Total synthesis requires the two key intermediates and
Total synthesis requires the two key intermediates 3 and 7, which were synthesized using the route described in below. Phenylethylamine 3 was readily synthesized from vanillin or isovanillin; the overall yield was 50–52%. To prepare the intermediates 7, bromination of 2-(4-hydroxyphenyl) acetate with bromine was followed by protection with benzyl bromide or methyl iodide in acetone to give the intermediate 4. Phenols 5 were synthesized from halogen benzene 4 with (bis(8-quinolinolate)copper(II)) catalyst with a chemical yield of 90%. Intermediate 5 was subjected to hydroxymethylation with phenylboric AZ 628 and paraformaldehyde to afford the lactone 6. Finally, the lactone 6 was methylated with iodomethane to provide the intermediates 7. The method for the preparation of Intermediate 5 has been reported by Weller et al., but the reaction conditions require concentrated sodium hydroxide as base and CuSO as a catalyst, reacted at 150°C for 36h in stainless steel cannula. The change was made to simplify the reaction conditions by choosing bis(8-quinolinolate)copper(II) as the catalyst. The reaction was done at 120°C for 6h without nitrogen protection in sealed stainless steel cannula, and the yield was increased to 85–90%. At the same time, the bis(8-quinolinolate)copper (II) catalyst was recovered by filtration and washing. Intermediates 8 was prepared by the condensation of lactone 7 with phenethylamine 3 in ethanol (see ). The benzyl alcohol group was converted to the corresponding acetate 9 with acetic anhydride/triethylamine. The Bischler-Napieralski reaction was done in the presence of phosphorus oxychloride in acetonitrile, and the imine 10 was obtained in approximately quantitative yield. Freshly prepared imine 10 was used directly for the next reduction without further purification. Asymmetric transfer hydrogenation was done under argon in DMF, in the presence of Noyori’s catalyst and formic acid/triethylamine (v/v=5/2) as the hydrogen source. Intermediates 13 was prepared by the chlorination of the benzyl alcohol group in 12 with thionyl chloride in methylene chloride at 0°C. The ring closure reaction was accomplished by basifying the methylene chloride solution with saturated sodium bicarbonate. Deprotection was achieved by refluxing 14 in concentrated hydrochloric acid, with ee values up to 99.6%. Dopamine receptor binding was performed according to published procedures., For D1 and D5 receptors, [H]SCH23390 was used as the radiolabeled ligand and fluphenazine (10μM) was used to define nonspecific binding. For D2, D3, and D4 receptors, [H]methylspiperone and (+)-butaclamol (4μM) were used, respectively. Membranes were prepared from transfected HEK293 or CHO cells as described previously. Saturation binding of [H]SCH23390 or [H]methylspiperone to the D1 and D5 receptors or D2, D3, and D4 receptors, respectively, was done with at least 8 concentrations of [H]SCH23390 (ranging from 20pM to 20nM) or [H]methylspiperone (ranging from 20pM to 10nM). Binding was done in 50mM Tris-HCl buffer containing 120mM NaCl, 5mM KCl, 2mM CaCl, and 1mM MgCl (pH 7.4) at room temperature for 1h in duplicate in a volume of 250μL with 10–200μg membrane protein depending on receptor expression level. Incubations were terminated by filtration through Whatman GF/B filters under vacuum, and radioactivity on filters was measured. Competitive inhibition of [H]SCH23390 (2nM) binding to the D1 and D5 receptors or [H]methylspiperone (1nM) binding to the D2, D3, and D4 receptors by tetrahydroprotoberberine derivatives was performed with various concentrations (10M to 10M). Binding data were analyzed with the Prism program (GraphPad, San Diego, CA) and K, Bmax, and Ki values were determined. shows competitive inhibition by -THP and its demethylated analogs of radioligand binding to dopamine receptors. -THP has a low affinity for D1 and D5 (Ki of 140nM and 305nM) receptors and does not appear to bind to D2, D3, or D4 receptors (Ki>1000nM). -THB, and -DTHB showed high to moderate affinity for D1, D2 and D5, low affinity for D3 and D4. -ICP showed high to moderate affinity for D1, D2, D3, D4 and D5. -CP and -CD have similar agonists activity with -THP.