(S)-Willardiine ((-)-Willardiine)

Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine

Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5-bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5-iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5-iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)-willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.

Binding of the new radioligand (S)-[3H]AMPA to rat brain synaptic membranes: effects of a series of structural analogues of the non-NMDA receptor agonist willardiine

This study examined the binding of (S)-[3H]AMPA, the radiolabelled active isomer of AMPA, to rat brain synaptic membranes. Under non-chaotropic conditions specific binding of 10 nM (S)-[3H]AMPA represented 33 +/- 2% of the total; this increased to 74 +/- 1% in the presence of 100 mM KSCN. (S)-[3H]AMPA binding was inhibited by non-NMDA receptor agonists and the antagonists NBQX and CNQX, with the following rank order of potency: NBQX > (S)-AMPA > or = quisqualate > CNQX > L-glutamate > domoate > or = kainate > (R)-AMPA. NMDA, and the metabotropic glutamate receptor agonist (1S,3R)-ACPD, up to 100 microM, did not inhibit (S)-[3H]AMPA binding. A number of willardiine analogues all effectively inhibited (S)-[3H]AMPA binding with the rank order of potency: (S)-5-fluorowillardiine > (S)-5-nitrowillardiine > (S)-5-trifluoromethylwillardiine > (S)-5-bromowillardiine approximately (S)-5-chlorowillardiine > (S)-5-cyanowillardiine > (S)-willardiine > (S)-5-iodowillardiine > (S)-6-methylwillardiine > (S)-5-methylwillardiine. This rank order closely reflects data from equilibrium measurements made, under voltage clamp, on cultured hippocampal neurons. In contrast the respective (R)-enantiomers and the racemate mixtures of (R,S)-3, 5 and 6-isowillardiine were relatively inactive. Similar IC50 values and thus rank orders of potency for the willardiines were observed in the presence of 100 mM KSCN.

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.

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.

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.