2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and willardiine

Some N3-substituted analogues of willardiine such as 11 and 13 are selective kainate receptor antagonists. In an attempt to improve the potency and selectivity for kainate receptors, a range of analogues of 11 and 13 were synthesized with 5-substituents on the uracil ring. An X-ray crystal structure of the 5-methyl analogue of 13 bound to GLUK5 revealed that there was allowed volume around the 4- and 5-positions of the thiophene ring, and therefore the 4,5-dibromo and 5-phenyl (67) analogues were synthesized. Compound 67 (ACET) demonstrated low nanomolar antagonist potency on native and recombinant GLUK5-containing kainate receptors (KB values of 7 +/- 1 and 5 +/- 1 nM for antagonism of recombinant human GLUK5 and GLUK5/GLUK2, respectively) but displayed IC50 values >100 microM for antagonism of GLUA2, GLUK6, or GLUK6/GLUK2.

Topics: Alanine; Animals; Animals, Newborn; Binding Sites; Calcium; Cell Line; Crystallography, X-Ray; Humans; In Vitro Techniques; Ligands; Models, Molecular; Motor Neurons; Nerve Fibers, Unmyelinated; Protein Conformation; Pyrimidinones; Rats; Receptors, AMPA; Receptors, Kainic Acid; Recombinant Proteins; Spinal Nerve Roots; Stereoisomerism; Structure-Activity Relationship; Uracil

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.

Topics: Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Binding Sites; Cell Line; Drug Evaluation, Preclinical; Humans; In Vitro Techniques; Molecular Structure; Pyrimidinones; Rats; Receptors, Kainic Acid; Recombinant Proteins; Structure-Activity Relationship; Time Factors; Uracil

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.

Topics: Alanine; Animals; Animals, Newborn; Calcium; Cell Line; Humans; In Vitro Techniques; Long-Term Potentiation; Mossy Fibers, Hippocampal; Nerve Fibers, Unmyelinated; Protein Subunits; Pyrimidinones; Radioligand Assay; Rats; Receptors, AMPA; Receptors, Kainic Acid; Recombinant Proteins; Spinal Cord; Spinal Nerve Roots; Stereoisomerism; Structure-Activity Relationship; Uracil

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.

Topics: Alanine; Animals; Animals, Newborn; Benzodiazepines; Benzyl Compounds; Dose-Response Relationship, Drug; Evoked Potentials; Excitatory Amino Acid Antagonists; Motor Neurons; Nerve Fibers; Pyrimidinones; Quinoxalines; Rats; Receptors, AMPA; Receptors, Kainic Acid; Spinal Nerve Roots; Time Factors; Uracil

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.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain; Cycloleucine; Male; Pyrimidinones; Quinoxalines; Radioligand Assay; Rats; Rats, Wistar; Receptors, AMPA; Stereoisomerism; Synaptic Membranes; Tritium; Uracil