EP2013226A2

EP2013226A2 - Processes for the preparation of octreotide

Info

Publication number: EP2013226A2
Application number: EP07734124A
Authority: EP
Inventors: Kumar Kamlesh Laxmi Singh; Siddiqui Mohammed Jaweed Mukarram; Deshpande Amol Ashok; Roy Anupam Badal Chandra
Original assignee: Wockhardt Ltd
Current assignee: Wockhardt Ltd
Priority date: 2006-03-28
Filing date: 2007-03-28
Publication date: 2009-01-14
Also published as: WO2007110765A3; WO2007110765A2

Abstract

The invention relates to processes for the preparation of pure octreotide or pharmaceutically acceptable salts thereof. The invention also relates to an amorphous form of octreotide and processes for the preparation of amorphous form of octreotide or pharmaceutically acceptable salts thereof. The invention also relates to pharmaceutical compositions that include the pure octreotide or the amorphous octreotide or pharmaceutically acceptable salts thereof.

Description

PROCESSES FOR THE PREPARATION OF OCTREOTIDE

Field of the Invention

The field of the invention relates to processes for the preparation of pure octreotide or pharmaceutically acceptable salts thereof. The invention also relates to an amorphous form of octreotide and processes for the preparation of amorphous form of octreotide or pharmaceutically acceptable salts thereof. The invention also relates to pharmaceutical compositions that include the pure octreotide or the amorphous octreotide or pharmaceutically acceptable salts thereof.

Background of the Invention

Octreotide is a cyclic octapeptide. Chemically, octreotide is D-Phenylalanyl-L-cysteinyl- L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-iV- [2-hydroxy- 1 -(hydroxymethyl) propyl] -L-cysteinamide cyclic (2D7)-disulfide having the structural Formula I. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Octreotide is indicated to reduce blood levels of growth hormone and IGF-I in acromegaly patients. It is also indicated for the symptomatic treatment of patients with metastatic carcinoid tumors and Vasoactive Intestinal Peptide Tumors (VIPomas).

FORMULA I U.S. Patent No 4,395,403 and European Patent No 029,579 disclose solution synthesis for the preparation of octreotide.

Several solid phase synthetic procedures have been subsequently reported for example, in European Patent No. 953,577; U.S. Patent No. 5,889,146; and in various research publications. 12th American Peptide Symposium discloses aminomethyl resin and Fmoc- butyl protection scheme for the synthesis of octreotide. Tetrahedron Letters 1997, 38, 883 discloses an active carbonate resin and Boc-Bzl protection scheme, necessitating the use of hydrogen fluoride/anisole for final deprotection. Journal of Medicinal Chemistry 1994, 37, 3749 discloses synthesis using Fmoc-butyl protection and HMP resin, and European Patent No. 953, 577 discloses a synthesis using 2-chlorotrityl-type resin and Fmoc-butyl protection scheme.

All the solid phase synthetic procedures disclosed are not suitable for commercial manufacture of octreotide because they use costly resins and Fmoc-butyl protected amino acids in 2 to 4 times excess at every step. Some synthetic procedures use destructive and hazardous reagents for final deprotection and the product is not obtained in high purity, thus making the procedures commercially difficult to implement. The present invention provides a process which is efficient and does not result in impure product; rather pure product having purity more than 99% is obtained. The product when made by the process of the present invention is easy to isolate and handle thus making the process amenable for commercial scale use.

Summary of the Invention

hi one general aspect there is provided a process for the preparation of R₁-(D) Phe- Cys(Acm)-Phe-OH, wherein Ri is amino or suitable amino protecting group used in peptide chemistry. The process includes the steps of: a) hydrolyzing R₁-(D)PhC-CyS(ACm)-PhC-OMe in one or more solvents by maintaining a pH of about 9.0 to about 10 of reaction mass; b) acidifying the reaction mass after completion of hydrolysis; and c) isolating the R₁-(D)PhB-CyS(ACm)-PlIe-OH from the reaction mass thereof.

Embodiment of the process may include one or more of the following features. For example, the hydrolysis may be carried out in the presence of one or more bases. The bases include hydroxides, carbonates or bicarbonates of alkali or alkali earth metal compound. The solvent may include one or more of lower alkanols. The lower alkanol may include one or more of primary, secondary and tertiary alcohols having from one to six carbon atoms. The lower alkanol may include one or more of methanol, ethanol, n- propanol, and isopropanol.

The process may include protection of amino group with a suitable protecting group and deprotection of amino protecting group prior to isolating the R₁-(D) Phe-Cys(Acm)-Phe- OH.

Li another general aspect there is provided a process for the preparation of L-Thr-OMe. The process includes the steps of: a) treating L-Thr-OH with methanol and thionyl chloride at a temperature above

45⁰C; b) removing excess thionyl chloride from reaction mass; and c) isolating the L-Thr-OMe from the reaction mass thereof.

In another general aspect there is provided a process for removal of inorganic impurities (de-salting) OfR₁-CyS(ACm)-ThT-OL, wherein R₁ is amino or a suitable amino protecting group used in peptide chemistry. The process includes the steps of: a) reducing R₁-CyS(ACm)-ThT-OMe in one or more solvents in the presence of sodium borohydride; b) extracting the resultant mass in dichloromethane; and c) isolating the R₁-CyS(ACm)-ThT-OL from the extract thereof. The solvent may be, for example, one or more of lower alkanols, lower aliphatic acids, water, or mixtures thereof. The lower alkanol may include one or more of methanol, ethanol, isopropanol and n-propanol. The lower aliphatic acid may include formic acid, acetic acid, propionic acid, or mixtures thereof.

In another general aspect there is provided a process for the preparation of Cys (Acm)- Phe-OMe or a salt thereof. The process includes the steps of: a) treating t-BOC-Cys(Acm)-Phe-OMe or a salt thereof with less than 15 moles of trifluoroacetic acid; b) removing the excess trifluoroacetic acid from reaction mass; and c) isolating the Cys(Acm)-Phe-OMe or a salt thereof from the reaction mass thereof.

In another general aspect there is provided a process for the preparation of Cys(Acm)- Thr-OL or a salt thereof. The process includes the steps of: a) treating t-BOC-Cys(Acm)-Thr-OL or a salt thereof with less than 15 moles of trifluoroacetic acid; b) removing the excess trifluoroacetic acid from reaction mass; and c) isolating the Cys(Acm)-Thr-OL or a salt thereof from the reaction mass thereof.

Embodiment of the process may include one or more of the following features. For example, removing the excess trifluoroacetic acid may include distillation, distillation under vacuum and evaporation. The process may also include non-polar solvent like ether, hexane or mixture thereof.

The intermediates thus obtained can be converted to octreotide or salts thereof by processes known in the art.

hi another general aspect there is provided a process for the preparation of trifluoroacetate salt of 8P-OL. The process includes the steps of: a) coupling t-BOC-6P-OH and Cys(Acm)-Thr-OL in one or more suitable organic solvent to give t-BOC-8P-OL; b) removing the t-BOC protection from the product t-BOC-8P-OL using trifluoroacetic acid to give trifluoroacetate salt of 8P-OL; c) extracting the trifluoroacetate salt of 8P-OL in water to get an aqueous solution; d) removing undissolved dicyclohexylcarbodiimide-urea complex from the aqueous solution; and e) isolating the trifluoroacetate salt of 8P-OL from the aqueous solution by lyophilization.

Embodiment of the process may include one or more of the following features. For example, suitable organic solvent may include polar aprotic solvent. Polar aprotic solvent may include one or more of N, N-dimethylformamide, N,N-dimethylacetarnide, dimethylsulphoxide, N-methyl pyrrolidone, tetrahydrofuran or acetonitrile. The process may include one or more catalysts and coupling reagents in the coupling reaction. The catalyst may include 1-hydroxybenzotriazole (HOBt), and the like. The coupling reagent may include dicyclohexylcarbodiimide (DCC), and the like.

The process may also include adding one or more scavengers at step (b) for deprotecting the t-BOC protection. The scavengers may include thioanisole, anisole, mercaptoethanol, and the like.

The process may produce the trifluoroacetate salt of 8P-OL having less than 1% of dicyclohexylcarbodiimide -urea.

In another general aspect there is provided a process for the preparation of octreotide or a salt thereof. The process includes the steps of: a) treating trifluoroacetate salt of 8P-OL in methanol with iodine; b) drying the resultant mixture; c) treating the resultant mass with one or more bases; d) acidifying the solution of step (c) with acetic acid; and e) isolating the octreotide mixture by desalting. The base may include one or more of sodium hydroxide, potassium hydroxide, carbonates or bicarbonates of alkali or alkali earth metal compounds, and the like.

Octreotide or salts thereof so obtained may be further purified. It may be purified by preparative chromatography.

In another general aspect there is provided a process for the preparation of octreotide acetate by preparative chromatography. The process includes the steps of: a) charging octreotide or a salt thereof over preparative chromatography column with 3% ammonium acetate and 1% acetic acid; b) collecting desired elute; c) concentrating the desired elute; and d) isolating the octreotide or a salt thereof from the elute.

The preparative chromatography may be performed by using YMC ODS-A column having length 50 x 500 mm and particle size 15 micron having 12nm pore size.

In another general aspect there is provided octreotide or salt or derivative with a purity of greater than about 98.5% by HPLC.

In another general aspect there is provided amorphous octreotide acetate.

The amorphous form of octreotide acetate may have, for example, the X-ray diffraction pattern of Figure 1.

In another general aspect there is provided a pharmaceutical composition that includes a therapeutically effective amount of the amorphous form of octreotide acetate; and one or more pharmaceutically acceptable carriers, excipients or diluents.

In another general aspect there is provided a process for the preparation of amorphous form of octreotide acetate. The process includes the steps of: a) charging octreotide or a salt thereof, wherein the salt is other than acetate, over preparative chromatography column; b) eluting the column with 0.1 to 4% ammonium acetate and 0.1-2% acetic acid; c) eluting the column with a mixture comprising of acetic acid and methanol to get desired fraction; d) concentrating the desired fraction; and e) isolating the octreotide acetate from the concentrate of step d).

The process may include further drying of the product obtained. The process may include isolating the amorphous form of octreotide acetate by lyophilization.

The process may produce the amorphous form of the octreotide having the X-ray diffraction pattern of Figure 1.

The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the description and claims.

Description of the Drawings

Figure 1 is an X-Ray Diffraction Pattern of amorphous form of octreotide.

Figure 2 is a Fourier Transform Infrared (FTIR) spectrum of amorphous octreotide.

Detailed Description of the Invention

The inventors have developed processes for the preparation of pure octreotide . or pharmaceutically acceptable salts having a purity of more than 99% by HPLC. The inventors also have developed processes for the preparation of intermediates of octreotide or pharmaceutically acceptable salts thereof. The inventors have found a new form of octreotide acetate, the amorphous form and, in particular, the amorphous octreotide acetate. The new form is characterized by its X-ray powder diffraction pattern and infrared spectrum as shown in Figures 1 and 2, respectively. The inventors also have developed a process for the preparation of the pure amorphous form of octreotide acetate, from trifluoroacetate salt of H-(D)-PlIe¹- Cys(Acm)²-Phe³-(D)-Trp⁴-Lys⁵-Thr⁶-Cys(Acm)⁷-Thr⁸-OL (i.e. 8P-OL). The amorphous form of octreotide acetate offers several advantages in terms of enhanced solubility, greater stability and ease in incorporation to dosage form. The inventors also have developed pharmaceutical compositions that contain the amorphous form of the octreotide acetate, in admixture with one or more solid or liquid pharmaceutical diluents, carriers, and/or excipients.

The process for the preparation of R₁-(D)PhC-CyS(ACm)-PhC-OMe is well known to those skilled in the art. The prior art mentions use of sodium hydroxide for hydrolysis of R₁-(D)PlIe-CyS(ACm)-PlIe-C)H which gives racemization of up to 35% resulting in the reduction of yield of the desired product. When potassium hydroxide was used instead of sodium hydroxide, the racemization was reduced only up to 25%. Surprisingly, the present inventors found that the use of lithium hydroxide reduced racemization up to 15% thus resulting in increase in desired product yield. It was further observed that when hydrolysis of Rr(D)Phe-Cys(Acm)-Phe-OMe was carried out at a pH of from about 9.0 to about 10, yield of the product and quality was greatly improved. This was achieved by adding small quantities of alkali at regular intervals to maintain the pH of the reaction. The addition of controlled quantities of alkali reduces the presence of excess alkali which is required for racemization and thus improves the yield and quality of the product.

R₁-(D)PhC-CyS(ACm)-PhC-OMe and L-Thr-OH can be prepared by any of the processes known in the art._Commercially available raw materials can also be employed. The treatment of L-Thr-OH with methanol and thionyl chloride at a temperature above 45⁰C facilitates the rate of reaction thus requiring less reaction time resulting in good yield and quality of the product. The earlier known processes operate on lower temperatures and therefore are more time consuming and the reaction do not go to completion. R₁-CyS(ACm)-ThT-OMe can also be prepared by the procedures well known to those skilled in peptide chemistry. According to the prior art, in case Of R₁-CyS(ACm)-ThT-OL dipeptide purification, desalting was carried out with the help of HPLC followed by collection of desired product in the form of aqueous solution; this aqueous solution was further lyophilized to get the resultant mass. This process takes about 40 hours. The present inventors have now found out that when desalting was carried out by extracting the resultant mass in dichloromethane, all the undesired inorganic salts remain in aqueous solution and the desired product component gets selectively extracted in the organic phase, which can be isolated in purified form. Thus, there is no need of lyophilization, which saves time, cost and improves the product quality and yield.

The t-BOC-Cys(Acm)-Phe-OMe can be prepared by the procedures well known to those skilled in the art of peptide chemistry. The prior art uses 25 to 32 moles of trifluoroacetic acid for the preparation of Cys(Acm)-Phe-OMe but the present inventors have now found that when less than 10 moles of trifluoroacetic acid was employed, the impurities in the reaction mixture were greatly reduced and further helped to remove the product from the reaction mixture with greater ease. The excess trifluoroacetic acid was removed from the reaction mixture after completion of the reaction by vacuum distillation and the residue was triturated with mixture of ether and hexane to get the product.

t-BOC-Cys(Acm)-Thr-OL can be prepared by the procedures well known to those skilled in the art of peptide chemistry. After the deprotection, the mass was concentrated under vacuum and the product was triturated with non-polar hydrocarbon solvent to get the desired product.

The present inventors have observed that when trifluoroacetate salt of 8P-OL remains in contact with urea, complete degradation of the product occurs in two to three days. However, removal of DCC-Urea complex from the crude trifluoroacetate salt of 8P-OL provides excellent stability to the product and the product remains stable for 55 days. The yield and the quality of the product was also improved. It was also noticed that if DCC- Urea complex was not removed at this stage, it continues in the next stages of the octreotide preparation till final purification of the product on preparative chromatography leading to damage of the costly separation columns.

The process further includes coupling of two starting materials t-BOC-6P-OH and Cys(Acm)-Thr-OL , which can be prepared by processes known in the art. The two starting materials were coupled in the presence of DCC and 1-hydroxybenzotriazole (HOBt) in the presence of polar aprotic solvent such as N,N-dimethylformamide, N₅N- dimethylacetamide, dimethylsulphoxide, N-mythylpyrrolidone, tetrahydrofuran, acetonitrile, and the like. After completion of the reaction, the mass was concentrated and the residue was treated with non-polar solvent such as ether, hexane etc. The separated solids were filtered and extracted in ethyl acetate and the suspension was warmed, cooled and filtered to get the desired t-BOC-8P-OL.

For removal of t-BOC protection, the product was treated with trifluoroacetic acid in dichloromethane in the presence of thioanisole, anisole and mercaptoethanol as scavengers. After completion of reaction, the mixture was concentrated and the residue was treated with ether. The separated product was filtered and suspended in water. The insoluble particles of DCC-Urea were filtered and the resultant aqueous solution was lyophilized to get the desired product.

The trifluoroacetate salt of 8P-OL can be prepared by the procedures well known to those skilled in the art. Octreotide or salts or derivatives thereof can be prepared by treating trifluoroacetate salt of 8P-OL to the iodine containing alkanol at ambient temperature, further drying this resultant mixture, followed by basifying the reaction mixture with base or alkaline agents. Suitable basifying agents can be selected from sodium hydroxide, potassium hydroxide, carbonate or bicarbonate of alkali or alkali earth metal compounds, and the like. Further, the resultant solution was treated with acetic acid and the crude octreotide was isolated by desalting. The Preparative HPLC is an efficient tool for separation of one or several components from a complex mixture. It helps in the isolation of the desired product with defined purity, in maximum amounts and with minimum time. The present inventors now observed that when preparative chromatography was used for isolation and purification of octreotide or salt or derivative thereof from the reaction mixture, it yields product with better yield and more than 98.5% purity.

The purification of octreotide or salt or derivative thereof by preparative chromatography was performed by using YMC ODS-A column having length 50 x 500 mm and particle size 15 micron having 12nm pore size and 0.5% -10% ammonium acetate and 0.1%-5% acetic acid as solvent.

Following abbreviations are employed in coming invention and are well known to those skilled in the art. Abbreviations:

Acm=Acetamidomethyl

Boc=tert.-Butyloxycarbonyl

BTFA=Boron-tris-trifluoroacetate tBu=tert-Butyl DCC=Dicyclohexylcarbodiimide

DCM=Dichloromethane

DMAc=Dimethylacetamide

DMSO=Dimethylsulfoxide

HOBt=I -Hydroxybenzotriazole IBCF=Isobutylchloroformate

NMM=N-methylmorpholine

TEA=Triethylamine

TFA=Trifluoroacetic acid

THF=Tetrahydrofuran Powder XRD of the samples were determined by Rigaku X-Ray diffractometer model no. 2200-v Japan.

FT IR of the samples were determined by Perkin Elmer, Spectrum One FT-IR spectrometer in KBr pellets.

The present invention is illustrated by the following examples which are provided merely to be exemplary of the invention and do not limit the scope of the invention. Certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Example 1; Preparation of Boc-fDVPhe-CvsfACMVPhe-OH

Lithium hydroxide solution (140 ml, IM) was added drop wise to the solution of R₁- (D)Phe-Cys(Acm)-Phe-OMe (112 g) in methanol (2500 ml) at 20- 25 ⁰C. The solution was stirred at 3O⁰C till completion of the reaction and the reaction was monitored by HPLC. The pH was brought to 2-3 by addition of hydrochloric acid (584 ml, IN) at 5- 1O⁰C. The solution was concentrated under vacuum. The concentrate was saturated with solid sodium chloride and diluted with ethyl acetate (1000 ml). Ethyl acetate layer was separated and the aqueous layer was extracted with ethyl acetate (1000 ml). The combined extracts were evaporated under vacuum and to the residue was added ether (1000 ml). The separated solids were filtered and dried to get the title compound. Yield = 78g Calculated Mass = 586 Obtained mass =587

Example 2: Preparation of L-Thr-OMe.HCl

The amino acid H-Thr-OH (35.7 g) was added in portions to methanol (200 ml) and thionyl chloride (109.2 ml) under stirring at temperature 10-20⁰C. After complete addition, the reaction mass was refluxed till the reaction was complete as monitored by TLC. The reaction mixture was evaporated under vacuum at 45 ⁰C. Ether (100 ml x 3) was added to the residue and triturated it. The precipitated solids were filtered and dried under vacuum at 40⁰C for 1 hour. Yield = 50 g Calculated mass = 133 Obtained mass = 134

Example 3: Preparation of Boc-Cvs (Acm)-Thr-OL

Sodium borohydride (14 g) was added to a solution of Boc-Cys (Acm)-Thr-OMe (75.4 g) in aqueous ethanol (452.4 ml, 90%) at 5⁰C. The stirring was continued for further about 2 hours till the reaction was complete as monitored by HPLC. The reaction mass was slowly added to aqueous acetic acid (942.5 ml, 4% w/w) under stirring at 5°C. The reaction mixture was saturated with sodium chloride and extracted in dichloromethane (3 x 500 ml). The dichloromethane layer was dried on sodium sulfate and concentrated under vacuum to get the desired product as syrupy oil. Yield =70 g Calculated Mass = 379 Obtained Mass = 380.3

Example 4: Preparation of H-CysfAcmVPhe-OMe trifluroacetate salt t-BOC-Cys(Acm)-Phe-OMe (181 g) was suspended in dichloromethane (271 ml) and stirred at 5⁰C. It was followed by the addition of trifluoroacetic acid (245 ml). The resultant mass after completion of the reaction was distilled under vacuum to remove trifluoroacetic acid and dichloromethane. Ether (180 ml x 3) and hexane (360 ml x 3) was added to the residue, the mixture was triturated and allowed to settle under ice cooling.

The ether-hexane layer was discarded and the residue was further concentrated under vacuum to get the title product as syrupy oil. Yield= 186 g on anhydrous and dry basis.

Calculated mass = 353

Obtained mass= 354.1 Example 5: Preparation of t-BOC- 8P-OL (BOC- (D)- Phe-Cvs(Acm)- Phe- (D)-Trp- Lvs (BOC)-Thr-Cvs (Acm)-Thr-OL)

A solution of protected t-BOC-6P-OH (100 g) and HOBt (15.4 g) in tetrahydrofuran (600 ml) and N,N-dimethylacetamide (60 ml) was added to a solution of trifluoroacetate salt of Cys(Acm)-Thr-OL (71.4g) and triethylamine (32.72 ml) in tetrahydrofuran (100 ml) and

N₅N-dimethylacetamide (100 ml) and stirred at 5⁰C. DCC (20.73g) and triethylamine

(7.1 ml) in tetrahydrofuran (104 ml) was added at 4°C and the resultant mass was stirred at ambient temperature for 12 hours, followed by concentration under vacuum. To the residue so obtained, ether (1600 ml) was added and the separated product was filtered and the solid was washed with ether (470 ml). Solid was suspended into ethyl acetate

(1600 ml), warmed to 6O⁰C for half an hour and brought to room temperature under stirring followed by filtration of the separated product. The ethyl acetate washing was repeated twice. The product obtained was dried under vacuum to get the title compound.

Yield = 116 g Calculated mass = 1362

Mass obtained = 1363.8

Example 6: Preparation of trifluoroacetate salt of 8P-OL (H-(D) - Phe -Cvs (Acm)-

Phe- (D)- Trp-Lvs- Thr-Cvs (Acm)-Thr-OL.2TFA)

A mixture of t-BOC-8P-OL (100 g) and mercaptoethanol (20.66 ml), anisole (32 ml) and thioanisole (34.53 ml) in dichloromethane (200 ml) was cooled to 5°C and trifluoroacetic acid (400 ml) was added followed by stirring for one hour. The resultant mass was , concentrated under vacuum at 3O⁰C and ether (1500 ml) was added to the residue. The separated solids were filtered and dried under high vacuum to get the title product having

DCC-Urea in it. Yield = 96 g

Solid product obtained above was suspended into water (1000 ml) and stirred for one hour. The undissolved product (DCC-Urea) was filtered and discarded. The filtrate was charged for lyophilization and dried for about 16hrs.

Yield= 67.3 g Calculated mass = 1390

Obtrained mass = 1163.4 Example 7; Preparation of Octreotide acetate

H-(D)-Phe¹-Cys²-Phe³-(D)-Tφ⁴-Lys⁵-Thr⁶-Cys⁷-Thr⁸-OL

A solution of linear octapeptide H-(D)-Phe¹-Cys(Acm)²-Phe³-(D)-Trp⁴-Lys⁵-Thr⁶- Cys(Acm)⁷-Thr⁸-OL.2TFA (25 g, 18 mmol) in 90% methanol (150 ml) was added drop wise to a solution of 90% methanol (600 ml) containing iodine (23g, 90 mmol) over a period of 2 lirs time at ambient temperature. Stirring was continued at room temperature till the completion of reaction, monitored by HPLC. The reaction mixture was cooled to 5⁰C and solution of sodium sulphite (135ml, IN) was added, followed by the addition of sodium hydroxide (45 ml, 4N) and acetic acid (19 ml). The reaction mass was diluted with 0.5% acetic acid (5.5 lit) in water. The solution was filtered through celite bed on sinter funnel and the bed was washed with 0.5% acetic acid (500ml) in water. The clear solution so obtained was subjected to preparative HPLC for desalting and lyophilization. The crude octreotide thus obtained was purified by preparative HPLC and converted into acetate salt with 3% ammonium acetate and 1% acetic acid. It was dried by lyophilization to yield the desired product. Yield = 1O g HPLC purity = 99.16% ESMS = 1019 (M+H) [Ot]²⁵ _D= -66.8° (C=I, Glacial Acetic Acid)

Example 8: Preparation of amorphous octreotide acetate

Octreotide-trifluoroacetate salt (16 g) was charged on preparative HPLC and trifluoroacetatic acid was removed by eluting with 3% ammonium acetate. The column was further eluted with 1% acetic acid to convert octreotide base to octreotide acetate, which was then eluted from the column using a mixture of methanol and acetic acid. The desired fraction containing octreotide acetate was concentrated and the concentrated mass was lyophilized to yield the titled product. Yield = 10 g (XRD as per Figure 1 showed it to be an amorphous material)

HPLC purity = 99.16%

ESMS = 1019 (M+H)

[α]²⁵D= -66.8° (C=I, Glacial Acetic Acid)

While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Claims

We Claim:

1. A process for the preparation Of R₁-(D)PlIe-CyS(ACm)-PlIe-OH, wherein R₁ is amino or a suitable amino protecting group, the process comprising: a) hydrolyzing R₁-(D)PtLe-CyS(ACm)-PlIe-OMe in one or more solvents by maintaining a pH of about 9.0 to about 10 of reaction mass; b) acidifying the reaction mass after completion of hydrolysis; and c) isolating the R₁-(D)PhC-CyS(ACm)-PhC-OH from the reaction mass thereof.

2. The process of claim 1, wherein the isolation comprises one or more of acidification of reaction mass and extraction in a suitable organic solvent.

3. The process of claim 2, further comprising concentrating the organic extracts and treating residue with ether.

4. The process of claim 1, wherein the pH is adjusted by adding a base.

5. The process of claim 4, wherein the base comprises one or more of hydroxides, carbonates or bicarbonates of alkali or alkali earth metal compounds.

6. A process for the preparation of L-Thr-OMe, the process comprising: a) treating L-Thr-OH with methanol and thionyl chloride at a temperature above 45⁰C; b) removing excess thionyl chloride from reaction mass; and c) isolating the L-Thr-OMe from the reaction mass thereof.

7. The process of claim 6, wherein the reaction is carried out at a temperature of about 6O⁰C or above.

8. A process for removal of inorganic impurities from R₁-CyS(ACm)-ThT-OL, wherein R₁ is amino or a suitable amino protecting group, the process comprising: a) reducing R₁-CyS(ACm)-ThT-OMe in one or more solvents in the presence of sodium borohydride; b) extracting the resultant mass in dichloromethane; and c) isolating the R₁-CyS(ACm)-ThT-OL from the extract thereof.

9. The process of claim 8, wherein the solvent comprises one or more of lower alkanols, lower aliphatic acids, water, or mixtures thereof.

10. The process of claim 9, wherein the lower alkanol comprises one or more of methanol, ethanol, isopropanol, and n-propanol.

11. A process for the preparation of Cys(Acm)-Phe-OMe or a salt thereof, the process comprising: a) treating t-BOC-Cys(Acm)-Phe-OMe or a salt thereof with less than 15 moles of trifluoroacetic acid; b) removing the excess trifluoroacetic acid from reaction mass; and c) isolating the Cys(Acm)-Phe-OMe or a salt thereof from the reaction mass thereof.

12. A process for the preparation of Cys(Acm)-Thr-OL or a salt thereof, the process comprising: a) treating t-BOC-Cys(Acm)-Thr-OL or a salt thereof with less than 15 moles of trifluoroacetic acid; b) removing the excess trifluoroacetic acid from reaction mass; and c) isolating the Cys(Acm)-Thr-OL or a salt thereof from the reaction mass thereof.

13. The process of claim 11 or 12, wherein the amount of trifluoroacetic acid required is less than 8 moles.

14. A process for the preparation of trifluoroacetate salt of 8P-OL, the process comprising: a) coupling t-BOC-6P-OH and Cys(Acm)-Thr-OL in one or more suitable organic solvent to give t-BOC-8P-OL; b) removing the t-BOC protection from the product t-BOC-8P-OL using trifluoroacetic acid to give trifluoroacetate salt of 8P-OL; c) extracting the trifluoroacetate salt of 8P-OL in water to get an aqueous solution; d) removing undissolved dicyclohexylcarbodiimide-urea complex from the aqueous solution; and e) isolating the trifluoroacetate salt of 8P-OL from the aqueous solution by lyophilization.

15. The process of claim 14, wherein the suitable organic solvent comprises one or more of polar aprotic solvents.

16. The process of claim 15, wherein the polar aprotic solvent comprises one or more of N, N-dimethylformamide, N, N-dimethylacetamide, dimethylsulphoxide, N-methyl pyrrolidone, tetrahydrofuran, and acetonitrile.

17. The process of claim 14, wherein one or more catalysts and coupling reagent s are used in step a).

18. The process of claim 17, wherein the catalyst is 1-hydroxybenzotriazole and the coupling reagent is dicyclohexylcarbodiimide.

19. Trifluoroacetate salt of 8P-OL' having less than 1% of dicyclohexylcarbodiimide - urea.

20. A process for the preparation of octreotide or a salt thereof, the process comprising: a) treating trifluoroacetate salt of 8P-OL in methanol with iodine; b) drying the resultant mixture; c) treating the resultant mass with one or more bases; d) acidifying the solution of step (C) with acetic acid; and e) isolating the octreotide mixture by desalting.

21. The process of claim 20, wherein the base comprises one or more of hydroxides, carbonates or bicarbonates of alkali or alkali earth metal compounds.

22. A process for the preparation of octreotide acetate by preparative chromatography, the process comprising: a) charging octreotide or a salt thereof over preparative chromatography column with 3% ammonium acetate and 1% acetic acid; b) collecting desired elute; c) concentrating the desired elute; and d) isolating the octreotide or a salt thereof from the elute.

23. The process of claim 21, wherein the octreotide or a salt thereof is isolated by lyophilization.

24. Octreotide or a salt or a derivative thereof having purity more than 98.5% by HPLC.

25. The octreotide or a salt or a derivative thereof of claim 24 having purity more than 99.16% by HPLC.

26. An amorphous form of octreotide acetate.

27. The amorphous form of octreotide acetate of claim 26, wherein the octreotide acetate has the X-Ray diffraction pattern of Figure 1.

28. The amorphous form of octreotide acetate of claim 26, wherein the octreotide acetate has the infrared spectrum of Figure 2.

29. A pharmaceutical composition comprising a therapeutically effective amount of an amorphous form of octreotide acetate; and one or more pharmaceutically acceptable carriers, excipients or diluents.

30. A process for the preparation of amorphous form of octreotide acetate, the process comprising: a) charging octreotide or a salt thereof wherein the salt is other than acetate, over preparative chromatography column; b) eluting the column with 0.1 to 4% ammonium acetate and 0.1-2% acetic acid; c) eluting the column with a mixture comprising acetic acid and methanol to get desired fraction; d) concentrating the desired fraction; and e) isolating the amorphous form of octreotide acetate from the concentrate of step d).

31. The process of claim 30, wherein the amorphous form of octreotide acetate is isolated from the concentrate by lyophilization.