RESUMEN
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.
Asunto(s)
Agonistas Muscarínicos/síntesis química , Pirimidinas/química , Receptores Muscarínicos/metabolismo , Animales , Arecolina/farmacología , Encéfalo/metabolismo , Carbacol/farmacología , Línea Celular , Modelos Moleculares , Agonistas Muscarínicos/química , Agonistas Muscarínicos/metabolismo , Fosfatidilinositoles/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Quinuclidinil Bencilato/metabolismo , Ratas , Receptor Muscarínico M1 , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Amidinas/química , Receptores Muscarínicos/metabolismo , Amidinas/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Cinética , Modelos Moleculares , Quinuclidinil Bencilato/metabolismo , Ratas , Receptor Muscarínico M1RESUMEN
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.
Asunto(s)
Corteza Cerebral/metabolismo , Parasimpaticomiméticos/química , Fosfatidilinositoles/metabolismo , Piridinas/química , Pirimidinas/química , Receptores Muscarínicos/fisiología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Corteza Cerebral/efectos de los fármacos , Diseño de Fármacos , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Inositol/metabolismo , Isomerismo , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Parasimpaticomiméticos/síntesis química , Parasimpaticomiméticos/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Ratas , Receptores Muscarínicos/efectos de los fármacos , Espectrofotometría Infrarroja , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Oxadiazoles/síntesis química , Parasimpaticomiméticos/síntesis química , Pirimidinas/síntesis química , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Oxadiazoles/química , Oxadiazoles/farmacología , Parasimpaticomiméticos/química , Parasimpaticomiméticos/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Quinuclidinil Bencilato/metabolismo , Ratas , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Oxotremorina/análogos & derivados , Parasimpaticomiméticos/metabolismo , Pirrolidinas/metabolismo , Receptores Muscarínicos/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Autorradiografía , Encéfalo/metabolismo , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Cinética , Parasimpaticomiméticos/química , Pirrolidinas/química , Ratas , EstereoisomerismoRESUMEN
The relative proportions of the phospholipid fatty acids of erythrocyte membranes in mice were changed by chronic ethanol treatment and were not related to effects of the drug on nutrition, body temperature or experimental stress. Similar changes were observed using two different routes of ethanol administration and they did not reflect the metabolic effects of ethanol seen in the phospholipid fatty acids of whole liver. The observed increased content of saturated fatty acids and decreased content of polyunsaturated acids support the concept of adaptive changes taking place in the membrane during tolerance development to compensate for an increased membrane fluidity caused by ethanol. However, an increased content of the mono-unsaturated acid, octadecenoic (oleic), was found and there was no change in the cholesterol/phospholipid ratio. Other contrasting types of plasma membrane in mice showed different patterns of change in their phospholipid fatty acids during chronic ethanol administration. It is suggested that changes in membrane lipid composition could only partly account for an adaptation to ethanol-induced membrane disordering.