Benztropine is a dopamine uptake inhibitor, equipotent to cocaine, but without appreciable abuse in humans or a cocaine-like behavioral profile in animal models. Benztropine's interesting neurochemical and behavioral profile, as well as its chemical structure, which shares some components (tropane-based ring) with cocaine and yet is different (lacks a 2-substituent and possesses a 3-diphenylmethoxy substituent) provided a promising lead compound. The goal of the initial studies was 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. 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). Molecular modeling studies using Conformational Molecular Field Analysis (CoMFA) have been performed that provide models for both the DAT and muscarinic receptors and demonstrate a divergence in the optimal structural components for these molecular targets as well as clues to further separability. These studies have also led to the 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. Behavioral evaluation of many of the BZT analogs have all demonstrated that, with few exceptions, despite high affinity and selectivity for the DAT, the BZTs are not efficacious locomotor stimulants. In vivo microdialysis studies on 4'Cl-BZT demonstrated that this compound, at a dose equivalent to cocaine (40 mg/kg, i.p.) significantly increased dopamine efflux over baseline, demonstrating that the drug penetrates the blood brain barrier and blocks dopamine uptake, in vivo. Despite this neurochemical profile, 4'Cl BZT demonstrated a behavioral profile that was distinctive from cocaine. Upon further behavioral evaluation, 3'Cl and 4'Cl 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',4""""""""diF analogs were not self administered in this model. A second class of dopamine uptake inhibitors, based on rimcazole, were discovred to bind to DAT equipotently to cocaine (Ki=100 nM) but these compounds are not efficacious locomotor stimulants. In order to better characterize the structural features that were required for DAT binding and to further assess whether or not these compounds were accessing a low affinity binding site on the DAT, an expanded series of rimcazole analogs were prepared where 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. SAR revealed that increased potency at the DAT, in general, resulted as the compounds more closely resembled GBR 12909. In addition, the rimcazole analogs, like the parent compound, monophasically displaced [3H]WIN 35,428 from the DAT and inhibited dopamine uptake. Further, 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. The role of sigma receptors and other neurochemical substrates for these analogs in their behavioral profile is currently under study. 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. Meperidine, a mu-opioid receptor agonist, binds to the DAT and exhibits exaggerated biphasic inhibition of dopamine uptake in a chopped tissue preparation, as compared to cocaine. Based on the neurochemical and behavioral profile of meperidine, it was suggested that this drug may be interacting primarily with a high affinity component or conformational state of the DAT that is responsible for its psychomotor stimulant actions. It was recognized that meperidine shared some structural features of WIN 35,065, a cocaine analog. Based on the observation that the tropane ring in WIN 35,065 provides a conformationally rigid piperidine, the structurally rigid analogs of meperidine were initially targeted as starting points for SAR at the DAT. The tropane-ring based meperidine analogs, where the piperidine ring is set in a chair conformation, were obtained commercially. However, the azabicyclo[2.2.1]heptane system, where the piperidine is set in a boat conformation, had to be prepared. Thus, a stereoselective and high yielding synthesis of one of the stereoisomers was achieved and the synthesis of the opposite stereoisomer is underway.
Showing the most recent 10 out of 62 publications
