# Patent

## Title
Process for the manufacture of Degarelix and its intermediates

## Abstract

The present invention relates to a liquid (or solution)-phase manufacturing process for preparing the decapeptide Degareiix, its amino-protected precursor, and other useful intermediates. The invention further relates to polypeptides useful in the solution-phase manufacturing process and to the purification of Degareiix itself. The manufacturing process comprises the step of cleaving the ε-amino protecting group Pε from a Degareiix precursor according to formula {P4)(Pε)Ac-AA1-AA10-NH2 in an organic solvent comprising the precursor and a cleaving agent dissolved therein: wherein P4 is a hydroxyl-protecting group or hydrogen, preferably hydrogen.

## Background
This is a national stage entry application of International Patent Application No. PCT/EP2011/068733, filed Oct. 26, 2011, which claims the benefit of priority of European Patent Application No. 10189011.9, filed Oct. 27, 2010, both of which are incorporated herein by reference in their entireties.
TECHNICAL FIELD
The present invention relates to a liquid (or solution)-phase manufacturing process for preparing the decapeptide Degarelix, its amino-protected precursor, and other useful intermediates. The invention further relates to polypeptides useful in the solution-phase manufacturing process and to the purification of Degarelix itself.
BACKGROUND OF THE INVENTION
Prostate cancer is a leading cause of morbidity and mortality for men in the industrialised world. Degarelix, also known as FE200486, is a third generation gonadotropin releasing hormone (GnRH) receptor antagonist (a GnRH blacker) that has been developed and recently approved for prostate cancer patients in need of androgen ablation therapy (Doehn et al., Drugs 2006, vol. 9, No. 8, pp. 565-571; WO 09846634). Degarelix acts by immediate and competitive blockade of GnRH receptors in the pituitary and, like other GnRH antagonists, does not cause an initial stimulation of luteinizing hormone production via the hypothalamic-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare (Van Poppel, Cancer Management and Research, 2010:2 39-52; Van Poppel et al., Urology, 2008, 71(6), 1001-1006); James, E. F. et al., Drugs, 2009, 69(14), 1967-1976).
Degarelix is a synthetic linear decapeptide containing seven unnatural amino acids, five of which are D-amino acids. It has ten chiral centers in the back bone of the decapeptide. The amino acid residue at position 5 in the sequence has an additional chiral center in the side-chain substitution giving eleven chiral centers in total. Its CAS registry number is 214766-78-6 (of free base) and it is commercially available under the Trademark FirmagonTM. The drug substance is chemically designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-diaxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl- and is represented by the chemical structure below:




The structure of Degarelix can also be represented as:

Ac-D-2NaI-D-4Cpa-D-3PaI-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 

where Ac is acetyl, 2NaI is 2-naphthylalanine, 4 Cpa is 4-chlorophenylalanine, 3 PaI is 3-pyridylalanine, Ser is serine, 4Aph is 4-aminophenylalanine, Hor is hydroorotyl, Cbm is carbamoyl, Leu is leucine, Lys(iPr) is N6-isopropyllysine, Pro is proline and Ala is alanine.

For the purposes of describing this invention, each amino acid in Degarelix will be given the shorthand notation as follows:
AA1 is D-2NaI, AA2 is D-4 Cpa, AA3 is D-3 PaI, AA4 is Ser, AA5 is 4Aph(L-Hor), AA6 is D-Aph(Cbm), AA7 is Leu, AA8 is Lys(iPr), AA9 is Pro and AA10 is D-Ala.
Thus, as an example, Degarelix can be represented as Ac-AA1-AA10-NH2, the tetrapeptide Ac-D-2NaI-D-4 Cpa-D-3 PaI-Ser can be represented as Ac-AA1-AA4 and the hexapeptide 4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 as AA5-AA10-NH2.
Degarelix has previously been prepared using Boc-solid phase peptide synthesis (SPPS) methodology as reported in WO 98/46634 and Jiang et al., J. Med. Chem. 2001, 44, 453-467. Basically, Boc-protected D-Ala is first coupled to MBNA resin in dimethylformamide (DMF)/CH2Cl2 using diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) as activating or coupling agents. Once D-Ala is coupled to the resin, synthesis proceeds by washing, deblocking and then coupling the next amino acid residue until the decapeptide has been completed. The side chain primary amino groups of 4Aph in the 5-position and of D-4Aph in the 6-position are protected by Fmoc when they are added and modified with L-Hor and Cbm respectively before the next amino acid in the chain is added. This requires the additional steps of first removing the side-chain protection with piperidine, reacting the newly freed amino group on the peptidoresin with tert-butyl isocyanate or L-hydroorotic acid, ensuring that the reaction is complete with a ninhydrin test and then washing the peptidoresin before adding the next amino acid residue (see also Sorbera et al., Drugs of the Future 2006, Vol. 31, No. 9, pp 755-766).
While Boc-SPPS methodology has afforded sufficient quantities of Degarelix until now, the growing demand for this polypeptide means that ever larger quantities are required. Boc-SPPS, which requires HF cleavage, is not suited to large scale industrial synthesis. Indeed, WO 98/46634 mentions that SPPS is only suitable for limited quantities of up to 1 kg while classical peptide solution synthesis, or liquid phase peptide synthesis (LPPS), is preferred for larger quantities of product. WO 98/46634 does not specify how such synthesis should be performed. Further, expense attributable the large excess of coupling reagents, additives, and amino acids required for the SPPS. While the existence of a liquid phase peptide synthesis of Degarelix has been reported [EMEA Report: Assessment Report for FirmagonTM (Degarelix): Doc. Ref. EMEA/CHMP/635761/2008], as of now no details of such a process have been publically disclosed.
WO 97/34923 and WO 99/26964 are international Application Publications which are concerned with liquid phase processes for the preparation of biologically active peptides. WO 99/26964 is particularly concerned with the liquid phase synthesis of decapeptides having activity as GnRH antagonists. WO 99/26964 lists a number of inherent limitations of the SPPS methodology for producing GnRH antagonists including the limited capacity of the resin, the large excess of reagents and amino acids required, as well as the need to protect all reactive side chains such as the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph, the ε-i-propylamino group in Lys(i-Pr).
International Application Publication No. WO 99/26964 describes a liquid phase process which involves first preparing the central peptide fragments of the 5 and 6 positions of a decapeptide with the side chains fully elaborated and then assembling the peptide through a “4-2-4”, “3-3-4” or “3-4-3” fragment assembly pattern. For example, in the preparation of the GnRH antagonist Azaline B, a tetrapeptide is coupled with a hexapeptide to form the desired decapeptide. When the same fragment assembly pattern is attempted for Degarelix, racemisation of the Serine amino acid (AA4) occurs resulting in about 20% impurity of L-Ser. This impurity carries over into the final decapeptide and is difficult to remove. Furthermore, when preparing the tetrapeptide AA1-AA4 by adding the Ser unit to the tripeptide AA1-AA3 following the procedure described in WO 99/26964, tetrabutylammonium ions from the hydrolysis of the benzyl ester group could not be removed completely during the subsequent operations and were carried through to the final product. It was further found that in the Degarelix synthesis, the L-hydroorotyl group rearranges to its hydantainacetyl analogue when L-dihydroorotic acid is coupled with 4 Amp to prepare AA5. These and other problems with the solution-phase synthesis of Degarelix have now been overcome and a new solution-phase polypeptide synthesis of this decapeptide is disclosed herein for the first time.
SUMMARY OF THE INVENTION
The problems of SSPS methods for preparing Degarelix and the drawbacks of LLPS methods as described in WO 97/34923 and WO 99/26964 have now been overcome and are the subject of this invention.
In general, this invention relates to a liquid-phase synthesis of the decapeptide Degarelix.
In one aspect, the invention relates to a liquid-phase process for preparing Degarelix having the formula Ac-AA1-AA10-NH2 or a pharmaceutically acceptable salt or solvate thereof, comprising the step of cleaving the ε-amino protecting group Pε from a Degarelix precursor according to formula (P4)(Pε)Ac-AA1-AA10-NH2 in an organic solvent comprising the precursor and a cleaving agent dissolved therein:




P4 is a hydroxyl-protecting group or hydrogen, preferably, tBu, (ψPro) (i.e. pseudo-proline), or hydrogen. If P4 is a hydroxyl-protecting group, the process also comprises the step of cleaving the hydroxyl-protecting group P4 from the Degarelix precursor. The protecting group P4 is preferably selected in such a way that this cleavage step can be carried out simultaneously with the cleavage of the amino-protecting group Pε. This is for example the case if both P4 and Pε are BOC.
In a second aspect, the invention also relates to a liquid-phase process for preparing a protected Degarelix precursor having the formula (P4)(Pε)Ac-AA1-AA10-NH2 or a pharmaceutically acceptable salt or solvate thereof, comprising the step of coupling (P4)Ac-AA1-AA4 with (Pε)AA5-AA10NH2 or coupling Ac-AA1-AA3 with (P4)(Pε)AA4-AA10NH2 in an organic solvent comprising the two peptides, a peptide coupling reagent and an organic amine base dissolved therein wherein Pε is an amino protecting group. The peptides are represented below:







A third aspect concerns the liquid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA1-AA4:




or a pharmaceutically acceptable salt or solvate thereof, comprising the step of hydrolyzing a compound having the formula (P4)Ac-AA1-AA4-R with an alkaline hydroxide, wherein R represents a carboxyl protecting group, preferably C1-C4 alkyl or benzyl:





A fourth aspect concerns a process for preparing the compound (P4)Ac-AA3-AA4-R by coupling Ac-AA1-AA3 with (P4)AA4-R or coupling Ac-AA1-AA2 with (P4)AA3-AA4-R, the peptides being represented below




In each of the formulae described above, AA1 to AA10, P4 and Pε have the same meanings as in formula II, and R represents a carboxyl protecting group, preferably C1-C4 alkyl or benzyl
In a fifth aspect, the tetrapeptide (P4)Ac-AA1-AA4 is prepared not by liquid phase synthesis, but by solid phase synthesis. This invention thus also relates to a solid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA1-AA4:




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

    
    
        a) reacting (PN)AA2 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-AA1-AA4.

wherein P4 is H or a hydroxyl protecting group on AA4, and PN is an amino protecting group.

    
    


A sixth aspect of the invention concerns liquid-phase process for preparing the hexapeptide (Pε)AA5-AA10NH2 comprising the coupling of (Pε)AA6-AA10NH2 and (PX)AA5, wherein PX is an amino protecting group and AA5 to AA10 and Pε have the same meaning as above, to provide (PX)(Pε)AA5-AA10NH2, and cleaving Px with TFA to provide (Pε)AA5-AA10NH2, the peptides being represented below:




A seventh aspect of the invention concerns a liquid-phase process for preparing the hexapeptide (Px)(Pε)AA5-AA10NH2 comprising the coupling of (Px)AA5-AA7 with (Pε)AA8-AA10NH2.
An eighth aspect of the invention concerns processes for purifying Degarelix, such as by preparative HPLC using PLRP—S and/or C8 and C18 columns.
It should be understood that in the process of preparing Degarelix according to this invention, any of the above process steps may be combined. For example, this invention also embodies a process in which (P4)Ac-AA1-AA4 is first prepared from (P4)Ac-AA1-AA4-R according to the third aspect of the invention or according to the fifth aspect of the invention before being coupled with (Pε)AA5-AA10-NH2 to form the protected precursor (P4)(Pε)Ac-AA1-AA10-NH2 according to the second aspect of the invention. The precursor (P4)(Pε)Ac-AA1-AA10-NH2 formed by such a process may then be deprotected according to the first aspect of the invention ultimately giving a single process for preparing Degarelix that incorporates the first, second and third or fifth aspects of the invention.
Naturally, any of the purification steps for Degarelix that are described herein may be incorporated into any process in which Degarelix is the final product.



FIGURES
FIG. 1. Liquid phase preparation of the tetrapeptide Ac-AA1-AA4 
FIG. 2. Preparation of the Pε protected hexapeptide AA5-AA10NH2 where Pε is Fmoc.
FIG. 3. Segment condensation and deprotection to yield Degarelix.
FIG. 4: Solid phase preparation of the tetrapeptide Ac-AA1-AA4 with a pseudoproline protecting group on AA4.
FIG. 5: Solid phase preparation of the tetrapeptide Ac-AA1-AA4 with a tBu protecting group on AA4.
FIG. 6: Solid phase preparation of BOC-protected AA5-AA7 
FIG. 7: Solid phase preparation of Fmoc-protected AA8-AA10NH2.



DETAILED DESCRIPTION OF THE INVENTION  
The present invention will now be described in more detail.
Deprotection of the Degarelix Precursor
In a first aspect, the present invention relates to a liquid-phase process for preparing Degarelix having the formula Ac-AA1-AA10-NH2 or a pharmaceutically acceptable salt or solvate thereof. The process comprises the step of cleaving an ε-amino protecting group Pε from a Degarelix precursor according to formula (P4)(Pε)AA1-AA10 in an organic solution comprising the precursor and a cleaving agent dissolved therein.
In this case, Pε is any side chain protecting group known in the art such as those described in E. Gross & J. Meienhofer, The Peptides: Analysis, Structure, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Academic Press, N.Y., 1981), Suitable examples include 9-fluorenylmethyloxycarbonyl (Fmoc), CBZ, and substituted CBZ, such as, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and the like; aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(p-biphenylyl)-isopropyloxycarbonyl, and the like; cycloalkyl urethane-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclo hexyloxycarbonyl; allyloxycarbonyl (Mac). Preferred protecting groups are Fmoc, Boc and Alloc with Fmoc being most preferred.
If required, the hydroxyl group of Ser may also be protected, although this is not preferred. In this case, P4 is not hydrogen, but a hydroxyl protecting group such as for example, a C4-C6 alkyl (e.g. t-butyl, cyclohexyl), trityl, benzyl, a benzyl ether such as p-methoxybenzyl, or other substituted benzyls such as p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, 2,6-dichlorobenzyl, or (ψPro), (pseudoproline). If Ser is protected, particularly preferred is t-butyl, benzyl and 9-fluorenylmethyl ethers, t-butyl being most preferred. P4 is H, tBu, or (ψPro), preferably tBu or (ψPro),
The cleaving agent used to remove the ε-amino protecting group or the Ser hydroxyl protecting group depends on the nature of the protecting group and are well known in the art. In a preferred embodiment, the same cleaving agent is used for both the ε-amino protecting group and the Ser hydroxyl protecting group, if present.
Preferred cleaving agents for the Ser hydroxyl protecting group are:

    
    
        trifluoracetic acid (TFA), HCl, or methanesulfonic acid, particularly for t-butyl ether as a protecting group
        H2/Pd—C, HF, or trifluoromethane sulfonic acid, particularly for benzyl ether as a protecting group, and
        SiCl4/anisol, particularly for 2-(methylsulfinyl)benzylether as a protecting group;
    
    


Preferred cleaving agents for the ε-amino protecting group are:

    
    
        trifluoracetic acid (TFA), HCl, or methanesulfonic acid, particularly for t-butyl carbamates as protecting group
        H2/Pd—C, HF, or trifluoromethane sulfonic acid, particularly for benzyl carbamates as protecting group, and
        Piperidine, DBU and DEA, particularly for Fmoc as protecting group
    
    


Preferred solvents include DCM, DMF, NMP, dioxane, EtOH, Neat HF, and TFA,
Particularly preferred are the different cleavage conditions indicated in the following table 1:







TABLE 1







Cleavage conditions










Protecting group
Protected













Abbreviation
Name
group
Cleavage reagent
Solvent





t-Bu
t-Butyl ethers and esters
—OH and
TFA
DCM




—CO2H
HCl
Dioxane





Methanesulfonic
DCM





acid



Bzl
Benzyl ethers and esters
—OH
H2/Pd—C
EtOH/water




and
HF
Neat




—CO2H
Trifluoromethane-
DCM





sulfonic acid



MsOb
4-(Methylsulfinyl)-benzyl
—OH
SiCl4/anisol
TFA



ether





Tce
2,2,2-Trichloroethyl esters
—CO2H
Zn
AcOH/H2O


Cbz or Z
Benzyloxycarbonyl
—NH2
H2/Pd—C
EtOH/Water/acid





HF
Neat





Trifluoromethane-
DCM





sulfonic acid



Boc
tert-Butoxy-carbonyl
—NH2
TFA
DCM





HCl
Dioxane





Methanesulfonic
DCM





acid



Fmoc
9-Fluorenylmethoxy-
—NH2
piperidine
DMF



carbonyl

DBU (1,8-
DMF





diazabicyclo[5.4.0]-






undec-7-ene)






DEA (diethylamine)
DMF


Trt
Trityl (Trt)
—OH
1% TFA—DCM
DCM




—NH2




TBDMS
Tert-butyl-dimethyl-silyl
—OH
TFA
THF





ACOH—THF—H2O






(3:1:1), 18 h






0.1 MTBAF in THF



Cyclohexyl 
Cyclohexyl
—OH
HF or TFSMA
Neat HF or


(CHX or CHx)



DCM


Troc
2,2,2-Trichloroethoxy-
—NH2
Zn
AcOH/H2O



carbonyl





Reference: Chem. Rev. 2009, 109, 2465-2504 (by Albert Isidro-Llobet et al.)






Typically, a cleaving agent such as piperidine is dissolved in an organic solvent such as DMF, NMP under an inert atmosphere such as N2 or argon and cooled to a temperature between −20 and 0° C., preferably −10 and −2° C., e.g. about −5° C. The protected intermediate (Pε)AA1-AA10 is added and the reaction mixture is then stirred at a temperature of between −20 and 25° C., preferably −10 and 10° C. and more preferably 0 and 5° C. When the protecting group has been removed (preferably the yield is >95% yield, most preferably >99%), the crude Degarelix can be precipitated, filtered and then washed with ether. For example, the crude Degarelix can be precipitated by adding it to ether, such as methyl t-butyl ether (MTBE) or DIPE, and stirring for 10 to 30 minutes. The precipitate can then be washed with ether (preferably DIPE). Subsequently, the solid may be taken up in e.g. ethyl acetate and stirred for some time at room temperature. The fine solid obtained may then be filtered, washed (e.g. with ethyl acetate) and dried under vacuum.
It has been found that extended reaction periods are not detrimental to the quality of the reaction and that no significant (<0.03% yield as determined by HPLC) increase in hydantoin impurity is observed if the reaction is allowed to proceed for up to 24 hours. Furthermore, particularly in the case of piperidine as a cleaving agent, no hydantoin impurity is observed if the reaction is performed in the presence of 5 Vol % water per volume of solvent (e.g. 0.1 ml of water per 2 ml solvent such as DMF) for up to 20 hours. This demonstrates the robustness of this deprotecting reaction for the PPS of Degarelix.
4+6 coupling and 3+7 coupling
In a second aspect, the invention relates to a liquid-phase process for preparing the protected Degarelix precursor having the formula (P4)(Pε)AA1-AA10 or a pharmaceutically acceptable salt or solvate thereof. This process may comprise the step of coupling a tetrapeptide intermediate according to formula (P4)Ac-AA1-AA4 with a hexapeptide intermediate according to formula (Pε)AA5-AA10. It may also comprise the step of coupling a tripeptide intermediate according to formula Ac-AA1-AA3 with a heptapeptide intermediate according to formula (P4)(Pε)AA4-AA10. In either case, the protecting group Pε may be any ε-amino protecting group as discussed previously. The hydroxyl group of Ser may also be protected if required (i.e., in this case P4 is not hydrogen, but a hydroxyl protecting group). The coupling reaction is performed in an organic solution where the two peptides, a peptide coupling reagent and an organic amine base are dissolved therein. A peptide coupling additive may also be present.
The organic solvent, peptide coupling reagent, peptide coupling additive and organic amine base may be any of those known in the art of LPPS.
Typical organic solvents are THF, NMP (N-methylpyrrolidone), Davi, DMF, DMSO, and mixtures thereof.
Typical peptide coupling reagents are one or more of o-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), o-(benzotriazol-1-0)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP—Cl), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), 1,3 dicyclohexylcarbodiimide (DCC), 1,3-diisopropyl-carbodiimide (DIC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCDl), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isopropylchloroformate (IPCF), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA) and 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). Preferred coupling reagents are DIC, HATU, HBTU, and BOP.
Typical peptide coupling additives are 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-1H-benzotriazole (HOBt), 6-chloro-HOBt, and 1-hydroxy-7-azabenzotriazole (HOAt). Particularly preferred are HOBt and HOAt.
Typical organic amine bases are NMM, DIPEA, TEA, and collidine. Particularly preferred is DIPEA.
Particularly preferred is the combined use of HATU, HOAt, and DIPEA. Another preferred embodiment relates to the combined use of DIC, 6-chloro-HOBt, optionally in combination with copper salts.
Surprisingly, it has been found that the choice of organic solvent, peptide coupling reagent/additive and organic amine base has an effect on the yields of the desired products and on the racemisation of the Ser amino acid in the polypeptide backbone.
For instance, while THF, NMP, DCM, DMF and mixtures thereof can be used as solvents for these coupling reactions, the use of DMF, either alone or in a mixture (e.g. with DCM), increases the yield of the desired final product while at the same time reducing any D-Ser impurity. The use of DMF apparently increases the yield and reduces D-Ser impurities. The activation is rapid in a polar solvent such as DMF but slow in a non-polar solvent such as DCM. The HATU/HOAt combination leads to a highly efficient coupling partly because of rapid activation by a base such as DIEA or NMM followed by rapid coupling in the presence of the same base in a polar solvent like DMF. The effect of different solvents is illustrated in Table 2.







TABLE 2







Screening of different solvents during the coupling of AA1-AA4 and AA5-AA10 segment












Coupling

Purity by HPLC (%)
















Reagent &
Yield
Product
Impurity



Entry
Solvent
additive
(%)
CDEG-1
(D-ser)
Remarks
















1
THF
EDC•HCl
35
62.42
16.29
NMM as a base; CLEU-15








content 26.5%


2
NMP
EDC•HCl
35
62.42
16.29
NMM as a base; CLEU-15








content 0.17%


3
DCM-DMF
EDC•HCl
53
47.81
10.39
CLEU-15 content 25.54%


4
DMF
EDC•HCl
65
72
9.2
Collidine as a base


5
DMF
HATU/HOAt
74
84.4
1.91
3.0 eq. of DIEA is used


6
DMF
HATU/HOAt
74
85.3
1.44
3.0 eq. of DIEA is used


7
DMF
HATU/HOAt
87
83.65
1.44
3.0 eq. of DIEA is used









The choice of coupling reagent and additive also has a large effect on the yield and degree of racemisation. Coupling reactions using HBTU, HCTU, TBTU, BOP and HATU were performed and found with HBTU and HATU giving the best overall yields. However, it was found that coupling reactions using BOP or HATU as coupling reagent led to much less racemisation of the Ser amino acid.
The addition of a coupling additive significantly improves the yield of the desired polypeptide. In many cases the coupling additive also reduces the degree of racemisation of the Ser amino acid even further thus leading to a product with fewer impurities. Combinations of coupling reagent and additive which increased yield are TBTU/HOAt, HATU/HOAt and HATU/HOBt. Surprisingly, the combination of TBTU/HOAt or HATU/HOAt increased yield while at the same time reducing racemisation, with HATU/HOAt performing best out of all the combinations tested.
The effect of different coupling agents and additives is illustrated in the following Tables 3 and 4.







TABLE 3







Screening of different coupling reagents to control the racemization











Coupling
Purity by HPLC (%)
















Reagent &
CDEG-1

Impurity



Entry
Base
additive
Yield (%)
CDEG-1
(D-ser)
Remarks
















1
NMM
EDC•HCl/HOBt
67
65
20
Racemization is more


2
NMM
EDC•HCl/HOBt
67
65
18.9
Racemization is more


3
DIEA
TBTU
39
67.93
13.34
1.3% of CLEU-15 remains


4
DIEA
HBTU
53
54.9
20.49
1.9% of CLEU-15 remains


5
DIEA
EDC•HCl
51
—
—
68.9% of CLEU-15 remains


6
NMM
HBTU
78
61.42
15.16
9.8% of CLEU-15 remains


7
NMM
HCTU
60
51.63
15.92
7.32% of CLEU-15 remains


8
NMM
TBTU
53
62.78
14.73
3.75% of CLEU-15 remains


9
NMM
BOP
51
46.3
8.92
11.93% of CLEU-15 remains


10
NMM
HATU
74
65.97
10.62
4.52% of CLEU-15 remains


11
DIEA
TBTU/HOAt
98
76.26
6.8
0.14% of CLEU-15 remains


12
DIEA
DIC/HOAt
—
20.49
1.18
45.9% of CLEU-15 remains


13
DIEA
HATU/HOAt
74
83.16
2.9
No unreacted CLEU-15


14
NMM
HATU/HOAt
81
64.0
13.32
7.1% of CLEU-15 remains


15
NMM
HATU/HOBt
89
65.0
13.14
4.35% of CLEU-15 remains


16
NMM
EDC•HCl/HOAt
74
70.38
13.66
No unreacted CLEU-15


17
DIEA
HATU/HOAt
79
79.79
2.3
This reaction was repeated








in larger scale


18
DIEA
TBTU/HOAt
84
75.8
7.2
Reaction was repeated in








larger scale.


19
DIEA
HATU/HOAt
74
84.4
1.91
Reaction was repeated in








larger scale.


20
DIEA
HATU/HOAt
74
85.3
1.44
Reaction was repeated in








larger scale.


21
DIEA
HATU/HOAt
87
84
1.44
Reaction was repeated in








larger scale.


22
DIEA
HATU/HOAt
79
84.5
1.37
Reaction was repeated in








larger scale.
















TABLE 4







Screening of different additives to control the racemization during the coupling of AA1-AA4


and AA5-AA10 segment












Coupling
CDEG-1
Purity by HPLC (%)















Entry
Reagent
Additive
Base
Yield (%)
CDEG-1
(D-ser)
Remarks

















1
TBTU
HOAt
DIEA
98
76.26
6.8
0.14% of CLEU-15









remains


2
DIC
HOAt
DIEA
—
20.49
1.18
45.9% of CLEU-15 remains


3
HATU
HOAt
DIEA
74
83.16
2.9
Completion of reaction


4
HATU
HOAt
NMM
80
64.0
13.32
7.1% of CLEU-15









remains


5
HATU
HOBt
NMM
89
65.0
13.14
4.3% of CLEU-15









remains


6
EDC•HCl
HOAt
NMM
74
70.38
13.66
No unreacted CLEU-15


7
HATU
HOAt
DIEA
79
79.79
2.3
Repeat reaction


8
TBTU
HOAt
DIEA
84
75.8
7.2
Repeat reaction


9
HATU
HOAt
DIEA
74
84.4
1.91
3.0 eq. of base is used


10
HATU
HOAt
DIEA
74
85.3
1.44
3.0 eq. of base is used


11
HATU
HOAt
DIEA
87
83.65
1.44
3.0 eq. of base is used


12
EDC•HCl
HOBt
NMM
67
65
20
Racemization is more


13
EDC•HCl
HOBt
NMM
64
65
17.9
Racemization is more


14
EDC•HCl
HOBt
NMM
67
65
18.9
Racemization is more


15

HOSu
NMM
—
—
—
Reaction did not proceed









The choice of organic amine base also affects the reaction. For the present invention, NMM and DIEA are preferred as they allow the desired polypeptide to be obtained in the best yields. DIEA is more preferred since this base reduces the degree of Ser racemisation. It has also been found that the amount of base affects the reaction. When a base such as DIEA is used, it was found that the more base present, the lower the yield and higher the degree of racemisation. For example, six equivalents of base (with respect to A51-A10) lead to a two-fold increase of the racemisation product as when three equivalents of base are used. Thus, it is preferred to use 1-5 equivalents of base, more preferably 2-4 equivalents of base and most preferably 2.5 to 3.5 equivalents of base in these coupling reactions. The effect of different bases and their amounts is shown in the following tables 5 and 6.







TABLE 5







Screening of different bases to control the racemization during the coupling of AA1-AA4 and


AA5-AA10 segment












Coupling
Yield (%)
Purity by HPLC (%)















Entry
Reagent
Additive
Base
CDEG-1
CDEG-1
(D-ser)
Remarks

















1
TBTU
HOAt
DIEA
98
76.26
6.8
0.14% of CLEU-15









remains


2
DIC
HOAt
DIEA
—
20.49
1.18
45.9% of CLEU-15









remains


3
HATU
HOAt
DIEA
74
83.16
2.9
No CLEU-15 remains


4
HATU
HOAt
NMM
80
64.0
13.32
7.1% of CLEU-15









remains


5
HATU
HOBt
NMM
89
65.0
13.14
4.35% of CLEU-15









remains


6
EDC•HCl
HOAt
NMM
74
70.38
13.66
No CLEU-15 remains


7
HATU
HOAt
DIEA
79
79.79
2.3


8
TBTU
HOAt
DIEA
84
75.8
7.2


9
HATU
HOAt
DIEA
74
84.4
1.91
3.0 eq. of base is used


10
HATU
HOAt
DIEA
74
85.3
1.44
3.0 eq. of base is used


11
HATU
HOAt
DIEA
87
83.65
1.44
3.0 eq. of base is used


12
EDC•HCl
HOBt
Collidine
65
72
9.2


13
EDC•HCl
HOBt
NMM
67
65
20
Racemization is more


14
EDC•HCl
HOBt
NMM
64
65
17.9
Racemization is more


15
EDC•HCl
HOBt
NMM
67
65
18.9
Racemization is more
















TABLE 6







Screening of base equivalence to control the racemization during the 


coupling of AA1-AA4 and AA5-AA10 segment












DIEA Qty
Coupling Reagent &
CDBG-1
Purity by HPLC (%)












Entry
(eq.)
additive
Yield (%)
Product CDEG-1
Impurity (D-ser)















1
6.0
HATU/HOAt
62
74.19
1.5


2
5.0
HATU/HOAt
69
79.00
3.88


3
4.5
HATU/HOAt
68
84.12
1.18


4
4.0
HATU/HOAt
70
82.64
1.19


5
3.0
HATU/HOAt
74
84.4
1.91


6
3.0
HATU/HOAt
74
85.3
1.44


7
3.0
HATU/HOAt
87
83.65
1.44









The temperature that the coupling reaction is performed also influences the yield and the degree of racemisation of the final product. It was found that a reaction carried out at −15° C. gives higher yield, higher purity and less racemisation of the final product than the equivalent reaction carried out at −5° C. Thus, it is preferable to carry out these coupling reactions at temperatures lower than −5° C., preferably lower than −10° C. and most preferably at −15° C. or lower. The reaction time of these coupling reactions is usually 2-3 hours.
It should be noted that by controlling the reaction temperature and the amount of base added, it is also possible to reduce any hydantoin impurity formation. The hydantoin content is preferably less than 0.5 wt. %, more preferably less than 0.3 wt. %. Thus, it is preferred to use 2.5 to 3.5 equivalents of base at temperatures of −10° C. or lower in these reactions.
Finally, the order of addition of the various reagents also plays a role in the final yield, purity and amount of racemisation. If the peptides and coupling additive are first dissolved in the organic solvent before the coupling reagent and the organic amine are added, the overall yield of the desired product is significantly higher. Furthermore, the amount of racemisation is drastically reduced.
Fragment (P4)Ac-AA1-AA4 
The present invention provides different methods for preparing (P4)Ac-AA1-AA4.
In a third aspect, the invention relates to a liquid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA1-AA4:




or a pharmaceutically acceptable salt or solvate thereof, wherein P4 is hydrogen or a hydroxyl-protecting group, preferably hydrogen.

When preparing (P4)Ac-AA1-AA4, an ester having the formula (P4)Ac-AA1-AA4-R is first prepared, wherein R is a carboxyl protecting group, such as a benzyl group, preferably however a C1-C4 alkyl group. Normally, a benzyl ester of serine is used, the benzyl group being then removed by hydrolysis with tetrabutylammonium hydroxide (see for example WO 99/26964). However, it was found that in the preparation of Degarelix, the tetrabutylammonium ions were not removed completely during subsequent operations and were carried through to the final product. This problem was overcome by using a C1-C4 alkyl ester of serine (e.g. serine methyl ester). It was found that the alkyl ester could be easily hydrolyzed using an alkali hydroxide such as LiOH. The yield and quality of the tetrapeptide was not affected by this change and the problem of tetrabutylammonium ion impurities was eliminated.
For example, a compound according to formula (P4)Ac-AA1-AA4-R may be suspended in an organic solvent such as THF and then stirred and cooled to a temperature of between −20 and 5° C., and more preferably −5 and 0° C. An aqueous solution of LiOH is then added to the cooled solution. The aqueous LiOH is added at a rate that maintains the temperature of the cooled solution in the range of −5 to 0° C. or below. The solution (oftentimes turbid) is stirred for up to 12 hours, preferably up to 3 hours, before being added, with good stirring, to water with a temperature of 5° C. or below, or preferably a mixture of ice and water. Any precipitate at this point is removed by filtration. The pH is then adjusted to pH 4.1-4.3, preferably about 4.2 using any known pH adjusting agent. Preferred is HCl, for example 2M HCl. The precipitate that forms after adjusting the pH is collected by filtration. The precipitate can be further purified by washing it with water, and/or stirring a slurry of it in refluxing MeOH and/or a MeOH/MeCN mixture before collecting it by filtration and then drying it to yield (P4)Ac-AA1-AA4.
A fourth aspect of the invention concerns a process for preparing the compound (P4)Ac-AA1-AA4-R by coupling Ac-AA1-AA3 with (P4)AA4-R or coupling Ac-AA1-AA2 with (P4)AA3-AA4-R, wherein R is a C1-C4-alkyl and P4 is hydrogen or a hydroxyl-protecting group, preferably hydrogen. For the coupling reaction, essentially the same reagents and conditions as those described above can be used.
In a fifth aspect, this invention relates to a solid-phase process for preparing a Degarelix intermediate having the formula (P4)Ac-AA1-AA4:




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

    
    
        a) reacting (PN)AA2 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-AA1-AA4.

wherein P4 is H or a hydroxyl protecting group on AA4, and PN is an amino protecting group.

    
    


PN is preferably Fmoc, which is preferably removed with piperidine/NMP.
P4 is preferably tBu or (WPro). Particularly preferred is the combination of (ψPro) for P4 and Fmoc for PN.
Each coupling step is preferably carried out in a manner known per se, preferably however using HATU (or HBTU) and DIPEA and coupling additives.
The starting material




starting can be prepared by coupling (PN, P4)AA3-AA4 to a resin, for example to a 2-CITrt resin, and then removing PN, or by coupling (PN, P4)AA4 to





to obtain





removing PN, and then reacting (PN)AA3 with





to provide





and then removing PN. This is illustrated in FIGS. 4 and 5.

Suitable resins




include trityl, 2-CITrt and SASRIN.

In the case P4 is (ψPro) and PN is Fmoc, (PN, P4)AA3-AA4 can be prepared following J. Am, Chem. Soc. 1996, 118, 9218-9227. That is, Fmoc protected AA3 is activated; reacted with serine or a salt thereof, and subsequently reacted with acetone or acetone dimethylketal, as illustrated below. Fmoc-D3 PaI-Ser((ψMe,MePro)-OH is particularly preferred as (PN, P4)AA3-AA4.




Fragment (PX)(Pε)AA5-AA10NH2 

A sixth aspect of the invention concerns liquid-phase process for preparing the hexapeptide (Pε)AA5-AA10NH2 comprising the coupling of (Pε)AA6-AA10NH2 and (PX)AA5, wherein PX is an amino protecting group and AA5 to AA10 and Pε have the same meaning as above, to provide (PX)(Pε)AA5-AA10NH2, and cleaving Px with TFA to provide (Pε)AA5-AA10NH2.
A seventh aspect of the invention concerns a liquid-phase process for preparing the hexapeptide (Pε)AA5-AA10NH2 by coupling (P5)AA5-AA7 with (Pε)AA8-AA10NH2 to provide (P5, Pε)AA5-AA10NH2, and subsequently cleaving P5 to provide (Pε)AA5-AA10NH2 (wherein P5 is an amino-protecting group on AA5).
P5 protecting group is preferably BOC. Pε is preferably 9-fluorenylmethyloxycarbonyl (Fmoc). The coupling reaction is preferably carried out in the presence of HATU.
(P5)AA5-AA7 and (Pε)AA5-AA10NH2 are preferably synthesized by solid phase peptide synthesis, e.g. as illustrated in FIGS. 6 and 7, respectively. The following combinations of P5 and Pε are preferred:














P5
Pε









Boc
Fmoc



Cbz
Boc



Troc
Boc










That is, (P5)AA5-AA7OH can be prepared by coupling protected AA7 to a resin; removing the protecting group (e.g. Fmoc); reacting protected AA6 with the obtained product; removing the protecting group (e.g. Fmoc); reacting protected AA5 (e.g. BOC protected) with the obtained product; and cleavage from the resin. This is illustrated in FIG. 6.
(Pε)AA5-AA10NH2 can be prepared by coupling protected AA10 to a resin, e.g. a Rink amide resin; removing the protecting group (e.g. Fmoc); reacting protected AA9 with the obtained product; removing the protecting group (e.g. Fmoc); reacting protected AA8 (preferably Boc-protected on the alpha-amino group and Fmoc-protected on the side chain) with the obtained product; removing the alpha-amino protecting group; and cleavage from the resin. This is illustrated in FIG. 7.
(Pε)AA8-AA10NH2 can also be prepared by reacting AA10 NH2 with protected (e.g. BOC) AA9; removing the protecting group; reacting AA9-AA10 NH2 with protected AA8 (preferably Boc-protected on the alpha-amino group and Fmoc-protected on the side chain); and removing the protecting group on the alpha-amino group.
In an eighth aspect, the invention relates to the purification of Degarelix. The purification can be carried out in a manner known to the skilled person, e.g. by preparative chromatography.
For example, first purification of Degarelix is achieved with a PLRP—S stationary phase, pH 2.25 using TEAP as a buffer and MeCN (75:25) as mobile phase. Purity of up to 95% can be obtained with this step. If required, a second purification can be carried out using a combination of C8 and C18 columns (e.g. Zorbax) to achieve purity of 99% and above.
EXPERIMENTAL
The following examples are intended to illustrate a process for the LPPS synthesis of Degarelix. Reference is made to FIGS. 1 and 2 for the structures of each peptide and polypeptide described herein.
CLEU-2:
L-Cbz-proline (50.0 g) was dissolved in 2-propanol (500 ml{circumflex over (=)}10 V) and the solution was cooled to 15° C. N-Methylmorpholine (25 ml{circumflex over (=)}0.5 V) was then added slowly. After stirring the solution for 15 minutes, 28.49 g (1.04 eq.) iso-butyl chloroformate was added dropwise at −15° C. A solution of D-ala-NH2.HCl (27.48 g, 1.1 eq) and NMM (25 ml{circumflex over (=)}0.5 V) in water (250 ml{circumflex over (=)}5 V) was added to the reaction mixture at −15° C. The mixture was stirred for 30 minutes at the same temperature and then warmed to 25° C. and stirred for 3-5 hrs. The reaction mixture was quenched by adding ethyl acetate (1000 ml{circumflex over (=)}20 V) and water (10 V) containing NaCl (25 g) and NaHCO3 (25 g). The organic layers were separated, washed with water (2×500 ml{circumflex over (=)}10 V) and then dried over sodium sulphate. The organic layer was concentrated to 4 volumes under vacuum below 40° C. The solution was diluted with ethyl acetate (250 ml{circumflex over (=)}5 V) and n-hexane (375 ml{circumflex over (=)}7.5 V) was added dropwise to obtain a white solid. The solid was filtered off and dried to afford the product.
Output: 35.2 g; 54.7%; [α]25D: −13.0° [CHCl3, Literature report: −11.2° (U.S. Pat. No. 5,710,246)]). HPLC purity: 99.44%
CLEU-4:
CLEU-2 (19.5 g) was taken in 2-propanol (129 ml{circumflex over (=)}6.5 V) into a Parr hydrogenation flask and a solution of p-toluenesuiphanic acid in water (20 ml{circumflex over (=)}1.0 V) was added to it. 10% Pd/C (5% w/w) was added to the reaction mixture and the mixture then hydrogenated at 40 psi for 2 hrs. When TLC showed the disappearance of the starting material, the catalyst was filtered and washed with 2-propanol (79 ml{circumflex over (=)}4 V) and water (8.75 ml{circumflex over (=)}0.5 V). The filtrate was concentrated under vacuum and stripped off with acetonitrile (4×254 ml{circumflex over (=)}13 V). The residue was taken in a flask and acetonitrile (215 mL{circumflex over (=)}11 V) was added followed by Boc-Lys(Cbz)-OH (25.5 g{circumflex over (=)}1.1 equiv) and HOBt (9.9 g{circumflex over (=)}1.2 equiv). The suspension was cooled to −5° C. and NMM (14.95 g{circumflex over (=)}2.45 equiv) was added slowly. Finally, a solution of EDC.HCl (15.3 g{circumflex over (=)}1.3 equiv) in acetonitrile (120 ml{circumflex over (=)}6 V) was added. The reaction mixture was then warmed to 25° C. and stirred for 10-12 hrs at the same temperature. The solvent was removed under vacuum below 40° C. and diluted with water (120 ml{circumflex over (=)}6 V). The product was extracted with ethyl acetate (878 ml{circumflex over (=)}45 V), water (120 ml{circumflex over (=)}6 V), and 10% sodium carbonate (110 ml{circumflex over (=)}5.7 V). The aqueous layer was extracted with ethyl acetate (2×430 ml{circumflex over (=)}2×22 V) and the organic layers combined and washed with 10% citric acid solution (2×105 ml), 10% sodium carbonate solution (2×110 ml{circumflex over (=)}2×5 V), water (120 ml{circumflex over (=)}6 V) and dried over sodium sulphate. The organic layer was concentrated to 10-12 volumes under vacuum below 40° C., stripped off with ethyl acetate (3×20 V) and maintained the final 10-12 volumes in each stripping. N-hexane (14 V) was added dropwise to the concentrate mass to get a white solid. The solid was filtered off and dried to afford the product.
Output 29.2 g; Yield: 86.6%; Purity 99.18%, [α]25D: −26.0° (c 1, CHCl3)
CLEU-5:
CLEU-4 (16.3 g) was taken in a mixture of methanol (163 ml{circumflex over (=)}10 V), acetone (22 ml{circumflex over (=)}1.4 V) in a stainless steel Parr hydrogenation flask. 10% Pd/C (10% w/w) was added and the mixture was hydrogenated (60 psi) for 8-12 hrs at 25° C. After the starting material had disappeared (TLC), the catalyst was filtered through celite and was washed with methanol (163 ml{circumflex over (=)}10 V). The filtrate was concentrated under vacuum below 40° C. and the residue was stripped off with ethyl acetate (3×143 ml{circumflex over (=)}3×9 V). The residue was then taken in ethyl acetate (51 ml{circumflex over (=)}3 V) and n-hexane (20.4 ml{circumflex over (=)}1.25 V) added. The mixture was stirred for 2-3 hrs to obtain a free solid. The solid was filtered, washed with n-hexane (38 ml{circumflex over (=)}2 V), and dried under vacuum below 40° C.
Output: 12.3 g; Yield: 90.8%; Purity: 98.9%
CLEU-6:
CLEU-5 (12.2 g) was added to THF (40 ml{circumflex over (=)}3.3 V) and the mixture was cooled to 0° C. 10% sodium carbonate solution (34 ml{circumflex over (=)}2.7 V) was added to the mixture over 20 minutes. Fmoc-Cl (8.63 g{circumflex over (=)}1.2 equiv) in THF (12.2 ml{circumflex over (=)}1.0 V) was then added slowly over 15-20 minutes at 0° C. The reaction mixture was stirred for 1 hr at same temperature and diluted with water (134.2 ml{circumflex over (=)}11 V). The product was extracted with ethyl acetate (269 ml{circumflex over (=)}22 V). The organic layer was washed with water (134.2 ml{circumflex over (=)}11 V), 10% citric acid solution (2×134 ml{circumflex over (=)}2×11 V) and water (134.2 ml{circumflex over (=)}11 V). The organic layer was concentrated under vacuum below 40° C. and the crude product was purified by column chromatography.
Output: 13.3 g; Yield: 73.2%; Purity: 98.2%
CLEU-7:
CLEU-6 (9.0 g) was charged to a TFA (61 ml{circumflex over (=)}6.75 V) and m-cresol (0.61 ml) solution at −5° C. The reaction mixture was stirred for 2 hrs at 0° C. and then concentrated under vacuum below 35° C. Traces of TFA were removed by co-distillation with toluene (2×45 ml). The product was crystallized from a mixture of MTBE (9 ml{circumflex over (=)}1 V), and DIPE (90 ml{circumflex over (=)}10 V). The solid was filtered off under nitrogen, washed with DIPE (180 ml{circumflex over (=)}20 V) and dried under vacuum to get pure CLEU-7.
Output: 8.8 g; Yield: 90.4%; Purity: 98.3%.
Comment: CLEU-7 material is hygroscopic in nature and thus should be handled with care.
CLEU-8:
CLEU-7 (8.5 g) was taken in acetonitrile (85 ml{circumflex over (=)}10 V). Boc-Leu-OH (3.12 g{circumflex over (=)}1.1, equiv), HOBt (2.31 g{circumflex over (=)}1.39 equiv) and NMM (1.4 ml{circumflex over (=)}1.03 equiv) were added to the solution. The solution was cooled to −2° C. and treated with NMM (1.4 ml{circumflex over (=)}1.03 equiv) and EDC.HCl (2.58 g{circumflex over (=)}1.1 equiv). The reaction mixture was stirred for 2-3 hrs at 0° C. and the solvent was removed by distillation under vacuum. 10% citric acid (85 ml{circumflex over (=)}10 V) and ethyl acetate (213 ml{circumflex over (=)}25 V) were added to the residue. The organic layer was separated and washed with 10% citric acid solution (2×85 ml{circumflex over (=)}2×10.0 V), DM water (85 ml{circumflex over (=)}10 V), and 5% sodium bicarbonate solution (3×85 ml{circumflex over (=)}3×10.0 V) and again with DM water (85 ml{circumflex over (=)}10 V). Finally, the organic layer was dried over sodium sulphate and concentrated under vacuum below 35° C. to obtain the crude product. The crude product was crystallized from MTBE (68 ml{circumflex over (=)}8 V) and n-hexane (34 ml{circumflex over (=)}4 V). The solid was dried under vacuum at below 35° C.
Output: 7.8 g; Yield: 81.2%; Purity: 97.5%.
CLEU-9:
CLEU-8 (7.5 g) was charged to a TFA (54 ml{circumflex over (=)}7.2 V) and m-cresol (0.27 ml) solution at −5° C. The reaction mixture was stirred for 2.0 hrs at 0° C. and then concentrated under vacuum below 35° C. Traces of TFA were removed by co-distillation with toluene (2×38 ml the product was crystallized from MTBE (75 ml{circumflex over (=)}10 V) and n-hexane (413 ml{circumflex over (=)}15 V). The solid was filtered under nitrogen, washed with n-hexane (37.5 ml{circumflex over (=)}5 V), and dried under vacuum to yield pure CLEU-9.
Output: 7.2 g; Yield: 94.8%; Purity: 97.2%
CLEU-12:
CLEU-9 (10.5 g) was taken in acetonitrile (158 ml{circumflex over (=)}15 V) under nitrogen atmosphere. CMAP-5A (4.2 g{circumflex over (=)}1.0 equiv), HOBt (2.11 g{circumflex over (=)}1.2 equiv) were added into the suspension and the mixture was cooled to 0° C. EDC.HCl (2.73 g{circumflex over (=)}1.1 equiv) was added into the suspension followed by slow addition of NMM (1.38 g{circumflex over (=)}1.05 equiv). The reaction mixture was stirred for 0.5 hr at 0° C., warmed to ambient conditions and then stirring was continued for 3 hrs. The solvent was removed under vacuum below 35° C. and the residue was taken in a mixture of 10% citric acid (105 ml{circumflex over (=)}10 V) and ethyl acetate (263 ml{circumflex over (=)}25 V). The organic layer was separated and washed with 10% citric acid solution (105 ml{circumflex over (=)}10 V), DM water (105 ml{circumflex over (=)}10 V), 5% sodium bicarbonate solution (3×105 ml{circumflex over (=)}3×10 V), and DM water (105 ml{circumflex over (=)}10 V). The organic layer was dried over sodium sulphate and concentrated under vacuum below 35° C. The crude product was precipitated with n-hexane (84 ml{circumflex over (=)}8 V), washed with n-hexane (2×84 ml{circumflex over (=)}2×8 V), and dried under vacuum at 35° C.
Output: 10.5 g; Yield: 80.7%; Purity: 92.1%
CLEU-13:
CLEU-12 (10.5 g) was charged to a TFA (78.8 ml{circumflex over (=)}7.5 V) and m-cresol (0.4 ml) solution at −5° C. The reaction mixture was stirred for 2 hrs at 0° C. and then concentrated under vacuum below 35° C. Traces of TFA were removed by co-distillation with toluene (2×52 ml). The residue was taken up in ethyl acetate (105 ml{circumflex over (=)}10 V) and n-hexane (158 ml{circumflex over (=)}15 V) is added to precipitate the product. The solid was filtered off under nitrogen and washed with n-hexane (53 ml{circumflex over (=)}5 V). The compound was dried under vacuum at 35° C.
Output: 9.6 g; Yield: 90.5%; Purity: 91.9%.
CLEU-14:
CLEU-13 (9.5 g) was dissolved in DMF (95 ml{circumflex over (=)}10 V) under a nitrogen atmosphere. NMM (1.0 g{circumflex over (=)}1.05 equiv), CSER-2 (3.95 g{circumflex over (=)}1.0 equiv) and HOBt (1.4 g{circumflex over (=)}1.1 equiv) were added to the solution and the reaction mass was cooled to 0° C. EDC.HCl (2.0 g{circumflex over (=)}1.1 equiv) and NMM (1.0 g{circumflex over (=)}1.05 equiv) were added subsequently into the mixture. The reaction mass was stirred for 5 hrs at 0° C. and then poured into ice cooled water (950 ml{circumflex over (=)}100 V) and stirred for 30 minutes. The precipitated solid was filtered off and washed with water (20 V), 10% citric acid (10 V), again with water (190 ml{circumflex over (=)}20 V), 5% NaHCO3 solution (95 ml{circumflex over (=)}10 V), and water (95 ml{circumflex over (=)}10 V). The product was dried under vacuum below 35° C.
Output: 10.3 g; Yield: 84.40%; Purity: 90.4%.
CLEU-15:
CLEU-14 (10.0 g) was charged to a TFA (75 ml{circumflex over (=)}7.5 g) and m-cresol (0.37 ml) solution at −5° C. The reaction mixture was stirred for 2 hrs at 0° C. and then concentrated under vacuum below 35° C. Traces of TFA were removed by co-evaporation with toluene (2×50 ml). The residue was taken up in ethyl acetate (100 ml{circumflex over (=)}10 V) and n-hexane (60 ml{circumflex over (=)}6 V) was added to precipitate the product. The solid was filtered off under nitrogen, washed with n-hexane (2×30 ml{circumflex over (=)}6 V) and then dried under vacuum at 35° C.
Output: 9.6 g; Yield: 95.0%; Purity: 91.5%.
CSER-2:
To a stirred solution of L-hydroorotic acid (23.4 g, 148 mmol) and N-hydroxysuccinimide (18.14 g{circumflex over (=)}1.1 equiv) in dry DMF (585 ml{circumflex over (=)}25 V) was added DIC (20.5 g{circumflex over (=)}1.1 equiv) with external ice water cooling. The reaction mixture was stirred at room temperature for 13-14 hrs. The precipitate was filtered off and the filtrate was evaporated. The oily residue was washed with diisopropyl ether (94 ml{circumflex over (=)}4 V) and dissolved in dry DMF (293 ml{circumflex over (=)}12.5 V). N-Boc-L-4-aminophenylalanine (41.5 g{circumflex over (=)}1.0 equiv) was added to the above solution. DIEA (22.97 g{circumflex over (=)}1.2 equiv) was added at 0° C. and the reaction mixture was stirred for 22 hrs and the solvent then evaporated. The residue was mixed with water (702 ml{circumflex over (=)}30 V) and the pH of the resulting suspension was adjusted to 9.0 with saturated sodium bicarbonate solution. The precipitate of diisopropylurea was filtered off and the filtrate was washed with ethyl acetate (70.2 ml{circumflex over (=)}3 V). The aqueous layer was acidified to pH 2.5 with 6 N HCl and the resulting precipitate collected by filtration. The product was obtained as a yellow solid.
Output: 40.0 g; Yield 64%; mp-270° C., [α]25D=+61.5 (c 1.0, 1% NaHCO3); Purity 95%.
CBBC-2B:
Fragment-A (20.0 g) (purchased from Chirotech, UK) was dissolved in DMF (300 ml{circumflex over (=)}15 V) at 30-35° C. and then cooled −10° C. to 5° C. wherein HOBt (5.06 g{circumflex over (=)}1.1 equiv) was added to the mixture, stirred for 30 minutes before L-serine methyl ester (5.29 g{circumflex over (=)}1.0 equiv) was added to the suspension. The mixture was stirred for 30 minutes, treated with NMM (7.23 g{circumflex over (=)}2.1 equiv) and then stirred for 30 minutes. EDC.HCl (7.18 g{circumflex over (=)}1.1 equiv) was then added to the suspension and the reaction mixture was stirred for 5 hrs at −5° C. to 0° C. The reaction mixture was poured into chilled DM water (151 or 1500 ml{circumflex over (=)}75 V) and stirred for 30 minutes. The product was precipitated by stirring at 0-5° C. After 1 hr the resulting solid was collected by filtration. The filter cake was washed with water (200 ml{circumflex over (=)}10 V), 10% citric acid (200 ml{circumflex over (=)}10 V), again with water (200 ml{circumflex over (=)}10 V), 5% NaHCO3 solution (200 ml{circumflex over (=)}10 V), water (200 ml{circumflex over (=)}10 V) and then the solid was dried under vacuum for 4 hrs. The product was slurried in methanol (300 ml{circumflex over (=)}15 V) and stirred for 1 hr. The suspension was filtered and the cake washed with methanol (100 ml{circumflex over (=)}5 V) then dried under vacuum at 30-35° C. to a constant weight.
Output: 22 g; Yield: 93.8%.
CBBC-3B:
CBBC 2B (20.0 g) was suspended in THF (500 ml{circumflex over (=)}25 V) and then stirred and cooled to −5° C. to 0° C. To the cooled solution was added an aqueous solution of lithium hydroxide (3.65 g{circumflex over (=)}3.0 equiv) at such a rate that the reaction temperature was maintained at between −5° C. and 0° C. (about 30 mins). The solid dissolved after the base was added to afford a slightly turbid solution. Stirring was continued for another 2 hrs at below −5° C. to 0° C. After 3 hrs the turbid reaction mixture was added slowly, with good stirring, into ice/water (700 ml{circumflex over (=)}35 V) at 5° C. whereby any undissolved particles were filtered under vacuum. While stirring, the pH was adjusted to 4.2 using 2M HCl (≈40 ml{circumflex over (=)}≈2 V). The thick white precipitate was collected by filtration and the damp cake washed with 200 ml{circumflex over (=)}10 V of water, air dried under vacuum briefly, slurried in 400 ml{circumflex over (=)}20 V of methanol and then stirred under reflux. The suspension was filtered and the damp cake washed again with 200 ml 10 V of methanol then dried under vacuum. The wet cake was then taken in methanol (400 ml{circumflex over (=)}20 V) and acetonitrile (200 ml{circumflex over (=)}10 V) and stirred under reflux. The suspension was filtered hot and the damp cake washed with 200 ml{circumflex over (=)}10 V of methanol then dried under vacuum. The product was dried under vacuum at 35° C. to a constant weight to afford the tetrapeptide acid Ac-(AA1-AA4).
Output: 120 g; Yield: 62.0%; Purity: 97.1%.
CDEG-1:
CLEU-15 (4.3 g), CBBC-3A (2.24 g{circumflex over (=)}1.0 equiv), and HOAt (0.56 g{circumflex over (=)}1.2 equiv) were charged into an RBF containing DMF ( ̃2.6 ml{circumflex over (=)}6 V). The mixture was stirred for 15-30 minutes to yield a clear solution at 25° C. and then treated with DIPEA (1.71 g{circumflex over (=)}4.0 equiv) The reaction was then cooled to −15° C. and HATU (1.57 g{circumflex over (=)}1.25 equiv) was added to the mixture. The reaction mixture was stirred for 2 hrs at −10° C., warmed to 20° C. and then stirred for 1 hr at the same temperature. The reaction mass was added to 10% citric acid solution (270 ml{circumflex over (=)}60 V) and stirred for 30 minutes at 10° C. The precipitated solid was filtered, washed with water (270 ml{circumflex over (=)}30 V), 5% NaHCO3 solution (130 m{circumflex over (=)}30 V) and again with water (270 ml{circumflex over (=)}60 V). The solid was dried under vacuum at 35° C.
Output: 4.0 g; Yield: 65.1%; Purity 79.25%.
CDEG:
20% piperidine in DMF (25 ml{circumflex over (=)}5 V) was charged into an RBF under a nitrogen atmosphere. The solution was cooled to −5° C. and CDEG-1 (5.0 g) was added. The reaction mixture was stirred for 45 minutes at 0° C. The reaction mixture was poured into DIPE (250 ml{circumflex over (=)}50 V) and then stirred for 15 minutes. The precipitated solid was filtered off under nitrogen and washed with DIPE (50 ml{circumflex over (=)}10 V). The solid was then taken up in ethyl acetate (125 ml{circumflex over (=)}25 V) and stirred for 1 hr at 25° C. The fine solid obtained was filtered, washed with ethyl acetate (50 ml{circumflex over (=)}10 V), and then dried under vacuum.
Output: 4.7 g; Yield: Quantitative, Purity: 87.6%, D-Ser impurity 1.5%, hydantoin impurity 0.16%.
HPLC Condition for CDEG:
Column: YMC basic (250 mm×3.0 mm), 5μ
Mobile phase A: 0.1% TFA in ACN
Mobile phase B: 0.1% TFA in H2O
Wave length: 226 nm, Diluent: Mobile phase A: M.P.B=27:73
Column tem: 50° C., inject.vol. 50 μl
Gradient T/% A=0/73, 18/70, 41/30, 43/73, 50/73
Flow rate 0.5 ml/min

## Claims

1. A liquid-phase process for preparing Degarelix having the formula Ac-AA1-AA10-NH2:



or a pharmaceutically acceptable salt or solvate thereof; comprising the steps of:
coupling peptides (P4)Ac-AA1-AA4 with (Pε)AA5-AA10NH2 or coupling peptides Ac-AA1-AA3 with (P4)(Pε)AA4-AA10NH2 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 (P4)Ac-AA1-AA4 with (Pε)AA5-AA10NH2 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 (P4)(Pε)Ac-AA1-AA10-NH2:




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-AA1-AA3 with P4)(Pε)AA4-AA10NH2, 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 AA5-AA10 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 (P4)Ac-AA1-AA4:



or a pharmaceutically acceptable salt or solvate thereof,
comprising the step of hydrolyzing a compound having the formula (P4)Ac-AA1-AA4-R with an alkaline hydroxide, wherein R is a carboxyl protecting group, and P4 is hydrogen or a hydroxyl protecting group, wherein the compound (P4)Ac-AA1-AA4-R has the formula:

17. A liquid-phase process for preparing a hexapeptide (Pε)AA5-AA10NH2 comprising:
coupling (Pε)AA6-AA10NH2 and (PX)AA5, wherein PX is an amino protecting group, AA5 is 4Aph(L-Hor), AA6 is D-Aph(Cbm), AA7 is Leu, AA8 is Lys(iPr), AA9 is Pro, AA10 is D-Ala, and Pε is an ε-amino protecting group, to provide (PX)(Pε)AA5-AA10NH2, and
cleaving Px with TFA to provide (Pε)AA5-AA10NH2, wherein (PX)(Pε)AA5-AA10NH2, (Pε)AA6-AA10NH2 and (PX)AA5 have the following structures:

18. The process according to claim 16, wherein the compound having the formula Ac-AA1-AA4-R is first prepared by coupling Ac-AA1-AA3 with (P4)AA4-R or coupling Ac-AA1-AA2 with (P4)AA3-AA4-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-AA1-AA4:



or a pharmaceutically acceptable salt or solvate thereof, comprising the steps:
a) reacting (PN)AA2 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-AA1-AA4,

wherein P4 is H or a hydroxyl protecting group on AA4, and PN is an amino protecting group.

23. A liquid-phase process for preparing a hexapeptide (Pε)AA5-AA10NH2 comprising:
coupling (P5)AA5-AA7 with (Pε)AA8-AA10NH2 to provide (P5, Pε)AA5-AA10NH2, and
subsequently cleaving P5 to provide (Pε)AA5-AA10NH2, wherein P5 is an amino-protecting group on AA5 and Pε is a side chain amino protecting group on AA6 and wherein AA5 is 4Aph(L-Hor), AA6 is D-Aph(Cbm), AA7 is Leu, AA8 is Lys(iPr), AA9 is Pro, and AA10 is D-Ala.

24. The liquid-phase process of claim 23, followed by coupling (P4)Ac-AA1-AA4 to (Pε)AA5-AA10NH2.

25. The process according to claim 16, wherein the carboxyl protecting group is chosen from a C1-C4 alkyl and benzyl group.

26. The polypeptide according to claim 21, wherein the carboxyl protecting group is chosen from a C1-C4 alkyl and benzyl group.