Rational Design of Betulin-Based Hybrids as Multi-Target Inhibitors of Anti-Apoptotic BCL Proteins ^†

Triterpenes such as betulin are intensively studied for their intrinsic pro-apoptotic effects in cancer, providing a privileged scaffold for anti-tumor drug design [1,2]. As part of this study, we built a focused virtual library in which the 3β- and 28-hydroxyl groups of betulin were tethered via ω-chloro-alkyl linkers of variable length and then esterified with either hydroxybenzotriazole or 1,2,4-triazole-3-thiol and 1,3,4-oxadiazole-2-thiol fragments bearing diverse 5-substituted groups. Docking analysis against the anti-apoptotic proteins BCL-2 (PDB 4LVT), BCL-XL (2YXJ), and MCL-1 (5LOF) was carried out using both AutoDock Vina [3] and Glide scoring functions. Six independent rankings (3 targets × 2 scoring functions) were generated; compounds appearing in the top 20 (by binding score) of at least four lists were advanced. This consensus ranking yielded five hits: two hydroxybenzotriazole esters connected through two- and four-carbon spacers, two 5-(2-naphthyl)-1,3,4-oxadiazole-2-thiol esters with analogous spacers, and one 5-(4-chlorophenyl)-1,3,4-oxadiazole-2-thiol linked by a single-carbon bridge. All candidates underwent 100 ns molecular dynamics simulations in an explicit solvent with each protein partner. Root-mean-square deviation trajectories, hydrogen-bond occupancy, and MM/GBSA free-energy decomposition confirmed conformational stability and persistent anchoring within the BH3-binding grooves. Our results support the capacity of tailored betulin derivatives to engage multiple anti-apoptotic BCL family members, which is a strategy expected to circumvent resistance mediated by functional redundancy. The selected hybrids can then be synthesized and their pro-apoptotic activity validated in relevant cancer cell models.