A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures.

Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.

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).

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 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.

Stereochemistry and molecular pharmacology of (S)-thio-ATPA, a new potent and selective GluR5 agonist.

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isothiazolyl)propionic acid (thio-ATPA), a 3-isothiazolol analogue of (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), has previously been shown to be a relatively weak AMPA receptor agonist at native (S)-glutamic acid ((S)-Glu) receptors (EC(50)=14 microM), comparable in potency with ATPA (EC(50)=34 microM). Recent findings, that (S)-ATPA is a potent (EC(50)=0.48 microM) and selective agonist at homomerically expressed ionotropic GluR5, prompted us to resolve thio-ATPA using chiral chromatography and pharmacologically characterize the two enantiomers at native as well as cloned ionotropic glutamate receptors. The enantiomers, (S)- and (R)-thio-ATPA, were obtained in high enantiomeric excess, and their absolute stereochemistry established by an X-ray crystallographic analysis. Electrophysiologically, the two enantiomers were evaluated in the rat cortical wedge preparation, and the S-enantiomer was found to be an AMPA receptor agonist (EC(50)=8.7 microM) twice as potent as the racemate, whereas the R-enantiomer was devoid of activity. In accordance with this, (S)-thio-ATPA proved to be an agonist at homomerically expressed recombinant AMPA receptors (GluR1o, GluR3o, and GluR4o) with EC(50) values of 5, 32 and 20 microM, respectively, producing maximal steady state currents of 78--168% of those maximally evoked by kainic acid, and 120-1600% of those maximally evoked by (S)-ATPA. At homomerically expressed GluR5, (S)-thio-ATPA was found to be a potent agonist (EC(50)=0.10 microM), thus being approximately five times more potent than (S)-ATPA. (R)-Thio-ATPA induced saturating currents with an estimated EC(50) value of 10 microM, most likely due to a contamination with (S)-thio-ATPA. At heteromerically expressed GluR6+KA2 receptors, (S)-thio-ATPA showed relatively weak agonistic properties (EC(50)=4.9 microM). Thus, (S)-thio-ATPA has been shown to be a very potent agonist at GluR5, and may be a valuable tool for the investigation of desensitization properties of AMPA receptors.

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist.

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.

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.

UV-guided screening of benzodiazepine producing species in Penicillium.

The benzodiazepine sclerotigenin (auranthine B) recently described as a metabolite of Penicillium sclerotigenum, has been isolated as the major metabolite from an isolate of P. commune. The structure of sclerotigenin was established by a single-crystal X-ray diffraction study and by NMR spectroscopy. UV-guided screening for benzodiazepine production by other penicillia revealed that sclerotigenin was also produced by isolates of P. clavigerum, P. lanosum, P. melanoconidium, P. sclerotigenum and P. verrucosum. Sclerotigenin was detected both intra- and extracellularly. Apparently, P. aurantiogriseum is the only auranthine producing species in genus Penicillium.

Synthesis and muscarinic receptor pharmacology of a series of 4,5,6,7-tetrahydroisothiazolo[4,5-c]pyridine bioisosteres of arecoline.

A series of O- and ring-alkylated derivatives of 4,5,6,7-tetrahydroisothiazolo[4,5-c]pyridin-3-ol was synthesized via treatment of appropriately substituted 4-benzylamino-1,2,5,6-tetrahydropyridine-3-carboxamides with hydrogen sulfide and subsequent ring closure by oxidation with bromine. The muscarinic receptor affinity as well as estimated relative efficacy and subtype selectivity of this series of bicyclic arecoline bioisosteres were determined using rat brain membranes and a number of tritiated muscarinic receptor ligands. The effects at the five cloned human muscarinic receptor subtypes of a selected series of chiral analogues, with established absolute stereochemistry, were studied using receptor selection and amplification technology (R-SAT). The potency, relative efficacy, and receptor subtype selectivity of these compounds were related to the structure of the O-substituents and the position and stereochemical orientation of the piperidine ring methyl substituents.

Selective inhibitors of glial GABA uptake: synthesis, absolute stereochemistry, and pharmacology of the enantiomers of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and analogues.

3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a), or the corresponding 3-ethoxy analogue (20b), and 3-chloro-4,5,6, 7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized by regioselective chromic acid oxidation of the respective bicyclic tetrahydrobenzenes 19a,b and 50, and they were used as key intermediates for the syntheses of the target zwitterionic 3-isoxazolols 8-15 and 3-isothiazolols 16 and 17, respectively. These reaction sequences involved different reductive processes. Whereas (RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-benzisoxazole (8, exo-THPO) was synthesized via aluminum amalgam reduction of oxime 22a or 22b, compounds 9, 11-13, and 15-17 were obtained via reductive aminations. Compound 10 was synthesized via N-ethylation of the N-Boc-protected primary amine 25. The enantiomers of 8 were obtained in high enantiomeric purities (ee >/= 99.1%) via the diastereomeric amides 32 and 33, synthesized from the primary amine 23b and (R)-alpha-methoxyphenylacetyl chloride and subsequent separation by preparative HPLC. The enantiomers of 9 were prepared analogously from the secondary amine 27. On the basis of X-ray crystallographic analyses, the configuration of oxime 22a was shown to be E and the absolute configurations of (-)-8 x HCl and (+)-9 x HBr were established to be R. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation and primary cultures of mouse cortical neurons and glia cells (astrocytes). Whereas the classical GABA uptake inhibitor, (R)-nipecotic acid (2), nonselectively inhibits neuronal (IC(50) = 12 microM) and glial (IC(50) = 16 microM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo¿4,5-cpyridin-3-ol (1, THPO) shows some selectivity for glial (IC(50) = 268 microM) versus neuronal (IC(50) = 530 microM) GABA uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC(50) = 200 microM) rather than neuronal (IC(50) = 900 microM) GABA uptake. This selectivity was more pronounced for 9, which showed IC(50) values of 40 and 500 microM as an inhibitor of glial and neuronal GABA uptake, respectively. These effects of 8 and 9 proved to be enantioselective, (R)-(-)-8 and (R)-(+)-9 being the active inhibitors of both uptake systems. The selectivity of 9 as a glial GABA uptake inhibitor was largely lost by replacing the N-methyl group of 9 by an ethyl group, compound 10 being an almost equipotent inhibitor of glial (IC(50) = 280 microM) and neuronal (IC(50) = 400 microM) GABA uptake. The remaining target compounds, 11-17, were very weak or inactive as inhibitors of both uptake systems. Compounds 9-13 and 15 were shown to be essentially inactive against isoniazide-induced convulsions in mice after subcutaneous administration. The isomeric pivaloyloxymethyl derivatives of 9, compounds 43 and 44, were synthesized and tested as potential prodrugs in the isoniazide animal model. Both 43 (ED(50) = 150 micromol/kg) and 44 (ED(50) = 220 micromol/kg) showed anticonvulsant effects, and this effect of 43 was shown to reside in the (R)-(+)-enantiomer, 45 (ED(50) = 44 micromol/kg). Compound 9 also showed anticonvulsant activity when administered intracerebroventricularly (ED(50) = 59 nmol).

Antiprotozoal properties of 16,17-dihydrobrachycalyxolide from Vernonia brachycalyx.

Extracts of the leaves from Vernonia brachycalyx showed in vitro activity against Plasmodium falciparum and promastigotes of Leishmania major. The germacrane dilactone 16,17-dihydrobrachycalyxolide (1) which was previously isolated from the aerial parts of the plant was shown to be the major antiplasmodial principle. An X-ray crystallographic analysis established the absolute configuration and some signals in the NMR spectra were reassigned. 16,17-Dihydrobrachycalyxolide (1) elicited a strong antiplasmodial and antileishmanial activity but also a high toxicity against human lymphocytes.

UV-Guided isolation of alantrypinone, a novel Penicillium alkaloid.

Fumiquinazoline F (1) and alantrypinone (2) have been isolated as the two major metabolites of Penicillium thymicola. The structure of 2, which contains a new ring structure, was elucidated by analysis of spectroscopic data including 2D NMR. The absolute configuration of 2 was established by a single-crystal X-ray diffraction study.

Antiprotozoal compounds from Asparagus africanus.

Two antiprotozoal compounds have been isolated from the roots of Asparagus africanus Lam. (Liliaceae), a new sapogenin, 2 beta, 12 alpha-dihydroxy-(25R)-spirosta-4,7-dien-3-one (1), which was named muzanzagenin, and the lignan (+)-nyasol (2), (Z)-(+)-4,4'-(3-ethenyl-1-propene-1,3-diyl)-bisphenol. The structure of the sapogenin was elucidated by MS and by 1D and 2D NMR methods and established by a single crystal X-ray analysis. (+)-Nyasol potently inhibits the growth of Leishmania major promastigotes, the IC50 being 12 microM, and moderately inhibits Plasmodium falciparum schizonts with the IC50 49 microM. These concentrations only moderately affect the proliferation of human lymphocytes. Muzanzagenin showed a moderate in vitro activity in all three tests, the IC50 against leishmania promastigotes was 70 microM, and against four different malaria schizont strains the IC50 values were 16, 163, 23, and 16 microM, respectively.

Excitatory amino acid receptor antagonists: resolution, absolute stereochemistry, and pharmacology of (S)- and (R)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA).

We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation using N-BOC protected ATAA and (R)- and (S)-phenylethylamine. Enantiomeric purities (ee > 98%) of (R)- and (S)-ATAA were determined using the Crownpak CR(-) and CR(+) columns, respectively. The absolute configuration of (R)-ATAA was established by an X-ray crystallographic analysis of the (R)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation (Ki > 1,000 microM).

Conformational analysis of acetylcholine and related choline esters.

The crystal structures of carbamoylcholine [2-(carbamoyloxy)-N,N,N-trimethylethanaminium] chloride, bromide and iodide, methoxycarbonylcholine [2-(methoxycarbonyloxy)-N,N,N-trimethylethanaminium] iodide, acetylcholine [2-(acetyloxy)-N,N,N-trimethylethanaminium] chloride and succinylcholine ¿2,2'-[(1,4-dioxo-1,4-butanediyl)bis(oxy)]bis(N,N,N-trimethylet hanaminium)¿ iodide have been redetermined at 105 K in order to obtain detailed and accurate information on the geometry of choline esters and to elucidate the conformationally dependent changes of geometry. The conformational flexibility and the preferred conformations are elucidated based on results obtained from X-ray crystallographic studies and molecular mechanics (MM2) calculations. The usefulness of molecular mechanics calculations for quaternary ammonium ions is discussed.

N-methyl-D-aspartic acid receptor agonists: resolution, absolute stereochemistry, and pharmacology of the enantiomers of 2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid.

(R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy)-5-methyl-4-isoxazolyl]N-tert-butyl-2- [N-[(S)-1-phenylethyl]benzamido]-acetamide (16 and 17, respectively) were synthesized and separated chromatographically. The absolute stereochemistry of 16 was confirmed by an X-ray analysis. Deprotection of these intermediates did, however, provide (R)- (8) and (S)- (9) AMAA, respectively, in extensively racemized forms. N-BOC-protected (R,S)-AMAA (21) was successfully resolved via diastereomeric salt formation using cinchonidine. The stereochemical purity and stability of 8 and 9 obtained via this resolution were determined using chiral HPLC. (R)-AMAA (8) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly. In electrophysiological studies using rat brain tissue, 8 (EC50 = 7.3 +/- 0.3 microM) was 1 order of magnitude more potent than 9 (EC50 = 75 +/- 9 microM) as an NMDA receptor agonist.

Synthesis and structure-activity studies on acidic amino acids and related diacids as NMDA receptor ligands.

The 3-isoxazolol amino acids (S)-2-amino-3-(3-hydroxy-5-methyl-4- isoxazolyl)propionic acid [(S)-AMPA, 2] and (R,S)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA, 5a) (Figure 1) are potent and specific agonists at the AMPA and N-methyl-D-aspartic acid (NMDA) subtypes, respectively, of (S)-glutamic acid (1) receptors. A number of amino acids and diacids structurally related to AMAA were synthesized and tested electrophysiologically and in receptor-binding assays. The hydroxymethyl analogue 7c of AMAA was an NMDA agonist approximately equipotent with AMAA in the [3H]CPP-binding assay (IC50 = 7 +/- 3 microM) and electropharmacologically in the rat cortical wedge model (EC50 = 8 +/- 2 microM). In contrast to this, the tertbutyl analogue 7a of AMAA turned out to be an antagonist at NMDA and AMPA receptors. The conformational characteristics of AMAA and 7a, c were studied by molecular mechanics calculations. Compound 7a possesses extra steric bulk and shows significant restriction of conformational flexibility compared to AMAA and 7c, which may be determining factors for the observed differences in biological activity. Although the nitrogen atom of quinolinic acid (6) has very weak basic character, 6 is a, perhaps subtype-selective, NMDA receptor agonist and a potent neurotoxic agent. These aspects prompted us to synthesize and test the diacids 8a, b, in which the amino group of AMAA has been replaced by a methylthio and methoxy group, respectively. Neither compound showed significant affinity for nor depolarizing effects at NMDA receptors. The hydroxymethyl AMPA analogue 3c showed no interaction with NMDA receptors and only weak AMPA agonist effects.

GABAB antagonists: resolution, absolute stereochemistry, and pharmacology of (R)- and (S)-phaclofen.

Phaclofen, which is the phosphonic acid analogue of the GABAB agonist (RS)-3-(4-chlorophenyl)-4-aminobutyric acid (baclofen), is a GABAB antagonist. As part of our studies on the structural requirements for activation and blockade of GABAB receptors, we have resolved phaclofen using chiral chromatographic techniques. The absolute stereochemistry of (-)-(R)-phaclofen was established by X-ray crystallographic analysis. (-)-(R)-Phaclofen was shown to inhibit the binding of [3H]-(R)-baclofen to GABAB receptor sites on rat cerebellar membranes (IC50 = 76 +/- 13 microM), whereas (+)-(S)-phaclofen was inactive in this binding assay (IC50 > 1000 microM). (-)-(R)-Phaclofen (200 microM) was equipotent with (RS)-phaclofen (400 microM) in antagonizing the action of baclofen in rat cerebral cortical slices, while (+)-(S)-phaclofen (200 microM) was inactive. The structural similarity of the agonist (R)-baclofen and the antagonist (-)-(R)-phaclofen suggests that these ligands interact with the GABAB receptor sites in a similar manner. Thus, it may be concluded that the different pharmacological effects of these compounds essentially result from the different spatial and proteolytic properties of their acid groups.

Structures of acetylcholine picrate and methoxycarbonylcholine picrate hemihydrate.

Acetylcholine picrate, C7H16NO2+.C6H2N3-O7-, Mr = 374.3, orthorhombic, Pbca, at 105 K: a = 18.799 (4), b = 7.726 (2), c = 22.878 (4) A, V = 3323 (2) A3, Z = 8, Dm(295 K, flotation) = 1.44, D chi(105 K) = 1.496 Mg m-3, mu(Mo K alpha) = 0.120 mm-1, F(000) = 1568, m.p. (hot-stage microscope) 381-382 K, R = 0.048 for 1049 observed [I greater than or equal to 3.0 sigma(I)] reflections. Methoxycarbonylcholine picrate hemihydrate, C7H16NO3+.C6H2N3O7-.1/2H2O, Mr = 399.3, monoclinic, P2/n, at 105 K: a = 11.337 (16), b = 7.279 (2), c = 21.424 (13) A, beta = 103.01 (7) degrees, V = 1723 (4) A3, Z = 4, Dm(295 K, flotation) = 1.49, D chi(105 K) = 1.539 Mg m-3, mu(Mo K alpha) = 0.126 mm-1, F(000) = 836, m.p. (hot-stage microscope) 391-391.5 K, R = 0.033 for 6359 observed [I greater than or equal to 3.0 sigma(I)] reflections. The acetylcholine ion as well as the methoxycarbonylcholine ion have as first neighbours a great number of oxygen atoms. Contacts to the quaternary ammonium group do not seem to be more important than contacts to the acetyl or methoxy-carbonyl moieties. No direct contacts between aromatic rings and quaternary ammonium groups are found.