Characterisation and autoradiographic localisation of 5-HT3 receptor recognition sites identified with [3H]-(S)-zacopride in the forebrain of the rat

The pharmacological characterisation and topographical distribution of [3H]-(S)-zacopride recognition sites in the forebrain of the rat was studied using homogenate and autoradiographic radioligand binding techniques. [3H]-(S)-Zacopride labelled a single, saturable, specific binding site (defined by 10.0 microM granisetron) in homogenates prepared from the entorhinal cortex of the rat (pKD = 9.51 +/- 0.08; Bmax = 104 +/- 7 fmol mg-1 protein; mean +/- SEM, n = 8). Pharmacological characterisation of the recognition site, within the entorhinal cortex, suggested that [3H]-(S)-zacopride selectively labelled the recognition site of the 5-HT3 receptor. Specific binding of [3H]-(S)-zacopride (defined by 1.0 microM granisetron) was differentially distributed throughout the forebrain of the rat; highest densities were located within sub-nuclei of the amygdala (cortical amygdaloid nucleus, amygdalohippocampal area, posterior medial cortical amygdaloid nucleus, posterior lateral amygdaloid nucleus), cortical areas (primary olfactory cortex, entorhinal cortex) and hippocampus. Non-specific binding was distributed homogeneously, although lower in myelinated structures. It is concluded that [3H]-(S)-zacopride selectively labels 5-HT3 receptor recognition sites within the forebrain of the rat; the topographical distribution of these sites, within the limbic nuclei, is consistent with the behavioural actions in animal models of the selective 5-HT3 receptor antagonists.     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The potent 5-HT3 receptor antagonist (R)-zacopride labels an additional high affinity site in the central nervous system.

The binding characteristics of [3H](R)- and [3H](S)-zacopride were investigated in membranes from the rat entorhinal cortex and NG 108-15 clonal cells. In contrast to [3H](S)-zacopride which bound solely to 5-HT3 receptors, [3H](R)-zacopride recognized another class of binding sites, called the (R)-sites, in both membrane preparations. In addition to (R)-zacopride (Ki = 3-11 nM), only (R)-iodo-zacopride, (R)-dechloro-zacopride, prazosin and mianserin exhibited high to moderate affinity for the (R)-sites, whose possible functions remain to be established.     

2-(Carboxycyclopropyl)glycines: binding, neurotoxicity and induction of intracellular free Ca2+ increase.

The excitatory actions of the eight stereoisomers of 2-(carboxycyclopropyl)glycine (CCG), conformationally rigid glutamate analogues, were analyzed for the glutamate receptor subtypes by means of binding assays with rat brain membranes. All CCG isomers inhibited the binding of [3H]3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid ([3H]CPP) to N-methyl-D-aspartate (NMDA) receptors. The (2S,3R,4S) isomer (L-CCG-IV) was the most potent agonist for the NMDA receptor and its binding potency was 17- and 790-fold higher than that of L-glutamate and NMDA, respectively. The (2S,3S,4R) isomer (L-CCG-III) showed a potent inhibitory activity for [3H]D-aspartate uptake. Further, L-CCG-IV caused a marked increase of intracellular free Ca2+ concentration [( Ca2+]i) and potent neurotoxicity in the single rat cerebral cortical neurons in vitro, and both were blocked effectively by the NMDA antagonists. Significant correlations were observed between neurotoxicity and the increase of [Ca2+]i and [3H]CPP binding affinity to the NMDA receptor.     

Profiles of interaction of R(+)/S(-)-zacopride and anxiolytic agents in a mouse model.

The mouse black and white test box was used to measure changes in behaviour in an aversive situation where the administration of R(+)-zacopride (but not S(-)-zacopride) alone decreased aversive responding to the white area. A similar anxiolytic profile of action was observed using parachlorophenylalanine (PCPA), whose effects were antagonised by a co-treatment with R(+)-zacopride and reversed by S(-)-zacopride to an exacerbation of the aversive response. An anxiolytic profile of action was also observed using ondansetron, granisetron, chlordiazepoxide, diazepam, ritanserin, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), E4424 (2-[4-[4-(4-chloro-l-pyrazoyl)butyl]-l-piperazinyl]-pyrimidine), umepsirone, DuP753 (2-n-butyl-4-chloro-5-hydroxy-methyl-1-[2(1H-tetrazol-5-yl) biphenyl-4-yl)methyl)]-imidazole), SQ29,852 ((S)-1-[6-amino-2[hydroxy)(4-phenyl-butyl)phosphinyl]-oxy)-1- nexy]-2-proline), devazepide and guanfacine, and this was retained following co-treatment with PCPA. The anxiolytic profile of action of PCPA was also retained following co-treatment with renzapride which when administered alone failed to modify behaviour. However, the ability of chlordiazepoxide, diazepam, ondansetron and E4424 (but not devazepide, DuP753 or SQ29,852) to reduce aversive responding was inhibited by co-treatment with R(+) and/or S(-)-zacopride. It is concluded that the reduction in aversive responding caused by pharmacological manipulation at the benzodiazepine, 5-HT receptor subtypes 5-HT1A, 5-HT1C/5-HT2 and 5-HT3 (but not at the cholecystokin CCKA or angiotensin receptors or inhibition of angiotensin converting enzyme) can be inhibited by R(+) and S(-)-zacopride. The data is discussed in terms of zacopride having an agonist or partial agonist effect at the 5-HT3 receptor.     

Aporphines. 48. Enantioselectivity of (R)-(-)- and (S)-(+)-N-n-propylnorapomorphine on dopamine receptors

The enantiomers [(S)-(+) and (R)-(-)] of N-n-propylnorapomorphine (NPA) were synthesized. (R)-NPA was obtained by the acid-catalyzed rearrangement of N-n-propylnormorphine. (R)-NPA also was converted to (RS)-N-n-propylnorapomorphine dimethyl ether by dehydrogenation of the 10,11-O,O'-dimethyl ether of (R)-NPA with 10% palladium on carbon in acetonitrile, followed by reduction with sodium cyanoborohydride under acidic conditions. Alternatively (RS)-NPA 10,11-O,O'-dimethyl ether was obtained via total synthesis. (+)-Dibenzoyl-D-tartaric acid was used to resolve (RS)-NPA dimethyl ether. Ether cleavage gave (S)-NPA isolated as the hydrochloride salt in greater than 99.9% enantiomeric purity, as determined by circular dichroism (CD) spectra. The pharmacological activities of (S)- and (R)-NPA were evaluated with subnanomolar concentrations of 3H-labeled apomorphine (APO), ADTN, and spiroperidol (SPR) for competition for binding to a membrane-rich subsynaptosomal fraction of calf caudate nucleus. IC50 (nM) values for (R)-NPA vs. (S)-NPA were as follows: [3H]APO, 2.5 vs. 66; [3H]ADTN, 2.0 vs. 60; [3H]SPR, 174 vs. 1400. The efficacy of (R)- and (S)-NPA in stimulating dopamine-sensitive adenylate cyclase from both homogenates of rat corpus striatum and pieces of intact carp retina was also evaluated. Three behavioral effects in the rat (stereotyped behavior, sedation, and catalepsy) were also examined. Only (R)-NPA induced stereotypy; (S)-NPA failed to antagonize this action of the R isomer. The effects of (R)- and (S)-NPA on adenylate cyclase agreed with the behavioral effects and radioreceptor binding assays in that the R isomer was the strongly preferred enantiomer at dopamine receptors. The S enantiomer of NPA was, however, the weakly preferred configuration for rat liver catechol O-methyltransferase. A dopamine-receptor model accommodates the configuration of NPA and related aporphines.     

Possible existence of a novel receptor for uridine analogues in the central nervous system using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridines.

Uridine analogue binding sites, the so-called uridine receptor, were observed in the experiments on specific [3H]N3-phenacyluridine binding to bovine synaptic membranes using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, as ligands. The potent hypnotic, N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not the (R)-isomer, strongly inhibited [3H]N3-phenacyluridine binding. The racemate had inhibitory activity intermediate between that of the two alpha-hydroxy-beta-phenethyluridines ((R)- or (S)-isomers). The inhibitory constants of these compounds were determined. The Ki values of N3-phenacyluridine, alpha-hydroxy-beta-phenethyluridine (racemate), N3-(R)-(-)-, and N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine were 0.65, 397.4, 1908, and 10.2 nM, respectively. The present results indicate the existence of uridine receptors in the central nervous system in relation to their hypnotic activities reported previously.     

Structure-activity studies on the activity of a series of cyclopentane GABA analogues on GABAA receptors and GABA uptake

A series of analogues of gamma-aminobutyric acid (GABA) has been investigated for GABA mimetic activity on the isolated guinea-pig ileum, facilitation of [3H]diazepam binding in rat brain membranes and inhibition of [3H]GABA uptake from rat brain cortical slices. The derivatives tested include six racemic amino acids, all of which contain a 'GABA backbone' with the conformation restricted by a cyclopentane or cyclopentene ring system. From the more potent analogues, five optically pure compounds, including (+)-(4S)-4-aminocyclopent-1-ene-1-carboxylic acid and its (-)-4R enantiomer, have also been assessed as GABA agonists. Of the racemic analogues, 4-aminocyclopent-1-ene-1-carboxylic acid and trans-3-aminocyclopentane-1-carboxylic acid were the most potent at GABAA receptors, while most of the analogues had considerable activity on GABA uptake. The individual resolved isomers of 4-aminocyclopent-1-ene-carboxylic acid and of trans-3-aminocyclopentane-1-carboxylic acid displayed great specificity for GABA receptor and GABA uptake sites. For example (+)-(4S)-4-aminocyclopent-1-ene-1-carboxylic acid was approximately twice as potent as GABA and about 600 times more active than the (-)-4R isomer on the guinea-pig ileum, while it was not significantly active as a GABA uptake inhibitor at 500 microM. On the other hand, its (-)-4R isomer was selective for inhibiting GABA uptake with an IC50 equal to that of racemic nipecotic acid.     

Effects of racemic, (S)- and (R)-methylenedioxyamphetamine on synaptosomal uptake and release of tritiated norepinephrine

Racemic, (S)- and (R)-methylenedioxyamphetamine (MDA) were potent competitive inhibitors of [³H]norepinephrine (NE) uptake by rat hypothalamic synaptosomes. (S)- and (R)-α-methyldopamine were extremely potent competitive inhibitors of [³H]norepinephrine uptake, establishing these compounds as the most potent inhibitors of brain NE uptake reported to date. Racemic. (S)- and (R)-MDA were slightly less potent as releasing agents than (±)-, (+)- and (-)amphetamine respectively. (S)- and (R)-α-methyldopamine were the most potent releasing agents examined.     

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.].The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.     

The interaction of R(+)- and S(-)-zacopride with PCPA to modify rodent aversive behaviour.

The interaction of R(+)- and S(-)zacopride (0.00001-10 mg/kg i.p.) with parachlorophenylalanine (PCPA, 3 day treatment 100 mg/kg i.p.) to modify behaviour in an aversive situation was investigated in the mouse black and white test box and rat social interaction test. R(+)-Zacopride (but not S(-)zacopride) and PCPA had an anxiolytic profile of action to reduce aversive responding in both species. Their established anxiolytic profiles were abolished by a subsequent treatment with S(-)zacopride. In contrast, S(-)-zacopride was less or ineffective if administered simultaneously with R(+)-zacopride. A co-treatment of PCPA with R(+)-zacopride also inhibited the anxiolytic profiles observed to the individual treatments. It is concluded that there is a complex interaction between the isomers of zacopride to modify responding to an aversive situation that is dependent on the temporal sequence of drug administration, and which may be modified by changes in basal 5-hydroxytryptamine (5-HT) tone and agonist, partial agonist and antagonist effects at the 5-HT3 receptor.     

Differential modulation of extracellular levels of 5-hydroxytryptamine in the rat frontal cortex by (R)- and (S)-zacopride.

1. The ability of various anxiolytic and potential anxiolytic agents to modify 5-hydroxytryptamine (5-HT) release in the frontal cortex of the rat was assessed by the microdialysis technique. 2. The benzodiazepine receptor agonist, diazepam (2.5 mg kg-1, i.p.), the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.32 mg kg-1, s.c.) and the 5-HT1A receptor partial agonist buspirone (4.0 mg kg-1, i.p.) maximally reduced extracellular levels of 5-HT in the rat frontal cortex by approximately 50-60%, 70-80% and 30-40%, respectively. 3. (R)-zacopride (1.0-100 micrograms kg-1, i.p.) dose-dependently reduced extracellular levels of 5-HT in the rat frontal cortex (approximately 80% maximal reduction) whereas the other 5-HT3 receptor antagonists ondansetron (10 micrograms kg-1, i.p.) and (S)-zacopride (10-100 micrograms kg-1, i.p.) were ineffective. 4. In contrast to (S)-zacopride (100 nM; administered via the microdialysis probe), (R)-zacopride (1.0-100 nM; administered via the microdialysis probe) induced a concentration-dependent reduction in extracellular levels of 5-HT in the rat frontal cortex (approximately 70% maximal reduction). 5. In contrast to ondansetron (100 micrograms kg-1, i.p.), (S)-zacopride (10-100 micrograms kg-1, i.p.) dose-dependently reversed the (R)-zacopride (10 micrograms kg-1, i.p.) induced reduction in extracellular levels of 5-HT in the rat frontal cortex. The highest dose of (S)-zacopride (100 micrograms kg-1, i.p.) completely prevented the (R)-zacopride response.(ABSTRACT TRUNCATED AT 250 WORDS)     

(+)-Hydrastine, a potent competitive antagonist at mammalian GABAA receptors.

1. (+)-Hydrastine is a phthalide isoquinoline alkaloid, isolated from Corydalis stricta. It has the same 1S,9R configuration as the competitive GABAA receptor antagonist bicuculline and is the enantiomer of the commercially available (-)-hydrastine. 2. (+)-Hydrastine (CD50 0.16 mg kg-1, i.v.) was twice as potent as bicuculline (CD50 0.32 mg kg-1, i.v.) as a convulsant in mice. This action was stereoselective in that (+)-hydrastine was 180 times as potent as (-)-hydrastine. 3. (+)-Hydrastine was a selective antagonist at bicuculline-sensitive GABAA receptors in the guinea-pig isolated ileum. It did not influence phaclofen-sensitive GABAB receptors or acetylcholine receptors in this tissue. (+)-Hydrastine was a competitive antagonist of GABAA responses (pA2 6.5) more potent than bicuculline (pA2 6.1). 4. When tested against the binding of [3H]-muscimol to high affinity GABAA binding sites in rat brain membranes, (+)-hydrastine (IC50 2.37 microM) was 8 times more potent than bicuculline (IC50 19.7 microM). 5. As an antagonist of the activation of low affinity GABAA receptors as measured by the stimulation by GABA of [3H]-diazepam binding to rat brain membranes, (+)-hydrastine (IC50 0.4 microM) was more potent than bicuculline (IC50 2.3 microM). 6. (+)-Hydrastine, 10 nM to 1 mM, did not inhibit the binding of [3H]-(-)-baclofen to GABAB binding sites in rat brain membranes.     

Antagonism of [3H]zacopride binding to 5-HT3 recognition sites by its (R) and (S) enantiomers

[3H]Zacopride exhibits high affinity (Kd less than or equal to 1 nM) for 5-HT3 binding sites (inhibited by ICS 205-930) in rabbit intestinal muscularis and vagus nerve, human jejunum, rat intestinal muscularis and rat brain cortex. Its binding was inhibited by several 5-HT3 antagonists that displayed similar rank orders of potency in the tissues examined. Zacopride's (S) enantiomer was significantly more potent than its (R) enantiomer (21- to 42-fold in rabbit and human; 8- to 12-fold in rat) as an inhibitor of [3H]zacopride binding. These studies indicate that the utility of [3H]zacopride as a high affinity 5-HT3 ligand resides with the (S) enantiomer.     

Synthesis, molecular modeling studies, and muscarinic receptor activity of azaprophen analogues

Synthesis, radioligand binding, and pharmacologic activities of a series of muscarinic receptor ligands including and related to azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3 alpha-ol 2,2-diphenylpropionate, 1) have been measured to determine activity and selectivity for muscarinic receptor subtypes. Pharmacologic affinities of antagonists were determined as pA2 values for antagonism of methacholine-induced tension responses in guinea pig ileum. Binding affinities were measured by competition against [3H]QNB binding in guinea pig ileum, rat heart and brain, and m1- or m3-transfected Chinese hamster ovary (CHO) cells. The efficacies of muscarinic agonists in brain were determined by the ratio of binding affinities against [3H]QNB or [3H]NMS and [3H]oxotremorine-M ([3H]Oxo-M). Nine muscarinic antagonists, including azaprophen, did not discriminate significantly between the subtypes of muscarinic receptors. KI values for receptor binding for azaprophen (1) were between 8.81 x 10(-11) and 4.72 x 10(-10) M in ileum, heart, brain, and m1- or m3-transfected CHO cells. The alpha- and beta-benzilate esters 5 and 6 are as potent as azaprophen, and diphenylacetate esters 3 and 4 and N-(6)-benzyl alpha-isomer 7 are less potent than azaprophen. Significant stereoselectivity was exhibited with (+)-azaprophen being approximately 200 times more potent than the (-)-enantiomers and the 3 beta-ol isomer 2 being ca. 50 times less potent than azaprophen in all systems. A molecular modeling-molecular mechanics study was conducted to account for the difference. Putative muscarinic agonists (analogues and isomers of 6-methyl-6-azabicyclo[3.2.1]octan-3-ol acetate) did not discriminate muscarinic receptor subtypes with KI values between 2.77 x 10(-6) and 4.33 x 10(-5) M without significant stereoselectivity in the systems examined. The most active analogue was (1R,3R,5S)-6-[1(R)-phenylethyl]-6-azabicyclo[3.2.1]octan-3 alpha-ol acetate. However, efficacies of these putative agonists were in general very low.     

Effects of (+)- and (-)-mianserin on alpha-adrenoceptors and tyramine-induced tachycardia in rats

The individual stereoisomers of mianserin were tested in pithed normotensive rats for their antagonistic activity at vascular postjunctional alpha 1- and alpha 2-adrenoceptors as well as their interaction with the 'amine pump receptor'. (+)-Mianserin was found approximately five times more potent than (-)-mianserin in antagonizing the increase in diastolic pressure brought about by either selective alpha 1 (agonist: methoxamine)- or alpha 2 (agonist: B-HT 920)-adrenoceptor stimulation. (+)-Mianserin also behaved as the more effective displacer of [3H]prazosin and [3H]clonidine binding to rat brain membranes. Tyramine, an indirectly acting sympathomimetic agent, increased the resting heart rate of pithed rats. This effect was competitively antagonised by the noradrenaline uptake inhibitor desipramine. (-)-Mianserin was inactive in inhibiting the tyramine-induced increase in heart rate. (+)-Mianserin showed some activity, but was much less potent than desipramine. The present results clearly indicate that (+)-mianserin is the pharmacologically more active isomer.     

Probes for narcotic receptor mediated phenomena. 15. (3S,4S)-(+)-trans-3-methylfentanyl isothiocyanate, a potent site-directed acylating agent for the delta opioid receptors in vitro

Recently we reported the synthesis of the first enantiomeric pair of irreversible opioid ligands [(3S,4R)-(-)- and (3R,4S)-(+)-cis-4, SUPERFIT] and specific interaction of the latter with the delta receptor. Here we report another enantiomeric pair of irreversible opioid ligands, (+)-trans- and (-)-trans-3-methylfentanyl isothiocyanates [(3S,4S)-(+)-trans- and (3R,4R)-(-)-trans-4]. A single-crystal X-ray analysis of the 2,4,6-trinitrobenzenesulfonic acid salt of (+)-trans-3-methyl-N-phenyl-4-piperidinamine [(+)-trans-8] revealed it (and, therefore, 4) to have the trans configuration and the absolute configuration of (+)-trans-8 to be 3S,4S. The (+)-trans enantiomer of 4 was shown to be highly potent and about 10-fold more selective as an acylating agent than (-)-trans-4 for the higher affinity [3H]DADL (delta) binding site in rat brain membranes. In that assay, (+)-trans-4 and (+)-cis-4 were essentially equipotent as affinity ligands, and the levo enantiomers were considerably less potent. (+)-trans-4 was, thus, a potent, subtype-selective acylating agent for the delta opioid receptor in vitro. With membranes from NG108-15 neuroblastoma x glioma hybrid cells, containing only delta receptors, (+)-cis-4 was found to be a little more potent than (+)-trans-4. Similarly, (+)-cis-4 is the most effective inhibitor of adenylate cyclase in these membranes, (+)-trans-4 has weak activity, and the levo enantiomers are inactive. Only (+)-cis-4 was found to have antinociceptive activity in vivo.     

Probes for narcotic receptor mediated phenomena. 17. Synthesis and evaluation of a series of trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacet amide (U50,488) related isothiocyanate derivatives as opioid receptor affinity ligands

A series of U50,488 related isothiocyanates was synthesized from enantiomerically pure (S,S)-(+)-trans-2-pyrrolidinyl-N-methylcyclohexylamine [(+)-7] and (R,R)-(-)-trans-2-pyrrolidinyl-N-methylcyclohexylamine [(-)-7]. DCC coupling of (+)- and (-)-7 with nitrophenylacetic acids followed by catalytic hydrogenation and treatment with thiophosgene afforded a series of six isomeric aryl isothiocyanate analogues of U50,488. Similarly, DCC coupling of (+)- and (-)-7 with (+)- and (-)-N-t-Boc-protected phenylglycines afforded four isomeric alkyl isothiocyanates. Evaluation of the isothiocyanates for their capacity to produce wash-resistant inhibition of mu, delta, and kappa sites in vitro was performed using rat and guinea pig brain membranes. None of the compounds was able to irreversibly inhibit binding of [3H]bremazocine to guinea pig and rat brain membranes (depleted of functional mu and delta receptors by pretreatment with acylating agents BIT and FIT). However, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-1] was able to specifically and irreversibly inhibit kappa receptors labeled by [3H]-U69,593: Incubation of rat brain membranes for 60 min at 25 degrees C with 1 microM of (-)-1 resulted in a wash-resistant reduction of the binding to 11.2 +/- 2.5% of the control. Binding analysis revealed the wash-resistant reduction in [3H]-U69,593 binding by (-)-1 to be through an increase in the Kd without effect on the Bmax. (-)-1 failed to effect mu or delta binding in rat or guinea pig brain under the same conditions. The enantiomer of (-)-1, (1R,2R)-trans-2-isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(+)-1], failed to affect kappa receptors labeled by [3H]-U69,593 under the same conditions as for (-)-1. (1S,2S)-trans-3-Isothiocyanato-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-2] inhibited to 49.6 +/- 5.1% of the control, in a wash-resistant manner, kappa receptors labeled by [3H]-U69,593. However, (-)-2 was not as selective as (-)-1 since it also reduced [3H]DADLE (delta) binding to 82.4 +/- 8.0% of the control value. (1S,2S)-trans-4-Isothiocyanato-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide [(-)-3] exhibited selective wash-resistant inhibition of delta receptors labeled by [3H]DADLE resulting in a reduction in binding to 42.9 +/- 4.2% of control.(ABSTRACT TRUNCATED AT 400 WORDS)     

(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPA receptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rat hippocampal and frontal cortex slices

The present study describes the effect of (S)-2,3-dihydro-[3, 4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986-1), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with cognitive-enhancing effects, on (S)-AMPA-induced [3H]noradrenaline release in rat hippocampal and frontal cortex slices. (S)-AMPA significantly increased [3H]noradrenaline release in rat hippocampus and frontal cortex slices, whereas S18986-1 (3-1000 microM) alone, was inactive. However, S18986-1 between 30 and 1000 microM potently enhanced (+200%) (S)-AMPA-mediated [3H]noradrenaline release in both hippocampal and frontal cortex slices. The capacity of S18986-1 to potentiate [3H]noradrenaline release was specific for AMPA receptors as S18986-1 failed to potentiate either kainate and N-methyl-D-aspartate (NMDA)-mediated release of [3H]noradrenaline in rat hippocampal slices. Moreover, 1, 2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) and 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3, 4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-53655) but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801), inhibited (S)-AMPA and S18986-induced stimulation of (S)-AMPA-mediated [3H]noradrenaline release. In addition, S18986-1-induced stimulation of (S)-AMPA-evoked [3H]noradrenaline release was markedly attenuated in the presence of tetrodotoxin (1 microM) and in Ca(2+)-free buffer. S18986-1 enhanced (S)-AMPA-mediated [3H]noradrenaline release to a greater extent than its corresponding (R)-enantiomer S19024-1 and racemic mixture S17951-1. However, positive allosteric modulators of AMPA receptors such as aniracetam failed to potentiate AMPA-mediated noradrenaline release in hippocampal slices, whereas cyclothiazide potently enhanced (S)-AMPA-mediated [3H]noradrenaline release. These results suggest that the capacity of S18986-1 to enhance AMPA receptor-mediated release of noradrenaline in rat hippocampus and frontal cortex, could contribute to the cognition enhancing mechanisms of S18986-1.     

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.