The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord. The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLUK5.

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1 The natural product willardiine is a selective AMPA receptor agonist. We report that an N 3 -substituted analogue of willardiine, (S)-3-(4-carboxybenzyl)willardiine (3-CBW), antagonizes AMPA and kainate receptors expressed on motoneurones and dorsal root C-®bres, respectively. 2 Reduction of the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) has been used as a novel method to compare AMPA receptor antagonists. 3-CBW, NBQX and GYKI53655 depressed the fDR-VRP with IC 50 values of 10.3+2.4, 0.214+0.043 and 4.03+0.31 mM, respectively. That 3-CBW depressed the fDR-VRP by acting at AMPA and not metabotropic glutamate receptors was demonstrated by the lack of eect of LY341495 (100 mM). 3 The Schild plot for antagonism of responses to (S)-5-¯uorowillardiine on motoneurones by 3-CBW had a slope of 1.11+0.13 giving a pA 2 value of 4.48. The Schild plot for antagonism of kainate responses on the dorsal root by 3-CBW had a slope of 1.05+0.05 giving a pA 2 value of 4.96. 4 On neonatal rat motoneurones 3-CBW (200 mM) almost completely abolished responses to AMPA while responses to NMDA, kainate and DHPG were 101.6+11.6%, 39.4+5.8% and 110.5+9.0% of control, respectively. 3-CBW can therefore be used to isolate kainate receptor responses from those mediated by AMPA receptors. 5 3-CBW antagonized kainate-induced responses on dorsal root C-®bres with a pA 2 value of 4.96 whereas kainate receptor mediated responses (isolated by including GYKI53655 in the medium) on motoneurones were not completely blocked by 200 mM 3-CBW, substantiating evidence that kainate receptors on neonatal rat motoneurones dier from those on dorsal root C-®bres.

N3-substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU(K5)-containing kainate receptors (K(D) 0.105 +/- 0.007 microM vs kainate on native GLU(K5); K(D) 71.4 +/- 8.3 microM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant human GLU(K5), GLU(K5)/GLU(K6), and GLU(K5)/GLU(K2), K(B) values of 0.12 +/- 0.03, 0.12 +/- 0.01, and 0.18 +/- 0.02 microM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU(K6) or GLU(K7) kainate receptors or homomeric GLU(A1-4) AMPA receptors (IC(50) values > 100 microM). Thus, 43 is a potent and selective GLU(K5) receptor antagonist.