Reversible Small Molecule Inhibitors of MAO A and MAO B with Anilide Motifs

Abstract

Background: Ligands consisting of two aryl moieties connected via a short spacer were shown to be potent inhibitors of monoamine oxidases (MAO) A and B, which are known as suitable targets in treatment of neurological diseases. Based on this general blueprint, we synthesized a series of 66 small aromatic amide derivatives as novel MAO A/B inhibitors.

Methods: The compounds were synthesized, purified and structurally confirmed by spectroscopic methods. Fluorimetric enzymological assays were performed to determine MAO A/B inhibition properties. Mode and reversibility of inhibition was determined for the most potent MAO B inhibitor. Docking poses and pharmacophore models were generated to confirm the in vitro results.

Results: N-(2,4-Dinitrophenyl)benzo[d][1,3]dioxole-5-carboxamide (55, ST-2043) was found to be a reversible competitive moderately selective MAO B inhibitor (IC₅₀ = 56 nM, K_i = 6.3 nM), while N-(2,4-dinitrophenyl)benzamide (7, ST-2023) showed higher preference for MAO A (IC₅₀ = 126 nM). Computational analysis confirmed in vitro binding properties, where the anilides examined possessed high surface complementarity to MAO A/B active sites.

Conclusion: The small molecule anilides with different substitution patterns were identified as potent MAO A/B inhibitors, which were active in nanomolar concentrations ranges. These small and easily accessible molecules are promising motifs, especially for newly designed multitargeted ligands taking advantage of these fragments.

Keywords: Parkinson’s disease; enzyme inhibitor; molecular modeling; pharmacophore; salicylic acid derivatives; structure-activity relationships.

Figure 1

Synthetic route to the amide analogs 1–4, 6, 7, 9–24, 26–66. Reagents and conditions: (A) PCl₃, toluene, reflux. (B) CDI, THF, reflux. (C) mw, 160 °C. For R¹, R², and R³ see Table 1.

Figure 2

Compounds with anilide motifs taken to MAO A and B screening.

Figure 3

Visualization of 55 (ST-2043, A₁, A₂) and 7 (ST-2023, B₁, B₂) in the binding pockets of MAO A and B. Compound 55 (ST-2043) binding to the crystal structure of A₁) hMAO B (PDB: 6FVZ) and A₂) hMAO A (PDB: 2Z5X). Compound 7 (ST-2023) binding to the crystal structure of B₁) hMAO B (PDB: 6FVZ) and B₂) hMAO A (PDB: 2Z5X). Surface coloring: white: neutral, green: lipophilic; magenta: hydrophilic.

Figure 4

ROC-selected pharmacophores of MAO A and MAO B. (A) Overlay of compound 55 (ST-2043) and MAO B pharmacophore model. (B) Overlay of compound 7 (ST-2013) and MAO A pharmacophore model. Color code: green vectored sphere: hydrogen bond acceptor; orange vectored sphere: aromatic feature; blue sphere: hydrophobic feature.

Figure 5

Reversibility of inhibition after preincubation of 55 (ST-2043) and L-deprenyl with MAO B. Inhibitors (10 ⨰ IC₅₀) were preincubated with enzyme for 0, 30, 60 or 90 min prior to 50-fold dilution in assays mixture containing kynuramine as substrate (10 ⨰ K_M). Data represent mean values ± SD of n independent experiments, each performed in duplicates.

Figure 6

Lineweaver–Burk plots of compound 55 (ST-2043) (12.5 to 100 nM) with seven suitable concentrations of kynuramine. Data represent mean values ± SD of one representative experiment performed in duplicates.