A_2A receptor antagonist 4-(2-((6-Amino-9-ethyl-8-(furan-2-yl)-9H-purin-2-yl)amino)ethyl)phenol, a promising adenosine derivative for glioblastoma treatment

Adenosine, a pervasive signaling molecule mediated by its interaction with G-protein-coupled receptor subtypes, especially the A_2A adenosine receptor (A_2AAR), plays a crucial role in cancer treatment. Recently, A_2AAR targeting adenosine analogs have been proposed as a potential therapeutic target for cancer treatment. However, the molecules targeting A_2AAR and their mode of action in inhibiting glioblastoma cell progression remain unknown. We synthesized six adenosine derivatives substituted at the 9-, 2- and/or N^6- and/or 8- positions, and their anti-proliferative efficacy against the GBM cell lines LN229 and SNB19 was assessed. Molecular dynamic simulation integrated with experimental analyses, including cell cycle arrest, apoptosis assay, ligand binding assay, absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling, PAMPA assay, and 3D spheroid analysis, were performed to identify the interaction efficacy of the potential derivative with A_2AAR and its ability to prevent GBM cell progression. The most potent A_2AAR derivative (ANR), 4-(2-((6-Amino-9-ethyl-8-(furan-2-yl)-9H-purin-2-yl)amino)ethyl)phenol (ANR 672) inhibits 5′-N-Ethylcarboxamidoadenosine (NECA)-induced cAMP validating the antagonistic property with higher cytotoxicity effect against GBM cells. ANR 672 showed higher affinity toward A_2AAR (K_i = 1.02 ± 0.06 nM) and exhibited significant IC₅₀ concentrations of ∼ 60–80 µM, than FDA approved drug istredefylline. The A_2AAR-ANR 672 interaction profile showed well-defined hydrogen bonds and hydrophobic contacts, indicating a typical binding mechanism inside the receptor pocket and a higher degree of conformational flexibility than the A_2AAR-Istradefylline complex. The antagonist effect of ANR 672 blocked the A_2AAR signaling pathway, leading to necrosis-mediated cell death and cell cycle arrest at the S-phase in both the GBM cells. ANR 672 treated 3D tumour spheroids analysis with real-time spheroid volume and cell proliferation analysis revealed the potential ability of ANR 672 against GBM cell growth. Drug-likeness assessments also showed favorable pharmacokinetic profiles for ANR 672. Further validation of blood-brain barrier crossing potential revealed that ANR 672 possesses moderate permeability. Our findings shed light on how ANR 672 exerts its GBM-suppressive effect through the interaction of A_2AAR. These preclinical results suggest that A_2AAR blockade could be a unique strategy for treating GBM.