Chemical synthesis of 7-oxygenated 12α-hydroxy steroid derivatives to enable the biochemical characterization of cytochrome P450 8B1, the oxysterol 12α-hydroxylase enzyme implicated in cardiovascular health and obesity

Abstract

Cholic acid is the endogenous 12α-hydroxylated bile acid, which possesses enhanced cholesterol absorption properties compared to its 12-desoxy counterpart, chenodeoxycholic acid. The oxysterol 12α-hydroxylase enzyme is cytochrome P450 8B1 (P450 8B1), which regioselectively and stereoselectively incorporates the 12α-hydroxy group in 7α-hydroxycholest-4-en-3-one, the biosynthetic precursor of cholic acid. Despite the vital role of P450 8B1 activity in cardiovascular health, research studies of other 12α-hydroxy steroid derivatives are rare. A synthetic route to incorporate a C12α-hydroxy group into the C12-methylene (–CH2–) in dehydroepiandrosterone derivatives is disclosed. The incorporation of the C12-oxygen was accomplished through a copper mediated Schönecker oxidation of an imino-pyridine intermediate, introducing the 12β-hydroxy group. The resulting 12β-hydroxy steroid derivative was oxidized to the C12-ketone, which was stereoselectively reduced with lithium tri-sec-butylborohydride to afford the 12α-hydroxy stereochemistry. The C7-position was oxidized to yield the various 7-keto, 7β-hydroxy, and 7α-hydroxy derivatives. Furthermore, 7-ketodehydroepiandrosterone and 12 α-hydroxy-7-ketodehydroepiandrosterone both displayed NMDA receptor antagonistic activities at 10 μM concentrations. These C12α-hydroxy steroids will be used as tools to identify new biochemical properties of the enzymatic products of P450 8B1, the oxysterol 12α-hydroxylase.