KR100998175B1 - Process for the Preparation of Somatostatin - Google Patents

Abstract

The present invention relates to a method for producing somatostatin. In the preparation method of the present invention, in the process of synthesizing the peptide through a solid-phase synthesis method, the disulfide bond formation reaction of the peptide from which the resin is removed is usually performed in water, but the present invention is disulfide in an organic solvent. Since the bond formation reaction is performed, the separation and purification of the target substance is easy after the reaction is completed, and commercial mass production is possible.

Somatostatin, Solid Synthesis, Disulfide Bond, Organic Solvent

Description

Process for the Preparation of Somatostatin

The present invention relates to a method for producing somatostatin.

Somatostatin, a 14-amino acid peptide, was extracted from the hypothalamus of sheep in 1973 by Girman in the United States. Somatostatin inhibits the secretion of growth hormone somatotropin in the sense of growth hormone inhibiting factor (GIF), growth hormone inhibiting hormone (GIH), and somatotropin release inhibitory factor ( somatotropin release inhibiting factor (SRIF). In addition, it suppresses the secretion of thyroxine stimulation hormone (TSH) from the pituitary gland, and also inhibits the secretion of hormones from the pancreas or digestive tract such as insulin, glucagon or gastrin. Somatostatin has been proved to be widely present in the hypothalamus, as well as in the whole brain, peripheral nerves, thyroid, pancreas, stomach or intestine, and is involved in the digestion, absorption, and regulation of blood sugar levels of nutrients.

Conventional methods for preparing somatostatin are as follows.

U. S. Patent No. 3,862, 925 describes a technique for preparing somatostatin peptide by liquid phase synthesis. However, the manufacturing method has a number of manufacturing steps, a manufacturing process is not only difficult, and the manufacturing yield is low, and thus, there is a disadvantage in that the price competitiveness is low in commercial mass production.

U.S. Patent No. 4,337,194 describes a technique for preparing somatostatin peptides by sequentially binding each amino acid using t-butylcarbonyl protection synthetic techniques using phenylazobenzylsulfonylethanol. However, in the case of the manufacturing method, when removing the t- butylcarbonyl protecting group, trifluoroacetic acid (TFA), which is difficult to apply to commercial mass production, has a disadvantage that must be used in each step.

In the above-mentioned manufacturing method of U.S. Patent No. 3,862,925 and U.S. Patent No. 4,337,194, since the formation reaction of disulfide bond is performed in water, it is not easy to remove water and inorganic salts after the reaction is completed, so that the separation of the target object is not easy. And as purification becomes difficult, there are many limitations in the application of commercial mass production.

As mentioned above, the conventional techniques for preparing somatostatin peptides include many problems that need to be improved in their application to commercial mass production. Therefore, the research on how to effectively prepare somatostatin peptide is a very important development task in the pharmaceutical industry.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

The inventors have found that a suitable method for producing large amounts of somatostatin by finding out that it is suitable for mass production stably when disulfide bond formation reaction is carried out in an organic solvent during the synthesis of somatostatin through a solid-phase synthesis method. By this, the present invention was completed.

It is therefore an object of the present invention to provide a method for producing somatostatin.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for preparing somatostatin, comprising the following steps: (a) obtaining a peptide to which a resin represented by the following formula (I) is attached by a solid-phase synthesis method; ; (b) removing the resin from the peptide obtained in step (a) to obtain a peptide represented by the following formula (II); (c) oxidizing the peptide obtained in step (b) in an organic solvent to perform a disulfide bond formation reaction to obtain a peptide represented by Formula III below; And (d) deprotecting the peptide obtained in step (c) to obtain somatostatin.

Formula I

Formula II

Formula III

In the above formula, R is α-amino protecting group, R ¹ is thio protecting group, R ² is ε-amino protecting group, R ³ is hydrogen or amide protecting group, R ⁴ is hydrogen or indole protecting group, and R ⁵ is hydrogen or hydroxyl protecting group .

Summary of Acronyms

Unless otherwise specified herein, abbreviations used to designate amino acids and protecting groups are based on the terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry , 11: 1726-1732 (1972)). ).

Abbreviations for protecting groups used herein are as follows:

t-butylcarbonyl: Boc

t-butyl: tBu

9-fluorenyloxycarbonyl: Fmoc

Triphenylmethyl: Trt

R in formulas (I) to (III) is an α-amino protecting group and includes any protecting group commonly used in the art. Preferably, the α-amino protecting group is (1) an acyl type protecting group, which is a formyl group, trifluoroacetyl group, phthalyl group, toluenesulfonyl group (tosyl group), benzenesulfonyl group, dinitrophenylsulfonyl group, tri Tylsulphenyl group, o-nitrophenoxyacetyl group, chloroacetyl group, acetyl group, γ-chlorobutylyl group, (2) aromatic urethane type protecting group, benzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-nitrobenzyl Oxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2- (p-biphenyl) isopropoxycarbonyl, iphenylmethoxycarbonyl, (3) aliphatic urethane protecting groups , tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, (4) cycloalkyl urethane type protecting group, cyclopentyloxycarbonyl, adamant Tyloxycarbonyl, cyclohexyloxycarbonyl, (5) Thio urethane type protecting group , Phenylthiocarbonyl, (6) an aralkyl type protecting group, triphenylmethyl (trityl), benzyl, (7) trialkylsilane group, trimethylsilane, more preferably t-butyloxycarbonyl group or 9- It is a fluorenyl methoxycarbonyl group, Most preferably, it is a t-butyloxy carbonyl group.

In Formulas (I) to (III), R ¹ is a thio protecting group commonly used in the art. Preferably, the thio protecting group is benzoyl, trityl, benzyl, p-methoxybenzyl, benzyloxycarbonyl, p-nitrobenzyl, acetamidomethyl, diphenylmethyl, triphenylmethyl group or acetaminomethyl group Preferably it is a triphenylmethyl group.

In Formulas (I) to (III), R ² is an ε-amino protecting group commonly used in the art. Preferably, the ε-amino protecting group is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group, t-butyldimethylsilyl group, triphenylsilyl Group or triisopropylsilyl group, more preferably t-butyloxycarbonyl group or benzyloxycarbonyl group, and most preferably t-butyloxycarbonyl group.

R ³ in Formulas (I) to (III) includes hydrogen or amide protecting groups commonly used in the art. Preferably, the hydrogen or amide protecting group is a methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, t-butyldimethylsilyl group, triphenylsilyl group or triisopropylsilyl group, more preferably benzyloxymethyl group or tri It is a phenylmethyl group, Most preferably, an amide protecting group is a triphenylmethyl group.

In Formulas (I) to (III), R ⁴ is hydrogen or an indole protecting group commonly used in the art. Preferably, the hydrogen or indole protecting group is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group or allyl group, more preferably t-butyloxycarbono And a benzyloxycarbonyl group, most preferably a t-butyloxycarbonyl group.

In Formulas (I) to (III), R ⁵ may be a hydrogen or hydroxyl protecting group commonly used in the art. Preferably, the hydrogen or hydroxyl group protecting group is a tosyl group, trityl group, p-methoxybenzyl group, methoxymethyl group, benzyloxymethyl group, tetrahydropyran group, tetrahydrofuran group, t-butyl group, triphenylmethyl group, Benzyl, allyl, trimethylsilyl, t-butyldimethylsilyl, triphenylsilyl, triisopropylsilyl, t-butylcarbonyl, acetyl or benzoyl, more preferably t-butyl or tri It is a phenylmethyl group, Most preferably, a hydroxyl group protecting group is a t-butyl group.

Protector for the functional group is Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991).

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.

The manufacturing method of the present invention will be described in detail for each step as follows:

(a) to give the peptide of formula Ⅰ

Peptides represented by Formula I are prepared by solid-phase synthesis methods commonly used in the art (Merrifield, RB, J. Am . Chem . Soc . , 85: 2149-2154 (1963), Kaiser , E., Colescot, RL, Bossinger , CD, Cook, PI, Anal Biochem, 34:.. 595-598 (1970)). In other words, after the α-amino and side chain functional groups are protected to bind the resin to the resin, the α-amino protecting group is removed, and the remaining α-amino and side chain functional groups are protected in a step-by-step manner in order to intermediate the intermediate. Get

Selection of the appropriate protecting group depends on the functional group being protected, the conditions under which the protecting group is exposed and other functional groups that may be present in the molecule. The protecting group must be stable for the reaction conditions and reagents selected to remove the α-aminoprotecting group at each step of the synthesis, the deprotection should not occur in the coupling reaction, and the synthesis containing the desired amino acid chains has been completed. It should be stable under decomposition conditions with resin.

According to a preferred embodiment of the invention, the resin is used in the process of synthesizing the peptide of formula (I). Resin that can be used may use a conventional resin that can be easily degraded under mildly acidic conditions that can completely preserve the side chain protecting groups of the prepared peptides. Preferably, the resin is tritylchloride resin, 2-chlorotrityl resin, 4-methyltrityl resin or 4-methoxytrityl resin, more preferably tritylchloride resin or 2-chlorotrityl resin And most preferably 2-chlorotrityl resin.

(b) to give the peptide of formula Ⅱ

The compound represented by the formula (II) can be obtained by removing the resin under mild acidic conditions from the peptide obtained in step (a). At this time, the acidic conditions that can be used should be a mild condition in which the side chain protecting group of the amino acid chain can be maintained completely.

According to a preferred embodiment of the invention, the process of removing the resin is carried out under acidic conditions. Preferably, the acidic conditions are carried out under a solution of dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 or containing 0.5-5% trifluoroacetic acid. It is performed under dichloromethane solution.

(c) obtain a peptide represented by the formula Ⅲ

The compound represented by the formula (III) can be obtained by performing a disulfide bond formation reaction using a suitable oxidizing agent under an organic solvent from the peptide obtained in step (b).

According to a preferred embodiment of the present invention, the oxidizing agent available in step (c) is I ₂ , oxygen, perisignide, dimethylsulfoxide, glutathione, peroxynitride or hydrogen peroxide, more preferably I ₂ Or dimethyl sulfoxide, most preferably I ₂ .

According to a preferred embodiment of the present invention, an organic solvent is used in the reaction for forming disulfide bonds. Preferably, the organic solvent is a halogenated hydrocarbon solvent or an alcohol solvent, more preferably dichloromethane, dichloroethane, chloroform, methanol, ethanol or isopropanol, most preferably dichloromethane, acetic acid and trifluoroethanol Mixed solvent.

The disulfide bond formation reaction concentration of the mixed solvent is preferably 0.01-0.0001 M, most preferably 0.005-0.0005 M, based on the compound represented by the formula (III).

(d) to give the somatostatin

Somatostatin can be obtained from the peptide obtained in step (b) by performing a deprotection reaction under reaction conditions commonly used in the art.

The reaction conditions of the deprotection reaction are preferably trifluoroacetic acid, water, phenol, thioanisole and mixture of ethanedithiol, trifluoroacetic acid, triisopropylsilane and water mixture or trifluoroacetic acid, triiso It is possible under a mixture of propylsilane, water and ethanedithiol, most preferably under a mixture of trifluoroacetic acid, triisopropylsilane and water.

Based on the above contents, the entire process of preparing somatostatin is summarized as follows (Scheme 1).

As described in detail above, the present invention provides a method for producing somatostatin. In particular, in the process of synthesizing the peptide through the solid-phase synthesis method, the disulfide bond formation reaction of the resin-removed peptide is generally carried out in water, whereas the present invention provides a disulfide bond formation reaction in an organic solvent. Since the reaction is completed, the separation and purification of the target object is easy after the reaction is completed, there is an advantage that commercial mass production is possible.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Throughout this specification, “%” used to indicate the concentration of a particular substance, unless otherwise stated, is solid / solid (% by weight), solid / liquid is (weight / volume)%, and Liquid / liquid is (volume / volume)%.

Example  1: represented by the formula (IV) Peptide  Produce

Formula IV

9- Fluorenyloxycarbonyl - Cys ( Triphenylmethyl ) -O-2- Chlorotrityl  Manufacture of Resin

2-chlorotrityl chloride resin (23.6 g, f = 1.27 mmol / g resin, 30 mmol) and dichloromethane (200 mL) were added to a solid-phase synthesis reactor equipped with a filtration membrane. After expansion, the solvent was removed through the filtration membrane under reduced pressure. Dichloromethane with 9-fluorenyloxycarbonyl-Cys (triphenylmethyl) -OH (26.4 g, 45 mmol, 1.5 equiv) and 1-hydroxybenzotriazole (6.08 g, 45 mmol, 1.5 equiv) (200 mL) was added, followed by addition of diisopropylethylamine (6.59 g, 51 mmol, 1.7 equiv) and reaction at room temperature for 4 hours. The reaction solution was removed by filtration under reduced pressure, and the resin was washed once with dichloromethane. Then, dichloromethane: methanol: diisopropylethylamine = 17: 2: 1 (200 mL) was added to the resin and stirred for 20 minutes. The reaction solution was removed by filtration under reduced pressure, the resin was washed three times with dichloromethane, and then dried under vacuum to give 40.5 g 9-fluorenyloxycarbonyl-Cys (triphenylmethyl) -2-chlorotrityl resin. . The substitution rate was 0.56 mmol / g.

9- Fluorenyloxycarbonyl -amino acid- OH  Coupling reaction

(a) Obtaining H- Cys ( triphenylmethyl ) -2 -chlorotrityl resin

9-Fluorenyloxycarbonyl-Cys (triphenylmethyl) -2-chlorotrityl resin (17.9 g, f = 0.56 mmol / g, 10 mmol) and N, N-dimethyl in a high synthesis reactor equipped with a filtration membrane Formamide (150 mL) was added thereto, the resin was expanded for 15 minutes, and the solvent was removed through a filtration membrane under reduced pressure. N, N-dimethylformamide (150 ml) containing 20% (v / v) piperidine was added thereto, followed by removal of 9-fluorenyloxycarbonyl for 15 minutes, followed by filtration under reduced pressure. Removed. The removal reaction of 9-fluorenyloxycarbonyl was repeated, and the resin was then washed once with N, N-dimethylformamide, twice with dichloromethane and twice with N, N-dimethylformamide, and then H- Cys (triphenylmethyl) -2-chlorotrityl resin was obtained.

(b) Obtaining 9- Fluorenyloxycarbonyl - Ser (t-butyl) -Cys ( triphenylmethyl ) -2 -chlorotrityl resin

To H-Cys (triphenylmethyl) -2-chlorotrityl resin (10 mmol) obtained in the reaction (a), 9-fluorenyloxycarbonyl-Ser (t-butyl) -OH (11.5 g, 30 mmol) , 3.0 equiv) and 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3.0 equiv) were added N, N-dimethylformamide (120 mL), followed by N, N containing diisopropylcarbodiimide. -Dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv) was added followed by reaction at room temperature for 4 hours. The reaction solution was removed by filtration under reduced pressure, and the resin was washed twice with N, N-dimethylformamide to give 9-fluorenyloxycarbonyl-Ser (t-butyl) -Cys (triphenylmethyl) -2-chlorotrityl. Obtained Resin.

(c) Obtaining H- Ser (t-butyl) -Cys ( triphenylmethyl ) -2 -chlorotrityl resin

The 9-fluorenyloxycarbonyl-Ser (t-butyl) -Cys (triphenylmethyl) -2-chlorotrityl resin obtained in the reaction (b) contained 20% (v / v) piperidine. N, N-dimethylformamide (150 mL) was added thereto, followed by a reaction for removing 9-fluorenyloxycarbonyl for 15 minutes, followed by filtration under reduced pressure to remove the reaction solution. The removal reaction of 9-fluorenyloxycarbonyl was repeated, and the resin was then washed once with N, N-dimethylformamide, twice with dichloromethane and twice with N, N-dimethylformamide, followed by H- Ser (t-butyl) -Cys (triphenylmethyl) -2-chlorotrityl resin was obtained.

(d) coupling of 9- fluorenyloxycarbonyl -amino acid- OH and obtaining the final material

The following amino acid derivatives were sequentially coupled while repeating the reactions (c) and (d) above.

9-Fluorenyloxycarbonyl-Thr (t-butyl) -OH (11.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Thr (t-butyl) -OH (11.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Lys (t-butylcarbonyl) -OH (14.1 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Trp (t-butylcarbonyl) -OH (15.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzo triazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Asn (triphenylmethyl) -OH (17.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Lys (t-butylcarbonyl) -OH (14.1 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Cys (triphenylmethyl) -OH (17.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Gly-OH (8.92 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Ala-OH (9.3 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

Finally, after carrying out the coupling reaction of 9-fluorenyloxycarbonyl-Ala-OH, the resin is washed three times with N, N-dimethylformamide and three times with dichloromethane, which is represented by Chemical Formula IV. Peptides were obtained.

Example  2: represented by the formula (V) Peptide  Produce

Formula V

To the peptide of Formula IV obtained in Example 1 was added a mixed solution (300 ml) of dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 and stirred for 2 hours. The resin was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain 29.8 g of crude peptide represented by the above formula (V). The yield of crude was 97% and the LC purity was 76%.

Example  3: Formula VI to  Displayed Peptide  Produce

Formula VI

Dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 (1000 mL) was added to 3.0 g of the crude peptide represented by Chemical Formula V obtained in Example 2, and I ₂ (0.25 g, 10 mmol) was added. Dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 mixed solution (12.5 mL) was slowly added dropwise, followed by further stirring for 15 minutes. 1 N Na ₂ S ₂ O ₃ aqueous solution (500 mL) was added to remove excess I ₂ , the layers were separated, and the organic layer was washed with 1 N citric acid aqueous solution. The organic layer was dried over Na ₂ SO ₄ , filtered and distilled under reduced pressure to obtain 2.1 g of the peptide represented by Chemical Formula VI.

Example  4: represented by the formula Somatostatin  Produce

Formula Ⅶ

Ethyl acetate 2.1 g of the peptide represented by Formula (VI) obtained in Example 3 It was dissolved in (20 mL), tris (2-aminoethyl) amine (TAEA) (0.22 g, 1.5 mmol) was added at room temperature, and 9-fluorenyloxycarbonyl was removed for 1 hour. The resulting solid was removed by filtration, the organic layer was washed with water three times and then distilled under reduced pressure to remove ethyl acetate. Subsequently, a mixed solution of t-butylmethylether: triisopropylsilane: water = 95: 2.5: 2.5 (20 ml) was added thereto, followed by a deprotection reaction for 2 hours. The solvent was distilled off under reduced pressure, and t-butyl methyl ether (30 mL) was added to the obtained solid. The solid was filtered to obtain 1.3 g of crude somatostatin. 0.6 g of somatostatin represented by the formula VII by purification by reverse phase HPLC (220 nm, 10 ml / min, increased from 20% to 40% acetonitrile in 0.1% trifluoroacetic acid in 20 minutes on a 5 micron C18 column) Got it. Total yield was 36% and HPLC purity was 99%.

Example  5: represented by the formula Peptide  Produce

Formula Ⅷ

9- Fluorenyloxycarbonyl -amino acid- OH  Coupling reaction

(a) Obtaining H- Cys ( triphenylmethyl ) -2 -chlorotrityl resin

9-Fluorenyloxycarbonyl-Cys (triphenylmethyl) -2-chlorotrityl resin (17.9 g, f = 0.56 mmol / g, 10 mmol) and N, N-dimethyl in a high synthesis reactor equipped with a filtration membrane Formamide (150 mL) was added thereto, the resin was expanded for 15 minutes, and the solvent was removed through a filtration membrane under reduced pressure. N, N-dimethylformamide (150 ml) containing 20% (v / v) piperidine was added thereto, followed by removal of 9-fluorenyloxycarbonyl for 15 minutes, followed by filtration under reduced pressure. Removed. The removal reaction of 9-fluorenyloxycarbonyl was repeated, and the resin was then washed once with N, N-dimethylformamide, twice with dichloromethane and twice with N, N-dimethylformamide, followed by H- Cys (triphenylmethyl) -2-chlorotrityl resin was obtained.

(b) Obtaining 9- Fluorenyloxycarbonyl - Ser (t-butyl) -Cys ( triphenylmethyl ) -2 -chlorotrityl resin

To H-Cys (triphenylmethyl) -2-chlorotrityl resin (10 mmol) obtained in the reaction (a), 9-fluorenyloxycarbonyl-Ser (t-butyl) -OH (11.5 g, 30 mmol) , 3.0 equiv) and N, N-dimethylformamide (120 mL) containing 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3.0 equiv) was added followed by diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv) was added, followed by reaction at room temperature for 4 hours. The reaction solution was removed by filtration under reduced pressure, and the resin was washed twice with N, N-dimethylformamide to give 9-fluorenyloxycarbonyl-Ser (t-butyl) -Cys (triphenylmethyl) -2-chlorotrityl. Obtained Resin.

(c) Obtaining H- Ser (t-butyl) -Cys ( triphenylmethyl ) -O-2 -chlorotrityl resin

The 9-fluorenyloxycarbonyl-Ser (t-butyl) -Cys (triphenylmethyl) -2-chlorotrityl resin obtained in the reaction (b) contained 20% (v / v) piperidine. N, N-dimethylformamide (150 mL) was added thereto, followed by a reaction for removing 9-fluorenyloxycarbonyl for 15 minutes, followed by filtration under reduced pressure to remove the reaction solution. The removal reaction of 9-fluorenyloxycarbonyl was repeated, and the resin was then washed once with N, N-dimethylformamide, twice with dichloromethane and twice with N, N-dimethylformamide, followed by H- Ser (t-butyl) -Cys (triphenylmethyl) -O-2-chlorotrityl resin was obtained.

(d) coupling of 9- fluorenyloxycarbonyl -amino acid- OH and obtaining the final material

The following amino acid derivatives were sequentially coupled while repeating the reactions (b) and (c) above.

9-Fluorenyloxycarbonyl-Thr (t-butyl) -OH (11.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Thr (t-butyl) -OH (11.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Lys (t-butylcarbonyl) -OH (14.1 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Trp (t-butylcarbonyl) -OH (15.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Phe-OH (11.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Asn (triphenylmethyl) -OH (17.9 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Lys (t-butylcarbonyl) -OH (14.1 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diiso N, N-dimethylformamide solution with propylcarbodiimide (15 mL, 2 M solution, 3.0 equiv)

9-Fluorenyloxycarbonyl-Cys (triphenylmethyl) -OH (17.6 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcart N, N-dimethylformamide solution with bodyimide (15 mL, 2 M solution, 3.0 equiv)

9-fluorenyloxycarbonyl-Gly-OH (8.92 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide N, N-dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

N, N with t-butylcarbonyl-Ala-OH (5.68 g, 30 mmol, 3 equiv), 1-hydroxybenzotriazole (4.05 g, 30 mmol, 3 equiv) and diisopropylcarbodiimide Dimethylformamide solution (15 mL, 2 M solution, 3.0 equiv)

Finally, after carrying out the coupling reaction of t-butylcarbonyl-Ala-OH, the resin was washed three times with N, N-dimethylformamide and three times with dichloromethane in order to obtain a peptide represented by the above formula (VII). .

Example  6: represented by the formula Peptide  Produce

Formula Ⅸ

To the peptide obtained in Formula 5, dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 was added (300 ml), followed by stirring for 2 hours. The resin was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain 29.5 g of crude peptide represented by the above formula (VI). The yield of crude was close to 100% and the purity of LC was 78%.

Example  7: chemical formula to  Displayed Peptide  Produce

Formula Ⅹ

Dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 (2000 mL) was added to the crude peptide (5.87 g) obtained in Formula 6, and I ₂ (0.5 g, 20 mmol) was added. Dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1 mixed solution (25 mL) was slowly added dropwise, followed by further stirring for 15 minutes. 1 N Na ₂ S ₂ O ₃ aqueous solution (1000 mL) was added to remove excess I ₂ , and the layers were separated, and then the organic layer was washed with 1 N aqueous citric acid solution. The organic layer was dried over Na ₂ SO ₄ , filtered and distilled under reduced pressure to obtain 4.73 g of the peptide represented by the formula (VII).

Example  8: represented by the formula Somatostatin  Produce

Formula Ⅶ

4.73 g of the peptide represented by Formula (VII) obtained in Example 7 was added to a t-butylmethylether: triisopropylsilane: water = 95: 2.5: 2.5 (40 mL) mixed solution, and deprotected for 2 hours. It was. The solvent was distilled under reduced pressure, t-butylmethylether (60 mL) was added thereto, and the solid obtained was filtered to obtain 2.82 g of crude somatostatin. Purification by reverse phase HPLC (220 nm, 10 mL / min, increased from 20% to 40% acetonitrile in 0.1% trifluoroacetic acid in 20 minutes on a 5 micron C18 column) gave 1.24 g of somatostatin represented by the formula: . Total yield was 38% and HPLC purity was 99%.

Claims (12)

Method for preparing somatostatin comprising the following steps: (a) obtaining a peptide to which a resin represented by the following formula (I) is attached by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a peptide represented by the following formula (II); (c) oxidizing the peptide obtained in step (b) in an organic solvent to perform a disulfide bond formation reaction to obtain a peptide represented by Formula III below; And (d) performing deprotection on the peptide obtained in step (c) to obtain somatostatin. Formula I

Formula II

Formula III

In the above formula, R is α-amino protecting group, R ¹ is thio protecting group, R ² is ε-amino protecting group, R ³ is hydrogen or amide protecting group, R ⁴ is hydrogen or indole protecting group, and R ⁵ is hydrogen or hydroxyl protecting group . The α-amino protecting group according to claim 1, wherein the α-amino protecting group is t-butyloxycarbonyl group, 9-fluorenylmethoxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group and t-butyldimethylsilyl group, triphenylsilyl group or triisopropylsilyl group. The method for producing somatostatin according to claim 1, wherein the thio protecting group is a triphenylmethyl group or an acetaminomethyl group. The ε-amino protecting group is a t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group, t-butyldimethylsilyl group, tri A method for producing somatostatin, which is a phenylsilyl group or a triisopropylsilyl group. The method for producing somatostatin according to claim 1, wherein the hydrogen or amide protecting group is a methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, t-butyldimethylsilyl group, triphenylsilyl group or triisopropylsilyl group. The method for producing somatostatin according to claim 1, wherein the hydrogen or indole protecting group is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group or allyl group. The method of claim 1, wherein the hydrogen or hydroxyl group protecting group is a methoxymethyl group, benzyloxymethyl group, tetrahydropyran group, tetrahydrofuran group, t-butyl group, triphenylmethyl group, benzyl group, allyl group, trimethylsilyl group, t -A butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group, t-butylcarbonyl group, acetyl group or benzoyl group. The method of claim 1, wherein the resin in step (a) is 2-chlorotrityl chloride, trityl chloride, 4-methyltrityl chloride or 4-methoxytrityl chloride. The method of claim 1, wherein the step of removing the resin in step (b) is performed under acidic conditions. 10. The method of claim 9, wherein the acidic condition is a solution of dichloromethane: acetic acid: trifluoroethanol = 7: 2: 1. 10. The method of claim 9, wherein the acidic condition is 0.5 to 5% trifluoroacetic acid containing A method for producing somatostatin, which is a dichloromethane solution. The method for producing somatostatin according to claim 1, wherein the organic solvent in step (c) is a mixed solvent of dichloromethane, acetic acid and trifluoroethanol.