Structure-activity relationship studies on N3-substituted willardiine derivatives acting as AMPA or kainate receptor antagonists.

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.

Synthesis and pharmacology of willardiine derivatives acting as antagonists of kainate receptors.

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.

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist.

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Structural requirements for novel willardiine derivatives acting as AMPA and kainate receptor antagonists.

1. The natural product willardiine is an AMPA receptor agonist. We have examined the structural changes required to convert willardiine into an antagonist at AMPA and kainate receptors. Structure-activity analysis has been carried out to discover the structural features required to increase the potency and/or selectivity of the antagonists at AMPA or kainate receptors. 2. Reduction of the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) has been used to investigate AMPA receptor antagonist activity. To examine antagonist activity at kainate receptors, the ability of compounds to depress kainate-induced depolarisations of dorsal root fibres was assessed. 3. Blocking ionisation of the uracil ring by adding a methyl group to the N(3) position was not sufficient to convert willardiine into an antagonist. However, willardiine derivatives with a side-chain bearing a carboxylic acid group at the N(3)-position of the uracil ring could antagonise AMPA and kainate receptors. 4. S stereochemistry was optimal for antagonism. When compounds with differing interacidic group chain lengths were compared, a group chain length of two methylene groups was preferable for AMPA receptor antagonism in the series of compounds bearing a carboxyalkyl side chain (UBP275, UBP277 and UBP279 reduced the fDR-VRP with IC(50) values of 287+/-41, 23.8+/-3.9 and 136+/-17 micro M, respectively). For kainate receptor antagonism, two or three methylene groups were almost equally acceptable (UBP277 and UBP279 reduced dorsal root kainate responses with apparent K(D) values of 73.1+/-4.5 and 60.5+/-4.1 micro M, respectively). 5. Adding an iodo group to the 5-position of UBP277 and UBP282 enhanced activity at kainate receptors (UBP291 and UBP301 antagonised kainate responses on the dorsal root with apparent K(D) values of 9.83+/-1.62 and 5.94+/-0.63 micro M, respectively). 6. The most useful antagonist identified in this study was UBP301, which was a potent and approximately 30-fold selective kainate receptor antagonist. UBP282 may also be of use in isolating a non-GluR5-mediated kainate response.

The novel antagonist 3-CBW discriminates between kainate receptors expressed on neonatal rat motoneurones and those on dorsal root C-fibres.

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-fibres, 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 micro M, respectively. That 3-CBW depressed the fDR-VRP by acting at AMPA and not metabotropic glutamate receptors was demonstrated by the lack of effect of LY341495 (100 micro M). 3. The Schild plot for antagonism of responses to (S)-5-fluorowillardiine 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 micro M) 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-fibres 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 micro M 3-CBW, substantiating evidence that kainate receptors on neonatal rat motoneurones differ from those on dorsal root C-fibres.