Constitutional isomers of Reactive Blue 2 – selective P2Y-receptor antagonists?

Abstract

The anthraquinone derivative Reactive Blue 2 (RB 2) is one of the most widely used P2-receptor antagonists, still claimed to be P2Y-selective. RB 2 is defined as a mixture of two constitutional isomers and commercially available in different identity and purity. A sample of RB 2, offered for sale by RBI, purchased from Biotrend, Köln, Germany, was chromatographically purified and identified by 1H- and 13C-NMR studies as a 35:65 mixture of the terminal ring F meta and para constitutional isomers. The two constitutional isomers of RB 2 were synthesised and tested alongside with the ortho isomer Cibacron Blue 3GA (CB 3GA) on contractions of the rat vas deferens (RVD) elicited by α,β-methylene ATP (α,β-MeATP), mediated by P2X 1-receptors, and relaxations of the carbachol-precontracted guinea pig taenia coli elicited by adenosine 5′- O-(2-thiophosphate) (ADPβS), mediated by P2Y 1-like-receptors. All compounds inhibited the α,β-MeATP induced contraction of the RVD and the ADPβS induced relaxation of the carbachol precontracted guinea-pig taenia coli. The IC 50-values at P2X 1-R were 9.1 μM for CB 3GA, 28.4 μM for RB 2, 19.7 μM for RB 2 meta, and 35.5 μM for RB 2 para. The IC 50-values at P2Y 1-like-R were 17.4 μM for CB 3GA, 7.7 μM for RB 2, 12.0 μM for RB 2 meta, and 2.6 μM for RB 2 para. The results clearly show that neither RB 2 as a mixture nor the pure ortho and meta isomer are P2Y 1-like- versus P2X 1-selective antagonists. In contrast the pure para-isomer of RB 2 is a moderately P2Y 1-like- versus P2X 1-selective antagonist.