Novel Dopamine D3 Receptor Ligands

National Institute On Drug Abuse

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Abstract

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 abuse liability of this drug of abuse. In this pursuit, the dopamine D3 receptor subtype has been recently targeted. However, definitive behavioral investigations have been hampered by the lack of highly selective D3 agonists and antagonists. We have prepared the known D3 antagonist NGB 2904 in multi-gram quantities for acute and chronic behavioral testing. This compound demonstrated in vivo D3 antagonist actions and attenuated cocaine self-administration under a progressive ratio schedule. NGB 2904 also blocked cocaine-induced reinstatement of drug seeking behavior, an animal model of relapse. We then designed and synthesized novel series of compounds, based on NGB 2904. All the compounds 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 aryl amide. 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 analogs in this series, demonstrated D3/D2 selectivity of >200 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 provides 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 transmembrane region that appears to differ between D3 and D2. Further 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 physico-chemical 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. In addition, another of these highly selective 3-OH analogues, GCC 3-09 was recently tritiated. 3HGCC 3-09 is currently being developed as a potential radioligand for both rat brain tissue and cell-based binding assays. 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 vivo, with D3/D2-selectivites >200-fold. In addition, we have embarked on an SAR study of novel D2 antagonists, based on the parent ligand L741,626. Structural modification of this molecule has revealed important structural features that impart high affinity for D2 receptors, although improving D2/D3 selectivity has thus far remained elusive. Nevertheless, 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.