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

BACKGROUND OF THE INVENTION
Lanreotide acetate (1) is 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 ranges from 1.6 to 3.4. Lanreotide is a synthetic, cyclical octapeptide analog of the natural hormone, somatostatin and the amino acid sequence for the octapeptide is represented as follows,

Lanreotide acetate is indicated for long-term treatment of acromegaly and in the treatment of patients with locally advanced or metastatic gastroenteropancreatic neuroendocrine tumors.


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 strength of 60 mg/0.2 ml or 90 mg/0.3 ml.

Lanreotide acetate was first disclosed in US 4,853,371 wherein the synthetic process comprised treating benzhydryl amine-polystyrene resin (neutralized in the chloride ion form) with Boc-O-benzyl-threonine in presence of diisopropylcarbodiimide and the resulting amino acid resin is then 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-ß-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 provides 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 wherein the resin, Fmoc-Thr(Resin)-NH2 « DIPEA Fmoc-Thr-NH2 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)-Thr(Resin)-NH2 which after the deprotection reaction followed by cleavage from the resin and simultaneous iodine oxidation yielded the desired compound.

CN104497130 discloses a process wherein a combination of solid and liquid phase peptide synthesis methods was 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 a 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 synthetic methods for peptides, on the other hand, comprises independent synthesis of amino acids segments or blocks having the desired sequence, followed by condensation of these segments 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. Hence, there is a need for a convenient and economical process which involves utilization of peptide fragments that is developed in a facile manner using specific, selective, easily detachable, bulky protecting groups, as well as mild and selective reagents for coupling and deprotection to achieve the desired conversions. Use of bulky protecting groups gives solid intermediates which are easily isolated from the reaction mixtures and purified using simple techniques. Further, it was found that by a combination of different fragment blocks and liquid phase synthesis lead to reduced formation of associated impurities as compared to prior art methods.

The present inventors have developed an economical and convenient process for solution phase synthesis of Lanreotide acetate (1) which provides the desired molecule in good yield overcoming 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 and a facile condensation of the fragments facilitates in obtaining the desired molecule in fewer synthetic steps with significant yield improvement as compared to prior art processes.

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 elaborate protection, deprotection, purification methods.

Another object of the invention relates to a 4+4 solution phase synthesis of Lanreotide acetate comprising mild reagents and with facile functional group protection and deprotection in moderate reaction conditions to provide the intermediates and finally Lanreotide having desired purity.

SUMMARY OF THE INVENTION
An aspect of the invention relates to a 4+4 solution phase synthetic process for Lanreotide acetate (1) comprising coupling of two suitably protected tetrapeptide fragments, followed by deprotection, oxidation and acetic acid treatment to give Lanreotide acetate having desired purity.

Yet another aspect of the invention relates to synthesis of Lanreotide acetate comprising reaction of tetrapeptide H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (fragment A) with Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (fragment B) in presence of a coupling agent, a base and 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, the present inventors unexpectedly found that synthesis of two tetrapeptide fragments followed by a facile condensation reaction provided the desired octapeptide, Lanreotide in good yield.

The inventors also serendipitously found that most of the intermediates in the said strategy were obtained as solids, due to which various laborious and cumbersome intermediate isolation and purification steps were avoided. This not only ensured notably 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. Further, during the synthesis of tetrapeptide fragment B, the allyl (-CH2-CH=CH2) protection of the tryptophan carboxyl was easily deprotected by using Palladium (0) catalyst and avoiding 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 utilization of selective and specific, yet labile protecting groups at different stages, which are deprotected using mild acids, that do not adversely affect the chirality of the amino acids and intermediates in the synthetic sequence.

The synthetic process for obtaining Lanreotide acetate (1) is provided in Scheme-1 (tetrapeptide fragment A), Scheme-2 (tetrapeptide fragment B) and Scheme-3 (condensation of fragments A and B followed by deprotection and oxidation).

ABBREVIATIONS
Fmoc = Flourenylmethoxycarbonyl
Trt = Triphenyl methyl (Trityl)
Tbu = Tert-butyl
THF = Tetrahydrofuran
DMF = N, N- Dimethylformamide
NMM = N-methylmorpholine
TEA = Triethylamine
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
DTT =Dithiothreitol

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

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

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

In an embodiment, L-Threonine amide (2) was coupled with Fmoc-Cys (Trt)-OH (3) in a suitable solvent in presence of a coupling agent and a base such as NMM to give Fmoc-Cys (Trt)-Thr-NH2 (4). The coupling reaction was carried out in the temperature range of 0 to 30°C and in a solvent selected from polar aprotic solvents like DMSO, DMF, DMAc etc. After completion, the reaction mass was quenched using mineral acid like hydrochloric acid to precipitate the intermediate (4), which was filtered and optionally treated with water and a hydrocarbon solvent prior to drying. The hydrocarbon solvent was selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof.

Compound (4) was treated with a suitable base like TEA in an organic solvent for deprotection of the Fmoc group to afford H-Cys(Trt)-Thr-NH2 (5). The solvent was selected from polar aprotic solvents like DMSO, DMF, and DMAc while the reaction was carried out in the temperature range of 0 to 30°C. After completion, the reaction mass was quenched using mineral acid followed by filtration, neutralization and extraction with a water immiscible organic solvent. The water immiscible organic solvent was selected from ethers such as MTBE, diethyl ether, diisopropyl ether, halogenated hydrocarbons such as dichloromethane, ethylene dichloride and esters such as ethyl acetate, butyl acetate. Separation of the organic layer and concentration provided compound (5).

Coupling of (5) with Fmoc-Val-OH (6) in an organic solvent in presence of a coupling agent and a base like NMM gave Fmoc-Val-Cys(Trt)-Thr-NH2 (7). The reaction was carried out in the temperature range of 0 to 30°C. After completion, the reaction mass was quenched using mineral acid to precipitate the intermediate, which was filtered and treated with water and a hydrocarbon solvent prior to drying. The hydrocarbon solvent was selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof.
Fmoc deprotection of (7) using a suitable base like TEA in an organic solvent such as DMF afforded H-Val-Cys (Trt)-Thr-NH2 (8). The reaction was carried out in temperature range of 0 to 30°C. After completion, the reaction mass was quenched using mineral acid followed by filtration, neutralization, extraction with a water immiscible organic solvent selected from ethers such as MTBE, diethyl ether, diisopropyl ether, halogenated hydrocarbons such as dichloromethane, ethylene dichloride and esters such as ethyl acetate, butyl acetate. Separation of the aqueous layer and concentration provided (8).

Further coupling of compound (8) with Fmoc-Lys (Boc)-OH (9) in an organic solvent selected from DMF, DMSO etc., in presence of a coupling agent and a base such as NMM gave Fmoc-Lys(Boc)-Val-Cys(Trt)-Thr-NH2 (10). After completion, the reaction mass was quenched using mineral acid to precipitate the intermediate, which was filtered and treated with water and a hydrocarbon solvent prior to drying. The hydrocarbon solvent was selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof. Compound (10) was subjected to Fmoc deprotection using a suitable base like TEA in an organic solvent selected from DMF, DMSO etc., to afford H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (Fragment A). The reaction was carried out in the temperature range of 20 to 50°C. After completion of the reaction, the reaction mass was quenched with mineral acid such as hydrochloric acid and the resultant mass was filtered. Separation of the solid and drying provided Fragment A.

In a further embodiment, Boc-Tyr-OH (12) was coupled with allyl ester of D-Tryptophan, H-D-Trp-OAll (11) in presence of a coupling agent and a base such as NMM, and a suitable organic solvent selected from DMF, DMSO, DMAc etc., to give Boc-Tyr-D-Trp-OAll (13). After completion, the reaction mass was quenched using mineral acid like HCl to precipitate the intermediate, which was washed with aqueous alkali solution, followed by water washing, filtered and dried to give (13).
Boc deprotection of (13) using acid mixture such as anhydrous HCl in acetonitrile or ethyl acetate, or trifluoroacetic acid in dichloromethane afforded H-Tyr-D-Trp-OAll (14). The reaction was carried out at ambient temperature using anhydrous HCl in ethyl acetate and after completion, concentration of the reaction mixture provided a residue containing compound (14) as HCl salt.
Coupling of (14) with Fmoc-Cys(Trt)-OH (3) using an organic solvent selected from DMF, DMSO, DMAc etc., in presence of a coupling agent and a base like NMM at 0 to 30°C gave Fmoc-Cys (Trt)-Tyr-D-Trp-OAll (15). After completion, the reaction mass was quenched with a mineral acid to precipitate the solid, which was filtered, optionally treated with alkali solution, water and dried to give (15).
Fmoc deprotection of (15) in a halogenated hydrocarbon solvent like dichloromethane, using a suitable base such as TEA afforded H-Cys(Trt)-Tyr-D-Trp-OAll (16). The reaction was carried out at 0 to 30°C and after completion, quenching with water, followed by separation and concentration of the organic layer gave a residue. Treatment of the residue with hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof, followed by separation of solvent gave compound (16) as a solid.

Coupling of (16) with Boc-D-Nal-OH (17) in presence of a coupling agent and a base such as NMM or DIPEA in a suitable organic solvent furnished Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (18). The solvent was selected from polar aprotic solvents like acetonitrile, DMF, DMSO etc. and the reaction was carried out between 0 to 30°C. After completion, the reaction mass was filtered, quenched with a mineral acid to precipitate the intermediate, which was filtered, treated with alkali solution, followed by optional treatment with hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof, Further removal of solvent and drying gave compound (18).

In a further embodiment, allyl deprotection of (18) using the catalyst tetrakis(triphenylphosphine)palladium, morpholine and an organic solvent such as DMSO, DMF or DMAc at 0 to 30°C provided Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (Fragment B).

After completion of reaction, filtration, followed by treatment of filtrate with a mineral acid gave a solid which, after filtration, was washed with a hydrocarbon solvent such as toluene, cyclohexane and dried to provide fragment B.

In yet another embodiment, coupling of Fragment A and Fragment B in presence of a coupling agent, a base such as NMM and a suitable organic solvent like DMF furnished the octapeptide, Boc-D-Nal-Cys(Trt)-Tyr-D-Trp-Lys(Boc)-Val-Cys(Trt)-Thr-NH2 (19). After completion, the reaction mass was treated with a mineral acid and the precipitated solid was filtered. Treatment of the solid with a solvent selected from ethers such as MTBE, diethyl ether, diisopropyl ether and mixtures thereof provided compound (19).

Compound (19) was dissolved in a halogenated hydrocarbon solvent like dichloromethane and was treated with TFA, DTT and TES, in presence of anisole at 0 to 30°C to give (20). After completion, concentration of the reaction mixture gave a residue which was further treated with ether solvent like methyl tertiary butyl ether (MTBE) to give a solid after filtration. The solid was treated with aqueous acetic acid and iodine in presence of acetonitrile, followed by treatment with L-ascorbic acid to yield Lanreotide acetate (1).

The organic solvents were selected from the group comprising chlorinated hydrocarbons, aprotic solvents, ethers, esters and nitriles. Examples of these solvents are methylene chloride, chloroform, dichloroethane (EDC), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), ethyl acetate, N-methyl-2-pyrrolidinone (NMP), acetonitrile, and combinations thereof.

The coupling agent was selected from the group comprising substituted carbodiimides such as diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), BOP(Benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yloxy-tripyrrolidino-phosphonium-hexafluoro phosphate), PyBrOP (Bromotripyrrolidino phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate), DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one), TBTU (2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium tetrafluoroborate), HBTU (2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium hexafluoroborate), HATU (2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium hexafluorophosphate), COMU (1-[1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uroniumhexafluorophosphate), HCTU (2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminiumhexafluorophosphate) and TFFH (Tetramethylfluoroformamidinium hexafluorophosphate).

The base was selected from the group comprising diisopropylethylamine (DIEA), N-methylmorpholine (NMM), triethyl amine (TEA), diethyl amine (DEA), piperidine, l-methyl-2-pyrrolidinone (NMP). The acid employed for deprotection was selected from the group comprising trifluoroacetic acid either neat or in dichloromethane (DCM), 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-NH2 (4)
The solution of Fmoc-Cys (Trt)-OH (3, 100 g) in DMF (350 ml) was stirred under nitrogen atmosphere. The reaction mixture was cooled to 0 to 10°C and HOBt (32.30 g) and L-Threoninamide (2, 34.29 g), were added to it, followed by addition of EDAC.HCl (42.50 g) and N-methylmorpholine (45.76 g). Reaction mixture was stirred in the temperature range of 0 to 25°C. After completion of the reaction, as monitored by TLC, the stirred reaction mixture was quenched with dilute hydrochloric acid. The precipitated solid was filtered, optionally treated with water, cyclohexane and dried to give Fmoc-Cys (Trt)-Thr-NH2 (4).
Yield: 108 g, (92.3%)
Purity: > 95% (HPLC)

Example 2: Preparation of Fmoc-Val-Cys (Trt)-Thr-NH2 (7)
Triethylamine (36.88 g) was added to the solution of (4, 100 g) in DMF (300 ml) and the reaction mass was stirred between 25 and 45°C. After reaction completion, as monitored by TLC, the mixture was quenched by gradually adding 1N hydrochloric acid. Filtration, neutralization, extraction of the aqueous layer with ethyl acetate, followed by separation and concentration of the organic layer gave H-Cys (Trt)-Thr-NH2 (5).
The solution of compound (5) in DMF (150 ml) was gradually added to the mixture of Fmoc-Val-OH (6, 51.5 g) in DMF (50 ml), HOBt (27.24 g), EDAC.HCl (36.84 g) and N-methylmorpholine (20.40 g), stirred at 0 -10 °C. The resulting reaction mass was stirred at 0-30°C, till reaction completion, as monitored by TLC. The mass was quenched with 0.5 N hydrochloric acid with continued stirring. Filtration, neutralization of the precipitated solid, optional treatment with water, cyclohexane and drying gave Fmoc-Val-Cys (Trt)-Thr-NH2 (7).
Yield: 103.2 g, (91.07%)
Purity: > 95% (HPLC)

Example 3: Preparation of Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (10)
Triethylamine (32.22 g) was added to the solution of (7, 100 g) in DMF (300 ml) and the reaction mass was stirred between 25 and 45°C. After completion of reaction, as monitored by TLC, the reaction mass was quenched by gradually adding dilute hydrochloric acid (50 ml HCl 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-NH2 (8).
N-methylmorpholine (13.54 g) was added to the mixture of Fmoc-Lys(Boc)-OH (9, 45.88 g), in DMF (116 ml), HOBT (18.1 g), EDAC.HCl (23.7 g) and stirred at 0-100C. The solution of Val-Cys (Trt)-Thr-NH2 (8, 58.0 g) in DMF (150 ml) was further added to the resulting mass and the reaction mixture was stirred at 20-400C till completion of reaction, as monitored by TLC. After completion, the reaction mixture was quenched with dilute hydrochloric acid. Filtration of the precipitated solid, optional treatment with water, cyclohexane and drying gave Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (10).
Yield: 98 g, (94.44%)
Purity: > 85% (HPLC)

Example 4: Preparation of H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (Fragment A)
Triethylamine (22.46 g) was added to a mixture of (10, 90 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, the reaction mass was quenched by gradual addition of 1.0 N hydrochloric acid. The reaction mixture was filtered, and the filtrate was neutralized. Extraction with ethyl acetate and concentration of the organic layer gave H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (Fragment A).
Yield: 70.0 g (91.90%)
Purity: > 85% (HPLC)

Example 5: Preparation of Boc-Tyr-D-Trp-OAll (13)
D-Tryptophan (100 g), and PTSA.H2O (186.28 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 80-950C till completion of reaction, as monitored by TLC. 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 (11).
Yield: 108.01 g (90.3%)
Purity: > 95% (HPLC)

Boc-Tyr-OH (12, 115.15 g) was added to a stirred mixture of H-D-Trp-OAll (11, 100 g) and DMF (300 ml) at 25 to 35°C, followed by addition of HOBt (69.11g). NMM (50.0 g) and EDAC.HCl (94.06 g) were further added to it and the reaction mixture was stirred at 10 to 30°C, till completion, as monitored by TLC. The reaction mass was quenched with 0.5N HCl. The precipitated solid was filtered, treated with 5% aqueous sodium carbonate solution, filtered again and dried to give Boc-Tyr-D-Trp-OAll (13).
Yield: 190 g, 91%.
Purity: > 90% (HPLC)

Example 6: Preparation of Fmoc-Cys (Trt)-Tyr-D-Trp-OAll (15)
Compound (13, 100 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, the reaction mixture was concentrated and the obtained solid was dried to give H-Tyr-D-Trp-OAll (14) as its HCl salt.

Compound (14) was dissolved in DMF (260 ml) and Fmoc-Cys(Trt)-OH (3, 104.0 g). HOBT (33.2 g), NMM (44.1 g) were added to the mixture stirred at 25 to 30°C. EDAC.HCl (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. The reaction mixture was added to the cooled solution of 0.5M hydrochloric acid and stirred at 0 to 5°C. The solid was filtered, treated with 5% aqueous sodium carbonate solution, followed by water washing and dried to give Fmoc-Cys(Trt)-Tyr-D-Trp-OAll (15).
Yield: 154.01 g, (80.03%)
Purity: > 90% (HPLC)

Example 7: Preparation of Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (18)
Triethylamine (83 g) was added to the mixture of (15, 100 g) in MDC (1050 ml) and the reaction mass was stirred at 20 to 30°C till completion of the reaction, as monitored by TLC. After completion, the reaction mass was quenched with water and the organic layer was separated. Concentration of the organic layer, followed by treatment of resultant solid using toluene: cyclohexane mixture gave H-Cys (Trt)-Tyr-D-Trp-OAll (16) as solid.
Yield: 65.6 g (85%)
Purity: > 90% (HPLC)

Boc-D-Nal-OH (17, 22.6 g) was added to the stirred mixture of (16, 60 g) in DMF (180 ml) at 0-30°C, followed by addition of HOBT (11.8 g) and NMM (18.0 g). EDAC.HCl (17.0 g) was then added to the reaction mixture and stirring was continued at 0-30°C till completion of the reaction, as monitored by TLC. After completion, the reaction mixture was filtered, added to the cooled solution of 0.5M hydrochloric acid and stirred at 25 to 30°C. The solid was filtered, treated with 5% aqueous sodium carbonate solution, followed by treatment with toluene: cyclohexane (30:70) mixture and dried to give Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (18).

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 (25.05 g) and tetrakis (triphenylphosphine)palladium (3.3g) were added to the mixture of (18, 60 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 hydrochloric acid. The obtained solid was filtered, and the wet cake was treated with water, followed by treatment with toluene: cyclohexane mixture. The solid was treated with cyclohexane and dried 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-NH2 (19)
The mixture of Fragment B (45.0 g) in DMF (100 ml) was stirred at 0 -100C and HOBt (9.13 g), EDAC.HCl (11.1 g), NMM (6.5 g) were added to it with continued stirring. A mixture of Fragment A (35 g) in DMF (100 ml) was cooled to 0 -100C and added to the mixture and stirring was continued at 10-300C till completion of the reaction, as monitored by TLC. The reaction mixture was then quenched with cooled solution of 0.5N hydrochloric acid. The stirring was continued at 15-250C and the precipitated solid was filtered. MTBE treatment of the solid at 30-400C, filtration and drying provided the octapeptide, Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (19).
Yield : 64.3 g, 81.4%
Purity : > 80% (HPLC)

Example 10: Preparation of Lanreotide acetate (1)
DTT (17.31 g), TES (50 ml), TFA (300 ml), anisole (24.3 g) were added to the stirred mixture of compound 19 (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 (23.75 lit.) was added to the solution of above solid in 50% acetonitrile:water (2500 ml) which was stirred at 15-300C, 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)

,CLAIMS:
1. A process for the solution phase synthesis of lanreotide acetate (1), comprising reaction of H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (fragment A) with Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (fragment B) in an organic solvent in presence of a coupling agent and a base to give the octapeptide Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (19) which was then converted to lanreotide acetate by subsequent deprotection, oxidation, followed by treatment with acetic acid.
2. A process for the solution phase synthesis of H-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (fragment A) comprising reaction of Threoninamide with Fmoc-Cys (Trt)-OH to give Fmoc-Cys (Trt)-Thr-NH2 (4), deprotection followed by reaction with Fmoc-Val-OH to afford Fmoc-Val-Cys (Trt)-Thr-NH2 (7), deprotection followed by reaction with Fmoc-Lys (Boc)-OH to give Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (10), which on subsequent deprotection gave 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-OAll with Boc-Tyr-OH to give Boc-Tyr-D-Trp-OAll (13), deprotection followed by reaction with Fmoc-Cys (Trt)-OH to give Fmoc-Cys(Trt)- Tyr-D-Trp-OAll (15), deprotection followed by reaction with Boc-D-Nal-OH to give Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (18) which on subsequent allyl deprotection gave Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OH (fragment B).
4. Compound of formula, H-Lys (Boc)-Val-Cys (Trt)-Thr-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-NH2 (19).
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, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), BOP(Benzotriazol-1-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-NH2 (4), H-Cys (Trt)-Thr-NH2 (5)
Fmoc-Val-Cys (Trt)-Thr-NH2 (7),
H-Val-Cys (Trt)-Thr-NH2 (8), and
Fmoc-Lys (Boc)-Val-Cys (Trt)-Thr-NH2 (10).
12. Compounds of formulae Boc-Tyr-D-Trp-OAll (13),
H-Tyr-D-Trp-OAll (14),
Fmoc-Cys(Trt)- Tyr-D-Trp-OAll (15),
H-Cys(Trt)- Tyr-D-Trp-OAll (16) and
Boc-D-Nal-Cys (Trt)-Tyr-D-Trp-OAll (18).