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 Gαi-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., 5j, 5o) 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.

Synthesis, pharmacological characterization, and structure-activity relationship studies of small molecular agonists for the orphan GPR88 receptor.

GPR88 is an orphan G-protein-coupled receptor (GPCR) enriched in the striatum. Genetic deletion and gene expression studies have suggested that GPR88 plays an important role in the regulation of striatal functions and is implicated in psychiatric disorders. The signal transduction pathway and receptor functions of GPR88, however, are still largely unknown due to the lack of endogenous and synthetic ligands. In this paper, we report the synthesis of a GPR88 agonist 2-PCCA and its pure diastereomers, which were functionally characterized in both transiently and stably expressing GPR88 HEK293 cells. 2-PCCA inhibited isoproterenol-stimulated cAMP accumulation in a concentration-dependent manner in cells expressing GPR88 but not in the control cells, suggesting that the observed cAMP inhibition is mediated through GPR88 and that GPR88 is coupled to Gαi. 2-PCCA did not induce calcium mobilization in GPR88 cells, indicating no Gαq-mediated response. A structure-activity relationship (SAR) study of 2-PCCA was also conducted to explore the key structural features for GPR88 agonist activity.

Effects of p-chloroamphetamine on brain serotonin neurons.

p-Chloroamphetamine (PCA) is a useful pharmacologic tool for selectively increasing brain serotonin function acutely by release of serotonin into the synaptic cleft. PCA produces behavioral, neurochemical and neuroendocrine effects believed due to serotonin release after doses in the range of 0.5-5 mg/kg. At higher doses and at longer times, PCA causes depletion of brain serotonin. The mechanisms of this depletion are not well understood but require the serotonin uptake carrier. Antagonism of PCA-induced depletion of brain serotonin is a useful means of assessing the ability of a compound to block the serotonin uptake carrier on brain serotonin neurons. PCA can also be used as a neurotoxic agent to deplete brain serotonin in functional studies, apparently by destroying some serotonergic nerve terminals. Used in this way, PCA has an advantage over 5,6- and 5,7-dihydroxytryptamines in being effective by systemic injection, and it affects brain serotonergic projections with a different neuroanatomic specificity than the dihydroxytryptamines.

Analysis of p-chloroamphetamine and a side-chain monofluorinated analogue in rat brain.

p-Chloro-alpha-fluoromethylphenylethylamine (fluoro-p-chloroamphetamine) (FpCA) has been shown in acute studies to be a less potent depletor of the neurotransmitter amine 5-hydroxytryptamine (5-HT) in brain than is p-chloroamphetamine (pCA). Gas chromatographic assay procedures for FpCA and PCA have been developed in our laboratories and applied to preliminary measurements in brain tissue from rats injected intraperitoneally with pCA or FpCA. Groups of male Sprague-Dawley rats were injected with pCA (0.03 mmol/kg) or FpCA (0.05 or 0.1 mmol/kg) and killed 1, 2, or 4 hr later. The brains were analyzed for the halogenated amphetamines by gas chromatography with electron-capture detection (GC-ECD) following derivatization with pentafluorobenzenesulfonyl chloride (for pCA) or trichloroacetic anhydride (for FpCA). At the 0.05-mmol/kg dose, FpCA attained lower brain concentrations at 1, 2, and 4 hr after injection than did pCA at a considerably lower dose (0.03 mmol/kg). Even at the higher dose of FpCA used (0.10 mmol/kg), where concentrations of FpCA were higher than those of pCA initially, concentrations of FpCA had dropped below those of pCA by 4-hr. These preliminary results indicate that FpCA attains lower brain concentrations and is eliminated from the brain more rapidly than is the parent drug, pCA. However, differences in potency between FpCA and pCA on 5-HT depletion cannot be explained fully on the basis of obtained brain levels of the drug as even at time intervals where FpCA levels were higher than or equal to those of pCA, there was less depletion of 5-HT by the former drug.