title: 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.
abstract: 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)-alpha-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 x HCl and (+)-9 x 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 (IC(50) = 12 microM) and glial (IC(50) = 16 microM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo¿4,5-cpyridin-3-ol (1, THPO) shows some selectivity for glial (IC(50) = 268 microM) versus neuronal (IC(50) = 530 microM) GABA uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC(50) = 200 microM) rather than neuronal (IC(50) = 900 microM) GABA uptake. This selectivity was more pronounced for 9, which showed IC(50) values of 40 and 500 microM 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 (IC(50) = 280 microM) and neuronal (IC(50) = 400 microM) GABA uptake. The remaining target compounds, 11-17, were very weak or inactive as inhibitors of both uptake systems. Compounds 9-13 and 15 were shown to be essentially inactive against isoniazide-induced convulsions in mice after subcutaneous administration. The isomeric pivaloyloxymethyl derivatives of 9, compounds 43 and 44, were synthesized and tested as potential prodrugs in the isoniazide animal model. Both 43 (ED(50) = 150 micromol/kg) and 44 (ED(50) = 220 micromol/kg) showed anticonvulsant effects, and this effect of 43 was shown to reside in the (R)-(+)-enantiomer, 45 (ED(50) = 44 micromol/kg). Compound 9 also showed anticonvulsant activity when administered intracerebroventricularly (ED(50) = 59 nmol).
authors.affiliation: Centre for Drug Design and Transport, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Falch
authors.forename: J
authors.initials: J
authors.lastname: Perregaard
authors.forename: B
authors.initials: B
authors.lastname: FrŁlund
authors.forename: B
authors.initials: B
authors.lastname: SŁkilde
authors.forename: A
authors.initials: A
authors.lastname: Buur
authors.forename: L M
authors.initials: LM
authors.lastname: Hansen
authors.forename: K
authors.initials: K
authors.lastname: Frydenvang
authors.forename: L
authors.initials: L
authors.lastname: Brehm
authors.forename: T
authors.initials: T
authors.lastname: Bolvig
authors.forename: O M
authors.initials: OM
authors.lastname: Larsson
authors.forename: C
authors.initials: C
authors.lastname: Sanchez
authors.forename: H S
authors.initials: HS
authors.lastname: White
authors.forename: A
authors.initials: A
authors.lastname: Schousboe
authors.forename: P
authors.initials: P
authors.lastname: Krogsgaard-Larsen
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007555:Isoxazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D015394:Molecular Structure; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D007555:Isoxazoles; C026804:4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol; D005680:gamma-Aminobutyric Acid