Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2‑PCCA: Synthesis, Structure–Activity Relationships, and Molecular Modeling Studies

GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal ganglia-associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS investigation is required. We previously reported that 2-PCCA was able to modulate GPR88-mediated cAMP production through a Gα_i-coupled pathway. Early structure–activity relationship (SAR) studies suggested that the aniline moiety of 2-PCCA is a suitable site for diverse modifications. Aimed at elucidating structural requirements in this region, we have designed and synthesized a series of analogues bearing a variety of substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., <b>5j</b>, <b>5o</b>) showed improved or comparable potency, but have lower lipophilicity than 2-PCCA (clogP 6.19). These compounds provide the basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR trends and supported the notion that 4′-substituents on the biphenyl ring exit through a largely hydrophobic binding site to the extracellular loop

Additional file 1: of Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88

Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88. (DOC 316 kb

Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake

The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (<b>6</b>). RTI-13951-33 exhibited an EC₅₀ of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1<i>R</i>,2<i>R</i>)-2-PCCA (<b>2</b>) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally

Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake

The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (<b>6</b>). RTI-13951-33 exhibited an EC₅₀ of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1<i>R</i>,2<i>R</i>)-2-PCCA (<b>2</b>) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally

Design, Synthesis, and Structure–Activity Relationship Studies of Novel GPR88 Agonists (4-Substituted-phenyl)acetamides Based on the Reversed Amide Scaffold

The development of synthetic agonists for the orphan receptor GPR88 has recently attracted significant interest, given the promise of GPR88 as a novel drug target for psychiatric and neurodegenerative disorders. Examination of structure–activity relationships of two known agonist scaffolds 2-PCCA and 2-AMPP, as well as the recently resolved cryo-EM structure of 2-PCCA-bound GPR88, led to the design of a new scaffold based on the “reversed amide” strategy of 2-AMPP. A series of novel (4-substituted-phenyl)acetamides were synthesized and assessed in cAMP accumulation assays as GPR88 agonists, which led to the discovery of several compounds with better or comparable potencies to 2-AMPP. Computational docking studies suggest that these novel GPR88 agonists bind to the same allosteric site of GPR88 that 2-PCCA occupies. Collectively, our findings provide structural insight and SAR requirement at the allosteric site of GPR88 and a new scaffold for further development of GPR88 allosteric agonists

Design, Synthesis, and Structure–Activity Relationship Studies of Novel GPR88 Agonists (4-Substituted-phenyl)acetamides Based on the Reversed Amide Scaffold

The development of synthetic agonists for the orphan receptor GPR88 has recently attracted significant interest, given the promise of GPR88 as a novel drug target for psychiatric and neurodegenerative disorders. Examination of structure–activity relationships of two known agonist scaffolds 2-PCCA and 2-AMPP, as well as the recently resolved cryo-EM structure of 2-PCCA-bound GPR88, led to the design of a new scaffold based on the “reversed amide” strategy of 2-AMPP. A series of novel (4-substituted-phenyl)acetamides were synthesized and assessed in cAMP accumulation assays as GPR88 agonists, which led to the discovery of several compounds with better or comparable potencies to 2-AMPP. Computational docking studies suggest that these novel GPR88 agonists bind to the same allosteric site of GPR88 that 2-PCCA occupies. Collectively, our findings provide structural insight and SAR requirement at the allosteric site of GPR88 and a new scaffold for further development of GPR88 allosteric agonists

Discovery of <i>N</i>‑{4-[(3-Hydroxyphenyl)-3-methylpiperazin-1-yl]methyl-2-methylpropyl}-4-phenoxybenzamide Analogues as Selective Kappa Opioid Receptor Antagonists

There is continuing interest in the discovery and development of new κ opioid receptor antagonists. We recently reported that N-substituted 3-methyl-4-(3-hydroxyphenyl)­piperazines were a new class of opioid receptor antagonists. In this study, we report the syntheses of two piperazine JDTic-like analogues. Evaluation of the two compounds in an in vitro [³⁵S]­GTPγS binding assay showed that neither compound showed the high potency and κ opioid receptor selectivity of JDTic. A library of compounds using the core scaffold <b>21</b> was synthesized and tested for their ability to inhibit [³⁵S]­GTPγS binding stimulated by the selective κ opioid agonist U69,593. These studies led to <i>N</i>-[(1<i>S</i>)-1-{[(3<i>S</i>)-4-(3-hydroxyphenyl)-3-methylpiperazin-1-yl]­methyl}-2-methylpropyl]-4-phenoxybenzamide (<b>11a</b>), a compound that showed good κ opioid receptor antagonist properties. An SAR study based on <b>11a</b> provided 28 novel analogues. Evaluation of these 28 compounds in the [³⁵S]­GTPγS binding assay showed that several of the analogues were potent and selective κ opioid receptor antagonists