DESC:
FORM 2

THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention – IMPROVED PROCESS FOR PRODUCTION OF DEGARELIX ACETATE

2. Applicant(s)

NAME : OMGENE LIFE SCIENCES PVT. LTD

NATIONALITY: an Indian Company

ADDRESS: 333-334, GIDC Makarpura,
Vadodara- 390 010 Gujarat, India

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION:

The present invention relates to improved manufacturing process for preparing Degarelix Acetate.

BACKGROUND OF INVENTION:

Prostate cancer is a leading cause of morbidity and mortality for men in the industrialised world. Degarelix Acetate is the acetate form of a long-acting, synthetic peptide with gonadotrophin-releasing hormone (GnRH) antagonistic properties.

Degarelix targets and blocks GnRH receptors located on the surfaces of gonadotroph cells in the anterior pituitary, thereby reducing secretion of luteinizing hormone (LH) by pituitary gonadotroph cells and so decreasing testosterone production by interstitial (Leydig) cells in the testes. Degarelix acts by immediate and competitive blockade of GnRH receptors in the pituitary and, like other GnRH antagonists, does not cause an initial stimulation of luteinizing hormone production via the hypothalamic-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare.

Degarelix is chemically designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl -3-(3-pyridinyl)-D-alanyl-L-seryl-L-tyrosyl-N5- (aminocarbonyl)-D-ornithyl-L-leucyl-L-arginyl-L-prolyl, acetate (salt) and is represented by the chemical structure below:

Degarelix can also be represented as:

Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 where Ac is acetyl, 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Ser is serine, 4Aph is 4-aminophenylalanine, Hor is hydroorotyl, Cbm is carbamoyl, Leu is leucine, Lys(iPr) is N6-isopropyllysine, Pro is proline and Ala is alanine.

Degarelix has previously been prepared using Boc-solid phase peptide synthesis (SPPS) methodology as reported in WO 98/46634. WO 97/34923 and WO 99/26964 are International Application Publications which are concerned with liquid phase processes for the preparation of biologically active peptides. WO 99/26964 is particularly concerned with the liquid phase synthesis of decapeptides having activity as GnRH antagonists. WO 99/26964 lists a number of inherent limitations of the SPPS methodology for producing GnRH antagonists including the limited capacity of the resin, the large excess of reagents and amino acids required, as well as the need to protect all reactive side chains such as the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph, the e-i-propyiamino group in Lys (i-Pr). WO 99/26964 describes a liquid phase process which involves first preparing the central peptide fragments of the 5 and 6 positions of a decapeptide with the side chains fully elaborated and then assembling the peptide through a "4-2-4", "3-3-4" or "3-4-3" fragment assembly pattern.

An improved method for synthesis of this decapeptide is disclosed herein.

SUMMARY OF INVENTION:

The present invention is a stepwise, and specific synthetic strategy of N- protected amino acids followed by protection and deprotection to get degarelix acetate.

The general procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions, with resin washes between each step. In the first step, the first amino acid is coupled to the resin. Subsequently, the amine is deprotected, and then coupled with the free acid of the second amino acid. This cycle repeats until the desired sequence has been synthesized.

Deprotection is generally performed with piperidine in DMF at concentration of about 15 to 25% v/v. Such high concentration of piperidine shall lead to larger amounts of industrial waste and is a cost burden. Hence there is a need to improve the procedure. Deprotection according to present invention is achieved by bases such as Pyrrolidine, Ethanolamine, Piperazine, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) or mixture of any of these, preferably Pyrrolidine at a concentration of 0.5% v/v to 8.0% v/v in DMF; most preferably 2.0% v/v. Pyrrolidine, also known as tetrahydropyrrole, is an organic compound with the molecular formula (CH2)4NH. It is a cyclic secondary amine, also classified as a saturated heterocycle.

According to the present aspect of the invention is an improved process for synthesis of degarelix acetate using Fmoc synthesis comprising following steps:
Deprotecting the resin by treating with Pyrrolidine in Dimethylformamide (DMF).
Washing with at least one solvent to remove all soluble compounds from the resin; preferably N,N-Dimethylformamide (DMF) and N-methyl pyrrolidinone (NMP)
Loading of the first Fmoc-protected amino acid;
Repetitive cycles of the following:
Deprotecting to remove the Fmoc group by reacting it with Pyrrolidine in Dimethylformamide (DMF);
Washing with at least one solvent to remove all soluble compounds from the resin; preferably with N,N-Dimethylformamide (DMF) and/or N-methyl pyrrolidinone (NMP)
Coupling of a suitable Fmoc-protected amino acid (according to the required sequence) to the terminal amino group residue attached to the resin using a suitable coupling reagent to form an Fmoc-protected peptide fragment attached to the resin; and
Washing the product of step (iii) with at least one solvent to remove all soluble compounds from the resin;
deprotection of the last amino acid Fmoc-D-Nal and acetylating the N- terminus;
washing and drying the peptide-resin;
cleaving and deprotection of the protected peptide intermediate from the resin using trifluoro acetic acid with/without various scavengers;
isolating the crude Degarelix from the cleavage solution;
purifying the crude Degarelix by a suitable separation method followed by counter-ion exchange to acetate to form a Degarelix acetate solution; and
isolating Degarelix product as Degarelix acetate by drying the Degarelix acetate solution by means of any suitable procedure; preferably lyophilization.

DETAILED DESCRIPTION OF THE INVENTION:

As used herein, the terms below have the meanings indicated.

The singular forms "a," "an," and "the" may refer to plural articles unless specifically stated otherwise.

The term "about," as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term "about" should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.

For the purpose of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are defined below:

ACN: acetonitrile

BOC: tert-butoxycarbonyl

DIPEA: N,N-Diisopropylethylamine

DMF: N,N-Dimethylformamide

ES/MS: Electron Spray Mass Spectrometry

Fmoc: 9-fluorenylmethoxycarbonyl

TBTU: 2-[lH-benzotriazole- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium tetrafluoroborate

HOBt: N-hydroxybenzotriazole

HPLC: High Performance Liquid Chromatography

SPPS: solid phase peptide synthesis

TFA: trifluoroacetic acid

t-Bu: tert-butyl

TIS: Triisopropylsilane

DIPC: N,N-Diisopropylcarbodiimide

EDT: Ethanedithiol

Trt: trityl

MDC: Methylene dichloride

DIPE: Diisopropyl ether

In the first embodiment the present invention provides process for preparation of Degarelix acetate compound of formula I which includes the step of:

Synthesis of the protected peptide was carried out by a stepwise Fmoc SPPS (solid phase peptide synthesis) procedure using Fmoc protected Rink amide AM resin. The resin was first deprotected by removing the Fmoc group by washing with Pyrrolidine/DMF solution.

After washing with DMF, the first amino acid was loaded by a regular coupling procedure in the presence of DIPC/HOBt. After washing of the resin with DMF, the Fmoc protecting group was removed by treatment with Pyrrolidine in DMF. After washing of residual reagents with DMF, the second amino acid (Fmoc-Pro-OH) was introduced to start the second coupling step. Fmoc protected amino acids were activated in situ using TBTU/HOBt or DIPC/HOBt, and subsequently coupled to the resin. After washing of the resin, the Fmoc protecting group on the -amine was removed with Pyrrolidine in DMF. These steps were repeated each time with the next amino acid according to peptide sequence. After deblocking of the last amino acid, the peptide was acetylated on its N-terminus by reaction with acetic anhydride in presence of DIPEA. All amino acids used were Fmoc- protected. At the end of the synthesis the peptide-resin was washed with suitable solvent, followed by MDC and followed by DIPE, and dried under vacuum to obtain the dry peptide-resin. The cleavage and deprotection of peptide is achieved by suitable cleavage solution. The crude degarelix obtained is subjected to column purification followed by salt exchange and lyophilization to give Degarelix acetate, API.

The above process is represented stepwise as shown below:

SCHEME-1

The resin could be, but is not limited to, Rink amide resin, Rink amide AM resin, Rink amide MBHA resin, and the permanently stable protecting group should be compatible with Fmoc strategy. In a preferred embodiment, the resin is Rink amide AM resin.

Typical peptide coupling reagents are one or more of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-Cl), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), iso-butylchloroformate (IBCF), 1,3 dicyclohexylcarbodiimide (DCC), 1,3-diisopropyl-carbodiimide (DIC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCDI), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isopropylchloroformate (IPCF),2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA) and 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU).

A preferred coupling agent is DIPC, which is preferably used in combination with HOBt,

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

Typical organic amine bases are NMM, DIPEA, TEA, and collidine.

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

In one of the embodiment of the present invention of improved process whereby the deprotection is completed in shorter duration when the base is added. Typically, a 0.5%v/v to about 8.0%v/v solution of base (Pyrrolidine) in solvent is used for the deprotection step. Excess base concentration can increase base-catalyzed side reactions affecting the quality of crude.

Deprotection is achieved by base, such as Pyrrolidine. Pyrrolidine, also known as tetrahydropyrrole, is an organic compound with the molecular formula (CH2)4NH. It is a cyclic secondary amine, also classified as a saturated heterocycle. Deprotection is achieved by base, such as Pyrrolidine in range of about 0.5%v/v to about 8.0%v/v in DMF. Deprotection is achieved by base, most preferably Pyrrolidine in DMF and is preferably used in an amount of 1.5%v/v to 4.0%v/v, most preferably 2%v/v in DMF.

Example-1:
Charged 50 g of Rink amide AM resin and 500 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 500 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged solution of 2% of Pyrrolidine in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 32.68 g of Fmoc-D-Ala-OH, 16.08 g of HOBt.H2O and 16.44 mL of DIPC into 500 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 500 mL of solution of 2% Pyrrolidine in DMF into peptide vessel and stirred for 5 min. Removed the solvent by applying vacuum and repeated second deprotection for 10 min. Washed the resin with 4x500 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 12.5 mL of acetic anhydride and 10 mL of DIPEA in 500 mL of DMF into peptide vessel and stirred for 30 min. Removed the solvent by applying vacuum. Washed the resin with 3x500 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 500 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 80g to 90g. (purity by HPLC: 88.04%)

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-2:
Charged 100 g of Rink amide AM resin and 1000 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 1000 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged 1000 mL solution of 2% of Pyrrolidine in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Wash resin with 4x1000 mL of DMF. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 65.36 g of Fmoc-D-Ala-OH, 32.16 g of HOBt.H2O and 32.88 mL of DIPC into 1000 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 1000 mL of solution of 2% Pyrrolidine in DMF into peptide vessel and stirred for 5 min. removed the solvent by applying vacuum and repeated second deprotection for 10 min. washed the resin with 4x1000 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 25.0 mL of acetic anhydride and 20 mL of DIPEA in 1000 mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x1000 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 1000 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 160 to 180 g. (purity by HPLC: 88.24%)

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-3:
Charged 10 g of Rink amide AM resin and 100 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 100 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged 100 mL solution of 3% of DBU in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Wash resin with 4x100 mL of DMF. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 6.54 g of Fmoc-D-Ala-OH, 3.22 g of HOBt.H2O and 3.3 mL of DIPC into 100 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 100 mL of solution of 3% DBU in DMF into peptide vessel and stirred for 5 min. removed the solvent by applying vacuum and repeated second deprotection for 10 min. washed the resin with 4x100 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 2.5 mL of acetic anhydride and 2.0 mL of DIPEA in 100mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x100 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 100 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 16.0 to 18.0 g. (purity by HPLC: 66.15%)

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-4:
Charged 0.5 g of Rink amide AM resin and 5.0 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 5.0 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged solution of 5% Piperazine, 1% DBU and 1% formic acid in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 0.32 g of Fmoc-D-Ala-OH, 0.16 g of HOBt.H2O and 0.16 mL of DIPC into 5.0 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 5.0 mL of solution of 5% Piperazine, 1% DBU and 1% formic acid in DMF into peptide vessel and stirred for 5 min. Removed the solvent by applying vacuum and repeated second deprotection for 10 min. Washed the resin with 4x5.0 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 0.12 mL of acetic anhydride and 0.1 mL of DIPEA in 5.0 mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x5.0 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 5.0 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 0.8g to 0.9g. (Purity by HPLC:74.27%)
Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-5:
Charged 0.5 g of Rink amide AM resin and 5.0 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 5.0 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged solution of 5% ethanolamine in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 0.32 g of Fmoc-D-Ala-OH, 0.16 g of HOBt.H2O and 0.16 mL of DIPC into 5.0 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 5.0 mL of solution of 5% ethanolamine in DMF into peptide vessel and stirred for 5 min. Removed the solvent by applying vacuum and repeated second deprotection for 10 min. Washed the resin with 4x5.0 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 0.12 mL of acetic anhydride and 0.1 mL of DIPEA in 5.0 mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x5.0 mL of DMF and tested resin bead for coupling.
Cleavage of Degarelix form Resin: Charged 5.0 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 0.8g to 0.9g. (Purity by HPLC:60.79%)

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-6:
Charged 40 g of Rink amide AM resin and 400 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 400 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged 400 mL solution of 20% of Piperidine in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Wash resin with 4x400 mL of DMF. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 26.16 g of Fmoc-D-Ala-OH, 12.88 g of HOBt.H2O and 13.2 mL of DIPC into 400 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.

Deprotection of Amino acid: Charged 400 mL of solution of 20% Piperidine in DMF into peptide vessel and stirred for 5 min. removed the solvent by applying vacuum and repeated second deprotection for 10 min. washed the resin with 4x400 mL of DMF and tested resin bead for deprotection.
Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 10.0 mL of acetic anhydride and 8.0 mL of DIPEA in 400mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x400 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 400 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 50 to 55 g (purity by HPLC: 83%)

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

Example-7:
Charged 1.0 g of Rink amide AM resin and 10 mL of DMF into the Peptide vessel. Stirred the resin for 10 min and solvent is removed by applying vacuum. For swelling of resin charged 10 mL of DMF and allowed resin to stir for 45 min. Removed solvent by applying vacuum. Charged 10 mL solution of 2% of Azepine in DMF into peptide vessel and stir for 10 min to deprotect the Fmoc of Resin. Wash resin with 4x100 mL of DMF. Kaiser test indicated complete deprotection.

Coupling of Amino acid: Charged solution of 0.65 g of Fmoc-D-Ala-OH, 0.32 g of HOBt.H2O and 0.33 mL of DIPC into 10 mL of DMF into peptide vessel and stirred for 1 hr at 25±5°C. Kaiser test indicated complete coupling of N-protected amino acid.
Deprotection of Amino acid: Charged 10 mL of solution of 2% Azepine in DMF into peptide vessel and stirred for 5 min. removed the solvent by applying vacuum and repeated second deprotection for 10 min. washed the resin with 4x10 mL of DMF and tested resin bead for deprotection.

Repeated above coupling and deprotection procedure for sequential loading of Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph(tBu-cbm)-OH, Fmoc-L-4Aph(L-Hor)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-3-Pal-OH, Fmoc-D-Phe(4-Cl)-OH, Fmoc-D-2-Nal-OH.

Acetylation: After deprotection of Fmoc-D-2-Nal-OH, charged solution of 0.25 mL of acetic anhydride and 0.20 mL of DIPEA in 10 mL of DMF into peptide vessel and stirred for 30 min. removed the solvent by applying vacuum. Washed the resin with 3x10 mL of DMF and tested resin bead for coupling.

Cleavage of Degarelix form Resin: Charged 10 mL of TFA into a reaction vessel and cooled to 15±5°C. Charged resin slowly into the reaction vessel and stirred for 30 to 40 hrs at 25±5°C. Filtered the resin and the filtrate was concentrated under vacuum at 25±5°C. Charged concentrated residue into pre-chilled Di-isopropyl ether and stir for 1 hr at 25±5°C. Filtered the solid under vacuum and washed the solid with Di-isopropyl ether and the wet cake was dried under vacuum for 3 hrs. at 30°C. Obtained weight of Degarelix crude is between 0.7 to 1.0 g. (purity by HPLC: 66.15%).

Purification of Degarelix Crude: Degarelix crude is purified and salt exchange on preparative HPLC column packed with C18 reverse phase material using gradient elution programme of Acetonitrile and water.

The following table summarizes the impact of various based used for Fmoc deprotection, on the yield and quality of degarelix crude. 2.0% v/v of pyrrolidine in DMF is shown to provide better results.
Exp.
Sr. No. Organic base for Fmoc-deprotection % v/v of base in DMF Yield factor HPLC purity of Degarelix crude after cleavage from resin
1 Piperidine 20.0 1.4 83.0 %
2 Pyrrolidine 2.0 1.9 88.24 %
3 DBU 3.0 1.8 66.15 %
4 Piperazine:DBU: Formic acid 5.0:1:1 1.8 74.27 %
5 Ethanolamine 5.0 1.8 60.79 %
7 Azepine 2.0 1.0 66.15 %
8 Piperidine 2.0 Experiment abandoned as each
F-moc deprotection step took more than 20 hours

,CLAIMS:CLAIMS:

We Claim,

An improved process for synthesis of degarelix acetate using method of deprotection in solid phase peptide synthesis in which the improvement comprises: treating with Pyrrolidine in Dimethylformamide in small volume to the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle.

An improved process for synthesis of degarelix according to claim 1 wherein, Pyrrolidine in small volume is about 0.5% to about 8.0% solution in solvent.

An improved process for synthesis of degarelix according to claim 2 wherein, Pyrrolidine in small volume preferably used in an amount of 1.5%v/v to 4.0%v/v, most preferably 2%v/v solution in solvent.

An improved process for synthesis of degarelix according to claim 1 wherein, deprotection reaction time is about 1min to about 30min.

An improved process for synthesis of degarelix according to claim 4 wherein, deprotection reaction time preferably is about 5min to about 20min.

An improved process for synthesis of degarelix according to claim 1 wherein, synthesis of degarelix acetate using Fmoc synthesis comprising following steps:
deprotecting the resin by treating with Pyrrolidine in Dimethylformamide (DMF).
washing with solvent to remove all soluble compounds from the resin;
Loading of the first Fmoc-protected amino acid;
Repetitive cycles of the Coupling suitable Fmoc-protected amino acid and deprotecting following:
deprotecting to remove the Fmoc group by reacting it with Pyrrolidine in small volume is about 0.5% to about 8.0% in Dimethylformamide (DMF);
washing with solvent to remove all soluble compounds from the resin;
coupling of a suitable Fmoc-protected amino acid (according to the required sequence) to the terminal amino group residue attached to the resin using a suitable coupling reagent to form an Fmoc-protected peptide fragment attached to the resin; and
washing the product of step (iii) with at least one solvent to remove all soluble compounds from the resin;
deprotection of the last amino acid Fmoc-D-Nal and acetylating the N- terminus;
washing and drying the peptide-resin;
cleaving and deprotection of the protected peptide intermediate from the resin using trifluoro acetic acid;
isolating the crude Degarelix from the cleavage solution;
purifying the crude Degarelix; and
isolating Degarelix.

An improved process for synthesis of degarelix according to claim 1 to 6 wherein, solvent are preferably N,N-Dimethylformamide (DMF) and N-methyl pyrrolidinone (NMP).

An improved process for synthesis of degarelix according to claim 6, wherein the amino group linked to the support is a-amino group of a fragment of degarelix linked to the support.

An improved process for synthesis of degarelix according to claim 6, wherein the support is selected from Rink amide AM resin, Rink amide MBHA resin.

An improved process for synthesis of degarelix according to claim 6, wherein Degarelix acetate is isolated by drying the Degarelix acetate solution by means of any suitable procedure; preferably lyophilization.

An improved process for synthesis of degarelix according to claim 6, wherein Degarelix crude is purified by a separation method and counter-ion exchange to acetate to form a Degarelix acetate solution

Dated this 28th day of May 2018