Selective A₃ Adenosine Receptor Antagonist Radioligand for Human and Rodent Species

Abstract

The A 3 adenosine receptor (A 3 AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure−activity relationship of a previously reported A 3 AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [ 3 H]MRS7799. A 3 AR K d values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.

Structure–Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators

“…The selected hA 3 AR pose of compound 17 (Figure ) was compared to other known A 3 AR ligands. In the suggested binding mode, the compound’s 1 H -imidazo­[4,5- c ]­pyridin-4-amine moiety mimics the adenine scaffold of adenosine-like agonists and the [1,2,4]­triazolo­[1,5- c ]­pyrimidin-5-amine scaffold of the known antagonist N -(9-chloro-2-(furan-2-yl)-[1,2,4]­triazolo­[1,5- c ]­quinazolin-5-yl)-2-phenyl­acetamide ( 64 , MRS1220) in its previously predicted pose . A conserved π–π stacking interaction is observed between the aromatic scaffold of 17 , similar to 64 , and F168 EL2 , while nitrogen N3 and the exocyclic amino group at position 4 are engaged in a bidentate H-bond with N250 6.55 .…”

“…Conversely, we could model the ability of 17 to be accommodated in hA 3 AR orthosteric site, which is well known for ARs. Given the lack of an experimental hA 3 AR structure, we employed a previously reported homology model built on the experimental structure of an antagonist-bound hA 1 AR X-ray structure . The compound was docked to the orthosteric site through an induced fit docking (IFD) procedure and refined by minimization through molecular mechanics with a generalized Born and surface area solvation (MM-GBSA) .…”

“…Ligands were drawn and minimized with OPLS4 force field with water solution model. The model of hA 3 AR was generated in a previous work, using an antagonist-bound hA 1 AR X-ray structure as a template and a refinement with Induced Fit Docking of the antagonist 64 . The following tautomeric states were employed for histidines: protonation at N δ nitrogen for H272 7.43 (HSD according to the CHARMM nomenclature) and at the Nε nitrogen for H79 3.21 , H95 3.37 , H124 4.39 , H158 EL2 , and H304 8.65 .…”

“…Compound 48 was synthesized following the stannylation procedure described above using 39 (49.0 mg, 0.10 mmol) and hexamethylditin (105 μL, 0.50 mmol). The crude was purified by silica gel column (20% ethyl acetate in hexane) to afford 5.0 mg (10%) of a white solid: 1 [4,5-c]quinolin-4-amine (49). Compound 49 was synthesized following the stannylation procedure described above using 39 (50.0 mg, 0.10 mmol) and hexabutylditin (300 μL, 0.50 mmol).…”

“…In the ) in its previously predicted pose. 49 A conserved π−π stacking interaction is observed between the aromatic scaffold of 17, similar to 64, and F168 EL2 , while nitrogen N3 and the exocyclic amino group at position 4 are engaged in a bidentate H-bond with N250 6.55 . The 2-cyclopropyl moiety is located deep into the orthosteric binding pocket, surrounded by T94 3.36 , M177 5.38 , S181 5.42 , I186 5.47 , W243 6.48 , L246 6.51 , and N250 6.55 (residues located within 3 Å), while the 3,4-dichlorophenyl group points toward the extracellular portion of the receptor, surrounded by the tip of TM6, TM7, EL2, and EL3, and specifically by residues Q167 EL2 , F168 EL2 , I249 6.54 , N250 6.55 , I253 6.58 , V259 EL3 , L264 7.35 , and I268 7.39 .…”