Investigation of Novel Thiazole Derivatives Bearing the Benzenesulfonamide Moiety as MAO Inhibitors with a Promising Activity Profiles

1. Introduction

The monoamine oxidase (MAO) enzymes are flavin adenine dinucleotide (FAD) dependent enzymes that are bound to the outer membranes of mitochondria (Youdim et al., 2006). The MAOs consist of two isoforms, MAO-A and MAO-B, which are products of different genes (Shih et al., 1999; Shih et al., 2018). The two MAO isoforms are similar in their three-dimensional structures and have approximately 70% homology of their amino acid sequences (Bach et al., 1988; Son et al., 2008; Binda et al., 2002). Among the 16 amino acid residues that comprise the active sites, only 6 differ between the MAO isoforms (Son et al., 2008). Due to these similarities, significant overlap in substrate and inhibitor specificities exist between MAO-A and MAO-B. For example, epinephrine, norepinephrine, dopamine, tyramine and tryptamine are metabolized efficiently by both MAO-A and MAO-B, while several clinically used inhibitors (e.g., tranylcypromine, phenelzine) inhibit both isoforms (Youdim et al., 2006). In spite of this, certain substrates (e.g., serotonin) are specific for MAO-A, while exogenous amines such as phenethylamine and benzylamine are specifically metabolized by MAO-B (Youdim et al., 2006). In addition, numerous clinically used and experimental inhibitors display specificity for a specific isoform. For example, harmine and safinamide are highly specific reversible inhibitors of MAO-A and MAO-B, respectively (Son et al., 2008; Binda et al., 2007). The molecular basis for the inhibitor specificities of MAO-A and MAO-B may be explained by the reported X-ray crystal structures of the MAOs (Son et al., 2008; Binda et al., 2002; Hubálek et al., 2005). The active site of MAO-A consists of a single cavity, while the active site of MAO-B consists of a larger substrate cavity where catalysis takes place, as well as a smaller entrance cavity that provides access from the protein surface to the site of catalysis. The aromatic residue Phe208 in MAO-A restricts the binding of larger inhibitors (e.g., safinamide) to MAO-A, while in MAO-B, the analogous residue is Ile199, the gating residue that normally separates the substrate and entrance cavities (Son et al., 2008; Binda et al., 2007). Since Ile199 is smaller in size, it can rotate into an alternate conformation to allow the MAO-B substrate and entrance cavities to fuse. When this occurs, larger cavity-spanning inhibitors bind to MAO-B while not being able to productively bind to the MAO-A active site. Similarly, Tyr326 in MAO-B would sterically prevent certain inhibitors (e.g., harmine) from binding to MAO-B, while the analogous residue in MAO-A (Ile335) is smaller and permits the binding of such inhibitors (Son et al., 2008; Binda et al., 2007).

The rapid degradation of neurotransmitter and dietary amines is critical for the regulation of synaptic neurotransmission and to prevent molecules from acting as false neurotransmitters. Imbalances of neurotransmitter levels has been associated with various neurogenic disorders (Kumar et al., 2017). For example, the monoamine theory of depression states that decreased concentrations of centrally available monoamines (e.g., serotonin, norepinephrine and dopamine) may precipitate depressive disorders (Mulinari, 2012). Although this may be an oversimplification of a complex mental disorder, it has been found that MAO-A levels in depression patients were elevated by approximately 34% in all regions of the brain compared to healthy individuals (Meyer et al., 2006). It may be concluded that elevated MAO-A density might be the primary monoamine lowering process during major depression, and that inhibitors of MAO-A would be effective antidepressant agents. Nonselective and selective MAO-A inhibitors have thus been used for the treatment of atypical depressions and phobic anxiety for several decades (Zisook, 1985; Lum & Stahl, 2012; Suchting et al., 2021).

Parkinson’s disease is another disorder where the symptoms are caused by abnormal neurotransmitter levels in the brain. In Parkinson’s disease, progressive degeneration of dopaminergic neurons in the nigrostriatal pathway results in diminished dopamine levels in this pathway. This affects the extrapyramidal system and ultimately gives rise to the characteristic motor symptoms (Przedborski, 2017; Chinta & Andersen, 2005). MAO-B inhibitors are of therapeutic value in Parkinson’s disease since they may prevent central dopamine degradation. Irreversible (e.g., selegiline and rasagiline) and reversible (e.g., safinamide) inhibitors of MAO-B are established therapy in Parkinson’s disease and although providing only moderate symptomatic benefit, these agents are used as monotherapy or in combination with levodopa in late-stage Parkinson’s disease while having few pharmacological adverse effects and safety concerns (Alborghetti et al., 2024; Murakami et al., 2023). MAO-mediated metabolism is also a prominent source of hydrogen peroxide production in the central nervous system, which may be converted to highly reactive hydroxyl free radicals if not detoxified (Pizzinat et al., 1999; Edmondson, 2014). The destructive processes associated with these reactive oxygen species have the potential to contribute to neurodegeneration in Parkinson’s disease. By decreasing the production of hydrogen peroxide, MAO-B inhibitors may have the potential to act as disease modifying agents in neurodegenerative disorders (Edmondson, 2014; Youdim & Bakhle, 2006). It is particularly relevant to note that the density of the MAO-B isoform increases in the brain with age, which provides a further rationale for MAO-B inhibitors as neuroprotective agents in aged-related neurodegenerative disorders (Fowler et al., 1997; Fowler et al., 1980). In contrast, the density of MAO-A in the brain remains unchanged with age.

Similar to the prooxidant role of MAO-B in the brain, MAO-A have been identified as a significant source of hydrogen peroxide in the heart, and the resultant oxidative damage to cardiac tissue might lead to cardiovascular diseases such as congestive heart failure (Kaludercic et al., 2014; Manni et al., 2016; Umbarkar et al., 2015). MAO-A inhibitors might therefore have a future role as cardioprotective agents. Interestingly MAO inhibitors are also under investigation for the treatment of other disease states such as prostate cancer and rheumatoid arthritis (Han et al., 2023; Bala, 2023).

Since MAO has a significant role in the regulation of neurotransmitter levels and is a source of hydrogen peroxide and oxidative injury in certain tissues, inhibitors of these enzymes are of much scientific and medicinal interest. The discovery novel MAO inhibitors with favourable specificity and activity profiles are pursued by researchers in the academia and industry (Guglielmi et al., 2022). We have recently reported that a series of 1,3,4-oxadiazol-2-ylbenzenesulfonamides act as potent MAO-B inhibitors with a high degree of isoform specificity (Shetnev et al., 2019). For example, the most potent compound (A) presented with an IC₅₀ value of 0.0027 µM. In a related study, we have discovered additional compounds with MAO inhibition properties among a series of pyridazinone-substituted benzenesulfonamides. Two compounds (B, C) exhibited specific inhibition of MAO-B with IC₅₀ values of 2.90 and 4.36 μM, respectively (Petzer et al., 2023). An earlier study has shown that the sulfonamide compound, zonisamide, also inhibits MAO-B, although with moderate potency (IC₅₀ = 24.8 µM) (Sonsalla et al., 2010). The crystal structure of zonisamide in complex with human MAO-B showed that zonisamide binds within the substrate cavity, leaving the entrance cavity unoccupied (Binda et al., 2011). While the sulfonamide functional group undergoes important interactions in the substrate cavity, the absence of interaction with the residues of the entrance cavity might explain the relatively lower MAO-B inhibition potency of zonisamide. In contrast, modelling suggested that for the 1,3,4-oxadiazol-2-ylbenzenesulfonamides (e.g., A), the benzenesulfonamide group binds in the substrate cavity in proximity to the FAD, while the dichlorophenyl extends into the entrance cavity (Petzer et al., 2023). These compounds are thus cavity-spanning inhibitors and the additional interactions afforded by the residues of the entrance cavity may explain, in part, the high MAO-B inhibition potencies of these compounds. The present study aimed to expand on these findings by replacing the 1,3,4-oxazole moiety with a 1,3-thiazole heterocycle. This study thus synthesized a series of 1,3-thiazolylbenzenesulfonamides by reaction of an α-haloketone with a substituted carbothioamide, and the resulting compounds were evaluated in vitro as inhibitors of the human MAOs. The results showed that these compounds display specificity for the MAO-B isoform with some compounds exhibiting nanomolar potencies. Besides the 1,3,4-oxadiazol-2-ylbenzenesulfonamides reported earlier, this study is only the second example of sulfonamide compounds that exhibit such high potency MAO inhibition (Petzer et al., 2023).

Figure 1. The structures of isoform-selected MAO inhibitors, 1,3,4-oxadiazol-2-ylbenzenesulfonamide A and pyridazinone-substituted benzenesulfonamides B and C.

Figure 1. The structures of isoform-selected MAO inhibitors, 1,3,4-oxadiazol-2-ylbenzenesulfonamide A and pyridazinone-substituted benzenesulfonamides B and C.

2. Results and Discussion

2.1. Chemistry

For the synthesis of the 1,3-thiazolylbenzenesulfonamide derivatives, primary benzenesulfonamide fragments 2a,b and 4 were used building blocks (Figure 2). These are easily synthesized according to the methods described in literature (Scobie, 2018; Kaboudin, & Elhamifar, 2006). This strategy made it possible to avoid the use of dangerous and corrosive reagents, and reagents with low selectivity (e.g., chlorosulfonic acid and its derivatives) for the synthesis of the benzenesulfonamide derivatives.

The target 1,3-thiazolylbenzenesulfonamide derivatives were synthesized by reaction of thioamides with the corresponding α-haloketone, with the reactions being heated in isopropyl alcohol (i-PrOH) without any acceptor of the evolved hydrogen bromide. Remarkably, during the cyclization reaction to form 1,3-thiazole ring used (Figure 3 and Figure 4), the nucleophilic substitution reaction of the sulfur-containing nucleophile proceeded regioselectively without reaction at the second nucleophilic center, the primary sulfonamide moiety. By reacting a benzenesulfonamide-containing α-haloketone (2a,b) with a substituted carbothioamide derivative (1a–o), 1,3-thiazoles 3a–t were synthesized in yields of 40–91%. In a similar manner, benzenesulfonamide-containing carbothioamide (4) underwent a cyclocondensation reaction with a substituted α-haloketone (5, 7) to yield the corresponding 1,3-thiazoles 6a–b and 8 in yields of 44–72%. In most instances, the reaction products did not require additional purification and precipitated from the reaction medium after 24 h of boiling in i-PrOH.

2.2. In vitro biological evaluation – MAO inhibition

The 1,3-thiazolylbenzenesulfonamide derivatives (3, 6, 8) were evaluated as in vitro inhibitors of MAO by using the recombinant human MAO enzymes (Novaroli et al., 2005). Kynuramine was used as enzyme substrate for both MAO isoforms and is metabolized to give 4-hydroxyquioline. In this study the formation of 4-hydroxyquinoline was monitored by fluorescence spectrophotometry. MAO catalytic activity was thus measured in the presence of a range of concentrations (0.003–100 µM) of the test inhibitors and sigmoidal graphs of enzyme activity versus inhibitor concentration (Log[I]) were constructed from the activity measurements. These graphs were used to estimate IC₅₀ values for the inhibition of MAO, which are reported as the mean ± standard deviation (SD) of triplicate measurements. The sigmoidal graphs for the inhibition of MAO-B by 3j, 3l and 3m are presented in Figure 5 as examples.

The potencies of MAO inhibition by the 1,3-thiazolylbenzenesulfonamide derivatives (3, 6, 8) are presented in Table 1. From the inhibition data it is evident that the derivatives are weak MAO-A inhibitors with most compounds exhibiting no inhibition at a maximal tested concentration of 100 µM. The most potent MAO-A inhibitors were 3a, 3f and 3m with IC₅₀ values of 11.9, 18.7 and 17.2 µM, respectively. While clear structure-activity relationships (SARs) are not apparent, is may be noted that among the compounds with IC₅₀ < 30 µM (e.g., 3a, 3b, 3f, 3j, 3l, 3m and 3t) the benzenesulfonamide group was substituted on C4 rather than C2 (6a,b) of the 1,3-thiazole moiety. Among these were compounds with substitution of the sulfonamide on both the meta and para positions of the phenyl.

In contrast, some of the 1,3-thiazolylbenzenesulfonamide derivatives were found to be potent inhibitors of MAO-B with the most potent inhibitors being 3j, 3l and 3m with IC₅₀ values of 0.103, 0.193 and 0.172 µM, respectively. In fact, seven derivatives in total exhibited IC₅₀ < 1 µM. SARs that were note included the following: (a) With the exception of 3j and 3q, all submicromolar inhibitors contained a halogen (F, Cl) on the phenyl ring; (b) For the submicromolar inhibitors, substitution of the benzenesulfonamide group occurred on either C4 or C2 (6b); (c) The most potent inhibitors (3j, l, m) all contained substitution of the sulfonamide on the meta position of the phenyl rather than the and para position. Most of the meta substituted compounds were more potent than the corresponding para substituted homologue (3j vs. 3b; 3l vs. 3f; 3m vs. 3d; 3n vs. 3k; 3q vs. 3c); (d) An aromatic substituent is a requirement for good potency MAO-B inhibition since derivatives with methyl substitution (3e, 3i, 8) were weak inhibitors.

2.3. Lineweaver-Burk graphs for the inhibition of MAO-B by 3j

This study has identified compound 3j as the most potent MAO-B inhibitor of the series. The mode of inhibition by this compound was further investigated by constructing sets of Lineweaver-Burk graphs. Six Lineweaver-Burk graphs were constructed at the following inhibitor concentrations: 0 × IC₅₀, ¼ × IC₅₀, ½ × IC₅₀, ¾ × IC₅₀, 1 × IC₅₀ and 1¼ × IC₅₀. For each graph, eight kynuramine concentrations (15 to 250 μM) were used to measure MAO-B activity. The Lineweaver-Burk graphs are shown in Figure 6 and suggest that compound 3j is a competitive inhibitor since the lines intersect on the y-axis. The enzyme-inhibitor dissociation constant (K_i) may be determined from a graph of the slopes of the Lineweaver-Burk graphs versus inhibitor concentration, and was estimated to be 0.21 µM.

2.4. Cytotoxicity in vitro

The effect of compounds 3j and 3m on human cell cultures were investigated with two cell lines, IMR-32 (neuroblastoma) and HS-5 (bone marrow stroma). In this respect, the cell viability after treatment with 0.5–100 µM of the test compounds were evaluated using the MTT assay (Gradiz et al., 2016; Mosmann, 1983)

The results of these experiments showed that compound 3j did not exhibit toxicity at the concentration range used for this study (Figure 7). Compound 3m, however, exhibited a TC₅₀ value of approximately 4 µM for both cell lines evaluated.

2.5. Molecular docking

This investigation found that certain 1,3-thiazolylbenzenesulfonamide derivatives act as potent MAO-B inhibitors. A molecular docking study was carried out to determine potential binding orientations and interactions of a selected derivative (3m) with the MAO-B enzyme. Molecular docking was carried out according to the literature procedure (Mostert et al., 2015) using Discovery Studio 3.1 and the published X-ray crystal structure of MAO-B in complex with zonisamide (PDB code: 3PO7) (Binda et al., 2011). As anticipated, compound 3m binds with the benzenesulfonamide moiety in the substrate cavity, in proximity to the FAD where the sulfonamide group occupies the same space as the sulfonamide of zonisamide (Figure 8). The sulfonamide is involved in hydrogen bonding with Gln206 and a water molecule as well as a pi-sulfur interaction with the FAD. The dichlorophenyl substituent extends beyond Ile199 and Tyr326 into the entrance cavity where numerous van der Waals interactions occur with the surrounding amino acid residues. Interestingly, the thiazole ring forms a pi-pi interaction with Tyr326 and pi-sulfur interactions with Cys172 and Phe168. This analysis underscores the significant interactions of the sulfonamide and thiazole moieties with MAO-B, while further inhibitor stabilization is provided by interactions between the dichlorophenyl and the entrance cavity. A similar binding mode to MAO-B has been predicted for A (Shetnev et al., 2019).

3. Conclusion

The present study investigated the MAO inhibition properties of a series of 1,3-thiazolylbenzenesulfonamides. The compounds were found to be specific inhibitors of MAO-B, with three derivatives (3j, 3l and 3m) exhibiting IC₅₀ values in the low nanomolar range. In contrast, the derivatives were much weaker MAO-A inhibitors with the most potent inhibition observed for 3a (IC₅₀ = 11.9 µM). A molecular docking study predicts that both the sulfonamide and thiazole moieties productively interact with the MAO-B active site and thus contribute to inhibitor stabilization. This investigation is an extension of our previous study that showed 1,3,4-oxadiazol-2-ylbenzenesulfonamides to act as potent MAO-B inhibitors and is the second report of sulfonamide compounds with nonomolar inhibition potencies. Potent MAO-B inhibitors such as those identified here may be considered to be good leads for the development of new therapies for disorders such as Parkinson’s disease.

4. Materials and Methods

4.1. Chemicals and instrumentation

All reagents and solvents were obtained from commercial sources (Aldrich, Fluka, Merck) and were used without further purification. Reactions were monitored with silica gel TLC using a mixture of toluene, acetone and petroleum ether (60:100:100) as eluent. Melting points were measured with a Stuart SMP30 apparatus in capillary tubes and are uncorrected. Electrospray ionization (ESI) mass spectra were obtained on a Bruker maXis spectrometer equipped with an ESI source. The instrument was operated in positive ion mode using an m/z range of 50–1200. The flow rate of the nebulizer gas was set to 1.0 bar, and that of the drying gas was set to 4.0 L/min. NMR spectra were recorded on a Bruker Avance 400 at ambient temperature, and were referenced to the residual solvent signal. ¹H NMR spectra were recorded at 400 MHz while ¹³C NMR spectra were recorded at 100 MHz. Chemical shifts are reported in parts per million (ppm).

Recombinant human MAO-A and MAO-B, kynuramine and reference MAO inhibitors used for the biological experiments were obtained from Sigma-Aldrich. A SpectraMax iD3 multi-mode microplate reader (Molecular Devices) was used to record the fluorescence measurements of the enzymatic reactions.

4.2. General procedure for the preparation of thiazoles (3) (GP1).

Thioamide 1 (0.9 mmol, 1 equiv.) was dissolved in i-PrOH (2.5 mL) and sulfonamide 2 (0.9 mmol, 1 equiv.) was added. The reaction mixture was stirred at room temperature for 1 h and then stirred overnight at 82.5 °C. The solvent was evaporated under reduced pressure and the reaction mixture was diluted with cold water (5 mL). The resulting precipitate was collected by filtration, washed with water (15 mL) and air-dried at 50 °C.

4-(2-(Thiophen-2-yl)thiazol-4-yl)benzenesulfonamide (3a). The reaction of 2-thiophenecarbothioamide (0.9 mmol, 0.13 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.24 g (83%) of 3a isolated as the beige solid. m.p. 248-250 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H), 8.17 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 5.0 Hz, 1H), 7.76 (dd, J = 3.7, 1.0 Hz, 1H), 7.42 (s, 2H), 7.21 (dd, J = 5.0, 3.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO- d₆) δ 161.98, 153.75, 144.13, 137.18, 136.84, 129.94, 129.18, 128.49, 127.04, 126.98, 116.99. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₃H₁₀N₂O₂S₃ 322.9977; found 322.9978.

4-(2-Phenylthiazol-4-yl)benzenesulfonamide (3b). The reaction of thiobenzamide (1.8 mmol, 0.26 g) and 4-(2-bromoacetyl)benzenesulfonamide (1.8 mmol, 0.25 g) according to general procedure GP1 afforded 0.51 g (89%) of 3b isolated as the beige solid. m.p. 215-217 °C. ¹H NMR (400 MHz, DMSO- d₆) δ 8.37 (s, 1H), 8.24 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 1.7 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.56 (s, 1H), 7.54 (t, J = 2.2 Hz, 2H), 7.41 (s, 2H). ¹³C NMR (101 MHz, DMSO- d₆) δ 168.12, 154.35, 144.10, 137.53, 133.45, 131.25, 129.97, 127.09, 126.97 (2С), 117.62. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₂N₂O₂S₂ 317.0413; found 317.0410.

4-(2-(4-(Dimethylamino)phenyl)thiazol-4-yl)benzenesulfonamide (3c). The reaction of 4-(dimethylamino)benzenecarbothioamide (0.9 mmol, 0.162 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.29 g (91%) of 3c isolated as the grey solid. m.p. 254-256 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 8.4 Hz, 2H), 8.15 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.40 (s, 2H), 6.81 (d, J = 8.9 Hz, 2H), 3.01 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.96, 153.77, 152.34, 143.81, 137.85, 128.13, 126.95, 126.89, 121.19, 115.07, 112.51, 39.58. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₇H₁₇N₃O₂S₂ 360.0835; found 360.0839.

4-(2-(3,4-Dichlorophenyl)thiazol-4-yl)benzenesulfonamide (3d). The reaction of 3,4-dichlorobenzenecarbothioamide (0.9 mmol, 0.19 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.30 g (86%) of 3d isolated as the beige solid. m.p. 189-191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.02 (dd, J = 8.4, 2.0 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 1H), 7.41 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.26, 154.55, 144.23, 137.18, 133.79, 133.61, 132.83, 132.18, 128.26, 127.18, 127.09, 126.97, 118.83. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₀Cl₂N₂O₂S₂ 384.9634; found 384.9632.

4-(2-Methylthiazol-4-yl)benzenesulfonamide (3e). The reaction of thioacetamide (1.9 mmol, 0.15 g) and 4-(2-bromoacetyl)benzenesulfonamide (1.9 mmol, 0.5 g) according to general procedure GP1 afforded 0.41 g (89%) of 3e isolated as the white solid. m.p. 250-252 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J = 3.4 Hz, 2H), 8.11 (s, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.37 (s, 2H), 2.74 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.81, 152.99, 143.75, 137.74, 126.91, 126.86, 116.83, 19.59. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₀H₁₀N₂O₂S₂ 255.0256; found 255.0257.

4-(2-(4-Fluorophenyl)thiazol-4-yl)benzenesulfonamide (3f). The reaction of 4-fluorothiobenzamide (1.8 mmol, 0.29 g) and 4-(2-bromoacetyl)benzenesulfonamide (1.8 mmol, 0.5 g) according to general procedure GP1 afforded 0.46 g (77%) of 3f isolated as the beige solid. m.p. 203-205 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.36 (s, 1H), 8.23 (d, J = 8.3 Hz, 2H), 8.11 (d, J = 5.4 Hz, 1H), 8.09 (d, J = 5.6 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.41 (s, 2H), 7.38 (d, J = 8.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.90, 164.01, 162.78, 154.26, 144.06, 137.41, 130.08, 129.34, 129.25, 127.06, 126.94, 117.71, 117.10, 116.88. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₁FN₂O₂S₂ 335.0319; found 335.0317.

4-(2-(5-Methylthiophen-2-yl)thiazol-4-yl)benzenesulfonamide (3g). The reaction of 5-methyl-2-thiophenecarbothioamide (0.9 mmol, 0.14 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.24 g) according to general procedure GP1 afforded 0.23 g (79%) of 3g isolated as the beige solid. m.p. 241-243 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.15 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 3.6 Hz, 1H), 7.40 (s, 2H), 6.91 (d, J = 3.0 Hz, 1H), 2.51 (s, 3H). ¹³C NMR (101 MHz, DMSO- d₆) δ 162.07, 153.53, 144.03, 143.77, 137.22, 134.44, 128.51, 127.60, 126.96 (2С), 116.42, 15.79. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₄H₁₂N₂O₂S₃ 337.0134; found 337.0131.

4-(2-(4-(Trifluoromethoxy)phenyl)thiazol-4-yl)benzenesulfonamide (3h). The reaction of 4-(trifluoromethoxy)benzenecarbothioamide (0.9 mmol, 0.2 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.22 g (61%) of 3h isolated as the beige solid. m.p. 194-196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.42 (s, 1H), 8.24 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.42 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.46, 154.52, 150.29, 144.20, 137.36, 132.57, 129.02, 127.11, 126.98, 122.37, 118.28. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₆H₁₁F₃N₂O₃S₂ 401.0236; found 401.0233.

3-(2-Methylthiazol-4-yl)benzenesulfonamide (3i). The reaction of thioacetamide (0.9 mmol, 0.07 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.17 g (74%) of 3i isolated as the beige solid. m.p. 168-170 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 8.07 (s, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.41 (s, 2H), 2.75 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.81, 152.95, 145.46, 135.43, 130.13, 129.51, 125.49, 123.78, 115.97, 19.57. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₀H₁₀N₂O₂S₂ 255.0256; found 255.0257.

3-(2-Phenylthiazol-4-yl)benzenesulfonamide (3j). The reaction of thiobenzamide (0.9 mmol, 0.12 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.18 g (64%) of 3j isolated as the brown solid. m.p. 180-181 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.33 (s, 1H), 8.25 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 6.7 Hz, 2H), 7.84 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 5.9 Hz, 3H), 7.47 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.14, 154.35, 145.59, 135.28, 133.49, 131.24, 130.24, 129.99, 129.79, 126.95, 125.86, 123.94, 116.85. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₂N₂O₂S₂ 317.0413; found 317.0410.

4-(2-(4-Isopropylphenyl)thiazol-4-yl)benzenesulfonamide (3k). The reaction of 4-isopropylthiobenzamide (0.9 mmol, 0.16 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.25 g (78%) of 3k isolated as the beige solid. m.p. 177-179 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (d, J = 2.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 3.2 Hz, 2H), 3.02 – 2.91 (m, 1H), 1.30 – 1.20 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.23, 154.25, 151.87, 144.07, 137.60, 131.28, 127.89, 127.07 (2С), 126.96, 117.17, 34.02, 24.28. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₈H₁₈N₂O₂S₂ 359.0882; found 359.0883.

3-(2-(4-Fluorophenyl)thiazol-4-yl)benzenesulfonamide (3l). The reaction of 4-fluorothiobenzamide (0.9 mmol, 0.14 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.26 g (87%) of 3l isolated as the beige solid. m.p. 189-191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.31 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.10 (dd, J = 8.6, 5.5 Hz, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.45 (s, 2H), 7.39 (t, J = 8.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.94, 164.04 (d, J = 248.4 Hz), 154.31, 145.57, 135.18, 130.00 (d, J = 46.0 Hz), 129.28 (d, J = 8.5 Hz), 125.86, 123.92, 117.12, 116.91. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₁FN₂O₂S₂ 335.0319; found 335.0322.

3-(2-(3,4-Dichlorophenyl)thiazol-4-yl)benzenesulfonamide (3m). The reaction of 3,4-dichlorobenzenecarbothioamide (0.9 mmol, 0.19 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.3 g (86%) of 3m isolated as the beige solid. m.p. 177-179 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.40 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.23 (s, 1H), 8.02 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 1.4 Hz, 1H), 7.82 (t, J = 9.5 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.47 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.28, 154.59, 145.61, 134.96, 133.84, 133.64, 132.87, 132.21, 130.27, 129.87, 128.23, 127.05, 126.00, 123.95, 118.09. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₀Cl₂N₂O₂S₂ 384.9634; found 384.9637.

3-(2-(4-Isopropylphenyl)thiazol-4-yl)benzenesulfonamide (3n). The reaction of 4-isopropylthiobenzamide (0.9 mmol, 0.16 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.17 g (65%) of 3n isolated as the beige solid. m.p. 140-142 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.28 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 7.7 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.46 (s, 2H), 7.42 (d, J = 8.2 Hz, 2H), 2.97 (dt, J = 13.8, 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.24, 154.25, 151.88, 145.57, 135.33, 131.30, 130.23, 129.77, 127.90, 127.05, 125.81, 123.91, 116.40, 34.02, 24.28. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₈H₁₈N₂O₂S₂ 359.0882; found 359.0880.

3-(2-(3,4-Dimethoxyphenyl)thiazol-4-yl)benzenesulfonamide (3o). The reaction of 3,4-dimethoxybenzenecarbothioamide (0.9 mmol, 0.18 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.29 g (85%) of 3o isolated as the beige solid. m.p. 139-141 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (s, 1H), 8.24 (d, J = 10.7 Hz, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.47 (s, 2H), 7.11 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.23, 154.06, 151.65, 149.80, 145.53, 135.36, 130.19, 129.81, 126.44, 125.73, 123.84, 120.28, 115.91, 112.74, 110.07, 56.40 (2C). HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₇H₁₆N₂O₄S₂ 377.0624; found 377.0621.

4-[2-(3,4-Dimethoxyphenyl)-1,3-thiazol-4-yl]benzenesulfonamide (3p). The reaction of 3,4-dimethoxybenzenecarbothioamide (0.9 mmol, 0.18 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.25 g (74%) of 3p isolated as the beige solid. m.p. 185-187 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.21 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 6.3 Hz, 2H), 7.39 (s, 2H), 7.07 (d, J = 8.8 Hz, 1H), 3.84 (d, J = 20.4 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.19, 154.04, 151.63, 149.79, 144.02, 137.64, 127.07 (2C), 126.92 (2C), 126.40, 120.29, 116.69, 112.68, 109.98, 56.38 (2C). HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₇H₁₆N₂O₄S₂ 377.0624; found 377.0628.

3-{2-[4-(Dimethylamino)phenyl]-1,3-thiazol-4-yl}benzenesulfonamide (3q). The reaction of 4-(dimethylamino)benzenecarbothioamide (0.9 mmol, 0.16 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.25 g (78%) of 3q isolated as the green solid. m.p. 134-136 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.10 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.46 (s, 2H), 6.81 (d, J = 8.8 Hz, 2H), 3.00 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.89, 153.78, 152.12, 145.49, 135.55, 130.12, 129.67, 128.13, 125.58, 123.86, 121.64, 114.37, 112.85, 39.82. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₇H₁₇N₃O₂S₂ 360.0835; found 360.0836.

3-(2-(4-(Trifluoromethoxy)phenyl)thiazol-4-yl)benzenesulfonamide (3r). The reaction of 4-(trifluoromethoxy)benzenecarbothioamide (0.9 mmol, 0.2 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.14 g (40%) of 3r isolated as the beige solid. m.p. 146-148 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.38 (s, 1H), 8.25 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.46 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.49, 154.53, 150.31, 145.61, 135.10, 132.61, 130.28, 129.82, 129.01, 125.96, 123.94, 122.41, 121.97, 119.41, 117.57. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₆H₁₁F₃N₂O₃S₂ 401.0236; found 401.0233.

4-(2-(3-(Trifluoromethyl)phenyl)thiazol-4-yl)benzenesulfonamide (3s). The reaction of 3-(trifluoromethyl)benzenecarbothioamide (0.9 mmol, 0.18 g) and 4-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.3 g (87%) of 3s isolated as the white solid. m.p. 158-160 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.37 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.18 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.47 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.48, 154.52, 150.30, 145.59, 135.09, 132.61, 130.29, 129.81, 129.01, 125.95, 123.92, 122.43, 117.59. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₆H₁₁F₃N₂O₃S₂ 401.0236; found 401.0237.

3-(2-(4-Methoxyphenyl)thiazol-4-yl)benzenesulfonamide (3t). The reaction of 4-methoxybenzothioamide (0.9 mmol, 0.15 g) and 3-(2-bromoacetyl)benzenesulfonamide (0.9 mmol, 0.25 g) according to general procedure GP1 afforded 0.27 g (87%) of 3u isolated as the yellow solid. m.p. 160-162 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.23 (d, J = 4.5 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.46 (s, 2H), 7.11 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.03, 161.79, 154.05, 145.53, 135.37, 130.19, 129.74, 128.56, 126.32, 125.73, 123.90, 115.82, 115.34, 56.13. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₆H₁₄N₂O₃S₂ 347.0519; found 347.0515.

4.3. Preparation and characterization of isomeric thiazoles (6)

General procedure for the preparation of isomeric thiazoles (6) (GP2). 4-(Aminosulfonyl)benzenecarbothioamide 4 (1.2 mmol, 1 equiv.) was dissolved in i-PrOH (2.5 mL) and bromide 5 (1.2 mmol, 1 equiv.) was added. The reaction mixture was stirred at room temperature for 1 h and then stirred overnight at 82.5 °C. The solvent was evaporated under reduced pressure and the reaction mixture was diluted with cold water (5 mL). The resulting precipitate was collected by filtration, washed with water (15 mL) and air-dried at 50 °C.

4-(4-(Benzo[d][1,3]dioxol-5-yl)thiazol-2-yl)benzenesulfonamide (6a). The reaction of 1-(1,3-benzodioxol-5-yl)-2-bromoethanone (0.9 mmol, 0.22 g) and 4-(aminosulfonyl)benzenecarbothioamide (0.9 mmol, 0.19 g) according to general procedure GP2 afforded 0.14 g (44%) of 6a isolated as the white solid. m.p. 181-183 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 8.3 Hz, 1H), 8.15 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 6.8 Hz, 2H), 7.50 (s, 2H), 7.02 (d, J = 8.6 Hz, 2H), 6.08 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.69, 156.00, 148.50, 148.10, 145.87, 136.32, 128.79, 127.30 (2C), 120.92, 115.10, 109.24, 107.17, 101.94. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₆H₁₂N₂O₄S₂ 361.0311; found 361.0310.

4-(4-(4-Chlorophenyl)thiazol-2-yl)benzenesulfonamide (6b). The reaction of 2′-bromo-4-chloroacetophenone (0.9 mmol, 0.21 g) and 4-(aminosulfonyl)benzenecarbothioamide (0.9 mmol, 0.19 g) according to general procedure GP2 afforded 0.18 g (60%) of 6b isolated as the white solid. m.p. 158-160 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (s, 1H), 8.22 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 8.3 Hz, 2H), 7.97 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.52 (s, 2H). ¹³C NMR (101 MHz, DMSO- d₆) δ 166.29, 154.99, 146.05, 136.19, 133.62, 133.29, 129.58, 128.60, 127.37 (2C), 117.31. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₅H₁₁ClN₂O₂S₂ 351.0023; found 351.0020.

4.4. Preparation and characterization of the thiazole 8

General procedure for the preparation of thiazole 8 (GP3). Following the previously reported procedure (Yamane et al., 2004), 4-(aminosulfonyl)benzenecarbothioamide 4 (0.9 mmol, 1 equiv.) was dissolved in i-PrOH (2 mL) and 3-chlorobutan-2-one 7 (1.4 mmol, 1.5 equiv.) was added. The reaction mixture was stirred at room temperature for 1 h and then stirred overnight at 82.5 °C. The solvent was evaporated under reduced pressure and the reaction mixture was diluted with cold water (5 mL). The resulting precipitate was collected by filtration, washed with water (15 mL) and air-dried at 50 °C.

4-(4,5-Dimethylthiazol-2-yl)benzenesulfonamide (8). Beige solid, yield 72%. m.p. 211-213 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.45 (s, 2H), 2.41 (s, 3H), 2.34 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.97, 150.43, 145.19, 136.61, 129.11, 127.24, 126.57, 15.24, 11.77. HRMS (ESI), m/z: [M+H]⁺ calcd. for C₁₁H₁₂N₂O₂S₂ 269.0413; found 269.0415.

4.7. Procedure for measuring MAO inhibition potency

The catalytic activities of MAO-A and MAO-B were measured by following the literature procedure (Mostert et al., 2015; Weissbach et al., 1960). Commercially available recombinant human MAO-A and MAO-B served as enzyme sources while kynuramine was used as a mixed MAO-A/B substrate. The MAO enzymes oxidize kynuramine to yield 4-hydroxyquinoline as the ultimate product. Since 4-hydroxyquinoline is fluorescent in basic medium, the reactions were terminated the end-point with the addition of sodium hydroxide and fluorescence spectrophotometry was used to quantitate 4-hydroxyquinoline.

4.8. Procedure for measuring cytotoxicity

The human cell culture lines, IMR-32 (neuroblastoma) and HS-5 (bone marrow stroma), were grown in a mixture (1:1) of RPMI-1640 and Ham's F12 media without glutamine. To the media was added FBS (10%), L-glutamine (2 mM), penicillin (50 IU/ml) and streptomycin (50 μg/mL). The test compounds were dissolved in DMSO and added to the culture media to yield a final concentration of 0.1% DMSO. As positive control (0% cell viability) the cells were exposed to sodium azide (0.1%). As negative control, cell viability was measured in the absence of the test compounds. The cytotoxicity of the test compounds was determined by the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] protocol (Gradiz et al., 2016; Mosmann, 1983). Cells were seeded at a concentration of 1×10⁴ cells /200 μL in a 96-well plate and cultured at 37 °C in a humid atmosphere with 5% CO₂. After 24 h of incubation, the test compounds were added to the wells at final concentrations that ranged from 0.03–100 µM and the cells were cultured under the same conditions for a further 72 h. Each test concentration was performed in triplicate. All substances were dissolved in DMSO and the final concentration of DMSO in the wells did not exceed 0.1%. For the negative control wells, DMSO was added to also yield a final concentration of 0.1%. After incubation, 20 μL of MTT (5 mg/mL) was added to each well and the plates were incubated for 2 h. The growth media was removed from the wells and 100 μL of DMSO was added to each well to dissolve the resulting formazan crystals. The absorbance was measured at a wavelength of 590 nm with a CLARIOstar (BMG Labtech) spectrophotometer. The blank consisted of culture medium.

4.9. Procedure for performing molecular docking

Molecular docking was carried out according to the procedure reported in literature (Mostert et al., 2015; Shetnev et al., 2019). The Discovery Studio 3.1 suite of life sciences software was used for all simulations and the X-ray crystal structures of MAO-A (PDB code: 2Z5X) and MAO-B (PDB code: 3PO7) bound to harmine and zonisamide, respectively, were selected as the protein models (Son et al., 2008; Binda et al., 2011).