KR102342942B1 - Method for preparing leuprolide using solid-phase synthesis - Google Patents

Abstract

An embodiment of the present invention relates to a method for producing leuprolide using a solid-phase synthesis method that uses a polymer support resin, separates the resin from the peptide-resin, and couples ethylamine at the same time. Separation and purification It has the advantage of being easy.

Description

Manufacturing method of leuprolide using solid phase synthesis method {METHOD FOR PREPARING LEUPROLIDE USING SOLID-PHASE SYNTHESIS}

The present invention relates to a method for producing leuprolide using a solid-phase synthesis method, and more particularly, by using a polymer support resin, separating the resin from the peptide-resin and coupling ethylamine, It relates to a method for producing leuprolide using a solid-phase synthesis method capable of simplification of steps and increasing yield and purity.

Luteinizing Hormone Releasing Hormone (LHRH), also known as GnRH (Gonadotropin Releasing Hormone), is a hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly) that regulates the reproductive system of vertebrates. -Leu-Arg-Pro-Gly-NH2). LH-RH agonists deplete LHRH receptors in the pituitary gland and induce desensitization of the pituitary receptors in response to LHRH stimulation, thereby inhibiting LH secretion. In addition, LHRH agonists and antagonists are indicated for the treatment of endometriosis, fibroids, polycystic ovarian cancer, breast cancer, ovarian and endometrial cancer in women, gonadotropin pituitary desensitization during medical-assisted birth protocols, benign prostate and polymorphism in men. and prostate cancer, and for the treatment of precocious puberty in men or women. Such LH-RH agonists typically include leuprolide.

Methods for chemically synthesizing peptides can be largely divided into solution-phase and solid-phase. The solution phase synthesis method is a classical chemical synthesis method in which all reagents are dissolved in a solution, and has the advantage of low cost and fast reaction rate. There is a disadvantage that separation and purification are difficult due to the possibility of occurrence. The solid phase synthesis method has been developed since R. B. Merrifield proposed a theory on solid phase peptide synthesis, and has the advantage of simple separation and purification and automation.

The conventional method for producing leuprolide was using only a solution-phase synthesis method or a method combining solution-phase synthesis and solid-phase synthesis. U.S. Patent No. 4,008,209, U.S. Patent No. 6,448,031, and European Patent No. 1790656 describe a technology for producing leuprolide by a solution-phase synthesis method. However, the manufacturing method has a number of manufacturing steps and a complicated manufacturing process, as well as a low manufacturing yield, so that price competitiveness is lowered in commercial mass production. U.S. Patent No. 20130060004 and Chinese Patent No. 101195653 describe a technology for producing leuprolide by combining solid phase synthesis and liquid phase reaction. However, these methods have a disadvantage in that the liquid phase reaction is implied and the productivity is lowered in commercial mass production. U.S. Patent No. 5,602,231, U.S. Patent No. 6,897,289, European Patent No. 0518655, and European Patent No. 1777232 describe a technology for preparing LH-RH peptides such as leuprolide by a solid-phase synthesis method. are doing In the case of synthesizing leuprolide by the method described in European Patent No. 1777232, there is a one-step liquid phase reaction as well as a lot of impurities around the peak of leuprolide on HPLC, so purification is very difficult. .

Accordingly, development of a method for producing leuprolide by a solid phase synthesis method is required, but impurities that are difficult to separate, for example, leuprolide-proline in which proline is added to leuprolide are generated. The impurity shows a peak at a position close to the peak of leuprolide on HPLC, so it is difficult to purify and the yield is very low to obtain high-purity leuprolide. it was

Therefore, the present inventors use a polymer support resin during research to solve the above problems, and at the same time coupling the ethylamine while separating the resin from the peptide-resin, simplify the synthesis step and increase the yield and purity. A method for preparing leuprolide using a solid-phase synthesis method was proposed.

The technical problem to be achieved by the present invention is a solid-phase synthesis method that uses a polymer support resin and can simplify the synthesis step and increase the yield and purity by separating the resin from the peptide-resin and coupling ethylamine at the same time To provide a method for manufacturing a leuprolide using

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above technical problem, an embodiment of the present invention,

A first step of preparing a peptide-resin represented by the following Chemical Formula 1 by sequentially binding amino acids or amino acid derivatives to a polymer support resin;

a second step of treating the peptide-resin obtained in the first step with ethylamine to separate the peptide from the resin and at the same time obtaining a peptide represented by Formula 2 to which ethylamine is coupled;

Including a third step of deprotecting the peptide obtained in the second step,

Provided is a method for preparing leuprolide using a solid-phase synthesis method.

[Formula 1]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-O- resin (resin)

[Formula 2]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-NHEt

In the above formula, R ¹ is hydrogen or a protecting group selected from the group consisting of t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group and allyl group,

R ² is hydrogen or a protecting group selected from the group consisting of t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group and allyl group,

R ³ is hydrogen, p-methoxybenzyl group, methoxymethyl group, benzyloxymethyl group, tetrahydropyran group, tetrahydrofuran group, t-butyl group, triphenylmethyl group, benzyl group, allyl group, trimethylsilyl group, It is a protecting group selected from the group consisting of t-butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group, t-butylcarbonyl group, acetyl group and benzoyl group,

R ⁴ is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group, t-butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group , nitro group, 2,2,5,7,8-pentamethylchromene-6-sulfonyl group, 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, 2,2,4,6,7- It is a protecting group selected from the group consisting of a pentamethyl-dihydrobenzofuran-5-sulfonyl group and a toluenesulfonyl group.

In an embodiment of the present invention, the resin in the first step may be a PAM resin, a Wang resin, or a Hydroxymethyl resin.

In an embodiment of the present invention, R ¹ , R ² , R ³ , and R ⁴ are each a triphenylmethyl group, t-butyloxycarbonyl group, t-butyl group, and 2,2,4,6,7- It may be a pentamethyl-dihydrobenzofuran-5-sulfonyl group.

In an embodiment of the present invention, the first step may include reacting Boc-Pro-OH with the polymer support resin and then removing the Boc group to obtain a peptide-resin.

In an embodiment of the present invention, the first step may include the step of removing Fmoc at least once after reacting the _{peptide-resin with Fmoc-(AA)n-OH (n is 1 to 8).} .

In an embodiment of the present invention, the second step may be characterized in that the peptide-resin is treated with a mixture of THF and an aqueous solution of ethylamine.

In an embodiment of the present invention, the volume ratio of the THF and the aqueous ethylamine solution may be 1:2 to 2:1.

According to an embodiment of the present invention, the present invention provides a novel manufacturing method capable of manufacturing leuprolide by a solid-phase synthesis method.

In addition, according to an embodiment of the present invention, the leuprolide prepared by the present invention may have the advantage of being able to be easily separated and purified, so that commercial mass production is possible.

In addition, according to an embodiment of the present invention, the present invention can exhibit a very economical effect in terms of production cost by providing a simpler manufacturing process than the looprolide manufacturing method used in the art. In particular, since leuprolide can be separated from the leuprolide-resin by a single process and ethylamine can be added at the same time, production efficiency can be increased.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 shows an overall schematic diagram of the novel leuprolide manufacturing process of the present invention.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in several different forms, and thus is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is said to be “connected (connected, contacted, coupled)” with another part, it is not only “directly connected” but also “indirectly connected” with another member interposed therebetween. "Including cases where In addition, when a part "includes" a certain component, this means that other components may be further provided without excluding other components unless otherwise stated.

The terminology used herein is used only to describe specific embodiments, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The first aspect of the present application is

Provided is a method for preparing leuprolide using a solid-phase synthesis method.

Hereinafter, the manufacturing method of leuprolide using the solid-phase synthesis method according to the first aspect of the present application will be described in detail step by step.

[Formula 1]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro- resin (resin)

[Formula 2]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-NHEt

summary of abbreviations

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

Abbreviations for amino acids and protecting groups used herein are as follows:

PAM: 4-Hydroxymethyl-phenylacetamidomethyl resin

(4-Hyroxymethyl-phenylacetamidomethyl Resin)

Tyr: Tyrosine

Glu: Glutamic acid

pGlu: Pyroglutamic acid

Ser: Serine

Arg: Arginine

Pro: Proline

Leu: Leucine

His: Histidine

Ala: Alanine

Trp: Tryptophane

Et: ethyl (Ethyl)

Boc: t-butyloxycarbonyl (t-butyloxycarbonyl)

tBu: t-butyl (t-butyl)

Fmoc: 9-fluorenyl methoxyoxy carbonyl (9-Fluorenylmethyloxycarbonyl)

Trt: triphenylmethyl

Mtr: 4-methoxy-2,3,6-trimethylbenzenesulfonyl (4-methoxy-2,3,6-trimethylbenzenesulfonyl)

Pmc: 2,2,5,7,8-pentamethylchromen-6-sulfonyl (2,2,5,7,8-pentamethyl-croman-6-sulfonyl)

Tos: toluensulfonyl

Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)

Step 1

First, in one embodiment of the present application, amino acids or amino acid derivatives are sequentially bound to a polymer support resin to prepare a peptide-resin represented by the following formula (1):

[Formula 1]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-O- resin (resin)

As used herein, the term “polymer support” refers to a substrate that is substantially insoluble in the medium in which it is used, as well as inert to the reagents and reaction conditions described herein. Representative solid supports include diatomaceous earth, silica gel and controlled pore glass; organic polymers, for example polystyrene, polypropylene, polyethylene glycol, polyacrylamide, comprising 1-2% copolystyrene divinyl benzene (in gel form) and 20-40% copolystyrene divinyl benzene (in macroporous form); cellulose and the like; and polyacrylamide supported within a matrix of composite inorganic/polymeric compositions, such as diatomaceous earth particles. See J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd, Ed., Pierce Chemical Co. (Chicago, IL, 1984)].

As used herein, the term "resin" refers to a solid support modified with a reactive group that modulates coupling with the carboxy or N-terminus of an amino acid, peptide or cyclic peptide fragment as defined herein. . Representative resins include Merrifield resin (chloromethylated polystyrene), hydroxymethyl resin, 2-chlorotrityl chloride resin, trityl chloride resin, Rink acid resin (4-benzyloxy-2',4' -dimethoxybenzhydrol resin), trityl alcohol resin, PAM resin (4-hydroxymethylphenylacetamidomethyl resin), Wang resin (p-benzyloxybenzyl alcohol resin), MBHA resin (p-methyl Benzhydrylamine resin), BHA resin (benzhydrylamine resin), Link amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin) and PAL resin (5-( 4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid-MBHA resin). Preferred resins may be selected from PAM resins, Wang resins, and hydroxymethyl resins, which are resins containing a hydroxy group, specifically hydroxymethyl benzene, among resins used in the solid phase synthesis method, and most preferably PAM resin. have. When the preferred resin is used, the resin removal and coupling reaction using ethylamine can be performed in high yield, and high-purity peptides can be obtained.

According to one embodiment of the present application, the PAM resin may be loaded at 0.8 to 1.2 mmol/g, preferably at 1.0 to 1.1 mmol/g.

As used herein, the terms “amino acid” and “amino acid derivative” refer to amino acids and amino acid derivatives constituting leuprolide. Specifically, the leuprolide of the present invention has a formula of pGlu-His(R1)-Trp(R2)-Ser(R3)-Tyr(R3)-D-Leu-Leu-Arg(R4)-Pro-NHEt. Here, pGlu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, D-Leu is D-type optically active leucine, Leu is leucine, Arg is arginine, and Pro is proline. means As used herein, the term amino acid "derivative" includes amino acids having substitutions or modifications by covalent attachment of the parent amino acid, eg, by alkylation, glycosylation, acetylation, phosphorylation, and the like. For example, one or more analogs of amino acids having substituted linkages as well as other modifications known in the art are further included within the definition of "derivative".

As used herein, the term “protecting group” refers to a moiety that, when attached to a reactive group in a molecule, masks, reduces, or prevents its reactivity.

In one embodiment of the present application, all or part of the N-amino group and the condensed chain functional group may be protected in the amino acids and amino acid derivatives. On the other hand, selection of an appropriate protecting group may vary depending on the functional group to be protected, the conditions to which the protecting group is exposed, and other functional groups that may exist in the molecule. The protecting group must be stable to the reaction conditions and reagents selected to remove the α-amino protecting group in each step of synthesis, ㈁ must not undergo deprotection reaction in the coupling reaction, and ㈂ the synthesis including the desired amino acid chain is complete It should be stable under decomposition conditions with resin. Since the protecting group affects the determination of the yield and purity of the preparation method of the present invention, it is necessary to select an appropriate protecting group.

In addition, in one embodiment of the present application, the amino acid and its derivatives may have a side chain protecting group. In Formulas 1 and 2, R ¹ is hydrogen or an imidazole protecting group commonly used in the art, R ² is a hydrogen or indole protecting ^{group commonly used in the art, R 3} is a hydrogen or hydroxyl protecting group commonly used in the art, and R ⁴ may be a guanidinenidinnidin protecting group commonly used in the art. Specifically, the imidazole protecting group is a t-butyloxycarbonyl group (Boc), a benzyloxycarbonyl group (“Cbz” or “Z”), a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group (or trityl, Trt ), a protecting group selected from the group consisting of a benzyl group and an allyl group. Preferably, it may be a triphenylmethyl group or a t-butyloxycarbonyl group, and more preferably a triphenylmethyl group. The indole protecting group is a protecting group selected from the group consisting of t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group and allyl group, preferably t-butyloxycarbonyl group or a benzyloxycarbonyl group, more preferably a t-butyloxycarbonyl group. The hydroxyl group protecting group is p-methoxybenzyl group, methoxymethyl group, benzyloxymethyl group, tetrahydropyran group, tetrahydrofuran group, t-butyl group, triphenylmethyl group, benzyl group, allyl group, trimethylsilyl group, t- It is a protecting group selected from the group consisting of butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group, t-butylcarbonyl group, acetyl group and benzoyl group, preferably t-butyl group or triphenylmethyl group, more preferably may be a t-butyl group. The guanidinenidinnidin protecting group is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group, t-butyldimethylsilyl group, triphenylsilyl group, triiso Propylsilyl group, nitro group, 2,2,5,7,8-pentamethylchromene-6-sulfonyl group (Pmc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl group (Mtr), 2 ,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl group (Pbf) and toluenesulfonyl group (Tos) a protecting group selected from the group consisting of, more preferably a Pbf or Mtr group; , most preferably a Pbf group. According to the selection of the preferred protecting group, the production method of the present invention can achieve high yield and high purity leuprolide production.

In one embodiment of the present application, amino acids and amino acid derivatives may have a protecting group at the N-terminus. The N-terminal protecting group is formyl group, acetyl group, trifluoroacetyl group, benzyl group, benzyloxycarbonyl group (CBZ), tert-butoxycarbonyl group (Boc), trimethyl silyl group (TMS), 2-trimethylsilyl- an ethanesulfonyl group (SES), a trityl (Trt) group and a substituted trityl group, an allyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group (FMOC), a protecting group of a nitro-veratryloxycarbonyl group (NVOC), Preferably, it is characterized in that it is protected with a Fmoc or Boc protecting group. Fmoc can be utilized as a particularly useful N-terminal protecting group in connection with the solid-phase synthesis of the leuprolide of the present invention.

The peptide-resin is one in which the C-terminus of a peptide composed of one or more amino acids or amino acid derivatives is bonded to a resin, and can be prepared by a solid-phase synthesis method commonly used in the art. Specifically, a peptide comprising one or more amino acids or amino acid derivatives protected with an α-amino group and optionally a side chain functional group is bound to a resin or a peptide-resin, and then the step of removing the α-amino protecting group is sequentially performed. A peptide-resin can be prepared. The first step includes a reaction for obtaining a peptide-resin by first binding an amino acid or a derivative thereof protected with an α-amino group and optionally a side chain functional group to a solid support resin, followed by deprotection. Then, the C-terminal carboxy group of the next amino acid with the N-terminus protected by the amine group of the peptide-resin reacts. It is then deprotected to expose the N-terminus so that the next amino acid can be reacted.

In solid phase peptide synthesis, two methods are mainly used, Fmoc and Boc protecting group. Preferably, the Boc protecting group is used when first binding an amino acid to a resin, and the Fmoc protecting group is used when sequentially binding the next amino acid. Specifically, the first step may include first reacting the polymer support resin with Boc-Pro-OH and then removing the Boc group to obtain a peptide-resin. The first step may include at least one step of removing Fmoc after reacting the peptide-resin with Fmoc-(AA) _n -OH (n is 1 to 8, AA is an amino acid or an amino acid derivative) have.

Depending on the protecting group of Fmoc-AA or Boc-AA, the deprotection process is different. Among them, in the case of the Boc protecting group according to an embodiment of the present invention, deprotection is possible under acidic conditions, for example, weakly acidic cleavage such as TFA (trifluoroacetic acid)/DCM (dichloromethane) solution can be added. In the case of the Fmoc protecting group, deprotection is possible under the conditions of a base, for example, 1,8-diazabicyclo[5.4.0]andek-7-ene (1,8-Diazabicyclo[5.4.0] undec- A solution such as N,N-dimethylformamide containing 7-ene) may be added.

Step 2

The method for producing leuprolide of the present invention includes a second step of treating the obtained peptide-resin with ethylamine to separate the peptide from the resin and at the same time obtaining a peptide represented by Formula 2 to which ethylamine is coupled. :

[Formula 2]

^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-NHEt

According to one embodiment of the present application, ethylamine acts as a nucleophile to selectively attack and cut the bond between the peptide and the resin, and at the same time, it is possible to obtain a peptide represented by Formula 2 in which ethylamine is linked. In the present invention, since the second step does not include the step of cleaving the bond between the peptide and the resin and the step of binding ethylamine to the peptide as separate processes, it is possible to provide a method for producing leuprolide with high efficiency.

In one embodiment of the present application, in the method for producing leuprolide using the solid-phase synthesis method, an aqueous solution of tetrahydrofuran (THF) and ethylamine is added to the peptide-resin obtained in the first step. ethylamine can be coupled at the same time as the peptide is separated from the resin by treating the mixed solution of the

In one embodiment of the present application, when coupling ethylamine in the first step, the ethylamine may be treated with a mixed solution of tetrahydrofuran (THF) and an aqueous ethylamine solution, preferably THF and an aqueous ethylamine solution The volume ratio may be 1:2 to 2:1, and more preferably, the volume ratio of THF to the aqueous ethylamine solution may be 1:1. If the volume of THF in the volume ratio of THF and aqueous ethylamine solution is higher than the above range, there may be a problem in that the resin separation reaction is not completed. This may cause a problem in that the yield is reduced.

Step 3

The method for producing leuprolide of the present invention includes a third step of deprotecting the peptide obtained in the second step. Leuprolide can be obtained from the peptide obtained in the second step by performing a deprotection reaction under reaction conditions commonly used in the art. In the deprotection process, the reaction is carried out under acidic conditions, and in the peptide obtained in step 2, the protecting groups of Trt, Boc, tBu and pbf, which are easily removed from acid, protect the side chain, so that the deprotection can be performed well.

In one embodiment of the present application, the reaction conditions for the deprotection reaction are acidic conditions, and the acids that can be used are not particularly limited, but preferably trifluoroacetic acid, water, phenol, thioanisole and ethanedithiol. a mixture of trifluoroacetic acid, triisopropylxylene and water or a mixture of trifluoroacetic acid, triisopropylxylene, water and ethanedithiol, most preferably trifluoroacetic acid, triisopropylxylene and a mixture of water.

When the deprotection reaction is completed, organic impurities may be removed by an extraction method using an organic solvent, and preferably, a filtration method using t-butylmethyl ether may be used. Additionally, the purity of leuprolide may be increased through additional separation and purification after the extraction, and preferably, a reverse phase HPLC purification method may be used.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Example: Preparation of leuprolide

Step 1: Preparation of the peptide-resin represented by the formula (I)

[Formula I]

pGlu-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(pbf)-Pro-PAM resin

Step a: Preparation of Boc-Pro-PAM resin

19 g of PAM resin (f=1.05 mmol/g of resin, 20 mmol) and 200 ml of dichloromethane were put into a solid-phase synthesis reactor equipped with a filtration membrane, and the resin was expanded for 30 minutes, followed by a filtration membrane under reduced pressure. The solvent was removed through 200 ml of dichloromethane containing 21.53 g of t-butyloxycarbonyl-Pro-OH, (100 mmol, 5.0 equiv) was added to the treated resin, followed by 23.73 g of pyridine (300 mmol, 15 equiv) And 2,6-dichlorobenzoyl chloride (2,6-dichlorobenzoyl chloride) 20.9 g (100 mmol, 5 equivalents) was added, and then reacted at room temperature for 5 hours. The reaction solution was removed by filtration under reduced pressure, and the resin was washed with dichloromethane three times, and then dried under vacuum to obtain a t-butyloxycarbonyl-Pro-PAM resin. The substitution rate was 0.76 mmol/g.

Step b: Removal of the Boc group of the resin

13.16 g (0.76 mmol/g, 10 mmol) of t-butyloxycarbonyl-Pro-PAM resin and 100 ml of dichloromethane were put into a solid-state reactor equipped with a filtration membrane, and the resin was expanded for 15 minutes, followed by a filtration membrane under reduced pressure. The solvent was removed through 100 ml of dichloromethane containing 50% (v/v) trifluoroacetic acid was added to the resin, followed by a reaction to remove t-butyloxycarbonyl for 2 hours, followed by filtration under reduced pressure to remove the reaction solution. Then, the resin was sequentially treated with dichloromethane once, once with N,N-dimethylformamide, twice with N,N-dimethylformamide containing 5% N,N-diisopropylethylamine, N,N- The H-Pro-PAM resin was obtained by washing with dimethylformamide 5 times.

Step c: Treating Fmoc-Leu-Arg(pbf)-OH to prepare Fmoc-Leu-Arg(pbf)-Pro-PAM resin

To the H-Pro-PAM resin (10 mmol) obtained in step b, 22.8 g of 9-fluorenyloxycarbonyl-Leu-Arg(Pbf)-OH, (30 mmol, 3 equivalents) and 1-hydroxy 100 ml of N,N-dimethylformamide of 4.45 g (33 mmol, 3.3 equiv) of benzotriazole was added, followed by the addition of 4.65 ml of diisopropylcarbodiimide (30 mmol, 3 equiv), followed by 4 hours at room temperature reacted while The reaction product was filtered under reduced pressure to remove the reaction solution, and the resin was washed twice with N,N-dimethylformamide to obtain 9-fluorenyloxycarbonyl-Leu-Arg(pbf)-Pro-PAM resin.

Step d: Remove Fmoc from Fmoc-Leu-Arg(pbf)-Pro-Pam resin obtained in step c

2% (v/v) 1,8-diazabicyclo[5.4.0]andek-7 to the 9-fluorenyloxycarbonyl-Leu-Arg(pbf)-Pro-PAM resin obtained in step c above -ene (1,8-Diazabicyclo[5.4.0] undec-7-ene) containing 100 ml of N,N-dimethylformamide was added and the reaction of 9-fluorenyloxycarbonyl was removed for 5 minutes. , the reaction solution was removed by filtration under reduced pressure. The reaction to remove 9-fluorenyloxycarbonyl was repeated, and then the resin was washed 6 times with N,N-dimethylformamide to obtain H-Leu-Arg(pbf)-Pro-PAM resin.

Step e: sequentially coupling the amino acid derivative to the resin obtained in step d

Steps c and d were performed in the same manner except that the following amino acid derivative was used instead of Fmoc-Leu-Arg(pbf)-OH for the resin obtained in step d. The following amino acid derivatives were sequentially used and steps c and d were repeatedly performed to sequentially couple the following amino acid derivatives.

[Amino acid derivatives]

9-fluorenyloxycarbonyl-D-Leu-OH 10.6 g (30 mmol, 3 equiv)

9-fluorenyloxycarbonyl-Tyr(t-butyl)-OH 13.8 g (30 mmol, 3 equiv)

11.5 g (30 mmol, 3 eq) of 9-fluorenyloxycarbonyl-Ser(t-butyl)-OH

9-fluorenyloxycarbonyl-Trp(t-butyloxycarbonyl)-OH 15.8 g (30 mmol, 3 equiv)

9-fluorenyloxycarbonyl-His(triphenylmethyl)-OH 18.6 g (30 mmol, 3 equiv)

pGlu-OH 3.88 g (30 mmol, 3 equiv)

Step f: washing the resin obtained in step e

The resin obtained in step e was sequentially washed 3 times with N,N-dimethylformamide and 3 times with tetrahydrofuran to obtain a peptide represented by Formula 1 above.

Step 2: Preparation of the peptide represented by Formula II by coupling ethylamine with the removal of the resin from the peptide-resin

[Formula II]

pGlu-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(pbf)-Pro-NHEt

150 ml of a 70% ethylamine aqueous solution: tetrahydrofuran = 1:1 mixture was added to the peptide-resin represented by the formula (I) obtained in the first step and stirred for 24 hours. The resin was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain 16 g of the crude peptide represented by Formula II.

Step 3: Obtaining leuprolide after deprotection and purification of the peptide

[Formula III]

pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

500 ml of a trifluoroacetic acid:triisopropylsilane:water=95:2.5:2.5 mixed solution was added to 16 g of the peptide represented by Formula II obtained in the second step, and deprotection reaction was performed for 2 hours. . The solvent was distilled under reduced pressure, and 500 ml of t-butylmethyl ether was added, and the obtained solid was filtered to obtain 10.2 g of crude leuprolide. Purification by reversed-phase high performance liquid chromatography (HPLC) gave 4.8 g of leuprolide represented by Formula III. The total yield was 40% and the purity of HPLC was 99%.

A summary of the entire process of manufacturing leuprolide based on the above is shown in FIG. 1 .

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (7)

A first step of preparing a peptide-resin represented by the following Chemical Formula 1 by sequentially binding amino acids or amino acid derivatives to a polymer support resin including a hydroxyl group;
a second step of treating the peptide-resin obtained in the first step with ethylamine to separate the peptide from the resin and at the same time obtaining a peptide represented by Formula 2 to which ethylamine is coupled;
Including a third step of deprotecting the peptide obtained in the second step,
Method for preparing leuprolide using solid-phase synthesis:
[Formula 1]
^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-O- resin (resin)
[Formula 2]
^{pGlu-His (R 1) -Trp} (R 2) -Ser (R 3) -Tyr (R 3) -D-Leu-Leu-Arg (R 4) -Pro-NHEt
In the above formula, R ¹ is hydrogen or a protecting group selected from the group consisting of t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group and allyl group,
R ² is hydrogen or a protecting group selected from the group consisting of t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group and allyl group,
R ³ is hydrogen, p-methoxybenzyl group, methoxymethyl group, benzyloxymethyl group, tetrahydropyran group, tetrahydrofuran group, t-butyl group, triphenylmethyl group, benzyl group, allyl group, trimethylsilyl group, It is a protecting group selected from the group consisting of t-butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group, t-butylcarbonyl group, acetyl group and benzoyl group,
R ⁴ is t-butyloxycarbonyl group, benzyloxycarbonyl group, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, benzyl group, allyl group, t-butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group , nitro group, 2,2,5,7,8-pentamethylchromene-6-sulfonyl group, 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, 2,2,4,6,7- It is a protecting group selected from the group consisting of a pentamethyl-dihydrobenzofuran-5-sulfonyl group and a toluenesulfonyl group.
The method according to claim 1, wherein the resin in the first step is a PAM resin, a Wang resin, or a Hydroxymethyl resin.
The method according to claim ¹ , wherein R 1 , R ² , R ³ , and R ⁴ are each a triphenylmethyl group, t-butyloxycarbonyl group, t-butyl group, and 2,2,4,6,7-pentamethyl- A method of producing a dihydrobenzofuran-5-sulfonyl group, leuprolide.
The method of claim 1, wherein the first step comprises reacting Boc-Pro-OH with the polymer support resin and then removing the Boc group to obtain a peptide-resin.
5. The method of claim 4, wherein the first step is _{a step of removing Fmoc after reacting the peptide-resin with Fmoc-(AA) n} -OH (n is 1 to 8, AA is an amino acid or an amino acid derivative). The method for producing a leuprolide, comprising the above.
The method according to claim 1, wherein in the second step, the peptide-resin is treated with a mixed solution of THF and an aqueous ethylamine solution.
The method of claim 6, wherein the volume ratio of the THF and the aqueous ethylamine solution is 1:2 to 2:1.

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