N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamides as Selective Dopamine D3 Receptor Ligands: Critical Role of the Carboxamide Linker for D3 Receptor Selectivity

Abstract

N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)-butyl)-aryl carboxamides were prepared and evaluated for binding and function at dopamine D3 (D3R) and D2 receptors (D2R). In this series, we discovered some of the most D3R selective compounds reported to date, (e.g. 8d and 8j >1000-fold D3R-selective over D2R.) In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g. D3RKi for 15b = 393 v. for 8j = 2.6 nM) resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.

A Single Glycine in Extracellular Loop 1 Is the Critical Determinant for Pharmacological Specificity of Dopamine D2 and D3 Receptors

“…The current results detail the specific contribution of EL1 to the D3R over D2R selectivity of R-22 and C4 analog, and identify Gly94 as the critical residue in D3R for this selectivity, through its ability to modulate the size and shape of the SBP and allow the SP to bind in Ptm23. The combination of EL1 and EL2 led to a slightly greater D3R selectivity (Table 2), consistent with the higher affinity seen for the F-analog of R-22 in the D2R chimera that included both EL1 and EL2 of D3R (Banala et al, 2011). EL2 may act synergistically with EL1, given the extensive interactions in our models between EL1 and EL2 that involve divergent residues in D3R and D2R.…”

“…Similarly, modeling studies of the A 3 adenosine receptor in complex with an agonist containing rigid C2 extensions have suggested that an outward displacement of TM2 is required to accommodate such a bulky ligand (Tosh et al, 2012). Prior chimeric receptor and mutagenesis studies were consistent with a contribution of a region containing EL1 and, to a much lesser degree, EL2, in D3R over D2R selectivity for R-22 (Newman et al, 2009) and another highly D3R-selective F-analog of R-22 (Banala et al, 2011). The current results detail the specific contribution of EL1 to the D3R over D2R selectivity of R-22 and C4 analog, and identify Gly94 as the critical residue in D3R for this selectivity, through its ability to modulate the size and shape of the SBP and allow the SP to bind in Ptm23.…”

“…Wild-type human D3R and D2R were tagged with a signal peptide (Guo et al, 2003) followed by a hemagglutinin epitope and SNAP tag, and cloned into pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA) using standard molecular biology procedures. Chimeric receptors were generated starting with constructs provided by Robert Luedtke (University of North Texas Health Science Center, Fort Worth, TX) (Banala et al, 2011). The N terminus-TM1-intracellular loop 1 (NT-TM1-IL1) (residues 1-67), EL1 (residues 98-102), and EL2 (residues 173-185) of D2R alone or in combination were replaced with that of D3R (residues 1-62, 93-98, and 171-184, respectively).…”

“…These D3R-selective compounds are characterized by a 4-phenylpiperazine primary pharmacophore (PP) and an extended aryl amide secondary pharmacophore (SP) connected by a 4-carbon linking chain (Heidbreder and Newman, 2010). Previous structure-activity relationship studies have attributed the subtype selectivity for D3R over D2R to the substituents on the 4-phenylpiperazine, an extended aryl amide ring system, and the length and functionalization of the linking chain (Grundt et al, 2007;Newman et al, 2009;Banala et al, 2011). Based on the crystal structure of D3R, we recently demonstrated that D3R over D2R selectivity mainly arises from divergent interactions of the SP within a second binding pocket (SBP) lined by residues from TMs 1, 2, 3, and 7, and extracellular loops 1 and 2 (EL1 and EL2, respectively) (Newman et al, 2012a).…”

Discovery and Characterization of a G Protein–Biased Agonist That Inhibits β-Arrestin Recruitment to the D2 Dopamine Receptor

“…Control experiments determined that this PathHunter receptor construct will couple to G protein-mediated signaling with similar efficacy as an unmodified construct (data not shown). Assays were conducted, with minor modifications, as previously published by our laboratory (Banala et al, 2011;Bergman et al, 2013). In brief, CHO-K1 cells expressing D2R long isoform (DiscoveRx) were seeded in Cell Plating Media 2 (DiscoveRx) at a density of 2625 cells/well in 384-well black, clear-bottom plates.…”