Dual functionalization of steviol enables mitochondrial targeting and redox modulation in antitumor therapy

Mitochondria are essential for cancer cell survival, with the thioredoxin/thioredoxin reductase 2 (Trx/TrxR2) system acting as a key redox regulator. Steviol, an abundant natural ent-kaurane diterpenoid, exhibits negligible cytotoxicity, while most active ent-kaurane analogs depend on a reactive exo-methylene cyclopentanone moiety, raising selectivity and safety concerns. To address these limitations, 28 triphenylphosphonium (TPP)-conjugated steviol derivatives were synthesized to enhance mitochondrial accumulation and modulate mitochondrial signaling. SAR analysis revealed that dual functionalization at C-13 (TPP) and C-19 (esterification) markedly improved potency and selectivity. Conjugate 23d (C-13 TPP, C-19 benzyl ester) was the most potent (IC₅₀ = 0.19 μM, SI = 15.42) and significantly suppressed Huh7 xenografts growth with favorable safety. Mechanistic studies demonstrated mitochondrial accumulation, TrxR2 inhibition, ROS elevation, and ASK1-mediated apoptosis. To our knowledge, 23d is the first non-electrophilic ent-kaurane derivative to combine mitochondrial targeting with TrxR2 inhibition and in vivo antitumor efficacy, highlighting dual modification as a promising strategy integrating biodistribution engineering with activity optimization for anticancer drug development.