piperidines and 5-((4-(4-(diethylamino)butyl)-1-piperidinyl)acetyl)-10-11-dihydrobenzo(b-e)(1-4)diazepine-11-one

A series of new dibenzodiazepinone-type muscarinic receptor ligands, including two homo-dimeric compounds, was prepared. Sixteen representative compounds were characterized in equilibrium binding studies with [(3)H]N-methylscopolamine ([(3)H]NMS) at the muscarinic receptor subtype M2, and seven selected compounds were additionally investigated at M1, M3, M4 and M5 with respect to receptor subtype selectivity. The side chain of the known M2 preferring muscarinic receptor antagonist DIBA was widely varied with respect to chain length and type of the basic group (amine, imidazole, guanidine and piperazine). Most of the structural changes were well tolerated with respect to muscarinic receptor binding, determined by displacement of [(3)H]NMS. Compounds investigated at all subtypes shared a similar selectivity profile, which can be summarized as M2>M1≈M4>M3≈M5 (46, 50, 57, 62-64) and M2>M1≈M4>M3>M5 (1, 58). The homo-dimeric dibenzodiazepinone derivatives UNSW-MK250 (63) and UNSW-MK262 (64) exhibited the highest M2 receptor affinities (pIC50=9.0 and 9.2, respectively). At the M2 receptor a steep curve slope of -2 was found for the dimeric ligand 63, which cannot be described according to the law of mass action, suggesting a more complex mechanism of binding. In addition to equilibrium binding studies, for selected ligands, we determined pEC50,diss, an estimate of affinity to the allosteric site of M2 receptors occupied with [(3)H]NMS. Compounds 58 and 62-64 were capable of retarding [(3)H]NMS dissociation by a factor >10 (Emax,diss >92%), with highest potency (pEC50,diss=5.56) residing in the dimeric compound 64. As the monomeric counterpart of 64 was 100 times less potent (62: pEC50,diss=3.59), these data suggest that chemical dimerization of dibenzodiazepinone-type M receptor ligands can enhance allosteric binding.

Topics: Allosteric Site; Animals; Benzodiazepinones; Chemistry Techniques, Synthetic; CHO Cells; Cricetulus; Dimerization; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Ligands; N-Methylscopolamine; Piperidines; Radioligand Assay; Receptor, Muscarinic M2; Structure-Activity Relationship

When cells are acutely exposed to the oxonol dye, bis(1,3-dibutylbarbituric acid)pentamethine oxonol (diBA), at 0 degrees C, the concentration that gives half inhibition of Cl- exchange (IC50) is 0.146 +/- 0.013 microM (n = 12) initially, but the inhibition increases with time. These characteristics indicate that a rapid initial binding is followed by a slow conformational change that makes the binding tighter. If diBA is allowed to equilibrate with band 3, the IC50 is only 1.05 +/- 0.13 nM (n = 5), making diBA a more potent inhibitor than 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), for which the IC50 under similar conditions is 31 +/- 6 nM [T. Janas, P. J. Bjerrum, J. Brahm, and J. O. Wieth. Am. J. Physiol. 257 (Cell Physiol. 26): C601-C606, 1989]. Inhibition by diBA is very slowly reversible at 0 degrees C (t1/2 > 50 h), but the effect is more readily reversible at higher temperatures. DiBA competes with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) for inhibition, suggesting an external site of action. In contrast to DIDS and DNDS, however, increasing Cl- concentrations do not decrease the inhibitory effect of diBA, indicating that the inhibition is not competitive. Thus diBA may be useful for investigating conformational changes during anion exchange and for stopping transport without preventing substrate binding. However, when diBA and other oxonols are used to sense membrane potential, they may have undesirable side effects on anion transport processes.

Topics: Anion Exchange Protein 1, Erythrocyte; Anions; Benzodiazepinones; Chlorides; Humans; Ion Exchange; Models, Biological; Piperidines

A series of 5-[[[(dialkylamino)alkyl]-1-piperidinyl]acetyl]- 10,11-dihydro-5H-dibenzo[b,e][1,4]-diazepin-11-ones were prepared as potential M2-selective ligands. The compounds were evaluated for their affinity and selectivity for the muscarinic cholinergic receptor. The best M2-selective antimuscarinic agent studied is 5-[[4-[4-diethylamino)butyl]-1- piperidinyl]acetyl]-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11- one, which is approximately 10 times more potent at M2 receptors than previously known compounds such as 11-[[4-[4-(diethylamino)butyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one (AQ-RA 741).

Topics: Animals; Benzodiazepinones; Brain; Mice; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Structure-Activity Relationship

Although several m2-selective muscarinic antagonists have been described, they are not particularly potent. Thus, the development of potent m2-selective compounds remains an important goal. We now report that a bio-isoster of AQ-RA 741 is both one order of magnitude more potent and slightly more selective than previously described compounds. DIBA, a di-benzo derivative of AQ-RA 741, in which the pyridine of the tricycle is replaced with a benzene ring, had Ki values of 4, 0.3, 11 and 2 nM at m1 through m4 receptors, respectively. These values were determined in competition studies with [3H]N-methylscopolamine ([3H]NMS) in membranes from transfected A9 L cells (m1 and m3), rat heart (m2) and NG108-15 cells (m4). AQ-RA 741 had Ki values of 34, 4, 86 and 15 nM at each of these receptors. The autoradiographic distribution of DIBA binding sites was determined by competition studies of [3H]NMS in rat brain. At low concentration, DIBA reduced [3H]NMS binding most significantly from superior colliculi, thalamus, hypothalamus, pontine nucleus, and interpeduncular nucleus, and not appreciably from caudate nucleus, cerebral cortical regions, or hippocampus, consistent with its binding to m2 receptors. These data indicate that DIBA is the most potent, m2-selective muscarinic antagonist yet described. DIBA should therefore become a useful probe in future studies of muscarinic function.

Topics: Animals; Autoradiography; Benzodiazepinones; Brain; Cell Membrane; Male; Membranes; Molecular Structure; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic