JP3249915B2 - Method for producing LH-RH derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for efficiently producing LH-RH and / or a derivative thereof, which are peptides useful as medicines, using an enzyme.

[0002]

2. Description of the Related Art Luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released from the anterior pituitary gland under the control of luteinizing hormone releasing hormone LH-RH produced in the hypothalamus. LH-RH and its derivatives have gonadotropin-secreting activity and have a phenomenon of suppressing gonadal function by continuous administration. For example, endometriosis, central precocious puberty, infertility, prostate cancer, etc. Has been applied as a prophylactic and / or therapeutic agent. Buserelin (Japanese Patent Publication No. 60-9519), Goserelin (Japanese Patent Publication No. 61-13480), leuprorelin (Japanese Patent Publication No. 53-14072), nafarelin (Japanese Patent Publication No. 63-56238)
Publication).

As a method for producing the above LH-RH and its derivatives, a peptide fragment having a partial sequence corresponding to the polypeptide is formed by a liquid phase method or a solid phase method.
Chemical synthesis by a liquid phase synthesis method in which each fragment is further coupled in a liquid phase (JP-B-56-47175, JP-A-49-117468, JP-B-57-29462, JP-B-57-29462)
No. 7-25540, Japanese Patent Publication No. 63-17839, Japanese Patent Publication No. 63-4539
No. 8, JP-A-48-40770, JP-A-50-88069, JP-A-49-41375, JP-A-49-41376, JP-A-48-99170, JP-B-52-20996, JP-A-49
-35381, JP-B-52-8831, JP-B-57-61268
JP, JP-B-53-14072, JP-B-57-26506, JP-B-60-22720, JP-B-61-13480, JP-B3-71439, etc.) are known. .

However, in the liquid phase synthesis method, the solubility slightly changes as the number of amino acid residues of the peptide increases, so that it becomes increasingly difficult to find a suitable solvent.
Accordingly, the difficulty in separating the target peptide from unreacted products and by-products increases. In particular, the serine residue at the 4-position of LH-RH and its derivatives is easily racemized and therefore remains as a contaminant, and the post-reaction treatment is difficult and uneconomical, such as inability to recover the raw materials. It is not industrially satisfactory.

[0005]

The object of the present invention is to provide an LH
An object of the present invention is to provide a method for efficiently producing RH and / or its derivative in large quantities at low cost.

[0006]

As a result of intensive studies to solve the above problems, the present inventors have found a method for producing LH-RH and / or a derivative thereof suitable for industrial production by an enzyme synthesis technique and completed the present invention. did.

That is, the present invention provides a peptide fragment represented by the general formula (1): pGlu-His-Trp-OR ¹ (1) (wherein R ¹ represents lower alkyl); 2): H-Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X is D-Leu, D-Ser (But), D-Trp, (2-naphthyl)
Represents an amino acid selected from the group consisting of -D-Ala and Gly, and Y represents Gly-NH ₂ , azaglycine or NHR ² (R ² is lower alkyl). ) In the presence of chymotrypsin or a chymotrypsin-like enzyme in a reaction medium consisting of a buffer itself or from dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, ethanol, butanol and ethyl acetate. Wherein the reaction is carried out at a temperature in the range of 0 to 50 ° C. in a reaction medium obtained by mixing an organic solvent selected from the group consisting of water and a buffer solution, wherein pGlu-His is used. -Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (wherein X and Y are as described above) and / or a derivative thereof. is there. Here, the production method of the present invention is characterized by further comprising a step of purifying the peptide of the general formula (3) by partition chromatography. Further, R ¹ is an alkyl group having 1 to 3 carbon atoms, and R ² is an alkyl group having 1 to 3 carbon atoms.
Wherein the alkyl group is The present invention also provides
A peptide fragment represented by the general formula (1) pGlu-His-Trp-OR ¹ (1) (wherein R ¹ represents lower alkyl); and a general formula (2): H-Ser-Tyr-X -Leu-Arg-Pro-Y (2) (where X is D-Leu, D-Ser (But), D-Trp, (2-naphthyl)
Represents an amino acid selected from the group consisting of -D-Ala and Gly, and Y represents Gly-NH ₂ , azaglycine or NHR ² (R ² is lower alkyl). ) In the presence of an immobilized enzyme to which chymotrypsin or a chymotrypsin-like enzyme is immobilized, in a reaction medium consisting of a buffer solution itself, or dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, A general formula characterized by reacting at a temperature in the range of 0 to 50 ° C. in a reaction medium obtained by mixing an organic solvent selected from the group consisting of ethanol, butanol and ethyl acetate with water or a buffer. 3): LH-RH represented by pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (wherein X and Y are as defined above) and / or This is a method for producing the derivative. Here, the production method of the present invention is characterized by further comprising a step of purifying the peptide of the general formula (3) by partition chromatography. The above R ¹ is an alkyl group having 1 to 3 carbon atoms, and the above R ² is an alkyl group having 1 carbon atom.
-3 alkyl groups.

[0008] In the present specification, "lower alkyl" means alkyl having 1 to 3 carbon atoms, and methyl,
Ethyl, propyl and isopropyl. Further, in the present specification, amino acids, peptides, protecting groups,
Abbreviations for solvents and others refer to the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry (IU
The provisions of B) or conventional symbols in the field shall be followed. An example is shown below. However, when there is an optical isomer with respect to an amino acid or the like, the L-form is indicated unless otherwise specified.

Tyr: Tyrosine residue Gly: Glycine residue Azgly: Azaglycine residue Glu: Glutamic acid residue pGlu: Pyroglutamic acid residue Ser: Serine residue Arg: Arginine residue Asp: Aspartic acid residue Pro: Proline residue Leu: Leucine residue His: Histidine residue Ala: Alanine residue Trp: Tryptophan residue Et: Ethyl Boc: t-butoxycarbonyl Aoc: t-amyloxycarbonyl Bz: benzyl Z: benzyloxycarbonyl Tos: tosyl OMe: methyl Ester OBz: benzyl ester OSu: N-hydroxysuccinimide ester TFA: trifluoroacetic acid THF: tetrahydrofuran DMF: dimethylformamide DCC: dicyclohexylcarbodiimide WSC: N-ethyl-N'-dimethylaminopropyl-carbodiimide HOSu: N-hydroxysuccinimide Acid imide HOBt: 1-hydro Xybenzotriazole MeOH: methanol EtOH: ethanol AcOH: acetic acid

The peptide fragment represented by the general formula (1) corresponds to the first to third amino acid residues of the amino acid sequence of the LH-RH derivative represented by the general formula (3). The peptide fragment represented by the general formula (2) corresponds to the amino acid residue at the 4-position or lower in the amino acid sequence of the LH-RH derivative represented by the general formula (3).

The peptide fragment represented by the general formula (1) or the peptide fragment represented by the general formula (2) is
Each of them can be synthesized according to known methods for peptide synthesis. For example, "The Peptides" Vol. 1
(1966) [Schreder and Luhke, Academic Press, New
York, USA] or “Peptide synthesis” [Izumiya et al., Maruzen Co., Ltd. (1975)], for example, azide method, acid chloride method, acid anhydride method,
Mixed acid anhydride method, DCC method, active ester method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carboimidazole method, redox Method, a DCC-additive (HONB, HOBt, HOSu) method, a solid phase method and the like. According to the general peptide synthesis method as described above, for example, the so-called stepwise extension method in which amino acids are sequentially condensed one by one in accordance with the amino acid sequence to the C-terminal amino acid, or each fragment is divided into several fragments. It can be produced by a fragment condensation method of synthesizing and coupling them.

In the step of synthesizing the peptide fragment represented by the above general formula (1) or (2), functional groups that should not be involved in the reaction are protected by ordinary protecting groups, Is detached. In addition, the functional groups involved in the reaction are usually activated. Each of these reaction methods is known, and the reagents and the like used therefor can be appropriately selected from known ones.

The protecting groups for the amino group include benzyloxycarbonyl (Z) and t-butyloxycarbonyl (Boc)
, T-amyloxycarbonyl (Aoc), isobonyloxycarbonyl, p-methoxybenzyloxycarbonyl,
2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl,
Formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl and the like.

Examples of the carboxyl-protecting group include alkyl esters (eg, methyl ester, ethyl ester, propyl ester, butyl ester, tert-butyl ester, etc.), benzyl ester, p-nitrobenzyl ester, methylbenzyl ester, p-benzyl ester and the like. Chlorobenzyl ester, benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide and the like can be mentioned.

Examples of activated carboxyl groups involved in peptide bonds include, for example, corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (eg, pentachlorophenol, p-nitrophenol) , N-hydroxysuccinimide, N-hydroxybenztriazole, esters with N-hydroxy-5-norbornene-2,3-dicarboximide) and the like.

In some cases, the peptide bond formation reaction can be carried out in the presence of a condensing agent, for example, a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole, or tetraethylpyrophosphate.

Conventionally, proteolytic enzymes have been mainly used for cleaving peptide bonds, but it has long been known that they can also participate in the formation of peptide bonds, which is the reverse reaction. Enzymatic synthesis of long-chain peptides is limited to amino acids with limited substrate specificity in the structure or to enzymes with limited substrate specificity. Rare enzymes are rarely used in peptide formation reactions. Therefore, the enzymatic reaction is mainly used for peptide bonds of relatively short chains such as oligopeptides.However, since it takes time to study synthesis conditions, chemical synthesis is generally used when producing peptides. Enzyme synthesis is tried when there are many side reactions or when the reaction is difficult in the synthesis. Enzyme synthesis may solve the above-mentioned problems in chemical synthesis depending on the conditions, and may be a very useful method when performed under mild conditions that enable mass production.

According to the present invention, as a result of intensive studies, the general formula
By reacting the peptide fragment represented by (1) and the peptide fragment represented by the general formula (2) in the presence of chymotrypsin or a chymotrypsin-like enzyme to bind them, the general formula (3) It has succeeded in efficiently producing the represented LH-RH and / or derivative thereof.

The chymotrypsin used in the present invention has been registered with the International Union of Biochemistry (IUB) Enzyme Committee as enzyme number EC.3.4.21.1 and is an enzyme obtained from bovine pancreas. Chymotrypsin is commercially available from SIGMA and others. This reaction is usually carried out at pH 5-10, preferably at pH 6-9.
The reaction is performed in a medium containing a buffer solution.

The type of the buffer is not particularly limited as long as the pH is within the above range, and various types can be used. For example, Tris-HCl buffer,
Macilbain buffer, phosphate buffer, ammonium acetate buffer, Atkins & Pantin buffer, Veronal buffer and the like.

When the above-mentioned buffer is used as a reaction medium, the buffer is usually used by mixing with a water-miscible organic solvent. Examples of the water-miscible organic solvent include dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylimidazolidinone (DMI), hexamethylphosphoryltriamide (HMPA), methanol (MeOH), ethanol (EtOH) and the like. Is mentioned. Preferred are dimethylformamide, methanol, and ethanol. A solvent that separates from butanol, ethyl acetate and the like can also be used. These organic solvents can be used alone or in combination of two or more. The mixing ratio of the water-miscible organic solvent is generally
The range is 70% by volume or less, preferably 50% by volume or less.

The reaction is usually carried out in a temperature range in which chymotrypsin or a chymotrypsin-like enzyme acts, that is, generally in the range of about 0 to about 50 ° C, preferably about 0 ° C to about 20 ° C. The amount of chymotrypsin or chymotrypsin-like enzyme used is not particularly limited, and can be appropriately changed depending on reaction conditions.

The amount of the peptide fragment represented by the general formula (1) is usually 1 to 5 mol, preferably 2 to 4 mol per mol of the peptide fragment represented by the general formula (2).
Use moles. The reaction can be performed using an immobilized enzyme obtained by immobilizing chymotrypsin or a chymotrypsin-like enzyme by a general method, for example, a carrier binding method, a cross-linking method, an entrapment method, or another method. Examples of the carrier used in the carrier binding method include polysaccharide derivatives such as cellulose, dextran, and agarose, polyacrylamide gel, and porous glass. As a crosslinking reagent used in the crosslinking method, for example, glutaraldehyde,
Bisdiazobenzidine, N, N-polymethylenebisiodoacetamide, N, N-ethylenebismaleimide and the like can be mentioned. Materials used in the entrapment method include polyacrylamide gel, polyacryl alcohol gel, starch, konjac powder, nylon, polyurea, polystyrene, ethyl cellulose, collodion, cellulose nitrate and the like. However, the immobilization method is not limited to these.

LH-R prepared by the method of the present invention
The H derivative can be desalted and purified according to a usual method. For example, ion exchange chromatography such as DEAE-cellulose, partition chromatography such as Sephadex LH-20 and Sephadex G-25, normal phase chromatography such as silica gel, reverse phase chromatography such as ODS-silica gel, high performance liquid chromatography And the like.

The method of the present invention has the following advantages as compared with the known methods for producing LH-RH derivatives, and is extremely useful industrially. B) Due to the nature of the enzymatic reaction, it can be synthesized without a side reaction such as racemization, so that purification and separation are easy. B) The yield is high, and unreacted fragments can be recovered and reused, which is economically advantageous. The LH-RH derivative can be converted into a pharmaceutically acceptable salt, for example, an acetate, a hydrochloride, a phosphate and the like, if necessary.

[0026]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. In the following Examples, the obtained pure peptide was identified by measuring the retention time of high performance liquid chromatography (HPLC), measuring the optical rotation, and analyzing amino acids. These measurements were performed by the following measurement methods and measurement conditions unless otherwise specified.

High performance liquid chromatography (HPL)
C) For high performance liquid chromatography analysis, use LC-Module-1
(Manufactured by Nippon Waters Limited). (HPLC analysis conditions) Column: TSK gel ODS-120T (4.6 × 250 mm) Solvent: 0.1% TFA-acetonitrile (linear gradient gradient changing acetonitrile from 20% to 50% at 1% per minute) Flow rate: 1 ml / min Detection wavelength: 220nm

Optical rotation The optical rotation was measured using DIC-370 (manufactured by JASCO Corporation). (Measurement of optical rotation) Ray: Na lamp 589 nm Temperature: 20 ° C. Layer length: 100 mm Concentration: 5 mg / ml ・ Amino acid analysis In the amino acid analysis, the obtained peptide was prepared by adding 6N hydrochloric acid (0.1
% Phenol) at 110 ° C. for 20 hours, and then performed using a Hitachi amino acid analyzer L-8500 (manufactured by Hitachi, Ltd.).

Example 1 pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt
Production (1-1) Production of Boc-Arg (Tos) -Pro-NHEt 214.3 g of Boc-Arg (Tos) -OH was added to 150 ml of THF and 100 ml of DMF.
dissolved in a mixed solvent of dry ice and ethanol.
Cooled to 20 ° C. Next, add N-methylmorpholine
35 ml was added dropwise, followed by isobutyl chloroformate 66
The resulting mixture was added dropwise and stirred at -20 ° C for 1 minute to prepare the corresponding mixed acid anhydride. The obtained reaction solution was H-Pro-NHEt 71.
1 g was mixed with a solution of 300 ml of THF and stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes. Thereafter, the obtained reaction mixture was concentrated under reduced pressure. Ethyl acetate (1200 ml, twice) was added to the residue, and the mixture was washed twice with water (500 ml), and the ethyl acetate layer was concentrated under reduced pressure. The residue was solidified by treatment with ether and dried. Thus, Boc-Arg (Tos) -Pro-NHEt 221.18 g
(80.0% yield) was obtained. The melting point, optical rotation, Tf Rf and elemental analysis of Boc-Arg (Tos) -Pro-NHEt are shown below.

Mp: 100-102 ° C. [α] _D : -30.3 (c = 1.0, MeOH) Rf: 0.69 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₂₅ H ₄₀ N) ₆ O ₆ as S · H ₂ O) theory C: 52.61% H: 7.42% N: 14.73% measured value C: 52.85% H: 7.30% N: 14.41%

(1-2) Production of Z-Leu-Arg (Tos) -Pro-NHEt 110.54 g of Boc-Arg (Tos) -Pro-NHEt produced in the above (1-1)
Was dissolved in 150 ml of dichloromethane (DCM). To this, 150 ml of TFA was added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. The obtained reaction solution was concentrated under reduced pressure, and ether 15 was added to the residue.
00 ml was added to solidify and dried. In this way, H-Arg
(Tos) -Pro-NHEt.TFA was obtained. This deprotected form H-Arg (Tos) -P
ro-NHEt.TFA was dissolved in a mixed solvent of 80 ml of DMF and 200 ml of THF, and neutralized with N-methylmorpholine while cooling. Next, Z-Leu-OH 53.06g and HOSu 23.02g
In 200 ml of THF and 36.4 ml of WSC
Was added and the mixture was stirred at 0 ° C. for 5 minutes and at room temperature overnight. After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure.

To the residue was added 1500 ml of ethyl acetate, and 500 ml of water.
And twice with 500 ml of saturated saline. Next, the ethyl acetate layer was concentrated under reduced pressure, the residue was treated with ether, solidified, and dried. Thus, Z-Leu-Arg (Tos) -Pro
119.5 g (85.1% yield) of -NHEt was obtained. Z-Leu-Arg (To
The melting point, optical rotation, Tf Rf and elemental analysis of s) -Pro-NHEt are shown below.

Mp: 99-103 ° C. [α] _D : -40.6 (c = 1.0, MeOH) Rf: 0.75 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis (C ₃₄ H ₄₉ N) _{(As 7} O ₇ S) Theoretical value C: 58.35% H: 7.06% N: 14.01% Measurement value C: 58.40% H: 7.26% N: 13.78%

(1-3) Production of ZD-Ser (But) -Leu-Arg-Pro-NHEt 20.99 g of Z-Leu-Arg (Tos) -Pro-NHEt produced in the above (1-2)
Was added to the mixture, and about 200 ml of HF was added thereto while cooling to -70 ° C with dry ice-ethanol.
Stirred at C for 1 hour. Thereafter, the mixture was concentrated under reduced pressure, and the residue was treated with ether. The precipitated product was collected by filtration and dried in vacuo over sodium hydroxide to obtain H-Leu-Arg-Pro-NHEt.HF.

8.86 g of ZD-Ser (But) -OH was dissolved in 100 ml of THF. This was cooled to -20 ° C with dry ice-ethanol, 3.3 ml of N-methylmorpholine was added dropwise, and then 3.96 ml of isobutyl chloroformate was added dropwise.
The mixture was stirred at -20 ° C for 1 minute to produce the corresponding mixed anhydride. The obtained reaction solution was subjected to H-Leu-Arg-Pro-NHEt.HF DM
F. The solution was mixed with 200 ml of a solution of F (neutralized with N-methylmorpholine) and stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes.
Thereafter, the mixture was concentrated under reduced pressure, 500 ml of water was added to the residue, and extracted five times with 200 ml of butanol. After the extract was washed five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, ZD-Ser (But) -Leu-Arg-P
22.89 g of ro-NHEt (90.5% yield) was obtained. ZD-Ser (Bu
The melting point, optical rotation, Tf Rf and elemental analysis values of t) -Leu-Arg-Pro-NHEt are shown below.

Mp: 121-123 ° C. [α] _D : -49.0 (c = 1.0, MeOH) Rf: 0.51 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₃₄ H ₅₆ N ₈ O ₇ ) Theoretical C: 59.28% H: 8.19% N: 16.27% Measured C: 59.01% H: 8.15% N: 16.19%

(1-4) Production of Z-Ser-Tyr-OMe 23.9 g of Z-Ser-OH and 23.2 g of H-Tyr-OMe were dissolved in 50 ml of DMF, and 12.7 g of HOSu and 20 ml of WSC were added. Stirred overnight at room temperature for minutes. After confirming with ninhydrin,
The reaction mixture was concentrated under reduced pressure. 500 ml of ethyl acetate was added to the residue, twice with 200 ml of 1N hydrochloric acid and twice with 200 ml of saturated aqueous sodium hydrogencarbonate solution.
Washing was performed twice in the order of a saturated saline solution. The ethyl acetate layer was concentrated under reduced pressure, and the residue was solidified by treatment with hexane. In this way, 38.44 g (92.3% yield) of Z-Ser-Tyr-OMe was obtained. Z
The melting point, optical rotation, Tf Rf and elemental analysis of -Ser-Tyr-OMe are shown below.

Mp: 49 to 53 ° C. (decomposition) [α] _D : +2.3 (c = 1.0, MeOH) Rf: 0.89 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₂₁ H ₂₄ N as ₂ O ₇₎ theory C: 60.57% H: 5.81% N: 6.73% measured value C: 60.88% H: 5.88% N: 6.99%

(1-5) Production of Z-Ser-Tyr-NHNH ₂ 38.0 g of Z-Ser-Tyr-OMe produced in the above (1-4) was dissolved in 200 ml of methanol, and 50 g of hydrazine was added. And left overnight. Thereafter, the mixture was concentrated under reduced pressure and washed with methanol. Thus, 34.2 g of Z-Ser-Tyr-NHNH ₂ (yield 90.0
%)was gotten. Melting point, optical rotation, TL of Z-Ser-Tyr-NHNH ₂
The Rf and elemental analysis value of C are shown below.

Mp: 194 to 197 ° C. [α] _D : +9.9 (c = 1.0, DMF) Rf: 0.64 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis (C ₂₀ H ₂₄ N) ₄ O ₆ as) theory C: 57.69% H: 5.81% N: 13.45% measured value C: 57.85% H: 5.83% N: 13.49%

(1-6) Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro
Production of -NHEt ZD-Ser (But) -Leu-Arg-Pro-NHEt produced in (1-3) above
28.0 g was dissolved in 300 ml of methanol and hydrogen was added in the presence of 2.5 g of 10% palladium / carbon. After stirring at room temperature for 7 hours, the catalyst was removed, concentrated under reduced pressure, and the residue was treated with ether. In this way, HD-Ser (But) -Leu-Arg-Pro
-NHEt was obtained.

The Z-Ser-Tyr-NHNH ₂ 2 produced in the above (1-5)
0.1 g was dissolved in 150 ml of DMF and cooled to -20 ° C with dry ice-ethanol. 43.5 ml of 4N HCl-dioxane and 6.5 ml of isoamyl nitrite were added to the solution to obtain an azide compound. Further, 24.4 ml of triethylamine was added for neutralization. HD-Ser (But) -Leu-Arg-Pro
-NHEt was transferred to DMF 150ml solution, -20 ℃ for 2 hours, 4 ℃
After stirring for 17 hours, the mixture was concentrated. Add 500 ml of water to the residue,
The mixture was extracted five times with 200 ml of butanol. After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, Z-Ser-Tyr-D-Ser (But) -Leu-Arg
33.2 g (87.5% yield) of -Pro-NHEt was obtained. Z-Ser-Tyr-
Melting point, optical rotation, TLC of D-Ser (But) -Leu-Arg-Pro-NHEt
The Rf and elemental analysis values of are shown below.

Mp: 115-118 ° C. (decomposition) [α] _D : -27.6 (c = 1.0, DMF) Rf: 0.36 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₄₆ H ₇₀ N ₁₀ O ₁₁ as a) theory C: 58.83% H: 7.51% N: 14.91% measured value C: 58.88% H: 7.55% N: 14.90%

(1-7) Production of Z-pGlu-His-OH 52.6 g of Z-pGlu-OH was dissolved in 400 ml of THF. The solution was cooled to −20 ° C. with dry ice-ethanol, 22.0 ml of N-methylmorpholine and 26.4 ml of isobutyl chloroformate were added dropwise, and the mixture was stirred at −20 ° C. for 1 minute to obtain the corresponding mixed anhydride. Was manufactured. In the resulting reaction mixture
25.3 g of HOSu was added and stirred for 30 minutes. Then add HH
62.9 g of is-OH.HCl and triethylamine (TEA) 28.
A solution of 0 ml in 400 ml of water was added. 1 hour at 0 ° C,
After stirring at room temperature overnight, the mixture was concentrated under reduced pressure. The remaining aqueous solution was washed with ethyl acetate, and the aqueous layer was extracted with butanol. After washing several times with water, the butanol layer was concentrated under reduced pressure. After solidifying with ether, it was dissolved in water and adjusted to pH5. This aqueous solution was injected into a column (5.0 × 20 cm) packed with HP-20,
The effluent was washed with water until the Poury reaction became ±, and then eluted with methanol. The eluted methanol solution was concentrated under reduced pressure, and the residue was treated with ether. It was then recrystallized from methanol-ether. Thus, Z-pGlu-His-OH
59.5 g (74.3% yield) was obtained. The melting point, optical rotation, Tf Rf and elemental analysis values of Z-pGlu-His-OH are shown below.

Mp: 146 to 149 ° C. (decomposition) [α] _D : -0.91 (c = 1.0, DMF) Rf: 0.14 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₁₉ H ₂₀ N ₄ O as ₆₎ theory C: 57.00% H: 5.03% N: 13.99% measured value C: 57.23% H: 5.05% N: 14.05%

(1-8) Production of Z-pGlu-His-Trp-OMe 40.0 g of Z-pGlu-His-OH produced in the above (1-7), H-Trp-OMe
19.6 g of HCl and 12.7 g of HOSu were dissolved in 100 ml of DMF. Next, the solution was neutralized with N-methylmorpholine while cooling, and 2.0 ml of WSC was added, followed by stirring at 0 ° C. for 5 minutes and at room temperature overnight. After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure. 200 ml of ethyl acetate-n-butanol (1: 1) was added to the residue, and the organic layer was washed three times with 100 ml of water. The organic layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, 48.5 g (92.3% yield) of Z-pGlu-His-Trp-OMe was obtained. The melting point, optical rotation, Tf Rf and elemental analysis values of Z-pGlu-His-Trp-OMe are shown below.

Mp: 112-115 ° C. (decomposition) [α] _D : -1.5 (c = 1.0, DMF) Rf: 0.3 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₃₁ H ₃₂ N ₆ O ₇ ) Theoretical C: 61.99% H: 5.37% N: 13.99% Measured C: 61.54% H: 5.33% N: 13.80%

(1-9) Production of pGlu-His-Trp-OMe 40.0 g of Z-pGlu-His-Trp-OMe produced in the above (1-8) was added to DM
F. in 50 ml and hydrogenated in the presence of 500 mg of 10% palladium / carbon. After stirring at room temperature for 7 hours, the catalyst is removed and the mixture is concentrated under reduced pressure. The residue was solidified by treatment with ether.
Thus, 28.5 g of pGlu-His-Trp-OMe (91.8% yield)
was gotten. Melting point, optical rotation, TLC of pGlu-His-Trp-OMe
The Rf and elemental analysis values of are shown below.

Mp: 132 to 137 ° C. (decomposition) [α] _D : +4.1 (c = 1.0, DMF) Rf: 0.18 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₂₃ H ₂₆ N ₆ O ₅ ) Theoretical C: 59.22% H: 5.62% N: 18.02% Measured C: 59.01% H: 5.55% N: 17.89%

(1-10) H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pr
Production of o-NHEt Z-Ser-Tyr-D-Ser (But) -Leu-Arg-P produced in (1-6) above
93.0 g of ro-NHEt was dissolved in 800 ml of methanol, and hydrogen was added in the presence of 5.0 g of 10% palladium / carbon. After stirring at room temperature for 7 hours, the catalyst was removed and the mixture was concentrated under reduced pressure. The residue was solidified by treatment with ether. In this way H-Ser-Ty
74.0 g of rD-Ser (But) -Leu-Arg-Pro-NHEt were obtained (yield 90.3%). The melting point, optical rotation, Tf Rf and elemental analysis values of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt are shown below.

Mp: 124 to 128 ° C. (decomposition) [α] _D : +10.1 (c = 1.0, DMF) Rf: 0.16 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₃₈ H ₆₄ N ₁₀ O ₉ as) theory C: 56.70% H: 8.01% N: 17.40% measured value C: 56.55% H: 7.97% N: 17.18%

(1-11) pGlu-His-Trp-Ser-Tyr-D-Ser (But)
Production of -Leu-Arg-Pro-NHEt pGlu-His-Trp-OMe 30 g produced in (1-9) above and (1-9)
H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHE prepared in -10)
t22g was dissolved in butanol saturated water 1300ml. To the solution, add 78 mg of 1000 U / mg chymotrypsin to butanol-saturated water.
A solution dissolved in 2.5 ml was added, the pH was adjusted to 7.8, and the mixture was stirred at 10 ° C for 1 hour. 300 ml of n-butanol was added to the reaction mixture, and the partition chromatography of n-butanol (200 ml) and water was repeated. Then, with 100 ml of n-butanol, 5
Extracted times. Butanol was washed with water, the organic layer and the aqueous layer were separately concentrated, and each was solidified with ether. H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt recovered from the aqueous layer
Is used again for the reaction. The organic layer (n-butanol) was concentrated, and ether was added to the residue to make a powder. The powder obtained from the organic layer was dissolved in a 0.01 M ammonium acetate aqueous solution and injected onto a column (4 × 30 cm) filled with CM-cellulose. 0.01M ammonium acetate aqueous solution (pH 4.4) 5
00ml-0.1M ammonium acetate aqueous solution (pH4.4) 500ml
Was performed (60 ml / hour), and the eluate was fractionated and collected in 10 ml portions. The eluate was analyzed by high performance liquid chromatography, and the target fraction was collected and freeze-dried. Thus, pGlu-His-Trp-Ser-Tyr-D-Ser (Bu
22.4 g of t) -Leu-Arg-Pro-NHEt were obtained. pGlu-His-Trp-
The optical rotation, elemental analysis value, and amino acid analysis value of Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt are shown below.

[0053] _{[α] D: -46.0 (C} = 1, H 2 O) Elemental analysis _{(C 60 H 86 N 16 O} 13 · CH 3 as COOH · 2H ₂ O) theory C, 55.76% H, 7.09% N, 16.78% measured value C, 55.60% H, 7.10% N, 16.79% amino acid composition; Ser 2.05 (2), Glu 1.08 (1), Pro 0.99 (1), Leu 1.09 (1), Ty
r 0.92 (1), His 0.93 (1), Arg 1.06 (1)

[Embodiment 2] In this embodiment, Embodiment 1
ZD-Ser (But) -L manufactured according to (1-1) to (1-3)
pGlu-His-Trp-Ser was prepared in the same manner as in Example 1 except that eu-Arg-Pro-NHEt was produced according to the following (2-1) to (2-3).
-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt was produced.

(2-1) Production of Z-Leu-Arg-NHNH ₂ 172.5 g of Z-Leu-OH and 178.9 g of H-Arg-OEt.2HCl were added to DMF.
/ THF (1/1) mixed solution in 800 ml, and N-
The mixture was neutralized by adding 143 ml of methylmorpholine. Then D
147.5 ml of CC was added, and the mixture was stirred at 0 ° C for 5 minutes and at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure.
After adding 00 ml and washing twice with 500 ml of ethyl acetate, the aqueous layer was extracted three times with 1000 ml of butanol. The resulting butanol layer was concentrated under reduced pressure to obtain Z-Leu-Arg-OEt.

Z-Leu-Arg-OEt was dissolved in 500 ml of methanol, and 150 ml of NH ₂ NH ₂ .H ₂ O was added under ice-cooling.
Stirred for hours. The reaction solution was concentrated under reduced pressure, and water was added to the residue obtained.
500 ml was added, and the mixture was extracted five times with 1000 ml of butanol. The extract was washed four times with 500 ml of water and concentrated under reduced pressure. The resulting residue was solidified by treatment with ether and dried. Thus, 209.7 g (yield: 74.1%) of Z-Leu-Arg-NHNH ₂ was obtained. The melting point of _{Z-Leu-Arg-NHNH 2} , optical rotation, Rf value of TLC,
And the elemental analysis values are shown below.

Mp: 105-107 ° C. [α] _D : -30.6 (c = 1.0 MeOH) Rf value: 0.60 (BuOH / AcOH / H ₂ O = 4/1 /
5) Elemental analysis (C ₂₀ H ₃₃ N ₇ as O ₄₎ theory C: 55.61% H: 7.64% N: 22.51% measured value C: 55.84% H: 7.42% N: 22.41%

(2-2) Production of Z-Leu-Arg-Pro-NHEt 130.8 g of Z-Leu-Arg-NHNH ₂ produced in the above (2-1) was added to DM
F dissolved in 500ml, dry ice-ethanol -20 ℃
And cooled. To the solution, 4N HCl-dioxane 225 ml,
And 40.5 ml of isoamyl nitrite were added to obtain an azide compound. Further, 126 ml of triethylamine was added for neutralization. H-Pro-NHEt 42.6g DMF 200ml
The mixture was transferred to a solution, stirred at -20 ° C for 2 hours and at 4 ° C for 16 hours, and then concentrated. 800 ml of water was added to the residue, and ethyl acetate 4
After washing twice with 00 ml, the aqueous layer was washed with 800 ml of butanol.
Extracted once and washed with 400 ml of water. The obtained butanol layer was concentrated under reduced pressure. The resulting residue was solidified by treatment with ether and dried. Thus, Z-Leu-Arg-Pro-
154.5 g (94.3% yield) of NHEt was obtained. Z-Leu-Arg-Pro-NH
The melting point, optical rotation, Tf Rf value, and elemental analysis value of Et are shown below.

Mp: 125 to 126 ° C. [α] _D : -66.6 (c = 1.0 MeOH) Rf: 0.45 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₂₇ H ₄₃ N ₇ O as ₅₎ theory C: 59.43% H: 7.94% N: 17.97% measured value C: 59.31% H: 7.78% N: 18.13%

(2-3) Production of ZD-Ser (But) -Leu-Arg-Pro-NHEt 154.5 g of Z-Leu-Arg-NHEt produced in the above (2-2) was dissolved in 1500 ml of methanol. Hydrogen was added in the presence of 14 g of palladium / carbon. After stirring at room temperature for 16 hours, the catalyst was removed and concentrated under reduced pressure to obtain H-Leu-Arg-Pro-NHEt.

82.3 g of ZD-Ser (But) -OH was dissolved in 200 ml of THF. This was cooled to -20 ° C with dry ice-ethanol, 30.7 ml of N-methylmorpholine was added dropwise, and then 36.3 ml of isobutyl chloroformate was added dropwise. The mixture was stirred at -20 ° C for 1 minute and the corresponding mixed acid anhydride was added. Was manufactured. The obtained reaction solution was subjected to H-Leu-Arg-Pro-NHEt DM
The mixture was mixed with 700 ml of F solution and stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes. Thereafter, the mixture was concentrated under reduced pressure, 1000 ml of water was added to the residue, and the mixture was washed twice with 500 ml of ethyl acetate. The extract was washed 5 times with 500 ml of water, concentrated under reduced pressure, treated with ether to solidify, and dried.
In this way, D-Ser (But) -Leu-Arg-Pro-NHEt 178.3 g
(91.2% yield) was obtained. Incidentally, the melting point of the obtained compound,
Optical rotation, Rf of TLC, and elemental analysis values were as in (1-3) of Example 1.
And D-Ser (But) -Leu-Arg-Pro-NHEt.

Example 3 Production of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (3-1) Boc-D-Leu-Leu-Arg (Tos) -Pro Preparation of -NHEt Z-Leu-Arg (Tos) -Pro-NHEt prepared in (1-2) of Example 1
Dissolve 20.99g in methanol 200ml, 10% palladium /
Hydrogen was added in the presence of 1.5 g of carbon. After stirring at room temperature for 7 hours, the catalyst was removed and the mixture was concentrated under reduced pressure. The residue was solidified by treatment with ether. In this way, H-Leu-Arg (To
s) -Pro-NHEt was obtained.

H-Leu-Arg (Tos) -Pro-NHEt was converted to DMF 80m
l and 200 ml of THF. The solution was neutralized with N-methylmorpholine while cooling. Next, 8.86 g of Boc-D-Leu-OH and 23.02 g of HOSu were added to this solution for T times.
A solution dissolved in 200 ml of HF and 36.4 ml of WSC were added, and the mixture was stirred at 0 ° C. for 5 minutes and at room temperature overnight. After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure. 500 m of water in the residue
l, and extracted 5 times with 200 ml of butanol. After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, Boc-D-Leu-Leu-Ar
21.5 g (92.1% yield) of g (Tos) -Pro-NHEt was obtained. Boc
-D-Leu-Leu-Arg (Tos) -Pro-NHEt melting point, optical rotation, TL
The Rf and elemental analysis value of C are shown below.

Mp: 119-121 ° C. [α] _D : -45.2 (c = 1.0, MeOH) Rf: 0.50 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis (C ₃₀ H ₅₆ N) ₈ O ₆ ) Theoretical C: 57.67% H: 9.03% N: 17.93% Measured C: 57.43% H: 8.89% N: 17.80%

(3-2) Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro
Production of -NHEt Boc-D-Leu-Leu-Arg (Tos) -Pro-NHE produced in (3-1) above
25.8 g of t was dissolved in 10 ml of DCM. Under ice cooling, TFA 1
0 ml was added and the mixture was stirred at room temperature for 30 minutes. Then, the mixture was concentrated under reduced pressure, and the residue was solidified by adding 200 ml of ether and dried.
Thus, HD-Leu-Leu-Arg (Tos) -Pro-NHEt.TF
A got.

16.6 g of Z-Ser-Tyr-NHNH ₂ was dissolved in 100 ml of DMF and cooled to −20 ° C. with dry ice-ethanol. 29.8 ml of 4N HCl-dioxane and 5.3 ml of isoamyl nitrite were added to the solution to obtain an azide compound. Further, the mixed solution neutralized by adding 16.7 ml of triethylamine was added to HD-Leu-Leu-Arg (Tos) -Pro-NHEt.TFA DMF 20
The solution was transferred to a 0 ml solution, stirred at -20 ° C for 2 hours and at 4 ° C for 17 hours, and then concentrated. Add 500 ml of water to the residue, 200 ml of butanol
And extracted 5 times. After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro-NHEt 25.0
g (83.2% yield) was obtained. Z-Ser-Tyr-D-Leu-Leu-Arg
The melting point, optical rotation, Tf Rf and elemental analysis of (Tos) -Pro-NHEt are shown below.

Mp: 114 to 118 ° C. (decomposition) [α] _D : -30.1 (c = 1.0, DMF) Rf: 0.32 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₄₅ H ₆₈ N ₁₀ O ₁₀ ) Theoretical C: 59.45% H: 7.54% N: 15.41% Measured C: 59.33% H: 7.52% N: 15.32%

(3-3) H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt
Production of Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -P produced in (3-2) above
To 20.4 g of ro-NHEt, 32 ml of anisole was added, and the mixture was cooled to -70 ° C with dry ice-ethanol. 200ml HF
Was added and stirred at 0 ° C. for 1 hour. Then, concentrated under reduced pressure,
The residue was treated with ether, and the precipitated product was collected by filtration and dried in vacuo over sodium hydroxide. In this way, H-
16.0 g (92.4% yield) of Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt was obtained. Melting point of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt,
The optical rotation, Rf of TLC, and elemental analysis values are shown below.

Mp: 121-123 ° C. [α] _D : +11.0 (c = 1.0, DMF) Rf: 0.20 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis (C ₃₇ H ₆₂ N) ₁₀ as O ₈₎ theory C: 57.35% H: 8.06% N: 18.07% measured value C: 57.21% H: 8.01% N: 18.10%

(3-4) pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-A
Production of rg-Pro-NHEt Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt,
H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NH produced in (3-3) above
PGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHE by the same method as (1-11) in Example 1 except that Et 1.30 g was used.
811 mg were obtained. pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg
The optical rotation, elemental analysis value and amino acid analysis value of -Pro-NHEt are shown below.

[Α] _D : -32.0 (C = 0.52, 5% AcOH) Elemental analysis (as C ₅₉ H ₈₄ N ₁₆ O ₁₂ ) Theoretical value C: 58.59% H: 7.00% N: 18.53% Measured value C: 58.40% H: 7.10% N: 18.31% Amino acid composition; Ser 1.05 (1), Glu 1.08 (1), Pro 0.99 (1),
Leu 2.09 (2), Tyr 0.92 (1), His 0.93 (1), Arg 1.06 (1)

Example 4 pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-
Production of NH ₂ (4-1) Production of Z-Pro-Azgly-NH ₂ 24.9 g of Z-Pro-OH, 11.2 g of semicarbazide hydrochloride and 14.5 ml of triethylamine were dissolved in 200 ml of DMF. 20.6 g of DCC was added to the solution under cooling to 0 ° C., and the mixture was stirred at 4 ° C. for 16 hours. Thereafter, dicyclohexylurea (DCU) was removed from the reaction mixture by filtration and concentrated under reduced pressure. 500 ml of ethyl acetate was added to the residue, and the mixture was washed twice with 200 ml of water. After concentration under reduced pressure, the residue was treated with ether and solidified.
Thus, 16.7 g of Z-Pro-Azgly-NH ₂ (yield 54.3%)
was gotten. Melting point, optical rotation, TLC of Z-Pro-Azgly-NH ₂
The Rf and elemental analysis values of are shown below.

Mp: 189 to 190 ° C. [α] _D : -43.6 (c = 1.4, DMF) Rf: 0.62 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₁₄ H ₁₈ N) ₄ O ₄ ) Theoretical C: 54.89% H: 5.95% N: 18.29% Measured C: 54.41% H: 7.74% N: 15.60%

[0074] (4-2) Boc-Arg (NO 2) was prepared in -Pro-Azgly-NH ₂ of manufacturing the (4-1) Z-Pro-Azgly -NH 2 16.5g of DMF
It was dissolved in 500 ml and hydrogen was added in the presence of 1.1 g of 10% palladium / carbon. After stirring at room temperature for 7 hours, the catalyst was removed and the mixture was concentrated under reduced pressure. The residue was solidified by adding 1500 ml of ether and dried. Thus, H-Pro-Azgly-NH ₂ .TFA was obtained.

13.5 g of Boc-Arg (NO ₂ ) -OH was dissolved in 100 ml of THF, and cooled to -20 ° C. with dry ice-ethanol.
To this, 3.3 ml of N-methylmorpholine was added dropwise, followed by 3.96 ml of isobutyl chloroformate.
The mixture was stirred at -20 ° C for 1 minute to produce the corresponding mixed anhydride. The obtained reaction solution was washed with H-Pro-Azgly-NH ₂ TFA in DMF.
200 ml solution (neutralized with N-methylmorpholine)
After stirring at 0 ° C. for 5 minutes and at room temperature for 30 minutes, the mixture was concentrated under reduced pressure. Add 500 ml of water to the residue and add 200 ml of butanol
And extracted 5 times. After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. In this way, Boc-Arg (NO ₂ ) -Pro-Azgly-NH ₂ 16.7 g (88.
6%). The melting point, optical rotation, Tf Rf and elemental analysis values of Boc-Arg (NO ₂ ) -Pro-Azgly-NH ₂ are shown below.

Mp: 135-137 ° C. (decomposition) [α] _D : + 35.3 ° (c = 1.0, DMF) Rf: 0.49 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value ( (As C ₁₇ H ₃₁ N ₉ O ₇ ) Theoretical C: 43.12% H: 6.60% N: 26.62% Measured C: 54.41% H: 7.74% N: 15.60%

(4-3) Production of Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH ₂ 110.54 g of Boc-Arg (NO ₂ ) -Pro-NHEt produced in the above (4-2)
Was dissolved in 150 ml of DCM. Add T
150 ml of FA was added, and the mixture was stirred at room temperature for 30 minutes. Then, the reaction mixture was concentrated under reduced pressure, and the residue was solidified by adding 1500 ml of ether and dried. Thus, H-Arg (NO ₂ ) -Pro
-NHEt • TFA was obtained.

H-Arg (NO ₂ ) -Pro-NHEt · TFA was added to DMF 80m
l and THF in 200 ml of a mixed solvent. The solution was neutralized with N-methylmorpholine while cooling. Then, 53.06 g (0.2 mole) of Z-Leu-OH and HOS
A solution of 23.02 g (0.22 mole) of u in 200 ml of THF and 36.4 ml (0.2 mole) of WSC were added, and the mixture was stirred at 0 ° C. for 5 minutes and at room temperature overnight. After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure. 1500 ml of ethyl acetate was added to the residue, and the mixture was washed twice with 500 ml of water and twice with 500 ml of saturated saline.
Thereafter, the ethyl acetate layer was concentrated under reduced pressure, the residue was treated with ether, solidified and dried. In this way, Z-Leu-Arg
27.52 g (98.6% yield) of (NO ₂ ) -Pro-Azgly-NH ₂ were obtained. _{Z-Leu-Arg (NO 2} ) -Pro-Azgly-NH 2 of the melting point, optical rotation,
The Rf and elemental analysis values of TLC are shown below.

Mp: 88-90 ° C. [α] _D : -30.2 (c = 1.5, DMF) Rf: 0.57 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₂₆ H ₄₀ N) ₁₀ as O ₈₎ theory C: 50.31% H: 6.50% N: 22.57% measured value C: 50.24% H: 6.41% N: 22.45%

(4-4) ZD-Ser (But) -Leu-Arg-Pro-Azgly-NH
Production of ₂ Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH ₂ 1 produced in (4-3) above
Dissolve 6.5 g in 500 ml of DMF and add 10% palladium / carbon 1.
Hydrogen was added in the presence of 1 g. After stirring at room temperature for 7 hours, the catalyst was removed and the mixture was concentrated under reduced pressure. Ether 1500 in the residue
The mixture was solidified and dried. Thus, H-Leu-Arg-
Pro-Azgly-NH ₂ was obtained.

8.86 g of ZD-Ser (But) -OH was dissolved in 100 ml of THF and cooled to -20 ° C. with dry ice-ethanol. To this solution, 3.3 ml of N-methylmorpholine was added dropwise, and then 3.96 ml of isobutyl chloroformate was added dropwise, followed by stirring at -20 ° C for 1 minute to produce the corresponding mixed acid anhydride. The obtained reaction solution was H-Leu-Arg-Pro-Azgly-
It was mixed with 200 ml of a solution of NH ₂ in DMF (neutralized with N-methylmorpholine), stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes, and concentrated under reduced pressure. 500 ml of water was added to the residue, and the mixture was extracted five times with 200 ml of butanol. After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. Thus, ZD-Ser (But) -Leu-Arg-Pro-Azgly
16.9 g (yield 78.3%) of -NH ₂ was obtained. ZD-Ser (But) -L
The melting point, optical rotation, Tf Rf and elemental analysis values of eu-Arg-Pro-Azgly-NH ₂ are shown below.

Mp: 115-118 ° C. [α] _D : -45.8 (c = 1.0, DMF) Rf: 0.51 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₃₃ H ₅₄ N) ₁₀ as O ₈₎ theory C: 55.14% H: 7.57% N: 19.48% measured value C: 55.01% H: 7.42% N: 19.33%

(4-5) Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-
Production of Azgly-NH ₂ ZD-Ser (But) -Leu-Arg-Pro-Azgly produced in (4-4) above
26.7 g of -NH ₂ was dissolved in 500 ml of methanol, and hydrogen was added in the presence of 1.9 g of 10% palladium / carbon. After stirring at room temperature for 7 hours, the catalyst was removed and concentrated under reduced pressure. The residue was treated with ether, and the precipitated product was collected by filtration and dried over sodium hydroxide in vacuo. In this way, HD-Ser (Bu
t) were obtained -Leu-Arg-Pro-Azgly- NH 2.

Z-Ser-Tyr-NH prepared in (1-5) of Example 1
18.5 g of NH ₂ was dissolved in 150 ml of DMF and cooled to -20 ° C with dry ice-ethanol. 4N-HCl in the solution
-33.4 ml of dioxane and 6.0 ml of isoamyl nitrite were added to form an azide compound. Further triethylamine 1
8.7 ml were added for neutralization. HD-Ser (Bu
t) -Leu-Arg-Pro-Azgly-NH ₂ transferred to a DMF 150 ml solution,
After stirring at -20 ° C for 2 hours and 4 ° C for 17 hours, the mixture was concentrated. 500 ml of water was added to the residue, and the mixture was extracted five times with 200 ml of butanol.
After washing five times with water, the butanol layer was concentrated under reduced pressure, and the residue was treated with ether and solidified. In this way, Z-Ser-Ty
rD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ 28.3 g (yield 78.
6%). Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-A
The melting point, optical rotation, Tf Rf and elemental analysis of zgly-NH ₂ are shown below.

Mp: 110-113 ° C. (decomposition) [α] _D : -28.2 (c = 1.0, DMF) Rf: 0.36 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₄₅ H ₆₈ N ₁₂ O ₁₂ ) Theoretical C: 55.77% H: 7.07% N: 17.34% Measured C: 55.64% H: 7.01% N: 17.29%

(4-6) H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-
Production of Azgly-NH ₂ Z-Ser-Tyr-D-Ser (But) -Leu-Arg-P produced in (4-5) above
Dissolve 25.4 g of ro-Azgly-NH ₂ in 500 ml of methanol and add 10%
Hydrogen was added in the presence of 1.3 g of palladium / carbon. 7
After stirring at room temperature for an hour, the catalyst was removed and the mixture was concentrated under reduced pressure.
The residue was solidified by treatment with ether. In this way,
H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ 20.7 g
(94.6% yield) was obtained. H-Ser-Tyr-D-Ser (But) -Leu-
The melting point, optical rotation, Tf Rf and elemental analysis values of Arg-Pro-Azgly-NH ₂ are shown below.

Mp: 120 to 122 ° C. [α] _D : +11.2 (c = 1.0, DMF) Rf: 0.31 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₃₇ H ₆₂ N) ₁₂ O ₁₀ as a) theory C: 53.22% H: 7.48% N: 20.13% measured value C: 53.19% H: 7.43% N: 20.01%

(4-7) pGlu-His-Trp-
Ser-Tyr-D-Ser (But) -Leu-Ar
Preparation of g-Pro-Azgly-NH ₂ Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt,
H-Ser-Tyr-D-Ser (But) -Leu-Arg-P produced in (4-6) above
Except that 1.30 g of ro-Azgly-NH ₂ was used, pGlu-His-Trp-Ser-Tyr-D-Ser (But)-was obtained in the same manner as in (1-11) of Example 1.
812 mg of Leu-Arg-Pro-Azgly-NH ₂ were obtained. pGlu-His-Trp-S
er-Tyr-D-Ser ( But) optical rotation of -Leu-Arg-Pro-Azgly- NH 2,
Elemental analysis values and amino acid analysis values are shown below.

[Α] _D : -52.4 (C = 1.0, DMF) Elemental analysis (as C ₅₉ H ₈₄ N ₁₈ O ₁₄ ) Theoretical value C: 55.82% H: 6.67% N: 19.86% Measured value C: 55.70% H: 6.55% N: 19.72% Amino acid composition; Ser 2.02 (2), Glu 1.07 (1), Pro 1.00 (1),
Leu 1.03 (1), Tyr 0.92 (1), His 0.94 (1), Arg 1.02 (1)

Example 5 Preparation of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂
Concrete (5-1) was subjected to solid phase synthesis using H-Ser-Tyr-D- Trp-Leu-Arg-Pro-Gly-NH Milligen Bioreserch Inc. Peptide Synthesizer 9600 as _second manufacturing solid-phase synthesizer. First,
694 mg of p-methylbenzhydrylamine (MBHA) resin (manufactured by Peptide Research Laboratories, amino group 0.72 mmol / g) is placed in a reaction vessel for solid phase peptide synthesis, and 8 ml of DCM (4 times, 1 each)
Min), 8 ml of DCM solution containing 60% TFA (20 min), D
DMF containing 4 ml of CM (3 times, 15 seconds each) and 1 ml of DIEA
The solution was treated in the order of 3 ml of the solution (2 times, 1 minute each) and 8 ml of DMF (6 times, 40 seconds each) in a stream of argon gas under stirring. In addition, filtration was performed for each treatment.

On the other hand, pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-A
Boc-Gly-OH 2 mmole corresponding to the tenth amino acid residue in the amino acid sequence of rg-Pro-Gly-NH ₂ was dissolved in 4 ml of DCM. This solution is placed in an amino acid activation vessel and DCC
(0.5M-DCM solution) 3ml and HOBt (0.5M-D
(CM solution) 4 ml was added and reacted for 30 minutes. The reaction was filtered and transferred to a concentration vessel. Add 3ml of DMF to this,
DCM was distilled off under a stream of argon gas. Then DMF
3 ml was added, and the mixture was transferred to the above-described reaction vessel for solid phase peptide synthesis and reacted for 30 minutes. It was then washed with 8 ml of DCM (6
Times for 20 seconds each). Further, 2 mmol of Boc-Gly-OH was added to DC
M 4 ml, and a so-called double coupling method in which the same operation was repeated in an amino acid activation reaction vessel was performed, followed by filtration to obtain a Boc-Gly-MBHA resin.

Next, the obtained Boc-Gly-MBHA resin was washed with 8 ml of DCM (4 times, 1 minute each) and filtered.
To this, 8 ml of a DCM solution containing 60% TFA (for 20 minutes), D
DMF containing 4 ml of CM (3 times, 15 seconds each) and 1 ml of DIEA
3 ml of solution (2 times, 1 minute each), 8 ml of DMF (6 times, 40 minutes each)
For a second) in the order of argon gas stream, with stirring,
In addition, filtration was performed for each treatment.

Further, pGlu-His-Trp-Ser-Tyr-D-Trp-Leu
Corresponding to the ninth amino acid residues of the amino acid sequence of _{-Arg-Pro-Gly-NH 2} Boc-Pro-OH 2mmole the DCM 4
dissolved in DCC (0.5M-
(DCM solution) (1.5 ml) and reacted for 7 minutes. Thereafter, the reaction mixture was filtered and transferred to a concentration vessel, where the DM
3 ml of F was added, and DCM was distilled off under a stream of argon gas.
To this, 3 ml of DMF was added, transferred to the reaction vessel for peptide solid phase synthesis described above, and reacted for 30 minutes. It was then washed with 8 ml of DCM (6 times, 20 seconds each), filtered and Boc-Pro-G
A ly-MBHA resin was obtained. Hereinafter, the 8th to 4th amino acids were sequentially coupled using the amino group-protected amino acids shown below.

Amino-protected amino acid Amount used Acid sequence (mmol) 8 Boc-Arg (Tos) -OH 2 × 27 Boc-Leu-OH 26 Boc-D-Trp-OH 25 Boc-Tyr (Bzl) -OH 24 Boc-Ser (Bzl) -OH 2 In the above solid phase synthesis, double coupling was performed when Arg was used. Thus, the protected peptide-M
BHA resin, Boc-Ser (Bzl) -Tyr (Bzl) -D-Trp-Leu-Arg (To
2.76 g of s) -Pro-Gly-MBHA resin was obtained. 5 ml of anisole was added to 2.76 g of the protected peptide-MBHA resin, and 25 ml of anhydrous hydrogen fluoride was further added thereto, followed by stirring at 0 ° C. for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ether. Thus, H-Ser-Tyr-D-Tr
130 g of p-Leu-Arg-Pro-Gly-NH ₂ were obtained.

The optical rotation of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ , Rf of TLC, and elemental analysis values are shown below. [α] _D : -50.4 (c = 1.0, MeOH) Rf: 0.13 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (C ₅₉ H ₈₄ N ₁₆ O ₁₂ .AcOH.2H ₂ O As) Theoretical value C: 54.31% H: 7.04% N: 17.27% Measurement value C: 54.20% H: 6.89% N: 16.97%

(5-2) pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Ar
Production of g-Pro-Gly-NH ₂ Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt,
H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gl produced in (5-1) above
pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro by the same method as in (1-11) of Example 1 except that 1.30 g of y-NH ₂ was used.
It was obtained -Gly-NH ₂ 808mg. pGlu-His-Trp-Ser-Tyr-D-Trp-L
The optical rotation, elemental analysis value, and amino acid analysis value of eu-Arg-Pro-Gly-NH ₂ are shown below.

[Α] _D : -56.6 (C = 1.0, H ₂ O) Elemental analysis (as C ₆₄ H ₈₂ N ₁₈ O ₁₃ .AcOH.2H ₂ O) Theoretical value C: 56.32% H: 6.44% N: 17.91% measured value C: 56.20% H: 6.38% N: 16.97% amino acid composition; Ser 0.98 (1), Glu 1.07 (1), Gly 1.01 (1),
Pro 0.99 (1), Leu 1.00 (1), Tyr 0.92 (1), His 0.95 (1), Ar
g 1.08 (1)

Example 6 pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pr
Production of o-Gly-NH ₂ (6-1) H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly
Preparation of -NH ₂ Example 5 was repeated except that Boc-D- (2-naphthyl) -D-Ala-OH was used instead of Boc-D-Trp-OH corresponding to the sixth amino acid residue. The same treatment as in 5-1) was performed to obtain 0.98 g of H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ . HS
The optical rotation, Tf Rf and elemental analysis of er-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ are shown below.

[Α] _D : -44.1 (C = 1.0, MeOH) Rf: 0.15 Elemental analysis value (as C ₄₇ H ₆₆ N ₁₂ O ₁₀ ) Theoretical value C: 58.86% H: 6.94% N: 17.52% Measurement value C: 58.72% H: 6.88% N: 17.49%

(6-2) Production of pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ H-Ser-Tyr-D-Ser ( But) -Leu-Arg-Pro-NHEt
H-Ser-Tyr- (2-naphthyl) -D-Ala-L produced in (6-1) above
Example 1 was repeated except that 1.30 g of eu-Arg-Pro-Gly-NH ₂ was used.
By the same method as (1-11), pGlu-His-Trp-Ser-Tyr- (2-
802 mg of (naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ were obtained.
pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pr
The optical rotation, elemental analysis value, and amino acid analysis value of o-Gly-NH ₂ are shown below.

[Α] _D : -50.3 (C = 1.0, H ₂ O) Elemental analysis (as C ₆₉ H ₈₈ N ₁₈ O ₁₄ ) Theoretical value C: 59.47% H: 6.37% N: 18.09% Measured value C: 59.40% H: 6.33% N: 17.98% Amino acid composition; Ser 0.98 (1), Glu 1.05 (1), Gly 1.00 (1),
Pro 1.03 (1), Leu 1.00 (1), Tyr 0.93 (1), His 0.94 (1), A
rg 1.07 (1)

Example 7 Production of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (7-1) H-Ser-Tyr-Gly-Leu-Arg-Pro Production of -Gly-NH ₂ The same treatment as (5-1) of Example 5 except that Boc-Gly-OH was used instead of Boc-D-Trp-OH corresponding to the sixth amino acid residue. H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ 1.
12 g were obtained. Showing optical rotation of H-Ser-Tyr-Gly- Leu-Arg-Pro-Gly-NH 2, the Rf and elemental analysis values of TLC below.

[Α] _D : -40.1 (c = 1.0, MeOH) Rf: 0.10 (BuOH / AcOH / H ₂ O = 4/1/5) Elemental analysis value (as C ₃₃ H ₅₃ N ₁₁ O ₉ ) Theory Value C: 53.00% H: 7.14% N: 20.60% Measured value C: 53.20% H: 6.89% N: 20.97%

(7-2) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg
Preparation of -Pro-Gly-NH ₂ Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt,
H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- produced in (6-1) above
Except that 1.30 g of NH ₂ was used, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly was obtained in the same manner as in (1-11) of Example 1.
To obtain a -NH ₂ 811mg. pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg
Elemental analysis values and amino acid analysis values of -Pro-Gly-NH ₂ are shown below.

Elemental analysis (as C ₅₅ H ₇₅ N ₁₇ O ₁₃ .AcOH.2H ₂ O) Theoretical value C: 53.55% H: 6.54% N: 18.63% Measurement value C: 53.85% H: 6.89% N: 18.97% Amino acid composition; Ser 1.01 (1), Glu 1.03 (1), Gly 2.01 (2),
Pro 0.98 (1), Leu 1.01 (1), Tyr 0.94 (1), His 0.94 (1), A
rg 1.08 (1)

[0106]

The method of the present invention utilizes an enzymatic reaction and does not involve side reactions such as racemization, so that the LH-RH derivative can be easily separated and purified, and the yield is high.
Since unreacted peptide fragments can be recovered and reused, they are extremely useful industrially.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Kinya Tomisaki 1-2-2 Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki Pref. Central Research Laboratory of Itoham Co., Ltd. (72) Akiko Ohata Kubo, Moriya-cho, Kitasoma-gun, Ibaraki 1-2 Kagaoka Ito Ham Co., Ltd. Central Research Laboratory (56) References Tetrahedron Letters, Vol. 33, No. 20, p. 2799 -2802 (1992)

Claims (8)

(57) [Claims] 1. A peptide fragment represented by the general formula (1): pGlu-His-Trp-OR ¹ (1) (wherein R ¹ represents lower alkyl), and a general formula (2): H -Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X is D-Leu, D-Ser (But), D-Trp, (2-naphthyl)
Represents an amino acid selected from the group consisting of -D-Ala and Gly, and Y represents Gly-NH ₂ , azaglycine or NHR ² (R ² is lower alkyl). ) In the presence of chymotrypsin in a reaction medium obtained by mixing butanol with water or a buffer solution, without adding KCl to the reaction medium, at 0 to 50 ° C. Wherein the reaction is carried out at a temperature in a range of: General formula (3): pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (wherein X and Y are as defined above) The method for producing LH-RH and / or a derivative thereof represented by the formula: 2. The method for producing LH-RH and / or a derivative thereof according to claim 1, further comprising a step of purifying the peptide of the general formula (3) by partition chromatography. 3. The LH-R according to claim 1, wherein R ¹ is an alkyl group having 1 to 3 carbon atoms.
A method for producing H and / or a derivative thereof. 4. The method for producing LH-RH and / or a derivative thereof according to claim 1, wherein R ² is an alkyl group having 1 to 3 carbon atoms. 5. A peptide fragment represented by the general formula (1): pGlu-His-Trp-OR ¹ (1) (wherein R ¹ represents lower alkyl), and a general formula (2): H -Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X is D-Leu, D-Ser (But), D-Trp, (2-naphthyl)
Represents an amino acid selected from the group consisting of -D-Ala and Gly, and Y represents Gly-NH ₂ , azaglycine or NHR ² (R ² is lower alkyl). ) In the presence of immobilized enzyme immobilized chymotrypsin in the reaction medium of butanol and water or buffer, without adding KCl to the reaction medium Wherein the reaction is carried out at a temperature in the range of 0 to 50 ° C., wherein pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) , X and Y are as defined above.) A method for producing LH-RH and / or a derivative thereof represented by the formula: 6. The method for producing LH-RH and / or a derivative thereof according to claim 5, further comprising a step of purifying the peptide of the general formula (3) by partition chromatography. 7. The LH-R according to claim 5, wherein said R ¹ is an alkyl group having 1 to 3 carbon atoms.
A method for producing H and / or a derivative thereof. 8. The method for producing LH-RH and / or a derivative thereof according to claim 5, wherein R ² is an alkyl group having 1 to 3 carbon atoms.