Med Chem Res MEDICINAL

DOI 10.1007/s00044-017-1875-0
CHEMISTRY
RESEARCH
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 1
Received: 31 December 2016 / Accepted: 10 March 2017
© Springer Science+Business Media New York 2017

Abstract An interesting hybrid molecular framework interactions with the key amino acid residues Phe118,
comprising of benzylidenethiazolidin-4-one, chalcone and His440, and Tyr464 at the active site of PPARα receptor,
fibrate was designed and synthesized (BRF1–12) in order to was assessed by molecular docking studies.
develop safe and efficacious compounds for the treatment of
dyslipidemia, and related complications such as athero- Keywords Anti-hyperlipidemic Rhodanine Microwave
● ●

sclerosis. The synthesized derivatives were characterized by assisted synthesis Molecular docking
● ●

Fourier transform infrared spectroscopy, mass, and nuclear Benzylidenethiazolidin-4-one Fibrates

●

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 Introduction
compounds of the series, BRF4 and BRF6, demonstrated
significant antidyslipidemic profile by lowering low density Dyslipidemia, a common metabolic syndrome, remains one
lipoprotein cholesterol, very low density lipoprotein cho- of the leading causes of many pathological conditions
lesterol, and triglyceride and increasing the level of high related to insulin resistance, type 2 diabetes, obesity,
density lipoprotein cholesterol, thereby decreasing the atherosclerosis and thereby enhanced risk of coronary heart
atherogenic index. Overall, these effects of BRF4 and disease (CHD) (Sashidhara et al. 2013). Several studies
BRF6 were found to be more potent than fenofibrate, in have demonstrated the relationship between plasma cho-
lipid lowering activity and reducing atherogenic index. lesterol levels and the development of CHD (Tiwari et al.
Structure–activity relationship studies conclusively estab- 2006). A 1% drop in serum cholesterol reduces the risk of
lished that the presence of N-acetic acid methyl ester at 3rd CHD by 2% (Mc Gill 1985). Currently, the most common
position of the thiazolidin-4-one nucleus, and a C-3 fibric method to treat dyslipidemia is the use of statins, a HMG-
acid moiety at benzene nucleus were instrumental for CoA reductase inhibitor. The widespread clinical use of the
enhanced biological activity. The binding mode of statins is accompanied by potential dose-limiting hepato-
benzylidenethiazolidin-4-one fibrate class of compounds, toxicity and myotoxicity, which may be due to the reduced
showing crucial hydrogen bonds and pi–pi stacking levels of essential isoprenoid precursors, the antioxidant
ubiquinone, or dolichols (Sashidhara et al. 2014). More-
over, statins can hardly normalize the high density lipo-
* Shailendra K. Saraf protein (HDL) abnormality and significant residual
[email protected] cardiovascular risk remains in many patients despite statins
1 therapy. In view of the recent warning by the US-FDA that
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Babu Banarasi Das Northern India Institute of Technology, statins may enhance the risk of diabetes mellitus (Graham
Lucknow 226028 Uttar Pradesh, India et al. 2004; US Food and Drug Administration 2012),
 Med Chem Res

search is on the better therapeutic strategies for the devel- 2013) followed by indole-chalcone-fibrate (Sashidhara et al.
opment of safe and effective antidyslipidemic drugs. 2014) with promising lipid lowering activity. Moreover,
The peroxisome proliferator activating receptors thorough literature survey revealed that the benzylide-
(PPARs) are a subfamily of ligand-activated nuclear hor- nethiazolidinone served as a privileged scaffold in drug
mone receptors that are highly expressed in metabolically discovery and exhibited anti-hyperlipidemic activity with
active tissues which regulate genes encoding lipid and atherogenesis, caused by low density lipoprotein (LDL)
glucose metabolism, and overall energy homeostasis (Varga oxidation (Jeong et al. 2004).
et al. 2011). More specifically, PPARα plays a critical role Inspired by the beneficial effect of PPAR-α/γ dual ago-
in lipid metabolism by decreasing both serum triglycerides nistic action, the hybridization concept was used to develop
(TG) and free fatty acid levels, and increasing HDL level lipid lowering agents that also have the potential to reduce
(Fruchart 2009). Apart from this, the PPARγ has a pivotal atherogenic index. A novel series of compounds containing
role in fatty acid storage and glucose metabolism by coor- fibrates (active part of PPAR-α agonist, fenofibrate), thia-
dinating the expression of genes involved in lipid metabo- zolidinone (active part of PPAR-γ agonist, rosiglitazone),
lism, adipogenesis, and inflammation (Lehrke and Lazar and chalcone (active part of some naturally occurring lipid
2005). Two classes of compounds, namely fibrates as lowering agents like licochalcone, xanthohumol etc.) in a
antihyperlipedimic agents (Lalloyer and Staels 2010; single molecular frame have been designed and synthesized
Wierzbicki 2009), and thiazolidinediones as antidiabetic (Fig. 1). Furthermore, these compounds were evaluated for
agents (Quinn et al. 2008; Rizos et al. 2008; Tripathi et al. their lipid profile activity via in vivo and in silico approa-
2013; Verma and Saraf 2008) are currently marketed as ches. An environmentally benign microwave facilitated
PPARα and γ agonists, respectively. However, due to some green approach was employed for the synthesis of the
unavoidable limitations over the use of these drugs a proposed derivatives. Few representative chemical struc-
number of emerging approaches, directed towards the tures of important compounds possessing thiazolidin-4-one,
development of new potent combined agonists of different chalcone, fibrate and our synthesized prototype containing
subtype receptors, have represented the logical evolution for fragments have been presented in Fig. 1, which endow
the efficient treatment of metabolic syndromes. For exam- possibly better complementarity with the receptor molecule.
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). Results and discussion
Many traditional thoughts of medicine have claimed that
a balanced modulation of several targets can provide a Chemistry
superior therapeutic effect, and decrease in side effect pro-
file compared to the action of a single selective ligand, The microwave assisted synthetic route to the designed
especially in the treatment of metabolic syndromes like compounds is outlined in Scheme 1. By adopting the
dyslipidemia, which have multi-factorial basis of their reported procedures (Bruno et al. 2002; Rakowitz et al.
development in the body. Effort is being devoted to find 2006; Redemann et al. 1955; Sortino et al. 2007), 3- and 4-
new therapeutics, aiming at multiple targets, as an innova- hydroxybenzaldeydes underwent Knoevenagel condensa-
tive paradigm in drug discovery. To achieve them, two tion with the thiazolidin-4-one derivatives in presence of the
strategies are conceivable; the first attempt is to employ a catalytic amounts of piperidine and glacial acetic acid to
single compound to hit multiple targets; and the other is to afford corresponding benzylidenethiazolidin-4-ones (BR1–
employ two or more active ingredients in one drug (Morphy BR4). This was followed by the consequences of reactions
et al. 2004; Zhang 2005). Alternatively, in search for novel at N-3 position, to form N-methyl ester derivatives (BR5–
drug leads, the hybrid approach is a promising one since it BR8), and their subsequent hydrolysis to form N-acetic acid
can effectively target multi factorial diseases like metabolic derivatives (BR9–BR12).
syndromes. Hybrid molecules that contain multiple struc- In order to attach the fibrate moiety with the aforemen-
tural units of different nature generally possess improved tioned compounds (BR1–BR12), the electrophilic sub-
biological activities (Sashidhara et al. 2013). To date, stitution of these compounds with ethyl α-bromoisobutyrate
numerous admirable researches, based on the hybrid con- in presence of potassium carbonate and methyl isobutyl
cept of lipid lowering agents, have been reported in the ketone were carried out to accomplish the desired
literature. Shukla et al. synthesized some chalcone-fibrates benzylidenethiazolidin-4-one fibrate derivatives (BRF1–
and claimed them to be useful in treating dyslipidemia BRF12). The possible mechanism of the reaction is deli-
(Shukla et al. 2011). Two years later, Sashidhara et al. neated in Scheme 2. Finally, structures of the synthesized
worked on coumarin-chalcone-fibrate (Sashidhara et al. compounds were established by infrared (IR), 1H 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 HO EtO N H
HO N H S O
+ S S
X O X
X
BR1-BR4 BRF1-BRF4
(b)

O O
Series X Point of attachment O
of fibrate ester O (d) EtO N
N O
BRF1 S 4 HO S O
BRF2 S 3
S O O X
BRF3 O 4 X
BRF4 O 3
BRF5 S 4 BR5 -BR8 BRF5 -BRF8
BRF6 S 3
BRF7 O 4 (c)
BRF8 O 3
BRF9 S 4 O
BRF10 S 3 O
BRF11 O 4 OH (d) OH
BRF12 O 3 N EtO N
HO O
S S O
O O
X X

BR9 -BR12 BRF9 -BRF12

Scheme 1 The synthetic route to the title compounds (BRF1-BRF12). (20 min, 140 W); d Ethyl α-bromo isobutyrate, K2CO3, methyl iso-
a Piperidine, AcOH, M.W. (20 min, 140 W); b BrCH2COOCH3, butyl ketone, M.W. (20 min, 460 W)
K2CO3, Acetone, M.W. (14 min, 560 W); c AcOH, HCl, M.W.

13
magnetic resonance (NMR), and C NMR spectroscopy permeability of the active compounds in drug discovery.
and mass spectrometry. Very high or very low log P values generally affect ADME
properties of molecules to a great extent. The result of
Partition coefficient partition coefficient study indicated that the compound
BRF6 was the most lipophilic among the synthesized series,
Lipophilicity is represented by the descriptors log P (also where presence of S at the rhodanine nucleus significantly
known as Kow or Pow) and is used to predict in vivo increases the log P value of BRF6 (2.09). Compound
 Med Chem Res

O H
O
- C 5 H 11 NH
HO
N R + C 5 H 11 N N R
S S
Proton abstraction Condensation
X by piperidine base X
Carbanion

O OH O O O
-H 2 O H+
N R N R N R
S Dehydration S Protonation S
O X HO
X X
H Enolate

Electrophilic substitution, -HBr N R

EtO S
O O
X
Br O
EtO

Scheme 2 The plausible reaction mechanism

BRF12 was the least lipophilic in the series, with a log P benzene ring was the most hydrolyzed (28.64%) and com-
value of 1.65. pound BRF4, having no any substitution on N-3 position of
thiazolidine-2,4-dione along with the fibric acid moiety on
In vitro hydrolysis 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 stability at physiological pH (acidic and basic) is of the probable bioavailability through the intestine.
prime importance for the drugs intended for oral adminis-
tration. It has been observed that many drugs have low In vivo antidyslipidemic activity
bioavailability owing to the degradation at the low pH (1–2)
of stomach. The simulated gastric fluid (SGF) mimics the The anti-hyperlipidemic activity of the synthesized com-
gastric fluid in terms of the acidity and molarity and the pounds was determined using high fat dietary model (Lee
simulated intestinal fluid (SIF) mimics the intestinal fluid in et al. 2010) for inducing hypercholesterolemia in rats.
terms of basicity. These fluids are the perfect media to Feeding of the rats resulted in the alteration of body weight
determine the stability of drug candidates in vitro. In the as well as the serum lipid profile (Tables 1 and 2). It was
present study, the synthesized compounds were tested evident from the observation that continuous post feeding of
in vitro using the SGF and SIF. The results of in vitro the synthesized compounds, at the dose of 30 mg/kg for 7
hydrolysis showed that compound BRF6, having N-acetic days, resulted in an appreciable improvement of body
acid methyl ester at N-3 position of rhodanine along with weights and hyperlipidemia. Interestingly, the treatment
the fibric acid moiety on the C-3 position of the benzene with the synthesized compounds significantly reduced the
ring, was least hydrolyzed (2.25%), thereby indicating its levels of TG, LDL, VLDL and elevated the levels of HDL,
stability in the stomach and the probable bioavailability. with a reduction in body weight. The results of the study are
However, compound BRF11, having N-acetic acid on N-3 shown in Tables 1 and 2. All the synthesized compounds
position of thiazolidine-2,4-dione along with the fibric acid exhibited significant lowering in the serum TG and VLDL
moiety on the C-4 position of the benzene ring was the most concentration. Compounds BRF1–11 showed significant
hydrolyzed (24.77%) in SGF. The compound BRF7, hav- reduction of the serum total cholesterol (TC). The com-
ing N-acetic acid methyl ester on N-3 position of rhodanine pounds BRF4, 6, 7, 11, and 12 also lowered the LDL. The
along with the fibric acid moiety on the C-4 position of the compounds BRF4, 6, 9, 10, and 12 elevated the level of
Table 1 Effect of the synthesized derivatives (BRF1-12) on the body weight of rats fed with HFD
Treatment groups Dose (mg/kg) Initial weight (g) Weight on 30th day Weight gain (g) Weight on 37th day (g) Weight reduction (g) Food intake (g) Food efficiency ratio
[A] (g) (after HFD) [B] [B−A] (after 7 days treatment) (after treatment) [C] [(B−A)/C] *100
Med Chem Res

ND control (Only normal 148 ± 2.86 156 ± 2.75 8 ± 0.78 158 ± 2.58 −2 ± 0.32 425 ± 13.75 1.9 ± 0.03
diet)
HFD control (HFD + 1% 168 ± 3.53 195 ± 4.24 27 ± 1.63 200 ± 4.24 −5 ± 0.71 310 ± 15.62 8.7 ± 0.16
Gum Acacia)
Standard (HFD + 30 218 ± 1.41 256 ± 4.14 38 ± 2.68 240 ± 2.12 16 ± 3.91* 390 ± 6.32 9.7 ± 0.18
Fenofibrate)
BRF1 30 130 ± 3.53 165 ± 2.52 35 ± 1.24 150 ± 14.1 15 ± 1.96* 280 ± 17.34 12.5 ± 0.14*
BRF2 30 164 ± 7.07 182 ± 5.14 18 ± 3.19 175 ± 3.53 7 ± 0.05** 265 ± 8.38 6.7 ± 0.08
BRF3 30 112 ± 2.12 140 ± 3.53 28 ± 2.34 135 ± 4.24 5 ± 1.25 270 ± 14.14 10.3 ± 0.25
BRF4 30 115 ± 2.52 138 ± 3.50 23 ± 1.18 122 ± 7.07 16 ± 0.49* 325 ± 5.80 7.0 ± 0.89
BRF5 30 172 ± 3.53 194 ± 4.24 22 ± 1.22 185 ± 3.53 9 ± 3.53* 385 ± 17.5 5.7 ± 0.52**
BRF6 30 125 ± 4.24 162 ± 3.50 37 ± 0.84 145 ± 14.1 17 ± 2.52* 345 ± 15.62 10.7 ± 0.76
BRF7 30 150 ± 4.10 176 ± 3.53 26 ± 3.21 170 ± 7.07 6 ± 0.29 210 ± 6.41 12.3 ± 0.94*
BRF9 30 124 ± 2.52 154 ± 4.24 30 ± 0.34 146 ± 3.53 8 ± 3.92** 295 ± 14.14 10.2 ± 1.01
BRF10 30 126 ± 3.5 143 ± 3.53 17 ± 1.11 130 ± 2.52 13 ± 3.82* 215 ± 12.06 7.9 ± 0.62
BRF11 30 173 ± 4.14 213 ± 5.07 40 ± 0.94 200 ± 3.53 13 ± 4.70* 415 ± 13.05 9.6 ± 0.45
BRF12 30 132 ± 8.5 163 ± 8.50 31 ± 1.76 155 ± 2.12 8 ± 2.08** 320 ± 15.62 9.7 ± 0.93
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 compounds among the series
*P ≤ 0.01 and **P ≤ 0.05 (When HFD control was compared with ND control)
 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) AI (TC-HDL)/HDL
TC HDL-c LDL-c TG VLDL-c

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

BRF1 351.435 −5.82972 1 5.75 3.526 2 0.192 100 1105.14 82.408 0

BRF2 351.435 −6.28481 1 5.75 3.531 2 0.193 100 1105.363 82.385 0
BRF3 335.374 −6.06512 1 5.75 2.542 1 0.091 84.326 1046.644 112.585 0
BRF4 335.374 −7.98248 1 5.75 2.539 1 0.089 84.319 1045.7 112.562 0
BRF5 423.498 −6.20834 0 7.25 4.011 2 0.197 100 1329.118 113.177 0
BRF6 423.498 −7.16932 0 7.25 4.011 2 0.196 100 1328.66 113.153 0
BRF7 407.437 −3.74430 0 7.75 3.019 2 −0.028 85.563 1297.707 132.495 0
BRF8 407.437 −6.91927 0 7.75 3.025 2 −0.027 85.607 1298.036 132.471 0
BRF9 409.471 −5.92356 1 7.25 3.771 2 0.045 78.558 1243.827 126.899 0
BRF10 409.471 −2.60685 1 7.25 3.806 2 0.059 78.695 1249.866 126.883 0
BRF11 393.411 −5.16139 1 7.75 2.854 2 −0.139 66.116 1212.722 146.529 0
BRF12 393.411 −5.43395 1 7.75 2.907 2 −0.118 66.301 1222.081 146.503 0
Co-crystal ligand GW735 478.485 −11.5622 2 6.75 5.457 4 0.618 82.532 1402.252 99.785 1
Standard values/range 130 to 725 – 0–6 2–20 −2.0–6.5 1–8 −1.5–1.5 >80% is high<20% is poor 500–2000 7–200 Maximum is 4
Number of violations of Lipinski’s rule of five
DonarHB hydrogen bond donar, AccptHB hydrogen bond acceptor, Metab number of likely metabolic reactions, QPlogKhsa prediction of binding to human serum albumin, Volume total solvent
accessible volume in cubic angstrom using a probe with 1.4 Ǻ radius, PSA Vander Waals polar surface area of nitrogen and oxygen atoms
 Med Chem Res

Fig. 2 Design of PPAR-α agonist on the basis of rosiglitazone molecule

(3D-QSAR) studies and crystal structure information hypolipidemic activity on the basis of higher negative value
revealed that the pharmacophoric features of these agents of Glide XP gscore. The docking results showed a good
essentially consists of three parts: (i) an acidic head, (ii) ligand–protein interaction, coverage of contacts and parti-
central aromatic region, and (iii) a lipophilic side chain cipation of the active site residues His440 and Tyr464 in H-
(Sundriyal et al. 2008) (Fig. 2). On the basis of these bonding, which contributed to stabilize the ligand with
findings, molecular structures based on fibric acid and gli- PPARα receptor. The fibric acid moiety in position 4
tazones have been designed and docked into the binding demonstrated negative role on the basis of the same para-
pocket of PPARα protein (PDB-ID: 2P54), to gain a cor- meters, as observed with the compounds BRF1, 3, 5, and 7.
relation with the hypolipidemic activity. Additionally, it was observed that in derivatives BRF 5–8,
Generally, it has been reported that three important both thiazolidinone (TZD) as well as fibric acid moiety were
ligand–receptor interactions, with His323, Tyr473, and buried well inside the hydrophobic pocket of PPARα while
His449 are imperative for the activation of the receptor in case of BRF9–12, TZD moiety was mostly extruded out
PPAR-γ, whereas interaction with His440 and Tyr464 of the hydrophobic pocket of PPARα. This revealed that the
residue is crucial for the activation of PPAR-α (da Costa replacement via –CH2COOCH3 at the position N-3 of TZD
Leite et al. 2007). As the consequence of the same, it has ring, was found to be more favorable than that of the
been observed that compounds BRF4 and BRF6 showed a replacement via –CH2COOH for hypolipidemic activity.
key interaction with the His440 and Tyr464 residues of the The fibric acid and TZD moieties of BRF4 and BRF6 were
PPARα receptor protein. This also gave strong evidence oriented in such a way so that the favorable π–π and
that these compounds must have some beneficial effects hydrogen bond interactions was developed with active site
through the PPARα receptor. residues Phe118, His440, and Tyr464 (Fig. 3). Thus, they
After performing docking simulations, it became possi- attained the maximum docking scores of −7.96678 and
ble to conclude that the presence of the fibric acid moiety at −7.16932 respectively, anticipating that BRF4 and BRF6
the third position of the phenyl ring, as observed in the may be the most active molecules through PPARα. In vivo
BRF2, 4, 6, and 8 played an important role in eliciting the activity also indicated similar findings that the BRF4 and
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 chalcone-fibrate, indole chalcone-fibrates, in the literatures,
synthesized derivatives. On the other hand, the compounds however, following details are summarized for the first time
BRF7 and BRF10 showed the least docking scores of in the present research on thiazolidin-4-one chalone-fibrates
−3.74430 and −2.60685, respectively, because of the towards antidyslipidemic activity-
maximum four polar atom burial and desolvation penalties, ● Hybridization of thiazolidin-4-one, chalcone and fibrates
and penalty for intra-ligand contacts. Moreover, Compound
in a single moiety to reinforce the antidyslipidemic
BRF-12 exhibited significant in vivo activity, whereas the
effect through PPARα/γ dual agonistic action.
same exhibited moderate activity in the in silico assessment. ● Synthesis of all the compounds via knoevenagel reaction
The reason for the activity may be due to the esterification
and thereafter simple electrophilic substitution with the
at N–H reactive site of rhodanine.
help of microwave assisted organic synthesis.
To date, despite wealth of the information available on ● Development of N-methyl ester and N-methyl acid
the therapeutic importance of chalcone-fibrates, coumarin
 Med Chem Res

derivatives of benzalidinethiazolidin-2,4-dione fibrate EL III Elemental analyzer (Elementar, Germany) at CDRI,

and benzalidine-2-thioxo-4-thiazolidinone fibrate to Lucknow.
enhance the activity.
● Significant lowering of bad cholesterol levels like LDLc, General procedure for the synthesis of
VLDLc, and TG and significant elevation in the good benzylidenethiazolidin-4-one fibrates (BRF1–12)
cholesterol level like HDL-c as compared with reference
drug, fenofibrate at the same dose level. A mixture of appropriately substituted benzilidinethiazoli-
● More reduction of AI and thereby atherogenesis as dine-4-one (BR1–12) (1.71 mmol), ethyl α-bromoisobuty-
compared with reference compound, fenofibrate at same rate (0.30 ml, 2.05 mmol), potassium carbonate (0.471 g,
dose level. 3.42 mmol) and methyl isobutyl ketone (10 ml) was placed
● Significant reduction of the body weight with minimal in a round bottom flask and irradiated for 20 min in a
food efficiency ratio. microwave synthesizer at 560 W. The reaction was mon-
● Molecular docking of the synthesized molecules onto itored by TLC, using 40% ethyl acetate: n-hexane as the
PPAR-α receptor using Glide-XP present in Maestro solvent system. The reaction mixture was cooled to room
user interface of Schrödinger Inc. showing better temperature, the inorganic salts were filtered off; then the
complementarity and binding affinity of some of the solvent was evaporated under reduced pressure, the solid
molecules with the receptor, showing a good correlation residue was collected and recrystallized from ethanol to
of the in silico results with the in vivo studies. obtain the title compounds (BRF1–12).

2-Methyl-2-[4-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-
phenoxy]-propionic acid ethyl ester (BRF1)
Experimental
Dark orange solid; yield: 71.07%; melting range (°C):
General 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,
The chemicals and reagents were procured from Sigma 928.86 C–O str (ether), 1749.48 C=O str (ester); 1H NMR
Aldrich Chemicals, Mumbai and were used without further [(CD3OD, 300 MHz) δ in ppm: 1.276 (t, 3H) 1.768 (s, 6H),
purification. Microwave assisted synthesis was performed 3.306–3.315 (m, 2H) 6.890–6.910 (d, 2H, aromatic),
using Raga’s Scientific Microwave Systems (Ragatech, 7.383–7.705 (d, 4H, aromatic); 13C NMR (CD3OD, 75
Pune, Maharashtra, India). Progress of the reaction was MHz) δ: 23.24 (2CH3 aliphatic), 48.30 (CH2 aliphatic),
monitored by thin layer chromatography on silica gel G 126.23–134.20 (2CH aromatic), 154.20 (C aromatic),
plates using iodine vapors and UV light as visualizing 161.92 (C amide), 171.52 (C carboxyl) 196.91 (C thioa-
agents. Melting points were determined by open capillary mide); ESI-MS: [M + 1]+ at m/z 352.02. Anal. calcd. for
method and are uncorrected. After physical characterization, C16H17NO4S2: C, 54.68; H, 4.88; N, 3.99. Found: C, 54.71;
the compounds were subjected to spectral analysis. UV H, 4.90; N, 4.03.
spectra were recorded on a Double Beam spectrophotometer
(Shimadzu-1700). The IR spectra were recorded on Perkin
2-Methyl-2-[3-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-
Elmer RX1 FTIR spectrophotometer using KBr disks and
phenoxy]-propionic acid ethyl ester (BRF2)
the values are expressed in cm−1 and only noteworthy
absorption levels (reciprocal centimeters) are listed. The
Dark yellow solid; yield: 71.18%; melting range (°C):
mass spectra were recorded on JEOL-Accu TOF JMS-
218–220; IR (KBr) (ν cm−1): 1638.97 C=O str (amide),
T100LC mass spectrometer and ESI-MS-Accu TOF JMS-
1329.41 C–N str, 1215.68 C=S str, 1628.97 C=C str,
T100LC mass spectrometer at CDRI, Lucknow. The
928.86 C–O str (ether), 1749.56 C=O str (ester); 1H NMR
nuclear magnetic resonance (1H and 13C NMR) spectra
[(CD3OD, 300 MHz) δ in ppm: 1.153–1.176 (t, 3H)
were recorded at 300 and 75 MHz on a Bruker DRX
1.200–1.269 (s, 6H), 3.299–3.321 (m, 2H), 6.863–6.940 (s,
spectrometer (Bruker Instruments Inc., USA) at CDRI,
3H, aromatic) 7.002–7.330 (s, 1H, aromatic), 7.499–7.596
Lucknow. CD3OD was taken as the solvent and the che-
(s, 2H, Ar); 13C NMR (CD3OD, 75 MHz) δ: 23.42 (2CH3
mical shifts are reported in parts per million (δ values),
aliphatic), 48.30 (CH2 aliphatic), 127.26 (2CH aromatic),
using TMS (δ 0 ppm for 1HNMR) as the internal standard.
136.06 (C aromatic), 159.53 (C amide), 171.17 (C car-
The spin multiplicities in 1H NMR are indicated as follows:
boxyl), 196.95 (C thioamide); ESI-MS: [M + 1]+ at m/z
s (singlet), d (doublet), dd (double doublet), t (triplet) and m
352.13. Anal. calcd. for C16H17NO4S2: C, 54.68; H, 4.88;
(multiplet). Elemental analysis was performed on a Vario
N, 3.99. Found: C, 54.75; H, 4.89; N, 4.00.
Med Chem Res

2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2- 2-[3-(3-Methoxycarbonylmethyl-4-oxo-2-thioxo-thiazolidin-
methyl-propionic acid ethyl ester (BRF3) 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), Greenish yellow solid; yield: 58.98%; melting range (°C):
1215.56 C–N str, 1645.76 C=C str, 1025.88 C–O str 286–288; IR (KBr) (ν cm−1): 1683.79 C=O str (amide),
(ether), 1745.63 C=O str (ester); 1H NMR [(CD3OD, 300 1215.72 C–N str, 1215.73 C=S str, 1612.37 C=C str,
MHz) δ in ppm: 1.153 (t, 3H) 1.269 (s, 6H), 3.299–3.321 1025.48 C–O str (ether), 1744.83 C=O str (ester), 1411.98
(m, 2H) 6.748–6.751 (d, 2H, aromatic), 7.014–7.016 (d, 2H, CH2 bend; 1H NMR [(CD3OD, 300 MHz) δ in ppm: 1.283
aromatic), 7.259–7.430 (s, 2H); 13C NMR (CD3OD, 75 (t, 3H) 2.839 (s, 6H), 3.188–3.319 (m, 2H), 3.481 (s, 2H),
MHz) δ: 22.56 (2CH3 aliphatic), 48.27 (CH2 aliphatic), 4.315 (s, 3H), 6.854–6.857 (d, 3H, aromatic), 7.000–7.015
122.56 (2CH aromatic), 138.09 (CH aromatic), 159.10 (C (t, 1H, aromatic), 7.807 (s, 1H); 13C NMR (CD3OD, 75
amide), 185.97 (C thioamide); ESI-MS: [M + 1]+ at m/z MHz) δ: 23.24 (CH3 aliphatic) 26.23 (2CH3 aliphatic),
336.97. Anal. calcd. for C16H17NO4S2: C, 57.30; H, 5.11; 48.30 (CH2 aliphatic) 49.72 (CH2 aliphatic), 50.00 (CH3
N, 4.18. Found: C, 57.26; H, 5.09; N, 4.20. 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;
2-[3-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-
H, 5.01; N, 3.32.
methyl-propionic acid ethyl ester (BRF4)
2-[4-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-
Crystalline yellow solid; yield: 46.75%; melting range (°C):
ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
250–252; IR (KBr) (ν cm−1): 1645.34 C=O str (amide),
ester (BRF7)
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):
0.898–1.178 (t, 3H), 1.285 (s, 6H), 3.299–3.321 (m, 2H)
298–300; IR (KBr) (ν cm−1): 1686.35 C=O str (amide),
6.873–6.945 (s, 2H, Ar) 7.000 (t, 1H, aromatic), 7.521 (s,
1323.73 C–N str, 1607.03 C=C str, 935.39 C–O str (ether),
H, aromatic); 13C NMR (CD3OD, 75 MHz) δ: 23.36 (2CH3
1741.69 C=O str (ester), 1441.30 CH2 bend; 1H NMR
aliphatic), 48.30 (CH2 aliphatic), 123.36 (2CH aromatic),
[(CD3OD, 300 MHz) δ in ppm: 1.235 (t, 3H), 1.618 (s, 6H),
133.22 (CH aromatic), 159.58 (C amide), 196.87 (C
3.299–3.320 (m, 2H), 3.774 (s, 2H), 3.798 (s, 3H),
thioamide); ESI-MS: [M + 1]+ at m/z 320.34. Anal. calcd.
6.878–6.881 (d, 2H, aromatic), 7.318–7.344 (d, 2H, aro-
for C15H14NO5S: C, 56.41; H, 4.41; N, 4.37. Found: C,
matic), 7.839 (s, 1H); 13C NMR (CD3OD, 75 MHz) δ:
56.70; H, 4.35; N, 4.34.
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-
2-[4-(3-Methoxycarbonylmethyl-4-oxo-2-thioxo-thiazolidin- boxyl); ESI-MS: [M + 1]+ at m/z 408.10. Anal. calcd. for
5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl C19H21NO7S: C, 56.01; H, 5.20; N, 3.44. Found: C, 56.16;
ester (BRF5) H, 5.21; N, 3.42.

Brick red solid; yield: 61.72%; melting range (°C): 2-[3-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-

282–284; IR (KBr) (ν cm−1): 1639.09 C=O str (amide), ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl
1329.01 C–N str, 1215.57 C=S str, 1639.09 C=C str, ester (BRF8)
927.90 C–O str (ether), 1747.43 C=O str (ester), 1408.18
CH2 bend; 1H NMR [(CD3OD, 300 MHz) δ in ppm: 1.271 Cream white solid; yield: 65.95%; melting range (°C):
(t, 3H) 2.338 (s, 6H), 3.299–3.321 (m, 3H), 3.745–3.776 (s, 302–305; IR (KBr) (ν cm−1): 1686.67 C=O str (Amide),
2H), 6.863–6.873 (d, 2H, aromatic), 7.364 (d, 2H, aro- 1323.13 C–N str, 1607.64 C=C, 1022.46 C–O str (Ether),
matic), 7.698 (s, 1H); 13C NMR (CD3OD, 75 MHz) δ: 1741.07 C=O str (Ester), 1441.83 CH2 bend; 1H NMR
17.57 (CH3 aliphatic) 20.66 (2CH3 aliphatic), 48.30–49.72 [(CD3OD, 300 MHz) δ in ppm: 1.235 (t, 3H) 1.618 (s, 6H),
(CH2 aliphatic) 120.66 (2CH aromatic), 134.43 (2CH aro- 3.299–3.320 (m, 3H), 4.500 (s, 4H), 6.878–6.881 (d, 2H,
matic), 161.54 (C amide), 171.48 (C carboxyl), 196.87 (C aromatic), 7.025 (t, 1H, aromatic), 7.839 (s, 1H); 13C NMR
thioamide); ESI-MS: [M + 1]+ at m/z 424.97. Anal. calcd. (CD3OD, 75 MHz) δ: 17.57 (CH3 aliphatic), 20.66 (CH3
for C19H21NO6S2: C, 53.88; H, 5.00; N, 3.31. Found: C, aliphatic), 48.87 (CH2 aliphatic), 53.41 (CH3 aliphatic),
53.80; H, 4.97; N, 3.34. 117.45 (2CH aromatic), 135.84 (CH aromatic), 159.53 (C
 Med Chem Res

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

Creamy white solid; yield: 68.06%; melting range (°C): Conc: of compound in nocatanol
Log P ¼ log
348–350; IR (KBr) (ν cm−1): 1686.35 C=O str (amide), Conc: of compound in water
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
Med Chem Res

Hydrolysis studies Table 4 Composition of the HFD (%)

Components Composition (g/1000 g of diet)
In vitro hydrolysis studies were performed spectro-
Casein 20
photometrically using SGF/SIF (Ashford 2002; Tripathi
D,L-methionine 0.3
et al. 2014) and the per cent hydrolysis was calculated using
the following formulae. Corn starch 15
Sucrose 27.5
% Hydrolized in SGF Cellulose powder 5
ðAbs: at 0 min  Abs: at 90 minÞ Mineral mixa 3.5
¼  100;
Abs: at 0 min Vitamin mix b
1
Choline bitartrate 0.2
% Hydrolized in SGF
Corn oil 9.9
ðAbs: at 0 min  Abs: at 120 minÞ
¼  100: Lard 17.6
Abs :at 0 min Total (%) 100
kcal/100 g diet 498.7
Calories from fat (%) 49.6
Pharmacology Calories from carbohydrate (%) 34.1
Calories from protein (%) 16.3
Male Wistar rats, weighing 125–225 gm, were used for a
AIN-76 mineral mixture contained (in g/kg of mixture): calcium
pharmacological studies of all the newly synthesized deri- phosphate, dibasic 500.0; sodium chloride, 74.0; potassium citrate
vatives. The animals were housed in polypropylene cages monohydrate, 220.0; potassium sulphate, 52.0; magnesium oxide,
with steel net and maintained under standard living condi- 24.0; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6;
tions of temperature 24 ± 5 °C under controlled humidity of cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01;
chromium potassium sulphate, 0.55; sucrose, finely powdered, 118.03
55 ± 5%, with regular 12 h light/dark cycle, and allowed b
AIN-76A vitamin mixture contained (in g/kg of mixture): thiamine
free access to laboratory food and water. All the animals HCl, 0.6; riboflavin, 0.6; pyridoxine HCl, 0.7: niacin, 3.0; D-calcium
were treated morally in accordance with the guidelines laid pantothenate, 1.6; folic acid, 0.2: D-biotin, 0.02; cyanocobalamin
down by the Institutional Animal Ethics Committee (IAEC, (vitamin B12), 1.0; dry vitamin A palmitate (500,000 U/g), 0.8;
vide protocol approval no. BBDNIIT/IAEC/001/2014). dry vitamin E acetate (500 U/g), 10.0; vitamin D3 trituration
(400,000 U/g), 0.25; menadione sodium bisulphite complex, 0.15;
Experiments were performed after the approval by the sucrose, fine powder, 981.08
IAEC, strictly adhering to the ethical guidelines.

In vivo screening group was compared with that of the control group. The
reduction in atherogenesis is expressed in terms of AI,
The high fat diet (HFD) induced hyperlipidemic model (Lee which was determined using the following equation:
et al. 2010) was used to determine antihyperlipidemic
TC  HDL
activity of the series of compounds in male Wistar rats. A Atherogenic index ðAIÞ ¼
HDL
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- In silico screening
nely. The test compounds were administered at a dose of 30
mg/kg body weight. Fenofibrate, at a dose of 30 mg/kg was To study the ligand–protein interactions and to establish a
taken as the reference drug. The synthesized compounds correlation with biological activity, the docking analyses of
and standard drug were suspended in 1% gum acacia and all the derivatives were carried out, using Glide-XP protocol
administrated orally (p.o.). At the end of the experiment, the of Schrödinger Inc (Maestro, version 9.3, Schrödinger,
blood sample was withdrawn from the retro-orbital plexus LLC, New York, NY, 2012). The molecular target taken
of eye of the rats for the estimation of TC, serum TG, (LDL- into consideration was the nuclear PPARα receptor, whose
c), HDL-c and VLDL-c. A significant reduction in TG, crystallographic structure was procured from Protein Data
LDL-c, and VLDL-c levels, a significant increase in HDL-c Bank (PDB-ID: 2P54). The crystal structure was subse-
levels and a significant reduction in body weight after drug quently optimized and minimized with the “protein pre-
treatments, as compared to control animals, were considered paration wizard” workflow, as implemented in the
as a positive antidyslipidemic response. The mean value of Schrödinger 2012 package. The ligands were built using
lipid profile and weight reduction for each experimental Maestro 9.3 build panel and prepared by LigPrep 2.5
 Med Chem Res

version v25111(Schrödinger, LLC, USA) application that Compounds BRF4 and BRF6 were found to be the most
uses optimized potential liquid simulations 2005 force field, active compounds among all the synthesized derivatives
and it gave the corresponding energy minima 3D con- in vivo, and exhibited anti-dyslipidemic activity by low-
formers of the ligands. The default settings were used for all ering LDL-c, VLDL-c, and TG, and increasing the level of
other parameters. The active site was considered as a rigid HDL-c, thereby decreasing the AI. Altogether, these effects
molecule, whereas the ligands were treated as being flex- of BRF4 and BRF6 were found to be more potent as
ible, i.e. all non-ring torsions were allowed. The ligand compared with the standard drug, in either case of lipid
interaction affinities of the synthesized compounds were lowering activity or reducing AI. The interesting feature of
compared with the re-docking results of the native bioactive these compounds was to increase the level of good cho-
co-crystal ligand GW735 in the 2P54 protein crystal lesterol, i.e. HDL-c, and thus reduction in AI to a level more
structure. than that by the standard drug, fenofibrate. The reason for
Nearly 40% of drug candidates fail in clinical trials the higher activity in case of HDL-c and AI may be due to
due to poor absorption, distribution, metabolism, and the attachment of both, the benzylidenethiazolidin-4-one
excretion (ADME) properties. These late-stage failures and fibrate moiety in a single frame. Benzylidenethiazoli-
contribute significantly to the rapidly escalating cost of new din-4-one individually has already been reported to reduce
drug development. The ability to detect the problematic atherosclerosis caused by LDL oxidation. The results sug-
candidates early can dramatically reduce the amount of gest that these thiazolidine-chalcone-fibrates constitute a
wasted time and resources, and streamline the overall new prototype of lipid lowering agents that might act by
development process. QikProp, version 3.5 (Schrödinger, lowering bad cholesterol and elevating good cholesterol,
LLC, New York, NY, 2012) program was used for in silico and thus decreasing the AI. However, this newly synthe-
prediction of pharmacokinetic properties of the synthesized sized pharmacophoric framework needs to go through fur-
compounds. 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-
Statistical analysis
effects.
All the values of the experimental results are expressed as
Acknowledgements We express our sincere gratitude to Central
mean ± SEM and statistical significance between the groups Drugs Research Institute (CDRI), Lucknow, India for providing the
was calculated by one way analysis of variance (ANOVA) library and sophisticated analytical instrument facilities. Authors are
followed by Dunett’s multiple comparison tests. P ≤ 0.01 thankful to the All India Council for Technical Education (AICTE),
was considered statistically significant. Statistical analysis New Delhi, India, for providing grant under the Research Promotion
Scheme (RPS), through which the computational software facility has
was carried out using Graph Pad Instat 3.0 (Graph Pad been made available at the host institute. We also acknowledge the
Software). 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

Conclusion interests.

While considering all the newly synthesized compounds

together, it may be concluded that the hybridization of fibric References
acid analog with a special type of chalcones, namely ben-
zylidinethiazolidin-2,4-diones and benzylidine-rhodanines, Adeghate E, Adem A, Hasan MY, Tekes K, Kalasz H (2011) Med-
establish an important pharmacophore and the positions N- icinal chemistry and actions of dual and pan PPAR modulators.
Open Med Chem J 5:93–98
3, 4, and 5 of the thiazolidin-4-one moiety are the key
Ashford M (2002) Assessmemt of biopharmaceutical properties. In:
reactive sites, which could be altered with different groups Aulton ME (ed) Pharmaceutics—the science of dosage form
to elicit valuable hypolipidemic profiles. More precisely, design, 2 edn. Churchill Livingstone, Edinburg, pp 257–258
based on the in vivo and in silico activity results, the Balakumar P, Rose M, Ganti SS, Krishan P, Singh M (2007) PPAR
dual agonists: are they opening Pandora’s Box? Pharmacol Res
esterification of N–H functionality in thiazolidin-4-one
56:91–98
moiety, to yield N-acetic acid methyl ester derivatives, Bruno G, Costantino L, Curinga C, Maccari R, Monforte F, Nicolo F,
appeared to be significant in activity demonstrating pro- Ottana R, Vigorita MG (2002) Synthesis and aldose reductase
nounced anti-hyperlipidemic activity. In addition, substitu- inhibitory activity of 5-arylidene-2,4-thiazolidinediones. Bioorg
Med Chem 10:1077–1084
tion with fibric acid moiety in C-3 position of benzene ring,
da Costa Leite LF, Veras Mourao RH, de Lima Mdo C, Galdino SL,
attached with thiazolidin-4-one moiety, was also respon- Hernandes MZ, de Assis Rocha Neves F, Vidal S, Barbe J, da
sible for the enhancement of activity. Rocha Pitta I (2007) Synthesis, biological evaluation and
Med Chem Res

molecular modeling studies of arylidene-thiazolidinediones with Sashidhara KV, Dodda RP, Sonkar R, Palnati GR, Bhatia G (2014)
potential hypoglycemic and hypolipidemic activities. Eur J Med Design and synthesis of novel indole-chalcone fibrates as lipid
Chem 42:1263–1271 lowering agents. Eur J Med Chem 81:499–509
Fruchart JC (2009) Peroxisome proliferator-activated receptor-alpha Sashidhara KV, Palnati GR, Sonkar R, Avula SR, Awasthi C, Bhatia
(PPARalpha): at the crossroads of obesity, diabetes and cardio- G (2013) Coumarin chalcone fibrates: a new structural class of
vascular disease. Atherosclerosis 205:1–8 lipid lowering agents. Eur J Med Chem 64:422–431
Graham DJ, Staffa JA, Shatin D, Andrade SE, Schech SD, La Grenade Shukla P, Srivastava SP, Srivastava R, Rawat AK, Srivastava AK,
L, Gurwitz JH, Chan KA, Goodman MJ, Platt R (2004) Incidence Pratap R (2011) Synthesis and antidyslipidemic activity of chal-
of hospitalized rhabdomyolysis in patients treated with lipid- cone fibrates. Bioorg Med Chem Lett 21:3475–3478
lowering drugs. Jama 292:2585–2590 Sortino M, Delgado P, Juarez S, Quiroga J, Abonia R, Insuasty B,
Jeong TS, Kim JR, Kim KS, Cho KH, Bae KH, Lee WS (2004) Nogueras M, Rodero L, Garibotto FM, Enriz RD, Zacchino SA
Inhibitory effects of multi-substituted benzylidenethiazolidine- (2007) Synthesis and antifungal activity of (Z)-5-arylidenerho-
2,4-diones on LDL oxidation. Bioorg Med Chem 12:4017–4023 danines. Bioorg Med Chem 15:484–494
Lalloyer F, Staels B (2010) Fibrates, glitazones, and peroxisome Sundriyal S, Viswanad B, Ramarao P, Chakraborti AK, Bharatam PV
proliferator-activated receptors. Arterioscler Thromb Vasc Biol (2008) New PPARgamma ligands based on barbituric acid: vir-
30:894–899 tual screening, synthesis and receptor binding studies. Bioorg
Lee JS, Bok SH, Jeon SM, Kim HJ, Do KM, Park YB, Choi MS Med Chem Lett 18:4959–4962
(2010) Antihyperlipidemic effects of buckwheat leaf and flower Tiwari P, Puri A, Chander R, Bhatia G, Misra AK (2006) Synthesis
in rats fed a high-fat diet. Food Chem 119:235–240 and antihyperlipidemic activity of novel glycosyl fructose deri-
Lehrke M, Lazar MA (2005) The many faces of PPARgamma. Cell vatives. Bioorg Med Chem Lett 16:6028–6033
123:993–999 Tripathi AC, Gupta SJ, Fatima GN, Sonar PK, Verma A, Saraf SK
Mc Gill HC (1985) Geographical pathology of athersclerosis. Wil- (2013) 4-Thiazolidinones: the advances continue. Eur J Med
liams and Wilkins Co, Baltimore Chem 72C:52–77
Morphy R, Kay C, Rankovic Z (2004) From magic bullets to designed Tripathi P, Tripathi AC, Chawla V, Saraf SK (2014) Syntheses,
multiple ligands. Drug Discov Today 9:641–651 characterization and evaluation of novel 2,6-diarylpiperidin-4-
Pirat C, Farce A, Lebegue N, Renault N, Furman C, Millet R, Yous S, ones as potential analgesic-antipyretic agents. Eur J Med Chem
Speca S, Berthelot P, Desreumaux P, Chavatte P (2012) Target- 82:439–448
ing peroxisome proliferator-activated receptors (PPARs): devel- US Food and Drug Administration (2012) Statin drugs_drug safety
opment of modulators. J Med Chem 55:4027–4061 communication class labelling change. http://www.fda.gov/
Quinn CE, Hamilton PK, Lockhart CJ, McVeigh GE (2008) Thiazo- safety. Accessed Feb 2014
lidinediones: effects on insulin resistance and the cardiovascular Varga T, Czimmerer Z, Nagy L (2011) PPARs are a unique set of fatty
system. Br J Pharmacol 153:636–645 acid regulated transcription factors controlling both lipid meta-
Rakowitz D, Maccari R, Ottana R, Vigorita MG (2006) In vitro aldose bolism and inflammation. Biochim Biophys Acta 1812:
reductase inhibitory activity of 5-benzyl-2,4-thiazolidinediones. 1007–1022
Bioorg Med Chem 14:567–574 Verma A, Saraf SK (2008) 4-Thiazolidinone—a biologically active
Redemann CE, Icke RN, Alles GA (eds) (1955) Organic Synthesis vol scaffold. Eur J Med Chem 43:897–905
3. Organic Syntheses Inc., New York, Wiley Wierzbicki AS (2009) Fibrates in the treatment of cardiovascular risk
Rizos CV, Liberopoulos EN, Mikhailidis DP, Elisaf MS (2008) and atherogenic dyslipidaemia. Curr Opin Cardiol 24:372–379
Pleiotropic effects of thiazolidinediones. Expert Opin Pharmac- Zhang HY (2005) One-compound-multiple-targets strategy to combat
other 9:1087–1108 Alzheimer’s disease. FEBS Lett 579:5260–5264