Peptide synthesis method that suppresses defects caused by diketopiperazine formation

Abstract

The present invention addresses the problem of a desired elongation reaction not progressing as a result of a 6-membered cyclic amidine skeleton structure and a diketopiperazine formed when removing an N-terminated protective group during solid-phase peptide synthesis. The inventors have discovered that it is possible to solve said problem by modifying the production of a peptide via the solid-phase method so as to treat a peptide protected by an N-terminated amino group in a protective group having an Fmoc skeleton by using a base which has a pKa of 23 or higher in an acetonitrile of a conjugate acid in a specific solvent, and next, to perform peptide chain elongation.

Description

[Technical Field]

[0001] The present invention relates to a novel method for synthesizing peptides by solid phase synthesis, which enables the synthesis of peptides with high purity and high efficiency. [Background technology]

[0002] The Fmoc method is a widely used peptide synthesis method due to its reliability and mild deprotection conditions (Fmoc deprotection conditions). Recently, there has been active research into the application of peptides containing many N-alkylamino acids, such as N-methylamino acids, to pharmaceuticals. However, the acid instability of such molecular species is known (Non-Patent Document 1, Patent Document 1). Therefore, these documents employ the Fmoc method, which allows deprotection under basic conditions, rather than the Boc method, which requires acidic conditions for N-terminal deprotection.

[0003] Diketopiperazine (DKP) formation has long been recognized as a problem in peptide synthesis. Diketopiperazine formation can occur when the N-terminal protecting group of a dipeptide supported on a solid phase via an ester bond is removed, exposing a free amino group. Several approaches have been reported to alleviate this problem in peptide synthesis using the Fmoc method.

[0004] Specifically, a method is known in which diketopiperazine formation is suppressed by using an Alloc group, which undergoes a deprotection reaction under neutral conditions without using a base, as a protecting group at a site where diketopiperazine formation is likely to occur (Non-Patent Document 2).

[0005] In addition, in order to avoid the N-terminus becoming a free amino group at a site where diketopiperazine formation is likely to proceed, a method is known in which a dipeptide containing such a sequence is synthesized in advance and used to elongate the peptide chain (Non-Patent Document 3).

[0006] Furthermore, a method is known in which, instead of piperidine, which is commonly used in the deprotection of Fmoc, stronger bases such as DBU or TBAF are used to extremely shorten the time required for deprotection, thereby suppressing the formation of diketopiperazine (Non-Patent Document 4).

[0007] All of these methods address the diketopiperazine formation of dipeptides supported on a solid phase via an ester bond. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO 2018 / 225851 A1 [Non-patent literature]

[0009] [Non-Patent Document 1] J. Peptide Res., 2005, 65, 153-166. [Non-patent document 2] Org. Lett., 2012, 14, 612-615. [Non-patent document 3] Published by Bachem Solid Phase Peptide Synthesis(https: / / www.bachem.com / fileadmin / user_upload / pdf / Catalogs_Brochures / Solid_Phase_Peptide_Synthesis.pdf) [Non-patent document 4] Org. Lett., 2008, 10, 3857-3860. Summary of the Invention [Problem to be solved by the invention]

[0010] As described above, it is known that when a dipeptide is supported on a solid phase via an ester bond, the ester bond is cleaved to form a diketopiperazine. However, when an N-substituted amino acid is contained in the peptide sequence, the amide bond, which is stronger than the ester bond, is cleaved to form a diketopiperazine, which may result in the dipeptide containing the N-substituted amino acid being removed from the peptide sequence. This removal can occur at any position in the peptide sequence where the N-substituted amino acid is present, and therefore it has been found that it can occur not only in dipeptides supported on a solid phase.

[0011] Furthermore, the present inventors have clarified that when an N-substituted amino acid is contained in a peptide sequence, in addition to the problem of diketopiperazine formation, the desired elongation reaction does not proceed, and instead of the target product, an impurity having a six-membered cyclic amidine skeleton at the N-terminus (six-membered cyclic amidine skeleton structure) is formed. No method for solving these problems has been reported so far.

[0012] In order to suppress not only the diketopiperazine elimination that occurs at the ester bond of a dipeptide supported on a solid phase but also the diketopiperazine elimination that occurs at the amide bond and the formation of the six-membered cyclic amidine skeleton structure, it is conceivable to apply the above-mentioned conventional techniques. However, the scope of application of these techniques is limited, and they cannot be said to provide a sufficient solution.

[0013] For example, the method described in Non-Patent Document 2 requires Alloc amino acids, which are less common than Fmoc amino acids in terms of availability. Furthermore, there is a concern that the peptide chain elongation method using a dipeptide described in Non-Patent Document 3 may cause racemization of the amino acid at the C-terminus of the dipeptide. Therefore, it is difficult to apply the method described in this document except when the amino acid at the C-terminus of the dipeptide fragment is achiral (e.g., glycine) or when racemization is difficult to proceed (e.g., proline). Furthermore, the method described in Non-Patent Document 4 is characterized by the extremely short time required for the Fmoc removal step, requiring the Fmoc removal step and solid-phase washing operation to be completed within 1 minute, for example, 10 to 20 seconds. It is impossible to apply such extremely short operations to scale-up synthesis, including industrialization.

[0014] As described above, no practical methodology for suppressing diketopiperazines has yet been presented. The present invention was made in light of these circumstances, and in one aspect, the present invention aims to provide a peptide synthesis method capable of suppressing diketopiperazine formation, which (i) uses a protecting group containing an Fmoc skeleton as the N-terminal protecting group, (ii) is not subject to sequence application restrictions from the standpoint of racemization, and (iii) allows for scale-up synthesis, such as industrialization. In another aspect, the present invention aims to provide a peptide synthesis method capable of suppressing the formation of a 6-membered cyclic amidine skeleton structure. [Means for solving the problem]

[0015] The present inventors have discovered that in peptide production by solid-phase methods, the formation of diketopiperazine and 6-membered cyclic amidine skeleton structures can be suppressed throughout the process from removal of the protecting group to peptide chain elongation by treating a peptide having a protecting group containing an Fmoc skeleton with a base whose conjugate acid in acetonitrile has a pKa of 23 or higher in a specific solvent, and then reacting the peptide with an active elongation species such as an activated ester generated using an acid chloride or a condensing agent, thereby completing the present invention.

[0016] That is, the present invention includes the following. [1] A method for producing a peptide by a solid phase method, comprising the steps of: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a resin for solid phase synthesis; (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents; and (3) The production method as described above, which comprises, after the step (2), a step of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. [2] The method according to [1], which does not include a step of treating the first peptide with piperidine as a single base prior to step (2). [3] The method according to [1], which does not include a step of treating the first peptide with a single base having a pKa in acetonitrile of less than 23 for the conjugate acid prior to step (2). [4] The method according to any one of [1] to [3], which does not include a step of adding an acid to neutralize the remaining base between the step (2) and the step (3). [5] The method according to any one of [1] to [4], wherein the solvent in step (2) is a solvent having a swelling capacity of the solid phase resin of 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more. [6] The method according to any one of [1] to [5], wherein at least a portion of the first peptide obtained in the step (2) is in the form of a carbamate. [7] The method according to [6], wherein the carbamate is a salt with a base whose conjugate acid has a pKa in acetonitrile of 23 or more. [8] The method according to either [6] or [7], wherein the carbamate is a DBU salt, a TMG salt, a HP1(dma) salt, a MTBD salt, a P1-tBu salt, or a P2-Et salt. [9] In the step (2), 1The method according to any one of [6] to [8], wherein the molar ratio of the carbamate to the amine formed by further decarboxylation (carbamate / amine), determined from the proton integral ratio in H-NMR, is 0.6 or more, 0.8 or more, 1.0 or more, 2.0 or more, 3.0 or more, 4.0 or more, 4.6 or more, 5.0 or more, 6.0 or more, 8.0 or more, or 10.0 or more.

[10] The method according to any one of [1] to [9], wherein the solvent in the step (2) contains 25 v / v % or more of at least one solvent selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[11] The method according to any one of [1] to

[10] , wherein the solvent in the step (2) contains 50 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[12] The method according to any one of [1] to

[11] , wherein the solvent in the step (2) contains 75 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[13] The method according to any one of [1] to

[12] , wherein the solvent in the step (2) further comprises one or more solvents selected from an amide solvent, a urea solvent, or a sulfone solvent.

[14] The method according to any one of [1] to

[12] , wherein the solvent in the step (2) is at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[15] The aromatic hydrocarbon solvent is one or more selected from the group consisting of toluene, benzene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene; the halogen-based solvent is one or more selected from the group consisting of dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride; the ether-based solvent is one or more selected from the group consisting of tetrahydrofuran, diethyl ether, 2-methyltetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxolane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme; the ester solvent is one or more selected from the group consisting of methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone; the ketone solvent is one or more selected from the group consisting of acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone; The carbonate solvent is one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, and The method according to any one of [1] to

[14] , wherein the phosphate ester solvent is one or more selected from the group consisting of trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[16] The method according to any one of [1] to

[15] , wherein the solvent in step (2) has a donor number value of 26 or less. [16.1] The method according to any one of [1] to

[16] , wherein the aromatic hydrocarbon solvent, halogen-based solvent, ether-based solvent, ester-based solvent, ketone-based solvent, carbonate-based solvent, or phosphate ester-based solvent in step (2) has a donor number value of 26 or less.

[17] The method according to any one of [1] to

[16] , wherein the solvent in step (2) has a donor number value of 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. [17.1] The method according to any one of [1] to

[17] , wherein the aromatic hydrocarbon solvent, halogen-based solvent, ether-based solvent, ester-based solvent, ketone-based solvent, carbonate-based solvent, or phosphate ester-based solvent in step (2) has a donor number value of 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[18] The method according to any one of [1] to [9], wherein the solvent in the step (2) is toluene or cumene.

[19] The method according to any one of [1] to [9], wherein the solvent in the step (2) is dichloromethane.

[20] The method according to any one of [1] to [9], wherein the solvent in the step (2) is tetrahydrofuran, 1,2-dimethoxyethane, 1,3-dioxolane, or 2-methyltetrahydrofuran.

[21] The method according to any one of [1] to [9], wherein the solvent in the step (2) is methyl propionate or butyl acetate.

[22] The method according to any one of [1] to [9], wherein the solvent in the step (2) is methyl ethyl ketone or diethyl ketone.

[23] The method according to any one of [1] to [9], wherein the solvent in the step (2) is dimethyl carbonate.

[24] The method according to any one of [1] to [9], wherein the solvent in the step (2) is tributyl phosphate.

[25] The method according to any one of [1] to

[24] , wherein the base in the step (2) is at least one selected from the group consisting of amidines, guanidines, and phosphazenes.

[26] The method according to any one of [1] to

[25] , wherein the base in the step (2) is at least one selected from the group consisting of DBU, MTBD, TMG, P1tBu, P2Et, and HP1(dma).

[27] The method according to any one of [1] to

[26] , wherein the base in the step (2) is DBU, MTBD, TMG, P1tBu, P2Et, or HP1(dma).

[28] The method according to any one of [1] to

[26] , wherein the base in the step (2) is (i) a combination of DBU and piperidine, or (ii) a combination of DBU and MTBD, HP1(dma), P1tBu, or P2Et.

[29] The method according to any one of [1] to

[28] , wherein the base in the step (2) is contained in the solvent at a concentration of 1 to 8 v / v %.

[30] The method according to any one of [1] to

[29] , wherein the step (2) further comprises a step of contacting the solvent with CO2.

[31] The method according to any one of [1] to

[30] , wherein the step (2) further comprises a step of bubbling CO2 into the solvent.

[32] The method according to any one of [1] to

[29] , wherein the solvent in the step (2) is a solvent that has been contacted with CO2 in advance.

[33] The method according to any one of [1] to

[29] , wherein the solvent in the step (2) is a solvent into which CO2 has been bubbled in advance.

[34] The method according to any one of [1] to

[33] , wherein step (2) is repeated multiple times.

[35] The method according to

[34] , wherein when step (2) is repeated multiple times, the same solvent is used each time step (2) is repeated.

[36] The method according to

[34] , wherein when step (2) is repeated multiple times, a different solvent is used each time step (2) is repeated.

[37] The method according to any one of

[34] to

[36] , wherein when step (2) is repeated multiple times, the same base is used in each step (2).

[38] The method according to any one of

[34] to

[36] , wherein when step (2) is repeated multiple times, a different base is used in each step of step (2).

[39] The method according to any one of

[34] to

[38] , further comprising the step of discharging the solution between each repetition of step (2) when step (2) is repeated multiple times.

[40] The carboxylic acid or carboxylic acid analog is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkylcarboxylic acid, or a C6-C 10 an aryl carboxylic acid, or a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 an activated ester of an aryl carboxylic acid, or a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 The acid halide of an arylcarboxylic acid is a C1-C8 alkylcarboxylic acid and a C6-C 10 The method according to any one of [1] to

[39] , wherein the arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl, and dialkylaminocarbonyl.

[41] The method according to

[40] , wherein the protecting group is a carbamate-based protecting group, a sulfonyl-based protecting group, or an acyl-based protecting group.

[42] The method according to

[41] , wherein the carbamate-based protecting group is a protecting group containing an Fmoc skeleton, Alloc, Teoc, Boc, or Cbz, the sulfonyl-based protecting group is Ns, and the acyl-based protecting group is Tfa.

[43] The carboxylic acid or carboxylic acid analogue is a C1-C8 alkyl carboxylic acid or a C6-C 10aryl carboxylic acid or its activated ester or acid halide, and the C1-C8 alkyl carboxylic acid and C6-C 10 The method according to

[40] , wherein the arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, and halogen.

[44] The method according to

[40] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkylcarboxylic acid optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, and halogen, or an activated ester or acid halide thereof.

[45] The method according to

[40] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkylcarboxylic acid, or an activated ester or acid halide thereof.

[46] The method according to any one of [1] to

[45] , wherein the first peptide contains 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues.

[47] The method according to any one of [1] to

[46] , wherein the first peptide is a dipeptide.

[48] ​​The method according to any one of [1] to

[47] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group, or an activated ester of an amino acid or a second peptide having a protecting group, or an acid halide of an amino acid or a second peptide having a protecting group, and the first peptide and / or the second peptide having a protecting group contains one or more N-substituted amino acids, and / or the amino acid having a protecting group is an N-substituted amino acid.

[49] The method according to any one of [1] to

[48] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, or an activated ester of an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, or an acid halide of an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, and the first peptide and / or the second peptide having a protecting group containing an Fmoc skeleton contains one or more N-substituted amino acids, and / or the amino acid having a protecting group containing an Fmoc skeleton is an N-substituted amino acid.

[50] The method according to any one of [1] to

[49] , wherein the second amino acid residue from the N-terminus of the first peptide is an N-substituted amino acid.

[51] The condensing agent in the step (3) is in the form of a salt, and its counter anion is PF6 - or BF4 - The method according to any one of [1] to

[50] ,

[52] The method according to any one of [1] to

[51] , wherein the condensing agent in the step (3) comprises at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, and TOTU.

[53] The method according to any one of [1] to

[52] , wherein the condensing agent in step (3) is PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, or TOTU.

[54] The method according to any one of [1] to

[53] , wherein the step (3) is carried out in the presence of a base.

[55] The method according to any one of [1] to

[54] , wherein the step (3) is carried out in the presence of a base having a pKa of 5 to 12 in water for the conjugate acid.

[56] The method according to

[54] or

[55] , wherein the base in step (3) is DIPEA.

[57] The method according to any one of [1] to

[56] , wherein the protecting group containing an Fmoc skeleton is represented by the following formula (1): TIFF0007808554000001.tif35170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl).

[58] The method according to

[57] , wherein the protecting group containing an Fmoc skeleton is an Fmoc group, an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br) group, a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, an sm-Fmoc group, or an rm-Fmoc group.

[59] The method according to

[57] , wherein the protecting group containing an Fmoc skeleton is an Fmoc group.

[60] The method according to any one of [1] to

[59] , further comprising a step of washing the resin for solid phase synthesis between the step (2) and the step (3).

[61] The washing step i) washing the resin with a solvent that has been previously contacted with CO2; and / or ii) washing the resin with a solvent that has not been previously exposed to CO2; The method according to

[60] , comprising:

[62] The washing step i) washing the resin for solid phase synthesis with a solvent that has been previously bubbled with CO2; and / or ii) washing the resin with a solvent that has not been previously bubbled with CO2; The method according to

[60] , comprising:

[63] The method according to any one of [1] to

[62] , wherein the solvent in the step (3) is the same as the solvent in the step (2).

[64] The method according to any one of [1] to

[63] , wherein the solvent in the step (3) is a solvent in which the condensing agent can be dissolved.

[65] The method according to any one of [1] to

[64] , wherein the solvent in the step (3) comprises at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[66] The method according to any one of [1] to

[64] , wherein the solvent in the step (3) contains 25 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[67] The method according to any one of [1] to

[64] , wherein the solvent in the step (3) contains 50 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[68] The method according to any one of [1] to

[64] , wherein the solvent in the step (3) contains 75 v / v% or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[69] The method according to any one of [1] to

[68] , wherein the solvent in the step (3) is at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[70] The method according to any one of [1] to

[69] , wherein the solvent in the step (3) is an aromatic hydrocarbon solvent, a halogen-based solvent, an ether-based solvent, an amide-based solvent, a sulfoxide-based solvent, a sulfone-based solvent, a urea-based solvent, an ester-based solvent, a ketone-based solvent, a carbonate-based solvent, or a phosphate ester-based solvent.

[71] The aromatic hydrocarbon solvent is one or more selected from the group consisting of benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzeneanisole, ethylbenzene, nitrobenzene, and cumene; the halogen-based solvent is one or more selected from the group consisting of dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride; the ether solvent is one or more selected from the group consisting of diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,3-dioxolane, 1,4-dioxane, 1,2-dimethoxyethane, diisopropyl ether, t-butyl methyl ether, diglyme, triglyme, and tetraglyme; the amide solvent is one or more selected from the group consisting of DMF, NMP, DMA, NEP, NBP, and formamide; the sulfoxide solvent is one or more selected from the group consisting of DMSO and methyl phenyl sulfoxide; the sulfone solvent is one or more selected from the group consisting of diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone; the urea-based solvent is one or more selected from the group consisting of DMI and DMPU, the ester solvent is one or more selected from the group consisting of methyl acetate, ethyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone; the ketone solvent is one or more selected from the group consisting of acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, diethyl ketone, and cyclopentanone; the carbonate solvent is one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate, and dibutyl carbonate; The method according to any one of

[65] to

[70] , wherein the phosphate ester solvent is one or more selected from the group consisting of trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[72] The method according to any one of [1] to

[71] , wherein the carboxylic acid used in the step (3) is an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, and the protecting group containing an Fmoc skeleton carried by the amino acid or the second peptide is the same as or different from the protecting group containing an Fmoc skeleton carried by the first peptide used in the step (1).

[73] The carboxylic acid analog used in the step (3) is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkylcarboxylic acid, or a C6-C 10 the carboxyl group of an amino acid having a protecting group containing an Fmoc skeleton, the C-terminal carboxyl group of a second peptide having a protecting group containing an Fmoc skeleton, the carboxyl group of the C1-C8 alkylcarboxylic acid, or the C6-C 10 The method according to any one of [1] to

[72] , wherein a carboxyl group of the arylcarboxylic acid is converted to an acid halide group.

[74] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in peptide production by a solid-phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents; The method comprising: [Effects of the Invention]

[0017] According to the present invention, even in the case of an amino acid sequence in which the desired elongation reaction does not proceed sufficiently under conventional conditions due to the formation of a diketopiperazine and / or a six-membered cyclic amidine backbone structure in solid-phase peptide synthesis, the formation of such impurities can be significantly reduced, thereby enabling efficient peptide chain elongation to obtain a peptide having the desired amino acid sequence. The method of the present invention does not require the use of a special protecting group such as Alloc, which is not common, and is not subject to operational restrictions such as extremely short reaction times, making it a highly versatile and practical synthesis method that can be scaled up. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the H-NMR spectra between 3.6 and 6.8 ppm before and after the reaction of Fmoc-Phe-NMe2 (compound 1-3-1) with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). It also shows the characteristic H signals observed in this range for compounds 1-3-1, 1-3-2, 1-3-3a, and dibenzofulvene. Figure 1 (a) shows the H-NMR spectrum of the solution obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). Figure 1 (b) shows the H-NMR spectrum of a solution of compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7). [Figure 2] Figure 2 shows the H-NMR spectrum around 5.4-3.5 ppm, which indicates that compound 1-3-2 and compound 1-3-3a, produced by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7), undergo chemical exchange in a ratio of 82:18. In Figure 2, a)-1 shows the H-NMR spectrum of the solution obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). In Figure 2, a)-2 shows a 1D-ROESY spectrum obtained by selectively exciting the 1H signal at 4.82 ppm of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7). In Figure 2, a)-3 shows a 1D-ROESY spectrum obtained by selectively exciting the 1H signal at 3.95 ppm of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7). In Figure 2, b) is a 1H-NMR spectrum showing the integral ratio of the 1H signal at 4.85 ppm to the 1H signal at 3.95 ppm of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7). [Figure 3]Figure 3 shows the H-NMR spectrum around 5.2-3.5 ppm, indicating that compound 1-3-2 was produced by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). In Figure 3, a)-1 shows the H-NMR spectrum of an authentic sample of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7). In Figure 3, a)-2 shows the H-NMR spectrum of a solution obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). In Figure 3, a)-3 shows the H-NMR spectrum of a solution obtained by treating Compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of authentic Compound 1-3-2. In Figure 3, a)-4 shows the 1D-NOESY spectrum of a solution obtained by treating Compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of authentic Compound 1-3-2, with selective excitation of the H signal at 4.84 ppm. In Figure 3, a)-5 shows a 1D-NOESY spectrum obtained by selectively exciting the 1H signal at 3.95 ppm of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-3-2. In Figure 3, b) shows the ratio of compounds 1-3-3 and 1-3-2 in the solution calculated from the integrals of the proton signals at 4.84 ppm and 3.95 ppm in the H-NMR spectrum of the solution obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-3-2. [Figure 4]Figure 4 shows H-NMR and 1D-NOESY spectra around 5.1-3.7 ppm, indicating that compound 1-3-3a was produced by bubbling CO2 into compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) followed by the addition of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). Figure 4 (a) shows the H-NMR spectrum of an authentic sample of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7). Figure 4 (b) shows the H-NMR spectrum of a solution obtained by bubbling CO2 into an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute. In Figure 4, c) shows a 1D-NOESY spectrum of a solution obtained by bubbling CO2 through an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute, with selective excitation of the H signal at 4.77 ppm. In Figure 4, d) shows a 1D-NOESY spectrum of a solution obtained by bubbling CO2 through an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute, with selective excitation of the H signal at 3.96 ppm. In Figure 4, e) shows a H-NMR spectrum of a solution obtained by bubbling CO2 through an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute, followed by the addition of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). In Figure 4, f) is a diagram of the H-NMR spectrum of a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) after bubbling CO2 for 1 minute, showing the integral ratio of the H signal at 4.77 ppm to the H signal at 3.96 ppm. In Figure 4, e) is a diagram of the H-NMR spectrum of a solution obtained by bubbling CO2 for 1 minute in an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2, followed by the addition of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), showing an enlarged view of the H signal at 4.83 ppm. Note that to improve resolution, this diagram was converted using a Sinusoidal window function. [Figure 5]Figure 5 shows a C-NMR spectrum around 164-124 ppm, indicating that compound 1-3-3a was produced by bubbling CO2 through compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) followed by the addition of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). Figure 5 (a) shows the C-NMR spectrum of the solution obtained by bubbling CO2 through compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7). Figure 5 (b) shows the C-NMR spectrum of the solution obtained by bubbling CO2 through compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) followed by the addition of DBU. [Figure 6]Figure 6 shows the H-NMR spectrum around 6.9-3.6 ppm, indicating the presence of compound 1-3-3a in a solution (Solution B, Sol B) obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). In Figure 6, a) shows the H-NMR spectrum of a solution (Solution B) obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). In Figure 6, b) shows the H-NMR spectrum of a solution obtained by mixing a solution (Solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7) with a solution (Solution A) obtained by bubbling CO into a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). In Figure 6, c) shows the H-NMR spectrum of a solution (solution A) obtained by bubbling CO into a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). [Figure 7]Figure 7 shows a solution (Solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7), and a solution (Solution C) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) after bubbling 13CO2 for 1 minute. 1-3-3a is present in the solution obtained by mixing the solution A) with the compound 1-3-3a. The NH-HSQC (N[F1]: around 97-85 ppm, H[F2]: around 5.7-4.7 ppm), NH-HMBC (N[F1]: around 101-83 ppm, H[F2]: around 3.04-2.64 ppm), and CH-HMBC (C[F1]: around 165-158 ppm, H[F2]: around 5.7-4.5 ppm) spectra are shown. In Figure 7, a) shows the NH-HSQC spectrum of a solution obtained by mixing a solution (solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7) with a solution (solution A) obtained by bubbling CO into an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). In Figure 7, b) shows the NH-HMBC spectrum of a solution obtained by mixing a solution (solution B) obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7) with a solution (solution A) obtained by bubbling CO into an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU).In Figure 7, c) shows the CH-HMBC spectrum of a solution obtained by mixing a solution (Solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-3-1 in N,N-dimethylformamide-d7 (DMF-d7) with a solution (Solution A) obtained by bubbling CO into a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). [Figure 8] FIG. 8 shows the identification of all 1H, 13C, and 15N signals of the carbamate moiety of compound 1-3-3a present in a solution obtained by mixing a solution (solution B) obtained by reacting compound 1-3-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7) with a solution (solution A) obtained by bubbling 13CO2 into an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-3-2 for 1 minute and then reacting it with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). DETAILED DESCRIPTION OF THE INVENTION

[0019] (Definition of terms) As used herein, the term "halogen atom" includes, for example, F, Cl, Br, or I.

[0020] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a hydrocarbyl or hydrocarbon group structure subset containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q " means that the number of carbon atoms is p to q), and preferably C1-C10 Alkyl is preferably C1-C6 alkyl. Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc.

[0021] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP 2 Alkenyl is a monovalent group having 2-4 carbon atoms. Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl includes not only straight chains but also branched chains. Alkenyl is preferably C2-C 10 Alkenyl, more preferably C2-C6 alkenyl, is exemplified, and specific examples include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, etc.

[0022] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Alkynyl is preferably C2-C 10Alkynyl, more preferably C2-C6 alkynyl, is included, and specific examples include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, and the like.

[0023] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferred examples of cycloalkyl include C3-C8 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl.

[0024] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

[0025] As used herein, the term "heterocyclyl" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl, and may be a single ring or a condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, and isothiazolidinyl. Examples include thiadiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, and 2-oxaspiro[3.3]heptyl.

[0026] As used herein, "heteroaryl" refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a single ring or a condensed ring with other rings, and may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0027] As used herein, "alkoxy" refers to an oxy group bonded to an "alkyl" as defined above, and preferably includes C1-C6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0028] As used herein, "alkenyloxy" refers to an oxy group bonded to the above-defined "alkenyl," and preferably includes C2-C6 alkenyloxy. Specific examples of alkenyloxy include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0029] As used herein, "cycloalkoxy" refers to an oxy group bonded to a "cycloalkyl" as defined above, and preferably includes C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0030] As used herein, "aryloxy" refers to an oxy group to which the above-defined "aryl" is bonded, and preferably has a C6-C 10 Specific examples of the aryloxy include phenoxy, 1-naphthyloxy, and 2-naphthyloxy.

[0031] As used herein, the term "heteroaryloxy" refers to an oxy group bonded to the above-defined "heteroaryl", and preferably includes 5- to 10-membered heteroaryloxy.

[0032] As used herein, "amino" refers to -NH2 in a narrow sense and -NRR' in a broad sense, where R and R' are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R and R' together with the nitrogen atom to which they are attached form a ring. Preferred amino groups include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and 4- to 8-membered cyclic amino.

[0033] As used herein, "monoalkylamino" refers to a group in which R is hydrogen and R' is an "alkyl" as defined above, among the "amino" groups defined above, and preferably includes mono-C1-C6 alkylamino. Specific examples of monoalkylamino include methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, and t-butylamino.

[0034] As used herein, "dialkylamino" refers to a group in which R and R' are independently "alkyl" as defined above, among the "amino" groups defined above, and preferably includes diC1-C6 alkylamino. Specific examples of dialkylamino include dimethylamino and diethylamino.

[0035] As used herein, "cyclic amino" refers to the above-defined "amino" in which R and R' form a ring together with the nitrogen atom to which they are attached, and preferably includes 4- to 8-membered cyclic amino. Specific examples of cyclic amino include 1-azetidyl, 1-pyrrolidyl, 1-piperidyl, 1-piperazyl, 4-morpholinyl, 3-oxazolidyl, 1,1-dioxidethiomorpholinyl-4-yl, and 3-oxa-8-azabicyclo[3.2.1]octan-8-yl.

[0036] As used herein, "haloalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been substituted with halogen, and is preferably C1-C8 haloalkyl, more preferably C1-C6 haloalkyl.

[0037] As used herein, "fluoroalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been substituted with fluorine atoms, and C1-C8 fluoroalkyl is preferred. Specific examples of haloalkyl include monofluoromethyl, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl.

[0038] As used herein, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is substituted with an "aryl" as defined above, and is a C7-C 14 Aralkyl is preferred, C7-C 10Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0039] As used herein, "aralkyloxy" refers to an oxy group to which the above-defined "aralkyl" is bonded, and is a C7-C 14 Aralkyloxy is preferred, C7-C 10 Aralkyloxy is more preferred. Specific examples of aralkyloxy include benzyloxy, phenethyloxy, and 3-phenylpropoxy.

[0040] As used herein, the term "peptide chain" refers to a peptide chain in which one or more natural amino acids and / or unnatural amino acids are linked by amide bonds and / or ester bonds. The peptide chain preferably contains 1 to 15 amino acid residues, and more preferably contains 5 to 12 amino acid residues.

[0041] The "peptide compound" of the present invention is not particularly limited as long as it is a peptide compound in which natural amino acids and / or unnatural amino acids are linked by amide bonds or ester bonds, but is preferably a peptide compound of 5 to 30 residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. The peptide compound synthesized in the present invention preferably contains at least three N-substituted amino acids, and more preferably at least five or more N-substituted amino acids in one peptide. These N-substituted amino acids may be present consecutively or discontinuously in the peptide compound. The peptide compound of the present invention may be linear or cyclic, with cyclic peptide compounds being preferred. In this specification, "peptide compound" may also be referred to as "peptide."

[0042] The "cyclic peptide compound" of the present invention is a cyclic peptide compound obtainable by cyclizing the N-terminal group and the C-terminal group of a linear peptide compound. The cyclization may be via any form, such as cyclization via a carbon-nitrogen bond such as an amide bond, cyclization via a carbon-oxygen bond such as an ester bond or an ether bond, cyclization via a carbon-sulfur bond such as a thioether bond, cyclization via a carbon-carbon bond, or cyclization via a heterocyclic ring structure. Among these, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxylic acid group in a side chain and an amino group in the N-terminal main chain is more preferred. The position of the carboxylic acid group or amino group used for cyclization may be on either the main chain or the side chain, and is not particularly limited as long as it is in a position that allows cyclization.

[0043] As used herein, the term "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0044] As used herein, the term "solid-phase synthesis resin" is not particularly limited as long as it can be used in the synthesis of peptide compounds by solid-phase synthesis. Specific examples of such solid-phase synthesis resins include CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt), which are removable under acidic conditions. The resin can be appropriately selected depending on the functional group of the amino acid used. For example, when a carboxylic acid (main-chain carboxylic acid or side-chain carboxylic acid, such as Asp or Glu) or a hydroxy group on an aromatic ring (phenol group, such as Tyr) is used as the functional group of the amino acid, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin). When an aliphatic hydroxy group (an aliphatic alcohol group such as Ser or Thr) is used as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin), or 4-methyltrityl chloride resin (Mtt resin). In this specification, resin may also be referred to as "resin."

[0045] The type of polymer constituting the resin is not particularly limited. In the case of a resin made of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The cross-linking rate is also not particularly limited, but 1% DVB (divinylbenzene) cross-linking is preferred. Examples of the type of polymer constituting the resin include Tentagel and Chemmatrix.

[0046] In the preparation of the compounds described herein, if a defined group undergoes an undesired chemical transformation under the conditions of the method, the compound can be prepared by, for example, using means such as protection and deprotection of the functional group. Here, the selection and deprotection of the protecting group can be performed using, for example, the methods described in "Greene's, 'Protective Groups in Organic Synthesis' (5th ed., John Wiley & Sons 2014)," which can be used appropriately depending on the reaction conditions. Furthermore, the order of reaction steps such as the introduction of substituents can also be changed as necessary.

[0047] In the present specification, when the modifier "optionally substituted" is used, examples of the substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, and the like.

[0048] Furthermore, each of these may be given a substituent, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom. Examples of such substituents include optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl.

[0049] The compounds described herein may be in the form of salts or solvates thereof. Examples of the salts described herein include hydrochlorides, hydrobromides, hydroiodides, phosphates, phosphonates, sulfates, sulfonates such as methanesulfonates and p-toluenesulfonates, carboxylates such as acetates, citrates, malates, tartrates, succinates, and salicylates, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and ammonium salts such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts. These salts can be prepared, for example, by contacting the compound with an acid or a base. The solvate of the compound described herein refers to a phenomenon in which solute molecules strongly attract solvent molecules in a solution, forming a molecular cluster. When the solvent is water, it is called a hydrate. The compounds described herein may be solvated with a single solvent or multiple solvents selected from organic solvents such as alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), dimethylformamide, or diglyme, or water.

[0050] As used herein, "amino acid" includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives). As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Unnatural amino acids (amino acid derivatives) are not particularly limited, but examples include β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains differ from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids may have any stereoconfiguration. The side chain of an amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-linked cycloalkyl groups. Each of these may have a substituent, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. Examples of such substituents include optionally substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., as well as oxo, aminocarbonyl, and halogen atoms. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the definition of amino acids).

[0051] In this specification, examples of the substituent containing a halogen atom include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, each of which has a halogen atom as a substituent, and more specific examples thereof include a fluoroalkyl, a difluoroalkyl, a trifluoroalkyl, and the like.

[0052] Examples of the substituent containing an O atom include hydroxy (-OH), oxy (-OR), carbonyl (-C=OR), carboxy (-COH), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO-R), aminosulfonyl (-SO-NHR), sulfamoylamino (-NH-SO-NHR), thiocarboxyl (-C=O-SH), and carboxylcarbonyl (-C=O-COH).

[0053] Examples of oxy (—OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among these, methoxy or ethoxy is preferred.

[0054] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0055] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0056] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0057] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0058] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0059] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl (e.g., C1-C6 or C1-C4 alkylaminocarbonyl, particularly ethylaminocarbonyl, methylaminocarbonyl, etc.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in -C=O-NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0060] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-C=OR may be further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0061] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0062] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0063] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0064] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0065] Examples of substituents containing an S atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-SO2-R), and sulfo (-SO3H).

[0066] Examples of thio (-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0067] Examples of sulfonyl (-SO2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0068] Examples of substituents containing an N atom include azide (-N3, also referred to as an "azido group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also referred to as monosubstituted amino), tertiary amino (-NR(R'; also referred to as disubstituted amino), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridyl, piperidino, morpholino, and azetidinyl.

[0069] Examples of secondary amino (-NH-R; monosubstituted amino) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0070] Examples of tertiary amino (-NR(R'); disubstituted amino) include an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring. Specific examples include dialkylamino, particularly C1-C6 dialkylamino, C1-C4 dialkylamino, dimethylamino, diethylamino, etc. In the present specification, the term "C p -C q "Dialkylamino group" means an amino group with C p -C q A group substituted with two alkyl groups, both C p -C q The alkyl groups may be the same or different.

[0071] Examples of substituted amidino (-C(=NR)-NR'R") include groups in which the three substituents R, R', and R" on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0072] Examples of substituted guanidino (-NR-C(=NR''')-NR'R") include groups in which R, R', R", and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0073] Examples of aminocarbonylamino (-NR-CO-NR'R") include groups in which R, R', and R" are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0074] In this specification, the "amino acid residues" that constitute a peptide compound may be simply referred to as "amino acids".

[0075] In the present invention, the term "N-substituted amino acid" refers to an amino acid defined above in which the main chain amino group is N-substituted. Specific examples of N-substituted amino acids include those in which the main chain amino group of the amino acid is an NHR group, where R is any group other than hydrogen, such as an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group, and cyclic amino acids such as proline in which the carbon atom bonded to the N atom and the carbon atom from the α-position form a ring. Examples include prolines that form a five-membered ring, azetidines that form a four-membered ring, and piperidines that form a six-membered ring. The substituents of each optionally substituted group are not particularly limited and include, for example, a halogen group, an ether group, and a hydroxyl group.

[0076] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36Includes Cl etc.

[0077] As used herein, the term "prolines" refers to a group of compounds comprising proline and one or more optional substituents bonded to any carbon atom forming the five-membered ring of the proline. When multiple substituents are present on the five-membered ring, these substituents may be combined to form a ring, which may be any aromatic ring. Specific examples of "prolines" include proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-4-fluoro-L-proline, cis-4-fluoro-L-proline, and 2-methyl-L-proline. When prolines contain a hydroxy group, the hydroxy group may be protected with a protective group, or an ether or ester bond may be formed between the optional substituent and the oxygen atom of the hydroxy group. Furthermore, any one of the carbon atoms forming the five-membered ring may be replaced with an oxygen atom or a sulfur atom. Specific examples of such compounds include L-thioproline. Furthermore, prolines may contain an unsaturated bond within the five-membered ring they form. A specific example of such a compound is 3,4-dehydro-L-proline, which may be in the D-form as well as the L-form.

[0078] As used herein, "azetidines" refers to cyclic amino acids based on a compound in which one nitrogen atom and three carbon atoms form a four-membered ring, with a carboxyl group bonded to the carbon atom adjacent to the nitrogen atom. Any carbon atom forming the four-membered ring may be bonded with one or more optional substituents. When multiple substituents are present on the four-membered ring, these substituents may be joined together to form a ring. Specific examples of "azetidines" include (S)-azetidine-2-carboxylic acid and (R)-azetidine-2-carboxylic acid. Compounds in which a carboxyl group is bonded to the second carbon atom, counting from the nitrogen atom forming the four-membered ring, are also included in the "azetidines," and examples of such compounds include azetidine-3-carboxylic acid.

[0079] As used herein, "piperidines" refers to cyclic amino acids based on a compound in which one nitrogen atom and five carbon atoms form a six-membered ring, with a carboxyl group bonded to the carbon atom adjacent to the nitrogen atom. Any carbon atom forming the six-membered ring may be bonded to one or more optional substituents. When multiple substituents are present on the six-membered ring, these substituents may be joined together to form a ring, which may be any aromatic ring. Specific examples of "piperidines" include (R)-piperidine-2-carboxylic acid, (S)-piperidine-2-carboxylic acid, (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0080] In this specification, examples of aromatic hydrocarbon solvents include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene.

[0081] In this specification, examples of halogen-based solvents include dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride.

[0082] In this specification, examples of ether solvents include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme.

[0083] In this specification, examples of amide solvents include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide.

[0084] In this specification, examples of sulfoxide solvents include dimethyl sulfoxide (DMSO) and methyl phenyl sulfoxide.

[0085] In this specification, examples of sulfone solvents include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0086] In this specification, examples of urea-based solvents include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0087] In this specification, examples of the ester solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone.

[0088] In this specification, examples of ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone.

[0089] In this specification, examples of carbonate solvents include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate.

[0090] In this specification, examples of phosphate ester solvents include trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[0091] As used herein, the term "amidines" refers to bases represented by the following formula B1: TIFF0007808554000002.tif33170[In the formula, RB1 and RB4 are each independently C1-C4 alkyl, or RB1 and RB4 together with the nitrogen atom to which RB1 is bonded and the carbon atom to which RB4 is bonded form a 5- to 8-membered ring; RB2 and RB3 are each independently C1-C4 alkyl, or RB2 and RB3 together with the nitrogen atom to which RB2 is bonded and the nitrogen atom to which RB3 is bonded and the carbon atom to which the nitrogen atoms are bonded form a 5- to 8-membered ring.

[0092] When RB1 to RB4 are C1-C4 alkyl, preferred examples of the C1-C4 alkyl include methyl and ethyl. When RB1 and RB4 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. When RB2 and RB3 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0093] Specific examples of amidines include DBU: 1,8-diazabicyclo[5.4.0]-7-undecene and DBN: 1,5-diazabicyclo[4.3.0]-5-nonene.

[0094] As used herein, the term "guanidines" refers to bases represented by the following formula B2: TIFF0007808554000003.tif33170[In the formula, RB6 is hydrogen or C1-C4 alkyl; RB5 and RB7 each independently represent C1-C4 alkyl, or form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the carbon atoms to which they are bonded; RB8 is C1-C4 alkyl and RB9 is hydrogen, C1-C4 alkyl or phenyl, or RB8 and RB9 together with the nitrogen atoms to which they are bonded and the carbon atoms to which they are bonded form a 5- to 8-membered ring; When RB9 is phenyl, two benzene rings of the phenyl groups in the two formulae B2 may be fused to form a naphthalene.]

[0095] When RB5 to RB8 are C1-C4 alkyl, the C1-C4 alkyl is preferably methyl, and when RB9 is C1-C4 alkyl, the C1-C4 alkyl is preferably t-butyl. When RB5 and RB7 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably an imidazolidine ring, a hexahydropyrimidine ring, or a 1,3-diazepane ring. When RB8 and RB9 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0096] Specific examples of guanidines include TMG: 1,1,3,3-tetramethylguanidine, TBD: 1,5,7-triazabicyclo[4.4.0]dec-5-ene, and MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene.

[0097] As used herein, the term "phosphazenes" refers to bases represented by the following formula B3 or B4:

[0098] TIFF0007808554000004.tif38170[In the formula, RB 10 is C1-C4 alkyl, or RB 10 and R.B. 11 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 11 is RB 10 and R.B. 11 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 11 and R.B. 12 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, RB12 is RB 11 and R.B. 12 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 12 and R.B. 13 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 13 is RB 12 and R.B. 13 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 13 and R.B. 14 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, RB 14 is RB 13 and R.B. 14 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 14 and R.B. 15 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 15 is RB 14 and R.B. 15 is C1-C4 alkyl, except when it forms a 5- to 8-membered ring; RB 16 is hydrogen, C1-C8 alkyl, or C6-C 10 It is aryl.

[0099] RB 10 ~RB 15 is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and RB 16 When is C1-C8 alkyl, the C1-C8 alkyl is preferably t-butyl or t-octyl. RB 10 and R.B. 11、 RB 12 and R.B. 13 , and / or R.B. 14 and R.B. 15When forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. RB 11 and R.B. 12 , and / or R.B. 13 and R.B. 14 When R forms a 5- to 8-membered ring, the 5- to 8-membered ring is 11 , R.B. 12 , R.B. 13 , and R.B. 14 is preferably a 5- to 8-membered saturated ring that does not contain any heteroatoms other than the nitrogen atoms to which each is bonded and the phosphorus atom to which each nitrogen atom is bonded.

[0100] TIFF0007808554000005.tif50170[In the formula, RB 17 are independently C1-C4 alkyl, or R 17 and R.B. 18 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 18 is RB 17 and R.B. 18 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 18 and R.B. 19 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, RB 19 is RB 18 and R.B. 19 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 19 and R.B. 20 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 20 is RB 19 and R.B. 20 is C1-C4 alkyl, except when it forms a 5- to 8-membered ring; RB 21 is C1-C4 alkyl, or RB 21 and R.B. 22form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 22 is RB 21 and R.B. 22 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 22 and R.B. 23 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, RB 23 is RB 22 and R.B. 23 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 23 and R.B. 24 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 24 is RB 23 and R.B. 24 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 24 and R.B. 25 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, RB 25 is RB 24 and R.B. 25 is C1-C4 alkyl, except when R forms a 5- to 8-membered ring, or R 25 and R.B. 26 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, RB 26 is RB 25 and R.B. 26 is C1-C4 alkyl, except when it forms a 5- to 8-membered ring; RB 27 is C1-C4 alkyl, or C6-C 10 It is aryl.

[0101] RB 17 ~RB 26is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and RB 27 When is C1-C4 alkyl, the C1-C4 alkyl is preferably ethyl or t-butyl. RB 17 and R.B. 18、 RB 19 and R.B. 20 , R.B. 21 and R.B. 22、 RB 23 and R.B. 24、 RB 25 and R.B. 26 When forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. RB 17 and R.B. 18 are both C1-C4 alkyl, 19 and R.B. 20 Preferably, both R and R are C1-C4 alkyl. 17 and R.B. 18 forms a 5- to 8-membered ring, RB 19 and R.B. 20 It is preferable that the ring also forms a 5- to 8-membered ring. RB 21 and R.B. 22 are both C1-C4 alkyl, 23 and R.B. 24 and R.B. 25 and R.B. 26 Preferably, both R and R are C1-C4 alkyl. 21 and R.B. 22 forms a 5- to 8-membered ring, RB 23 and R.B. 24 and R.B. 25 and R.B. 26 It is preferable that the ring also forms a 5- to 8-membered ring. RB 18 and R.B. 19 , and / or R.B. 22 and R.B. 23 When R forms a 5- to 8-membered ring, the 5- to 8-membered ring is 11 , R.B. 12 , R.B. 13 , and R.B. 14is preferably a 5- to 8-membered saturated ring that does not contain any heteroatoms other than the nitrogen atoms to which each is bonded and the phosphorus atom to which each nitrogen atom is bonded.

[0102] Specific examples of phosphazenes include P2tBu: 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 -catenadi(phosphazene), P2Et: tetramethyl(tris(dimethylamino)phosphoranylidene)phosphoric acid triamide-ethylimine, HP1(dma): imino-tris(dimethylamino)phosphorane, BTPP: tert-butylimino-tri(pyrrolidino)phosphorane, P1tBu: tert-butylimino-tris(dimethylamino)phosphorane, BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine, and the like.

[0103] As used herein, the term "and / or" includes any combination of "and" and "or." Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, (vii) A, B, and C.

[0104] (Manufacturing method) In one aspect, the present invention relates to a method for producing a peptide by a solid phase method, comprising the following steps (1) to (3): (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a resin for solid phase synthesis; (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents; and (3) After the step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0105] Process (1) Step (1) of the present invention is a step of providing a first peptide having a protecting group containing an Fmoc skeleton supported on a resin for solid phase synthesis.

[0106] The "first peptide having a protecting group containing an Fmoc skeleton supported on a solid-phase synthesis resin" in step (1) is usually supported on the solid-phase synthesis resin via a functional group (e.g., a carboxyl group) in its C-terminal amino acid residue, but may also be supported on the solid-phase synthesis resin via a functional group (e.g., a carboxyl group) of an amino acid residue other than the C-terminus. The "first peptide having a protecting group containing an Fmoc skeleton supported on a solid-phase synthesis resin" has the amino group of its N-terminal amino acid residue protected by a protecting group containing an Fmoc skeleton.

[0107] The resin for solid-phase synthesis carrying the first peptide may be any known in the art, and is generally linked to a specific amino acid residue of the first peptide (e.g., the C-terminal amino acid residue) via an ester bond. The resin preferably has a resin-binding group that is acid-sensitive and is determined to be "H (<5% TFA in DCM)" as described in the Solid Phase Synthesis Handbook (published by Merck Ltd., May 1, 2002), and can be appropriately selected depending on the functional group of the amino acid used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid such as Asp or Glu) or a hydroxy group on an aromatic ring (phenol group such as Tyr) is used as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (Clt resin).When an aliphatic hydroxy group (aliphatic alcohol group such as Ser or Thr) is used as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (Clt resin), or 4-methyltrityl chloride resin (Mtt resin).

[0108] In the present invention, the term "protecting group containing an Fmoc skeleton" refers to an Fmoc group or a group in which an arbitrary substituent has been introduced at an arbitrary position of the structural skeleton of the Fmoc group. Specific examples of such a protecting group containing an Fmoc skeleton include a protecting group represented by the following formula (1): TIFF0007808554000006.tif36170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl).

[0109] More specifically, examples of the protecting group containing an Fmoc skeleton include a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, a 1-methyl-Fmoc (Fmoc(1Me)) group, a 2-fluoro-Fmoc (Fmoc(2F)) group, a 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, a 2-monoisooctyl-Fmoc (mio-Fmoc) group, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, a 2,7-(3,3,4 ,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc (tdf-Fmoc) group, 2,7-bis(trimethylsilyl)-Fmoc (Fmoc(2TMS)) group, (2-sulfo-9H-fluoren-9-yl)methoxycarbonyl group (Fmoc(2so3h)), [(1S)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (sm-Fmoc), [(1R)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (rm-Fmoc), etc. The Fmoc group is preferred as a protecting group containing the Fmoc skeleton. These protecting groups containing the Fmoc skeleton can be introduced by known methods using commercially available reagents, etc.

[0110] In the present invention, the type and number of amino acid residues constituting the first peptide are not limited, and any peptide having an amino acid sequence containing two or more amino acid residues, for example, 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, can be used as the first peptide. The amino acids can be natural amino acids and / or unnatural amino acids. The first peptide preferably contains one or more N-substituted amino acids.

[0111] The first peptide having a protecting group containing an Fmoc backbone supported on a solid-phase synthesis resin provided in step (1) can be obtained from a commercial supplier or can be produced by applying a method known in the art, for example, a method described in WO2013 / 100132 or WO2018 / 225864.

[0112] In one embodiment, the first peptide is a dipeptide. In this case, the C-terminal amino acid residue of the dipeptide is typically supported on a solid-phase synthesis resin via an ester bond. As shown in the following scheme, the free amino group resulting from removal of the protecting group of the N-terminal amino acid residue may react with the carbonyl group in the ester bond to produce undesired DKPs. The method of the present invention can prevent a decrease in the yield of the target peptide due to DKP elimination. TIFF0007808554000007.tif64170 (wherein R1 to R4 represent any atom or group, and * represents the bonding site with the resin for solid phase synthesis.)

[0113] In one embodiment, the first peptide is a peptide in which the second amino acid residue from the N-terminus is an "N-substituted amino acid." In this case, as shown in the following scheme, the free amino group resulting from removal of the protecting group of the N-terminal amino acid residue may react with the carbonyl group in the peptide bond between the second and third amino acids from the N-terminus, resulting in the formation of undesired DKPs, DKP elimination products, and impurities having an amidine skeleton (6-membered cyclic amidine skeleton structures). By using the method of the present invention, the formation of such DKPs, DKP elimination products, and 6-membered cyclic amidine skeleton structures can be suppressed, thereby preventing a decrease in the yield of the target peptide. TIFF0007808554000008.tif89170 (In the formula, R4 represents any atom or group other than hydrogen, R1 to R3 and R5 to R8 represent any atoms or groups, and * represents the bonding site with the adjacent amino acid residue or the resin for solid-phase synthesis. When R6 is hydrogen, either Route A or Route B in the above scheme can be taken, and when R6 is other than hydrogen, only Route A in the above scheme can be taken.)

[0114] Preferred examples of the "N-substituted amino acid" in the present invention include N-alkylamino acids that are N-alkylated, such as N-methylated, and cyclic amino acids such as prolines and azetidines.

[0115] Process (2) Step (2) of the present invention is a step of treating the first peptide provided in step (1) with one or more bases including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more in acetonitrile in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents.

[0116] Step (2) of the present invention involves the removal of the protecting group from the N-terminal amino acid residue. Without being bound by any particular theory, it is believed that the formation of the DKP elimination product and the six-membered cyclic amidine skeletal structure occurs via a six-membered cyclic intermediate, as described above. Energy calculations of the process from the six-membered cyclic intermediate to DKP elimination suggest that the higher the hydrogen-accepting capacity of the solvent, the greater the stabilization due to coordination. The donor number (DN) value, which is an index of the solvent's ability to donate unshared electron pairs, is believed to be an important parameter. It is believed that solvents with a DN value of 26 or less, or containing such solvents, tend to suppress the reaction via the six-membered cyclic intermediate, thereby reducing the formation of the DKP elimination product and the six-membered cyclic amidine skeletal structure. Therefore, it is preferred that the solvent used in step (2) has a donor number (DN) value of 26 or less. In some embodiments, the DN value can be 26 or less, 25 or less, 24 or less, 23 or less, or 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. As an indicator of the DN value of each solvent, values ​​described in "Eur. Chem. Bull., 2015, 4(2), 92-97" can be used. In addition, values ​​such as those in the table described in "JP 2010-50089" or the table described in "Chemical Dictionary 1989, 1584-1585" can also be used. The DN value can also be measured by the methods described in "Journal of the American Chemical Society 1972, 94(5), 1431-1434" and "Chemistry A European Journal 2012, 18(35), 10969-10982". Furthermore, the DN value of the mixed solvent can be measured by the method described in "Chemistryselect 2021, 6(4), 600-608".Specifically, the DN values ​​are toluene: 0.1, DCM: 1, cumene: 6, methyl propionate: 11, sulfolane: 14.8, diethyl ketone: 15, butyl acetate: 15, propyl acetate: 16, diethyl carbonate: 16, acetone: 17, dimethyl carbonate: 17.2, methyl ethyl ketone: 17.4, 2-methyltetrahydrofuran: 18, diethyl ether: 19.2, tetrahydrofuran: 20, 1,2-dimethoxyethane: 20, 1,3-dioxolane: 21.2, trimethyl phosphate: 23, tributyl phosphate: 23.7, DMF: 26.6, NMP: 27.3, DMA: 27.8, DMI: 27.8.

[0117] In one embodiment, at least a portion of the first peptide obtained in step (2) is in the form of a carbamate. Without being bound by a particular theory, in step (2), when the protecting group containing the Fmoc skeleton is removed, dibenzofulvene is generated in at least a portion of the first peptide without decarboxylation, and the N-terminal amino group is converted to COO derived from the protecting group. - together with the carbamate ion (-NRC(=O)O - , where R is hydrogen or any substituent of an amino group), which forms a carbamate with a protonated base in the system (below is a scheme showing the formation of a carbamate when Fmoc is used as a protecting group containing an Fmoc skeleton and DBU is used as a base). TIFF0007808554000009.tif51170The formation of a carbamate salt can suppress the intramolecular nucleophilic reaction of the terminal amino group, and it is presumed that this suppresses the formation of six-membered ring intermediates even under basic deprotection conditions, thereby reducing the by-production of DKP elimination products and six-membered cyclic amidine skeleton structures.

[0118] In the method of the present invention, in which a base having a pKa of 23 or higher in acetonitrile is used in step (2), the first peptide can be present as a carbamate in an amount sufficient to significantly reduce the formation of DKP elimination products and 6-membered cyclic amidine backbone structures. For example, in the method of the present invention, the carbamate can be formed in an amount such that the molar ratio of the carbamate to the amine produced by further decarboxylation (carbamate / amine) is 0.6 or higher, 0.8 or higher, 1.0 or higher, 2.0 or higher, 3.0 or higher, 4.0 or higher, 4.6 or higher, 5.0 or higher, 6.0 or higher, 8.0 or higher, or 10.0 or higher. Such a molar ratio can be, for example, 1 It can be determined from the proton integral ratio in H-NMR.

[0119] The carbamate may be a salt with any base whose conjugate acid has a pKa in acetonitrile of 23 or higher. Specific preferred examples of such salts include DBU salt, TMG salt, HP1(dma) salt, MTBD salt, P1-tBu salt, and P2-Et salt.

[0120] In one embodiment, the solvent used in step (2) may be a single solvent or a mixed solvent, as long as it contains at least one of an aromatic hydrocarbon solvent, a halogenated solvent, an ether solvent, an ester solvent, a ketone solvent, a carbonate solvent, and a phosphate ester solvent. In the case of a mixed solvent, the total amount of the aromatic hydrocarbon solvent, the halogenated solvent, the ether solvent, the ester solvent, the ketone solvent, the carbonate solvent, and the phosphate ester solvent is preferably 25 v / v% or more, 50 v / v% or more, or 75 v / v% or more of the total solvent, and more preferably 25 v / v% or more, 50 v / v% or more, or 75 v / v% or more of at least one solvent selected from the group consisting of an aromatic hydrocarbon solvent, a halogenated solvent, an ether solvent, an ester solvent, a ketone solvent, a carbonate solvent, and a phosphate ester solvent.

[0121] When an aromatic hydrocarbon solvent is used in step (2), specific examples of the solvent include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene, and preferred are toluene and cumene.

[0122] When a halogenated solvent is used in step (2), specific examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride, with dichloromethane being preferred.

[0123] When an ether solvent is used in step (2), specific examples of the solvent include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane (DME), diisopropyl ether, cyclopentyl methyl ether (CPME), t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme, and preferred are tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, and 1,3-dioxolane.

[0124] When an ester solvent is used in step (2), specific examples of the solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone, and preferred are butyl acetate and methyl propionate.

[0125] When a ketone solvent is used in step (2), specific examples of the solvent include acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone (3-pentanone), and preferred are methyl ethyl ketone and diethyl ketone.

[0126] When a carbonate solvent is used in step (2), specific examples of the solvent include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, with dimethyl carbonate being preferred.

[0127] When a phosphate ester solvent is used in step (2), specific examples of the solvent include trimethyl phosphate, triethyl phosphate, and tributyl phosphate, with tributyl phosphate being preferred.

[0128] When the solvent used in step (2) is a mixed solvent, the mixed solvent may be composed of at least one solvent, or two or more solvents, selected from aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate-based solvents, and may contain, in addition to aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate-based solvents, one or more solvents selected from amide-based solvents, urea-based solvents, and sulfone-based solvents.

[0129] When an amide solvent is used in step (2), specific examples of the solvent include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide.

[0130] When a urea-based solvent is used in step (2), specific examples of the solvent include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0131] When a sulfone solvent is used in step (2), specific examples of the solvent include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0132] When the solvent used in step (2) is a single solvent, the solvent is preferably toluene, cumene, 1,2-dichlorobenzene, benzene, anisole, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxolane, ethyl acetate, isopropyl acetate, butyl acetate, methyl propionate, acetone, methyl ethyl ketone, diethyl ketone, dimethyl carbonate, diethyl carbonate, trimethyl phosphate, or tributyl phosphate.

[0133] In one embodiment, the solvent used in step (2) preferably has a swelling capacity of the solid phase resin of 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more. As an index of the swelling capacity of the solid phase resin of each solvent, values ​​described in "Tetrahedron Lett., 1998, 39, 8951-8954", "Org. Process Res Dev. 2018, 22, 494-503", and "RSC Adv., 2020, 10, 42457-42492" can be used. Specifically, the swelling capacity (mL / g) of the solid phase resin is as follows: DMI: 8.5, DMPU: 8.0, NMP: 6.4, THF: 6.0, DMA: 5.8, CHCl3: 5.6, CPME: 5.6, CHCl: 5.4, 2-methyltetrahydrofuran: 5.4, DMF: 5.2, butyl acetate: 5.2, DME: 4.8, MEK: 4.4, toluene: 4.0, AcOEt: 4.2, xylene: 3.0, EtO: 2.8, dimethyl carbonate: 2.8, MTBE: 2.4, CHCN: 2.0, tributyl phosphate: 1.6, MeOH: 1.6, HO: 1.6. In addition, the judgments in the table in "Green Chem., 2017, 19, 952-962" or table 2 in "Green Chem., 2020, 22, 996-1018" can also be used.

[0134] In step (2), one or more bases are used, including at least one base whose conjugate acid has a pKa of 23 or more in acetonitrile. When only one base is used, that base is a base whose conjugate acid has a pKa of 23 or more in acetonitrile. On the other hand, when multiple bases are used, at least one of the bases is a base whose conjugate acid has a pKa of 23 or more in acetonitrile, and the other bases may be bases whose conjugate acid has a pKa of 23 or more in acetonitrile, or may be bases whose pKa is less than 23.

[0135] The base having a pKa of 23 or more in acetonitrile of the conjugate acid used in step (2) may be a base having a pKa of 34 or less, preferably 30 or less, more preferably 28 or less.

[0136] pKa is an index that indicates the degree of basicity, and the larger the pKa, the higher the basicity. In the present invention, the pKa (CH3CN) of the conjugate acid of the organic base in acetonitrile is calculated based on the formula in "New J. Chem. 2009, 33, 588" 1) or "Sigma-Aldrich, Phosphazene Bases: https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html" 2) Alternatively, values ​​such as those shown in "Eur. J. Org. Chem. 2019, 6735-6748" may be adopted.

[0137] Examples of bases whose conjugate acids have a pKa of 23 or more in acetonitrile include amidines, guanidines, and phosphazenes. For example, the pKa of the bases listed below is 23 or more. DBU:pKa 1) =24.3 DBN:pKa 1) =23.8 TMG:pKa 1) =23.3 TBD:pKa 1) =26.0 MTBD:pKa 1) =25.5 P2tBu:pKa 1) =33.5 P2Et:pKa 1) =32.9 HP1(dma):pKa 1) =25.9 BTPP:pKa 2) =28.4 P1tBu:pKa 1) =27.0 BEMP:pKa 2) =27.6

[0138] In this step, when multiple bases having a pKa of 23 or more in acetonitrile of the conjugate acid are used, multiple bases, for example, two bases selected from the amidines, guanidines, or phosphazenes described above can be used in combination. Specific preferred combinations of such bases include DBU and MTBD, DBU and HP1(dma), DBU and P1tBu, and DBU and P2Et.

[0139] In this step, when a base having a pKa of 23 or more in acetonitrile and a base having a pKa of less than 23 are used in combination, the base having a pKa of 23 or more in acetonitrile can be selected from the amidines, guanidines, or phosphazenes described above. Furthermore, the base having a pKa of less than 23 in acetonitrile is preferably selected from piperidine, piperazine, and morpholine. A preferred example of a specific combination of such bases is DBU and piperidine.

[0140] In some embodiments, step (2) can be repeated multiple times before step (3). In this case, the same solvent and / or the same base may be used each time step (2), or different solvents and / or different bases may be used. In the present invention, the term "same solvent" means that the type of solvent and the mixing ratio of the solvents are the same, and the term "different solvents" means that either or both of the type of solvent and the mixing ratio of the solvents are different. Furthermore, in the present invention, the term "same base" means that the type of base and the concentration of the base in the solvent are the same, and the term "different bases" means that either or both of the type of base and the concentration of the base in the solvent are different.

[0141] In one embodiment, when step (2) is included multiple times, the method of the present invention may include a step of discharging the solution between each step (2), thereby replacing the solvent and / or base used in the previous step (2) with the solvent and / or base used in the next step (2).

[0142] In certain embodiments, the method of the present invention does not include a step of treating the first peptide with a single base, such as piperidine, whose conjugate acid has a pKa of less than 23 in acetonitrile, prior to step (2). Specifically, the present invention excludes an embodiment in which the first peptide provided in step (1) having a protecting group containing an Fmoc skeleton is treated, for example, in any solvent (e.g., DMF) with a base (e.g., piperidine) whose conjugate acid has a pKa of less than 23 in acetonitrile, and then step (2) of the present invention is performed (e.g., treatment with DBU or a combination of DBU and piperidine in a solvent containing toluene).

[0143] In one embodiment, the base used in step (2) can be contained in the solvent at any concentration that allows the reaction of this step to proceed. Specific examples of such a concentration range include 0.25 to 20 v / v%, 0.5 to 20 v / v%, 1 to 20 v / v%, 1 to 15 v / v%, 1 to 10 v / v%, 1 to 9 v / v%, 1 to 8 v / v%, 1 to 7 v / v%, 1 to 6 v / v%, 1 to 5 v / v%, 1 to 4 v / v%, 1 to 3 v / v%, and 1 to 2 v / v%, with 1 to 8 v / v% being preferred.

[0144] In some embodiments, step (2) can further comprise a step of contacting the solvent with CO2. In some embodiments, the solvent used in step (2) can be a solvent that has been contacted with CO2 prior to step (2). Contact of the solvent with CO2 can be carried out, for example, by bubbling CO2 into the solvent. CO2 can be included in the reaction system by contacting the solvent with CO2 during or before step (2). Without being bound by a particular theory, in step (2), the N-terminal amino group of at least a portion of the first peptide is converted to COO derived from the protecting group. - By adding CO2 to the reaction system, the amine in the reaction system can be more easily converted to a carbamate. Therefore, by adding CO2 to the reaction system, the amount of amine in the reaction can be further reduced, and the intramolecular nucleophilic reaction of the terminal amino group can be further suppressed. This further suppresses the formation of the six-membered ring intermediate, even under basic deprotection conditions, and further reduces the by-production of the DKP elimination product and the six-membered cyclic amidine skeleton structure.

[0145] In one embodiment, the reaction in step (2) can be carried out at a temperature of from −100° C. to a temperature near the boiling point of the solvent, preferably 0 to 50° C. The reaction in step (2) can be carried out for a reaction time of 1 minute to 1 week, preferably 1 minute to 3 hours, 3 minutes to 3 hours, or 5 minutes to 3 hours, and more preferably 1 minute to 20 minutes, 3 minutes to 20 minutes, or 5 minutes to 20 minutes.

[0146] Process (3) Step (3) of the present invention is a step following step (2) in which the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. In this step, the N-terminal amino group of the first peptide is condensed with the carboxyl group of the carboxylic acid to synthesize the third peptide.

[0147] In some embodiments, the carboxylic acid used in step (3) is a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 It can be an aryl carboxylic acid.

[0148] In certain embodiments, the carboxylic acid analog used in step (3) can be an activated ester of the carboxylic acid or an acid halide of the carboxylic acid.

[0149] a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 By using the activated ester of each arylcarboxylic acid, the condensation reaction of step (3) can be carried out without a condensing agent. Specific examples of activated esters include those having a group capable of promoting the reaction with an amine, such as an O-(benzotriazol-1-yl) group (OBt), an O-(7-azabenzotriazol-1-yl) group (OAt), an N-hydroxysuccinimide group (OSu), a pentafluorophenoxy group (OPfp), or an SR group, at the carbonyl group in these compounds, particularly the carbonyl group contained in the C-terminal amino acid residue in the case of the second peptide. 11 (R 11 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, an optionally substituted heteroaryl group, an optionally substituted alkenyl group, or an optionally substituted alkylene group.

[0150] a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 By using an acid halide of each arylcarboxylic acid, the condensation reaction of step (3) can be carried out without a condensing agent. Specific examples of acid halides include those in which the carboxyl group in these compounds, particularly the carboxyl group contained in the C-terminal amino acid residue in the case of the second peptide, has been converted to a chlorocarbonyl group, a fluorocarbonyl group, a bromocarbonyl group, an iodocarbonyl group, or the like.

[0151] When the carboxylic acid or carboxylic acid analog used in step (3), such as an amino acid or a second peptide, has a protecting group, the protecting group can be a carbamate-based protecting group, a sulfonyl-based protecting group, or an acyl-based protecting group.

[0152] As used herein, the term "carbamate protecting group" refers to a protecting group that comprises a carbamate structure, and includes, for example, a protecting group having an Fmoc skeleton, Alloc, Teoc, Boc, Cbz, and a 2,2,2-trichloroethoxycarbonyl group (Troc). The term "sulfonyl protecting group" refers to a protecting group that contains a sulfonyl structure, and includes, for example, a methanesulfonyl (Ms) group, a p-toluenesulfonyl (Ts) group, a 2-nitrobenzenesulfonyl (Ns) group, a trifluoromethanesulfonyl (Tf) group, and a (2-trimethylsilyl)-ethanesulfonyl (SES) group. The term "acyl protecting group" refers to a protecting group that contains an acyl structure, and includes, for example, an acetyl group, a pivaloyl group, a benzoyl group, and a trifluoroacetyl (Tfa) group.

[0153] In one embodiment, when the carboxylic acid or carboxylic acid analog used in step (3) is an amino acid having a protecting group containing an Fmoc backbone, the amino acid having a protecting group containing an Fmoc backbone can be any natural or unnatural amino acid protected with a protecting group containing an Fmoc backbone.

[0154] In one embodiment, when the carboxylic acid or carboxylic acid analog used in step (3) is a second peptide having a protecting group containing an Fmoc backbone, the second peptide having a protecting group containing an Fmoc backbone is not limited in type or number of amino acid residues constituting the peptide, as long as its N-terminal amino acid is protected with a protecting group containing an Fmoc backbone, and a peptide having any amino acid sequence containing two or more natural and / or unnatural amino acid residues can be used. Preferably, the second peptide contains one or more N-substituted amino acids.

[0155] In some embodiments, the carboxylic acid or carboxylic acid analog used in step (3) is a C1-C8 alkyl carboxylic acid, a C6-C 10 In the case of arylcarboxylic acids, or their activated esters or acid halides, they may be substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl, and dialkylaminocarbonyl. C1-C8 alkylcarboxylic acids, or their activated esters or acid halides, are preferably C1-C4 alkylcarboxylic acids, or their activated esters or acid halides, and specific examples thereof include acetic acid, propanoic acid, butanoic acid, or pentanoic acid, or their activated esters or acid halides. C6-C 10 Specific examples of the arylcarboxylic acid, or its active ester, or its acid halide include benzoic acid, naphthoic acid, or its active ester, or its acid halide.

[0156] In step (3), 1 to 20 equivalents, preferably 2 to 10 equivalents, of an amino acid having a protecting group containing an Fmoc skeleton or a second peptide having a protecting group containing an Fmoc skeleton can be used relative to the first peptide.

[0157] In one embodiment, the first peptide and / or the second peptide having a protecting group containing an Fmoc backbone preferably contains at least one N-substituted amino acid, and the amino acid having a protecting group containing an Fmoc backbone is preferably an N-substituted amino acid.

[0158] In one embodiment, the protecting group containing an Fmoc backbone carried by the amino acid or second peptide used in step (3) may be the same as or different from the protecting group containing an Fmoc backbone carried by the first peptide used in step (1).

[0159] In one embodiment, the solvent used in step (3) may be a single solvent or a mixed solvent, as long as it contains at least one of an aromatic hydrocarbon solvent, a halogen-based solvent, an ether-based solvent, an amide-based solvent, a sulfoxide-based solvent, a sulfone-based solvent, a urea-based solvent, an ester-based solvent, a ketone-based solvent, a carbonate-based solvent, or a phosphate-based solvent. In the case of a mixed solvent, the solvent used in step (3) preferably contains at least one of an aromatic hydrocarbon solvent, a halogen-based solvent, an ether-based solvent, an amide-based solvent, a sulfoxide-based solvent, a sulfone-based solvent, a urea-based solvent, an ester-based solvent, a ketone-based solvent, a carbonate-based solvent, or a phosphate-based solvent in an amount of 25 v / v % or more, 50 v / v % or more, or 75 v / v % or more.

[0160] When an aromatic hydrocarbon solvent is used in step (3), specific examples of the solvent include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene, and preferred are toluene and cumene.

[0161] When a halogen-based solvent is used in step (3), specific examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, carbon tetrachloride, etc., and preferably dichloromethane.

[0162] When an ether solvent is used in step (3), specific examples of the solvent include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme, and preferred are tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, and 1,3-dioxolane.

[0163] When an amide solvent is used in step (3), specific examples of the solvent include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide, and preferably DMF or NMP.

[0164] When a sulfoxide solvent is used in step (3), specific examples of the solvent include dimethyl sulfoxide (DMSO) and methyl phenyl sulfoxide, and preferably DMSO.

[0165] When a sulfone solvent is used in step (3), specific examples of the solvent include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0166] When a urea-based solvent is used in step (3), specific examples of the solvent include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), and preferably DMI.

[0167] When an ester solvent is used in step (3), specific examples of the solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone, and preferred are ethyl acetate, butyl acetate, and methyl propionate.

[0168] When a ketone solvent is used in step (3), specific examples of the solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone, and preferred are methyl ethyl ketone and diethyl ketone.

[0169] When a carbonate solvent is used in step (3), specific examples of the solvent include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, with dimethyl carbonate being preferred.

[0170] When a phosphate ester solvent is used in step (3), specific examples of the solvent include trimethyl phosphate, triethyl phosphate, and tributyl phosphate, with tributyl phosphate being preferred.

[0171] When the solvent used in step (3) is a mixed solvent, the mixed solvent may consist of two or more solvents selected from aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents, or may contain any other solvent.

[0172] When the solvent used in step (3) is a single solvent, the solvent is preferably toluene, cumene, 1,2-dichlorobenzene, benzene, anisole, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxolane, DMF, NMP, DSMO, DMI, DMPU, ethyl acetate, isopropyl acetate, butyl acetate, methyl propionate, acetone, methyl ethyl ketone, diethyl ketone, dimethyl carbonate, diethyl carbonate, trimethyl phosphate, tributyl phosphate, sulfolane, or 3-methylsulfolane.

[0173] In some embodiments, the solvent used in step (3) can be the same as the solvent used in step (2).

[0174] The solvent used in step (3) is preferably one in which the condensing agent can be dissolved. In the present invention, the base used in step (2) may remain, and this remaining base may cause by-products in which amino acids or peptides are over-elongated. When a solvent in which the condensing agent can be dissolved is used in step (3), the formation of such over-elongated products can be suppressed.

[0175] In one embodiment, the condensing agent used in step (3) is in the form of a salt, the counter anion of which is PF6 - or BF4 - Specific examples of such condensing agents include PyOxim ([ethylcyano(hydroxyimino)acetato-O 2]tri-1-pyrrolidinylphosphonium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1-yloxy)trispirolizinophosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yloxy)trispirolizinophosphonium hexafluorophosphate), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) salt), HATU (O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate), HCTU (O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), TDBTU (O-(3,4 -dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate), TATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate) ), TBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate), TCTU (O-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), and TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate).

[0176] The condensing agent used in step (3) preferably includes at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, and TOTU, and it is more preferable to use any one of these.

[0177] In step (3), the condensing agent can be used in an amount of 1 to 20 equivalents, preferably 2 to 10 equivalents, relative to the first peptide.

[0178] In one embodiment, the condensation reaction in step (3) can be carried out in the presence of a base. The type of base is not limited as long as it contributes to the progress of the condensation reaction. However, a base with a pKa of 5 to 12 in water is preferred. Specific examples of such bases include DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine. In step (3), 1 to 20 equivalents, preferably 2 to 10 equivalents, of base can be used relative to the first peptide. The pKa of the conjugate acid of the base in water can be calculated, for example, using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((c)1994-2020 ACD / Labs).

[0179] In some embodiments, the present invention does not include a step of neutralizing the remaining base by adding an acid between steps (2) and (3). When elongating a peptide chain using a solid-phase method, it is generally considered undesirable to leave the base used in the deprotection step prior to the elongation step. Therefore, the remaining base may be neutralized with an acid between the deprotection step and the elongation step. However, unexpectedly, in the present invention, it was found that such neutralization actually leads to the formation of DKP compounds, and the neutralization step does not contribute to efficient reaction progress. Without being bound by theory, it is presumed that the addition of an acid decomposes the formed carbamate salt and converts it to an amine, which then forms a six-membered ring intermediate, increasing the by-production of DKP elimination products and six-membered cyclic amidine framework structures.

[0180] In one embodiment, the present invention may further comprise a step of washing the resin for solid phase synthesis between steps (2) and (3). Any solvent can be used to wash the resin, and for example, it is preferable to use the solvent used in step (2). The resin can be washed multiple times with the same solvent or different solvents.

[0181] In one embodiment, in the step of washing the resin for solid phase synthesis, a solvent that has been contacted with CO2 prior to the step (e.g., a solvent bubbled with CO2 gas) may be used, a solvent that has not been contacted with CO2 may be used, or a combination of a solvent that has been contacted with CO2 and a solvent that has not been contacted with CO2 may be used. When washing is performed multiple times, it is preferable to wash the resin once or multiple times with a solvent that has been contacted with CO2 in advance, and then wash the resin once or multiple times with a solvent that has not been contacted with CO2 in advance.

[0182] In one embodiment, the reaction in step (3) can be carried out at a temperature of from −100° C. to about the boiling point of the solvent, preferably from 0 to 60° C. The reaction in step (3) can be carried out for a reaction time of from 1 minute to 1 week, preferably from 30 minutes to 20 hours.

[0183] In one embodiment, the peptide produced by the method of the present invention comprises a third peptide produced via steps (1) to (3). In this case, the peptide produced by the method of the present invention can further comprise any number of amino acids in addition to the third peptide.

[0184] In one embodiment, the peptide produced by the method of the present invention may be the third peptide produced via the above steps (1) to (3).

[0185] In some embodiments, the peptide produced by the method of the present invention can contain at least one, or two or more, three or more, four or more, or five or more N-substituted amino acids, which can be N-alkyl amino acids, preferably N-methyl amino acids.

[0186] In one embodiment, the method of the present invention may include a step of cleaving the peptide from the solid-phase synthesis resin after the peptide chain elongation step. For the cleavage step, a method known in the art or the method described in this Example can be used.

[0187] In one aspect, the present invention also relates to a method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in peptide production by a solid-phase method, the method comprising the steps (1) and (2).

[0188] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0189] The present invention is further illustrated, but not limited, by the following examples, comparative examples, and reference examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0190] The following abbreviations are used in the examples: AA: Ammonium acetate Alloc: allyloxycarbonyl group Boc: tert-butoxycarbonyl group Cbz: benzyloxycarbonyl group COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate DBU: 1,8-diazabicyclo[5.4.0]-7-undecene DCM: dichloromethane DCE: 1,2-dichloroethane DMA: Dimethylacetamide DME: 1,2-dimethoxyethane DMF: N,N-dimethylformamide DMI: 1,3-dimethyl-2-imidazolidinone DKP: Diketopiperazine DIC: N,N'-diisopropylcarbodiimide DIPEA: N,N-diisopropylethylamine DMSO: dimethyl sulfoxide EDCI·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc: ethyl acetate FA: Formic acid Fmoc: 9-fluorenylmethyloxycarbonyl group HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxybenzotriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU: O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HCTU: O-(1H-6-chloro-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOTU:O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate HP1(dma): iminotris(dimethylamino)phosphorane MEK: Methyl ethyl ketone MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene NMP: N-methyl-2-pyrrolidone Ns: 2-nitrobenzenesulfonyl group Oxyma: Ethyl cyanohydroxyiminoacetate pip: piperidine P1tBu: t-butylimino-tris(dimethylamino)phosphorane P2Et: Tetramethyl(tris(dimethylamino)phosphoranylidene)phosphate triamide-ethylimine PyAOP: (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate PyBOP: Benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate PyOxim: [Ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate TBME: t-butyl methyl ether Teoc: 2-trimethylsilylethoxycarbonyl group TES: Triethylsilane Tfa: trifluoroacetyl group TFA: Trifluoroacetic acid TFE: 2,2,2-trifluoroethanol THF: tetrahydrofuran THP: tetrahydropyranyl group TMG: 1,1,3,3-tetramethylguanidine TATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate TBTU: 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate TCTU: O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TDBTU: O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TOTU: O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0191] The LCMS analysis conditions are shown in Table 1. [Table 1]

[0192] Example 1: Preparation of amino acids and resin-supported peptides used in this example Example 1-1: Fmoc-amino acids used in peptide synthesis using a peptide synthesizer In the peptide synthesis described herein, the Fmoc-amino acids listed in Tables 2 to 4 were used in the synthesis using a peptide synthesizer. The Fmoc-amino acids listed in Table 2 were synthesized according to the methods described in WO2018 / 225864 and WO2021 / 090856. The Fmoc-amino acids listed in Table 3 were purchased from commercial suppliers. The Fmoc-amino acids listed in Table 4 were synthesized according to the scheme shown below.

[0193] [Table 2]

[0194] [Table 3] TIFF0007808554000013.tif221170

[0195] [Table 4]

[0196] Synthesis of compound aa3-1, (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid (Fmoc-MeAla(3-Pyr)-OH) TIFF0007808554000015.tif32170 Under a nitrogen atmosphere, a solution of commercially available (2S)-2-[9H-fluoren-9-ylmethoxycarbonylamino]-3-pyridin-3-ylpropanoic acid (Fmoc-Ala(3-Pyr)-OH, compound aa3-1-a) (3 g, 7.72 mmol) in toluene (10.5 mL) was added with paraformaldehyde (696 mg, 23.17 mmol) and trifluoroacetic acid (TFA) (5.36 mL, 69.5 mmol), and the mixture was stirred at 40 ° C for 2 hours. The reaction mixture was concentrated under reduced pressure, diluted with dichloromethane (DCM), washed with saturated aqueous sodium bicarbonate, dried over anhydrous magnesium sulfate, and then filtered. The resulting solution was concentrated under reduced pressure to give the crude product, compound aa3-1-b (2.95 g, 95%). This was used in the next reaction without further purification. LCMS(ESI)m / z=401(M+H)+ Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0197] Under a nitrogen atmosphere, triethylsilane (TES) (10.59 mL, 66.3 mmol) and trifluoroacetic acid (TFA) (15.32 mL, 199 mmol) were added to a solution of the crude product compound aa3-1-b (2.95 g) in 1,2-dichloroethane (DCE) (15.5 mL) at room temperature, and the mixture was stirred at 70 °C for 5 hours. The reaction mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The resulting crude product was dissolved in ethyl acetate, and t-butyl methyl ether / n-hexane = 9 / 1 (40 mL) was added and stirred. After cooling the mixture in a refrigerator for 30 minutes, the resulting solid was filtered, washed three times with t-butyl methyl ether / n-hexane = 9 / 1 (30 mL), and then dried under reduced pressure. The resulting crude product was suspended in DCM (50 mL), 3 mol / L aqueous sodium dihydrogen phosphate (45 mL, containing 1.5 mol / L sodium chloride) was added, and the mixture was stirred. After removing the aqueous layer, the organic layer was washed twice with 3 mol / L aqueous sodium dihydrogen phosphate (45 mL, containing 1.5 mol / L sodium chloride). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by reverse-phase column chromatography (water / acetonitrile) to give compound aa3-1, (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid, Fmoc-MeAla(3-Pyr)-OH (2.67 g, 90%). LCMS(ESI)m / z=403(M+H)+ Retention time: 0.53 minutes (Analysis conditions SQDFA05)

[0198] Synthesis of compound aa3-2, (2S)-3-[4-(trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxycarbonylamino)propanoic acid (Teoc-Phe(4-CF3)-OH) TIFF0007808554000016.tif38170

[0199] Under a nitrogen atmosphere, commercially available (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoic acid (H-Phe(4-CF3)-OH, compound aa3-2-a) (2.00 g, 8.58 mmol) was dissolved in water (12.0 mL), followed by the addition of a mixture of triethylamine (2.38 mL, 17.15 mmol) and 1,4-dioxane (10.0 mL) and stirring at room temperature for 10 minutes. 2,5-Dioxopyrrolidin-1-yl(2-(trimethylsilyl)ethyl)carbonate (2.45 g, 9.43 mmol) was then added and stirred at room temperature for 1 hour. 2-Methyltetrahydrofuran (5 mL) was added to the reaction mixture, which was then washed twice with 10% aqueous sodium carbonate solution (10 mL). The resulting organic layer was washed with saturated aqueous potassium hydrogen sulfate (10 mL), then with 10% aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The resulting solution was concentrated under reduced pressure to give compound aa3-2, 2S)-3-[4-(trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxycarbonylamino)propanoic acid (Teoc-Phe(4-CF3)-OH) (2.88 g, 89%). LCMS(ESI)m / z=376(MH)- Retention time: 0.87 minutes (Analysis conditions SQDFA05)

[0200] Synthesis of compound aa3-3, (2S)-2-(allyloxycarbonylamino)-3-phenylpropanoic acid (Alloc-Phe-OH) TIFF0007808554000017.tif33170

[0201] L-Phenylalanine (1.00 g, 6.05 mmol) was mixed with 50% aqueous 1,4-dioxane (20 mL) and then mixed. Sodium bicarbonate (1.27 g, 15.1 mmol, 2.5 equiv.) was added. While the mixture was cooled in an ice bath, allyl chloroformate (0.97 mL, 9.1 mmol, 1.5 equiv.) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours, then diluted with dichloromethane (10 mL, 10 v / w). The aqueous layer was acidified with phosphoric acid (0.53 mL, 1.5 equiv.) and separated into organic and aqueous layers. The aqueous layer was further washed with dichloromethane (5 mL, 5 v / w), and the resulting organic layer was washed with half-saturated brine (15 mL, 15 v / w). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was purified by reverse-phase column chromatography (acetonitrile containing 0.1% formic acid / distilled water containing 0.1% formic acid), and the collected fractions were purified by reverse-phase column chromatography (acetonitrile / distilled water without addition) to obtain compound aa3-3, (2S)-2-(allyloxycarbonylamino)-3-phenylpropanoic acid (Alloc-Phe-OH) (0.52 g, 35%). LCMS(ESI)m / z=248(MH)- Retention time: 0.58 minutes (Analysis conditions SQDFA05)

[0202] Synthesis of compound aa3-4, 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propanoic acid (Ns-Aib-OH) TIFF0007808554000018.tif29170

[0203] A solution of commercially available 2-amino-2-methylpropanoic acid methyl ester hydrochloride (4.0 g, 26.0 mmol) and DIPEA (10.92 mL, 62.5 mmol) in DCM (22.1 mL) was added to a solution of 2-nitrobenzenesulfonyl chloride (6.93 g, 31.2 mmol) in DCM (30.0 mL) while cooling in an ice bath, and the mixture was stirred at 0° C. for 5 minutes and then at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give crude product aa3-4-b. LCMS(ESI)m / z=301(M−H)- Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0204] To a solution of the resulting crude product aa3-4-b (3.63 g) in THF / methanol (1 / 1, 52 mL), 5N aqueous sodium hydroxide (26 mL) was added while cooling in an ice bath. The mixture was stirred at 0 °C for 5 minutes and then at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in 4N hydrochloric acid (30 mL), and the solvent was evaporated. The mixture was extracted twice with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and filtered. The solution was concentrated under reduced pressure. The resulting crude product was purified twice by reverse-phase column chromatography (0.1% formic acid in acetonitrile / 0.1% formic acid in distilled water) to give compound aa3-4, 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propanoic acid (Ns-Aib-OH) (2.46 g, 71%). LCMS(ESI)m / z=287(MH)- Retention time: 0.53 minutes (Analysis conditions SQDFA05)

[0205] Example 1-2: Preparation of resin-supported amino acids and peptides used in this example Example 1-2-1: Synthesis of (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1) TIFF0007808554000019.tif50170

[0206] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin site may be represented by a circle. Furthermore, to clarify the reactive site of the resin site, the chemical structure of the reactive site may be represented by connecting it to a circle. For example, in the above structure (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1)), the 2-chlorotrityl group of the resin is bonded to the side-chain carboxylic acid of Asp via an ester bond. Note that pyrro refers to pyrrolidine, and in the above structure, the C-terminal carboxylic acid group forms an amide bond with the pyrrolidine. TIFF0007808554000020.tif81170

[0207] Under a nitrogen atmosphere, EDCI·HCl (67.1 g, 350 mmol), HOBt (43.3 g, 321 mmol), and Fmoc-Asp(OtBu)-OH (120 g, 2929 mmol) were added sequentially to DMF (600 mL) at 0°C and stirred at 0°C for 1 hour. Ethyl acetate (10 v) and 0.5 mol / L aqueous hydrochloric acid (2 v) were added to the reaction mixture at 0°C, and the organic layer was separated. The resulting organic layer was washed sequentially with 0.5 mol / L aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate / water (1 / 1 (v / v)), and saturated brine / water (1 / 1 (v / v)). After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain compound 1-2-1-a as a crude product (137.1 g, quant.). LCMS(ESI)m / z=465(M+H)+ Retention time: 1.05 minutes (Analysis conditions SQDAA05)

[0208] Under ice cooling, TFA (271 mL) was slowly added to a solution of compound 1-2-1-a (137 g, 395 mmol) in DCM (137 mL) so that the internal temperature did not exceed 10°C. After stirring at room temperature for 1 hour, diisopropyl ether (3.4 L) was added in four portions, and the precipitated solid was collected by filtration and dried to obtain compound 1-2-1-b ((3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid, Fmoc-Asp-pyrro). (108.4 g, 90%) LCMS(ESI)m / z=409(M+H)+ Retention time: 0.83 minutes (Analysis conditions SQDAA05)

[0209] The Fmoc amino acid resin loading reaction was carried out according to the method described in WO2013 / 100132 or WO2018 / 225864. 2-Chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, 48.7 g) and dehydrated dichloromethane (500 mL) were placed in a filter-equipped reaction vessel and shaken at room temperature for 20 minutes. After removing the dichloromethane under nitrogen pressure, a mixture of compound 1-2-1-b (15.91 g), dehydrated dichloromethane (350 mL), dehydrated methanol (12.63 mL), and diisopropylethylamine (DIPEA) (32.6 mL) was added to the reaction vessel and shaken for 60 minutes. After removing the reaction mixture under nitrogen pressure, a mixture of 350 mL of dehydrated dichloromethane, 97.3 mL of dehydrated methanol, and 32.6 mL of diisopropylethylamine (DIPEA) was added to the reaction vessel and shaken for 1 hour and 30 minutes. After removing the reaction mixture under nitrogen pressure, 350 mL of dichloromethane was added and shaken for 5 minutes, followed by removal of the reaction mixture under nitrogen pressure. The resin was washed five times with dichloromethane, and the resulting resin was dried overnight under reduced pressure to yield (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoate-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1, 59.79 g).

[0210] To confirm the loading rate, the obtained compound 1-2-1 (12.6 mg) was placed in a reaction vessel, DMF (2 mL) was added, and the mixture was shaken at room temperature for 1 hour. Then, DBU (40 μL) was added, and the mixture was shaken at 30 °C for 30 minutes. Then, DMF (8 mL) was added to the reaction mixture, and 1 mL of the resulting solution was diluted with DMF (11.5 mL). The absorbance (294 nm) of the resulting diluted solution was measured (using a Shimadzu UV-1600PC (cell length 1.0 cm)), and the loading amount of compound 1-2-1 was calculated to be 0.464 mmol / g. In addition, other lots synthesized in the same manner but with different loading amounts were also used for peptide synthesis and investigation.

[0211] Example 1-2-2: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip, compound 1-2-2) TIFF0007808554000021.tif35170 (S)-3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate-2-chlorotrityl resin (compound 1-2-2, Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) was synthesized as described in the literature (International Publication No. WO 2013 / 100132 A1). Note that pip refers to piperidine, and in the above structure, the C-terminal carboxylic acid group forms an amide bond with the piperidine.

[0212] Example 1-2-3: Synthesis of (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin, compound 1-2-3) Compound 1-2-3, (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-OH) (7.1 g, 21.82 mmol) purchased from a commercial supplier and 2-chlorotrityl chloride resin (1.6 mmol / g, 100-200 mesh, 1% DVB, 27.25 g, 43.6 mmol) were used to obtain compound 1-2-3, (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin) (33.44 g, loading 0.598 mmol / g) in a similar manner to the synthesis of compound 1-2-1. In addition, a different lot synthesized in the same manner but with a different loading amount was also used for peptide synthesis in this example.

[0213] Example 1-2-4: Synthesis of (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid-2-chlorotrityl resin (Fmoc-MeAla-O-Trt(2-Cl)-resin, compound 1-2-4) Compound 1-2-4, (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid-2-chlorotrityl resin (Fmoc-MeAla-O-Trt(2-Cl)-resin), was obtained in the same manner as in the synthesis of compound 1-2-1, using (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid (Fmoc-MeAla-OH) (1.0 g, 3.07 mmol) purchased from a commercial supplier and 2-chlorotrityl chloride resin (1.25 mmol / g, 100-200 mesh, 1% DVB, 4.92 g, 6.15 mmol). (5.42 g, loading: 0.514 mmol / g)

[0214] Example 1-2-5: Preparation of solid-supported peptide compounds (compounds 1-2-5 to 1-2-12) used in this example Compounds 1-2-5 to 1-2-12 used in this example were prepared by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by Intavis). Detailed procedures were described in the manual attached to the synthesizer.

[0215] Solution 1 was prepared by dissolving the Fmoc-protected amino acids (0.6 mol / L) constituting the target peptide and HOAt, Oxyma, or HOOBt (0.375 mol / L) as a carboxylic acid activator in NMP. If the Fmoc-protected amino acids or HOOBt were poorly soluble, DMSO was added to prepare solution 1 to a concentration of 20%. Solution 2 was prepared by mixing N,N'-diisopropylcarbodiimide (DIC) (10 v / v%) and N,N-dimethylformamide (DMF).

[0216] The 2-chlorotrityl resins (compounds 1-2-1 to 1-2-3) (100 mg per column) bound to the side chain carboxylic acid moiety of aspartic acid whose N-terminus is protected with an Fmoc group or the main chain carboxylic acid moiety of an amino acid whose N-terminus is protected with an Fmoc group, as prepared in Examples 1-2-1 to 1-2-3, were added to a solid-phase reaction vessel and placed in a peptide synthesizer. Dichloromethane (DCM) (1.0 mL) was added to the resin (100 mg) and allowed to stand for approximately 30 minutes to allow the resin to swell. Solutions 1 and 2 were placed in the peptide synthesizer, and automated synthesis using the peptide synthesizer was initiated. The solutions were then discharged through the frit, and the resin was washed twice with DMF (0.7 mL per column).

[0217] Fmoc removal process A DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and the Fmoc group was deprotected at room temperature. The reaction was allowed to proceed for 4.5 minutes for the first deprotection and 10 minutes for the second and subsequent deprotections. The solution was then drained through the frit. The column was then washed twice with DMF (0.7 mL per column), once with a solution of DIPEA (0.14 mol / L) and HOAt (0.14 mol / L) in DMF (0.7 mL per column), and twice with DMF (0.7 mL per column).

[0218] Extension process Solution 1 (0.3 mL per column) and Solution 2 (0.36 mL per column) were mixed in the synthesizer's mixing vial and then added to the resin. The solid-phase reaction vessel was heated from 40°C to 60°C for difficult-to-extend sequences, and the reaction time was 2.5 hours to 14 hours for difficult-to-extend sequences, allowing the condensation reaction of the amino groups on the resin with the Fmoc-protected amino acids. The solution was then drained through the frit. If the elongation efficiency was low, this condensation reaction of the Fmoc-protected amino acids was repeated one or two more times. The resin was then washed three times with DMF (0.7 mL per column).

[0219] This Fmoc deprotection reaction followed by the Fmoc amino acid condensation reaction constituted one cycle, and this cycle was repeated to elongate the peptide on the resin surface. After the last amino acid was elongated, the column was washed four times with DCM (1.0 mL per column) without the Fmoc deprotection step, dried, and then used for further studies. Using this method as the standard condition, the following solid-phase-supported peptide compounds (compounds 1-2-5 to 1-2-12) were prepared. As described above, 100 mg of Compounds 1-2-1 to 1-2-3 were used per column for preparation, but multiple columns were arranged per array and similar preparation was performed.

[0220] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pyrro (SEQ ID NO: 1) (Compound 1-2-5) TIFF0007808554000024.tif32170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.552 mmol / g).

[0221] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pip (SEQ ID NO: 2) (Compound 1-2-6) TIFF0007808554000025.tif37170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0222] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(O-Trt(2-Cl)-resin)-pip(SEQ ID NO: 3) (Compound 1-2-7) TIFF0007808554000026.tif52170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0223] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (SEQ ID NO: 4) (Compound 1-2-8) TIFF0007808554000027.tif48170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0224] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (SEQ ID NO: 5) (Compound 1-2-9) TIFF0007808554000028.tif44170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0225] Fmoc-Ser(nPr)-MeAla-Phe-Asp(O-Trt(2-Cl)-resin)-pip (SEQ ID NO: 6) (Compound 1-2-10) TIFF0007808554000029.tif38170 Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0226] Fmoc-Aib-Pro-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-11) TIFF0007808554000030.tif51170 Prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.519 mmol / g).

[0227] Fmoc-cLeu-MeVal-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-12) TIFF0007808554000031.tif51170 Prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.519 mmol / g).

[0228] Fmoc-Ala-MeAla-O-Trt(2-Cl)-resin (compound 1-2-13) Using a similar method to that used for compounds 1-2-5 to 1-2-12, instead of a peptide synthesizer, 1 g of Fmoc-MeAla-O-Trt(2-Cl)-resin (compound 1-2-4, 0.514 mmol / g) was added to a 20 mL filter column to carry out a solid-phase reaction. During the Fmoc removal process, the resin was washed four times with DMF (7 mL) to prepare compound 1-2-13 (Fmoc-Ala-MeAla-O-Trt(2-Cl)-resin, 0.408 mmol / g).

[0229] As an example, to confirm the production of compound 1-2-5, a portion of the resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS, confirming the production of the target peptide (compound 1-2-5*). The production of target peptides (compounds 1-2-6* to 1-2-12*) was also confirmed for compounds 1-2-6 to 1-2-12 using a method similar to that for compound 1-2-5. In this example, when an * is attached to a compound number, it indicates a compound that was confirmed by cleaving the peptide from the resin to confirm the reaction. Compound 1-2-5* refers to a peptide compound obtained by cleaving the bond between the carboxylic acid of the peptide contained in compound 1-2-5 and the 2-chlorotrityl group of the resin.

[0230] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 9) (Compound 1-2-5*) TIFF0007808554000033.tif32170LCMS (ESI) m / z=928.79(M+H)+ Retention time: 0.88 minutes (Analysis conditions SQDFA05)

[0231] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip (array number: 10) (compound 1-2-6*) TIFF0007808554000034.tif35170LCMS (ESI) m / z=928.84(M+H)+ Hold time: 0.92 minutes (analysis condition SQDFA05)

[0232] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip (Allocation No.: 11) (Compound 1-2-7*) TIFF0007808554000035.tif39170LCMS (ESI) m / z=1003.82(M+H)+ Hold time: 1.11 minutes (Analysis condition SQDFA05)

[0233] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip(Allocation number: 12)(Compound 1-2-8*) TIFF0007808554000036.tif41170LCMS (ESI) m / z=855.83(M+H)+ Hold time: 0.80 minutes (Analysis condition SQDFA05)

[0234] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-pip (array number: 13) (compound 1-2-9*) TIFF0007808554000037.tif45170LCMS (ESI) m / z=888.78(M+H)+ Hold time: 1.10 minutes (Analysis condition SQDFA05)

[0235] Fmoc-Ser(nPr)-MeAla-Phe-Asp-pip (Allocation number: 14) (Compound 1-2-10*) TIFF0007808554000038.tif36170LCMS (ESI) m / z=784.75(M+H)+ Hold time: 0.91 minutes (analysis condition SQDFA05)

[0236] Fmoc-Aib-Pro-D-3-Abu-OH (compound 1-2-11*) TIFF0007808554000039.tif31170LCMS (ESI) m / z=508.60(M+H)+ Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0237] Fmoc-cLeu-MeVal-D-3-Abu-OH (compound 1-2-12*) TIFF0007808554000040.tif33170LCMS (ESI) m / z=326.56(MH)- Detected as MS ((MH-Fmoc)-) of the Fmoc-deprotected fragment Retention time: 0.85 minutes (Analysis conditions SQDFA05)

[0238] Fmoc-Ala-MeAla-OH (compound 1-2-13*) TIFF0007808554000041.tif37170LCMS(ESI)m / z=397(M+H)+ Retention time: 0.69 minutes (Analysis conditions SQDFA05)

[0239] Example 2: Experiments to confirm the effects of reagents and solvents on the Fmoc removal step, resin washing step, and elongation step in solid-phase peptide synthesis In solid-phase peptide synthesis, the Fmoc deprotection and resin washing are followed by amino acid elongation. In this example, compound 1-2-5 was used as the solid-phase-supported peptide sequence, and the reagents and solvents used in each step were varied to compare the degree of reduction in impurities (diketopiperazine elimination products, 6-membered cyclic amidine framework structures, and over-elongation products) generated by peptide synthesis, thereby identifying the range of appropriate conditions.

[0240] Basic operation A (Table 5, run1~24) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-purged glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc deprotection (0.7 mL per column). The Fmoc deprotection solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base listed in Table 5 in the Fmoc deprotection solvent at the volume percent (v / v%) listed in Table 5. After draining the solution through the frit, the resin was washed six times with the washing solvent (0.7 mL per column).

[0241] The resin obtained above was mixed with a solution of Fmoc-MeLeu-OH (0.6 mol / L) and Oxyma (0.375 mol / L) in an elongation solvent (0.3 mL per column) and a solution of DIC (10 v / v%) in an elongation solvent (0.36 mL per column) and shaken at 40 °C for 2.5 h. When DCM was used as the solvent, the column was shaken at room temperature for 2.5 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and then dried.

[0242] For example, run 3 in Table 5 was carried out as follows based on basic operation A. 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5, prepared in Example 1-2-5 from 0.497 mmol / g of Compound 1-2-1) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMF (0.7 mL per column). A DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and reacted at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMF (0.7 mL per column).

[0243] A mixture of 0.3 mL of a DMF solution of Fmoc-MeLeu-OH (0.6 mol / L), 0.375 mol / L of Oxyma (0.375 mol / L), and 0.36 mL of a DMF solution of DIC (10 v / v%) was added to the resin obtained above, and the mixture was shaken at 40°C for 2.5 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0244] To monitor the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A 2% (v / v) DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added and incubated at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a 1 / 1 (v / v) TFE / DCM solution containing DIPEA (0.045 mol / L). LCMS analysis of the cleaved solution confirmed the formation of the target peptide (TM) (2-1*), a diketopiperazine (DKP) elimination product (2-2*), a six-membered cyclic amidine backbone structure (2-3*), and an over-extended MeLeu product (2-4*).

[0245] Target peptide compound (TM), H-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 15) (Compound 2-1*) TIFF0007808554000042.tif51170LCMS (ESI) m / z=833.79(M+H)+ Retention time: 2.64 minutes (Analysis conditions SQDAA05long)

[0246] Diketopiperazine elimination product, H-MeLeu-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 16) (Compound 2-2*) TIFF0007808554000043.tif47170LCMS (ESI) m / z=635.58(M+H)+ Retention time: 2.12 minutes (Analysis conditions SQDAA05long)

[0247] Six-membered cyclic amidine skeleton structure (compound 2-3*) TIFF0007808554000044.tif44170LCMS (ESI) m / z=688.65(M+H)+ Retention time: 2.49 minutes (Analysis conditions SQDAA05long)

[0248] MeLeu-overstretched form, H-MeLeu-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 17) (Compound 2-4*) TIFF0007808554000045.tif45170LCMS (ESI) m / z=960.90(M+H)+ Retention time: 3.04 minutes (Analysis conditions SQDAA05long)

[0249] Basic operation B (Table 5, run25~54) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-purged glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc deprotection (0.7 mL per column). The Fmoc deprotection solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base listed in Table 5 in the Fmoc deprotection solvent at the volume percent (v / v%) listed in Table 5. After draining the solution through the frit, the resin was washed six times with the washing solvent (0.7 mL per column).

[0250] To the resin obtained above, a solution (0.25 mL) of Fmoc-MeLeu-OH (4 equiv.) in an elongation solvent, a solution (0.25 mL) of a uronium or phosphonium condensing agent (4 equiv.) in an elongation solvent, and DIPEA (6 equiv.) were mixed and left to stand for 1-2 minutes, then the mixture was added and shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0251] For example, run 26 in Table 5 was carried out as follows based on basic operation B. 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5, prepared in Example 1-2-5 from 0.552 mmol / g of Compound 1-2-1) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with toluene (0.7 mL per column). A toluene solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and reacted at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with toluene (0.7 mL per column).

[0252] The resin was then mixed with a DMF solution (0.25 mL) of Fmoc-MeLeu-OH (0.221 mmol, 4 equiv.), a DMF solution (0.25 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equiv.), and DIPEA (0.057 mL, 0.331 mmol, 6 equiv.) for 1-2 min, and then shaken at room temperature for 4 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0253] To monitor the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A 2% (v / v) DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a 1 / 1 (v / v) TFE / DCM solution containing 0.045 mol / L DIPEA. The cleaved solution was analyzed by LCMS (SQDAA05long).

[0254] Basic operation C (Table 5, run55) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5, prepared in Example 1-2-5 from 0.552 mmol / g of Compound 1-2-1) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with toluene (0.7 mL per column). A toluene solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with toluene (0.7 mL per column).

[0255] To the resin obtained above, a mixture of Fmoc-MeLeu-Cl (described below) (0.221 mmol, 4 equivalents) in NMP (0.7 mL) and 2,4,6-trimethylpyridine (0.55 mmol, 10 equivalents) was added, and the mixture was shaken at 40°C for 1.5 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried. Fmoc-MeLeu-Cl was prepared as follows. (2S)-2-[9H-Fluoren-9-ylmethoxycarbonyl(methyl)amino]-4-methylpentanoic acid (Fmoc-MeLeu-OH) (80.8 mg, 0.22 mmol) was dissolved in dichloromethane (0.44 mL). DMF (17.0 μL, 0.22 mmol) was added dropwise to the solution while cooling in an ice bath, followed by the addition of thionyl chloride (24.1 μL, 0.33 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. Toluene (0.81 mL, 10 v / w) was added to the resulting residue, and this process of concentration under reduced pressure was repeated twice. The resulting crude product was dissolved in NMP (0.7 mL) and used in the above reaction.

[0256] To monitor the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A 2% (v / v) DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a 1 / 1 (v / v) TFE / DCM solution containing 0.045 mol / L DIPEA. The cleaved solution was analyzed by LCMS (SQDAA05long).

[0257] The formation of diketopiperazine elimination products is a commonly known synthetic problem in peptide synthesis. The formation of six-membered cyclic amidine skeletal structures is unprecedented and represents a new challenge in peptide synthesis discovered by the present inventors. Without being limited to a particular theory, the formation of diketopiperazine elimination products proceeds through nucleophilic attack by the N-terminal amino group on the carbonyl group forming the second amide bond from the N-terminus to form a six-membered ring, followed by elimination of the amino group forming the original amide bond. On the other hand, the formation of six-membered cyclic amidine skeletal structures is thought to similarly proceed through the formation of a six-membered ring followed by elimination of a hydroxyl group derived from the carbonyl group generated by nucleophilic attack. In other words, the formation of diketopiperazine elimination products and six-membered cyclic amidine skeletal structures both share a common peptide synthesis challenge resulting from the formation of a six-membered ring.

[0258] The results obtained (relative ratios of UV areas in LCMS of Compound 2-1*, Compound 2-2*, Compound 2-3*, and Compound 2-4* under each condition, expressed as percentages) are shown in Table 5 below.

[0259] [Table 5] TIFF0007808554000047.tif238170

[0260] Run 1 (comparison experiment) covers the Fmoc removal, resin washing, and elongation steps commonly performed in standard peptide synthesis. Piperidine (pip) (pKa 19.35 in acetonitrile; see Eur. J. Org. Chem. 2019, 6735-6748) was used as the base for Fmoc removal. Under these conditions, the formation of a diketopiperazine (DKP) elimination product (with two missing residues) and the formation of a six-membered cyclic amidine framework were confirmed at approximately 8% each. In contrast, runs 2 to 5 demonstrate that the formation of both the diketopiperazine elimination product and the six-membered cyclic amidine framework can be suppressed by using a stronger base with a conjugate acid pKa of 23 or higher.

[0261] Next, we fixed the base for the Fmoc removal step as DBU and investigated the solvents for the Fmoc removal step, resin washing step, and elongation step. Compared to run 3, we confirmed that the formation of diketopiperazine elimination products and six-membered cyclic amidine framework structures could be suppressed by changing the solvent from DMF to halogenated solvents (DCM), aromatic hydrocarbon solvents (toluene, cumene), ether solvents (THF, DME, 1,3-dioxolane, 2-methyltetrahydrofuran), phosphate ester solvents (tributyl phosphate), ester solvents (methyl propionate, butyl acetate), ketone solvents (diethyl ketone, methyl ethyl ketone), and carbonate solvents (dimethyl carbonate) in runs 6 to 18. This result contrasts with the results in runs 19 to 21, where the use of amide solvents other than DMF (DMA, NMP) or DMI failed to suppress the formation of diketopiperazine elimination products and amidine framework structures. The solvents in which the formation of the diketopiperazine elimination product and the six-membered cyclic amidine skeletal structure was confirmed to be suppressed all had a DN (donor number) value of 26 or less. On the other hand, the amide-based and urea-based solvents, which do not exhibit a formation-suppressing effect, all had DN (donor number) values ​​greater than 26. Without being bound by any particular theory, energy calculations of the diketopiperazine elimination process via a six-membered cyclic intermediate showed that the higher the hydrogen-accepting capacity of the solvent, the greater the stabilization due to coordination. It is therefore presumed that the donor number (DN), which is an indicator of the solvent's ability to donate unshared electron pairs, is an important parameter. Considering the results of the solvent study based on this, it is believed that the suppression of the formation of the diketopiperazine elimination product and the six-membered cyclic amidine skeletal structure in solvents with a DN value of 26 or less is due in part to the suppression of the reaction via the six-membered cyclic intermediate. It is therefore presumed that a wide range of solvents, not just the solvents listed above, can be used in the present invention as long as they have a DN value of 26 or less. Furthermore, as shown in runs 22 to 24, when the solvent in the Fmoc removal step and resin washing step was fixed to toluene and the elongation solvent was changed to ethyl acetate, methyl ethyl ketone, or 2-methyltetrahydrofuran, it was confirmed that the formation of diketopiperazine elimination products and amidine products could be suppressed. Furthermore, from the results of this series of investigations, it was confirmed that the formation of excess elongation products can sometimes be another issue that reduces the purity of the peptide product.

[0262] In addition to the Fmoc removal step and resin washing step, the elongation step was performed under conditions discovered by the inventors of the present invention. The results are shown in runs 25 to 43, 45, 47, 48, and 50. Under these conditions, the production of diketopiperazine elimination products, 6-membered cyclic amidine skeleton structures, and excess elongation products were suppressed, demonstrating that the target peptide could be obtained with high purity.

[0263] As shown in the results of runs 44, 46, and 49, it was confirmed that even when the solvent for Fmoc removal discovered by the present inventors was used in the resin washing step and the elongation step under the conditions discovered by the present inventors, the formation of diketopiperazine elimination products and 6-membered cyclic amidine skeleton structures could not be suppressed when piperidine, which is the most commonly used base for Fmoc removal, was used.

[0264] In the present invention, two or more types of bases may be present at the same time in the Fmoc removal step. For example, in the case of two types of bases being present, as long as one of them is an optimal base (here, DBU) as shown in run 51, the formation of diketopiperazine elimination products and six-membered cyclic amidine skeleton structures can be suppressed even in the presence of piperidine, which is widely known to give adducts to dibenzofulvene generated in the Fmoc removal step.

[0265] Furthermore, in the presence of HP1(dma), P1tBu, and P2Et, which are even stronger bases than DBU, a slight progression of epimerization of the target peptide was observed, but it was confirmed that the production of diketopiperazine elimination products and six-membered cyclic amidine structures could be suppressed (runs 52 to 54).

[0266] In the present invention, an acid chloride may be used in the elongation step. For example, as in run 55, the solvent used in the Fmoc removal discovered by the present inventors was used, and after a resin washing step, a separately prepared acid chloride was reacted in NMP in the presence of 2,4,6-trimethylpyridine, and it was confirmed that the formation of diketopiperazine elimination products and 6-membered cyclic amidine skeleton structures could be suppressed.

[0267] As described above, the outline of the scope of each step (Fmoc removal step, resin washing step, and elongation step) capable of suppressing the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures was identified from the results of Example 2. Note that the suppression of the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures is not limited to the combination of reaction conditions shown in Example 2.

[0268] Example 3: Experiment on solid-phase peptide synthesis in which a base neutralization step was added to the Fmoc removal step In the literature, there are examples in which the base used in the Fmoc removal reaction is neutralized during the Fmoc removal step or the subsequent resin washing step. For example, in solid-phase peptide synthesis, a rapid Fmoc removal reaction using DBU / DMF followed by neutralization with HOBt is known to suppress diketopiperazine elimination (Org. Lett., 2008, 10, 3857-3860). In addition, in peptide synthesis using an anchor group, a case has been reported in which Fmoc removal using DBU is followed by neutralization using hydrochloric acid or the like in order to perform Fmoc removal and subsequent elongation in one pot (WO 2012165546 A1). Experiments were carried out to confirm the effect of these neutralization steps on the production of diketopiperazine elimination products.

[0269] Basic operations (Table 6) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5 prepared in Example 1-2-5 from 0.552 mmol / g of Compound 1-2-1) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the vessel was left standing for 10 minutes to allow the resin to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc deprotection listed in Table 6 (0.7 mL per column). The Fmoc deprotection solution (0.7 mL per column) was added, and the vessel was left standing for 10 minutes at room temperature to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base listed in Table 6 in the Fmoc deprotection solvent at the volume percent (v / v%) listed in Table 6. Next, as a neutralization step, 0.4 mL of a DMF solution of HOAt (0.25 mol / L or 0.5 mol / L) (equivalent to approximately 2 or 4 equivalents of the peptide supported on the solid phase) was added and the column was left to stand for an additional 5 minutes. As a control experiment, a control experiment was also performed in which the Fmoc removal step was performed for 10 minutes without the neutralization step, followed by the addition of DMF (0.4 mL) and the column was left to stand for an additional 5 minutes. After the solution was drained from the frit, the resin was washed six times with the washing solvent (0.7 mL per column) listed in Table 6.

[0270] The resin was then added to a solution of Fmoc-MeLeu-OH (0.221 mmol, 4 equiv.) in DCM (0.3 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equiv.) in DCM (0.3 mL), and DIPEA (0.057 mL, 0.331 mmol, 6 equiv.) for 1–2 min, and then shaken at room temperature for 2 or 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0271] For example, run 2 in Table 6 was carried out as follows based on the basic operations. 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5, prepared in Example 1-2-5 from Compound 1-2-1 at 0.552 mmol / g) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit. A solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in DCM (2% v / v, 0.7 mL per column) was then added to the resin and allowed to stand at room temperature for 10 minutes to remove Fmoc. Subsequently, 0.4 mL of a solution of 0.5 mol / L HOAt in DMF (approximately 4 equivalents relative to the peptide loaded on the solid phase) was added and allowed to stand for an additional 5 minutes as a neutralization step. After draining the solution through the frit, the resin was washed six times with DCM (0.7 mL per column).

[0272] The resin was then mixed with a solution of Fmoc-MeLeu-OH (0.221 mmol, 4 equiv.) in DCM (0.3 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equiv.) in DCM (0.3 mL), and DIPEA (0.057 mL, 0.331 mmol, 6 equiv.) for 1-2 min, and then shaken at room temperature for 4 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0273] To confirm the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A DMF solution (2% v / v) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDAA05long) with the results shown in Table 6.

[0274] [Table 6]

[0275] As described above, the results of Example 3 confirmed that when the neutralization step is performed, the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures increases compared to when the neutralization step is not performed, which causes a decrease in the purity of the target peptide.

[0276] Example 4: Experiment to confirm the generality of the peptide sequence of the effect of reducing diketopiperazine elimination during peptide synthesis under the discovered conditions In this example, for various peptide sequences prepared in Example 1-2-5 (peptide sequences extended to Core 2 in Table 7, Compounds 1-2-6 to 1-2-12), the amino acids listed in Core 1 were extended by a general conventional method (Condition-1) and the method of the present invention (Condition-2), and the amounts of diketopiperazine elimination products produced were compared.

[0277] Condition 1 (conventional method) 100 mg of the peptide-loaded solid-phase resin (Compounds 1-2-6 to 1-2-12 (corresponding to runs 1 to 7 in Table 7, respectively)) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMF (0.7 mL per column). A piperidine DMF solution (20% v / v, 0.7 mL per column) was added, and the reaction was continued at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed eight times with DMF (0.7 mL per column).

[0278] To the resin obtained above, a mixture of a DMF solution (0.3 mL) of Fmoc-protected amino acid (0.6 mol / L), HOAt (0.375 mol / L), and a DMF solution (0.36 mL) of DIC (10 v / v%) was added, and the mixture was shaken for 4 hours at 40°C. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0279] Condition 2 (method of the present invention) 100 mg of the peptide-loaded solid-phase resin (Compounds 1-2-6 to 1-2-12, corresponding to runs 1 to 7 in Table 7, respectively) prepared in Example 1-2-5 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with toluene (0.7 mL per column). A toluene solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed twice with toluene (0.7 mL per column) and then twice with DCM (0.7 mL per column).

[0280] To the resin obtained above, a solution of 0.25 mL of Fmoc-protected amino acid (4 equiv.) in DCM, 0.25 mL of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (4 equiv.) in DCM, and 6 equiv. of DIPEA was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0281] To confirm the progress of the reaction, a portion of each resin was taken, swollen in DCM, and then the peptides were cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long), and the formation of the target peptides (TM) (compounds 4-1-1 to 4-1-7) and diketopiperazine elimination products (compounds 4-2-1 to 4-2-7) was confirmed.

[0282] The amount of diketopiperazine (DKP) elimination product produced for each sequence and each condition is shown in Table 7. The amount of diketopiperazine (DKP) elimination product shown in Table 7 is the UV area percentage of the diketopiperazine (DKP) elimination product when the total UV area percentage of the target peptide (TM) and the diketopiperazine (DKP) elimination product in LCMS is taken as 100%.

[0283] Target peptide compound (TM), Fmoc-MeLeu-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip (SEQ ID NO: 18) (run 1) (compound 4-1-1*) TIFF0007808554000049.tif37170LCMS (ESI) m / z=1056.10(M+H)+ Retention time: 3.11 minutes (Analysis conditions SQDFA05long)

[0284] Diketopiperazine elimination product, Fmoc-MeLeu-Aze(2)-MeCha-MeGly-Asp-pip (SEQ ID NO: 19) (run 1) (Compound 4-2-1*) TIFF0007808554000050.tif40170LCMS (ESI) m / z=871.94(M+H)+ Retention time: 2.99 minutes (Analysis conditions SQDFA05long)

[0285] Target peptide compound (TM), Fmoc-Thr(tBu)-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip (SEQ ID NO: 20) (run 2) (compound 4-1-2*) TIFF0007808554000051.tif45170LCMS (ESI) m / z=1160.90(M+H)+ Retention time: 3.81 minutes (Analysis conditions SQDFA05long)

[0286] Diketopiperazine eliminator, Fmoc-Thr(tBu)-Cys(StBu)-Phe-Asp-pip (SEQ ID NO: 21) (run 2) (Compound 4-2-2*) TIFF0007808554000052.tif51170LCMS (ESI) m / z=918.77(M+H)+ Retention time: 3.47 minutes (Analysis conditions SQDFA05long)

[0287] Target peptide compound (TM), Fmoc-Nle-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip (SEQ ID NO: 22) (run 3) (compound 4-1-3*) TIFF0007808554000053.tif50170LCMS (ESI) m / z=968.94(M+H)+ Retention time: 2.49 minutes (Analysis conditions SQDFA05long)

[0288] Diketopiperazine eliminator, Fmoc-Nle-Ser(iPen)-Asp-pip(run3) (compound 4-2-3*) TIFF0007808554000054.tif47170LCMS (ESI) m / z=693.72(M+H)+ Retention time: 2.88 minutes (Analysis conditions SQDFA05long)

[0289] Target peptide compound (TM), Fmoc-Nle-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-pip (SEQ ID NO: 23) (run 4) (compound 4-1-4*) TIFF0007808554000055.tif49170LCMS (ESI) m / z=1001.86(M+H)+ Retention time: 3.55 minutes (Analysis conditions SQDFA05long)

[0290] Separate body, Fmoc-Nle-Ser(iPen)-Asp-pip(run4)(compound 4-2-4*) TIFF0007808554000056.tif43170LCMS (ESI) m / z=693.72(M+H)+ Duration: 2.88 minutes (Analysis condition: SQDFA05long)

[0291] Target ペプチド compound (TM), Fmoc-Pro-Ser(nPr)-MeAla-Phe-Asp-pip (array number: 24) (run5) (compound 4-1-5*) TIFF0007808554000057.tif40170LCMS (ESI) m / z=881.78(M+H)+ Duration: 2.61 minutes (Analysis condition: SQDFA05long)

[0292] ジケトピペラジン detachment, Fmoc-Pro-Phe-Asp-pip (run5) (compound 4-2-5*) TIFF0007808554000058.tif36170LCMS (ESI) m / z=667.64(M+H)+ Hold time: 2.44 minutes (Analysis condition: SQDFA05long)

[0293] Target compound (TM), Fmoc-MeIle-Aib-Pro-D-3-Abu-OH (array number: 25) (run6) (compound 4-1-6*) TIFF0007808554000059.tif34170LCMS (ESI) m / z=635.71(M+H)+ Hold time: 2.35 minutes (Analysis condition: SQDFA05long)

[0294] Separate body of ジケトピペラジン, Fmoc-MeIle-D-3-Abu-OH (run6) (compound 4-2-6*) TIFF0007808554000060.tif33170LCMS (ESI) m / z=453.55(M+H)+ Retention time: 2.46 minutes (Analysis conditions SQDFA05long)

[0295] Target peptide compound (TM), Fmoc-Pro-cLeu-MeVal-D-3-Abu-OH (SEQ ID NO: 26) (run7) (compound 4-1-7*) TIFF0007808554000061.tif35170LCMS (ESI) m / z=645.79(MH)- Retention time: 2.33 minutes (Analysis conditions SQDFA05long)

[0296] Diketopiperazine eliminator, Fmoc-Pro-D-3-Abu-OH (run 7) (compound 4-2-7*) TIFF0007808554000062.tif33170LCMS (ESI) m / z=423.53(M+H)+ Retention time: 1.87 minutes (Analysis conditions SQDFA05long)

[0297] [Table 7]

[0298] As described above, the results of Example 4 confirmed that, in the synthesis of various peptides, not limited to specific peptide sequences, the method of the present invention is more effective in suppressing the production of diketopiperazine elimination products than general conventional methods when elongation is performed using the method of the present invention.

[0299] Example 5: Experiment to confirm the generality of carboxylic acid substrates that improves the diketopiperazine elimination reduction effect during peptide synthesis under the discovered conditions In this example, the peptide sequence (compound 1-2-5) prepared in Example 1-2-5 was elongated with various carboxylic acids described in Core 1 using the method of the present invention (condition-3), and the amounts of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures produced were confirmed.

[0300] Condition 3 (method of the present invention) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5, prepared in Example 1-2-5 from 0.552 mmol / g of Compound 1-2-1) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with toluene (0.7 mL per column). A toluene solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and reacted at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with toluene (0.7 mL per column).

[0301] The resin obtained above was mixed with a DMF solution (0.25 mL) of the elongating carboxylic acid (0.221 mmol, 4 equiv.) listed in Table 8, a DMF solution (0.25 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equiv.) and DIPEA (0.057 mL, 0.331 mmol, 6 equiv.) for 1-2 minutes, and then shaken at room temperature for 2 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and then dried.

[0302] To confirm the progress of the reaction, a portion of each resin was taken, swollen in DCM, and then the peptides were cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long or SQDAA05long), and the formation of the target peptides (TM) (compounds 5-1-1 to 5-1-6), diketopiperazine elimination products (compounds 5-2-1 to 5-2-6), and the six-membered cyclic amidine framework (compound 2-3) was confirmed. The amounts of diketopiperazine elimination product (TM-DKP) and six-membered cyclic amidine skeletal structure produced for each sequence are shown in Table 8. The amounts of diketopiperazine elimination product (TM-DKP) and six-membered cyclic amidine skeletal structure produced shown in Table 8 are the UV area percentages of the diketopiperazine elimination product (TM-DKP) and six-membered cyclic amidine skeletal structure when the total UV area percentages by LCMS of the target peptide (TM), diketopiperazine elimination product (TM-DKP), and six-membered cyclic amidine skeletal structure are taken as 100%.

[0303] Target peptide compound (TM), Teoc-Phe(4-CF3)-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 34) (run 1) (compound 5-1-1*) TIFF0007808554000064.tif42170LCMS (ESI) m / z=1065.7(M+H)+ Retention time: 2.90 minutes (Analysis conditions SQDFA05long)

[0304] Diketopiperazine elimination product, Teoc-Phe(4-CF3)-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 35) (run 1) (Compound 5-2-1*) TIFF0007808554000065.tif44170LCMS (ESI) m / z=867.5(M+H)+ Retention time: 2.85 minutes (Analysis conditions SQDFA05long)

[0305] Target peptide compound (TM), Boc-MeAla-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 36) (run 2) (compound 5-1-2*) TIFF0007808554000066.tif36170LCMS (ESI) m / z=891.7(M+H)+ Retention time: 3.07 minutes (Analysis conditions SQDAA05long)

[0306] Diketopiperazine elimination product, Boc-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 37) (run 2) (Compound 5-2-2*) TIFF0007808554000067.tif40170LCMS (ESI) m / z=691.6(MH)- Retention time: 2.77 minutes (Analysis conditions SQDAA05long)

[0307] Target peptide compound (TM), Alloc-Phe-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 38) (run 3) (compound 5-1-3*) TIFF0007808554000068.tif36170LCMS (ESI) m / z=937.6(M+H)+ Retention time: 3.02 minutes (Analysis conditions SQDAA05long)

[0308] Diketopiperazine elimination product, Alloc-Phe-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 39) (run 3) (Compound 5-2-3*) TIFF0007808554000069.tif39170LCMS (ESI) m / z=739.5(M+H)+ Retention time: 2.90 minutes (Analysis conditions SQDAA05long)

[0309] Target peptide compound (TM), Ac-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 40) (run 4) (compound 5-1-4*) The notation Ac means an acetyl group formed by extending acetic acid. TIFF0007808554000070.tif40170LCMS (ESI) m / z=748.6(M+H)+ Retention time: 1.53 minutes (Analysis conditions SQDFA05long)

[0310] Diketopiperazine elimination product, Ac-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 41) (run 4) (Compound 5-2-4*) TIFF0007808554000071.tif39170LCMS (ESI) m / z=548.5(MH)- Retention time: 1.22 minutes (Analysis conditions SQDFA05long)

[0311] Target peptide compound (TM), Tfa-Aib-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 42) (run 5) (compound 5-1-5*) TIFF0007808554000072.tif36170LCMS (ESI) m / z=887.6(M+H)+ Retention time: 2.75 minutes (Analysis conditions SQDAA05long)

[0312] Diketopiperazine elimination product, Tfa-Aib-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 43) (run 5) (Compound 5-2-5*) TIFF0007808554000073.tif39170LCMS (ESI) m / z=687.6(MH)- Retention time: 2.42 minutes (Analysis conditions SQDAA05long)

[0313] Target peptide compound (TM), Ns-Aib-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 44) (run 6) (compound 5-1-6*) TIFF0007808554000074.tif36170LCMS (ESI) m / z=976.6(M+H)+ Retention time: 2.08 minutes (Analysis conditions SQDFA05long)

[0314] Diketopiperazine elimination product, Ns-Aib-Aze(2)-MeCha-MeGly-Asp-pyrro (SEQ ID NO: 45) (run 6) (Compound 5-2-6*) TIFF0007808554000075.tif39170LCMS (ESI) m / z=776.5(MH)- Retention time: 1.85 minutes (Analysis conditions SQDFA05long)

[0315] [Table 8]

[0316] As described above, the results of Example 5 confirmed that, in the elongation of various carboxylic acids, not limited to specific Fmoc amino acids, the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures can be suppressed by performing the elongation using the method of the present invention, thereby enabling the synthesis of target peptides.

[0317] Example 6: Confirmation of the effect of the discovered conditions on the formation of diketopiperazines from dipeptides supported on a solid phase via ester bonds Diketopiperazine elimination generally becomes a problem when the ester bond of a solid-phase-supported dipeptide is cleaved to form a diketopiperazine. In this example, a dipeptide supported on the solid phase via an ester bond as prepared in Example 1-2-4 was subjected to Fmoc-MeLeu-OH elongation using a general conventional method (Condition-4) and the method of the present invention (Condition-5), and a confirmation experiment was conducted to determine the effect of the method of the present invention on diketopiperazine formation.

[0318] Condition-4 (conventional method) (run1) 100 mg of the dipeptide-loaded solid-phase resin (Compound 1-2-13) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMF (0.7 mL per column). A piperidine DMF solution (20% v / v, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMF (0.7 mL per column).

[0319] A mixture of 0.3 mL of a DMF solution of Fmoc-MeLeu-OH (0.6 mol / L), 0.375 mol / L of Oxyma (0.375 mol / L), and 0.36 mL of a DMF solution of DIC (10 v / v%) was added to the resin obtained above, and the mixture was shaken at 40°C for 2.5 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0320] Condition-5 (method of the present invention) (run 2 and run 3) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-13) previously prepared in Example 1-2-5 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-purged glove box and allowed to stand for 10 minutes to allow the resin to swell. The solution was then drained through the frit, and the resin was washed twice with toluene (0.7 mL per column). A toluene solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with toluene (0.7 mL per column).

[0321] The resin was then added to a solution of Fmoc-MeLeu-OH (0.163 mmol, 4 equiv.) in DMF or DCM (0.25 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.163 mmol, 4 equiv.) in DMF or DCM (0.25 mL), and DIPEA (0.040 mL, 0.245 mmol, 6 equiv.) for 1–2 min, and then shaken at room temperature for 2 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0322] To confirm the progress of the reaction, a portion of each resin was taken, swollen in DCM, and then the peptide was cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long), and the production of the target peptide (TM) (compound 6-1*) was confirmed.

[0323] Target peptide compound (TM), Fmoc-MeLeu-Ala-MeAla-OH (compound 6-1*) TIFF0007808554000077.tif35170LCMS (ESI) m / z=524.5(M+H)+ Retention time: 2.48 minutes (Analysis conditions SQDFA05long)

[0324] Fmoc quantitative method To confirm the loading amount, the resulting resin (run 1, 10.64 mg) was placed in a reaction vessel, DMF (4.0 mL) was added, and the mixture was shaken at room temperature for 30 minutes. Then, DBU (40 μL) was added and the mixture was shaken at 30°C for 15 minutes. DMF was then added to the reaction mixture to make a 10.0 mL solution, and 80 μL of the resulting solution was diluted with DMF (920 μL). The resulting diluted solution was analyzed by LCMS (injection volume: 5 μL). Based on the UV area values ​​of dibenzofulvene (294 nm: 88.61, 304 nm: 72.01), the loading amount of the resulting resin was calculated to be 0.021 mmol / g. (A calibration curve was prepared based on the UV area values ​​of dibenzofulvene at wavelengths of 294 nm and 304 nm on each measurement day, using a mixed solution of Fmoc-Gly-OH (purchased from a commercial supplier) and DBU of known concentrations as the standard substance, and the average of the loading amounts calculated at each wavelength was used as the loading amount on the resin.)

[0325] In run 2, the loading amount was calculated using the same Fmoc quantification method and was found to be 0.321 mmol / g (UVarea value at 294 nm: 1432.15, UVarea value at 304 nm: 1217.70).

[0326] In run 3, the loading amount was calculated using the same Fmoc quantification method and was found to be 0.306 mmol / g (UVarea value at 294 nm: 1364.67, UVarea value at 304 nm: 1162.58).

[0327] Recovery rate calculation method In Example 6, the recovery rate was defined as follows, and the rate of inhibition of diketopiperazine elimination from the resin during the solid-phase reaction was evaluated. Recovery rate = reaction product loading amount (mmol / g) ÷ loading amount (mmol / g) of resin when 100% of the target product is produced (Equation 1)

[0328] The amount of resin supported (mmol / g) when 100% of the target product is produced is calculated as follows: Resin loading amount (mmol / g) when 100% of the target substance is produced = Loading amount of raw resin (mmol / g) × Weight of raw resin (g) ÷ Weight of resin when 100% of the target substance is produced (g) (Equation 2)

[0329] The weight (g) of resin when 100% of the desired product is produced is calculated as follows: Weight of resin (g) when 100% of the target substance is produced = Weight of raw resin (g) - Weight of peptide component on raw resin (g) + Weight of peptide component on resin when 100% of the target substance is produced (g) (Equation 3)

[0330] The weight (g) of the peptide component on the raw resin is calculated as follows: Weight of peptide component on raw resin (g) = Weight of raw resin (g) × Amount supported on raw resin (mmol / g) × Molecular weight of peptide component on raw resin (g / mol) × 0.001 (mol / mmol) (Equation 4)

[0331] When 100% of the target product is produced, the weight (g) of the peptide component on the resin is calculated as follows: Weight of peptide component on resin when 100% of target product is produced (g) = Weight of raw resin (g) × Amount supported on raw resin (mmol / g) × Molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol) (Equation 5) Substituting equations 3, 4, and 5 into equation 2, we get Resin loading amount (mmol / g) when 100% of the target substance is produced = loading amount of raw resin (mmol / g) ÷ (1 - loading amount of raw resin (mmol / g) × molecular weight of peptide component on raw resin (g / mol) × 0.001 (mol / mmol) + loading amount of raw resin (mmol / g) × molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol)) = 1 ÷ (1 ÷ loading amount of raw resin (mmol / g) - molecular weight of peptide component on raw resin (g / mol) × 0.001 (mol / mmol) + molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol)) (Equation 6)

[0332] Substituting Equation 6 into Equation 1, the recovery rate is calculated using the following formula: Recovery rate = reaction product loading amount (mmol / g) × (1 ÷ loading amount of raw resin (mmol / g) - molecular weight of peptide component on raw resin (g / mol) × 0.001 (mol / mmol) + molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol))

[0333] Elongation was performed using the conventional method (condition-4) and the method of the present invention (condition-5), and the amount of loading calculated by the above-mentioned Fmoc quantification method and the recovery rate calculated by the recovery rate calculation method for each obtained resin are shown in Table 9 below. [Table 9]

[0334] From the results of Example 6, it was confirmed that the formation of diketopiperazine can be suppressed by elongating a dipeptide supported on a solid phase via an ester bond using the method of the present invention, and the target peptide can be synthesized in high yield.

[0335] Example 7: Confirmation of the presence of carbamate in the Fmoc removal step and confirmation of its effect on diketopiperazine elimination The compounds described in the examples are shown in the table below. [Table 10]

[0336] The following abbreviations are used in the examples: NMR: nuclear magnetic resonance spectroscopy ROESY: Rotating Frame Overhauser Effect Spectroscopy NOESY: Nuclear Overhauser Effect Spectroscopy HSQC: Heterogeneous nuclear single quantum correlation HMBC: Heteronuclear multibond correlation cpd.: Compound

[0337] NMR measurements were carried out at 298 K using a Bruker AVANCE III 600 Cryo-TCI or AVANCE III HD 600 BBFO.

[0338] When a base containing multiple nitrogen atoms is protonated to form a cation and forms a salt with an anion in the system, any of the protonated nitrogen atoms can serve as the cation source. For example, in the case of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), the following three are possible cation sources. In this specification, we refer to any cation and its salt with the corresponding anion as a DBU salt (Figure 1). Other bases, such as 1,1,3,3-tetramethylguanidine (TMG) and iminotris(dimethylamino)phosphouran (HP1(dma)), are similarly referred to as TMG salts or HP1(dma) salts. TIFF0007808554000080.tif18170

[0339] Compound 1-3-1, 9H-fluoren-9-ylmethyl N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl]carbamate (Fmoc-Phe-NMe 2 ) synthesis To a solution of commercially available N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-Phe-OH, CAS: 35661-40-6, compound aa2-12) (2.0 g, 5.16 mmol) in a mixture of N,N-dimethylformamide (DMF) (14 mL) and dichloromethane (DCM) (4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (1.19 g, 6.19 mmol) and 1-hydroxybenzotriazole (HOBt) (0.767 g, 5.68 mmol) at room temperature. Then, a solution of dimethylamine in tetrahydrofuran (THF) (2.0 M, 2.84 mL, 5.68 mmol) was added under ice cooling and the mixture was stirred for 15 minutes. Isopropyl acetate (20 mL) was added to the solution, and the organic phase was washed twice with 1 M hydrochloric acid (20 mL), once with water (50 mL), twice with half-saturated aqueous sodium bicarbonate (20 mL), and twice with half-saturated aqueous sodium chloride (20 mL). The resulting organic phase was dried over sodium sulfate, filtered, and the solvent in the resulting mother liquor was evaporated under reduced pressure. The resulting crude product was azeotroped twice with toluene and then allowed to cool at 5 °C, yielding compound 1-3-1 (1.88 g, 88%) as white crystals. TIFF0007808554000081.tif35170 1 H-NMR(600MHz,toluene-d8,298K)δ7.51(d,2H,J=7.2Hz),7.43(d,2H,J=7.3Hz),7.21-7.16(m,2 H),7.14-7.07(m,2H),7.05-7.00(m,4H),7.00-6.95(m,1H),6.00(d,1H,J=8.4Hz,NH),4.88(ddd ,1H,J=8.4,8.4,6.0Hz),4.32(dd,1H,J=10.5,7.4Hz),4.23(dd,1H,J=10.5,7.4Hz),4.03(t,1H, J=7.4Hz), 2.93(dd,1H,J=13.0,8.4Hz),2.86(dd,1H,J=13.0,6.0Hz),2.49(s,3H),2.02(s,3H). 13C-NMR(151MHz,toluene-d8,298K)δ171.1,155.9,144.6,141.8,129.9,128.5,127.8(overlapped with solvent peak,detected by CH-HSQC ),127.3,127.0,125.6,125.4,120.2,67.2,52.3,47.7,40.2,36.0,35.0. LCMS (ESI) m / z=415.44(M+H)+ Retention time: 0.89 minutes (Analysis conditions SQDFA05)

[0340] Example 7-1: DBU·HOCO-Phe-NMe when compound 1-3-1 is treated with DBU 2 Checking for the existence of Example 7-1-1: Product obtained by treating compound 1-3-1 with DBU in N,N-dimethylformamide-d7 (DMF-d7) solution Compound 1-3-1 (12.9 mg, 0.031 mmol) in N,N-dimethylformamide-d7 (DMF-d7) solution (0.5 mL) 1 After measuring H-NMR (Figure 1-b), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (5.11 μL, 0.034 mmol) was added to the solution and stirred vigorously. 1 H-NMR (Figure 1-a) and 1D-ROESY (Figures 2-a-2 and 2-a-3) were measured. The NMR spectra confirmed that three compounds were produced. Specifically, dibenzofulvene, compound 1-3-2 (H-Phe-NMe2), and N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl]carbamate DBU salt (compound 1-3-3a, DBU·HOCO-Phe-NMe2) were present in the solution, confirming that chemical exchange had occurred between compound 1-3-2 and compound 1-3-3a. The identification of compound 1-3-2 (H-Phe-NMe2) in the solution was described in detail in Example 7-1-1, and the identification of compound 1-3-3a was described in detail in Example 7-1-3.

[0341] In DMF-d7, compound 1-3-1 was reacted with DBU to produce compound 1-3-2 (H-Phe-NMe 2 ) and compound 1-3-3a undergo chemical exchange and their ratios The 1D-ROESY of the solution was measured (4.84 ppm and 3.95 ppm). 1 The H signal was selectively excited, as shown in Figure 2-a)-2 and Figure 2-a)-3. 1 H signal at 3.95 ppm, which corresponds to the α-methine proton of compound 1-3-2 1 It was confirmed that the H signal was undergoing chemical exchange in the system (Figure 2-a-2, Figure 2-a-3). 1 When the H signal was selectively excited, the peak was 3.95 ppm. 1 From the H signal (Figure 2-a)-2), 3.95 ppm 1 When the H signal was selectively excited, the peak was 4.85 ppm. 1 From the H signal (Figure 2-a)-3), ROESY was observed in phase with the selectively excited signal, confirming that the respective signals were chemically exchanged (Figure 2-a). 1 H-NMR 1 The integral ratio of the H signal confirmed that the abundance ratio of compound 1-3-3a to compound 1-3-2 was 82:18 (Figure 2-b).

[0342] Compound 1-3-2 (H-Phe-NMe 2 ) existence check 3 μL of a separately prepared sample of compound 1-3-2 was mixed with the solution. 1 H-NMR (Fig. 3-a)-3, Fig. 3-b)) and 1D-NOSEY (Fig. 3-a)-4, Fig. 3-a)-5) were measured. 1) Add 3 μL of compound 1-3-2 to the solution and 1 As a result of measuring H-NMR, 1 3.95 ppm observed by H-NMR 1 H signal (Figure 3-a)-2) and the signal at 3.94 ppm observed in the standard sample of compound 1-3-2. 1 The triplet H signal (Figure 3-a) was observed at 3.95 ppm without separation. 1 The H signal was observed in perfect agreement (Figure 3-a)-3). 2) 1D-NOESY was measured for the solution obtained by adding 3 μL of the compound 1-3-2 standard to the solution (4.84 ppm and 3.95 ppm). 1 H signal (selective excitation), 4.84 ppm 1 When the H signal was selectively excited, the peak was 3.95 ppm. 1 From the H signal (Figure 3-a)-4), 3.95 ppm 1 When the H signal was selectively excited, the peak was 4.85 ppm. 1 From the H signal (Figure 3-a)-5), NOESY (4.85 ppm: double triplet, 3.95 ppm: triplet) was observed, which was in phase with the selectively excited signal and had the same shape as the original signal. 3) When 3 μL of the compound 1-3-2 preparation was added to the solution, 4.84 ppm of compound 1-3-3a was detected. 1 H signal and 3.95 ppm of compound 1-3-2 1 The integral ratio of the H signals changed from 82:18 (integral ratio before the addition of compound 1-3-2 [Figure 2-b]) to 61:39 (Figure 3-b). The presence of compound 1-3-2 in the solution was confirmed from the above three points 1) to 3).

[0343] Example 7-1-2: Compound 1-3-2 13 CO 2 Identification of compound 1-3-3a produced when sprayed with DBU All of the following experimental procedures in Example 7-1-2 were carried out in a glove box (under nitrogen atmosphere and dehydrated conditions). N,N-dimethylformamide-d7 (DMF-d7) was used as a solvent, which had been dried for 12 hours or more with MS4A that had been activated by heating to a high temperature using microwaves and then cooling under vacuum (x2). The samples were then sampled in airtight NMR tubes with valves in a glove box, and NMR measurements were performed on samples that were completely isolated from the outside air.

[0344] Preparation of Solution A (SolA) To a solution of compound 1-3-2 (4.64 mg, 0.024 mmol) in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13After bubbling CO2 for 1 minute, 1 H-NMR (Figure 4-b) 13 C-NMR (Figure 5-a), 1D-NOESY (4.77 ppm and 3.96 ppm) 1 The H signals were selectively excited and measured (Figure 4-c, Figure 4-d). Then, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (3.97 μL, 0.027 mmol) was added to prepare solution A containing compound 1-3-3a (DBU·HOCO-Phe-NMe2). 1 H-NMR (Figure 4-e) 13 C-NMR (Figure 5-b) was measured.

[0345] Identification of Compound 1-3-3a in Solution A The structure of compound 1-3-3a is: 1 H-NMR, 13 This can be explained without contradiction by the results of C-NMR and 1D-NOESY measurements. 4) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13 CO2 was bubbled through the solution for 1 minute. 1 As a result of H-NMR measurement, the raw material compound 1-3-2 1 A new peak at 4.76 ppm was not observed in H-NMR (Figure 4-a). 1 H signal was confirmed (Figure 4-b) (the formation of carbamate anion or carbamic acid, see Figure 5-a)). 5) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13 CO2 was bubbled through the solution for 1 minute. 1 4.76 ppm observed by H-NMR 1 H signal and 3.96 ppm corresponding to the α-methylene of compound 1-3-2 1 The integrated value of the H signal was measured and the ratio was confirmed to be 52:48 (Figure 4-f). 6) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 131D-NOESY of the solution after bubbling CO2 for 1 minute revealed a concentration of 4.76 ppm. 1 3.96 ppm when the H signal is selectively excited 1 From the H signal (Figure 4-c), and also from 3.96 ppm 1 When the H signal was selectively excited, the peak was 4.76 ppm. 1 The in-phase NOESY was observed from the H signal (Figure 4-d), confirming that chemical exchange between the two compounds was occurring. 7) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13 After bubbling CO2 for 1 minute, DBU was added to the solution. 1 As a result of H-NMR measurement, the peak of 4.76 ppm observed before adding DBU was 1 When DBU is added, the H signal , Shi It was confirmed that the concentration of the compound 1-3-3a was reduced to 4.83 ppm (Figure 4-e) (confirmation of the production of compound 1-3-3a). 8) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13 After bubbling CO2 for 1 minute, DBU was added to the solution, and the 1H-NMR measurement revealed a peak of 4.83 ppm for α-methine. 1 H signal and 13 C-labeled carbonyl at 161.5 ppm 13 C signal (Figure 5-b)) 3 J CH The coupling was confirmed (Figure 4-g).

[0346] 9) To a solution of compound 1-3-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), 13 CO2 was bubbled for 1 minute. 13 C-NMR was measured (Figure 5-a), and then DBU was added to the resulting solution. 13 As a result of comparing C-NMR measurements (Figure 5-b), 13 C corresponds to the labeled carbonyl 13C signal increased from 157.1 ppm to 161.5 ppm with the addition of DBU Heshi It was confirmed that the compound 1-3-3a was produced (confirmation of production of compound 1-3-3a).

[0347] By preparing Solution A (SolA) from the six points 4) to 9), the primary amine of compound 1-3-2 and 13 It was confirmed that CO2 reacted to form carbamate or carbamic acid, and then 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added to produce compound 1-3-3a (DBU·HOCO-Phe-NMe2). The above experiment does not contradict the formation of compound 1-3-3a (DBU·HOCO-Phe-NMe2). This structure was identified in more detail in Example 7-1-3.

[0348] Example 7-1-3: Proof of structure of compound 1-3-3a present in solution A + solution B (SolA + SolB) The following experimental procedures described in Example 7-1-3 were all carried out in a glove box (under nitrogen atmosphere and dehydrated conditions). N,N-dimethylformamide-d7 (DMF-d7) was used as a solvent, which had been dried for 12 hours or more with MS4A that had been activated by heating to a high temperature using microwaves and then cooling under vacuum (x2). The samples were then sampled in airtight NMR tubes with valves in a glove box, and NMR measurements were performed on samples that were completely isolated from the outside air.

[0349] Preparation of Solution B (SolB) Solution B was prepared by adding 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (4.56 μL, 0.031 mmol) to a solution (0.5 mL) of compound 1-3-1 (11.5 mg, 0.028 mmol) in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR was measured (SolB, Figure 6-a).

[0350] Preparation of Solution A + Solution B (SolA + SolB) The above solution B (SolB) (0.15 mL, Figure 6-a), 0.0084 mmol) and solution A (SolA) (0.35 mL, SolA, Figure 6-c), 0.0168 mmol) were mixed. 1 H-NMR (Figure 6-b) 13 C-NMR, CH-HMBC (Figure 7-c), CH-HSQC, NH-HMBC (Figure 7-b), and NH-HSQC (Figure 7-a) were measured.

[0351] Confirmation of the presence of compound 1-3-3a in solution B (SolB) Solution A 1 H-NMR observed a concentration of 4.83 ppm, which corresponds to the α-methine of compound 1-3-3a. 1 H signal (Figure 6-c)) and solution B 1 4.85 ppm observed by H-NMR 1 The H signal (Figure 6-a) was 4.84 ppm. 1 The H signal shifted to the 4.85 ppm observed in solution B, and the H signal was found to match perfectly (Figure 6-b). 1 The H signal has a peak at the low magnetic field side without coupling (the peak enclosed by the dotted line in Figure 6-a) and a peak at 4.83 ppm observed in solution A. 1 H signal 13 It was confirmed that the peak on the low magnetic field side (circled peak in Figure 6-b) where coupling with C (2.7 Hz) can be recognized shifts relative to the peak when solution B and solution A are mixed, and is converted into an intermediate broad peak (circled peak in Figure 6-b). From these results, 1 The H signal confirmed the presence of compound 1-3-3a in (SolB).

[0352] Proof of structure of compound 1-3-3a in solution A + solution B (SolA + SolB) The structure of compound 1-3-3a in solution A + solution B (SolA + SolB) is: 1 H-NMR (Figure 6-b) 13 Using C-NMR, CH-HMBC (Figure 7-c), CH-HSQC, NH-HMBC (Figure 7-b), and NH-HSQC (Figure 7-a), it can be consistently explained that this is a carbamic acid DBU salt. 10) The CH-HMBC of the mixed solution of Solution A (SolA) and Solution B (SolB) was measured, and the concentration of α-methine was 4.84 ppm. 1 H signal and solvent 13 Higher than C signal 13 C labeled 161.5 ppm 13 It was confirmed that there was a CH-HMBC correlation between the C signal and the CH-HMBC signal (Figure 7-c). 13 C signal value, experimentally 13 The origin of C bubbled up 13 CO2, so 161.5 ppm 13 The C signal is consistent with the identification of the carbamate carbonyl. 11) NH-HSQC of the mixed solution of Solution A (SolA) and Solution B (SolB) was measured. 1 H signals at 5.30 ppm and 91.4 ppm were identified. 15 The NH-HSQC correlation was observed in the N signal, indicating that the 15 The N signal was identified as the nitrogen atom of the carbamate (Figure 7-a). 1 H signal at 91.4 ppm, which corresponds to the nitrogen atom of the carbamate. 15 We also confirmed the NH-HMBC correlation between the N signal (Figure 7-b). 15 The N signal value is 91.4 ppm. 15 This can be explained without contradiction by the N signal being the nitrogen atom of the carbamate.

[0353] When Solution A + Solution B (SolA + SolB) was prepared from the two points 10) and 11), the presence of compound 1-3-3a was confirmed in the system. Furthermore, as shown below, all NMR signals of the carbamate moiety of compound 1-3-3a could be assigned (Figure 8), and the values ​​of all NMR signals can consistently explain the structure of compound 1-3-3a. TIFF0007808554000082.tif42170 1H-NMR(600MHz,DMF-d7,298K)δ7.29-7.23(m,4H),7.22-7.11(m,1H),5.30(brs,1H,NH),4.90(ddd,1H,J=6.8,6.2Hz, 3 J CH = 2.7Hz),2.91(dd,1H,J=13.3,6.8Hz),2.87(s,3H),2.83(dd,1H,J=13.3,6.2Hz),2.78(s,3H). 13 C-NMR(151MHz,DMF-d7,298K)δ173.6(qC),161.5(qC),139.4(qC),129.7(CH x2),128.3(CH x2),126.3(CH),53.0(CH),40.0(CH2),36.4(CH3),34.8(CH3). 15 N-NMR(61MHz,DMF-d7,298K,assigned by NH-HSQC and NH-HMBC)δ96,91.

[0354] From the above results, it was confirmed that when Fmoc-protected amino groups were treated with DBU, the amino groups were not completely exposed through the elimination of dibenzofulvene followed by decarboxylation, but rather the majority of the amino groups existed as DBU·HOCO-Phe-NMe2. 1 This was confirmed by H-NMR.

[0355] Example 7-2: Confirmation of the presence of carbamate when compound 1-3-1 is treated with DBU or piperidine in various solvents Basic operations After preparing a solution of compound 1-3-1 (approximately 25 mg) in a heavy solvent (1.25 mL, tetrahydrofuran-d8 (THF-d8) [Entry 1], toluene-d8 [Entry 2], dichloromethane-d2 (DCM-d2) [Entry 3], N,N-dimethylformamide-d7 (DMF-d7) [Entry 4]), 1.0 mL of the solution was divided into two (0.5 mL), one was treated with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL) and the other with piperidine (67.5 μL), and the NMR of each was measured to determine the ratio of compound 1-3-3a to compound 1-3-2. 1 The amounts of compound 1-3-1 used in this experiment were 25.1 mg for entry 1, 24.8 mg for entry 2, 25.0 mg for entry 3, and 25.3 mg for entry 4. TIFF0007808554000083.tif45170 [Table 11]

[0356] These results confirmed that when the Fmoc-protected amino group was treated with DBU, carbamate salts were present in a majority ratio in all solvents. On the other hand, when the Fmoc-protected amino group was treated with piperidine, no carbamate salts were detected in any solvent, confirming that decarboxylation had progressed to complete exposure of the amino group.

[0357] When considering the results of solid-phase experiments based on this finding, and without being bound by any particular theory, it can be explained that the reduction effect is due to the presence of carbamate, which prevents the formation of a six-membered ring that causes diketopiperazine elimination, as shown in the following formula. TIFF0007808554000085.tif30170

[0358] For example, deprotection with DBU, which confirmed the formation of carbamate salts by NMR, was compared with deprotection with piperidine, which did not, in a solid-phase experiment to confirm the effects. Reactions were carried out using the same method as in Basic Procedure B described in Example 2, using the reagents and solvents listed in Table 12, runs 1 and 2. The results (relative ratios of UV areas in LCMS for Compounds 2-1*, 2-2*, 2-3*, and 2-4* under each condition, expressed as percentages) are shown in Table 12 below. [Table 12]

[0359] Without being bound by any particular theory, when comparing run 1 (2% DBU) and run 2 (20% piperidine) in Table 12, the fact that treatment with DBU significantly suppresses the production of diketopiperazine elimination products and six-membered cyclic amidine skeletal structures can be explained without contradiction by the fact that the presence of carbamate prevents the formation of a six-membered ring that causes diketopiperazine elimination, etc.

[0360] Example 7-3: Confirmation of the presence of compound 1-3-3 when compound 1-3-1 is treated with various bases in THF Basic operations Four tetrahydrofuran-d8 (THF-d8) solutions (0.5 mL) of compound 1-3-1 (approximately 10 mg, 0.24 mmol) were prepared, and then treated with each of the four bases: piperidine (67.5 μL, Entry 1), 1,1,3,3-tetramethylguanidine (TMG) (13.5 μL, Entry 2), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL, Entry 3), and iminotris(dimethylamino)phosphouran (HP1(dma)) (13.5 μL, Entry 4). After 30 minutes, NMR was measured for each solution to determine the ratio of compound 1-3-3 to compound 1-3-2. 1 The values ​​were determined from the proton integral ratios of H-NMR (Table 13). In this experiment, 10.0 mg of compound 1-3-1 was used in entry 1, 10.0 mg in entry 2, 10.0 mg in entry 3, and 10.1 mg in entry 4. TIFF0007808554000087.tif34170 [Table 13]

[0361] From the above results, it was confirmed that when an Fmoc-protected amino group is treated with a base whose conjugate acid has a pKa of 23 or more, carbamate salts are present in the majority. When considering the results of solid-phase experiments based on this finding, without being limited to a specific theory, it is believed that the reduction in diketopiperazine elimination and other problems in runs 47 and 48 of Example 2 is due to the fact that the presence of carbamate prevents the formation of a six-membered ring that causes diketopiperazine elimination and other problems, as described above.

[0362] Example 7-4: Confirmation of behavior when compound 1-3-1 is treated with DBU and then acid (HOAt) is added 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (9.0 μL) was added to a deuterated solvent solution (1.25 mL, tetrahydrofuran-d8 (THF-d8) [Entry 1], toluene-d8 [Entry 2], dichloromethane-d2 (DCM-d2) [Entry 3], N,N-dimethylformamide-d7 (DMF-d7) [Entry 4]) of compound 1-3-1 (approximately 25 mg), and then 1.0 mL of the solution was divided into two portions (0.5 mL). 1-hydroxybenzotriazole (HOAt) (3.3 mg, with HOAt) was added to one portion, and nothing was added to the other portion (without HOAt). NMR of each portion was measured, and the ratio of compound 1-3-3a to compound 1-3-2 was determined. 1 The amounts of compound 1-3-1 used in this experiment were 24.9 mg for entry 1, 25.3 mg for entry 2, 25.2 mg for entry 3, and 24.9 mg for entry 4. TIFF0007808554000089.tif40170 [Table 14]

[0363] From the above results, it was confirmed that when the Fmoc-protected amino group was treated with DBU and then the acid HOAt (pKa in water: 3.28) was added, the proportion of carbamate present was significantly reduced. When the results of the solid phase experiments are considered based on this finding, and without being limited to a particular theory, it can be consistently explained that the increase in the ratio of diketopiperazine elimination products and amidine products when neutralization was attempted by adding HOAt in Example 3 is partly due to a decrease in the abundance ratio of carbamate. As described above, without being limited to a particular theory, when Fmoc protection is treated with a base, the N-terminal amino group exists as a carbamate, which can reduce diketopiperazine elimination, etc. Furthermore, the results of Example 7-3 identified the range of bases that can exist as carbamates.

[0364] Example 7-5: In the solid-phase synthesis of peptides, CO was used in the Fmoc removal step. 2 Peptide synthesis experiments using bubbling solvents. In Examples 7-1 to 7-4, it was explained that the presence of carbamates prevents the formation of a six-membered ring that causes diketopiperazine elimination, leading to the suppression of diketopiperazine formation. Therefore, to stabilize the carbamates during the Fmoc elimination reaction, we conducted an experiment to confirm the effect of using a solvent bubbled with CO2 as a carbonate source on the production of the diketopiperazine elimination product.

[0365] Condition A (Table 15, run 1, no CO2 bubbling) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-11, prepared in Example 1-2-5 from Compound 1-2-3 at 0.567 mmol / g) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMF (0.7 mL per column). A solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in DMF (2% v / v, 0.7 mL per column) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMF (0.7 mL per column).

[0366] The resin was mixed with a DMF solution (0.25 mL) of Fmoc-MeIle-OH (0.227 mmol, 4 equiv.), a DMF solution (0.25 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.227 mmol, 4 equiv.), and DIPEA (0.056 mL, 0.340 mmol, 6 equiv.) for 1-2 min, and then shaken at room temperature for 2 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0367] Condition B (Table 15, run 2, CO2-bubbled solvent) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-11, prepared in Example 1-2-5 from Compound 1-2-3 at 0.567 mmol / g) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMF (0.7 mL per column). A CO₂-DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added and reacted at room temperature for 10 minutes to deprotect the Fmoc group. The CO₂-DMF solvent was prepared by bubbling CO₂ gas through 50 mL of DMF in a 100 mL glass bottle for 5 minutes. After the solution was drained through the frit, the resin was washed six times with DMF (0.7 mL per column).

[0368] The resin was mixed with a DMF solution (0.25 mL) of Fmoc-MeIle-OH (0.227 mmol, 4 equiv.), a DMF solution (0.25 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.227 mmol, 4 equiv.), and DIPEA (0.056 mL, 0.340 mmol, 6 equiv.) for 1-2 min, and then shaken at room temperature for 2 h. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0369] To confirm the progress of the reaction, a portion of each resin was taken, swollen in DCM, and then the peptide was cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long), and the production of the target peptide (TM) (compound 4-1-6*), diketopiperazine elimination product (compound 4-2-6*), and MeIle overextension product (compound 7-5-1*) was confirmed.

[0370] MeIle overextension, Fmoc-MeIle-MeIle-Aib-Pro-D-3-Abu-OH (compound 7-5-1*) TIFF0007808554000091.tif35170LCMS (ESI) m / z=538.7(MH)- Detected as MS ((MH-Fmoc)-) of the Fmoc-deprotected fragment Retention time: 2.63 minutes (Analysis conditions SQDFA05long)

[0371] The results obtained (relative ratios of UV areas in LCMS of Compound 4-1-6*, Compound 4-2-6*, and Compound 7-5-1* under each condition, expressed as percentages) are shown in Table 15 below. [Table 15]

[0372] From the results of Run 7-5, it was confirmed that the production of diketopiperazine elimination products can be suppressed when a solvent with CO2 bubbling is used during the Fmoc removal reaction (Run 2) compared to when a solvent without CO2 bubbling is used (Run 1). [Industrial Applicability]

[0373] The present invention has revealed that in peptide production using a solid-phase method, the formation of by-products such as diketopiperazine and 6-membered cyclic amidine structures can be suppressed, and peptide chains can be efficiently elongated. The present invention is useful in the field of peptide synthesis using a solid-phase method.

Claims

1. A method for producing a peptide by a solid phase method, comprising the steps of: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a resin for solid phase synthesis; (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents; and (3) after the step (2), condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide; The method does not include a step of treating the first peptide with piperidine as a single base prior to the step (2), The aforementioned production method, which does not include a step of adding an acid to neutralize the remaining base between the step (2) and the step (3).

2. 2. The method of claim 1, wherein at least a portion of the first peptide obtained by step (2) is in the form of a carbamate.

3. 3. The method according to claim 1, wherein the solvent in step (2) contains 25 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

4. the aromatic hydrocarbon solvent is one or more selected from the group consisting of toluene, benzene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene; the halogen-based solvent is one or more selected from the group consisting of dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride; the ether-based solvent is one or more selected from the group consisting of tetrahydrofuran, diethyl ether, 2-methyltetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxolane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme; the ester solvent is one or more selected from the group consisting of methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone; the ketone solvent is one or more selected from the group consisting of acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone; The carbonate solvent is one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, and The method according to any one of claims 1 to 3, wherein the phosphate ester solvent is one or more selected from the group consisting of trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

5. The method according to any one of claims 1 to 4, wherein the solvent in step (2) has a donor number value of 26 or less.

6. The method according to any one of claims 1 to 5, wherein the base in the step (2) is at least one selected from the group consisting of amidines, guanidines, and phosphazenes.

7. The method according to any one of claims 1 to 6, wherein the base in step (2) is at least one selected from the group consisting of 1,8-diazabicyclo[5.4.0]-7-undecene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,1,3,3-tetramethylguanidine, t-butylimino-tris(dimethylamino)phosphorane, tetramethyl(tris(dimethylamino)phosphoranylidene)phosphoric acid triamide-ethylimine, and imino-tris(dimethylamino)phosphorane.

8. The step (2) is a step of adding CO ２ The method of any one of claims 1 to 7, further comprising contacting

9. The carboxylic acid or carboxylic acid analogue may comprise an amino acid having a protecting group, a second peptide having a protecting group, C １ -C ８ Alkyl carboxylic acid, or C ６ -C １０ an amino acid which is an aryl carboxylic acid or has a protecting group; a second peptide which has a protecting group; C 1 -C 8 Alkyl carboxylic acid, or C 6 -C 10 an amino acid which is an activated ester of an aryl carboxylic acid or which has a protecting group; a second peptide which has a protecting group; C 1 -C 8 Alkyl carboxylic acid, or C 6 -C 10 an acid halide of an arylcarboxylic acid, １ -C ８ Alkyl carboxylic acids and C ６ -C １０ 9. The method of any one of claims 1 to 8, wherein the arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl, and dialkylaminocarbonyl.

10. The method of any one of claims 1 to 9, wherein the first peptide is a dipeptide.

11. The method according to any one of claims 1 to 10, wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group comprising an Fmoc backbone, or an active ester of an amino acid or a second peptide having a protecting group, or an acid halide of an amino acid or a second peptide having a protecting group, and the first peptide and / or the second peptide having a protecting group comprising an Fmoc backbone comprises one or more N-substituted amino acids, and / or the amino acid having a protecting group comprising an Fmoc backbone is an N-substituted amino acid.

12. The method according to any one of claims 1 to 11, wherein the second amino acid residue from the N-terminus of the first peptide is an N-substituted amino acid.

13. The condensing agent in the step (3) is in the form of a salt, and its counter anion is PF ６ － or BF ４ － The method according to any one of claims 1 to 12, wherein

14. The condensing agent in the step (3) is selected from the group consisting of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate, O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(1H-6-chloro-1-yl)-1,1,3,3 -tetramethyluronium hexafluorophosphate, O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate 16. The method of any one of claims 1 to 15, comprising at least one selected from the group consisting of benzotriazolium 3-oxide tetrafluoroborate, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate, O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, and O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate.

15. 1. A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in peptide production by a solid phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing at least one selected from the group consisting of aromatic hydrocarbon solvents, halogenated solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, and phosphate ester solvents; Including, The method does not include a step of treating the first peptide with piperidine as a single base prior to the step (2), The above method, which does not include a step of adding an acid to neutralize the remaining base after the step (2).

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