FIGURE 1.

The monocarboxylic acids A_2ARPAM-1 and A_2ARPAM-2 are positive adenosine A_2A receptor allosteric modulators. (A) Chemical synthesis of A_2ARPAM-2. A_2ARPAM-2 was produced by combining 4-bromo-2-pyridinecarboxylic acid (1) and 4-propylaniline (2). (B,C) Dose-dependent changes of cAMP levels in mA_2AR-expressing Chinese hamster ovary cells after treatment with various adenosine (AD) and A_2ARPAM-1 (B) or A_2ARPAM-2 (C) concentrations. Experiments were performed in triplicate wells for each condition and data are presented as mean ± SEM. (D,E) Antagonistic activity of A_2ARPAM-1 and A_2ARPAM-2 against human orexin A (OXA) at orexin-1 receptors (D, OX₁R) and orexin-2 receptors (E, OX₂R) in a cell-based calcium assay. Experiments were performed in duplicate wells for each condition and data are presented as mean ± SEM. (F,G) UPLC-MS/MS analysis of brain samples from mice injected intraperitoneally with 75 mg kg^-1 A_2ARPAM-1. Single-ion (m/z 392) signals (F) and total brain concentrations (G) of A_2ARPAM-1 in the brain samples. Data (n = 5) are presented as mean ± SEM.