We previously identified phenylquinoxalinone CFTR_act-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC₅₀ of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC₅₀ down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTR_act-J027, activated CFTR with EC₅₀ ∼ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTR_act-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTR_act-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTR_act-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTR_act-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation.