The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles in several physiological pathways including stress and pain response, appetite and lifespan. Recently, we reported the crystal structure of human NAPE-PLD and discovered specific binding sites for the bile acid deoxycholic acid. In this study we demonstrate that in the presence of this secondary bile acid, the stiffness of the protein measured by elastic neutron scattering increases, and NAPE-PLD results ~7 times faster to catalyze the hydrolysis of the more unsaturated substrate N-arachidonyl-phosphatidylethanolamine, compared with N-palmitoyl-phosphatidylethanolamine. Chenodeoxycholic acid and glyco- or tauro-dihydroxy conjugates can also bind to NAPE-PLD and drive its activation. The only natural monohydroxy bile acid, lithocholic acid, shows an affinity of ~20 μM and acts instead as a reversible inhibitor (IC50 ≈ 68 μM). Overall, these findings provide important insights into the allosteric regulation of the enzyme mediated by bile acid cofactors, and reveal that NAPE-PLD responds primarily to the number and position of their hydroxyl groups.

As a continuation of previous efforts in mapping functional hot spots on the bile acid scaffold, we here demonstrate that the introduction of a hydroxy group at the C11β position affords high selectivity for FXR. In particular, the synthesis and FXR/TGR5 activity of novel bile acids bearing different hydroxylation patterns at the C ring are reported and discussed from a structure-activity standpoint. The results obtained led us to discover the first bile acid derivative endowed with high potency and selectivity at the FXR receptor, 3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (TC-100, 7) which also shows a remarkable physicochemical and pharmacological profile. Compound 7 combines the excellent physicochemical properties of hydrophilic bile acids such as ursodeoxycholic acid, with the distinct ability to specifically bind and regulate FXR activity in vivo, thus providing a bona fide novel therapeutic agent to treat enterohepatic disorders such as cholestasis, NASH, and inflammatory bowel disease.

The integration of flow systems with statistical design of experiments is emerging as a valuable strategy to develop new synthetic routes towards relevant building blocks, chemical probes, and drug compounds. Optimization by experimental design incorporates statistical algorithms, mathematical models and equations, predicting tools, feedback control, and validation to generate new optimal conditions. Continuous-flow chemistry is ideally suited for this scope, as the integration of in-line analysis is simple; experimental parameters such as temperature, pressure, and flow rate can be easily controlled and fine-regulated; and automation of reaction screening can be accomplished with software assistance. This review article aims to illustrate how the combination of flow synthesizers and design of experiments can be profitable to speed up the development and optimization of more efficient, safer, and reproducible protocols for modern synthetic methods and manufacturing processes.

Bile acids are a peculiar class of steroidal compounds that never cease to amaze. From being simple detergents with a primary role in aiding the absorption of fats and fat-soluble vitamins, bile acids are now widely considered as crucial hormones endowed with genomic and non-genomic functions that are mediated by their interaction with several proteins including the nuclear receptor Farnesoid X Receptor (FXR). Taking advantages of the peculiar properties of bile acids in interacting with the FXR receptor, several biliary derivatives have been synthesized and tested as FXR ligands. The availability of these compounds has contributed to characterize the receptor from a structural, patho-physiological and therapeutic standpoint. Among these, obeticholic acid is a first-in-class FXR agonist that is demonstrating hepatoprotective effects upon FXR activation in patients with liver diseases such as primary biliary cirrhosis and nonalcoholic steatohepatitis. This review provides an historical overview of the rationale behind the discovery of obeticholic acid and chemical tools generated to depict the molecular features and bio-pharmacological relevance of the FXR receptor, as well as to summarize structure-activity relationships of bile acid-based FXR ligands so far reported.

Continuous processing has been demonstrated to be a superior approach when applied to fast and energetic chemical transformations. Indeed, whereas classical batch or semi-batch methods require cryogenic conditions and slow addition rates of reactive species, flow technologies enable rapid mixing of synthetic partners in a highly controlled environment. As a result, low yielding and dangerous processes in batch can be performed at scale in a cost competitive and safer continuous manner. Despite the advantages of higher quality and safety, the perennial problems of solids build-up and pipe fouling threaten the robustness and reliability of flow processes. In this contribution, a new methodology to prevent reactor fouling is reported and discussed. The implementation of this methodology has been decisive in solving fouling issues encountered during the piloting of an organolithium based flow process.