FIELD OF THE INVENTION

The present invention relates to an improved process for preparing LH-RH agonists, which is a polypeptide having a C-terminal alkylated amide group.

The present invention relates to an invention disclosed in our co-pending application IN 23/CHE/2013, wherein present invention is an improvement for the preparation of LH-RH agonists by using a novel solid support linker of Formula (I).

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 generally 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) or 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 CJ represents resin.

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, Rg, 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 N-alkylated polypeptide amides in high purity and yield. We have now found a linker, which is easy to manufacture and cost effective. Further, by using this linker as a solid support, we have now prepared polypeptide with C-terminal N-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 of Formula (I), which is easy to manufacture, industrially applicable and robust.

where R| and R2 are CH3, C2H5, C3H7 and (CH2)n COOH with a proviso that any one of
the Ri or R2 is (CH2)n COOH; wherein n is 1 to 7; R3 is H, amino protecting group like
Fmoc (9-fluorenylmethoxy carbonyl), Boc(tert-butoxy carbonyl),
Cbz(benzyloxycarbonyl), Alloc(allyloxycarbonyl), 2-(4-biphenylyl)-2-
propyloxycarbonyl (Bpoc), and propargyloxycarbonyl (Poc) group most preferably Fmoc.

In another objective of the present invention is use of the linker of Formula (I) in the preparation of polypeptide having C-terminal alkylated amide, which is a LH-RH-agonists.

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 agonists by a cost-effective and reproducible process.

Yet another objective of the present invention is to provide an improved process for preparing a polypeptide having C-terrhinal alkylated amide group, which is LH-RH agonists, in high yield and high purity.

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 of Formula I.

where R) and R2 are CH3, C2H5, C3H7 and (CH2)n COOH with a proviso that any one of
the R| or R2 is (CH2)n COOH; wherein n is 1 to 7; R3 is H, amino protecting group like
Fmoc (9-fluorenylmethoxy carbonyl), Boc(tert-butoxy carbonyl),
Cbz(benzyloxycarbonyl), Alloc(allyloxycarbonyl), 2-(4-biphenylyl)-2-
propyloxycarbonyl (Bpoc), and propargyloxycarbonyl (Poc) group most preferably Fmoc.

Another aspect of the present invention is to provide a process for the preparation of Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (la).

Yet Another aspect of the present invention is to provide a process for the preparation of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb).

In still another embodiment, the present invention also relates to the use of Fmoc protected 4-(4-(ethylaminq)methyl)-3-methoxyphenoxy)butyric acid (la) and Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb), prepared by the present invention in the preparation of LH-RH agonists.

BRIEF DESCRIPTION OF FIGURES

Figure-1: Process to prepare 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyramide (linker with solid support) of Formula (II).

Figure-2: Process to prepare Leuprolide acetate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improved process for preparing LH-RH agonists, 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 Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid of Formula la, 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-(4-((ethylamino)methyl)-3-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-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid ;

b) reacting the protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid with 4-methylbenzhydrylamine resin (MBHA resin or Amino methyl resin) to obtain protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyramide! (linker with solid support); and

c) deprotecting the protected 4-(4-((ethylamino)methyl)-3-
methoxyphenoxy)butyramide (linker with solid support) with a base, selected
from piperidine, diethyamine, l,8-diazobicyclo(5,4.0)-7-undecene-(DBU) or a
combination thereof, to obtain 4-(4-((ethyIamino)methyI)-3-
methoxyphenoxy)butyramide (linker with solid support) of Formula II.

Another aspect of the present invention is to provide a process for preparing LH-RH agonists, 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 a mixture of trifluoroacetic acid: triisopropyl silane : 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 acetate, 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); Fmot-Ser(t-Bu); Fmoc-Trp(Boc)-OH; Fmoc-His(Ttr)-OH and pGlu-OH as steps a) and b);

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

g) purifying the crude Leuprolide by reverse phase HPLC to obtain Leuprolide acetate.
The coupling reagent is selected from a group comprising N,N diisopropyl carbodiimide
(DIC) /1-hydroxybenzotriazole (HOBT), 0-(benzotriazole-l-yl-l,1,3,3-
tetramethyluronium tetrafluoroborate(TBTU)/HOBT/N,N-diisopropylethylamine
(DIEA), dicyclohexyl carbodiimide (DCC)/1-hydroxybenzotriazole (HOBT), l-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 cyanoglyoxylate-2-oxime (Oxyma Pure), 0-(6-
chloro-1 -hydrocibenzotriazol-1 -yl)-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, dimethylformamide, N-methylpyrrolidone and at the temperature ranging from about 10°C to 50°C.

Secondary base for deprotection of amino protecting group is selected from a group comprising piperidine, diethyamine, l,8-diazobicyclo(5,4.0)-7-undecene-(DBU) or 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, thioanisole or mixtures thereof.

In an embodiment solvent used for deprotection is selected from a group comprising water, dichloromethane, dichloroethane or 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 carried 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.

Another aspect of the present invention is to provide a compound of Formula (I), adopting solid state polypeptide synthesis of LH-RH agonists.

! wherein R| and R2 are CH3, C2H5, C3H7 and (CH2)n COOH with a proviso that any one
of the Ri or R2 is (CH2)n COOH; wherein n is 1 to 7; R3 is H, amino protecting group
like Fmoc (9-fluorenylmethoxy carbonyl), Boc(tert-butoxycarbonyl),
Cbz(benzyloxycarbonyl), Alloc(allyloxycarbonyl), 2-(4-biphenylyl)-2-

propyloxycarbonyl (Bpoc), and propargyloxycarbonyl (Poc) group most preferably Fmoc.

Another aspect of the present invention is to provide a process for the preparation of Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (la);
which comprises:

a) reacting 4-hydroxy-2-methoxy benzaldehyde (III) with ethyl-4-halo-butyrate in presence of inorganic compound to produce ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate (IV); wherein ethyl-4-halo-butyrate is ethyl-4-chloro-butyrate or ethyl-4-bromo-butyrate,

b) reacting ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate (IV) with ethyl amine or its salt in presence of reducing agent to produce ethyl 4-(4-((ethylamino)methyl)-3-methoxy phenoxy) butanoate (V); and

c) hydrolysis of ethyl 4-(4-((ethylamino)methyl)-3-methoxyphenoxy) butanoate (V) with a base to produce 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (VI); and

d) protecting 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (VI) with a protecting group Fmoc (9-fluorenylmethoxy carbonyl) to produce Fmoc protected 4-(4-((ethylamino)methyl)-3-rnethoxyphenoxy)butyric acid (la).

The process is shown in Scheme-I as given below:

the reaction in step-(a) is carried out in the presence of inorganic compound comprises potassium iodide, potassium phosphate, sodium iodide, sodium phosphate or mixtures thereof.

The reaction in step-(a) is also carried out in the presence of phase transfer catalyst comprises tetra methyl ammonium bromide, tetra ethyl ammonium bromide, tetra butyl ammonium bromide, methyl tributyl ammonium bromide, tetra methyl ammonium chloride,, tetra ethyl ammonium chloride , tetra butyl ammonium chloride, methyl tributyl ammonium chloride or mixtures thereof.

The solvent is used in step-(a) comprises N,N-dimethyl formamide (DMF), N,N-dimethyl acetamide (DMA), N-methylpyrrolidine (NMP), formamide, acetamide, propanamide, water, ethyl acetate, n-propyl acetate, n-butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, methyl acetate, methyl propanoate, ethyl propanoate, or mixtures thereof.

The reaction in step-(a) is performed at a temperature ranging between about 0°C to about 100°C for a period of about 1 to 24 hrs.

After completion of the reaction step, the compound of Formula (IV) is isolated by conventional methods such as extraction and/or distillation. Alternately the compound of Formula (IV) is used as such in the next step without isolation.

The reducing agent is used in step-(b) includes but not limited to borohydride compounds, such as NaBH(OAc)3, KBH(OAc)3 sodium cyanoborohydride, sodium borohydride or mixtures thereof.

The solvent is used in step-(b) comprises methanol, ethanol, 2-nitroethahol, 2-fluroethanol, 1-propanol, 2-propanol (isopropyl alcohol), 1-butanol, 2-butanol, benzyl alcohol, phenol, glycerol, water, ethyl acetate, n-propyl acetate, n-butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, methyl acetate, methyl propanoate, ethyl propanoate or mixtures thereof.

The reaction in step-(b) is performed at a temperature ranging between about 0°C to about 100°C for a period of about 1 to 24 hrs.

After completion of the reaction step, the compound of Formula (V) is isolated by conventional methods such as extraction and/or distillation. Alternately the compound of Formula (V) is used as such in the next step without isolation.

The base is used in step-(c) is selected from the group comprising sodamide, potassium amide, potassium. bis(trimethylsilyl)amide (KHDMS), sodium methoxide, sodium ethoxide, potassium isopropoxide, sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, cesium hydroxide, ammonia or mixture thereof.

The solvent is used in step-(c) comprises 1,4-dioxane, diethyl ether, diisopropyl ether, methyl t-butyl ether, glyme, tetrahydrofuran, dimethyl furan, anisole, water methanol, ethanol, 2-nitroethanol, 2-fluroethanol, 1-propanol, 2-propanol (isopropyl alcohol), 1-butanol, 2-butanol, benzyl alcohol, phenol, glycerol, ethyl acetate, n-propyl acetate, n-butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, methyl acetate, methyl propanoate, ethyl propanoate, or mixtures thereof.

The reaction in step-(c) is performed at a temperature ranging between about 0°C to about 50°C for a period of about 1 to 24 hrs.

After completion of the reaction step, the compound of Formula (VI) is isolated by conventional methods such as extraction and/or distillation. Alternately the compound of Formula (VI) is used as such in the next step without isolation.

The base is used in step-(d) is selected from the group comprising potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate sodium hydroxide, potassium hydroxide , Di-isopropyl ethylamine ,tri-ethyl amine etc or mixtures thereof.

The solvent is used in step-(d) comprises 1,4-dioxane, acetone, tetrahydrofuran diethyl ether, diisopropyl ether, methyl t-butyl ether, glyme, dimethyl furan, anisole, water methanol, ethanol, 2-nitroethanol, 2-fluroethanol, 1-propanol, 2-propanol (isopropyl alcohol), 1-butanol, 2-butanol, benzyl alcohol, phenol, glycerol, ethyl acetate, n-propyl acetate, n-butyl acetate, isobutyl acetate, t-butyl acetate, ethyl formate, methyl acetate, methyl propanoate, ethyl propanoate, or mixtures thereof.

The reaction in step-(d) is performed at a temperature ranging between about 0°C to about 50°C for a period of about 1 to 24 hrs.

The compound of Formula (la) is isolated by conventional techniques such as extraction, filtration, crystallization and dried the product.

Another aspect of the present invention is to provide a process for the preparation of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb),

which comprises: a) reacting 2-hydroxy-4-methoxy benzaldehyde (VII) with ethyl-4-halo butyrate in presence of inorganic compound to. produce ethyl 4-(2-formyl-5-methoxy phenoxy) butanoate (VIII);

wherein ethyl-4-halo-butyrate is ethyl-4-chloro-butyrate or ethyl-4-bromo-butyrate, b) reacting ethyl 4-(2-formyl-5-methoxy phenoxy) butanoate (VIII) with ethyl amine or its salt in presence of reducing agent to produce ethyl 4-(2-((ethylamino)methyl)-5-methoxyphenoxy) butanoate (IX); and c) hydrolysis of ethyl 4-(2-((ethylamino)methyl)-5-methoxyphenoxy) butanoate (IX) with a base to produce 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (X). d) protecting 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (X) with a protecting group Fmoc (9-fiuorenylmethoxy carbonyl) to produce Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb).

The process is shown in Scheme-II as given below:

The reagents, conditions and solvents employed in the preparation of Fmoc protected 4-(4-((ethylamino)methyI)-3-methoxyphenoxy)butyric acid (la) are employed in the preparation of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb).

In another embodiment, the present invention also relates to the use of Fmoc protected 4-(4-(ethylamino)methyl)-3-methoxyphenoxy)butyric acid (la) and Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb), prepared by the present invention in the preparation of LH-RH agonists.

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

EXAMPLES:

Example 1: Preparation of 4-(4-((Ethylamino)methyl)-3-methoxyphenoxy)
butyramide resin.

Step A: 4-Methylbenzhydrylamine resin (MBHA resin; 250 gm substitution 1.2
mmol/gm) was taken in a SPPS reactor and 1.5 L of dichloromethane was added. The
resin was allowed to swell for 20 min and thereafter drained.

Step B: Fmoc protected 4-(4-((ethylamino)methyl)-3^methoxyphenoxy)butyric acid
(200 gm, 1.5 eq) was dissolved in 1.5 L of DMF. TBTU (130 gm, 1.5 eq) / HOBT H20
(60 gm, 1.5 eq) and DIEA (140 ml, 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 3 hrs at room
temperature. The progress of coupling was monitored by Chloranil and Kaiser Tests.
After completion of the reaction the resin was drained and washed with DMF. 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 DMF (20 mL), followed by
DCM. It was then isolated and dried.

The above resin was deblocked with one bed volume of 20% piperidine in DMF for 15
min and thereafter the resin was drained and washed with DMF, IPA and DMF.
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-(4-((ethylamino)methyl)-3-methoxyphenoxy)
butyramide resin.

Fmoc-Pro-OH (2 eq) and HOBT (2 eq) were dissolved in 1.5 L DMF and cooled to 0-
5°C while stirring. DIC (2 eq) was added and stirred for 5 min, followed by addition to
the resin and kept for two hours at room temperature and washed with DMF (20 mL).
The coupling was monitored by Kaiser Test.

The Fmoc protecting group in above resin was deblocked with one bed volume of 20%
piperidine in DMF for. 15 min and thereafter the resin was drained and washed with
DMF, IPA and DMF.

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.
Yield: 1.1 Kg

Example 3: Preparation of crude Leuprolide.
The obtained resin of example-2 was treated with a mixture TFA+Water+TIS
(95%+2.5%+2.5%) for 2.5 hrs at room temperature. The crude peptide (Leuprolide) was
isolated by precipitating with MTBE.
Yield: 400 gm

Example 4: Purification of Leuprolide.
Crude Leuprolide was purified by reverse phase HPLC using 18% acetonitrile 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: 120 gm
Purity: 99.5%

Example-5: Synthesis of Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxy
phenoxy) butyric acid (la).

Step-(I): Synthesis of Ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate.
To DMF (850 ml), 4-hydroxy -2-methoxy benzaldehyde (200 gm, 1 eq), potassium
phosphate (552 gm, 2eq), Me4NBr (40.5 gm, 0.2 eq), potassium iodide (43.6 gm,
0.2eq), and ethyl-4-bromo butyrate (245 ml, 1.3eq) were added and stirred at room.
temperature. The progress of the reaction was monitored by TLC. After completion of
the reaction, DM water was added to the above reaction mass and extracted with ethyl
acetate. Layers were separated. The organic layer was dried with anhydrous sodium
sulphate and concentrated on rota-vac until approximately 80% of ethyl acetate was
distilled off. To the reaction mass, n-heptane was added and stirred to obtain off-white
precipitate. The precipitate was filtered and washed with n-Heptane and dried to give
ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate.
Yield: (301 gm) 86%
TLC: -Single spot

Step-(II): Synthesis of ethyl 4-(4-((ethylamino)methyl)-3-methoxyphenoxy) butanoate.
A mixture of ethylamine hydrochloride (255.4 gm, 3eq) and MeOH (.1.5 L) was stirred
in 10 L dried reactor at room temperature for 15 min, clear solution was observed and
cooled to 0 to 5°C, TEA (437 mL, 3eq) was added drop wise using dropping funnel.
Ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate (278 gm) was added in one lot. This
reaction mixture was cooled to -40°C, NaBH(OAc)3 (663.8 gm) was added portion wise
and stirred for 12 hrs at room temperature. The progress of the reaction was monitored
by TLC. After completion of the reaction, the reaction mixture was cooled to 0 to 5° C,
20% KHCO3 solution was added slowly over a period of 30 min and stirred. Methanol
was evaporated completely under reduced pressure, and the product was extracted with
ethyl acetate. Layers were separated, and the organic layer was washed with 20%
KHCO3 solution and water. The organic layer was dried with anhydrous sodium
sulphate and concentrated on rota-vac to obtain oily compound.
Yield: 215 gm (70%)
TLC: ~ Single spot.-
Step-(IH): Synthesis of 4-(4-((ethylamino) methyl)-3-methoxy phenoxy) butyric
acid.
To the solution of linker (obtained from Step-(II); 215 gm, leq) in 1, 4-dioxane, ,
aqueous NaOH solution (64 gm in 900 mL water) was added at 0 to 5° C and stirred for
2 hrs at room temp. The pH was adjusted to 7-8 with 6N HC1. This solution was used as
such for next step.
Theoretical yield: 210 gm (100%)
TLC: -Single spot
Step-(IV): Synthesis of Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxy
phenoxy) butyric acid.

To the solution of Step-(III) (-210 gm, 1 eq), potassium carbonate (200 gm, 2 eq) was. added at 0 to. 5° C and stirred for 10 min. A solution of Fmoc-OSu in 1, 4-Dioxane (227.8 gm in 1.26 L of dioxane) was added drop wise to the above reactor (under ice cooling) over a period of 20 min and stirred. The progress of the reaction was monitored by TLC. After completion of the reaction DM water was added to the above reaction mixture and reaction mixture was washed with MTBE. PH of the aqueous solution was adjusted to ~2 to 3 with 6N HC1 under ice cooling and the compound was extracted with MTBE.The organic layer was washed with IN HC1 and water. The MTBE layer was . dried with anhydrous sodium sulphate and concentrated on rota-vac till -90% MTBE was distilled. To this oily product n-heptane was added and stirred to obtain off-white precipitate. The precipitate was filtered and washed with n-heptane and dried to give Fmoc protected 4-(4-((ethylamino) methyl)-3-methoxy phenoxy) butyric acid. Yield: 292 gm (82.5%) Purity by HPLC. 98.7%.

Example-6: Synthesis of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxy phenoxy) butyric acid (lb).

Step-(I): Synthesis of Ethyl 4-(2-formyl-5-methoxy phenoxy) butanoate. To DMF (850 ml), 2-hydroxy -4-methoxy benzaldehyde (200 gm, 1 eq), potassium phosphate (552 gm, 2eq), Me4NBr (40.5 gm, 0.2 eq), potassium iodide (43.6 gm, 0.2eq), and ethyl-4-bromo butyrate (245 ml, 1.3eq) were added and stirred at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, DM water was added to the above reaction mass and extracted with ethyl acetate. Layers were separated. The organic layer was dried with anhydrous sodium sulphate and concentrated on rota-vac until approximately 80% of ethyl acetate was distilled off. To the reaction mass, n-heptane was added and stirred to obtain off-white precipitate. The precipitate was filtered and washed with n-Heptane and dried to give ethyl 4-(2-formyl-5-methoxy phenoxy) butanoate. Yield: (294gm) 84% TLC: -Single spot

Step-(II): Synthesis of ethyl 4-(2-i((ethylamino)methyl)-5-methoxyphenoxy) butanoate.

A mixture of ethylamine hydrochloride (255.4 gm, 3eq) and MeOH (1.5 L) was stirred in 10 L dried reactor at room temperature for 15 min, clear solution was observed and cooled to 0 to 5°C, TEA (437 mL, 3eq) was added drop wise using dropping funnel. Ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate (278 gm) was added in one lot. This reaction mixture was cooled to -40°C, NaBH(OAc)3 (663.8 gm) was added portion wise and stirred for .12 hrs at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to 0 to 5° C, 20% KHCO3 solution was added slowly over a period of 30 min and stirred. Methanol was evaporated completely under reduced pressure, and the product was extracted with ethyl acetate. Layers were separated, and the organic layer was washed with 20% KHCO3 solution and water. The organic layer was dried with anhydrous sodium sulphate and concentrated on rota-vac to obtain oily compound. Yield: 215 gm (70%) TLC: ~ Single spot

Step-(III): Synthesis of 4-(2-((ethylamino) methyl)-5-methoxy phenoxy) butyric acid. To the solution of linker (obtained from Step-(II); 215 gm, leq) in 1, 4-dioxane, aqueous NaOH solution (64 gm in 900 mL water) was added at 0 to 5° C and stirred for 2 hrs at room temp. The pH was adjusted to 7-8 with 6N HC1. This solution was used as such for next step.Theoretical yield: 210 gm (100%)TLC: ~Single spot

Step-(IV): Synthesis of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxy phenoxy) butyric acid.
To the solution of Step-(III) (-210 gm, 1 eq), potassium carbonate (200 gm, 2 eq) was. added at 0 to 5° C and stirred for 10 min. A solution of Fmoc-OSu in 1, 4-Dioxane (227.8 gm in 1.26 L of dioxane) was added drop wise to the above reactor (under icecooling) over a period of 20 min and stirred. The progress of the reaction was monitoredby TLC. After completion of the reaction DM water was added to the above reaction mixture and reaction mixture was washed with MTBE. PH of the aqueous solution was adjusted to ~2 to 3 with 6N HC1 under ice cooling and the compound.was extracted with MTBE. The organic layer was washed with IN HC1 and water. The MTBE layer was dried with anhydrous sodium sulphate and concentrated on rota-vac till -90% MTBE was distilled. To this oily product n-heptane was added and stirred to obtain off-white precipitate. The precipitate was filtered and washed with n-heptane and dried to give Fmoc protected 4-(2-((ethylamino) methyl)-5-methoxy phenoxy) butyric acid. Yield: 292 gm (82.5%) Purity by HPLC: 98.3%

WE CLAIM

1) A process for making a solid support for the synthesis of polypeptides, which comprises,

a) protecting 4-(4-((ethylamino)methyl)-3-methbxyphenoxy)butyric acid of Formula VI, 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 to obtain protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid;

b) reacting protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid with 4-methylbenzhydrylamine resin (MBHA resin) or amino methyl resin to obtain protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyramide (linker with solid support); and

c) deprotecting the protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyramide (linker with solid support) with a base to obtain 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyramide (linker with solid support) of Formula II.

2) 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.

3) A process for preparing LH-RH agonists, 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; and

e) purifying the obtained peptide by reverse phase HPLC.

4) A process for preparing Leuprolide acetate, which comprises:

a) coupling Na 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 to give crude Leuprolide; and

e) purifying the crude Leuprolide by reverse phase HPLC to obtain Leuprolide acetate.

5) The process according to claims 3 and 4, wherein the coupling reagent is selected from a group comprising of N,N Diisopropyl carbodiimide (DIC) /l-hydroxybenzotriazole (HOBT), 0-(benzotriazole-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/HOBT/N,N-Diisopropylethylamine (DIEA), Dicyclohexyl carbodiimide (DCC)/l-hydroxybenzotriazole (HOBT), l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/HOBT,. 2-( 1 H-benzotriazole-1 -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 cyanoglyoxylate-2-oxime (Oxyma Pure) and 0-(6-chloro-1 -hydrocibenzotriazol-1 -yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU) and the like.

6) The process according to claims 3 and 4, wherein the deblocking and deprotection is carried out using a mixture of acid, scavenger and a solvent.

7) A process for the preparation of Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (la);
which comprises:
a) reacting 4-hydroxy-2-methoxy benzaldehyde (III) with ethyl-4-halo-butyrate in
presence of inorganic compound to produce ethyl 4-(4-formyl-3-methoxy
phenoxy) butanoate (IV);

wherein ethyl-4-halo-butyrate is ethyl-4-chloro-butyrate or ethyl-4-bromo-butyrate, b) reacting ethyl 4-(4-formyl-3-methoxy phenoxy) butanoate (IV) with ethyl amine or its salt in presence of reducing agent to produce ethyl 4-(4-((ethylamino)methyl)-3-methbxy phenoxy) butanoate (V); and

c) hydrolysis of ethyl 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butanoate (V) with a base to produce 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (VI); and

d) protecting 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (VI) with a protecting group Fmoc (9-fluorenylmethoxy carbonyl) to produce Fmoc protected 4-(4-((ethylamino)methyl)-3-methoxyphenoxy)butyric acid (la).

8) A process for the preparation of Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb),
which comprises:

a) reacting 2-hydroxy-4-methoxy benzaldehyde (VII) with ethyl-4-halo butyrate in
presence of inorganic compound to produce ethyl 4-(2-formyl-5-methoxy
phenoxy) butanoate (VIII);

wherein ethyl-4-halo-butyrate is ethyl-4-chloro-butyrate or ethyl-4-bromo-butyrate, b) reacting ethyl 4-(2-formyl-5-methoxyphenoxy) butanoate (VIII) with ethyl amine or its salt in presence of reducing agent to produce ethyl 4-(2-((ethylamino)methyl)-5-methoxyphenoxy) butanoate (IX); and

c) hydrolysis of ethyl 4-(2-((ethylamino)methyl)-5-methoxyphenoxy) butanoate (IX) with a base to . produce 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (X); and

d) protecting 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (X) with a protecting group Fmoc (9-fluorenylmethoxy carbonyl) to produce Fmoc protected 4-(2-((ethylamino)methyl)-5-methoxyphenoxy)butyric acid (lb).

9) A solid support linker of Formula (I).

where R| and R2 are CFf3, C2H5, C3H7 and (CH2)n COOH with a proviso that any one of
the R| or R2 is (CH2)n COOH; wherein n is 1 to 7; R3 is H, amino protecting group like
Fmoc (9-fluorenylmethoxy carbonyl), Boc(tert-butoxy carbonyl),
Cbz(benzyloxycarbonyl), Alloc(allyloxycarbonyl), 2-(4-biphenylyl)-2-
propyloxycarbonyl (Bpoc), and propargyloxycarbonyl (Poc) group most preferably Fmoc.

10) A solid support linker of Formula (II).