Iodine → HCID 131724574, compound

To a stirring suspension of 3′-deoxy-2′-acetoxycytidine (15.0 mg, 0.056 mmol) in dry DMF (0.60 ml) was added dry pyridine (0.20 ml) followed by a freshly prepared solution of 2-chloro-4 H-1,3,2-benzodioxaphosphorin-4-one 0.5 M in 1,4-dioxane (111 μl, 0.056 mmol). The mixture was stirred 30 minutes at room temperature, then tributylamine (36 μl, 0.152 mmol) and a solution of tributylammonium pyrophosphate 0.5 M in DMF (101 μl, 0.051 mmol) were added simultaneously. The mixture was stirred another 30 minutes. A solution of I2 1% in pyridine/H2O (98:2) (1.01 ml, 0.081 mmol of I) was added and the mixture was stirred 30 minutes. The excess of iodine was destroyed by adding 0.2 ml of aqueous sodium bisulfite 5%. The mixture was stirred 15 minutes, then it was concentrated under reduced pressure to remove all solvents. The residue was dissolved in water, washed two times with methylene chloride and once with ethyl acetate. The aqueous layer was concentrated and purified by charcoal column as follow: about 400 mg of charcoal, placed over a thin layer of Celite in a funnel with fritted disk, was prewashed by passing methanol, then deionized water (by vaccuum). The crude residue was diluted in a minimum of water, acidified to pH 1-2 by adding few drops of HCl 1N, then placed on the top of the charcoal column. The column was eluted with deionized water (35 ml) in order to remove inorganic salts, then 0.5 N ammonia (15 ml) to collect the desired triphosphate. The collected triphophate was concentrated and diluted in deionized water (1 ml) and concentrated NH4OH (2 ml). The mixture was stirred one hour at room temperature to cleave the acetyl group, then concentrated to dryness. The residue was purified on a pad of C18 RP silica gel eluting with deionized water (the desired triphosphate comes out fast). The fractions containing the desired triphosphate were collected and lyophilized to give the 3′-deoxycytidine 5′-triphosphate triammonium salt as a yellowish solid (18 mg, 69% yield, purity >85% evaluated by 1H and 31P-NMR). 1H NMR (400 MHz, D2O) δ: 7.90 (d, 1 H, 7.5 Hz), 5.99 (d, 1 H, 7.5 Hz), 5.73 (s, 1 H), 4.55 (s, 1 H), 4.35 (d, 1 H, 5.0 Hz), 4.26 (m, 1 H), 4.04 (m, 1 H), 2.05 (m, 1 H), 1.94 (m, 1 H) ppm. 31P NMR (162 MHz, D2O) δ: −5.9 (br.s), −10.4 (d, 19 Hz), −21.5 (br.s) ppm. In a similar manner, the compounds of the invention can be obtained.

1. stirringThe mixture was stirred another 30 minutes
2. stirringthe mixture was stirred 30 minutes
3. customThe excess of iodine was destroyed
4. additionby adding 0.2 ml of aqueous sodium bisulfite 5%
5. stirringThe mixture was stirred 15 minutes
6. concentrationit was concentrated under reduced pressure
7. customto remove all solvents
8. dissolutionThe residue was dissolved in water
9. washwashed two times with methylene chloride
10. concentrationThe aqueous layer was concentrated
11. custompurified by charcoal column
12. customabout 400 mg of charcoal, placed over a thin layer of Celite in a funnel with fritted disk
13. additionThe crude residue was diluted in a minimum of water
14. additionby adding few drops of HCl 1N
15. washThe column was eluted with deionized water (35 ml) in order
16. customto remove inorganic salts
17. custom0.5 N ammonia (15 ml) to collect the desired triphosphate
18. concentrationThe collected triphophate was concentrated
19. additiondiluted in deionized water (1 ml)
20. stirringThe mixture was stirred one hour at room temperature
21. concentrationconcentrated to dryness
22. customThe residue was purified on a pad of C18 RP silica gel eluting with deionized water (the desired triphosphate
23. additionThe fractions containing the desired triphosphate
24. customwere collected

ORD record

ord-0783e534046546d288dc02b59f432f09

Source dataset

uspto-grants-2004_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06784161B2