Structure-activity relationships for SNC80 and related compounds at cloned human delta and mu opioid receptors

The racemic compound (+/-)-BW373U86 ¿(+/-)-4-((alpha R*)- alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxy- benzyl)-N,N-diethylbenzamide dihydrochloride} is a potent delta opioid receptor agonist in the mouse vas deferens assay with little mu or kappa opioid receptor activity in the guinea pig ileum tissue preparation. In contrast, radioligand binding studies show that (+/-)-BW373U86 is only about 10-fold selective for delta over mu opioid receptors. Studies of the enantiomeric forms of (+/-)-BW373U86 and derivatives (SNC80 and related compounds) show that some of these isomers are significantly better in both receptor binding and pharmacological selectivity than (+/-)-BW373U86. In this study we have determined the binding affinities of 10 different SNC80-related compounds at cloned human delta and mu opioid receptors and measured the potency of SNC80 for the inhibition of forskolin-stimulated adenylyl cyclase. The most selective delta receptor ligand (SNC162) differed from SNC80 by the absence of the 3-methoxy substitution of the benzyl ring. The Ki for SNC162 at the delta receptor (0.625 nM) was over 8700-fold lower than that at the mu receptor (5500 nM), making this the most selective delta receptor ligand available. Reduction of the allyl side chain of SNC80 to produce radiolabeled [3H]SNC121 allowed direct measurement of the association and dissociation rate constants. SNC80 was 26-fold less potent than [D-Pen2, pCI-Phe4, D-Pen5]enkephalin in the delta receptor adenylyl cyclase inhibition assay, but showed full agonist activity with an EC50 value of 9.2 nM. The regulation of SNC80 binding affinity to the delta receptor by GTP analogs is undetectable in [3H]naltrindole binding inhibition studies, but direct binding studies with [3H]SNC121 in the presence of 100 microM 5'-guanylylimidotriphosphate show a 55% reduction in maximum binding site density consistent with a lower affinity for a part of the receptor population. Addition of 120 mM sodium chloride reduces SNC80 affinity nearly 40-fold in [3H]naltrindole binding inhibition studies. The results of these studies define specific structural features of these compounds responsible for opioid receptor interactions and suggest a possibly novel mechanism for delta receptor activation.