Chemical and instrumentsOrganic chemicals and solvents were sourced from reputable companies and used without additional purification. Analytical TLC was conducted with pre-coated aluminum sheets and silica gel to check reaction completion and compound purity. Melting points were measured using an Electrothermal 9100 apparatus. 1H NMR and 13C NMR spectra were recorded on Bruker 400–500 MHz instruments with TMS as the internal standard and CDCl3 and DMSO-d6 as solvents. Elemental analysis for C, H and N was performed using a Costech model 4010 instrument, and the obtained values closely matched theoretical values within ± 0.4% deviation. Electrospray ionization mass spectra (ESI–MS) were obtained using Agilent 6410 Triple Quad. LC/MS. LC chromatograms were obtained using HPLC system (YL9100) equipped with UV detector, isocratic mobile phase was CH3OH: H2O (80:20 v/v).Synthesis of 1-(1-methyl-1H-indol-3-yl)ethanone (1)A mixture of 3-acetylindole (1.0 mmol) and K2CO3 (1.5 mmol) in DMF (5 ml) was stirred at room temperature for 15 min. After that the methyl iodide (1.1 mmol) was added and the reaction was stirred for 4 h at 80 ̊C. The reaction mixture was added into ice-cold water, the precipitates were collected by filtration, and used for the next step without further purification. Yield: 81%; white crystals; m.p. 106–108 °C. FT-IR (KBr) cm−1: 3114, 1676, 1261. 1H NMR (500 MHz, CDCl3-d6): δ 8.36 (s, 1H, Indole H4), 7.69 (s, 1H, ‒N = CH-), 7.32 (m, 3H, Indole H5, H6, H7), 3.84 (s, 3H, N‒CH3), 2.52 (s, 3H, CH3). ESI-Mass m/z: 174 [M + H]+. Anal. Calcd. for C11H11NO: C, 76.28; H, 6.40; N, 8.09. Found: C, 75.97; H, 6.08; N, 8.32.Synthesis of ethyl 4-(1-methyl-1H-indol-3-yl)-2,4-dioxobutanoate (2)The mixture of freshly prepared NaEtO 10% (10 mL) and diethyloxalate (1.5 mmol) was added dropwise into the stirring compound 1 (1.0 mmol) in ethanol at 0 °C. Then, the reaction mixture was stirred at 75 °C for 2 h to appear orange precipitate. To quench the reaction, 2 mL glacial acetic acid (HOAc) was added and stirred for 3 h to give a yellow precipitate. After filtration, the precipitates were washed thoroughly with water and compound 2 was obtained. Yield: 81%; Yellow solid; m.p. 124- 125 °C. FT-IR (KBr) cm−1: 3114, 2977, 2929, 1738. 1H NMR (500 MHz, CDCl3): δ 15.48 (bs, 1H, OH), 8.37 (d, 1H, J = 6.8 Hz, Indole H4), 7.85 (s, 1H, Indole H2), 7.37- 7.35 (m, 3H, Indole H5, H6, H7), 6.84 (s, 1H, = CH—OH), 4.39 (q, 2H, J = 7.0 Hz, CH2), 3.88 (s, 3H, CH3), 1.42 (t, 3H, J = 7.0 Hz, CH3).13C NMR (125 MHz, DMSO-d6): 189.0, 162.7, 162.2, 139.9, 138.3, 126.0, 124.1, 123.4, 122.1, 114.0, 111.7, 101.0, 62.3, 34.0, 14.4. ESI-Mass m/z: 274 [M + H]+. Anal. Calcd. for C15H15NO4: C, 65.92; H, 5.53; N, 5.13. Found: C, 65.68; H, 5.28; N, 5.40.Synthesis of ethyl 5-(1-methyl-1H-indol-3-yl)isoxazole-3-carboxylate (3)Compound 2 (1.0 mmol) reacted with hydroxylamine hydrochloride (1.0 mmol) in ethanol and the mixture was refluxed for 6 h. Then, the reaction was cooled and poured into the ice-cold water and the resulting pale green precipitates (3) were collected after filtration. Yield: 95%; Pale-green solid; m.p: 141–143 °C. FT-IR (KBr) cm−1: ν 3166, 3119, 1726 (C = O). 1H NMR (500 MHz, DMSO-d6): δ 8.21 (s, 1H, Indole H2), 8.01 (d, 1H, J = 8.0 Hz, Indole H4), 7.60 (d, 1H, J = 8.0 Hz, Indole H7), 7.31 (dt, 2H, J = 24.0, 8.0 Hz, Indole H5, H6), 7.11 (s, 1H, Isoxazole), 4.40 (dd, 2H, J = 12.0, 8.0 Hz, CH2), 3.90 (s, 3H, N‒CH3), 1.36 (t, 3H, J = 8.0 Hz, CH3). 13C NMR (125 MHz, DMSO-d6): 168.9, 160.2, 156.8, 137.4, 131.3, 124.3, 123.2, 121.9, 120.2, 111.4, 102.2, 97.5, 62.2, 33.5, 14.5. ESI-Mass m/z: 271 [M + H]+. Anal. calcd. for C15H14O3N2: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.73; H, 5.56; N, 10.03.Synthesis of 5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (4)To a solution of compound (3) (1.0 mmol) in ethanol, hydrazine monohydrate 95% (1.5 mmol) was added and stirred at 50 °C for 2 h. The mixture was cooled down by the addition of a water/ice mixture and compound 4 was collected by filtration. Yield: 81%; off-withe solid; m.p. 237- 239 °C. FT-IR (KBr) cm−1: ν 3257.64 (NH), 1675.00, 1626.22. 1H NMR (500 MHz, DMSO-d6): δ 13.31 (s, 1H, CONH), 9.37 (s, 1H, Indole H2), 7.87 (d, 1H, J = 8.0 Hz, Indole H4), 7.78 (s, 1H, Isoxazole), 7.53 (d, 1H, J = 8.0 Hz Indole H7), 7.26 (t, 1H, J = 8.0 Hz Indole H5), 7.18 (t, 1H, J = 8.0 Hz Indole H6), 4.42 (s, 2H, NH2), 3.85 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6): 165.2, 162.1, 149.1, 137.3, 128.0, 122.5, 121.1, 120.7, 119.8, 110.8, 104.5, 101.6, 33.2. ESI-Mass m/z: 257 [M + H]+. Anal. calcd. for C15H14N4O2: C, 60.93; H, 4.72; N, 21.86. Found: C, 61.22; H, 4.66; N, 22.13.General procedure for the synthesis of compound 5a-mA mixture of compound 4 (1.0 mmol) and the appropriate aromatic aldehydes (1.0 mmol) in the presence of a catalytic amount of glacial acetic acid (HOAc, 0.5 mL) was heated at reflux in the absolute ethanol for 4 h and then left to cool. The resulting precipitates were filtered, dried, and finally recrystallized from ethanol to afford desired compounds 5a-m.N’-Benzylidene-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5a)Yield: 57%; light-brown solid; m.p. 219–222 ℃. 1H NMR (400 MHz, DMSO-d6): δ 8.55 (s, 1H, CONH), 8.15 (bs, 1H, ‒N = CH‒), 7.99–7.73 (m, 4H, ‒N = CH‒C6H5 H2, H4, H6, Indole H2), 7.54–7.48 (m, 4H, Indole H4, H7, ‒N = CH‒C6H5 H3, H5), 7.29–7.23 (m, 2H, Indole H5, H6), 7.08 (s, 1H, Isoxazole), 3.87 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 166.6, 159.1, 149.9, 143.7, 139.0, 137.3, 136.0, 132.0, 131.0, 130.9, 126.5, 125.1, 123.1, 121.9, 120.1, 110.9, 102.6, 100.0, 98.5, 26.0. ESI-Mass m/z: 345 [M + H]+. Anal. calcd. for C20H16N4O2: C, 69.76, H, 4.68; N, 16.27. Found: C, 69.36; H, 4.91; N, 16.02.N’-(2-Hydroxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5b)Yield: 43%; light-gray solid; m.p. 283–285 ℃. 1H NMR (400 MHz, DMSO-d6): δ 12.09 (s, 1H, CONH), 11.47 (s, 1H, ‒N = CH‒), 9.01 (s, 1H, OH), 8.72 (s, 1H, ‒N = CH‒C6H5 H3), 7.90 (d, 1H, J = 8.0 Hz, Indole H4), 7.85 (s, 1H, Indole H2), 7.70 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H5), 7.55 (d, 1H, J = 8.0 Hz, Indole H7), 7.48 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H6), 7.30 (m, 2H, Indole H5, ‒N = CH‒C6H5 H4 ), 7.21 (t, 1H, J = 8.0 Hz, Indole H6) 7.07 (s, 1H, Isoxazole), 3.88 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 167.5, 159.1, 158.2, 156.9, 149.7, 141.3, 138.2, 131.0, 129.4, 128.4, 123.2, 121.4, 120.0, 119.8, 117.9, 116.9, 110.1, 102.6, 98.2, 38.5. ESI-Mass m/z: 361 [M + H]+. Anal. calcd. for C20H16N4O3: C, 66.66, H, 4.48; N, 15.55. Found: C, 66.32; H, 4.27; N, 15.74.N’-(2-Methoxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5c)Yield: 61%; cream-white solid; m.p. 160–161 ℃. 1H NMR (400 MHz, DMSO-d6): δ 8.87 (s, 1H, CONH), 7.91–7.89 (m, 2H, ‒N = CH‒, Indole H4), 7.83 (s, 1H, Indole H2), 7.72 (m, 1H, ‒N = CH‒C6H5 H6), 7.56–7.49 (m, 1H, ‒N = CH‒C6H5 H4), 7.47–7.34 (m, 2H, Indole H5, H7), 7.28–7.21 (m, 2H, ‒N = CH‒C6H5 H5, Indole H6) 7.19–7.04 (m, 2H, ‒N = CH‒C6H5 H3, Isoxazole) 3.87 (s, 6H, CH3). 13C NMR (100 MHz, DMSO-d6): 168.7, 159.5, 159.1, 150.9, 149.3, 139.2, 138.0, 137.9, 129.5, 128.9, 124.2, 123.8, 122.5, 121.6, 119.9, 110.7, 104.5, 101.7, 100.2, 56.9, 38.4. ESI-Mass m/z: 375 [M + H]+. Anal. calcd. for C21H18N4O3: C, 67.37, H, 4.85; N, 14.96. Found: C, 67.02; H, 4.86; N, 15.14.N’-(3-Methoxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5d)Yield: 83%; light-brown solid; m.p. 183–185 ℃. 1H NMR (400 MHz, DMSO-d6): δ 8.52 (s, 1H, CONH), 8.15 (bs, 1H, ‒N = CH‒), 7.90 (d, 1H, J = 8.0 Hz, Indole H4), 7.85 (s, 1H, Indole H2), 7.56 (d, 1H, J = 8.0 Hz, Indole H7), 7.39 (t, 1H, J = 8.0 Hz, Indole H5), 7.30- 7.27 (m, 3H, Indole H6, ‒N = CH‒C6H5 H2, H6), 7.21 (t, 1H, ‒N = CH‒C6H5 H5), 7.06–7.01 (m, 2H, ‒N = CH‒C6H5 H4, Isoxazole), 3.88 (s, 3H, CH3), 3.83 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 168.6, 159.0, 159.0, 149.7, 148.1, 144.7, 139.0, 138.1, 137.3, 134.8, 128.3, 127.9, 123.5, 120.0, 114.0, 110.5, 104.5, 102.1, 100.5, 56.7, 34.9. ESI-Mass m/z: 375 [M + H]+. Anal. calcd. for C21H18N4O3: C, 67.37, H, 4.85; N, 14.96. Found: C, 67.52; H, 4.63; N, 15.24.N’-(2,3-Dimethoxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5e)Yield: 69%; cream-white solid; m.p. 232–234 ℃. 1H NMR (400 MHz, DMSO-d6): δ 11.79 (s, 1H, CONH), 8.84 (s, 1H, ‒N = CH‒), 7.91 (d, 1H, J = 8.0 Hz, Indole H4), 7.85 (s, 1H, Indole H2), 7.57 (d, 1H, J = 8.0 Hz, Indole H7), 7.50 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H6 ), 7.28 (t, 1H, J = 8.0 Hz, Indole H5), 7.23 (t, 1H, J = 8.0 Hz, Indole H6), 7.17–7.11 (m, 2H, ‒N = CH‒C6H5 H4, H5), 7.07 (s, 1H, Isoxazole), 3.91 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.85 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 158.8, 153.2, 148.5, 146.9, 143.4, 139.1, 137.3, 128.3, 125.1, 124.8, 122.5, 120.7, 119.8, 119.4, 117.5, 114.5, 110.8, 104.0, 102.5, 61.7, 56.5, 33.3. ESI-Mass m/z: 405 [M + H]+. Anal. calcd. for C22H20N4O4: C, 65.34, H, 4.98; N, 13.85. Found: C, 65.59; H, 5.30; N, 13.92.N’-(2,5-Dimethoxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5f)Yield: 43%; off-white solid; m.p. 166–168 ℃. 1H NMR (400 MHz, DMSO-d6): δ 8.85 (s, 1H, CONH), 8.10 (s, 1H, ‒N = CH‒), 7.90- 7.83 (m, 3H, Indole H2, H4, H7), 7.72 (s, 1H, ‒N = CH‒C6H5 H6), 7.54–7.22 (m, 4H, Indole H5, H6, ‒N = CH‒C6H5 H3, H4), 7.07 (s, 1H, Isoxazole), 3.84 (s, 3H, CH3) 3.78 (s, 6H, CH3, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 169.5, 160.0, 157.1, 154.4, 152.0, 143.9, 137.8, 129.0, 128.0, 124.5, 121.7, 120.2, 118.8, 114.7, 112.8, 112.8, 109.7, 106.6, 98.8, 58.1, 0.54.8, 33.6. ESI-Mass m/z: 405 [M + H]+. Anal. calcd. for C22H20N4O4: C, 65.34, H, 4.98; N, 13.85. Found: C, 65.21; H, 5.23; N, 13.47.N’-(2-Hydroxy-3-methoxybenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5g)Yield: 54%; ligt-gray solid; m.p. 268–269 ℃. 1H NMR (400 MHz, DMSO-d6): δ 12.07 (s, 1H, ‒N = CH‒C6H5 OH), 11.22 (s, 1H, CONH), 8.72 (s, 1H, ‒N = CH‒), 7.90–7.83 (m, 2H, Indole H2, H4), 7.54 (d, 1H, J = 8.0 Hz, Indole H7), 7.28–7.18 (m, 2H, Indole H5, H6), 7.09–7.03 (m, 2H, ‒N = CH‒C6H5 H4, H6), 7.09 (s, 1H, Isoxazole), 6.88 (t, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H5), 3.87 (s, 3H, N‒CH3), 3.82 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): 165.5, 154.0, 149.0, 148.4, 148.6, 141.35, 137.3, 134.4, 129.1, 128.2, 122.6, 121.9, 119.9, 119.5, 114.2, 112.7, 110.9, 107.4, 102.6, 56.3, 33.2. ESI-Mass m/z: 391 [M + H]+. Anal. calcd. for C21H18N4O4: C, 64.61, H, 4.56; N, 14.35. Found: C, 64.30; H, 4.82; N, 14.72.N’-(2-Fluorobenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5h)Yield: 64%; cream-white solid; m.p. 258–260 ℃. 1H NMR(400 MHz, DMSO-d6): δ 11.92 (s, 1H, CONH), 8.81 (s, 1H, ‒N = CH‒), 7.98 (t, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H4), 7.91 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H6), 7.85 (s, 1H, Indole H2), 7.56–7.49 (m, 2H, Indole H4, H7), 7.33- 7.27 (m, 3H, ‒N = CH‒C6H5 H3, H5, Indole H5), 7.21 (t, 1H, J = 8.0 Hz, Indole H6), 7.07 (s, 1H, Isoxazole), 3.88 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6):168.3 (d, J C-F = 245.3 Hz), 168.1, 160.0, 159.1, 149.9, 146.5, 140.2 (d, J C-F = 8.0 Hz), 139.2, 137.3, 132.2 (d, J C-F = 8.0 Hz), 128.4, 126.8, 125.4, 122.5, 120.7, 119.7 (d, J C-F = 21.5 Hz), 116.8 (d, J C-F = 21.0 Hz), 110.9, 102.6, 33.4. ESI-Mass m/z: 363 [M + H]+. Anal. calcd. for C20H15FN4O2: C, 66.29, H, 4.17; N, 15.46. Found: C, 66.51; H, 4.25; N, 15.83.N’-(2-Chlorobenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5i)Yield: 72%; off-white solid; m.p. 280–283 ℃. 1H NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H, CONH), 8.97 (s, 1H, ‒N = CH‒), 8.05 (s, 1H, ‒N = CH‒C6H5 H3), 7.90 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H6), 7.84 (s, 1H, Indole H2), 7.56- 7.53 (m, 2H, Indole H4, H7), 7.46 (m, 2H, ‒N = CH‒C6H5 H4, H5), 7.28 (t, 1H, J = 8.0 Hz, Indole H5), 7.21 (t, 1H, J = 8.0 Hz, Indole H6), 7.08 (s, 1H, Isoxazole), 3.88 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 159.0, 146.6, 143.5, 139.3, 137.3, 134.9, 132.8, 131.8, 131.4, 128.9, 128.4, 126.0, 125.1, 122.6, 120.7, 119.8, 110.9, 104.0, 102.6, 33.3. ESI-Mass m/z: 379 [M + H]+. Anal. calcd. for C20H15ClN4O2: C, 63.41, H, 3.99; N, 14.79. Found: C, 63.73; H, 4.06; N, 14.51.N’-(3-Bromobenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5j)Yield: 52%; off-white solid; m.p. 152–157 ℃. 1H NMR (400 MHz, DMSO-d6): δ 11.87 (s, 1H, CONH), 8.52 (s, 1H, ─N═CH─), 7.91–7.90 (m, 2H, Indole H4, ─N═CH─C6H4 H2), 7.85 (s, 1H, Indole H2), 7.70 (d, 1H, J = 8.0 Hz, ─N═CH─C6H4 H4), 7.64 (d, 1H, J = 8.0 Hz, Indole H7), 7.56 (d, 1H, J = 8.0 Hz, ─N═CH─C6H4 H6), 7.44 (t, 1H, J = 8.0 Hz, ─N═CH─C6H4 H5), 7.29 (t, 1H, J = 8.0 Hz, Indole H5), 7.21 (t, 1H, J = 8.0 Hz, Indole H6), 7.07 (s, 1H, Isoxazole H4), 3.88 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): 168.2, 158.8, 155.0, 145.7, 139.8, 137.9, 137.8, 131.5, 128.0, 126.7, 126.1, 125.1, 124.7, 122.7, 120.8, 120.7, 110.5, 102.6, 101.2, 33.7. ESI-Mass m/z: 424 [M + H]+. Anal. calcd. for C20H15BrN4O2: C, 56.75, H, 3.57; N, 13.24. Found: C, 56.89; H, 3.29; N, 13.44.N’-(3-Nitrobenzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5k)Yield: 64%; yellow solid; m.p. 268–270 ℃. 1H NMR (400 MHz, DMSO-d6): δ 11.99 (s, 1H, CONH), 8.66 (s, 1H, ‒N = CH‒), 8.53 (s, 1H, ‒N = CH‒C6H5 H2), 8.27 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H4), 8.14 (d, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H6), 7.90 (d, 1H, J = 8.0 Hz, Indole H4), 7.84 (s, 1H, Indole H2), 7.77 (t, 1H, J = 8.0 Hz, ‒N = CH‒C6H5 H5), 7.55 (d, 1H, J = 8.0 Hz, Indole H7), 7.28 (t, 1H, J = 8.0 Hz, Indole H5), 7.22 (t, 1H, J = 8.0 Hz, Indole H4), 7.08 (s, 1H, Isoxazole), 3.88 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 168.8, 159.8, 159.1, 148.7, 146.3, 136.4, 134.5, 131.1, 128.4, 125.1, 122.2, 120.7, 119.8, 119.7, 110.9, 108.7, 102.7, 100.1, 99.2, 33.3. ESI-Mass m/z: 390 [M + H]+. Anal. calcd. for C20H15N5O4: C, 61.69, H, 3.88; N, 17.99. Found: C, 61.61; H, 4.13; N, 17.69.N’-(4-(Methylthio)benzylidene)-5-(1-methyl-1H-indol-3-yl)isoxazole-3-carbohydrazide (5l)Yield: 54%; light-gray solid; m.p. 254–255 ℃. 1H NMR (400 MHz, DMSO-d6): δ 8.50 (s, 1H, CONH), 8.10 (bs, 1H, ‒N = CH‒), 7.89–7.83 (m, 2H, Indole H2, H4), 7.66–7.54 (m, 3H, Indole H5, H6, H7), 7.34–7.22 (m, 4H, ‒N = CH‒C6H5 H2, H3, H5, H6), 7.05 (s, 1H, Isoxazole), 3.87 (s, 3H, N‒CH3), 2.37 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): 169.9, 160.0, 154.5, 152.7, 142.7, 138.8, 134.8, 130.2, 128.9, 128.0, 127.4, 120.9, 119.9, 116.6, 113.0, 110.1, 106.4, 102.8, 98.3, 33.9, 14.7. ESI-Mass m/z: 391 [M + H]+. Anal. calcd. for C21H18N4O2S: C, 64.60, H, 4.65; N, 14.35. Found: C, 64.75; H, 4.73; N, 14.54.5-(1-Methyl-1H-indol-3-yl)-N’-((pyridin-4-yl)methylene)isoxazole-3-carbohydrazide (5m)Yield: 75%; light-brown solid; m.p. 225–227 ℃. 1H NMR (400 MHz, DMSO-d6): δ 9.39 (s, 1H, CONH), 8.58 (bs, 1H, ‒N = CH‒), 7.92- 7.67 (m, 4H, Indole H4, H7, Pyr. H3, H5), 7.51 (d, 2H, J = 8.0 Hz, ‒N = CH‒Pyr. H2, H6), 7.24 (t, 1H, J = 8.0 Hz, Indole H5), 7.16 (t, 1H, J = 8.0 Hz, Indole H6), 7.08–6.99 (m, 2H, Isoxazole, Indole H2), 3.89 (s, 3H, N‒CH3). 13C NMR (100 MHz, DMSO-d6): 169.9, 158.6, 154.1, 151.9, 148.9, 141.0, 137.3, 136.2, 131.7, 127.9, 122.0, 120.0, 118.3, 115.1, 112.8, 110.5, 101.6, 100.0, 33.1. ESI-Mass m/z: 346 [M + H]+. Anal. calcd. for C19H15N5O2: C, 66.08, H, 4.38; N, 20.28. Found: C, 65.75; H, 4.39; N, 20.60.In vitro cholinesterase inhibitory activityThe anti-cholinesterase activity of synthesized compounds was performed according to our previous study32,33.Kinetic characterization of AChE inhibitionTo elucidate the mechanism of action of compound 5d, reciprocal plots of 1/velocity versus 1/[substrate] were conducted using Ellman’s method. The substrate, acetylthiocholine, was tested at different concentrations (187.5, 750, 1500 and 3000 μM). In the assay, various concentrations of compound 5d (0, 13.36, 53.47 and 106.95 μM) were introduced into the reaction mixture, which was then preincubated with the enzyme at room temperature for 10 min before adding the substrate. Kinetic measurements were recorded at 412 nm while monitoring the enzyme catalyzed reaction. A parallel control experiment was conducted without the presence of the inhibitor34.BACE1 inhibitory activityThe investigation of the inhibitory activity of the selected compounds was performed using a FRET-based assay kit provided by Invitrogen (previously known as Pan Vera, Madison, WI). As a reference inhibitor, OM99-2 was employed. Detailed information on the methodology can be found in our previously published studies28,35.Metal Chelating ActivityMetal binding studies were conducted using an Agilent UV-2450 spectrophotometer in a 1 cm quartz cuvette, employing the mole ratio method. Investigating the metal binding capability of the chosen derivative involved recording the UV absorption of the 5d (dissolved in MeOH) with and without Fe3+ ions in the solution. The UV absorption was measured within the wavelength range of 200 to 600 nm after incubation at room temperature for 30 min. All substances were dissolved in absolute methanol, with the 5d concentration set at 20 μM and the Fe3+ concentration varying from 0 to 50 μM. The stoichiometry of the complex was determined by gradually adding FeCl3 to the methanol solution of the tested compound.Molecular dockingThe molecular docking of the synthesized molecules was conducted using the Schrödinger Suites Maestro molecular modeling platform36. X-ray crystallographic structures of AChE and BACE1 were obtained from the RCSB Protein Data Bank (www.rcsb.org), with PDB IDs of 4EY7 and 1W51, respectively37. The protein preparation wizard was used to remove co-crystallized atoms and water molecules from the proteins to construct the protein structures38. The prime tool was used to add any missing loops, and the terminals were then sealed39. Additionally, pH 7.4 was used to create hetero atom states using EPIK, and pH 7.4 was used to assign H-bonding interactions using PROPKA. ChemDraw (ver. 16) was used to create the 2D structures of the ligands, which were then exported as SDF files. The SDF files were created using the OPLS3e force field and the ligand preparation wizard. Using an induced-fit docking approach40, the interactions of the ligands were studied. The receptor and ligand’s van der Waals radii were set at 0.7 and 0.5, respectively.Molecular dynamics simulationsThe starting model was obtained by imposing the induced fit docking to BACE1 (PDB ID: 1W51) and AChE (4EY7). MD simulations were conducted using the Desmond v5.3 of Schrodinger’s suit maestro according to previously reported procedures41,42.