**1. Introduction**

Monoamine oxidases (EC.1.4.3.4; MAO-A and -B) are FAD-dependent enzymes that are responsible for the metabolism of neurotransmitters such as dopamine, adrenaline, serotonin, and noradrenaline, and also for the inactivation of exogenous arylalkyl amines [1–3]. Due to their vital role in neurotransmitter metabolism, these enzymes signify attractive drug targets for the pharmacological therapy of neurodegenerative diseases and neurological disorders [4–7]. Recent efforts toward the

development of MAO inhibitors have been focused on selective, reversible MAO-A or MAO-B inhibitors. The identification of MAO inhibitors could be helpful for numerous aspects of new drug discovery. Selective MAO-A inhibitors are effective in the treatment of depression and anxiety [8–11]. By contrast, MAO-B inhibitors are suitable for the treatment of the neurodegenerative diseases Alzheimer's disease and Parkinson's disease [5,7,8,11,12]. Historically, monoamine oxidase inhibitors (MAOI's) have been used to treat neurological disorders including depression [10]. Presently, MAO-A inhibitors play an important role in the control of neurological disorders, anxiety, and depression, while MAO-B inhibitors could potentially be used as therapeutic agents for Parkinson's and Alzheimer's diseases [13]. Pharmacotherapeutic limitations and adverse effects of the currently available MAO inhibitor drugs require the discovery of new MAO inhibitors with selective inhibition profiles and multi-target neuropharmacological profiles [12]. Natural products provide useful sources for MAO inhibitors combined with neuroprotective actions [3,14,15]. Several plant extracts and bioactive natural product metabolites with catecholaminergic neuropharmacological properties [16] have shown promising utility for the treatment of Alzheimer's disease and Parkinson's disease [17].

Recent studies from our lab have reported selective inhibition of MAO with flavonoid natural products [18–20]. In continuation of these studies on selected classes of flavonoids as well as other related published reports on various flavonoids [21,22], we have selected a series of methoxylated flavones and chalcones from our repository for further evaluation to explore their activities. The structure of chalcone is different from flavone/flavonol/flavanone, but they are biogenetically correlated. However, from our previous studies on flavone/flavanone [18,19], and those reported for chalcones [23] as valid MAO inhibitors, the difference in structures did not explain the structure–activity relationship for the inhibition of MAO A/B. Therefore, we chose to study the MAO inhibitory activity of these two types of flavonoids (flavone/flavonol and chalcone), including their docking studies. Therefore, these studies were further extended to test a set of related *O*-methylated flavonoids isolated from different plants, namely, *Senecio roseiflorus* (3,4- -di-*O*-methylkaempferol; **1**) [24], *Polygonum senegalense* (2- -hydroxy-4- ,6- -dimethoxy-chalcone; **2** and 2- ,4- -dihydroxy-6- -methoxy-chalcone; **3**) [25], *Bhaphia macrocalyx* (8-demethylsideroxylin; **4**) [26] *Gardenia ternifolia* (4- -*O*-methylkaempferol; **5**) [27], and *Psiadia punctulata* (5,7-dihydroxy-2,3,4,5-tetramethoxyflavone; **6**), [28] for experimental activities against MAO-A and -B. The isolated compounds are all non-polar chalcones (di-*O*-methylated, **2**, and **3**) and flavones exhibiting mono-*O*-methylation (**4** and **5**) or di-*O*-methylation (**1**) or tetra-*O*-methylation (**6**). There was one (**4**) which even showed ring A C-methylation (**4**) (Figure 1). This study was also extended to determine enzyme kinetics and the mechanism of inhibition of the compounds which showed the best IC50 values in the recombinant human monoamine oxidases assays (MAO-A and -B). Furthermore, molecular docking simulations were performed to understand the putative binding modes of the best compounds to MAO-A and -B.

**Figure 1.** Chemical structures of *O*-methylated flavonoids **1**–**6**.

#### **2. Results**

#### *2.1. Isolation, Purification, and Characterization of O-Methylated Flavonoids*

The *O*-methylated flavonoids reported in this paper were isolated from various plants, using general methods reported earlier [25,27]. Aerial parts (leaves and branches) were dipped in a non-polar solvent for short periods to wash off the exudates into the solvent (without affecting cell vacuole compounds). The solvents used were normally medium polarity solvents such as acetone or ethyl acetate. The solvent was removed using a rotary evaporator and the remaining solid materials were subjected to column chromatography using silica gel as a stationary phase and eluting with hexane/dichloromethane in a gradient fashion continuously increasing polarity followed by dichloromethane/methanol. Compounds **1**–**6** were isolated from different plants, namely, *S. roseiflorus* (**1**) [24], *P. senegalense* (**2** and **3**) [25], *G. ternifolia* (**5**) [27], *P. punctulata* (**6**) [28], and *B. macrocalyx* (**4**). Compound **4** is the first report from the genus *Baphia*. The isolation of compound **4** was not reported in the literature by us, therefore, its structure was determined using 1D and 2D NMR spectral data and TOF-MS (see Material and Methods section for details).

#### *2.2. Enzyme Inhibition and Kinetics Mechanism of MAO-A and -B with Compounds 1–6*

The inhibition (IC50) of the MAO-A and -B enzymes by compounds **1**–**6** are shown in Table 1. Compounds **1**, **2**, and **5** showed selective potent inhibition of MAO-A compared to compound **3**, which was potent at MAO-A but only slightly selective for MAO-A over -B. Compounds **4** and **6** were more potent than **3** at MAO-B and were selective for MAO-B over -A.



<sup>a</sup> The IC50 values computed from the dose–response inhibition curves are mean ± S.D. of at least triplicate observations. <sup>b</sup> SI Selectivity index: MAO-A IC50/MAO-B IC50. <sup>c</sup> Selectivity Index: MAO-A IC50 / MAO-B IC50. bClorgyline and cL-deprenyl were used as positive controls for MAO-A and -B, respectively.

Furthermore, the MAO-A inhibition mechanisms of compounds **1**–**3** and **5** were studied, using varying concentrations of kynuramine, a nonselective substrate, to investigate the nature of inhibition of the enzymes. Based on dose–response inhibition results, at least two concentrations of **1**–**3** and **5** were selected for the inhibition kinetics assay—one below and another above the IC50 value. Three sets of assays were completed at varying concentrations of the substrate for each experiment, one control without inhibitor and the others with two different concentrations of the inhibitor. The data were evaluated by double reciprocal Lineweaver-Burk plots for determination of the Ki (i.e., inhibition/binding affinity) values. Binding of compounds **1**–**3** and **5**, with human MAO-A, yielded the Km value (i.e., the affinity of the substrate for the enzyme) as well as Vmax (maximum enzyme activity) (Figure 2A–D). Ki values were computed from the double reciprocal plots (Table 2). Binding of compounds **3**, **4**, and **6** to human MAO-B yielded the Km value (i.e., the affinity of the substrate for the enzyme) as well as Vmax (maximum enzyme activity) (Figure 3A–C). Ki values were computed from the double reciprocal plots (Table 2). Compounds **3**, **4**, and **6** showed inhibitory activity of MAO-B with substantially high affinity (Ki = 1.242, 0.809, and 0.874 μM, respectively) (Table 2).

**Figure 2.** Kinetics characteristics of the inhibition mechanism of recombinant human MAO-A by compounds (**A**) **1**; (**B**) **2**; (**C**) **3**; (**D**) **5**; and (E) Clorgyline. Each point shows the mean value of three observations.

**Table 2.** Inhibition/binding affinity constants (Ki) for inhibition of recombinant human MAO-A by compounds **1**–**3** and **5** and of MAO-B by compounds **3**, **4**, and **6.**


<sup>a</sup> The results are presented as the mean ± SD of three observations; <sup>b</sup> Clorgyline and l-deprenyl were used as positive controls for MAO-A and -B, respectively.

**Figure 3.** Kinetics characteristics of the inhibition mechanism of recombinant human MAO-B by compounds (**A**) **3**; (**B**) **4**; (**C**) **6**; and (**D**) l-Deprenyl. Each point shows the mean value of three observations
