HK1167865B - Method for the manufacture of degarelix - Google Patents

Description

Method for producing degarelix

Technical Field

The present invention relates to a method for producing a synthetic peptide, and more particularly to a method for producing a decapeptide degarelix (degarelix).

Background

There are many known methods for peptide synthesis. One typical method is Liquid Phase Peptide Synthesis (LPPS), which is the preferred method for mass production of peptides. Another currently used method of polypeptide synthesis is Solid Phase Peptide Synthesis (SPPS), in which growing peptide chains are covalently bound to a resin on a solid support until the desired length and sequence is reached and then cleaved. In these methods, in order to prevent reactions other than the formation of new peptide bonds required for the growing peptide, it is necessary to protect the reactive side chains of the incorporated amino acids. In addition, in order to prevent side reactions between the added amino acids and to incorporate a plurality of amino acids in each step, the added amino acids are generally protected by an α -amino group. Thus, the α -amino group synthesized as a solid phase bound peptide is deprotected and then coupled to a repeated cycle of α -amino protected amino acid units.

Degarelix is a GnRH antagonist used in the treatment of prostate cancer. Degarelix acts rapidly, inhibiting gonadotropins, testosterone and Prostate Specific Antigen (PSA). Degarelix is a compound of formula Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-4Aph (hor) -D-4Aph (cbm) -Leu-ILys-Pro-D-Ala-NH₂Synthetic decapeptides as shown.

The fifth amino acid portion from the amino terminus in degarelix is the unnatural amino acid Aph (L-hor). Aph (L-hor) denotes (L-hydroorotic acid) -4-amino-phenylalanine. It is well known in the art (Koedjikov, A.H. et al, J.chem.Soc.Perkin, trans.2, 1984, page 1077-1081; Kaneti, J.et al, org.Biomol.chem., 2004, page 1098-1103) that compounds comprising a dihydrouracil moiety rearrange under basic conditions to compounds comprising a hydantoin moiety. The corresponding rearrangement of Aph (L-Hor) is shown below (upper left: dihydrouracil moiety N-4- (L-hydroorotylamino) -phenylalanine I, R ═ CH₂CHNH₂COOH; right lower: hydantoin moiety II, N-4- [2- (5-hydantoinyl) -acetyl) -phenylalanine).

In the rearrangement, the dihydrouracil moiety I is converted into a hydantoin moiety II. When the L-Hor moiety of 4Aph (L-Hor) is of the dihydrouracil type, it is desirable that such rearrangement occurs during the manufacture of degarelix using basic conditions. The applicant has demonstrated by the following meansThe above result is understood, i.e. contacting a peptide synthesis intermediate comprising a terminal 4aph (hor) with an α -amino group protected by Fmoc with NaOH or the organic base Dicyclohexylamine (DCHA). The resulting deprotected product was found to be contaminated with up to several weight% of the corresponding hydantoin rearrangement product. It is therefore expected that during the synthesis of degarelix, the intermediate Fmoc-4Aph (hor) -4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH-resin undergoes partial rearrangement during deprotection under basic conditions to give Fmoc-X-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH-resin, X being 4- ([2- (5-hydantoin)]-acetylamino) -phenylalanine. It is therefore foreseen that the degarelix product obtained from Fmoc-4Aph (hor) -4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH-resin is purified by a corresponding amount of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH₂And (4) pollution. Degarelix is the active ingredient of a drug administered to humans. It cannot be contaminated with any impurities in an amount exceeding 0.3% by weight of the product. Therefore, the amount of hydantoin byproducts in degarelix suitable for human use is not allowed to be more than 0.3 wt%. Since the hydantoin moiety-containing by-products are very similar in structure to degarelix, it is difficult to separate them. If attempted, separation can be expected to result in significant loss of product. Therefore, basic conditions should be avoided in the manufacturing process of pharmaceutical grade degarelix using the protecting group Fmoc.

The synthesis of degarelix is disclosed in US 5925730 a. The preferred alpha-amino protecting group in this synthesis is tert-butoxycarbonyl (Boc), which was used in all examples. Also, for this purpose, a variety of other well-known protecting groups are disclosed, such as fluorenylmethyloxycarbonyl (Fmoc). The Boc group has the advantage that the α -amino group protected by it can be deblocked by standard treatment with trifluoroacetic acid (TFA) under acidic conditions. TFA has the disadvantage that it is highly toxic to humans, which puts the manufacturer at risk. Another disadvantage of TFA is that it is toxic to the environment, which results in high disposal costs and environmental pollution once improperly disposed.

Object of the Invention

It is an object of the present invention to provide a process for the manufacture of degarelix which does not pose a risk to human health, and in particular is less harmful to human health than the process disclosed in US 5925730 a.

It is a further object of the present invention to provide a process for the manufacture of degarelix which does not pose a risk to the environment, and in particular is less harmful to the environment than the process disclosed in US 5925730 a.

It is a further object of the present invention to provide a method of manufacturing degarelix at a lower cost than known methods in the art.

Other objects of the present invention will become apparent from the following summary of the invention, a number of preferred embodiments disclosed by way of example, and the appended claims.

Summary of The Invention

The present inventors have surprisingly found that pharmaceutically pure degarelix can be manufactured by solid phase synthesis using Fmoc as the alpha-amino protecting group. By "pharmaceutically pure" is meant that the product does not contain any more than 0.3% by weight of any impurity. Unexpectedly, the Aph (L-Hor) moiety does not undergo rearrangement in solid phase synthesis even when multiple cycles of Fmoc protection and deprotection are performed under basic conditions.

The Fmoc-protected amino acid of the alpha-amino group is coupled to a resin and then coupled to another Fmoc-protected amino acid of the alpha-amino group in a step-wise, cyclic, sequence-dependent manner. Each amino acid coupling step is followed by a one-step deprotection to remove the Fmoc protecting group to enable coupling of the next amino acid. Deprotection is carried out with a base. Deprotection is preferably carried out using a base such as piperidine or an alkyl substituted piperidine in an organic medium.

Protection of the side chains is preferably included, with the aim of protecting the side chains of particularly reactive or particularly labile amino acids against side reactions and/or branching of the growing molecule. Once the growing peptide reaches full length, the side chain protecting groups are removed.

Accordingly, the present invention discloses degarelix Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-4Aph (hor) -D-4Aph (cbm) -Leu-ILys-Pro-D-AIa-NH₂The degarelix comprises 0.3 wt% or less, particularly preferably 0.1 wt% or less, most preferably 0.01 wt% or less of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH₂Wherein X is 4- ([2- (5-hydantoin)]-acetylamino) -phenylalanine, the method comprising a stepwise synthesis on a solid support having an amino group attached thereto, wherein the steps comprise: providing a solution of an amino acid or peptide having an α -amino group protected by Fmoc; contacting said support with said solution in the presence of an agent that forms a peptide bond between the carboxyl group of said dissolved amino acid or peptide and said amino group attached to said support for a time sufficient to form said peptide bond; contacting the support with an organic base in an organic solvent to remove the Fmoc. The preferred organic base is piperidine. Another preferred organic base is a C-alkyl-substituted piperidine, particularly preferably a 2-alkylpiperidine, a 3-alkylpiperidine, a 2, 4-dialkylpiperidine, a 2, 5-dialkylpiperidine, a 2, 6-dialkylpiperidine, wherein the alkyl is a branched or straight-chain alkyl group of 1 to 6 carbons, particularly preferably a methyl or ethyl group, most preferably a methyl group. The preferred solvent is dimethylformamide. Another preferred solvent is diethylformamide. Other preferred solvents are NMP or DMA. Preferred reagents for the formation of peptide bonds include N, N' -diisopropylcarbodiimide. The support-attached amino groups are preferably alpha-amino groups of the support-attached fragments of degarelix. The Fmoc-protected peptide is also preferably a fragment of degarelix. The preferred support is one selected from Rink amide AM resin and Rink amide MBHA resin. A preferred method of releasing degarelix from the support is acid treatment.

A preferred aspect of the present invention discloses degarelix prepared by the process of the invention comprising 0.3% by weight or less, particularly preferably 0.1% by weight or less, most preferably 0.01% by weight or less of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH₂Wherein X is 4- ([2 ]- (5-hydantoin group)]-acetylamino) -phenylalanine.

Another preferred aspect of the invention discloses the use of Fmoc in the preparation of degarelix by solid phase synthesis comprising less than 0.3% by weight, particularly preferably less than 0.1% by weight, most preferably less than 0.01% by weight, of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH₂Wherein X is 4- ([2- (5-hydantoin)]-acetylamino) -phenylalanine.

The invention will be described in more detail below with reference to a number of preferred embodiments, which are illustrated in the accompanying drawings and examples.

Detailed Description

Abbreviations

4- (2 ', 4' -Dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamidomethyl polystyrene resin [ Fmoc-Rink amide AM-resin ]

4- (2 ', 4' -Dimethoxyphenyl-Fmoc-aminomethyl) -phenoxyacetamido-4-methylbenzhydrylamine polystyrene resin [ Fmoc-Rink amide MBHA-resin ]

9-Fmoc-D-4-chlorophenylalanine [ Fmoc-D-Phe- (4Cl) -OH ]

9-Fmoc-D-2-naphthylalanine [ Fmoc-D-2Nal-OH ]

9-Fmoc-D-3-pyridylalanine [ Fmoc-D-3Pal-OH ]

9-Fmoc-N (4) - (tert-butylcarbamoyl) -D-4-aminophenylalanine [ Fmoc-D-4Aph (tBuCbm) -OH ]

9-Fmoc-N (4) - (L-Hydroorotic acid) -4-aminophenylalanine [ Fmoc-Aph (L-Hor) -OH ]

9-Fmoc-leucine-OH [ Fmoc-L-Leu-OH ]

9-Fmoc-O-tert-butyl-serine [ Fmoc-Ser (tBu) -OH ]

9-Fmoc-L-proline [ Fmoc-Pro-OH ]

9-Fmoc-D-alanine [ Fmoc-D-Ala-OH ]

9-Fmoc-N (epsilon) -isopropyl-N (epsilon) -Boc-lysine [ Fmoc-L-ILys (Boc) -OH ]

Acetonitrile

2-propanol (isopropanol) (IPA)

Ethanol, 99.9% (EtOH)

Methanol (MeOH)

Pure water (water)

Ethyl acetate (AcOEt)

Acetic acid (AcOH)

Aqueous ammonium hydroxide solution (Aq. NH3)

Ammonium acetate (AcONH4)

Acetyl imidazole

N-methylmorpholine (NMM)

N-methylpyrrolidone (NMP)

N, N' -Diisopropylcarbodiimide (DIC)

N, N-Dimethylformamide (DMF)

N, N-Dimethylacetamide (DMA)

Dimethyl sulfoxide (DMSO)

Dicyclohexylamine (DCHA)

1-hydroxybenzotriazole (HOBt)

Aqueous sodium hydroxide solution (Aq. NaOH)

Aqueous hydrochloric acid (Aq. HCl)

Phosphoric acid (H)₃PO₄)

Trifluoroacetic acid (TFA)

Diisopropylethylamine (DIEA)

Ethanedithiol (EDT)

Isopropyl ethyl ether (IPE)

Control In Processing (IPC)

Benzyloxycarbonyl (Z)

1, 8-diazabicyclo [5.4.0] -undec-7-ene (DBU)

Example 1

Hydantoin formation in the synthesis of degarelix. During the production of degarelix, rearrangement of the hydrogenated orotic groups to hydantoin acetyl groups was observed in two stages and under two alkaline conditions.

The first rearrangement occurs in the fragment Z-Ser (tBu) -4Aph (hor) -D-4Aph (tBu-Cbm) -Leu-ILys (Boc) -Pro-D-Ala-NH₂In the alkaline extraction process. The pH was adjusted to 9.1 with concentrated NaOH solution in an organic/aqueous biphasic system, thus forming 4.5 wt% hydantoin analog. The mechanism is seen to consist of two steps: (a) the six-membered hydrogenated orotic acid moiety is hydrolyzed under alkaline conditions and then ring-closed under acidic conditions to form a five-membered hydantoin analog.

The second rearrangement occurs during the evaporation of the fragment Z-Ser (tBu) -4Aph (hor) -D-4Aph (tBu-Cbm) -Leu-OHDHA. After the above extraction, Z-Ser (tBu) -4Aph (hor) -D-4Aph (tBu-Cbm) -Leu-OH was dissolved in a mixture of ethyl acetate and 2-butanol. To isolate the fragments as DCHA salts after solvent evaporation followed by a precipitation step, DCHA (2.5 equivalents) was added. In a particular batch, the hydantoin analogs described above, as well as the hydrolyzed forms described above, may be observed. Hydantoin quantification was not possible because hydantoin was difficult to separate from other products by HPLC; the hydrolyzed form was formed in an amount of 1.34 wt.% of the combined product. Experimental evidence suggests that the amount of rearrangement/hydrolysis is related to the amount of DCHA used in the above process.

The following experiment provides further evidence of the instability of the hydrogenated orotic acid moiety under alkaline conditions. Z-Ser (tBu) -4Aph (hor) -D-4Aph (tBu-Cbm) -Leu-OHDHA (67mM) and 167mM (2.5 equivalents) DCHA were dissolved together in aqueous 2-BuOH at 31.0 ℃. After 25 hours, 1.3% hydantoin analogs and 0.3% hydrolysis intermediates were formed.

Example 2

Stability of degarelix in DBL VDMF and piperidine/DMF. The stability of degarelix was determined under conditions corresponding to those used for the Fmoc group elimination during SPPS. Hydrogenated orotate groups on the side chain of the 5 th amino acid residue in the degarelix sequence, i.e. 4aph (hor), are known to be sensitive to bases and to rearrange to hydantoin acetyl groups. All SPPS procedures known to the present inventors are based on Boc-chemistry.

Degarelix samples were dissolved in 20% piperidine/DMF, 2% DBU in DMF and 2% DBU + 5% water in DMF, respectively. After 20 hours the samples were analyzed by HPLC to determine the amount of hydantoin analogues.

2% DBU/DMF resulted in the formation of 1.8% hydantoin. If 5% water is also present (simulated aqueous DMF), the amount increases to 7%. Surprisingly, the use of 20% piperidine in DMF did not result in the formation of any hydantoin analogs, indicating that this mixture may be used for SPPS of Fmoc-based degarelix.

Example 3

Synthesis and purification of degarelix using Fmo-/Rink amide AM resin

Step 1. Fmoc-Rink amide AM resin (64 g; substitution 0.67mmol/g) was placed in a reactor and washed with 1.9L of DMF. To 250ml of the swollen resin was added 20% piperidine in DMF and the mixture was stirred for 20 minutes. The reactor was evacuated to allow the reactor to vent through the bottom filter and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 2L of DMF. The reactor was then evacuated and vented. The peptide resin was ready for use in step 2.

Step 2. A solution of 27.0g Fmoc-D-AIa-OH (2 equiv.), 14.3g HOBt and 13.2ml DIC was dissolved in 250ml DMF, activated for 15 minutes and then poured into a reactor containing peptide resin. After the reaction time reached 1 hour, 2.2ml of NMM was added to the solution and the reaction was continued for another 1 hour. Then, 30ml of acetic anhydride and 2ml of NMM were added to the mixture, and the mixture was stirred for 15 minutes. The reactor was then vented using vacuum. The peptide resin was washed with 2L of DMF. After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 250ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 2L of DMF. I.e. can be used in step 3.

Step 3. A solution of 29g of Fmoc-L-Pro-OH (2 equiv.), 14.3g of HOBt and 13.2ml of DIC was dissolved in 250ml of DMF, activated for 25 minutes and then poured into a reactor containing a peptide resin. After 75 minutes of reaction, 2.2ml of NMM was added to the solution and the reaction was continued for another 1 hour. Then, 30ml of acetic anhydride and 2ml of NMM were added to the mixture, and the mixture was stirred for 15 minutes. The reactor was then vented using vacuum. The peptide resin was washed with DMF (2.6L). After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 250ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 2L of DMF. I.e. can be used in step 4.

Step 4. A solution of 33g of Fmoc-L-ILys (Boc) -OH (1.5 equiv.), 10.7g of HOBt and 10.1ml of DIC was dissolved in 250ml of DMF, allowed to react for 0.5 hour, and then poured into a reactor containing a peptide resin. After 2 hours of reaction, 2.2ml of NMM was added to the solution and the reaction was continued for another 1 hour. Then 30ml of acetic anhydride and 2.2ml of NMM were added to the mixture, which was stirred for 15 minutes, and then the reactor was vented using vacuum. The peptide resin was washed with DMF (3L). After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 250ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 3.5L of DMF. I.e. can be used in step 5.

Step 5. A solution of 38g of Fmoc-L-Leu-OH (2.5 equiv.), 18g of HOBt and 16.8ml of DIC was dissolved in 250ml of DMF, activated for 0.5 hour and then poured into a reactor containing a peptide resin. After 2 hours of reaction, 2.2ml of NMM was added to the solution and the reaction was continued for another 50 minutes. Then, 30ml of acetic anhydride and 2ml of NMM were added to the mixture, and the mixture was stirred for 15 minutes. The reactor was then vented using vacuum. The peptide resin was washed with DMF (2.6L). After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 250ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 2.5L of DMF. I.e. can be used in step 6.

Step 6. A solution of 32g of Fmoc-D-4Aph (tBu-Cbm) -OH (1.5 equiv.), 10.7g of HOBt and 10.1ml of DIC was dissolved in 250ml of DMF, activated for 1 hour and then poured into a reactor containing a peptide resin. After 20 minutes of reaction, 22ml of NMM was added to the solution and the reaction was continued for another 20 hours. Then, 30ml of acetic anhydride and 2ml of NMM were added to the mixture, and the mixture was stirred for 15 minutes. The reactor was then vented using vacuum. The peptide resin was washed with 4L of DMF. After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 250ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 250ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 3.4L of DMF. I.e. can be used in step 7.

Step 7. A solution of 35g of Fmoc-L-4Aph (L-Hor) -OH (1.5 equiv.), 11g of HOBt and 10.1ml of DIC was dissolved in 350ml of DMF, activated for 1 hour, and then poured into a reactor containing a peptide resin. After 50 minutes of reaction, 2.2ml of NMM was added to the solution and the reaction was continued for another 21.5 hours. The reactor was vented using vacuum. The peptide resin was washed with 4.4L of DMF. After removal of DMF by applying vacuum to the reactor, the peptide resin was treated with 350ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 350ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 4.4L of DMF. I.e. can be used in step 8.

Step 8 Fmoc-L-Ser (tBu) -OH (2.5 equiv.) (41g), 17.9g HOBt, 16.8ml DIC and 4.9ml NMM were dissolved in 500ml DMF and poured into a reactor containing peptide resin. The reaction was carried out for 3.5 hours. The reactor was then vented using vacuum. The peptide resin was washed with 4.2L of DMF. After the DMF was removed by applying a vacuum to the reactor, the peptide resin was treated with 375ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 375ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 4.2L of DMF. I.e., can be used in step 9.

Step 9. A solution of 25g Fmoc-D-3Pal-OH (1.5 equiv.), 10.7g HOBt, 10.1ml DIC and 4.9ml NMM was dissolved in 400ml DMF and poured into a reactor containing peptide resin. The reaction was carried out for 4.5 hours. The reactor was then vented using vacuum. The peptide resin was washed with 4.2L of DMF. After the DMF was removed by applying a vacuum to the reactor, the peptide resin was treated with 375ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 375ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 4.2L of DMF. I.e., may be used in step 10.

Step 10. A solution of 27g Fmoc-D-Phe (4Cl) -OH (1.5 equiv.), 10.7g HOBt, 10.1ml DIC and 4.9ml NMM was dissolved in 400ml DMF and poured into a reactor containing peptide resin. The reaction was carried out for 10 hours. The reactor was vented using vacuum. The resin was washed with 5.5L of DMF. After the DMF was removed by applying a vacuum to the reactor, the peptide resin was treated with 375ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 375ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 5L of DMF. I.e., may be used in step 11.

Step 11. A solution of 28g Fmoc-D-2Nal-OH (1.5 equiv.), 10.7g HOBt, 10.1ml DIC and 4.9ml NMM was dissolved in 400ml DMF and poured into a reactor containing peptide resin. The reaction was carried out for 2.5 hours. The reactor was vented using vacuum. The peptide resin was washed with 5.2L of DMF. After the DMF was removed by applying a vacuum to the reactor, the peptide resin was treated with 375ml of 20% piperidine in DMF for 20 minutes. The reactor was vented by applying vacuum and a second treatment with 375ml of 20% piperidine in DMF was carried out for 20 minutes. The reactor was again evacuated to empty it and the peptide resin was washed with 5L of DMF. I.e., may be used in step 12.

Step 12 acetylimidazole (3 equivalents) (14.5g) and 4.9ml of NMM were dissolved in 400ml of DMF and poured into the reactor. After 1.5 hours, the reactor was vented by pulling a vacuum on the reactor. The peptide resin was washed with 5L of DMF and the reactor was vented using vacuum.

Step 13. washing the peptide resin with IPA and drying under vacuum. The peptide resin was isolated (129.8 g; yield 96%).

Step 14. Dry peptide resin (60g) was suspended in 600ml TFA for 25 hours at room temperature. It is then poured into a mixture of 2.4L of water, 620g of ammonium acetate, 600ml of ethanol and 600ml of acetic acid. The mixture is adjusted to a pH between 3 and 4 with TFA and filtered.

Step 15. purify the product using a two-step purification scheme. In the first step, a column (2.5cm x 34cm) packed with reversed phase C-18 material was used, the buffer system comprising buffer a (0.12% aqueous TFA) and buffer B (99.9% ethanol). A volume of the filtrate from step 14 corresponding to 1.6g of product was added to the column. Purification was performed using a step gradient starting with 10% B for 2-3 column volumes, 29% B for 5-7 column volumes, followed by a gradient from 29% B to 50% B over 3 column volumes at a flow rate of 70 ml/min. The process continues until all of the filtrate from step 14 has been treated. All fractions collected were analyzed by analytical HPLC. The product-containing fractions with a purity of more than 94% were combined. In the second purification step, a column (2.5cm x 34cm) packed with reversed phase C-18 material was used, the buffer system comprising buffer a (1% aqueous acetic acid), buffer B (99.9% ethanol) and buffer C (0.5M aqueous ammonium acetate). An amount corresponding to 1.3g of product was taken from the combined fractions containing product and fed to the column and purified using a step gradient starting with 10% B + 90% C for 2-3 column volumes and then 90% A + 10% B for 2-3 column volumes. The product was eluted with 24% B + 76% a. The product-containing fractions of acceptable purity were combined and desalted using the same column.

Desalting was performed using buffer A (1% aqueous acetic acid) and buffer B (99.9% ethanol). A volume corresponding to 1.6g of product was removed from the combined purified fractions and added to the column, and all ammonium acetate in the product was washed off with 2-3 column volumes of buffer A. The product was then eluted with 50% a + 50% B. The solution of the purified product containing 50% ethanol was concentrated in a rotary evaporator. After all ethanol was removed, the remaining solution containing the product was lyophilized. A total of 11.8g (37% overall yield) of degarelix was obtained as a fluffy solid. 4- ([2- (5-hydantoinyl) ] -acetylamino) -phenylalanine could not be detected in the product (HPLC).

Example 4

Synthesis and purification of degarelix using Fmo-/Rink amide MBHA

The synthesis and purification were performed substantially in the same manner as in example 1. The difference from the process of example 1 is:

a) Fmoc-D-Aph (tBu-Cbm) -OH was replaced by Fmoc-D-Aph (Cbm) -OH;

b) acetylation of the N-terminus of the H-D-2-Nal-peptide-resin was performed with acetic anhydride instead of acetylimidazole.

c) Acetonitrile was used instead of ethanol during the purification.

4- ([2- (5-hydantoinyl) ] -acetylamino) -phenylalanine could not be detected in the product by HPLC.

Claims (13)

1. Degarelix Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-4Aph (hor) -D-4Aph (cbm) -Leu-ILys-Pro-D-AIa-NH₂The degarelix comprises 0.3 wt% or less of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4Aph (Cbm) -Leu-ILys-Pro-D-Ala-NH₂Wherein X is 4- ([2- (5-hydantoin)]-acetylamino) -phenylalanine, the method comprising a stepwise synthesis on a solid support having an amino group attached thereto, wherein the steps comprise: providing a solution of an amino acid or peptide having an α -amino group protected by Fmoc; after the carboxyl group of the dissolved amino acid or peptide is bonded to the branchContacting said support with said solution in the presence of an agent that forms a peptide bond between said amino groups of the support for a time sufficient to form said peptide bond; contacting the support with an organic base selected from piperidine or C-alkyl substituted piperidine wherein alkyl is a branched or straight chain alkyl of 1 to 6 carbons in an organic solvent to remove Fmoc.

2. The method of claim 1, wherein the degarelix comprises less than 0.1% by weight of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4aph (cbm) -Leu-ILys-Pro-D-Ala-NH₂。

3. The method of claim 1, wherein the degarelix comprises less than 0.01% by weight of Ac-D-2Nal-D-Phe (4Cl) -D-3Pal-Ser-X-D-4aph (cbm) -Leu-ILys-Pro-D-Ala-NH₂。

4. The method of claim 1, wherein the organic base is selected from the group consisting of: 2-alkylpiperidine, 3-alkylpiperidine, 2, 4-dialkylpiperidine, 2, 5-dialkylpiperidine and 2, 6-dialkylpiperidine, wherein alkyl is a branched or straight chain alkyl group of 1 to 6 carbons.

5. The method of claim 1 or 4, wherein the alkyl group is a methyl group or an ethyl group.

6. The method of claim 1 or 4, wherein the alkyl group is a methyl group.

7. The method of claim 1, wherein the organic base is piperidine.

8. The method of claim 1, wherein the organic solvent is dimethylformamide.

9. The method of claim 1, wherein the agent for forming a peptide bond comprises N, N' -diisopropylcarbodiimide.

10. The method of claim 1, wherein the amino groups attached to the support are α -amino groups of a fragment of degarelix attached to the support.

11. The method of claim 1, wherein the Fmoc-protected peptide is a fragment of degarelix.

12. The method of claim 1, wherein the support is selected from Rink amide AM resin or Rink amide MBHA resin.

13. The method of claim 1, wherein degarelix is released from the support by acid treatment.