Synthesis, biological evaluation and molecular docking studies of novel 3,5-disubstituted 2,4-thiazolidinediones derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.102993

A series of thirteen novel 2,4-thiazolidinedione derivatives were synthesized through three step reaction procedure. The title compounds were synthesized by Knoevenagel condensation at the 5th position of the 2,4-thiazolidinedione ring. Various physicochemical and spectral studies were conducted to characterize the synthesized derivatives including- IR, Mass, ¹H NMR, ¹³C NMR and elemental analysis. The derivatives were screened for in vivo anti diabetic, in vivo anti-inflammatory and in vitro free radical scavenging activities by carrageenan induced rat paw edema method, alloxan induced diabetes in wistar rats method and FRAP (ferric reducing antioxidant power) method respectively. Some of the derivatives emerged out as potent antidiabetic, anti inflammatory and free radical scavenging agents. Molecular docking was carried out to investigate some possible structural insights into the potential binding patterns of the most potent anti-diabetic molecules NB7,NB12 and NB13 with the active sites of target PPARγ (PDB ID: 2PRG) using MOE software. Dichloro derivative compound NB-7 has shown great potential in the present study as it not only has maximum antidiabetic activity but also possess excellent anti-inflammatory and antioxidant potential.

Full text of DOI:10.1016/j.bioorg.2019.102993

Accepted Manuscript
Synthesis, biological evaluation and molecular docking studies of novel 3,5-
disubstituted 2,4-thiazolidinediones derivatives
Alok Ranjan Srivastava, Rohit Bhatia, Pooja Chawla
PII:
S0045-2068(18)31300-2
https://doi.org/10.1016/j.bioorg.2019.102993
102993
DOI:
Article Number:
Reference:
YBIOO 102993
Bioorganic Chemistry
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Accepted Date:
10 November 2018
26 March 2019
17 May 2019
Please cite this article as: A. Ranjan Srivastava, R. Bhatia, P. Chawla, Synthesis, biological evaluation and molecular
docking studies of novel 3,5-disubstituted 2,4-thiazolidinediones derivatives, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.102993
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Synthesis, biological evaluation and molecular docking studies of novel 3,5-
disubstituted 2,4-thiazolidinediones derivatives
Alok Ranjan Srivastavaa, Rohit Bhatiab, Pooja Chawlaa,b*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Babu Banarasi Das National
Institute of Technology and Management, Lucknow-226028, Uttar Pradesh, India.
b
Department of Pharmaceutical Chemistry, Indo-Soviet Friendship College of Pharmacy, Moga-
142001, Punjab, India
* Corresponding author: Prof. (Dr.) Pooja Chawla, Department of Pharmaceutical Chemistry,
Indo-Soviet Friendship College of Pharmacy, Moga-142001, Punjab, India.
Voice Contact: +91-1636-324201 (office), +918057952150 (mobile),
Fax: +91-1636-324201
E-mail: pvchawla@gmail.com
ABSTRACT
A series of thirteen novel 2,4-thiazolidinedione derivatives were synthesized through three step
reaction procedure. The title compounds were synthesized by Knoevenagel condensation at the
5th position of the 2,4-thiazolidinedione ring. Various physicochemical and spectral studies were
1
13
conducted to characterize the synthesized derivatives including- IR, Mass, H-NMR, C-NMR
and elemental analysis. The derivatives were screened for in vivo anti diabetic, in vivo anti-
inflammatory and in vitro free radical scavenging activities by carrageenan induced rat paw
edema method, alloxan induced diabetes in wistar rats method and FRAP (ferric reducing
antioxidant power) method respectively. Some of the derivatives emerged out as potent
antidiabetic, anti inflammatory and free radical scavenging agents. Molecular docking was
carried out to investigate some possible structural insights into the potential binding patterns of
the most potent anti-diabetic molecules NB7,NB12 and NB13 with the active sites of target
PPARγ (PDB ID: 2PRG) using MOE software. Dichloro derivative compound NB-7 has shown
great potential in the present study as it not only has maximum antidiabetic activity but also
possess excellent anti-inflammatory and antioxidant potential.
Keywords: Thiazolidinedione, anti diabetic, anti inflammatory, anti oxidant, structure activity
relationship, docking

GRAPHICAL ABSTRACT
1. Introduction
Diabetes is rapidly spreading life threatening disease with a global prevalence of 171 million in
year 2000 and is expected to increase to 366 million by 2030 [1]. Diabetes is a group of diseases
characterized by chronic hyperglycemia due to deficiency of insulin action. The deficiency of
insulin action, a common basis of diabetes, leads to characteristic abnormalities in the
metabolism of carbohydrates, lipids, and proteins which results in chronic complications. Type 2
diabetes (T2D) results from insulin resistance, a condition in which cells fail to use insulin
properly, sometimes combined with an absolute insulin deficiency. Despite the abundance of
available anti-diabetes therapies, a considerable number of patients with type 2 diabetes continue
to have relatively poor glycemic control and are at risk for macrovascular and microvascular
disease [2]. Inflammation and injury to the arterial wall in the peripheral or coronary vascular
system is thought to result in atherosclerosis which affects the arteries that supply the heart, brain
and lower extremities. These also contribute in increased risk of developing cardiovascular
disease (CVD) [3]. High glucose results in oxidative stress which leads to formation of free
radicals mainly reactive oxygen species (ROS). This results in microvascular complications
(damage to smaller blood vessels throughout the body) in retina, renal glomerulus and peripheral
nerves [4]. So in hyperglycemic condition increased level of inflammation and oxidative stress
are chief contributing factors in development of diabetic complications. Many researchers have
proposed that the treatment with antioxidants ameliorates diabetic complications (Fig.1) [5-8].
Contribution of inflammation in development of insulin resistance (IR) [9] and other
complications like diabetic nephropathy (DN) [10] and diabetic retinopathy (DR) [11] has been
proved by many researchers. So if the drug which is used for antidiabetic therapy possesses anti-
inflammatory activity as well as is capable of scavenging free radicals generated during

hyperglycemic conditions, can prove highly effective not only in treatment of diabetes but also
for complications arising due to high glucose level in the body.
Fig. 1. High glucose level in diabetes and its complications.
Thiazolidnediones (TZDs) constitute an important class of heterocyclic compounds which
possess significant anti-diabetic activity along with various other activities like
immunostimulatory, antiarthritic, oncostatic, anti-inflammatory, antioxidant etc. TZD is insulin
sensitizing moiety that acts by selectively binding to the nuclear transcription factor peroxisome-
proliferator–activated receptor γ (PPAR γ). Studies have shown the association of PPAR γ with
the inflammation [12-14] and free radical generation [15-16]. Even some glitazones are shown to
have potential to check the inflammation and free radical generation [12,17-18]. So targeting
inflammation and oxidative stress generated due to free radicals can prove beneficial in anti-
diabetic therapy as well as to increase the efficacy of treatment.
Our study is based on developing new compounds by utilizing the multifunctionality of TZD
nucleus for diabetes which may prove more efficacious than currently available drugs for
diabetes. Pharmacophore thiazolidine-2,4-dione is essential for activity; Linker region: flat linker
region is favourable for activity; Aryl substituent: group attached to left side of linker provides
large area for stearic interaction with the hydrophobic region of receptor. N-terminal (head
portion) of the molecule is hydrophilic/cationic site as it forms several H-bonds with nuclear
receptor PPARγ; tail portion having arylidine substituent is responsible for stearic interaction
with the receptor due to its large area, lipophilicity is favoured in this portion of molecule (Fig.
2). As the developed compound also targets the inflammation along with oxidative stress
generated inside cell, it may be of high benefit in diabetes treatment. N-terminal of TZD nucleus
is cationic/hydrophilic site which forms hydrogen-bond with Ser 289 of PPARγ receptor, it also
forms several hydrogen bonds with critical residues of the activation function helix 2 (AF-2
helix) of PPARγ including His 323, His 449 and Tyr 473 [19-21]. So hydrogen presence is
necessary near N-terminal for agonistic activity of PPARγ-ligand. N-terminal of TZD is
substituted with allyl group (R1) by replacing the solitary hydrogen present at 3rd position, as it is

more acidic owing to presence of two carbonyl groups at 2nd and 4th position of TZD which may
contribute to gastric irritation. It is evident that substitution of alkyl group at N-terminal results
in decreased activity of compound [22]. Allyl group is selected for replacing the hydrogen at 3rd
position of TZD as it decreases the acidity of hydrogen alongwith increase in the lipophilicity by
maintaining the hydrophilicity as well. The log p value of N-allyl group is in between N-H and
N-C6H5 group. Selected allyl group may increase the solubility profile of compound as well as its
activity profile should also increase as PPARγ receptors are mostly present in adipose tissue [23-
24]. As observed during literature review arylidene substitution on 5th position is most beneficial
21, 25-26
for plasma glucose (PG) level reduction [21,25-26].
Electron donating groups at 4th
position in arylidene substituted thiazolidinediones (ATZDs) produce a significant reduction in
PG [25]. Various substitutents (R2) (electron donating as well as withdrawing) like -OCH3, -Cl, -
F, -NO2, -OH are attached at different positions in arylidene group attached to 5th position of
TZD to check the activity profile of the compounds.
Fig. 2. Regions of molecule required for binding to PPARγ

S
S
S
O
y
x
O
C
+
ClCH2COOH
H2N NH2
HN
N
RBr
O
O
R
(II)
thiazolidine-2,4-dione
2-chloroacetic acid
thiourea
(R= CH2=CHCH2-)
(I)
(b)
(a)
ArCHO
O
z
Ar
S
N
O
R
(III)
Scheme 1. Reagents: (x) HCl; (y) EtOH+water; (z) sodium acetate, hot glacial acetic acid.
2. Results and discussion
2.1 Chemistry
The chemicals required were obtained from Hi-media Chem. Ltd, SD-Fine Ltd. and Sigma
Aldrich Pvt. Ltd and were used as such. A total of thirteen derivatives (NB1-NB13) were
synthesized via three step reaction process. The synthetic strategy utilized for preparation of
TZD derivatives is outlined in scheme 1. Thiourea gives thiazoldine-2,4-dione (I) with
chloroacetic acid [27]. N-allylation of thiazoldine-2,4-dione (I) with allylbromide furnished 3-
allylthiazoldine-2,4-dione (II) [28], which on which on Knoevenagel condensation with various
aromatic aldehydes yielded 5-arylidene derivatives (III)[29] (figure 3). Melting points were
determined using open capillary tube melting point apparatus and are uncorrected. Reaction
progress was monitored by performing thin layer chromatography on silica gel G plates, using
iodine vapours and UV chamber as visualizing agents. After physical characterization, the
compounds were subjected to spectral analysis. Proton Nuclear Magnetic resonance spectra were
recorded on Bruker ultra shield (400 MHz) spectrometer and chemical shifts are reported in parts
1
per million (δ value) from TMS (δ 0 ppm for H NMR) as an internal standard. Coupling
constant are given in Hertz. Mass spectra were recorded on a JEOL GC-HRMS instrument using
ESI. Infrared spectra were taken on Perkin Elmer (FTIR) spectrometer and values are expressed
in cm-1.

H3CO
H3CO
H3CO
O
O
O
H3CO
N
CH2CHCH2
N CH2CHCH2
O
N
CH2CHCH2
S
S
H3CO
S
O
O
NB3
NB1
NB2
O
N
O
N
O
O
HO
CH2CHCH2
CH2CHCH2
N CH2CHCH2
O
H3CO
OCH3S
NB4
Cl
S
S
O
NB5
NB6
H3CO
HO
O
N
O
O
O
Cl
F
CH2CHCH2
N CH2CHCH2
O
N CH2CHCH2
O
S
NB7
NO2
S
S
NB8
NB9
OCH3
Cl
O
O
O
N CH2CHCH2
O
N CH2CHCH2
O
N CH2CHCH2
O
S
S
S
NB10
NB11
NB12
O
Cl
N CH2CHCH2
O
S
NB13
Fig. 3. Structures of synthesized final compounds III (NB1-NB13).
2.2. Pharmacological studies
The synthesized derivatives of thiazolidinedione were subjected to pharmacological screening
for anti-diabetic activity against alloxan induced diabetes in wistar rats [30-33] (Table 1), anti-
inflammatory activity using carrageenan induced rat paw edema model [34-35] (Table 2) and in
vitro antioxidant activity using FRAP method [36] (Table 3).
2.2.1. Antidiabetic Activity
Results of antidiabetic activity show that halogen containing compound showed excellent
activity than others, especially compounds having chloro group at ortho and para position of
arylidene ring showed best activity among all the derivatives. NB7 is found to possess maximum

antidiabetic activity among all derivatives. NB2 having three –OCH3 groups showed least
potency in lowering the blood glucose level (Fig. 4). From the result it may be concluded that
groups like halide attached to molecule which may contribute in lipophilicity of the molecule
showed excellent activity. This may be due to the fact that increased lipophilicity may help in
easy penetration of compound into PPARγ receptor which is mainly present in high
concentration in adipose tissue.
Table 1
Blood glucose levels of alloxan induced diabetic rats.
Treatment
(mg/kg, p.o.)
Blood Glucose Level (mg/dL)
0 hour 1 hour 3 hour
251±3.13 253±4.72 252±4.40 254±6.14
6 hour
Group I (Control)
Group II (Pioglitazone) 276±4.84 213±3.44 114±5.49 89±3.26
Group III (NB1)
Group III (NB2)
Group III (NB3)
Group III (NB4)
Group III (NB5)
Group III (NB6)
Group III (NB7)
Group III (NB8)
Group III (NB9)
Group III (NB10)
Group III (NB11)
Group III (NB12)
Group III (NB13)
248±6.39 222±6.36 189±7.29 138±9.49
260±4.24 225±7.38 191±5.93 157±6.71
308±6.25 241±6.83 201±7.35 150±4.19
305±9.72 205±5.72 194±3.79 155±8.48
281±7.39 233±4.59 181±5.04 143±5.93
253±5.62 232±6.52 185±6.86 152±9.11
301±5.43 211±4.74 166±7.08 108±3.95
264±5.73 225±6.14 189±4.93 145±7.39
245±7.30 208±5.93 161±4.73 129±8.61
244±7.28 208±6.92 178±5.62 133±4.05
261±3.87 230±6.49 172±5.26 149±4.11
298±6.34 213±7.39 170±4.82 122±9.24
283±8.32 238±6.43 167±10.32 111±5.48

350
300
250
200
150
100
50
0 hour
1 hour
3 hour
6 hour
0
Fig. 4. Blood glucose level of different compounds at 0, 1, 3 and 6 hours
2.2.2. Antiinflammatory Activity
The entire series of investigated compounds exhibited moderate to good anti-inflammatory
activity comparable to reference drug diclofenac sodium (10 mg/kg). Compounds NB7, NB13
and NB9 showed significant anti-inflammatory activity at third hour being 59.5%, 57.1% and
54.7% respectively closer to that of standard drug diclofenac sodium (64.2%).
The data of in vivo anti-inflammatory activity of synthesized novel compound evidence that
presence of group at 4th position of arylidene substituent attached to TZD is necessary to exhibit
good anti-inflammatory activity. Halide derivative compounds of TZD showed good activity
among all the derivatives. Even compounds NB7 and NB13 showed more activity than standard
compound during 0, 1 and 2 hrs. Compound NB7 showed maximum activity (59.5%) among all
the synthesized compounds quite closer to standard compound (64.2%). (Fig. 5).
2.2.3. Antioxidant Activity
Data obtained for antioxidant activity of synthesized compounds shows that compounds bearing
electron donating groups like –OH and –OCH3 showed greater reducing power in comparison to
other derivatives. NB8 showed good reduction potential which may be attributed to presence of –
OH and –OCH3 groups which are capable of donating electrons to electron deficient species
(Table 3). The absorption at 100 µg/ml for the thirteen samples was compared with ascorbic
acid for the plot was calculated by the equation: y = 0.0084x + 0.0025 (R2 = 0.994) (Fig. 6),
it was expressed as AAE (ascorbic acid equivalent) means that reducing power of
100µg/ml of each compound is equivalent to reducing power of µg of ascorbic acid or
expressed as µg AAE/mg of compound. Data obtained reveals that compound bearing electron
donating groups like –OH and –OCH3 showed greater reducing power in comparison to other
derivatives. NB8 showed good reduction potential which may be attributed to presence of –OH
and –OCH3 groups which are capable of donating electrons. Compounds bearing electron
withdrawing group (NB10) and unsubstituted derivative (NB5) showed least potency which may
be outcome of their least ability to donate electron.

Anti-inflammatory activity of compounds
70
60
50
40
30
20
10
0
Fig. 5. Anti-inflammatory activity profile of compounds
Table 2
Percent Inhibition value of compounds against carrageenan-induced edema in rats.
Compounds
Control
Normal
Volume(ml)
Paw Mean Paw Volume ± SEM(ml) and % inhibition
Time After Carrageenan Injection
0 hr
1hr
2hr
3hr
0.015±0.005
0.012±0.005
0.011±0.004
0.011±0.005
0.010±0.005
0.010±0.005
0.011±0.004
0.011±0.005
0.010±0.006
0.010±0.005
0.011±0.004
0.048±0.008
0.048±0.008 0.045±0.005 0.042±0.005
Diclofenac
sodium
NB1
0.042±0.004
(12.5%)
0.042±0.005
(12.5%)
0.045±0.005
(6.2%)
0.043±0.006
(10.4%)
0.040±0.005
(16.7%)
0.041±0.005
(14.6%)
0.037±0.005
(19.9%)
0.038±0.005
(20.8%)
0.036±0.006 0.033±0.004 0.015±0.004
(25.0%)
0.037±0.005 0.032±0.004 0.026±0.006
(22.9%) (28.9%) (38.1%)
0.039±0.007 0.030±0.004 0.027±0.005
(18.7%) (33.3%) (35.7%)
0.039±0.005 0.032±0.005 0.031±0.008
(18.7%) (28.9%) (26.2%)
0.038±0.005 0.035±0.003 0.029±0.004
(20.8%) (22.2%) (31.0%)
0.036±0.005 0.032±0.005 0.027±0.005
(25.1%) (28.9%) (35.7%)
0.035±0.006 0.027±0.007 0.024±0.005
(27.1%) (40.0%) (42.8%)
0.032±0.005 0.026±0.005 0.017±0.004
(33.3%) (42.2%) (59.5%)
0.037±0.004 0.029±0.005 0.023±0.005
(22.9%) (35.6%) (45.2%)
0.038±0.005 0.034±0.005 0.019±0.005
(20.8%) (24.4%) (54.7%)
(26.7%)
(64.2%)
NB2
NB3
NB4
NB5
NB6
NB7
NB8
NB9
0.040±0.005
(16.7%)
0.040±0.005
(16.7%)

NB10
NB11
NB12
NB13
0.011±0.006
0.010±0.004
0.011±0.005
0.010±0.005
0.041±0.006
(14.6%)
0.040±0.005
(16.7%)
0.040±0.006
(16.7%)
0.043±0.004
(10.4%)
0.032±0.005 0.027±0.004 0.024±0.003
(33.3%) (40.0%) (42.9%)
0.038±0.005 0.033±0.006 0.028±0.004
(20.8%) (26.7%) (33.3%)
0.035±0.005 0.030±0.007 0.022±0.005
(27.1%) (33.3%) (47.6%)
0.033±0.005 0.028±0.006 0.018±.007
(31.2%) (37.8%) (57.1)
Table 3
Concentration of samples (100 µg/ml) equivalent to ascorbic acid (µg/ml).
S. No.
Test Sample
NB1
Abs. at 700 nm
(100µg/ml)
0.206
Concentration Equivalent to
AAE
24.35
1
2
NB2
0.184
0.123
0.155
0.107
0.305
0.283
0.361
0.117
0.071
0.180
0.210
0.222
21.73
14.35
18.15
12.56
36.01
33.39
42.68
13.63
8.03
3
NB3
4
NB4
5
NB5
6
NB6
7
NB7
8
NB8
9
NB9
10
11
12
13
NB10
NB11
NB12
NB13
21.13
24.70
26.13

Standard ascorbic acid curve
1
0.5
0
0
20
40
60
80
100
120
Concentration(µg/mL)
Fig. 6. Standard linearity curve of ascorbic acid.
3. Molecular Docking Studies
Molecular docking was carried out to investigate some possible structural insights into
the potential binding patterns of the most potent anti-diabetic molecules NB7,NB12 and NB13
with the active sites of target PPARγ (PDB ID: 2PRG) using MOE software. The various
possible interactions and orientations were investigated and compared with the binding patterns
of pioglitazone. Compounds NB7, NB12 and NB13 displayed docking scores with values of -
11.6930, -10.1553 and -11.1008 which were almost comparable to the reference drug
pioglitazone (-12.8116). Compounds NB7 showed two hydrogen bond interactions with amino
acid residues Ser A289 (2.86 Å) and His A323 (2.39 Å). The 2D and 3D binding patterns of
compound NB 7 have been depicted in Fig. 7. One keto group of compound NB12 showed
hydrogen bonding interaction (2.86 Å) with water (HOH 604) in association to Ser 342 (3.02 Å);
whereas the other keto group showed hydrogen bonding interaction (2.17 Å) with another water
molecule (HOH 496) in association to Arg (2.84 Å) (Fig. 8). Compound NB13 forms hydrogen
bond with Ser A342 (2.54 Å) as depicted in Fig. 9. Pioglitazone showed a binding pattern by
making a hydrogen bond with Glu A343 (2.81 Å) and another interaction with HOH 604 (3.02
Å) associated with Ser 342. The interactions and biding pattern of pioglitazone to the receptor
has been presented in Fig. 10.
From the findings it is evident that the pattern of binding in the active site of PPARγ is almost
similar to that of pioglitazone which is a well-established therapeutic candidate against PPARγ.
Further it is also supported by the in vivo activity results in rats where almost equal activity to
that of reference drug has been observed. Therefore, the synthesized compound may serve as
new leads for the drug development against diabetes.

(a)

(b)

(c)
Fig. 7. (a) 2D interactions of NB 7 with PPARγ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor.
(a)

(b)
(c)

Fig. 8. (a) 2D interactions of NB 12 with PPARγ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor.
(a)

(b)

(c)
Fig. 9. (a) 2D interactions of NB 13 with PPARγ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor.

(a)

(b)
(c)

Fig. 10. (a) 2D interactions of pioglitazone with PPARγ (b) 3D pose of embedded ligand inside
the pocket (c) Possible interactions between ligand and receptor.
4. Conclusion
Despite the abundance of available antidiabetes therapies, a considerable number of patients with
type 2 diabetes continue to have relatively poor glycemic control and are at risk for
macrovascular and microvascular disease. This study can provide an excellent diabetic control to
the patient as well as the synthesized compounds can also check the complications arising due to
diabetes which is proved to be result of increased inflammation and oxidative stress in
hyperglycemic condition. Dichloro derivative compound NB-7 has shown great potential in the
present study as it not only possesses maximum antidiabetic activity but also has excellent anti-
inflammatory and antioxidant potential. Docking studies were carried out on the most potent
compounds to get an insight to the drug-receptor interactions. The compound NB7 (docking
score -11.6930) revealed similar fashion of binding pattern as in case of marketed anti-diabetic
drug pioglitazone (docking score -12.8116). A general SAR is prepared for the synthesized
thiazoldinedione derivative on the basis of data obtained from various activities. Compounds
bearing halide group at arylidine substituent attached to 5th position of TZD pharmacophore
showed best antidiabetic activity while those having groups like –OCH3 and –OH showed least
activity. Presence of groups at 4th position of arylidene substituent is essential for reduction in
level of inflammation. Compounds having methoxy group at 2nd or 3rd position in arylidine
substituent results in loss of anti-inflammatory activity. Electron donating groups like -OH and -
OCH3 play a central in radical scavenging potential of compounds, while those having electron
withdrawing or unsubstituted derivative showed least activity. Lipophilicity of compounds
played a major role in reduction of blood glucose level. Molecular docking studies were
performed using MOE version 2008.10 software. The 3D structure of PPARγ (PDB ID: 2PRG)
was procured from protein data bank and was prepared by deletion of ligand, addition of
hydrogens, polar hydrogens followed by preparation of dummies using site finder wizard. The
ligands were energy minimized by selecting force field MMFF94x, Austin model 1 (AM 1) with
gradient value of 0.0001 kcal/mol and were saved as mdb format. The docking simulations were
predicted by docking the prepared ligands in the binding pocket of PPARγ and results were
displayed in database viewer. 2D and three 3D interactions of ligands with the receptor was
predicted using compute tool. The docking results were compared to the standard drug
pioglitazone. The employed docking protocol was validated by calculating the RMSD value.
5. Experimental section
Melting points were determined by open capillary method with electric melting point apparatus
and are uncorrected. All the reagents for synthesis were obtained from Sigma Aldrich Pvt. Ltd.,
Rankem, Thomas Baker, SD fine chemical Ltd., Himedia and were used as such. Thin layer
chromatography was used to monitor the progress of reactions, and the spots on TLC plates were
visualized by iodine vapours, and UV irradiation at 254 nm. IR spectra were recorded on a FT-IR
(Shimadzu-8400S) spectrometer using KBr pellets from 400-4000 cm-1. 1H NMR spectra were
recorded on JEOL AL 300 FT-NMR spectrophotometer (400 MHz) in CDCl3 using
tetramethylsilane as an internal standard. The chemical shifts were expressed in δ ppm. The
following notations expressed the peak types in the spectra: singlet (s), doublet (d), triplet (t) and
multiplet (m). ESI mass spectra were recorded using Agilent 6530 Accurate-Mass Q-TOF mode.

Elemental analysis (C, H, N) was carried out using X-SUPREME 8000-EDXRF analyser and
were found within range of ±0.4% of theoretical values.
5.1 Chemistry
5.1.1 Preparation of thiazolidine-2,4-dione (I)
A mixture of 56.5 gm (0.6 mol) of 2-chloroacetic acid in 60 ml of water and 45.6 gm (0.6 mol)
of thiourea in 60 ml of water was stirred for 15 min. The reaction mixture was cooled and white
precipitates were formed. To this reaction mixture 60 ml of concentrated hydrochloric acid was
added with the help of dropping funnel. Then the mixture was refluxed for 30-40 hrs. After
completion of reaction, the whole content was transferred in to crushed ice, white solid product
was obtained. Filtered and washed the solid with water. Recrystallized from ethanol and thus
obtained a pure thiazolidine-2,4-dione (I). The progress of reaction was monitored by TLC using
chloroform and ethylacetate (60:40). Yield = 89%; IR (KBr) 1737.74, 1670.24, 3481.72,
1340.43, 3051.18, 1390.85 cm-1; MS, ESI+: 117.01[M+H]+.
5.1.2 Preparation of 3-allylthiazolidine-2,4-dione (II)
Mixture of 60 % ethyl alcohol (EtOH) and 40 % water was stirred for 10 min. Thiazolidine-2,4-
dione (0.01 mol) (I) was added to above mixture and stirred for 30 min. Allylbromide (0.012
mol) was added to resulting mixture and stirred for 20 hrs at room temperature. Product was
separated as oil which was taken up by chloroform added to it and washed with water. Obtained
product was dried over sodium sulphate. Resulting product was recrystallized from ethanol to
obtain 3-allylthiazolidine-2,4-dione (II). The progress of reaction was monitored by TLC using
ethylacetate and toluene (7:3). Yield = 73%; IR (KBr) 1742.32, 1671.28, 1337.41, 3068.12,
3123.37, 984.13, 923.57, 1637.19 cm-1; MS, ESI+: 157.49 [M+H]+.
5.1.3 General method for preparation of compound (III)
Refluxed the mixture of 1.57 gm of 3-allylthiazolidine-2,4-dione (II) and 0.01 mol of substituted
benzaldehyde on an oil bath in the presence of 3.38 gm (0.04 mol) of sodium acetate and 10 ml
of hot glacial acetic acid. The reaction was completed within 30 min. Filtered and washed it with
water. On drying the solid product was obtained and recrystallized from ethanol. The reaction
was monitored by using thin layer chromatography in chloroform and methanol (90:10) as
solvent and iodine vapours as visualizing agent.
5.1.3.1. 3-allyl-5-(4-methoxybenzylidine)thiazolidine-2,4-dione (NB1)
Yield = 81%; IR (KBr) 1728.42, 1668.56, 1335.82, 2965.37, 3122.56, 825.73, 1635.10 cm-1; 1H
NMR (DMSO) δ 3.834 (s, 3H, CH3), 5.194-5.191 (d, 1H,CH, J=1.2), 5.216-5.213 (d, 2H,CH,
J=1.2), 5.227-5.224 (d, 1H,CH, J=1.2), 5.879-5.858 (m, 1H,CH), 6.942-6.939 (d, 1H,ArH,
J=1.2), 7.622-7.619 (d, 1H,ArH, J=1.2), 7.954 (s, 1H, CH); MS, ESI+: 275.05 [M+H]+.
5.1.3.2. 3-allyl-5-(3,4,5-trimethoxybenzylidine)thiazolidine-2,4-dione (NB2)
Yield = 85%; IR (KBr) 1726.77, 1669.42, 1369.73, 2971.77, 3129.63, 883.10, 1622.44 cm-1; 1H
NMR (DMSO) δ 3.834 (s, 3H, CH3), δ 5.194-5.191 (d, 1H,CH, J=1.2), δ 5.216-5.213 (d, 2H,CH,

J=1.2), δ 5.227-5.224 (d, 1H,CH, J=1.2), δ 5.879-5.858 (m, 1H,CH), δ 6.784-6.781 (d, 1H, ArH,
J=1.2), δ 7.954 (s, 1H, CH); MS, ESI+: 335.10 [M+H]+.
5.1.3.3. 3-allyl-5-(3-methoxybenzylidine)thiazolidine-2,4-dione (NB3)
Yield = 79%; IR (KBr) 1719.57, 1678.32, 1342.45, 2973.62, 3133.49, 784.95, 1635.48 cm-1; 1H
NMR (DMSO) δ 3.834 (s, 3H, CH3), δ 5.194-5.191 (d, 1H,CH, J=1.2), δ 5.216-5.213 (d, 2H,CH,
J=1.2), δ 5.227-5.224 (d, 1H,CH, J=1.2), δ 5.879-5.858 (m, 1H,CH), δ 6.874-6.869 (d, 1H, ArH,
J=2.0), δ 7.164-7.158 (d, 1H, ArH, J=2.4), δ 7.592-7.585 (t, 1H, ArH), δ 7.954 (s, 1H, CH); MS,
ESI+: 275.00 [M+H]+.
5.1.3.4. 3-allyl-5-(2,3-dimethoxybenzylidine)thiazolidine-2,4-dione (NB4)
Yield = 83%; IR (KBr) 1743.30, 1681.44, 1348.39, 2960.48, 3115.94, 819.21, 1632.26 cm-1; 1H
NMR (DMSO) δ 3.834 (s, 3H, CH3), δ 5.194-5.191 (d, 1H,CH, J=1.2), δ 5.216-5.213 (d, 2H,CH,
J=1.2), δ 5.227-5.224 (d, 1H,CH, J=1.2), δ 5.879-5.858 (m, 1H,CH), δ 6.763-6.760 (d, 1H, ArH,
J=1.2), δ 7.157-7.151 (t, 1H, ArH), δ 7.224-7.221 (d, 1H, ArH, J=1.2), δ 8.220 (s, 1H, CH); MS,
ESI+: 305.07 [M+H]+.
5.1.3.5. 3-allyl-5-benzylidinethiazolidine-2,4-dione (NB5)
1
Yield = 74%; IR (KBr) 1709.28, 1664.58, 1338.70, 3126.76, 859.84, 1625.72 cm-1; H NMR
(DMSO) δ 5.194-5.191 (d, 1H,CH, J=1.2), δ 5.216-5.213 (d, 2H,CH, J=1.2), δ 5.227-5.224 (d,
1H,CH, J=1.2), δ 5.879-5.858 (m, 1H,CH), δ 7.329-7.326 (d, 1H, ArH, J=1.2), δ 7.481-7.475 (t,
1H, ArH), δ 7.621-7.618 (d, 1H, ArH, J=1.2), δ 7.954 (s, 1H, CH); MS, ESI+: 245.10 [M+H]+.
5.1.3.6. 3-allyl-5-(4-hydroxybenzylidine)thiazolidine-2,4-dione (NB6)
Yield = 80%; IR (KBr) 1722.35, 1670.26, 1328.21, 3372.84, 3124.38, 862.74, 1624.83, 1253.89
1
cm-1; H NMR (DMSO) δ 5.198-5.195 (d, 1H,CH, J=1.2), δ 5.213-5.210 (d, 2H,CH, J=1.2), δ
5.221-5.219 (d, 1H,CH, J=1.2), δ 5.876-5.855 (m, 1H,CH), δ 6.658-6.655 (d, 1H, ArH, J=1.2), δ
7.561-7.558 (d, 1H, ArH, J=1.2), δ 9.496 (s, 1H, OH); ESI+: 261.07. [M+H]+.
5.1.3.7. 3-allyl-5-(2,4-dichlorobenzylidine)thiazolidine-2,4-dione (NB7)
1
Yield = 89%; IR (KBr) 1726.52, 1659.20, 1328.46, 3141.97, 831.06, 1632.71 cm-1; H NMR
(DMSO) δ δ 5.193-5.190 (d, 1H,CH, J=1.2), δ 5.217-5.214 (d, 2H,CH, J=1.2), δ 5.223-5.220 (d,
1H,CH, J=1.2), δ 5.882-5.861 (m, 1H,CH), δ 7.324-7.321 (d, 1H, ArH, J=1.2), δ 7.489 (s, 1H,
ArH), δ 7.619-7.616 (d, 1H, ArH, J=1.2), δ 8.227 (s, 1H, CH); MS, ESI+: 313.03 [M+H]+.
5.1.3.8. 3-allyl-5-(4-hydroxy-3-methoxybenzylidine)thiazolidine-2,4-dione (NB8)
Yield = 89%; IR (KBr) 1738.41, 1672.86, 1341.26, 3368.11, 3109.28, 860.24, 1629.78, 1251.80
1
cm-1; H NMR (DMSO) δ 3.834 (s, 3H, CH3), δ 5.197-5.194 (d, 1H,CH, J=1.2), δ 5.216-5.213
(d, 2H,CH, J=1.2), δ 5.222-5.219 (d, 1H,CH, J=1.2), δ 5.874-5.853 (m, 1H,CH), δ 6.993-6.990
(d, 1H, ArH, J=1.2), δ 7.123 (s, 1H, ArH), δ 7.167 (s, 1H, ArH), δ 7.954 (s, 1H, CH), δ 9.834 (s,
1H, OH); MS, ESI+: 291.23 [M+H]+.
5.1.3.9. 3-allyl-5-(4-fluorobenzylidine)thiazolidine-2,4-dione (NB9)

1
Yield = 86%; IR (KBr) 1728.31, 1666.10, 1362.24, 3100.37, 841.08, 1642.57 cm-1; H NMR
(DMSO) δ 5.197-5.194 (d, 1H,CH, J=1.2), δ 5.213-5.210 (d, 2H,CH, J=1.2), δ 5.222-5.219 (d,
1H,CH, J=1.2), δ 5.874-5.853 (m, 1H,CH), δ 7.193-7.190 (d, 1H, ArH, J=1.2), δ 7.723-7.720 (d,
1H, ArH, J=1.2), δ 7.951 (s, 1H, CH); MS, ESI+: 263.03 [M+H]+.
5.1.3.10. 3-allyl-5-(2-nitrobenzylidine)thiazolidine-2,4-dione (NB10)
Yield = 92%; IR (KBr) 1731.78, 1658.65, 1365.52, 3048.06, 782.53, 1636.11, 1518.13 cm-1; 1H
NMR (DMSO) δ 5.193-5.189 (d, 1H,CH, J=1.6), δ 5.216-5.213 (d, 2H,CH, J=1.2), δ 5.228-5.223
(d, 1H,CH, J=2.0), δ 5.873-5.852 (m, 1H,CH), δ 7.793-7.877 (t, 1H, ArH), δ 7.895-7.887 (t, 1H,
ArH), δ 8.009-8.004 (d, 1H, ArH, J=2.0), δ 8.214-8.210 (d, 1H, ArH, J=1.6), δ 8.513 (s, 1H,
CH); MS, ESI+: 290.06 [M+H]+.
5.1.3.11. 3-allyl-5-(2-methoxybenzylidine)thiazolidine-2,4-dione (NB11)
Yield = 84%; IR (KBr) 1741.35, 1668.42, 1340.17, 2972.18, 3113.47, 879.01, 1640.34 cm-1; 1H
NMR (DMSO) δ 3.834 (s, 3H, CH3), δ 5.198-5.195 (d, 1H,CH, J=1.2), δ 5.213-5.210 (d, 2H,CH,
J=1.2), δ 5.221-5.219 (d, 1H,CH, J=1.2), δ 5.876-5.855 (m, 1H,CH), δ 6.941-6.937 (d, 1H, ArH,
J=1.6), δ 6.968-6.963 (d, 1H, ArH, J=2.0), δ 7.227-7.221 (t, 1H, ArH), δ 7.663-7.657 (d, 1H,
ArH, J=2.4), δ 8.228 (s,1H, CH); MS, ESI+: 275.09 [M+H]+.
5.1.3.12. 3-allyl-5-(2-chlorobenzylidine)thiazolidine-2,4-dione (NB12)
1
Yield = 90%; IR (KBr) 1729.11, 1655.38, 1336.42, 3108.42, 758.09, 1632.24 cm-1; H NMR
(DMSO) δ 5.194-5.191 (d, 1H,CH, J=1.6), δ 5.216-5.212 (d, 2H,CH, J=1.6), δ 5.227-5.222 (d,
1H,CH, J=2.0), δ 5.879-5.858 (m, 1H,CH), δ 7.274-7.268 (t, 1H, ArH), δ 7.305-7.299 (t, 1H,
ArH), δ 7.360-7.357 (d, 1H, ArH, J=1.2), δ 7.457-7.453 (d, 1H, ArH, J=1.6), δ 8.219 (s, 1H,
CH); MS, ESI+: 279.24 [M+H]+.
5.1.3.13. 3-allyl-5-(4-chlorobenzylidine)thiazolidine-2,4-dione (NB13)
1
Yield = 88%; IR (KBr) 1727.25, 1653.82, 1341.19, 3128.10, 791.49, 1634.24 cm-1; H NMR
(DMSO) δ 5.193-5.180 (d, 1H,CH, J=1.2), δ 5.219-5.214 (d, 2H,CH, J=2.0), δ 5.224-5.220 (d,
1H,CH, J=1.6), δ 5.882-5.861 (m, 1H,CH), δ 7.450-7.446 (d, 1H, ArH, J=1.6), δ 7.689-7.684 (d,
1H, ArH, J=2.0), δ 7.951 (s, 1H, CH); MS, ESI+: 279.40 [M+H]+.
5.2. Molecular docking studies
Molecular docking studies were performed using MOE version 2008.10 software. The 3D
structure of PPARγ (PDB ID: 2PRG) was procured from protein data bank and was prepared by
deletion of ligand, addition of hydrogens, polar hydrogens followed by preparation of dummies
using site finder wizard. The ligands were energy minimized by selecting force field MMFF94x,
Austin model 1 (AM 1) with gradient value of 0.0001 kcal/mol and were saved as mdb format.
The docking simulations were predicted by docking the prepared ligands in the binding pocket of
PPARγ and results were displayed in database viewer. 2D and three 3D interactions of ligands
with the receptor was predicted using compute tool. The docking results were compared to the
standard drug pioglitazone. The employed docking protocol was validated by calculating the
RMSD value.

5.3. Biological activity
5.1 Biology
Rats were housed in groups of six in clean poly acrylic cages. Rice husk as bedding material of
the cages was changed every day. The animals were maintained under natural day and night
cycle. Animals were acclimatized for one week to the laboratory conditions before starting the
experiment. Animals were given standard pellet diet and allowed water ad libitum. All the
animal studies were conducted in accordance with the guidelines for animal care. The protocol
was approved by the Institutional Animal Ethics Committee (IAEC) (Reg No.
BBDNITM/IAEC/Clear/04/2012).
5.1.1 Antidiabetic Activity
Male Albino Wistar rats, weighing about 150-250gm were used for antidiabetic studies. Animals
were divided into following groups consisting of six rats. Group 1: Consisted of healthy rats and
received 0.5 ml of 0.9% normal saline; Group 2: Treated with standard drug Pioglitazone at a
dose of 10 mg/kg; Group 3-15: All the test compounds (NB1-NB13) were given as oral dose of
10 mg/kg suspended in carboxy methyl cellulose (CMC) by using oral gastric gavages. Rats
were fasted overnight and next day anaesthetized with ethyl ether and hyperglycemia was
induced by a single intra peritoneal injection of freshly prepared Alloxan (120 mg/kg body
weight dissolved in 0.9% w/v saline) solution. Diabetes (blood glucose level more than 250
mg/dl) was confirmed on 4th day by determining the blood glucose concentration using a
Glucometer (Accu Chek). The blood glucose levels of the animals were measured at 0, 1st, 3rd
and 6th hour by withdrawing 0.1-0.2 ml of blood from tail vein under mild ether anesthesia [30-
33].
5.1.2 Anti-inflammatory Activity
Male Albino Wistar rats, weighing about 110-170g were used for anti-inflammatory studies.
Animals were divided in three groups each containing 6 animals. Animals in group 1 recieves the
vehicle CMC, group 2 was given Diclofenac sodium (10 mg/kg, p.o.) and test compounds NB1-
NB13 in group 3 has given 10mg/kg through oral route. All the compounds were administered
30 minutes prior to carrageenan injection. A mark was put on the leg at the mallaleus region to
facilitate the dipping of the leg to the same level at the second and subsequent times. Acute
inflammation was induced by injecting carrageenan (0.l ml of 1% suspension in 0.5% CMC) in
sub-plantar region and paw volume was measured at 0, 1, 2 and 3hours with the help of
plethysmometer. The mean paw volume at different times was calculated for all groups [34-35].
Percentage reduction in edema volume was calculated by using the formula,
푉푡
푉푐
Percentage reduction = 1 -
× 100
Where, Vc = Volume of the paw of control at time‘t’ and Vt = Volume of the paw of drug treated
at time‘t’.

5.1.3 In vitro Antioxidant Activity
The in vitro antioxidant activities were carried out with FRAP models. In this method four
concentrations (25, 50, 75, 100 µg/ml) of each sample and standard in DMSO were prepared and
mixed (2.5 ml) with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1.0 % potassium ferricyanide
(2.5 ml). The mixture was incubated at 50 ºC for 20 minutes. Aliquots of 10 % trichloro acetic
acid (2.5 ml) were added to the mixture, centrifuged at 5000 rpm for 10 min. The upper layer of
solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride
solution (0.5 ml, 0.1 %) and allowed to stand for 30 minutes in dark to complete the reaction.
The control solution was prepared as above, taking water in place of samples. The absorbance
was measured at 700 nm [36]. All tests and analysis were run in triplicates and the result
obtained was averaged and expressed as mean ± standard deviation.
5.1.4 Molecular Docking Studies
Molecular docking studies were performed using MOE version 2008.10 software. The 3D
structure of PPARγ (PDB ID: 2PRG) was procured from protein data bank and was prepared by
deletion of ligand, addition of hydrogens, polar hydrogens followed by preparation of dummies
using site finder wizard. The ligands were energy minimized by selecting force field MMFF94x,
Austin model 1 (AM 1) with gradient value of 0.0001 kcal/mol and were saved as mdb format.
The docking simulations were predicted by docking the prepared ligands in the binding pocket of
PPARγ and results were displayed in database viewer. 2D and three 3D interactions of ligands
with the receptor was predicted using compute tool. The docking results were compared to the
standard drug pioglitazone. The employed docking protocol was validated by calculating the
RMSD value.
ACKNOWLEDGEMENTS
Authors would like to acknowledge Babu Banarasi Das National Institute of Technology and
Management, Lucknow for providing necessary facilities during research. The authors
acknowledge Central Drugs Research Institute, Lucknow, India for providing the library
facilities and sophisticated analytical instrument facilities.
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