FIELD OF THE INVENTION
The present invention relates to an improved process for preparing LH-RH agonist, which is a polypeptide having a C-terminal alkylated amide group.
BACKGROUND OF THE INVENTION
The LH-RH (luteinizing hormone-releasing hormone) is a neurohormone produced in the hypothalamus, which stimulates the secretion of the pituitary hormones, LH (luteinizing hormone) and FSH (follicle-stimulating hormone), which in turn act on the gonads to stimulate the synthesis of steroid hormones. The pulsatile release of LH-RH, and the consequent release of LH and FSH, controls the reproductive cycle in mammals.
LH-RH analogs are nona- or decapeptides, which are structurally related to LH-RH and exhibit biological activity similar to that of LH-RH by acting on the pituitarygonadal axis in competition with naturally occurring LH-RH.
LH-RH agonist such as Leuprolide, Buserelin, Deslorelin. Alarelin and the like, all differ from naturally occurring LH-RH by replacement of at least the glycine residue at the 6-position with a D-amino acid. The synthetic agonists then have, in common with the naturally occurring hormone, histidine at position 2, serine at position 4, tyrosine at position 5, and arginine at position 8, all of which have reactive side chains.
LH-RH analogs may be synthesized by various methods, such as are taught by J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, 1969; J. Meinenhofer, Hormonal Proteins and Peptides, Vol. 2, page 46, Academic Press (New York), 1973; and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press (New York), 1965. The methods may be broadly characterized as either solution phase or solid phase techniques. Both

methods involve the sequential addition of amino acids to a growing peptide chain; however, the more rigorous conditions of solid phase synthesis generally require that any reactive side chains on the amino acids be protected during formation of the amide linkage. The side chain protecting groups may be removed concurrently with the cleavage of the completed polypeptide from the inert support on which it is made or in a separate deprotection step.
US 4,234,571 discloses the synthesis of a-amino (Na) function of each amino acid is protected by an acid sensitive group, such as t-butoxycarbonyl (Boc); any reactive side chains, as are present on arginine, serine, histidine and tyrosine, are also protected with strongly bound groups which require treatment with hydrogen fluoride or similarly drastic procedures for their removal. This use of Na-Boc protection and HF labile side chain protection, while adequate for the preparation of research quantities of peptides, is not entirely satisfactory for large scale production. The fully protected amino acids are expensive and require a separate Step for deprotection. Further, the use of hydrogen fluoride for the final deprotection, in addition to posing serious environmental hazards, contributes to commercially unacceptable yield losses.
To overcome the problems associated with Boc-SPPS synthesis of peptides, Fmoc-SPPS has gained in popularity because of its use of environmentally safer reagents and comparatively milder reaction conditions. In particular, Fmoc-SPPS peptide synthesis avoids the use of HF.
CN 101407540 A discloses the preparation of decapeptide using solid phase synthesis. The linker used is
wherein

By using this linker as solid support the required polypeptide chain is prepared.
US 7,714,063 B2 discloses the preparation of decapeptide using solid phase synthesis. The linker used here is wherein R2 and R4 are hydrocarbyl; R3 is selected from the group consisting of hydrogen, a protecting group, an amino acid residue, and a peptide; R5 is a solid support comprising at least one polymer; and R6, R7, Rs, and R9 are independently selected from the group consisting of hydrogen, hydrocarbyl, and substituted hydrocarbyl; by using this linker as solid support the required polypeptide chain is prepared.
A need therefore exists for a solid support that is economical to produce, and that can be used for Fmoc-SPPS synthesis of polypeptides in high purity and yield. We have now found a linker, which is easy to manufacture and economic cost effective. Further, by using this linker as a solid support, we have new prepared polypeptide with C-terminal alkylated amide in high yield and purity and also this process is more convenient and cost effective for large scale synthesis of Leuprolide.
OBJECTIVE OF THE INVENTION
An objective of the present invention is to provide a linker, which is easy to manufacture. In another objective of the present invention is use of the linker in the preparation of polypeptide having C-terminal alkylated amide, which is a LH-RH-agonist.
Another objective of the present invention is to provide an improved process for preparing a polypeptide having C-terminal alkylated amide group, which is LH-RH agonist by a cost-effective and reproducible process. Another objective of the present invention is to provide an improved process for preparing a polypeptide having C-terminal alkylated amide group, which is LH-RH agonist, in high yield and high purity.

BRIEF DESCRIPTION OF FIGURES
Figure-1: Process to prepare 4-(2-((ethylamino)methyl)-5- methoxyphenoxy)butyramide (linker with solid support) of Formula II.
Figure-2: Process to prepare Leuprolide.
SUMMARY OF THE INVENTION
The present invention relates to an improved process for preparing LH-RH agonist, which is a polypeptide having C-terminal alkylated amide group, selected from Leuprolide, Buserelin, Deslorelin, Alarelin and the like, adopting solid state polypeptide synthesis by using a linker 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid of Formula I.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an improved process for preparing LH-RH agonist, the polypeptide which has C-terminal alkylated amide group, selected from Leuprolide, Buserelin, Deslorelin, Alarelin and the like, adopting solid state polypeptide synthesis by using a linker 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid of Formula I, which is anchored to polymeric resin.
Another aspect of the invention encompasses a process for making a solid support for the synthesis of polypeptides. The process comprises,

a) protecting 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid with a protecting group selected from Fmoc (9-fluorenylmethoxy carbonyl), Boc(tert-butoxycarbonyl), Cbz(benzyloxycarbonyl), Alloc(allyloxycarbonyl), 2-(4-biphenylyl)-2-propyloxycarbonyl (Bpoc), and propargyloxycarbonyl (Poc) group preferably Fmoc, to obtain protected 4- (2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid;

b) reacting the protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric
acid with 4-methylbenzhydrylamine resin (MBHA resin) to obtain protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyramide; and

c) deprotecting the protected 4-(2-((ethylamino)rnethyl)-5-
methoxyphenoxy)butyramide with a base, selected from piperidine,
diethyamine, l,8-diazobicyclo(5,4.0)-7-undecene-(DBU) or a combination
thereof, to obtain 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyramide
(linker with solid support) of Formula II.

Another aspect of the present invention is to provide a process for preparing LH-RH agonist, which comprises:
a) coupling Na-protected amino acid (side chain protected with acid labile group) with linker supported on resin of Formula II using a coupling reagent;
b) deprotecting with secondary amine in a solvent;
c) optionally repeating the steps a and b till the desired protected peptide is obtained;
d) deblocking and deprotecting the peptide using mixture of trifiuoroacetic acid : triisoprepyl si lane : water; and
e) purifying the obtained peptide by reverse phase HPLC.
Another aspect of the present invention is to provide a process for preparing Leuprolide, which comprises:
a) coupling Na-Fmoc protected Pro-OH with linker supported on resin of Formula II using a coupling reagent;
b) deprotecting with secondary amine in a solvent;
c) coupling Fmoc protected Arg(Pbf) using a coupling reagent;
d) deprotecting with secondary amine in a solvent;
e) coupling and deprotecting the Fmoc-Leu-OH; Fmoc-D-Leu-OH; Fmoc-Tyr(t-Bu); Fmoc-Ser(t-Bu); Fmoc-Trp(Boe)-OH; Fmoc-His(Ttr)-OH and pGlu-OH as steps a and b;
f) deblocking and deprotecting the protected Leuprolide using mixture of trifiuoroacetic acid : triisopropyl silane : water to give Leuprolide; and
g) purifying the obtained Leuprolide by reverse phase HPLC.

The coupling reagent is selected from a group comprising N,N Diisopropyl
carbodiimide (DIC) /1-hydroxybenzotriazole (HOBT), 0-(benzotriazole-l-yl~
1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU)/HOBT/N,N-
Diisopropylethylamine (DIEA), Dicyclohexyl carbodiimide (DCC)/1-
hydroxybenzotriazole (HOBT), l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCVHOBT, 2-(lH-benzotriazole-l-yl)-l,l ,3,3-tetramethyluronium (HBTU)
/HOBT/DIEA, benzotriazole-l-yl-oxy-tris(dimethylamino)phosphonium (BOP),
benzotriazole-1 -yl-oxy-tris-(pyrrolidino)phosphonium (PyBOP), bromo-tris-
pyrrolidino-phosphoniumhexafluorophosphate(PyBrOP), tris(pyrrolidino)
phosphonium hexafluorophosphate (PyCOP), Ethyl cyanoglyoxylate-2-oxime
(Oxyma Pure), 0-(6-chloro-1 -hydrocibenzotriazol-1 -y 1)-1,1,3,3 -
tetramethyluronium tetrafluoroborate (TCTU) and the like.

The coupling reagent used in the above reaction may range from about 1 to about 5 molar equivalents, per molar equivalent of resin with respect to resin loading capacity. Preferably, 2 molar equivalents of individual coupling agents per molar equivalent of the resin with respect to resin loading capacity are used. The coupling reaction may be carried out in a suitable solvent selected from the following dichloromethane, tetrahydrofuran, dimethylformamjde, N-methylpyrroiidone and at the temperature ranging from about 10°C to 50°.

Secondary base for deprotection is selected from a group comprising piperidine, diethyamine, l,8-diazobicyclo(5,4.0)-7~undeeene-(DBU) and mixtures thereof. Deblocking and deprotecting is carried out using a mixture of acid, scavenger and solvents.
In an embodiment scavenger is selected from a group comprising Ethanedithiol (EDT), Tri-isopropyl silane (TIS), Triethyl silane (TES), Phenol, Tliioanisole and mixtures thereof. In an embodiment solvent used for deprotection is selected from a group comprising water, dichloromethane, dichloroethane and mixtures thereof. In an embodiment acid is selected from a group comprising trifluoroacetic acid, hydrobromic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid and trifluoromethanesulfonic acid.
Purification of crude Leuprolide is earned out using preparative RP-HPLC in presence of buffer and a solvent.
In an embodiment the solvent used for purification is selected from a group comprising ethanol, methanol, isopropanol, acetonitrile and mixtures thereof as eluting agents. In an embodiment the buffer solution used for purification is prepared by mixing acid in water in an amount of 0.01 to 1% of acid, selected from a group comprising of aqueous ortho phosphoric acid, aqueous triflouroacetic acid, aqueous formic acid, aqueous acetic acid and aqueous ammonium acetate. 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.
EXAMPLE 1

PREPARATION OF 4-(2-((ETHYLAMINO)METHYL)-5-
ETHOXYPHENOXY)BUTYRAMIDE RESIN

Step A MBHA resin (3gm) was taken in a SPPS reactor and 30ml of diehloromethane was added. The resin was allowed to swell for 20 min and thereafter drained.

StepB Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (2gm, 1.5eq) was dissolved in 10ml of DMF, TBTU (1.3 gm, 1.5 eq) / HOBT (0.55gm, 1.5eq) and DIEA (1.5ml, 3eq) were added and the reaction mixture was stirred for 5 min.
Step B reaction mass was added to Step A resin and stirred for 4 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 20ml of DMF (3 times). The resin was then capped with acetic anhydride (3 eq) and DIEA (3eq) solution in DCM for 20 min and drained. Thereafter the resin was
washed with 20ml of DMF (2 times), followed by DCM (2 times). It was then isolated and dried. The above resin was deblocked with 30ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 20ml of DMF (2 times), IP A (2 times) and DMF (2 times).

EXAMPLE 2

PREPARATION OF pGLU-HIS(Trt)-TRP(Boc)~SER(t-Bu)-TYR(t-Bu)-D-LEU-LEU-ARG(Pbf)-PRO-NEt-RESIN USING 4-(2-((ETHYL AMINO) METHYL)-5- ETHOXY PHENOXY) BUTYRAMIDE RESIN

Fmoc-Pro-OH (2eq.) and HOBT (2eq) were dissolved in 10ml DMF and cooled to 0-5°C while stirring. DIC (2eq) was added and stirred for 5 min, followed by addition to the resin and kept for two hours at room temperature and washed with 20ml of DMF (3 times). The coupling was monitored by Kaiser Test.
The above resin was deblocked with 30ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 20 ml DMF (2 times), IPA (2 times) and DMF (2 times). The repeated cycles of operation (Amino acid coupling and Fmoc
deprotection) were performed for Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-D-
Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-His(Trt)-OH, pGlu-OH. Finally the peptide resin was isolated and dried.

EXAMPLE 3

PREPERATION OF CRUDE LEUPROLIDE

Cleavage and deblocking of peptide was performed with a mixture TFA+Water+TIS (95%+2.5%+2.5%) for 2.5 hrs at room temp. The crude peptide (Leuprolide) was isolated by precipitating with MTBE. Yield: 10.3gm
Chromatographic Purity (by HPLC): 84.4%

EXAMPLE 4

PURIFICATION OF LEUPROLIDE

Crude Leuprolide was purified by reverse phase HPLC using 18% ethanol in aqueous acetic acid (1%). The fractions containing pure Leuprolide acetate were pooled, the organic modifier was removed under reduced pressure. The resulting peptide solution was freeze-dried to isolate fluffy material.

EXAMPLE 5

PREPARATION OF 4-(2-((ETHYLAMINO)METHYL)-5-
ETHOXYPHENOXY)BUTYRAMIDE RESIN

Step A MBHA resin (30gm) was taken in a SPPS reactor and 300ml of dichloromethane was added. The resin was allowed to swell for 20 min and thereafter drained.
StepB Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (19.8gm, 1.5eq) was dissolved in 100ml DMFSTBTU (13gm, 1.5eq) / HOBT (5.6gm, 1.5 eq) and DIEA (15ml, 3 eq) was added and the reaction mixture was stirred for 5 min.
Step B reaction mass was added to Step A resin and stirred for 4 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. Thereafter the resin was washed with one bed volume of DMF (2 times), DCM (2 times) and MTBE (2 times). It was then isolated and dried.

The above resin was deblocked with 300ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 200ml of DMF (2 times), IPA (2 times) and DMF (2 times).
EXAMPLE 6

PREPERATION OF pGLU-HIS(trt)-TRP(Boc)-SER(t-Bu)-TYR(t~Bu)-D-LEU-LEU~ARG(Pbf)-PRO-NEt-RESIN USING 4-(2-((ETHYL AMINO) METHYL>5- ETHOXY PHENOXY) BUTYRAMIDE RESIN

Fmoc-Pro-OH (2eq) and HOBT (2eq) were dissolved in 100ml of DMF and cooled to 0-5°C while stirring. D1C (2eq) was added and stirred for 5 min, followed by addition to the resin and kept for two to four 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 deblocked with 300ml of 20% piperidine in DMF for 15 min and thereafter the resin was drained and washed with 200ml of DMF (2 times), IPA (2 times) and DMF (3 times). The repeated cycles of operation (Amino acid coupling and Fmoc
deprotection) were performed for Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-D-
Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-His(Trt)-QH, pGlu-OH. Finally the peptide resin was isolated and dried. Yield: 114gm

EXAMPLE 7

PREPERATION OF CRUDE LEUPROLIDE

Cleavage and deblocking of peptide was performed with a mixture TFA+Water+TIS (95%+2.5%+2.5%) for 2.5 hrs at room temp. The crude peptide (Leuprolide) was isolated by precipitating with MTBE.
Yield: 40gm

Chromatographic Purity (by HPLC): 84%

EXAMPLE 8

PURIFICATION OF LEUPROLIDE

Crude Leuprolide was purified by reverse phase HPLC using 18% ethanol in
aqueous acetic acid (1%). The fractions containing pure Leuprolide acetate were
pooled, the organic modifier was removed under reduced pressure. The resulting
peptide solution was freeze-dried to isolate fluffy material.

Yield: 14.5gm (45%)

Chromatographic Purity (by HPLC): 99.7 %

1) A process for making a solid support for the synthesis of polypeptides, which comprises,
a) protecting 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid of Formula I,
with a protecting group to obtain protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid;
b) reacting protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid with 4-methylbenzhydrylamine resin (MBHA resin) to obtain protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyramide; and
c) deprotecting the protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyramide with a base to obtain 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyramide (linker with solid support) of Formula II.

2) The process according to claim 1, wherein the protecting group is selected from a group comprising of Fmoc (9-fluorenylmethoxy carbonyl), Boc (tert- butoxycarbonyl), Cbz (benzyloxycarbonyl). Alloc (allyloxycarbonyl), Bpoc (2-(4-biphenylyl)-2-propyloxycarbonyl) and propargyloxycarbonyl (Poc).

3) The process according to claim 1, wherein the base is selected from a group
comprising of Piperidine, Diethyamine, l,8-diazobicyclo(5,4.0)-7-undecene-(DBU) or mixture thereof.

4) A process for preparing LH-RH agonist, which comprises:

a) coupling Na-protected amino acid (side chain protected with acid labile group) with linker supported on resin of Formula 11,
using a coupling reagent;

b) deprotecting with secondary amine in a solvent;

c) optionally repeating the steps a and b, till the desired protected peptide is obtained;

d) deblocking and deprotecting the peptide using mixture of trifluoroacetic acid : triisopropyl silane : water; and

e) purifying the obtained peptide by reverse phase HPLC.

5) A process for preparing Leuprolide, which comprises:

a) coupling N" protected Pro-OH with linker supported on resin of Formula II using a coupling
reagent;

b) deprotecting with secondary amine in a solvent;

c) coupling and deprotecting the protected Arg(Pbf); protected-Leu-OH; protected-D-Leu-OH; protected-Tyr(t-Bu); protected-Ser(t-Bu); protected-Trp(Boc)-OH; protected-His(Ttr)-OH and pGlu-OH one after the other sequentially,

d) deblocking and deprotecting the protected Leuprolide using mixture of trifluoroacetic acid : triisopropyl silane : water to give Leuprolide; and

e) purifying the obtained Leuprolide by reverse phase HPLC.

6) The process according to claims 4 and 5, wherein the coupling reagent is selected from a group comprising of N,N Diisopropyl carbodiimide (DIG) /l-hydroxybenzotriazole (HOBT), 0-(benzotriazole-l-yl-l, 1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/HOBT/N,N-Diisopropylethylamine (DIEA), Dicyclohexyl carbodiimide (DCC)/1-hydroxybenzotriazole (HOBT), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/HOBT, 2-(lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium (HBTU)/HOBT/DIEA, benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium (BOP), benzotriazole-1 -yl-oxy-tris-(pyrrolidino)phosphonium (PyBOP), bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate(PyBrOP), tris(pyrrolidino) phosphonium hexafluorophosphate (PyCOP), Ethyl cyanoglyoxyIate-2-oxime (Oxyma Pure) and 0-(6-chloro-1 -hydrocibenzotriazol-1 -yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU) and the like.

7) The process according to claims 4 and 5, wherein the deblocking and deprotecting is earned out using a mixture of acid, scavenger and solvent.

8) The process according to claim 7, wherein the scavenger is selected from a group comprising Ethanedithiol (EDT), Tri-isopropyl silane (TIS), Triethyl silane (TES), phenol, thioanisole and mixtures thereof.

9) The process according to claim 7, wherein the solvent is selected from a group
comprising of water, Dichloromethane, Dichloroethane and mixtures thereof;
acid is selected from a group comprising of trifluoroacetic acid, Hydrobromic
acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid and
trifluoromethanesulfonic acid.

10) A solid support linker of formula II.