Charge-balanced chemoselective linkers

• US 20050158370A1
• Filed: 04/07/2003
• Published: 07/21/2005
• Est. Priority Date: 04/08/2002
• Status: Abandoned Application

• Current Assignee: Amura Therapeutics Ltd. (Amura Holdings Ltd.)
• Original Assignee: Amura Therapeutics Ltd. (Amura Holdings Ltd.)
• Inventors: Turnell, William Gordon, Flinn, Nicholas Sean, Quibell, Martin, Ramjee, Manoj Kumar
• Application Number:
  US10/510,893
• Publication Number:
  US 20050158370A1
• Time in Patent Office: Days
• US Class Current: 424/445
• CPC Class Codes: A61K 47/646 the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: B01D 67/0093 Chemical modification
• CPC Class Codes: B01D 67/00931 by introduction of specific groups after membrane formation, e.g. by grafting
• CPC Class Codes: C07C 59/74 containing —CHO groups
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids

Abstract

Compounds according to general formulae (Ia to Ie) wherein: X═O or S; Y is O, S or CH₂, CHR, CRR, where R is C_1-7 alkyl; Z is O or S; R₁ is H or C_1-7 alkyl; R₂ is H or C_1-7 alkyl; R₄ is H or C_1-7 alkyl at any vacant position on the aromatic ring; R₃ is C_1-7 alkyl-L₁-R₅-L₂-R₆—COOH, C_3-10 cycloalkyl-L₁-R₅-L₂-R₆—COOH or Ar—C_0-7 alkyl-L₁-R₅-L₂-R₆—COOH; each of L₁ and L₂ is absent or a suitable linker such as an amide CONH; or an ether —O—, or a thioether —S— or a sulphone —SO₂—; R₅ is C_1-7 alkyl, C_3-10 cycloalkyl or Ar—C_0-7 alkyl each of which is substituted with either NR₈R₉, where the nitrogen atom is capable of being protonated in solution to give N⁺HR₈R₉; or a quaternary nitrogen atom N⁺R₈R₉R₁₀, such that R₅ contains a positive charge; each of R₈, R₉ and R₁₀ is independently C_1-7 alkyl, C_3-10 cycloalkyl or Ar—C_0-7 alkyl, or any two or more of R₈, R₉ and R₁₀ together form an alicyclic or arylalicyclic ring system; R₆ is C_1-7 alkyl, C_3-10 cycloalkyl or Ar—C_0-7 alkyl; and their salts, hydrates, solvates, complexes or prodrugs are of use as linkers for conjugating an epitope to a carrier protein.

Claims

1. A positive charge-balanced linker according to general formulae (Ia to Ie):

2. A compound as claimed in claim 1 wherein, independently or in any combination:

X is oxygen;

Y is oxygen;

R₁ is hydrogen, methyl or ethyl;

R₂ is hydrogen or C_1-4 alkyl;

L₁ is an amide (CONH); and

L₂ is an amide (CONH).

3. A compound as claimed in claim 1, wherein R₁ is hydrogen.

4. A compound as claimed in claim 1, wherein R₂ is hydrogen or methyl.

5. A compound as claimed in claim 1 wherein R₃ comprises

6. A compound as claimed in claim 1, wherein L₁-R₅-L₂ is CO—

NHR₅CO—

NH and wherein NHR₅CO comprises a simple amino acid residue that contains a side-chain protonatable amine functionality.

7. A compound as claimed in claim 6 wherein NHR₅CO is represented by the formula:

—

NH—

CH[(CH₂)_pN⁺R₈R₉R₁₀]CO—

wherein p is 1 to 5 and R₈, R₉ and R₁₀ are as defined above.

8. A compound as claimed in claim 7, wherein p is 1 to 4.

9. A compound as claimed in claim 1 wherein R₈, R₉ and R₁₀ are each independently C_1-4 alkyl.

10. A compound as claimed in claim 1 wherein L₂ is CONH;

wherein R₆ is

—

(CH₂)_q-A_s-(CH₂)_r—

;

where q and r are each 0 to 3, provided that both q and r are not both 0;

s is 0 or 1; and

A is a 5-10 membered stable monocyclic or bicyclic aromatic ring or a 3-6 membered carbocyclic or alicyclic ring.

11. A compound as claimed in claim 10 wherein r and s are 0 and q is 1 or 2.

12. A compound of general formula (Ia) as defined in claim 1.

13. A compound as claimed in claim 12,

wherein

X and Y are O,

R₁ is H;

and

R₂ and R₃ are as defined in claim 1.

14. A compound as claimed in claim 13,

wherein:

R₃ is —

(CH₂)_o—

C(O)—

NH—

CH(—

(CH₂)_p—

N⁺(R₈)(R₉)(R₁₀))(—

C(O)—

R₆—

COOH)

o is an integer from 2-6;

p is an integer from 1 to 5; and

R₆, R₈, R₉ and R₁₀ are as defined in claim 1.

15. A compound as claimed in claim 14, wherein p is an integer from 1 to 4.

16. A compound as claimed in claim 15,

wherein

R₃ is —

(CH₂)_o—

C(O)—

NH—

CH(—

(CH₂)_p—

N⁺(R₈)(R₉)(R₁₀))(—

C(O)—

R₆—

COOH)

o is an integer from 3 to 6;

p is an integer from 2 to 4;

R₈ and R₉ are methyl; and

R₁₀ ═

H or methyl.

17. A process for the preparation of a compound of general formula (I) in which L₁ and L₂ are CONH, the process comprising:

(i) reacting a compound of general formula V;

H₂N—

R₆—

COOH 

 

(V)

wherein R₆ is as defined for general formula (I) in claim 1;

and

wherein the compound of general formula (V) is bound at its C-terminus to a solid support;

with a compound of general formula (VI);

W—

NH—

R₅—

COOH 

 

(VI)

wherein;

R₅ is as defined in claim 1;

and

W is a protecting group;

(ii) removal of the protecting group W and reaction with a compound of general formula (VII);

wherein

X, Y, Z, R₁, R₂, and R₄ are as defined in claim 1;

R₁₁ is C_1-7 alkyl-COOH, C_3-10 cycloalkyl-COOH or Ar—

C_0-7 alkyl-COOH; and

(iii) removal of the product from the solid support.

18. A process as claimed in claim 17 wherein, in the compound of general formula (VI), W is a urethane protecting group.

19. A compound of general formula (XIV):

20. A compound as claimed in claim 19 wherein Q is part of a proteinaceous molecule, a polysaccharide, cellulose beads, a polymeric amino acid, a polymer, which may be a copolymer, an inactive virus particle or attenuated bacteria.

21. A process for the preparation of a compound as claimed in claim 19, the process comprising reacting a compound of general formula (I) as defined above with a carrier.

22. A compound of general formula (XV):

23. A compound as claimed in claim 22 which comprises E or G groups derived from two or more active moieties.

24. A process for the preparation of a compound of general formula (XV) as defined in claim 22, the process comprising reacting a compound of general formula (XIV) as defined above with a compound of general formula (XVIa), (XVIb) or (XVIc):

E-NH—

CO—

(CH₂)_tCONHNH₂ 

 

(XVIa)

E-NH—

CO—

NHNH₂ 

 

(XVIb)

G-CO—

NHNH₂ 

 

(XVIc)

where E, G and t are as defined in claim 22.

25. A compound as claimed in claim 22 which is soluble in aqueous solution.

26. A compound as claimed in claim 25 wherein E or G is derived from an epitope or mimotope.

27. A compound as claimed in claim 26 wherein the epitope is a fragment derived from a protein or peptide molecule or a variant thereof.

28. A compound as claimed in claim 26 wherein the epitope is a B cell or T cell epitope.

29. A compound as claimed in claim 25 which includes another active moiety comprising an immunomodulating compound attached to the carrier protein.

30. A method for raising specific antibodies against an epitope or mimotope, the method comprising immunizing a subject with a compound as claimed in claim 26 comprising E or G derived from said epitope or mimotope.

34. A pharmaceutical composition comprising a compound as claimed in claim 26 together with a pharmaceutically acceptable excipient.

35. A vaccine composition comprising a compound as claimed in claim 26 together with a pharmaceutically acceptable excipient and a pharmaceutically acceptable adjuvant.

36. A non-destructive method of quantifying the extent and/or rate of reaction of a protein with a linker of general formula (I) as defined in claim 1 in which R₂ is H and X is O, the method comprising either:

a) measuring the intensity of the absorbance spectrum at a wavelength above 300 nm and at a pH greater than 7 in order to detect the formation of a compound of general formula (XIV) in which R₂ is H and X is O;

or

b) measuring the fluorescence emission upon excitation at a selected wavelength in order to detect the formation of a compound of general formula (XIV) as defined in claim 19 in which R₂ is Hand X is O.

37. A non-destructive method for quantifying the extent and/or rate of reaction of a linker-protein of general formula (XIV) as defined in claim 19 wherein R₂ is H and X is O, with an active moiety hydrazide, the process comprising measuring the intensity of the absorbance spectrum at a wavelength above 300 nm and a pH less than 7.

38. A process for the preparation of a compound of general formula (XV) as defined in claim 22 in which:

R₂ is Hand X is O;

the carrier has more than one Q;

a first selected percentage of the Q groups is derivatized with a first active moiety; and

, optionally

further selected percentages of the Q groups are derivatized with further active moieties;

the process comprising;

a) reacting a compound of general formula (XIV) as defined in claim 19 in which R₂ is H and X is O with a first compound of general formula (XVI) as defined in claim 24 at a pH less than 7;

b) monitoring the progress of the reaction by measuring the intensity of the absorbance spectrum at a wavelength of above 300 nm and stopping the reaction when the intensity of the absorbance spectrum reaches the first selected percentage of the known maximum intensity; and

optionally

c) reacting the product of steps (a) and (b) with one or more further compounds of general formula (XVI), monitoring the progess of the reaction by measuring the intensity of the absorbance spectrum at a wavelength of above 300 nm and stopping the reaction when the intensity of the absorbance spectrum reaches further selected percentages of the known maximum intensity.

39. A method for quantifying the extent and/or rate of release of an active moiety hydrazide from a compound of general formula (XV) as defined in claim 22 in which R₂ is H and X is O, the method comprising the measurement of the absorbance spectrum maximum at a wavelength above 300 nm and at pH less than 7.

40. A compound as claimed in claim 22 wherein E or G is a labelling moiety.

41. A compound as claimed in claim 22 which is insoluble in aqueous solution.

42. A compound as claimed in claim 41 wherein E or G is a ligand which is specific for a compound to be separated.

43. A compound as claimed in claim 42 which contains additional E or G groups derived from labelling molecules.

44. A method of separating a compound from a mixture, the method comprising contacting the mixture with a compound of claim 42 wherein E or G is a ligand which is specific for said compound and the carrier is a solid support.

45. An assay method comprising contacting a mixture suspected of containing an analyte with a compound of claim 55 in which E or G is a ligand for said analyte and the carrier is a solid support.

46. A wound dressing comprising a compound as claimed in claim 41.

50. A compound as claimed in claim 41 wherein the carrier is a polymer suitable for use in dialysis tubing and E or G is heparin for use in the preparation of dialysis tubing.

51. Dialysis tubing comprising a compound as claimed in claim 50.

52. A compound as claimed in claim 26 wherein the epitope is an antigenic determinant derived from a protein or peptide molecule.

53. A compound as claimed in claim 29 wherein the immunomodulating compound is a lipid, an adjuvant, an immunostimulating DNA sequence or cytokine.

54. A compound as claimed in claim 25 wherein E or G is a labelling moiety.

55. A compound as claimed in claim 41 wherein E or G is a ligand which is specific for an analyte.

56. A compound as claimed in claim 41 wherein the carrier is a functionalized polymer of the type commonly used in wound dressings and E or G is a peptide growth factor, a chemo-attractant protein, a ligand or an analogue of one of these.

31. (canceled)

32. (canceled)

33. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)