Design and microwave facilitated green synthesis of 2-[4-(3-carboxymethyl, methoxy carbonylmethyl-2,4-dioxo and 4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2 and 3-methyl propionic acid ethyl ester derivatives: a novel structural class of antidys

Article abstract of DOI:10.1007/s00044-017-1875-0

An interesting hybrid molecular framework comprising of benzylidenethiazolidin-4-one, chalcone and fibrate was designed and synthesized (BRF1–12) in order to develop safe and efficacious compounds for the treatment of dyslipidemia, and related complicatio

Full text of DOI:10.1007/s00044-017-1875-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1875-0
ORIGINAL RESEARCH
Design and microwave facilitated green synthesis of 2-[4-(3-
carboxymethyl, methoxy carbonylmethyl-2,4-dioxo and 4-oxo-2-
thioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2 and 3-methyl
propionic acid ethyl ester derivatives: a novel structural class of
antidyslipidemic agents
Ashok Kumar Singh1 Avinash C. Tripathi1 Aseem Tewari1 Viney Chawla1
Shailendra K. Saraf
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1
Received: 31 December 2016 / Accepted: 10 March 2017
© Springer Science+Business Media New York 2017
Abstract An interesting hybrid molecular framework
comprising of benzylidenethiazolidin-4-one, chalcone and
fibrate was designed and synthesized (BRF1–12) in order to
develop safe and efficacious compounds for the treatment of
dyslipidemia, and related complications such as athero-
sclerosis. The synthesized derivatives were characterized by
Fourier transform infrared spectroscopy, mass, and nuclear
magnetic resonance spectral studies and evaluated for their
antihyperlipidemic potential, using in vivo and in silico
methods. All the synthesized compounds exhibited pro-
mising antidyslipidemic activity comparable to, and some-
times better than that of, the standard drug-fenofibrate at the
tested dose of 30 mg/kg body weight. The most active
compounds of the series, BRF4 and BRF6, demonstrated
significant antidyslipidemic profile by lowering low density
lipoprotein cholesterol, very low density lipoprotein cho-
lesterol, and triglyceride and increasing the level of high
density lipoprotein cholesterol, thereby decreasing the
atherogenic index. Overall, these effects of BRF4 and
BRF6 were found to be more potent than fenofibrate, in
lipid lowering activity and reducing atherogenic index.
Structure–activity relationship studies conclusively estab-
lished that the presence of N-acetic acid methyl ester at 3rd
position of the thiazolidin-4-one nucleus, and a C-3 fibric
acid moiety at benzene nucleus were instrumental for
enhanced biological activity. The binding mode of
benzylidenethiazolidin-4-one fibrate class of compounds,
showing crucial hydrogen bonds and pi–pi stacking
interactions with the key amino acid residues Phe118,
His440, and Tyr464 at the active site of PPARα receptor,
was assessed by molecular docking studies.
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Keywords Anti-hyperlipidemic Rhodanine Microwave
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●
assisted synthesis Molecular docking
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Benzylidenethiazolidin-4-one Fibrates
Introduction
Dyslipidemia, a common metabolic syndrome, remains one
of the leading causes of many pathological conditions
related to insulin resistance, type 2 diabetes, obesity,
atherosclerosis and thereby enhanced risk of coronary heart
disease (CHD) (Sashidhara et al. 2013). Several studies
have demonstrated the relationship between plasma cho-
lesterol levels and the development of CHD (Tiwari et al.
2006). A 1% drop in serum cholesterol reduces the risk of
CHD by 2% (Mc Gill 1985). Currently, the most common
method to treat dyslipidemia is the use of statins, a HMG-
CoA reductase inhibitor. The widespread clinical use of the
statins is accompanied by potential dose-limiting hepato-
toxicity and myotoxicity, which may be due to the reduced
levels of essential isoprenoid precursors, the antioxidant
ubiquinone, or dolichols (Sashidhara et al. 2014). More-
over, statins can hardly normalize the high density lipo-
protein (HDL) abnormality and significant residual
cardiovascular risk remains in many patients despite statins
therapy. In view of the recent warning by the US-FDA that
statins may enhance the risk of diabetes mellitus (Graham
et al. 2004; US Food and Drug Administration 2012),
* Shailendra K. Saraf
dirpharmniec@gmail.com
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Babu Banarasi Das Northern India Institute of Technology,
Lucknow 226028 Uttar Pradesh, India

Med Chem Res
search is on the better therapeutic strategies for the devel-
opment of safe and effective antidyslipidemic drugs.
2013) followed by indole-chalcone-fibrate (Sashidhara et al.
2014) with promising lipid lowering activity. Moreover,
thorough literature survey revealed that the benzylide-
nethiazolidinone served as a privileged scaffold in drug
discovery and exhibited anti-hyperlipidemic activity with
atherogenesis, caused by low density lipoprotein (LDL)
oxidation (Jeong et al. 2004).
The peroxisome proliferator activating receptors
(PPARs) are a subfamily of ligand-activated nuclear hor-
mone receptors that are highly expressed in metabolically
active tissues which regulate genes encoding lipid and
glucose metabolism, and overall energy homeostasis (Varga
et al. 2011). More specifically, PPARα plays a critical role
in lipid metabolism by decreasing both serum triglycerides
(TG) and free fatty acid levels, and increasing HDL level
(Fruchart 2009). Apart from this, the PPARγ has a pivotal
role in fatty acid storage and glucose metabolism by coor-
dinating the expression of genes involved in lipid metabo-
lism, adipogenesis, and inflammation (Lehrke and Lazar
2005). Two classes of compounds, namely fibrates as
antihyperlipedimic agents (Lalloyer and Staels 2010;
Wierzbicki 2009), and thiazolidinediones as antidiabetic
agents (Quinn et al. 2008; Rizos et al. 2008; Tripathi et al.
2013; Verma and Saraf 2008) are currently marketed as
PPARα and γ agonists, respectively. However, due to some
unavoidable limitations over the use of these drugs a
number of emerging approaches, directed towards the
development of new potent combined agonists of different
subtype receptors, have represented the logical evolution for
the efficient treatment of metabolic syndromes. For exam-
ple, “glitazars” have been recognized as very attractive
candidates in case of metabolic syndromes, by combining
the beneficial effects of PPARα/γ dual agonists (Adeghate
et al. 2011; Balakumar et al. 2007; Pirat et al. 2012).
Many traditional thoughts of medicine have claimed that
a balanced modulation of several targets can provide a
superior therapeutic effect, and decrease in side effect pro-
file compared to the action of a single selective ligand,
especially in the treatment of metabolic syndromes like
dyslipidemia, which have multi-factorial basis of their
development in the body. Effort is being devoted to find
new therapeutics, aiming at multiple targets, as an innova-
tive paradigm in drug discovery. To achieve them, two
strategies are conceivable; the first attempt is to employ a
single compound to hit multiple targets; and the other is to
employ two or more active ingredients in one drug (Morphy
et al. 2004; Zhang 2005). Alternatively, in search for novel
drug leads, the hybrid approach is a promising one since it
can effectively target multi factorial diseases like metabolic
syndromes. Hybrid molecules that contain multiple struc-
tural units of different nature generally possess improved
biological activities (Sashidhara et al. 2013). To date,
numerous admirable researches, based on the hybrid con-
cept of lipid lowering agents, have been reported in the
literature. Shukla et al. synthesized some chalcone-fibrates
and claimed them to be useful in treating dyslipidemia
(Shukla et al. 2011). Two years later, Sashidhara et al.
worked on coumarin-chalcone-fibrate (Sashidhara et al.
Inspired by the beneficial effect of PPAR-α/γ dual ago-
nistic action, the hybridization concept was used to develop
lipid lowering agents that also have the potential to reduce
atherogenic index. A novel series of compounds containing
fibrates (active part of PPAR-α agonist, fenofibrate), thia-
zolidinone (active part of PPAR-γ agonist, rosiglitazone),
and chalcone (active part of some naturally occurring lipid
lowering agents like licochalcone, xanthohumol etc.) in a
single molecular frame have been designed and synthesized
(Fig. 1). Furthermore, these compounds were evaluated for
their lipid profile activity via in vivo and in silico approa-
ches. An environmentally benign microwave facilitated
green approach was employed for the synthesis of the
proposed derivatives. Few representative chemical struc-
tures of important compounds possessing thiazolidin-4-one,
chalcone, fibrate and our synthesized prototype containing
fragments have been presented in Fig. 1, which endow
possibly better complementarity with the receptor molecule.
Results and discussion
Chemistry
The microwave assisted synthetic route to the designed
compounds is outlined in Scheme 1. By adopting the
reported procedures (Bruno et al. 2002; Rakowitz et al.
2006; Redemann et al. 1955; Sortino et al. 2007), 3- and 4-
hydroxybenzaldeydes underwent Knoevenagel condensa-
tion with the thiazolidin-4-one derivatives in presence of the
catalytic amounts of piperidine and glacial acetic acid to
afford corresponding benzylidenethiazolidin-4-ones (BR1–
BR4). This was followed by the consequences of reactions
at N-3 position, to form N-methyl ester derivatives (BR5–
BR8), and their subsequent hydrolysis to form N-acetic acid
derivatives (BR9–BR12).
In order to attach the fibrate moiety with the aforemen-
tioned compounds (BR1–BR12), the electrophilic sub-
stitution of these compounds with ethyl α-bromoisobutyrate
in presence of potassium carbonate and methyl isobutyl
ketone were carried out to accomplish the desired
benzylidenethiazolidin-4-one fibrate derivatives (BRF1–
BRF12). The possible mechanism of the reaction is deli-
neated in Scheme 2. Finally, structures of the synthesized
1
compounds were established by infrared (IR), H nuclear

Med Chem Res
Fig. 1 Chemical structures of
representative fibrate,
thiazolidinone, chalcone, and the
synthesized prototype
O
O
O
O
(a)
N
H
(d)
H
EtO
N H
HO
N
H
HO
O
S
+
S
S
O
X
X
X
BR1-BR4
(b)
BRF1-BRF4
O
O
N
Series
X
Point of attachment
of fibrate ester
O
O
(d)
EtO
N
O
HO
BRF1
BRF2
BRF3
BRF4
BRF5
BRF6
BRF7
BRF8
BRF9
BRF10
BRF11
BRF12
S
S
O
O
S
4
3
4
3
4
3
4
3
4
3
4
3
S
O
S
O
O
X
X
BR5 -BR8
(c)
BRF5 -BRF8
S
O
O
S
S
O
O
O
O
OH
(d)
OH
EtO
N
N
O
HO
S
O
S
O
O
X
X
BR9 -BR12
BRF9 -BRF12
Scheme 1 The synthetic route to the title compounds (BRF1-BRF12).
a Piperidine, AcOH, M.W. (20 min, 140 W); b BrCH2COOCH3,
K2CO3, Acetone, M.W. (14 min, 560 W); c AcOH, HCl, M.W.
(20 min, 140 W); d Ethyl α-bromo isobutyrate, K2CO3, methyl iso-
butyl ketone, M.W. (20 min, 460 W)
magnetic resonance (NMR), and 13C NMR spectroscopy
and mass spectrometry.
permeability of the active compounds in drug discovery.
Very high or very low log P values generally affect ADME
properties of molecules to a great extent. The result of
partition coefficient study indicated that the compound
BRF6 was the most lipophilic among the synthesized series,
where presence of S at the rhodanine nucleus significantly
increases the log P value of BRF6 (2.09). Compound
Partition coefficient
Lipophilicity is represented by the descriptors log P (also
known as Kow or Pow) and is used to predict in vivo

Med Chem Res
O
H
O
O
-
C5H11NH
HO
Condensation
N
R
+
C5H11
N
N
R
S
S
Proton abstraction
by piperidine base
X
X
Carbanion
O
O
O
OH
O
H+
Protonation
-H2O
N
R
N
R
N
R
S
S
S
Dehydration
O
H
HO
X
X
X
Enolate
O
-HBr
Electrophilic substitution,
O
N
R
S
EtO
O
X
O
Br
EtO
Scheme 2 The plausible reaction mechanism
BRF12 was the least lipophilic in the series, with a log P
value of 1.65.
benzene ring was the most hydrolyzed (28.64%) and com-
pound BRF4, having no any substitution on N-3 position of
thiazolidine-2,4-dione along with the fibric acid moiety on
the C-3 position of the benzene ring, was the least hydro-
lyzed (2.40%) in SIF, which is indicative of its stability and
the probable bioavailability through the intestine.
In vitro hydrolysis
The stability at physiological pH (acidic and basic) is of
prime importance for the drugs intended for oral adminis-
tration. It has been observed that many drugs have low
bioavailability owing to the degradation at the low pH (1–2)
of stomach. The simulated gastric fluid (SGF) mimics the
gastric fluid in terms of the acidity and molarity and the
simulated intestinal fluid (SIF) mimics the intestinal fluid in
terms of basicity. These fluids are the perfect media to
determine the stability of drug candidates in vitro. In the
present study, the synthesized compounds were tested
in vitro using the SGF and SIF. The results of in vitro
hydrolysis showed that compound BRF6, having N-acetic
acid methyl ester at N-3 position of rhodanine along with
the fibric acid moiety on the C-3 position of the benzene
ring, was least hydrolyzed (2.25%), thereby indicating its
stability in the stomach and the probable bioavailability.
However, compound BRF11, having N-acetic acid on N-3
position of thiazolidine-2,4-dione along with the fibric acid
moiety on the C-4 position of the benzene ring was the most
hydrolyzed (24.77%) in SGF. The compound BRF7, hav-
ing N-acetic acid methyl ester on N-3 position of rhodanine
along with the fibric acid moiety on the C-4 position of the
In vivo antidyslipidemic activity
The anti-hyperlipidemic activity of the synthesized com-
pounds was determined using high fat dietary model (Lee
et al. 2010) for inducing hypercholesterolemia in rats.
Feeding of the rats resulted in the alteration of body weight
as well as the serum lipid profile (Tables 1 and 2). It was
evident from the observation that continuous post feeding of
the synthesized compounds, at the dose of 30 mg/kg for 7
days, resulted in an appreciable improvement of body
weights and hyperlipidemia. Interestingly, the treatment
with the synthesized compounds significantly reduced the
levels of TG, LDL, VLDL and elevated the levels of HDL,
with a reduction in body weight. The results of the study are
shown in Tables 1 and 2. All the synthesized compounds
exhibited significant lowering in the serum TG and VLDL
concentration. Compounds BRF1–11 showed significant
reduction of the serum total cholesterol (TC). The com-
pounds BRF4, 6, 7, 11, and 12 also lowered the LDL. The
compounds BRF4, 6, 9, 10, and 12 elevated the level of

Med Chem Res

Med Chem Res
Table 2 Effects of the synthesized derivatives (BRF1–12) on TC, HDL-c, LDL-c, TG, VLDL-c, and AI in rats fed with HFD
Treatment groups
Dose (mg/kg) Blood cholesterol level (mg/dl)
TC HDL-c
AI (TC-HDL)/HDL
LDL-c
TG
93.8 37.69
VLDL-c
ND control
Only Normal
Diet
72.8 10.66 29.1 3.78
92.8 4.87
35.4 3.64
18.7 7.54
1.37 0.17
HFD control
(HFD + 1%
16.2 2.54*** 61.7 7.14*** 144.3 9.05*** 29.8 1.43
4.72 0.08***
3.80 0.06*
gum acacia)
Standard (HFD +
Fenofibrate)
30
70.9 1.05* 14.7 1.12
43.0 2.64*
57.3 4.32*
11.0 0.03*
BRF1
BRF2
BRF3
BRF4
BRF5
BRF6
BRF7
BRF9
BRF10
BRF11
BRF12
30
30
30
30
30
30
30
30
30
30
30
73.2 1.45* 16.6 0.27
71.6 0.92* 18.2 0.32
77.8 3.94* 17.6 1.10
81.7 0.76* 27.3 1.54*
79.1 0.89* 14.6 3.20
82.6 1.67** 29.3 1.23*
77.3 1.32* 15.7 0.59
74.1 2.46* 23.4 0.12*
72.6 1.04* 23.3 0.97*
75.4 0.32* 12.3 0.10
53.7 0.59
51.7 2.30
54.8 1.53
42.1 2.40*
49.8 1.18**
41.1 2.60*
42.9 2.78*
59.3 0.74
53.8 0.98
41.9 1.82*
48.2 1.34*
78.2 5.81*
97.5 4.32*
86.5 6.07*
43.8 5.32*
51.3 4.31*
43.9 4.32*
80.4 5.16*
103.7 9.1*
50.1 9.42*
62.3 5.20*
74.1 3.90*
16.9 0.25*
19.3 0.56*
17.8 0.34*
9.3 0.14*
3.40 0.10*
2.93 0.09*
3.40 0.19*
1.99 0.28*
4.40 0.04
10.2 0.96*
10.7 0.31*
16.2 1.08*
21.9 0.23*
10.3 0.75*
13.2 1.02*
15.7 1.01*
1.81 0.09*
3.90 0.08*
2.16 0.05*
2.11 0.06*
2.30 0.21*
2.60 0.08*
89.8 3.02
24.6 0.34*
Data are expressed as Mean SEM, n = 5, P ≤ 0.01 and P ≤ 0.05 compared with HFD control group using one way analysis of variance (ANOVA)
followed by Dunnet’s test
Bold values are the most active derivatives of the series
*P ≤ 0.01, **P ≤ 0.05, and ***P ≤ 0.01 (When HFD control was compared with ND control)
good cholesterol, i.e. high density lipoprotein cholesterol
(HDL-c) even more than that by the standard drug. The
compounds BRF4 and BRF6, at a dose of 30 mg/kg,
reduced the body weight to a level, which was more than
that of the other test and standard drug-treated groups. After
an analysis of body weight reduction and lipid profile, it can
be concluded that the compounds BRF4 and BRF6 showed
comparable effect in terms of TC, TG, low density lipo-
protein cholesterol (LDL-c), and very low density lipopro-
tein cholesterol (VLDL-c) to that of the standard drug
fenofibrate. The interesting feature of these compounds was
the increase in the level of good cholesterol, i.e. HDL-c, and
therefore reduction in atherogenic index (AI) more than that
of the standard drug, fenofibrate. The reason for the higher
activity than that of the standard in case of HDL-c and AI
may be attributed to the attached benzylidenethiazolidin-4-
one moiety along with fibrates. It is also evident from the
results that the presence of ester functionality at the N-H
reactive site of the thiazolidin-4-one nucleus significantly
increased the antihyperlipidemic activity when compared to
the unsubstituted or carboxyl substituted moieties.
module present in the Maestro user interface of Schrödinger
Inc. (Maestro, version9.3, Schrödinger, LLC, New York,
NY, 2012). The molecular target taken into consideration
was the nuclear PPAR-α receptor, whose crystallographic
structure was procured from PDB database (PDB-ID:
2P54). The ligand interaction affinities of the synthesized
compounds were compared with the re-docking results of
the native bioactive co-crystal ligand GW735 in the 2P54
protein structure. The data of molecular docking studies are
given in Table 3.
Also, all the synthesized derivatives showed promising
pharmacokinetic (ADME) properties and druggability in the
in silico studies, when predicted by QikProp module of the
Schrodinger Software package and the results are sum-
marized in Table 3.
Furthermore, the molecules belonging to the fibrate class
of drugs, such as fenofibrate, are known to act as PPARα
agonists. The molecules belonging to glitazone class of
drugs, such as rosiglitazone, are known to act as PPARγ
agonists. Since the synthesized compounds showed struc-
tural resemblance close to that of glitazones as well as
fibrates, results of this study strongly suggested that these
compounds should have some effects through the PPARα/γ.
Moreover, the docking procedure for the PPAR-α binding
site basically followed the same setup as shown for PPAR-
γ. A survey of various classes of PPARγ agonists, three
dimensional-quantitaive structure activity relationship
Molecular docking studies
To see the ligand–protein interaction and to establish a
correlation with the biological activity, the docking analyses
of compounds BRF1–12 were carried out, using Glide-XP

Med Chem Res

Med Chem Res
Fig. 2 Design of PPAR-α agonist on the basis of rosiglitazone molecule
(3D-QSAR) studies and crystal structure information
revealed that the pharmacophoric features of these agents
essentially consists of three parts: (i) an acidic head, (ii)
central aromatic region, and (iii) a lipophilic side chain
(Sundriyal et al. 2008) (Fig. 2). On the basis of these
findings, molecular structures based on fibric acid and gli-
tazones have been designed and docked into the binding
pocket of PPARα protein (PDB-ID: 2P54), to gain a cor-
relation with the hypolipidemic activity.
Generally, it has been reported that three important
ligand–receptor interactions, with His323, Tyr473, and
His449 are imperative for the activation of the receptor
PPAR-γ, whereas interaction with His440 and Tyr464
residue is crucial for the activation of PPAR-α (da Costa
Leite et al. 2007). As the consequence of the same, it has
been observed that compounds BRF4 and BRF6 showed a
key interaction with the His440 and Tyr464 residues of the
PPARα receptor protein. This also gave strong evidence
that these compounds must have some beneficial effects
through the PPARα receptor.
hypolipidemic activity on the basis of higher negative value
of Glide XP gscore. The docking results showed a good
ligand–protein interaction, coverage of contacts and parti-
cipation of the active site residues His440 and Tyr464 in H-
bonding, which contributed to stabilize the ligand with
PPARα receptor. The fibric acid moiety in position 4
demonstrated negative role on the basis of the same para-
meters, as observed with the compounds BRF1, 3, 5, and 7.
Additionally, it was observed that in derivatives BRF 5–8,
both thiazolidinone (TZD) as well as fibric acid moiety were
buried well inside the hydrophobic pocket of PPARα while
in case of BRF9–12, TZD moiety was mostly extruded out
of the hydrophobic pocket of PPARα. This revealed that the
replacement via –CH2COOCH3 at the position N-3 of TZD
ring, was found to be more favorable than that of the
replacement via –CH2COOH for hypolipidemic activity.
The fibric acid and TZD moieties of BRF4 and BRF6 were
oriented in such a way so that the favorable π–π and
hydrogen bond interactions was developed with active site
residues Phe118, His440, and Tyr464 (Fig. 3). Thus, they
attained the maximum docking scores of −7.96678 and
−7.16932 respectively, anticipating that BRF4 and BRF6
may be the most active molecules through PPARα. In vivo
activity also indicated similar findings that the BRF4 and
After performing docking simulations, it became possi-
ble to conclude that the presence of the fibric acid moiety at
the third position of the phenyl ring, as observed in the
BRF2, 4, 6, and 8 played an important role in eliciting the

Med Chem Res
Fig. 3 3D-Ligand protein
interaction diagram of most
active derivatives a BRF4 and
b BRF6, showing H-bond and
π–π staching interactions at the
receptor site of PPARα protein
(PDB-ID: 2P54)
BRF6 were the most active compounds, among all the
synthesized derivatives. On the other hand, the compounds
BRF7 and BRF10 showed the least docking scores of
−3.74430 and −2.60685, respectively, because of the
maximum four polar atom burial and desolvation penalties,
and penalty for intra-ligand contacts. Moreover, Compound
BRF-12 exhibited significant in vivo activity, whereas the
same exhibited moderate activity in the in silico assessment.
The reason for the activity may be due to the esterification
at N–H reactive site of rhodanine.
chalcone-fibrate, indole chalcone-fibrates, in the literatures,
however, following details are summarized for the first time
in the present research on thiazolidin-4-one chalone-fibrates
towards antidyslipidemic activity-
●
Hybridization of thiazolidin-4-one, chalcone and fibrates
in a single moiety to reinforce the antidyslipidemic
effect through PPARα/γ dual agonistic action.
Synthesis of all the compounds via knoevenagel reaction
●
and thereafter simple electrophilic substitution with the
help of microwave assisted organic synthesis.
Development of N-methyl ester and N-methyl acid
To date, despite wealth of the information available on
the therapeutic importance of chalcone-fibrates, coumarin
●

Med Chem Res
derivatives of benzalidinethiazolidin-2,4-dione fibrate
and benzalidine-2-thioxo-4-thiazolidinone fibrate to
enhance the activity.
Significant lowering of bad cholesterol levels like LDLc,
VLDLc, and TG and significant elevation in the good
cholesterol level like HDL-c as compared with reference
drug, fenofibrate at the same dose level.
More reduction of AI and thereby atherogenesis as
compared with reference compound, fenofibrate at same
dose level.
Significant reduction of the body weight with minimal
food efficiency ratio.
Molecular docking of the synthesized molecules onto
PPAR-α receptor using Glide-XP present in Maestro
user interface of Schrödinger Inc. showing better
complementarity and binding affinity of some of the
molecules with the receptor, showing a good correlation
of the in silico results with the in vivo studies.
EL III Elemental analyzer (Elementar, Germany) at CDRI,
Lucknow.
●
●
General procedure for the synthesis of
benzylidenethiazolidin-4-one fibrates (BRF1–12)
A mixture of appropriately substituted benzilidinethiazoli-
dine-4-one (BR1–12) (1.71 mmol), ethyl α-bromoisobuty-
rate (0.30 ml, 2.05 mmol), potassium carbonate (0.471 g,
3.42 mmol) and methyl isobutyl ketone (10 ml) was placed
in a round bottom flask and irradiated for 20 min in a
microwave synthesizer at 560 W. The reaction was mon-
itored by TLC, using 40% ethyl acetate: n-hexane as the
solvent system. The reaction mixture was cooled to room
temperature, the inorganic salts were filtered off; then the
solvent was evaporated under reduced pressure, the solid
residue was collected and recrystallized from ethanol to
obtain the title compounds (BRF1–12).
●
●
2-Methyl-2-[4-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-
phenoxy]-propionic acid ethyl ester (BRF1)
Experimental
General
Dark orange solid; yield: 71.07%; melting range (°C):
215–217; IR (KBr) (ν cm−1): 1638.52 C=O str (amide),
1330.80 C–N str, 1215.51 C=S str, 1629.31 C=C str,
1
The chemicals and reagents were procured from Sigma
Aldrich Chemicals, Mumbai and were used without further
purification. Microwave assisted synthesis was performed
using Raga’s Scientific Microwave Systems (Ragatech,
Pune, Maharashtra, India). Progress of the reaction was
monitored by thin layer chromatography on silica gel G
plates using iodine vapors and UV light as visualizing
agents. Melting points were determined by open capillary
method and are uncorrected. After physical characterization,
the compounds were subjected to spectral analysis. UV
spectra were recorded on a Double Beam spectrophotometer
(Shimadzu-1700). The IR spectra were recorded on Perkin
Elmer RX1 FTIR spectrophotometer using KBr disks and
the values are expressed in cm−1 and only noteworthy
absorption levels (reciprocal centimeters) are listed. The
mass spectra were recorded on JEOL-Accu TOF JMS-
T100LC mass spectrometer and ESI-MS-Accu TOF JMS-
T100LC mass spectrometer at CDRI, Lucknow. The
nuclear magnetic resonance (1H and 13C NMR) spectra
were recorded at 300 and 75 MHz on a Bruker DRX
spectrometer (Bruker Instruments Inc., USA) at CDRI,
Lucknow. CD3OD was taken as the solvent and the che-
mical shifts are reported in parts per million (δ values),
928.86 C–O str (ether), 1749.48 C=O str (ester); H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.276 (t, 3H) 1.768 (s, 6H),
3.306–3.315 (m, 2H) 6.890–6.910 (d, 2H, aromatic),
7.383–7.705 (d, 4H, aromatic); 13C NMR (CD3OD, 75
MHz) δ: 23.24 (2CH3 aliphatic), 48.30 (CH2 aliphatic),
126.23–134.20 (2CH aromatic), 154.20 (C aromatic),
161.92 (C amide), 171.52 (C carboxyl) 196.91 (C thioa-
mide); ESI-MS: [M + 1]+ at m/z 352.02. Anal. calcd. for
C16H17NO4S2: C, 54.68; H, 4.88; N, 3.99. Found: C, 54.71;
H, 4.90; N, 4.03.
2-Methyl-2-[3-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-
phenoxy]-propionic acid ethyl ester (BRF2)
Dark yellow solid; yield: 71.18%; melting range (°C):
218–220; IR (KBr) (ν cm−1): 1638.97 C=O str (amide),
1329.41 C–N str, 1215.68 C=S str, 1628.97 C=C str,
1
928.86 C–O str (ether), 1749.56 C=O str (ester); H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.153–1.176 (t, 3H)
1.200–1.269 (s, 6H), 3.299–3.321 (m, 2H), 6.863–6.940 (s,
3H, aromatic) 7.002–7.330 (s, 1H, aromatic), 7.499–7.596
(s, 2H, Ar); 13C NMR (CD3OD, 75 MHz) δ: 23.42 (2CH3
aliphatic), 48.30 (CH2 aliphatic), 127.26 (2CH aromatic),
136.06 (C aromatic), 159.53 (C amide), 171.17 (C car-
boxyl), 196.95 (C thioamide); ESI-MS: [M + 1]+ at m/z
352.13. Anal. calcd. for C16H17NO4S2: C, 54.68; H, 4.88;
N, 3.99. Found: C, 54.75; H, 4.89; N, 4.00.
1
using TMS (δ 0 ppm for HNMR) as the internal standard.
The spin multiplicities in 1H NMR are indicated as follows:
s (singlet), d (doublet), dd (double doublet), t (triplet) and m
(multiplet). Elemental analysis was performed on a Vario

Med Chem Res
2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-
methyl-propionic acid ethyl ester (BRF3)
2-[3-(3-Methoxycarbonylmethyl-4-oxo-2-thioxo-thiazolidin-
5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF6)
Crystal white solid; yield: 69.41%; melting range (°C):
248–250; IR (KBr) (ν cm−1): 1645.76 C=O str (amide),
1215.56 C–N str, 1645.76 C=C str, 1025.88 C–O str
Greenish yellow solid; yield: 58.98%; melting range (°C):
286–288; IR (KBr) (ν cm−1): 1683.79 C=O str (amide),
1215.72 C–N str, 1215.73 C=S str, 1612.37 C=C str,
1025.48 C–O str (ether), 1744.83 C=O str (ester), 1411.98
1
(ether), 1745.63 C=O str (ester); H NMR [(CD3OD, 300
MHz) δ in ppm: 1.153 (t, 3H) 1.269 (s, 6H), 3.299–3.321
(m, 2H) 6.748–6.751 (d, 2H, aromatic), 7.014–7.016 (d, 2H,
aromatic), 7.259–7.430 (s, 2H); 13C NMR (CD3OD, 75
MHz) δ: 22.56 (2CH3 aliphatic), 48.27 (CH2 aliphatic),
122.56 (2CH aromatic), 138.09 (CH aromatic), 159.10 (C
amide), 185.97 (C thioamide); ESI-MS: [M + 1]+ at m/z
336.97. Anal. calcd. for C16H17NO4S2: C, 57.30; H, 5.11;
N, 4.18. Found: C, 57.26; H, 5.09; N, 4.20.
1
CH2 bend; H NMR [(CD3OD, 300 MHz) δ in ppm: 1.283
(t, 3H) 2.839 (s, 6H), 3.188–3.319 (m, 2H), 3.481 (s, 2H),
4.315 (s, 3H), 6.854–6.857 (d, 3H, aromatic), 7.000–7.015
(t, 1H, aromatic), 7.807 (s, 1H); 13C NMR (CD3OD, 75
MHz) δ: 23.24 (CH3 aliphatic) 26.23 (2CH3 aliphatic),
48.30 (CH2 aliphatic) 49.72 (CH2 aliphatic), 50.00 (CH3
aliphatic), 124.84 (3CH aromatic), 136.09 (C aromatic),
159.47 (C amide), 169.20 (C carboxyl), 169.67 (C thioa-
mide); ESI-MS: [M + 1]+ at m/z 424.12. Anal. Calcd. for
C19H21NO6S2: C, 53.88; H, 5.00; N, 3.31. Found: C, 53.86;
H, 5.01; N, 3.32.
2-[3-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-
methyl-propionic acid ethyl ester (BRF4)
2-[4-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF7)
Crystalline yellow solid; yield: 46.75%; melting range (°C):
250–252; IR (KBr) (ν cm−1): 1645.34 C=O str (amide),
1215.64 C–N str, 1025.59 C–O str (ether), 1746.74 C=O str
(ester); 1H NMR [(CD3OD, 300 MHz)
δ in ppm:
Cream white solid; yield: 65.15%; melting range (°C):
298–300; IR (KBr) (ν cm−1): 1686.35 C=O str (amide),
1323.73 C–N str, 1607.03 C=C str, 935.39 C–O str (ether),
1741.69 C=O str (ester), 1441.30 CH2 bend; 1H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.235 (t, 3H), 1.618 (s, 6H),
3.299–3.320 (m, 2H), 3.774 (s, 2H), 3.798 (s, 3H),
6.878–6.881 (d, 2H, aromatic), 7.318–7.344 (d, 2H, aro-
matic), 7.839 (s, 1H); 13C NMR (CD3OD, 75 MHz) δ:
25.90 (CH3 aliphatic), 42.95 (CH2 aliphatic) 48.87 (CH2
aliphatic), 53.41 (CH3 aliphatic), 117.45 (2CH aromatic),
135.84 (2CH aromatic), 159.53 (C amide), 168.94 (C car-
boxyl); ESI-MS: [M + 1]+ at m/z 408.10. Anal. calcd. for
C19H21NO7S: C, 56.01; H, 5.20; N, 3.44. Found: C, 56.16;
H, 5.21; N, 3.42.
0.898–1.178 (t, 3H), 1.285 (s, 6H), 3.299–3.321 (m, 2H)
6.873–6.945 (s, 2H, Ar) 7.000 (t, 1H, aromatic), 7.521 (s,
H, aromatic); 13C NMR (CD3OD, 75 MHz) δ: 23.36 (2CH3
aliphatic), 48.30 (CH2 aliphatic), 123.36 (2CH aromatic),
133.22 (CH aromatic), 159.58 (C amide), 196.87 (C
thioamide); ESI-MS: [M + 1]+ at m/z 320.34. Anal. calcd.
for C15H14NO5S: C, 56.41; H, 4.41; N, 4.37. Found: C,
56.70; H, 4.35; N, 4.34.
2-[4-(3-Methoxycarbonylmethyl-4-oxo-2-thioxo-thiazolidin-
5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF5)
Brick red solid; yield: 61.72%; melting range (°C):
282–284; IR (KBr) (ν cm−1): 1639.09 C=O str (amide),
1329.01 C–N str, 1215.57 C=S str, 1639.09 C=C str,
927.90 C–O str (ether), 1747.43 C=O str (ester), 1408.18
2-[3-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF8)
1
CH2 bend; H NMR [(CD3OD, 300 MHz) δ in ppm: 1.271
Cream white solid; yield: 65.95%; melting range (°C):
302–305; IR (KBr) (ν cm−1): 1686.67 C=O str (Amide),
1323.13 C–N str, 1607.64 C=C, 1022.46 C–O str (Ether),
1741.07 C=O str (Ester), 1441.83 CH2 bend; 1H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.235 (t, 3H) 1.618 (s, 6H),
3.299–3.320 (m, 3H), 4.500 (s, 4H), 6.878–6.881 (d, 2H,
aromatic), 7.025 (t, 1H, aromatic), 7.839 (s, 1H); 13C NMR
(CD3OD, 75 MHz) δ: 17.57 (CH3 aliphatic), 20.66 (CH3
aliphatic), 48.87 (CH2 aliphatic), 53.41 (CH3 aliphatic),
117.45 (2CH aromatic), 135.84 (CH aromatic), 159.53 (C
(t, 3H) 2.338 (s, 6H), 3.299–3.321 (m, 3H), 3.745–3.776 (s,
2H), 6.863–6.873 (d, 2H, aromatic), 7.364 (d, 2H, aro-
matic), 7.698 (s, 1H); 13C NMR (CD3OD, 75 MHz) δ:
17.57 (CH3 aliphatic) 20.66 (2CH3 aliphatic), 48.30–49.72
(CH2 aliphatic) 120.66 (2CH aromatic), 134.43 (2CH aro-
matic), 161.54 (C amide), 171.48 (C carboxyl), 196.87 (C
thioamide); ESI-MS: [M + 1]+ at m/z 424.97. Anal. calcd.
for C19H21NO6S2: C, 53.88; H, 5.00; N, 3.31. Found: C,
53.80; H, 4.97; N, 3.34.

Med Chem Res
amide), 168.94 (C carboxyl); ESI-MS: [M + 1]+ at m/z
408.09. Anal. calcd. for C19H21NO7S: C, 56.01; H, 5.20; N,
3.44. Found: C, 56.12; H, 5.24; N, 3.40.
C=O str (carboxylic), 1441.30 CH2 bend; 1H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.281 (t, 3H) 3.302–3.307
(s, 6H), 3.318–3.323 (m, 2H), 4.445 (s, 2H) 6.873–6.875 (d,
2H, aromatic), 7.014–7.040 (d, 2H, aromatic), 7.824 (s,
1H), 10.476 (s, 1H); 13C NMR (CD3OD, 75 MHz) δ: 19.23
(CH3 aliphatic), 21.16 (2CH3 aliphatic), 43.07 (CH2 ali-
phatic) 48.58 (CH2 aliphatic), 117.42 (2CH aromatic),
135.85 (2CH aromatic), 159.46 (C amide), 167.10–168.90
(C carboxyl); ESI-MS: [M + 1]+ at m/z 394.06. Anal. calcd.
for C18H19NO7S: C, 54.95; H, 4.87; N, 3.56. Found: C,
54.98; H, 4.90; N, 3.52.
2-[4-(3-Carboxymethyl-4-oxo-2-thioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF9)
Light yellow solid; yield: 58.06%; melting range (°C):
310–312; IR (KBr) (ν cm−1): 1632.42 C=O str (Amide),
1215.32 C–N str, 1215.32 C=S str, 928.28 C–O str (Ether),
1731.04 C=O str (Ester), 1731.04 C=O str (Carboxylic
acid), 1404.62 CH2 bend; 1H NMR [(CD3OD, 300 MHz) δ
in ppm: 1.281 (t, 3H) 3.302–3.307 (s, 6H), 3.318–3.323 (m,
2H), 4.445 (s, 2H) 6.873–6.875 (d, 3H, aromatic),
7.014–7.040 (d, 2H, aromatic), 7.824 (s, 1H), 10.681 (s,
1H); 13C NMR (CD3OD, 75 MHz) δ: 17.42 (CH3 aliphatic),
22.60 (2CH3 aliphatic), 43.07 (CH2 aliphatic) 48.86 (CH2
aliphatic), 117.42 (C aromatic), 131.53 (2H aromatic),
159.46 (C amide), 167.10 (C thaimide), 168.90 (2C car-
boxyl); ESI-MS: [M + 1]+ at m/z 410.10. Anal. Calcd. for
C18H19NO6S2: C, 52.80; H, 4.68; N, 3.42. Found: C, 52.72;
H, 4.77; N, 3.38.
2-[3-(3-Carboxymethyl-2,4-dioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF12)
Greenish yellow solid; yield: 71.09%; melting range (°C):
355–357; IR (KBr) (ν cm−1): 1681.76 C=O str (amide),
1324.75 C–N str, 1216.74 C=S str, 1607.85 C=C str,
928.39 C–O str (ether), 1741.87 C=O str (ester), 1712.32
C=O str (carboxylic), 1440.70 CH2 bend; 1H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.899 (t, 3H) 3.767 (s, 6H),
3.308–3.318 (m, 2H), 4.476 (s, 2H), 6.865–6.873 (d, 2H,
aromatic), 6.893 (s, 1H, aromatic) 7.374–7.438 (t, 1H,
aromatic), 7.819 (s, 1H), 10.580 (s, 1H); 13C NMR
(CD3OD, 75 MHz) δ: 19.23 (CH3 aliphatic), 42.87 (CH2
aliphatic) 48.58 (CH2 aliphatic), 117.34 (2CH aromatic),
133.72 (2CH aromatic), 161.50 (C amide), 167.22 (C ester),
169.03 (C thiamide), 170.04 (C carboxyl); ESI-MS: [M +
1]+ at m/z 394.06. Anal. calcd. for C18H19NO7S: C, 54.95;
H, 4.87; N, 3.56. Found: C, 55.01; H, 4.88; N, 3.55.
2-[3-(3-Carboxymethyl-4-oxo-2-thioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF10)
Yellowish green solid; yield: 59.20%; melting range (°C):
306–308; IR (KBr) (ν cm−1): 1631.03 C=O str (amide),
1215.74 C–N str, 1215.74 C=S str, 1631.03 C=C str,
928.39 C–O str (ether), 1732.04 C=O str (ester), 1732.04
C=O str (carboxylic), 1408.31 CH2 bend; 1H NMR
[(CD3OD, 300 MHz) δ in ppm: 1.899 (t, 3H) 3.767 (s, 6H),
3.308–3.318 (m, 2H), 4.476 (s, 2H), 6.865–6.873 (d, 2H,
aromatic), 6.893 (s, 1H, aromatic) 7.374–7.438 (t, 1H,
aromatic), 7.819 (s, 1H), 10.679 (s, 1H); 13C NMR
(CD3OD, 75 MHz) δ: 17.90 (CH3 aliphatic), 20.97 (2CH3
aliphatic), 42.87 (t, CH2 aliphatic) 48.87 (t, CH2 aliphatic),
53.39 (C aliphatic), 117.34 (2CH aromatic), 136.00 (2CH
aromatic), 161.50 (C amide), 167.22 (C ester), 169.64 (C
thiamide), 170.04 (C carboxyl); ESI-MS: [M + 1]+ at m/z
410.10. Anal. calcd. for C18H19NO6S2: C, 52.80; H, 4.68;
N, 3.42. Found: C, 52.75; H, 4.80; N, 3.37.
In-vitro studies
The in vitro studies included determination of partition
coefficient and hydrolysis profile of the synthesized deri-
vatives in SGF and SIF .
Determination of partition coefficient
Partition coefficient of the synthesized compounds was
determined by the “Shake Flask Method” using reported
procedure (Ashford 2002; Tripathi et al. 2014), and the log
P values of the synthesized compounds were determined by
applying the formula, given below:
2-[4-(3-Carboxymethyl-2,4-dioxo-thiazolidin-5-
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
ester (BRF11)
Conc: of compound in nꢀocatanol
Log P ¼ log
Creamy white solid; yield: 68.06%; melting range (°C):
348–350; IR (KBr) (ν cm−1): 1686.35 C=O str (amide),
1323.73 C–N str, 1216.32 C=S str, 1607.03 C=C str,
928.28 C–O str (ether), 1741.69 C=O str (ester), 1711.04
Conc: of compound in water

Med Chem Res
Table 4 Composition of the HFD (%)
Hydrolysis studies
Components
Composition (g/1000 g of diet)
In vitro hydrolysis studies were performed spectro-
photometrically using SGF/SIF (Ashford 2002; Tripathi
et al. 2014) and the per cent hydrolysis was calculated using
the following formulae.
Casein
20
0.3
D,L-methionine
Corn starch
15
Sucrose
27.5
5
% Hydrolized in SGF
Cellulose powder
Mineral mixa
Vitamin mixb
Choline bitartrate
Corn oil
ðAbs: at 0 min ꢀ Abs: at 90 minÞ
3.5
¼
ꢁ 100;
Abs: at 0 min
1
0.2
% Hydrolized in SGF
9.9
ðAbs: at 0 min ꢀ Abs: at 120 minÞ
Abs :at 0 min
Lard
17.6
100
498.7
49.6
34.1
16.3
¼
ꢁ 100:
Total (%)
kcal/100 g diet
Calories from fat (%)
Calories from carbohydrate (%)
Calories from protein (%)
a
Pharmacology
Male Wistar rats, weighing 125–225 gm, were used for
pharmacological studies of all the newly synthesized deri-
vatives. The animals were housed in polypropylene cages
with steel net and maintained under standard living condi-
tions of temperature 24 5 °C under controlled humidity of
55 5%, with regular 12 h light/dark cycle, and allowed
free access to laboratory food and water. All the animals
were treated morally in accordance with the guidelines laid
down by the Institutional Animal Ethics Committee (IAEC,
vide protocol approval no. BBDNIIT/IAEC/001/2014).
Experiments were performed after the approval by the
IAEC, strictly adhering to the ethical guidelines.
AIN-76 mineral mixture contained (in g/kg of mixture): calcium
phosphate, dibasic 500.0; sodium chloride, 74.0; potassium citrate
monohydrate, 220.0; potassium sulphate, 52.0; magnesium oxide,
24.0; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6;
cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01;
chromium potassium sulphate, 0.55; sucrose, finely powdered, 118.03
b
AIN-76A vitamin mixture contained (in g/kg of mixture): thiamine
HCl, 0.6; riboflavin, 0.6; pyridoxine HCl, 0.7: niacin, 3.0; D-calcium
pantothenate, 1.6; folic acid, 0.2: D-biotin, 0.02; cyanocobalamin
(vitamin B12), 1.0; dry vitamin A palmitate (500,000 U/g), 0.8;
dry vitamin
E acetate (500 U/g), 10.0; vitamin D3 trituration
(400,000 U/g), 0.25; menadione sodium bisulphite complex, 0.15;
sucrose, fine powder, 981.08
In vivo screening
group was compared with that of the control group. The
reduction in atherogenesis is expressed in terms of AI,
which was determined using the following equation:
The high fat diet (HFD) induced hyperlipidemic model (Lee
et al. 2010) was used to determine antihyperlipidemic
activity of the series of compounds in male Wistar rats. A
Group of five animals each were used as control and treated
mice. Male Wistar rats were fed with HFD (Table 4) for a
period of 1 month for the development of hyperlipidemia.
Feed consumption and body weights were measured routi-
nely. The test compounds were administered at a dose of 30
mg/kg body weight. Fenofibrate, at a dose of 30 mg/kg was
taken as the reference drug. The synthesized compounds
and standard drug were suspended in 1% gum acacia and
administrated orally (p.o.). At the end of the experiment, the
blood sample was withdrawn from the retro-orbital plexus
of eye of the rats for the estimation of TC, serum TG, (LDL-
c), HDL-c and VLDL-c. A significant reduction in TG,
LDL-c, and VLDL-c levels, a significant increase in HDL-c
levels and a significant reduction in body weight after drug
treatments, as compared to control animals, were considered
as a positive antidyslipidemic response. The mean value of
lipid profile and weight reduction for each experimental
TC ꢀ HDL
Atherogenic index ðAIÞ ¼
HDL
In silico screening
To study the ligand–protein interactions and to establish a
correlation with biological activity, the docking analyses of
all the derivatives were carried out, using Glide-XP protocol
of Schrödinger Inc (Maestro, version 9.3, Schrödinger,
LLC, New York, NY, 2012). The molecular target taken
into consideration was the nuclear PPARα receptor, whose
crystallographic structure was procured from Protein Data
Bank (PDB-ID: 2P54). The crystal structure was subse-
quently optimized and minimized with the “protein pre-
paration wizard” workflow, as implemented in the
Schrödinger 2012 package. The ligands were built using
Maestro 9.3 build panel and prepared by LigPrep 2.5

Med Chem Res
version v25111(Schrödinger, LLC, USA) application that
uses optimized potential liquid simulations 2005 force field,
and it gave the corresponding energy minima 3D con-
formers of the ligands. The default settings were used for all
other parameters. The active site was considered as a rigid
molecule, whereas the ligands were treated as being flex-
ible, i.e. all non-ring torsions were allowed. The ligand
interaction affinities of the synthesized compounds were
compared with the re-docking results of the native bioactive
co-crystal ligand GW735 in the 2P54 protein crystal
structure.
Nearly 40% of drug candidates fail in clinical trials
due to poor absorption, distribution, metabolism, and
excretion (ADME) properties. These late-stage failures
contribute significantly to the rapidly escalating cost of new
drug development. The ability to detect the problematic
candidates early can dramatically reduce the amount of
wasted time and resources, and streamline the overall
development process. QikProp, version 3.5 (Schrödinger,
LLC, New York, NY, 2012) program was used for in silico
prediction of pharmacokinetic properties of the synthesized
compounds.
Compounds BRF4 and BRF6 were found to be the most
active compounds among all the synthesized derivatives
in vivo, and exhibited anti-dyslipidemic activity by low-
ering LDL-c, VLDL-c, and TG, and increasing the level of
HDL-c, thereby decreasing the AI. Altogether, these effects
of BRF4 and BRF6 were found to be more potent as
compared with the standard drug, in either case of lipid
lowering activity or reducing AI. The interesting feature of
these compounds was to increase the level of good cho-
lesterol, i.e. HDL-c, and thus reduction in AI to a level more
than that by the standard drug, fenofibrate. The reason for
the higher activity in case of HDL-c and AI may be due to
the attachment of both, the benzylidenethiazolidin-4-one
and fibrate moiety in a single frame. Benzylidenethiazoli-
din-4-one individually has already been reported to reduce
atherosclerosis caused by LDL oxidation. The results sug-
gest that these thiazolidine-chalcone-fibrates constitute a
new prototype of lipid lowering agents that might act by
lowering bad cholesterol and elevating good cholesterol,
and thus decreasing the AI. However, this newly synthe-
sized pharmacophoric framework needs to go through fur-
ther structural modification on the basis of the structural
activity relationship, molecular docking or QSAR approach
to improve potency, lipophilicity and to minimize the side-
effects.
Statistical analysis
All the values of the experimental results are expressed as
mean SEM and statistical significance between the groups
was calculated by one way analysis of variance (ANOVA)
followed by Dunett’s multiple comparison tests. P ≤ 0.01
was considered statistically significant. Statistical analysis
was carried out using Graph Pad Instat 3.0 (Graph Pad
Software).
Acknowledgements We express our sincere gratitude to Central
Drugs Research Institute (CDRI), Lucknow, India for providing the
library and sophisticated analytical instrument facilities. Authors are
thankful to the All India Council for Technical Education (AICTE),
New Delhi, India, for providing grant under the Research Promotion
Scheme (RPS), through which the computational software facility has
been made available at the host institute. We also acknowledge the
technical support team/application scientists of Schrodinger Inc. for
their help during computational studies.
Conflict of interest The authors declare that they have no competing
interests.
Conclusion
While considering all the newly synthesized compounds
together, it may be concluded that the hybridization of fibric
acid analog with a special type of chalcones, namely ben-
zylidinethiazolidin-2,4-diones and benzylidine-rhodanines,
establish an important pharmacophore and the positions N-
3, 4, and 5 of the thiazolidin-4-one moiety are the key
reactive sites, which could be altered with different groups
to elicit valuable hypolipidemic profiles. More precisely,
based on the in vivo and in silico activity results, the
esterification of N–H functionality in thiazolidin-4-one
moiety, to yield N-acetic acid methyl ester derivatives,
appeared to be significant in activity demonstrating pro-
nounced anti-hyperlipidemic activity. In addition, substitu-
tion with fibric acid moiety in C-3 position of benzene ring,
attached with thiazolidin-4-one moiety, was also respon-
sible for the enhancement of activity.
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