Identification of potent pyrazole based APELIN receptor (APJ) agonists
Graphical abstract
Introduction
The apelinergic system comprises the endogenous apelin peptides and its cognate G-protein coupled apelin receptor, encoded by the gene AGTRL1 (angiotensin receptor like 1).1 The apelin receptor is considered physiologically important due to its presence across many species including human, monkey, pig, rodents, frog and zebrafish.2 The apelin receptor is a 380 amino acid protein having ~33% sequence identity to the AT1 receptor.1 Apelin-36 was the first endogenous peptide identified along with other smaller peptides (apelin-17, apelin-13, apelin-12, pyr-apelin-13) that were derived from the cleavage of paired basic residues (Arg-Arg and Arg-Lys) of the 77 amino acid precursor protein3, 4, 5, 6, 7 Known apelin peptides are readily hydrolyzed in vivo (half-life ~5 min).8 These proteins belong to an understudied component of the renin-angiotensin system with angiotensin-converting-enzyme II (ACE II) being involved in degradation of apelin in vivo.8, 9 However, none of the known angiotensin receptor ligands or peptides activate apelin receptor.1 Recently, Elabela/Toddler was identified as the second endogenous ligand of this receptor.10, 11, 12 Identification of these two sets of endogenous ligands facilitated molecular characterization of apelin receptor. Apelin receptors are primarily coupled to inhibitory Gαi proteins and their activation is associated with inhibition of forskolin-stimulated cAMP production.3 Additionally, apelin receptor can also activate PLC mediated calcium flux through Gαq coupling13, 14 and recruit β-arrestin that desensitizes the receptor utilizing clathrin mediated endocytosis.15 Apelin and apelin receptor are widely expressed across tissues in both human and rodents. In rat CNS, apelin mRNA is expressed within the hypothalamus where it is co-expressed with vasopressin mRNA in paraventricular (PVN) and supraoptic nuclei (SON).16, 17 Peripherally, apelin receptor is expressed in many organs including kidney, lungs, adipose tissue, liver and heart.18, 19, 20 Activation of the apelinergic system has many beneficial effects including protection against hypertensive disorders, excitotoxic neuronal damage, pulmonary arterial hypertension, metabolic syndrome and heart failure.21, 22 The receptor also plays an important role during embryonal development through regulation of vasculogenesis.23
Efforts are underway to produce small molecule agonists of apelin receptor with improved drug-like properties because the endogenous peptide ligands have short half-lives and limited utility as therapeutics. A number of small molecules have been reported. Iturrioz and colleagues reported a non-peptide functional apelin receptor agonist E339-3D6 (Fig. 1) that exhibited an EC50 of 90 nM (FRET) and Ki value of ~400 nM in both rat and human apelin receptor.24
The ML233 core (Fig. 1) ((E)-2-cyclohexyl-5-methyl-4-(phenylsulfonyloxyimino)cyclohexa-2,5-dienone) was identified through a high throughput screen of ~330 600 compounds from the NIH small molecule library.25 ML233 is a full agonist and exhibits an EC50 of 3.7 µM (β-arrestin), but it displayed poor solubility and stability in microsomal fractions. In addition, ML233 had significant off-target binding at several other receptors including the 5-HT1A, α2C adrenergic, mu-opioid, benzylpiperazine receptors and norepinephrine transporter. A number of patent disclosures have also emerged. Sanofi Aventis,26 and Sanford-Burnham Medical Research Institute,27 have disclosed small molecule agonists based on the substituted benzimidazole (US20140094450 A1) and triazole core (WO 2015/184011 A2) respectively (Fig. 1). In addition, Bristol-Myers Squibb and Amgen have disclosed small molecules based on dihydropyrimidine-4-one core (WO 2017/096130A1)28 and triazole core (WO 2017/192485A1)29 respectively. A small molecule benzimidazole derivative related to the Sanofi Aventis scaffold CMF-019 (Fig. 1) was reported to be G protein biased small molecule apelin receptor agonist. CMF-019 was ~400 fold bias towards Gαi signaling over β-arrestin recruitment and ~6000 fold bias over receptor internalization. Studies using these biased agonists that preferentially stimulate G-protein activation over β-arrestin recruitment have demonstrated improved in vivo efficacy (vasodilation and inotropic actions) without β-arrestin dependent cardiac hypertrophy.30, 31, 32
Our laboratory has previously reported a novel pyrazole scaffold 8 (EC50 = 21.5 µM (calcium), Ki = 5.2 µM) identified through focused screening, which was further optimized to an initial lead 9 (EC50 = 0.800 µM (calcium), Ki = 1.3 µM) (Fig. 2).33 In our efforts to identify more potent agonists as tool compounds and to explore the structural features important for apelin receptor agonism, we carried out modifications at N1 position of the pyrazole core as well as the amino acid side-chain of the lead compound 9. Synthesized compounds were characterized using calcium mobilization, inhibition of forskolin-induced cAMP accumulation and β- arrestin recruitment assays. Considering the potential importance of selectivity over the β-arrestin pathway,30, 31 bias factors for the most promising compounds were calculated.
Section snippets
Chemistry
Scheme 1 depicts the synthetic route employed to prepare target compounds 9–38. Synthesis of analogs 25 and 37 are shown as representative examples. 2, 6-Dimethoxy acetophenone 39 was condensed with diethyl oxalate to afford the sodium salt of diketone 40 in quantitative yield. Reaction of 40 with isobutylhydrazine or cyclopentylhydrazine trifluoroacetate in refluxing ethanol provided 1, 5-pyrazoles 41a, 42a and 1,3-pyrazoles 41b, 42b in a ratio of 4:1. Transformation of 41a and 42a to acids 43
Results and discussion
Our laboratory has previously reported the novel pyrazole based small molecule agonist hit 8 (Ca2+ EC50 = 21.5 µM, Ki = 5.2 µM) identified through focused screening that was further optimized to an initial lead 9 (Ca2+ EC50 = 0.800 µM, cAMP EC50 = 844 nM, Ki = 1.3 µM). In addition to improving potency and affinity, compound 9 exhibited significantly reduced off-target NTR2 activity and did not activate or inhibit the AT1 receptor (Table 1).33 Considering the structural similarity with the
Conclusions
Systematic modifications at N1 position of the pyrazole core as well as the amino acid side chain resulted in potent small molecule agonists exhibiting EC50 values of ≤100 nM. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. Modification at N1 position of the pyrazole core resulted in compounds 11, 13, 14 (n-propyl, cyclopentyl, cyclohexyl substitutions) that exhibited enhanced potency and functional selectivity
Experimental
Reagents and starting materials were obtained from commercial suppliers and were used without purification. Reactions were conducted under N2 atmosphere using oven-dried glassware. All solvents and chemicals used were reagent grade. Anhydrous tetrahydrofuran (THF), dichloromethane (DCM), and N, N-dimethylformamide (DMF) were purchased from Fisher Scientific and used as such. Flash column chromatography was carried out using a Teledyne ISCO Combiflash Rf system and Redisep Rf gold pre-packed HP
Lance™ cAMP accumulation assay
Stimulation buffer containing 1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated to pH 7.4 at room temperature. Serial dilutions of the test compounds and 1 µM forskolin, both prepared at 4x the desired final concentration in stimulation buffer, were added to a 96-well white ½ area microplate (PerkinElmer). A cAMP standard curve prepared at 4x the desired final concentration in stimulation buffer was added to the assay
Bias factor calculation
Data from technical replicates on the same day were baseline-corrected and normalized relative to the reference agonist Pyr-Apelin-13. Data from multiple days were then combined and fit to logistic functions with Hill coefficient equal to 1 to determine Emax, EC50 and their standard errors. Bias factors, which quantify the degree of signaling through pathway 1 compared to pathway 2 for a ligand relative to a reference agonist on a logarithmic scale, were calculated as previously described.35, 36
X-ray crystallographic data
Atomic coordinates for 43 and 44 have been deposited with the Cambridge Crystallographic Data Centre (deposition numbers 1946460, 1946461). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)-1223–336033 or e-mail: [email protected]].
Funding
This work was supported by NIH grants: 1R01HD079547-01A1 and 1R01DK103625-01A1.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgements
Authors wish to thank Dr. Elaine Gay, Dr. Ann Decker for help with various assays and Dr. Thomas Prisinzano, Victor Day, and the University of Kansas X-Ray Crystallography Laboratory for the crystal structures.
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