Cyclic peptide production method

Abstract

To provide a production method that can efficiently remove multimeric impurity by-produced during a cyclization reaction, improve the purity of the obtained cyclic peptide, and reduce the burden on the purification step.SOLUTION: A method for producing a cyclic peptide includes the following step (1) and step (2): (1) a step of cyclizing a linear peptide; and (2) a step of obtaining a cyclic peptide by adding a poor solvent to a mixture of the cyclic peptide and multimeric impurity, which mixture is obtained in the above-mentioned step, and filtering off the multimeric impurity as an insoluble material.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a cyclic peptide. [Background technology]

[0002] In recent years, active drug development has been conducted to improve blood stability by cyclizing peptides, and various cyclic peptide shapes have been reported. Somatostatin, octreotide, linaclotide, plecanatide, ziconotide, atosiban, eptifibatide, etc. are known as SS-type cyclic peptides that are intramolecularly SS-cyclized by linking the SH groups in the side chains of the constituent amino acids cysteine ​​and cysteine ​​derivatives with each other through disulfide bonds, or by linking the SH group extending from the N-terminus of the peptide chain with the SH group in the side chain of cysteine ​​or cysteine ​​derivatives through disulfide bonds. In addition, cyclosporine and cyclic RGD peptides are known as lactam-type cyclic peptides in which the amino group and carboxyl group at the end or side chain of the peptide chain are linked by an amide bond. Furthermore, peptide compounds having a cyclic thioether bond, such as carbetocin, barsiban, and merotocin, are known as CS-type cyclic peptides in which a carbonyl alkylene group is mediated between the N-terminus of the peptide chain and the side chain SH group of the constituent amino acid cysteine ​​or a cysteine ​​derivative.

[0003] However, in the conventional manufacturing method of any cyclic peptide, intermolecularly bonded polymeric impurities such as dimers, trimers, and oligomers are by-produced during the cyclization reaction, which reduces the yield of the target cyclic peptide, and it is difficult to remove these polymeric impurities. For this reason, in order to suppress the by-production of polymeric impurities, a method with extremely low production efficiency, such as performing the cyclization reaction under dilute conditions, is generally adopted. In addition, in order to remove polymeric impurities, Patent Document 1 describes that in a peptide having two or more SH groups in the molecule, after deprotecting all protecting groups in the N-terminus, C-terminus, and side chains of the constituent amino acids of the peptide chain, intramolecular SS cyclization is performed in an aqueous solvent under oxidizing conditions, and in order to precipitate non-polar high molecular weight impurities such as dimers and trimers, the impurities are removed by centrifugation or filtration. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017 / 134687 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a method for producing a highly pure cyclic peptide by efficiently removing polymerized impurities produced as a by-product during a cyclization reaction. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above problems, the present inventors have succeeded in efficiently obtaining a cyclic peptide with high purity by precipitating the polymerized impurities generated during cyclization of a linear peptide with a poor solvent and filtering out the insoluble matter. The inventors found that this solved the above problems and thus completed the present invention. That is, the present invention has the following characteristics.

[0007] [1] A method for producing a cyclic peptide, comprising the following steps (1) and (2): (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of the cyclic peptide and polymerized impurities obtained in the above step, thereby filtering out the polymerized impurities as insoluble matter, thereby obtaining the cyclic peptide. [2] The method according to [1], further comprising adding a good solvent before, simultaneously with, or after the addition of the poor solvent. [3] The method according to [1] or [2], wherein the cyclic structure of the cyclic peptide is any one of a) SS type, b) lactam type, c) CS type, d) CC type, or e) lactone type. [3'] The method according to [1] or [2], wherein the cyclic structure of the cyclic peptide is any one of a) SS type, b) lactam type, c) CS type, or d) CC type. [4] The method for producing a linear peptide according to any one of [1] to [3] and [3'], wherein the C-terminus of the linear peptide is protected by a protecting group and the linear peptide is cyclized either i) between the side chains of the constituent amino acids or ii) between the N-terminus and the side chain of the constituent amino acid. [5] The method according to [4], wherein the protecting group at the C-terminus of the linear peptide and / or at the side chain of the constituent amino acid is either a liquid-phase protecting group or a pseudo-solid-phase protecting group. [6] The method according to [4], further comprising the step of deprotecting only the solid phase support prior to the step (2), wherein in the step (1), the protecting group at the C-terminus of the linear peptide is a solid phase support. [7] The method according to any one of [4] to [6], wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as a by-product when the linear peptide is cyclized to obtain a cyclic peptide. [8] The method according to [7], wherein the good solvent is a solvent capable of dissolving the target cyclic peptide. [9] The method for producing a linear peptide according to any one of [1] to [3] and [3'], wherein the C-terminus of the linear peptide is not protected, and the portions other than the site to be cyclized are protected, and the linear peptide is cyclized either i) between the side chains of the constituent amino acids, ii) between the N-terminus and the side chain of the constituent amino acid, iii) between the C-terminus and the side chain of the constituent amino acid, or iv) between the N-terminus and the C-terminus. [9A] Between step (1) and step (2), A step of removing all protecting groups from the cyclic peptide obtained in step (1). The method according to [4] or [9], further comprising:

[10] The method according to [9], wherein the protecting groups of the side chains of the constituent amino acids of the linear peptide are either liquid-phase protecting groups or pseudo-solid-phase protecting groups.

[11] The method according to [9] or

[10] , wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities that are by-produced when the linear peptide is cyclized to obtain a cyclic peptide.

[12] The method according to

[11] , wherein the good solvent is a solvent capable of dissolving the target cyclic peptide.

[13] The method for producing a linear peptide according to any one of [1] to [3] and [3'], wherein the linear peptide is unprotected at any of its C-terminus, N-terminus and side chains of the constituent amino acids, and is cyclized either i) between the side chains of the constituent amino acids, ii) between the N-terminus and the side chains of the constituent amino acids, iii) between the C-terminus and the side chains of the constituent amino acids, or iv) between the N-terminus and the C-terminus.

[14] Between step (1) and step (2), A step of isolating the cyclic peptide obtained in step (1). The method according to

[13] , further comprising:

[15] The method according to

[13] or

[14] , wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when the linear peptide is cyclized to obtain a cyclic peptide.

[16] The method according to

[15] , wherein the good solvent is a solvent capable of dissolving the desired cyclic peptide.

[17] After the above step (2), (3) Removal of all protecting groups The method according to any one of [1] to

[16] and [3'], further comprising:

[18] A method for producing a peptide having a cyclic thioether bond, comprising any one of the following steps: (A) a step of cyclizing a linear peptide, the C-terminus of which is protected or unprotected with a protecting group, the N-terminus of which is modified with an alkylene carbonyl group having a leaving group, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative of which is unprotected, at the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative; (B) a step of deprotecting a linear peptide in which the C-terminus is protected or not protected by a protecting group, the N-terminus is protected or not protected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative is modified with an alkylene group having a carboxy group, and then cyclizing the linear peptide at the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative, if the N-terminus is protected, or (C) A step of deprotecting the amino group of a linear peptide in which the C-terminus is protected or not protected by a protecting group, the amino groups of the side chains of the constituent amino acids are protected or not protected, and the side chain of the constituent amino acid, cysteine ​​or a cysteine ​​derivative, is modified with an alkylene group having a carboxy group, and then cyclizing the linear peptide with the side chain of the constituent amino acid having an amino group and the side chain of the cysteine ​​or cysteine ​​derivative. [18'] A method for producing a peptide having a cyclic thioether bond, comprising any one of the following steps: (A) a step of cyclizing a linear peptide, the C-terminus of which is protected with a protecting group, the N-terminus of which is modified with an alkylene carbonyl group having a leaving group, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative of which is not protected, at the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative; (B) a step of cyclizing a linear peptide, the C-terminus of which is protected with a protecting group, the N-terminus of which is not protected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative of which is modified with an alkylene group having a carboxy group, at the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative; or (C) A process of cyclizing a linear peptide in which the C-terminus of the linear peptide is protected with a protecting group, the amino groups of the side chains of the constituent amino acids are unprotected, and the side chains of the constituent amino acids, cysteine ​​or cysteine ​​derivatives, are modified with an alkylene group having a carboxy group, via the side chains of the constituent amino acids having an amino group and the side chains of cysteine ​​or cysteine ​​derivatives.

[19] The method according to

[18] or [18'], wherein the protecting group at the C-terminus is a pseudo solid-phase protecting group, a liquid-phase protecting group, or a solid-phase support.

[20] Step (B) comprises: (B-1) modifying the side chain of a cysteine ​​or cysteine ​​derivative of a linear peptide in which the C-terminus is protected or not protected by a protecting group, the N-terminus is protected or not protected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is not protected, with an alkylene group having a carboxy group, to produce a linear peptide in which the C-terminus is protected or not protected by a protecting group, the N-terminus is protected or not protected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is modified with an alkylene group having a carboxy group; or the step (C) comprises: (C-1) a step of modifying the side chain of a cysteine ​​or cysteine ​​derivative of a linear peptide in which the C-terminus of the linear peptide is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is unprotected, with an alkylene group having a carboxy group, to produce a linear peptide in which the C-terminus of the linear peptide is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is modified with an alkylene group having a carboxy group; The manufacturing method according to

[18] or

[19] .

[21] The method according to any one of

[18] to

[20] , wherein step (B) comprises the step of (B-2) deprotecting all protecting groups of the obtained protected cyclic peptide. Effect of the Invention

[0008] According to the present invention, a method for producing a cyclic peptide can be provided that can efficiently remove polymerized impurities produced as a by-product during a cyclization reaction, improve the purity of the resulting cyclic peptide, and reduce the burden on the purification process. [Brief description of the drawings]

[0009] [Figure 1] Fig. 1 shows an outline of an embodiment of the present invention. Steps (1) to (3) in "Embodiment 1", "Embodiment 2" and "Embodiment 3" in the figure correspond to steps (1) to (3) in the present invention. In addition, the embodiment of the known example corresponds to the embodiment of Patent Document 1 of this patent specification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Unless otherwise specified in the context, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that can be used in connection with the described invention.

[0011] The "cyclic peptide" in the present invention is a peptide having a cyclic chemical structure in which constituent amino acids are bonded, and may have a partial structure other than that derived from the constituent amino acids in a part of the cyclic structure. Examples of the partial structure other than that derived from the constituent amino acids include carbonyl alkylene and carbonyl alkylene thio. In carrying out the present invention, the type of the cyclic peptide to be used is not particularly limited, and may be, for example, a pharmaceutical product. The cyclic peptide may also be a natural product or a non-natural product. Examples of such cyclic peptides include, but are not limited to, SS-type cyclic peptides such as somatostatin, octreotide, linaclotide, plecanatide, ziconotide, atosiban, eptifibatide, etc.; lactam-type cyclic peptides such as cyclosporin; and CS-type cyclic peptides such as carbetocin, barusiban, and merotocin.

[0012] The amino acids that are the building blocks of the peptides produced by the method of the present invention are compounds having an amino group and a carboxy group in the same molecule, and may be natural or unnatural amino acids, and may be L-, D- or racemic. Furthermore, the peptides are synthesized by repeating a dehydration condensation step (condensation step) between the amino group of one amino acid component and the carboxy group of another amino acid component according to the amino acid sequence.

[0013] Next, the production method of the present invention will be described. The production method of the cyclic peptide of the present invention is characterized by comprising the following steps (1) and (2). (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of the cyclic peptide and polymerized impurities obtained in the above step, thereby filtering out the polymerized impurities as insoluble matter, thereby obtaining the cyclic peptide.

[0014] Step (1): Cyclizing a linear peptide The "linear peptide" in the present invention is not particularly limited as long as it has a protecting group and / or a pseudo solid-phase protecting group at the N-terminus, C-terminus and / or the side chain of the constituent amino acid, and has an unprotected N-terminus, C-terminus and / or the side chain of the constituent amino acid that can undergo a cyclization reaction. A partially cyclized linear peptide is also included. Cyclization occurs when unprotected N-terminuses, C-terminuses and / or side chains of constituent amino acids that can undergo cyclization are bonded to each other to form, for example, -SS- bonds, -CO-NH- bonds, -CS- bonds, -CC- bonds, -CO-O- bonds, etc.

[0015] Examples of -SS- bonds include bonds between "thiol groups in the side chains of constituent amino acids," bonds between "an N-terminus modified with an alkylene carbonyl group having a thiol group" and "a thiol group in the side chain of a constituent amino acid," and bonds between "a side chain amino group of a constituent amino acid modified with an alkylene carbonyl group having a thiol group" and "a thiol group in the side chain of a constituent amino acid." Here, the "alkylenecarbonyl group having a thiol group" is, for example, a group that reacts with a thiol group in the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative. The alkylene in the alkylenecarbonyl group having a thiol group includes an alkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 3 carbon atoms, and includes a methylene group, an ethylene group, and a propylene group.

[0016] Examples of the -CO-NH- bond include a bond between the N-terminus and the C-terminus, a bond between the N-terminus and the "side chain carboxyl group of a constituent amino acid", a bond between the "side chain amino group of a constituent amino acid" and the C-terminus, and a bond between the "side chain amino group of a constituent amino acid" and the "side chain carboxyl group of a constituent amino acid".

[0017] Examples of -CS- bonds include a bond between a "thiol group in the side chain of a constituent amino acid" and an "N-terminus modified with an alkylene carbonyl group having a leaving group", and a bond between a "thiol group in the side chain of a constituent amino acid" and a "side chain amino group of a constituent amino acid modified with an alkylene carbonyl group having a leaving group". Here, the "alkylenecarbonyl group having a leaving group" is, for example, a group that reacts with a thiol group in the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative, and includes a halogenoalkylenecarbonyl group, a tosyloxyalkylenecarbonyl group, a mesyloxyalkylenecarbonyl group, etc. Examples of the halogenoalkylenecarbonyl group include a chloroalkylenecarbonyl group, a bromoalkylenecarbonyl group, an iodoalkylenecarbonyl group, etc., and among these, a chloroalkylenecarbonyl group is more preferred. Examples of the alkylene group in the halogenoalkylenecarbonyl group, the tosyloxyalkylenecarbonyl group, and the mesyloxyalkylenecarbonyl group include an alkylene group having 1 to 6 carbon atoms, and more preferably an alkylene group having 1 to 3 carbon atoms, and include a methylene group, an ethylene group, and a propylene group.

[0018] An example of a -CC- bond is a bond between "side chains of constituent amino acids modified with a terminal olefin group".

[0019] Examples of the -CO-O- bond include a bond between the C-terminus and a "side chain hydroxy group of a constituent amino acid" and a bond between a "side chain hydroxy group of a constituent amino acid" and a "side chain carboxyl group of a constituent amino acid".

[0020] The cyclization reaction can be carried out under conditions commonly used in the art.

[0021] In the case of a -SS- bond, for example, conditions that are usually used for the oxidation reaction between thiol groups can be mentioned.

[0022] In the case of a -CO-NH- bond, for example, conditions usually used for forming an intramolecular amide bond (lactam bond) can be mentioned.

[0023] In the case of a -CS- bond, for example, conditions that are usually used when reacting an alkyl group having a leaving group with a thiol group can be mentioned.

[0024] In the case of a -CC- bond, for example, conditions that are commonly used when utilizing an olefin metathesis reaction (for example, Org. Lett., 2015, 17(3), 696) and the like can be mentioned.

[0025] In the case of a -CO-O- bond, for example, conditions usually used for forming an intramolecular ester bond (lactone bond) can be mentioned.

[0026] By the above, it is possible to produce a cyclic peptide whose cyclic structure is any one of a) SS type, b) lactam type, c) CS type, d) CC type, and e) lactone type.

[0027] The reaction solvent in the cyclization reaction is preferably one capable of dissolving the cyclic peptide.

[0028] In the linear peptide of step (1), in the "fully protected peptide" in which the N-terminus, C-terminus and / or side chains of the constituent amino acids are all protected, it is necessary to deprotect the site to be cyclized in this step (1). To deprotect such a site to be cyclized in this step (1), a deprotection method known per se can be adopted without particular limitation depending on the type of protecting group to be deprotected. When deprotecting the site to be cyclized in this step (1), it is necessary to deprotect only the site to be cyclized in this step (1) in the "fully protected peptide", and deprotection conditions having such selectivity in deprotection can be appropriately selected. A person skilled in the art can appropriately select appropriate conditions based on the overall synthesis strategy. The conditions described in the deprotection step of step (3) below can be used as the conditions for each deprotection.

[0029] Cyclization of a linear peptide results in a mixture of the cyclic peptide and its by-product multimeric impurities. Here, the "cyclic peptide" refers to the target peptide that is cyclized intramolecularly. In addition, the "polymerized impurities" refer to peptide polymers (dimers, trimers, oligomers, polymers, etc.) cyclized by intermolecular bonds, which are by-products produced when a cyclic peptide is obtained by cyclizing a linear peptide, and their precursors, linear peptide polymers (dimers, trimers, oligomers, polymers, etc.) bound by intermolecular bonds.

[0030] Step (2): A step of adding a poor solvent to a mixture of the cyclic peptide and polymerized impurities, filtering off the polymerized impurities as insoluble matter, and obtaining the cyclic peptide. The "poor solvent" is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when a cyclic peptide is obtained by cyclizing a linear peptide, and is not particularly limited. Examples of the poor solvent include acetonitrile, IPE (diisopropyl ether), diethyl ether, toluene, hexane, heptane, methanol, ethanol, isopropyl alcohol, THF (tetrahydrofuran), water, etc., and these may be used alone or in combination of two or more.

[0031] A good solvent may be further added before, simultaneously with, or after the addition of the poor solvent. The "good solvent" is a solvent capable of dissolving the target cyclic peptide, and is not particularly limited. Examples include chloroform, dichloromethane, DMF (dimethylformamide), N-methylpyrrolidone, methanol, ethanol, isopropyl alcohol, THF (tetrahydrofuran), etc., and these may be used alone or in combination of two or more. The good solvent is preferably the reaction solvent used in the cyclization step in step (1). In addition, it is preferable to add the good solvent before or at the same time as adding the poor solvent in step (2). When adding the poor solvent, it is desirable that the target cyclic peptide is completely dissolved, but it may also be in the form of a slurry. The combination of the good solvent and the poor solvent can be selected so as to generate a larger difference in solubility between the target cyclic peptide and the by-product polymerized impurity. It is preferable that the "poor solvent" and the "good solvent" are not the same solvent.

[0032] (Protecting group at the C-terminus of the peptide) In the present specification, examples of the protecting group for the C-terminus of a peptide include a liquid-phase protecting group and a pseudo-solid-phase protecting group. Examples of the protecting group include, but are not limited to, protecting groups commonly used in the art, such as an ester-type protecting group, an amide-type protecting group, and a hydrazide-type protecting group.

[0033] As the ester-type protecting group, a substituted or unsubstituted alkyl ester or a substituted or unsubstituted aralkyl ester is preferably used. As the substituted or unsubstituted alkyl ester, a methyl ester, an ethyl ester, a tert-butyl ester, a cyclohexyl ester, a trichloroethyl ester, a phenacyl ester, etc. are preferably used. As the substituted or unsubstituted aralkyl ester, a benzyl ester, a p-nitrobenzyl ester, a p-methoxybenzyl ester, a diphenylmethyl ester, a 9-fluorenylmethyl (Fm) ester, a 4-picolyl (Pic) ester, etc. are preferably used.

[0034] Preferred examples of amide-type protecting groups include unsubstituted amides, primary amides such as N-methylamide, N-ethylamide, and N-benzylamide, and secondary amides such as N,N-dimethylamide, pyrrolidinylamide, and piperidinylamide.

[0035] As the hydrazide type protecting group, unsubstituted hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide, and the like are preferably used.

[0036] (Protecting group at the N-terminus of a peptide) In the present specification, the protecting group for the N-terminus of a peptide is not particularly limited, and examples thereof include protecting groups commonly used in the art, such as 9-fluorenylmethyloxycarbonyl group (Fmoc group), benzyloxycarbonyl group (Cbz group), tert-butoxycarbonyl group (Boc group), etc. The Fmoc group is preferred.

[0037] (Protecting groups for functional groups on peptides) In this specification, the protecting group for the side chain of the peptide is not particularly limited, and examples thereof include protecting groups described in Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1985), Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons (1999), etc.

[0038] When the side chain is a carboxy group, examples of the protecting group at the C-terminus include the same protecting groups as those mentioned above, such as a liquid-phase protecting group, a pseudo-solid-phase protecting group, and a solid-phase carrier.

[0039] When the side chain is an amino group, examples of the protecting group include a urethane type protecting group, an acyl type protecting group, and a sulfonyl type protecting group. Examples of urethane-type protecting groups include a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl (Boc) group, a benzyloxycarbonyl (Z) group, etc., and preferred are a methoxycarbonyl group, an ethoxycarbonyl group, a Boc group, etc. As the acyl-type protecting group, for example, a formyl group, an acetyl group, a trifluoroacetyl group, etc. are preferably used. As the sulfonyl-type protecting group, for example, a p-toluenesulfonyl (Ts) group, a p-tolylmethanesulfonyl group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, and the like are preferably used.

[0040] When the functional group on the peptide is a hydroxy group (including a phenolic hydroxy group), examples of the protecting group include an alkyl type protecting group, an alkoxyalkyl type protecting group, an acyl type protecting group, and an alkylsilyl type protecting group.

[0041] Examples of the alkyl-type protecting group include a methyl group, an ethyl group, and a tert-butyl group.

[0042] Examples of alkoxyalkyl-type protecting groups include a methoxymethyl group (MOM group), a 2-tetrahydropyranyl group (THP group), and an ethoxyethyl group (EE group).

[0043] Examples of the acyl-type protecting group include an acetyl group, a pivaloyl group, and a benzoyl group.

[0044] Examples of alkylsilyl-type protecting groups include trimethylsilyl group (TMS group), triethylsilyl group (TES group), tert-butyldimethylsilyl group (TBS group or TBDMS group), triisopropylsilyl group (TIPS group), and tert-butyldiphenylsilyl group (TBDPS group).

[0045] Other functional groups can also be protected by protective groups commonly used in the art. For example, the guanidino group of arginine can be protected by a p-toluenesulfonyl group. The imidazole group of histidine can be protected by a trityl group, a benzyloxymethyl group, etc. Also, the indole group of tryptophan can be protected by a formyl group.

[0046] Although the protecting groups for the functional groups on the peptide have been described above, those skilled in the art can carry out this step by appropriately selecting a protecting scheme in the art (e.g., Fmoc / tBu strategy, Boc / Bzl strategy, Bzl / tBu strategy, etc.) that is selected along with the overall synthetic strategy when carrying out the present invention. Among them, the Fmoc / tBu strategy is preferred.

[0047] (liquid phase protecting group) When the present invention is carried out under liquid phase conditions, it is desirable that the C-terminus and, when the functional group on the peptide is a carboxyl group, at least one of the carboxyl groups is protected. Examples of the protecting group for the carboxyl group include the protecting groups listed in the above "protecting group for the C-terminus" (ester type protecting group, amide type protecting group, hydrazide type protecting group, etc.). Among these, an ester type protecting group is preferable. As the ester type protecting group, a substituted or unsubstituted alkyl ester and a substituted or unsubstituted aralkyl ester are preferably used. As the substituted or unsubstituted alkyl ester, a methyl ester, an ethyl ester, a tert-butyl ester, a cyclohexyl ester, a trichloroethyl ester, a phenacyl ester, etc. are preferably used. As the substituted or unsubstituted aralkyl ester, a benzyl ester, a p-nitrobenzyl ester, a p-methoxybenzyl ester, a diphenylmethyl ester, a 9-fluorenylmethyl (Fm) ester, a 4-picolyl (Pic) ester, etc. are preferably used. In particular, a tert-butyl ester, a benzyl ester, etc. are preferable.

[0048] (pseudo solid phase protecting group) When the present invention is carried out under liquid phase conditions, in order to facilitate purification, the C-terminus and, if the functional group on the peptide is a carboxy group, at least one of the carboxy groups may be protected by a pseudo solid-phase protecting group (hereinafter sometimes referred to as an "anchor" in this specification) as necessary. The purification method of the peptide using the pseudo solid-phase protecting group is not particularly limited, but can be performed according to a method known per se (see JP 2000-44493 A, WO 2006 / 104166, WO 2007 / 034812, WO 2007 / 122847, WO 2010 / 113939, WO 2010 / 104169, WO 2011 / 078295, WO 2012 / 029794, WO 2016 / 140232, WO 2003 / 018188, WO 2017 / 038650, WO 2019 / 009317, etc.) or a method similar thereto. Here, the pseudo solid-phase protecting group refers to a group that is soluble in halogen-based solvents or ether-based solvents and insoluble in polar solvents, contains an anchor having a molecular weight of 300 or more (e.g., a benzyl compound, a diphenylmethane compound, or a fluorene compound), and can be condensed with a carboxy group.

[0049] In the present specification, the pseudo solid-phase protecting group is not particularly limited, and examples thereof include pseudo solid-phase protecting groups commonly used in the art.

[0050] (Solid Phase Support) The "solid phase support" may be any solid phase support known in the art suitable for use in solid phase synthesis. The term "solid phase" as used herein includes peptides attached or linked to said solid phase support via conventional functional linkers or handle groups, and such linkers are also implied when referring to "solid phase" in this context. Examples of solid phases are, for example, polystyrene supports (which may be further functionalized, for example, with p-methylbenzyl-hydrylamine), or rigid functionalized supports such as diatomaceous earth-encapsulated polydimethylacrylamide (pepsin K), silica, or microporous glass. The resin matrix of the solid phase may be composed of amphiphilic polystyrene-PEG resins or PEG-polyamide or PEG-polyester resins. Solid phase supports also include, for example, Wang-PEG resin and Rink-amide PEG resin.

[0051] (Isolation process) Between the above steps (1) and (2), a step of isolating the cyclic peptide obtained in step (1) can be further included. The isolation of the cyclic peptide obtained in step (1) can be performed by a method commonly used in the art, such as filtration. For example, a solvent that can be used as a poor solvent can be added to filter the precipitate. Examples of the solvent for filtration include acetonitrile, IPE (diisopropyl ether), diethyl ether, toluene, hexane, heptane, methanol, ethanol, isopropyl alcohol, THF (tetrahydrofuran), water, etc., and these may be used alone or in a mixture of two or more. Preferred examples include IPE (diisopropyl ether) and diethyl ether. In particular, when the linear peptide is unprotected at any of its C-terminus, N-terminus and side chains of the constituent amino acids and is cyclized at either i) the side chains of the constituent amino acids, ii) the N-terminus and the side chains of the constituent amino acids, iii) the C-terminus and the side chains of the constituent amino acids, or iv) the N-terminus and the C-terminus, it is preferable to further include a step of isolating the cyclic peptide obtained in step (1) between the above steps (1) and (2).

[0052] In the case where the linear peptide has its C-terminus protected by a protecting group and is cyclized either i) between the side chains of the constituent amino acids, or ii) between the N-terminus and the side chain of a constituent amino acid, or in the case where the linear peptide has its C-terminus unprotected and is protected other than the site to be cyclized and is cyclized either i) between the side chains of the constituent amino acids, ii) between the N-terminus and the side chain of a constituent amino acid, iii) between the C-terminus and the side chain of a constituent amino acid, or iv) between the N-terminus and the C-terminus, the linear peptide may further include a step of removing all protecting groups from the cyclic peptide obtained in step (1) between steps (1) and (2).

[0053] (Step of Deprotecting All Protective Groups) Step (3): Deprotecting all protecting groups Except when the linear peptide used in step (1) is not protected at its C-terminus, N-terminus, and all of the side chains of the constituent amino acids, the method may further include a step of removing all protecting groups after the above step (2).

[0054] For example, in the case of a lower alkyl group such as Me or Et, the group can be deprotected by reacting with a base such as sodium hydroxide or potassium hydroxide in a solvent such as an aqueous organic solvent or a polar organic solvent. In the case of tBu, the protection can be removed by reacting with an acid such as trifluoroacetic acid (TFA) or hydrochloric acid in a solvent such as chloroform or ethyl acetate. In the case of Bzl, the deprotection can be carried out in a solvent such as methanol or DMF, or by reaction with a strong acid such as hydrogen fluoride, trifluoromethanesulfonic acid, or HBr.

[0055] The acid that can be used for deprotecting the Boc group is not particularly limited, and examples of the acid that can be used include mineral acids such as hydrogen chloride, sulfuric acid, and nitric acid, carboxylic acids such as formic acid and trifluoroacetic acid (TFA), sulfonic acids such as methanesulfonic acid and p-toluenesulfonic acid, and mixtures thereof. Examples of mixtures that can be used include hydrogen bromide / acetic acid, hydrogen chloride / dioxane, and hydrogen chloride / acetic acid.

[0056] The organic base that can be used for deprotection of the Fmoc group is not particularly limited, and examples thereof include secondary amines such as diethylamine, piperidine, and morpholine, and tertiary amines such as diisopropylethylamine, dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN).

[0057] More preferably, the deprotection of the Fmoc group is carried out by treating with a non-nucleophilic organic base in a halogenated or ethereal solvent, in a solvent that does not affect the reaction.

[0058] Examples of the non-nucleophilic base include 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), among which DBU and DBN are preferred, and DBU is more preferred.

[0059] The deprotection of the pseudo solid-phase protecting group is preferably carried out by acid treatment. Examples of the acid used for deprotection include trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, etc., and among these, TFA is preferred. Examples of the solvent used for deprotection include chloroform, dichloromethane, 1,2-dichloroethane, and a mixed solvent thereof. The concentration of the acid used for deprotection is, for example, 0.1 w / v% to 5 w / v%.

[0060] The deprotection of the pseudo solid-phase protecting group can be performed simultaneously with the deprotection of the protecting groups of other functional groups in the peptide. In that case, a conventional method performed in the field, particularly in peptide synthesis, is used, but a method of adding an acid or the like is preferably adopted. As the acid, trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, mesylic acid, tosylic acid, trifluoroethanol, hexafluoroisopropanol, etc. are used. Among them, TFA is particularly preferable. The amount of the acid used is appropriately set depending on the type of acid used, and an appropriate amount for removing the anchor group is used. The amount of the acid used is preferably 3 moles or more, more preferably 5 moles or more, and preferably 100 moles or less, more preferably 50 moles or less, relative to 1 mole of peptide. In addition to the use of these, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, BF3·etherate, etc. can also be added as an additional strong acid source.

[0061] The conditions for deprotecting the above pseudo solid-phase protecting groups can be appropriately selected by those skilled in the art depending on the type of protecting group used.

[0062] Other protecting groups can be appropriately deprotected according to the methods commonly used in the art or the deprotection methods for protecting groups described herein, depending on the type of protecting group.

[0063] The above steps (1) to (3) can be carried out under liquid phase conditions. In this case, those skilled in the art can appropriately select the liquid phase conditions depending on the synthesis strategy such as the structure of the desired cyclized peptide and the purpose of production (production scale, etc.).

[0064] The above steps (1) to (3) can also be carried out under pseudo solid-phase conditions using a pseudo solid-phase protecting group. Specifically, this method can be applied when the C-terminus of a linear peptide is protected by a protecting group and cyclized at either i) the side chains of the constituent amino acids, or ii) the N-terminus and the side chain of the constituent amino acid, and the protecting group of the C-terminus and / or the side chain of the constituent amino acid of the linear peptide is a pseudo solid-phase protecting group. This method can also be applied when the C-terminus of a linear peptide is not protected, and the portions other than the portion to be cyclized are protected, and the protecting group of the side chain of the constituent amino acid of the linear peptide is a pseudo solid-phase protecting group, and the linear peptide is also applied when the C-terminus is not protected, and the portions other than the portion to be cyclized are protected, and the protecting group of the side chain of the constituent amino acid of the linear peptide is a pseudo solid-phase protecting group. In the above, those skilled in the art can appropriately select pseudo solid-phase conditions using pseudo solid-phase protecting groups depending on the synthesis strategy such as the structure of the desired cyclized peptide and the purpose of production (production scale, etc.).

[0065] Step (1) can also be carried out under solid-phase conditions. Specifically, this method can be applied to the case where the C-terminus of a linear peptide is protected by a protecting group and cyclized either i) between the side chains of the constituent amino acids or ii) between the N-terminus and the side chain of the constituent amino acid, and the protecting group of the C-terminus of the linear peptide is a solid-phase support. In this case, the method further includes a step of deprotecting only the solid-phase support prior to the above step (2). In this case, a person skilled in the art can appropriately select solid-phase conditions (including deprotection conditions for the solid-phase support) depending on the synthesis strategy, such as the structure of the desired cyclized peptide and the purpose of production (production scale, etc.).

[0066] (Final purification process) Step (4): Final purification step When the cyclic peptide obtained in the above steps (1) to (3) is obtained under liquid phase conditions, it can be purified by a method commonly used in the art.

[0067] More specifically, the present invention can take the following embodiments 1 to 3. Embodiment 1: A method for producing a cyclic peptide, comprising the steps of: (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of the cyclic peptide and polymerized impurities obtained in the above step, and filtering out the polymerized impurities as insoluble matter to obtain the cyclic peptide, Examples of such peptides include a linear peptide whose C-terminus is protected with a protecting group and which is cyclized either i) between the side chains of the constituent amino acids or ii) between the N-terminus and a side chain of a constituent amino acid. In the above, the protecting group at the C-terminus of the linear peptide and / or the side chain of the constituent amino acid is preferably either a liquid-phase protecting group or a pseudo-solid-phase protecting group. In the above, the poor solvent is a solvent capable of precipitating / depositing polymerized impurities that are by-produced when obtaining a cyclic peptide by cyclizing the linear peptide, and is preferably at least one selected from acetonitrile, methanol, and water. In the above, a good solvent may be added before, simultaneously with, or after the addition of the poor solvent. The good solvent is a solvent capable of dissolving the target cyclic peptide, and is preferably at least one selected from chloroform, dichloromethane, DMF (dimethylformamide), and THF (tetrahydrofuran). Another aspect of embodiment 1 is a case where in step (1), the protecting group at the C-terminus of the linear peptide is a solid phase support, and the method further comprises a step of deprotecting only the solid phase support prior to the above step (2).

[0068] Embodiment 2: A method for producing a cyclic peptide, comprising the steps of: (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of the cyclic peptide and polymerized impurities obtained in the above step, and filtering out the polymerized impurities as insoluble matter to obtain the cyclic peptide, Examples of such cases include linear peptides in which the C-terminus is unprotected and the rest of the peptide is protected, and the peptide is cyclized either i) between the side chains of the constituent amino acids, ii) between the N-terminus and the side chain of a constituent amino acid, iii) between the C-terminus and the side chain of a constituent amino acid, or iv) between the N-terminus and the C-terminus. In the above, the protecting groups of the side chains of the constituent amino acids of the linear peptide are preferably either liquid-phase protecting groups or pseudo-solid-phase protecting groups. In the above, the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as a by-product when obtaining a cyclic peptide by cyclizing the linear peptide, and is preferably at least one selected from IPE (diisopropyl ether), diethyl ether, toluene, hexane, and heptane. In the above, a good solvent may be added before, simultaneously with, or after the addition of the poor solvent. The good solvent is a solvent capable of dissolving the target cyclic peptide, and it is preferable that the good solvent is at least one selected from chloroform, dichloromethane, N-methylpyrrolidone, and DMF (dimethylformamide).

[0069] Embodiment 3: A method for producing a cyclic peptide, comprising the steps of: (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of the cyclic peptide and polymerized impurities obtained in the above step, and filtering out the polymerized impurities as insoluble matter to obtain the cyclic peptide, Examples of such cases include linear peptides that are not protected at any of their C-terminus, N-terminus, or side chains of constituent amino acids, and that are cyclized either i) between the side chains of the constituent amino acids, ii) between the N-terminus and the side chains of the constituent amino acids, iii) between the C-terminus and the side chains of the constituent amino acids, or iv) between the N-terminus and the C-terminus. In the above, it is preferable to further include a step of isolating the cyclic peptide obtained in step (1) between the steps (1) and (2). In the above, the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when obtaining a cyclic peptide by cyclizing the linear peptide, and is preferably at least one selected from water, IPE (diisopropyl ether), acetonitrile, ethanol, isopropyl alcohol, and THF (tetrahydrofuran). In the above, a good solvent may be added before, simultaneously with, or after the addition of the poor solvent. The good solvent is a solvent capable of dissolving the target cyclic peptide, and is preferably at least one selected from DMF (dimethylformamide), methanol, and N-methylpyrrolidone.

[0070] The production method of the present invention will now be described. The method for producing a peptide having a cyclic thioether bond (CS type cyclic peptide) of the present invention is characterized by comprising any one of the following steps: (A) a step of cyclizing a linear peptide, the C-terminus of which is protected or unprotected by a protecting group, the N-terminus of which is modified with an alkylene carbonyl group having a leaving group, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative of which is unprotected, at the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative (embodiment A); (B) a step of deprotecting a linear peptide in which the C-terminus is protected or not protected by a protecting group, the N-terminus is protected or not protected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative is modified with an alkylene group having a carboxy group, and then cyclizing the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative (embodiment B), if the N-terminus is protected, or (C) A step of cyclizing a linear peptide in which the C-terminus is protected or not protected by a protecting group, the amino groups of the side chains of the constituent amino acids are protected or not protected, and the side chains of the constituent amino acids, cysteine ​​or a cysteine ​​derivative, are modified with an alkylene group having a carboxy group, by deprotecting the amino groups of the side chains of the constituent amino acids if they are protected, and then cyclizing the side chains of the constituent amino acids having an amino group and the side chains of the cysteine ​​or cysteine ​​derivative (embodiment C).

[0071] As used herein, "cysteine ​​derivatives" include homocysteine ​​and the like.

[0072] In the method for producing a peptide having a cyclic thioether bond of the present invention, the protecting group at the C-terminus of the linear peptide may be a pseudo-solid-phase protecting group, a liquid-phase protecting group, or a solid-phase carrier. Among these, a pseudo-solid-phase protecting group is preferred.

[0073] The "alkylenecarbonyl group having a leaving group" in the present invention is, for example, a group that reacts with a thiol group in the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative, and includes a halogenoalkylenecarbonyl group, a tosyloxyalkylenecarbonyl group, a mesyloxyalkylenecarbonyl group, etc. Examples of the halogenoalkylenecarbonyl group include a chloroalkylenecarbonyl group, a bromoalkylenecarbonyl group, an iodoalkylenecarbonyl group, etc., and among these, a chloroalkylenecarbonyl group is more preferred. Examples of the alkylene group in the halogenoalkylenecarbonyl group, the tosyloxyalkylenecarbonyl group, and the mesyloxyalkylenecarbonyl group include an alkylene group having 1 to 6 carbon atoms, and more preferably an alkylene group having 1 to 3 carbon atoms, and include a methylene group, an ethylene group, and a propylene group.

[0074] In the present invention, the "alkylene group having a carboxy group" is, for example, a group that reacts with the N-terminus of a peptide or a group that reacts with an amino group in the side chain of a constituent amino acid. The alkylene group of the above-mentioned "alkylene group having a carboxy group" includes alkylene groups having 1 to 6 carbon atoms, more preferably alkylene groups having 1 to 3 carbon atoms, and includes a methylene group, an ethylene group, and a propylene group.

[0075] Step (B) may further include a step of (B-1) modifying the side chain of a cysteine ​​or a cysteine ​​derivative of a linear peptide having a C-terminus protected or unprotected by a protecting group, a protected or unprotected N-terminus, and an unprotected side chain of a constituent amino acid, cysteine ​​or a cysteine ​​derivative, with an alkylene group having a carboxy group to produce a linear peptide having a C-terminus protected or unprotected by a protecting group, a protected or unprotected N-terminus, and a side chain of a constituent amino acid, cysteine ​​or a cysteine ​​derivative, modified with an alkylene group having a carboxy group.

[0076] Step (B) may further include the step (B-2) of deprotecting all protecting groups of the obtained protected cyclic peptide.

[0077] Step (C) may further comprise the step of: (C-1) modifying the side chain of cysteine ​​or a cysteine ​​derivative of a linear peptide in which the C-terminus is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative is unprotected, with an alkylene group having a carboxy group, to produce a linear peptide in which the C-terminus is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative is modified with an alkylene group having a carboxy group. EXAMPLES

[0078] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples, and can of course be practiced with appropriate modifications within the scope of the above and below aims, all of which are included in the technical scope of the present invention. Furthermore, reagents, devices, and materials used in the present invention are commercially available unless otherwise specified. Furthermore, when amino acids and the like are represented by abbreviations in this specification, each representation is based on the abbreviations according to the IUPAC-IUB Commission on Biochemical Nomenclature or the commonly used abbreviations in the relevant field.

[0079] Production Example 1: Linear peptide A (fully protected) Using Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(Me)-OH, and chlorobutyric acid as raw materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, linear peptide A (fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0080] Linear peptide A (fully protected) Cl-C3H6CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-NH-Dpm(4,4'-OPy)

[0081] Production Example 2: Linear peptide B (fully protected) Using Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(Me)-OH, and chloroacetic acid as raw materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, linear peptide B (fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0082] Linear peptide B (fully protected) Cl-CH2CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-NH-Dpm(4,4'-OPy)

[0083] Production Example 3: Linear peptide C (fully protected) 3-Mercapto(Trt)propionic acid, Fmoc- D Using -Tyr(Et)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Orn(Boc)-OH, and Fmoc-Gly-OH as starting materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, linear peptide C (fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0084] Linear peptide C (fully protected) 3-Mercapto(Trt)propionyl- D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-NH-Dpm(OPy)

[0085] Production Example 4: Linear peptide D (fully protected) Fmoc-Val-OH, Fmoc- D Using -Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, and Fmoc-Arg(Pbf)-OH as starting materials and 2-(3'-4'-5'-tri(2'',3''-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol (referred to as HO-MTB(OPhy)) as a pseudo solid-phase protecting group, a linear peptide D (fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0086] Linear peptide D (fully protected) Fmoc-Arg(Pbf)-Gly-Asp(OtBu)- D -Phe-Val-Arg(Pbf)-Gly-Asp(OtBu)- D -Phe-Val-O-MTB(OPhy)

[0087] Production Example 5: Linear peptide E (protected form with only the N-terminus free) Using Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, and Fmoc-Tyr(Me)-OH as starting materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, a linear peptide E (only the N-terminus is free and protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0088] Linear peptide E (protected form with only the N-terminus free) H-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-NH-Dpm(4,4'-OPy)

[0089] (HPLC measurement method) The compounds obtained in the following Examples were subjected to HPLC measurement under the following conditions. Column: YMC-Pack ODS-AM 150×4.6mm I.D. S-5μm 12nm Mobile phase A: Water containing 0.05% TFA (trifluoroacetic acid) Mobile phase B: acetonitrile containing 0.05% TFA Temperature: 40℃ Flow rate: 1.0mL / min Detection wavelength: 220nm Time program (ratio of mobile phase A): 0-25 min 80%→20% 25-30 minutes 1%

[0090] (Pretreatment method before HPLC measurement) Before the above-mentioned HPLC measurement, pretreatment was carried out to remove the side chain protecting groups and the pseudo solid-phase protecting groups, as necessary. The pretreatment method is shown below. A part of the compound obtained in each of the following Examples was deprotected by adding 0.2 ml of a mixed solution of TFA / water / TIPS (triisopropylsilane) = 95.0 / 2.5 / 2.5 and stirring for 1 to 3 hours. Then, 0.8 ml of 50% acetonitrile water was added to the solution to prepare a sample for HPLC.

[0091] Example 1 5.0ml of chloroform, 0.9ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 500mg of linear peptide A (fully protected form) having a chlorobutyryl group at the N-terminus of the linear peptide and a methoxytrityl protecting group at the Cys residue in the peptide chain, which was synthesized in Production Example 1, and only the methoxytrityl group was removed in an ice bath. After stirring for 30 minutes, 0.98 equivalents of pyridine were added to the reaction solution to neutralize it, and the same amount of pure water as chloroform was added to separate the liquid. The obtained organic layer was concentrated with an evaporator, 5.0ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 307mg of deprotected form. To the obtained deprotected product (102 mg), 4.1 ml of chloroform and 1 equivalent of DBU (1,8-diazabicyclo[5.4.0]-7-undecene) were added to carry out cyclization between the terminal chlorobutyryl group and the SH group. After stirring overnight, 4.0 ml of 20% NaCl (sodium chloride) aqueous solution was added, and the mixture was separated, concentrated, and dried to obtain cyclic peptide A (100 mg). To the obtained cyclic peptide A (100 mg), 0.3 ml of chloroform was added and stirred well. Then, 2.7 ml of acetonitrile was added, stirred well, and the insoluble matter was removed by filtration. To the obtained insoluble matter, 0.2 ml of chloroform was added and stirred well. Then, 1.4 ml of acetonitrile was added, stirred well, and the insoluble matter was removed by filtration again. This was repeated twice, and the mother liquors obtained were mixed and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide A had improved from 74% to 89%. (85% yield vs. cyclic peptide A before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 988.3

[0092] Example 2 10.0 ml of chloroform, 0.5 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.0 g of linear peptide B (fully protected form) having a chloroacetyl group at the N-terminus of the linear peptide and a methoxytrityl protecting group at the Cys residue in the peptide chain, which was synthesized in Production Example 2, and only the methoxytrityl group was removed in an ice bath. After stirring for 30 minutes, 0.98 equivalents of pyridine were added to the reaction solution to neutralize it, and the same amount of pure water as chloroform was added and separated. The obtained organic layer was concentrated with an evaporator, 5.0 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 780 mg of deprotected form. To the obtained deprotected product (195 mg), 2.0 ml of chloroform and 1 equivalent of DBU were added to carry out cyclization between the terminal chloroacetyl group and the SH group. After stirring for 1 hour, the mixture was neutralized with 0.9 equivalents of methanesulfonic acid, and 2.0 ml of 20% NaCl aqueous solution was added. The mixture was separated, concentrated, and dried to obtain 206 mg of cyclic peptide B. To the obtained cyclic peptide B, 0.4 ml of chloroform was added and stirred well. Then, 4.3 ml of acetonitrile was added, stirred well, and the insoluble matter was removed by filtration. Then, the mixture was concentrated and dried to obtain 147 mg of cyclic peptide B. To the obtained insoluble matter, 0.3 ml of chloroform was added and stirred well. Then, 3.2 ml of acetonitrile was added, stirred well, and the insoluble matter was removed by filtration again. The obtained mother liquors were mixed and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide B had improved from 85% to 94%. (99% yield vs. cyclic peptide B before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 960.7

[0093] Example 3 6.8 ml of CPME (cyclopentyl methyl ether), 1.2 ml of methanol, and 1 equivalent of iodine were added to 200 mg of linear peptide C (fully protected form) having a mercaptopropionyl group protected by a trityl group at the N-terminus of the linear peptide and a trityl protecting group at the Cys residue, synthesized in Production Example 3, and cyclization between SH groups was carried out at room temperature. After stirring for 4 hours, the mixture was separated twice with an aqueous solution in which 133 mg of ascorbic acid was dissolved in 8 ml of water, and then washed twice with a 20% aqueous NaCl solution. The obtained organic layer was concentrated and dried using an evaporator to obtain 195 mg of cyclic peptide C. 0.6 ml of chloroform was added to 98 mg of the obtained cyclic peptide C and stirred well. Then, 3.0 ml of acetonitrile was added, stirred well, and insoluble matter was removed by filtration. 0.6 ml of chloroform was added to the obtained insoluble matter and stirred well. Then, 3.0 ml of acetonitrile was added, and the mixture was thoroughly stirred, after which the insoluble matter was filtered off again. The resulting mother liquors were combined and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide C had improved from 67% to 87%. (96% yield vs. cyclic peptide C before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 994.2

[0094] Example 4 24.0 ml of chloroform was added to 1.20 g of linear peptide D (fully protected) in which the N-terminus of the linear peptide was protected with an Fmoc group and the C-terminus was protected with a pseudo solid-phase protecting group, synthesized in Production Example 4, and 3 equivalents of thiomalic acid and 11 equivalents of DBU were added in an ice bath to remove only the Fmoc group at the N-terminus under room temperature conditions. After stirring for 2 hours, a mixed solution of 112 mg of acetic acid and 0.6 ml of chloroform was added in an ice bath. Then, the mixture was separated twice with an aqueous solution of 2.6 ml of DMF and 10.6 ml of 5% sodium carbonate solution, and then separated twice with 3.8 ml of DMF and 5.8 ml of 20% NaCl aqueous solution, and once with 24 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated and dried using an evaporator to obtain 1.07 g of a solid. To the obtained solid, 10.7 ml of HFIP (hexafluoroisopropanol) was added, and only the C-terminal pseudo solid-phase protecting group was removed at room temperature. After stirring for 5 hours, the mixture was concentrated with an evaporator. Then, 64 ml of cyclohexane was added to isolate the product, and the solid was dried to obtain 0.65 g of linear peptide D with no N-terminal protection and no C-terminal protection. 1.5 ml of chloroform, 0.5 equivalents of HOBt (1-hydroxybenzotriazole), and 1.1 equivalents of EDC.HCl (N-ethyl-N'-3-dimethylaminopropylcarbodiimide hydrochloride) were added to 150 mg of the linear peptide D, and cyclization was performed by intramolecular amide bond (lactam bond) at room temperature. After stirring for 4 hours, the mixture was washed once with 1.5 ml of 20% NaCl aqueous solution. The resulting organic layer was concentrated and dried using an evaporator to obtain 142 mg of cyclic peptide D. 1.7 ml of chloroform was added to 142 mg of the resulting cyclic peptide D, and the mixture was stirred well. Then, 4.0 ml of IPE (diisopropyl ether) was added, and the mixture was stirred well, and the insoluble matter was removed by filtration. 1.7 ml of chloroform was added to the resulting insoluble matter, and the mixture was stirred well. Then, 4.0 ml of IPE was added, and the mixture was stirred well, and the insoluble matter was removed by filtration again. This was repeated three times. The resulting mother liquors were combined and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide D had improved from 35% to 75%. (Yield 86% vs. cyclic peptide D before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 1149.4

[0095] Example 5 15.0 ml of chloroform, 7.4 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.57 g of linear peptide E (protected form with only the N-terminus free) synthesized in Production Example 5, which has an unprotected N-terminus and a methoxytrityl protecting group on the Cys residue in the peptide chain, and only the methoxytrityl group was removed in an ice bath. After stirring for 7 hours, 0.98 equivalents of pyridine was added to the reaction solution to neutralize it, and the same amount of pure water as the chloroform was added to separate the liquid. The resulting organic layer was concentrated with an evaporator, 15.7 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 1.04 g of a deprotected product. 1.0 ml of chloroform was added to 100 mg of the obtained deprotected product, and 2.1 equivalents of chloroacetic acid and 6.0 equivalents of DBU were added in an ice bath, and the chloroacetic acid was subjected to a nucleophilic substitution reaction with the SH group of the Cys residue of the linear peptide under room temperature conditions. After stirring for 4 hours, the mixture was washed twice with 1.0 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated and dried using an evaporator to obtain an oily substance. 4.1 ml of chloroform, 2.5 equivalents of HOBt, and 1.1 equivalents of EDC.HCl were added to the obtained oily substance, and cyclization between the N-terminal amino group and the side chain carboxyl group was carried out at room temperature. After stirring for 17 hours, the mixture was washed twice with 4.1 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated and dried using an evaporator to obtain cyclic peptide E. 0.6 ml of chloroform was added to the obtained cyclic peptide E and stirred well. Then, 6.9 ml of acetonitrile was added, stirred well, and insoluble matter was removed by filtration. 0.1 ml of chloroform was added to the obtained insoluble matter and stirred well. Then, 1.6 ml of acetonitrile was added, stirred well, and insoluble matter was removed by filtration again. The obtained mother liquors were combined and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide E had improved from 73% to 83%. (Yield 89% vs. cyclic peptide E before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 960.7

[0096] Example 6 To 388 mg of cyclic peptide A synthesized in Example 1 before filtering off the insoluble matter, 7.8 ml of a mixed solution of TFA (trifluoroacetic acid) / water / TIPS (triisopropylsilane) = 95.0 / 2.5 / 2.5 and 10 equivalents of mercaptopropionic acid were added for final deprotection so that all protective groups were deprotected. After stirring for 2 hours, 38.8 ml of IPE (diisopropyl ether) was added, the precipitate was filtered, and dried to obtain 157 mg of cyclic peptide A' (unprotected form). 30.0 μl of DMF was added to 5.0 mg of the obtained cyclic peptide A' and stirred well. Then, 60.0 μl of water was added next, and after stirring well, the insoluble matter was filtered off. The obtained insoluble matter was washed with a mixed solution of 100.0 μl of DMF and 200.0 μl of water. The obtained mother liquors were mixed and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide A' had improved from 66% to 88%. (Yield 81% vs. Cyclic Peptide A' before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 988.3

[0097] Example 7 To 98 mg of cyclic peptide C synthesized in Example 3 before filtering out the insoluble matter, 2.0 ml of a mixed solution of TFA / water = 97.5 / 2.5 and 10 equivalents of p-cresol were added for final deprotection so that all protective groups were deprotected. After stirring for 16 hours, 10.0 ml of IPE was added, the precipitate was filtered, and dried to obtain 49 mg of cyclic peptide C' (unprotected form). 45.0 μl of methanol was added to 5.0 mg of the obtained cyclic peptide C' and stirred well. Then, 45.0 μl of IPE was added, stirred well, and the insoluble matter was filtered out. The obtained insoluble matter was washed with a mixed solution of 250.0 μl of methanol and 250.0 μl of IPE. The obtained mother liquors were mixed and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide C' had improved from 75% to 89%. (91% yield vs. cyclic peptide C' before filtering out the insoluble matter) TOF-MS: m / z [M+H] + 994.2

[0098] Example 8 2.6 ml of a mixed solution of TFA / water / TIPS=95.0 / 2.5 / 2.5 was added to 128 mg of cyclic peptide D synthesized in Example 4 before filtering out the insoluble matter, and the final deprotection was performed so that all the protecting groups were deprotected. After stirring for 3 hours, 13.0 ml of IPE was added, the precipitate was filtered, and dried to obtain 79 mg of cyclic peptide D' (unprotected form). 60.0 μl of DMF was added to 10.0 mg of the obtained cyclic peptide D' and stirred well. Then, 240.0 μl of water was added, and after stirring well, the insoluble matter was filtered out. The obtained insoluble matter was washed with a mixed solution of 40.0 μl of DMF and 160.0 μl of water. The obtained mother liquors were mixed and analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide D' had improved from 47% to 68%. (76% yield vs. cyclic peptide D' before filtering out the insoluble matter) TOF-MS: m / z [M+H] + 1149.4

[0099] The above Example 1 is an example in which cyclic peptide A (CS type) is produced using chloroform as a good solvent and acetonitrile as a poor solvent in embodiment 1 of the present application. Also, the above Example 1 corresponds to embodiment A. The above Example 2 is an example of producing cyclic peptide B (CS type) in embodiment 1 of the present application using chloroform as a good solvent and acetonitrile as a poor solvent. The above Example 2 corresponds to embodiment A. The above Example 3 is an example in which cyclic peptide C (SS type) is produced in accordance with embodiment 1 of the present application using chloroform as a good solvent and acetonitrile as a poor solvent. The above Example 4 is an example in which cyclic peptide D (lactam type) is produced in accordance with embodiment 2 of the present application using chloroform as a good solvent and IPE (diisopropyl ether) as a poor solvent. The above Example 5 is an example in which cyclic peptide E (CS type) is produced using chloroform as a good solvent and acetonitrile as a poor solvent in embodiment 1 of the present application. Moreover, the above Example 5 corresponds to embodiment B. The above Example 6 is an example in which cyclic peptide A (CS type) is produced using DMF as a good solvent and water as a poor solvent in embodiment 1 of the present application. Moreover, the above Example 6 corresponds to embodiment A. The above Example 7 is an example in which cyclic peptide C (SS type) is produced in accordance with embodiment 1 of the present application using methanol as a good solvent and IPE as a poor solvent. The above Example 8 is an example in which cyclic peptide D (lactam type) is produced in accordance with embodiment 2 of the present application using DMF as a good solvent and water as a poor solvent.

[0100] Example 9 15.0 ml of chloroform, 7.4 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.57 g of linear peptide E (protected form only at the N-terminus) synthesized in Production Example 5, which has an unprotected N-terminus and a methoxytrityl protecting group at the Cys residue in the peptide chain, and only the methoxytrityl group was removed in an ice bath. After stirring for 7 hours, 0.98 equivalents of pyridine was added to the reaction solution to neutralize it, and the same amount of pure water as chloroform was added to separate the mixture. The resulting organic layer was concentrated with an evaporator, 15.7 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 1.04 g of a deprotected product. 8.8 ml of chloroform was added to 875 mg of the obtained deprotected product, and 2.1 equivalents of chloroacetic acid and 6.0 equivalents of DBU were added in an ice bath, and the chloroacetic acid was subjected to a nucleophilic substitution reaction with the SH group of the Cys residue of the linear peptide under room temperature conditions. After stirring for 2 hours, a mixture of 4.5 equivalents of acetic acid and 0.5 ml of chloroform was added to neutralize the mixture, and the mixture was washed once with 8.8 ml of 20% NaCl aqueous solution. 0.5 equivalents of HOBt and 1.6 equivalents of EDC.HCl were added to the obtained organic layer, and cyclization between the N-terminal amino group and the side chain carboxyl group was carried out at room temperature. After stirring for 18 hours, the mixture was washed once with 10.0 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated and dried using an evaporator to obtain a solid. The obtained solid was added with 9.5 ml of a mixture of TFA / water / TIPS=95.0 / 2.5 / 2.5 and 10 equivalents of mercaptopropionic acid for final deprotection so that all protecting groups were deprotected. After stirring for 20 hours, 47.0 ml of IPE was added, the precipitate was filtered, and dried to obtain 375 mg of cyclic peptide E' (completely unprotected) before insoluble matter was removed by filtration. 12.1 ml of methanol was added to 152.0 mg of the obtained cyclic peptide E' before filtering off the insoluble matter, and the mixture was thoroughly stirred. Then, 18.2 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off. 12.1 ml of methanol was added to the obtained insoluble matter, and the mixture was thoroughly stirred. Then, 18.2 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. The obtained mother liquors were mixed together and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide E' had improved from 47% to 86%. (90% yield vs. cyclic peptide E' before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 960.5

[0101] Production Example 6: Linear peptide F (N-terminal Boc fully protected) The linear peptide F (fully N-terminal Boc-protected) having the following sequence was synthesized according to a conventional method using Boc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, and Fmoc-Gly-OH as raw materials and Siber Amide resin as a solid-phase protecting group.

[0102] Linear peptide F (N-terminal Boc fully protected) Boc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-NH-Siber resin

[0103] Example 10 6.3 ml of chloroform and 126 μl of trifluoroacetic acid were added to 632 mg of linear peptide F (N-terminal Boc fully protected) having a Boc protecting group at the N-terminus, which was synthesized in Production Example 6, and only the solid-phase protecting group was removed under room temperature conditions. After stirring for 1 hour, the deprotected solid-phase protecting group was filtered to obtain a filtrate. 6.3 ml of chloroform and 126 μl of trifluoroacetic acid were added again to the filtered solid-phase protecting group, and after stirring for 1 hour under room temperature conditions, the deprotected solid-phase protecting group was filtered to obtain a filtrate. The obtained filtrate was mixed, neutralized by adding 147 μl of piperidine in an ice bath, concentrated with an evaporator, and then 25.2 ml of IPE was added in an ice bath, and the precipitate was filtered and dried to obtain 697 mg of a protected peptide amide. 12.8 ml of chloroform, 2.3 ml of methanol, and 3.0 equivalents of iodine were added to 300 mg of the obtained protected peptide amide, and cyclization between SH groups was performed at room temperature. After stirring for 30 minutes, the mixture was separated twice with an aqueous solution of 148.6 mg of ascorbic acid dissolved in 5.0 ml of water, and then washed with a 20% NaCl (sodium chloride) aqueous solution. The obtained organic layer was concentrated with an evaporator, and 10.0 ml of IPE was added in an ice bath, and the precipitate was filtered and dried to obtain 177 mg of cyclic peptide F before insoluble matter was removed by filtration. 2.2 ml of THF was added to 100.3 mg of the obtained cyclic peptide F before insoluble matter was removed by filtration, and the mixture was stirred well. Then, 2.8 ml of hexane was added in sequence, and the insoluble matter was removed by filtration after stirring well. 2.2 ml of THF was added to the obtained insoluble matter, and the mixture was stirred well. Then, 2.8 ml of hexane was added in sequence, and the insoluble matter was removed by filtration again after stirring well. The obtained mother liquors were combined and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide F had improved from 49% to 70%. (Yield 78% vs. cyclic peptide F before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 1007.4

[0104] Example 11 5.6 ml of chloroform and 111 μl of trifluoroacetic acid were added to 556 mg of linear peptide F (N-terminal Boc fully protected) having a Boc protecting group at the N-terminus, which was synthesized in Production Example 6, and only the solid-phase protecting group was removed at room temperature. After stirring for 1 hour, 111 μl of piperidine was added in an ice bath for neutralization, and the deprotected solid-phase protecting group was filtered to obtain a filtrate. 5.6 ml of chloroform and 111 μl of trifluoroacetic acid were added again to the filtered solid-phase protecting group, and the mixture was stirred for 1 hour at room temperature, and then 111 μl of piperidine was added in an ice bath for neutralization, and the deprotected solid-phase protecting group was filtered to obtain a filtrate. The obtained filtrates were mixed, concentrated and dried in an evaporator to obtain a protected peptide amide. 13.1 ml of chloroform, 2.5 ml of methanol, and 3.0 equivalents of iodine were added to the obtained protected peptide amide, and cyclization between SH groups was performed at room temperature. After stirring for 30 minutes, the mixture was separated twice with an aqueous solution of 152.2 mg of ascorbic acid dissolved in 5.0 ml of water, and then washed with 10.0 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated with an evaporator, and then 10.0 ml of IPE was added in an ice bath, the precipitate was filtered, and dried to obtain 163 mg of cyclic peptide F'. 163 mg of the obtained cyclic peptide F' was added with 3.3 ml of a mixed solution of TFA / water / p-cresol = 97.5 / 2.5 / 2.5 for final deprotection so that all protecting groups were deprotected. After stirring for 3 hours, 9.5 ml of IPE was added in an ice bath, the precipitate was filtered, and dried to obtain 82 mg of cyclic peptide F'' (completely unprotected) before insoluble matter was removed by filtration. To 63 mg of the obtained cyclic peptide F'' before filtering off the insoluble matter, 1.3 ml of methanol was added and thoroughly stirred. Then, 1.9 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off. To the obtained insoluble matter, 0.6 ml of methanol was added and thoroughly stirred. Then, 0.9 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. The obtained mother liquors were combined and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide F'' had improved from 43% to 81%. (Yield 97% vs. cyclic peptide F before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 1007.3

[0105] Production Example 7: Linear peptide G (N-terminal Ac fully protected) Using Fmoc-Lys(Mtt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(OPis)-OH, and Fmoc-Leu-OH as raw materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, linear peptide G (N-terminal Ac fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803). The Ac group at the N-terminus of the following peptide was converted to Ac using acetic anhydride and N-ethyldiisopropylamine.

[0106] Linear peptide G (N-terminal Ac fully protected) Ac-Lys(Mtt)-Tyr(tBu)-Ile-Pro-Gln(Trt)-Ala-Ala-Gln(Trt)-Ala-Ala-Glu(OPis)-Ile-Pro-Leu-NH-Dpm(4,4'-OPy)

[0107] Example 12 14.8 ml of chloroform and 225 μl of trifluoroacetic acid were added to 489 mg of linear peptide G (N-terminal Ac complete protection) having an Ac protecting group at the N-terminus, a methyltrityl protecting group at the Lys residue in the peptide chain, and a 2-phenylisopropyl protecting group at the Glu residue in the peptide chain, and the methyltrityl group and the 2-phenylisopropyl group were removed at room temperature. After stirring for 2 hours, the mixture was neutralized by adding 237 μl of piperidine in an ice bath, and then separated twice with 10.0 ml of 5% Na2CO3 (sodium carbonate) aqueous solution, and washed twice with 10.0 ml of 20% NaCl (sodium chloride) aqueous solution. The obtained organic layer was concentrated with an evaporator, 10.0 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 410 mg of deprotected G. 25.0 ml of DMF, 1.0 equivalent of HOBt, and 3.0 equivalent of EDC.HCl were added to the obtained deprotected product G (410 mg), and cyclization was performed by intramolecular amide bond (lactam bond) at room temperature. After stirring overnight, 50.0 ml of CPME and 50.0 ml of 20% NaCl aqueous solution were added and the mixture was separated. The obtained organic layer was concentrated with an evaporator, 10.0 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 426 mg of cyclic peptide G before insoluble matter was removed by filtration. 2.3 ml of chloroform was added to 198 mg of the obtained cyclic peptide G before filtering off the insoluble matter, and the mixture was thoroughly stirred. Then, 57.2 ml of acetonitrile was added in sequence, and after thorough stirring, the insoluble matter was filtered off. 1.2 ml of chloroform was added to the obtained insoluble matter, and the mixture was thoroughly stirred. Then, 58.3 ml of acetonitrile was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. The obtained mother liquors were mixed together and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide F had improved from 40% to 71%. (90% yield vs. cyclic peptide G before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 1535.8

[0108] Production Example 8: Linear peptide H (fully protected) The linear peptide H (fully protected) having the following sequence was synthesized according to a conventional method using Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and chloroacetic acid as raw materials and Siber Amide resin as a solid-phase protecting group.

[0109] Linear peptide H (fully protected) Cl-CH2CO-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-NH-Siber resin

[0110] Example 13 12.5 ml of chloroform, 656 μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 658 mg of linear peptide H (fully protected form) having a chloroacetyl group at the N-terminus of the linear peptide and a methoxytrityl protecting group at the Cys residue in the peptide chain, which was synthesized in Production Example 8, and the methoxytrityl group and the solid-phase protecting group were removed in an ice bath. After stirring for 30 minutes, 694 μl of pyridine was added to the reaction solution to neutralize. Then, the deprotected solid-phase protecting group was filtered to obtain a filtrate. 12.5 ml of chloroform, 656 μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added again to the filtered solid-phase protecting group, and the methoxytrityl group and the solid-phase protecting group were removed in an ice bath. After stirring for 30 minutes, 694 μl of pyridine was added to the reaction solution to neutralize. Then, the deprotected solid-phase protecting group was filtered to obtain a filtrate. The filtrates were mixed, and then 13.2 ml of pure water was added to separate the layers. The organic layer was concentrated using an evaporator, and 32.9 ml of IPE was added in an ice bath. The precipitate was filtered and dried to obtain 203 mg of a protected peptide amide in which only the SH group of the Cys residue in the peptide chain was deprotected. 2.0 ml of chloroform and 1.5 equivalents of DBU were added to 203 mg of the protected peptide amide in which only the SH group of the Cys residue in the obtained peptide chain was deprotected, and cyclization between the terminal chloroacetyl group and the SH group was performed. After stirring for 30 minutes, 1.4 equivalents of acetic acid were added in an ice bath to neutralize. Then, 8.0 ml of 20% NaCl aqueous solution was added, and the mixture was separated twice, concentrated and dried to obtain 199 mg of cyclic peptide H. 7.2 ml of chloroform was added to 199 mg of the obtained cyclic peptide H and stirred well. Then, 12.7 ml of IPE was added in sequence, and after stirring well, the insoluble matter was filtered off. 7.2 ml of chloroform was added to the obtained insoluble matter and stirred well. Then, 12.7 ml of IPE was added in sequence, and after stirring well, the insoluble matter was filtered off again. This was repeated four times, and the mother liquors obtained were mixed and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide H had improved from 55% to 79%. (70% yield vs. cyclic peptide H before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 946.4

[0111] Production Example 9: Linear peptide I (protected form with only the N-terminus free) Using Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH as raw materials and Siber Amide resin as a solid-phase protecting group, linear peptide F (only the N-terminus is free and protected) having the following sequence was synthesized according to a conventional method.

[0112] Linear peptide I (protected form with only the N-terminus free) H-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-NH-Siber resin

[0113] Example 14 10.1 ml of chloroform, 0.5 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 517 mg of linear peptide I (protected only at the N-terminus) synthesized in Production Example 9, which has an unprotected N-terminus and a methoxytrityl protecting group at the Cys residue in the peptide chain, and the methoxytrityl group and the solid-phase protecting group were removed in an ice bath. After stirring for 30 minutes, 533 μl of pyridine was added to the reaction solution to neutralize. Then, the deprotected solid-phase protecting group was filtered to obtain a filtrate. 10.1 ml of chloroform, 0.5 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added again to the filtered solid-phase protecting group, and the methoxytrityl group and the solid-phase protecting group were removed in an ice bath. After stirring for 30 minutes, 533 μl of pyridine was added to the reaction solution to neutralize. Then, the deprotected solid-phase protecting group was filtered to obtain a filtrate. After mixing the obtained filtrate, 20.2 ml of pure water was added and the mixture was separated three times. The obtained organic layer was concentrated with an evaporator, and then 25.3 ml of IPE was added in an ice bath, and the precipitate was filtered and dried to obtain 188 mg of a protected peptide amide in which only the N-terminus and the SH group of the Cys residue in the peptide chain were deprotected. 1.9 ml of chloroform was added to the obtained 188 mg of a protected peptide amide in which only the N-terminus and the SH group of the Cys residue in the peptide chain were deprotected, and 5.0 equivalents of chloroacetic acid and 9.0 equivalents of DBU were added in an ice bath, and the chloroacetic acid was subjected to a nucleophilic substitution reaction with the SH group of the Cys residue of the linear peptide under room temperature conditions. After stirring for 1 hour, the mixture was separated twice with 7.5 ml of 5% Na2CO3 aqueous solution, and then separated and washed twice with 20% NaCl aqueous solution. To the obtained organic layer, 20.0 ml of chloroform, 2.0 equivalents of HOBt, and 1.1 equivalents of EDC.HCl were added, and cyclization between the N-terminal amino group and the side chain carboxyl group was carried out at room temperature. After stirring for 1 hour, the mixture was washed once with 20.0 ml of 20% NaCl aqueous solution. The obtained organic layer was concentrated and dried using an evaporator to obtain 183 mg of cyclic peptide I' before filtering out insoluble matter. 3.7 ml of chloroform was added to 101 mg of the obtained cyclic peptide I' before filtering off the insoluble matter, and the mixture was thoroughly stirred. Then, 6.4 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off. 3.7 ml of chloroform was added to the obtained insoluble matter, and the mixture was thoroughly stirred. Then, 6.4 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. This was repeated four times. The obtained mother liquors were mixed together and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide I' had improved from 38% to 70%. (Yield 87% vs. cyclic peptide I' before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 946.4

[0114] Example 15 14.3 ml of chloroform, 975 μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 489 mg of linear peptide G (N-terminal Ac complete protection) having an Ac protecting group at the N-terminus, a methyltrityl protecting group at the Lys residue in the peptide chain, and a 2-phenylisopropyl protecting group at the Glu residue in the peptide chain, which was synthesized in Production Example 7, and the methyltrityl group and the 2-phenylisopropyl group were removed in an ice bath. After stirring for 2 hours, 1.0 g of piperidine was added in an ice bath for neutralization, and then the mixture was separated twice with 15.0 ml of pure water. The obtained organic layer was concentrated with an evaporator, 10.0 ml of acetonitrile was added, and the precipitate was filtered and dried to obtain 404 mg of deprotected form G'. 18.0 ml of DMF, 1.0 equivalent of HOBt, and 3.0 equivalent of EDC.HCl were added to 300 mg of the obtained deprotected form G', and cyclization was performed by intramolecular amide bond (lactam bond) at room temperature. After stirring overnight, 36.0 ml of CPME and 36.0 ml of 20% NaCl aqueous solution were added and the mixture was separated. The obtained organic layer was concentrated with an evaporator, 10.0 ml of acetonitrile was added, the precipitate was filtered, and dried to obtain 276 mg of cyclic peptide G'. 5.5 ml of a mixed solution of TFA / water / TIPS=95.0 / 2.5 / 2.5 and 10 equivalents of mercaptopropionic acid were added to the obtained cyclic peptide G' 276.0 mg for final deprotection so that all protecting groups were deprotected. After stirring for 17 hours, 27.6 ml of IPE was added, the precipitate was filtered, and dried to obtain 146 mg of cyclic peptide G'' (completely unprotected form) before insoluble matter was removed by filtration. To 58 mg of the obtained cyclic peptide G'' before filtering off the insoluble matter, 0.9 ml of methanol was added and thoroughly stirred. Then, 2.0 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off. To the obtained insoluble matter, 0.9 ml of methanol was added and thoroughly stirred. Then, 2.0 ml of IPE was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. This was repeated three times. The obtained mother liquors were combined and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide G' had improved from 40% to 70%. (Yield 92% vs. cyclic peptide G' before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 1535.7

[0115] Production Example 10: Linear peptide J (fully protected) Using Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, and chloroacetic acid as raw materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, a linear peptide G (fully protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem. Int. Ed. 2017. 27, (56), 7803).

[0116] Linear peptide J (fully protected) Cl-CH2CO-Tyr(tBu)-Ile-Pro-Tyr(tBu)-Gln(Trt)-Ala-Ala-Cys(Mmt)-Ile-Pro-Leu-NH-Dpm(4,4'-OPy)

[0117] Example 16 30.0ml of chloroform, 525μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.50g of linear peptide J (fully protected form) having a chloroacetyl group at the N-terminus of the linear peptide and a methoxytrityl protecting group at the Cys residue in the peptide chain, which was synthesized in Production Example 10, and only the methoxytrityl group was removed in an ice bath. After stirring for 30 minutes, 536μl of pyridine was added to the reaction solution to neutralize it. Then, 30.0ml of pure water was added and the mixture was separated twice. The obtained organic layer was concentrated with an evaporator, 30.0ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain 1.11g of a deprotected form in which only the SH group of the Cys residue in the peptide chain was deprotected. To 300 mg of the deprotected product in which only the SH group of the Cys residue in the obtained peptide chain was deprotected, 6.0 ml of chloroform and 1.5 equivalents of DBU were added to carry out cyclization between the terminal chloroacetyl group and the SH group. After stirring for 3 hours, 0.5 equivalents of acetic acid was added in an ice bath to neutralize. Then, 6.0 ml of 20% NaCl aqueous solution was added and separated twice. The obtained organic layer was concentrated with an evaporator, and 30.0 ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain cyclic peptide J. 2.4 ml of chloroform was added to the obtained cyclic peptide J and stirred well. Then, 48.1 ml of acetonitrile was added in sequence, and after stirring well, the insoluble matter was filtered off. 0.9 ml of chloroform was added to the obtained insoluble matter and stirred well. Then, 18.9 ml of acetonitrile was added in sequence, and after stirring well, the insoluble matter was filtered off again. The obtained mother liquors were combined and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide H had improved from 51% to 88%. (Yield 83% vs. cyclic peptide J before removing insoluble matter by filtration) TOF-MS: m / z [M+H] + 1290.6

[0118] Production Example 11: Linear peptide K (protected form with only the N-terminus free) Using Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Tyr(tBu)-OH as starting materials and (4,4'-bishydrophytyloxy)benzhydrylamine (referred to as NH2-Dpm(4,4'-OPhy)) as a pseudo solid-phase protecting group, linear peptide K (only the N-terminus is free and protected) having the following sequence was synthesized according to a standard method (see International Publication No. 2012 / 029794 and Angew Chem.Int.Ed. 2017. 27, (56), 7803).

[0119] Linear peptide K (protected form with only the N-terminus free) H-Tyr(tBu)-Ile-Pro-Tyr(tBu)-Gln(Trt)-Ala-Ala-Cys(Mmt)-Ile-Pro-Leu-NH-Dpm(4,4'-OPy)

[0120] Example 17 30.0ml of chloroform, 525μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.50g of linear peptide K (protected form with only the N-terminus free) synthesized in Production Example 11, which has an unprotected N-terminus and a methoxytrityl protecting group on the Cys residue in the peptide chain, and only the methoxytrityl group was removed in an ice bath. After stirring for 30 minutes, 536μl of pyridine was added to the reaction solution to neutralize it. Then, 30.0ml of pure water was added and the mixture was separated twice. The obtained organic layer was concentrated with an evaporator, 30.0ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain 1.11g of a deprotected form in which only the SH group of the Cys residue in the peptide chain was deprotected. 5.0 ml of chloroform was added to 500 mg of the deprotected product in which only the SH group of the Cys residue in the obtained peptide chain was deprotected, and 5.0 equivalents of chloroacetic acid and 9.0 equivalents of DBU were added in an ice bath, and chloroacetic acid was subjected to a nucleophilic substitution reaction with the SH group of the Cys residue of the linear peptide under room temperature conditions. After stirring for 1 hour, the mixture was separated twice with 5.0 ml of 5% Na2CO3 aqueous solution, and then separated and washed with 20% NaCl aqueous solution. The obtained organic layer was concentrated with an evaporator, and 15.0 ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain a solid. 17.4 ml of chloroform, 2.0 equivalents of HOBt, and 2.0 equivalents of EDC.HCl were added to the obtained solid, and cyclization between the N-terminal amino group and the side chain carboxyl group was performed at room temperature. After stirring for 1 hour, the mixture was washed once with 20.0 ml of 20% NaCl aqueous solution. The resulting organic layer was concentrated and dried using an evaporator to obtain 322 mg of cyclic peptide K before insoluble matter was removed by filtration. 1.9 ml of chloroform was added to 200 mg of the obtained cyclic peptide K before filtering off the insoluble matter, and the mixture was thoroughly stirred. Then, 38.1 ml of acetonitrile was added in sequence, and after thorough stirring, the insoluble matter was filtered off. 1.9 ml of chloroform was added to the obtained insoluble matter, and the mixture was thoroughly stirred. Then, 38.1 ml of acetonitrile was added in sequence, and after thorough stirring, the insoluble matter was filtered off again. The obtained mother liquors were mixed together and quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide K had improved from 48% to 87%. (95% yield vs. cyclic peptide K before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 1290.6

[0121] Example 18 60.0 ml of chloroform, 1.35 ml of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 3.0 g of linear peptide J (fully protected form) having a chloroacetyl group at the N-terminus of the linear peptide and a methoxytrityl protecting group at the Cys residue in the peptide chain, which was synthesized in Production Example 10, and only the methoxytrityl group was removed in an ice bath. After stirring for 3 hours, 1.4 ml of pyridine was added to the reaction solution to neutralize it. Then, 60.0 ml of pure water was added and the mixture was separated twice. The obtained organic layer was concentrated using an evaporator, and 60.0 ml of acetonitrile was added at room temperature to filter the precipitate and dry it to obtain 2.70 g of a deprotected form in which only the SH group of the Cys residue in the peptide chain was deprotected. To the resulting deprotected product (794 mg) in which only the SH group of the Cys residue in the peptide chain was deprotected, 15.9 ml of chloroform and 1.5 equivalents of DBU were added to carry out cyclization between the terminal chloroacetyl group and the SH group. After stirring for 3 hours, 0.5 equivalents of acetic acid was added in an ice bath to neutralize. Then, 50.0 ml of 20% NaCl aqueous solution was added and separated twice. The resulting organic layer was concentrated using an evaporator, and 50.0 ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain 669 mg of cyclic peptide J'. To the resulting cyclic peptide J' (669 mg), 12.7 ml of a mixed solution of TFA / water / TIPS=95.0 / 2.5 / 2.5 and 10 equivalents of mercaptopropionic acid were added for final deprotection so that all protecting groups were deprotected. After stirring for 6 hours, 66.9 ml of IPE was added, and the precipitate was filtered and dried to obtain 379 mg of cyclic peptide J'' (completely unprotected) before removing insoluble matter by filtration. To 201 mg of the obtained cyclic peptide J'', 28.7 ml of methanol was added and thoroughly stirred. Then, 71.6 ml of IPE was added in sequence, thoroughly stirred, and insoluble matter was removed by filtration. The obtained mother liquor was quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide J'' had improved from 51% to 77%. (Yield 93% vs. cyclic peptide J'' before filtration of insoluble matter) TOF-MS: m / z [M+H] + 1290.5

[0122] Example 19 30.0ml of chloroform, 525μl of trifluoroacetic acid, and 10 equivalents of mercaptopropionic acid were added to 1.50g of linear peptide K (protected form with only the N-terminus free) synthesized in Production Example 11, which has an unprotected N-terminus and a methoxytrityl protecting group on the Cys residue in the peptide chain, and only the methoxytrityl group was removed in an ice bath. After stirring for 30 minutes, 536μl of pyridine was added to the reaction solution to neutralize it. Then, 30.0ml of pure water was added and the mixture was separated twice. The obtained organic layer was concentrated with an evaporator, 30.0ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain 1.11g of a deprotected form in which only the SH group of the Cys residue in the peptide chain was deprotected. 5.0 ml of chloroform was added to 500 mg of the deprotected product in which only the SH group of the Cys residue in the obtained peptide chain was deprotected, and 5.0 equivalents of chloroacetic acid and 9.0 equivalents of DBU were added in an ice bath, and chloroacetic acid was subjected to a nucleophilic substitution reaction with the SH group of the Cys residue of the linear peptide under room temperature conditions. After stirring for 1 hour, the mixture was separated twice with 5.0 ml of 5% Na2CO3 aqueous solution, and then separated and washed with 20% NaCl aqueous solution. The obtained organic layer was concentrated with an evaporator, and 15.0 ml of acetonitrile was added at room temperature, and the precipitate was filtered and dried to obtain a solid. 17.4 ml of chloroform, 2.0 equivalents of HOBt, and 2.0 equivalents of EDC.HCl were added to the obtained solid, and cyclization between the N-terminal amino group and the side chain carboxyl group was performed at room temperature. After stirring for 1 hour, the mixture was washed once with 20.0 ml of 20% NaCl aqueous solution. The resulting organic layer was concentrated and dried using an evaporator to obtain 322 mg of cyclic peptide K'. 5.7 ml of a mixed solution of TFA / water / TIPS = 95.0 / 2.5 / 2.5 and 10 equivalents of mercaptopropionic acid were added to the resulting cyclic peptide K' (285 mg) for final deprotection so that all protecting groups were deprotected. After stirring for 5 hours, 28.5 ml of IPE was added, the precipitate was filtered, and dried to obtain 154 mg of cyclic peptide K'' (completely unprotected) before insoluble matter was removed by filtration. To 101 mg of the obtained cyclic peptide K'' before filtering off the insoluble matter, 8.6 ml of methanol was added and thoroughly stirred. Then, 21.6 ml of IPE was added in sequence, and after thoroughly stirring, the insoluble matter was filtered off. The obtained mother liquor was quantitatively analyzed by HPLC, and it was confirmed that the HPLC purity of cyclic peptide K'' had improved from 47% to 85%. (Yield 87% vs. cyclic peptide K'' before filtering off the insoluble matter) TOF-MS: m / z [M+H] + 1290.5

[0123] The above Example 9 is an example in which cyclic peptide E (CS type) is produced using methanol as a good solvent and IPE as a poor solvent in embodiment 1 of the present application. The above Example 9 corresponds to embodiment B. The above Example 10 is an example in which cyclic peptide F (SS type) is produced in accordance with embodiment 2 of the present application using THF as a good solvent and hexane as a poor solvent. The above Example 11 is an example in which cyclic peptide F (SS type) is produced in accordance with embodiment 2 of the present application using methanol as a good solvent and IPE as a poor solvent. The above Example 12 is an example in which cyclic peptide G (lactam type) is produced in accordance with embodiment 1 of the present application using chloroform as a good solvent and acetonitrile as a poor solvent. The above Example 13 is an example of producing cyclic peptide H (CS type) in embodiment 2 of the present application using chloroform as a good solvent and IPE as a poor solvent. The above Example 13 corresponds to embodiment A. The above Example 14 is an example of producing cyclic peptide I (CS type) in embodiment 2 of the present application using methanol as a good solvent and IPE as a poor solvent. The above Example 14 corresponds to embodiment B. The above Example 15 is an example in which cyclic peptide G (lactam type) is produced in accordance with embodiment 1 of the present application using methanol as a good solvent and IPE as a poor solvent. The above Example 16 is an example in which cyclic peptide J (CS type) is produced using chloroform as a good solvent and acetonitrile as a poor solvent in embodiment 1 of the present application. Moreover, the above Example 16 corresponds to embodiment A. The above Example 17 is an example of producing cyclic peptide K (CS type) using chloroform as a good solvent and acetonitrile as a poor solvent in embodiment 1 of the present application. Moreover, the above Example 17 corresponds to embodiment B. The above Example 18 is an example of producing cyclic peptide J (CS type) using methanol as a good solvent and IPE as a poor solvent in the embodiment 1 of the present application. The above Example 18 corresponds to embodiment A. The above Example 19 is an example of producing cyclic peptide K (CS type) using methanol as a good solvent and IPE as a poor solvent in the embodiment 1 of the present application. The above Example 19 corresponds to embodiment B. [Industrial Applicability]

[0124] The method for producing a cyclic peptide of the present invention can efficiently remove polymerized impurities produced as by-products during the cyclization reaction, improve the purity of the resulting cyclic peptide, and reduce the burden on the purification step.

[0125] This application is based on patent application No. 2019-122174 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing a cyclic peptide, comprising the following steps (1) and (2): (1) cyclizing a linear peptide; and (2) A step of adding a poor solvent to the mixture of cyclic peptide and polymerized impurities obtained in the above step, thereby filtering out the polymerized impurities as insoluble matter, thereby obtaining a cyclic peptide.

2. The method according to claim 1 , further comprising adding a good solvent before, simultaneously with, or after the addition of the poor solvent.

3. The method according to claim 1 or 2, wherein the cyclic structure of the cyclic peptide is any one of a) S-S type, b) lactam type, c) C-S type, d) C-C type, and e) lactone type.

4. The method according to any one of claims 1 to 3, wherein the linear peptide has a C-terminus protected by a protecting group and is cyclized either i) between the side chains of the constituent amino acids or ii) between the N-terminus and a side chain of the constituent amino acid.

5. The protecting group at the C-terminus of the linear peptide is a substituted or unsubstituted alkyl ester, a substituted or unsubstituted aralkyl ester, an unsubstituted amide, a primary amide, a secondary amide, an unsubstituted hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide, (4,4'-bishydrophytyloxy)benzhydrylamine or 2-(3'-4'-5'-tri(2",3''-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol, and / or the protecting group at the side chain of the constituent amino acid is a methoxy The method according to claim 4, wherein the aryl group is a carbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a formyl group, an acetyl group, a trifluoroacetyl group, a p-toluenesulfonyl group, a p-tolylmethanesulfonyl group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, (4,4'-bishydrophytyloxy)benzhydrylamine, or 2-(3'-4'-5'-tri(2",3"-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol.

6. The method according to claim 4, wherein in the step (1), the protecting group at the C-terminus of the linear peptide is a solid support, and the method further comprises a step of deprotecting only the solid support prior to the step (2).

7. The method according to any one of claims 4 to 6, wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when the linear peptide is cyclized to obtain a cyclic peptide.

8. The method according to claim 7, wherein the good solvent is a solvent capable of dissolving the cyclic peptide of interest.

9. The method according to any one of claims 1 to 3, wherein the linear peptide is not protected at its C-terminus, is protected at a site other than the site to be cyclized, and is cyclized at either i) the side chains of the constituent amino acids, ii) the N-terminus and the side chain of the constituent amino acid, iii) the C-terminus and the side chain of the constituent amino acid, or iv) the N-terminus and the C-terminus.

10. The method according to claim 9, wherein the protecting group of the side chain of the constituent amino acid of the linear peptide is a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a formyl group, an acetyl group, a trifluoroacetyl group, a p-toluenesulfonyl group, a p-tolylmethanesulfonyl group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, (4,4'-bishydrophytyloxy)benzhydrylamine or 2-(3'-4'-5'-tri(2",3''-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol.

11. The method according to claim 9 or 10, wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when the linear peptide is cyclized to obtain a cyclic peptide.

12. The method according to claim 11, wherein the good solvent is a solvent capable of dissolving the cyclic peptide of interest.

13. The method according to any one of claims 1 to 3, wherein the linear peptide is unprotected at any of its C-terminus, N-terminus and side chains of constituent amino acids, and is cyclized at either i) the side chains of the constituent amino acids, ii) the N-terminus and the side chains of the constituent amino acids, iii) the C-terminus and the side chains of the constituent amino acids, or iv) the N-terminus and the C-terminus.

14. Between step (1) and step (2), A step of isolating the cyclic peptide obtained in step (1). The method of claim 13 further comprising:

15. The method according to claim 13 or 14, wherein the poor solvent is a solvent capable of precipitating / depositing polymerized impurities produced as by-products when the linear peptide is cyclized to obtain a cyclic peptide.

16. The method according to claim 15, wherein the good solvent is a solvent capable of dissolving the cyclic peptide of interest.

17. After the above step (2), (3) Removal of all protecting groups The method of any one of claims 1 to 16, further comprising:

18. A method for producing a peptide having a cyclic thioether bond, comprising any one of the following steps: (B) a step of deprotecting a linear peptide in which the C-terminus is protected or not protected by a protecting group, the N-terminus is protected or not protected, and the side chain of the constituent amino acid cysteine ​​or a cysteine ​​derivative is modified with an alkylene group having a carboxy group, and then cyclizing the N-terminus and the side chain of the cysteine ​​or cysteine ​​derivative, if the N-terminus is protected, or (C) a step of deprotecting the amino group of a linear peptide in which the C-terminus is protected or not protected by a protecting group, the amino groups of the side chains of the constituent amino acids are protected or not protected, and the side chain of the constituent amino acid, cysteine ​​or a cysteine ​​derivative, is modified with an alkylene group having a carboxy group, and then cyclizing the linear peptide with the side chain of the constituent amino acid having an amino group and the side chain of the cysteine ​​or cysteine ​​derivative.

19. The method according to claim 18, wherein the protecting group at the C-terminus of the linear peptide is a substituted or unsubstituted alkyl ester, a substituted or unsubstituted aralkyl ester, an unsubstituted amide, a primary amide, a secondary amide, an unsubstituted hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide, (4,4'-bishydrophytyloxy)benzhydrylamine, 2-(3'-4'-5'-tri(2",3''-dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol, or a solid phase support.

20. Step (B) comprises: (B-1) a step of modifying the side chain of a cysteine ​​or cysteine ​​derivative of a linear peptide in which the C-terminus is protected or unprotected with a protecting group, the N-terminus is protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is unprotected, with an alkylene group having a carboxy group, to produce a linear peptide in which the C-terminus is protected or unprotected with a protecting group, the N-terminus is protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is modified with an alkylene group having a carboxy group; or the step (C) comprises: (C-1) a step of modifying the side chain of a cysteine ​​or cysteine ​​derivative of a linear peptide in which the C-terminus of the linear peptide is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is unprotected, with an alkylene group having a carboxy group, to produce a linear peptide in which the C-terminus of the linear peptide is protected or unprotected with a protecting group, the amino groups of the side chains of the constituent amino acids are protected or unprotected, and the side chain of the constituent amino acid cysteine ​​or cysteine ​​derivative is modified with an alkylene group having a carboxy group; The method according to claim 18 or 19.

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