Design, synthesis, in silico molecular docking and biological evaluation of novel oxadiazole based thiazolidine-2,4-diones bis-heterocycles as PPAR-γ agonists

Article abstract of DOI:10.1016/j.ejmech.2014.09.010

A library of novel 1,3,4-oxadiazole and 2-4-thiazolidinedione based bis-heterocycles 7 (a-r) has been synthesized which exhibited significant PPAR-?3 transactivation and blood glucose lowering effect comparable with the standard drugs Pioglitazone and Rosiglitazone. Compounds 7m and 7r did not cause body weight gain and were found to be free from hepatotoxic and cardiotoxic side effects. Compounds 7m and 7r increased PPAR-?3 gene expression by 2.10 and 2.00 folds, respectively in comparison to the standard drugs Pioglitazone (1.5 fold) and Rosiglitazone (1.0 fold). Therefore the compounds 7m and 7r may be considered as potential candidates for development of new antidiabetic agents.

Full text of DOI:10.1016/j.ejmech.2014.09.010

European Journal of Medicinal Chemistry 87 (2014) 175e185
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, in silico molecular docking and biological evaluation
of novel oxadiazole based thiazolidine-2,4-diones bis-heterocycles as
PPAR-g agonists
,
Syed Nazreen a, Mohammad Sarwar Alam a *, Hinna Hamid a, Mohammad Shahar Yar b,
Syed Shafi a, Abhijeet Dhulap c, Perwez Alam d, M.A.Q. Pasha d, Sameena Bano a,
Mohammad Mahboob Alam a, Saqlain Haider a, Yakub Ali a, Chetna Kharbanda a,
K.K. Pillai e
a Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
c CSIR Unit for Research and Development of Information Products, Pune 411038, India
d Functional Genomics Unit, CSIR-Institute of Genomics & Integrative Biology, New Delhi, India
e Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University) New Delhi 110 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of novel 1,3,4-oxadiazole and 2-4-thiazolidinedione based bis-heterocycles 7 (aer) has been
Received 30 May 2014
Received in revised form
24 August 2014
Accepted 4 September 2014
Available online 16 September 2014
synthesized which exhibited significant PPAR-
g transactivation and blood glucose lowering effect
comparable with the standard drugs Pioglitazone and Rosiglitazone. Compounds 7m and 7r did not
cause body weight gain and were found to be free from hepatotoxic and cardiotoxic side effects. Com-
pounds 7m and 7r increased PPAR-g gene expression by 2.10 and 2.00 folds, respectively in comparison
to the standard drugs Pioglitazone (1.5 fold) and Rosiglitazone (1.0 fold). Therefore the compounds 7m
and 7r may be considered as potential candidates for development of new antidiabetic agents.
Keywords:
2,4-Thiazolidinedione
1,3,4-Oxadiazole
Antidiabetic
© 2014 Elsevier Masson SAS. All rights reserved.
PPAR-
g
Hepatotoxicity
Cardiotoxicity
1. Introduction
promote glucose utilization in peripheral tissues [4]. These are
high-affinity ligands of peroxisome proliferator activated receptor-
Type 2 diabetes is a metabolic disorder which due to insulin
resistance and impaired insulin secretion leads to hyperglycemia.
Patients with type 2 diabetes suffers from several complications
such as neuropathy, nephropathy, retinopathy, cardiovascular dis-
eases and atherosclerosis [1,2]. In normal humans, up to 80% of
insulin-stimulated glucose disposal occurs in the skeletal muscle, a
major site of insulin resistance in type 2 diabetes [3].
g [5]. PPAR-g, a member of a large family of ligand-activated nu-
clear hormone receptors is an important drug target for regulating
glucose metabolism [6e9]. It increases insulin sensitivity in the
adipose, muscle and hepatic tissues [10,11]. It leads to the chan-
neling of fatty acids into adipose tissue and reducing their con-
centration in the plasma and thus alleviating insulin resistance and
improving plasma glucose levels effectively [12e14]. 1,3,4-
oxadiazoles are another important class of heterocyclic com-
pounds which are known for a number of important pharmaco-
logical activities like hypoglycaemic, hypolipidemic, antimicrobial,
antiinflammatory, analgesic, antimitotic and anticonvulsive activ-
ities [15e22].
2,4-thiazolidinediones (TZDs) are an important class of com-
pounds that enhances insulin action (insulin sensitizers) and
Abbreviations: TZD, thiazolidinediones; STZ, streptozotocin; ALT, alanine
transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; RT, reverse
transcription; PCR, polymerase chain reaction.
* Corresponding author.
E-mail addresses: msalam@jamiahamdard.ac.in, msalam5555@gmail.com
(M.S. Alam).
Considering the biological importance of 2,4-thiazolidinediones
and 1,3,4-oxadiazoles, we have conjugated thiazolidinediones with
1,3,4-oxadiazole under one construct through a methylene linkage
to enhance the blood glucose lowering effect and minimize the side
http://dx.doi.org/10.1016/j.ejmech.2014.09.010
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

176
S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
Scheme 1. Synthetic route for novel 1,3,4-oxadiazole-thiazolidine-2,4-diones based bis-heterocycles.
effects. We herein report for the first time, the synthesis and in silico
their cardiotoxic risk evaluation as well as effect on PPAR-
g gene
molecular docking studies of oxadiazole and thiazolidinedione
expression.
based bis-heterocycles 7 (aer) against PPAR-
pounds showing good docking score (>ꢀ7.20) were screened for
their in vitro PPAR- transactivation activity. Compounds 7b, 7h, 7k,
7m and 7r showing significant PPAR- transactivation activity were
further evaluated for their in vivo antidiabetic activity and hepa-
totoxicity. Two compounds 7m and 7r showing the most potent
in vitro and in vivo antidiabetic activity were finally evaluated for
g target. The com-
2. Results and discussion
g
g
2.1. Chemistry
Treatment of different aromatic acids 1 (aee) with MeOH in
presence of a few drops of conc. H2SO4 yielded methyl esters of

S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
177
aromatic acids 2 (aee) which on further reaction with hydrazine
2.3. Biological activities
monohydrate in presence of absolute alcohol afforded aromatic
hydrazides 3 (aee). Knoevenagel condensation of different hydroxy
benzaldehydes 4 (aef) and 2,4-thiazolidinedione (5) in presence of
absolute alcohol and NaOH at ꢀ5 to 0 ꢁC for 10e12 h yielded in-
termediate phenoxy acetic acid based thiazolidinediones 6 (aef).
The compounds showing good glide scores (>ꢀ7.20) in molec-
ular docking study were screened for in vitro PPAR- trans-
activation activity in order to confirm their mode of action. It was
observed from Fig. 2 that compounds 7b, 7m and 7r exhibited
g
Reaction of different aromatic hydrazides
3
(aee) with in-
significant PPAR-g transactivation of 59.81%, 63.78% and 64.67%
termediates 6 (aef) in presence of POCl3 gave corresponding 1,3,4-
oxadiazole-thiazolidinediones based bis-heterocycles 7 (aer) in
good yields (Scheme 1).
respectively in comparison to standard drugs Pioglitazone and
Rosiglitazone which showed 71.94% and 85.27% activation
respectively. Compounds 7h (52.32%), 7i (49.78%), 7k (52.96%) and
1H NMR, 13C NMR and mass spectral data were found to be in
agreement with the proposed structures of all the newly synthe-
sized compounds. Formation of aromatic hydrazides 3 (aee) was
7q (52.41%) showed moderate in vitro PPAR-g transactivation ac-
tivity whereas compound 7c, 7l and 7p did not show good activity.
The active compounds (7b, 7h, 7k, 7m, 7r) were then evaluated for
the blood glucose lowering effect in STZ induced diabetic rats. As
observed from the data of Fig. 3, compounds 7b (139.7 6.12), 7h
confirmed by the presence of signals in a range of
d 9.7e10.12
(eCONHe, s) and
d
4.33e4.52 (eNH2e, s) in the 1H NMR spectra.
The absence of methoxy signals in a range of 3.32e3.91 in 3 (aee)
as seen in the 1H NMR spectra of 2 (aee) further confirmed their
formation. Formation of intermediates 6 (aef) was confirmed by
(135.2
(134.0
6.16), 7k (142.2
5.09) also caused significant lowering in blood glucose
7.18), 7m (139.6
6.40) and 7r
level comparable to standard drugs Pioglitazone (132.0 5.20) and
Rosiglitazone (144.2 6.12). It was observed that the compounds
the presence of the singlets in a range of
d 4.62e4.89, d 7.72e7.74
and
d
11.02e13.07 for OeCH2, exocyclic olefinic and carboxylic
7b, 7h, 7k, 7m and 7r exerted much lower PPAR-g transactivation
activity than Pioglitazone and Rosiglitazone but similar or lower
blood glucose levels than Pioglitazone and Rosiglitazone. The
protons, respectively in their 1H NMR spectra. This data was sup-
ported by their 13C NMR spectral data. The formation of target
compounds 7 (aer) was confirmed by the presence of additional
absence of linear correlation between in vitro PPAR-
g trans-
aromatic protons in a range of
d
7.02e8.35 and OeCH2 protons in a
activation assay and in vivo pharmacological profile in albino rats
may be attributed to several reasons. For example, the test com-
pounds 7b, 7h, 7k, 7m and 7r are administered orally and therefore
absorption, metabolism, excretion, etc. of the test compounds
might have contributed in exerting the significant lowering in
blood glucose level [23]. Also, compounds 7b, 7h, 7k, 7m and 7r
might be exhibiting their antihyperglycemic activity through other
range of
d
4.87e5.67 in their 1H NMR spectra. The absence of car-
boxylic proton signals as seen in the 1H NMR spectra of 6 (aef)
further confirmed their formation. Further structural confirmation
of the target compounds 7 (aer) was provided by the presence of
absorption bands in a range of 1575e1596 cmꢀ1 for C]N stretching
of oxadiazole ring in the IR spectra and the signals in a range of
d
161.80e165.89 for C]N carbons in the 13C NMR spectra of these
mechanisms, in addition to binding to PPAR-g. Similar kind of re-
compounds. Further structural confirmation was done by their
mass spectral data.
sults have been reported by other groups working with TZDs, e.g.,
Reddy et al. (1999) have synthesized TZDs which show superior
euglycemic and hypolipidemic profiles in db/db mice but not sig-
nificant in vitro PPAR-g transactivation activity than the standard
drug troglitazone [23].
The most active compound 7r from the in vivo study was further
tested for oral glucose tolerance as well as for insulin tolerance test
(Fig. 4). Oral glucose tolerance test of compound 7r showed that the
administration of compound 7r causes significant decrease in the
blood glucose levels of diabetic rats at 120 min when compared to
diabetic control rats indicating that glucose tolerance were
2.2. Molecular docking studies
The 3D structures of 7 (aer) were generated using ligand
preparation module of Schrodinger suite and molecular docking
was carried out using the Glide software. Molecular docking studies
were done to provide insights of molecular binding modes of
Table 1
molecules inside the large pocket of PPAR-g receptors. The com-
Docking score and predicted ADME of the synthesized compounds 7 (aer).
pounds were docked against the grid generated by Schrodinger
glide software. Rosiglitazone has been reported to show H-bonding
with TYR 473, HIS 449 & CYS 285. Docking of Rosiglitazone against
the generated grid showed similar docking mode and hydrogen
bonds with RMSD value of 2.8 and hence the generated grid was
validated. In order to analyse the binding pattern and energies of
synthesized compounds, they were docked individually against the
generated grid. All the synthesized molecules docked showed good
binding energies ranging from ꢀ89.4 to ꢀ44.5 kcal/mol. All the
molecules except 7d showed good glide score higher than the
standard drug Rosiglitazone (¡5.77) Compound 7c was found to
form hydrogen bonding with ALA 292, 7l with SER 342, 7r with LYS
261, 7p with ARG 288 and7i and 7q with the water molecule of the
protein residue. Whereas the compounds 7b, 7f, 7h, 7j, 7k and 7m
were found to be aligned perfectly with the hydrophobic pocket of
the protein. The in silico ADME (Absorption, Distribution, Meta-
bolism and Excretion) prediction of the 7(aer) were found to be
within the acceptable range. The calculated glide score, binding
energies and predicted ADME of all the synthesized molecules are
represented in Table 1 and Fig. 1.
S.No.
Compd
s-Score
e-Energy
Log PO/W
PSA
Log S
1
2
3
4
5
6
7
8
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
¡6.79
¡7.43
¡7.72
ꢀ5.70
¡6.92
¡7.12
¡6.65
¡7.75
¡7.74
¡7.07
¡7.20
¡7.26
¡8.29
¡6.80
¡6.89
¡7.31
¡7.70
¡8.42
¡5.77
ꢀ50.37
ꢀ73.83
ꢀ53.76
ꢀ46.35
ꢀ76.51
ꢀ73.69
ꢀ49.26
ꢀ77.95
ꢀ52.27
ꢀ75.60
ꢀ89.45
ꢀ60.13
ꢀ54.49
ꢀ46.68
ꢀ49.79
ꢀ44.54
ꢀ69.34
ꢀ60.76
ꢀ71.55
2.94
2.98
3.48
3.54
3.54
3.48
3.45
3.56
3.69
3.88
4.95
3.81
3.09
3.57
3.64
3.55
3.35
3.91
3.47
116.31
122.21
124.61
115.69
116.93
122.13
116.90
121.74
114.90
117.32
121.67
126.44
114.76
115.26
114.14
121.02
119.39
118.85
94.37
ꢀ4.62
ꢀ4.91
ꢀ5.35
ꢀ5.45
ꢀ5.28
ꢀ5.65
ꢀ5.35
ꢀ5.78
ꢀ5.40
ꢀ5.43
ꢀ6.84
ꢀ5.46
ꢀ5.01
ꢀ5.81
ꢀ5.90
ꢀ5.27
ꢀ5.36
ꢀ5.81
ꢀ4.49
9
10
11
12
13
14
15
16
17
18
19
Rosi
The bold values signify the glide score of those compounds are higher than that of
the standard drug Rosiglitazone.

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S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
Fig. 1. Molecular docking of compounds against the PPAR-g target.
improved by administration of compound 7r. Insulin tolerance test
of compound 7r showed that the blood glucose levels of compound
7r treated diabetic rats were significantly lowered after 90 min of
insulin administration as compared to diabetic rats indicating in-
sulin resistance was improved by compound 7r. These results
suggest that the antihyperglycemic activity of 7r may result from
enhanced insulin and glucose resistance.
As the PPAR-g agonists are associated with side effect of body
weight gain [24], 7r was tested for body weight gain study. Com-
pound 7r treated normal rats did not show any significant increase
in the body weight as compared to normal control rats. However,
oral administration of 7r to diabetic rats for 15 days caused a sig-
nificant improvement in body weight of diabetic rats Fig. 5.
From the above results, the structure activity relationship (SAR)
can be drawn as follows. Compounds having oxadiazole ring
attached to o-position of TZD attached aromatic ring (7m) were
more active in lowering the blood glucose level than the com-
pounds having oxadiazole ring attached to p-position (7m>7a).
Methoxy substitution on the TZD attached aromatic ring (7b)
increased the activity. Compounds with halogen on oxadiazole

S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
179
Fig. 2. In vitro PPAR-
g
transactivation assay of compounds. Values are expressed as mean SE from three experiments conducted in triplicate at 10 mM.
attached aromatic ring (7h) exhibited promising activity. Presence
of phenyl group on oxadiazole attached aromatic ring (7k)
increased the activity. Dichloro substituted compounds (7r) resul-
ted in enhanced activity.
expression supports the results of in vitro PPAR-
study. It is thus clear that the in vitro PPAR- transactivation and
in vivo blood glucose lowering activity of compound may be due to
increase in the PPAR- gene expression. It has been reported that
g transactivation
g
g
PPAR-
g
agonists are also reported to cause hepatotoxicity which
TZDs improve insulin action by effects on gene transcription in the
fat cell that lead to diminished plasma levels of free fatty acids
(FFAs) and an increase in the level of adiponectin, an adipokine that
activates AMPK [28e30]. The increase in gene expression by com-
pounds 7m and 7r might be due to the activation of AMPK. The
is the other major drawback encountered with this class of drug
[25,26]. Compounds 7b, 7h, 7k, 7m and 7r have therefore been
assayed for AST, ALT and ALP levels in liver. It has been reported that
the levels of these enzymes are significantly increased in STZ rats
indicating the toxic effect of STZ on liver [27]. The elevation of these
enzymes might be due to increased protein catabolism followed by
gluconeogenesis as ALT and AST are directly involved in the amino
acid conversion to keto acids. It was observed that the levels of
serum AST, ALT and ALP increased in STZ treated rats were signif-
icantly decreased to near normal level after treatment with the
active compounds 7b, 7h, 7k, 7m and 7r (Fig. 6). Compounds 7m
and 7r were found to be more potent in lowering the AST, ALT and
ALP level more than the standard drug Pioglitazone. Remaining
compounds were as potent as the standard drug Pioglitazone in
lowering the AST, ALT and ALP levels to normal level. Therefore,
these compounds exhibited a protective effect on liver.
overexpression of PPAR-g in mature 3T3-L1 adipocytes increases
the amount of the mRNA for the ubiquitous GLUT1, whose
expression is reported to be downregulated during adipocyte dif-
ferentiation [31]. The reduction of insulin-stimulated glucose
transport in 3T3-L1 adipocytes overexpressing PPAR-g may be due
to the reduced expression of IR, IRS1, IRS2, and GLUT4. Thus,
compounds 7m and 7r increase the gene expression by maintain-
ing insulin sensitivity in mature 3T3-L1 adipocytes by regulating
the expression of genes that encode components of the insulin
signaling pathway as well as by increasing the expression levels of
GLUT1 and GLUT4 in these cells.
Histopathological study of the liver of the treated animals also
showed that the compounds 7b, 7h, 7m and 7r did not cause any
damage to the liver. Compound 7k caused insignificant inflamma-
tion in portal vein whereas Pioglitazone caused significant damage
i.e. significant dilation in sinusoidal space and inflammation in
centrizonal vein of the liver (Table 3, Supplementary data).
TZDs are associated with cardiovascular risks [16] and are the
leading cause for the withdrawal of these drugs from the market.
We therefore tested compound 7m and 7r for hERG inhibition. It
3. Conclusion
A library of eighteen novel conjugates of oxadiazoles and thia-
zolidinediones have been synthesized. Three compounds, 7b, 7m
and 7r exhibited significant in vitro PPAR-g transactivation activity
and in vivo blood glucose lowering effect. Compounds 7b, 7m and
7r recovered the activity of serum AST, ALT and ALP and did not
cause any damage to the liver. Compounds 7m and 7r were also
found to be free from cardiotoxicity. Compounds 7m and 7r
has been reported that compounds with an IC50 > 10
inhibit hERG significantly and hence have no cardiotoxicity. Since
compound 7m and 7r were found to have an IC50 of 41 and 29 M,
mM do not
increased the PPAR-
g gene expression by 2.10 and 2.0 fold,
respectively in comparison to standard drugs Rosiglitazone (1.0
fold) and Pioglitazone (1.5 fold). Compounds 7b, 7m and 7r may be
considered as potential candidates for the development of new
antidiabetic agents.
m
respectively, indicating that compound 7m and 7r would not be
associated with cardiotoxicity.
Since compounds 7m and 7r were found to be the most PPAR-
active, they were further subjected to PPAR- gene expression
study. The PPAR- gene expression study was done to know the
impact of compound 7m and 7r on the expression of the PPAR-
gene. It was observed that the PPAR- expression was significantly
g
g
4. Experimental protocols
g
g
4.1. Materials and methods
g
increased in presence of compound 7m (2.10 fold) and 7r (2.00
fold) in comparison to the standard drugs Pioglitazone (1.5 fold)
All chemicals (reagent grade) used were commercially available.
Melting points were measured on a VEEGO-VMP-DS melting point
apparatus and are uncorrected. 1H NMR was recorded on a Bruker
and Rosiglitazone (1.0 fold). The increase in PPAR-
g gene

180
S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
Fig. 3. In vivo antidiabetic activity of the active compounds in STZ induced diabetic rats. Data is analysedby one way ANOVA followed by Dunnett's ‘t’ test and expressed as
mean SEM from five observations; ** indicates p < 0.01 vs diabetic control. Piog: Pioglitazone; Rosi: Rosiglitazone; ctrl: normal control; Diab. ctrl: diabetic control.
DPX 400, 300 instruments in CDCl3/DMSO-d6 using TMS as internal
standard. Chemical shifts and coupling constants J are given in ppm
and Hz respectively. Mass spectra were recorded on a Jeol JMS-D
300 instrument fitted with a JMS 2000 data system at 70 eV.
Mass-spectrometric (MS) data is reported in m/z. Elemental anal-
ysis was carried out using Elemental Vario EL III elemental analyser.
Elemental analysis data is reported in % standard.
C20H15N3O5S: C, 58.67; H, 3.69; N, 10.26; S, 7.83. Found: C, 58.63; H,
3.3.71; N, 10.23; S, 7.81%.
4.2.3. 5-[4-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}-3-
ethoxybenzylidene]thiazolidine-2,4-dione (7c)
White crystals; yield: 65%; m.p. 202e203 ꢁC; IR (KBr):
n
(cmꢀ1
1.90
)
3040, 1715, 1620, 1541, 1092; 1H NMR (300 MHz, DMSO-d6):
d
4.2. General procedures for the synthesis of oxadiazole based 2,4-
thiazolidinediones 7(aer)
(t, 3H, J ¼ 7.1 Hz, CH3), 4.08 (q, 2H, J ¼ 6.4 Hz, OeCH2), 5.55 (s, 2H,
OeCH2), 7.06e8.32 (m, 8H, AreH), 7.69 (s,1H, C]CeH),12.01 (s,1H,
NH); 13C NMR (75 MHz, DMSO-d6):
d 14.97, 59.21, 64.78, 113.28,
To 10e15 ml POCl3, 2 mmol of aromatic hydrazides 3 (aee) and
2 mmol of different aromatic acids 6 (aef) were added, the reaction
mixture was kept for stirring at 60e70 ꢁC for 8e14 h. After
completion of reaction, monitored by TLC, the reaction mixture was
concentrated under reduced pressure, poured on crushed ice and
neutralized by sodium bicarbonate. The precipitate so obtained was
filtered, washed with cold water, dried and purified by column
chromatography using n-hexane:EtOAc as an eluent.
115.89, 122.12, 123.23, 123.64, 127.12, 128.28, 130.02, 132.14, 143.71,
148.52, 162.75, 165.50, 168.12, 168.64; MS ES (þve): 423 (M)þ, 424
(Mþ1)þ; Anal. Calcd. for C21H17N3O5S: C, 59.57; H, 4.05; N, 9.92; S,
7.57. Found: C, 59.59; H, 4.02; N, 9.90; S, 7.58%.
4.2.4. 5-[4-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}-3,5-
dimethylbenzylidene]thiazolidine-2,4-dione (7d)
Yellow crystals; yield: 70%; m.p. 195e197 ꢁC; IR (KBr):
n
(cmꢀ1
d 2.12
)
4.2.1. 5-[4-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}benzylidene]
3043, 1700, 1636, 1556, 1141; 1H NMR (300 MHz, DMSO-d6):
thiazolidine-2,4-dione (7a)
(s, 6H, 2 ꢂ CH3), 4.87 (s, 2H, OeCH2), 6.92 (s, 2H, AreH), 7.28 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.24e7.81 (m, 3H, AreH), 7.38 (s, 1H, C]CeH),
Yellow crystals; yield: 60%; m.p. 206e207 ꢁC; IR (KBr):
n
(cmꢀ1
5.60
)
3100, 1715, 1683, 1573, 1141; 1H NMR (300 MHz, DMSO-d6):
d
11.87 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 15.73, 62.69,
(s, 2H, OeCH2), 7.28 (d, 2H, J ¼ 8.4 Hz, AreH), 7.60e7.66 (m, 5H,
122.26, 122.63, 126.23, 128.52, 129.24, 130.35, 131.11, 131.19, 131.50,
155.86, 161.56, 164.97, 167.07, 167.56; MS ES (þve): 408 (Mþ1)þ;
Anal. Calcd. for C21H17N3O4S: C, 61.90; H, 4.21; N, 10.31; S, 7.87.
Found: C, 61.91; H, 4.19; N, 10.33; S, 7.89%.
AreH), 7.77 (s, 1H, C]CeH), 8.02 (d, 2H, J ¼ 7.2 Hz, AreH), 12.62 (s,
1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 60.22, 116.23, 121.87,
123.42, 127.16, 127.22, 130.01, 131.79, 132.51, 132.81, 159.27, 162.80,
165.30, 168.13, 168.52; MS ES (þve): 380 (Mþ1)þ; Anal. Calcd. for
C
19H13N3O4S: C, 60.15; H, 3.45; N, 11.08; S, 8.45. Found: C, 60.11; H,
3.42; N, 11.10; S, 8.47%.
4.2.5. 5-[4-[{5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl}methoxy]
benzylidene]thiazolidine-2,4-dione (7e)
4.2.2. 5-[4-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}-3-
methoxybenzylidene]thiazolidine-2,4-dione (7b)
White crystals; yield: 60%; m.p. 225e226 ꢁC; IR (KBr):
n
(cmꢀ1
5.59
)
3042, 1734, 1684, 1595, 1144; 1H NMR (300 MHz, DMSO-d6):
d
White crystals; yield: 60%; m.p. 214e216 ꢁC; IR (KBr):
n
(cmꢀ1
3.83
)
(s, 2H, OeCH2), 7.28 (d, 2H, J ¼ 8.7 Hz, AreH), 7.61 (d, 2H, J ¼ 9.0 Hz,
AreH), 7.70 (d, 2H, J ¼ 8.4 Hz, AreH), 7.77 (s, 1H, C]CeH), 8.03 (d,
2H, J ¼ 8.7 Hz, AreH), 12.51 (s, 1H, NH); 13C NMR (75 MHz, DMSO-
3044, 1715, 1681, 1590, 1139; 1H NMR (300 MHz, DMSO-d6):
d
(s, 3H, OeCH3), 5.54 (s, 2H, OeCH2), 7.18e8.15 (m, 8H, AreH), 7.75
(s, 1H, C]CeH), 12.60 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d6):
d 60.18, 114.21, 122.36, 122.54, 126.41, 127.32, 129.18, 132.15,
d
56.19, 60.88, 114.42, 115.27, 122.30, 123.45, 123.60, 127.16, 128.03,
132.89, 133.14, 159.18, 160.25, 163.98, 168.24, 168.40; MS ES (þve):
413 (M)þ, 415 (Mþ2)þ; Anal. Calcd. for C19H12ClN3O4S: C, 55.14; H,
2.92; N, 10.15; S, 7.75. Found: C, 55.15; H, 2.94; N, 10.12; S, 7.77%.
130.00, 132.09, 132.78, 148.82, 149.92, 162.85, 165.33, 168.05,
168.47; MS ES (þve): 410 (Mþ1)þ, 411 (Mþ2) þ; Anal. Calcd. for

S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
181
Fig. 4. Effect of compound 7r on OGTT and ITT. Data is analysed by one way ANOVA followed by Dunnett's ‘t’ test and expressed as mean SEM from five observations; * indicates
p < 0.001 vs diabetic control. # indicates p < 0.05 vs diabetic control. NC: normal control; NCþ7r: normal control þ 7r; STZ: diabetic control; STZþ7r: 7r treated diabetic rats.
AreH), 7.76 (s, 1H, C]CeH), 7.83 (d, 2H, J ¼ 8.4 Hz, AreH), 7.95 (d,
2H, J ¼ 8.4 Hz, AreH), 12.09 (s, 1H, NH); 13C NMR (75 MHz, DMSO-
d6):
d 60.20, 116.23, 121.82, 122.65, 126.45, 127.22, 129.10, 131.84,
132.50, 133.10, 159.26, 162.98, 164.69, 168.29, 168.47; MS ES (þve):
457.75 (M)þ, 459.91 (Mþ2)þ; Anal. Calcd. for C19H12BrN3O4S: C,
49.80; H, 2.64; N, 9.17; S, 7.00. Found: C, 49.82; H, 2.62; N, 9.19; S,
7.02%.
4.2.8. 5-[4-[{5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl}methoxy]-3-
methoxybenzylidene] thiazolidine ꢀ2,4-dione (7h)
White crystals; yield: 68%; m.p. 199e200 ꢁC; IR (KBr):
n
(cmꢀ1
3.82
)
3098, 1720, 1675, 1583, 1151; 1H NMR (300 MHz, DMSO-d6):
d
(s, 3H, OeCH3), 5.59 (s, 2H, OeCH2), 6.98e7.41 (m, 3H, AreH), 7.76
(s,1H, C]CeH), 8.02 (d, 2H, J ¼ 7.5 Hz, AreH), 8.13 (d, 2H, J ¼ 8.4 Hz,
AreH), 12.52 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 55.35,
61.52, 113.21, 120.32, 122.18, 128.26, 128.95, 129.18, 133.62, 134.43,
148.26, 149.81, 161.28, 165.88, 168.92, 169.43; MS ES (þve): 486
(M)þ, 488 (Mþ2)þ; Anal. Calcd. for C20H14BrN3O5S: C, 49.19; H,
2.89; N, 8.61; S, 6.57. Found: C, 49.21; H, 2.87; N, 8.63; S, 6.55%.
Fig. 5. Effect of compound 7r on body weight in albino wistar rats. Data is analyzed by
one way ANOVA followed by Dunnett's ‘t’ test and expressed as mean SEM from five
observations; * indicates p < 0.01 vs diabetic control. NC: Normal control; NCþ7r:
normal control þ 7r; STZ: diabetic control; STZþ7r: 7r treated diabetic rats.
4.2.9. 5-[4-[{5-(2-Ethoxyphenyl)-1,3,4-oxadiazol-2-yl}methoxy]
benzylidene]thiazolidine-2,4-dione (7i)
Yellow crystals; yield: 62%; m.p. 175e177 ꢁC; IR (KBr):
n
(cmꢀ1
d 1.47 (t,
)
3041,1734,1682,1557,1112; 1H NMR (300 MHz, DMSO-d6):
4.2.6. 5-[4-[{5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl}methoxy]-3-
3H, J ¼ 6.0 Hz, CH3), 4.12 (q, 2H, J ¼ 6.5 Hz, OeCH2), 5.37 (s, 2H,
methoxybenzylidene]thiazolidin eꢀ2,4-dione (7f)
White crystals; yield: 60%; m.p. 212e213 ꢁC; IR (KBr):
n
(cmꢀ1
3.86
)
OeCH2), 7.15 (d, 2H, J ¼ 8.1 Hz, AreH), 7.48 (d, 2H, J ¼ 7.8 Hz, AreH),
7.73 (s, 1H, C]CeH), 7.89e8.0 (m, 4H, AreH), 11.87 (s, 1H, NH); 13
C
3048, 1716, 1685, 1595, 1176; 1H NMR (300 MHz, DMSO-d6):
d
NMR (75 MHz, DMSO-d6):
d 14.44, 59.64, 63.56, 114.77, 115.28,
(s, 3H, OeCH3), 5.54 (s, 2H, OeCH2), 7.19e7.40 (m, 3H, AreH), 7.72
(s, 1H, C]CeH), 8.04 (d, 2H, J ¼ 6.9 Hz, AreH), 8.15 (d, 2H,
J ¼ 9.2 Hz, AreH), 12.31 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
115.73, 122.47, 122.82, 125.40, 125.72, 128.68, 128.96, 132.18, 156.27,
158.58, 162.82, 164.78, 167.41, 168.54; MS ES (þve): 424 (Mþ1)þ,
425 (Mþ2)þ; Anal. Calcd. for C21H17N3O5S: C, 59.57; H, 4.05; N,
9.92; S, 7.57. Found: C, 59.59; H, 4.07; N, 9.90; S, 7.57%.
d
55.32, 60.16, 114.24, 121.36, 121.54, 127.11, 127.72, 129.17, 132.72,
132.89, 133.44, 152.60, 153.21, 160.28, 164.78, 168.44, 168.47; MS ES
(þve): 443 (M)þ, 445 (Mþ2)þ; Anal. Calcd. for C20H14ClN3O5S: C,
54.12; H, 3.18; N, 9.47; S, 7.22. Found: C, 54.14; H, 3.20; N, 9.45; S,
7.24%.
4.2.10. 5-[4-[{5-(2-Ethoxyphenyl)-1,3,4-oxadiazol-2-yl}methoxy]-
3-methoxybenzylidene]thiazolidi ne-2,4-dione (7j)
White crystals; yield: 70%; m.p. 178e179 ꢁC; IR (KBr):
n
(cmꢀ1
1.36
)
3040, 1735, 1685, 1575, 1152; 1H NMR (300 MHz, DMSO-d6):
d
4.2.7. 5-[4-[{5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl}methoxy]
(t, 3H, J ¼ 6.0 Hz, CH3), 3.82 (s, 3H, OeCH3), 4.13 (q, 2H, J ¼ 6.9 Hz,
benzylidene]thiazolidine-2,4-dione (7g)
OeCH2), 5.50 (s, 2H, OeCH2), 7.12e7.33 (m, 7H, AreH), 7.71 (s, 1H,
White crystals; yield: 72%; m.p. 228e229 ꢁC; IR (KBr):
n
(cmꢀ1
5.59
(s, 2H, OeCH2), 7.27 (d, 2H, J ¼ 9.0 Hz, AreH), 7.60 (d, 2H, J ¼ 8.4 Hz,
)
C]CeH), 12.51 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 14.11,
55.24, 60.62, 63.64, 113.25, 114.89, 114.96, 123.77, 125.48, 126.71,
128.90, 133.69, 148.27, 149.12, 155.24, 163.86, 164.76, 167.42, 169.36;
3044, 1715, 1685, 1575, 1152; 1H NMR (300 MHz, DMSO-d6):
d

182
S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
d
5.36 (s, 2H, OeCH2), 5.42 (s, 2H, OeCH2), 7.06e7.55 (m, 13H,
AreH), 7.70 (s, 1H, C]CeH), 12.16 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6):
d 60.22, 62.53, 116.22, 122.57, 123.22, 128.15, 128.23,
130.12, 131.53, 132.55, 132.98, 158.64, 159.34, 162.84, 165.15, 168.26,
168.46; MS ES (þve): 486 (Mþ1)þ; Anal. Calcd. for C26H19N3O5S: C,
64.32; H, 3.94; N, 8.65; S, 6.60. Found: C, 64.34; H, 3.96; N, 8.64; S,
6.62%.
4.2.12. 5-[4-[[5-{(2,4-Dichlorophenoxy)methyl}-1,3,4-oxadiazol-2-
yl]methoxy]benzylidene]thiazolidi ne-2,4-dione (7l)
Yellow crystals; yield: 60%; m.p. 238e240 ꢁC; IR (KBr):
n
(cmꢀ1
5.57
)
3041, 1732, 1684, 1572, 1150; 1H NMR (300 MHz, DMSO-d6):
d
(s, 2H, OeCH2), 5.65 (s, 2H, OeCH2), 7.17e7.60 (m, 7H, AreH), 7.93
(s, 1H, C]CeH), 12.62 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
60.46, 61.88, 114.21, 121.25, 122.62, 129.11, 129.73, 130.42, 131.83,
132.58, 133.19, 149.29, 158.24, 163.52, 164.57, 168.86, 169.36; MS ES
(þve): 477 (M)þ, 479 (Mþ2)þ; Anal. Calcd. for C20H13Cl2N3O5S: C,
50.22; H, 2.74; N, 8.79; S, 6.70. Found: C, 50.24; H, 2.76; N, 8.81; S,
6.67%.
4.2.13. 5-[2-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}benzylidene]
thiazolidine-2,4-dione(7m)
White crystals; yield: 65%; m.p. 192e193 ꢁC; IR (KBr):
n
(cmꢀ1
5.66
(s, 2H, OeCH2), 7.17e8.02 (m, 9H, AreH), 7.66 (s,1H, C]CeH),12.60
(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
61.33, 114.62, 122.97,
)
3014, 1733, 1650, 1588, 1157; 1H NMR (300 MHz, DMSO-d6):
d
d
123.41, 127.13, 127.28, 129.20, 129.98, 132.58, 132.88, 159.25, 164.63,
165.89, 169.86, 169.91; MS ES (þve): 380 (Mþ1)þ; Anal. Calcd. for
C19H13N3O4S: C, 60.15; H, 3.45; N, 11.08; S, 8.45. Found: C, 60.12; H,
3.47; N, 11.06; S, 8.47%.
4.2.14. 5-[2-[{5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl}methoxy]
benzylidene]thiazolidine-2,4-dione (7n)
White crystals; yield: 70%; m.p. 207e208 ꢁC; IR (KBr):
n
(cmꢀ1
5.65
)
3038, 1715, 1699, 1574, 1152; 1H NMR (300 MHz, DMSO-d6):
d
(s, 2H, OeCH2), 7.16e7.49 (m, 4H, AreH), 7.57 (s, 1H, C]CeH), 7.65
(d, 2H, J ¼ 8.0 Hz, AreH), 8.01 (d, 2H, J ¼ 8.2 Hz, AreH), 11.94 (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6):
d 61.13, 114.45, 116.78, 122.31,
122.95, 128.94, 130.13, 131.18, 132.36, 134.78, 137.52, 156.53, 163.06,
164.48; 169.89, 169.92; MS ES (þve): 413.74 (M)þ, 415.58 (Mþ2)þ;
Anal. Calcd. for C19H12ClN3O4S: C, 55.14; H, 2.92; N, 10.15; S, 7.75.
Found: C, 55.16; H, 2.95; N, 10.13; S, 7.77%.
4.2.15. 5-[2-[{5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl}methoxy]
benzylidene]thiazolidine-2,4-dione (7o)
White crystals; yield: 70%; m.p. 182e183 ꢁC; IR (KBr):
n
(cmꢀ1
5.65
)
3100, 1716, 1685, 1574, 1152; 1H NMR (300 MHz, DMSO-d6):
d
(s, 2H, OeCH2), 7.16e7.69 (m, 4H, AreH), 7.36 (s, 1H, C]CeH), 7.82
(d, 2H, J ¼ 8.7 Hz, AreH), 7.96 (d, 2H, J ¼ 8.4 Hz, AreH), 12.58 (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6):
d 60.05, 113.90, 122.21, 122.46,
123.20, 123.56, 126.01, 128.42, 128.63, 131.58, 132.64, 156.01, 162.68,
164.17, 168.81, 169.71; MS ES (þve): 457.80 (M)þ, 459.76 (Mþ2)þ;
Anal. Calcd. for C19H12BrN3O4S: C, 49.80; H, 2.64; N, 9.17; S, 7.00.
Found: C, 49.83; H, 2.62; N, 9.19; S, 7.02%.
Fig. 6. Effect of compounds on serum AST, ALT and ALP activities. Values are given as
mean S.D.
4.2.16. 5-(2-((5-(2-Ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)
MS ES (þve): 453 (M)þ; Anal. Calcd. for C22H19N3O6S: C, 58.27; H,
benzylidene)thiazolidine-2,4-dione (7p)
White crystals; yield: 55%; m.p. 169e171 ꢁC; IR (KBr):
n
(cmꢀ1
1.36
)
4.22; N, 9.27; S, 7.07. Found: C, 58.29; H, 4.23; N, 9.25; S, 7.09%.
3042, 1715, 1682, 1557, 1154; 1H NMR (300 MHz, DMSO-d6):
d
(t, 3H, J ¼ 6.9 Hz, CH3), 4.12 (q, 2H, J ¼ 6.9 Hz, OeCH2), 5.63 (s, 2H,
4.2.11. 5-[2-[[5-{(4-Phenylphenoxy)methyl}-1,3,4-oxadiazol-2-yl]
OeCH2), 7.02e7.97 (m, 8H, AreH), 8.02 (s,1H, C]CeH),12.62 (s,1H,
methoxy]benzylidene]thiazolidine-2,4-dione (7k)
NH); 13C NMR (75 MHz, DMSO-d6):
d 14.51, 60.65, 63.61, 114.04,
115.10, 115.33, 122.49, 122.52, 124.40, 125.82, 128.56, 128.60, 132.28,
156.17, 161.58, 161.80, 164.77, 167.44, 168.04; MS ES (þve): 424
White crystals; yield: 70%; m.p. 231e233 ꢁC; IR (KBr):
n
(cmꢀ1
)
3162, 3057, 1718, 1623, 1156, 761; 1H NMR (300 MHz, DMSO-d6):

S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
183
(Mþ1)þ; Anal. Calcd. for C21H17N3O5S: C, 59.57; H, 4.05; N, 9.92; S,
4.4. Pharmacology
4.4.1. PPAR- transactivation assay
7.57. Found: C, 59.59; H, 4.08; N, 9.94; S, 7.59%.
g
4.2.17. 5-[2-{(5-Phenyl-1,3,4-oxadiazol-2-yl)methoxy}-5-
bromobenzylidene]thiazolidine-2,4-dione (7q)
Human embryonic kidney (HEK) 293 cells were cultured in
DMEM with 10% heat inactivated fetal bovine serum in a humidi-
fied 5% CO2 atmosphere at 37 ꢁC. Cells were seeded in 6-well plates
the day before transfection to give a confluence of 70e80% at
transfection. Cells grown in DMEM were inoculated in 96-well plate
White crystals; yield: 70%; m.p. 216e218 ꢁC; IR (KBr):
n
(cmꢀ1
5.68
(s, 2H, OeCH2), 7.41e8.03 (m, 8H, AreH), 7.89 (s,1H, C]CeH),12.71
(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
61.34, 114.31, 116.80,
)
3040, 1700, 1636, 1585, 1152; 1H NMR (300 MHz, DMSO-d6):
d
d
containing 60,000 cells/well. Cells were transfected with 2.5
PPRE-Luc, 6.67 L of PPAR- , 1.0 L of Renilla and 20 L of Lip-
ofectamine. Following 5 h after transfection, cells were treated with
compound (10 M) for 24 h and then collected with cell culture
mL of
123.40, 124.91, 125.29, 126.84, 127.14, 130.01, 131.16, 132.84, 134.82,
155.64, 162.60, 165.27, 167.69, 168.03; MS ES (þve): 457.80 (M)þ,
459.76 (Mþ2)þ; Anal. Calcd. for C19H12 BrN3O4S: C, 49.80; H, 2.64;
N, 9.17; S, 7.00. Found: C, 49.82; H, 2.67; N, 9.19; S, 7.02%.
m
g
m
m
m
lysis buffer. Luciferase activity was monitored on luminometer
using the luciferase assay kit according to the manufacturer's in-
structions. Rosiglitazone and Pioglitazone were used as standard
drugs. The results are summarized in Fig. 2.
4.2.18. 5-[2-[[5-{(2,4-Dichlorophenoxy)methyl}-1,3,4-oxadiazol-2-
yl]methoxy]benzylidene]thiazoli dine-2,4-dione (7r)
Yellow crystals; yield: 65%; m.p. 222e223 ꢁC; IR (KBr):
n
(cmꢀ1
5.48
)
3048, 1715, 1675, 1570, 1124; 1H NMR (300 MHz, DMSO-d6):
d
4.4.2. Antidiabetic activity
(s, 2H, OeCH2), 5.23 (s, 2H, OeCH2), 7.08e7.68 (m, 7H, AreH), 7.67
The antidiabetic activity was performed according to the re-
ported method [32] using streptozotocin induced diabetic model.
Albino Wistar rats of either sex, 150e200 g were obtained from
Central Animal House, Jamia Hamdard University, New Delhi. The
rats were kept in cages at the room temperature and fed with food
and water ad libitum. The experiments were performed in accor-
dance with the rules of Institutional Animals Ethics Committee
(registration number 173-CPCSEA). The rats were fasted overnight
and diabetes was induced by injecting streptozotocin (STZ) (60 mg/
kg body weight) intraperitoneally. STZ was prepared freshly in
0.1 M citrate buffer (pH 4.5). The animals were allowed to drink 5%
glucose solution overnight to overcome the drug-induced hypo-
glycemia. The rats were considered as diabetic, if their blood
glucose values were above 250 mg/dL on the 3rd day after STZ
injection. The rats were divided into five groups comprising of six
animals in each group. Control rats receiving 0.1 M citrate buffer
(Group I), Diabetic rats received STZ injection (Group II), Diabetic
rats orally fed with Pioglitazone (as 0.25% carboxymethyl cellulose
suspension) at a dose of 36 mg/kg (Group III), Diabetic rats orally
fed with Rosiglitazone (as 0.25% carboxymethyl cellulose suspen-
sion) at a dose of 36 mg/kg (Group IV), Diabetic rats orally fed with
synthesized compounds (as 0.25% carboxymethyl cellulose sus-
pension) at an equimolar dose of the standard drug Pioglitazone
(Group V). The blood glucose level of each group was checked at 0,
1, 7 and 15 day by glucose oxidase method [33]. The results are
summarized in Fig. 3.
(s, 1H, C]CeH), 12.60 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
60.42, 61.75, 114.25, 121.78, 123.24, 129.15, 129.63, 130.72, 131.42,
132.65, 133.43, 156.23, 158.15, 163.71, 164.49, 168.17, 169.23; MS ES
(þve): 477.63 (M)þ, 479.54 (Mþ2)þ; Anal. Calcd. for
C
20H13Cl2N3O5S: C, 50.22; H, 2.74; N, 8.79; S, 6.70. Found: C, 50.24;
H, 2.77; N, 8.81; S, 6.73%.
4.3. Molecular docking
Molecular docking studies involve mainly protein selection &
preparation, grid generation, ligand preparation, docking & further
analysis of docking studies. Schrodinger software was mainly used
for all the above steps.
4.3.1. Protein selection & preparation
Protein with Accession number 3CS8 was selected and down-
loaded from Protein Data Bank. This protein is reported to bind with
drug Rosiglitazone. The protein was imported, optimized, mini-
mized by removing unwanted molecules and other defects re-
ported by the software. PPAR-g receptor is a dimer which has two
monomers chains (A & B). For the purpose of studies, chain B was
deleted and water molecules near the ligands were retained. Finally
a low energy minimized protein structure was obtained and used
for further docking studies.
4.3.2. Grid generation
Minimized protein was used for grid generation which involves
selected ligand as the reference as it signifies the binding sites of
drug with respect to the target. The generated grid was used for
further docking of new molecules.
4.4.3. Oral glucose tolerance test (OGTT) and insulin tolerance test
(ITT)
OGTT and ITT were performed as reported previously [34]. The
results are summarized in Fig. 4.
4.3.3. Ligand preparation
4.4.4. Biochemical parameters
Molecules drawn in 3D form were refined by LigPrep module.
The molecules were subjected to OPLS-2005 force field to generate
single low energy 3-D structure for each input structure. During
this step chiralities were maintained.
Serum AST and ALT were assayed according to the reported
method of Reitman and Frankel method [35] and ALP assay was
done according to method of Walter and Schult [36] using p-
nitrophenyl phosphate as the substrate. The results are summa-
rized in Fig. 6.
4.3.4. Docking studies
Docking studies was carried using Glide software. It was carried
using Extra precision and write XP descriptor information. This
generates favourable ligand poses which are further screened
through filters to examine spatial fit of the ligand in the active site.
Ligand poses which pass through initial screening are subjected to
evaluation and minimization of grid approximation. Scoring was
then carried on energy minimized poses to generate Glide score.
The results are summarized in Table 1 and Fig. 1.
4.4.5. Hepatotoxicity studies
The hepatotoxicity was performed according to the reported
method [37]. For the study, rats were sacrificed under light
anaesthesia after 5 h of the administration of the tested drugs (3
times to the dose used for antidiabetic activity) and their liver
specimens were removed and put into 10% formalin solution.
Morphological examination was performed with Haematoxylin and

184
S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
eosin staining to analyze histological changes and examined under
microscope. The results are summarized in Fig. 7.
4.5.2. RNA extraction, reverse transcription and gene expression
analysis
After 24 h cells were scrapped and collected in 1.5 ml micro
centrifuge tubes. The total RNA was isolated by TRI Reagent®
(Molecular Research Centre). RNA quantity and quality were
determined on a NanoDrop ND-2000c spectrophotometer and
integrity was checked on a 1.5% agarose gel. Total RNA (1 mg) was
used to generate cDNA using an EZ-first strand cDNA synthesis kit
for RT (reverse transcription)ePCR (Biological Industries). Primers
4.4.6. hERG inhibition assay
The activity was performed according to the reported method of
Cheng et al., 2002 [38].
for real-time PCR were designed for PPAR-g and b-actin using the
Pearl Primer software and are listed in Table 2 (Supplementary
data). Reactions were run at 95 ꢁC for 10 min followed by 40 cy-
cles of 95 ꢁC for 15 s and 60 ꢁC for 1 min. Real-time PCR was per-
formed on an ABI Prism 7300 Sequence Detection System (Applied
Biosystems) using the SYBR Green PCR Master Mix (Applied Bio-
systems). PCR was performed in triplicate and was repeated two
times for each gene and each sample. Relative transcript quantities
4.5. PPAR-g gene expression study
4.5.1. Cell culture experiments
3T3-L1 cells (ATCC) were seeded in 24 well plate 24 h before
treatment in DMEM containing 10% calf serum (Invitrogen). After
24 h cells were treated with compound 7m and 7r (10
standard drugs, Pioglitazone and Rosiglitazone (10 M) as positive
mM) and
m
were calculated using the Ct method with
b-actin as the endoge-
control and DMSO as negative control, followed by 24 h of incu-
bation of cells in CO2 incubator at 37 ꢁC and 5% CO2.
nous reference gene. The results are summarized in Fig. 8.
Fig. 7. Haematoxylin and eosin immunohistochemical staining of liver after administration of synthesized drugs. Histopathology report of rat liver. As illustrated in above figure,
Low and high power photomicrograph of liver from animal treated groups 7b, 7h, 7k, 7m, 7r and standard. 10ꢂ. Low power photomicrograph of liver from corresponding animal
7b, treated groups showing normal arrangement of cells in the liver lobule. PT ¼ portal triad and CV ¼ central vein. 40ꢂ. Compound 7b, 7h, 7m and 7r treated groups showing
normal arrangement of cells in the liver lobule and normal arrangement of hepatocytes in the centrizonal area. 7k treated group showing insignificant inflammation in portal vein
whereas Pioglitazone treated groups showing a mild dilatation of sinusoidal spaces and mild inflammation in centrizonal vein.

S. Nazreen et al. / European Journal of Medicinal Chemistry 87 (2014) 175e185
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Acknowledgements
The author thanks Vice Chancellor, Dr. G. N. Qazi for providing
necessary facilities to the Department of Chemistry. Syed Nazreen
acknowledges University Grants Commission, New Delhi, for
financial assistance.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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