Evaluation and initial in vitro and ex vivo characterization of the potential positron emission tomography ligand, BU99008 (2-(4,5-dihydro-1H-imidazol-2-yl)-1- methyl-1H-indole), for the imidazoline2 binding site.

The density of the Imidazoline2 binding site (I2BS) has been shown to change in psychiatric conditions such as depression and addiction, along with neurodegenerative disorders such as Alzheimer's disease and Huntington's chorea. The presence of I2BS on glial cells and the possibility that they may in some way regulate glial fibrillary acidic protein has led to increased interest into the role of I2BS and I2BS ligands in conditions characterized by marked gliosis. In addition, it has been suggested that I2BS may be a marker for human glioblastomas. Therefore, the development of a positron emission tomography (PET) radioligand for the I2BS would be of major benefit in our understanding of these conditions. We now report the successful synthesis and initial pharmacological evaluation of potential PET radioligands for the I2BS as well as the tritiation and characterization of the most favorable of the series, BU99008 (6), both in vitro and ex vivo in rat. The series as a whole demonstrated excellent affinity and selectivity for the I2BS, with BU99008 (6) selected as the lead candidate to be taken forward for in vivo assessment. BU99008 (6) showed very good affinity for the I2BS (K(i) of 1.4 nM; K(d) = 1.3 nM), good selectivity compared with the α2 -adrenoceptor (909-fold). In addition, following peripheral administration, [³H]BU99008 demonstrated a heterogenous uptake into the rat brain consistent with the known distribution of the I2BS in vivo. This, and the amenability of BU99008 (6) to radiolabeling with a positron-emitting radioisotope, indicates its potential as a PET radioligand for imaging the I2BS in vivo.

Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors.

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.

BU74, a complex oripavine derivative with potent kappa opioid receptor agonism and delayed opioid antagonism.

In the search for opioid agonists with delayed antagonist actions as potential treatments for substance abuse, the bridged morphinan BU74 (17-cyclopropylmethyl-3-hydroxy-[5beta,7beta,3',5']-pyrrolidino-2'[S]-phenyl-7alpha-methyl-6,14-endoetheno morphinan) (3f) was synthesized. In isolated tissue and [35S]GTPgammaS opioid receptor functional assays BU74 was shown to be a potent long-lasting kappa opioid receptor agonist, delta opioid receptor partial agonist and mu opioid receptor antagonist. In antinociceptive tests in the mouse, BU74 showed high efficacy and potent kappa opioid receptor agonism. When its agonist action had waned BU74 became an antagonist of kappa and mu opioid receptor agonists in the tail flick assay and of delta, kappa and mu opioid receptor agonists in the acetic acid writhing assay. The slow onset, long-duration kappa opioid receptor agonist effects of BU74 suggests that it could be a lead compound for the discovery of a treatment for cocaine abuse.