Synthesis of novel GABA uptake inhibitors. Part 6: preparation and evaluation of N-Omega asymmetrically substituted nipecotic acid derivatives.

In a previous series of potent GABA uptake inhibitors published from this laboratory, we noticed that asymmetry in the substitution pattern of the bis-aromatic moiety in known GABA uptake inhibitors such as 4 [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid] and 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid] was beneficial for high affinity. This led us to investigate asymmetric analogues of known symmetric GABA uptake inhibitors in which one of the aryl groups has been exchanged with an alkyl, alkylene or cycloalkylene moiety as well as other modifications in the lipophilic part. The in vitro values for inhibition of [(3)H]-GABA uptake in rat synaptosomes was determined for each compound, and it was found that several of the novel compounds inhibit GABA uptake as potently as their known symmetrical reference analogues. Several of the novel compounds were also evaluated for their ability to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in vivo. Some of the compounds, for example 18 [(R)-1-(2-(((1,2-bis(2-fluorophenyl)ethylidene)amino)oxy)ethyl)-3-piperidinecarboxylic acid], show a high in vivo potency and protective index comparable with that of our recently launched anticonvulsant product, 5 [(R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid], and may therefore serve as second-generation drug candidates.

Synthesis of novel gamma-aminobutyric acid (GABA) uptake inhibitors. 5.(1) Preparation and structure-activity studies of tricyclic analogues of known GABA uptake inhibitors.

On the basis of the SAR of a series of known gamma-aminobutyric acid (GABA) uptake inhibitors, including 4 (SKF 89976), new tricyclic analogues have been prepared. These novel compounds are derivatives of nipecotic acid, guvacine, and homo-beta-proline, substituted at the nitrogen of these amino acids by various lipophilic moieties such as (10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)alkoxyalkyl or (10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)alkoxyalkyl. The in vitro values for inhibition of [(3)H]-GABA uptake in rat synaptosomes was determined for each compound in this new series, and it was found that several of the novel compounds showed a high potency comparable with that of the reference compounds 4, 5 (tiagabine), and 6 (CI-966). Several of the novel compounds were also evaluated for their ability in vivo to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). One compound, (R)-1-(2-(2-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)ethoxy)ethyl)-3-piperidinecarboxylic acid (23), was selected for further biological investigations and showed a protective index comparable to or slightly better than that of the recently launched anticonvulsant product 5 ((R)-1-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid).

Systemic administration of the immunophilin ligand GPI 1046 in MPTP-treated monkeys.

Systemic administration of immunophilin ligands provides trophic influences to dopaminergic neurons in rodent models of Parkinson's disease (PD) resulting in the initiation of clinical trials in patients with Parkinson's disease. We believe that prior to clinical trials, novel therapeutic strategies should show safety and efficacy in nonhuman models of PD. The present study assessed whether oral administration of the immunophilin 3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrollidinecarboxylate (GPI 1046) could prevent the structural and functional consequences of n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in nonhuman primates. Twenty-five rhesus monkeys received daily oral administration of vehicle (n = 5) or one of four doses of GPI 1046 (0.3 mg/kg, n = 5; 1.0 mg/kg, n = 5; 3.0 mg/kg, n = 5; 10.0 mg/kg, n = 5). Two weeks after starting the drug treatment, all monkeys received a unilateral intracarotid injection of MPTP-HCl (3 mg). Daily drug administration continue for 6 weeks postlesion after which time the monkeys were sacrificed. Monkeys were assessed for performance on a hand reach task, general activity, and clinical dysfunction based on a clinical rating scale. All groups of monkeys displayed similar deficits on each behavioral measure as well as similar losses of tyrosine hydroxylase (TH)-immunoreactive (ir) nigral neurons, TH-mRNA, and TH-ir striatal optical density indicating that in general treatment failed to have neuroprotective effects.

Synthesis of novel GABA uptake inhibitors. 4. Bioisosteric transformation and successive optimization of known GABA uptake inhibitors leading to a series of potent anticonvulsant drug candidates.

By bioisosteric transformations and successive optimization of known GABA uptake inhibitors, several series of novel GABA uptake inhibitors have been prepared by different synthetic approaches. These compounds are derivatives of nipecotic acid and guvacine, substituted at the nitrogen of these amino acids by various lipophilic moieties such as diarylaminoalkoxyalkyl or diarylalkoxyalkyl. The in vitro values for inhibition of [(3)H]GABA uptake in rat synaptosomes was determined for each compound, and it was found that the most potent compound from this series, (R)-1-(2-(3,3-diphenyl-1-propyloxy)ethyl)-3-piperidinecarboxyli c acid hydrochloride (29), is so far the most potent parent compound inhibiting GABA uptake into synaptosomes. Structure-activity results confirm our earlier observations, that an electronegative center in the chain connecting the amino acid and diaryl moiety is very critical in order to obtain high in vitro potency. Several of the novel compounds were also evaluated for their ability in vivo to inhibit clonic seizures induced by a 15 mg/kg (ip) dose of methyl 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). Some of the compounds tested show a high in vivo potency comparable with that of the recently launched anticonvulsant product 6 ((R)-1-(4, 4-bis(3-methyl-2-thienyl)-3-butenyl)-3-piperidinecarboxylic acid).

Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether derivatives of nipecotic acid and guvacine as anticonvulsant agents.

(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.

Neural actions of immunophilin ligands.

Immunophilins, protein receptors for immunosuppressant drugs such as cyclosporin A and FK506, are enriched far more in the brain than in the immune system. Drug-immunophilin complexes bind to calcineurin, inhibiting its phosphatase activity and leading to immunosuppressant effects. The immunophilin FKBP-12 (FK506 binding protein, 12 kDa) forms a complex with the ryanodine and inositol (1,4,5) trisphosphate (IP3) receptors to regulate their physiological release of intracellular Ca2+. Here, Solomon Snyder and colleagues describe how non-immunosuppressant as well as immunosuppressant immunophilin ligands are neurotrophic for numerous classes of damaged neurones, both in culture systems and intact animals. Their ability to stimulate functional regrowth of damaged sciatic, cortical cholinergic, dopamine and 5-HT neurones may have therapeutic relevance.

M1 receptor agonist activity is not a requirement for muscarinic antinociception.

The analgesic effects of a series of muscarinic agonists were investigated by use of the mouse acetic acid writhing, grid-shock, hot-plate and tail-flick tests. The compounds tested were oxotremorine, pilocarpine, arecoline, aceclidine, RS86 and four 3-3(substituted-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahy-dro-1 -methyl pyridines (substituted TZTP), these being propoxy-TZTP, 3-Cl-propylthio-TZTP, xanomeline (hexyloxy-TZTP) and hexylthio-TZTP. These agonists were also assayed for their ability to displace [3H]oxotremorine-M and [3H]pirenz-epine binding and for their functional selectivity at pharmaco-logic M1, M2 and M3 receptors. These compounds all produced dose-dependent antinociceptive effects in all of the mouse analgesia tests. The effects of oxotremorine in the writhing test were fully antagonized by the muscarinic antagonist scopolamine (0.1 mg/kg), but only partially antagonized by methsco-polamine (10 mg/kg) and unaffected by the opioid antagonist naltrexone. 3-Cl-propylthio-TZTP and propoxy-TZTP had virtually no effect at the M1 receptor subtype as measured by the human m1 clone expressed in baby hamster kidney cells or the rabbit vas deferens assay. These compounds, however, were more potent in the analgesia tests than the selective M1 agonists xanomeline and hexylthio-TZTP. These data suggest that muscarinic analgesia is mediated by central muscarinic receptors. However, activity at the M1 receptor subtype is not a requirement for antinociceptive activity.

Butylthio[2.2.2] (NNC 11-1053/LY297802): an orally active muscarinic agonist analgesic.

Butylthio[2.2.2] ((+)-(S)-3-(4-(Butylthio)-1,2,5-thiadiazol-3-yl)-1-azabicyclo[2.2. 2] octane) is an agonist/antagonist at muscarinic receptors. The analgesic potential of butylthio[2.2.2] was assessed in the mouse by use of the grid-shock, tail-flick, hotplate and writhing tests. The ED50 values ranged from 0.19 to 1.47 mg/kg and 1.51 to 12.23 mg/kg 30 min after s.c. and p.o. administration, respectively, yielding p.o./s.c. ratios ranging from 7 to 27. The ED50 values for salivation and tremor were > 30 and 12.31 mg/kg s.c., and > 60 and > 60 mg/kg p.o., yielding therapeutic windows > 130 and 54, and, > 40 and > 40, after s.c. and p.o. administration, respectively. Motor impairment or lethality were only seen at doses 116 and 254 times higher than the antinociceptive doses. Butylthio[2.2.2] was equieffective to, and 3- to 24-fold more potent than morphine. The duration of action was similar to that of morphine. The dose-response curve was shifted dose dependently to the right by the muscarinic antagonist scopolamine but not by the opioid antagonist naltrexone. The antinociceptive effect of butylthio[2.2.2] was reversed by the centrally acting muscarinic antagonist scopolamine but not by the peripherally acting muscarinic antagonist methscopolamine. After 6.5 days repeated dosing in mice, morphine produced marked tolerance, whereas butylthio[2.2.2] produced minimal, if any, tolerance. In the rat grid-shock test, ED50 values of 0.26 mg/kg s.c. and 25.28 mg/kg p.o. were obtained. These data show that butylthio[2.2.2] is a potent and efficacious antinociceptive with a very favorable therapeutic window after s.c. and p.o. administration in mice, and with good efficacy in rats.

Pharmacology of butylthio[2.2.2] (LY297802/NNC11-1053): a novel analgesic with mixed muscarinic receptor agonist and antagonist activity.

Butylthio[2.2.2], ((+)-(S)-3-(4-butylthio-1,2,5-thiadiazol-3-yl)-1-azabicyclo[2.2.2] octane; LY297802/NNC11-1053) is a muscarinic receptor ligand which is equiefficacious to morphine in producing antinociception. In vitro, butylthio[2.2.2] had high affinity for muscarinic receptors in brain homogenates, but had substantially less or no affinity for several other neurotransmiter receptors and uptake sites. In isolated tissues, butylthio[2.2.2] was an agonist with high affinity for M1 receptors in the rabbit vas deferens (IC50 = 0.33 nM), but it was an antagonist at M2 receptors in guinea pig atria (pA2 = 6.9) and at M3 receptors in guinea pig urinary bladder (pA2 = 7.4) and a weak partial agonist in guinea pig ileum, which contains a heterogeneous population of muscarinic receptors. In vivo, butylthio[2.2.2] was without effect on acetylcholine, dopamine and serotonin levels in rat brain. Moreover, butylthio[2.2.2] did not decrease charcoal meal transit in mice, nor did it significantly alter heart rate in rats. Further, butylthio[2.2.2] did not produce parasympathomimetic effects such as salivation or tremor in mice, but it antagonized salivation and tremor produced by the nonselective muscarinic agonist oxotremorine. The present data demonstrate that butylthio[2.2.2] is a novel muscarinic receptor mixed agonist/antagonist and its pharmacological profile suggests that it may have clinical utility in the management of pain as an alternative to opioids.

Neurotrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models.

Although immunosuppressant immunophilin ligands promote neurite outgrowth in vitro, their neurotrophic activities are clearly independent of their immunosuppressive activity. In the present report, a novel nonimmunosuppressive immunophilin ligand, GPI-1046 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate+ ++) is described. In vitro, GPI-1046 bound to FK506 binding protein-12 and elicited neurite outgrowth from sensory neuronal cultures with picomolar potency with maximal effects comparable to nerve growth factor. In vivo, GPI-1046 stimulated the regeneration of lesioned sciatic nerve axons and myelin levels. In the central nervous system, GPI-1046 promoted protection and/or sprouting of serotonin-containing nerve fibers in somatosensory cortex following parachloroamphetamine treatment. GPI-1046 also induced regenerative sprouting from spared nigrostriatal dopaminergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice or 6-hydroxydopamine (6-OHDA) toxicity in rats. The rotational abnormality in 6-OHDA treated rats was alleviated by GPI-1046. These neurotrophic actions in multiple models suggest therapeutic utility for GPI-1046 in neurodegenerative diseases.

PET study of the M1-agonists [11C]xanomeline and [11C]butylthio-TZTP in monkey and man.

Xanomeline, a substituted TZTP, is a new M1 selective muscarinic agonist in clinical trials for Alzheimer's disease. The brain uptake of [11C]xanomeline and the analog [11C]butylthio-TZTP was examined by positron emission tomography (PET). Radioactivity accumulated most markedly in the neocortex and the striatum. Pharmacological characterization in vitro and in cynomolgus monkeys in vivo by PET indicated specific [11C]butylthio-TZTP binding to muscarinic receptors and to sigma-1 recognition sites. More than 5% of the radioactivity was in the human brain 5 min after i.v. injection of [11C]xamomeline or [11C]butylthio-TZTP. This high brain uptake may be clinically advantageous in the sense that substituted TZTP may induce central muscarinic agonist effects at a dose level for which there is a low risk of peripheral side-effects.

Anticonvulsant profile of the imidazoquinazolines NNC 14-0185 and NNC 14-0189 in rats and mice.

The anticonvulsant effects of NNC 14-0185 (3-(3-cyclopropyl-5-isoxazolyl)-6-fluoro-5-morpholino-imidazo[1,5- a] quinazoline) and NNC 14-0189 (3-(5-cyclopropyl-1,2, 4-oxadiazol-3-yl)-7-fluoro-5-(4-methyl-1-piperazinyl)-imidazo[1,5- a] quinazoline) in mice and rats were evaluated and compared with those of diazepam, clonazepam and the novel beta-carboline, abecarnil. Following i.p. administration, NNC 14-0185 and NNC 14-0189 prevented audiogenic seizures in DBA/2 mice and the clonic convulsions induced in mice by pentylenetetrazole, DMCM (methyl 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), 3-mercaptopropionic acid and a low dose of bicuculline. NNC 14-0185 and NNC 14-0189 prevented seizures induced by pentylenetetrazole in rats and were also effective anticonvulsants in amygdala-kindled rats. In general, the anticonvulsant potencies of NNC 14-0185 and NNC 14-0189 were comparable to those of the reference benzodiazepines. However, like abecarnil, they were not effective against the seizures induced in mice by maximal electroshock and a high dose of bicuculline. The anticonvulsant effects of NNC 14-0185 and NNC 14-0189 against pentylenetetrazole-induced seizures were apparent within 5 min of i.p. injection and persisted for at least 2 h. These effects appeared to be mediated by benzodiazepine receptors since they were inhibited by concurrent administration of flumazenil. Both NNC 14-0185 and NNC 14-0189 showed greater separation between their anticonvulsant and muscle relaxant effects (measured as impaired rotarod performance) than did diazepam. In this respect, their therapeutic windows were similar (NNC 14-0185) to or better (NNC 14-0189) than that of abecarnil. Tolerance did not develop to the anticonvulsant effects of NNC 14-0185 and NNC 14-0189 over a 4-day test. In comparison, the anticonvulsant effects of diazepam and abecarnil were attenuated by repeated drug administration. Thus, NNC 14-0185 and NNC 14-0189 have a promising anticonvulsant and side-effect profile in comparison with diazepam, clonazepam and abecarnil. The potential use of these compounds in the treatment of epilepsy should be explored further.

Synthesis and binding properties of [3H]NNC 12-0781, a new radioligand for the dopamine reuptake system.

The tritiated dopamine reuptake inhibitor [3H]NNC 12-0781 ([1-[2-(bis(4-fluorophenyl)-methoxy)-ethyl]-4-(3-(2-furanyl)-2,3-[3H] - propyl)-piperazine) was radiolabelled in one step starting from 1-[2-(bis(4-fluorophenyl)-methoxy)-ethyl]-4-(3-(2-furanyl)-2-propenyl)- piperazine, using tritium gas and PdO as catalyst. The radiochemical purity of [3H]NNC 12-0781 was higher than 99% after HPLC purification with a specific radioactivity of 21 Ci/mmol. [3H]NNC 12-0781 bound specifically to rat striatum in vitro at +4 degrees C with a Kd of 1.76 nM and Bmax of 587 fmol/mg tissue. The nonspecific binding was about 10% at Kd. At +37 degrees C no acceptable binding was observed. The association of [3H]NNC 12-0781 thus has the characteristics of a radioligand for the dopamine transporter in vitro at +4 degrees C.

The effect of lorazepam tolerance and withdrawal on metabotropic glutamate receptor function.

Mice were exposed to lorazepam (4 mg/kg) or vehicle by continuous infusion by implanted (s.c.) osmotic minipumps that were removed after 7 days. Dose-response curves for stimulation of phosphoinositide (PI) hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist, (1S,3R)-1-aminocyclopentane dicarboxylic acid [(1S,3R)-ACPD] were performed with cortical slices from mice treated with lorazepam or vehicle for 7 days and subject to 0, 1, 2, 3, 4 and 7 days of withdrawal. The efficacy of (1S,3R)-ACPD to stimulate PI hydrolysis was increased significantly at 2 and 3 days of lorazepam withdrawal when compared with responses in control slices. The effect was blocked by the mGluR antagonist, L-2-amino-3-phosphonopropionate (L-AP3). Enhancement of PI hydrolysis in cortical slices from mice at 2 days of discontinuation from 7 days exposure to lorazepam was also observed with agonists of alpha 1 adrenergic and histamine receptors, but not with agonists of muscarinic or serotonin receptors when compared with responses in control slices. Intracerebroventricular infusion of L-AP3 significantly increased pentylenetetrazol-seizure threshold in mice withdrawn for 2 days from 7 days of exposure to lorazepam, but showed no effect in comparable vehicle-exposed mice. These data suggest that PI-coupled mGluRs may be implicated in regulation of GABAergic functionality as observed after withdrawal from prolonged exposure to lorazepam.

A review of the preclinical pharmacology of tiagabine: a potent and selective anticonvulsant GABA uptake inhibitor.

We review the neurochemical and behavioral profile of the selective gamma-aminobutyric acid (GABA) uptake inhibitor, (R)-N-(4,4-di-(3-methylthien-2-yl)but-3-enyl) nipecotic acid hydrochloride [tiagabine (TGB), previously termed NNC 05-0328, NO 05-0328, and NO-328], which is currently in phase III clinical trials for epilepsy. TGB is a potent, and specific GABA uptake inhibitor. TGB lacks significant affinity for other neurotransmitter receptor binding sites and/or uptake sites. In electrophysiological experiments in hippocampal slices in culture, TGB prolonged the inhibitory postsynaptic potentials (IPSP) and inhibitory postsynaptic currents (IPSC) in the CA1 and CA3 produced by the addition of exogenous GABA. In vivo microdialysis shows that TGB also increases extracellular GABA overflow in a dose-dependent manner. Together these biochemical data suggest that the in vitro and in vivo mechanism of action of TGB is to inhibit GABA uptake specifically, resulting in an increase in GABAergic mediated inhibition in the brain. TGB is a potent anticonvulsant agent against methyl-6,7-dimethyoxy-4-ethyl-B-carboline-3-carboxylate (DMCM)-induced clonic convulsions (mice), subcutaneous pentylenetetrazol (PTZ)-induced tonic convulsions (mice and rats), sound-induced convulsions in DBA/2 mice and genetically epilepsy-prone rats (GEPR), and electrically induced convulsions in kindled rats. TGB is partially efficacious, against subcutaneous PTZ-induced clonic convulsions, and photically induced myoclonus in Papio papio. TGB is weakly efficacious in the intravenous PTZ seizure threshold test and the maximal electroshock seizure (MES) test and produces only partial protection against bicuculline (BIC)-induced convulsions in rats. The overall biochemical and anticonvulsant profile of TGB suggests potential utility in the treatment of chronic seizure disorders such as generalized clonic-tonic epilepsy (GTCS), photomyoclonic seizures, myoclonic petit mal epilepsy, and complex partial epilepsy.

Effects of chronic tiagabine treatment on [3H]GABAA, [3H]GABAB and [3H]tiagabine binding to sections from mice brain.

(R)-N-(4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl)nipecotic acid (tiagabine) is a potent inhibitor of gamma-aminobutyric acid (GABA) uptake, which maintains its initial anticonvulsant effects when administered to mice for a prolonged period (21 days). In the present study, mice received chronic (21 days) p.o. administration of tiagabine (15 mg/kg, twice daily) or vehicle alone and the densities of GABAA and GABAB receptors and of [3H]tiagabine recognition sites were measured in several brain regions. The following changes were observed following chronic administration of tiagabine as compared to vehicle: significant reduction (18-37%) in [3H]tiagabine binding in the temporal and entorhinal cortex and in the molecular and granular layer of the cerebellar cortex; increases in the number of GABAA sites (22-44%) in various regions of the frontal cortex, in caudate putamen and in the lateral septum; decreases in the numbers of GABAB sites (18-42%) in the motor cortex, the more dorsal parts of cortex, the anteroventral thalamic nucleus, medial geniculate, superior colliculus and the molecular layer of the cerebellar cortex. Such data suggest that the GABAergic system is differentially modulated in a regional specific manner in response to chronic elevation of the extracellular levels of GABA. The significance of these findings in relation to the reported lack of development of tolerance to the anticonvulsant effects of tiagabine is discussed.

Characterization of novel ligands for wild-type and natural mutant diazepam-insensitive benzodiazepine receptors.

A series of benzodiazepine receptor ligands with different chemical structures were evaluated for their affinities at diazepam-sensitive and diazepam-insensitive binding sites for [3H]Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5a][1,4] benzodiazepine-3-carboxylate) in cerebellar GABAA receptors. Rats of Wistar strain and of alcohol-sensitive (ANT) and alcohol-insensitive (AT) lines were used. The ANT rats possess a single point mutation in their GABAA receptor alpha 6 subunit, which makes their diazepam-insensitive sites sensitive to benzodiazepine agonists, unlike those of AT and Wistar rats. All compounds evaluated displayed high-affinity binding to diazepam-sensitive sites (Ki < 50 nM). In contrast, a wider range of affinities were observed at diazepam-insensitive sites which depended upon the basic structure and substitutions. The 7- and 8-halogen substituted imidazobenzodiazepines and 12-halogen substituted diimidazoquinazolines displayed the highest affinities (Ki < 15 nM), while intermediate to low affinities (100 < Ki < 4000 nM) were displayed by imidazoquinazolines, thienopyrimidines, one oxoimidazoquinoxaline, and some cyclopyrrolones. The imidazoquinoxalines evaluated displayed the lowest affinity (Ki > 10000 nM). The oxoimidazoquinoxaline, 6-chloro-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5-dihydro-5-isop ropyl-4-oxo-imidazo[1,5-a]quinoxaline (NNC 14-0578) and suriclone represent the first benzodiazepine receptor full agonists to bind with relatively high affinity (Ki approximately 100 nM) to diazepam-insensitive sites. The 5 position substituted methoxybenzyl, dimethylallyl, and 4-fluorobenzyl oxoimidazoquinoxaline analogs demonstrated a 58-336-fold higher affinity for ANT than AT diazepam-insensitive sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic agonists as analgesics. Antinociceptive activity versus M1 activity: SAR of alkylthio-TZTP's and related 1,2,5-thiadiazole analogs.

Alkylthio-TZTPs (3-(3-alkylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridines) and corresponding azabicyclic analogs were tested for m1 efficacy in cloned human m1 receptors and for antinociceptive activity in the mouse grid shock assay. The m1 (%PI) SAR were distinctly different from the analgesia and the salivation SAR, suggesting that analgesia is mediated by neither m1 nor M3 muscarinic receptors.

L-glutamate uptake inhibitors may stimulate phosphoinositide hydrolysis in baby hamster kidney cells expressing mGluR1a via heteroexchange with L-glutamate without direct activation of mGluR1a.

The functional efficacies of inhibitors of L-glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. L-Serine-O-sulfate was an agonist at mGluR1a (EC50 = 70 microM), mGluR2 (EC50 = 25 microM), and mGluR4 (EC50 = 324 microM). L-Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, L-cysteine, and DL-threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC50 values of 43, 64, 463, and 488 microM, respectively, and displaced L-[3H]glutamate binding from membranes prepared from these cells with respective IC50 values of 48, 44, 79, and 139 microM. However, D-aspartate, L-trans-pyrrolidine-2,4-dicarboxylate, L-threo-3-hydroxyaspartate, and L-aspartate-beta-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC50 values of 73, 54, 57, and 430 microM) but did not displace L-[3H]glutamate binding. These compounds inhibited Na(+)-dependent L-glutamate uptake into baby hamster kidney cells with IC50 values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this L-glutamate transporter, was significantly attenuated in the presence of L-glutamate decarboxylase (EC 4.1.1.15) or L-alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mM L-trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 +/- 0.2 microM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous L-glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.

Effects of bromohomoibotenate on metabotropic glutamate receptors.

(S)-Bromohomoibotenic acid [(S)-BrHIbo] stereoselectively antagonized glutamate-stimulated phosphoinositide (PI) hydrolysis in baby hamster kidney (BHK) cells expressing mGluR1a in a competitive manner with an IC50 of 250 microM. However, (S)-BrHIbo did not inhibit (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]-induced PI hydrolysis in rat hippocampal slices (S)- or (R)-BrHIbo did not show any effects on forskolin-stimulated cAMP-formation in BHK cells expressing mGluR2 or mGluR4 but did displace [3H]2-amino-4-phosphonobutyrate ([3H]AP4) binding from rat corticalmembranes with high affinities (IC50 = 1.0 microM and 1.1 microM, respectively). These data suggest that (S)-BrHIbo may interest with multiple PI-coupled glutamate receptors, however, at concentrations that are several fold higher than for displacement of [3H]AP4 binding from rat cortical membranes.