Materials and instrumentation

The reagents required for the chemical synthesis were purchased from Sigma-Aldrich while 2-methylbenzo[d]thiazol-6-ol (6) and 2-methylbenzo[d]thiazol-5-ol (7) were from Ambeed. Merck provided deuterated dimethyl sulfoxide (DMSO-d6) for use in nuclear magnetic resonance (NMR) spectroscopy. 1H and 13C NMR spectra were recorded on a Bruker Avance III 600 spectrometer at frequencies of 600 MHz and 151 MHz, respectively. Each sample was analysed using dimethyl sulfoxide (DMSO-d6) as solvent. Chemical shifts (δ) are reported in parts per million (ppm) and were referenced to the solvent signal at 2.5 and 39.5 ppm for 1H and 13C NMR, respectively. Multiplicities are abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet). The coupling constant (J) is given in hertz (Hz). A Bruker micrOTOF-Q II mass spectrometer, performing in the atmospheric-pressure chemical ionisation (APCI) mode, was used to record high resolution mass spectra (HRMS). A Büchi melting point B-545 instrument was used to measure melting points. TLC (silica gel 60 F254; Merck) was used to monitor the progress and completion of the chemical reactions. The mobile phase consisted of a mixture of n-hexane and ethyl acetate (3:2). HPLC analyses were carried out to determine the purities of the synthesised compounds according to the published procedure [28].

All the reagents used for the biochemical analyses, including the enzymes and substrates, were obtained from Sigma-Aldrich. A Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) and a SpectraMax iD3 multi-mode microplate reader (Molecular Devices) were used to measure fluorescence intensities.

The synthesis of 2-methylbenzo[d]thiazole derivatives (4a–f, 5a–g)

The protocol for the synthesis of 2-methylbenzo[d]thiazole derivatives (4a–f, 5a–g) has been reported [23]. 2-Methylbenzo[d]thiazol-6-ol (6) or 2-methylbenzo[d]thiazol-5-ol (7) (3.026 mmol) was dissolved in N,N-dimethylformamide (DMF; 10 mL) and the appropriate benzyl bromide derivative (4.539 mmol) was added. Anhydrous potassium carbonate (K2CO3; 6.05 mmol) was added and the reaction was stirred for 24 h at room temperature. Ethanol was finally added to the reaction mixture (±50 mL) and the solvent was removed under reduced pressure. Ethyl acetate was added to the residue and the solution was allowed to recrystallise. The crystals were collected by filtration, washed with n-hexane, and air-dried.

6-(Benzyloxy)-2-methylbenzo[d]thiazole (4a)

The title compound (dark grey crystal shards) was produced from 2-methylbenzo[d]thiazol-6-ol and benzyl bromide. Yield: 62%, mp: 70–74 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.80 (d, J = 8.7 Hz, 1H), 7.72–7.65 (m, 1H), 7.48 (d, J = 7.5 Hz, 2H), 7.44–7.37 (m, 2H), 7.37–7.30 (m, 1H), 7.14 (dd, J = 8.8, 2.7 Hz, 1H), 5.16 (s, 2H), 2.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.70, 156.38, 147.95, 137.32, 136.92, 128.91, 128.37, 128.27, 122.86, 115.99, 106.43, 70.28, 20.03. Purity: 97%. APCI-HRMS m/z Calc. for C15H14NOS: 256.0791, found 256.0780 (MH+).

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

The title compound (light purple powder-like crystals) was produced from 2-methylbenzo[d]thiazol-6-ol and 4-bromobenzyl bromide. Yield: 61%, mp: 113–115 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.80 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.14 (dd, J = 8.9, 2.4 Hz, 1H), 5.15 (s, 2H), 2.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.81, 156.16, 148.04, 136.92, 136.81, 131.84, 130.35, 122.89, 121.48, 115.97, 106.52, 69.46, 20.04. Purity: 95%. APCI-HRMS m/z Calc. for C15H13BrNOS: 333.9896, found 333.9885 (MH+).

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

The title compound (dark purple crystal shards) was produced from 2-methylbenzo[d]thiazol-6-ol and 4-(bromomethyl)benzonitrile. Yield: 80%, mp: 134–136 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.88 (d, J = 7.8 Hz, 2H), 7.81 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 2.3 Hz, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.17 (dd, J = 8.9, 2.3 Hz, 1H), 5.29 (s, 2H), 2.75 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.95, 155.98, 148.14, 143.15, 136.93, 132.89, 128.62, 122.95, 119.20, 115.93, 111.01, 106.58, 69.31, 20.04. Purity: 100%. APCI-HRMS m/z Calc. for C16H13N2OS: 281.0743, found 281.0729 (MH+).

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

The title compound (yellowish crystal shards) was produced from 2-methylbenzo[d]thiazol-6-ol and 4-nitrobenzyl bromide. Yield: 32%, mp: 150–152 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.28 (d, J = 7.8 Hz, 2H), 7.82 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 2.6 Hz, 1H), 7.19 (dd, J = 8.8, 2.7 Hz, 1H), 5.35 (s, 2H), 2.75 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 165.00, 155.94, 148.19, 147.51, 145.27, 136.94, 128.78, 124.09, 122.97, 115.94, 106.62, 69.07, 20.04. Purity: 100%. APCI-HRMS m/z Calc. for C15H13N2O3S: 301.0641, found 301.0644 (MH+).

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

The title compound (light purple powder-like crystals) was produced from 2-methylbenzo[d]thiazol-6-ol and 4-chlorobenzyl bromide. Yield: 65%, mp: 101–103 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.80 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 2.6 Hz, 1H), 7.51 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.14 (dd, J = 8.9, 2.5 Hz, 1H), 5.17 (s, 2H), 2.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.80, 156.18, 148.03, 136.92, 136.39, 132.95, 130.06, 128.92, 122.89, 115.97, 106.51, 69.42, 20.04. Purity: 96%. APCI-HRMS m/z Calc. for C15H13ClNOS: 290.0401, found 290.0409 (MH+).

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

The title compound (sparkling purple crystals) was produced from 2-methylbenzo[d]thiazol-6-ol and 4-methylbenzyl bromide. Yield: 77%, mp: 123–125 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.79 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.36 (d, J = 7.7 Hz, 2H), 7.21 (d, J = 7.6 Hz, 2H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H), 5.11 (s, 2H), 2.74 (s, 3H), 2.31 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 164.64, 156.40, 147.90, 137.62, 136.90, 134.27, 129.45, 128.37, 122.83, 116.01, 106.42, 70.18, 21.24, 20.03. Purity: 97%. APCI-HRMS m/z Calc. for C16H16NOS: 270.0947, found 270.0958 (MH+).

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

The title compound (beige powder-like crystals) was produced from 2-methylbenzo[d]thiazol-5-ol and benzyl bromide. Yield: 78%, mp: 76–78 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.89 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 2.5 Hz, 1H), 7.51–7.47 (m, 2H), 7.43–7.37 (m, 2H), 7.36–7.31 (m, 1H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 5.20 (s, 2H), 2.76 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.56, 157.83, 154.67, 137.43, 128.90, 128.30, 128.19, 127.53, 122.72, 115.22, 106.83, 70.06, 20.26. Purity: 88%. APCI-HRMS m/z Calc. for C15H14NOS: 256.0791, found 256.0784 (MH+).

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

The title compound (small yellow/beige crystals) was produced from 2-methylbenzo[d]thiazol-5-ol and 4-bromobenzyl bromide. Yield: 78%, mp: 121–123 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.89 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.10 (dd, J = 8.8, 2.5 Hz, 1H), 5.18 (s, 2H), 2.77 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.65, 157.60, 154.64, 136.93, 131.83, 130.29, 127.68, 122.77, 121.40, 115.20, 106.89, 69.22, 20.27. Purity: 85%. APCI-HRMS m/z Calc. for C15H13BrNOS: 333.9896, found 333.9899 (MH+).

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

The title compound (small, thin off-white crystal shards) was produced from 2-methylbenzo[d]thiazol-5-ol and 4-(bromomethyl)benzonitrile. Yield: 26%, mp: 162–164 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.91 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 2.5 Hz, 1H), 7.13 (dd, J = 8.8, 2.5 Hz, 1H), 5.32 (s, 2H), 2.77 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.74, 157.46, 154.65, 143.30, 132.87, 128.55, 127.89, 122.84, 119.18, 115.14, 110.97, 106.95, 69.13, 20.27. Purity: 92%. APCI-HRMS m/z Calc. for C16H13N2OS: 281.0743, found 281.0739 (MH+).

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

The title compound (small yellow crystal shards) was produced from 2-methylbenzo[d]thiazol-5-ol and 4-nitrobenzyl bromide. Yield: 9%, mp: 178–180 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.27 (d, J = 7.1 Hz, 2H), 7.92 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.55 (s, 1H), 7.15 (d, J = 8.7 Hz, 1H), 5.38 (s, 2H), 2.77 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.80, 157.39, 154.63, 147.47, 145.42, 128.73, 127.92, 124.08, 122.89, 115.14, 106.92, 68.86, 20.28. Purity: 98%. APCI-HRMS m/z Calc. for C15H13N2O3S: 301.0641, found 301.0627 (MH+).

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

The title compound (large yellow/white crystal) was produced from 2-methylbenzo[d]thiazol-5-ol and 4-chlorobenzyl bromide. Yield: 81%, mp: 102–106 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.90 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.49–7.44 (m, 2H), 7.10 (dd, J = 8.8, 2.5 Hz, 1H), 5.20 (s, 2H), 2.76 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.65, 157.61, 154.63, 136.49, 132.87, 130.00, 128.91, 127.65, 122.78, 115.19, 106.84, 69.16, 20.27. Purity: 90%. APCI-HRMS m/z Calc. for C15H13ClNOS: 290.0401, found 290.0412 (MH+).

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

The title compound (white crystal shards) was produced from 2-methylbenzo[d]thiazol-5-ol and 4-methylbenzyl bromide. Yield: 31%, mp: 91–93 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.88 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 7.37 (d, J = 7.5 Hz, 2H), 7.20 (d, J = 7.6 Hz, 2H), 7.08 (dd, J = 8.8, 2.7 Hz, 1H), 5.14 (s, 2H), 2.77 (s, 3H), 2.31 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.53, 157.83, 154.65, 137.54, 134.37, 129.45, 128.30, 127.43, 122.69, 115.25, 106.81, 69.93, 21.24, 20.26. Purity: 90%. APCI-HRMS m/z Calc. for C16H16NOS: 270.0947, found 270.0933 (MH+).

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

The title compound (grey powder-like granules) was produced from 2-methylbenzo[d]thiazol-5-ol and 3-(bromomethyl)benzonitrile. Yield: 82%, mp: 105–107 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.85–7.80 (m, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.13 (dd, J = 8.7, 2.5 Hz, 1H), 5.27 (s, 2H), 2.77 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 168.73, 157.49, 154.65, 139.22, 132.88, 132.10, 131.49, 130.20, 127.88, 122.82, 119.11, 115.18, 111.94, 106.95, 68.91, 20.27. Purity: 93%. APCI-HRMS m/z Calc. for C16H13N2OS: 281.0743, found 281.0756 (MH+).

MAO activity measurements

MAO activity measurements and inhibition studies were carried out according to the method described in literature [24, 25]. For this purpose, the commercially available recombinant human MAO-A and MAO-B enzymes were used.

Protocol for molecular docking

Molecular docking was carried out according to the protocol previously described [24]. The Discovery Studio 3.1 suite (Accelrys) was used for the simulations and 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 [22, 26]. Illustrations were created with the PyMOL molecular graphics system [29].