6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors.

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-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 M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.

6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors, and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6beta-carbomethoxy analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did 6beta-carbomethoxynortropane (13) and 6beta-acetoxytropane (4). In transfected CHO cells, 6beta-acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central alpha4beta2-nicotinic receptors with 6beta-carbomethoxynortropane (13) having the highest affinity, which was still 150-fold less than that of nicotine. 6beta-Acetoxynortropane (5) represents a potent muscarinic agonist with apparent selectivity toward M2-receptors.