The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl Ϫ channel in the apical membrane of epithelial cells for cAMP-dependent Cl Ϫ secretion. Here we report 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 These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMPindependent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as deter-

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Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GPinh5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.

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