Dopamine has been implicated as the primary neurotransmitter associated with the psychomotor stimulant and reinforcing effects of cocaine. These findings have resulted in intensive efforts to characterize and elucidate the roles of the various dopamine receptor subtypes in the pharmacology and addiction liability of cocaine and other drugs of abuse. In this pursuit, the dopamine D3 receptor subtype has been intensively targeted. However, definitive behavioral investigations have been hampered by the lack of highly selective D3 agonists and antagonists. We have used the classic D3 antagonist NGB 2904 as the template for our structural modifications to elucidate SAR and develop novel and selective D3 antagonists and partial agonists with drug-like physicochemical properties. NGB 2904 blocks cocaine-induced reinstatement of drug seeking behavior, an animal model of relapse. All NGB 2904 analogues included either a 2,3-dichloro- or 2-methoxy-substituted-phenylpiperazine, a four carbon linking chain with varying saturation (butyl, hydroxybutyl, and trans butenyl) and a terminal arylcarboxamide. Evaluation for in vitro binding in HEK 293 cells transfected with human D2, D3, or D4 receptor cDNAs resulted in D3 binding affinities ranging from Ki=0.3-500 nM. The most potent and selective analogs in this series, initially demonstrated D3/D2 selectivity of >100 and a D3/D4 selectivity of >1000. Functional evaluation in vitro using a mitogenesis assay in D3 or D2 receptor transfected CHO cells demonstrated that these compounds were either potent antagonists or partial agonists at D3 receptors and were selective over D2 receptors, in this function. However, a functional comparison of a series of butenyl and saturated butyl analogues showed that these compounds generally exhibited higher intrinsic activity in the adenylate cyclase assay than in the mitogenesis assay, suggesting the potential of functional selectivity. Furthermore, structure-activity relationships (SAR) were deduced based on function, which was both instructive and provided additional functional data to be compared to in vivo activities for the identification of underlying mechanisms, at the G-protein level. In binding studies, SAR demonstrated that the trans-butenyl linker provided additional D3 selectivity as compared to the saturated linking chain. Moreover, addition of a hydroxy (OH) group in the 2- or 3-position of the butyl linker also gave several highly selective and potent D3 antagonists or partial agonists. Further, replacement of the sterically bulky aryl ring system with various heteroaryl groups served to retain high affinity and selectivity for D3, while decreasing lipophilicity. To this end we have recently discovered very selective D3 antagonists and partial agonists with D3/D2-selectivites reaching 400-fold. In addition, several of these analogues have been further screened for binding in 60 additional receptor and ion channel assays and did not show significant binding affinities at any of these other targets, highlighting that these agents are some of the most potent and selective D3-antagonists and partial agonists reported to date. Further, the (+)- and (-)-enantiomers of one of these 3-OH analogues, PG648, were synthesized and demonstrated enantioselectivity at D3 (>15-fold), but not significantly (<2-fold) at D2 receptors. This was the first demonstration of enantioselectivity of a D3 antagonist and further chimera studies, with these enantiomers, identified a extracellular loop (E2) region that appears to differ between D3 and D2. Further pharmacological characterization of these enantiomers and the synthesis of others are currently underway. The latter goal of reducing lipophilicity of the most potent agents was to improve physicochemical properties that would provide a more favorable pharmacokinetic/bioavailability profile than the currently existing D3 agents. Ten of these analogues are currently being evaluated for pharmacokinetics, blood brain barrier penetration, and for potential metabolic pathways for degradation in vivo, in rats. These compounds are all being tested in a D3-agonist induced yawning model, in rats, to compare their pharmacological and bioavailability profiles in vivo. In addition, several of the most potent and selective compounds of this series have been synthesized in multi-gram quantities and are currently being evaluated in numerous animal models of cocaine and methamphetamine abuse, in both rodents and primates. Chronic studies in these and additional rodent and monkey models of drug abuse and impulsivity are underway, with PG01037 and several other butenyl and hydroxy-butyl linked analogues. Our newest series of analogues replaces the 3-OH group in the butyl linking chain with a F-group, and several of these compounds show favorable pharmacological profiles in vitro, with several compounds demonstrating D3/D2-selectivites >1000-fold. Exceptionally high D2/D3 receptor affinities with some structural motifs have led to the hypothesis that our highly D3-selective ligands maybe bitopic and thus accessing both orthosteric and allosteric binding sites on the D3 receptor. Current synthesis and pharmacological assay development to test this hypothesis is underway. Recently, the D3 receptor protein was crystallized and a computational model was created using the crystal coordinates. R-PG 648 has been docked in this D3 receptor model and the homologous D2 receptor model and significant binding domain differences have been identified that suggest the D3 receptor has a secondary binding pocket that includes the extracellular loops E1 and E2 that is distinct from D2 and appears to be responsible for the binding selectivities of PG 648 and analogues thereof. Attempts to crystallize the D3 receptor with one of our novel D3-selective ligands are underway as well as numerous computation and empirical studies using D2/D3 chimeras, single point mutations and molecular modeling studies. In addition, we have embarked on an SAR study of novel D2 agonist sumanirole. It is anticipated that structural modification of this molecule will reveal important structural features that impart high affinity for D2 receptors. These studies will undoubtedly provide new insight into novel drug design.
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