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-[(aminocarbony])amino]-D-phenylalanyl-L-leucyl-A?5-(l-methylethy])-L-]ysyl-L-proly].
The structural formula of Degarelix is:

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

Lys(iPr)8,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 methylbenzhydrylamine resin (MBHA resin) using diisopropylcarbodiimide (DIC) and 1-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:

After, 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). Further coupling with N-alpha-Boc-L-4-(Fmoc-amino)phenylalanine (XIV), followed by Fmoc deprotection with piped dine 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.

US 2013/0281662 by Ferring discloses a process for the preparation of Degarelix using liquid phase peptide synthesis. The process comprising the coupling of tripeptide (XXXII) with heptapeptide (XXXIII) to produce protected Degarelix (XXXI), which was de-protected using piperidine to produce Degarelix.

The process of 3+7 coupling as shown in Scheme-V below:

The main drawback of the above process is the cost of reagents used during fragment coupling prohibits manufacturing of Degarelix on large scale. Hence, there is a need for a method of manufacturing of Degarelix a more efficiently and economically. Hence it was felt by the present inventors to overcome the above 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.

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 a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof using liquid phase peptide synthesis (LPPS) by azide method proceeds via N-terminal tripeptide hydrazide with C-terminal heptapeptide (3+7 fragment protocol).

In another embodiment, the present invention relates to a process for the preparation of N-terminal tripeptide hydrazide by liquid phase peptide synthesis (LPPS), and its use in the preparation of Degarelix or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates a process for the preparation of C-terminal heptapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy and its use in the synthesis of Degarelix or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a process for the preparation of Degarelix or a pharmaceutically acceptable salt thereof using liquid phase peptide synthesis (LPPS) by azide method proceeds via the coupling of N-terminal dipeptide hydrazide with C-terminal octapeptide (2+8 fragment protocol).

In another embodiment, the present invention relates to a process for the preparation of N-terminal dipeptide hydrazide by liquid phase peptide synthesis (LPPS), and its use in the preparation of Degarelix or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to a process for the preparation of C-terminal octapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy and its use in the synthesis of Degarelix or a pharmaceutically acceptable salt thereof.

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 using liquid phase peptide synthesis (LPPS) by azide method proceeds via the coupling of N-terminal tripeptide hydrazide with C-terminal heptapeptide (3+7 fragment protocol), wherein said process comprising the steps of:

(i) coupling of N-terminal tripeptide hydrazide with C-terminal heptapeptide in the presence of an alkyl nitrite in a solvent and a base to produce protected Degarelix;

(ii) de-protection of protected Degarelix using acidic composition to produce crude Degarelix; and

(iii) Purifying and isolating the crude Degarelix of step-ii to produce Degarelix or 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 leusine, 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.

In another embodiment of the present invention, the coupling reaction is performed in an organic solution where the two peptides, a reagent and an organic amine base are dissolved therein.

In another embodiment of the present invention, the alkyl nitrite reagent used in the coupling of N-terminal peptide hydrazide with C-terminal peptide comprises tert-butyl nitrite, isoamyl nitrite, sodium nitrite in acidic conditions or mixtures thereof.

In another embodiment of the present invention, the base is organic amine base, which comprises N-methylmorpholine (NMM), diisopropylamine, N,N-diisopropylethylamine (DIPEA), triethylamine, dimethylamine, trimethyl amine, pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and/or mixtures thereof. 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.

In another embodiment of the present invention, the coupling reaction is carried out at a temperature range of -40°C to reflux temperature, based on the solvent or mixture of solvents used for the reaction.

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 maybe 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 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 comprises 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 another embodiment, the present invention relates to a process for the preparation of N-terminal tripeptide hydrazide by liquid phase peptide synthesis (LPPS), and its use in the preparation of Degarelix or a pharmaceutically acceptable salt thereof, wherein said process comprising the steps of:

(i) coupling of BOC-D-Phe(4Cl)-OH with H-D-3Pal-OMe.2HCl in the presence of a coupling reagent and a base to produce BOC-D-Phe(4Cl)-D-3Pal-OMe;

(ii) deprotection of BOC protecting group from BOC-D-Phe(4Cl)-D-3Pal-OMe of step-i using mild or strong acidic condition to produce H-D-Phe(4Cl)-D-3Pal-OMe.TFA;

(iii) D-Phe(4Cl)-D-3Pal-OMe.TFA of step-ii is coupled with Ac-D-2Nal-OH in the presence of a coupling reagent to produce Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe;

(iv) Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe of step-iii is reacted with hydrazine hydrate in a solvent to produce N-terminal tripeptide hydrazide (Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-N2H3); and

(v) (v) optionally converting the N-terminal tripeptide hydrazide to Degarelix or a pharmaceutically acceptable salt thereof.
(vi)

The process as summarized below:

In another embodiment of the present invention, the coupling reagent used in the above processcomprises o-(7-azabenzotriazol-l -yl)-l ,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), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), 1,3 dicyclohexylcarbodiimide (DCC) or 1,3-diisopropyl-carbodiimide (DIC) and like.

In another embodiment of the present invention, the peptide coupling additive comprises 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-benzotriazine (HOOBt), 1-hydroxy-1 H-benzotriazole (HOBt), 6-chloro-HOBt orl-hydroxy-7-azabenzotriazole (HOAt).

In yet another embodiment of the present invention, the reaction conditions for the above said process such as the use of base, solvent are described above.

In yet another embodiment of the present invention, the mild or strong acidic solution used in the above process comprises different concentrations of TFA. The mild acidic solution may be, for example, 1 % TFA in dichloromethane. The strong acidic solution may be, for example, 95% TFA in 5% water.

In another embodiment, the present invention relates to a process for the preparation of N-terminal tripeptide hydrazide intermediate of Degarelix by liquid phase peptide synthesis (LPPS), and its use in the synthesis of Degarelix or a pharmaceutically acceptable salt thereof, wherein said process comprising the steps of:
(i) coupling of BOC-D-Phe(4Cl)-OH with H-D-3Pal-OMe.2HCl in the presence of a coupling reagent and a base to produce BOC-D-Phe(4Cl)-D-3Pal-OMe;

(ii) deprotection of BOC protecting group from BOC-D-Phe(4Cl)-D-3Pal-OMe of step-i using mild or strong acidic conditions to produce D-Phe(4Cl)-D-3Pal-OMe.TFA;

(iii) D-Phe(4Cl)-D-3Pal-OMe.TFA of step-ii is coupled with Z-D-2Nal-OH, in the presence of a coupling agent, wherein Z is a protecting group that can be removed by hydrogenation, reacted with Pd/C in a solvent, followed by acetylation to produce Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe;

(iv) Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe of step-iii is reacted with hydrazine hydrate in a solvent to produce N-terminal tripeptide hydrazide (Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-N2H3); and

(v) optionally converting the N-terminal tripeptide hydrazide to Degarelix or a pharmaceutically acceptable salt thereof.

The process as summarized below:

In another embodiment of the present invention, the reaction conditions for the above said process such as the use of coupling reagent, peptide coupling additive, base and solvent mild or strong acidic conditions are described above.

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 another embodiment, the present invention relates to a process for the preparation of C-terminal heptapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy and its use in the synthesis of Degarelix or a pharmaceutically acceptable salt thereof, wherein the said process comprising the steps of:

(i) coupling of Fmoc-D-Ala-OH with a resin in the presence of a coupling reagent and a base in a solvent to produce Fmoc-D-Ala-NH-resin;

(ii) de-protection of Fmoc-D-Ala-NH-resin of step-i in the presence of a base in a solvent to produce H-D-Ala-NH-resin;

(iii) sequential couplings of the desired amino acids in the presence of a coupling reagent, and a base to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin;

(iv) de-protection of Boc and cleavage of resin from Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin of step-(iii) using mild orstrong acidic conditions to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2;

(v) Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2 of step-(iv) is treated with Boc-anhydride (BOC2O) in the presence of a base and a solvent to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu- Lys(Ipr,boc)-Pro-D-Ala-NH2;

(vi) removal of Fmoc from the Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu- Lys(Ipr,boc)-Pro-D-Ala-NH2 of step-(v) using a base in a solvent to produce heptapeptide; and

(vii) optionally, converting the heptapeptide to Degarelix or a pharmaceutically acceptable salt thereof.

The process as summarized below:

In another embodiment of the present invention, Suitable resins for use in the process comprises, Rink amide methylbenzhydrylamine (MBHA) resin or Rink amide aminomethyl (AM) resin.

In another embodiment of the present invention, the reaction conditions for the above said process such as the use of coupling reagent, peptide coupling additive, base, solvent and mild or strong acidic conditions are described above.

In another embodiment, the present invention relates to a process for the preparation of Degarelix or a pharmaceuticaliy acceptable salt thereof using liquid phase peptide synthesis (LPPS) by azide method proceed via the coupling of N-terminal dipeptide hydrazide with C-terminal octapeptide, wherein the said process comprising the steps of: (i) coupling of N-terminal dipeptide hydrazide with C-terminal octapeptide in the presence of an alkyl nitrile in a solvent and a base to produce protected Degarelix; (ii) de-protection of protected Degarelix to produce crude Degarelix; and (iii) purifying and isolating the crude Degarelix of step (ii) in to pharmaceuticaliy acceptable salts of Degarelix.
The process as summarized below:

In another embodiment of the present invention, the coupling reaction is performed in an organic solution where the two peptides, and a reagent and an organic amine base are dissolved therein.

In another embodiment of the present invention, the reaction conditions for the above said process such as the use of alkyl nitrite reagent, base and solvent are described above.

Cleavage of the protecting groups from the peptide may be affected by addition of a strong acidic composition. The acidic composition is described above.

The crude peptide product may be purified by any known method, which is described above.

In another embodiment, the present invention relates to a process for the preparation of N-terminal dipeptide hydrazide by liquid phase peptide synthesis (LPPS), and its use in the preparation of Degarelix or a pharmaceutically acceptable salt thereof, wherein said process comprising the'step's of:

(i) coupling of Ac-D-2Nal with H-D-Phe(4Cl)-OMe. HC1 in the presence of acoupling reagent and a base to produce Ac-D-2Nal-D-Phe(4Cl)-OMe;

(ii) Ac-D-2Nal-D-Phe(4Cl)-OMe of step-i is reacted with hydrazine hydrate in a solvent to produce N-terminal dipeptide hydrazide (Ac-D-2Nal-D-Phe(4Cl)-N2H3); and

(iii) optionally converting the N-terminal dipeptide hydrazide to Degarelix or pharmaceutically acceptable salt thereof.

The process as summarized below:

In another embodiment of the present invention, the reaction conditions for the above said process such as the use of coupling reagent, peptide coupling additive, base and solvent are described above.

In another embodiment, the present invention relates to a process for the preparation of C-terminal octapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy and its use in the synthesis of Degarelix or a pharmaceutically acceptable salt thereof, wherein the said process comprising the steps of:

(i) coupling of Fmoc-D-Ala-OH with a resin in the presence of a coupling reagent and a base in a solvent to produce Fmoc-D-Ala-NH-resin;

(ii) de-protection of Fmoc-D-Ala-NH-resin of step-i using a base in a solvent to produce H-D-Ala-NH-resin;

(iii) sequential couplings of the desired amino acids in the presence of a coupling reagents, a solvent to produce Fmoc-P-3Pal-Ser(tbu)-4Aph(Hor)-D- 4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin;

(iv) de-protection of Boc and cleavage of resin from Fmoc-D-3Pal-Ser(tbu)- 4Aph(Hor)- -4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin of step-(iii) using mild or strong acidic conditions to produce Fmoc-D-3Pal-Ser(tbu)- 4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2;

(v) Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala- NH2 of step-(iv) is treated with protecting agent to produce Fmoc-D-3Pal- Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2;

(vi) removal of Fmoc from the Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)- Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2 of step-(v) using a base in a solvent to produce octapeptide; and

(vii) optionally converting the octapeptide to Degarelix or a pharmaceutically acceptable salt thereof.

The process as summarized below:

In another embodiment of the present invention, Suitable resins for use in the process comprises, Rink amide methylbenzhydrylamine (MBHA) resin, Rink amide aminomethyl (AM) resin.

In another embodiment of the present invention, the reaction conditions for the above said process such as the use of coupling reagent, peptide coupling additive, base, solvent and mild or strong acidic conditions are described above.

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: Synthesis of Degarelix acetate using azide method proceeding via 3+7 fragment protocol:

Step-i: Synthesis of protected Degarelix

Ac-D-2Nal-D-Phe (4C1)-D- 3Pal-N2H3 (1.0 eq) was taken in a clean and dry 100 ml round bottom flask containing DMF (40 ml) cooled to -15 to -20°C, HCl in ethyl acetate was added (pH) followed by tert butyl nitrite (l.leq) and stirred for 40 min and then DIEA was added to adjust pH to 6.5.

A solution of H-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2 was added to the above reaction mixture and stirred for 48 hrs. The progress of coupling was monitored by HPLC. After completion of the reaction 100 ml of MTBE was added to obtain off-white precipitate as protected Degarelix.

Step-ii: 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.

Step-iii: 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.

Example 1A: Synthesis of hydrazide of N-terminal tripeptide (Ac-D-2Nal-D-Phe (4C1)-D- 3Pal-N2H3):

Step-i: Synthesis of BOC-D-Phe (4Cl)-D-3Pal-OMe:

Boc-D-Phe(4Cl)-OH (1.0 eq) was taken in a clean and dry 100 ml round bottom flask containing DMF (20 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-3-Pal-OMe.2HCl (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 solution and stirred the reaction mixtures for 5 min. The reaction mixtures so obtained was added to above reaction mass and stirred for 1-2 hrs at room temperature. The progress of coupling was monitored by TLC. 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 n-hexane to give Boc-D-Phe(4Cl)-D- 3Pal-OMe.

Step-ii: Synthesis of H-D-Phe(4Cl)-D-3Pal-OMe. TFABoc-D-Phe(4Cl)-D-3Pal-OMe was taken in a clean and dry 100 ml round bottom flask containing 30% TFA in DCM and stirred the reaction mixtures for 30 min. The progress of de-protection was monitored by TLC. After completion of the reaction, evaporate the solvent under reduced pressure, H-D-Phe(4Cl)-D-3Pal-OMe.TFA was isolated by precipitating with MTBE.

Step-iii: Synthesis of Ac-D-2NaI-D-Phe (4CI)-D- 3Pal-N2H3

Ac-D-2Nal-OH (1.0 eq) was taken in a clean and dry 100 ml round bottom flask containing DMF (20 ml), TBTU (1.1 eq) / HOBT (1.0 eq) and DIEA (2.2 eq) were added and stirred the reaction mixture for 5 min.

H-D-Phe(4Cl)-D-3Pal-OMe.TFA (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 solution and stirred the reaction mixtures for 5 min. The reaction a mixture so obtained was added to the above reaction mass (Ac-D-2Nal-OH solution) and stirred for 1 -2 hrs at room temperature. The progress of coupling was monitored by TLC 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 Ac-D-2Nal-D-Phe (4C1)-D- 3Pal-OMe.

Ac-D-2Nal-D-Phe (4Cl)-D-3Pal-OMe was suspended in methanol, hydrazine hydrate was added and stirred for overnight, filtered and washed with methanol, and isopropyl ether, compound was dried under high vacuum to obtain Ac-D-2Nal-D-Phe(4Cl)-D- 3Pal-N2H3.

Example IB: Synthesis of C-terminal heptapeptide (H-Ser(tbu)-4Aph(Hor)-D-4Aph (Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH2):

Step-i: Rink amide MBHA resin (5 gm) was taken in a SPPS reactor, 50ml of DCM was added and allowed it to swell for 20 min and thereafter drained. The Fmoc group of above resin was de-blocked with 60 ml of 20% piperidine in DMF for 15 min and drained and washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (2 times).

Fmoc-D-Ala-OH (2 eq) and HOBT (2 eq) were dissolved in DMF (20 ml) and cooled to 0-5°C while stirring. DIC (2 eq) was added and stirred for 5 min. The reaction mixture so obtained was added to the above resin and. stirred for two to three hours at room temperature. The progress of the 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 resin was then capped with acetic anhydride and DIE A 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.

Step-ii: The above resin obtained in the step-i was de-blocked with 60 ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 200 ml of DMF (2 times), IPA (2 times) and DMF (3 times).

Step-iii: 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, and Fmoc-Ser(tbu)-OH to obtain Fmoc-Ser (tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-Resin. Selective cleavage of rink amide MBHA resin from the peptide was performed with a mixture of 3% TFA in DCM at 0-5°C.

Step-iv: The above peptidyl obtained in the step-iii was taken in SPPS reactor and treated with a solution of 3% TFA in DCM for 5 min at room temp and drained. The filtrate was immediately neutralized with DIEA (15% in methanol) 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 and precipitating with MTBE to obtain Fmoc-Ser(tbu)-4Aph (Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2.

Step-v & Step-vi: The peptide obtained in the step-iv is reacted with Boc-anhydride in the presence of catalytic amount of DMAP in DCM, followed by deblocking of Fmoc group with diethylamine in DCM to obtain Heptapeptide.

Example 2: Synthesis of Degarelix acetate using azide method proceeding via 2+8 fragment protocol:

Step-i: Synthesis of protected Degarelix

Ac-D-2Nal-D-Phe(4Cl)-N2H3 (1.0 eq) was taken in a clean and dry 100 ml round bottom flask containing DMF (40 ml) and cooled to -15 to -20°C, HC1 in ethyl acetate was added (pH ) followed by tert butyl nitrite (1.1 eq) and stirred for 40 min then DIEA was added to adjust pH to 6.5.

A solution of H-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH2 was added to the above reaction mixture and stirred for 48 hrs. The progress of coupling was monitored by HPLC. After completion of the reaction 100 ml of MTBE was added to obtain off-white precipitate as protected Degarelix.

Step-ii: 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.

Step-iii: 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.

Example 2A: Synthesis of hydrazide of N-terminal dipeptide (Ac-D-2NaI-D-Phe(4Cl)-N2H3)

Step-i: Synthesis of Ac-D-2Nal-D-Phe(4Cl)-OMe

Ac-D-2Nal-OH (l.Oeq) was taken in a clean and dry 100 ml round bottom flask containing DMF (20 ml), TBTU (1.1 eq) / HOBT (1.0 eq) and DIEA (2.2 eq) were added and stirred the reaction mixture for 5 min.

H-D-Phe(4Cl)-OMe .HO (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 solution and stirred the reaction mixture for 5 min. The reaction mixture so obtained was added to the above reaction mixture (Ac-D-2Nal-OH solution) and stirred for 1-2 hrs at room temperature. The progress of coupling was monitored by TLC 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 Ac-D-2Nal-D-Phe (4Cl)-OMe.

Step-ii: Synthesis of Ac-D-2NaI-D-Phe(4Ci)-N2H3

Ac-D-2Nal-D-Phe(4Cl)-OMe was suspended in methanol, hydrazine hydrate was added and stirred for overnight, filtered and washed with methanol, and isopropyl ether, compound was dried under high vacuum to obtain Ac-D-2Nal-D-Phe(4Cl)-N2H3.

Example 2B: Synthesis of C-terminal Octapeptide (H-D-3Pal-Ser(tbu)-4Aph (Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2)

Step-i: Rink amide MBHA resin (5 gm) was taken in a SPPS reactor, 50 ml of DCM was added and allowed it to swell for 20 min and thereafter drained. The Fmoc group of above resin was de-blocked with 50 ml of 20% piperidine in DMF for 15 min and drained and washed with 40 ml of DMF (2 times), IPA (2 times) and DMF (2 times).

Fmoc-D-Ala-OH (2 eq.) and HOBT (2 eq) were dissolved in DMF (20 ml) and cooled to 0-5°C, while stirring DIC (2 eq) was added and stirred for 5 min. The reaction mixture so obtained was added to the resin and stirred for two to three hours at room temperature. The progress of the 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 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.

Step-ii: The above resin obtained in the step-i was de-blocked with 60 ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 200 ml of DMF (2 times), IPA (2 times) and DMF (3 times).

Step-iii: 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 and Fmoc-D-3Pal-OH to obtain Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,Boc)-Pro-D-Ala-NH-resin. Selective cleavage of rink amide MBHA resin from the peptide was performed with a mixture of 3% TFA in dichloromethane at 0-5°C.

Step-iv: The above peptidyl resin obtained in the step-iii was taken in SPPS reactor and treated with a solution of 3% TFA in DCM for 5 min at room temp and drained. The filtrate was immediately neutralized with DIEA (15% in methanol) 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 and precipitating with MTBE to obtain Fmoc-D-3Pal-Ser (tbu)-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (Ipr)-Pro-D-Ala-NH2.

Step-v & Step-vi: The peptide obtained in the step-iv was reacted with Boc-anhydride in presence of catalytic amount of DMAP in DCM, followed by de-blocking of Fmoc group with diethylamine in DCM to obtain Octapeptide.

WE CLAIM:

1. A process for the preparation of Degarelix or a pharmaceutical^ acceptable salt thereof using liquid phase peptide synthesis (LPPS) by azide method proceeds via the coupling of suitable N-terminal peptide hydrazide with suitable C-terminal peptide, wherein said process comprising the steps of:

(i) coupling of suitable N-terminal peptide hydrazide is selected from the following formula; in the presence of an alkyl nitrite in a solvent and a base to produce protected Degarelix;

(ii) de-protection of protected Degarelix using acidic composition to produce crude Degarelix;

(iii) optionally purifying the Degarelix of step (ii); and (iv) isolating Degarelix or its pharmaceutically acceptable salt thereof.

2. The process according to claim 1, alkyl nitrite is selected from the reagent comprising, tert-butyl nitrite, isoamyl nitrite, sodium nitrite or mixtures thereof.

3. The process according to claim 1, wherein the base is selected from the reagent comprising N-methylmorpholine (NMM), diisopropylamine, N,N-diisopropylethylamine (DIPEA), triethylamine, dimefhylamine, 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 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).

5. A process for the preparation of N-terminal tripeptide hydrazide by liquid phase peptide synthesis (LPPS), wherein said process comprising the steps of:

(i) coupling of BOC-D-Phe(4Cl)-OH with H-D-3Pal-OMe.2HCl in the presence of a coupling reagent and a base to produce BOC-D-Phe(4Cl)-D-3Pal-OMe;

(ii) deprotection of BOC protecting group from BOC-D-Phe(4Cl)-D-3Pal-OMe of step-i using mild or strong acidic condition to produce H-D-Phe(4CI)-D-3Pal- Me.TFA;

(iii) D-Phe(4Cl)-D-3Pal-OMe.TFA of step-ii is coupled with Ac-D-2Nal-OH in the presence of a coupling reagent to produce Ac-D-2Nal-D-Phe(4Cl)-D-3Pal- OMe;

(iv) Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe of step-iii is reacted with hydrazine hydrate in a solvent to produce N-terminal tripeptide hydrazide (Ac-D-2Nal-D- Phe(4Cl)-D-3Pal-N2H3);

(v) optionally converting the N-terminal tripeptide hydrazide to Degarelix or a pharmaceutically acceptable salt thereof.

6. A process for the preparation of N-terminal tripeptide hydrazide intermediate of Degarelix by liquid phase peptide synthesis (LPPS), wherein said process comprising the steps of:

(i) coupling of BOC-D-Phe(4Cl)-OH with H-D-3Pal-OMe.2HCl in the presence of a coupling reagent and a base to produce BOC-D-Phe(4Cl)-D-3Pal-OMe;

(ii) deprotection of BOC protecting group from BOC-D-Phe(4Cl)-D-3Pal-OMe of step-i using mild or strong acidic conditions to produce D-Phe(4Cl)-D-3Pal-OMe.TFA;

(iii) D-Phe(4Cl)-D-3Pal-OMe.TFA of step-ii is coupled with Z-D-2Nal-OH, in the presence of a coupling agent, wherein Z is a protecting group that can be removed by hydrogenation, reacted with Pd/C in a solvent, followed by acetylation to produce Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe;

(iv) Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-OMe of step-iii is reacted with hydrazine hydrate in a solvent to produce N-terminal tripeptide hydrazide (Ac-D-2Nal-D-Phe(4Cl)-D-3Pal-N2H3); and

(v) optionally converting the N-terminal tripeptide hydrazide to Degarelix or a pharmaceutically acceptable salt thereof.

7. A process for the preparation of C-terminal heptapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy, wherein the said process comprising the steps of:
(i) coupling of Fmoc-D-Ala-OH with a resin in the presence of a coupling reagent and a base in a solvent to produce Fmoc-D-Ala-NH-resin;

(ii) de-protection of Fmoc-D-Ala-NH-resin of step-i in the presence of a base in a solvent to produce H-D-Ala-NH-resin;

(iii) sequential couplings of the desired amino acids in the presence of a coupling reagent and a base to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin;

(iv) de-protection of Boc and cleavage of resin from Fmoc-Ser(tbu)-4Aph(Hor)-D- 4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin of step-(iii) using mild or strong acidic conditions to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)- Leu-Lys(Ipr)-Pro-D-Ala-NH2;

(v)Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2 of step-

(vi)The process as summarized below; is treated with Boc-anhydride (BOC2O) in the presence of a base and a solvent to produce Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu- Lys(Ipr,boc)-Pro-D-Ala-NH2;

(vi) removal of Fmoc from the Fmoc-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2 of step-(v) using a base in a solvent to produce heptapeptide; and

(vii) optionally converting the heptapeptide to Degarelix or a pharmaceutically acceptable salt thereof.

8. A process for the preparation of N-terminal dipeptide hydrazide by liquid phase
peptide synthesis (LPPS), wherein said process comprising the steps of:

(i) coupling of Ac-D-2Nal with H-D-Phe(4Cl)-OMe. HC1 in the presence of a coupling reagent and a base to produce Ac-D-2Nal-D-Phe(4Cl)-OMe;

(ii) Ac-D-2Nal-D-Phe(4Cl)-OMe of step-i is reacted with hydrazine hydrate in a solvent to produce N-terminal dipeptide hydrazide (Ac-D-2Nal-D-Phe(4Cl)- N2H3); and

(iii) optionally converting the N-terminal dipeptide hydrazide to Degarelix or pharmaceutically acceptable salt thereof.

9. A process for the preparation of C-terminal octapeptide, by solid phase peptide synthesis (SPPS) using Fmoc strategy, wherein the said process comprising thesteps of:

(i) coupling of Fmoc-D-Ala-OH with a resin in the presence of a coupling reagent and a base in a solvent to produce Fmoc-D-Ala-NH-resin;

(ii) de-protection of Fmoc-D-Ala-NH-resin of step-i using a base in a solvent to produce H-D-Ala-NH-resin;

(iii) sequential couplings of the desired amino acids in the presence of a couplingreagent in a solvent to produce Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D- 4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin;

(iv) de-protection of Boc and cleavage of resin from Fmoc-D-3Pal-Ser(tbu)- 4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH-resin of step-(iii)using mild or strong acidic conditions to produce Fmoc-D-3Pal-Ser(tbu)- 4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala-NH2;

(v) Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr)-Pro-D-Ala- NH2 of step-(iv) is treated with Boc-anhydride (BOC2O) to produce Fmoc-D- 3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D-Ala-NH2;

(vi) removal of Fmoc from the Fmoc-D-3Pal-Ser(tbu)-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(Ipr,boc)-Pro-D~Ala-NH2 of step-(v) using a base in a solvent to produce octapeptide; and

(vii) optionally converting the octapeptide to Degarelix or a pharmaceutically acceptable salt there of.

10. 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-0)-l, 1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP).