Selective isoxazolopyrimidine PAT1 (SLC26A6) inhibitors for therapy of intestinal disorders

A loss of prosecretory Cl − channel CFTR activity in the intestine is considered as the key cause of gastrointestinal problems in cystic fibrosis (CF): meconium ileus, distal intestinal obstruction syndrome (DIOS) and constipation. Since CFTR modulators have minimal effects on gastrointestinal symptoms, there is an unmet need for novel treatments for CF-associated gastrointestinal disorders. Meconium ileus and DIOS mainly affect the ileum (distal small intestine). SLC26A6 (putative anion transporter 1, PAT1) is a Cl − /HCO 3 − exchanger at the luminal membrane of small intestinal epithelial cells which facilitates Cl − and fluid absorption. We recently identified first-in-class PAT1 inhibitors by high-throughput screening. Isoxazolopyrimidine PAT1 inh -A01 was a hit compound, which had low potency (IC 50 5.2 μM) for SLC26A6 inhibition precluding further preclinical development. Here we performed structure-activity relationship studies to optimize isoxazolopyrimidine SLC26A6 inhibitors and tested a potent inhibitor in mouse models of intestinal fluid absorption. Structure-activity studies of 377 isoxazolopyrimidine analogs identified PAT1 inh -A0030 (ethyl 4-(benzyl(methyl)amino)-3-methylisoxazolo[5,4- d ]pyrimidine-6-carboxylate) as the most potent SLC26A6 inhibitor with a 1.0 μM IC 50 . Selectivity studies showed that PAT1 inh -A030 has no activity on relevant ion transporters/channels (SLC26A3, SLC26A4, SLC26A9, CFTR, TMEM16A). In a closed-loop model of intestinal fluid absorption, intraluminal PAT1 inh -A0030 treatment inhibited fluid absorption in the ileum of wild-type and CF mice ( Cftr delF508/delF508 ) with >90% prevention of a decrease in loop fluid volume and loop weight/length ratio at 30 minutes. These results suggest that SLC26A6 is the key transporter mediating Cl − and fluid absorption in the ileum and SLC26A6 inhibitors are novel drug candidates for treatment of CF-associated small intestinal disorders. This study reports identification of selective isoxazolopyrimidine inhibitors of intestinal anion exchanger SLC26A6 (PAT1) with improved potency and good in vivo efficacy. PAT1 inhibitors are novel drug candidates for intestinal diseases.