Deprenyl

The two compounds were synthesized, purified and characterized as recently reported [6 (link)], whereas the two approved hMAO-B inhibitors (deprenyl and safinamide) were provided by Sigma-Aldrich (Milan, Italy). CLogP values for the newly-synthesized compounds were generated by ChemBioDraw Ultra 12.0.

Dimethyl sulfoxide (DMSO) (Sigma, Rehovot, Israel) and polyethylene glycol (PEG) (CS Chemicals, Haifa, Israel) were mixed in a 1:1 ratio and used as a vehicle to emulsify rotenone. Rotenone was used emulsified in a 6.7 mg/mL DMSO/PEG mixture and injected into an Alzet minipump until the pump was full. For vehicle-treated rats, the 1:1 DMSO/PEG mixture was used alone. Deprenyl (Sigma, Rehovot, Israel) was prepared in a concentration of 1.75 mg/mL in 0.9% sodium chloride for injection.

Autoradiography competition binding assay of [¹⁸F]flortaucipir binding to normal human brain was used to determine the IC₅₀ values of flortaucipir, MAO-A inhibitors clorgyline (Sigma-Aldrich) and fluoroethyl-harmol (prepared by alkylation of harmol [Alfa Aesar] with 1-bromo-2-fluoroethane as described by Ng et al. [21 (link)]), and MAO-B inhibitors deprenyl (Sigma-Aldrich) and safinamide (TCI). Adjacent frozen sections (10 μm thick) from a normal human temporal cortex (sourced from National Disease Research Interchange) were incubated with a fixed concentration of [¹⁸F]flortaucipir (0.74MBq [20 μCi] in 500 μL, 1.4 nM each slide) with varying concentrations of competing compound (log serial dilution from 10 μM to 0.001 μM) in 2.5% DMSO/2.5% EtOH/PBS, pH 7.4. After 60-min incubation at room temperature, PBS washes (4 × 2 min) were used to remove any unbound ligand. After drying under ambient conditions, the sections were placed in a cassette and exposed to a phosphor imaging plate overnight. The plate was then scanned with a GE Typhoon FLA7000 Bio-Imaging System. Signal intensity (counts/pixel) on each section was measured using Multi Gauge V3.0 Imaging software. Total binding was evaluated by nonlinear regression using GraphPad Prism v8 to determine IC₅₀ values.

Oleanolic Acid (OA), kynuramine, clorgyline, deprenyl, buspirone, Tween 80 (2% polyoxyethylenesorbitan monooleate), p-chlorophenylalanine (PCPA), N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclohexane carboxamide (WAY-100635 or WAY), Tris-HCl, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), polyethylenimine, 8-OH-DPAT, [³⁵S]-GTPγS and dimethyl Sulfoxide (DMSO) were purchased from Sigma-Aldrich, St Louis, MO, USA. Diazepam (DZP), imipramine (IMI) and prazosin (PRAZ) were purchased from Cristália, Itapira, SP, Brazil. Recombinant Human Monoamine Oxidase-A and -B (MAO-A and -B) were purchased from BD Biosciences Bedford, MA, USA. Oleanolic Acid derivatives (OAD); Oleanolic Acid acrylate (D1), Oleanolic Acid methacrylate (D2), Oleanolic Acid methyl fumarate (D3) and Oleanolic Acid ethyl fumarate (D4) were synthesized by esterification of OA with corresponding acyl chlorides. For in vivo assay, drugs were dissolved in a vehicle [a mixture of 0.9% NaCl and 5% Tween-80 (v/v)] and administered orally (p.o.) or intraperitonealy (i.p) in a volume of 0.1 mL per 10 g of mice body weight. All control animals received vehicle on the same regimen as the treated groups. For in vitro assay, drugs were dissolved in DMSO to yield a final DMSO concentration of 1.0% in the reaction mixture.