Twenty-four 4-alkylidene glutamic acids were synthesised and tested as potential subtype selective GluR5 and 6 ligands. It was found that a critical size of alkylidene group gave potent and selective GluR5 receptor agonists. LY339624 had Kis of 0.0326 and >100 μM on GluR5 and 6 receptors, respectively.

Enantiomerically pure (2S,4R)-4-substituted glutamic acids were prepared and tested for homomeric GluR5 and GluR6 kainate subtype receptor affinity. Some of the 4-cinnamyl analogues showed high selectivity and potency (K(i) < 25 nM) for the GluR5 receptors. The greatest selectivity and potency were achieved with the 3-(2-naphthyl)prop-2-enyl compound. This compound, LY339434, has negligible activity at the AMPA and kainate receptors GluR1, -2, -4 and -6. Although, LY339434 shows agonist activity at NMDA receptors in cultural hippocampal neurons (approximate EC(50) of 2.5 microM), we consider that LY339434 should be a useful pharmacological tool for the investigation of the functional role of GluR5 kainate receptors.

A general and efficient method for the stereoselective synthesis of racemic 5 beta- and 5 alpha-substituted kainic acids 3 and 4 has been developed starting from the bicyclic pyroglutamate derivative 6, readily available on a large scale. Compound 6 proved to be a versatile synthon from which straightforward functionalizations at both C-5 beta and C-5 alpha were accomplished in a stereoselective manner without compromising the stereogenic integrity of the potentially labile C-2 center. The key steps involved the stereoselective nucleophilic addition of organocopper reagents to the N-acyliminium ion I, and the stereoselective hydrogenation of the cyclic imine 9 derived from 6. Transformations of the bicyclic intermediates 7 and 8 into the final substituted kainic acids 3 and 4 were accomplished via stepwise sequences that avoid the facile and undesirable intramolecular Claisen and epimerization reactions. Compounds 3 and 4 have shown no significant binding affinity for the kainate receptors, which reflects the sensitivity of the recognition site to steric and conformational factors.

The activity of a γ-substituted glutamate analogue, (2S, 4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) and (2S,4R)-4-methylglutamic acid at ionotropic glutamate receptors has been examined. Ligand binding studies were performed using [3H] AMPA binding to membranes expressing either homomeric recombinant GluR1, GluR2, GluR4 receptors, and [3H] kainate binding to GluR5 and GluR6 kainate receptors. LY339434 and (2S,4R)-4-methylglutamic acid showed selectivity in ligand binding studies for kainate receptors over AMPA receptors. Within the kainate class of glutamate receptors, LY339434 showed selectivity for GluR5 over GluR6 whereas (2S,4R)-4-methylglutamic acid showed high affinity for both GluR5 and GluR6 kainate receptors. Examination of the functional activity of LY339434 and (2S,4R)-4-methylglutamic acid showed that both compounds evoked inward currents in dorsal root ganglion neurons (DRG) with estimated EC50 values of 0.8±0.2 μM and 0.17±0.04 μM, respectively. In GluR5 expressing HEK 293 cells, LY339434 evoked inward currents with an estimated EC50 value of 2.5±0.9 μM but had little effect on GluR6 expressing cells at concentrations less than 100 μM. LY339434 was a weak AMPA receptor agonist (EC50 values>300 μM) as determined by activity in acutely isolated cerebellar Purkinje neurons. LY339434 and (2S,4R)-4-methylglutamic acid had agonist activity at NMDA receptors studied in cultured hippocampal neurons with EC50s of 2.5 μM and 11.7 μM, respectively. These results indicate that both LY339434 and (2S,4R)-4-methyl glutamic acid may be useful pharmacological tools for the examination of kainate receptors.

Racemic 4-substituted kainic acids 4a,b have been synthesized from ethyl (1SR, 2SR, 5RS, 6RS)-N-(benzyloxycarbonyl)-3-aza-6-hydroxy-6-methylbicyclo[3.3.0]octane-2-carboxylate (7) after its transformation in the pyroglutamate derivative 6. The key step in the synthesis has been the regioselective alkylation of 6 to obtain 11a,b, without compromising the stereogenic integrity of the potentially labile C-2 center. The elaboration of the C-3 and C-4 substituents of kainic acid over 11 was achieved after double bond isomerization of 11, oxidative double bond cleavage of 5, and chemoselective aldehyde reduction of 16 followed by Wittig olefination reaction of 17 and final alcohol oxidation and hydrolysis over 18 to the corresponding 4-methyl kainic acid 4a and 4-benzyl kainic acid 4b. The relatively small changes introduced in the structure of kainic acid 1 have been found to lead to a loss of affinity for kainate receptors.