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

1<i>H</i>-Imidazo[4,5-<i>c</i>]quinolin-4-amines are lipid-facing, positive allosteric modulators (PAMs) of the G_i-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 <i>N</i>1-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 <i>N</i>1-benzylation to reduce undesired orthosteric site binding. Fluorophores, biotin, click moieties, chemically reactive, and photouncaging groups were included. Compound <b>38</b> (MRS8435, 9-methylenes, 0.1-10 μM) achieved ∼300% agonist <i>E</i>_max without ago-PAM activity. 4-Methyl <b>36</b> and 4-iodo <b>46</b> substitution of <i>N</i>1-benzyl increased Cl-IB-MECA potency by 14.7- and 30.5-fold, respectively. 9-Methylene <i>N</i>1-benzyl derivatives <b>35</b> and <b>42</b> achieved high ago-PAM efficacy (∼77% Cl-IB-MECA <i>E</i>_max). 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.