New class of thio/semicarbazide-based benzyloxy derivatives as selective class of monoamine oxidase-B inhibitors

Chemicals

For the synthesis of 4-(3-fluorobenzyloxy)benzaldehyde, 4-benzyloxy benzaldehyde, 3-benzyloxy benzaldehyde, and 2-benzyloxy benzaldehyde were purchased from TCI Chemicals (Chennai, Tamil Nadu, India). Thiosemicarbazide, methyl thiosemicarbazide, semicarbazide hydrochloride, phenyl thiosemicarbazide, recombinant human MAO-A, MAO-B, benzylamine, kynuramine, safinamide mesylate salt, pargyline, clorgyline, and toloxatone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate (dibasic and monobasic anhydrous) was purchased from Daejung Chemicals and Metals Co. Ltd. (Siheung, Korea). The DiaEasy™ Dialysis kit (6–8 kDa) was purchased from BioVision (St. Louis, MA, USA)³¹. Melting point (Digital Melting Point Apparatus, Amtech India, Ambala Cantt Haryana, India, 133001), 1H nuclear magnetic resonance (NMR), 13C NMR (Bruker Advance Neo 500 MHz NMR spectrometer, Billerica, MA, USA), and mass spectra (Waters Xevo G2-XS QTOF, Milford, MA, USA) were recorded to characterize the synthesized compounds.

Chemical synthesis

Benzyloxybenzene-derived thio-/semicarbazide-based derivatives were synthesized in a microwave reactor Monowave 50 (Anton Paar, Graz, Austria) by reacting benzyloxybenzene derivatives (1 equivalent) with thio-/semicarbazide derivatives (1.3 equivalent) using ethanol as the solvent and glacial acetic acid as the catalyst. The preparation was maintained at 80 ֯C for 5 min for thio derivatives and between 80 and 120 ֯C for 6 min for semicarbazide derivatives (Scheme 1). The solid product obtained as a precipitate was washed with ethanol and the dried product was purified by flash chromatography. Thin-layer chromatography (TLC) was performed to confirm each reaction using precoated TLC plates (silica gel, 60–120 #). Hexane: ethyl acetate (2:1) was used as the solvent system for the thio derivatives, whereas hexane: ethyl acetate (1:2) was used for the semicarbazide derivatives.

2-(4-((3-fluorobenzyl)oxy)benzylidene)hydrazine-1-carbothioamide (BT1)

Pale yellow; Percentage Yield: 86%; MP: 154–156 °C; Rf: 0.67. ¹H NMR (500 MHz, CDCl₃) δ: 10.03 (s, 1 H, –NH-N=), 7.88 (s, 1 H, -N = CH-), 7.60–7.57 (d, 2 H, J = 15 Hz, Ar-H), 7.37–7.32 (m, 1 H, J = 25 Hz, Ar-H), 7.20–7.17 (m, 2 H, J = 15 Hz, Ar-H), 7.15–7.13 (d, 1 H, J = 10 Hz, Ar-H), 7.04-7.00 (m, 1 H, J = 20 Hz, Ar-H), 6.98–6.95 (d, 2 H, J = 15 Hz, Ar-H), 6.50 (s, 1 H, H₂N-C = S), 5.14 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 178.02, 163.98, 162.02, 160.59, 143.97, 138.98, 138.92, 130.28, 130.21, 129.20, 126.12, 122.73, 115.14, 69.26, 29.70. Molecular formula: C₁₅H₁₄FN₃OS, (ESI) Calculated MW = 303.35, observed MW = 303.3498.

2-(4-((3-fluorobenzyl)oxy)benzylidene)-N-methylhydrazine-1-carbothioamide (BT2)

White; Percentage Yield: 82%; MP: 135–137 °C; Rf: 0.70. ¹H NMR (500 MHz, CDCl₃) δ: 9.23 (s, 1 H, –NH-N=), 7.73 (s, 1 H, -N = CH-), 7.60–7.57 (d, 2 H, J = 15 Hz, Ar-H), 7.43 (s, 1 H, CH₃–NH-), 7.37–7.33 (m, 1 H, J = 20 Hz, Ar-H), 7.19–7.18 (d, 1 H, J = 5 Hz, Ar-H), 7.16–7.14 (d, 1 H, J = 10 Hz, Ar-H), 7.08-7.00 (m, 1 H, J = 40 Hz, Ar-H), 6.99–6.96 (d, 2 H, J = 15 Hz, Ar-H), 5.10 (s, 2 H, -O-CH₂-Ph), 3.26–3.25 (d, 3 H, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ: 178.26, 164.08, 162.12, 160.46, 142.17, 139.09, 132.11, 130.36, 129.03, 126.40, 122.80, 115.33, 114.23, 69.36, 32.01, 31.28. Molecular formula: C₁₆H₁₆FN₃OS, (ESI) Calculated MW = 317.38, observed MW = 317.3799.

2-(4-((3-fluorobenzyl)oxy)benzylidene)hydrazine-1-carboxamide (BT3)

White; Percentage Yield: 81%; MP: 1110 –112 °C; Rf: 0.58. ¹H NMR (500 MHz, DMSO) δ: 10.12 (s, 1 H, -N = CH-), 7.79 (s, 1 H, –NH-N=), 7.67–7.65 (d, 2 H, J = 10 Hz, Ar-H), 7.46–7.42 (m, 1 H, J = 10 Hz, Ar-H), 7.30–7.27 (m, 2 H, J = 15 Hz, Ar-H), 7.18–7.14 (m, 1 H, J = 10 Hz, Ar-H), 7.04–7.02 (d, 2 H, J = 10 Hz, Ar-H), 6.43 (s, 2 H, H₂N-C = O), 5.16 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, DMSO) δ: 163.05, 161.12, 158.74, 156.74, 139.77, 138.93, 130.40, 127.93, 127.73, 123.42, 114.83, 114.41, 114.22, 114.05, 68.31. Molecular formula: C₁₅H₁₄FN₃O₂, (ESI) Calculated MW = 287.28, observed MW = 287.2798.

2-(4-((3-fluorobenzyl)oxy)benzylidene)-N-phenylhydrazine-1-carbothioamide (BT4)

Pale brown; Percentage Yield: 85%; MP: 1124 –126 °C; Rf: 0.63. ¹H NMR (500 MHz, DMSO) δ: 11.72 (s, 1 H, –NH-N=), 10.04 (s, 1 H, Ph-NH-C = S), 8.12 (s, 1 H, -N = CH-), 7.87–7.85 (d, 2 H, J = 10 Hz, Ar-H), 7.59–7.57 (d, 2 H, J = 10 Hz, Ar-H), 7.46–7.42 (m, 1 H, J = 20 Hz, Ar-H), 7.38–7.35 (m, 2 H, J = 15 Hz, Ar-H), 7.31–7.28 (m, 2 H, J = 15 Hz, Ar-H), 7.21–7.18 (m, 1 H, J = 15 Hz, Ar-H), 7.16–7.14 (m, 1 H, J = 10 Hz, Ar-H), 7.08–7.06 (d, 2 H, J = 10 Hz, Ar-H), 5.19 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, DMSO) δ: 175.55, 163.07, 161.13, 159.58, 142.63, 139. 66, 139.02, 130.43, 130.36, 129.21, 127.91, 126.85, 125.66, 125.10, 123.48, 123.46, 114.92, 114.63, 114.47, 114.10, 68.39. Molecular formula: C₂₁H₁₈FN₃OS, (ESI) Calculated MW = 379.45, observed MW = 379.4498.

2-(4-(benzyloxy)benzylidene)hydrazine-1-carbothioamide (BT5)

Pale yellow; Percentage Yield: 86%; MP: 132–134 °C; Rf: 0.51. ¹H NMR (500 MHz, CDCl₃) δ: 9.86 (s, 1 H, –NH-N=), 7.85 (s, 1 H, -N = CH-), 7.59–7.57 (d, 2 H, J = 10 Hz, Ar-H), 7.42–7.38 (m, 4 H, J = 20 Hz, Ar-H), 7.37–7.32 (m, 1 H, J = 25 Hz, Ar-H), 7.08–7.07 (d, 1 H, J = 5 Hz, Ar-H), 6.99–6.97 (d, 2 H, J = 10 Hz, Ar-H), 6.43 (s, 1 H, H₂N-C = S), 5.09 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 178.14, 161.02, 143.98, 136.40, 132.09, 129.23, 128.81, 128.75, 128.41, 128.27, 127.54, 125.89, 115.34, 115.24, 70.20. Molecular formula: C₁₅H₁₅N₃OS, (ESI) Calculated MW = 285.36, observed MW = 285.3599.

2-(4-(benzyloxy)benzylidene)-N-methylhydrazine-1-carbothioamide (BT6)

Pale yellow; Percentage Yield: 81%; MP: 139–141 °C; Rf: 0.55. ¹H NMR (500 MHz, DMSO) δ: 11.36 (s, 1 H, –NH-N=), 8.43–8.42 (d, 1 H, J = 10 Hz, Ar-H), 8.00 (s, 1 H, -N = CH-), 7.76–7.73 (d, 2 H, J = 15 Hz, Ar-H), 7.47–7.45 (d, 2 H, J = 10 Hz, Ar-H), 7.41–7.38 (m, 2 H, J = 15 Hz, Ar-H), 7.35–7.32 (m, 1 H, –NH-), 7.07–7.04 (d, 2 H, J = 15 Hz, Ar-H), 5.15 (s, 2 H, -O-CH₂-Ph), 3.02–3.01 (d, 3 H, CH₃). ¹³C NMR (125 MHz, DMSO) δ: 177.42, 159.57, 141.44, 136.66, 128.66, 128.32, 127.79, 127.64, 126.96, 114.87, 69.24, 30.65. Molecular formula: C₁₆H₁₇N₃OS, (ESI) Calculated MW = 299.39, observed MW = 299.3899.

2-(4-(benzyloxy)benzylidene)hydrazine-1-carboxamide (BT7)

White; Percentage Yield: 78%; MP: 117–119 °C; Rf: 0.56. ¹H NMR (500 MHz, DMSO) δ: 10.11 (s, 1 H, -N = CH-), 7.79 (s, 1 H, –NH-N=), 7.66–7.64 (d, 2 H, J = 10 Hz, Ar-H), 7.46–7.44 (d, 2 H, J = 10 Hz, Ar-H), 7.40–7.38 (m, 2 H, J = 10 Hz, Ar-H), 7.35–7.31 (m, 1 H, J = 20 Hz, Ar-H), 7.03-7.00 (d, 2 H, J = 15 Hz, Ar-H), 6.43 ( s, 2 H, H₂N-C = O), 5.13 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, DMSO) δ: 158.99, 156.75, 138.99, 136.76, 128.32, 127.91, 127.76, 127.62, 127.55, 114.81, 69.18. Molecular formula: C₁₅H₁₅N₃O₂, (ESI) Calculated MW = 269.29, observed MW = 269.2898.

2-(4-(benzyloxy)benzylidene)-N-phenylhydrazine-1-carbothioamide (BT8)

Pale brown; Percentage Yield: 79%; MP: 130–132 °C; Rf: 0.50. ¹H NMR (500 MHz, CDCl₃) δ: 10.28 (s, 1 H, –NH-N=), 9.17 (s, 1 H, Ph-NH-C = S), 7.91 (s, 1 H, -N = CH-), 7.66–7.64 (d, 2 H, J = 10 Hz, Ar-H), 7.61–7.59 (d, 2 H, J = 10 Hz, Ar-H), 7.42–7.37 (m, 6 H, J = 25 Hz, Ar-H), 7.34–7.31 (t, 1 H, J = 15 Hz, Ar-H), 7.25–7.22 (t, 1 H, J = 15 Hz, Ar-H), 7.00-6.98 (d, 2 H, J = 10 Hz, Ar-H), 5.09 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 175.41, 160.88, 143.11, 137.89, 136.35, 129.15, 128.78, 128.72, 128.67, 128.19, 127.48, 126.15, 125.97, 124.59, 115.27, 70.13. Molecular formula: C₂₁H₁₉N₃OS, (ESI) Calculated MW = 361.46, observed MW = 361.4598.

2-(3-(benzyloxy)benzylidene)hydrazine-1-carbothioamide (BT9)

Pale yellow; Percentage Yield: 79%; MP: 127–129 °C; Rf: 0.63. ¹H NMR (500 MHz, CDCl₃) δ: 10.18 (s, 1 H, –NH-N=), 7.89 (s, 1 H, -N = CH-), 7.44–7.42 (d, 3 H, J = 10 Hz, Ar-H), 7.41–7.38 (m, 3 H, J = 15 Hz, Ar-H), 7.34–7.31 (m, 2 H, J = 15 Hz, Ar-H), 7.30–7.28 (d, 2 H, J = 10 Hz, Ar-H), 7.20–7.19 (d, 2 H, J = 5 Hz, Ar-H), 7.03–7.01 (d, 1 H, J = 10 Hz, Ar-H), 6.55 (s, 1 H, H₂N-C = S), 5.08 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 159.18, 143.99, 136.65, 134.42, 130.02, 128.72, 128.21, 127.57, 120.98, 117.66, 113.01, 70.28. Molecular formula: C₁₅H₁₅N₃OS, (ESI) Calculated MW = 285.36, observed MW = 285.3598.

2-(3-(benzyloxy)benzylidene)-N-methylhydrazine-1-carbothioamide (BT10)

Pale yellow; Percentage Yield: 80%; MP: 145–146 °C; Rf: 0.65. ¹H NMR (500 MHz, CDCl₃) δ: 9.79 (s, 1 H, –NH-N=), 7.80 (s, 1 H, -N = CH-), 7.47–7.45 (d, 3 H, J = 10 Hz, Ar-H), 7.44–7.41 (m, 2 H, J = 15 Hz, Ar-H), 7.40–7.38 (d, 1 H, J = 10 Hz, Ar-H), 7.35–7.33 (d, 1 H, J = 10 Hz, Ar-H), 7.32–7.28 (m, 1 H, –NH-), 7.20–7.18 (d, 1 H, J = 10 Hz, Ar-H), 7.02-7.00 (m, IH, J = 10 Hz, Ar-H), 5.09 (s, 2 H, -O-CH₂-Ph), 3.26–3.25 (d, 3 H, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ: 178.29, 159.14, 142.26, 136.63, 134.71, 129.93, 128.66, 128.15, 127.53, 120.70, 117.06, 112.93, 70.25, 31.19. Molecular formula: C₁₆H₁₇N₃OS, (ESI) Calculated MW = 299.39, observed MW = 299.3899.

2-(3-(benzyloxy)benzylidene)hydrazine-1-carboxamide (BT11)

White; Percentage Yield: 80%; MP: 102–104 °C; Rf: 0.52. ¹H NMR (500 MHz, DMSO) δ: 10.27 (s, 1 H, -N = CH-), 7.82 (s, 1 H, –NH-N=), 7.48–7.46 (m, 3 H, J = 10 Hz, Ar-H), 7.41–7.38 (m, 2 H, J = 15 Hz, Ar-H), 7.35–7.33 (d, 1 H, J = 10 Hz, Ar-H), 7.32–7.28 (m, 1 H, J = 20 Hz, Ar-H), 7.22–7.20 (d, 1 H, J = 10 Hz, Ar-H), 7.00-6.97 (m, 1 H, J = 15 Hz, Ar-H), 6.55 (s, 2 H, H₂N-C = O), 5.14 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, DMSO) δ: 158.59, 156.74, 139.06, 136.96, 136.19, 129.60, 128.35, 127.79, 127.75, 119.85, 115.95, 111.44, 69.25. Molecular formula: C₁₅H₁₅N₃O₂, (ESI) Calculated MW = 269.29, observed MW = 269.2899.

2-(3-(benzyloxy)benzylidene)-N-phenylhydrazine-1-carbothioamide (BT-12)

Pale yellow; Percentage Yield: 77%; MP: 156–158 °C; Rf: 0.62. ¹H NMR (500 MHz, CDCl₃) δ: 10.40 (s, 1 H, –NH-N=), 9.15 (s, 1 H, Ph-NH-C = S), 7.93 (s, 1 H, -N = CH-), 7.64–7.63 (d, 2 H, J = 5 Hz, Ar-H), 7.44–7.40 (m, 3 H, J = 20 Hz, Ar-H), 7.39–7.35 (m, 3 H, J = 20 Hz, Ar-H), 7.33–7.29 (t, 3 H, J = 20 Hz, Ar-H), 7.27–7.21 (m, 2 H, J = 30 Hz, Ar-H), 7.04–7.02 (m, 1 H, J = 10 Hz, Ar-H), 5.09 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 175.83, 159.19, 143.10, 137.80, 136.66, 134.54, 130.03, 128.89, 128.71, 128.18, 127.53, 126.41, 124.82, 120.93, 117.54, 113.05, 70.28. Molecular formula: C₂₁H₁₉N₃OS, (ESI) Calculated MW = 361.46, observed MW = 361.4599.

2-(2-(benzyloxy)benzylidene)hydrazine-1-carbothioamide (BT13)

Pale yellow; Percentage Yield: 86%; MP: 125–127 °C; Rf: 0.48. ¹H NMR (500 MHz, CDCl₃) δ: 9.84 (s, 1 H, –NH-N=), 8.34 (s, 1 H, -N = CH-), 7.80–7.79 (d, 1 H, J = 5 Hz, Ar-H), 7.41–7.39 (d, 4 H, J = 10 Hz, Ar-H), 7.36–7.32 (m, 2 H, J = 20 Hz, Ar-H), 7.14 (s, 1 H, Ar-H), 6.99–6.93 (m, 2 H, J = 30 Hz, Ar-H), 6.56 (s, 1 H, H₂N-C = S), 5.06 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 178.23, 157.62, 140.32, 136.29, 131.99, 128.77, 128.23, 127.43, 126.59, 121.96, 121.13, 112.68, 70.45. Molecular formula: C₁₅H₁₅N₃OS, (ESI) Calculated MW = 285.36, observed MW = 285.3598.

2-(2-(benzyloxy)benzylidene)-N-methylhydrazine-1-carbothioamide (BT14)

Pale yellow; Percentage Yield: 76%; MP: 142–144 °C; Rf: 0.66. ¹H NMR (500 MHz, CDCl₃) δ: 9.36 (s, 1 H, –NH-N=), 8.23 (s, 1 H, -N = CH-), 7.83–7.81 (d, 1 H, J = 10 Hz, Ar-H), 7.42–7.39 (d, 5 H, J = 15 Hz, Ar-H), 7.37–7.32 (m, 2 H, J = 25 Hz, Ar-H), 7.00-6.97 (t, 2 H, J = 15 Hz, Ar-H), 5.09 (s, 2 H, -O-CH₂-Ph), 3.21–3.20 (d, 3 H, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ: 178.34, 157.46, 138.70, 136.31, 131.67, 128.75, 128.24, 127.42, 126.44, 122.17, 121.12, 112.67, 70.49, 31.14. Molecular formula: C₁₆H₁₇N₃OS, (ESI) Calculated MW = 299.39, observed MW = 299.3898.

2-(2-(benzyloxy)benzylidene)hydrazine-1-carboxamide (BT15)

White; Percentage Yield: 77%; MP: 119–121 °C; Rf: 0.56. ¹H NMR (500 MHz, CDCl₃) δ: 9.04 (s, 1 H, -N = CH-), 8.19 (s, 1 H, -NH-N=), 7.81–7.80 (d, 1 H, J = 5 Hz, Ar-H), 7.41–7.36 (m, 4 H, J = 25 Hz, Ar-H), 7.33–7.28 (m, 2 H, J = 25 Hz, Ar-H), 6.98–6.96 (d, 1 H, J = 10 Hz, Ar-H), 6.95–6.92 (t, 1 H, J = 15 Hz, Ar-H), 5.19 (s, 2 H, H₂N-C = O), 5.09 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 157.55, 156.98, 137.88, 136.59, 130.94, 128.66, 128.12, 127.38, 126.21, 122.89, 121.10, 112.69, 70.46. Molecular formula: C₁₅H₁₅N₃O₂, (ESI) Calculated MW = 269.29, observed MW = 269.2897.

2-(2-(benzyloxy)benzylidene)-N-phenylhydrazine-1-carbothioamide (BT16)

Pale yellow; Percentage Yield: 80%; MP: 145–147 °C; Rf: 0.67. ¹H NMR (500 MHz, CDCl₃) δ: 9.39 (s, 1 H, –NH-N=), 9.18 (s, 1 H, Ph-NH-C = S), 8.31 (s, 1 H, -N = CH-), 7.88–7.87 (d, 1 H, J = 5 Hz, Ar-H), 7.66–7.64 (d, 2 H, J = 10 Hz, Ar-H), 7.42–7.32 (m, 8 H, Ar-H), 7.25–7.21 (m, 1 H, J = 20 Hz, Ar-H), 7.03–6.99 (m, 2 H, J = 20 Hz, Ar-H), 5.12 (s, 2 H, -O-CH₂-Ph). ¹³C NMR (125 MHz, CDCl₃) δ: 175.73, 157.67, 139.11, 137.92, 136.21, 132.07, 128.78, 128.76, 128.31, 127.40, 126.46, 126.05, 124.29, 121.90, 121.22, 112.74, 70.56. Molecular formula: C₂₁H₁₉N₃OS, (ESI) Calculated MW = 361.46, observed MW = 361.4598.

Enzyme assays and kinetics

The activities of MAO-A and MAO-B were assayed using kynuramine (0.06 mM) and benzylamine (0.30 mM), respectively, by continuously measuring the absorbance changes at 316 and 250 nm, respectively³². K_m values were determined by assaying five substrate concentrations and analyzing Lineweaver–Burk (LB) plots.

Inhibition studies of MAO-A and MAO-B

As an initial screening step for inhibition study and inhibition kinetics, residual activity was analyzed by measuring the absorbance change in the presence of 10 µM of the inhibitor. IC₅₀ values were determined for potential compounds with residual activity 50 was set to 40 µM. The selectivity index (SI) for MAO-B was calculated by IC₅₀ of MAO-A the IC₅₀ of MAO-B³³. The inhibition types of the leading compounds, BT1 and BT5, for MAO-B were determined at three inhibitor concentrations (~ 1/2×, 1×, and 2× IC₅₀) as well as five different substrate concentrations ranging from 0.0375 to 0.6 µM³⁴. Inhibition patterns and K_i values were determined by comparing the Lineweaver–Burk lots with their secondary plots, respectively³⁵. Toloxatone, clorgyline, safinamide, and pargyline were used as the reference compounds.

Reversibility studies

The reversibilities of BT1 and BT5 for MAO-B were evaluated by comparing the undialyzed (A_U) and dialyzed (A_D) residual activities at a concentration of about 2-times the IC₅₀ after pre-incubation for 30 min prior to measurement. Two reference inhibitors were used: a reversible MAO-B inhibitor, safinamide mesylate, and an irreversible MAO-B inhibitor pargyline³⁶.

PAMPA for blood–brain barrier (BBB) permeation study

Early drug research employed a PAMPA to predict passive drug transcellular permeability across the BBB. A sandwich-like structure was established in the PAMPA using a 96-well microtiter plate and 96-well filter plate (IPVH, 125 μm thick filter, 0.45 μm pore; Millipore, Billerica, MA, USA). The plates were submerged in 0.1 mL of n-dodecane. Stock solutions of drug samples in Dimethyl sulfoxide (DMSO) were prepared at 10 mM dosages and stored at 0 °C before use. The stock solution was further diluted in a buffer (pH 7.4) before being added to a 96-well filter plate. This allowed for the attainment of a final sample concentrations of 0.01, 0.1, 0.5, and 1 mM and the restriction of the DMSO content to 1% (v/v). Two hundred microliters of a pH 7.4 buffer were added to the acceptor well and the final diluted solutions were transferred in 270 µL aliquots to the donor wells. In order for the plates to create a “sandwich,” the donor plate had to be precisely positioned over the acceptor filter plate. The donor plate contains an artificial lipid barrier in the middle, an aqueous recipient on top, and an aqueous donor carrying an analyte on bottom. Via the lipid membrane, the test material diffuses from the donor well and into the acceptor well. At the time of the breach, the “sandwich” was allegedly intact. Ultraviolet spectroscopy was used to determine drug concentrations in the donor, recipient, and reference wells. The degree of penetration was calculated as follows³⁷:

Log Pe = -ln [1-C_A /E_quilibrium]/A× (1/V_D + 1/V_A) × t.

where Pe is permeability (cm. s^− 1), C_A is the receptor concentration, A is the area of effective filtering (4.8 cm²), V_D is the donor volume (mL), V_A is the acceptor volume (mL), t is the time of incubation (s), and E_quilibrium= (C_D×V_D + C_A×V_A)/(V_D+V_A).

In-vitro cytotoxicity and antioxidant assays

The cytotoxicity of the lead molecules BT1, BT3, BT5, BT6, and BT7 were analyzed using the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The antioxidant effects were evaluated by determining the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) in IMR 32 cells^38,39,40. The detailed procedure is described in the Supporting Information.

MAO-B prediction

The recently created web program MAO-B-pred (https://mao-b-pred.streamlit.app/) was used to evaluate the bioactivity of each compound against MAO-B. The expected values for the compounds were compared with those of standard MAO-B inhibitors (Safinamide and Pargyline)⁴¹.

Molecular docking

The promising compounds, BT1, BT3, BT5, BT6, and BT7 were subjected to molecular docking analysis using the Schrödinger Glide docking module within the hMAO-B enzyme (PDB ID: 2V5Z) -crystallized with safinamide⁴². The protein crystal structure was generated using a three-step Protein Preparation Wizard, which includes preprocessing, optimization, and protein energy minimization phases⁴³. LigPrep was used to prepare the ligands for the docking studies. A receptor grid-generating module and grid file were generated⁴⁴. The extra-precision (XP) mode was employed for ligand docking.

MD simulation

MD studies were performed for the top-docking postures (lowest negative scores) of the lead compound BT1 by applying the Desmond MD simulation program and NVIDIA Quadro 6000 graphics processing unit⁴⁵. Using the default values for the systems under investigation, 1000 frame trajectories were created to investigate interactions between the protein and ligand. The MD generation was 100 ns, with coordinates captured at 100 ps. Prior research has indicated the value of additional information for MD investigations, such as box type, estimates of long- and short-range interactions, and thermostat and barometer settings^46,47,48,49.