Method for analyzing oligomer contained in peptide compound

Abstract

The present invention provides a method for analyzing a peptide compound in a sample and an oligomer derived from said peptide compound, said method comprising using a reversed-phase column to perform liquid chromatography in which a solvent containing a C2-3 alcohol is used an eluent.

Description

Method for analyzing oligomers contained in peptide compounds

[0001] The present invention relates to a method for analyzing oligomers contained in peptide compounds.

[0002] It is known that when a linear peptide is cyclized intramolecularly to produce a cyclic peptide, in addition to the desired cyclic peptide, oligomers such as dimers and trimers of the linear peptide are by-produced as a result of intermolecular bonds formed between linear peptides (Non-Patent Document 1).

[0003] Size exclusion chromatography (SEC) using an aqueous mobile phase is used as a standard separation method for quantifying protein oligomers (Non-Patent Document 2). SEC using an aqueous mobile phase is also used in the analysis of oligomers produced during the synthesis of low molecular weight compounds (Non-Patent Document 3). Furthermore, in the analysis of peptides and their oligomers, it has been disclosed that peptides and their oligomers are separated using reversed-phase column chromatography (ODS column) as the stationary phase and acetonitrile and water as the mobile phase (Patent Documents 1 and 2).

[0004] International Publication No. WO 2022 / 234864 International Publication No. WO 2024 / 080333

[0005] McMurray, JS et al. Peptide Res., 1994, 7, 195-206.Fekete, S. et al. J. Pharm. Biomed. Anal., 2014, 101, 161-173.Kim, J et al. Org. Process Res. Dev. 2021, 25, 2249-2259.

[0006] If the cyclic peptide and its oligomers could be simultaneously analyzed when a cyclic peptide is produced by intramolecular cyclization of a linear peptide, it would be easier to control the reaction rate, side reactions, and the quality of the target peptide. However, the present inventors have found that oligomers produced during the production of highly lipophilic cyclic peptides are even more lipophilic, and that under conditions generally used for high-performance liquid chromatography (HPLC) analysis, the oligomers may be insoluble in the solvent, or may adsorb to the stationary phase of the column, making it difficult to elute all of the oligomers. Therefore, under conditions generally used for HPLC analysis, the amount of oligomers appears to be small, and the purity of the target cyclic peptide may not be accurately determined.

[0007] The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a means for simultaneously analyzing a target peptide, particularly a cyclic peptide and its oligomer. Another objective of the present invention is to provide a method for producing a peptide using the analytical means.

[0008] As a result of extensive research to solve the above problems, the present inventors discovered that a target peptide and its oligomers can be simultaneously analyzed by using a stationary phase column with appropriate retention power in reversed-phase liquid chromatography and (normal-phase) size exclusion chromatography (SEC) using an organic solvent as the mobile phase, and eluting the peptides with a specific solvent for analysis, thereby completing the present invention.

[0009] That is, in one aspect of the present invention, the following inventions are provided. [A1] A method for analyzing a peptide compound and an oligomer derived from the peptide compound in a sample, comprising performing liquid chromatography using a reverse-phase column and an eluent containing a solvent containing an alcohol having 2 to 3 carbon atoms. [A2] The method of [A1], in which the peptide compound and the oligomer derived from the peptide compound are separated and analyzed. [A3] The method of [A1], in which the peptide compound and the oligomer derived from the peptide compound are simultaneously analyzed. [A4] The method of [A1], in which the peptide compound and the oligomer derived from the peptide compound are separated and analyzed simultaneously. [A5] The method of any of [A1] to [A4], in which the alcohol having 2 to 3 carbon atoms is one or more selected from the group consisting of ethanol, 1-propanol, and 2-propanol. [A5-1] The method of any of [A1] to [A4], in which the alcohol having 2 to 3 carbon atoms is one or more selected from the group consisting of ethanol and 2-propanol. [A6] The method according to any one of [A1] to [A4], wherein the alcohol having 2 to 3 carbon atoms is an alcohol having 3 carbon atoms. [A7] The method according to [A6], wherein the alcohol having 3 carbon atoms is 1-propanol or 2-propanol. [A7-1] The method according to [A6], wherein the alcohol having 3 carbon atoms is 2-propanol. [A8] The method according to any one of [A1] to [A4], wherein the alcohol having 2 to 3 carbon atoms is ethanol. [A9] The method according to any one of [A1] to [A8], wherein the solvent further contains an organic acid. [A10] The method according to [A9], wherein the organic acid is formic acid or trifluoroacetic acid. [A11] The reversed-phase column is a C ６ Aryl C ２ -C １０ Alkyl silica gel column or C ４ -C １８ [A12] The method according to any one of [A1] to [A10], wherein the reversed-phase column is an alkyl silica gel column. ６ Aryl C ２ -C １０The method according to any one of [A1] to [A10], wherein the column is an alkyl silica gel column. ６ Aryl C ２ -C １０ The method according to [A11] or [A12], wherein the alkyl silica gel column is one selected from the group consisting of a phenylhexyl silica gel column, a pentafluorophenylpropyl silica gel column, and a phenethyl silica gel column. ６ Aryl C ２ -C １０ The alkyl silica gel column is ２ -C １０ The method according to any one of [A11] to [A13], wherein the phenyl C is an alkyl silica gel column. ２ -C １０ [A15] The method according to [A14], wherein the alkyl silica gel column is a phenylhexyl silica gel column. ４ -C １８ The method according to any one of [A1] to [A10], wherein the column is an alkyl silica gel column. ４ -C １８ The alkyl silica gel column is １８ [A16] The method according to [A11] or [A15], wherein the reversed-phase column is a phenylhexyl silica gel column, a pentafluorophenylpropyl silica gel column, and an octadecyl (C １８ [A17] The method according to any one of [A1] to [A10], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is one selected from the group consisting of a phenylhexyl silica gel column, a pentafluorophenylpropyl silica gel column, and an octadecyl (C １８[A18] The method according to any one of [A1] to [A3], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column or a pentafluorophenylpropyl silica gel column. [A18-1] The method according to any one of [A1] to [A3], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column or a pentafluorophenylpropyl silica gel column. [A18-2] The method according to any one of [A1] to [A3], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column or a pentafluorophenylpropyl silica gel column. [A18-3] The method according to any one of [A1] to [A3], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column or a pentafluorophenylpropyl silica gel column. [A18-4] The method according to any one of [A1] to [A3], wherein the alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column or a pentafluorophenylpropyl silica gel column. １８) The method according to any one of [A1] to [A3], wherein the column is a silica gel column. [A19] The method according to any one of [A1] to [A18], wherein the Clog P of the peptide compound is 7 to 20. [A20] The method according to any one of [A1] to [A18], wherein the Clog P of the peptide compound is 8 to 20. [A21] The method according to any one of [A1] to [A18], wherein the Clog P of the peptide compound is 11 to 20. [A22] The method according to any one of [A1] to [A21], wherein the Clog P / total aa of the peptide compound is 1.0 to 1.8. [A23] The method according to any one of [A1] to [A22], wherein the number of amino acid residues constituting the peptide compound is 7 to 20. [A24] The method according to any one of [A1] to [A22], wherein the number of amino acid residues constituting the peptide compound is 7 to 14. [A25] The method according to any one of [A1] to [A22], wherein the number of amino acid residues constituting the peptide compound is 10 to 14. [A26] The method according to any one of [A1] to [A22], wherein the number of amino acid residues constituting the peptide compound is 11. [A27] The method according to any one of [A1] to [A26], wherein the number of N-substituted amino acids contained in the peptide compound is 3 or more. [A28] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion. [A29] The method according to [A28], wherein the number of amino acid residues constituting the cyclic portion is 7 to 14. [A30] The method according to [A28], wherein the number of amino acid residues constituting the cyclic portion is 8 to 14. [A31] The method according to [A28], wherein the number of amino acid residues constituting the cyclic portion is 11. [A32] The method according to any one of [A28] to [A31], wherein the number of amino acid residues constituting the cyclic portion is 3 or more. [A33] The method according to any one of [A28] to [A32], wherein the side chain of the cyclic moiety does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A34] The method according to any one of [A28] to [A32], wherein the side chain of the cyclic moiety does not have a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group.[A35] The method according to any one of [A28] to [A34], wherein, when the cyclic moiety has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10. [A36] The method according to any one of [A28] to [A35], wherein, when the cyclic moiety has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10. [A37] The method according to [A27] or [A32], wherein the N-substituted amino acid is an N-alkyl amino acid. [A38] The method according to [A37], wherein the N-alkyl amino acid is an N-methyl amino acid. [A39] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic moiety, the number of amino acid residues constituting the cyclic moiety is 7 to 14, and the ClogP is 7 to 20. [A40] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 8 to 14, and wherein the ClogP is 8 to 20. [A41] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 11, and wherein the ClogP is 11 to 20. [A42] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 7 to 14, and wherein the ClogP / total aa is 1.0 to 1.8. [A43] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 8 to 14, and wherein the ClogP / total aa is 1.0 to 1.8. [A44] The method according to any of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the cyclic portion comprising 11 amino acid residues, and ClogP / total aa is 1.0 to 1.8.[A45] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP is 7 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A46] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, the ClogP is 8 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A47] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, the ClogP is 11 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A48] The method according to any of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP is 7 to 20, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10. [A49] The method according to any of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, the ClogP is 8 to 20, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.[A50] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, the ClogP is 11 to 20, the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10. [A51] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A52] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A53] The method according to any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [A54] The method according to any of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.[A55] The method according to any of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10. [A56] The method of any one of [A1] to [A27], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10. [A57] The method of any one of [A1] to [A27], wherein the cyclic peptide compound is one selected from the group consisting of peptide compounds listed in Tables 5 to 7. [A58] The method of any one of [A1] to [A57], wherein the oligomer comprises a dimer of the peptide compound. [A59] The method of any one of [A1] to [A57], wherein the oligomer comprises a dimer and a trimer of the peptide compound. [A60] The method of any one of [A1] to [A57], wherein the oligomer comprises a dimer, a trimer, and a tetramer or higher oligomer of the peptide compound. [A61] The method of any one of [A1] to [A57], wherein the oligomer is one or more selected from the group consisting of a dimer and a trimer of the peptide compound. [A62] The method of any one of [A1] to [A57], wherein the oligomer is a dimer of the peptide compound. [A63] The method of any one of [A1] to [A57], wherein the oligomer is a trimer of the peptide compound. [A64] The method of any one of [A1] to [A57], wherein the oligomer is a cyclic oligomer produced as a by-product under peptide cyclization conditions.[A65] The method of any one of [A1] to [A57], wherein the oligomer comprises a cyclic dimer of the peptide compound. [A66] The method of any one of [A1] to [A57], wherein the oligomer comprises a cyclic dimer and a cyclic trimer of the peptide compound. [A67] The method of any one of [A1] to [A57], wherein the oligomer comprises a cyclic dimer, a cyclic trimer, and a cyclic tetramer or higher cyclic oligomer of the peptide compound. [A68] The method of any one of [A1] to [A57], wherein the oligomer is one or more selected from the group consisting of a cyclic dimer and a cyclic trimer of the peptide compound. [A69] The method of any one of [A1] to [A57], wherein the oligomer is a cyclic dimer of the peptide compound. [A70] The method of any one of [A1] to [A57], wherein the oligomer is a cyclic trimer of the peptide compound. [B1] A method for analyzing a peptide compound and an oligomer derived from the peptide compound in a sample, comprising performing size exclusion chromatography using a styrenedivinylbenzene column and an organic solvent as an eluent. [B2] The method of [B1], wherein the peptide compound and the oligomer derived from the peptide compound are separated and analyzed. [B3] The method of [B1], wherein the peptide compound and the oligomer derived from the peptide compound are simultaneously analyzed. [B4] The method of [B1], wherein the peptide compound and the oligomer derived from the peptide compound are separated and analyzed simultaneously.[B5] The organic solvent is tetrahydrofuran (THF), 1,4-dioxane, n-hexane, cyclohexane, dodecane, toluene, benzene, xylene, 1-methyl-2-pyrrolidone (NMP), pyridine, quinoline, acetone, methyl ethyl ketone (MEK), o-dichlorobenzene (ODCB), 1,2,4-trichlorobenzene (TCB), 1-chloronaphthalene, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), chloroform, dichloromethane, tetrahydrofuran ... The method according to any one of [B1] to [B4], wherein the organic solvent is one selected from the group consisting of carbon chloride, dichloroethane, trichloroethane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / chloroform, m-cresol / chloroform, o-chlorophenol / chloroform, methanol / chloroform, ethanol, and ethyl acetate. [B6] The method according to any one of [B1] to [B4], wherein the organic solvent is tetrahydrofuran (THF). [B7] The method according to any one of [B1] to [B6], wherein the styrene divinylbenzene column has an average particle size of 1 to 5 μm. [B8] The method according to any one of [B1] to [B7], wherein the styrene divinylbenzene column has an average pore size of 1 to 10 nm. [B9] The method according to any one of [B1] to [B4], wherein the styrene divinylbenzene column has an average particle size of 1 to 5 μm and an average pore size of 1 to 10 nm, and the organic solvent is tetrahydrofuran (THF). [B10] The method according to any one of [B1] to [B4], wherein the styrene divinylbenzene column has an average particle size of 3 to 5 μm and an average pore size of 1.5 to 7.5 nm, and the organic solvent is tetrahydrofuran (THF). [B11] The method according to any one of [B1] to [B4], wherein the styrene divinylbenzene column has an average particle size of 3 μm and an average pore size of 7.5 nm, and the organic solvent is tetrahydrofuran (THF). [B12] The method according to any one of [B1] to [B11], wherein the peptide compound has a ClogP of 7 to 20.[B13] The method of any one of [B1] to [B11], wherein the ClogP of the peptide compound is 8 to 20. [B14] The method of any one of [B1] to [B11], wherein the ClogP of the peptide compound is 11 to 20. [B15] The method of any one of [B1] to [B14], wherein the ClogP / total aa of the peptide compound is 1.0 to 1.8. [B16] The method of any one of [B1] to [B15], wherein the number of amino acid residues constituting the peptide compound is 7 to 20. [B17] The method of any one of [B1] to [B15], wherein the number of amino acid residues constituting the peptide compound is 7 to 14. [B18] The method of any one of [B1] to [B15], wherein the number of amino acid residues constituting the peptide compound is 10 to 14. [B19] The method of any one of [B1] to [B15], wherein the number of amino acid residues constituting the peptide compound is 11. [B20] The method according to any one of [B1] to [B19], wherein the number of N-substituted amino acids contained in the peptide compound is 3 or more. [B21] The method according to any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion. [B22] The method according to [B21], wherein the number of amino acid residues constituting the cyclic portion is 7 to 14. [B23] The method according to [B21], wherein the number of amino acid residues constituting the cyclic portion is 8 to 14. [B24] The method according to [B21], wherein the number of amino acid residues constituting the cyclic portion is 11. [B25] The method according to any one of [B21] to [B24], wherein the number of N-substituted amino acids contained in the cyclic portion is 3 or more. [B26] The method according to any one of [B21] to [B25], wherein the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain. [B27] The method according to any one of [B21] to [B25], wherein the side chain of the cyclic moiety does not have a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group. [B28] The method according to any one of [B21] to [B27], wherein, when the cyclic moiety has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10.[B29] The method of any one of [B21] to [B28], wherein, when the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10. [B30] The method of [B20] or [B25], wherein the N-substituted amino acid is an N-alkyl amino acid. [B31] The method of [B30], wherein the N-alkyl amino acid is an N-methyl amino acid. [B32] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 7 to 14, and wherein the ClogP is 7 to 20. [B33] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 8 to 14, and wherein the ClogP is 8 to 20. [B34] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 11, and wherein ClogP is 11 to 20. [B35] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 7 to 14, and wherein ClogP / total aa is 1.0 to 1.8. [B36] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 8 to 14, and wherein ClogP / total aa is 1.0 to 1.8. [B37] The method according to any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 11, and ClogP / total aa being 1.0 to 1.8. [B38] The method according to any one of [B1] to [B20], wherein the cyclic peptide compound is one selected from the group consisting of the peptide compounds listed in Tables 5 to 7.[B39] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP is 7 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B40] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, the ClogP is 8 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B41] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, the ClogP is 11 to 20, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B42] The method according to any of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP is 7 to 20, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10. [B43] The method according to any of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, the ClogP is 8 to 20, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.[B44] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, the ClogP is 11 to 20, the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10. [B45] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, the ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B46] The method according to any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B47] The method according to any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, ClogP / total aa is 1.0 to 1.8, and the side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group. [B48] The method according to any of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.[B49] The method according to any of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 8 to 14, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10. [B50] The method of any one of [B1] to [B20], wherein the peptide compound is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in its side chain, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10. [B51] The method of any one of [B1] to [B50], wherein the oligomer comprises a dimer of the peptide compound. [B52] The method of any one of [B1] to [B50], wherein the oligomer comprises a dimer and a trimer of the peptide compound. [B53] The method of any one of [B1] to [B50], wherein the oligomer comprises a dimer, trimer, and tetramer or higher oligomer of the peptide compound. [B54] The method of any one of [B1] to [B50], wherein the oligomer is one or more selected from the group consisting of a dimer and a trimer of the peptide compound. [B55] The method of any one of [B1] to [B50], wherein the oligomer is a dimer of the peptide compound. [B56] The method of any one of [B1] to [B50], wherein the oligomer is a trimer of the peptide compound. [B57] The method of any one of [B1] to [B50], wherein the oligomer is a cyclic oligomer generated as a by-product under peptide cyclization conditions. [B58] The method of any one of [B1] to [B50], wherein the oligomer comprises a cyclic dimer of the peptide compound.[B59] The method of any one of [B1] to [B50], wherein the oligomer comprises a cyclic dimer and a cyclic trimer of the peptide compound. [B60] The method of any one of [B1] to [B50], wherein the oligomer comprises a cyclic dimer, a cyclic trimer, and a cyclic oligomer of tetramer or higher of the peptide compound. [B61] The method of any one of [B1] to [B50], wherein the oligomer is one or more selected from the group consisting of a cyclic dimer and a cyclic trimer of the peptide compound. [B62] The method of any one of [B1] to [B50], wherein the oligomer is a cyclic dimer of the peptide compound. [B63] The method of any one of [B1] to [B50], wherein the oligomer is a cyclic trimer of the peptide compound. [C1] A method for producing a composition containing a peptide compound, comprising: i) providing a composition containing a desired peptide compound, and ii) analyzing the peptide compound and oligomers derived from the peptide compound in the composition by a method described in any of [A1] to [A70] or [B1] to [B63]. [C2] The method according to [C1], further comprising, before step i), a step of cyclizing a precursor of the desired peptide compound. [C3] The method according to [C1] or [C2], wherein the content of the peptide compound in the composition in step ii) is 90% or more, 95% or more, 98% or more, or 99% or more, wherein the content of the peptide compound is determined by the UV area value at 220 nm by HPLC analysis based on the total amount of the composition. [C4] The method according to any one of [C1] to [C3], wherein the content of the peptide compound-derived oligomer in the composition in step ii) is less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 1%, or an undetectable amount, wherein the content of the peptide compound-derived oligomer is determined by the UV area value at 220 nm by HPLC analysis based on the total amount of the composition. [C5] The method according to any one of [C1] to [C4], wherein the composition is a pharmaceutical composition.[C6] A method for producing a pharmaceutical preparation containing a peptide compound, comprising the steps of: i) providing a composition containing a desired peptide compound; ii) analyzing the peptide compound and oligomers derived from the peptide compound in the composition by a method described in any of [A1] to [A70] or [B1] to [B63]; and iii) formulating the composition to provide a pharmaceutical preparation. [C7] The method according to [C6], further comprising a step of cyclizing a precursor of the desired peptide compound before step i). [C8] The method according to [C6] or [C7], wherein the content of the peptide compound in the composition in step ii) is 90% or more, 95% or more, 98% or more, or 99% or more, wherein the content of the peptide compound is determined by the UV area value at 220 nm by HPLC analysis based on the total amount of the composition. [C9] The method according to any one of [C6] to [C8], wherein the content of the peptide compound-derived oligomer in the composition in step ii) is less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 1%, or an undetectable amount, wherein the content of the peptide compound-derived oligomer is determined by a UV area value at 220 nm by HPLC analysis based on the total amount of the composition. [C10] The method according to any one of [C1] to [C9], wherein the peptide compound is one selected from the group consisting of the peptide compounds listed in Tables 5 to 7. [C11] The peptide compound is (1S,4S,10S,13S,17S,20S,26S,28R,32S,38S,42Z)-20-cyclopentyl-28-ethoxy-32-[2-[3-methoxy-4-(trifluoromethyl)phenyl]ethyl]-N,N,2,14,18,21,24,36-octamethyl-10-[(1S)-1-methylpropyl]-3,9,12,15,19,22,25,31,34,37,45-undecaoxo-13-propyl-38-[[4-(trifluoromethyl)phenyl]methyl]spiro[2,8,11,14,18,21,24,30,33,36,39-undecazatetracyclo[37.5.1.0. 4,8 .0 26,30The method for producing the compound according to any one of [C1] to [C9], wherein the compound is pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide (compound 9). In the above numbering scheme, the numbers referred to in the dependent claims include their subnumbers unless otherwise specified. For example, [A5] referred to in the dependent claims indicates that it includes not only [A5] but also its subnumbers, such as [A5-1]. The same applies to other numbering schemes.

[0010] A means for simultaneously analyzing a target peptide, particularly a cyclic peptide and its oligomer, is provided, as well as a method for producing a peptide using the analytical means.

[0011] FIG. 1 shows a basic method for synthesizing cyclic peptides. FIG. 2 shows a chromatogram of Compound 1 obtained under analytical condition-11. FIG. 3 shows the chemical structure of a C-dimer derived from Compound 1. FIG. 4 shows the chemical structure of a C-trimer derived from Compound 1. FIG. 5 shows a chromatogram of Compound 1 obtained under analytical condition-12. FIG. 6 shows a chromatogram of Compound 2 obtained under analytical condition-11. FIG. 7 shows a chromatogram of Compound 2 obtained under analytical condition-12. FIG. 8 shows a chromatogram of Compound 3 obtained under analytical condition-11. FIG. 9 shows a chromatogram of Compound 3 obtained under analytical condition-12. FIG. 10 shows a chromatogram of Compound 9 obtained under analytical condition-12. FIG. 11 shows a chromatogram of Compound 1 obtained under analytical condition-14. FIG. 12 shows a chromatogram of Compound 2 obtained under analytical condition-14. FIG. 13 shows a chromatogram of Compound 1 obtained under Analysis Condition-15. FIG. 14 shows a chromatogram of Compound 2 obtained under Analysis Condition-15. FIG. 15 shows a chromatogram of Compound 3 obtained under Analysis Condition-15. FIG. 16 shows a chromatogram of Compound 1 obtained under Analysis Condition-17. FIG. 17 shows a chromatogram of Compound 1 obtained under Analysis Condition-18. FIG. 18 shows a chromatogram of Compound 1 obtained under Analysis Condition-19. FIG. 19 shows a chromatogram of Compound 2 obtained under Analysis Condition-17. FIG. 20 shows a chromatogram of Compound 2 obtained under Analysis Condition-18. FIG. 21 shows a chromatogram of Compound 2 obtained under Analysis Condition-19.

[0012] The present invention will be described in detail below by showing definitions of symbols, terms, etc. used in this specification, and embodiments of the present invention.

[0013] In this specification, "alkyl" refers to a linear or branched monovalent saturated hydrocarbon group derived from an aliphatic saturated 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 atoms and carbon atoms. Alkyl includes not only linear ones but also branched ones. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C １ -C ２０ , hereinafter referred to as “C ｐ -C ｑ " means that the number of carbon atoms is p to q. 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, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-detradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-heptadecyl, and n-octadecyl.

[0014] In the present specification, the term "aryl" refers to a monovalent aromatic hydrocarbon ring group consisting of a single ring or fused rings that exhibits monovalent aromaticity. In the present specification, an aryl consisting of a single ring is referred to as a monocyclic aryl, and an aryl consisting of a fused ring is referred to as a fused ring aryl. Examples of aryl include C ６ -C １４ aryl, C ６ Aryl, C １０ Aryl and C １４ Examples of the aryl include phenyl, 1-naphthyl, and 2-naphthyl. These aryl groups may further have a substituent, and examples thereof include tolyl, xylyl, and 2,3,4,5,6-pentafluorophenyl.

[0015] As used herein, "aralkyl (arylalkyl)" refers to a group in which one or more hydrogen atoms of an "alkyl" are substituted with an "aryl". Examples of aralkyl include C ７ -C ２０ Aralkyl, C ７ -C １８ Aralkyl, C ７ -C １６ Aralkyl, or C ７ -C １４ An example is aralkyl. ７ -C ２０ Examples of aralkyl include C ６ -C １０ Aryl C １ -C １０ alkyl, C ６ -C １０ Aryl C １ -C ８ Alkyl is preferred, C ６ Aryl C １ -C １０ Alkyl or C １０ Aryl C １ -C １０ Alkyl is more preferred, C ６ Aryl C １ -C １０ Alkyl is more preferred. ７ -C １８ Examples of aralkyl include C ６ -C １０ Aryl C １ -C ８ alkyl, C ６ -C １０ Aryl C １ -C ６ Alkyl is preferred, C ６ Aryl C １ -C ６ Alkyl or C １０ Aryl C １ -C ６ Alkyl is more preferred, C ６ Aryl C １ -C ６ Alkyl is more preferred. ７ -C １６ Examples of aralkyl include C ６ -C１０ Aryl C １ -C ６ alkyl, C ６ -C １０ Aryl C １ -C ４ Alkyl is preferred, C ６ Aryl C １ -C ４ Alkyl or C １０ Aryl C １ -C ４ Alkyl is more preferred, C ６ Aryl C １ -C ４ Alkyl is more preferred. ７ -C １４ Examples of aralkyl include C ６ -C １０ Aryl C １ -C ４ alkyl, C ６ -C １０ Aryl C １ -C ３ Alkyl is preferred, C ６ Aryl C １ -C ３ Alkyl or C １０ Aryl C １ -C ３ Alkyl is more preferred, C ６ Aryl C １ -C ３ Specific examples of aralkyl include benzyl, phenethyl, 3-phenylpropyl, and phenylhexyl.

[0016] As used herein, a "peptide compound" refers to two or more amino acids linked by an amide bond. Peptide compounds may contain bonds other than amide bonds. For example, peptides containing bonds selected from the group consisting of COC bonds, C(O)-O bonds, C(S)-O bonds, C(O)-S bonds, C(S)-S bonds, C-S-C bonds, C-S-C bonds, C-S-C bonds, C-S-C bonds, C-N-C bonds, C-N-C bonds, C-N-C bonds, and C-C bonds using nitrogen atoms are also included in the term "peptide compound" herein. As used herein, "peptide compounds" include linear peptide compounds and cyclic peptide compounds. As used herein, the "amino acids" constituting a peptide compound may be referred to as "amino acid residues." As used herein, a peptide compound having a cyclic portion may be referred to as a "cyclic peptide compound." As used herein, the "cyclic portion" of a peptide compound refers to a cyclic portion formed by linking four or more amino acid residues. As used herein, the term "linear portion" used to refer to a partial structure of a cyclic peptide compound refers to a portion that is not included in the main chain structure of the cyclic portion and that has at least one amide bond and / or ester bond in the chain of the portion. Furthermore, the term "peptide compound" as used herein may also include pharmaceutically acceptable salts thereof.

[0017] As used herein, the "number of amino acid residues" refers to the number of amino acid residues (amino acid units) constituting a peptide, and means the number of amino acid units generated when the amide bonds, ester bonds, and cyclized bond linking the amino acids are cleaved. Therefore, the number of amino acid residues in a peptide compound or peptide moiety (also referred to as the number of amino acid residues constituting the peptide compound or peptide moiety) refers to the number of amino acid units generated when the amide bonds, ester bonds, and cyclized bond contained in the entire peptide moiety, including the cyclic and linear portions, are cleaved.

[0018] As used herein, "amino acid residues constituting the cyclic portion" (also referred to as "amino acid residues of the cyclic portion") refers to amino acid residues present on the main chain of the cyclic portion among amino acid residues generated when an amide bond, an ester bond, or a bond in the cyclized portion present in the peptide portion of a cyclic peptide compound is cleaved, and does not include amino acid residues constituting the linear portion. As used herein, "the number of amino acid residues constituting the cyclic portion" refers to the number of amino acid units present on the main chain of the cyclic portion.

[0019] As used herein, the term "side chain" refers to the side chain of an amino acid or the side chain of the cyclic portion of a cyclic peptide compound, and refers to the portion not included in the respective main chain structures. In the case of α-amino acids, it refers to a group and / or atom bound to the carbon (α-carbon) to which the amino group and carboxyl group are bonded. For example, the methyl group of alanine (Ala) is the side chain of an amino acid. In the case of β-amino acids, the group and / or atom bound to the α-carbon and / or β-carbon can be the side chain of an amino acid, and in the case of γ-amino acids, the group and / or atom bound to the α-carbon, β-carbon, and / or γ-carbon can be the side chain of an amino acid.

[0020] As used herein, "amino acid" includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives). Furthermore, as used herein, "amino acid" may refer to an amino acid residue. As used herein, "natural amino acid" refers to any L-amino acid selected from the group consisting of Gly (glycine), L-Ala (alanine), L-Ser (serine), L-Thr (threonine), L-Val (valine), L-Leu (leucine), L-Ile (isoleucine), L-Phe (phenylalanine), L-Tyr (tyrosine), L-Trp (tryptophan), L-His (histidine), L-Glu (glutamic acid), L-Asp (aspartic acid), L-Gln (glutamine), L-Asn (asparagine), L-Cys (cysteine), L-Met (methionine), L-Lys (lysine), L-Arg (arginine), and L-Pro (proline). As used herein, L-amino acids included in natural amino acids may be referred to without the "L-". That is, the natural amino acid L-Ala may be referred to as Ala. As used herein, "unnatural amino acids" refer to amino acids other than natural amino acids. Examples of unnatural amino acids include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids other than Pro, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids. Any configuration is acceptable for amino acids herein. The side chains of amino acids are not particularly limited and may be freely selected from groups such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and spiro-linked cycloalkyl, and / or atoms such as hydrogen atoms. Each group and / or atom may be further substituted. In a non-limiting embodiment, the amino acids herein may be compounds having a carboxy group and an amino group in the same molecule. Even in this case, amino acids also include proline, hydroxyproline, azetidine-2-carboxylic acid, and the like, in which the nitrogen atom of the amino group and any atom in the side chain of the amino acid form a ring.Furthermore, the nitrogen atom of the amino group may form a ring together with the side chain of another amino acid, and such a partial structure may be a structure represented by the following formula (* indicates the point of attachment to the adjacent atom):

[0021] As used herein, the term "main chain of an amino acid" refers to the chain portion composed of an amino group, an α-carbon, and a carboxyl group in the case of an α-amino acid, the chain portion composed of an amino group, a β-carbon, an α-carbon, and a carboxyl group in the case of a β-amino acid, and the chain portion composed of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group in the case of a γ-amino acid.

[0022] The main chain amino group of the amino acid is unsubstituted (-NH ２ ) or may be substituted (i.e., -NHR). Here, R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the α-position, as in proline, may form a ring. Such amino acids in which the main chain amino group is substituted may be referred to as "N-substituted amino acids" or "N-substituted amino acid residues" in this specification. As used herein, "N-substituted amino acids" or "N-substituted amino acid residues" include N-alkyl amino acids, N-C １ -C ６ Alkyl amino acids, N-C １ -C ４ Alkyl amino acids, N-methyl amino acids, N-ethyl amino acids, N-C ７ -C １４ Examples include, but are not limited to, aralkyl amino acids, N-benzyl amino acids, N-phenethyl amino acids, proline, hydroxyproline, and azetidine-2-carboxylic acid.

[0023] In one aspect, the present invention provides a method for analyzing a peptide compound and an oligomer derived from the peptide compound in a sample, which comprises performing liquid chromatography using a reverse-phase column and an eluent containing a solvent containing an alcohol having 2 to 3 carbon atoms (hereinafter also referred to as "Analysis Method 1").

[0024] In one embodiment, analytical method 1 may separate and analyze a peptide compound and an oligomer derived from the peptide compound in a sample, may simultaneously analyze a peptide compound and an oligomer derived from the peptide compound in a sample, or may separate and simultaneously analyze a peptide compound and an oligomer derived from the peptide compound in a sample. Here, "simultaneous analysis" means that a target peptide compound and one or more oligomers derived from the peptide compound are detected by injecting a sample into liquid chromatography once.

[0025] The eluent used in Analysis Method 1 contains an alcohol having 2 to 3 carbon atoms. Examples of alcohols having 2 to 3 carbon atoms include ethanol, 1-propanol, or 2-propanol, and mixtures thereof. Ethanol, 1-propanol, or 2-propanol is preferred, 1-propanol or 2-propanol is more preferred, and 2-propanol is even more preferred. Examples of alcohols having 2 to 3 carbon atoms include ethanol or 2-propanol, and mixtures thereof.

[0026] In one embodiment, the eluent used in analytical method 1 comprises ethanol.

[0027] In one embodiment, the eluent used in Analysis Method 1 contains an alcohol having a carbon number of 3. Examples of the alcohol having a carbon number of 3 include 1-propanol and 2-propanol. Among these, 2-propanol is preferred.

[0028] In one embodiment, the eluent used in Analysis Method 1 may contain an organic acid in addition to the alcohol having 2 to 3 carbon atoms. Examples of organic acids include formic acid, acetic acid, trifluoroacetic acid, phosphoric acid, citric acid, tartaric acid, and boric acid. Among these, formic acid and trifluoroacetic acid are preferred. In another embodiment, the eluent used in Analysis Method 1 may contain a salt in addition to the alcohol having 2 to 3 carbon atoms. Examples of salts include sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, ammonium acetate, sodium carbonate, sodium citrate, ammonium bicarbonate, and ammonium formate.

[0029] In one embodiment, the eluent used in Analysis Method 1 may further contain water.

[0030] The column used in Analysis Method 1 is a reversed-phase column. Liquid chromatography using a reversed-phase column uses a packing material with low polarity (e.g., a silica gel column) as the stationary phase and a polar solvent as the mobile phase (eluent), and separates peptide compounds or oligomers derived from the peptide compounds contained in a sample by utilizing the difference in hydrophobic interaction between the stationary phase and the peptide compounds.

[0031] The reversed-phase column used in the analysis method 1 is, for example, C ４ -C １８ Alkyl silica gel column, C ６ Aryl C ２ -C １０ Alkyl silica gel column, phenyl C ２ -C １０ Alkyl silica gel column. ４ -C １８ Examples of alkyl silica gel columns include butyl (C ４ ) silica gel column, octyl (C ８ ) silica gel column, octadecyl (C １８ ) silica gel column. ６ Aryl C ２ -C １０Examples of alkyl silica gel columns include phenethyl (C ２ ) silica gel column, phenylpropyl (C ３ ) silica gel column, pentafluorophenylpropyl (C ３ ) silica gel column, or phenylhexyl (C ６ ) silica gel column. Phenyl C ２ -C １０ Examples of alkyl silica gel columns include phenethyl (C ２ ) silica gel column, phenylpropyl (C ３ ) silica gel column, or phenylhexyl (C ６ Among these, C ６ Aryl C ２ -C １０ Alkyl silica gel column or phenyl C ２ -C １０ Alkyl silica gel columns are preferred, and phenylhexyl (C ６ ) silica gel column or pentafluorophenylpropyl (C ３ ) silica gel column is more preferred, and phenylhexyl (C ６ ) A silica gel column is more preferred.

[0032] The reversed-phase column used in the analysis method 1 can be a commercially available product, for example, octadecyl (C １８ ) Examples of silica gel columns include ACQUITY UPLC CSH C18 Column (Waters), phenylhexyl silica gel columns include InertSustain Phenylhexyl (GL Sciences), pentafluorophenylpropyl silica gel columns include InertSustain PFP (GL Sciences), and phenethyl silica gel columns include Inertsil Ph (GL Sciences).

[0033] In one aspect, the present invention provides a method for analyzing peptide compounds and oligomers derived from the peptide compounds in a sample, which comprises performing size exclusion chromatography using a styrene divinylbenzene column and an organic solvent as an eluent (hereinafter also referred to as "Analysis Method 2").

[0034] In one embodiment, analytical method 2 may separate and analyze a peptide compound and an oligomer derived from the peptide compound in a sample, may simultaneously analyze a peptide compound and an oligomer derived from the peptide compound in a sample, or may separate and simultaneously analyze a peptide compound and an oligomer derived from the peptide compound in a sample. Here, "simultaneous analysis" means that a target peptide compound and one or more oligomers derived from the peptide compound are detected by injecting a sample into size exclusion chromatography once.

[0035] The eluent used in Analysis Method 2 is an organic solvent. Examples of organic solvents include tetrahydrofuran (THF), 1,4-dioxane, n-hexane, cyclohexane, dodecane, toluene, benzene, xylene, 1-methyl-2-pyrrolidone (NMP), pyridine, quinoline, acetone, methyl ethyl ketone (MEK), o-dichlorobenzene (ODCB), 1,2,4-trichlorobenzene (TCB), 1-chloronaphthalene, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), chloroform, dichloromethane, carbon tetrachloride, dichloroethane, trichloroethane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / chloroform, m-cresol / chloroform, o-chlorophenol / chloroform, methanol / chloroform, ethanol, and ethyl acetate. Among these, tetrahydrofuran (THF) is preferable. In this specification, the expression "A / B" in the organic solvent means a mixed solvent of A and B, and there is no limitation on the ratio thereof.

[0036] Analysis method 2 involves size exclusion chromatography (SEC). Size exclusion chromatography separates peptide compounds or oligomers derived from the peptide compounds in a sample based on differences in molecular size. The column used in size exclusion chromatography has many pores, and as each compound passes through the column, small molecules penetrate the pores, while large molecules flow to the column outlet without penetrating the pores. As a result, larger molecules elute faster.

[0037] The column used in Analysis Method 2 is a styrene-divinylbenzene column based on a styrene-divinylbenzene copolymer. The average particle size of the styrene-divinylbenzene column is preferably 1 to 5 μm, more preferably 3 to 5 μm, and even more preferably 3 μm. The average pore size of the styrene-divinylbenzene column is preferably 1 to 10 nm, more preferably 1.5 to 7.5 nm, and even more preferably 7.5 nm. Commercially available styrene-divinylbenzene columns can be used, such as TSKgel (registered trademark) SuperH (Tosoh Bioscience) or PLgel (Agilent Technologies).

[0038] Analysis methods 1 and 2 are suitable for use in analyzing highly lipophilic peptide compounds. The ClogP of the peptide compound to be analyzed may be, for example, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, or 20 or less, 19 or less, 18 or less, or 17 or less. The ClogP of the peptide compound may also be, for example, 7 to 20, 8 to 20, 9 to 20, 10 to 20, or 11 to 20. The ClogP of the peptide compound is preferably 7 to 20, and more preferably 8 to 20, 9 to 20, 10 to 20, or 11 to 20. Here, ClogP is a partition coefficient calculated by a computer, and can be determined in accordance with the principles described in "CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )". As an example of a method for calculating ClogP, the CLOGP is calculated using the CLOGP Reference Manual published by Daylight Chemical Information Systems, Inc. Examples of calculations include using Daylight Version 4.95 (release date: August 1, 2011, ClogP algorithm version 5.4, database version 28, https: / / www.daylight.com / dayhtml / doc / release_notes / index.html) by Yahoo!

[0039] The principles described in the CLOGP Reference Manual Daylight Version 4.9 (release date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / ) are as follows: 1. Introduction The CLOGP Reference Manual willhelp the user understand why the CLOGP program calculates logP(ow) the way itdoes. Although the procedure cannot be derived from first principles, we havetried to make the rules consistent with solvation theory, if for no other reason than they are more easily remembered. The method simply adds togethervalues ​​for structural parts of a solute molecule and correction factors dependent upon the particular way the parts are put together. The CLOGP EXAMPLES sectioncontains example CLOGP calculations for a variety of chemicals and is designedas a companion to the CLOGP Reference Manual. An asterisk (*) appears in theCLOGP Reference Manual when one or more examples are provided in CLOGP ExampleCalculations to illustrate aspects of the CLOGP computation. Examples alsodemonstrate DEPICT (chemical metaphor).Funding for the development ofCLOGP was provided by the U.S. Environmental Protection Agency throughCooperative Agreement No. 809295, and we wish to acknowledge the encouragementand support of the project officer, Dr. Gilman Veith of ERL-Duluth. 1.1 Measurement and Past Uses ofPartition Coefficients The partition coefficient is theequilibrium concentration of solute in a non-polar solvent divided by theconcentration of the same species in a polar solvent. In this and most otherapplications, the polar solvent is water. The logarithm of the partitioncoefficient, log P, has been successfully used as a hydrophobic parameter in'extrathermo-dynamic' Hammett methodology.1-octanol has much to recommend itas the choice for the non-polar phase (1) and logP(ow) has been usedsuccessfully in Quantitative Structure Activity Relationships (QSAR) in thefollowing special fields: drug and pesticide design (2,3); pharmacokinetics(4); anaesthesiology (5); environmental transport and soil binding (6,7);toxicology (8); bioaccumulation (9); protein folding (10); enzyme binding(11,12); enzymic reactions in non-aqueous solvents (13); and host-guestcomplexation (14a,b). In principle, the measurement ofthe equilibrium concentration of solute in the octanol and water phases, aftershaking in a separatory funnel, is very simple, and since good measured valuesare always to be preferred over calculated ones, it would seem that thereshould be little need for a procedure to calculate them. As it turns out,reliable shake-flask measurements are time-consuming and often difficult tomake.The criteria for high reliability are: measurements over a 10-fold concentrationrange (with upper concentration no more than 75% of solubility or CMC, or nomore than 5% of the aqueous phase, whichever is lower) and standard deviationof 0.03 or less in log terms. This often requires working at sub-micromolarconcentrations, and so, with either UV spectrophotometry or gas chromatography,it means that the standard curves must be established with utmost care.Radiotracer methods seem well-suited for analyses at these low concentrations,but impurities as well as adsorption at phase boundaries (including containerwalls) can introduce significant errors. HPLC procedures provide a wayaround this bottleneck(15,16) and can save time if there is a limited varietyof structural types, and the log P values fall in the range of 0.5 to 4.0.MostHPLC procedures which are used to develop log P(ow) values do no use octanoland thus have to be referred to that system by standard curves which can bedifferent depending on whether the solutes do or do not contain certain basicfragments, such as pyridine nitrogen. If the solutes do not absorb well in theUV, difficulties in detecting the elution time eliminates any advantage HPLCmay have over the shake-flask method. Procedures which employ filterprobes (17) or solubility columns(18) speed up partition coefficientmeasurements by eliminating centrifugation as the means of phase separation.However, each has its own set of disadvantages and limit its acceptance as amethod for establishing the standard values for a calculation procedure. More efficient methods ofmeasurement of octanol / water partition coefficients are certain to be developedin the future, but no conceivable 'breakthrough' is likely to eliminate theneed for logP calculation.To put the problem in proper prospective, one needonly imagine some dedicated synthetic chemist making all possibletri-substituted benzoic acids with the methods commonly available today. Whenfinished, there would be five million analogs for which partition coefficientscould be determined. And of course only by calculation is one going to have anestimate of hydrophobicity before synthesis. The Pomona MedChem Project sawthese and other arguments as reason enough to develop a method to calculate logP(ow) from structure by an additive-constitutive procedure. As it turns out,the 'constitutive' portion of the procedure was, by the very nature of the twocompeting solvation equilibria, very complex, and the manual method requiredconsiderable effort before it could be applied with confidence.It is the aimof the second-generation program, CLOGP, to take most of the routinecalculation burden from the user but still encourage him to study the interplayof hydrophobic and polar solvation forces which can be so crucial to the designof bioactive chemicals. 1.2 How to Understand CLOGPCalculations The first published method forcalculating log P(ow) from structure (19) was based on a 'substitution'procedure and was developed with substituent pi constants for aromatic rings inmind. Of course this method was limited to deriving a new log P from a 'parent'structure whose log P was already known. Rekker(20) was the first to publish aprocedure which was more general in that it assigned 'fragmental constants' toa variety of structural pieces, and the calculated log P was the sum of thevalues appropriate for the molecule in question. The original pi system can beexpressed as:. while the expression for Rekker'sfragment system is: The method developed by PomonaMedChem (21) follows Rekker's general formulation, but there are some importantdifferences in the approach used to derive the actual working constants. Rekkerused a 'reductionist' approach - deriving the constants for carbon and hydrogenas well as those for polar fragments from a statistical treatment of a largebody of log P data which contained numerous interaction factors. Both thefragment values (f) and interaction factors (F) had to be identified andevaluated concurrently. Also, Rekker neglected to clearly define just whatconstitutes a fragment. Instead he provides a table in which the knownconstants can be found (see footnote). Rekker also treats all correctionfactors as some multiple of a 'Magic Number' (+0.28), but the selection ofmultiples was not made clear in his published work. Although his method gainedsome acceptance for manual calculation, we considered it too seriously flawedto serve as the basis for a computer method.In order to construct adependable, verifiable algorithm suitable for log P calculations used in developingQSAR at Pomona College, we first elected to clearly define what constitutes afragment. Next we chose a 'constructionist' approach to evaluate them; that is,we accepted as axiomatic that the hydrophobic portions of solutes were thosemost 'hydrocarbon-like', and defined these carbons and hydrogen fragment valuesas being truly constant. We gave very heavy emphasis to the carefully-measuredvalues for three solutes; molecular hydrogen, methane and ethane, because fromthese we could derive fragment constants for carbon and hydrogen, which wouldbe free of obscuring interactions. For all hydrocarbon structures more complexthan these, whose measured values were NOT the sum of fragment values, weattempted to define the difference in terms of universally-applicablecorrection factors.It appears that this approach has led not only to aworkable algorithm, but has highlighted the importance of certain types ofpolar solvation forces which have received insufficient attention in the past. The first attempt to reduce the'Pomona Method' of log P calculation to computer algorithm was made incollaboration with Dr. Jack Chou and Dr. Peter Jurs of Pennsylvania StateUniversity (22). It was called CLOGP. A great deal was learned in the processof developing this first version, and it certainly established the real needfor a 'stand-alone' program to make these calculations. Nevertheless, CLOGP wasdifficult to install and modify, and many well-known correction factors couldnot be implemented due to programming difficulties. In light of thisexperience, we deemed it essential to incorporate, in the second generationprogram, design features which would encourage its continuing evolution.Toachieve that objective, the program had to be conceived as a 'modeling system'which could operate from one or more easily-revised 'value files'. As anexample of the ease of updating, the largest fragment encountered to date is:. It took less than two minutes toenter it into the database and begin to use it in calculations. A number of significantimprovements have been made to CLOGP over the past few years. The mostsignificant improvement is the ability to estimate a polar fragment value whichhas not appeared in a solute having a measure log P (oct). This type of estimationis designated as 'calculated'. If the fragment in question has appeared in ameasured solute, but in a different bonding environment (e.g., aromaticattached when aliphatic attached is needed), the new 4.0 version allows for allextrapolations and designates the value as 'derived'. The methodology for the'No Missing Fragments' algorithm is explained in more detail in ref. 23. Earlier versions of CLOGP wereable to assign corrections to polar fragments interacting over two IsolatingCarbons (see Section 2. following). In the latest version, this distance hasbeen extended to three I.C.s.A significant improvement in steroid calculationsrecognizes the unique contribution of polar groups at the 11 position as wellas some long-range intramolecular hydrogen bonds. These and a few other minorimprovements will be noted in the changed values in the CLOGP ExampleCalculations, 6.3. 2. Fundamental Fragments In view of the decision to makealkane carbons and hydrogens the most fundamental fragments in the system, itis necessary to define these very carefully before defining the polar, morehydrophilic fragments. 2.1. Isolating Carbons An 'Isolating Carbon' (I.C.) atomis carbon which is NOT doubly- or triply- bonded to a hetero atom. An I.C. maybe bonded to a hetero atom by a single or an aromatic bond. This definition canbe made clearer from the following two examples:. In an earlier version of themanual calculation procedure (Ref. 21 p. 34) the Kekule structures forpyrimidine was considered; the earlier rule is now superceded. In coumarin,both rings are designated as aromatic, and the only carbon which is notisolating is the one in the carbonyl group, because it is doubly bonded to ahetero outside the ring. Although the hydrophobic value ofan I.C. is constant, several types must still be identified; the degree towhich they delocalize electrons in any polar fragments attached to them has agreat influence on overall log P. The types of I.C.s presently identified arelisted below with appropriate symbols: To be completely characterized, apolar fragment must have each of its 'valence bonds' designated with one of theabove symbols (see section "Fragment Valence Types"). The numericalvalue of the fragment will increase roughly in the order 'A' to 'a', but mustbe experimentally determined for high reliability. All hydrogens bonded to I.C.s arefragments. These two kinds of fundamental fragments are the most importantmembers of the non-polar class. A comparison of their relative values (C = 0.2;H = 0.225) is a reminder that the measure of effective cavity size may not beas simple as using van der Waals radii or CPK models. 2.2. PolarFragments A fragment is any atom or group ofatoms bounded by Isolating Carbon atoms, and all except hydrogen are consideredpolar. A fragment may have many internal bonds but those connecting it to I.C.sare called 'valence bonds'. Valence bonds are most often single, but can bearomatic, as in the case of the N fragments in pyrimidine shown above.Eachhydrogen in methane is a fragment, but the hydrogens in formaldehyde are notbecause the carbon to which they are bonded is not isolating. This is veryimportant to remember, for one frequently sees published calculations in whichone fragment value is obtained from another by the replacement of a fragmenthydrogen with another fragment of known value. At the present time a good ruleto follow is: "Never break up a Fragment; estimations can be made fromvalues measured for different bond environments (see below) but a Fragmentcannot be constructed from parts." Examples of fragments which cannot be"broken down" further are:. As will become evident in thefollowing sections, polar fragments can interact in various ways. To quantitatethis interaction it is necessary to define several types of polar fragments: (A) X = any halogen, but for onetype of interaction fluorine must be assigned to a special subclass,'F'. (B) Y= all non-X fragments; these are further subdivided according to: sensitivity to halogen interactionas 'Y-1', 'Y-2', and 'Y-3' containing '-OH' or not. 2.3.Intrinsic Values Here the term, 'intrinsic'fragment values, means those which would, if summed, yield the correct log Pwithout any correction factors. It is worthwhile to examine some of theaccepted hypotheses as to what solvation forces or other phenomena determinethese intrinsic values. It takes more energy to form acavity in water than in octanol. One would predict, therefore, that increasingthe size of a solute would increase its log P. Other factors being equal, thisappears to be the case.However, other features of the solute can partly orcompletely override the effect of its size. Water is much more capable than isoctanol of accommodating localized dipoles, and it contains, on a molar basis,more hydrogen bond accepting and donating groups. So it is these three factors-size, localized dipole strength, and H-bonding ability - which largelydetermine the sign and magnitude of any fragment value. 2.3.1Halogens Halogens form an intense localizeddipole when bonded to an aliphatic carbon atom.* This intensity is somewhatlessened if the I.C. is benzyl and greatly lessened if it is vinyl, styryl oraromatic.* Fluorine has a negative fragment value when attached to an aliphaticcarbon, because the dipole effect outweighs the effect of size. Size is ofgreater importance with chlorine and bromine, but even bromine is lesshydrophobic than a hydrogen in an aliphatic setting.As will become evident inthe following section, much of this hydrophilic polar effect can be lostthrough 'shielding' by other halogens, or by electronic interaction with 'Y'type polar groups. 2.3.2H-Polar Fragments H-Polar Fragments ('Y') almostuniversally form some sort of hydrogen bonds with the donor (H) or acceptor (O)of the aqueous phase. This is thought to interrupt the peculiar 'ice-like'water shell which forms around the non-polar, hydrocarbon-like portions of eachsolute molecule, and thereby effectively reduces cavity size. As noted above,this should reduce log P. 2.3.3Ions Octanol can accept some largersolutes containing a full formal charge in sufficient concentration formeasurement. However, one must be careful that the species measured is thesame, because water easily supports complete ionization while ion-pairing isthe usual condition in octanol except at the very lowest concentrations.Consistent values can be obtained if 'standard conditions' are adhered to: 0.1M small counter-ion (Na+ or Cl-) and extrapolation to infinite dilution. Measuredin this way a carboxylate ion is about 4.1 log units lower than theundissociated acid. No single value can be given to the positive charge on aprotonatedamine or quaternary ammonium, because the charge is delocalized alongthe hydrocarbon chain and thus the effect is dependent on chain-length. Itshould be emphasized at this point that, except for zwitterions, CLOGPcalculates values for the neutral solute only.* 2.3.4Unsaturations Double bonds in isolation have aslightly negative effect on log P.* This effect may arise from the polarity ofthe pi electrons or else it may be due to the shorter bond length reducingcavity size. At any rate, it disappears if the double bonds are conjugated.*Triple bonds are decidedly hydrophilic and require a large negative correctionfactor. 3.Correction Factors 3.1Structural Factors 3.1.1Bonds To properly perform itscalculations, CLOGP needs to know the number and types of certain bonds in thesolute structure. There is some reason to believe that, for the bonds inquestion, factors other than bond length affect the size of the solvent cavityneeded to contain the hydrophobic portions of the solute molecule. The effect of all bonds within anyfragment is taken care of by the fragment value, and so it is NOT necessary tokeep track of them, NOR of any bonds to hydrogen. And, as explained below, itis convenient to allow for the bond effect in aromatic rings by including it ina special aromatic I.C. type, 'aromatic carbon'; therefore, aromatic bonds alsoare NOT given special attention. Bonds which DO need to beidentified are the following: 3.1.1.1Chain bonds Chain bonds are non-ring bondsbetween I.C.s plus any valence bonds to fragments*. 3.1.1.2Ring Bonds Ring bonds are non-aromatic ringbonds between I.C.s plus any valence bonds to fragments*. 3.1.1.3Branch Bonds Branch bonds are chain bondsemanating from 'Branched Fragments' (a fragment type presently limited tophosphate esters) and counted to the last I.C. preceding any polar fragment.* A separate count of each of thesebonds types must be made, and a negative correction applied. For chain bondsonly, this correction applies to bonds AFTER the first in each chain. Forexample, there is no net bond correction for ethane but there is one for propane.This suggests that the correction accounts for flexing of the chain which isnot possible in methane or ethane. 1,2-diethylbenzene gets only a net of twobond corrections, because each chain is counted separately. Also compatiblewith a 'flexing' hypothesis is the fact that the correction is greater forchains than for aliphatic rings. As noted above, an isolated doublebond is assigned a negative correction factor (-0.09). This factor actuallybecomes slightly positive if the double bonds are conjugated in a ring such asbenzene.Since it is much more convenient to assign all bonds in large fusedring systems as aromatic type, rather than using the Kekule system ofalternating doubles and singles, it is worthwhile to assign a special fragmentvalue to an aromatic carbon and include all the necessary bonding effectstherein. The value of aliphatic carbon is +0.20;the value for aromatic carbonwhich includes all bond effects associated with the aromatic ring system is+0.13.* 3.1.2Branching at Isolating Carbons 3.1.2.1Chain Branch It is well-known that iso-alkanesare more water-soluble than their n-isomers.* This branching evidently does notproduce a corresponding solubility increase in the octanol phase in thepartitioning process, because the correction required in CLOGP is negative insign. In CLOGP, the concept of branchingwas expanded, and now replaces the earlier use of the 'ring' cluster'correction(21).Fusion carbons in non-aromatic rings are considered as branchedand given the same correction factor as chains; i.e., -0.13.* They aredesignated cluster branches. 3.1.2.2Group Branch If an H-Polar group branches froman I.C. the increase in water solubility, compared to the n-isomer, is evengreater than with chain branching. Again this carries over to partitioningequilibrium; H-Polar group branching requires a larger correction than doeschain branching. For this reason isopropyl alcohol is given one group branchcorrection and no chain branch is considered.* Tertiary butyl alcohol gets oneof each type.* If a fragment has more than twoexternal(valence) bonds, it could be considered a branching point. However, inall cases except the 'Branched Fragments' noted above (t-amines and phosphateesters), the entire negative branching effect is included in the fragment valueitself. Only in the case of the 'Branched Fragments' is the effect chain-lengthdependent. 3.2Interaction Factors 3.2.1Aliphatic Proximity (Measured Topologically) 3.2.1.1Halogen vs. Halogen (X vs. X) The positive correction to log Pfor this interaction is thought to result from dipole shielding and is limitedto halogens on the same (geminal) or adjacent (vicinal) I.C.s. The geminalinteraction is designated 'X-C-X', and the corrections can be thought to ariseas follows: adding a second halogen to an I.C. which already has one createsthe first X-C-X pair, and the correction required is +0.60.* Adding the thirdhalogen to the same I.C. creates two more such paintings, each of whichrequires a correction of +0.5.* If the fourth halogen is added, the dipole isalmost completely shielded, and the three additional pairings requirecorrections of +0.40 each.* For carbon tetrachloride the total geminal halogencorrection would be: EQ on pg. 12 For the vicinal halogencorrection, X-C-C-X, the bond between carbons must not be double.* Thecorrection is evaluated by subtracting one from the number of halogens meetingthe structural requirement and multiplying by the factor 0.28. (Again Rekker'sMagic Constant pops up!) 3.2.1.2H-Polar vs. H-Polar (Y vs. Y) As noted in the section on'Intrinsic Values', the negative sign on the 'Y' fragments is thought to resultfrom their 'structure-breaking' (and thus cavity-reducing) ability in the waterphase. 'Y' fragments appear to eliminate the cavity requirement for two or moreI.C.s to which they are attached. Obviously if two 'Y' fragments are located onthe same or adjacent I.C.s to which they are attached. Obviously if two 'Y'fragments are located on the same or adjacent I.C.s some of this cavityreduction is going to be counted twice. Thus a positive correction factor iscalled for when the topological separation is less than three I.C.s.The CLOGPalgorithm is an improvement over the original Rekker procedure(20) in that, inplace of the same correction for every Y-C-Y or Y-C-C-Y, it makes thecorrection proportional to how much hydrophilic character (negative fragmentvalue) is involved. This proportionality appears to apply even if one of thefragments is charged and has a highly negative value, but as previously noted,the CLOGP algorithm currently does not treat ions. If one of the 'Y' fragments in aY-C-Y interaction contains an -OH moiety (e.g. -NHOH, -COOH, or -OH itself), agreater proportion of the hydrophilic character of the pair is lost. Thecoefficient by which the fragment sum is multiplied increases from 0.32 to0.42.* If both the 'Y' fragments and thecarbons of Y-C-C-Y are in a ring, the hydrophilicity loss is not as great as ifthey are all in a chain (coefficient 0.26 vs. 0.20).* If one 'Y' is asubstituent on the ring while the other is in the ring, the correctioncoefficients are averaged (0.23).* If one of the carbons has two 'Y' fragments,the geminal correction is applied first; then, for the (Y-C-C,Y'Y'')correction, both pairings are calculated and averaged.* If both I.C.s havegeminal 'Y' fragments, then the vicinal correction is not applicable.* (Seepenicillin in EXAMPLES). 3.2.1.3Halogen vs. H-Polar (X vs. Y) The interaction being consideredat this point is limited to that which takes place across single bonds. It istherefore, probably due to an inductive or field effect. (The electronicinteraction between fragments on or in aromatic rings is discussed in thefollowing section.) In evaluating the X-C-Y correction factor, all halogens canbe treated alike. However, there are at least three levels of sensitivity shownby 'Y' type fragments. In CLOGP the most sensitive class, 'Y-3', is restrictedto the structural type: -SO2-R.* 'Y-2' consists of the types: -CONH-R, -O-R,-S-R, and -NH-R; and 'Y-1' of all other, H-polar fragments.* The correctionfactor for the first alpha-halogen (i.e. X-C-Y) is the same for all threeY-types (+0.9). For 'Y-3' fragments, this correction factor is doubled whenthere are two alpha halogens (X{2}-C-Y3) and tripled when there are three(X{3}-C-Y3). For 'Y-2' fragments, the second and third alpha-halogens need muchless correction, and for 'Y-1', virtually none. In the case of multiplehalogenation, the X-C-X and the X-C-Y corrections are additive. The CLOGP algorithm makes noseparation of 'Y' types to make the X-C-C-Y correction, but needs todistinguish fluorine from the other 'X' halogens.* 3.2.2Electronic (through Pi-bonds) 3.2.2.1Fragment Valence Type As previously noted, all fragments(X or Y type) are assigned the most negative values when bonded to aliphaticI.C.s (designated as 'A'). This can be considered as the 'base' or 'intrinsic'level. If the fragment value when attached to a vinyl I.C.(V) has not beenmeasured it can be estimated as the average of the base and aromatic-bonded (a)values.* Likewise the value for the styryl-attached fragment can be estimatedas two-thirds the way from the base to the aromatic value.* CLOGP will onlymake these estimations when measured values have not been entered in thedatabase. 3.2.2.2Extension of Aromaticity The extension of the aromatic ringsystem through fusion (as in naphthalene or direct substitution (as inbiphenyl)) appears to increase log P, especially if the heteroaromatic atom isnext to the juncture.* If the ringjoining carbons are attached only to otheraromatic carbons, electron delocalization is minimal and so is the correction:+0.10 for each I.C. If the I.C.s are also attached to a polar (fused-in)fragment, such as in quinoline or 2-phenylpyrimidine, the correction isgreater, +0.31.* 3.2.2.3Sigma / Rho Fragment Interaction When two or more X and / or Y typefragments are attached to an aromatic ring system, the correction factors canbe calculated by a method very similar to that used by Hammett(23) to calculatethe electronic effects in other equilibria, such as acid ionization.* Thisrequires the assignment of a measure of electronic 'strength' (sigma) and'susceptibility' (rho). In dealing with electronic effects on partitioningequilibria, a few fragments appear to act 'bidirectionally' and require bothsigma and rho values, although they cannot, of course, act upon themselves.Most of the details of sigma and rho assignment to X and Y type fragments canbe found elsewhere (24), but it should be pointed out that the latest versionof the program (CLOGP) follows a newer procedure for 'fused-in' fragments.Fragments fused in aromatic rings(e.g.-N= or >C=O) may also be assignedboth rho and sigma constants and treated together with 'on-ring' fragmentsinstead of requiring a separate treatment.* Fragments on different rings in anaromatic ring system interact with one another but the effect is attenuated.*If the two rings in the system are fused, as in 5-acetyl-1-naphthylamine, the'intrinsic' effect is only half the sigma rho product just as it is in thebiphenyl system such as in 4-(m-chlorophenyl)aniline. One frequently encounters aromaticring systems containing several fragments with rho and sigma values assigned,and the potential correction from all cross products of sigma / rho could be verylarge. Since these multiple effects are NOT additive, some scaling downprocedure was indicated. The one chosen for CLOGP takes the following steps: a) The full potential sigma / rhoproduct for each possible interaction is calculated and placed in descendingvalue order, AFTER considering if the fragment pair are on the same or separaterings.b) Except for the sigma for apyridine type nitrogen, each use of sigma or rho causes it to 'age'. The firstinteraction at the top of the list is entered at full potential because thecurrent age of its sigma and rho components is 'zero' for each. Each usereduces the effective sigma or rho value to 1 / 2 its previous value, and so ifeach were at 'age 1', the increment to the correction would only be 1 / 4 as muchas a 'fresh' interaction. The mechanics of this computation are best understoodby looking at the detailed output of some complex structures. Two such examplesare provided in the example section.* 3.2.3Special Ortho As noted in the previous section,aromatic substituent (fragment) pairs, if they have sigma and rho valuesassigned to them, are given the same correction factor regardless of theirrelative position on the ring. It is important to keep in mind that if thefragment pair are on adjacent positions (i.e. ortho), an additional correctionmay be required. 3.2.3.1Crowding 'Crowding' of certain fragmenttypes can effectively lower their aromatic-attached values. This is mostapparent in the case of fragments attached to the aromatic ring through ahetero atom which possesses an electron pair, such as -NHCOCH3.* A reasonableexplanation of this observation is that the lone pair can no longer remain inthe plane of the ring, making the fragment attachment resemble aliphatic (A)rather than true aromatic (a). The magnitude of the correction appears todepend on both steric and electronic (field) effects(25). If this explanation is valid, onewould expect the correction to vary continuously up to a maximum characteristicof each fragment type. It was surprising, therefore, to find that the ratherlarge data set used in the original evaluation of the 'negative ortho' effect(24) seemed to fit multiples of Rekker's Magic Constant(20). This is handled inCLOGP by assigning integers to a matrix which has generalized fragment typesfor coordinates.More recent data provides manyexamples which do not support this 'quantized' correction. Nevertheless, it isbeing retained for the present because of simplicity and because its maximum isonly 0.14. 3.2.3.2Intra-Molecular Hydrogen Bonding Hydrogen bonding is known to occurintramolecularly between two ortho subsituents if one is a donor and the otheran acceptor. A classical example of such an H-bond is that in o-nitrophenol. Asmight be expected, an intramolecular H-bond reduces water's ability toaccommodate that solute, and the log P of o-nitrophenol is over two log unitshigher than the m- and p-isomers in the heptane and carbon tetrachloridesolvent systems. One must always keep in mind, however, that the octanol phasepossesses both H-donor and H-acceptor capability, not only because it is analcohol, but because of the 2M water present at saturation.In actuality, the presenceof the intramolecular H-bond in o-nitrophenol penalizes solvation in octanolslightly more than it does solvation in water, and its log P is 0.09 log unitslower than the m- and p-isomers. In terms of intramolecularH-bonding between aromatic ortho substituents, the octanol / water system appearsto be sensitive to a very restricted class. The only clear-cut cases seem toresult from a carbonyl group directly attached to the ring acting as acceptor,and a directly-attached -OH or -NH- acting as donor.* In all of the casesobserved so far, the correction is very close to +0.63, and is stored in thesame matrix used for the 'negative ortho' corrections. Thus the 'crowding' andH-bonding ortho effects never are applied simultaneously, but a sigma / rho correctioncannot be added to either. 4. Summary The fragment method of calculatinglog P(ow) has been proved valuable in many fields, including drug design andhazard assessment.However, manual calculations require a great deal ofinstructions and become very lengthy for complex structures and thus areerror-prone. The computer program, CLOGP, enables the method to be applied bynon-experts and includes an estimate of error, which is not possible in thefuture. Regular users can avail themselves of an annual update which will bringthem current with all newly measured fragment values and improved correctionfactors. Versions with the Unified Driver compare the calculation fromstructure with a measured value from log P(ow) for neutral solutes. Starlist isalso included in the annual updating service. We plan to make available in thenear future a searching program, GENIE, which will extend the search ofStarlist to close analogs. The current literature containsmany examples of QSAR accompanied by calculations of hydrophobicity which havenot been made according to a consistent application of the rules they purportto follow.This has caused some confusion and cast doubt upon the entireapproach. Perhaps, if the use of CLOGP becomes more widespread, published calculationswill become more comparable, especially if reference is made to the programversion. Any prediction of the future isrisky, but, judging from the recent past, we can expect an increasing demandfor logP(ow) values. It is inconceivable that CLOGP will be perfected to suchan extent that it supplants partition coefficient measurement. The two methodsshould remain as they are now: mutually complimentary. With the STARLIST module in CLOGP,the user will be able to check the calculated value against an acceptablemeasured value if the solute structure entered is one of over 4,000 containedin that special file which is limited to non-tautomeric structures measured ata pH where the neutral form predominates. 5.Bibliography 1) Smith, R., Hansch, C. Ames, M.,J. Pharm. Sci., 64, 599 (1975). 2) Martin, Y., Quantitative DrugDesign, Medicinal Research Series No. 8, Marcel Dekker, New York, 1978. 3) Magee, P., Chemtech, 11, 378(1981). 4) Smyth, R., Pfeffer, M., VanHarken, D., Cohen, A. and Hottendorf, G. Antimicrob. Agents Chemother., 10,1004, (1981). 5) Koblin, D., Eger II,E.,Johnson, B.,Collins, P., Terrell, R., and Spears, L., Anesth. Analg., 60,464 (1981). 6) Brown, D. and Flagg, E., J.Environ. Qual., 10, 382 (1981). 7) Ellinghausen, H.,Guth, J. andEser, H., Ecotox. Environ. Safety, 4, 26 (1980). 8) Levitan, H., Proc. Nat. Acad.Sci. USA, 74, 2914 (1977). 9) Neeley, W., Branson, D. andBlau, G., Environ. Sci. Technol., 8, 1113 (1974). 10) Tanford, C., The HydrophobicEffect, Wiley, New York, 1980, 2nd Ed., Ch. 13. 11) Coats, E., Genther, G., Dietrich,S., Guo, Z., and Hansch, C., J. Med. Chem., 24, 1422 (1981). 12) Smith, R., Hansch, C. andLangridge, R., Arch. Giochem. Biophys., 215, 319 (1982). 13) Martinek, K., and Semenov,A.,J. Appl. Biochem., 3, 93 (1981). 14a) Newcomb, M., Moore, S.andCram, D., J. Am. Chem.Soc.,99, 6405 (1977). 14b) Hansch, C., J. Org. Chem., 43, 4889 (1978). 15) Unger, S. and Feuerman, T.,J.Chromatog., 176, 426 (1979). 16) Mirrlees, R. and Taylor, P.,J. Med. Chem., 19, 615 (1976). 17) Tomlinson, E., Proceedings ofA.Ph.A., Symposium on Partition Coefficients, San Diego, CA, (1982). 18) Tewari, Y., Miller, M., Wasik,S. and Martire, D., J. Chem. Eng. Data, 27, 451 (1982). 19) Fujita, T., Iwasa, J. and Hansch,C.,J. Am. Chem. Soc.,86, 5157(1964). 20) Rekker, R., The HydrophobicFragmental Constant, Elsevier, Amsterdam (1977). 21) Hansch, C. and Leo, A.,Substituent Constants for Correlation Analysis in Chemistry and Biology, WileyInterscience New York (1979). 21) Leo., A. and Hoekman, D.,Perspect. in Drug Discov. & Design, 18, 19 (2000). 22) Chou, J. and Jurs, P., J.Chem. Inf. Comput. Sci., 19, 172 (1979). 23) Hammett, L., Physical OrganicChemistry: Reaction Rates, Equilibria and Mechanism, McGrawHill, New York, 1970,2nd Ed. 24) Leo, A., J. Chem. Soc., PerkinTrans. II, 825 (1983). 25) Ogino, A., Matsumura, S. andFujita, T., J. Med. Chem., 23, 437 (1980). 26) Leo, A. and Hansch, C.,J. Org.Chem., 36, 1539, (1971).

[0040] The ClogP / total aa of the peptide compound to be analyzed may be, for example, 1.0 or more, 1.1 or more, or 1.2 or more, or 1.8 or less, 1.7 or less, 1.6 or less, or 1.5 or less. Furthermore, the ClogP / total aa of the peptide compound may be, for example, 1.0 to 1.8, 1.0 to 1.7, 1.1 to 1.6, or 1.1 to 1.5. The ClogP / total aa of the peptide compound is preferably 1.0 or more, more preferably 1.1 or more, preferably 1.8 or less, or 1.7 or less, more preferably 1.6 or less, or 1.5 or less, preferably 1.0 to 1.8 or 1.0 to 1.7, and more preferably 1.1 to 1.6 or 1.1 to 1.5. As used herein, "total aa" refers to the total number of amino acids constituting the peptide compound. For example, the total aa of a cyclic peptide compound in which the cyclic portion consists of 10 amino acids and the linear portion consists of 1 amino acid is 11. ClogP / total aa is calculated by dividing ClogP calculated by the above method by total aa.

[0041] The number of amino acid residues constituting the peptide compound to be analyzed is not limited, and may be, for example, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, or 20 or less, 14 or less, 13 or less, 12 or less, or 11 or less. The number of amino acid residues constituting the peptide compound may be, for example, 7 to 20, 7 to 14, 10 to 14, 10 to 11, or 11. The number is preferably 7 to 14, more preferably 10 to 14 or 10 to 11, and even more preferably 11.

[0042] The number of N-substituted amino acids contained in the peptide compound to be analyzed is not limited, and may be, for example, 3 or more, 4 or more, or 5 or more, or 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, 9 or less, or 8 or less. The number of N-substituted amino acids contained in the peptide compound is preferably 3 or more, more preferably 4 or more or 5 or more, and also preferably 20 or less or 15 or less, more preferably 14 or less, 13 or less, 12 or less, 10 or less, 9 or less, or 8 or less. Here, the N-substituted amino acid is preferably an N-alkyl amino acid, and N-C １ -C ６ An alkyl amino acid is more preferred, and an N-methyl amino acid or an N-ethyl amino acid is even more preferred.

[0043] The peptide compound to be analyzed is preferably a cyclic peptide compound having a cyclic portion. The number of amino acid residues constituting the cyclic portion is not limited, and may be 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, or 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less. The number of amino acid residues constituting the cyclic portion may be, for example, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 11 to 15, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 11 to 13, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 11 to 12, 7 to 11, 8 to 11, 9 to 11, 10 to 11, or 11. The number of amino acid residues constituting the cyclic portion is preferably 7 or more, more preferably 8 or more or 9 or more, and preferably 15 or less, more preferably 14 or less or 13 or less. The number of amino acid residues constituting the cyclic portion is preferably 7 to 15 or 7 to 14, more preferably 8 to 14, 8 to 13, 9 to 14, or 10 to 14, and even more preferably 11. Furthermore, the number of N-substituted amino acids contained in the cyclic portion is not limited, and may be, for example, 3 or more, 4 or more, or 5 or more, or 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, 9 or less, or 8 or less. The number of N-substituted amino acids contained in the cyclic portion is preferably 3 or more, more preferably 4 or more, or 5 or more, and preferably 20 or less or 15 or less, more preferably 14 or less, 13 or less, 12 or less, 10 or less, 9 or less, or 8 or less. Here, the N-substituted amino acid is preferably an N-alkyl amino acid, and N-C １ -C ６ An alkyl amino acid is more preferred, and an N-methyl amino acid or an N-ethyl amino acid is even more preferred.

[0044] In one aspect, when the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, it is preferable that the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in the side chain thereof.

[0045] As used herein, the term "substituted hydroxyphenyl group" refers to a group in which at least one hydrogen atom on the aromatic ring of a hydroxyphenyl group is substituted with a substituent. Although not intended to be limiting, an example of a substituted hydroxyphenyl group is a 3-fluoro-4-hydroxyphenyl group. Note that the hydrogen atoms on the aromatic ring in this specification do not include the H of the hydroxy group (-OH) in the hydroxyphenyl group. For example, a methoxyphenyl group is not included in either the "substituted hydroxyphenyl group" or the "unsubstituted hydroxyphenyl group" in this specification.

[0046] As used herein, the term "unsubstituted hydroxyphenyl group" refers to a hydroxyphenyl group that has no substituent. Although not intended to be limiting, an example of an unsubstituted hydroxyphenyl group is a 4-hydroxyphenyl group. Furthermore, substituted hydroxyphenyl groups and unsubstituted hydroxyphenyl groups may be collectively referred to as "substituted or unsubstituted hydroxyphenyl groups."

[0047] In one aspect, when the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, it is preferable that the cyclic peptide compound does not have a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group in the side chain of the cyclic portion.

[0048] In one embodiment, when the cyclic peptide compound has an acidic side chain at the cyclic moiety, the pKa of the acidic side chain may be 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more, or may be 10 or less. The pKa of the acidic side chain may also be 3.5 to 10, 4.0 to 10, 4.5 to 10, or 5.0 to 10.

[0049] In one embodiment, when the cyclic peptide compound has a basic side chain at the cyclic moiety, the basic pKa of the basic side chain may be 10 or less, 9.5 or less, 9.0 or less, or 8.5 or less, or may be 4.0 or more. The basic pKa of the basic side chain may be 4.0 to 10, 4.0 to 9.5, 4.0 to 9.0, or 4.0 to 8.5.

[0050] As used herein, an "acidic side chain" refers to a side chain having a pKa of 10 or less, and a "basic side chain" refers to a side chain having a basic pKa of 4 or more. As used herein, a side chain having a pKa of more than 10 and a side chain having a basic pKa of less than 4 are defined as neutral side chains. In addition, as used herein, the pKa and basic pKa of the side chain of the cyclic portion of a cyclic peptide compound can be calculated using ADMET Predictor (Simulations Plus Inc., ver. 8.0) using a partial structure obtained by extracting the side chain portion from the side chain β position (carbon directly connected to the main chain). The calculated pKa and basic calculated pKa are calculated using a partial structure obtained by extracting the side chain portion from the side chain β position (carbon directly connected to the main chain). As an example, the case of Lys is shown below. The basic calculated pKa was 10.5 when calculated using a partial structure containing the side chain β-position (carbon directly bonded to the main chain). Similarly, for acids, the calculated pKa of the side chain carboxy group of Asp was 4.3, the calculated pKa of the side chain phenolic hydroxyl group of Tyr was 9.9, the calculated pKa of the side chain phenolic hydroxyl group of 3-fluorotyrosine (Tyr(3-F)) was 8.7, and the calculated pKa of tetrazole was 3.7. On the other hand, for bases, the basic calculated pKa of the side chain guanidino group of Arg was 12.7, the basic calculated pKa of the imidazolyl group of His was 7.6, and the basic calculated pKa of pyridine was 5.4.

[0051] Some of the amino acids with basic side chains for which the Basic calculated pKa values ​​were calculated using the method described herein are shown in the table below.

[0052] Some of the amino acids with acidic side chains for which pKa values ​​have been calculated using the methods described herein are listed in the table below.

[0053] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 7 to 14, and the ClogP being 7 to 20.

[0054] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, and the ClogP being 8 to 20.

[0055] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 11, and the ClogP being 11-20.

[0056] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 7 to 14, and ClogP / total aa being 1.0 to 1.8.

[0057] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, and ClogP / total aa being 1.0 to 1.8.

[0058] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, and ClogP / total aa is 1.0 to 1.8.

[0059] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 7 to 14, the ClogP being 7 to 20, and the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0060] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, the ClogP being 8 to 20, and the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0061] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 11, the ClogP being 11 to 20, and the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0062] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 7 to 14, the ClogP being 7 to 20, the side chain of the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10.

[0063] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, the ClogP being 8 to 20, the side chain of the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic side chain has a basic pKa of 4.0 to 10.

[0064] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, the ClogP is 11 to 20, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.

[0065] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 7 to 14, ClogP / total aa being 1.0 to 1.8, and the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0066] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, ClogP / total aa being 1.0 to 1.8, and the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0067] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 11, ClogP / total aa being 1.0 to 1.8, and the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof.

[0068] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 7 to 14, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.

[0069] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion being 8 to 14, ClogP / total aa being 1.0 to 1.8, the cyclic portion not having an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.

[0070] In one embodiment, the peptide compound to be analyzed is a cyclic peptide compound having a cyclic portion, the number of amino acid residues constituting the cyclic portion is 11, ClogP / total aa is 1.0 to 1.8, the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and if the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10, and if the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.

[0071] In some embodiments, the cyclic peptide compound to be analyzed is one selected from the group consisting of the peptide compounds set forth in Tables 5-7.

[0072] In one embodiment, the oligomer derived from the peptide compound to be analyzed may include a dimer of the peptide compound, a dimer and a trimer of the peptide compound, or a dimer, trimer, tetramer or higher oligomer of the peptide compound.

[0073] In one embodiment, the oligomer derived from the peptide compound to be analyzed may be one or more selected from the group consisting of a dimer and a trimer of the peptide compound, and may be a dimer of the peptide compound or a trimer of the peptide compound.

[0074] In one embodiment, the oligomer derived from the peptide compound to be analyzed is a cyclic oligomer generated as a by-product under peptide cyclization conditions. In this case, the cyclic oligomer may include a cyclic dimer of the peptide compound, a cyclic dimer and a cyclic trimer of the peptide compound, or a cyclic dimer, a cyclic trimer, a cyclic tetramer or higher cyclic oligomer of the peptide compound.

[0075] In one embodiment, the oligomer derived from the peptide compound to be analyzed is a cyclic oligomer generated as a by-product under peptide cyclization conditions. In this case, the cyclic oligomer may be one or more selected from the group consisting of a cyclic dimer and a cyclic trimer of the peptide compound, or may be a cyclic dimer or a cyclic trimer of the peptide compound.

[0076] Production Method In one aspect, the present invention is a method for producing a composition containing a peptide compound, comprising the steps of: i) providing a composition containing a desired peptide compound; and ii) analyzing the peptide compound and oligomers derived from the peptide compound in the composition using the analytical method of the present invention (hereinafter also referred to as "Production Method 1").

[0077] In one aspect, the present invention provides a method for producing a pharmaceutical formulation containing a peptide compound, comprising the steps of: i) providing a composition containing a desired peptide compound; ii) analyzing the peptide compound and oligomers derived from the peptide compound in the composition using the analytical method of the present invention (hereinafter also referred to as "Production Method 1"); and iii) formulating the composition to provide a pharmaceutical formulation (hereinafter also referred to as "Production Method 2").

[0078] In some embodiments, the production methods 1 and 2 may further comprise a step of cyclizing the precursor of the desired peptide compound before step i). As used herein, "cyclization" refers to forming a cyclic moiety at the peptide portion of the peptide compound.

[0079] In some embodiments, the content of the peptide compound in the composition in step ii) of Production Methods 1 and 2 is 90% or more, 95% or more, 98% or more, or 99% or more, based on the total amount of the composition, as determined by the UV area value at 220 nm by HPLC analysis.

[0080] In some embodiments, the content of oligomers derived from peptide compounds in the composition in step ii) of Production Methods 1 and 2 is less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 1%, or an undetectable amount, based on the total amount of the composition, as determined by the UV area value at 220 nm by HPLC analysis.

[0081] In some embodiments, the composition in Production Method 1 may be a pharmaceutical composition.

[0082] For the formulation in step iii) of production method 2, a formulation method known to those skilled in the art can be used.

[0083] In some embodiments, the peptide compounds in Production Methods 1 and 2 may be any of the peptide compounds listed in Tables 5-7.

[0084] In this specification, the term "to" indicating a numerical range includes both ends of the range. For example, "A to B" means a numerical range that is equal to or greater than A and equal to or less than B.

[0085] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus and minus 10% of that numerical value.

[0086] 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, and (vii) A, B, and C.

[0087] The present invention is further illustrated, but not limited, in the following examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0088] The following abbreviations are used in the examples: AA: Amino acid DBU: 1,8-diazabicyclo[5.4.0]-7-undecene DCM: Dichloromethane DIPEA: N,N-diisopropylethylamine DME: Dimethytoxyethane, ethylene glycol dimethyl ether DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride FA: Formic acid HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HFIP: 1,1,1,3,3,3-hexafluoroisopropyl alcohol HMDS: Hexamethyldisilazane HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxybenzotriazole HPLC: High performance liquid chromatography IPAC: Isopropyl acetate MEK: Methyl ethyl ketone MeCN: Acetonitrile, CH ３ CN MeOH: Methanol MeTHF: 2-Methyltetrahydrofuran MS: Mass spectrometer NMI: N-methylimidazole NMM: N-methylmorpholine NMR: Nuclear magnetic resonance SEC: Size exclusion chromatography TCFH: Chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate TFA: Trifluoroacetic acid TFE: 2,2,2-trifluoroethanol THF: Tetrahydrofuran TIPS: Triisopropylsilane TMSOTf: Trimethylsilyl trifluoromethanesulfonate UV: Ultraviolet-visible absorption

[0089] The cyclic peptides used in this example are shown in Tables 5 to 7.

[0090] Example 1 Synthesis of Cyclic Peptide Compounds In Example 1, high-performance liquid chromatography (HPLC) analysis, liquid chromatography mass spectrometry (LCMS), and ion chromatography (IC) analysis were performed using any of the analytical conditions described below. Each compound was detected using a variable UV detector or a mass spectrometer, but other methods such as a photodiode array detector may also be used.

[0091] Analysis condition-1 (HPLC-UV) Instrument: Waters ACQUITY UPLC H-Class system Column: Waters ACQUITY UPLC CSH C18, 1.7 μm, 2.1 mm x 150 mm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.3 mL / min Column temperature: 50°C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 20% B (0 min) → 100% B (24 min) → 100% B (29 min) → 20% B (29.1 min) → 20% B (34 min)

[0092] Analysis condition-2 (HPLC-MS) Instrument: ThermoVanquish HPLC system + Thermo Q Exactive orbitrap mass spectrometer Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm x 150 mm Mobile phase A: 0.1% formic acid / water Mobile phase B: 0.1% formic acid / 2-propanol Ion mode: Negative Flow rate: 0.2 mL / min Column temperature: 50°C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0093] Analysis condition 3 (HPLC-UV) Instrument: Waters ACQUITY UPLC H-Class system Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm x 150 mm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / 2-propanol Flow rate: 0.2 mL / min Column temperature: 50°C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0094] Analysis condition-4 (HPLC-UV) Apparatus: Waters Alliance HPLC system Column: TOSOH TSKgel SuperH3000, 6.0 mmID x 15 cm, 3 μm Mobile phase: Tetrahydrofuran Flow rate: 0.45 mL / min Column temperature: 35°C Detection wavelength: 220 nm Elution method: Isocratic mode Analysis time: 30 min

[0095] Analysis Conditions - 5 (IC) Instrument: 930compact IC flex system (Metrohm) Column: Metrosep C4-150 / 4.0, 4.0 mmID x 15 cm, 5 μm Mobile phase: 2 mM HNO3 - 1 mM Oxalic acid - 2 mM Crown / 50% MeCN solution Flow rate: 1.0 mL / min Column temperature: 35°C Detector: Electrical conductivity detector Elution method: Isocratic mode Analysis time: 15 min

[0096] Analytical conditions - SQFA05 (HPLC-MS) Instrument: Acquity UPLC / SQD or Acquity UPLC / SQD2 Column: Ascentis Express C18, 2.1 mm ID x 50 mm, 2.7 μm Mobile phase A: 0.1% formic acid / water Mobile phase B: 0.1% formic acid / acetonitrile Flow rate: 1.0 mL / min Column temperature: 35°C Detection wavelength: 210-400 nm Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 100% B (1 min) → 100% B (1.4 min)

[0097] Analytical conditions - SQFA05Long (HPLC-MS) Instrument: Acquity UPLC / SQD or Acquity UPLC / SQD2 Column: Ascentis Express C18, 2.1 mm ID x 50 mm, 2.7 μm Mobile phase A: 0.1% formic acid / water Mobile phase B: 0.1% formic acid / acetonitrile Flow rate: 1.0 mL / min Column temperature: 35°C Detection wavelength: 210-400 nm Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 100% B (4.5 min) → 100% B (5 min)

[0098] Analysis Conditions - M Apparatus: Waters ACQUITY UPLC H-Class system Column: Ascentis Express 90A C18, 2.1 mm ID x 50 mm, 2.7 μm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA) Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 100% B (10 min) → 5% B (10.1 min) → 5% B (12 min)

[0099] Analysis conditions - K-1 (HPLC-MS) Apparatus: Waters Acquity UPLC / QDa Column: CAPCELL CORE ADME, 2.1 mm x 50 mm, 2.7 μm (Osaka Soda) Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA) Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 100% B (10 min) → 5% B (10.1 min) → 5% B (12 min)

[0100] Analysis conditions - K-2 (HPLC-MS) Apparatus: Waters Acquity UPLC / QDa Column: CAPCELL CORE ADME, 2.1 mm x 50 mm, 2.7 μm (Osaka Soda) Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210 nm (PDA) Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 100% B (5 min) → 5% B (5.1 min) → 5% B (7 min)

[0101] Analytical conditions - FC2 (HPLC-MS) Apparatus: Waters UPLC Column: Waters ACQUITY UPLC CSH C18, 1.7 μm, 2.1 mm x 100 mm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.3 mL / min Column temperature: 50°C Detection wavelength: 210 nm (PDA) Elution method: Gradient mode Gradient conditions: 20% B (0 min) → 100% B (10 min) → 100% B (13.5 min) → 20% B (13.6 min) → 20% B (15.5 min)

[0102] Analysis conditions - cycle (HPLC) Apparatus: Waters UPLC Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm x 150 mm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.3 mL / min Column temperature: 50°C Detection wavelength: 220 nm (PDA) Elution method: Gradient mode Gradient conditions: 20% B (0 min) → 100% B (24 min) → 100% B (29 min) → 20% B (29.1 min) → 20% B (34 min)

[0103] Analysis conditions - H (HPLC) Apparatus: Waters UPLC Column: Ascentis Express RP-Amide, 3.0 mm ID × 50 mm, 2.7 μm Mobile phase A: 0.05% trifluoroacetic acid / water Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile Flow rate: 0.7 mL / min Column temperature: 30°C Detection wavelength: 210 nm (PDA) Elution method: Gradient mode Gradient conditions: 5% B (0 min) → 95% B (10 min) → 95% B (12 min) → 5% B (12.1 min) → 5% B (15 min)

[0104] The qNMR measurement was carried out by dissolving the residue containing the target compound and the internal standard in CDCl3 or DMSO-d6 under the following analytical conditions.

[0105] Measurement equipment: Bruker Avance III 400 Internal standard substance: 3,5-bis(trifluoromethyl)benzoic acid Measurement conditions (19F-NMR): CDCl3 or DMSO-d6, 24.8°C, pulse angle 90°, digital resolution 0.24Hz, relaxation time 15 seconds, no spin, number of accumulations 64

[0106] Measurement equipment: JEOL JNM-ECZ500R / S1 Measurement conditions (1H-NMR): methanol-d4, 25.3°C, pulse angle 45°, digital resolution 0.76Hz, relaxation time 5 seconds, with spin, number of accumulations 8

[0107] <General Method for Synthesizing Amino Acids> The Fmoc-protected amino acids (Fmoc-amino acids) used in this example can be produced by general amino acid synthesis methods, for example, by the method described in WO 2021 / 090855. Furthermore, Fmoc-protected amino acids are available from commercial suppliers. In this example, commercially available amino acids with an Fmoc group introduced at the N-terminus or commercially available amino acids with their C-terminus deprotected can also be used in the elongation reaction of the target peptide. The protecting group deprotection reaction can be performed, for example, by the method described in "Greene's, 'Protective Groups in Organic Synthesis' (5th Edition, John Wiley & Sons 2014)." Amino acids with side chains different from those of naturally occurring amino acids (functional groups on the α-carbon of the amino acid) can be produced by appropriately converting the side chain of an amino acid (naturally occurring amino acid) available from a commercial supplier into the desired side chain. For side chain transformations, see Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3 rd Edition, by RC Larock, or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 th Edition, by MB March, etc. An amino acid alkylated at the N-terminus (N-alkylamino acid) can be produced by reacting an amino acid not alkylated at the N-terminus with an aldehyde according to the method of Freidinger et al. (J. Org. Chem., 1983, 48(1), 77-81) to obtain an oxazolidinone derivative having a cyclic protecting group introduced therein, followed by a ring-opening reaction of the cyclic protecting group.

[0108] The Fmoc-amino acids used in the solid-phase synthesis of this example are shown in Tables 8 to 10. The Fmoc-amino acids listed in Tables 8 to 10 were either purchased from commercial suppliers or synthesized with reference to the methods described in WO 2021 / 090855.

[0109]

[0110]

[0111]

[0112] <General Peptide Synthesis Method> The peptide compounds used in this example were synthesized by solid-phase synthesis or liquid-phase synthesis. Examples of solid-phase synthesis methods include Amino Acids, 2018, 50, 39-68, or Solid Phase Peptide Synthesis (published by Bachem) [searched June 14, 2024], and the internet URL: https: / / www.Bachem.com / wpfd_file / solid-phase-peptide-synthesis / . In this example, the peptide synthesis method using the Fmoc method described in International Publication No. 2013 / 100132 or International Publication No. 2018 / 225864 was used as a reference. In this example, peptide elongation was performed using the basic cyclic peptide synthesis method shown in Figure 1. The basic synthesis method for cyclic peptides shown in Figure 1 comprises five steps: 1) peptide elongation from the N-terminus of an amino acid by the Fmoc method using a 2-chlorotrityl resin with the carboxyl group of the Asp side chain or the carboxyl group of the peptide main chain supported thereon (peptide chain elongation reaction using an Fmoc-protected amino acid as a starting material); 2) peptide cleavage from the 2-chlorotrityl resin; 3) amide cyclization by condensation of the Asp side chain carboxyl group or the peptide main chain carboxyl group generated by cleavage from the 2-chlorotrityl resin with the amino group of the N-terminus of the peptide chain (triangle unit); 4) deprotection of protecting groups of side chain functional groups contained in the peptide chain, if necessary; and 5) pretreatment of the compound by liquid-liquid separation or the like. In this example, unless otherwise noted, cyclic peptides were synthesized using the basic synthesis method for cyclic peptides shown in Figure 1.

[0113] <Method for synthesizing peptide compounds containing N-alkylamino acids> Peptide compounds containing N-alkylamino acids can be synthesized according to the "<General method for peptide synthesis>" described in this example, using as starting materials N-alkylamino acids protected with an Fmoc group shown in Tables 8 to 10.

[0114] Deprotection of the Side Chain Functional Groups of Cyclic Peptides: For sequences synthesized using Fmoc-protected amino acids bearing THP-protected hydroxyl groups in the side chains, such as Fmoc-Thr(THP)-OH and Fmoc-Ser(THP)-OH, 4 mL of tetramethylammonium hydrogen sulfate (0.05 M) in HFIP (containing 2% (v / v) TIPS and 1% (v / v) 1,2-dichloroethane) was added to the resulting cyclic peptide residue (14 reaction vessels), dissolving the residue, and then allowing to stand at room temperature for 4 hours to deprotect the THP groups. After confirming completion of the reaction by LC / MS (Waters SQ Detector 2), DIPEA (70 μL) was added to the reaction solution (14 reaction vessels), and the solvent was evaporated under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12), yielding the cyclic peptide.

[0115] [Example 1-1: (5S,8S,11S,15S,18S,23aS,29S,35S,37aS)-8-((S)-sec-butyl)-29-(3-chloro-4-(trifluoromethyl)phenethyl)-35-(cyclohexylmethyl)-18-cyclopentyl-11-isobutyl-5,6,12,16,19,33,36-heptamethyl-15-(morpholine-4 Synthesis of (14H,22H)-undecaone (compound 1)] <Synthesis of compound 1-a (cyclization precursor)> Compound 1-c was derived in the same manner as described in WO 2022 / 234852, and the crude product of compound 1-a was obtained by deprotecting Fmoc after cleavage from the resin. Specifically, a mixture of TFE (28 mL), Toluene (28 mL), and DIPEA (0.94 mL) was added to the resin-containing compound 1-c (10.017 g) in a reaction vessel and the mixture was shaken and stirred for 4 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. MeTHF (100 mL) was added to the concentrated residue, and the resulting organic phase was washed with 5% aqueous KHSO4 (28 mL), 5% aqueous Na2HPO4 (28 mL), and 5% aqueous NaCl (28 mL). The organic phase was dried under reduced pressure to obtain compound 1-b (5.517 g). Compound 1-b (1 g) was dissolved in acetonitrile (5 mL) in a reaction vessel, and DBU (0.3 mL) was added. After confirming the completion of the reaction by HPLC analysis, the reaction solution was concentrated under reduced pressure. The resulting crude compound 1-a was purified by reverse-phase silica gel chromatography (acetonitrile / water) to obtain compound 1-a. Similarly, compound 1-b (0.3 g) was dissolved in acetonitrile (1.5 mL) and DBU (80 μL) was added. After confirming the completion of the reaction by HPLC measurement, the reaction solution was concentrated under reduced pressure, and the resulting crude product of compound 1-a was purified by reverse-phase silica gel chromatography (acetonitrile / water). This was combined with the previously purified product to obtain compound 1-a (0.6538 g). HPLC: Retention time: 12.29 minutes (analysis condition-1).

[0116]

[0117] <Synthesis of Compound 1 by Cyclization Reaction>

[0118] Compound 1-a (300 mg) obtained by column purification was added to a reaction vessel with acetonitrile (30 mL) and DIPEA (81.5 μL) and stirred at 25 °C. HATU (147 mg) was added to acetonitrile (15 mL) to prepare a solution, which was then added dropwise to the reaction solution over 33 minutes. The vessel containing the HATU acetonitrile solution was washed twice with acetonitrile (3 mL), and the washings were added dropwise to the reaction solution. After the completion of the addition, a sample was taken at 1 hour, and the reaction completion was confirmed by HPLC analysis (analysis condition-1). The reaction solution was concentrated under reduced pressure and subjected to azeotropic distillation with ethyl acetate (20 mL) twice. Ethyl acetate (9 mL) was added to the concentrated residue to form the organic phase, which was then washed sequentially with 2.5% aqueous ammonia (6 mL), 5% aqueous potassium hydrogen sulfate (6 mL), 5% aqueous disodium hydrogen phosphate (6 mL), 5% aqueous sodium chloride (6 mL), and 0.5% aqueous sodium chloride (6 mL). Ethyl acetate (3 mL) was added to the organic phase, which was then washed with 0.5% aqueous sodium chloride solution (6 mL). Each aqueous phase discharged from the previous wash was extracted with ethyl acetate (6 mL). The resulting organic phases were collected in a recovery flask, concentrated under reduced pressure, and then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The above reaction and post-treatment steps were carried out twice in total, and the resulting filtrates were combined, concentrated under reduced pressure, and further dried in vacuo to obtain a concentrated, dry product containing compound 1 (0.5563 g). LCMS (ESI) m / z = 1501 LCMS elution time: 2.6 min (analysis condition-2)

[0119] [Example 1-2: (5S,8S,11S,15S,18S,23aS,29S,35S,37aS)-8,18-di((S)-sec-butyl)-29-(3-chloro-4-(trifluoromethyl)phenethyl)-11-isobutyl-N,N,5,6,12,16,19,33,36-nonamethyl-35-(4-methylbenzyl)-4,7,10,13 Synthesis of 17,20,23,28,31,34,37-undecaoxotetratriacontahydro-2H,4H-spiro[azeto[2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16,19,22,25,28,31]undecaazacyclotetratriacontin-21,1'-cyclopentane]-15-carboxamide (Compound 2) Synthesis of Compound 2-a (Cyclization Precursor) Compound 2-a, the cyclization precursor of Compound 2, was synthesized using a method similar to that described in International Publication No. 2021 / 90855. The crude product of Compound 2-a was purified by reverse-phase silica gel chromatography (acetonitrile / water) to obtain Compound 2-a. HPLC: Retention time: 11.82 minutes (Analysis Condition-1)

[0120] <Synthesis of Compound 2 by Cyclization Reaction>

[0121] The concentrated solid (300 mg), containing compound 2 obtained by column purification, acetonitrile (30 mL), and DIPEA (84.2 μL) were added to a reaction vessel and stirred at 25 °C. HATU (152 mg) was added to acetonitrile (15 mL) to prepare a solution, which was then added dropwise to the reaction solution over 30 minutes. The vessel containing the HATU-acetonitrile solution was washed twice with acetonitrile (3 mL), and the washings were added dropwise to the reaction solution. After the completion of the addition, a sample was taken at 1 hour, and the reaction completion was confirmed by HPLC analysis (analysis condition-1). The reaction solution was concentrated under reduced pressure and subjected to azeotropic distillation with ethyl acetate (20 mL) twice. Ethyl acetate (9 mL) was added to the concentrated residue to form the organic phase, which was then washed sequentially with 2.5% aqueous ammonia (6 mL), 5% aqueous potassium hydrogen sulfate (6 mL), 5% aqueous disodium hydrogen phosphate (6 mL), 5% aqueous sodium chloride (6 mL), and 0.5% aqueous sodium chloride (6 mL). Ethyl acetate (6 mL) was added to the organic phase and washed with 0.5% aqueous sodium chloride (6 mL). The resulting organic phase was collected in a recovery flask and concentrated under reduced pressure. The mixture was then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to obtain a concentrated, dry product. Each aqueous phase removed during the previous wash was extracted with ethyl acetate (6 mL). The resulting organic phase was collected in a recovery flask and concentrated under reduced pressure. The mixture was then subjected to azeotropic distillation with acetonitrile (10 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo. The resulting concentrated, dry product was dissolved in acetonitrile, added to the previously obtained concentrated, dry product, and concentrated under reduced pressure. Further drying in vacuo yielded a concentrated, dry product containing compound 2 (0.2875 g). LCMS(ESI) m / z =1454 LCMS elution time: 2.4 min (Analysis conditions-2)

[0122] Example 1-3: (6S,9S,14S,17S,20S,24S,27S,33S)-27-cyclopentyl-7-ethyl-20-isobutyl-N,N,4,14,15,21,25,28-octamethyl-17-[(1S)-1-methylpropyl]-2,5,8,13,16,19,22,26,29,32-decaoxo-6-(p-trimethyl)spiro[1,4,7,12,15,18,21,25,28,31-decazatricyclo[31,3,0,09,12]hexatriacontane-30,1′-cyclopentane]-24-carboxamide (Compound 3). Synthesis of Compound 3-a (Cyclization Precursor) Compound 3-a, a cyclization precursor of compound 3, was synthesized by a method similar to that described in WO 2022 / 234864.

[0123] <Synthesis of Compound 3 by Cyclization Reaction>

[0124] The concentrated solid (300 mg), acetonitrile (60 mL), and DIPEA (101 μL) containing compound 3-a were added to a reaction vessel and stirred at 25 °C. A solution was prepared by adding acetonitrile (15 mL) to HATU (182 mg), and then added dropwise to the reaction solution over 33 minutes. The vessel containing the HATU acetonitrile solution was washed twice with acetonitrile (1.8 mL), and the washings were added dropwise to the reaction solution. After the completion of the addition, the reaction solution was sampled at 1 hour, and the completion of the reaction was confirmed by HPLC analysis (analysis condition-1). The reaction solution was concentrated under reduced pressure and subjected to azeotropic distillation twice with ethyl acetate (20 mL). Ethyl acetate (9 mL) was added to the concentrated residue to form an organic phase, which was then washed sequentially with 2.5% aqueous ammonia (6 mL), 5% aqueous potassium hydrogen sulfate (6 mL), 5% aqueous disodium hydrogen phosphate (6 mL), 5% aqueous sodium chloride (6 mL), 0.5% aqueous sodium chloride (6 mL), and 0.5% aqueous sodium chloride (6 mL). The resulting organic phase was concentrated under reduced pressure and then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to obtain a concentrated, dry product containing compound 3 (0.2470 g). LCMS (ESI) m / z = 1217 LCMS elution time: 2.3 min (analysis condition-2).

[0125] [Synthesis of Compound 3] HATU (182 mg) and acetonitrile (7.5 mL) were added to a reaction vessel and stirred at 25 °C. Acetonitrile (7.5 mL) and DIPEA (101 μL) were added to the residue containing compound 3-a (300 mg) to prepare a solution, which was then added dropwise to the aforementioned solution over 17 minutes. The vessel containing the acetonitrile solution of compound 3-a and DIPEA was washed twice with acetonitrile (1.5 mL), and the washings were added dropwise to the reaction solution. After the completion of the addition, a sample was taken at 1 hour, and the completion of the reaction was confirmed by HPLC analysis (analysis condition-1). The reaction solution was concentrated under reduced pressure and subjected to azeotropic distillation with ethyl acetate (20 mL) twice. Ethyl acetate (9 mL) was added to the concentrated residue to form an organic phase, which was then washed sequentially with 2.5% aqueous ammonia (6 mL), 5% aqueous potassium hydrogen sulfate (6 mL), 5% aqueous disodium hydrogen phosphate (6 mL), 5% aqueous sodium chloride (6 mL), 0.5% aqueous sodium chloride (6 mL), and 0.5% aqueous sodium chloride (6 mL). The resulting organic phase was concentrated under reduced pressure and then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to yield a concentrated solid containing compound 3 (0.2472 g).

[0126] [Example 1-4: (3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39R,43S)-21,36-dibenzyl-27-((S)-sec-butyl)-6-((R)-1-hydroxyethyl)-9-(hydroxymethyl)-3,12,18,24,30-pentaisobutyl-4,15,16,19,25 Synthesis of ,31,33,34,37,39,40-undecamethyl-43-(piperidine-1-carbonyl)-1,4,7,10,13,16,19,22,25,28,31,34,37,40-tetradecaazacyclotritetracontane-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecaone (compound 4)

[0127] Compound 4-a was synthesized according to the method described in WO 2013 / 100132. The peptide was elongated using the Fmoc method, cleaved from the resin, and then cyclized. The THP group was then deprotected to obtain a concentrated, dry product of compound 4. Ethyl acetate (9.2 mL) was added to the concentrated, dry product (460 mg) containing compound 4 to form the organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride (9.2 mL). Each of the drained aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previously prepared organic phase. The organic phase was concentrated under reduced pressure and then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to obtain a concentrated, dry product containing compound 4 (0.1089 g). LCMS(ESI) m / z =1714 LCMS elution time: 2.8 min (Analysis conditions-2)

[0128] [Example 1-5: (3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,41S)-6,24-dibenzyl-33-((S)-sec-butyl)-21-((R)-1-hydroxymethyl)-3,9,12,18,30-pentaisobutyl-4,10,13,15,16,19,25, Synthesis of 27,28,31,38-undecamethyl-41-(piperidine-1-carbonyl)-1,4,7,10,13,16,19,22,25,28,31,34,38-tridecaazacyclohentetracontane-2,5,8,11,14,17,20,23,26,29,32,35,39-tridecaone (compound 5)

[0129] Compound 4-a was synthesized according to the method described in WO 2013 / 100132. The peptide was elongated using the Fmoc method, cleaved from the resin, and then cyclized. The THP group was then deprotected to yield a concentrated, dry product of compound 5. Ethyl acetate (9.2 mL) was added to the concentrated, dry product (352.9 mg) containing compound 5 to form the organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride (9.2 mL). Each of the drained aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previously prepared organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations with acetonitrile (20 mL). Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to yield a concentrated, dry product (0.1106 g) containing compound 5. LCMS(ESI) m / z =1641 LCMS elution time: 3.1 min (Analysis conditions-2)

[0130] [Example 1-6: (3S,6S,9S,12S,15S,18S,21S,27S,30S,33S,41S)-6,9-dibenzyl-15-((S)-sec-butyl)-27-((R)-1-hydroxyethyl)-12,18,21,30,33-pentaisobutylbutyl-3,7,13,19,22,25,31,38-octamethyl-41-(piperidine-1-carbonyl)-1,4,7,10,13,16,19,22,25,28,31,34,38-tridecaazacyclohentetracontane-2,5,8,11,14,17,20,23,26,29,32,35,39-tridecaone (Compound 6)]

[0131] Compound 4-a was synthesized according to the method described in WO 2013 / 100132. The peptide was elongated using the Fmoc method, cleaved from the resin, and then cyclized. The THP group was then deprotected to yield a concentrated, dry product of compound 6. Ethyl acetate (9.2 mL) was added to the concentrated, dry product containing compound 6 (421.1 mg) to form the organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride (9.2 mL). Each of the drained aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previously prepared organic phase. The organic phase was concentrated under reduced pressure and then subjected to azeotropic distillation with acetonitrile (20 mL) twice. Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to yield a concentrated, dry product containing compound 6 (0.1305 g). LCMS(ESI) m / z =1598 LCMS elution time: 2.7 min (Analysis conditions-2)

[0132] Example 1-7: Synthesis of (3S,6S,9S,12S,18S,21S,24S,27S,30R,34S)-6,27-dibenzyl-18-(R)-1-hydroxyethyl)-3,12,21,24-tetraisobutyl-30-isopropyl-4,7,13,16,22,28-hexamethyl-34-(piperidine-1-carbonyl)-9-(propoxymethyl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotetratriacontane-2,5,8,11,14,17,20,23,26,29,32-undecaone (Compound 7)

[0133] Compound 4-a was synthesized according to the method described in WO 2013 / 100132, and THP-protected cyclic peptide compound 7-a was obtained by synthesizing it according to the method described in WO 2021 / 090855. Subsequently, the THP group was deprotected to obtain a concentrated dry product of compound 7. Ethyl acetate (9.2 mL) was added to the concentrated dry product (483 mg) containing compound 7 to form the organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride (9.2 mL). Each of the drained aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the aforementioned organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations with acetonitrile (20 mL). Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to obtain a concentrated dry product (0.1066 g) containing compound 7. LCMS(ESI) m / z =1444 LCMS elution time: 2.9 min (Analysis conditions-2)

[0134] Example 1-8: Synthesis of (S)-1-((2S,5S,8S,11S,14S,17S,20S,23S)-14,20-dibenzyl-8-((S)-secbutyl)-5,17-diisobutyl-11-isopropyl-2,7,13,19-tetramethyl-3,6,9,12,15,18,21,25)-octaoxy-1,4,7,10,13,16,19,22-octaazacyclopentacosane-23-carbonyl]-N-((2S,3S)-3-methyl-1-oxo-1-(piperidin-1-yl)pentan-2-yl)pyrrolidine-2-carboxamide (Compound 8)

[0135] Synthesis of Compound 8-b (Fmoc-Ile-pip) Under a nitrogen atmosphere, EDCI·HCl (3.25 g, 17.0 mmol) was mixed with DMF (30 mL) in a reaction vessel. The mixture was stirred at room temperature for 10 minutes and then cooled to 0°C. HOBt (2.10 g, 3.25 mmol) and compound 8-a (Fmoc-Ile-OH, CAS: 71989-23-6) (15.00 g, 14.15 mmol) were added in sequence and stirred at 0°C for 1 hour. Piperidine (1.27 g, 14.9 mmol) was slowly added to the resulting reaction mixture and stirred at 0°C for 1 hour. Ethyl acetate (10 v / w) and 0.5 mol / L aqueous hydrochloric acid solution (10 v / w) were added to the reaction mixture, and the organic phase was separated. The resulting organic phase was washed sequentially with 0.5 mol / L aqueous hydrochloric acid, water, 5% aqueous sodium carbonate, and saturated aqueous sodium chloride / water (1 / 1 (v / v)). The washed organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give compound 8-b (Fmoc-Ile-pip) as a crude product (6.11 g). LCMS (ESI) m / z = 421 (M+H). ＋ Retention time: 0.98 minutes (Analysis conditions - SQDFA05)

[0136] Synthesis of Compound 8-c (Fmoc-Pro-Ile-pip) To a DMF solution (50 mL) of compound 8-b (Fmoc-Ile-pip), DBU (2.21 g, 14.53 mmol) was added and stirred at room temperature for 10 minutes. Triethylamine hydrochloride (2.00 g, 14.53 mmol), DIPEA (2.54 mL, 14.53 mmol), Fmoc-Pro-OH (CAS: 719890-31-6) (4.90 g, 14.53 mmol), EDCI·HCl (3.90 g, 20.35 mmol), and HOAt (2.37 g, 17.43 mmol) were added sequentially to the reaction solution and stirred at room temperature for 1 hour. Ethyl acetate (10 v / w) and 0.5 mol / L aqueous hydrochloric acid (10 v / w) were added to the reaction solution, and the organic phase was separated. The resulting organic phase was washed sequentially with 0.5 mol / L aqueous hydrochloric acid, water, 5% aqueous sodium carbonate, and saturated aqueous sodium chloride / water (1 / 1 (v / v)). The washed organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain compound 8-c as a crude product. DMSO (5 mL) and ethyl acetate (20 mL) were added to the resulting crude product, and the resulting solution was filtered. The ethyl acetate was then removed by distillation under reduced pressure. The resulting residue was purified by reverse-phase medium-pressure column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain compound 8-c (Fmoc-Pro-Ile-pip) (2.41 g, 32%, purity 97% [peak area percentage]). LCMS (ESI) m / z = 518 (M+H) + , retention time: 0.94 min (analysis condition - SQDFA05).

[0137] Synthesis of compound 8-d (Fmoc-Asp(OAl)-Pro-Ile-pip): Compound 8-c (Fmoc-Pro-Ile-pip) (2.41 g, 4.66 mmol) in DMF (23 mL) was added to a reaction vessel and stirred at room temperature for 10 minutes. Triethylamine hydrochloride (0.641 g, 4.66 mmol), DIPEA (0.813 mL, 4.66 mmol), Fmoc-Asp(OAl)-OH (CAS: 146982-24-3) (2.41 g, 4.66 mmol), EDCI.HCl (1.25 g, 6.52 mmol), and HOAt (0.760 g, 5.59 mmol) were added sequentially to the reaction solution and stirred at room temperature for 1 hour and 30 minutes. Ethyl acetate (10 v / w) and 0.5 mol / L aqueous hydrochloric acid (10 v / w) were added to the reaction mixture, and the organic phase was separated. The resulting organic phase was washed sequentially with 0.5 mol / L aqueous hydrochloric acid, water, 5% aqueous sodium carbonate, and saturated aqueous sodium chloride / water (1 / 1 (v / v)). The washed organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give compound 8-d as a crude product. The resulting crude product was dissolved in DMSO and purified by reverse-phase medium-pressure column chromatography (acetonitrile / water containing 0.1% formic acid) to give compound 8-d (Fmoc-Asp(OAl)-Pro-Ile-pip) (1.71 g, 55%, purity 84% [peak area percentage]). LCMS (ESI) m / z = 674 (M+H) + retention time: 2.94 min (analysis condition - SQDFA05Long).

[0138] Synthesis of compound 8-e (Fmoc-Asp-Pro-Ile-pip) Tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3, Pd(Pphe3)4, 0.294 g, 0.254 mmol) and phenylsilane (CAS: 694-53-1, 0.313 mL, 2.54 mmol) were added to a reaction vessel containing a dichloromethane solution (5.08 mL) of compound 8-d (Fmoc-Asp(OAl)-Pro-Ile-pip) (1.71 g, purity 84%), and the mixture was stirred at 0°C for 15 minutes. The solvent of the resulting reaction mixture was evaporated under reduced pressure, and the concentrated residue was purified by reverse-phase medium-pressure column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain compound 8-e (Fmoc-Asp-Pro-Ile-pip) (1.11 g, 69%, purity 99% [peak area percentage]). LCMS (ESI) m / z = 633 (M + H) + Retention time: 0.80 minutes (Analysis conditions - SQDFA05)

[0139] Compound 8-f (Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-Ile-pip) was synthesized according to the method described in International Publication WO 2013 / 100132. Subsequent peptide elongation using the Fmoc method, cleavage from the resin, and peptide cyclization afforded compound 8. Ethyl acetate (9.2 mL) was added to the concentrated, dried product (214.7 mg) containing compound 8 to form the organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride (9.2 mL). Each of the drained aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previously prepared organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations with acetonitrile (20 mL). Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo. Ethyl acetate (9.2 mL) was added to the resulting concentrated, dried product to form the organic phase. The organic phase was washed three times with 0.5% aqueous sodium chloride (9.2 mL). Each of the discarded aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previous organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations with acetonitrile (20 mL). Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure. Ethyl acetate (9.2 mL) was added to the resulting concentrated residue to form the organic phase. The organic phase was washed four times with 0.5% aqueous sodium chloride (9.2 mL). Each of the discarded aqueous phases was extracted with ethyl acetate (9.2 mL), and the resulting organic phase was combined with the previous organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations with acetonitrile (20 mL). Acetonitrile was added to the concentrated residue, and insoluble matter was removed using a syringe filter. The filtrate was concentrated under reduced pressure and further dried in vacuo to obtain a concentrated dry product (87.3 mg) containing compound 8. LCMS (ESI) m / z = 1283 LCMS elution time: 2.9 min (analysis condition-2).

[0140] [Examples 1-9: (1S,4S,10S,13S,17S,20S,26S,28R,32S,38S,42Z)-20-cyclopentyl-28-ethoxy-32-[2-[3-methoxy-4-(trifluoromethyl)phenyl]ethyl]-N,N,2,14,18,21,24,36-octamethyl-10-[(1S)-1-methylpropyl]-3,9,12,15,19,22,25,31,34,37,45-undecaoxo-13-propyl-38-[[4-(trifluoromethyl)phenyl]methyl]spiro[2,8,11,14,18,21,24,30,33,36,39-undecazatetracyclo[37.5.1.0 4,8 .0 26,30 Synthesis of ]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide (compound 9)

[0141] Synthesis of Compound 9-a: 9H-fluoren-9-ylmethyl(4S)-5-oxo-4-[[4-(trifluoromethyl)phenyl]methyl]oxazolidine-3-carboxylic acid

[0142] A nitrogen-purged reactor was charged with DCM (45 L) and (2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-[4-(trifluoromethyl)phenyl]propionic acid (3.05 kg) at room temperature and stirred. Paraformaldehyde (0.90 kg) and MgSO (2.02 kg) were then added and stirred at 20°C for 10 minutes. The external temperature of the reactor was set to 15°C, and BF OEt (0.95 kg) was slowly added dropwise at an internal temperature of 15-20°C. The reaction mixture was stirred at 20-25°C for 12 hours, then filtered through a filter lined with silica gel (3.05 kg). The silica gel was washed twice with DCM (15.3 L). The filtrate was concentrated under reduced pressure at an external temperature of 30°C, and the crude product containing compound 9-a was purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1) and concentrated under reduced pressure to obtain compound 9-a (2.76 kg).

[0143] Synthesis of Compound 9-b: (2S)-2-[but-3-enyl(9H-fluoren-9-ylmethoxycarbonyl)amino]-3-[4-(trifluoromethyl)phenyl]propionic acid

[0144] Toluene (22.4 L) and compound 9-a (2.80 kg) were added to a nitrogen-purged reactor at room temperature and stirred. Allyltrimethylsilane (1.37 kg) and ZnBr (1.35 kg) were then added and stirred at 20°C for 10 minutes. The internal temperature of the reactor was set to 40-45°C, and the reaction mixture was stirred for 10 hours. The reaction mixture was added to ice water (28.0 L) at an internal temperature of 10±5°C and stirred. The aqueous phase was discharged, and the organic phase was washed with 5% brine (28.0 L). The resulting organic phase was concentrated under reduced pressure at an external temperature of 45-50°C to yield compound 9-b (2.44 kg).

[0145] Synthesis of Compound 9-c: tert-butyl 2-[[(2S)-2-[but-3-enyl (9H-fluoren-9-ylmethoxycarbonyl)amino]-3-[4-(trifluoromethyl)phenyl]propanoyl]-methyl-amino]acetic acid

[0146] N-methyl-2-pyrrolidone (17.0 L) and compound 9-b (2.44 kg) were added to a nitrogen-purged reactor at room temperature and stirred. Sarcosine tert-butyl ester hydrochloride (0.87 kg) and HATU (2.18 kg) were then added at 20 °C and stirred for 30 minutes. DIPEA (1.85 kg) was added dropwise over 60 minutes at an internal temperature of 15-20 °C. The reaction mixture was stirred at 20-25 °C for 3 hours and then diluted with methyl tert-butyl ether (48.8 L). The organic phase was washed with water (48.8 L x 2) and 5% brine (24.4 L) and then concentrated under reduced pressure to obtain a concentrated, dry product containing compound 9-c. The resulting concentrated, dry product was purified using a column (petroleum ether / ethyl acetate = 4 / 1) and concentrated under reduced pressure at 35 °C to obtain compound 9-c (2.79 kg) as a dark yellow oil.

[0147] Synthesis of Compound 9-d: tert-butyl 2-[[(2S)-2-(but-3-enylamino)-3-[4-(trifluoromethyl)phenyl]propanoyl]-methyl-amino]acetate

[0148] Toluene (28.0 L) and compound 9-c (2.79 kg) were added to a nitrogen-purged reactor at room temperature and stirred. DBU (0.67 kg) was then added at an internal temperature of 20°C and stirred for 2 hours. The reaction mixture was added to ice water (27.9 L) at an internal temperature of 20±5°C and stirred. Ethyl acetate (14.0 L) was added and stirred, and the aqueous phase was then discarded. The organic phase was washed with 5% brine (28.0 L) and concentrated under reduced pressure at 45±5°C. The concentrate containing compound 9-d was purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1) and then concentrated under reduced pressure to yield compound 9-d (1.49 kg) as a pale yellow oil.

[0149] Synthesis of Compound 9-e: 9H-Fluoren-9-ylmethyl N-[(1S)-1-chlorocarbonylbut-3-enyl]-N-methyl-carbamate

[0150] In a nitrogen-purged reactor, DCM (10 L), (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]pent-4-enoic acid (1.7 kg), and DMF (0.02 kg) were added at room temperature and stirred at an internal temperature of 20°C. Oxalyl chloride (1.84 kg) was added over 2 hours at an internal temperature of 10°C, and the mixture was stirred for 2 hours while maintaining the internal temperature at 10°C. The mixture was concentrated under reduced pressure at 30-35°C. DCM (3.4 L) was added to the concentrate and concentrated under reduced pressure twice to obtain compound 9-e (1.65 kg) as a yellow oil.

[0151] Synthesis of Compound 9-f: tert-butyl 2-[[(2S)-2-[but-3-enyl-[(2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]pent-4-enoyl]amino]-3-[4-(trifluoromethyl)phenyl]propanoyl]-methyl-amino]acetic acid

[0152] DCM (15.0 L) and compound 9-d (1.49 kg) were added to a nitrogen-purged reactor at room temperature and stirred. Compound 9-e (1.72 kg) dissolved in DCM (2.96 L) was then added dropwise over 1 hour at an internal temperature of 0-10°C. The mixture was stirred at an internal temperature of 10°C for 30 minutes, followed by the dropwise addition of DIPEA (0.926 kg) over 1 hour at an internal temperature of 0-10°C. After stirring at 20°C for 2 hours, the reaction mixture was added to ice water (14.8 L) at an internal temperature of 20°C and stirred. Organic phase 1 was separated and set aside. The aqueous phase was extracted with DCM (7.4 L) and combined with the set aside organic phase 1. The combined organic phase was washed with 5% brine (14.9 L x 2) and concentrated under reduced pressure at 30±5°C. The concentrate containing compound 9-f was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) and concentrated under reduced pressure to give compound 9-f (1.97 kg) as a colorless powder.

[0153] Synthesis of Compound 9-g: tert-butyl 2-[[(2S)-2-[(4Z,7S)-7-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-8-oxo-2,3,6,7-tetrahydroazocin-1-yl]-3-[4-(trifluoromethyl)phenyl]propanoyl]-methyl-amino]acetate

[0154] Toluene (59.0 L) and compound 9-f (0.655 kg) were added to a nitrogen-purged reactor at room temperature and stirred. p-Benzoquinone (28.4 g) was then added and the mixture heated to 100°C. To the mixture heated to 100°C, first-generation Hoveyda-Grubbs catalyst (42.0 g) dissolved in toluene (100 mL) was added dropwise over 20 minutes. The above reaction was repeated in three batches. The three batches of reaction mixture were combined and concentrated under reduced pressure at 45°C. The concentrate containing compound 9-g was purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1) and concentrated under reduced pressure to yield compound 9-g (1.05 kg) as a yellow oil.

[0155] Compound 9-h: 2-[[(2S)-2-[(4Z,7S)-7-[9H-fluoren-9-ylmethoxycarbonyl (methyl)amino]-8-oxo-2,3,6,7-tetrahydroazocin-1-yl]-3-[4-(trifluoromethyl)phenyl]propanyl]-methyl-amino]acetic acid

[0156] A solution of compound 9-h (3.89 kg) in MeTHF (20.5 kg) was added to the reaction vessel at room temperature, cooled to an external temperature of 15°C, and HMDS (2.19 kg) was added and stirred. Subsequently, TMSOTf (1.80 kg) was slowly added to the mixture at an internal temperature below 25°C, and the mixture was stirred at 25°C for 2 hours. MeTHF (16.6 kg) and acetonitrile (4.60 kg) were added, and the mixture was cooled to an internal temperature below 15°C. 5% aqueous sodium bicarbonate solution (27.3 kg) was slowly added, stirred at room temperature, and the aqueous phase was discharged by liquid-liquid separation. The organic phase was washed with 5% aqueous sodium hydrogen sulfate solution (27.2 kg) and 5% brine (27.2 kg x 3) and concentrated to 10 L at an external temperature of 40°C. Toluene (56.0 kg) was added to the concentrated solution, which was then concentrated to 55 L at an external temperature of 40 °C. Toluene (15.6 kg) was then added and concentrated to 54 L at an external temperature of 40 °C twice. After confirming the precipitation of crystals, cyclohexane (14.0 kg) was added and the mixture was stirred overnight. The crystals were filtered and washed with a 3:1 toluene / cyclohexane mixture (15.2 kg). The wet crystals obtained at an external temperature of 50 °C were dried under reduced pressure to obtain compound 9-h monotoluene solvate (3.21 kg) as a white solid.

[0157] Synthesis of Compound 9-i: tert-butyl N-[(benzyloxy)carbonyl]-L-isoleucyl-L-prolinate

[0158] N-Cyclohexylcyclohexanaminium (2S,3S)-2-{[(benzyloxy)carbonyl]amino}-3-methylpentanoate (135 g) was added to toluene (583 g) and 5% aqueous sodium hydrogen sulfate (2066 g), and the mixture was stirred at room temperature for 10 minutes. The organic phase was separated. The resulting organic phase was washed sequentially with 5% aqueous sodium hydrogen sulfate (2066 g) and 5% brine (1397 g), and the solvent was evaporated under reduced pressure. Toluene (55 mL), 1,3-dimethyl-2-imidazolidinone (270 mL), (2S)-2-(tert-butoxycarbonyl)pyrrolidin-1-ium chloride (75.3 g), MeTHF (540 mL), and 4-methylmorpholine (133 mL) were added to the resulting residue and stirred at an external temperature of 15 °C. To this mixture, a 50% solution of propylphosphonic anhydride in MeTHF (370 mL) was added dropwise over approximately 1 hour, and the resulting reaction mixture was stirred at an external temperature of 20°C for 1 hour. 5% aqueous sodium bicarbonate (993 g) and 1-methylimidazole (24 mL) were added to the reaction mixture at an internal temperature of 20°C or below, followed by stirring at an external temperature of 20°C for approximately 30 minutes, after which the organic phase was separated. The resulting organic phase was washed sequentially with 10% aqueous sodium hydrogen sulfate (709 g), 10% aqueous sodium hydrogen sulfate (639 g), and 10% aqueous sodium bicarbonate (710 g), and the solvent was removed under reduced pressure to obtain a solution containing compound 9-i (137 g). LCMS (ESI) of compound 9-i: retention time: 4.21 min, m / z = 419 [M+H]+ (analysis condition -M).

[0159] Synthesis of Compound 9-j: tert-butyl L-isoleucyl-L-prolinate

[0160] Pd / C (50% wet, 36.7 g) and MeTHF (253 mL) were stirred at an external temperature of 25 °C under hydrogen pressure (0.4 MPa) for 2 hours. To the resulting mixture, a solution containing compound 9-i (137 g) and MeTHF (495 mL) were added, and the mixture was stirred at an external temperature of 25 °C under hydrogen pressure (0.2 MPaG) for 2 hours. The reaction mixture was filtered, and the solid was washed three times with MeTHF (127 mL). The filtrate was combined and the solvent was distilled off under reduced pressure to obtain a solution containing compound 9-j (160 g). LCMS (ESI) of compound 9-j: retention time: 2.40 min, m / z = 285 [M+H] + (analysis condition -M).

[0161] Synthesis of Compound 9-k: tert-butyl N-[(benzyloxy)carbonyl]-N-methyl-L-norvalyl-L-isoleucyl-L-prolinate

[0162] A mixture of a solution containing compound 9-j (120 g), N-[(benzyloxy)carbonyl]-N-methyl-L-norvaline (72.12 g), MeTHF (257 mL), and 4-methylmorpholine (100 mL) was stirred at an external temperature of 15°C. A 50% solution of propylphosphonic anhydride in MeTHF and methyltetrahydrofuran (277 mL) was added dropwise to the mixture over approximately 40 minutes, and the resulting reaction mixture was stirred at an external temperature of 20°C for 1 hour. A 5% aqueous solution of sodium bicarbonate (472 g) and 1-methylimidazole (18 mL) were added to the reaction mixture at an internal temperature below 30°C. After stirring at an external temperature of 15°C for approximately 30 minutes, the organic phase was separated. The resulting organic phase was washed with 10% aqueous sodium hydrogen sulfate (338 g), 10% aqueous sodium hydrogen sulfate (338 g), and 10% aqueous sodium hydrogen carbonate (340 g) in that order at an external temperature of 20°C, and the solvent was removed under reduced pressure to give a solution containing compound 9-k (219 g). LCMS (ESI) of compound 9-k: retention time: 4.58 min, m / z = 532 [M+H] (analysis condition -M).

[0163] Synthesis of Compound 9-l: tert-butyl N-methyl-L-norvalyl-L-isoleucyl-L-prolinate

[0164] Pd / C (50% wet, 22.9 g) and MeTHF (400 mL) were stirred at an external temperature of 25 °C under hydrogen pressure (0.4 MPaG) for 2 hours. To the resulting mixture, a solution containing compound 9-k (182 g) and MeTHF (50 mL) were added, and the mixture was stirred at an external temperature of 25 °C under hydrogen pressure (0.4 MPaG) for 2 hours. The reaction mixture was filtered, and the solid was washed three times with MeTHF (100 mL). The combined filtrate was concentrated under reduced pressure to give a solution containing compound 9-l (109 g). The solvent was removed from 9.5346 g of this solution under reduced pressure, and heptane (100 mL) was added to the resulting residue. The mixture was dissolved at an external temperature of 50 °C, and seed crystals (11.0 mg) were added at an internal temperature of 40 °C. The mixture was stirred at an external temperature of 42°C for 15 minutes, at an external temperature of 43°C for 13 minutes, and at an external temperature of 44°C for 17 minutes, after which the external temperature was cooled to 0°C at a rate of 12°C per hour and further stirred at an external temperature of 0°C for 1.5 hours. The resulting solid was collected by filtration, washed with cold heptane (25 mL), and dried under reduced pressure at an external temperature of 30°C to 40°C to obtain compound 9-l (4.8474 g). LCMS (ESI) of compound 9-l: retention time: 2.56 minutes, m / z = 398 [M+H]+ (analysis condition -M).

[0165] Compound 9-l: Seed crystal synthesis of tert-butyl N-methyl-L-norvalyl-L-isoleucyl-L-prolinate. A portion of the solution containing compound 9-l obtained in the previous reaction was concentrated under reduced pressure, and heptane (7622 μL) was added to the resulting residue (0.3811 g). The resulting solid was dissolved at an external temperature of 50°C and then cooled to room temperature with stirring. Heptane (3811 μL) was added to the resulting slurry, and stirring was continued. The solid was collected by filtration, washed with heptane (1906 μL), and then dried at room temperature under reduced pressure to obtain the title compound 9-l seed crystal. LCMS (ESI) of compound 9-l seed crystal: retention time: 2.60 min, m / z = 398 [M+H]+ (analysis condition -M).

[0166] Synthesis of Compound 9-m: (tert-butyl (3S)-3-[benzyloxycarbonyl(methyl)amino]-4-(dimethylamino)-4-oxo-butanoate)

[0167] (2S)-2-(((benzyloxy)carbonyl)(methyl)amino)-4-tert-butoxy-4-oxobutanoic acid dicyclohexylamine salt (CAS number 42417-70-9, 25.00 g, 48.2 mmol) and MeTHF (126 g) were added to a reaction vessel at 25 °C. The mixture was washed twice with 10% aqueous sodium hydrogen sulfate monohydrate (150 g) and then with 5% aqueous sodium chloride (150 g). The resulting organic phase was concentrated under reduced pressure. MeTHF (95 g) was added to the resulting residue, and this process of concentration under reduced pressure was repeated twice. MeTHF (95 g), acetonitrile (75 g), DIPEA (35.46 g, 274 mmol), and dimethylamine hydrochloride (7.88 g, 96.6 mmol) were added to the resulting residue (47.91 g) at 25 °C. A solution of propylphosphonic anhydride in MeTHF (50.4 wt%, 61.33 g, 97.1 mmol) was added dropwise over 1 hour and 30 minutes. After stirring for 1 hour, the mixture was sampled and analyzed by HPLC to confirm completion of the reaction. 2M aqueous sodium hydroxide (150 g) was added. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous phase was removed. The resulting organic phase was washed with 2M aqueous sodium hydroxide (150 g), 13% aqueous sulfuric acid (150 g), 10% aqueous sodium hydrogen sulfate monohydrate (150 g), and 5% aqueous sodium carbonate (150 g) and then concentrated under reduced pressure. This process of adding MeTHF (125 g) and concentrating under reduced pressure was repeated twice to obtain a solution containing compound 9-m (42.39 g). LCMS (ESI) of compound 9-M: retention time: 3.37 minutes, m / z = 387 [M+Na]+ (analysis condition -K-2).

[0168] Synthesis of Compound 9-n: (tert-butyl (3S)-4-(dimethylamino)-3-(methylamino)-4-oxo-butanoate)

[0169] A reaction vessel was charged with 10% Pd / C (54.33% wet, 3.39 g, 1.45 mmol, 3 mol% on Pd metal basis) and MeTHF (75 g). The atmosphere was replaced with nitrogen and then hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. A solution of compound 9-m (42.39 g) and MeTHF (22 g) were added. After stirring for 1 hour and 30 minutes under a hydrogen atmosphere (0.20 MPaG), a sample was taken and HPLC analysis confirmed the completion of the reaction. The reaction mixture was filtered, and the cake was washed twice with MeTHF (75 g). The mixture of the filtrate and washings was concentrated under reduced pressure to give a solution containing compound 9-n (30.76 g). LCMS (ESI) of compound 9-n: retention time: 1.44 minutes, m / z = 231 [M+H]+ (analysis conditions -K-2).

[0170] Synthesis of Compound 9-o: (tert-butyl (3S)-3-[[(2S)-2-[benzyloxycarbonyl(methyl)amino]-2-cyclopentyl-acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate)

[0171] A solution of compound 9-n (30.76 g), (S)-2-(((benzyloxy)carbonyl)(methyl)amino)-2-cyclopentylacetic acid (CAS number 2411591-78-9, 16.96 g, 58.2 mmol), MeTHF (40 g), acetonitrile (17 g), and DIPEA (27.74 g, 215 mmol) were added to a reaction vessel at 25 °C. HATU (27.49 g, 72.3 mmol) was added over 10 min. After stirring for 3 h, the mixture was sampled and analyzed by HPLC to confirm completion of the reaction. Toluene (30 g), 5% aqueous potassium carbonate solution (23 g), and 1-methylimidazole (3.97 g, 48.4 mmol) were added and stirred for 30 min. 2.5% aqueous ammonia solution (88 g) and MeTHF (25 g) were added. After stirring for 10 min, the mixture was allowed to stand and the aqueous phase was removed. The resulting organic phase was washed with 2.5% aqueous ammonia (113 g), 10% aqueous sodium hydrogen sulfate monohydrate (113 g, twice), and 5% aqueous potassium carbonate (113 g), and then concentrated under reduced pressure. MeTHF (42 g) was added and the mixture was concentrated under reduced pressure to give a solution containing compound 9-o (52.78 g). LCMS (ESI) of compound 9-o: retention time: 4.10 min, m / z = 526 [M+Na] (analysis condition -K-2).

[0172] Synthesis of Compound 9-p: (tert-butyl (3S)-3-[[(2S)-2-cyclopentyl-2-(methylamino)acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate)

[0173] A reaction vessel was charged with 10% Pd / C (54.33% wet, 3.39 g, 1.45 mmol, 3 mol% on Pd metal basis) and MeTHF (75 g). The atmosphere was replaced with nitrogen at 25 °C, then replaced with hydrogen, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. Compound 9-0 solution (52.78 g) and MeTHF (15 g) were added, and the temperature was raised to 30 °C. After stirring under a hydrogen atmosphere (0.20 MPaG) for 2 hours, a sample was taken and HPLC analysis confirmed the completion of the reaction. The reaction mixture was filtered, and the cake was washed twice with MeTHF (75 g). The mixture of the filtrate and washings was concentrated under reduced pressure, followed by the addition of acetonitrile (75 g) and concentration under reduced pressure twice to obtain a solution containing compound 9-p (42.5 mL). LCMS (ESI) of compound 9-p: Retention time: 2.96 minutes, m / z = 370 [M+H]+ (Analysis conditions-K-1)

[0174] Synthesis of Compound 9-q: (tert-butyl (3S)-3-[[(2S)-2-cyclopentyl-2-(methylamino)acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate) hydrochloride

[0175] A solution of compound 9-p (42.5 mL) and acetonitrile (8.0 g) were added to a reaction vessel. MTBE (65 g) was added at 40°C, followed by the dropwise addition of a solution of pyridine hydrochloride in acetonitrile (16.94 w / w%, 4.50 g) over 30 minutes. After stirring for 1 hour, a solution of pyridine hydrochloride in acetonitrile (16.94 w / w%, 31.34 g) was added dropwise over 3 hours and 30 minutes, followed by the addition of acetonitrile (14 g). After stirring for 1 hour, the mixture was cooled to 10°C over 6 hours. After stirring at 10°C for an additional 11 hours, the slurry was filtered. The resulting solid was washed twice with MTBE (38 g) and then dried under reduced pressure to obtain compound 9-q (15.68 g). LCMS (ESI) of compound 9-q: retention time: 2.92 minutes, m / z = 370 [M+H] (analysis condition -K-1).

[0176] Synthesis of Compound 9-r: (tert-butyl (S)-3-((S)-2-(1-(((benzyloxy)carbonyl)(methyl)amino)-N-methylcyclobutane-1-carboxamido)-2-cyclopentyl-N-methylacetamido)-4-(dimethylamino)-3-oxo-butanoate)

[0177] Acetonitrile (224 mL), DIPEA (150 g), compound 9-q (74.56 g), and 1-(benzyloxycarbonyl(methyl)amino)cyclobutanoic acid (CAS number 1408729-60-1, 131 g) were placed in a nitrogen-purged reactor and stirred at room temperature for 10 minutes. After confirming complete dissolution, a 50 wt% solution of propylphosphonic anhydride in MeTHF (339 g) was added to the mixture over 15 minutes. After the addition was complete, the internal temperature was raised to 60°C and stirred for 2 hours. HPLC analysis confirmed that the reaction conversion was 99% or higher. 5% aqueous potassium carbonate (447 mL) and N,N-dimethyl-4-aminopyridine (90 g) were added, and the mixture was stirred for an additional 1 hour and 30 minutes. The internal temperature was cooled to 25°C, toluene (373 mL) was added, and the aqueous phase was removed by separation. The organic phase was washed with 4% aqueous sulfuric acid (447 mL x 2) and 5% aqueous sodium carbonate (447 mL x 2). The resulting organic phase was concentrated under reduced pressure to 226 mL at an external temperature of 60 °C. After concentration under reduced pressure, THF (447 mL) was added, and the concentration under reduced pressure to 226 mL was repeated twice to obtain a THF solution of compound 9-r (167.76 g). LCMS (ESI) of compound 9-r: retention time: 7.02 min, m / z = 637.43 [M + Na] + (analysis condition -K-1).

[0178] Synthesis of Compound 9-s: (tert-butyl (S)-3-((S)-2-cyclopentyl-N-methyl-2-(N-methyl-1-(methylamino)cyclobutane-1-carboxamido)acetamido)-4-(dimethylamino)-4-oxo-butanoate)

[0179] A pressure-resistant reactor was charged with a THF solution of compound 9-r (163.97 g), THF (665 mL), and 4.57 wt% Pd / C (14.7 g). The external temperature was set to 25 °C and the mixture was stirred. The atmosphere was purged with nitrogen at 0.20 MPaG three times, then with hydrogen at 0.20 MPaG three times, and the mixture was stirred under a hydrogen atmosphere at 0.20 MPaG for 1 hour. HPLC analysis confirmed that the reaction conversion was >99%. The atmosphere in the reactor was purged with nitrogen at 0.20 MPaG three times, and the Pd / C was removed by filtration. The removed Pd / C was washed three times with THF (444 mL). The filtrate and washings were combined and concentrated under reduced pressure to 174 mL at an external temperature of 50 °C. Acetonitrile (444 mL) was added, and the mixture was concentrated under reduced pressure to 174 mL twice, yielding an acetonitrile solution of compound 9-s (100.91 g). LCMS (ESI) of compound 9-s: Retention time: 2.63 minutes, m / z = 481.43 [M+H]+ (Analysis conditions -K-2)

[0180] Synthesis of Compound 9-t: (benzyl (2S,4R)-2-((1-(((S)-2-(((S)-4-(tert-butoxy)-1-(dimethylamino)-1,4-dioxabutan-2-yl)(methyl)amino)-1-cyclopentyl-2-oxoethyl)(methyl)carbamoyl)cyclobutyl)(methyl)carbamoyl)-4-ethoxypyrrolidine-1-carboxylate)

[0181] (2S,4R)-1-((benzyloxy)carbonyl)-4-ethoxypyrrolidine-2-carboxylic acid dicyclohexylamine salt (227 g) and MeTHF (767 mL) were added to a reactor and stirred at room temperature. 4% aqueous sulfuric acid (1151 mL) was added, and the aqueous phase was separated and discarded. The organic phase was washed with 4% aqueous sulfuric acid (1151 mL) and 5% aqueous sodium chloride (1151 mL). The resulting organic phase was concentrated under reduced pressure to 153 mL at an external temperature of 50 °C. Acetonitrile (384 mL) was added to the resulting concentrate and concentrated under reduced pressure to 153 mL. Reaction mixture 9-s (89.31 g), acetonitrile (384 mL), and DIPEA (103 g) were added and stirred at room temperature. TCFH (121 g) was added, and the external temperature of the reactor was raised to 50 °C and stirred for 5 hours. HPLC analysis confirmed that the reaction conversion was over 99%. Water (307 mL) and NMI (52.4 g) were added and stirred for 30 minutes. Stirring was stopped and the mixture was stored at room temperature overnight. Toluene (767 mL) was added at room temperature, and the aqueous phase was removed by separation. The organic phase was washed with 10% aqueous ammonia (767 mL x 2), 4% aqueous sulfuric acid (767 mL x 2), and 5% aqueous sodium carbonate (767 mL). The resulting organic phase was concentrated under reduced pressure to 242 mL at an external temperature of 50 °C. After concentration under reduced pressure, THF (537 mL) was added, and the concentration was repeated twice to 242 mL to obtain a THF solution of compound 9-t (195.54 g). LCMS (ESI) of compound 9-t: retention time: 4.30 min, m / z = 778.59 [M + Na] + (analysis condition -K-2).

[0182] Synthesis of Compound 9-u: (tert-butyl (S)-3-((S)-2-cyclopentyl-2-(1-((2S,4R)-4-ethoxy-N-methylpyrrolidine-2-carboxamido)-N-methylcyclobutane-1-carboxamido)-N-methylacetamido)-4-(dimethylamino)-4-oxo-butanoate)

[0183] A pressure-resistant reactor was charged with 4.57 wt% Pd / C (8.53 g) and THF (388.5 mL). The external temperature was set to 25 °C and the mixture was stirred. The atmosphere was purged with 0.20 MPaG nitrogen three times, then with 0.20 MPaG hydrogen three times, and the mixture was stirred under a 0.40 MPaG hydrogen atmosphere for 3 hours. A THF solution of compound 9-t (179.08 g) and THF (531 mL) were added. The atmosphere was purged with 0.20 MPaG nitrogen three times, then with 0.20 MPaG hydrogen three times, and the mixture was stirred under a 0.20 MPaG hydrogen atmosphere for 6 hours. HPLC analysis confirmed that the reaction conversion was >99%. The atmosphere inside the reactor was purged with 0.20 MPaG nitrogen three times, and the Pd / C was removed by filtration. The removed Pd / C was washed three times with MeTHF (232 mL). The filtrate and washings were combined and concentrated under reduced pressure to 182 mL at an external temperature of 50 °C. MeTHF (774 mL) was added and concentrated under reduced pressure to 182 mL. MeTHF (55.2 mL) was added to the resulting concentrate, and the temperature was raised to 54 °C and stirred. 158 mL of heptane was added, and seed crystals of compound 9-t (0.396 g) were suspended in heptane (15.85 mL) and added to the reactor. The mixture was stirred at an external temperature of 54 °C for 3 hours, cooled to 22 °C over 6.5 hours, and stirred for an additional 10 hours. Heptane (143 mL) was added over 1 hour and stirred for 1 hour. Heptane (634 mL) was added over 4 hours and stirred for 1 hour. The mixture was cooled to 10 °C over 2.5 hours and stirred for 14 hours. The reaction mixture was filtered through a separatory funnel, and the resulting crystals were washed twice with heptane (396 mL). The obtained crystals were dried under reduced pressure for 2.5 hours at an external temperature of 40°C. The dried powder was collected to obtain compound 9-u as a white powder (72.9 g). LCMS (ESI) of compound 9-u: retention time: 4.50 min, m / z = 622.53 [M+H] (analysis condition - K-1).

[0184] Compound 9-v: Synthesis of benzyl (S)-2-((tert-butoxycarbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate

[0185] A reaction vessel (1000 L) was charged with a mixture of nickel bromide trihydrate (1.23 kg) and 1,3-dimethyl-2-imidazolidinone (98.9 kg) as a slurry, followed by 4,4'-di-tert-butyl-2,2-dipyridyl (1.21 kg) and stirring at 20.8 to 22.7°C for 28 minutes. 1-Benzyl 5-(1,3-dioxoisoindolin-2-yl)(tert-butoxycarbonyl)-L-glutamate (31.0 kg), synthesized according to WO 2020 / 189540, 4-bromo-2-methoxy-1-(trifluoromethyl)benzene (24.57 kg), 1,3-dimethyl-2-imidazolidinone (230 kg), and N-methylmorpholine (3.25 kg) were charged, then cooled to an internal temperature of 8.7°C. Then, zinc (12.6 kg) was added. After stirring and stabilizing the temperature, chlorotrimethylsilane (21.0 kg) was added dropwise over 3 hours at an internal temperature ranging from 8.7 to 11.9°C. HPLC analysis confirmed a reaction conversion of 99% or greater. After cooling the internal temperature to 2.6°C, 15% aqueous ammonium chloride solution (332 kg) was added dropwise. Toluene (135 kg) was added to the resulting mixture, which was stirred for at least 30 minutes and then filtered through Celite. The Celite was washed with toluene (135 kg), and the aqueous phase was discarded. The organic phase was washed with a mixed solution (465 kg) of disodium ethylenediaminetetraacetate (24.3 kg) and 2.5% aqueous ammonia solution, followed by 10% aqueous sodium chloride solution (332 kg). The resulting organic phase was concentrated to 93 L at an external temperature of 40°C, yielding 87.3 kg of a solution containing the title compound 9-v. LCMS (ESI) of compound 9-v: retention time: 7.23 min, m / z = 368.38 [M-Boc+H]+ (analysis conditions -H).

[0186] Synthesis of Compound 9-w: (S)-2-((tert-butoxycarbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoic acid

[0187] A pressure-resistant reactor (150 L) was charged with a solution containing compound 9-v (43.7 kg) while washing with toluene (40.2 kg). 10% Pd / C (8.37 kg) was added and stirring was initiated. The pressure was increased to 0.15 MPaG with hydrogen, and the gas phase was replaced with hydrogen. The mixture was stirred for 2 hours and 2 minutes at an internal temperature of 22.6-29.2°C. HPLC analysis confirmed a reaction conversion of 99% or higher. The mixture was filtered, and the Pd / C residue was washed with toluene (26.7 kg). The filtrate was collected again using the same procedure. All the filtrates were combined and concentrated to 155 L at an external temperature of 40°C to obtain 155 kg of a solution containing compound 9-w. LCMS (ESI) of compound 9-w: retention time: 5.72 min, m / z = 278.37 [M-Boc+H]+ (analysis conditions -H).

[0188] Synthesis of Compound 9-x: (S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate

[0189] A solution containing compound 9-w (155 kg) was added to a 1000 L reactor, and methanesulfonic acid (24.7 kg) was added dropwise over 32 minutes at an internal temperature of 20.4°C to 27.6°C. The temperature was then raised. HPLC analysis confirmed a reaction conversion of 99% or higher. The reaction mixture was cooled, and water (186 kg) was added dropwise over 80 minutes at an internal temperature of 25.9°C to 32.0°C. After removing the organic phase, the mixture was washed three times with toluene (124 kg) and then adjusted to pH 6.52 with 40% aqueous potassium phosphate (95.7 kg). Acetonitrile (61.2 kg) was then added, and the pH was adjusted to 7.83 again with 40% aqueous potassium phosphate (25.4 kg). To the pH-adjusted reaction mixture, N-(benzyloxycarbonyloxy)succinimide (16.0 kg) was added. HPLC analysis confirmed that the reaction conversion was greater than 99%. The pH of the resulting reaction mixture was adjusted to 7.73 using 40% aqueous potassium phosphate (44.7 kg), followed by the addition of methyl tert-butyl ether (46.3 kg) and heptane (42.4 kg). Separation into three layers was confirmed. The upper layer was discarded, and methyl tert-butyl ether (115 kg) was added to the lower two layers. A 24% aqueous sodium hydroxide solution (2.1 kg), sodium chloride (12.4 kg), and water (110.0 kg) mixture was then added, and the aqueous phase was discarded. The resulting organic phase was washed four times with a 24% aqueous sodium hydroxide solution (2.1 kg), sodium chloride (12.4 kg), and water (110.0 kg) mixture. The resulting organic phase was washed with 0.2 M aqueous sodium hydroxide (62.1 kg) and then with a mixed solution of 24% aqueous sodium hydroxide (2.1 kg), sodium chloride (3.1 kg), and water (72.3 kg). The resulting organic phase was washed with 1 M hydrochloric acid (310 kg). The resulting organic phase was washed with 10% brine (166 kg) and concentrated to 62 L. Toluene (80.9 kg) was added and the mixture was concentrated again to 62 L, thereby carrying out solvent substitution with toluene. Toluene (204 kg) was added to the resulting concentrated residue to obtain a solution containing compound 9-x (260 kg).LCMS (ESI) of compound 9-x: Retention time: 5.913 min, m / z = 368.39 [M-C02+H]+ (Analysis conditions -H).

[0190] Synthesis of Compound 9-y: ((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoic acid dicyclohexylamine salt)

[0191] A solution containing compound 9-x (260 kg) was added to a 1000 L reactor, and the internal temperature was raised to 59.5°C. Dicyclohexylamine (16.1 kg) was then added. The reaction mixture was heated to 66.3°C, and heptane (49.9 kg) was added dropwise over 1 hour and 7 minutes. Compound 9-y (91.0 g) was suspended in a toluene (2.51 kg)-heptane (511 g) mixed solution as seed crystals and charged. The internal temperature was cooled from 67.0°C to 52.1°C over 1 hour and 38 minutes, after which stirring was continued for over 1 hour. The internal temperature was then cooled from 50.1°C to 23.0°C over 1 hour and 55 minutes. After cooling and stirring for 14 hours, the resulting slurry was filtered and washed with a toluene (47.4 kg)-heptane (37.2 kg) mixed solution to obtain a wet powder. The obtained wet powder was dried at an external temperature of 38°C for 50 hours to obtain compound 9-y (25.6 kg). LCMS (ESI) of compound 9-y: retention time: 5.89 minutes, m / z = 368.41 [M-CO2-DCHA+H]+ (analysis conditions -H).

[0192] Synthesis of Compound 9-z: ((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate)

[0193] Compound 9-y (57.8 kg) and MeTHF (253 mL) were added to a reaction vessel (2 L) at room temperature and stirred. 4% aqueous sulfuric acid (253 mL) was added, and the aqueous phase was removed by separation. The organic phase was washed with 4% aqueous sulfuric acid (253 mL) and 5% aqueous sodium chloride (253 mL). The resulting organic phase was concentrated under reduced pressure to 100.8 mL at an external temperature of 40°C. MeTHF (253 mL) was added to the resulting concentrated solution, and the concentration was repeated twice to 100.8 mL, yielding a solution containing compound 9-z (94.29 g). LCMS (ESI) of compound 9-z: retention time: 5.92 min, m / z = 368.18 [M-CO2 + H]+ (analysis condition -K-1).

[0194] Synthesis of Compound 9-aa: (tert-butyl-(S)-3-((S)-2-(1-((2S,4R)-1-((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoyl)-4-ethoxy-N-methylpyrrolidine-2-carboxamido)-N-methylcyclobutane-1-carboxamido)-2-cyclopentyl-N-methylacetamido)-4-(dimethylamino)-4-oxo-butanoate

[0195] Compound 9-u (94.29 g), MeTHF (253 mL), DIPEA (46.2 g), and compound 9-z (50.5 g) were added to a reaction vessel, cooled to 10 °C, and stirred. A 50 wt% propylphosphonic anhydride MeTHF solution (124 g) was added to the mixture over 15 min. The reaction vessel was heated to 25 °C and stirred for 1 h. HPLC analysis confirmed that the reaction conversion was >99%. A 5% aqueous potassium carbonate solution (303 mL) and NMI (6.67 g) were added and stirred for 30 min. Stirring was stopped and the mixture was stored at room temperature overnight. The aqueous phase was removed by separation, and the organic phase was washed with 4% aqueous sulfuric acid (303 mL x 2). Heptane (182 mL), MTBE (121 mL), acetonitrile (116 mL), and 2.5% aqueous potassium carbonate (288 mL) were added to the organic phase, and the aqueous phase was removed by separation. Acetonitrile (172 mL), MeTHF (101 mL), and 2.5% aqueous potassium carbonate solution (434 mL) were added to the resulting organic phase, and the aqueous phase was removed by separation. Acetonitrile (172 mL), MeTHF (101 mL), and 2.5% aqueous potassium carbonate solution (434 mL) were then added to the resulting organic phase, and the aqueous phase was removed by separation. The organic phase was concentrated under reduced pressure to 167 mL at an external temperature of 50°C. After concentration under reduced pressure, IPAC (354 mL) was added, and the concentration under reduced pressure to 167 mL was repeated twice to obtain a solution containing compound 9-aa (170.11 g). LCMS (ESI) of compound 9-aa: retention time: 8.10 min, m / z = 1037.62 [M+H]+ (analysis condition -K-1).

[0196] Synthesis of compound 9-ab: (3S)-3-[[(2S)-2-[[1-[[(2S,4R)-1-[(2S)-2-(benzyloxycarbonylamino)-4-[3-methoxy-4-(trifluoromethyl)phenyl]butanoyl]-4-ethoxy-pyrrolidine-2-carbonyl]-methyl-amino]cyclobutanecarbonyl]-methyl-amino]-2-cyclopentyl-acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoic acid

[0197] A solution of compound 9-aa (47.8 g) in IPAC (53.1 g) and IPAC (186 mL) were added to a nitrogen-purged reactor at room temperature and stirred. HMDS (19.0 g) was then added, and the reactor's external temperature was set to 0 °C and stirred for 15 minutes. TMSOTf (15.4 g) was added dropwise at an internal temperature below 20 °C. The reactor's external temperature was set to 20 °C, and the reaction mixture was stirred for 1 hour. After that, it was diluted with MeTHF (244 mL), and the reactor's external temperature was set to 10 °C. 5% aqueous potassium hydrogen phosphate (478 mL) was added dropwise, and stirring was stopped. The aqueous phase was then discharged. The resulting organic phase was washed with 5% sodium dihydrogen phosphate (478 mL), followed by the addition of diisopropylethylamine (9.0 mL), and concentrated under reduced pressure to yield a MeTHF solution of compound 9-ab (120.1 g). LCMS (ESJ) of compound 9-ab: retention time: 4.30 minutes, m / z = 981.66 [M+Na]+ (analysis condition -K-2)

[0198] Synthesis of Compound 9-ac: tert-butyl (2S)-1-[(2s,3s)-2-[[(2S)-2-[[(3S)-3-[[(2S)-2-[[1-[[(2S,4R)-1-[(2S)-2-(benzyloxycarbonylamino)-4-[3-methoxy-4-(trifluoromethyl)phenyl]butanoyl]-4-ethoxy-pyrrolidine-2-carbonyl]-methyl-amino]cyclobutanecarbonyl]-methyl-amino]-2-cyclopentyl-acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoyl]-methyl-amino]pentanoyl]amino]-3-methyl-pentanoyl]pyrrolidine-2-carboxylate

[0199] MeTHF (70 mL) and a MeTHF solution (113.85 g) of compound 9-ab (40.6 g) were added to a nitrogen-purged reactor at room temperature and stirred. Compound 9-l (21.9 g) dissolved in diisopropylethylamine (25.3 mL) and MeTHF (180 mL) was then added, followed by acetonitrile (40 mL) and stirring. HATU (24.2 g) was suspended in acetonitrile (53 mL) and added to the reactor and stirred for 1 hour. Acetic acid (1.2 mL) was added, stirred for 20 minutes, and then allowed to stand overnight. Stirring was resumed, the mixture was diluted with MeTHF (122 mL), and 10% aqueous ammonia (282 mL) was added. After stopping the stirring and discharging the aqueous phase, the resulting organic phase was washed with 10% aqueous ammonia (280 mL), 4% diluted sulfuric acid (280 mL), 4% diluted sulfuric acid (280 mL), and 5% aqueous sodium carbonate (280 mL), and concentrated under reduced pressure to give a solution of compound 9-ac in MeTHF (132.34 g).

[0200] Synthesis of Compound 9-ad: tert-butyl (2S)-1-[(2S,3S)-2-[[(2S)-2-[[(3S)-3-[[(2S)-2-[[1-[[(2S,4R)-1-[(2S)-2-amino-4-[3-methoxy-4-(trifluoromethyl)phenyl]butanoyl]-4-ethoxy-pyrrolidine-2-carbonyl]-methyl-amino]cyclobutanecarbonyl]-methyl-amino]-2-cyclopentyl-acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoyl]-methyl-amino]pentanoyl]amino]-3-methyl-pentanoyl]pyrrolidine-2-carboxylate

[0201] MeTHF (1.0 mL) and 10% palladium on activated carbon (90.3 mg) were added to a nitrogen-purged reaction vessel at room temperature. The reaction vessel was purged with hydrogen and stirred under hydrogen pressure (0.40 MPaG) for 1 hour. After venting the hydrogen and replacing it with nitrogen, compound 9-ac (514 mg) dissolved in MeTHF (1.5 mL) was added and stirred. After venting the nitrogen and replacing it with hydrogen, the mixture was stirred under hydrogen pressure (0.20 MPaG) for a total of 8 hours and 30 minutes. After venting the hydrogen and replacing it with nitrogen, the palladium on activated carbon was removed by filtration. The cake was washed three times with MeTHF (500 μL), and the filtrate was combined and concentrated to dryness under reduced pressure to give compound 9-ad (503 mg). LCMS (ESI) of compound 9-ad: retention time: 8.04 min, m / z = 1205.0 [M+H]+ (analysis condition -FC2).

[0202] Synthesis of Compound 9-ae: tert-butyl [(S)-2-[(S)-3-[(S)-2-(1-[(2S,4R)-1-[(S)-2-amino-[3-methoxy-4-(trifluoromethyl)phenyl]butanoyl]-4-ethoxy-N-methylpyrrolidine-2-carboxamido]-N-methylcyclobutane-1-carboxamido)-2-cyclopentyl-N-methylacetamido]-4-[dimethylamino]-N-methyl-4-oxobutanamido]pentanoyl]-L-isoleucyl-L-prophosphate tert-butyl

[0203] A MeTHF solution (17.6 kg) of compound 9-ad (3.41 kg) was added to the reactor at room temperature, followed by MeTHF (12.3 kg), compound 9-h mono-toluene solvate (2.38 kg), and acetonitrile (6.20 kg), sequentially. The external temperature was set to 5°C, and NMM (1.26 kg) and HATU (1.65 kg) were added at an internal temperature below 30°C. The mixture was stirred at 25°C for 1 hour. After confirming a reaction conversion of 99% or higher, 5% aqueous potassium carbonate (23.9 kg) and 1-methylimidazole (234 g) were added and stirred at room temperature for 30 minutes. The aqueous phase was then separated and discharged. The resulting organic phase was washed with 10% aqueous ammonia (23.0 kg), 5% aqueous sodium hydrogen sulfate (23.9 kg), and 5% aqueous sodium carbonate (23.9 kg) and then concentrated to 10 L at an external temperature of 40°C. Acetonitrile (16.2 kg) was added to the concentrated solution, and the mixture was concentrated twice to 10 L to obtain an acetonitrile solution of compound 9-ae (19.4 kg).

[0204] Synthesis of compound 9-af: (S)-2-[(S)-3-[(S)-2-cyclopentyl-2-[1-[(2S,4R)-4-ethoxy-1-[(S)-4-[3-methoxy-4-(trifluoromethyl)phenyl]-2-[(2-[(S)-N-methyl-2-[(R,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl]-3-[4-(trifluoromethyl)phenyl]propanamido]acetamido)butanoyl]-N-methylpyrrolidine-2-carboxamido]-N-methylcyclobutane-1-carboxamido]-N-methylacetamido]-4-(dimethylamino)-N-methyl-4-oxobutanamido]pentanoyl]-L-isoleucyl-L-prophosphate tert-butyl ester

[0205] A solution of compound 9-ae (4.67 kg) in acetonitrile (14.9 kg) was added to the reactor, and acetonitrile (4.53 kg) was added. The mixture was cooled to an internal temperature of below 15°C. DBU (1.65 kg) was added at an internal temperature of below 30°C. After stirring at 25°C for 1 hour and confirming a reaction conversion of 99% or higher, the mixture was cooled again to an internal temperature of below 15°C. Triethylamine (1.02 kg), water (1.14 kg), and sodium bisulfite (925 g) were added sequentially, with the internal temperature not exceeding 30°C, and the mixture was stirred for 1 hour. After stirring, toluene (20.4 kg) and 10% aqueous ammonia (46.7 kg) were added to the reaction mixture, and the mixture was stirred. The aqueous phase was then removed by liquid-liquid separation. Acetonitrile (18.4 kg) and 10% aqueous ammonia (46.7 kg) were added to the organic phase, and the mixture was stirred again. This process of removing the aqueous phase by liquid-liquid separation was repeated twice. The organic phase was washed with 5% brine (23.4 kg) and separated into liquids, and then the organic phase was concentrated to 9.3 L at an external temperature of 60° C. MeTHF (16.0 kg) was added to the concentrated solution, and concentration to 9.3 L at an external temperature of 60° C. was repeated twice to obtain a MeTHF solution (7.72 kg) of compound 9-ae.

[0206] Synthesis of Compound 9-ag: (S)-2-[(S)-3-[(S)-2-cyclopentyl-2-[1-[(2S,4R)-4-ethoxy-1-[(S)-4-[3-methoxy-4-(trifluoromethyl)phenyl]-2-[(2-[(S)-N-methyl-2-[(R,Z)-3-(methylamino)-2-oxo-3,4,7,8-tetrahydroazocin-1(2H)-yl]-3-[4-(trifluoromethyl)phenyl]propanamido]acetamido)butanoyl]-N-methylpyrrolidine-2-carboxamido]-N-methylcyclobutane-1-carboxamido]-N-methylacetamido]-4-(dimethylamino)-N-methyl-4-oxobutanamido]pentanoyl]-L-isoleucyl-L-proline

[0207] A solution of compound 9-af (3.74 kg) in MeTHF (11.9 kg) was added to the reactor, followed by additional MeTHF (7.78 kg) and HMDS (1.12 kg). After cooling to below 5°C, TMSOTf (1.03 kg) was added at below 30°C, and the mixture was heated to 25°C and stirred for 2 hours. After confirming a reaction conversion of 99% or higher, MeTHF (25.6 kg) and acetonitrile (5.88 kg) were added. The mixture was then cooled to below 5°C and 5% dipotassium hydrogen phosphate (26.2%) was added at below 30°C. The aqueous phase was discharged by liquid-liquid separation, and the organic phase was washed sequentially with 5% sodium hydrogen sulfate (26.2 kg), 5% sodium carbonate (26.2 kg), and 5% saline (26.2 kg). The organic phase obtained at an external temperature of 50 °C was concentrated to 9.4 L, and acetonitrile (7.36 kg) was added. This process was repeated twice. Acetonitrile (8.82 kg) and heptane (25.6 kg) were added to the concentrated solution and stirred. The liquid was separated, and the lower layer was collected to obtain an acetonitrile solution of compound 9-ag (17.7 kg). Quantitative analysis by qNMR indicated that the yield of compound 9-ag over the three steps was 93.4%.

[0208] Synthesis of compound 9

[0209] A solution of HATU in acetonitrile was prepared by dissolving 949 g of HATU in acetonitrile (25.6 kg) in a reactor. A solution of compound 9-ag in acetonitrile (5.52 kg), acetonitrile (19.1 kg), and N-methylmorpholine (504 g) was added to another vessel. The acetonitrile solution of compound 9-ag was added dropwise to the acetonitrile solution of HATU at an external temperature of 25 °C over 4 hours and 22 minutes, followed by rinsing with acetonitrile (1.04 kg). After the addition, the mixture was stirred for an additional hour. HPLC analysis confirmed a reaction conversion of 99% or greater. The external temperature was set to 40 °C, and the reaction mixture was concentrated to approximately 12.5 L. MTBE (14.5 kg) and 2.5% aqueous ammonia (13.3 kg) were added to the resulting concentrate at room temperature and stirred for 34 minutes. After discharging the aqueous phase by liquid-liquid separation, the resulting organic phase was washed sequentially with 5% sulfuric acid (13.1 kg), 5% dipotassium hydrogen phosphate aqueous solution (13.1 kg), and 0.5% sodium chloride aqueous solution (13.07 kg). Acetonitrile (2.00 kg) was added to the resulting organic phase, which was then washed with 0.5% sodium chloride aqueous solution (13.1 kg). After solvent substitution with ethanol, the resulting organic phase was analyzed by HPLC (analysis conditions - cyc) using a standard sample. Compound 9 was obtained in an amount of 1.14 kg (88.3% yield). HPLC of compound 9: retention time: 18.67 (analysis conditions - cyc).

[0210] Evaluation of Lipid Solubility of Cyclic Peptide Compounds Table 11 shows the ClogP and ClogP / total amino acid of compounds 1 to 9.

[0211] Example 2: Analysis of Oligomers of Cyclic Peptide Compounds In Example 2, high-performance liquid chromatography (HPLC) analysis was performed using one of the analytical conditions described below. Each compound was detected using a variable UV detector or a mass spectrometer, but other methods such as a photodiode array detector may also be used.

[0212] Analysis Condition-11 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / 2-propanol Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0213] Analysis Conditions-12 (HPLC-UV) Apparatus: Waters Alliance HPLC system Column: TOSOH TSKgel SuperH3000, 3 μm, 6.0 mm ID x 150 mm Mobile phase: Tetrahydrofuran Flow rate: 0.45 mL / min Column temperature: 35 °C Detection wavelength: 220 nm Elution method: Isocratic mode Analysis time: 30 min

[0214] Analysis Condition-13 (HPLC-MS) Apparatus: Thermo Vanquish HPLC system - Thermo Q Exactive orbitrap MS Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.1% FA / water Mobile phase B: 0.1% FA / 2-propanol Flow rate: 0.2 mL / min Column temperature: 50 °C Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0215] Analysis Condition-14 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / methanol Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0216] Analysis Condition-15 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0217] Analysis Conditions-16 (HPLC-UV) Apparatus: Waters Alliance HPLC system Column: TOSOH TSKgel G3000 PWXL, 7 μm, 7.8 mm ID x 300 mm Mobile phase: 20% acetonitrile / 80% water Flow rate: 0.5 mL / min Column temperature: 35 °C Detection wavelength: 220 nm Elution method: isocratic mode Analysis time: 120 min

[0218] Analysis Condition-17 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH C18, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / 2-propanol Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0219] Analysis Condition-18 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH Fluoro-Phenyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / 2-propanol Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0220] Analysis Condition-19 (HPLC-UV) Apparatus: Waters, ACQUITY UPLC H-Class system Column: Waters, ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm ID x 150 mm Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / ethanol Flow rate: 0.2 mL / min Column temperature: 50 °C Detection wavelength: 220 nm Elution method: Gradient mode Gradient conditions: 70% B (0 min) → 70% B (3 min) → 100% B (3.1 min) → 100% B (5 min) → 70% B (5.1 min) → 70% B (12 min)

[0221] Example 2-1: Oligomer analysis of Compound 1 Compound 1 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.5 minutes. The area values ​​of the peaks detected in the elution time range from 2.6 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 2 and Table 12.

[0222] The above-mentioned oligomer is defined as a polymer containing the constituent components of the target cyclic peptide. Oligomers include cyclic dimers (C-dimers) and cyclic trimers (C-trimers) derived from the process of producing the target cyclic peptide. Examples of the chemical structures of the C-dimer and C-trimer derived from Compound 1 are shown in Figures 3 and 4. The C-dimer and C-trimer in Example 2-1 and subsequent examples refer to the C-dimer and C-trimer of the cyclic peptide appearing in that example.

[0223] Compound 1 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.1 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 6.0 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 5 and Table 12.

[0224] Compound 1 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0225] Compound 1 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-17. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 2.1 minutes. The area values ​​of the peaks detected in the elution time range from 2.2 minutes to 8.0 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 16 and Table 12.

[0226] Compound 1 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-18. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 2.3 minutes. The area values ​​of the peaks detected in the elution time range from 2.4 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 17 and Table 12.

[0227] Compound 1 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-19. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 4.9 minutes. The area values ​​of the peaks detected in the elution time range from 5.0 to 9.0 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 18 and Table 12.

[0228] Example 2-2: Oligomer Analysis of Compound 2 Compound 2 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.3 minutes. The area values ​​of the peaks detected in the elution time range from 2.4 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 6 and Table 12.

[0229] Compound 2 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.1 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 6.0 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 7 and Table 12.

[0230] Compound 2 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0231] Compound 2 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-17. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 2.0 minutes. The area values ​​of the peaks detected in the elution time range from 2.1 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 19 and Table 12.

[0232] Compound 2 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-18. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.3 minutes. The area values ​​of the peaks detected in the elution time range from 2.4 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 20 and Table 12.

[0233] Compound 2 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-19. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 3.9 minutes. The area values ​​of the peaks detected in the elution time range from 4.0 minutes to 9.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 21 and Table 12.

[0234] Example 2-3: Oligomer analysis of compound 3 Compound 3 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-11. The chromatogram obtained was used for evaluation after subtracting a chromatogram of a blank solution using analysis software. The target cyclic peptide was detected at an elution time of 2.2 minutes. The area values ​​of the peaks detected in the elution time range from 2.3 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 8 and Table 12.

[0235] Compound 3 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.4 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 6.3 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 9 and Table 12.

[0236] Compound 3 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0237] Example 2-4: Oligomer analysis of compound 4 Compound 4 obtained in Example 1 was diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.7 minutes. The area values ​​of the peaks detected in the elution time range from 2.8 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0238] Compound 4 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.0 minutes. The area values ​​of the peaks detected in the elution time range of 3.5 to 5.9 minutes were integrated and estimated to represent a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0239] Compound 4 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0240] Example 2-5: Oligomer analysis of compound 5 Compound 5 obtained in Example 1 was diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 3.0 minutes. The area values ​​of the peaks detected in the elution time range from 3.1 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0241] Compound 5 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.0 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 5.9 minutes were integrated, and the peaks were estimated to be compounds other than the target cyclic peptide, including oligomers. The results are shown in Table 12.

[0242] Compound 5 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0243] Example 2-6: Oligomer analysis of compound 6 Compound 6 obtained in Example 1 was diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.5 minutes. The area values ​​of the peaks detected in the elution time range from 2.6 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0244] Compound 6 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.0 minutes. The area values ​​of the peaks detected in the elution time range of 3.5 to 5.9 minutes were integrated and estimated to represent a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0245] Compound 6 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0246] Example 2-7: Oligomer analysis of compound 7 Compound 7 obtained in Example 1 was diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.8 minutes. The area values ​​of the peaks detected in the elution time range from 2.9 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0247] Compound 7 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.1 minutes. The area values ​​of the peaks detected in the elution range from 3.5 to 6.0 minutes were integrated, and the peaks were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0248] Compound 7 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0249] Example 2-8: Oligomer analysis of compound 8 Compound 8 obtained in Example 1 was diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-11. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. The target cyclic peptide was detected at an elution time of 2.8 minutes. The area values ​​of the peaks detected in the elution time range from 2.9 minutes to 8.0 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0250] Compound 8 obtained in Example 1 was diluted with THF to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.3 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 6.2 minutes were integrated, and the compounds were estimated to be compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Table 12.

[0251] Compound 8 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0252] Example 2-9: Oligomer Analysis of Compound 9 Compound 9 obtained in Example 1 was diluted with THF to a target cyclic peptide concentration of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-12. The target cyclic peptide was detected at an elution time of 6.0 minutes. The area values ​​of the peaks detected in the elution time range from 3.5 minutes to 5.9 minutes were integrated, and the compounds were estimated to be a group of compounds other than the target cyclic peptide, including oligomers. The evaluation results are shown in Figure 10 and Table 12.

[0253] Compound 9 obtained in Example 1 was diluted with acetonitrile to a concentration of approximately 1 mg / mL of the target cyclic peptide to prepare a sample solution, which was then analyzed under analysis condition 13. The structure was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes. The name of the estimated compound, MS elution time (minutes), ESI-MS observed value (ESI (m / z)), and estimated ion charge of the analytical results are shown in Table 13.

[0254] Comparative Example 2-1: Oligomer Analysis Using Methanol as Eluent. Compounds 1 and 2 obtained in Example 1 were each diluted with acetonitrile to a target cyclic peptide concentration of approximately 1 mg / mL to prepare a sample solution, which was then analyzed under analysis condition-14. The data obtained after subtracting a chromatogram of a blank solution using analysis software from the resulting chromatogram was used for evaluation. As a result of the analysis of Compound 1, the target cyclic peptide was detected at 8.0 minutes. The area values ​​of the peaks detected in the elution time range from 8.1 to 9.0 minutes were integrated and estimated as a group of compounds other than the target cyclic peptide, including oligomers. As a result of the analysis of Compound 2, the target cyclic peptide was detected at 7.7 minutes, but no other major peaks were observed, suggesting that oligomers were not eluted from the analytical column. The evaluation results are shown in Figures 11, 12, and Table 12.

[0255] When the area percentage of the target cyclic peptide was compared under analytical conditions -11, -12, and -14 in the analysis results for Compound 1, the result under analytical condition -14 was the highest (Table 12). The 0.05% TFA / methanol used as mobile phase B under analytical condition -14 had low elution power for the target cyclic peptide and oligomers, suggesting that not all components were completely eluted from the analytical column. Therefore, it was demonstrated that separation and simultaneous detection of the target cyclic peptide and oligomers is difficult using conventional analytical methods that use methanol as an eluent.

[0256] Comparative Example 2-2: Oligomer Analysis Using Acetonitrile as Eluent Compounds 1, 2, and 3 obtained in Example 1 were each diluted with acetonitrile to a target cyclic peptide concentration of approximately 1 mg / mL to prepare a sample solution, which was then analyzed according to analysis condition-15. The data obtained after subtracting a blank solution chromatogram from the resulting chromatogram using analysis software was used for evaluation. The analysis results for Compounds 1, 2, and 3 showed that the target cyclic peptides were detected at 4.4 minutes, 3.8 minutes, and 2.9 minutes, respectively. The area values ​​of the peaks observed at elution times slower than the elution times of the respective target cyclic peptides were integrated and estimated to represent a group of compounds other than the target cyclic peptides, including oligomers. The evaluation results are shown in Figures 13, 14, 15, and Table 12.

[0257] The area percentages of the target cyclic peptides were compared for Compounds 1, 2, and 3 under Analysis Conditions 11, 12, and 15. Analysis Condition 15 showed the highest values ​​for all compounds (Table 12). The 0.05% TFA / acetonitrile used in Mobile Phase B under Analysis Condition 15 had low elution power for the target cyclic peptides and oligomers, suggesting that not all components were completely eluted from the analytical column. This indicates that conventional analytical methods using acetonitrile as an eluent are difficult to separate and simultaneously detect the target cyclic peptides and oligomers.

[0258] Comparative Example 2-3: Oligomer analysis by SEC method using aqueous mobile phase Compound 1 and Compound 2 obtained in Example 1 were each diluted with acetonitrile to a concentration of the target cyclic peptide of approximately 1 mg / mL to prepare a sample solution, which was analyzed according to Analysis Condition-16.

[0259] The analytical results for Compound 1 and Compound 2 showed no peaks, making analytical evaluation impossible. The combination of column and eluent under Analysis Condition-16 had a low elution power for the target cyclic peptide and oligomers, suggesting that not all components were completely eluted from the analytical column. This demonstrates the difficulty of separating and simultaneously detecting the target cyclic peptide and oligomers using conventional SEC methods using an aqueous mobile phase.

[0260]

[0261]

[0262] The present invention provides a means for simultaneously analyzing a target peptide, particularly a cyclic peptide and its oligomer, and also provides a method for producing a peptide using the analytical means.

Claims

A method for analyzing a peptide compound and an oligomer derived from said peptide compound in a sample, comprising: The aforementioned analytical method comprises carrying out liquid chromatography using a reverse phase column and an eluent containing a solvent containing an alcohol having 2 to 3 carbon atoms. 　 The method of claim 1 , wherein the peptidic compounds and oligomers derived from the peptidic compounds are separated and analyzed. 　 The method of claim 1 , wherein the peptidic compound and oligomers derived from the peptidic compound are analyzed simultaneously. 　 The method according to any one of claims 1 to 3, wherein the alcohol having 2 to 3 carbon atoms is one or more selected from the group consisting of ethanol, 1-propanol, and 2-propanol. 　 The method of any one of claims 1 to 4, wherein the solvent further comprises an organic acid. 　 The reverse phase column is C ６ Aryl C ２ -C １０ Alkyl silica gel column or C ４ -C １８ The method according to any one of claims 1 to 5, wherein the column is an alkyl silica gel column. 　 The alcohol having 2 to 3 carbon atoms is 2-propanol, and the reversed-phase column is a phenylhexyl silica gel column, a pentafluorophenylpropyl silica gel column, and an octadecyl (C １８ 4. The method according to claim 1, wherein the column is one selected from the group consisting of: a silica gel column; 　 The method according to any one of claims 1 to 7, wherein the ClogP of the peptide compound is 7 to 20. 　 The method according to any one of claims 1 to 8, wherein the ClogP / total aa of the peptide compound is 1.0 to 1.

8. The method according to any one of claims 1 to 9, wherein the number of amino acid residues constituting the peptide compound is 7 to 20. 　 The method according to any one of claims 1 to 10, wherein the peptide compound is a cyclic peptide compound having a cyclic portion. 　 The method of any one of claims 1 to 11, wherein the oligomer comprises a dimer of the peptide compound.

1. A method for producing a composition comprising a peptide compound, comprising: i) providing a composition comprising a desired peptide compound; and ii) analyzing the peptide compound and oligomers derived from the peptide compound in the composition by the method according to any one of claims 1 to 12; The manufacturing method comprising: 　 The method of claim 13, further comprising the step of cyclizing the precursor of the desired peptide compound prior to step i). 　 The method of claim 13 or 14, wherein the composition is a pharmaceutical composition.

1. A method for producing a pharmaceutical formulation comprising a peptide compound, comprising: i) providing a composition comprising a desired peptide compound; ii) analyzing the peptide compounds and oligomers derived from the peptide compounds in the composition by the method of any one of claims 1 to 12; and iii) formulating said composition to provide a pharmaceutical formulation; The manufacturing method comprising:

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