Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D.

Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane. The four human receptor subtypes, which we assign to group 5 of family C GPCRs, show 31-42% amino acid sequence identity to each other and 20-25% sequence identity to the transmembrane domains of metabotropic glutamate receptor subtypes 2 and 3 and other family C members. In contrast to the remaining family C members, the group 5 receptors have short amino terminal domains of some 30-50 amino acids. GPRC5D was shown to be clustered with RAIG1 on chromosome 12p13.3 and like RAIG1 and GPRC5B to consist of three exons, the first exon being the largest containing all seven transmembrane segments. GPRC5D mRNA is widely expressed in the peripheral system but all four receptors show distinct expression patterns. Interestingly, mRNA levels of all four group 5 receptors were found in medium to high levels in the kidney, pancreas and prostate and in low to medium levels in the colon and the small intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1, and the seven transmembrane domain of the orphan receptors were constructed and tested in binding and functional assays.

Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists.

Development of new selective ligands for excitatory amino acid receptors has been fundamental in supporting this rapidly developing field. Some of the most important ligands have come from the laboratories of Jeff Watkins, Povl Krogsgaard-Larsen and Tage Honoré, who collaborate in this double-length review to describe the chemical features and SARs of agonists and antagonists, particularly those features associated with subtype selectivity.

Inhibitors of AMPA and kainate receptors.

The glutamate receptor system is implicated in the development and maintenance of epileptic seizures, and animal studies have disclosed potent anticonvulsant activity of a number of inhibitors of AMPA and/or kainate (KA) receptor activity. These results make such inhibitors potential future antiepileptic drugs. Different series of compounds with inhibitory activity towards AMPA receptors have been developed. Most of these inhibitors are structurally derived from AMPA, quinoxalinedione or 2,3-benzodiazepine. In contrast, only a limited number of inhibitors of KA receptor activity have been developed, most of which contain quinoxalinedione or decahydroisoquinoline skeletons. In spite of promising anticonvulsant activity in various animal model studies, no AMPA/KA receptor inhibitors are in clinical use against epilepsy today. Based on molecular biology studies, AMPA and KA receptors are at present divided into four and five subtypes, respectively, and attempts to develop subtype selective compounds have been initiated. Future studies and development of such compounds will indicate whether AMPA/KA receptor inhibition is a feasible therapeutic strategy for the treatment of epilepsy.

GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects.

In the mid seventies a drug design programme using the Amanita muscaria constituent muscimol (7) as a lead structure, led to the design of guvacine (23) and (R)-nipecotic acid (24) as specific GABA uptake inhibitors and the isomeric compounds isoguvacine (10) and isonipecotic acid (11) as specific GABAA receptor agonists. The availability of these compounds made it possible to study the pharmacology of the GABA uptake systems and the GABAA receptors separately. Based on extensive cellular and molecular pharmacological studies using 23, 24, and a number of mono- and bicyclic analogues, it has been demonstrated that neuronal and glial GABA transport mechanisms have dissimilar substrate specificities. With GABA transport mechanisms as pharmacological targets, strategies for pharmacological interventions with the purpose of stimulating GABA neurotransmission seem to be (1) effective blockade of neuronal as well as glial GABA uptake in order to enhance the inhibitory effects of synaptically released GABA, or (2) selective blockade of glial GABA uptake in order to increase the amount of GABA taken up into, and subsequently released from, nerve terminals. The bicyclic compound (R)-N-Me-exo-THPO (17) has recently been reported as the most selective glial GABA uptake inhibitor so far known and may be a useful tool for further elucidation of the pharmacology of GABA transporters. In recent years, a variety of lipophilic analogues of the amino acids 23 and 24 have been developed, and one of these compounds, tiagabine (49) containing (R)-nipecotic acid (24) as the GABA transport carrier-recognizing structure element, is now marketed as an antiepileptic agent.

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors.

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50 > 300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.

Anticonvulsant activity of the glial-selective GABA uptake inhibitor, THPO.

The intramuscular administration of 4,5,6,7-tetrahydroisoxazolo [4,5-c] pyridin-3-ol (THPO) delayed the onset of isonicotinic acid hydrazide-induced seizures in very young chicks but not in adult mice, the difference being due to the state of development of the blood-brain-barrier which controls access of the drug to the brain tissue. THPO was also effective in preventing seizures induced in epileptic chicks by intermittent photic stimulation. The anticonvulsant action after combined administration of THPO and gabaculine, an inhibitor of GABA-alpha-oxoglutarate aminotransferase activity, was no greater than the anticonvulsant action of gabaculine alone.

[3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

Modification of the philanthotoxin-343 polyamine moiety results in different structure-activity profiles at muscle nicotinic ACh, NMDA and AMPA receptors.

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.

Ibotenic acid analogues. Synthesis, molecular flexibility, and in vitro activity of agonists and antagonists at central glutamic acid receptors.

The syntheses of (RS)-alpha-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (9, ATPA), (alpha-RS, beta-RS)-alpha-amino-beta-methyl-3-hydroxy-5-isoxazolepropionic acid (8), (RS)-alpha-amino-3-hydroxy-5-isoxazolebutyric acid (15a), and (RS)-alpha-amino-3-hydroxy-5-isoxazolevaleric acid (15b) are described. The compounds were tested in vitro together with (RS)-alpha-amino-3-hydroxy-5-(bromomethyl)-4-isoxazolepropionic acid (ABPA) as inhibitors of the binding of radioactive-labeled (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to rat brain synaptic membranes. These data were compared with the earlier reported effects of the compounds on single neurons in the feline spinal cord obtained by microelectrophoretic techniques. The three compounds AMPA, ATPA, and ABPA are agonists at the class of receptors assumed to represent a subtype of physiological (S)-glutamic acid (Glu) receptors. Inhibition of [3H]AMPA binding by ATPA was 1 order of magnitude weaker than that of AMPA, in agreement with the relative potency of these compounds in vivo. ABPA proved to be equipotent with AMPA both as an inhibitor of AMPA binding and as a neuronal excitant. The compounds 8, 15a, and 15b have no effect as inhibitors of AMPA binding, in agreement with in vivo studies that have shown that 8 does not affect the firing of central neurons whereas 15a and 15b are antagonists at NMDA receptors, a subpopulation of excitatory receptors not affected by AMPA. Molecular mechanical calculations on AMPA, ATPA, and ABPA using the program MM2 showed that conformations of AMPA, ABPA, and especially ATPA by rotation of the amino acid side chain have energy barriers. A possible receptor-active conformation is suggested.

Esters of isoguvacine as potential prodrugs.

The syntheses of the methyl ester, butyl ester, (ethoxycarbonyl)methyl ester, and 11 (acyloxy)methyl esters of the potent gamma-aminobutyric acid agonist isoguvacine (1,2,3,6-tetrahydropyridine-4-carboxylic acid) and described. The chemical stability of the esters and their in vitro rates of hydrolysis under approximately physiological conditions by nonspecific esterases from human serum were examined. A selected number of the esters were tested for antagonism of convulsions induced by bicuculline, isoniazide, and by electroschock. While in the compounds showed only weak activities in the bicuculline and isoniazide tests, a good correlation between in vitro rates of enzymatic hydrolysis and the time of onset of the antagonism of the electroschock-induced convulsions could be found.

GABA agonists and uptake inhibitors. Synthesis, absolute stereochemistry, and enantioselectivity of (R)-(-)- and (S)-(+)-homo-beta-proline.

The cyclic analogue of 4-aminobutyric acid (GABA), 3-pyrrolidineacetic acid (homo-beta-proline), is a potent agonist at GABAA receptors, it interacts effectively with GABA-uptake mechanisms, and it is a moderately potent inhibitor of GABAB receptor binding. (R)-(-)- (10) and (S)-(+)-homo-beta-proline (15) were synthesized via methyl (3S)-1-[(R)-1-phenylethyl]-5-oxo-3-pyrrolidinecarboxylate (5) and its 3R diastereomer (4), respectively. The mixture 3 consisting of 4 and 5 was synthesized via addition-cyclization reactions between (R)-1-phenylethylamine and itaconic acid (1). The diastereomers 5 and 4, which were separated chromatographically, were converted into (R)- (10) and (S)-homo-beta-proline (15), respectively. The absolute stereochemistry of 10 and 15 was established on the basis of an X-ray analysis of compound 5. The enantiomers 10 and 15 were shown to bind to GABAA and GABAB receptor sites with opposite stereoselectivity. Thus, (R)-homo-beta-proline (10) proved to be more than 1 order of magnitude more potent than the S enantiomer (15) as an inhibitor of GABAA receptor binding, whereas the GABAB receptor affinity of homo-beta-proline was shown to reside exclusively in (S)-homo-beta-proline (15). In contrast to the stereoselective receptor affinities of 10 and 15, these enantiomers were approximately equieffective as inhibitors of synaptosomal GABA uptake.

Enzymic resolution and binding to rat brain membranes of the glutamic acid agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.

The enantiomers of the glutamic acid central nervous system receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were prepared via kinetic resolution of the racemic N-acetylated 3-methoxy derivative by reusable, immobilized aminoacylase. L-AMPA was more effective (IC50 = 0.6 microM) than D-AMPA (IC50 = 4.8 microM) in displacing racemic [3H]AMPA from binding sites on rat brain synaptic membranes in agreement with their relative in vivo excitatory potencies.

A novel class of conformationally restricted heterocyclic muscarinic agonists.

A series of conformationally restricted compounds containing the 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) skeleton, including O-methyl-THPO (10a) and O,5-dimethyl-THPO (11a), were synthesized. The compounds were designed by bioisosteric replacement of the methyl ester groups of the muscarinic cholinergic agonists norarecoline and arecoline by the 3-methoxyisoxazole group, and their interactions with central and peripheral muscarinic receptors were tested in vitro. The compounds 10a, 11a, O-ethyl-THPO (10b), O-propargyl-THPO (10j), and O-ethyl-5-methyl-THPO (11b) were inhibitors of the binding of the muscarinic mustard [3H]PrBCM to rat brain membranes with an increasing order of potency. There was, however, a very low degree of correlation between these binding data and the effects of the compounds on peripheral (ileal) muscarinic receptors, where 11a, 10j, 11b, and 10a were agonists with a decreasing order of potency, whereas O-isopropyl-THPO (10e) showed antagonistic effects. The relatively low pKa values of the compounds (7.5-7.7 for compounds with secondary and 6.1-7.0 for compounds with tertiary amino groups) are likely to allow the compounds to penetrate the blood-brain barrier.

GABA agonists. Resolution, absolute stereochemistry, and enantioselectivity of (S)-(+)- and (R)-(-)-dihydromuscimol.

(RS)-5-(Aminomethyl)-2-isoxazolin-3-ol (dihydromuscimol, DHM) is a potent 4-aminobutyric acid (GABA) agonist, the inhibitory effects of which on neurons are sensitive to the antagonist bicuculline methochloride (BMC), and it also interacts with the GABA uptake system in vitro. (S)-(+)-DHM (4) and (R)-(-)-DHM (5) were obtained in optically pure forms via resolution of tert-butyloxycarbonyl-protected DHM (1) using cinchonidine as the only resolving agent. The optical purity and absolute stereochemistry of 4 and 5 were established by chemical correlation to the (S)-(+) enantiomer of 3-hydroxy-4-aminobutyric acid (GABOB). While 4 was a specific and potent BMC-sensitive GABA agonist in vivo and in vitro, possibly the most potent GABA agonist so far described, the inhibition of GABA uptake by DHM proved to reside exclusively in the (R)-(-) enantiomer (5). The affinity of 5 for BMC-sensitive GABA receptor sites in vitro was some 50 times lower than that of 4. Compounds 4 and 5 can be considered semirigid isosteres of the conformationally flexible GABA analogues (S)-(+)- and (R)-(-)-GABOB, respectively, which show a very low degree of enantioselectivity with respect to GABA synaptic mechanisms. This correlation between the degree of enantioselectivity and conformational mobility of chiral GABA analogues might be of importance for the design of new drugs with specific actions at synapses at which GABA is the transmitter.

Ibotenic acid analogues. Synthesis and biological and in vitro activity of conformationally restricted agonists at central excitatory amino acid receptors.

A number of analogues of ibotenic acid [(RS)-3-hydroxy-5- isoxazoleglycine ] were synthesized; they were tested as excitants on neurons in the cat spinal cord, by using microelectrophoretic techniques, and as inhibitors of the binding of kainic acid (KA) in vitro, by using synaptic membranes prepared from rat brains. The excitatory effects of the 3- isoxazolol amino acids (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5, 4-c]pyridine-7-carboxylic acid (4, 7- HPCA ), (RS)-alpha-amino-3-hydroxy-5,6-dihydro-4H- cyclohept [1,2-d] isoxa zole - 8-propionic acid (8, 8- AHCP ), (RS)-alpha-amino-3- hydroxy-7,8-dihydro-6H- cyclohept [1,2-d] isoxazole -4-propionic acid (12, 4- AHCP ), and (RS)-alpha-(methylamino)-3-hydroxy-5-methyl- 4- isoxazolepropionic acid (15, N-Me-AMPA) were shown to be sensitive to (S)-glutamic acid diethyl ester (GDEE), an antagonist at quisqualic acid ( QUIS ) receptors, and insensitive to (RS)-2-amino-5-phosphonovaleric acid ( 2APV ), an antagonist at N-methyl-(R)-aspartic acid (NMDA) receptors. The compounds 4 and 12 proved to be particularly potent agonists at the former class of receptor, assumed to represent physiological glutamic acid receptors. The amino acids (RS)-beta-(2-carboxyphenyl)alanine (19), an analogue of 12, and (RS)-2-(3-carboxyphenyl) glycine were weak GDEE-sensitive excitants with potencies comparable with that of 8. All of the compounds were tested as inhibitors of KA binding. With the exception of 12 and 19, which showed very low affinity for the KA binding sites, the compounds studied were inactive in this in vitro test system.

Neuroactive polyamine wasp toxins: nuclear magnetic resonance spectroscopic analysis of the protolytic properties of philanthotoxin-343.

Acid-base properties (pKa values and proton distribution patterns) of philanthotoxin-343(PhTX-343) were investigated by 1H and 13C NMR titration. Chemical shift data and the total ionization shifts were used to assign carbon atoms of the polyamine chain. Nonlinear analysis of the 13C NMR titration curves gave four pKa values (pK1 8.5, pK2 9.5, pK3 10.4, pK4 11.4) and the intrinsic chemical shifts of the non-, mono-, di-, tri-, and tetraprotonated forms. The changes of intrinsic chemical shifts enabled analysis of the deprotonation sequence of fully protonated PhTX-343. The results of analysis of the 13C NMR titration curves were supported by 1H NMR data obtained from two-dimensional 1H, 13C chemical shift correlation experiments. Thus, the first deprotonation mainly takes place at the inner amino group. The phenol group is deprotonated in the second and third deprotonation steps. The preferential deprotonation of the inner amino group is also apparent in the deprotonated form. The monoprotonated form carries a practically fully ionized phenol group and the proton shared between the three amino groups. This characteristic is in agreement with existing data on polyamines. At physiological pH, the tetraprotonated form of PhTX-343 predominates, but the proportion of the triprotonated form becomes significant at low ionic strength. The terminal, primary amino group, which has been shown to be essential for biological activity, remains practically fully protonated at biologically relevant pH values, and this charge is likely to participate in the receptor-binding event. Protonation of the central amino group does not appear to be necessary for biological activity.

Excitatory amino acid agonists. Enzymic resolution, X-ray structure, and enantioselective activities of (R)- and (S)-bromohomoibotenic acid.

The enantiomers of alpha-amino-4-bromo-3-hydroxy-5-isoxazolepropionic acid (4-bromohomoibotenic acid, Br-HIBO, 1) a selective and potent agonist at one class of the central (S)-glutamic acid receptors, were prepared with an enantiomeric excess higher than 98.8% via stereoselective enzymic hydrolysis of (RS)-alpha-(acetylamino)-4-bromo-3-methoxy-5-isoxazolepropionic acid (4) using immobilized aminoacylase. The absolute configuration of the enantiomers of Br-HIBO was established by X-ray crystallographic analysis, which confirmed the expected preference of the enzyme for the S form of the substrate 4. (S)- and (RS)-Br-HIBO were potent neuroexcitants on cat spinal neurones in vivo, while (R)-Br-HIBO was a very weak excitant. Correspondingly, the S enantiomer of Br-HIBO (IC50 = 0.34 microM) was considerably more potent than the R form (IC50 = 32 microM) as an inhibitor of [3H]-(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([ 3H]AMPA) binding to rat brain synaptic membranes in vitro. In contrast, (S)- and (R)-Br-HIBO were approximately equipotent (IC50 values of 0.22 and 0.15 microM, respectively) as inhibitors of [3H]-(S)-glutamic acid binding in the presence of CaCl2. The enantiomers of Br-HIBO showed no significant affinity for those binding sites on rat brain membranes which are labeled by [3H]kainic acid or [3H]-(R)-aspartic acid.

Heterocyclic muscarinic agonists. Synthesis and biological activity of some bicyclic sulfonium arecoline bioisosteres.

A number of S-methylsulfonium analogues of the conformationally restricted muscarinic agonists of the 3-alk-oxy-4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine (O-alkyl-THPO) type have been synthesized. The effects on muscarinic receptors of these 3-alkoxy-5-methyl-6,7-dihydro-4H-thiopyrano[3,4-d]isoxazol-5 -ium (O-alkyl-S-methyl-DHTO) analogues (7a-d) were assessed in receptor-binding experiments with tritiated oxotremorine M, pirenzepine, and quinuclidinyl benzilate as ligands and were supported by studies on the isolated guinea pig ileum. The degree of muscarinic agonist activity of the compounds (M-agonist index) and their selectivity for M-1 or M-2 muscarinic receptor subtypes (M-2/M-1 index) were estimated on the basis of receptor-binding studies. The in vitro pharmacological profiles of the compounds were compared with those of arecoline and its sulfonium and 3-methoxyisoxazole isosteres, sulfoarecoline and O,5-dimethyl-THPO, respectively. While O-methyl-DHTO (5a) and N-methyl-DHTO (6a) were inactive, all of the sulfonium analogues 7a-d were muscarinic agonists with the exception of O-ethyl-S-methyl-DHTO (7b), which showed a muscarinic antagonist profile.

AMPA receptor agonists: synthesis, protolytic properties, and pharmacology of 3-isothiazolol bioisosteres of glutamic acid.

A number of 3-isothiazolol bioisosteres of glutamic acid (1) and analogs of the AMPA receptor agonist, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 2a), including (RS)-2-amino-3-(3-hydroxy-5-methylisothiazol-4-yl)propionic acid (thio-AMPA, 2b), were synthesized. Comparative in vitro pharmacological studies on this series of 3-isothiazolol and the corresponding 3-isoxazolol amino acids were performed using a series of receptor binding assays (IC50 values) and the electrophysiological rat cortical slice model (EC50 values). Whereas 2a (IC50 = 0.04 +/- 0.005 microM, EC50 = 3.5 +/- 0.2 microM) is markedly more potent than the tert-butyl analog ATPA (3a) (IC50 = 2.1 +/- 0.16 microM, EC50 = 34 +/- 2.4 microM) in [3H]AMPA binding and electrophysiological studies, 2b (IC50 = 1.8 +/- 0.13 microM, EC50 = 15.0 +/- 2.4 microM) was approximately equipotent with thio-ATPA (3b) (IC50 = 0.63 +/- 0.07 microM, EC50 = 14 +/- 1.3 microM). (RS)-2-Amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (HIBO, 4a) was approximately equipotent with its thio analog 4b, whereas 4-Br-HIBO (5a) (IC50 = 0.65 +/- 0.12 microM, EC50 = 22 +/- 0.6 microM) turned out to be much more potent than the corresponding 3-isothiazolol 5b (IC50 = 17 +/- 2.2 microM, EC50 = 500 +/- 23 microM). 2b (ED50 = 130 mumol/kg) was more potent than 2a (220 mumol/kg) as a convulsant after subcutaneous administration in mice. The protolytic properties of 2a,b-4a,b were determined using 13C NMR spectroscopy. For each pair of compounds, the alpha-amino acid groups showed similar protolytic properties, whereas the 3-isoxazolol moieties typically showed pKa values 2 units lower than those of the 3-isothiazolols. Accordingly, calculations of ionic species distributions revealed pronounced differences between 3-isoxazolol and 3-isothiazolol amino acids. No simple correlation between activity as AMPA agonists in vitro and pKa values of these compounds was apparent. On the other hand, the relative potencies of AMPA (2a) and thio-AMPA (2b) in vitro and in vivo may reflect that these compounds predominantly penetrate the blood-brain barrier as net uncharged diprotonated ionic species.

Relationship between structure, conformational flexibility, and biological activity of agonists and antagonists at the N-methyl-D-aspartic acid subtype of excitatory amino acid receptors.

The relationship between conformational flexibility and agonist or antagonist actions at the N-Methyl-D-aspartic acid (NMDA) subtype of central L-glutamic acid (GLU) receptors of a series of racemic piperidinedicarboxylic acids (PDAs) was studied. The conformational analyses were based on 1H NMR spectroscopy and supported by computer simulations and molecular mechanics calculations. While the trans forms of 2,3-PDA and 2,4-PDA and cis-2,5-PDA show NMDA receptor agonist activities, cis-2,3-PDA and cis-2,4-PDA are NMDA antagonists. The compounds trans-2,5-PDA and cis-2,6-PDA did not interact with NMDA receptors. Each of the three cyclic acidic amino acids showing NMDA agonist activities was found to exist as an equilibrium mixture of two conformers in aqueous solution. In contrast, the NMDA antagonists cis-2,3-PDA and cis-2,4-PDA as well as the inactive compounds trans-2,5-PDA and cis-2,6-PDA were shown to exist predominantly in a single conformation. These results seem to indicate that a certain degree of conformational flexibility of analogues of GLU is a prerequisite for activation of, but not for binding to, the NMDA receptor.