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

Abstract

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.

Figure 1

Structure of willardiine and a number of new derivatives.

Figure 2

Concentration–response curves for the reduction of the fDR-VRP by UBP275, UBP277 and UBP279. The IC₅₀ values were 287±41, 23.8±3.9 and 136±17 μM, respectively (n=3; mean±s.e.m.).

Figure 3

(a) Concentration–response curves for the reduction of the fDR-VRP by UBP290, UBP291 and UBP277. The IC₅₀ values were 79.9±15.9, 13.7±1.7 and 23.8±3.9 μM, respectively (n=3; mean±s.e.m.). (b) Concentration-response curves for the reduction of the fDR-VRP by UBP282 and UBP301. The IC₅₀ values were 10.3±2.4 and 164±25 μM, respectively (n=3; mean±s.e.m.).

Figure 4

(a) Concentration–response curves for kainate on the dorsal root in the absence and presence of 100 μM UBP277. The apparent K_D value was 73.1±4.5 μM (n=3; mean±s.e.m.). (b) Concentration–response curves for kainate in the absence and presence of 100 μM UBP279. The apparent K_D value was 60.5±4.1 μM (n=3; mean±s.e.m.). To normalise the data, responses were analysed as the percentage of the response to 30 μM kainate in the absence of antagonist.

Figure 5

Concentration–response curves for kainate on the dorsal root in the absence and presence of 100 μM UBP291. The apparent K_D value was 9.83±1.62 μM (n=3; mean±s.e.m.). To normalise the data, responses were analysed as the % of the response to 30 μM kainate in the absence of antagonist.

Figure 6

Concentration–response curves for kainate on the dorsal root in the absence and presence of 50 μM UBP301. The apparent K_D value was 5.94±0.63 μM (n=3; mean±s.e.m.). To normalise the data, responses were analysed as the percentage of the response to 30 μM kainate in the absence of antagonist.