Materials and instrumentation

Reagents required for the chemical synthesis were purchased from Merck. Deuterated dimethyl sulfoxide (DMSO-d6) for use in NMR spectroscopy was supplied by Merck. A Bruker Avance III 600 spectrometer was used to record 1H and 13C NMR spectra at 600 and 150 MHz, respectively. DMSO-d6 was used as the solvent for NMR analysis. The multiplicities of NMR signals are given as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), and multiplet (m) while the chemical shift (δ) is given in parts per million (ppm), referenced to the solvent signal at 2.5 and 39.5 ppm for 1H and 13C NMR, respectively. The coupling constant (J) is reported in hertz (Hz). The chemical reactions were monitored with thin layer chromatography (TLC). Acetone was used to dissolve the reagents and products before applying them to the TLC sheets. Compounds 1a–f and 2a–g were analysed using aluminium TLC sheets coated with silica gel 60 containing UV254 fluorescent indicator (Merck). The mobile phase consisted of n-hexane and ethyl acetate (3:2). An ultraviolet (UV) light at a wavelength of 254 nm was used to visualise the developed TLC sheets. Melting points (mp) were measured using a Büchi melting point B-545 instrument and are reported uncorrected. High resolution mass spectra (HRMS) were recorded using a Bruker micrOTOF-Q II mass spectrometer operating in the atmospheric-pressure chemical ionisation (APCI) mode.

HPLC analysis was performed to estimate the purities of the synthesised compounds. This was done according to the published protocol [31]. Enzymes and substrates that were required for the biochemical studies were purchased from Merck. A SpectraMax iD3 multi-mode microplate reader (Molecular Devices) and Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) were used to measure the fluorescence intensities.

Single crystal X-ray diffraction analysis

X-ray diffraction analysis was carried out with a Bruker D8 Quest Eco single crystal diffractometer (SC-XRD) with graphite monochromated MoKα radiation (λ = 0.71076 Å). A plate-like specimen of compound 2b, measuring approximately 0.200 mm × 0.200 mm × 0.200 mm was used and the X-ray intensity data were measured at room temperature. A total of 726 frames were collected and the total exposure time was 2.02 h. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The structure was solved and refined using the Bruker SHELXTL Software Package [32, 33]. The supplementary crystallographic data (CCDC 2408388) for this paper can be found at the Cambridge Crystallographic Data Centre (CCDC).

The synthesis of 2-methylbenzo[d]oxazole derivatives (1a–f, 2a–g)

The procedure for the synthesis of 2-methylbenzo[d]oxazole derivatives (1a–f, 2a–g) has been reported [34]. DMF (10 mL) was used as solvent. 2-Methylbenzo[d]oxazol-6-ol (4) or 2-methylbenzo[d]oxazol-5-ol (5) (3.35 mmol) was dissolved in DMF in a round bottom flask and the appropriate substituted benzyl bromide (4.54 mmol) was added. Following the addition of anhydrous potassium carbonate (K2CO3; 6.05 mmol), the reaction was stirred for 24 h at room temperature. After completion of the reaction, 50 mL of ethanol was added to the reaction mixture and the solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue and the resulting solution was allowed to recrystallise. After filtering and washing with n-hexane, the crystals were allowed to air dry.

6-(Benzyloxy)-2-methylbenzo[d]oxazole (1a)

The title compound (thin white crystal shards) was afforded from 2-methylbenzo[d]oxazol-6-ol and benzyl bromide. Yield: 14%, mp: 57–59 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.57–7.51 (m, 1H), 7.48 (d, J = 7.2 Hz, 2H), 7.44–7.38 (m, 2H), 7.38–7.29 (m, 2H), 7.00 (d, J = 7.9 Hz, 1H), 5.16 (s, 2H), 2.56 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.21, 156.70, 151.61, 137.30, 135.31, 128.90, 128.35, 128.25, 119.49, 113.25, 97.18, 70.46, 14.48. Purity: 98%. APCI-HRMS m/z [MH+] 240.1011 (Calc. for C15H14NO2: 240.1019).

6-((4-Bromobenzyl)oxy)-2-methylbenzo[d]oxazole (1b)

The title compound (small white crystal shards) was afforded from 2-methylbenzo[d]oxazol-6-ol and 4-bromobenzyl bromide. Yield: 37%, mp: 75–77 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.60 (d, J = 7.4 Hz, 2H), 7.53 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 7.4 Hz, 2H), 7.39–7.33 (m, 1H), 7.00 (d, J = 8.7 Hz, 1H), 5.14 (s, 2H), 2.56 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.29, 156.46, 151.58, 136.80, 135.43, 131.83, 130.34, 121.46, 119.53, 113.25, 97.27, 69.63, 14.48. Purity: 99%. APCI-HRMS m/z [MH+] 318.0126 (Calc. for C15H13BrNO2: 318.0124).

4-(((2-Methylbenzo[d]oxazol-6-yl)oxy)methyl)benzonitrile (1c)

The title compound (yellow/beige powder-like crystals) was afforded from 2-methylbenzo[d]oxazol-6-ol and 4-(bromomethyl)benzonitrile. Yield: 77%, mp: 120–122 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.88 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 8.5 Hz, 1H), 7.37 (s, 1H), 7.02 (d, J = 8.5 Hz, 1H), 5.28 (s, 2H), 2.56 (d, J = 2.8 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.38, 156.28, 151.56, 143.14, 135.57, 132.88, 128.61, 119.59, 119.19, 113.20, 111.00, 97.32, 69.47, 14.48. Purity: 100%. APCI-HRMS m/z [MH+] 265.1002 (Calc. for C16H13N2O2: 265.0990).

2-Methyl-6-((4-nitrobenzyl)oxy)benzo[d]oxazole (1d)

The title compound (small yellow/beige granules) was afforded from 2-methylbenzo[d]oxazol-6-ol and 4-nitrobenzyl bromide. Yield: 82%, mp: 135–137 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.27 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 2.3 Hz, 1H), 7.04 (dd, J = 8.8, 2.3 Hz, 1H), 5.34 (s, 2H), 2.56 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.42, 156.24, 151.56, 147.50, 145.25, 135.61, 128.78, 124.07, 119.62, 113.22, 97.36, 69.25, 14.48. Purity: 100%. APCI-HRMS m/z [MH+] 285.0865 (Calc. for C15H13N2O4: 285.0870).

6-((4-Chlorobenzyl)oxy)-2-methylbenzo[d]oxazole (1e)

The title compound (yellowish clumpy crystals) was afforded from 2-methylbenzo[d]oxazol-6-ol and 4-chlorobenzyl bromide. Yield: 28%, mp: 66–68 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.56–7.48 (m, 3H), 7.48–7.44 (m, 2H), 7.39–7.34 (m, 1H), 7.00 (d, J = 8.7 Hz, 1H), 5.16 (s, 2H), 2.56 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.29, 156.48, 151.58, 136.37, 135.42, 132.93, 130.05, 128.91, 119.53, 113.25, 97.26, 69.59, 14.48. Purity: 98%. APCI-HRMS m/z [MH+] 274.0618 (Calc. for C15H13ClNO2: 274.0629).

2-Methyl-6-((4-methylbenzyl)oxy)benzo[d]oxazole (1f)

The title compound (sparkly white dense powder-like granules) was afforded from 2-methylbenzo[d]oxazol-6-ol and 4-methylbenzyl bromide. Yield: 58%, mp: 90–92 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.52 (d, J = 8.2 Hz, 1H), 7.40–7.32 (m, 3H), 7.20 (d, J = 7.5 Hz, 2H), 6.98 (d, J = 8.6 Hz, 1H), 5.10 (s, 2H), 2.56 (s, 3H), 2.31 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 163.16, 156.72, 151.60, 137.59, 135.26, 134.25, 129.44, 128.35, 119.46, 113.27, 97.16, 70.37, 21.23, 14.47. Purity: 100%. APCI-HRMS m/z [MH+] 254.1177 (Calc. for C16H16NO2: 254.1176).

5-(Benzyloxy)-2-methylbenzo[d]oxazole (2a)

The title compound (fluffy off-white crystal shards) was afforded from 2-methylbenzo[d]oxazol-5-ol and benzyl bromide. Yield: 36%, mp: 88–90 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.54 (d, J = 8.8 Hz, 1H), 7.49–7.45 (m, 2H), 7.42–7.37 (m, 2H), 7.35–7.31 (m, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.00 (dd, J = 8.8, 2.6 Hz, 1H), 5.15 (s, 2H), 2.57 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.12, 156.09, 145.49, 142.46, 137.54, 128.86, 128.25, 128.17, 113.63, 110.97, 104.42, 70.47, 14.63. Purity: 98%. APCI-HRMS m/z [MH+] 240.1017 (Calc. for C15H14NO2: 240.1019).

5-((4-Bromobenzyl)oxy)-2-methylbenzo[d]oxazole (2b)

The title compound (off-white clumpy crystals) was afforded from 2-methylbenzo[d]oxazol-5-ol and 4-bromobenzyl bromide. Yield: 51%, mp: 91–93 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.59 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 2.5 Hz, 1H), 6.99 (dd, J = 8.9, 2.5 Hz, 1H), 5.13 (s, 2H), 2.57 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.18, 155.85, 145.56, 142.45, 137.03, 131.79, 130.27, 121.35, 113.64, 111.01, 104.51, 69.64, 14.64. Purity: 94%. APCI-HRMS m/z [MH+] 318.0137 (Calc. for C15H13BrNO2: 318.0124).

4-(((2-Methylbenzo[d]oxazol-5-yl)oxy)methyl)benzonitrile (2c)

The title compound (off-white crystal shards) was afforded from 2-methylbenzo[d]oxazol-5-ol and 4-(bromomethyl)benzonitrile. Yield: 75%, mp: 124–126 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.86 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 2.5 Hz, 1H), 7.02 (dd, J = 8.8, 2.5 Hz, 1H), 5.27 (s, 2H), 2.57 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.26, 155.69, 145.66, 143.39, 142.47, 132.84, 128.55, 119.19, 113.61, 111.08, 110.93, 104.53, 69.50, 14.63. Purity: 97%. APCI-HRMS m/z [MH+] 265.0981 (Calc. for C16H13N2O2: 265.0972).

2-Methyl-5-((4-nitrobenzyl)oxy)benzo[d]oxazole (2d)

The title compound (yellow/orange crystal shards) was afforded from 2-methylbenzo[d]oxazol-5-ol and 4-nitrobenzyl bromide. Yield: 20%, mp: 153–156 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.26 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H), 7.04 (dd, J = 8.8, 2.6 Hz, 1H), 5.33 (s, 2H), 2.58 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.28, 155.65, 147.47, 145.70, 145.50, 142.48, 128.71, 124.03, 113.62, 111.11, 104.56, 69.27, 14.63. Purity: 100%. APCI-HRMS m/z [MH+] 285.0879 (Calc. for C15H13N2O4: 285.0870).

5-((4-Chlorobenzyl)oxy)-2-methylbenzo[d]oxazole (2e)

The title compound (light pink crystal clumps) was afforded from 2-methylbenzo[d]oxazol-5-ol and 4-chlorobenzyl bromide. Yield: 42%, mp: 77–79 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.55 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.9, 2.4 Hz, 1H), 5.15 (s, 2H), 2.58 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.18, 155.86, 145.55, 142.44, 136.59, 132.84, 129.98, 128.88, 113.64, 111.02, 104.46, 69.57, 14.65. Purity: 98%. APCI-HRMS m/z [MH+] 274.0626 (Calc. for C15H13ClNO2: 274.0629).

2-Methyl-5-((4-methylbenzyl)oxy)benzo[d]oxazole (2f)

The title compound (off-white crystal clumps) was afforded from 2-methylbenzo[d]oxazol-5-ol and 4-methylbenzyl bromide. Yield: 63%, mp: 93–95 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.53 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 2.5 Hz, 1H), 7.20 (d, J = 7.8 Hz, 2H), 6.97 (dd, J = 8.8, 2.5 Hz, 1H), 5.09 (s, 2H), 2.57 (s, 3H), 2.31 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.10, 156.08, 145.43, 142.42, 137.49, 134.47, 129.41, 128.29, 113.64, 110.95, 104.38, 70.33, 21.23, 14.65. Purity: 96%. APCI-HRMS m/z [MH+] 254.1179 (Calc. for C16H16NO2: 254.1176).

3-(((2-Methylbenzo[d]oxazol-5-yl)oxy)methyl)benzonitrile (2g)

The title compound (off-white powder-like granules) was afforded from 2-methylbenzo[d]oxazol-5-ol and 3-(bromomethyl)benzonitrile. Yield: 61%, mp: 88–90 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.97–7.92 (m, 1H), 7.85–7.79 (m, 2H), 7.62 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 7.03 (dd, J = 8.8, 2.6 Hz, 1H), 5.22 (s, 2H), 2.58 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.26, 155.70, 145.63, 142.44, 139.29, 132.90, 132.08, 131.50, 130.18, 119.14, 113.64, 111.89, 111.09, 104.50, 69.23, 14.65. Purity: 96%. APCI-HRMS m/z [MH+] 265.0964 (Calc. for C16H13N2O2: 265.0972).

Monoamine oxidase inhibition

The experimental protocol described in literature was used to evaluate MAO inhibitory potencies [29, 30]. Kynuramine was used as a non-selective MAO-A and MAO-B substrate, and commercially available recombinant human MAO-A and MAO-B served as enzyme sources. MAO-A and MAO-B oxidise kynuramine to produce the fluorescent metabolite 4-hydroxyquinoline and fluorescence spectroscopy (λex = 310 nm; λem = 400 nm) was used to measure concentrations of 4-hydroxyquinoline. Using this approach, catalytic activities of MAO-A and MAO-B were measured in the presence and absence of the test inhibitors and the rate data were fitted to the one-site competition model with the Prism 5 software package (GraphPad). Sigmoidal curves of catalytic rate vs the logarithm of inhibitor concentration (log[I]) were thus obtained from which the IC50 values were estimated. The IC50 values were measured in triplicate and were reported as the mean ± standard deviation (SD). The inhibitor concentration range that was used for these studies was 0.003–100 µM for all compounds except 2b and d for which the range was 0.0003–1 µM when the inhibition of MAO-B was evaluated.

Protocol for molecular docking

Molecular docking was carried out according to the previously reported procedure [29]. The X-ray crystal structures of MAO-A (PDB code: 2Z5X) and MAO-B (PDB code: 2V5Z) bound to harmine and safinamide, respectively, were selected as the protein models [3, 22]. The preparation of the protein models and docking simulations were performed with Discovery Studio 3.1 (Accelrys). The PyMOL molecular graphics system was used to create the illustrations [35].

Supplementary information

1H NMR and 13C NMR spectra and mass spectra for the synthesised compounds, HPLC traces for purity estimation and crystallographic data for 2b. Sigmoidal plots for the inhibition of MAO-A and MAO-B by the 2-methylbenzo[d]oxazole derivatives. These data are provided in the online version at https://doi.org.