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Title:
METHOD OF PREPARING OLIGOMERIC COMPOUNDS USING MODIFIED CAPPING PROTOCOLS
Document Type and Number:
WIPO Patent Application WO/2014/028739
Kind Code:
A4
Abstract:
Provided herein are methods for the solid phase synthesis of oligomeric compounds wherein at least one of the capping steps has been modified. More particularly, methods are provided wherein one or more of the capping steps is omitted or performed using reduced equivalents of acetic anhydride. In certain embodiments, the methods provide an enhanced purity profile. In certain embodiments, the methods provide an increased yield. The methods provided herein also provide at least an economic advantage over currently used methods in that reduced amounts of the mixture of capping reagents are required.

Inventors:
CEDILLO, Isaiah, E. (2855 Gazelle Court, Carlsbad, CA, 92010, US)
JANCZAK, Darren (2855 Gazelle Court, Carlsbad, CA, 92010, US)
WEAVER, Phillip, Michael (55 Corporate Drive, Mail Code: 55A-505ABridgewater, NJ, 08807, US)
Application Number:
US2013/055146
Publication Date:
April 10, 2014
Filing Date:
August 15, 2013
Export Citation:
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Assignee:
ISIS PHARMACEUTICALS, INC. (2855 Gazelle Court, Carlsbad, CA, 92010, US)
GENZYME CORPORATION (500 Kendall Street, Cambridge, MA, 02142, US)
International Classes:
C07H19/04; C07K1/04
Attorney, Agent or Firm:
ANDREWS, Robert, S. et al. (Isis Pharmaceuticals, Inc.2855 Gazelle Cour, Carlsbad CA, 92010, US)
Download PDF:
Claims:
AMENDED CLAIMS

received by the International Bureau on 16 February 2014 (16.02.2014)

What is Claimed is:

1 . A method of preparing an oiigomeric compound comprising:

a) provid ing a solid support having a plurality of monomer subunits l inked thereto wherein each of the monomer subunits comprises a blocked hydroxy! group;

b) deblocking the blocked hydroxy! groups to provide free hydroxy! groups;

c) coupling further monomer subunits to the free hydroxyi groups, wherein each further monomer subunit comprises a phosphoramidite group and a blocked hydroxyi group, to the free hydroxy! groups to provide phosphite triester linked monomer subunits;

d) oxidizing or su!furizing the phosphite triester linked monomer subursits to provide phosphate triester or thiophosphate triester linked monomer subunits;

e) optionally treating the phosphate triester or thiophosphate triester linked monomer subunits with a mixture of capping reagents to block any unreacied free hydroxyi groups;

f) iteratively repeating steps h) through e) a predetermined number of times to provide the oiigomeric compound: and

wherein:

the last iterative step e) is omitted and at least one iterative step e) is performed using a mixture of capping reagents having less than about 8 equivalents of acetic anhydride based on the loading of the solid support; or

the last iterative step e) is omitted and each of the remaining iterative steps e) is performed using a mixture of capping reagents having less than about 8 equivalents of acetic anhydride based on the loading of the sol id support.

2. The method of claim 1 wherein the plurality of monomer subunits linked to the solid support are each ! inked by a 3 '-ester linkage.

3. The method of claim 2 wherein the 3 '-ester linkage is a S'-suceinyl group,

4. T he method of claim 1 wherein the plurality of monomer subunits linked to the solid support are each attached to a Unylinker11"1 fimetionalized solid support by a 3"-phospbite triester linkage further comprising; oxidizing or sulfurizing each 3'-phosphite triester linkage to either a phosphate triester or a thiophosphate triester; and

treating the resulting sol id support l inked monomer subun its with a m ixture of capping reagents having 90 equivalents or less of acetic anhydride based on the loading of the solid support.

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5. The method of claim 4 wherein each phosphite triesier is sulfurized to a thiophosphate triester.

6. The method of any of claims 4 or 5 wherein the plurality of monomer subunits linked to the solid support is treated with a mixture of capping reagents having from about 50 equivalents to about 90 equivalents of acetic anhydride based on the loading of the solid support.

7. The method of any of claims 4 or 5 wherein the plurality of monomer subunits linked to the solid support is treated with a mixture of capping reagents having from about 20 equivalents to about 50 equivalents of acetic anhydride based on the loading of the solid support.

8. The method of any of claims 1 to 7 wherein each monomer subunit is, independently, a nucleoside or a modified nucleoside,

9. The method of claim 8 wherein each modified nucleoside independently comprises a furanose or modified furanose sugar group.

10. The method of claim 9 wherein each modified nucleoside independently comprises a substituted nucleoside, a 4'-S-modified nucleoside or a bicyclic modified nucleoside.

1 1. The method of claim 8 wherein each modified nucleoside independently comprises a 2'-substituted sugar, a 5'-substituted sugar, a ?.' and 5'-substiiuied sugar or a 2'-4' bridged bicyclic sugar.

12. The method of claim 8 wherein at least one modified nucleoside comprises a sugar surrogate group.

13. The method of any of claims 1 to 12 wherein each monomer subunit comprises a heterocyclic base moiety that is optionally protected and is independently selected from a purine, substituted purine, pyrimidine and substituted pyrimidine.

14. The method of any of claims 3 to 13 wherein each monomer subunit comprises a heterocyclic base moiety independently selected from uracil, thymine, cytosine, 4-N-benzoylcytosine> 5-met.hyicytosine. 4-N- benzoyi-S-methylcyiosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyryiguanine.

15. The method of any of claims 1 to 14 wherein each of the blocked hydroxy! groups is, independently, blocked with a hydroxy! blocking group selected from a substituted or unsubstituted trityi group.

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16. The method of claim 15 wherein each hydroxy! blocking group is 4,4 '-dimethoxytritylv

17. The method of any of claims 1 to 1 6 wherein each phosphoramidite group is a diisopropyi- eyanoerhoxy phosphoramidite (~P(N[(CH)C1'] ]2)(0(CH2)2CN).

18. The method of any of claims 1 to 17 wherein dichloroacetic acid in toluene is used to deblock blocked hydroxy! groups.

19. The method of any of claims 1 to 18 further comprising treatment of the oligomeric compound with triethvlamine in acetoniirile to remove phosphorus protecting groups thereby providing linkages between monomer subunits that are independently selected from phosphodiesier and phosphorothioate.

20. The method of any of claims 1 to 19 further comprising treatment of the oligomeric compound with ammonium hydroxide to remove further protecting groups and cleave the oligomeric compound from the solid support.

21. The method of any of claims 1 to 20 wherein the solid support is crosslinked polystyrene.

22. The method of any of claims 1 to 21 wherein the solid support is a crosslinked polystyrene selected from Primer Support 5G or NittoPhaseHL.

23. The method of any of claims 1 to 22 wherein at least one iterative step e) in addition to the last one is omitted.

24. The method of any of claims 1 to 23 wherein iterative step e) is performed for about the first 50% of the iterative steps h) through e) and omitted for the remaining iterative steps b) through e),

25. The method of any of claims 1 to 23 wherein iterative step e) is performed for about the first 75% of the iterative steps b) through e) and omitted for the remaining iterative steps h) through e).

26. The method of any of c!aims 1 to 22 wherein the last iterative step e) ss omitted and each of the remaining iterative steps e) is performed.

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27. The method of any of claims 1 to 26 wherein the m ixture of capping reagents used for essential ly each capping step that is performed comprises about 8 equivalents of acetic anhydride based on the loading of the solid support.

28. The method of any of claims 1 to 26 wherein the mixture of capping reagents used for each capping step that is performed comprises about 6 equivalents of acetic anhydride based on the loading of the solid support.

29. The method of any of claims 1 to 26 wherein the mixture of capping reagents used for each capping step that is performed comprises about 4 equivalents of acetic anhydride based on the loading of the solid support.

30. The method of any of claims 1 to 26 wherein the mixture of capping reagents used for each capping step comprises about 2 equivalents of acetic anhydride based on the loading of the solid support.

3 1 . The method of any of claims 1 to 26 wherein the mixture of capping reagents used for each capping step that is performed comprises less than 1 equivalent of acetic anhydride based on the loading of the solid support.

32. The method of any of claims 1 to 26 wherein the volume of the mixture of capping reagents is modified independently for each cycle of steps b) through e) such that about 1 7 equivalents of acetic anhydride are used for the first cycle and over each successive cycle that includes the capping step e) the equivalents of acetic anhydride are serially reduced to about 1 equivalent based on the loading of the solid support.

33. The method of any of claims 3 to 26 wherein the volume of the mixture of capping reagents is mod ified independently for each cycle of steps b) through e) such that about 8 equivalents of acetic anhydride are used for the first cycle and over each successive cycle that includes the capping step e) the equivalents of acetic anhydride are serially reduced to about 1 equivalent based on the loading of the solid support.

34. The method of any of claims 1 to 26 wherein the volume of the mixture of capping reagents is modified independently for each cycle of steps b) through e) such that about 4 equivalents of acetic anhydride are used for the first cycle and over each successive cycle thai includes the capping step e) the equ ivalents of acetic anhydride are serial ly reduced to about I equivalent based on the load ing of the olid support.

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35. The method of any of claims 3 to 34 wherein the mixture of capping reagents comprises from about 5% to about 10% acetic anhydride, from about 5% to about 10% N-methylitrtidazole and from about 5% to about 1 5% pyridine or from about 5% to about 10% 2,6-iutidine dissolved in tetrahydrofuran. toiuene or acetomtriie.

36. The method of any of claims 1 to 34 wherein the mixture of capping reagents comprises from about 5% to about 30% acetic anhydride, from about 5% to about 10% N-methylimadazoIe and from about 5% to about 35% pyridine in toluene.

37. The method of any of claims I to 34 wherein the mixture of capping reagents comprises from about 10% acetic anhydride, about 10% N-rnethylimidazoie and about 35% pyridine in toluene.

38. The method of any of claims 1 to 37 wherein the oligomeric compound comprises from about 10 to about 40 monomer subunits in length.

39. The method of any of claims 1 to 37 wherein the oligomeric compound comprises from about 12 to about 30 monomer subunits in length.

40. The method of any of claims 1 to 37 wherein the oligomeric compound comprises from about 14 to about 20 monomer subunits in length.

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