[Technical field]

The present invention relates to a liquid (or solutionj-phase manufacturing process for preparing the decapeptide Degarelix, its 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 blocker) 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 hypothaiamtc-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare (Van Poppei, 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 Firmagon™. The drug substance is chemically designated as D-Aianinamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl- 4-ch!oro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[ [[(4S)-hexahydro-2,6-dioxo-4- pyrimidinyl]carbonyl]amino]-L-phenylalany!-4-[(aminocarbonyl )amino]-D-phenyiaianyl-L- leucy[-N6-(l-methylethyl)-L-lysyl-L-prolyl- and is represented by the chemical structure below (in the following also referred to as Formula I):

The structure of Degarelix can also be represented as:

Ac-D-2Nai-D-4Cpa-D-3Pal- Ser-4Aph(L-Hor)-D-4Ap (Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH ₂ where Ac is acetyl, 2Nai is 2-naphthyla!anine, 4Cpa is 4-chloropbenylalanine, 3Pal is 3- pyridyialanine, Ser is serine, 4Aph is 4-aminophenyiaianine, 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:

AAi is D-2Nal, AA ₂ is D-4Cpa, AA ₃ is D-3Pai, AA ₄ is Ser, AA _S 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 _i0 is D-Ala.

Thus, as an example, Degarelix can be represented as Ac-AAi-AAi ₀ -NH ₂ , the tetrapeptide Ac- D-2Nal-D-4Cpa-D-3Pal-Ser can be represented as Ac-AAi-AA ₄ and the hexapepttde 4Aph{L- Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH ₂ as AA ₅ -AA ₁₀ -NH ₂ .

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 MBHA resin in dimethylform amide

(DMF)/CH ₂ CI ₂ 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 piperdine, 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 ai., Drugs of the Future 2006, Vol. 31, No. 9, pp 755-766).

While Boc-SPPS methodology has afforded sufficient quantities of Degareiix 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. While the existence of a liquid phase peptide synthesis of Degareiix has been reported [EMEA Report: Assessment Report for Firmagon™ (Degareiix): Doc. Ref.

EMEA/CHMP/635761/2008], as of now no details of such a process have been pubiically disclosed.

WO 97/34923 and WO 99/26964 are documents 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). WO 99/26964 proposes a liquid phase process which involves first preparing the centra! 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 Ser amino acid (AA ₄ ) 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 AA ₂ -AA ₄ 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 hydantoinacetyl analogue when L-dihydroorotic acid is coupled with 4Amp to prepare AA ₅ . 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-AA AA _l0 - H ₂ or a pharmaceutically acceptable salt or solvate thereof, comprising the step of subjecting a Degarelix precursor according to the following formula S, or a salt or solvate thereof to a treatment with a cleaving agent:

The compound of Formula II thus corresponds to

{Pi)AA AA2-AA ₃ -AA4{p4}-AA5-AA ₆ (P ₆ )-AA7-AA8(P8}-AA9-AA ₁₀ -NH2 (I I)

where AAi to AA ₁₀ are the same as for formula (I),

Pi is an amino protecting groups or acetyl;

P ₄ is hydrogen or a hydroxy! protecting group, preferably a hydroxy! protecting group;

P ₆ is hydrogen or an amino protecting groups; preferably an amino protecting groups; and P _g is an amino protecting group.

Preferably, the protecting group Pi, if present, is orthogonal to g , i.e. both protecting groups can be cleaved independently.

!n a preferred embodiment, Pi is acetyl, and each of P ₄ and P ₈ are protecting groups that can be cleaved in a single step, and Pe is hydrogen or a protecting group that can be cleaved together with the protecting groups P ₄ and P _s . The cleavage of the protecting groups is preferably carried out by treating the precursor of formuia il with trifluoroacetic acid (TFA). It is particularly preferred that P ₄ , P ₆ , and P ₈ are protecting groups selected from tert-butyl (tBu) and t-butyloxycarbonyl (Boc), and most preferred is a precursor of formula II wherein P ₄ is tBu, P ₆ is hydrogen or tBu, and P ₈ is Boc.

The present invention also relates to a process for the liquid-phase manufacture of the intermediate represented by formuia (il) or pharmaceutically acceptable salt or solvate thereof, comprising the step of coupling a first polypeptide represented by formula (Hi) (Ρι)ΑΑι-ΑΑ ₂ -ΑΑ ₃ (111) or a salt thereof, with a second polypeptide represented by formula (IV)

AA4(p4)-AA ₅ -AA ₆ (P ₆ )-AA ₇ -AA _s (P ₈ }-AA ₉ -AA ₁ o-N H ₂ (IV) or salt thereof, in a liquid reagent medium in the presence of a peptide coupling reagent, optionally together with an organic amine base, to form a decapeptide represented by formula (II). In this case AAi to ΑΑι ₀ , P ₄ , Ρό and P _g are the same as for formula (II).

The salts include acid salts such as hydrochlorides and basic salts such as alkali metal salts, alkaline earth metal salts, and ammonium salts.

According to the invention, the second polypeptide represented by form ula (IV) may be prepared by eliminating the protective group P _N from the following compound (!Va):

(PN)AA4(P4)-AAS-AA6(P6)-AA ₇ -AA ₈ { ₈ )-AA9-AA ₁ O- H2 (IVa)

PIM is preferably an N-terminai amino protecting group and more preferably a protecting group that can be eliminated by hydrogenation such as benzyloxycarbonyl.

The compound of formula (IVa) may be obtained by coupling a polypeptide represented by formula (V) with a polypeptide represented by formula (VI)

(P _N )AA ₄ (P ₄ )-AA _S -AA ₆ (P ₆ )-AA ₇ (V)

AA8{Ps)-AA ₉ -AAio-N H ₂ (VI) or salts of these compounds, where AA ₄ to AA ₁₀ , P , P , Pe and Pg are the same as for formula (IVa).

The invention also relates to the polypeptides represented by formulae (II) to (VI) which are useful in the liquid-phase manufacturing process of the invention. [Figures]

Figure 1 shows a flow diagram for the synthesis of a derivatives of AA^ and AA2 for the peptide synthesis.

Figure 2 shows a flow diagram for the synthesis of a derivative of AA2-AA3 for the peptide synthesis.

Figure 3 shows a flow diagram for the synthesis of Ac-AA^-AA3.

Figure 4 shows a flow diagram for the synthesis of a derivative of AA5-AA7.

Figure 5 shows a flow diagram for the synthesis of a derivative of AA5-AA7.

Figure 6 shows a flow diagram for the synthesis of Z-AA4-AA7.

Figure 7 shows a flow diagram for the synthesis of AA9-AA20-

Figures 8 and 9 show a flow diagram for the synthesis of Z-AAg-AA^o-

Figure 10 shows a flow diagram for the synthesis of Z-AA4-AA1Q.

Figure 11 shows a flow diagram for the synthesis of the precursor of formula II.

Figure 12 shows a flow diagram for the synthesis of Degarelix from the precursor of formula II and the purification and lyophilisation.

[Detailed Description of the Invention]

The present invention will now be described in more detail. Deprotection step

In a first aspect, the present invention relates to a liquid-phase process for preparing Degarelix having the formula Ac~AAi-AA ₁₀ - H ₂ or a pharmaceutically acceptable salt or solvate thereof. The process comprises the step of cleaving the protecting groups (P ₄ ), (P ₆ ), and (P ₈ ), if present, from the Degarelix precursor according to formula M {{Pi)AA ₁ -AA ₂ ~AA ₃ - AA4(p4)-AA5-AA ₆ { ₆ )-AA7-AAs(Ps)-AA9-AA ₁₀ -NH ₂ ), or a salt or solvate thereof, in an organic solution comprising the precursor and a cleaving agent dissolved therein.

AAi to A A 10 in formula II have the same meaning as in formula (I), and Ρχ is an amino protecting groups or acetyl, preferably acetyl.

P ₈ is an amino protecting group. Preferably, P ₈ 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), benzyloxycarbonyl (Cbz or Z), , and substituted Cbz, such as, e.g., 2-bromo-benzyloxycarbonyl (2-Br-Z), 2-chloro- benzyloxycarbonyl (2-CI-Z), p-ch!orobenzyioxycarbonyl, 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, isopropyloxycarbony!, 2-(p-biphenylyl)-isopropyioxycarbonyl, and the like; cycloalkyl urethane-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyciohexyloxycarbonyl; aliytoxycarbony! (Alloc) acetyl (Ac), benzoyl (Bz), trifluoroacetyl (Tfa), toluenesulfonyl (Tos), benzyl (Bn), triphenylmethyl (Trt), o-nitrophenyl-sulfenyl (Nps), t-butyl-dimethylsilyloxycarbonyl, [2-(3,5-dimethoxyphenyl)-propyI-2-oxycarbonyl] (Ddz), 2,2,2-trichloroethyloxycarbonyl (Troc), biphenylylisopropyloxycarbonyl (Bpoc), and o- nitrobenzyloxycarbonyl. Preferred protecting groups are Fmoc, Boc and Alloc with Boc being most preferred.

P ₄ is hydrogen or a hydroxyl protecting group, preferably a hydroxyl protecting group. The hydroxy! group of Ser (P ₄ ) is preferably a C ₄ -C ₆ alkyl (e.g. t- butyl, cyclohexyl), acetyl (Ac), trity!, benzyl, a benzyl ether such as p-methoxybenzyS, or other substituted benzyls such as p- nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl, tetrahydropyranyl, tri(Ci-C ₆ )alkylsityl, 2-methoxyethoxymethyl (M EM), 4-dimethylcarbamoyfbenzyi and O- phenoxyacetyl ethers. Particularly preferred are t-butyl, benzyl and 9-fluorenylmethyl ethers, t-butyl being most preferred.

P ₆ is hydrogen or an amino protecting groups, preferably an amino protecting groups.

Preferred protecting groups include C ₄ -C ₆ alkyl {e.g. t-butyl, cyclohexyl), acetyl (Ac), trityl, benzyl, a benzyl ether such as p-methoxybenzy!, or other substituted benzyls such as p- nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichtorobenzyl, tetrahydropyranyl, tri{C C6)alkyisilyl, 2-methoxyethoxymethyl (MEM ), 4-dimethylcarbamoylbenzyl and O- phenoxyacetyl ethers. Particularly preferred are t-butyl, benzyl and 9-fluorenylmethyl ethers, t-butyl being most preferred.

The cleaving agent used to remove the protecting groups depends on the nature of the protecting group and are well known in the art.

Preferred cleaving agents for the Ser hydroxy! protecting group P4 are:

• trifluoracetic acid (TFA), HCI, or methanesulfonic acid, particularly for t-butyl ether as a protecting group

• H ₂ /Pd-C, HF, or trifluoromethane-sulfonic acid, particularly for benzyl ether as a

protecting group, and

• SiCI ₄ /anisol, particularly for 2~(rnethyfsulfinyi)benzylether as a protecting group;

Preferred cleaving agents for the amino protecting group Pg are:

• trifluoracetic acid (TFA), HCI, or methanesulfonic acid, particularly for t-butyl

carbamates as protecting group

• H ₂ /Pd-C, HF, or trifluoromethane-sulfonic acid, particularly for benzyl carbamates as protecting group, and

• Piperidine, D8U and DEA, particulary for Fmoc as protecting group

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

Table 1: Cleavage conditions

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

Typically, the precursor of formula II is dissolved in a cleaving agent, preferably TFA, with or without an additional solvent, at room temperature (20 to 25 °C) for 20 to 30 hours, preferably 24 hours. When the protecting group has been removed (preferably the conversion is > 95% yield, most preferably > 99%), the crude Degarelix is then preferably poored into a buffered water-ethanol mixture to provide a buffered solution of crude degarelix for subsequent purification. The preferred pH is preferably in the range of 2 to 4, more preferably in the range of 2.5 to 3.5, and most preferably approximately 3.

Specific embodiments of the deprotection step are shown in Figure 12 and Example 4 (see Step 12).

Purification and lyophilization

The solution of crude degarelix is preferably purified using chromatographic techniques such as preparative reverse phase chromatography ( PC).

The resulting product is then preferably lyophilized.

3 + 7 coupling

In a further aspect the present ^" invention also relates to a process for the liquid-phase manufacture of the ^" intermediate represented by formula (II) or pharmaceutically acceptable salt or solvate thereof, comprising the step of coupling a first polypeptide represented by formula (III) or a salt thereof, with a second polypeptide represented by formula (IV)

AA4( p4)-AA5~AA ₆ {P ₆ )-AA7-AA ₈ (P _g )-AA9-AA ₁₀ -NH2 (IV) or a salt thereof, in a liquid reagent medium in the presence of a peptide coupling reagent and optionally an organic amine base, to form a decapeptide represented by formula (il). In this case AAi to AA ₁₀ , Pi, P ₄ , Pe and P _s are the same as for formula (II). The salts include acid salts such as hydrochlorides and basic salts such as alkali metal salts, a!kaline earth metal salts, and ammonium salts.

The coupling reaction is performed in an organic solution where the two peptides and a peptide coupling reagent and optionaiiy an organic amine base are dissolved therein. A peptide coupling additive and/or an organic amine may also be present.

The organic soivent, 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-methyl pyrrolidone), DCM, DMF, DMSO, and mixtures thereof. The most preferred solvent is DMSO.

Typical peptide coupling reagents are one or more of o-(7-azabenzotriazoi-l-yl)-l, 1,3,3- tetramethyluronium hexafiuorophosphate (HATU), o-(benzotriazoi-l-yi)-l,l,3,3- tetramethyluronium hexafiuorophosphate (HBTU), o-(benzotriazol-l-yl)-l,l,3,3- tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-l-yl-oxy- tris(dimethylamino)phosphonium hexafiuorophosphate {BOP), benzotriazole-l-y!-oxy-tris- pyrrolidinophosphonium hexafiuorophosphate (PyBOP), N,N-bis-(2-oxo-3- oxazolidinyl)phosphonic dichloride (BOP-Ci), bromo-tris-pyrrolidino-phosphonium hexafiuorophosphate (PyBroP), iso-butyichioroformate (IBCF), 1,3 dicyciohexyicarbodiimide (DCC), 1,3-di ^' tsopropyl-carbodiimide (DIC), l-(dimethyiaminopropyl)-3-ethylcarbodiirnide hydrochloride (WSCDI), N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoiine (EEDQ), isopropylchloroformate (IPCF), 2-{5-norbornen-2,3-dicarboximido}- 1,1,3,3- tetramethyiuronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA) and 2-succinimido-l,l,3,3-tetramethyluronium tetrafluoroborate (TSTU).

A preferred coupling agent is DCC,. DCC is preferably used without an organic amine. In a particularly preferred embodiment, DCC is used in combination with DMSO. DCC is preferably used in an amount of 1.3 to 2, most preferably 1.4 to 1.6 equivalents with respect to the tripeptide. Another preferred coupling agent is DIC, which is preferably used in combination with 6- chloro-HOBt, optionally together with copper salts.

Typical peptide coupling additives are 1-hydroxy-lH-benzotriazole (HOBt), 6-chloro-HOBt, and l-hydroxy-7-azabenzotriazole (HOAt).

Typical organic amine bases are NM , DiPEA, TEA, and coilidine.

It is particularly preferred to carry out the coupling reaction using DMSO as solvent and DCC as coupling agent.

Specific embodiments of the coupling step are shown in Figure 11 and Example 4 (see Step 11).

Synthesis of the heptapeptide

The compound of formula IV, i.e. AA4(P ₄ )-AA5-AA ₆ (P ₆ }-AA ₇ -AA8(P8)-AA9-AA ₁ o- H ₂ , is preferably obtained by eliminating the protective group Pp of the following compound !Va:

(P )AA ₄ { P4)-AA5-AA ₆ (P ₅ }-AA ₇ -AA ₈ (P ₈ )-AA ₉ -AA ₁₀ -N H ₂ (IVa)

While AA4 to AA^Q, P4, e _> ^an< ^ ^P S ^{nave tne same} meaning as above, jj is an N-terminal amino protecting group. In a preferred embodiment, P[\j is a protecting group that can be eliminated by hydrogenation, e.g. using hydrogen and a palladium catalyst (such as Pd/C). The most preferred protecting group PJ\J is benzyloxycarbonyl (Z).

Specific embodiments of the elimination step of Pj\j are shown in Figure 11 and Example 4 (see Step 11).

The compound of formula (IVa) may be obtained by coupling a polypeptide represented by formula (V) with a polypeptide represented by formula (VI) (P _N )AA ₄ (P ₄ )-AA ₅ -AA ₆ (P ₆ )-AA ₇ (V)

AA ₈ { P8)-AA ₉ -AA ₁₀ -NH2 (VI) or salts of one or both of these compounds, where AA ₄ to AAi ₀ , P ₄ , P ₆ and P ₈ are the same as for formula (IVa). The coupling reaction is performed in an organic solution where the two peptides and a peptide coupling reagent are dissoived therein. A peptide coupling additive and/or an organic amine may also be present. The same peptide coupling reagents, organic solvents, peptide coupling additives and organic amines as described above may be used, !n a preferred embodiment, DCC is used as a coupling reagent, optionally with ethyl acetate as solvent.

Synthesis of the tetrapeptide of Formula V

In a preferred embodiment, the tetrapeptide of formula V, i.e. ( _N )AA ₄ { p4}-AA5~AA ₆ {P ₆ }-AA7 _i is prepared by a process comprising the following steps:

(a) providing (P _N2 )AA5-AA ₆ (P ₆ )-AA ₇ (Pc), wherein P ₆ has the same meaning as above in formula IVa, (P _N2 ) is an N-terminal amino protecting group or hydrogen, and (P _c ) is a C-terminal carboxyi protecting group that can be cleaved by hydrogenation;

(b) removing the amino protecting group (PN2) _> if present;

(c) hydrogenating H-AA ₅ -AA ₆ {P ₆ )-AA ₇ (P _C ) to obtain H-AA _S -AA ₆ (P ₆ )-AA ₇ ; and

(d) reacting H-AAs-AAefPeW with ah activated ester of ( N)AA ₄ ( 4) to provide

(P _N )AA4(P )-AA5-AA ₆ ( 6)-AA7, wherein (P4) is a hydroxy! protecting group or hydrogen, P _$ is hydrogen or an amino protecting group, and P| j is a protecting group that can preferably be eliminated by hydrogenation, e.g. using hydrogen and a palladium catalyst (such as Pd/C).

The most preferred protecting group Pj j is benzyloxycarbonyi (Z).

The preferred activated ester of (P _N )AA ₄ ( 4) is a 4-nitrophenyl ester (ONp). Hence, preferably, the benzyl ester is removed in the AA5-AA7 intermediate to provide both N- and C- unprotected AA5-AA7. This tripeptide is then reacted with preactivated serine (e.g. Z-Ser(tBu)-ONp).

Degarelix manufacture

The present invention thus relates to the manufacture of Degarelix, the above-discussed deprotection step being an essential step of this manufacture, in a preferred embodiment, this deprotection step follows the 3 + 7 coupling discussed above. In an even more preferred embodiment, the 3 + 7 coupling follows the synthesis of the heptapeptide discussed above, i.e. the process comprises the following steps: a) Synthesis of AA4(p4)-AA ₅ -AA ₆ (P6}-AA7-AA ₈ (P ₈ )-AA9-AA ₁₀ - H2 , or a salt or solvate thereof;

b) Coupling of AA ₄ (P ₄ hAA ₅ -AA ₆ (P ₆ }-AA7-AA ₈ (P ₈ }-AA _! rAA _1(r NH ₂ and {Pi)AAi-AA ₂ -AA ₃ to provide (Ρι}ΑΑ ₁ -ΑΑ2-ΑΑ3-ΑΑ ₄ { Ρ4)-ΑΑ5-ΑΑ ₆ (Ρ6)-ΑΑ7-ΑΑ ₈ (Ρ ₈ }-ΑΑ9-ΑΑ ₁ ο- Η ₂ ), or a salt or solvate thereof;

c} Deprotection of (Pi}AA AA ₂ -AA3-AA ₄ (P ₄ )-AA ₅ -AA ₆ (P _s }-AA ₇ -AA ₈ { p8)-AA9-AAio- H ₂ }, or a salt or solvate thereof to provide degarelix, or a solvate or salt thereof.

AAi to ΑΑ ο and ?\ _t P4, P , and Pg have the same meanings as defined above.

In a particularly preferred embodiment,

• Pi is acetyl;

• P4 is a protecting group that is cleavabie with TFA, preferably tBu;

• P5 is a hydrogen or a protecting group that is cleavabie with TFA, preferably tBu;

• Pg is a protecting group that is cleavabie with TFA, preferably Boc.

In the most preferred embodiment, the heptapeptide is produced using the heptapeptide synthesis described above. Moreover, the deprotection step is preferably followed by the purification and lyophilisation methods described above. in the synthesis of degarelix or its precursors, and particularly in ail steps containing a peptide with the hydroorotyl moiety, the pH is preferably kept below 9, preferabiy betow 8.5, even more preferred being below 8. It is preferred to use a weak base such as NaHC03 for pH adjustment. It is particularly preferred that all extractions after the coupling steps are carried out within a pH range of 2 to 9, preferabiy 2.5 to 8 (see steps 6, 7, 10, and 11 in the experimental section), !t is additionally preferred to add and C4-5 aliphatic alcohol such as n-butanoi or 2-butanol prior to the extraction or washing step.

Intermediates

The invention also relates to the polypeptides represented by formulae (11} to (V!) which are useful in the liquid-phase manufacturing process of the invention.

Preferred embodiments of formula (11)

(Pi)AA _r AA2-AA3-AA4(P4)-AA ₅ -AA ₆ (P ₆ )-AArAA ₈ ( p8)-AA ₉ -AAao-NH ₂ (II)

Table 2 lib Ac tBu tBu Boc

He Ac H H Alloc

Sid Ac H H Boc lie Ac tBu H Boc

Hf Boc tBu tBu Fmoc llg Fmoc tBu tBu Boc i!h Boc tBu H Fmoc

!li Fmoc tBu H Boc llj Ac H tBu Boc

Ilk Ac H tBu Fmoc in Ac H H Fmoc

Preferred embodiments include salts of these com pou

Preferred embodiments of formula (III)

(P _N )AAi-AA ₂ -AA ₃ (111)

Table 3

Compound P

liia Ac

1Mb Boc

Preferred embodiments include salts of these compounds.

is Preferred embodiments of formula (IV)/(IVA)

{P _N ) AA ₄ {P ₄ )-AA ₅ -AA ₆ (P ₆ )-AA ₇ -AA ₈ (P ₈ )-AArAA ₁₀ -N H ₂ (IV)

Table 4

Preferred embodiments include salts of these compounds.

Preferred embodiments of formula (V)

(Ρ _Ν )ΑΑ ₄ (Ρ ₄ }-ΑΑ ₅ ~ΑΑ ₆ (Ρ ₆ }-ΑΑ ₇ <ν)

Table s

Preferred embodiments include salts and solvates of these compou Preferred embodiments of formula (VI)

AA ₈ (P ₈ )-AA9-AAio-NH ₂ (VI)

Table 6

When a protecting group is not present, the functional group is the deprotected group (e.g. >IMH).

Preferred embodiments include salts of these compounds.

Experimental Section

Materials Used in the Experimental Section

The materials used in the experimental section are listed below.

Chemicals:

aqeuous ammonia NH ₃ (aq)

Acetonitrile C ₂ H ₃ N

D-Butanol C ₄ H ₁₀ 0

2-Butanol C ₄ H ₁₀ 0

Isopropanol (isopropanol) C ₃ H ₈ 0

Butyl acetate C5 H 12O2

Ethanol, 99.9% C ₂ H ₆ 0

Methanol CH ₄ 0

Heptane C ₇ H ₁₅

Purified water H ₂ 0

Ethyl acetate C ₃ H ₈ 0 ₂

Acetic acid C ₂ H ₄ G ₂

Ammonium acetate C ₂ H ₇ N0 ₂

Acetyl imidazole C ₅ H ₆ N ₂ 0

Triethylamine C ₆ Hi ₅ N

W-Methylmorpholine C ₅ HnNO

W-Methylpyrro!idone C ₅ H ₉ NO

Λ/,Λ/'-Dicyclohexylcarbodiimide Ci ₃ H ₂₂ N ₂

Dicyc!ohexylamine Ci ₂ H ₂ 3

Λ/,Λ/'-Diisopropylcarbodiim!de C ₇ Hi ₄ N ₂

/,/V-Dimethylethylenediamine C ₄ H _i2 N

Λ/,/V-Dimethylformamide C3H7NO

Dimethyl sulphoxide C ₂ H ₆ OS

l-Hydroxybenzotriazoie C ₆ H ₅ N ₃ 0

p-Nitropheno! C ₆ H ₅ N0 ₃

/V-Hydroxysuccinimide C ₄ H ₅ N0 ₃

Isobutyl chloroformate C5H9OO2

Sodium chloride NaCi

Sodium hydroxide, aqueous NaOH (aq) Hydrochloric acid, aqueous HC! (aq)

Phosphoric acid H3PO4

Sodium hydrogensulphate NaHS0 ₄

Sodium hydrogencarbonate NaHC0 ₃

Methanesulphonic acid CH4SO3

Trifluoroacetic acid C2H F3O2

Palladium on charcoal, 5% Pd-C

Hydrogen H ₂

Toluene C ₇ H ₈

Starting materials:

N-t-Butyloxycarbonyl-D-4-chlorophenylalanine Boc-D-4Cpa-OH C Hi ₈ N0 ₄

N-t-Butyloxycarbonyl-D-2-naphtylalanine Boc-D-2Nal-OH C ₁₈ H ₂₁ N0 ₄

D-3-PyridylaSanine hydrochloride H-D-3Pal-OH x 2HCI C ₈ H ₁₂ Cl ₂ N ₂ 0 ₂

N-a-t-Butyloxycarbonyl-N-4-(t-Butylcarbamoyl)- D-4-Aminophenylalanine Boc-D-4Aph(tBuCbm)-OH C19H29N3O5

N-a-t-Butyloxycarbonyl-N-4-(L-Hydroorotyl)

-4-Aminophenylalanine Boc-4Aph(L-Hor}-OH C ₁₉ H ₂ 4lM40 ₇

Leucine benzyl ester p-tosyiaie H-Leu-OBzi x TOS C ₂ oH ₂₇ NOs

N-Benzyloxycarbonyl-O-t-butyl-serine Z-Ser(tBu)-OH C _s H ₁₅ N0 ₅

N-t-Butyioxycarbonyl-proline Boc-Pro-OH C _l0 H ₁₇ NO ₄

D-Alaninamide hydrochloride H-D-Ala-N H ₂ x HCI C ₃ H ₈ CIN0 ₂

N-a-Benzyloxycarbonyl-N-£-t-butyloxycarbonyl-N- ε-isopropyl-lysine,

dicyclohexylamine salt Z~Lys(iPr,Boc)-OH x DCHA C ₃₄ H ₅₇ I\

Example 1: Synthesis of Intermediate Ac(l-3)ONa: Ac-D-2Nal-D-4Cpa-D-3Pal-ONaf7l

Activation of Boc-D-4Cpa-OH and isolation

Step 1 (Reaction step)

8oc-D-4Cpa-OH ( 299.75 g) is dissolved in iPrOH ( 3.53 kg), the mixture is stirred and HOSu (0.184 kg) and DIC (0.164 kg) are added, and stirred at 0°C for 1 hour. The precipitate is filtered off and washed with iPrOH. The solid is dried under reduced pressure to yield Boc-D- 4Cpa-OSu[l].

Activation of Boc-D-2Nal-OH and isolation

Step 2 (Reaction step)

Boc-D-2Nal-OH (315.38 g) is dissolved in iPrOH (5.35 kg) and IBC (157.07 g) and MM (116.7 g) are added. A mixture of water (42 mL), iPrOH {1.1 kg) and HOSu (230.14 g) is added after cooling to -10°C together with additional NMM (lO.llg), and the mixture stirred 30 min. Water (0.82 L) is added and the precipitate is filtered off, and washed with iPrOH and dried under reduced pressure to yield Boc-D-2Nal-OSu[2].

Synthesis of Boc(2-3)OH: Boc-D-4Cpa-D-3Pal-OH

Step 3 (Reaction step)

H~D-3Pal-OH x 2 HCI (0.251 kg) and Boc-D-4Cpa-OSu [1] (0.397 kg) from step 1 are dissolved in D SO (3.33 L) and NMM (318.8 g) is added. The mixture is stirred at 20°C for 6 hours. Water (17 L) is added and pH is adjusted by adding HCI to pH 4.25. The precipitate is filtered off, and dispersed in water. The obtained slurry is then filtered and washed with water. The solid is dried under reduced pressure to yield Boc-D-4Cpa-D-3Pal-OH[3],

Synthesis of Intermediate Ac(l-3)ONa: Ac-D-2Nal-D-4Cpa-D-3Pal-ONa[7] (Compound of formula Ilia)

Step 4 (Reaction step)

Boc-D-4Cpa-D-3Pal-OH [3] (447.93 g) from step 3 is dissolved in a mixture of AcOEt {3.4 L) and AcOH ( 675 mL), the mixture is coo!ed at 5°C where after MSA { 672.77 g) is added. The reaction continues at 10°C for 2 hours and to the solution is added TEA { 1214.28 g) to yield H-D-4Cpa-D-3Pai-OH[4].

Boc-D-2Nal-OSu [2] (412.44 g) from step 2 is added to H-D-4Cpa-D-3Pai-OH [4], stirred for 24 hours at 20°C. 25% aqueous NH ₃ (0.154L)and n-butanol (4.5L) are added, and the mixture is stirred at 45°C for 1 hour.

The solution is washed with:

• Water

• Water at pH 9.5 (pH is adjusted while stirring with aq. NaOH)

• Water AcOH (4.5 L) is added to the organic phase and the solution is concentrated to an oil under reduced pressure. The oil is re-dissolved in AcOH (4.5 L) and re-concentrated under reduced pressure to yield Boc-D-2Nal-D-4Cpa-D-3Pal-OH[5] as an oil.

Boc-D-2Nal-D-4Cpa-D-3Pal-OH [5] is dissolved in water (0.09 L) and AcOH ( 1.8 L). MSA ( 672.77 g} is added and the mixture is stirred at below 35°C for 2 hours. The solution is neutralised with TEA (779.16 g). The solution is concentrated under reduced pressure to an oil. The oil is re- dissolved in toluene (2.5 L) and re-concentrated under reduced pressure to an oil. The last step ts repeated to yield H-D-2Nal-D-4Cpa-D-3Pal-OH[6].

H-D-2Nal-D-4Cpa-D-3Pal-OH [6] is dissolved in toluene (2.0 L) and a solution of acetyl imidazole (132.14 g} in toluene (0.25 L) is added. The solution is stirred at 20°C for 2 hours, and water (0.1 L} is added,

n-Butanoi (4.5 L) is added and the organic mixture is washed at 35° C with:

• 5% aqueous NaCl

• Methanol and water at acidic pH 5.5 (pH is adjusted while stirring with aq, NaOH)

« Methanol and water at pH 11 (pH is adjusted while stirring with aqueous NaOH)

• Methanol and 10% aqueous NaCl

To the stirred organic phase from the extractions, heptane (15 L) is added at 20°C for 1 hour, and the resulting suspension is left with stirring at 20"C for 1 hour. The precipitate is isolated by filtration, and suspended in heptane (3.5 L). The suspension is filtered again. The last washing step with heptane and the filtration is repeated. The solid is then dried under reduced pressure to yield Ac-D-2Nal-D-4Cpa-D-3Pat-O a[7j.

Specifications for kev intermediates

Step 4 Intermediate Ac(l-3 )O.Na [7]

Quality control Acceptance criteria

Description "White to slightly yellow powder (visual inspection)"

Identification (1) "587.2--0.4Da. (MS)"

Identification (2) "2Naf 0.9-1. ! , 4Cpa 0.9-1.1 , 3Pal 0.9- 1. 1 ( ^' AAA)"

Chiral purity L-2NaI <1.3%, L-4Cpa <0.7%, L-3Pal <2.0% (GC-MS)

Purity > 0% (HPLC, Area ¾)

Example 2: Synthesis of Intermediate Z(4-7)OH x DCHA: Z-Ser(tBu)-4Aph(L-Hor)-D- 4Aph(tBuCbm)-Leu-OH x PCHAfl5l

Synthesis of Intermediate Boc(6-7)OBzl: Boc-D-4Aph(tBuCbm)-Leu-OBzl

Step 5 (Reaction step)

Boc-D-4Aph(tBuCbm)-OH (379.45 g) is dissolved in NMP (0.76 L) and AcOEt (4.4 kg). After cooling at - 4°C, 1BC (150.2 g) and NMM (101.1 g) are added, and the solution stirred at ~7°C for 0.5 hour to yield Boc-D-4Aph(tBuCbm)-OAct[8],

H~Leu-OBzl x TOS (491.88 g) is dissolved in NMP (1.5 L) and AcOEt (2.7 kg) is added, followed by NMM (126.4 g). This solution is subsequently transferred to Boc-D-4Aph(tBuCbm)-OAct [8], and stirred at - 10°C for 1 hour. Then, water (0.5 L) is added.

The reaction mixture is washed at 20"C with:

• Water at pH 8.5 (pH is adjusted white stirring with aq. NaOH)

• Water at pH 2.0 (pH is adjusted while stirring with aq. HCI)

• Water The organic phase is concentrated under reduced pressure to an oil. The oil is re-dtssolved in AcOEt (0.6 kg) and re-concentrated under reduced pressue to an oil. The remaining oil is dissolved in AcOEt (0.6 kg). Heptane (15.5 L) is added while stirring at 20°C. The precipitate is isolated by filtration, and washed with heptane and subsequently dried under reduced pressure at to yield Boc-D-4Aph(tBuCbm)-Leu-OBzl[9],

Synthesis of Boc-(5-7)-OBzl: Boc-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OBzl

Step 6 (Reaction step)

Boc-D-4Aph(tBuCbm)-Leu-OBzi [9] (582.7 g) from step 5 is dissolved in AcOEt (3.15 kg). MSA (481 g) is added, and stirred below 15°C for 5 hours, and TEA (406 g) is added. DMF (0.333 kg) is added followed by TEA (101 g) and NMM (51 g) to yield H-D-4Aph(tBuCbm)-Leu- OBzl[10].

Boc-4Aph(L-Hor)-OH (462.46 g) is dissolved in DMF (2.09 kg) and AcOEt (1.44 kg). IBC (150.24 g) and NMM (111.27 g) are added, and stirred at -10"C for 0.5 h to yield Boc-4Aph(L-Hor)- OActfllJ.

H-D-4Aph(tBuCbm)-Leu-OBzl [10] is added to Boc-4Aph(L-Hor)-OAct [11] and stirred at -10°C for 1.5 hours. Then, AcOEt (5.4 kg) and n-butanol (6.0 L) are added.

The organic phase is washed at 20°C with:

• 5% aqueous NaHC0 ₃ at about pH 8 (pH is adjusted while stirring with aq NaHC0 ₃ )

• 10% aqueous. NaCI at pH 2.5 (pH is adjusted while stirring with aq.H ₃ P0 ₄ )

DMF (0.9 L) is added to the organic phase, which is then concentrated under reduced pressure to an oil. The solution is poured into water (14 L) while stirring. The precipitate is isolated on a filter, and washed with water. The solid is dried under reduced pressure to yield Boc-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu~OBzl[12].

Synthesis of Intermediate Z(4-7)OH x DCHA: Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu- OH x DCHA (Compound of formula Va)

Step 7 (Reaction step)

Boc-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OBzl [12] (885.02 g) from step 6 is added to a mixture of MSA (961.1 g) and AcOEt (7.2 kg) and 2-butanol (2 L) is added, and the resulting mixture stirred at 0°C for 6 hours. MSA is then neutralised with TEA (909.0 g).

5% Pd/C (88.5 g) dispersed in 2-butanol (1 L) is added and the mixture is hydrogenated under pressure at 20°C for 3 hours. Then, the Pd/C is filtered off, and washed with 2-butanoi to yield the solution containing H-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH [13].

Z-Ser(tBu)-OH (413.5 g) is dissolved in MeCN (2.5 L) and the solution is cooled to -5°C.

HONp (195 g) is added followed by DCC (278.5 g), and the mixture stirred at 20°C for 24 hours. The mixture is then filtered, and washed with MeCN to yield Z-Ser(tBu)-ONp [14]. NMM (354.2 g), DMF (4.75 kg) and Z-Ser(tBu)~ONp [14] is added to the solution of H- 4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH [13] and the mixture is left with stirring at 20°C for 3 days.

The resulting mixture is washed with:

• 10% aqueous NaCI at pH 2.5 (pH is adjusted while stirring with aqueous HCI)

• Water at acidic pH (pH 2.5) (pH is adjusted while stirring with aqueous HCI) • 7.5% aqueous NaHC0 ₃

• 5% aqueous. NaCI at (pH 2.5) (pH is adjusted while stirring with aqueous HCI)

• 10% aqeuous NaCI

To the final organic phase DCHA (181 g) is added and the organic phase is concentrated under reduced pressure to an oil. The oil is re-dissolved in iPrOH (3.14 kg) and re- concentrated under reduced pressure to an oil. The remaining oil is re-dissolved in iPrOH (3.14 kg) and while stirring the solution is poured into AcOEt (31.5 kg). Stirring is continued at 20°C for 1 hour until precipitation and the precipitate is then isolated by filtration, and washed with AcOEt. The solid is dried under reduced pressure at 30°C for 30 hours to yield Z- Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA[15]. Purity of intermediate Z(4-7)OH x DCHA[15] is >80% (HPLC).

Step 7 Intermediate Z(4-7)GH [15]

Quality control Acceptance, criteria

Description "While to yellow powder (visual inspection)"

Identification (I) "972.5±0.4Da (MS)"

Identification (2) "Ser 0.9-1 , 1, 4Aph 1.8-2.2, Leu 0.9-1.1 (AAA)"

Clnral purity D-Ser <2.0%, D-4Aph 47-53%, D-Leu <0.7% (GC-MS)

Purity (1) [4Aph ⁵ (Hydantoinacetyl)] Z(4-7)OH DCHA <0.5% (HPLC, Area%) Purity (2) >S0 % (HPLC, Area %)

Example 3: Synthesis of Intermediate H(8-10)NHh>: H-Lvs(iPr.Bod-Pro-P-Aia-fSIH _z i211

Synthesis of Boc{9-10)NH ₂ : Boc-Pro-D-Ala-NH ₂

Step 8 (Reaction step)

Boc-Pro-OH (226.02 g) is dissolved in iPrOH (1.73 kg). The reaction mixture is cooled to -5°C. IBC (143.4 g) and NMM (106.2 g) are added and the mixture is stirred at 5°C for 0.5 hour to yield Boc-Pro-OAct[16].

H-D-Ala-NH ₂ x HCI (124.57 g) is suspended in a mixture of iPrOH (1.57 kg) and NMM (106.2 g). The suspension is added to Boc-Pro-OAct [16]. The reaction mixture is left with stirring at 10°C for 3 hours. Then DMEDA (10.6 ml) is added. The mixture is filtered, and the filtrate is concentrated under reduced pressure to an oil. The oil is re-dissolved and re-concentrated with AcOEt (1.125 kg).

The residual oil is dissolved in a mixture of AcOEt (1.8 kg) and n-butanol (0.6 L). The organic phase is washed with:

® 15% aqueous. NaCI at pH 2.5 (pH is adjusted while stirring with aqeuous HCI)

• 15% aqueous NaCI at pH 9.5 (pH is adjusted while stirring with aqeuous NaOH)

The organic phase is concentrated under reduced pressur , re-dissolved in AcOEt (1.08 kg) and re-concentrated to an oil.

A mixture of AcOEt (0.33 kg) and heptane (0.75 L) is added at 20°C and stirred for 16 hours. The resulting precipitate is filtered and washed with a mixture of AcOEt and heptane on the filter. The solid is then dried under reduced pressure to yield Boc-Pro-D-A!a-NH ₂ [17). Synthesis of Intermediate H(8-10)NH ₂ : H-Lvs(iPr,Boc)-Pro-D-Ala-NH ₂ (Compound of formula Via)

Step 9 (Reaction step)

Boc-Pro-D-Ala-NH? [17] (313.89 g) from Step 8 is dissolved in a mixture of MSA (528.61 g) and iPrOH (0.785 kg) and the solution is stirred at 45°C for 1 hour. The mixture then is neutralised with TEA (607.14 g) to yield H-Pro-D-Ala-NH ₂ [18],

Z-Lys(iPr,Boc)-OH x DCHA (603.83 g) is suspended in AcOEt (1.17 kg) and washed with:

• 12% aqeuous NaHS0 ₄

• Water

• 15% aqeuous aCi

The organic phase of Z-Lys(iPr,Boc)-OH [19] from the extractions is added to H-Pro-D-Ala- NH ₂ [18]. HOBt (183.79 g) and DCC (227.0 g) dissolved in AcOEt (0.135 kg) are added, and the mixture stirred at 20°C for 0.5 hours. Then, water (0.2 L) is added. The mixture is filtered and washed with AcOEt. The combined filtrates are concentrated under reduced pressure to an oil. The oil is dissolved in AcOEt (0.9 kg), filtered and the solution is washed with:

• Water at pH 2.5 (pH is adjusted while stirring with aqueous HCI)

• Water at pH 9 (pH is adjusted while stirring with aqueous NaOH)

• 10 % aqueous NaCl at pH 7 (pH is adjusted while stirring with aqueous HCI or aqeuous NaOH)

The organic phase is concentrated under reduced pressure to yield Z-Lys(iPro,Boc)-Pro-D- Ala-NH ₂ [20].

Z-Lys(iPro,Boc)-Pro-D-Ala-NH ₂ [20] is dissolved in ethanol (0.04 kg) and water (0.5 L), and 5% Pd/C (50 g) is added. The slurry is acidified to pH 2.5 by addition of 6 M HCI and

hydrogenated at 20°C. After completed reaction the catalyst is removed by filtration and pH is raised to pH 7.0 by addition of 32 % NaOH. The ethanol is subsequently removed by evaporation under reduced pressure . n-Butanol (1 L) is added to the resulting aqueous phase and the pH is adjusted to alkaline pH 9 with aqueous NaOH and the extraction starts. This step is repeated. The combined organic phases are concentrated under reduced pressure to an oil.

The oil is dissolved in AcOBu (0.5 L), concentrated under reduced pressure at 20"C and re- dissolved in AcOBu (0.5 L). Then, heptane (2 L) is added at 50°C for 1 hour. The suspension is left with stirring at 0°C for 16 hours. The precipitate is isolated by filtration and washed with heptane. Finally, the solid is dried under reduced pressure at to yield H-Lys(iPr,Boc)~Pro-D- Ala-NH ₂ [21], Purity of intermediate H{8-10)NH ₂ [21] is >95% (HPLC).

Step 9 intermediate H(8-10)NH2 [21]

Quality control Acceptance criteria.

Description "White to slightly yellow powder (visual inspection)"

Identification ( 1 ) "456.3±0.4Da (MS)"

Identification (2) "Lys(iPr) 0.9-1 .1, Pro 0.9- 1 .1, Ala 0.9-1. 1 (AAA) ^* "

Chiral purity D-Lys(iPr) <0.3%, D-Pro <0.3%, L-Ala <0.5% (GC-MS)

Purity >95 % (HPLC, Area %) Example 4: Segment Condensations to Final Intermediate (Compound of Formula II)

Intermediate Z(4-10)NH ₂ : Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D - Ala-NH ₂ [22] (Compound of formula IVg)

Step 10 (Reaction step)

Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA [15] (1153.41 g) from Step 7 is dissolved in DMF (2.1 kg). Then, HOBt (153.2 g) is added together with AcOEt (6.9 kg) and H- Lys(iPr,Boc)-Pro-D-Ala-NH ₂ [21] (569.5 g) from step 9. When all solids are dissolved MSA (96.1 g) is added. The solution is cooled below 5°C and DCC (309.5 g) dissolved in AcOEt (0.810 kg) is added. The temperature is raised to 20°C and the reaction continues for 24 hours. Conversion of Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA [15] is >96 % (HPLC). AcOEt (4.95 kg) and water (5.5 L) are added, and the mixture is stirred, and filtered. While stirring, 7.5% NaHC0 ₃ (aq) (35L) is added to the filtrate. The phases are separated and the organic layer is further washed with:

7.5% NaHC0 ₃

• Water at pH 3 (pH is adjusted while stirring with aqueous HCI)

• Water

The final organic phase is concentrated under reduced pressure to an oil. The oil is re- concentrated with EtOH (0.405 kg) and subsequently with AcOEt (0.45 kg). The remaining oil is dissolved in EtOH (0.405 kg}, and AcOEt (0.45 kg) and AcOBu (4.6 L) are added. The solution is added to heptane (27.6 L) at 20°C for 1 hour. Then, the precipitate is filtered, and washed with heptane. The solid is dried under reduced pressure at maxiumum to yield Z- Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-A la-NH ₂ [22]. Purity of Z- Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuC m)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH ₂ [22] is >70 % (HPLC).

Final Intermediate Ac(l-10)NH ₂ : Ac-D-2Nai-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-

4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH ₂ [24]

Step 11 (Reaction step)

Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pr o-D"Ala~NH ₂ [22] (1409.67 g) from Step 10 is added to a mixture of EtOH (10.98 kg) and water (3.2 L) and stirred until the solution is homogenous. 5 % Pd/C (211 g) is added. The mixture is hydrogenated at 20°C with pH-controi at pH 2.5 ,with aqueous HCI.

The catalyst is removed by filtration and the pH is adjusted to pH 3.8 ,with aqueous NaOH. The filtrate is concentrated under reduced pressure to an oil. EtOH (4.7 kg) is added to the oil and re-concentrated. Then, AcOEt (5.4 kg) is added to the oil and re-concentrated and this process is repeated again to yield H-Ser(tBu)-4Aph{L-Hor)-D-4Aph(tBuCbm)-leu- Lys(iPr,Boc)-Pro-D-A!a-NH ₂ [23].

H-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D -Ala-NH ₂ [23] is dispersed in AcOEt (1.125 kg), then HOBt {153.16 g) is added and the mixture is cooied to 0°C. Ac-D-2Nal- D-4Cpa-D-3Pal-ONa [7] (609.05 g) from Step 4 is dissolved in D SO (2.5 L), this solution is mixed with the slurry containing H-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)- Pro-D-Ala-NH ₂ [23] and DCC (309.5 g) dissolved in AcOEt (0.45 kg) is added. The mixture is stirred at 5°C for 24 hours. Conversion of [23] is >96% (HPLC).

Water (150 mL) and DMSO (0.5 L) are added and the stirring is continued at 20°C for more than 3 hours . The precipitate is filtered, and washed with a mixture of AcOEt and DMSO. The filtrates are combined, and n-butanol (17 L) is added. The organic solution is washed with:

• Water at pH 2.5 (pH is adjusted while stirring with aqueous HCI)

7% NaHC0 ₃ (aq)

• 10% aqueous NaCl (pH in the mixture is neutralised to pH 7.0, if necessary, while stirring with aqueous HCI)

DMF (4.75 kg) is added and the organic phase is concentrated under reduced pressure to an oil. The oil is slowly added to water (50 L) at 20°C for 1 hour under vigorous stirring. The precipitate is isolated on a filter, and washed twice with water. The solid is subsequently dried under reduced pressure to yield Ac-D-2Nal-D-4Cpa-D-3Pal-Ser{tBu)-4Aph(i_-Hor}-D- 4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH ₂ [24] [Final intermediate]. Purity of [24] is >70 % (HPLC)

Example 5: Deprotection of Final Intermediate Ac(l-10)NH? to Crude Degarelixf25] Step 12 (Reaction step)

Ac-D-2Nal-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCb m)-Leu-Lys(iPr,Boc)-Pro-D-Ala- NH ₂ [24] (Compound of formula lib}(1844.59 g) from step 11 is dissolved in TFA (28.3 kg) at 20°C. The solution is stirred at 20°C (removal of 3 protection groups) for 24 hours.

Conversion of [24] is >99 % (HPLC).

The reaction mixture is then mixed with a cold solution (below 10°C ) of water (74 L), AcONH ₄ (19.1 kg), AcOH (18.4 L) and EtOH (14.52 kg). During mixing of the two solutions the temperature is kept below 25°C. The pH of the final solution is adjusted to pH 3 with TFA or AcONH ₄ , if necessary, to yield the solution of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D- 4Aph{Cbm)-Leu-Lys(iPr)™Pro-D~AIa-l\IH ₂ [25][Crude Degarelix].

Step 13 {Purification and lyophilLsation)

The solution of crude degarelix is pumped through a reversed phase column. Degarelix is eluted from the column with a gradient of EtOH/ 0.1.2 % TFA in water. Fractions with a purity >95% are repurified on a reversed phase column using a gradient of EtOH/ 1 % AcOH in water. Fractions of high purity are lyophilised.