Preparation of phosphorothioate and boranophosphate oligomers

• US 6,160,109 A
• Filed: 06/30/1998
• Issued: 12/12/2000
• Est. Priority Date: 10/20/1995
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated, McGill University
• Original Assignee: ISIS Pharmaceuticals Incorporated, McGill University
• Inventors: Just, George, Manoharan, Muthiah, Xin, Zhili, Jin, Yi, Marsault, Eric, Wang, Jianchao
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): OWENS JR, HOWARD V
• Application Number:
  US09/109,215
• Time in Patent Office: 896 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.32
• US Class Current: 536/25.3
• CPC Class Codes: C07F 9/1411 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/65846 the phosphorus atom being part of a six-membered ring which may be condensed with another ring system
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 9/04 Cyclic acetals
• CPC Class Codes: C07H 9/06 the hetero ring containing nitrogen as ring hetero atoms

Abstract

Methods and intermediates for the preparation of oligomers containing diastereomerically enriched phosphorothioate and boranophosphate linkages are disclosed.

Claims

1. A method for synthesizing an oligomer having a boranophosphate linkage comprising the steps of:

reacting a first synthon of Formula I;

##STR118## wherein;

Q is independently O or S;

R¹ is a hydroxyl protecting group;

R² is a chiral auxiliary of formula

--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ;

R³ is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl, trihalomethyl,

--CH₂ R₄,

--CH₂ Si(R⁴)₃, or --CH₂ --SO_k R⁴ where k is 0, 1 or 2;

R⁴ is independently alkyl, aryl, aralkyl or alkaryl having up to 15 carbon atoms, --N(R₇₀)--C(═

O)--R₇₁, --S--C(═

O)--R₇₀, or --O--C(═

O)--O--N(R₇₀)(R₇₁);

R₇₀ and R₇₁ are each independently alkyl, α

-halo substituted alkyl, aralkyl, α

-halo substituted aralkyl, or aryl substituted with up to three electronegative groups;

R⁵ is H, --CN, --Si(R⁴)₃, SO_k R⁴ or halogen;

R⁸ is H or methyl;

R¹⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

or when R⁸ and R¹⁶ are each H, and R³ and R⁵, together, form one of the structures ##STR119## wherein;

R¹⁰ and R¹¹ are H, alkyl having from 1 to about 10 carbons, --CH₂ C(═

O)OR²², --CH₂ CN, --CH₂ Si(CH₃)₃,

or o- or p--C₆ H₄ --R²¹ ;

R²¹ is hydrogen, --O--C(═

O)CH₃, alkoxy having from 1 to about 10 carbons, --NO₂, or --N(R²²)₂ ;

R²² is independently H or alkyl having from one to about 10 carbon atoms;

p is 1 or 2;

Z¹ and Z² are independently halogen, CN, --Si(CH₃)₃, and --C(═

O)OR²² ;

R³⁰ is hydrogen, --O--C(═

O)CH₃, alkoxy having from 1 to about 10 carbons, or --O--Si(R₄)₃ ;

R⁶ is H, alkyl or aralkyl having up to 15 carbon atoms;

or R⁵ and R⁶, together with the atoms to which they are attached, form a 5 or 6 membered ring;

R⁷ is alkyl or aralkyl having up to 15 carbon atoms;

or R⁶ and R⁷, together, form one of the structures ##STR120## wherein V, T, and Z are independently CH or N;

R₄₁, R₄₂, R₄₃, and R₄₄ are each independently H or an electronegative group;

B is a nucleobase; and

n is an integer from 0 to 50;

with a second synthon of Formula II;

##STR121## wherein;

R⁹ is a hydroxyl protecting group or a linker connected to a solid support; and

m is an integer from 0 to 50;

in the presence of a catalyst, for a time and under reaction conditions effective to form a third synthon of Formula III;

##STR122## and contacting said third synthon with a boronating agent to form an oligomer of Formula IV;

##STR123## wherein D is a boranophosphate linkage having the formula;

##STR124## wherein G is a borano group.

2. The method of claim 1, wherein said borano group G has the formula BH(R'"'"') wherein each R'"'"' is independently selected from the group consisting of H;

CN;

carboxyl;

carboxyl salts;

C_1-6 alkyl;

alkaryl;

aryl;

alkoxycarbonyl and alkylaminocarbonyl each optionally substituted with OH, halogen, amino or CN and said boronating agent is a compound having the formula R"₂ S--BH(R'"'"')₂ wherein each R" is independently C_1-6 alkyl.

3. The method of claim 2, wherein said boronating agent is selected from the group consisting of BH₃ --Me₂ S, BH(Me)₂ --Me₂ S, BH₂ Me--Me₂ S and BH₂ CN--Me₂ S.

4. The method of claim 2, wherein said boronating agent is BH₃ --Me₂ S.

5. The method of claim 1 wherein said boranophosphate linkage is diastereomerically enriched.

6. The method of claim 1 wherein about 75% of said boranophosphate linkage is in a single stereoisomeric form.

7. The method of claim 1 wherein about 85% of said boranophosphate linkage is in a single stereoisomeric form.

8. The method of claim 1 wherein about 95% of said boranophosphate linkage is in a single stereoisomeric form.

9. The method of claim 1 wherein said boranophosphate linkage is in a single stereoisomeric form.

10. The method of claim 1 wherein n is 0.

11. The method of claim 1 wherein said first synthon is in a single stereoisomeric form.

12. The method of claim 1 further comprising removing said R¹ group from said boranophosphate.

13. The method of claim 1 further comprising removing said chiral auxiliaries.

14. The method of claim 1 wherein said compound of Formula IV contains a plurality of said boranophosphate linkages.

15. The method of claim 1 wherein said first and second synthons are reacted at a temperature of from about -20°

C. to about 40°

C.

16. The method of claim 1 wherein said first and second synthons are reacted at a temperature of from about -15°

C. to about 0°

C.

17. The method of claim 1 wherein said first synthon is formed by reacting a compound of Formula V:

##STR125## with an azaphospholane of Formula VIa;

##STR126## wherein R³ -R⁸ are as defined above; and

X is halogen, dialkylamino, imidazole, triazole or substituted phenoxy wherein said substituents are electron withdrawing and wherein R¹, R², and R¹⁶ are as defined above.

18. The method of claim 17 wherein said electron withdrawing substituents are halogen or nitro.

19. The method of claim 17 wherein said azaphospholane is produced by reacting a reagent of formula HO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ and a phosphorus trihalide, phosphorus tri(dialkylamide), phosphorus triphenoxide, or phosphorus tri(imidazolides).

20. The method of claim 1 wherein said first synthon is formed by reacting a compound of Formula VII:

##STR127## and a γ

-amnino alcohol of formula HO--C(R⁸)R³ --C(R¹⁶)R⁵ --CHR⁶ --NHR⁷ ;

wherein X and R¹ -R⁸ and R¹⁶ are as defined above.

21. The method of claim 20 wherein X is chlorine.

22. The method of claim 20 wherein said reaction is stereoselective.

23. The method of claim 22 wherein said first synthon is in a single stereoisomeric form.

24. The method of claim 1 wherein said catalyst has one of the Formulas VIII or IX:

##STR128## wherein;

R¹² and R¹³ are independently hydrogen, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, an ester group, or R¹² and R¹³ together with the carbon atoms to which they are attached, form a substituted or unsubstituted phenyl ring where said substituents are electron withdrawing; and

R¹⁴ is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, norbornyl, substituted norbornyl, aryl, substituted aryl wherein said substituents are electron withdrawing, or has the formula;

##STR129## wherein L is protecting group.

25. The method of claim 24 wherein R¹⁴ is halogen or nitro, and wherein R¹² and R¹³ are each halogen or each cyano.

26. The method of claim 25 wherein R¹⁴ is bromine, and R¹² and R¹³ are each cyano.

27. The method of claim 25 wherein R¹⁴, R¹² and R¹³ are each bromo.

28. The method of claim 1 wherein R³ is cyanomethyl or --CH₂ --SO_k R⁴ where k is 0, 1 or 2.

29. The method of claim 28 wherein R⁷ is lower alkyl or aralkyl.

30. The method of claim 1 further comprising removing said R⁹ groups.

31. The method of claim 1 further comprising removing said R² groups.

32. The oligomer produced by the method of claim 1.

33. The method of claim 13 wherein the removing of said chiral auxiliaries is via β

-elimination.

34. The method of claim 13 wherein the removing said chiral auxiliaries is by treatment with acidic reagents.

45. The method of claim 1 wherein said first synthon has one of the Formulas Xa, XIa, XIIa, XIIIa or XXa:

wherein W has the formula;

##STR133## and R¹ -R¹⁶, V, T and Z are as defined above.

46. The method of claim 24 wherein R¹⁴ has one of the formulas:

##STR134## wherein R¹⁵ is H, methyl, trialkylsilyl or acetyl.

49. The method of claim 45 wherein said first synthon has the Formula XIa;

R³ is --CH₂ R₄ ; and

R⁴ is independently --N(R₇₀)--C(═

O)--R₇₁, --S--C(═

O)--R₇₀, or --O--C(═

O)--O--N(R₇₀)(R₇₁) and wherein R₇₀ and R₇₁ are as defined above.

50. The method of claim 49 wherein R₇₀ and R₇₁ are independently alkyl or α

-halo substituted alkyl.

51. The method of claim 45 wherein said first synthon has the Formula Xb or Xc:

52. The method of claim 45 wherein said first synthon has the Formula XIVa:

wherein p, Z¹, Z², R⁶, R⁷, and R⁸ are as defined above.

53. The method of claim 52 wherein said first synthon has the Formula XVa or XVIa:

##STR136## wherein Y, R⁷, Z¹, Z², and p are as defined above.

54. The mnethod of claim 45 wherein said first synthon has the Formula XVIIa or XVIIIa:

##STR137## wherein W, R⁷, R¹⁰ and R¹¹ are as defined above.

35. A method of synthesizing an oligomeric compound having a chiral boranophosphate linkage of the formula (XXXI) ##STR130## wherein R₂ is a chiral auxiliary;

and G is a borano group;

comprising reacting a chiral oligomeric precursor of formula (XXXII);

##STR131## with a boronating agent capable of yielding a boranophosphate in the presence of a catalyst.

36. The method according to claim 35, wherein G is a borano group having the formula BH(R'"'"')₂ wherein each R'"'"' is independently selected from the group consisting of H;

CN;

carboxyl;

carboxyl salts;

C_1-6 alkyl, alkaryl, aryl, alkoxycarbonyl and alkylaminocarbonyl each optionally substituted with OH, halogen, amino or CN; and

said boronating agent is a compound having the general formula R"₂ S--BH(R'"'"')₂ wherein each R" is independently C_1-6 alkyl.

37. The method according to claim 36, wherein said borano group G is selected from BH₃, BHMe₂, BH₂ COOH, BH₂ COOMe and BH₂ CN.

38. The method according to claim 36, wherein said boronating agent is selected from the group consisting of BH₃ --Me₂ S, BH(Me)₂ --Me₂ S, BH₂ Me--Me₂ S and BH₂ CN--Me₂ S.

39. The method according to claim 35, wherein said oligomeric compound is an oligonucleotide.

40. The method according to claim 39, wherein said oligonucleotide comprises ribonucleotides and deoxyribonucleotides.

41. The method according to claim 35, wherein said chiral boranophosphate linkage of formula (XXXI) has Rp configuration.

42. The method according to claim 35, wherein said chiral boranophosphate linkage of formula (XXXI) has Sp configuration.

43. The method according to claim 42, wherein R₂ is a chiral auxiliary selected form the group consisting of:

##STR132##

44. The method of claim 35, further comprising reacting said oligomeric compound under conditions suitable to remove said chiral auxiliary R².

47. The method according to claim 35, wherein R² is a chiral auxiliary selected from the group consisting of ##STR135##

48. the method according to claim 42, wherein R² is a chiral auxiliary selected from the group consisting of: