Process for the synthesis of pyrazolopyrimidines

• US 6,962,991 B2
• Filed: 09/12/2001
• Issued: 11/08/2005
• Est. Priority Date: 09/12/2001
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Dempcy, Robert O., Reed, Michael W., Belousov, Yevgeniy S., Adams, A. David
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Henry, Michael C.
• Application Number:
  US09/954,624
• Publication Number:
  US 20030078413A1
• Time in Patent Office: 1,518 Days
• Field of Search: 536/26.1, 536/22.1, 536/18.7, 536/27.14, 536/124, 544/253, 544/245
• US Class Current: 536/26.1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/044 Pyrrole radicals

Abstract

The present invention provides a nucleoside comprising a pyrazolopyrimidine base and a process for producing the same. In particular, the processes of the present invention comprises using a halogenated pyrazolopyrimidine base and removing the halogen after the base is coupled to a sugar moiety. The presence of the halogen on the nucleoside base allows facile and economical production of a large quantity of nucleosides.

Claims

1. A PPG phosphoramidite comprising a photolabile hydroxy protecting group, wherein said phosphoramidite nucleoside is of the formula:

wherein

R¹ is selected from the group consisting of hydrogen and alkyl;

R² is selected from the group consisting of hydrogen, alkyl, and an amine protecting group, or R¹ and R² together form an amine protecting group;

each of Z¹, Z², Z⁴, and Z⁶ is independently selected from the group consisting of hydrogen, halide, alkyl, —

OR¹¹, wherein each R¹¹ is independently selected from the group consisting of hydrogen, alkyl, and a hydroxy protecting group or two R¹¹ groups form a diol protecting group, or Z² and Z⁴ together with the carbon atoms to which they are attached and C-3 carbon atom of the carbohydrate ring form a five-to seven membered ring; and

one of Z³ or Z⁵ is —

OR¹² and the other is —

OR¹³, where R¹² is a photolabile hydroxy protecting group and R¹³ is a phosphoramidite.

2. The PPG phosphoramidite according to claim 1 of the formula:

wherein

R¹, R², Z³ and Z⁵ are those defined in claim 1.

3. The PPG phosphoramidite according to claim 2, wherein Z³ is —

OR¹³ and Z⁵ is —

OR¹², where R¹² is a photolabile hydroxy protecting group and R¹³ is a phosphoramidite.

4. The PPG phosphoramidite according to claim 3, wherein the photolabile hydroxy protecting group is selected from the group consisting of α

-methyl-6-nitropiperonyloxycarbonyl, 2-(2-nitrophenyl)-2-methylethoxycarbonyl, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl, and 3′

,5′

-dimethoxybezoinoxycarbonyl.

5. The PPG phosphoramidite according to claim 4, wherein R¹ and R² together form an amine protecting group.

6. The PPG phosphoramidite according to claim 5, wherein R¹ and R² together form an amine protecting group of the formula:

═

CH—

N(CH₃)₂.

44. The PPG phosphoramidate according to claim 1, wherein R₂ is a photolabile amine protecting group, or R¹ and R² together form a photolabile amine protecting group.

45. The PPG phosphoramidate according to claim 2, wherein R₂ is a photolabile amine protecting group, or R¹ and R² together form a photolabile amine protecting group.

46. The PPG phosphoramidate according to claim 3, wherein R₂ is a photolabile amine protecting group, or R¹ and R² together form a photolabile amine protecting group.

7. A process for producing a non-halogenated nucleoside base containing nucleoside comprising:

(a) contacting a halogenated nucleoside base with an activated sugar under conditions sufficient to produce a halogenated nucleoside base containing nucleoside; and

(b) reducing said halogenated nucleoside base containing nucleoside under conditions sufficient to produce said non-halogenated nucleoside base containing nucleoside.

8. The process of claim 7, wherein said non-halogenated nucleoside base containing nucleoside is purified by recrystallization.

9. The process of claim 7, wherein the yield of said non-halogenated nucleoside base containing nucleoside from said halogenated nucleoside base is at least about 50%.

10. The process of claim 7, wherein said halogenated nucleoside base containing nucleoside reducing step comprises hydrogenation of said halogenated nucleoside base containing nucleoside in the presence of a hydrogenation catalyst.

11. The process of claim 7, further comprising protecting amine and hydroxy groups of the non-halogenated nucleoside base and reacting the resulting product with an activated phosphoramidite to produce a -phosphoramidite nucleoside.

12. The process of claim 11, further comprising incorporating said phosphoramidite nucleoside in an oligonucleoside or an oligonucleotide.

13. A process for producing a nucleoside comprising a hydropyrazolopyrimidine nucleoside base, said process comprising hydrolyzing and reducing or reducing and hydrolyzing an iodopyrazolopyrimidine nucleoside of the formula:

under conditions sufficient to produce a hydropyrazolopyrimidine nucleoside of the formula;

wherein

R¹ is selected from the group consisting of hydrogen and alkyl;

R² is selected from the group consisting of hydrogen, alkyl, and an amine protecting group, or R¹ and R² together form an amine protecting group;

R³ is selected from the group consisting of alkyl, and a hydroxy protecting group; and

each of Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ is independently selected from the group consisting of hydrogen, halide, alkyl, —

OR⁴, wherein each R⁴ is independently selected from the group consisting of hydrogen, alkyl, and a hydroxy protecting group or two R⁴ groups form a diol protecting group, or Y² and Y⁴ together with the carbon atoms to which they are attached to and C-3 carbon atom of the carbohydrate ring form a five-to seven membered ring.

14. The process of claim 13, wherein R¹, R², Y¹, Y², Y⁴, and Y⁶ are hydrogen, and Y³ and Y⁵ are —

OR⁴.

15. The process of claim 14, wherein R⁴ are hydrogen.

16. The process of claim 15 further comprising producing a PPG phosphoramidite of the formula:

from said hydropyrazolopyrimidine nucleoside,

wherein

R¹ is hydrogen and R² is an amine protecting group or R¹ and R² together form an amine protecting group; and

one of R⁹ and R¹⁰ is a phosphoramidite and the other is a hydroxy protecting group,

said PPG phosphoramidite producing step comprises;

(a) (i) contacting said hydropyrazolopyrimidine nucleoside with an amine protecting reagent under conditions sufficient to produce an amine-protected nucleoside of the formula;

(ii) contacting said amine-protected nucleoside with a hydroxy protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula;

 

or

i) contacting said hydropyrazolopyrimidine with a hydroxy protecting reagent under conditions sufficient to produce a monohydroxy protected nucleoside of the formula;

(ii) contacting said monohydroxy protected nucleoside with an amine protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula;

 

wherein

R¹ is hydrogen and R² is an amine protecting group or R¹ and R² together form an amine protecting group; and

one of R⁷ and R⁸ is hydrogen and the other is a hydroxy protecting group; and

(b) contacting said amine/monohydroxy protected nucleoside with an activated phosphoramidite under conditions sufficient to produce said PPG phosphoramidite.

17. The process of claim 16, wherein said amine protecting reagent is selected from the group consisting of N,N-dialkylformamide dialkylacetal, and N,N-dialkylacetamide dialkylacetal.

18. The process of claim 16, wherein said hydroxy protecting reagent is a photolabile hydroxy protecting reagent.

19. The process of claim 18, wherein said photolabile hydroxy protecting reagent is selected from the group consisting of 1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate, 2-(2-nitrophenyl)-2-methylethyl chloroformate, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl chloride and 3′

,5′

-dimethoxybezoinoxyl chloroformate.

20. The process of claim 16, wherein said hydroxy protecting reagent is an acid labile hydroxy protecting reagent.

21. The process of claim 20, wherein said acid labile hydroxy protecting reagent is selected from the group consisting of trityl halide, monomethoxytrityl halide and dimethoxytrityl halide.

22. The process of claim 16, wherein said activated phosphoramidite is of the formula:

wherein

X² is a leaving group.

23. The process of claim 22, wherein X² is selected from the group consisting of halide and diisopropylamino.

24. The process of claim 22, wherein R⁹ is dimethoxytrityl and R¹⁰ is a phosphoramidite moiety of the formula —

P[N(i-Pr)₂]OCH₂CH₂CN.

25. The process of claim 13 further comprising producing said nucleoside of Formula I, wherein said nucleoside of Formula I producing step comprises:

contacting an iodopyrazolopyrimidine of the formula;

with an activated sugar of the formula;

under conditions sufficient to produce said nucleoside of Formula I,

wherein

R¹, R², R³, Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are those defined claim 13; and

X¹ is a leaving group.

26. The process of claim 25 further comprising producing said iodopyrazolopyrimidine nucleoside of Formula I from a pyrimidinone of the formula:

said iodopyrazolopyrimidine nucleoside producing process comprising;

(i) contacting said pyrimidinone with a halogenating agent and a formylating agent under conditions sufficient to produce a dihalopyrimidine carboxyaldehyde of the formula;

wherein

each X³ is independently selected from the group consisting of F, Cl, Br and I;

(ii) contacting said dihalopyrimidine carboxyaldehyde with hydrazine under conditions sufficient to produce a halopyrazolopyrimidine of the formula;

(iii) contacting said halopyrazolopyrimidine with an alkoxide of the formula R³—

OM, wherein R³ is alkyl and M is a metal, to produce an alkoxypyrazolopyrimidine of the formula;

and

(iv) iodinating said alkoxypyrazolopyrimidine with an iodinating agent under conditions sufficient to produce said iodopyrazolopyrimidine.

27. The process of claim 26, wherein said halogenating agent is selected from the group consisting of POCl₃, iodine monochloride, N-iodosuccinamide and SOCl₂.

28. The process of claim 26, wherein said formylating agent is a compound comprising a formyl group attached to a secondary amino group.

29. The process of claim 28, wherein said formylating agent is selected from the group consisting of dimethyl formamide, 1-formylpiperidine, 1-formylmorpholine and triformamide.

30. The process of claim 26, wherein said iodinating agent is selected from the group consisting of iodine monochloride and N-iodosuccinimide.

31. A process for producing a nucleoside comprising:

(a) contacting an iodopyrazolopyrimidine of the formula;

with an activated sugar of the formula;

under conditions sufficient to produce an deoxy iodopyrazolopyrimidine nucleoside of the formula;

(b) producing an amino dihydro hydropyrazolopyrimidine nucleoside from said deoxy iodopyrazolopyrimidine nucleoside, wherein said amino dihydro hydropyrazolopyrimidine nucleoside is of the formula;

wherein

R³ is alkyl;

R⁵ and R⁶ are hydroxy protecting groups; and

X¹ is a leaving group.

32. The process of claim 31, wherein said step of producing said amino dihydro hydropyrazolopyrimidine nucleoside comprises removing said hydroxy protecting groups R⁵ and R⁶;

hydrolyzing —

OR³ group; and

reducing the iodine.

33. The process of claim 31 further comprising:

(c) contacting said amino dihydro hydropyrazolopyrimidine nucleoside with an amine protecting reagent under conditions sufficient to produce an amine protected nucleoside of the formula;

(d) contacting said amine protected nucleoside with a hydroxy protecting reagent under conditions sufficient to produce an amine/monohydroxy protected nucleoside of the formula;

and

(e) contacting said amine/monohydroxy protected nucleoside with an activated phosphoramidite of the formula;

under conditions sufficient to produce a PPG phosphoramidite of the formula;

wherein

R¹ is hydrogen;

R² is an amine protecting group;

or R¹ and R² together form an amine protecting group;

R⁴ is a hydroxy protecting group; and

X² is a leaving group.

34. The process of claim 33, wherein X² is selected from the group consisting of halide, and —

N(i-Pr)₂.

35. The process of claim 33, wherein R¹ and R² together form a nitrogen protecting group of the formula:

═

CH—

N(CH₃)₂.

36. The process of claim 35, wherein R⁴ is selected from the group consisting of an acid labile hydroxy protecting group and a photolabile hydroxy protecting group.

37. The process of claim 36, wherein R⁴ is selected from the group consisting of dimethoxytrityl, trityl, pixyl, 1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl, α

-methyl-6-nitropiperonyloxycarbonyl, 2-(2-nitrophenyl)-2-methylethoxycarbonyl, 2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl and 3′

,5′

-dimethoxybezoinoxycarbonyl.

38. The process of claim 31, wherein said step (b) comprises reducing the iodide by hydrogenation.

39. The process of claim 31, wherein said iodopyrazolopyrimidine is produced from a pyrimidinone of the formula:

said iodopyrazolopyrimidine producing step comprising;

(i) contacting said pyrimidinone with a halogenating agent and a formylating agent under conditions sufficient to produce a dihalopyrimidine carboxyaldehyde of the formula;

wherein each X³ is independently selected from the group consisting of F, Cl, Br and I;

(ii) contacting said dihalopyrimidine carboxyaldehyde with hydrazine under conditions sufficient to produce a halopyrazolopyrimidine of the formula;

(iii) contacting said halopyrazolopyrimidine with an alcohol of the formula R³—

OH to produce an alkoxypyrazolopyrimidine of the formula;

and

(iv) iodinating said alkoxypyrazolopyrimidine with an iodinating agent under conditions sufficient to produce said iodopyrazolopyrimidine.

40. The process of claim 39, wherein said halogenating agent is selected from the group consisting of POCl₃, iodine monochloride, N-idosuccinamide and SOCl₂.

41. The process of claim 40, wherein said halogenating agent is selected from the group consisting of POCl₃ and SOCl₂.

42. The process of claim 39, wherein said formylating agent is selected from the group consisting of dimethyl formamide, 1-formylpiperidine, 1-formylmorpholine and triformamide.

43. The process of claim 39, wherein said iodinating agent is selected from the group consisting of iodine monochloride and N-iodosuccinimide.

47. A PPG phosphoramidite comprising a hydroxy protecting group, wherein said phosphoramidite nucleoside is of the formula:

wherein R¹ is selected from the group consisting of hydrogen and alkyl;

R² is selected from the group consisting of hydrogen, alkyl, and an amine protecting group, or R¹ and R² together form an amine protecting group;

each of Z¹, Z², Z⁴, and Z⁶ is independently selected from the group consisting of hydrogen, halide, alkyl, —

OR¹¹, wherein each R¹¹ is independently selected from the group consisting of hydrogen, alkyl, and a hydroxy protecting group or two R¹¹ groups form a diol protecting group, or Z² and Z⁴ together with the carbon atoms to which they are attached and C-3 carbon atom of the carbohydrate ring form a five-to seven membered ring; and

Z³ is —

OR¹² and Z⁵ is —

OR¹³, where R¹² is a photolabile hydroxy protecting group and R¹³ is a phosphoramidite.

48. The PPG phosphoramidite according to claim 47 of the formula:

wherein R¹, R², Z³ and Z⁵ are those defined in claim 47.

49. The PPG phosphoramidate according to claim 47, wherein R² is a photolabile amine protecting group, or R¹ and R² together form a photolabile amine protecting group.

50. The PPG phosphoramidate according to claim 48, wherein Z³ is OR¹² and R¹² is a photolabile hydroxy protecting group.

51. The PPG phosphoramidite according to claim 50, wherein R¹ and R², taken together with the nitrogen atom to which they are bonded, form a dimethylaminoformamidine group.

52. The PPG phosphoramidate having the formula:

wherein R¹ is hydrogen and R² is an amine protecting group, or R¹ and R² together form an amine protecting group, R⁹ is a phosphoramidite, and R¹⁰ is a hydroxy protecting group.

53. The PPG phosphoramidite according to claim 52, wherein the amine protecting group is an acid-labile protecting group.

54. The PPG phosphoramidite according to claim 53, wherein R¹ and R² together form an acid-labile amine protecting group, and R¹⁰ is an acid-labile hydroxy protecting group.

55. The PPG phosphoramidate according to claim 54, wherein R¹ and R², taken together with the nitrogen atom to which they are bonded, form a dimethylaminoformamidine group.

56. The PPG phosphoramidate according to claim 55, having the formula:

wherein “

DMTr”

represents a dimethoxytrityl group.