Biomedical subjects

P Krogsgaard-Larsen

Publications and source records attributed to P Krogsgaard-Larsen.

At least 73 records · Page 4Linked to original sources

PubMed1997

AMPA receptor agonists: resolution, configurational assignment, and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA).

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee > or = 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [3H]AMPA binding (IC50 = 0.19 +/- 0.06 microM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC50 = 4.5 +/- 0.3 microM) comparable with AMPA (IC50 = 0.040 +/- 0.01 microM; EC50 = 3.5 +/- 0.2 microM), but much more potent than (+)-(S)-APPA (IC50 = 5.5 +/- 2.2 microM; EC50 = 230 +/- 12 microM). Like (-)-(R)-APPA (IC50 > 100 microM), (-)-(R)-2-Py-AMPA (IC50 > 100 microM) did not significantly affect [3H]AMPA binding, and both compounds were weak AMPA receptor antagonists (Ki = 270 +/- 50 and 290 +/- 20 microM, respectively).

Alanine↗

PubMed1997

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).

Animals↗

PubMed1997

Bioisosterically modified dipeptide excitatory amino acid receptor antagonists containing 3-oxygenated isothiazole ring systems.

The AMPA receptor agonist Thio-AMPA, the 3-isothiazolol analogue of AMPA was converted into the selective NMDA antagonist, 2, in which a 3-isothiazolone unit is a bioisosteric analogue of the peptide bond of the NMDA antagonist, gamma-(R)-Glu-Gly. The isomeric 3-oxygenated isothiazole amino acid, 3, and the corresponding isothiazole phosphono amino acid 4 were also synthesized, and were shown to be selective AMPA receptor antagonists. Compound 1, in which the peptide bond of gamma-(R)-Glu-Gly is replaced by an ester group, was synthesized and shown to be unstable in the test buffer system.

Animals↗

PubMed1997

Differences in agonist/antagonist binding affinity and receptor transduction using recombinant human gamma-aminobutyric acid type A receptors.

Using human gamma-aminobutyric acid type A (GABAA) receptor subunit combinations, expressed in cell lines and Xenopus laevis oocytes, the pharmacology of a number of ligands interacting directly with the GABA recognition site has been studied in [3H]muscimol binding and electrophysiologically. The binding affinity of GABAA agonist and antagonist ligands showed small but statistically significant dependence on the subunit composition of receptors that include gamma 2 and different alpha and beta subunits. The potency of antagonist ligands was largely independent of receptor subunit composition, whereas the composition of receptors expressed in oocytes strongly influenced the EC50 value of agonists. An apparent reciprocal correlation between subunits favoring agonist binding and antagonist binding, respectively, was observed. Whereas antagonists showed comparable potencies in binding and functional studies, the potency of agonists in binding studies was generally two to three orders of magnitude higher than the agonist potencies measured electrophysiologically. 5-(4-Piperidyl)isothiazol-3-ol, which behaves as a low efficacy partial agonist at GABAA receptors in cultured cortical neurons, showed no efficacy in oocytes, but produced pure antagonist effects with a binding/functional affinity ratio between those observed for the agonists and antagonists. It is concluded that the GABAA receptor mechanisms transducing binding into physiological response, but not the binding per se, is dependent on the receptor subunit composition.

Animals↗

PubMed1996

Functional partial agonism at cloned human muscarinic acetylcholine receptors.

We have previously defined the concept of functional partial agonism as the partial agonist responses recorded in brain slices after administration of full ionotropic glutamate receptor agonists and competitive antagonists at fixed ratios. Functional partial agonism can be established at any level of maximal response, depending on the molar ratio of agonist and antagonist used. Using recombinant human muscarinic acetylcholine receptors (m1 and m5) and the functional assay, receptor selection and amplification technology (R-SAT), we have now shown that co-administration of the full agonist, carbachol, and a competitive antagonist, atropine or pirenzepine, at fixed ratios display functional partial agonism. The levels of apparent intrinsic activity of the functional partial agonist responses were shown to be dependent of the receptor density and G-protein concentration in the same manner as that determined for the true partial muscarinic agonist, 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butynyltrimethylammonium chloride (McN A-343). Thus, functional as well as true partial agonist responses became more efficacious and potent with increasing receptor and G-protein levels. The level of maximal functional partial agonist response, which is dependent on the agonist/antagonist ratio, is predictable from the Waud equation, describing competitive receptor/ligand interactions. In agreement with the relative antagonist potencies of pirenzepine at m1 and m5, a 10:1 ratio of carbachol and pirenzepine produced very low-efficacy functional partial agonism, approaching full antagonism, at m1 but virtually full agonism at the m5 subtype.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗

PubMed1996

A new highly selective metabotropic excitatory amino acid agonist: 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid.

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.

Alanine↗

PubMed1996

Different characteristics of AMPA receptor agonists acting at AMPA receptors expressed in Xenopus oocytes.

A series of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) analogues were evaluated for activity at homo-oligomeric glutamate1-flop (Glu1-flop) receptors expressed in Xenopus oocytes, using the two-electrode voltage clamp technique. (RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) (EC50, 2.4 microM), a homologue of AMPA having a carboxyl group as the terminal acidic functionality, was five times more potent than AMPA (EC50, 12 microM) and 20 times more potent than kainate (EC50, 46 microM). (RS)-2-Amino-3(3-hydroxy-5-trifluoromethyl-4-isoxazolyl)propionic acid (Tri-F-AMPA), in which an electronegative trifluoromethyl group is substituted for the methyl group on the isoxazole ring in the AMPA structure, was three times more potent than AMPA, whereas (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA), a bicyclic analogue of AMPA with highly restricted conformational flexibility was 10 times less potent than AMPA. The limiting slope of log-log plots of Glu1-flop receptor currents versus low agonist concentrations had a value of 1.7 for ACPA and kainate compared to 1.5 for Tri-F-AMPA and 1.3 for 5-HPCA and AMPA. The amplitude of responses evoked by near saturating concentrations of the agonists varied more than 7-fold. The sequence of efficacy was ACPA = kainate > Tri-F-AMPA > AMPA > 5-HPCA. Moreover, when saturating concentrations of Tri-F-AMPA and kainate were co-applied, the response was significantly greater than when each of the agonists was applied separately. The potency of the antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) (estimated KB, approximately 200 nM), to block currents mediated by Glu1-flop receptors was similar for all of the agonists tested in this study. These results indicate that relatively minor changes in the molecular structure of AMPA are associated with marked effects on potency and efficacy. In particular, it is suggested that the acidity of the terminal group plays a major role in determining the degree of receptor activation in the steady state.

Animals↗

PubMed1996

Synthesis and pharmacology of highly selective carboxy and phosphono isoxazole amino acid AMPA receptor antagonists.

(RS)-2-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 5) and the selective AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA, 7) have been used as leads for the design and synthesis of a number of potential AMPA receptor antagonists. Two parallel series of AMOA analogs were synthesized, containing either a distal carboxylic acid (compounds 8b-g and 11b) or a phosphonic acid (compounds 9a-g, 10c, and 11c). Pharmacological characterization of the synthesized compounds was carried out using a series of receptor binding assays and by in vitro electrophysiological experiments using the rat cortical slice model. The two analogs with a tert-butyl substituent, (RS)-2-amino-3-[5-tert-butyl-3-(carboxymethoxy)-4-isoxazolyl]pr opi onic acid (ATOA, 8b) and the corresponding phosphonic acid analog ATPO (9b), were the most potent and selective AMPA antagonists within each series. ATOA and ATPO showed IC50 values of 150 and 28 microM, respectively, toward AMPA-induced depolarizations in the cortical slice model compared to IC50 = 320 microM for the parent compound, AMOA. These two new competitive AMPA antagonists were significantly more selective than AMOA, showing no antagonism (up to 1 mM) toward NMDA-induced responses, whereas AMOA (at 1mM) showed weak (19%) inhibition toward NMDA-induced responses. The structure-activity relationships for the two series of compounds revealed considerable differences with respect to the substituents effects, and the phosphonic acid analogs generally exhibited significantly higher potencies compared to the carboxylic acid analogs.

Animals↗

PubMed1996

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.

Carbon Isotopes↗

PubMed1996

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.

Acetates↗

PubMed1996

Analogues of carbacholine: synthesis and relationship between structure and affinity for muscarinic and nicotinic acetylcholine receptors.

A series of acyclic and heterocyclic analogues of carbacholine (1) was synthesized using N-methylcarbacholine (MCC, 2), N,N-dimethylcarbacholine (DMCC, 3), and the corresponding tertiary amine (4) as leads. Whereas nicotinic acetylcholine receptor affinity was determined using [3H]nicotine as the radioactive ligand, [3H]oxotremorine-M ([3H]Oxo-M) and [3H]quinuclidinyl benzilate ([3H]QNB), in some cases supplemented with [3H]pirenzepine ([3H]PZ), were used as radioligands for muscarinic acetyicholine receptors on rat brain membranes. On the basis of receptor binding data, nicotinic/muscarinic (N/M) selectivity factors were determined, and muscarinic receptor efficacy (M agonist index) and M1 selectivity (M2/M1 index) estimated. In most cases, quaternized analogues showed higher affinity than the corresponding tertiary amines for muscarinic and, in particular, nicotinic receptor sites. Among the new compounds, N,N-diethylcarbacholine (9e) (IC50 = 0.046 microM), (S)-1-methyl-2-(N,N- dimethyl-aminocarbonyloxymethyl)pyrrolidine (17k) (IC50 = 0.068 microM), and the corresponding quaternized analogue, 18k (IC50 = 0.018 microM) showed the highest nicotinic receptor affinity. The tertiary amine, 17k showed much higher nicotinic receptor affinity than the acyclic analogue, 4 (IC50 = 5.7 microM), and the N/M selectivity factor determined for 17k (150) is an order of magnitude lower than that of nicotine (1400). THe N/M selectivity factors for MCC (2) and DMCC (3), previously reported to be highly selective nicotinic receptor ligands, were shown to be 6.5 and 60, respectively, the latter value being comparable with that of 18k (89).

Animals↗

PubMed1996

Functional partial agonism at ionotropic excitatory amino acid receptors.

(RS)-2-Amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid (APPA), which is an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), shows the characteristics of a partial AMPA receptor agonist. Since (S)-APPA is a full AMPA agonist and (R)-APPA a competitive antagonist, the partial agonism observed for APPA, which is a 1:1 mixture of (S)- and (R)-APPA, is only apparent. These observations have prompted comparative pharmacological studies of different molar ratios of a series of AMPA and N-methyl-D-aspartic acid (NMDA) agonists and respective competitive antagonists, and of these agonists in the presence of fixed concentrations of antagonist. Using the rat cortical wedge preparation, the latter series of experiments showed the expected rightward parallel shifts of the dose-response curves. The former type of experiments, on the other hand, produced dose-response curves at different levels of maximal response, depending on the molar ratios of agonist and antagonist used. This phenomenon, which is in agreement with the theory for competitive receptor interaction, has been termed functional partial agonism, a new pharmacological concept of potential therapeutic utility. These results were obtained using AMPA, the AMPA agonist (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), the competitive AMPA antagonists (RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid (AMOA) and 6-nitro-7-sulfamoylbenzo[f] quinoxalin-2,3-dione (NBQX), NMDA, and the competitive NMDA antagonist (RS)-3-(2-carboxy-4-piperazinyl)propyl-1-phosphonic acid (CPP).

Alanine↗

PubMed1995

Differentiation of in vivo effects of AMPA and NMDA receptor ligands using drug discrimination methods and convulsant/anticonvulsant activity.

The discriminative stimulus properties of the AMPA ((RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid) receptor agonist ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid) and NMDA (N-methyl-D-aspartic acid) in rats have been characterized. It is suggested that the cues are mediated by separate mechanisms in the central nervous system. The ATPA cue is not mimicked by NMDA or an NMDA receptor agonist, and is inhibited by the AMPA receptor antagonist (R)-APPA ((R)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid) but not the AMPA receptor antagonist ATOA ((RS)-2-amino-3-(3-carboxymethoxy-5-tert-butylisoxazol-4-yl)propio nic acid) or the NMDA receptor antagonist CPP ((RS)-3-(2-carboxypiperazin-4-yl)propyl)phosphonic acid). The ATPA cue is not mimicked by AMPA which is believed not to penetrate the blood-brain barrier. In contrast, ATPA does not generalize to the NMDA cue, which is mimicked by some NMDA receptor agonists (tetrazol-5-yl-glycine and AMAA ((RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid)) and is inhibited by the NMDA receptor antagonist CPP. Highly potent convulsant activity was demonstrated in mice with all AMPA and NMDA receptor agonists after intracerebroventricular (i.c.v.) injection, whereas weaker or no effects were found after subcutaneous (s.c.) or intravenous injection. Only (RS)-tetrazol-5-yl-glycine had a potent effect after s.c. administration. I.c.v. ATOA and CPP inhibited convulsions induced by i.c.v. injection of AMPA or NMDA, while (R)-APPA was ineffective. These results indicate that there are differences in the structure-activity relations in the drug discrimination and convulsant/anticonvulsant models, even when effects after i.c.v. and s.c. injection are taken into consideration. The convulsion models are rapid tests which can give an indication of central nervous system penetration, but are less pharmacologically specific with respect to differentiation between AMPA and NMDA ligands where cue models demonstrate clear differences in effects of ligands with selectivity for receptor subtypes.

Animals↗

PubMed1995

Partial GABAA receptor agonists. Synthesis and in vitro pharmacology of a series of nonannulated analogs of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol.

5-(4-Piperidyl)isoxazol-3-ol (4-PIOL, 10), a structural analog of 4-aminobutanoic acid (GABA, 1) and the GABAA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 5), is a low-efficacy partial GABAA agonist. A number of compounds bioisosterically derived from 10, including 5-(4-piperidyl)isothiazol-3-ol (11), 3-(4-piperidyl)isoxazol-5-ol (12), 5-(1,2,3,6-tetrahydropyrid-4-yl)isoxazol-3-ol (13), and 5-(1,2,3,6-tetrahydropyrid-4-yl)isothiazol-3-ol (14), were synthesized and tested as GABAA receptor ligands. Whereas none of these compounds significantly affected GABAB receptor binding or GABA uptake, they showed affinities for GABAA receptor sites in the low-micromolar range. Using cultured cerebral cortical neurons and whole-cell patch-clamp techniques, the efficacies of these compounds relative to that of the full GABAA agonist, isoguvacine (8) (20 microM), were determined. The relative efficacy of 11, which has a higher receptor affinity (IC50 = 1.3 +/- 0.3 microM) than 10 (IC50 = 9.3 +/- 2.6 microM), was comparable with that of 10 (30-35%). The tetrahydropyridine analog of 10, compound 13, showed a markedly lower receptor affinity (IC50 = 32 +/- 10 microM) and apparently a lower relative efficacy than 10. The corresponding unsaturated analog of 11, compound 14, showed a slightly weaker receptor affinity (IC50 = 4.0 +/- 2.0 microM) but a significantly higher relative efficacy (50-55%) than 11. The 5-isoxazolol isomer of 10, compound 12, showed a reduced receptor affinity (IC50 = 26 +/- 7 microM) and a very low relative efficacy. Substitution of propanoic or propenoic acid moieties for the acidic heterocyclic units of these compounds gave the monocyclic amino acids 15-18, which have very little or no affinity for GABAA receptor sites.

Animals↗

PubMed1995

Annulated heterocyclic bioisosteres of norarecoline. Synthesis and molecular pharmacology at five recombinant human muscarinic acetylcholine receptors.

A series of O-alkylated analogs of 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) were synthesized and characterized as ligands for muscarinic acetylcholine receptors (mAChRs). O-Methyl-THAO (4a), O-ethyl-THAO (4b), O-isopropyl-THAO (4c), and O-propargyl-THAO (4d) were shown to be potent inhibitors of the binding of tritiated quinuclidinyl benzilate (QNB), pirenzepine (PZ), and oxotremorine-M (Oxo-M) to tissue membrane preparations. In the [3H]-Oxo-M binding assay, receptor affinities in the low nanomolar range were measured for 4a (IC50 = 0.010 microM), 4b (IC50 = 0.003 microM), 4c (IC50 = 0.011 microM), and 4d (IC50 = 0.0008 microM). Pharmacological effects (EC50 or Ki values) and intrinsic activities (per cent of maximal carbachol responses) were determined using five recombinant human mAChRs (m1-m5) and the functional assay, receptor selection and amplification technology (R-SAT). Compound 4c antagonized carbachol-induced responses at m1, m3, and m5. With the exception of 4b, which was an antagonist at m5, 4a,b,d showed partial agonism at m1-m5 with very similar subtype selectivity (m2 > m4 > m1 > or = m3 > m5). Agonist index values for 4a-d, which were calculated from [3H]QNB (brain) and [3H]Oxo-M (brain) binding data, were shown to be predictive of pharmacologically determined intrinsic activities at m1-m5, the same rank order of intrinsic activity being observed at all five mAChRs (4a > 4d > 4b > 4c). It is concluded that within this class of high-affinity mAChR (m1-m5) ligands, containing secondary amino groups, minor changes of the bioisosteric ester alkyl groups have marked effects on potency and, in particular, intrinsic activity.

3T3 Cells↗

PubMed1995

Thiomuscimol, a new photoaffinity label for the GABAA receptor.

Thiomuscimol inhibits [3H]muscimol binding to brain GABAA receptors. Exposure of Ag(+)-treated membrane preparations to UV radiation at 254 nm for 40 min in the presence of thiomuscimol (10(-5) M) produced a 20-30% irreversible decrease in high-affinity [3H]muscimol binding sites. The photoaffinity labeling of thiomuscimol was inhibited by GABA (10(-4) M) added prior to exposure to UV light. The data show that thiomuscimol can label the GABAA receptor site and that the ligand can be used as a photoaffinity label for purification and identification of GABA binding sites within the GABAA receptor complex.

Affinity Labels↗

PubMed1995

Ketobemidone, methadone and pethidine are non-competitive N-methyl-D-aspartate (NMDA) antagonists in the rat cortex and spinal cord.

The opiate agonists, ketobemidone, methadone and pethidine, were evaluated as N-methyl-D-aspartate (NMDA) receptor antagonists using the rat cortical wedge preparation and the neonatal rat spinal cord preparation for electrophysiological studies and [3H](RS)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine ([3H]MK-801) binding experiments using rat forebrain homogenates. Ketobemidone, methadone and pethidine were inhibitors of [3H]MK-801 binding with Ki values of 26 microM, 0.85 microM and 47 microM, respectively. In the cortex, 1 mM ketobemidone and 1 mM methadone reduced NMDA responses, but not (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) or kainate responses in an use-dependent manner, whereas 1 mM pethidine was devoid of antagonist activity. In the spinal cord preparation, the activities of ketobemidone and methadone were weaker than in cortex. In contrast, pethidine was equipotent with ketobemidone in the spinal cord. These results suggest that ketobemidone and methadone may be useful therapeutic agents in conditions where a combined opiate agonist and NMDA antagonist treatment is desired.

Analgesics, Opioid↗

PubMed1995

GABAA agonists: resolution and pharmacology of (+)- and (-)-isoguvacine oxide.

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABAA receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABAA receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyl-oxycarbonyl)-3,4-epoxypiperidin e-4-carboxylate (1) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee > or = 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [3H]GABA to GABAB receptor sites (IC50 > 100 microM), (+)-isoguvacine oxide (IC50 = 0.20 +/- 0.03 microM) and (-)-isoguvacine oxide (IC50 = 0.32 +/- 0.05 microM) showed comparable potencies as inhibitors of the binding of [3H]GABA to GABAA receptor sites. Furthermore, (+)-isoguvacine oxide (EC50 = 6 microM; 33% relative efficacy) and (-)-isoguvacine oxide (EC50 = 5 microM; 38% efficacy relative to 10 microM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [3H]diazepam to the GABAA receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABAA agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABAA recognition site(s), derived from extensive structure-activity studies on GABAA agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗