Dopamine has been implicated as the primary neurotransmitter associated with the psychomotor stimulant and reinforcing effects of many drugs of abuse, such as cocaine, methamphetamine and the opioids. 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 these abued drugs. In this pursuit, the dopamine D3R subtype has been intensively targeted. However, definitive behavioral investigations have been hampered by the lack of highly selective D3R agonists and antagonists. We initially used the classic D3R antagonist NGB 2904 as the template for our structural modifications to elucidate SAR and develop novel and selective D3R antagonists and partial agonists with drug-like physicochemical properties. In binding studies, structure activity relationships (SAR )demonstrated that the trans-butenyl linker provided additional D3R 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 D3R 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 D3R, while decreasing lipophilicity. To this end we discovered very selective D3R antagonists and partial agonists with D3R/D2R-selectivites reaching 400-fold. In addition, several of these analogues have been further screened for binding in additional receptors and ion channels and did not show significant binding affinities at any of these other (off) targets, highlighting that these agents are some of the most potent and selective D3R-antagonists and partial agonists reported to date. Moreover, the (+)- and (-)-enantiomers of one of these 3-OH analogues, PG648, were synthesized and demonstrated enantioselectivity at D3R (>10-fold), but not significantly (<2-fold) at D2Rs. This was the first demonstration of enantioselectivity of a D3R antagonist and further chimera studies, with these enantiomers, identified an extracellular loop (E1) region that appears to differ between D3R and D2R. 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 D3R agents. One new series of analogues replaced the 3-OH group in the butyl linking chain with a F-group, and many of these compounds show favorable pharmacological profiles in vitro, with several compounds demonstrating D3R/D2R-selectivites >1000-fold. The enantiomers of our lead F-analogue, BAK2-66, were prepared, although robust enantioselectivity was not apparent. A chimera study showed the EL1 appears to play an important role in both binding and D3R selectivity. Recently, the D3R protein was crystallized and a computational model was created using the crystal coordinates. R-PG648 was docked in this D3R model and the homologous D2R model and significant binding domain differences have been identified that suggest the D3R has a secondary binding pocket that includes the extracellular loops EL1 and EL2. This distinction from D2Rs appears to be responsible for the binding selectivities of PG648 and its F-analogues. In an effort to further explore the role of the pharmacophoric components of R-PG648 in D3R binding selectivity and efficacy, we systematically deconstructed the full-length molecules into components that we refer to as synthons. We hypothesized that the 2,3-diCl- or 2-OCH3-substituted-4-phenylpiperazine terminus (head group) defined as the primary pharmacophore (PP), bind within the orthosteric binding site (OBS) of both the D2R and D3Rs, while the indole amide terminus termed as the secondary pharmacophore (SP), binds in a secondary binding pocket(SBP) at the interface of transmembrane domains (TMs) 1, 2, and 7 and the EL1, EL2, that significantly differ from the D2R. Site-directed mutagenesis studies have identified a single amino acid (Gly94) in the EL1 that differs between D2 and D3 receptors and is critically important for subtype selectivity. These studies have provided a structural basis for the contribution of the each component in these molecules to the binding and functional efficacy at D3R, and to the relative orientation of the primary and secondary pharmacophores for optimal D3R binding affinity, selectivity and efficacy. For example, when the D3R-selective antagonist, (R)-PG648, was docked into the D3R crystal structure, the 2,3-diCl-phenylpiperazine overlayed onto eticlopride and confirmed that this was the PP that binds to the OBS in the D3R. We reasoned that if we replaced the 2,3-diCl-phenylpiperazine with substitutions borrowed from the eticlopride structure that the new templates might serve as PPs with potentially improved D3R affinities, selectivities and/or metabolic stability. Hence, we designed a hybrid PP, using the 2-Cl substituent from the 2,3-dichlorophenylpiperazine and the 3-ethyl group that gives rise to high affinity binding at D2R and D3R to give 2-chloro-3-ethylphenylpiperazine. In addition, we incorporated another privileged D3R PP, the 2-methoxyphenylpiperazine, into our design to make the 3-chloro-5-ethyl-2-methoxyphenylpiperazine, which includes all but the 2-OH substituent from eticlopride and the 5-chloro-6-methoxy substituent found in both eticlopride and raclopride. Further, we explored the SP with different heteroaryl amides, as well as investigating the 2- or 3-OH substituted butyl linking chain. A series of 20 analogues, including their PPs, were synthesized. In vitro binding and functional profiles have been assessed and SAR developed that demonstrates these new PPs can yield highly selective D3R antagonists, (e.g. VK4-116) that show promising behavioral results in rodent models of oxycodone abuse. VK4-116 is also more metabolically stable than our previous lead compound, PG648, and appears to reduce acquisition to oxycodone self administration suggesting that it might be useful as a treatment for opioid dependence, but also may be useful in preventing prescription opioid addiction when long term treatment is necessary for chronic pain.
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