Discovery of novel cinnamylidene-thiazolidinedione derivatives as PTP-1B inhibitors for the management of type 2 diabetes

Article abstract of DOI:10.1039/c6ra24501c

Protein tyrosine phosphatase-1B (PTP-1B) inhibition is a legitimate approach to combat type 2 diabetes and obesity as it corrects the insulin and leptin signalling cascades. In pursuing this, our goal is to discover compounds bearing small molecular scaffolds, and able to inhibit PTP-1B in a selective manner. In the present work, we have synthesized N³-substituted cinnamylidene-thiazolidinediones (4p-4x), and evaluated in vitro PTP-1B inhibitory activity, and in vivo anti-hyperglycaemic potential in streptozotocin-nicotinamide induced diabetic mice. Among various synthesized compounds, 4w exhibited the most potent in vitro PTP-1B inhibitory activity (IC₅₀ ～ 6.52 μM) along with excellent in vivo anti-hyperglycaemic activity. Furthermore, molecular docking assisted 3D-QSAR study was performed on the synthesized compounds (4p-4x) for the exploration of the binding mode of interactions, prediction of the binding affinity, and identification of 3D-pharmacophoric features (steric and electrostatic) responsible for inhibitory activity. The docking results were in agreement with the biological activity results i.e. compound 4w showed the highest binding affinity with PTP-1B (MolDock score -123.715), which is comparable with ertiprotafib (MolDock score -125.183). The lead discovered can be used for the further development of cinnamylidene-thiazolidinedione derivatives as antidiabetic agents.

Full text of DOI:10.1039/c6ra24501c

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Discovery of novel cinnamylidene-
thiazolidinedione derivatives as PTP-1B inhibitors
Cite this: RSC Adv., 2016, 6, 108928
for the management of type 2 diabetes†‡
ab
Sant K. Verma,a Diksha Haksar,b Tilak R. Bhardwajb
*
Suresh Thareja,
and Manoj Kumarb
Protein tyrosine phosphatase-1B (PTP-1B) inhibition is a legitimate approach to combat type 2 diabetes and
obesity as it corrects the insulin and leptin signalling cascades. In pursuing this, our goal is to discover
compounds bearing small molecular scaffolds, and able to inhibit PTP-1B in a selective manner. In the
present work, we have synthesized N3-substituted cinnamylidene-thiazolidinediones (4p–4x), and
evaluated in vitro PTP-1B inhibitory activity, and in vivo anti-hyperglycaemic potential in streptozotocin-
nicotinamide induced diabetic mice. Among various synthesized compounds, 4w exhibited the most
potent in vitro PTP-1B inhibitory activity (IC50
ꢀ
6.52 mM) along with excellent in vivo anti-
hyperglycaemic activity. Furthermore, molecular docking assisted 3D-QSAR study was performed on the
synthesized compounds (4p–4x) for the exploration of the binding mode of interactions, prediction of
the binding affinity, and identification of 3D-pharmacophoric features (steric and electrostatic)
responsible for inhibitory activity. The docking results were in agreement with the biological activity
results i.e. compound 4w showed the highest binding affinity with PTP-1B (MolDock score ꢁ123.715),
which is comparable with ertiprotafib (MolDock score ꢁ125.183). The lead discovered can be used for
the further development of cinnamylidene-thiazolidinedione derivatives as antidiabetic agents.
Received 1st October 2016
Accepted 26th October 2016
DOI: 10.1039/c6ra24501c
www.rsc.org/advances
million by the year 2035.3,5 The present threatening data creates
urge for pharmacological agents that can be able to manage
Introduction
diabetes and contribute to health scenario and quality of life.
Protein tyrosine phosphatase-1B (PTP-1B) is a cytosolic non
trans-membranous prototypic enzyme that belongs to PTPs
superfamily,1a,b,3 and expressed in the cells of the liver, adipocytes
and muscles.6 Biochemical and cellular studies, chemical and
genetic experiments on mouse and human have strongly estab-
lished this enzyme as a central negative regulator of insulin and
leptin signalling cascades.7a–g Mice lacking protein tyrosine
phosphatase-1B (PTP-1B) demonstrated enhanced insulin sensi-
tivity with diminished plasma glucose and insulin levels. Further-
more, these mice also exhibit lower adiposity without producing
anomalies in growth or fertility or other pathogenetic effects.7a,b,8a,b
PTP-1B inhibition is the legitimate approach to combat type 2
diabetes and obesity as it corrects the insulin and leptin signalling
cascades.7c–f,9 A number of PTP-1B inhibitors have been reported
over the last decades, although no commercial drugs have been
approved till date.7c,10a–e However, ertiprotab and trodusquemine
(Fig. 1) are the only two drug candidates that have entered into
clinical trials. Due to side effects and low rate of in vivo efficacy,
ertiprotab was withdrawn in phase II clinical trials.11a–c Tro-
dusquemine is an allosteric, noncompetitive and highly selective
inhibitor of PTP-1B currently in phase I clinical trials. Recent
study demonstrated that it might also provide a promising way to
combat anxiety disorders along with obesity.12a,b
Diabetes mellitus (DM) is a metabolic disorder characterized by
long-lasting hyperglycaemia.1a,b Type
2 diabetes mellitus
(T2DM) is the most common form of diabetes with a share of
90–95% of total cases of diabetes around the world.2 The
underlying pathogenesis behind T2DM includes ineffective
biological responses to sufficiently produce insulin i.e. insulin
resistance.3 Diabetes is associated with typical long-term dia-
betic complications namely cardiac abnormalities, atheroscle-
rosis, microangiopathy, nephropathy, neuropathy, retinopathy
and cataracts.4 The frequency of diabetes is also increasing in
parallel with obesity; therefore diabetes is also known as ‘dia-
besity’ or ‘obesity dependent diabetes mellitus’.1b The preva-
lence of diabetes worldwide was 285 million in the year 2010
while present data showed that 387 million people worldwide
have diabetes in the year 2014, and it is estimated to affect 592
aSchool of Pharmaceutical Sciences, Guru Ghasidas Central University, Bilaspur-495
009, C.G., India. E-mail: sureshthareja@gmail.com
bUniversity Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160
014, India
† The authors declare that in the present study none of the experimentation was
performed using the human subjects.
‡ Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra24501c
108928 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
derivatives with N-substitution are of particular interest as
PTP-1B inhibitors and this structural amendment making them
devoid of side effects associated with glitazones.15d,e,17 We have
also performed molecular docking assisted 3D-QSAR study to
map the spatial ngerprints of arylidine-2,4-TZDs for potent
PTP-1B inhibitory activity.3
Fig. 1 PTP-1B inhibitors entered in clinical trials (ertiprotafib and tro-
Taking into consideration above aspects, a series of compounds
bearing 2,4-TZD scaffold in conjugation with cinnamaldehyde i.e.
N3-substituted cinnamylidene-thiazolidinedione derivatives (4)
(Scheme 1) was designed, synthesized and evaluated for their in
vitro PTP-1B inhibitory activity as well as in vivo anti hyper-
glycaemic potential. In addition, a molecular docking assisted 3D-
QSAR model was developed on synthesized derivatives (4) for the
identication of binding mode of interactions, determination of
binding affinity, and exploration of 3D-pharmacophoric (steric and
electrostatic) features of cinnamylidene-thiazolidinediones for
PTP-1B inhibitory activity.
dusquemine) and marketed TZD derivative (pioglitazone).
Development of an orally bioavailable and specic PTP-1B
inhibitors bearing small molecular scaffold is not an easy task
due to poor cell permeability of the small molecules exhibiting
high affinity with PTP-1B due to their hydrophilic nature. A rst-
in-class PTP-1B inhibitor has yet to be discovered; however,
extensive research is under way to develop a potential block-
buster drug. Small molecule PTP-1B inhibitors that not only
serve as powerful tools to dene the physiological roles of PTP-
1B in vivo, but also as excellent lead compounds for the devel-
opment of new therapeutic agents for the management of
T2DM and obesity.10c–e,13
Pioglitazone (Fig. 1), a PPAR-g agonist, is the only molecule
bearing benzyl-2,4-thiazolidinedione (benzyl-2,4-TZD) scaffold
that is available in the market to improve glycaemic control by
ameliorating insulin resistance and show efficient insulin
sensitization both in peripheral tissues and liver in type 2 dia-
betic patients.14a,b Various researchers around the world re-
ported that 2,4-thiazolidinedione (2,4-TZD) derivatives exhibit
inhibitory effects against PTP-1B.15a–i
Results and discussion
Chemistry
Compounds (4p–x) were synthesized according to the general
method depicted in Scheme 1. The condensation of commercially
available 2,4-TZD (1) with cinnamaldehyde (2) in the presence of
piperidine provided (5Z)-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-
dione (3a). The reaction between compound 3a with different alkyl
bromide, substituted alkyl bromide and substituted aryl bromide
(R1–8) using anhydrous potassium carbonate as base in reuxing
acetone, followed by a workup and recrystallization from meth-
anol, provided pure compounds 4p–4w (Scheme 1). Further, the
reaction of compound 3a was carried out with ethyl chloroacetate
It has been reported that naturally occurring cinnmaldehyde
derivatives possess potent PTP-1B inhibitory activities.16a–c
Recently, it has also been reported that arylidene-2,4-TZD
Scheme 1 Reagents and conditions: (a) piperidine, ethanol, reflux for 26 h; (b) R-Br, acetone, anhydrous K2CO3, reflux for 26 h; (c) ethyl
chloroacetate, anhydrous K2CO3, acetone, reflux for 24 h; (d) glacial acetic acid, hydrochloric acid, reflux for 2 h.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108929

View Article Online
RSC Advances
Paper
in presence of anhydrous potassium carbonate as base, aer
removal of solvent the recovered product recrystallized from
methanol to yield pure ethyl 2-[(5Z)-5-{(E)-3-phenylallylidene}-
thiazolidine-2,4-dione-3-yl]acetate (3b). Acidic hydrolysis of 3b
was carried out in reuxing glacial acetic acid, followed by
a workup and recrystallization from methanol to yield pure 2-[(5Z)-
{(E)-3-phenylallylidene}-thiazolidine-2,4-dione-3-yl]-acetic acid (4x)
(Scheme 1).
Biological evaluation
In vitro PTP-1B inhibitory activity. The synthesized
compounds (4p–4x) were evaluated in vitro for their ability to
inhibit PTP-1B using PTP-1B drug discovery assay kit. Suramin
served as reference standard for comparison. Compounds (4p–
4x) bearing various substituents (R1–8) at N3 exhibited moderate
PTP-1B inhibitory activities (Table 1). Among the series,
compound 4w bearing methyl benzoic acid exhibited most
potent inhibitory activity with IC50 ꢀ 6.52 mM. Increase in chain
length from 4p to 4u resulted in compounds with marginal
decrease in potency. Compounds bearing bromo group on alkyl
chain were found to be more potent as compared to their chloro
derivatives. Compound 4x bearing acetic acid substituent at N3
also exhibited good inhibitory activity.
In vivo anti-hyperglycaemic activity. Anti-hyperglycaemic
activity of compounds (4p–4x) was carried in STZ-NA induced
diabetic mice. Pioglitazone was taken as reference standard. All
Fig.
2
(A) Comparative anti-hyperglycaemic activities of N3-
substituted cinnamylidene-thiazolidinediones (4p–x). (B) Anti-hyper-
glycaemic activities of control, pioglitazone and compound 4w.
the compounds as well as reference standard were administered
at a xed dose of 30 mg kgꢁ1
body weight orally as
19a,b
suspension in carboxy methyl cellulose (CMC). Administration
of compounds (4p–x) reduced serum glucose level (SGL)
signicantly (P < 0.001) in diabetic mice as compared to control
group (Fig. 2A).
during seven days treatment period which can be attributed to
T2DM. Pioglitazone treatment resulted in increase in the body
weight. Effect on body weight aer treatment with cinnamylidene-
thiazolidinedione derivatives (4p–x) in diabetic mice are pre-
sented in Fig. 3. Results indicate that 4w administration has
marginally increased in body weight but weight gain was less as
compared to reference standard pioglitazone.
Histopathology of pancreas. Histological analysis by Gomori
staining of non-diabetic mouse pancreas showed normal
histological structure, depicted average sized islets and normal
sized b cells (Fig. 4A). Enlarged and inamed cells of islets,
destruction of the b cells, damaged cell membrane, along with
necrotic cells were seen in diabetic control animals (Fig. 4B).
However, intact cell membrane and normal sized cells were
observed in pioglitazone treatment group which indicated
a signicant protection against diabetes induced histopatho-
logical changes (Fig. 4C). Furthermore, the most potent
compound 4w also showed similar patterns to that of pioglita-
zone i.e. intact cell membrane and nucleus were clearly visible
(Fig. 4D) indicated similar behaviour in terms of protection
against diabetes induced histopathological changes.
Compound 4w exhibited activity comparable to reference
standard pioglitazone. The in vivo activity data of control, pio-
glitazone and compound 4w was compared separately and is
presented in Fig. 2B. Introducing alkyl substituent (4u) resulted
in compound with decreased activity while alkyl chain bearing
chloro and bromo substituent resulted in compounds with better
anti-hyperglycaemic activity. Compound 4x bearing free carbox-
ylic group also exhibited good anti-hyperglycaemic activity.
Effect on body weight. During the study period of seven days,
the mice were weighed and their body weights were recorded. In
control mice, a trend of decrease in body weight was observed
Table 1 In vitro PTP-1B inhibitory activities of N3-substituted cinna-
mylidene-thiazolidinediones (4p–x)
Compound
IC50 (mM)
Compound
IC50 (mM)
4p
4q
4r
4s
4t
30.770
20.458
33.271
32.829
20.916
33.607
4v
4w
4x
32.006
6.522
21.476
11.100
1.4a
Suramin
Ertiprotab
Pioglitazone
Molecular docking assisted 3D-QSAR
4u
220b
In order to shed light on binding affinities as well as binding
interactions of cinnamylidene-thiazolidinedione derivatives
(4p–x) within the active site of PTP-1B, and generation of 3D-
a
b
Data taken from the work of Shrestha et al.18 Data taken from the
work of Bhattarai et al.15f
108930 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
Fig. 3 Graphical presentation of change in body weight after treat-
ment with cinnamylidene-thiazolidinedione derivatives (4p–x) in dia-
betic mice.
Fig. 5 Secondary structure of PTP-1B with mapped binding cavities 1,
2 and 3 (green).
potential binding modes (poses), and a re-ranking score is the
indicative of highest ranked poses to further increase docking
accuracy.20 It is observed that the most active compound (4w)
exhibits highest MolDock score (ꢁ123.715) among the series of
synthesized compounds, which is comparable with ertiprotab
(ꢁ125.183). Compound 4w also exhibits highest re-rank score
(ꢁ102.381) (Table 3). In particular, the electrostatic interactions
and hydrophobicity of the compounds 4w anchored in the cavity
1 of PTP-1B are shown in Fig. 7. The weak hydrogen bond (1) is
formed between oxygen of 4-keto present at TZD ring of
compound 4w with Asp 48, and the strong hydrogen bond (2) is
formed between –OH group of methyl benzoic acid present at N3
of compound 4w with Arg 45 (Fig. 8A). The properties of hydrogen
bonds such as H-bond donor/acceptor, energy and length are
presented in Table 4. In Fig. 8B the steric interactions of
compound 4w with PTP are demonstrated. The phenyl ring of
methyl benzoic acid present at N3 of TZD ring in compound 4w
showed simultaneous steric interactions with Arg 47 and Tyr 46,
and the phenyl ring of phenyl allylidene group present at h
position of TZD ring in compound 4w showed steric interaction
with Cys 215. The root mean square deviation (RMSD) between
Fig. 4 Histopathological analysis of pancreas of non-diabetic group
(A), diabetic control group (B), pioglitazone treated group (C), and
treated group with compound 4w (D).
QSAR model for the exploration of pharmacophoric require-
ments that are mandatory for a chemical entity to be an
imminent, potent, selective and bioavailable inhibitor of PTP-
1B with reduced toxicity, molecular docking simulations
based 3D-QSAR study was performed.
Three distinct cavities (Fig. 5) with different surface area and
volume have been mapped within PTP-1B (PDB entry: 2QBS). The
volume and surface area of these cavities are depicted in Table 2.
The key residues of cavity 1 included Ala 217, Arg 45, Arg 47, Arg
221, Asp 48, Asp 181, Cys 215, Gln 262, Gln 266, Gly 183, Gly 218,
Gly 220, Gly 259, Ile 219, Phe 182, Ser 216, Ser 222, Tyr 46 and Val
49 (Fig. 6). The docking simulations in the active site 1 (cavity 1)
of PTP-1B with cinnamylidene-thiazolidinedione derivatives (4p–
x) were performed by the MVD program, provided excellent result
in accordance with the biological activity. The docking scores
(MolDock, re-rank and H-bond scores) of compounds (4p–4x) are
depicted in Table 3. MolDock, a docking scoring function that
takes hydrogen bond directionality into account, combines the
differential evolution optimization technique with a cavity
prediction algorithm, the use of predicted cavities during the
search process allows for a fast and accurate identication of
ꢀ
co-crystallized and re-docked conformation was found 0.99 A
suggesting high docking reliability.
Table 2 The volume and surface area of cavities (1–3) detected within
PTP-1B
ꢀ3
ꢀ2
Cavity No.
Volume (A )
Surface area (A )
1
2
3
53.760
15.360
14.336
165.120
66.560
70.400
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108931

View Article Online
RSC Advances
Paper
Fig. 6 Cavity 1 (green) of PTP-1B with its constituting amino acid
residues.
Table 3 Docking scores (MolDock, Re-rank and H-bond scores) of
compounds (4p–4x) and ertiprotafib with PTP-1B
MolDock score
Re-rank score
H-Bond score
Compound
(kcal molꢁ1
)
(kcal molꢁ1
)
(kcal molꢁ1
)
4p
4q
4r
4s
4t
4u
4v
4w
ꢁ112.227
ꢁ112.153
ꢁ110.484
ꢁ116.670
ꢁ116.214
ꢁ117.467
ꢁ117.597
ꢁ123.715
ꢁ120.287
ꢁ125.183
ꢁ93.2859
ꢁ92.3753
ꢁ94.2814
ꢁ93.4941
ꢁ91.6772
ꢁ95.5568
ꢁ96.6064
ꢁ97.6551
ꢁ102.381
ꢁ77.276
ꢁ2.5337
ꢁ2.6035
ꢁ2.4132
ꢁ3.3635
ꢁ3.5233
ꢁ3.3398
ꢁ1.9144
ꢁ1.8259
ꢁ8.7196
ꢁ4.0603
Fig. 7 Electrostatic interactions (A) (red: electronegative interactions,
blue: electropositive interactions) and hydrophobicity (B) (red:
hydrophilic interactions, blue: hydrophobic interactions) of compound
4w anchored in the cavity 1 of PTP-1B.
moderate difference between actual and predicted values (<1)
(Table 6) are the indicator of a good linear correlation of the
compounds selected in present 3D-QSAR data set. Further,
results from the 3D-QSAR study indicated that the contribu-
tion of steric feature is of slight higher signicance (51%) as
compare to electrostatic feature (49%) for PTP-1B inhibitory
potential.
4x
Ertiprotab
3D-QSAR model was generated from a series of nine
synthesized cinnamylidene-thiazolidine-
N3-substituted
diones (4p–4x) acting as inhibitors of PTP-1B by using
conformational alignment schemes depicted in Fig. 9. As
pIC50 ¼ 0.0005 + 0.5005 shape + 0.4999 electrostatic
(1)
ꢀ
evident from the literature, only 0.5 A grids spacing has been
opted for the present 3D-QSAR investigation. 3D-QSAR
calculation for both electrostatic and shape potentials were
performed and combined using PLS technique to generate
3D-QSAR model (eqn (1)).3,21 Table 5 demonstrates a statis-
tical summary of the generated 3D-QSAR model. The devel-
oped 3D-QSAR model showed excellent cross validated
correlation coefficient Q2 (0.828), non-cross validated corre-
lation coefficient r2 (0.866), standard error of estimate S-value
(0.089) and high Fisher test (F-test) value (45.405) against
PTP-1B. Due to signicant statistical measures and robust-
ness of the developed 3D-QSAR model, it is used for the
prediction of PTP-1B inhibitory potential of the synthesized
compounds (4p–4x) (Table 6). The smaller residual values i.e.
The contribution of shape and electrostatic features of the
cinnamylidene-thiazolidinedione derivatives required for
potent PTP-1B inhibitory activity are displayed in the form of
master grid maps (Fig. 10), which also provide a direct visual
indication regarding structural features responsible for desired
biological activity of molecules under study. The master grid
maps also offered an interpretation as to design and optimize
novel molecules with superior biological activities. Here, we
used most potent compound 4w as reference structure for
visualization and interpretation of electrostatic and steric
master grid maps (Fig. 10).
108932 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
Fig.
9
Alignment scheme for development of 3D-QSAR model
(substituents R1 and R2 used for alignment purpose).
Table 5 PLS statistics of 3D-QSAR model
Statistical parameters
Values
Q2
0.828
0.866
r2
S
0.089
F-Test
45.405
51.000
49.000
Steric contribution (%)
Electrostatic contribution (%)
Table 6 Actual and 3D-QSAR predicted PTP-1B inhibitory activities of
compounds 4p–x
Actual activity
(pIC50
Predicted activity
(pIC50
Compound
)
)
Residual activity
4p
4q
4r
4s
4t
4u
4v
4w
4x
ꢁ1.488
ꢁ1.310
ꢁ1.522
ꢁ1.516
ꢁ1.320
ꢁ1.526
ꢁ1.505
ꢁ0.814
ꢁ1.332
ꢁ1.473
ꢁ1.476
ꢁ1.456
ꢁ1.405
ꢁ1.403
ꢁ1.460
ꢁ1.504
ꢁ0.832
ꢁ1.325
ꢁ0.015
0.165
ꢁ0.066
ꢁ0.111
0.082
ꢁ0.066
ꢁ0.001
0.018
ꢁ0.007
Fig. 8 Hydrogen bond (A) (1 and 2, yellow dotted bonds) and steric (B)
(red dotted lines) interactions of compound 4w within the cavity 1 of
PTP-1B.
A cluster of red points around the hydrogen atom of –OH group
of the same functionality strongly support requirement of
electropositive atoms or groups attached with oxygen of –OH
group for favourable PTP-1B inhibitory activity. Furthermore,
few red points around third and h position of phenyl ring of
the methyl benzoic acid indicate the presence of electropositive
atoms or groups for potent PTP-1B inhibitory activity.
In 3D-QSAR steric master grid map (Fig. 10B), presence of red
and blue points indicate sterically favored (more steric bulk
increases activity) and un-favored (more steric bulk decreases
activity) area, respectively.3,21 Presence of cluster of blue points
surrounded by red points around substituent ‘R1’ of
cinnamylidene-thiazolidinedione scaffold indicates that ‘R1’ should
bear bulky moiety in the core with less branching for favourable
steric interaction desired for potent PTP-1B inhibitory activity.
In electrostatic grid maps (Fig. 10A), electrostatic favoured
areas have been exemplied by red points (more positive charge
increases activity, while more negative charge decreases
activity), and un-favoured areas by blue points (more negative
charge increases activity, while more positive charge decreases
activity).3,21 In the electrostatic master grid map (Fig. 10A),
presence of a high density of blue points around the pC]O
group of methyl benzoic acid present at N3 position of
cinnamylidene-thiazolidinedione scaffold indicates existence of
electronegative groups favourable for PTP-1B inhibitory activity.
Table 4 Properties of hydrogen bonds formed between compound
4w and PTP-1B
Experimental
Chemistry
Energy (kcal
ꢁ1 ꢀꢁ1
ꢀ
H-Bond ID
H-Bond donor
mol
A
)
Length (A)
All chemicals and solvents were obtained from commercial
sources and puried using standard procedures whenever
1
2
Target (PTP-1B)
Ligand (compound 4w)
0.332
1.494
3.492
3.301
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108933

View Article Online
RSC Advances
Paper
CH]CH; J ¼ 15.28 Hz), 7.33 (t, 3H, –CH arom), 7.42 (d, 1H,
C6H5–CH]CH–CH]; J ¼ 11.44 Hz), 7.54 (d, 2H, –CH arom) and
12.21 ppm (br s, NH, exchangeable with D2O), 13C NMR (DMSO-
d6): d 167.61 (–C]O, TZD), 167.03 (–C]O, TZD), 143.12 (CH]
CH–CH]), 132.06 (C6H5–CH]CH), 125.42 (C6H5–CH]CH),
122.22 (–C–C]O), 135.67, 129.83, 129.04, 127.74 and
123.28 ppm (C, arom).
General method for the synthesis of (5Z)-3-(2-alkyl/substituted
alkyl/substituted aryl)-5-[(E)-3-phenylallylidene]-thiazolidine-
2,4-dione (4p–4w)
A
mixture of (5Z)-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-
dione (3a) (0.46 g, 2.0 mmol), alkyl bromide/substituted alkyl
bromide/substituted aryl bromide (R1–8Br) (3.0 mmol) and
anhydrous potassium carbonate (1.38 g, 10.0 mmol) in acetone
(50.0 mL) was reuxed for 24 h. The solvent was evaporated
under reduced pressure to obtain the residue. The residue was
stirred in crushed ice and the precipitated product was ltered,
washed with water and dried. It was crystallised from methanol
to yield compounds 4p–4w.
(5Z)-3-(2-Chloroethyl)-5-[(E)-3-phenylallylidene]-thiazolidine-
2,4-dione (4p). Yield 83.5%; mp 165–168 ꢂC; Rf-value: 0.54
(CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3018 (CH arom),
2954 (CH aliph), 1738 (4-CO), 1673 (2-CO), 1605 (C]C) and 737
(C–Cl); 1H NMR (CDCl3): d 3.48 (t, 2H, –NCH2CH2–), 4.10 (t, 2H,
–NCH2CH2–), 6.68 (m, 1H, C6H5–CH]CH–CH–, J ¼ 11.48 Hz),
7.04 (d, 1H, C6H5–CH]CH–CH–, J ¼ 15.24 Hz), 7.36 (m, 3H, CH
arom), 7.49 (m, 2H, CH arom) and 7.58 ppm (d, 1H, C6H5–CH]
CH–CH], J ¼ 11.40 Hz); 13C NMR (CDCl3): d 167.06 (–C]O,
TZD), 165.22 (–C]O, TZD), 144.28 (C6H5–CH]CH–CH]),
135.36 (C6H5–CH]CH–CH–), 134.09 (C6H5–CH]CH–CH),
122.22 (–C–C]O), 42.60 (–NCH2CH2–), 25.80 (–NCH2CH2–),
130.16, 129.05, 127.75 and 122.69 ppm (C, arom); mass: 293
(M+) and 295 (M+ + 2).
ꢀ
Fig. 10 Electrostatic (A) and steric (B) master grid maps at 0.5 A grid
resolutions.
required. Melting points were recorded on Veego-540 melting
point apparatus and are uncorrected. The structures of the
nally synthesized compounds were conrmed by IR, 1H-NMR,
13C-NMR and mass spectrometry. 1H and 13C nuclear magnetic
resonance spectra were obtained using Bruker AC-400F, 400
MHz spectrometer for solutions in deuteriochloroform (CDCl3),
deuterated dimethylsulfoxide (DMSO-d6) and were reported in
parts per million (ppm), downeld from tetramethylsilane
(TMS) as internal standard. The spin multiplicities are indi-
cated by the symbols, s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet) and br s (broad singlet). Infrared (IR)
spectra were obtained with Perkin-Elmer 882 Spectrum and RXI,
FT-IR model using potassium bromide pellets (cmꢁ1). Mass
spectra were obtained with electrospray ionization (ESI),
atmospheric pressure chemical ionization (APCI) and matrix
assisted laser desorption ionization-time of ight (MALDI-TOF)
mass spectrometer. Reactions were monitored and the homo-
geneity of the products was checked by thin layer chromatog-
raphy (TLC). For TLC, glass plates coated with silica gel G (E.
Synthesis of (5Z)-3-(2-bromoethyl)-5-[(E)-3-phenylallylidene]-
thiazolidine-2,4-dione (4q). Yield: 80.3%; mp 175–177 ꢂC; Rf-
value: 0.68 (CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3017 (CH
arom), 2932 (CH aliph), 1734 (4-CO), 1673 (2-CO), 1605 (C]C)
ꢂ
Merck) were used. The TLC plates were activated at 110 C for
30 min and visualized by exposure to iodine vapour. Pre-coated
aluminium sheets coated with Silica Gel 60 F254, 0.2 mm
thickness were used for nal monitoring.
1
and 736 (C–Br); H NMR (CDCl3): d 3.55 (t, 2H, –NCH2CH2–),
4.10 (t, 2H, –NCH2CH2–), 6.68 (m, 1H, C6H5–CH]CH–CH–, J ¼
11.64 Hz), 7.04 (d, 1H, C6H5–CH]CH–CH–, J ¼ 15.24 Hz), 7.36
(m, 3H, CH arom), 7.49 (m, 2H, CH arom) and 7.58 ppm (d, 1H,
C6H5–CH]CH–CH], J ¼ 11.44 Hz); 13C NMR (CDCl3): d 167.06
(–C]O, TZD), 165.22 (–C]O, TZD), 144.28 (C6H5–CH]CH–
Synthesis of (5Z)-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-
dione (3a)
A mixture of 2,4-thiazolidinedione (1.17 g, 10.0 mmol) and CH]), 135.36 (C6H5–CH]CH–CH–), 134.08 (C6H5–CH]CH–
cinnamaldehyde (1.32 mL, 10.0 mmol) was reuxed in presence CH–), 122.23 (–C–C]O), 42.60 (–NCH2–CH2–), 26.98 (–NCH2–
of catalytic amount of piperidine (0.25 mL) for 26 h in absolute CH2–), 130.16, 129.05, 127.75 and 122.70 ppm (C, arom); mass:
ethanol (50.0 mL). The solvent was evaporated under reduced 336 (M+) and 338 (M+ + 2).
pressure to obtain the residue. The residue was poured onto
(5Z)-3-Propyl-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-dione
crushed ice with stirring. The separated product was ltered, (4r). Yield: 82.4%; mp 125–127 ꢂC; Rf-value: 0.68 (CHCl3-
washed with hydrochloric acid (5%) and dried. It was crystal-
lized from methanol to yield pure 3a.
: MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3021 (CH arom), 2961 (CH
aliph), 1736 (4-CO), 1672 (2-CO) and 1605 (C]C); 1H NMR
Yield 79.0%; mp 202–204 ꢂC;22a,b Rf-value: 0.58 (CHCl3- (CDCl3): d 0.92 (t, 3H, –NCH2CH2CH3), 1.63 (m, 2H, –NCH2-
: MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3189 (NH), 3026 (CH arom), CH2CH3), 3.67 (t, 2H, –NCH2CH2CH3), 6.69 (m, 1H, C6H5–CH]
1722 (4-CO), 1683 (2-CO) and 1607 (C]C); 1H NMR (DMSO-d6): CH–CH–, J ¼ 11.44 Hz), 7.02 (d, 1H, C6H5–CH]CH–CH–, J ¼
d 6.69 (t, 1H, C6H5–CH]CH, J ¼ 11.48 Hz), 7.05 (d, 1H, C6H5– 15.28 Hz), 7.33 (m, 3H, CH arom), 7.49 (m, 2H, CH arom) and
108934 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
7.59 ppm (d, 1H, C6H5–CH]CH–CH], J ¼ 11.64 Hz); 13C NMR 1.83 (m, 2H, –NCH2CH2CH2CH2–), 3.41 (t, 2H, –NCH2CH2CH2-
(CDCl3): d 167.39 (–C]O, TZD), 165.82 (–C]O, TZD), 143.56 CH2–), 3.73 (t, 2H, –NCH2CH2CH2CH2–), 6.68 (m, 1H, C6H5–
(C6H5–CH]CH–CH]), 135.50 (C6H5–CH]CH–CH–), 133.17 CH]CH–CH–, J ¼ 11.48 Hz), 7.02 (d, 1H, C6H5–CH]CH–CH–, J
(C6H5–CH]CH–CH–), 122.93 (–C–C]O), 43.79 (–NCH2CH2- ¼ 15.24 Hz), 7.26 (m, 3H, CH arom), 7.49 (m, 2H, CH arom) and
CH3), 21.17 (–N–CH2CH2CH3), 11.20 (–NCH2CHCH3), 129.96, 7.58 ppm (d, 1H, C6H5–CH]CH–CH], J ¼ 11.64 Hz); 13C NMR
129.01, 127.64 and 123.09 ppm (C, arom); mass: 274 (M+ + 1).
(CDCl3): d 167.27 (–C]O, TZD), 165.62 (–C]O, TZD), 143.82
(5Z)-3-(3-Chloropropyl)-5-[(E)-3-phenylallylidene]-thiazolidine- (C6H5–CH]CH–CH]), 135.38 (C6H5–CH]CH–CH–), 133.46
2,4-dione (4s). Yield: 84.0%; mp 144–146 ꢂC; Rf-value: 0.60 (C6H5–CH]CH–CH–), 122.71 (–C–C]O), 40.83 (–NCH2CH2-
(CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3012 (CH arom), CH2CH2Br), 32.68 (–NCH2CH2CH2CH2–), 29.62 (–NCH2CH2-
2860 (CH aliph), 1719 (4-CO), 1682 (2-CO), 1605 (C]C) and 735 CH2CH2), 26.44 (–NCH2CH2CH2CH2–), 129.99, 128.97, 128.84,
(C–Cl); 1H NMR (CDCl3): d 2.13 (m, 2H, –NCH2CH2CH2–), 3.55 (t, 127.64, and 122.77 ppm (C, arom); mass: 365 (M+) and 367 (M+ +
2H, –NCH2CH2CH2–), 3.87 (t, 2H, –NCH2CH2CH2–), 6.69 (t, 1H, 2).
C6H5–CH]CH, J ¼ 11.42 Hz), 7.04 (d, 1H, C6H5–CH]CH–, J ¼
4-[(5Z)-5-{(E)-3-Phenylallylidene}-thiazolidine-2,4-dione-3-yl-
15.24 Hz), 7.26 (t, 3H, CH arom), 7.50 (d, 2H, CH arom) and methyl]benzoic acid (4w). Yield: 75.4%; mp 216–220 ꢂC; Rf-
7.58 ppm (d, 1H, –CH]CH–CH], J ¼ 11.60 Hz); 13C NMR value: 0.45 (CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3447
(CDCl3): d 167.28 (–C]O, TZD), 165.62 (–C]O, TZD), 143.99 (OH), 3035 (CH arom), 2918 (CH aliph), 1739 (CO carboxy), 1686
(C6H5–CH]CH–CH]), 135.42 (C6H5–CH]CH–), 133.65 (4-CO) and 1599 (2-CO); 1H NMR (CDCl3): d 4.62 (s, 2H, –NCH2–),
(C6H5–CH]CH–), 122.78 (–C–C]O), 41.84 (–NCH2CH2CH2–), 6.74 (m, 1H, C6H5–CH]CH–CH–, J ¼ 11.44 Hz), 7.00 (d, 1H,
39.59 (–NCH2CH2CH2–),30.68 (–NCH2CH2CH2–), 130.07, C6H5–CH]CH–CH–, J ¼ 15.24 Hz), 7.32 (m, 3H, CH arom), 7.44
129.02, 127.69 and 122.64 ppm (C, arom); mass: 307 (M+) and (m, 2H, CH arom), 7.47 (t, 2H, –CH arom), 7.50 ppm (d, 1H,
309 (M+ + 2).
C6H5–CH]CH–CH], J ¼ 11.68 Hz), 7.55 (d, 2H, –CH arom),
(5Z)-3-(3-Bromoopropyl)-5-[(E)-3-phenylallylidene]-thiazolidine- 7.94 (d, 2H, –CH arom) and 12.21 ppm (br s, 1H, –COOH,
2,4-dione (4t). Yield: 79.5%; mp 180–182 ꢂC; Rf-value: 0.68 exchangeable with D2O); 13C NMR (CDCl3): d 167.16 (–COOH),
(CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3016 (CH arom), 166.85 (–C]O, TZD), 166.54 (–C]O, TZD), 142.52 (C6H5–CH]
2953 (CH aliph), 1736 (4-CO), 1673 (2-CO), 1601 (C]C) and 738 CH–CH]), 135.29 (C6H5–CH]CH–CH–), 131.68 (C6H5–CH]
1
(C–Br); H NMR (CDCl3): d 2.17 (m, 2H, –NCH2CH2CH2–), 3.38 CH–CH–), 122.91 (–C–C]O), 32.63 (–NCH2–), 142.25, 130.53,
(t, 2H, –NCH2CH2CH2–), 3.86 (t, 2H, –NCH2CH2CH2–), 6.69 (m, 129.56, 129.10, 128.91, 128.61, 127.37 and 124.92 ppm (C,
1H, C6H5–CH]CH–CH–, J ¼ 11.44 Hz), 7.04 (d, 1H, C6H5–CH] arom); mass: 388 (M+ + Na).
CH–CH–, J ¼ 15.28 Hz), 7.36 (m, 3H, CH arom), 7.50 (m, 2H, CH
arom) and 7.58 ppm (d, 1H, C6H5–CH]CH–CH], J ¼ 11.64
Hz); 13C NMR (CDCl3): d 167.27 (–C]O, TZD), 165.59 (–C]O,
Ethyl 2-[(5Z)-5-{(E)-3-phenylallylidene}-thiazolidine-2,4-dione-
3-yl]acetate (3b)
TZD), 144.01 (C6H5–CH]CH–CH]), 135.38 (C6H5–CH]CH–
CH–), 133.67 (C6H5–CH]CH–CH–), 122.58 (–C–C]O), 40.50
A mixture of (5Z)-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-
(–NCH2CH2CH2–), 30.80 (–NCH2CH2CH2–), 29.69 (–NCH2CH2- dione (3a) (0.92 g, 4.0 mmol), ethyl chloroacetate (0.53 mL,
CH2–), 130.06, 129.01, 127.68 and 122.75 ppm (C, arom); mass: 5.0 mmol) and anhydrous potassium carbonate (2.76 g, 20.0
351 (M+) and 353 (M+ + 2).
mmol) in acetone (50.0 mL) was reuxed for 24 h. The solvent
(5Z)-3-Butyl-5-[(E)-3-phenylallylidene]-thiazolidine-2,4-dione was removed under reduced pressure to obtain the residue. The
(4u). Yield: 81.5%; mp 142–144 ꢂC; Rf-value: 0.68 (CHCl3- residue was poured onto crushed ice with stirring. The sepa-
: MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3018 (CH arom), 2929 (CH rated product was ltered, washed with water and dried. It was
aliph), 1734 (4-CO), 1672 (2-CO) and 1601 (C]C); 1H NMR crystallized from methanol to yield pure 3b.
Yield: 85.0%; mp 126–128 C;23 Rf-value: 0.44 (CHCl3-
ꢂ
(CDCl3): d 0.92 (t, 3H, –NCH2CH2CH2CH3),1.30 (m, 2H,
–NCH2CH2CH2CH3), 1.59 (m, 2H, –NCH2CH2CH2CH3), 3.70 (t,
: MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 2993 (CH aliph), 1737 (CO
2H, –NCH2CH2CH2CH3), 6.69 (m, 1H, C6H5–CH]CH–CH–, J ¼ ester), 1692 (4-CO), 1608 (2-CO) and 1216 (C–O); 1H NMR
11.48 Hz), 7.02 (d, 1H, C6H5–CH]CH–CH–, J ¼ 15.28 Hz), 7.33 (CDCl3): d 1.27 (t, 3H, –CH2CH3), 4.20 (q, 2H, –COOCH2), 4.43 (s,
(m, 3H, CH arom), 7.49 (m, 2H, CH arom) and 7.58 ppm (d, 1H, 2H, –CH2COO–), 6.69 (t, 1H, C6H5–CH]CH, J ¼ 11.46 Hz), 7.04
C6H5–CH]CH–CH], J ¼ 11.68 Hz); 13C NMR (CDCl3): d 167.34 (d, 1H, C6H5–CH]CH, J ¼ 15.28 Hz), 7.36 (t, 3H, CH arom), 7.50
(–C]O, TZD), 165.79 (–C]O, TZD), 143.52 (C6H5–CH]CH– (d, 2H, CH arom), 7.59 ppm (d, 1H, CH]CH–CH]; J ¼ 11.68
CH]), 135.48 (C6H5–CH]CH–CH–), 133.11 (C6H5–CH]CH– Hz); 13C NMR (CDCl3): d 166.90 (–CH2COO–), 166.32 (–C]O,
CH–), 122.91 (–C–C]O), 41.79 (–NCH2CH2CH2CH3), 29.82 TZD), 164.92 (–C]O, TZD), 144.28 (C6H5–CH]CH), 135.38
(–NCH2CH2CH2CH3), 19.97 (–NCH2CH2CH2CH3), 13.63 (CH]CH–CH]), 134.16 (C6H5–CH]CH), 122.36 (–C–C]O),
(–NCH2CH2CH2–CH3), 129.93, 128.98, 127.61 and 123.09 ppm 62.14 (–COOCH2–), 42.08 (–NCH2COO–), 14.11 (–COOCH2CH3),
(C, arom); mass: 288 (M+ + 1).
129.04, 127.75 and 122.69 ppm (C, arom).
(5Z)-3-(4-Bromobutyl)-5-[(E)-3-phenylallylidene]-thiazolidine-
2-[(5Z)-{(E)-3-Phenylallylidene}-thiazolidine-2,4-dione-3-yl]-
2,4-dione (4v). Yield: 82.0%; mp 99–101 ꢂC; Rf-value: 0.68 acetic acid (4x). A mixture of 3b (1.0 g, 3.15 mmol), glacial acetic
(CHCl3 : MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3021 (CH arom), acid (25.0 mL) and hydrochloric acid (12 N, 5.0 mL) was reuxed
2947 (CH aliph), 1737 (4-CO), 1672 (2-CO), 1605 (C]C) and 735 for 2 h. The solvent was evaporated under reduced pressure to
1
(C–Br); H NMR (CDCl3): d 1.78 (m, 2H, –NCH2CH2CH2CH2–), obtain the residue. The residue was poured onto crushed ice
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108935

View Article Online
RSC Advances
Paper
with stirring. The product obtained was ltered, dried and (IAEC) and conducted according to the ‘Committee for the
crystallized from methanol to yield pure compound 4x.
Purpose of Control and Supervision of Experiments on Animals’
Yield: 72.5%; mp 214–216 ꢂC;24 Rf-value: 0.40 (CHCl3- (CPCSEA) guidelines on the use and care of experimental
: MeOH::9 : 1); IR (KBr): nmax/cmꢁ1 3408 (OH), 2943 (CH aliph), animals.
1727 (CO carboxy), 1691 (4-CO) and 1608 (2-CO); 1H NMR
Drugs and reagents. STZ (Sigma Chemical Co., USA), NA (Hi-
(DMSO-d6): d 4.31 (s, 2H, –CH2COOH), 6.64 (t, 1H, C6H5–CH] Media), Tween-80 (Research-Lab, India), D-glucose (S. D. Fine-
CH–, J ¼ 11.68 Hz), 7.00 (d, 1H, C6H5–CH]CH–, J ¼ 15.24 Hz), Chem. Ltd., India), and glucose oxidase peroxidase diagnostic
7.29 (t, 3H, CH arom), 7.44 (d, 2H, CH arom), 7.51 (d, 1H, CH] enzyme kit (Span Diagnostic Chemicals, India) were used in
CH–CH], J ¼ 11.44 Hz) and 8.70 ppm (br s, 1H, –COOH); 13C anti-hyperglycemic activity. STZ was dissolved in citrate buffer
NMR (DMSO-d6): d 167.63 (–CH2COOH), 166.28 (–C]O, TZD), (pH 4.5) and NA in normal physiological saline.
164.32 (–C]O, TZD), 143.64 (C6H5–CH]CH), 134.84, (C6H5–
Induction of diabetes and determination of serum glucose.
CH]CH–), 133.35 (C6H5–CH]CH), 121.93 (–C–C]O), 41.61 T2DM was induced in overnight fasted mice by a single intra-
(–NCH2COO–), 129.56, 128.43 and 127.23 ppm (C, arom); mass: peritoneal (i.p.) injection of STZ (45 mg kgꢁ1 body weight)
312 (M+ + Na).
15 min aer the i.p. administration of 110 mg kgꢁ1 body weight
of NA. Animals were fed with glucose solution (5%) for 12 h to
avoid hypoglycaemia. The collected blood samples were placed
in eppendorffs tube (1.5 mL). The serum was separated by
centrifugation maintained at 4 ꢂC and run at speed of 7000 rpm
for 15 min. Serum (10.0 mL) and working reagent (GOD/POD)
Protein tyrosine phoshatase-1B (PTP-1B) assay
The PTP-1B drug discovery assay kit (Enzo Life Sciences) is
a colorimetric, non-radioactive assay designed to measure the
phosphatase activity of puried PTP-1B. The kit composed of
human, recombinant PTP-1B (residues 1–322; MW ¼ 37.4 kDa),
expressed in E. coli. The detection of free phosphate released
is based on the classic malachite green assay and offers
the advantages of convenient, single step detection and excel-
lent sensitivity, without radioactivity. The [pTyr 1146] phos-
phorylated-peptide, corresponding to the sequence 1142–1153
of the insulin receptor b-subunit domain that must be auto-
phosphorylated to achieve full receptor kinase activation, was
used as substrate for the inhibition assays. This ‘activation loop’
is the target for regulators of PTP-1B. All assays were performed
(1.0 mL) were mixed and incubated for 15 min at 37 C.26 The
ꢂ
absorbance of sample (As) and standard (Astd) provided by
manufacturer were measured against blank at 505 nm. Glucose
was estimated by using the formula:
Glucose (mg dLꢁ1) ¼ As/Astd ꢄ 100
whereas As ¼ sample reading; Astd ¼ standard reading.
The elevated glucose level in serum determined at 72 h
conrmed hyperglycaemia. Animals with blood glucose
concentration more than 250 mg dLꢁ1 were selected for the
study.27a,b
Acute study. In acute study, animals were fasted overnight
and the fasting serum glucose (SG), 0 h, levels were calculated.
All the compounds were administered at a xed dose of 30 mg
kgꢁ1 body weight orally (homogenized suspension in 0.5%
carboxy methyl cellulose (CMC) and permissible amounts of
Tween 80). Animals of vehicle treated group were given an equal
amount of 0.5% CMC and those of control group were kept as
such. Blood samples were removed from all animals at 2, 4, 6
and 24 h by retro orbital puncture method and percentage
reduction in SG was calculated with respect to control group. A
10% reduction in serum glucose level versus control group was
considered as a positive screening result.25
Sub-acute study. In sub-acute study, animals were fasted
overnight and the fasting SG, 0 day, levels were calculated. Now
the compounds were administered for a xed dose of 30 mg kgꢁ1
orally homogenized suspension in 0.5% CMC and permissible
amounts of Tween 80 for 7 days at a xed time. Aer 7th day,
blood samples were removed from all animals and percentage
change in SG was calculated. The data obtained were analyzed by
one-way ANOVA followed by Dunnett test. The results were
expressed as mean ꢃ standard error of mean (SEM) for each
group, p < 0.001 was considered as statistically signicant.
Effect on body weight in diabetic mice. During the study
period of 7 days, the mice were weighed and their body weights
were recorded. From this data, mean change in body weight and
standard error of the mean (SEM) were calculated.
at 30 C in a 96-well microtitre plate.24
ꢂ
Assay buffer (35.0 mL) was added to each well in microtitre
plate and warmed to 30 C. Test sample/inhibitors (10.0 mL) at
ꢂ
various concentrations were added to appropriate well. PTP-1B
enzyme solution (5.0 mL) was added to each well and enzymatic
reaction was initiated by adding warmed substrate (50.0 mL)
followed by incubation at 30 ꢂC for 30 min. Aer incubating
wells for desired duration, reaction was terminated by addition
of BIOMOL RED™ reagent (BML-KI468) (25.0 mL). Mixing was
carried out thoroughly by repeated pipetting with care to avoid
ꢂ
producing bubbles. Color was allowed to develop at 30 C for
20 min. The amount of inorganic phosphate released from the
peptide was determined by measuring the absorbance at
620 nm on a micro plate-reading spectrophotometer and the
IC50 values were calculated by a regression analysis of the linear
portion of the inhibition curves.25
Anti-hyperglycaemic activity using STZ-nicotinamide induced
diabetic mice model
Animals. Male albino mice of Laca strain bred were used in
the present anti-diabetic screening. They were housed (six mice
ꢂ
per cage) under standard (25 ꢃ 2 C, 60–70% humidity) labo-
ratory conditions, maintained on a 12 h natural day–night cycle,
with free access to standard food and water. Animals were
acclimatized to laboratory conditions before the experiment. All
the experimental protocols regarding animal studies were
approved by the ‘Institutional Animal Ethical Committee’
108936 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
Histopathology of mice pancreas. The isolated pancreas
RSC Advances
of them aer completion of docking and the similar poses were
removed keeping the best scoring one. The clusters were ranked
through compare the conformation of the lowest binding
energy in each cluster. The lowest binding free energy poses
were selected for the analysis of the docking results including
various intermolecular interactions with PTP-1B, and for
generation of 3D-QSAR model.1a,3,30 The lowest binding free
energy poses were aligned using substituent based alignment
techniques shown in Fig. 9. Among the synthesized
cinnamylidene-thiazolidinedione derivatives, compound 4w
showed most potent PTP-1B inhibitory potential. Therefore, it
was selected as standard reference compound for alignment
purpose. All the aligned structures were converted in .cssr le
were trimmed into small pieces and preserved in 10.0%
formalin for 24 h. Specimens were cut in section of 3–5 mm in
thickness and stained by hematoxyline–eosin stain. The spec-
imen was mounted by disterene phthalate xylene (DPX). The
photomicrographs of each tissue section were observed using
cell imaging soware for life science microscopy (Olympus so
imaging solution GmbH, Munster, Germany). Pancreatic tissue
was processed for Gomori staining for morphology of pancre-
atic b cells.28
3D-QSAR study
Data set and biological activity. The synthesized format using VEGA ZZ soware. SOMFA program readily takes
cinnamylidene-thiazolidinedione derivatives (4p–x) having up .cssr le format for developing QSAR models. Superimposi-
inhibitory activity against PTP-1B were taken to develop 3D- tion of all the aligned structures onto the reference compound
QSAR based pharmacophoric model. All the molecules were 4w is depicted in Fig. 11.
kept into training set. The negative logarithm of IC50 (desig-
nated as pIC50, values in mM) against PTP-1B was taken to model was developed using self-organizing molecular eld
arrange the data in ascending and linear manner (Table 6).
analysis (SOMFA) approach based on generation of grids from
Generation of 3D-QSAR model. In present work, 3D-QSAR
Molecular modelling, docking and alignment. For the the molecules under consideration. It is a validated 3D-QSAR
present study, Molegro Virtual Docker (MVD 2013.6.0),29a VLife technique developed by Robinson and co-workers.33 Master
MDS 3.5,29b SOMFA 2.0.029c and VEGA ZZ29d programs were grid maps generate intrinsic molecular properties, such as the
employed to develop 3D-QSAR model. The chemical structures molecular shape and electrostatic potential which is used in
of all the data set ligands along with ertiprotab were sketched, developing the nal model using suitable statistical analysis
subjected to energy minimization using molecular mechanics approach.3 Further, grid maps obtained from 3D-QSAR will be
(MM2) and adopted for re-optimization using the Hamiltonian helpful in identifying the fundamental structural features of the
approximations Austin model 1 (AM1) method available in the cinnamylidene-thiazolidinedione derivatives that will be help-
MOPAC module of Chem3D Ultra 8.0 until the root mean ful in the exploration of pharmacophoric requirements that are
square (RMS) gradient attains a value smaller than 0.001 kcal mandatory for a chemical entity to be an imminent, potent and
ꢀꢁ1
molꢁ1
A
in both the optimization techniques.1a,3,30 Geomet- specic inhibitor of PTP-1B with reduced toxicity and improved
rical optimizations included the solvent effect i.e. implicit bioavailability.
solvent environment.3 The nal active conformation search was
Initially, structures in .cssr formats were loaded in the 3D-
performed by docking all the compounds with PTP-1B (PDB QSAR soware along with biological activity (pIC50) against
ꢀ
entry: 2QBS) using MVD. The 2QBS is a well-established crystal PTP-1B. 3D-QSAR models with a 40 ꢄ 40 ꢄ 40 A grid originating
ꢀ
structure of PTP-1B given by W. Xu by using X-ray diffraction at (ꢁ20, ꢁ20, ꢁ20) with a resolution of 0.5 A were generated
method at 2.10 A resolutions.31 Structural evaluations as well as around the aligned molecules.3 Only training set molecules
ꢀ
structural validation1a,3,32a,b and the structural description1b of were used in developing the 3D-QSAR model. 3D-QSAR model
the target protein (PTP-1B) including its recognition sites have was generated for electrostatic and steric properties against
been reported in previous studies. While performing molecular PTP-1B using alignment scheme depicted in Fig. 9. The gener-
docking, both the protein and ligand molecules were imported ated model was used for predicting the electrostatic and steric
into the workspace. All the crystallographic water molecules properties of synthesized cinnamylidene-thiazolidinedione
were removed from the protein during import process. Further,
protein and ligands were subjected to molecules preparation.
The option ‘detect cavities’ in the preparation window was used
to identify cavities (active sites) within the enzyme PTP-1B.
During this computational procedure, the maximum numbers
ꢀ
of cavities were xed to 5, grid resolution 0.80 A and probe size
ꢀ
1.2 A. The binding radius, grid resolution and maximum iter-
ꢀ
ꢀ
ations parameters were set to 15 A, 0.3 A and 1500, respectively;
while the other parameters were set as default. The docking
algorithm was set to simplex evolution population size 50,
ꢀ
RMSD thresholds 1.00 A for cluster similar poses, RMSD
ꢀ
threshold 1.00 A for ignore similar poses (for multiple runs
only) and 10 independent runs were conducted, each of these
runs was returning to a single nal solution (pose). Only nega-
Fig. 11 Superimposition of all the molecules on the template
tive lowest-energy representative cluster was returned from each structure.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108937

View Article Online
RSC Advances
Paper
Table 7 ADME screening data of compounds 4p–x
derivatives. Electrostatic and steric properties were combined
using partial least square (PLS) technique to develop statisti-
cally reliable 3D-QSAR model.
H-Bond
acceptor
H-Bond
donor
Rotatable
Compound
Mol. wt
log P
bonds
Statistical analysis of generated 3D-QSAR model. PLS
methodology was used for the analysis of generated 3D-QSAR
model. PLS correlates the electrostatic and steric properties of
cinnamylidene-thiazolidinedione derivatives with the inhibi-
tory activities against PTP-1B. PLS algorithm was used in union
with leave one out cross validation to develop nal 3D-QSAR
model. The electrostatic and steric potentials were used inde-
pendent variables while pIC50 values were used as dependent
variables in user dened PLS regression analysis in the V-Life
package to derive 3D-QSAR model. Statistical measures ob-
tained from PLS analysis i.e. cross-validated correlation coeffi-
cient (Q2), correlation coefficient (r2), standard error of estimate
(S-value) and Fischer statistics (F-test) serves as an indicator for
a trustworthy QSAR model. Q2 an internal validation indicator
and r2 an external indicator takes up values in the range from 1,
suggesting a perfect model, to less than 0 where errors of
prediction are greater than the error from assigning each
compound mean activity of the model. For an acceptable and
reliable QSAR model, the value of Q2 and r2 should be greater
than 0.5 and 0.6, respectively. For statistically reliable model, S-
value should be closer to 0 but should not be greater than 0.5.
Further, the model is acceptable if the F-test is above
a threshold value. The larger the value of F, greater is the
probability that QSAR model is statistically signicant.3,34
Interpretation of above said statistical parameters of PLS model
is not informative in terms of explaining the behavior. 3D-QSAR
generated master grid maps best explain the behavior of the
molecule in terms of steric and electrostatic properties by Grid-
Visualizer program. These maps are three dimensional and
represent the region in space where steric and electrostatic eld
interactions are responsible for the observed variations in the
desired pharmacological activity. Individual compound in data
set can be visualized in these grids and variation in activity can
be best explained by the master grids.21
4p
4q
4r
4s
4t
4u
4v
4w
4x
293.78
338.23
273.37
307.81
352.26
287.40
366.29
365.42
289.32
2
2
2
2
2
2
2
4
4
0
0
0
0
0
0
0
1
1
2.76
2.82
2.86
2.81
2.87
3.26
3.32
3.52
1.59
6
6
6
7
7
7
8
7
6
mice pancreatic tissue. The binding affinities of compounds
(4p–x) with PTP-1B in terms of docking scores were determined
using molecular docking simulations and the in silico binding
affinities were in accordance with biological results i.e.
compound 4w showed highest MolDock as well as re-rank
scores. The various binding interactions (electrostatic, hydro-
phobic, H-bond and steric) of 4w within the vicinity of PTP-1B
were explored, most importantly it shows steric interaction
with Cys215 which is a part of signature motif of PTP-1B and
essential for catalysis of PTP-1B.
Although all the synthesized compounds (4p–x) were
screened for drug likeness or ADME screening based on “Lip-
inski's Rule of Five”, which state that for becoming a drug
candidate a molecule should have molecular weight < 500, H-
bond donor # 5, H-bond acceptor # 10, log P < 5, rotatable
bonds # 10 (Table 7), however further experiments are neces-
sary for evaluation of toxicity and bioavailability parameters of
compound 4w. Moreover, the lead generated from 3D-QSAR can
be used for further development of potent PTP-1B inhibitors
bearing cinnamylidene thiazolidinedione scaffold and devoid
of side effects associated with conventional therapies.
Acknowledgements
Conclusions
The authors gratefully acknowledge Indian Council of Medical
Research (ICMR) for providing the Senior Research Fellowship
(SRF). ST pleased to acknowledge University Grants Commis-
sion (UGC), New Delhi, Govt. of India for UGC-BSR Research
Startup Grant [No. F.20-16(3)/2012(BSR)]. We are also thankful
to Dr Rene Thomsen for permission to use MVD soware.
In search of PTP-1B inhibitors bearing small molecular scaffold
and devoid of side effects associated with glitazones; design,
synthesis, evaluation of in vitro PTP-1B inhibitory potential as
well as anti-hyperglycaemic potential of N3-substituted
cinnamylidene-thiazolidinedione derivatives (4p–x) in STZ-NA
induced diabetic mice model was performed. The in vitro
assay results show that compound bearing methyl benzoic acid
at N3 (4w) exhibits most potent PTP-1B inhibitory activity with
IC50 ꢀ 6.52 mM and comparable anti-hyperglycaemic potential
with insulin sensitizer pioglitazone. In addition, the effect of
treatment on body weight of diabetic mice with compounds 4p–
x was matched with pioglitazone treatment, and observed that
treatment with 4w has marginal increase in body weight but
weight gain was less as compared to pioglitazone. The potency
of compound 4w was tested by histopathological analysis of
mice pancreas; it shows similar pattern of intact cell membrane
and clearly visible nucleus as of pioglitazone treated diabetic
Notes and references
1 (a) S. K. Verma, T. Rajpoot, M. K. Gautam, A. K. Jain and
S. Thareja, Lett. Drug Des. Discovery, 2016, 13, 295–300; (b)
S. Thareja, S. Aggarwal, T. R. Bhardwaj and M. Kumar,
Med. Res. Rev., 2012, 32, 457–517.
2 Diagnosis and Classication of Diabetes Mellitus, http://
www.care.diabetesjournals.org/content/diacare/32/
Supplement_1/S62.full.pdf, accessed October 2015.
3 S. K. Verma and S. Thareja, RSC Adv., 2016, 6, 33857–33867.
108938 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
4 S. Suzen and E. Buyukbingol, Curr. Med. Chem., 2003, 10, 15 (a) M. S. Malamas, J. Sredy, I. Gunawan, B. Mihan,
1329–1352.
D. R. Sawicki, L. Seestaller, D. Sullivan and B. R. Flam, J.
Med. Chem., 2000, 43, 995–1010; (b) R. Maccari, R. Ottana,
C. Curinga, M. G. Vigorita, D. Rakowitz, T. Steindl and
T. Langer, Bioorg. Med. Chem., 2005, 13, 2809–2823; (c)
A. P. Combs, E. W. Yue, M. Bower, P. J. Ala, B. Wayland,
B. Douty, A. Takvorian, P. Polam, Z. Wasserman, W. Zhu,
M. L. Crawley, J. Pruitt, R. Sparks, B. Glass, D. Modi,
E. McLaughlin, L. Bostrom, M. Li, L. Galya, K. Blom,
M. Hillman, L. Gonneville, B. G. Reid, M. Wei, M. Becker-
Pasha, R. Klabe, R. Huber, Y. Li, G. Hollis, T. C. Burn,
R. Wynn, P. Liu and B. Metcalf, J. Med. Chem., 2005, 48,
6544–6548; (d) R. Maccari, P. Paoli, R. Ottana,
M. Jacomelli, R. Ciurleo, G. Manao, T. Steindl, T. Langer,
M. G. Vigorita and G. Camici, Bioorg. Med. Chem., 2007, 15,
5137–5149; (e) R. Ottana, R. Maccari, R. Ciurleo, P. Paoli,
M. Jacomelli, G. Manao, G. Camici, C. Laggner and
T. Langer, Bioorg. Med. Chem., 2009, 17, 1928–1937; (f)
B. R. Bhattarai, B. Kae, J. S. Hwang, D. Khadka,
S. M. Lee, J. S. Kang, S. W. Ham, I. O. Han, H. Park and
H. Cho, Bioorg. Med. Chem. Lett., 2009, 19, 6161–6165; (g)
B. R. Bhattarai, B. Kae, J. S. Hwang, S. W. Ham,
K. H. Lee, H. Park, I. O. Han and H. Cho, Bioorg. Med.
Chem. Lett., 2010, 20, 6758–6763; (h) Z. Liu, Q. Chai,
Y. Y. Li, Q. Shen, L. P. Ma, L. N. Zhang, X. Wang, L. Sheng,
J. Y. Li, J. Li and J. K. Shen, Acta Pharmacol. Sin., 2010, 31,
1005–1012; (i) Z. Wang, Z. Liu, W. Lee, S. N. Kim, G. Yoon
and S. H. Cheon, Bioorg. Med. Chem. Lett., 2014, 24, 3337–
3340.
5 J. E. Shaw, R. A. Sicree and P. Z. Zimmet, Diabetes Res. Clin.
Pract., 2010, 87, 4–14.
6 M. Feldhammer, N. Uetani, D. Miranda-Saavedra and
M. L. Tremblay, Crit. Rev. Biochem. Mol. Biol., 2013, 48,
430–445.
7 (a) L. D. Klaman, O. Boss, O. D. Peroni, J. K. Kim,
J. L. Martino, J. M. Zabolotny, N. Moghal, M. Lubkin,
Y. B. Kim, A. H. Sharpe, A. Stricker-Krongrad,
G. I. Shulman, B. G. Neel and B. B. Kahn, Mol. Cell. Biol.,
2000, 20, 5479–5489; (b) S. Vakili, S. S. Ebrahimi,
A. Sadeghi, S. Gorgani-Firuzjaee, M. Beigy, P. Pasalar and
R. Meshkani, Mol. Med. Rep., 2013, 7, 211–216; (c)
T. O. Johnson, J. Ermolieff and M. R. Jirousek, Nat. Rev.
Drug Discovery, 2002, 1, 696–709; (d) N. K. Tonks, FEBS
Lett., 2003, 546, 140–148; (e) J. Montalibet and
B. P. Kennedy, Drug Discovery Today: Ther. Strategies, 2005,
2, 129–135; (f) S. Koren and I. G. Fantus, Best Pract. Res.,
Clin. Endocrinol. Metab., 2007, 21, 621–640; (g) D. E. Moller,
Nature, 2001, 414, 821–827.
8 (a) M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish,
S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. Himms-
Hagen, C. Chan, C. Ramachandran, M. J. Gresser,
M. L. Tremblay and B. P. Kennedy, Science, 1999, 283,
1544–1548; (b) C.
M. Rondinone, J.
M. Trevillyan,
J. Clampit, R. J. Gum, C. Berg, P. Kroeger, L. Frost,
B. A. Zinker, R. Reilly, R. Ulrich, M. Butler, B. P. Monia,
M. R. Jirousek and J. F. Waring, Diabetes, 2002, 51, 2405–2411.
9 S. Zhang and Z. Y. Zhang, Drug Discovery Today, 2007, 12, 16 (a) B. S. Lakshmi, S. Sujatha, S. Anand, K. N. Sangeetha,
373–381.
R. B. Narayanan, C. Katiyar, A. Kanaujia, R. Duggar,
Y. Singh, K. Srinivas, V. Bansal, S. Sarin, R. Tandon,
S. Sharma and S. Singh, J. Diabetes, 2009, 1, 99–106; (b)
E. J. Moran, S. Sarshr, J. F. Cargill, M. M. Shahbaz, A. Lio
and A. M. M. Mjalli, J. Am. Chem. Soc., 1995, 117, 10787–
10788; (c) H. Fu, J. Park and D. Pei, Biochemistry, 2002, 41,
10700–10709.
10 (a) A. J. Nichols, R. D. Mashal and B. Balkan, Drug Dev. Res.,
2006, 67, 559–566; (b) S. Lee and Q. Wang, Med. Res. Rev.,
2007, 27, 553–573; (c) A. J. Barr, Future Med. Chem., 2010,
2, 1563–1576; (d) M. E. Sobhia, S. Paul, R. Shinde,
M. Potluri, V. Gundam, A. Kaur and T. Haokip, Expert
Opin. Ther. Pat., 2012, 22, 125–153; (e) R. He, L. F. Zeng,
Y. He, S. Zhang and Z. Y. Zhang, FEBS J., 2013, 280, 731–750. 17 R. Maccari, R. Ottana, R. Ciurleo, P. Paoli, G. Manao,
11 (a) Z. Y. Zhang and S. Y. Lee, Expert Opin. Invest. Drugs, 2003,
12, 223–233; (b) D. V. Erbe, S. Wang, Y. L. Zhang,
G. Camici, C. Laggner and T. Langer, ChemMedChem, 2009,
4, 957–962.
K. Harding, L. Kung, M. Tam, L. Stolz, Y. Xing, S. Furey, 18 S. Shrestha, B. R. Bhattarai, K. J. Chang, K. H. Lee and
A. Qadri, L. D. Klaman and J. F. Tobin, Mol. Pharmacol., H. Cho, Bioorg. Med. Chem. Lett., 2007, 17, 2760–2764.
2005, 67, 69–77; (c) S. Shrestha, B. R. Bhattarai, H. Cho, 19 (a) R. Sakhardande, V. Kulkarni, N. Sachan and
J. K. Choi and H. Cho, Bioorg. Med. Chem. Lett., 2007, 17,
2728–2730.
12 (a) M. Mclane, H. Wolfe, A. Turner and I. Ruiz-White,
M. Nimbalkar, Eur. Pat., WO2010082212A2, 2010; (b)
N. Sachan, S. Thareja, R. Agarwal, S. S. Kadam and
V. M. Kulkarni, Int. J. PharmTech Res., 2009, 1, 1625–1631.
Keystone Symposia on Molecular and Cellular Biology, 20 R. Thomsen and M. H. Christensen, J. Med. Chem., 2006, 49,
Silverthorne, Colorado, 2009; (b) Z. Qin, X. Zhou, 3315–3321.
N. R. Pandey, H. A. Vecchiarelli, C. A. Stewart, X. Zhang, 21 S. Thareja, T. Rajpoot and S. K. Verma, Steroids, 2015, 95, 96–
D. C. Lagace, J. M. Brunel, J. C. Beique, A. F. R. Stewart,
M. N. Hill and H. H. Chen, Neuron, 2015, 85, 1319–1331.
13 L. F. Iversen, K. B. Moller, A. K. Pedersen, G. H. Peters,
A. S. Petersen, H. S. Andersen, S. Branner, S. B. Mortensen
and N. P. Moller, J. Biol. Chem., 2002, 277, 19982.
103.
22 (a) Y. Hu, T. Xie, K. Fu, H. Kang, P. Wei and H. Huang,
Heterocycles, 2009, 78, 757–761; (b) D. Hardej, C. R. Ashby,
N. S. Khadtare, S. S. Kulkarni, S. Singh and T. Talele, Eur.
J. Med. Chem., 2010, 45, 5827–5832.
14 (a) S. Mudaliar and R. R. Henry, Annu. Rev. Med., 2001, 52, 23 S. M. Rida, H. M. Salama, I. M. Labouta and Y. S. A. Ghany,
239–257; (b) J. Patel, R. J. Anderson and E. B. Rappaport,
Diabetes, Obes. Metab., 1999, 1, 165–172.
Pharmazie, 1985, 40, 727–728.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 108928–108940 | 108939

View Article Online
RSC Advances
Paper
24 E. V. Tkachenko, R. B. Lesik and V. D. Lukyanchuk, Farming 30 S. K. Verma, S. K. Sharma and S. Thareja, Lett. Drug Des.
Magazine, 2004, 5, 88–93. Discovery, 2015, 12, 284–291.
25 PTP-1B Tyrosine Phosphatase Drug Discovery Kit Manual, 31 Protein data bank, http://www.rcsb.org/pdb/explore/
http://www.enzolifesciences.com/leadmin/enzo/BML/
ak822.pdf, accessed October 2014.
26 S. L. Badole and S. L. Bodhankar, Eur. J. Pharmacol., 2010,
632, 103–109.
27 (a) J. C. Huralikuppi, Phytother. Res., 1991, 5, 217–223; (b)
E. U. Etuk, Agric. Biol. J. North Am., 2010, 1, 130–134.
28 G. L. Gomori, Am. J. Clin. Pathol., 1950, 20, 665–666.
29 (a) Molegro Virtual Docker, version 6.0.0, CLC Bio, 8200
Aarhus N, Denmark, 2012; (b) VLife Molecular Design Suite
explore.do?structureId¼2QBS, accessed October 2015.
32 (a) D. P. Wilson, Z. K. Wan, W. X. Xu, S. J. Kirincich,
B. C. Follows, D. Joseph-McCarthy, K. Foreman,
A. Moretto, J. Wu, M. Zhu, E. Binnun, Y. L. Zhang,
M. Tam, D. V. Erbe, J. Tobin, X. Xu, L. Leung, A. Shilling,
S. Y. Tam, T. S. Mansour and J. Lee, J. Med. Chem., 2007,
50, 4681–4698; (b) R. Ottana, R. Maccari, J. Mortier,
A. Caselli, S. Amuso, G. Camici, A. Rotondo, G. Wolber and
P. Paoli, Eur. J. Med. Chem., 2014, 71, 112–127.
(VLife MDS), version 3.5, VLife Sciences Technologies Pvt. 33 D. D. Robinson, P. J. Winn, P. D. Lyne and W. G. Richards, J.
Ltd., Pune, India, 2013; (c) D. D. Robinso, SOMFA2, version Med. Chem., 1999, 42, 573–583.
2.0.0, Computational Chemistry Research Group, Oxford 34 A. Golbraikh and A. Tropsha, J. Mol. Graphics Modell., 2002,
University, UK, 2006; (d) VEGA ZZ, version 3.0.3.18, Drug
Design Laboratory, Department of Pharmaceutical
Sciences, University of Milan, Milan, Italy, 2014.
20, 269–276.
108940 | RSC Adv., 2016, 6, 108928–108940
This journal is © The Royal Society of Chemistry 2016