Rational design and structural insights of osthole-derived selective AKR1C3 inhibitors with cellular activity.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in androgen biosynthesis and a crucial driver of castration-resistant prostate cancer (CRPC), making it a promising therapeutic target. Unintended inhibition of its homologous isoforms AKR1C1 and AKR1C2 may cause off-target effects and is therefore highly undesirable. In this study, we rationally designed selective AKR1C3 inhibitors derived from the natural product osthole by optimizing the C8- and C7-substituents to target the SP1 and SP2 pockets, respectively, thereby achieving high selectivity for AKR1C3. Among the compounds synthesized, eleven exhibited potent inhibition of AKR1C3 (IC₅₀ < 0.1 μM) with high selectivity over AKR1C1. Notably, compounds 7k and 7o demonstrated significant antiproliferative activity in 22Rv1 cells (IC₅₀ = 3.05 μM and 5.45 μM, respectively). Crystallographic analysis revealed that the coumarin core anchors to the oxyanion site, while the C8-substitute interacts with the SP1 pocket, enhancing both potency and selectivity. In contrast to other coumarin-based inhibitors, osthole-derived compounds with extended C8-substitutions, interact with the SP1 pocket in a novel and distinct manner. These findings validate our design strategy of enhancing SP1 pocket binding to improve AKR1C3 selectivity over other AKR1C isoforms, and provide valuable structural insights for the further development of selective AKR1C3 inhibitors as potential therapeutics for CRPC.