Bitopic A3 Adenosine Receptor Molecular Probes: Positive Allosteric Modulation and Noncanonical Activation.

1H-Imidazo[4,5-c]quinolin-4-amines are lipid-facing, positive allosteric modulators (PAMs) of the Gi-coupled A3 adenosine receptor (A3AR). Elongated amino-alkyl chains anchor these bitopic PAMs secondarily to anionic phospholipids in the membrane's inner leaflet. Varied terminal functionalities and introduced reporter groups, as well as N1-alkylation on the core heterocycle, substantially enhanced human A3AR agonist (Cl-IB-MECA) potency and efficacy in [35S]GTPγS binding and revealed ago-PAM activity. Docking calculations predicted N1-benzylation to reduce undesired orthosteric site binding. Fluorophores, biotin, click moieties, chemically reactive, and photouncaging groups were included. Compound 38 (MRS8435, 9-methylenes, 0.1-10 μM) achieved ∼300% agonist Emax without ago-PAM activity. 4-Methyl 36 and 4-iodo 46 substitution of N1-benzyl increased Cl-IB-MECA potency by 14.7- and 30.5-fold, respectively. 9-Methylene N1-benzyl derivatives 35 and 42 achieved high ago-PAM efficacy (∼77% Cl-IB-MECA Emax). Molecular dynamics simulations detected stable electrostatic phospholipid interactions while maintaining A3AR allosteric binding. Thus, we rationally expanded SAR of bitopic A3AR PAMs, including molecular probes.