New C-30 secondary and tertiary amines of betulin with selective antiproliferative activity toward hematological malignancies - Synthesis and biological evaluation

A novel synthetic approach for preparation of secondary and tertiary amine derivatives of betulin was developed. The synthesis starts from commercially available betulin diacetate ( 1 ). The final step of the sequence relied on a Tsuji–Trost reaction. To explore the scope of this procedure, we prepared a variety of substituted secondary and tertiary amines. The reaction successfully afforded 31 out of 32 target products, with only one example failing to react. All the prepared compounds were tested for in vitro cytotoxic activity against 6 cancer cell lines and 2 non-cancer cell lines. Several derivatives exhibited pronounced and selective antiproliferative activity toward hematological malignancies, with compounds 5q , 5s , and 5t showing the most potent effects in CCRF-CEM and K562 cells while maintaining favorable selectivity over non-cancer fibroblasts. Mechanistic studies revealed that these compounds induce replication stress, leading to S-phase cell-cycle arrest and suppression of DNA synthesis. This was accompanied by activation of DNA damage response signaling, as indicated by increased γH2AX levels and upregulation of Cyclin E1 and p21. Further analysis demonstrated involvement of the intrinsic apoptotic pathway, as evidenced by mitochondrial membrane depolarization and increased expression of the pro-apoptotic protein BAX. In contrast, limited caspase activation and minimal PARP cleavage at equitoxic concentrations indicate that cells predominantly undergo early apoptotic commitment rather than full apoptotic execution. Collectively, these findings identify replication stress-mediated cell death as a key mechanism of action and highlight aminoderivatives of betulin diacetate as promising candidates for targeting proliferation-associated vulnerabilities in leukemia cells.