Natural Product-Inspired Bis(trifluoromethyl) Phenyl Hydroxycinnamate Derivatives as Promising Nonsteroidal Inhibitors of Human Steroid 5α-Reductase Type-1: Synthesis, In Vitro, and In Silico Studies

The development of steroid 5α-reductase inhibitors for the treatment of androgenetic alopecia remains challenging, primarily due to the unavailability of a crystal structure for human steroid 5α-reductase type-1 (SRD5A1) and the adverse effects associated with existing steroidal therapeutic agents such as finasteride and dutasteride. In this study, hydroxycinnamate derivatives were synthesized and evaluated for their inhibitory activity against SRD5A1 using an immortalized human keratinocyte cell-based assay coupled with nonradioactive high-performance thin-layer chromatography analysis. Three ferulate derivatives featuring 2',5'-, 3',4'-, and 3',5'-bis-(trifluoromethyl)-phenyl substituents (10a, 10b, and 10c) demonstrated inhibitory activity with half-maximal inhibitory concentrations (IC₅₀) of 8.50, 10.06, and 8.05 μM, respectively, exhibiting low cytotoxicity and favorable predicted lipophilicity. Compound 10a was chosen for further investigation to assess its effect on SRD5A1 protein expression. Treatment of HaCaT cells with compound 10a at concentrations of 10-15 μM for 24 h significantly downregulated SRD5A1 protein expression, suggesting its inhibitory effect on both SRD5A1 activity and expression. An in silico model identified key binding interactions between the AlphaFold-generated SRD5A1 catalytic pocket and the residues R98, M119, A120, E202, and F228, among which M119 is recognized as a crucial residue for the stereoselective and irreversible reduction catalyzed by SRD5A1. Structure-activity relationship analysis indicated that the 3-methoxy-4-hydroxy core facilitates hydrogen bonding with R98, whereas the cinnamoyl and bis-(trifluoromethyl)-phenyl moieties enhance hydrophobic and halogen interactions with key residues, improving binding affinity and molecular orientation. Thus, these bis-(trifluoromethyl)-substituted phenyl ferulate derivatives present a promising class of nonsteroidal inhibitors for SRD5A1, with potential applications in developing topical antiandrogenic agents for the treatment androgen-related conditions.