Identification of novel benzothiazole–thiadiazole-based thiazolidinone derivative: in vitro and in silico approaches to develop promising anti-Alzheimer’s agents

Abstract

Aim: The present study describes benzothiazole derived thiazolidinone based thiadiazole derivatives (1–16) as anti-Alzheimer agents.

Materials & methods: Synthesis of benzothiazole derived thiazolidinone based thiadiazole derivatives was achieved using the benzothiazole bearing 2-amine moiety. These synthesized compounds were confirmed via spectroscopic techniques (¹H NMR, ¹³C NMR and HREI-MS). These compounds were biologically evaluated for their anti-Alzheimer potential. Binding interactions with proteins and drug likeness of the analogs were explored through molecular docking and ADMET analysis, respectively. In the novel series, compound-3 emerged as the most potent inhibitor when compared with other derivatives of the series.

Conclusion: The present study provides potent anti-Alzheimer’s agents that can be further optimized to discover novel anti-Alzheimer’s drugs.

Graphical Abstract

Plain language summary

Article highlights.

Background

• Alzheimer’s disease is a neurodegenerative cholinergic impairment which is reported as the fourth major death causing disease. Thus, a new selective anti-Alzheimer’s agent is highly demanded across the world.

Synthesis

• In order to combat the global health challenge, a novel series of benzothiazole derived thiazolidinone based thiadiazole derivatives was designed and synthesized. Synthetic process is mainly composed of treating benzothiazole bearing 2-amine moiety with ammonium isothiocyanate to obtain benzothiazole derived thiazolidinone moiety. In the subsequent reactions with thiosemicarbazide and varied substituted benzaldehyde were added to afford novel compounds.

Spectroscopic analysis

• These derivatives were spectroscopically characterized through ¹H-NMR, ¹³C-NMR and HREI-MS.

Anti-Alzheimer’s potential

• Anti-Alzheimer’s potential of the synthesized scaffolds was evaluated in comparison to standard drug donepezil having IC₅₀ = 4.20 ± 0.10 and 5.30 ± 0.20 μM. Compounds 3, 6 and 10 having fluoro and chloro substituents exhibited excellent inhibition profile due to the presence of groups such as -F, -Cl, -OH and -CF₃ groups. These functional moieties interact with the target enzyme via number of different interactions. Among these interactions, hydrogen bonding plays a significant role in biological profile of the compound.

Structure–activity relationship

• Inhibitory potential of the compounds was demonstrated on the basis of structure–activity relationship interpretation which mainly depends on number, nature (electron-donating/ electron-withdrawing) and position of the substituents around the phenyl ring. It was found that the groups involved in formation of hydrogen bonding have a profound impact on the inhibitory activity, significantly influencing the ability of the compound to bind and interact with targets.

Molecular docking study

• Protein–ligand interaction profile of the potent analogs was also explored via molecular docking study. The compounds showing promising inhibition were selected for further investigation through in silico studies to elucidate their binding interactions with enzymes. Detailed 2D and 3D visualizations revealed a range of interactions, including hydrogen bonding and other contacts at various distances, providing valuable insights into their binding mechanisms.

Absorption, distribution, metabolism, excretion & toxicity analysis

• Additionally, an absorption, distribution, metabolism, excretion and toxicity (ADMET) analysis was performed on the lead compounds to assess their drug-like properties and evaluate the pharmacokinetic and safety profiles of the most promising analogs.

1. Background

Alzheimer’s disease (AD), a prolonged chronic neurodegenerative and irremediable disease results in degeneration of brain cells and disables the affected person’s ability to function independently. AD, the foremost cause of dementia has globally affected world’s population and an increase from 57.4 million cases globally in 2019 to 152.8 million cases in 2050 is expected, having a rise in its commonness with the age [1]. AD was ranked as the fourth major fatal disease in developed countries, subsequent to cancer, cerebral and cardiovascular abnormalities. The equilibrium of certain neurotransmission chemical messengers including acetylcholine (ACh), butyrylcholine (BuCh), dopamine, serotonin, noradrenaline, glutamate and gamma-aminobutyric acid is vital for the normal brain functioning [2]. A cognitive impairment occurs in adult-onset dementia characterized by the disorders inclusive of AD [3]. ACh is hydrolyzed into choline and acetic acid under the action of cholinergic enzymes acetylcholinesterase (AChE) which is mainly found in postsynaptic neuromuscular junctions, especially in muscles and nerves, and butyrylcholinesterase (BuChE). Loss of cholinergic tone which is caused by the neurodegeneration of basal forebrain, especially due to projections to the hippocampus and cortex, is considered to be the major cause of cognitive impairment known as AD [4–7]. The irreversible consequences of imbalanced cholinergic system include thinking problems, confusion, loss of memory, problem solving difficulties and cognitive impairment [8–10]. Therefore, the utmost goal is to synthesize highly potent and novel anti-Alzheimer drugs with significant inhibition against AChE and BuChE [11]. Acetylcholine has the major role in cognitive processes such as memory. A decline of cholinergic neurotransmission in brain is observed due to acetylcholine which gives rise to the development of AD [12,13]. BuChE enzyme is related to AChE and attracts the attention for its role in developing AD [14,15]. BuChE inhibits the enzyme involved in transmission of neurotransmitter which may induce many side effects such as nausea, fever, vomiting and even leading to death [16]. AChE enzyme is mainly located in cholinergic neurons, muscles and brain, whereas BuChE enzyme is located in kidneys, heart, liver, intestines and lungs [17,18]. The decline in cholinergic activity occurs due to the breakdown of ester-containing derivatives under the enzymatic activity of AChE in brain. Therefore, a highly effective drug is required for the treatment of AD by the inhibition of enzymes AChE and BuChE. Therapeutic drugs available in market for curing AD are reported in providing only the symptomatic relief [19]. US FDA approved medications for treating AD includes rivastigmine, donepezil, galantamine and tacrine. AChE and BuChE both are inhibited by rivastigmine and tacrine, whereas donepezil and galantamine are used to hinder AChE [20] (Figure 1).

Figure 1.

US FDA-approved drugs for treatment of Alzheimer’s disease.

Thiazolidinone scaffolds have been reported with significant inhibitory potential in various biologically potent drugs. Thiadiazole and its derivatives are used as anti-urease, antithymidine phosphorylase agents [21], antidiabetic [22] and anti-Alzheimer’s [23]. Furthermore, thiazole-based hybrids have compelling applications in medicines as anti-inflammatory, analgesic [24], anticancer [25], antihypertensive [26] and anti-asthmatic [24]. Thiadiazole derived thiazolidinone derivatives [27] and benzothiazole derivatives [28] shown in Figure 2, are previously reported with anti-Alzheimer’s activity. The current study aims to synthesize novel bis-thiazole-thiazolidinone hybrid derivatives as potent inhibitors against AChE and BuChE for the treatment of AD. In this approach, we have successfully synthesized 16 analogs having significant inhibitory profile against the enzymes acetylcholinesterase and butyrylcholinesterase. All the synthesized analogs expressed moderate to good inhibition except analog-3 which exhibited exceptional potency against both the targeted enzymes with IC₅₀ values 0.90 ± 0.20 and 1.10 ± 0.40 μM.

Figure 2.

Rationale of current study.

2. Materials & methods

2.1. Chemicals & standards

The chemicals and solvents used for the mentioned research were bought reputable suppliers, namely Sigma Aldrich (MO, USA) and Merck (Darmstadt, Germany). For characterization of the synthesized compounds, Bruker AM spectrometer (Bruker, MA, USA) having operating frequency of 600 MHz was used to obtain nuclear magnetic resonance (NMR) spectra. Mass spectra (HR-EI-MS) were captured using the spectrometer MAT 312, MAT 113D and JEOL JMS-600H (JEOL, Tokyo, Japan). For TLC, we have used precoated silica gel-254 from Merck and the spots were observed at the wavelength of 366 and 254 nm. Chemical shift values were measured in parts per million (ppm) and coupling constants were measured in frequency (Hz), using the reference DMSO-d₆ serving.

2.2. Chemistry

Benzothiazole bearing 2-amine moiety (1 mmol) was refluxed for about 3 h with ammonium isothiocyanate (1 mmol) using the catalyst triethylamine (Et₃N 3-drops) in ethanol to obtain benzothiazole based thiourea moiety (II) which was then cyclized in the presence of chloro acetic acid (1 mmol) followed by addition of Et₃N in ethanol under reflux for 4 h, yielded benzothiazole derived thiazolidinone moiety (III). Compound-III was again cyclized by using thiosemicarbazide (1 mmol) in ethanol in the presence of sodium acetate, benzothiazole derived thiazolidinone and thiadiazole bearing amine moiety as bis-cyclized product (IV) was achieved after refluxed about 8 h. The amine moiety of compound-IV was further used for the production of Schiff base derivatives by the reactions of varied substituted benzaldehyde (1 mmol), afforded the targeted moieties (1–16) as shown in Figure 3. Varied substituted compounds were found with different yields (61–87%). After washing and confirmation through thin layer chromatography (TLC), these synthesized moieties were further explored by spectroscopic techniques including ¹H NMR, ¹³C NMR and HREI-MS.

Figure 3.

Details mechanism of benzothiazole derived thiazolidinone based thiadiazole derivatives (1–16).

2.3. Assay protocol for acetylcholinesterase & butyrylcholinesterase assays

With a little bit modification, inhibitory potential against AChE and BuChE was investigated according to the reported protocol [29]. The total volume of the reaction mixture was kept at 100 μl, containing 60 μl of Na₂HPO₄ buffer with a concentration of 50 mM and a pH of 7.7. Added test compound (well-1) with volume of 10 μl and a concentration of 0.5 mM followed by the addition of an enzyme of 10 μl (0.005 unit well-1). Pre-incubation of substances at 37°C for 10 min was achieved. Reaction was started by addition of 10 μl of 0.5 mM well-1 substrate (acetylthiocholine iodide/butyrylthiochloine chloride) followed by addition of 10 μl DTNB (0.5 mM well-1). Absorbance at 405 nm was measured after 15 min of incubation at 37°C using the 96-well plate reader Synergy HT, BioTek (VT, USA). All experiments were done with their respective controls in triplicate. Standard drug used was donepezil. The percent inhibition was computed using the following equation

$$\text{Inhibition}(\%)=\frac{\text{Control}-\text{Test}}{\text{control}\times 100}$$

Control EZ-Fit Enzyme kinetics software (Perrella Scientific Inc., MA, USA) was used for the calculation of IC₅₀ values.

2.4. Assay protocol for molecular docking study

Protein Data Bank was used as a medium for retrieval of crystalline structure using the code 1ACL for AChE and 1P0P for BuChE. Optimization of the protein structure was completed by the removal water molecules, co-factors and hetero-atoms and computing hydrogen bonds, charges and the missing atoms. Benzothiazole derived thiazolidinone based thiadiazole derivatives used for docking studies were prepared and then optimized by the use of built and ligand preparation module implemented in Discovery Studio 2018 (Dassault Systemes, BIOVIA, CA, USA). AutoDock Vina and Pymol were used for docking analysis; Ligand preparation involves generating varied tautomer’s, bond order assigning and stereochemistry. Nine different poses of the ligand were generated and the one with lowest energy was selected for further docking procedure. Furthermore, AChE and BuChE active site was surrounded by the receptor grid choosing centroid of complexed ligand (Montbretin A). Radius of 12 Å around the Montbretin A binding site was defined for enzyme active site. Accomplishment of docking calculations was achieved using Chem PLP scoring function [30–39].

3. Results & discussion

3.1. Structural elucidation

3.1.1. Synthesis of benzothiazole derived thiazolidinone derivative

In step-I, Benzothiazole bearing 2-amine moiety (1 mmol) was refluxed continuing the stirring for about 3 h with ammonium isothiocyanate (1 mmol) in the presence of catalyst triethylamine (Et₃N 3-drops) in ethanol (15 ml) affording benzothiazole based thiourea moiety (II). In step-II, cyclization of intermediate II was achieved in the presence of chloro acetic acid (I mmol) by addition of Et₃N catalyst in ethanol (15 ml) under reflux for 4 h, yielded benzothiazole derived thiazolidinone moiety (III).

3.1.2. Synthesis of benzothiazole derived thiazolidinone based thiadiazole derivative

In step-III, compound-III was further cyclized using thiosemicarbazide (1 mmol) in ethanol (15 ml) in the presence of catalyst sodium acetate, benzothiazole derived thiazolidinone and thiadiazole bearing amine moiety as bis-cyclized product (IV) was achieved after stirring and reflux for a duration of about 8 h.

3.1.3. Synthesis of benzothiazole derived thiazolidinone based thiadiazole derivatives (1–16)

In step-IV, amine moiety of compound-IV was further used for the production of Schiff base derivatives by the reactions of varied substituted benzaldehyde (1 mmol), afforded the targeted moieties (1–16). Varied substituted compounds were found with different yields (61–87%). After washing than confirmation of TLC these synthesized moieties were further explored by spectroscopic techniques including ¹H NMR, ¹³C NMR and HREI-MS.

4. Experimental analysis

4.1. Synthesis

4.1.1. Spectral analysis

4.1.1.1. (Z)-2-((6-(5-(((E)-4-methylbenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (1)

FTIR (cm^-1) : 3407, 3215, 2972, 1730, 1560; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.98 (s, 1H, N-H), 10.04 (s, 1H, H_imine), 8.71 (s, 1H, H_Ar), 8.58 (d, J = 6.4 Hz,2H, H_Ar), 8.19 (d, J = 6.4 Hz, 2H, H_Ar), 8.12 (d, J = 7.2 Hz, H_Ar, 1H), 8.08 (d, J = 6.7 Hz, H_Ar, 1H), 4.09 (s, H_{thiazolidinone}, 2H), 2.30 (s, 3H, -CH₃); ¹³C-NMR (150 MHz, DMSO-d₆): δ 178.2, 177.4, 175.4, 163.4, 162.8, 154.3, 151.8, 150.2, 145.2, 145.1, 144.5, 144.1, 141.4, 141.2, 140.5, 132.1, 125.4,119.7, 35.2, 28.4; HREI-MS: m/z calcld for C₂₀H₁₄N₆OS₃, [M]⁺ 450.2850 Found 450.2843.

4.1.1.2. (Z)-2-((6-(5-(((E)-3,4-dimethylbenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (2)

FTIR (cm^-1) : 3439, 3230, 3009, 1745, 1530; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.91 (s, 1H, N-H), 10.12 (s, 1H, H_imine), 8.83 (s, 1H, H_Ar), 8.31 (d, J = 7.3 Hz, H_Ar, 1H), 8.26 (d, J = 6.9 Hz, H_Ar, 1H), 7.65 (s, 1H, H_Ar), 7.53 (d, 1H, J = 6.2 Hz, H_Ar), 7.48 (d, 1H, J = 6.9 Hz, H_Ar), 3.99 (s, H_{thiazolidinone}, 2H), 2.44 (s, H_CH3, 6H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 176.8, 176.2, 174.9, 161.5, 160.3, 156.8, 153.7, 152.1, 149.9, 146.2, 141.5, 134.2, 133.8, 131.5, 130.6, 126.1, 120.2, 115.4, 33.1, 29.6, 28.9; HREI-MS: m/z calcld for C₂₁H₁₆N₆OS₃, [M]⁺ 464.1765 Found 464.1753.

4.1.1.3. (Z)-2-((6-(5-(((E)-4-(trifluoromethyl)benzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (3)

FTIR (cm^-1) : 3297, 3193, 2812, 1780, 1540; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.76 (s, 1H, N-H), 8.89 (s, 1H, H_imine),8.32 (s, 1H, H_Ar), 8.08 (d, 1H, J = 7.4 Hz, H_Ar), 7.59 (d, J = 7.0 Hz, H_Ar, 2H), 6.64 (d, J = 7.2 Hz, H_Ar 2H), 6.55 (d, J = 7.0 Hz, H_Ar 1H), 4.33 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 171.1, 163.1, 159.0, 151.2 (q, J_CF = 37.8 Hz), 150.9, 145.6, 141.2 (d, J_CF = 4.7 Hz), 131.9, 130.4, 127.0, 126.9 (q, J_CF = 273.2 Hz), 122.3, 119.7, 119.7, 114.1, 114.0, 111.0, 104.6, 55.1; HREI-MS: m/z calcld for C₂₀H₁₁F₃N₆OS₃, [M]⁺ 504.9174, Found 504.9124.

4.1.1.4. (Z)-2-((6-(5-(((E)-4-chloro-3-methylbenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (4)

FTIR (cm^-1) : 3415, 3205, 2986, 1748, 1554; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.97 (s, 1H, N-H), 9.92 (s, 1H, H_imine), 8.78 (s, 1H, H_Ar), 8.49 (d, J = 7.3 Hz, H_Ar, 1H), 8.40 (d, J = 7.0 Hz, H_Ar, 1H), 8.09 (s, 1H, H_Ar), 7.91 (d, J = 7.2 Hz, H_Ar 1H), 7.83 (d, J = 7.3 Hz, H_Ar 1H), 4.08 (s, H_{thiazolidinone}, 2H), 3.95 (s, H_CH3, 3H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 181.8, 181.2, 179.4, 164.5, 163.9, 159.2, 157.8, 153.6, 153.4, 147.2, 146.2, 144.2, 140.8, 134.6, 133.9, 130.2, 121.2, 119.9, 39.6, 29.9; HREI-MS: m/z calcld for C₂₀H₁₃ClN₆OS₃, [M]⁺ 485.8854, Found 485.8843.

4.1.1.5. (Z)-2-((6-(5-(((E)-4-chloro-2-methylbenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (5)

FTIR (cm^-1) : 3391, 3223, 2993, 1730, 1521; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.05 (s, 1H, N-H), 10.00 (s, 1H, H_imine), 8.76 (s, 1H, H_Ar), 8.54 (d, J = 7.0 Hz, H_Ar, 1H), 8.45 (d, J = 6.7 Hz, H_Ar, 1H), 7.87 (s, 1H, H_Ar), 7.71 (d, J = 7.0 Hz, H_Ar 1H), 7.53 (d, J = 6.8 Hz, H_Ar 1H), 4.21 (s, H_{thiazolidinone}, 2H), 2.17 (s, 3H, H_CH3); ¹³C-NMR (150 MHz, DMSO-d₆): δ 171.2, 171.0, 170.6, 161.2, 160.9, 160.5, 154.2, 151.6, 151.0, 143.8, 142.9, 140.2, 140.0, 131.0, 130.9, 130.4, 127.5, 126.1, 120.1, 43.7, 29.4; HREI-MS: m/z calcld for C₂₀H₁₃ClN₆OS₃, [M]⁺ 484.8927, Found 484.8916.

4.1.1.6. (Z)-2-((6-(5-(((E)-3-(trifluoromethyl)benzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (6)

FTIR (cm^-1) : 3288, 3008, 2831, 1780, 1543; ¹H-NMR (600 MHz, DMSO-d₆): δ 13.34 (s, 1H, N-H), 9.95 (s, 1H, H_imine), 7.86 (d, J = 7.8 Hz, H_Ar 1H), 7.69 (t, J = 7.0 Hz, H_Ar 1H), 7.61 (s, 1H, H_Ar) 7.54 (d, J = 7.0 Hz, H_Ar 1H), 7.35 (dd, J = 7.1, 3.2 Hz, H_Ar, 1H), 7.10 (dd, J = 6.9, 1.7 Hz, H_Ar, 1H), 6.96 (s, 1H, H_Ar), 3.97 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 166.1, 163.1 (q, J_CF = 37.1 Hz), 149.8, 141.3 (d, J_CF = 3.9 Hz), 133.1, 132.2, 131.6, 131.6 (q, J_CF = 273.5 Hz), 131.5, 130.5, 127.8, 127.6, 122.4, 122.3 (q, J_CF = 273.5 Hz), 121.3, 120.9, 119.8 (d, J_CF = 23.2 Hz), 119.6, 111.0, 107.5, 55.2.8; HREI-MS: m/z calcld for C₂₀H₁₁F₃N₆OS₃, [M]⁺ 504.4968, Found 504.4742.

4.1.1.7. (Z)-2-((6-(5-(((E)-4-fluoro-2-hydroxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (7)

FTIR (cm^-1) : 3318, 3234, 3042, 1789, 1591; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.24 (s, 1H, N-H), 10.44 (s, 1H, H_OH), 7.84 (s, 1H, H_imine), 7.69(d, J = 6.7 Hz, H_Ar, 1H), 7.61 (d, J = 7.0 Hz, H_Ar, 1H), 7.54 (d, J = 7.0 Hz, H_Ar 1H), 7.08 (s, 1H, H_Ar),6.97 (s, 1H, H_Ar), 6.64 (d, J = 7.3 Hz, H_Ar 1H), 4.19 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 173.6, 165.8, 163.1 (d, J_CF = 249.3 Hz),158.7, 157.0, 153.0 (d, J_CF = 6.9 Hz),150.9, 147.7, 141.2, 137.2 (d, J_CF = 8.2 Hz), 131.8, 130.3, 122.8, 122.3, 119.7, 114.0, 111.8, 109.0, 109.0,55.4; HREI-MS: m/z calcld for C₁₉H₁₁FN₆O₂S₃, [M]⁺ 470.1743, Found 470.1311.

4.1.1.8. (Z)-2-((6-(5-(((E)-naphthalen-1-ylmethylene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (8)

FTIR (cm^-1) : 3443, 3216, 3067, 1781, 1540; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.16 (s, 1H, N-H), 9.72 (s, 1H, H_imine), 8.79 (dd, J = 7.3, 1.2 Hz, H_Ar 1H), 8.71 (s, 1H, H_Ar), 8.67 (dd, J = 7.0, 2.0 Hz, H_Ar 1H), 8.49 (dd, J = 7.0, 2.1 Hz, H_Ar, 1H), 8.32 (t, J = 6.9 Hz, H_Ar, 1H), 8.17 (dd, J = 7.3, 1.9 Hz, H_Ar 1H), 8.11 (dd, J = 6.8, 1.2 Hz, H_Ar 1H), 8.02 (d, J = 6.9 Hz, H_Ar, 1H), 7.99 (t, J = 7.1 Hz, H_Ar 1H), 7.81 (t, J = 7.5 Hz, H_Ar 1H), 4.20 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 176.8, 176.6, 175.2, 164.9, 161.2, 161.0, 159.2, 155.8, 152.9, 152.6, 150.8, 147.3, 142.3, 140.8, 139.2, 136.9, 133.4, 133.2, 126.5, 121.9, 120.0, 115.6, 44.3 HREI-MS: m/z calcld for C₂₃H₁₄N₆OS₃, [M]⁺ 486.0571, Found 486.0562.

4.1.1.9. (Z)-2-((6-(5-(((E)-4-phenoxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (9)

FTIR (cm^-1) : 3408, 3219, 3049, 1706, 1583; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.07 (s, 1H, N-H), 9.63 (s, 1H, H_imine), 8.61 (s, 1H, H_Ar), 8.37 (dd, J = 7.2 Hz, H_Ar, 2H), 8.31 (d, J = 7.3 Hz, H_Ar, 2H), 7.89 (d, J = 7.2 Hz, H_Ar 1H), 7.63 (d, J = 7.0 Hz, H_Ar 1H), 7.58 (dd, J = 7.2, 1.7 Hz, H_Ar 2H), 7.39 (t, J = 6.4 Hz, H_Ar 2H), 6.95 (m, J = 7.9 Hz, H_Ar 1H), 4.18 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 179.9, 178.8, 178.2, 163.8, 162.1, 160.7, 154.8, 153.9, 152.1, 143.8, 142.8, 136.9, 135.6, 133.7, 131.6, 123.7, 122.8, 117.8, 117.2, 116.8, 113.5, 106.8, 106.6, 42.4; HREI-MS: m/z calcld for C₂₅H₁₆N₆O₂S₃, [M]⁺ 528.6476, Found 528.6461.

4.1.1.10. (Z)-2-((6-(5-(((E)-3-chloro-4-fluorobenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (10)

FTIR (cm^-1) : 3435, 3291, 3076, 1749, 1532; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.10 (s, 1H, N-H), 9.54 (s, 1H, H_imine), 8.78 (s, 1H, H_Ar), 8.69 (d, J = 6.5 Hz, H_Ar, 1H), 8.54 (s, 1H, H_Ar), 8.50 (d, J = 6.7 Hz, H_Ar, 1H), 7.85 (d, J = 7.2 Hz, H_Ar 1H), 7.48 (d, J = 6.0 Hz, H_Ar 1H), 4.17 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 177.8, 177.0, 176.4, 165.7, 163.0 (d, J_CF = 247.6 Hz), 162.5, 159.4, 156.2, 154.3 (d, J_CF = 7.3 Hz), 149.8, 148.4, 143.2, 140.8, 139.0 (d, J_CF = 9.0 Hz), 135.3, 126.8, 123.9, 122.8, 42.8; HREI-MS: m/z calcld for C₁₉H₁₀FClN₆OS₃, [M]⁺ 488.4320, Found 488.4305.

4.1.1.11. (Z)-2-((6-(5-(((E)-3-bromo-4-chlorobenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (11)

FTIR (cm^-1) : 3468, 3309, 3041, 1752, 1545; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.13 (s, 1H, N-H), 10.09 (s, 1H, H_imine), 8.73 (s, 1H, H_Ar), 8.53 (d, J = 6.7 Hz, H_Ar, 1H), 8.38 (d, J = 7.0 Hz, H_Ar, 1H), 7.93 (d, J = 7.0 Hz, H_Ar 1H), 7.73 (s, 1H, H_Ar), 7.58 (d, J = 7.3 Hz, H_Ar 1H), 4.21 (s, H_{thiazolidinone}, 2H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 180.9, 180.4, 177.4, 165.8, 162.2, 160.4, 159.3, 155.2, 153.8, 150.3, 149.2, 141.8, 137.2, 136.9, 134.8, 126.1, 122.6, 118.1, 43.6; HREI-MS: m/z calcld for C₁₉H₁₀BrClN₆OS₃, [M]⁺ 549.8754, Found 549.8740.

4.1.1.12. (Z)-2-((6-(5-(((E)-2-bromo-4-chlorobenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (12)

FTIR (cm^-1) : 3383, 3234, 3032, 1781, 1540; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.90 (s, 1H, N-H), 9.72 (s, 1H, H_imine), 8.83 (s, 1H, H_Ar), 8.66 (s, 1H, H_Ar), 8.42 (d, J = 6.2 Hz, H_Ar, 1H), 8.27 (d, J = 7.0 Hz, H_Ar, 1H), 7.91 (d, J = 7.3 Hz, H_Ar 1H), 7.64 (d, J = 7.0 Hz, H_Ar 1H), 4.16 (s, H_{thiazolidinone}, 2H), 2.47 (s, H_CH3, 3H); ¹³C-NMR (150 MHz, DMSO-d₆): δ 178.2, 177.9, 177.2, 163.3, 161.8, 161.6, 155.2, 155.0, 153.1, 149.5, 149.1, 146.2, 139.8, 131.0, 130.8, 116.6, 114.9, 112.7, 41.8; HREI-MS: m/z calcld for C₁₉H₁₀BrClN₆OS₃, [M]⁺ 549.1361, Found 549.1349.

4.1.1.13. (Z)-2-((6-(5-(((E)-4-(dimethylamino)benzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (13)

FTIR (cm^-1) : 3412, 3245, 3072, 1743, 1521; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.11 (s, 1H, N-H), 9.96 (s, 1H, H_imine), 8.59 (s, 1H, H_Ar), 8.44 (d, J = 7.0 Hz, H_Ar, 2H), 8.36 (d, J = 6.4 Hz, 1H, H_Ar), 8.32 (d, J = 6.8 Hz, H_Ar, 1H), 8.09 (d, J = 7.1 Hz, H_Ar 2H), 3.88 (s, H_{thiazolidinone}, 2H), 3.05 (s, 6H, N(CH₃)₂); ¹³C-NMR (150 MHz, DMSO-d₆): δ 176.3, 175.9, 174.6, 164.2, 163.8, 162.9, 158.2, 153.4, 151.9, 151.6, 145.8, 139.2, 137.8, 132.9, 132.7, 129.1, 123.0, 119.2, 43.9, 36.5, 35.9; HREI-MS: m/z calcld for C₂₁H₁₇N₇OS₃, [M]⁺ 479.4379, Found 479.4361.

4.1.1.14. (Z)-2-((6-(5-(((E)-4-chloro-2-methoxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (14)

FTIR (cm^-1) : 3458, 3149, 3034, 1776, 1503; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.19 (s, 1H, N-H), 9.98 (s, 1H, H_imine), 8.68 (s, 1H, H_Ar), 8.42 (d, J = 6.2 Hz, H_Ar, 1H), 8.27 (d, J = 7.0 Hz, H_Ar, 1H), 7.91 (d, J = 7.3 Hz, H_Ar 1H), 7.73 (s, 1H, H_Ar), 7.64 (d, J = 7.0 Hz, H_Ar 1H), 4.32 (s, H_{thiazolidinone}, 2H); 3.66 (s, 3H, H_OCH3); ¹³C-NMR (150 MHz, DMSO-d₆): δ 180.7, 179.3, 178.9, 165.3, 163.7, 163.2, 157.5, 155.5, 154.4, 149.2, 146.3, 143.4, 137.8, 136.8, 136.2, 126.8, 124.1, 122.1, 41.3; HREI-MS: m/z calcld for C₂₀H₁₃ClN₆O₂S₃, [M]⁺ 500.8062, Found 500.8048.

4.1.1.15. (Z)-2-((6-(5-(((E)-4-bromo-2-methoxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (15)

FTIR (cm^-1) : 3431, 3247, 3095, 1745, 1541; ¹H-NMR (600 MHz, DMSO-d₆): δ 11.92 (s, 1H, N-H), 9.88 (s, 1H, H_imine), 8.81 (s, 1H, H_Ar), 8.67 (d, J = 7.2 Hz, H_Ar, 1H), 8.40 (d, J = 7.2 Hz, H_Ar, 1H), 8.13 (s, 1H, H_Ar),8.05 (d, J = 6.6 Hz, H_Ar 1H), 7.83 (d, J = 7.0 Hz, H_Ar 1H), 4.13 (s, H_{thiazolidinone}, 2H), 3.66 (s, 3H, H_OCH3); ¹³C-NMR (150 MHz, DMSO-d₆): δ 182.9, 180.3, 179.9, 165.5, 161.9, 161.0, 154.3, 153.9, 152.9, 144.8, 143.8, 141.9, 140.2, 134.7, 132.1, 127.4, 123.2, 117.2, 55.6, 44.3;HREI-MS: m/z calcld for C₂₀H₁₃BrN₆O₂S₃, [M]⁺ 545.1356, Found 545.1341.

4.1.1.16. (Z)-2-((6-(5-(((E)-2-methoxy-4-methylbenzylidene)amino)-1,3,4-thiadiazol-2-yl)benzo[d]thiazol-2-yl)imino)thiazolidin-4-one (16)

FTIR (cm^-1) : 3403, 3217, 3105, 1763, 1529; ¹H-NMR (600 MHz, DMSO-d₆): δ 12.10 (s, 1H, N-H), 10.14 (s, 1H, H_imine), 8.83 (s, 1H, H_Ar), 8.56 (d, J = 6.9 Hz, H_Ar, 1H), 8.38 (d, J = 7.2 Hz, H_Ar, 1H), 8.16 (s, 1H, H_Ar), 7.84 (d, J = 7.0 Hz, H_Ar 1H), 7.58 (d, J = 7.3 Hz, H_Ar 1H), 4.13 (s, H_{thiazolidinone}, 2H), 3.42 (s, 3H, H_OCH3), 2.36 (s, 3H, H_CH3); ¹³C-NMR (150 MHz, DMSO-d₆): δ 178.2, 177.9, 177.2, 163.3, 161.8, 161.6, 155.2, 155.0, 153.1, 149.5, 149.1, 146.2, 139.8, 131.0, 130.8, 116.6, 114.9, 112.7, 55.9, 41.8, 33.9; HREI-MS: m/z calcld for C₂₁H₁₆N₆O₂S₃, [M]⁺ 480.8034, Found 480.8021.

4.2. Biological activities

The synthesized compounds were investigated for their inhibitory potency against both AChE and BuChE enzymes and their binding interactions revealed effective inhibition. All the compounds were found with moderate to good inhibition (except non-active analog-8, 9, 11 and 12). Some of the derivatives revealed exceptional inhibitory potency, becoming the lead candidates. Structure–activity relationship for the synthesized derivatives was used as a medium to explain the binding interactions and their effectiveness which shows its dependence on nature, position, number, electron-donating and electron-donating substituents on the aromatic ring. Variation in biological potential of the analogs comes from the differently substituted phenyl ring is shown in the basic skeleton on the analogues. This substitution may be of -CF₃, -F, -OH, -Cl, -Br, -CH₃, -N(CH₃)₂ etc., which influence the inhibitory activity of the respective analogues against target enzymes as shown in Supplementary Table S1A.

4.3. In vitro acetylcholinesterase inhibition

All the synthesized analogs of thiazolidinone based thiadiazole derivatives were inquired for AChE inhibitory potentials by comparing IC₅₀ values in search of lead candidates. All the analogs displayed moderate to good potency against the enzyme. A range of IC₅₀ value from 0.90 ± 0.20 to 18.30 ± 0.10 μM was revealed by the analogs in series. Some of the derivatives were found with higher potential against AChE enzyme than the standard drug Donepezil (IC₅₀ = 4.20 ± 0.10 μM). The better potential was explained through structure–activity relationship which mainly depends on nature, number, position, electron-donating and electron-withdrawing groups substituted on the ring. Among all the synthesized analogs in the series, analog-3 (IC₅₀ = 0.90 ± 0.20 μM) was revealed to be at the top in inhibition against the enzyme. This effective inhibition of the analog-3 may be awarded due to the substitution of trifluoro moiety at the para position of the ring shown in Supplementary Figure S1A. The small sized and highly electronegative fluoro moieties withdraw electronic density from the ring which makes it more susceptible to be attacked by the electron rich moiety of the amino acid present on the enzyme. Analogue-3 binds via strong hydrogen bond interaction with the active site of the enzyme, which results in decline of enzyme activity. Thus, it was found to be the most potent derivative than the standard drug.

Analog-6 (IC₅₀ = 3.10 ± 0.70 μM) was at the second position in ranking in inhibitory potential as it bears trifluoro moiety at the meta-position shown in Supplementary Figure S1B. As the meta position of the ring has somewhat lower electronic density than the ortho and para position, as a result, inhibitory profile of the analogs also lowers.

Due to the presence of fluoro and chloro groups at para and meta-position respectively, as shown in Supplementary Figure S1C, analog-10 (IC₅₀ = 3.50 ± 0.20 μM) also showed better inhibitory potency as compared with the standard drug donepezil. Fluoro group present at the para position inhibits the enzyme through hydrogen bond interaction due to its high electronegativity and small size. Chloro group at the meta position also withdraws electronic density from the ring, which results in binding between the analogue and enzyme through effective interactions.

Moreover, among the fluoro substituted derivatives, analog-7 (IC₅₀ = +4.20 ± 0.2 μM) was also explored with better potential which may be attributed to the presence of fluoro moiety at para-position and hydroxyl group at ortho-position as shown in Supplementary Figure S1D, both functionalities involved in hydrogen bond and inhibit enzyme activity. Fluoro group (highly electronegative) at the meta position of the ring is capable of inhibiting the enzyme through hydrogen bond interaction, whereas, -OH group utilizes the active lone pair of electrons present on the oxygen atom and partial positively charged hydrogen atom for developing the inhibitory bindings (hydrogen bond).

Among the chloro-substituted derivatives, analog-5 (IC₅₀ = 7.60 ± 0.10 μM) displayed better potential due to presence of chloro moiety at para-position and methyl moiety at ortho-position than analog-4 (IC₅₀ = 8.20 ± 1.0 μM) which bears chloro group at para-position and bulky methyl group at meta-position as shown in Supplementary Figure S1E, gives rise to the steric hindrance and declines its inhibitory potential. As methyl group is an electron-donating moiety, the substitution of methyl group at the meta position is not very favorable due to lower electronic density at the meta position, thus activity of the analog is decreased.

Analog-2 (IC₅₀ = 9.40 ± 0.20 μM) bearing two methyl moieties, one at para and the other at meta-position was found with somewhat better potential than analog-1 (IC₅₀ = 11.10 ± 0.20 μM) which holds a single methyl moiety at para-position showed a declining potency. Methyl group is an electron donating functional moiety, the two methyl groups in compound-2 increase the electronic density on the ring which can bind with the electron deficient site of the amino acid and results in effective binding between the two. In case of compound-1, a single methyl group has lower potential to increase the electronic density on the ring, thus shows lower inhibition of the enzyme as shown in Supplementary Figure S1F.

Comparing the methoxy (-OCH₃) bearing derivatives, analog-14 (IC₅₀ = 13.70 ± 0.30 μM) revealed its better inhibition due to presence of methoxy group at ortho-position and chloro moiety at para position. Methoxy group is an electron-donating moiety which increases electronic cloud density on the phenyl ring. Besides, the oxygen atom of methoxy group can also inhibit the enzyme through hydrogen bond interaction. Electron-withdrawing chloro moiety also interacts via suitable interactions in order to lower the activity of enzyme. In analog-15 (IC₅₀ = 18.30 ± 0.10 μM) and analog-16 (IC₅₀ = 16.30 ± 1.30 μM), the presence of bromine and methyl moiety, respectively, lower the activity of the stated compounds due to the bulky substitution which hinders the interactions between the compound and the enzyme as shown in Supplementary Figure S1G.

4.4. In vitro butyrylcholinesterase inhibition

The synthesized compounds were also tested for inhibitory action against BuChE and were disclosed with effective and better inhibitory profile comparing with the standard drug Donepezil (IC₅₀ = 4.20 ± 0.10 μM). The inhibitory profile was ranging between IC₅₀ = 1.10 ± 0.40 μM and IC₅₀ = 18.70 ± 1.20 μM. Ranking at the top, analog-3 having IC₅₀ = 1.10 ± 0.40 μM was found highly potent in inhibition than the standard drug (IC₅₀ = 4.20 ± 0.10 μM). The better inhibition of analogue-3 is due to the formation of strong hydrogen bond between the active site of enzyme and trifluoro moiety at para-position of the ring.

Other compounds also displayed their inhibitory capabilities against the BuChE. Inhibitory potential of analog-10 (IC₅₀ = 4.20 ± 0.20 μM) was found to be the second higher than all other derivatives, which is due to presence of fluoro moiety at para-position involved in hydrogen bonding and a chloro moiety at the meta-position. Analog-6 having IC₅₀ = 4.50 ± 0.30 μM was also demonstrated as more effective than the standard drug, its effectiveness was declared due to presence of trifluoro moiety at the meta position which binds with the enzyme through hydrogen bond and lowers the enzyme activity. Among fluoro-substituted scaffolds, analog-7 with IC₅₀ = 4.50 ± 0.30 μM also exhibited better potency due to the presence of hydrogen bond making fluoro moiety at para-position and a hydroxyl (-OH) group at the ortho-position which increases the extent of hydrogen bonding interaction, elevating activity of the ring. Both fluoro and hydroxyl groups make the compound with better fitting into the active site of the enzyme. Chloro-substituted analog-5 (IC₅₀ = 8.20 ± 0.40 μM) was found better in inhibition than analog-4 (IC₅₀ = 11.20 ± 0.10 μM) due to the presence of bulky methyl group at ortho-position in prior and at meta-position in the latter one causing steric hindrance and decreasing the activity. Analog-2 with IC₅₀ = 10.20 ± 0.10 μM was found few folds better than analog-1 (10.20 ± 0.10 μM). The difference in inhibition was due to presence of two electron-donating methyl groups at meta- and para-position of the ring in analog-2 while a single methyl group only at the para -position of the ring showing a shrink in its activity. Analog-13 also manifested inhibitory potency against BuChE with an IC₅₀ = 14.20 ± 0.20 μM bearing a dimethylamino moiety at the para-position. Methoxy (-OCH₃) substituted analogs-14, 15 and 16 were also found active against the enzyme with IC₅₀ = 14.50 ± 0.20, 18.70 ± 1.20 and 17.70 ± 1.20 μM, respectively. Analog-14 was better due to the presence of methoxy moiety at ortho-position and chloro moiety at the para position.

Overall, it was figured out that analog-3 was highly potent against both AChE and BuChE enzymes than the standard drug Donepezil. The better inhibitory profile of the analog-3 is due to the presence of trifluoro functionality capable of making hydrogen bond which can inhibit the negative impact of enzymes, and a decline in enzyme activity was observed confirming the potential of the derivative.

4.5. In silico molecular docking studies

To explore the binding modes of newly afforded compounds with active site of AChE and BuChE enzymes, molecular docking study was conducted using different tools including Auto Dock Vina, Discovery studio Visualizer and Pymol. The proteins for docking study were retrieved from the online source RSCB Protein Data Bank using their codes. Docking procedure was completed after using the command prompt and nine different poses of all the ligands were obtained in a log file. The conformation with lowest binding affinity was made as a selection for complete visualization of protein–ligand interaction (PLI). Docking procedure and usage of docking tools adopted by our research group was elaborated in literature [29–38] (Figure 4).

Figure 4.

Basic skeleton of benzothiazole-based thiadiazole–thiazolidinone hybrid derivatives.

Docking results revealed the proper orientation of all the ligands in active site of both enzymes. Different substitutions on all the compounds had great impact on their inhibitory profile as shown in Supplementary Table S1B, and we found that compound-3 had the inhibitory potency at altitude in the entire series against AChE and BuChE. The PLI profile of analog-3 revealed the key interactions with catalytic residues which include Ile235, Glu233, Lys200, His201, Ala198, Leu162, Trp59 of against AChE (Figure 5) and catalytic residues including Ile235, Ala198, Leu162, Trp59, His201, Lys200, Glu233 against BuChE (Figure 6). The elevated inhibition of this analog might be due to the presence of hydrogen bond making trifluoro moiety having effective interaction with active site of the enzymes as shown in Figures 5 & 6.

Figure 8.

Protein–ligand interaction profile 3D (left) and 2D (right) of potent analog-6 against butyrylcholinesterase.

Figure 5.

Protein–ligand interaction profile 3D (left) and 2D (right) of potent analog-3 against acetylcholinesterase.

Figure 6.

Protein–ligand interaction profile 3D (left) and 2D (right) of potent analog-3 against butyrylcholinesterase.

Similarly key interactions of compound-6 were revealed in PLI profile, including catalytic residues Ala198, Leu162, Tyr151, His201, Lys200, Ile235, Trp59, Gln63 against AChE and catalytic residues including Ala198, Leu162, Tyr151, Lys200, His201, Ile235, Trp59, Gln63 against BuChE. The effective inhibition of compound-6 is due to the presence of trifluoro moiety on the ring developing hydrogen bond as shown in Figures 7 & 8.

Figure 7.

Protein–ligand interaction profile 3D (left) and 2D (right) of potent analog-6 against acetylcholinesterase.

The key interactions of compound-7 revealed in PLI profile, include catalytic residues Leu165, Trp59, Gln63, Ala198, Leu162, His201, against AChE and catalytic residues including Ala198, Leu162, His201, Leu 165, Trp59 against BuChE as shown in Supplementary Figure S1H & I.

Similarly, key interactions of compound-10 were revealed in PLI profile, including catalytic residues Glu233, His201, Ile235, Lys200, Tyr151, Leu162, Ala198, Trp59 against AChE and catalytic residues including His201, Ala198, Leu162, Leu165, Trp59, Glu63, Ile235 against BuChE as shown in Supplementary Figure S2C & D.

4.6. Absorption, distribution, metabolism & excretion-analysis

To assure certain feature such as absorption, distribution, metabolism, excretion and toxicity (ADMET) of the potent scaffolds, an online tool Swiss ADME was used. This research covers log Kp, Muegge violations, Lipinski, Brenk alerts, PAINS, Veber, gan, Ghose, Leadlikeness violations and Bioavailability score. We found significant findings for tested compounds, listed in Supplementary Graph I-1 to I-4

5. Conclusion

In this study we have synthesize benzothiazole derived thiazolidinone based thiadiazole derivatives (1–16). After spectroscopic confirmation these compounds were tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the presence of standard drug donepezil (IC₅₀ = 4.20 ± 0.10 and 5.30 ± 0.20 μM.). Few compounds were found with moderate to good inhibitory profiles but derivative 3 (IC₅₀ = 0.90 ± 0.20 and 1.10 ± 0.40 μM), 6 (IC₅₀ = 3.10 ± 0.70 and 4.50 ± 0.30 μM) and 10 (IC₅₀ = 3.50 ± 0.20 and 4.20 ± 0.20 μM) displayed excellent potentials which were further investigated through molecular docking studies to get their binding interaction with active sites of protein. ADMET analysis was studies for the selected compounds which show the drug likeness. Compound-3 emerged as the most potent inhibitor when compared with other derivatives of the series might be the due to trifluoro substituent which resist the enzyme activity by varied type of interactions.

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2366159

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.