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 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. 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 D3R binding affinities ranging from Ki=0.3-100 nM. The most potent and selective analogs in this series initially demonstrated D3R/D2R selectivity of >100 and a D3R/D4 selectivity of >1000. Functional evaluation in vitro using a mitogenesis assay in D3R or D2R transfected CHO cells demonstrated that these compounds were either potent antagonists or partial agonists at D3Rs and were selective over D2Rs, in this functional assay. In binding studies, 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 60 additional receptors and ion channels 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 D3R-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 D3R (>15-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. A subset of 15 compounds with high affinity (Ki<10 nM) at D3Rs and selectivity over D2Rs (>100-fold) are currently being tested in a D3R-agonist induced yawning model, in rats, to compare their pharmacological and bioavailability profiles in vivo. Several of these analogues have been evaluated for pharmacokinetics, blood brain barrier penetration, and for potential metabolic pathways for degradation in vivo, in rats. 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 non human primates. Chronic studies in these and additional models of drug abuse and impulsivity are underway, with PG01037, PG 619 and several other analogues. Our newest series of analogues replaces 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. A chimera study showed the EL1 appears to play an important role in both binding and D3R selectivity. Site-directed mutagenesis studies have identified a single amino acid in the EL1 that differs between D2 and D3 receptors and is critically important for subtype 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 the F analogues. Additional studies using D2R/D3R chimeras, single point mutations and molecular modeling are underway to characterize the binding domains of these compounds and identify drug molecule-protein interactions at the amino acid residue level. JJC 7-065 is a simple analogue of R-PG648, with the linking chain 3-OH group removed. As previously reported, the 3-OH substituent typically improves D3R selectivity, by significantly decreasing binding affinity at the D2R. JJC 7-082 is further modified by replacing the 2,3-diCl substitution of the pendant phenyl ring with a 2-OCH3 group, found in many classic D3R antagonists and partial agonists. As with JJC 7-065, D3R affinity is high, whereas D2R affinity is 100-fold lower. Thus, in an effort to further explore the role of the pharmacophoric components of R-PG648 and its close analogues JJC 7-065 and JJC 7-082 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 termini defined as the primary pharmacophore, bind within the orthosteric binding site of both the D2R and D3Rs, while the indole amide terminus termed as the second pharmacophore, binds in a second binding pocket at the interface of transmembrane domains (TMs) 1, 2, and 7 and the EL1, EL2, that significantly differ from the D2R. These studies have provided a structural basis for the contribution of the each component in these molecules to the binding and functional efficacy at the D3R, and to the relative orientation of the primary and secondary pharmacophores for optimal D3R binding affinity, selectivity and efficacy. In addition, we have embarked on an SAR study of novel analogues of the D2R agonist sumanirole. It is anticipated that structural modification of this molecule, coupled with analogous computational studies to the ones described above will reveal important structural features that impart high affinity for D2Rs. These studies will undoubtedly provide new insight into novel drug design.

Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2012
Total Cost
$1,505,719
Indirect Cost
Name
National Institute on Drug Abuse
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Kumar, Vivek; Moritz, Amy E; Keck, Thomas M et al. (2017) Synthesis and Pharmacological Characterization of Novel trans-Cyclopropylmethyl-Linked Bivalent Ligands That Exhibit Selectivity and Allosteric Pharmacology at the Dopamine D3 Receptor (D3R). J Med Chem 60:1478-1494
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Wang, Xiao-Fei; Bi, Guo-Hua; He, Yi et al. (2015) R-modafinil attenuates nicotine-taking and nicotine-seeking behavior in alcohol-preferring rats. Neuropsychopharmacology 40:1762-71
Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia et al. (2015) High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice. J Med Chem 58:6195-213
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