Synthesis and biological evaluation of novel C-30 modified lupane triterpenoids selectively cytotoxic against cancer cells

Abstract

In this work, we describe a new synthetic pathway to 30-methylidyne derivatives of lupane-triterpenoids that were consequently used as excellent substrates for the preparation of new compounds via Sonogashira coupling and via Cu^I catalyzed Azide-Alkyne 1,3-dipolar cycloadditions. Reactions had moderate to excellent yields. All prepared compounds were tested for their in vitro cytotoxicity on six cancer and two non-cancer cell lines. The most active compounds were triazoles with free 3-OH and 28-OH groups (betulin analogues) and among them, conjugate with triazole substituted by furan 16j was the best with IC₅₀ of 2.68 μM against CCRF-CEM and therapeutic index of 18.66. Mechanistic studies revealed that both 16g and 16j induce apoptosis in CCRF-CEM cells, as confirmed by annexin V/PI staining. Cell cycle analysis showed that 16j causes pronounced S phase arrest, while 16g modulates G1/S transition in a concentration-dependent manner. Moreover, 16g strongly suppressed DNA and RNA synthesis, whereas 16j paradoxically increased RNA synthesis despite replication inhibition. Both compounds triggered mitochondrial hyperpolarization, suggesting early mitochondrial involvement in their apoptotic mechanism. Western blot analysis supported these findings, revealing γH2AX induction and PARP cleavage, alongside distinct modulation of key regulators: 16j upregulated p21 and phospho-Chk1 (Ser345), while 16g downregulated both at high dose, consistent with checkpoint activation versus checkpoint bypass. These findings support 16g and 16j as promising candidates for further anticancer drug development.