Structure-activity relationships of novel P2-receptor antagonists structurally related to Reactive Blue 2.

P2 membrane receptors for nucleotides represent significant targets for experimental pharmacology and drug research. In earlier publications, we have shown that Reactive Blue 2 (RB 2), one of the most widely used P2-receptor antagonists, displays only moderate affinity and does not discriminate between native P2X- and P2Y-receptor subtypes. In the present study we have pharmacologically evaluated a series of 15 synthesized and re-evaluated four commercially obtained and chromatographically purified RB 2 type anthraquinone derivatives on contractions of the rat vas deferens (RVD) elicited by alpha,beta-methylene ATP (alpha,beta-meATP), mediated by P2X1-receptors, and relaxations of the carbachol-precontracted guinea-pig taenia coli (GPTC) elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), mediated by P2Y1-like receptors. Based on the structure-activity relationships (SAR) it is concluded that hydrophobic interactions of aromatic pi-electron systems, hydrogen bonds with nitrogen as donor and acceptor atoms, and, particularly, position, conformational distance and number of anionic sulfonate groups are of great importance for the blockade of the two native P2-receptor subtypes. We have also identified novel, for the most part reversible antagonists that bind with higher affinity and improved subtype selectivity in comparison to RB 2. In particular, 1-amino-4-{4-[4-chloro-6-(2-sulfonatophenylamino)-[1,3,5]triazine-2-ylamino]-2-sulfonatophenylamino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid trisodium salt (MG 50-3-1) is the most potent antagonist at the P2Y1-like-receptors of the GPTC reported so far (IC50=4.6 nM). It is significantly less potent as reversible antagonist at the P2X1-receptors of the RVD (IC50=2.8 microM). Thus, MG 50-3-1 represents a selective pharmacological tool and may be a lead compound for future investigations.

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

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 alpha,beta-methylene ATP (alpha,beta-MeATP), mediated by P2X(1)-receptors, and relaxations of the carbachol-precontracted guinea pig taenia coli elicited by adenosine 5'-O-(2-thiophosphate) (ADPbetaS), mediated by P2Y(1)-like-receptors. All compounds inhibited the alpha,beta-MeATP induced contraction of the RVD and the ADPbetaS induced relaxation of the carbachol precontracted guinea-pig taenia coli. The IC(50)-values at P2X(1)-R were 9.1 microM for CB 3GA, 28.4 microM for RB 2, 19.7 microM for RB 2 meta, and 35.5 microM for RB 2 para. The IC(50)-values at P2Y(1)-like-R were 17.4 microM for CB 3GA, 7.7 microM for RB 2, 12.0 microM for RB 2 meta, and 2.6 microM 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.