Synthesis and structure-activity relationships of a series of aminopyridazine derivatives of gamma-aminobutyric acid acting as selective GABA-A antagonists.

We have recently shown that an arylaminopyridazine derivative of GABA, SR 95103 [2-(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride], is a selective and competitive GABA-A receptor antagonist. In order to further explore the structural requirements for GABA receptor affinity, we synthesized a series of 38 compounds by attaching various pyridazinic structures to GABA or GABA-like side chains. Most of the compounds displaced [3H]GABA from rat brain membranes. All the active compounds antagonized the GABA-elicited enhancement of [3H]diazepam binding, strongly suggesting that all these compounds are GABA-A receptor antagonists. None of the compounds that displaced [3H]GABA from rat brain membranes interacted with other GABA recognition sites (GABA-B receptor, GABA uptake binding site, glutamate decarboxylase, GABA-transaminase). They did not interact with the Cl- ionophore associated with the GABA-A receptor and did not interact with the benzodiazepine, strychnine, and glutamate binding sites. Thus, these compounds appear to be specific GABA-A receptor antagonists. In terms of structure-activity, it can be concluded that a GABA moiety bearing a positive charge is necessary for optimal GABA-A receptor recognition. Additional binding sites are tolerated only if they are part of a charge-delocalized amidinic or guanidinic system. If this delocalization is achieved by linking a butyric acid moiety to the N(2) nitrogen of a 3-aminopyridazine, GABA-antagonistic character is produced. The highest potency (approximately equal to 250 times bicuculline) was observed when an aromatic pi system, bearing electron-donating substituents, was present on the 6-position of the pyridazine ring.

Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site.

An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA), SR 95103, has been shown to be a selective antagonist of GABA at the GABAA receptor site. Subsequent structure-activity studies showed that suppressing the methyl in the 4-position of the pyridazine ring, and substituting the phenyl ring at the para position with a chlorine (SR 42641) or a methoxy group (SR 95531) led to compounds which exhibited the highest affinities for the GABA receptor site in this series. In the present study we examined the biochemical interaction of these compounds with the GABA receptor as well as their biochemical selectivity for this receptor. SR 95531 and SR 42641 displaced [3H]GABA from rat brain membranes with apparent Ki values of 0.15 microM and 0.28 microM respectively and Hill numbers near 1.0. The two compounds antagonized the GABA-elicited enhancement of [3H]diazepam-binding in a concentration-dependent manner without affecting [3H]diazepam-binding per se. Scatchard and Lineweaver-Burk analysis of the interaction of the two compounds with the GABAA receptor sites, revealed that the compounds were competitive at the high affinity site, but non-competitive at the low affinity site. Neither compound interacted with other GABAergic processes or with a variety of central receptor sites. When administered intravenously, SR 95531 and SR 42641 elicited tonic-clonic seizures in mice. Based on these results, it is postulated that SR 95531 and SR 42641 are specific, potent and competitive GABAA antagonists.

Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.

We describe the characteristics of SR 48692, a selective, nonpeptide antagonist of the neurotensin receptor. In vitro, this compound competitively inhibits 125I-labeled neurotensin binding to the high-affinity binding site present in brain tissue from various species with IC50 values of 0.99 +/- 0.14 nM (guinea pig), 4.0 +/- 0.4 nM (rat mesencephalic cells), 7.6 +/- 0.6 nM (COS-7 cells transfected with the cloned high-affinity rat brain receptor), 13.7 +/- 0.3 nM (newborn mouse brain), 17.8 +/- 0.9 nM (newborn human brain), 8.7 +/- 0.7 nM (adult human brain), and 30.3 +/- 1.5 nM (HT-29 cells). It also displaces 125I-labeled neurotensin from the low-affinity levocabastine-sensitive binding sites but at higher concentrations (34.8 +/- 8.3 nM for adult mouse brain and 82.0 +/- 7.4 nM for adult rat brain). In guinea pig striatal slices, SR 48692 blocks K(+)-evoked release of [3H]dopamine stimulated by neurotensin with a potency (IC50 = 0.46 +/- 0.02 nM) that correlates with its binding affinity. In a cell line derived from a human colon carcinoma (HT-29), SR 48692 competitively antagonizes neurotensin-induced intracellular Ca2+ mobilization with a pA2 (-log Kapp) values of 8.13 +/- 0.03, which is consistent with results obtained in binding studies. Moreover, SR 48692 is devoid of any intrinsic agonist activity. This compound is also active in vivo, since it reverses at low dose (80 micrograms/kg) the turning behavior induced by intrastriatal injection of neurotensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes.

Anticonvulsant activity of 4,9-dioxo-5,10-diazatetradecane (CM 40142), a new GABA derivative, in mice and photosensitive baboons).

The authors describe the anticonvulsant activity of a new gamma-amino-butyric acid (GABA) derivative in several animal models of generalized epilepsy including photoepileptic baboons. In all the studies, 4,9-dioxo-5,10-diazatetradecane (CM 40 142) revealed potencies against chemically, electrically and photic-induced seizures very similar to those observed with sodium valproate. In chemically elicited seizures in mice, CM 40142 exhibited a higher potency than sodium valproate in antagonizing anti-GABAergic agents. Although CM 40142 was synthesized as a compound which would cross the blood-brain barrier and liberate GABA within the central nervous system, preliminary biochemical investigations in mice failed to demonstrate a rise in brain GABA levels after treatment with CM 40142. Furthermore, CM 40142 increased spontaneous motility in mice at anticonvulsant doses. The data suggest that CM 40142 could be a broad spectrum nonsedative antiepileptic agent.

SR146131: a new potent, orally active, and selective nonpeptide cholecystokinin subtype 1 receptor agonist. II. In vivo pharmacological characterization.

SR146131 is a potent and selective agonist at cholecystokinin subtype 1 (CCK1) receptors in vitro. The present study evaluates the activity of the compound in vivo. SR146131 completely inhibited gastric and gallbladder emptying in mice (ED50 of 66 and 2.7 micrograms/kg p.o., respectively). SR146131 dose dependently reduced food intake in fasted rats (from 0.1 mg/kg p.o.), in nonfasted rats in which food intake had been highly stimulated by the administration of neuropeptide Y (1-36) (from 0.3 mg/kg p.o.), in fasted gerbils (from 0.1 mg/kg p.o.), and in marmosets maintained on a restricted diet (from 3 mg/kg p.o.). SR146131 (10 mg/kg p.o.) also increased the number of Fos-positive cells in the hypothalamic paraventricular nucleus of rats. Locomotor activity of mice was reduced by orally administered SR146131 (from 0.3 mg/kg p.o.). When administered intrastriatally, SR146131 elicited contralateral turning behavior in mice. Furthermore, orally administered SR146131 (0.3-10 mg/kg), also reduced the levels of cerebellar cyclic GMP. Finally, SR146131 (0.1 microgram/kg to 1 mg/kg, p.o.) significantly and dose dependently antagonized fluphenazine-induced mouth movements in rats. The CCK1 antagonist SR27897B prevented all the effects of SR146131. Conversely, SR146131 was unable to elicit any agonist or antagonist effects in a model of CCK2 receptor stimulation in vivo. SR146131 is a very potent and selective nonpeptide CCK1 agonist in vivo. SR146131 is more potent than any other CCK1 agonists reported to date. Because pharmacodynamic studies suggest that SR146131 should have a high absolute bioavailability, it may be a promising drug for the treatment of eating and motor disorders in humans.

SR146131: a new potent, orally active, and selective nonpeptide cholecystokinin subtype 1 receptor agonist. I. In vitro studies.

SR146131 inhibited the binding of [125I]-Bolton Hunter (BH)-sulfated cholecystokinin octapeptide (CCK-8S) for the human recombinant cholecystokinin subtype 1 (CCK1) receptor (IC50 = 0.56 nM) with high (300-fold) selectivity to the CCK2 receptor. The biological activity of SR146131 was characterized in vitro in a NIH-3T3 cell line expressing the human recombinant CCK1 receptor (3T3-hCCK1). Measuring intracellular calcium release, SR146131 behaved as a full agonist with an efficacy comparable with that of CCK-8S (EC50 = 1.38 +/- 0.06 nM). On individual cells, SR146131 induced, like CCK-8S, Ca2+ oscillations at subnanomolar concentrations and sustained responses at higher concentrations. Like CCK-8S, SR146131 also fully stimulated inositol monophosphate formation (EC50 = 18 +/- 4 nM). SR146131 partially activated mitogen-activated protein kinase and enhanced the expression of the immediate early gene krox 24. In the human CHP212 and IMR32 neuroblastoma cell lines, which constitutively express the CCK1 receptor, SR146131 behaved as a partial agonist on intracellular calcium release and inositol monophosphate formation. All of these effects of SR146131 were inhibited by the CCK1 receptor antagonists SR27897B and devazepide, suggesting that the effects of SR146131 were entirely mediated by the CCK1 receptor. In contrast, high concentrations (>1 microM) of SR146131 had only minimal effects on CCK-8S-stimulated and unstimulated Chinese hamster ovary (CHO) cells expressing the human CCK2 receptor, indicating that SR146131 is functionally inactive on the CCK2 receptor. In conclusion, these in vitro experiments show that SR146131 is a highly potent and selective agonist of the CCK1 receptor.