*2.3. Binding and Time-Dependent Assays of MAO-A and -B with Compounds 1–6*

The characteristics of binding of compounds **1**–**3** and **5** with MAO-A were investigated by the equilibrium-dialysis assay. High concentrations of the compounds **1**–**3** and **5** (10.0, 25.0, 25.0, and 100.0 μM, respectively) were incubated with the MAO-A enzyme for 20 min and the resulting enzyme–inhibitor–complex preparation was dialyzed overnight against the 0.025 M phosphate buffer (pH 7.4). The activities of the enzyme were analyzed before and after the dialysis (Figure 4). The binding of compounds **1**–**3** with MAO-A was reversible and compound **5** showed partial reversibility (Table 2). Incubation of MAO-B with compounds **3**, **4**, and **6** (50.0, 50.0, and 50.0 μM, respectively) produced more than 60% inhibition of activity, and 80% of the activity of the enzyme was recovered after dialysis (Figure 5). Thus, the binding of compounds **3**, **4**, and **6** with MAO-B was reversible (Table 2). The selective MAO-B inhibitor deprenyl was confirmed to bind irreversibly with the enzyme (Table 2).

**Figure 4.** Binding modes of compounds **1** (10.0 μM), **2** (25.0 μM), **3** (25.0 μM), **5** (100.0 μM) and clorgyline (0.100 μM) with MAO-A.

**Figure 5.** Binding modes of compounds **3** (50.0 μM), **4** (50.0 μM), **6** (50.0 μM) and l-deprenyl (0.500 μM) with MAO-B.

Further investigation of the time dependence of the assay showed that the inhibition of MAO-A by compounds **1**–**3** and **5** was not time-dependent (Figure 6A). The compounds **3**, **4**, and **6** also did not show time-dependent inhibition of MAO-B (Figure 6B). For validation, we have run MAO-A and -B standards simultaneously for the time-dependent assay.

**Figure 6.** (**A**) Time-dependent inhibition of recombinant human MAO-A by compounds **1** (0.20 μM), **2** (1.6 μM), **3** (3.0 μM), **5** (16.0 μM) and l-deprenyl (0.010 μM). The remaining activity was expressed as % of initial activity. Each point represents the mean ± S.D. of triplicate values. (**B**) Time-dependent inhibition of recombinant human MAO-B by compounds **3** (3.0 μM), **4** (1.0 μM), **6** (2.0 μM) and l-deprenyl (0.070 μM). The remaining activity was expressed as % of the initial activity. Each point represents the mean ± S.D. of triplicate values.
