Substituted 4-methylcoumarin inhibitors of SLC26A3 (DRA) for treatment of constipation and hyperoxaluria†

Abstract

SLC26A3, also known as downregulated in adenoma (DRA), is an anion (Cl⁻, HCO₃⁻ and oxalate) exchanger in the luminal membrane of intestinal epithelial cells. Loss of DRA function in mice and humans causes congenital chloride-losing diarrhea and reduces urinary excretion of oxalate, a major constituent of kidney stones. Thus, inhibition of DRA is a potential treatment approach for constipation and calcium oxalate kidney stones. High-throughput screening previously identified 4,8-dimethylcoumarins (4a–4c) as DRA inhibitors, with lead candidate 4b having an IC₅₀ of 40–50 nM for DRA inhibition. Here, we explored the effects of varying substituents at the 8-position, and replacing 8-methyl by 5-methyl (4e–4h). A focused library of 17 substituted compounds (4d–4t) was synthesized with good yield and purity. Compounds were tested for DRA inhibition potency using Fischer rat thyroid cells stably expressing DRA and a halide-sensitive YFP. Structure–activity analysis revealed that 8-bromo- (4m–4p) and 8-fluoro-coumarins (4q–4t) were slightly less potent than the corresponding 8-chloro analogs, demonstrating that the size of methyl or chloro substituents at the coumarin 8 position affects the potency. An analog containing 8-chlorocoumarin (4k) had ∼2-fold improved potency (IC₅₀ 25 nM) compared with the original lead candidate 4b. 5,8-Dimethylcoumarins were active against DRA, but with much lower potency than 4,8-disubstituted coumarins. In mice, orally administered 4k at 10 mg kg⁻¹ reduced constipation and normalized stool water content in a loperamide-induced constipation model with comparable efficacy to 4b. Pharmacokinetic analysis of orally administered 4k at 10 mg kg⁻¹ in mice indicated serum levels of >10 μM for at least six hours after single dose. This study expands SAR knowledge of 4,8-disubstituted coumarin inhibitors of DRA as novel drug candidates for constipation and kidney stones.