Enantiomeric (ent-)Steroids and Bile Acids: Total Synthesis and Biomedical Research Applications

          Steroids such as bile acids (e.g. 2-3) elicit many important pharmacological effects through specific binding interactions with various nuclear receptors and GPCRs. Alternatively, other biological phenomena are mediated by the well-known detergent properties of bile acids. Cholesterol (the enantiomer of 1) is similar in the sense that some of its associated properties are affected by direct binding with biomolecules. On the other hand, due to its lipophilic physical characteristics, cholesterol also stabilizes mammalian membrane bilayers and mediates membrane fluidity. It is sometimes difficult for biochemists to delineate and assess the relative contribution of receptor-mediated properties of steroids separately from the detergent or lipid properties. With this goal in mind, organic chemists have synthesized the unnatural enantiomeric forms of both cholesterol (1) and certain bile acids such as chenodeoxycholic acid (2). Complementary pairs of enantiomers have identical physical properties but have markedly different three-dimensional structures due to opposite stereochemical configurations at asymmetric carbon positions. Enantiomers, therefore, almost always exhibit differential binding affinities with their protein targets due to the chiral three-dimensional environment of proteinogenic ligand binding pockets. Unnatural enantiomeric steroid derivatives can only be obtained by total (de novo) synthesis.

          The synthetic strategies used by chemists to access the enantiomeric steroids 1 and 2 will be the primary topic of the current post. However, the important potential therapeutic applications of the related semisynthetic bile acid 3 developed by Intercept Pharmaceuticals are worth mentioning in brief. INT-777 (3) acts in the periphery (outside of the enterohepatic system) through activation of TGR5, a GPCR that is also activated by chenodeoxycholic acid. TGR5 is implicated in a number of liver and metabolic diseases including obesity and type II diabetes. INT-777 exhibits relatively potent TGR5 agonist activity (EC₅₀ ~ 800 nM, 166% efficacy) and is able to induce the release of glucagon-like protein 1 (GLP-1) in enteroendocrine cells, an attractive in vitro property for a potential diabetes treatment. More recently, a new semisynthetic analogue of avicholic acid was disclosed by the same group with comparable TGR5 potency (EC₅₀ 650 nM) along with weak agonist activity at the farnesoid X nuclear receptor (FXR). Agonism of FXR prevents the accumulation of toxic concentrations of certain bile acids in cells and therefore may also be therapeutically useful. FXR activation promotes bile acid excretion and represses bile acid synthesis and import.

          The enantiomer of natural cholesterol, ent-cholesterol (1), was first prepared by Rychnovsky and co-workers in 1992. Their basic strategy was to synthesize 1 via the intermediacy of ent-testosterone (9) by employing a modified version of the well-known synthetic steroid technologies invented at Hoffmann-La Roche in the 1970’s and 80’s. The cholesterol C17 side chain was then appended to the steroid nucleus at a relatively late stage (cf. 9 à 1). The ‘unnatural’ ring junction configuration of the Hajos-Parrish ketone was obtained by executing the intramolecular aldol (H-P-E-S-W) reaction under enamine catalysis conditions mediated by D-proline. The initial stereogenic center (pro-C13) was used to control all of the remaining stereocenters of 1. Enone 4 was elaborated to the exocyclic enone 6 by treatment with the Stiles’ MMC reagent and then hydrogenation, followed by a decarboxylative aldol reaction with formaldehyde. Subsequent Robinson annulation between 6 and the b-keto ester according to the Hoffmann-La Roche protocol fashioned the tricyclic intermediate 7. Next, a brilliant implementation of the single-electron transfer (SET) reductive alkylation procedure of Stork installed the C19 angular methyl group in a stereoselective fashion as depicted in the bracketed structure above. Finally, exposure of 8 to Bronsted acidic conditions completed the first synthesis of ent-testosterone (9). Nine additional operations were required to elaborate the C17 side chain and establish the A/B ring fusion stereochemistry of ent-cholesterol (1).

          Covey and co-workers disclosed an alternate synthesis of 1 in 2002 that adhered to an ‘east to west’ approach that constructed the steroidal A-ring in the final stages of the total synthesis. The racemic bicyclic b-keto ester system of 10, previously described in 1997 by He et al., was ring-opened with an in situ-generated organocuprate to establish the intact C17 cholesterol side chain appended to a suitably functionalized steroid D-ring. The racemic cyclopentanone 11 was resolved by transesterification with an enantiopure alcohol and then further elaborated (à 12) by an intramolecular aldol cyclization/dehydration sequence. Robinson annulation of 13 followed by application Stork’s reductive alkylation chemistry (similar to above) provided the advanced intermediate 15. In this series of transformations, no serious complications imparted by the lipophilic C17 side chain were noted. Finally, reduction of the dienol acetate 16 afforded synthetic 1 along with a small amount (10%) of its corresponding C3 epimer.

          The laboratory of Douglas Covey at the Washington University School of Medicine has also described the total synthesis of several unnatural enantiomeric bile acids along with characterization of some of their physical properties and receptor interaction profiles. In their synthesis of ent-chenodeoxycholic acid (2), a key transformation, subsequent to oxygenation of C7 (9 à 18), was the stereocontrolled ene reaction of (Z)-olefin 19 with methyl propiolate to provide the diene 20 in moderate yield (47%). Hydrogenation of 20 from the b-face followed by base-mediated hydrolysis then completed the synthesis of 2. Not surprisingly, the unnatural bile acid enantiomer 2, due to its disparate three-dimensional structure (relative to naturally occurring steroids), was inactive at the TGR5 G protein-coupled receptor discussed above.
NOTE: Thanks to reader Bob Hanson for pointing out several structural errors within this post. Those errors have all been corrected. -BHH