Synthesis and pharmacology of 3-hydroxy-delta2-isoxazoline-cyclopentane analogues of glutamic acid.

The synthesis and pharmacology of two potential glutamic acid receptor ligands are described. Preparation of the bicyclic 3-hydroxy-delta2-isoxazoline-cyclopentane derivatives (+/-)-7 and (+/-)-8 was accomplished via 1,3-dipolar cycloaddition of bromonitrile oxide to suitably protected 1-amino-cyclopent-3-enecarboxylic acids. Their structure was established using a combination of 1H NMR spectroscopy and molecular mechanics calculations carried out on the intermediate cycloadducts (+/-)-11 and (+/-)-12. Amino acid derivatives (+/-)-7 and (+/-)-8 were assayed at ionotropic and metabotropic glutamic acid receptor subtypes and their activity compared with that of trans-ACPD and cis-ACPD. The results show that the replacement of the omega-carboxylic group of the model compounds with the 3-hydroxy-delta2-isoxazoline moiety abolishes or reduces drastically the activity at the metabotropic glutamate receptors. Conversely, on passing from cis-ACPD to derivative (+/-)-8, the agonist activity at NMDA receptors is almost unaffected.

Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA.

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid binding (IC(50) = 3.6 microM), and potent AMPA receptor agonist activity on cortical neurons (EC(50) = 0.25 microM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC(50) = 0.039 microM), but was found to be a relatively weak AMPA receptor agonist (EC(50) = 12 microM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC(50) = 220 microM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu(2) receptor subtype (K(B) = 148 microM).

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.

Unprecedented migration of N-alkoxycarbonyl groups in protected pyroglutaminol.

[figure: see text] Cleavage of an O-silyl ether in an N-BOC-protected pyroglutaminol using TBAF led to an unprecedented migration of the BOC group. An investigation of the mechanism, based on experimental data and quantum mechanical calculations, is presented. Similar migration was observed for N-Cbz and N-methoxycarbonyl groups.

Synthesis and receptor binding affinity of new selective GluR5 ligands.

Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2S,4R)-4-methylglutamic acid ((2S,4R)-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropic glutamic acid receptors. The (S)-enantiomers of E-4-(2,2-dimethylpropylidene)glutamic acid ((S)-1) and E-4-(3,3-dimethylbutylidene)glutamic acid ((S)-2) were shown to be selective and high affinity GluR5 ligands, with Ki values of 0.024 and 0.39 microM, respectively, compared to Ki values at GluR2 of 3.0 and 2.0 microM. respectively. Their affinities in the [3H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC50 > 100 microM).

Synthesis and pharmacology of 3-isoxazolol amino acids as selective antagonists at group I metabotropic glutamic acid receptors.

Using ibotenic acid (2) as a lead, two series of 3-isoxazolol amino acid ligands for (S)-glutamic acid (Glu, 1) receptors have been developed. Whereas analogues of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [AMPA, (RS)-3] interact selectively with ionotropic Glu receptors (iGluRs), the few analogues of (RS)-2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid [HIBO, (RS)-4] so far known typically interact with iGluRs as well as metabotropic Glu receptors (mGluRs). We here report the synthesis and pharmacology of a series of 4-substituted analogues of HIBO. The hexyl analogue 9 was shown to be an antagonist at group I mGluRs. The effects of 9 were shown to reside exclusively in (S)-9 (K(b) = 30 microM at mGlu(1) and K(b) = 61 microM at mGlu(5)). The lower homologue of 9, compound 8, showed comparable effects at mGluRs, but 8 also was a weak agonist at the AMPA subtype of iGluRs. Like 9, the higher homologue, compound 10, did not interact with iGluRs, but 10 selectively antagonized mGlu(1) (K(b) = 160 microM) showing very weak antagonist effect at mGlu(5) (K(b) = 990 microM). The phenyl analogue 11 turned out to be an AMPA agonist and an antagonist at mGlu(1) and mGlu(5), and these effects were shown to originate in (S)-11 (EC(50) = 395 microM, K(b) = 86 and 90 microM, respectively). Compound 9, administered icv, but not sc, was shown to protect mice against convulsions induced by N-methyl-D-aspartic acid (NMDA). Compounds 9 and 11 were resolved using chiral HPLC, and the configurational assignments of the enantiomers were based on X-ray crystallographic analyses.

Structural determinants for AMPA agonist activity of aryl or heteroaryl substituted AMPA analogues. Synthesis and pharmacology.

We have previously reported the synthesis and pharmacological characterization of analogues of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 1a), in which the methyl group was replaced by a phenyl group (APPA, 1b) or heteroaryl groups. While 2b and its 3-pyridyl analogue 2-amino-3-[3-hydroxy-5-(3-pyridyl)-4-isoxazolyl]propionic acid (3-Py-AMPA, 3) show very low affinity for AMPA receptors, introduction of heteroaryl substituents containing heteroatom in the 2-position provides potent AMPA receptor agonists. We here report the synthesis and pharmacology of 2-amino-3-(3-hydroxy-5-pyrazinyl-4-isoxazolyl)propionic acid (7) (IC50 = 1.2 microM), which is weaker as an AMPA agonist than AMPA (IC50 = 0.040 microM; EC50 = 3.5 microM) but comparable in potency with 2-Py-AMPA (4) (IC50 = 0.57 microM; EC50 = 7.4 microM), as determined in radioligand binding and electrophysiological experiments, respectively. The AMPA analogues 8a-c, containing 2-, 3-, or 4-methoxyphenyl substituents, respectively, and the corresponding hydroxyphenyl analogues, 9a-c, were also synthesized and evaluated pharmacologically. With the exception of 2-amino-3-[3-hydroxy-5-(2-hydroxyphenyl)-4-isoxazolyl]propionic acid (9a), which is a very weak AMPA agonist (IC50 = 45 microM; EC50 = 324 microM), none of these compounds showed detectable effect at AMPA receptors.

Structural determinants of AMPA agonist activity in analogues of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid: synthesis and pharmacology.

We have previously shown that the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA, 2), binds to AMPA receptors in a manner different from that of AMPA (1) itself and that 2, in contrast to 1, also binds to kainic acid receptor sites. To elucidate the structural requirements for selective activation of the site/conformation of AMPA receptors recognized by 2, a number of isosteric analogues of 2 have now been synthesized and pharmacologically characterized. The compound 2-amino-3-(5-carboxy-3-methoxy-4-isoxazolyl)propionic acid (3a) (IC(50) = 0.11 microM; EC(50) = 1.2 microM), which is a regioisostere of 2 with a methoxy group substituted for the methyl group, was approximately equipotent with 2 (IC(50) = 0.020 microM; EC(50) = 1.0 microM) as an inhibitor of [(3)H]AMPA binding and as an AMPA agonist, respectively, whereas the corresponding 3-ethoxy analogue 3b (IC(50) = 1.0 microM; EC(50) = 4.8 microM) was slightly weaker. The analogues 3c-e, containing C3 alkoxy groups, were an order of magnitude weaker than 3b, whereas the additional steric bulk of the alkoxy groups of 3f-i or the presence of an acidic hydroxyl group at the 3-position of the isoxazole ring of 3j prevented interaction with AMPA receptor sites. The 2-amino-3-(2-alkyl-5-carboxy-3-oxo-4-isoxazolyl)propionic acids 4a,b, i, which are regioisosteric analogues of 3a,b,i, showed negligible interaction with AMPA recognition sites. Similarly, replacement of the carboxyl group of 3b by isosteric tetrazolyl or 1,2,4-triazolyl groups to give 5 and 6, respectively, or conversion of 3b into analogue 7, in which the diaminosquaric acid group has been bioisosterically substituted for the alpha-aminocarboxylic acid unit, provided compounds completely devoid of effect at AMPA receptors. In contrast to the parent compound ACPA (2) (IC(50) = 6.3 microM), none of the analogues described showed detectable inhibitory effect on [(3)H]kainic acid receptor binding.

Agonist discrimination between AMPA receptor subtypes.

The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H]AMPA radioligand binding experiments. (S)-4-bromohomoibotenic acid (BrHIBO) exhibited a 126-fold selectivity for GluR1o compared to GluR3o. Xenopus laevis oocytes were used to express functional homomeric and heteromeric recombinant AMPA-R and to determine BrHIBO potency (EC50) at these channels. (R,S)-BrHIBO exhibited a 37-fold selectivity range amongst the AMPA-R. It is hoped that BrHIBO can be used as a lead structure for the development of other subtype-selective compounds.

Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist.

We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2), on the other hand, was shown to be a selective AMPA receptor antagonist (IC(50) = 73 microM), more potent in electrophysiological experiments than AMOA (IC(50) = 320 microM). The isomeric analogue of 2, compound 5, did not show AMPA antagonist effects, but was a weak NMDA receptor antagonist (IC(50) = 540 microM). Finally, compound 3, which is an isomer of ACPA, turned out to be a very weak NMDA antagonist, and an AMPA receptor agonist approximately 1000 times weaker than ACPA. None of the compounds showed agonist or antagonist effects at metabotropic EAA receptors.

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid.

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.

Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA.

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA). has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring (S)-glutamic acid ((S)-Glu) receptors. We report here the separation of (+)- and (-)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and > 99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of (S)-(+)-ATPA and (R)-(-)-ATPA. (S)- and (R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6 + KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, (S)-ATPA showed AMPA receptor agonist effects (EC50 = 23 microM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (Ki = 253 microM) and by (S)-ATPA (Ki = 376 microM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (Ki = 301 microM and 1115 microM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC50 values of approximately 8 microM and at GluR1 (EC50 = 22 microM), producing maximal steady state currents only 5.4-33% of those evoked by kainic acid. (R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with Ki values of 33-75 microM. (S)-ATPA produced potent agonist effects at GluR5 (EC50 = 0.48 microM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, (S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by (S)-ATPA and kainic acid were not significantly different. (R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither (S)- nor (R)-ATPA showed significant effects at GluR6. (S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6 + KA2 receptors, whereas (R)-ATPA was inactive. Thus, (S)- and (R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA (S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.

Synthesis and enantiopharmacology of new AMPA-kainate receptor agonists.

Regioisomeric 3-carboxyisoxazolinyl prolines [CIP-A (+/-)-6 and CIP-B (+/-)-7] and 3-hydroxyisoxazolinyl prolines [(+/-)-8 and (+/-)-9] were synthesized and assayed for glutamate receptor activity. The tests were carried out in vitro by means of receptor binding techniques, second messenger assays, and the rat cortical wedge preparation. CIP-A showed a good affinity for both 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and kainic acid (KAIN) receptors. These results were confirmed in the cortical slice model where CIP-A displayed an EC(50) value very close to that of AMPA. The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice at doses as low as 3.2 nmol/mouse. On the basis of the above-reported results we prepared and tested the enantiomers of CIP-A and CIP-B, obtained by reacting (S)-3,4-didehydroproline and (R)-3,4-didehydroproline, respectively, with ethoxycarbonylformonitrile oxide. In all the tests the S-form, CIP-AS [(-)-6], emerged as the eutomer evidencing common stereochemical requirements with the reference compounds AMPA and KAIN. Through modeling studies, carried out on CIP-A, AMPA, and KAIN, active conformations for CIP-AS and AMPA at AMPA receptors as well as for CIP-AS and KAIN at KAIN receptors are suggested.

Characterization of a new AMPA receptor radioligand, [3H]2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid.

(RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is a potent and selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, has previously been shown to desensitize AMPA receptors to a much lower degree than AMPA itself. We now report the synthesis of [3H]ACPA (32.5 Ci/mmol), the neurochemical and pharmacological characterization of [3H]ACPA binding, and a comparison of the distribution of [3H]ACPA, [3H]AMPA, and [3H](S)-5-fluorowillardiine binding sites in rat brain. Under equilibrium conditions, [3H]ACPA was shown to bind to a single population of receptor sites on rat brain membranes. [3H]ACPA was shown to bind with single and similar affinities (15-45 nM) to cloned AMPA receptor subunits (GluR1-4), expressed in insect cells, whereas a K(D) value of 330 nM was determined for the binding of [3H]ACPA to cloned kainic acid preferring GluR5 subunits. Whereas Bmax and K(D) values for [3H]ACPA binding, determined using filtration techniques, were different from such obtained in centrifugation assays, Bmax and K(D) values as well as association and dissociation constants were not significantly affected by the addition of the chaotropic agent KSCN. K(D) values, determined under equilibrium conditions, were, however, markedly different from K(D) values derived from kinetic data. Furthermore, the results of analyses of these kinetic data were consistent with the existence of two different populations of [3H]ACPA binding sites. The pharmacology of [3H]ACPA binding sites was characterized using a series of AMPA receptor agonists and antagonists. Whereas addition of KSCN had little effect on the affinities of AMPA receptor agonists for [3H]ACPA binding, this chaotropic agent reduced the affinities of AMPA receptor antagonists structurally related to AMPA. Based on these and previously reported data, the AMPA receptor agonists, ACPA, AMPA and (S)-5-fluorowillardiine, seem to bind to and activate AMPA receptors in a nonidentical fashion, and these three agonists together may be useful tools for studies of AMPA receptor mechanisms.

3-Pyrazolone analogues of the 3-isoxazolol metabotropic excitatory amino acid receptor agonist homo-AMPA. Synthesis and pharmacological testing.

We have previously shown that the higher homologue of (S)-glutamic acid [(S)-Glu], (S)-alpha-aminoadipic acid [(S)-alpha-AA] is selectively recognized by the mGlu(2) and mGlu(6) subtypes of the family of metabotropic glutamic acid (mGlu) receptors. Furthermore, a number of analogues of (S)-alpha-AA, in which the terminal carboxyl group has been replaced by various bioisosteric groups, such as phosphonic acid or 3-isoxazolol groups, have been shown to interact selectively with different subtypes of mGlu receptors. In this paper we report the synthesis of the 3-pyrazolone bioisosteres of alpha-AA, compounds (RS)-2-amino-4-(1,2-dihydro-5-methyl-3-oxo-3H-pyrazol-4-yl)butyric acid (1) and (RS)-2-amino-4-(1,2-dihydro-1,5-dimethyl-3-oxo-3H-pyrazol-4-yl)butyric acid (2). At a number of steps in the reaction sequences used, the reactions took unexpected courses and provided products which could not be transformed into the target compounds, and attempts to synthesize the 2,5-dimethyl isomer of 2, compound 3, failed. An X-ray crystallographic analysis of the intermediate 1,2-dihydro-4-(2-hydroxyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) confirmed the expected regioselectivity of the reaction between methylhydrazine and alpha-acetylbutyrolactone (4). Neither 1 nor 2 showed significant effects at the different types of ionotropic glutamic acid receptors or at mGlu(1a) (group I), mGlu(2) (group II), and mGlu(4a) and mGlu(6) (group III) receptors, representing the three indicated groups of mGlu receptors.

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.

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes.

The activity of the (R, S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist, (R,S) -2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO), at recombinant ionotropic glutamate receptors (GluRs) was evaluated using electrophysiological techniques. Responses at homo- or heterooligomeric AMPA-preferring GluRs expressed in human embryonic kidney (HEK) 293 cells (GluR1-flip) or Xenopus laevis oocytes (GluR1-4-flop or GluR1-flop + GluR2) were potently inhibited by ATPO with apparent dissociation constants (Kb values) ranging from 3.9 to 26 microM. A Schild analysis for kainate (KA)-activated GluR1 receptors showed ATPO to have a KB of 8.2 microM and a slope of unity, indicating competitive inhibition. The antagonism by ATPO at GluR1 was of similar magnitude at holding potentials between -100 mV and +20 mV. In contrast, ATPO (<300 microM), does not inhibit responses to kainate at homomeric GluR6 or heterooligomeric GluR6/KA2 expressed in HEK 293 cells but activated GluR5 and GluR5/KA2 expressed in X. laevis oocytes. ATPO produced <15% inhibition at the maximal concentration (300 microM) of current responses through NR1A + NR2B receptors expressed in X. laevis oocytes. Thus, ATPO shows a unique pharmacological profile, being an antagonist at GluR1-4 and a weak partial agonist at GluR5 and GluR5/KA2.

Excitatory amino acid receptor ligands: resolution, absolute stereochemistry, and enantiopharmacology of 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid.

(RS)-2-Amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (Bu-HIBO, 6) has previously been shown to be an agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors and an inhibitor of CaCl2-dependent [3H]-(S)-glutamic acid binding (J. Med. Chem. 1992, 35, 3512-3519). To elucidate the pharmacological significance of this latter binding affinity, which is also shown by quisqualic acid (3) but not by AMPA, we have now resolved Bu-HIBO via diastereomeric salt formation using the diprotected Bu-HIBO derivative 11 and the enantiomers of 1-phenylethylamine (PEA). The absolute stereochemistry of (S)-Bu-HIBO (7) (ee = 99.0%) and (R)-Bu-HIBO (8) (ee > 99.6%) were established by an X-ray crystallographic analysis of compound 15, a salt of (R)-PEA, and diprotected 8. Circular dichroism spectra of 7 and 8 were recorded. Whereas 7 (IC50 = 0.64 microM) and 8 (IC50 = 0.57 microM) were equipotent as inhibitors of CaCl2-dependent [3H]-(S)-glutamic acid binding, neither enantiomer showed significant affinity for the synaptosomal (S)-glutamic acid uptake system(s). AMPA receptor affinity (IC50 = 0.48 microM) and agonism (EC50 = 17 microM) were shown to reside exclusively in the S-enantiomer, 7. Compounds 7 and 8 did not interact detectably with kainic acid or N-methyl-D-aspartic acid (NMDA) receptor sites. Neither 7 nor 8 affected the function of the metabotropic (S)-glutamic acid receptors mGlu2 and mGlu4a, expressed in CHO cells. Compound 8 was shown also to be inactive at mGlu1 alpha, whereas 7 was determined to be a moderately potent antagonist at mGlu1 alpha (Ki = 110 microM) and mGlu5a (Ki = 97 microM). Using the rat cortical wedge preparation, the AMPA receptor agonist effect of 7 was markedly potentiated by coadministration of 8 at 21 degrees C, but not at 2-4 degrees C. These observations together indicate that the potentiation of the AMPA receptor agonism of 7 by 8 is not mediated by metabotropic (S)-glutamate receptors but rather by the CaCl2-dependent (S)-glutamic acid binding system, which shows the characteristics of a transport mechanism. After intravenous administration in mice, 7 (ED50 = 44 mumol/kg) was slightly more potent than AMPA (1) (ED50 = 55 mumol/kg) and twice as potent as Bu-HIBO (6) (ED50 = 94 mumol/kg) as a convulsant, whereas 8 was inactive. After subcutaneous administration in mice, Bu-HIBO (ED50 = 110 mumol/kg) was twice as potent as AMPA (ED50 = 220 mumol/kg) as a convulsant. Since 7 and Bu-HIBO (EC50 = 37 microM) are much weaker than AMPA (EC50 = 3.5 microM) as AMPA receptor agonists in vitro, the presence of a butyl group in the molecules of Bu-HIBO and 7 seems to facilitate the penetration of these compounds through the blood-brain barrier.