Fluorescent-Labeled Selective Adenosine A_2B Receptor Antagonist Enables Competition Binding Assay by Flow Cytometry

Fluorescent ligands represent powerful tools for biological studies and are considered attractive alternatives to radioligands. In this study, we developed fluorescent antagonists for A_2B adenosine receptors (A_2BARs), which are targeted by antiasthmatic xanthines and were proposed as novel targets in immuno-oncology. Our approach was to merge a small borondipyrromethene (BODIPY) derivative with the pharmacophore of 8-substituted xanthine derivatives. On the basis of the design, synthesis, and evaluation of model compounds, several fluorescent ligands were synthesized. Compound <b>29</b> (PSB-12105), which displayed high affinity for human, rat, and mouse A_2BARs (<i>K</i>_i = 0.2–2 nM) and high selectivity for this AR subtype, was selected for further studies. A homology model of the human A_2BAR was generated, and docking studies were performed. Moreover, <b>29</b> allowed us to establish a homogeneous receptor–ligand binding assay using flow cytometry. These compounds constitute the first potent, selective fluorescent A_2BAR ligands and are anticipated to be useful for a variety of applications

α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective <i>ecto</i>-5′-Nucleotidase (CD73) Inhibitors

<i>ecto</i>-5′-Nucleotidase (<i>e</i>N, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. <i>e</i>N inhibitors have potential for use as cancer therapeutics. The <i>e</i>N inhibitor α,β-methylene-ADP (AOPCP, adenosine-5′-<i>O</i>-[(phosphonomethyl)­phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant <i>e</i>N. 6-(Ar)­alkylamino substitution led to the largest improvement in potency. <i>N</i>⁶-Monosubstitution was superior to symmetrical <i>N</i>⁶,<i>N</i>⁶-disubstitution. The most potent inhibitors were <i>N</i>⁶-(4-chlorobenzyl)- (<b>10l</b>, PSB-12441, <i>K</i>_i 7.23 nM), <i>N</i>⁶-phenylethyl- (<b>10h</b>, PSB-12425, <i>K</i>_i 8.04 nM), and <i>N</i>⁶-benzyl-adenosine-5′-<i>O</i>-[(phosphonomethyl)­phosphonic acid] (<b>10g</b>, PSB-12379, <i>K</i>_i 9.03 nM). Replacement of the 6-NH group in <b>10g</b> by O (<b>10q</b>, PSB-12431) or S (<b>10r</b>, PSB-12553) yielded equally potent inhibitors (<b>10q</b>, 9.20 nM; <b>10r</b>, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other <i>ecto</i>-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent <i>e</i>N inhibitors described to date