WO2017175107A1

WO2017175107A1 - Process for preparation of octreotide acetate

Info

Publication number: WO2017175107A1
Application number: PCT/IB2017/051882
Authority: WO
Inventors: Mukund Keshav Gurjar; Narendra Kumar Tripathy; Chinmoy Mriganka PRAMANIK; Sanjay Shankar Deshmukh; Ulhas Supadu MAHAJAN; Ashish Pramod DESHPANDE
Original assignee: Emcure Pharmaceuticals Limited
Priority date: 2016-04-04
Filing date: 2017-04-01
Publication date: 2017-10-12

Abstract

Disclosed herein is an improved 4+4 solution phase synthesis of octreotide acetate. The process comprises coupling of two suitably protected tetrapeptide fragments which on deprotection, oxidation, and treatment with acetic acid provides octreotide acetate having desired purify.

Description

PROCESS FOR PREPARATION OF OCTREOTIDE ACETATE

This application claims the benefit of Indian Provisional Application No. 201621011904 filed on 4^th April 2016, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an improved process for the solution phase synthesis of an octapeptide like octreotide acetate (1) and its key intermediates by a process comprising coupling of suitably protected tetrapeptide fragments, followed by deprotection, oxidation to provide the octapeptide acetate ( 1 ) of desired purity.

BACKGROUND OF THE INVENTION

Octreotide acetate (1), chemically known as D-phenylalanyl-L-cysteinyl-L-phenylalanyl- D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy- l-(hydroxymethyl) propyl] -L-cysteinamide cyclic (2-7)-disulfide, is a cyclic octapeptide and a highly potent analog of somatostatin. The peptide, also represented as D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-Ol (Disulfide Bridge Cys2-Cys7); is a pharmacologically selective somatostatin analog which exhibits excellent in vitro and in vivo biological activity for inhibition of growth hormone for a longer duration. It is indicated for treating acromegaly for controlling and reducing the plasma level of growth hormone and for the symptomatic treatment of patients with metastatic carcinoid tumors and vasoactive intestinal peptide tumors.

The presence of a D-phenylalanine fragment in the N-terminal and an amino alcohol at the C-terminal end, along with a D-tryptophan residue and a disulfide bridge makes the molecule resistant to metabolic degradation.

Octreotide acetate (1) (X= 1.4 to 2.5)

Octreotide acetate developed by Novartis with proprietary name Sandostatin was first approved by USFDA on Oct. 21, 1988 as an injection with strength of 0.05-1.0mg base/ml. Later, octreotide acetate injection with a higher dose range of 10 to 30 mg base/vial was approved by USFDA on Nov. 25, 1998.

Conventional syntheses of peptides are divided into two major types, solid phase and solution -phase synthesis. Solid phase synthesis 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. US 6346601, KR 2009074316, WO 20081087794, CN 1837232, CN 1699404, CN 1810829, CA 2511711, CN 1569890, TW 519545, US 6476186, WO 2002081499 disclose solid phase synthesis of octreotide, while EP 953,577, US 5889146 disclose a method using 2-chlorotrityl resin and Fmoc -butyl protection. Solid phase peptide synthesis involves 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 the isolation of desired product render these processes uneconomical and unsuitable for large scale industrial production of the product. On the other hand, solution phase synthesis comprises synthesis of amino acids segments or blocks, followed by condensation in the desired sequence in solution. Such processes are comparatively economical and hence more suited for synthesis on industrial scale. The first solution phase synthesis of octreotide was disclosed in US 4,395,403 by a different synthetic process involving protection of cysteine thiol with methoxybenzyl group and subsequent deprotection with boron tris-trifluoroacetate in trifluoroacetic acid which however, causes degradation of the tryptophan moiety in the polypeptide sequence, thus causing considerable impurity formation and loss in yield due to the resulting purification.

Subsequently, various patent applications such as WO 2013132505, WO 2007110765, and WO 2003/097668 have disclosed solution phase synthesis of octreotide wherein 3+3+2 strategy was used for the polypeptide synthesis. Herein, the thiol group of cysteine is protected by acetamidomethyl (Acm) group and iodine is used for deprotection of acetamidomethyl group, which is followed by oxidative cyclization. The major drawback of this procedure involves racemization of phenylalanine methyl ester present in the fragment polypeptide Boc-D-Phe-Cys(Acm)-Phe-OMe. Further, the process involves purification of intermediate peptide segments by preparative HPLC, which lowers the yield and significantly increases cost of production for the desired product.

WO 2007/ 110765 discloses a solution phase synthesis for octreotide employing 3+5=6+2 strategy. The process includes coupling of two tripeptide segments, Boc-D-Phe- Cys(Acm)-Phe-OMe and Z-D-Trp-Lys(Boc)-Thr-OMe to provide a hexapeptide Boc-D- Phe-Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr-OMe, which is further condensed with the dipeptide H-Cys(Acm)-Thr-OMe to yield the desired octapeptide. Use of phenylalanine methyl ester and acetamidomethyl (Acm) group for thiol protection in cysteine are the major drawback of the procedure. While a relatively non -bulky group like Acm does not ensure that the intermediates are isolated as solids, the methyl ester in phenylalanine causes racemization at the hydrolysis stage, severely affecting the enantiomeric purity of the intermediates. As a consequence, the process involves purification of segments by preparative HPLC, which increases process time, use of solvents and ultimately, the production cost.

WO 2013132505 also discloses a 6+2 strategy for preparation of the desired octapepeptide. Although the synthesis involves use of bulky trityl moiety as a protecting group for cysteine, use of methyl ester of threonine is likely to result in epimerization and racemization and as already mentioned creates a negative impact on yield, and consequently, increases the project cost. It is now evident that most of the synthetic methods described in the aforementioned references resort to tedious synthesis of fragment blocks and their condensation involving expensive reagents and elaborate deprotection and separation procedures at various intermediate stages of synthesis. Hence, there is a need for a convenient and economical process which involves a synthetic route that utilizes a different fragment block that are developed in a facile manner using specific, easily detachable, bulky protecting groups which result in intermediates that are well characterized solid compounds and utilizes mild and selective deprotecting and coupling reagents to achieve the desired conversions. 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 octreotide acetate (1) which provides the desired molecule in good yield overcoming the problems faced in the prior art. The inventors have found that employing 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 viable, convenient process for synthesis of Octreotide acetate (1), which avoids use of lengthy reaction sequences and elaborate protection, deprotection and purification methods.

Another object of the invention relates to a 4+4 solution phase synthesis of Octreotide acetate comprising mild reagents and moderate reaction conditions to provide the desired purity. SUMMARY OF THE INVENTION

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

Yet another aspect of the invention relates to solution phase synthesis of octreotide acetate (1), comprising reaction of H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (fragment A) with Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH (fragment B) in presence of a coupling agent, a base and in a solvent to give the octapeptide Boc-D-Phe-Cys(Trt)-Phe-D-Trp-Lys(Boc)- Thr(OtBu)-Cys(Trt)-Thr-01 (19) which on subsequent deprotection, oxidation, followed by treatment with acetic acid gives octreotide acetate (1), having desired purity.

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 developing a convenient, industrially applicable solution phase synthetic strategy for octreotide, the present inventors surprisingly found that condensation of two suitably protected tetrapeptide (4+4) fragment combination was far superior to other fragment blocks reported in prior art. The condensation of the two tetrapeptide fragments was quite facile and there was greater control on the formation of associated impurities during the formation of the individual four amino acid fragments as well as during their condensation and subsequent reactions which resulted in a condensed product having impurities below desirable limits with higher yield, thereby making the process cost-effective.

The inventors also unexpectedly found that, owing to the specific protecting groups and nature of the peptide fragments, most of the intermediates in the said strategy were obtained as solids, which were purified easily with simple purification methods including recrystallization. Due to this, various laborious and cumbersome intermediate isolation and purification steps were avoided. This not only ensured notably higher yield for the desired octapeptide but also led to a convenient and economical synthetic process for octreotide which could easily be scaled up for commercial production. In particular, in the synthesis of tetrapeptide fragment B, allyl (-CH₂-CH=CH₂) protection of the tryptophan carbonyl , which could be deprotected using palladium (0) catalyst under neutral conditions resulted in circumventing use of bases like lithium hydroxide, thus significantly minimizing the problems of racemization which are very commonly observed in the synthesis of polypeptides. The strategy also comprises selective and specific, yet labile protecting groups at different stages, which are deprotected using mild acids, that did not adversely affect the chirality of the amino acids and intermediates in the synthetic sequence.

The synthesis of tetrapeptide fragments A and B is presented in Scheme- 1 and Scheme-2 respectively while condensation of fragments A and B followed by deprotection and oxidation to afford octreotide acetate is disclosed in Scheme-3.

ABBREVIATIONS

Fmoc = Fluorenylmethoxycarbonyl

Trt = Triphenyl methyl (Trityl)

Tbu = tert - Butyl

THF = Tetrahydrofuran

ACN = Acetonitrile

DMF = N, N- Dimethylformamide

DMSO = Dimethyl sulfoxide

DMAc = N, N dimethyl acetamide

NMM =N-methylmorpholine

TEA = Triethylamine

Bz = Benzyl

TFA = Trifluoroacetic acid

EDT = Ethanedithiol

TIS =Triisopropylsilane

TES=triethylsilane

HOBt = 1 -Hydroxybenzotriazole

DCM = Dichloromethane

EDAC= 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide HPLC= High performance liquid chromatography TLC= Thin layer chromatography

DIPEA= Diisopropylethylamine

MTBE = Methyl tertiary butyl ether

NMP= N-methylpyrolidine

Fmoc-Thr(OtBu)-Cys(Trt)-Thr-OI H-Thr(OtBu)-Cys(Trt)-Thr-OI

Fmoc-L s(Boc)-Thr(OtBu)-Cys(Trt)-Thr-OI (10)

H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-OI Fragment A

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

H-Phe-D-Trp-OAII (15) Fmoc-Cys(Trt)OH (3) Fmoc-Cys(Trt)-D-Phe-Trp-OAII (16)

H-Cys( -D-Phe-Trp-OAII ( ⁷) Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OAII (18)

Allyl

deprotection

Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH Fragment B

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

H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-OI Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH

Fragment A Fragment B

Boc-D-Phe-Cys(Trt)-P e-D-Trp-Lys (Boc)-Thr(OtBu)-Cys(Trt)-Thr-OI (19)

Octreotide acetate (1), X=1.4 to 2.5

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

In an embodiment, L-Threoninol (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-Ol (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 selected from hydrochloric, nitric, sulfuric acid, preferably hydrochloric acid to precipitate the intermediate, which was filtered and optionally treated with water prior to drying.

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-01 (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 addition of a water miscible organic solvent and treatment of the mixture with a water immiscible organic solvent. The water miscible organic solvent was selected from DMF, DMSO, ACN, THF and the like whereas the water immiscible organic solvent were 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. Further basification of the separated aqueous layer, extraction with a suitable solvent and concentration provided H-Cys(Trt)-Thr-01 (5). Coupling of (5) with Fmoc-Thr(OtBu)-OH (6) in an organic solvent in presence of a coupling agent and a base like NMM gave Fmoc-Thr (OtBu)-Cys (Trt)-Thr-Ol (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 prior to drying.

Fmoc deprotection of (7) using a suitable base like TEA in an organic solvent such as DMF afforded H-Thr(OtBu)-Cys(Trt)-Thr-01 (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 addition of a water miscible organic solvent and further treatment with water immiscible organic solvent. The water miscible organic solvent was selected from DMF, DMSO, ACN, THF and the like whereas the water immiscible organic solvent was selected from ethers such as MTBE, diethyl ether, diisopropyl ether etc., halogenated hydrocarbons such as dichloromethane, ethylene dichloride etc., esters such as ethyl acetate, butyl acetate etc. as well as mixtures thereof. Further basification of the separated aqueous layer, extraction with a suitable solvent and concentration provided H-Thr(OtBu)-Cys(Trt)-Thr- 01 (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)-Thr(OtBu)-Cys(Trt)-Thr-01 (10). After completion, the reaction mass was quenched using mineral acid to precipitate the intermediate, which was filtered and treated with water prior to drying. Compound (10) was subjected to Fmoc deprotection using a suitable base like DEA in an organic solvent to afford H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (Fragment A). The reaction was carried out in the temperature range of 0 to 30°C. The solvent was selected from halogenated hydrocarbons such as dichloromethane, ethylene dichloride. After reaction completion, the reaction mass was quenched with water, organic layer was separated and concentrated. Further washing of the residue with a water immiscible organic solvent selected from a group of ethers such as MTBE, diethyl ether, diisopropyl ether or halogenated hydrocarbons such as dichloromethane, ethylene dichloride, or esters like ethyl acetate, butyl acetate or mixtures thereof, followed by treatment with hydrocarbon solvent or mixtures thereof provided a precipitate which was filtered and dried to give fragment A. The hydrocarbon solvent was selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof. Preferably, mixtures of toluene: cyclohexane in proportions ranging from 1: 1 to 1:4 were used.

In a further embodiment, D-Tryptophan i.e. H-D-Trp-OH (11) was treated with allyl alcohol in a hydrocarbon solvent such as toluene. The reaction was carried out at 80 to 100°C. After completion, the reaction mass was quenched with mineral acid and a water miscible organic solvent selected from DMF, DMSO, DMAc etc. was added to it. Concentration of the reaction mixture, alkali treatment of the residue, followed by extraction with an organic solvent like methyl tert-butyl ether, ethyl acetate after concentration provided the desired allyl protected compound, H-D-Trp-OAll (12). Compound (12) was coupled with Boc-Phe-OH (13) using a polar aprotic solvent like acetonitrile, in presence of a coupling agent and a base such as NMM. The reaction was carried out at 0 to 30°C. After completion, the reaction mass was quenched using mineral acid like HC1 to precipitate the intermediate, which was filtered and dried to give Boc-Phe- D-Trp-OAll (14).

Boc deprotection of (14) using an acid mixture such as HC1 in acetonitrile or trifluoroacetic acid in dichloromethane afforded H-Phe-D-Trp-OAll (15). The reaction was carried out at ambient temperature and after completion, concentration of the reaction mixture provided a residue containing compound (15).

Coupling of (15) with Fmoc-Cys (Trt)-OH (3) using an organic solvent like acetonitrile, in presence of a coupling agent and a base such as DIPEA gave Fmoc-Cys(Trt)-Phe-D-Trp- OA11 (16). The reaction was carried out at 0 to 30°C. After completion, the reaction mass was quenched with a mineral acid to precipitate the intermediate, which was filtered, optionally treated with alkali solution, water and dried to give (16).

Fmoc deprotection of (16) in a halogenated hydrocarbon solvent like dichloromethane, using a suitable base such as TEA afforded H-Cys(Trt)-Phe-D-Trp-OAll (17). 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 (17).

Coupling of (17) with Boc-D-Phe-OH (13) using organic solvent like acetonitrile in presence of a coupling agent and a base such as NMM or DIPEA furnished Boc-D-Phe- Cys(Trt)-Phe-D-Trp-OAll (18). The reaction was carried out at 0 to 30°C. After completion, the reaction mass was treated with a mineral acid and the precipitated solid was separated. Further treatment of the solid using a halogenated hydrocarbon solvent selected from dichlorome thane, ethylene dichloride and the like followed by washing the resultant mixture with alkali solution, separation and concentration of the organic layer. The residue was triturated with a hydrocarbon solvent selected from pentane, n-hexane, cyclohexane, heptane, toluene and mixtures thereof to provide compound (18). Filtration, drying of the separated solid and an optional treatment with hydrocarbon solvent gave compound (18).

In a further embodiment, allyl deprotection of (18) using an organic solvent such as dimethyl sulphoxide or dimethylformamide in presence of morpholine and a catalyst tetrakis(triphenylphosphine)palladium at 0 to 30°C provided Boc-D-Phe-Cys(Trt)-Phe-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 with fragment B was carried out in a suitable organic solvent like DMF in presence of a coupling agent at 0 to 30°C and in presence of a base such as N-methyl morpholine, furnished the octapeptide Boc-D-Phe- Cys(Trt)-Phe-D-Trp-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (19). After completion, the reaction mass was treated with a mineral acid and the precipitated solid was filtered to provide (19).

Compound (19) was dissolved in a halogenated hydrocarbon solvent like dichloromethane was treated with TFA, and TES, in presence of anisole at 20 to 30°C to give (20). After completion, concentration of the reaction mixture gave an oily 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 octreotide acetate. 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 (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, l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (ED AC), BOP(Benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-l-yloxy-tripyrrolidino-phosphonium- hexafluoro phosphate), PyBrOP (Bromotripyrrolidino phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol- 1 -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- lH-benzotriazol-l-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate), COMU (1- [l-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]- uroniumhexafluorophosphate), HCTU (2-(6-Chloro- lH-benzotriazol- 1 -yl)-N,N,N' ,Ν' - 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, 1- 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 Fmoc-Cys (Trt)-Thr-Ol (4)

A mixture of Fmoc-Cys (Trt)-OH (3, 77 g) in DMF (154 ml) was stirred under nitrogen atmosphere and L-Threoninol (2, 13.82g), HOBt (20.12 g) were added to it. Reaction mixture was cooled to 0-5°C and NMM (51.95 g) and DMF (77 ml) were added to the mixture, followed by addition of EDAC.HC1 (30.23 g) in DMF (77ml). The reaction mixture was stirred at 15 to 30°C. After completion of the reaction, as monitored by HPLC, aqueous hydrochloric acid (385 ml) was added to the reaction mass with stirring and the precipitated solid was filtered. Optional treatment of the wet cake with water and drying gave Fmoc- Cys(Trt)-Thr-01 (4).

Yield: 80.05 g (90.53%),

Purity: >95% (HPLC) Example 2: Synthesis of Fmoc-Thr(OtBu)-Cys (Trt)-Thr-Ol (7)

TEA (77.7 ml) in DMF (75 ml) was added to the solution of compound (4) (75 g) in DMF (150 ml). The reaction mass was stirred at 25 to 30°C till completion, as monitored by TLC. After completion, the mixture was quenched with 0.5N hydrochloric acid (1921 ml) and DMF (672 ml) was added to it, followed by treatment with MTBE. The aqueous layer was separated, basified using 10% aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was separated, and concentrated to give a residue containing H-Cys(Trt)-Thr-01 (5). The residue was dissolved in DMF (150 ml) and Fmoc- Thr(OtBu)-OH (6, 44.3 g) and HOBt (17 g) were added to the mixture. The mass was then cooled to 0-5°C and NMM (13.5 g), EDAC.HC1 (25.6 g) and DMF (75 ml) were added to it. Reaction mixture was stirred at 20 to 30°C till completion of reaction as monitored by TLC. After completion, the stirred reaction mass was quenched with 0.5N hydrochloric acid and the precipitated solid was filtered. Optional treatment of the wet cake with water and drying gave Fmoc-Thr (OtBu)-Cys (Trt)-Thr-Ol (7).

Yield: 80.4 g (86.89 %),

Purity: >93% (HPLC) Example 3: Synthesis of Fmoc-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (10)

Triethylamine (71.7 ml) was added to the solution of compound (7) (75 g) in DMF (225 ml). The reaction mass was stirred at 25 to 30°C till completion of reaction, as monitored by TLC. After completion, the reaction mixture was quenched with 0.5N hydrochloric acid (1900 ml) and DMF (730 ml) was added to it, followed by treatment with MTBE. The aqueous layer was separated, basified using 10% aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was separated, and concentrated to give an oily residue containing H-Thr (OtBu)-Cys (Trt)-Thr-Ol (8).

The oily residue containing (8) was dissolved in DMF (225 ml) and Fmoc-Lys (Boc)-OH (9, 33.9 g), HOBt (13.8 g) were added to it. The reaction mixture was cooled to 10-15°C, and NMM (12.55 g), EDAC.HC1 (23.78 g) were added to it. The reaction mixture was stirred at 20 to 30°C till completion of reaction, as monitored by TLC. After completion, the mass was quenched with 0.5N hydrochloric acid and the precipitated solid was filtered.

Optional treatment of the wet cake with water, followed by treatment with cyclohexane and drying gave Fmoc-Lys (Boc)-Thr (OtBu)-Cys (Trt)-Thr-Ol (10).

Yield: 66.60 g (87.06 %),

Purity: >90%

Example 4: Synthesis of Fragment A

DEA (56.7 ml) in dichloromethane (60 ml) was added to the stirred solution of compound (10) (60 g) in dichloromethane (600 ml) and the resultant mixture was stirred at 20 to 30°C till completion of reaction, as monitored by TLC. After completion, water (300 ml) was added to the reaction mixture and the organic layer was separated. Concentration of the organic layer, followed by optional treatment of the residue with MTBE and further treatment with toluene: cyclohexane (1:3) mixture provided a precipitate, which was filtered and dried to give H-Lys (Boc)-Thr (OtBu)-Cys (Trt)-Thr-Ol, Fragment A.

Yield: 46.5 g (98 %), Purity: >95% (HPLC)

ESI MS: [M+23] 1081.0 Example 5: Synthesis of Boc-Phe-D-Trp-OAll (14)

Allyl alcohol (71.46 ml) was added to the solution of H-D-Trp-OH (11, 24.0 g) in toluene (214 ml) and the resulting mixture was stirred at 85-95°C till completion of reaction, as monitored by HPLC. After completion, the mass was quenched with 0.5N hydrochloric acid and DMF (204 ml) was added to it. The mass was cooled to 50-55°C and concentrated. The residue was treated with 5% aqueous sodium bicarbonate solution (238 ml) and 5% aqueous sodium hydroxide solution (119 ml), followed by extraction with ethyl acetate. Separation and concentration of the organic layer gave an oily residue containing H-D-Trp- OA11 (12).

The residue was dissolved in acetonitrile (95 ml) and Boc-Phe-OH (13, 34.02g), HOBt (17.89 g), EDAC.HC1 (3.59 g), NMM (23.58 g) were added to it at 0-30°C. The reaction mixture was stirred till completion, as monitored by HPLC. After completion, the mixture was quenched with 0.5N hydrochloric acid and the precipitated solid was filtered and dried to give Boc-Phe-D-Trp-OAll (14).

Yield: 45.26 g (71.80%),

Purity: >95% (HPLC)

Example 6: Synthesis of Fmoc-Cys(Trt)-Phe-D-Trp-0-All (16)

Compound 14 (40 g) was added to the mixture of ACN containing HC1, stirred at 0-5°C and the resulting mixture was stirred at 25-35°C till completion of reaction, as monitored by HPLC. After completion, the reaction mass was concentrated to give an oily residue containing H-Phe-D-Trp-OAll (15). The oily residue was dissolved in ACN (226 ml) and Fmoc-Cys (Trt)-OH (3, 42.9 g), HOBt (15.55 g), EDAC.HC1 (21.18 g) and DIPEA (23.75 g) were added to it. The reaction mixture was stirred at 0 to 30 °C till completion of the reaction, as monitored by HPLC. After completion, the stirred mass was quenched with 0.5N hydrochloric acid and was cooled to 0-5°C. The precipitated solid was filtered, washed with water, 5% aqueous sodium bicarbonate solution and dried to give Fmoc- Cys(Trt)-Phe-D-Trp-OAll (16).

Yield: 62 g (79%),

Purity: >95% (HPLC) Example 7: Synthesis of Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OAll (18)

TEA (50.6 g) was added to the stirred solution of compound (16) (60 g) in dichloromethane (850 ml) and the resultant mixture was stirred at 20 to 30°C till completion of reaction, as monitored by TLC. After completion, the reaction mixture was quenched with water and the organic layer was separated. Concentration of the organic layer, followed by treatment of the obtained mass with around 10% toluene in cyclohexane and decantation gave a residue containing H-Cys(Trt)-Phe-D-Trp-OAll (17).

The residue was dissolved in acetonitrile (296 ml) and Boc-D-Phe-OH (13, 16.8 g), HOBt (9.6 g), EDAC.HC1 (14.4 g), and NMM (12.7 g) were added to it. The reaction mixture was stirred at 0 to 30°C till completion of the reaction, as monitored by HPLC. After completion, the stirred mass was quenched with 0.5N hydrochloric acid and the obtained solid was filtered.

The solid was dissolved in dichloromethane (230 ml), and the solution was washed with 5% aqueous sodium bicarbonate solution. The organic layer was separated and concentrated. Treatment of the residue with 10% toluene in cyclohexane, followed by filtration of the precipitate, optional treatment with cyclohexane and drying gave Boc-D- Phe-Cys(Trt)-Phe-D-Trp-OAll (18).

Yield: 53.4 g (85.63%)

Purity: >90% (HPLC)

Example 8: Synthesis of Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH (Fragment B)

A mixture of 18, (50 g) in DMSO (200 ml) was stirred and morpholine (22.2 g), followed by tetrakis (triphenylphosphine)palladium (1.7 g) were added to it. The reaction mass was stirred at 25 to 30°C till completion of reaction as monitored by HPLC. After completion, the reaction mixture was filtered, and 0.5N hydrochloric acid was added to the filtrate with stirring. The obtained solid was filtered, wet cake was treated with toluene, cyclohexane and dried to give Fragment B.

Yield: 45 g (87%)

Purity: >95% (HPLC)

ESI MS: [M+23] 966.8, [M-l] 942.7 Example 9: Synthesis of Boc-D-Phe-Cys(Trt)-Phe-D-Trp-Lys(Boc)-Thr(OtBu)- Cys(Trt)-Thr-OH (19) - Condensation of Fragment A and B.

The solution of Fragment A (38 g) in DMF (114 ml) was added to the solution of Fragment B (42.9 g) in DMF followed by addition of HOBt (7.7 g) ED AC. HCl (4.2 g), and NMM (5.5 g). The reaction mass was stirred at 15 to 20°C till completion of reaction as monitored by HPLC. After completion, 0.5N aqueous hydrochloric acid was added to the reaction mass with stirring. The obtained solid was filtered, the wet cake was washed with aqueous 5% sodium bicarbonate solution, was further treated with toluene, cyclohexane and dried to give the octapeptide, Boc-D-Phe-Cys(Trt)-Phe-D-Trp-Lys(Boc)-Thr(OtBu)- Cys(Trt)-Thr-01 (19).

Yield: 62.7 g (78.29 %),

Purity: >85% (HPLC)

ESI MS: [M+l] 1762.4, [M+23] 1784.3 Example 10: Synthesis of Octreotide acetate (1)

Anisole (29.5 g) and TES (60 ml) were added to the stirred solution of compound 19 (60 g) in dichloromethane (180 ml) at 25 to 30°C. The reaction mixture was cooled to around 10 °C and TFA (180 ml) was gradually added to it. The resultant mass was stirred at 25 to 30°C till completion of reaction as monitored by HPLC. After completion, the reaction mixture was concentrated and the oily residue so obtained was treated with MTBE. The solid was filtered and the wet cake was dissolved in a mixture of acetonitrile and water. The solution was added to aqueous acetic acid (100 ml in 19900 ml water) with stirring. The solution of iodine (15 g) in methanol (400 ml) was added to it and the reaction mixture was stirred at 25 to 30°C till completion of the reaction as monitored by HPLC. After completion, L-ascorbic acid (13 g) was added to the reaction mass with stirring, followed by filtration to give octreotide acetate (1), which was purified by preparative HPLC.

Yield: 11.1 g (30.21%)

Purity: >99% (HPLC).

Claims

1. A process for the solution phase synthesis of octreotide acetate (1), comprising reaction of H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (fragment A) with Boc-D-Phe-Cys(Trt)-Phe- D-Trp-OH (fragment B) in an organic solvent in presence of a coupling agent and a base gave Boc-D-Phe-Cys(Trt)-Phe-D-Trp-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (19) which was then converted to octreotide acetate by subsequent deprotection, oxidation, followed by treatment with acetic acid.

2. A process for the solution phase synthesis of H-Lys (Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (fragment A) comprising reaction of L-Threoninol with Fmoc-Cys (Trt)-OH gave Fmoc- Cys(Trt)-Thr-01 (4), deprotection followed by reaction with Fmoc-Thr(OtBu)-OH gave Fmoc-Thr(OtBu)-Cys(Trt)-Thr-01 (7), deprotection followed by reaction with Fmoc-Lys (Boc)-OH gave Fmoc-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (10) which on subsequent deprotection gave H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (fragment A).

3. A process for the solution phase synthesis of Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH (fragment B) comprising reaction of H-D-Trp-OAll with Boc-Phe-OH gave Boc-Phe-D- Trp-OAll (14), deprotection followed by reaction with Fmoc-Cys (Trt)-OH gave Fmoc- Cys(Trt)-Phe-D-Trp-OAll (16), deprotection followed by reaction with Boc-D-Phe-OH gave Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OAll (18) which on subsequent deprotection gave Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH (fragment B).

4. Compound of formula, H-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (fragment A).

5. Compound of formula, Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OH (fragment B)..

6. Compound of formula Boc-D-Phe-Cys(Trt)-Phe-D-Trp-Lys(Boc)-Thr(OtBu)-Cys(Trt)- Thr-01 (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, 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-01 (4),

Fmoc-Thr(OtBu)-Cys(Trt)-Thr-01 (7),

H-Thr(OtBu)-Cys(Trt)-Thr-01 (8), and

Fmoc-Lys(Boc)-Thr(OtBu)-Cys(Trt)-Thr-01 (10).

12. Compounds of formulae Boc-Phe-D-Trp-OAll (14),

H-Phe-D-Trp-OAll (15),

Fmoc-Cys(Trt)-Phe-D-Trp-OAll (16),

H-Cys(Trt)-Phe-D-Trp-OAll (17) and

Boc-D-Phe-Cys(Trt)-Phe-D-Trp-OAll (18).