The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.

Background: Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications.

Objective: To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells.

Methods: In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100μM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically.

Results: The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7μM) and ornithine (IC50=10μM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain.

Conclusions: The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.