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  • All chemicals were purchased from

    2022-04-26

    All chemicals were purchased from Aladdin Chemical Co. All reac-tions were carried out under anhydrous conditions. Chromatography was performed on HPTLC silica gel 60 F254. HNMR spectra were re-corded on a Bruker AVANCE 300 MHz spectrometer, and CNMR spectra were recorded on Bruker AVANCE 400 MHz spectrometer. Mass spec-tral data were recorded on LCMS-2020 (SHIMADZU) mass spectro-meter.
    To a solution of N-Boc-L-threonine 2 (2.19 g, 10 mmol) in anhydrous THF (60 mL) 1-hydroxybenzotriazole hydrate (HOBt) (1.35 g, 10 mmol) was added at 0 °C. The pH value of the solution was adjusted to 8–9 with 4-methylmorpholine. After the reaction mixture was stirred for 5 min, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl) (2.20 g, 11 mmol) and a solution of alkyl amine in anhydrous THF (5 mL) was added. The reaction mixture was stirred at 0 °C for 2 h and at 20 °C overnight. After evaporation of the mixture in vacuo, the residue was dissolved in ethyl acetate (60 mL), and the solution was washed successively with saturated NaHCO3, 5% KHSO4, and saturated NaCl. The organic phase was separated and dried over anhydrous MgSO4. After filtration and evaporation in vacuo, desired (2S,3R)-tert-butyl-3-hydroxy-1-oxo-1-(alkyl)butan-2-ylcarbamate.3a-d was ob-tained. To a solution of (2S,3R)-tert-butyl-3-hydroxy-1-oxo-1-(alkyl) butan-2-ylcarbamate 3 in CH2Cl2 (70 mL) trifluoroacetic CFTRinh-172 (10.0 mL) was added at 0 °C. The reaction mixture was stirred at 20 °C for 4 h. After evaporation in vacuo, the residue was dripped into the 1 N NaOH (100 mL), and generate precipitation. Filtration and dried in vacuo led to desired L-threoninamide 4a-d.
    To a solution of carboxylic acid (1.0 mmol) in anhydrous THF
    methylmorpholine. The reaction mixture was stirred at 0 °C for 2 h and overnight at room temperature. After evaporation of the mixture in vacuo, the residue was dissolved in ethyl acetate (50 mL). The solution was washed successively with saturated NaHCO3, 5% KHSO4, and sa-turated NaCl. The organic phase was separated and dried over anhy-drous MgSO4. After filtration and evaporation in vacuo, residue was purified by recrystallization in petroleum ester/ethyl acetate to give the desired ceramide analogues 5xa-xj.
    At 0 °C, to a solution of 1.0 mmol of (2S,3R)-2-amino-3-hydroxy-N-alkylbutanamide (4a-4d) and 2.0 mmol of pyridine in anhydrous CH2Cl2 (20 mL), 10 mL CH2Cl2 solution of heterocyclic sulfonyl chloride or aryl sulfonyl chloride was dripped added. The mixture was stirred at 0 °C for 2 h and overnight at room temperature. After eva-poration in vacuo, the residue was dissolved in ethyl acetate (50 mL). The solution was washed successively with saturated NaHCO3, 5% KHSO4, and saturated NaCl, and the organic phase was separated and dried over anhydrous MgSO4 for 2 h. After filtration and evaporation in vacuo, residue was purified by recrystallization in petroleum ester/ethyl acetate to give the desired ceramide analogues 5xk-xm.
    2.1.4. Synthetic procedure for control compound 6
    For comparing carbon skeleton of L-threoninamide with L-ser-inamide, control compound 6 was prepared by the same protocol with ceramide analogues 5.
    2.2. Cell viability assay
    The effect of ceramide analogues on cell viability was measured by MTT assay according to the manufacturer’s instructions (ATCC, Manassas VA). Briefly, six human cancer cells (Liver Cancer HepG2, Squamous Carcinoma A431, Pancreatic Cancer SA, Breast Cancer MCF-7, Ovarian Cancer SiHa and Colon Cancer SW620) were seeded in 96-well plates (7 × 103 cells/well) for 24 h. 47 Ceramide analogues were then added to final concentrations of 0 to 200 μM, and the cells were cultured for another 48 h, followed by MTT assay.
    2.3. Apoptosis assay
    Human Colon Cancer cells SW620 were treated with the indicated ceramide analogues at doses of their IC50 for 8 h or 24 h. Cells were then fixed in ice-cold 70% ethanol for 15 min, washed with PBS and stained with Hoechst 33,342 (Sigma) for 20 min in the dark. Morphologic changes were analyzed under a fluorescence microscope. DMSO used as vehicle control.
    2.4. TRAIL-mediated apoptosis
    Five human cancer cells(Liver Cancer HepG2, Colon Cancer SW620、LS411N, Breast Cancer MCF-7, Cervical Cancer Hela) were seeded in 96-well plates and cultured for 24 h. Ceramide analogues, TRAIL (100 ng/ml) or both ceramide analogue and TRAIL were added to the culture for 48 h. Cell death was measured by MTT assay.
    2.5. Western blotting analysis
    Western blotting analysis was performed essentially as previously described.32 Anti-xIAP antibody was obtained from Cell signaling, anti-Bcl-xL was obtained from BD Biosciences, and anti-β-actin was from Sigma.