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The human Trace Amine-Associated Receptor subtype 1 (hTAAR1) is a G protein-coupled receptor that has therapeutic potential for multiple diseases, including schizophrenia, drug addiction, and Parkinson’s Disease. Although several potent agonists have been identified and have shown positive results in various clinical trials for schizophrenia, the discovery of potent hTAAR1 antagonists remains elusive. Herein, we report the results of structure-activity relationship studies that have led to the discovery of a potent hTAAR1 antagonist (RTI-7470–44, 34). RTI-7470–44 exhibited an IC(50) of 8.4 nM in an in vitro cAMP functional assay, a K(i) of 0.3 nM in a radioligand binding assay, and showed species selectivity for hTAAR1 over the rat and mouse orthologues. RTI-7470–44 displayed good blood-brain barrier permeability, moderate metabolic stability, and a favorable preliminary off-target profile. Finally, RTI-7470–44 increased the spontaneous firing rate of mouse VTA dopaminergic neurons and blocked the effects of the known TAAR1 agonist RO5166017. Collectively, this work provides a promising hTAAR1 antagonist probe that can be used to study TAAR1 pharmacology and potential therapeutic role in hypodopaminergic diseases such as Parkinson’s Disease.

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and [35S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940 binding assay, indicating greater CB1 receptor affinity and/or cooperativity.

Orexins are hypothalamic neuropeptides playing important roles in many functions including the motivation of addictive behaviors. Blockade of the orexin-1 receptor has been suggested as a potential strategy for the treatment of drug addiction. We have previously reported OX1 receptor antagonists based on the tetrahydroisoquinoline scaffold with excellent OX1 potency and selectivity; however, these compounds had high lipophilicity (clogP > 5) and low to moderate solubility. In an effort to improve their properties, we have designed and synthesized a series of analogs where the 7-position substituents known to favor OX1 potency and selectivity were retained, and groups of different nature were introduced at the 1-position where substitution was generally tolerated as demonstrated in previous studies. Compound 44 with lower lipophilicity (clogP = 3.07) displayed excellent OX1 potency (Ke = 5.7 nM) and selectivity (> 1,760-fold over OX2) in calcium mobilization assays. In preliminary ADME studies, 44 showed excellent kinetic solubility (> 200 μM), good CNS permeability (Papp = 14.7 × 10−6 cm/sec in MDCK assay), and low drug efflux (efflux ratio = 3.3).

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB(1) receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB(1) negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB(1) allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB(1) agonist CP55,940 stimulated calcium mobilization and [(35)S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [(3)H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [(3)H]CP55,940 and decrease binding of the antagonist [(3)H]SR141716. In saturation binding studies, only increases in [(3)H]CP55,940 B(max), but not K(d), were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [(35)S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [(3)H]CP5,5940 binding assay, indicating greater CB(1) receptor affinity and/or cooperativity.

Selective blockade of the orexin-1 receptor (OX1) has been suggested as a potential approach to drug addiction therapy because of its role in modulating the brain's reward system. We have recently reported a series of tetrahydroisoquinoline-based OX1 selective antagonists. Aimed at elucidating structure-activity relationship requirements in other regions of the molecule and further enhancing OX1 potency and selectivity, we have designed and synthesized a series of analogues bearing a variety of substituents at the 1-position of the tetrahydroisoquinoline. The results show that an optimally substituted benzyl group is required for activity at the OX1 receptor. Several compounds with improved potency and/or selectivity have been identified. When combined with structural modifications that were previously found to improve selectivity, we have identified compound 73 (RTIOX-251) with an apparent dissociation constant (Ke) of 16.1 nM at the OX1 receptor and620-fold selectivity over the OX2 receptor. In vivo, compound 73 was shown to block the development of locomotor sensitization to cocaine in rats.

The neuropeptide FF (NPFF) system has been implicated in a number of physiological processes including modulating the pharmacological activity of opioid analgesics and several other classes of drugs of abuse. In this study, we report the discovery of a novel proline scaffold with antagonistic activity at the NPFF receptors through a high throughput screening campaign using a functional calcium mobilization assay. Focused structure-activity relationship studies on the initial hit 1 have resulted in several analogs with calcium mobilization potencies in the submicromolar range and modest selectivity for the NPFF1 receptor. Affinities and potencies of these compounds were confirmed in radioligand binding and functional cAMP assays. Two compounds, 16 and 33, had good solubility and blood-brain barrier permeability that fall within the range of CNS permeant candidates without the liability of being a P-glycoprotein substrate. Finally, both compounds reversed fentanyl-induced hyperalgesia in rats when administered intraperitoneally. Together, these results point to the potential of these proline analogs as promising NPFF receptor antagonists.

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.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.