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.