Description:TECHNICAL FIELD

The present invention is in relation to pharmaceutical chemistry. In particular, the present invention is in relation to the preparation of Octreotide, in particular to its Acetate salt. The method adopts solid phase synthesis to beget Octreotide Acetate in high yield and purity.

BACKGROUND OF INVENTION
Octreotide is an octapeptide of formula A, it has been potentially adopted in the management of various disorders and diseases. It is used in the management of growth hormone related disorders and diseases like Tumours, Acromegaly. Also, the bleeding esophagealvarices, and gastrointestinal secretions and fistules are controlled by the use of said compound. It has also been adopted in radiolabelling techniques in the identification of tumours.

Formula A

The importance of Octreotide in medical domain has necessitated to develop simple and cost effective method for large scale production.
The patent document US 6,346,601 demonstrates a method of preparation of Octreotide and its salts based on solid phase synthesis. The disclosed method is not suitable for large scale preparation.

The patent document PCT/IN2003/00160 discloses a method of preparation of the Octreotide by solution phase peptide chemistry. The method involves condensation of dipeptides and use of reducing agents like sodium borohydride rendering the process to be expensive and unsuitable for large scale synthesis.

The patent document PCT/CA2005/000378 discloses a method of preparation of the Octreotide by a hybrid synthesis wherein the method comprises liquid phase condensation of two or three peptide blocks, of which at least one is synthesized by solid-phase method. Each block contains two or more amino acid residues. The method is cumbersome owing to multiple couplings and purification steps.

Patent document PCT/IN2012/000264 adopts liquid phase synthesis for the preparation of the peptide. The linear octapeptide alcohol is treated withcocktail mixture TFA /water/ TIS -9.0:0.5:0.25, to removes the Trt and Boc groups, followed by oxidation with hydrogenperoxide to obtain Octreotide in liquid phase.

Another document PCT/IN2012/000652 discloses a method of preparation of Octreotide by solid phase synthesis adopting 2-chloritrityl chloride resin by coupling appropriately protected amino acid, deprotection oxidation of the thiol to disulphide followed by purification.
The prior art processes require multiple purification steps and expensive reagents due to the steps adopted. The low yield is another disadvantage associated with the processes. It is necessary to develop a method involving fewer steps, high yield and purity, considering the importance of Octreotide in the medical field.

SUMMARY OF INVENTION
Accordingly, the present invention provides a method of preparation Octreotide Acetate comprising steps of-
a) preparing suitably protected heptapeptide- Cys-Phe-Trp-Lys-Thr-Cys-Thr on a resin by solid phase synthesis,
b) oxidizing the heptapeptide to obtain disulphide bridge between the two Cystiene ( Cys) amino acids by solid phase on the resin.
c) adding the protected amino acid Phenylalanine (Phe) to the heptapeptide and deprotecting the heptapeptide to obtain the Octreotide,
d) isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
e) purifying and lyophilizing to obtain Octreotide Acetate.
A method of preparation Octreotide Acetate comprising steps of-
a. preparing protected heptapeptide of formula B on 2-Chlorotrityl chloride resin by solid phase synthesis,

Formula B
b. oxidizing the heptapeptide of formula B with Iodine to obtain compound of formula C with disulphide bridge between the two Cystiene (Cys) amino acids,

Formula C
c. adding Fmoc protected amino acid Phenylalanine (Phe) to the compound of formula C obtain compound of formula D;

Formula D
d. deprotecting the heptapeptide of formula D to obtain the Octreotide,
e. isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
f. purifying and lyophilizing to obtain Octreotide Acetate.

A peptide of formula E,

Formula E
BRIEF DESCRIPTION OF FIGURES
The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of invention and are therefore not to be considered limiting of its scope for the invention.
Figure 1: illustrates mass spectra of the stage VIII compound of the Octreotide Acetate preparation process, with the S-S bridge.
Figure 2: illustrates Octreotide HPLC Chromatogram
Figure 3: illustrates the Octreotide Acetate HPLC Chromatogram
DETAILED DESCRIPTION OF INVENTION

The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the figures, description and claims.It may further be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.

Abbreviations:
MDC Dichloromethane
DIEPA N,N'-Diisopropylethylamine
MeOH Methanol
Fmoc 9-Fluorenylmethoxycarbonyl
Trt Trityl
HOBT N-Hydroxybenzotriazol
t-Bu tert-butyl
TFA Trifluoro Acetic acid
MTEB Methyl tert-butyl ether
DMF N,N-Dimethylformamide
HPLC High-performance liquid chromatography
DIC Diisopropylcarbodiimide
Lc-Ms Liquid chromatography–mass spectrometry
Cys L-Cysteine
Boc
RT
USP tert-butoxycarbonyl
Room Temperature ranging from 20? to 35?
US Pharmacopeia

The present invention is related to an economical and simple method of preparation of Octreotide and its salt. In particular to a method of preparation of Octreotide acetate by solid phase synthesis. The method yields Octreotide acetate of 99.9% purity.

The general method of preparation of Octreotide acetate involves the steps of -
a) preparing suitably protected heptapeptide- Cys-Phe-Trp-Lys-Thr-Cys-Thr on a resin by solid phase synthesis,
b) oxidizing the heptapeptide to obtain disulphide bridge between the two Cystiene ( Cys) amino acids by solid phase on the resin.
c) adding the protected amino acid Phenylalanine (Phe) to the heptapeptide and deprotecting the heptapeptide to obtain the Octreotide,
d) isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
e) purifying and lyophilizing to obtain Octreotide Acetate.

In an embodiment of the present invention the resin is 2-Chlorotrityl chloride.
In another embodiment of present invention, the solvent is selected from a group comprising Dichloromethane, N, N'-Diisopropylethylamine, Dimethyl Formamide, Methanol, Water, Acetonitrile, Acetic acid.

In another embodiment of present invention, the protecting group is selected from a group comprising tert-butyloxycarbonyl, 9-Fluorenylmethoxycarbonyl group, Trityl group, N-Hydroxybenzotriazol, tert-butyl group and combination thereof.

In an embodiment of present invention the Octreotide is purified by High performance liquid chromatography.

In an embodiment of the invention the protected heptapeptide Cys-Phe-Trp-Lys-Thr-Cys-Thr formed on the resin is of formula B

Formula B

In another embodiment of present invention, the protected peptide of formula B is oxidized to form disulphide bridge by Iodine in a solvent to obtain compound of formula C.

Formula C
In still another embodiment of present invention, the peptide of formula-E obtained by cleaving compound of formula C from the resin.

Formula- E

In still another embodiment of present invention, the protected amino acid Phenylalanine (Phe) is linked to the compound of formula C to obtain the protected octapeptide of formula D.

Formula D

In an embodiment of the present invention, the method of preparation of Octreotide acetate involves the steps of-
a) preparing protected heptapeptide of formula B on 2-Chlorotrityl chloride resin by solid phase synthesis,

Formual B
b) oxidizing the heptapeptide with Iodine to obtain disulphide bridge between the two Cystiene ( Cys) amino acids,

Formula C
c) adding the protected amino acid Phenylalanine (Phe) to compound of formula C obtain compound of formula D;

Formula D
d) deprotecting the heptapeptide of formula D to obtain the Octreotide,
e) isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
f) purifying and lyophilizing to obtain Octreotide Acetate.

In an embodiment of present invention the eluent in the High Performance Liquid Chromatography is Ammonium acetate with Buffer A of 0.5% Acetic acid in water and Buffer B with 70% Acetonitrile in Acetic acid.

The above said method of preparation is depicted in the flowchart -1 below.

FLOWCHART-1
Experimentation:
Example-1
Stage 1:
Solid phase synthesis is carried out in 250 ml reactor. Resin 2CTC, 10 gm is charged and swelled with MDC 100 ml for one hr and washed with MDC. Solution of Fmoc-Thr(tBu)-OL (3.0 equivalent) in MDC (1000 ml) and DIEPA ( 5.0 Equivalent) and the resin is kept under stirring for 10 hrs. After reaction, solution is filtered and washed with MDC (100 ml) and DMF (100 ml). End capping is performed with MDC (100 ml) and MeOH (2 ml) with stirring one hr.Solid resin is filtered, washed with DMF 200 ml X5. Dry weight of resin is 13 gm.

Example-2
De protection and Coupling
Stage 1 resin (13 gm )is added to DMF ( 130 ml ) and charged to reactor. Deportation is carried using DMF-Piperidine 20% (150 ml) stirring 20 min. Deprotection is confirmed by Kiser test. Reaction mass is washed with DMF (260 ml ) X5times.

Coupling reaction is carried out using Fmoc-Cys-Trt-OH(three equivalent), HOBT (three equivalent) and DIC (5.0 equivalent) in DMF 130 ml. Coupling reaction is confirmed by Kiser test after three hours. Reaction mass is filtered and washed with DMF 260ml.

Example-3
Stage –III to Stage VII
De protection and coupling stages are carried out same as example -2 by respective Fmoc-amino acid in the sequential order- Fmoc- Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys-Trt-OH.

Example-4
Stage –VIII
To product of stage VII, a solution of Iodine 4 gm in DMF 100 ml and MeOH 100 ml is added. The reaction mass stirred for 4 hrs at room temperature. Solid bed is filtered washed with DMF X5(100ml each time), MeOH 100 ml, MDC 100 ml. Disulphide Bridge is confirmed by LC-Ms and Ellman’s test.

Also, the product is analysed by mass spectra. Approximately, 100 mg,of compound istaken and cleaved the resin by TFA (20 ml),followed by crystallisation with MTEB. Dry product( compound of formula E) is analysed by Mass spectra. Mass spectral analysis is carried out with Liquid chromatography–mass spectrometer, Shimadzu Lc-Ms 8045. The major peak with mass –to-charge ratio is 1094 M+/M-( figure 1).

Formula –E (Stage VIII compound)

Example-5
Stage-IX
The above stage VIII product is deprotected by DMF-Piperidine 20% (150 ml) stirring for 30 min which is confirmed by Keiser test. Reaction mas is washed with DMF 250 ml. A solution of Fmoc D-Phe-OH (Three equivalent) in DMF (100ml ) DIC ( Five equivalent ) HOBT (Three equivalent )is added and stirred for four hrs. Coupling reaction is confirmed by kisers test. Another de protection is carried out by DMF-Piperidine 20% ( 150 ml) stirring for 30 min which is confirmed by Keiser test. Solid bed is filtered and washed with DMF 100 ml and MDC 100 ml. Unloaded solid resin weight is 18 gm.

Example-6
Stage-X
Stage IX ( 18 gm) is charged to RB Flask and TFA (100 ml) is added. The mass is stirred for 4 hrs at room temperature. Solid mass is filtered off and washed with TFA 50 ml. Combined filtrate is distilled under high vacuum below 30oC. Trace of TFA distilled out by adding 50 ml Toluene.

To this mass, MTEB (200 ml) is added and stirred for 2 hrs at RT. The off- white crystals are filtered, washed with MTEB 50 ml. Crude Octreotide obtained, which is dried under vacuum, dry weight 6.0 gm crude (HPLC purity 78%; Figure 2)

Example-7
Purification by Preparative HPLC
Preparative HPLC is carried out in Knaur Azura Prep HPLC equipment.
Octreotide crude (2 gm), is dissolved in 30 ml water loaded in Prep HPLC, using 50 mm C18- 10 µ column. Column bed is saturated with Ammonium Acetate 3%. Buffer A: 0.5% Acetic acid in water with solution in water. Buffer B: 70% Acetonitrile in 0.5% Acetic acid is passed by gradient at 30 ml/ min flow rate gradient (A:B::90:10, 80:20, 70:30. 70:30, 40:60, 10:90 ) at 210 nm wavelength. Fractions are collected and lyophilised to get dry pure Octreotide Acetate(800 mg)with HPLC purity 99.90%( Figure3).
The Specific Optical Rotation of the Octreotide Acetate is analysed by with Rudolph Autopol II and the same is observed to be [a] d20 (C= 2 inMeOH) - 14.850.
The amino acid analysis of the Octreotide Acetate carried out with Amino acid analyser SYKAM S433 is indicated in the Table 1.

Table 1: Amino acid analysis of Octreotide Acetate

SN Name
Nmol Result
(Mole ratio) Acceptance (USP Reference)
Criteria; Mole ratio

1 Threonine 125.794 0.84 0.7 - 1.1
2 Lysine 111.869 1.12 0.9 - 1.3
3 Phenylalanine 168.14 2.03 1.8 - 2.2
4 Tryptophan 70.45 0.47 0.4 - 1.1
5 Cystine 76.667 1.03 1.0 - 2.2
6 Threonin-ol 135.444 0.91 0.6 - 1.3

Thus the present invention provides an economical and facile method of preparation of Octreotide acetate. The other advantages of the invention involves–
1. Formation of Disulphide SS bridge is carried out in solid phase with Trityl / Trityl cysteine (2-7 sequence) without cleavage of resin which results clean process and better purity. In the said step, cleavage of trityl moietyand SS bridging are taking place in one step (insitu).
2. Purification of crude Octreotide is achieved up to 99.9% Octreotide acetate with high yield in single run by prep HPLC. Developed gradient run flow is cost effective and time saving process.
3. The new peptide fragment Fmoc-Cys-Phe-D-(Boc)Trp-(Boc)Lys-(tBu)Thr-Cys-(tBu)-Thr-Resin (Disulfide bridge Cys 2-Cys 7) is separately synthesised and coupled with Fmoc-D-Phe-OH on solid phase followed by global de protection and cleavage of resin. Themethod retains desired chirality and avoid racemisation of final compound- Octreotide.
, Claims:WE CLAIM
1. A method of preparation Octreotide Acetate comprising steps of-
a) preparing suitably protected heptapeptide- Cys-Phe-Trp-Lys-Thr-Cys-Thr on a resin by solid phase synthesis,
b) oxidizing the heptapeptide to obtain disulphide bridge between the two Cystiene ( Cys) amino acids by solid phase on the resin.
c) adding the protected amino acid Phenylalanine (Phe) to the heptapeptide and deprotecting the heptapeptide to obtain the Octreotide,
d) isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
e) purifying and lyophilizing to obtain Octreotide Acetate.

2. The method of preparation as claimed in claim 1, wherein the resin is 2-Chlorotrityl chloride.

3. The method of preparation as claimed in claim 1, wherein the solvent is selected from a group comprising Dichloromethane, N,N'-Diisopropylethylamine, Dimethyl Formamide, Methanol, Water, Acetonitrile, Acetic acid and combination thereof.

4. The method of preparation as claimed in claim 1, wherein the protecting group is selected from a group comprising tert-butyloxycarbonyl, 9-Fluorenylmethoxycarbonyl group, Trityl group, N-Hydroxybenzotriazol, tert-butyl group and combination thereof.
5. The method of preparation as claimed in claim 1, wherein the heptapeptide is oxidized with Iodine.

6. The method of preparation as claimed in claim 1, wherein the Octreotide is purified by High performance liquid chromatography.

7. A method of preparation Octreotide Acetate comprising steps of-
a. preparing protected heptapeptide of formula B on 2-Chlorotrityl chloride resin by solid phase synthesis,

Formula B
b. oxidizing the heptapeptide of formula B with Iodine to obtain compound of formula C with disulphide bridge between the two Cystiene (Cys) amino acids,

Formula C
c. adding Fmoc protected amino acid Phenylalanine (Phe) to the compound of formula C obtain compound of formula D;

Formula D
d. deprotecting the heptapeptide of formula D to obtain the Octreotide,
e. isolating the Octreotide from the resin and filtering to obtain crude Octreotide, and
f. purifying and lyophilizing to obtain Octreotide Acetate.
8. The method as claimed in claim 1 and 7, wherein the Octreotide acetate is of 99.9% purity.
9. A peptide of formula E,

Formula E