Discovery of 1,3-disubstituted pyrazole peripheral cannabinoid receptor partial agonists.

Partial agonists of peripheral cannabinoid receptors (CBRs) have potential therapeutic applications in several medical conditions. However, (-)-trans-Δ9-tetrahydrocannabinol (THC), the principal active component of marijuana, which is a partial agonist of CB1 and CB2 penetrates the central nervous system (CNS) and produces adverse effects. Peripherally restricted partial agonists of CBRs, particularly of CB1, can be used to treat illnesses safely and effectively with a better therapeutic index. Here, we report on our efforts to synthesize pyrazole partial CBR agonists with peripheral selectivity, resulting in lead compound 40. This compound is a potent partial agonist of CB1 with âˆ¼ 5-fold higher plasma biodistribution over brain and represents an early lead for optimization.

Synthesis and characterization of an orally bioavailable small molecule agonist of the apelin receptor.

The apelin receptor (APJ) is a target for cardiovascular indications. Previously, we had identified a novel pyrazole-based agonist 1 ((S)-N-(1-(cyclobutylamino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamide hydrochloride) of this GPCR. Systematic modification of 1 was performed to produce compounds with improved potency and ADME properties. Orally bioavailable compound 47 with favorable agonist potency (Ca2+EC50 = 24 nM, cAMPi EC50 = 6.5 nM) and pharmacokinetic properties (clearance ∼20 mL/min/kg in rats) was identified. This compound has vastly reduced brain penetration and is devoid of significant off-target liability. In summary, a potent and selective APJ agonist suitable for in vivo studies of APJ in peripheral tissues after oral administration has been identified.

Structure-Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59-3074.

Selective modulation of peripheral cannabinoid receptors (CBRs) has potential therapeutic applications in medical conditions, including obesity, diabetes, liver diseases, GI disorders and pain. While there have been considerable efforts to produce selective antagonists or full agonists of CBRs, there has been limited reports on the development of partial agonists. Partial agonists targeting peripheral CBRs may have desirable pharmacological profiles while not producing centrally mediated dissociative effects. Bayer reported that BAY 59-3074 is a CNS penetrant partial agonist of both CB1 and CB2 receptors with efficacy in rat models of neuropathic and inflammatory pain. In this report, we demonstrate our efforts to synthesize analogs that would favor peripheral selectivity, while maintaining partial agonism of CB1. Our efforts led to the identification of a novel compound, which is a partial agonist of the human CB1 (hCB1) receptor with vastly diminished brain exposure compared to BAY 59-3074.

Identification of potent pyrazole based APELIN receptor (APJ) agonists.

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC50 = 21.5 µM, Ki = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC50 = 0.800 µM, Ki = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC50 values of <100 nM. Recruitment of ß-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over ß-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.

Functionalized 6-(piperidin-1-yl)-8,9-diphenyl purines as inverse agonists of the CB1 receptor - SAR efforts towards selectivity and peripheralization.

Antagonists of type 1 cannabinoid receptors (CB1) may be useful in treating diabetes, hepatic disorders, and fibrosis. Otenabant (1) is a potent and selective CB1 inverse agonist that was under investigation as an anti-obesity agent, but its development was halted once adverse effects associated with another marketed inverse agonist rimonabant (2) became known. Non-tissue selective antagonists of CB1 that have high levels of brain penetration produce adverse effects in a small subset of patients including anxiety, depression and suicidal ideation. Currently, efforts are underway to produce compounds that have limited brain penetration. In this report, novel analogs of 1 are explored to develop and test strategies for peripheralization. The piperidine of 1 is studied as a linker, which is functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a connector in the form of an amine, amide, sulfonamide, sulfamide, carbamate, oxime, amidine, or guanidine. We also report more polar replacements for the 4-chlorophenyl group in the 9-position of the purine core, which improve calculated physical properties of the molecules. These studies resulted in compounds such as 75 that are potent inverse agonists of hCB1 with exceptional selectivity for hCB1 over hCB2. SAR studies revealed ways to adjust physical properties to limit brain exposure.

Synthesis and pharmacological characterization of functionalized 6-piperazin-1-yl-purines as cannabinoid receptor 1 (CB1) inverse agonists.

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki = 4 nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties.

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.

Pyrazole antagonists of the CB1 receptor with reduced brain penetration.

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse effects. In this study, novel analogs of rimonabant (1) were explored in which the 1-aminopiperidine group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further studied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharmacokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain penetration compared to rimonabant.

Discovery of a novel small molecule agonist scaffold for the APJ receptor.

The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attractive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists. Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC50 value of 21.5±5µM and binding affinity (Ki) of 5.2±0.5µM. Initial structure-activity studies afforded compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an EC50 value of 800±0.1nM and Ki of 1.3±0.3µM. Preliminary SAR, synthetic methodology, and in vitro pharmacological characterization indicate this scaffold will serve as a favorable starting point for further refinement of APJ potency and selectivity.

Modulation of pain in pediatric sickle cell disease: understanding the balance between endothelin mediated vasoconstriction and apelin mediated vasodilation.

Children with sickle cell disease (SCD) have painful vaso-occlusive episodes (VOEs), which often reoccur across the individual's lifespan. Vaso-constrictive and vaso-dilatory molecules have been hypothesized to play a role in VOEs. Endothelin-1 (ET-1) is a potent vasoconstrictor that is released during VOEs and is correlated with pain history. Apelin is a vaso-dilatory peptide that also has a modulatory role in pain processing. We hypothesize that the ratio between vaso-dilatory and vaso-constrictive tone in children with SCD may be a marker of pain sensitization and vaso-occlusion. Plasma endothelin and apelin levels were measured in 47 children with SCD. Procedural pain and baseline pain were assessed via child- and caregiver-reports and observational distress. Pain history was assessed using retrospective chart review. Plasma apelin was related to age, with decreased levels in older children. The ratio between apelin and ET-1 was negatively correlated to observational baseline pain. The ratio between apelin and Big ET was negatively correlated to caregiver ratings of baseline pain and positively correlated to history of VOEs, which is possibly due to hydroxyurea treatment. These results suggest that an imbalance in the apelin and endothelin systems may contribute to an increasing number of VOEs and baseline pain in children with SCD.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of the 3- and 4-methyl substituents.

The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure. In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs for antagonistic activity, compounds 4a-c, and 4d-f which have either the 3-methyl or both the 3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated for their in vitro opioid receptor antagonist activities using a [(35)S]GTPγS binding assay. Other ADME properties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective κ opioid receptor antagonists.

Identifying structural determinants of potency for analogs of apelin-13: integration of C-terminal truncation with structure-activity.

Apelin peptides function as endogenous ligands of the APJ receptor and have been implicated in a number of important biological processes. While several apelinergic peptides have been reported, apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe) remains the most commonly studied and reported ligand of APJ. This study examines the effect of C-terminal peptide truncations and comprehensive structure-activity relationship (SAR) for a series of analogs based on apelin-13 in an attempt to develop more potent and stable analogs. C-terminal truncation studies identified apelin-13 (N-acetyl 2-11) amide (9) as a potent agonist (EC50=4.4 nM). Comprehensive SAR studies also determined that Arg-2, Leu-5, Lys-8, Met-11, were key positions for determining agonist potency, whereas the hydrophobic volume of Lys-8 was a specific determinate of activity. Plasma stability studies on the truncated 10-mer peptide 28 (EC50=33 nM) indicated the primary sites of cleavage occurred between Nle-3 and Leu-4 and also between Ala-5 and Ala-6. These new ligands represent the shortest known apelin peptides with good functional potency.

Novel Peripherally Selective Cannabinoid Receptor 1 Neutral Antagonist Improves Metabolic Dysfunction-Associated Steatotic Liver Disease in Mice.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing globally. MASLD is characterized by clinically significant liver steatosis, and a subset of patients progress to more severe metabolic-disorder-associated steatohepatitis (MASH) with liver inflammation and fibrosis. Cannabinoid receptor 1 (CB1) antagonism is a proven therapeutic strategy for the treatment of the phenotypes that underlie MASLD, though work on early centrally penetrant compounds largely ceased following adverse psychiatric indications in humans. We present here preclinical testing of a CB1 neutral antagonist, N-[1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-phenylpiperidin-4l]methanesulfonamide (RTI-348), with minimal brain exposure when administered to mice. In a diet-induced model of MASLD-induced MASH, administration of RTI-348 decreased the total body and liver weight gain. Animals treated with RTI-348 showed reduced steatosis. Furthermore, they produced lower plasma alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), biomarkers associated with liver damage. Mice maintained on the MASH diet had elevated expression of genes associated with profibrogenesis, immune response, and extracellular matrix remodeling, and treatment with RTI-348 mitigated these diet-induced changes in gene expression. Using an intracranial electrical self-stimulation model, we also demonstrated that RTI-348 does not produce an anhedonia response, as seen with the first-generation CB1 inverse agonist rimonabant. Altogether, the results herein point to RTI-348 as a promising neutral antagonist for MASH.

The Monoacylglycerol Lipase Inhibitor ABX-1431 Does Not Improve Alcoholic Liver Disease.

Introduction: Excessive alcohol consumption can result in alcoholic liver disease (ALD). There is no FDA-approved drug to specifically treat ALD and current management approaches have limited efficacy. Past studies indicate that monoacylglycerol lipase (MAGL) inhibition can have a positive impact on nonalcoholic fatty liver disease. However, the effect of MAGL inhibition in ALD has not been reported. Materials and Methods: We tested the highly selective and clinically evaluated MAGL inhibitor ABX-1431 in the Lieber-DeCarli liquid alcohol diet-induced model of ALD in C57BL/6 mice. Results: ABX-1431 failed to reduce ALD-associated steatosis and elevated levels of liver enzymes associated with hepatic injury. Furthermore, survival rate declined with increasing doses of ABX-1431 when compared with mice administered vehicle only. Conclusion: These data suggest that MAGL inhibition does not improve ALD and is unlikely to be a good strategy for this condition.

Improvement of the Metabolic Stability of GPR88 Agonist RTI-13951-33: Design, Synthesis, and Biological Evaluation.

GPR88 is an orphan G protein-coupled receptor mainly expressed in the brain, whose endogenous ligand has not yet been identified. To elucidate GPR88 functions, our group has developed RTI-13951-33 (1b) as the first in vivo active GPR88 agonist, but its poor metabolic stability and moderate brain permeability remain to be further optimized. Here, we report the design, synthesis, and pharmacological characterization of a new series of RTI-13951-33 analogues with the aim of improving pharmacokinetic properties. As a result, we identified a highly potent GPR88 agonist RTI-122 (30a) (cAMP EC50 = 11 nM) with good metabolic stability (half-life of 5.8 h) and brain permeability (brain/plasma ratio of >1) in mice. Notably, RTI-122 was more effective than RTI-13951-33 in attenuating the binge-like alcohol drinking behavior in the drinking-in-the-dark paradigm. Collectively, our findings suggest that RTI-122 is a promising lead compound for drug discovery research of GPR88 agonists.

Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity.

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.

Peripherally Selective CB1 Receptor Antagonist Improves Symptoms of Metabolic Syndrome in Mice.

Metabolic syndrome (MetS) is a complex disorder that stems from the additive effects of multiple underlying causes such as obesity, insulin resistance, and chronic low-grade inflammation. The endocannabinoid system plays a central role in appetite regulation, energy balance, lipid metabolism, insulin sensitivity, and ß-cell function. The type 1 cannabinoid receptor (CB1R) antagonist SR141716A (rimonabant) showed promising antiobesity effects, but its use was discontinued due to adverse psychiatric events in some users. These adverse effects are due to antagonism of CB1R in the central nervous system (CNS). As such, CNS-sparing CB1R antagonists are presently being developed for various indications. In this study, we report that a recently described compound, 3-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[6-(difluoromethoxy)pyridin-3-yl]urea (RTI1092769), a pyrazole based weak inverse agonist/antagonist of CB1 with very limited brain exposure, improves MetS related complications. Treatment with RTI1092769 inhibited weight gain and improved glucose utilization in obese mice maintained on a high fat diet. Hepatic triglyceride content and steatosis significantly improved with treatment. These phenotypes were supported by improvement in several biomarkers associated with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). These results reinforce the idea that CB1 antagonists with limited brain exposure should be pursued for MetS and other important indications.

"Photo-Rimonabant": Synthesis and Biological Evaluation of Novel Photoswitchable Molecules Derived from Rimonabant Lead to a Highly Selective and Nanomolar "Cis-On" CB1R Antagonist.

Human cannabinoid receptor type 1 (hCB1R) plays important roles in the regulation of appetite and development of addictive behaviors. Herein, we describe the design, synthesis, photocharacterization, molecular docking, and in vitro characterization of "photo-rimonabant", i.e., azo-derivatives of the selective hCB1R antagonist SR1411716A (rimonabant). By applying azo-extension strategies, we yielded compound 16a, which shows marked affinity for CB1R (Ki (cis form) = 29 nM), whose potency increases by illumination with ultraviolet light (CB1R Kitrans/cis ratio = 15.3). Through radioligand binding, calcium mobilization, and cell luminescence assays, we established that 16a is highly selective for hCB1R over hCB2R. These selective antagonists can be valuable molecular tools for optical modulation of CBRs and better understanding of disorders associated with the endocannabinoid system.

Evaluation of Amide Bioisosteres Leading to 1,2,3-Triazole Containing Compounds as GPR88 Agonists: Design, Synthesis, and Structure-Activity Relationship Studies.

The orphan receptor GPR88 has been implicated in a number of striatal-associated disorders, yet its endogenous ligand has not been discovered. We have previously reported that the amine functionality in the 2-AMPP-derived GPR88 agonists can be replaced with an amide (e.g., 4) without losing activity. Later, we have found that the amide can be replaced with a bioisosteric 1,3,4-oxadiazole with improved potency. Here, we report a further study of amide bioisosteric replacement with a variety of azoles containing three heteroatoms, followed by a focused structure-activity relationship study, leading to the discovery of a series of novel 1,4-disubstituted 1H-1,2,3-triazoles as GPR88 agonists. Collectively, our medicinal chemistry efforts have resulted in a potent, efficacious, and brain-penetrant GPR88 agonist 53 (cAMP EC50 = 14 nM), which is a suitable probe to study GPR88 functions in the brain.

Aplnr knockout mice display sex-specific changes in conditioned fear.

The G-protein-coupled receptor APLNR and its ligands apelin and ELABELA/TODDLER/apela comprise the apelinergic system, a signaling pathway that is critical during development and physiological homeostasis. Targeted regulation of the receptor has been proposed to treat several important diseases including heart failure, pulmonary arterial hypertension and metabolic syndrome. The apelinergic system is widely expressed within the central nervous system (CNS). However, the role of this system in the CNS has not been completely elucidated. Utilizing an Aplnr knockout mouse model, we report here results from tests of sensory ability, locomotion, reward preference, social preference, learning and memory, and anxiety. We find that knockout of Aplnr leads to significant effects on acoustic startle response and sex-specific effects on conditioned fear responses without significant changes in baseline anxiety. In particular, male Aplnr knockout mice display enhanced context- and cue-dependent fear responses. Our results complement previous reports that exogenous Apelin administration reduced conditioned fear and freezing responses in rodent models, and future studies will explore the therapeutic benefit of APLNR-targeted drugs in rodent models of PTSD.