The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus or squirrel monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) will be directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics. In addition, the synthesis of two additional series (1) novel N-substituted 4',4""""""""-diF-BZTs, whose N-substituents were designed to be less lipophilic then previous generations of compounds and (2) novel 6,7-bridgehead-substituted BZTs to further explore SAR were prepared recently. In vitro evaluation of both series of compounds demonstrated that many were well tolerated at the DAT and showed both high affinity and selectivity for this target. SAR at the DAT further supported our previous findings wherein the BZTs do not share structural requirements nor the same structural modifications to optimize DAT binding, as compared to the cocaine class of tropane-based DAT inhibitors. Further investigation into correlating structure and pharmacological action of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Radioiodinated analogues of both azido (photoactivated) and isothiocyanato-derivatives of our tropane based DAT inhibitors have been synthesized and are currently being utilized in combination with proteolysis and MALDI MS to elucidate transmembrane domains at which these compounds bind covalently to both DAT and SERT. We have also synthesized high affinity fluorescent DAT ligands that are being used to characterize the DAT, at a cellular level. In another series of compounds, based on the sigma receptor antagonist, rimcazole, [3-cis-3,5-dimethyl-1-piperazinyl)alkyl] bis-(4'-fluorophenyl)amines were designed and synthesized. These compounds have been evaluated as both DAT and sigma receptor ligands. This series of compounds appear to have duaal actions at both DAT and sigma, which may be exploited in the development of agents to treat cocaine abuse. Currently, selected compounds are being evaluated for conditioned place preference in WT and DAT KO mice and in rats to further elucidate the mechanism of their actions. In addition to classical drug design and synthesis, we have continued our computational chemistry efforts with a three dimensional quantitative structure-activity relationship (3D-QSAR) study on both the rimcazole analogues as well as a series of 2-substituted 3-aryltropanes, using the comparative molecular field analysis (CoMFA) method with their corresponding DAT binding affinities. These studies have led to the design of novel 2-substituted tropane and rimcazole analogues with which we will continue to elucidate structure and function of the binding sites of the DAT.
Showing the most recent 10 out of 62 publications
