Integrated insight and in silico investigation of hybrid bis-thiazolidinone derivatives along with anti-Alzheimer activity

Abstract

In the current study, a novel route was established for the synthesis of hybrid benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone (1–15) scaffolds. These compounds were screened for their biological potential as anti-Alzheimer therapeutic agents by inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. The biological evaluation and molecular docking studies revealed that most of the synthesized compounds exhibited significant inhibitory activity against both enzymes, outperforming the standard drug, donepezil. Among them, Analog 15 demonstrated remarkable therapeutic potential, with IC₅₀ values of 3.30 ± 0.70 µM and 3.80 ± 0.90 µM, as well as strong binding affinities/docking scores of − 8.97 and − 12.84 kcal/mol for AChE and BuChE, respectively. Additionally, enzyme kinetics analysis using Lineweaver–Burk plots confirmed the mode of inhibition of the synthesized analogs. Pharmacokinetic predictions further supported the drug-like properties of these compounds, highlighting favorable pharmacological profiles, including good water solubility, non-carcinogenicity, and biological safety. The findings presented in this study provide compelling evidence for the anti-Alzheimer potential of these novel scaffolds, warranting further investigation through in vivo studies and clinical exploration to assess their full therapeutic applicability.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-025-04313-6.

Introduction

A progressive neurodegenerative disorder named as Alzheimer disease is characterized by disturbance in cortical cholinergic activities (cognitive disability) (Aggarwal et al. 2022; Wilkinson et al. 2004). Cognitive impairment gradually leads to various symptoms including disorientation, behavioral disturbance, memory loss, problem in decisions making, social instability and with time these symptoms become severe and lead to death of patient (Chochinov et al. 1997). According to current studies, 50 million people have severe dementia disorder and among them 60–70% cases are of AD and it is predicted that these number will almost be doubled in the next two decades (Khachaturian 1985) (Khachaturian 1985; Rocca et al. 2011). AD is considered as a most common multifactor syndrome in old age population (over 65 year) (Jack et al. 2010; Villemagne et al. 2013). According to cholinergic hypothetical statement, AD pathogenesis is mostly characterized by reduced acetylcholine level, results by impaired secretion of cholinesterase enzymes (AChE and BuChE). Acetylcholine (ACh), a neurotransmitter responsible for the proper cognitive functions such as learning, thinking and, decision making in healthy brain (Contestabile 2011; Hasselmo 2006). AChE enzyme is mostly present in brain, muscle tissues, cholinergic neurons, and considered as main factor for ACh metabolism, while butyrylcholunesterase is mostly found in brain, liver, kidneys of healthy person (Dinamarca et al. 2011; Ecobichon and Comeau 1973; He et al. 2007; Rahim et al. 2015a, b; Rahim et al. 2016). In case of AD affected brain, AChE activity declined and BuChE activity increases. The disturbance in AChE activity cause accumulation of Aβ amyloid protein and this leads to death of neural cells and form aggregate (neurotoxicity) (Fig. 3) (Desai and Mishra 2024; Gurjar et al. 2020; Kumar 2015). Therefore, targeting both cholinesterase enzymes will be affective to treat AD. For this purpose, various marketed drugs including donepezil, tacrine and galantamine were approved by FDA authorities for AChE inhibition (Liston et al. 2004), while BuChE inhibitors include benzofuran (Rizzo et al. 2008) and cymserine (Yu et al. 2001). These therapeutic drugs control the AD symptoms up to some extent by reducing enzymatic activity of both enzymes but also exhibit certain side effects (Xu et al. 2019). Therefore, synthesis and evaluation of novel potent compounds having minimal side effects is a main concern to control complications of AD.

Benzothiazole has been reported as significant biologically active moiety, having various pharmaceutical applications including antileishmanial (Rahim et al. 2015a, b), anti-cancer (Pathak et al. 2020), anti-tumor (Hussein et al. 2012), and anti-Alzheimer (Nasab et al. 2024). They were also used as potent inhibitors urease, β-glucoronidase and thymidine phosphorylase enzymes (Khan et al. 2011; Khan et al. 2023a, b, c; Khan et al. 2023a, b, c). Heterocyclic compounds having thiazole moiety displayed strong inhibitory profile as anti-fungal (Bharti et al. 2010), anti-inflammatory (Helal et al. 2013), anti-ulcer (Pallavi et al. 2023) and anti-neoplastic agent (PKN et al. 2016). Thiazolidinone based derivatives have pivotal role in various biological activities like anti-fungal (Mishra et al. 2012), bactericidal (Patel et al. 2010), anti-tuberculosis (Küçükgüzel et al. 2002), anti-Alzheimer (Khan et al. 2024a, b, c, d, e), anti-viral (Rao et al. 2003). Many scaffolds having benzothiazole, thiazole and thiazolidinone ring with strong biological profile against different enzymes, bacteria, fungus, viruses and cancer cell lines were designed and synthesized by researchers(Arshad et al. 2022; Bhat and Belagali 2020; Sahiba et al. 2020), as illustrated in Fig. 1.

Fig. 1.

Represents compounds containing benzothiazole, thiazole and thiazolidinone ring

The current research work aims to synthesize potent scaffolds and asses against acetylcholinesterase and butyrylcholinesterae to control the symptoms of AD. They were studied against these Alzheimer causing enzymes and further their types of interactions were explored by in silico docking. Rational studies of previously reported benzothiazole (Hafez et al. 2023), thiazole (Turan-Zitouni et al. 2013) and thiazolidinone (Khan et al. 2023a, b, c) derivatives as well as highly active analog among reported derivatives were illustrated in (Fig. 2).

Fig. 2.

Represents rational studies (comparative study) of previously reported benzothiazole, thiazole and thiazolidinone derivatives as well as highly active analog 15 among reported derivatives

Materials and methods

General information

To synthesize benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone derivatives, required reagents (optimized and pure reagents) were bought by only source Sigma Aldrich, USA. To validate and purify these derivatives, product confirmation was carried out by means of TLC plates (Merck, Germany). TLC was analyzed under UV rays (lamp) with 254 and 366 nm wavelength. Structures were elucidated by NMR procedure. For this purpose, ¹H-NMR at frequency of 600 MHz and ¹³CNMR at 125 MHz, respectively and mass spectrometry were used. Signals were recorded in hertz and for reference solvent DMSO-d6 were used. Boiling points were measured by means of Buchi M-560. Finnigan MAT-311A mass spectrometer used to obtain the mass spectra or molecular weight of synthesized compounds.

Synthetic approach for hybrid benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone derivatives (1–15) synthesis (systematic procedure)

In first step, 1 equivalent amount of 2-marcaptobenzothiazole bearing aldehyde was reacted with 1 equivalent chloro-substituted ethanone bearing thiazole, in 10 mL EtOH solvent and Et₃N (3 drops) were employed to yield substrate II (S-substituted benzothiazole based ketone bearing thiazole).

In next step, equal amount (1 equivalent) of substrate II was mixed with thiosemicarbazide, the reaction was refluxed for 4 h in 2 mL acetic acid (catalyst) and 10 mL ethanol solvent to obtain substrate III.

Furthermore, 1 equivalent of substrate III was cyclized to obtain intermediate (IV), when reacted with chloro acetic (2 mL) in 10 mL ethanol and 3 drops of Et₃N.

In final step, 1 equivalent of substrate IV was allowed to react with substituted benzaldehyde (versatile substitution) (each 1 equivalent) to afford desire product benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone derivatives (Table 1).

Table 1.

Represents key characteristics of synthesized analogues

S.No	Formula	Melting point	Physical state (color)	Yield (%)	Retardation factor (Rf)
					Compound distance (cm)	Solvent distance (cm)	Rf
1	C33H20Cl2N8O2S5	186–190 °C	Green	76	2.5	5	0.5
2	C33H18Cl4N8O4S5	187–191 °C	Yellow	65	2	5	0.4
3	C33H20N10O6S5	182–186 °C	Light yellow	72	3.1	5	0.62
4	C35H24N10O6S5	188–192 °C	White	81	2.2	5	0.44
5	C33H20Br2N8O2S5	190–194 °C	Light green	78	2.5	5	0.5
6	C33H20N10O6S5	185–189 °C	Yellow	82	2.5	5	0.5
7	C33H20N10O6S5	184–188 °C	Dark yellow	68	3.6	5	0.72
8	C35H26 N8O4S5	190–194 °C	Light brown	70	4	5	0.8
9	C35H20 Cl2N8O6S5	186–190 °C	Yellow	73	3.8	5	0.76
10	C35H24 Cl2N8O2S5	192–196 °C	Brown	76	2.6	5	0.52
11	C33H18 N12O10S5	194–198 °C	Yellow	80	3	5	0.6
12	C33H20 N10O6S5	182–186 °C	Light yellow	67	2.5	5	0.5
13	C35H26 N8O4S5	193–197 °C	Light green	68	2.2	5	0.44
14	C45H30 N8O4S5	186–190 °C	White	74	3.5	5	0.7
15	C33H18 F4N10O2S5	190–194 °C	Yellow	70	4	5	0.8

The pure dry product was obtained by removing impurities with nonpolar solvent which is n-hexane (washing) and drying at reduced pressure. The end product was further subjected to structural elucidation (¹³CNMR, ¹HNMR and HREI-MS).

Molecular docking assay protocol

Ligands and protein retrieval

To conduct molecular docking with high accuracy firstly we used different sources to collect ligand and targeted protein complex. For this purpose, among the synthesized compounds, potent ligands having leading potential were selected and their structures were drawn through chemBioDraw software and the targeted protein (AChE and BuChE) were collected from an online source (RSCB PDB) used for protein retrieval. To retrieve AChE enzymes, 1ACI code were used while 1POP was used for BuChE enzyme complex retrieval through PDB.

Optimization of ligands and targeted protein complex (preparation)

After collecting ligand structure and protein complex, the next step is optimization of both ligand and enzyme complex. Potent analogues of benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone series were accurately prepared for molecular docking studies by their optimization though built and Ligand Preparation module. This module is implemented in Discovery Studio 2018 (Dassault Systemes BIOVIA, USA). Ligand preparation also includes generating various tautomer’s, assigning stereochemistry and bond orders within a molecule. In a similar way, crystal protein complex structure was optimized by the computation of missing atoms, hydrogen bonds as well as charges. Moreover, heteroatoms, water molecules and cofactors were removed and protein structure will be completely optimized (Korb et al. 2009).

Defining active site and molecular docking

By selecting centroid of complexed ligand (Montbretin A), a receptor grid was generated around the active site of AChE and BuChE enzyme. Similarly, the active site was accurately defined having radius of 12 Å around the Montbretin A binding site. Gold docking tool was used to conduct the molecular docking. Ligand was docked in a protein complex structure and run for different poses. A pose having maximum binding affinity and binding interactions with minimum root means square deviation (RMSD) value were optimized and further docking calculations were carried out by means of Chem PLP scoring function. Pymol and DSV software packages were used for the 2D and 3D visualizations of docking outcomes.

Spectral analysis (Supplementary information)

AChE and BuChE assay protocol (Supplementary information).

Results and discussion

Chemistry

For the synthesis of hybrid benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone scaffolds, a unique efficient synthetic route was designed, which comprised of multistep process.

In initial step, 2-marcaptobenzothiazole having aldehyde moiety was treated with chloro-substituted ethanone bearing thiazole, in EtOH solvent and few drops of triethylamine were added to afford substrate II (S-substituted benzothiazole based ketone bearing thiazole). In next step, substrate II was mixed with thiosemicarbazide, the reaction was refluxed for 4 h in acetic acid (catalyst) and ethanol solvent to obtain substrate III. Furthermore, substrate III was cyclized to obtain intermediate (IV), when reacted with chloro acetic in ethanol and Et₃N. In final step, substrate IV was allowed to react with substituted benzaldehyde (versatile substitution) to afford desire product benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone derivatives (Scheme 1). The pure dry product was obtained by removing impurities with n-hexane (washing) and drying at reduced pressure.

Scheme 1.

Synthetic procedure for benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone scaffolds (1–15) along with the optimized reaction conditions

AChE and BuChE biological profile

All these novel reported derivatives (1–15) were evaluated for their anti-enzymatic profile for acetylcholinesterase and butyrylcholinesterase inhibition (Fig. 3) and to identify potent scaffolds having strong biological profile (lowest inhibitory concentration). To establish comparison criteria, marketed drug donepezil (Prajapati et al. 2025; Rai et al. 2020; Ramakrishna et al. 2024, 2023; Singh et al. 2024; Srivastava et al. 2019; Tripathi et al. 2024, 2019; Yadav et al. 2024) were used having inhibition zone of (10.10 ± 0.30 µM and 11.30 ± 0.10 µM) against both enzymes. Most of the compounds have lowest inhibition concentration and showed versatile biological potential for stated enzymes. The inhibitory concentration against AChE for all synthesized scaffolds ranged as (3.30 ± 0.70 to 22.20 ± 0.10) and against BuChE ranged as (3.80 ± 0.90 to 23.10 ± 0.20). Substituents nature, their position of attachment and their configuration have significant role in biological activity of all reported compounds. Subsequently, scaffolds having highly electronegative atoms or strong electron withdrawing groups have excellent inhibitory profile. In a same way compounds having bulky groups found inactive against both targeted enzymes. The enzymatic potential of reported compound was illustrated in Table 2.

Fig. 3.

Illustrates the level of damage caused by AChE and BuChE enzyme in the brain of Alzheimer patient

Table 2.

Enzymatic activity (IC₅₀ value in µM) of benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone scaffolds for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)

S. no.	R1	BuChE IC50 in µM	AChE IC50 in µM
1		11.10 ± 0.20	10.30 ± 0.10
2		6.10 ± 0.10	5.10 ± 0.20
3		14.90 ± 0.30	14.10 ± 0.20
4		16.20 ± 0.20	15.10 ± 0.20
5		22.10 ± 0.20	21.10 ± 0.20
6		11.30 ± 0.30	10.10 ± 0.10
7		4.10 ± 0.50	3.70 ± 0.10
8		12.70 ± 0.10	12.10 ± 0.10
9		5.90 ± 0.50	5.20 ± 0.10
10		12.10 ± 0.30	11.10 ± 0.20
11		10.70 ± 0.40	9.10 ± 0.20
12		14.40 ± 0.20	13.10 ± 0.30
13		13.30 ± 0.10	12.30 ± 0.10
14		23.10 ± 0.20	22.20 ± 0.10
15		3.80 ± 0.90	3.30 ± 0.70
Donepezil (Standard drug)		11.30 ± 0.10	10.10 ± 0.30

SAR of these novel hybrid derivatives revealed that analog 7 and 15 bearing highly electronegative fluorine atoms have maximum inhibitory profile and ranked as first and second most active analogues among the reported derivatives.

Analog 15 containing fluorine atoms at ortho and meta-position on both phenyl rings have lowest inhibitory concentration (3.30 ± 0.70 µM and 3.80 ± 0.90 µM) (Fig. 4) and have maximum biological potential against targeted enzymes in contrast to donepezil (control drug) and all remaining analogues. This might be due fluorine atoms as it has very small size and high electronegative nature, so, both fluorine atoms were capable to make striking H-Bonds with receptor residue of active sites. These fluorine atoms have ability to increase the electrophilic character of phenyl rings, making more reactive towards anionic components in amino acids of targeted enzymes. It also increases lipophilicity, which allows the molecule to cross cell membranes and specifically bind with the targeted enzymes by effective interactions. So, the superior inhibitory profile of this analog attributed to benzothiazole, thiazole and both thiazolidinone ring as well as both phenyl rings, as all these moieties contain interesting heteroatoms or attached substituents (di-flouro) having versatile interactive properties. Similarly, same highly electronegative flouro substituted analog, were second most potent analog among the synthesize derivatives. This analog 7 holds –F group on para-position of aromatic ring. This analog has biological potential of 3.70 ± 0.10 µM and 4.10 ± 0.50 µM, respectively (Fig. 4). The reason for maximum activity of this scaffold was flouro group at favorable para-position, it withdraw electrons from phenyl ring and make it polar (partial positive charge on phenyl rings), which further interact with amino acids (containing polar groups) of stated enzymes. The complete skeleton of molecule also has minimum effect on these fluorine atoms as they have favorable position of attachment and easily inhibit the enzyme active sites with it and impede their normal activity to bind with substrate. These fluorine atoms also contribute to the lipophilic nature of this molecule, to reach targeted sites. Analog 2 with inhibition value of 5.10 ± 0.20 µM were compared with analog 9 with inhibition value of 5.20 ± 0.10 µM to evaluate potent inhibitor of AChE (Fig. 4). Analog 2 exhibit better activity due to presence of two chloro moieties (meta-substitutions) as well as hydroxyl group (ortho-substitution) of aryl rings. These highly electronegative chlorine atoms have nucleophillic character and make ring unstable. This unstable ring interacts with different amino acids of AChE enzyme and regains stability. Hydroxyl groups were responsible for making hydrogen bonding by both oxygen (H-Bond acceptor) and hydrogen (H-Bond donor). However, when one chlorine atom was replaced with hydroxyl group, as in analog 9 its enzymatic activity against AChE retarted. Against BuChE, analog 9 exhibit maximum inhibition than analog 2. As, both hydroxyl groups have characteristics to inhibit enzyme activity by making molecular hydrogen bonds that may be donor H-Bonds and acceptor H-Bonds with BuChE complex. Moreover, chlorine atom also disturbed the polarity of molecule and this molecule strongly interacts with receptor residues (polar amino acids) of BuChE.

Fig. 4.

Represents the complete structure and enzymatic activity (IC₅₀ in µM) of Analog 2,7,9 and 15

Analog 3, 4, 6, 11 and 12 have nitro-substitution on different positions of phenyl ring. Among these four tested compounds, analog 11 having two –NO₂ moiety at ortho and meta-position was found as potent inhibitor of targeted enzymes and have inhibition concentration value of (9.10 ± 0.20 µM and 10.70 ± 0.40 µM) (Fig. 5). Greater number of strong electron accepting or withdrawing groups have pivotal role in their enhanced biological activity. They make phenyl rings cation, and they make several polar interactions. These functionalities have maximum capabilities to make promising binding pockets (hydrogen bonding) with active sites of stated enzymes. Similarly, analog 6 have equivalent potency to donepezil having inhibition zone of (10.10 ± 0.10 µM and 11.30 ± 0.30 µM). The –NO₂ moiety at favorable para-position polarize the aromatic ring (makes it partial positive), which further interact with polar amino acids which composed targeted enzymes. They also have much capability to make interactions, due to less impact of complete molecule on this substituent. However, analog 3 bearing nitro moiety on ortho-position has less ability to make interactions, as it is near to the bulk of molecule and this might be involved in making hydrogen bonds within the molecule and don’t inhibit enzyme activity upto larger extent. Similarly, analog 12 also have less inhibitory activity due to unfavorable attachment positions of nitro moiety on varied substituted ring. At meta-position, they have minimum holds on phenyl ring to make cationic and have reduced activity against targeted enzymes.

Fig. 5.

Represents the overall structure and enzymatic activity (IC₅₀ in µM) of Analog 3, 4, 6, 11 and 12

SAR of analog 1 and 10 having chloro substituent revealed that analog 1 (10.30 ± 0.10 µM and 11.10 ± 0.20 µM) (Fig. 6) bearing chloro group on para-position have better enzymatic activity than analog 10. The reason behind the promising efficacy of this analog is chloro group at this position, induce maximum negative charge on phenyl ring which further interact with polar groups or parts of different amino acids in the receptor sites of diabetic enzymes (polar amino acids) targeted enzymes. However for analog 10, addition of methyl group on ortho-position was responsible for declined in biological potential of compound 10. This methyl group has minimum ability to makes any effective interactions, as it has much hyper conjugation. It caused steric hindrance around phenyl rings due to its bulky nature. So, analog 10 has decline activity. Similarly, methoxy-substituted analogues 8 and 13 displayed moderate potency in contrast to donepezil. Analog 8 (12.10 ± 0.10 µM and 12.70 ± 0.10 µM) with –OMe at para-position have somewhat more efficacy than analog 13 (12.30 ± 0.10, 13.30 ± 0.10 µM). This might be due to para-substituted –OMe, at that position it has less impact (hydrophobic effect) on molecule. So, this analog exhibit better attachment with amino acids of stated enzymes (Fig. 6). But at meta-position, the methoxy moiety has less capability to allow the ring to undergo different interactions, and by itself make weak interactions with enzyme complex. So, analog 13 was less potent molecule in comparison to donepezil.

Fig. 6.

Represents the complete structure and enzymatic activity (IC₅₀ in µM) of Analog 1,8,10 and 13

However, among the synthesized derivatives (1–14), analog containing bulky substituents were found less potent analogues (analog 5 and 14). Analog 5 having bulky bromo group exhibit poor inhibition. This bulky –Br group surround the phenyl ring, and prevent it to bind with enzymes receptor sites. Similarly, analog 14 bearing benzoyl oxy moiety have hydrophobic nature, and displayed very weak interactions with receptor residues of stated enzymes (Fig. 7).

Fig. 7.

Inhibitory concentration graph for analog 15 against AChE

Inhibitory potential graphs for potent analogues were also plotter to understand the relations between the %age inhibition and inhibition concentration. It shows that %age inhibition varies with respect to concentration of inhibition, as illustrated in Figs. 8, 9 and Graph (S.1–S.4).

Fig. 8.

Inhibitory concentration graph for analog 15 against BuChE

Fig. 9.

Analog 15 as competitive or active inhibitor

Enzyme kinetics

Enzyme kinetic study was carried out to explore the inhibition of synthesized analogues and their competency level as competitive, uncompetitive and non-competitive inhibitors. The enzyme kinetics for given analogues were interpreted by plotting lineweaver–Burk graph. Figure 7 depict that analog 15 was a most potent inhibitor or has maximum competence level, as by varying concentration the 1/V value increases, as well as the Km constant also increases which decrease the substrate binding affinity for enzyme and ultimately reduced enzyme normal activity. On the other hand, analog 10 was recognized as uncompetitive inhibitor due to its moderate inhibitory activity as Km constant continuously declined (Fig. 10) as well as inhibition rate varies with concentration. For this case, the affinity of substrate increases for enzyme, so this analog 10 showed moderate inhibition. Furthermore, analog 14 was a least potent or non-competitive inhibitor of synthesized series, as it has poor inhibition. This analog binds with the allosteric sites of enzyme rather than active site and has minimum inhibitory potential. The inhibition rate varies for this analog at different concentrations, while the Km constant will remain same, as illustrated in Fig. 11.

Fig. 10.

Analog 10 as un-competitive or moderate inhibitor

Fig. 11.

Analog 14 as non-competitive or least potent inhibitor

Molecular docking

Many biologically active hybrid scaffolds were synthesized and their interactive assessment profile was evaluated by in silico molecular docking studies by our research group by using certain software tools (Khan et al. 2024a, b, c, d, e; Khan et al. 2025; Khan et al. 2024a, b, c, d, e; Khan, et al. 2024a, b, c, d, e). By similar protocols binding modes of lead candidates having maximum zone of inhibition were explored in this current research work. In this process software tools like Pymol, Discovery studio visualizer-DSV (to visualize the geometries of protein complex, in which ligand molecule were docked) and 1.5.7 version Autodock vina were used. By using their corresponding codes, crystal structures of targeted enzymes were collected from PDB. After optimization of enzyme complex and preparation of protein complex and potent molecules, binding folds of potent compounds with targeted enzyme were visualized. Results revealed that the potent analogues exhibit strong and effective forces such as H-Bonds, pi-pi T-Shaped, pi-alkyl with the receptor residues of stated enzymes (Tables 3 and S.1; Figs. 12, 13, 14, 15, 16, 17, 18, 19). These compounds have negative binding score and maximum binding affinity and strongly hold with enzyme complex via versatile interactions (stable binding folds). All moieties were involved in making promising binding interactions with different receptor residues of both diabetic enzymes. Substituted phenyl rings have maximum capability to make effective interactions and allow the overall compound to make possible interactions with molecule to completely fit into the active sites of targeted enzymes, and block each site from substrate and in this way inhibit the normal metabolic functions of both diabetic enzymes.

Table 3.

Mode of interaction of potent analogue 15 with amino acids of AChE and BChE enzymes

Potent scaffolds	Receptor	Type Of interactions	Distance	Docking score
Analog 15 in Acetylcholinesterase complex	TYR-A-334	Pi–Pi Stacked	5.16	− 8.97
	PHE-A-330	Pi–Pi Stacked	4.24
	TYR-A-70	C–H Bond	4.08
	PHE-A-331	H-F	3.56
	ILE-A-287	Pi–Alkyl	4.85
	SER-A-286	H–F	5.28
	HIS-A-440	H–F	4.85
	GLU-A-199	H–F	5.69
	GLY-A-441	C–H Bond	3.45
	TRP-A-84	Pi–Pi Stacked	3.93
Analog 15 in Butyrylcholinesterase complex	THR-A-122	H-Bond	4.61	− 12.84
	TRP-A-82	C–H Bond	5.38
	TRP-A-82	Pi–Pi Stacked	4.42
	TRP-A-82	Pi-Pi Stacked	3.71
	TRP-A-82	Pi–Pi Stacked	5.02
	TRP-A-82	Pi–Lone Pair	3.86
	TYR-A-128	H–Bond	6.84
	GLU-A-197	Pi–Anion	5.94
	GLY-A-116	H-Bond	3.47
	GLY-A-116	Pi–Pi Stacked	4.70
	LEU-A-286	H–F	3.69
	LEU-A-286	Pi-Sigma	5.02
	TRP-A-231	Pi–Pi Stacked	6.47
	PHE-A-329	Pi–Pi Stacked	6.51
	HIS-A-438	H-Bond	5.13
	HIS-A-438	Pi-Sulfur	5.54
	HIS-A-438	Pi-Sulfur	3.97
	SER-A-198	H-Bond	4.47
	SER-A-198	H-Bond	4.36
	GLU-A-325	Unfavorable bump	4.94
	GLU-A-325	Unfavorable bump	4.66
	TRP-A-430	Pi-Alkyl	5.85
	TRP-A-430	Pi-Sulfur	7.25
	MET-A-437	Pi-Alkyl	4.98
	MET-A-437	Pi-Alkyl	5.58
	TYR-A-440	Pi-Sulfur	6.02
	GLY-A-121	H-Bond	3.80

Fig. 12.

Analog 2 Binding Interactions with AChE Complex

Fig. 13.

Analog 2 Binding Interactions with BuChE Complex

Fig. 14.

Analog 7 binding interactions with AChE Complex

Fig. 15.

Analog 7 binding interactions with BuChE Complex

Fig. 16.

Analog 9 Binding Interactions with AChE Complex

Fig. 17.

Analog 9 Binding Interactions with BuChE Complex

Fig. 18.

Analog 15 binding interactions with AChE Complex

Fig. 19.

Analog 15 binding interactions with BuChE Complex

Molecular dynamic simulations

An advanced molecular dynamic simulation approach was employed to further explore the binding mechanism of potent analogue with targeted enzymes. This study helps to determine the conformational changes or structural deviations in a targeted protein due to binding interactions of active ligand with them. This process was run for both simple protein and protein–ligand complex over 100 ns time. This helps to determine the relative stability in protein and protein–ligand complex through their RMSD calculations. Both have RMSD values in a smaller range which shows that active compound effectively fit into the binding pocket of targeted enzymes without causing any much fluctuations or conformational changes. Results showed that many significant interactions between ligand and the protein receptors were responsible for their stability in dynamic conditions (Fig. 20). The ligand shape fit perfectively with a binding site having maximum affinity and therefore exhibit high efficacy. This study also highlights the significance of interactions like hydrogen bonding which contribute to maximum inhibition profile of active compound. In summary, molecular docking and MD simulations explores the underlying factors that were involved in binding of potent analogue with targeted protein complex which further suggest patent analog as effective anti-leukemia agent.

Fig. 20.

Molecular dynamic simulation of protein and protein–ligand complex showing the intermolecular interactions, conformational changes and binding affinity and stability overtime for analog 15

ADMET analysis

After confirmations of various interactions with enzyme complex, potent molecules were virtually analyzed by ADMET, to predict whether these compounds were considered as therapeutically safe drug candidates. Tool employed for the ADMET analysis was swissADME, in which lead molecules were examined under various ADMET guidelines, to observe their lipophilic, physiochemical, water solubility, pharmacokinetics and medicinal capabilities of these potent compounds. These guidelines consist of Lipinski, Bioavailability score, PAINS, Lead likeness violations, Brenk alerts, Ghose, log Kp, Veber, Muegge violations. Results revealed that all the active scaffolds didn’t display any violation of these rules, and they were recognized as non-toxic compounds. These results were mentioned in Table 4, Fig. 21 and Graphs (S.5-S.7).

Table 4.

ADMET analysis of analog 15 encompassing various physiochemical, medicinal and drug likeness attributes confirming the effective therapeutic profile

Physicochemical property
Molecular weight (MW)	794.010
Volume	689.069
Density	1.152
nHA	10
nHD	2
nRot	11
nRing	7
MaxRing	9
nHet	19
fChar	0
nRig	42
Flexibility	0.262
Stereo Centers	0
TPSA	137.440
logS	− 8.052
logP	7.596
logD	4.298
Medicinal Chemistry
Fsp3	0.030
MCE-18	78.000
NPscore	− 1.629
Pfizer Rule	Accepted
BMS Rule	0 alert(s)
Chelator Rule	0 alert(s)
Absorption
Caco-2 Permeability	− 5.415
MDCK Permeability	1.3e−05
Pgp-inhibitor	0.6
Pgp-substrate	0.1
HIA	0.1
F20%	0.6
F30%	0.9
Distribution
PPB	115.770%
VD	1.013
BBB Penetration	0.3
Fu	0.460%
Metabolism
CYP1A2 inhibitor	0.4
CYP1A2 substrate	0.1
CYP2C19 inhibitor	0.8
CYP2C19 substrate	0.9
CYP2C9 inhibitor	0.5
CYP2C9 substrate	0.6
CYP2D6 inhibitor	0.1
CYP2D6 substrate	0.8
CYP3A4 inhibitor	0.2
CYP3A4 substrate	0.2
Excretion
CL	1.564
T1/2	0.001
Toxicity
hERG blockers	0.1
H-HT	0.8
DILI	0.9
AMES toxicity	0.4
Rat oral acute toxicity	0.6
FDAMDD	0.8
Skin sensitization	0.8
Carcinogencity	0.9
Eye corrosion	0.1
Eye irritation	0.1
Respiratory toxicity	0.2

Fig. 21.

ADMET assessment profile of analog 15

SMILES representation and boiled egg notation for analog 15 was also illustrated in Figs. 22 and 23 to understand the molecular arrangement of given molecule and it’s binding into the active sites of targeted enzymes.

Fig. 22.

Analog 15 SMILES representation, which shows the molecular arrangement of analog 15

Fig. 23.

Boiled egg depiction for analog 15

Conclusion

In search of lead scaffolds having strong biological profiles against AChE and BuChE, hybrid benzothiazole derived thiazole bearing bis-thiazolidinone-chalcone compounds (1–15) were designed and synthesized. These compounds anti-Alzheimer potent were asses in comparison to donepezil and their in vitro biological screening revealed that that these compounds displayed versatile biological activity due to different attached substituents on phenyl rings, as well as biologically active moieties benzothiazole, thiazole, bis-thiazolidinone rings were also involved in inhibition of these enzymes. They have excellent to good biological activity against AChE and BuChE, and their biological profile was ranged as 3.30 ± 0.70 µM to 22.20 ± 0.10 µM and 3.80 ± 0.90 µM to 23.10 ± 0.20 µM in the comparison with donepezil (10.10 ± 0.30 µM and 11.30 ± 0.10 µM). Their biological potential mainly depends on number of substituent, their ability to activate or deactivate phenyl rings (nature) and position on these aryl part of molecules. Among these derivatives (1–15), analog 15 was found as most potent analog with lowest inhibitory concentration (3.30 ± 0.70 µM and 3.80 ± 0.90 µM). This might be due to 2 highly electronegative fluorine atoms on both phenyl rings. They have maximum capability to make stable molecular H-Bonds with receptor sites of stated AChE and BuChE enzymes. Inhibitory concentrations and Lineweaver–Burk graphs were also plotted to understand the inhibition rate and enzyme kinetics of these synthesized analogues. Binding modes and interactive characteristics of active scaffolds were investigated by docking and to predict drug likeness, pharmacokinetics as well as physiochemical characteristics of these analogues (lead candidates), ADMET analysis was conducted. Structures of these scaffolds (1–15) were validated by ¹³CNMR, ¹HNMR and HREI-MS techniques.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was funded by the Researchers Supporting Project Number (RSP2025R388), King Saud University, Riyadh, Saudi Arabia.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflicts of interest

It is declared that there is no conflict of interest between the authors of the current manuscript.

Research involving human participants and/or animals

This research work involves no human or animal participation.

Informed consent

This research work is entirely in silico and in vitro, eliminating the need for human or animal subjects and ensuring compliance with animal welfare and human ethics regulations.