Early- and Late-Stage Chemoenzymatic Functionalization of Steroid-Inspired Compounds.

Selective C-H functionalization of small molecule leads is a powerful approach for efficient chemical space exploration and medicinal chemistry optimization. Here, we report a screening strategy for early- and late-stage chemoenzymatic oxidation of steroid-inspired compounds using a panel of P450BM3 variants. Late-stage oxidation of sterol transport protein inhibitors and their intermediates revealed enzyme- and substrate-dependent regioselectivity, which was rationalized by docking studies, and yielded hydroxylated analogues that provided rapid structure-activity relationship insights into the spirooxepinoindole as a privileged scaffold and its potential metabolic products. Early-stage functionalization of steroidal building blocks, including the Wieland-Miescher ketone and acetyl-cis-decalone, furnished hydroxylated derivatives with high diastereoselectivity. Notably, both enantiomers of both diastereoisomers of the C6-hydroxylated Wieland-Miescher ketone were obtained in preparative quantities, providing access to valuable early-stage building blocks for further derivatization. This work highlights the potential of chemoenzymatic approaches to deliver regio- and stereoselective oxidations of challenging steroid-like scaffolds, complementing classical synthetic methods and expanding opportunities in medicinal chemistry and natural product synthesis.