title: Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues.
abstract: A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.
authors.affiliation: Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
authors.forename: Rasmus P
authors.initials: RP
authors.lastname: Clausen
authors.forename: Ejner K
authors.initials: EK
authors.lastname: Moltzen
authors.forename: Jens
authors.initials: J
authors.lastname: Perregaard
authors.forename: Sibylle M
authors.initials: SM
authors.lastname: Lenz
authors.forename: Connie
authors.initials: C
authors.lastname: Sanchez
authors.forename: Erik
authors.initials: E
authors.lastname: Falch
authors.forename: Bente
authors.initials: B
authors.lastname: Frølund
authors.forename: Tina
authors.initials: T
authors.lastname: Bolvig
authors.forename: Alan
authors.initials: A
authors.lastname: Sarup
authors.forename: Orla M
authors.initials: OM
authors.lastname: Larsson
authors.forename: Arne
authors.initials: A
authors.lastname: Schousboe
authors.forename: Povl
authors.initials: P
authors.lastname: Krogsgaard-Larsen
mesh_terms: D000818:Animals; D018756:GABA Antagonists / Q000138:chemical synthesis* / Q000494:pharmacology*; D007555:Isoxazoles / Q000138:chemical synthesis* / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D051381:Rats
chemical_list: D018756:GABA Antagonists; D007555:Isoxazoles