Synthesis and Molecular Docking Studies of Thiazolidineones with Peroxiredoxins as Antioxidant Agents

Jainey P. James¹*, Pavitra Pramod Bhandarkar¹, Pavan T. S¹, Zakiya Fathima C¹,

Sindhu T. J.¹, Treesa P. Varghese²

¹Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences,

NITTE (Deemed to be University), Paneer, Deralakatte, Mangalore - 575018, Karnataka, India.

²Department of Pharmacy Practice, Yenepoya Pharmacy College and Research Centre,

Yenepoya (Deemed to be University), Naringana, Mangalore.

*Corresponding Author E-mail: jaineyjames@gmail.com

ABSTRACT:

Antioxidants either slow or stop the oxidation processes that occur due to the presence of oxygen or reactive oxygen species in the environment. The schiff base was synthesised and cyclised with thioglycolic acid to yield thiazolidineones (B1-B9). Structural characterisation was performed by IR, Mass and ¹H NMR spectral studies. The compounds were checked for antioxidant activity by analysing the interactions with peroxiredoxins (1URM), and Among them, the top-scored compounds were B6 and B4, excellently interacting with the targets. According to the physicochemical and ADME properties of Qikprop, synthesised compounds can be considered druglike molecules. Antioxidant activity studies were conducted using the DPPH method, and the docking results were found from the in vitro studies. Based on these results, it is concluded that the synthesised compounds can be utilised as leads as antioxidant agents, which can be further verified by conducting pharmacological studies.

KEYWORDS: Thiazolidineone, molecular docking, peroxiredoxins, antioxidant activity.

INTRODUCTION: 

Antioxidants are crucial in slowing or preventing oxidation processes induced by oxygen or reactive oxygen species in various environments. They find applications in stabilising polymeric products, petrochemicals, foodstuffs, cosmetics, and medications. A sophisticated cellular antioxidant defence system exists within the human body, categorised into primary and secondary systems. Primary defences include superoxide and peroxidase, while secondary defences involve proteolytic and lipolytic enzymes. These systems work collaboratively to counteract oxidative stress and maintain cellular integrity¹.

In recent years, a surge in reported diseases has been attributed to an imbalance in the homeostatic regulation of pro-oxidants in the body.

Elevated pro-oxidants lead to an increased generation of reactive oxygen species (ROS) or free radicals. These free radicals, characterised by their instability and reactivity due to an unpaired electron in the outer shell, initiate attacks on specific biomolecules within the body. This results in significant cellular damage, including modifications to polypeptides and lipid peroxidation². The consequences of such oxidative stress contribute to the onset of various diseases, including cancer³, cardiovascular diseases⁴, neurodegenerative disorders^5,6, alcohol-induced liver diseases, ulcerative colitis, atherosclerosis⁷, and the ageing process.

Spices and herbs have undergone extensive scrutiny due to their potent antioxidant activity, contributing to human health benefits. Antioxidants are crucial in protecting food lipids and oils against oxidative degradation. When added to food, antioxidants prevent rancidity, delay the formation of toxic oxidation products, maintain nutritional quality, and extend the overall shelf life of products. Due to safety concerns, synthetic antioxidants are restricted to serving as food preservatives. Natural antioxidants from edible materials, such as spices and herbs, have become increasingly attractive. Spices inherently possess natural oxidants that aid in reducing oxidative stress. Various detrimental factors, including gamma, UV, and X-ray radiation, can induce oxidative stress by generating high concentrations of free radicals in cells and tissues ^9,10.

Ascorbic acid, uric acid, vitamin A, carotenes, and flavonoids represent some plant antioxidants. Meanwhile, synthetic antioxidants like phenolic antioxidants (such as probucol and nitecapone), butylated hydroxytoluene (BHT), and propyl gallate (PG) are currently under development as therapeutic agents against oxidative stress¹¹. In response to public interest in the potential health benefits of dietary antioxidants, food and beverage companies are emphasising incorporating antioxidants into their products. Antioxidants play a crucial role in the protective effects exerted by plant foods, and consuming fruits, vegetables, grains, and nuts has been linked to a lower incidence of chronic diseases¹². Peroxiredoxins (Prx) are crucial antioxidant enzymes that effectively reduce oxidative stress. They play a key role in catalysing the reduction of H₂O₂ and various peroxides, effectively clearing these reactive oxygen species¹³.

In the contemporary landscape, heterocyclic compounds have gained paramount importance due to their significant biological and pharmacological activities. These compounds possess diverse pharmacological properties and are promising lead molecules in drug discovery. Thiazolidineones, as oxo-derivatives of thiazolidine, a saturated form of thiazole, fall within the extensive category of sulfur- and nitrogen-containing compounds with a five-membered ring. Thiazolidineone is recognised for exhibiting a broad spectrum of biological activities^14-15. In silico studies^16-19 have enormously contributed to discovering new leads for many diseases. Therefore, thiazolidineone will be synthesised and checked for its antioxidant action and interaction with peroxiredoxins in this study.

MATERIALS AND METHODS:

Thiazolidinone derivatives were synthesised under standard laboratory conditions using readily available reagents and appropriate equipment. Characterisation involved identifying the molecular framework, the nature of functional groups, their location within the skeletal structure, and determining any existing stereochemical relationships. The study utilised the following techniques: IR Spectroscopy (Bruker FT-IR), frequencies expressed in cm⁻¹; 1H-NMR Spectroscopy (Agilent-NMR Spectrometer at 400MHz, DMSO); Mass Spectrometry (LC-MS Perkin Elmer Clarus 680 Spectrometer) and the method is Time-of-flight ionization. These analytical techniques were employed to characterise the synthesised compounds, providing valuable information about their structural features and confirming the success of the synthetic process.

Synthesis of Schiff base:

The synthetic procedure involved the following steps: An equimolar mixture of hydrazide and aromatic aldehydes was taken in a round-bottomed flask. The mixture was refluxed for 4 hours using ethanol as the solvent and a catalytic amount of concentrated H₂SO₄. The completion of the reaction was checked by TLC (Thin Layer Chromatography). The reaction mixture was allowed to cool. The separated solid was filtered and dried. A blend of diethyl ether and ethyl acetate was used for the recrystallisation of the final products. The overall yield of the process was reported to be 80-95%²⁰.

(E)-1-(4-bromobenzylidene)-2-phenylhydrazine (A1): yellow solid, m.p.145-147^oC; yield 67.2%; IR (cm^-1): 678 (C-Br), 3023 (aromatic C-H), 1676 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 7.21-8.51 (m, 7H, Ar-H), 4.72 (s, 1H, NH), 8.02 (s, 1H, CH); Mass m/z: (M+) 274.

(E)-1-(4-chlorobenzylidene)-2-phenylhydrazine (A2): orange solid, m.p.157-159^oC; yield 71.25%; IR (cm^-1): 669 (C-Cl), 3026 (aromatic C-H), 1681(imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm):6.71-8.21 (m, 7H, Ar-H), 4.74 (s, 1H, NH), 8.04 (s, 1H, CH); Mass m/z: (M+) 230.

(E)-1-(4-fluorobenzylidene)-2-phenylhydrazine (A3): white solid, m.p.141-143^oC; yield 73.58%; IR (cm^-1): 1020 (C-F), 3028 (aromatic C-H), 1674 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.81-8.31 (m, 7H, Ar-H), 4.73 (s, 1H, NH), 8.03 (s, 1H, CH); Mass m/z: (M+) 214.

(E)-1-(4-aminobenzylidene)-2-phenylhydrazine (A4): yellow solid, m.p.144-146^oC; yield 48.65%; IR (cm^-1): 3320 (C-NH₂), 3004 (aromatic C-H), 1650 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.91-8.41(m, 7H, Ar-H), 4.77 (s, 1H, NH), 8.06 (s, 1H, CH); Mass m/z: (M+) 211.

(E)-1-(4-hydroxybenzylidene)-2-phenylhydrazine (A5): yellow solid, m.p.172-174^oC; yield 52.78%; IR (cm^-1): 3325 (C-OH), 3015 (aromatic C-H), 1680 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.95-8.45 (m, 7H, Ar-H), 4.71 (s, 1H, NH), 8.05 (s, 1H, CH); Mass m/z: (M+) 212.

(E)-1-(4-nitrobenzylidene)-2-phenylhydrazine (A6): brown solid, m.p.167-169^oC; yield 48.45%; IR (cm^-1): 1525 (C-NO₂), 3010 (aromatic C-H), 1685 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.92-8.42 (m, 7H, Ar-H), 4.75 (s, 1H, NH), 8.01 (s, 1H, CH); Mass m/z: (M+) 241.

(E)-1-(4-methylbenzylidene)-2-phenylhydrazine (A7): white solid, m.p.167-169^oC; yield 37.21%; IR (cm^-1): 3028 (aromatic C-H), 1679 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.94-8.44 (m, 7H, Ar-H),4.76 (s, 1H, NH), 8.07 (s, 1H, CH); Mass m/z: (M+) 210.

(E)-1-(4-methoxybenzylidene)-2-phenylhydrazine (A8): white solid, m.p.176-178^oC; yield 34.29%; IR (cm^-1): 3021 (aromatic C-H), 1645 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.93-8.43 (m, 7H, Ar-H),4.70 (s, 1H, NH), 8.02 (s, 1H, CH); Mass m/z: (M+) 226.

(E)-1-(4-dimethylaminobenzylidene)-2-phenylhydrazine (A9): red solid, m.p.181-183^oC; yield 41.45%; IR (cm^-1): 3027 (aromatic C-H), 1650 (imine C=N); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.96-8.46 (m, 7H, Ar-H),4.78 (s, 1H, NH), 8.08 (s, 1H, CH); Mass m/z: (M+) 239.

Synthesis of thiazolidineone derivatives:

0.01 mol of the corresponding Schiff base was taken in a 250ml round bottom flask. N, N-Dimethyl formamide (DMF) was added sufficiently to dissolve the Schiff base. Thioglycolic acid (0.0125 mol) and a pinch of ZnCl₂ catalyst were added to the mixture. The reaction mixture was refluxed for 8 hours. The formation of a clear distillate marked the completion of the reaction. Excess DMF was distilled off and collected. The mixture was cooled and poured onto crushed ice in portions, with constant stirring. Solids precipitated and were filtered. The residue was washed with cold water and a 5% sodium bicarbonate solution. After filtration, the product was dried. The dried product was purified by recrystallisation from DMF, DMSO, ethanol, or mixtures of DMF or DMSO with water or toluene. The melting points of the pure compounds were determined. This synthesis process is a standard method for obtaining purified compounds through recrystallisation, ensuring the removal of impurities and obtaining a well-defined product. The melting point determination is a standard technique to assess the purity of the synthesised compounds²⁰ (Figure 1).

Spectral data:

2-(4-bromophenyl)-3-(phenylamino)thiazolidin-4-one (B1): brown solid, m.p. 253-255^oC;

IR in cm^-1: 630 (C-Br), 1530 (C-NO₂), 3312 (NH), 1653 (C=O), 820 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.71-8.21 (m, 7H, Ar-H), 4.71 (s, 1H, NH),3.84 (d, 2H, CH₂); Mass m/z: (M+) 349.

2-(4-chlorophenyl)-3-(phenylamino)thiazolidin-4-one (B2): yellow solid, m.p. 279-281^oC;

IR in cm^-1: 810 (C-Cl), 1535 (C-NO₂), 3320 (NH), 1660 (C=O), 825 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.75-8.25 (m, 7H, Ar-H), 4.72 (s, 1H, NH),3.85 (d, 2H, CH₂); Mass m/z: (M+) 288.

2-(4-fluorophenyl)-3-(phenylamino)thiazolidin-4-one (B3): red solid, m.p. 228-230^oC; IR in cm^-1: 1265 (C-F), 1540 (C-NO₂), 3315 (NH), 1655 (C=O), 830 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.76-8.26 (m, 7H, Ar-H), 4.73 (s, 1H, NH), 3.86 (d, 2H, CH₂); Mass m/z: (M+) 304.

2-(4-aminophenyl)-3-(phenylamino)thiazolidin-4-one (B4): brown solid, m.p. 257-259^oC;

 IR in cm^-1: 3312 (C-NH₂), 1545 (C-NO₂), 3325 (NH), 1656 (C=O), 831 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.77-8.27 (m, 7H, Ar-H), 4.74 (s, 1H, NH), 3.87 (d, 2H, CH₂); Mass m/z: (M+) 285.

2-(4-hydroxyphenyl)-3-(phenylamino)thiazolidin-4-one (B5): brown solid, m.p. 237-239^oC; IR in cm^-1: 3378 (C-OH), 1546 (C-NO₂), 3326 (NH), 1676 (C=O), 840 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.78-8.28 (m, 7H, Ar-H), 4.75 (s, 1H, NH), 3.88 (d, 1H, CH₂); Mass m/z: (M+) 286.

2-(4-nitrophenyl)-3-(phenylamino)thiazolidin-4-one (B6): cream solid, m.p. 264-266^oC; IR in cm^-1: 1524 (C-NO₂), 3330 (NH), 1677 (C=O), 845 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.79-8.29 (m, 7H, Ar-H), 4.76 (s, 1H, NH), 3.89 (d, 2H, CH₂); Mass m/z: (M+) 315.

2-(4-methylphenyl)-3-(phenylamino)thiazolidin-4-one (B7): yellow solid, m.p. 273-275^oC; IR in cm^-1: 1548 (C-NO₂), 3335 (NH), 1678 (C=O), 850 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.89-8.39 (m, 7H, Ar-H), 4.77 (s, 1H, NH), 3.83 (d, 2H, CH₂); Mass m/z: (M+) 284.

2-(4-methoxyphenyl)-3-(phenylamino)thiazolidin-4-one (B8): red solid, m.p. 245-245^oC; IR in cm^-1: 1532 (C-NO₂), 3340 (NH), 1679 (C=O), 855 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.99-8.49 (m, 7H, Ar-H), 4.70 (s, 1H, NH), 3.832 (d, 2H, CH₂); Mass m/z: (M+) 300.

2-(4-dimethylaminophenyl)-3-(phenylamino)thiazolidin-4-one (B9): cream solid, m.p. 289-291^oC; IR in cm^-1: 1550 (C-NO₂), 3345 (NH), 1661 (C=O), 860 (C-S-C); ¹H NMR (400 MHz, DMSO, δ/ppm): 7.61-8.28 (m, 7H, Ar-H), 4.78 (s, 1H, NH), 3.843 (d, 2H, CH₂); Mass m/z: (M+) 313.

In silico platform:

All computational analysis was carried out on Maestro 12.3 version (LigPrep, Glide XP docking, binding free energy calculations, ADMET, pharmacophore modelling) (Schrödinger 2021-2, LLC, New York). This software package is programmed on a DELL Inc.27" workstation machine with Linux–x86_64 as the operating system.

Molecular docking and binding free energy calculations:

The crystal structures of the selected targets (PDB ID: 1URM)²¹ are obtained from the Protein Data Bank (PDB) and are minimised using the Protein Preparation Wizard in Schrödinger software. The OPLS-2005 force field is applied for the minimisation process. Chemdraw Pro 12.0 is used for designing novel thiazolidineones. The designed molecules' SMILES (Simplified Molecular Input Line Entry System) are generated and imported into the Maestro workspace. LigPrep (Schrödinger, 2021-2) application is used for ligand preparation. The best conformations of ligands are selected for further analysis. A receptor grid box is generated at the active site of the protein targets using default parameters. This step defines the search space for ligand binding during molecular docking. Glide-XP (extra precision) (Schrödinger, 2021-2) is employed for molecular docking computations^22,23,24. The binding free energy (MMGBSA dGbind) between the receptor and ligands is calculated using the Prime module (Schrödinger, 2021-2). This step provides a quantitative measure of the stability of the protein-ligand complex. Binding poses and interactions between designed molecules and protein targets are generated. Designed molecules are ranked based on their docking scores and binding free energies. This computational approach allows for the in silico assessment of the potential interactions between the designed thiazolidineones and the selected protein targets, providing insights into their binding affinities and potential as antioxidants.

ADME and Physicochemical Properties:

ADME and physicochemical properties of designed ligands were determined using QikProp of the Schrodinger software (Schrödinger 2020-4: QikProp)²⁵. The prepared ligands were processed and incorporated into the QikProp tool. The ADME features include Caco-2 cell permeability, BBB permeability, % human oral absorption solvent accessible surface area (SASA) and physicochemical properties like molecular weight, log P, donor-HB and accept-HB analyses Lipinski Rule of five (Lipinski, 2004) were assessed.

In vitro antioxidant assay by DPPH Method:

Evaluating antioxidant activity is crucial in assessing compounds' potential health benefits. In this case, the DPPH (2,2-diphenyl-1-picrylhydrazyl) method was employed. A 0.2mM solution of DPPH in methanol was prepared. Various concentrations of the synthesised thiazolidineone derivatives (10-50µg/ml) were prepared. Ascorbic acid was used as the standard drug. 100µL of the prepared DPPH solution was added to each sample concentration and standard. The reaction mixture was allowed to stand for 30 minutes. After the incubation period, the absorbance of the resulting mixture was measured at 517nm. This measurement provides information about the amount of DPPH that the compounds in the sample have scavenged. The percentage inhibition was calculated by comparing the absorbance values of the control and test samples with the standard. The antioxidant activity is often expressed as the percentage of DPPH inhibition, where a higher percentage indicates a more potent antioxidant effect. Ascorbic acid serves as a reference standard with known antioxidant properties. The results of this assay provide insights into the ability of the synthesised thiazolidineone derivatives to scavenge free radicals, indicating their potential as antioxidants²⁶.

RESULTS AND DISCUSSION:

Chemistry:

Ten thiazolidineone Schiff base derivatives (B1-B9) have been synthesised following the reaction shown in Scheme 1 (Figure 1). IR, NMR and mass spectroscopic techniques were used to confirm the structures.

Molecular docking:

The ten thiazolidineone derivatives were docked with the target 1URM. Among them, the top compounds were B6 and B4, with docking scores of -4.545 and -4.665, respectively and binding energy of -60.57 and -59.01 kcal/mol while comparing with their co-crystal ascorbic acid, which exhibited docking scores of -7.043 and binding energy of -35.66 kcal/mol. In B6, amino acids that were responsible for hydrophobic interactions are Pro 40, Pro 45, Leu 149, Ile 119, and Phe 120 and amino acids Thr 147, Ser 47, Thr 44 made polar interactions and Arg 127, and Ser 47 make hydrogen bond interactions (Figure 2 and 3) (Table 1 and 2).

Figure 1. Scheme- Synthesis of thiazolidine ones

Figure 2. 2D and 3D Docking Interaction of B4 with 1URM

Figure 3. 2D and 3D Docking Interaction of B6 with 1URM

Table 1. Docking scores and binding free energies of thiazolidineones (B1-B10) with protein 1URM

S. No.	Compounds	G Score	MMGBSA dG Bind
1.	B1	-4.675	-60.43
2.	B2	-4.777	-57.23
3.	B3	-4.765	-57.16
4.	B4	-4.665	-59.01
5.	B5	-4.902	-60.60
6.	B6	-4.545	-60.57
7.	B7	-4.769	57.75
8.	B8	-5.031	-60.24
9.	B9	-4.836	-62.29
10.	Ascorbic acid	-7.043	-35.66

ADME properties:

QikProp was used to determine the ADME properties. It helps us establish the compound's absorption, distribution, metabolism, and elimination and provides information related to the onset of action and how the drug crosses the barrier. The ADMET properties help the medicinal chemist to make necessary modifications to improve the activity. QikProp determined the variables such as bioavailability, blood-brain barrier penetration, plasma-protein binding, metabolism, HERG K⁺ and solvent-accessible surface area (Tables 3 and 4).

Table 2. Docking interactions of thiazolidineones (B1-B10) with protein 1URM

Compound	Hydrophobic Interactions	Polar Interactions	Hydrogen Bonding
B1	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47	Arg 127, Ser 47
B2	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B3	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147,Ser 47, Thr 44	Arg 127, Ser 47
B4	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B5	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B6	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B7	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B8	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
B9	Pro 40, Pro 45, Leu 149, Ile 119, Phe 120	Thr 147, Ser 47, Thr 44	Arg 127, Ser 47
Ascorbic acid	Pro 40, Pro 45, Leu 149, Phe 120	Thr 147, Ser 47, Thr 44	Thr 147, Ser 47, Thr 44, Gly 46

Table 3. ADMET properties of thiazolidineones (B1-B10)

Compound	%Human Oral Absorption	#metab	QPlogS	QPlogHERG	QPP Caco	QPlog BB	QPlog Kp	QPlog KHSA	SASA
B1	100	3	-4.357	-4.612	2632.257	0.214	-1.498	0.224	505.833
B2	100	3	-4.027	-4.525	2625.966	0.162	-1.467	0.154	490.849
B3	92.647	4	-3.343	-4.511	783.028	-0.475	-2.413	-0.019	493.602
B4	85.611	4	-3.720	-4.659	312.099	-0.865	-3.244	0.071	520.297
B5	100	4	-3.495	-4.190	2879.545	0.036	-1.384	0.113	502.198
B6	100	3	-4.380	-4.621	2634.107	0.227	-1.504	0.245	509.415
B7	91.285	4	-3.071	-4.129	755.766	-0.444	-2.484	-0.064	500.515
B8	100	4	-4.094	-4.618	2646.355	0.037	-1.522	0.265	513.365
B9	100	4	-4.381	-4.808	2543.545	-0.059	-1.529	0.274	558.534
Ascorbic acid	47.989	5	-0.525	-2.898	47.148	-1.723	-5.393	-0.942	348.025

Table 4. Physicochemical properties of thiazolidineones (B1-B10)

Compound	Molecular Weight	Molecular Volume	LogP	Donor H	Acceptor H	PSA	RO5	RO3
B1	304.793	86.603	3.588	1.000	4.000	41.804	0	0
B2	288.339	868.831	3.394	1.000	4.000	41.752	0	0
B3	286.348	874.912	2.375	2.000	4.750	64.536	0	0
B4	315.346	925.206	2.395	1.000	5.000	86.844	0	0
B5	300.375	918.895	3.169	1.000	4.750	49.786	0	0
B6	349.244	904.469	3.651	1.000	4.000	41.805	0	0
B7	285.363	872.633	2.190	2.500	5.000	67.919	0	0
B8	284.375	912.406	3.400	1.000	4.000	41.718	0	0
B9	313.417	1004.799	3.533	1.000	5.000	45.248	0	0
Ascorbic acid	176.126	891.564	-1.521	3.000	6.150	124.205	0	0

Bioavailability prediction:

The parameters that assess oral absorption are the predicted aqueous solubility (logS), the predicted % human oral absorption and agreement to Jorgensen’s famous “Rule of Three (RO3). According to Jorgensen’s RO3, if a compound complies with all or some of the rules (logS > −5.7, Caco- >22 nm/s and # Primary Metabolites< 7), then it is more likely to be orally available. The non-active transport for the gut-blood barrier was assessed from Caco-2 cell permeability, and the studied compounds exhibited a wide range of values.

The non-active transport for the gut-blood barrier was assessed from Caco-2 cell permeability, and the examined ten derivatives showed a wide range of values. All synthesised compounds showed the maximum permeability to the gut-blood barrier and obeyed Jorgensen’s RO3 with no violations. All the substances are orally bioavailable, based on their analysis. The compounds' predicted aqueous solubility (logS) was within the range of -6.5 to -0.5mol dm^–3. The per cent human oral absorption parameter suggests that all the compounds have high human oral absorption.

Prediction of blood-brain barrier (BBB) penetration:

Blood/brain partition coefficients (QPlogBB) assessed the access to the central nervous system. QPlogBB of all compounds fall in the range of -3.0 -1.2, the recommended limit so that they can penetrate the blood-brain barrier.

Prediction of plasma-protein binding:

The binding of the drugs to plasma proteins decreases the amount of drug reaching the blood circulation, affecting drug efficiency. The plasma protein binding is determined by binding to human serum albumin (log KHSA) (recommended range is −1.5 to 1.5), and from the data, it was found that all the compounds were in the acceptable range. So, all the compounds are likely to reach the blood circulation freely and thus be more available at the target site.

Metabolism prediction:

All were within the recommended number (1 to 8 reactions).

Prediction of blockage of human ether-a-go-go-related gene potassium (HERG K+) channel:

HERG K⁺ channel blockers are potentially toxic, and the predicted IC₅₀ values often provide reasonable predictions for the cardiac toxicity of drugs in the early stages. All the compounds showed IC₅₀ values below -5 for HERG K+ channels.

Prediction of solvent accessible surface area (SASA):

The measure of the contact area between the solvent and molecule represents SASA, which is usually in the range of 300.0 – 1000.0 Å2, and all the compounds are within the standard limits. ADMET properties of the synthesised compounds are listed in Table 5.

Physicochemical properties:

The physicochemical properties determined by QikProp establish the drug-likeness property of the compound. The various physicochemical properties of the ten thiazolidineone derivatives are listed in Table 6. The lipophilicity QPlogPo/w of the ten compounds was within the permissible limit (–2.0 to 6.5), ranging from 2.19 to 3.65. The polar surface area correlating the Van der Waals surface area of polar nitrogen and oxygen atoms was calculated. It found that compounds were between 41.71 to 86.88 Å, within the standard limit of 7.0 – 200.0.  All the compounds obey Lipinski’s RO5 with no violations. From the above observations, the compounds were considered druglike molecules.

In vitro antioxidant assay by DPPH Method:

The DPPH method was used to study compounds' in vitro antioxidant inhibitory activity. Ascorbic acid was taken as the standard, which showed an IC50 value of 31.67 μM. The results reveal that B1, B4, and B6 were the potent enzyme inhibitor with an IC50 value of 3.5, 7.8 and 10.9 μM, respectively (Table 5).

Table 5. In vitro antioxidant activity of thiazolidineones (B1-B10) by DPPH method

Sample	% inhibition					IC50 (µg/ml)
	10	20	30	40	50
B1	64.85	33.14	9.57	39.57	39.71	3.5
B2	55.28	44.57	48.57	34.42	34.42	34.7
B3	79.857	46.857	52.714	52.285	57.857	55.7
B4	87.28	62.57	62	55.85	61.85	7.8
B5	79.285	65	64.857	55.714	51.428	43.7
B6	64.428	62.428	63.142	71.285	70.142	10.9
B7	72.857	59.285	53.857	48.857	52.857	19.6
B8	68.85	34.71	64	33.57	94.42	23.7
B9	68.857	50.571	54.428	54.857	65.428	34.8
Ascorbic acid	45.89	67.78	69.90	57.78	35.78	31.67

CONCLUSION:

Thiazolidineone derivatives were synthesised in two steps, involving the reaction of hydrazide and aromatic aldehydes followed by cyclisation with thioglycolic acid. The reaction conditions and yields were reported. The synthesised compounds were characterised using various spectroscopic techniques, including IR, Mass, and ¹H NMR. Spectral data provide information about the compounds' molecular structure, functional groups, and stereochemistry. Molecular docking studies were performed to assess the interactions of the synthesised compounds with the protein 1URM. The compounds B6 and B4 exhibited high scores, indicating favourable interactions with the target protein. QikProp software was employed to evaluate ADME (absorption, distribution, metabolism, and excretion) and physicochemical properties of the compounds. The analysis included Caco-2 cell permeability, BBB permeability, % human oral absorption, molecular weight, log P, and others. The DPPH method was used to assess the antioxidant activity of the synthesised compounds. Compounds B1, B4, and B6 showed potent enzyme inhibition with IC₅₀ values, indicating their potential as antioxidants. The top-scored compounds (B6 and B4) from computational analysis aligned with the in vitro studies, reinforcing their potential as antioxidant agents. As indicated by ADMET and physicochemical analyses, the synthesised compounds exhibit drug-like properties and bioavailability. The study concludes by suggesting that the synthesised compounds can serve as leads for antioxidant agents, with the recommendation for further validation through pharmacological studies. This multi-faceted approach thoroughly explains the synthesised compounds, bridging the gap between computational predictions and experimental validations. It sets the stage for potential drug development based on the antioxidant properties of the thiazolidineone derivatives.

REFERENCES:

1. Sachin Malik, Ashish Choudhary. Anti-Oxidant Activity of Novel 5-Substituted Arylidene-3-Substituted-Benzyl-Thiazolidine-2, 4-Diones. Asian J. Research Chem. 2011; January; 4(1): 120-122.

2. Sailaja Rao P, Kalva S, Yerramilli A, Mamidi S. Free Radicals and Tissue Damage: Role of Antioxidants. Free Radicals Antioxidants. 2011; 1(4): 2–7. Available from: http://dx.doi.org/10.5530/ax.2011.4.2

3. Fuchs-Tarlovsky V. Role of antioxidants in cancer therapy. Nutrition. 2013; 29(1): 15–21. Available from: http://dx.doi.org/10.1016/j.nut.2012.02.014

4. Dubois-Deruy E, Peugnet V, Turkieh A, Pinet F. Oxidative stress in cardiovascular diseases. Antioxidants. 2020; Sep 14; 9(9): 864.

5. Duthie GG, Wahle KW., James WP. Oxidants, antioxidants and cardiovascular disease. 1989; 5: 51–62.

6. Halliwell B. Role of Free Radicals in the Neurodegenerative Diseases. Drugs Aging. 2001; 18(9): 685–716.

7. Fang YZ, Yang S, Wu G. Free radicals, antioxidants, and nutrition. Nutrition. 2002; 18(10): 872-9. doi: 10.1016/s0899-9007(02)00916-4. PMID: 1236178.

8. Pinto-Basto D, Silva JP, Queiroz MJ, Moreno AJ, Coutinho OP. Antioxidant activity of synthetic diarylamines: a mitochondrial and cellular approach. Mitochondrion. 2009; 9(1): 17-26.

9. Chandrahas N Khobragade, Ragini G Bodade, Anand V. Manwar. Synthesis and Antioxidant Activity of Some Flavone Derivatives. Asian J. Research Chem. 2010; Jan.-Mar. 3(1): 139-141

10. Akshay R. Yadav, Shrinivas K. Mohite. Antioxidant Activity of Malvastrum coromandelianum Leaf extracts. Research J. Topical and Cosmetic Sci. 2020; 11(2): 59-61.

11. Salomi S., Muthukumaran P., Umamaheshwari R. In-vitro antioxidant activity of Azima tetracantha leaves . Research J. Science and Tech. 2012; 4(4): 148-151

12. Alam MN, Bristi NJ, Rafiquzzaman M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J. 2013; 21(2): 143–52.

13. James JP, Crasta L, Shetty V, Jyothi D, Jouhara M, Fathima ZC, Sindhu TJ. Tyrosinase and peroxiredoxin inhibitory action of ethanolic extracts of Memecylon malabaricum leaves. Research Journal of Pharmacy and Technology. 2024; 17(4): 1763-70.

14. Kunzler A, Neuenfeldt PD, Das Neves AM, Pereira CMP, Marques GH, Nascente PS, et al. Synthesis, antifungal and cytotoxic activities of 2-aryl-3-((piperidin-1- yl)ethyl)thiazolidinones. Eur J Med Chem. 2013; 64: 74–80.

15. Hu J, Wang Y, Wei X, Wu X, Chen G, Cao G, et al. Synthesis and biological evaluation of novel thiazolidinone derivatives as potential anti-inflammatory agents. Eur J Med Chem. 2013; 64: 292–301.

16. James JP, Bhat I, Jose N. Synthesis, in silico physicochemical properties and biological activities of some pyrazoline derivatives. Asian Journal of Pharmaceutical and Clinical Research. 2017: Apr 1: 456-9.

17. Kodical DD, James JP, Deepthi K, Kumar P, Cyriac C, Gopika KV. ADMET, Molecular docking studies and binding energy calculations of Pyrimidine-2-Thiol Derivatives as Cox Inhibitors. Research Journal of Pharmacy and Technology. 2020; 13(9): 4200-4206.

18. James JP, Paramatha, Jouhara M, D'Souza RR. Green synthesis, multitargeted molecular docking and ADMET studies of chalcones based scaffold as anti-breast cancer agents. Research Journal of Pharmacy and Technology. 2023; May 1; 16(5): 2215-22.

19. Bhat IS, Jainey PJ. Antimicrobial studies of some substituted pyrazoline derivatives derived from acetyl hydrazines. Asian J Pharm Clin Res. 2014; 7(4): 237-9.

20. Pavan TS, James JP, Dwivedi SRP, Priya S, Fathima CZ, Sindhu TJ. Synthesis, Molecular Docking and Molecular Dynamic Studies of Thiazolidineones as Acetylcholinesterase and Butyrylcholinesterase Inhibitors. Polycyclic Aromatic Compounds. 2023; Jul 15: 1-21.

21. Evrard C, Smeets A, Knoops B, Declercq JP. Crystal structure of the C47S mutant of human peroxiredoxin 5. Journal of Chemical Crystallography. 2004; Aug; 34: 553-8.

22. James JP, Sasidharan P, Mandal SP, Dixit SR. Virtual screening of alkaloids and flavonoids as acetylcholinesterase and MAO-B inhibitors by molecular docking and dynamic simulation studies. Polycyclic Aromatic Compounds. 2023; Jul 3; 43(6): 5453-77.

23. James JP, Devaraji V, Sasidharan P, Pavan TS. Pharmacophore Modeling, 3D QSAR, Molecular Dynamics Studies and Virtual Screening on Pyrazolopyrimidines as anti-Breast Cancer Agents. Polycyclic Aromatic Compounds. 2023; Sep 14; 43(8): 7456-73.

24. James JP, Aiswarya TC, Priya SN, Jyothi DI, Dixit SR. Structure based multitargeted molecular docking analysis of pyrazole condensed heterocyclics against lung cancer. Int J App Pharm. 2021; Nov 7; 13(6): 157-69.

25. James JP, Ail PD, Crasta L, Kamath RS, Shura MH, Sindhu TJ. In silico ADMET and molecular interaction profiles of phytochemicals from medicinal plants in Dakshina Kannada. Journal of Health and Allied Sciences NU. 2024; Apr; 14(02): 190-201.

26. Dubey SK, Batra A. Study of antioxidant and anti-inflammatory activity from ethanol fraction of Thuja occidentalis Linn. Research J. Science and Tech. 2009; 1(1): 39-42.

Received on 20.12.2023      Revised on 05.06.2024

Accepted on 25.09.2024   Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5855-5862.

DOI: 10.52711/0974-360X.2024.00889

© RJPT All right reserved