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.

Pyrazole Agonist of the Apelin Receptor Improves Symptoms of Metabolic Syndrome in Mice.

Apelin receptor agonism improves symptoms of metabolic syndrome. However, endogenous apelin peptides have short half-lives, making their utility as potential drugs limited. Previously, we had identified a novel pyrazole-based agonist scaffold. Systematic modification of this scaffold was performed to produce compounds with improved ADME properties. Compound 13 with favorable agonist potency (cAMPi EC50 = 162 nM), human liver microsome stability (T1/2 = 62 min), and pharmacokinetic profile in rodents was identified. The compound was tested in a mouse model of diet-induced obesity (DIO) and metabolic syndrome for efficacy. Treatment with 13 led to significant weight loss, hypophagia, improved glucose utilization, reduced liver steatosis, and improvement of disease-associated biomarkers. In conclusion, a small-molecule agonist of the apelin receptor has been identified that is suitable for in vivo investigation of the apelinergic system in DIO and perhaps other diseases where this receptor has been implicated to play a role.

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.

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.

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists.

Discovery of a novel trans-1,4-dioxycyclohexane GPR119 agonist series.

The design and optimization of a novel trans-1,4-dioxycyclohexane GPR119 agonist series is described. A lead compound 21 was found to be a potent and efficacious GPR119 agonist across species, and possessed overall favorable pharmaceutical properties. Compound 21 demonstrated robust acute and chronic regulatory effects on glycemic parameters in the diabetic or non-diabetic rodent models.

Regulation of the Apelinergic System and Its Potential in Cardiovascular Disease: Peptides and Small Molecules as Tools for Discovery.

Apelin peptides and the apelin receptor represent a relatively new therapeutic axis for the potential treatment of cardiovascular disease. Several reports suggest apelin receptor activation with apelin peptides results in cardioprotection as noted through positive ionotropy, angiogenesis, reduction of mean arterial blood pressure, and apoptosis. Considering the potential therapeutic benefit attainable through modulation of the apelinergic system, research is expanding to develop novel therapies that limit the inherent rapid degradation of endogenous apelin peptides and produce metabolically stable small molecule agonists and antagonists to more rigorously interrogate the apelin receptor system. This review details the structure-activity relationships for chemically modified apelin peptides and recent disclosures of small molecule agonists and antagonists and summarizes the peer reviewed and patented literature. Development of metabolically stable ligands of apelin receptor and their effects in various models over the coming years will hopefully lead to establishment of this receptor as a validated target for cardiovascular indications.

Discovery of 1a,2,5,5a-tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalen-4-carboxamides as potent and selective CB2 receptor agonists.

The design and synthesis of novel 1a,2,5,5a-tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalen-4-carboxamide CB2 selective ligands for the potential treatment of pain is described. Compound (R,R)-25 has good balance between CB2 agonist potency and selectivity over CB1, and possesses overall favorable pharmaceutical properties. It also demonstrated robust in vivo efficacy mediated via CB2 activation in the rodent models of inflammatory and osteoarthritis pain after oral administration.

Identification of N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (NTRC-808), a novel nonpeptide chemotype selective for the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesic effects in relevant animal models. Using a pharmacophore model based on known NT receptor nonpeptide compounds, we screened commercial databases to identify compounds that might possess activity at NTS2 receptor sites. Modification of our screening hit to include structural features known to be recognized by NTS1 and NTS2, led to the identification of the novel NTS2 selective nonpeptide, N-{[6-chloro-4-(2,6-dimethoxyphenyl)quinazolin-2-yl]carbonyl}-l-leucine (9). This compound is a potent partial agonist in the FLIPR assay with a profile of activity similar to that of the reference NTS2 analgesic nonpeptide levocabastine (5).

Discovery and optimization of 5-fluoro-4,6-dialkoxypyrimidine GPR119 agonists.

A series of 5-fluoro-4,6-dialkoxypyrimidine GPR119 modulators were discovered and optimized for in vitro agonist activity. A lead molecule was identified that has improved agonist efficacy relative to our clinical compound (APD597) and possesses reduced CYP2C9 inhibitory potential. This optimized lead was found to be efficacious in rodent models of glucose control both alone and in combination with a Dipeptidyl peptidase-4 (DPP-4) inhibitor.

Synthesis and pharmacological evaluation of 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), a cocaine antagonist, in rodents.

Cocaine interacts with monoamine transporters and sigma (σ) receptors, providing logical targets for medication development. In the present study, in vitro and in vivo pharmacological studies were conducted to characterize SN79, a novel compound which was evaluated for cocaine antagonist actions. Radioligand binding studies showed that SN79 had a nanomolar affinity for σ receptors and a notable affinity for 5-HT(2) receptors, and monoamine transporters. It did not inhibit major cytochrome P450 enzymes, including CYP1A2, CYP2A6, CYP2C19, CYP2C9*1, CYP2D6, and CYP3A4, suggesting a low propensity for potential drug-drug interactions. Oral administration of SN79 reached peak in vivo concentrations after 1.5 h and exhibited a half-life of just over 7.5 h in male, Sprague-Dawley rats. Behavioral studies conducted in male, Swiss Webster mice, intraperitoneal or oral dosing with SN79 prior to a convulsive or locomotor stimulant dose of cocaine led to a significant attenuation of cocaine-induced convulsions and locomotor activity. However, SN79 produced sedation and motor incoordination on its own at higher doses, to which animals became tolerant with repeated administration. SN79 also significantly attenuated the development and expression of the sensitized response to repeated cocaine exposures. The ability of SN79 to significantly attenuate the acute and subchronic effects of cocaine provides a promising compound lead to the development of an effective pharmacotherapy against cocaine.

Early development of sigma-receptor ligands.

Sigma receptors (σ-1 and σ-2) are non-opioid proteins implicated in the pathophysiology of various neurological disorders and cancer. The σ-1 subtype is a chaperon protein widely distributed in the CNS and peripheral tissues. These receptors are involved in the modulation of K(+)- and Ca(2+)-dependent signaling cascades at the endoplasmic reticulum and modulation of neurotransmitter release. σ-1 receptors are emerging targets for the treatment of neurophychiatric diseases (schizophrenia and depression) and cocaine addiction. σ-2 receptors are lipid raft proteins. They are highly expressed on many tumor cells and hence considered potential targets for anticancer drugs. σ receptors bind to a diverse class of pharmacological compounds like cocaine, methamphetamine, benzomorphans like (±)-pentazocine, (±)-SKF-10,047 and endogenous neurosteroids and sphingolipids. In this review we focus on the early development of σ receptor-specific ligands and radiolabeling agents.

Sigma receptor agonists: receptor binding and effects on mesolimbic dopamine neurotransmission assessed by microdialysis.

BACKGROUND: Subtypes of sigma (σ) receptors, σ1 and σ2, can be pharmacologically distinguished, and each may be involved in substance-abuse disorders. σ-Receptor antagonists block cocaine place conditioning and σ-receptor agonists are self-administered in rats that previously self-administered cocaine. Self-administration of abused drugs has been related to increased dopamine (DA) neurotransmission, however, σ-receptor agonist effects on mesolimbic DA are not fully characterized. METHODS: Receptor-binding studies assessed affinities of σ-receptor ligands for σ-receptor subtypes and the DA transporter; effects on DA transmission in the rat nucleus accumbens shell were assessed using in vivo microdialysis. RESULTS: Cocaine (.1-1.0 mg/kg intravenous [IV]), the nonselective σ(½)-receptor agonist DTG (1.0-5.6 mg/kg IV), and the selective σ1-receptor agonist PRE-084 (.32-10 mg/kg IV) dose-dependently increased DA to ∼275%, ∼150%, and ∼160% maxima, respectively. DTG-induced stimulation of DA was antagonized by the nonselective σ(½)-receptor antagonist BD 1008 (10 mg/kg intraperitoneal [IP]) and the preferential σ2-receptor antagonist SN 79 (1-3 mg/kg IP), but not by the preferential σ1-receptor antagonist, BD 1063 (10-30 mg/kg IP). Neither PRE-084 nor cocaine was antagonized by BD 1063 or BD 1008. CONCLUSIONS: σ-Receptor agonists stimulated DA in a brain area critical for reinforcing effects of cocaine. DTG effects on DA appear to be mediated by σ2-receptors rather than σ1-receptors. However, DA stimulation by cocaine or PRE-084 does not likely involve σ-receptors. The relatively low potency on DA transmission of the selective σ1-receptor agonist, PRE-084, and its previously reported potent reinforcing effects, suggest a dopamine-independent reinforcing pathway that may contribute to substance-abuse disorders.

Sigma receptors and cocaine abuse.

Sigma receptors have been well documented as a protein target for cocaine and have been shown to be involved in the toxic and stimulant actions of cocaine. Strategies to reduce the access of cocaine to sigma receptors have included antisense oligonucleotides to the sigma-1 receptor protein as well as small molecule ligand with affinity for sigma receptor sites. These results have been encouraging as novel protein targets that can attenuate the actions of cocaine are desperately needed as there are currently no medications approved for treatment of cocaine toxicity or addiction. Many years of research in this area have yet to produce an effective treatment and much focus was on dopamine systems. A flurry of research has been carried out to elucidate the role of sigma receptors in the blockade of cocaine effects but this research has yet to yield a clinical agent. This review summarizes the work to date on the linkage of sigma receptors and the actions of cocaine and the progress that has been made with regard to small molecules. Although there is still a lack of an agent in clinical trials with a sigma receptor mechanism of action, work is progressing and the ligands are becoming more selective for sigma systems and the potential remains high.

Conversion of a highly selective sigma-1 receptor-ligand to sigma-2 receptor preferring ligands with anticocaine activity.

Cocaine's toxicity can be mitigated by blocking its interaction with sigma-1 receptors. The involvement of sigma-2 receptors remains unclear. To investigate their potential role, we have designed compounds through a convergent synthesis utilizing a highly selective sigma-1 ligand and elements of a selective sigma-2 ligand. Among the synthesized compounds was produced a subnanomolar sigma-2 ligand with an 11-fold preference over sigma-1 receptors. These compounds may be useful in developing effective pharmacotherapies for cocaine toxicity.