Identification of novel ligands for the gabapentin binding site on the alpha2delta subunit of a calcium channel and their evaluation as anticonvulsant agents.

As part of a program to investigate the structure-activity relationships of Gabapentin (Neurontin), a number of alkylated analogues were synthesized and evaluated in vitro for binding to the Gabapentin binding site located on the alpha2delta subunit of a calcium channel. A number of other bridged and heterocyclic analogues are also reported along with their in vitro data. Two compounds showing higher affinity than Gabapentin were selected for evaluation in an animal model of epilepsy. One of these compounds, cis-(1S,3R)-(1-(aminomethyl)-3-methylcyclohexyl)acetic acid hydrochloride (19), was shown to be effective in this model with a profile similar to that of Gabapentin itself.

[3H]PD 140376: a novel and highly selective antagonist radioligand for the cholecystokininB/gastrin receptor in guinea pig cerebral cortex and gastric mucosa.

The specific binding characteristics of the novel cholecystokinin (CCK)B/gastrin receptor-selective peptoid antagonist radioligand [3H]PD 140376 were investigated using membrane homogenates prepared from guinea pig cerebral cerebral cortex and gastric fundic mucosa. [3H]PD 140376 (0.01-10 nM) bound to both cerebral cortex and gastric gland homogenates with comparable high affinity (Kd, 0.1-0.2 nM) and to an apparent single population of sites with Bmax values of 119 and 296 fmol/mg of protein, respectively. The level of specific binding, defined as that displaced by unlabeled CCK sulfated octapeptide, was routinely between 60 and 70% in the cortex and between 50 and 60% in the fundic mucosa. Pharmacological characterization of the [3H]PD 140376-labeled binding sites with a series of agonist and antagonist ligands selective for each of the CCK receptor subtypes demonstrated, in both preparations, an affinity profile consistent with that of the CCKB/gastrin receptor. However, Hill slopes for the competition curves for the unlabeled agonist ligands against specific [3H]PD 140376 binding were significantly less than unity, whereas those for the antagonist ligands, including unlabeled PD 140376, were close to unity. The affinity and Hill slope for PD 140376 and the related CCKB/gastrin antagonist CI-988 were unaffected by the presence of the nonhydrolyzable GTP analogue guanylyl-5'-imidodiphosphate. In contrast, guanylyl-5'-imidodiphosphate caused a characteristic decrease in affinity and an increase in the Hill slopes towards unity for the agonist ligands CCK sulfated octapeptide and pentagastrin. The binding characteristics of unlabeled PD 140376 were also unaffected by the presence of the monovalent cation sodium. In conclusion, the present study has demonstrated that [3H]PD 140376 is the most potent and selective antagonist radioligand yet described for the characterization of CCKB/gastrin receptors in the central and peripheral nervous systems.

Tetrahydropyridyloxadiazoles: semirigid muscarinic ligands.

Recent studies have described novel azabicycle-based muscarinic agonists which readily penetrate into the central nervous system and are capable of displaying high efficacy at cortical sites. The current paper describes the synthesis and biochemical assessment of semirigid muscarinic ligands which were used to map the requirements of the cortical muscarinic receptor and to study the degree of conformational flexibility required to cause receptor activation. Analogues 6 and 9 provide high-efficacy muscarinic agonists at cortical sites; however, C-alkylation on the tetrahydropyridine ring resulted in more rigid analogues and showed lower predicted efficacy. Molecular mechanics calculations indicated a preference for the E rotameric form. This conformation was also observed in the X-ray crystal structure of ethenyloxadiazole 12. The new compounds were tested in a biochemical assay designed to measure receptor affinity and to predict cortical efficacy.

Synthesis and biological activity of 1,2,4-oxadiazole derivatives: highly potent and efficacious agonists for cortical muscarinic receptors.

The synthesis and biochemical evaluation of novel 1,2,4-oxadiazole-based muscarinic agonists which can readily penetrate into the CNS is reported. Efficacy and binding of these compounds are markedly influenced by the structure and physicochemical properties of the cationic head group. In a series of azabicyclic ligands efficacy and affinity are influenced by the size of the surface area presented to the receptor, at the active site, and the degree of conformational flexibility. The exo-1-azanorbornane 16a represents the optimum arrangement, and this compound is one of the most efficacious and potent muscarinic agonists known. In a series of isoquinuclidine based muscarinic agonists efficacy and affinity are influenced by the geometry between the cationic head.group and hydrogen bond acceptor pharmacophore and steric bulk in the vicinity of the base. The anti configuration represented by 22a is optimal for muscarinic activity. Ligands with pKa below 6.5 show poor binding to the muscarinic receptor as exemplified by the diazabicyclic derivative 42.

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells.

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.

Inotropic activities of imidazopyridines.

A series of 2-substituted 1H-imidazo[4,5-b]pyridines and the isomeric 1H-imidazo[4,5-c]pyridine derivatives has been prepared and evaluated as inotropic agents. The 1H-imidazo-[4,5-b] derivatives were found to be consistently more potent than their isomers in the [4,5-c] series in isolated guinea pig papillary muscle preparations. Structure-activity relationships and the species-dependence of inotropic potencies are discussed.