SYNTHESIS OF SUBSTITUTED S-CARBAMOYLECGONINE METHYL-ESTER ANALOGS - IRREVERSIBLE AND PHOTOAFFINITY LIGANDS FOR THE COCAINE RECEPTOR DOPAMINE TRANSPORTER

Photoaffinity ligands are useful tools for the isolation, purification , and characterization of proteins. As a step toward the goal of produ cing a photoaffinity probe for the dopamine transporter, isocyanato an d azido derivatives of 3-[(phenylcarbamoyl)oxy]ecgonine methyl ester w ere synthesized and tested for their ability to interact with the coca ine receptor of mammalian brain via two different assays. The ability of two isothiocyanato (N=C=S) (para and meta) and two azido (N-3) (par a and meta) derivatives, as well as (-)-cocaine, to inhibit [H-3]cocai ne binding and [H-3]dopamine uptake and to covalently interact with th e cocaine-binding site was tested. The p-N=C=S was the most potent, wi th IC50 values of 0.23 and 0.49 mu M for [H-3] cocaine binding and [H- 3] dopamine uptake. The m-N-3 and p-N-3 inhibited [3H]cocaine binding with IC50 values of 0.63 and 1.00 mu M and inhibited [H-3] dopamine up take with IC50 values of 5.08 and 1.32 mu M, respectively. Preincubati on of synaptosomal membranes with the m- or p-N=C=S isomer either in r educed lighting or under ultraviolet light followed by two washes resu lted in inhibition of 70% and 85% of [3H]cocaine binding, respectively , indicating the highly reactive properties of these compounds. After preincubation in reduced lighting, m-N-3 and p-N-3 inhibited 0% and 13 % of [H-3] cocaine binding, while following preincubation under ultrav iolet light, the inhibition increased to 61% and 68%, respectively. Th us, the isothiocyanato derivatives appear to bind irreversibly to the cocaine receptor in the presence or absence of ultraviolet light, wher eas the azido derivatives are photoreactive compounds which may prove useful in the purification of the receptor.