A series of 4'-substituted and 4',4"-disubstituted 3 alpha-(diphenylmethoxy)tropane analogs were prepared as novel probes for the dopamine transporter. These compounds were evaluated in radiolabeled binding assays for the dopamine, norepinephrine, and serotonin transporters. All of these compounds monophasically displaced [3H]WIN 35,428 binding in rat caudate putamen with Ki values ranging from 11.8 to 2000 nM. The most potent compound in this series was 4',4"-difluoro 3 alpha-(diphenylmethoxy)tropane 7c with a Ki = 11.8 nM. All of the compounds inhibited dopamine uptake in rat caudate putamen (IC50 = 24-4456 nM) which correlated significantly (r = 0.907; p > 0.0001) with binding affinities at the dopamine transporter. None of the compounds demonstrated high-affinity binding at the norepinephrine (Ki > 4800 nM) or serotonin (Ki > 690 nM) transporters. Therefore, the most potent dopamine uptake inhibitors in this series were highly selective for the dopamine transporter. Preliminary behavioral studies of several of these analogs (7a-e) suggested that the compounds did not display a cocaine-like behavioral profile, despite their ability to inhibit dopamine uptake. The present data coupled with the observed differences from cocaine in structure-activity relationships suggested that the 3 alpha-(diphenylmethoxy)tropane analogs may be interacting at a different active site than cocaine on the dopamine transporter and that an additional binding domain might be exploited for the identification of potential therapeutics for the treatment of cocaine abuse.

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Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

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