JPH07118293A - Production of polypeptide - Google Patents

Abstract

PURPOSE:To easily obtain a polypeptide useful as e.g. a medicine for osteoporosis, etc., in a high yield using a small amount of solvent by cyclizing a specific peptide bound to an oxime resin along with eliminating the resin followed by, if needed, condensation with an amino acid or peptide. CONSTITUTION:A peptide, covalently bound to an oxime resin, of formula I (X is Val, such a peptide that, as shown in formula IV, a sequence of arbitrary number of amino acid from the Val at 7th order to the Pro at 31st order are mutually bound in succession with the Val as N-terminal, or (protected) OH; R is oxime resin; Ser and Thr may have protecting groups, respectively) or of formula II is subjected to intramolecular condensation to effect cyclization, along with eliminating the oxime resin, thus obtaining a cyclic peptide of formula III, followed by, if needed, condensation of an amino acid or peptide at the site after eliminating said oxime resin, and then elimination of the protecting group, thus obtaining the polypeptide of the formula IV.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a polypeptide represented by formula (1) or an acid addition salt thereof or a complex thereof, and more specifically, a polypeptide represented by formula (1) or acid addition thereof. The present invention relates to a method for producing a salt or a complex thereof by a solid phase synthesis method.

[0002]

[Chemical 4]

[0003]

The polypeptide represented by the above formula (1) has potent serum calcium and phosphorus lowering activity,
It is known to have excellent pharmacological actions such as bone formation promoting action, bone resorption suppressing action, and urinary phosphorus excretion promoting action,
For example, it is used as a prophylactic or therapeutic drug for hypercalcemia associated with abnormal calcium metabolism, Paget's disease of bone, osteoporosis, bone fracture, osteomalacia, rickets and the like.

As a method for producing the above-mentioned polypeptide, a liquid phase synthesis method (for example, a special method) in which a peptide fragment having a partial sequence corresponding to the polypeptide is formed in a liquid phase and each fragment is further coupled in the liquid phase Japanese Patent Publication No. 53-41677) is known. However, in the liquid-phase synthesis method, the solubility changes slightly as the number of amino acids increases, and it becomes increasingly difficult to find a suitable solvent, and the difficulty of separating it from unreacted products and by-products also increases. . Especially in the cyclization reaction, since the synthesis is performed while diluting with a solvent in order to suppress the formation of by-products as much as possible, a large excess of a solvent with a high boiling point and toxicity is used, and it is impossible to recover the raw materials. As a result, there is a drawback that the treatment after the reaction is difficult and uneconomical.

[0005]

Therefore, an object of the present invention is to provide a method for producing the polypeptide represented by the formula (1), which does not have the above-mentioned drawbacks.

[0006]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention have achieved the above-mentioned object by simultaneously performing a cyclization reaction of a peptide and a elimination reaction of the peptide from a resin by a solid phase synthesis method. The inventors have found that the above can be achieved and completed the present invention. That is, the present invention is a method for producing a polypeptide represented by formula (1) or an acid addition salt thereof or a complex thereof,

[0007]

[Chemical 5]

Covalent bond to the oxime resin represented by any of the formulas (2-1), (2-2), (2-3), (2-4), (2-5) and (2-6). The peptide is cyclized by intramolecular condensation, and the peptide is eliminated from the oxime resin,
(3) producing the cyclic peptide, optionally comprising the step of condensing an amino acid or peptide at the site eliminated from the oxime resin

[0009]

[Chemical 6]

(In the formula, X is Val and the seventh Val in the formula (1) is
To the 31st Pro in order from the N-terminal Val to a peptide to which an arbitrary number of consecutive amino acids are bound or an optionally protected hydroxyl group, R represents an oxime resin, and Ser and Thr are protective groups. May have. )

[0011]

[Chemical 7]

(In the formula, X is Val, and the seventh Val in formula (1) is
From the 31st to the 31st Pro, which represents a peptide having an arbitrary number of consecutive amino acids bounded to Val at the N-terminus or an optionally protected hydroxyl group, and Ser and
Thr may have a protecting group. ) Is provided.

In the present specification, amino acids, peptides,
When abbreviating symbols for protecting groups, solvents, etc., IUPA
It shall be in accordance with the provisions of C, IUB or the symbols conventionally used in the relevant field, and examples are given below. However, when an amino acid and the like may have optical isomers, the L form is shown unless otherwise specified. Tyr Tyrosine residue Ile Isoleucine residue Gly Glycine residue Ser Serine residue Arg Arginine residue Asp Aspartic acid residue Lys Lysine residue Pro Proline residue Leu Leucine residue Thr Threonine residue Lys Lysine residue Glu Glutamic acid residue Gln Glutamine residue Val Valine residue Asn Asparagine residue His Histidine residue Ala Alanine residue Met Methionine residue Asu Aminosuberic acid residue Boc t-butoxycarbonyl Fmoc 9-fluorenylmethyloxycarbonyl Bz benzyl Z benzyloxycarbonyl Tos Tosyl OMe methyl ester OBz benzyl ester OBut t-butyl ester But t-butyl Bom benzyloxymethyl ONP p-nitrobenzyl ester OS u N-hydroxysuccinimide ester TFA trifluoroacetic acid THF tetrahydrofuran DMF dimethylformamide DCC dicyclohexylcarbodiimide WSC N-ethyl-N'-dimethylaminopropyl-carbodiimide Bop benzotriazolyl-N-hydroxytrisaminophosphonyl hexafluorophosphide Salt HOSu N-Hydroxysuccinimide HOBt 1-Hydroxybenzotriazol DCM DCM Dichloromethane MeOH Methanol EtOH Ethanol AcOH Acetic Acid DIPEA N, N-Diisopropylethylamine First, in any of formulas (2-1) to (2-6) Prepare the peptide covalently bound to the indicated oxime resin.

[0014]

[Chemical 8]

(In the formula, X is Val, and the seventh Val of the formula (1) is Val.
To the 31st Pro in order from the N-terminal Val to a peptide to which an arbitrary number of consecutive amino acids are bound or an optionally protected hydroxyl group, R represents an oxime resin, and Ser and Thr are protective groups. May have. ) X in formulas (2-1) to (2-6) is an arbitrary number of consecutive amino acids with Val and Val from the 7th Val in formula (1) to the 31st Pro in order as the N-terminal. Representing a bound peptide or an optionally protected hydroxyl group, when X is a peptide, the amino acids that make up this peptide may have a protecting group. When X is a hydroxyl group, an alkyl group (eg, methyl, ethyl, propyl, butyl, tert-butyl, etc.), benzyl group, p-nitrobenzyl group, p-chlorobenzyl group, benzhydryl group, etc. May be protected by.

R in the formulas (2-1) to (2-6) represents an oxime resin. Oxime resin was used in 1980 by De Grade and Kaiser (J.Org.
Chem., 45,1295), which allows easy introduction of amino acids into oxime resins, and
It has an advantage that the introduced amino acid can be easily removed by a base. Further, the oxime resin itself has a function as an active ester. The introduction of the amino acid into the oxime resin is preferably carried out in a nonpolar solvent such as DCM, and the coupling of the second and subsequent residues is preferably carried out in a DMF solvent. The amount of amino acid introduced into the oxime resin is preferably as small as possible, and the amount of amino acid introduced per 1 g of oxime resin is preferably 0.1 mmole to 0.5 mmole.

Examples of the protecting groups for Ser and Thr in formulas (2-1) to (2-6) include a benzyl group, a tert-butyl ether group, a tetrahydropyranyl group and a tritylsilyl group. A benzyl group is preferred. Formula (2-1)
The peptide covalently bound to the oxime resin represented by any one of (2-6) is a so-called stepwise method of sequentially condensing amino acids according to a method for producing a peptide by a known solid-phase synthesis method, or several moieties. It can be produced by a method of coupling peptide fragments. In the solid phase synthesis of this partial peptide fragment, a resin other than oxime resin, for example,
Chlormethyl resin, oxymethyl resin, aminomethyl resin, benzhydrylamine resin, methylbenzhydryl resin, 4-aminomethylphenoxymethyl resin, 4-hydroxymethylphenoxymethyl resin, 4-oxymethylphenylacetamidomethyl resin, etc. Although it can be used, when Fmoc is used as a protecting group for the α-amino group, it is preferable to use one that can be eliminated from the resin with trifluoroacetic acid such as 4-hydroxymethylphenoxymethyl resin. When used, it is preferable to use 4-oxymethylphenylacetamide methyl resin or the like that can be eliminated from the resin with hydrogen fluoride or the like.

Alternatively, a peptide fragment produced by a known liquid phase synthesis method is covalently bonded to an oxime resin to thereby covalently bond the oxime resin represented by any one of the formulas (2-1) to (2-6). Peptides may be produced. The peptide bond-forming reaction in peptide synthesis can be carried out using a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole or a condensing agent such as tetraethylpyrophosphate or Bop.
Further, in peptide synthesis, it is preferable that functional groups that should not participate in the reaction in the amino acid be protected by ordinary protecting groups and that functional groups that should participate in the reaction be activated. The protection and activation of the functional groups in these amino acids can be performed according to a known method, and the reagents and the like used at that time can be appropriately selected from the known ones. Examples of the amino-protecting group include carbobenzoxy (hereinafter Z
Abbreviated), tert-butyloxycarbonyl (hereinafter abbreviated as Boc), tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl,
Examples include 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyldiphenylphosphinothioyl oil, and the like. Examples of the protective group for the carboxyl group include an alkyl ester (eg, methyl,
Ester groups such as ethyl, propyl, butyl, tert-butyl), benzyl ester, p-nitrobenzyl ester, p-chlorobenzyl ester, benzhydryl ester, carbobenzoxyhydrazide, tert-butyloxycarbonylhydrazide, tritylhydrazide, etc. can give. As the hydroxyl-protecting group, a benzyl group,
Examples thereof include a tert-butyl ether group, a tetrahydropyranyl group and a tritylsilyl group.

The carboxyl group in the amino acid is, for example, the corresponding acid chloride, acid anhydride or mixed acid anhydride, azide, active ester (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenzide). Triazole, N-hydroxy-5-norbornene
It can be activated by forming an ester with -2,3-dicarboximide or the like).

Then, the formulas (2-1), (2-2), (2-3), (2-4),
The peptide covalently bound to the oxime resin represented by any of (2-5) and (2-6) is cyclized by intramolecular condensation and the peptide is eliminated from the oxime resin to give the compound represented by the formula (3). Produce cyclic peptides that are

[0021]

[Chemical 9]

(In the formula, X is Val and the seventh Val in the formula (1) is
From the 31st to the 31st Pro, which represents a peptide having an arbitrary number of consecutive amino acids bounded to Val at the N-terminus or an optionally protected hydroxyl group, and Ser and
Thr may have a protecting group. ) By carrying out the peptide cyclization reaction on the resin in this manner, the amount of by-products such as dimer cyclized product is reduced, and the desired monomer cyclized product is obtained in high yield. In addition, purification of the target cyclized monomer becomes easy.

The above cyclization reaction and peptide elimination reaction can be carried out using a tertiary amine. As the tertiary amine, N, N-diisopropylethylamine, N, N-diisopropylpentylamine, triethylamine, N, N-diisopropylbutylamine, trimethylamine, N, N-diisopropylpropylamine, N-methylmorpholine and the like can be used. it can. Among these, N, N-diisopropylethylamine and N, N-diisopropylpentylamine are preferable. Further, lower fatty acid may be added to the tertiary amine as described above. The amount of the lower fatty acid added is 0.5 to 5 times the molar amount of the tertiary amine, preferably the equimolar amount. Examples of the lower fatty acid include acetic acid, propionic acid and butyric acid, of which acetic acid is preferable. The above cyclization reaction and peptide elimination reaction are represented by the formula (2-1), (2-
2), (2-3), (2-4), after removing the N-terminal protecting group of the peptide covalently bound to the oxime resin represented by (2-5) and (2-6) Add a solution containing a tertiary amine and optional lower fatty acid, -10 to 50 ° C, preferably 0 to 30 ° C
At 12 to 72 hours, preferably 24 to 48 hours. As a solvent for a solution containing a tertiary amine and optional lower fatty acid, DMF, DCM, THF, dimethylsulfoxide, dimethylimidazolidinone, hexamethylphosphoryltriamide, pyridine, butanol, methanol, ethanol, ether, acetone, etc. Mention may be made of these, of which DMF, DCM and THF are preferred. The amount of the tertiary amine in the solution is 1 to the peptide.
The amount is preferably 5 times, more preferably 2 to 3 times.

X in the formula (3) is 3 from the 7th Val in the formula (1).
If all the amino acids up to the 1st Pro are not bound peptides, the 31 amino acids that compose the polypeptide represented by the formula (1) are condensed by further condensing an amino acid or peptide at the site eliminated from the oxime resin. A cyclic peptide with all bonds is produced. The cyclic polypeptide having 31 amino acids bound thereto can be produced from the cyclic peptide represented by the formula (3) by the known liquid phase synthesis method, solid phase synthesis method, or a combination thereof as described above. In the amino acid or peptide to be condensed, as described above, it is preferable that the functional groups that should not participate in the reaction are protected and the functional groups that should participate in the reaction be activated.

When the cyclic peptide having 31 amino acids bound thereto as described above has a protecting group, the protecting group can be removed to produce the polypeptide represented by the formula (1). it can.

[0026]

[Chemical 10]

The removal of the protecting group can be carried out by a known method. For example, anisole is added to the above-mentioned protected polypeptide and HF and −20 to 10 ° C., preferably 0.
The reaction can be carried out at -5 ° C for 30 minutes to 3 hours, preferably 1 to 2 hours. Also, trifluoromethanesulfonic acid, methanesulfonic acid, sodium-
The protecting group can be removed with liquid ammonia or the like.

The polypeptide represented by the formula (1) can be separated and purified from the reaction product by a conventional method. Separation and purification are carried out by means of means utilizing the difference in solubility between the polypeptide represented by formula (1) and by-products, means utilizing the difference in adsorption affinity, and means utilizing the difference in molecular weight, respectively. Alternatively, they may be used in appropriate combination or repeatedly used.
Specifically, recrystallization, various ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like may be combined to separate and purify the polypeptide represented by the formula (1).

The polypeptide represented by the formula (1) produced by the method of the present invention can be prepared by using, for example, an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, propionic acid,
By reacting the salt with an organic acid such as glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, lower alkane sulfonic acid, benzene sulfonic acid, toluene sulfonic acid Can be formed.

The formula (1) produced by the method of the present invention is also
A certain inorganic substance or organic substance may be added to the polypeptide represented by to form a complex. As the above-mentioned inorganic substance, calcium, magnesium, aluminum,
Mention may be made of inorganic compounds derived from metals such as cobalt or zinc, especially phosphates, pyrophosphates or polyphosphates of these metals. Further, as the organic substance, non-antigenic gelatin, CMC, sulfonic acid ester or phosphoric acid ester of alginic acid, dextran,
Examples thereof include polyalcohol, phytic acid, polyglutamic acid and protamine.

[0031]

INDUSTRIAL APPLICABILITY The method of the present invention is industrially extremely useful because it has the following advantages as compared with the conventionally known polypeptide synthesis methods. B) The amount of solvent used for the cyclization reaction can be reduced, which simplifies the operation. (B) Since the cyclization reaction is carried out on the resin, the amount of by-products such as dimer cyclized product is small, and the desired monomer cyclized product can be easily purified.

Hereinafter, the present invention will be specifically described by way of examples, but the scope of the present invention is not limited to these. The resins and protected amino acids used in the following examples were obtained from Bachem Japan KK unless otherwise specified.

[0033]

【Example】

[Example 1] Boc-Asu-OMe (10 mmole) and oxime resin (0.45 mmole / g) 11.2 g were mixed with 10 ml of 1NDCC / DCM solution to give 18
After stirring for 2 hours, the reaction product was washed twice with DCM and DCM / EtOH (1: 1).
It was washed 4 times. 25% (v
/ V) TFA and deblocking solution containing 75% (v / v) DCM and 30
The Boc group was removed by contacting for a minute. After washing twice with DCM, once with isopropyl alcohol and three times with DMF, the above resin was washed with Boc-Thr (Bz) -OH 10mmole, equimolar Bop and
The reaction was carried out at room temperature for 1 hour in 50 ml of a DMF mixture containing 4.5 volumes of DIPEA. B sequentially washed with DMF and DCM
oc-Thr (Bz) -Asu (R) -OMe (in the formula, R represents an oxime resin) was obtained. Similarly, Boc-Ser (Bz) -OH, Boc-Leu-
OH and Boc-Asn-OH, Boc-Ser (Bz) -OH (10mmole each) were added sequentially to Bop 660mg, HOBt 2.3g and DIPEA 4.7ml, and DMF was added.
Coupling was performed in the above to obtain 13.8 g of the following peptide resin.

[0034]

[Chemical 11]

The obtained peptide resin was treated with 25% (v / v) TFA
Boc group was removed by contacting with a deblocking solution containing 75% (v / v) DCM for 30 minutes. After washing with DMF, 570 μl acetic acid and
The mixture was stirred with 150 ml of DMF solution containing 1.74 ml of DIPEA for 24 hours. The solvent is concentrated, solidified with ether, methanol-
Recrystallization from ether gave 2.1 g of a cyclic peptide represented by the following formula (I) (yield 62% based on resin).

[0036]

[Chemical 12]

Example 2 In the same manner as in Example 1, Boc-Ser (Bz) -OH (10 mmole) was mixed with oxime resin (0.45 mmo).
le / g) 11.2g, Boc-Asu-OMe, Boc-Thr (Bz) -OH,
Boc-Ser (Bz) -OH and Boc-Leu-OH, Boc-Asn-OH (each 10 mmol
e) are sequentially coupled and the following peptide resin 14.4g
Got

[0038]

[Chemical 13]

(In the formula, R represents an oxime resin.) Then, in the same manner as in the method of Example 1, the peptide of the above peptide resin was cyclized and simultaneously released from the resin to give the following formula (I 2.8 g of a cyclic peptide represented by () (yield 81% based on resin) was obtained.

[0040]

[Chemical 14]

[Example 3] In the same manner as in Example 1, Boc-Asn-OH (10 mmole) was mixed with oxime resin (0.45 mmole /
g) After introducing to 11.2g, Boc-Ser (Bz) -OH, Boc-Asu-OMe, Boc
-Thr (Bz) -OH, Boc-Ser (Bz) -OH and Boc-Leu-OH (10 mm each
ole) sequentially coupled to the following peptide resin 12.5
got g.

[0042]

[Chemical 15]

(In the formula, R represents an oxime resin.) Then, in the same manner as in the method of Example 1, the peptide of the above-mentioned peptide resin was cyclized and simultaneously released from the resin to give the following formula (I 0.3 g (yield 7.7% based on resin) of the cyclic peptide represented by

[0044]

[Chemical 16]

Example 4 In the same manner as in Example 1, Boc-Leu-OH (10 mmole) was mixed with oxime resin (0.45 mmole /
g) Boc-Asn-OH, Boc-Ser (Bz) -OH, Boc-
Asu-OMe, Boc-Thr (Bz) -OH and Boc-Ser (Bz) -OH (10m each
mole) in sequence and the following peptide resin 14.
I got 1g.

[0046]

[Chemical 17]

(In the formula, R represents an oxime resin.) Then, in the same manner as in the method of Example 1, the peptide of the above-mentioned peptide resin was cyclized and simultaneously released from the resin to give the following formula (I ) Was obtained (3.0% yield based on resin).

[0048]

[Chemical 18]

[Example 5] In the same manner as in Example 1, Boc-Ser (Bz) -OH (10mmole) was mixed with oxime resin (0.45mmo).
le / g) After introducing into 11.2g, Boc-Asn-OH, Boc-Leu-OH, Boc-S
er (Bz) -OH, Boc-Asu-OMe and Boc-Thr (Bz) -OH (each 10mmo
le) are sequentially coupled, and the following peptide resin 14.4 g
Got

[0050]

[Chemical 19]

(In the formula, R represents an oxime resin.) Then, in the same manner as in the method of Example 1, the peptide of the above-mentioned peptide resin was cyclized and simultaneously released from the resin to give the following formula (I 2.9 g (yield 85% based on resin) of a cyclic peptide represented by

[0052]

[Chemical 20]

Example 6 In the same manner as in Example 1, Boc-Thr (Bz) -OH (10 mmole) was mixed with oxime resin (0.45 mmo).
le / g) 11.2g, Boc-Ser (Bz) -OH, Boc-Asn-OH, B
oc-Leu-OH, Boc-Ser (Bz) -OH and Boc-Asu-OMe (10mmo each
le) are sequentially coupled, and 14.5 g of the following peptide resin
Got

[0054]

[Chemical 21]

(In the formula, R represents an oxime resin.) Then, in the same manner as in Example 1, the peptide of the above peptide resin was cyclized and simultaneously released from the resin to give the following formula (I ) Was obtained (3.0% yield based on resin).

[0056]

[Chemical 22]

Example 7 First, Boc-Ser (Bz) -Asn-Leu-
Ser (Bz) -Thr (Bz) -Asu-OMe was prepared as follows. Boc
-Thr (Bz) -OH 30.9 g was dissolved in THF 200 ml, cooled to -20 ° C with dry ice-ethanol, N-methylmorpholine 11.0 ml, and then isobutyl chloroformate 13.2 ml.
Was added dropwise, and the mixture was stirred at -20 ° C for 1 minute to prepare a corresponding mixed acid anhydride. This reaction solution was added to L-aminosuberic acid.
The mixture was mixed with a 200 ml solution of THF containing 20.3 g of -α-methyl ester, stirred at 0 ° C for 5 minutes and at room temperature for 20 minutes, and concentrated under reduced pressure. To the residue was added 400 ml of ethyl acetate, 200 ml of 1N hydrochloric acid.
2 times, once with 200 ml of saturated saline solution, and 2 times with 200 ml of saturated sodium bicarbonate solution.
It was washed twice with saturated saline. After drying over anhydrous magnesium sulfate, concentration under reduced pressure, Boc-Thr (Bz) -Asu-OMe oil 4
Obtained 4.5 g.

Boc-Thr (Bz) -Asu-OMe 38 g under ice cooling TFA 72
After adding ml to dissolve and stirring for 30 minutes, the mixture was concentrated under reduced pressure, and the oily substance was vacuum dried over sodium hydroxide. Dissolve the above product in 200 ml of DMF and add it to pH 4.0 with TEA under ice cooling.
After preparation in DMF 50 containing 30.1 g of Boc-Ser (Bz) -OSu
ml solution was added. The mixture was stirred at 0 ° C for 1 hour and at room temperature overnight, 2.64 ml of N, N-dimethylethylenediamine was added under ice cooling, and the mixture was stirred at 0 ° C for 1 hour and concentrated under reduced pressure. To the residue was added 600 ml of ethyl acetate, 200 ml of 1N hydrochloric acid was added twice, and 200 ml of water was added.
After being washed twice with water, it was concentrated under reduced pressure and Boc-Ser (Bz) -Thr (Bz)-
47.0 g (yield 91.1%) of Asu-OMe oil was obtained.

50 ml of TFA was added to 28.5 g of Boc-Ser (Bz) -Thr (Bz) -Asu-OMe under ice cooling, and the mixture was dissolved. After stirring for 30 minutes, the mixture was concentrated under reduced pressure and the oily substance was added to sodium hydroxide. Vacuum dried. The above product was dissolved in 200 ml of DMF, adjusted to pH 4.0 with TEA under ice-cooling, and then added with 50 ml of DMF containing 13.9 g of Boc-Leu-OSu. The mixture was stirred at 0 ° C for 1 hour and at room temperature overnight, and cooled under ice-cooling with N, N-dimethylethylenediamine.
1.62 ml was added, and the mixture was stirred at 0 ° C for 1 hr and concentrated under reduced pressure.
600 ml of ethyl acetate was added to the residue, and 200 ml of 1N hydrochloric acid was added twice.
After washing twice with 200 ml of water, concentration under reduced pressure, Boc-Leu-Ser (B
30.6 g (yield 92.8%) of an oily substance of z) -Thr (Bz) -Asu-OMe was obtained.

Boc-Leu-Ser (Bz) -Thr (Bz) -Asu-OMe 30.6 g
To the solution was added TFA (60 ml) under ice-cooling to dissolve it, and the mixture was stirred for 30 minutes and then concentrated under reduced pressure. The residue was treated with ether, the precipitated product was filtered off and dried over sodium hydroxide in vacuo.
Dissolve the above product in 200 ml of DMF and add it to TEA under ice cooling.
DMSO containing 13.7g of Boc-Asn-ONP after adjusting to pH 4.0 with
F 50 ml solution was added. The mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight, 1.02 ml of N, N-dimethylethylenediamine was added under ice cooling, and the mixture was stirred at 0 ° C. for 1 hour and concentrated under reduced pressure. To the residue was added 600 ml of ethyl acetate, 200 ml of 1N hydrochloric acid twice, and 200 ml of water
It was washed twice with and then concentrated under reduced pressure. The residue was treated with ether to solidify and recrystallized from methanol-ether to obtain 31.0 g (yield 88.1%) of Boc-Asn-Leu-Ser (Bz) -Thr (Bz) -Asu-OMe.

Mp 184.3 to 192 ° C. [α] _D = -9.8 (C = 1, DMF) Elemental analysis Calculated as C ₄₅ H ₆₆ N ₆ O ₁₃ .H ₂ O C, 58.94% H, 7.47% N, 9.16 % Measured value C, 58.93% H, 7.50% N, 8.88% Boc-Asn-Leu-Ser (Bz) -Thr (Bz) -Asu-OMe 15.8g under ice cooling
TFA (50 ml) was added and dissolved, and the mixture was stirred for 30 minutes and then concentrated under reduced pressure. The residue was treated with ether, the precipitated product was filtered off and dried over sodium hydroxide in vacuo.

The above product was dissolved in 200 ml of DMF and adjusted to pH 4.0 with TEA under ice cooling, and then Boc-Ser (Bz) -OSu.
A 50 ml DMF solution containing 6.91 g was added. The mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight, 0.32 ml of N, N-dimethylethylenediamine was added under ice cooling, and the mixture was stirred at 0 ° C. for 1 hour and concentrated under reduced pressure. 600 ml of ethyl acetate was added to the residue, and 1N hydrochloric acid was added to 200
It was washed twice with 100 ml of water and twice with 200 ml of water, and then concentrated under reduced pressure.
The residue was treated with ether to solidify and recrystallized from methanol-ether to give Boc-Ser (Bz) -Asn-Leu-Ser (Bz)-
15.0 g (yield 79.2%) of Thr (Bz) -Asu-OMe was obtained.

Mp 178 to 179 ° C. [α] _D = -5.3 (C = 1, DMF) Elemental analysis Calculated as C ₅₅ H ₇₇ N ₇ O ₁₅ .H ₂ O C, 60.37% H, 7.28% N, 8.96 % Measured value C, 60.11% H, 7.27% N, 8.68% Above Boc-Ser (Bz) -Asn-Leu-Ser (Bz) -Thr (Bz) -Asu-OMe
(10mmole) to oxime resin (0.45mmole / g) 11.2g 1NDCC
/ DCM solution (10 ml) was added and stirred for 18 hours, then twice with DCM, DCM
After washing 4 times with / EtOH (1: 1), 13.7 g of the following peptide resin was obtained.

[0064]

[Chemical 23]

(In the formula, R represents an oxime resin.) Then, in the same manner as in the method of Example 1, the peptide of the above-mentioned peptide resin was cyclized and simultaneously released from the resin to give the following formula (I ) Was obtained (2.1% yield based on resin).

[0066]

[Chemical 24]

[Embodiment 8] (1)

[0068]

[Chemical 25]

Formula (I) prepared in any of Examples 1-7
The cyclic peptide represented by 5.1 g was dissolved in methanol-DMF and treated with 1.1 ml of 90% saturated water hydrazine solution for 18 hours, then the solvent was concentrated, solidified and washed with water, and then washed with ether to obtain the above-mentioned product. Hydrazide compound represented by formula (II)
Obtained 5.1 g. mp 200 to 202 ° C (decomposition) [α] _D = -6.0 (C = 1, DMF) Elemental analysis C ₄₉ H ₆₇ N ₉ O ₁₁ Calculated value C, 61.43% H, 6.05% N, 13.16% Measured value C , 61.20% H, 6.32% N, 13.11% (2)

[0070]

[Chemical 26]

4.5 g of the hydrazide compound represented by the formula (II)
Dissolve in 120 ml of DMF and dry ice-with ethanol-
After cooling to 20 ° C., 2.35 ml of 6N hydrochloric acid-dioxane solution and 0.63 ml of isoamyl nitrite were added to give an azide compound. After neutralizing with 1.97 ml of triethylamine, H-Val-Leu-
Add Gly-OH (1.35g) to DMF solution and dissolve at -20 ℃ for 2
The mixture was stirred for 17 hours at 4 ° C. Concentrate under reduced pressure to remove DMF, add water to the residue to solidify, recrystallize twice with DMF-ethyl acetate to obtain the cyclic peptide represented by the above formula (III).
5.4 g (yield 94.7%) was obtained. mp 240 ° C (decomposition) [α] _D = -18.5 (C = 1, DMF) Elemental analysis Calculated as C ₆₂ H ₈₈ N ₁₀ O ₁₅ C, 61.37% H, 7.31% N, 11.54% Measured value C, 61.12 % H, 7.56% N, 11.27% amino acid composition; Asp 0.92 (1), Thr 0.98 (1), Ser 1.95 (2),
Gly 0.96 (1), Val 0.98 (1), Leu 1.09 (1), Asu 0.99 (1) (3)

[0072]

[Chemical 27]

The peptide represented by the formula (IV) is prepared as shown in Japanese Examined Patent Publication No. 53-
It was produced by a liquid phase synthesis method according to the method described in Japanese Patent No. 41677. Boc-Lys (Z) -Leu-Ser (Bz) -Gln-Glu (OBz) -Leu-His-Lys (Z)
-Leu-Gln-Thr (Bz) -Tyr (Bz) -Pro-Arg (Tos) -Thr (Bz) -Asp
(OBz) -Val-Gly-Ala-Gly-Thr (Bz) -Pro-NH ₂ (IV) Anisole 2 was added to 7.2 g of the peptide represented by the above formula (IV) under ice cooling.
ml and TFA 15 ml were added and dissolved, and the mixture was stirred for 30 minutes and then concentrated under reduced pressure. The residue is treated with ether, the precipitated product is filtered off, dried over sodium hydroxide in vacuo and H-
Lys (Z) -Leu-Ser (Bz) -Gln-Glu (OBz) -Leu-His-Lys (Z) -Leu
-Gln-Thr (Bz) -Tyr (Bz) -Pro-Arg (Tos) -Thr (Bz) -Asp (OBz)
-Val-Gly-Ala-Gly-Thr (Bz) -Pro-NH2.TFA was obtained.

The above product, a compound of formula (II) prepared in (2) above
IMF) peptide 2.9g and HOBt 0.33g were added to DMF 20
It was dissolved in 0 ml, adjusted to pH 4.0 with TEA under ice cooling, and 0.45 ml of WSC was added. After stirring at 0 ° C. for 1 hour and at room temperature overnight, the mixture was concentrated under reduced pressure. The residue was solidified by adding 1N hydrochloric acid and recrystallized once with DMF-ethyl acetate and twice with DMF-methanol to give the cyclic peptide represented by the formula (V) 9.0.
g (yield 96.1%) was obtained. mp 174-177 ° C [α] _D = -17.5 (C = 1, DMF) Elemental analysis Calculated as C ₂₄₁ H ₃₂₂ N ₄₂ O ₅₃ S ・ 2H ₂ O C, 60.59% H, 6.87% N, 11.87% measurement Value C, 60.35% H, 6.79% N, 11.69% amino acid composition; Asp 1.92 (2), Thr 3.88 (4), Ser 2.88 (3),
Glu 3.08 (3), Pro 1.99 (2), Gly 2.96 (3), Ala 0.98 (1), Va
l 1.98 (2), Leu 4.09 (4), Tyr 0.92 (1), His 0.93 (1), Lys
2.16 (2), Arg 1.06 (1), Asu 0.99 (1) (5)

[0075]

[Chemical 28]

4.7 g of the cyclic peptide represented by the formula (V) obtained in (4) above was reacted with 75 ml of hydrogen fluoride in 8 ml of anisole at 0 ° C. for 1 hour. After the reaction, hydrogen fluoride was distilled off, the residue was treated with ether, the precipitated product was filtered, and dried over sodium hydroxide in vacuo. This is 1M acetic acid solution 150m
It was dissolved in l, passed through Dowex 1-X4 (acetic acid type), and the effluent was freeze-dried to obtain 3.0 g of powder.

This powder was dissolved in a 0.05 M ammonium acetate aqueous solution and poured onto a column (4 × 30 cm) packed with CM-cellulose to prepare a 0.01 M ammonium acetate aqueous solution (pH 4.4) 500.
ml-0.11M ammonium acetate aqueous solution (pH 4.4) 500 ml linear concentration gradient elution (60 ml / hour), 10 ml fractions of eluate were collected and analyzed by high performance liquid chromatography to collect the target fractions. After freeze-drying, 601 mg of powder was obtained.

This powder was dissolved in 0.1 M acetic acid, poured into Sephadex G-10 (2.2 × 110 cm), eluted with 0.1 M acetic acid, and the target fraction was lyophilized to give the formula (1) above. 572 mg of cyclic polypeptide was obtained. mp 241 ° C (decomposition) [α] _D = -94.6 (C = 1,0.1MAcOH) Elemental analysis C ₁₄₈ H ₂₄₄ N ₄₂ O ₄₇・ 18H ₂ O ・ 2AcOH
Calculated value C, 47.94% H, 7.62% N, 15.45% Measured value C, 47.75% H, 7.35% N, 15.43% Amino acid composition; Asp 1.92 (2), Thr 3.88 (4), Ser 2.88 (3),
Glu 3.08 (3), Pro 1.99 (2), Gly 2.96 (3), Ala 0.98 (1), Va
l 1.98 (2), Leu 4.09 (4), Tyr 0.92 (1), His 0.93 (1), Lys
2.16 (2), Arg 1.06 (1), Asu 0.99 (1)

Claims (3)

[Claims] 1. A method for producing a polypeptide represented by formula (1), an acid addition salt thereof, or a complex thereof, which comprises: Formula (2-1), (2-2), (2-3), (2-4), (2-5) and a peptide covalently bonded to the oxime resin represented by (2-6) Cyclization by intramolecular condensation and elimination of the peptide from the oxime resin produce a cyclic peptide represented by formula (3), and if necessary, an amino acid or peptide is condensed at the site eliminated from the oxime resin. The above-mentioned manufacturing method including a step. [Chemical 2] (In the formula, X is Val, a peptide having an arbitrary number of consecutive amino acids bounded to Val from the 7th Val of the formula (1) to the 31st Pro in order to the N-terminal, or an optionally protected hydroxyl group. Represents a group, R represents an oxime resin, and Ser and Thr may have a protecting group.) (In the formula, X is Val, 31 from the 7th Val in formula (1)
It represents a peptide to which an arbitrary number of consecutive amino acids starting from Val to the Nth Pro are bound or a hydroxyl group which may be protected, and Ser and Thr may have a protecting group. ) 2. A tertiary amine is used to formula (2-1), (2-2),
(2-3), (2-4), (2-5) and (2-6) covalently bound to the oxime resin represented by any one of the peptides cyclized by intramolecular condensation and the peptide oxime The method according to claim 1, wherein the cyclic peptide is cleaved from the resin to produce the cyclic peptide represented by the formula (3). 3. The method according to claim 2, wherein a tertiary amine is used in the presence of a lower fatty acid.