Design, synthesis and biological evaluation of 5-benzylidene-2-iminothiazolidin-4-ones as selective GSK-3β inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.04.075

In this work, iminothiazolidin-4-one derivatives were explored as selective GSK-3β inhibitors. Molecular docking analysis was carried to design a series of compounds, which were synthesized using substituted thiourea, 2-bromoacetophenones and benzaldehydes. Out of the twenty five compounds synthesized during this work, the in?vitro evaluation against GSK-3 led to the identification of nine compounds with activity in lower nano-molar range (2–85?nM). Further, in?vitro evaluation against CDK-2 showed five compounds to be selective towards GSK-3.

Full text of DOI:10.1016/j.ejmech.2016.04.075

Accepted Manuscript
Design, synthesis and biological evaluation of 5-benzylidene-2-iminothiazolidin-4-
ones as selective GSK-3β inhibitors
Minhajul Arfeen, Shweta Bhagat, Rahul Patel, Shivcharan Prasad, Ipsita Roy, Asit K.
Chakraborti, Prasad V. Bharatam
PII:
S0223-5234(16)30392-0
10.1016/j.ejmech.2016.04.075
EJMECH 8604
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 March 2016
Revised Date: 26 April 2016
Accepted Date: 28 April 2016
Please cite this article as: M. Arfeen, S. Bhagat, R. Patel, S. Prasad, I. Roy, A.K. Chakraborti, P.V.
Bharatam, Design, synthesis and biological evaluation of 5-benzylidene-2-iminothiazolidin-4-ones
as selective GSK-3β inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.075.
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Graphical Abstract

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of 5-benzylidene-2-iminothiazolidin-
4-ones as selective GSK-3β Inhibitors
Minhajul Arfeen,a Shweta Bhagat,a Rahul Patel,a Shivcharan Prasad,b Ipsita Roy,b Asit K. Chakrabortia and
Prasad V. Bharatam*a
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar160062, Punjab, India
bDepartment of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar160062, Punjab, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
In this work, iminothiazolidin-4-one derivatives were explored as selective GSK-3β inhibitors.
Molecular docking analysis was carried to design a series of compounds, which were
synthesized using substituted thiourea, 2-bromoacetophenones and benzaldehydes. Out of the
twenty five compounds synthesized during this work, the in vitro evaluation against GSK-3 led
to the identification of nine compounds with activity in lower nano-molar range (2-85 nM).
Further, in vitro evaluation against CDK-2 showed five compounds to be selective towards
GSK-3.
Keywords:
GSK-3
CDK-2
2009 Elsevier Ltd. All rights reserved
.
Molecular Docking
Molecular Dynamics
SBDD
Alzheimer’s disease is a neurodegenerative disorder affecting
46.8 million people across the globe.[1] The disease is
characterized by progressive cognitive decline such as loss of
memory and orientation capability. Glycogen Synthase kinase-
3β (GSK-3β), also called as tau phosphorylating kinase, has
been targeted for development of therapeutic agents which can
be used for treatment of the Alzheimer’s disease.[2] GSK-3β, is
a protein kinase was originally identified and named for its
ability to inactivate the enzyme glycogen synthase.[3] It is a
proline directed serine threonine kinase found abundantly in the
neurons of the brain.[4] Besides treatment of Alzheimer’s
disease, GSK-3 has also been targeted for the development of
novel therapeutic agents for Type-II diabetes, CNS disorders,
chronic inflammatory disorders, malaria and cancer.[5]
2-thioxoimidazolidin-4-one (11), thiazolidin-2,4-dione (12), and
1,3,4-oxadiazole (13). Compounds containing ring systems (1-
7
) have been reported as substrate competitive or ATP non-
competitive inhibitors of GSK-3β by Martinez et al.[6]
Hydantoins ( ) were first reported as GSK-3β ATP competitive
8
inhibitors by Khanfar et al. through in silico screening. Further
they identified several potent compounds with five membered
heterocyclic scaffolds such as hydantoins (8), rhodanines (9)
and pyrrolidin-2-ones (10) through pharmacophore modeling
and virtual screening.[7] Saitoh et al. reported oxadiazoles as
potent GSK-3β inhibitors.[8] Taking clues from the above
mentioned reports, it was found worth exploring the chemical
space associated with five membered heterocyclic rings as head
groups to design new GSK-3 inhibitors[9].
Figure 2 Flow diagram representing the approach utilized for the design of 2-
iminothiazolidin-4-one as GSK-3β ATP competitive inhibitors.
Figure 1. Five membered rings reported as GSK-3β inhibitors (1-13).
Among the various strategies that have been used for the
inhibition of GSK-3β, ATP competitive inhibition has been
extensively explored. Several known GSK-3β inhibitors carry
5-membered heterocyclic rings (Figure 1) with hydrogen bond
acceptor and donor functionalities. These include 1,2,4-
Figure 3. Different subpockets in the ATP binding region of GSK-3β along
with AMP-PNP (Pdb code 1PYX).
Computer assisted structure based drug design
(SBDD) is a proven strategy for the rational development of
small molecules of therapeutic interest without necessitating its
synthesis at the preliminary stages. In this method, the available
data from X-ray co-crystallized structures is utilized to enhance
the pace of drug discovery. Till date, forty seven crystal
structures have been reported for GSK-3β. Among these, more
than forty co-crystallized structures are reported with ATP
dithiazolidin-3,5-dione (
pyrrol-2,5-dione ( ), 5-imino-1,2,4-thiadiazolidin-3-one (
1,2,4-thiadiazol-3,5-diamine ), 5-amino-1,2,4-thiadiazol-
3(2H)-one ( ), 1,2,4-thiadiaz-5(2H)-imine ( ) imidazolidin-2,4-
dione ( ), 2-thioxothiazolidin-4-one ( ), pyrrolidin-2-one (10),
1
), 1,2,4,-triazoldin-3,5-dione (
2
), 1H-
3
4
),
(
5
6
7
8
9
1

competitive inhibitors. Similarly, more than 150 co-crystallized
bond donor in cis orientation. In light of the above observations,
potential hinge binding fragments (HF-1 to HF-7) with
hydrogen bond acceptor and donor groups in cis orientation
were identified during the design, novel GSK-3β inhibitors
(Figure 4) were docked into the hinge region of the ATP
binding domain of GSK-3β using the protein structure with
PDB code 1Q5K. Figure S1 (see Supporting Information)
shows the docking results of the hinge binding fragments (HF-1
to HF-7). Top three binding poses of all the fragments were
considered for the analysis of docking results. Only those
fragments were considered for further evaluation which showed
a minimum of two hydrogen bond interactions with hinge
binding residues (either Val135 or both Asp133 and Val135).
ACCEPTED MANUSCRIPT
structures are reported for CDK-2. Despite this wealth of
structural information, the role of SBDD has been limited to
suggest the analogues of existing leads and to post-rationalize
the bioactivity data. Therefore, in this work, molecular docking
guided SBDD, synthesis, biological evaluation and molecular
dynamics simulations were carried out to identify 2-
iminothiazolidin-4-one as selective GSK-3β ATP competitive
inhibitors carrying heterocyclic five membered rings.
Rational Design: Figure 2 represents the strategy utilized to
identify the 2-iminothiazolidin-4-ones as selective GSK-3β
inhibitors. ATP binding pocket of the kinases can be generally
divided into four subpockets, namely: (i) solvent accessible
region, (ii) hinge region, (iii) hydrophobic back pocket, which is
guarded by a gate keeper residue and (iv) polar region
(phosphate binding region) as shown in Figure 3 along with
AMP-PNP.[10] The most important subpocket of ATP binding
domain is hinge region, which is responsible for molecular
recognition. Any side chain extending towards the polar region
contributes to an increase in potency, whereas side chains
extending to the solvent accessible region or towards the
hydrophobic back pocket contributes to the selective inhibition
of GSK-3β. Two factors were generally held responsible for
selective inhibition of GSK-3β in the literature (i) the
interactions with amino acids that are unique to GSK-3β active
site[11] and (ii) the larger size of the ATP binding cavity in
GSK-3β when compared to its homologous counterparts (≈25
kinases).[12] The residues reported for selective inhibition of
GSK-3β are Leu132 (Phe80 in CDK-2), Tyr134 (Phe82 in
CDK-2), Arg141 (Lys89 in CDK-2) and Cys199 (Ala144 in
CDK-2). The larger size of the ATP binding domain in GSK-3β
has been attributed to the presence of Leu132. The
corresponding residue in case of CDK-2 is Phe80, which does
not allow the binding of bulkier group in its surroundings.
Another factor contributing to the larger size is the orientation
of Arg141 side chain, which points away from the cavity. In
case of CDKs, Lys89 at a similar position points inwards and
interacts with Ile10 of the glycine rich loop. Similarly the
orientation of Asp200 side chain is away from the ATP binding
cavity of GSK-3β, whereas in case of CDKs the side chain of
Asp at a similar position is directed towards the cavity. As per
the literature reports, the selectivity of the 6-bromoindirubins
and thiazole-methoxybenzyl-thiourea is due to the presence of
Leu132 and orientation of Arg141 respectively.[13] Therefore,
the design strategy for the identification of novel GSK-3β
inhibitors was divided into three steps (i) identification of hinge
binding fragments, (ii) identification of fragments which can
access the polar region and (iii) identification of fragments that
can bind to solvent accessible region.
Therefore, -NH hydrogen of HF-1, HF-2, HF-3, and HF-5
showed hydrogen bond interaction with carbonyl oxygen of
Val135, whereas the carbonyl oxygen of these hinge binding
fragments showed hydrogen bond contact with NH of Val135.
For fragments HF-4, and HF-7 the two hydrogen bond contacts
were observed with the Asp133 and Val135 i.e. hydrogen of
‘NH’ (NH2 in case of HF-4) making hydrogen bond contact
with carbonyl oxygen of Asp133 and carbonyl oxygen of
fragment making hydrogen bond contact with the NH of
Val135. The fragment HF-6 showed only one desired hydrogen
bond contact i.e. hydrogen of the ring ‘NH’ making hydrogen
bond contact with carbonyl oxygen of Val135 and hence, it was
not considered for any further study. In addition, careful
examination of docking results of these fragments suggested
that HF-5 binds with a pose, in which the methylene carbon and
the imine nitrogen can be substituted to access the polar region
and solvent accessible regions of the ATP binding domain
respectively. Therefore, HF-5 was selected for the design of
novel lead compounds.
Figure 5. Fragments considered for the docking study into the polar binding
region of ATP competitive inhibitors.
Identification of Fragments Binding to Polar Region: For the
identification of fragments that can bind to polar region, phenyl ring
substituted with polar functionalities was chosen. Figure 5 and
Figure S2 (supporting information) show the fragments considered
and docking results of the fragments binding to the polar region
respectively. As mentioned in the previous section, top three binding
poses were considered for the docking result analysis. Only those
fragments were considered for evaluation which showed hydrogen
bond interaction with either the side chain of Lys85 and backbone
NH of Asp200 from the DFG loop or with Lys85 or -NH of Asp200
from DFG loop. Therefore, FG-2, FG-5, FG-8, FG-10, FG-13, FG-
15 and FG-23 showed hydrogen bond interaction with Lys85
whereas FG-6, FG-9, FG-14, FG-17, FG-18, FG-19 and FG-20
were found to be involved in hydrogen bond interaction with the side
chain of Lys85 and NH of Asp200. Furthermore, FG-1 and FG-25
showed hydrogen bond contact with Asp200, while FG-3, FG-4,
FG-11, FG-12, FG-16 and FG-21 did not show desired binding
pose. Finally, eighteen fragments (FG-1, FG-2, FG-5, FG-6, FG-8,
FG-9, FG-10, FG-13, FG-14, FG-15, FG-17, FG-18, FG-19, FG-
20, FG-22, FG-23, FG-24 and FG-25) were considered for further
design of GSK-3β ATP competitive inhibitors.
Figure 4. Fragments considered for the binding to hinge region. Fragments
were screened on the basis of interaction with Asp133 and Val135.
Identification of Hinge Binding Fragments: The crystal
structure analysis showed that in majority of co-crystallized
complexes the ATP competitive inhibitors formed hydrogen
bond interactions with two residues Asp133 and Val135 in the
hinge region.[13b, 14] Moreover, literature search on the ATP
competitive inhibitors of GSK-3β, CDK-2 and CDK-5
(phylogenetically most closely related kinase to GSK-3β)
showed requirement of hydrogen bond acceptor and hydrogen
Figure 6. Structures of the designed first and second series of
compounds.
2

Design of Novel GSK-3β Inhibitors: The molecular docking
However, regioselective formation of the
Z
isomer, was
confirmed through chemical shift values in 1H NMR (δH 7.53-
7.58 ppm). The expected chemical shift of the isomers is
6.20–6.30 ppm.[22] The downfield shift of ‘H’ in the -isomer
ACCEPTED MANUSCRIPT
studies in hinge binding fragments and polar region led to the
identification of the heterocycle HF-5 and eighteen polar fragments.
The identified fragments were joined with the exocyclic double bond
(Figure S3, supporting information) so as to maintain the co-
planarity of the rings, thus designing the first series of compounds
(14, Figure 6). The designed compounds were evaluated for their
binding pose using Glide and FlexX (implemented in SYBYL 7.1)
molecular docking programs using crystal structure with PDB code
1Q5K. The cross docking experiments using crystal structure with
PDB code 4ACC were also performed to reconfirm the reliability of
binding pose. The compounds were evaluated on the basis of
hydrogen bond interaction with hinge residues and polar region (as
mentioned above). The docking results of the designed compounds
showed that the binding pose of the fragment HF-5 was retained in
all the cases, while in case of fragments binding to the polar region
though the binding pose was retained, variation in terms of hydrogen
bonds was observed.
E
Z
is attributed to the anisotropic effect of the nearby C-4 carbonyl
group. The synthesized compounds were tested for their
inhibitory activity against GSK-3β using a commercially
available assay system, based on Z′-LYTE technology,
available from invitrogen Life Technologies using human
recombinant GSK-3β as the enzyme source.[7b, 23] Two (14a
,
14c and 14l) out of twelve synthesized compounds showed
more than 50% inhibitory activity at 0.5 ꢀM, and were
evaluated for their IC50 values. In this process, two compounds
(
14a and 14l), with IC50 values 80.5 and 84.8 nM respectively,
were identified as GSK-3β inhibitors.
Scheme 2. Reagents, conditions and yields (i) DCM, NH4OH, rt, 2 h, 81%
and 83% (ii) EtOH, CH3COONa, reflux, 6 h, 73% and 77% (iii) CH3COOH,
CH3COONa, reflux, 72 h, 30-40%.
The designed compounds carry 5-arylidene-2-iminothiazolidin-4-one
functional unit in the core of the molecules. A substructure of these
molecules i.e. 2-iminothiazolidin-4-one is expected to interact with
the hinge binding residues which are important for molecular
recognition. This functional unit is already reported to be of
considerable therapeutic importance and is known to exhibit anti-
inflammatory (I),[15] anti-microbial (II),[16] and anti-tumor
properties (III)[17] (Figure S7, supporting information). Several
tautomers of this functional unit are also known and reported for
various drug discovery efforts (IV-VII, Figure S7, Supporting
information).[18] It is interesting to note that compounds with both
these tautomeric scaffolds are of therapeutic importance. In the
designed compounds also the tautomerism is possible. To evaluate
this, quantum chemical calculations at B3LYP/6-31+G(d) level were
carried out which indicated imine tautomer is marginally (1.77
kcal/mol) less stable in 14 (vide infra). The molecular docking
results of the two tautomers indicated imine form to be more
favourable for interactions with Val135. Hence, substitution at sp2
nitrogen atom was considered for further evaluation. The quantum
chemical calculations showed that in case of phenyl and benzyl
substituted derivatives imine tautomer was favoured by 4.91 and
1.87 kcal/mol respectively (Figure S8, supporting information). The
crystal structure analysis further showed that only three classes of
molecules i.e. analinomaleimide (PDB code 1Q4L),[19] ARA014418
(PDB code 1Q5K)[13b] and pyrazines (4ACD, 4ACC, 4ACG,
4ACH)[20] efficiently exploited the solvent accessible region using
phenyl group. Therefore, phenyl and related benzyl group were
selected for the design of new series of compounds (15, Figure 6)
which can bind to three regions (hinge region, polar region and
solvent accessible region) of ATP binding domain.
Thirteen compounds were selected for synthesis from second
series (Scheme 2) after performing molecular docking analysis.
The hydrogen atom of the imino group in the new set of
compounds was substituted with phenyl or benzyl groups to
gain access to the solvent accessible region of the ATP binding
cavity. The compounds substituted with hydroxy, methoxy,
halogen and nitro at 3rd and 4th positions of the benzylidine
ring were considered for synthesis. The starting material for the
synthesis of the desired compounds was obtained by the
treatment of phenyl and benzyl isothiocynates (18) with 24%
solution of aqueous ammonia. The precipitated thiourea (19
)
was heated under reflux with α-chloroethylacetate (20) in the
presence of sodium acetate in ethanol to obtain the substituted
4-iminothiazolidinone derivatives (21). The substituted 4-
iminothiazolidinone was heated under reflux conditions with
substituted benzaldehydes and sodium acetate in acetic acid to
obtain the desired products. The synthesized compounds were
evaluated for their GSK-3β inhibitory activity as mentioned
above (Figure S11, supporting information). Seven out of
thirteen synthesized compounds showed more than 50%
inhibition at 0.5 ꢀM. The identified compounds were then
evaluated for their IC50 values (Table 1). The lowest value was
found to be 2.1 nM (15g) while the highest was found to be
51.7 nM (15k).
Table 1. IC50 values for GSK-3β and percentage residual activity of CDK-2
for seven compounds at 2 ꢀM Inhibitor concentration. The activity of CDK-2
in the absence of inhibitor was assigned the value of 100%.
Earlier, in 2007 Richardson et al. reported 2-iminothiazolidin-4-
ones as potent CDK-2 inhibitors. While screening for selective
inhibition of CDK-2, five compounds showed non-selective
inhibition and one compound showed selective inhibition for
Scheme 1. Reagents, conditions and yields (i) CH3COOH, CH3COONa,
reflux, 72 h, Yield 40-75%.
GSK-3β.[24] Similarly, in 2012, Bazureau et al. reported a
closely related 2-amino-thiazole-4-one derivative as possible
kinase inhibitor.[18a] The newly designed compounds (Table 1)
are significantly more active than the reported compounds.
Moreover, all the compounds reported from this work fit
comfortably into the ATP binding domain. CDK-2 is the most
closely related kinase to GSK-3β with an overall similarity of
33% (98% similarity in ATP binding domain). Even the most
selective inhibitors of GSK-3β reported so far inhibit CDK-2,
Synthesis and Biological Evaluation: Based on the docking
scores and binding mode analysis, twelve molecules (14a-l)
were selected for synthesis in the first series (Scheme 1). The
synthetic scheme utilizes sodium acetate catalyzed condensation
of substituted benzaldehydes
thiazolidinone (17) in acetic acid under reflux condition.[21]
Geometrical isomerism ( ) is possible because of the
restricted rotation around exocyclic C=C double bond.
(
16
)
with 2-imino-4-
E/Z
3

therefore, CDK-2 was chosen to evaluate the selectivity of
inhibitors. The identified hits should show the low inhibition of
CDK-2 to avoid possible adverse effects. The seven identified
position in case of CDK-2 are 0.057, 0.484, 0.194 (repulsive) and
0.73 kcal/mol respectively. The above analysis clearly indicates that
the residues like Arg141, Thr138, Cys199 and interaction with Lys85
in GSK-3β play deterministic role in selective inhibition of GSK-3β.
ACCEPTED MANUSCRIPT
compounds (15a, b, e, f, g, k, l) were evaluated for selectivity
against CDK-2 at 2 ꢀM concentration, five of them showed
significant selectivity for inhibition of GSK-3β (> 60% residual
activity for CDK-2), the highest being shown by 15g (Table 1).
The experimentally observed selective affinity of these
inhibitors for GSK-3β can be attributed to the following two
reasons (i) Arg141 forms salt bridge interaction with Glu137
and hence defines the periphery and entry region of the ATP
binding pocket. Residues at similar position in case of CDK-2,
involved in the salt bridge formation are Lys89 and Glu85. The
side chain of the Lys89 points inwards and interacts with the
backbone atom of Ile10. This arrangement would not allow the
occupancy of the phenyl or benzyl group in case of CDK-2. (ii)
Difference in the gate keeper residue which is Leu132 in case of
GSK-3 and Phe80 in case of CDK-2. The presence of Phe80
does not allow the accommodation of substituted benzylidene
ring around the polar region. However, the docking study
suggests that this group fits elegantly into the GSK-3β ATP
Discussion
SBDD methods were employed to design 2-iminothiazolidin-4-one
class of compounds as potent GSK-3 inhibitors. Synthesis and
biochemical evaluation using GSK-3 and CDK-2 assay system lead
to the identification of five selective GSK-3 inhibitors. MD
simulations on the 15g-GSK-3 and 15g-CDK-2 complexes provided
clues towards the observed selectivity. This molecular modelling,
synthesis and biochemical study provided an opportunity to explore
structure activity relation analysis as discussed below. The
comparative analysis of binding mode for 14a-14l and their
tautomers showed that imine tautomer (2-iminothiazolidin-4-one) is
essential for the fingerprint H-bond (Val135) for GSK-3β in the
hinge region, whereas the 2-aminothiazolidin-4-one was found to
make hydrogen bond contacts with Arg141 (solvent accessible
region, see Supporting Information). In vitro screening of the 12
synthesized compounds from the first series (14a-14l) resulted in two
compounds with 50% GSK-3β inhibitory activity below 500 nM.
The reason for the smaller number of successful compounds from
this series can be attributed to the preference of amine tautomer
which leads to the variability of binding mode in the ATP binding
cavity and thus leading to the instability of inhibitor-GSK-3β
complex. This assumption gains strength from the fact that in the
second series (15a-15m), seven out of the thirteen synthesised
compounds were found to show 50% GSK-3β inhibitory activity
below 500 nM. This increased number of successful molecules in the
second series can be credited to the N-substituted phenyl and benzyl
ring, which leads to the greater stability in the binding mode because
of the preference for the imine tautomer. Moreover, the above stated
substitution resulted into the compounds with higher affinity for
GSK-3β (ex. 15l in comparison to 14a) however, the high potency of
14a, 14l, 15a, 15b, 15e, 15f, 15g, 15k and 15l can be ascribed to the
occupancy of the hydrophobic pocket around polar region by the
benzylidene ring. It was further observed that groups capable of
making hydrogen bonds (15a, 15f, 15g, 15k and 15l) or halogen
bonds (15b and 15e) with Lys85 are preferred at the 4th position.
Interestingly both nitro (15e and 15g) and methoxy/ethoxy groups
(15k and 15l) were favoured at the 3rd position. Compounds with
nitro substitution (24.0 and 2.1 nM) showed higher potency in
comparison to compounds with methoxy substitution (51.7 and 44.3
nM). Relative investigation of binding mode revealed that nitro
group is involved in the polar interaction with Leu132, whereas
methoxy and ethoxy groups are involved in the weak steric clash
with Leu132. Besides, nitro group is also involved in polar
interaction with Val70 and Asp200. This observation is in
accordance with the MD simulation study where Leu132, Val70 and
Asp200 (Figure 7) showed interaction energies of ~ 0.58, 1.75 and
0.64 kcal/mol respectively. It is pertinent to mention that 15d did not
show significant GSK-3β inhibitory activity even at 500 nM. It was
observed that there exists a strong intramolecular hydrogen bond
between nitro and hydroxyl groups, which leads to the co-planarity
of nitro group with the benzylidene ring and thus loss of polar
contacts with Leu132, Val70 and Asp200. Similarly, 15h did not
show significant inhibitory activity at evaluatory concentration,
which can be ascribed to the strong steric clash with Gly65, Val70,
Lys85 and Asp200.
binding site (Figure
information).
7 and Figure S5, see supporting
Figure 7. Docked pose of 15g and bar diagram represents the per residue
wise decomposition analysis, residues on the X-axis, whereas Energy
(kcal/mol) is on Y-axis.
Molecular Dynamics Simulations: To gain further insights into the
selective inhibition of GSK-3β, molecular dynamics simulations for
24 ns were carried out for 15g (most active and selective) in complex
with GSK-3β and CDK-2. The MM-GBSA energy decomposition
analysis and residue wise energy decomposition were performed
using a python script of AMBER 11.0 program. The RMSD analysis
for the two complexes showed that systems attained equilibrium
within 2 ns of simulation run (Figure S9, supporting information).
The binding free energy calculations showed a difference of 4.10
kcal/mol (solvent phase) in favor of GSK-3β (Figure S10, Table S1,
supporting information) with respect to CDK-2. The energy
decomposition analysis showed that the van der Waals energy
favored the binding of 15g to GSK-3β by 3.22 kcal/mol.
The electrostatic energy resists the binding of 15g to GSK-3β by
2.30 kcal/mol under gas phase while the same favors binding of 15g
to GSK-3β under solvent condition by ~1 kcal/mol. Residue wise
energy decomposition analysis was also taken up to identify key
residues that play a detrimental role in selective inhibition of GSK-
3β over CDK-2 (Figure 7, Table S2, supporting information). The
interactions were quantified in terms of pair wise interaction
decomposition of free energy. The important residues found to be
participating in GSK-3β inhibitor interaction (cut off value 0.5
kcal/mol) are Ile62, Val70, Ala83, Lys85, Leu132, Tyr134, Val135,
Glu137, Thr138, Arg141, Asn186, Leu188, Cys199, and Asp200 (for
CDK-2, the equivalent amino acids are Ile10, Val18, Ala31, Lys33,
Phe80, Phe82, Leu83, Gln85, Asp86, Lys89, Asn132, Leu134,
Ala144 and Asp145). The amino acids Ile10 (Ile62), Val70 (Val18),
Ala83 (Ala31), Leu132 (Phe80), Tyr134 (Phe82), Val135 (Leu83),
Glu137 (Gln85), Thr138 (Asp86), Asn186 (Asn132), Leu188
(Leu134), Asp200 (Asp145) showed similar strength of interaction
(0.5 to 2 kcal/mol). The residues Lys85, Thr138, Arg141 and Cys199
showed the interaction energy of 0.97, 1.13, 1.11 and 1.371 kcal/mol
respectively. The interaction energies for the residues at the similar
4

1
3148, 2962, 1667, 1581 cm-1; H NMR (400 MHz, CD3SOCD3) δ
ACCEPTED MANUSCRIPT
9.31 (br s, 2H), 7.54 (s, 1H), 6.9 (s, 2H), 3.8 (s, 6H), 3.69 (s, 3H);
13C NMR (100 MHz, CD3SOCD3): δ 180.79, 175.88, 153.61,
139.11, 130.11, 129.85, 128.86, 107.38, 60.64, 56.37 HRMS (ESI-
TOF): m/z calculated for C13H14N2O4S [M+Na]+, 317.0572; found
317.0567.
Conclusions
In conclusion, SBDD using molecular docking methodology was
utilized to design 2-iminothiazolidinones as a new class of GSK-3β
inhibitors. The synthesis and in vitro biological evaluation resulted in
the identification of nine compounds, which showed GSK-3β
inhibition in lower nano molar range (lowest being 2.1 nM and
highest being 84.8 nM). Five compounds emerged as selective GSK-
3β inhibitors against CDK-2. MD simulation study of the most
potent as well as selective compound showed that the selectivity of
this class of compounds is determined by the residues Lys85,
Thr138, Arg141 and Cys199 of GSK-3. Further, this work proves
that the above mentioned residues can be effectively utilized to
design selective GSK-3β inhibitors.
5-(4-Hydroxy-3-methoxybenzylidene)-2-iminothiazolidin-4-one
(14c): Yellow solid; mp > 200 ºC; 215 mg, 43% yield; IR (neat) νmax
= 3364, 2955, 1735, 1680, 1584 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 9.76 (s, 1H), 9.29 (br s, 1H), 9.05 (s, 1H), 7.50 (s,
1H), 7.13 (s, 1H), 7.06 (d, J = 8.28, 1H), 6.9 (d, J = 7.92, 1H); 13
NMR (100 MHz, CD3SOCD3): δ 175.91, 148.92, 148.35, 130.35,
125.94, 125.81, 123.69, 116.49, 113.88, 56.00; HRMS (ESI-TOF):
m/z calculated for C11H10N2O3S [M+H]+, 251.0490; found 251.0491.
C
5-(4-Hydroxy-3-nitrobenzylidene)-2-iminothiazolidin-4-one
Experimental Section
(14d): Orange solid; mp > 200 ºC; 330 mg, 61% yield; IR (neat) νmax
1
= 3164, 2772, 1679, 1604 cm-1; H NMR (400 MHz, CD3SOCD3)
δ
General Information:
9.46 (br s, 1H), 9.18 (s, 1H), 8.05 (s, 1H), 7.75 (d, J = 8.74, 1H), 7.55
(s, 1H), 7.24 (d, J = 8.72, 1H); 13C NMR (100 MHz, CD3SOCD3): δ
180.71, 175.57, 153.52, 137.52, 136.46, 129.04, 127.52, 126.27,
125.66, 120.53 HRMS (ESI-TOF): m/z calculated for C10H7N3O4S
[M+Na]+, 288.0055; found 288.0046.
All starting materials were purchased from Sigma-Aldrich/Alfa
Aesar and used further without any additional purification. All
reactions were carried out in flame-dried glass wares under N2. All
the compounds (intermediates and final compounds) were purified
by column chromatography using silica gel (100-200 mesh) as
stationary phase and hexane-ethyl acetate as mobile phase. Proton
nuclear magnetic resonance spectra (1H NMR) were recorded on
Avance III 400 Bruker (400 MHz). Proton chemical shifts are
expressed in parts per million (ppm, δ scale) and are referenced to
residual proton in the NMR solvent (CDCl3, δ 7.26, CD3OD, δ 3.31
and CD3SOCD3, δ 2.49). The following abbreviations were used to
describe peak patterns when appropriate: br = broad, s = singlet, d =
doublet, t = triplet, m = multiplet. Coupling constants, J, were
reported in Hertz unit (Hz). Carbon 13 nuclear magnetic resonance
spectroscopy (13C NMR) was recorded on Avance III 400 Bruker
(100 MHz) and was fully decoupled by broad band decoupling.
Chemical shifts were reported in ppm referenced to the centre line at
a 77.0, 49.0 and 39.7 ppm of CDCl3, CD3OD and CD3SOCD3
respectively. High resolution mass spectra were taken with Maxis-
Bruker using ESI-TOF method.
5-(4-Bromo-3-nitrobenzylidene)-2-iminothiazolidin-4-one
(14e):Yellow solid; mp > 200 ºC; 334 mg, 51% yield; IR (neat) νmax
1
= 3197, 2761, 2302, 1666, 1613, 1610 cm-1; H NMR (400 MHz,
CD3SOCD3) δ 9.62 (s, 1H), 9.32 (s, 1H), 8.04 (d, J = 8.28, 1H), 7.74
(s, J = 8.28, 1H), 7.6 (s, J = 8.28, 1H); 13C NMR (100 MHz,
CD3SOCD3): δ 180.28, 175.47, 150.34, 136.00, 135.72, 133.96,
133.49, 126.23, 126.06, 114.00; HRMS (ESI-TOF): m/z calculated
for C10H6BrN3O3S [M+Na]+, 349.9211; found 349.9203.
5-(4-Methoxy-3-nitrobenzylidene)-2-iminothiazolidin-4-one
(14f): Orange solid; mp > 200 ºC; 301 mg, 54% yield; IR (neat) νmax
1
= 3236, 2951, 1682, 1608 cm-1; H NMR (400 MHz, CD3SOCD3) δ
9.49 (s, 1H), 9.21 (s, 1H), 8.11 (s, 1H), 7.86 (d, J = 8.00 1H), 7.58 (s,
1H) 7.51 (d, J = 8.84, 1H) 4.31 (s, 3H); 13C NMR (100 MHz,
CD3SOCD3): δ 180.62, 175.61, 153.00, 139.71, 135.70, 130.05,
127.13, 125.98, 115.70, 57.51: HRMS (ESI-TOF): m/z calculated for
C11H9N3O4S [M+Na]+, 302.0211; found 302.0244.
Experimental Procedures for Synthesized Compounds
5-(4-Formylbenzylidene)-2-iminothiazolidin-4-one (14g): Yellow
solid; mp > 200 ºC; 315 mg, 68% yield; IR (neat) νmax = 3194, 2995,
Preparation of compound 14a: To the magnetically stirred solution
of 17 (232 mg, 2 mmol) in HOAc (7 mL), was added NaOAc (500
mg, 6 mmol). After 15 min 3,4-dimethoxybenzaldhyde (16a, 400
mg, 2.4 mmol) was added and the reaction mixture was heated under
reflux for 72 h. The HOAc was removed under reduced pressure and
the resultant solid was washed successively with water, methanol
and EtOAc to obtain the desired products as solid.
1
1677, 1606 cm-1; H NMR (400 MHz, CD3SOCD3) δ 10.01 (s, 1H),
9.56 (s, 1H), 9.33 (s, 1H), 8.01 (d, J = 7.16, 2H), 7.77 (d, J = 7.40,
2H), 7.57 (s, 1H); 13C NMR (100 MHz, CD3SOCD3): δ 193.00,
180.49, 175.84, 140.23, 136.39, 133.08, 130.57, 130.34, 128.01;
HRMS (ESI-TOF): m/z calculated for C11H8N2O2S [M+Na]+,
255.0204; found 255.0249.
5-(3,4-Dimethoxybenzylidene)-2-iminothiazolidin-4-one
Yellow solid; mp > 200 ºC; 235 mg, 45% yield; IR (neat) νmax
(14a):
5-(4-Chloro-3-nitrobenzylidene)-2-iminothiazolidin-4-one
(14h):Orange solid; mp > 200 ºC; 338 mg, 60% yield; IR (neat) νmax
= 3214, 3009, 1965, 1693, 1667 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 9.58 (s, 1H), 9.33 (s, 1H) 8.20 (s, 1H), 7.82-7.89 (m,
2H), 7.62 (s, 1H); 13C NMR (100 MHz, CD3SOCD3): δ 180.28,
175.47, 148.21, 135.23, 134.26, 133.46 132.95, 126.39, 125.98,
125.79; HRMS (ESI-TOF): m/z calculated for C10H6ClN3O3S
[M+Na]+, 305.9716; found 305.9712.
=
3344, 2759, 1720, 1690, 1678 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ = 9.35 (s, 1H), 9.09 (s, 1H), 7.54 (s, 1H), 7.08-7.16
(m, 3H), 3.80 (s, 6H); 13C NMR (100 MHz, CD3SOCD3): δ =
180.52, 175.39, 149.98, 148.81, 129.42, 126.62, 126.51, 122.77,
112.63, 111.98, 55.57, 55.38; HRMS (ESI-TOF): m/z calculated for
C12H12N2O3S [M+Na]+, 287.0466; found 287.0461.
5-(3,4,5-Dimethoxybenzylidene)-2-iminothiazolidin-4-one (14b):
Yellow solid; mp > 200 ºC; 229 mg, 39% yield; IR (neat) νmax
=
5

5-(3-Carboxybenzylidene)-2-iminothiazolidin-4-one (14i): Yellow
7.32, 1H), 7.00(d, J = 7.21H), 4.05-3.96 (m, 2H); 13C NMR
(100 MHz, CD3SOCD3): δ 188.71, 178.71, 139.22, 129.77, 129.50,
125.16, 122.00, 120.71.
ACCEPTED MANUSCRIPT
solid; mp > 200 ºC; 218 mg, 44% yield; IR (neat) νmax = 3278, 3110,
2764, 2413, 1874, 1682, 1641, 1602 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 9.51 (br s, 1H) 9.21 (s, 1H), 8.51 (s, 1H), 7.97 (d, J =
7.60, 1H), 7.84 (d, J = 7.48, 1H), 7.64 (s, 1H), 7.62-7.60 (m, 1H);
13C NMR (100 MHz, CD3SOCD3): δ 180.65, 175.85, 167.26,
134.97, 134.91, 132.12, 130.97, 130.44, 130.07, 129.32, 128.38;
HRMS (ESI-TOF): m/z calculated for C11H8N2O3S [M+H]+,
249.0334; found 249.0328.
2-(Benzylimino)thiazolidin-4-one (21b):white solid; 359 mg, 87%
yield, 1H NMR (400 MHz, CD3SOCD3) δ = 7.37-7.25 (m, 5H),
4.61 (s, 2H), 3.90 (s, 2H); 13C NMR (100 MHz, CD3SOCD3): δ =
187.62, 182.69, 138.03, 137.57, 129.00, 128.94, 128.72, 128.18,
127.99, 127.95, 127.82, 127.79, 48.31.
Preparation of Compound 15a: To the stirred solution of
phenyliminothiazolidin-4-one (21a, 385 mg, 2 mmol) and 4-
hydroxybenzaldehyde (16a, 183 mg, 1.5 mmol) in HOAc (5 mL),
NaOAc (820 mg, 10 mmol) was added. The reaction mixture was
then heated under reflux for 72 h. The reaction mixture was then
allowed to cool to room temperature, resulting into the precipitation
of yellow solid which was filtered and washed with water, methanol
and EtOAc to purify.
5-(3-ethoxy-4-hydroxybenzylidene)-2-iminothiazolidin-4-one
(14j): Yellow solid; mp > 200 ºC, 216 mg, 41% yield; IR (neat) νmax
= 3395, 3120, 2759, 1689, 1585 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 12.50 (s, 1H) 9.88 (s, 1H), 7.69 (s, 1H), 7.13 (s, 1H),
7.04 (d, J = 7.98, 1H), 6.93 (d, J = 8.2, 1H), 4.06-4.05 (m, 2H), 2.07
(s, 1H), 1.34 (t, J = 6.92, 3H); 13C NMR (100 MHz, CD3SOCD3): δ
168.51, 167.92, 150.11, 147.56, 133.12, 124.80, 124.64, 119.56,
116.69, 115.62, 64.34, 15.07; HRMS (ESI-TOF): m/z calculated for
C12H12N2O3S [M+H]+, 265.0647; found 265.0637.
5-(4-Hydroxybenzylidene)-2-(phenylimino)thiazolidin-4-one
(15a): Pale yellow solid; mp > 200 ºC; 355 mg, 60% yield; IR
(neat) νmax = 2951, 2766, 2539, 1725, 1611, 1574, 1526 cm-1; 1H
NMR (400 MHz, CD3SOCD3) δ = 7.77 (s, 1H), 7.64-7.34 (m,
5H), 7.19 (t, J = 7.16, 1H), 7.05 (s, 1H), 6.9 (s, 1H), 6.86 (s,
1H); 13C NMR (100 MHz, CD3SOCD3) δ = 162.05, 158.12,
151.27, 137.34, 132.06, 130.28, 129.32, 128.65, 123.47, 122.66,
120.89, 115.77, 115.27; HRMS (ESI-TOF): m/z calculated for
C16H12N2OS [M+Na]+, 319.0517; found 319.0511.
5-(4-Carboxybenzylidene)-2-iminothiazolidin-4-one
Yellow solid; mp > 200 ºC; 233 mg, 47% yield; IR (neat) νmax
(14k):
=
3072, 2774, 2418, 1862, 1644 1585 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 9.52 (br s, 1H), 9.25 (s, 1H), 8.04 (d, J = 8.40, 2H),
7.68 (d, J = 8.36, 2H), 7.63 (s, 1H), 13C NMR (100 MHz,
CD3SOCD3): δ 180.52, 175.83, 167.17, 138.70, 132.28, 131.43,
130.44, 129.85, 128.15; HRMS (ESI-TOF): m/z calculated for
C11H8N2O3S [M+H]+, 249.0334; found 249.0329.
5-(4-Fluorobenzylidene)-2-(phenylimino)thiazolidin-4-one (15b):
5-(3-Carboxy-4-hydroxybenzylidene)-2-iminothiazolidin-4-one
(14l):Yellow solid; mp > 200 ºC; 153 mg, 29% yield; IR (neat) νmax
= 3243, 1654, 1580 cm-11H NMR (400 MHz, CD3SOCD3) δ 9.39 (br
s, 1H), 9.09 (s, 1H), 8.04 (s, 1H), 7.75 (d, J = 8.72, 1H), 7.55 (s, 1H),
7.09 (d, J = 8.64, 1H); 13C NMR (100 MHz, CD3SOCD3): δ 180.92,
175.77, 171.67, 162.23, 137.86, 132.73, 131.09, 128.66, 127.69,
125.67, 124.63, 118.69, 114.29; HRMS (ESI-TOF): m/z calculated
for C11H8N2O4S [M+H]+, 265.0283; found 265.0278.
Pale yellow solid; mp > 200 ºC 476 mg, 80% yield; IR (neat) νmax
=
3204, 2922, 2854, 1667, 1636, 1598, 1571 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ = 7.78 (s, 1H), 7.74 (s, 1H), 7.69-7.67, (m, 1H), 7.64
(s, 1H), 7.58-55 (m, 1H), 7.38-7.46 (m, 2H), 7.31 (t, J = 8.6, 1H),
7.21 (t, J = 7.2, 1H), 7.06 (d, J = 7.48, 1H); 13C NMR (100 MHz,
CD3SOCD3) δ = 164.18, 161.74, 132.53, 131.05, 129.98, 128.77, ,
127.63, 125.67, 121.89, 121.10, 116.93, 16.71; HRMS (ESI-TOF):
m/z calculated for C16H11FN2OS [M+Na]+, 321.0474; found
321.0439.
Preparation of compound 19a: To a magnetically stir solution of
phenyl-isothiocyanate (18-Ph, 239 ꢀL, 2 mmol) in toluene (5 mL)
was added aq. NH3 solution (417 ꢀL, 6 mmol). The reaction mixture
was allowed to stir for 3 h. The precipitated white solid was filtered
and washed with diethyl ether to directly use in the next step without
any further purification.
5-(4-Chlorobenzylidene)-2-(phenylimino)thiazolidin-4-one (15c):
Yellow solid; mp > 200 ºC; 544 mg, 86% yield; IR (neat) νmax
=
3261, 3201, 3140, 2921, 2852, 1665, 1635, 1601, 1588, 1570, 1535
1
cm-1; H NMR (400 MHz, CD3SOCD3) δ = 7.76 (s, 1H), 7.18 (s,
1H), 7.64-7.62 (m, 2H), 7.53 (s, 2H), 7.40-7.43 (m, 2H), 7.23-7.20
(m, 1H), 7.06-7.04 (m, 2H); 13C NMR (100 MHz, CD3SOCD3) δ =
173.97, 134.06, 133.02, 132.50, 131.16, 129.42, 129.28,
129.21, 129.07, 128.36, 127.32, 124.97, 124.82, 124.70,
121.46 120.83; HRMS (ESI-TOF): m/z calculated for
C16H11ClN2OS [M+Na]+, 337.0178; found 337.0142.
Preparation of compound 21a: To
a magnetically stirred
suspension of phenylthiourea (19a, 304 mg, 2 mmol) and anhydrous
NaOAc (820 mg, 10 mmol) in absolute ethanol (7 mL) was added
ethyl chloroacetate (20, 0.5 mL, 4 mmol) and the mixture was heated
under reflux for 6 h. The reaction mixture was concentrated under
reduced pressure. The residue was diluted with water (50 mL) and
extracted with EtOAc (3 15 mL). The combined EtOAc extracts
were dried (anhydrous Na2SO4), filtered and the filtrate was
concentrated under rotary vacuum evaporator to obtain a solid mass
which was recrystallized from EtOAc to obtain the desired product
as white solid.
5-(4-Hydroxy-3-nitrobenzylidene)-2-(benzylimino)thiazolidin-4-
one (15d): yellow solid; mp > 200 ºC; 217 mg, 30% yield; IR (neat)
1
νmax = 3269, 3057, 2757, 1691, 1622, 1602, 1535 cm-1; H NMR
(400MHz, CD3SOCD3) δ 10.10 (s, 1H), 8.11 (s, 1H) 7.77 (d, J =
8.78, 1H), 7.60 (s, 1H), 7.39-7.31 (m, 5H), 7.27 (d, J = 8.64, 1H),
4.75 (s, 2H); 13C NMR (100 MHz, CD3SOCD3) δ = 179.96,
173.81, 153.36, 137.58, 136.36, 129.10, 128.47, 128.18,
128.07, 127.78, 126.42, 125.79, 120.49, 48.28; HRMS (ESI-
TOF): m/z calculated for C17H13N3O4S [M+Na], 378.0524; found
378.0516.
2-(Phenylimino)thiazolidin-4-one (21a): White solid; 347 mg, 90%
yield; IR (neat) νmax = 3276, 3210, 3141, 3090, 3011, 2862, 1681,
1637, 1606, 1570 cm-1; 1H NMR (400 MHz, CD3SOCD3) δ =
7.69 (d, J = 7.52, 1H), 7.38(d, J = 7.24, 2H), 7.16 (t, J =
6

5-(4-Chloro-3-nitrobenzylidene)-2-(benzylimino)thiazolidin-4-
5-(3-ethoxy-4-hydroxybenzylidene)-2-(benzylimino)thiazolidin-4-
one (15k):Pale yellow solid; mp 178-180 ºC; 294 mg, 41% yield; IR
(neat) νmax = 3524, 3354, 3061, 2976, 2931, 2753, 1681, 1633, 1610,
1587 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 7.11-7.23 (m,
5H), 6.89-6.91 (m, 2H), 6.76 (d, J = 8.12, 1H). 4.61 (s, 2H), 4.05-
4.00 (m, 2H), 1.29 (m, 3H); 13C NMR (100 MHz, CD3SOCD3) δ =
186.08, 179.20, 152.90, 151.08, 140.54, 135.93, 132.51, 132.40,
131.61, 131.54, 131.26, 129.75, 127.83, 127.78, 119.52, 117.91,
68.25, 17.61; HRMS (ESI-TOF): m/z calculated for C19H18N2O3S
[M+Na]+, 377.0936; found 377.0928.
ACCEPTED MANUSCRIPT
one (15e): Yellow solid; mp > 200 ºC; 195 mg, 26% yield; IR (neat)
1
νmax = 3211, 3060, 2770, 1693, 1635, 1610, 1532 cm-1; H NMR
(400MHz, CD3SOCD3) δ 8.27 (s, 1H), 7.85-7.92 (m, 2H), 7.68 (s,
1H), 7.37-7.40 (m, 5H), 4.77 (s, 2H); 13C NMR (100 MHz,
CD3SOCD3) δ = 179.53, 173.64, 148.21, 137.37, 135.17,
134.16, 132.96, 132.79, 129.11, 128.21, 128.12, 126.49,
126.23, 125.82, 48.45; HRMS (ESI-TOF): m/z calculated for
C17H12ClN3O3S [M+Na]+, 396.0186; found 396.0177.
5-(4-Hydroxybenzylidene)-2-(benzylimino)thiazolidin-4-one
5-(3,4-Dimethoxybenzylidene)-2-(benzylimino)thiazolidin-4-one
(15l): Pale yellow solid; mp > 200 ºC; 270 mg, 38% yield; IR (neat)
νmax = 3524, 2753, 1679, 1624, 1593 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 7.58 (s, 1H), 7.38-7.28 (m, 5H), 7.16-7.12 (m, 2H),
7.06 (d, J = 8.28, 1H), 4.72 (s, 2H), 3.42 (s, 6H); 13C NMR (100
MHz, CD3SOCD3) δ = 180.31, 174.16, 150.71, 150.56, 149.35,
137.69, 137.55, 130.70, 130.20, 129.09, 129.06, 128.14, 128.07,
128.01, 127.93, 127.13, 126.92, 126.39, 123.26, 123.18, 113.55,
113.28, 112.50, 56.06, 56.00, 48.15; HRMS (ESI-TOF): m/z
calculated for C19H18N2O3S [M+Na]+, 377.0936; found 377.0936.
(15f): Pale yellow solid; mp > 200 ºC; 192 mg, 31%, yield; IR (neat)
1
νmax = 3161, 2805, 1710, 1677, 1616, 1574, 1536 cm-1; H NMR
(400 MHz, CD3SOCD3) δ 7.54 (s, 1H), 7.30-7.42 (m, 7H), 6.88 (d,
2H), 4.72 (s, 2H); 13C NMR (100 MHz, CD3SOCD3) δ = 180.44,
176.16, 159.52, 137.75, 132.13, 131.99, 130.27, 129.07, 128.17,
128.01, 127.79, 126.27, 124.99, 116.62, 48.10; HRMS (ESI-TOF):
m/z calculated for C17H14N2O2S [M+Na]+, 333.0674; found
333.0669.
5-(4-Methoxy-3-nitrobenzylidene)-2-(benzylimino)thiazolidin-4-
one (15g):Yellow solid; mp > 200 ºC; 280 mg, 38% yield; IR (neat)
5-(4-Methoxybenzylidene)-2-(benzylimino)thiazolidin-4-one
(15m): Pale yellow solid; mp > 200 ºC; 177 mg, 27% yield; IR (neat)
νmax = 1710, 1667, 1637, 1598, 1571 cm-1; 1H NMR (400 MHz,
1
νmax = 3216, 3018, 1710, 1677, 1616, 1574, 1536 cm-1; H NMR
(400 MHz, CD3SOCD3) δ 8.11 (s, 1H), 7.8803 (d, J = 8.86, 1H),
7.60 (s, 1H), 7.54 (d, J = 8.84, 1H), 7.30-7.37 (m, 5H), 4.73 (s, 2H),
3.97 (s, 3H); 13C NMR (100 MHz, CD3SOCD3) δ = 179.87,
173.68, 153.00, 139.71, 137.61, 135.65, 129.48, 129.10,
128.19, 128.06, 127.85, 127.25, 127.09, 126.14, 115.72,
57.52, 48.42; HRMS (ESI-TOF): m/z calculated for C18H15N3O4S
[M+Na]+, 392.0681; found 392.0673.
CD3SOCD3)
δ 7.54 (s, 1H), 7.39 (d, J = 8.68, 2H), 7.39-7.28 (m.
5H), 7.04 (d, J = 8.48, 2H), 4.75 (s, 2H), 3.75 (s, 3H); 13C NMR (100
MHz, CD3SOCD3) δ = 180.40, 174.21, 160.80, 137.64, 131.75,
129.86, 129.08, 128.15, 128.05, 127.78, 126.80, 126.14, 115.24,
55.85, 48.14; HRMS (ESI-TOF): m/z calculated for C18H16N2O2S
[M+Na]+, 347.0830; found 347.0864.
5-(4-Ethoxy-3-nitrobenzylidene)-2-(benzylimino)thiazolidin-4-
Acknowledgments
one(15h): Pale yellow solid; mp > 200 ºC; 347 mg, 44% yield; IR
1
(neat) νmax = 3454, 2769, 1691, 1631, 1606, 1523 cm-1; H NMR
The authors thank Department of Science and Technology
(DST), Govt. of India, New Delhi, India for financial support.
The authors thank NIPER, S. A. S. Nagar for infrastructural
Facilities and financial assistance. S.B thanks University Grant
Commission (UGC), Govt. of India, New Delhi, India for
financial assistance.
(400 MHz, CD3SOCD3) δ 8.09 (s, 1H), 7.84 (d, J = 8.88, 1H), 7.63
(s, 1H), 7.52 (d, J = 8.88, 1H) 7.39-7.31 (m, 5H). 4.75 (s, 2H), 4.31 -
4.25 (m, 2H), 1.37 (t, J = 6.93, 3H); 13C NMR (100 MHz,
CD3SOCD3) δ 179.87, 173.79, 152.22, 140.00, 137.54, 135.49,
129.25, 129.10, 128.20, 128.08, 127.86, 127.43, 126.92,
126.11, 116.39, 66.01, 48.32, 14.74; HRMS (ESI-TOF): m/z
calculated for C19H17N3O4S [M+Na]+, 406.0837; found 406.0832.
Supporting Information
Supporting information contains the docking pose of the hinge
binding fragments, docking pose of the fragments binding to the
polar region, binding pose of the compounds, quantum chemical
information for tautomeric analysis, details of molecular
5-(3-Hydroxybenzylidene)-2-(benzylimino)thiazolidin-4-one
(15i): Pale yellow solid; mp > 200 ºC; 186 mg, 31% yield; IR (neat)
1
νmax = 3278, 2813, 1715, 1686, 1624, 1609, 1590 cm-1; H NMR
1
dynamics, H NMR and 13C NMR spectra, experimental details
(400 MHz, CD3SOCD3) δ 7.50 (s, 1H), 7.38-7.27 (m, 6H), 6.99 (d, J
= 7.32, 1H), 6.95 (s, 1H), 6.82 (d, J = 8.04, 1H); 13C NMR (100
MHz, CD3SOCD3) δ = 180.09, 174.20, 158.30, 137.68, 135.70,
130.67, 129.87, 129.08, 128.97, 128.22, 128.04, 127.77,
121.21, 117.33, 116.00,115.90, 48.29; HRMS (ESI-TOF): m/z
calculated for C17H14N2O2S [M+Na]+, 333.0674; found 333.0665.
of biochemical assay
References and notes
[1] A.K. Acharya, Y.A. Chang, G.O. Jones, J.E. Rice, J.L. Hedrick, H.W.
Horn, R.M. Waymouth, Experimental and computational studies on the
mechanism of zwitterionic ring-opening polymerization of delta-
valerolactone with N-heterocyclic carbenes, J. Phys. Chem. B, 118 (2014)
6553-6560.
5-(4-Fluorobenzylidene)-2-(benzylimino)thiazolidin-4-one (15j):
Pale yellow solid; mp > 200 ºC; 232 mg, 37% yield; IR (neat) νmax
=
1710, 1686, 1627, 1610, 1597 cm-1; 1H NMR (400 MHz,
CD3SOCD3) δ 7.64-7.60 (m, 3H), 7.40-7.30 (m, 7H), 4.74 (s, 2H);
13C NMR (100 MHz, CD3SOCD3) δ = 180.03, 174.11, 164.08,
161.60, 137.55, 132.35, 132.21, 132.12, 131.12, 131.08, 129.10,
128.93, 128.90, 128.70, 128.22, 128.08, 127.80, 116.88, 116.67,
48.25; HRMS (ESI-TOF): m/z calculated for C17H13FN2OS
[M+Na]+, 335.0630; found 335.0623.
[2] a) A. Martinez, C. Gil, D.I. Perez, Glycogen synthase kinase 3 inhibitors
in the next horizon for Alzheimer's disease treatment, Int. J. Alzheimers Dis.,
2011
Article
ID
280502,
280507
pages,
282011.
doi:280510.284061/282011/280502; b) P. Cohen, M. Goedert, GSK-3
inhibitors: development and therapeutic potential, Nat. Rev. Drug. Discov., 3
(2004) 479-487; c) C.A. Grimes, R.S. Jope, The multifaceted roles of
glycogen synthase kinase 3β in cellular signaling, Prog. Neurobiol., 65 (2001)
391-426; d) T. Kramer, B. Schmidt, F. Lo Monte, Small-Molecule Inhibitors
of GSK-3: Structural Insights and Their Application to Alzheimer's Disease
Models, International Journal of Alzheimer's Disease, 2012 (2012) 32.
7

skeletal muscle. Separation from cyclic-AMP-dependeAnt CproCteiEn kPinTasEe aDnd MANUSCRPIoPlyTchronopoulos,
[3] N. Embi, D.B. Rylatt, P. Cohen, Glycogen synthase kinase-3 from rabbit
[12] a) P.
P. Magiatis, A.L. Skaltsounis, V.
Myrianthopoulos, E. Mikros, A. Tarricone, A. Musacchio, S.M. Roe, L.
Pearl, M. Leost, P. Greengard, L. Meijer, Structural Basis for the Synthesis of
Indirubins as Potent and Selective Inhibitors of Glycogen Synthase Kinase-3
and Cyclin-Dependent Kinases, J. Med. Chem., 47 (2004) 935-946; b) A.P.
Kozikowski, I.N. Gaisina, P.A. Petukhov, J. Sridhar, L.T. King, S.Y. Blond,
T. Duka, M. Rusnak, A. Sidhu, Highly Potent and Specific GSK-3β inhibitors
That Block tau Phosphorylation and Decrease alpha-Synuclein Protein
Expression in a Cellular Model of Parkinson's disease, ChemMedChem, 1
(2006) 256-266.
[13] a) L. Meijer, A.L. Skaltsounis, P. Magiatis, P. Polychronopoulos, M.
Knockaert, M. Leost, X.P. Ryan, C.A. Vonica, A. Brivanlou, R. Dajani, C.
Crovace, C. Tarricone, A. Musacchio, S.M. Roe, L. Pearl, P. Greengard,
GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins, Chem.
Biol., 10 (2003) 1255-1266; b) R. Bhat, Y. Xue, S. Berg, S. Hellberg, M.
Ormo, Y. Nilsson, A.C. Radesater, E. Jerning, P.O. Markgren, T. Borgegard,
M. Nylof, A. Gimenez-Cassina, F. Hernandez, J.J. Lucas, J. Diaz-Nido, J.
Avila, Structural Insights and Biological Effects of Glycogen Synthase
Kinase 3 Specific Inhibitor AR-A014418, J. Biol. Chem., 278 (2003) 45937-
45945; c) P.M. Fischer, CDK versus GSK-3 inhibition: a purple haze no
longer?, Chem. Biol., 10 (2003) 1144-1146.
[14] a) M. Arfeen, P.V. Bharatam, Design of glycogen synthase kinase-3
inhibitors: an overview on recent advancements, Curr. Pharm. Des., 19
(2013) 4755-4775; b) D.S. Patel, N. Dessalew, P. Iqbal, P.V. Bharatam,
Structure-based approaches in the design of GSK-3 selective inhibitors, Curr.
Protein Pept. Sci., 8 (2007) 352-364; c) M. Arfeen, R. Patel, T. Khan, P.V.
Bharatam, Molecular dynamics simulation studies of GSK-3β ATP
competitive inhibitors: understanding the factors contributing to selectivity, J.
Biomol. Struct. Dyn., 33 (2015) 2578-2593.
[15] a) N. Singh, A. Tripathi, A. Tewari, R. Kumar, S. Saraf, Ulcerogenicity
devoid novel non-steroidal anti-inflammatory agents (NSAIDS): syntheses,
computational studies, and activity of 5-aryliden-2-imino-4-thiazolidinones,
Med. Chem. Res., 24 (2015) 1927-1941; b) P. Eleftheriou, A. Geronikaki, D.
Hadjipavlou-Litina, P. Vicini, O. Filz, D. Filimonov, V. Poroikov, S.S.
Chaudhaery, K.K. Roy, A.K. Saxena, Fragment-based design, docking,
synthesis, biological evaluation and structure-activity relationships of 2-
benzo/benzisothiazolimino-5-aryliden-4-thiazolidinones as cycloxygenase-
lipoxygenase inhibitors, Eur. J. Med. Chem., 47 (2012) 111-124.
phosphorylase kinase, Eur. J. Biochem., 107 (1980) 519-527.
[4] J.R. Woodgett, P. Cohen, Multisite phosphorylation of glycogen synthase.
Molecular basis for the substrate specificity of glycogen synthase kinase-3
and casein kinase-II (glycogen synthase kinase-5), Biochim. Biophys. Acta.,
788 (1984) 339-347.
[5] a) E. Droucheau, A. Primot, V. Thomas, D. Mattei, M. Knockaert, C.
Richardson, P. Sallicandro, P. Alano, A. Jafarshad, B. Baratte, C. Kunick, D.
Parzy, L. Pearl, C. Doerig, L. Meijer, Plasmodium falciparum glycogen
synthase kinase-3: molecular model, expression, intracellular localisation and
selective inhibitors, Biochim. Biophys. Acta., 1697 (2004) 181-196; b) M.
Mora-Santos, M.C. Limon-Mortes, S. Giraldez, J. Herrero-Ruiz, C. Saez,
M.A. Japon, M. Tortolero, F. Romero, Glycogen synthase kinase-3beta
(GSK3beta) negatively regulates PTTG1/human securin protein stability, and
GSK3beta inactivation correlates with securin accumulation in breast tumors,
J. Biol. Chem., 286 (2012) 30047-30056; c) I. Collins, Targeted small-
molecule inhibitors of protein kinase B as anticancer agents, Anticancer
Agents Med. Chem., 9 (2009) 32-50; d) J. Lee, M.S. Kim, The role of GSK-3
in glucose homeostasis and the development of insulin resistance, Diabetes
Res. Clin. Pract., 77 Suppl 1 (2007) S49-57; e) T.D. Gould, Targeting
glycogen synthase kinase-3 as an approach to develop novel mood-stabilising
medications, Expert Opin. Ther. Targets, 10 (2006) 377-392; f) E.J.
Henriksen, B.B. Dokken, Role of glycogen synthase kinase-3 in insulin
resistance and type 2 diabetes, Curr. Drug. Targets, 7 (2006) 1435-1441; g)
R. Boulahjar, A. Ouach, S. Bourg, P. Bonnet, O. Lozach, L. Meijer, C.
Guguen-Guillouzo, R. Le Guevel, S. Lazar, M. Akssira, Y. Troin, G.
Guillaumet, S. Routier, Advances in tetrahydropyrido[1,2-a]isoindolone
(valmerins) series: Potent glycogen synthase kinase 3 and cyclin dependent
kinase 5 inhibitors, Eur. J. Med. Chem., 101 (2015) 274-287.
[6] a) V. Palomo, D.I. Perez, C. Perez, J.A. Morales, I. Soteras, S. Alonso-
Gil, A. Encinas, A. Castro, N.E. Campillo, A. Perez-Castillo, C. Gil, A.
Martinez, 5-Imino-1,2,4-Thiadiazoles: First Small Molecules as Substrate
Competitive Inhibitors of Glycogen Synthase Kinase 3, J. Med. Chem., 55
(2012) 1645-1661; b) A. Martinez, M. Alonso, A. Castro, C. Perez, F.J.
Moreno, First non-ATP competitive glycogen synthase kinase 3β (GSK-3β)
inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of
Alzheimer's disease, J. Med. Chem., 45 (2002) 1292-1299; c) A. Martinez,
M. Alonso, A. Castro, I. Dorronsoro, J.L. Gelpi, F.J. Luque, C. Perez, F.J.
Moreno, SAR and 3D-QSAR Studies on Thiadiazolidinone Derivatives:
Exploration of Structural Requirements for Glycogen Synthase Kinase 3
Inhibitors, J. Med. Chem., 48 (2005) 7103-7112.
[7] a) M.A. Khanfar, B.A. Asal, M. Mudit, A. Kaddoumi, K.A. El Sayed, The
marine natural-derived inhibitors of glycogen synthase kinase-3β
phenylmethylene hydantoins: In vitro and in vivo activities and
pharmacophore modeling, Bioorg. Med. Chem., 17 (2009) 6032-6039; b)
M.A. Khanfar, R.A. Hill, A. Kaddoumi, K.A. El Sayed, Discovery of novel
GSK-3β inhibitors with potent in vitro and in vivo activities and excellent
brain permeability using combined ligand- and structure-based virtual
screening, J. Med. Chem., 53 (2010) 8534-8545.
[8] a) M. Saitoh, J. Kunitomo, E. Kimura, Y. Hayase, H. Kobayashi, N.
Uchiyama, T. Kawamoto, T. Tanaka, C.D. Mol, D.R. Dougan, G.S. Textor,
G.P. Snell, F. Itoh, Design, synthesis and structure-activity relationships of
1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-
3β, Bioorg. Med. Chem. Lett., 17 (2009) 2017-2029; b) M. Saitoh, J.
Kunitomo, E. Kimura, H. Iwashita, Y. Uno, T. Onishi, N. Uchiyama, T.
Kawamoto, T. Tanaka, C.D. Mol, D.R. Dougan, G.P. Textor, G.P. Snell, M.
Takizawa, F. Itoh, M. Kori, 2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-
yl}-5-methyl-1,3,4-oxadiazol e derivatives as novel inhibitors of glycogen
synthase kinase-3β with good brain permeability, J. Med. Chem., 52 (2009)
6270-6286.
[16] a) P. Vicini, A. Geronikaki, M. Incerti, F. Zani, J. Dearden, M. Hewitt,
2-Heteroarylimino-5-benzylidene-4-thiazolidinones
analogues
of
2-
thiazolylimino-5-benzylidene-4-thiazolidinones with antimicrobial activity:
Synthesis and structure–activity relationship, Bioorg. Med. Chem., 16 (2008)
3714-3724; b) I. Kucukguzel, G. Satilmis, K.R. Gurukumar, A. Basu, E.
Tatar, D.B. Nichols, T.T. Talele, N. Kaushik-Basu, 2-Heteroarylimino-5-
arylidene-4-thiazolidinones as a new class of non-nucleoside inhibitors of
HCV NS5B polymerase, Eur. J. Med. Chem., 69 (2013) 931-941.
[17] A. Geronikaki, P. Eleftheriou, P. Vicini, I. Alam, A. Dixit, A.K. Saxena,
2-Thiazolylimino/Heteroarylimino-5-arylidene-4-thiazolidinones as New
Agents with SHP-2 Inhibitory Action, J. Med. Chem., 51 (2008) 5221-5228.
[18] a) W.K. Coulibaly, L. Paquin, A. Benie, Y.A. Bekro, E. Durieux, L.
Meijer, R.L. Guevel, A. Corlu, J.P. Bazureau, Synthesis of New N,N'-Bis(5-
arylidene-4-oxo-4,5-dihydrothiazolidin-2-yl)piperazine Derivatives Under
Microwave Irradiation and Preliminary Biological Evaluation, Sci. Pharm.,
80 (2012) 825-836; b) M. Sarkis, D.N. Tran, S. Kolb, M.A. Miteva, B.O.
Villoutreix, C. Garbay, E. Braud, Design and synthesis of novel bis-
thiazolone derivatives as micromolar CDC25 phosphatase inhibitors: Effect
of dimerisation on phosphatase inhibition, Bioorg. Med. Chem. Lett., 22
(2012) 7345-7350; c) A.A. Geronikaki, A.A. Lagunin, D.I. Hadjipavlou-
Litina, P.T. Eleftheriou, D.A. Filimonov, V.V. Poroikov, I. Alam, A.K.
Saxena, Computer-Aided Discovery of Anti-Inflammatory Thiazolidinones
with Dual Cyclooxygenase/Lipoxygenase Inhibition, J. Med. Chem., 51
(2008) 1601-1609.
[19] J.A. Bertrand, S. Thieffine, A. Vulpetti, C. Cristiani, B. Valsasina, S.
Knapp, H.M. Kalisz, M. Flocco, Structural characterization of the GSK-3β
active site using selective and non-selective ATP-mimetic inhibitors, J. Mol.
Biol., 333 (2003) 393-407.
[20] S. Berg, M. Bergh, S. Hellberg, K. Hogdin, Y. Lo-Alfredsson, P.
Soderman, S. von Berg, T. Weigelt, M. Ormo, Y. Xue, J. Tucker, J.
Neelissen, E. Jerning, Y. Nilsson, R. Bhat, Discovery of novel potent and
highly selective glycogen synthase kinase-3beta (GSK3beta) inhibitors for
Alzheimer's disease: design, synthesis, and characterization of pyrazines, J.
Med. Chem., 55 (2012) 9107-9119.
[9] a) S. Sestito, G. Nesi, S. Daniele, A. Martelli, M. Digiacomo, A. Borghini,
D. Pietra, V. Calderone, A. Lapucci, M. Falasca, P. Parrella, A. Notarangelo,
M.C. Breschi, M. Macchia, C. Martini, S. Rapposelli, Design and synthesis of
2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway
for the treatment of glioblastoma multiforme, Eur. J Med. Chem., 105 (2015)
274-288; b) E. Moine, I. Dimier-Poisson, C. Enguehard-Gueiffier, C. Logé,
M. Pénichon, N. Moiré, C. Delehouzé, B. Foll-Josselin, S. Ruchaud, S. Bach,
A. Gueiffier, F. Debierre-Grockiego, C. Denevault-Sabourin, Development of
new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma gondii
calcium-dependent protein kinase 1, Eur. J. Med. Chem., 105 (2015) 80-105.
[10] F. Zuccotto, E. Ardini, E. Casale, M. Angiolini, Through the "gatekeeper
door": exploiting the active kinase conformation, J. Med. Chem., 53 (2010)
2681-2694.
[21] a) M.H. Bolli, S. Abele, C. Binkert, R. Bravo, S. Buchmann, D. Bur, J.
Gatfield, P. Hess, C. Kohl, C. Mangold, B. Mathys, K. Menyhart, C. Müller,
O. Nayler, M. Scherz, G. Schmidt, V. Sippel, B. Steiner, D. Strasser, A.
Treiber, T. Weller, 2-Imino-thiazolidin-4-one Derivatives as Potent, Orally
Active S1P1 Receptor Agonists, J. Med. Chem., 53 (2010) 4198-4211; b)
G.H. Al-Ansary, M.A.H. Ismail, D.A. Abou El Ella, S. Eid, K.A.M. Abouzid,
[11] A.P. Kozikowski, I.N. Gaisina, H. Yuan, P.A. Petukhov, S.Y. Blond, A.
Fedolak, B. Caldarone, P. McGonigle, Structure-based design leads to the
identification of lithium mimetics that block mania-like effects in rodents.
possible new GSK-3β therapies for bipolar disorders, J. Am. Chem. Soc., 129
(2007) 8328-8332.
8

(2012) 1257-1267; c) F. Lo Monte, T. Kramer, J. Gu, U.R. Anumala, L.
Molecular design and synthesis of HCV inhibitors AbaCsedCoEn PthTiazEoloDne MANUSCRIPT
scaffold, Eur. J Med. Chem., 68 (2013) 19-32.
Marinelli, V. La Pietra, E. Novellino, B. Franco, D. Demedts, F. Van Leuven,
A. Fuertes, J.M. Dominguez, B. Plotkin, H. Eldar-Finkelman, B. Schmidt,
Identification of Glycogen Synthase Kinase-3 inhibitors with a selective sting
for glycogen synthase kinase-3alpha, J. Med. Chem., 55 (2012) 4407-4424.
[24] C.M. Richardson, C.L. Nunns, D.S. Williamson, M.J. Parratt, P.
Dokurno, R. Howes, J. Borgognoni, M.J. Drysdale, H. Finch, R.E. Hubbard,
P.S. Jackson, P. Kierstan, G. Lentzen, J.D. Moore, J.B. Murray, H.
Simmonite, A.E. Surgenor, C.J. Torrance, Discovery of a potent CDK2
inhibitor with a novel binding mode, using virtual screening and initial,
structure-guided lead scoping, Bioor. Med. Chem. Lett., 17 (2007) 3880-
3885.
[22] J.C. Thenmozhiyal, P.T.-H. Wong, W.-K. Chui, Anticonvulsant Activity
of Phenylmethylenehydantoins:ꢁ A Structure−Activity Relationship Study, J.
Med. Chem, 47 (2004) 1527-1535.
[23] a) S.M. Rodems, B.D. Hamman, C. Lin, J. Zhao, S. Shah, D. Heidary, L.
Makings, J.H. Stack, B.A. Pollok, A FRET-based assay platform for ultra-
high density drug screening of protein kinases and phosphatases, Assay Drug
Dev. Technol., 1 (2002) 9-19; b) C. Grutter, J.R. Simard, S.C. Mayer-
Wrangowski, P.H. Schreier, J. Perez-Martin, A. Richters, M. Getlik, O.
Gutbrod, C.A. Braun, M.E. Beck, D. Rauh, Targeting GSK3 from Ustilago
maydis: type-II kinase inhibitors as potential antifungals, ACS Chem. Biol., 7
9

List of Figures, Schemes and Table
Figure captions
ACCEPTED MANUSCRIPT
Figure 1. Five membered rings reported as GSK-3β inhibitors (1-13).
Figure 2 Flow diagram representing the approach utilized for the design of 2-iminothiazolidin-4-one as GSK-3β ATP competitive inhibitors.
Figure 3. Different subpockets in the ATP binding region of GSK-3β along with AMP-PNP (Pdb code 1PYX).
Figure 4. Fragments considered for the binding to hinge region. Fragments were screened on the basis of interaction with Asp133 and Val135.
Figure 5. Fragments considered for the docking study into the polar binding region of ATP competitive inhibitors.
Figure 6. Structures of the designed first and second series of compounds.
Figure 7. Docked pose of 15g and bar diagram represents the per residue wise decomposition analysis, residues on the X-axis, whereas Energy
(kcal/mol) is on Y-axis.
Scheme captions
Scheme 1. Reagents, conditions and yields (i) CH3COOH, CH3COONa, reflux, 72 h, Yield 40-75%.
Scheme 2. Reagents, conditions and yields (i) DCM, NH4OH, rt, 2 h, 81% and 83% (ii) EtOH, CH3COONa, reflux, 6 h, 73% and 77% (iii)
CH3COOH, CH3COONa, reflux, 72 h, 30-40%.
Table caption
Table 1. IC50 values for GSK-3β and percentage residual activity of CDK-2 for seven compounds at 2 ꢀM Inhibitor concentration. The activity of
CDK-2 in the absence of inhibitor was assigned the value of 100%.
10

Figures
Figure 1.
ACCEPTED MANUSCRIPT
Figure 2.
Figure 3.
Figure 4.
11

Figure 5.
ACCEPTED MANUSCRIPT
Figure 6.
Figure 7.
12

Schemes
ACCEPTED MANUSCRIPT
Scheme 1.
Scheme 2.
13

Table
ACCEPTED MANUSCRIPT
Table1.
Sr. No. Compound IC50 (nM) % Res. Act.
1
2
3
4
5
6
7
27.7
40.0
24.0
27.1
2.1
69.63
68.93
68.40
63.23
73.01
39.10
53.83
15a
15b
15e
15f
15g
15k
15l
51.7
44.3
14

ACCEPTED MANUSCRIPT
Highlights
1. A few selective GSK-3β Inhibitors were designed and synthesized: 5-benzylidene-2-
iminothiazolidin-4-ones.
2. Structure Based Drug Design techniques were utilized employed to highlight the
importance of 2-iminothialidin-4-one ring.
3. In vitro evaluation of 25 compounds resulted in the identification of nine compounds
with activity in the lower nanomolar range (2.1-84.8 nM).
4. Six compounds showed selective GSK-3β inhibitory activity against CDK-2 at 2µM
concentration.
5. MD simulation showed that residues like Lys85, Thr138, and Arg141 play crucial role in
selective inhibition.