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 , IC 50  = 0.14 μM). 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.