Mechanism of Inactivation of Monoamine Oxidase-B by the Anticonvulsant Agent Milacemide (2-(n-Pentylamino)acetamide)

The anticonvulsant agent milacemide (2-(n-pentylamino)acetamide) is known to inactivate monoamine oxidase-B (MAO-B). Various isotopically labeled analogues of milacemide are used to elucidate the mechanism of inactivation of MAO-B by this compound. The metabolites of the oxidation of milacemide by MAO-B (pentanoic acid, pentanal, and glycinamide) are shown not to be responsible for inactivation. MAO was inactivated with 2-(n-pentylamino)-acetamide(1a),2-(n-pentylamino)[2,2-²H₂]acetamide(1b),and 2-([1,1⁻²H₂]-n-pentylamino)acetamide(1c). Compound 1b exhibited little or no isotope effect on inactivation (k_inact/K₁) and 1c showed an isotope effect of 4.55 on k_inact/K₁. These compounds also were found to be excellent substrates for MAO-B; 1b showed no isotope effect, but 1c exhibited an isotope effect of 4.53 on k_cat/K_m. Incubation of MAO with 2-(n-pentylamino) [2-¹⁴C]acetamide followed by dialysis under denaturing conditions resulted in the incorporation of 0.7 equiv of radioactivity per enzyme molecule. The same treatment with 2-([1-¹⁴C]-n-pentylamino)acetamide led to the incorporation of 4 equiv of radioactivity into the enzyme. The excess radioactivity bound presumably arises from the [¹⁴C]pentanal that is generated during turnover. In order to test this, MAO-B was incubated with [1-¹⁴C]pentylamine under similar conditions and 5.9 equiv of radioactivity was incorporated into the denatured enzyme. Therefore, the entire molecule becomes attached to the enzyme during inactivation. By following changes in the flavin absorption spectrum during inactivation with milacemide, it was shown that the flavin becomes reduced; however, denaturation of the inactivation enzyme causes flavin reoxidation under conditions where radioactivity for 2-(n-pentylamino) [2-¹⁴C] acetamide remains bound. This suggests that milacemide is oxidized during inactivation and the adduct results from attachment of milacemide to an amino acid residue, not to the flavin cofactor. Inactivation with 2-([1-¹⁴C]-n-pentylamino)acetamide produced [¹⁴C]pentanoic acid and [¹⁴C]-pentylamine in the ratio of 92:8. Inactivation of MAO with 2-(n-pentylamino) [2-¹⁴C]acetamide gave [¹⁴C] glycinamide and [¹⁴C]oxamic acid, further supporting oxidation reactions at both the pentyl side chain and the acetamido methylene. All of these results indicate that milacemide is oxidized at both the pentyl methylene and the acetamido methylene. Pentyl oxidation leads to inactivation, but it is not clear if acetamido methylene oxidation also leads to inactivation (Scheme I).