Structure-activity relationship and biological evaluation of pyrrolo(2,3-b)pyrazines compounds as nanomolar activators of CFTR channel

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl- channel in the apical membrane of epithelial cells for cAMP-dependent Cl- secretion. Mutations of this channel causes cystic fibrosis disease ; thus discovery of pharmacological activators of CFTR is crucial to design future medicament for protein therapy. Recently, we reported on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3- b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR and F508del-CFTR Cl- channels (Noel et al, 2006). This preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR (RP- 107 and RP-108). Here we studied structure-function relationship of more than 190 compounds prepared by chemical synthesis, and the subsequent activation of CFTR channels. Within the 6-phenylpyrrolo[2,3-b]pyrazines family, we observed by iodide efflux technique that RP-173 bearing a 2- hydroxyphenyl substituant is more potent (EC50 = 16 nM) than RP-107 having 4-hydroxyphenyl substituant (EC50 = 150 nM). By whole-cell patch clamp recording analysis, we confirmed that nanomolar concentrations of RP-173 activate linear chloride current in CHO cells stably transfected with human wild-type CFTR. This current was inhibited by 10 μM of CFTRinh- 172. We also found significant stimulation of short circuit current (Isc) by RP-173 (EC50 = 9 nM) on colon of Cftr+/+ but not of Cftr-/- mice mounted in Ussing chamber. Stimulation of Isc by RP derivatives was inhibited by glibenclamide. The structural analogue RP-146 (3-chlorophenyl), was less potent (EC50 = 347 nM). As for RP-107 compound, we found that the 7-nbutyl chain is crucial for RP-146 and RP-173 activity, and that the 2-hydroxyphenyl compounds without 7-n-butyl chain are unactive on CFTR. In this study, we showed that the presence of an hydroxyl group at position 2, 3 or 4 of the pyrrolopyrazine cycle determined the highest activity on CFTR. The most potent compound is the 7-n-butyl-6-(2-hydroxyphenyl)5Hpyrrolo[2,3- b]pyrazine (RP-173). The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis. Supported by Vaincre La Mucoviscidose and MucoVie66