Glycoconjugation of Betulin Derivatives Using Copper-Catalyzed 1,3-Dipolar Azido-Alkyne Cycloaddition Reaction and a Preliminary Assay of Cytotoxicity of the Obtained Compounds

Pentacyclic lupane-type triterpenoids, such as betulin and its synthetic derivatives, display a broad spectrum of biological activity. However, one of the major drawbacks of these compounds as potential therapeutic agents is their high hydrophobicity and low bioavailability. On the other hand, the presence of easily transformable functional groups in the parent structure makes betulin have a high synthetic potential and the ability to form different derivatives. In this context, research on the synthesis of new betulin derivatives as conjugates of naturally occurring triterpenoid with a monosaccharide via a linker containing a heteroaromatic 1,2,3-triazole ring was presented. It has been shown that copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction (CuAAC) provides an easy and effective way to synthesize new molecular hybrids based on natural products. The chemical structures of the obtained betulin glycoconjugates were confirmed by spectroscopic analysis. Cytotoxicity of the obtained compounds was evaluated on a human breast adenocarcinoma cell line (MCF-7) and colorectal carcinoma cell line (HCT 116). The obtained results show that despite the fact that the obtained betulin glycoconjugates do not show interesting antitumor activity, the idea of adding a sugar unit to the betulin backbone may, after some modifications, turn out to be correct and allow for the targeted transport of betulin glycoconjugates into the tumor cells.


1,2,3,4,6-Penta-O-acetyl--D-glucopyranose 9a
Dry sodium acetate (36.3 mmol, 3.0 g) and anhydrous acetic anhydride (278.0 mmol, 26 mL) were placed in a three-necked flask equipped with a reflux condenser. Anhydrous glucose (27.8 mmol, 5.0 g) was added in portions and then the mixture was stirred and heated to reflux for 1 h. The reaction progress was monitored on TLC in an eluents system toluene:AcOEt (1:1). After that the mixture was cooled and poured into ice water and stirred vigorously until until the acetic anhydride was completely hydrolyzed and the product precipitated. The product was separated by filtration in vacuo.

1,2,3,4,6-Penta-O-acetyl--D-galactopyranose 9b
Dry sodium acetate (36.3 mmol, 3.0 g) and anhydrous acetic anhydride (278.0 mmol, 26 mL) were placed in a three-necked flask equipped with a reflux condenser. Anhydrous galactose (27.8 mmol, 5.0 g) was added in portions and then the mixture was stirred and heated to reflux for 1 h. The reaction progress was monitored on TLC in an eluents system toluene:AcOEt (1:1). After that the mixture was cooled and poured into ice water and stirred vigorously until until the acetic anhydride was completely hydrolyzed and the product precipitated. The product was separated by filtration in vacuo.

2,3,4,6-Tetra-O-acetyl--D-glucopyranosyl bromide 11a
1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose 9a (7.7 mmol, 3.0 g) was dissolved in glacial acetic acid (15 mL). The flask was placed into an ice bath, and then a 33 % solution of HBr in AcOH (158.0 mmol, 9 mL) was added dropwise to the mixture. Reaction was carried out on the magnetic stirrer for 1 h at room temperature. The reaction progress was monitored on TLC in an eluents system toluene:AcOEt (1:1). After the reaction was completed, the solution was diluted with CH2Cl2 (50 mL) and then poured into ice-water (70 mL). The two-phase system was separated, the aqueous phase was washed with CH2Cl2 (2 x 50 mL) and the combined organic phases were washed with saturated NaHCO3 solution (1 x 50 mL) and brine (2 x 40 mL). The organic phase was dried with MgSO4, filtered and the filtrate was evaporated in vacuo. The crude products were purified using column chromatography (toluene:AcOEt, gradient: 10:1 to 6:1).
Product 11a was obtained as a white solid (

2,3,4,6-Tetra-O-acetyl--D-galactopyranosyl bromide 11b
1,2,3,4,6-Penta-O-acetyl-β-D-galactopyranose 9b (7.7 mmol, 3.0 g) was dissolved in glacial acetic acid (15 mL). The flask was placed in an ice bath, and then a 33 % solution of HBr in AcOH (158.0 mmol, 9 mL) was added dropwise to the mixture. Reaction was carried out on the magnetic stirrer for 1 h at room temperature. The reaction progress was monitored on TLC in an eluents system toluene:AcOEt (1:1). After the reaction was completed, the solution was diluted with CH2Cl2 (50 mL) and then poured into ice-water (70 mL). The two-phase system was separated, the aqueous phase was washed with CH2Cl2 (2 x 50 mL) and the combined organic phases were washed with saturated NaHCO3 solution (1 x 50 mL) and brine (2 x 40 mL). The organic phase was dried with MgSO4, filtered and the filtrate was evaporated in vacuo. The crude products were purified using column chromatography (toluene:AcOEt, gradient: 10:1 to 6:1).