Process for the manufacture of Degarelix and its intermediates

1. A liquid-phase process for preparing Degarelix having the formula Ac-AA 1 -AA 10 -NH 2 :

or a pharmaceutically acceptable salt or solvate thereof; comprising the steps of:

coupling peptides (P 4 )Ac-AA 1 -AA 4 with (Pε)AA 5 -AA 10 NH 2 or coupling peptides Ac-AA 1 -AA 3 with (P 4 )(Pε)AA 4 -AA 10 NH 2 in an organic solvent,

wherein the organic solvent comprises the two peptides, a peptide coupling reagent and an organic amine base dissolved therein,

wherein Pε is an ε-amino protecting group and P4 is a hydroxyl protecting group or hydrogen, wherein the peptide coupling agent in the case of coupling (P 4 )Ac-AA 1 -AA 4 with (Pε)AA 5 -AA 10 NH 2 is selected from at least one of o-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexfluorophosphate (BOP), and

wherein the peptides have the following formulae:

to provide a protected Degarelix precursor having the formula (P 4 )(Pε)Ac-AA 1 -AA 10 -NH 2 :

and

cleaving the ε-amino protecting group Pε from the Degarelix precursor in an organic solvent, wherein the organic solvent comprises the precursor and a cleaving agent dissolved therein to provide Degarelix.

2. The process according to claim 1 , wherein the cleaving agent is chosen fromtrifluoroacetic acid and piperidine.

3. The process according to claim 1 , wherein Pε is chosen from t-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and allyloxycarbonyl (Alloc).

4. The process according to claim 1 , wherein the Pε protecting group is Fmoc.

5. The process according to claim 1 , wherein the organic solvent is DMF.

6. The process according to claim 1 , wherein in the case of coupling Ac-AA 1 -AA 3 with P 4 )(Pε)AA 4 -AA 10 NH 2 , the peptide coupling reagent is chosen from at least one of o-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlroride (EDC.HCl), (2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium)hexafluorophosphate (HCTU), 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexfluorophosphate (BOP), and diisopropylcarbodiimide (DIC).

7. The process according to claim 1 , wherein the organic amine base is chosen from at least one of N,N′-diisopropyl ethyl amine (DIPEA), N-methylmorpholine (NMM), triethyl amine (TEA) and 2,4,6-trimethylpyridine.

8. The process according to claim 1 , wherein the solution further comprises a coupling additive chosen from 3,4-dihydro-3-hydroxy4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-7-aza-benzotriazole (HOAt) and 1-hydroxybenzotriazole (HOBt) dissolved therein.

9. The process according to claim 1 , wherein the organic amine base is DIPEA and the peptide coupling reagent is HATU.

10. The process according to claim 8 , wherein the organic amine base is DIPEA and the peptide coupling additive is HOAt.

11. The process according to claim 8 , wherein the peptide coupling reagent is HATU and the peptide coupling additive is HOAt.

12. The process according to claim 8 , wherein the organic amine base is DIPEA, the peptide coupling reagent is HATU and the peptide coupling additive is HOAt.

13. The process according to claim 1 , wherein the organic amine base is used in an amount ranging from about 2.5 to about 3.5 molar equivalents of AA 5 -AA 10 hexapeptide.

14. The process according to claim 1 , wherein the organic solvent is cooled to a temperature of −10° C. or lower and the coupling reaction is then performed at that temperature.

15. The process according to claim 8 , wherein the peptides and coupling additive are first dissolved in the organic solvent before adding the coupling reagent and the organic amine.

16. A liquid-phase process for preparing a Degarelix intermediate having the formula (P 4 )Ac-AA 1 -AA 4 :

or a pharmaceutically acceptable salt or solvate thereof,

comprising the step of hydrolyzing a compound having the formula (P 4 )Ac-AA 1 -AA 4 -R with an alkaline hydroxide, wherein R is a carboxyl protecting group, and P 4 is hydrogen or a hydroxyl protecting group, wherein the compound (P 4 )Ac-AA 1 -AA 4 -R has the formula:

17. A liquid-phase process for preparing a hexapeptide (Pε)AA 5 -AA 10 NH 2 comprising:

coupling (Pε)AA 6 -AA 10 NH 2 and (P X )AA 5 , wherein P X is an amino protecting group, AA 5 is 4Aph(L-Hor), AA 6 is D-Aph(Cbm), AA 7 is Leu, AA 8 is Lys(iPr), AA 9 is Pro, AA 10 is D-Ala, and Pε is an ε-amino protecting group, to provide (P X )(Pε)AA 5 -AA 10 NH 2 , and

cleaving Px with TFA to provide (Pε)AA 5 -AA 10 NH 2 , wherein (P X )(Pε)AA 5 -AA 10 NH 2 , (Pε)AA 6 -AA 10 NH 2 and (P X )AA 5 have the following structures:

18. The process according to claim 16 , wherein the compound having the formula Ac-AA 1 -AA 4 -R is first prepared by coupling Ac-AA 1 -AA 3 with (P 4 )AA 4 -R or coupling Ac-AA 1 -AA 2 with (P 4 )AA 3 -AA 4 -R, the peptides having the following formulae:

19. The process according to claim 16 , wherein R is methyl or benzyl.

20. The process according to claim 16 , wherein the alkaline hydroxide is LiOH.

21. Intermediate polypeptides according to the formulae:

wherein R is a carboxyl protecting group, Pε is an amino protecting group, and P4 is hydrogen or a hydroxyl protecting group.

22. A solid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA 1 -AA 4 :

or a pharmaceutically acceptable salt or solvate thereof, comprising the steps:

a) reacting (PN)AA 2 with

 to provide

b) removal of PN from

 to provide

c) reacting (PN)AA1 with

 to provide

d) if PN is not acetyl, removal of PN from

 to provide

 and subsequently acetylating

 to provide

 and

e) cleaving

 to provide (P4)Ac-AA 1 -AA 4 ,

wherein P4 is H or a hydroxyl protecting group on AA 4 , and PN is an amino protecting group.

23. A liquid-phase process for preparing a hexapeptide (Pε)AA 5 -AA 10 NH 2 comprising:

coupling (P5)AA 5 -AA 7 with (Pε)AA 8 -AA 10 NH 2 to provide (P5, Pε)AA 5 -AA 10 NH 2 , and

subsequently cleaving P5 to provide (Pε)AA 5 -AA 10 NH 2 , wherein P5 is an amino-protecting group on AA 5 and Pε is a side chain amino protecting group on AA 6 and wherein AA 5 is 4Aph(L-Hor), AA 6 is D-Aph(Cbm), AA 7 is Leu, AA 8 is Lys(iPr), AA 9 is Pro, and AA 10 is D-Ala.

24. The liquid-phase process of claim 23 , followed by coupling (P4)Ac-AA 1 -AA 4 to (Pε)AA 5 -AA 10 NH 2 .

25. The process according to claim 16 , wherein the carboxyl protecting group is chosen from a C 1 -C 4 alkyl and benzyl group.

26. The polypeptide according to claim 21 , wherein the carboxyl protecting group is chosen from a C 1 -C 4 alkyl and benzyl group.