FIELD OF THE INVENTION

The present invention relates to a process for the preparation of Degarelix or pharmaceutically acceptable salts and intermediates thereof.

BACKGROUND OF THE INVENTION

Degarelix is a third generation gonadotropin releasing hormone (GnRH) antagonist (blocker). Degarelix is a synthetic linear decapeptide containing seven unnatural amino acids, five of which are D-amino acids and is chemically designated as D-Alaninamide, A^-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(45)-hexahydro-2,6-dioxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-iV6-(l-methylethyl)-L-lysyl-L-prolyl.
The structural formula of Degarelix is:

Its common name is: [Ac-D-2Nal', D-4Cpa2, D-3Pal , 4Aph(L-Hor) , D-4Aph(Cbm) , 10

Lys(iPr) ,D-Ala ]GnRH where: 2Nal is 2-Naphthylalanine, 4Cpa is 4-Chlorophenylalanine, 3Pal is 3-Pyridylalanine, Hor is hydroorotyl, Lys(iPr) is N6-Isopropyllysine, 4Aph is 4-Aminophenylalanine, and Cbm is the carbamoyl group.

Degarelix acetate is known to be therapeutically useful and marketed as subcutaneous injectable dosage forms under the brand name Firmagon® for treatment of patients with advanced prostate cancer.

Degarelix and its pharmaceutical^ acceptable salts are disclosed in US 5,925,730. The synthesis of Degarelix has been described in US '730 by solid phase synthesis using Boc chemistry.

N-Boc-D-alanine (I) was coupled to the 4-Methylbenzhydrylamine resin (MBHA resin) using diisopropylcarbodiimide (DIC) and l-hydroxybenzotriazole (HOBT) to afford resin (II). Subsequent cleavage of the Boc protecting group by means of trifluoroacetic acid (TFA) provided the D-alanine-bound resin (III). Sequential coupling and deprotection cycles were carried out with the following protected amino acids: N-Boc-L-proline (IV), N-alpha-Boc-N6-isopropyl-N6-carbobenzoxy-L-lysine (VI) and N-Boc-L-leucine (VIII) to afford the respective peptide resins (V), (VII) and (IX). N-alpha-Boc-D-4-(Fmoc-amino)phenylalanine (X) was coupled to (IX), yielding resin (XI). Cleavage of the side-chain Fmoc protecting group with piperidine DMF gave the aniline derivative (XII). The conversion of compound (XII) to the corresponding urea by treatment with tert-butyl isocyanate, the Boc group was cleaved with TFA to produce resin (XIII).

This portion of the synthesis is shown below in Scheme-I:

Further coupling with N-alpha-Boc-L-4-(Fmoc-amino)phenylalanine (XIV), followed by Fmoc deprotection with piperidine produced the aniline derivative (XV). The aniline derivative (XV) was acylated with L-hydroorotic acid (XVI), followed by Boc group cleavage to yield resin (XVII). Coupling of (XVII) with N-Boc-L-serine(O-benzyl) (XVIII) and subsequent deprotection gave peptide (XIX).

This portion of the synthesis is shown below in Scheme-II:

Peptide (XIX) was sequentially coupled with N-alpha-Boc-D-(3-pyridyl)alanine (XX) and N-Boc-D-(4-chlorophenyl)alanine (XXII), followed by deprotection cycles with TFA to produce corresponding resins (XXI) and (XXIII) respectively.

This portion of the synthesis is shown below in Scheme-Ill:

The coupling of resin (XXIII) with N-Boc-D-(2-naphthyl)alanine (XXIV), deprotection cycle with TFA to produce corresponding resin (XXV). The peptide resin (XXV) was acetylated with Ac20 and finally deprotected and cleaved from the resin by treatment with HF to provide Degarelix. This portion of the synthesis is shown below in Scheme-IV:

The main drawback of the prior-art is that the cleavage and deprotection of the peptide from the resin require treatment with hydrogen fluoride (HF) or similar drastic conditions. It is not only hazardous to handle HF but also limits the use of large quantities. Hence, the process is not scalable. It is also hazardous to the environment.

Alternatively, after coupling of the peptide resin (XIII) with alpha-Boc-L-4-(Fmoc-amino)-phenylalanine (XIV), the Fmoc protecting group was not removed, yielding resin (XXVI). Subsequent coupling cycles with amino acids (XVIII), (XX), (XXII) and (XXIV) as above finally produced resin (XXVII). The Fmoc group was then deprotected by treatment with piperidine, and the resulting aniline was acylated with L-hydroorotic acid (XVI) to provide resin (XXVIII). The Resin (XXVIII) was finally cleaved and deprotected by treatment with HF to produce Degarelix.

This portion of the synthesis is shown below in Scheme-V:

The main drawback of the prior-art is that the cleavage and deprotection of the peptide from the resin require treatment with hydrogen fluoride (HF) or similar drastic conditions. It is not only hazardous to handle HF but also limits the use of large quantities. Hence, the process is not scalable. It is also hazardous to the environment.

WO 2011/066386 of Teva discloses a process for the preparation of Degarelix using combined solid phase peptide synthesis (SPPS) and solution phase synthesis via a 9+1 fragment protocol. The process comprising all the Fmoc-protected amino acids are coupled sequentially on a 2-chlototrityl resin (CTC resin), followed by washing, de-protection and cleaving the resin using trifluoroacetic acid (TFA) in dichloromethane (DCM) to produce nine amino acid peptide (Ac-D-Nal-D-Phe(4Cl)-D-3Pal-Ser(Trt)-4- Aph(Hor)-4-Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-OH), wherein the serine is protected with trityl group. The obtained nine amino acid peptide is coupled with H-D-Ala-NH2 by solution phase to produce Degarelix.

The process is as shown in Scheme-VI below:

The main drawback of the above process is that the stability of Serine (Trt). Since Serine (Trt) is unstable, it is expected that it will get degraded after its incorporation in the peptide chain during synthesis. Consequently, there shall be some impurities formed during synthesis. This will lead to impure crude Degarelix and that leads to difficult purification and poor yield, perhaps, poor quality Degarelix.

US 2012/0041172 (IN 2192/MUMNP/2011 A) of Polypeptide Laboratories discloses a process for the preparation of Degarelix using solid phase peptide synthesis by Fmoc strategy. The process comprising a step-wise addition of an amino acid solution in which an of-amino group is protected by Fmoc; contacting a solid support having an amino group linked thereto with the solution in the presence of reagent which forms a peptide bond between a carboxyl group of the dissolved amino acid and the amino group linked to the support to form a peptide bond; removing Fmoc by contacting the support with an organic base selected from piperidine to produce Degarelix.

The process is as shown in Scheme-VII below:

The main drawback of the above process is that the deblocking of tert-butyl protecting group of 4-D-Aph(Cbm) and serine using 100%TFA without scavengers for 25 hrs ended up in undesired side reactions and degradation of peptide. This lead to impure crude Degarelix and resorted to difficult purification. The yield of the peptide was poor.

Hence, there is a need to develop a more efficient process was felt by the present inventors by overcoming the above said disadvantages. The present invention provides a better process, for the preparation of Degarelix acetate, which results in better yields and avoids the use of hazardous raw material and reagents as reported in prior art.

Thus by providing a new process for the preparation of Degarelix acetate suitable for industrial scale the present inventors have addressed the problems associated with the prior-art and have provided a cost effective, simple, scalable, robust process for preparation of Degarelix acetate. Furthermore, the process avoids the cumbersome and tedious procedures of isolation, crystallization and purification of intermediates and hence it is economical and convenient to operate on a commercial scale. Therefore the process of the present invention is simple, cost effective, and industrially viable over the prior art procedures.

OBJECTIVE OF THE INVENTION

The main objective of the present invention is to provide simple, cost effective, improved processes for the preparation of Degarelix or pharmaceutically acceptable salts and intermediates thereof.

SUMMARY OF THE INVENTION

In one embodiment of the present invention provides a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof using combined solid phase peptide synthesis (SPPS) and liquid phase peptide synthesis (LPPS) by Fmoc strategy, proceeding via 9+1 fragment protocol.

In another embodiment of the present invention provides a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof by the solid phase peptide synthesis (SPPS) using novel resin linkers.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention provides a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof which comprises the steps of:

(i) loading of Fmoc-Pro-OH on a resin in the presence of base and solvent to produce Fmoc-Pro-resin; (ii) removing the Fmoc protecting group from Fmoc-Pro-resin of step (i) to produce H-Pro-resin; (iii) sequentially coupling the following Fmoc protected a-amino acid Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4- Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH with H-Pro-resin in the presence of coupling reagent to produce Fmoc-protected peptide fragment; (iv) removing of Fmoc protecting group from the protected peptidoresin of step (iii), followed by acetylation to produce protected nine amino acid peptide resin; (v) deprotecting the resin from the peptide of step (iv) using mild acidic condition to produce protected nine amino acid peptide; (vi) coupling of protected nine amino acid peptide of step (v) with D-alaninamide (D-Ala-NH2) in the presence of coupling reagent to produce protected Degarelix; (vii) de-protection of protected. Degarelix using acidic composition to produce Degarelix; (viii) optionally purifying the Degarelix of step (vii); and (ix) isolating Degarelix or its pharmaceutically acceptable salt thereof.

The process as summarized below:

wherein, the Fmoc is 9-fluorenylmethyloxycarbonyl, Pro-OH is proline, Lys(Ipr) is lysine isopropyl, Boc is tert-butoxycarbonyl, Leu-OH is leucine, 4-Aph(Cbm) is 4-aminophenyl-alanine(carbamoyl), 4-Aph(Hor)-OH is 4-aminophenylalanine(hydroorotyl), Ser-OH is serine, t-bu is tert-butyl, 3-Pal-OH is 3-pyridylalanine, Phe(4Cl)-OH is 4-chlorophenylalanine, 2Nal is 2-naphthylalanine and D-Ala-NH2 is D-alaninamide. The present invention uses the tert-butyl group for the protection of serine, which is stable throughout the reaction and hence yield the Degarelix with more than 98.5 % purity by HPLC.

In another embodiment of the present invention, Suitable resins for use in the process is selected from chlorotrityl resin, Rink acid resin, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA include, most preferred super acid labile resin is 2-chlorotrityl resins.

In still another embodiment of the present invention, step (i) is carried out in presence of a base and in the presence of solvent. The base is organic or inorganic base. The inorganic base comprises potassium carbonate, lithium carbonate, sodium carbonate, sodium ethoxide, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and mixtures thereof; the organic base comprises diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine, piperidine, N,N-dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and/or mixtures thereof. The solvent comprises dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), methanol, isopropanol, dichloroethane, 1,4-dioxane, tetrahydrofuran (THF), ethyl acetate, acetonitrile, acetone, and/or mixtures thereof.

In still another embodiment of the present invention, the coupling reagent used in the above process comprises 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-C1), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), iso- butylchloroformate (IBCF), 1,3 dicyclohexylcarbodiimide (DCC), 1,3-diisopropyl- carbodiimide (DIC), l-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCD1), N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ), isopropylchloroformate (IPCF), 2-(5-norbornen-2,3-dicarboximido)-l,l ,3,3- tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU).

In yet another embodiment of the present invention, the amino acids introduced in step (iii) of the sequential synthesis described above are commercially available as protected amino acids that are stable to any reactions and modifications of the side chains during the synthesis that could result in derivatives of the constituent amino acids.

In yet another embodiment of the present invention, wherein the sequential coupling of Fmoc protected a-amino acid in step (iii) is carried out by coupling the first amino acid with H-Pro-resin followed by removing Fmoc protecting of resultant amino acid and repeating the cycles for the addition of subsequent amino acids

In still another embodiment of the present invention, wherein the removal of Fmoc protection is carried out using a secondary amine base. The secondary amine base comprises piperidine, dimethylamine, diethylamine, diphenylamine or mixtures thereof. Although one of ordinary skill in the art may substitute the reagents with other suitable reagents depending on the nature of the protecting group. In the case of Fmoc, beside piperidine, other reagents could be used such as l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine, piperazine, or dimethylethyl amine and the like.

In still another embodiment of the present invention, wherein the acetylating agent in step (iv) comprises acetic anhydride or acetyl chloride or mixtures thereof.

In still another embodiment of the present invention, wherein the resin is deprotected using a mild comprising about 0.1% to about 5% of TFA in an organic inert solvent or a mixture of acetic acid with trifluoroethanol and DCM.

In still another embodiment of the present invention, cleavage of the protecting groups from the peptide may be affected by addition of a strong acidic composition. The acidic composition is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, ethanedithiol (EDT), TIS (triisopropylsilane) and water. The relative ratio of acidic material to scavenger to water may be from about 85% to about 99% acidic material, from about 0.1% to about 15% scavenger, and from about 0.1% to about 15% water by weight. A preferred acidic composition comprises about 95% TFA, about 2.5% EDT, and about 2.5% water.

In still another embodiment of the present invention, coupling of protected nine amino acid peptide of step (v) with D-alaninamide (D-Ala-NH2) in the presence of a coupling reagent in a solvent to produce protected Degarelix.

In still another embodiment of the present invention, the crude peptide product may be purified by any known method. Preferably, the peptide is purified using HPLC on a reverse phase (RP) column. At the end of the purification process or as a part of the purification process the counter ion of the peptide may be exchanged by a suitable ion such as, but are not limited to, acetate ion. The counter-ion exchange can be done by any suitable method such as HPLC or ion exchange. Suitable HPLC method can be done for example by loading a solution of the peptide to the head of the column; washing the column by actate buffer to replace and remove TFA or other acids, after completion of washings the peptide is eluted from the column by addition of strong solvent such as acetonitrile to the acetate buffer; The ion- exchange can be done by attaching the peptide to the ion exchange column as a salt of the functional acidic residues of the ion-exchange resin, washing the column to remove TFA or other acids, releasing the peptide by gradient salt concentration increase. The resulting purified product is dried and may be lyophilized.

In still another embodiment of the present invention, all synthetic steps of the above described process are performed under mild conditions providing products containing a low content of by-products and producing a final product in high yield and high purity.

In another embodiment of the present invention provides a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof by the solid phase peptide synthesis (SPPS) using novel resin linkers, wherein said process comprises the steps of: (i) coupling of Fmoc-D-Ala-OH on resin linker in the presence of a base in a solvent to produce Fmoc-D-Ala-NH-linker-resin; (ii) removing of Fmoc from Fmoc-D-Ala-NH-linker-resin of step-(i) in the presence of secondary amine base to produce H- D-Ala-NH-linker-resin; (iii) (iii)sequentially coupling the following Fmoc protected ce-amino acid Fmoc-Pro-OH, Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH with H-D-Ala-NH-linker-resin in the presence of coupling reagent to produce Fmoc-protected deca-peptide fragment attached to the linker-resin; (iv) removing of Fmoc from Fmoc-protected deca-peptide fragment of step (iii), in the presence of a secondary amine base, followed by acetylation using acetylating agent to produce protected deca-peptide peptide fragment attached to the linker-resin; (v) concurrent cleavage and deblocking of the peptide from the resin of step-(iv) using acidic composition to produce crude Degarelix; (vi) purifying and isolating crude Degarelix of step (v) in to pharmaceutically acceptable salts of Degarelix.

The process as summarized below:

wherein, the resin linker used in the above process comprises 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA (XXXIV), 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA (XXXV) and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA (XXXVI), which are depicted by following structural formulae;

In yet another embodiment of the present invention, the reaction conditions for the above said process such as the use of base, solvent, secondary amine base, acetylating agent and coupling reagent are described above.

In yet another embodiment of the present invention, the amino acids introduced in step (iii) of the sequential synthesis described above are commercially available as protected amino acids that are stable to any reactions and modifications of the side chains during the synthesis that could result in derivatives of the constituent amino acids.

Concurrent cleavage and deblocking of the peptide from the resin may be affected by addition of a strong acidic composition. The acidic composition is preferably based on an acidic material such as TFA, and comprises scavenger reagents including, but not limited to, ethanedithiol (EDT), TIS (triisopropylsilane) and water. The relative ratio of acidic material to scavenger to water may be from about 85%> to about 99% acidic material, from about 0.1% to about 15% scavenger, and from about 0.1% to about 15%> water by weight. A preferred acidic composition comprises about 95% TFA, about 2.5% EDT, and about 2.5% water.

The crude peptide product may be purified by any known method. Preferably, the peptide is purified using HPLC on a reverse phase (RP) column. At the end of the purification process or as a part of the purification process the counter ion of the peptide may be exchanged by a suitable ion such as, but are not limited to, acetate ion. The counter-ion exchange can be done by any suitable method such as HPLC or ion exchange. Suitable HPLC method can be done for example by loading a solution of the peptide to the head of the column; washing the column by acetate buffer to replace and remove TFA or other acids, after completion of washings the peptide is eluted from the column by addition of strong solvent such as acetonitrile to the acetate buffer; The ion- exchange can be done by attaching the peptide to the ion exchange column as a salt of the functional acidic residues of the ion-exchange resin, washing the column to remove TFA or other acids, releasing the peptide by gradient salt concentration increase. The resulting purified product is dried and may be lyophilized.

In still another embodiment of the present invention, all synthetic steps of the above described process are performed under mild conditions providing products containing a low content of by-products and producing a final product in high yield and high purity.

The following examples illustrate the nature of the invention and are provided for illustrative purposes only and should not be construed to limit the scope of the invention.

EXAMPLES:

Example 1: Preparation of Degarelix acetate via (9+1) fragment protocol:

Step-i: 50 ml of dry dichloromethane (DCM) was added to 2-chlorotrityl resin (CTC resin) (6 gm) in a SPPS reactor, and allowed it to swell for 10 min and drained. A solution of Fmoc-Pro-OH (3.3 gm, 1.1 eq) and N,N-diisopropylethylamine (DIEA) (6.3 ml, 4 eq) in dry DCM (50 ml) was added to the above CTC resin and stirred for one to two hours at room temperature and drained. The resin was then capped with methanol (40%) and DIEA (10%) solution in DCM (50%) for 20 min and drained. Thereafter, washed the resin with one bed volume of DMF (2 times), DCM (2 times) and MTBE (2 times) isolated and dried. Yield: 8.8 gm Loading-0.75

Step-ii: The above resin obtained in the step-i was de-blocked with 60 ml of 20% piperidine in DMF for 10 min to 15 min and washed with 40 ml of DMF (2 times), IP A (2 times) and DMF (2 times).

Step-iii: Fmoc-Lys(Ipr,boc)-OH (6.7 gm, 2 eq.) and HOBT (1.8 gm, 2 eq) were dissolved in DMF (50 ml) and cooled to 0-5°C while stirring. N,N'-diisopropylcarbodiimide (DIC) (2 ml, 2 eq) was added and stirred the reaction mixture for 5 min. It was added to the resin in Step-ii and stirred for two to three hours at room temperature. The progress of coupling was monitored by Kaiser Tests. After completion of the reaction the resin was drained and washed with one bed volume of DMF (3 times). The above resin was de-blocked with 60 ml of 20% piperidine in DMF for 10 min and 15 min and washed with one bed volume of DMF (2 times), IPA (2 times) and DMF (2 times). The repeated cycles of operations (amino acid coupling and Fmoc de-protection) were performed for Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH until the desired peptidyl resin was obtained.

Step-iv: Synthesis of Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-CTC resin.

After coupling of the last amino acid (Fmoc-D-2Nal-OH), it was de-blocked with 60 ml of 20% piperidine in DMF for 15 min and drained. It was washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (3 times). The N-terminus of peptide resin was acetylated with acetic anhydride and diisopropyl ethyl amine in DCM. The resin was drained and washed with 50 ml of DMF (2 times) and DCM (2 times). Finally the peptide resin was isolated and dried. Yield 16 g.

Step-v: Synthesis of 9 amino acid peptide (Ac-D-2Nal-D-Phe(4CI)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-OH):

The above peptidyl resin was taken in SPPS reactor and treated with a solution of 0.6% TFA in DCM for 5 min at room temperature and drained. The filtrate was immediately neutralized with DIEA (15% in dichloromethane) under cooling. The above procedure was repeated twice to cleave the peptide from the resin completely. The DCM solution was washed with water (2 times), organic layer was dried and concentrated under reduced pressure. Crude protected peptide was isolated by precipitating with MTBE. Yield 9.8 g.

Step-vi: Preparation of protected Degarelix (Solution phase synthesis)

Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-OH (1.0 eq) was taken in a clean and dry 100 ml round bottom flask containing DMF (30 ml), TBTU ( 1.1 eq) / HOBT (1.0 eq) and DIEA (2.2 eq) were added and stirred the reaction mixtures for 5 min.

H-D-Ala-NH2.HC1 (1.1 eq) was taken in a separate clean and dry 100 ml round bottom flask containing DMF (10 ml) and cooled the solution to 0-5°C, DIEA (1.1 eq) was added to the above solution and stirred the reaction mixtures for 5 min. The reaction mixtures so obtained was added to the above reaction mass and stirred for 3-4 hrs at room temperature. The progress of coupling was monitored by HPLC. After completion of the reaction 100 ml of DM water was added to obtain off-white precipitate. The precipitate was filtered and washed with 50 ml DM water followed by MTBE to give protected Degarelix. Yield 10 g.

Step-vii: Preparation of crude Degarelix

De-blocking of protected Degarelix was performed with a mixture of TFA + Water +TIS (90%+5%+5%) for 2 hrs at room temperature. The crude peptide (Degarelix) was isolated by precipitating with MTBE. Yield 8.5 g. HPLC Purity 85.7 %.

Step-viii: Purification of Degarelix

Crude Degarelix was purified by reverse phase C-18 HPLC using 0.1% aqueous triflouroacetic acid (as buffer a) and 100% acetonitrile (as buffer b). The fractions containing pure Degarelix triflouroacetate were pooled; the organic modifier was removed under reduced pressure. Desalting was performed with 18% ethanol in aqueous acetic acid (1%). The fractions containing pure Degarelix acetate were pooled; the organic modifier was removed under reduced pressure. The resulting peptide solution was freeze-dried to isolate white fluffy material as Degarelix acetate. Yield 2.5 g. HPLC Purity 98.5%.

EXAMPLE 2: Synthesis of Degarelix acetate by solid phase synthesis using a new linker resin namely 4-(2-(aminomethyl)-5-methoxyphenoxy)butyramide resin

Step-i: Synthesis of 4-(2-(aminomethyl)-5-methoxyphenoxy)butyramide resin MBHA resin (6 gm) was taken in a SPPS reactor, 50 ml of DCM was added and allowed it to swell for 20 min and drained. Fmoc-4-(2-(aminomethyl)-5-methoxyphenoxy)butyric acid (3.7 gm, 1.5 eq), TBTU (2.6 gm, 1.5 eq) / HOBT (1.2 gm, 1.5 eq) were dissolved in DMF ( 20 ml). DIEA (3 ml, 3 eq) was added and stirred the reaction mixtures for 5 min. The reaction mixtures so obtained was added to the resin and stirred for 3 hrs at room temperature. The progress of the coupling was monitored by Chloranil and Kaiser Tests. After completion of the reaction, the resin was drained and washed with one bed volume of DMF (3 times). The resin was then capped with acetic anhydride and DIEA solution in DCM for 20 min and drained. The resin was washed with one bed volume of DMF (2 times), DCM (2 times) and MTBE (2 times). It was isolated and dried.

Yield: 8.5gm, Loading ~ 0.72

Step-ii: The above resin obtained in the step-I was de-blocked with 60 ml of 20% piperidine in DMF for 10 min to 15 min and washed with 40 ml of DMF (2 times), IP A (2 times) and DMF (2 times).

Step-iii: Synthesis of Fmoc-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-AMBA linker-MBHA resin.

Fmoc-D-Ala-OH (3.7 gm, 2eq.) and HOBT (1.6 gm, 2eq) were dissolved in 20 ml of DMF and cooled to 0-5°C while stirring. DIC (1.8 ml, 2 eq) was added and stirred for 5 min. The reaction mixture so obtained was added to the above resin and stirred for three hours at room temperature. It was drained and washed with one bed volume of DMF (3 times). The coupling was monitored by Kaiser Test. The above resin was de-blocked with 60 ml of 20% piperidine in DMF first for 10 min and then 15 min and drained. It was washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (3 times).

The repeated cycles of operations (amino acid coupling and Fmoc de-protection) were performed for Fmoc-Pro-OH, Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH to obtain the desired peptidyl resin.

Step-iv: Synthesis of Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph (Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-AMBA linker-MBHA resin.

After coupling of the last amino acid (Fmoc-D-2Nal-OH), it was de-blocked with 60 ml of 20% piperidine in DMF for 15 min and drained. It was washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (3 times). The N-terminus of peptide resin was acetylated with acetic anhydride and diisopropyl ethyl amine in DCM. The resin was drained and washed with 50 ml of DMF (2 times) and DCM (2 times). Finally the peptide resin was isolated and dried. Yield 19.2 g.

Step-v: Preparation of crude Degarelix

Cleavage and de-blocking of peptide was performed with a mixture of TFA + Water + TIS (90%+5%+5%) for 6 hrs at room temp. The crude peptide (Degarelix) was isolated by precipitating with MTBE. Yield 12 g. HPLC Purity 82.4%.

Step-v: Purification of Degarelix

Crude Degarelix was purified by reverse phase C-18 HPLC using 0.1% aqueous triflouroacetic acid (as buffer a) and 100%> acetonitrile (as buffer b). The fractions containing pure Degarelix triflouroacetate were pooled; the organic modifier was removed under reduced pressure. Desalting was performed with 18% acetonitrile in aqueous acetic acid (1%)). The fractions containing pure Degarelix acetate were pooled; the organic modifier was removed under reduced pressure. The resulting peptide solution was freeze-dried to isolate white fluffy material as Degarelix acetate. Yield 3.2 g. HPLC Purity 99.44 %.

EXAMPLE 3: Synthesis of Degarelix acetate by solid phase synthesis using a new linker resin namely 4-(4-(aminomethyl)-3-methoxyphenoxy)butyramide resin

Step-i: Synthesis of 4-(4-(aminomethyl)-3-methoxyphenoxy)butyramide resin

MBHA resin (6 gm) was taken in a SPPS reactor, 50 ml of DCM was added and allowed it to swell for 20 min and drained. Fmoc protected 4-(4-(aminomethyl)-3-methoxyphenoxy)butyric acid (3.7 gm, 1.5 eq) TBTU (2.6 gm, 1.5 eq) / HOBT (1.2 gm, 1.5 eq) were dissolved in DMF (20 ml). DIEA (3 ml, 3 eq) was added and stirred the reaction mixtures for 5 min. The reaction mixture so obtained was added to the resin and stirred for 3 hrs at room temperature. The progress of coupling was monitored by Chloranil and Kaiser Tests. After completion of the reaction the resin was drained and washed with one bed volume of DMF (3 times). The resin was then capped with acetic anhydride and DIEA solution in DCM for 20 min and drained. The resin was washed with one bed volume of DMF (2 times), DCM (2 times) and MTBE (2 times). It was isolated and dried. Yield: 8.6gm Loading ~ 0.74

Step-ii: The above resin obtained in the step-i was de-blocked with 60 ml of 20% piperidine in DMF for 10 min to 15 min and washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (2 times).

Step-iii: Synthesis of Fmoc-D-2NaI-D-Phe(4CI)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph (Cbm)-Leu-Lys (Ipr,Boc)-Pro-D-Ala-NH-AMPA linker-MBHA resin.

Fmoc-D-Ala-OH (3.9 gm, 2eq.) and HOBT (1.7 gm, 2 eq) were dissolved in 20 ml of DMF and cooled to 0-5°C while stirring, DIC (2 ml, 2 eq) was added and stirred for 5 min, The reaction mixture so obtained was added to the resin of step-ii and kept for two to three hours at room temperature and washed with one bed volume of DMF (3 times). The coupling was monitored by Kaiser Test. The above resin was de-blocked with 60 ml of 20% piperidine in DMF for 10 min 15 min and thereafter the resin was drained and washed with 40ml of DMF (2 times), IPA (2 times) and DMF (3 times).

The repeated cycles of operation (amino acid coupling and Fmoc de-protection) were performed for Fmoc-Pro-OH, Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH to obtain desired peptidyl resin.

Step-iv: Synthesis of Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-AMPA linker-MBHA resin.

After coupling of the last amino acid (Fmoc-D-2Nal-OH), it was de-blocked with 60 ml of 20% piperidine in DMF for 10 to 15 min and drained .It was washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (3 times). The N-terminus of peptide resin was acetylated with acetic anhydride and diisopropyl ethyl amine in DCM. The resin was drained and washed with 50 ml of DMF (2 times) and DCM (2 times). Finally the peptide resin was isolated and dried

Step-v: Preparation of crude Degarelix

Cleavage and de-blocking of peptide was performed with a mixture of TFA + Water + TIS (90%+5%+5%) for 2.5 hrs at room temperature. The crude peptide (Degarelix) was isolated by precipitating with MTBE.

Step-vi: Purification of Degarelix

Crude Degarelix was purified by reverse phase C-18 HPLC using 0.1% aqueous triflouroacetic acid (as buffer a) and 100% acetonitrile (as buffer b). The fractions containing pure Degarelix triflouroacetate were pooled; the organic modifier was removed under reduced pressure. Desalting was performed with 18% acetonitrile in aqueous acetic acid (1%). The fractions containing pure Degarelix acetate were pooled; the organic modifier was removed under reduced pressure. The resulting peptide solution was freeze-dried to isolate white fluffy material as Degarelix acetate.

WE CLAIM:

1. A process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof which comprises the steps of:

(i) loading of Fmoc-Pro-OH on a resin in the presence of base and solvent to produce Fmoc-Pro-resin;

(ii) removing the Fmoc protecting group from Fmoc-Pro-resin of step (i) to produce H-Pro-resin;

(iii) sequentially coupling the following Fmoc protected o-amino acid Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH with H-Pro-resin in the presence of coupling reagent to produce Fmoc-protected peptide fragment of formula; Fmoc-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro—Q

(iv) removing of Fmoc protecting group from the protected peptidoresin of step (iii), followed by acetylation to produce protected nine amino acid peptide resin of formula; Ac-D-2Nal-D-Phe(4Cl)-D-3PaI-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro—Q

(v) deprotecting the resin from the peptide of step (iv) using mild acidic condition to produce protected nine amino acid peptide of formula; Ac-D-2NaI-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr, Boc)-Pro-OH [Protected 9 amino acid peptide]

(vi) coupling of protected nine amino acid peptide of step (v) with D-alaninamide (D-Ala-NH2) in the presence of coupling reagent to produce protected Degarelix of formula; Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH2

[Protected Degarelix]

(vii) de-protection of protected Degarelix using acidic composition to produce Degarelix; (viii) optionally purifying the Degarelix of step (vii); and (ix) isolating Degarelix or its pharmaceutically acceptable salt thereof.

2. The process according to claim 1, wherein the resin is selected from chlorotrityl resin, Rink acid resin, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.

3. The process according to claim 1, wherein the base is selected from the reagent comprising diisopropylamine, N,N-diisopropylethylamine triethylamine, dimethylamine, trimethyl amine, pyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and/or mixtures thereof and the solvent is selected from the reagent comprising dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), methanol, isopropanol, dichloroethane, 1,4-dioxane, tetrahydrofuran (THF), ethyl acetate, acetonitrile, acetone, and/or mixtures thereof.

4. The process according to claim 1, wherein the sequential coupling of Fmoc protected a-amino acid in step (iii) is carried out by coupling the first amino acid with H-Pro-resin followed by removing Fmoc protecting of resultant amino acid and repeating the cycles for the addition of subsequent amino acids.

5. The process according to any of the proceeding claims, wherein the removal of Fmoc protection is carried out using a secondary amine base, comprises, piperidine, dimethylamine, diethylamine, diphenylamine or mixtures thereof.

6. The process according to any of the proceeding claims, coupling reagent comprises 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) or diisopropylcarbodiimide (DIC) or o-(7-azabenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate (HATU) or o-(benzotriazol-l-O)-!,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) or benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

7. The process according to claim 1, wherein the acetylation in step (iv) is carried out using an acetylating agent comprises acetic anhydride, acetyl chloride or its mixtures thereof.

8. The process according to claim 1, wherein the resin is deprotected using a mild acidic solution comprising about 0.1% to about 5% of TFA in an organic inert solvent or a mixture of acetic acid with trifluoroethanol and dichloromethane (DCM).

9. The process according to claim 1, wherein deprotection of protecting groups in protected Degarelix is carried out using an acidic solution comprising TFA in water, which may optionally contains scavengers selected from triisopropylsilane (TIS) and ethanedithiol (EDT).

10. A process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof by the solid phase peptide synthesis (SPPS) using novel resin linkers, wherein said process comprises the steps of:

(i) coupling of Fmoc-D-Ala-OH on resin linker to produce Fmoc-D-Ala-NH-linker-resin;

(ii) removing of Fmoc from Fmoc-D-Ala-NH-linker-resin of step-(i) to produce H-D-Ala-NH-linker-resin;

(iii) sequentially coupling the following Fmoc protected o-amino acid Fmoc-Pro-OH, Fmoc-Lys(Ipr,Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4-Aph(Cbm)-OH, Fmoc-4-Aph(Hor)-OH, Fmoc-Ser(tbu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH and Fmoc-D-2Nal-OH with H-D-Ala-NH-linker-resin in the presence of coupling reagent to produce Fmoc-protected deca-peptide fragment attached to the linker-resin of formula;

(iv) removing of Fmoc from Fmoc-protected deca-peptide fragment of step (iii), followed by acetylation using acetylating agent to produce protected deca-peptide peptide fragment attached to the linker-resin of formula; Ac-D-2Nal-D-Phe(4CI)-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-linker—O

(v) concurrent cleavage and deblocking of the peptide from the resin of step-(iv) using acidic composition to produce crude Degarelix;

(vi) purifying and isolating crude Degarelix of step (v) in to pharmaceutically acceptable salts of Degarelix, wherein the linker resin is selected from 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.