KR101454892B1

KR101454892B1 - Process for the Preparation of Exenatide

Info

Publication number: KR101454892B1
Application number: KR1020120122650A
Authority: KR
Inventors: 김종민; 김재일; 최동호; 반수호; 박진석; 김형식; 강성진; 박소영; 고아라
Original assignee: 애니젠 주식회사
Priority date: 2012-10-31
Filing date: 2012-10-31
Publication date: 2014-11-04
Also published as: KR20140056803A

Abstract

The present invention relates to a method for preparing exenatide (SPPS method). The production method of the present invention can be carried out by a sonication treatment under heating and a nitrogen gas stream in the synthesis instead of a conventional method of synthesizing a peptide through a solid phase synthesis method, And the effect of improving the purity. Therefore, the present invention can be applied to other peptides in the art, resulting in a very economical effect. The present invention also relates to a method for preparing a novel Exenatide which combines a solid-phase synthesis and a solution-phase reaction.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for preparing exenatide,

The present invention relates to a novel process for preparing exenatide.

Exenatide is a functional analogue of GLP-1 (glucagon-like peptide-1) isolated from salivary glands of the king lizard (Heloderma, suspectum) in the southwestern United States and is used as a treatment for Type II diabetes. Exendin-4, exendin-4, is a 39-amino acid physiologically active peptide that is 53% amino acid analogous to GLP-1 present in the human body and stable to degradative enzymes such as DPP-4 (Dipetidyl peptidase-4) Compared to GLP-1, it has excellent biostability.

The above-mentioned methods for synthesizing exenatide peptides generally include a solid-phase synthesis method and a solution-phase synthesis method. In the solid phase synthesis method, an amino acid sequence is attached to a solid support, and after the assembly is completed, the sequence is obtained from the support. 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.

As a prior art European patent (EP 1 773 870 B1) reported the preparation of exenatide using a solid phase reaction as in this patent, but a relatively expensive NovaSyn TGT resin and HMPB-AM resin were used and the yield was low It looked. In addition, another patent WO 2009/053315 describes a method for synthesizing exenatide using peptide synthesis of several fragments. Here, since synthesis of several peptides by a solid-phase reaction method is also low, the increase of impurities And the final overall yield is low.

As mentioned above, conventional techniques for the preparation of exenatide have a number of problems that must be improved in synthesis. Therefore, research on the method of effectively producing exenatide can be considered as a very important technology in the pharmaceutical industry.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to improve the problems of the conventional synthesis method of exenatide by the solid phase synthesis method. As a result, it has been found that the synthesis of exenatide can be carried out at a high yield and purity by ultrasonication, heating, and under nitrogen gas flow conditions, and a solid-phase synthesis and a solution- (Convergnet) Synthesis The process for synthesizing Exenatide was completed by identifying the synthesis method.

It is therefore an object of the present invention to provide a novel process for preparing exenatide.

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 process for preparing exenatide comprising the steps of:

(a) obtaining a peptide represented by Formula (I) having a resin attached thereto by a solid-phase synthesis method of applying ultrasound in a nitrogen stream;

(b) removing the resin and the protecting group through a deprotection reaction in the peptide obtained in the step (a) to obtain an exenatide represented by the following formula (II);

Formula I

^{H-His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 3) -Phe-Thr (R 3) -Ser (R 3) -Asp (R 2) -Leu-Ser (R 3 ^{) -Lys (R 4) -Gln (} R 6) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-Val-Arg (R 5) -Leu-Phe-Ile- ^{Glu (R 2) -Trp (R} 2) -Leu-Lys (R 4) -Asn (R 6) -Gly-Gly-Pro-Ser (R 4) -Ser (R 3) -Gly-Ala-Pro- Pro-Pro-Ser (R < ³ >) -NH-resin

(II)

Glu-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser

In Formula Ⅰ, R ¹ is hydrogen or an imidazole protecting group, R ² is hydrogen or a carboxylic acid protecting group, R ³ is a protecting group of hydrogen or hydroxyl, R ⁴ is hydrogen or an amine protecting group, R ⁵ is hydrogen or a guanidine protecting group, and R ⁶ is a hydrogen or amide 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 ( Biochemistry , 11: 1726-1732 (1972) ).

Abbreviations for the protecting groups used herein are:

Thr: Threonine

Glu: Glutamic acid < RTI ID = 0.0 >

Ser: Serine

Arg: Arginine

Pro: Proline

Leu: Leucine

His: Histidine

Ala: Alanine

Gly: Glycine

Phe: phenylalanine

Asp: Aspartic acid

Lys: Lyine

Gln: Glutamine

Met: Methionine

Ala: Alanine

Val: Valine

Ile: Isoleucine

Trp: tryptophan

Asn: Asparagine

Boc: t-butyloxycarbonyl < RTI ID = 0.0 >

tBu: t-butyl (t-butyl)

Fmoc: 9-Fluorenylmethyloxycarbonyl (9-fluorenylmethyloxycarbonyl)

Trt: triphenylmethyl (triphenylmethyl)

Mtt: 4-methyltriphenylmethyl (4-methyltriphenylmethyl)

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

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

In the above formula (II), R < ¹ ^> is a hydrogen or imidazole protecting group commonly used in the art. Preferably, the imidazole protecting group is a t-butyloxycarbonyl group, a benzyloxycarbonyl group, a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a benzyl group or an allyl group, more preferably a triphenylmethyl group or a t- Butyloxycarbonyl group, and most preferably a triphenylmethyl group.

In the above formula (II), R ² is a hydrogen or carboxylic acid protecting group commonly used in the art. Preferably, the carboxylic acid protecting group is a t-butyl group, a methyl group or a benzyl group, more preferably a t-butyl group or a benzyl group, and most preferably a t-butyl group.

In the above formula (II), R ³ is a hydrogen or hydroxyl protecting group commonly used in the art. Preferably, the hydroxyl protecting group is a protecting group selected from the group consisting of p-methoxybenzyl, methoxymethyl, benzyloxymethyl, tetrahydropyran, tetrahydrofuran, T-butyldimethylsilyl group, triphenylsilyl group, triisopropylsilyl group, t-butylcarbonyl group, acetyl group or benzoyl group, more preferably t-butyl group or triphenylmethyl group, and most preferably Is a t-butyl group.

In the above formula (II), R ⁴ is a hydrogen or amine protecting group commonly used in the art. Preferably a t-butyloxycarbonyl group, a methyltriphenyl group, a 9-fluorenylmethylcarbonyl group, a benzylcarbonyl group, a acetic acid group, a trifluoroacetic acid group, a p-toluenesulfonyl group or a methoxymethyl group, Is a t-butyloxycarbonyl group or a fluorenylmethylcarbonyl group, and most preferably a t-butyloxycarbonyl group.

In the above formula (II), R < ⁵ > is a guanidine protecting group commonly used in the art. Preferably, the guanidine protecting group is selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, methoxymethyl, benzyloxymethyl, triphenylmethyl, benzyl, allyl, t- A nitro group, a 2,2,5,7,8-pentamethylchromene-6-sulfonyl group (Pmc), a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group (Mtr) , 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl group (Pbf), fluorenylmethyl carbonate or torenesulfonyl group (Tos), more preferably Pbf or Pmc group And most preferably a Pbf group.

In the above formula (II), R < ⁶ > is a hydrogen or an amide protecting group commonly used in the art. It is preferably a triphenylmethyl group, a trimethoxybenzyl group or a methyltriphenyl group, more preferably a trimethoxybenzyl group or a triphenylmethyl group, and most preferably a triphenylmethyl group.

The protecting group 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 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:

SPPS (elegance Peptides Synthesis method) Exenatide Produce

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

First, a peptide represented by the following formula (I) having a resin attached thereto is obtained by a solid-phase synthesis method in which ultrasonic waves are applied under a nitrogen gas stream. 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? -Amino and side chain functional groups are protected is bound to the resin, the? -Amino protecting group is removed, and the remaining? -Amino and the amino acid protected with the side chain functional group are stepwise coupled in a 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 慣 -amino protecting group at each step of the synthesis, the deprotecting reaction should not occur in the ㈁ coupling reaction, and the synthesis involving the desired amino acid chain has been completed It should be stable under decomposition conditions with resin.

According to a preferred embodiment of the present invention, a resin is used in the synthesis of the peptide of formula (I). Resins that can be used can be conventional resins that are readily degradable under mildly acidic conditions that can fully preserve the side chain protecting groups of the peptides produced. Preferably, the resin is a link amide resin or a link amide MBHA resin, more preferably a link amide MBHA resin.

According to a preferred embodiment of the present invention, the synthesis method of step (a) is carried out at a temperature of 25-45 占 폚, more preferably 30-45 占 폚, even more preferably 30-40 占 폚 Preferably at 35-40 < 0 > C.

According to a preferred embodiment of the present invention, the synthesis method of step (a) is performed by applying ultrasonic waves for 3 to 6 hours, more preferably by applying ultrasonic waves for 4-5 hours.

Step (b): to give the Exenatide through the removal of the resin and deprotection reaction

Then, exenatide of formula (2) is obtained by removing the resin and protecting group from the mild compound of formula (I) by deprotection.

Reaction conditions for the deprotection reaction are preferably a mixture of trifluoroacetic acid, water, a mixture of thioanisole and ethanedithiol, a mixture of trifluoroacetic acid, water, phenol, thioanisole and ethanedithiol, trifluoroacetic acid It is possible to use a mixture of acetic acid, triisopropylsilane and water or a mixture of trifluoroacetic acid, triisopropylsilane, water and ethanedithiol, more preferably trifluoroacetic acid, water, thioanisole and ethanedithiol And more preferably in a mixture having a volume ratio of trifluoroacetic acid: thioenisole: ethanedithiol: water of 87.5: 12.5: 5: 5.

The present invention is capable of producing exenatide purified at high yields, especially at high purity (for example, at least 98% purity), compared with solid phase synthesis methods used to produce exenatide in the art. (Refer to the embodiments of the present invention). In addition, the present invention can bring about a far greater economic effect than the conventional production method in terms of production yield in the case of mass production of exenatide peptide.

The overall process for preparing exenatide based on the above description is summarized as follows:

Scheme 1

According to another aspect of the present invention, the present invention provides a process for preparing exenatide comprising the steps of:

(a) obtaining a peptide represented by the following general formula (VI) and (VII) to which a resin is attached by a solid-phase synthesis method in which ultrasonic waves are applied under a nitrogen stream;

(b) removing the resin from the peptide obtained in the step (a) to obtain a peptide represented by the following formula (III) and (IV);

(c) obtaining a peptide represented by the following formula (V) through a coupling reaction with H-Ser-NH ₂ by a solution-phase synthesis method of the peptide represented by the formula (IV) obtained in the step (b);

(d) obtaining a peptide represented by the following formula (I) by convergent reaction of the peptides represented by the formulas (III) and (V) obtained in the above steps (b) and (c); And

(e) obtaining an exenatide represented by the following formula (II) through deprotection in the peptide obtained in step (d);

Formula I

^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ^{2) -Lys (R 3) -Gln} (R 1) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-Val-Arg (R 4) -Leu-Phe-Ile ^{-Glu (R 2) -Trp (R} 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R 2) -Ser (R 2) -Gly-Ala-Pro -Pro-Pro-Ser (R ² ) -NH ₂

(II)

(III)

^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ² ) -Lys (R ³ ) -Gln (R ¹ ) -Met-Glu (R ² ) -Glu (R ² ) -Glu (R ² ) -Ala-OH

(IV)

^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-OH

Formula V

^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

(VI)

^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ^{2) -Lys (R 3) -Gln} (R 1) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-O- resin

Formula VII

^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-NH-

Wherein R ¹ is hydrogen or an imine protecting group, R ² is hydrogen or a carboxylic acid protecting group, R ³ is a hydrogen or hydroxyl protecting group, R ⁴ is hydrogen or an amine protecting group, R ⁵ is hydrogen Or a guanidine protecting group, and R < ⁶ > is a hydrogen or an amide protecting group.

R ¹ to R ⁶ included in the above-mentioned formulas (I) and (III) to (VII) are as described in the above-mentioned SPPS synthesis method, and redundant description is omitted.

convergence( Convergent ) Synthesis method Exenatide Produce

Step (a): to obtain the peptide of the formula and the formula Ⅵ Ⅶ

First, a peptide represented by the formula (VI) and the formula (VII) to which a resin is attached is obtained by a solid-phase synthesis method in which ultrasonic waves are applied under a nitrogen gas stream. The peptides represented by the formulas (VI) and (VII) are prepared by a solid-phase synthesis method commonly used in the art. That is, an amino acid having a protected α-amino group and a side chain functional group is bound to a resin, followed by stepwise coupling of the amino acid having an α-amino group and a protected side chain functional group in the desired order, .

According to a preferred embodiment of the present invention, a resin is used in the synthesis of the peptides of the formulas (VI) and (VII). Resins that can be used can be conventional resins that are readily degradable under mildly acidic conditions that can fully preserve the side chain protecting groups of the peptides produced. More preferably, the resin is a trityl chloride resin, 2-chlorotrityl resin, 4-methyltritile resin or 4-methoxytrityl resin, more preferably a trityl chloride resin or 2-chlorotri Tylresin, even more preferably 2-chlorotrityl resin.

According to a preferred embodiment of the present invention, the synthesis method of step (a) is carried out at a temperature of 25-45 캜, more preferably 30-45 캜, even more preferably 35-40 캜.

Step (b): to obtain the peptide of formula Ⅲ) and (Ⅳ

Subsequently, the compound represented by the formula (VI) and the compound represented by the formula (VII) is removed under mildly acidic conditions to obtain the peptide of the formula (III) or (IV). 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.

According to a preferred embodiment of the present invention, the process of removing the resin is carried out in the presence of a solution exhibiting acidity. Preferably, the acidic conditions are carried out in the presence of a solution of dichloromethane, acetic acid and trifluoroethanol in a volume ratio of 8: 1: 1 respectively, or in a dichloromethane solution containing 0.5-5 vol% trifluoroacetic acid Lt; / RTI >

Step (c): to obtain the peptide of the formula Ⅴ

The peptide represented by the formula (V) obtained in the step (b) is subjected to a coupling reaction with H-Ser-NH ₂ by a solution-phase synthesis method to obtain the peptide represented by the formula (V).

This process is one of the unique processes in the production process of the present invention. In the peptide represented by formula (IV), all sequences except Ser sequence are produced by solid phase synthesis, but the last C-terminal Ser residue is represented by formula Lt; / RTI >

According to a preferred embodiment of the present invention, the coupling reagents available in step (c) are N, N'-dicyclohexyl carbodiimide (DCC), N, N'-diisobutyl 1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (DIC), benzotriazole-1-yl-oxy- 1-yl-oxy-tris- (pyrrolidino) -phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy- (1H-benzotriazole-1,1, 3-tetramethyluronium hexafluorophosphate (PyBOP)), 2- (1H-benzotriazole- 3,3-tetramethyluronium-hexafluorophosphate (HBTU), 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate 1-yl) -1,1,3,3-tetramethyluronium tetraf luoroborate: TBTU), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O- (7-azabenzotriazol-1-yl) -N, N, N ' N, N'-carbonyldiimidazole (TATU), tetramethyluronium tetrafluoroborate (TATU), and N, N'-carbonyldiimidazole : CDI), 3- (diethoxyphosphoryloxy) -1,2,3-benzotriazin-4 (3H) -one (3- (Diethoxyphosphoryloxy) -1,2,3-benzotriazin- one: DEPBT), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP), 1-hydroxy-7-azabenzotriazole 7-azabenzotriazole HOAt), N, N, N ', N'-tetramethyl-O- (3,4-dihydro-4-oxo-1,2,3-benzotrazin- (N, N, N ', N'-Tetramethyl-O- (3,4- dihydro-4-oxo-1,2,3-benzotriazin-3-yl) uranium tetrafluoroborate: TDBTU), O- (N-succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoro (N-Succinimidyl) -1,1,3,3-tetramethyl uranium tetrafluoroborate (TSTU), 2- (6-chloro-1H-benzotriazol- Ethyl-3- (3-chloro-1H-benzotriazole-1-yl) -1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) (1-ethyl-3- (3-dimethyllaminopropyl) carbodiimide hydrochloride: EDC.HCl), preferably HBTU or EDC.HCl, and most preferably Preferably EDC.HCl.

According to a preferred embodiment of the present invention, the organic solvent used in the coupling liquid phase reaction is at least one solvent selected from the group consisting of dichloromethane, dichloroethane, chloroform and dimethylformamide, more preferably dichloromethane, dimethylformamide Or a mixed solvent of dichloromethane and dimethylformamide, and more preferably a mixed solvent of dichloromethane and dimethylformamide.

The reaction temperature of the coupling reaction is -20-50 ° C, preferably 0-25 ° C, more preferably 0-15 ° C, even more preferably 0-5 ° C.

Step (d): to obtain the peptide of the formula Ⅰ through the convergence (convergent) reaction

Subsequently, the peptides represented by the formulas (III) and (V) obtained in the steps (b) and (c) are subjected to a convergent reaction to obtain a peptide represented by the following formula (I).

According to a preferred embodiment of the present invention, the convergent reaction is carried out in a solvent of 1-hydroxy-7-azabenzotriazole (HOAt) and ethylene dichloride (EDC) Or less for 8-12 hours, more preferably for 5 hours or less for 9-11 hours.

Step (e): yield of Exenatide of the formula Ⅱ

The exenatide can be obtained from the peptide obtained in the above step (d) by carrying out a deprotection reaction under reaction conditions conventionally used in the art.

Reaction conditions for the deprotection reaction are preferably a mixture of trifluoroacetic acid, water, a mixture of thioanisole and ethanedithiol, a mixture of trifluoroacetic acid, water, phenol, thioanisole and ethanedithiol, trifluoroacetic acid It is possible to use a mixture of acetic acid, triisopropylsilane and water or a mixture of trifluoroacetic acid, triisopropylsilane, water and ethanedithiol, more preferably trifluoroacetic acid, water, thioanisole and ethanedithiol , More preferably in a mixture of trifluoroacetic acid: thioenisole: ethanedithiol: water in a volume ratio of 87.5: 12.5: 5: 5.

The present invention relates to a process for the preparation of exenatide, which is capable of producing exenatide with a much improved yield with fewer impurities than the liquid phase synthesis method or solid phase synthesis method used to produce exenatide in the art, Exenatide can be produced (see Examples of the present invention). In addition, the present invention can bring about a far greater economic effect than the conventional production method in terms of production yield in the case of mass production of exenatide peptide.

The overall process for preparing exenatide on the basis of the above contents is summarized as follows.

Scheme 2

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a process for producing solid-phase synthesis under sonication, heating, and under nitrogen gas stream.

(b) The present invention also provides a novel process for preparing exenatide, which is a mixture of solid-phase synthesis and solution-phase reaction.

(c) The present invention improves the separation and purification of synthesized exenatide, enabling commercial mass production.

(d) In addition, the present invention has improved yield and purity over exenatide synthesized by a method used in the related art, and thus has an economic effect in terms of production cost.

Figure 1 shows the chemical structure of exenatide prepared by the process of the present invention.
2 shows the SPPS method in the process of manufacturing exenatide. The English notation and expressions in FIG. 2 are interpreted with reference to the expressions disclosed in the description of the present invention.
FIG. 3 shows a convergent method during the process of manufacturing exenatide. 3 are interpreted with reference to the expressions set forth in the detailed description 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, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example 1: Preparation of Peptide Represented by Formula I

Formula I

(T-Bu) -Phe-Thr (t-Bu) -Ser (trt) -Asp (t-Bu) -Leu-Ser Glu (t-Bu) -Glu (t-Bu) -Ala-Val-Arg (Pbf) -Leu-Phe -Ile-Glu (t-Bu) -trp (Boc) -Leu-Lys (Boc) -Asn (Trt) -Gly-Gly- -Pro-Pro-Pro-Ser (t-Bu) -Link Amide MBHA Resin

9-Fluorenyloxycarbonyl-amino acid-OH coupling reaction

(a) Synthesis of 9-fluorenyloxycarbonyl-Ser (t-Bu) -linkamide MBH resin

Rink Amide MBHA resin (0.30 mmol / g, 30 mmol, GL biochem Ltd.) was added to a special preparation reactor (Dongsung Scientific, customized) equipped with a filtration membrane and capable of nitrogen bubbling and ultrasonic treatment N, N-Dimethylformamide (500 ml, purified water) was added and the resin was expanded for 15 minutes. Then, the solvent was removed through a filtration membrane under reduced pressure. 500 ml of N, N-dimethylformamide containing 20% piperidine was placed in the resin, followed by removal of 9-fluorenyloxycarbonyl for 15 minutes, followed by decompression to remove the reaction solution. The removal of the 9-fluorenylcarbonyl was repeated, and the resin was subsequently washed six times with N, N-dimethylformamide. To the resin obtained in the above reaction was added 9-fluorenyloxycarbonyl-Ser (t-Bu) -OH (57.5 g, 150 mmol, 5.0 eq.) And 1-hydroxybenzotriazole (22.3 g, 165 mmol, N, N-dimethylformamide solution (75 ml, 2 M solution, 5.0 equivalent) containing diisopropylcarbodiimide was added to the reaction mixture, followed by the addition of N, N-dimethylformamide After the addition, the reaction was carried out for 4 hours. The reaction was carried out in a stream of nitrogen. Ultrasonic treatment was carried out for at least 3 hours at the start of the reaction, and the temperature inside the reactor was maintained at 30-40 ° C.

(b) Obtaining H-Ser (t-Bu) -Linkamide MBH resin

500 ml of N, N-dimethylformamide containing 20% piperidine was added to the 9-fluorenyloxycarbonyl-Ser (t-Bu) -linkamide MBHA resin obtained in the above reaction (a) Then, 9-fluorenyloxycarbonyl removal reaction was performed for 15 minutes, and the reaction solution was removed by reduced pressure. The 9-fluorenylcarbonyl removal reaction was repeated and the resin was then washed six times with N, N-dimethylformamide to obtain H-Ser (t-Bu) -linkamide MBHA resin.

(c) Coupling of 9-fluorenyloxycarbonyl-amino acid -OH and obtaining final material

The following amino acid derivatives were sequentially coupled while repeating the above reaction (a) and (b). After repeating the above procedure, the peptide represented by formula (I) was obtained. The following is the composition of the amino acid and reaction reagent for each step:

(50.6 g, 150 mmol, 5 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem Ltd.), 9-fluorenyloxycarbonyl- , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(49.4 g, 150 mmol, 5 equiv., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 equiv. GL Biochem Ltd.), 9-fluorenyloxycarbonyl- , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(53.0 g, 150 mmol, 5 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem Ltd.), 9-fluorenyloxycarbonyl- , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., 5 eq., GL Biochem Ltd.), 9-fluorenyloxycarbonyl-Ser (t- Bu) GL Biochem Ltd.), N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

1-Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 equiv., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Pro-OH (50.6 g, 150 mmol, , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(89.5 g, 150 mmol, 5 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem) as 9-fluorenyloxycarbonyl- N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem.) Was added to a solution of 9-fluorenyloxycarbonyl-Lys (Boc) -OH N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Leu-OH (59.6 g, 150 mmol, , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 equiv., GL Biochem) was added to a solution of 9-fluorenyloxycarbonyl-Trp (Boc) -OH (79.0 g, 150 mmol, N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(44.6 g, 150 mmol, 5 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq. GL Biochem Ltd.), N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

1-Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 equiv., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Ile-OH (59.6 g, , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Phe-OH (70.3 g, 150 mmol, , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(97.3 g, 150 mmol, 5 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(50.9 g, 150 mmol, 5 equiv., GL Biochem Ltd.), 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 eq., GL Biochem Ltd.), 9-fluorenyloxycarbonyl-Val- , N, N-dimethylformamide solution (75 ml, 2 M solution, 5 equivalents) containing diisopropylcarbodiimide,

(62.4 g, 210 mmol, 7 equiv., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq. GL Biochem Ltd.), N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Met-OH (96.5 g, 210 mmol, , N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

(128.2 g, 210 mmol, 7 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem N, N-dimethylformamide solution (105 ml, 2 M solution, 7 eq.) Containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem) was added to a solution of 9-fluorenyloxycarbonyl-Lys (Boc) -OH (80.5 g, 210 mmol, N, N-dimethylformamide solution (105 ml, 2 M solution, 7 eq.) Containing diisopropylcarbodiimide,

(80.5 g, 210 mmol, 7 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq. GL Biochem Ltd.), N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Leu-OH (83.5 g, 210 mmol, , N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq.) Was added to a solution of 9-fluorenyloxycarbonyl-Asp (t-Bu) -OH (86.4 g, 210 mmol, 7 equiv, GL Biochem Ltd.) GL Biochem Ltd.), N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL) was added to a solution of 9-fluorenyloxycarbonyl-Thr (t-Bu) -OH (83.5 g, 210 mmol, 7 eq., GL Biochem Ltd.) Biochem Ltd.), N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

(98.4 g, 210 mmol, 7 equiv., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem Ltd.), 9-fluorenyloxycarbonyl- , N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

(83.5 g, 210 mmol, 7 equiv., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq. GL Biochem Ltd.), N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

Hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem Ltd.) was added to a solution of 9-fluorenyloxycarbonyl-Gly-OH (74.2 g, 210 mmol, , N, N-dimethylformamide solution (105 ml, 2 M solution, 7 equivalents) containing diisopropylcarbodiimide,

(78.0 g, 210 mmol, 7 eq., GL Biochem Ltd.), 1-hydroxybenzotriazole (31.1 g, 230 mmol, 7.7 eq., GL Biochem N, N-dimethylformamide solution (105 ml, 2 M solution, 7 eq.) Containing diisopropylcarbodiimide,

700 ml of N, N-dimethylformamide containing 20% piperidine was added to the 9-fluorenyloxycarbonyl-AA (39 mer) -linkamide MBHA resin obtained in the above procedure, After the removal reaction of fluorenyloxycarbonyl was performed, the reaction solution was removed by decompression. The 9-fluorenylcarbonyl removal reaction was repeated, followed by washing the resin three times with N, N-dimethylformamide three times and with dichloromethane three times to obtain a peptide represented by the formula (I).

Example 2: to Displayed Exenatide Produce

(II)

500 ml of N, N-dimethylformamide containing 20% piperidine was added to the peptide represented by the above formula (I) obtained in Example 1, and then the removal reaction of 9-fluorenyloxycarbonyl was carried out for 15 minutes After that, the reaction solution was removed by decompression. The removal of the 9-fluorenylcarbonyl was repeated, and the resin was subsequently washed six times with N, N-dimethylformamide.

Then, a mixed solution of trifluoroacetic acid: thioenisole: 2,2-ethanedithiol: water = 87.5: 12.5: 5: 5 (1500 ml) containing 1.5% ammonium iodide was added and deprotection . Subsequently, the reaction solution was filtered to remove the degassed resin, and 6000 ml (purified gold) of diethyl ether cooled in the filtrate was added to form a solid. The obtained solid was filtered and washed with 2000 ml of diethyl ether. This procedure was repeated three times and dried to obtain 138 g (yield: 110%, HPLC purity: 30-40%) of exenatide containing an impurity represented by the formula (II) &Lt; / RTI > The crude product was then purified by reverse phase HPLC (230 nm, 10 ml / min, increased to 40% from 20% initial concentration of acetonitrile in 0.1% trifluoroacetic acid in 30 min on a 10 micron C18 column) 26.2 g of tide (yield 19%, HPLC purity 98%) was obtained.

Example 3: a compound represented by the formula (III) and a compound represented by the formula (IV) Of peptide Produce

(III)

(T-Bu) -Phe-Thr (t-Bu) -Ser (trt) -Asp (t-Bu) -Leu-Ser (t-Bu) -Lys (Boc) -Gln (Trt) -Met-Glu (t-Bu) -Glu

(IV)

(Boc) -Leu-Lys (Boc) -Asn (trt) -Gly-Gly-Pro-Ser (t-Bu) -Trp ) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Pro-OH

(a-1) 9- Fluorenyloxycarbonyl - Ala -2- Chlorotrityl Manufacture of resin

Chlorotrityl chloride resin (resin = f = 1.40 mmol / g, 30 mmol, Bidtech) and dichloromethane (600 mL, purified water = 100 mL) were added to a solid-phase synthesis reactor equipped with a filtration membrane ), The resin was expanded for 15 minutes, and then the solvent was removed through a filtration membrane under reduced pressure. Dichloromethane (400 mL) containing 9-fluorenyloxycarbonyl-Ala-OH (14.9 g, 45 mmol, 1.5 eq., GL Biochem Ltd.) was added to the treated resin followed by diisopropylethyl Amine (45 ml, 90 mmol, 3.0 equivalents of a 0.2 M solution, purified water), and the mixture was reacted at room temperature for 8 hours. The reaction product was filtered under reduced pressure to remove the reaction solution. The resin was washed three times with dichloromethane, and dichloromethane: methanol: diisopropylethylamine = 17: 2: 1 (volume ratio, 600 ml) was added to resin, And stirred for 20 minutes. The reaction product was filtered under reduced pressure to remove the reaction solution, and the resin was washed three times with dichloromethane and then dried under vacuum to obtain 9-fluorenyloxycarbonyl-Ala-2-chlorotrityl resin. The replacement ratio was 0.70 mmol / g.

(a-2) 9- Fluorenyloxycarbonyl - Pro -2- Chlorotrityl Manufacture of resin

Chlorotrityl chloride resin (resin = f = 1.40 mmol / g, 30 mmol, Bidtech) and dichloromethane (600 mL, purified water = 100 mL) were added to a solid-phase synthesis reactor equipped with a filtration membrane ), The resin was expanded for 15 minutes, and then the solvent was removed through a filtration membrane under reduced pressure. Dichloromethane (500 mL) containing 9-fluorenyloxycarbonyl-Pro-OH (20.2 g, 60 mmol, 2.0 eq., GL Biochem Ltd.) was added to the treated resin followed by diisopropylethyl Amine (60 ml, 120 mmol, 4.0 equivalents of a 0.2 M solution, purified water), and the mixture was reacted at room temperature for 8 hours. The reaction product was filtered under reduced pressure to remove the reaction solution. The resin was washed three times with dichloromethane, and dichloromethane: methanol: diisopropylethylamine = 17: 2: 1 (volume ratio, 600 ml) was added to resin, And stirred for 20 minutes. The reaction product was filtered under reduced pressure to remove the reaction solution, and the resin was washed three times with dichloromethane and then dried under vacuum to obtain 9-fluorenyloxycarbonyl-Ala-2-chlorotrityl resin. The replacement ratio was 0.60 mmol / g.

(b) 9- Fluorenyloxycarbonyl -amino acid- OH Coupling reaction

H- Ala (or Pro )-2- Chlorotrityl Acquisition of resin

Fluorenyloxycarbonyl-Ala (or Pro) -2-chlorotrityl and N, N-dimethylformamide (500 ml, purified water) were placed in a solid-phase synthesis reactor equipped with a filtration membrane, And the solvent was removed through a filtration membrane under reduced pressure. N, N-Dimethylformamide (500 ml) containing 20% (v / v) piperidine was placed in the resin, followed by removal of 9-fluorenyloxycarbonyl for 15 minutes, The reaction solution was removed by filtration. The 9-fluorenyloxycarbonyl removal reaction was repeated, followed by washing the resin one time with N, N-dimethylformamide, three times with dichloromethane and three times with N, N-dimethylformamide to obtain H- To obtain Ala (or Pro) -2-chlorotrityl resin.

9- Fluorenyloxycarbonyl (or Boc )-amino acid- OH &Lt; / RTI > and the final product

To the resin obtained in the above reaction was added N-fluorenyloxycarbonyl-AA-OH (150 mmol, 5.0 equivalents) and 1-hydroxybenzotriazole (22.3 g, 165 mmol, 5.5 equiv., GL Biochem Ltd.) , And N-dimethylformamide (500 ml) were added. Then, N, N-dimethylformamide solution (75 ml, 2M solution, 5.0 equivalent) containing diisopropylcarbodiimide was added and the mixture was reacted for 6 hours . The reaction was carried out in a stream of nitrogen. Ultrasonic treatment was carried out for at least 3 hours at the start of the reaction, and the temperature inside the reactor was maintained at 30-40 ° C. The above procedure was repeated according to the amino acid sequence (see Example 1) to obtain a product represented by the following formula.

Formula 1

(T-Bu) -Phe-Thr (t-Bu) -Ser (trt) -Asp (t-Bu) -Leu-Ser (t-Bu) -Lys (Boc) -Gln (Trt) -Met-Glu (t-Bu)

(2)

(Boc) -Leu-Lys (Boc) -Asn (trt) -Gly-Gly- (Trp) -Leu- Ala-Pro-Pro-Pro-2-Chlorotrityl Resin (Pro-Ser)

A mixed liquid (600 ml) of dichloromethane: acetic acid: trifluoroethanol = 8: 1: 1 was added to the resin of Formulas (1) and (2) and stirred for 2 hours. The resin was removed by filtration under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain a peptide represented by the formula (III) in which the tritylazine was removed in the formula (1) and the peptide represented by the formula (IV) in which the resin was removed in the formula (2).

Example 4: A compound represented by the formula Of peptide Produce

Formula V

(Boc) -Leu-Lys (Boc) -Asn (trt) -Gly-Gly- (Trp) -Leu- Pro-Ser (t-Bu) -Ser (t-Bu) -Gly-Ala-Pro-Pro-Pro-Ser-NH2

(30 mmol) was dissolved in 300 ml of dimethylformamide, and H-Ser-NH2 (45 mmol, 1.5 equivalent), 1-hydroxybenzotriazole (60 mmol, 2.0 equivalent ). EDC.HCl (60 mmol, 1.5 eq., GL Biochem Ltd.) was then slowly added thereto, and the reaction was allowed to proceed at 0 ° C for 10 hours. After the completion of the reaction, purified water was added to precipitate a solid, and the solid was separated by centrifugation. Thereafter, washing with purified water was performed twice, followed by washing and freeze-drying to obtain the peptide represented by the above formula (IV).

After drying the stomach product, N, N-dimethylformamide (500 ml) containing 20% (v / v) piperidine was added, followed by removal of 9-fluorenyloxycarbonyl for 15 minutes After that, purified water was added to precipitate a solid, followed by centrifugation to separate the solid. Thereafter, purified water washing was further carried out and then lyophilized to obtain a peptide (Formula V) in which 9-fluorenyloxycarbonyl was removed in the above formula (IV).

Example 5: Exenatide Produce

Hydroxy-7-azabenzotriazole (HOAt) (60 mmol, 2 equivalents), EDC (10 mmol) and the like were added to a solution obtained by dissolving 30 mmol of the final product peptide (peptide represented by the formula III) in 400 ml of dimethylformamide in Example 3 . HCl (60 mmol, 2 eq.) Was added and stirred. Thereafter, a solution obtained by dissolving the final product peptide (30 mmol) obtained in Example 4 in 250 ml of dimethylformamide was slowly added to the reactor. The reaction was maintained at 5 DEG C or lower for 10 hours. After completion of the reaction, purified water was added to precipitate a solid, which was then centrifuged to separate the solid. Thereafter, the purified water was washed twice more and then lyophilized to obtain a peptide.

To the obtained peptide was added a mixed solution of trifluoroacetic acid: thioenisole: 2,2-ethanedithiol: water = 87.5: 12.5: 5: 5 (1500 ml) containing 1.5% ammonium iodide, The deprotection reaction was carried out for a period of time. The reaction solution was then filtered to remove the degassed resin, and 6000 ml of cold diethyl ether was added to the filtrate to form a solid. The solid was filtered and washed with 2000 ml of diethyl ether. The procedure was repeated three times and dried to obtain 110.5 g (85% yield, HPLC purity 65%) of exenatide containing the impurity represented by the formula (II) Respectively. It was then purified by reverse phase HPLC (230 nm, 10 ml / min, increased to 40% from 20% initial concentration of acetonitrile in 0.1% trifluoroacetic acid in 30 min in a 10 micron C18 column) and the purification was replaced with acetate To obtain 22.1 g (20% yield, HPLC purity 99%) of exenatide represented by the formula (II).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> ANYGEN CO., LTD. <120> Process for the Preparation of Exenatide <130> PN120591 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Chemical I <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Ser 35 <210> 2 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Chemical II <400> 2 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Ser 35 <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Chemical III <400> 3 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Chemical IV <400> 4 Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly 1 5 10 15 Ala Pro Pro Pro 20 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Chemical V <400> 5 Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly 1 5 10 15 Ala Pro Pro Pro Ser 20 <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Chemical VI <400> 6 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Chemical VII <400> 7 Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly 1 5 10 15 Ala Pro Pro Pro 20

Claims

A process for preparing exenatide comprising the steps of:
(a) obtaining a peptide represented by Formula (I) having a resin attached thereto by a solid-phase synthesis method of applying ultrasound in a nitrogen stream; And
(b) obtaining an exenatide represented by the following formula (II) through a deprotection reaction for removing the resin and a protecting group from the peptide obtained in the step (a);

Formula I
^{H-His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 3) -Phe-Thr (R 3) -Ser (R 3) -Asp (R 2) -Leu-Ser (R 3 ^{) -Lys (R 4) -Gln (} R 6) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-Val-Arg (R 5) -Leu-Phe-Ile- ^{Glu (R 2) -Trp (R} 2) -Leu-Lys (R 4) -Asn (R 6) -Gly-Gly-Pro-Ser (R 4) -Ser (R 3) -Gly-Ala-Pro- Pro-Pro-Ser (R < ³ >) -NH-resin

(II)
Glu-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser
In Formula Ⅰ, R ¹ is hydrogen or an imidazole protecting group, R ² is hydrogen or a carboxylic acid protecting group, R ³ is a protecting group of hydrogen or hydroxyl, R ⁴ is hydrogen or an amine protecting group, R ⁵ is hydrogen or a guanidine protecting group, and R ⁶ is a hydrogen or amide protecting group.

The method of claim 1, wherein the synthesis of step (a) is carried out at a temperature of 25-45 ° C.

The method for preparing exenatide according to claim 1, wherein the synthesis of step (a) is conducted by applying ultrasonic waves for 3 to 6 hours.

The method of claim 1, wherein the resin of step (a) is a link amide resin or a link amide MBHA resin.

The method of claim 1, wherein the step of removing the resin and the protecting group in step (b) is carried out in the presence of an acidic solution.

6. The method according to claim 5, wherein the acidic solution is a mixed solution of trifluoroacetic acid, water, thioanisole and ethanedithiol, a mixed solution of trifluoroacetic acid, triisopropylsilane and water, or a mixed solution of trifluoroacetic acid, Propylsilane, water, and ethanedithiol in the presence of a catalyst.

7. The method according to claim 6, wherein the acidic solution is a mixed solution having a volume ratio of trifluoroacetic acid: thioenisole: 2,2-ethanedithiol: water of 87.5: 12.5: 5: 5 Way.

A process for preparing exenatide comprising the steps of:
(a) obtaining a peptide represented by the following general formula (VI) and (VII) to which a resin is attached by a solid-phase synthesis method in which ultrasonic waves are applied under a nitrogen stream;
(b) removing the resin from the peptide obtained in the step (a) to obtain a peptide represented by the following formula (III) and (IV);
(c) obtaining a peptide represented by the following formula (V) through a coupling reaction with H-Ser-NH ₂ by a solution-phase synthesis method of the peptide represented by the formula (IV) obtained in the step (b);
(d) obtaining a peptide represented by the following formula (I) by convergent reaction of the peptides represented by the formulas (III) and (V) obtained in the above steps (b) and (c); And
(e) obtaining an exenatide represented by the following formula (II) through deprotection in the peptide obtained in step (d);
Formula I
^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ^{2) -Lys (R 3) -Gln} (R 1) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-Val-Arg (R 4) -Leu-Phe-Ile ^{-Glu (R 2) -Trp (R} 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R 2) -Ser (R 2) -Gly-Ala-Pro -Pro-Pro-Ser (R ² ) -NH ₂

(II)
Glu-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser

(III)
^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ² ) -Lys (R ³ ) -Gln (R ¹ ) -Met-Glu (R ² ) -Glu (R ² ) -Glu (R ² ) -Ala-OH

(IV)
^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-OH

Formula V
^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

(VI)
^{R 5 -His (R 1) -Gly} -Glu (R 2) -Gly-Thr (R 2) -Phe-Thr (R 2) -Ser (R 1) -Asp (R 2) -Leu-Ser (R ^{2) -Lys (R 3) -Gln} (R 1) -Met-Glu (R 2) -Glu (R 2) -Glu (R 2) -Ala-O- resin

Formula VII
^{R 5 -Val-Arg (R 4} ) -Leu-Phe-Ile-Glu (R 2) -Trp (R 3) -Leu-Lys (R 3) -Asn (R 1) -Gly-Gly-Pro-Ser (R ² ) -Ser (R ² ) -Gly-Ala-Pro-Pro-Pro-NH-
Wherein R ¹ is hydrogen or an imine protecting group, R ² is hydrogen or a carboxylic acid protecting group, R ³ is a hydrogen or hydroxyl protecting group, R ⁴ is hydrogen or an amine protecting group, R ⁵ is hydrogen Or a guanidine protecting group, and R < ⁶ > is a hydrogen or an amide protecting group.

9. The method of claim 8, wherein the synthesis of step (a) is carried out at a temperature of 25-45 < 0 > C.

The method of claim 8, wherein the synthesis of step (a) is conducted by applying ultrasonic waves for 3 to 6 hours.

The method of claim 8, wherein the resin in step (a) is selected from the group consisting of trityl chloride resin, 2-chlorotrityl resin, 4-methyltritile resin, or 4-methoxytrityl resin. Gt;

9. The method of claim 8, wherein the step of removing the resin in step (b) is carried out in the presence of an acidic solution.

13. The method of claim 12, wherein the acidic solution is a solution of dichloromethane, acetic acid, and trifluoroethanol in a volume ratio of 8: 1: 1 or a solution of dichloromethane containing 0.5-5 vol% trifluoroacetic acid A process for preparing exenatide.

9. The method of claim 8, wherein the coupling reagent used in step (c) is selected from the group consisting of N, N'-Dicyclohexylcarbodiimide, N, N'-diisopropylcarbodiimide N, N'-diisopropylcarbodiimide, Benzotriazole-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate, Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, 2- (1H-benzotriazole- 1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, 2 (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, - (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (2- (1H- -tetramethyluronium tetrafluoroborate), 2- (7-aza-1H-benzotriazole-1 - (7-Aza-1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), O- (7-azabenzotriazole-1-yl) -N, N, N ', N'- tetramethyluronium tetrafluoroborate (O- N, N'-carbonyldiimidazole, 3- (diethoxyphosphoryloxy) -1,2,3-benzotriazine-N, N'-tetramethyluronium tetrafluoroborate, 4 (3H) -one, (bromo-tris-pyrrole dino-phosphonium hexafluorophosphate (Bromo-tris pyrrolidino-phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole, N, N, N ', N'-tetramethyl- N, N'-tetramethyl-O- (3,4-dihydro-4-oxo-1,2,3-benzotriazin- oxo-1,2,3-benzotriazin-3-yl) uranium tetrafluoroborate), O- (N- (N-Succinimidyl) -1,1,3,3-tetramethyl uranium tetrafluoroborate), 2- (6-chlorophenyl) -1,3,3-tetramethyluronium tetrafluoroborate, -1H-benzotriazole-1-yl) -1,1, 3-tetramethylaminium hexafluorophosphate (2- 3-tetramethylaminium hexafluorophosphate) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. &Lt; / RTI >

The method of claim 8, wherein the solvent used in step (c) is at least one solvent selected from the group consisting of dichloromethane, dichloroethane, chloroform, and dimethylformamide.

9. The method of claim 8, wherein the reaction temperature in step (c) is between -20 < 0 > C and 50 < 0 > C.

9. The method of claim 8, wherein the convergent reaction of step (d) is carried out in a solvent of 1-hydroxy-7-azabenzotriazole and ethylene dichloride.

The method of claim 1 or claim 8, wherein R ¹ is selected from imidazole already t- butyloxy carbonyl, benzyloxy carbonyl group, methoxymethyl group, benzyloxy group, triphenylmethyl group, a benzyl group and an allyl group consisting of groups Wherein the protecting group is a protecting group.

The method of claim 1 or 8, wherein R ² is a carboxylic acid protecting group selected from the group consisting of t-butyl, methyl and benzyl.

9. The compound according to claim 1 or 8, wherein R ³ is selected from the group consisting of a p-methoxybenzyl group, a methoxymethyl group, a benzyloxymethyl group, a tetrahydropyran group, a tetrahydrofuran group, Is a hydroxyl group protecting group selected from the group consisting of a chlorotrityl group, a benzyl group, an allyl group, a t-butyldimethylsilyl group, a triphenylsilyl group, a triisopropylsilyl group, a t-butylcarbonyl group, an acetyl group and a benzoyl group A process for preparing exenatide.

The method of claim 1 or 8, wherein R ⁴ is a group selected from the group consisting of a t-butyloxycarbonyl group, a methyltriphenyl group, a 9-fluorenylmethylcarbonyl group, a benzylcarbonyl group, a acetic acid group, a trifluoroacetic acid group, A sulfonyl group and a methoxymethyl group. &Lt; RTI ID = 0.0 > 8. < / RTI >

9. The compound according to claim 1 or 8, wherein R < ⁵ > is selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, methoxymethyl, benzyloxymethyl, triphenylmethyl, benzyl, allyl, , Triphenylsilyl group, triisopropylsilyl group, nitro group, 2,2,5,7,8-pentamethylchromene-6-sulfonyl group, 4-methoxy-2,3,6-trimethylbenzenesulfonyl group , A 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl group, a fluorenylmethyl carbonate and a toluenesulfonyl group. &Lt; / RTI >

9. The process of claim 1, wherein R < ⁶ > is an amide protecting group selected from the group consisting of triphenylmethyl group, trimethoxybenzyl group and methyltriphenyl group.