Design, synthesis, and neurochemical evaluation of 5-(3-alkyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines as M1 muscarinic receptor agonists.

A series of 5-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines+ ++ (7a-h) was synthesized for biological evaluation as selective agonists for M1 receptors coupled to phosphoinositide (PI) metabolism in the central nervous system. Each ligand bound with high affinity to muscarinic receptors from rat brain as measured by inhibition of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) binding. 5-(3-Methyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine+ ++ trifluoroacetate (CDD-0098-J;7a) displayed high affinity (IC50 = 2.7 +/- 0.69 microM) and efficacy at muscarinic receptors coupled to PI metabolism in the rat cortex and hippocampus. Increasing the length of the alkyl substituent increased affinity for muscarinic receptors yet decreased activity in PI turnover assays. The hippocampal PI response of 7a was blocked by lower concentrations of pirenzepine (8) or by higher concentrations of either AF-DX 116 (9) or p-fluorohexahydrosiladifenidol (10), suggesting that at low concentrations 7a selectively stimulates PI turnover through M1 receptors.

Synthesis and biological characterization of 1,4,5,6-tetrahydropyrimidine and 2-amino-3,4,5,6-tetrahydropyridine derivatives as selective m1 agonists.

Previous studies identified several novel tetrahydropyrimidine derivatives exhibiting muscarinic agonist activity in rat brain. Such compounds might be useful in treating cognitive and memory deficits associated with low acetylcholine levels, as found in Alzheimer's disease. To determine the molecular features of ligands important for binding and activity at muscarinic receptor subtypes, the series of tetrahydropyrimidines was extended. Several active compounds were examined further for functional selectivity through biochemical studies of muscarinic receptor activity using receptor subtypes expressed in cell lines. Several amidine derivatives displayed high efficacy at m1 receptors and lower activity at m3 receptors coupled to phosphoinositide (PI) metabolism in A9 L cells. Four ligands, including 1b, 1f, 2b, and 7b, exhibited marked functional selectivity for m1 vs m3 receptors. Compound 1f also exhibited low activity at m2 receptors coupled to the inhibition of adenylyl cyclase in A9 L cells. Molecular modeling studies also were initiated to help understand the nature of the interaction of muscarinic agonists with the m1 receptor using a nine amino model of the m1 receptor. Several important interactions were identified, including interactions between the ester moiety and Thr192. Additional interactions were found for oxadiazoles and alkynyl derivatives with Asn382, suggesting that enhanced potency and selectivity may be achieved by maximizing interactions with Asp105, Thr192, and Asn382. Taken together, the data indicate that several amidine derivatives display functional selectivity for m1 muscarinic receptors, warranting further evaluation as therapeutic agents for the treatment of Alzheimer's disease. In addition, several amino acid residues were identified as potential binding sites for m1 agonists. These data may be useful in directing efforts to develop even more selective m1 agonists.

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.