Synthesis of Willardiine and 6-Azawillardiine Analogs:  Pharmacological Characterization on Cloned Homomeric Human AMPA and Kainate Receptor Subtypes

Abstract

Both willardiine and azawillardiine analogs (18-28) have been reported to be potent and selective agonists for either AMPA or kainate receptors. We report here the novel synthesis and pharmacological characterization of a range of willardiine (18-23) and 6-azawillardiine (24-28) analogs on cells individually expressing human homomeric hGluR1, hGluR2, hGluR4, or hGluR5 receptors. Reaction of the sodium salts of substituted uracils (7-12) or 6-azauracils (13-16) with (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one (17) in dry DMF, subsequent deprotection in TFA, and purification by ion-exchange chromatography gave mainly the willardiine analog in which alkylation took place on N1 of the uracil ring. We have investigated the subtype selectivity of these compounds by examining their binding affinity for homomeric hGluR1, -2, -4, or -5 (and hGluR6 in the case of 5-iodowillardiine (22)). From this study we have demonstrated that 22 has high affinity for hGluR5 and, compared to kainate, displays excellent selectivity for this receptor over both the AMPA receptor subtypes and the homomeric kainate receptor, hGluR6. 5-Fluorowillardiine (19) has higher affinity than AMPA for both homomeric hGluR1 and hGluR2 and compared to AMPA displays greater selectivity for AMPA receptor subtypes over the kainate receptor, hGluR5. Some structural features required for optimal activity at homomeric AMPA or kainate receptor subtypes have also been identified. It would appear that quite large lipophilic substituents at the 5-position of the uracil ring not only are accommodated by hGluR5 receptors but also lead to enhanced affinity for these receptors. In contrast to this, for optimal binding affinity to hGluR1, -2, or -4, smaller, electron-withdrawing substituents are required. For optimal activity at hGluR4 receptors a 6-aza-substituted willardiine is favored. The subtype-selective compounds described here are likely to be useful tools to probe the distribution and the physiological roles of the various glutamate receptor subunits in the central nervous system.

The Selective Activation of the Glutamate Receptor GluR5 by ATPA Is Controlled by Serine 741

“…Studies performed on cortical wedges, which mainly reflect activation of the AMPA receptors, showed that the 5-ethyl analog of AMPA was more potent than AMPA, whereas the propyl and butyl analogs showed decreased potencies (Sløk et al, 1997). These observations and studies of willardiine analogs (Wong et al, 1994;Jane et al, 1997;Swanson et al, 1998) have resulted in a hypothesis proposing that the AMPA and kainate receptors might contain a hydrophobic cavity that can accommodate hydrophobic substituents to a certain size at the 5-position of the isoxazole ring of the AMPA molecule (Krogsgaard-Larsen et al, 1996).…”

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

“…Quisqualate, aniracetam, AMPA, and CNQX were obtained from Tocris. The willardiine compounds were synthesized as described previously (41). The GluR2 S1S2J construct was obtained from Eric Gouaux (Vollum Institute; Ref.…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…propionic acid, (S)-5-iodowillardiine, and (4R)-isopentyl glutamate are potent agonists at homomeric GluK1 receptors but have little to no activity at GluK2 receptors (Clarke et al, 1997;Jane et al, 1997;Zhou et al, 1997;Small et al, 1998;Brehm et al, 2003;). GluK1 selectivity arises from the larger GluK1 binding cavity, which relieves steric occlusion of the bulky tert-butyl group of ATPA and halogen atom of (S)-5-iodowillardiine (Mayer, 2005).…”