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

Abstract

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 D(3) receptors versus dopamine D(2) 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 [(3)H]WC-10 binding to genetically cloned dopamine D(2L) and D(3) receptors was evaluated in vitro. [(3)H]WC-10 binds with a 66-fold higher affinity to human HEK D(3) than HEK D(2L) receptors, with a dissociation constant (K(d)) of 1.2 nM at HEK D(3) receptors. However, [(3)H]WC-10 binds to rat Sf9 rD(3) receptors with a K(d) of 3.9 nM, a value that is 3-fold lower than binding to human HEK D(3) 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 [(3)H]WC-10 to D(3) receptors was in agreement with previously reported data. In vitro autoradiography studies of rat and monkey brains show that [(3)H]WC-10 labeled D(3) sites in the striatal region.

Fig. 1. Chemical structure of [³H]WC-10

The structure of [³H]WC-10 are shown, detailed synthesis scheme was reported previously (Xu et al., 2009).

Fig. 2. Saturation analysis of the binding of [³H]raclopride to cloned D₂-like dopamine receptors

Direct binding analysis was performed to determine the equilibrium binding affinity of [³H]raclopride for human hD₃ and hD₂ (A, B) or rat rD₃ and rD₂ (C, D) receptors. Human receptors were expressed in HEK 293 cells and the rat receptors were expressed in Sf9 cells. Scatchard plots were used to determine the dissociation constants (K_d values). The inset graphs are the Hill plots for determining the Hill coefficient (n_H values). K_d and n_H are presented as mean values ± S.E.M for n = 3.

Fig. 3. Saturation analysis of the binding of [³H]WC-10 to rhesus monkey and rat brain

Direct binding analysis was performed to determine the equilibrium binding affinity of [³H]WC-10 binding sites in the rhesus monkey caudate (graph A), putamen (graph B) and rat striatum (graph C). Nonspecific binding was determined from samples which contained 1 uM S(−)-Eticlopride. Scatchard plots were used to determine the dissociation constants (K_d values). The inset graphs are the Hill plots for determining the Hill coefficient (n_H values). K_d and n_H are presented as mean values ± S.E.M for n = 3.

Fig. 4. Quantitative autoradiographic analysis of the binding of [³H]WC-10 and [³H]raclopride to rat and rhesus monkey brain

Autoradiograms show neuroanatomical localization of [³H]WC-10 and [³H]raclopride binding sites in rhesus monkey (A, B) and Sprague-Dawley rat (C, D) brain sections. For this study [³H]WC-10 was used at a concentration of 4 nM (A, C) and [³H]raclopride was used at a concentration of 10 nM (B, D). The numbers 1 through 4 in panels A and C designate the following CNS anatomical regions: 1) cortex, 2) primate caudate, 3) primate putamen and 4) rat striatum. Panel E shows autoradiographic image of a [³H]Microscale, which was counted for 24 hours along with the brain sections for the purpose of quantitative analysis.

Fig. 5. Quantitative calibration of in vitro autoradiography

Calibrated autoradiography standard typical curve obtained by counting a series of tritium standards of a [³H]Microscale, digitalized image was used to analyze the region of interest. This curve was used to convert cpm/mm² to nCi/mg to quantify autoradiography.

Fig. 6. Film autoradiography of the binding of [³H]WC-10 to rat brain

Autoradiograms obtained with the traditional film exposure techniques with different exposure time, A. 1 month; B, 3 months. The numbers 1 and 2 designate the following CNS anatomical regions: 1) cortex, 2) rat striatum. For this study [³H]WC-10 was used at a concentration of 4 nM.