Selective Inhibitors of Glial GABA Uptake:  Synthesis, Absolute Stereochemistry, and Pharmacology of the Enantiomers of 3-Hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and Analogues

3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a), or the corresponding 3-ethoxy analogue (20b), and 3-chloro-4,5,6,7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized by regioselective chromic acid oxidation of the respective bicyclic tetrahydrobenzenes 19a,b and 50, and they were used as key intermediates for the syntheses of the target zwitterionic 3-isoxazolols 8 − 15 and 3-isothiazolols 16 and 17, respectively. These reaction sequences involved different reductive processes. Whereas (RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-benzisoxazole (8, exo-THPO) was synthesized via aluminum amalgam reduction of oxime 22a or 22b, compounds 9, 11 − 13, and 15 − 17 were obtained via reductive aminations. Compound 10 was synthesized via N-ethylation of the N-Boc-protected primary amine 25. The enantiomers of 8 were obtained in high enantiomeric purities (ee ≥ 99.1%) via the diastereomeric amides 32 and 33, synthesized from the primary amine 23b and (R)-α-methoxyphenylacetyl chloride and subsequent separation by preparative HPLC. The enantiomers of 9 were prepared analogously from the secondary amine 27. On the basis of X-ray crystallographic analyses, the configuration of oxime 22a was shown to be E and the absolute configurations of (−)-8·HCl and (+)-9·HBr were established to be R. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation and primary cultures of mouse cortical neurons and glia cells (astrocytes). Whereas the classical GABA uptake inhibitor, (R)-nipecotic acid (2), nonselectively inhibits neuronal (IC50 = 12 μM) and glial (IC50 = 16 μM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (1, THPO) shows some selectivity for glial (IC50 = 268 μM) versus neuronal (IC50 = 530 μM) GABA uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC50 = 200 μM) rather than neuronal (IC50 = 900 μM) GABA uptake. This selectivity was more pronounced for 9, which showed IC50 values of 40 and 500 μM as an inhibitor of glial and neuronal GABA uptake, respectively. These effects of 8 and 9 proved to be enantioselective, (R)-(−)-8 and (R)-(+)-9 being the active inhibitors of both uptake systems. The selectivity of 9 as a glial GABA uptake inhibitor was largely lost by replacing the N-methyl group of 9 by an ethyl group, compound 10 being an almost equipotent inhibitor of glial (IC50 = 280 μM) and neuronal (IC50 = 400 μM) GABA uptake. The remaining t