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67651099452
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note
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4 solvent was removed. Subsequent column chromatography on silica gel using eluents mixture chloroform/ethyl acetate (4/1) give desired compounds (in the case of betulin some of the disubstituted products were isolated). Whether to obtain disubstituted betulin derivatives exactly same protocol was applied, but 4 molar excess of the Boc-amino acid and CDI was used toward betulin. In case of the small side chains of amino acids (Gly, Ala) this allows to obtain only disubstituted derivatives, whereas in the case of more bulky substituents mixture of mono- and disubstituted derivatives was obtained. Reaction time in this case was 48 h. Their separation was achieved using above eluent system.
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10
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67651103310
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note
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We have found that application of the 25% TFA in dichloromethane or 3 M HCl in MeOH results in the partial decomposition of the compounds or/and formation of unidentified side products. Compounds after deprotection were suspended in diethyl ether, sonicated and filtered. It allows removing of all unreacted Boc protected derivatives as they are very well soluble in ether.
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12
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67651096464
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note
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Cell cytotoxicity sulforhodamine B (SRB) assay: SRB method was used for the toxicity screening of all compounds to adherent cells in a 96-well format, based on the measurement of cellular protein content. After an incubation period for 24 h, cell monolayers were fixed with 50% (wt/vol) trichloroacetic acid for 1 h in 4 °C and subsequently washed (five times) in a cold water and desiccated. Cells staining was carried out using 0.4% SRB in 1% acetic acid for 30 min in room temperature. After incubation, excess of the dye was removed by repeatedly washing (four times) with 1% (vol/vol) acetic acid. Plates were desiccated and the protein-bound dye was dissolved in 10 mM Tris base (pH 10.5) solution for absorbance determination at 562 nm using a microplate reader (Tecan, Infinite M200).
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13
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66349104551
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67651087821
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16
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67651125427
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note
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2 for 20 min, and then silver staining was performed. For scoring the comet pattern, 100-200 nuclei on each slide were counted and assigned to a category of apoptotic or normal cells.
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