Synthesis characterization and evaluation of novel triazole based analogs as a acetylcholinesterase and α-glucosidase inhibitors

2.1 Chemicals and instruments

All the experiments were carried out under aerobic conditions unless stated otherwise. Alfa Aesar, Sigma Aldrich and Merck were contacted for analytical grade chemicals and solvents through local suppliers and forwarded to reaction as such. Melting points of the synthesized compounds (Acetylcholinesterase and α-Glucosidase inhibitors) were computed through Griffin and George apparatus. IR spectral patterns were recorded by Jasco-320A spectrometer using the KBr disc method. FT NMR (¹H and ¹³C) spectra of the synthesized compounds (10a-k) were collected by Bruker 600 MHz in deuterated dimethyl sulfoxide (DMSO‑d₆). JMS-HX-110 spectrometer was a tool for recording EI-MS spectra. The TLC plates were observed in UV light at 254 nm or iodine vapors and were made of aluminum plates pre-coated by silica gel.

2.2 Synthesis of 1-(4-toluenesulfonyl)-4-(ethoxycarbonyl) piperidine (3)

1,2,4 Trizole was synthesized by following the scheme-1 described by previously reported methods of (Bitla et al., 2021) 4-Ethoxycarbonylpiperidine (2; 0.05 mol) was mixed with 20 % Na₂CO_3(aq) in 500 mL round bottom (RB) flask. 4-Toluenesulfonyl chloride (1; 0.05 mol) was added by parts on stirring Na₂CO_3(aq) was used to maintain PH = 10 TLC was monitored to confirm completion. The pH of the medium was moved to 6 by dilute HCl_(aq). The title compound was collected as precipitates, washed, and dried.

2.3 Synthesis of 1-(4-toluenesulfonyl)-4-(2-hydrazinocarbonyl)piperidine (5)

Hydrazine monohydrate (4; 0.045 mol) and equimolar compound 3 were shaken together in methanol (150 mL) in RB flask (500 mL). The contents of the flask were refluxed for 4 h and monitored through TLC. The precipitates were collected after distillation of methanol, washed with n-hexane and dried in air.

2.4 Synthesis of 1-(4-toluenesulfonyl)-4-[1-(methylamino thiocarbonyl)-2 hydrazinocarbonylpiperidine(7)

Equimolar compound 5 and methyl isothiocyanate (6) were shaken together in ethanol (150 mL) in RB flask (500 mL). The contents of the flask were refluxed for 2 h and monitored through TLC. The precipitates were collected after the distillation of the ethanol. Formed precipitates were washed through distilled water and dried in air.

2.5 Procedure for 1-(4-toluenesulfonyl)-4-(3-mercapto-4-methyl-4H-1,2,4-triazol-5-yl)piperidine (8)

Compound 7 (0.04 mol) was refluxed with 15 % KOH_(aq) for 2 h and monitored through TLC. The pH of the medium was moved to 6 by dilute HCl_(aq). The title compound was collected as precipitates, washed and dried.

2.6 Procedure for 1-(4-toluenesulfonyl)-4-(3-aralkylthio-4-methyl-4H-1,2,4-triazol-5-yl)piperidine (10a-k)

Compound 8 (2 mmol) and LiH (2 mmol) were shaken together in DMF (15 mL) in RB flask (100 mL) and stirred for 0.5 h. Equimolar aralkyl halides (9a-k) were added and the contents of the flask were stirred for 3–5 h. TLC was performed and precipitates were collected after the addition of ice-cold distilled water. The title compounds were collected as precipitates, washed and dried.

2.7 1-(4-Toluenesulfonyl)-4-(3-benzylthio-4-methyl-4H-1,2,4-triazol-5-yl)piperidine (10a)

Amorphous white solid;; M.P.: 186–188 °C; Yield: 86 %; IR (υ_max, cm⁻¹): 3061, 1670, 1548, 1367, 694;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.66 (2H, J = 7.6 Hz, d,2′' & 6′'), 7.48 (2H, J = 7.6 Hz, d,3′' & 5′'), 7.27–7.25 (3H, m, 3′'' to 5′''), 7.22 (2H, J = 6.8 Hz, d,2′'' & 6′''), 4.24 (2H, s, 7′''), 3.64–3.62 (2H_e, m, 2′ & 6′), 3.19 (3H, s, 6), 2.81–2.77 (1H, m, 4′), 2.42 (3H, s, 7′'), 2.42–2.39 (2H_a, m, 2′ & 6′), 1.89–1.87 (2H_e, m, 3′ & 5′), 1.74–1.68 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.7 (5), 148.4 (3), 143.4 (1′'), 137.2 (1′''), 132.3 (4′'), 129.8 (3′' & 5′'), 128.8 (2′'' & 6′''), 128.3 (3′'' & 5′''), 127.5 (2′' & 6′'), 127.3 (4′''), 45.4 (2′ & 6′), 37.5 (4′), 30.4 (7′''), 29.6 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₆N₄O₂S₂; M.W.: 442 gmol⁻¹; EI-MS (m/z): 442 [M]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 287 [C₁₅H₁₉N₄S]⁺, 264 [C₁₃H₁₆N₂O₂S]⁺, 204 [C₁₀H₁₀N₃S]⁺, 155 [C₇H₇O₂S]⁺, 149 [C₈H₇NS]⁺, 91 [C₇H₇]⁺, 65 [C₅H₅]⁺.

2.8 1-(4-Toluenesulfonyl)-4-[3-(2-methylbenzyl) thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10b

Amorphous white solid;; M.P.: 178–180 °C; Yield: 88 %;; IR (υ_max, cm⁻¹): 3069, 1656, 1577, 1374, 689;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.6 Hz, d, 2′' & 6′'), 7.46 (2H, J = 7.5 Hz, d, 3′' & 5′'), 7.02–6.95 (4H, m,3′'' to 6′''), 4.19 (2H, s, 7′''), 3.63–3.61 (2H_e, m, 2′ & 6′), 3.20 (3H, s, 6), 2.80 (1H, br.s, 4′), 2.42 (3H, s, 7′'), 2.42–2.40 (2H_a,m, 2′ & 6′), 2.24 (3H, s, 8′''), 1.89–1.85 (2H_e, m, 3′ & 5′), 1.74–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.6 (5), 148.6 (3), 143.4 (1′'), 137.8 (1′''), 132.3 (4′'), 131.4 (2′''), 130.2 (3′''), 129.8 (3′' & 5′'), 128.3 (4′''), 127.9 (6′''),127.5 (2′' & 6′'), 127.3 (5′''), 45.4 (2′ & 6′), 37.4 (4′), 30.4 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'), 20.6 (8′''); M.F.: C₂₃H₂₈N₄O₂S₂; M.W.: 456 gmol⁻¹. EI-MS (m/z): 456 [M]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 301 [C₁₆H₂₁N₄S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 218 [C₁₁H₁₂N₃S]⁺, 163 [C₉H₉NS]⁺, 155 [C₇H₇O₂S]⁺, 105 [C₈H₉]⁺, 65 [C₅H₅]⁺.

2.9 1-(4-Toluenesulfonyl)-4-[3-(3-methylbenzyl)thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10c)

Amorphous white solid; M.P.: 170–172 °C; Yield: 79 %;;IR (υ_max, cm⁻¹):3063, 1659, 1570, 1371, 682;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.3 Hz, d, 2′' & 6′'), 7.47 (2H, J = 7.5 Hz, d, 3′' & 5′'), 7.15 (1H, J = 7.4 Hz, t, 5′''), 7.06 (1H, J = 7.4 Hz, d, 6′''), 7.01 (1H, s, 2′''), 7.01–6.99 (1H, m, 4′''), 4.18 (2H, s, 7′''), 3.63–3.62 (2H_e, m, 2′ & 6′), 3.19 (3H, s, 6), 2.79 (1H, br.s, 4′), 2.41 (3H, s, 7′'), 2.41–2.39 (2H_a, m, 2′ & 6′), 2.23 (3H, s, 8′''), 1.89–1.86 (2H_e, m, 3′ & 5′), 1.73–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.7 (5), 148.5 (3), 143.4 (1′'), 137.5 (1′''), 137.0 (3′''), 132.3 (4′'), 129.8 (3′' & 5′'), 129.3 (5′''), 128.3 (4′''), 128.0 (2′''), 127.5 (2′' & 6′'), 125.9 (6′''), 45.4 (2′ & 6′), 37.6 (4′), 30.4 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'), 20.8 (8′''); M.F.: C₂₃H₂₈N₄O₂S₂; M.W.: 456 gmol^−1. EI-MS (m/z): 456 [M]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 301 [C₁₆H₂₁N₄S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 218 [C₁₁H₁₂N₃S]⁺, 163 [C₉H₉NS]⁺, 155 [C₇H₇O₂S]⁺, 105 [C₈H₉]⁺, 65 [C₅H₅]⁺.

2.10 1-(4-Toluenesulfonyl)-4-[3-(4-methylbenzyl) thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10d)

Amorphous white solid; M.P.: 186–188 °C; Yield: 87 %; IR (υ_max, cm⁻¹):3073, 1659, 1580, 1382, 687;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.4 Hz, d, 2′' & 6′'), 7.47 (2H, J = 7.5 Hz, d, 3′' & 5′'), 7.10 (2H, J = 7.4 Hz, d, 2′'' & 6′''), 7.07 (2H, J = 7.4 Hz, d, 3′'' & 5′''), 4.19 (2H, s, 7′''), 3.63–3.62 (2H_e, m, 2′ & 6′), 3.20 (3H, s, 6), 2.79 (1H, br.s, 4′), 2.41 (3H, s, 7′'), 2.41–2.39 (2H_a, m, 2′ & 6′), 2.25 (3H, s, 8′''), 1.89–1.86 (2H_e, m, 3′ & 5′), 1.71–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.7 (5), 148.5 (3), 143.4 (1′'), 136.6 (1′''), 134.0 (4′''), 132.3 (4′'), 129.8 (3′' & 5′'), 128.9 (3′'' & 5′''), 128.7 (2′'' & 6′''), 127.5 (2′' & 6′'), 45.4 (2′ & 6′), 37.2 (4′), 30.4 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'), 20.6 (8′''); M.F.: C₂₃H₂₈N₄O₂S₂; M.W.: 456 gmol⁻¹; EI-MS (m/z): 456 [M]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 301 [C₁₆H₂₁N₄S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 218 [C₁₁H₁₂N₃S]⁺, 163 [C₉H₉NS]⁺, 155 [C₇H₇O₂S]⁺, 105 [C₈H₉]⁺, 65 [C₅H₅]⁺.

2.11 1-(4-Toluenesulfonyl)-4-[3-(2-chlorobenzyl)thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10e)

Amorphous white solid; M.P.: 168–170 °C; Yield: 83 %;;IR (υ_max, cm⁻¹):3087, 1666, 1589, 1396, 702, 691;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.66 (2H, J = 8.1 Hz, d, 2′' & 6′'), 7.47 (2H, J = 8.0 Hz, d, 3′' & 5′'), 7.44 (1H, J = 7.9 Hz, d, 6′''), 7.30 (1H, J = 2.9, 6.2 Hz, dt, 4′''), 7.24–7.22 (2H, m, 3′'' & 5′''), 4.29 (2H, s, 7′''), 3.65–3.63 (2H_e, m, 2′ & 6′), 3.36 (3H, s, 6), 2.82–2.78 (1H, m, 4′), 2.42 (3H, s, 7′'), 2.42–2.40 (2H_a, m, 2′ & 6′), 1.89–1.87 (2H_e, m, 3′ & 5′), 1.75–1.68 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 158.0 (5), 147.8 (3), 143.4 (1′'), 134.6 (1′''), 133.0 (5′''), 132.3 (4′'), 131.2 (3′''), 129.7 (3′' & 5′'), 129.5 (4′''), 129.4 (6′''), 127.5 (2′' & 6′'), 127.2 (2′''), 45.4 (2′ & 6′), 35.7 (4′), 30.5 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅ClN₄O₂S₂; M.W.: 477 gmol⁻¹ EI-MS (m/z): 479 [M + 2]⁺, 477 [M]⁺, 321 [C₁₅H₁₈ClN₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 238 [C₁₀H₉ClN₃S]⁺, 183 [C₈H₆ClNS]⁺, 155 [C₇H₇O₂S]⁺, 125 [C₇H₆Cl]⁺, 65 [C₅H₅]⁺.

2.12 1-(4-Toluenesulfonyl)-4-[3-(4-chlorobenzyl) thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10f)

Amorphous white solid; M.P.: 174–176 °C; Yield: 90 %; IR (υ_max, cm⁻¹):3081, 1660, 1584, 1399, 705, 688;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.66 (2H, J = 7.5 Hz, d, 2′' & 6′'), 7.48 (2H, J = 7.6 Hz, d, 3′' & 5′'), 7.34 (2H, J = 7.9 Hz, d, 2′'' & 6′''), 7.27 (2H, J = 7.4 Hz, d, 3′'' & 5′''), 4.25 (2H, s, 7′''), 3.64–3.62 (2H_e, m, 2′ & 6′), 3.26 (3H, s, 6), 2.81 (1H, br.s, 4′), 2.42 (3H, s, 7′'), 2.42–2.39 (2H_a, m, 2′ & 6′), 1.90–1.87 (2H_e, m, 3′ & 5′), 1.73–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.8 (5), 148.2 (3), 143.4 (1′'), 136.4 (4′''), 132.3 (4′'), 131.9 (1′''), 130.7 (3′'' & 5′''), 129.8 (3′' & 5′'), 128.3 (2′'' & 6′''), 127.5 (2′' & 6′'), 45.4 (2′ & 6′), 36.4 (4′), 30.4 (7′''), 29.6 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅ClN₄O₂S₂; M.W.: 477 gmol⁻¹; EI-MS (m/z): 479 [M + 2]⁺, 477 [M]⁺, 321 [C₁₅H₁₈ClN₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 238 [C₁₀H₉ClN₃S]⁺, 183 [C₈H₆ClNS]⁺, 155 [C₇H₇O₂S]⁺, 125 [C₇H₆Cl]⁺, 65 [C₅H₅]⁺.

2.13 1-(4-Toluenesulfonyl)-4-[3-(2-bromobenzyl) thio-4-methyl-4H-1,2,4-triazol-5-yl] piperidine (10 g)

Amorphous white solid; M.P.: 178–180 °C; Yield: 75 %; IR (υ_max, cm⁻¹):3098, 1673, 1591, 1394, 653, 682;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 8.0 Hz, d, 2′' & 6′'), 7.61 (1H, J = 7.7 Hz, d, 6′''), 7.46 (2H, J = 8.1 Hz, d, 3′' & 5′'), 7.32 (1H, J = 7.2 Hz, t, 4′''), 7.26 (1H, J = 6.9 Hz, t, 5′''), 6.91 (1H, J = 7.6 Hz, d, 3′''), 4.29 (2H, s, 7′''), 3.65–3.61 (2H_e, m, 2′ & 6′), 3.20 (3H, s, 6), 2.79 (1H, br.s, 4′), 2.42 (3H, s, 7′'), 2.42–2.41 (2H_a, m, 2′ & 6′), 1.90–1.87 (2H_e, m, 3′ & 5′), 1.74–1.71 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ,ppm): 158.0 (5), 147.8 (3), 143.4 (1′'), 136.2 (1′''), 132.3 (4′'), 131.2 (3′''), 129.8 (3′' & 5′'), 129.5 (4′''), 128.6 (6′''), 127.8 (5′''), 127.5 (2′' & 6′'), 121.9 (2′''), 45.4 (2′ & 6′), 38.2 (4′), 30.5 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅BrN₄O₂S₂; M.W.: 521 gmol⁻¹; EI-MS (m/z): 523 [M + 2]⁺, 521 [M]⁺, 366 [C₁₅H₁₈BrN₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 283 [C₁₀H₉BrN₃S]⁺, 264 [C₁₃H₁₆N₂O₂S]⁺, 228 [C₈H₆BrNS]⁺, 170 [C₇H₆Br]⁺, 155 [C₇H₇O₂S]⁺, 65 [C₅H₅]⁺.

2.14 1-(4-Toluenesulfonyl)-4-[3-(3-bromobenzyl)thio-4-methyl-4H-1,2,4-triazol-5-yl] piperidine (10 h)

Amorphous white solid; M.P.: 172–174 °C; Yield: 79 %;; IR (υ_max, cm⁻¹):3086, 1681, 1595, 1391, 649, 688;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.6 Hz, d, 2′' & 6′'), 7.47 (2H, J = 7.5 Hz, d, 3′' & 5′'), 7.33 (1H, s, 2′''), 7.17–7.13 (2H, m, 5′'' & 6′''), 6.97 (1H, J = 7.5 Hz, d, 4′''), 4.25 (2H, s, 7′''), 3.63–3.61 (2H_e, m, 2′ & 6′), 3.20 (3H, s, 6), 2.79 (1H, br.s, 4′), 2.41 (3H, s, 7′'), 2.41–2.39 (2H_a, m, 2′ & 6′), 1.89–1.85 (2H_e, m, 3′ & 5′), 1.74–1.68 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.9 (5), 148.2 (3), 143.4 (1′'), 140.2 (1′''), 132.3 (4′'), 131.4 (4′''), 130.4 (2′''), 130.1 (5′''), 129.8 (3′' & 5′'), 127.9 (6′''), 127.5 (2′' & 6′'), 121.3 (3′''), 45.4 (2′ & 6′), 36.5 (4′), 30.4 (7′''), 29.5 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅BrN₄O₂S₂; M.W.: 521 gmol⁻¹ EI-MS (m/z): 523 [M + 2]⁺, 521 [M]⁺, 366 [C₁₅H₁₈BrN₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 283 [C₁₀H₉BrN₃S]⁺, 264 [C₁₃H₁₆N₂O₂S]⁺, 228 [C₈H₆BrNS]⁺, 170 [C₇H₆Br]⁺, 155 [C₇H₇O₂S]⁺, 65 [C₅H₅]⁺.

2.15 1-(4-Toluenesulfonyl)-4-[3-(4-bromobenzyl)thio-4-methyl-4H-1,2,4-triazol-5-yl] piperidine (10i)

Amorphous white solid; M.P.: 182–184 °C; Yield: 75 %; IR (υ_max, cm⁻¹):3076, 1679, 1597, 1388, 647, 685;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.3 Hz, d, 2′' & 6′'), 7.48–7.46 (4H, m, 4H, 3′' & 5′', 2′'' & 6′''), 7.20 (2H, J = 7.2 Hz, d, 3′'' & 5′''), 4.23 (2H, s, 7′''), 3.64–3.62 (2H_e, m, 2′ & 6′), 3.26 (3H, s, 6), 2.82–2.79 (1H, m, 4′), 2.42 (3H, s, 7′'), 2.42–2.40 (2H_a, m, 2′ & 6′), 1.90–1.88 (2H_e, m, 3′ & 5′), 1.74–1.68 (2H_a, m, 3′ & 5′);¹³C NMR (125 MHz, DMSO‑d₆, δ ppm): 157.8 (5), 148.2 (3), 143.4 (1′'), 136.8 (1′''), 132.3 (4′'), 131.2 (3′'' & 5′''), 131.0 (2′'' & 6′''), 129.8 (3′' & 5′'), 127.5 (2′' & 6′'), 120.5 (4′''), 45.4 (2′ & 6′), 36.4 (4′), 30.4 (7′''), 29.6 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅BrN₄O₂S₂; M.W.: 521 gmol⁻¹; EI-MS (m/z): 523 [M + 2]⁺, 521 [M]⁺, 366 [C₁₅H₁₈BrN₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 283 [C₁₀H₉BrN₃S]⁺, 264 [C₁₃H₁₆N₂O₂S]⁺, 228 [C₈H₆BrNS]⁺, 170 [C₇H₆Br]⁺, 155 [C₇H₇O₂S]⁺, 65 [C₅H₅]⁺.

2.16 1-(4-Toluenesulfonyl)-4-[3-(4-flourobenzyl) thio-4-methyl-4H-1,2,4-triazol-5-yl]piperidine (10j)

Amorphous white solid; M.P.: 170–172 °C; Yield: 81 %; IR (υ_max, cm⁻¹):3096, 1669, 1584, 1389, 1057, 676;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.65 (2H, J = 7.6 Hz, d, 2′' & 6′'), 7.45 (2H, J = 7.6 Hz, d, 3′' & 5′'), 7.21 (2H, J = 8.1 Hz, d, 3′'' & 5′''), 7.02 (2H, J = 8.0 Hz, d, 2′'' & 6′''), 4.24 (2H, s, 7′''), 3.64–3.61 (2H_e, m, 2′ & 6′), 3.21 (3H, s, 6), 2.80 (1H, br.s, 4′), 2.42 (3H, s, 7′'), 2.42–2.39 (2H_a, m, 2′ & 6′), 1.90–1.88 (2H_e, m, 3′ & 5′), 1.72–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (600 MHz, DMSO‑d₆, δ ppm): 158.1 (5), 150.5 (4′''), 148.1 (3), 143.4 (1′'), 132.1 (4′'), 131.8 (1′''), 130.6 (2′'' & 6′''), 129.8 (3′' & 5′'), 127.5 (2′' & 6′'), 127.1 (3′'' & 5′''), 45.4 (2′ & 6′), 36.4 (4′), 30.4 (7′''), 29.6 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₅FN₄O₂S₂; M.W.: 460 gmol⁻¹; EI-MS (m/z): 460 [M]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 305 [C₁₅H₁₈FN₄S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 264 [C₁₃H₁₆N₂O₂S]⁺, 222 [C₁₀H₉FN₃S]⁺, 167 [C₈H₆FNS]⁺, 155 [C₇H₇O₂S]⁺, 109 [C₇H₆F]⁺, 65 [C₅H₅]⁺.

2.17 1-(4-Toluenesulfonyl)-4-[3-(3,4-dichlorobenzyl)thio-4-methyl-4H-1,2,4-triazol-5-yl] piperidine (10 k)

Amorphous white solid; M.P.: 176–178 °C; Yield: 78 %; IR (υ_max, cm⁻¹):3066, 1658, 1579, 1388, 707, 686;¹H NMR (600 MHz, DMSO‑d₆, δ ppm): 7.66 (2H, J = 7.4 Hz, d, 2′' & 6′'), 7.59 (1H, s, 2′''), 7.47 (2H, J = 7.5 Hz, d, 3′' & 5′'), 7.33 (1H, J = 8.3 Hz, t, 5′''), 7.30 (1H, J = 8.0 Hz, d, 6′''), 4.28 (2H, s, 7′''), 3.65–3.63 (2H_e, m, 2′ & 6′), 3.28 (3H, s, 6), 2.84–2.80 (1H, m, 4′), 2.42 (3H, s, 7′'), 2.42–2.40 (2H_a, m, 2′ & 6′), 1.90–1.88 (2H_e, m, 3′ & 5′), 1.75–1.69 (2H_a, m, 3′ & 5′);¹³C NMR (600 MHz, DMSO‑d₆, δ ppm): 158.0 (5), 147.6 (3), 143.4 (1′'), 133.99 (1′''), 133.94 (4′''), 133.0 (3′''), 132.4 (5′''), 132.3 (4′'), 129.7 (3′' & 5′'), 128.8 (2′''), 127.5 (2′' & 6′'), 127.3 (6′''), 45.4 (2′ & 6′), 34.7 (4′), 30.5 (7′''), 29.6 (6), 28.8 (3′ & 5′), 20.9 (7′'); M.F.: C₂₂H₂₄Cl₂N₄O₂S₂; M.W.: 511 gmol⁻¹; EI-MS (m/z): 515 [M + 4]⁺, 513 [M + 2]⁺, 511 [M]⁺, 356 [C₁₅H₁₇Cl₂N₄S]⁺, 319 [C₁₅H₁₉N₄O₂S]⁺, 293 [C₁₄H₁₉N₃O₂S]^.+, 273 [C₁₀H₈Cl₂N₃S]⁺, 264 [C₁₃H₁₆N₂O₂S]⁺, 218 [C₈H₅Cl₂NS]⁺, 160 [C₇H₅Cl₂]⁺, 155 [C₇H₇O₂S]⁺, 65 [C₅H₅]⁺.

2.18 In-vitro acetylcholinesterase (AChE) inhibition assay

Acetylcholinesterase (3.1.1.7) inhibition activity was measured by modifying the reported methods of Ellman using 96-wells plate readers (Synergy HT, BioTek, USA) (Mitsumori et al., 2003). Acetylcholine Iodide (AChEI) was used as the substrate, 5,5ʹ-dithio bis (2-nitro benzoic) acid (DTNB) to estimate the activity of AChE and Eserine as reference. The reaction mixture containing 10 μL cholinesterase enzyme (Sigma, USA), tested compounds (0.05 mM; 10 to 100 mM), 60 μL phosphate buffer (pH 7.7; 0.05 mM), and 10 μL DTNB (0.05 mM) was incubated at 37 °C for 15–30 min. The change in optical density (O.D.) at zero, 15, and 30 min was monitored at 405 nm after the initiation of the reaction by the addition of acetyl thiocholine iodide (10 μL) while Eserine (0.5 mM) was employed as a positive control. The percentage inhibition was noted as, $$\mathbf{I}\mathbf{n}\mathbf{h}\mathbf{i}\mathbf{b}\mathbf{i}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}(\%)=\frac{\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}-\mathbf{T}\mathbf{e}\mathbf{s}\mathbf{t}}{\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}}\times 100$$

The Ez-Fit software (Perrella Scientific Inc. Amherst, USA) and computing data in Graph Pad Prism 5 software were used to calculate IC₅₀ values. Inhibition parameters and Vmax were determined from Lineweaver-Burk plots (Duyckaerts et al., 2009).

2.19 In-vitro α-Glucosidase assay

α-Glucosidase inhibition assay was performed following the reported methods of with minor modifications using acarbose as a reference standard (Tao et al., 2013). Tested compounds (10 to 100 μL; 0.05 mM), α-glucosidase enzyme (10 μL) (Sigma, USA) and phosphate buffers (70 μL; pH 6.8; 0.05 M) were mixed and pre-incubated at 37 °C for 10 min followed by pre-reading at 400 nm. Change in absorbance was monitored by adding p-nitrophenyl glucopyranoside (10 μL; 0.5 mM). The previously mentioned formula was used to assess the % inhibition and IC₅₀ values.

2.20 Molecular docking simulations

The inhibitory potential of produced compounds was determined theoretically by virtual molecular docking software by Molegro Virtual Docker version 2010. Proteins for anti-acetylcholine esterase were saved as pdb after downloading from an online protein data bank. Chemdarw (Pro; 12.0.2; 875–317589-4732; Cambridgesoft) was used to draw the structures of produced compounds (10a-k) and saved as a MOL2 file by copying it into 3D chem draw. Files of pdb proteins were imported in the docking software to prepare a template then all produced compounds were run one by one.

3.1 Chemistry

The compound 10d has been explicated as a single compound discussion from this alkyl halide series (10a-k). The compounds, 10a-c,e-k, have been structurally elucidated similarly. The white amorphous solid, 10d (C₂₃H₂₈N₄O₂S₂, 456 g mol⁻¹, justified through mass spectrum), possessed a melting point of 186–188 °C with 87 % yield. The IR data was known to possess the following stretching frequencies for prominent functionalities, 3073, 1659, 1580, 1382 and 687. 4-Methylphenylsulfonyl moiety was justified through three signals in the ¹H NMR spectrum (Figs. 1, 2) at δ 7.65 (2H, J = 7.4 Hz, d, 2′' & 6′'), 7.47 (2H, J = 7.5 Hz, d, 3′' & 5′') and 2.41 (3H, s, 7′'); and five signals in the ¹³C NMR spectrum at δ 143.4 (1′'), 132.3 (4′'), 129.8 (3′' & 5′'), 127.5 (2′' & 6′') and 20.9 (7′'). 4-Methylbenzyl moiety was justified through four signals in ¹H NMR spectrum (Figs. 1, 3) at δ 7.10 (2H, J = 7.4 Hz, d, 2′'' & 6′''), 7.07 (2H, J = 7.4 Hz, d, 3′'' & 5′''), 4.19 (2H, s, 7′'') and 2.25 (3H, s, 8′''); and six signals in ¹³C NMR spectrum (Figure-4, Figure-5) at δ 136.6 (1′''), 134.0 (4′''), 128.9 (3′'' & 5′''), 128.7 (2′'' & 6′''), 30.4 (7′'') and 20.6 (8′''). 3,5-Disubstituted-6-methyl-1,2,4-triazole moiety was justified through one signal in the ¹H NMR spectrum (Fig. 2) at δ 3.20 (3H, s, 6); and three signals in the ¹³C NMR spectrum (Figs. 4, 5) at δ 157.7 (5), 148.5 (3) and 29.5 (6). Piperidine moiety was justified through five signals in the ¹H NMR spectrum (Fig. 2) at δ 3.63–3.62 (2H_e, m, 2′ & 6′), 2.79 (1H, br.s, 4′), 2.41–2.39 (2H_a, m, 2′ & 6′), 1.89–1.86 (2H_e, m, 3′ & 5′) and 1.71–1.69 (2H_a, m, 3′ & 5′); and three signals in the ¹³C NMR spectrum (Fig. 5) at δ 45.4 (2′ & 6′), 37.2 (4′) and 28.8 (3′ & 5′).

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Fig. 1

¹H NMR spectrum (aromatic) of compound 10d.

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Fig. 2

¹H NMR spectrum (shielded aliphatic) of compound 10d.

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Fig. 3

¹H NMR spectrum (deshielded aliphatic) of compound 10d.

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Fig. 4

¹³C NMR spectrum (aromatic) of compound 10d.

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Fig. 5

¹³C NMR spectrum (aliphatic) of compound 10d.

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3.2 Biological activities

The biological activities of the prepared compounds were evaluated, which are important for practical applications (Muhammad et al., 2020; Suhail and Ali, 2020; Asif and Abida, 2019; Deeba et al., 2018; Pathak et al., 2021). The 1,2,4-triazole analogs (10a-k) were further evaluated for their inhibitory activities against AChE and α-glucosidase enzymes (Table 1, 2). It is investigated that AD and DM have an elevated activity of AChE (E.C. 3.1.1.7) which leads to alteration in cholinergic neurotransmission (Gulçin et al., 2018). Any alteration in the expression and activity of AChE that’s a prime cause of neurodegenerative disorders may act as a biomarker for neurotoxicity determination (Isik et al., 2015) Inhibitors of AChE are widely recommended to eliminate neurotoxic effects produced by the change of the cholinergic hypothesis. Inhibitors of AChE may reduce the multiple β-amyloid neurotoxic products and protects the cell from oxidative damage (Taslimi et al., 2021). AChE inhibitors commonly used in treatment slow down excessive ACh hydrolysis and avert AD progression which leads to relaxation and improves cognitive functions (Isik et al., 2019)The structure–activity relationship (SAR) analysis revealed that the presence of electron-donating or electron-withdrawing groups at meta, ortho, or para positions of the whole series of derivatives (1,2,4-triazole analogs 10a-k) presented a vital role in enhancing their anti-enzymatic role against AChE. The whole series of derivatives (1,2,4-triazole analogs 10a-k) were active against the AChE enzyme (Table 1, 2, Figs. 6a & 6b). The excellent activity was possessed by four compounds, 10b (bearing 2-methyl benzyl moiety), 10c (bearing 3-methylbenzyl moiety), 10j (bearing 4-fluorobenzyl moiety), and 10 k (bearing 3,4-dichlorobenzyl moiety). among methyl-benzyl substituents, the most active ones were ortho and meta-substituted molecules, ortho substituted in the chlorobenzyl substituents, meta substituted in the bromobenzyl substituents, while ortho and meta substituted in the mono-halo-benzyl substituents whereas 4-fluorobenzyl bearing molecules were the exception. The only molecule bearing dichloro benzyl moiety remained the most efficient compound against AChE.

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Fig. 6a

AChE inhibition of synthesized compounds (10a-10c).

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Fig. 6b

AChE inhibition of synthesized compounds (10d-10 k).

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Lineweaver-Burk and Michaelis Menten equation was used to determine the value of K_i and type of inhibition at eight different concentrations both in the presence and in absence of triazole derivatives (Table 2 and Figs. 6a & 6b). It can be suggested that derivative 10 k inhibited the AChE un-competitively by binding AChE and AChEI complex reversibly with weak interactions. Furthermore, its K_i value was determined as 0.084 ± 0.05 µM. Literature reported that many neurodegenerative disorders such as Parkinson's disease, Lewy body dementia, myasthenia gravis, and glaucoma especially AD could be treated by cholinesterase inhibitors In another study, (Shelke et al., 2015) investigated water-soluble triazole substituted metal-free and metallo-phthalocyanines for AChE inhibition potential. In line with our findings, 4a phthalocyanine having 0.040 µM IC₅₀ was active against AChE which was 1.5-fold active as compared to standard.

The whole series of derivatives were active against the α-glucosidase enzyme (Table 2, Fig. 7). Most of the compounds among the series were active and were better inhibitors than that of the reference standard. The excellent activity was possessed by two compounds, 10b (bearing 2-methylbenzyl moiety) and 10c (bearing 3-methylbenzyl moiety). Among the methyl benzyl substituents, the most active ones were ortho and meta-substituted molecules while ortho-substituted in chlorobenzyl substituents, meta-substituted in bromobenzyl substituents, ortho and meta in the mono-halo benzyl substituents whereas derivatives bearing unsubstituted benzyl moiety and di chlorobenzyl moiety were found to be the least active ones. Triazole due to its poor basicity as compared to other azaheterocycles is not protonated at physiological pH. The better inhibition potential of derivative compounds might be due to excellent mimic partial positive charge at the anomeric carbon in the transition state of glucosidase catalyzed reaction in non-protonated sp²-hybridized nitrogen atoms of triazole derivative than corresponding basic nitrogen of iminosugars. Similar to our findings, it has been reported that several triazole derivatives showed an inhibitory effect against α-glucosidase among which 10b showed promising results and was considered as the most active analog with IC₅₀ value of 14.2 µM (Taslimi et al., 2017). Whereas compound 6 showed contrary results due to a low IC₅₀ value of 218.1 µM. Similarly, (Ye et al., 2019) determined the antidiabetic potential of xanthone triazole derivatives by inhibiting the α-glucosidase and glucose uptake in HepG₂ cells. They reported that compound 5e was found to be most potent having 2.06 µM IC₅₀ than parental (1,3-dihydroxyxanthone IC₅₀ = 160.8 µM) and 1-deoxynojirimycin (Std. IC₅₀ = 59.5 µM). The position of different substituents in 1,2,4-triazole analogs (10a-k) helped to establish SAR. In-vitro studies have indicated that chloro and fluoro groups present at R₂ and R₃ positions in derivatives best inhibit the activities of both AChE and α-glucosidase. The reason might be due to the presence of chloro and fluoro groups as a substituent on triazole ring have a negative inductive effect causing destabilization of the triazole ring resulting in enhanced activity of 1,2,4-triazole analogs. The compounds 10 k, 10j, 10b, and 10c are the most active and presented the most potent inhibitory potential as compared to others. When all 1,2,4-triazole analogs (10a-k) were examined, it was observed that chloro, fluoro and methyl groups containing 1,2,4-triazole analogs (10a-k) are the most active compounds. The findings revealed that the triazole analogs prepared in the present investigation showed promising biological activity (Hassan and Arshad, 2022; Tegegne et al., 2020; Shadrach et al., 2020), which have potential for partical application.

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Fig. 7

α-Glucosidase inhibition of synthesized compounds (10a-k).

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3.3 Molecular docking simulation

Docking imitations of applicant ligands with AChE co-crystal structure (PDB ID:1NEN) using Auto dock tools predicted that the compound has a value of binding affinity of −9.9 kcal/mol which is the best binding score (Taslimi et al., 2017; Pradhan and Vishwakarma, 2020). The detailed analysis along with types of bonds with their distances formed between the amino acids was provided in Table 3. Drug-target interactions were assumed in the provision of interfacing amino acids fragments, hydrogen bonding, docking energy investigation, presumed binding sites and peculiarities of active site amino acid residues (Fig. 8). Total binding strength is a consequence of different kinds of bonds inclusive of ionic, hydrophobic interactions, Vander Waals forces and hydrogen bonds which are the promoter. Compound (10j) showed conventional hydrogen bonding interactions with amino acid HIS 45, THR 46, ASN 218, ALA 201 and electrostatic interaction with HIS 354, ARG 399, GLU 388 (Virk et al., 2019). It has convenient van der Waals interactions with HIS 354, ALA 49, LEU 252, LYS 38, HIS 354, ALA49, LYS 38 (Ye et al., 2016). In the form of numerous types such as Pi-Pi T-shaped, Alkyl and Pi-Alkyl (Yin et al., 2009). All these interactions with certain amino acids occurred at different distances are shown in Table 3.

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Fig. 8

Binding modes of compound 10j in acetyl cholinesterase (PDB ID:1NEN). Dotted lines presented the hydrogen bonds.

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