The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant actions. In order to understand further the molecular mechanisms underlying the pharmacological actions of cocaine, as well as mechanisms that underlie its 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 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 effected. 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 (+)-2-substituted-4',4"-diF BZTs and (2) novel N-substituted 4',4"-diF-BZTs, whose N-substituents were designed to be less lipophilic then previous generations of compounds were prepared. 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 supports 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. Nevertheless, for the first time, the (+)-2-substituted BZTs did demonstrate cocaine-like actions in both locomotor activity and drug discrimination. 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. We have also synthesized the first cysteine-selective MTS analog of MFZ 2-12, that is being used in combination with cysteine-directed mutagenesis studies of the DAT to characterize the binding domain of this cocaine-like irreversible ligand. 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. We found that substitution of the carbazole ring system of rimcazole with bis-(4'-fluorophenyl)amine improves binding affinity and selectivity for the DAT. The most potent compound in this series showed a DAT binding affinity (Ki=17.6 nM) comparable to GBR 12909. Despite high affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest that this compound behaves more like rimcazole than GBR 12909 and does not demonstrate a cocaine-like behavioral profile in mice. Another series of analogues were then prepared in which lipophilicity was decreased and binding affinity of the lead compound improved to Ki = 8.5 nM. These compounds have also been evaluated as sigma ligands and 3D-QSAR studies using CoMFA and comparing DAT and sigma receptor requirements have been undertaken. This series of compounds appear to have dual actions at both DAT and sigma, which may be exploited in the development of agents to treat cocaine abuse. 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 a series of mazindol analogues using the comparative molecular field analysis (CoMFA) method with their corresponding DAT binding affinities. The cross-validated CoMFA models were derived from a training set of 50 compounds, and the predictive ability of the resulting CoMFA models was evaluated against a test set of 21 compounds. A set of alignment rules was derived to superimpose these compounds onto a template structure, mazindol. These CoMFA models yielded significant cross-validated r2cv values. The best QSAR model was selected based on the predictive ability of the activity on the external test set of compounds. The analysis of coefficient contour maps provided further insight into the binding interactions of mazindol analogues with the DAT. A similar study was undertaken with a series of nicotinic receptor ligands, recently synthesized by Horti and colleagues (NIDA-IRP).