Azido- and isothiocyanato-substituted aryl pyrazoles bind covalently to the CB1 cannabinoid receptor and impair signal transduction

3-Azidophenyl- and 3-isothiocyanatophenyl- and 2-(5'-azidopentyl)- and 2-(5 '-isothiocyanatopentyl)pyrazoles were synthesized to determine whether thes e compounds could behave as covalently binding ligands for the CB1 cannabin oid receptor in rat brain membranes. Heterologous displacement of [H-3]CP55 940 indicated that the apparent affinity of these compounds for the CB1 rec eptor was similar to that of the parent compound, SR141716A, with the excep tion of the 3-isothiocyanato derivatives, which showed a 10-fold loss of af finity. The 3-azidophenyl and 3-isothiocyanatophenyl compounds behaved as a ntagonists against the cannabinoid agonist desacetyllevonantradol in activa tion of G proteins [guanosine 5'-O-(gamma-[S-35]thio)triphosphate ([S-35]GT P gamma S) binding] and regulation of adenylyl cyclase. The 2-(5'-azidopent yl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were poor antagonists for [S- 35]GTP gamma S binding, and both compounds failed to antagonize the cannabi noid regulation of adenylyl cyclase. After incubation with the isothiocyana to analogues or UV irradiation of the azido analogues, the 3-substituted ar yl pyrazoles formed covalent bonds with the CB1 receptor as evidenced by th e loss of specific binding of [H-3]CP55940. In the case of the isothiocyana to analogues, the log concentration-response curve for cannabinoid-stimulat ed [S-35]GTP gamma S binding was shifted to the right, indicating that loss of receptors compromised signal transduction capability. These irreversibl y binding antagonists might be useful tools for the investigation of tolera nce and receptor down-regulation in both in vitro and in vivo studies.