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Title:
PROCESS FOR PREPARATION OF LANREOTIDE ACETATE
Document Type and Number:
WIPO Patent Application WO/2019/077507
Kind Code:
A1
Abstract:
The invention relates to an improved method for the solution phase synthesis of Lanreotide acetate (1) comprising coupling of two suitably protected tetrapeptide fragments wherein the threonine hydroxyl is protected, to give an octapeptide, which on deprotection, oxidation, followed by treatment with acetic acid provides Lanreotide acetate (1) having desired purity.

Inventors:
GURJAR, Mukund Keshav (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
DESHMUKH, Sanjay Shankar (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
HONPARKHE, Ramchandra Birappa (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
HINGMIRE, Vaibhav Shivaji (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
KAPE, Sandeep Ashok (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
Application Number:
IB2018/058034
Publication Date:
April 25, 2019
Filing Date:
October 17, 2018
Export Citation:
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Assignee:
EMCURE PHARMACEUTICALS LIMITED (Emcure House, T-184 M.I.D.C., Bhosari, Pune 6, 411026, IN)
International Classes:
C07K5/10
Foreign References:
IN201621012581A
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Claims:
CLAIMS

We claim,

1. A process for the solution phase synthesis of lanreotide acetate (1), comprising reaction of H-Lys(Boc)-Val-Cys(Trt)-Thr(t-Bu)-NH2 (Fragment A) with Boc-D-Nal-Cys (Trt)- Tyr-D-Trp-OH (Fragment B) in an organic solvent, in presence of a base and a coupling agent to give the octapeptide, Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-Lys(Boc)-Val-Cys (Trt)- Thr(t-Bu)-NH2 (22), which is converted to lanreotide acetate by subsequent deprotection, followed by oxidation and treatment with acetic acid to provide lanreotide acetate (1) having desired purity.

2. A process for the solution phase synthesis of H-Lys(Boc)-Val-Cys(Trt)-Thr(t-Bu)-NH2 (Fragment A), comprising reaction of H-L-Thr(t-Bu)-NH2 (4) with Fmoc-Cys(Trt)-OH (5) to give Fmoc-Cys(Trt)-Thr(t-Bu)-NH2 (6), deprotection, followed by reaction with Fmoc-Val-OH (8) to give Fmoc-Val-Cys (Trt)-Thr(t-Bu)-NH2 (9), which on deprotection, followed by reaction with Fmoc-Lys (Boc)-OH (11) gave Fmoc-Lys (Boc)- Val-Cys (Trt)-Thr(t-Bu)-NH2 (12), which on subsequent deprotection provided Fragment A.

3. A process for the solution phase synthesis of Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-OH (Fragment B) comprising reaction of H-D-Trp-O-All (14) with Boc-Tyr-OH (15) to give Boc-Tyr-D-Trp-OAll (16), deprotection, followed by reaction with Fmoc-Cys (Trt)-OH (5) to give Fmoc-Cys (Trt)-Tyr-D-Trp-OAll (18), which on deprotection, followed by reaction with Boc-D-Nal-OH (20) gave Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (21), which on removal of the allyl group gave Fragment B.

4. Compound of formula, H-Lys(Boc)-Val-Cys(Trt)-Thr(t-Bu)-NH2 (Fragment A).

5. Compound of formula, Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-OH (Fragment B).

6. Compound of formula Boc-D-Nal-Cys(Trt)-Tyr-(D)-Trp-Lys(Boc)-Val-Cys-(Trt)-Thr(t- Bu)-NH2 (22).

7. The process as claimed in claim 1, wherein the solvent is selected from methylene chloride, chloroform, dichloroethane, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, N-methyl-2-pyrrolidinone, acetonitrile, and combinations thereof.

8. The process as claimed in claim 1, wherein the coupling agent is selected from diisopropylcarbodiimide, dicyclohexylcarbodiimide, l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (ED AC), BOP(Benzotriazol-l-yloxy-tris(dimethylamino) -phosphonium hexafluorophosphate).

9. The process as claimed in claim 1, wherein the base is selected from diisopropylethylamine, N-methylmorpholine, triethylamine, diethylamine, piperidine and N-methylpyrrolidine.

10. The process as claimed in claim 3, wherein the deprotection of allyl group is carried out using tetrakis (triphenylphosphine) palladium.

11. Compounds of formulae Fmoc-Cys(Trt)-Thr(t-Bu)-NH2 (6),

H-Cys(trt)-Thr(t-Bu)-NH2 (7)

Fmoc-Val-Cys (Trt)-Thr(t-Bu)-NH2 (9),

H-Val-Cys (Trt)-Thr(t-Bu)-NH2 (10), and

Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)-NH2 (12).

12. Compounds of formulae Boc-Tyr-D-Trp-OAll (16),

H-Tyr-D-Trp-OAll (17),

Fmoc-Cys(Trt)- Tyr-D-Trp-OAll (18),

H-Cys(Trt)- Tyr-D-Trp-OAll (19) and

Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (21).

Description:
'PROCESS FOR PREPARATION OF LANREOTIDE ACETATE'

This application claims the priority of Indian Provisional Application No. 201721036985 filed on 18 th October 2017, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an improved process for solution phase synthesis of an octapeptide, Lanreotide acetate and its key intermediates comprising coupling of suitably protected tetrapeptide fragments A and B, followed by steps of deprotection, oxidation and acetic acid treatment to provide Lanreotide acetate (1) having desired purity.

BACKGROUND OF THE INVENTION

Lanreotide acetate (1), chemically known as [cyclo S-S]-3-(2-naphthyl)-D-alanyl-L- cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl -L-threoninamide acetate salt wherein the acetic acid range is 1.6 to 3.4, is a synthetic, cyclical octapeptide analog of the natural hormone, somatostatin. The amino acid sequence for the octapeptide is represented as follows,

S S

D-pNai-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH 2> x(CM 3 COOH) where x = 1.6 to 3.4

Lanreotide acetate (1) x= 1.6 to 3.4

Lanreotide acetate, developed by Ipsen with proprietary name Somatulin (depot) was first approved by USFDA on August 30, 2007 as an injection with a strength of 60 mg/0.2 ml or 90 mg/0.3 ml. It is indicated for long-term treatment of acromegaly and in the treatment of patients with locally advanced or metastatic gastroenteropancreatic neuroendocrine tumors.

Lanreotide acetate was first disclosed in US 4,853,371 wherein the synthetic process comprised treating benzhydrylamine -polystyrene resin (neutralized in the chloride ion form) with Boc-O-benzyl-threonine in presence of diisopropylcarbodiimide with the resulting amino acid resin coupled successively with Boc-S-methylbenzyl-Cys, Boc-Val, Boc-Ne-benzyloxycarbonyl-lysine, Boc-D-Trp, Boc-Tyr, Boc-S- methylbenzyl-Cys, and Boc-D-P-naphthylalanine. Further treatment of the resin with anisole, anhydrous hydrogen fluoride and precipitation in ether provided the crude peptide, which when treated with acetic acid, iodine in methanol, followed by HPLC purification and lyophilization provided the desired octapeptide, Lanreotide acetate.

Later. EP 389180, WO 8904666 disclosed a similar synthetic process for Lanreotide. However, in these methods, the resultant octapeptide is iodinated using reagents such as Chloramine-T/ sodium iodide; Lactoperoxidase-glucose oxidase (LP-GO)/ sodium iodide; Iodine/ potassium iodide; Iodine monochloride etc. followed by purification using preparative HPLC.

WO 2013098802 discloses a solid phase peptide synthesis of Lanreotide comprising use of resin-bound Thr-amide. The resin, Fmoc-Thr(Resin)-NH2 « DIPEA Fmoc-Thr- NH 2 was subjected to seven cycles of sequential deprotection and coupling steps to give Boc-D-2-Nal-Cys(Trt)-Tyr(Clt)-D-Trp-Lys(Mtt)-Val-Cys(Trt)-Th r(Resin)-NH 2 which, after the deprotection reaction followed by cleavage from the resin and simultaneous iodine oxidation yielded the desired compound.

CN 104497130 discloses a process wherein a combination of solid and liquid phase peptide synthesis methods is used to obtain Lanreotide.

The conventional synthesis of peptides is divided into two major types, solid phase and solution-phase peptide synthesis. Solid phase peptide synthesis methods, as mentioned above, comprises attachment of a C-terminal amino acid to resin, with step by step building up of the peptide chain by utilizing pre-activated amino acids. These methods involve use of expensive resins and Fmoc/tert-butyl protected amino acids in three to four fold excess, necessitating complex purification procedures to separate the product from the impurities. These additional steps before isolation render these processes unsuitable for large scale industrial production of the product.

Solution phase synthesis methods for peptides, on the other hand, comprise independent synthesis of amino acids segments or blocks, followed by condensation of various segments in the desired sequence in solution. Such processes are comparatively economical and hence more suited for synthesis on industrial scale.

It is now evident that most of the synthetic methods disclosed in the aforementioned references involve use of expensive resins, costly reagents, elaborate deprotection and separation procedures at various intermediate stages of synthesis, purification at various stages and inability for removal of impurities at work-up stage. Hence, there is a need for a convenient and economical synthetic process for Lanreotide acetate which involves solution phase synthetic approach comprising synthesis of fragments followed by condensation reaction with specific, easily detachable protecting groups and mild selective reagents to achieve the desired conversions.

Based on the shortcomings faced by inventors while trying out prior art processes with the amino acid fragments having different lengths of sequences, the present inventors developed an economical and convenient process for solution phase synthesis of Lanreotide acetate ( 1 ) which provides the desired molecule in good yield and overcame the problems faced in the prior art. The use of 4+4 strategy comprising synthesis of two tetrapeptide fragments, clubbed with highly specific protection and deprotection methods, convenient processes for isolation, purification of the intermediates and a facile condensation of the fragments facilitated in obtaining the desired molecule in fewer synthetic steps with significant yield improvement as compared to prior art processes. It was also found that in prior art, the threonine moiety which was used without protection of the hydroxy group, gave rise to unidentified impurities which were difficult to remove during work up and subsequently required several purifications. Simultaneously, the inventors also worked on the processes for preparation of intermediates at each step, whereby impurities were removed during the isolation. These steps resulted in a yield increase of about 10-15% which helped the commercial scale up and reduced the overall cost considerably.

OBJECT OF THE INVENTION

An objective of the present invention is to provide an industrially applicable, convenient process for solution phase synthesis of Lanreotide acetate (1), which avoids use of expensive resins, costly reagents in solid phase peptide synthesis and also lengthy reaction sequences and relies on protection of key functional groups for obtaining a product having high purity.

Another object of the invention relates to a 4+4 solution phase synthesis of Lanreotide acetate comprising suitably protected tetrapeptide fragments, mild reagents and moderate reaction conditions for synthesis and coupling of fragments to provide the desired compound possessing purity conforming to regulatory specifications. SUMMARY OF THE INVENTION

An aspect of the invention relates to a solution phase synthetic process for Lanreotide acetate (1) comprising coupling of two suitably protected tetrapeptide fragments, to give an octapeptide intermediate, which, when subjected to deprotection, oxidation and acetic acid treatment provides Lanreotide acetate having desired purity.

Yet another aspect of the invention relates to synthesis of the tetrapeptide fragments, H-Lys (Boc)-Val-Cys(Trt)-Thr(t-Bu)-NH 2 (Fragment A) and Boc-D-Nal-Cys (Trt)- Tyr-D-Trp-OH (Fragment B) followed by their coupling in presence of a suitable coupling agent in an organic solvent to give the octapeptide, which on subsequent deprotection, oxidation, followed by treatment with acetic acid gives Lanreotide acetate (1) having purity conforming to regulatory specifications.

The objectives of the present invention will become more apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

While carrying out extensive experimentation aimed at designing a convenient, industrially applicable solution phase synthetic strategy for Lanreotide acetate, the present inventors surprisingly found that synthesis of two suitably protected tetrapeptide fragments followed by a facile condensation reaction provided the desired octapeptide, which after further reactions provided Lanreotide acetate in good yield.

It was also found that in prior art, the threonine moiety which was used without protecting the hydroxyl group, gave rise to unidentified impurities which were difficult to remove during work up and subsequently required several purifications. Simultaneously, the inventors also worked on the processes at each step whereby impurities were removed during isolation. These steps resulted in an increase of about 10-15% in yield which helped the commercial scale up and reduced the overall cost considerably.

The inventors also unexpectedly found that most of the intermediates in the said strategy were obtained as solids, due to which several laborious and cumbersome intermediate isolations and purification steps were avoided. This not only ensured significantly higher yield for the desired compound, but also led to a convenient and economical synthetic process for Lanreotide acetate which could easily be scaled up for commercial production. In the synthesis of tetrapeptide Fragment B, allyl (-CH 2 - CH=CH 2 ) protection of the tryptophan carbonyl which could be deprotected using Palladium (0) catalyst avoided use of bases like lithium hydroxide, thus significantly minimizing the problems of racemization which are very commonly observed in the polypeptide solution phase synthesis.

The present strategy also comprises selective and specific, yet labile protecting groups at different stages, which are deprotected easily using acids, that do not adversely affect the chirality of the amino acids and intermediates in the synthetic sequence.

Further advantages of the process are as given below. a) Use of tertiary butyl protection for hydroxyl group in Threonine avoids side reactions, reduces impurity formation at intermediate levels, and consequently in the final product. The significant reduction in the level of unidentified impurities caused the improved the yield by 10 to 15%.

b) An effective method for Fmoc deprotection ensures significant reduction in the associated by-products, especially in case of intermediates such as H- Cys(Trt)-Thr(t-Bu)-NH 2 , obtained from Fmoc-Cys(Trt)-Thr(t-Bu)-NH 2 .

c) Due to appropriate selection of protecting groups, majority of the intermediates are solids which can be conveniently isolated and purified, avoiding steps of solvent addition, precipitation as in case of oily, semi-solid intermediates.

d) Treatment of intermediates using various solvent combinations provides easy method for purification, which results in reduction of associated impurities during their respective coupling reactions and also in the final product.

e) Selection of solvents such as NMP during coupling reaction decreases racemization, reduces impurity formation and hence provides advantages in terms of purity and increased yield,

f) Use of bases such as TEA, diisopropylethylamine, N-methylmorpholine is avoided during coupling reactions.

g) The process provides crude lanreotide having more than 90% purity, which is further purified to possess purity greater than 99%.

In addition, the present invention discloses a process for preparation of Fmoc-Thr(t- Bu)-NH 2 from Fmoc-Thr(t-Bu)-OH using inorganic reagents such as ammonium carbonate, ammonium acetate etc. wherein the amination reaction is carried out without deprotecting the N-Fmoc group. The details of synthetic process are provided in Scheme- 1, Scheme-2 and Scheme-3. The syntheses of tetrapeptide Fragments A and B are depicted in Scheme- 1 and Scheme-2 while condensation of Fragments A and B followed by deprotection and oxidation to afford Lanreotide acetate is presented in Scheme-3.

ABBREVIATIONS

Fmoc = Flourenylmethoxycarbonyl

Trt = Triphenyl methyl (Trityl)

Tbu = Tert-butyl

THF = Tetrahydrofuran

DMF = N, N- Dimethylformamide

DMSO = Dimethyl sulfoxide

DMAc = N, N- Dimethylacetamide

NMM = N-methylmorpholine

TEA = Triethylamine

DEA = Diethylamine

Bz = Benzyl

TFA = Trifluoroacetic acid

EDT = Ethanedithiol

TIS = Triisopropylsilane

HOBt = 1-Hydroxybenzotriazole

DCM = Dichloromethane

EDAC= 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide

HPLC= High performance liquid chromatography

TLC = Thin layer chromatography

PTSA= p-toluene sulfonic acid

CSA = camphorsulfonic acid

DTT =dithiothreitol

MTBE =Methyl tertiary butyl ether

NMP = l-methyl-2-pyrrolidinone

DMAP =dimethylamino pyridine

HC1= Hydrochloric acid 2 (4)

Fmoc-Cys(Trt)-Thr(t-Bu)NH 2 (6) H-Cys(Trt)-Thr(t-Bu)NH.

Fmoc-Val-Cys(Trt)-Thr(t-Bu)NH 2 (9) H-Val-Cys(Trt)-Thr(t-Bu) N H 2

NHFmoc

HOOC "NHBoc

Fmoc-Lys-(Boc)-OH (11) coupling

Fmoc-Lys-(Boc)-Val-Cys(Trt)-Thr(t-Bu)NH 2 (12)

H-Lys-(Boc)-Val-Cys(Trt)-Thr(t-Bu)NH 2 (Fragment A)

Scheme 1: Method embodied in the present invention for preparation of Fragment A

H-Cys(Trt)-Tyr-D-Trp-OAII (19)

Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-OAII

Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-OH Fragment B

Scheme 2: Method embodied in the present invention for preparation of Fragment B

Boc-D-Nal-Cys(Trt)-Tyr-D-Trp

-Lys-(Boc)-Val-Cys(Trt)-Thr(t-Bu)NH,

Lanreotide acetate (1 ) x= 1.6 to 3.4

Scheme 3: Method embodied in the present invention for preparation of Lanreotide acetate (1)

In an embodiment, Fmoc-Thr(t-Bu)-OH (2) was treated with an ammonium salt in an organic solvent and in presence of EDAC.HC1, HOBt to give Fmoc-Thr(t-Bu)-NH 2 (3). The solvent was selected from polar aprotic solvents such as DMF, DMSO etc., esters like ethyl acetate, butyl acetate etc. and mixtures thereof. The ammonium salt was selected from ammonium carbonate, ammonium acetate, ammonium formate, ammonium chloride etc. Compound (3) was further subjected to Fmoc deprotection using a suitable base like TEA in a solvent such as acetonitrile, DMF etc. to give H- L-Thr(t-Bu)-NH 2 (4), which was optionally treated with an organic solvent selected from hydrocarbons such as pentane, n-hexane, cyclohexane, heptane, toluene, ethers like diethyl ether, methyl tertiary butyl ether etc. and mixtures thereof.

Coupling of (4) with Fmoc-Cys (Trt)-OH (5) in presence of a coupling agent, base in a suitable solvent selected from aprotic solvents like DMF, acetonitrile gave Fmoc- Cys(Trt)-Thr(t-Bu)-NH 2 (6). The coupling reaction was carried out in the temperature range of 0 to 30°C and the resulting product was optionally treated with water, hydrocarbons such as pentane, n-hexane, cyclohexane, heptane, toluene, etc. and mixtures thereof.

Fmoc deprotection of (6) using a suitable base like DEA, TEA and an organic solvent afforded H-Cys(Trt)-Thr(t-Bu)-NH 2 (7). The solvent was selected from aprotic solvents like DMF, DMSO, acetonitrile or esters such as ethyl acetate, butyl acetate etc., while the reaction was carried out in the temperature range of 10 to 45°C. The product was optionally treated with organic solvent selected from hydrocarbons such as pentane, n-hexane, cyclohexane, heptane and toluene; esters like ethyl acetate, butyl acetate etc. and mixtures thereof.

Compound (7) on further coupling with Fmoc-Val-OH (8) in an organic solvent in presence of a coupling agent and base gave Fmoc-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (9). The reaction was carried out in the temperature range of 0 to 30°C and the solvent was selected from aprotic solvents like DMF, DMSO, acetonitrile etc. The product was optionally treated with water, hydrocarbon selected from pentane, n-hexane, cyclohexane, heptane, toluene, etc. and mixtures thereof.

Fmoc deprotection of (9) using a suitable base like DEA, TEA and an organic solvent afforded H-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (10). The solvent was selected from aprotic solvents like DMF, DMSO, acetonitrile etc., ethers such as diethyl ether, methyl tertiary butyl ether etc. Compound (10) was optionally treated with water, a hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, and toluene, esters like ethyl acetate, butyl acetate etc. and mixtures thereof.

Further coupling of (10) with Fmoc-Lys(Boc)-OH (11) in an organic solvent selected from aprotic solvents like DMF, DMSO, acetonitrile etc., in presence of a coupling agent and base gave Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (12). Compound (12) was optionally purified using solvents such as methanol, acetonitrile, water, hydrocarbon selected from pentane, n-hexane, cyclohexane, heptane, toluene and combinations thereof.

Fmoc deprotection of (12) using a suitable base like DEA, TEA in an organic solvent provided H-Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (Fragment A). The solvent was selected from aprotic solvents like DMF, DMSO, acetonitrile etc. Fragment A was optionally treated with water, a hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, and toluene, esters like ethyl acetate, butyl acetate etc. and mixtures thereof.

In a further embodiment, D-Tryptophan (H-D-Trp-OH, 13) was treated with allyl alcohol in presence of an acid to give the allyl ester, H-D-Trp-OAll (14). The acid was selected from PTSA, CSA and the like. In case of CSA, it was optionally used in combination with DMAP. The allyl ester (14) was optionally treated with a solvent selected from ethers like diethyl ether, methyl tertiary butyl ether etc., esters like ethyl acetate, butyl acetate etc. and combinations thereof.

Further coupling of (14) with Boc-Tyr-OH (15) in presence of a coupling agent and a suitable organic solvent selected from esters such as ethyl acetate, butyl acetate etc., polar aprotic solvents like DMF, DMSO, NMP etc. and combinations thereof gave Boc-Tyr-D-Trp-O-All (16).

Boc deprotection of (16) using suitable acid like acetic acid, hydrochloric acid, trifluoroacetic acid and combinations thereof in organic or aqueous solvent afforded H-Tyr-D-Trp-OAll (17) as its acid addition salt. The solvent was selected from esters like ethyl acetate, butyl acetate etc. and combinations thereof. Coupling of compound (17) with Fmoc-Cys (Trt)-OH (5) using an organic solvent selected from DMF, DMSO, DMAc etc. in presence of a coupling agent and a base at 0 to 30°C gave Fmoc-Cys (Trt)-Tyr-D-Trp-OAll (18).

Fmoc deprotection of (18) using a suitable base like DEA, TEA and an organic solvent selected from halogenated hydrocarbons, esters such as ethyl acetate, butyl acetate etc. at 25 to 50°C afforded H-Cys (Trt)-Tyr-D-Trp-O-All (19), which was optionally treated with water, a hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof.

Compound (19), when coupled with Boc-D-Nal-OH (20) in presence of a coupling agent and a base in a suitable organic solvent provides Boc-D-Nal-Cys (Trt)-Tyr-D- Trp-OAll (21). The solvent was selected from polar aprotic solvents like acetonitrile, DMF, DMSO etc. and the reaction was carried out between 0 to 30°C.

In a further embodiment, allyl deprotection of (21) using a zero valent palladium reagent like tetrakis (triphenylphosphine)palladium, optionally in presence of morpholine, in an organic solvent such as DMSO, DMF or DMAc at 0 to 30°C furnished Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (Fragment B).

In yet another embodiment, coupling of Fragment A and Fragment B in presence of a coupling agent, in a suitable organic solvent furnished the octapeptide, Boc-D-Nal- Cys (Trt)-Tyr-D-Trp- Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (22). The solvent was selected from aprotic solvents like DMF, DMSO, acetonitrile etc. Compound (22) on treatment with an acid like trifluoroacetic acid, triethylsilane (TES), dithiothreitol (DTT) and anisole in a suitable organic solvent at 0 to 30°C, followed by reaction with iodine and acetic acid afforded the final product which was purified to yield Lanreotide acetate (1). The solvent was selected from halogenated hydrocarbons such as dichlorome thane, ethylene dichloride, methylene chloride etc.

Organic solvents that can be used were selected from the group comprising chlorinated hydrocarbons, aprotic solvents, ethers, esters and nitriles. Examples of these solvents are methylene chloride, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, tetrahydrofuran, ethyl acetate, l-methyl-2- pyrrolidinone, acetonitrile, or combinations thereof. The coupling agents were selected from the group comprising of substituted carbodiimides such as diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1 -Ethyl - 3-(3-dimethylaminopropyl)carbodiimide (ED AC), BOP (Benzotriazol- 1 -yloxy- tris(dimethylamino)phosphoniumhexafluorophosphate), PyBOP(Benzotriazol-l- yloxy-tripyrrolidino-phosphoniumhexafluorophosphates), PyBrOP (Bromotripyrrolidino phosphoniumhexafluorophosphate), PyAOP (7-Aza- benzotriazol-l-yloxy-tripyrrolidinophosphonium hexafluorophosphate), DEPBT (3- (Diethoxyphosphoryloxy)-l,2,3-benzo[d]triazin-4(3H)-one), TBTU (2-(lH- Benzotriazol-l-yl)-N,N,N',N' -tetramethylaminium tetrafluoroborate), HBTU (2-(lH- Benzotriazol-l-yl)-N,N,N',N' -tetramethylaminium hexafluoroborate), HATU (2-(7- Aza- 1 H-benzotriazol- 1 -yl)-N,N,N' ,Ν' -tetramethylaminium hexafluorophosphate), COMU ( 1 -[ 1 -(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino- morpholino]-uroniumhexafluorophosphate), HCTU(2-(6-Chloro- 1 H-benzotriazol- 1- yl)-N,N,N',N' -tetramethylaminium hexafluorophosphate) and TFFH (Tetramethylfluoroformamidinium hexafluorophosphate).

The bases were selected from the group comprising of Diisopropyl ethyl amine (DIPEA), N-methylmorpholine (NMM), triethyl amine, diethyl amine, N- methylmorpholine, piperidine, N-methylpyrrolidine. The acid employed for deprotection was selected from the group comprising of trifluoroacetic acid; either neat or in solvent, hydrogen chloride gas dissolved in ethyl acetate, acetonitrile or dioxane. The following examples are meant to be illustrative of the present invention. These examples exemplify the invention and are not to be construed as limiting the scope of the invention. EXAMPLES

Example 1: Synthesis of of Fmoc-Cys (Trt)-Thr(t-Bu)-NH 2 (6)

Fmoc-Thr(t-Bu)-OH (2) (1 mmol) was treated with EDAC.HC1 (1.4mmol), HOBt (1.3mmol) and ammonium acetate, using DMF(6 volume) and ethyl acetate (5 volume) as solvents to give Fmoc -Thr(t-Bu)- NH 2 (3). Further reaction of (3) with TEA (3 mmol), followed by treatment with MTBE hexane gave H-L-Thr(t-Bu)-NH 2 (4).

The solution of Fmoc-Cys (Trt)-OH (5, 100.0 g) in DMF (350 ml) was stirred under nitrogen atmosphere, the reaction mixture was cooled to 0 to 10°C and H-L-Thr(t- Bu)-NH 2 (4, 48.5 g), and HOBt (32.3 g) were added to it, followed by addition of EDAC.HC1 (42.5 g) and NMM (45.8 g). The resulting mass was stirred in the temperature range of 0 to 25°C. After completion of the reaction, as monitored by TLC, HPLC, the stirred reaction mixture was quenched with dilute HC1. The precipitated solid was filtered, optionally treated with water and cyclohexane to give Fmoc-Cys(Trt)-Thr(t-Bu)-NH 2 (6).

Yield: 116.91 g, (92.3%), Purity: > 95% (HPLC)

Example 2: Preparation of Fmoc-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (9)

TEA (31.7 g) was added to the solution of Fmoc-Cys (Trt)-Thr(t-Bu)-NH 2 (6, 100.0 g) in DMF (300 ml) and the reaction mass was stirred between 25 and 45 °C. After completion of reaction, as monitored by TLC, HPLC, the mixture was quenched by gradually adding IN HC1. Filtration, neutralization, extraction of the aqueous layer with ethyl acetate, followed by separation and concentration of the organic layer gave H-Cys (Trt)-Thr(t-Bu)-NH 2 (7). The solution of compound (7) in DMF (150 ml) was gradually added to the mixture of Fmoc-Val-OH (8, 47.6 g) in DMF (50 ml), HOBt (25.5 g), EDAC.HCl (34.0 g) and NMM (19.1 g); stirred at 0 to 10 °C. The resulting reaction mass was stirred at 0 to 30°C, till completion of reaction, as monitored by TLC, HPLC. The mass was quenched with 0.5 N HC1 with continued stirring. Filtration, neutralization of the precipitated solid, optional treatment with water and cyclohexane gave Fmoc-Val- Cys (Trt)-Thr(t-Bu)-NH 2 (9).

Yield: 103.2 g, (91.07%), Purity: > 95% (HPLC) Example 3: Preparation of Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (12)

TEA (29.3 g) was added to the solution of (9, 100.0 g) in DMF (300 ml) and the reaction mass was stirred between 25 and 45°C. After completion of reaction, as monitored by TLC, HPLC, the reaction mass was quenched by gradually adding dilute HC1 (50 ml HC1 in 450 ml water). Filtration, followed by neutralization, extraction of the filtrate with ethyl acetate gave an organic layer which was concentrated to give H-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (10).

NMM (11.4 g) was added to the mixture of Fmoc-Lys(Boc)-OH (11, 42.8 g), in DMF (116 ml), HOBt (16.8 g), EDAC.HCl (23.9 g) and stirred at 0 to 10°C. The solution of H-Val-Cys (Trt)-Thr(t-Bu)-NH 2 (10, 71.0 g) in DMF (150 ml) was further added to the resulting mass and the reaction mixture was stirred at 20 to 40°C till completion of reaction, as monitored by TLC, HPLC. After completion, the reaction mixture was quenched with dilute HC1. Filtration of the precipitated solid, optional treatment with water and cyclohexane gave Fmoc-Lys (Boc)-Val-Cys(Trt)-Thr(t-Bu)- NH 2 (12).

Yield: 120 g, (94.44%), Purity: > 85% (HPLC) Example 4: Preparation of H-Lys(Boc)-Val-Cys(Trt)-Thr(t-Bu)-NH 2 (Fragment

A)

TEA (21.3 g) was added to a mixture of Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr(t-Bu)- NH 2 (12, 90.0 g) in DMF (450 ml) at 20 to 30°C and the reaction mass was stirred between 30 and 45°C. After completion of reaction, as monitored by TLC, HPLC, the reaction mass was quenched by gradual addition of IN HCl. The reaction mixture was filtered, and the filtrate was neutralized. Extraction with ethyl acetate and concentration of the organic layer gave Fragment A.

Yield: 72.80 g (91.90%), Purity: > 85% (HPLC)

Example 5: Preparation of Boc-Tyr-D-Trp-OAll (16)

D-Tryptophan (H-D-Trp-OH, 13, 100.0 g), and PTSA.H 2 0 (186.3 g) were added to allyl alcohol (1000 ml) stirred at 25 to 35°C, followed by addition of toluene (500ml). The resulting mixture was stirred at 80 to 95°C till completion of reaction, as monitored by TLC, HPLC. After completion, the mass was cooled and 5% aqueous sodium bicarbonate solution was added to it. Extraction with ethyl acetate followed by separation and concentration of the organic layer gave a residue containing H-D-Trp-OAll (14).

Yield: 108.01 g (90.3%), Purity: > 95% (HPLC)

Boc-Tyr-OH (15, 115.2 g) was added to a stirred mixture of H-D-Trp-OAll (14, 100.0 g) and DMF (300 ml) at 25 to 35°C, followed by addition of HOBt (69. lg). NMM (50.0 g) and EDAC.HC1 (94.1 g). The reaction mixture was stirred at 10 to 30°C, till completion, as monitored by TLC, HPLC. The reaction mass was quenched with 0.5N HCl. The precipitated solid was filtered, treated with 5% aqueous sodium carbonate solution, filtered to give Boc-Tyr-D-Trp-OAll (16).

Yield: 190 g, 91%, Purity: > 90% (HPLC) Example 6: Preparation of Fmoc-Cys (Trt)-Tyr-D-Trp-OAll (18)

Boc-Tyr-D-Trp-OAll (16, 100.0 g) was added to the cooled mixture of anhydrous HCl in ethyl acetate (400 ml) under stirring and the reaction mass was stirred at 10 to 30°C till completion of the reaction as monitored by TLC, HPLC, the reaction mixture was concentrated to give H-Tyr-D-Trp-OAll (17) as its HCl salt.

Compound (17) was dissolved in DMF (260 ml) and Fmoc-Cys(Trt)-OH (5, 104.0 g). HOBt (33.2 g), NMM (44.1 g) were added to the mixture stirred at 25 to 30°C. EDAC.HC1 (42.0 g) was then added to the reaction mixture and stirring was continued at 0 to 30°C till completion, as monitored by TLC, HPLC. The reaction mixture was added to the cooled solution of 0.5M HCl and stirred at 0 to 5°C. The solid was filtered, treated with 5% aqueous sodium carbonate solution, followed by water washing to give Fmoc-Cys(Trt)-Tyr-D-Trp-OAll (18).

Yield: 154.01 g, (80.03%), Purity: > 90% (HPLC)

Example 7: Preparation of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (21)

TEA (83.0 g) was added to a mixture of Fmoc-Cys(Trt)-Tyr-D-Trp-OAll (18, 100.0 g) in DCM (1050 ml) and the reaction mass was stirred at 20 to 30°C till completion of the reaction, as monitored by TLC, HPLC. After completion, the reaction mass was quenched with water and the organic layer was separated. Concentration of the organic layer, followed by optional treatment of resultant solid using toluene: cyclohexane mixture gave H-Cys (Trt)-Tyr-D-Trp-OAll (19) as solid.

Yield: 65.6 g (85%), Purity: > 90% (HPLC). Boc-D-Nal-OH (20, 22.6 g) was added to the stirred mixture of H-Cys (Trt)-Tyr-D- Trp-OAll (19, 60.0 g) in DMF (180 ml) at 0 to 30°C, followed by addition of HOBt (11.8 g) and NMM (18.0 g). EDAC.HC1 (17.0 g) was then added to the reaction mixture and stirring was continued at 0 to 30°C till completion of the reaction, as monitored by TLC, HPLC. After completion, the reaction mixture was filtered, added to the cooled solution of 0.5M HC1 and stirred at 25 to 30°C. The solid was filtered, treated with 5% aqueous sodium carbonate solution, followed by an optional treatment with toluene: cyclohexane (30:70) mixture to give Boc-D-Nal-Cys (Trt)- Tyr-D-Trp-OAll (21).

Yield: 66.2 g, (79.06%), Purity: > 80% (HPLC)

Example 8: Preparation of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (Fragment B)

Morpholine (25.1 g) and tetrakis (triphenylphosphine)palladium (3.3g) were added to the mixture of Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-OAll (21 , 60.0 g) in DMSO (240 ml). The reaction mixture was stirred at 15 to 30°C, till completion of the reaction, as monitored by TLC. After completion, the reaction mass was filtered and quenched with dilute HC1. The obtained solid was filtered, and the wet cake was treated with water, followed by an optional treatment with toluene: cyclohexane mixture. The solid thus obtained was optionally further treated with cyclohexane to give Boc-D- Nal-Cys (Trt)-Tyr-D-Trp-OH (Fragment B).

Yield: 48.0 g (83.02%), Purity: > 90% (HPLC)

Example 9: Preparation of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-Lys (Boc)-Val-Cys (Trt)-Thr (t-Bu)-NH 2 (22)

The mixture of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (Fragment B, 45.0 g) in DMF (100 ml) was stirred at 0 tol0°C and HOBt (9.13 g), EDAC.HC1 (11.1 g), NMM (6.5 g) were added to it with continued stirring. A mixture of H-Lys (Boc)-Val-Cys (Trt)- Thr(t-Bu)-NH 2 (Fragment A, 37.7 g) in DMF (100 ml) was cooled to 0 to 10°C and added to the mixture and stirring was continued at 10 to 30°C till completion of the reaction, as monitored by TLC, HPLC. The reaction mixture was then quenched with cooled solution of 0.5N HC1. The stirring was continued at 15 to 25°C and the precipitated solid was filtered. MTBE treatment of the solid at 30-40°C and filtration provided the octapeptide, Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-Lys (Boc)-Val-Cys (Trt)- Thr(t-Bu)-NH 2 (22).

Yield: 66.69 g, 81.4%, Purity: > 80% (HPLC). Example 10: Preparation of Lanreotide acetate (1)

DTT (17.3 g), TES (50 ml), TFA (300 ml), and anisole (24.3 g) were added to the stirred mixture of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-Lys (Boc)-Val-Cys (Trt)-Thr(t- Bu)-NH 2 (22, 50.0 g) in MDC (300 ml). The resultant mass was stirred at 25 to 30°C till completion of the reaction, as monitored by TLC. After completion, the reaction mass was concentrated, the obtained oily residue was treated with MTBE under stirring and the solid was filtered.

A mixture of acetic acid (60 ml), water (2380 ml) was added to the solution of the filtered solid in 50% aqueous acetonitrile (2500 ml) which was stirred at 15-30°C, followed by addition of iodine (12.7 g) in methanol (290 ml). The stirring was continued till completion of the reaction, as monitored by HPLC.

After completion, L-ascorbic acid (9.5 g) was added to the mixture. The resultant mass was filtered and purified using preparative HPLC to give Lanreotide acetate (1). Yield: 9.7 g (29.9%), Purity: > 99 (HPLC).