Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925

Abstract

The G q protein-coupled, ATP-and UTP-activated P2Y 2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y 2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y 2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y2 antagonist

“…Kemp et al () reported that 10‐μM AR‐C118925XX had no effect at 37 other GPCR and ion channels. The only clear indication of an off‐target action of sub‐μM concentrations is at P2X3 receptors, with an IC 50 of 819 nM (Rafehi, Burbiel, et al, ). The K B was not calculated, however.…”

“…The biggest methodological difference between our study and those of Rafehi, Burbiel, et al (2017), Rafehi, Neumann, et al (2017), and Kindon et al (2017) is that they used multi-well plates and a microplate reader to record changes in cytoplasmic Ca 2+ levels. To generate a CRC, multiple populations of cells were stimulated once only, with a single concentration of UTP.…”

“…While this report was in preparation, Rafehi, Burbiel, Attah, Abdelrahman, and Müller (2017) published an improved procedure for synthesis of AR-C118925XX and details of its pharmacological actions at hP2Y 2 receptors expressed in 1321N1 cells. It is notable that the reported potency of AR-C118925XX (pA 2 = 7.43, K B = 37.2 nM) is an order of magnitude lower than ours.…”

Update of P2Y receptor pharmacology: IUPHAR Review 27

“…AR‐C118925 (5‐[[5‐(2,8‐dimethyl‐5 H ‐dibenzo[a,d]cyclohepten‐5‐yl)‐3,4‐dihydro‐2‐oxo‐4‐thioxo‐1(2 H )‐pyrimidinyl]methyl]‐ N ‐2 H ‐tetrazol‐5‐yl‐2‐furancarboxamide, 16 ) is a potent and selective antagonist for P2Y 2 receptors (Kindon et al, 2017; Rafehi, Burbiel, Attah, Abdelrahman, & Müller, 2017; Rafehi, Neumann, et al, 2017).…”

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice

“…To support the specificity of the UTP stimulus over P2Y2 receptor, we used the selective P2Y2R antagonist ARC118925 [13]; thus, ARC abolished the ERK phosphorylation induced by UTP (277.1% ± 37.3% and 130.2% ± 43.3 % of basal, respectively, p = 0.04, Student's t-test), showing that P2Y2R is mediating the UTP actions in primary cultures of mice hepatocytes ( Figure 4B). The exclusive role of P2Y2R was supported by PCR analysis (Figure 2, Section 2.2) since no transcript of P2ry4 was amplified in cDNAs of primary cultured hepatocytes, suggesting that P2Y4 is not expressed in this cell type.…”