurement
of the duration of immobility when rodents are exposed to an inescapable situation. These
tests have good predictive validity and allow rapid and economical detection of
substances with potential antidepressant activity. The results as depicted in Fig. 1 show
the effect on immobility in the tail suspension test. The mean duration of immobility was
significantly reduced with the standard drug fluoxetine, which exhibited an immobility
time of 153 sec. The test compounds 4f, 4j, 5b, 5g and 5i resulted in immobility times of
120, 174, 168, 135 and 105 sec respectively as compared to the normal saline, which
showed an immobility time of 183 sec (P < 0.001). Decrease in immobility shown by 5i
and 4f was found to be significant in comparison with the standard drug fluoxetine,
furthermore decrease in immobility time on administration of 4f, 5g and 5i was also
statistically significant in comparison to normal saline. The results indicate that the

compounds 4f and 5i showing a potent GSK-3β inhibition also possessed a significant
antidepressant potential.
Figure 1: Tail suspension test. Data is represented as Mean± SEM and analyzed by one
way ANOVA followed by Tukey Kramer multiple comparison test, *P < 0.05, ** P <
#
0.01, ***P < 0.001, P < 0.05, ##P < 0.01, ###P < 0.001 when compared with normal
control and standard (fluoxetine) respectively.
The forced swim test (FST) or despair swim test has been proposed as model to test
for antidepressant activity by Vincent Castagne and co-workers’s method.30 The effect of
immobility in the forced swim test results are shows in the Fig. 2. Significant decreases
in immobility time were observed on administration of the compounds 4f, 5g and 5i in
comparison to normal saline (P < 0.01). Furthermore the compounds 4f, 5g and 5i were
also found to exhibit significant activity in comparison to the standard drug fluoxetine.
These observations are in agreement with the results of TST, corroborating that
compounds 4f and 5i possess significant antidepressant potential.

Figure 2: Forced swim test. Data is represented as Mean± SEM and analyzed by one
way ANOVA followed by Tukey Kramer multiple comparison test, *P < 0.05, ** P <
n
0.01, ***P < 0.001, P < 0.05, ##P < 0.01, ###P < 0.001 when compared with normal
control and standard (fluoxetine) respectively.
Reported in-silico studies for GSK-3β protein shows that the key residues in the
ATP binding pocket namely ASP133 & VAL133 form hydrogen bonds with
Staurosporine (PDB: 1Q3D). Furthermore within the ATP binding site, hydrophobic
interaction with residues like CYS-199, LEU-132, VAL-110, ALA-83, LEU-188, TYR-
134, VAL-135 & ILE-62 contributes to overall efficacy of binding drug molecule. The
binding interactions of Staurosporine within the ATP binding pocket of GSK-3β protein
is as shown in Fig. 3. The synthesised molecules were docked against the GSK-3β target
protein. The Glide score of the molecules ranged from -7.69 to -4.50 in comparison to
staurosporine with Glide score of -10.01, as shown in Table S.2 & S.3. The highly
ranked molecule 5i exhibiting a Glide score of -7.69 was found to show Hydrogen bond
interaction with key residues ASP-133 and LYS-183. The other high scoring molecule 5g
with Glide score -7.64 formed Hydrogen bonds with PRO-136 & VAL-135 residues and
side chains of TYR-134 & LYS-60 residues. The binding interactions of these molecules
(5i, 5g & 4f) are shown in Fig. 4-6. The results from the in silico studies were found to be

in agreement with the in vitro GSK-3β assay as the top-ranked ligands (5i & 5g) which
showed better binding affinity in silico studies also exhibited highest in vitro activity.
Figure 3: In silico molecular docking of standard (staurosporine) against GSK-3β.
Staurosporine is reported to show H-bond formation with Val-135 & Asp-133 residues.

Figure 4: In silico molecular docking of ligand 5i against GSK-3β
Figure 5: In silico molecular docking of ligand 5g against GSK-3β

Figure 6: In silico molecular docking of ligand 4j against GSK-3β
General approaches were used to achieve diversity of the synthesised compounds.
From the results, the following structural activity relationship could be drawn. Among the
two classes of compounds, carbohydrazides conjugates of benzimidazole were found to
be more active when compared with thiadiazole conjugates of benzimidazole.
Furthermore, nature and position of the substitution on the aromatic ring of
carbohydrazide conjugates of benzimidazole greatly influenced the GSK-3 inhibitory
activity. Glyoxalic acid conjugates were found to be most active among the
carbohydrazide conjugates of benzimidazole. Hetero aromatic/aromatic carboxylic acid
conjugates were found to be more active when compared to aromatic/hetero aromatic
acetic acid conjugates. Amongst the hetero aromatic and aromatic carboxylic acid
conjugates, hetero aromatic rings led to greater activity over the aromatic ring. In the
thiadiazole conjugates of benzimidazole series, the activity of the compounds is
influenced by the nature of the substituents on aromatic ring. Electron donating groups

were found to confer better activity on compounds as compared to the electron
withdrawing group.
In summary, this work focused on the development of new potentially active
GSK-3β and anti-depressant benzimidazole based thiadiazole and carbohydrazide
conjugates. Among the tested compounds, compound 5i was found to be the most active
(IC50 = 72 nM), while compounds 4f, 4j, 5b, 5g and 5i were also found to exhibit
significant inhibition of GSK-3β in comparison to staurosporine, used as standard. The
structure activity relationships and molecular modelling study provided considerable
insight into interactions between the enzyme and these ligands. The compounds 4f, 4j,
5b, 5g and 5i were further evaluated for their antidepressant potential and compound 5i
was found to significantly reduce the duration of immobility at 50 mg/Kg, when
compared with fluoxetine, a known antidepressant drug. Phosphorylation of GSK3β
predominantly mediates the acute antidepressant-like effect of fluoxetine. It could be thus
concluded that molecule 5i can act as a potential lead candidate for development of
potent GSK-3β inhibitors with antidepressant potential.
Acknowledgements
The authors wish to express their thanks to Dr. G. N. Qazi, Vice-Chancellor,
Jamia Hamdard for providing necessary facilities to carry out this work. The authors
thank DST-SERB, Govt. of India for awarding research project to HH and fellowship to
IK.

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Graphical Abstract
N
N
H
S
N
Benzimidazole backbone
with drug like properties
N
N
NO2
IC50 = 107 nM, GSK-3β
N
GSK-3β pharmacophore fragments
GSK-3β inhibition
N
H
H
N
N
O
Anti-depressant activity
H
N
O
N
N
H
O
IC50 = 72 nM, GSK-3β