All the chemicals used in the synthesis were purchased from Sigma Aldrich and used without any further purification or distillation. Synthesized 5-substituted 2,4-Thiazolidinedione derivatives were purified by recrystallization in appropriate solvents and purity was checked by thin layer chromatography using silica gel 60 F₂₅₄ Merck using ethyl acetate: petroleum ether (1:1) as solvent system. Melting points were determined in open glass capillaries by using Digital Gallenhamp (SANYO) model MPD.BM 3.5 apparatus and are uncorrected. Characterization of all the newly synthesized arylidene derivatives was done by using spectrophotometric analysis i.e. FTIR (Thermo-scientific NICOLET IS10 Spectrophotometer) and ¹HNMR (Bruker AM-300 Spectrophotometer) using DMSO-d₆ as solvent. Chemical shifts (δ) were indicated in parts per million downfield from tetramethylsilane, and the coupling constants (J) were recorded in Hertz. Molecular docking studies were carried out using AutoDock Vina software.

2,4 Thiazolidinedione was synthesized following the standard procedure [17Nawale, S.L.; Dhake, A.S. Synthesis and evaluation of novel thiazolidinedione derivatives for antibacterial activity. Pharma Chem., 2012, 4, 2270-2277.].

Yield 62%, m.p (obs) 123-125^oC, m.p (lit) 123-125^oC, R f = 0.34 (ethyl acetate: pet. ether 1:1), IR (cm^-1): 3217 (N-H), 1680 (C=O), 1648 (C=O), 1472 (CH₂).

A mixture of 2,4-Thiazolidinedione (3.0 mmol), diethyl amine (3.0 mmol) and respective aldehydes (3.0 mmol) in 20-25 ml of distilled water was stirred at room temperature for 3-5 hours. The progress of the reaction was monitored by thin layer chromatography (ethyl acetate: pet. ether 1:1). After reaction completion as indicated by TLC, the crude product separated was filtered and recrystallized from ethanol [18Barakat, A.; Al-Majid, A.M.; AL-Najjar, H.J.; Mabkhot, Y.N.; Ghabbour, H.A.; Fun, H-K. An efficient and green procedure for synthesis of rhodanine derivatives by aldol-thia-Michael protocol using aqueous diethylamine medium. RSC Adv., 2014, 4, 4909-4916.
[http://dx.doi.org/10.1039/c3ra46551a] ] (Table 1).

2.3.1. 5-(3-Phenylallylidene) Thiazolidine-2,4-Dione (1a)

Yield 67%, m.p. 215-217°C, Rf = 0.25 (ethyl acetate: pet. Ether 1:1), IR (cm^-1): 3217 (N-H), 1656 (C=O), 1648 (C=O), 1511 (C=C), ¹HNMR (300 MHz, DMSO): 12.37 (s, 1H, NH), 7.26-7.42 (m, 5H, Aryl-H), 7.99 (d, 1H, J = 14.5 Hz, HC=CH), 7.02 (d, 1H, J = 13.0 Hz, C=CH), 6.71 (d, 1H, J = 14.5 Hz, HC=CH). ¹³CNMR (100 MHz, CCl₄): 169.3 (C=O), 167.1 (C=O), 140.2 (CH), 136.6, 131.2, 131.9, 127.4 (Aryl-C), 128.7 (C). Elemental Anal. Calc.: C = 62.26%, N = 6.05%, H = 3.92%, Found: C = 62.32%, N = 6.13%, H = 4.03%.

Table 1
Chemical structures of the compounds 1(a-g).

To a suspension of 6.0 mmoles of the respective arylidene derivative in ethanol, 7.0 mmol of potassium hydroxide was added and the mixture was stirred for two hours at room temperature. The precipitates of corresponding potassium salt were separated, filtered and washed with ethanol. In the next step, a mixture of 0.02 mole of respective salt and 0.03 mole of sodium chloroacetate in ethanol was refluxed for 5-6 hours. The reaction mixture was then diluted with 40 ml of water and acidified with dilute hydrochloric acid to pH 3. The precipitates formed were separated by filtration and washed with ether. The crude product was recrystallized from DMF-ethanol [19Zimenkovskii, B.S.; Kutsyk, R.V.; Lesyk, R.B.; Matyichuk, V.S.; Obushak, N.D.; Klyufinska, T.I. Synthesis and antimicrobial activity of 2,4-dioxothiazolidine-5-acetic acid amides. Pharm. Chem. J., 2006, 40, 303-306.
[http://dx.doi.org/10.1007/s11094-006-0115-6] ] (Table 2).

2.4.1. 2-(2,4-dioxo-5-(3-phenylallylidene) thiazolidin-3-yl) acetic acid (2a)

Yield 42%, m.p. 218-220°C, R_f = 0.34 (ethyl acetate: pet. Ether 1:1), IR (cm^-1): 3560 (O-H), 1710 (C=O), 1676 (C=O), 1640 (C=O), 1500(C=C), ¹HNMR (300 MHz, DMSO): 12.52 (s, 1H, COOH), 7.99 (d, 1H, J = 12.4 Hz, C=CH), 7.33-7.46 (m, 5H, Aryl-H), 7.02 (d, 1H, J = 13.5 Hz HC=CH), 6.71 (d, 1H, J = 13.4 Hz, HC=CH), 4.65 (s, 2H, CH₂-CO). ¹³CNMR (100 MHz, CCl₄): 173.5 (COOH), 170.0 (C=O), 165.7 (C=O), 138.9, 133.8, 132.7 (CH), 136.5-127.1 (Aryl C). 52.6 (CH₂). Elemental Anal. Calc.: C = 58.06%, N = 4.83%, H = 3.83%, Found: C = 58.12%, N = 4.77%, H = 3.80%.

Table 2
Chemical structures of the synthesized compounds 2(a-d).

In-vitro α-amylase inhibitory activity was performed by following reported procedure. 100 µL reaction mixture was prepared using 70 µL of 50 mM phosphate buffer pH 6.8, 50 µL of test compound followed by the addition of 10 µL (0.057 units) enzyme solution in the buffer. The contents were mixed, pre-incubated for 10 minutes at 25°C and absorbance measured at 400nm. The 10 µL of 0.5 mM p-nitrophenyl glucopyranoside and incubated at 25°C, the absorbance of p-nitrophenol was measured at 400nm using the 96-well plate reader. Acarbose was used as standard. All the experiments were carried out in triplicates. The percent inhibition was calculated by the given formula:

Active compounds were suitably diluted and their inhibition studies were determined. Results were obtained as %inhibition. The IC₅₀ values of samples were calculated by linear regression analysis.

Exploration of mechanism of action and molecular interaction of synthesized derivatives with α-amylase was performed by molecular docking studies using AutoDock Vina software. To study the molecular interaction of compounds, crystallographic data of human pancreatic α-amylase (PDB:1HNY) was retrieved from Protein Data Bank. Retrieved crystal structure of α-amylase was cleaned and hydrogen atoms were added. All the heteroatoms were removed before docking study. The samples used in the molecular docking studies were compounds 1(a-g) and 2(a-d) (Fig. 2). The 2D structures were sketched using ChemDraw-ultrav14.0 followed by 3D structure conversion and energy minimization by ChemBioOffice modelling software (Cambridgesoft, UK). The 2D and 3D interactions were visualized using discovery studio 4.0.

In our study, synthesis of 2,4-thiazolidinedione was carried out by reacting chloroacetic acid and thiourea in the presence of concentrated HCl (Scheme 1). Formation of thiazolidinedione nucleus was confirmed by comparing its melting point with the reported literature [13Marshall, P.G.; Vallance, D.K. Anticonvulsant activity; derivatives of succinimide, glutarimide, thiazolidinedione and methanol, and some miscellaneous compounds. J. Pharm. Pharmacol., 1954, 6(10), 740-746.
[http://dx.doi.org/10.1111/j.2042-7158.1954.tb11011.x] [PMID: 13212661] ]. In the next step, 2,4-thiazolidinedione was reacted with various aldehydes in the presence of diethyl amine and distilled water [14Ceriello, A. Thiazolidinediones as anti-inflammatory and anti-atherogenic agents. Diabetes Metab. Res. Rev., 2008, 24(1), 14-26.
[http://dx.doi.org/10.1002/dmrr.790] [PMID: 17990280] ] to get 5-arylidene derivatives of 2,4-thiazolidinediones 1 (a-g). The synthesized compounds were characterized by FTIR and NMR spectroscopic data. The yields of compounds were obtained in the range of 40-70%. Purity in each case was established by thin layer chromatography. In the IR spectra characteristic peaks of amide carbonyl of thiazolidinedione moiety were observed in the range 1648-1686 cm^-1. NH stretchings were present at 3121-3266 cm^-1. Aromatic C=C stretchings were obsereved at 1500-1594 cm^-1 which indicated the condensation of aromatic aldehydes with thiazolidinedione nucleus. In the ¹HNMR spectra of 1 (a-g) characteristic singlet peaks of methine protons were observed downfield in the range 7.02-8.22 ppm confirming the formation of arylidene derivatives. In case of 1a having cinnamoyl moiety two doublets of methine protons appeared at 6.71 and 7.99 ppm with trans coupling constant of 14.0 Hz. NH singlet peak was observed in all derivatives at 12.31-12.58 ppm. The ¹³CNMR data showed two peaks for carbonyl carbons of thiazolidinedione nucleus. Methine carbon of arylidene moiety appeared at 143.3 ppm as expected.

In the last step compounds 2(a-d) were synthesized by the reaction of respective arylidene derivative of thiazolidinedione 1(a-d) with sodium chloroacetate [19Zimenkovskii, B.S.; Kutsyk, R.V.; Lesyk, R.B.; Matyichuk, V.S.; Obushak, N.D.; Klyufinska, T.I. Synthesis and antimicrobial activity of 2,4-dioxothiazolidine-5-acetic acid amides. Pharm. Chem. J., 2006, 40, 303-306.
[http://dx.doi.org/10.1007/s11094-006-0115-6] ]. In case of other three arylidene derivatives 1(e-g) the reaction was not successful due to very low solubility of these compounds. The yields of isolated acetic acid derivatives were also low. The target compounds 2(a-d) were characterized by FTIR and NMR spectroscopic data. The IR data exhibited characteristic carbonyl stretching of carboxylic acid moiety in the range 1732-1750 cm^-1, while amide cabonyl stretchings were observed at lower frequency. The ¹HNMR data of these derivatives exhibited characteristic singlet peak for methylene protons of acetic acid moiety in the range 3.38-4.75 ppm. Also, NH singlets were absent in all the spectra. Singlets of carboxylic OH were observed downfield at 12.52-12.60 ppm. In the ¹³CNMR spectra carboxylic carbon resonated at 169.0 ppm while carbonyl groups of thiazolidinedione moiety appeared at 164.4 and 173.4 ppm. Methylene carbon appeared upfield at 47.9 ppm. All the other carbons resonated in the expected region.

The synthesized compounds were tested for their inhibitory potential against α-amylase enzyme. Different concentrations of the test compounds i.e. 20 µg/mL, 30 µg/mL, 40 µg/mL and 50 µg/mL were used for the assay. The concentrations were plotted against % inhibition and IC₅₀ of each compound was calculated by non-linear regression method by using Graphpad Prism V6.0. All tested compounds showed good to excellent inhibitory potential with IC₅₀ values ranging from 2.03 µg/mL to 32.16 µg/mL (Fig. 1). Compounds 1c, 1d and 1g were found to be most potent inhibitors among all the tested compounds with IC₅₀ value of 6.59 µg/mL, 2.03 µg/mL and 3.14 µg/mL respectively. The inhibition was significant (P < 0.01) when compared to positive control. It can be concluded from the results that unsubstituted benzene derivative exhibits the lowest IC₅₀ value, whereas 3-hydroxy and 3-methoxy derivatives were also more active as compared to the standard Acarbose. This may be due to hydrogen bond formation capacity of these derivatives in the active site of the enzyme. In case of 2-hydroxy and 4-hydroxy arylidene derivatives the inhibitory potential was slightly lower as compared to the standard. All of the 5-arylidene of 2,4-thiazolidinediones showed good inhibition as compared to acetic acid derivatives of 5-arylidene 2,4 thiazolidinediones. The inhibitory potential of all the acetic acid derivatives of 2,4-thiazolidinediones was low. This can be attributed to the polar nature of these derivatives due to the carboxylic group present in 2(a-d) which might result in less permeability across cell membranes.

Scheme 1

Fig. (1) Comparison of IC50 of the test compounds. Significance vs. Positive control group: **P < 0.01.

Molecular docking study was carried out to determine the potential interactions and binding affinity between the ligand and the target enzyme, α-amylase. All the ligands tend to bind in the same binding site of α-amylase within an RMSD of 1.5Å when compared with acarbose. Three significant residues i.e., Asp300, Glu233 and Asp197 are present at the active site cleft which are mainly involved in the hydrolysis of glycosidic bonds present in carbohydrates. The most acceptable docked poses of ligands with the best binding affinity for human pancreatic α-amylase at the active site were selected and are shown in Fig. (2).

Fig. (2) Binding cavity of α-amylase with docked ligands and the interacting residues around.

The binding affinities of the synthesized compounds and acarbose against α-amylase enzyme were determined using Autodock vina. All the compounds gave good binding affinities ranging from -7.2 to -6.3 KJ/mol due to hydrogen bond formation capacity at multiple sites especially the carbonyl groups present in the ring are involved in hydrogen bonding in the active site. Acetic acid derivatives have good binding affinity due to extra pi-anion interactions with the aspartic acid residues present in the enzyme’s active site. IC₅₀ value of 1d is lowest whereas it showed lower binding affinity due to absence of any hydrogen bond formation group on benzene ring. Acarbose showed binding affinity of -7.5 KJ/mol. The binding affinities of the compounds and the interactions with the protein are given in Table 3.

Table 3
Binding affinity and binding interactions of synthesized compounds.

The compound 2a showed the lowest binding affinity of -7.2 KJ/mol (Fig. 3). The binding affinities of acetic acid derivatives were found to be better as compared to 1(a-g) but inhibitory potential was low which can be attributed to the polar carboxylic group present in 2(a-d) making them less permeable across cell membranes.

Fig. (3) The ligand-protein interactions of compound 2a with the active site of α-amylase generated by using Discovery Studio 4.0. The (A) shows the two-dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The (B) show the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate the interactions between the ligand and the amino acids of the receptor.