Allobetulin 3-benzoate was obtained as an unexpected byproduct during the acid-mediated protection–deprotection of betulin. Single-crystal X-ray diffraction analysis shows that it crystallizes in the triclinic space group P1 with Z = 2 and contains two crystallographically independent molecules in the asymmetric unit. Compared with allobetulin (P2₁, Z = 4) and allobetulin 3-acetate (C2, Z = 4), the benzoyl substitution at C-3 induces distinct molecular packing and intermolecular interaction patterns, highlighting the influence of steric and electronic effects on crystal architecture.

In this study, we isolated and structurally characterized a diastereoisomer (1- R) of the betulonic acid–zidovudine (AZT) conjugates 1- S, a previously reported anticancer lead. The absolute configuration at the C-2 position was determined via 1D/2D nuclear magnetic resonance (NMR) and optical rotatory dispersion (ORD). Both diastereomers exhibited potent anticancer activity against a panel of human cancer cell lines, with particular efficacy against SMMC-7721 (hepatoma) and A549 (lung carcinoma) cells (IC50 = 3.38–4.09 μM). Mechanistic investigations revealed that 1- R induces cell death primarily through a caspase-dependent apoptotic pathway. Notably, the necroptosis inhibitor necrostatin-1 (Nec-1, an inhibitor of necroptosis) provided substantial protection, suggesting that necroptosis may play a more prominent role in 1- R compared to that of 1- S, although this requires direct comparative validation in future studies. The autophagy inhibitor 3-methyladenine (3-MA, an autophagy inhibitor) partially attenuated cell death, indicating a modulatory role for autophagy. These findings highlight 1- R as a promising anticancer agent. Despite its initial isolation as a low-yield byproduct (3.2%), the comparable activity of 1- R to 1- S, coupled with the conformational plasticity of the C-2 linker, simplifies synthetic requirements and supports its further optimization.