Affinity ligands and related agents for brain muscarinic and nicotinic cholinergic receptors

This study describes the chemical synthesis and receptor binding characteristics of various affinity ligands and related ligands for brain muscarinic and nicotinic cholinergic receptors, including the 4-bromoacetamidobenzoic acid esters of dimethylaminoethanol (DMBAB) and choline (BABC) and 4-iodoacetamidobenzoylcholine (IABC). The reversible binding of [3H]3-quinuclidinylbenzilate ([3H]QNB) to calf brain membranes was inhibited in a concentration-dependent and saturable manner by DMBAB, BABC, and IABC with Ki values of 8 x 10(-7), 3 x 10(-7) and 8 x 10(-7) M, respectively; the Ki values for inhibition of reversible binding of the nicotinic ligand, [3H]methylcarbamylcholine ([3H]-MCC), were 1 x 10(-6), 6 x 10(-8), and 1 x 10(-6) M, respectively. The Ki values for irreversible inhibition of [3H]QNB binding were 8 x 10(-7), 1 x 10(-7), and 2 x 10(-7) M for DMBAB, BABC, and IABC, respectively, and for [3H]MCC binding, 8 x 10(-5), 1 x 10(-5), and 2 x 10(-5) M, respectively. Although DMBAB was found to inhibit the QNB-induced hyperactivity in mice, it did not antagonize the toxic or other pharmacologic effects of oxotremorine. Structure-activity studies with various non-affinity analogues of the 4-aminobenzoate ester of dimethylaminoethanol and choline revealed that removal of the NH2 moiety from the phenyl group increased affinity for the muscarinic but not the nicotinic cholinergic site, and quaternization of the ester side chain greatly increased affinity for the muscarinic site. Dimethylation of NH2 in 4-aminobenzoylcholine decreased the affinity for both cholinergic sites. Replacement of NH2 by NO2 increased affinity for the muscarinic but not the nicotinic site, whereas quaternization of the 4-nitrobenzoyl ester markedly increased affinity for the nicotinic site while diminishing affinity for the muscarinic site. The findings indicate that DMBAB and its analogues are useful affinity ligands for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nanomolar concentration range.