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.

Synthesis and biochemical activity of novel amidine derivatives as m1 muscarinic receptor agonists.

As part of a continuing effort aimed at the development of selective, efficacious, and centrally active m1 muscarinic agonists for the treatment of Alzheimer's disease, a series of amide and hydrazide amidine derivatives (2a-e and 3b-d) was synthesized and examined for muscarinic agonist activity. Preliminary biochemical studies indicated that 2b, 2d, and 3d bound to muscarinic receptors in rat brain and stimulated phosphoinositide (PI) metabolism in rat cerebral cortex. Compounds 2b and 2d were also highly efficacious at m1 muscarinic receptors expressed in cultured A9 L cells. Molecular modeling studies suggest slightly different modes of interaction with m1 receptors for the ester and amide derivatives. Also, hydrogen-bond formation with a Thr residue may be important for m1 muscarinic agonist potency. The data suggest that the amide moiety can replace the ester group found in muscarinic agonists and provide further support for the utility of amidine derivatives in the development of efficacious m1 agonists.

Design, synthesis, and neurochemical evaluation of 2-amino-5-(alkoxycarbonyl)-3,4,5,6-tetrahydropyridines and 2-amino-5-(alkoxycarbonyl)-1,4,5,6-tetrahydropyrimidines as M1 muscarinic receptor agonists.

Four regioisomers of 2-amino-(methoxycarbonyl)-3,4,5,6-tetrahydropyridine (2a-5a) were synthesized as the racemates to evaluate the utility of exocyclic amidines in the development of novel agonists for M1 muscarinic receptors. Of the four regioisomers, only racemic 2-amino-5-(methoxycarbonyl)-3,4,5,6-tetrahydropyridine (4a; CDD-0075-A) displayed high affinity (IC50 = 10 +/- 3.0 microM) and activity at muscarinic receptors coupled to PI metabolism in the rat cortex (260 +/- 4.5% stimulation above basal levels at 100 microM). A series of 2-amino-5-(alkoxycarbonyl)-3,4,5,6-tetrahydropyridines then was synthesized for further evaluation as M1 agonists. Only the propargyl derivative (4d) retained substantial agonist activity (120 +/- 14% at 100 microM) in this series. On the basis of the activity of the 5-(alkoxycarbonyl)-1,4,5,6- tetrahydropyrimidines (1a and 1d) and the 2-amino-5-(alkoxycarbonyl)-3,4,5,6-tetrahydropyridines, the corresponding cyclic guanidine derivatives were synthesized and tested. 2-Amino-5-(methoxycarbonyl)-1,4,5,6-tetrahydropyrimidine (7a) displayed a modest affinity for muscarinic receptors in the CNS (22 +/- 5.3 microM) and an ability to stimulate PI turnover in rat cerebral cortex (81 +/- 16% at 100 microM). The propargyl derivative (7d) also had modest binding affinity (31 +/- 15 microM) and high activity (150 +/- 8.5% at 100 microM), as expected based on the activity of propargyl esters of 1,4,5,6-tetrahydropyrimidine and 2-amino-3,4,5,6-tetrahydropyridine. Computational chemical studies revealed five distinct minimum-energy conformations for 1a, (R)-4a, and 7a, and three for 1d, (R)-4d, and 7d, each with a unique orientation of the ester moiety. Each of the five conformations for 1a could be superimposed upon a unique conformer of (R)-4a and 7a, suggesting that the compounds interact with muscarinic receptors in a similar fashion. Taken together, the data indicate the general utility of amidine systems as suitable replacements for the ammonium group of acetylcholine in developing ligands with activity at M1 muscarinic receptors in the central nervous system. Such compounds might be useful in the treatment of patients with Alzheimer's disease.