Benztropine is a dopamine uptake inhibitor, equipotent to cocaine, but without appreciable abuse in humans or a cocaine-like behavioral profile in animal models. The goal of the current studies has been to identify optimum structural components for high affinity binding at the DAT, improve selectivity over the other monoamine transmitter transporters and reduce or eliminate muscarinic receptor binding, which for the parent compound was 100-fold more potent than at the DAT. Previously, the design, synthesis and in vitro evaluation of over 100 3-substituted and N-substituted BZT analogs led to the discovery of compounds with high affinity at the DAT (Ki=10-30 nM), selectivity over serotonin and norepinephrine transporters (100-2000-fold) and selectivity over muscarinic receptors (>50-fold). Recently we have discovered that chiral N-substituents, such as N-valine, provided enantioselectivity at the DAT that was opposite to that found at muscarinic sites. The result was a compound with 140-fold selectivity for DAT over muscarinic receptors which is the most selective compound in our series, thus far. Molecular modeling studies using Conformational Molecular Field Analysis (CoMFA) have been performed in the 3-substituted series of benztropine analogs as well as the N-substituted series. These 3D-Quantitative Structure Activity Relationship (QSAR) studies have provided highly predictive (q2=0.78) molecular models for the DAT and may be used in future drug design. Furthermore, these models have provided topological information about the binding site at which this series of dopamine uptake inhibitors interact. These studies, when compared to other studies in the cocaine-class of dopamine uptake inhibitors further support the divergence in SAR between these two classes of tropane-based DAT ligands and the likelihood that their binding sites on DAT are distinct. These studies have also led to the design and synthesis of a radioiodinated photoaffinity ligand which has been demonstrated to covalently attach to the DAT. Proteolytic and immunological peptide mapping studies have shown that this ligand labels a binding domain on the DAT that is near transmembrane spanning regions 1-2. This site appears to be similar to that labeled by the GBR 12935-based photoaffinity label [125I]DEEP and in contrast, is different from the binding domain of the cocaine-related photoaffinity label [125I]RTI 82. These studies are the first to demonstrate that these tropane-based photoaffinity labels covalently link to different binding sites on the DAT and support the divergent SAR reported by our group and others between these classes of dopamine uptake inhibitors. We have evaluated >20 BZT analogs as locomotor stimulants in mice and in a drug discrimination paradigm wherein rats are trained to discriminate 10 mg of cocaine from saline. With few exceptions, these BZT analogs have demonstrated behavioral profiles that were distinctive from cocaine. In general the BZT analogs were more potent at the DAT (in terms of binding affinity and potency for inhibition of dopamine uptake, in vitro) and were much more selective over the other monoamine transporters than cocaine. Our results suggest that either the inhibition of dopamine uptake may not be entirely responsible for the reinforcing effects of cocaine or that subtle differences in the binding interaction on the DAT can result in distinctive pharmacological profiles. Upon further behavioral evaluation, 3Cl and 4Cl BZT were found to be self administered in rhesus monkeys, trained to self administer cocaine. However, these compounds did not appear to be as strongly reinforcing as cocaine. The 3,4-diCl and the N-butylphenyl-4,4diF analogs were not self administered in this model. Various N-substituted analogs of the 4Cl, 3Cl, 4,4diCl and 3,4-diCl BZTs have recently been synthesized and are being evaluated both neurochemically and behaviorally in order to further probe the relationships between structure and function in this class of compounds. Taken together, it is anticipated that the combination of all these studies will allow the quantitation of the pharmacokinetics and pharmacodynamics of these compounds and will provide tools for the further examination of their divergent behavioral effects as compared to cocaine and their interactions with cocaine in these animal models. Additional computational drug design efforts at the DAT have been undertaken with the recent development of a 2D QSAR model. This model was initially developed using the genetic algorithms-partial least squares (GA-PLS) methodology with 70 structurally diverse DAT ligands, all tested for DAT binding in our laboratory. This highly predictive model (q2=0.69) has been used to screen the NCI database which contains >400,000 compounds. The model has successfully identified hits and these compounds have been obtained from NCI. Upon evaluation of these compounds in the DAT binding assay it is anticipated that a new lead for chemical modification will be identified. A second class of dopamine uptake inhibitors, based on rimcazole, have been explored. In order to better characterize the structural features that are required for DAT binding and to further assess whether or not these compounds are accessing a low affinity binding site on the DAT, a series of rimcazole analogs were prepared. Chemical modifications were made at the terminal piperazine nitrogen and at the carbazole ring structure. These compounds were evaluated in vitro for binding at the monoamine transporters and sigma receptors. The rimcazole analogs, like the parent compound, monophasically displaced [3H]WIN 35,428 from the DAT and inhibited dopamine uptake. The behavioral profile of the two most potent compounds, in this series, closely resembled the parent compound rimcazole, in animal models of cocaine abuse, despite structural similarity to GBR 12909. At this time it is unclear whether or not these rimcazole analogs bind to a binding domain on the DAT which is accessed by the GBR analogs. In order to further explore the binding domain of this class of compounds and to further investigate the role of sigma receptors in the behavioral actions of these compounds, an extended series of compounds are being prepared and evaluated. In an effort to further characterize the binding site of these rimcazole analogs, an N-alkylisothiocyanato-derivative of rimcazole has been discovered to be a novel irreversible ligand for the DAT. In addition, it appears that this analog has selectivity for the low affinity binding site, labeled by [3H]WIN 35,428. This finding suggests that this rimcazole analog may serve to discriminate functional correlates to the high and low affinity binding sites on the DAT. Additional binding studies in rat caudate putamen tissue as well as in a hDAT transfected cell line are being pursued. In addition, other potential irreversible ligands based on rimcazole and benztropine are being synthesized so as to compare the binding domains of these structurally diverse ligands at DAT. In summary, the development of compounds that are selective for the DAT will provide highly valuable tools with which to study this neurochemical target and its role in the reinforcing effects of cocaine. It is anticipated that as we elucidate the neurochemical mechanisms underlying the reinforcing effects of cocaine and compare those to drugs that have neurochemical similarities but behavioral differences, we will ultimately identify leads toward potential cocaine abuse pharmacotherapeutics.
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