[³H]4-(dimethylamino)-N-[4-(4-(2-methoxyphenyl)piperazin-1-yl) butyl]benzamide, a selective radioligand for dopamine D₃ receptors. I. In vitro characterization

4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzamide (WC-10), a N-phenyl piperazine analog, has been shown to have high affinity and selectivity for dopamine D₃ receptors versus dopamine D₂ receptors (Chu et al. [2005] Bioorg Med Chem 13:77-87). In this study, WC-10 was radiolabeled with tritium (specific activity = 80 Ci/mmol) and [ ³H]WC-10 binding to genetically cloned dopamine D_2L and D₃ receptors was evaluated in vitro. [³H]WC-10 binds with a 66-fold higher affinity to human HEK D₃ than HEK D_2L receptors, with a dissociation constant (K_d) of 1.2 nM at HEK D ₃ receptors. However, [³H]WC-10 binds to rat Sf9 rD ₃ receptors with a Kd of 3.9 nM, a value that is 3-fold lower than binding to human HEK D₃ receptors and 40-fold value higher than binding to rat Sf9 rD_2L receptors. The K_d values obtained from saturation binding experiments were consistent with the results determined from kinetic (k_on and k_off) studies. The pharmacologic profiles of a series of dopaminergic drugs for inhibiting the binding of [ ³H]WC-10 to D₃ receptors was in agreement with previously reported data. In vitro autoradiography studies of rat and monkey brains show that [³H]WC-10 labeled D₃ sites in the striatal region.