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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. [Display omitted] • The process optimization and scale-up synthesis of INT-767 is provided. • The synthesized INT-767 metabolites are weaker FXR/TGR5 agonists. • Computational analysis provides novel insights into the structure-activity relationships of bile acid derivatives. [ABSTRACT FROM AUTHOR]

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists. [Display omitted] • The synthesis of a collection of novel semisynthetic cholic acid derivatives is reported. • The C 7 β-butyl-CA (8) and the C 23 -difluoro-CA (19) were identified as selective low micromolar TGR5 agonists. • Biological data and computational modelling evidenced for the first time the negative impact of the 6α-ethyl group in the TGR5 activity of bile acids. • Unprecedented insights into the functional role of TGR5 hot-spots are reported and may be explored to design potent and selective TGR5 agonists. [ABSTRACT FROM AUTHOR]