Synthesis of enantiomerically pure HIP-A and HIP-B and investigation of their activity as inhibitors of excitatory amino acid transporters

The present report deals with the synthesis of the two couples of enantiomers (+)-(3aS,4R,6aS)-/(−)-(3aR,4S,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-4-carboxylic acid [(+)-HIP-A and (−)-HIP-A] and (+)-(3aS,6S,6aS)-/(−)-(3aR,6R,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-HIP-B and (−)-HIP-B], and the investigation of their inhibitory activity at EAATs. When tested in a [3H]d-aspartate uptake assay on native EAATs present in rat brain synaptosomal fractions, (−)-HIP-A and (+)-HIP-B turned out to be considerably more potent than their enantiomers; in particular, (−)-HIP-A showed an eudismic ratio (ER) higher than 100. Molecular modeling investigations gave clues with regard to the key amino acid residues involved in the binding with our inhibitors and provided explanations for the observed stereoselectivity.

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(−)-(3aR,4S,6aR)-3-Hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-4-carboxylic acidC6H8N2O4[α]D20=-9.0 (c 0.14, H2O)ee >99.5%Source of chirality: (−)-(S)-N-Boc-3,4-didehydroproline methyl ester

(+)-(3aS,4R,6aS)-3-Hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-4-carboxylic acidC6H8N2O4[α]D20=+8.0 (c 0.1, H2O)ee >99.5%Source of chirality: (+)-(R)-N-Boc-3,4-didehydroproline methyl ester

(+)-(3aS,6S,6aS)-3-Hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-6-carboxylic acidC6H8N2O4[α]D20=+66.6 (c 0.105, H2O)ee >99.5%Source of chirality: (−)-(S)-N-Boc-3,4-didehydroproline methyl ester

(−)-(3aR,6R,6aR)-3-Hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-6-carboxylic acidC6H8N2O4[α]D20=-73.0 (c 0.112, H2O)ee >99.5%Source of chirality: (+)-(R)-N-Boc-3,4-didehydroproline methyl ester