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34 mL of BSTFA are added to a suspension of 6.4 g of azacytosine in 300 mL of acetonitrile. The mixture is brought to 50C and it is stirred for 90 minutes, It is cooled to ambient temperature, 6.3 mL of trimethylsilyltriflate and 11.1 g of 1-chloro-3,5-di-p-toluoyl-2-deoxy-D-ribofuranose are added to the solution, in about ten minutes. It is stirred for 140 minutes, 400 mL of methanol are added controlling the temperature in an ice bath. Concentration is carried out at low pressure until a viscous residue is obtained, 400 mL of methanol are added, it is heated to 50C and 36 mL of 30% solution of sodium methylate in methanol are added. It is stirred for 1 hour, it is cooled to 0C, it is stirred to complete precipitation and the solid is filtered by washing it on the filter with methanol. 2.3 g of decitabine with HPLC purity (UV 254 nm) of 99.2% are obtained after drying at low pressure. After recrystallisation from methanol the HPLC purity (UV 254 nm) is 99.8%.
Example 9 Preparation of Decitabine 34 mL of BSTFA are added to a suspension of 6.4 g of azacytosine in 300 mL of acetonitrile. The mixture is brought to 50 C. and it is stirred for 90 minutes. It is cooled to ambient temperature, 6.3 mL of trimethylsilyltriflate and 11.1 g of 1-chloro-3,5-di-p-toluoyl-2-deoxy-D-ribofuranose are added to the solution, in about ten minutes. It is stirred for 140 minutes, 400 mL of methanol are added controlling the temperature in an ice bath. Concentration is carried out at low pressure until a viscous residue is obtained, 400 mL of methanol are added, it is heated to 50 C. and 36 mL of 30% solution of sodium methylate in methanol are added. It is stirred for 1 hour, it is cooled to 0 C., it is stirred to complete precipitation and the solid is filtered by washing it on the filter with methanol. 2.3 g of decitabine with HPLC purity (UV 254 nm) of 99.2% are obtained after drying at low pressure. After recrystallisation from methanol the HPLC purity (UV 254 nm) is 99.8%.
Step 2. Creating the nucleoside analog in preparation for addition of a side chain (FIG. 4). To a mixture of powdered KOH (2.6 g) in acetonitrile (150 mL) was added TDA-1 (0.27 mL) and the mixture was stirred for 5 min. To this mixture was added the amino chloro 7-deazapurine (3, 3.6 g) and the mixture was stirred for 5 min; the chloro-sugar (4, 9.1 g) was then added to the mixture. Stirring was continued for another 40 min (reaction complete by TLC). The mixture was adsorbed on silica gel and eluted with MC/EA until all the product was eluted. The filtrate was evaporated with silica gel and purified on silica column (MC:EA=20:1 to 10:1) to give a white solid (9.0 g, 17.3 mmol, 80%) which contains small amount of impurities. It was used for the next reaction without further purification (The actual yield could be around 75%) (0034) A mixture of the starting material (9.6 g) and pivaloyl chloride (11.5 mL) in pyridine (95 mL) was stirred at rt for 2 h. TLC showed reaction was complete (Almost one spot, which is higher than starting material). Volatiles were removed by rotary evaporation and the residue was dissolved in dichloromethane/aq. HCl and extracted with dichloromethane. The organic layer was dried and purified on silica column to give a white solid (solvent is not easily removed by evaporation). It was dissolved in DMF (150 mL) and treated with N-iodosuccinimide (NIS, 6.2 g) and stirred at rt overnight. The mixture was mixed with water and extracted with dichloromethane. The organic layer (contained some DMF) was evaporated and purified on silica column. (0035) Rf=0.3 (MC:Hx:EA=1:1:0.2) After column chromatography, the appropriate fractions were combined and evaporated to give white solid. It was treated with ethyl acetate/hexane and the resulting solid was filtered and dried. The filtrate was evaporated and stored at rt overnight. This was treated with ethyl acetate/hexane and the resulting solid was filtered. The filtered solids were combined. The filtrate was analyzed by TLC to confirm the existence of the product. The total yield was around 9.7 g, 13.3 mmol, 72%.