PROBES FOR NARCOTIC RECEPTOR MEDIATED PHENOMENA .17. SYNTHESIS AND EVALUATION OF A SERIES OF TRANS-3,4-DICHLORO-N-METHYL-N-[2-(1-PYRROLIDINYL)CYCLOHEXYL]BENZENEACETAMIDE (U50,488) RELATED ISOTHIOCYANATE DERIVATIVES AS OPIOID RECEPTOR AFFINITY LIGANDS

A series of U50,488 related isothiocyanates was synthesized from enantiomerically pure (S,S)-(+)-trans-2- pyrrolidinyl-N-methylcyclohexylamine {(+)-7} and (R,R)-(-)-trons-2-pyrrolidinyl-N-methylcyclohexylamine {(-)-7}. DCC coupling of (+)- and (-)-7 with nitrophenylacetic acids followed by catalytic hydrogenation and treatment with thiophosgene afforded a series of six isomeric aryl isothiocyanate analogues of U50,488. Similarly, DCC coupling of (+)- and (-)-7 with (+)- and (-)-N-t-Boc-protected phenylglycines afforded four isomeric alkyl isothiocyanates. Evaluation of the isothiocyanates for their capacity to produce wash-resistant inhibition of μ,δ and κ sites in vitro was performed using rat and guinea pig brain membranes. None of the compounds was able to irreversibly inhibit binding of [3H]bremazocine to guinea pig and rat brain membranes (depleted of functional μ and δ receptors by pretreatment with acylating agents BIT and FIT). However, (1S,2S)-trans-2-isothiocyanato-N-methyl-iV-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide [(—)-l] was able to specifically and irreversibly inhibit κ receptors labeled by [3H]-U69,593: Incubation of rat brain membranes for 60 min at 25 °C with 1 μM of (-)-1 resulted in a wash-resistant reduction of the binding to 11.2 ± 2.5% of the control. Binding analysis revealed the wash-resistant reduction in [3H]-U69,593 binding by (-)-1 to be through an increase in the Kdwithout effect on the Bmax. (-)-1 failed to effect μ or δ binding in rat or guinea pig brain under the same conditions. The enantiomer of (-)-1, (1R,2R)-trans-2- isothiocyanato-Ar-methyl-Af-[2-(l-pyrrolidinyl)cyclohexyl]benzeneacetamide [(+)-1], failed to affect ? receptors labeled by [3H]-U69,593 under the same conditions as for (~)-l. (1S,2S)-trans-3-Isothiocyanato-N-methyl-/V-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide [(—)-2] inhibited to 49.6 ± 5. 1% of the control, in a wash-resistant manner, κ receptors labeled by [3H]-U69,593. However, (-)-2 was not as selective as (-)-1 since it also reduced [3H]DADLE (δ) binding to 82.4 ± 8.0% of the control value. (1S,2S)-ira/is-4-Isothiocyanato-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide [(-)-3] exhibited selective wash-resistant inhibition of δ receptors labeled by [3H]DADLE resulting in a reduction in binding to 42.9 ± 4.2% of control. In the alkyl isothiocyanate series, (1S,2S)-trans-N- methyl-N-[2-(l-pyrrolidinyl)cyclohexyl]-(S)-2-phenyl-2-isothiocyanatoacetamide [(-)-11] also showed the capacity to selectively inhibit [3H]-U69,593-sensitive κ sites, resulting in a reduction in binding to 72.2 ± 2.54% of control at 1 μM while (+)-11 was inactive. None of the amino precursors (—)-8, (+)-8, (-)-9, (+)-9, H-10, (+)-10, (-)-15, (+)-15, (-)-16, and (+)-16 of the isothiocyanates exhibited the capacity for wash-resistant inhibition of any of the receptor systems tested. Although intracerebroventricular (icv) injection of the most potent compound (-)-1 into guinea pig brain failed to produce any irreversible inhibition of κ receptors, icv injection of the less potent (-)-2 into guinea pig brain resulted in a significant reduction in the κ receptors that bind [3H]-U69,593, but not those that bind [3H]bremazocine. All of the compounds that showed the capacity to irreversibly inhibit κ receptors labeled by [3H]-U69,593 in vitro possessed the 1S,2S absolute configuration. © 1990, American Chemical Society. All rights reserved.