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The first representatives of F16-conjugated pentacyclic triterpenoids, betulin and betulinic, ursolic, oleanolic, and glycyrrhetic acid derivatives, were synthesized. The triterpene core was linked, at the С-3, С-28, or С-30 position, to one or two mitochondria-targeting delocalized lipophilic cations F16 via butane or triethylene glycol spacer. The human cancer cell lines U937 (leukemic monocyte lymphoma), K562 (chronic myeloid leukemia), and Jurkat (T-lymphoblastic leukemia), and a human nonmalignant fibroblast cell line were used to evaluate the cytotoxic activities of the products. Most of the obtained conjugates showed considerable enhancement of the antitumor action in comparison with the parent betulinic acid (≈100−200-fold) and a markedly higher cytotoxic effect against tumor cell lines over healthy fibroblast cells. In the series of test compounds, F16 conjugates with betulin and betulinic acid 6, 8, and 11 were most selective, showing acceptable values of selectivity index (≥10).

The paper examines the molecular mechanisms of the cytotoxicity of conjugates of betulinic acid with the penetrating cation F16. The in vitro experiments on rat thymocytes revealed that all the obtained F16-betulinic acid derivatives showed more than 10-fold higher cytotoxicity as compared to betulinic acid and F16. In this case, 0.5–1 μM of all conjugates showed mitochondria-targeted action, inducing superoxide overproduction and reducing the mitochondrial potential of cells. Experiments on isolated rat liver mitochondria revealed the ability of conjugates to dose-dependently reduce the membrane potential of organelles, as well as the intensity of respiration and oxidative phosphorylation, which is also accompanied by an increase in the production of hydrogen peroxide by mitochondria. It was shown that these actions of derivatives may be due to several effects: the reversion of ATP synthase, changes in the activity of complexes of the respiratory chain and permeabilization of the inner mitochondrial membrane. All compounds also demonstrated the ability to induce aggregation of isolated rat liver mitochondria. Using the model of lecithin liposomes, we found that the F6 conjugate (2 μM) induces the permeability of vesicle membranes for the fluorescent probe sulforhodamine B. High concentrations (25 μM) of the F6 derivative have been found to induce dynamic processes in the liposome membrane leading to aggregation and/or fusion of vesicle membranes. The paper discusses the relationship between the mitochondria-targeted effects of F16-betulinic acid conjugates and their cytotoxicity.

The BODIPY family fluorophores have been covalently linked to numerous classes of biomolecules, including proteins, carbohydrates, fatty acids, and steroids. Still, only two research papers on the synthesis and fluorescent biological analysis of BODIPY-labeling of triterpenoid compounds have been reported so far. In these works, fluorescent pentacyclic triterpene conjugates have been prepared by covalent binding to the known BODIPY-FL fluorophore through the 3-OH and 17-COOH functional groups. Here we aimed to work out a new approach for the synthesis of BODIPY-triterpenoid acids conjugates avoiding covalent binding of the triterpene core to the BODIPY-platform at the C-3 and C-17 positions. Novel BODIPY-lupane triterpenoid conjugates bearing a fluorescent marker at the C-2 position of ring A of the triterpene core were obtained via the Sonogashira reaction as a key step. The starting compounds in the cross-coupling reaction were C-2 propynyl derivatives of betulinic or betulonic acids and fluorescent dyes with an iodo-group at C-2 or meso position of BODIPY-platform. The elaborated coupling procedure might have applicability in the synthesis of fluorescent-labeled triterpenoid conjugates suitable for biological assays