A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were synthesized and tested for their ability to preferentially inhibit the obligate cysteine-glutamate transporter system xc− versus the vesicular glutamate transporter (VGLUT). Several promising candidate molecules were identified: R/S-4-[4′-carboxyphenyl]-phenylglycine, a biphenyl substituted analog of 4-carboxyphenylglycine and 2-thiopheneglycine-5-sulfonic acid both of which reduced glutamate uptake at system xc− by 70–75% while having modest to no effect on glutamate uptake at VGLUT.

The condensation between dienophiles and α,β-unsaturated hydrazone azadienes was previously reported to afford piperidines. During an attempt to adapt this reaction to the preparation of piperidine-based conformationally-restricted analogs of glutamate, it was discovered that the electrophile, dimethyl oxoglutaconate (DOG) led to highly substituted dihydropyrans in 20–50% yield. The unexpected pyran product likely results from an initial 1,4-addition of the hydrazone to the oxoglutaconate followed by intramolecular cyclization of the resultant enolate oxygen to the α,β-unsaturated iminium ion. Further manipulations afford substituted tetrahydropyran 6-methamino-2,4-dicarboxylic acids.

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pKa ~ 9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system xc−). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5 mM but few inhibited system xc− greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system xc− inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT.