Advertisement

Medicinal Chemistry Research

, Volume 26, Issue 7, pp 1535–1549 | Cite as

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 Singh
  • Avinash C. Tripathi
  • Aseem Tewari
  • Viney Chawla
  • Shailendra K. Saraf
Original Research
  • 158 Downloads

Abstract

An interesting hybrid molecular framework comprising of benzylidenethiazolidin-4-one, chalcone and fibrate was designed and synthesized (BRF112) in order to develop safe and efficacious compounds for the treatment of dyslipidemia, and related complications such as atherosclerosis. 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 promising antidyslipidemic activity comparable to, and sometimes 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 cholesterol, 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 established 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.

Keywords

Anti-hyperlipidemic Rhodanine Microwave assisted synthesis Molecular docking 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 cholesterol 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 hepatotoxicity and myotoxicity, which may be due to the reduced levels of essential isoprenoid precursors, the antioxidant ubiquinone, or dolichols (Sashidhara et al. 2014). Moreover, statins can hardly normalize the high density lipoprotein (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), search is on the better therapeutic strategies for the development of safe and effective antidyslipidemic drugs.

The peroxisome proliferator activating receptors (PPARs) are a subfamily of ligand-activated nuclear hormone 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 coordinating the expression of genes involved in lipid metabolism, 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 example, “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 profile 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 innovative 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 structural units of different nature generally possess improved biological activities (Sashidhara et al. 2013). To date, numerous admirable researches, based on the hybrid concept 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. 2013) followed by indole-chalcone-fibrate (Sashidhara et al. 2014) with promising lipid lowering activity. Moreover, thorough literature survey revealed that the benzylidenethiazolidinone 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).

Inspired by the beneficial effect of PPAR-α/γ dual agonistic 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), thiazolidinone (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 approaches. An environmentally benign microwave facilitated green approach was employed for the synthesis of the proposed derivatives. Few representative chemical structures 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.
Fig. 1

Chemical structures of representative fibrate, thiazolidinone, chalcone, and the synthesized prototype

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 condensation with the thiazolidin-4-one derivatives in presence of the catalytic amounts of piperidine and glacial acetic acid to afford corresponding benzylidenethiazolidin-4-ones (BR1BR4). This was followed by the consequences of reactions at N-3 position, to form N-methyl ester derivatives (BR5BR8), and their subsequent hydrolysis to form N-acetic acid derivatives (BR9BR12).
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 isobutyl ketone, M.W. (20 min, 460 W)

In order to attach the fibrate moiety with the aforementioned compounds (BR1BR12), the electrophilic substitution 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 (BRF1BRF12). The possible mechanism of the reaction is delineated in Scheme 2. Finally, structures of the synthesized compounds were established by infrared (IR), 1H nuclear magnetic resonance (NMR), and 13C NMR spectroscopy and mass spectrometry.
Scheme 2

The plausible reaction mechanism

Partition coefficient

Lipophilicity is represented by the descriptors log P (also known as Kow or Pow) and is used to predict in vivo 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 BRF12 was the least lipophilic in the series, with a log P value of 1.65.

In vitro hydrolysis

The stability at physiological pH (acidic and basic) is of prime importance for the drugs intended for oral administration. 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, having N-acetic acid methyl ester on N-3 position of rhodanine along with the fibric acid moiety on the C-4 position of the benzene ring was the most hydrolyzed (28.64%) and compound 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 hydrolyzed (2.40%) in SIF, which is indicative of its stability and the probable bioavailability through the intestine.

In vivo antidyslipidemic activity

The anti-hyperlipidemic activity of the synthesized compounds 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 BRF111 showed significant reduction of the serum total cholesterol (TC). The compounds BRF4, 6, 7, 11, and 12 also lowered the LDL. The compounds BRF4, 6, 9, 10, and 12 elevated the level of 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 lipoprotein cholesterol (LDL-c), and very low density lipoprotein 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.
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) [A]

Weight on 30th day (g) (after HFD) [B]

Weight gain (g) [B−A]

Weight on 37th day (g) (after 7 days treatment)

Weight reduction (g) (after treatment)

Food intake (g) [C]

Food efficiency ratio [(B−A)/C] *100

ND control

(Only normal diet)

148 ± 2.86

156 ± 2.75

8 ± 0.78

158 ± 2.58

−2 ± 0.32

425 ± 13.75

1.9 ± 0.03

HFD control

(HFD + 1% Gum Acacia)

168 ± 3.53

195 ± 4.24

27 ± 1.63

200 ± 4.24

−5 ± 0.71

310 ± 15.62

8.7 ± 0.16

Standard (HFD + Fenofibrate)

30

218 ± 1.41

256 ± 4.14

38 ± 2.68

240 ± 2.12

16 ± 3.91*

390 ± 6.32

9.7 ± 0.18

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)

Table 2

Effects of the synthesized derivatives (BRF112) 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 Diet

72.8 ± 10.66

29.1 ± 3.78

35.4 ± 3.64

93.8 ± 37.69

18.7 ± 7.54

1.37 ± 0.17

HFD control

(HFD + 1% gum acacia)

92.8 ± 4.87

16.2 ± 2.54***

61.7 ± 7.14***

144.3 ± 9.05***

29.8 ± 1.43

4.72 ± 0.08***

Standard (HFD + Fenofibrate)

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*

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)

Molecular docking studies

To see the ligand–protein interaction and to establish a correlation with the biological activity, the docking analyses of compounds BRF112 were carried out, using Glide-XP 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.
Table 3

Molecular docking and in silico ADME prediction of synthesized compounds (BRF112) 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

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

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 summarized 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 structural 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 (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 glitazones have been designed and docked into the binding pocket of PPARα protein (PDB-ID: 2P54), to gain a correlation with the hypolipidemic activity.
Fig. 2

Design of PPAR-α agonist on the basis of rosiglitazone molecule

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.

After performing docking simulations, it became possible 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 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 participation 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 parameters, as observed with the compounds BRF1, 3, 5, and 7. Additionally, it was observed that in derivatives BRF 58, both thiazolidinone (TZD) as well as fibric acid moiety were buried well inside the hydrophobic pocket of PPARα while in case of BRF912, 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 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.
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)

To date, despite wealth of the information available on the therapeutic importance of chalcone-fibrates, coumarin 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 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.

Experimental

General

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 chemical shifts are reported in parts per million (δ values), using TMS (δ 0 ppm for 1HNMR) 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 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 benzilidinethiazolidine-4-one (BR112) (1.71 mmol), ethyl α-bromoisobutyrate (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 monitored 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 (BRF112).

2-Methyl-2-[4-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-propionic acid ethyl ester (BRF1)

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, 928.86 C–O str (ether), 1749.48 C=O str (ester); 1H 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 thioamide); 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, 928.86 C–O str (ether), 1749.56 C=O str (ester); 1H 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 carboxyl), 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.

2-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF3)

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 (ether), 1745.63 C=O str (ester); 1H 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.

2-[3-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF4)

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: 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 CH2 bend; 1H NMR [(CD3OD, 300 MHz) δ in ppm: 1.271 (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, aromatic), 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 aromatic), 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.

2-[3-(3-Methoxycarbonylmethyl-4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF6)

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 CH2 bend; 1H 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 thioamide); 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-[4-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF7)

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, aromatic), 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 carboxyl); 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.

2-[3-(3-Methoxycarbonylmethyl-2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF8)

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 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.

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 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, 52.72; H, 4.77; N, 3.38.

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.

2-[4-(3-Carboxymethyl-2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxy]-2-methyl-propionic acid ethyl ester (BRF11)

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 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 aliphatic) 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-[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.

In-vitro studies

The in vitro studies included determination of partition coefficient and hydrolysis profile of the synthesized derivatives 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:
$${\rm{Log}}\,P\,{\rm{ = }}\,{\rm{log}}\frac{{{\rm{Conc}}{\rm{.}}\,{\rm{of}}\,{\rm{compound}}\,{\rm{in}}\,n{\rm{ - ocatanol}}}}{{{\rm{Conc}}{\rm{.}}\,{\rm{of}}\,{\rm{compound}}\,{\rm{in}}\,{\rm{water}}}}$$

Hydrolysis studies

In vitro hydrolysis studies were performed spectrophotometrically using SGF/SIF (Ashford 2002; Tripathi et al. 2014) and the per cent hydrolysis was calculated using the following formulae.
$$ \% \,{\rm{Hydrolized}}\,{\rm{in}}\,{\rm SGF} \\ = \frac{{\left( {{\rm{Abs}}{\rm{.}}\,{\rm{at}}\,{\rm{0}}\,{\rm{min}} - {\rm{Abs}}{\rm{.}}\,{\rm{at}}\,9{\rm{0}}\,{\rm{min}}} \right)}}{{{\rm{Abs}}{\rm{.}}\,{\rm{at}}\,{\rm{0}}\,{\rm{min}}}} \times 100,$$
$$ \% \,{\rm{Hydrolized}}\,{\rm{in}}\,{\rm{SGF}} \\ = \frac{{\left( {{\rm{Abs}}{\rm{.}}\,{\rm{at}}\,{\rm{0}}\,{\rm{min}} - {\rm{Abs}}{\rm{.}}\,{\rm{at}}\,12{\rm{0}}\,{\rm{min}}} \right)}}{{{\rm{Abs}}\,{\rm{.at}}\,{\rm{0}}\,{\rm{min}}}} \times 100.$$

Pharmacology

Male Wistar rats, weighing 125–225 gm, were used for pharmacological studies of all the newly synthesized derivatives. The animals were housed in polypropylene cages with steel net and maintained under standard living conditions 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.

In vivo screening

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 routinely. 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 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:
$${\rm{Atherogenic}}\,{\rm{index}}\,{\rm{(AI)}}\,{\rm{ = }}\,\frac{{{\rm{TC - HDL}}}}{{{\rm{HDL}}}}$$
Table 4

Composition of the HFD (%)

Components

Composition (g/1000 g of diet)

Casein

20

d,l-methionine

0.3

Corn starch

15

Sucrose

27.5

Cellulose powder

5

Mineral mixa

3.5

Vitamin mixb

1

Choline bitartrate

0.2

Corn oil

9.9

Lard

17.6

Total (%)

100

kcal/100 g diet

498.7

Calories from fat (%)

49.6

Calories from carbohydrate (%)

34.1

Calories from protein (%)

16.3

a 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 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 subsequently optimized and minimized with the “protein preparation 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 version v25111(Schrödinger, LLC, USA) application that uses optimized potential liquid simulations 2005 force field, and it gave the corresponding energy minima 3D conformers 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 flexible, 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.

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).

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 benzylidinethiazolidin-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 pronounced anti-hyperlipidemic activity. In addition, substitution with fibric acid moiety in C-3 position of benzene ring, attached with thiazolidin-4-one moiety, was also responsible for the enhancement of activity.

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 lowering 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 cholesterol, 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. Benzylidenethiazolidin-4-one individually has already been reported to reduce atherosclerosis caused by LDL oxidation. The results suggest 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 synthesized pharmacophoric framework needs to go through further 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.

Notes

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.

References

  1. Adeghate E, Adem A, Hasan MY, Tekes K, Kalasz H (2011) Medicinal chemistry and actions of dual and pan PPAR modulators. Open Med Chem J 5:93–98CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ashford M (2002) Assessmemt of biopharmaceutical properties. In: Aulton ME (ed) Pharmaceutics—the science of dosage form design, 2 edn. Churchill Livingstone, Edinburg, pp 257–258Google Scholar
  3. Balakumar P, Rose M, Ganti SS, Krishan P, Singh M (2007) PPAR dual agonists: are they opening Pandora’s Box? Pharmacol Res 56:91–98CrossRefPubMedGoogle Scholar
  4. Bruno G, Costantino L, Curinga C, Maccari R, Monforte F, Nicolo F, Ottana R, Vigorita MG (2002) Synthesis and aldose reductase inhibitory activity of 5-arylidene-2,4-thiazolidinediones. Bioorg Med Chem 10:1077–1084CrossRefPubMedGoogle Scholar
  5. da Costa Leite LF, Veras Mourao RH, de Lima Mdo C, Galdino SL, Hernandes MZ, de Assis Rocha Neves F, Vidal S, Barbe J, da Rocha Pitta I (2007) Synthesis, biological evaluation and molecular modeling studies of arylidene-thiazolidinediones with potential hypoglycemic and hypolipidemic activities. Eur J Med Chem 42:1263–1271CrossRefPubMedGoogle Scholar
  6. Fruchart JC (2009) Peroxisome proliferator-activated receptor-alpha (PPARalpha): at the crossroads of obesity, diabetes and cardiovascular disease. Atherosclerosis 205:1–8CrossRefPubMedGoogle Scholar
  7. Graham DJ, Staffa JA, Shatin D, Andrade SE, Schech SD, La Grenade L, Gurwitz JH, Chan KA, Goodman MJ, Platt R (2004) Incidence of hospitalized rhabdomyolysis in patients treated with lipid-lowering drugs. Jama 292:2585–2590CrossRefPubMedGoogle Scholar
  8. Jeong TS, Kim JR, Kim KS, Cho KH, Bae KH, Lee WS (2004) Inhibitory effects of multi-substituted benzylidenethiazolidine-2,4-diones on LDL oxidation. Bioorg Med Chem 12:4017–4023CrossRefPubMedGoogle Scholar
  9. Lalloyer F, Staels B (2010) Fibrates, glitazones, and peroxisome proliferator-activated receptors. Arterioscler Thromb Vasc Biol 30:894–899CrossRefPubMedPubMedCentralGoogle Scholar
  10. Lee JS, Bok SH, Jeon SM, Kim HJ, Do KM, Park YB, Choi MS (2010) Antihyperlipidemic effects of buckwheat leaf and flower in rats fed a high-fat diet. Food Chem 119:235–240CrossRefGoogle Scholar
  11. Lehrke M, Lazar MA (2005) The many faces of PPARgamma. Cell 123:993–999CrossRefPubMedGoogle Scholar
  12. Mc Gill HC (1985) Geographical pathology of athersclerosis. Williams and Wilkins Co, BaltimoreGoogle Scholar
  13. Morphy R, Kay C, Rankovic Z (2004) From magic bullets to designed multiple ligands. Drug Discov Today 9:641–651CrossRefPubMedGoogle Scholar
  14. Pirat C, Farce A, Lebegue N, Renault N, Furman C, Millet R, Yous S, Speca S, Berthelot P, Desreumaux P, Chavatte P (2012) Targeting peroxisome proliferator-activated receptors (PPARs): development of modulators. J Med Chem 55:4027–4061CrossRefPubMedGoogle Scholar
  15. Quinn CE, Hamilton PK, Lockhart CJ, McVeigh GE (2008) Thiazolidinediones: effects on insulin resistance and the cardiovascular system. Br J Pharmacol 153:636–645CrossRefPubMedGoogle Scholar
  16. Rakowitz D, Maccari R, Ottana R, Vigorita MG (2006) In vitro aldose reductase inhibitory activity of 5-benzyl-2,4-thiazolidinediones. Bioorg Med Chem 14:567–574CrossRefPubMedGoogle Scholar
  17. Redemann CE, Icke RN, Alles GA (eds) (1955) Organic Synthesis vol 3. Organic Syntheses Inc., New York, WileyGoogle Scholar
  18. Rizos CV, Liberopoulos EN, Mikhailidis DP, Elisaf MS (2008) Pleiotropic effects of thiazolidinediones. Expert Opin Pharmacother 9:1087–1108CrossRefPubMedGoogle Scholar
  19. Sashidhara KV, Dodda RP, Sonkar R, Palnati GR, Bhatia G (2014) Design and synthesis of novel indole-chalcone fibrates as lipid lowering agents. Eur J Med Chem 81:499–509CrossRefPubMedGoogle Scholar
  20. Sashidhara KV, Palnati GR, Sonkar R, Avula SR, Awasthi C, Bhatia G (2013) Coumarin chalcone fibrates: a new structural class of lipid lowering agents. Eur J Med Chem 64:422–431CrossRefPubMedGoogle Scholar
  21. Shukla P, Srivastava SP, Srivastava R, Rawat AK, Srivastava AK, Pratap R (2011) Synthesis and antidyslipidemic activity of chalcone fibrates. Bioorg Med Chem Lett 21:3475–3478CrossRefPubMedGoogle Scholar
  22. Sortino M, Delgado P, Juarez S, Quiroga J, Abonia R, Insuasty B, Nogueras M, Rodero L, Garibotto FM, Enriz RD, Zacchino SA (2007) Synthesis and antifungal activity of (Z)-5-arylidenerhodanines. Bioorg Med Chem 15:484–494CrossRefPubMedGoogle Scholar
  23. Sundriyal S, Viswanad B, Ramarao P, Chakraborti AK, Bharatam PV (2008) New PPARgamma ligands based on barbituric acid: virtual screening, synthesis and receptor binding studies. Bioorg Med Chem Lett 18:4959–4962CrossRefPubMedGoogle Scholar
  24. Tiwari P, Puri A, Chander R, Bhatia G, Misra AK (2006) Synthesis and antihyperlipidemic activity of novel glycosyl fructose derivatives. Bioorg Med Chem Lett 16:6028–6033CrossRefPubMedGoogle Scholar
  25. Tripathi AC, Gupta SJ, Fatima GN, Sonar PK, Verma A, Saraf SK (2013) 4-Thiazolidinones: the advances continue. Eur J Med Chem 72C:52–77Google Scholar
  26. Tripathi P, Tripathi AC, Chawla V, Saraf SK (2014) Syntheses, characterization and evaluation of novel 2,6-diarylpiperidin-4-ones as potential analgesic-antipyretic agents. Eur J Med Chem 82:439–448CrossRefPubMedGoogle Scholar
  27. US Food and Drug Administration (2012) Statin drugs_drug safety communication class labelling change. http://www.fda.gov/safety. Accessed Feb 2014
  28. Varga T, Czimmerer Z, Nagy L (2011) PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation. Biochim Biophys Acta 1812:1007–1022CrossRefPubMedPubMedCentralGoogle Scholar
  29. Verma A, Saraf SK (2008) 4-Thiazolidinone—a biologically active scaffold. Eur J Med Chem 43:897–905CrossRefPubMedGoogle Scholar
  30. Wierzbicki AS (2009) Fibrates in the treatment of cardiovascular risk and atherogenic dyslipidaemia. Curr Opin Cardiol 24:372–379CrossRefPubMedGoogle Scholar
  31. Zhang HY (2005) One-compound-multiple-targets strategy to combat Alzheimer’s disease. FEBS Lett 579:5260–5264CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Pharmaceutical Chemistry, Faculty of PharmacyBabu Banarasi Das Northern India Institute of TechnologyLucknowIndia

Personalised recommendations