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

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Abstract
The isothiocyanate analog (1S,2S‐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 (1S,2S)‐(+)‐trans‐2‐pyrrolidinyl‐N‐methyl‐cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to κ receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 ± 0.36 nM, whereas 827.42 ± 5.88 and 115.10 ± 1.23 nM were obtained for the IC50 value of the 1R,2R 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.