Synthesis, docking, in vitro and in vivo antidiabetic activity of pyrazole-based 2,4-thiazolidinedione derivatives as PPAR-γ modulators

Article abstract of DOI:10.1002/ardp.201700223

The design, synthesis, structure–activity relationship, and biological activity of 2,4-thiazolidinedione derivatives as peroxisome proliferator-activated receptor-γ (PPAR-γ) modulators for antidiabetic activity are reported. Fifteen 2,4-thiazolidinedione

Full text of DOI:10.1002/ardp.201700223

Received: 10 July 2017
Revised: 9 January 2018
Accepted: 10 January 2018
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DOI: 10.1002/ardp.201700223
FULL PAPER
Synthesis, docking, in vitro and in vivo antidiabetic activity of
pyrazole-based 2,4-thiazolidinedione derivatives as PPAR-γ
modulators
Mohd. Javed Naim1
Ozair Alam1
Md. Jahangir Alam1
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Mohammad Shaquiquzzaman1
Md. Mumtaz Alam1
Vegi Ganga Modi Naidu2
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1 Medicinal Chemistry and Molecular
Abstract
Modelling Lab, Department of Pharmaceutical
Chemistry, School of Pharmaceutical
Education and Research, Jamia Hamdard, New
Delhi, India
The design, synthesis, structure–activity relationship, and biological activity of 2,4-
thiazolidinedione derivatives as peroxisome proliferator-activated receptor-γ (PPAR-γ)
modulators for antidiabetic activity are reported. Fifteen 2,4-thiazolidinedione derivatives
clubbed with pyrazole moiety were docked into the ligand binding domain of PPAR-γby the
Glide XP module of Schrodinger. Eight derivatives (5a, 5b, 5d, 5f, 5i, 5l, 5n, 5o) having Glide
XP scores > −8 as compared to the standard drug, rosiglitazone (Glide XP score = −9.165),
showed almost similar interaction with the amino acids such as HIS 449, TYR 473, TYR 327,
HIS 323, and SER 289 in the molecular docking studies. These eight derivatives were further
screened for PPAR-γ transactivation and in vivo blood glucose lowering activity in the
streptozotocin-induced diabetic rat model. Compounds 5o, 5n, 5a, 5i, and5b showed 52.06,
51.30, 48.65, 43.13, and 40.36% PPAR-γ transactivation as compared to the reference
drugs rosiglitazone and pioglitazone with 85.30 and 65.22% transactivation, respectively.
The data analysis showed significant blood glucose lowering effects (hypoglycemia) of
compounds 5o, 5n, and 5a (140.1 4.36, 141.4 6.15, and 150.7 4.15, respectively),
along with reference drugs pioglitazone (135.2 4.91) and rosiglitazone (141.1 5.88) as
compared to the diabetic control. Furthermore, the most potent compound 5o also elevated
the PPAR-γ gene expression by 2.35-fold as compared to rosiglitazone (1.27-fold) and
pioglitazone (1.6-fold). It also significantly lowered the AST, ALT, and ALP levels and caused
no damage to the liver.
2 Department of Pharmacology and
Toxicology, National Institute of
Pharmaceutical Education and Research,
Hyderabad, India
Correspondence
Dr. Ozair Alam, Medicinal Chemistry and
Molecular Modelling Lab, Department of
Pharmaceutical Chemistry, School of
Pharmaceutical Education and Research,
Jamia Hamdard, New Delhi-110062, India.
Email: dr.ozairalam@gmail.com
Funding information
DST-SERB, New Delhi, Grant number: SB/
FT/LS-203-2012
K E Y W O R D S
diabetes, molecular docking, PPAR-γ, thiazolidinedione
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1
INTRODUCTION
Abbreviations: 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; ALP, alkaline phospha-
tase; ALT, alanine transaminase; AMPK, 5′-adenosine monophosphate-activated protein
kinase; AST, aspartate transaminase; DMEM, Dulbecco's Modified Eagle's Medium; DMF,
N,N-dimethyl formamide; ESI-MS, electrospray ionization mass spectrometry; FBS, fetal
bovine serum; FFA, free fatty acid; FT-IR, Fourier transform infrared spectroscopy; HEK,
human embryonic kidney; IRAD, insulin resistance associated disorders; LBD, ligand binding
domain; mRNA, messenger RNA; NIDDM, non-insulin dependent diabetes mellitus; NMR,
nuclear magnetic resonance; PCR, polymerase chain reaction; POCl3, phosphoryl
oxychloride; PPAR, peroxisome proliferator-activated receptor; SAR, structure–activity
relationship; STZ, streptozotocin; TZD, thiazolidinedione; US FDA, Food and Drug
Administration of the United States.
Diabetes has reached an epidemic level by becoming a major health
issue round the globe.[1] Peroxisome proliferator-activated receptor-γ
(PPAR-γ) agonists have come up with a leading role as an orally
effective antidiabetic agent.[2] Diabetes or diabetes mellitus is a
multifactorial metabolic disease/polygenic disorder characterized by
increased glucose levels/insulin resistance over prolonged periods in
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liver and peripheral tissues.[3] It is categorized as Type I, Type II, and
gestational diabetes.[4] Type II diabetes (non-insulin-dependent
diabetes mellitus) occurs in 80–90% of the patients,[5] often
characterized by hyperglycemia and insulin resistance in the initial
stages leading finally to serious complications such as stroke,
cardiovascular diseases, atherosclerosis, chronic eye damage, kidney
failure, obesity, etc.[6–8] collectively known as insulin resistance
associated disorders (IRAD).[9]
replaced by pyrazole ring which was then attached with the TZD ring
through the linker. Further a lipophilic fragment was directly attached
(without having the oxymethyl linker as in the pioglitazone and
rosiglitazone) to the central pyrazole, and another phenyl group was
attached through N-1 atom of the pyrazole in such a way that the
designed molecules can efficiently accommodate in the Y-shaped
PPAR-γ-LBD. Here, we report the design and synthesis of newer 2,4-
thiazolidinedione derivatives (5a–o), which were then evaluated for in
vitro PPAR-γ transactivation and gene expression studies and in vivo
blood glucose lowering and hepatotoxicity studies with a hope to
overcome the complications/side effects as reported in earlier
marketed drugs. The derivatives (5a–o) were then characterized by
1H NMR, 13C NMR, IR and mass spectral analysis.
Glitazones (TZDs or thiazolidinediones), a new class of drug was
approved by US FDA in late 1990s for treatment of diabetes which
shares common partial structures (rosiglitazone and pioglitazone) and
by mid-1990s PPAR-γ was found to be molecular target of these
glitazones (Figure 1). Thiazolidinedione (TZD) is an important class of
anti-diabetic agent that has attracted attention round the globe for its
excellent anti-hyperglycemic profile without causing hypoglycemia. It
exhibits a diverse biological profile which includes anti-hyperglyce-
mic,[10] anti-proliferative,[11] antitumor,[12] antioxidant,[13] antimicro-
bial,[14] anti-inflammatory,[15] antimalarial,[16] cytotoxic,[17] 15-PGDH
inhibitors,[18] aldose reductase inhibitor,[19] anti-obesity,[20] histone
deacetylase inhibitor,[21] tyrosinase inhibitor,[22] and GPR40 ago-
nist.[23] They have been considered as the high affinity agonist of
PPARs (peroxisome proliferator-activated receptors) with maximum
selectivity for PPAR-γ instead of PPAR-α and PPAR-β. PPAR-γ also
known as glitazone receptor belongs to nuclear receptor family
(NR1C3) which regulates the expression of genes involved in glucose
metabolism. PPAR-γ is a significant element in adipogenesis which also
plays an important role in insulin sensitivity, cell cycle regulation, and
cell differentiation. It is the most widely studied PPAR subtype which is
further classified as PPAR-γ agonists, PPAR-γ partial agonists, PPAR-
α/γ dual agonists, and PPAR-γ antagonists.[24–27] In recent years, we
have seen exhaustive research on TZD moiety as an excellent
antidiabetic agent due to its attractive features of improving insulin
sensitivity and lack of hypoglycemia. However, despite their excellent
therapeutic effect, they possess many deleterious target-related side
effects which includes weight gain, risk of congestive heart failure,
peripheral edema, and increased chances of bone-related disorders.
These safety concerns have not only restricted clinical use of these
PPAR-γ agonists but also led to failed development of many agonists.
Some of the marketed drugs include ciglitazone, troglitazone,
pioglitazone, and rosiglitazone of which troglitazone was removed
due to its hepatotoxicity.[28] Now new TZD derivatives are being
worked on to overcome this complication[29–31] for which we hunt
surrogate lipophilic templates to take over larger lipophilic core of
earlier representative agonists. Furthermore the PPAR-γ LBD (ligand-
binding domain) is Y-shaped cavity and the docking studies of new lead
(compound, 5o) efficiently fill this cavity.[32] This approach has led to
convincing improvement of PPAR-γ activity. Further optimization to
balance in vitro activity and metabolic stability allowed the discovery of
selective, potential, and orally efficacious PPAR-γ agonist. Structure–
activity relationship (SAR) study as well as thorough exploration of the
binding mode of compounds to the PPAR-γ-LBD revealed the
necessary structural features of these lead molecules as mentioned
in Figure 1. The central phenyl ring of pioglitazone and rosiglitazone is
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The title compounds 5a–o were synthesized as per the route outlined in
Scheme 1. The compounds 3a–o were synthesized by treating
substituted acetophenones/aryl ketones (1a–o) with phenyl hydrazine
(2) which then undergoes Vilsmeier–Haack reaction in the presence of
POCl3 and N,N-dimethyl formamide (DMF) to give pyrazole carbalde-
hydes 4a–o. These resulting carbaldehydes were finally treated with
2,4-thiazolidinedione to give the final derivatives 5a–o. The structures
of TZD derivatives (5a–o) were characterized by FT-IR, 1H and13C NMR
(Bruker-400 NMR spectrometers) and mass spectrometry (ESI-MS,
water). Elemental analysis (C, H, and N) data were within 0.4% of the
theoretical values. The formation of TZD derivatives was confirmed by
the appearance of a singlet for ─CHC─ protons at δ 7.607–8.311 ppm
and a broad singlet for NH-TZD protons at δ 12.337–12.564 in 1H NMR
spectra followed by the presence of peaks 3347–3379 cm−1 (NHstr),
  
1723–1773 (C Ostr), 1649–1681 (─CH Cstr), 1548–1582 (C Nstr),
and 642–679 (C─S) in the IR spectrum.
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2.2 Pharmacological activities
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2.2.1 In vitro PPAR-γ transactivation assay
The derivatives with good Glide scores (>8) in molecular docking
studies were further screened for PPAR-γ transactivation assay so as
to determine their mode of action as compared to the reference drug
rosiglitazone with a Glide score of −9.165. Compounds 5a, 5b, 5i, 5n,
and 5o were found to be PPAR-γ active. Compounds 5o, 5n, 5a, 5i, and
5b showed 52.06, 51.30, 48.65, 43.13, and 40.36% PPAR-γ trans-
activation as compared to reference drugs rosiglitazone and pioglita-
zone with 85.30 and 65.22% transactivation, respectively, while
compounds 5d, 5f, and 5l showed moderate results (Figure 2).
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2.2.2 In vivo blood glucose lowering effect
Streptozotocin (STZ)-induced diabetic model is the most preferred
model because of its lengthier half-life and hyperglycemic interval

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FIGURE 1 Reported thiazolidinedione containing derivatives showing anti-diabetic activity along with designed molecule
followed by reduced risk of ketosis and mortality. The TZD derivatives
5a, 5b, 5d, 5f, 5i, 5l, 5n, and 5o were evaluated on STZ-induced diabetic
rats for their blood glucose lowering effect. A single dose of these
compounds was administered and the blood glucose levels were
monitored as per standard protocols on 1st, 7th, and 15th day of the
commencement of experiment. The data analysis showed significant
blood glucose lowering effect (hypoglycemia) with compounds 5o, 5n,
and 5a showing 140.1 4.36, 141.4 6.15, and 150.7 4.15, respec-
tively, along with reference drugs pioglitazone (135.2 4.91) and
rosiglitazone (141.1 5.88) as compared to diabetic control. Therefore

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SCHEME 1 Reagents and conditions: (a) Abs. ethanol, conc. HCl, reflux for 6–8 h; (b) DMF, POCl3, reflux for 6–8 h; (c) TZD, abs. ethanol,
piperidine, reflux for 3–5 h
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these synthesized compounds showed almost identical blood glucose
lowering effect as reference drugs. Compounds 5i and 5b showed
moderate results whereas compounds 5d, 5f, and 5l were found to be
least active as compared to reference drugs (Figure 3).
2.2.4 Hepatotoxicity studies
The derivatives 5a–o were synthesized with a view to overcome the
reported hepatotoxic effects of TZDs. The compounds with good in
vivo results 5a, 5b, 5i, 5n, and 5o were further analyzed for increase or
decrease in the levels of aspartate transaminase (AST), alanine
transaminase (ALT), and alkaline phosphatase (ALP) followed by
histopathological study of liver. Due to toxic effects of the STZ on rats
liver, the level of these enzymes were significantly increased but
decreased to almost normal after administration of the respective
compounds. The compound 5o was found to be the most potent in
lowering down the levels of AST, ALT, and ALP as compared to
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2.2.3 Body weight gain study
Compound 5o was further analyzed for body weight gain study for
15 days. No significant body weight change was observed in 5o-treated
normal rats and normal control rats. However, diabetic rats showed
significant improvement in weight gain which is shown in Figure 4.[2]

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FIGURE 2 In vitro PPAR-γ transactivation assay of the
compounds. Values are expressed as mean SE from three
experiments conducted in triplicate at 10 mM
FIGURE 4 Effect of compound 5o on body weight in albino
Wistar rats. Data are analyzed by one-way ANOVA followed by
Dunnett's t-test and expressed as mean SEM from five
observations; *p < 0.01 versus diabetic control. NC, normal control;
NC + 5o, normal control + 5o; STZ, diabetic control; STZ 5o, 5o-
treated diabetic rats
pioglitazone. Compounds 5i and 5b were found to be equally potent as
the reference drug in reducing the level of these enzymes to normal
(Figure 5). Histopathological studies also confirmed that 5b, 5i, 5n, and
5o caused no damage to liver. Compound 5a was found to decrease
the levels of AST and ALT and found to be hepato-protective but in
contrast it showed slight dilation of sinusoidal space in microscopical
image of the liver (exact mechanism unknown) along with pioglitazone
(Figure 6).
(Figure 7). Thus, the increase in gene expression due to compound 5o
supports and is responsible for blood glucose lowering effect and
PPAR-γ transactivation. This increase in expression of PPAR-γ genes
might be due to AMPK activation. TZDs have been reported to
improve action of insulin by effecting gene transcription in fat cells
leading to decreased free fatty acids (FFAs) level in plasma followed by
increase in adiponectin level. The overexpression of PPAR-γ in 3T3-L1
adipocytes increases mRNA content for ubiquitous GLUT1 which is
down regulated during differentiation of adipocytes. The insulin-
stimulated glucose transport in 3T3-L1 was found to be reduced due to
diminished expression of IR, IRS1, IRS2, and GLUT4. As a result,
compound 5o increases gene expression and levels of GLUT1 and
GLUT4 while maintaining insulin sensitivity in mature 3T3-L1
adipocytes.[33,34]
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2.2.5 PPAR-γ gene expression study
PPAR-γ gene expression study was carried out to determine the effect
of the most potent derivative (5o) on PPAR-γ gene. The gene analysis
includes compound 5o and reference drugs rosiglitazone and
pioglitazone in 10 μM concentrations and it was observed that the
expression of the PPAR-γ gene was significantly increased by 2.35-
fold for compound 5o as compared to the standard drugs rosiglitazone
and pioglitazone where expression was increased by 1.27- and 1.6-fold
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2.3 Molecular docking
The molecular docking study of the designed and synthesized
compounds into the LBD (ligand binding domain) of PPAR-γ was
carried out to explore the possible binding interactions as well as to
compare the binding pattern of these synthesized compounds to
the well-known PPAR-γ modulator, rosiglitazone. The compounds
5a, 5b, 5i, 5n, and 5o showing good overall results were then
selected for interaction with LBD of PPAR-γ which has a Y-shaped
cavity composed of a hydrophobic gate (ARM 3) which is divided
into two sub-arms, ARM 1 (essentially polar), which is lengthened
toward helix 12 (H 12), and ARM 2 (predominantly hydrophobic),
which is placed between helix 3 (H 3) and a β-sheet, as shown in
Figure 8.
The molecular docking studies showed that compounds 5a, 5b, 5i,
5n, and 5o were well accommodated in the PPAR-γ active site. The
compound 5o interacted well with PPAR-γ-LBD in a manner similar to
known PPAR-γ modulator, rosiglitazone. The 3D binding mode of
docked compound 5o showed six hydrogen bond interactions with the
FIGURE 3 Antidiabetic activity of compounds in STZ-induced
diabetic rats. Data are analyzed by one-way ANOVA followed by
Dunnett's t-test and expressed as mean SEM from five
observations; ##p < 0.01 versus control; **p < 0.01 and *p < 0.05
versus diabetic control

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FIGURE 5 Effect of compounds on serum AST, ALT, and ALP activities. Values are given as mean SD
FIGURE 6 Photomicrograph of rat liver stained with hematoxylin and eosin (H & E) is shown in the above figure. Histopathology study of
rat liver was illustrated in animal-treated groups 5a, 5b, 5i, 5n, and 5o, normal control and standard. Photomicrographs of liver of control
group are showing normal arrangement of innumerable lobe and consisting of central vein and portal vein. There were no pathological
changes of lobe in group treated with our synthesized drug (5b, 5i, 5n, and 5o), when treated groups were compared with the control group.
5a and pioglitazone-treated groups showed mild dilatation of sinusoidal spaces around the central vein and mild inflammation in portal vein

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FIGURE 7 Effect of compound 5o on PPAR-γ gene expression.
The experiments were conducted at 10 μM. PCR was performed in
triplicate and was repeated two times for each gene and sample.
Relative transcript quantities were calculated using the Ct method
with β-actin as the endogenous reference gene

side chain residues SER 289 (C O...HO, 3.03 Å), TYR 327


(C O. . .HO, 2.91 Å), HIP 323 (C O...HN, 3.26 Å), TYR 473 (NH...
OH, 3.97 Å), and HIP 449 (CO...HN, 3.86 Å) as displayed in
Figure 10a and an additional hydrogen bonding and π-cation
interaction with ARG 288 (pyrazole-N. . .HN, 3.92 Å and phenyl. . .CN,
4.17 Å, respectively). The hydrophilic acidic head group 2,4-thiazoli-
dinedione ring of compound 5o binds to AF-2 (activation factor 2) helix
to form a network of hydrogen bonds with HIP 449 (Helix 11), TYR 473
(Helix 12), SER 289 (Helix 3), TYR 327 (Helix 4), and HIP 323 (Helix 4),
shown in Figure 9 and occupy the right arm of Y-shaped cavity, as
shown in Figure 8. These interactions of 2,4-thiazolidinedione ring
FIGURE 9 Insight of the PPAR-γ protein complexes with
compounds 5o (turquoise color) showing interaction with different
helixes. The protein is represented as cartoon model
stabilize helix 12 (H12) and are crucial for the transactivation activity as
well as agonistic activity of PPAR-γ. Compound 5n binds with the
acidic TZD moiety in a fashion similar to that observed in compound 5o

and showed hydrogen bonding with SER 289 (C O. . .HO, 2.99 Å),

TYR 327 (NH. . .OH, 3.05 Å), HIP 323 (C O. . .HN, 3.00 Å), TYR 473

(NH. . .OH, 3.56 Å), and HIP 449 (C O. . .HN, 3.74 Å), as shown in
Figure 8b. Moreover, 5n also forms additional hydrogen bonding with
ARG 288 (pyrazole-N. . .HN, 3.98 Å). However, the TZD moiety of
compounds 5a and 5i as shown in Figure 10c and d interacted with the
PPAR-γ in a similar fashion as 5o. Compound 5a binds to the LBD of

PPAR-γ through hydrogen bonding with SER 289 (C O. . .HO,

3.00 Å), TYR 327 (NH. . .OH, 2.89 Å), HIP 323 (C O. . .HN, 3.17 Å),

TYR 473 (NH. . .OH, 3.76 Å), and HIP 449 (C O. . .HN, 3.95 Å), as
shown in Figure 10c. In addition, 5a also forms hydrogen bonding with
ARG 288 (pyrazole-N. . .HN, 3.88 Å). Compound 5i (containing the
meta methyl and para fluoro group) forms a network of hydrogen

bonds with SER 289 (C O. . .HO, 3.03 Å), TYR 327 (NH. . .OH, 2.86 Å),

HIP 323 (C O. . .HN, 3.19 Å), TYR 473 (NH. . .OH, 3.79 Å), and HIP

449 (C O. . .HN, 3.96 Å), as shown in Figure 10d whereas the
pyrazole ring in 5i formed a hydrogen bond with ARG 288 (pyrazole-
N. . .HN, 3.92 Å).
It was clearly observed that compounds containing small
heterocyclic ring such as thiophene (5b) attached to the pyrazole
scaffold enhances the interaction of TZD moiety to TYR 473, on the
other hand 5b also formed
a π–π interaction with TYR 327
(thiophene. . .phenyl, 4.86 Å). The acidic TZD moiety of compound
5b fitted nicely into the LBD site of the PPAR-γ making five hydrogen
FIGURE 8 Structure of the Y-shaped LBD of PPAR-γ forming a
complex with compounds 5o (yellow color)

bond interactions with HIP 449 (C O. . .HN, 2.81 Å), GLN 286

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FIGURE 10 Docked pose of compounds (a) 5o (yellow color), (b) 5n (green color), (c) 5a (turquoise color), and (d) 5i (plum color) showing
hydrogen bond interaction (red-dashed lines) with amino acids in the binding site of PPAR-γ. The ligand and amino acids are represented as
ball and stick and tube model, respectively

(C O. . .H2N, 2.33 Å), TYR 473 (NH. . .OH, 3.52 Å), HIP 323
Figure 12a. The docked compound 5o superimposed well with the co-
crystal ligand rosiglitazone and all common interactions (HIP 449, TYR
473, HIP 323, TYR 327, and SER 289) were consistent with the ones in
the crystal complexes of ligand binding domain (LBD) of PPAR-γ, as
shown in Figure 12b. The 5o derivative is enclosed by hydrophobic
amino acids such as ILE 326, TYR 327, LEU 330, VAL 339, ILE 341, LEU
353, ILE 281, and PHE 363 shown in Figure 13a, while co-crystal ligand
rosiglitazone is surrounded by hydrophobic amino acids such as ILE
281, LEU 353, VAL 339, and ILE 341, represented in Figure 13b.

(CO. . .HN, 1.67 Å), and SER 289 (C O. . .HO, 1.77 Å) as shown in
Figure 11a. The receptor surface model of compound 5o in the binding
site of PPAR-γ is also depicted in Figure 11b. It was established that the
compounds having bulkier hydrophobic moiety/group such as
naphthalene and isobutyl phenyl were the most active in the series.
The co-crystal ligand, rosiglitazone assumed to occupy the LBD of
PPAR-γ and established hydrogen bonding interactions with HIP 323,
SER 289, TYR 327, TYR47, HIP 449, and GLN 286, as depicted in
FIGURE 11 (a) Docked pose of 5b (plum color) showing hydrogen bond interaction (red-dashed lines) with amino acids in the binding site
of PPAR-γ. (b) Receptor surface model of 5o (turquoise color) in the binding site of PPAR-γ

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FIGURE 12 (a) Docked pose of rosiglitazone showing hydrogen bond interaction (red-dashed lines) with amino acids in the binding site of
PPAR-γ. (b) Superimposed structure of compound 5o (turquoise color) with rosiglitazone (yellow color) at the binding site of PPAR-γ
The molecular docking protocol was validated by re-docking of
the co-crystallized ligand rosiglitazone back into the same active site
of PPAR-γ and found to follow the same interaction pattern and
results with RMSD value of 1.9 and, therefore, docking protocol was
validated by the generated grid. The information achieved from this
work must be useful to compare the binding modes of designed
compound 5o to the known PPAR-γ modulator, i.e., rosiglitazone
and pioglitazone, and expedient for further designing and develop-
ment of newer PPAR-γ modulators with advantages over current
marketed TZD derivatives.
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2.4 SAR
The SAR of the derivatives in relation to PPAR-γ transactivation
revealed that compounds containing bulkier aryl groups at N-position
of the pyrazole core as in 5n (51.30%) and 5o (52.06%) showed good in
vitro results. Introduction of a halo atom at the phenyl ring resulted in
enhanced antidiabetic activity. Among the halo derivatives, di-
substitutions at the meta and para positions in 5a (48.65%) showed
significant lowering in blood glucose level than halogen at other
positions. Compounds 5i (43.13%) with smaller halogen atom F at para
position and an electron releasing group (methyl group) at meta
position was found more potent than the larger bromo derivatives. The
replacement of aromatic ring with thiophene in 5b (40.36%) resulted in
moderate activity, despite having marginally poor aromatic character
and very good interaction of thiophene ring with the PPAR-γ receptor.
However, compound 5l showed moderate activity due to steric
hindrance caused by the larger size of the 2-bromo biphenyl group.
Compound 5f containing electron releasing group at meta and para
positions on the aryl ring observed less lowering in blood glucose level.
Interestingly, incorporation of an additional methyl group at the meta
position along with para fluoro in compound 5i was also able to
produce significant lowering in blood glucose with respect to
compound 5f having methyl group at meta position and hydroxyl at
para position on aryl ring. Compound 5d, with the larger iodo group in
the para position of phenyl ring demonstrated low activity.
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3
CONCLUSION
A series of 15 TZD derivatives have been synthesized among which
compounds 5a, 5b, 5i, 5n, and 5o exhibited significant in vitro PPAR-γ
transactivation and in vivo antidiabetic potency. Among these
derivatives, compound 5o (equipped with naphthalene moiety)
FIGURE 13 XP visualization pose of (a) compound 5o and (b)
rosiglitazone, showing major hydrophobic interactions with the
PPAR-γ LBD

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appeared as the most potential antidiabetic agent with no noteworthy
weight gain and showed no signs of hepatotoxicity. Compound 5o
exhibited 2.35-fold increase in PPAR-γ gene expression against
pioglitazone (1.6-fold) and rosiglitazone (1.27-fold) as standard drugs.
Compound 5o may be considered as a potential and safe candidate
which may undergo modifications for the design and development of
newer antidiabetic agents/PPAR-γ modulator.
drop-wise for 15–20 min (Vilsmeier–Haack reaction). Substituted
hydrazone (3a–o, 0.00925 mol) was then added and stirred for few
minutes and then heated at 70–80°C for 6–8 h. Cool it, pour in
minimum amount of crushed ice and basify it with saturated solution of
NaHCO3, filtered, dried, and recrystallized from ethanol.[35]
Synthesis of TZD derivatives 5a–o
Equimolar quantities of substituted pyrazole carbaldehydes (4a–o) and
TZD (earlier prepared from thiourea and chloroacetic acid) in absolute
ethanol with few drops of glacial acetic acid and piperidine was
refluxed for 2–3 h. The solvent was removed under reduced pressure;
residue left behind was poured in crushed ice, filtered, washed with ice
cold water, dried, and recrystallized from ethanol to yield the final
derivatives[36,37] (Scheme 1).
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
All required chemicals, solvents, and reagents were purchased from
Sigma–Aldrich and Merck India. Progress of the reaction was
monitored on pre-coated thin layer chromatographic aluminum
sheets Silica Gel Merck 60 F254 (Merck) using toluene/ethyl
acetate/formic acid (5:4:1), benzene/acetone (8:2) and TLC spot
visualization was done by using UV lamp and iodine chamber.
Melting points of the synthesized compounds were determined by
open glass capillary tubes and are uncorrected. Fourier transform
infrared spectra were recorded on (IR Affinity SHIMADZU) FTIR-
spectrophotometer using KBr pellets and λmax values were given in
cm−1. The 1H NMR and 13C NMR spectra were recorded on Bruker
model DRX-400 and 100 MHz, respectively, in CDCl3 and DMSO-d6
solvents using tetramethylsilane (TMS) as the internal standard.
Chemical shift values are given in δ (ppm) scale and the signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet) whereas coupling constants (J) are expressed in Hz. Mass
spectra (ESI-MS) were recorded on a Waters mass spectrometer
UPLC-MS/MS and elemental analysis was undertaken with Perkin-
Elmer model 240C analyzer. Elemental analysis was 0.4%, i.e.
within the theoretical values.
|
4.1.3 Characterization of synthesized compounds
5-((3-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5a)
Yield = 59%, m.p. 256–258°C, Rf = 0.63. FT-IR (KBr, vmax cm−1): 3349


(NH), 3067, 3044 (C─H, Ar), 1751, 1739 (C O), 1655 (─CH C─),
1568 (C N), 1386 (C─N), 775 (C─Cl), 650 (C─S). 1H NMR (CDCl3,

400 MHz), δ ppm: 7.424–7.443 (t, 1H, Ar─H, J = 7.6 Hz), 7.517–7.580
(m, 4H, Ar─H), 7.788–7.797 (m, 3H, Ar─H), 8.073 (s, 1H, ─CHC),
8.536 (s, 1H, CH, pyrazole-H), 12.536 (bs, 1H, NH-TZD). 13C NMR
(CDCl3, 75 MHz): 114.8, 119.5, 121.1, 123.7, 126.3, 128.1, 128.5,
129.2, 129.7, 130.2, 131.3, 132.9, 139.0, 142.3, 152.3, 167.0, 167.6.
ESI-m/z: 417.73 [M+H]+. Anal. calcd. for C19H11Cl2N3O2S: C, 54.82; H,
2.66; N, 10.09. Found: C, 54.85; H, 2.62; N, 10.13%.
5-((1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl)methylene)-
thiazolidine-2,4-dione (5b)
The InChI codes of the investigated compounds are provided as
Supporting Information.
Yield = 73%, m.p. 230–232°C, Rf = 0.79. FT-IR (KBr, vmax cm−1): 3367

(NH), 3085, 3044 (C─H, Ar), 1767, 1752 (C O), 1661 (─CHC─), 1579
(CN), 1376 (C─N), 651 (C─S). 1H NMR (DMSO-d6, 400 MHz), δ ppm:
7.258–7.269 (t, 1H, J = 4.4 Hz, ─CH-thiophene), 7.409–7.428 (t, 2H,
J = 7.6 Hz, ─CH-thiophene), 7.555–7.574 (t, 2H, Ar─H, J = 7.6 Hz),
7.740–7.750 (d, 2H, Ar─H, J = 4 Hz), 7.964–7.984 (m, 2H, Ar─H,
|
4.1.2 General procedures for the synthesis of TZD
derivatives (5a–o)

─CH C), 8.681 (s, 1H, CH, pyrazole-H), 12.560 (1H, bs, NH-TZD).
Synthesis of substituted hydrazones 3a–o
13C NMR (DMSO-d6, 75 MHz): 115.5, 119.7, 121.7, 123.7, 127.9,
128.0, 128.3, 128.6, 128.7, 130.0, 133.1, 138.9, 147.9, 167.5, 167.9.
ESI-m/z: 353.94 [M+H]+. Anal. calcd. for C17H11N3O2S2: C, 57.77; H,
3.14; N, 11.89. Found: C, 57.82; H, 3.12; N, 11.93%.
To a solution of (1a–o) substituted acetophenones/aryl ketones (0.01
moles) and (2) phenyl hydrazine (0.02 moles) in absolute ethanol (25–
30 mL) were added few drops of concentrated HCl in a 100 mL round
bottom flask and refluxed for 6–7 h. The solvent (abs. ethanol) was
evaporated under reduced pressure; the residue left was poured into
crushed ice with continuous stirring, filtered, washed with ice cold
water, dried, and recrystallized from ethanol.
5-((3-(2-Bromo-4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)-thiazolidine-2,4-dione (5c)
Yield = 76%, m.p. 263–265°C, Rf = 0.61. FT-IR (KBr, vmax cm−1): 3365


(NH), 3056, 3041 (C─H, Ar), 1757, 1743 (C O), 1667 (─CH C─),
1

Synthesis of substituted pyrazole carbaldehydes 4a–o
1582 (C N), 1379 (C─N), 628 (C─Br), 665 (C─S). H NMR (DMSO-d6,
To
a
solution of DMF (0.188 mol; ∼14.5 mL) at 0–5°C under
400 MHz), δ ppm: 3.663 (s, 3H, OCH3), 7.403–7.425 (t, 1H, Ar─H,
J = 8.8 Hz), 7.513–7.550 (m, 3H, Ar─H), 7.589–7.611 (d, 2H, Ar─H,
continuous stirring was added POCl3 (0.0385 mol; ∼3.6 mL) slowly

NAIM ET AL.
11 of 15
|


J = 8.8 Hz), 7.626–7.647 (d, 2H, Ar─H, J = 8.4 Hz), 7.971 (s, 1H,
(─CH C─), 1565 (C N), 1459 (NO2), 1373 (C─N), 669 (C─S).
1H NMR (CDCl3, 400 MHz), δ ppm: 3.813 (s, 3H, OCH3), 7.330–7.367
(m, 2H, Ar─H), 7.456 (s, 1H, Ar─H), 7.698–7.718 (d, 2H, Ar─H,
J = 8 Hz), 7.887–7.919 (m, 2H, Ar─H), 7.967–7.996 (m, 2H, Ar─H,

─CH C), 8.716 (s, 1H, CH, pyrazole H), 12.553 (bs, 1H, NH-TZD).
13C NMR (DMSO-d6, 75 MHz): 55.2, 114.9, 115.7, 116.1, 118.3,
119.2, 121.1, 121.9, 128.1, 128.6, 129.2, 131.4, 139.5, 152.7, 159.3,
162.9, 167.4, 167.9. ESI-m/z: 457.03 [M+H]+. Anal. calcd. for

─CH C), 8.683 (s, 1H, CH, pyrazole-H), 9.535 (s, 1H, OH), 12.534 (bs,
C
20H14BrN3O3S: C, 52.64; H, 3.09; N, 9.21. Found: C, 52.61; H,
1H, NH-TZD). 13C NMR (CDCl3, 75 MHz): 57.3, 107.5, 115.7, 119.3,
122.0, 125.9, 128.2, 129.4, 129.9, 130.3, 133.0, 139.7, 142.8, 147.8,
148.6, 151.3, 167.4, 167.9. ESI-m/z: 394.55 [M+H]+. Anal. calcd. for
3.07; N, 9.25%.
5-((3-(4-Iodophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-
thiazolidine-2,4-dione (5d)
C20H15N3O4S: C, 61.06; H, 3.84; N, 10.68. Found: C, 61.10; H, 3.87; N,
10.65%.
Yield = 66%, m.p. 247–249°C, Rf = 0.79. FT-IR (KBr, vmax cm−1): 3357


(NH), 3077, 3053 (C─H, Ar), 1759, 1748 (C O), 1649 (─CH C─),
5-((3-(2-Chloro-3,4-dihydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)-thiazolidine-2,4-dione (5h)
Yield = 68%, m.p. 269–271°C, Rf = 0.67. FT IR (KBr, vmax cm−1): 3656
1

1561 (C N), 1377 (C─N), 653 (C─S), 536 (C─I). H NMR (DMSO-d6,
400 MHz), δ ppm: 7.410–7.428 (t, 1H, Ar─H, J = 7.2 Hz), 7.555–7.574
(t, 3H, Ar─H, J = 7.6 Hz), 7.619–7.677 (m, 4H, Ar─H), 7.993–8.013 (m,

(OH), 3371 (NH), 3085, 3043 (C─H, Ar), 1765, 1747 (C O), 1653
1


2H, Ar─H, ─CH C), 8.715 (s, 1H, CH, pyrazole-H), 12.564 (bs, 1H,
(─CHC─), 1577 (C N), 1361 (C─N), 787 (C─Cl), 644 (C─S). H NMR
NH-TZD). 13C NMR (DMSO-d6, 75 MHz): 96.3, 116.0, 119.8, 121.0,
123.6, 128.0, 128.6, 129.4, 130.0, 130.6, 130.7, 139.1, 152.6, 167.5,
167.9. ESI-m/z: 473.86 [M+H]+. Anal. calcd. for C19H12IN3O2S: C,
48.22; H, 2.56; N, 8.88. Found: C, 48.24; H, 2.61; N, 8.97%.
(DMSO-d6, 400 MHz), δ ppm: 7.421–7.439 (t, 1H, Ar─H, J = 7.2 Hz),
7.539–7.612 (m, 2H, Ar─H), 7.757–7.791 (m, 4H, Ar─H), 7.957 (s, 1H,

─CH C), 8.477 (s, 1H, CH, pyrazole-H), 9.231 (s, 1H, OH), 10.219 (s,
1H, OH), 12.520 (bs, 1H, NH-TZD). 13C NMR (DMSO d6, 75 MHz):
114.6, 116.7, 119.2, 121.3, 123.2, 126.0, 128.3, 129.2, 129.8, 130.4,
131.3, 142.5, 146.2, 148.5, 151.6, 167.1, 167.6. ESI-m/z: 414.77
[M+H]+. Anal. calcd. for C19H12ClN3O4S: C, 55.14; H, 2.92; N, 10.15.
Found: C, 55.18; H, 2.90; N, 10.21%.
5-((3-(2,4-Dihydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5e)
Yield = 77%, m.p. 221–223°C, Rf = 0.68. FT-IR (KBr, vmax cm−1): 3633

(OH), 3347 (NH), 3069, 3057 (C─H, Ar), 1763, 1739 (C O), 1662
1


(─CH C─), 1548 (C N), 1371 (C─N), 679 (C─S). H NMR (DMSO-d6,
5-((3-(4-Fluoro-3-methylphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5i)
400 MHz), δppm: 6.45 (s, 1H, Ar─H), 6.610–6.812 (d, 1H, Ar─H, J = 8 Hz),
Yield = 72%, m.p. 174–176°C, Rf = 0.66. FT-IR (KBr, vmax cm−1): 3348

7.012–7.563 (m, 6H, Ar─H), 7.861 (s, 1H, ─CH C─), 8.536 (s, 1H,



─CH C─), 9.65 (s, 1H, ─OH), 10.96 (s, 1H, ─OH), 12.337 (bs, 1H, NH-
(NH), 3064, 3057 (C─H, Ar), 1767, 1752 (C O), 1656 (─CH C─),
1573 (CN), 1368 (C─N), 1234 (C─F), 654 (C─S). 1H NMR (DMSO-d6,
400 MHz), δ ppm: 2.61 (s, 3H, CH3), 7.40–7.422 (t, 3H, Ar─H,
J = 8.8 Hz), 7.551–7.570 (t, 3H, Ar─H, J = 7.6 Hz), 7.663–7.698 (m, 2H,
TZD). 13C NMR (DMSO-d6, 75 MHz): 106.3, 111.7, 115.4, 115.8, 120.6,
123.7, 127.1, 129.9, 131.0, 135.4, 141.2, 146.6, 150.0, 156.1, 161.7,
167.2, 167.8.ESI-m/z: 380.65 [M+H]+. Anal. calcd. for C19H13N3O4S: C,
60.15; H, 3.45; N, 11.08. Found: C, 60.21; H, 3.41; N, 11.12%.

Ar─H), 7.987–8.007 (m, 2H, Ar─H, ─CH C), 8.689 (s, 1H, CH,
pyrazole-H), 12.527 (bs, 1H, NH-TZD). 13C NMR (DMSO-d6, 75 MHz):
17.8, 115.9, 116.5, 119.7, 122.2, 123.2, 124.6, 126.5, 127.9, 128.4,
130.0, 131.1, 131.3, 139.1, 152.9, 164.6, 167.5, 167.9. ESI-m/z:
379.93 [M+H]+. Anal. calcd. for C20H14FN3O2S: C, 63.31; H, 3.72; N,
11.08. Found: C, 63.28; H, 3.77; N, 11.12%.
5-((3-(4-Hydroxy-3-methylphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5f)
Yield = 59%, m.p. 228–230°C, Rf = 0.79. FT-IR (KBr, vmax cm−1): 3623

(OH), 3349 (NH), 3066, 3051 (C─H, Ar), 1761, 1743 (C O), 1653
1

(─CH C─), 1557 (CN), 1367 (C─N), 673 (C─S). H NMR (DMSO-d6,
400 MHz), δ ppm: 2.35 (s, 3H, CH3), 6.912–6.933 (d, 2H, Ar─H,
J = 8.4 Hz), 7.385–7.404 (t, 1H, Ar─H, J = 7.6 Hz), 7.422–7.443 (d, 2H,
5-((3-(3,4-Dimethoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5j)
Ar─H, J = 8.4 Hz), 7.540–7.552 (m, 3H, Ar─H), 8.038 (s, 1H, ─CH C),
Yield 59%, m.p. 250–252°C, Rf = 0.71. FT-IR (KBr, vmax cm−1): 3362


8.636 (s, 1H, CH, pyrazole-H), 9.827 (s, 1H, OH), 12.507 (bs, 1H, NH-
TZD). 13C NMR (DMSO-d6, 75 MHz): 15.7, 115.5, 116.7, 120.1, 122.3,
123.5, 125.1, 127.6, 128.1, 130.2, 130.5, 131.7, 133.0, 139.1, 151.6,
155.3, 167.3, 167.8. ESI-m/z: 378.06 [M+H]+. Anal. calcd. for
(NH), 3077, 3048 (C─H, Ar), 2871 (C─H, OCH3), 1745, 1723 (C O),
1675 (─CH C─), 1566 (C N), 1361 (C─N), 658 (C─S). 1H NMR
(DMSO-d6, 400 MHz), δ ppm: 3.827 (s, 6H, ─OCH3), 7.098–7.143 (m,
2H, Ar─H), 7.202 (s, 1H, Ar─H), 7.396–7.414 (t, 1H, J = 7.2 Hz), 7.548–



C20H15N3O3S: C, 63.65; H, 4.01; N, 11.13. Found: C, 63.72; H,
7.567 (t, 2H, Ar─H, J = 7.6 Hz), 7.607 (s, 1H, ─CH C), 7.988–8.008 (d,
3.98; N, 11.16%.
2H, Ar─H, J = 8 Hz), 8.651 (s, 1H, CH, pyrazole-H), 12.497 (bs, 1H, NH-
TZD). 13C NMR (DMSO-d6, 75 MHz): 56.1, 115.9, 117.6, 119.5, 121.1,
122.8, 126.3, 128.7, 129.0, 129.5, 130.4, 139.1, 146.7, 151.6, 153.8,
159.2, 167.3, 167.8. ESI-m/z: 408.27 [M+H]+. Anal. calcd. for
5-((3-(4-Hydroxy-3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5g)
Yield = 78%, m.p. 243–245°C, Rf = 0.66. FT-IR (KBr, vmax cm−1): 3644
C21H17N3O4S: C, 61.90; H, 4.21; N, 10.31. Found: C, 61.87; H,

(OH), 3362 (NH), 3068, 3047 (C─H, Ar), 1761, 1742 (C O), 1657
4.26; N, 10.33%.

12 of 15
NAIM ET AL.
|
5-((3-(2,4-Dimethoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5k)
Yield = 55%, m.p. 241–243°C, Rf = 0.57. FT-IR (KBr, vmax cm−1): 3371
129.2, 130.1, 132.8, 139.2, 141.6, 142.9, 149.6, 167.6, 168. ESI-m/z:
404.36 [M+H]+. Anal. calcd. for C23H21N3O2S: C, 68.46; H, 5.25; N,
10.41. Found: C, 68.52; H, 5.21; N, 10.45%.

(NH), 3079, 3053 (C─H, Ar), 2857 (C─H, OCH3), 1754, 1736 (C O),
1673 (─CH C─), 1577 (C N), 1365 (C─N), 647 (C─S). 1H NMR
(DMSO-d6, 400 MHz), δ ppm: 3.812 (s, 6H, ─OCH3), 7.116–7.253 (m,
3H, Ar─H), 7.366–7.617 (m, 4H, Ar─H), 7.983–8.005 (m, 2H, Ar─H,
5-((3-(Naphthalen-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)-
thiazolidine-2,4-dione (5o)
Yield = 69%, m.p. 281–283°C, Rf = 0.81. FT-IR (KBr, vmax cm−1): 3371



 
(NH), 3058, 3042 (C─H, Ar), 1771, 1747 (C O), 1672 (─CH C─),
─CH C), 8.636 (s, 1H, CH, pyrazole-H), 12.541 (bs, 1H, NH-TZD).
13C NMR (DMSO-d6, 75 MHz): 55.3, 56.4, 101.4, 111.7, 119.2, 119.4,
121.3, 126.1, 128.3, 129.1, 129.5, 130.1, 139.2, 146.5, 153.1, 157.6,
159.3, 167.1, 167.5. ESI-m/z: 408.11 [M+H]+. Anal. calcd. for
1579 (C N), 1375 (C─N), 645 (C─S). 1H NMR (CDCl3, 400 MHz), δ


ppm: 7.521–7.956 (m, 12H, Ar─H), 8.256 (s, 1H, ─CH C), 8.656 (s,
1H, CH, pyrazole-H), 12.426 (bs, 1H, NH-TZD). 13C NMR (CDCl3,
100 MHz): 116.2, 120.3, 123.5, 126.1, 126.4, 126.9, 128.8, 129.1,
129.7, 130.2, 130.9, 133.6, 134.4, 141.5, 145.3, 152.8, 167.1, 167.8.
ESI-m/z: 398.36 [M+H]+. Anal. calcd. for C23H15N3O2S: C, 69.50; H,
3.80; N, 10.57. Found: C, 69.56; H, 3.83; N, 10.62%.
C
21H17N3O4S: C, 61.90; H, 4.21; N, 10.31. Found: C, 61.93; H,
4.25; N, 10.36%.
5-((3-(3-Bromo-[1,1′-biphenyl]-4-yl)-1-phenyl-1H-pyrazol-4-yl)-
methylene)thiazolidine-2,4-dione (5l)
Yield = 62%, m.p. 209–211°C, Rf = 0.76. FT-IR (KBr, vmax cm−1): 3367
|
4.2 Molecular docking studies

(NH), 3077, 3061 (C─H, Ar), 2932 (C─H, CH3), 1757, 1734 (C O),


1663 (─CH C─), 1582 (C N), 1355 (C─N), 633 (C─Br), 643 (C─S).
1H NMR (CDCl3, 400 MHz), δ ppm: 7.309–7.329 (d, 2H, Ar─H,
J = 8 Hz), 7.391–7.415 (m, 5H, Ar─H), 7.513–7.532 (t, 2H, Ar─H,
J = 7.6 Hz), 7.710–7.730 (d, 2H, Ar─H, J = 8 Hz), 7.787–7.807 (d, 2H,
Molecular docking study was carried out to assess their interaction and
binding modes with target receptor using Glide 5.5 extra precision (XP)
Maestro 10.1 Schrodinger,[38] running on Linux 64 operating system. It
comprised of a series of steps which includes selection of protein and
its preparation, receptor grid generation, preparation of ligand and its
docking to the receptor. Protein with PDB ID-2PRG was downloaded
from Protein Data Bank at a resolution of 2.3 Å, which was used in
molecular docking studies. Peroxisome proliferator-activated recep-
tor-γ (PPAR-γ) is a nuclear receptor consisting of two monomer chains
(A and B). Due to the presence of our TZD moiety in chain A, chain B
was deleted along with residual water molecules which were beyond
5 Å, leaving behind water molecules near the ligand to yield energy
minimized protein structure. These energy minimized protein struc-
tures were then used to generate grid which used reference drug as a
ligand to signify the binding sites within the receptor target. The ligand
preparation involves 2D or 3D structures and producing their low
energy states in Maestro format using OPLS 2005 force field, with the
possibilities to extend each input structure by generating variation on
ionization states. Finally docking was carried out using Glide software
with extra precision and write XP descriptor information. During this
procedure, favorable ligand poses were then generated to determine
their spatial fit into the active site of receptor and those who fitted best
were then evaluated and minimized for generating glide scores. The
Glide score, hydrogen bonds, and π–π interaction formed with the
surrounding amino acids were used to predict the binding affinities and
proper alignment of these compounds at the active site of the receptor
or enzyme (Table 1).

Ar─H, J = 8 Hz), 8.016 (s, 1H, ─CH C), 8.538 (s, 1H, CH, pyrazole-H),
12.508 (bs, 1H, NH-TZD). 13C NMR (CDCl3,75 MHz): 131.4, 119.2,
120.5, 122.3, 126.1, 126.7, 127.2, 127.9, 128.9, 129.4, 129.8, 130.6,
134.5, 138.4, 138.7, 139.3, 141.1, 144.0, 150.6, 167.5, 168.1. ESI-m/z:
503.45 [M+H]+. Anal. calcd. for C25H16BrN3O2S: C, 59.77; H, 3.21; N,
8.36. Found: C, 59.73; H, 3.18; N, 8.41%.
5-((1-Phenyl-3-(pyridin-3-yl)-1H-pyrazol-4-yl)-methylene)-
thiazolidine-2,4-dione (5m)
Yield = 63%, m.p. 235–237°C, Rf = 0.58. FT-IR (KBr, vmax cm−1): 3379

(NH), 3047, 3031 (C─H, Ar), 1770, 1745 (C O), 1681 (─CHC─), 1567
(C N), 1355 (C─N), 667 (C─S). 1H NMR (CDCl3, 400 MHz), δ ppm:

6.679–6.697 (t, 1H, Ar─H, J = 7.2 Hz), 6.908–6.944 (d, 2H, Ar─H,
J = 14.4 Hz), 7.028–7.110 (m, 4H, Ar─H), 7.913–7.933 (d, 2H, Ar─H,

J = 8 Hz), 8.311 (s, 1H, ─CH C), 8.749 (s, 1H, CH, pyrazole-H), 12.463
(bs, 1H, NH-TZD). 13C NMR (CDCl3, 75 MHz): 115.8, 119.6, 122.6,
123.1, 125.3, 127.1, 130.3, 132.6, 132.9, 133.6, 138.5, 141.7, 147.9,
148.3, 167.1, 167.7. ESI-m/z: 349.13 [M+H]+. Anal. calcd. for
C
18H12N4O2S: C, 62.06; H, 3.47; N, 16.08. Found: C, 62.02; H,
3.45; N, 16.11%.
5-((3-(4-Isobutylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-
thiazolidine-2,4-dione (5n)
Yield = 75%, m.p. 221–223°C, Rf = 0.74. FT-IR (KBr, vmax cm−1): 3365


(NH), 3056, 3038 (C─H, Ar), 1773, 1741 (C O), 1663 (C C), 1582
|
4.3 Biological activity
(C N), 1367 (C─N), 642 (C─S). 1H NMR (CDCl3, 400 MHz), δ ppm:

1.238 (s, 6H, CH3), 1.815 (s, 1H, CH, isobutyl), 2.215 (m, 2H, CH2,
|
4.3.1 In vitro PPAR-γ transactivation assay
isobutyl), 7.398–7.417 (t, 1H, Ar─H, J = 7.6 Hz), 7.460–7.537 (m, 5H,

Ar─H), 7.795–7.882 (m, 4H, Ar─H), 8.181 (s, 1H, ─CH C), 8.550 (s,
1H, CH, pyrazole-H), 12.513 (bs, 1H, NH-TZD). 13C NMR (CDCl3,
Human embryonic kidney (HEK) 293 cells were cultured with 10% heat
inactivated fetal bovine serum (FBS) in Dulbecco's Modified Eagle's
Medium (DMEM) under humid conditions with 5% CO2 at 37°C. The
75 MHz): 23.2, 31.7, 43.3, 115.4, 119.7, 121.8, 126.1, 127.3, 129.0,

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TABLE 1 Glide score, Glide energy, and ADME prediction of the synthesized compounds, 5a–o
Comp.
Glide score
−9.363
−8.896
−5.751
−8.153
−6.865
−8.027
−6.407
−6.902
−8.986
−6.962
−6.726
−8.781
−6.496
−9.526
−9.706
−9.165
Glide energy
−73.293
−64.599
−66.265
−69.927
−72.633
−70.468
−73.449
−76.981
−64.253
−69.935
−66.977
−74.327
−58.219
−65.668
−81.249
−87.861
Log Po/wa
4.712
4.001
4.207
4.688
2.392
3.398
3.142
2.678
4.297
3.889
3.910
5.767
3.059
2.805
4.747
3.594
PSAb
Log Sc
Rule of fived
5a
89.383
88.440
94.932
89.375
130.195
109.976
119.870
130.020
89.377
106.045
102.887
86.452
133.358
102.247
89.372
94.178
−6.755
−5.813
−5.995
−6.784
−4.737
−5.522
−4.980
−5.140
−6.165
−5.350
−5.642
−7.854
−5.600
−4.777
−6.621
−4.988
0
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
Rosiglitazone
aPredicted octanol/water partition coefficient (<5).
bVan der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms (range 7–200).
cPredicted aqueous solubility, log S. S in mol/dm3 is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid (range
−6.5 to 0.5).
dLipinski's violations (≤1).
day before transfection, six-well plates were seeded with cells to give
70–80% confluence at transfection. 96-well plates containing 60000
cells/well were inoculated with cells grown in DMEM and were
transfected with 2.5 μL of PPRE-luciferase, 6.67 μL of PPAR-γ, 1.0 μL
of Renilla, and 20 μL of lipofectamine. Five hours after transfection, the
cells were treated with synthesized analogues (10 μM) for 24 h and
then collected with cell culture lysis buffer. Pioglitazone and
rosiglitazone were used as reference drug and luciferase activity
was monitored using the luciferase assay kit (Promega) on lumin-
ometer (Perkin Elmer, USA) according to instructions cited by
manufacturer.
into five groups containing six animals in each group. Group I (control)
received 0.1 M citrate buffer, diabetic rats received STZ injection
(Group II), diabetic rats orally fed with pioglitazone (as 0.25% CMC
suspension) at a dose of 36 mg/kg (Group III), diabetic rats orally fed
with rosiglitazone (as 0.25% CMC suspension) at a dose of 36 mg/kg
(Group IV), diabetic rats orally fed with synthesized compounds, 5a–o
(as 0.25% CMC suspension) at an equimolar dose of the reference drug
pioglitazone (Group V). The blood glucose level of each group was
checked at 0, 1, 7, and 15 days through glucose oxidase method. The
experiments performed followed the rules of Institutional Animals
Ethics Committee (registration number 173-CPCSEA).[39,40]
|
|
4.3.2 Antidiabetic activity
4.3.3 Biochemical parameters
In vivo antidiabetic study was determined by studying the effects of
orally administered different TZD analogues on glucose tolerance in
normal and streptozotocin (STZ) induced non-insulin-dependent
diabetes mellitus (NIDDM) in rats. Healthy Albino Wistar rats of
either sex (180–250 g) were acquired from Central Animal House
Facility, Jamia Hamdard, New Delhi, and kept at room temperature
with food and water ad libitum. STZ was freshly prepared in 0.1 M
citrate buffer solution (pH 4.5) for inducing diabetes at a dose of
60 mg/kg body weight intraperitoneally to overnight-fasted rats. To
overcome drug-induced hypoglycemia, the animals were permitted to
drink only 5% glucose solution whole night. If the blood glucose level of
animals was found to be 250 mg/dL or above on third day after STZ
injection, they were considered as diabetic. The animals were classified
Serum aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) were performed according to Reitman and Frankel method[41]
and alkaline phosphatase (ALP) was assayed according to Walter and
Schult method[42] using 4-nitrophenyl phosphate as substrate.
|
4.3.4 Hepatotoxicity studies
The hepatotoxicity determination of synthesized analogues was
carried out according to the reported method of Lambert et al.[43]
The animals were classified into five groups containing six animals in
each group. Group I (control) received 0.1 M citrate buffer, diabetic
rats received STZ injection at a dose of 60 mg/kg (Group II), diabetic
rats orally fed with pioglitazone (as 0.25% CMC suspension) at a dose

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NAIM ET AL.
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of 108 mg/kg (Group III), diabetic rats orally fed with rosiglitazone (as
0.25% CMC suspension) at a dose of 108 mg/kg (Group IV), diabetic
rats orally fed with synthesized compounds 5a, 5b, 5i, 5n, and 5o (as
0.25% CMC suspension) at an equimolar dose of the reference drug
pioglitazone (Group V). After 5 h of the test drug administration in rats
at a dose three times higher than the dose for anti-diabetic activity, rats
were sacrificed using low level of anesthesia to remove their liver
specimens which were then preserved in formalin solution (10%).
Morphological examination was conducted to analyze histological
changes in liver using hematoxylin and eosin dye staining and then
examined under microscope.
ORCID
Ozair Alam
http://orcid.org/0000-0003-0322-2509
Md. Mumtaz Alam
http://orcid.org/0000-0001-6420-8757
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ACKNOWLEDGMENTS
The authors are thankful to Jamia Hamdard, New Delhi, India, for
providing facilities for research work, spectral and elemental studies.
The corresponding author (Dr. Ozair Alam) is also thankful to DST-
SERB (SB/FT/LS-203-2012), New Delhi, for providing funds to
purchase molecular modeling software (Schrödinger, USA) under
Fast track young scientist.
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CONFLICT OF INTEREST
The authors declare no conflict of interest.

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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
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How to cite this article: Naim MJ, Alam O, Alam MJ,
Shaquiquzzaman M, Alam MM, Naidu VGM. Synthesis,
docking, in vitro and in vivo antidiabetic activity of pyrazole-
based 2,4-thiazolidinedione derivatives as PPAR-γ
modulators. Arch Pharm Chem Life Sci. 2018;1–15.
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