Introduction

Various neurological disorders have been attributed to dysfunctional neurotransmission and include Parkinson’s disease, major depressive disorder and schizophrenia [1,2,3]. The observation that inhibition of enzymes such as monoamine oxidase (MAO; E.C. 1.4.3.4) and D-amino acid oxidase (DAAO; E.C. 1.4.3.3) increase monoaminergic and glutamatergic function, respectively, has led to an interest in the pharmacological potential of inhibitors of these enzymes in the palliative treatment of neuropsychiatric and neurodegenerative disorders [4, 5].

MAO is a mitochondrial bound flavoenzyme that is widely distributed throughout the body with high expression levels in both peripheral and nervous system tissues [6, 7]. These proteins are the primary degradative enzymes of monoamine neurotransmitters as well as various endogenous and exogenous amines [8]. MAO consists of two isozymes termed MAO-A and MAO-B. Even though these isozymes share 70% amino acid sequence homogeneity, MAO-A and MAO-B exhibit different substrate and inhibitor specificities [9, 10]. MAO-A metabolizes bulkier neurotransmitter amines such as serotonin, whereas MAO-B prefers smaller, non-hydroxylated amines as substrates (e.g., phenethylamine and benzylamine). Certain amines including dopamine, epinephrine and norepinephrine, tyramine and tryptamine are oxidized by both isozymes [5]. Primary amine substrates undergo MAO-catalyzed oxidative deamination to yield the corresponding aldehyde species with ammonia and hydrogen peroxide forming as by-products [11].

MAO regulates neurotransmitter levels and prevents certain extraneous molecules (e.g., dietary amines) from acting as false neurotransmitters, and metabolism facilitated by MAO is thus essential for the correct functioning at synaptic junctions [12]. Overactivity of MAO has been implicated in various psychiatric disorders. Elevated MAO-A density in different brain regions may be responsible for reducing monoamine levels (e.g., serotonin, norepinephrine and dopamine) during major depression and inhibitors of MAO-A have been used for the treatment of depression [13,14,15]. Similarly, MAO-B expression increases with age in the human brain, and MAO-B inhibitors have been used to treat age-related neurodegenerative disorders such as Parkinson’s disease [16, 17]. The rationale behind the use of MAO-B specific inhibitors is that these agents maintain synaptic dopamine levels by preventing its metabolism (or indirectly by elevating phenethylamine levels), thereby alleviating the motor symptoms of Parkinson’s disease [18, 19]. When combined with levodopa, MAO-B inhibitors may improve motor fluctuations and allow for a reduction of the levodopa dose [19]. Furthermore, MAO-mediated metabolism is a prominent source of hydrogen peroxide in the central nervous system and this by-product may contribute to neurodegeneration in Parkinson’s disease [20]. Under certain conditions, hydrogen peroxide might be converted to hydroxyl free radicals that cause oxidative stress and neuronal damage. This process has been linked with neurodegeneration observed in Parkinson’s disease and can potentially form part of the disease pathology [6, 21]. By attenuating hydrogen peroxide production, MAO inhibitors may thus possess neuroprotective effects and MAO-B inhibitors have been advocated as potential disease modifying agents [8, 22,23,24]. MAO-B inhibitors are a safe and well-tolerated approach in the symptomatic treatment of early or mild Parkinson’s disease and although these agents offer only modest symptomatic benefit, they reduce motor fluctuations caused by long-term use of levodopa, slow the rate of clinical decline and improve the efficacy of a lower levodopa dosage [19, 25, 26].

DAAO is a flavoenzyme that metabolizes D-amino acids such as D-alanine and D-serine [27, 28]. DAAO serves a regulatory function in the brain since it metabolizes the endogenous gliotransmitter, D-serine, which acts as a co-agonist that potentiates N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission by binding to its strychnine-insensitive glycine site [29, 30]. Hypofunction of the NMDA receptor has been implicated in the pathophysiology of schizophrenia, and D-serine and DAAO inhibitors have therefore been investigated for the treatment of schizophrenia [4, 31]. As adjuvant to antipsychotic drugs, D-serine has been shown to reduce the neurocognitive symptoms in schizophrenia [32, 33]. The poor blood-brain barrier permeability of D-serine, however, requires high doses to be administered which may lead to nephrotoxicity [34, 35]. DAAO inhibitors have been investigated as a potential treatment strategy for the positive and negative symptoms associated with schizophrenia. DAAO inhibitors would reduce the central metabolism of D-serine and thus enhance NMDA receptor-mediated neurotransmission [4, 36]. Most synthetic DAAO inhibitors are small molecules that contain the carboxylic acid group or isosteres thereof [31, 37, 38]. Several good potency DAAO inhibitors have been reported as exemplified by 3-hydroxycoumarin (1) and 3-hydroxyquinolin-2(1H)-one (2) (Fig. 1) [27, 31].

Fig. 1
figure 1

The structures of 3-hydroxycoumarin (1), 3-hydroxyquinolin-2(1H)-one (2) and 7-nitroindazole (3)

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In previous studies, the neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (3), also demonstrated moderate MAO-B inhibition activity (Ki = 4 µM) [39, 40]. As with most small molecule inhibitors, 7-nitroindazole is expected to bind to the substrate cavity of the MAO-B enzyme, leaving the entrance cavity unoccupied [41,42,43]. In general, larger inhibitors that span both cavities are more potent due to the additional interactions formed between the inhibitor and the hydrophobic environment of the entrance cavity [43]. Appropriate substitution with a benzyloxy moiety has been reported to enhance the inhibitory efficacy of similar small molecules by enabling the compound to span both the entrance and substrate cavities of MAO-B [42, 44]. Based on these considerations, a series of C5- and C6-substituted indazole derivatives was synthesized in an attempt to improve the inhibition potency of 7-nitroindazole. The C5 and C6 positions were selected for substitution since literature showed that the MAO-B inhibition potency of another small molecule inhibitor, isatin, was significantly enhanced by substitution at the analogous positions [44, 45]. Substitution at these positions would direct the substituent into the entrance cavity. The series of indazole derivatives was evaluated as potential in vitro inhibitors of MAO-A, MAO-B and DAAO. Although there is no structural rationale for these compounds to act as DAAO inhibitors, the evaluation was used for comparison with the MAO inhibition potencies and for the generation of selectivity data.

Results and discussion

Chemistry

The indazole derivatives (4 and 5) were synthesized according to the method reported in literature. In the presence of K2CO3, commercially available 1H-indazol-5-ol (6) or 1H-indazol-6-ol (7) was dissolved in DMF and reacted with the appropriate alkyl bromides to produce the desired derivatives (Fig. 2). The yields of the reactions ranged from 3.1 to 22.8% (4a–i) and 10.2 to 18.4 (5a–f). The products were characterized by NMR and mass spectrometry (see supplementary material).

Fig. 2
figure 2

Synthetic route to C5- and C6-substituted indazoles 4a–i and 5a–f. Key: (i) DMF, K2CO3, rt, 24 h

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Monoamine oxidase inhibition

The C5- and C6-substituted indazole derivatives synthesized in this study were evaluated as potential in vitro inhibitors of human MAO-A and MAO-B. The recombinant MAO enzymes were utilized to determine the inhibition properties of the compounds, while kynuramine served as a mixed MAO-A/B substrate. MAO metabolizes kynuramine to yield 4-hydroxyquinoline as final product. The production of 4-hydroxyquinoline was monitored by fluorescence spectrophotometry since the compound fluoresces in an alkaline environment. The enzyme reactions were thus carried out in the presence of a range of inhibitor concentrations (0.003–100 µM). After 20 min have elapsed, the reactions were terminated by the addition of sodium hydroxide and the formation of 4-hydroxyquinoline was quantitated. The enzyme catalytic activities were calculated and sigmoidal graphs of activity versus the logarithm of inhibitor concentration (log[I]) were constructed from triplicate experiments. These graphs were used to estimate IC50 values, which are reported in Table 1. Examples of sigmoidal graphs for the estimation of IC50 values are presented in Fig. 3.

Table 1 The potencies (IC50 values) of the inhibition of human MAO-A and MAO-B, and porcine DAAO by the indazole derivatives (4a–i, 5a–f) and reference inhibitors
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Fig. 3
figure 3

A Sigmoidal graphs for the inhibition of MAO-A by 5c (filled circles), 5f (open circles) and toloxatone (triangles). B Sigmoidal graphs for the inhibition of MAO-B by 4d (filled circles), 5f (open circles) and safinamide (triangles)

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The results of the inhibition studies showed that most of the indazole derivatives inhibited MAO-A with the most potent inhibition recorded for 5c (IC50 = 0.745 µM). This compound was the only submicromolar MAO-A inhibitor of the series. Among the test series, nine compounds exhibited IC50 < 10 µM and thus possessed potencies that were similar to the reference inhibitors isatin (IC50 = 8.29 µM) and toloxatone (IC50 = 1.67 µM). The following structure-activity relationships were noted for the inhibition of MAO-A: (a) The C6-substituted derivatives were more potent than the corresponding C5-substituted homologs (e.g., 4a vs. 5a; 4b vs. 5b; 4c vs. 5c; 4d vs. 5d; 4h vs. 5f). (b) For both the C5- and C6-substituted derivatives, the 4-chloro substituted compounds (4c and 5c) were the most potent MAO-A inhibitors while the methyl substituted compounds (4e, 4i, 5e) did not inhibit MAO-A at a maximal tested concentration of 100 µM. (c) It was interesting to note that neither 1H-indazol-5-ol (6) nor 1H-indazol-6-ol (7) inhibited MAO-A which demonstrated the requirement of a C5 or C6 substituent for inhibition.

The indazole derivatives were significantly more potent inhibitors of MAO-B compared to the MAO-A isoform, with all derivatives exhibiting IC50 < 1 µM. Based on their potency ranges, it was clear that the C5-substituted derivatives were more potent MAO-B inhibitors compared to the C6-substituted derivatives. For the C6-substituted derivatives, the IC50 values ranged from 0.118–0.979 µM while those of the C5-substituted derivatives ranged from 0.0025–0.024 µM, a difference of one to two orders of magnitude. The C5-substituted derivatives were therefore significantly more potent than the reference inhibitor safinamide (IC50 = 0.240 µM). The following structure-activity relationships were noted for the inhibition of MAO-B: (a) Among the C5-substituted derivatives, the benzyloxy substituted compound 4a (IC50 = 0.024 µM) was the weakest MAO-B inhibitor, which indicated that a substituent (F, Cl, Br, CH3, NO2) on the benzyloxy ring enhanced MAO-B inhibition. (b) For the C5-substituted derivatives, substitution on both the C4 and C3 positions of the benzyloxy ring (e.g., 4c vs. 4g; 4d vs. 4h; 4e vs. 4i) resulted in high potency inhibition, while for the C6-substituted derivatives (5a–f) substitution on C3 of the benzyloxy ring (5f) yielded the most potent inhibitor. In fact, 5f (IC50 = 0.118 µM) was approximately fivefold more potent than the C4-substituted homolog 5d (IC50 = 0.565 µM). (c) As for MAO-A, neither 1H-indazol-5-ol (6) nor 1H-indazol-6-ol (7) inhibited MAO-B which demonstrated the requirement of a C5 or C6 substituent for inhibition.

Since 7-nitroindazole is a reversible competitive inhibitor of MAO-B, it is likely that the indazole derivatives would also act as competitive inhibitors of the MAO enzymes. To further investigate the inhibition properties of the indazole derivative, sets of Lineweaver-Burk graphs were constructed for the inhibition of MAO-A and MAO-B by 5c. This compound represents a potent inhibitor of the respective MAO isoforms. To construct the Lineweaver-Burk graphs, MAO activities were measured in the absence and presence of the following set of inhibitor concentrations: ¼ × IC50, ½ × IC50, ¾ × IC50, 1 × IC50 and 1¼ × IC50. Each Lineweaver-Burk graph was prepared with substrate concentrations ranging from 15 to 250 μM. As shown in Fig. 4, the lines of the Lineweaver-Burk graphs intersected on the y-axis for both MAO-A and MAO-B, which was indicative of competitive inhibition. Replots of the slopes of the Lineweaver-Burk graphs versus inhibitor concentration also yielded linear lines from which the enzyme-inhibitor dissociation constants, Ki, were estimated (–Ki = x-axis intercept). Ki values of 0.40 and 0.16 µM were estimated for the inhibition of MAO-A and MAO-B, respectively, by 5c.

Fig. 4
figure 4

Lineweaver-Burk graphs for the inhibition of MAO-A (A) and MAO-B (B) by 5c. The insets are replots of the slopes of the Lineweaver-Burk plots versus inhibitor concentration, from which Ki values were estimated. For MAO-A the inhibitor concentration ranged from 0.186 to 0.931 µM, while for MAO-B, the inhibitor concentration ranged from 0.1 to 0.5 µM

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Molecular docking

A molecular docking study was undertaken to investigate the potential binding modes and interactions of selected indazole derivatives (4a and 5a) in the active sites of MAO-A and MAO-B. For the docking study, the crystal structures of human MAO-A and MAO-B bound to harmine and safinamide, respectively, were used [42, 46]. The CDOCKER module of the Discovery Studio 3.1 suite of software was used and the protocol reported in literature was followed [47]. The predicted binding modes of 4a and 5a to MAO-A are presented in Fig. 5 and show that the inhibitors adopted virtually identical orientations in the active site, with the only difference being that the indazole ring is rotated through 180 ° between the two derivatives. As a result, 5a formed a hydrogen bond interaction with Asn-181 in addition to that observed with Tyr-444 for both derivatives. Other stabilizing interactions included pi interactions of the benzyloxy ring of the inhibitors with Phe-208 and both indazole rings with Tyr-407.

Fig. 5
figure 5

The predicted binding of 4a (A) and 5a (B) to the active site of human MAO-A. Hydrogen bonding is indicated by dashed lines. Residues Ile-199 and Tyr-326 in MAO-B, which correspond to Phe-208 and Ile-335 in MAO-A, respectively, are shown in green with the labels bracketed

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The predicted binding modes of 4a and 5a to MAO-B are presented in Fig. 6. As for MAO-A, the inhibitors adopted similar binding orientations with the only difference being that the indazole ring was rotated through 180 °. A hydrogen bond interaction was observed between the inhibitors and Tyr-435. The MAO-B active site consists of two cavities, an entrance cavity which leads to a substrate cavity. For the inhibitors, the indazole ring was located in the substrate cavity while the benzyloxy ring extended into the entrance cavity. These compounds were thus cavity spanning inhibitors of MAO-B. Such compounds are reported to possess higher affinity for MAO-B due to the additional interactions provided by the entrance cavity compared to compounds that bind only to the substrate cavity [43]. Other interactions between the derivatives and MAO-B included a pi interaction between the phenyl of the indazole ring system and Tyr-326 and a pi-sulfur interaction between both rings of the indazole and Cys-172.

Fig. 6
figure 6

The predicted binding of 4a (A) and 5a (B) to the active site of human MAO-B. Hydrogen bonding is indicated by dashed lines. Residues Phe-208 and Ile-335 in MAO-A, which correspond to Ile-199 and Tyr-326 in MAO-B, respectively, are shown in green with the labels bracketed

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D-Amino acid oxidase inhibition

DAAO from porcine kidney was used to investigate the inhibition properties of the indazole derivatives, while D-serine served as the substrate. The catalytic activity of DAAO was determined by measuring the production of H2O2 via continuous fluorescence spectrophotometry [37, 48]. The formation of H2O2 was measured in a peroxidase-coupled assay, where Amplex red is oxidized to produce the fluorescent compound, resorufin. DAAO and D-serine were incubated in the presence of various inhibitor concentrations (0.003–100 µM) while the fluorescence intensities were continuously monitored. Sigmoidal graphs of the rate of change in fluorescence vs. inhibitor concentration (log [I]) were constructed from triplicate experiments, and the IC50 values were calculated using these plots. The IC50 values are reported in Table 1 while Fig. 7 presents examples of sigmoidal graphs for the estimation of IC50 values.

Fig. 7
figure 7

Sigmoidal graphs for the inhibition of DAAO by 1H-indazol-5-ol (6) (filled circles) and 3-methylpyrazole-5-carboxylic acid (open circles)

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The results showed that only five of the indazole derivatives exhibited weak DAAO inhibition with the most potent inhibition recorded for 4h (IC50 = 60.5 µM). Interestingly, all five of the compounds that showed inhibition were C5-substituted indazoles. However, 1H-indazol-5-ol (6) was found to be a good potency inhibitor with an IC50 value of 2.03 µM, whereas 1H-indazol-6-ol (7) did not inhibit the enzyme. The inhibition potency of 6 was thus similar to that of the reference inhibitor 3-methylpyrazole-5-carboxylic acid (IC50 = 2.04 µM).

There existed the possibility that a test compound may suppress the fluorescent signal produced by the peroxidase-coupled spectrofluorometric assay system, which may be incorrectly interpreted as DAAO inhibition. To investigate if this may have occurred for 1H-indazol-5-ol (6), the effect of the inhibitor on the fluorescence signal generated from hydrogen peroxide was measured. As shown in in Table 2, compound 6 decreased the fluorescent signal slightly at concentrations of 1 and 10 µM (92–96% residual signal), while significant suppression of the signal was observed at 100 µM (24% residual signal). Since the IC50 value for the inhibition of DAAO was well below 10 µM, it may be concluded that this compound was a true inhibitor and did not merely interfere with the assay system. For 3-methylpyrazole-5-carboxylic acid, only slight suppression of the fluorescence signal was observed at all tested concentrations.

Table 2 Percentage of the fluorescence signal generated from H2O2 by the peroxidase-coupled spectrofluorometric assay system in the presence of a test inhibitor (1, 10 and 100 µM) compared to the signal generated in the absence of inhibitor (100%)
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The observation of the weak inhibition potencies of these compounds is consistent with reports that the binding site of DAAO prefers small and highly polar ligands [49]. Most of the known, good potency DAAO inhibitors are mono- or bicyclic compounds that consist of an acidic functionality (or isostere thereof) without large substituents or side-chains as exemplified by 3-hydroxycoumarin (1) and 3-hydroxyquinolin-2(1H)-one (2) [27, 31]. Despite the fact that the C5- and C6-substituted indazole derivatives are bicyclic, the bulky side-chains most likely prevented the compounds from binding and inhibiting DAAO.

Conclusion

It has been reported that 7-nitroindazole is a moderate MAO-B inhibitor. Since the inhibitory potencies of small molecule compounds can be enhanced with the addition of appropriate substituents, the present study synthesized and evaluated a series of C5 and C6-substituted indazole derivatives as in vitro inhibitors of MAO-A and MAO-B. The most potent inhibitors presented with IC50 values of 0.745 µM (5c) and 0.0025 µM (4d) for MAO-A and MAO-B, respectively. These compounds were more potent inhibitors than the MAO-A and MAO-B reference inhibitors, toloxatone (IC50 = 1.67 µM) and safinamide (IC50 = 0.240 µM), respectively. The results documented that the indazole derivatives were more potent MAO-B inhibitors than MAO-A inhibitors. Furthermore, the C5-substituted compounds displayed higher MAO-B inhibition potencies than the corresponding C6-substituted analogs. It may be concluded that the MAO-B inhibitors identified in this study could serve as lead compounds for the development of drugs for the treatment of Parkinson’s disease. However, none of the indazole derivatives exhibited good potency inhibition of DAAO. This might be attributed to the bulky side-chains resulting from substitution with the benzyloxy moiety that hindered the analogs from binding to the DAAO enzyme.

Experimental

Materials and instrumentation

Chemical 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].