Materials and instrumentationChemical reagents

The solvents and reagents used for the synthetic procedures were obtained from Sigma-Aldrich. Deuterated dimethylsulfoxide (DMSO-d6) used for nuclear magnetic resonance (NMR) spectroscopy was procured from Merck.

NMR

A Bruker Avance III 600 spectrometer was used to record proton (1H) and carbon (13C) NMR spectra at frequencies of 600 MHz and 150 MHz, respectively. The integration, multiplicity, and coupling constants (J), which are given in hertz (Hz), are included in the notation of the spectra. Chemical shifts (δ) are reported in parts per million (ppm) and were referenced to the residual solvent (DMSO-d6) signal at 2.5 ppm for 1H NMR and 39.5 ppm for 13C NMR. Spin multiplicities are denoted as s (singlet), d (doublet), dd (doublet of doublets), t (triplet) or m (multiplet).

Mass spectrometry

High resolution mass spectra (HRMS) were obtained with a Bruker micrOTOF-Q II mass spectrometer using atmospheric-pressure chemical ionization (APCI) in the positive mode.

TLC (thin layer chromatography)

The progression and completion of the chemical reactions were monitored by TLC. Silica gel 60 aluminum coated TLC sheets (containing UV254 fluorescent indicator) were used with a mobile phase consisting of ethyl acetate and hexane (3:2).

Biology

Enzymes, substrates and reference inhibitors used for the biological experiments 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 record fluorescence measurements of all enzymatic reactions.

The synthesis of C5- (4a–i) and C6-substituted (5a–f) indazoles

1H-Indazol-5-ol or 1H-indazol-6-ol (4 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 10 mL) at room temperature, after which potassium carbonate (K2CO3, 8 mmol) was added. The appropriate alkyl bromide (5 mmol for 4a–f and 5a–f, 4.5 mmol for 4g–i) was subsequently added and the reaction was stirred at 0 °C for 2 h. The reaction was allowed to warm to room temperature and stirring was continued for 24 h. Upon completion, water (50 mL) was added and the resulting precipitate was collected by filtration and dried under vacuum. Compounds 5a–c were purified by recrystallisation from ethanol. Silica gel column chromatography (ethyl acetate:hexane, 4:1) was used to purify compounds 5d–f and 4a–i.

5‐(Benzyloxy)‐1H‐indazole (4a)

Yield: 10.0%; mp 175–177.4 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.94 (s, 1H), 7.95 (s, 1H), 7.50 – 7.44 (m, 3H), 7.43 – 7.37 (m, 2H), 7.36–7.31 (m, 1H), 7.29 – 7.24 (m, 1H), 7.08 (dd, J = 9.0, 2.3 Hz, 1H), 5.12 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 153.24, 137.77, 136.25, 133.29, 128.86, 128.20, 128.14, 123.51, 118.82, 111.53, 101.78, 70.09; APCI-HRMS m/z: calculated for C14H13N2O (MH+), 225.1022, found 225.1018.

5‐[(4‐Fluorophenyl)methoxy]‐1H‐indazole (4b)

Yield: 22.8%; mp 177.7-179.5 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.95 (s, 1H), 7.56 – 7.50 (m, 2H), 7.46 (d, J = 8.9 Hz, 1H), 7.29 – 7.25 (m, 1H), 7.25 – 7.19 (m, 2H), 7.08 (dd, J = 8.9, 2.3 Hz, 1H), 5.10 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 162.98, 161.37, 153.15, 136.32, 134.02, 134.00, 133.30, 130.36, 130.31, 123.51, 118.80, 115.72, 115.58, 111.52, 101.97, 69.47; APCI-HRMS m/z: calculated for C14H12FN2O (MH+), 243.0928, found 243.0924.

5‐[(4‐Chlorophenyl)methoxy]‐1H‐indazole (4c)

Yield: 19.0%; mp 179-181 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.94 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.48 – 7.44 (m, 3H), 7.30 – 7.22 (m, 1H), 7.08 (dd, J = 8.9, 2.3 Hz, 1H), 5.13 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 153.05, 136.87, 136.34, 133.31, 132.75, 129.90, 128.85, 123.50, 118.76, 111.55, 102.03, 69.34; APCI-HRMS m/z: calculated for C14H12ClN2O (MH+), 259.0633, found 259.0626.

5‐[(4‐Bromophenyl)methoxy]‐1H‐indazole (4d)

Yield: 13.9%; mp 184–187 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.90 (s, 1H), 7.94 (s, 1H), 7.63 – 7.57 (m, 2H), 7.50 – 7.40 (m, 3H), 7.29 – 7.23 (m, 1H), 7.08 (dd, J = 8.9, 2.3 Hz, 1H), 5.11 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 153.04, 137.30, 136.33, 133.31, 131.78, 130.20, 123.50, 121.26, 118.75, 111.55, 102.05, 69.37; APCI-HRMS m/z: calculated for C14H12BrN2O (MH+), 303.0128, found 303.0142.

5‐[(4‐Methylphenyl)methoxy]‐1H‐indazole (4e)

Yield: 15.2%; mp 187-189.9 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.94 (s, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.28 – 7.22 (m, 1H), 7.20 (d, J = 7.8 Hz, 2H), 7.06 (dd, J = 8.9, 2.3 Hz, 1H), 5.06 (s, 2H), 2.31 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 153.26, 137.40, 136.24, 134.73, 133.28, 129.40, 128.22, 123.52, 118.84, 111.48, 101.83, 70.03, 21.23; APCI-HRMS m/z: calculated for C15H15N2O (MH+), 239.1179, found 239.1168.

5‐[(4‐Nitrophenyl)methoxy]‐1H‐indazole (4f)

Yield: 5.8%; mp 191–193 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.95 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.95 (s, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 9.0 Hz, 1H), 7.30 – 7.24 (m, 1H), 7.13 (dd, J = 9.0, 2.3 Hz, 1H), 5.31 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 152.81, 147.41, 145.81, 136.39, 133.34, 128.65, 124.05, 123.45, 118.68, 111.67, 102.09, 68.97; APCI-HRMS m/z: calculated for C14H12N3O3 (MH+), 270.0873, found 270.0863.

5‐[(3‐Chlorophenyl)methoxy]‐1H‐indazole (4g)

Yield: 3.1%; mp 163.5–165 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.93 (s, 1H), 7.95 (s, 1H), 7.56 – 7.53 (m, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.45 – 7.41 (m, 2H), 7.41–7.38 (m, 1H), 7.29 – 7.23 (m, 1H), 7.10 (dd, J = 9.0, 2.3 Hz, 1H), 5.15 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 152.98, 140.42, 136.33, 133.55, 133.32, 130.80, 128.10, 127.70, 126.59, 123.47, 118.75, 111.59, 101.96, 69.20; APCI-HRMS m/z: calculated for C14H12ClN2O (MH+), 259.0633, found 259.0637.

5‐[(3‐Bromophenyl)methoxy]‐1H‐indazole (4h)

Yield: 7.0%; mp 165–167 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.95 (s, 1H), 7.70 – 7.66 (m, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.37 (t, J = 7.8 Hz, 1H), 7.29 – 7.24 (m, 1H), 7.10 (dd, J = 9.0, 2.3 Hz, 1H), 5.14 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 153.00, 140.69, 136.36, 133.33, 131.08, 131.00, 130.57, 126.98, 123.49, 122.14, 118.74, 111.58, 102.03, 69.20; APCI-HRMS m/z: calculated for C14H12BrN2O (MH+), 303.0128, found 303.0142.

5‐[(3‐Methylphenyl)methoxy]‐1H‐indazole (4i)

Yield: 5.6%; mp 144–147 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.92 (s, 1H), 7.94 (s, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.31–7.28 (m, 2H), 7.28 – 7.24 (m, 2H), 7.14 (d, J = 7.1 Hz, 1H), 7.08 (dd, J = 8.9, 2.3 Hz, 1H), 5.07 (s, 2H), 2.33 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 153.30, 138.00, 137.69, 136.26, 133.30, 128.82, 128.76, 128.68, 125.22, 123.52, 118.81, 111.51, 101.76, 70.17, 21.47; APCI-HRMS m/z: calculated for C15H15N2O (MH+), 239.1179, found 239.1181.

6‐(Benzyloxy)‐1H‐indazole (5a)

Yield: 18.4%; mp 161–163 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.78 (s, 1H), 7.93 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.52–7.46 (m, 2H), 7.41 (t, J = 7.9 Hz, 2H), 7.34 (t, J = 7.6 Hz, 1H), 7.05 – 6.99 (m, 1H), 6.83 (dd, J = 8.7, 2.1 Hz, 1H), 5.18 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 158.05, 141.45, 137.53, 133.82, 128.89, 128.24, 128.05, 121.72, 118.23, 113.12, 92.81, 69.94; APCI-HRMS m/z: calculated for C14H13N2O (MH+), 225.1022, found 225.1032.

6‐[(4‐Fluorophenyl)methoxy]‐1H‐indazole (5b)

Yield: 13.4%; mp 132–134 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.94 (s, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.57 – 7.50 (m, 2H), 7.23 (t, J = 8.9 Hz, 2H), 7.04 – 6.99 (m, 1H), 6.82 (dd, J = 8.8, 2.1 Hz, 1H), 5.16 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 163.03, 161.41, 157.94, 141.43, 133.83, 133.75, 133.73, 130.33, 130.27, 121.74, 118.27, 115.76, 115.62, 113.09, 92.84, 69.23; APCI-HRMS m/z: calculated for C14H12FN2O (MH+), 243.0928, found 243.0919.

6‐[(4‐Chlorophenyl)methoxy]‐1H‐indazole (5c)

Yield: 11.9%; mp 142.5–148 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.94 (s, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.54 – 7.49 (m, 2H), 7.48 – 7.44 (m, 2H), 7.03 – 6.98 (m, 1H), 6.83 (dd, J = 8.8, 2.2 Hz, 1H), 5.18 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 157.85, 141.41, 136.60, 133.83, 132.84, 129.84, 128.89, 121.77, 118.31, 113.06, 92.90, 69.11; APCI-HRMS m/z: calculated for C14H12ClN2O (MH+), 259.0633, found 259.0643.

6‐[(4‐Bromophenyl)methoxy]‐1H‐indazole (5d)

Yield: 10.6%; mp 167–169 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.94 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.03 – 6.96 (m, 1H), 6.82 (dd, J = 8.7, 2.1 Hz, 1H), 5.16 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 157.80, 141.36, 137.01, 133.84, 131.83, 130.18, 121.79, 121.35, 118.26, 113.07, 92.79, 69.06; APCI-HRMS m/z: calculated for C14H12BrN2O (MH+), 303.0128, found 303.0115.

6‐[(4‐Methylphenyl)methoxy]‐1H‐indazole (5e)

Yield: 10.2%; mp 147–149 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.94 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 7.02 – 6.97 (m, 1H), 6.80 (dd, J = 8.7, 2.1 Hz, 1H), 5.12 (s, 2H), 2.31 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 158.03, 141.41, 137.50, 134.43, 133.81, 129.45, 128.19, 121.71, 118.12, 113.19, 92.65, 69.75, 21.24; APCI-HRMS m/z: calculated for C15H15N2O (MH+), 239.1179, found 239.1169.

6‐[(3‐Bromophenyl)methoxy]‐1H‐indazole (5f)

Yield: 12.4%; mp 146.7–149 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.94 (s, 1H), 7.71 – 7.68 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.57 – 7.47 (m, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.03 – 6.98 (m, 1H), 6.84 (dd, J = 8.7, 2.1 Hz, 1H), 5.20 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 157.77, 141.37, 140.40, 133.84, 131.13, 131.08, 130.56, 126.94, 122.16, 121.82, 118.31, 113.06, 92.83, 68.91; APCI-HRMS m/z: calculated for C14H12BrN2O (MH+), 303.0128, found 303.0129.

MAO activity measurements

The activities of MAO-A and MAO-B were measured by following the procedure as reported in literature [47, 50]. For this purpose, kynuramine was used as a mixed substrate and commercially available recombinant human MAO-A and MAO-B served as enzyme sources. Kynuramine is oxidized by MAO to produce 4-hydroxyquinoline as final product. Fluorescence spectrophotometry was used to measure 4-hydroxyquinoline following the alkalinization of the reactions at the end-point.

DAAO activity measurements

To measure the activity of DAAO, a previously reported protocol was followed [37, 48, 51]. D-Serine served as the substrate and porcine kidney DAAO was used as enzyme source. H2O2, a by-product of the catalytic cycle of DAAO, was measured in a peroxidase-coupled assay system using the reagent, Amplex red. In the presence of H2O2, horseradish peroxidase catalyzes the oxidation of Amplex red to yield the fluorescent compound, resorufin. Fluorescence spectrophotometry was used to continuously measure resorufin formation.

To investigate the possibility that a test inhibitor may suppress the fluorescence signal produced in the peroxidase-coupled assay system, the test inhibitor at 1, 10 and 100 µM was incubated with horse radish peroxidase, Amplex red and hydrogen peroxide and the fluorescence intensities were measured. These data were compared to control experiments conducted in the absence of inhibitor. The protocol for this experiment has been reported [51].

Molecular docking

Molecular docking was performed according to the protocol previously described [47]. The Discovery Studio 3.1 suite was used 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 [42, 46]. Illustrations were created with the PyMOL molecular graphics system [52].