Method for Preparing Branched Functionalized Polymers Using Branched Polyol Cores

• US 20070031371A1
• Filed: 07/18/2006
• Published: 02/08/2007
• Est. Priority Date: 07/18/2005
• Status: Active Grant

• Current Assignee: Nektar Therapeutics
• Original Assignee: Nektar Therapeutics Al Corporation (Nektar Therapeutics)
• Inventors: Kozlowski, Antoni, McManus, Samuel
• Granted Patent: US 8,568,705 B2
• Time in Patent Office: Days
• US Class Current: 424/78.370
• CPC Class Codes: A61K 31/74 Synthetic polymeric materials
• CPC Class Codes: A61K 31/765 Polymers containing oxygen
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/34 from hydroxy compounds or their metallic derivatives C08G65/26 takes precedence
• CPC Class Codes: C08G 65/48 Polymers modified by chemical after-treatment
• CPC Class Codes: C08G 71/04 Polyurethanes
• CPC Class Codes: C08L 2203/02 for biomedical use
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

A method of preparing a multiarm polymer includes reacting a branched polyol with one or more functionalizing reagents to effect substitution of an ionizable functional group or a protected ionizable functional group, Y, to form a mixture comprising (i) unsubstituted branched polyol containing no Y groups; (ii) a monosubstituted polyol comprising one Y group, and (iii) a multisubstituted polyol (e.g., a disubstituted polyol comprising two Y groups); followed by purifying the mixture to separate the monosubstituted polyol from other species Thereafter, a water-soluble and non-peptidic polymer segment is attached to the monosubstituted branched polyol at the site of at least one of the hydroxyl groups. The invention also provides purified monosubstituted branched polyols and multiarm polymers prepared by the method and polyol precursors for use in the method.

Claims

1. A method of preparing a multiarm polymer, comprising:

(a) optionally, to a branched polyol bearing at least three hydroxyl groups or a mixture of branched polyols comprising a branched polyol bearing at least three hydroxyl groups, attaching a water-soluble and non-peptidic polymer segment to the branched polyol at the site of at least one of the hydroxyl groups;

(b) reacting the branched polyol, in one or more reaction steps, with one or more functionalizing reagents to effect substitution of an ionizable functional group or a protected ionizable functional group, —

Y, to form a mixture comprising (i) unsubstituted branched polyol containing no —

Y groups;

(ii) a monosubstituted polyol comprising a single —

Y group, and (iii) a multisubstituted polyol comprising at least two —

Y groups;

(c) where —

Y is a protected ionizable functional group, deprotecting the ionizable functional group;

(d) purifying the mixture to separate the monosubstituted polyol from the unsubstituted and multisubstituted polyol species; and

(e) if optional step (a) is absent, attaching a water-soluble and non-peptidic polymer segment to the monosubstituted branched polyol at the site of at least one of the hydroxyl groups either before or after said purifying step (d).

2. The method of claim 1, wherein the branched polyol of step (a) has the structure R(OH)_p, wherein R is a branched hydrocarbon, optionally including one or more ether linkages, and p is at least 3.

3. The method of claim 2, wherein p is 3 to about 25.

4. The method of claim 2, wherein p is 3 to about 10.

5. The method of claim 2, wherein the R group includes a poly(alkylene glycol) chain of 1 to about 25 monomer units attached to each terminal hydroxyl group.

6. The method of claim 1, wherein the branched polyol is selected from the group consisting of pentaerythritol, oligomers of pentaerythritol, polymers of pentaerythritol, oligomers of glycerol, polymers of glycerol, and sugar-derived polyols.

7. The method of claim 1, wherein the water-soluble and non-peptidic polymer is selected from the group consisting of poly(alkylene glycol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharide), poly(α

-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), and copolymers, terpolymers, and mixtures thereof.

8. The method of claim 1, wherein the water-soluble and non-peptidic polymer is poly(ethylene glycol).

9. The method of claim 1, wherein the total molecular weight of the water-soluble and non-peptidic polymer portion of the multiarm polymer is from about 44 Da to about 100,000 Da.

10. The method of claim 9, wherein the total molecular weight of the water-soluble and non-peptidic polymer portion of the multiarm polymer is from about 1,000 Da to about 80,000 Da.

11. The method of claim 10, wherein the total molecular weight of the water-soluble and non-peptidic polymer portion of the multiarm polymer is from about 2,000 Da to about 40,000 Da.

12. The method of claim 1, wherein said reacting step (b) is carried out under conditions effective to form no more than about 50 percent of the multisubstituted polyol.

13. The method of claim 12, wherein said reacting step (b) is carried out under conditions effective to form no more than about 30 percent of the multisubstituted polyol.

14. The method of claim 13, wherein said reacting step (b) is carried out under conditions effective to form no more than about 18 percent of the multisubstituted polyol.

15. The method of claim 14, wherein said reacting step (b) is carried out under conditions effective to form no more than about 7 percent of the multisubstituted polyol.

16. The method of claim 15, wherein said reacting step (b) is carried out under conditions effective to form no more than about 4 percent of the multisubstituted polyol.

17. The method of claim 1, wherein said reacting step (b) is conducted under conditions effective to form a ratio of monosubstituted polyol to multisubstituted polyol that is about 1:

1 to about 50;

1.

18. The method of claim 17, wherein said reacting step (b) is conducted under conditions effective to form a ratio of monosubstituted polyol to multisubstituted polyol that is about 2:

1 to about 40;

1.

19. The method of claim 18, wherein said reacting step (b) is conducted under conditions effective to form a ratio of monosubstituted polyol to multisubstituted polyol that is about 4:

1 to about 20;

1.

20. The method of claim 1, wherein —

Y is selected from the group consisting of aldehyde hydrate, ketone hydrate, amine, hydrazine, hydrazide, thiol, carboxylic acid, sulfonic acid, primary amide, secondary amide, amidate, 2-substituted-1,3-oxazoline, 2-substituted 1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline, 2-substituted 1,3-(4H)-dihydrothiazines, dithiopyridine, vinylpyridine, hydroxylamine, and oxime.

21. The method of claim 1, wherein —

Y is amine or carboxylic acid.

22. The method of claim 1, wherein —

Y is a protected ionizable functional group.

23. The method of claim 22, wherein —

Y is a protected carboxylic acid and said deprotecting step (e) comprises hydrolyzing said protected carboxylic acid to thereby form a carboxylic acid.

24. The method of claim 23, wherein said protected carboxylic acid is selected from the group consisting of esters, thiolesters, amides, amidates, thioamidates and hydrazides.

25. The method of claim 23, wherein said protected carboxylic acid is an ortho ester.

26. The method of claim 22, wherein —

Y is a protected amine.

27. The method of claim 26, wherein —

Y is a carbonitrile or amide and said deprotecting step comprises reducing the carbonitrile or amide to thereby form an amine.

28. The method of claim 1, wherein said reacting step (b) comprises a nucleophilic substitution or a nucleophilic addition reaction.

29. The method of claim 1, wherein and said purifying step comprises purifying the mixture by ion exchange chromatography.

30. The method of claim 29, wherein said purifying step comprises:

passing the mixture through a first ion exchange column to provide an eluate, wherein said passing the mixture step is carried out under conditions effective to adsorb substantially all of said multisubstituted polyol onto the first column;

passing the eluate through a second ion exchange column under conditions effective to adsorb substantially all of the monosubstituted polyol onto said second column;

washing the second column with a water or a solution having low ionic strength to remove unsubstituted polyol; and

passing a solution having high ionic strength through the second column to desorb the monosubstituted polyol.

31. The method of claim 30, wherein said second ion exchange column is connected in series to one or more additional ion exchange columns, and said washing step further comprises washing said second and one or more additional ion exchange columns and said passing the eluate step further comprises passing the solution having high ionic strength through said second and one or more additional columns.

32. The method of claim 29, wherein said purifying step comprises:

passing the mixture through a first ion exchange column to provide an eluate, wherein said passing the mixture step is carried out under conditions effective to adsorb substantially all of said multisubstituted polyol onto the first column;

passing said eluate through a second ion exchange column connected in series to one or more additional ion exchange columns under conditions effective to adsorb a fraction of the monosubstituted polyol onto said second column and onto each of the one or more additional columns;

washing the second column and one or more additional ion exchange columns with a solution having low ionic strength to remove unsubstituted polyol;

and passing a solution having high ionic strength through the second and one or more additional ion exchange columns to desorb the monosubstituted polyol.

33. The method of claim 1, wherein attaching step comprises ethoxylation of the branched polyol or attachment of a preformed polymer segment via a nucleophilic substitution or a nucleophilic addition reaction.

34. The method of claim 1, further comprising, prior to said attaching step (e), converting functional group —

Y to a hydroxyl group.

35. The method of claim 1, further comprising, prior to said attaching step (e), transforming hydroxyl groups of the branched polyol to a different reactive moiety.

36. The method of claim 35, wherein said reactive moiety comprises a functional group selected from the group consisting of active ester, active carbonate, ortho ester, acetal, aldehyde, aldehyde hydrate, ketone, ketone hydrate, oxime, alkenyl, acrylate, methacrylate, nitrile, primary or secondary amide, imide, acrylamide, active sulfone, amine, hydrazide, thiol, carboxylic acid, isocyanate, isothiocyanate, maleimide, succinimide, vinylsulfone, dithiopyridine, vinylpyridine, amidate, 2-substituted-1,3-oxazoline, 2-substituted 1,3-(4H)-dihydrooxazines, 2-substituted-1,3-thiazoline, 2-substituted 1,3-(4H)-dihydrothiazines, hydroxylamine, iodoacetamide, orthopyridyl disulfide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

37. The method of claim 1, further comprising, prior to said purifying step (d), reacting the hydroxyl groups, optionally in anionic form, of the branched polyol with a reagent comprising two or more protected hydroxyl groups, and thereafter deprotecting the protected hydroxyl groups, thereby increasing the number of hydroxyl groups of the branched polyol.

38. The method of claim 37, wherein the reagent comprises two or more cyclic acetal or ketal groups.

39. The method of claim 38, wherein the reagent is a chloroethylated alkyl diacetal.

40. The method of claim 1, further comprising, prior to said reacting step (b), reacting the branched polyol, in one more reaction steps, with one or more hydroxyl-blocking reagents under conditions sufficient to convert at least one hydroxyl group of the branched polyol to a protected hydroxyl group;

and deprotecting the protected the at least one protected hydroxyl groups either before or after said purifying step (d).

41. The method of claim 40, wherein said step of reacting the branched polyol, in one more reaction steps, with one or more hydroxyl-blocking reagents comprises reacting under conditions sufficient to convert enough hydroxyl groups to protected hydroxyl groups such that no more than one-third of the hydroxyl groups remain in unprotected form.

42. The method of claim 40, wherein the protected hydroxyl groups are cyclic acetal or ketal groups.

43. The method of claim 40, wherein the protected hydroxyl groups are benzyl, diphenylmethyl, or trityl esters.

44. A multiarm polymer prepared by the method of claim 1.

45. A branched polyol molecule comprising protected hydroxyl groups and at least one ionizable functional group, the branched polyol molecule having the structure:

46. The branched polyol molecule of claim 45, wherein X is Y.

47. The branched polyol molecule of claim 45, wherein X is hydroxyl.

48. The branched polyol molecule of claim 45, wherein m is 0-10.

49. The branched polyol molecule of claim 48, wherein m is 0-3.

50. The branched polyol molecule of claim 45, wherein Y is a carboxylic acid or amine.

51. A chromatographically purified branched polyol comprising at least two hydroxyl groups and a single ionizable functional group, optionally in protected form.

52. The branched polyol of claim 51, wherein the polyol has a purity of at least about 83%.

53. The branched polyol of claim 52, wherein the polyol has a purity of at least about 91%.

54. The branched polyol of claim 53, wherein the polyol has a purity of at least about 97%.

55. The branched polyol of claim 54, wherein the polyol has a purity of about 100%.

56. The branched polyol of claim 51, having the structure Y—

R(—

OH)_p, wherein R is a branched hydrocarbon, optionally including one or more ether linkages, and p is at least 2.

57. The branched polyol of claim 56, wherein p is 3 to about 25.

58. The branched polyol of claim 56, wherein p is 3 to about 10.

59. The branched polyol of claim 56, wherein the R group includes a poly(alkylene glycol) chain of 1 to about 25 monomer units attached to each terminal hydroxyl group.

60. The branched polyol of claim 51, wherein the branched polyol is derived from a branched polyol core selected from the group consisting of pentaerythritol, oligomers of pentaerythritol, polymers of pentaerythritol, oligomers of glycerol, polymers of glycerol, and sugar-derived polyols.

61. The branched polyol of claim 51, having the structure Y—

R(—

OH)_p, wherein R comprises an amino acid bearing the ionizable functional group —

Y, and further comprises one or more hydroxyl-bearing, branched hydrocarbons covalently attached to the amino acid, the hydroxyl-bearing, branched hydrocarbons optionally including one or more ether linkages and bearing a total of p hydroxyl groups, wherein p is at least 4.

62. The branched polyol of claim 61, wherein the amino acid is lysine and the —

Y group is carboxylic acid.

63. A chromatographically purified multiarm polymer comprising a single ionizable functional group, having the structure Y—

POLY′

_0,1-L′

_0,1-R(-L_0,1-POLY-Z)p, wherein —

Y is the single ionizable functional group, L and L′

are optional spacers, POLY′

is an optional water soluble and non-peptidic polymer, each POLY is a water-soluble and non-peptidic polymer, R is a core molecule, each Z is an independently-selected non-ionizable functional group, and p is at least 2.

64. The multiarm polymer of claim 63, wherein R is a branched hydrocarbon, optionally including one or more ether linkages.

65. The multiarm polymer of claim 63, wherein p is 3 to about 25.

66. The multiarm polymer of claim 65, wherein p is 3 to about 10.

67. The multiarm polymer of claim 63, wherein the R group includes a poly(alkylene glycol) chain of 1 to about 25 monomer units at each terminus thereof.

68. The multiarm polymer of claim 63, wherein the R core molecule is derived from a branched polyol selected from the group consisting of pentaerythritol, oligomers of pentaerythritol, polymers of pentaerythritol, oligomers of glycerol, polymers of glycerol, and sugar-derived polyols.

69. The multiarm polymer of claim 63, wherein R comprises an amino acid bearing the ionizable functional group —

Y, and further comprises one or more branched hydrocarbons covalently attached to the amino acid, the branched hydrocarbons optionally including one or more ether linkages and including p termini, wherein p is at least 4.

70. The multiarm polymer of claim 69, wherein the amino acid is lysine and the —

Y group is carboxylic acid.

71. The multiarm polymer of claim 63, wherein each Z is hydroxyl or an ionizable functional group in protected form.

72. A conjugate comprising a multiarm polymer of claim 63 or a derivative thereof covalently attached to at least one biologically active molecule.