Synthesis and beta-adrenoceptor activity of the erythro and threo isomers of substituted alpha-hydroxytrimetoquinol.

The synthesis and pharmacological activity of erythro and threo isomers of 1-(3',4',5'-trimethoxy-alpha-hydroxy-benzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, 2 and 3, are reported. The structural assignments of 2 and 3 are based upon NMR spectra of the 6,7-dibenzyl precursors, 6 and 10, and of the synthetic derivatives of 13alpha- and 13beta-hydroxy-2,3-(dibenzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine, 8 and 12, respectively. The erythro isomer 2 was a more potent beta-adrenoceptor stimulant than the threo isomer 3 in guinea pig atrial, guinea pig tracheal, and rat adipocyte preparations. The differential activity of these compounds on lipolysis was favorably correlated to changes in the stimulation of adenylate cyclase activity and cAMP accumulation in rat adipocytes.

Selective and potent beta 2-adrenoceptor agents within the tetrahydroisoquinoline class: effect of methyl substitution at the benzylic carbon of the 1-(3,4,5-trimethoxybenzyl) group of trimetoquinol.

A systematic series of methyl (2 and 3) and dimethyl (4) analogues of trimetoquinol (1) were synthesized and evaluated for their beta 1 (atria) and beta 2 (trachea) and adrenoceptor activities. Structural assignments for the erythro (2) and the threo (3) diastereoisomers of 1-(3,4,5-trimethoxy-alpha-methylbenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were based on NMR spectra of the 6,7-dibenzyl precursors 15 and 16, respectively, and on the synthetic derivatives of cis- and trans-13-methyl-2,3-bis(benzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine (18 and 17). The rank order of beta 2-agonist activity for these compounds was 3 greater than 1 greater than 2 greater than 4. The rank order of activity as beta 1 agonists on the guinea pig atria is 1 greater than 3 greater than 2, and 4 was inactive. The methylated analogues show selectivity for beta 2 receptors in our preliminary pharmacological studies. The threo isomer 3 is the most potent and selective beta 2 stimulant reported to date in the tetrahydroisoquinoline class.

Inhibition of synaptosomal uptake of norepinephrine and dopamine by conformationally restricted sympathomimetic amines.

The conformationally restricted cis and trans isomer of substituted cyclobutanes were examined for their ability to inhibit 3H-norepinephrine and 3H-dopamine accumulation by synaptosomes prepared from the cortex and corpus striatum, respectively. The drugs were more effective in preventing the accumulation of 3H-norepinephrine by cortical synaptosomes than 3H-dopamine by striatal synaptosomes. However, in the synaptosomes isolated from both regions, the trans isomers were more potent inhibitors of accumulation than the cis isomers. The greatest stereoselectivity was exhibited by the isomers of 2-amino-1-phenylcyclobutanol. The accumulation of 3H-norepinephrine by cortical synaptosomes and the accumulation of 3H-dopamine by striatal synaptosomes were inhibited 50% by concentrations of the trans isomer of 7.4 X 10(-6) M and 1.7 X 10(-4) M, respectively. The cis isomer was inactive. In separate experiments, the releasing capabilities of the restricted analogs were determined by superfusing cortical and striatal synaptosomes labelled in vitro with 3H-catecholamines. The trans and cis isomers elicited a trivial release of 3H-norepinephrine and 3H-dopamine from cortical and striatal synaptosomes, respectively. The results indicate that the decreased synaptosomal accumulation of 3H-catecholamines caused by the analogs was due mainly to inhibition of uptake. The influence of dihydral angle between phenyl--NH2 on the inhibition of uptake is discussed. It is concluded that the anti conformation of sympathomimetic amines is the preferred conformation at the noradrenergic amine pump.

Binding of [3H]dihydroazapetine to alpha-adrenoreceptor-related proteins from rat vas deferens.

The potent alpha-adrenoreceptor blocking agent, azapetine, has been catalytically reduced with tritium gas to form [3H]dihydroazapetine. [3H]Dihydroazapetine retains significant ability to block alpha-adrenoreceptors and has been used as a ligand to study the receptor in a subcellular fraction containing membrane fragments from rat vas deferens. Specific binding of [3H]dihydroazapetine rapidly reaches equilibrium and is also reversible and saturable with a dissociation constant similar to that determined pharmacologically. The binding capacity is approximately 40 pmol/mg of protein. All alpha-adrenergic blockers tested were able to inhibit specific binding. High concentrations of alprenolol, atropine, or chlorpheniramine had no effect. In addition, all alpha-adrenergic agonists of the imidazoline class inhibit binding in low concentrations, whereas soterenol or carbamylcholine did not. There is good correlation (r=0.84) between blockade or stimulation of the receptor in intact tissues and inhibition of binding of [3H]dihydroazapetine to the subcellular fraction. These findings suggest that the fraction contains alpha-adrenoreceptor-related proteins. Alpha-adrenergic agonists structurally related to norepinephrine caused a stereoselective increase in binding in favor of the (-)-isomer, possibly reflecting an allosteric interaction at a different binding site on the receptor protein. The possibility of two different modes of binding for structurally dissimilar agonists is suggested.

Kinetics of accumulation, efflux and the pharmacological effects of tritiated dihydroazapetine on the rabbit aorta.

Azepatine, a potent alpha adrenergic antagonist, was catalytically reduced with tritium and hydrogen gas to form dihydroazapetine. The pA2 azapetine was 7.9 whereas that of dihydroazapetine was 6.6, corresponding to a 20-fold decrease in potency. 3H-dihydroazapetine is accumulated into three kinetically distinct compartment in the denervated rabbit aorta. Likewise, efflux of the labeled antagonist occurs from three compartments. The rat constant for onset of alpha adrenoreceptor blockade is 3.15 min-1 which is nearly identical to the rate constant for entry of 3H-dihydroazapetine into a rapidly filling compartment (3.86 min-1) possibly representing the extracellular space. These data are consistent with the hypothesis that onset of alpha adrenoreceptor blockade by dihydroazapetine is diffusion limited.

Synthesis and application of 13C enriched phenylisothiocyanate as a 13C NMR protein probe.

Phenylisothiocyanate, enriched with 13C at the isothiocyanate carbon, has been synthesized and utilized as a 13C NMR probe of proteins for the first time. The reagent has been used to label the amino groups of oxidized glutathione, and the resulting 13C NMR spectrum shows a prominent thiocarbonyl peak after a single NMR scan. The reagent is also capable of differentiating amino groups on the insulin molecule with distinct peaks corresponding to the amino groups on the A and B chains of insulin. This study illustrates the potential of using a new 13C label to functionalize amino groups of proteins and to study the labeled proteins with 13C NMR.

NMR studies of retinoid-protein interactions: the conformation of [13C]-beta-ionones bound to beta-lactoglobulin B.

PURPOSE: Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein beta-lactoglobulin B (beta-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. beta-LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle. METHODS: To examine the conformation of the model retinoid beta-ionone both in solution and when bound to beta-LG, NMR and computational methods have been employed. RESULTS: Taken together, NMR studies of beta-ionone in solution measuring scalar and dipolar coupling, as well as CHARMm calculations, suggest beta-ionone prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR studies of 13C-labeled beta-ionones bound to beta-LG, primarily employing the HMQC-NOE experiment, suggest beta-ionone also binds to beta-LG in its 6-s-cis conformation. CONCLUSIONS: The methods employed here allow estimates of protein-bound ligand conformation. However, additional sites of ligand labeling will be necessary to aid in binding site localization.