Synthesis of an affinity ligand (‘UPHIT’) for in vivo acylation of the κ-opioid receptor

Abstract

The isothiocyanate analog (1 S,2 S- trans-2-isothiocyanato-4,5-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective κ-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of κ-opioid receptors in vivo. The isothiocyanate ( 3a) which we have designated UPHIT and its enantiomer ( 3b) were synthesized in 3 steps starting from optically pure (1 S,2 S)-(+)- trans-2-pyrrolidinyl- N-methyl-cyclohexylamine ( 4a) and its enantiomer ( 4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1 S,2 S enantiomer 3a to κ receptors labelled by [ 3H]U69,593 was shown to occur with an IC 50 value of 25.92 ± 0.36 nM, whereas 827.42 ± 5.88 and 115.10 ± 1.23 nM were obtained for the IC 50 value of the 1 R,2 R enantiomer ( 3b) and (±)- 3 respectively. Intracerebroventricular (ICV) injection of 100 μg of (±)- 3 into guinea-pig brain followed by analysis of remaining κ-binding sites 24 h later revealed that (±)- 3 depleted 98% of the κ receptors that bind [ 3H]U69,593 and 40% of those that bind [ 3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the κ-opioid receptor.