Synthesis of aryl anilinomaleimide based derivatives as glycogen synthase kinase-3β inhibitors with potential role as antidepressant agents

Article abstract of DOI:10.1039/c5nj02896e

A series of aryl anilinomaleimide based derivatives has been synthesized and evaluated for in vitro glycogen synthase kinase-3β (GSK-3β) inhibitory activity. A large number of compounds from the series exhibited moderate to potent inhibitory activity against GSK-3β, with more than one-third of the compounds showing inhibition with IC₅₀ values <1 μM. The molecular docking studies against GSK-3β (PDB: 1Q3D) revealed multiple H-bonding interactions of the synthesized molecules with important amino acid residues on the receptor site, in addition to H-bonding with water residues and π-cation interactions by some compounds. Given the potential role of GSK-3β inhibition in the treatment of depression, compounds 8j, 8b, 8i, 8l, 8a and 8n, exhibiting significant GSK-3β inhibition (IC₅₀ values of 0.09, 0.12, 0.17, 0.19, 0.21 and 0.23 μM respectively), were further investigated for antidepressant activity by the widely accepted forced swim test and tail suspension test (FST and TST) models. All the tested compounds displayed antidepressant-like effects, particularly compounds 8j and 8b, which exhibited significant antidepressant activity, about 1.4-fold higher than fluoxetine, a standard antidepressant drug in both FST and TST. Preliminary structure-activity relationships have also been generated based on the experimental data obtained.

Full text of DOI:10.1039/c5nj02896e

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Synthesis of Aryl anilinomaleimide based derivatives as glycogen synthase kinase-3β
DOI: 10.1039/C5NJ02896E
inhibitors with potential role as antidepressant agents
Mushtaq A Tantraya, Imran Khana, Hinna Hamida*, Mohammad Sarwar Alama, Abhijeet
Dhulapb, Abul Kalamc
aDepartment of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New
Delhi 110062, India.
bCSIR- Unit for Research and Development of Information Products (URDIP), Pune 411038,
India
cDepartment of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi 110062, India.
Correspondence to Dr. Hinna Hamid, Jamia Hamdard (Hamdard University) Hamdard Nagar,
New Delhi 110062, Email ID. hhamid@jamiahamdard.ac.in. Phone: +91 11 26059688 (5559).
Abstract
A series of aryl anilinomaleimide based derivatives has been synthesized and evaluated for in
vitro glycogen synthase kinase-3β (GSK-3β) inhibitory activity. Large number of compounds
from the series exhibited moderate to potent inhibitory activity against GSK-3β, with more
than one-third of the compounds showing inhibition with IC50 values <1 µM. The molecular
docking studies against GSK-3β (PDB: 1Q3D) revealed multiple H-bonding interactions by
the synthesized molecules with important amino acid residues on the receptor site in addition
to H-bond with water residue and π-cation interactions by some compounds. Given the
potential role of GSK-3β inhibition in treatment of depression, compounds 8j, 8b, 8i, 8l, 8a
and 8n exhibiting significant GSK-3β inhibition (IC50 values of 0.09, 0.12, 0.17, 0.19, 0.21 and
0.23 µM respectively) were further investigated for antidepressant activity by the widely
accepted forced swim test and tail suspension test (FST and TST) models. All the tested
compounds displayed antidepressant-like effects, particularly compounds 8j and 8b which
exhibited significant antidepressant activity, about 1.4-fold higher than fluoxetine, a standard
antidepressant drug in both FST and TST. Preliminary structure-activity relationships have also
been generated based on the experimental data obtained.

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DOI: 10.1039/C5NJ02896E
Introduction
Glycogen synthase kinase-3 (GSK-3), is a serine-threonine protein kinase, constitutively active
and ubiquitously expressed in all mammalian tissues and subcellular organelles1. GSK-3 is
involved in a multitude of key cellular and physiological events ranging from metabolism to
immunity and behaviour by virtue of regulating more than 70 diverse substrates1, 2. Besides
being involved in a number of human pathological conditions, including Alzheimer’s disease,
cancer and diabetes3-5, GSK-3 dysfunction has been implicated in psychiatric disorders like
bipolar disorder, mood disorders, depression and schizophrenia1, 6, 7. GSK-3 is present in two
major isoforms, GSK-3α and GSK-3β. Among the two isoforms, GSK-3β is the most often
investigated isoform and has received more interest from investigators studying mood
disorders8. For example, lithium, a well-known GSK-3β inhibitor, used as a mood stabiliser in
bipolar disorder as well as adjunct therapy for depression and schizophrenia for nearly 50 years
has been proposed to exert its therapeutic action by inhibiting GSK-3β directly as well as
indirectly through Akt/β-arrestin/protein phosphatase 2A complex7, 9, 10
.
The involvement of GSK-3β in depression has been linked to deficient serotonergic
neurotransmission observed in depression, considering that serotonergic activity contributes to
the inhibitory control of GSK-3β in mammalian brain in vivo8. The regulation of behaviour by
various psychoactive medications including selective serotonin reuptake inhibitors,
monoamine oxidase inhibitors, tricyclic antidepressant and second-generation antipsychotics,
involve a shared action of inhibition of GSK-3β in either the direct or downstream mechanism
of action11, 12. GSK-3β is further validated as a viable target for antidepressant action by the
reported anti-depressive effects of GSK-3β inhibitors in animal models. The substrate-
competitive peptide inhibitor L803-mts displayed antidepressant effect in the mouse FST13,
AR-A014418 showed similar effects in rats14 while as the thiadiazolidinone NP031115 also
displayed antidepressant activity in the mouse FST after systemic administration15.
Furthermore, pharmacological or genetic inhibition of GSK-3β was found to replicate
behavioural effects of these drugs in rodents16. Given its potential role in psychiatric disorders
and their treatment, pharmacological inhibition of GSK-3β represents a fascinating therapeutic
strategy for treatment of human neurodegenerative and psychiatric conditions.
Among the GSK-3β inhibitors identified from remarkably different structural classes,
maleimides like SB415286 (anilinomaleimide) and SB216763 (arylindolemaleimide) are well
known representative compounds of potential importance17-19. Their applications in many
pathologies including diabetes, Alzheimer’s disease, cancer, inflammation, neuropathological
and neuropsychiatric disorders have been explored20. LY2090314, a maleimide based GSK-3β

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New Journal of Chemistry
inhibitor, is in clinical trial stages for the treatment of cancer21. Anilinomaleimides have VbieeweAnrticle Online
DOI: 10.1039/C5NJ02896E
reported as potent and selective GSK-3 inhibitors18, 20, 22 and are amenable for further structural
modifications in order to search for the development of effective chemotherapeutics.
Encouraged by the need to develop new GSK-3β inhibitors with manifold therapeutic
applications, we thought it worthwhile to synthesize new small molecules based on aryl
anilinomaleimide scaffold as GSK-3β inhibitors, focusing in particular on their application in
depression. A library of thirty-two compounds has been synthesized and evaluated for in vitro
GSK-3β inhibition. The compounds which exhibited significant GSK-3β inhibition profile
were further subjected for in vivo evaluation of antidepressant activity using forced swim test
and tail swim test models. In silico molecular docking studies of all the synthesized compounds
have also been performed against GSK-3β to understand the binding interactions of these
compounds with its active site.
Results and Discussion
Chemistry
The compounds were synthesized as illustrated in Scheme 1. Aryl acetamides 3a-h were
synthesized from corresponding acids 1a-d by esterification followed by treatment with
ammonium hydroxide or methyl amine. The aryl acetamides 3a-f on condensation with diethyl
oxalate in the presence of potassium tert-butoxide formed 3-hydroxymaleimide derivatives 4a-
h which were then treated with oxalyl chloride to form 3-chloromaleimide derivatives 5a-h.
The 3-chloromaleimide derivatives were subsequently treated with 4-aminobenzoic acid in N-
methyl pyrrolidine (NMP) to obtain 4-carboxyanilinomaleimide derivatives 6a-h which on
treatment with appropriate anilines in presence of EDC.HCl and HOBT afforded the target
compounds 7a-7p and 8a-8p (Table 1).

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DOIO: 10.1039/C5NJ02896E
R1
R1
R1
O
O
a
b
R2
N
OH
O
H
1a-d
2a-d
3a-h
1a, 2a R1 = H
1b, 2b R1 = OCH3
1c, 2c R1 = F
1d, 2d R1 = Cl
c
R2
R2
R2
N
N
O
N
O
O
O
O
O
e
d
OH
HN
Cl
OH
O
R1
R1
R1
6a-h
5a-h
4a-h
f
3a, 4a, 5a, 6a R1 = H, R2 = CH3
3b, 4b, 5b, 6b R1 = OCH3, R2 = CH3
3c, 4c, 5c, 6c R1 = F, R2 = CH3
3d, 4d, 5d, 6d R1 = Cl, R2 = CH3
3e, 4e, 5e, 6e R1 = H, R2 = H
R2
N
O
O
HN
O
R3
3f, 4f, 5f, 6f
R1 = OCH3, R2 = H
HN
3g, 4g, 5g, 6g R1 = F, R2 = H
3h, 4h, 5h, 6h R1 = Cl, R2 = H
R1
7a-7p (R2= -CH3)
8a-8p (R2= -H)
Reagents and conditions: (a) Methanol, H2SO4, Reflux, 12 h (b) R-NH2, Methanol, RT, 15 h
0
(c) Diethyl oxalate, t-BuOK, THF/DMF, 0 0C-RT, 6 h (d) Oxalyl chloride, THF/DMF, 0 C-
RT, 4 h (e) 4-Aminobenzoic acid, NMP, 90 0C, 24 h (f) Anilines, EDC.HCl, HOBT, DMF, RT,
8 h.
Scheme 1 - Synthesis of aryl anilinomaleimide based derivatives.

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Table 1
Structural data and GSK-3β inhibitory activity of the synthesized compounds.
R2
N
O
O
HN
O
R3
HN
R1
7a-7p (R2= -CH3)
8a-8p (R2= -H)
Compd.
IC50 (µM) Compd.
± SEMa
(R2=H)
IC50 (µM)
± SEMa
R1
R3
R1
R3
(R2=CH3)
7a
-H
-H
-H
16.49±0.57
-H
-H
-H
-OCH3
-F
8a
8b
8c
8d
8e
8f
0.21±0.03
0.12±0.01
0.51±0.06
0.34±0.03
0.56±0.03
-OCH3 25.70±2.16
7b
-H
-F
-Cl
-H
23.82±1.97
19.05±2.34
20.71±0.92
-H
7c
-H
-H
-Cl
7d
-Cl
-Cl
-H
7e
-Cl
-OCH3 18.24±1.24
-Cl
-OCH3 0.62±0.05
7f
-Cl
-F
-Cl
-H
>50
>50
-Cl
-F
-Cl
-H
2.57±0.10
3.78±0.46
0.17±0.02
0.09±0.01
0.76±0.04
0.19±0.01
0.32±0.02
0.23±0.02
1.82±0.12
6.86±0.32
7g
8g
8h
8i
-Cl
-Cl
7h
-OCH3
14.73±0.83
-OCH3
7i
-OCH3 -OCH3 15.37±1.05
-OCH3 -OCH3
7j
8j
-OCH3
-OCH3
-NO2
-F
-Cl
-H
16.81±1.01
16.34±1.13
>50
-OCH3
-OCH3
-NO2
-F
-Cl
7k
8k
8l
7l
-H
7m
7n
8m
8n
8o
8p
-NO2
-OCH3 23.72±1.72
-NO2
-OCH3
-F
-NO2
-F
>50
-NO2
7o
-NO2
-Cl
14.40±0.78
0.04±01
-NO2
-Cl
7p
Staurosporine
a SEM: Standard Error Mean.

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DOI: 10.1039/C5NJ02896E
GSK-3β inhibitory activity
All the 32 synthesized aryl anilinomaleimide based derivatives were evaluated for
GSK-3β inhibitory activity by non-radioactive based Kinase-GloTM luminescence assay23.
Staurosporine, a known kinase inhibitor was taken as standard. The in vitro assay results are
summarized in Table 1.
As illustrated in Table 1, many compounds from the series exhibited moderate to potent
inhibitory activity against GSK-3β, with more than one-third of the compounds showing
inhibition with IC50 < 1 µM. Compound 8j (R1 = R3 = OCH3) displayed highest GSK-3β
inhibition of 0.09 µM. The inhibitory potency was found to be influenced both by the
substitutions on the maleimides nitrogen (R2) as well as on the aryl rings (R1 and R3). For
compound 8a, with NH-maleimide and both aryl rings unsubstituted (R1= R2= R3= H),
significant GSK-3β inhibition was observed with IC50 of 0.21 µM. Replacement of hydrogen
on the maleimide core by methyl group led to drastic loss of activity, as observed in 7a (R2=
CH3; R1 = R3= H) exhibiting IC50 of 16.49 µM. Similar trend was displayed by all the other
compounds with R2 = H (8b-8p) in comparison to their respective counterparts with R2 = CH3
(7b-7p), signifying the importance of NH of the imide motif (R2 = H) as an essential
requirement of the pharmacophore for exhibiting GSK-3β inhibitory activity.
A preliminary structure-activity relationship has also been generated around aryl rings
by introducing different substitutions (R1 and R3), to have an understanding of the resulting
impact on the efficiency of GSK-3β inhibition. The presence of methoxy or hydrogen at either
or both R1 and R3 positions were found to be more favourable for exhibiting GSK-3β inhibitory
activity than either chloro or nitro at R1 and chloro or fluoro at R3. For example, replacement
of H at R3 in 8a by methoxy resulted in increase in GSK-3β inhibitory activity to 0.12 µM in
8b, while as substitution by chloro (8d) or fluoro (8c) at R3 resulted in decrease in activity (8d,
IC50 = 0.34 µM; 8c, IC50 = 0.51 µM) in comparison to 8a. Similarly, replacing H at R1 in 8a by
methoxy caused an increase in GSK-3β inhibition in 8i (IC50 = 0.17 µM) while presence of
nitro (8m) or chloro (8e) resulted in lowering of GSK-3β inhibition (8m, IC50 = 0.32 µM; 8e,
IC50 = 0.56 µM) in comparison to 8a. Similar trend was observed by introducing hydrogen,
methoxy, chloro or fluoro at R3 while substituting either methoxy, nitro or chloro group at R1.
The correlation can be summarized as shown in Figure 1.

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DOI: 10.1039/C5NJ02896E
R2
N
H > CH3
O
O
HN
O
R3
HN
R1
H or OCH3 > Cl or F
H or OCH3
> NO2 or Cl
Figure 1: Structure-Activity Relationship against GSK-3β
Docking studies:
In order to gain a better insight of the binding interactions of synthesized maleimide derivatives
against GSK-3β, molecular docking studies were performed. Compounds were docked
individually against GSK-3β protein target (PDB: 1Q3D) containing Staurosporine as binding
ligand. Staurosporine displays key interactions by forming two hydrogen bonds with carbonyl
oxygen of ASP-133 and backbone nitrogen of VAL-135 residues respectively, besides showing
hydrophobic interactions with ILE-62, VAL-70 and PHE-67 residues within the ATP binding
site of GSK-3β protein contributing to enhance inhibitory activity of Staurosporine ligand. The
glide score along with the glide energies of all the synthesized compounds and the standard
Staurosporine are shown in Table 2. The docking results revealed the synthesized molecules
exhibited multiple H-bonding interactions with important amino acid residues on the receptor
site in addition to H-bond with water residue and π-cation interactions by some compounds.
The N-H of maleimide core (R2= H, compounds 8a-8p) shows H-bonding interaction with
ASP-133 residue in the hinge region. Substitution of H by -CH3 (R2= CH3, compounds 7a-7p)
at this position prevents the H-bond formation with ASP-133 which probably results in lower
dock scores displayed by these molecules and lower activity in in vitro assays. Compound 8j
was found to be the most active molecule with significant binding affinity towards GSK-3β
(Glide score -8.77) closest to reference Staurosporine with Glide score -10.01, forming
hydrogen bond between the nitrogen atom (maleimide ring) and carbonyl oxygen of ASP-133
residue and a similar interaction between oxygen atom (maleimide ring) and backbone nitrogen

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of VAL-135 residue. Additionally, one amide nitrogen and one of the methoxy group show H-
DOI: 10.1039/C5NJ02896E
bond interactions with the side chain residue of ASN-186 and water molecule in the system
respectively. Furthermore, π-cation interaction between the terminal benzene ring and LYS-
183 residue is also observed. This finding justifies the in vitro study results, wherein compound
8j has been found to exhibit least IC50 value of 0.09 µM (maximum GSK-3β inhibition).
Likewise, multiple hydrogen bond interactions of 8b with VAL-135, ASP-133 and ASP-200
residues and the π-cation interaction between the terminal benzene ring and LYS-183 residue
resulted in high binding efficiency with glide score of -8.32, thereby supporting its significant
in vitro GSK-3β inhibitory activity (IC50 = 0.12 µM). The snapshots of Staurosporine and top
inhibitory molecules 8j and 8b, with their binding mode and molecular interactions are shown
in Figure 2a-f.
Table-2
Molecular docking study results of the synthesized compounds against GSK-3β.
Compd.
Glide Score Glide Energy Compd. Glide Score Glide Energy
-5.61
-6.16
-5.57
-5.03
-6.11
-6.12
-6.05
-5.95
-6.38
-4.36
-6.44
-6.33
-2.4
-48.23
-40.88
-45.88
-45.8
-7.93
-8.32
-7.86
-7.85
-7.84
-7.77
-7.75
-7.72
-7.96
-8.77
-7.79
-8.19
-7.87
-7.91
-7.69
-7.05
-50.77
-51.03
-50.36
-50.89
-50.5
7a
8a
8b
8c
8d
8e
8f
7b
7c
7d
-45.92
-47.99
-46.79
-46.8
7e
-51.63
-50.74
-51.11
-51.93
-53.65
-52.84
-47.72
-53.65
-53.33
-52.12
-53.43
7f
7g
8g
8h
8i
7h
-48.61
-47.39
-49.06
-49.87
-46.01
-41.44
-48.53
-49.74
-59.09
7i
7j
8j
7k
8k
8l
7l
7m
8m
8n
8o
8p
-4.52
-6.18
-6.46
-10.01
7n
7o
7p
Staurosporine

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2a
2b

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2c
2d

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2e
2f
Figure 2: Docking poses and interaction of ligands with respect to GSK-3β protein.
(a) Docking pose of Staurosporine (b) Ligplot of Staurosporine (c) Docking pose of 8j
(d) Ligplot of 8j
(e) Docking pose of 8b
(f) Ligplot of 8b.

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Antidepressant activity
Considering the close association of GSK-3β inhibition with antidepressant activity,
the compounds 8j, 8b, 8i, 8l, 8a and 8n exhibiting potent GSK-3β inhibitory activity were
further investigated for antidepressant activity by the widely accepted forced swim test and tail
suspension test (TST and FST) models.
Forced swim test
The results of the effect of test compounds 8j, 8b, 8i, 8l, 8a and 8n on immobility in
the forced swim test are depicted in Figure 3. Except 8n and 8a, all the other test compounds
significantly reduced the immobility time in experimental rats compared to normal saline and
fluoxetine treated groups (P < 0.05). Administration of compound 8j or 8b registered an almost
1.6-fold decrease in immobility time in comparison to normal saline group and 1.4-fold
decrease in comparison to standard drug fluoxetine treated group.
Figure 3: 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 < 0.01, ***P <
0.001 when compared with normal control; #P < 0.05, ##P < 0.01, ###P < 0.001 when compared
with standard fluoxetine.
Tail suspension test
The results from tail suspension test illustrating the effect of test compounds 8j, 8b, 8i,
8l, 8a and 8n on immobility are shown in Figure 4.Compounds 8j, 8b and 8i significantly

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shortened the immobility time periods in experimental rats compared to both normal saline and
DOI: 10.1039/C5NJ02896E
fluoxetine treated groups (P < 0.05), while as compound 8l showed significant effect with
respect to normal saline treated group only. Compounds 8j and 8b were particularly most
active. Pre-treatment with compound 8j or 8b resulted in a considerable decrease in mean
duration of immobility by 1.75 to 1.8-fold relative to normal control group and by about 1.4 to
1.5-fold relative to standard drug fluoxetine.
Among the compounds tested, compounds 8j and 8b thus exhibited the maximum
antidepressant-like effects in both forced swim test and tail suspension test (TST and FST)
models. Furthermore, the antidepressant activity of the tested compounds was observed
consistent with respect to their GSK-3β inhibitory profile. These results reinforce the notion
that GSK-3β inhibitors hold a significant potential for treatment of depression and related
disorders.
Figure 4: 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 when compared with normal control; #P < 0.05, ##P < 0.01, ###P < 0.001 when compared
with standard fluoxetine.
Conclusion
A novel series of aryl anilinomaleimide based derivatives has been synthesized and tested for
in vitro GSK-3β inhibition potential. Large number of compounds displayed moderate to potent
inhibitory activity against GSK-3β, with more than one-third showing inhibition with IC50 < 1

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µM. The molecular docking studies showed multiple H-bonding interactions formed by the
DOI: 10.1039/C5NJ02896E
synthesized molecules with important amino acid residues on the receptor site in addition to
H-bond with water residue and π-cation interactions by some compounds. Compound 8j and
8b were found to have the closest binding efficiency (G score of -8.77 and -8.32 respectively)
relative to Staurosporine (G score of -10.01). Compounds 8j, 8b, 8i, 8l, 8a and 8n exhibiting
potent GSK-3β inhibition were further assessed for in vivo antidepressant activity by the widely
accepted forced swim test and tail suspension test models. Among the tested compounds, 8j
and 8b exhibited significant antidepressant activity in comparison to normal control group as
well as fluoxetine, a standard antidepressant drug, treated group in both forced swim test and
tail suspension test models. Furthermore, the antidepressant activity of the tested compounds
correlated well with respect to their GSK-3β inhibitory profile signifying the role of GSK-3β
inhibitors for the treatment of depression and related disorders. The results of our study suggest
that compounds 8j and 8b may serve as valuable candidates for further exploration of
pathological implications of GSK-3β inhibition on depression and related abnormalities.
Experimental
Materials and Methods
The chemicals used as starting materials and reagents were of analytical grade and purchased
from Spectrochem and Sigma Aldrich. Human recombinant GSK-3β and prephosphorylated
polypeptide substrate GS-2 were purchased from Merck-Millipore Corporation (India).
Kinase-Glo Luminescent Kinase Assay (catalog number V6713) was obtained from Promega
Corporation (Madison, WI). Staurosporine and ATP were purchased from Sigma-Aldrich.
Luminescence was recorded on Infinite F200® PRO (Tecan) instrument. Fluoxetine
hydrochloride was purchased from the local market.
All the melting points have been measured using Veego VMP-DS apparatus and are
uncorrected. IR spectra were recorded on Perkin Elmer 1650 spectrophotometer (USA). NMR
spectra (1H and 13C) were determined on a Bruker (300 MHz or 400 MHz) spectrometer and
chemical shifts are expressed as ppm against TMS as internal reference. Mass spectra were
recorded on 70 eV (EI Ms-QP 1000EX, Shimadzu, Japan). Elemental analysis was carried out
using Elementar Vario EL III elemental analyzer. Elemental analysis data is reported in %
standard.
Synthesis

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Procedure for the synthesis of intermediates (1a-d to 6a-h)
Intermediates 1a-d to 6a-h have been synthesized as per the procedure reported in the
literature18, 24
.
General procedure for the synthesis of target compounds (7a-7p and 8a-8p)
Appropriate acid (6a-h) was dissolved in dry DMF. EDC.HCl and HOBT were added and
stirring was continued for 30 min at room temperature followed by addition of appropriate
anilines. The reaction mixture was allowed to stir for 8-10 h with regular monitoring by TLC.
After completion of the reaction, the reaction mixture was poured onto crushed ice and the
solid obtained was filtered and air dried. The compounds were then crystallized in ethyl acetate
to afford purified target compounds (7a-7p and 8a-8p).
4-(2,5-dihydro-1-methyl-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-phenylbenzamide (7a)
ο
Yellow-orange powder; yield: 80%; m. p. 259-261 C; IR (KBr): ν (cm-1) 3390, 3285, 1754,
1703, 1632, 1596, 1523, 1503, 1440, 1385, 1314, 1262, 1241, 1162, 1062, 986, 825; 1H NMR
(DMSO-d6, 400 MHz): δ 2.99 (s, 3H), 6.72 (d, 2H, J = 8.8 Hz), 6.98-7.01 (m, 2H), 7.03-7.09
(m, 4H), 7.22-7.26 (m, 2H), 7.50-7.54 (m, 4H), 9.76 (s, 1H), 10.11 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): δ 23.77, 104.95, 120.05, 120.28, 123.45, 127.29, 127.49, 128.41, 128.50,
129.11, 129.81, 136.68, 139.13, 140.81, 164.41, 167.89, 171.61; ESI-MS: 398 (M+1)+; Anal.
Calcd. for C24H19N3O3: C, 72.53; H, 4.82; N, 10.57; O, 12.08%; Found: C, 72.57; H, 4.81; N,
10.53%.
4-(2,5-dihydro-1-methyl-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (7b)
ο
Yellow-orange powder; yield: 81%; m. p. 240-241 C; IR (KBr): ν (cm-1) 3383, 3278, 1755,
1
1695, 1642, 1600, 1513, 1453, 1381, 1312, 1270, 1243, 1168, 1106, 991, 824; H NMR
(DMSO-d6, 400 MHz): δ 3.02 (s, 3H), 3.72 (s, 3H), 6.81 (d, 2H, J = 8.8 Hz), 6.89-6.91 (m,
2H), 7.03-7.06 (m, 2H), 7.12-7.15 (m, 3H), 7.52-7.57 (m, 4H), 9.85 (s, 1H), 10.19 (s, 1H); 13C
NMR (DMSO-d6, 100 MHz): δ 24.27, 55.61, 105.31, 114.12, 120.56, 122.39, 127.77, 127.85,
129.06, 129.61, 130.32, 132.68, 137.21, 141.12, 155.88, 164.48, 168.38, 172.12; ESI-MS: 428
(M+1)+; Anal. Calcd. for C25H21N3O4: C, 70.25; H, 4.95; N, 9.83; O, 14.97%; Found: C, 70.29;
H, 4.92; N, 9.85%.

New Journal of Chemistry
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DOI: 10.1039/C5NJ02896E
4-(2,5-dihydro-1-methyl-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (7c)
ο
Yellow-orange powder; yield: 83%; m. p. 233-234 C; IR (KBr): ν (cm-1) 3386, 3299, 1758,
1
1703, 1634, 1600, 1515, 1453, 1390, 1309, 1263, 1246, 1181, 1126, 994, 829; H NMR
(DMSO-d6, 400 MHz): δ 3.00 (s, 3H), 6.82 (d, 2H, J = 7.6 Hz), 7.05-7.06 (m, 2H), 7.14-7.17
(m, 5H), 7.62 (d, 2H, J = 7.6 Hz), 7.74 (dd, 2H, J = 7.6 Hz and 5.2 Hz), 9.87 (s, 1H), 10.06 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.76, 105.00, 115.06 (d, J = 22.0 Hz), 120.05, 122.08
(d, J = 8.0 Hz), 127.28, 127.47, 128.22, 129.11, 129.81, 135.48, 135.50, 136.66, 140.87, 158.14
(d, J = 239.0 Hz), 164.34, 167.88, 171.60; ESI-MS: 416 (M+1)+; Anal. Calcd. for
C24H18FN3O3: C, 69.39; H, 4.37; F, 4.57; N, 10.12; O, 11.55%; Found: C, 69.44; H, 4.39; N,
10.11%.
4-(2,5-dihydro-1-methyl-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (7d)
Yellow-orange powder; yield: 84%; m. p. 242-243οC; IR (KBr): ν (cm-1) 3386, 3301, 1756,
1702, 1639, 1599, 1524, 1494, 1444, 1389, 1305, 1284, 1239, 1179, 1090, 993, 817;1H NMR
(DMSO-d6, 400 MHz): δ 2.99 (s, 3H), 6.71 (d, 2H, J = 8.0 Hz), 6.98-7.07 (m, 5H), 7.22 (d, 2H,
J = 9.2 Hz), 7.50-7.55 (m, 4H), 9.87 (s, 1H), 10.06 (s, 1H); 13C NMR (DMSO-d6, 100 MHz):
δ 24.26, 105.64, 120.54, 122.26, 127.54, 127.79, 128.04, 128.59, 128.90, 129.61, 130.29,
137.13, 138.63, 141.48, 165.01, 168.37, 172.08; ESI-MS: 432 (M+1)+; Anal. Calcd. for
C24H18ClN3O3: C, 66.75; H, 4.20; Cl, 8.21; N, 9.73; O, 11.11%; Found: C, 66.69; H, 4.23; N,
9.72%.
4-(4-(4-chlorophenyl)-2,5-dihydro-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-
phenylbenzamide (7e)
ο
Yellow-orange powder; yield: 86%; m. p. 216-217 C; IR (KBr): ν (cm-1) 3385, 3301,
1757, 1701, 1639, 1599, 1524, 1494, 1444, 1389, 1305, 1285, 1239, 1175, 1090, 993, 820; 1H
NMR (DMSO-d6, 400 MHz): δ 3.04 (s, 3H), 6.86 (d, 2H, J = 8.4 Hz), 7.05-7.10 (m, 3H), 7.23
(d, 2H, J = 8.8 Hz), 7.31-7.35 (m, 2H), 7.69-7.74 (m, 4H), 9.93 (s, 1H), 9.99 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): δ 23.61, 102.90, 120.13, 120.33, 123.27, 127.06, 127.53, 128.18,
128.37, 128.89, 130.47, 132.00, 136.76, 138.98, 140.34, 164.36, 167.49, 171.22; ESI-MS: 432
(M+1)+; Anal. Calcd. for C24H18ClN3O3: C, 66.75; H, 4.20; Cl, 8.21; N, 9.73; O, 11.11%;
Found: C, 66.70; H, 4.21; N, 9.72%.

Page 17 of 31
New Journal of Chemistry
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DOI: 10.1039/C5NJ02896E
4-(4-(4-chlorophenyl)-2,5-dihydro-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (7f)
ο
Yellow-orange powder; yield: 79%; m. p. 212-213 C; IR (KBr): ν (cm-1) 3392, 3261,
1750, 1671, 1649, 1610, 1590, 1506, 1454, 1392, 1307, 1290, 1234, 1175, 1093, 961, 820; 1H
NMR (DMSO-d6, 300 MHz): δ 3.04 (s, 3H), 3.70 (s, 3H), 6.60 (d, 2H, J = 8.4 Hz), 6.79 (d,
2H, J = 9.0 Hz), 6.95 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 8.7 Hz), 7.39 (d, 2H, J = 9.0 Hz),
7.51 (d, 2H, J = 8.4 Hz), 9.93 (s, 1H), 9.99 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 24.28,
60.23, 103.74, 114.13, 120.82, 122.45, 127.79, 127.97, 129.23, 129.36, 131.17, 132.17, 132.65,
137.68, 141.07, 155.92, 164.43, 168.15, 171.93; ESI-MS: 462 (M+1)+; Anal. Calcd. for
C25H20ClN3O4: C, 65.01; H, 4.36; Cl, 7.68; N, 9.10; O, 13.86%; Found: C, 65.06; H, 4.34; N,
9.07%.
4-(4-(4-chlorophenyl)-2,5-dihydro-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (7g)
ο
Yellow-orange powder; yield: 82%; m. p. 206-207 C; IR (KBr): ν (cm-1) 3382, 3291,
1
1753, 1697, 1644, 1612, 1587, 1505, 1454, 1387, 1319, 1176, 1084, 993, 828; H NMR
(DMSO-d6, 400 MHz): δ 2.98 (s, 3H); 6.84 (d, 2H, J = 8.4 Hz); 7.04 (d, 2H, J = 8.4 Hz); 7.13-
7.18 (m, 2H); 7.22 (d, 2H, J = 8.4 Hz); 7.67 (d, 2H, J = 8.4 Hz); 7.75-7.71 (m, 2H); 9.96 (s,
1H); 10.06 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 24.27, 103.97, 115.56 (d, J = 22.0 Hz),
120.80, 122.64 (d, J = 8.0 Hz), 127.80, 128.09, 129.04, 129.16, 131.18, 132.24, 135.96, 137.55,
141.21, 158.68 (d, J = 239.0 Hz), 164.78, 168.15, 171.91; ESI-MS: 450 (M+1)+; Anal. Calcd.
for C24H17ClFN3O3: C, 64.08; H, 3.81; Cl, 7.88; F, 4.22; N, 9.34; O, 10.67%; Found: C, 64.13;
H, 3.83; N, 9.31%.
4-(4-(4-chlorophenyl)-2,5-dihydro-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (7h)
ο
Orange powder; yield: 85%; m. p. 257-259 C; IR (KBr): ν (cm-1) 3396, 3298, 1753,
1689, 1632, 1603, 1587, 1495, 1434, 1387, 1306, 1291, 1229, 1178, 1083, 990, 825; 1H NMR
(DMSO-d6, 400 MHz): δ 3.00 (s, 3H), 6.86 (d, 2H, J = 8.4 Hz), 7.06 (d, 2H, J = 8.4 Hz), 7.23
(d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 9.2 Hz), 7.69 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.8 Hz),
9.95 (s, 1H), 10.13 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.80, 103.65, 120.29, 121.80,
127.08, 127.33, 127.67, 128.41, 128.66, 130.69, 131.77, 137.04, 138.10, 140.83, 164.45,
167.66, 171.41; ESI-MS: 466 (M+1)+; Anal. Calcd. for C24H17Cl2N3O3: C, 61.82; H, 3.67; Cl,
15.21; N, 9.01; O, 10.29%; Found: C, 61.76; H, 3.69; N, 9.04%.

New Journal of Chemistry
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DOI: 10.1039/C5NJ02896E
4-(2,5-dihydro-4-(4-methoxyphenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-
phenylbenzamide (7i)
ο
Orange powder; yield: 86%; m. p. 211-212 C; IR (KBr): ν (cm-1) 3384, 3295, 1756, 1688,
1
1658, 1603, 1581, 1512, 1435, 1395, 1338, 1294, 1246, 1173, 1090, 992, 829; H NMR
(DMSO-d6, 400 MHz): δ 2.99 (s, 3H), 3.68 (s, 3H), 6.76 (d, 2H, J = 8.4 Hz), 6.82 (d, 2H, J =
8.4 Hz), 7.02-7.09 (m, 3H), 7.32 (d, 2H, J = 7.6 Hz), 7.66 (d, 2H, J = 8.4 Hz), 7.72 (d, 2H, J =
13
7.6 Hz), 9.72 (s, 1H), 9.96 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 23.73, 55.05, 106.05,
112.91, 119.54, 120.31, 122.04, 123.42, 127.59, 128.01, 128.49, 130.45, 135.21, 139.17,
141.05, 158.44, 164.49, 168.13, 171.79; ESI-MS: 428 (M+1)+; Anal. Calcd. for C25H21N3O4:
C, 70.25; H, 4.95; N, 9.83; O, 14.97%; Found: C, 70.18; H, 4.98; N, 9.81%.
4-(2,5-dihydro-4-(4-methoxyphenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (7j)
ο
Orange powder; yield: 79%; m. p. 213-214 C; IR (KBr): ν (cm-1) 3385, 3282, 1756, 1695,
1643, 1596, 1503, 1454, 1388, 1315, 1294, 1246, 1179, 1092, 993, 825; 1H NMR (DMSO-d6,
400 MHz): δ 2.99 (s, 3H), 3.68 (s, 3H), 3.73 (s, 3H), 6.75 (d, 2H, J = 8.8 Hz), 6.81 (d, 2H, J =
8.4 Hz), 6.90 (d, 2H, J = 9.2 Hz), 7.03 (d, 2H, J = 8.8 Hz), 7.60-7.66 (m, 4H), 9.70 (s, 1H),
9.86 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.73, 55.05, 55.10, 105.88, 112.89, 113.61,
119.56, 121.92, 122.05, 127.44, 128.15, 130.45, 132.21, 135.23, 140.85, 155.37, 158.42,
164.08, 168.13, 171.80; ESI-MS: 458 (M+1)+; Anal. Calcd. for C26H23N3O5: C, 68.26; H, 5.07;
N, 9.19; O, 17.49%; Found: C, 68.20; H, 5.09; N, 9.22%.
4-(2,5-dihydro-4-(4-methoxyphenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (7k)
ο
Orange powder; yield: 83%; m. p. 202-203 C; IR (KBr): ν (cm-1) 3390, 3271, 1760, 1695,
1
1650, 1603, 1581, 1504, 1435, 1392, 1309, 1294, 1235, 1175, 1096, 990, 818; H NMR
(DMSO-d6, 400 MHz): δ 2.99 (s, 3H); 3.68 (s, 3H); 6.75 (d, 2H, J = 8.4 Hz); 6.82 (d, 2H, J =
8.4 Hz), 7.03 (d, 2H, J = 8.4 Hz), 7.14-7.18 (m, 2H), 7.65 (d, 2H, J = 8.4 Hz), 7.72-7.75 (m,
13
2H), 9.72 (s, 1H), 10.05 (s, 1H); C NMR (DMSO-d6, 100 MHz): 23.73, 55.05, 106.11,
112.91, 115.05 (d, J = 22.0 Hz), 119.54, 122.04, 122.10 (d, J = 8.0 Hz), 127.57, 127.81, 130.45,
135.18, 135.51, 140.10, 158.13 (d, J = 239.0 Hz), 158.44, 164.43, 168.12, 171.78; ESI-MS:
446 (M+1)+; Anal. Calcd. for C25H20FN3O4: C, 67.41; H, 4.53; F, 4.27; N, 9.43; O, 14.37%;
Found: C, 67.38; H, 4.52; N, 9.43%.

Page 19 of 31
New Journal of Chemistry
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DOI: 10.1039/C5NJ02896E
4-(2,5-dihydro-4-(4-methoxyphenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (7l)
ο
Orange powder; yield: 86%; m. p. 208-209 C; IR (KBr): ν (cm-1) 3389, 3263, 1750, 1672,
1
1650, 1612, 1591, 1506, 1455, 1394, 1307, 1290, 1236, 1176, 1093, 961, 820; H NMR
(DMSO-d6, 400 MHz): δ 2.99 (s, 3H), 3.68 (s, 3H), 6.76 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J =
8.4 Hz), 7.03 (d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.8 Hz), 7.66 (d, 2H, J = 8.4 Hz), 7.77 (d,
2H, J = 8.4 Hz), 9.74 (s, 1H), 10.11 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.74, 55.05,
106.25, 112.92, 119.52, 121.76, 122.02, 127.00, 127.65, 128.41, 130.45, 135.14, 138.17,
140.23, 158.45, 164.59, 168.13, 171.17; ESI-MS: 462 (M+1)+; Anal. Calcd. for C25H20ClN3O4:
C, 65.01; H, 4.36; Cl, 7.68; N, 9.10; O, 13.86%; Found: C, 65.05; H, 4.34; N, 9.08%.
4-(2,5-dihydro-4-(4-nitrophenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-
phenylbenzamide (7m)
ο
Orange powder; yield: 87%; m. p. 231-232 C; IR (KBr): ν (cm-1) 3396, 3288, 1763, 1695,
1632, 1591, 1519, 1456, 1341, 1306, 1291, 1228, 1174, 1111, 990, 820; 1H NMR (DMSO-d6,
400 MHz): δ 3.01 (s, 3H), 6.84 (d, 2H, J = 8.4 Hz), 7.06-7.09 (m, 1H), 7.23 (d, 2H, J = 8.0 Hz),
7.58 (d, 2H, J = 8.4 Hz), 7.67-7.71 (m, 4H), 7.98 (d, 2H, J = 8.4 Hz), 9.74 (s, 1H), 10.11 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.85, 101.72, 120.93, 121.46, 122.65, 124.05,
128,15, 128.99, 129.97, 130.49, 137.42, 139.52, 139.64, 140.93, 145.91, 164.80, 168.78,
172.51; ESI-MS: 443 (M+1)+; Anal. Calcd. for C24H18N4O5: C, 65.15; H, 4.10; N, 12.66; O,
18.08%; Found: C, 65.09; H, 4.12; N, 12.63%.
4-(2,5-dihydro-4-(4-nitrophenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (7n)
Red-orange powder; yield: 85%; m. p. 227-229 οC; IR (KBr): ν (cm-1) 3396, 3307, 1763, 1685,
1648, 1598, 1511, 1434, 1410, 1340, 1320, 1236, 1175, 1093, 960, 820; 1H NMR (DMSO-d6,
400 MHz): δ 2.95 (s, 3H), 3.66 (s, 3H), 6.80-6.83 (m, 4H), 7.17 (d, 2H, J = 8.8 Hz), 7.52 (d,
2H, J = 8.8 Hz), 7.60 (d, 2H, J = 9.2 Hz), 7.92 (d, 2H, J = 8.8 Hz), 9.81 (s, 1H), 10.19 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): δ 23.86, 55.09, 101.20, 113.62, 121.11, 122.12, 122.22,
127.53, 129.67, 129.79, 131.92, 136.85, 139.27, 140.19, 145.32, 155.48, 163.95, 167.15,
171.08; ESI-MS: 473 (M+1)+; Anal. Calcd. for C25H20N4O6: C, 63.56; H, 4.27; N, 11.86; O,
20.32%; Found: C, 63.60; H, 4.26; N, 11.83%.

New Journal of Chemistry
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4-(2,5-dihydro-4-(4-nitrophenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-DOI: 10.1039/C5NJ02896E
fluorophenyl)benzamide (7o)
ο
Yellow-orange powder; yield: 86%; m. p. 265-267 C; IR (KBr): ν (cm-1) 3398, 3291, 1743,
1
1684, 1626, 1614, 1594, 1501, 1458, 1399, 1339, 1249, 1205, 1094, 990, 825; H NMR
(DMSO-d6, 400 MHz): δ 2.95 (s, 3H), 6.82 (d, 2H, J = 8.4 Hz), 7.07-7.11 (m, 2H), 7.18 (d, 2H,
J = 8.8 Hz), 7.59-7.66 (m, 4H), 7.92 (d, 2H, J = 8.8 Hz), 9.98 (s, 1H); 10.20 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): δ 23.88, 101.43, 115.09 (d, J = 21.00 Hz), 121.04, 122.21(d, J = 9.00
Hz), 127.67, 129.38, 129.78, 132.49, 135.34, 135.45, 136.85, 139.21, 145.35, 158.19 (d, J =
239.00 Hz), 164.22, 167.16, 171.05; ESI-MS: 461(M+1)+; Anal. Calcd. for C24H17FN4O5: C,
62.61; H, 3.72; F, 4.13; N, 12.17; O, 17.38%; Found: C, 62.64; H, 3.71; N, 12.18%.
4-(2,5-dihydro-4-(4-nitrophenyl)-1-methyl-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (7p)
Orange powder; yield: 88%; m. p. 280-282οC; IR (KBr): ν (cm-1) 3390, 3294, 1760, 1696,
1
1643, 1591, 1517, 1501, 1454, 1384, 1346, 1302, 1238, 1173, 1090, 986, 848; H NMR
(DMSO-d6, 400 MHz): δ 3.02 (s, 3H), 6.90 (d, 2H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.0 Hz), 7.38
(d, 2H, J = 8.8 Hz), 7.68 (d, 2H, J = 8.4 Hz), 7.74 (d, 2H, J = 8.8 Hz), 8.00 (d, 2H, J = 8.8 Hz),
10.11 (s, 1H); 10.27 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 23.88, 101.54, 121.02, 122.85,
127.26, 127.14, 127.75, 128.42, 129.26, 129.80, 136.81, 138.01, 139.15, 140.52, 145.37,
164.39, 167.16, 171.04; ESI-MS: 477 (M+1)+; Anal. Calcd. for C24H17ClN4O5: C, 60.45; H,
3.59; Cl, 7.43; N, 11.75; O, 16.78%; Found: C, 60.41; H, 3.61; N, 11.78%.
4-(2,5-dihydro-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-phenylbenzamide (8a)
ο
Orange powder; yield: 81%; m. p. 276-278 C; IR (KBr): ν (cm-1) 3391, 3286, 1754, 1701,
1
1630, 1594, 1521, 1501, 1440, 1385, 1314, 1260, 1241, 1162, 1062, 985, 820; H NMR
(DMSO-d6, 400 MHz): δ 6.81 (d, 2H, J = 8.8 Hz); 7.03-7.08 (m, 3H); 7.14-7.18 (m, 3H); 7.29-
7.33 (m, 2H); 7.63 (d, 2H, J = 8.8 Hz); 7.72 (d, 2H, J = 7.6 Hz); 9.70 (s, 1H); 9.76 (s, 1H);
13
10.88 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 106.29, 120.45, 120.81, 123.94, 127.72,
127.99, 128.82, 128.99, 129.75, 130.37, 137.15, 139.65, 141.38, 164.94, 169.54, 173.09; ESI-
MS: 384 (M+1)+; Anal. Calcd. for C23H17N3O3: C, 72.05; H, 4.47; N, 10.96; O, 12.52%; Found:
C, 72.12; H, 4.49; N, 10.94%.
4-(2,5-dihydro-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-methoxyphenyl)benzamide
(8b)

Page 21 of 31
New Journal of Chemistry
ο
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Yellow-orange powder; yield: 85%; m. p. 290-292 C; IR (KBr): ν (cm-1) 3385, D3O3I3: 160.,103329/8C05N,J02896E
1758, 1697, 1669, 1634, 1595, 1504, 1475, 1410, 1349, 1301, 1249, 1237, 1175, 1103, 1027,
829; 1H NMR (DMSO-d6, 400 MHz): δ 3.72 (s, 3H), 6.80 (d, 2H, J = 8.8 Hz), 6.88-6.90 (m,
2H), 7.02-7.05 (m, 2H), 7.13-7.16 (m, 3H), 7.59-7.62 (m, 4H), 9.68 (s, 1H), 9.85 (s, 1H), 10.87
13
(s, 1H); C NMR (DMSO-d6, 100 MHz): δ 55.62, 106.12, 114.13, 120.46, 122.42, 127.71,
127.84, 128.95, 129.75, 130.38, 132.70, 137.17, 141.19, 155.89, 164.52, 169.54, 173.11; ESI-
MS: 414 (M+1)+; Anal. Calcd. for C24H19N3O4: C, 69.72; H, 4.63; N, 10.16; O, 15.48%; Found:
C, 69.74; H, 4.61; N, 10.15%.
4-(2,5-dihydro-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-fluorophenyl)benzamide (8c)
Red-orange powder; yield: 86%; m. p. 292-293 οC; IR (KBr): ν (cm-1) 3392, 3284, 1757, 1699,
1
1632, 1596, 1521, 1501, 1439, 1384, 1314, 1260, 1241, 1162, 1062, 985, 825; H NMR
(DMSO-d6, 400 MHz): δ 6.81 (d, 2H, J = 8.8 Hz), 7.03-7.06 (m, 2H), 7.12-7.19 (m, 5H), 7.62
13
(d, 2H, J = 8.8 Hz), 7.71-7.76 (m, 2H), 9.70 (s, 1H), 10.03 (s, 1H), 10.88 (s, 1H); C NMR
(DMSO-d6, 100 MHz): δ 106.33, 115.55 (d, J = 22.0 Hz), 120.45, 122.57, 122.68 (d, J = 8.0
Hz), 127.96, 128.62, 129.74, 130.36, 136.01, 137.13, 141.43, 158.65 (d, J = 238.0 Hz), 164.87,
169.53, 173.09; ESI-MS: 402 (M+1)+; Anal. Calcd. for C23H16FN3O3: C, 68.82; H, 4.02; F,
4.73; N, 10.47; O, 11.96%; Found: C, 68.77; H, 3.99; N, 10.43%.
4-(2,5-dihydro-2,5-dioxo-4-phenyl-1H-pyrrol-3-ylamino)-N-(4-chlorophenyl)benzamide (8d)
Red-orange powder; yield: 82%; m. p. 274-275 οC; IR (KBr): ν (cm-1) 3381, 3342, 3303, 3145,
1759, 1696, 1668, 1634, 1599, 1582, 1505, 1432, 1415, 1308, 1296, 1240, 1179, 1088, 998,
829; 1H NMR (DMSO-d6, 400 MHz): δ 6.81 (d, 2H, J = 8.8 Hz), 7.03-7.05 (m, 2H), 7.14-7.16
(m, 3H), 7.36-7.38 (m, 2H), 7.61 (d, 2H, J = 8.8 Hz), 7.75-7.77 (m, 2H), 9.71 (s, 1H), 10.09 (s,
1H), 10.88 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 106.45, 120.43, 122.27, 127.53, 127.73,
128.03, 128.46, 128.90, 129.73, 130.35, 137.10, 138.64, 141.56, 165.03, 169.52, 173.07; ESI-
MS: 418 (M+1)+; Anal. Calcd. for C23H16ClN3O3: C, 66.11; H, 3.86; Cl, 8.48; N, 10.06; O,
11.49%; Found: C, 66.17; H, 3.89; N, 10.04%.
4-(4-(4-chlorophenyl)-2,5-dihydro-2,5-dioxo-1H-pyrrol-3-ylamino)-N-phenylbenzamide (8e)
ο
Yellow-orange powder; yield: 85%; m. p. 258-260 C; IR (KBr): ν (cm-1) 3387, 3343, 3301,
3145, 1758, 1696, 1668, 1632, 1597, 1582, 1506, 1433, 1415, 1308, 1296, 1240, 1180, 1088,
998, 829; 1H NMR (DMSO-d6, 400 MHz): δ 6.85 (d, 2H, J = 8.4 Hz), 7.04-7.09 (m, 3H), 7.22
(d, 2H, J = 8.4 Hz), 7.31-7.34 (m, 2H), 7.68-7.74 (m, 4H), 9.81 (s, 1H), 10.00 (s, 1H), 10.94

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13
View Article Online
(s, 1H); C NMR (DMSO-d6, 100 MHz): δ 104.25, 120.21, 120.36, 123.49, 127D.2O4I:,101.120379./6C15N,J02896E
128.50, 128.61, 128.74, 130.85, 131.72, 137.05, 139.11, 140.73, 164.37, 168.82, 172.43; ESI-
MS: 418 (M+1)+; Anal. Calcd. for C23H16ClN3O3: C, 66.11; H, 3.86; Cl, 8.48; N, 10.06; O,
11.49%; Found: C, 66.05; H, 3.89; N, 10.03%.
4-(4-(4-chlorophenyl)-2,5-dihydro-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (8f)
ο
Yellow-orange powder; yield: 83%; m. p. 282-284 C; IR (KBr): ν (cm-1) 3390, 3344, 3301,
3189, 1757, 1666, 1698, 1639, 1602, 1514, 1488, 1391, 1355, 1303, 1242, 1169, 1094, 1013,
826; 1H NMR (DMSO-d6, 400 MHz): δ 3.74 (s, 3H), 6.83 (d, 2H, J = 8.4 Hz), 6.90 (d, 2H, J =
8.4 Hz), 7.04 (d, 2H, J = 8.0 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.61-7.69 (m, 4H), 9.79 (s, 1H),
9.90 (s, 1H), 10.94 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 55.11, 104.09, 113.62, 120.22,
121.98, 127.22, 127.46, 128.74, 130.85, 131.70, 132.14, 137.07, 140.53, 155.42, 163.95,
168.82, 172.44; ESI-MS: 448 (M+1)+; Anal. Calcd. for C24H18ClN3O4: C, 64.36; H, 4.05; Cl,
7.92; N, 9.38; O, 14.29%; Found: C, 64.31; H, 4.08; N, 9.35%.
4-(4-(4-chlorophenyl)-2,5-dihydro-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (8g)
Red-orange powder; yield: 83%; m. p. 272-273 οC; IR (KBr): ν (cm-1) 3402, 3334, 3300, 3197,
1761, 1697, 1661, 1633, 1601, 1505, 1487, 1401, 1347, 1302, 1249, 1209, 1091, 1012, 829;
1H NMR (DMSO-d6, 400 MHz): 6.84 (d, 2H, J = 8.4 Hz), 7.05 (d, 2H, J = 8.4 Hz), 7.12-7.17
(m, 2H), 7.23 (d, 2H, J = 8.4 Hz), 7.65 (d, 2H, J = 8.4 Hz), 7.76-7.71 (m, 2H), 9.85 (s, 1H),
13
10.11 (s, 1H), 10.81 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 104.30, 115.07 (d, J = 22.0
Hz), 120.21, 122.16 (d, J = 8.0 Hz), 127.25, 127.58, 128.41, 128.73, 130.84, 131.73, 135.45,
137.03, 140.78, 158.17 (d, J = 238.0 Hz), 164.30, 168.82, 172.42; ESI-MS: 436 (M+1)+; Anal.
Calcd. for C23H15ClFN3O3: C, 63.38; H, 3.47; Cl, 8.13; F, 4.36; N, 9.64; O, 11.01%; Found: C,
63.44; H, 3.45; N, 9.62%.
4-(4-(4-chlorophenyl)-2,5-dihydro-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (8h)
Red-orange powder; yield: 85%; m. p. 290-292 οC; IR (KBr): ν (cm-1) 3392, 3319, 3281, 3155,
1759, 1694, 1661, 1633, 1602, 1504, 1487, 1395, 1353, 1303, 1283, 1237, 1176, 1086, 1012,
829; 1H NMR (DMSO-d6, 400 MHz): δ 6.85 (d, 2H, J = 7.6 Hz), 7.05 (d, 2H, J = 7.6 Hz), 7.23
(d, 2H, J = 7.2 Hz), 7.39 (d, 2H, J = 8.0 Hz), 7.68 (d, 2H, J = 7.6 Hz), 7.77 (d, 2H, J = 7.6 Hz),

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New Journal of Chemistry
9.82 (s, 1H), 10.14 (s, 1H), 10.96 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 104D.4O0I: ,101.120309./1C95N,J02896E
121.81, 127.07, 127.26, 127.66, 128.26, 128.42, 128.71, 130.84, 131.75, 136.99, 138.11,
140.89, 164.47, 168.82, 172.42; ESI-MS: 452 (M+1)+; Anal. Calcd. for C23H15Cl2N3O3: C,
61.08; H, 3.34; Cl, 15.68; N, 9.29; O, 10.61%; Found: C, 61.13; H, 3.31; N, 9.26%.
View Article Online
4-(2,5-dihydro-4-(4-methoxyphenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-phenylbenzamide
(8i)
Yellow-orange powder; yield: 86%; m. p. 268-270οC; IR (KBr): ν (cm-1) 3386, 3338, 3281,
1759, 1696, 1669, 1635, 1596, 1506, 1472, 1410, 1349, 1302, 1249, 1237, 1172, 1103, 1028,
831; 1H NMR (DMSO-d6, 400 MHz): δ 3.68 (s, 3H), 6.74-6.76 (m, 2H), 6.81 (d, 2H, J = 8.0
Hz), 7.02-7.09 (m, 3H), 7.30-7.34 (m, 2H), 7.66 (d, 2H, J = 8.8 Hz), 7.73 (d, 2H, J = 8.8 Hz),
13
9.59 (s, 1H), 9.98 (s, 1H), 10.83 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 55.56, 107.34,
113.36, 119.94, 120.83, 122.60, 123.92, 128.08, 128.40, 128.98, 131.08, 135.75, 139.68,
141.62, 158.94, 165.01, 169.77, 173.28; ESI-MS: 414 (M+1)+; Anal. Calcd. for C24H19N3O4:
C, 69.72; H, 4.63; N, 10.16; O, 15.48%; Found: C, 69.77; H, 4.61; N, 10.15%.
4-(2,5-dihydro-4-(4-methoxyphenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (8j)
ο
Orange powder; yield: 84%; m. p. 274-276 C; IR (KBr): ν (cm-1) 3382, 3318, 3223, 3198,
1760, 1694, 1650, 1630, 1601, 1517, 1497, 1411, 1361, 1303, 1295, 1259, 1174, 1104, 1026,
820; 1H NMR (DMSO-d6, 400 MHz): δ 3.68 (s, 3H), 3.73 (s, 3H), 6.73-6.76 (m, 2H), 6.80 (d,
2H, J = 8.8 Hz), 6.88-6.91 (m, 2H), 7.01-7.03 (m, 2H), 7.61-7.65 (m, 4H), 9.57 (s, 1H), 9.87
(s, 1H), 10.83 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 55.55, 55.61, 107.14, 113.34, 114.12,
119.97, 122.45, 122.62, 127.94, 128.55, 131.08, 132.72, 135.75, 141.42, 155.89, 158.92,
164.61, 169.78, 173.30; ESI-MS: 444 (M+1)+; Anal. Calcd. for C25H21N3O5: C, 67.71; H, 4.77;
N, 9.48; O, 18.04%; Found: C, 67.66; H, 4.74; N, 9.49%.
4-(2,5-dihydro-4-(4-methoxyphenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (8k)
Red-orange powder; yield: 85%; m. p. 271-272 οC; IR (KBr): ν (cm-1) 3383, 3349, 3281, 3166,
1758, 1696, 1666, 1634, 1601, 1504, 1470, 1410, 1349, 1303, 1249, 1173, 1104, 1025, 830;
1H NMR (DMSO-d6, 400 MHz): δ 3.68 (s, 3H), 6.74-6.76 (m, 2H), 6.81 (d, 2H, J = 8.8 Hz),
7.02-7.04 (m, 2H), 7.14-7.18 (m, 2H), 7.65 (d, 2H, J = 8.8 Hz), 7.72-7.76 (m, 2H), 9.59 (s,
13
1H), 10.04 (s, 1H), 10.84 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 55.55, 107.38, 113.36,

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115.54 (d, J = 22.0 Hz), 119.95, 122.62 (d, J = 8.0 Hz), 122.87, 128.06, 128.21, 131.08, 135.70,
DOI: 10.1039/C5NJ02896E
136.04, 141.68, 158.64 (d, J = 239.0 Hz), 158.94, 164.95, 169.77, 173.28; ESI-MS: 432
(M+1)+; Anal. Calcd. for C24H18FN3O4: C, 66.82; H, 4.21; F, 4.40; N, 9.74; O, 14.83%; Found:
C, 66.86; H, 4.23; N, 9.71%.
4-(2,5-dihydro-4-(4-methoxyphenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (8l)
ο
Orange powder; yield: 88%; m. p. 286-288 C; IR (KBr): ν (cm-1) 3375, 3339, 3292, 3166,
1757, 1695, 1667, 1633, 1601, 1504, 1411, 1350, 1302, 1249, 1173, 1105, 1026, 825; 1H NMR
(DMSO-d6, 400 MHz): δ 3.68 (s, 3H), 6.74-6.76 (m, 2H), 6.82 (d, 2H, J = 8.8 Hz), 7.02-7.04
(m, 2H), 7.37-7.39 (m, 2H), 7.65 (d, 2H, J = 8.8 Hz), 7.76-7.78 (m, 2H), 9.59 (s, 1H), 10.10 (s,
1H), 10.84 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): δ 55.56, 107.52, 113.37, 119.93, 122.28,
122.59, 127.51, 128.07, 128.14, 128.89, 131.08, 135.67, 138.68, 141.80, 158.96, 165.11,
169.76, 173.26; ESI-MS: 448 (M+1)+; Anal. Calcd. for C24H18ClN3O4: C, 64.36; H, 4.05; Cl,
7.92; N, 9.38; O, 14.29%; Found: C, 64.41; H, 4.07; N, 9.40%.
4-(2,5-dihydro-4-(4-nitrophenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-phenylbenzamide (8m)
Red-orange powder; yield: 81%; m. p. 281-282 οC; IR (KBr): ν (cm-1) 3386, 3349, 3294, 3187,
1758, 1703, 1667, 1632, 1597, 1514, 1442, 1402, 1341, 1317, 1248, 1184, 1110, 1024, 854;
1H NMR (DMSO-d6, 400 MHz): δ 6.89 (d, 2H, J = 8.8 Hz), 7.06-7.09 (m, 1H), 7.26 (d, 2H, J
= 8.4 Hz), 7.30-7.34 (m, 2H), 7.67-7.71 (m, 4H), 7.99 (d, 2H, J = 8.8 Hz), 9.98 (s, 1H), 10.11
13
(s, 1H), 11.07 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 102.68, 120.92, 121.46, 122.65,
124.05, 128.17, 128.99, 129.97, 130.48, 137.43, 139.52, 139.64, 140.93, 145.90, 164.80,
168.79, 172.52; ESI-MS: 429 (M+1)+; Anal. Calcd. for C23H16N4O5: C, 64.48; H, 3.76; N,
13.08; O, 18.67%; Found: C, 64.53; H, 3.74; N, 13.11%.
4-(2,5-dihydro-4-(4-nitrophenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
methoxyphenyl)benzamide (8n)
Red-orange powder; yield: 84%; m. p. 294-296 οC; IR (KBr): ν (cm-1) 3378, 3349, 3294, 3164,
1770, 1715, 1651, 1633, 1601, 1513, 1412, 1342, 1315, 1247, 1179, 1107, 1026, 820; 1H NMR
(DMSO-d6, 400 MHz): δ 3.73 (s, 3H), 6.86-6.90 (m, 4H), 7.24 (d, 2H, J = 8.8 Hz), 7.59 (d, 2H,
J = 8.8 Hz), 7.66 (d, 2H, J = 8.8 Hz), 7.98 (d, 2H, J = 8.8 Hz), 9.86 (s, 1H), 10.09 (s, 1H), 11.06
13
(s, 1H); C NMR (DMSO-d6, 100 MHz): δ 55.62, 102.56, 114.13, 121.49, 122.56, 122.63,
128.03, 130.13, 130.48, 132.56, 137.44, 139.68, 140.75, 145.90, 155.98, 164.40, 168.79,

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New Journal of Chemistry
172.52; ESI-MS: 459 (M+1)+; Anal. Calcd. for C24H18N4O6: C, 62.88; H, 3.96; N, 12.22; O,
View Article Online
DOI: 10.1039/C5NJ02896E
20.94%; Found: C, 62.92; H, 3.94; N, 12.23%.
4-(2,5-dihydro-4-(4-nitrophenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
fluorophenyl)benzamide (8o)
Red-orange powder; yield: 77%; m. p. 292-294 οC; IR (KBr): ν (cm-1) 3378, 3345, 3301, 3106,
1766, 1714, 1643, 1609, 1596, 1531, 1509, 1405, 1345, 1311, 1261, 1208, 1135, 1007, 832;
1H NMR (DMSO-d6, 400 MHz): δ 6.88 (d, 2H, J = 8.8 Hz), 7.14-7.18 (m, 2H), 7.26 (d, 2H, J
= 8.4 Hz), 7.66-7.73 (m, 4H), 7.99 (d, 2H, J = 8.8 Hz), 10.04 (s, 1H), 10.11 (s, 1H), 11.06 (s,
13
1H); C NMR (DMSO-d6, 100 MHz): δ 102.69, 115.55 (d, J = 22.0 Hz), 121.47, 122.65,
122.74 (d, J = 8.0 Hz), 128.15, 129.78, 130.45, 135.89, 137.46, 139.68, 141.05, 145.88, 158.71
(d, J = 239.0 Hz), 164.75, 168.77, 172.51; ESI-MS: 447 (M+1)+; Anal. Calcd. for
C23H15FN4O5: C, 61.88; H, 3.39; F, 4.26; N, 12.55; O, 17.92%; Found: C, 61.93; H, 3.42; N,
12.52%.
4-(2,5-dihydro-4-(4-nitrophenyl)-2,5-dioxo-1H-pyrrol-3-ylamino)-N-(4-
chlorophenyl)benzamide (8p)
Red-orange powder; yield: 83%; m. p. 280-282 οC; IR (KBr): ν (cm-1) 3343, 3266, 3222, 3116,
1769, 1709, 1646, 1621, 1589, 1538, 1496, 1398, 1336, 1307, 1255, 1188, 1112, 1012, 821;
1H NMR (DMSO-d6, 400 MHz): δ 6.89 (d, 2H, J = 8.8 Hz); 7.25 (d, 2H, J = 8.8 Hz); 7.36-7.39
(m, 2H); 7.67 (d, 2H, J = 8.8 Hz); 7.73-7.76 (m, 2H); 7.98-8.00 (m, 2H); 10.10 (s, 1H); 10.11
13
(s, 1H); 11.07 (s, 1H); C NMR (DMSO-d6, 100 MHz): δ 102.83, 121.45, 122.37, 122.66,
127.65, 128.22, 128.90, 129.65, 130.48, 137.41, 138.53, 139.60, 141.09, 145.92, 164.91,
168.79, 172.51; ESI-MS: 463 (M+1)+; Anal. Calcd. for C23H15ClN4O5: C, 59.68; H, 3.27; Cl,
7.66; N, 12.10; O, 17.28%; Found: C, 59.65; H, 3.28; N, 12.11%.
GSK-3β inhibitory activity
The synthesized compounds were screened for their GSK-3β inhibition potentialby
Kinase-Glo assay luminescence assay23. This assay determines the kinase activity by
quantifying the remaining amount of ATP in solution following a kinase reaction. In a
conventional assay, 10 μL of test compound of different concentrations (dissolved in dimethyl
sulfoxide [DMSO] and diluted with assay buffer) and 10μL (20 ng) of enzyme were added to

New Journal of Chemistry
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each well succeeded by 20μL of assay buffer containing substrate and ATP DtOoI: 1o0b.1t0a39in/C5aNJ02896E
concentration of 25 μM substrate and 1 μM ATP per well. The final DMSO concentration in
the reaction mixture was kept below 1%. The enzymatic reaction,after half-an-hour of
incubation at 30°C, was quenched with 40 μL of Kinase-Glo reagent. Luminescence was then
recorded after 10 min using Infinite F200®PRO multimode reader (Tecan). The activity is
proportional to the difference of the total and consumed ATP. Staurosporine, a well-known
kinase inhibitor, was used as a standard GSK-3β inhibitor.
In silico molecular docking studies
To provide more insights at molecular level, the synthesized molecules are virtually
prepared and docked against GSK-3β protein target (PDB: 1Q3D) using Schrodinger Maestro
suite (Version 10.2). The ligands were virtually prepared by Lig-Prep module (Version 3.3).
The GSK-3β protein was prepared by Protein Preparation Wizard through Maestro Interface
(Version 10.2). The binding pocket was generated by selecting a grid with respect to reference
ligand staurosporine. The prepared molecules were docked against the generated grid using
Glide Module (Version 6.6) with Extra Precision (XP) mode. The binding pocket which is
defined by important residues like ASP-133, VAL-135 residues and other residues like ILE-
62, VAL70 and PHE-67 form the hydrophobic pocket. The docked molecules were ranked with
respect to Glide score.
Antidepressant Activity
Antidepressant activity was evaluated by the two well-known behavioural models- Forced
swim test and Tail suspension test.
Animals
Adult male wistar rats (180-200 g) were used for antidepressant studies. The rats were procured
from Central Animal House, Hamdard University (Jamia Hamdard), New Delhi, kept incages
at room temperature and fed with food and water ad libitum. The experiments were performed
in compliance with Institutional Animals Ethics Committee guidelines, approved by the
Institutional Animal Ethics Committee of the University, Jamia Hamdard, New Delhi, India
(Registration No. 1103-CPSCEA). All the compounds were either suspended in 0.5%
methylcellulose (Methocel) or dissolved in distilled water when soluble and administered
orally 1 h before behavioural testing.

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DOI: 10.1039/C5NJ02896E
Forced Swim Test:
The FST is the most widely used pre-clinic behavioural test for screening of novel compounds
for potential antidepressant activity25. The test was performed following the procedure
described by Vincent Castagne et al26. Rats were moved from the housing colony room to the
testing laboratory and kept undisturbed for at least 1 h before testing. They were provided free
access to standard rodent diet and tap water, except during the test. A controlled temperature
0
of 21±3 C and standard light/dark cycle with illumination from 0700 to 1900 h were
maintained. Each experimental group consisted of 6 rats. The rats were individually placed to
swim in a plexiglas cylinders (20 cm in diameter × 40 cm high) filled with water (25°C) to a
depth of 30 cm. A prior habituation session of 15-min was conducted prior to drug
administration and without behavioural recording. After 24 h, each group were administered
with the appropriate treatment of fluoxetine (30 mg/kg BW) or test compounds (20 mg/kg BW)
or their vehicles followed 1 h later by a 6-min test session. The total immobility time, defined
by lack of activity, except for necessary movements to keep their heads above water, was
scored during this session. Data was compared with the data from the control and the standard
group.
Tail suspension test (TST)
The tail suspension test as a model of behavioural despair is conceptually related to the forced
swimming test, except that immobility is induced by suspending the rat by its tail 27, 28. Rats
were moved from the housing colony room to the testing laboratory and kept undisturbed for
at least 1 h before testing. The dosage, temperature and other environmental conditions were
provided same as in forced swim test. The test was performed using an automated apparatus.
Rats were individually suspended upside-down using an adhesive scotch tape to fix its tail to a
hook at 60 cm connected to a strain gauge. A square platform was temporarily positioned 20
cm below the apparatus, just under the rat’s forepaws, in order to minimize the weight sustained
by the animal's tail until the beginning of the recording session. The movements of the rat were
picked up by the strain gauge and transmitted there from to a central unit that calculated the
total duration of immobility during a 6-min test with the help of a maze software version 3.0.
The rat was considered immobile when it was not making any movements of struggling,
attempting to catch the adhesive tape, body torsions, or jerks. Data collected were expressed as
a mean of immobility time (in second ± the standard error mean: S.E.M).
Statistical analysis

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Results are expressed as the mean ± SEM, and different groups were compared using
DOI: 10.1039/C5NJ02896E
one way analysis of variance (ANOVA) followed by Tukey Kramer test for multiple
comparisons.
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. The author
MAT is thankful to HNF for providing research assistance.
References
1.
2.
A. R. Cole, The FEBS journal, 2013, 280, 5213-5227.
T. Del’Guidice, C. Latapy, A. Rampino, J. Khlghatyan, M. Lemasson, B. Gelao, T.
Quarto, G. Rizzo, A. Barbeau, C. Lamarre, A. Bertolino, G. Blasi and J.-M. Beaulieu,
Proceedings of the National Academy of Sciences, 2015, 112, E4610-E4619.
E.-M. Hur and F.-Q. Zhou, Nature reviews. Neuroscience, 2010, 11, 539-551.
A. Martinez, A. Castro, I. Dorronsoro and M. Alonso, Medicinal Research Reviews,
2002, 22, 373-384.
3.
4.
5.
Z. Wang, K. S. Smith, M. Murphy, O. Piloto, T. C. Somervaille and M. L. Cleary,
Nature, 2008, 455, 1205-1209.
6.
X. Li and R. S. Jope, Neuropsychopharmacology : official publication of the American
College of Neuropsychopharmacology, 2010, 35, 2143-2154.
T. D. Gould, C. A. Zarate and H. K. Manji, The Journal of clinical psychiatry, 2004,
65, 10-21.
7.
8.
X. Li, W. Zhu, M. S. Roh, A. B. Friedman, K. Rosborough and R. S. Jope,
Neuropsychopharmacology, 2004, 29, 1426-1431.
9.
T. D. Gould, J. A. Quiroz, J. Singh, C. A. Zarate and H. K. Manji, Molecular psychiatry,
2004, 9, 734-755.
10.
J. M. Beaulieu, S. Marion, R. M. Rodriguiz, I. O. Medvedev, T. D. Sotnikova, V. Ghisi,
W. C. Wetsel, R. J. Lefkowitz, R. R. Gainetdinov and M. G. Caron, Cell, 2008, 132,
125-136.
11.
12.
X. Li, M. A. Frye and R. C. Shelton, Neuropsychopharmacology, 2012, 37, 77-101.
J. M. Beaulieu, Journal of psychiatry & neuroscience : JPN, 2012, 37, 7-16.

Page 29 of 31
New Journal of Chemistry
View Article Online
13.
14.
O. Kaidanovich-Beilin, A. Milman, A. Weizman, C. G. Pick and H. EldarD-FOiI:n10k.1e0l3m9/aCn5N,J02896E
Biological psychiatry, 2004, 55, 781-784.
T. D. Gould, H. Einat, R. Bhat and H. K. Manji, The international journal of
neuropsychopharmacology / official scientific journal of the Collegium Internationale
Neuropsychopharmacologicum (CINP), 2004, 7, 387-390.
15.
A. O. Rosa, M. P. Kaster, R. W. Binfare, S. Morales, E. Martin-Aparicio, M. L.
Navarro-Rico, A. Martinez, M. Medina, A. G. Garcia, M. G. Lopez and A. L.
Rodrigues, Progress in neuro-psychopharmacology & biological psychiatry, 2008, 32,
1549-1556.
16.
17.
J. M. Beaulieu, R. R. Gainetdinov and M. G. Caron, Annual review of pharmacology
and toxicology, 2009, 49, 327-347.
Q. Ye, W. Mao, Y. Zhou, L. Xu, Q. Li, Y. Gao, J. Wang, C. Li, Y. Xu, Y. Xu, H. Liao,
L. Zhang, J. Gao, J. Li and T. Pang, Bioorganic & medicinal chemistry, 2015, 23, 1179-
1188.
18.
19.
20.
D. G. Smith, M. Buffet, A. E. Fenwick, D. Haigh, R. J. Ife, M. Saunders, B. P. Slingsby,
R. Stacey and R. W. Ward, Bioorganic & medicinal chemistry letters, 2001, 11, 635-
639.
H. Gunosewoyo, A. Midzak, I. N. Gaisina, E. V. Sabath, A. Fedolak, T. Hanania, D.
Brunner, V. Papadopoulos and A. P. Kozikowski, Journal of Medicinal Chemistry,
2013, 56, 5115-5129.
M. P. Coghlan, A. A. Culbert, D. A. E. Cross, S. L. Corcoran, J. W. Yates, N. J. Pearce,
O. L. Rausch, G. J. Murphy, P. S. Carter, L. Roxbee Cox, D. Mills, M. J. Brown, D.
Haigh, R. W. Ward, D. G. Smith, K. J. Murray, A. D. Reith and J. C. Holder, Chemistry
& Biology, 2000, 7, 793-803.
21.
M. J. Zamek-Gliszczynski, T. L. Abraham, J. J. Alberts, P. Kulanthaivel, K. A. Jackson,
K. H. Chow, D. J. McCann, H. Hu, S. Anderson, N. A. Furr, R. J. Barbuch and K. C.
Cassidy, Drug metabolism and disposition: the biological fate of chemicals, 2013, 41,
714-726.
22.
23.
24.
J. A. Bertrand, S. Thieffine, A. Vulpetti, C. Cristiani, B. Valsasina, S. Knapp, H. M.
Kalisz and M. Flocco, Journal of molecular biology, 2003, 333, 393-407.
A. Baki, A. Bielik, L. Molnár, G. Szendrei and G. M. Keserü, Assay and Drug
Development Technologies, 2007, 5, 75-83.
G. Xu, Q. He, B. Yang and Y. Hu, Letters in Drug Design and Discovery, 2009, 6, 51-
55.