The title compound ([ 3 H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K 2 CO 3 . Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164 Ci/mmol. The new radioligand was suitable for labeling human 5-HT 2A receptors in two heterologous cell lines and had about 20-fold higher affinity than [ 3 H]ketanserin.

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The in vitro and in vivo binding characteristics of [125I]iodomethyllycaconitine ([125I]iodoMLA) were determined in the rat. [125I]iodoMLA binding to rat cerebral cortex membranes was saturable and reversible and its specific binding represented approximately 70-80% of the total binding. [125I]iodoMLA labeled a single site with Kd = 1.8 +/- 0.4 nM and Bmax = 68 +/- 3 fmol/mg protein. Kinetic analysis revealed a t1/2 for association and dissociation of 10.5 +/- 3.1 and 10.3 +/- 1.6 min, respectively. Pharmacological characterization of [125I]iodoMLA binding indicated that it was specific for the alpha7 nAChR. In vitro brain region binding studies revealed greater binding in regions known to contain high numbers of alpha7 nAChRs. The analysis of the biodistribution of intravenously administered [125I]iodoMLA indicated that it was rapidly cleared and exhibited poor brain penetration; nevertheless, the levels of [125I]iodoMLA in alpha7 nAChR-rich target regions were significantly increased compared to the nontarget region (cerebellum) 60-120 min after administration. No metabolism of MLA by human liver S9 fraction was detected. Our results suggest that [125I]iodoMLA will be a useful radioligand to study the alpha7 nAChR in vitro and in vivo.

The nociceptin/orphanin FQ opioid peptide (NOP) receptor is a widely expressed GPCR involved in the modulation of pain, anxiety, and motor behaviors. Dissecting the functional properties of this receptor is limited by the lack of systemically active ligands that are brain permeant. The small molecule NOP receptor-selective, full agonist 8-[(1S,3aS)-2,3,3a,4,5,6-hexahydro- 1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198) hydrochloride is an active, brain penetrant ligand, but its difficult and cost-prohibitive synthesis limits its widespread use and availability for animal studies. Here, we detail a more efficient and convenient method of synthesis, and use both in vitro and in vivo pharmacological assays to fully characterize this ligand. Specifically, we characterize the pharmacodynamics of Ro 64-6198 in cAMP and G-protein coupling in vitro and examine, for the first time, the effects of nociceptin/orphanin FQ and Ro 64-6198 in arrestin recruitment assays. Further, we examine the effects of Ro 64-6198 on analgesia, anxiety, and locomotor responses in vivo. This new synthesis and pharmacological characterization provide additional insights into the useful, systemically active, NOP receptor agonist Ro 64-6198.