Synthesis, Molecular Docking and Molecular Dynamic Studies of Thiazolidineones as Acetylcholinesterase and Butyrylcholinesterase Inhibitors

Abstract Neurodegenerative diseases are chronic, progressive, age-related, and characterized by the loss of function of neurons caused by the accumulation of free radicals and oxidative stress. Although the prevalence of neuro disorders is rising, therapeutic efficacy is still limited due to various variables, including the blood-brain barrier. Hence, to identify molecules targeting different enzymes like acetylcholinesterase, butyrylcholinesterase and peroxiredoxins, a series of thiazolidineone derivatives were designed and synthesized. Schiff base was synthesized and cyclised with thioglycolic acid to yield thiazolidineones (T1-T10). Structural characterization was performed by IR, Mass and 1H NMR spectral studies and then subjected to in silico analysis against acetylcholinesterase (6O4W) and butyrylcholinesterase (1P0P). Compound T-9 (–10.10 kcal/mol) and T-8 (–7.65 kcal/mol) have shown excellent binding with 6O4W and 1P0P, respectively, compared with other derivatives. In addition, the compounds were checked for antioxidant activity by analyzing the interactions with peroxiredoxins (1URM), and compound T-4 was active. According to the physicochemical and ADME properties of Qikprop, synthesized compounds can be considered druglike molecules. In vitro, acetylcholinesterase inhibitory activity reveals compound T-8 as the most potent AChE inhibitor. In vitro, antioxidant activity found that compound T-4 has significant antioxidant activity. The compound T-8, with better docking scores and decisive acetylcholinesterase inhibitory action, was further explored to validate the molecular interactions through molecular dynamics studies. It was observed that compounds with the benzyl sulfonyl group (T-6 to T-10) showed higher AChE inhibitory potency than derivatives with phenyl substituents in place of the benzyl sulfonyl group (T-1 to T-5). Therefore, it is inferred that the sulfonyl group and substituents at the para position are essential for the higher inhibitory activity of compounds. Thus, there is plenty of scope for further study in developing these as promising lead compounds.


Introduction
Neurodegenerative diseases (NDD) are complex deteriorating illnesses related to age with dysfunction of neurons with progressive loss of cognition, which includes Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). 1 Oxidative stress and glutamate receptors' excessive stimulation contribute to these diseases' progression. 2When the reactive oxygen and nitrogen species formation exceeds the cellular and mitochondrial antioxidant enzymes, it reduces antioxidant defence, resulting in oxidative stress. 3DD therapy mainly involves the usage of cholinesterase inhibitors.They act by inhibiting the enzymes catalyzing acetylcholine (ACh) or butyrylcholine (BuCh) hydrolysis.Their action elevates the availability of ACh and augments synaptic transmission.The drugs inhibiting acetylcholinesterase (AChE) include Rivastigmine, donepezil or galantamine. 4However, they cannot stop the neurodegenerative process; therefore, a neuroprotective therapy which aims at etiopathogenesis modification is required.A favorable method for delaying NDD disease development is to remove primary oxidative stress sources and provide neuroprotection simultaneously by gradually using neuroprotective antioxidants. 5nly limited research has been conducted on the consumption of the antioxidant due to the experimental irregularities, blood-brain barrier poor permeation and poorer efficacy per dose.Antioxidant enzymes, such as superoxide dismutase, superoxide reductase, catalase, and peroxiredoxins (Prxs), protect the cells against extreme oxidation.They support NDD treatment with their neuroprotective action and help to retard the disease progression.Prxs scavenge H 2 O 2 and control the transduction of numerous cellular signals by oxidative signaling pathways. 6,72][13] With the structural modification in nitrogen and sulfur-containing heterocycles such as thiazolidinone scaffolds, 14 results in acetylcholinesterase, 15 aldose reductase, 16 and alpha-amylase and urease inhibitors, 17 anti-cancer 18 action.
Aromatic and heterocyclic groups with carbon linkers are the established pharmacophores for AChE inhibition.This concept encouraged us to screen the molecules against AChE.This drug design combines in silico techniques [19][20][21] to analyze their molecular interactions with various enzymes and develop thiazolidinones-based inhibitors.
The complex pathophysiology of NDD requires multi-target agents which can act simultaneously at multiple pathways.Introducing more pharmacophoric groups into one scaffold will result in multi-target agents targeting numerous enzymes. 22,23Based on the kinds of literature reported for neuroprotective properties, [24][25][26][27] we have incorporated dinitro and sulfonyl hydrazine into Schiff base and further cyclised to thiazolidinones scaffold to target cholinesterase and peroxiredoxins enzymes, simultaneously (Figure 1).And we have synthesized these designed compounds, analyzed their molecular interactions with AChE, BChE and Prx using in silico tools, and tested their AChE inhibitory activity.

Methods
All the reactions were undertaken as per standard laboratory conditions.The study was carried out using available laboratory reagents and suitable types of equipment.The following steps are involved in synthesizing thiazolidinone derivatives in the present work.The characterization requires identifying molecular framework, functional groups' nature and location within the skeletal structure and establishing any stereo-chemical relationship that might exist.The current work records IR spectra using BRUKER FT-IR, and frequencies are expressed in cm À1 . 1 H-NMR spectra were recorded at 400 MHz "AGILENT-NMR" SPECTROMETER obtained in DMSO solution.The mass spectrum was recorded on LC-MS PERKIN ELMER CLARUS 680 SPECTROMETER obtained by the time-of-flight ionization method.The chemical shift values were reported as values in ppm relative to TMS (d ¼ 0) as an internal standard.

Synthesis of Schiff base
An equimolar mixture of hydrazide (1 mmol) and aromatic aldehydes (1 mmol) was taken in a round-bottomed flask and refluxed for 6 to 8 hrs hours using ethanol as the solvent and a catalytic amount of conc.H 2 SO 4 .TLC checked the completion of the reaction.The mixture was allowed to cool, and the separated solid was filtered and dried.Ethanol was used for the recrystallisation of the final products.(Yield: 80-95%). 28nthesis of thiazolidinone 0.01 mol of the corresponding Schiff base was taken in a 250 ml round bottom flask containing sufficient N, N-dimethyl formamide (DMF) to dissolve the Schiff base.Further, thioglycolic acid (0.0125 mol) and a pinch of ZnCl 2 catalyst were added.This mixture was refluxed for 8 h.The formation of a clear distillate marked the completion of the reaction.The mixture is poured onto crushed ice in portions, with constant stirring.Solids get precipitated and filtered.The residue was washed with cold water, followed by a 5% sodium bicarbonate solution, and filtered.After drying, it was purified by recrystallisation from solvents (DMF/DMSO/ethanol).The reaction progress was determined by TLC (Figure 2).

Molecular docking and MM-GBSA binding free energy
Molecular docking was made to understand the docking interactions of compounds with AChE and BuChE.The Ligprep module 30 was used to prepare all compounds.The RCSB Protein Data Bank was used to download the three-dimensional crystal structures of AChE (PDB: 604 W), 31 BuChE (PDB: 1P0P) 32 and human peroxidase enzyme (1URM). 33The protein preparation wizard of Schrodinger was used for AChE and BuChE preparation.Receptor Grid Generation was used to generate the grid for docking in the 604 W and 1P0P.Finally, these ligands were docked using Glide extra precision (XP) scoring function. 34The receptor binding free energy and a set of ligands were predicted using the calculation of the Prime module in Schrodinger. 35, 36 57 The software estimates the total free energy of binding as dGbind (kcal/mol).

MD Simulation study
Gromacs version 2022.1 (https://www.gromacs.org/)was used to perform the MD Simulations.The topology of the protein was generated by applying the Charmm36 ForceField using the PDB2gmx module of Gromacs.The proteins were solvated using a three-point water model in a dodecahedron box with dimensions of 1 nm in all directions.The model system was neutralized by adding sodium (Na þ ) and Chloride (Cl -) ions as counter ions.Energy minimization was performed using the steepest descent integrator with a verlet cutoff scheme for a maximum of 50,000 steps to achieve the slightest energy confirmation.The system was equilibrated using Canonical (NVT) and Isobaric (NPT) for 100 picoseconds.V-rescale, a modified Berendsen thermostat, was applied to maintain constant volume and temperature at 300 K. Similarly, a Crescale pressure coupling algorithm was used to maintain constant pressure at 1 bar.Particle Mesh Ewald was applied for long-range computing electrostatics, coulomb, and Vander Waals with a cutoff of 1.2 nm.The LINCS algorithm was used to constrain bond length.Each complex was subjected to an MD run for 50 ns; the coordinates and energies were saved at every ten picoseconds.The trajectories generated were analyzed using in-built Gromacs utilities.On completion of the molecular dynamic(MD) run, root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent assessable surface area (SASA), Radius of gyration (RoG), and the number of hydrogen bonds were analyzed. 37hysicochemical and ADMET properties Physical-chemical and ADMET properties describe a biomolecule's characteristics in living things, including bioavailability, oral absorption rate, BBB and CNS crossability, etc. Schr€ odinger software's Qikprop module was used to estimate the compound's physicochemical and ADMET properties. 38 vitro studies

Acetylcholinesterase inhibitory activity
AChE Inhibitory activity of newly synthesized thiazolidinone derivatives was evaluated by Ellman's method, where donepezil was used as the standard.Acetylthiocholine iodide was taken as the substrate.This method was based on the substrate's color formed by the thio group with the chromogenic 5,5 0 -dithio-bis(2-nitrobenzoic) acid (DTNB), also called the Ellman reagent.A series of concentration (100-500 mg/mL) test compounds were prepared using DMSO.150 ml sodium phosphate buffer (pH 8.0), 10 ml DTNB solution, 10 ml test solution, and 20 ml AChE solution were added to a 96-well microtitre plate and incubated (25 C, 15 min).10 ml of substrate acetylthiocholine iodide to initiate the reaction was added.The hydrolysis of acetylthiocholine iodide was monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB at a wavelength of 412 n m.Percentage inhibition is calculated by using the below-mentioned equation. 39

% inhibition
Where, A0 ¼ Absorbance of Control and A1 ¼ Absorbance of Test

DPPH method
The antioxidant activity of synthesized thiazolidinone derivatives was evaluated using DPPH (2,2diphenyl-1-picrylhydrazyl) radical scavenging method.0.2 mM solution of DPPH in methanol was prepared, and 100 lL of this solution was added to 100 lL of various concentrations of the samples (100-500 lg/ml) in a 96-well microtiter plate.The reaction mixture was then allowed to stand for 30 min in the dark.The absorbance of the resulting mixture was measured at 517 nm.Ascorbic acid was used as the standard drug.The percentage inhibition was calculated by comparing the absorbance values of both control and test samples with the standard. 40hibition Where, A0 ¼ Absorbance of Control and A1 ¼ Absorbance of Test

Chemistry
The study involves the synthesis of thiazolidinone by cyclising thioglycolic acid with Schiff base intermediates.The first step consists of forming Schiff bases by reacting the primary amine group of the hydrazides with the carbonyl group of the different aldehydes taken.The Schiff bases are refluxed in the second step using thioglycolic acid to give aryl hydrazide-bearing thiazolidinone.FTIR spectra of compounds (T-1 to T-10) showed characteristic amide (C¼O) stretching between 1611 and 1685 cm À1 and amide (N-H) stretching between 3413 and 3512 cm À1 , confirming the formation of the amide linkage and C-S-C stretching around 810-930 cm À1 , thus ensuring the appearance of thiazolidinone.FTIR spectra of T-1 show C¼O at 1672 cm À1 , NH at 3413 cm À1 , and C-S-C at 892 cm À1 . 41Additional information about the number of protons in the structure of the thiazolidinone derivatives was obtained from the 1 H NMR spectrum. 1H NMR spectrum of thiazolidinone (T-1) shows the signals of aromatic protons at d 7.61-8.28,singlet proton of amine at 8.77 ppm.The disappearance of azomethine proton (N¼CH) further confirms the formation of thiazolidinones.Further evidence of the structure (T-1) of the compound was obtained by recording the mass spectra of the compounds.The spectra showed a molecular ion peak at 467(Mþ), which agrees with the molecular formula.

Molecular docking
After the synthesis, in silico analysis was done to identify the detailed intermolecular interaction between synthesized thiazolidinone derivatives and targeted enzymes such as acetylcholinesterase, butyrylcholinesterase and peroxiredoxins.All the synthesized thiazolidinone products (T-1 to T-10) have been docked in the active sites of proteins 6O4W, 1P0P and 1URM.The synthesized derivatives showed significant binding affinity toward proteins compared to standard drugs.Acetylcholinesterase and butyrylcholinesterase inhibitory activity of thiazolidinones range from À8.51 to À10.10 kcal/mol and À5.81 to À7.65, respectively (Table 1).Apart from the docking score, docking energy, binding modes and interaction of each ligand with the functional residues of targeted protein (hydrogen bonding interactions, hydrophobic interactions, p-p stacking, polar interaction, p-p staking) were also analyzed in detail (Table 2).
Compound T-9 have shown excellent binding with 6O4W with a docking score of À10.10 kcal/mol, followed by T-8 (-9.96 kcal/mol).T-8 produced the highest dock score (-7.65 kcal/mol) by interacting with 1P0P, followed by T6 (-7.23 kcal/mol) and T9 (-6.88 kcal/mol), which is greater than the co-crystal butrylcholine's docking score of À5.In addition, the compounds were checked for antioxidant activity by analyzing the interactions with 1URM.The compound T-4 was active, and the amino acids Arg 127, Ser 47, Thr 147, and  4(a and b)).

Binding free energy calculations
The binding free energy of the ligand-protein complexes predictions by the MM-GBSA method validated the docking results.DGbind value of thiazolidinones ranges from À60.37 to À81.91 kcal/mol for 6O4W and À40.36 to À72.36 kcal/mol for 1P0P (Table 3).
The higher negative value implies that it is more active than the co-crystal.The electrostatic interaction (DGCoul) moderately favors some of the derivatives.Paradox to this, covalent  (DGCoul) and electrostatic solvation (DGsolv) strongly disfavor the binding of these compounds with enzymes.Overall, the practical binding energy terms are DGbind, DGvdW and DGLipo in comparison with DGCoul and DGSolv, which proves that van der Waals and non-polar solvation energy terms are the dynamic forces essential for the compounds to interact with the protein mentioned above. 42,43

MD For complex with 6O4W
The compound T-8, with better docking scores and potent acetylcholinesterase inhibitory action, was further explored to validate the molecular interactions through molecular dynamics studies.The RMSD for the 6O4W complex was analyzed for both the c-alpha atoms and backbone and was observed to be stable at $12 ns of MD run, and after that, it displayed slight fluctuations in the range of $0.5 Ð; this indicates the stability of the complex after 12 ns (Figure 5(a)).The RMSD of the ligand displayed fluctuation in the range of $1Ð to $4Ð, which indicates minor changes in the acceptable content (Figure 5(b)).The RMSF was analyzed for c-alpha atoms and complex; the RMS fluctuation ranged between 0.05 nm to 0.5 nm with a change of $3.5 Ð. Threonine 543, glutamic acid 4, and threonine 262 were observed to possess the highest RMSF of 0.53, 0.42, and 0.39 nm, respectively (Figure 5(c)).The radius of gyration (RoG) was analyzed for the backbone and complex, where it is perceived that the RoG became stable at 12 ns of MD run with a fluctuation of less than 0.3 Ð (Figure 5(d)).
Similarly, SASA was calculated for the protein and complex where there was an observed increase in the SASA at the initial stage of simulation and became stable after $15 ns of MD run, indicating the complex to be stable (Figure 5(e)).The analysis for the number of hydrogen bonds displayed a maximum of 3 bonds; the number of hydrogen bonds was regular for five ns at different time intervals (Figure 5(f)).This indicates the interaction of the ligand with the protein for short breaks, which other weak interactions may accomplish (Figure 7(a); Supplementary File).

MD For complex with 1POP
MD simulations help us to analyze and validate the results obtained via molecular docking at a given time gradient.The RMSD of the c-alpha was seen to be fluctuating till $10 ns; after that, it became stable.After stabilization, the deviation was displayed to be less than 0.5 Ð indicating suitable interaction of the ligand with the protein.The difference in RMSD between the c-alpha and backbone was $0.5 Ð, showing the structural flexibility of the complex (Figure 6(a)).The RMSD of the ligand displayed fluctuations in the range of $0.5 nm to $5 nm indicating the unstable interaction between the ligand and complex (Figure 6(b)).The RMSF was calculated for complex and c-alpha atoms, where the RMSF plot for both displayed variation of $3Ð.This indicates the crucial amino acids interacting with the ligand in contrast with the innate amino acids of the protein; histidine 77, glutamine 380, and 484 were the residues with the highest RMSF of 0.42, 0.38, and 0.33 nm, respectively (Figure 6(c)).The RoG defines the varied masses calculated to the root mean square distances from the center of the protein.The RoG value was calculated for the backbone and complex.The RoG for the complex displayed a slight fluctuation of less than $0.5 Ð in contrast with the RoG for the backbone (Figure 6(d)).Solvent-accessible surface area helps us to assess the surface available for interaction; a high SASA value indicates a more excellent free surface for the ligand to bind.The SASA was analyzed for the complex and protein, and fluctuation was in the range of $215 nm 2 to $235 nm 2 .The change in SASA may be due to the hydrogen bonds forming and deforming throughout the simulation (Figure 6(e)).The number of hydrogen bonds indicates the interaction between the protein and ligand in terms of hydrogen bonds which are known to possess more excellent stability in the biological system.The hydrogen bonds were analyzed between the ligand and protein (1POP), where it was perceived that there were fewer hydrogen bond interactions till $38 ns of MD run, after that one hydrogen bond was visible, which was seen to be forming and deforming throughout the simulation; this may be due to the instability of the complex (Figure 6(f)).However, other weak interactions like Vander Waal forces, hydrophobic interactions, and p-p exchanges may be present as other parameters displayed stable plots (Figure 7(b); Supplementary File).

Physicochemical properties
QikProp calculated the physicochemical properties, which assessed the drug-likeness property, stating that all compounds have obeyed the Lipinski rule of five.The lipophilicity QPlogPo/w of the synthesized derivatives was within the permissible limit (-2.0 to 6.5). 38,44The polar surface area correlating the Van der Waals surface area of polar nitrogen and oxygen atoms was calculated.Standard ascorbic acid and donepezil obtained a PSA value below seven; all derivatives were in the normal limit of 7.0 À 200.0 Å.The estimated number of hydrogen bond donors/acceptors was within the limit (0.0-6.0 donors and 2.0 to 20 acceptors). 38,44All derivatives, including the standard drugs, have been found to obey Lipinski's RO5 with no violations.On analyzing all the compounds, T-4 and T-5 have violated one rule in RO5.Overall, it is inferred that all the synthesized compounds can be considered druglike molecules 45 (Table 4).

ADME properties
QikProp was used to determine the pharmacokinetic properties.It helps us establish the compound's absorption, distribution, metabolism, and elimination and provides information about the onset of action and how the drug crosses the barrier.The ADME properties help the medicinal chemist to make necessary modifications to improve the activity.QikProp tool determined the variables such as bioavailability, blood-brain barrier penetration, plasma-protein binding, metabolism, HERG K þ and solvent-accessible surface area 46 (Table 5).

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 this rule, if a compound complies with all or some of the rules (logS > À5.7, Caco-2 > 22 nm/s and # Primary Metabolites< 7), then it is more likely to be orally available.The nonactive 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 synthesized compounds showed the maximum permeability to the gut-blood barrier and obeyed Jorgensen's RO3.On the analysis of all the derivatives, T-5 violated one rule, excluding that all products followed RO3, indicating that they are orally bioavailable. 47ediction of blood-brain barrier (BBB) penetration QPlogBB assessed the access to the central nervous system.All derivatives were able to penetrate the blood-brain barrier.The compounds T-6, T-7 and T-8 were expected to be moderately active in the CNS, each with a predicted CNS value of À1 on a scale of À2 to þ2.Madin-Darby canine kidney (MDCK) monolayers are used as an added measure in BBB prediction, as they mimic the BBB (for non-active transport), and the recommended scale is <25 poor > 500 great nm/sec.T-4 and T-5 showed insufficient mimicking activity, and the remaining derivatives showed moderate activity.

Prediction of plasma-protein binding
The binding of the drugs to plasma proteins decreases the number of medications reaching the blood circulation, affecting drug efficiency.The plasma protein binding is determined by binding to human serum albumin (log KHSA) (recommended range is À1.5 to 1.5).The derivatives are likely to reach the blood circulation freely, thus more available to the target site. 48

Metabolism prediction
All the thiazolidinone derivatives are predicted to undergo metabolism below eight; hence the metabolic rates are within the recommended number (1 to 8 reactions).
Prediction of blockage of human ether-a-go-go-related gene potassium (HERG Kþ) channel HERG K þ channel blockers are potentially toxic, and the predicted IC 50 values often provide reasonable predictions for the cardiac toxicity of drugs in the early stages.T-5 showed an expected IC 50 value below À5 for HERG K þ channels, which does not comply with the standard range. 49,50ediction of solvent-accessible surface area (SASA, FOSA, FISA) The solvent and molecule measure contact area represents SASA, usually 300.0 À 1000.0Å 2 ; all derivatives are within the standard limits.The hydrophobic component of the SASA (saturated carbon and attached hydrogen) represents FOSA (0-750), and the values reported are within the permissible limit.The hydrophilic component of the SASA (SASA on N, O, and H on heteroatoms) represents FISA, and the values fall within the limit (7.0 À 330.0). 51,52 vitro acetylcholinesterase studies Alzheimer's and Parkinson's disease affect the older population primarily.Due to a drop in the acetylcholine levels and subsequent cholinergic neuron degeneration in the basal forebrain, AD is characterized mainly by cognitive impairments.Various research carried out serves as evidence in proving the role of oxidative stress in neurotoxicity. 53,54The activity of AChE increases by an increase in calcium influx followed by oxidative stress, which results in the cytotoxicity of cholinergic neurons.AChE inhibitors are now the most effective treatment for alleviating AD symptoms as they promote neuronal transmission. 55,56The acetylcholinesterase inhibitory activity of  6).
In vitro analysis assisted in deriving the SAR of the designed compounds (T-1 to T-10).Compounds T-8, T-10 and T-9 showed the highest activity against AChE among the synthesized compounds.On analyzing the most active compound, T-8, which has better anticholinesterase action than the standard, the presence of electron-withdrawing group fluoro at the para position and benzene sulfonyl ring might have contributed to obtaining the highest activity.T-10 and T-9, which have hydroxyl and nitro groups at para position respectively, along with benzene sulfonyl ring, attributed to the prominent biological action compared with others.
Compounds T-1 to T-5 with a phenyl substituent in the place of the benzyl sulfonyl group showed lesser AChE inhibitory potency compared to compounds T-6 to T-10, which possess a benzyl sulfonyl group.Hence, it may be concluded that the sulfonyl group and substituent at the para position are essential for the higher inhibitory activity of compounds.

In vitro antioxidant assay
Considerable evidence has made it clear that oxidative stress-mediated damage is crucial in the etiology of various neurodegenerative illnesses, including Alzheimer's.Antioxidants have been recognized as a component of multiple therapies for AD; therefore, developing methods to stop or lessen oxidative damage may have therapeutic value.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 24.96 lM.The results reveal that T-4 was the potent enzyme inhibitor with an IC50 value of 23.44 lM.At the same time, compounds T-7 and T-5 showed similar inhibitory activity to the standard (Table 7).

Conclusion
This study synthesized novel thiazolidinone derivatives by cyclising Schiff bases using thioglycolic acid.The structure of the synthesized products was characterized by IR, 1 HNMR, and MASS spectroscopy.The synthesized compounds were subjected to in silico studies to determine the binding interaction and affinity of compounds against acetylcholinesterase (6O4W) and butyrylcholinesterase (1P0P) enzymes.Compounds showed good docking scores compared to the standard drug donepezil and butrylcholine.Compound T-9 (-10.10 kcal/mol) and T-8 (-7.65 kcal/mol) have shown excellent binding with 6O4W and 1P0P, respectively, compared with other derivatives.According to physicochemical properties and ADME properties synthesized compounds can be considered druglike molecules.The acetylcholinesterase inhibitory activity of compounds was evaluated by Ellman's method.In vitro results reveal compound T-8 obtained IC 50 as 8.96 lM, indicating it as the most potent AChE inhibitor than donepezil.The compound T-8, with better docking scores and decisive acetylcholinesterase inhibitory action, was further explored to validate the molecular interactions through molecular dynamics studies.Compounds T-1 to T-5 with a phenyl substituent in the place of the benzyl sulfonyl group showed lesser AChE inhibitory potency compared to compounds T-6 to T-10, which possess a benzyl sulfonyl group.Therefore, it may be inferred that the sulfonyl group and substituent at the para position are essential for the higher inhibitory activity of compounds.Hence, it is concluded that there is plenty of scope for further study in developing these as promising lead compounds.

Figure 5 .
Figure 5. Analysis of RMSD, RMSF, RoG, SASA, and number of hydrogen bonds, of ligand (T-8) with 6O4W for a 50 ns of MD run.where, (a) RMSD of c-alpha atoms (black) and backbone (red) (b) RMSD of ligand (c) RMSF of complex (black) and c-alpha atoms (red) (d) Radius of gyration (RoG) of the protein backbone (black) in its free and complex (red) (e) SASA of complex for T-8-6O4W and protein (red), (f) number of hydrogen bonds formed for T-8 and 6O4W.

Figure 7 .
Figure 7.The interaction of ligand T-8 with receptors (a) 6O4W, and (b) 1POP at the lowest binding energy at frames (a) 2501 and (b) 4201 displaying the hydrogen bond residues.

Figure 6 .
Figure 6.Analysis of RMSD, RMSF, RoG, SASA, and number of hydrogen bonds, of ligand (T-8) with 1P0P for a 50 ns of MD run.where, (a) RMSD of c-alpha atoms (black) and backbone (red) (b) RMSD of ligand (c) RMSF of complex (black) and c-alpha atoms (red) (d) Radius of gyration (RoG) of the protein backbone (black) in its free and complex (red) (e) SASA of complex for T-8-1P0P and protein (red), (f) number of hydrogen bonds formed for T-8 and 1P0P.

Table 6 .
Data of Acetylcholinesterase inhibitory activity of thiazolidinones (T-1 to T-10).Ellman spectroscopic method.The standard drug taken was donepezil which showed an IC 50 value of 14.6 lM.In vitro results reveal compound T-8 obtained IC50 as 8.96 lM, indicating it is the more potent AChE inhibitor than donepezil.Thiazolidinones T-10, and T-9, showed moderate inhibitory activity with IC 50 values of 22.59 and 25.75 lM (Table