KR20190003913A - Process for the Preparation of Goserelin - Google Patents

Abstract

The present invention relates to a method for preparing goserelin which is obtained by performing a hydriding reaction after preparing linear peptides by performing a solid-phase synthesis and solution-phase synthesis concurrently. The preparing method of the present invention facilitates separation and refinement of a target substance after finishing reactions by performing a solid-phase reaction, a convergence reaction, and a hydriding reaction in order, which is suitable for commercial mass production of goserelin.

Description

The present invention relates to a process for preparing goserelin,

The present invention relates to a method for producing goserelin.

Goserelin is a derivative of the gonadotrophin releasing hormone (GnRH) that continuously suppresses the production of pituitary gonadotropin and gonadal steroids, and inhibits prostate cancer, premenopausal and postmenopausal women Advanced breast cancer, endometriosis, and the like.

There are convergent synthesis methods and liquid phase synthesis methods for synthesis of goserelin. Convergent synthesis is a method of attaching an amino acid sequence to a solid support to complete assembly and then to obtain the sequence from the support, followed by liquid phase reaction to obtain goserelin. This method is advantageous in that the reaction speed is fast, the byproducts are small, and the automation is easy, but there is a disadvantage that it is necessary to use an excessive amount of raw materials. On the other hand, the liquid phase synthesis method has a disadvantage in that it is difficult to purify because it is advantageous in that the cost of reagents and materials is low as a conventional organic synthesis method, but the number of reaction steps is large and the intermediates are advantageous for each step and isomers are generated.

A conventional preparation method for preparing goserelin is as follows.

Korean Patent No. 10-2008-0033120 describes a method for peeling a peptide from a resin after solid phase reaction and obtaining goserelin after hydrogen reaction under palladium. However, there is a drawback to use expensive link-resin.

 U.S. Patent No. 6,448,031 describes a technique for preparing goserelin using a liquid phase synthesis method. However, the manufacturing method is disadvantageous in that the production process is difficult such as the use of a reagent which is industrially unavailable in the final process due to a large number of production steps, and the production yield is low, so that the price competitiveness is inferior in commercial mass production.

 U.S. Patent No. 6,897,289 discloses a technique for sequestering each amino acid in a solid-phase reaction, removing the resin, and producing goserelin through a convergence reaction. However, this method has disadvantages in that a large amount of impurities are generated by conducting the reaction in a state in which the protecting group is removed, and purification is difficult.

As mentioned above, conventional techniques for the preparation of goserelin have many problems that must be improved in commercial mass production applications. Therefore, studies on the effective production of goserelin can be a very important development task in the production of peptide API.

The present inventors have made extensive efforts to improve the problem that it is difficult to stably produce goserelin in a large quantity. As a result, it was clarified that goserelin can be stably mass-produced by carrying out a deprotection reaction and a hydrogen reaction of a peptide after combining a solid-phase synthesis method and a solution-phase synthesis method, The inventors have completed the present invention by developing a method for producing the present invention.

Accordingly, an object of the present invention is to provide a novel method for producing goserelin.

One aspect of the present invention relates to a method of making goserelin comprising the steps of:

(a) obtaining a peptide represented by the following formula (I) to which a resin is attached by a solid-phase synthesis method;

(b) removing the resin from the peptide represented by formula (I) to obtain a peptide represented by formula (II);

(c) obtaining a peptide represented by the following formula (III) by carrying out a coupling reaction with H-Pro-NHNHCONH ₂ .HCl by a solution-phase synthesis method of the peptide represented by the formula (II);

(d) subjecting the peptide represented by the formula (III) to a deprotection reaction to obtain a peptide represented by the following formula (IV); And

(e) hydrogenating a peptide represented by the formula (IV) to obtain goserelin represented by the following formula (v).

(I)

^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -O-Resin

[Formula II]

^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -OH

[Formula (III)

^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -Pro-NHNHCONH 2

(IV)

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg (R 3) -Pro-NHNHCONH 2

(V)

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHNHCONH 2

In the above general formulas (I) to (III)

R < ^{1 >} is hydrogen or an imidazole protecting group,

R ² is hydrogen or a hydroxyl protecting group,

R ³ is a guanidine protecting group.

Acronym

Unless otherwise indicated herein, the abbreviations used in the designation of amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Bihemistry , 11: 1726-1732 (1972) ; Pure & Appl . Chem ., Vol. 56, No. 5, pp. 595-624, 1984).

The abbreviations of the protecting groups and amino acids used herein are as follows:

tBu: tert-Butyl (tert-butyl)

Fmoc: 9-fluorenyloxycarbonyl (9-fluorenyloxycarbonyl)

Trt: Triphenylmethyl (or trityl) (Triphenylmethyl or Trityl)

Mtt: 4-Methyl trityl

Bzl: Benzyl (benzyl)

Arg: Arginine

Pro: Proline

Leu: Leucine

Ser: Serine

D-Ser: D-Serine

Tyr: Tyrosine

Trp: Tryptophan

His: Histidine

Pyr: Pyroglutamic acid

In the above general formulas (I) to (III), R ¹ may be an imidazole protecting group commonly used in the art.

In the above general formulas (I) to (III), R ¹ may be an imidazole protecting group commonly used in the art, for example, hydrogen (H) or a 4-methyltrityl group.

In the above formulas (I) to (III), R ² may be a hydrogen or hydroxyl protecting group commonly used in the art.

The hydroxyl protecting group may be a triphenylmethyl group or a benzyl group.

In the above general formulas (I) to (IV), R ³ may be a guanidine protecting group ordinarily used in the art. For example, the guanidine protecting group may be hydrogen (H), hydrochloric acid or nitro (NO ₂ ) group.

Protecting groups for these functional groups are described in detail in 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 peptide bonds and amino acid residues joined together.

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

(a) obtaining a peptide represented by the formula (I )

The peptide represented by formula (I) is prepared by a solid-phase synthesis method 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)).

That is, after the amino acid in which the alpha-amino and side chain functional groups are protected is bound to the resin, the alpha-amino protecting group is removed, and the remaining amino acid with the alpha-amino and side chain functional groups is bound stepwise in the desired order to obtain an intermediate .

The choice of a suitable protecting group depends on the protecting group, 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 to the reaction conditions and reagents selected to remove the alpha-amino protecting group at each step of the synthesis, and the deprotection reaction should not occur in the coupling reaction. When the synthesis involving the desired amino acid chain is complete, Should be stable under decomposition conditions.

According to one embodiment of the present invention, a resin is used in the synthesis of the peptide of formula (I).

The resin may be a conventional resin which can be easily degraded under mildly acidic conditions which can completely preserve the side chain protecting group of the prepared peptide. For example, 2-chlorotrityl resin, trityl but is not limited to, one or more resins selected from the group consisting of trityl resin, 4-methyl trityl resin and 4-methoxy trityl resin. .

(b) to give the peptide of formula Ⅱ

The peptide represented by the above formula (II) can be obtained by removing the resin from the peptide represented by the formula (I) under mild acidic conditions.

The acidic condition should be a mild condition in which the side chain protecting group of the amino acid chain can be maintained.

The removal of the resin can be carried out using a mixed solution of dichloromethane, acetic acid and trifluoroethanol, for example, by mixing dichloromethane: acetic acid: trifluoroethanol = 8: 1: 1 (by volume) Solution. ≪ / RTI >

(c) obtain a peptide represented by the formula Ⅲ

The compound represented by the formula (III) can be obtained by subjecting the peptide obtained in the step (b) to a liquid phase reaction in the presence of a coupling reagent with H-Pro-NHNHCONH ₂ .HCl.

The coupling reagent may be selected from the group consisting of N, N'-dicyclohexyl carbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) 1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxy- Benzotriazol-1-yl-oxy-tris- (pyrrolidino) -phosphonium hexafluorophosphate (PyBOP), 2- (1H-benzotriazole-1,1,3,3-tetramethyluronium-hexafluorophosphate (HBTU), 2- (1H-benzo 1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (2- (1H-Benzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium tetrafluoroborate : TBTU), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3- (HATU), O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluroniumhexafluorophosphate ), N, N, N ', N'-tetramethyluronium tetrafluoroborate (TATU), N, N'-carbonyldiimidazole (CDI) and N- (3-dimethylaminopropyl) -N '-carbonyldiimidazole (N- -ethylcarbodiimide hydrochloride: EDC.HCl), and may be, for example, EDC.HCl.

According to one embodiment of the present invention, the coupling liquid phase reaction may be carried out in an organic solvent.

The organic solvent is at least one solvent selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform and N, N-dimethylformamide, preferably a mixture of dichloromethane and N, N-dimethylformamide Can be every day.

The binding reaction may be performed at a temperature of -20 to 50 캜, -20 캜 to 40 캜, -20 캜 to 3 캜, -20 캜 to 20 캜, -20 캜 to 10 캜, -20 캜 to 4 캜, 10 ° C to 40 ° C, -10 ° C to 30 ° C, -10 ° C to 20 ° C, -10 ° C to 10 ° C, -10 ° C to 4 ° C, 0 ° C to 50 ° C, 0 ° C to 30 ° C, 0 ° C to 20 ° C, or 0 ° C to 10 ° C, for example, 0 ° C to 4 ° C.

When the coupling reaction is carried out at a low temperature of 0 to 4 캜, generation of impurities can be reduced.

(d) to give the peptide of formula Ⅳ

The peptide represented by the formula (IV) can be obtained by carrying out a deprotection reaction on the peptide represented by the formula (III) under an acidic condition.

The deprotection reaction can be carried out in a mixed solution of dichloromethane, acetonitrile and trifluoroacetic acid.

(e) Obtaining goserelin

Goserelin can be obtained from a peptide represented by the formula (IV) by carrying out a hydrogen reaction under the reaction conditions commonly used in the art.

The hydrogen reaction can be performed using H ₂ / Pd (5% palladium carbon in H ₂ gas) as a catalyst.

The overall process for preparing goserelin based on the above contents is summarized as follows.

[Reaction Scheme 1]

The present invention relates to a method for producing goserelin, which comprises combining solid-phase synthesis with a solution-phase reaction, followed by deprotection of the peptide and hydrogenation to obtain goserelin, It is easy to separate and purify the target product, and there is an advantage that commercial mass production is possible.

1 is a schematic diagram showing an entire process for preparing goserelin according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, the term "%" used to denote the concentration of a specific substance is intended to include solids / solids (weight / weight), solid / liquid (weight / volume) / Liquid is (volume / volume)%.

Example 1: Preparation of peptide represented by formula (VI)

(VI)

Pyr-His (Mtt) -Trp- Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -O- Resin

1-1. H-Arg (NO ₂ ) -2-Chlorotrityl resin

To the reaction vessel, 47.2 g of 2-chlorotrityl chloride resin (with a substitution ratio of 1.27 mmole / g) was added. 399.0 g of dichloromethane (hereinafter referred to as DCM) was added, followed by stirring for 30 minutes to swell and completely drain the DCM.

523.0 g Fmoc-Arg (NO ₂ ) was dissolved in 399.0 g DCM. 23.3 g of N, N-diisopropylethylamine (hereinafter referred to as DIEA) was completely dissolved in the above solution, which was slowly added to a reaction vessel containing the resin and stirred at 25 ± 5 ° C. for 4 hours. After the reaction solution was completely drained, 399.0 g of DCM was added, stirred for 2 minutes and then drained.

The resin was further washed one more time with the addition of 399.0 g DCM and a solution of DCM / methanol (hereinafter MeOH) / DIEA (17: 2: 1 ( V / V / V% )) (255.0 mL (339.2 g) / 30.0 mL g) /15.0 mL (11.1 g)) was added thereto. The mixture was stirred at 25 ± 5 ° C. for 10 minutes, and the solution was drained. The resin was further added one more time to give 255.0 mL (339.2 g) / 30.0 mL (23.8 g) / 15.0 mL (11.1 g) of DCM / MeOH / DIEA (17: 2: 1 (V / V / V%) ) And treated in the same manner. 399.0 g of DCM was added, stirred for 2 minutes and then drained. The resin was washed one more time using 399.0 g DCM and completely drained. Then, 283.2 g of N, N-dimethylformamide (hereinafter referred to as DMF) was added, stirred for 2 minutes and then drained. The resin was further washed twice with 283.2 g of DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL In a (226.6 g) solution and a drain and stirred for 15 minutes and then, 20% (V / V% ) piperidine 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated one more time. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-2. H-Leu-Arg (NO ₂ ) -2-Chlorotrityl resin

31.8 g Fmoc-Leu / 13.4 g 1-Hydroxybenzotriazole (hereinafter, referred to as HOBt) was dissolved in DMF (240.7 g) and added to the reaction vessel. 11.4 g of N, N'-diisopropylcarbodiimide (hereinafter referred to as DIC) was diluted in 29.2 g of DMF, and the mixture was stirred at 25 ± 5 ° C for 4 hours. Ninhydrin (Ninhydrin) test was conducted to confirm the progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-3. H-D-Ser (tBu) -Leu-Arg (NO ₂ ) -2-Chlorotrityl resin

34.5 g Fmoc-D-Ser (tBu) / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reaction vessel. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-4. H-Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) -2-Chlorotrityl resin

36.3 g Fmoc-Tyr / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reaction vessel. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-5. H-Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) -2-Chlorotrityl resin

51.32 g Fmoc-Ser (Trt) / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reaction vessel. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-6. H-Trp- Ser ( Trt ) - Tyr -D- Ser ( tBu ) -Leu- Arg (NO ₂ )-2- Chlorotrityl  Manufacture of resin

38.4 g Fmoc-Trp / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reaction vessel. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-7. H-His ( Mtt ) -Trp- Ser ( Trt ) - Tyr -D- Ser ( tBu ) -Leu- Arg (NO ₂ )-2- Chlorotrityl  Manufacture of resin

57.0 g Fmoc-His (Mtt) / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reaction vessel. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 20% (V / V%) piperidin 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) a one The solution was added, and then stirred for 15 minutes and drain, and then, 20% (V / V% ) piperidine A solution of 60.0 mL (51.7 g) / DMF 240.0 mL (226.6 g) was further treated once more. The reaction solution was completely drained, 283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed five more times with 283.2 g DMF and the following procedure was followed (yield> 98%).

1-8. Pyr -His ( Mtt ) -Trp- Ser ( Trt ) - Tyr -D- Ser ( tBu ) -Leu- Arg (NO ₂ ) -2-Chlorotrityl resin

11.6 g Pyr / 13.4 g HOBt was dissolved in 240.7 g of DMF and added to the reactor. 11.4 g of the DIC solution was diluted in 29.2 g of DMF, and the mixture was stirred at 25 占 폚 for 4 hours. Ninhydrin test was carried out to confirm progress of the reaction. When the reaction was completed, the reaction solution was completely drained.

283.2 g of DMF was added, stirred for 2 minutes and then drained. The resin was further washed one more time with 283.2 g DMF. 399.0 g of DCM was added, stirred for 2 minutes and then drained. The resin was further washed two more times with 399.0 g DCM.

Example 2: Preparation of peptide represented by formula (VII)

[Formula VII]

Pyr-His (Mtt) -Trp- Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -OH

In DCM / 2,2,2- trifluoro-ethanol (hereinafter referred to as TFE) / acetic acid (hereinafter AcOH) (8: 1: 1 (V / V / V%)) (600.0 mL (798.0 g)) /75.0 mL (104.3 g) /750.0 mL (78.7 g) was added thereto and stirred at 25 ± 5 ^{° C} for 2 hours, and the discharged liquid was transferred to another reactor. (20.0 g) / 15.0 mL (15.7 g) of DCM / TFE / AcOH (8: 1: 1 ( V / V / V% )) After stirring for 5 minutes, the drain solution was transferred to another reactor and combined. The remaining resin was washed with 199.5 g DCM and drained to combine with the resin cleavage solution. The reaction solution was added to 3552.0 g of tert- butyl methylether (hereinafter referred to as MTBE), stirred, and the solid was crystallized and filtered. 444.0 g of MTBE was added to the solid, which was then stirred and filtered. This was followed by further drying and drying. The dried solid was dissolved in DCM / DMF (270.0 mL (359.1 g) / 30.0 mL (28.3 g)) mixed solvent.

The mixed solvent dissolved in 2700.0 mL (1998.0 g) of MTBE was added, and the precipitated solid was filtered after stirring. The filtered solid was dissolved in 300.0 mL (222.0 g) of MTBE, stirred, filtered and dried.

The dried solid was dissolved in a mixed solvent of DCM / DMF (270.0 mL (359.1 g) / 30.0 mL (28.3 g)) and the mixed solvent dissolved in 2700.0 mL (1998.0 g) of MTBE was added to the reaction mixture, The solid was dissolved in 300.0 mL (222.0 g) of MTBE and stirred, followed by filtration and drying. As a result, 54.7 g (yield> 100%) of the peptide represented by the formula (VII) was obtained.

Example 3: Preparation of peptide represented by formula (VIII)

(VIII)

Pyr-His (Mtt) -Trp- Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -Pro-NHNHCONH 2

0.4 g of Fmoc-Pro / 24.5 g HOAt / semicarbazide hydrochloride 20.1 g was dissolved in 798.0 g DCM / 283.2 g DMF and added to the reaction vessel. The reaction vessel was cooled to 0 ± 5 ° C with gentle stirring.

34.5 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) was dissolved in 300.0 mL (283.2 g) of DMF and added dropwise to the reaction vessel for 140 minutes. When the dropping was completed, the temperature was raised to 25 ± 5 ° C, and the reaction was continued until Fmoc-Pro was present at a maximum of 2.0% or less at this temperature. After completion of the reaction, the reaction solution was washed with 600.0 g of water and then the DCM layer was separated. The DCM layer was separated by washing with 10% NaCl solution (NaCl 60.0 g, purified water 600.0 g) and MTBE 3600.0 mL (2664.0 g) was added to precipitate Fmoc-Pro-NHNHCONH ₂ , and the layers were separated to remove the supernatant. To the precipitated crystals, 300.0 mL (222.0 g) of MTBE was added dropwise thereto, followed by washing and layering to remove the supernatant. To the precipitated crystals, 300.0 mL (222.0 g) of MTBE was added dropwise thereto, followed by washing and filtration. After completion of the filtration, the filtrate was dried under reduced pressure at 40 ± 5 ° C. for 12 hours or more to obtain 35.5 g (yield> 100%) of Fmoc-Pro-NHNHCONH ₂ .

The 35.5 g Fmoc-Pro-NHNHCONH ₂ was dissolved in 42.4 g DEA / 566.4 g DMF, and stirred for 2 hours. When the reaction was completed, 3000.0 mL (2220.0 g) of MTBE was added dropwise to precipitate a solid. After stirring, the layers were separated and the supernatant was removed. 300.0 mL (222.0 g) of MTBE was added, and the mixture was stirred and then layered to remove the supernatant. The solid was filtered and dried at 40 +/- 5 DEG C for over 12 hours. The dried Pro-NHNHCONH ₂ was dissolved in 85.0 g DMF and added dropwise to 399.6 g MTBE to precipitate a solid. The MTBE was removed by filtration. 44.4 g MTBE was stirred and filtered to dryness. The dried Pro-NHNHCONH ₂ was dissolved again in 85.0 g of DMF and added dropwise to 399.6 g of MTBE to precipitate a solid. The MTBE was removed by filtration. 44.4 g MTBE was stirred and filtered to dryness.

The dried Pro-NHNHCONH ₂ was dissolved in 71.3 g of MeOH and added dropwise to 399.6 g of MTBE to precipitate a solid. The MTBE was removed by filtration. 44.4 g MTBE was stirred and filtered. After completion of filtration, it was dried under reduced pressure at 40 ± 5 ° C for 12 hours or longer to obtain Pro-NHNHCONH ₂ 15.5 g (yield > 100%) was obtained.

54.7 g of Pyr-His (Mtt) -Trp-Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) 8.2 g of 1-hydroxy-7-azabenzotriazole (hereinafter referred to as HOAt) was dissolved in 195.0 mL (259.4 g) of DCM / 60.0 mL (56.6 g) of DMF, charged into the reaction vessel, and 10.4 g of H-Pro-NHNHCONH ₂ Lt; / RTI > The reaction vessel was cooled to 0 ± 5 ° C with gentle stirring. 11.5 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) was dissolved in 45.0 mL (42.5 g) of DMF and added dropwise to the reaction vessel for 140 minutes. (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) is increased to a maximum of 2.0% at this temperature, Lt; / RTI > After completion of the reaction, the reaction mixture was washed with 300.0 mL of water and the DCM layer was separated. The DCM layer was washed once with 10% NaCl solution (30.0 g of NaCl and 300.0 g of purified water) MTBE separated and put into the 1200.0 mL (888.0 g) Pyr- His (Mtt) -Trp-Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -Pro-NHNHCONH to precipitate the ₂ And the supernatant was removed. To the precipitated crystals, 150.0 mL (111.0 g) of MTBE was added dropwise thereto, followed by washing and layering to remove the supernatant. To the precipitated crystals, 150.0 mL (111.0 g) of MTBE was added dropwise thereto, followed by washing and filtration. After filtration, Pyr-His (Mtt) -Trp-Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) -Pro-NHNHCONH ₂ 57.1 g (yield > 100%).

Example 4: Preparation of Peptide Represented by Formula IX

[Formula IX]

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -Pro-NHNHCONH 2

57.1 g of dried Pyr-His (Mtt) -Trp-Ser (Trt) -Tyr-D-Ser (tBu) -Leu-Arg (NO ₂ ) -Pro- NHNHCONH ₂ were added to the reactor and then DCM 750.0 mL The mixture was stirred at 25 占 5 占 폚 for 24 hours with addition of 1530.0 mL of a mixed solution of 750.0 mL (589.5 g) of acetonitrile (hereinafter ACN) and 30.0 mL (44.7 g) of TFA. A small amount of the solution was taken, crystallized with tert-butyl methyl ether and analyzed by HPLC to confirm final cleavage. When the reaction was completed, the solution was added dropwise to 7500.0 mL (5550.0 g) of MTBE to precipitate a solid.

After stirring, the layers were separated and the supernatant was removed. 600.0 mL (444.0 g) of MTBE was added, and the mixture was stirred and then layered to remove the supernatant. The solid was filtered and dried at 40 +/- 5 DEG C for over 12 hours.

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -Pro-NHNHCONH 2 39.4 g was obtained (yield > 90%).

Example 5: Preparation of goserelin represented by the formula (v)

(V)

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHNHCONH 2

The dried Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg (NO 2) -Pro-NHNHCONH 2 39.4 g of ethanol, 900.0 mL (710.1 g) / H 2 O 600.0 mL (600.0 g ), And the mixture was charged into a reactor. 60.0 mL (73.2 g) of formic acid was added, and 19.3 g of 5% Pd (in carbon) was added thereto. A small amount of the solution was taken, crystallized with tert-butyl methyl ether and analyzed by HPLC to confirm final cleavage. When the reaction was completed, the reaction solution was filtered to remove 5% Pd (in carbon).

Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHNHCONH 2 (Yield> 90%, purity> 70%).

(27000.0 mL (21210.0 g)) / H ₂ O 3000.0 (2700.0 g) was added to purified solvent A as purified solvent B, and purified water (67500.0 mL (67500.0 g)) / ACN (7500.0 mL mL (3000.0 g) / AcOH (31.5 g). Crude goserelin was concentrated and then filtered through a 36.2 g / 600.0 mL H ₂ O 0.45 μm filter.

The column temperature was separated and purified in purified solvent A and B gradient conditions at room temperature, UV wavelength 230 nm, flow rate 1.0 L / min.

The concentrate was transferred to a tray. The refrigerant was circulated and the temperature of the trap was set at -50 ° C and the temperature was lowered over 5 hours. The product solution was completely frozen in the tray, and then the vacuum pump was turned on to gradually reduce the pressure, and then the solution was sequentially adjusted and dried for about 3 days.

After drying, the product was dissolved in 500.0 mL (500.0 g) of purified water. The solution was filtered through 0.2 μm and loaded into a tray. The refrigerant was circulated and the temperature of the trap was set at -50 ° C and the temperature was lowered over 5 hours. The product solution was completely frozen in the tray, and then the vacuum pump was turned on to gradually reduce the pressure, and then the solution was sequentially adjusted and dried for about 3 days. The vacuum was discarded and a sample was taken to determine residual solvent, moisture and AcOH content (residual solvent, ACN <410 ppm; moisture, <10.0%; AcOH content, 4.5-15.0%). If the specifications are not met, vacuum is applied to continue drying. If IPC results in residual solvent, moisture, and AcOH content within the specified range, it is taken out and analyzed for final packaging and test. Final lyophilization proceeded to obtain 10.3 g of goserelin acetate acetate (lyophilization yield> 90%) (purity> 99%, total yield> 30%).

<110> ANYGEN CO., LTD. <120> Process for the Preparation of Buserelin <130> PN170176 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> Buserelin <220> <221> SITE <222> (1) <223> Pyroglutamic acid <220> <221> SITE <222> (6) <223> D-Serine <220> <221> SITE <222> (6) &Lt; 223 > (tert-Butyl) <220> <221> SITE <222> (9) <223> -NHNHCONH2 <400> 1 Glu His Trp Ser Tyr Ser Leu Arg Pro   1 5

Claims (12)

A method for preparing goserelin comprising the steps of:
(a) obtaining a peptide represented by the following formula (I) to which a resin is attached by a solid-phase synthesis method;
(b) removing the resin from the peptide represented by formula (I) to obtain a peptide represented by formula (II);
(c) obtaining a peptide represented by the following formula (III) by carrying out a coupling reaction with H-Pro-NHNHCONH ₂ .HCl by a solution-phase synthesis method of the peptide represented by the formula (II);
(d) subjecting the peptide represented by the formula (III) to a deprotection reaction to obtain a peptide represented by the following formula (IV); And
(e) hydrogenating a peptide represented by the formula (IV) to obtain goserelin represented by the following formula (v).
(I)
^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -O- Resin
[Formula II]
^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -OH
[Formula (III)
^{Pyr-His (R 1) -Trp} -Ser (R 2) -Tyr-D-Ser (tBu) -Leu-Arg (R 3) -Pro-NHNHCONH 2
(IV)
Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg (R 3) -Pro-NHNHCONH 2
(V)
Pyr-His-Trp-Ser- Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHNHCONH 2
In the above general formulas (I) to (III), R ¹ is hydrogen or an imidazole protecting group,
R ² in the formulas (I) to (III) is a hydrogen or hydroxyl protecting group,
R ³ in the above general formulas (I) to (IV) is a guanidine protecting group. The method for producing goserelin according to claim 1, wherein R ¹ in the general formulas (I) to (III) is hydrogen (H) or 4-methyltrityl The method according to claim 1, wherein R ² in the above formulas (I) to (III) is selected from the group consisting of hydrogen (H), triphenylmethyl and benzyl groups. The method according to claim 1, wherein R ³ in the formulas (I) to (IV) is selected from the group consisting of hydrogen (H), hydrochloric acid and nitro (NO ₂ ) groups. The method of claim 1, wherein the resin in step (a) is selected from the group consisting of 2-chlorotrityl resin, trityl resin, 4-methyl trityl resin, Wherein the resin is at least one selected from the group consisting of 4-methoxy trityl resin. The method for producing goserelin according to claim 1, wherein the removal of the resin is carried out in the presence of an acidic solution. 7. The method of claim 6, wherein the acidic solution is a mixed solution of at least two selected from the group consisting of dichloromethane, acetic acid and trifluoroethyl alcohol. The method according to claim 7, wherein the acidic solution is a mixed solution of dichloromethane: acetic acid: trifluoroethyl alcohol in a volume ratio of 8: 1: 1 . 2. The method of claim 1, wherein the coupling reaction of step (c) is performed using N, N'-dicyclohexyl carbodiimide (DCC), N, N'-diisopropylcarbodiimide N, N'-diisopropylcarbodiimide: DIC), benzotriazole-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris- (pyrrolidino) -phosphonium hexafluorophosphate (PyBOP) , 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (2- (1H-Benzotriazole-1,1,3,3-tetramethyluronium- hexafluorophosphate (HBTU), 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate , 1,3,3-tetramethyl-uronium tetrafluoroborate (TBTU), 2- (7-aza-1H-benzoate 1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (2- (7-Aza-1H-benzotriazol- tetramethyluronium hexafluorophosphate (HATU), O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate ), N, N'-carbonyldiimidazole (CDI), and N- (3-dimethylaminopropyl) -N-tetramethyluronium tetrafluoroborate (TATU) Wherein the at least one binding reagent selected from the group consisting of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC.HCl) is used. The method according to claim 1, wherein the step (c) is performed in a solvent selected from the group consisting of dichloromethane, 1,2-dichloroethane, chloroform, and N, N-dimethylformamide &Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; The method of claim 1, wherein the binding reaction is performed at -20 캜 to 50 캜. The method according to claim 1, wherein the hydrogenation reaction is performed in a mixed solvent of water and ethanol.

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