JPH0745517B2 - Peptides and methods for producing cyclic peptides - Google Patents

Description

TECHNICAL FIELD The present invention relates to a peptide and a method for producing a cyclic peptide. More specifically, the present invention relates to a cyclic peptide, for example, a peptide having the following formula: The present invention relates to a novel method for producing a peptide and a cyclic peptide which are useful for obtaining a synthetic calcitonin derivative (elcatonin) represented by the formula: or an acid addition salt or complex thereof.

BACKGROUND ART Calcitonin has been known as a polypeptide with excellent pharmacological effects, such as potent serum calcium and phosphorus reduction, promoting bone formation and inhibiting bone resorption, and promoting urinary phosphorus excretion. Calcitonin is extracted from the thyroid gland of various mammals, including humans, or from the ultimobranchial body of fish, cyclostomes, and birds, and its primary amino acid sequence has been elucidated. Many synthetic calcitonins with similar structures based on this sequence have also been reported. All of these animal-derived calcitonins are polypeptides consisting of 32 constituent amino acids, the first and seventh amino acids of which are L-amino acids.
The two mercapto groups form a disulfide bond, and the carboxyl terminus is a proline amide. On the other hand, these natural calcitonins have a disulfide bond in the molecule,
Not stable enough in solution.

Therefore, as synthetic calcitonin, the first and seventh L
- Cysteine in the following formula: A method for producing a polypeptide in which the amino acid sequence is replaced with α-L-aminosuberic acid of the formula: (wherein X represents a hydroxyl group, a carboxyl-protecting group commonly used in peptide chemistry, or an amino acid residue or peptide residue corresponding to calcitonin, and each amino acid residue may be protected by a protecting group commonly used in peptide chemistry) is subjected to a cyclization reaction in liquid phase, and each fragment is further coupled in liquid phase (hereinafter referred to as "liquid phase synthesis") to synthesize the desired calcitonin derivative [see JP-B Nos. 53-41677, 61-112099, and 63-203699, and Pharmacia Review No. 3, "Biologically Active Peptides in Search of New Drugs" (edited by Pharmacia Review Editorial Board), pp. 153-154].

However, in these methods, as the number of amino acids increases, the solubility of the peptide changes slightly, making it increasingly difficult to find a suitable solvent. This also increases the difficulty of separating the target polypeptide from unreacted materials and by-products. In particular, it is necessary to minimize the formation of by-products in the cyclization reaction. Therefore, conventional methods for producing synthetic calcitonin derivatives using liquid-phase synthesis have low yields and are not industrially satisfactory.

Accordingly, an object of the present invention is to provide a peptide or an acid addition salt or complex thereof that is useful for producing a cyclic peptide, particularly elcatonin, a calcitonin derivative, in good yield.

Another object of the present invention is to provide a novel and industrially useful method for producing a cyclic peptide, particularly elcatonin, a calcitonin derivative, or an acid addition salt or complex thereof, with high yield.

DISCLOSURE OF THE INVENTION In order to achieve the above object, the present inventors have conducted extensive research and have found that (1) instead of directly condensing the side chain carboxyl group of α-L-aminosuberic acid with the amino group of the N-terminal amino acid to form a cyclization reaction between the C-terminal carboxyl group and the N-terminal amino group, a peptide in which an amino acid or peptide is condensed with at least the side chain carboxyl group of α-L-aminosuberic acid, or an acid addition salt or complex thereof, is used, (2) the cyclization reaction is carried out in the presence of an alkali metal salt, and (3) a liquid phase synthesis method and a solid phase synthesis method are effectively combined to form a cyclization reaction.
The present invention was completed based on the discovery that the above object could be achieved.
That is, the present invention provides a peptide represented by the following general formula (I) or an acid addition salt or complex thereof:

[Wherein A and B are the following (1) to (5)] (1) A: Ser(X1)-Asn-Leu-Ser(X1)-Thr(X1)
-OH B:X2 (2) A:Ser(X1)-Asn-Leu-Ser(X1)-OH B:X2-Thr(X1) (3) A:Ser(X1)-Asn-Leu-OH B:X2-Ser(X1)-Thr(X1) (4) A:Ser(X1)-Asn-OH B:X2-Leu-Ser(X1)-Thr(X1) (5) A:Ser(X1)-OH B:X2-Asn-Leu-Ser(X1)-Thr(X1) (wherein X1 represents a hydrogen atom or a protecting group for a hydroxyl group, and X2 represents a hydrogen atom or a protecting group for an amino group). X represents a hydroxyl group, a protecting group for a carboxyl group, an amino acid residue represented by Val-OH or Met-OH, or a group represented by the following general formula (VII)-A1-Leu-A2-OH (VII) (wherein A1 represents Val or Met, and A2 represents Ser or Gly).
or a lower peptide residue represented by the following general formula (VIII):
1−A12−A13−A14−A15−A16−A17−A18−A19−Gly−A2
0-A21-A22-Pro- _NH2 (VIII) (wherein A1 and A2 are the same as above, and A3 to A22 represent the following amino acid residues.)

A3: Lys, Thr, or Ala A4: Leu or Tyr A5: Ser, Thr, or Trp A6: Gln, Lys, or Arg A7: Glu, Asp, or Asn A8: Leu or Phe A9: His or Asn A10: Lys or Asn A11: Leu, Phe, or Tyr A12: Gln or His A13: Thr or Arg A14: Tyr or Phe A15: Pro or Ser A16: Arg, Gly, or Gln A17: Thr or Met A18: Asp, Ala, Asn, or Gly A19: Val, Ile, Thr, or Phe A20: Ala, Val, Pro, or Ser A21: Gly or Glu A22: Thr or Ala or a peptide residue represented by the general formula (VIII) containing at least -A1-Leu-A2-A3 and A1 to A22
The present invention relates to a peptide fragment having a structure represented by the following general formula (II): [wherein D-E represents Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group; X is the same as above; provided that when X is the amino acid residue, lower peptide residue, peptide residue, or peptide fragment, the carboxyl group of the C-terminal amino acid in X and the reactive groups of each amino acid residue are protected with protecting groups (hereinafter the same applies to general formulas (III) and (IV))], or an acid addition salt or complex thereof, is produced by the following method.

(1) The following general formula (III) (Wherein D represents Ser-OH, Ser-Asn-OH, Ser-Asn-Leu
-OH, Ser-Asn-Leu-Ser-OH or Ser-Asn-Leu-Ser
-Thr-OH, E represents H-Asn-Leu-Ser-Thr, H
1. A method for producing a peptide represented by the formula (III) (III-Leu-Ser-Thr, H-Ser-Thr, H-Thr or a hydrogen atom, and each amino acid residue may be protected by a protecting group; X is as defined above), by subjecting the peptide to a cyclization reaction by chemical condensation reaction (excluding cyclization reactions using proteases).

(2) The following general formula (IV) (wherein F represents a hydroxyl group or an active ester residue, G
represents H-Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group. X is the same as above.) A method for producing a peptide represented by the following formula (X represents H-Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group. X is the same as above.) is subjected to a cyclization reaction in the presence of an alkali metal salt.
The side chain carboxyl group of aminosuberic acid does not necessarily have to be condensed with an amino acid or peptide.

The present invention also provides a compound represented by the following general formula (IX): (wherein A1 to A22 are as defined above, and each amino acid residue may be protected with a protecting group) or an acid addition salt or complex thereof, the method comprising: (1) producing a cyclic peptide represented by the following general formula (IIIa): wherein D is Ser-OH, Ser-Asn-OH, Ser-Asn-Leu
-OH, Ser-Asn-Leu-Ser-OH or Ser-Asn-Leu-Ser
-Thr-OH, E represents H-Asn-Leu-Ser-Thr, H
-Leu-Ser-Thr, H-Ser-Thr, H-Thr or a hydrogen atom, and each amino acid residue may be protected with a protecting group; W represents a hydroxyl group or a lower peptide residue represented by the general formula (VII) -A1-Leu-A2-OH (VII) (A1 and A2 are as defined above), and in the lower peptide residue, the carboxyl group of the C-terminal amino acid and the reactive group of each amino acid residue are protected with a protecting group), by a chemical condensation reaction (excluding cyclization reactions using proteases), or (2) a peptide represented by the following general formula (IVa): (wherein F represents a hydroxyl group or an active ester residue, G
represents H-Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group. W is the same as above.) is subjected to a cyclization reaction in the presence of an alkali metal salt to obtain a peptide represented by the following general formula (V): [wherein W is as defined above], then the protecting group of the carboxyl group of the C-terminal amino acid of the peptide represented by general formula (V) is removed, followed by condensation with a peptide-resin represented by the following general formula (VI) synthesized on a solid-phase reaction resin by a solid-phase method, and then removing the resin.

Y-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13
−A14−A15−A16−A17−A18−A19−Gly−A20−A21−A22
-Pro-Resin (VI) [Y represents a hydrogen atom or H-Al-Leu-A2- (A1 and A2 are the same as above), and each amino acid residue may be protected with a protecting group. However, when W is a hydroxyl group, Y is H-
When A1-Leu-A2- and W is -A1-Leu-A2-OH, Y is a hydrogen atom. Resin represents a solid-phase reaction resin. A3 to A22 are the same as above. In this specification, when amino acids, peptides, protecting groups, solvents, etc. are represented by abbreviations, they are based on the IUPAC, IUB
or symbols commonly used in the art. Examples are given below. However, when optical isomers are possible for amino acids, etc., the L-isomer is indicated unless otherwise specified.

Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Gln: Glutamine Glu: Glutamic acid Gly: Glycine His: Histidine Ile: Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Tyr: Tyrosine Val: Valine Boc: tert-Butoxycarbonyl Bzl: Benzyl Z : benzyloxycarbonyl Tos: tosyl Cl-z: 2-chlorobenzyloxycarbonyl Br-z: 2-bromobenzyloxycarbonyl OMe: methyl ester OPac: phenacyl ester OBzl: benzyl ester OSu: N-hydroxysuccinimide ester HOSu: N-hydroxysuccinimide TFA: trifluoroacetic acid THF: tetrahydrofuran DMF: N,N-dimethylformamide DMSO: dimethyl sulfoxide NMP: N-methylpyrrolidone DCC: dicyclohexylcarbodiimide EDC: N-ethyl-N'-dimethylaminopropylcarbodiimide HOBt: 1-hydroxybenzotriazole Detailed Description of the Invention The peptide of the present invention represented by the general formula (I) is an α-
An amino acid or peptide is condensed to at least the side chain carboxyl group of L-aminosuberic acid.

The combinations of A and B in the peptide represented by the general formula (I) are as follows:

(1) A:Ser(X1)−Asn−Leu−Ser(X1)−Thr(X1)
-OH B:X2 (2) A:Ser(X1)-Asn-Leu-Ser(X1)-OH B:X2-Thr(X1) (3) A:Ser(X1)-Asn-Leu-OH B:X2-Ser(X1)-Thr(X1) (4) A:Ser(X1)-Asn-OH B:X2-Leu-Ser(X1)-Thr(X1) (5) A:Ser(X1)-OH B:X2-Asn-Leu-Ser(X1)-Thr(X1) (wherein X1 and X2 are as defined above) Furthermore, the preferred combinations of D and E in general formula (III) and F and G in general formula (IV) also form a peptide represented by Ser-Asn-Leu-Ser-Thr, similar to the combination of A and B. However, F in general formula (IV) may be different from D in general formula (III) and may be a hydroxyl group or an active ester residue.

In the general formulas (I) to (IV), the carboxyl-protecting group represented by X is not particularly limited as long as it is a group commonly used in peptide chemistry as a carboxyl-protecting group. Examples of the carboxyl-protecting group include a methoxy group,
Alkoxy groups such as ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, scc-butoxy, and tert-butoxy; cycloalkyloxy groups such as cyclohexyloxy; benzyloxy, p-
an aralkyloxy group which may have a substituent such as a methoxybenzyloxy group, a p-nitrobenzyloxy group, a p-chlorobenzyloxy group, a p-bromobenzyloxy group, or a benzhydryloxy group; a carbobenzoxyhydrazino group, a tert-butoxycarbonylhydrazino group,
Examples include substituted hydrazino groups such as tritylhydrazino groups, and phenacyl groups.

In the general formulas (I) to (IV), among the amino acid residues represented by X, examples of the amino acid residues constituting calcitonin include Val-OH and Met-OH. Furthermore, the peptide residue represented by X is not particularly limited as long as it is a peptide residue constituting calcitonin. Examples of the peptide residue include a lower peptide residue represented by the general formula (VII) -A1-Leu-A2-OH (VII);
1−A12−A13−A14−A15−A16−A17−A18−A19−Gly−A2
0-A21-A22-Pro-NH ₂ (VIII) (wherein A1 to A22 are the same as above), or a fragment thereof.

The amino acid residues constituting the calcitonin are represented by the general formula (VI)
The peptide residue -A1-Leu-A2 represented by formula (I) and peptides having a fragment of the peptide residue represented by formula (VIII) are suitable as intermediates for the synthesis of calcitonin.

In the lower peptide residues represented by the general formula (VII), preferred peptide residues constituting calcitonin are:
In the peptide residues represented by the general formula (VIII), the preferred peptide residues constituting calcitonin are: -Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His
−Lys−Leu−Gln−Thr−Tyr−Pro−Arg−Thr−Asp−Val
-Gly-Ala-Gly-Thr-Pro- _NH2 .

In the peptides represented by the above general formulae (I) to (VI) and (IX), the protecting groups for protecting each amino acid residue can be any protecting groups commonly used in peptide chemistry.

Among the protecting groups commonly used in peptide chemistry, the protecting group for a carboxyl group includes the same groups as the protecting group for a carboxyl group represented by X above.

Examples of the protecting group for the amino group include an alkoxycarbonyl group which may have a substituent such as a Boc group, a trichloroethyloxycarbonyl group, or a tert-amyloxycarbonyl group; a cycloalkyloxycarbonyl group which may have a substituent such as a cyclopentyloxycarbonyl group or a cyclohexyloxycarbonyl group; a Z group, a p-
Methoxybenzyloxycarbonyl group, Cl-Z group, p-
an aralkyloxycarbonyl group which may have a substituent such as a chlorobenzyloxycarbonyl group, a Br-Z group, a p-bromobenzyloxycarbonyl group, a p-nitrobenzyloxycarbonyl group, or an adamantyloxycarbonyl group; an aralkyl group which may have a substituent such as a Bzl group, a benzhydryl group, or a trityl group; a trifluoroacetyl group, a phthalyl group, a formyl group, a benzenesulfonyl group, a Tos group, or an o-nitrophenylsulfenyl group;
Examples include acyl groups which may have a substituent such as a 2,4-dinitrophenylsulfenyl group or a diphenylphosphinothioyl group.

The guanidino group of Arg can be protected, for example, with a nitro group, a Z group, a Tos group, etc. This guanidino group does not necessarily have to be protected.

The imidazolyl group of His may not be protected, but may be protected, for example, by a Bzl group, a Tos group, a Z group, a trityl group, an adamantyloxycarbonyl group, a 2,2,2-trifluoro-1-tert-
It can be protected with a butoxycarbonylaminoethyl group, a 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, or the like.

The hydroxy group of Ser, Thr, and Tyr can be, for example, a Bzl group, 4-
It can be protected with a methoxybenzyl group, a 4-methylbenzyl group, a 3,4-dimethylbenzyl group, a 4-chlorobenzyl group, a 2,6-dichlorobenzyl group, a 4-nitrobenzyl group, a benzhydryl group, a Z group, a Cl-Z group, a Br-Z group, a tetrahydropyranyl group, etc. The hydroxy group does not have to be protected.

The peptides represented by the general formulae (I), (III), (IIIa), (IV), and (IVa) can be obtained by repeating the attachment and detachment of protecting groups and condensation reactions according to a conventional method for peptide synthesis. That is, the protecting groups of the raw materials and intermediates used in the present invention can be selected from those known in peptide synthesis, for example,
A protecting group that can be easily removed by hydrolysis, acidolysis, reduction, aminolysis, hydrazinolysis, or the like is used.

Among the peptides represented by the general formulae (I), (III) and (IV), the peptide in which X is a protecting group for a hydroxyl group or a carboxyl group is, for example, L-aminosuberic acid-α-
These can be obtained by sequentially condensing the corresponding amino acids one by one to the side chain carboxyl and/or amino groups of a lower alkyl ester, or by condensing a peptide consisting of 2 to 5 amino acids. However, when B and E are hydrogen atoms in the peptides represented by general formulas (I) and (III), or when G is a hydrogen atom in the peptide represented by general formula (IV), it is necessary to protect the amino group of the L-aminosuberic acid-α-lower alkyl ester. Furthermore, when F is a hydroxyl group in the peptide represented by general formula (IV), it is preferable to protect the side chain carboxyl group of the L-aminosuberic acid-α-lower alkyl ester.

More specifically, among the peptides represented by the general formula (I), those represented by the following general formula: (wherein X1 represents a Bzl group and X represents a protecting group for a carboxyl group) can be synthesized, for example, as follows: N-protected leucylserine is substituted for threonine by the active ester method, the mixed acid anhydride method, or the DC
The tripeptide is condensed according to a conventional peptide synthesis method such as the C-HOBt method. Then, N-protected seryl asparagine and N-protected aminosuberic acid-α-lower alkyl ester are sequentially condensed with the resulting tripeptide, followed by elimination of the protecting group from the N-protected aminosuberic acid-α-lower alkyl ester. However, it is preferable to protect the carboxyl group of threonine with a protecting group, and then elimination of the protecting group after condensation of the amino acid or peptide as described above.

Among the peptides represented by the general formula (III) used in the present invention, those represented by the following general formula: (wherein X represents a protecting group for the carboxyl group) can be synthesized, for example, as follows:
N-protected threonine is condensed with a lower alkyl ester by the active ester method, the mixed acid anhydride method, or the like, and then the side chain carboxyl group of L-aminosuberic acid is activated, followed by condensation with seryl asparagyl leucine. N-protected serine is condensed with the resulting pentapeptide by a conventional peptide synthesis method such as the mixed acid anhydride method or the active ester method, to obtain N-protected threonine.
It can be obtained by removing the protecting group from the protected serine.

Of the peptides represented by the general formulae (I), (III) and (VI), the peptides in which X is an amino acid residue or a peptide residue can be prepared by, for example, eliminating the alkoxy group, which is the protecting group of the L-aminosuberic acid-α-lower alkyl ester obtained by condensing an amino acid or a peptide as described above, activating the carboxyl group, and then condensing the corresponding amino acid in one
The amino acid can be obtained by sequentially condensing one or more amino acids at a time, or by condensing a peptide consisting of two or more amino acids.

In the condensation reaction, (a) an amino acid or peptide having an activated terminal carboxyl group and an α-amino group protected with a protecting group may be reacted with an amino acid or peptide having a free α-amino group and a carboxyl group protected with a protecting group, or (b) an amino acid or peptide having an activated α-amino group and a protected carboxyl group may be reacted with an amino acid or peptide having a free carboxyl group and a protected α-amino group.

The activation of the carboxyl group can be achieved by, for example, using an acid azide, an acid anhydride, an acid imidazolide, an active ester, a carbodiimide,
This can be carried out by treating with N,N'-carbonyl-diimidazole or an isoxazolium salt, for example, a Woodward reagent, etc. Examples of active esters include cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester,
Examples thereof include OSu, N-hydroxyphthalimide ester, 8-hydroquinoline ester, and N-hydroxypiperidine ester.

Preferred condensation reactions are the carbodiimide method, the azide method, the active ester method, and the mixed acid anhydride method. More preferred condensation reactions are those that suppress racemization during the condensation process,
For example, the active ester method, the carbodiimide method, or a combination thereof, a method using carbodiimide and hydroxysuccinimide such as the DCC-HOSu method, and the DCC-HOBt method are known.
The WSC-HOBt method and other methods use a carbodiimide and hydroxybenzotriazole. WSC refers to a water-soluble carbodiimide such as EDC. More preferred methods include the so-called carbodiimide-additive method, which uses a hydroxybenzotriazole and a carbodiimide, or a hydroxysuccinimide and a carbodiimide.

The method for producing a cyclic peptide represented by general formula (II) from a peptide represented by general formula (III) is characterized by the cyclization of the activated carboxyl group of the C-terminal amino acid of the peptide represented by general formula (III) with the amino group of the N-terminal amino acid. The cyclization reaction for obtaining a peptide represented by general formula (II) from a peptide represented by general formula (III) can be carried out according to a conventional chemical condensation reaction. This cyclization reaction is preferably carried out in the presence of an alkali metal salt.

The method for producing a cyclic peptide represented by general formula (II) from a peptide represented by general formula (IV) is characterized in that the peptide represented by general formula (IV) is subjected to a cyclization reaction in the presence of an alkali metal salt to obtain a peptide represented by general formula (II). When F in the peptide represented by general formula (IV) is a hydroxyl group or an activated ester residue, this cyclization reaction can be carried out by a condensation reaction between the activated carboxyl group in the side chain of α-L-aminosuberic acid and the amino group of the N-terminal amino acid.
It is preferable to protect the hydroxyl groups of Ser and Thr. When the reaction is carried out in the presence of an alkali metal salt, the cyclization reaction can be carried out smoothly even when F is a hydroxyl group or an active ester residue in the peptide represented by the general formula (IV), and the yield of the peptide can be increased.

Examples of alkali metal salts include halides of lithium, sodium, potassium, rubidium, and cesium, such as fluorides, chlorides, bromides, and iodides.
These alkali metal salts can be used alone or in suitable combination.

The amount of alkali metal salt used is not particularly limited as long as it does not impair smooth cyclization reaction, but is usually about 0.01 to 400 equivalents, preferably about 0.01 to 100 equivalents, of the alkali metal salt relative to the peptide to be cyclized. More specifically,
When the alkali metal salt is a mixture of lithium chloride, sodium chloride, potassium chloride, and cesium chloride, each of these salts may be added in an amount of about 0.01 to 100 equivalents, preferably about 0.1 to 20 equivalents, relative to the peptide to be cyclized.

In the synthesis of the cyclic peptide represented by the general formula (II), when an amino acid or peptide in which an active group is protected is used, the resulting peptide protected with a protecting group is
By subjecting the compound to a conventional method for removing a protecting group, preferably removal with an acid, for example, a removal reaction with hydrogen fluoride,
A cyclic peptide is obtained.

W in the cyclic peptide represented by the general formula (V),
In addition, Y in the peptide-resin represented by the general formula (VI) preferably constitutes elcatonin. That is, (a) when W is a hydroxyl group, Y is H-Val-L
(b) W is -Val-Leu
When W is -Gly-OH, Y is preferably a hydrogen atom. A preferred combination of W and Y is the combination (b) above. Furthermore, a preferred peptide-resin represented by general formula (VI) is Y-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-G
ln−Thr−Tyr−Pro−Arg−Thr−Asp−Val−Gly−Ala−G
ly-Thr-Pro-Resin.

The characteristic of this production method is that the peptide represented by the general formula (IIIa) is cyclized by a chemical condensation reaction (excluding cyclization reactions using proteases), or the peptide represented by the general formula (IVa) is subjected to a cyclization reaction in the presence of an alkali metal salt to obtain the peptide represented by the general formula (V), and then the protecting group of the carboxyl group of the C-terminal amino acid of the peptide represented by the general formula (V) is eliminated, and the resulting product is reacted on a solid-phase resin with the peptide represented by the general formula (VI) synthesized by a solid-phase reaction to obtain a cyclic peptide,
In particular, the object is to obtain elcatonin. More specifically, the peptide represented by general formula (IIIa) is cyclized according to the method for cyclizing the peptide represented by general formula (III), or the peptide represented by general formula (IVa) is cyclized according to the method for cyclizing the peptide represented by general formula (I).
The resulting cyclic peptide represented by general formula (V), i.e., a fragment consisting of amino acids 1 to 6 or 1 to 9, is cyclized into the peptide on the solid-phase reaction resin represented by general formula (VI), i.e., a fragment consisting of amino acids 7 to 31 or 10.
The fragment represented by general formula (V) is condensed with a peptide consisting of amino acids 1 to 31 by solid-phase condensation. The C-terminal amino acid of the fragment represented by general formula (V) is preferably glycine for condensation reactivity and to prevent racemization. Therefore, elcatonin is preferably synthesized by condensing a peptide consisting of amino acids 1 to 9 of the cyclized peptide represented by general formula (V) with a peptide consisting of amino acids 10 to 31 of the peptide on the solid-phase reaction resin represented by general formula (VI).

The peptide on the solid-phase reaction resin represented by the general formula (VI) was prepared by R.B. Merrifield
According to the method developed by [D., J. Am. Chem. Soc., 85, 2149 (1963)], the compound can be synthesized by sequentially condensing protected amino acids, whose functional groups are protected with appropriate protecting groups, from the carboxyl terminal using the symmetric acid anhydride method, the activated ester method, or the like.

The resin used as the carrier is not particularly limited, but examples thereof include benzhydrylamine resin, p-methylbenzhydrylamine resin, and p-hydroxybenzoic acid resin, since the carboxyl terminus of the target peptide is an acid amide.

The method of reacting a cyclic peptide on a solid-phase reaction resin can be carried out in the same manner as in conventional solid-phase methods, and the amount of cyclic peptide represented by general formula (V) that can be used is, for example, approximately 1.0 to 3.0 equivalents relative to the solid-phase reaction resin.

The peptide having a protecting group synthesized on the solid-phase reaction resin is subjected to a conventional method for removing the protecting group, preferably an acid-based removal reaction, for example, an removal reaction using hydrogen fluoride, to obtain a cyclic peptide or elcatonin.

The reaction product can be isolated and purified by conventional separation and purification means such as gel filtration, ion exchange chromatography, partition chromatography, high performance liquid chromatography, reversed-phase high performance liquid chromatography, and electrophoresis.

The cyclic peptides and elcatonin obtained by the method of the present invention are obtained in the form of free peptides or their salts, depending on the reaction conditions. Free peptides and their salts can be mutually converted by conventional methods. To convert free peptides into pharmacologically acceptable salts, they can be reacted with, for example, inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, and phosphoric acid; or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid. Peptides and synthetic elcatonin form complexes with inorganic or organic substances. Examples of such substances include inorganic compounds derived from metals such as calcium, magnesium, aluminum, cobalt, and zinc, particularly slightly soluble salts of these metals, such as phosphates, pyrophosphates, and polyphosphates, hydroxides, and alkali metal polyphosphates.

Furthermore, to exert their pharmacological effects over a long period of time, cyclic peptides and synthetic elcatonin may be used in combination with organic substances, such as non-antigenic gelatin, carboxymethylcellulose, sulfonate or phosphate esters of alginic acid, dextran, glycols such as polyethylene glycol, phytic acid, polyglutamic acid, and protamine.

INDUSTRIAL APPLICABILITY The peptides of the present invention and the cyclic peptides obtained by the production method, particularly elcatonin, can be used for the treatment of hypercalcemia, osteoporosis, etc.

Examples and Reference Examples of the present invention will be shown below, but the present invention is not limited to these examples.

The amino acid analyses in the Examples and Reference Examples were all
The specimen was hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours.
This was carried out after drying under reduced pressure.

Example 1 1.15 g of the product was dissolved in 5 ml of TFA under ice cooling, and the mixture was left to stand for 30 minutes, then treated with ether, and the precipitated product was collected by filtration.
After drying under vacuum over potassium hydroxide, (CH ₂ ) ₅ CO−Ser(Bzl)−Asn−OH | TFA·H−Leu−Ser(Bzl)−Thr(Bzl)−HNCH−COOCH ₃
1.09 g (yield 97.1%) was obtained.

mp: 85-88°C ▲[α] ²⁴ _D ▼: 14.7° (C=1, DMF) Rf: 0.17 (chloroform:methanol:water=40:10:1) The resulting product was dissolved in 1 ml of DMF and neutralized with 0.12 ml of N-methylmorpholine under ice cooling, followed by addition of 70.1 mg of NaCl and 89.
5mg, LiCl50.9mg, CsCl202mg, EDC・HCl0.45g, HOBt・H
0.35 g of _{H 2} O was added. After stirring at room temperature for 96 hours, the mixture was concentrated under reduced pressure. 100 ml of H ₂ O was added to the residue, and the resulting oil was dissolved in 50 ml of H ₂ O.
After washing with 1 ml of 50% methanol, 50 ml of 50% methanol was added and the mixture was heated at 40°C for 30 minutes.
The mixture was incubated for 10 minutes. The resulting insoluble matter was filtered, and the filtrate was passed through a column (0.8 x 5 cm) packed with ion exchange resin. The eluate was concentrated under reduced pressure, and the residue was dried in vacuo to obtain 0.72 g (70.3% yield) of the desired product as a powder.

mp: 201-201°C ▲[α] ²⁵ _D ▼: -24.7° (C=1, DMF) Rf: 0.50 (chloroform:methanol=9:1) Amino acid analysis values: Asp 1.09 (1), Thr 0.97 (1), Ser 1.63 (2), Leu 1.
22(1), aminosuberic acid 1.09(1) Example 2 Boc−Lys(Cl−Z)−Leu−Ser(Bzl)−Gln−Glu(OBz
l) −Leu−His(Tos)−Lys(Cl−Z)−Leu−Gln−Thr
(Bzl)−Tyr(Br−Z)−Pro−Arg(Tos)−Thr(Bz
l) −Asp(OBzl)−Val−Gly−Ala−Gly−Thr(Bzl)−
756 mg of Pro-NH-resin was mixed with 20 ml of 30% TFA in methylene chloride.
The resin was diluted with methylene chloride 2 l and stirred at room temperature for 30 minutes.
The resulting solution was washed six times with 20 ml of 7% N-methylmorpholine in methylene chloride for 1.5 minutes, twice with 20 ml of 7% N-methylmorpholine in methylene chloride for 1.5 minutes, and then six times with 20 ml of methylene chloride for 1.5 minutes to obtain H-Lys(C1-Z)-Leu-Ser(Bzl)-Gln-Glu(OBzl).
l) −Leu−His(Tos)−Lys(Cl−Z)−Leu−Gln−Thr
(Bzl)−Tyr(Br−Z)−Pro−Arg(Tos)−Thr(Bz
l) −Asp(OBzl)−Val−Gly−Ala−Gly−Thr(Bzl)−
Pro-NH-resin was obtained.

Add the above resin to 20 ml of NMP and Add 215 mg of HOBt.H ₂ O, 64.5 mg of HOBt.H 2 O, and 86.8 mg of DCC, and let the mixture stand for 4 minutes at room temperature.
After the reaction was completed, the resin was washed with NMP, DMF, methanol, and methylene chloride in that order, and then dried under reduced pressure to obtain the target compound. Lys(C1−Z)−Leu−Ser(Bzl)−Gln−Glu(OBzl)−
Leu−His(Tos)−Lys(Cl−Z)−Leu−Gln−Thr(Bz
l) −Tyr(Br−Z)−Pro−Arg(Tos)−Thr(Bzl)−A
sp(OBzl)−Val−Gly−Ala−Gly−Thr(Bzl)−Pro−N
834 mg of H-resin was obtained.

Example 3 To 834 mg of resin, 0.9 ml of anisole and 9.0 ml of hydrogen fluoride were added and stirred at 0°C for 1 hour. After that, excess hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ethyl ether, extracted with 50 ml of 1M aqueous acetic acid solution, and freeze-dried to obtain crude elcatonin 35%.
2 mg was obtained.

The resulting crude elcatonin (352 mg) was loaded onto a column packed with cation exchange resin and eluted with a linear gradient of ammonium acetate buffer. The eluate was analyzed by high-performance liquid chromatography, and fractions containing high concentrations of the target elcatonin were collected and lyophilized to obtain 100 mg of intermediately purified elcatonin.

100 mg of the obtained intermediate purified elcatonin was subjected to high performance liquid chromatography (column: TSK GEL ODS manufactured by Tosoh Corporation).
120T, 2.15 x 30 cm, elution: 0.1% TFA/CH ₃ CN = 63/37, flow rate: 8 ml/min), and 70 mg of elcatonin TFA salt was obtained.
obtained.

The elcatonin TFA salt was injected onto a column packed with a cation exchange resin and then eluted with an aqueous ammonium acetate solution.
pH gradient elution was performed. Fractions with an absorption wavelength of 280 nm were collected and lyophilized to obtain 64 mg of elcatonin acetate powder.

mp: 240°C (decomposition) ▲[α] ²⁰ _D ▼: -93.1° (C = 0.25, 0.1M aqueous acetic acid solution) Rf: 0.39 (upper layer of water: n-butanol: acetic acid = 5:4:1) Amino acid analysis values: Asp 2.07 (2), Thr 3.69 (4), Ser 2.59 (3), Glu 3.
03 (3), Gly3.13 (3), Ala1.10 (1), Val2.06
(2), Leu5.29 (5), Tyr1.03 (1), Lys2.08
(2), His0.84 (1), Arg1.02 (1), Pro2.03
(2), aminosuberic acid 1.05 (1) Reference Example 1 7.74 g of Boc-Thr(Bzl)-OH was dissolved in 40 ml of THF and heated at −15° C.
The mixture was cooled to 100°C, and 2.56 ml of N-methylmorpholine and then 3.30 ml of isobutyl chloroformate were added dropwise.
The reaction mixture was stirred for 1 minute to prepare the corresponding mixed acid anhydride.
g and 3.50 ml of triethylamine in DMF solution,
After stirring at 0°C for 5 minutes and at room temperature for 1 hour, the mixture was concentrated under reduced pressure. 400 ml of ethyl acetate was added to the residue, and 100 ml of 10% citric acid was added.
twice with 100ml of saturated saline, twice with 100ml of cooled 5% sodium bicarbonate solution
The mixture was washed five times with 100 ml of saturated saline solution and twice with 100 ml of saturated saline solution.
Each wash was extracted twice with 300 ml of ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
11.56 g of an oily product consisting of the target substance was obtained.

Reference example 2 6.58 g of the oily product was dissolved in 40 ml of THF, and the resulting mixture was cooled with ice and then cooled with HO.
A THF solution of 1.53 g of Su and 2.74 g of DCC was added, and the mixture was stirred for 20 hours at 4° C. A white substance consisting of dicyclohexylurea (DCU) was removed, and THF was distilled off to obtain 7.87 g of an oily substance consisting of the target product.

Reference Example 3: Preparation of Boc-Ser(Bzl)-Asn-OH 4.50 g of H-Asn-OH·H ₂ O was suspended in 80 ml of H ₂ O, and the suspension was cooled with ice and added with 4.20 ml of triethylamine and 14.13 g of Boc-Ser(Bzl)-OSu.
80 ml of a THF solution of the above was added, and the mixture was stirred at room temperature for 20 hours, after which the THF was distilled off. The remaining aqueous solution was adjusted to pH 3 with 1 M citric acid under ice-cooling.
After adjusting the concentration to 100%, the resulting oil was extracted with 200 ml of ethyl acetate and washed twice with 50 ml of saturated saline. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The residue was treated with ether/petroleum ether (2/1 mixture) to solidify, and reprecipitated from methanol-isopropyl ether to give Boc-Ser.
8.74 g (71.2% yield) of (Bzl)-Asn-OH was obtained.

mp: 135-139°C ▲[α] ²² _D ▼: 10.2° (C=1, DMF) Rf: 0.54 (chloroform:methanol:acetic acid=7:2:1) Amino acid analysis values: Asp 1.14 (1), Ser 0.86 (1) Reference Example 4 5.45 g of Boc-Ser(Bzl)-Asn-OH was dissolved in 35 ml of TFA under ice cooling, and after standing for 30 minutes, it was treated with ether/petroleum ether (1/2 mixture). The precipitated product was collected by filtration and dried in vacuo over potassium hydroxide to give H-Ser(Bzl)-
Asn-OH·TFA was obtained.

The resulting product was dissolved in 35 ml of DMF, neutralized with triethylamine under ice cooling, and then 35 ml of a DMF solution containing 7.87 g of the mixture was added. The mixture was stirred at 0°C for 1 hour and at 4°C for 40 minutes.
After stirring for 2 hours, a small amount of insoluble matter was filtered off and the mixture was concentrated under reduced pressure. 300 ml of ethyl acetate was added to the residue, and the mixture was washed with 50 ml of cold 10% citric acid three times and with 50 ml of H ₂ O twice in this order.
The residue was concentrated under reduced pressure, and the addition and distillation of 30 ml of ethanol was repeated three times to dehydrate it, then treated with ether to solidify it, and reprecipitated from methanol-ether to obtain 9.07 g (yield 86.8%) of the target compound.

mp: 126-128°C ▲[α] ^26D _▼ : 4.6° (C=1, DMF) Rf: 0.49 (chloroform:methanol:water=70:30:3) Amino acid analysis values: Asp 1.11 (1), Thr 0.97 (1), Ser 0.81 (1), aminosuberic acid 1.11 (1) Reference Example 5 8.88 g of this was dissolved in 60 ml of TFA under ice cooling, and after standing for 40 minutes, it was treated with ether/hexane (1/2 mixture). The resulting oil was washed with hexane under cooling and then dried in vacuo over potassium hydroxide.

The resulting product was dissolved in 60 ml of DMF, neutralized with triethylamine under ice cooling, and then added to Boc-Ser(Bzl)-OSu5.
A solution of 34 g of the filtrate in 40 ml of DMF was added. The mixture was stirred at 0°C for 30 minutes and at room temperature for 12 hours, and then concentrated under reduced pressure. The residue was added to 300 ml of ethyl acetate.
The residue was washed twice with 50 ml of 10% citric acid and twice with 50 ml of _H2O , and then concentrated under reduced pressure. The residue was dehydrated by adding 30 ml of ethanol and distilling off the ethanol three times, and then treated with isopropyl ether to solidify it. The solid was then reprecipitated from methanol-isopropyl ether to give 8.60 g of the target compound (yield 7.60).
9.0%).

mp: 132-133°C ▲[α _]D ^▼ : 6.5° (C=1, DMF) Rf: 0.56 (chloroform:methanol:water=70:30:3) Amino acid analysis values: Asp 1.17 (1), Thr 1.01 (1), Ser 1.70 (2), aminosuberic acid 1.12 (1) Example 4 8.38 g of the product was dissolved in 50 ml of TFA under ice cooling, and after standing for 30 minutes, it was treated with ether-petroleum ether (1:2 mixture). The resulting oil was washed with petroleum ether under cooling and then dried under vacuum over potassium hydroxide.

The resulting product was dissolved in 60 ml of DMF, neutralized with triethylamine under ice cooling, and then added to a DMF solution of 3.00 g of Boc-Leu-OSu.
After stirring at 0°C for 30 minutes and at room temperature for 16 hours, the mixture was concentrated under reduced pressure. 300 ml of ethyl acetate was added to the residue, and 10
The residue was washed twice with 50 ml of 5% citric acid and twice with 50 ml of _H2O , and then concentrated under reduced pressure. The residue was dehydrated by adding 30 ml of ethanol and distilling off the ethanol three times, then treated with ether to solidify the solid, and reprecipitated from methanol-isopropyl ether to obtain 7.92 g of the target compound (yield 84.6%).

mp: 150-151°C ▲[α] ²⁶ _D ▼: 3.3° (C=1, DMF) Rf: 0.60 (chloroform:methanol:water=70:30:3) Amino acid analysis values: Asp 1.11 (1), Thr 0.98 (1), Ser 1.67 (2), Leu 1.
14 (1), aminosuberic acid 0.91 (1) Reference Example 6 0.76 g of this product was dissolved in 40 ml of DMF, and 0.78 ml of 2N aqueous sodium hydroxide solution was added under ice cooling. After stirring at 0°C for 2 hours, 0.40 ml of 2N aqueous sodium hydroxide solution was added. After stirring at 0°C for 1 hour, the product was neutralized with 2N hydrochloric acid and concentrated under reduced pressure. The residue was treated with H ₂
20 ml of 0 was added, and the mixture was adjusted to pH 3 with 2N hydrochloric acid under ice cooling, and the precipitated product was collected by filtration to obtain 0.70 g (yield 94.6%) of the target compound.

mp: 166-169°C ▲[α] ²⁵ _D ▼: -19.4° (C=1, DMF) Rf: 0.64 (chloroform:methanol:water=70:30:3) Amino acid analysis values: Asp 1.10 (1), Thr 0.98 (1), Ser 1.62 (2), Leu 1.
19(1), aminosuberic acid 1.10(1) Reference Example 7 Preparation of Boc-Leu-Gly-OPac 9.87 g of TosOH·H-Gly-OPac was dissolved in 30 ml of DMF and heated at -15°C.
The mixture was cooled to 50°C and adjusted to pH 7 with N-methylmorpholine.

On the other hand, 7.48 g of Boc-Leu-OH·H ₂ O and 4.59 g of HOBt·H ₂ O were dissolved in 1 mL of DMF.
The mixture was cooled to 0°C and heated to 6.1°C.
20 ml of a THF solution of 9 g of the mixture was added, and the mixture was stirred for 30 minutes while being cooled to 0° C. This solution was added to the above solution, and the mixture was stirred at −15° C. for 1 hour at 0° C.
The mixture was stirred at 4° C. for 2 hours and at 4° C. overnight. After the reaction was completed, the precipitate was collected by filtration and concentrated under reduced pressure.
The residue was concentrated under reduced pressure, and petroleum ether was added to the residue. The resulting precipitate was collected by filtration to give 8.93 g (yield 81.3%) of the desired product, Boc-Leu-Gly-OPac.

mp: 113-113.5°C ▲[ _α]20D ^▼ : -29.4°C (C=1, methanol) Rf: 0.66 (ethyl acetate:hexane=2:1) Amino acid analysis: Gly 0.95 (1), Leu 1.05 (1) Reference Example 8 Preparation of Boc-Val-Leu-Gly-OPac 4.00 g of Boc-Leu-Gly-OPac was dissolved in 50 ml of 4N hydrochloric acid/dioxane under cooling, allowed to stand for 1 hour, and then concentrated under reduced pressure to half the volume. Ether-petroleum ether was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The resulting dried product was dissolved in 40 ml of DMF and adjusted to pH 7 with N-methylmorpholine under cooling to -5°C. Meanwhile, Boc
Dissolve 2.57 g of −Val−OH and 1.81 g of HOBt·H ₂ O in 20 ml of DMF.
Under ice cooling, 15 ml of a THF solution of 2.44 g of DCC was added, and the mixture was stirred for 30 minutes under ice cooling and then for 20 minutes at -5°C. This solution was added to the above solution, and the mixture was stirred for 1 hour at -4°C and then for 3 days at 4°C. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was extracted with 150 ml of ethyl acetate.
The ethyl acetate solution was washed with 1N aqueous citric acid, saturated aqueous sodium bicarbonate, and saturated brine in this order, and then dried over anhydrous magnesium sulfate. After concentrating under reduced pressure, ethyl ether was added to the residue, and the resulting precipitate was collected by filtration to obtain the desired Boc-Val-Leu-G
4.12 g (yield 84.4%) of ly-OPac was obtained.

mp: 148.5-151°C ▲[α] ^26D _▼ : -21.5° (C=1, DMF) Rf: 0.51 (chloroform:methanol:acetic acid=90:10:1) Reference Example 9 506 mg of Boc-Val-Leu-Gly-OPac was dissolved in 5 ml of TFA under cooling, and the solution was left to stand for 30 minutes. Petroleum ether was added to the solution.
The resulting oil was washed several times with petroleum ether and dried over potassium hydroxide under reduced pressure. The resulting dried product was dissolved in 20 ml of DMF and the pH was adjusted to 7 with N-methylmorpholine under ice-cooling.

113 mg of HOBt·H ₂ O and 141 mg of EDC·HCl were added to the solution;
The mixture was stirred at room temperature for 31 hours. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was solidified by adding 10% aqueous citric acid solution under ice cooling, and then washed with 1N aqueous citric acid solution, 5% aqueous sodium bicarbonate solution, and H ₂ O in that order, and concentrated under reduced pressure. The dried product was further reprecipitated with methanol-ethyl acetate, and the precipitate was collected by filtration to obtain the target compound. 296 mg (yield 45.3%) was obtained.

mp: 263°C (field) ▲[α] ²⁵ _D ▼: -17.9° (C=1, DMSO) Rf: 0.57 (chloroform:methanol=6:1) Amino acid analysis values: Asp 1.04 (1), Thr 0.93 (1), Ser 1.51 (2), Gly 1.
10 (1), Val 1.10 (1), Leu 2.29 (2), aminosuberic acid 1.04 (1) Reference Example 10 296 mg of the product was dissolved in 15 ml of DMF, and 7 ml of acetic acid and 131 ml of zinc powder were added.
After the reaction was completed, the mixture was filtered, the filtrate was concentrated, water was added to the residue, and the precipitate was collected by filtration, washed with water, and dried under reduced pressure in the presence of diphosphorus pentoxide.
The dried product was further reprecipitated with methanol-ethyl ether, and the precipitate was collected by filtration to obtain the target product. 233 mg (yield 86.4%) was obtained.

mp: 265°C (field) ▲[α] ²⁵ _D ▼: -17.9° (C=1, DMSO) Rf: 0.46 (chloroform:methanol:water=70:30:3) Amino acid analysis values: Asp 1.05 (1), Thr 0.93 (1), Ser 1.53 (2), Gly 1.
12 (1), Val 1.11 (1), Leu 2.24 (2), aminosuberic acid 1.02 (1) Reference Example 11 Boc-Lys(C1-Z)-Leu-Ser(Bzl)-Gln-Glu(OBz
l) −Leu−His(Tos)−Lys(Cl−Z)−Leu−Gln−Thr
(Bzl)−Tyr(Br−Z)−Pro−Arg(Tos)−Thr(Bz
l) −Asp(OBzl)−Val−Gly−Ala−Gly−Thr(Bzl)−
Preparation of Pro-NH resin Paramethylbenzhydrylamine polystyrene resin (0.
42 mmol _NH2 /g resin, 1% divinylbenzene, 100-20
Using 1.19 g of 100 mesh (Peptide Institute, Inc.) as a starting carrier, the following amino acid derivatives (all manufactured by Peptide Institute, Inc.) were sequentially subjected to condensation and Boc removal reactions by the solid phase method.

Boc−Pro−OH 430mg Boc−Thr(Bzl)−OH 618mg Boc−Gly−OH 350mg Boc−Ala−OH 378mg Boc−Gly−OH 350mg Boc−Val−OH 434mg Boc−Asp(OBzl)−OH 647mg Boc−Thr(Bzl)−OH 618mg Boc−Arg(Tos)−OH 1008mg×2 Boc−Pro−OH 430mg Boc−Tyr(Br−Z)−OH 989mg Boc−Thr(Bzl)−OH 618mg Boc−Gln−OH 492mg×2 Boc−Leu−OH 500mg Boc−Lys(Cl−Z)−OH 830mg Boc−His(Tos)−OH 818mg Boc−Leu−OH 500mg Boc-Glu(OBzl)-OH 674 mg Boc-Gln-OH 492 mg x 2 Boc-Ser(Bzl)-OH 590 mg Boc-Leu-OH 500 mg Boc-Lys(Cl-Z)-OH 830 mg Of these amino acid derivatives, Boc-Arg(Tos)-OH
For the condensation of Boc-Gln-OH, the HOBt ester of the amino acid derivative was formed by the DCC-HOBt method and then subjected to the condensation reaction. However, this condensation reaction was repeated twice. For other amino acid derivatives, the symmetric acid anhydride of the amino acid derivative was formed by the DCC method and then subjected to the condensation reaction.

In this way, Boc-Lys(C1-Z)-Leu-Ser(Bz
l) −Gln−Glu(OBzl)−Leu−His(Tos)−Lys(Cl−
Z) −Leu−Gln−Thr(Bzl)−Tyr(Br−Z)−Pro−Ar
g(Tos)−Thr(Bzl)−Asp(OBzl)−Val−Gly−Ala−
2.18 g of Gly-Thr(Bzl)-Pro-NH-resin was obtained.

Example 5 108 mg of the product was dissolved in 0.4 ml of TFA and allowed to stand at room temperature for 1 hour. After distilling off the TFA under reduced pressure, the product was treated with isopropyl ether, and the precipitate was collected by filtration, washed with isopropyl ether, and dried over potassium hydroxide under reduced pressure to obtain 102 mg of the target compound (yield 97.1%).

mp: 170-172°C ▲[α] ²⁴ _D ▼: 3.7° (C=1, DMF) Rf: 0.19 (chloroform:methanol:water=40:10:1) Reference Example 12 (1) Preparation of Boc-Ser(Bzl)-OPac: 14.77 g of Boc-Ser(Bzl)-OH and 9.95 g of phenacyl bromide
The product was dissolved in 100 ml of ethyl acetate, and 7 ml of triethylamine was added under ice cooling, followed by stirring for 1 hour and then at room temperature for 4 hours.
The reaction mixture was washed with saturated aqueous sodium bicarbonate and then saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure.
The residue was treated with n-hexane to solidify it, and ethyl acetate-
Reprecipitation from n-hexane gave 18.1 g of the target compound (yield 87.6%).
%) was obtained.

mp: 67-68°C ▲[α] ^24D _▼ : -9.8° (C=1, methanol) Rf: 0.87 (chloroform:methanol:water=90:10:1) (2) Preparation of Boc-Leu-Ser(Bzl)-OPac 9.5 g of Boc-Ser(Bzl)-OPac was dissolved in 20 ml of TFA under ice cooling and allowed to stand at room temperature for 1 hour. The solution was again ice-cooled and
5.8 ml of 4N hydrochloric acid/dioxane was added, and the mixture was stirred and shaken.
n-Hexane was added, and the precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The dried product was dissolved in 20 ml of DMF.
and cooled to -10°C or below, and
2.35 ml was added and the pH was adjusted to 7.

On the other hand, 5.73 g of Boc-Leu-OH·H ₂ O and 3.87 g of HOBt·H ₂ O were
Dissolve in 10 ml of DMF, cool to below -10°C, and then add EDC/HCl.
A solution of 4.85 g of the mixture in cold DMF (50 ml) was added, and the mixture was stirred under ice-cooling for 30 minutes.

This solution was cooled again to below -10°C, then added to the DMF solution, and stirred at -15°C to -10°C for 2 hours, then at 4°C for 20 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The extract was washed five times with 1N aqueous citric acid solution, five times with saturated saline, ten times with saturated sodium bicarbonate water, and five times with saturated saline, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Diethyl ether/petroleum ether (1/4 mixture) was added to the residue to solidify it, and ethyl acetate-
The product was reprecipitated from diethyl ether/petroleum ether (1/3 mixture) to obtain 9.71 g (yield 80.1%) of the target product.

mp: 100-101°C ▲[α] ²⁴ _D ▼: -17.1° (C=1, methanol) Rf: 0.66 (chloroform:methanol:water=90:10:1) (3) Preparation of Boc-Leu-Ser(Bzl)-OH 9.71 g of Boc-Leu-Ser(Bzl)-OPac was dissolved in 100 ml of 90% aqueous acetic acid.
The resulting solution was dissolved in 1 ml of ethanol, and 36.1 g of zinc powder was added under ice cooling. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated and then extracted with ethyl acetate. The extract was washed five times with 1N aqueous citric acid and five times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Petroleum ether was added to the residue to solidify it, and the solid was reprecipitated with ethyl acetate-petroleum ether to obtain 6.97 g of the desired product (yield 92.7%).

mp: 73-75°C ▲[α] ²⁴ _D ▼: 3.0° (C=1, methanol) Rf: 0.31 (chloroform:methanol:water=85:15:1) 2.0 g of Boc-Ser(Bzl)-OPac was dissolved in 5 ml of 4N hydrochloric acid/dioxane under ice cooling, and the mixture was left at room temperature for 1 hour.
The precipitated product was collected by filtration, dried in vacuo over potassium hydroxide, and purified by HCl.H-Ser(Bzl)-O
The resulting product was dissolved in 5 ml of DMF and heated at −10° C.
The mixture was then neutralized with N-methylmorpholine under cooling.

Dissolve 2.61g of HCl and _0.89g of HOBt.H2O in 10ml of DMF and heat at -10°C.
After cooling to below 30 ml, a cold DMF solution of 1.11 g of EDC·HCl was added, and the mixture was stirred for 30 minutes under ice cooling.

This solution was cooled again to below -10°C, then added to the DMF solution, and stirred at -15°C to -10°C for 2 hours, then at 4°C for 18 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate/diethyl ether (1/1 mixture). The extract was washed five times with 1N aqueous citric acid solution, five times with saturated saline, ten times with saturated sodium bicarbonate water, and then five times with saturated saline.
After washing twice, the mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was diluted with diethyl ether/petroleum ether (1/4
The solid was solidified by treatment with ethyl acetate-diethyl ether/petroleum ether (1/1 mixture) and reprecipitated with ethyl acetate-diethyl ether/petroleum ether to obtain 2.44 g (yield 64.4%) of the target product.

mp: 65-67°C ▲[α] ²⁴ _D ▼: -5.2° (C=1, methanol) Rf: 0.64 (chloroform:methanol:water=90:10:1) 2.3 g of the product was dissolved in 5 ml of TFA under ice cooling, and the mixture was allowed to stand at room temperature for 1 hour. The solution was again ice-cooled, and 2.2 ml of 4N hydrochloric acid/dioxane was added.
After stirring and shaking, diethyl ether/petroleum ether (1/1 mixture) was added. The precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The dried product was dissolved in 10 ml of DMF, cooled to below -10°C, and then N-
Neutralized with methylmorpholine.

On the other hand, 1.61 g of Boc-Leu-Ser(Bzl)-OH and 1.61 g of HOBt.H
_2000g (0.66g) was dissolved in 10ml of DMF and cooled to below -10℃.
30 ml of a cold DMF solution of 0.82 g of EDC·HCl was added and stirred under ice cooling for 30 minutes. This solution was cooled again to below -10°C and then added to the previous DMF solution. The mixture was stirred at -15°C to -10°C for 2 hours, then at 4°C for 56 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The extract was washed five times with 1N aqueous citric acid, five times with saturated saline, and five times with saturated sodium bicarbonate water.
The residue was washed 10 times and then 5 times with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was solidified by adding diethyl ether/petroleum ether (1/1 mixture). The product was reprecipitated from ethyl acetate/diethyl ether to obtain 1.47 g (46.8% yield).

mp: 96-98°C ▲[α] ²⁴ _D ▼: -11.2° (C=1, methanol) Rf: 0.68 (chloroform:methanol:water=90:10:1) 1.35 g of the product was dissolved in 5 ml of TFA under ice-cooling and left at room temperature for 1 hour. The solution was ice-cooled again, and 0.94 g of 4N hydrochloric acid/dioxane was added. After stirring and shaking, the mixture was treated with diethyl ether. The precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The dried product was dissolved in 10 ml of DMF and diluted to -10
After cooling to below 100°C, the mixture was neutralized with N-methylmorpholine.

On the other hand, 0.44 g of Boc-Asn-OH and 0.32 g of HOBt·H ₂ O were dissolved in 10 ml of DMF.
After cooling to below -10°C, 20 ml of a cold DMF solution of 0.40 g of EDC·HCl was added and stirred for 30 minutes under ice-cooling. This solution was cooled again to below -10°C, added to the previous DMF solution, and stirred at -10°C to -5°C for 30 minutes, then for 2 hours under ice-cooling, and then for 21 hours at 4°C. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified by adding 1N aqueous citric acid under ice-cooling. The precipitate was collected by filtration, and then 1N aqueous citric acid, H ₂
The residue was washed with 200 ml of saturated sodium bicarbonate solution, followed by H ₂ O, and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was suspended in methanol, and a 1/2 mixture of ethyl acetate and diethyl ether was added. The precipitate was collected by filtration to obtain 1.22 g of the desired product (yield: 81.9%).

mp: 177-179°C ▲[α] ²⁴ _D ▼: -12.7° (C=1, DMF) Rf: 0.54 (chloroform:methanol:water=85:15:1) Dissolve 0.63 g of zinc powder in 7 ml of 90% acetic acid solution and add 1.
04g was added and stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was filtered, the filtrate was concentrated, and _H2O was added to the residue to solidify it. The precipitate was collected by filtration and diluted with _H2O , 50mM ammonium bicarbonate buffer (pH 8.0) containing 25mM EDTA, _H2O , 1N
The mixture was washed with an aqueous solution of citric acid and then with H ₂ O, and dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated with DMF-diethyl ether to obtain 0.41 g (yield 71.9%) of the target compound.

mp: 174-176°C ▲[α] ²⁴ _D ▼: -8.7° (C=1, DMF) Rf: 0.35 (chloroform:methanol:water=40:10:1) Amino acid analysis values: Asp 1.13 (1), Thr 0.95 (1), Ser 1.52 (2), Leu 1.
21(1), aminosuberic acid 1.18(1) Example 6 108 mg of the product was dissolved in 0.4 mL of TFA and allowed to stand at room temperature for 1 hour. The TFA in the solution was removed by distillation under reduced pressure, and the solution was then treated with isopropyl ether. The precipitated product was collected by filtration, washed with isopropyl ether, and dried under reduced pressure over potassium hydroxide to obtain 101 mg of the desired product (yield 96.2%).

mp: 94-97°C ▲[α] ²⁴ _D ▼: 3.3° (C=1, DMF) Rf: 0.20 (chloroform:methanol:water=40:10:1) Reference Example 13 (1) Preparation of Boc-Leu-OPac 4.99 g of Boc-Leu-OH·H ₂ O and 3.98 g of phenacyl bromide were dissolved in 100 ml of ethyl acetate, and the solution was cooled with ice and added with triethylamine.
2.8 ml of the mixture was added, and the mixture was stirred for 1 hour and then at room temperature for 4 hours.
The reaction mixture was washed with saturated sodium bicarbonate water and then with saturated saline.
The residue was treated with n-hexane to solidify it, and the solid was extracted with ethyl acetate-n-
Reprecipitation from hexane gave 6.6 g of the target product (yield 94.4%).

mp: 71-72°C ▲[α] ^D _▼ : -47.5°C (C=1, methanol) Rf: 0.80 (chloroform:methanol:water=90:10:1) (2) Preparation of Boc-Ser(Bzl)-Asn-Leu-OPac 2.0 g of Boc-Leu-OPac was dissolved in 5 ml of TFA under ice-cooling and allowed to stand at room temperature for 1 hour. The solution was ice-cooled again, and then 1.43 ml of 4N hydrochloric acid/dioxane was added. The mixture was stirred and shaken, and then treated with n-hexane. The resulting precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The resulting dried product was dissolved in 5 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

On the other hand, 2.34 g of Boc-Ser(Bzl)-Asn-OH and HOBt·H ₂ O
0.96 g was dissolved in 1.0 ml of DMF, cooled to below −10°C, and then
30 ml of a cold DMF solution containing 1.21 g of DC·HCl was added, and the mixture was stirred for 30 minutes under ice cooling.
Add to F solution and incubate at -10 to -5°C for 30 minutes under ice-cooling.
After stirring for 1 hour at 4° C., the reaction mixture was stirred for 16 hours at 4° C. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified by adding 1N aqueous citric acid solution. The precipitate was collected by filtration, and the resulting mixture was diluted with 1N aqueous citric acid solution, H ₂ O,
The mixture was washed with saturated aqueous sodium bicarbonate and then with H ₂ O, and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated with ethyl acetate-diethyl ether to obtain 2.33 g (yield 63.5%) of the target compound.

mp: 133-135°C ▲[α] ²⁴ _D ▼: -9.6° (C=1, DMF) Rf: 0.33 (chloroform:methanol:water=90:10:1) 2.11 g of Boc-Ser(Bzl)-Asn-Leu-OPac was added to TF
The mixture was dissolved in 7 ml of DMF and allowed to stand at room temperature for 1 hour. The solution was ice-cooled again, and 2.5 ml of 4N hydrochloric acid/dioxane was added. After stirring and shaking, the mixture was treated with diethyl ether. The precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The resulting dried product was dissolved in 5 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

on the other hand, Dissolve 2.44g of HCl and _0.84g of HOBt.H2O in 12ml of DMF and heat at -10°C.
After cooling to below -10°C, 20 ml of a cold DMF solution of 1.04 g of EDC·HCl was added and stirred for 30 minutes under ice-cooling. The solution was cooled again to below -10°C and added to the previous DMF solution. The mixture was stirred at -10°C to -5°C for 30 minutes, then under ice-cooling for 2 hours, and then at 4°C for 60 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified by adding 1N aqueous citric acid. The precipitate was collected by filtration and washed with 1N aqueous citric acid, _H2O , saturated aqueous sodium bicarbonate, and then _H2O .
The precipitate was dried under reduced pressure in the presence of diphosphorus pentoxide, and the dried product was washed with methanol to obtain 2.45 g (yield 77.5%) of the target product.

mp: 184-186°C ▲[α] ²⁴ _D ▼: -8.4° (C=1, DMF) Rf: 0.39 (chloroform:methanol:water=90:10:1) 2.3 g of this product was dissolved in 6 ml of TFA under ice-cooling and allowed to stand at room temperature for 1 hour. The solution was ice-cooled again, and 1.7 ml of 4N hydrochloric acid/dioxane was added. After stirring and shaking, the mixture was treated with diethyl ether. The precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The resulting dried product was dissolved in 10 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

On the other hand, 1.0 g of Boc-Ser(Bzl)-OH and 0.57 g of HOBt·H _{2 O}
Dissolve in 10 ml of DMF, cool to below -10°C, and then add EDC.
A cold DMF solution of 10.71 g of 10.71 g was added to 20 ml of the mixture and stirred under ice-cooling for 30 minutes. This solution was cooled again to below -10°C and added to the DMF solution. The mixture was stirred at -15°C to -10°C for 2 hours, then at 4°C for 20 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified with 1N citric acid solution, and the precipitate was collected by filtration. 1N citric acid solution, _H2O , saturated sodium bicarbonate solution, and then _H2O were added.
After washing with HCl, the mixture was dried under reduced pressure in the presence of diphosphorus pentoxide.
The dried product was suspended in methanol, and ethyl acetate/diethyl ether (1/2 mixture) was added. The precipitate was collected by filtration, and the target product was obtained.
1.91 g (70.7% yield) was obtained.

mp: 178-180°C ▲[α] ²⁴ _D ▼: -4.1° (C=1, DMF) Rf: 0.51 (chloroform:methanol:water=85:15:1) Dissolve 1.58 g of zinc powder in 20 ml of 90% acetic acid solution and add it to the
2.59g of EDTA was added and stirred at room temperature for 3 hours. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. _H2O was added to the residue to solidify it. The precipitate was collected by filtration and diluted with 50mM EDTA containing _H2O and 25mM EDTA.
The residue was washed with ammonium bicarbonate buffer (pH 8.0), _H2O , 1N aqueous citric acid solution, and then _H2O , and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated with DMF-diethyl ether to obtain 1.20 g of the target compound (yield 84.5%).

mp: 159-161°C ▲[α] ²⁴ _D ▼: 1.0° (C=1, DMF) Rf: 0.30 (chloroform:methanol:water 40:10:1) Amino acid analysis values: Asp 1.16 (1), Thr 0.93 (1), Ser 1.52 (2), Leu 1.
21(1), aminosuberic acid 1.17(1) Example 7 108 mg of the product was dissolved in 0.4 mL of TFA and allowed to stand at room temperature for 1 hour. The TFA in the solution was removed by distillation under reduced pressure, and the solution was then treated with isopropyl ether. The precipitated product was collected by filtration, washed with isopropyl ether, and dried under reduced pressure over potassium hydroxide to obtain 104 mg of the desired product (yield 99.0%).

mp: 157-160°C ▲[α] ²⁴ _D ▼: -7.5° (C=1, DMF) Rf: 0.30 (chloroform:methanol:water=40:10:1) Reference Example 14 (1) Boc−Ser(Bzl)−Asn−Leu−Ser(Bzl)−OPac
1.8 g of Boc-Leu-Ser(Bzl)-OPac was dissolved in 5 ml of TFA under ice-cooling and allowed to stand at room temperature for 1 hour.
2.6 ml of 4N hydrochloric acid/dioxane was added, and the mixture was stirred and shaken.
Diethyl ether was added, and the precipitate was collected by filtration.
The dried product was dried under reduced pressure over potassium hydroxide.
The solution was dissolved in 5 ml of F, cooled to below -10°C, and neutralized with N-methylmorpholine.

On the other hand, 1.47 g of Boc-Ser(Bzl)-Asn-OH and HOBt·H ₂ O
0.60 g was dissolved in 10 ml of DMF, cooled to below -10°C, and then
30 ml of a cold DMF solution containing 0.76 g of C.HCl was added, and the mixture was stirred for 30 minutes under ice cooling. This solution was cooled again to below -10°C, and the same DMF was added.
The mixture was added to the solution and stirred at -10°C to -5°C for 30 minutes, then under ice-cooling for 1 hour, and then at 4°C for 20 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified with 1N citric acid solution. The precipitate was collected by filtration, washed with 1N citric acid solution, _H2O , saturated sodium bicarbonate solution, and then _H2O , and then dried under reduced pressure in the presence of diphosphorus pentoxide. Reprecipitation from methanol-diethyl ether gave 2.38 g of the desired product (yield: 85.3%).

mp: 156-158℃ ▲[α] ²⁴ _D ▼: -10.4° (C=1, DMF) Rf: 0.34 (chloroform:methanol:water=90:10:1) Boc−Ser(Bzl)−Asn−Leu−Ser(Bzl)−OPac2.2g
The residue was dissolved in 7 ml of TFA under ice-cooling and allowed to stand at room temperature for 1 hour.
The solution was cooled again on ice, and 2.0 ml of 4N hydrochloric acid/dioxane was added.
After stirring and shaking, the mixture was treated with diethyl ether, and the resulting precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide.
The resulting dried product was dissolved in 10 ml of DMF, cooled to -10°C or below, and then neutralized with N-methylmorpholine.

on the other hand, 2.0 g of EDC·HCl and 0.68 g of HOBt·H ₂ O were dissolved in 12 ml of DMF and cooled to below −10° C., and then 20 ml of a cold DMF solution of 0.85 g of EDC·HCl was added and stirred for 30 minutes under ice cooling. After cooling again, this solution was added to the previous DMF solution and stirred at −10° C. to −5° C. for 30 minutes.
The mixture was stirred for 1 hour under ice-cooling and then for 62 hours at 4°C. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and 1N aqueous citric acid was added to the residue to solidify it. The precipitate was collected by filtration, washed with 1N aqueous citric acid, _H2O , saturated aqueous sodium bicarbonate, and then _H2O , and dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was washed with methanol to obtain 2.5 g of the desired product (yield 78.1%).

mp: 178-180°C ▲[α] ²⁴ _D ▼: -6.5° (C=1, DMF) Rf: 0.53 (chloroform:methanol:water=85:15:1) Dissolve 2.4 g of zinc powder in 30 ml of 90% acetic acid solution and add 3.
94g of ammonium bicarbonate was added and stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was filtered, the filtrate was concentrated, and _H2O was added to the residue to solidify it. The precipitate was collected by filtration and washed with _H2O , 50mM ammonium bicarbonate buffer (pH 8.0) containing 25mM EDTA, _H2O ,
The mixture was washed with 1N citric acid solution and then with H ₂ O, and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated with DMF-diethyl ether to obtain 1.95 g of the target product (yield: 90.1%).

mp: 175-177°C ▲[α] ²⁴ _D ▼: -1.6° (C=1, DMF) Rf: 0.32 (chloroform:methanol:water=40:10:1) Amino acid analysis values: Asp 1.19 (1), Thr 0.86 (1), Ser 1.64 (2), Leu 1.
26(1), aminosuberic acid 1.05(1) Example 8 108 mg of the product was dissolved in 0.4 ml of TFA and allowed to stand at room temperature for 1 hour. After distilling off the TFA under reduced pressure, the product was treated with isopropyl ether. The precipitated product was collected by filtration, washed with isopropyl ether, and then dried under reduced pressure over potassium hydroxide.
102 mg (yield 97.1%) of the target product was obtained.

mp: 165-167°C ▲[α] ²⁴ _D ▼: 3.6° (C=1, DMF) Rf: 0.28 (chloroform:methanol:water=40:10:1) Reference Example 15 (1) Preparation of Boc-Thr(Bzl)-OPac: 6.19 g of Boc-Thr(Bzl)-OH and 3.98 g of phenacyl bromide
The product was dissolved in 100 ml of ethyl acetate, and 2.8 ml of triethylamine was added under ice cooling, followed by stirring for 1 hour and then at room temperature for 4 hours.
The reaction mixture was washed with saturated aqueous sodium bicarbonate and then saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 8.44 g of the desired product (yield 98.7%).

Dissolve 6.1 g of this in 50 ml of H ₂ O, add 4.2 ml of triethylamine under ice cooling, and
A solution of 7.2 g of t-butyl dicarbonate in cold dioxane/H ₂ O (4/1 mixture) was added, and the mixture was stirred for 15 minutes under ice-cooling, and then for 30 minutes at room temperature. After the reaction was completed, the dioxane in the reaction mixture was distilled off, and the aqueous layer was adjusted to pH 10 with triethylamine under ice-cooling, washed with ethyl acetate, and then the aqueous layer was
The pH was adjusted to 2 with 2M aqueous citric acid, and extracted with ethyl acetate. The extract was washed with 1N aqueous citric acid and then saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. n-Hexane was added to the residue, and the precipitate was collected by filtration and reprecipitated with ethyl acetate-n-hexane to obtain the target compound.
8.03 g (yield 88.2%) was obtained.

mp: 50-51°C ▲[α] ²⁴ _D ▼: -17.6° (C=1, methanol) Rf: 0.38 (chloroform:methanol:water=90:10:1) (3) Preparation of Boc-Leu-Ser(Bzl)-Thr(Bzl)-OPac. 2.0 g of Boc-Thr(Bzl)-OPac was dissolved in 5 ml of 4N hydrochloric acid/dioxane under ice cooling, allowed to stand at room temperature for 1 hour, and then treated with n-hexane. The resulting precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The resulting dried product was dissolved in 5 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

On the other hand, 2.01 g of Boc-Leu-Ser(Bzl)-OH and HOBt·H ₂ O
0.83 g was dissolved in 10 ml of DMF, cooled to below -10°C, and then
30 ml of a cold DMF solution containing 1.04 g of C.HCl was added, and the mixture was stirred for 30 minutes under ice cooling. This solution was cooled again to below -10°C, and the same DMF was added.
The solution was then heated at -15°C to -10°C for 2 hours, and then at 4°C.
The mixture was stirred for 20 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ethyl acetate. The extract was washed with 1N aqueous citric acid solution, saturated brine, saturated aqueous sodium bicarbonate, and then saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Petroleum ether was added to the residue to solidify it, and the precipitate was collected by filtration and reprecipitated from ethyl acetate-diethyl ether/petroleum ether (1/4 mixture) to give 2.54 g of the target product (yield 75.6%).
%) was obtained.

mp: 53-55°C ▲[α] ²⁴ _D ▼: -14.2° (C=1, methanol) Rf: 0.68 (chloroform:methanol:water=90:10:1) (4) Boc-Ser(Bzl)-Asn-Leu-Ser(Bzl)-Thr
Preparation of (Bzl)-OPac: 2.4 g of Boc-Leu-Ser(Bzl)-Thr(Bzl)-OPac was dissolved in 7 ml of TFA under ice cooling and allowed to stand at room temperature for 1 hour. The solution was ice-cooled again, and 2.5 ml of 4N hydrochloric acid/dioxane was added. After stirring and shaking, diethyl ether/petroleum ether (1/1 mixture) was added. The precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. This dried product was dissolved in 10 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

On the other hand, 2.12 g of Boc-Ser(Bzl)-Asn-OH and HOBt·H ₂ O
0.87 g was dissolved in 20 ml of DMF, cooled to below -10°C, and then
40 ml of a cold DMF solution containing 1.09 g of C.HCl was added, and the mixture was stirred for 30 minutes under ice cooling. This solution was cooled again to below -10°C, and the same DMF was added.
The resulting mixture was added to the solution and stirred at -10°C to -5°C for 30 minutes, then under ice-cooling for 2 hours, and then at 4°C for 45 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was solidified with 1N citric acid solution. The precipitate was collected by filtration and washed with 1N citric acid solution, _H2O , saturated sodium bicarbonate solution, and then _H2O , and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated from DMF-diethyl ether to obtain 2.83 g of the desired product (yield: 84.0%).

mp: 154-156℃ ▲[α] ²⁴ _D ▼: -6.2° (C=1, DMF) Rf: 0.36 (chloroform:methanol:water=90:10:1) Boc−Ser(Bzl)−Asn−Leu−Ser(Bzl)−Thr(Bzl)
2.7 g of OPac was dissolved in 8 ml of TFA under ice-cooling and left at room temperature for 1 hour. The solution was again ice-cooled and added with 2.0 ml of 4N hydrochloric acid/dioxane.
After stirring and shaking, diethyl ether was added and the precipitate was collected by filtration and dried under reduced pressure over potassium hydroxide. The dried product was dissolved in 10 ml of DMF, cooled to below -10°C, and neutralized with N-methylmorpholine.

on the other hand, 1.8g of EDC·HCl and 1.0g of HOBt· _H2O were dissolved in 30ml of DMF and cooled to -10°C or below, then 40ml of a cold DMF solution of 1.24g of EDC·HCl was added and stirred for 30 minutes under ice cooling. This solution was cooled again to -10°C or below, then added to the previous DMF solution and stirred at -10°C to -5°C.
The mixture was stirred at 4° C. for 30 minutes, under ice-cooling for 2 hours, and then at 4° C. for 65 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the residue was diluted with 1N
Add aqueous citric acid solution to solidify, filter the precipitate, and
The mixture was washed with an aqueous solution of citric acid, H ₂ O, saturated aqueous sodium bicarbonate, and then H ₂ O, and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was suspended in methanol, and ethyl acetate/diethyl ether (1/2 mixture) was added. The precipitate was collected by filtration to give 2.77 g of the target compound.
(yield 86.6%).

mp: 165-167°C ▲[α] ²⁴ _D ▼: -8.8° (C=1, DMF) Rf: 0.50 (chloroform:methanol:water=85:15:1) Dissolve 1.44 g of the solution in 15 ml of 90% acetic acid solution, and add zinc powder under ice cooling.
2.37g of EDTA was added and stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated. _H2O was added to the residue to solidify it. The precipitate was collected by filtration and diluted with _H2O , 50mM ammonium bicarbonate buffer (pH 8.0) containing 25mM EDTA, and H2O.
The resulting mixture was washed with ₂₀ , 1N citric acid, and then _H2O , and then dried under reduced pressure in the presence of diphosphorus pentoxide. The dried product was reprecipitated with DMF-diethyl ether to give 1.13 g of the target compound (yield 86.8%).
obtained.

mp: 183-185°C ▲[α] ²⁴ _D ▼: -2.5° (C=1, DMF) Rf: 0.40 (chloroform:methanol:water=40:10:1) Amino acid analysis values: Asp 1.13 (1), Thr 0.93 (1), Ser 1.54 (2), Leu 1.
21(1), aminosuberic acid 1.19(1) Example 9 101 mg of the solution was dissolved in 100 ml of DMF, and 10.2 μl of N-methylmorpholine was added under ice cooling to neutralize _the solution.
g, EDC·HCl 21.1 mg was added, and the mixture was stirred at room temperature for 24 hours to obtain the desired cyclized product. 69 mg was obtained.

Claims (19)

[Claims] Claim 1: A compound represented by the following general formula (I): [Wherein A and B are the following (1) to (5)] (1) A: Ser(X1)-Asn-Leu-Ser(X1)-Thr(X1)
-OH B:X2 (2) A:Ser(X1)-Asn-Leu-Ser(X1)-OH B:X2-Thr(X1) (3) A:Ser(X1)-Asn-Leu-OH B:X2-Ser(X1)-Thr(X1) (4) A:Ser(X1)-Asn-OH B:X2-Leu-Ser(X1)-Thr(X1) (5) A:Ser(X1)-OH B:X2-Asn-Leu-Ser(X1)-Thr(X1) (wherein X1 represents a hydrogen atom or a protecting group for a hydroxyl group, and X2 represents a hydrogen atom or a protecting group for an amino group). X represents a hydroxyl group, a protecting group for a carboxyl group, an amino acid residue represented by Val-OH or Met-OH, or a group represented by the following general formula (VII)-A1-Leu-A2-OH (VII) (wherein A1 represents Val or Met, and A2 represents Ser or Gly).
or a lower peptide residue represented by the following general formula (VIII):
1−A12−A13−A14−A15−A16−A17−A18−A19−Gly−A2
0-A21-A22-Pro- _NH2 (VIII) (wherein A1 and A2 are the same as above. A3 to A22 represent the following amino acid residues: A3: Lys, Thr or Ala A4: Leu or Tyr A5: Ser, Thr or Trp A6: Gln, Lys or Arg A7: Glu, Asp or Asn A8: Leu or Phe A9: His or Asn A10: Lys or Asn A11: Leu, Phe or Tyr A12: Gln or His A13: Thr or Arg A14: Tyr or Phe A15: Pro or Ser A16: Arg, Gly or Gln A17: Thr or Met A18: Asp, Ala, Asn or Gly A19: Val, Ile, Thr or Phe A20 A21: Ala, Val, Pro or Ser; A21: Gly or Glu; A22: Thr or Ala), or a peptide residue represented by the general formula (VIII) containing at least -A1-Leu-A2-A3 and A1 to A22
wherein each amino acid residue may be protected with a protecting group], or an acid addition salt or complex thereof. Claim 2: The carboxyl-protecting group represented by X is
2. The peptide according to claim 1, wherein the amino group is an alkoxy group, a cycloalkyloxy group, an aralkyloxy group which may have a substituent, a substituted hydrazino group, or a phenacyl group, or an acid addition salt or complex thereof. 3. X is -Val-Leu-Gly-OH; or -Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His
−Lys−Leu−Gln−Thr−Tyr−Pro−Arg−Thr−Asp−Val
2. The peptide according to claim 1, wherein the peptide residue is -Gly-Ala-Gly-Thr-Pro- _NH2 , or an acid addition salt or complex thereof. 4. The peptide or its acid addition salt or complex according to claim 1, wherein the protecting group for the carboxyl group of the amino acid residue is an alkoxy group, a cycloalkyloxy group, an aralkyloxy group which may have a substituent, a substituted hydrazino group, or a phenacyl group. 5. The peptide or its acid addition salt or complex according to claim 1, wherein the protecting group for the amino group of the amino acid residue is an alkoxycarbonyl group which may have a substituent, a cycloalkyloxycarbonyl group which may have a substituent, an aralkyloxycarbonyl group which may have a substituent, an aralkyl group which may have a substituent, or an acyl group which may have a substituent. Claim 6: A compound represented by the following general formula (II): [wherein D-E represents Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group; X represents a protecting group for a hydroxyl group, a carboxyl group, Val-OH, or Met-
an amino acid residue represented by OH, the following general formula (VII) -A1-Leu-A2-OH (VII) (wherein A1 represents Val or Met, and A2 represents Ser or Gly);
or a lower peptide residue represented by the following general formula (VIII):
1−A12−A13−A14−A15−A16−A17−A18−A19−Gly−A2
0-A21-A22-Pro- _NH2 (VIII) (wherein A1 and A2 are the same as above. A3 to A22 represent the following amino acid residues: A3: Lys, Thr or Ala A4: Leu or Tyr A5: Ser, Thr or Trp A6: Gln, Lys or Arg A7: Glu, Asp or Asn A8: Leu or Phe A9: His or Asn A10: Lys or Asn A11: Leu, Phe or Tyr A12: Gln or His A13: Thr or Arg A14: Tyr or Phe A15: Pro or Ser A16: Arg, Gly or Gln A17: Thr or Met A18: Asp, Ala, Asn or Gly A19: Val, Ile, Thr or Phe A20 A21: Ala, Val, Pro or Ser; A21: Gly or Glu; A22: Thr or Ala), or a peptide residue represented by the general formula (VIII) containing at least -A1-Leu-A2-A3 and A1 to A22
and a peptide fragment in which the amino acid residues, lower peptide residues, peptide residues, and peptide fragments are arranged in the order of: (Wherein D represents Ser-OH, Ser-Asn-OH, Ser-Asn-Leu
-OH, Ser-Asn-Leu-Ser-OH or Ser-Asn-Leu-Ser
-Thr-OH, E represents H-Asn-Leu-Ser-Thr, H
A method for producing a cyclic peptide or an acid addition salt or complex thereof, comprising cyclizing a peptide represented by the formula (III) (III-Leu-Ser-Thr, H-Ser-Thr, H-Thr or a hydrogen atom, each amino acid residue optionally being protected by a protecting group; X is as defined above) by a chemical condensation reaction (excluding cyclization reactions using proteases). 7. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 6, wherein the cyclization reaction is carried out in the presence of an alkali metal salt. Claim 8: A compound represented by the following general formula (II): [wherein D-E represents Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group; X represents a protecting group for a hydroxyl group, a carboxyl group, Val-OH, or Met-
an amino acid residue represented by OH, the following general formula (VII) -A1-Leu-A2-OH (VII) (wherein A1 represents Val or Met, and A2 represents Ser or Gly);
or a lower peptide residue represented by the following general formula (VIII):
1−A12−A13−A14−A15−A16−A17−A18−A19−Gly−A2
0-A21-A22-Pro- _NH2 (VIII) (wherein A1 and A2 are the same as above. A3 to A22 represent the following amino acid residues: A3: Lys, Thr or Ala A4: Leu or Tyr A5: Ser, Thr or Trp A6: Gln, Lys or Arg A7: Glu, Asp or Asn A8: Leu or Phe A9: His or Asn A10: Lys or Asn A11: Leu, Phe or Tyr A12: Gln or His A13: Thr or Arg A14: Tyr or Phe A15: Pro or Ser A16: Arg, Gly or Gln A17: Thr or Met A18: Asp, Ala, Asn or Gly A19: Val, Ile, Thr or Phe A20 A21: Ala, Val, Pro or Ser; A21: Gly or Glu; A22: Thr or Ala), or a peptide residue represented by the general formula (VIII) containing at least -A1-Leu-A2-A3 and A1 to A22
and a peptide fragment in which the amino acid residues, lower peptide residues, peptide residues, and peptide fragments are arranged in the order of: (wherein F represents a hydroxyl group or an active ester residue, G
represents H-Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected by a protecting group. X is as defined above.) is subjected to a cyclization reaction in the presence of an alkali metal salt. 9. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 8, wherein the alkali metal salt is a halide of lithium, sodium, potassium, rubidium or cesium. 10. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 8, wherein the amount of the alkali metal salt relative to the peptide to be cyclized is 0.01 to 400 equivalents. 11. (1) Carbodiimide, (2) Hydroxybenzotriazole and Carbodiimide, or (3)
11. A method for producing the cyclic peptide or its acid addition salt or complex according to any one of claims 6 to 10, which comprises reacting the cyclic peptide using hydroxysuccinimide and carbodiimide. Claim 12: A compound represented by the following general formula (IX): [wherein A1 to A22 represent the following amino acid residues, and each amino acid residue may be protected with a protecting group.] A1: Val or Met A2: Ser or Gly A3: Lys, Thr, or Ala A4: Leu or Tyr A5: Ser, Thr, or Trp A6: Gln, Lys, or Arg A7: Glu, Asp, or Asn A8: Leu or Phe A9: His or Asn A10: Lys or Asn A11: Leu, Phe, or Tyr A12: Gln or His A13: Thr or Arg A14: Tyr or Phe A15: Pro or Ser A16: Arg, Gly, or Gln A17: Thr or Met A18: Asp, Ala, Asn, or Gly A19: Val, Ile, Thr, or Phe A20: Ala, Val, Pro, or Ser A21: Gly or Glu A22: Thr or Ala or an acid addition salt or complex thereof, comprising: (1) a compound represented by the following general formula (IIIa): wherein D is Ser-OH, Ser-Asn-OH, Ser-Asn-Leu
-OH, Ser-Asn-Leu-Ser-OH or Ser-Asn-Leu-Ser
-Thr-OH, E represents H-Asn-Leu-Ser-Thr, H
-Leu-Ser-Thr, H-Ser-Thr, H-Thr or a hydrogen atom, and each amino acid residue may be protected with a protecting group; W represents a hydroxyl group or a lower peptide residue represented by the general formula (VII) -A1-Leu-A2-OH (VII) (A1 and A2 are as defined above), and in the lower peptide residue, the carboxyl group of the C-terminal amino acid and the reactive group of each amino acid residue are protected with a protecting group), by a chemical condensation reaction (excluding cyclization reactions using proteases), or (2) a peptide represented by the following general formula (IVa): (wherein F represents a hydroxyl group or an active ester residue, G
represents H-Ser-Asn-Leu-Ser-Thr, and each amino acid residue may be protected with a protecting group. W is the same as above.) is subjected to a cyclization reaction in the presence of an alkali metal salt to obtain a peptide represented by the following general formula (V): [wherein W is as defined above], then the protecting group of the carboxyl group of the C-terminal amino acid of the peptide represented by general formula (V) is removed, followed by condensation with a peptide-resin represented by the following general formula (VI) synthesized on a solid-phase reaction resin by a solid-phase method, and then removing the resin.
−A14−A15−A16−A17−A18−A19−Gly−A20−A21−A22
-Pro-Resin (VI) [Y represents a hydrogen atom or H-Al-Leu-A2- (A1 and A2 are the same as above), and each amino acid residue may be protected with a protecting group. However, when W is a hydroxyl group, Y is H-
When A1-Leu-A2- and W is -A1-Leu-A2-OH, Y is a hydrogen atom. Resin represents a solid-phase reaction resin. A3 to A22 are the same as above. 13. The peptide-resin represented by the general formula (VI) is Y-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-G
ln−Thr−Tyr−Pro−Arg−Thr−Asp−Val−Gly−Ala−G
ly-Thr-Pro-Resin (wherein Y is a hydrogen atom or H-Al-Leu-Al- (Al is Val)
A2 represents a peptide residue represented by the formula (I) or (II), and A3 represents a peptide residue represented by the formula (I) or (II), and A4 represents a peptide residue represented by the formula (I) or (II), and A5 represents a peptide residue represented by the formula (I) or (II), and A6 represents a peptide residue represented by the formula (I) or (II), and A7 represents a peptide residue represented by the formula (I) or (II), and A8 represents a peptide residue represented by the formula (I) or (II), and A9 represents a peptide residue represented by the formula (I) or (II), and A1 represents a peptide residue represented by the formula (I) or (II), and A2 ...2 represents 14. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 12, wherein W is -Val-Leu-Gly-OH and Y is a hydrogen atom. 15. The solid-phase reaction resin is a benzhydrylamine resin, a p-methylbenzhydrylamine resin, or a p-
The method for producing the cyclic peptide or its acid addition salt or complex according to claim 12, which is a hydroxybenzoic acid resin. 16. The method according to claim 1, wherein the amount of the cyclic peptide represented by general formula (V) relative to the solid-phase reaction resin is 1.0 to 3.0 equivalents.
13. A method for producing the cyclic peptide or its acid addition salt or complex according to 12. 17. The method for producing the cyclic peptide or its acid addition salt or complex according to claim 12, wherein the peptide having a protecting group synthesized on a solid phase reaction resin is subjected to a reaction for removing the protecting group. 18. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 17, wherein the elimination reaction is an elimination reaction with an acid. 19. The method for producing a cyclic peptide or an acid addition salt or complex thereof according to claim 17, wherein the elimination reaction is an elimination reaction with hydrogen fluoride.