BAR-502

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

Herein we report our synthetic efforts in supporting the development of the bile alcohol sulfate INT-767, a FXR/TGR5 dual agonist with remarkable therapeutic potential for liver disorders. We describe the process development to a final route for large scale preparation and analogues synthesis. Key sequences include Grignard addition, a one-pot two-step shortening-reduction of the carboxylic side chain, and the final sulfation reaction. The necessity for additional steps such as the protection/deprotection of hydroxyl groups at the steroidal body was also evaluated for step-economy and formation of side-products. Critical bottlenecks such as the side chain degradation have been tackled using flow technology before scaling-up individual steps. The final synthetic route may be successfully employed to produce the amount of INT-767 required to support late-stage clinical development of the compound. Furthermore, potential metabolites have been synthesized, characterized and evaluated for their ability to modulate FXR and TGR5 receptors providing key reference standards for future drug investigations, as well as offering further insights into the structure-activity relationships of this class of compounds.