Method for purifying cyclic peptide and method for producing cyclic peptide

Abstract

The present disclosure relates to a method for purifying a cyclic peptide, said method comprising a step for separating, from a mixture containing a cyclic peptide that is a purification target and a peptide that is an impurity, the cyclic peptide that is the purification target or the peptide that is an impurity, as a complex with a divalent metal ion or a metal salt containing a divalent metal ion.

Description

Method for purifying and producing cyclic peptides

[0001] The present invention relates to a method for purifying a cyclic peptide and a method for producing a cyclic peptide using the same.

[0002] Conventionally, purification by column chromatography has been known as a method for purifying cyclic peptides (Non-Patent Document 1). On the other hand, Patent Document 1 describes that "cyclic peptides produced without isolating and purifying intermediates can be isolated and purified by crystallization without relying on column chromatography to obtain crystals of the cyclic peptide."

[0003] International Publication No. 2022 / 234864 International Publication No. 2002 / 055537 Japanese Patent Publication No. 2008-214348 Chinese Patent Application Publication No. 103059108

[0004] K. Nomura et al. , “Broadly Applicable and Comprehensive Synthetic Method for N-Alkyl-Rich Drug-like Cyclic Peptides”, J. Med. Chem. , 2022, 65, 19, 13401-13412

[0005] However, particularly in the case of industrial-scale peptide synthesis, purification by column chromatography tends to be inefficient. Furthermore, many cyclic peptides have physical properties that make them difficult to crystallize, and some cyclic peptides are difficult to purify by crystallization.

[0006] An object of the present invention is to provide a method for easily purifying and producing a cyclic peptide, which may be applicable to cyclic peptides that are difficult to crystallize.

[0007] The present invention relates to, for example, the following inventions. [1] A method for purifying a cyclic peptide, comprising a step of separating the cyclic peptide to be purified or the peptide as an impurity from a mixture containing the cyclic peptide to be purified and the peptide as an impurity as a complex with a divalent metal ion or a metal salt containing a divalent metal ion. [1-1] The method for purifying a cyclic peptide according to [1], comprising a step of separating the peptide as an impurity from a mixture containing the cyclic peptide to be purified and the peptide as an impurity as a complex with a divalent metal ion or a metal salt containing a divalent metal ion. [2] The method according to [1], comprising (1) a step of mixing the mixture containing the cyclic peptide to be purified and the peptide as an impurity with the divalent metal ion or the metal salt containing a divalent metal ion, as a pre-step of the separation step. [3] The method according to [2], wherein the step (1) is carried out in a first solvent. [4] A method for purifying a cyclic peptide, comprising the step (1)' of mixing, in a first solvent, a mixture containing the cyclic peptide to be purified and peptides as impurities with a divalent metal ion or a metal salt containing a divalent metal ion. [5] The method according to [4], wherein, in the step (1)', a complex is formed between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion in the first solvent. [5-1] The method according to [4], wherein, in the step (1)', a complex is formed between the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion in the first solvent. [6] The method according to [5], further comprising the step (2) of separating the complex from the mixture containing the cyclic peptide to be purified and peptides as impurities. [7] The purification method according to any one of [2] to [6], wherein in the step (1) or the step (1)', the metal salt containing the divalent metal ion is 10 wt % to 300 wt % with respect to the mixture containing the cyclic peptide to be purified and peptides as impurities.[8] The purification method according to any one of [2] to [7], wherein in step (1) or step (1)', the metal salt containing a divalent metal ion is present in an amount of 20 wt % to 100 wt % relative to the mixture containing the cyclic peptide to be purified and peptides as impurities. [9] The purification method according to any one of [2] to [8], wherein in step (1) or step (1)', the metal salt containing a divalent metal ion is present in an amount of 30 wt % to 50 wt % relative to the mixture containing the cyclic peptide to be purified and peptides as impurities.

[10] The purification method according to any one of [3] to [9], wherein in step (1) or step (1)', the mixture containing the cyclic peptide to be purified and peptides as impurities is present in an amount of 0.1 mg / mL to 1000 mg / mL relative to the first solvent.

[11] The purification method according to any one of [3] to

[10] , wherein in the step (1) or the step (1)', the mixture containing the cyclic peptide as the purification target and peptides as impurities has a concentration of 1.0 mg / mL to 500 mg / mL relative to the first solvent.

[12] The purification method according to any one of [3] to

[11] , wherein in the step (1) or the step (1)', the mixture containing the cyclic peptide as the purification target and peptides as impurities has a concentration of 10 mg / mL to 100 mg / mL relative to the first solvent.

[13] The purification method according to any one of [3] to

[12] , wherein in the step (1) or the step (1)', the first solvent has a liquid temperature of -20 to 100°C.

[14] The purification method according to any one of [3] to

[13] , wherein in the step (1) or the step (1)', the first solvent has a liquid temperature of 0 to 60°C. [14-1] The purification method according to any one of [3] to

[13] , wherein in the step (1) or the step (1)', the first solvent has a liquid temperature of 10 to 30° C.

[15] The purification method according to any one of [2] to

[14] , wherein in the step (1) or the step (1)', a mixture containing the cyclic peptide to be purified and peptides as impurities is mixed with a divalent metal ion or a metal salt containing a divalent metal ion for 0.1 to 192 hours.[15-1] The purification method according to any one of [2] to

[15] , wherein in the step (1) or the step (1)', a mixture containing the cyclic peptide to be purified and the peptide impurity is mixed with a divalent metal ion or a metal salt containing a divalent metal ion for 0.5 to 72 hours.

[16] The purification method according to any one of [2] to

[15] , wherein in the step (1) or the step (1)', a mixture containing the cyclic peptide to be purified and the peptide impurity is mixed with a divalent metal ion or a metal salt containing a divalent metal ion for 1 to 30 hours.

[17] The purification method according to any one of [1], [2], [3], and [6] to

[16] , wherein separation of the complex is carried out by solid-liquid separation or liquid-liquid separation.

[18] The purification method according to any one of [1], [2], [3], and [6] to

[17] , wherein separation of the complex is carried out by solid-liquid separation.

[19] The purification method according to any one of [1], [2], [3], and [6] to

[17] , wherein separation of the complex is carried out by liquid-liquid separation.

[20] The purification method according to any one of

[17] or

[18] , wherein the solid-liquid separation is centrifugation or filtration.

[21] The purification method according to any one of [1] to

[20] , further comprising (3) a step of removing the divalent metal ion or a metal salt containing a divalent metal ion from a mixture of the cyclic peptide as a purification target and the first solvent.

[22] The purification method according to

[21] , wherein the step (3) comprises a step of mixing a mixture of the cyclic peptide as a purification target and the first solvent with a second solvent and an aqueous solution, and separating the mixture into an aqueous phase and an organic phase, thereby removing the divalent metal ion or a metal salt containing a divalent metal ion from the mixture.

[23] The purification method according to

[22] , wherein the step (3) further comprises a step of removing the first solvent and the second solvent from an organic phase containing the cyclic peptide to be purified, thereby obtaining the cyclic peptide to be purified.

[24] The purification method according to

[23] , wherein the removal of the first solvent and the second solvent from the organic phase is carried out by vacuum distillation.

[25] (4) The purification method according to any one of [1] to

[24] , further comprising a step of confirming that the cyclic peptide as the purification target or the peptide as an impurity forms a complex with the divalent metal ion or a metal salt containing a divalent metal ion.

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

[25] , further comprising a step of confirming that the peptide as an impurity forms a complex with the divalent metal ion or a metal salt containing a divalent metal ion.

[28] The purification method according to any one of [1] to [3], [5], and [6] to

[27] , wherein the complex is a complex of the cyclic peptide as the purification target and the divalent metal ion or a metal salt containing a divalent metal ion.

[29] The purification method according to any one of [1] to [3], [5], and [6] to

[27] , wherein the complex is a complex of the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion.

[30] The purification method according to any one of [1] to [3], [5], and [6] to

[29] , wherein the complex is a complex obtained by contacting the cyclic peptide to be purified or the peptide as an impurity with the divalent metal ion or a metal salt containing a divalent metal ion.

[31] The purification method according to any one of [1] to [3], [5], and [6] to

[30] , wherein the complex is an adsorption complex. [31-1] The purification method according to any one of [1] to [3], [5], and [6] to

[30] , wherein the complex is a complex.

[32] The purification method according to

[31] , wherein the complex comprises a solid, a crystal, a liquid, or an amorphous form.

[33] The purification method according to

[31] or

[32] , wherein the complex comprises a crystal.

[34] The purification method according to

[31] or

[32] , wherein the complex comprises an amorphous form.

[35] The purification method according to any one of [1] to

[34] , wherein the mixture containing the cyclic peptide to be purified and peptides as impurities is a crude product obtained by producing the cyclic peptide to be purified.

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

[33] , wherein the cyclic peptide to be purified has 7 to 20 amino acid residues.

[35] The purification method according to any one of [1] to

[34] , wherein the cyclic peptide to be purified has 7 to 14 amino acid residues.

[36] The purification method according to any one of [1] to

[35] , wherein the number of amino acid residues in the cyclic portion of the cyclic peptide to be purified is 10 to 14.

[39] The purification method according to any one of [1] to

[38] , wherein the number of amino acid residues in the cyclic portion of the cyclic peptide to be purified is 11.

[40] The purification method according to any one of [1] to

[39] , wherein the cyclic peptide to be purified contains an unnatural amino acid.

[41] The purification method according to any one of [1] to

[40] , wherein the cyclic peptide to be purified contains an N-substituted amino acid.

[42] The purification method according to any one of [1] to

[41] , wherein the cyclic peptide to be purified contains three or more N-substituted amino acids.

[43] The purification method according to any one of [1] to

[42] , wherein the cyclic peptide to be purified contains five or more N-substituted amino acids.

[44] The purification method according to any one of [1] to

[43] , wherein the cyclic peptide to be purified contains six or more N-substituted amino acids.

[45] The purification method according to any one of [1] to

[44] , wherein the number of N-substituted amino acid residues in the cyclic peptide to be purified is 45% or more of the number of amino acid residues in the cyclic portion of the cyclic peptide.

[46] The purification method according to any one of

[41] to

[45] , wherein the N-substituted amino acid is at least one selected from the group consisting of N-methyl amino acid, N-ethyl amino acid, and N-propyl amino acid.

[47] The purification method according to any one of

[41] to

[46] , wherein the N-substituted amino acid is an N-methyl amino acid.

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

[47] , wherein the cyclic peptide to be purified contains an N-unsubstituted amino acid.

[49] The purification method according to

[48] , wherein the N-unsubstituted amino acid is an unnatural amino acid.

[50] The purification method according to

[48] or

[49] , wherein the ratio of the number of N-unsubstituted amino acids in the cyclic peptide to the total number of amino acid residues in the cyclic peptide is 55% or less. [50-1] The purification method according to any one of [1] to

[50] , wherein the cyclic peptide to be purified has a ClogP of 7 to 20. [50-2] The purification method according to any one of [1] to

[50] , wherein the cyclic peptide to be purified has a ClogP of 8 to 20.[50-3] The purification method according to any one of [1] to

[50] , wherein the ClogP of the cyclic peptide to be purified is 11 to 20.

[51] The purification method according to any one of [1] to

[50] , wherein the ClogP / total aa of the cyclic peptide to be purified is 1.0 or more and 1.8 or less. [51-1] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in the side chain of the cyclic portion. [51-2] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified 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. [51-3] The method according to any one of [1] to

[51] , wherein, when the cyclic peptide to be purified has an acidic side chain at its cyclic moiety, the pKa of the acidic side chain is 3.5 to 15 or less. [51-4] The method according to any one of [1] to

[51] , wherein, when the cyclic peptide to be purified has an acidic side chain at its cyclic moiety, the pKa of the acidic side chain is 6.0 to 15. [51-5] The method according to any one of [1] to

[51] , wherein, when the cyclic peptide to be purified has an acidic side chain at its cyclic moiety, the pKa of the acidic side chain is 10 to 15. [51-6] The method according to any one of [1] to

[51] , wherein, when the cyclic peptide to be purified has a basic side chain at its cyclic moiety, the basic pKa of the basic side chain is 3.0 to 15. [51-7] The method according to any one of [1] to

[51] , wherein, when the cyclic portion of the product to be purified has a basic side chain, the basic side chain has a basic pKa of 4.0 to 13. [51-8] The method according to any one of [1] to

[51] , wherein, when the cyclic portion of the cyclic peptide to be purified has a basic side chain, the basic side chain has a basic pKa of 4.0 to 11. [51-9] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 side chain of the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group.[51-10] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof. [51-11] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof. [51-12] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 side chain has a basic pKa of 4.0 to 10. [51-13] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 side chain has a basic pKa of 4.0 to 10.[51-14] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 11, wherein the ClogP is 11 to 20, wherein the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof, and wherein, 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. [51-15] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide having a cyclic portion, wherein the number of amino acid residues constituting the cyclic portion is 7 to 14, wherein the ClogP / total aa is 1.0 to 1.8, and wherein the cyclic portion does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in a side chain thereof. [51-16] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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. [51-17] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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. [51-18] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 side chain has a basic pKa of 4.0 to 10.[51-19] The method according to any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 side chain has a basic pKa of 4.0 to 10. [51-20] The method of any one of [1] to

[51] , wherein the cyclic peptide to be purified is a cyclic peptide 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 the side chain of the cyclic portion, 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. [51-21] The method of any one of [1] to

[51] , wherein the cyclic peptide to be purified is a compound that does not have an acidic functional group. [51-22] The method of any one of [1] to

[51] , wherein the cyclic peptide to be purified is a compound that does not have a carboxy group. [51-23] The cyclic peptide to be purified 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 ２６，３０]pentatetracont-42-ene-23,1′-cyclobutane]-17-carboxamide (compound 9), (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-carbonyl)docosahydro-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]-4,7,10,13,17,20,23,28,31,34,37(14H,22H)-undecaone (Compound 1), (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)-4,7,10,13,17,20,23,28,31,34,37-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), (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), (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-4,15,16,19,25,31,33,34,37,39,40-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), (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-4,10,13,15,16,19,25,27,28,31,38-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), (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-tridecaone azacyclohentetracontane-2,5,8,11,14,17,20,23,26,29,32,35,39-tridecaone (compound 6), (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), and (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,

[51] . The method according to any one of [1] to

[51] , wherein the compound is one selected from the group consisting of: [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). [51-24] The cyclic peptide to be purified 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 ２６，３０

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

[51] , wherein the impurity peptide is a cyclic peptide or a linear peptide produced during the synthesis of the cyclic peptide to be purified.

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

[52] , wherein the impurity peptide is a cyclic peptide different from the cyclic peptide to be purified.

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

[53] , wherein the impurity peptide is a cyclic peptide having 1.1 to 10 times the number of amino acid residues (wherein the number of amino acid residues is an integer) of the number of amino acid residues contained in the cyclic peptide to be purified.

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

[54] , wherein the peptide as an impurity is a cyclic peptide having two, three, four, five, six, seven, or eight times the number of amino acid residues contained in the cyclic peptide to be purified.

[56] The purification method according to any one of [1] to

[55] , wherein the peptide as an impurity is a cyclic peptide having two, three, or four times the number of amino acid residues contained in the cyclic peptide to be purified. [56-1] The purification method according to any one of [1] to

[55] , wherein the peptide as an impurity is a cyclic peptide having 10 to 300 amino acid residues. [56-2] The purification method according to any one of [1] to

[55] , wherein the peptide as an impurity is a cyclic peptide having 10 to 100 amino acid residues. [56-3] The purification method according to any one of [1] to

[55] , wherein the peptide that is an impurity is a cyclic peptide having 10 to 50 amino acid residues.

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

[56] , wherein the peptide that is an impurity is a cyclic peptide having twice the number of amino acid residues as the cyclic peptide that is the target of purification.

[58] The purification method according to any one of [1] to

[57] , wherein the peptide that is an impurity is a multimer of the cyclic peptide that is the target of purification.[58-1] The purification method according to any one of [1] to

[57] , wherein the peptide as an impurity is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer of the cyclic peptide to be purified.

[59] The purification method according to any one of [1] to

[58] , wherein the peptide as an impurity is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of the cyclic peptide to be purified.

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

[59] , wherein the peptide as an impurity is a dimer, trimer, or tetramer of the cyclic peptide to be purified.

[58] The purification method according to any one of [1] to

[57] , wherein the peptide as an impurity is a cyclic oligomer of the cyclic peptide to be purified.

[58] The purification method according to any one of [1] to

[57] , wherein the peptide impurity is a linear oligomer of the cyclic peptide to be purified.

[61] The purification method according to any one of [1] to

[60] , wherein the divalent metal ion is at least one selected from the group consisting of calcium ion, magnesium ion, strontium ion, and barium ion.

[62] The purification method according to any one of [1] to

[61] , wherein the divalent metal ion is at least one selected from the group consisting of calcium ion, magnesium ion, and barium ion.

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

[62] , wherein the divalent metal ion is a calcium ion.

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

[63] , wherein the divalent metal ion is an ion generated from a metal salt.

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

[64] , wherein the metal salt is at least one selected from the group consisting of chloride salts, fluoride salts, bromide salts, iodide salts, perchlorate salts, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, and sulfate salts.

[66] The purification method according to any one of

[64] and

[65] , wherein the metal salt is at least one selected from the group consisting of chloride salts, fluoride salts, bromide salts, carbonate salts, acetate salts, and sulfate salts.

[67] The purification method according to any one of

[64] to

[66] , wherein the metal salt is at least one selected from the group consisting of a chloride salt, a fluoride salt, and a bromide salt.

[68] The purification method according to any one of

[64] to

[67] , wherein the metal salt is a chloride salt.

[69] The purification method according to

[68] , wherein the chloride salt is calcium chloride or magnesium chloride.

[70] The purification method according to

[68] or

[69] , wherein the chloride salt is calcium chloride.

[71] The purification method according to any one of

[64] to

[67] , wherein the metal salt is a fluoride salt.

[72] The purification method according to

[69] , wherein the fluoride salt is calcium fluoride.

[73] The purification method according to any one of

[64] to

[67] , wherein the metal salt is a bromide salt.

[74] The purification method according to

[73] , wherein the bromide salt is at least one selected from the group consisting of calcium bromide, magnesium bromide, and zinc bromide.

[75] The purification method according to any one of

[64] to

[66] , wherein the metal salt is an iodide salt.

[76] The purification method according to

[75] , wherein the iodide salt is at least one selected from the group consisting of magnesium iodide, calcium iodide, strontium iodide, and zinc iodide.

[77] The purification method according to

[64] or

[65] , wherein the metal salt is a perchlorate.

[78] The purification method according to

[77] , wherein the perchlorate is at least one selected from the group consisting of barium perchlorate, magnesium perchlorate, calcium perchlorate, and zinc perchlorate.

[79] The purification method according to

[64] or

[65] , wherein the metal salt is an oxide salt.

[80] The purification method according to

[79] , wherein the oxide salt is magnesium oxide.

[81] The purification method according to

[64] or

[65] , wherein the metal salt is a trifluoromethanesulfonate.

[82] The purification method according to

[81] , wherein the trifluoromethanesulfonate is at least one selected from the group consisting of magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[83] The purification method according to

[64] or

[65] , wherein the metal salt is a toluenesulfonate.

[84] The purification method according to

[83] , wherein the toluenesulfonate is zinc(II) toluenesulfonate.

[85] The purification method according to

[64] or

[65] , wherein the metal salt is isopropylsulfonate.

[86] The purification method according to

[85] , wherein the isopropylsulfonate is zinc(II) isopropylsulfonate.

[87] The purification method according to

[64] or

[65] , wherein the metal salt is methanesulfonate.

[88] The purification method according to

[87] , wherein the methanesulfonate is zinc difluoromethanesulfonate.

[89] The purification method according to

[64] or

[65] , wherein the metal salt is a carbonate.

[90] The purification method according to

[89] , wherein the carbonate is at least one selected from the group consisting of calcium carbonate and zinc carbonate.

[91] The purification method according to either

[64] or

[65] , wherein the metal salt is an acetate.

[92] The purification method according to

[91] , wherein the acetate is magnesium acetate.

[93] The purification method according to

[64] or

[65] , wherein the metal salt is a sulfate.

[94] The purification method according to

[93] , wherein the sulfate is magnesium sulfate.

[95] The purification method according to any one of [1] to

[64] , wherein the metal salt is at least one selected from the group consisting of calcium chloride, calcium fluoride, calcium bromide, barium iodide, barium perchlorate, magnesium oxide, magnesium bromide, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, zinc trifluoromethanesulfonate, zinc perchlorate, zinc difluoromethanesulfonate, zinc(II) isopropylsulfonate, zinc(II) toluenesulfonate, zinc chloride, zinc carbonate, zinc iodide, manganese trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[96] The purification method according to any one of [1] to

[64] and

[95] , wherein the metal salt is at least one selected from the group consisting of calcium chloride, calcium fluoride, calcium bromide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, and magnesium chloride.

[97] The purification method according to any one of [1] to

[64] ,

[95] , and

[96] , wherein the metal salt is at least one selected from the group consisting of calcium chloride and calcium bromide.

[98] The purification method according to any one of [1] to

[64] ,

[95] ,

[96] , and

[97] , wherein the metal salt is calcium chloride.

[99] The purification method according to any one of [3] to

[98] , wherein the first solvent is a solvent capable of forming a complex between the cyclic peptide as a purification target or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion.

[100] The purification method according to any one of [3] to

[99] , wherein the first solvent comprises at least one solvent selected from the group consisting of nitrile solvents, ether solvents, ketone solvents, ester solvents, benzene solvents, halogenated solvents, and alcohol solvents.

[101] The purification method according to any one of [3] to

[100] , wherein the first solvent is an alcohol solvent.

[102] The purification method according to

[101] , wherein the alcohol solvent is at least one solvent selected from the group consisting of 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, ethanol, and methanol.

[103] The purification method according to

[101] or

[102] , wherein the alcohol solvent is at least one selected from the group consisting of 2-propanol, tert-butanol, and 2,2,2-trifluoroethanol.

[104] The purification method according to any one of [3] to

[100] , wherein the first solvent is a nitrile solvent.

[105] The purification method according to

[104] , wherein the nitrile solvent is acetonitrile.

[106] The purification method according to any one of [3] to

[100] , wherein the first solvent is a benzene-based solvent.

[107] The purification method according to

[106] , wherein the benzene-based solvent is toluene or xylene.

[108] The purification method according to any one of [3] to

[100] , wherein the first solvent is an ether-based solvent.

[109] The purification method according to

[108] , wherein the ether-based solvent is at least one selected from the group consisting of tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, MTBE (tert-butyl methyl ether), DME (dimethoxyethane), and CPME (cyclopentyl pentyl ether).

[110] The purification method according to any one of [3] to

[100] , wherein the first solvent is a ketone-based solvent.

[111] The purification method according to

[110] , wherein the ketone-based solvent is acetone or methyl ethyl ketone.

[112] The purification method according to any one of [3] to

[100] , wherein the first solvent is a halogenated solvent.

[113] The purification method according to

[112] , wherein the halogenated solvent is dichloromethane.

[114] The purification method according to any one of [3] to

[100] , wherein the first solvent is an ester-based solvent.

[115] The purification method according to

[114] , wherein the ester-based solvent is ethyl acetate or isopropyl acetate.

[116] The purification method according to any one of [3] to

[100] , wherein the first solvent is at least one selected from the group consisting of acetonitrile, DME (dimethoxyethane), tetrahydrofuran, acetone, MEK (methyl ethyl ketone), 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, toluene, 1,4-dioxane, dichloromethane, ethanol, methanol, ethyl acetate, and isopropyl acetate.

[117] The purification method according to any one of [3] to

[100] and

[116] , wherein the first solvent is at least one selected from the group consisting of acetonitrile, DME (dimethoxyethane), tetrahydrofuran, acetone, and MEK (methyl ethyl ketone).

[118] The purification method according to any one of

[22] to

[117] , wherein the second solvent is at least one selected from the group consisting of acetonitrile, MTBE (tert-butyl methyl ether), 2-methyltetrahydrofuran, tetrahydrofuran, dimethyl carbonate, anisole, isopropyl acetate, ethyl acetate, diethyl ether, dichloromethane, chloroform, DME (dimethyl ether), CPME (cyclopentyl methyl ether), 4-methyltetrahydropyran, heptane, and toluene.

[119] The purification method according to any one of

[22] to

[118] , wherein the second solvent is at least one selected from the group consisting of acetonitrile and MTBE (tert-butyl methyl ether).

[120] The purification method according to any one of [1] to

[119] , wherein the purification method is a method in which the purity (area %) of the cyclic peptide to be purified is higher than the purity (area %) of the cyclic peptide to be purified before the purification operation.

[121] The method of

[120] , wherein the purity (area %) of the cyclic peptide to be purified is determined by measuring the UV spectrum of a mixture containing the cyclic peptide to be purified and peptide impurities using HPLC and a PDA (photodiode array detector), and determining the ratio of the peak area of ​​the cyclic peptide to the sum of the peak areas of the entire mixture containing the cyclic peptide to be purified and peptide impurities at 220 nm.

[122] The purification method of any of [1] to

[121] , wherein the purification method is a method in which the ratio (%) of the total impurities is lower than the ratio (%) of the total peptide impurities before the purification procedure.

[123] The method of

[122] , wherein the ratio (%) of the total impurities is calculated using the following formula: Ratio (%) of total impurities = A. ｉｍｐ / A ＴＭ x 100 A ＴＭ : Target peak area (220 nm) A ｉｍｐ: sum of impurity peak areas (220 nm)

[124] A method for producing a composition containing a cyclic peptide, comprising the purification method according to any one of [1] to

[123] .

[125] The production method according to

[124] , further comprising the step of obtaining the cyclic peptide to be purified by liquid phase synthesis.

[126] The production method according to

[125] , further comprising the step of obtaining the cyclic peptide to be purified by solid phase synthesis.

[127] The production method according to

[125] , further comprising the step of obtaining the cyclic peptide to be purified by a culture method.

[128] A method for purifying a cyclic peptide, comprising separating the cyclic peptide to be purified as a complex with a divalent metal ion or a metal salt containing a divalent metal ion from a mixture containing the cyclic peptide to be purified and peptides as impurities, thereby improving the purity of the cyclic peptide compared to when a complex with a divalent metal ion or a metal salt containing a divalent metal ion is not formed.

[129] A method for purifying a cyclic peptide, comprising separating the cyclic peptide to be purified from a mixture containing the cyclic peptide to be purified and peptide impurities as a complex with a divalent metal ion or a metal salt containing a divalent metal ion, thereby reducing the content of peptide impurities compared to when a complex with a divalent metal ion or a metal salt containing a divalent metal ion is not formed.

[130] (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]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide, or a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of Compound 9, or a pharmaceutically acceptable salt thereof. [130-1] (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 ]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide, or a dimer or trimer of Compound 9, or a pharmaceutically acceptable salt thereof. [130-2] (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]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide trimer of Compound 9, or a pharmaceutically acceptable salt thereof.

[131] (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

[132] The composition according to

[131] , wherein the multimer is a dimer or trimer of compound 9.

[133] The composition according to

[132] , wherein the multimer is a dimer or trimer of compound 9.

[134] The composition according to

[133] , wherein the multimer is a dimer or trimer of compound 9.

[135] The composition according to

[135] , wherein the multimer is a dimer or trimer of compound 9.

[136] The composition according to

[136] , wherein the multimer is a dimer or trimer of compound 9.

[137] The composition according to

[137] , wherein the multimer is a dimer or trimer of compound 9.

[138] The composition according to

[138] , wherein the multimer is a dimer or trimer of compound 9.

[139] The composition according to

[140] , wherein the multimer is a dimer or trimer of compound 9.

[141] The composition according to

[142] , wherein the multimer is a dimer or trimer of compound 9.

[139] The composition according to

[143] , wherein the multimer is a dimer or trimer of compound 9.

[139] The composition according to

[144] , wherein the multimer is a dimer or trimer of compound 9.

[145] The composition according to

[145] , wherein the dimer or trimer of compound 9 is a dimer or trimer of compound 9.

[139] The composition according to

[146] , wherein the dimer or trimer of compound 9 is a dimer or trimer of compound 9.

[146] The composition according to

[146] , wherein the dimer or trimer of compound 9 is a dimer or trimer of compound 9.

[147] The composition according to

[147] , wherein the dimer or trimer of compound 9 is a dimer or trimer of compound 9.

[148] The composition according to

[148] , wherein the di

[133] (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

[134] A composition comprising compound 9, which is

[134] pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide, or a pharmaceutically acceptable salt thereof, together with a multimer of compound 9, wherein the content of the multimer of compound 9 is 3.0% or less relative to compound 9 or the pharmaceutically acceptable salt thereof.

[135] The composition according to

[134] , wherein the multimer is a dimer or trimer of compound 9.

[0008] In the above numbering scheme, unless otherwise specified, the numbers referred to in the dependent claims include numbers with different subnumbers. For example,

[20] referred to in the dependent claims includes

[20] as well as [20-1]. The same applies to other numbering schemes.

[0009] According to the present invention, a method for easily purifying and producing a cyclic peptide can be provided, which may be applicable to cyclic peptides that are difficult to crystallize. Although some cyclic peptides are difficult to crystallize, it has been found that even such cyclic peptides can be easily purified by forming a complex with a divalent metal ion or a metal salt containing a divalent metal ion.

[0010] Figure 1 is a schematic diagram showing a basic synthesis method for a cyclic peptide. Figure 2 is a diagram showing an LC chart of Compound 1 measured in Example 1-1. Figure 3 is a diagram showing the CaCl 2 concentration measured in Run 1 of Example 1-3. ２ Fig. 4 is an LC chart of Compound 2 measured in Example 2-1. Fig. 5 is an LC chart of CaCl measured in Example 2-2. ２ Fig. 6 is an LC chart of Compound 3 measured in Example 3-1. Fig. 7 is an LC chart of CaCl measured in Example 3-2. ２ 8 shows LC charts before and after treatment. ２ 9 shows LC charts before and after treatment. Fig. 9 shows LC charts of Compound 9 measured in Example 9-2.

[0011] The method of the present invention will now be described in detail.

[0012] In the present invention, "mixing" may refer to the "operation" of mixing one substance with another, and does not necessarily mean only bringing one substance into a mixed state. In this specification, "mixing (i) and (ii)" includes any of the following: adding (i) to (ii), adding (ii) to (i), and adding (i) and (ii) simultaneously. In this specification, the term "to" indicating a range includes both ends of the range; for example, "A to B" means a range equal to or greater than A and equal to or less than B. In this specification, the term "about," when used in combination with a numerical value, means a range of +10% and -10% of the numerical value. In this specification, the term "and / or" means 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.

[0013] As used herein, "purification of a cyclic peptide" means, for example, improving the proportion (purity) of the cyclic peptide to be purified in the entire mixture containing the cyclic peptide to be purified and peptides as impurities.

[0014] One embodiment of the present invention relates to a method for purifying a cyclic peptide, which may include a step of separating the cyclic peptide to be purified or the peptide as an impurity from a mixture containing the cyclic peptide to be purified and the peptide as an impurity as a complex with a divalent metal ion or a metal salt containing a divalent metal ion.

[0015] The separation step may include, as a pre-step, (1) a step of mixing the mixture containing the cyclic peptide to be purified and the peptide as an impurity with the divalent metal ion or a metal salt containing the divalent metal ion. The step (1) may be carried out in a first solvent.

[0016] Another embodiment of the purification method of the present invention relates to a method for purifying a cyclic peptide, and the purification method may include a step (1)' of mixing, in a first solvent, a mixture containing a cyclic peptide as the target of purification and a peptide as an impurity, with a divalent metal ion or a metal salt containing a divalent metal ion.

[0017] In step (1)', a complex may be formed in a first solvent between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal containing a divalent metal ion.

[0018] In the above step (1) or the above step (1)', the amount of metal salt containing the above divalent metal ion used may be 10 wt % or more, 20 wt % or more, or 30 wt % or more relative to the total amount of the mixture containing the cyclic peptide to be purified and the peptides as impurities, since this can further improve the purity of the cyclic peptide after purification.

[0019] In the above step (1) or the above step (1)', the amount of metal salt containing the above divalent metal ion used may be 300 wt % or less, 100 wt % or less, or 75 wt % or less relative to the total amount of the mixture containing the cyclic peptide to be purified and the peptides as impurities, since this can further improve the recovery rate of the cyclic peptide after purification.

[0020] In the step (1) or the step (1)', the amount of the metal salt containing the divalent metal ion used may be 10 wt % to 300 wt %, 20 wt % to 100 wt %, or 30 wt % to 50 wt % relative to the total amount of the mixture containing the cyclic peptide to be purified and the peptides as impurities, since this will result in both good purity and recovery rate of the cyclic peptide after purification.

[0021] In the step (1) or the step (1)', the liquid temperature of the first solvent may be -20°C or higher, 0°C or higher, or 10°C or higher, and may be 100°C or lower, 60°C or lower, or 60°C or lower. In the step (1) or the step (1)', the liquid temperature of the first solvent is preferably -20 to 100°C, more preferably 0 to 60°C, and most preferably 10 to 60°C.

[0022] In the step (1) or the step (1)', the time for mixing the mixture containing the cyclic peptide to be purified and the peptide impurities with the divalent metal ion or the metal salt containing the divalent metal ion in the first solvent may be 0.1 hours or more, 0.5 hours or more, or 1.0 hour or more, and may be 192 hours or less, 72 hours or less, or 30 hours or less. In the step (1) or the step (1)', the time for mixing the mixture containing the cyclic peptide to be purified and the peptide impurities with the divalent metal ion or the metal salt containing the divalent metal ion in the first solvent is preferably 0.1 to 192 hours, or 1 to 30 hours.

[0023] In the step (1) or the step (1)', the concentration of the cyclic peptide to be purified in the first solvent may be 0.1 mg / mL or more, 1.0 mg / mL or more, or 10 mg / mL or more, and may be 1000 mg / mL or less, 500 mg / mL or less, or 100 mg / mL or less. In the step (1) or the step (1)', the concentration of the cyclic peptide to be purified in the first solvent may be 0.1 mg / mL to 1000 mg / mL, 1.0 mg / mL to 500 mg / mL, or 10 mg / mL to 100 mg / mL. In the step (1) or the step (1)', the concentration of the cyclic peptide to be purified in the first solvent is preferably 0.1 mg / mL to 1000 mg / mL, more preferably 1.0 mg / mL to 500 mg / mL, and most preferably 10 mg / mL to 100 mg / mL.

[0024] In yet another embodiment of the present invention, the purification method may further comprise, after step (1)', a step (2) of separating the complex from a mixture containing the cyclic peptide to be purified and peptides as impurities.

[0025] In this specification, separation of the complex may be performed by solid-liquid separation or liquid-liquid separation, with solid-liquid separation being preferred from the viewpoint of ease of operation and stability of the cyclic peptide to be purified. Examples of solid-liquid separation methods include centrifugation and filtration. Examples of liquid-liquid separation methods include centrifugation and decantation.

[0026] In this specification, the method for purifying a cyclic peptide may include (3) a step of removing divalent metal ions or metal salts containing divalent metal ions from a mixture of the cyclic peptide, which is the target of purification, and the first solvent.

[0027] The step (3) may include a step of mixing a mixture of the cyclic peptide as the target of purification and a first solvent with a second solvent and an aqueous solution, and separating the mixture into an aqueous phase and an organic phase, thereby removing the divalent metal ion or a metal salt containing the divalent metal ion from the mixture. The aqueous solution may be saline.

[0028] The step (3) may further include a step of removing the first solvent and the second solvent from the organic phase containing the cyclic peptide, which is the target of purification, to obtain the cyclic peptide, which is the target of purification.

[0029] Specific examples of the method for removing the first solvent and the second solvent from the organic phase include distillation under atmospheric pressure, distillation under heating, and distillation under reduced pressure. The removal of the first solvent and the second solvent from the organic phase may be performed by distillation under reduced pressure.

[0030] In the present specification, the method for purifying a cyclic peptide may further include a step (4) of confirming that the divalent metal ion forms a complex with the cyclic peptide to be purified or the peptide to be an impurity. The step (4) may further include a step of confirming that the divalent metal ion or the metal salt containing a divalent metal ion forms a complex with the peptide to be an impurity. The step (4) may be performed by comparing the NMR peak of the cyclic peptide to be purified or the peptide to be an impurity with the NMR peak of a mixture of the cyclic peptide to be purified or the peptide to be an impurity and the divalent metal ion or the metal salt containing a divalent metal ion. NMR measurements can be performed using methods well known to those skilled in the art. For example, a solution of the compound to be measured dissolved in a solvent suitable for NMR measurement can be added to an NMR measurement sample tube, which can then be placed in the measurement device and measured. The solvent suitable for NMR measurement is preferably a solvent capable of dissolving the compound to be measured, and commercially available deuterated solvents can also be used. The NMR measurement conditions can be those known to those skilled in the art or those described in the manual for the measurement device. The measurement conditions are not particularly limited as long as the target peak component can be measured. For example, the measurement temperature can be in the range of 273 K to 320 K, the accumulation time can be in the range of 1 second to 7 days, and the rotation speed of the sample tube can be in the range of 0 to 20 Hz. Specifically, the step (4) is carried out as follows: (A) the cyclic peptide or the peptide impurity is dissolved in acetonitrile-d3 (deuterated acetonitrile, CD ３ (B) a solution obtained by dissolving the divalent metal ion or the metal salt containing a divalent metal ion and the cyclic peptide or the peptide that is an impurity in acetonitrile-d3 (heavy acetonitrile, CD4) in an amount of 1 to 6 molar equivalents of the divalent metal ion or the metal salt containing a divalent metal ion relative to the cyclic peptide or the peptide that is an impurity. ３ CN, CAS: 2206-26-0) to obtain a solution (the concentration of the cyclic peptide or the peptide as an impurity: 1.4 mM) １H-NMR may be measured at 278 K or 298 K, respectively. If a new peak related to the cyclic peptide to be purified or the peptide as an impurity is observed in the peak component (B) compared with the peak component (A), or if a change in the peak intensity ratio is observed by comparing the peak components (A) and (B), or if a sharpening or broadening of the peak is observed in the peak component (B) compared with the peak component (A), it may be determined that the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or the metal salt containing a divalent metal ion have formed the complex.

[0031] As used herein, the term "complex" may be a complex of the cyclic peptide to be purified and the divalent metal ion or a metal salt containing a divalent metal ion, or a complex of the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion. The term "complex" may be a complex obtained by contacting the cyclic peptide or the peptide as an impurity with the metal. As used herein, the term "complex" refers to a compound in which the cyclic peptide to be purified or the cyclic peptide as an impurity interacts with the divalent metal ion or a metal salt containing a divalent metal ion. The complex may be solid, crystalline, liquid, or amorphous. The complex is preferably an adsorption complex or complex. The complex may be an adsorption complex. An "adsorption complex" is a complex in which the cyclic peptide to be purified as an adsorbate or the peptide as an impurity is adsorbed to a metal salt containing a divalent metal ion. The state in which the cyclic peptide to be purified as an adsorbate or the peptide to be an impurity is adsorbed to a metal salt containing a divalent metal ion can be confirmed, for example, by removing a portion of the adsorption complex from the reaction vessel, adding an alcohol such as methanol, mixing, and observing the presence of the cyclic peptide to be purified or the peptide to be an impurity in the resulting mixed solution. Alternatively, the state can be confirmed by removing a portion of the adsorption complex from the reaction vessel, adding water and an organic solvent, mixing, obtaining an organic phase from the mixture by liquid-liquid separation, and observing the presence of the cyclic peptide to be purified or the peptide to be an impurity in the organic phase. The presence of the cyclic peptide to be purified or the peptide to be an impurity in the mixed solution or organic phase can be measured, for example, by HPLC.

[0032] The conjugate may be a complex. In this specification, a complex of the cyclic peptide to be purified or the peptide to be an impurity with a divalent metal ion is a compound in which the cyclic peptide to be purified or the peptide to be an impurity is coordinated to the divalent metal ion. The state in which the cyclic peptide to be purified or the peptide to be an impurity is coordinated to the divalent metal ion is determined, for example, by X-ray crystal structure analysis or changes in absorption spectrum (see "Coordination Chemistry - Basics and Latest Topics," edited by the Basic Complex Engineering Research Group, Kodansha Scientific, 1994, pp. 39-49). The complex may be solid, crystalline, liquid, or amorphous.

[0033] In this specification, the mixture containing the cyclic peptide to be purified and peptides as impurities may be a crude product obtained by producing the cyclic peptide to be purified.

[0034] In this specification, the number of amino acid residues in the cyclic peptide to be purified may be 5 or more, 7 or more, 9 or more, or 10 or more, and may be 20 or less, 18 or less, 16 or less, or 14 or less. The number of amino acid residues in the cyclic peptide to be purified may be 5 to 20, 5 to 14, 10 to 20, or 10 to 14.

[0035] In this specification, the number of amino acid residues in the cyclic portion of the cyclic peptide to be purified may be 5 or more, 7 or more, 8 or more, or 10 or more, and may be 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less. The number of amino acid residues in the cyclic portion of the cyclic peptide to be purified may be 5 to 15, 5 to 14, 10 to 15, or 8 to 14, or may be 8, 11, 13, or 14.

[0036] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. Furthermore, as used herein, "amino acid" may refer to amino acid residues. As used herein, "natural amino acids" refer to Gly, L-Ala, L-Ser, L-Thr, L-Val, L-Leu, L-Ile, L-Phe, L-Tyr, L-Trp, L-His, L-Glu, L-Asp, L-Gln, L-Asn, L-Cys, L-Met, L-Lys, L-Arg, and L-Pro. Examples of "unnatural amino acids" include, but are not limited to, β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids with side chains different from those found in nature. As used herein, amino acids may have any configuration. The side chain of the amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, or a spiro-linked cycloalkyl group. Each of these groups may be substituted, and the substituents are not limited, and may be independently selected from any substituent containing, for example, a halogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a boron atom, a silicon atom, or a phosphorus atom. Examples of such substituents include optionally substituted alkyl groups, alkoxy groups, alkoxyalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups, as well as oxo, aminocarbonyl, and halogen atoms.

[0037] As used herein, the term "side chain of an amino acid" refers to, in the case of an α-amino acid, an atomic group bound to the carbon (α-carbon) to which the amino group and carboxy group are bonded. For example, the methyl group of Ala is the side chain of an amino acid. In the case of a β-amino acid, the atomic group bound to the α-carbon and / or the β-carbon can be the side chain of the amino acid, and in the case of a γ-amino acid, the atomic group bound to the α-carbon, the β-carbon, and / or the γ-carbon can be the side chain of the amino acid.

[0038] 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 carboxy 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 carboxy 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 carboxy group in the case of a γ-amino acid.

[0039] 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, and proline.

[0040] In a non-limiting embodiment, the amino acid herein may be a compound having a carboxy group and an amino group in the same molecule. The nitrogen atom of the amino group may be combined with any atom in the side chain of the amino acid to form a 4- to 7-membered saturated heterocycle. Examples of such amino acids include proline, hydroxyproline, and azetidine-2-carboxylic acid. The nitrogen atom of the amino group of an amino acid may also be combined with the side chain of another amino acid to form a ring, and such a partial structure may be a structure represented by the following formula (* indicates the point of attachment to the adjacent atom):

[0041] In this specification, the term "substituent containing a halogen atom" includes fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) and the like.

[0042] In this specification, the "substituent containing an oxygen atom" includes hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxy (-CO ２ H), oxycarbonyl (-C(=O)-OR), carbonyloxy (-O-C(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio group (-S-C(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO ２ -R), aminosulfonyl (-SO ２ -NHR), sulfamoylamino (-NH-SO ２ -NHR), thiocarboxy (-C(=O)-SH), carboxycarbonyl (-C(=O)-CO ２ H) is exemplified.

[0043] As used herein, the term "substituent containing a nitrogen atom" includes azide (-N ３ , also called "azido group"), cyano (-CN), primary amino (-NH ２ ), secondary amino (—NH—R), tertiary amino (—NR(R′)), amidino (—C(═NH)—NH ２ ), substituted amidino (—C(═NR)—NR′R″), guanidino (—NH—C(═NH)—NH ２ ), substituted guanidino (-NR-C(=NR'")-NR'R"), and aminocarbonylamino (-NR-CO-NR'R").

[0044] As used herein, the term "sulfur atom-containing substituent" includes thiol (-SH), thio (-S-R), sulfinyl (-S=O-R), sulfonyl (-S(O) ２ -R), sulfo (-SO ３ H), pentafluorosulfanyl (-SF ５ ) is an example.

[0045] In this specification, the term "substituent containing a boron atom" refers to boryl (-BR(R')), dioxyboryl (-B(OR)(OR')), and trifluoroborate (-BF ３ － Specific examples include "boron atom-derived substituents" in which these two substituents R and R' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or "boron atom-derived substituents" in which these two substituents R and R' form a ring together with the atoms to which R and R' are bonded, i.e., a cyclic boryl group.

[0046] As used herein, the term "substituent containing a phosphorus atom" includes phosphoryl (-P(O)-R １ R ２ ), phosphonyl (—O—P(O)—R １ R ２ ), phospho(-PO ３ H ２ ) is an example.

[0047] Herein, the cyclic peptide to be purified may contain at least one unnatural amino acid. Herein, the cyclic peptide to be purified may contain an N-substituted amino acid. The cyclic peptide to be purified may contain three or more, four or more, five or more, or six or more N-substituted amino acids, and may contain 15 or fewer, 13 or fewer, or 10 or fewer N-substituted amino acids. The number of N-substituted amino acid residues in the cyclic peptide to be purified may be 45% or more, 50% or more, 55% or more, or 60% or more of the number of amino acid residues in the cyclic portion of the cyclic peptide, and may be 80% or fewer, 75% or fewer, 70% or fewer, or 65% or fewer, with 45% to 80% being preferred.

[0048] In the present specification, the N-substituted amino acid may be one selected from the group consisting of N-methyl amino acid, N-ethyl amino acid and N-propyl amino acid, and may be an N-methyl amino acid.

[0049] Herein, an amino acid whose main chain amino group is unsubstituted is referred to as an "N-unsubstituted amino acid." Herein, the cyclic peptide to be purified may contain an N-unsubstituted amino acid. The N-unsubstituted amino acid may be an unnatural amino acid. The ratio of the number of N-unsubstituted amino acids in the cyclic peptide to be purified to the total number of amino acid residues in the cyclic peptide to be purified may be 55% or less, 50% or less, 45% or less, or 40% or less, or may be 20% or more, 25% or more, 30% or more, or 35% or more, with 20% to 55% being preferred.

[0050] The compounds described herein may contain unnatural proportions of isotope atoms in one or more atoms constituting such compounds. The present invention also includes compounds in which any atom in a compound is substituted with another isotope atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons), thereby substituting isotopes with an abundance ratio different from the abundance ratio of isotopes in nature, i.e., compounds labeled with isotope atoms. Examples of isotope elements contained in the compounds of the present specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., and each of these isotopes is ２ H. ３ H. １３ C. １４ C. １５ N. １７ O. １８ O. ３２ P. ３５ S. １８ F. ３６ Cl, etc. Compounds labeled with isotope atoms are useful as therapeutic agents, preventive agents, research reagents (e.g., assay reagents), or diagnostic agents (e.g., in vivo imaging diagnostic agents). Compounds herein containing radioactive or non-radioactive isotopes in all proportions are encompassed within the scope of the present invention. Compounds labeled with isotope atoms can be produced using reagents and solvents containing the corresponding isotope atoms in the same manner as for producing unlabeled compounds.

[0051] In the present specification, the ClogP of the cyclic peptide to be purified may be 7-20, 8-20, or 11-20.

[0052] 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!

[0053] 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 will help the user understand why theCLOGP program calculates logP(ow) the way it does. Although the procedurecannot be derived from first principles, we have tried to make the rulesconsistent with solvation theory, if for no other reason than they are more easily remembered. The method simply adds together values ​​for structural partsof a solute molecule and correction factors dependent upon the particular waythe parts are put together. The CLOGP EXAMPLES section contains example CLOGP calculations for a variety of chemicals and is designed as a companion to the CLOGP Reference Manual. An asterisk (*) appears in the CLOGP Reference Manual when one or more examples are provided in CLOGP Example Calculations to illustrate aspects of the CLOGP computation. Examples also demonstrate DEPICT (chemical metaphor).Funding for the development of CLOGP was provided by the U.S.Environmental Protection Agency through Cooperative Agreement No. 809295, andwe wish to acknowledge the encouragement and support of the project officer,Dr. Gilman Veith of ERL-Duluth. 1.1 Measurement and Past Uses of Partition Coefficients The partition coefficient is the equilibrium concentration of solutein a non-polar solvent divided by the concentration of the same species in apolar solvent. In this and most other applications, the polar solvent is water.The logarithm of the partition coefficient, log P, has been successfully usedas a hydrophobic parameter in 'extrathermo-dynamic' Hammett methodology.1-octanol has much to recommend it as the choice for the non-polar phase (1)and logP(ow) has been used successfully in Quantitative Structure ActivityRelationships (QSAR) in the following special fields: drug and pesticide design(2,3); pharmacokinetics (4); anaesthesiology (5); environmental transport andsoil binding (6,7); toxicology (8); bioaccumulation (9); protein folding (10);enzyme binding (11,12); enzymic reactions in non-aqueous solvents (13); andhost-guest complexation (14a,b). In principle, the measurement of the equilibrium concentration ofsolute in the octanol and water phases, after shaking in a separatory funnel,is very simple, and since good measured values are always to be preferred overcalculated ones, it would seem that there should be little need for a procedureto calculate them. As it turns out, reliable shake-flask measurements aretime-consuming and often difficult to make.The criteria for high reliabilityare: measurements over a 10-fold concentration range (with upper concentrationno more than 75% of solubility or CMC, or no more than 5% of the aqueous phase,whichever is lower) and standard deviation of 0.03 or less in log terms. Thisoften requires working at sub-micromolar concentrations, and so, with either UVspectrophotometry or gas chromatography, it means that the standard curves mustbe established with utmost care. Radiotracer methods seem well-suited foranalyses at these low concentrations, but impurities as well as adsorption atphase boundaries (including container walls) can introduce significant errors. HPLC procedures provide a way around this bottleneck(15,16) and cansave time if there is a limited variety of structural types, and the log Pvalues fall in the range of 0.5 to 4.0.Most HPLC procedures which are used todevelop log P(ow) values do no use octanol and thus have to be referred to thatsystem by standard curves which can be different depending on whether the solutesdo or do not contain certain basic fragments, such as pyridine nitrogen. If thesolutes do not absorb well in the UV, difficulties in detecting the elutiontime eliminates any advantage HPLC may have over the shake-flask method. Procedures which employ filter probes (17) or solubility columns(18)speed up partition coefficient measurements by eliminating centrifugation asthe means of phase separation. However, each has its own set of disadvantagesand limit its acceptance as a method for establishing the standard values for acalculation procedure. More efficient methods of measurement of octanol / water partitioncoefficients are certain to be developed in the future, but no conceivable'breakthrough' is likely to eliminate the need for logP calculation.To put theproblem in proper prospective, one need only imagine some dedicated syntheticchemist making all possible tri-substituted benzoic acids with the methodscommonly available today. When finished, there would be five million analogsfor which partition coefficients could be determined. And of course only bycalculation is one going to have an estimate of hydrophobicity beforesynthesis. The Pomona MedChem Project saw these and other arguments as reasonenough to develop a method to calculate log P(ow) from structure by anadditive-constitutive procedure. As it turns out, the 'constitutive' portion ofthe procedure was, by the very nature of the two competing solvationequilibria, very complex, and the manual method required considerable effortbefore it could be applied with confidence.It is the aim of thesecond-generation program, CLOGP, to take most of the routine calculationburden from the user but still encourage him to study the interplay ofhydrophobic and polar solvation forces which can be so crucial to the design ofbioactive chemicals. 1.2 How to Understand CLOGP Calculations The first published method for calculating log P(ow) from structure(19) was based on a 'substitution' procedure and was developed with substituentpi constants for aromatic rings in mind. Of course this method was limited toderiving a new log P from a 'parent' structure whose log P was already known.Rekker(20) was the first to publish a procedure which was more general in thatit assigned 'fragmental constants' to a variety of structural pieces, and thecalculated log P was the sum of the values appropriate for the molecule inquestion. The original pi system can be expressed as:. while the expression for Rekker's fragment system is: The method developed by Pomona MedChem (21) follows Rekker's generalformulation, but there are some important differences in the approach used toderive the actual working constants. Rekker used a 'reductionist' approach -deriving the constants for carbon and hydrogen as well as those for polarfragments from a statistical treatment of a large body of log P data whichcontained numerous interaction factors. Both the fragment values (f) andinteraction factors (F) had to be identified and evaluated concurrently. Also,Rekker neglected to clearly define just what constitutes a fragment. Instead heprovides a table in which the known constants can be found (see footnote).Rekker also treats all correction factors as some multiple of a 'Magic Number'(+0.28), but the selection of multiples was not made clear in his publishedwork. Although his method gained some acceptance for manual calculation, weconsidered it too seriously flawed to serve as the basis for a computer method.In order to construct a dependable, verifiable algorithm suitablefor log P calculations used in developing QSAR at Pomona College, we firstelected to clearly define what constitutes a fragment. Next we chose a'constructionist' approach to evaluate them; that is, we accepted as axiomaticthat the hydrophobic portions of solutes were those most 'hydrocarbon-like',and defined these carbons and hydrogen fragment values as being truly constant.We gave very heavy emphasis to the carefully-measured values for three solutes;molecular hydrogen, methane and ethane, because from these we could derivefragment constants for carbon and hydrogen, which would be free of obscuringinteractions. For all hydrocarbon structures more complex than these, whosemeasured values were NOT the sum of fragment values, we attempted to define thedifference in terms of universally-applicable correction factors.It appearsthat this approach has led not only to a workable algorithm, but hashighlighted the importance of certain types of polar solvation forces whichhave received insufficient attention in the past. The first attempt to reduce the 'Pomona Method' of log P calculationto computer algorithm was made in collaboration with Dr. Jack Chou and Dr.Peter Jurs of Pennsylvania State University (22). It was called CLOGP. A greatdeal was learned in the process of developing this first version, and itcertainly established the real need for a 'stand-alone' program to make thesecalculations. Nevertheless, CLOGP was difficult to install and modify, and manywell-known correction factors could not be implemented due to programmingdifficulties. In light of this experience, we deemed it essential toincorporate, in the second generation program, design features which wouldencourage its continuing evolution.To achieve that objective, the program hadto be conceived as a 'modeling system' which could operate from one or moreeasily-revised 'value files'. As an example of the ease of updating, thelargest fragment encountered to date is:. It took less than two minutes to enter it into the database andbegin to use it in calculations. A number of significant improvements have been made to CLOGP overthe past few years. The most significant improvement is the ability to estimatea polar fragment value which has not appeared in a solute having a measure logP (oct). This type of estimation is designated as 'calculated'. If the fragmentin question has appeared in a measured solute, but in a different bondingenvironment (e.g., aromatic attached when aliphatic attached is needed), the new4.0 version allows for all extrapolations and designates the value as'derived'. The methodology for the 'No Missing Fragments' algorithm isexplained in more detail in ref. 23. Earlier versions of CLOGP were able to assign corrections to polarfragments interacting over two Isolating Carbons (see Section 2. following). Inthe latest version, this distance has been extended to three I.C.s.Asignificant improvement in steroid calculations recognizes the uniquecontribution of polar groups at the 11 position as well as some long-rangeintramolecular hydrogen bonds. These and a few other minor improvements will benoted in the changed values in the CLOGP Example Calculations, 6.3. 2. Fundamental Fragments In view of the decision to make alkane carbons and hydrogens themost fundamental fragments in the system, it is necessary to define these verycarefully before defining the polar, more hydrophilic fragments. 2.1. Isolating Carbons An 'Isolating Carbon' (I.C.) atom is carbon which is NOT doubly- ortriply- bonded to a hetero atom. An I.C. may be bonded to a hetero atom by asingle or an aromatic bond. This definition can be made clearer from thefollowing two examples:. In an earlier version of the manual calculation procedure (Ref. 21p. 34) the Kekule structures for pyrimidine was considered; the earlier rule isnow superceded. In coumarin, both rings are designated as aromatic, and theonly carbon which is not isolating is the one in the carbonyl group, because itis doubly bonded to a hetero outside the ring. Although the hydrophobic value of an I.C. is constant, several typesmust still be identified; the degree to which they delocalize electrons in anypolar fragments attached to them has a great influence on overall log P. Thetypes of I.C.s presently identified are listed below with appropriate symbols: To be completely characterized, a polar fragment must have each ofits 'valence bonds' designated with one of the above symbols (see section"Fragment Valence Types"). The numerical value of the fragment willincrease roughly in the order 'A' to 'a', but must be experimentally determinedfor high reliability. All hydrogens bonded to I.C.s are fragments. These two kinds offundamental fragments are the most important members of the non-polar class. Acomparison of their relative values (C = 0.2; H = 0.225) is a reminder that themeasure of effective cavity size may not be as simple as using van der Waalsradii or CPK models. 2.2. Polar Fragments A fragment is any atom or group of atoms bounded by Isolating Carbonatoms, and all except hydrogen are considered polar. A fragment may have manyinternal bonds but those connecting it to I.C.s are called 'valence bonds'.Valence bonds are most often single, but can be aromatic, as in the case of theN fragments in pyrimidine shown above.Each hydrogen in methane is a fragment,but the hydrogens in formaldehyde are not because the carbon to which they arebonded is not isolating. This is very important to remember, for one frequentlysees published calculations in which one fragment value is obtained fromanother by the replacement of a fragment hydrogen with another fragment ofknown value. At the present time a good rule to follow is: "Never break upa Fragment; estimations can be made from values measured for different bondenvironments (see below) but a Fragment cannot be constructed from parts."Examples of fragments which cannot be "broken down" further are:. As will become evident in the following sections, polar fragmentscan interact in various ways. To quantitate this interaction it is necessary todefine several types of polar fragments: (A) X = any halogen, but for one type of interaction fluorine mustbe assigned to a special subclass,'F'. (B) Y = all non-X fragments; these arefurther subdivided according to: sensitivity to halogen interaction as '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 P without any correction factors. It isworthwhile to examine some of the accepted hypotheses as to what solvationforces or other phenomena determine these intrinsic values. It takes more energy to form a cavity in water than in octanol. Onewould predict, therefore, that increasing the size of a solute would increaseits log P. Other factors being equal, this appears to be the case.However,other features of the solute can partly or completely override the effect ofits size. Water is much more capable than is octanol of accommodatinglocalized dipoles, and it contains, on a molar basis, more hydrogen bondaccepting and donating groups. So it is these three factors -size, localizeddipole strength, and H-bonding ability - which largely determine the sign andmagnitude of any fragment value. 2.3.1 Halogens Halogens form an intense localized dipole when bonded to analiphatic carbon atom.* This intensity is somewhat lessened if the I.C. isbenzyl and greatly lessened if it is vinyl, styryl or aromatic.* Fluorine has anegative fragment value when attached to an aliphatic carbon, because thedipole effect outweighs the effect of size. Size is of greater importance withchlorine and bromine, but even bromine is less hydrophobic than a hydrogen inan aliphatic setting.As will become evident in the following section, much ofthis hydrophilic polar effect can be lost through 'shielding' by otherhalogens, or by electronic interaction with 'Y' type polar groups. 2.3.2 H-Polar Fragments H-Polar Fragments ('Y') almost universally form some sort ofhydrogen bonds with the donor (H) or acceptor (O) of the aqueous phase. This isthought to interrupt the peculiar 'ice-like' water shell which forms around thenon-polar, hydrocarbon-like portions of each solute molecule, and thereby effectivelyreduces cavity size. As noted above, this should reduce log P. 2.3.3 Ions Octanol can accept some larger solutes containing a full formalcharge in sufficient concentration for measurement. However, one must becareful that the species measured is the same, because water easily supportscomplete ionization while ion-pairing is the usual condition in octanol exceptat the very lowest concentrations. Consistent values can be obtained if'standard conditions' are adhered to: 0.1 M small counter-ion (Na+ or Cl-) andextrapolation to infinite dilution. Measured in this way a carboxylate ion isabout 4.1 log units lower than the undissociated acid. No single value can begiven to the positive charge on a protonatedamine or quaternary ammonium,because the charge is delocalized along the hydrocarbon chain and thus theeffect is dependent on chain-length. It should be emphasized at this pointthat, except for zwitterions, CLOGP calculates values for the neutral soluteonly.* 2.3.4 Unsaturations Double bonds in isolation have a slightly negative effect on log P.*This effect may arise from the polarity of the pi electrons or else it may bedue to the shorter bond length reducing cavity size. At any rate, it disappearsif the double bonds are conjugated.* Triple bonds are decidedly hydrophilic andrequire a large negative correction factor. 3. Correction Factors 3.1 Structural Factors 3.1.1 Bonds To properly perform its calculations, CLOGP needs to know the numberand types of certain bonds in the solute structure. There is some reason tobelieve that, for the bonds in question, factors other than bond length affectthe size of the solvent cavity needed to contain the hydrophobic portions ofthe solute molecule. The effect of all bonds within any fragment is taken care of by thefragment value, and so it is NOT necessary to keep track of them, NOR of anybonds to hydrogen. And, as explained below, it is convenient to allow for thebond effect in aromatic rings by including it in a special aromatic I.C. type,'aromatic carbon'; therefore, aromatic bonds also are NOT given specialattention. Bonds which DO need to be identified are the following: 3.1.1.1 Chain bonds Chain bonds are non-ring bonds between I.C.s plus any valence bondsto fragments*. 3.1.1.2 Ring Bonds Ring bonds are non-aromatic ring bonds between I.C.s plus anyvalence bonds to fragments*. 3.1.1.3 Branch Bonds Branch bonds are chain bonds emanating from 'Branched Fragments' (afragment type presently limited to phosphate esters) and counted to the lastI.C. preceding any polar fragment.* A separate count of each of these bonds types must be made, and anegative correction applied. For chain bonds only, this correction applies tobonds AFTER the first in each chain. For example, there is no net bondcorrection for ethane but there is one for propane. This suggests that thecorrection accounts for flexing of the chain which is not possible in methaneor ethane. 1,2-diethylbenzene gets only a net of two bond corrections, becauseeach chain is counted separately. Also compatible with a 'flexing' hypothesisis the fact that the correction is greater for chains than for aliphatic rings. As noted above, an isolated double bond is assigned a negativecorrection factor (-0.09). This factor actually becomes slightly positive ifthe double bonds are conjugated in a ring such as benzene.Since it is muchmore convenient to assign all bonds in large fused ring systems as aromatictype, rather than using the Kekule system of alternating doubles and singles, itis worthwhile to assign a special fragment value to an aromatic carbon andinclude all the necessary bonding effects therein. The value of aliphaticcarbon is +0.20;the value for aromatic carbon which includes all bond effectsassociated with the aromatic ring system is +0.13.* 3.1.2 Branching at Isolating Carbons 3.1.2.1 Chain Branch It is well-known that iso-alkanes are more water-soluble than theirn-isomers.* This branching evidently does not produce a correspondingsolubility increase in the octanol phase in the partitioning process, becausethe correction required in CLOGP is negative in sign. In CLOGP, the concept of branching was expanded, and now replacesthe earlier use of the 'ring' cluster' correction(21).Fusion carbons innon-aromatic rings are considered as branched and given the same correctionfactor as chains; i.e., -0.13.* They are designated cluster branches. 3.1.2.2 Group Branch If an H-Polar group branches from an I.C. the increase in watersolubility, compared to the n-isomer, is even greater than with chainbranching. Again this carries over to partitioning equilibrium; H-Polar groupbranching requires a larger correction than does chain branching. For thisreason isopropyl alcohol is given one group branch correction and no chain branchis considered.* Tertiary butyl alcohol gets one of each type.* If a fragment has more than two external(valence) bonds, it could beconsidered a branching point. However, in all cases except the 'BranchedFragments' noted above (t-amines and phosphate esters), the entire negativebranching effect is included in the fragment value itself. Only in the case ofthe 'Branched Fragments' is the effect chain-length dependent. 3.2 Interaction Factors 3.2.1 Aliphatic Proximity (MeasuredTopologically) 3.2.1.1 Halogen vs. Halogen (X vs. X) The positive correction to log P for this interaction is thought toresult from dipole shielding and is limited to halogens on the same (geminal)or adjacent (vicinal) I.C.s. The geminal interaction is designated 'X-C-X', andthe corrections can be thought to arise as follows: adding a second halogen toan I.C. which already has one creates the first X-C-X pair, and the correctionrequired is +0.60.* Adding the third halogen to the same I.C. creates two moresuch paintings, each of which requires a correction of +0.5.* If the fourthhalogen is added, the dipole is almost completely shielded, and the threeadditional pairings require corrections of +0.40 each.* For carbontetrachloride the total geminal halogen correction would be: EQ on pg. 12 For the vicinal halogen correction, X-C-C-X, the bond betweencarbons must not be double.* The correction is evaluated by subtracting onefrom the number of halogens meeting the structural requirement and multiplyingby the factor 0.28. (Again Rekker's Magic Constant pops up!) 3.2.1.2 H-Polar vs. H-Polar (Y vs. Y) As noted in the section on 'Intrinsic Values', the negative sign onthe 'Y' fragments is thought to result from their 'structure-breaking' (andthus cavity-reducing) ability in the water phase. 'Y' fragments appear toeliminate the cavity requirement for two or more I.C.s to which they areattached. Obviously if two 'Y' fragments are located on the same or adjacentI.C.s to which they are attached. Obviously if two 'Y' fragments are located onthe same or adjacent I.C.s some of this cavity reduction is going to be countedtwice. Thus a positive correction factor is called for when the topologicalseparation is less than three I.C.s.The CLOGP algorithm is an improvement overthe original Rekker procedure(20) in that, in place of the same correction forevery Y-C-Y or Y-C-C-Y, it makes the correction proportional to how muchhydrophilic character (negative fragment value) is involved. Thisproportionality appears to apply even if one of the fragments is charged andhas a highly negative value, but as previously noted, the CLOGP algorithmcurrently does not treat ions. If one of the 'Y' fragments in a Y-C-Y interaction contains an -OHmoiety (e.g. -NHOH, -COOH, or -OH itself), a greater proportion of thehydrophilic character of the pair is lost. The coefficient by which thefragment sum is multiplied increases from 0.32 to 0.42.* If both the 'Y' fragments and the carbons of Y-C-C-Y are in a ring,the hydrophilicity loss is not as great as if they are all in a chain(coefficient 0.26 vs. 0.20).* If one 'Y' is a substituent on the ring while theother is in the ring, the correction coefficients are averaged (0.23).* If oneof 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 andaveraged.* If both I.C.s have geminal 'Y' fragments, then the vicinalcorrection is not applicable.* (See penicillin in EXAMPLES). 3.2.1.3 Halogen vs. H-Polar (X vs. Y) The interaction being considered at this point is limited to thatwhich takes place across single bonds. It is therefore, probably due to aninductive or field effect. (The electronic interaction between fragments on orin aromatic rings is discussed in the following section.) In evaluating theX-C-Y correction factor, all halogens can be treated alike. However, there areat least three levels of sensitivity shown by 'Y' type fragments. In CLOGP themost sensitive class, 'Y-3', is restricted to the structural type: -SO2-R.*'Y-2' consists of the types: -CONH-R, -O-R, -S-R, and -NH-R; and 'Y-1' of allother, H-polar fragments.* The correction factor for the first alpha-halogen(i.e. X-C-Y) is the same for all three Y-types (+0.9). For 'Y-3' fragments, thiscorrection factor is doubled when there are two alpha halogens (X{2}-C-Y3) andtripled when there are three (X{3}-C-Y3). For 'Y-2' fragments, the second andthird alpha-halogens need much less correction, and for 'Y-1', virtually none.In the case of multiple halogenation, the X-C-X and the X-C-Y corrections areadditive. The CLOGP algorithm makes no separation of 'Y' types to make theX-C-C-Y correction, but needs to distinguish fluorine from the other 'X'halogens.* 3.2.2 Electronic (through Pi-bonds) 3.2.2.1 Fragment Valence Type As previously noted, all fragments (X or Y type) are assigned themost negative values when bonded to aliphatic I.C.s (designated as 'A'). Thiscan be considered as the 'base' or 'intrinsic' level. If the fragment valuewhen attached to a vinyl I.C.(V) has not been measured it can be estimated asthe average of the base and aromatic-bonded (a) values.* Likewise the value forthe styryl-attached fragment can be estimated as two-thirds the way from thebase to the aromatic value.* CLOGP will only make these estimations whenmeasured values have not been entered in the database. 3.2.2.2 Extension of Aromaticity The extension of the aromatic ring system through fusion (as innaphthalene or direct substitution (as in biphenyl)) appears to increase log P,especially if the heteroaromatic atom is next to the juncture.* If theringjoining carbons are attached only to other aromatic carbons, electrondelocalization is minimal and so is the correction: +0.10 for each I.C. If theI.C.s are also attached to a polar (fused-in) fragment, such as in quinoline or2-phenylpyrimidine, the correction is greater, +0.31.* 3.2.2.3 Sigma / Rho Fragment Interaction When two or more X and / or Y type fragments are attached to anaromatic ring system, the correction factors can be calculated by a method verysimilar to that used by Hammett(23) to calculate the electronic effects inother equilibria, such as acid ionization.* This requires the assignment of ameasure of electronic 'strength' (sigma) and 'susceptibility' (rho). In dealingwith electronic effects on partitioning equilibria, a few fragments appear toact 'bidirectionally' and require both sigma and rho values, although theycannot, of course, act upon themselves. Most of the details of sigma and rhoassignment to X and Y type fragments can be found elsewhere (24), but it shouldbe pointed out that the latest version of the program (CLOGP) follows a newerprocedure for 'fused-in' fragments. Fragments fused in aromatic rings(e.g.-N=or >C=O) may also be assigned both rho and sigma constants and treatedtogether with 'on-ring' fragments instead of requiring a separate treatment.* Fragments on different rings in an aromatic ring system interactwith one another but the effect is attenuated.* If the two rings in the systemare fused, as in 5-acetyl-1-naphthylamine, the 'intrinsic' effect is only halfthe sigma rho product just as it is in the biphenyl system such as in4-(m-chlorophenyl)aniline. One frequently encounters aromatic ring systems containing severalfragments with rho and sigma values assigned, and the potential correction fromall cross products of sigma / rho could be very large. Since these multipleeffects are NOT additive, some scaling down procedure was indicated. The onechosen for CLOGP takes the following steps: a) The full potential sigma / rho product for each possibleinteraction is calculated and placed in descending value order, AFTERconsidering if the fragment pair are on the same or separate rings.b) Except for the sigma for a pyridine type nitrogen, each use ofsigma or rho causes it to 'age'. The first interaction at the top of the listis entered at full potential because the current age of its sigma and rhocomponents is 'zero' for each. Each use reduces the effective sigma or rhovalue to 1 / 2 its previous value, and so if each were at 'age 1', the incrementto the correction would only be 1 / 4 as much as a 'fresh' interaction. Themechanics of this computation are best understood by looking at the detailedoutput of some complex structures. Two such examples are provided in theexample section.* 3.2.3 Special Ortho As noted in the previous section, aromatic substituent (fragment)pairs, if they have sigma and rho values assigned to them, are given the samecorrection factor regardless of their relative position on the ring. It isimportant to keep in mind that if the fragment pair are on adjacent positions(i.e. ortho), an additional correction may be required. 3.2.3.1 Crowding 'Crowding' of certain fragment types can effectively lower theiraromatic-attached values. This is most apparent in the case of fragmentsattached to the aromatic ring through a hetero atom which possesses an electronpair, such as -NHCOCH3.* A reasonable explanation of this observation is thatthe lone pair can no longer remain in the plane of the ring, making thefragment attachment resemble aliphatic (A) rather than true aromatic (a). Themagnitude of the correction appears to depend on both steric and electronic(field) effects(25). If this explanation is valid, one would expect the correction tovary continuously up to a maximum characteristic of each fragment type. It wassurprising, therefore, to find that the rather large data set used in theoriginal evaluation of the 'negative ortho' effect (24) seemed to fit multiplesof Rekker's Magic Constant(20). This is handled in CLOGP by assigning integersto a matrix which has generalized fragment types for coordinates.More recent data provides many examples which do not support this'quantized' correction. Nevertheless, it is being retained for the presentbecause of simplicity and because its maximum is only 0.14. 3.2.3.2 Intra-Molecular Hydrogen Bonding Hydrogen bonding is known to occur intramolecularly between twoortho subsituents if one is a donor and the other an acceptor. A classicalexample of such an H-bond is that in o-nitrophenol. As might be expected, anintramolecular H-bond reduces water's ability to accommodate that solute, andthe log P of o-nitrophenol is over two log units higher than the m- andp-isomers in the heptane and carbon tetrachloride solvent systems. One mustalways keep in mind, however, that the octanol phase possesses both H-donor andH-acceptor capability, not only because it is an alcohol, but because of the 2Mwater present at saturation.In actuality, the presence of the intramolecularH-bond in o-nitrophenol penalizes solvation in octanol slightly more than itdoes solvation in water, and its log P is 0.09 log units lower than the m- andp-isomers. In terms of intramolecular H-bonding between aromatic orthosubstituents, the octanol / water system appears to be sensitive to a veryrestricted class. The only clear-cut cases seem to result from a carbonyl groupdirectly attached to the ring acting as acceptor, and a directly-attached -OHor -NH- acting as donor.* In all of the cases observed so far, the correctionis very close to +0.63, and is stored in the same matrix used for the 'negativeortho' corrections. Thus the 'crowding' and H-bonding ortho effects never areapplied simultaneously, but a sigma / rho correction cannot be added to either. 4. Summary The fragment method of calculating log P(ow) has been provedvaluable in many fields, including drug design and hazard assessment.However,manual calculations require a great deal of instructions and become verylengthy for complex structures and thus are error-prone. The computer program,CLOGP, enables the method to be applied by non-experts and includes an estimateof error, which is not possible in the future. Regular users can availthemselves of an annual update which will bring them current with all newlymeasured fragment values and improved correction factors. Versions with theUnified Driver compare the calculation from structure with a measured valuefrom log P(ow) for neutral solutes. Starlist is also included in the annualupdating service. We plan to make available in the near future a searchingprogram, GENIE, which will extend the search of Starlist to close analogs. The current literature contains many examples of QSAR accompanied bycalculations of hydrophobicity which have not been made according to aconsistent application of the rules they purport to follow.This has causedsome confusion and cast doubt upon the entire approach. Perhaps, if the use of CLOGPbecomes more widespread, published calculations will become more comparable,especially if reference is made to the program version. Any prediction of the future is risky, but, judging from the recentpast, we can expect an increasing demand for logP(ow) values. It isinconceivable that CLOGP will be perfected to such an extent that it supplantspartition coefficient measurement. The two methods should remain as they arenow: mutually complimentary. With the STARLIST module in CLOGP, the user will be able to checkthe calculated value against an acceptable measured value if the solutestructure entered is one of over 4,000 contained in that special file which islimited to non-tautomeric structures measured at a pH where the neutral formpredominates. 5. Bibliography 1) Smith, R., Hansch, C. Ames, M., J. Pharm. Sci., 64, 599 (1975). 2) Martin, Y., Quantitative Drug Design, Medicinal Research SeriesNo. 8, Marcel Dekker, New York, 1978. 3) Magee, P., Chemtech, 11, 378 (1981). 4) Smyth, R., Pfeffer, M., Van Harken, D., Cohen, A. and Hottendorf,G. Antimicrob. Agents Chemother., 10, 1004, (1981). 5) Koblin, D., Eger II, E.,Johnson, B.,Collins, P., Terrell, R., andSpears, L., Anesth. Analg., 60, 464 (1981). 6) Brown, D. and Flagg, E., J. Environ. Qual., 10, 382 (1981). 7) Ellinghausen, H.,Guth, J. and Eser, H., Ecotox. Environ. Safety,4, 26 (1980). 8) Levitan, H., Proc. Nat. Acad. Sci. USA, 74, 2914 (1977). 9) Neeley, W., Branson, D. and Blau, G., Environ. Sci. Technol., 8,1113 (1974). 10) Tanford, C., The Hydrophobic Effect, Wiley, New York, 1980, 2ndEd., 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. and Langridge, R., Arch. Giochem.Biophys., 215, 319 (1982). 13) Martinek, K., and Semenov, A.,J. Appl. Biochem., 3, 93 (1981). 14a) Newcomb, M., Moore, S. and Cram, 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 of A.Ph.A., Symposium on PartitionCoefficients, 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 Hydrophobic Fragmental Constant, Elsevier,Amsterdam (1977). 21) Hansch, C. and Leo, A., Substituent Constants for CorrelationAnalysis in Chemistry and Biology, Wiley Interscience 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 Organic Chemistry: Reaction Rates, Equilibriaand Mechanism, McGrawHill, New York, 1970, 2nd Ed.24) Leo, A., J. Chem. Soc., Perkin Trans. II, 825 (1983). 25) Ogino, A., Matsumura, S. and Fujita, T., J. Med. Chem., 23, 437(1980). 26) Leo, A. and Hansch, C.,J. Org. Chem., 36, 1539, (1971).

[0054] The ClogP / total aa of the cyclic peptide to be purified 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 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 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. For example, the total aa of a cyclic peptide 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.

[0055] In one aspect, when the cyclic peptide to be purified is a cyclic peptide having a cyclic portion, it is preferable that the cyclic peptide does not have an indole skeleton or a substituted or unsubstituted hydroxyphenyl group in the side chain of the cyclic portion.

[0056] 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.

[0057] In this specification, the term "unsubstituted hydroxyphenyl group" refers to a hydroxyphenyl group that has no substituent. In addition, substituted hydroxyphenyl groups and unsubstituted hydroxyphenyl groups may be collectively referred to as "substituted or unsubstituted hydroxyphenyl groups."

[0058] In one aspect, when the cyclic peptide to be purified is a cyclic peptide having a cyclic portion, it is preferable that the cyclic peptide 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.

[0059] In some embodiments, when the cyclic peptide 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. Alternatively, the pKa of the acidic side chain may be 3.5 to 10, 4.0 to 10, 4.5 to 10, or 5.0 to 10.

[0060] In one embodiment, when the cyclic peptide 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 also be 4.0 to 10, 4.0 to 9.5, 4.0 to 9.0, or 4.0 to 8.5.

[0061] As used herein, "acidic side chain" refers to a side chain having a pKa of 10 or less, and "basic side chain" refers to a side chain having a basic pKa of 4 or more. In this specification, 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 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 attached to the main chain). The pKa and basic pKa are calculated using a partial structure obtained by extracting the side chain portion from the side chain β position (carbon directly attached to the main chain) (hereinafter also referred to as calculated pKa and basic calculated pKa). 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.

[0062] Some of the amino acids with basic side chains for which the Basic calculated pKa values ​​have been calculated using the method described herein are shown in the table below.

[0063] 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.

[0064] The cyclic peptide to be purified may be a compound that does not have a carboxy group.

[0065] The cyclic peptide to be purified was (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]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide (compound 9), (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-carboxamide) (14H,22H)-docosahydro-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]-4,7,10,13,17,20,23,28,31,34,37(14H,22H)-undecaone (compound 1), (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)-4,7,10,13,1 7,20,23,28,31,34,37-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), (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), (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-4,15,16,19,25,31,33,34,37,39,40-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), (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-4,10,13,15,16,19,25,27,28,31,38-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), (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), (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), and (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).

[0066] In the present specification, the impurity peptide may be a cyclic peptide or a linear peptide produced during the synthesis of the cyclic peptide to be purified, or may be a cyclic peptide different from the cyclic peptide to be purified.

[0067] The peptide that is an impurity may be a cyclic peptide having 1.1 to 10 times the number of amino acid residues (here, the number of amino acid residues is an integer) as the number of amino acid residues contained in the cyclic peptide that is the target of purification.

[0068] The impurity peptide may be a cyclic peptide having two, three, four, five, six, seven, or eight times the number of amino acid residues contained in the cyclic peptide to be purified.

[0069] The impurity peptide may be a cyclic peptide having 10 to 300, 10 to 100, or 10 to 50 amino acid residues.

[0070] The peptide that is an impurity may be a multimer of the cyclic peptide that is the target of purification.

[0071] As used herein, the term "multimer" (oligomer) refers to a polymer containing the constituent components of a cyclic peptide, which is the target of purification. The multimer may be a cyclic oligomer or a linear oligomer that is a by-product under the cyclization conditions of the cyclic peptide, which is the target of purification, and includes a cyclic dimer (C-dimer) and a cyclic trimer (C-trimer). For example, when the cyclic peptide to be purified is Compound 1 below, the chemical structures of the C-dimer and C-trimer are assumed to be as follows: Compound 1 C-dimer of Compound 1

[0072] C-trimer of Compound 1 The peptide impurity may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer of the cyclic peptide to be purified.

[0073] In this specification, the divalent metal ion may be an alkaline earth metal ion, and may be at least one selected from the group consisting of calcium ions, magnesium ions, strontium ions, and barium ions, and is preferably at least one selected from the group consisting of calcium ions, magnesium ions, and barium ions, and is preferably a calcium ion.

[0074] The divalent metal ion may be a metal ion formed from a metal salt. Metal salts include solvates thereof. In the present disclosure, a "solvate" refers to a solvate with, for example, water, alcohol (e.g., methanol, ethanol, 1-propanol, or 2-propanol), acetone, dimethylformamide, or dimethylacetamide. The solvate may be with a single solvent or multiple solvents.

[0075] The metal salt and the metal salt containing a divalent metal ion may be at least one selected from the group consisting of a halide salt (e.g., a chloride salt, a fluoride salt, a bromide salt, an iodide salt), a perchlorate salt, an oxide salt, a trifluoromethanesulfonate salt, a toluenesulfonate salt, an isopropylsulfonate salt, a methanesulfonate salt, a carbonate salt, an acetate salt, and a sulfate salt, or may be at least one selected from the group consisting of a chloride salt, a fluoride salt, a bromide salt, a carbonate salt, an acetate salt, and a sulfate salt.

[0076] The metal salt may be at least one selected from the group consisting of chloride salts and bromide salts, as this provides a more excellent effect according to the present invention. The metal salt is more preferably a chloride salt, as this provides a better purity and recovery rate of the purified cyclic peptide.

[0077] The chloride salt may be calcium chloride or magnesium chloride.

[0078] The fluoride salt may be calcium fluoride.

[0079] The iodide salt may be at least one selected from the group consisting of lithium iodide, potassium iodide, barium iodide, magnesium iodide, calcium iodide, strontium iodide, samarium (III) iodide, zinc iodide, and indium iodide.

[0080] The bromide salt may be at least one selected from the group consisting of lithium bromide, magnesium bromide, calcium bromide, samarium (III) bromide, zinc bromide, and indium bromide.

[0081] The perchlorate may be at least one selected from the group consisting of barium perchlorate, magnesium perchlorate, calcium perchlorate, and zinc perchlorate.

[0082] The oxide salt may be magnesium oxide.

[0083] The trifluoromethanesulfonate may be at least one selected from the group consisting of magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[0084] The toluenesulfonate may be zinc(II) toluenesulfonate.

[0085] The isopropyl sulfonate may be zinc(II) isopropyl sulfonate.

[0086] The methanesulfonate salt may be zinc difluoromethanesulfonate.

[0087] The carbonate may be at least one selected from the group consisting of calcium carbonate and zinc carbonate.

[0088] The acetate may be magnesium acetate.

[0089] The sulfate may be magnesium sulfate.

[0090] The divalent metal ion may be an ion generated from at least one selected from the group consisting of calcium chloride, calcium fluoride, calcium bromide, barium iodide, barium perchlorate, magnesium oxide, magnesium bromide, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, zinc trifluoromethanesulfonate, zinc perchlorate, zinc difluoromethanesulfonate, zinc (II) isopropylsulfonate, zinc (II) toluenesulfonate, zinc chloride, zinc carbonate, zinc iodide, manganese trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[0091] The divalent metal ion may be an ion derived from at least one metal salt selected from the group consisting of calcium chloride, calcium fluoride, calcium bromide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, and magnesium chloride.

[0092] The divalent metal ion is preferably an ion generated from at least one metal salt selected from the group consisting of calcium chloride and calcium bromide, since this results in good purity and recovery rate of the cyclic peptide after purification, and more preferably an ion generated from calcium chloride.

[0093] The first solvent may be a solvent capable of forming a complex between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion. The first solvent may comprise at least one solvent selected from the group consisting of nitrile solvents, ether solvents, ketone solvents, ester solvents, benzene solvents, halogenated solvents, and alcohol solvents. When the first solvent is a solvent described in this paragraph, a complex between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion is more likely to be formed.

[0094] Specific examples of the nitrile solvent include chain nitriles such as acetonitrile, propionitrile, and acrylonitrile; and cyclic nitriles such as benzonitrile.

[0095] Specific examples of ether solvents include diethyl ether, DME (dimethyl ether), tetrahydrofuran, glyme, 1,4-dioxane, 2-methyltetrahydrofuran, MTBE (tert-butyl methyl ether, methyl tert-butyl ether, 2-methoxy-2-methylpropane, CAS: 1634-04-4), CPME (cyclopentyl methyl ether), tetrahydropyran, and dimethoxyethane.

[0096] Specific examples of ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone.

[0097] Specific examples of ester solvents include methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, γ-butyrolactone (GBL), ethylene carbonate (EC), propylene carbonate, butylene carbonate, dimethyl carbonate, diethyl carbonate (DEC), di-n-propyl carbonate, ethyl methyl carbonate (EMC), methyl-n-propyl carbonate, and ethyl-n-propyl carbonate.

[0098] Specific examples of benzene-based solvents include toluene, o-dichlorobenzene, 1,2,4-trichlorobenzene, and xylene.

[0099] Specific examples of halogen-based solvents include haloalkanes such as dichloromethane and dichloroethane, and haloarenes such as chlorobenzene.

[0100] Specific examples of alcohol solvents include methanol, ethanol, 1-propanol, 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, and ethylene glycol.

[0101] The first solvent may be at least one selected from the group consisting of acetonitrile, DME (dimethoxyethane), tetrahydrofuran, acetone, MEK (methyl ethyl ketone), 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, toluene, 1,4-dioxane, dichloromethane, ethanol, methanol, ethyl acetate, and isopropyl acetate.

[0102] The first solvent is preferably at least one selected from the group consisting of acetonitrile, DME (dimethoxyethane), tetrahydrofuran, acetone, and MEK (methyl ethyl ketone), as this can further improve the purity of the cyclic peptide after purification, and more preferably at least one selected from the group consisting of acetonitrile, DME (dimethoxyethane), tetrahydrofuran, and acetone, from the viewpoint of improving both the purity and recovery rate of the cyclic peptide after purification and the stability of the cyclic peptide that is the target of purification. The first solvent is preferably used for purifying a cyclic peptide that is highly lipid-soluble and is the target of purification.

[0103] Herein, the second solvent may be a water-immiscible solvent (e.g., a solvent with low solubility in water, a solvent with a large octanol / water partition coefficient (log Kow), or a solvent with a large predicted octanol / water partition coefficient). The second solvent may contain the above-mentioned first solvent in addition to the water-immiscible solvent or as a water-immiscible solvent. The second solvent may contain the same type of solvent as the first solvent and a water-immiscible solvent.

[0104] The octanol / water partition coefficient (log Kow) can be determined by any method known in the art or described herein. The predicted octanol / water partition coefficient (Log Kow) can be determined by known means, such as, but not limited to, a database search or literature search, or by separate explicit measurements. The octanol / water partition coefficient can be measured by, for example, but not limited to, a method in accordance with Japanese Industrial Standard JIS 7260-107:2000, Determination of Partition Coefficient (1-Octanol / Water) - Shake Flask Method (https: / / kikakurui.com / z7 / Z7260-107-2000-01.html [Accessed June 25, 2024]).

[0105] In certain embodiments, the water-immiscible organic solvent includes, but is not limited to, an organic solvent having low water solubility (e.g., solubility in water of 200 g / L or less, preferably 150 g / L or less). The water-immiscible organic solvent may contain a trace amount of another water-miscible organic solvent, for example, 0.01 wt % or less. The water solubility can be determined by any method known in the art or described herein. An exemplary method for determining the solubility includes, but is not limited to, gas chromatography, which can be determined by measuring the concentration of the organic solvent in water prepared by mixing equal volumes of the organic solvent and water at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C).

[0106] In one embodiment, the miscibility of an organic solvent with water can be determined, for example, by the separation of the solvent and water into two layers when equal volumes of the solvent and water are mixed in a container at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C). Whether the solvent and water separate into two layers can be determined, for example, by visual inspection or by sampling the upper and lower liquids in the container. When it is confirmed by such a method that the solvent and water have separated into two layers, the solvent may be referred to as a water-immiscible solvent. However, even a water-miscible solvent may form an interface with water and separate into two layers depending on the solute in the solvent and the salt concentration in the water.

[0107] In some embodiments, the water-immiscible solvent can be characterized as an ester having from 3 to 10 carbon atoms, such as ethyl acetate, isopropyl acetate, n-propyl acetate, t-butyl acetate, methyl propionate, or ethyl propionate.

[0108] In certain embodiments, the water-immiscible solvent can be characterized as a cyclic ether having 4 or more and 10 or less carbon atoms, and specific examples thereof include 2-methyltetrahydrofuran, tetrahydrofuran, 4-methyltetrahydropyran, or 1,4-dioxane. In certain embodiments, the water-immiscible solvent can be characterized as an acyclic ether having 4 or more and 10 or less carbon atoms, and specific examples thereof include MTBE (tert-butyl methyl ether), diisopropyl ether, or diethyl ether.

[0109] In one embodiment, the water-immiscible solvent can be characterized as an ether having both cyclic and acyclic alkyl groups having 6 or more and 10 or less carbon atoms, such as CPME.

[0110] In one embodiment, the water-immiscible solvent can be characterized as a carbonate ester having 3 or more and 10 or less carbon atoms, such as dimethyl carbonate, diethyl carbonate, or diisopropyl carbonate.

[0111] In some embodiments, the water-immiscible solvent can be characterized as a hydrocarbon having 5 or more and 10 or less carbon atoms, such as pentane, hexane, or heptane.

[0112] In certain embodiments, the water-immiscible solvent can be characterized as an aromatic hydrocarbon ring having at least 6 and at most 10 carbon atoms, such as toluene, xylene, or chlorobenzene.

[0113] In some embodiments, the water-immiscible solvent can be characterized as having a low boiling point at ambient pressure (about 1 atmosphere). In some embodiments, a low boiling point at atmospheric pressure (near 1 atmosphere) is exemplified as a temperature between 35°C and 140°C.

[0114] The water-immiscible solvent is preferably at least one selected from the group consisting of 2-methyltetrahydrofuran, tetrahydrofuran, dimethyl carbonate, anisole, isopropyl acetate, ethyl acetate, MTBE (tert-butyl methyl ether), diethyl ether, dichloromethane, chloroform, DME (dimethyl ether), CPME (cyclopentyl methyl ether), 4-methyltetrahydropyran, heptane, and toluene.

[0115] The second solvent may be at least one selected from the group consisting of acetonitrile, MTBE (tert-butyl methyl ether), 2-methyltetrahydrofuran, tetrahydrofuran, dimethyl carbonate, anisole, isopropyl acetate, ethyl acetate, diethyl ether, dichloromethane, chloroform, DME (dimethyl ether), CPME (cyclopentyl methyl ether), 4-methyltetrahydropyran, heptane, and toluene, or may be at least one selected from the group consisting of acetonitrile and MTBE (tert-butyl methyl ether). The second solvent may, for example, contain acetonitrile and a solvent that is immiscible with water, or may contain acetonitrile and MTBE (tert-butyl methyl ether).

[0116] The method for purifying a cyclic peptide in this specification may be a method in which the purity (area%) of the cyclic peptide to be purified is higher than the purity (area%) of the cyclic peptide to be purified before the purification operation.

[0117] The purity (area%) of the cyclic peptide that is the target of purification after the purification operation may be higher than the purity (area%) of the cyclic peptide that is the target of purification before the purification operation by 0.2 area% or more, 1.0 area% or more, 2.0 area% or more, 4.0 area% or more, 6.0 area% or more, 8.0 area% or more, 10.0 area% or more, 12.0 area% or more, 14.0 area% or more, 16.0 area% or more, or 18.0 area% or more, with 100.0 area% as the upper limit.

[0118] The purity (area%) of the cyclic peptide that is the target of purification after the purification operation may be 1.003 times or more, 1.01 times or more, 1.05 times or more, 1.10 times or more, 1.15 times or more, or 1.20 times or more of the purity (area%) of the cyclic peptide that is the target of purification before the purification operation, with an upper limit of 100.0 area%.

[0119] The purity (area%) of the cyclic peptide, which is the target of purification after the purification operation, may be 40.0 area% or more, 60.0 area% or more, 80.0 area% or more, 85.0 area% or more, 90.0 area% or more, 95.0 area% or more, or 97.0 area% or more, with an upper limit of 100.0 area%.

[0120] The purity (area %) of the cyclic peptide to be purified can be determined by measuring the UV spectrum of a mixture containing the cyclic peptide to be purified and impurity peptides using HPLC and a PDA (photodiode array detector), and calculating the proportion of the peak area of ​​the cyclic peptide to be purified to the sum of the peak areas of the entire mixture containing the cyclic peptide to be purified and impurity peptides at 220 nm.

[0121] The method for purifying a cyclic peptide herein may be a method that makes the total percentage of impurities lower than the total percentage of impurities before the purification procedure.

[0122] The ratio (%) of the total impurities after the purification operation may be lower than the ratio (%) of the total impurities before the purification operation by 1.0% or more, 5.0% or more, 10.0% or more, 15.0% or more, 20.0% or more, or 25.0% or more.

[0123] The ratio (%) of the total impurities after the purification operation may be 0.97 times or less, 0.95 times or less, 0.90 times or less, 0.80 times or less, 0.70 times or less, 0.60 times or less, 0.50 times or less, 0.40 times or less, 0.30 times or less, 0.30 times or less, 0.20 times or less, or 0.10 times or less of the purity (area%) of the cyclic peptide that is the purification target before the purification operation, with 100 area% as the upper limit.

[0124] The ratio (%) of the total impurities after the purification operation may be 120.0 area% or less, 100.0 area% or less, 80.0 area% or less, 60.0 area% or less, 40.0 area% or less, 25.0 area% or less, 20.0 area% or less, 15.0 area% or less, 10.0 area% or less, 8.0 area% or less, 6.0 area% or less, 4.0 area% or less, or 3.0 area% or less.

[0125] The total impurity ratio (%) can be calculated by the following formula: Total impurity ratio (%) = A ｉｍｐ / A ＴＭ x 100 A ＴＭ : Purified target product peak area (220 nm) A ｉｍｐ : Sum of impurity peak areas (220 nm) The peak area of ​​the target substance and the sum of the impurity peak areas can be measured by measuring the UV spectrum of a mixture containing the cyclic peptide, which is the target substance to be purified, and peptides, which are impurities, using HPLC and a PDA (photodiode array detector).

[0126] Yet another embodiment of the present invention relates to a method for producing a composition containing a cyclic peptide, including the method for purifying a cyclic peptide according to the present invention. In this specification, the method for producing a composition containing a cyclic peptide may further include a step of obtaining the cyclic peptide to be purified by liquid-phase synthesis, a step of obtaining the cyclic peptide to be purified by solid-phase synthesis, or a step of obtaining the cyclic peptide to be purified by a culture method. The step of obtaining the cyclic peptide to be purified by liquid-phase synthesis means that the step of obtaining the cyclic peptide to be purified includes liquid-phase synthesis. The step of obtaining the cyclic peptide to be purified by solid-phase synthesis means that the step of obtaining the cyclic peptide to be purified includes solid-phase synthesis. The liquid-phase synthesis method used in this specification includes a method for synthesizing a peptide using an organic tag (also called a hydrophobic tag). Methods for synthesizing peptides using organic tags include, but are not limited to, the methods described in WO 2012 / 029794, WO 2007 / 122847, and WO 2019 / 009317.

[0127] Yet another embodiment of the present invention relates to a method for purifying a cyclic peptide, characterized in that the cyclic peptide to be purified is separated from a mixture containing the cyclic peptide to be purified and peptides as impurities as a complex with a divalent metal ion or a metal salt containing a divalent metal ion, thereby improving the purity of the cyclic peptide compared to when a complex with a divalent metal ion or a metal salt containing a divalent metal ion is not formed.

[0128] Yet another embodiment of the present invention relates to a method for purifying a cyclic peptide, characterized in that the cyclic peptide to be purified is separated from a mixture containing the cyclic peptide to be purified and peptides as impurities as a complex with a divalent metal ion or a metal salt containing a divalent metal ion, thereby reducing the content of impurities compared to when a complex with a divalent metal ion or a metal salt containing a divalent metal ion is not formed.

[0129] Yet another embodiment of the present invention relates to a multimer of the above-mentioned compounds 1 to 9, or a pharmaceutically acceptable salt of the multimer of compounds 1 to 9. The multimer of compounds 1 to 9 may be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of compounds 1 to 9.

[0130] The multimers of Compounds 1 to 9 and the pharmaceutically acceptable salts of the multimers of Compounds 1 to 9 each include their pharmaceutically acceptable solvates.

[0131] Yet another embodiment of the present invention relates to a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of compound 9, or a pharmaceutically acceptable salt thereof.

[0132] The impurity peptide is useful, for example, as a standard for calculating the amount of the remaining impurity peptide contained in the cyclic peptide to be purified.

[0133] Yet another embodiment of the present invention relates to a composition comprising the above-mentioned compound 9 or a pharmaceutically acceptable salt thereof together with a multimer of compound 9, wherein the content of the multimer of compound 9 is 3.0 area% or less relative to the entire composition.

[0134] The content of the multimer of compound 9 may be 3.0 area % or less, 2.0 area % or less, or 1.0 area % or less, based on the entire composition, and may be above the detection limit.

[0135] The multimers may be dimers and trimers of compound 9.

[0136] The content (area %) of the multimer of compound 9 can be determined, for example, by measuring the UV spectrum of a mixture containing compound 9 and a multimer of compound 9 using HPLC and a photodiode array detector (PDA) and calculating the ratio of the peak area of ​​the multimer of compound 9 to the sum of the peak areas of the entire mixture containing compound 9 and a multimer of compound 9 at 220 nm.

[0137] Yet another embodiment of the present invention relates to a composition comprising the above-mentioned compound 9 or a pharmaceutically acceptable salt thereof together with a multimer of compound 9, wherein the content of the multimer of compound 9 is 3.0 area% or less relative to compound 9 or a pharmaceutically acceptable salt thereof.

[0138] The content of the multimer of compound 9 may be 3.0 area% or less, 2.0 area% or less, or 1.0 area% or less, relative to the content of compound 9 or the salt of compound 9, and may be above the detection limit.

[0139] The multimers may be dimers and trimers of compound 9.

[0140] The content (area %) of the multimer of compound 9 can be determined, for example, by measuring the UV spectrum of a mixture containing compound 9 and a multimer of compound 9 using HPLC and a PDA (photodiode array detector) and calculating the ratio of the peak area of ​​the multimer of compound 9 to the peak area of ​​compound 9 at 220 nm.

[0141] In this specification, examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.; sulfonates such as methanesulfonate, benzenesulfonate, toluenesulfonate, etc.; carboxylates such as formate, acetate, oxalate, maleate, fumarate, citrate, malate, succinate, malonate, gluconate, mandelate, benzoate, salicylate, fluoroacetate, trifluoroacetate, tartrate, propionate, glutarate, etc.; alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as magnesium salt, calcium salt, etc.; and ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, tetraalkylammonium salt, etc. Among them, hydrochloride, methanesulfonate, sodium salt, potassium salt, etc. are preferred, and hydrochloride, sodium salt, and potassium salt are more preferred.

[0142] In the present disclosure, a pharmaceutically acceptable solvate is, for example, a solvate with water, an alcohol (e.g., ethanol, 1-propanol, or 2-propanol), or acetone. The solvate may be with a single solvent or multiple solvents.

[0143] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety, including the following: WO 2013 / 100132, WO 2018 / 225851, WO 2018 / 225864, WO 2019 / 117274, WO 2020 / 111238, WO 2020 / 12 2182, WO 2021 / 075478, WO 2021 / 090856, WO 2021 / 132545, WO 202 / 1246471, WO 2022 / 097540, WO 2022 / 138891, WO 2022 / 145444, WO 2022 / 234864, WO 2023 / 127869.

[0144] The present invention is further illustrated by, but not limited to, the following examples and reference examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0145] The following abbreviations are used in the examples: HPLC: High performance liquid chromatography DIPEA: N,N-diisopropylethylamine HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate CaCl ２ : Calcium chloride CaF ２ : Calcium fluoride CaBr ２ : Calcium bromide MeCN: Acetonitrile, CH ３ CN DME: Dimethytoxyethane, ethylene glycol dimethyl ether THF: Tetrahydrofuran MEK: Methyl ethyl ketone NMM: N-methylmorpholine IPAC: Isopropyl acetate TIPS: Triisopropylsilane HFIP: 1,1,1,3,3,3-hexafluoroisopropyl alcohol MeTHF: 2-methyltetrahydrofuran TFE: 2,2,2-trifluoroethanol DBU: 1,8-diazabicyclo[5.4.0]-7-undecene DMF: N,N-dimethylformamide HOBt: 1-hydroxybenzotriazole EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride HOAt: 1-hydroxy-7-azabenzotriazole DMSO: Dimethyl sulfoxide DCM: Dichloromethane HMDS: Hexamethyldisilazane TMSOTf: trimethylsilyl trifluoromethanesulfonate TCFH: chloro-N,N,N':,N'-tetramethylformamidinium hexafluorophosphate NMI: N-methylimidazole NMR: nuclear magnetic resonance MTBE: tert-butyl methyl ester ESI: electrospray ionization

[0146] In the following examples, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), and ion chromatography (IC) analyses were performed using one of the analytical conditions described below. Each compound was detected using a variable UV detector or a mass spectrometer, but other techniques such as a photodiode array detector may also be used.

[0147] Analysis condition-1 (HPLC-UV) Apparatus: Waters ACQUITY UPLC H-Class system Column: Waters ACQUITY UPLC CSH C18, 1.7 μm, 2.1 mm × 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)

[0148] Analysis Condition-2 (HPLC-MS) Apparatus: Thermo Vanquish HPLC system + Thermo Q Exactive orbitrap mass spectrometer Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm × 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)

[0149] Analysis Condition-3 (HPLC-UV) Apparatus: Waters ACQUITY UPLC H-Class system Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm × 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)

[0150] Analysis Condition-4 (HPLC-UV) Apparatus: Waters Alliance HPLC system Column: TOSOH TSKgel Super H3000, 6.0 mm ID 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

[0151] Analysis conditions-5 (IC) Equipment: 930 compact IC flex system (Metrohm) Column: Metrosep C4-150 / 4.0, 4.0mm ID x 15cm, 5μm Mobile phase: 2mM HNO ３ -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

[0152] Analysis conditions - SQFA05 (HPLC-MS) Apparatus: Acquity UPLC / SQD or Acquity UPLC / SQD2 Column: Ascentis Express C18, 2.1 mm 1d 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)

[0153] Analysis conditions - SQFA05Long (HPLC-MS) Apparatus: Acquity UPLC / SQD or Acquity UPLC / SQD2 Column: Ascentis Express C18, 2.1 mm 1d 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)

[0154] Analysis Conditions - M Apparatus: Waters ACQUITY UPLC H-Class system Column: Ascentis Express 90A C18, 2.1 mm 1d 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)

[0155] 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)

[0156] 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)

[0157] Analysis conditions - FC2 (HPLC-MS) Apparatus: Waters UPLC Column: Waters ACQUITY UPLC CSH C18, 1.7 μm, 2.1 mm × 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)

[0158] Analysis conditions - cyc (HPLC) Apparatus: Waters UPLC Column: Waters ACQUITY UPLC CSH Phenyl-Hexyl Column, 1.7 μm, 2.1 mm × 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)

[0159] Analysis Conditions - H (HPLC) Apparatus: Waters UPLC Column: Ascentis Express RP-Amide, 3.0 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: 0.7 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)

[0160] The qNMR measurement method was carried out by dissolving the residue containing the target compound and the internal standard in CDCl. ３ or DMSO-d ６ The analysis was carried out under the following conditions.

[0161] Measurement device: Bruker Avance III 400 Internal standard substance: 3,5-bis(trifluoromethyl)benzoic acid Measurement conditions ( １９ F-NMR): CDCl ３ or DMSO-d ６ , 24.8°C, pulse angle 90°, digital resolution 0.24Hz, relaxation time 15 seconds, no spin, number of accumulations 64

[0162] Measuring device: JEOL JNM-ECZ500R / S1 Measuring conditions ( １ H-NMR): methanol-d ４ , 25.3℃, pulse angle 45°, digital resolution 0.76Hz, relaxation time 5 seconds, with spin, number of accumulations 8

[0163] <HPLC sample preparation and HPLC measurement in the examples> CaCl ２ Unless otherwise specified, the HPLC samples in this example were adjusted so that the concentration of the concentrated dry product was approximately 1 mg / mL. For example, in Run 1 of Example 1-2, the concentrated dry product (25 mg) was dissolved in acetonitrile (0.250 mL) to prepare the HPLC sample. 10 μL of the reaction solution was sampled and diluted with acetonitrile (0.990 mL) to prepare the HPLC sample. CaCl ２The filtrate after the treatment was also diluted to prepare an HPLC sample at the same dilution ratio. The HPLC sample was subjected to HPLC measurement under analytical condition-3, and the chromatogram obtained was used for evaluation after subtracting the chromatogram of the blank solution using analytical software.

[0164] <Calculation of Recovery Rate (%) 1> In the present examples except for Examples 1-5 and 2-2, the recovery rate (%) was calculated using the following formula based on the peak area of ​​the target compound obtained by HPLC measurement (Analysis Condition-3 or Analysis Condition-4). Recovery rate (%) = A ｆ ÷A ｉ x 100 A ｉ : CaCl ２ Peak area (220 nm) of the target compound in the reaction solution before addition A ｆ : CaCl ２ Peak area of ​​the target compound in the treated filtrate (220 nm)

[0165] <Calculation of Recovery Rate (%) 2> In Examples 1-5 and 2-2, the recovery rate (%) was calculated by the following formula: Recovery rate (%) = W ｐ ÷W ｉ x 100 W ｉ W: Amount (mg) of the target compound calculated by HPLC measurement using a standard solution. ｉ is CaCl ２ A portion of the reaction solution before the addition was diluted with acetonitrile to prepare a sample solution for HPLC. The sample solution for HPLC was subjected to HPLC measurement (analysis condition-3) together with the standard solution, and the W was calculated using the following formula. ｉ = A Ｓａｍｐｌｅ ÷A ＳＴＤ ×C ＳＴＤ ×V÷W ｓａｍｐｌｅ ×W ｓｏｌｎ x 1000 A Ｓａｍｐｌｅ A: Peak area of ​​the target compound in the sample solution (220 nm) ＳＴＤ C: Peak area of ​​the target compound in the standard solution (220 nm) ＳＴＤ : Standard solution concentration (mg / mL) V: Volume of solution (mL) W ｓａｍｐｌｅ W: Weight (mg) of reaction solution used in sample preparation ｓｏｌｎ : CaCl ２Weight of reaction solution before addition (g) W ｐ W: The amount (mg) of the target compound recovered calculated by HPLC measurement using the standard solution. ｐ is CaCl ２ The target compound was concentrated to dryness after treatment, followed by filtration, solid washing, concentration, and separation. The entire amount was dissolved in acetonitrile (20 mL), and a portion was diluted to prepare a sample solution for HPLC. The sample solution for HPLC was subjected to HPLC measurement (analysis condition-3) together with a standard solution, and the W was calculated using the following formula. ｐ = A Ｓａｍｐｌｅ ÷A ＳＴＤ ×C ＳＴＤ ×B×20 A Ｓａｍｐｌｅ A: Peak area of ​​the target compound in the sample solution (220 nm) ＳＴＤ C: Peak area of ​​the target compound in the standard solution (220 nm) ＳＴＤ : Standard solution concentration (mg / mL) B: Dilution ratio

[0166] <Calculation of Purity (area%) of Target Compound and Ratio (%) of Sum of Impurities to Target Compound> In this example, the purity (area%) was defined as the peak area percentage (220 nm) of the target compound, and the ratio (%) of the sum of impurities was calculated using the following formula: Ratio (%) of Sum of Impurities = A ｉｍｐ / A ＴＭ x 100 A ＴＭ : Peak area of ​​target compound (220 nm) A ｉｍｐ : Sum of impurity peak areas (220 nm)

[0167] The cyclic peptides used in this example are shown in the table below.

[0168] Reference Example 1: Synthesis of Cyclic Peptides General Methods for Synthesizing Amino Acids The amino acids protected with an Fmoc group (Fmoc-amino acids) used in the present Examples and Reference Examples can be produced by general amino acid synthesis methods, for example, by the method described in WO 2021 / 090855. Amino acids protected with an Fmoc group are also available from commercial suppliers. In the present Examples and Reference Examples, commercially available amino acids with an Fmoc group introduced at the N-terminus, or commercially available amino acids with the C-terminus deprotected can also be used in the elongation reaction of the target peptide. The deprotection reaction of the protecting group can be carried out, for example, by the method described in "Greene's, 'Protective Groups in Organic Synthesis' (5th Edition, John Wiley & Sons 2014)." Amino acids having side chains different from those of naturally occurring amino acids (functional groups on the α-carbon of amino acids) can be produced by appropriately converting the side chains of amino acids (naturally occurring amino acids) available from commercial suppliers into the desired side chains. The conversion of side chains is described in "Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3" (pp. 111-115). ｒｄ Edition, R. C. Or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 ｔｈ Edition, by M. B. 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 in accordance with 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, and then carrying out a ring-opening reaction of the cyclic protecting group.

[0169] The Fmoc-amino acids used in the solid-phase synthesis of this Reference Example are shown in Tables 4 to 6. The Fmoc-amino acids listed in Tables 4 to 6 were either purchased from commercial suppliers or synthesized with reference to the method described in WO 2021 / 090855.

[0170]

[0171] <General Peptide Synthesis Method> The peptides used in the Examples and Reference Examples were synthesized by solid-phase synthesis or liquid-phase synthesis. Examples of solid-phase synthesis 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 the Examples and Reference Examples, reference was made to the peptide synthesis method using the Fmoc method described in WO 2013 / 100132 or WO 2018 / 225864. In this Reference Example, peptide elongation was performed using the basic synthesis method for cyclic peptides shown in Figure 1. The basic synthesis method for cyclic peptides shown in Figure 1 comprises five steps: 1) a peptide elongation reaction from the N-terminus of an amino acid by the Fmoc method using a 2-chlorotrityl resin in which the carboxy group of the Asp side chain or the carboxy group of the peptide main chain is supported (a peptide chain elongation reaction using an amino acid protected with an Fmoc group as a starting material); 2) a process of cleaving the peptide from the 2-chlorotrityl resin; 3) an amide cyclization by condensation of the carboxy group of the Asp side chain or the carboxy group of the peptide main chain, which is generated by cleavage from the 2-chlorotrityl resin during the cleavage process, 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, as necessary; and 5) pretreatment of the compound by liquid-liquid separation or the like. In this Example and Reference Example, unless otherwise specified, cyclic peptides were synthesized using the basic synthesis method for cyclic peptides shown in Figure 1.

[0172] <Method for synthesizing peptides containing N-alkylamino acids> Peptides containing N-alkylamino acids can be synthesized according to the "<General method for peptide synthesis>" described in the present Examples and Reference Examples, using as starting materials N-alkylamino acids protected with an Fmoc group shown in Tables 4 to 6.

[0173] <Deprotection of Protecting Groups of Side Chain Functional Groups of Cyclic Peptides> In sequences synthesized using Fmoc-protected amino acids having a hydroxyl group protected with THP in the side chain, such as Fmoc-Thr(THP)-OH and Fmoc-Ser(THP)-OH, 4 mL of a HFIP solution (containing 2% (v / v) TIPS and 1% (v / v) 1,2-dichloroethane) of tetramethylammonium hydrogen sulfate (0.05 M) was added to the residue of the resulting cyclic peptide (14 reaction vessels) to dissolve the residue, and the mixture was allowed to stand at room temperature for 4 hours to deprotect the THP groups. After confirming the 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) to obtain a cyclic peptide.

[0174] Reference Example 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 resulting compound was cleaved from the resin and then deprotected by Fmoc to obtain a crude product of Compound 1-a. Specifically, a mixture of TFE (28 mL), toluene (28 mL), and DIPEA (0.94 mL) was added to compound 1-c (10.017 g) containing resin in a reaction vessel, and the mixture was shaken and stirred for 4 hours. 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 dissolved in 5% KHSO 4 . ４ Aqueous solution (28 mL), 5% Na ２ HPO ４The resulting solution was washed with an aqueous solution (28 mL) and a 5% NaCl aqueous solution (28 mL). The organic phase was dried under reduced pressure to obtain compound 1-b (5.517 g). DBU (0.3 mL) was added to a reaction vessel containing compound 1-b (1 g) dissolved in acetonitrile (5 mL). After confirming the completion of the reaction by HPLC measurement, the reaction solution was concentrated under reduced pressure. The resulting crude product of 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, combined with the purified product described above, gave compound 1-a (0.6538 g). HPLC: retention time: 12.29 minutes (analysis condition-1).

[0175] <Synthesis of Compound 1 by Cyclization Reaction> Compound 1-a (300 mg) obtained by column purification was added to a reaction vessel along with acetonitrile (30 mL) and DIPEA (81.5 μL), and the mixture was stirred at 25°C. A solution was prepared by adding acetonitrile (15 mL) to HATU (147 mg), and this solution was added dropwise to the reaction solution over 33 minutes. The vessel containing the acetonitrile solution of HATU 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 prepared, and the completion of the reaction was confirmed by HPLC measurement (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), 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 (6 mL). Each aqueous phase discharged from the previous washes 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 performed twice in total. 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 conditions-2)

[0176] [Reference Example 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 by a method similar to that described in WO 2021 / 90855. The crude product of Compound 2-a obtained was purified by reverse-phase silica gel chromatography (acetonitrile / water) to obtain Compound 2-a. HPLC: Retention time: 11.82 minutes (Analysis Condition-1) <Synthesis of Compound 2 by Cyclization Reaction> A concentrated, dried product (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. Acetonitrile (15 mL) was added to HATU (152 mg) 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 to prepare a sample, 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), and 0.5% aqueous sodium chloride (6 mL). Ethyl acetate (6 mL) was added to the organic phase, which was then washed with 0.5% aqueous sodium chloride solution (6 mL). The resulting organic phase was collected in a recovery flask and concentrated under reduced pressure. Then, azeotropic distillation with acetonitrile (20 mL) was performed 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 discharged in the above washings was extracted with ethyl acetate (6 mL). The resulting organic phase was collected in a recovery flask and concentrated under reduced pressure. Then, azeotropic distillation with acetonitrile (10 mL) was performed 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 vacuum drying yielded a concentrated, dry product (0.2875 g) containing Compound 2. LCMS (ESI) m / z = 1454 LCMS elution time: 2.4 min (Analysis conditions-2)

[0177] [Reference Example 3-1: (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. <Synthesis of compound 3 by cyclization reaction> A concentrated, dried product containing compound 3-a (300 mg), acetonitrile (60 mL), and DIPEA (101 μL) were added to a reaction vessel and stirred at 25°C. Acetonitrile (15 mL) was added to HATU (182 mg) 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 (1.8 mL), and the washings were added dropwise to the reaction solution. After the completion of the dropwise 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 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 azeotroped twice 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 (0.2470 g) containing compound 3. LCMS (ESI) m / z = 1217 LCMS elution time: 2.3 min (analysis condition-2).

[0178] Reference Example 3-2: 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 (300 mg) containing compound 3-a to prepare a solution, which was then added dropwise to the aforementioned solution over 17 minutes. The vessel in which the acetonitrile solution of compound 3-a and DIPEA was prepared 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 to prepare a sample, and the completion of the reaction was confirmed by HPLC measurement (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 azeotroped twice 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 containing compound 3 (0.2472 g).

[0179] [Reference Example 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)] Compound 4-a was synthesized according to the description of WO 2013 / 100132. The peptide was elongated using the Fmoc method, the elongated peptide was cleaved from the resin, and then cyclized. The THP group was then deprotected, yielding 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 an 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 using 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 (0.1089 g) containing compound 4. LCMS (ESI) m / z = 1714 LCMS elution time: 2.8 min (Analysis conditions-2)

[0180] Reference Example 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-4,10,13,15,16,19,25,2 Synthesis of 7,28,31,38-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)] Compound 4-a was synthesized according to the description of WO 2013 / 100132. The peptide was elongated using the Fmoc method, the elongated peptide was cleaved from the resin, and then cyclized. The THP group was then deprotected to obtain 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 an 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 using 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 (0.1106 g) containing compound 5. LCMS (ESI) m / z = 1641 LCMS elution time: 3.1 min (Analysis conditions-2)

[0181] [Reference Example 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)] Compound 4-a was synthesized according to the description of WO 2013 / 100132. The peptide was elongated using the Fmoc method, the elongated peptide was cleaved from the resin, and then cyclized. The THP group was then deprotected to obtain a concentrated dry product of Compound 6. Ethyl acetate (9.2 mL) was added to the concentrated dry product (421.1 mg) containing Compound 6 to form an 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 using 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 (0.1305 g) containing Compound 6. LCMS (ESI) m / z = 1598 LCMS elution time: 2.7 min (Analysis conditions-2)

[0182] Reference Example 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) Compound 4-a was synthesized according to the description in WO 2013 / 100132, and THP-protected cyclic peptide 7-a was obtained by synthesizing it according to the description 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 an organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride solution (9.2 mL). Each of the discharged 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, and then azeotroped twice 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)

[0183] Reference Example 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)

[0184] <Synthesis Method 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, 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 sequentially mixed with the reaction solution, and stirred at 0°C for 1 hour. Piperidine (1.27 g, 14.9 mmol) was slowly added to the resulting reaction solution, and the mixture was 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 solution, and the organic phase was separated. The resulting organic phase was washed successively 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 distilled off under reduced pressure to obtain 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)

[0185] <Method for synthesizing compound 8-c (Fmoc-Pro-Ile-pip)> DBU (2.21 g, 14.53 mmol) was added to a reaction vessel containing a DMF solution (50 mL) of compound 8-b (Fmoc-Ile-pip), followed by stirring 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 this reaction solution, followed by stirring 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 this 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 distilled off 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, after which the ethyl acetate was distilled off 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 minutes (Analysis conditions - SQDFA05)

[0186] <Method for synthesizing compound 8-d (Fmoc-Asp(OAl)-Pro-Ile-pip)> DBU (0.702 mL, 4.66 mmol) was added to a reaction vessel containing a DMF solution (23 mL) of compound 8-c (Fmoc-Pro-Ile-pip) (2.41 g, 4.66 mmol), followed by stirring 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 this reaction solution, followed by stirring 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 this reaction solution, and the organic phase was separated. The obtained organic phase was washed successively 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 distilled off under reduced pressure to obtain compound 8-d as a crude product. The obtained crude product was dissolved in DMSO and purified by reverse-phase medium-pressure column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain 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 minutes (Analysis conditions - SQDFA05Long)

[0187] <Synthesis of Compound 8-e (Fmoc-Asp-Pro-Ile-pip)> Tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3, Pd(Pphe ３ ) ４, 0.294 g, 0.254 mmol) and phenylsilane (CAS: 694-53-1, 0.313 mL, 2.54 mmol) were added and stirred at 0°C for 15 minutes. The solvent of the resulting reaction solution 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)

[0188] 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. Compound 8 was obtained through subsequent peptide elongation by the Fmoc method, cleavage from the resin, and peptide cyclization. Ethyl acetate (9.2 mL) was added to the concentrated, dried product (214.7 mg) containing compound 8 to form an organic phase. The organic phase was washed twice with 0.5% aqueous sodium chloride solution (9.2 mL). Each of the discharged 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, and then azeotroped twice 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 solid to dryness to form an organic phase. The organic phase was washed three times with 0.5% aqueous sodium chloride solution (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 previously prepared organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations using 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 an organic phase. The organic phase was washed four times with 0.5% aqueous sodium chloride solution (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 previously prepared organic phase. The organic phase was concentrated under reduced pressure, followed by two azeotropic distillations using 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).

[0189] [Reference Example 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)

[0190] Synthesis of Compound 9-a: 9H-fluoren-9-ylmethyl (4S)-5-oxo-4-[[4-(trifluoromethyl)phenyl]methyl]oxazolidine-3-carboxylic acid DCM (45 L) and (2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-[4-(trifluoromethyl)phenyl]propionic acid (3.05 kg) were added to a reaction vessel purged with nitrogen at room temperature and stirred, followed by paraformaldehyde (0.90 kg) and MgSO ４ (2.02 kg) was added and stirred at 20° C. for 10 minutes. The external temperature of the reactor was set to 15° C., and BF ３ OEt ２ (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 fitted with silica gel (3.05 kg), and 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).

[0191] Synthesis of Compound 9-b: (2S)-2-[but-3-enyl(9H-fluoren-9-ylmethoxycarbonyl)amino]-3-[4-(trifluoromethyl)phenyl]propionic acid

[0192] Toluene (22.4 L) and compound 9-a (2.80 kg) were added to a reaction vessel purged with nitrogen at room temperature and stirred. ２ (1.35 kg) was added and stirred at 20°C for 10 minutes. The internal temperature of the reaction vessel was set to 40-45°C, and the reaction mixture was stirred for 10 hours. The reaction solution 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 obtained organic phase was concentrated under reduced pressure at an external temperature of 45-50°C to obtain compound 9-b (2.44 kg).

[0193] 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]acetate

[0194] 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. Subsequently, sarcosine tert-butyl ester hydrochloride (0.87 kg) and HATU (2.18 kg) were 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 by column chromatography (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.

[0195] Synthesis of Compound 9-d: tert-butyl 2-[[(2S)-2-(but-3-enylamino)-3-[4-(trifluoromethyl)phenyl]propanoyl]-methyl-amino]acetate

[0196] Toluene (28.0 L) and compound 9-c (2.79 kg) were added to a reaction vessel purged with nitrogen at room temperature and stirred. Subsequently, DBU (0.67 kg) was 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 then the aqueous phase was discharged. 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 obtain compound 9-d (1.49 kg) as a pale yellow oil.

[0197] Synthesis of Compound 9-e: 9H-fluoren-9-ylmethyl N-[(1S)-1-chlorocarbonylbut-3-enyl]-N-methyl-carbamate

[0198] Into a reaction vessel purged with nitrogen, 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 at an internal temperature of 10°C over 2 hours, and the mixture was stirred for 2 hours while maintaining the internal temperature at 10°C. The mixed solution was concentrated under reduced pressure at 30-35°C. DCM (3.4 L) was added to the concentrate and concentrated under reduced pressure twice, yielding compound 9-e (1.65 kg) as a yellow oil.

[0199] 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]acetate

[0200] DCM (15.0 L) and compound 9-d (1.49 kg) were added to a reaction vessel purged with nitrogen at room temperature and stirred. Subsequently, compound 9-e (1.72 kg) dissolved in DCM (2.96 L) was added dropwise over 1 hour at an internal temperature of 0-10°C. Stirring was continued for 30 minutes at an internal temperature of 10°C, followed by dropwise addition of DIPEA (0.926 kg) over 1 hour at an internal temperature of 0-10°C. After stirring for 2 hours at 20°C, 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 the organic phase was 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 obtain compound 9-f (1.97 kg) as a colorless powder.

[0201] 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

[0202] Toluene (59.0 L) and compound 9-f (0.655 kg) were added to a nitrogen-purged reactor at room temperature and stirred. Subsequently, p-benzoquinone (28.4 g) was added, and the mixture was heated to an internal temperature of 100°C. To the mixed solution heated to an internal temperature of 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 reaction solutions from the three batches were mixed 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 obtain compound 9-g (1.05 kg) as a yellow oil.

[0203] 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

[0204] A solution of compound 9-h (3.89 kg) in MeTHF (20.5 kg) was added to a reaction vessel at room temperature, followed by cooling to an external temperature of 15°C. HMDS (2.19 kg) was then added and stirred. Subsequently, TMSOTf (1.80 kg) was slowly added to the mixture at an internal temperature of 25°C or below, and the mixture was stirred at an internal temperature of 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 of 15°C or below. 5% aqueous sodium bicarbonate solution (27.3 kg) was slowly added, the mixture was 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% saline solution (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, and the mixture was concentrated to 55 L at an external temperature of 40°C. Then, toluene (15.6 kg) was added and the mixture was concentrated to 54 L at an external temperature of 40°C. This process was repeated twice. After confirming the precipitation of crystals, cyclohexane (14.0 kg) was added and the mixture was stirred overnight. The mixture was filtered, and the crystals were 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.

[0205] Synthesis of Compound 9-i: tert-butyl N-[(benzyloxy)carbonyl]-L-isoleucyl-L-prolinate

[0206] Toluene (583 g) and 5% aqueous sodium hydrogen sulfate solution (2066 g) were added to N-cyclohexylcyclohexanaminium (2S,3S)-2-{[(benzyloxy)carbonyl]amino}-3-methylpentanoate (135 g), and the mixture was stirred at room temperature for 10 minutes. The organic phase was then separated. The resulting organic phase was washed with 5% aqueous sodium hydrogen sulfate solution (2066 g) and 5% brine (1397 g), successively, 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 the mixture was 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. To this reaction mixture, 5% aqueous sodium hydrogen carbonate solution (993 g) and 1-methylimidazole (24 mL) were added at an internal temperature of 20°C or less, and the mixture was stirred at an external temperature of 20°C for approximately 30 minutes, after which the organic phase was separated. The resulting organic phase was washed successively with 10% aqueous sodium hydrogen sulfate solution (709 g), 10% aqueous sodium hydrogen sulfate solution (639 g), and 10% aqueous sodium hydrogen carbonate solution (710 g), and the solvent was distilled off under reduced pressure to obtain a solution containing compound 9-i (137 g). LCMS (ESI) of compound 9-i: retention time: 4.21 minutes, m / z = 419 [M+H] ＋ (Analysis conditions-M)

[0207] Synthesis of Compound 9-j: tert-butyl L-isoleucyl-L-prolinate

[0208] 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 MPaG) 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 minutes, m / z = 285 [M+H] ＋ (Analysis conditions-M)

[0209] Synthesis of Compound 9-k: tert-butyl N-[(benzyloxy)carbonyl]-N-methyl-L-norvalyl-L-isoleucyl-L-prolinate

[0210] 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. To this mixture, a 50% solution of propylphosphonic anhydride in MeTHF and methyltetrahydrofuran (277 mL) was added dropwise over approximately 40 minutes, and the resulting reaction mixture was stirred at an external temperature of 20°C for 1 hour. To this reaction mixture, a 5% aqueous solution of sodium bicarbonate (472 g) and 1-methylimidazole (18 mL) were added at an internal temperature of 30°C or lower, followed by stirring for approximately 30 minutes at an external temperature of 15°C, and then the organic phase was separated. The obtained organic phase was washed with 10% aqueous sodium hydrogen sulfate solution (338 g), 10% aqueous sodium hydrogen sulfate solution (338 g), and 10% aqueous sodium hydrogen carbonate solution (340 g) in this order at an external temperature of 20°C, and the solvent was distilled off under reduced pressure to obtain a solution containing compound 9-k (219 g). LCMS (ESI) of compound 9-k: retention time: 4.58 minutes, m / z=532 [M+H] ＋ (Analysis conditions-M)

[0211] Synthesis of Compound 9-l: tert-butyl N-methyl-L-norvalyl-L-isoleucyl-L-prolinate

[0212] 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 filtrate was combined and concentrated under reduced pressure to obtain a solution containing compound 9-l (109 g). The solvent was distilled off 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, then 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 conditions-M)

[0213] 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 above 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 seed crystals of the title compound 9-l. LCMS (ESI) of compound 9-l seed crystals: retention time: 2.60 minutes, m / z = 398 [M+H] ＋ (Analysis conditions-M)

[0214] Synthesis of Compound 9-m: (tert-butyl (3S)-3-[benzyloxycarbonyl(methyl)amino]-4-(dimethylamino)-4-oxo-butanoate)

[0215] (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 separated and washed twice with a 10% aqueous solution of sodium hydrogen sulfate monohydrate (150 g), and then washed with a 5% aqueous solution of sodium chloride (150 g). The resulting organic phase was concentrated under reduced pressure. MeTHF (95 g) was added to the resulting residue, and the operation of concentrating under reduced pressure was repeated twice. To the resulting residue (47.91 g), MeTHF (95 g), acetonitrile (75 g), DIPEA (35.46 g, 274 mmol), and dimethylamine hydrochloride (7.88 g, 96.6 mmol) were added at 25°C. A MeTHF solution of propylphosphonic anhydride (50.4 wt%, 61.33 g, 97.1 mmol) was added dropwise over 1 hour and 30 minutes. After the addition was completed, the mixture was stirred for 1 hour, then sampled and analyzed by HPLC to confirm completion of the reaction. 2 M aqueous sodium hydroxide solution (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 2 M aqueous sodium hydroxide solution (150 g), 13% aqueous sulfuric acid solution (150 g), 10% aqueous sodium hydrogen sulfate monohydrate solution (150 g), and 5% aqueous sodium carbonate solution (150 g), and then concentrated under reduced pressure. The procedure 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 conditions-K-2)

[0216] Synthesis of Compound 9-n: (tert-butyl (3S)-4-(dimethylamino)-3-(methylamino)-4-oxo-butanoate)

[0217] 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 the completion of the reaction was confirmed by HPLC analysis. 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 obtain 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)

[0218] Synthesis of Compound 9-o: (tert-butyl (3S)-3-[[(2S)-2-[benzyloxycarbonyl(methyl)amino]-2-cyclopentyl-acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate)

[0219] 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 minutes. After the addition was completed and the mixture was stirred for 3 hours, sampling was performed, and completion of the reaction was confirmed by HPLC analysis. Toluene (30 g), a 5% aqueous potassium carbonate solution (23 g), and 1-methylimidazole (3.97 g, 48.4 mmol) were added, and the mixture was stirred for 30 minutes. A 2.5% aqueous ammonia solution (88 g) and MeTHF (25 g) were 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 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 obtain a solution containing compound 9-o (52.78 g). LCMS (ESI) of compound 9-o: retention time: 4.10 minutes, m / z=526 [M+Na] ＋ (Analysis conditions-K-2)

[0220] Synthesis of Compound 9-p: (tert-butyl (3S)-3-[[(2S)-2-cyclopentyl-2-(methylamino)acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate)

[0221] 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 hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. A solution of compound 9-0 (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, the mixture was sampled and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with MeTHF (75 g). The mixed solution of the filtrate and washings was concentrated under reduced pressure, and then acetonitrile (75 g) was added and the mixture was concentrated under reduced pressure. This procedure was repeated 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)

[0222] Synthesis of Compound 9-q: (tert-butyl (3S)-3-[[(2S)-2-cyclopentyl-2-(methylamino)acetyl]-methyl-amino]-4-(dimethylamino)-4-oxo-butanoate) hydrochloride

[0223] 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, and then a solution of pyridine hydrochloride in acetonitrile (16.94 w / w%, 4.50 g) was added dropwise 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, and acetonitrile (14 g) was added. After stirring for 1 hour, the mixture was cooled to 10°C over 6 hours. After stirring for an additional 11 hours at 10°C, 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 conditions-K-1)

[0224] 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)

[0225] Acetonitrile (224 mL), DIPEA (150 g), compound 9-q (74.56 g), and 1-(benzyloxycarbonyl(methyl)amino)cyclobutanoic acid (CAS No. 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 the mixture was stirred for 2 hours. HPLC analysis confirmed that the reaction conversion was 99% or higher. A 5% aqueous potassium carbonate solution (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 discharged by liquid separation. The organic phase was washed with a 4% aqueous sulfuric acid solution (447 mL x 2) and a 5% aqueous sodium carbonate solution (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 minutes, m / z = 637.43 [M+Na] ＋ (Analysis conditions-K-1)

[0226] 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) A THF solution of compound 9-r (163.97 g), THF (665 mL), and 4.57 wt % Pd / C (14.7 g) were placed in a pressure-resistant reactor, and the external temperature was set to 25°C and stirred. The atmosphere was purged with nitrogen at 0.20 MPaG three times, then with hydrogen at 0.20 MPa three times, and the mixture was stirred for 1 hour under a hydrogen atmosphere at 0.20 MPaG. HPLC analysis confirmed that the reaction conversion was 99% or higher. The atmosphere in the reaction vessel 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 mixed 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 to obtain 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)

[0227] 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)

[0228] (2S,4R)-1-((benzyloxy)carbonyl)-4-ethoxypyrrolidine-2-carboxylic acid dicyclohexylamine salt (227 g) and MeTHF (767 mL) were added to a reaction vessel at room temperature and stirred. 4% aqueous sulfuric acid solution (1151 mL) was added, and the aqueous phase was removed by separation. The organic phase was washed with 4% aqueous sulfuric acid solution (1151 mL) and 5% aqueous sodium chloride solution (1151 mL). The resulting organic phase was concentrated under reduced pressure to 153 mL with an external temperature of 50°C. Acetonitrile (384 mL) was added to the resulting concentrate, and the mixture was concentrated under reduced pressure to 153 mL. Reaction vessel compound 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 reaction vessel was raised to 50°C and stirred for 5 hours. HPLC analysis confirmed that the reaction conversion was 99% or more. 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 mixture was concentrated under reduced pressure twice to 242 mL, yielding 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 conditions-K-2)

[0229] 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)

[0230] 4.57 wt% Pd / C (8.53 g) and THF (388.5 mL) were placed in a pressure-resistant reactor, and the external temperature was set to 25°C and stirred. The pressure was purged with 0.20 MPa nitrogen three times, then purged with 0.20 MPaG hydrogen three times, and stirred for 3 hours under a 0.40 MPa hydrogen atmosphere. A THF solution of compound 9-t (179.08 g) and THF (531 mL) were added. The pressure was purged with 0.20 MPaG nitrogen three times, then purged with 0.20 MPaG hydrogen three times, and stirred for 6 hours under a 0.20 MPaG hydrogen atmosphere. HPLC analysis confirmed that the reaction conversion was 99% or higher. 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 the mixture was concentrated under reduced pressure to 182 mL. MeTHF (55.2 mL) was added to the resulting concentrated solution, 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 reaction vessel. 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 further added over 4 hours and stirred for 1 hour. The external temperature was cooled to 10°C over 2.5 hours and stirred for 14 hours. The reaction mixture was filtered through a Kiriyama funnel, and the resulting crystals were washed twice with heptane (396 mL). The resulting crystals were dried for 2.5 hours under reduced pressure with 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 minutes, m / z = 622.53 [M+H] ＋ (Analysis conditions-K-1)

[0231] Synthesis of Compound 9-v: benzyl (S)-2-((tert-butoxycarbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate

[0232] 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 and 4-bromo-2-methoxy-1-(trifluoromethyl)benzene (24.57 kg), 1,3-dimethyl-2-imidazolidinone (230 kg), N-methylmorpholine (3.25 kg) were charged, and then the internal temperature was cooled to 8.7 ° C., and zinc (12.6 kg) was charged. After stirring and the temperature stabilized, chlorotrimethylsilane (21.0 kg) was added dropwise over 3 hours at an internal temperature in the range of 8.7 to 11.9 ° C. The reaction conversion rate was confirmed to be 99% or more by HPLC analysis. After the internal temperature was cooled 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 30 minutes or more 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 of disodium ethylenediaminetetraacetate (24.3 kg) and 2.5% aqueous ammonia (465 kg), followed by a 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 minutes, m / z=368.38 [M-Boc+H] ＋ (Analysis conditions-H)

[0233] Synthesis of Compound 9-w: (S)-2-((tert-butoxycarbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoic acid

[0234] A pressure-resistant reactor (150 L) was charged with a solution (43.7 kg) containing compound 9-v while washing it in with toluene (40.2 kg), and 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 then stirred for 2 hours and 2 minutes at an internal temperature of 22.6 to 29.2°C. HPLC analysis confirmed that the reaction conversion was 99% or higher. The mixture was filtered, and the Pd / C residue was washed with toluene (26.7 kg). After obtaining another filtrate using the same procedure, all the filtrates were combined and concentrated to 155 L at an external temperature of 40°C, yielding 155 kg of a solution containing compound 9-w. LCMS (ESI) of compound 9-w: retention time: 5.72 minutes, m / z = 278.37 [M-Boc + H] ＋ (Analysis conditions-H)

[0235] Synthesis of Compound 9-x: ((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate)

[0236] A solution (155 kg) containing compound 9-w was added to a reaction vessel (1000 L), and methanesulfonic acid (24.7 kg) was added dropwise over 32 minutes at an internal temperature range of 20.4°C to 27.6°C, after which the temperature was raised. HPLC analysis confirmed that the reaction conversion was 99% or higher. The reaction mixture was cooled, and water (186 kg) was added dropwise over 80 minutes at an internal temperature range 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 the pH was adjusted to 6.52 with 40% aqueous tripotassium phosphate solution (95.7 kg). Acetonitrile (61.2 kg) was then added, and the pH was again adjusted to 7.83 using 40% aqueous tripotassium phosphate solution (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 99% or more. The pH of the resulting reaction mixture was adjusted to 7.73 using 40% aqueous potassium phosphate solution (44.7 kg), and then methyl tert-butyl ether (46.3 kg) and heptane (42.4 kg) were added. It was confirmed that the mixture separated into three layers. 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) mixed solution 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) mixed solution. The obtained organic phase was washed with 0.2 M aqueous sodium hydroxide solution (62.1 kg), and then washed with a mixed solution of 24% aqueous sodium hydroxide solution (2.1 kg) - sodium chloride (3.1 kg) - water (72.3 kg). The obtained organic phase was washed with 1 M hydrochloric acid (310 kg). The obtained organic phase was washed with 10% brine (166 kg) and concentrated to 62 L, after which 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 obtained concentrated residue to obtain a solution (260 kg) containing compound 9-x. LCMS (ESI) of compound 9-x: retention time: 5.913 minutes, m / z = 368.39 [M-CO2 + H] ＋(Analysis conditions-H)

[0237] Synthesis of Compound 9-y: ((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoic acid dicyclohexylamine salt)

[0238] A solution (260 kg) containing compound 9-x was added to a reaction vessel (1000 L), 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 1 hour or more, and then the internal temperature was 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 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)

[0239] Synthesis of Compound 9-z: ((S)-2-(((benzyloxy)carbonyl)amino)-4-(3-methoxy-4-(trifluoromethyl)phenyl)butanoate)

[0240] 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 solution (253 mL) was added, and the aqueous phase was discharged by a separation operation. The organic phase was washed with 4% aqueous sulfuric acid solution (253 mL) and 5% aqueous sodium chloride solution (253 mL), respectively. The obtained organic phase was concentrated under reduced pressure to 100.8 mL with the external temperature set to 40°C. MeTHF (253 mL) was added to the obtained concentrate, and the mixture was concentrated under reduced pressure 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 minutes, m / z = 368.18 [M-CO2 + H] + (analysis condition -K-1).

[0241] 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)

[0242] 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, and the external temperature was cooled to 10°C and stirred. A 50 wt% solution of propylphosphonic anhydride in MeTHF (124 g) was added to the mixed solution over 15 minutes. The external temperature of the reaction vessel was raised to 25°C and stirred for 1 hour. HPLC analysis confirmed that the reaction conversion was 99% or higher. A 5% aqueous potassium carbonate solution (303 mL) and NMI (6.67 g) were added and stirred for 30 minutes. Stirring was stopped and the mixture was stored at room temperature overnight. The aqueous phase was discharged by separation, and the organic phase was washed with a 4% aqueous sulfuric acid solution (303 mL x 2). Heptane (182 mL), MTBE (121 mL), acetonitrile (116 mL), and 2.5% aqueous potassium carbonate solution (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 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 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 minutes, m / z = 1037.62 [M+H] ＋ (Analysis conditions-K-1)

[0243] 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

[0244]

[0245] A solution of compound 9-aa (47.8 g) in IPAC (53.1 g) and IPAC (186 mL) were added to a reaction vessel purged with nitrogen at room temperature and stirred. Subsequently, HMDS (19.0 g) was added, and the external temperature of the reaction vessel was set to 0°C and stirred for 15 minutes. TMSOTf (15.4 g) was added dropwise at an internal temperature of 20°C or less. The external temperature of the reaction vessel was set to 20°C, and the reaction mixture was stirred for 1 hour, then diluted with MeTHF (244 mL), and the external temperature of the reaction vessel was set to 10°C. After adding 5% aqueous potassium dihydrogen phosphate (478 mL) dropwise, stirring was stopped and the aqueous phase was discharged. The resulting organic phase was washed with 5% sodium dihydrogen phosphate (478 mL), and then diisopropylethylamine (9.0 mL) was added. The mixture was concentrated under reduced pressure to obtain a MeTHF solution (120.1 g) of compound 9-ab. LCMS (ESJ) of compound 9-ab: Retention time: 4.30 min, m / z = 981.66 [M+Na] ＋ (Analysis conditions-K-2)

[0246] 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

[0247] MeTHF (70 mL) and a MeTHF solution (113.85 g) of compound 9-ab (40.6 g) were added to a reaction vessel purged with nitrogen at room temperature and stirred. Subsequently, diisopropylethylamine (25.3 mL) and compound 9-l (21.9 g) dissolved in MeTHF (180 mL) were added, followed by the addition of acetonitrile (40 mL) and stirring. HATU (24.2 g) was suspended in acetonitrile (53 mL), added to the reaction vessel, and stirred for 1 hour. Acetic acid (1.2 mL) was added, stirred for 20 minutes, and then allowed to stand overnight. After resuming stirring, the mixture was diluted with MeTHF (122 mL), and 10% aqueous ammonia solution (282 mL) was added. After stopping the stirring and discharging the aqueous phase, the resulting organic phase was washed successively with 10% aqueous ammonia solution (280 mL), 4% diluted sulfuric acid (280 mL), 4% diluted sulfuric acid (280 mL), and 5% aqueous sodium carbonate solution (280 mL). The resulting organic phase was concentrated under reduced pressure to give a MeTHF solution (132.34 g) of compound 9-ac.

[0248] 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

[0249] MeTHF (1.0 mL) and 10% palladium on activated carbon (90.3 mg) were added to a reaction vessel purged with nitrogen at room temperature. The reaction vessel was purged with hydrogen and stirred for 1 hour under hydrogen pressure (0.40 MPaG). After venting the hydrogen and replacing with nitrogen, compound 9-ac (514 mg) dissolved in MeTHF (1.5 mL) was added and stirred. After venting the nitrogen, the mixture was replaced with hydrogen and stirred for a total of 8 hours and 30 minutes under hydrogen pressure (0.20 MPaG). After venting the hydrogen and replacing 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 obtain compound 9-ad (503 mg). LCMS (ESI) of compound 9-ad: retention time: 8.04 minutes, m / z = 1205.0 [M+H] ＋ (Analysis conditions - FC2)

[0250] 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

[0251] A MeTHF solution (17.6 kg) of compound 9-ad (3.41 kg) was added to the reaction vessel at room temperature, followed by the sequential addition of MeTHF (12.3 kg), compound 9-h monotoluene solvate (2.38 kg), and acetonitrile (6.20 kg). The external temperature was set to 5°C, and NMM (1.26 kg) and HATU (1.65 kg) were added at an internal temperature of 30°C or less, followed by stirring at an internal temperature of 25°C for 1 hour. After confirming that the reaction conversion rate was 99% or higher, a 5% aqueous potassium carbonate solution (23.9 kg) and 1-methylimidazole (234 g) were added, followed by stirring at room temperature for 30 minutes, and the aqueous phase was discharged by liquid-liquid separation. The obtained organic phase was washed with a 10% aqueous ammonia solution (23.0 kg), a 5% aqueous sodium hydrogen sulfate solution (23.9 kg), and a 5% aqueous sodium carbonate solution (23.9 kg), and then concentrated at an external temperature of 40° C. to 10 L. Acetonitrile (16.2 kg) was added to the concentrated solution, and concentration to 10 L was repeated twice to obtain an acetonitrile solution of compound 9-ae (19.4 kg).

[0252] 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

[0253] A solution of compound 9-ae (4.67 kg) in acetonitrile (14.9 kg) was added to the reaction vessel, and acetonitrile (4.53 kg) was added. The mixture was cooled to an internal temperature of 15°C or below. DBU (1.65 kg) was then added at an internal temperature of 30°C or below. After stirring at 25°C for 1 hour and confirming that the reaction conversion rate was 99% or higher, the mixture was cooled again to an internal temperature of 15°C or below. Triethylamine (1.02 kg), water (1.14 kg), and sodium hydrogen sulfite (925 g) were added sequentially so that the internal temperature did not exceed 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 stirred. The aqueous phase was then discharged 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. The aqueous phase was then discharged by liquid-liquid separation. This process was repeated twice. The organic phase was washed with 5% brine (23.4 kg) and separated into liquid and liquid, 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.

[0254] 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 A solution of compound 9-af (3.74 kg) in MeTHF (11.9 kg) was added to the reaction vessel, followed by additional MeTHF (7.78 kg) and HMDS (1.12 kg). After cooling to an internal temperature of 5°C or below, TMSOTf (1.03 kg) was added at an internal temperature of 30°C or below, and the mixture was heated to 25°C and stirred for 2 hours. After confirming that the reaction conversion was 99% or higher, MeTHF (25.6 kg) and acetonitrile (5.88 kg) were added, the internal temperature was cooled to 5°C or below, and 5% aqueous potassium hydrogen phosphate solution (26.2%) was added at an internal temperature of 30°C or below. The aqueous phase was discharged by liquid-liquid separation, and the organic phase was washed sequentially with 5% aqueous sodium hydrogen sulfate solution (26.2 kg), 5% aqueous sodium carbonate solution (26.2 kg), and 5% saline solution (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, followed by another concentration to 9.4 L. This process was repeated twice. Acetonitrile (8.82 kg) and heptane (25.6 kg) were added to the concentrated solution, and the mixture was stirred. The liquid was separated, and the lower layer was recovered, yielding an acetonitrile solution (17.7 kg) of compound 9-ag. Quantitative analysis by qNMR showed that the yield of compound 9-ag over the three steps was 93.4%.

[0255] Synthesis of Compound 9

[0256] HATU (949 g) and acetonitrile (25.6 kg) were added to a reaction vessel and dissolved to prepare a solution of HATU in acetonitrile. A solution of compound 9-ag in acetonitrile (5.52 kg), acetonitrile (19.1 kg), and N-methylmorpholine (504 g) were added to another vessel to prepare a solution of compound 9-ag in acetonitrile. The prepared 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 dropwise addition, the mixture was stirred for an additional hour. A reaction conversion of 99% or greater was confirmed by HPLC analysis. 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 solution (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 successively with 5% sulfuric acid (13.1 kg), 5% aqueous dipotassium hydrogen phosphate (13.1 kg), and 0.5% aqueous sodium chloride (13.07 kg). Acetonitrile (2.00 kg) was added to the resulting organic phase, which was then washed with 0.5% aqueous sodium chloride (13.1 kg). The resulting organic phase was subjected to solvent substitution with ethanol, and HPLC analysis (analysis conditions - cyc) using a standard sample revealed that 1.14 kg (88.3% yield) of Compound 9 was obtained. HPLC of Compound 9: Retention time: 18.67 (analysis conditions - cyc).

[0257] Example 1-1: Analysis of Compound-1 A sample solution of Compound 1 obtained in Reference Example 1 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the chromatogram obtained using analysis software was used for evaluation. Compound 1 was detected at an elution time of 2.5 minutes. The area values ​​of peaks eluting later than Compound 1 were integrated, and this was estimated to be impurities including oligomers.

[0258] 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 C-dimers and C-trimers derived from Compound 1 are shown below. In Example 2 and subsequent examples, C-dimer and C-trimer refer to the C-dimer and C-trimer of the target cyclic peptide appearing in that example.

[0259] C-dimer of Compound 1

[0260] C-trimer of Compound 1

[0261] The sample solution of Compound 1 obtained in Reference Example 1 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 1 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0262] [Example 1-2: Confirmation of Purifiable Reagents] Acetonitrile (3.96 mL) was added to a concentrated, dried product (0.1585 g) containing Compound 1 to prepare a reaction solution (3.2795 g). The reaction solution was sampled, and an HPLC sample was prepared using acetonitrile, followed by HPLC measurement (Analysis Condition-3). The reaction solution (414 mg, containing 20 mg of concentrated, dried product, the number of moles of compound 1 when the content of the concentrated, dried product is 100%) was transferred to a reaction vessel (Runs 1 to 3). CaCl ２ (Run 1, 8 mg, 72 μmol) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25° C. for 1 day. ２ , CaBr ２ Regarding CaCl ２The resulting solution was weighed out so that the molar ratio was the same as that of the CaCl solution, added to each reaction solution, and after confirming that the solution was suspended, the solution was stirred at 25°C for 1 day. Each suspension was centrifuged, and the filtrate was sampled and used to prepare an HPLC sample using acetonitrile, followed by HPLC analysis (analysis condition 3). The results are shown in Table 7. ２ showed the best results in terms of purity and recovery rate.

[0263] [Example 1-3: Confirmation of Solvent Range for Purification] <Basic Procedure> A concentrated dry solid containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in various solvents shown in the table below. The reaction solution was sampled, and an HPLC sample was prepared using acetonitrile, followed by HPLC measurement (Analysis Condition-3). CaCl 40 weight % relative to the concentrated dry solid was added. ２ (10 mg) was added to each reaction solution, and after confirming that the suspension was formed, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and an HPLC sample was prepared using acetonitrile, followed by measurement by HPLC (analysis condition-3). The purity (area%), recovery rate (%), and ratio (%) of impurities including oligomers to Compound 1 were calculated, and the results are shown in Table 9.

[0264]

[0265] Run 1 in Table 9: Acetonitrile A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetonitrile (0.25 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and similarly analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 98.59 area%, and the recovery rate was 56%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0266] Run 2 in Table 9: Ethylene glycol dimethyl ether A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in ethylene glycol dimethyl ether (0.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 96.70 area%, and the recovery rate was 36%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0267] Run 3 in Table 9: Ethylene glycol dimethyl ether A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in ethylene glycol dimethyl ether (1.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-2). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 89.42 area%, and the recovery rate was 76%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0268] Run 4 in Table 9: Tetrahydrofuran A concentrated dry solid containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in tetrahydrofuran (0.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 95.52 area%, and the recovery rate was 83%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0269] Run 5 in Table 9: Acetone A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetone (0.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 100 area%, and the recovery rate was 35%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0270] Run 6 in Table 9: Acetone A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetone (1.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 99.72 area%, and the recovery rate was 66%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0271] Run 7 in Table 9: Methyl ethyl ketone A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in methyl ethyl ketone (0.250 mL) to prepare a reaction solution. A sample of the reaction solution was taken and subjected to HPLC analysis (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 100 area%, and the recovery rate was 3%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0272] [Examples 1-4: Purifiable CaCl ２Confirmation of the amount of addition and the range of solvent amount]

[0273] Runs 1 and 2 in Table 10: A concentrated dry product containing Compound 1 (90 mg) was added to a container and dissolved in acetonitrile (0.900 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC analysis (analysis condition-3). The reaction solution (223 mg, containing 25 mg of concentrated dry product) was transferred to each reaction container. CaCl ２ (Run 1 2.5 mg, 10 weight%, Run 2 5 mg, 20 weight%) was added to each reaction solution, and after confirming that the suspension was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 74.40 area% in Run 1 and 82.59 area% in Run 2, and the recovery rate was 99% in Run 1 and 94% in Run 2. It was confirmed that these operations reduced the amount of impurities including oligomers, and Compound 1 in the filtrate was purified.

[0274] Run 3 in Table 10: Same as Run 1 in Example 1-2.

[0275] Run 4 in Table 10: A concentrated dry product containing Compound 1 (40 mg) was added to a vessel and dissolved in acetonitrile (0.400 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC analysis (analysis condition-3). The reaction solution (265 mg, containing 30 mg of concentrated dry product) was transferred to a reaction vessel. CaCl ２ (15 mg, 50 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and similarly analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 100 area%, and the recovery rate was 49%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0276] Run 5 in Table 10: A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetonitrile (0.625 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２(10 mg, 40 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and similarly subjected to HPLC analysis (analysis condition-3). The purity of Compound 1 in the filtrate was 97.94 area%, and the recovery rate was 75%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0277] Run 6 in Table 10: A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetonitrile (1.25 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２ (10 mg) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of Compound 1 in the filtrate was 94.96 area%, and the recovery rate was 90%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0278] Run 7 in Table 10: A concentrated dry solid containing Compound 1 (12.5 mg) was added to a reaction vessel and dissolved in acetonitrile (1.25 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２ (12.5 mg, 100 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of Compound 1 in the filtrate was 89.21 area%, and the recovery rate was 87%. It was confirmed that these operations reduced the amount of impurities including oligomers, and Compound 1 in the filtrate was purified.

[0279] [Example 1-5: Confirmation of stirring time allowing purification] A concentrated dry product containing Compound 1 (25 mg) was added to a reaction vessel and dissolved in acetonitrile (0.250 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２(10 mg, 40 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 7 days. The suspension was centrifuged, and the filtrate was sampled and similarly analyzed by HPLC (analysis condition-3). The purity of Compound 1 in the filtrate was 99.81 area%, and the recovery rate was 52%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 1 in the filtrate was purified.

[0280] [Example 1-6: Scale-up experiment] A concentrated dry product containing Compound 1 (0.1299 g) was added to a vessel and dissolved in acetonitrile (3.25 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). The reaction solution (2.08 g, containing 0.1000 g of concentrated dry product) was transferred to a reaction vessel, and CaCl ２ (40 mg, 40 weight%) was added, and after confirming suspension, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the collected solid was washed three times with acetonitrile (0.5 mL) and centrifuged. The collected solid was further washed three times with acetonitrile (1 mL) and twice with acetonitrile (1.5 mL), and centrifuged. The obtained filtrate was collected and concentrated to dryness. Acetonitrile (1 mL), MTBE (0.5 mL), and 0.5% aqueous NaCl solution (1 mL) were added to the concentrated dryness, and the organic phase was washed. After removing the aqueous phase, the organic phase was concentrated to dryness. Acetonitrile was added to the concentrated dryness, and insoluble matter was removed by filtration. The filtrate was concentrated to dryness and then dried under reduced pressure to obtain a concentrated dryness of Compound 1. The entire amount of the obtained concentrated dryness was dissolved in 20 mL of acetonitrile. A sample of the acetonitrile solution was taken and subjected to HPLC analysis (analysis condition-3). The obtained Compound 1 had a purity of 99.43% and a recovery rate of 78%. ２ It was confirmed that the amount of impurities including oligomers was reduced by a series of operations including the treatment, and Compound 1 was purified. In addition, as a result of ion chromatography analysis (analysis condition-5), the amount of Ca contained in the obtained Compound 1 was ２＋ was 0.05 eq. relative to Compound 1, and residual calcium was also sufficiently suppressed.

[0281] Example 2-1: Analysis of Compound 2 A sample solution of Compound 2 obtained in Reference Example 2 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the obtained chromatogram using analysis software was used for evaluation. Compound 2 was detected at an elution time of 2.3 minutes. The area values ​​of peaks with elution times later than Compound 2 were integrated, and impurities other than Compound 2, including oligomers, were estimated.

[0282] The sample solution of Compound 2 obtained in Reference Example 2 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 2 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0283] [Example 2-2: Example using a cyclic peptide with different amino acid residues in the cyclic portion] A concentrated dry product containing Compound 2 (0.090 g) was added to a vessel and dissolved in acetonitrile (0.900 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). The reaction solution (0.221 g, equivalent to 25 mg of the concentrated dry product) was transferred to a reaction vessel and diluted with CaCl ２ (10 mg, 40 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of Compound 2 in the filtrate was 85.70%, and the recovery rate was 76%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that Compound 2 in the filtrate was purified.

[0284] [Example 2-2: Scale-up experiment] A concentrated dry product containing Compound 2 (0.1565 g) was added to a vessel and dissolved in acetonitrile (1.57 mL) to prepare a reaction solution. 20 μL of the reaction solution was diluted to 2 mL with acetonitrile to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). The reaction solution (1.07 g, containing 0.12 g of concentrated dry product) was transferred to a reaction vessel, and CaCl ２ (48 mg, 40 weight%) was added, and after confirming suspension, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the collected solid was washed three times with acetonitrile (0.6 mL) and centrifuged. The collected solid was further washed three times with acetonitrile (1.2 mL) and four times with acetonitrile (1.8 mL), and centrifuged. The obtained filtrate was collected and concentrated to dryness. Acetonitrile (1.2 mL), MTBE (0.6 mL), and 0.5% aqueous NaCl solution (1.2 mL) were added to the concentrated dryness, and the organic phase was washed. After removing the aqueous phase, the organic phase was concentrated to dryness. Acetonitrile was added to the concentrated dryness, and insoluble matter was removed by filtration. The filtrate was concentrated to dryness and then dried under reduced pressure to obtain a concentrated dryness of compound 2. The entire amount of the obtained concentrated dryness was dissolved in 20 mL of acetonitrile. The acetonitrile solution was sampled and subjected to HPLC analysis (analysis condition 3). The obtained compound 2 had a purity of 89.45% and a recovery rate of 73%. ２ It was confirmed that the amount of impurities including oligomers was reduced by a series of operations including the treatment, and Compound 2 was purified. ２＋ was 0.05 eq. relative to Compound 2, and residual calcium was also sufficiently suppressed.

[0285] Example 2-1: Analysis of Compound-1 A sample solution of Compound 3 obtained in Reference Example 3-1 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the obtained chromatogram using analysis software was used for evaluation. Compound 3 was detected at an elution time of 2.2 minutes. The area values ​​of peaks with elution times later than Compound 3 were integrated, and these were estimated to be impurities other than Compound 3, including oligomers.

[0286] The sample solution of Compound 3 obtained in Reference Example 3-1 was analyzed under Analysis Condition-2. Referring to the UV chromatogram results under Analysis Condition-3, the structures of Compound 3 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0287] [Example 3-2: Example using cyclic peptides with different numbers of amino acid residues in the cyclic portion] A reaction vessel was charged with the concentrated dry product (20 mg) containing Compound 3 synthesized in Reference Example 3-1, and the concentrate was dissolved in acetonitrile (0.200 mL) to prepare a reaction solution. 10 μL of the reaction solution was diluted with acetonitrile (0.490 mL) to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). CaCl ２ (5 mg, 25 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and 10 µL of the filtrate was sampled and diluted with acetonitrile (0.490 mL) in the same manner as above to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). The purity of compound 3 in the filtrate was 49.81 area%, and the recovery rate was 67%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that compound 3 in the filtrate was purified.

[0288] [Example 3-3: Example using cyclic peptides with different numbers of amino acid residues in the cyclic portion] A concentrated dry product (25 mg) containing compound 3 synthesized in Reference Example 3-2 was added to a reaction vessel and dissolved in acetonitrile (0.250 mL) to prepare a reaction solution. 10 μL of the reaction solution was diluted with acetonitrile (0.490 mL) to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). CaCl ２(5 mg, 20 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and 20 µL of the filtrate was sampled and diluted with acetonitrile (0.990 mL) in the same manner as above to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). The purity of compound 3 in the filtrate was 90.52%, and the recovery rate was 73%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that compound 3 in the filtrate was purified.

[0289] Example 3-4: Confirmation of stirring time for purification A concentrated dry product (20 mg) containing Compound 3 synthesized in Reference Example 3-2 was added to a reaction vessel and dissolved in acetonitrile (1.00 mL) to prepare a reaction solution. 50 μL of the reaction solution was diluted with acetonitrile (0.450 mL) to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). CaCl ２ (6 mg, 30 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 50°C for 1 day. The suspension was centrifuged, and 50 µL of the filtrate was sampled and diluted with acetonitrile (0.450 mL) in the same manner as above to prepare an HPLC sample, which was then subjected to HPLC measurement (analysis condition-3). The purity of compound 3 in the filtrate was 89.05 area%, and the recovery rate was 80%. These operations reduced the amount of impurities, including oligomers, and it was confirmed that compound 3 in the filtrate was purified.

[0290] Example 4-1: Analysis of Compound 4 A sample solution of Compound 4 obtained in Reference Example 4 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the obtained chromatogram using analysis software was used for evaluation. Compound 4 was detected at an elution time of 2.7 minutes. The area values ​​of peaks with elution times later than Compound 4 were integrated, and these were estimated to be impurities other than Compound 4, including oligomers.

[0291] The sample solution of Compound 4 obtained in Reference Example 4 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 2 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0292] [Example 4-2: Purification of Compound 4] A concentrated dry product containing Compound 4 (40 mg) was added to a vessel and dissolved in acetonitrile (0.400 mL) to prepare a reaction solution. The reaction solution was sampled, and an HPLC sample was prepared using acetonitrile, followed by HPLC measurement (analysis condition-3). The reaction solution (263 mg, containing 30 mg of concentrated dry product) was transferred to a reaction vessel. CaCl ２ (5 mg, 16.7 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of compound 4 in the filtrate was 75.62 area%, and the recovery rate was 60%. These operations reduced the amount of impurities including oligomers, and it was confirmed that compound 4 in the filtrate was purified.

[0293] Example 5-1: Analysis of Compound 5 A sample solution of Compound 5 obtained in Reference Example 5 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the chromatogram using analysis software was used for evaluation. Compound 5 was detected at an elution time of 3.0 minutes. The area values ​​of peaks with elution times later than Compound 5 were integrated, and these were estimated to be impurities other than Compound 4, including oligomers.

[0294] The sample solution of Compound 5 obtained in Reference Example 5 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 5 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0295] [Example 5-2: Purification of Compound 5] A concentrated dry product containing Compound 5 (40 mg) was placed in a vessel and dissolved in acetonitrile (0.400 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). The reaction solution (263 mg, containing 30 mg of concentrated dry product) was transferred to a reaction vessel. CaCl ２ (5 mg, 16.7 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of compound 5 in the filtrate was 49.87% area%, and the recovery rate was 89%. These operations reduced the amount of impurities including oligomers, and it was confirmed that compound 5 in the filtrate was purified.

[0296] Example 6-1: Analysis of Compound 6 A sample solution of Compound 6 obtained in Reference Example 6 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the chromatogram obtained using analysis software was used for evaluation. Compound 6 was detected at an elution time of 2.5 minutes. The area values ​​of peaks with elution times later than Compound 6 were integrated, and impurities other than Compound 6, including oligomers, were estimated.

[0297] The sample solution of Compound 6 obtained in Reference Example 6 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structure of Compound 6 and its oligomers was confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0298] [Example 6-2: Purification of Compound 6] A concentrated dry solid containing Compound 6 (13 mg) was placed in a container and dissolved in acetonitrile (1.300 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２ (1.3 mg, 10 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC measurement in the same manner (analysis condition-3). The purity of Compound 6 in the filtrate was 83.38 area%, and the recovery rate was 76%. It was confirmed that these operations reduced the amount of impurities including oligomers, and Compound 6 in the filtrate was purified.

[0299] Example 7-1: Analysis of Compound 7 A sample solution of Compound 7 obtained in Reference Example 7 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the obtained chromatogram using analysis software was used for evaluation. Compound 7 was detected at an elution time of 2.8 minutes. The area values ​​of peaks with elution times later than Compound 7 were integrated, and impurities other than Compound 7, including oligomers, were estimated.

[0300] The sample solution of Compound 7 obtained in Reference Example 7 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 2 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range of 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0301] [Example 7-2: Purification of Compound 7] A concentrated dry solid containing Compound 7 (30 mg) was placed in a container and dissolved in acetonitrile (0.300 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２ (6 mg, 20 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-3). The purity of compound 7 in the filtrate was 79.83 area%, and the recovery rate was 75%. These operations reduced the amount of impurities including oligomers, and it was confirmed that compound 7 in the filtrate was purified.

[0302] Example 8-1: Analysis of Compound 8 A sample solution of Compound 4 obtained in Reference Example 8 was prepared using acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-3. The data obtained after subtracting a chromatogram of a blank solution from the obtained chromatogram using analysis software was used for evaluation. Compound 8 was detected at an elution time of 2.8 minutes. The area values ​​of peaks with elution times later than Compound 8 were integrated, and impurities other than Compound 8, including oligomers, were estimated.

[0303] The sample solution of Compound 8 obtained in Reference Example 8 was analyzed under Analysis Condition-2. With reference to the UV chromatogram results under Analysis Condition-3, the structures of Compound 2 and its oligomers were confirmed by ESI-MS in mass spectrometry for the elution time range from 2 to 8 minutes, in which elution was observed. The names of the estimated compounds, MS elution times (minutes), ESI-MS observed values ​​(ESI (m / z)), and estimated ion valences from the analysis results are shown in the table below.

[0304] [Example 8-2: Purification of Compound 8] A concentrated dry solid containing Compound 8 (25 mg) was placed in a container and dissolved in acetonitrile (1.250 mL) to prepare a reaction solution. The reaction solution was sampled and subjected to HPLC measurement (analysis condition-3). CaCl ２ (5 mg, 20 weight%) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 1 day. The suspension was centrifuged, and the filtrate was sampled and similarly measured by HPLC (analysis condition-3). The purity of compound 8 in the filtrate was 47.49 area%, and the recovery rate was 72%. These operations reduced the amount of impurities including oligomers, and it was confirmed that compound 8 in the filtrate was purified.

[0305] Example 9-1: Analysis of Compound 9 A sample solution of Compound 9 obtained in Reference Example 9 was prepared using tetrahydrofuran to a concentration of approximately 1 mg / mL, and analyzed under Analysis Condition-4. Compound 9 was detected at an elution time of 6.0 minutes. The peak eluting earlier than Compound 9 was presumed to be an oligomer.

[0306] A sample solution was prepared by diluting Compound 9 synthesized in Reference Example 9 with acetonitrile to a concentration of approximately 1 mg / mL, and analyzed under analysis condition-2. 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 valence of the analytical results are shown in Table 29.

[0307] Example 9-2: Purification of Compound 9 2.41 g of an EtOH solution containing Compound 9 synthesized in Reference Example 9 (containing 0.1 g of Compound 9) was concentrated under reduced pressure. After azeotropy with acetonitrile and vacuum drying, 1 mL of acetonitrile was added to form a solution. 0.30 g of the resulting acetonitrile solution (containing 30 mg of Compound 9) was added to the reaction solution. 10 μL of the reaction solution was diluted with THF (0.990 mL) to prepare a sample for HPLC, which was then subjected to HPLC measurement (analysis condition-4). CaCl ２ (13 mg, 6 eq) was added to the reaction solution, and after confirming that it was suspended, the mixture was stirred at 25°C for 4 days. The suspension was subjected to centrifugal filtration, and the filtrate was sampled and subjected to HPLC analysis in the same manner (analysis condition-4). The purity of compound 9 in the filtrate was 98.85 area%, and the recovery rate was 97%. These operations reduced the amount of impurities including oligomers, and it was confirmed that compound 9 in the filtrate was purified.

Claims

1. A method for purifying a cyclic peptide, comprising a step of separating the cyclic peptide to be purified or the peptide as an impurity from a mixture containing the cyclic peptide to be purified and an impurity peptide as a complex with a divalent metal ion or a metal salt containing a divalent metal ion.

2. (1) The purification method according to claim 1, which includes a step of mixing a mixture containing the cyclic peptide as the target of purification and the peptide as an impurity with the divalent metal ion or a metal salt containing a divalent metal ion, as a pre-step of the separation step.

3. The purification method according to claim 1 or 2, wherein step (1) is carried out in a first solvent.

4. A method for purifying a cyclic peptide, comprising the step of: (1) mixing, in a first solvent, a mixture containing a cyclic peptide to be purified and peptide impurities, with a divalent metal ion or a metal salt containing a divalent metal ion.

5. The purification method according to claim 4, wherein in step (1)', a complex is formed in the first solvent between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion.

6. (2) A purification method according to claim 5, further comprising a step of separating the complex from a mixture containing the cyclic peptide to be purified and peptide impurities.

7. (3) A purification method according to any one of claims 3 to 6, further comprising a step of removing the divalent metal ion or a metal salt containing a divalent metal ion from a mixture of the cyclic peptide, which is the object of purification, and the first solvent.

8. A purification method according to any one of claims 1 to 3, 5 and 6, wherein the complex is an adsorption complex.

9. A purification method according to any one of claims 1 to 8, wherein the cyclic peptide to be purified has 7 to 20 amino acid residues.

10. A purification method according to any one of claims 1 to 9, wherein the impurity peptide is a cyclic peptide different from the cyclic peptide to be purified.

11. The purification method according to any one of claims 1 to 10, wherein the metal salt is at least one selected from the group consisting of chloride salts, fluoride salts, bromide salts, iodide salts, perchlorate salts, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, and sulfate salts.

12. The purification method according to any one of claims 3 to 6, wherein the first solvent is a solvent capable of forming a complex between the cyclic peptide to be purified or the peptide as an impurity and the divalent metal ion or a metal salt containing a divalent metal ion.

13. A method for producing a composition containing a cyclic peptide, comprising the purification method according to any one of claims 1 to 12.

14. (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 ]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide, or a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer of Compound 9, or a pharmaceutically acceptable salt thereof.

15. (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 ]pentatetracont-42-ene-23,1'-cyclobutane]-17-carboxamide or a pharmaceutically acceptable salt thereof, together with a multimer of said compound 9, wherein the content of said multimer of compound 9 is 3.0% or less relative to compound 9 or the pharmaceutically acceptable salt thereof.

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