Design and synthesis of new 1,2,3-triazole-methoxyphenyl-1,3,4-oxadiazole derivatives: selective butyrylcholinesterase inhibitors against Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) remains a significant public health challenge due to its progressive cognitive impairment and the absence of proven treatments. In this study, several novel 1,2,3-triazole-methoxyphenyl-1,3,4-oxadiazole derivatives were synthesized and evaluated for their ability to inhibit key enzymes associated with AD: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Structure-activity relationship (SAR) analysis revealed that derivatives featuring electron-withdrawing groups, particularly nitro and fluorine substituents, exhibited remarkable inhibitory activity against BChE while showing minimal effectiveness against AChE. Among these, compound 13s (R = 4-CH₃, R’ = 4-NO₂) demonstrated the highest potency, selectively targeting BChE with an IC₅₀ value of 11.01 µM. Molecular docking and molecular dynamics (MD) simulations provided deeper insights into the favorable interactions between these compounds and BChE. Additionally, cytotoxicity studies confirmed the active compound’s limited toxicity toward normal cells, indicating a promising therapeutic profile. These findings suggest that the synthesized selective anti-BChE compounds hold potential for consideration in the later stages of AD treatment.

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder primarily affecting the elderly, imposing profound burdens on patients, families, and healthcare systems. It is clinically characterized by memory loss, cognitive decline, and behavioral changes. Pathologically, AD is marked by the formation of neurofibrillary tangles, composed of hyperphosphorylated tau proteins, which lead to synaptic dysfunction and neuronal death [1, 2]. Another hallmark of the disease is the accumulation of amyloid-β (Aβ) plaques, produced by the cleavage of amyloid precursor protein (APP) via BACE1 and gamma-secretase [3].

The etiology of AD remains elusive, although factors such as genetics, aging, oxidative stress, and inflammation have been implicated [4]. The cholinergic hypothesis, one of the earliest theories in AD research, posits that a deficiency in acetylcholine (ACh), a neurotransmitter essential for learning and memory, plays a critical role in cognitive deficits observed in AD. This theory arose from evidence linking cholinergic deficits to the severity of cognitive impairment in AD patients [5, 6]. The loss of cholinergic neurons in regions critical for memory and learning, such as the basal forebrain and hippocampus, is a hallmark of AD. Acetylcholinesterase (AChE), which rapidly degrades ACh in the synaptic cleft, is central to the termination of neurotransmission [7, 8]. In contrast, butyrylcholinesterase (BChE), although less studied, becomes increasingly significant in the later stages of AD, regulating ACh levels in the brain.

In advanced AD, AChE activity decreases to 55–67% of normal levels, while BChE activity rises to 165% of normal levels in the brain [9]. Studies using AChE knockout mouse models suggest that BChE can compensate for AChE, maintaining normal cholinergic pathways [10]. Consequently, BChE inhibitors represent a promising therapeutic approach for restoring cholinergic function, potentially offering a synergistic benefit while minimizing peripheral side effects [11,12,13]. Based on this hypothesis, therapeutic strategies have focused on increasing ACh levels to mitigate cognitive deficits, leading to the development of AChE inhibitors such as donepezil, rivastigmine, and galantamine. These drugs aim to enhance cholinergic neurotransmission by preventing ACh degradation [14]. While they show modest benefits in stabilizing or improving cognitive function, they do not alter the underlying progression of AD and are associated with side effects like loss of appetite, nausea, diarrhea, and vomiting [15, 16].

Both natural products, such as alkaloids, coumarins, diarylheptanoids, flavonoids, phenanthrenes, terpenes, and xanthonoids [17,18,19,20,21], and synthetic compounds, including 1,2,3-triazoles [22], chromenones [23], indolinones [24], isoxazoles [25], and pyrazoles [26], have shown potential as cholinesterase inhibitors. This study investigates novel 1,2,3-triazole-methoxyphenyl-1,3,4-oxadiazole derivatives as dual inhibitors of AChE and/or BChE, aiming to enhance cholinergic function and provide innovative therapeutic options for Alzheimer’s disease (AD) management. The most potent compound was further examined for its kinetic properties, toxicity, and in silico evaluations, underscoring the importance of addressing cholinergic deficits that underlie cognitive impairments in AD.

Materials and methods

Synthesis

Melting points were determined using a Kofler hot stage apparatus and are uncorrected values. ¹H and ¹³C NMR spectra were recorded on a Bruker FT-500, using TMS as an internal standard. IR spectra were obtained using a Nicolet Magna FTIR 550 spectrophotometer (KBr disks). Elemental analysis was performed using an Elementar Analysensystem GmbH VarioEL CHNS mode.

General procedure for the preparation of Methyl 4-hydroxybenzoate (2)

A mixture of 4-hydroxybenzoic acid (10 mmol, 1) and sulfuric acid (1 mL) was refluxed in methanol (MeOH) for 24 h. Upon completion of the reaction, as confirmed by thin-layer chromatography (TLC), water was added to the reaction mixture. The resulting precipitate was filtered, washed with water, and dried to afford methyl 4-hydroxybenzoate (2) in a 92% yield.

General procedure for the preparation of Methyl 4-(prop-2-yn-1-yloxy)benzoate (4)

A mixture of methyl 4-hydroxybenzoate (5 mmol, 2), propargyl bromide (6 mmol, 3), and potassium carbonate (K₂CO₃, 5 mmol) was stirred in DMF at 80 °C for 24 h. Upon completion of the reaction, the mixture was poured into a water/ice mixture. The resulting precipitate was filtered, washed with water, and dried to yield compound 4 with an 85% yield.

General procedure for the preparation of compound 8

Initially, a mixture of benzyl bromide derivative (1.1 mmol, 5), sodium azide (NaN₃, 1 mmol, 6), and triethylamine (NEt₃, 1 mmol) was stirred in water and tert-butanol (t-BuOH) at room temperature for 1 h to generate the azide derivative 7 in situ. Subsequently, compound 4 (0.5 mmol), CuSO₄·5H₂O (10 mol%), and sodium ascorbate (20 mol%) were added to the reaction mixture, which was then stirred at room temperature for 24 h. The reaction’s progress was monitored by thin-layer chromatography (TLC). Upon completion, water was added to the mixture, and the precipitate was filtered, washed with water, and dried to yield product 8 with a 70% yield.

General procedure for the preparation of compound 10

A mixture of compound 8 (1 mmol) and hydrazine hydrate (1.5 mmol, 9) was refluxed in ethanol (12 mL) for 24 h. Upon completion, the reaction mixture was poured into a water/ice mixture. The resulting precipitate was filtered, washed with water, and dried to afford compound 10 with a yield of 67%.

General procedure for the preparation of compound 12

A mixture of the benzaldehyde derivative (1 mmol, 11), compound 10 (1 mmol), and a catalytic amount of acetic acid was refluxed in ethanol (12 mL) for 24 h. Upon completion, the reaction mixture was poured into a water/ice mixture. The resulting precipitate was filtered, washed with water, and dried to yield compound 12 with a 65% yield.

General procedure for the preparation of compound 13

A mixture of compound 12 (1 mmol), iodine (1.2 mmol), and K₂CO₃ (3 mmol) was stirred in dry DMSO at 100 °C for 24–48 h [27]. After completion, as confirmed by thin-layer chromatography (TLC), sodium thiosulfate solution (10%) was added to the reaction mixture. The product was then extracted using ethyl acetate, and the organic phase was dried over Na₂SO₄. The solvent was evaporated under vacuum, and the resulting precipitate was recrystallized from a mixture of n-hexane and ethyl acetate to yield compound 13 with a yield of 70%.

2-(4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-phenyl-1,3,4-oxadiazole (13a)

Yield: 94%; mp = 176–180 °C; IR (KBr): 3135, 2910, 1572 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.12 (d, J = 8.4 Hz, 2 H, H2′, H6′), 8.07 (d, J = 8.4 Hz, 2 H, H2, H6), 7.64–7.62 (m, 3 H, H3′, H4′, H5′), 7.37 (d, J = 7.0 Hz, 2 H, H3′′, H5′′), 7.33–7.32 (m, 3 H, H2′′, H4′′, H6′′), 7.28 (d, J = 8.4 Hz, 2 H, H3, H5), 5.67 (s, 2 H, CH₂), 5.27 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.3, 164.0, 161.3, 142.9, 136.4, 132.3, 129.8, 129.2, 129.0, 128.6, 128.4, 127.0, 125.3, 123.9, 116.4, 116.1, 61.8, 53.3 ppm. Anal. Calcd for C₂₄H₁₉N₅O₂: C, 70.40; H, 4.68; N, 17.10. Found: C, 70.51; H, 4.81; N, 17.26.

2-(4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (13b)

Yield: 83%; mp = 201–204 °C; IR (KBr): 3024, 2891, 1608, 1553, 1341 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.44 (d, J = 8.5 Hz, 2 H, H3′, H5′), 8.38 (d, J = 8.5 Hz, 2 H, H2′, H6′), 8.35 (s, 1H, triazole), 8.11 (d, J = 8.4 Hz, 2 H, H2, H6), 7.38 (d, J = 7.0 Hz, 2 H, H3′′, H5′′), 7.36–7.33 (m, 3 H, H2′′, H4′′, H6′′), 7.29 (d, J = 8.4 Hz, 2 H, H3, H5), 5.64 (s, 2 H, CH₂), 5.29 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.2, 162.8, 161.6, 149.5, 136.4, 129.5, 129.3, 129.2, 129.1, 128.6, 128.4, 128.3, 125.4, 125.1, 116.2, 116.1, 61.9, 53.3 ppm. MS (m/z, %): 91 (100), 144 (100), 172 (48), 283 (50), 454 (M⁺., 23). Anal. Calcd for C₂₄H₁₈N₆O₄: C, 63.43; H, 3.99; N, 18.49. Found: C, 63.17; H, 3.81; N, 18.28.

2-(4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (13c)

Yield: 85%; mp = 198–202 °C; IR (KBr): 3091, 2887, 1593 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.17 (dd, J = 8.7, 5.2 Hz, 2 H, H2′, H6′), 8.06 (d, J = 8.1 Hz, 2 H, H2, H6), 7.47 (t, J = 8.7 Hz, 2 H, H3′, H5′), 7.37 (d, J = 6.8 Hz, 2 H, H2′′, H6′′), 7.34–7.31 (m, 3 H, H3′′, H4′′, H5′′), 7.27 (d, J = 8.1 Hz, 2 H, H3, H5), 5.62 (s, 2 H, CH₂), 5.27 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.5, 164.4, 163.5, 162.3 (d, J_C−F = 247.2 Hz), 142.9, 136.4, 129.7 (d, J_C−F = 9.0 Hz), 129.2, 129.0, 128.6, 128.4, 125.4, 120.6, 117.1 (d, J_C−F = 22.3 Hz), 116.4, 116.1, 61.8, 53.3 ppm. Anal. Calcd for C₂₄H₁₈FN₅O₂: C, 67.44; H, 4.24; N, 16.38. Found: C, 67.16; H, 4.10; N, 16.45.

2-(4-((1-Benzyl-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (13d)

Yield: 86%; mp = 185–187 °C; IR (KBr): 3117, 2914, 1581 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.34 (s, 1H, triazole), 8.07–8.05 (m, 4 H, H2, H6, H2′, H6′), 7.38 (d, J = 7.1 Hz, 2 H, H3′′, H5′′), 7.35–7.32 (m, 3 H, H2′′, H4′′, H6′′), 7.27 (d, J = 8.5 Hz, 2 H, H3′, H5′), 7.17 (d, J = 8.4 Hz, 2 H, H3, H5), 5.63 (s, 2 H, CH₂), 5.27 (s, 2 H, CH₂), 3.87 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.3, 163.4, 162.5, 161.2, 140.8, 135.3, 129.5, 129.3, 128.9, 128.7, 128.4, 125.5, 116.3, 116.1, 115.9, 115.4, 61.8, 56.1, 53.3 ppm. Anal. Calcd for C₂₅H₂₁N₅O₃: C, 68.33; H, 4.82; N, 15.94. Found: C, 68.47; H, 4.70; N, 15.75.

2-(4-((1-(4-Fluorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-phenyl-1,3,4-oxadiazole (13e)

Yield: 82%; mp = 175–178 °C; IR (KBr): 3111, 2863, 1605 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.32 (s, 1H, triazole), 8.11 (d, J = 7.6 Hz, 2 H, H2′, H6′), 8.06 (d, J = 8.8 Hz, 2 H, H2, H6), 7.63–7.61 (m, 3 H, H3′, H4′, H5′), 7.40 (dd, J = 8.7, 5.6 Hz, 2 H, H2′′, H6′′), 7.26 (d, J = 8.8 Hz, 2 H, H3, H5), 7.20 (t, J = 8.7 Hz, 2 H, H3′′, H5′′), 5.61 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.4, 164.0, 162.3(d, J_C−F = 243.4 Hz), 161.3, 143.0, 132.7, 132.4, 130.7 (d, J_C−F = 8.2 Hz), 129.8, 129.0, 127.0, 125.3, 123.9, 116.5, 116.2, 116.1 (d, J_C−F = 21.5 Hz), 61.8, 52.5 ppm. Anal. Calcd for C₂₄H₁₈FN₅O₂: C, 67.44; H, 4.24; N, 16.38. Found: C, 67.62; H, 4.51; N, 16.11.

2-(4-((1-(4-Fluorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (13f)

Yield: 78%; mp = 190–193 °C; IR (KBr): 3110, 2903, 1597 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.34 (s, 1H, triazole), 8.18 (dd, J = 8.5, 5.1 Hz, 2 H, H2′, H6′), 8.07 (d, J = 8.3 Hz, 2 H, H2, H6), 7.47 (t, J = 8.5 Hz, 2 H, H3′, H5′), 7.42 (dd, J = 8.5, 5.5 Hz, 2 H, H2′′, H6′′), 7.28 (d, J = 8.3 Hz, 2 H, H3, H5), 7.22 (t, J = 8.5 Hz, 2 H, H3′′, H5′′), 5.63 (s, 2 H, CH₂), 5.28 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.5, 164.4, 163.5, 162.4 (d, J_C−F = 244.0 Hz), 162.3 (d, J_C−F = 248.5 Hz), 143.0, 132.7, 130.8 (d, J_C−F = 8.2 Hz), 129.7 (d, J_C−F = 8.9 Hz), 129.0, 125.3, 124.5, 117.1 (d, J_C−F = 22.4 Hz), 116.4, 116.2, 116.0 (d, J_C−F = 8.7 Hz), 61.8, 52.5 ppm. MS (m/z, %): 91 (100), 144 (100), 172 (27), 256 (26), 283 (32), 445 (M⁺., 29). Anal. Calcd for C₂₄H₁₇F₂N₅O₂: C, 64.72; H, 3.85; N, 15.72. Found: C, 64.86; H, 3.61; N, 15.83.

2-(4-((1-(4-Fluorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (13 g)

Yield: 82%; mp = 232–234 °C; IR (KBr): 3098, 2871, 1586 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.04–8.03 (m, 4 H, H2, H6, H2′, H6′), 7.41 (t, J = 7.0 Hz, 2 H, H3′′, H5′′), 7.26–7.19 (m, 4 H, H3′, H5′, H2′′, H6′′), 7.14 (d, J = 8.3 Hz, 2 H, H3, H5), 5.61 (s, 2 H, CH₂), 5.29 (s, 2 H, CH₂), 3.85 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.9, 164.3, 162.9, 160.3, 158.1 (d, J_C−F = 271.7 Hz), 143.0, 135.2, 132.8, 130.8, 130.7, 128.9, 125.3, 116.3, 116.2, 116.1, 115.3, 61.8, 56.0, 52.5 ppm. Anal. Calcd for C₂₅H₂₀FN₅O₃: C, 65.64; H, 4.41; N, 15.31. Found: C, 65.33; H, 4.30; N, 15.17.

2-(4-((1-(4-Chlorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-phenyl-1,3,4-oxadiazole (13 h)

Yield: 76%; mp = 160–163 °C; IR (KBr): 3115, 2910, 1604 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.35 (s, 1H, triazole), 8.14–8.08 (m, 3 H, H2′, H4′, H6′), 7.64 (d, J = 7.0 Hz, 2 H, H2, H6), 7.46–7.35 (m, 6 H, H3′, H5′, H2′′, H3′′, H5′′, H6′′), 7.29 (d, J = 7.0 Hz, 2 H, H3, H5), 5.64 (s, 2 H, CH₂), 5.29 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.1, 163.4, 161.3, 143.1, 135.4, 133.4, 132.4, 130.4, 129.9, 129.2, 129.0, 127.1, 125.5, 125.4, 116.5, 116.1, 61.8, 52.5 ppm. Anal. Calcd for C₂₄H₁₈ClN₅O₂: C, 64.94; H, 4.09; N, 15.78. Found: C, 64.81; H, 4.24; N, 15.63.

2-(4-((1-(4-Chlorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (13i)

Yield: 77%; mp = 223–227 °C; IR (KBr): 3104, 2895, 1612, 1551, 1348 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.42 (d, J = 8.3 Hz, 2 H, H3′, H5′), 8.36–8.33 (m, 3 H, H2′, H6′, triazole), 8.08 (d, J = 8.3 Hz, 2 H, H2, H6), 7.45 (d, J = 8.0 Hz, 2 H, H3′′, H5′′), 7.36 (d, J = 8.0 Hz, 2 H, H2′′, H6′′), 7.27 (d, J = 8.3 Hz, 2 H, H3, H5), 5.65 (s, 2 H, CH₂), 5.28 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.2, 163.8, 161.6, 149.6, 138.6, 135.4, 133.4, 130.4, 129.3, 128.3, 125.5, 125.0, 121.3, 119.2, 118.5, 116.1, 61.8, 52.5 ppm. Anal. Calcd for C₂₄H₁₇ClN₆O₄: C, 58.96; H, 3.50; N, 17.19. Found: C, 58.71; H, 3.27; N, 17.04.

2-(4-((1-(4-Chlorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (13j)

Yield: 81%; mp = 199–203 °C; IR (KBr): 3117, 2884, 1572 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.35 (s, 1H, triazole), 8.18 (dd, J = 8.4, 5.4 Hz, 2 H, H2′, H6′), 8.07 (d, J = 8.5 Hz, 2 H, H2, H6), 7.49–7.45 (m, 4 H, H3′, H5′, H3′′, H5′′), 7.37 (d, J = 8.1 Hz, 2 H, H2′′, H6′′), 7.28 (d, J = 8.5 Hz, 2 H, H3, H5), 5.65 (s, 2 H, CH₂), 5.29 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.5, 164.4, 163.5, 162.3 (d, J_C−F = 249.3 Hz), 143.1, 135.4, 133.4, 130.4, 129.7 (d, J_C−F = 9.0 Hz), 129.2, 129.1, 125.4, 120.6 (d, J_C−F = 3.2 Hz), 117.1 (d, J_C−F = 22.3 Hz), 116.4, 116.1, 61.8, 52.5 ppm. Anal. Calcd for C₂₄H₁₇ClFN₅O₂: C, 62.41; H, 3.71; N, 15.16. Found: C, 62.16; H, 3.87; N, 15.03.

2-(4-((1-(4-chlorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (13k)

Yield: 88%; mp = 202–205 °C; IR (KBr): 3095, 2873, 1581 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.06–8.03 (m, 4 H, H2, H6, H2′, H6′), 7.44 (d, J = 8.1 Hz, 2 H, H3′′, H5′′), 7.34 (d, J = 8.1 Hz, 2 H, H2′′, H6′′),7.25 (d, J = 8.4 Hz,2 H, H3′, H5′), 7.15 (d, J = 8.3 Hz, 2 H, H3, H5), 5.62 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂), 3.85 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.0, 163.9, 162.4, 161.2, 143.0, 135.4, 133.3, 130.4, 129.2, 128.9, 128.8, 125.4, 116.6, 116.3, 116.0, 115.3, 61.8, 56.0, 52.5 ppm. Anal. Calcd for C₂₅H₂₀ClN₅O₃: C, 63.36; H, 4.25; N, 14.78. Found: C, 63.20; H, 4.08; N, 14.96.

2-(4-((1-(4-Bromobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-phenyl-1,3,4-oxadiazole (13 L)

Yield: 84%; mp = 167–170 °C; IR (KBr): 3079, 2910, 1594 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.30 (s, 1H, triazole), 8.11 (d, J = 6.3 Hz, 2 H, H2, H6), 8.06 (d, J = 7.6 Hz, 2 H, H2′, H6′), 7.62–7.56 (m, 4 H, H3′, H5′, H3′′, H5′′), 7.37–7.27 (m, 5 H, H3, H5, H4′, H2′′, H6′′), 5.61 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.4, 164.0, 161.2, 142.9, 136.4, 135.8, 132.2, 130.7, 129.8, 129.2, 128.9, 128.4, 127.0, 123.9, 116.4, 116.1, 61.8, 52.6 ppm. Anal. Calcd for C₂₄H₁₈BrN₅O₂: C, 59.03; H, 3.72; N, 14.34. Found: C, 59.27; H, 3.50; N, 14.12.

2-(4-((1-(4-Bromobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (13 m)

Yield: 79%; mp = 226–228 °C; IR (KBr): 3096, 2859, 1597, 1554, 1347 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.42 (d, J = 8.4 Hz, 2 H, H3′, H5′), 8.36–8.33 (m, 3 H, H2′, H6′, triazole), 8.08 (d, J = 8.3 Hz, 2 H, H2, H6), 7.57 (d, J = 7.4 Hz, 2 H, H3′′, H5′′), 7.29–7.27 (m, 4 H, H3, H5, H2′′, H6′′), 5.61 (s, 2 H, CH₂), 5.27 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.2, 162.7, 161.1, 149.5, 142.9, 135.8, 132.2, 130.7, 129.5, 129.3, 128.3, 125.4, 125.0, 121.9, 116.2, 116.1, 61.8, 52.6 ppm. Anal. Calcd for C₂₄H₁₇BrN₆O₄: C, 54.05; H, 3.21; N, 15.76. Found: C, 54.19; H, 3.09; N, 15.89.

2-(4-((1-(4-Bromobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (13n)

Yield: 80%; mp = 191–195 °C; IR (KBr): 3112, 2915, 1581 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.17–8.15 (m, 2 H, H2′, H6′), 8.05 (d, J = 8.0 Hz, 2 H, H2, H6), 7.56 (d, J = 7.9 Hz, 2 H, H3′′, H5′′), 7.45 (t, J = 8.5 Hz, 2 H, H3′, H5′), 7.24–7.28 (m, 4 H, H3, H5, H2′′, H6′′), 5.60 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.5, 164.3, 163.5, 162.3 (d, J_C−F = 249.5 Hz), 143.1, 135.8, 132.2, 130.7, 129.7 (d, J_C−F = 8.7 Hz), 128.5, 125.4, 121.9, 120.6, 117.1 (d, J_C−F = 22.2 Hz), 116.4, 116.1, 61.8, 52.6 ppm. Anal. Calcd for C₂₄H₁₇BrFN₅O₂: C, 56.93; H, 3.38; N, 13.83. Found: C, 56.71; H, 3.49; N, 13.70.

2-(4-((1-(4-Bromobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (13o)

Yield: 73%; mp = 197–200 °C; IR (KBr): 3086, 2854, 1602 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.33 (s, 1H, triazole), 8.06–8.04 (m, 4 H, H2, H6, H2′, H6′), 7.58 (d, J = 8.0 Hz, 2 H, H3′′, H5′′), 7.30–7.26 (m, 4 H, H3′, H5′, H2′′, H6′′), 7.17 (d, J = 8.4 Hz, 2 H, H3, H5), 5.62 (s, 2 H, CH₂), 5.28 (s, 2 H, CH₂), 3.87 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.0, 163.9, 162.5, 161.2, 143.0, 135.8, 132.2, 130.7, 128.9, 128.8, 125.4, 122.0, 116.6, 116.3, 116.1, 115.3, 61.8, 56.0, 52.6 ppm. Anal. Calcd for C₂₅H₂₀BrN₅O₃: C, 57.93; H, 3.89; N, 13.51. MS (m/z, %): 91 (100), 144 (100), 171 (38), 268 (43), 454 (14), 517 (M⁺., 9), 519 ([M + 2]⁺., 9). Found: C, 57.82; H, 3.65; N, 13.79.

2-(4-((1-(4-Fluorobenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (13p)

Yield: 88%; mp = 220–224 °C; IR (KBr): 3088, 2875, 1610, 1550, 1347 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.45 (d, J = 8.7 Hz, 2 H, H3′, H5′), 8.38 (d, J = 8.7 Hz, 2 H, H2′, H6′), 8.35 (s, 1H, triazole), 8.11 (d, J = 8.7 Hz, 2 H, H2, H6), 7.42 (dd, J = 8.7, 5.5 Hz, 2 H, H2′′, H6′′), 7.29 (d, J = 8.7 Hz, 2 H, H3, H5), 7.22 (t, J = 8.7 Hz, 2 H, H3′′, H5′′), 5.63 (s, 2 H, CH₂), 5.29 (s, 2 H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.2, 162.8, 162.4 (d, J_C−F = 248.1 Hz), 161.6, 149.6, 143.0, 132.7, 130.8 (d, J_C−F = 8.5 Hz), 129.5, 129.3, 128.4, 125.3, 125.1, 116.2, 116.1, 116.0 (d, J_C−F = 17.2 Hz), 61.9, 52.5 ppm. Anal. Calcd for C₂₄H₁₇FN₆O₄: C, 61.02; H, 3.63; N, 17.79. Found: C, 61.25; H, 3.74; N, 17.61.

2-(4-((1-(4-Methylbenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-phenyl-1,3,4-oxadiazole (13q)

Yield: 82%; mp = 223–226 °C; IR (KBr): 3104, 2886, 1571 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.30 (s, 1H, triazole), 8.13–8.12 (m, 2 H, H2′, H6′), 8.07 (d, J = 7.0 Hz, 2 H, H2, H6), 7.64–7.63 (m, 3 H, H3′, H4′, H5′), 7.27 (d, J = 8.5 Hz, 2 H, H2′′, H6′′), 7.23 (d, J = 8.5 Hz, 2 H, H3′′, H5′′), 7.18 (d, J = 7.0 Hz, 2 H, H3, H5), 5.57 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂), 2.27 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.4, 164.0, 161.3, 138.0, 133.4, 132.3, 129.8, 129.7, 129.0, 128.5, 127.0, 126.5, 125.2, 123.9, 116.4, 116.1, 61.8, 53.1. 21.1 ppm. Anal. Calcd for C₂₅H₂₁N₅O₂: C, 70.91; H, 5.00; N, 16.54. Found: C, 71.03; H, 5.15; N, 16.41.

2-(4-Fluorophenyl)-5-(4-((1-(4-methylbenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-1,3,4-oxadiazole (13r)

Yield: 80%; mp = 204–207 °C; IR (KBr): 3103, 2879, 1573 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.26 (s, 1H, triazole), 7.83 (d, J = 8.0 Hz, 2 H, H2′, H6′), 7.22–7.16 (m, 8 H, H2, H6, H3′, H5′, H2′′, H3′′, H5′′, H6′′), 7.06 (d, J = 8.3 Hz, 2 H, H3, H5), 5.54 (s, 2 H, CH₂), 5.18 (s, 2 H, CH₂), 2.26 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 167.8, 164.8, 162.5 (d, J_C−F = 247.5 Hz), 160.8, 142.6, 138.0, 133.4, 129.8, 129.7, 128.5, 127.3, 125.1, 124.4, 116.8, 116.3, 114.6, 61.6 53.1, 21.1 ppm. Anal. Calcd for C₂₅H₂₀FN₅O₂: C, 68.02; H, 4.57; N, 15.86. Found: C, 68.31; H, 4.42; N, 15.67.

2-(4-((1-(4-Methylbenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (13s)

Yield: 80%; mp = 207–209 °C; IR (KBr): 3108, 2874, 1612, 1553, 1351 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.41(d, J = 8.4 Hz, 2 H, H3′, H5′), 8.33 (d, J = 8.4 Hz, 2 H, H2′, H6′), 8.30 (s, 1H, triazole), 8.06 (d, J = 8.1 Hz, 2 H, H2, H6), 7.26 (d, J = 8.1 Hz, 2 H, H3, H5), 7.23 (d, J = 7.3 Hz, 2 H, H2′′, H6′′), 7.17 (d, J = 7.3 Hz, 2 H, H3′′, H5′′), 5.57 (s, 2 H, CH₂), 5.26 (s, 2 H, CH₂), 2.26 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 165.2, 162.7, 161.6, 149.5, 138.0, 133.4, 132.4, 129.8, 129.5, 129.3, 128.5, 128.3, 125.2, 125.0, 116.1, 116.0, 61.9, 53.2, 21.1 ppm. MS (m/z, %): 91 (100), 144 (100), 171 (27), 268 (35) 454 (12), 468 (M⁺., 5). Anal. Calcd for C₂₅H₂₀N₆O₄: C, 64.10; H, 4.30; N, 17.94. Found: C, 64.35; H, 4.17; N, 17.81.

2-(4-Methoxyphenyl)-5-(4-((1-(4-methylbenzyl)-1 H-1,2,3-triazol-4-yl)methoxy)phenyl)-1,3,4-oxadiazole (13t)

Yield: 82%; mp = 177–179 °C; IR (KBr): 3112, 2895, 1574 cm^− 1. ¹H NMR (500 MHz, DMSO-d₆): 8.29 (s, 1H, triazole), 8.06–8.03 (m, 4 H, H2, H6, H2′, H6′), 7.27–7.22 (m, 4 H, H2′′, H3′′, H5′′, H6′′), 7.18–7.15 (m, 4 H, H3, H5, H3′, H5′), 5.56 (s, 2 H, CH₂), 5.25 (s, 2 H, CH₂), 3.86 (s, 3 H, CH₃), 2.27 (s, 3 H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d₆): 164.0, 163.9, 162.5, 161.2, 142.9, 138.0, 133.4, 129.8, 128.9, 128.8, 128.5, 125.2, 116.6, 116.3, 116.1, 115.3, 61.8, 56.0, 53.1, 21.1 ppm. Anal. Calcd for C₂₆H₂₃N₅O₃: C, 68.86; H, 5.11; N, 15.44. Found: C, 68.59; H, 5.01; N, 15.53.

AChE and BChE inhibition assay

Acetylthiocholine iodide (ATCI), butyrylcholinesterase (BChE, E.C. 3.1.1.8, from horse serum), acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 units), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), potassium hydroxide, sodium hydrogen carbonate, potassium dihydrogen phosphate, and potassium hydrogen phosphate were procured from Sigma-Aldrich for use in Ellman’s test. The synthesized compounds were dissolved in DMSO and further diluted with methanol. In a 96-well plate, 25 µL of the compound solution, 50 µL of potassium phosphate buffer (0.1 M, pH 8), and 25 µL of enzyme solution (final concentration of 0.22 U/mL) were added to each well. The plate was incubated at room temperature for 15 min. Subsequently, 125 µL of DTNB (3 mM in buffer) and either acetylthiocholine iodide or butyrylthiocholine iodide (3 mM in water) were added. Finally, after another 15 min, the absorbance at 405 nm was measured, and the IC₅₀ values were determined using inhibition curves [28].

Kinetic studies

Ellman’s approach for the inhibition assay was followed in the kinetic investigation of compound 13s inhibition of BChE, using four different inhibitor doses. For the BChE kinetic investigation, compound 13s was used at 0, 5, 20, and 40 µM dosages. The Lineweaver–Burk reciprocal plot was created by plotting 1/V against 1/[S] at different substrate butyrylthiocholine iodide dosages (187.5, 750, 1500, and 3000 µM) [28].

Molecular docking

Induced fit docking of compound 13s was carried out using the Schrödinger Suites Maestro molecular modeling platform. X-ray crystallographic structures of BChE (4BDS) were retrieved from the RCSB Protein Data Bank (www.rcsb.org). Protein preparation was conducted using the Protein preparation Wizard, where co-crystallized atoms and water molecules were removed to refine the protein structures. Missing loops were added, and terminals were sealed using the Prime tool. Heteroatom states were generated at pH 7.4 using EPIK, and hydrogen-bonding interactions were assigned at the same pH using PROPKA.

The ligand structure was created in ChemDraw and saved as SDF files, which were optimized using the OPLS3e force field and the LigPrep tool. The docking box was set to a size of 20 Å, with an energy window of 2.5 kcal/mol. Up to 20 poses were generated with receptor and ligand van der Waals radii set to 0.7 and 0.5, respectively. Residues within an 8 Å radius of the crystallographic ligands at the active site were refined, followed by side-chain optimization [29].

MD simulation

The best pose of the induced fit docking of BChE was selected for molecular dynamics (MD) simulation using Desmond v5.3 from Schrödinger’s suite Maestro. The 13s–ligand complex was solvated with explicit SPC water molecules and positioned at the center of an orthorhombic box under periodic boundary conditions. Counterions from a 0.15 M NaCl solution were added to neutralize the system, mimicking real cellular ionic concentrations.

The MD protocol consisted of three steps: minimization, pre-production, and production MD simulations. The system was first relaxed by performing energy minimization using the steepest descent approach for 2500 steps. To prevent abrupt changes, a small force constant was applied to the enzyme as the system temperature was gradually increased from 0 to 300 K. MD simulations were conducted under the NPT ensemble, maintaining a constant number of atoms, pressure (1.01325 bar), and temperature (300 K), utilizing the Nose–Hoover chain thermostat as the default.

Long-range electrostatic interactions were calculated using the particle-mesh Ewald method, with the cutoff radius for Coulombic interactions set to 9.0 Å. The protein-ligand complex underwent 100 ns of production MD simulations [26, 30].

Cell viability

SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle Medium with Ham’s F12 medium (DMEM/F12), supplemented with 15% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were seeded into flasks containing the medium and maintained at 37 °C in a humidified atmosphere with 5% CO₂. Cell viability was assessed using a quantitative colorimetric MTT assay. Cells were plated in 96-well microplates at 100 µL per well and incubated overnight at 37 °C. After incubation, half of the growth medium was replaced with 50 µL of fresh medium containing various concentrations of synthetic compounds dissolved in DMSO. The cells were then incubated for an additional 72 h [31]. At the end of this period, MTT reagent was added to each well at a final concentration of 0.5 mg/mL. The plate was further incubated in a humidified atmosphere for an additional 2 h.

Metabolically active cells reduced the yellow MTT tetrazolium compound to purple formazan crystals. Subsequently, the insoluble formazan was dissolved using dimethyl sulfoxide (DMSO), and the colorimetric determination of MTT reduction was performed by measuring absorbance at 540 nm. Control cells treated with media alone were considered as 100% viability [28, 32].

Prediction of ADME descriptors

The ADME properties were calculated using online servers, https://preadmet.bmdrc.kr and https://biosig.lab.uq.edu.au/pkcsm/.

Result and discussion

Design of 1,2,3-triazole-methoxyphenyl-1,3,4-oxadiazole derivatives

Aryloxadiazole derivatives are among the most selective cholinesterase (ChE) inhibitors. N-benzylpiperidines fused with 1,3,4-oxadiazole were developed as multi-target agents against Alzheimer’s disease (AD), demonstrating high potency against both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The presence of the 1,3,4-oxadiazole moiety enhanced binding affinity to the peripheral anionic site (PAS). Moreover, these compounds exhibited no toxicity against SH-SY5Y neuroblastoma cell lines in MTT assays and showed favorable blood-brain barrier permeability.

Compound A (Fig. 1) outperformed donepezil in efficacy at tested doses in the Morris water maze test using an Aβ-induced AD phenotypic model. Its pharmacokinetic evaluation revealed excellent oral absorption characteristics, encouraging further exploration of aryl 1,3,4-oxadiazole derivatives in subsequent studies [33]. Building on these findings, researchers designed a potent cholinesterase inhibitor, compound B, by substituting piperidine with pyridine. This modification enabled compound B to disaggregate Aβ plaques effectively. Ex vivo studies on hippocampal rat brain homogenates confirmed its AChE inhibitory activity and notable antioxidant potential. In silico molecular docking and molecular dynamics (MD) simulations supported its strong binding affinity and interactions with active site residues [34].

A pyridine-oxadiazole scaffold demonstrated exceptional in vitro enzyme inhibitory activity at nanomolar concentrations against both AChE and BChE, surpassing rivastigmine. In vivo and ex vivo assessments of lead compound C highlighted significant anti-AD effects, including reduced levels of lipid peroxidation and glutathione (GSH), normalized levels of 8-OHdG, and substantial decreases in β-amyloid protein, a hallmark of AD. Among the compounds studied, compound C emerged as the most effective dual inhibitor of AChE (IC₅₀ = 50.87 nM) and BChE (IC₅₀ = 4.77 nM) [35].

Fig. 1

Structural examples and rationale for phenyl oxadiazole–phenoxy methylene 1,2,3-triazole hybrid design

The potency of aryl 1,2,3-triazoles has been demonstrated in numerous studies. Notably, 1,2,3-triazole–arylcoumarin derivatives have shown exceptional BChE inhibitory activity, with one of the most potent inhibitors (Compound D) exhibiting neuroprotective effects that surpassed the standard drug quercetin in the PC12 cell model injured by H₂O₂. In silico studies further validated the interaction between the phenoxy-1,2,3-triazole linker and the catalytic triad of BChE [36]. Additionally, the high potency of phenoxy-methylene-1,2,3-triazole derivatives has been confirmed in other studies, including compounds E [37] and F [38].

In the current study, a molecular hybridization strategy was utilized to design and synthesize novel compounds by linking phenoxy-methylene-triazole with aryl-oxadiazole scaffolds. To comprehensively investigate the structure-activity relationship (SAR) against AChE and BChE, strategic substitutions were introduced at two key positions: the aryl group attached to the oxadiazole moiety, and the benzyl group connected to the 1,2,3-triazole.

Chemistry

Scheme 1

Synthesis of compound 13

The synthesis of the target compound 13 began with 4-hydroxybenzoic acid (1), which underwent esterification under acidic conditions followed by propargylation under basic conditions to yield compounds 3 and 4, respectively. Subsequently, a click reaction between compound 4 and freshly prepared benzyl azides 7-synthesized from the reaction of benzyl bromide derivative (5) and sodium azide (6)-was carried out in the presence of catalytic amounts of CuSO₄·5H₂O, leading to the formation of compound 8.

The reaction of compound 8 with hydrazine hydrate (9) under refluxing ethanol conditions produced the hydrazide derivative 10, which was further reacted with various aromatic aldehydes 11 in the presence of acetic acid under reflux in ethanol to give compound 12. Finally, cyclization and the formation of the 1,3,4-oxadiazole moiety were achieved via treatment with iodine and potassium carbonate in DMSO at 100 ˚C, yielding the corresponding derivatives 13 (Scheme 1).

The target compounds were obtained in excellent yields in the final step (73–94%), and their structures were confirmed using IR, NMR spectroscopy, and mass spectrometry. In the IR spectra, key peaks were observed around 3100 and 2850 cm⁻¹, corresponding to the C–H stretching vibrations of sp²- and sp³-hybridized carbons, respectively.

The ¹H NMR spectra typically exhibited a singlet peak around 8.30 ppm, indicative of the proton on the 1,2,3-triazole ring. Phenoxy protons (H2, H3, H5, and H6) were represented by two doublets with an integration of 2. H2 and H6 appeared near 8.00 ppm, while H3 and H5 were observed around 7.25 ppm, with the latter being more shielded due to resonance with the ortho-positioned oxygen. Protons from two aryl moieties appeared within the range of 7.00–8.50 ppm, depending on the electronic properties of the substituents. Furthermore, the two methylene group protons linked to the 1,2,3-triazole ring were identified at approximately 5.00 ppm. In the ¹³C NMR spectra, the most deshielded signals were associated with the isoxazole carbons, the carbon linked to the oxygen in the phenoxy moiety, and C4 of the 1,2,3-triazole ring, appearing in the range of 160–140 ppm. Other carbons were detected within the range of 115–139 ppm, while the signals for the two methyl groups were observed around 50–60 ppm. Additionally, the molecular weights of the synthesized compounds were confirmed by mass spectrometry.

In vitro cholinesterase inhibitory activity

The data for the series of compounds 13a–t provides valuable insights into the structure-activity relationship (SAR) regarding their inhibitory activities against AChE and BChE (Table 1). The SAR analysis focuses on the impact of various substitutions on the aromatic rings (R and R’) on the potency of these compounds, with particular emphasis on their efficacy against BChE.

The AChE inhibition results for this series indicate weak or negligible activity, with IC₅₀ values exceeding 100 µM. This finding suggests that the structural modifications in these derivatives are not conducive to AChE inhibition. In contrast, several compounds exhibit moderate to significant BChE inhibition, with IC₅₀ values ranging from 11.01 µM to over 100 µM. The most potent analogs identified in the series are 13s, 13f, and 13b.

BChE inhibitory activity

Substituents with hydrogen at R (R = H)

Compounds 13a (R’ = H) and 13d (R’ = 4-MeO) demonstrated no significant inhibition of BChE, with IC₅₀ values exceeding 100 µM. This indicates that the electron-donating methoxy group at R’ is not favorable for BChE inhibition.

In contrast, the introduction of fluorine, an electron-withdrawing and small substituent, in compound 13c (R’ = 4-F) led to enhanced BChE inhibitory activity, achieving an IC₅₀ value of 33.62 µM. Furthermore, the presence of a nitro group, another electron-withdrawing substituent, in compound 13b (R’ = 4-NO₂) significantly improved inhibitory potency. This enhancement is likely attributed to stronger interactions with the enzyme, suggesting that benzyl groups with electron-withdrawing substitutions are more effective in inducing potent anti-BChE activity.

Substituents with fluorine at R (R = 4-F)

Similar to the previous set, both the unsubstituted derivative 13e (R’ = H) and the electron-donating methoxy group in compound 13g (R’ = 4-MeO) exhibited no significant BChE inhibition (IC₅₀ > 100 µM). This result underscores that fluorine alone at R is insufficient to induce potent inhibition without a complementary electron-withdrawing substituent at R’. In contrast, compound 13f (R’ = 4-F) demonstrated significant BChE inhibition, achieving an IC₅₀ value of 12.86 µM. The presence of fluorine at both R and R’ appeared to facilitate favorable interactions with BChE, likely due to the chemical properties of fluorine, such as its electron-withdrawing capability and small size, enhancing binding efficiency at the enzyme’s active site.

Substituents with Chlorine at R (R = 4-Cl)

Both 13h (R’ = H) and 13k (R’ = 4-MeO) were inactive against BChE, with IC₅₀ values exceeding 100 µM. This finding indicates that chlorine alone at R is insufficient to drive significant BChE inhibition without the presence of a strong electron-withdrawing group at R’. In contrast, compounds 13j (R’ = 4-F; IC₅₀ = 28.43 µM) and 13i (R’ = 4-NO₂; IC₅₀ = 30.50 µM) exhibited notable BChE inhibitory activity. Among these, the fluorine substituent at R’ demonstrated slightly higher potency than the nitro group, suggesting its favorable effect on enzyme interactions.

Substituents with bromine at R (R = 4-Br)

Interestingly, 13l (R’ = H) exhibited no significant BChE inhibition (IC₅₀ > 100 µM), indicating that the absence of substituents on the aryl group connected to the oxadiazole moiety is unfavorable for activity. As anticipated, 13n (R’ = 4-F) demonstrated good inhibitory activity with an IC₅₀ of 20.32 µM, suggesting that the presence of fluorine enhances BChE interactions when paired with bromine.

A similar trend was observed for 13m (R’ = 4-NO₂), which showed potent BChE inhibition with an IC₅₀ of 16.63 µM, emphasizing the significant role of the strong electron-withdrawing nitro group at R’. Notably, 13o (R’ = 4-MeO) was the only active derivative featuring an electron-donating group at R’. While methoxy groups typically show lower efficacy in other sets, their combination with bromine in this case yielded noteworthy BChE inhibition with an IC₅₀ of 14.56 µM.

Substituents with Methyl at R (R = 4-CH₃)

Notably, compound 13s (R’ = 4-NO₂) emerged as the most potent BChE inhibitor in the series, with an IC₅₀ of 11.01 µM. This low IC₅₀ value highlights that the combination of a methyl group at R and a nitro group at R’ is particularly effective for enhancing BChE inhibitory activity. In contrast, other analogs, such as 13r (R’ = 4-F), 13q (R’ = H), and 13t (R’ = 4-MeO), exhibited poor inhibition (IC₅₀ > 100 µM), suggesting that these substitutions are less effective when paired with a methyl group at R.

The observed variations in potency underscore the crucial influence of specific substitutions at positions R and R’ on BChE inhibitory activity. In most cases, electron-withdrawing groups at R’ significantly enhanced potency. A notable exception was compound 13s, which, despite bearing an electron-donating nitro group at R’, displayed remarkable potency.

Given the limited AChE inhibition across the series, these compounds appear to exhibit greater selectivity towards BChE. This selectivity may hold therapeutic value in the later stages of AD, where BChE activity becomes more pronounced. While the current series demonstrated minimal AChE activity, the structural framework could be expanded to incorporate functional groups that might engage both AChE and BChE, offering the potential for dual inhibition and broader therapeutic applicability.

The synthesized compounds exhibited low activity towards AChE, highlighting their selectivity for BChE. Selective BChE inhibition could provide therapeutic advantages, particularly in late-stage AD, where AChE levels decline while BChE activity becomes significantly elevated. Additionally, BChE has been linked to abnormal Aβ deposition, and immunohistochemical analyses have revealed Aβ plaques in regions with elevated BChE concentrations. These findings emphasize the importance of targeting BChE as a potential therapeutic strategy [39].

It is important to consider whether dual inhibition of both AChE and BChE might be more advantageous in the early stages of AD, where AChE activity predominates. Selective BChE inhibition may have limited effectiveness at this stage, suggesting that dual AChE/BChE inhibition could offer a more effective therapeutic approach [40]. Furthermore, BChE plays a role in drug metabolism by hydrolyzing esters and xenobiotics, meaning its inhibition might lead to potential side effects. Thus, achieving an optimal dosage and designing inhibitors with lower IC₅₀ values and appropriate potency represent more agreeable therapeutic strategies. Tacrine, an earlier reversible inhibitor of both AChE and BChE, was among the first drugs targeting AD. Rivastigmine, an FDA-approved dual AChE/BChE inhibitor, has demonstrated efficiency in the later stages of AD, where elevated BChE activity becomes significant [41].

Table 1 In vitro cholinesterase inhibitory activity of compound 13a-t^a

Kinetic study

The kinetic study was conducted to elucidate the mechanism of BChE inhibition by compound 13s. Graphical analysis using the reciprocal Lineweaver–Burk plot (Fig. 2) revealed a competitive inhibition pattern. This was evidenced by an increase in the K_m value with rising concentrations of the inhibitor, indicating that compound 13s competes with the substrate for binding to the active site.

Fig. 2

Lineweaver-Burk plot for the inhibition of BChE by compound 13s at different concentrations of the substrate (ATCh). The secondary plot of slopes of the lines vs. various concentrations of 13s

Molecular docking study

Given the high potency of compound 13s, a molecular docking study was conducted to elucidate its mode of action and interactions within the active site of BChE. The binding site of BChE comprises the peripheral anionic site (PAS), including residues Asp70 and Tyr332, and the acyl binding pocket at the end of the site, formed by Leu286 and Val288. Additionally, the catalytic triad consists of Ser198, Glu325, and His438, while the oxyanion hole in the middle of the binding site is formed by Ala199, Gly117, and Gly116. Targeting these critical pockets is believed to enhance inhibitor potency.

The docking protocol was validated by re-docking the co-crystallized ligand into the enzyme’s binding site, achieving a root-mean-square deviation (RMSD) of less than 2 Å for the lowest energy pose, confirming the reliability of the methodology.

To gain further insights into the interactions of the most potent analog, molecular docking was also performed for donepezil. Donepezil was positioned in the BChE active site with a binding energy of -7.243 kcal/mol. The methoxy group attached to 2,3-dihydro-1H-inden-1-one formed a hydrogen bonding interaction with His438 of the catalytic triad. The piperidine-4-yl moiety established a salt bridge and pi-cation interactions with Asp70 and Tyr332 in the PAS. Additionally, the terminal benzyl group exhibited pi-pi stacking interactions with Trp82 (Fig. 3.

Fig. 3

3D and 2D interaction pattern of donepezil within the active site of BChE

Fig. 4

3D and 2D interaction pattern of 13s within the active site of BChE

The results of the molecular docking of compound 13s are presented in Fig. 4, showing a docking score of -9.955 kcal/mol. The nitro group formed two pi-cation interactions with Trp231 and Phe329. Additionally, the nitroaryl group established two pi-pi stacking interactions with Trp231. The 1,3,4-oxadiazole ring contributed two hydrogen bonding interactions: one with Gly117 in the oxyanion hole and another with Ser198 in the catalytic triad. Furthermore, this ring exhibited a pi-pi stacking interaction with His438 of the catalytic triad, a feature also observed in the interaction between donepezil and the BChE complex.

The phenoxy linker in compound 13s engaged in several pi-pi stacking interactions with His438 and Trp82, as well as a pi-cation interaction with His438. On the opposite side of the molecule, the 4-methylbenzylthiazole-1,2,3-triazole moiety exhibited two pi-pi stacking interactions with Trp82, a feature also observed in the interaction of donepezil with BChE. These findings highlight that all critical pockets of BChE were effectively engaged by the potent inhibitor, thereby justifying its high potency.

Considering the similar binding pose of compound 13s and donepezil in the active site, along with their shared ability to interact with the catalytic triad, 13s demonstrates significant potential as a potent anti-BChE agent.

Given that compound 13s is not classified as a potent AChE inhibitor, a molecular docking study was conducted to validate this observation. The docking study yielded a score of -7.268 kcal/mol. The analysis revealed that compound 13s exhibited only a π-π stacking interaction with Tyr124 in the peripheral anionic site (PAS) pocket of AChE (Fig. 5).

Fig. 5

3D and 2D interaction pattern of 13s within the active site of AChE

Molecular dynamics study

A molecular dynamics (MD) study was performed to compare the stability of BChE in complex with compound 13s against BChE in its unbound state. The root mean square deviation (RMSD) of backbone atoms served as an indicator of stability for both systems. As illustrated in Fig. 6, RMSD values were tracked over the course of a 100 ns simulation.

The unbound BChE maintained an RMSD value of approximately 1.6 Å throughout the MD simulation, indicating consistent stability. In comparison, the compound 13s-BChE complex displayed initial instability during the first 20 ns of the simulation. However, after this phase, the RMSD value stabilized at around 0.6 Å, confirming the remarkable stability of the complex for the remainder of the simulation.

Fig. 6

Superimposed RMSD of Cα atoms of BChE in complex with compound 13s (orange) and BChE (blue)

Fig. 7

Compound 13s RMSF during MD run

The root mean square fluctuation (RMSF) of the ligand is illustrated in Fig. 7. All ligand atoms displayed RMSD values below 2 Å, demonstrating consistent and favorable binding interactions throughout the simulation. These findings confirm that the ligand establishes and sustains stable interactions within the active site of the BChE enzyme, further validating its potency.

Fig. 8

Superimposed RMSF of Cα atoms of BChE in complex with compound 13s (orange) and BChE (blue) plus interactions with the protein residues that occur more than 30.0% of the simulation time

The RMSF plot for the protein was analyzed to evaluate the flexibility and mobility of the protein structure in both the apo form (unbound) and the ligand-BChE complex (Fig. 8). A comparative analysis of these fluctuations revealed key residues involved in ligand interactions. The ligand-BChE complex exhibited lower fluctuations compared to the unbound enzyme, particularly in the PAS and catalytic triad regions, confirming robust interactions with these critical residues. The reduced RMSF values in these crucial regions further support the high potency of compound 13s, attributed to its stable interactions within the active site. Moreover, the interactions observed during the MD simulation validate our design strategy. The nitroaryl group engaged in two pi-pi stacking interactions with Trp231 and Phe329, as well as a pi-cation interaction with Trp231. The 1,3,4-oxadiazole ring formed three hydrogen bonds with Gly116, Gly119, and Ala199 in the oxyanion hole. Additionally, the 1,2,3-triazole moiety and phenoxy linker established two pi-pi stacking interactions with Trp82 in the omega loop. The terminal 4-methylbenzyl moiety also displayed an additional pi-pi stacking interaction with Trp82. These findings underscore the strong and consistent interactions of compound 13s with BChE, further explaining its exceptional potency.

For comparison, the RMSD values of AChE and the AChE-13s complex were monitored over a 100 ns simulation to investigate the behavior of 13s as an inactive AChE inhibitor. The unbound AChE showed a slight increase in RMSD to 1.5 Å within the first 35 ns, maintaining this average value until the end of the simulation. In contrast, the AChE-13s complex exhibited a sharp increase in RMSD to 2.5 Å after 25 ns. This higher RMSD value indicates that 13s destabilizes the AChE system (Fig. 9).

Fig. 9

Superimposed RMSD of Cα atoms of AChE in complex with compound 13s (orange) and AChE (green)

The RMSF plot for AChE-13s, compared to the apo form (unbound), was analyzed (Fig. 10). A comparative assessment of these fluctuations indicated that compound 13s was not effective in reducing the fluctuation of the AChE enzyme. On the contrary, it induced some instability, particularly in the regions spanning residues 15–28, 70–84, and 338–349. Additionally, the 2D interaction analysis revealed two π-π stacking interactions with Trp86 and Tyr337, as well as a hydrogen bond with Ser125 (Fig. 10). These findings suggest a less stable binding pose for compound 13s within the AChE active site, reinforcing its classification as an inactive AChE inhibitor.

Fig. 10

Superimposed RMSF of Cα atoms of BChE in complex with compound 13s (orange) and BChE (green) plus interactions with the protein residues that occur more than 30.0% of the simulation time

Cytotoxicity study

The neurotoxicity of the most potent compound was evaluated against the SH-SY5Y neuroblastoma cell line. As depicted in Fig. 11, the IC₅₀ of the compound was determined to be 11.01 µM. Toxicity assessments revealed that cell viability remained at approximately 100% with no observed toxicity up to a concentration of 25 µM. At higher concentrations, cell viability reduced to around 81%, which is considered to be within a safe range.

Fig. 11

Cytotoxic studies of compound 13s on the SH-SY5Y neuroblastoma cell line after 72-h treatment

In Silico ADME evaluation

ADME properties, a crucial aspect of drug discovery research, were evaluated and summarized in Table 2. Additionally, blood-brain barrier (BBB) permeability was predicted. The synthesized derivatives 13 demonstrated generally satisfactory pharmacokinetic profiles, indicating their potential as viable drug candidates.

Table 2 Calculated molecular profile for compound 13

Conclusion

In this study, a series of novel 1,2,3-triazole-methoxyphenyl-1,3,4-oxadiazole derivatives were synthesized and evaluated for their inhibitory activity against BChE and AChE. The results demonstrated significant selectivity towards BChE, with several derivatives showing promising inhibition profiles. Compounds featuring electron-withdrawing groups, such as nitro and fluorine at the R’ positions, emerged as the most potent BChE inhibitors, emphasizing the importance of electronic effects in enzyme interactions.

Among these, compound 13s (R = 4-CH₃, R’ = 4-NO₂) stood out as the most potent competitive BChE inhibitor, with an IC₅₀ value of 11.01 µM, highlighting the effectiveness of the methyl-nitro substitution pattern. Docking studies further revealed that the incorporation of 4-nitroaryl, oxadiazole, phenoxy linker, and 4-methylbenzylthiazole-1,2,3-triazole moieties facilitated strong interactions with BChE. Notably, the phenoxy linker and triazole-thiazole moiety mimicked the interactions of donepezil within the enzyme’s active site. Additionally, compound 13s exhibited no toxicity up to 25 µM against the neuroblastoma SH-SY5Y cell line and demonstrated favorable ADME properties at a therapeutic dose.

The selective inhibition of BChE over AChE by these derivatives underscores their potential as therapeutic agents for AD, particularly in its later stages where BChE activity is more pronounced. The study’s structure-activity relationship (SAR) findings provided valuable guidance for designing more potent cholinesterase inhibitors, emphasizing the strategic selection of electron-withdrawing groups at critical positions.

Future efforts should focus on optimizing these compounds to enable dual inhibition of both BChE and AChE, which could offer broader therapeutic advantages in addressing neurodegenerative conditions like AD.