Design, synthesis, and biological evaluation of thiazolidine-2,4-dione conjugates as PPAR-γ agonists

Article abstract of DOI:10.1002/ardp.201400280

A library of synthesized conjugates of phenoxy acetic acid and thiazolidinedione 5a-m showed potent peroxisome proliferator activated receptor-γ (PPAR-γ) transactivation as well as significant blood glucose lowering effect comparable to the standard drugs pioglitazone and rosiglitazone. Most of the compounds showed higher docking scores than the standard drug rosiglitazone in the molecular docking study. Compounds 5l and 5m exhibited PPAR-γ transactivation of 54.21 and 55.41%, respectively, in comparison to the standard drugs pioglitazone and rosiglitazone, which showed 65.94 and 82.21% activation, respectively. Compounds 5l and 5m significantly lowered the blood glucose level of STZ-induced diabetic rats. Compounds 5l and 5m lowered the AST, ALT, and ALP levels more than the standard drug pioglitazone. PPAR-γ gene expression was significantly increased by compound 5m (2.00-fold) in comparison to the standard drugs pioglitazone (1.5-fold) and rosiglitazone (1.0-fold). Compounds 5l and 5m did not cause any damage to the liver and could be considered as promising candidates for the development of new antidiabetic agents.

Full text of DOI:10.1002/ardp.201400280

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
Archiv der Pharmazie
Full Paper
Design, Synthesis, and Biological Evaluation of
Thiazolidine-2,4-dione Conjugates as PPAR-g Agonists
Syed Nazreen1, Mohammad Sarwar Alam1, Hinna Hamid1, Mohammad Shahar Yar2, Abhijeet Dhulap3,
Perwez Alam4, Mohammad Abdul Qadar Pasha4, Sameena Bano1, Mohammad Mahboob Alam5,
Saqlain Haider1, Chetna Kharbanda1, Yakub Ali1, and Kolakappi Pillai6
1
Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
2
New Delhi, India
3
CSIR Unit for Research and Development of Information Products, Pune, India
Functional Genomics Unit, CSIR-Institute of Genomics & Integrative Biology, Delhi, India
Teva API India Ltd., Greater Noida, India
Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi,
4
5
6
India
A library of synthesized conjugates of phenoxy acetic acid and thiazolidinedione 5a–m showed potent
peroxisome proliferator activated receptor-g (PPAR-g) transactivation as well as significant blood glucose
lowering effect comparable to the standard drugs pioglitazone and rosiglitazone. Most of the
compounds showed higher docking scores than the standard drug rosiglitazone in the molecular docking
study. Compounds 5l and 5m exhibited PPAR-g transactivation of 54.21 and 55.41%, respectively, in
comparison to the standard drugs pioglitazone and rosiglitazone, which showed 65.94 and 82.21%
activation, respectively. Compounds 5l and 5m significantly lowered the blood glucose level of STZ-
induced diabetic rats. Compounds 5l and 5m lowered the AST, ALT, and ALP levels more than the standard
drug pioglitazone. PPAR-g gene expression was significantly increased by compound 5m (2.00-fold) in
comparison to the standard drugs pioglitazone (1.5-fold) and rosiglitazone (1.0-fold). Compounds 5l and
5m did not cause any damage to the liver and could be considered as promising candidates for the
development of new antidiabetic agents.
Keywords: Antidiabetic / Hepatotoxicity / Phenoxy acetic acid / PPAR-g / 2,4-Thiazolidinedione
Received: July 14, 2014; Revised: February 21, 2015; Accepted: February 27, 2015
DOI 10.1002/ardp.201400280
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
and promote glucose utilization in peripheral tissue [1, 2]. The
chemistry of TZDs has attracted attention as they have been
Introduction
2,4-Thiazolidinediones (TZDs) are an important class of
compounds that enhance insulin action (insulin sensitizers)
found to reveal several biological activities [3], such as
antihyperglycemic [1], anti-inflammatory [4, 5], antimalari-
al [6], antioxidant [7], antitumor [8], cytotoxic [9], antimicro-
bial [10], and antiproliferative [11]. Unlike sulfonylurea and
biguanides which lead to severe hypoglycemia, TZDs are not
associated with such hypoglycemic incidents.
TZDs are high-affinity ligands of peroxisome proliferator
activated receptor-g (PPAR-g) [12]. PPAR-g, a member of a large
family of ligand-activated nuclear hormone receptors, is an
important drug target for regulating glucose metabolism [13].
Correspondence: Prof. Mohammad Sarwar Alam, Department of
Chemistry, Faculty of Science, Jamia Hamdard (Hamdard Uni-
versity), New Delhi 110 062, India.
E-mail: msalam@jamiahamdard.ac.in
Fax: þ91 11 26059663
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
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It exists in two forms, mainly PPAR-g1 found in tissues except
muscles and secondly, PPAR-g2 found in adipose tissues and
intestine. These are encoded by PPAR-g gene in humans. It
increases insulin sensitivity at adipose, muscle, and hepatic
tissues [14, 15]. It leads to the channeling of fatty acids into
adipose tissue and reduces their concentration in the plasma.
Reducing the fatty acid availability for muscles alleviates insulin
resistance, thereby, improving plasma glucose levels effective-
ly [16, 17]. Keeping in view the biological importance of
thiazolidinediones as antidiabetic agents, we herein report the
synthesis and in silico molecular docking study of thiazolidine-
dione and phenoxy acetic acid conjugates 5a–m. These
conjugates have been evaluated for in vitro PPAR-g trans-
activation activity, in vivo antidiabetic activity with hepatotox-
icity risk evaluation, and PPAR-g gene expression.
studies were done to provide insights of molecular binding
modes of molecules inside the large pocket of PPAR-g
receptors. The compounds were docked against grid gener-
ated by Schrodinger glide software. In order to analyze the
binding pattern and energies of new molecules and reference
ligand (rosiglitazone), they were docked individually against
the generated grid. Initial docking of reference ligand
(rosiglitazone) against generated grid helped in validating
the grid generated by software. Rosiglitazone is reported to
have H-bonding with TYR 473, HIS 449, and CYS 285 [20, 21].
Docking of rosiglitazone against generated grid showed
similar docking mode and H-bonds with RMSD value of 2.8
and hence, validated the generated grid. The synthesized
molecules docked against the grid showed good binding
energies ranging from ꢀ59.9 to ꢀ31.6 kcal/mol. All the
molecules showed good glide score but the most promising
molecules were 5b, 5c, 5g, 5h, and 5j–m with glide score of
ꢀ7.15, ꢀ6.36, ꢀ6.50, ꢀ6.54, ꢀ6.57, ꢀ6.57, ꢀ7.04, and ꢀ7.03,
respectively, whereas glide score of rosiglitazone was
ꢀ5.77 kcal/mol with respect to the same grid. Compounds
5c and 5j showed hydrogen bonding with SERB 342 residue of
the protein whereas compounds 5b, 5l, and 5m were found to
be aligned perfectly with the hydrophobic pocket of the
protein (Fig. 1). The docking score and in silico ADME
(absorption, distribution, metabolism, and excretion) predic-
tion of the synthesized library are found to be within the
acceptable range (Table 1).
Results and discussion
Chemistry
Treatment of different hydroxy benzaldehydes 1a–m with
chloroacetic acid in the presence of diethyl ether and NaOH
solution yielded different substituted formyl phenoxy acetic
acids 2a–m [18] which upon Knoevenagel condensation with
thiazolidine-2,4-dione 3 in the presence of absolute alcohol and
NaOH at ꢀ5 to 0°C for 12–14 h yielded intermediates 4a–m [19].
Reduction of intermediates 4a–m with sodium borohydride in
the presence of CoCl2–DMG complex in DMF for 5–6 h resulted in
the hydrogenation of exocyclic double bond between TZD and
aromatic ring leading to the formation of target compounds 5a–
m (Scheme 1). A library of 13 new conjugates of phenoxy acetic
acid and TZD has been synthesized in good yields.
Biological activities
In vitro PPAR-g transactivation assay
The compounds showing good glide scores (>ꢀ6.50) in
molecular docking study were screened for in vitro PPAR-g
transactivation activity in order to confirm their mode of
action. It was observed from Fig. 2 that compounds 5b, 5g,
5h, and 5j–m were found to be PPAR-g active. Compounds 5b,
5l, and 5m exhibited PPAR-g transactivation of 48.35, 54.21,
and 55.41%, respectively, in comparison to standard drugs
pioglitazone and rosiglitazone which showed 65.94 and
82.21% activation, respectively. Compounds 5g, 5h, 5j, and
5k showed moderate in vitro PPAR-g transactivation activity.
The most active compound 5m was further tested for in vitro
PPAR-a and PPAR-d assay. It was observed that compound 5m
was found to be inactive PPAR-a and PPAR-d (Table 2).
Compound 5m showed PPAR-g of EC50 of 0.77 in comparison
to standard drug rosiglitazone which showed EC50 of 0.058.
1H NMR, 13C NMR, and mass spectral data were found to be
in agreement with the proposed structures of all the newly
synthesized compounds. Formation of substituted formyl
phenoxy acetic acids 2a–m was confirmed by the presence of a
singlet at d 4.62–4.89 for O–CH2 protons and carboxylic acid
protons appeared as a broad singlet at d 11.02–13.07 in the 1H
NMR spectra. Formation of condensed product 4a–m was
confirmed by the absence of aldehydic protons at d 9.85–10.73
and the presence of exocyclic olefinic proton as a singlet at d
7.72–7.74 in the 1H NMR spectra. Formation of target
compounds 5a–m was confirmed by the absence of exocyclic
olefinic proton singlet and the presence of characteristic three
double doublets at d 4.78–4.98, 3.18–3.63, and 2.94–3.19 due
to the newly generated methine and methylene protons in
the 1H NMR spectra. In the 13C NMR spectrum of 5a–m,
appearance of peaks at d 50.06–57.26 for newly generated
methine carbon and d 31.29–38.16 for methylene carbon
further confirmed the reduction of exocyclic olefinic bond.
Further confirmatory evidence was obtained from the mass
spectral data of the compounds.
In vivo blood glucose lowering effect
These compounds (5b, 5g, 5h, and 5j–m) were then
evaluated for the blood glucose lowering effect in STZ-
induced (streptozotocin) diabetic rats. STZ-induced diabetic
model was used for the in vivo blood glucose lowering effect
as it is a more preferred diabetic model than other standard
rodent diabetes models, due to its advantages such as longer
half-life time and hyperglycaemia duration and better
established diabetic complications with lower possibility of
ketosis or mortality incidents [22]. The synthesized
Molecular docking studies
The above synthesized compounds 5a–m were docked for
in silico studies against PPAR-g target. Molecular docking
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2,4-Thiazolidinedione Conjugates as PPAR-g Agonists
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O
H
O
H
O
(a)
Cl
R1
OH
S
R1
HO
R
2a–m
1a–m
S
O
O
(b)
(c)
NH
R
NH
S
O
R
R1
5a–m
O
O
R1
4a–m
N
H
O
3
Compds
Compds
R1
H
R1
H
R
R
5a 4-CH2COOH
5b 4-CH2COOH
5h
2-CH2COOH
2-CH2COOH
2-CH2COOH
3-OMe
5i
5j
5-NO2
3-OEt
5-Cl
5c 4-CH2COOH 3-OEt
5d 4-CH2COOH 3,5-DiOMe 5k 2-CH2COOH
5e 4-CH2COOH 5-Me 5l 2-CH2COOH
5f 4-CH2COOH 3,5-DiMe
5g 4-CH2COOH 3-Br
5-Br
5m 2-CH2COOH 5-Br, 3-OMe
Scheme 1. Synthesis of compounds 5a–m.
compounds 5a–m were administered a single dose on the first
day and the blood glucose level was measured on the 1st, 7th,
and 15th day of the experiment as per standard protocols. As
observed from the data of Fig. 3, it was found that these
compounds significantly lowered the blood glucose level in
diabetic rats. Compounds 5b, 5l, and 5m significantly lowered
blood glucose level to 157.5 ꢁ 5.01, 158.8 ꢁ 7.75, and
159.2 ꢁ 5.50, respectively, comparable to standard drugs
pioglitazone (134.2 ꢁ 4.52) and rosiglitazone (142.2 ꢁ 6.12).
Compounds 5j and 5k showed moderate blood glucose
lowering whereas compounds 5g and 5h did not show
promising lowering in blood glucose level. It was found that
the results of in vitro study were consistent with in vivo results
indicating that these compounds have exerted the blood
glucose lowering effect by activating PPAR-g receptors.
As the PPAR-g agonists are associated with body weight gain,
5m was also tested for body weight gain study. Compound
5m treated normal rats did not show any significant change in
body weight as compared to normal control rats. However,
oral administration of 5m to diabetic rats for 15 days caused a
significant improvement in body weight of diabetic rats
(Fig. 4).
Hepatotoxicity studies
TZDs are reported to cause hepatotoxicity which is the major
drawback encountered with this class of drug [23]. The active
compounds (5b, 5j–m) have, therefore, been assayed for
aspartate transaminase (AST), alanine transaminase (ALT),
and alkaline phosphatase (ALP) levels and histopathological
study of the liver. It has been reported that the levels of these
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Figure 1. Molecular docking of the compounds against the PPAR-g target.
enzymes are significantly increased in STZ rats indicating the
toxic effect of STZ on liver [24]. 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 ketoacids. It was
observed that the levels of serum AST, ALT, and ALP
significantly increased in STZ treated rats were significantly
decreased to near normal level after treatment with the
compounds 5b and 5j–m. Compounds 5l and 5m were found
to be more potent in lowering the AST, ALT, and ALP levels
than the standard drug pioglitazone. Compounds 5b, 5j, and
5k were equally potent to standard drug pioglitazone in
lowering the AST, ALT, and ALP levels to normal level,
thereby, suggesting that these compounds have protective
effect on liver (Fig. 5).
Histopathological study of the liver of the treated animals
also showed that the compounds 5b, 5j, 5l, and 5m did not
cause any damage to the liver whereas compound 5k and
standard drug pioglitazone caused mild dilation in sinusoidal
space (Fig. 6).
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Table 1. Docking score and ADME prediction of the synthesized compounds 5a–m.
No.
Compound
G-Score
G-Energy
Log Po/w
PSA
Log S
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
Rosi
ꢀ5.30
ꢀ7.15
ꢀ6.36
ꢀ4.25
ꢀ5.68
ꢀ5.42
ꢀ6.50
ꢀ6.54
ꢀ5.81
ꢀ6.57
ꢀ6.57
ꢀ7.04
ꢀ7.03
ꢀ5.77
ꢀ40.60
ꢀ59.96
ꢀ42.90
ꢀ37.54
ꢀ31.78
ꢀ31.67
ꢀ45.97
ꢀ37.95
ꢀ41.56
ꢀ57.66
ꢀ47.33
ꢀ43.91
ꢀ42.38
ꢀ71.55
1.10
1.58
1.61
1.46
1.37
1.81
1.62
1.27
0.52
1.28
1.56
1.91
1.71
3.47
130.11
137.25
137.48
143.65
128.76
123.66
132.207
128.13
176.27
131.10
130.64
125.88
138.83
94.37
ꢀ2.33
ꢀ2.93
ꢀ2.95
ꢀ2.48
ꢀ2.57
ꢀ3.05
ꢀ3.00
ꢀ2.51
ꢀ2.75
ꢀ2.70
ꢀ2.72
ꢀ3.3
9
10
11
12
13
14
ꢀ3.17
ꢀ4.49
The bold values signify the glide score of these compounds are higher than that of the standard drug Rosiglitazone.
PPAR-g gene expression activity
study. The PPAR-g gene expression study was done to know
the impact of compound 5m on PPAR-g gene. It was observed
that the PPAR-g expression was significantly increased in
presence of compound 5m (2.00-fold) in comparison to the
standard drugs rosiglitazone (1.0-fold) and pioglitazone (1.5-
fold) (Fig. 7). The increase in PPAR-g gene expression supports
the results of in vitro PPAR-g transactivation study. It is thus
clear that the in vitro PPAR-g transactivation and in vivo
blood glucose lowering activity of compound may be due to
increase in the PPAR-g gene expression. It has been reported
that 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 [25–27]. The
increase in gene expression by compound 5m might be due
to the activation of AMPK. The 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 differentiation [28]. 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, compound 5m
increases the gene expression by maintaining insulin sensitiv-
ity 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.
Since compound 5m was found to be most PPAR-g active,
it was further subjected to PPAR-g gene expression study.
Compound 5m (10 mM) and pioglitazone (10 mM) and
rosiglitazone (10 mM) were used for the gene expression
A
90
80
70
60
50
40
30
20
10
0
10 μM
Control Rosi
Piog
5b
5g
5h
5j
5k
5l
5m
B
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
From the biological data, the structure–activity relationship
(SAR) can be drawn as follows. Compounds having o-
substituted phenoxy acetic acid were more active in lowering
the blood glucose level than p-substituted compounds (5h
> 5a). Compounds containing OCH3 and halogens (Cl, Br) on
the aromatic ring showed promising activity (5b, 5l, 5m)
whereas compound with NO2 group decreased the activity
(5i). Increase in number of methoxy groups on the aromatic
ring decreased the activity (5d). Increase in number of alkyl
group decreased the activity (5f).
BL
Rosi
5m
Figure 2. (A) In vitro PPAR-g transactivation assay of the
compounds. Values are expressed as mean ꢁ SE from three
experiments conducted in triplicate at 10 mM. (B) PPAR-g
binding data of compound 5m. Activity was measured using
Lantha Screen TM TR-FRET PPAR-g competitive binding assay. BL
(blank control, without PPAR-g LBD), Rosi (rosiglitazone).
Activity was assayed at 100 mM. The fluorescence ratio at 520/
495 nm is shown.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
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Table 2. In vitro data of compound 5m.
PPAR-g
Compound
PPAR-a (10 mM)
PPAR-d (10 mM)
EC50 (mM)
%max
5m
IA
IA
0.77
ND
0.058
ND
65
ND
100
ND
WY14643
Rosiglitazone
GW-501516
4.3
ND
ND
ND
ND
5.2
IA, inactive; ND, not determined.
DS melting point apparatus and are uncorrected. 1H NMR
was recorded on Bruker DPX 400, 300 instruments in CDCl3/
DMSO-d6 using TMS as internal standard. Chemical shifts and
coupling constants J are given in parts per million and hertz,
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 are reported in m/z. Elemental analysis
was carried out using Elemental Vario EL III elemental analyzer.
Elemental analysis data are reported in percent standard.
Conclusion
A library of 13 novel conjugates of phenoxy acetic acid and
thiazolidinedione have been synthesized, out of which two
compounds, 5l and 5m, exhibited significant in vitro PPAR-g
transactivation and in vivo blood glucose lowering effect,
comparable to standard drugs rosiglitazone and pioglitazone.
Compounds 5l and 5m recovered the activity of serum AST,
ALT, and ALP levels and did not cause any damage to the liver.
Compound 5m increases the PPAR-g expression by 2.0-fold in
comparison to standard drug rosiglitazone (1.0-fold) and
pioglitazone (1.5-fold). Compounds 5l and 5m may be
considered as promising candidate for development of new
antidiabetic agents.
Synthesis of compounds 2a–m
To a solution of substituted hydroxy benzaldehyde (0.01 M) in
100 mL of ether, 25 g of sodium hydroxide dissolved in 50 mL
of water was added slowly while the solution was cooled and
stirred. To this mixture, a solution of chloroacetic acid (0.01 M)
in 30 mL of water was added in the same way. The ether was
removed and the mixture was refluxed for 2 h at 120–125°C.
The resulting solution was cooled, diluted with water,
acidified with concentrated HCl to Congo red, and allowed
to stand for 2–3 h at room temperature. The precipitate so
formed was filtered, dissolved in sodium bicarbonate
Experimental
Chemistry
All chemicals (reagent grade) used were commercially avail-
able. Melting points were measured on a VEEGO-VMP-
Figure 3. Antidiabetic activity of the 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 and *p < 0.05 versus diabetic control, Diab. Ctrl,
diabetic control; Ctrl, normal control; Piog, pioglitazone; Rosig, rosiglitazone.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–12
2,4-Thiazolidinedione Conjugates as PPAR-g Agonists
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reaction mixture was cooled and filtered. The filtrate so
obtained was acidified with 1N HCl to bring the pH 3 and
stirred for 30 min. The solid so obtained was filtered and dried
to yield the pure target compounds 5a–m. The target
compounds were crystallized in MeOH.
1 day
15 day
250
200
150
100
50
5-(4-Carboxymethoxybenzyl)-2,4-thiazolidinedione (5a)
White crystals; yield: 70%; m.p. 115–117°C; IR (KBr): n (cmꢀ1
)
3421, 3325, 1699, 1684, 1541, 1145; 1H NMR (300MHz, DMSO-
d6):d3.05(dd,1H, J ¼ 8.8,14.1 Hz),3.30(dd,1H, J ¼ 4.2,14.1 Hz),
4.64 (s, 2H O–CH2), 4.87 (dd, 1H, J ¼ 4.2, 9.1 Hz), 6.84 (d, 2H,
J ¼ 8.5 Hz, Ar-H), 7.15 (d, 2H, J ¼ 8.6 Hz, Ar-H), 12.46 (s, 2H, NH,
OH); 13C NMR (75 MHz, DMSO-d6): d 36.33, 53.08, 69.45, 114.36,
128.52, 130.44, 157.80, 170.34, 171.83, 175.82; MS FAB (þve):
282 (Mþ1). Anal. calcd. for C12H11NO5S: C, 51.24; H, 3.94;
N, 4.98; S, 11.40. Found: C, 51.20; H, 3.89; N, 4.94; S, 11.42%.
0
NC
NC+5m
STZ
STZ+5m
Figure 4. Effect of compound 5m 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 þ 5m, normal control þ 5m; STZ, diabetic control;
STZ þ 5m, 5m treated diabetic rats.
5-(4-Carboxymethoxy-3-methoxybenzyl)-2,4-
thiazolidinedione (5b)
White crystals; yield: 80%; m.p. 161–162°C; IR (KBr): n (cmꢀ1
)
3446, 3181, 1649, 1518, 1149; 1H NMR (300 MHz, DMSO-d6): d
3.03(dd, 1H, J ¼ 8.0, 12.0 Hz), 3.33(dd, 1H, J ¼ 4.1, 14.0 Hz), 3.77
(s, 3H, O–CH3), 4.63(2H, s, O–CH2), 4.91(dd, 1H, J ¼ 4.0, 12.0 Hz),
6.73(d,1H, J ¼ 8.0 Hz, Ar-H), 6.79(d,1H, J ¼ 8.0 Hz, Ar-H), 6.91(s,
1H, Ar-H), 12.10 (s, 2H, NH, OH); 13C NMR (75 MHz, DMSO-d6):
37.88, 53.49, 55.93, 65.36, 113.33, 113.61, 121.42, 130.44,
146.65, 149.03, 170.72, 172.24, 176.2; MS FAB (þve): 312 (Mþ1).
Anal. calcd. for C13H13NO6S: C, 50.16; H, 4.21; N, 4.50; S, 10.30.
Found C, 50.10; H, 4.18; N, 4.47; S, 10.33%.
solution, and extracted with ether. The aqueous layer was
acidified with concentrated HCl to Congo red and left for 2–
3 h at 5–10°C. The precipitate so formed was filtered and
crystallized from methanol. The experimental data of 2a–m
were compared with the reported literature data [18].
Synthesis of chalcones 4a–m [19]
5-(4-Carboxymethoxy-3-ethoxybenzyl)-2,4-
To a stirred solution of the substituted phenoxy acetic acid 2a–m
(0.01 M) and the appropriate aldehyde (0.01 M) in ethanol (30 mL),
40%aqueoussodiumhydroxide(20 mL)wasaddedportionwiseat
room temperature for 10 min. The mixture was kept overnight at
room temperature and then it was poured into crushed ice and
acidified with concentrated HCl. The solid separated was filtered
and crystallized from ethanol. The experimental data of 4a–m
were compared with the reported literature data [29].
thiazolidinedione (5c)
Yellow crystals; yield: 65%; m.p. 158–159°C; IR (KBr): n (cmꢀ1
)
3440, 3241, 1715, 1659, 1524, 1157; 1H NMR (300 MHz, DMSO-
d6): d 1.70 (t, 3H, J ¼ 7.2 Hz), 3.01 (dd, 1H, J ¼ 8.2, 12.1 Hz), 3.32
(dd, 1H, J ¼ 4.6, 14.1 Hz), 4.07 (q, 2H, J ¼ 6.6 Hz, O–CH2), 4.34
(2H, s, O–CH2), 4.78 (dd, 1H, J ¼ 4.5, 12.2 Hz), 6.48 (d, 1H,
J ¼ 8.8 Hz, Ar-H), 6.54 (d, 1H, J ¼ 8.4 Hz, Ar-H), 6.65 (s, 1H, Ar-
H), 12.10 (s, 2H, NH, OH); 13C NMR (75 MHz, DMSO-d6): d 15.10,
36.25, 53.52, 64.28, 66.15, 113.66, 114.94, 121.61, 130.10,
147.17, 148.18, 170.58, 171.28, 175.33; MS FAB (þve): 326
(Mþ1). Anal. calcd. for C14H15NO6S: C, 51.68; H, 4.65; N, 4.31;
S, 9.86. Found: C, 51.64; H, 4.63; N, 4.28; S, 9.81%.
Synthesis of target compounds 5a–m
In a 100 mL 3-neck RBF equipped with a thermometer and
mechanical stirrer, compounds 4a–m (0.01 mol) were charged.
Then 3 mL of H2O, 2 mL of MeOH, 3 mL of 1N NaOH were
added to the reaction mixture with constant stirring for
15 min. After 15 min, freshly prepared 1.5 mL CoCl2–DMG
complex solution (prepared by dissolving 42 mg CoCl2ꢂ6H2O
þ 250 mg DMG in 5 mL DMF) was added dropwise to the
reaction mixture and the stirring was continued for 15 min at
25°C. Freshly prepared sodium borohydride solution (eight
equivalents) was added at once to the reaction mixture and
temperature was increased to 35°C. On addition of sodium
borohydride solution, the color of the reaction mixture
changed to deep violet for seconds and then to gray. The
reaction was continued for 3 h at 35°C with constant stirring.
After the completion of the reaction, monitored by TLC, the
5-(4-Carboxymethoxy-3,5-dimethoxybenzyl)-2,4-
thiazolidinedione (5d)
White crystals; yield: 70%; m.p. 168–169°C; IR (KBr): n (cmꢀ1
)
1
3452, 3179, 3067, 1673, 1692, 1124, 1031; H NMR (400 MHz,
DMSO-d6): d 3.10 (dd, 1H, J ¼ 8.8, 14.08 Hz), 3.35 (dd, 1H,
J ¼ 4.32, 14.2 Hz), 3.75 (6H, s, O–CH3), 4.43 (2H, s, O–CH2), 4.89
(dd, 1H, J ¼ 4.36, 8.68 Hz), 6.58 (s, 2H, Ar-H), 12.10 (s, 1H, NH),
12.72 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6): d 38.16, 53.27,
56.46, 69.18, 106.94, 133.36, 135.06, 152.66, 170.60, 172.23,
176.24; MS FAB (þve): 342 (Mþ1). Anal. calcd. for C14H15NO7S:
C, 49.26; H, 4.43; N, 4.10; S, 9.39. Found: C, 49.24; H, 4.40; N,
4.14; S, 9.42%.
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ALT
AST
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Control STZ
5b
5j
5k
5l
5m
Piog
Control STZ
5b
5j
5k
5l
5m
Piog
ALP
140
120
100
80
60
40
20
0
Control STZ
5b
5j
5k
5l
Figure 5. Effect of compounds on serum AST, ALT, and ALP activities. Values are given as mean ꢁ SD.
5-(4-Carboxymethoxy-3-methylbenzyl)-2,4-
DMSO-d6): d 2.19 (s, 6H), 2.95 (dd, 1H, J ¼ 9.9, 14.1 Hz), 3.29
(dd, 1H, J ¼ 3.9, 13.8 Hz), 4.34 (2H, s, O–CH2), 4.85 (dd, 1H,
J ¼ 4.2, 9.9 Hz), 6.86 (s, 2H, Ar-H), 12.18 (s, 2H, NH, OH); 13C
NMR (75 MHz, DMSO-d6): d 16.12, 36.77, 52.89, 68.77, 129.47,
130.36, 132.57, 154.25, 170.31, 171.77, 175.79; MS ES (ꢀve):
309 (M)þ. Anal. calcd. for C14H15NO5S: C, 54.36; H, 4.89; N,
4.53; S, 10.37. Found: C, 54.30; H, 4.91; N, 4.55; S, 10.33%.
thiazolidinedione (5e)
White crystals; yield: 75%; m.p. 162–163°C; IR (KBr): n (cmꢀ1
)
1
3445, 3177, 3043, 1725, 1676, 1520, 1073; H NMR (300 MHz,
DMSO-d6): d 2.12 (s, 3H), 3.02 (dd, 1H, J ¼ 8.2, 12.4 Hz), 3.31 (dd,
1H, J ¼ 4.8, 14.0 Hz), 4.46 (2H, s, O–CH2), 4.82 (dd, 1H, J ¼ 4.5,
12.3 Hz), 6.72 (d, 1H, J ¼ 8.4 Hz, Ar-H), 6.78 (d, 1H, J ¼ 8.8 Hz, Ar-
H), 6.92 (s, 1H, Ar-H), 12.12 (s, 2H, NH, OH); 13C NMR (75 MHz,
DMSO-d6): d 15.10, 36.73, 56.63, 67.96, 114.63, 128.56, 129.64,
130.33, 132.51, 155.36, 170.33, 171.78, 175.63; MS FAB (þve):
296(Mþ1).Anal.calcd. forC13H13NO5S:C,52.87;H,4.44;N,4.74;
S, 10.86. Found: C, 52.83; H, 4.46; N, 4.72; S, 10.83%.
5-(4-Carboxymethoxy-3-bromobenzyl)-2,4-
thiazolidinedione (5g)
White crystals; yield: 70%; m.p. 188–190°C; IR (KBr): n (cmꢀ1
)
3450, 3176, 1705, 1680, 1230, 1110; 1H NMR (300 MHz, DMSO-
d6): d 2.96 (dd, 1H, J ¼ 8.4, 14.2 Hz), 3.34 (dd, 1H, J ¼ 4.5,
14.8 Hz), 4.77 (2H, s, O–CH2), 4.92 (dd, 1H, J ¼ 4.8, 12.5 Hz),
6.72 (d, 1H, J ¼ 8.4 Hz, Ar-H), 6.85 (d, 1H, J ¼ 8.8 Hz, Ar-H), 6.96
(s, 1H, Ar-H), 12.10 (s, 2H, NH, OH); 13C NMR (75 MHz, DMSO-
d6): d 36.34, 57.26, 67.16, 114.78, 122.28, 129.56, 130.58,
5-(4-Carboxymethoxy-3,5-dimethylbenzyl)-2,4-
thiazolidinedione (5f)
White crystals; yield: 72%; m.p. 172–174°C; IR (KBr): n (cmꢀ1
)
1
3450, 3179, 1699, 1682, 1521, 1145, 1031. H NMR (300 MHz,
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Figure 6. Hematoxylin and eosin immunohistochemical staining of liver after administration of synthesized drugs. Histopathology
report of rat liver. As illustrated in the above figure, low and high power photomicrograph of liver from animal treated groups 5b,
5j–5m, and standard (10ꢃ). Low power photomicrograph of liver from corresponding animal 5b, 5j–5m, and standard treated groups
showing arrangement of cells in the liver lobule. PT ¼ portal triad and CV ¼ central vein (40ꢃ). High power photomicrograph of liver
from animal treated groups 5b, 5j–5m, and standard showing arrangement of hepatocytes in the centrizonal area. CV ¼ central vein.
Compounds 5b, 5j, 5l, and 5m treated groups showing normal arrangement of cells in the liver lobule and normal arrangement of
hepatocytes in the centrizonal area. 5k and pioglitazone treated groups showing a mild dilatation of sinusoidal spaces around the
central vein and mild inflammation in portal vein.
133.57, 156.24, 170.23, 171.47, 175.46; MS FAB (þve): 359
(Mþ1), 361 (Mþ3). Anal. calcd. for C12H10BrNO5S: C, 40.02; H,
2.80; N, 3.89; S, 8.90. Found: C, 40.06; H, 2.82; N, 3.85; S, 8.87%.
J ¼ 7.5, 14.16 Hz), 4.78 (2H, s, O–CH2), 4.97 (dd, 1H, J ¼ 4.8,
9.9 Hz), 6.90 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.12–7.57 (m, 2H, Ar-H),
12.05 (s, 1H, NH), 12.35 (s, 1H, OH). 13C NMR (75 MHz, DMSO-
d6): d 31.29, 50.06, 63.62, 113.28, 113.89, 129.05, 132.19,
133.50, 149.56, 170.32, 172.51, 175.89; MS FAB (þve): 326
(M)þ. Anal. calcd. for C12H10N2O7S: C, 44.17; H, 3.09; N, 8.59; S,
9.83. Found: C, 44.13; H, 3.08; N, 8.63; S, 9.80%.
5-(2-Carboxymethoxybenzyl)-2,4-thiazolidinedione (5h)
White crystals; yield: 75%; m.p. 98–99°C; IR (KBr): n (cmꢀ1) 3446,
3176, 1680, 1651, 1558, 1160, 1074; 1H NMR (400MHz, DMSO-
d6): d 2.94 (dd, 1H, J ¼ 8.9, 14.10Hz), 3.59 (dd, 1H, J ¼ 4.5,
14.16 Hz), 4.75 (2H, s, O–CH2), 4.98 (dd, 1H, J ¼ 4.4, 10.56 Hz),
5-(2-Carboxymethoxy-3-ethoxybenzyl)-2,4-
6.89–7.23 (m, 4H, Ar-H), 12.03 (s, 1H, NH), 12.88 (s, 1H, OH). 13
C
thiazolidinedione (5j)
NMR (75 MHz, DMSO-d6): d 36.31, 56.25, 68.85, 114.16, 128.32,
129.32, 131.21, 156.70, 170.14, 170.83, 174.92; MS FAB (þve):
282(Mþ1).Anal.calcd. forC12H11NO5S:C,51.24;H,3.94;N,4.98;
S, 11.40. Found: C, 51.20; H, 3.93; N, 4.96; S, 11.38%.
Brown crystals; yield: 70%; m.p. 115–116°C; IR (KBr): n (cmꢀ1
)
1
3421, 3131, 1702, 1675, 1541, 1457, 1206; H NMR (300 MHz,
DMSO-d6): d 1.54 (t, 3H, J ¼ 7.2 Hz), 3.19 (dd, 1H, J ¼ 10.2,
14.1 Hz), 3.63(dd, 1H, J ¼ 4.8, 13.8 Hz), 4.04(q, 2H, J ¼ 6.9 Hz, O–
CH2), 4.72 (2H, s, O–CH2), 4.91 (dd, 1H, J ¼ 4.8, 9.9 Hz), 6.76–7.00
(m, 3H, Ar-H), 12.05 (s, 1H, NH), 12.33 (s, 1H, OH); 13C NMR
(75 MHz, DMSO-d6): d 15.05, 35.84, 55.52, 63.42, 69.25, 113.38,
122.21, 124.17, 130.85, 145.85, 150.96, 171.19, 172.29, 176.39;
MS FAB (þve): 326 (Mþ1). Anal. calcd. for C14H15NO6S: C, 51.68;
H,4.65;N,4.31;S,9.86.Found:C,51.65;H,4.62;N,4.30;S,9.89%.
5-(2-Carboxymethoxy-5-nitrobenzyl)-2,4-
thiazolidinedione (5i)
Yellow crystals; yield: 65%; m.p. 172–173°C; IR (KBr): n (cmꢀ1
3421, 3307, 1682, 1650, 1520, 1338, 1073; H NMR (300 MHz,
)
1
DMSO-d6): d 2.99 (dd, 1H, J ¼ 10.5, 13.5 Hz), 3.44 (dd, 1H,
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12.05(s, 1H, NH), 12.78(s, 1H, OH); 13CNMR(75 MHz, DMSO-d6):
d 32.48, 51.58, 56.68, 69.11, 115.65, 115.74, 124.78, 132.96,
145.14, 152.74, 170.92, 172.08, 176.20; MS FAB (þve): 389
(Mþ1), 391 (Mþ3). Anal. calcd. for C13H12BrNO6S: C, 40.01; H,
3.10; N, 3.59; S, 8.22. Found: C, 39.98; H, 3.12; N, 3.56; S, 8.20%.
2.5
2
1.5
1
Molecular docking study
Molecular docking studies involve mainly protein selection
and preparation, grid generation, ligand preparation, dock-
ing, and further analysis of docking studies. Schrodinger
software was mainly used for all the above steps.
0.5
0
Protein selection and preparation
Protein with Accession Number 3CS8 was selected and
downloaded from Protein Data Bank. This protein is reported
to bind with drug rosiglitazone. The protein was imported,
optimized, and minimized by removing unwanted molecules
and other defects reported by the software. PPAR-g receptor
is a dimer which has two monomers chains (A and B). For the
purpose of studies, chain B was deleted and water molecules
DMSO
Rosi
piog
5m
Figure 7. Effect of compound 5m on PPAR-g gene expression.
The experiments were conducted at 10 mM. Rosi, rosiglitazone;
piog, pioglitazone. PCR was performed in triplicate and was
repeated two times for each gene and each sample. Relative
transcript quantities were calculated using the Ct method with
b-actin as the endogenous reference gene.
near the ligands were retained. Finally
a low energy
minimized protein structure was obtained and used for
further docking studies.
5-(2-Carboxymethoxy-5-chlorobenzyl)-2,4-
thiazolidinedione (5k)
Grid generation
Yellow crystals; yield: 80%; m.p. 182–183°C; IR (KBr): n (cmꢀ1
)
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.
1
3420, 3129, 2925, 1699, 1684, 1152, 1070; H NMR (300 MHz,
DMSO-d6): d 2.97 (dd, 1H, J ¼ 9.8, 12.5 Hz), 3.18 (dd, 1H, J ¼ 7.3,
13.10 Hz), 4.70 (2H, s, O–CH2), 4.82 (dd, 1H, J ¼ 5.4, 10.25 Hz),
6.75 (d, 1H, J ¼ 8.8 Hz, Ar-H), 7.22–7.58 (m, 2H, Ar-H), 11.98 (s,
1H, NH), 12.60 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6): d 34.25,
52.76, 66.35, 113.23, 114.42, 123.85, 131.50, 132.27, 148.08,
170.48, 172.09, 176.14; MS FAB (þve): 315 (M)þ, 317 (Mþ2).
Anal. calcd. for C12H10ClNO5S: C, 45.65; H, 3.19; N, 4.44; S, 10.16.
Found: C, 45.61; H, 3.16; N, 4.41; S, 10.18%.
Ligand preparation
Molecules drawn in 3D form were refined by LigPrep module.
The molecules were subjected to OPLS-2005 force field to
generate single low energy 3D structure for each input
structure. During this step chiralities were maintained.
5-(2-Carboxymethoxy-5-bromobenzyl)-2,4-
Docking studies
thiazolidinedione (5l)
Docking studies were carried using Glide software. It was
carried using Extra precision and write XP descriptor
information. This generates favorable 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.
White crystals; yield: 85%; m.p. 105–106°C; IR (KBr): n (cmꢀ1
)
1
3342, 3241, 1698, 1675, 1547, 1105, 1029; H NMR (300 MHz,
DMSO-d6): d 2.99 (dd, 1H, J ¼ 10.5, 13.5 Hz), 3.44 (dd, 1H,
J ¼ 7.5, 14.16 Hz), 4.81 (2H, s, O–CH2), 4.97 (dd, 1H, J ¼ 4.8,
9.9 Hz), 6.90 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.12–7.57 (m, 2H, Ar-H),
12.35 (s, 2H, NH, OH); 13C NMR (75 MHz, DMSO-d6): d 33.35,
51.02, 65.31, 112.28, 114.52, 128.65, 131.08, 133.47, 155.68,
170.42, 172.12, 176.24. MS FAB (þve): 359 (Mþ1), 361 (Mþ3).
Anal. calcd. for C12H10BrNO5S: C, 40.02; H, 2.80; N, 3.89; S, 8.90.
Found: C, 40.00; H, 2.82; N, 3.86; S, 8.87%.
Biological activities
In vitro PPAR-g transactivation assay
5-(2-Carboxymethoxy-5-bromo-3-methoxybenzyl)-2,4-
Human embryonic kidney (HEK) 293 cells were cultured in
DMEM with 10% heat inactivated fetal bovine serum in a
humidified 5% CO2 atmosphere at 37°C. Cells were seeded
in six-well plates the day before transfection to give a
confluence of 70–80% at transfection. Cells grown in DMEM
were inoculated in 96-well plates containing 60000 cells/well.
Cells were transfected with 2.5 mL of PPRE-Luc, 6.67 mL of
thiazolidinedione (5m)
White crystals; yield: 70%; m.p. 135–136°C; IR (KBr): n (cmꢀ1
)
3450, 3177, 1684, 1521, 1419, 1216, 1151, 1051; 1H NMR
(300 MHz, DMSO-d6): d 3.13 (dd, 1H, J ¼ 9.6, 13.8 Hz), 3.59 (dd,
1H, J ¼ 5.4, 14.10 Hz), 3.86 (s, 3H, O–CH3), 4.59 (2H, s, O–CH2),
4.93 (dd, 1H, J ¼ 5.1, 9.6 Hz), 7.16 (s, 1H, Ar-H), 7.01 (s, 1H, Ar-H),
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PPAR-g, 1.0 mL of Renilla, and 20 mL of Lipofectamine.
Following 5 h after transfection, cells were treated with
compound (10 mM) for 24 h and then collected with cell
culture lysis buffer. Luciferase activity was monitored on
luminometer (Perkin Elmer, USA) using the luciferase assay kit
(Promega) according to the manufacturer’s instructions.
Rosiglitazone and pioglitazone were used as standard drug.
The results are summarized in Fig. 2.
PPAR-g gene expression study
Cell culture experiments: 3T3-L1 cells (ATCC) were seeded in
24-well plates 24 h before treatment in DMEM containing
10% calf serum (Invitrogen). After 24 h, cells were treated
with compound 5m (10 mM), and pioglitazone (10 mM), and
rosiglitazone (10 mM) as positive control and DMSO as
negative control followed by a 24 h of incubation of cells in
CO2 incubator at 37°C and 5% CO2.
RNA extraction, reverse transcription (RT), and gene
expression analysis: After 24 h, cells were scraped off and
collected in 1.5 mL microcentrifuge tubes. The total RNA
was isolated by TRI Reagent1 (Molecular Research Center).
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)-polymerase chain reaction (PCR) (Bio-
logical Industries). Primers for real-time PCR were designed
for PPAR-g and b-actin using the Pearl Primer software.
Reactions were run at 95°C for 10 min followed by 40 cycles
of 95°C for 15 s and 60°C for 1 min. Real-time PCR was
performed on an ABI Prism 7300 Sequence Detection System
(Applied Biosystems) using the SYBR Green PCR Master Mix
(Applied Biosystems). PCR was performed in triplicate and was
repeated two times for each gene and each sample. Relative
transcript quantities were calculated using the Ct method
with b-actin as the endogenous reference gene [30]. The
results are summarized in Fig. 7.
In vivo antidiabetic activity
The antidiabetic activity was performed according to the
reported method [30] using STZ-induced diabetic model.
Albino Wistar rats of either sex, 150–200 g, were obtained
from Central Animal House, Jamia Hamdard University, New
Delhi. The rats were kept in cages at room temperature and
fed with food and water ad libitum. The experiments were
performed in accordance with the rules of Institutional
Animals Ethics Committee (registration number 173-CPCSEA).
The rats were fasted overnight and diabetes was induced by
injecting 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 hypoglycemia. The rats were
considered as diabetic if their blood glucose values were
above 250 mg/dL on the third day after STZ injection. The rats
were divided into five groups comprising 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% carboxy-
methyl cellulose suspension) at a dose of 36 mg/kg (Group IV),
diabetic rats orally fed with synthesized compounds 5a–m
(as 0.25% carboxymethyl cellulose suspension) 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 days by glucose oxidase method [31]. The results
are summarized in Fig. 3.
The authors thank 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.
The authors have declared no conflict of interest.
References
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method of Reitman and Frankel method [32] and ALP assay
was done according to method of Walter and Schult [33] using
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their liver specimens were removed and put into 10%
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formed with hematoxylin and eosin staining to analyze
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results are summarized in Fig. 6.
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