Development of 3-(4-Chlorophenyl)-1-(phenethyl)urea Analogues as Allosteric Modulators of the Cannabinoid Type-1 Receptor: RTICBM-189 is Brain Penetrant and Attenuates Reinstatement of Cocaine-Seeking Behavior.

We have shown that CB1 receptor negative allosteric modulators (NAMs) attenuated the reinstatement of cocaine-seeking behaviors in rats. In an effort to further define the structure-activity relationships and assess the druglike properties of the 3-(4-chlorophenyl)-1-(phenethyl)urea-based CB1 NAMs that we recently reported, we introduced substituents of different electronic properties and sizes to the phenethyl group and evaluated their potency in CB1 calcium mobilization, cAMP, and GTPγS assays. We found that 3-position substitutions such as Cl, F, and Me afforded enhanced CB1 potency, whereas 4-position analogues were generally less potent. The 3-chloro analogue (31, RTICBM-189) showed no activity at >50 protein targets and excellent brain permeation but relatively low metabolic stability in rat liver microsomes. Pharmacokinetic studies in rats confirmed the excellent brain exposure of 31 with a brain/plasma ratio Kp of 2.0. Importantly, intraperitoneal administration of 31 significantly and selectively attenuated the reinstatement of the cocaine-seeking behavior in rats without affecting locomotion.

Discovery of Arylsulfonamides as Dual Orexin Receptor Agonists.

Loss of orexin-producing neurons results in narcolepsy with cataplexy, and orexin agonists have been shown to increase wakefulness and alleviate narcolepsy symptoms in animal models. Several OX2R agonists have been reported but with little or no activity at OX1R. We conducted structure-activity relationship studies on the OX2R agonist YNT-185 (2) and discovered dual agonists such as RTOXA-43 (40) with EC50's of 24 nM at both OX2R and OX1R. Computational modeling studies based on the agonist-bound OX2R cryogenic electron microscopy structures showed that 40 bound in the same binding pocket and interactions of the pyridylmethyl group of 40 with OX1R may have contributed to its high OX1R potency. Intraperitoneal injection of 40 increased time awake, decreased time asleep, and increased sleep/wake consolidation in 12-month old mice. This work provides a promising dual small molecule agonist and supports development of orexin agonists as potential treatments for orexin-deficient disorders such as narcolepsy.

Rational design of cannabinoid type-1 receptor allosteric modulators: Org27569 and PSNCBAM-1 hybrids.

Allosteric modulation offers an alternate approach to target the cannabinoid type-1 receptor (CB1) for therapeutic benefits. Examination of the two widely studied prototypic CB1 negative allosteric modulators (NAMs) Org27569 and PSNCBAM-1 revealed structural resemblance and similar structure-activity relationships (SARs). In silico docking and dynamics simulation studies using the crystal structure of CB1 co-bound with CP55,940 and Org27569 suggested that Org27569 and PSNCBAM-1 occupied the same binding pocket and several common interactions were present in both series with the CB1 receptor. A new scaffold was therefore designed by merging the key structural features from the two series and the hybrids retained these binding features in the in silico docking studies. In addition, one such hybrid displayed similar functions to Org27569 in dynamic simulations by preserving a key R2143.50-D3386.30 salt bridge and maintaining an antagonist-like Helix3-Helix6 interhelical distance. Based on these results, a series of hybrids were synthesized and assessed in calcium mobilization, [35S]GTPγS binding and cAMP assays. Several compounds displayed comparable potencies to Org27569 and PSNCBAM-1 in these assays. This work offers new insight of the SAR requirement at the allosteric site of the CB1 receptor and provides a new scaffold that can be optimized for the development of future CB1 allosteric modulators.

Design, Synthesis, and Structure-Activity Relationship Studies of (4-Alkoxyphenyl)glycinamides and Bioisosteric 1,3,4-Oxadiazoles as GPR88 Agonists.

Increasing evidence implicates the orphan G protein-coupled receptor 88 (GPR88) in a number of striatal-associated disorders. In this study, we report the design and synthesis of a series of novel (4-alkoxyphenyl)glycinamides (e.g., 31) and the corresponding 1,3,4-oxadiazole bioisosteres derived from the 2-AMPP scaffold (1) as GPR88 agonists. The 5-amino-1,3,4-oxadiazole derivatives (84, 88-90) had significantly improved potency and lower lipophilicity compared to 2-AMPP. Compound 84 had an EC50 of 59 nM in the GPR88 overexpressing cell-based cAMP assay. In addition, 84 had an EC50 of 942 nM in the [35S]GTPγS binding assay using mouse striatal membranes but was inactive in membranes from GPR88 knockout mice, even at a concentration of 100 µM. In vivo pharmacokinetic testing of 90 in rats revealed that the 5-amino-1,3,4-oxadiazole analogues may have limited brain permeability. Taken together, these results provide the basis for further optimization to develop a suitable agonist to probe GPR88 functions in the brain.

Synthesis and Pharmacological Evaluation of 1-Phenyl-3-Thiophenylurea Derivatives as Cannabinoid Type-1 Receptor Allosteric Modulators.

We previously reported diarylurea derivatives as cannabinoid type-1 receptor (CB1) allosteric modulators, which were effective in attenuating cocaine-seeking behavior. Herein, we extended the structure-activity relationships of PSNCBAM-1 (2) at the central phenyl ring directly connected to the urea moiety. Replacement with a thiophene ring led to 11 with improved or comparable potencies in calcium mobilization, [35S]GTPγS binding, and cAMP assays, whereas substitution with nonaromatic rings led to significant attenuation of the modulatory activity. These compounds had no inverse agonism in [35S]GTPγS binding, a characteristic that is often thought to contribute to adverse psychiatric effects. While 11 had good metabolic stability in rat liver microsomes, it showed modest solubility and blood-brain barrier permeability. Compound 11 showed an insignificant attenuation of cocaine seeking behavior in rats, most likely due to its limited CNS penetration, suggesting that pharmacokinetics and distribution play a role in translating the in vitro efficacy to in vivo behavior.

Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation.

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.

Novel Diarylurea Based Allosteric Modulators of the Cannabinoid CB1 Receptor: Evaluation of Importance of 6-Pyrrolidinylpyridinyl Substitution.

Allosteric modulators of the cannabinoid CB1 receptor have recently been reported as an alternative approach to modulate the CB1 receptor for therapeutic benefits. In this study, we report the design and synthesis of a series of diarylureas derived from PSNCBAM-1 (2). Similar to 2, these diarylureas dose-dependently inhibited CP55,940-induced intracellular calcium mobilization and [35S]GTP-γ-S binding while enhancing [3H]CP55,940 binding to the CB1 receptor. Structure-activity relationship studies revealed that the pyridinyl ring of 2 could be replaced by other aromatic rings and the pyrrolidinyl ring is not required for CB1 allosteric modulation. 34 (RTICBM-74) had similar potencies as 2 in all in vitro assays but showed significantly improved metabolic stability to rat liver microsomes. More importantly, 34 was more effective than 2 in attenuating the reinstatement of extinguished cocaine-seeking behavior in rats, demonstrating the potential of this diarylurea series as promising candidates for the development of relapse treatment of cocaine addiction.

Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists.

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.

Identification of the first biased NPS receptor agonist that retains anxiolytic and memory promoting effects with reduced levels of locomotor stimulation.

The neuropeptide S system has been implicated in a number of centrally mediated behaviors including memory consolidation, anxiolysis, and increased locomotor activity. Characterization of these behaviors has been primarily accomplished using the endogenous 20AA peptide (NPS) that demonstrates relatively equal potency for the calcium mobilization and cAMP second messenger pathways at human and rodent NPS receptors. This study is the first to demonstrate that truncations of the NPS peptide provides small fragments that retain significant potency only at one of two single polymorphism variants known to alter NPSR function (NPSR-107I), yet demonstrate a strong level of bias for the calcium mobilization pathway over the cAMP pathway. We have also determined that the length of the truncated peptide correlates with the degree of bias for the calcium mobilization pathway. A modified tetrapeptide analog (4) has greatly attenuated hyperlocomotor stimulation in vivo but retains activity in assays that correlate with memory consolidation and anxiolytic activity. Analog 4 also has a bias for the calcium mobilization pathway, at the human and mouse receptor. This suggests that future agonist ligands for the NPS receptor having a bias for calcium mobilization over cAMP production will function as non-stimulatory anxiolytics that augment memory formation.

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

BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.

Design, synthesis and pharmacological evaluation of 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives as GPR88 agonists.

The orphan receptor GPR88 is an attractive therapeutic target because of its implications in a number of basal ganglia-associated disorders. To date, pharmacological characterization of GPR88 has been limited due to the lack of potent and selective agonists and antagonists appropriate for CNS investigations. We have previously reported that GPR88 couples to Gαi proteins and modulates cAMP levels upon treatment with a small molecule agonist 2-PCCA. Recently, another chemotype of GPR88 agonist, represented by 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-phenyl)ethyl)-2-phenylpropanamide], has also been discovered. In this report, a new series of 2-AMPP structurally related 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives have been designed and evaluated for agonist activity at GPR88. The structure-activity relationship (SAR) studies suggest that the amine group in 2-AMPP can be replaced by hydroxyl, ester and amide groups, resulting in analogues with good to moderate potency, whereas the phenyl group on the amide cap is essential for activity and has limited size, shape and electronic tolerance.