N‑Substituted Phenoxazine and Acridone Derivatives: Structure–Activity Relationships of Potent P2X4 Receptor Antagonists

P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was <i>N</i>-(benzyloxycarbonyl)­phenoxazine (<b>26</b>, PSB-12054) with an IC₅₀ of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. <i>N</i>-(<i>p</i>-Methylphenylsulfonyl)­phenoxazine (<b>21</b>, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC₅₀: 0.928–1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure–activity relationship analysis of P2X4 antagonists

Development of Potent and Selective Antagonists for the UTP-Activated P2Y₄ Receptor

P2Y₄ is a G_q protein-coupled receptor activated by uridine-5′-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y₄ receptor antagonist has been described so far. Therefore, we developed and optimized P2Y₄ receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescence-based assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y₄ receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4-dimethylphenylthio)­phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, <b>61</b>) exhibited an IC₅₀ value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand–receptor interactions. Compound <b>64</b> (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)­phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most selective P2Y₄ receptor antagonist known to date. Compounds <b>61</b> and <b>64</b> are therefore anticipated to become useful tools for studying this scarcely investigated receptor

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

Potent Suppressive Effects of 1‑Piperidinylimidazole Based Novel P2X7 Receptor Antagonists on Cancer Cell Migration and Invasion

The P2X7 receptor (P2X7R) has been reported as a key mediator in inflammatory processes and cancer invasion/metastasis. In this study, we report the discovery of novel P2X7R antagonists and their functional activities as potential antimetastatic agents. Modifications of the hydantoin core-skeleton and the side chain substituents of the P2X7R antagonist <b>7</b> were performed. The structure–activity relationships (SAR) and optimization demonstrated the importance of the sulfonyl group at the R₁ position and the substituted position and overall size of R₂ for P2X7R antagonism. The optimized novel analogues displayed potent P2X7 receptor antagonism (IC₅₀ = 0.11–112 nM) along with significant suppressive effects on IL-1β release (IC₅₀ = 0.32–210 nM). Moreover, representative antagonists (<b>12g</b>, <b>13k</b>, and <b>17d</b>) with imidazole and uracil core skeletons significantly inhibited the invasion of MDA-MB-231 triple negative breast cancer cells and cancer cell migration in a zebrafish xenograft model, suggesting the potential therapeutic application of these novel P2X7 antagonists to block metastatic cancer