6β-Acyloxy(nor)tropanes:  Affinities for Antagonist/Agonist Binding Sites on Transfected and Native Muscarinic Receptors

A series of esters of 6β-hydroxynortropane and the N-methyl analogue 6β-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m₁-, m₂-, m₃-, and m₄-muscarinic receptors in transfected cell membranes and on native M₁-muscarinic receptors in rat brain membranes and native M₂-muscarinic receptors in rat heart membranes. Most 6β-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m₁- and native M₁-receptors, indicative of agonist activity. 6β-Acetoxynortropane had K_i values versus oxotremorine-M binding at m₁-, m₂-, and m₄-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6β-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6β-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M₂-muscarinic receptors of heart, but not at transfected m₂-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m₃-receptors, since significant oxotremorine-M binding could not be detected. 6β-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m₄-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.