Structure-Activity Relationship Studies on N 3 -Substituted Willardiine Derivatives Acting as AMPA or Kainate Receptor Antagonists Nigel P. Dolman, †,| Julia C. A. More, †,| Andrew Alt, § Jody L. Knauss, § Helen M. Troop, † David Bleakman, § Graham L. Collingridge, ‡ and David E. Jane †, * Department of Pharmacology, MRC Centre for Synaptic Plasticity, School of Medical Sciences, UniVersity Walk, UniVersity of Bristol, Bristol, BS8 1TD, UK, Department of Anatomy, MRC Centre for Synaptic Plasticity, School of Medical Sciences, UniVersity of Bristol, BS8 1TD, UK, and Neuroscience Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA ReceiVed October 26, 2005 N 3 -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 µM vs kainate on native GLU K5 ; K D 71.4 ( 8.3 µM 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 µM, 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 µM). Thus, 43 is a potent and selective GLU K5 receptor antagonist. Introduction (S)-Glutamate (1) can activate a range of glutamate receptor subtypes in the vertebrate central nervous system (CNS). There are two general classes of glutamate receptors, the ionotropic glutamate (iGlu) receptors and the metabotropic glutamate (mGlu) receptors. 1,2 The iGlu receptors are ligand-gated ion channels, which mediate fast synaptic responses in the CNS. The iGlu receptors were divided into three groups based on their pharmacology and are referred to as the (S)-2-amino-3-hydroxy- 5-methyl-4-isoxazolepropanoic acid (AMPA, 2), (2S,3S,4S)-3- carboxymethyl-4-isopropenylpyrrolidine-2-carboxylic acid (kain- ate, 3), and N-methyl-D-aspartic acid (NMDA, 4) receptor subtypes. AMPA receptors are tetrameric assemblies of a combination of GLU A1-4 subunits (IUPHAR nomenclature of the receptors that are also known as GluR1-4 or GluRA-D). 3c Kainate receptors are tetrameric assemblies of GLU K5-7 , GLU K1 , and GLU K2 subunits (IUPHAR nomenclature of the receptors that are also known as GluR5-7, KA1, and KA2). 3c Subunit- selective agonists and antagonists would facilitate the investiga- tion of the roles of the various subunits that make up AMPA and kainate receptors in central nervous system function. Despite considerable interest in the discovery of selective AMPA and kainate receptor antagonists, very few subunit-selective com- pounds have emerged. 1,2 A series of decahydroisoquinolines, including LY382884 (5) and 6, has been reported to selectively antagonize GLU K5 receptors. 4,5 The use of these antagonists has provided evidence to support the role of GLU K5 in hippocampal synaptic plasticity and in a number of CNS disorders such as epilepsy, chronic pain, ischaemia, and migraine. 4,5 Recent reports have highlighted the discovery of the first noncompetitive antagonists for GLU K5 , for example NS3763 (7). 6 We have previously reported that 5-substituted analogues of the natural product willardiine (8-10) exhibit selective agonist activity at either AMPA or kainate receptors. 7 We have also demonstrated that the agonist action of the willardiines can be converted to antagonism by N 3 -subtitution with carboxyalkyl or carboxybenzyl substituents. 8 In particular, the carboxyethyl and carboxybenzyl groups have been highlighted as useful N 3 - substituents for obtaining AMPA and kainate antagonist activity. Thus, 11 was a moderately potent AMPA receptor antagonist with weaker activity at kainate receptors. 8b p-Carboxy substitu- tion of the phenyl ring led to a mixed AMPA/kainate receptor antagonist (12), while o-carboxy substitution resulted in a selective GLU K5 antagonist (13). 8 While 11-13 were moder- ately potent AMPA and kainate receptor antagonists there is a need for more potent and selective antagonists. Herein we report the synthesis and pharmacological characterization of a new series of N 3 -substituted willardiine analogues in which the terminal acid group and the nature of the linker were varied in an attempt to enhance the potency at AMPA or kainate receptors. This novel series of compounds was pharmacologically char- acterized on both cloned and native AMPA and kainate receptors. Results Chemistry. The N 3 -substituted uracil analogues 14, 16a, and 16b were prepared as described previously. 8d Conversion of these nitrile-substituted compounds into the corresponding tetrazole-substituted analogues (15, 17a, and 17b) was effected using sodium azide and ammonium chloride in DMF (Scheme 1). The N-Boc protecting group was removed using TFA and the amino acids purified using ion-exchange resin chromatog- raphy. A considerable amount of racemization occurred during the tetrazole formation with ee values of 75, 84, and 60% for 15, 17a, and 17b, respectively, as adjudged by chiral HPLC analysis. The iodination of the uracil ring of 17b was carried out using iodine monochloride in aqueous hydrochloric acid 9 to give 18 (Scheme 1). * To whom correspondence should be addressed. Phone: +44 (0)117 9546451. Fax: +44 (0)117 9250168. E-mail: david.jane@bristol.ac.uk. † Department of Pharmacology, University of Bristol. ‡ Department of Anatomy, University of Bristol. § Eli Lilly and Company. | These authors contributed equally to this work. 2579 J. Med. Chem. 2006, 49, 2579-2592 10.1021/jm051086f CCC: $33.50 © 2006 American Chemical Society Published on Web 03/22/2006