Method for purifying and producing cyclic peptides

Abstract

The invention discloses a purification method of cyclic peptide. The purification method comprises the following separation step (i) or (ii). (i) A cyclic peptide as a purification target substance is separated from a mixture containing the cyclic peptide as the purification target substance in the form of a complex with a metal atom or a metal ion. And (ii) separating the cyclic peptide as a purification target substance or a peptide as an impurity from a mixture containing the cyclic peptide as the purification target substance and the peptide as the impurity in the form of a complex with a metal atom or a metal ion. With respect to this purification method, the metal atoms and the metal ions are at least one atom and ion selected from the group consisting of atoms and ions of alkali metals, atoms and ions of alkaline earth metals, atoms and ions of transition metals, atoms and ions of lean metals, and atoms and ions of rare earth metals.

Description

Technical Field

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

[0002] Conventionally, purification by column chromatography (Non-Patent Document 1) is known as a method for purifying cyclic peptides. On the other hand, Patent Document 1 describes that "isolating and purifying the resulting cyclic peptide compound without separating and purifying the intermediate by crystallization can provide crystals of the cyclic peptide compound without using column chromatography."

[0003] [Citation List]

[0004] [Patent Document]

[0005] [Patent Document 1] International Publication No. WO 2022 / 234864

[0006] [Non-Patent Document]

[0007] [Non-Patent Document 1] 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 Summary of the Invention

[0008] [Technical Problem]

[0009] However, purification by column chromatography is often inefficient, especially for peptide synthesis on an industrial scale. In addition, many cyclic peptides have physical properties that make crystallization difficult, and there are some cyclic peptides that are difficult to purify by crystallization.

[0010] An object of the present invention is to provide a method for easily purifying and producing a cyclic peptide, wherein the method can be applied to cyclic peptides that are difficult to crystallize.

[0011] [Solution to the Problem]

[0012] The present invention relates to aspects of the following inventions [1] to

[150] , for example. [1]

[0014] A method for purifying a cyclic peptide,

[0015] The purification method includes the following separation step (i) or (ii):

[0016] (i) Separating a cyclic peptide as a purification target in the form of a complex with a metal atom or metal ion from a mixture containing the cyclic peptide as the purification target, or

[0017] (ii) Separating a cyclic peptide as a purification target or a peptide as an impurity in the form of a complex with a metal atom or metal ion from a mixture containing the cyclic peptide as the purification target and the peptide as the impurity, wherein

[0018] the metal atom is at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom, and

[0019] the metal ion is at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion, and a rare earth metal ion. [2]

[0021] The purification method according to [1], the purification method comprising step (1): mixing a mixture containing the cyclic peptide as the purification target or a mixture containing the cyclic peptide as the purification target and the peptide as the impurity with the metal atom or the metal ion, as a preliminary step to the separation step. [3]

[0023] The purification method according to [2], wherein the step (1) is carried out in a first solvent. [4]

[0025] A method for purifying a cyclic peptide, the purification method comprising

[0026] step (1)': mixing a mixture containing the cyclic peptide as the purification target or a mixture containing the cyclic peptide as the purification target and the peptide as the impurity with a metal atom or a metal ion in a first solvent, wherein

[0027] the metal atom is at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom, and

[0028] the metal ion is at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion, and a rare earth metal ion. [5]

[0030] The purification method according to [4], wherein in the step (1)', a complex of the cyclic peptide as the purification target or the peptide as the impurity with the metal atom or the metal ion is formed in the first solvent. [6]

[0032] The purification method according to any one of [3] to [5], further comprising step (2): mixing the mixture obtained in step (1) or step (1)' with a second solvent, wherein

[0033] the first solvent and the second solvent are different solvents from each other. [7]

[0035] The purification method according to [6], comprising step (2)': removing at least a portion of the first solvent as a pre-step to step (2). [8]

[0037] The purification method according to any one of [2] to [7], wherein in step (1) or step (1)', the metal atom or metal ion is 0.2 to 12 molar equivalents relative to the cyclic peptide to be purified. [9]

[0039] The purification method according to any one of [2] to [8], wherein in step (1) or step (1)', the metal atom or metal ion is 0.5 to 4 molar equivalents relative to the cyclic peptide to be purified.

[10]

[0041] The purification method according to any one of [x] to [9], wherein in step (1) or step (1)', the metal atom or metal ion is 0.8 to 1.2 molar equivalents relative to the cyclic peptide to be purified.

[0042] [10-1]

[0043] The purification method according to any one of [2] to [9], wherein in step (1) or step (1)', the cyclic peptide to be purified is 0.1 mg / mL to 1000 mg / mL relative to the first solvent.

[0044] [10-2]

[0045] The purification method according to any one of [2] to [9], wherein in step (1) or step (1)', the cyclic peptide to be purified is 1.0 mg / mL to 500 mg / mL relative to the first solvent.

[0046] [10-3]

[0047] The purification method according to any one of [2] to [9], wherein in step (1) or step (1)', the cyclic peptide to be purified is 2.7 mg / mL to 150 mg / mL relative to the first solvent. It should be noted that in the original text, there is an unclear "x" in the description of "[x] to [9]" in step (23). This may be an error in the original text. The above translation is based on the existing content as accurately as possible.

[11]

[0049] The purification method according to any one of [6] to

[10] , wherein, in step (2), the mixture obtained in step (1) or step (1)' is mixed with the second solvent at a liquid temperature of -20°C to 100°C.

[12]

[0051] The purification method according to any one of [6] to

[11] , wherein, in step (2), the mixture obtained in step (1) or step (1)' is mixed with the second solvent at a liquid temperature of 20°C to 30°C.

[13]

[0053] The purification method according to any one of [6] to

[12] , wherein, in step (2), the mixture obtained in step (1) or step (1)' is mixed with the second solvent for 0.5 to 72 hours.

[14]

[0055] The purification method according to any one of [6] to

[13] , wherein, in step (2), the mixture obtained in step (1) or step (1)' is mixed with the second solvent for 1 to 2 hours.

[15]

[0057] The purification method according to any one of [6] to

[14] , wherein, in step (2), the concentration of the complex in the second solvent is 0.1 mg / mL to 200 mg / mL.

[0058] [15-1]

[0059] The purification method according to any one of [6] to

[14] , wherein, in step (2), the concentration of the complex in the second solvent is 0.5 mg / mL to 50 mg / mL.

[16]

[0061] The purification method according to any one of [6] to

[15] , wherein, in step (2), the concentration of the complex in the second solvent is 1.0 mg / mL to 30 mg / mL.

[17]

[0063] The purification method according to any one of [6] to

[16] , the purification method further comprising step (3): separating the complex from the mixed solution of the mixture obtained in step (1) or step (1)' and the second solvent.

[18]

[0065] A purification method for a cyclic peptide, the purification method comprising

[0066] Step (4): Mix the complex of the cyclic peptide as the purification target or the peptide as the impurity with a metal atom or metal ion with a second solvent, where

[0067] the metal atom is at least one selected from the group consisting of: alkali metal atoms, alkaline earth metal atoms, transition metal atoms, poor metal atoms, and rare earth metal atoms, and

[0068] the metal ion is at least one selected from the group consisting of: alkali metal ions, alkaline earth metal ions, transition metal ions, poor metal ions, and rare earth metal ions.

[19]

[0070] According to the purification method described in

[18] , the purification method further includes step (5): separating the complex from the mixed solution of the complex and the second solvent.

[20]

[0072] According to the purification method described in [1], [2], [3],

[17] , or

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

[0073] [20-1]

[0074] According to the purification method described in [1], [2], [3],

[17] , or

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

[0075] [20-2]

[0076] According to the purification method described in [1], [2], [3],

[17] , or

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

[21]

[0078] According to the purification method described in

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

[22]

[0080] According to the purification method described in [1], [2], [3],

[17] ,

[19] ,

[20] , or

[21] , the purification method further includes step (6): removing the metal atom or the metal ion from the separated complex.

[23]

[0082] According to the purification method described in

[22] , wherein step (6) includes the following steps: mixing the separated complex, a third solvent, and water and separating them into an aqueous phase and an organic phase to remove the metal atom or the metal ion present in the aqueous phase.

[24]

[0084] The purification method according to

[23] , wherein step (6) further comprises the step of removing the third solvent from the organic phase and obtaining the cyclic peptide as the purification target.

[25]

[0086] The purification method according to

[24] , wherein removing the third solvent from the organic phase is carried out by distillation under reduced pressure.

[26]

[0088] The purification method according to

[22] , wherein step (6) further comprises the following steps: mixing the separated complex, a fourth solvent, and a ligand or a compound that generates a ligand or an anion or a compound that generates an anion, and forming a first complex or a first metal salt between the metal atom or the metal ion and the ligand or the anion.

[27]

[0090] The method according to

[26] , wherein step (6) further comprises the step of removing the first complex or the first metal salt.

[28]

[0092] The purification method according to any one of [1] to [4] and [6] to

[27] , the purification method further comprising step (7): confirming that the cyclic peptide as the purification target or the peptide as an impurity forms the complex with the metal atom or the metal ion.

[29]

[0094] The purification method according to

[28] , wherein step (7) is carried out by: comparing the NMR peaks of the cyclic peptide as the purification target or the peptide as an impurity with the NMR peaks of a mixture obtained by mixing the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion.

[30]

[0096] The purification method according to any one of [1] to [4] and [6] to

[29] , wherein the complex is a complex of the cyclic peptide as the purification target and the metal atom or the metal ion. <{

[31]

[0098] The purification method according to any one of [1] to [4] and [6] to

[29] , wherein the complex is a complex of the peptide as an impurity and the metal atom or the metal ion.

[32]

[0100] The purification method according to any one of [1] to [4] and [6] to

[31] , wherein the complex is a complex obtained by contacting the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion.

[33]

[0102] The purification method according to any one of claims [1] to [4] and [6] to

[32] , wherein the complex is a coordination complex.

[34]

[0104] The purification method according to

[33] , wherein the coordination complex includes a solid, crystal, liquid or amorphous.

[35]

[0106] The purification method according to

[33] or

[34] , wherein the coordination complex includes a crystal.

[36]

[0108] The purification method according to

[33] or

[34] , wherein the coordination complex includes an amorphous.

[37]

[0110] The purification method according to any one of [1] to

[17] , wherein the mixture containing the cyclic peptide as the purification target or the mixture containing the cyclic peptide as the purification target and the peptide as the impurity is a crude product obtained by producing the cyclic peptide as the purification target.

[38]

[0112] The method according to any one of [1] to

[37] , wherein the number of amino acid residues of the cyclic peptide as the purification target is 5 to 20.

[39]

[0114] The method according to any one of [1] to

[38] , wherein the number of amino acid residues of the cyclic peptide as the purification target is 10 to 14.

[40]

[0116] The purification method according to any one of [1] to

[39] , wherein the number of amino acid residues of the cyclic part of the cyclic peptide as the purification target is 5 to 15.

[41]

[0118] The purification method according to any one of [1] to

[40] , wherein the number of amino acid residues of the cyclic part of the cyclic peptide as the purification target is 8 to 14.

[42]

[0120] The purification method according to any one of [1] to

[41] , wherein the number of amino acid residues of the cyclic part of the cyclic peptide as the purification target is 8, 11, 13 or 14.

[43]

[0122] The purification method according to any one of [1] to

[42] , wherein the number of amino acid residues in the cyclic moiety of the cyclic peptide as the purification target is 11.

[44]

[0124] The purification method according to any one of [1] to

[43] , wherein the cyclic peptide as the purification target contains unnatural amino acids.

[0125] [44-1]

[0126] The purification method according to any one of [1] to

[43] , wherein the cyclic peptide as the purification target contains N-substituted amino acids.

[45]

[0128] The purification method according to any one of [1] to

[44] , wherein the cyclic peptide as the purification target contains three or more N-substituted amino acids.

[46]

[0130] The purification method according to any one of [1] to

[45] , wherein the cyclic peptide as the purification target contains five or more N-substituted amino acids.

[47]

[0132] The purification method according to any one of [1] to

[46] , wherein the cyclic peptide as the purification target contains six or more N-substituted amino acids.

[48]

[0134] The purification method according to any one of [1] to

[47] , wherein the number of N-substituted amino acid residues in the cyclic peptide as the purification target is 45% or more relative to the number of amino acid residues in the cyclic moiety of the cyclic peptide.

[49]

[0136] The purification method according to any one of

[44] to

[48] , 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.

[50]

[0138] The purification method according to any one of

[44] to

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

[51]

[0140] The purification method according to any one of [1] to

[50] , wherein the cyclic peptide as the purification target contains N-unsubstituted amino acids.

[52]

[0142] The purification method according to

[51] , wherein the N-unsubstituted amino acid is a non-natural amino acid.

[53]

[0144] The purification method according to

[50] or

[51] , wherein the ratio of the number of N-unsubstituted amino acids in the cyclic peptide as a purification target to the total number of amino acid residues in the cyclic peptide as a purification target is 55% or less.

[54]

[0146] The purification method according to any one of [1] to

[53] , wherein the ClogP / number of amino acid residues of the cyclic peptide as the purification target is 1.0 or more and 1.8 or less.

[55]

[0148] The purification method according to any one of [1] to

[54] , wherein the peptide as an impurity is a peptide produced during the synthesis of the cyclic peptide as a purification target.

[56]

[0150] The purification method according to any one of [1] to

[55] , wherein the peptide as an impurity is a cyclic peptide different from the cyclic peptide as a purification target.

[57]

[0152] The purification method according to any one of [1] to

[56] , wherein the peptide as an impurity is a cyclic peptide having a number of amino acids twice the number of amino acids of the cyclic peptide as a purification target, a cyclic peptide having a number of amino acids three times the number of amino acids of the cyclic peptide as a purification target, or an isomer of the cyclic peptide as a purification target.

[58]

[0154] The purification method according to any one of [1] to

[57] , wherein the metal atom or the metal ion is an atom or ion of an alkali metal.

[59]

[0156] The purification method according to

[58] , wherein the alkali metal is at least one selected from the group consisting of lithium, sodium, potassium, rubidium and cesium.

[60]

[0158] The purification method according to

[58] or

[59] , wherein the alkali metal is lithium or potassium.

[61]

[0160] The purification method according to any one of [1] to

[57] , wherein the metal atom or the metal ion is an atom or ion of an alkaline earth metal.

[62]

[0162] The purification method according to

[61] , wherein the alkaline earth metal is at least one selected from the group consisting of: magnesium, calcium, strontium, and barium.

[63]

[0164] The purification method according to

[60] or

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

[64]

[0166] The purification method according to any one of [1] to

[57] , wherein the metal atom or the metal ion is an atom or an ion of a transition metal.

[65]

[0168] The purification method according to

[64] , wherein the transition metal is at least one selected from the group consisting of: scandium, manganese, iron, zinc, and tungsten.

[66]

[0170] The purification method according to

[64] or

[65] , wherein the transition metal is at least one selected from the group consisting of: scandium, manganese, and zinc.

[67]

[0172] The purification method according to any one of [1] to

[57] , wherein the metal atom or the metal ion is an atom or an ion of a rare earth metal.

[68]

[0174] The purification method according to

[67] , wherein the rare earth metal is at least one selected from the group consisting of: cerium, samarium, and ytterbium.

[69]

[0176] The purification method according to any one of [1] to [�7], wherein the metal atom or the metal ion is an atom or an ion of a poor metal.

[70]

[0178] The purification method according to

[69] , wherein the poor metal is at least one selected from the group consisting of: bismuth and indium.

[71]

[0180] The purification method according to any one of [1] to

[57] , wherein the metal atom is at least one selected from the group consisting of: a magnesium atom, a scandium atom, and a samarium atom; and

[0181] The metal ion is at least one selected from the group consisting of: a magnesium ion, a scandium ion, and a samarium ion.

[72]

[0183] The purification method according to any one of [1] to

[57] , wherein the metal atom comprises a magnesium atom, and

[0184] the metal ion comprises a magnesium ion.

[73]

[0186] The purification method according to any one of [1] to

[72] , wherein the metal atom or the metal ion is an atom or ion derived from a metal salt or its solvate or a second complex.

[74]

[0188] The purification method according to any one of [1] to

[73] , wherein the metal atom or the metal ion is an atom or ion derived from a metal salt or its solvate.

[75]

[0190] The purification method according to

[73] or

[74] , wherein the metal salt is at least one selected from the group consisting of: iodide salts, bromide salts, chloride salts, perchlorates, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, bis(trifluoromethanesulfonic acid) imide salts, ethylmalonate salts, nitrite salts, and sulfate salts.

[0191] [75-1]

[0192] The purification method according to

[73] or

[74] , wherein the metal salt is at least one selected from the group consisting of: iodide salts, bromide salts, chloride salts, perchlorates, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, bis(trifluoromethanesulfonic acid) imide salts, ethylmalonate salts, and nitrite salts.

[76]

[0194] The purification method according to any one of

[73] to

[75] , wherein the metal salt is at least one selected from the group consisting of: iodide salts, bromide salts, perchlorates, and trifluoromethanesulfonate salts.

[77]

[0196] The purification method according to any one of

[73] to

[76] , wherein the metal salt is a perchlorate or a trifluoromethanesulfonate salt.

[78]

[0198] The purification method according to any one of

[73] to

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

[79]

[0200] The purification method according to

[78] , wherein the iodide salt is 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.

[80]

[0202] The purification method according to any one of

[73] to

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

[81]

[0204] The purification method according to

[80] , wherein the bromide salt is at least one selected from the group consisting of lithium bromide, magnesium bromide, calcium bromide, samarium(III) bromide, zinc bromide, and indium bromide.

[82]

[0206] The purification method according to any one of

[73] to

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

[83]

[0208] The purification method according to

[82] , wherein the chloride salt is samarium chloride, cerium chloride, or magnesium chloride.

[0209] [83-1]

[0210] The purification method according to

[82] , wherein the chloride salt is samarium chloride or cerium chloride.

[84]

[0212] The purification method according to any one of

[73] to

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

[85]

[0214] The purification method according to

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

[86]

[0216] The purification method according to any one of

[73] to

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

[87]

[0218] The purification method according to

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

[88]

[0220] The purification method according to any one of

[73] to

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

[89]

[0222] The purification method according to

[88] , wherein the trifluoromethanesulfonate is at least one selected from the group consisting of magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, scandium trifluoromethanesulfonate, samarium(III) trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, iron(III) trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[90]

[0224] The purification method according to any one of

[73] to

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

[91]

[0226] The purification method according to

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

[92]

[0228] The purification method according to any one of

[73] to

[75] , wherein the metal salt is an isopropylsulfonate.

[93]

[0230] The purification method according to

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

[94]

[0232] The purification method according to any one of

[73] to

[75] , wherein the metal salt is a methanesulfonate.

[95]

[0234] The purification method according to

[94] , wherein the methanesulfonate is at least one selected from the group consisting of cerium(III) methanesulfonate and zinc difluoromethanesulfonate.

[96]

[0236] The purification method according to any one of

[73] to

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

[97]

[0238] The purification method according to

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

[98]

[0240] The purification method according to any one of

[73] to

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

[99]

[0242] The purification method according to

[98] , wherein the acetate is at least one selected from the group consisting of potassium acetate and magnesium acetate.

[100]

[0244] The purification method according to any one of

[73] to

[75] , wherein the metal salt is bis(trifluoromethanesulfonyl)imide salt.

[101]

[0246] The purification method according to

[100] , wherein the bis(trifluoromethanesulfonyl)imide salt is at least one selected from the group consisting of magnesium bis(trifluoromethanesulfonyl)imide, zinc bis(trifluoromethanesulfonyl)imide, and iron(II) bis(trifluoromethanesulfonyl)imide.

[102]

[0248] The purification method according to any one of

[73] to

[75] , wherein the metal salt is ethyl malonate.

[103]

[0250] The purification method according to

[102] , wherein the ethyl malonate is magnesium ethyl malonate.

[104]

[0252] The purification method according to any one of

[73] to

[75] , wherein the metal salt is nitrite.

[105]

[0254] The purification method according to

[104] , wherein the nitrite is potassium nitrite.

[0255] [105-1]

[0256] The purification method according to any one of

[73] to

[75] , wherein the metal salt is sulfate.

[0257] [105-2]

[0258] The purification method according to any one of [105-1], wherein the sulfate is magnesium sulfate.

[106]

[0260] The purification method according to any one of [1] to

[74] , wherein the metal atom or the metal ion is an atom or an ion derived from at least one selected from the group consisting of lithium iodide, lithium perchlorate, lithium tetrafluoroborate, lithium bromide, lithium chloride, potassium iodide, lithium fluoride, potassium carbonate, potassium nitrite, potassium acetate, potassium tetrafluoroborate, potassium hexafluorophosphate, barium iodide, barium perchlorate, magnesium ethylmalonate, magnesium bis(trifluoromethanesulfonate)imide, magnesium oxide, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium bromide, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, scandium trifluoromethanesulfonate, scandium chloride, samarium(III) trifluoromethanesulfonate, samarium(III) iodide, samarium(III) bromide, samarium(III) chloride, cerium(III) methanesulfonate, cerium(III) chloride, cerium(III) chloride heptahydrate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, zinc(II) tetrafluoroborate, zinc perchlorate, zinc difluoromethanesulfonate, zinc(II) isopropylsulfonate, zinc(II) toluenesulfonate, zinc bis(trifluoromethanesulfonate)imide, zinc bromide, zinc chloride, zinc carbonate, zinc fluoride, zinc iodide, manganese trifluoromethanesulfonate, manganese chloride, indium trifluoromethanesulfonate, indium bromide, indium chloride, indium iodide, iron(III) trifluoromethanesulfonate, iron(II) bis(trifluoromethanesulfonate)imide, tungsten(VI) fluoride, tungsten(VI) chlorofluoride, and copper trifluoromethanesulfonate.

[107]

[0262] The purification method according to any one of [1] to

[74] and

[106] , wherein the metal atom or the metal ion is an atom or an ion derived from at least one metal salt or its solvate selected from the group consisting of lithium iodide, lithium perchlorate, potassium iodide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, scandium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, and magnesium chloride.

[0263] [107-1]

[0264] The purification method according to any one of [1] to

[74] and

[106] , wherein the metal atom or the metal ion is an atom or an ion derived from at least one metal salt or its solvate selected from the group consisting of lithium iodide, lithium perchlorate, potassium iodide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, scandium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, and indium trifluoromethanesulfonate.

[108]

[0266] The purification method according to any one of [1] to

[74] ,

[106] and

[107] , wherein the metal atom or the metal ion is an atom or ion derived from at least one metal salt or its solvate selected from the group consisting of: lithium perchlorate, potassium iodide, magnesium bromide, magnesium perchlorate, magnesium iodide and manganese trifluoromethanesulfonate.

[109]

[0268] The purification method according to any one of [1] to

[73] , wherein the metal atom or the metal ion is an atom or ion derived from the second complex.

[110]

[0270] The purification method according to

[73] or

[109] , wherein the ligand of the second complex is at least one selected from the group consisting of: olefinic compounds, carbonyl-based compounds, phosphine-based compounds and carbon monoxide.

[111]

[0272] The purification method according to

[73] ,

[109] or

[110] , wherein the ligand of the second complex is at least one selected from the group consisting of: ethylene, dibenzylideneacetone, acetylacetone, triphenylphosphine and carbon monoxide.

[112]

[0274] The purification method according to

[73] and any one of

[109] to

[111] , wherein the second complex is at least one selected from the group consisting of: iron(III) tris(acetylacetonate), ferrocene, zinc(II) acetylacetonate and tungsten hexacarbonyl.

[113]

[0276] The purification method according to any one of [3] to

[17] and

[20] to

[112] , wherein the first solvent is a solvent capable of forming a complex of the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion.

[0277] [113-1]

[0278] The purification method according to any one of [3] to

[17] and

[20] to

[112] , wherein the first solvent comprises at least one selected from the group consisting of: alcohol-based solvents, nitrile-based solvents, benzene-based solvents, ether-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, sulfoxide-based solvents and amide-based solvents.

[0279] [113-2]

[0280] The purification method according to any one of [3] to

[17] and

[20] to

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

[114]

[0282] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is an alcohol-based solvent.

[115]

[0284] The purification method according to any one of [113-1], [113-2], and

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

[0285] [115-1]

[0286] The purification method according to any one of [113-1], [113-2], and

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

[116]

[0288] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is a nitrile-based solvent.

[117]

[0290] The purification method according to

[116] , wherein the nitrile-based solvent is acetonitrile.

[118]

[0292] The purification method according to any one of [3] to

[17] and

[20] to

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

[119]

[0294] The purification method according to

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

[120]

[0296] The purification method according to any one of [3] to

[17] and

[20] to

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

[121]

[0298] The purification method according to

[120] , 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 (dimethyl ether), and CPME (cyclopentyl pentyl ether).

[122]

[0300] The purification method according to any one of [3] to

[17] and

[20] to

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

[123]

[0302] The purification method according to

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

[124]

[0304] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is a halogen-based solvent.

[125]

[0306] The purification method according to

[124] , wherein the halogen-based solvent is dichloromethane.

[0307] [125-1]

[0308] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is a sulfoxide-based solvent.

[0309] [125-2]

[0310] The purification method according to [125-1], wherein the sulfoxide-based solvent is dimethyl sulfoxide.

[0311] [125-3]

[0312] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is an amide-based solvent.

[0313] [125-4]

[0314] The purification method according to [125-3], wherein the amide-based solvent is dimethylformamide or dimethylacetamide.

[126]

[0315] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is at least one selected from the group consisting of: 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, acetonitrile, tetrahydrofuran, toluene, 1,4-dioxane, acetone, dichloromethane, ethanol, methanol, dimethyl sulfoxide, dimethylformamide, and dimethylacetamide.

[0316] [126-1]

[0317] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the first solvent is at least one selected from the group consisting of: 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, acetonitrile, tetrahydrofuran, toluene, 1,4-dioxane, acetone, and dichloromethane.

[127]

[0319] The purification method according to any one of [3] to

[17] ,

[20] to

[113] , and

[126] , wherein the first solvent is at least one selected from the group consisting of: 2,2,2-trifluoroethanol, acetonitrile, tetrahydrofuran, and acetone.

[128]

[0321] The purification method according to any one of [6] to

[127] , wherein the second solvent includes at least one selected from the group consisting of: ether-based solvents, benzene-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, nitrile-based solvents, hydrocarbon-based solvents, alcohol-based solvents, sulfoxide-based solvents, and amide-based solvents.

[0322] [128-1]

[0323] The purification method according to any one of [6] to

[127] , wherein the second solvent includes at least one selected from the group consisting of: ether-based solvents, benzene-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, nitrile-based solvents, and hydrocarbon-based solvents.

[129]

[0325] The purification method according to any one of [6] to

[128] , wherein the second solvent is an ether-based solvent.

[130]

[0327] The purification method according to

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

[131]

[0329] The purification method according to any one of [6] to

[128] , wherein the second solvent is a benzene-based solvent.

[132]

[0331] The purification method according to

[128] or

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

[133]

[0333] The purification method according to any one of [6] to

[128] , wherein the second solvent is a ketone-based solvent.

[134]

[0335] The purification method according to

[128] or

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

[135]

[0337] The purification method according to any one of [6] to

[128] , wherein the second solvent is a halogen-based solvent.

[136]

[0339] The purification method according to

[128] or

[135] , wherein the halogen-based solvent is dichloromethane.

[137]

[0341] The purification method according to any one of [6] to

[128] , wherein the second solvent is a hydrocarbon-based solvent.

[138]

[0343] The purification method according to

[128] or

[137] , wherein the hydrocarbon-based solvent is at least one selected from the group consisting of hexane, heptane, cyclohexane, methylcyclohexane, or isooctane.

[139]

[0345] The purification method according to any one of [6] to

[128] , wherein the second solvent is an ester-based solvent.

[0346] [139-1]

[0347] The purification method according to

[128] or

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

[140]

[0349] The purification method according to any one of [6] to

[128] , wherein the second solvent is a nitrile-based solvent.

[0350] [140-1]

[0351] The purification method according to

[128] or

[140] , wherein the nitrile-based solvent is acetonitrile.

[0352] [140-2]

[0353] The purification method according to any one of [6] to

[128] , wherein the second solvent is an alcohol-based solvent.

[0354] [140-3]

[0355] The purification method according to

[128] or [140-2], wherein the alcohol-based solvent is at least one selected from the group consisting of methanol, ethanol, butanol, or benzyl alcohol.

[0356] [140-4]

[0357] The purification method according to any one of [6] to

[128] , wherein the second solvent is a sulfoxide-based solvent.

[0358] [140-5]

[0359] The purification method according to [140-4], wherein the sulfoxide-based solvent is dimethyl sulfoxide.

[0360] [140-6]

[0361] The purification method according to any one of [6] to

[128] , wherein the second solvent is an amide-based solvent.

[0362] [140-7]

[0363] The purification method according to [140-6], wherein the amide-based solvent is dimethylformamide.

[0364] [140-8]

[0365] The purification method according to any one of [3] to

[17] and

[20] to

[113] , wherein the second solvent is isopropyl acetate or a combination of methyl ethyl ketone and heptane, or a combination of acetonitrile and MTBE.

[141]

[0367] The purification method according to any one of

[23] to

[140] , wherein the third solvent is a solvent immiscible with water.

[142]

[0369] The purification method according to any one of

[23] to

[141] , wherein the third solvent is 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.

[143]

[0371] The purification method according to any one of

[26] to

[142] , wherein the fourth solvent is at least one selected from the group consisting of: methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butanol, ethylene glycol, cyclohexane, methylcyclohexane, heptane, isooctane, toluene, xylene, dimethyl sulfoxide, and acetonitrile.

[144]

[0373] The purification method according to any one of [1] to

[143] , wherein the purification method is a method for purifying the cyclic peptide as the purification target to have a purity of 85% or higher, wherein the purity (%) of the cyclic peptide as the purification target is the ratio of the peak area of the cyclic peptide as the purification target at 220 nm to the sum of the peak areas of each component in the entire mixture containing the cyclic peptide as the purification target, and wherein the UV spectrum of the mixture containing the cyclic peptide as the purification target is measured using HPLC and PDA (photodiode array detector).

[0374] [144-1]

[0375] The purification method according to any one of [1] to

[143] , wherein the purification method is a method for purifying the cyclic peptide as the purification target to have a purity of 85% or higher, wherein the purity (%) of the cyclic peptide as the purification target is the ratio of the peak area of the cyclic peptide as the purification target at 225 nm to the sum of the peak areas of each component in the entire mixture containing the cyclic peptide as the purification target, and wherein the UV spectrum of the mixture containing the cyclic peptide as the purification target is measured using HPLC and PDA (photodiode array detector).

[0376] [144-2]

[0377] The purification method according to any one of [1] to

[143] , wherein the purification efficiency of the impurities contained in the mixture containing the cyclic peptide as the purification target is a positive value.

[0378] [144-3]

[0379] According to the method described in [144-2], wherein the purification efficiency of the impurity is determined by the following formula:

[0380] Purification efficiency of impurities (%) = {1 - (ratio of the cyclic peptide as the purification target to impurities after the purification operation) / (ratio of the cyclic peptide as the purification target to impurities before the purification operation)} × 100.

[145]

[0382] A production method of a cyclic peptide, the production method comprising the purification method according to any one of [1] to

[144] .

[146]

[0384] According to the production method described in

[145] , the production method further comprises the step of obtaining the cyclic peptide as the purification target by a liquid phase synthesis method.

[147]

[0386] According to the production method described in

[145] , the production method further comprises the step of obtaining the cyclic peptide as the purification target by a solid phase synthesis method.

[148]

[0388] According to the production method described in

[145] , the production method further comprises the step of obtaining the cyclic peptide as the purification target by a cultivation method.

[149]

[0390] A method for improving the purity of a cyclic peptide as compared with not forming a complex with a metal atom or metal ion in the purification method of the cyclic peptide, the method being characterized in that the cyclic peptide as the purification target is separated from a mixture containing the cyclic peptide as the purification target as a complex with a metal atom or metal ion.

[150]

[0392] A method for reducing the impurity content as compared with not forming a complex with a metal atom or metal ion in the purification method of the cyclic peptide, the method being characterized in that the cyclic peptide as the purification target is separated from a mixture containing the cyclic peptide as the purification target as a complex with a metal atom or metal ion.

[0393] In the above numbers, unless otherwise mentioned, the numbers cited in the dependent claims include the same numbers but with different branch numbers. For example,

[20] cited in the dependent claims means not only including

[20] , but also including [20-1]. The same applies to other numbers.

[0394] [Advantageous effects of the present invention]

[0395] According to the present invention, a method for easily purifying and producing a cyclic peptide can be provided, and the method can be applied to cyclic peptides that are difficult to crystallize. Some cyclic peptides are difficult to crystallize, but it has been found that even such cyclic peptides can be easily purified by forming a complex of the cyclic peptide with a metal atom and a metal ion. Brief Description of the Drawings

[0396] Figure 1 is a schematic diagram showing the basic synthesis method of the cyclic peptide.

[0397] Figure 2 is a sketch of the 1 1H-NMR spectrum measured in Run 1 of Reference Example 2.

[0398] Figure 3 is a sketch of the 1 1H-NMR spectrum measured in Run 2 of Reference Example 2.

[0399] Figure 4 is a sketch of the 1 1H-NMR spectrum measured in Run 3 of Reference Example 2.

[0400] Figure 5 is a sketch of the 1 1H-NMR spectrum measured in Run 4 of Reference Example 2.

[0401] Figure 6 is a sketch of the 1 1H-NMR spectrum measured in Run 5 of Reference Example 2.

[0402] Figure 7 is a sketch of the 1 1H-NMR spectrum measured in Run 6 of Reference Example 2.

[0403] Figure 8 is a sketch of the LC chromatogram measured in Run 29 of Example 2-1.

[0404] Figure 9 is a sketch of the LC chromatogram measured in Run 2 of Example 2-3-1.

[0405] Figure 10 is a sketch of the [[ID=�3]] 1 1H-NMR spectrum measured in Example 3-1. Detailed Description of the Invention

[0406] Hereinafter, the method of the present invention will be described in detail.

[0407] In the present invention, the term "mixing" sometimes means the "operation" of mixing one substance with another substance, and does not necessarily mean only the miscible state of one substance with another substance. As used herein, the term "mixing (i) and (ii)" includes any embodiment of adding (i) to (ii), adding (ii) to (i), and adding (i) and (ii) simultaneously. As used herein, the term "to" indicates that the range includes the values at both ends thereof. For example, "A to B" means a range of A or more and B or less. As used herein, when used in combination with a numerical value, the term "about" means a range of +10% and -10% of the numerical value. In the present invention, the meaning of the term "and / or" includes any combination of the appropriate combinations of "and" and "or". Specifically, for example, the term "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.

[0408] As used herein, "purification of a cyclic peptide" means, for example, increasing the proportion (purity) of the cyclic peptide as the purification target relative to the entire mixture containing the cyclic peptide as the purification target.

[0409] One embodiment of the present invention relates to a method for purifying a cyclic peptide, wherein the purification method may include the step of separating the cyclic peptide as the purification target from the mixture of the cyclic peptide as the purification target in the form of a complex with a metal atom or metal ion; or separating the cyclic peptide as the purification target or the peptide as an impurity in the form of a complex with a metal atom or metal ion from the mixture containing the cyclic peptide as the purification target and the peptide as an impurity, wherein the metal atom may be at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom, and the metal ion may be at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion, and a rare earth metal ion.

[0410] The separation step includes step (1): mixing the mixture containing the cyclic peptide as the purification target or the mixture containing the cyclic peptide as the purification target and the peptide as an impurity with a metal atom or metal ion as a preliminary step. Step (1) can be carried out in a first solvent.

[0411] A purification method according to another embodiment of the present invention relates to a method for purifying a cyclic peptide, wherein the purification method may include step (1)': mixing a mixture containing the cyclic peptide as the purification target or a mixture containing the cyclic peptide as the purification target and a peptide as an impurity with a metal atom or a metal ion in a first solvent, wherein the metal atom may be at least one selected from the group consisting of: alkali metal atoms, alkaline earth metal atoms, transition metal atoms, poor metal atoms, and rare earth metal atoms, and the metal ion may be at least one selected from the group consisting of: alkali metal ions, alkaline earth metal ions, transition metal ions, poor metal ions, and rare earth metal ions.

[0412] In this step (1)', a complex of the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion may be formed in the first solvent.

[0413] The purification method according to another embodiment of the present invention may further include step (2): mixing the mixture obtained in step (1) or step (1)' with a second solvent. In the mixture obtained in step (1) or step (1)', before mixing with the second solvent, a complex of the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion may be formed. The mixture obtained in step (1) or step (1)' is "a mixture containing the cyclic peptide as the purification target or a mixture containing the cyclic peptide as the purification target and a peptide as an impurity and a metal atom or a metal ion". The first solvent and the second solvent are different solvents from each other. For example, when the first solvent is a good solvent for the complex, the second solvent may be a poor solvent for the complex. In contrast, when the first solvent is a poor solvent for the complex, the second solvent may be a good solvent for the complex. Step (2) may include step (2)': removing at least a part of the first solvent as a preliminary step of step (2).

[0414] In step (1) or step (1)', the metal atom or metal ion may be 0.2 molar equivalent or more, 0.3 molar equivalent or more, 0.4 molar equivalent or more, 0.5 molar equivalent or more, 0.6 molar equivalent or more, 0.7 molar equivalent or more, 0.8 molar equivalent or more, 0.9 molar equivalent or more, or 1 molar equivalent or more, and 12 molar equivalents or less, 10 molar equivalents or less, 8 molar equivalents or less, 6 molar equivalents or less, 4 molar equivalents or less, 2 molar equivalents or less, or 1.2 molar equivalents or less, relative to the cyclic peptide as the purification target. In step (1) or step (1)', the metal atom or metal ion may be 0.2 to 12 molar equivalents, 0.2 to 4 molar equivalents, 0.2 to 1.2 molar equivalents, 0.5 to 12 molar equivalents, 0.5 to 4 molar equivalents, 0.5 to 1.2 molar equivalents, 0.8 to 12 molar equivalents, 0.8 to 4 molar equivalents, or 0.8 to 1.2 molar equivalents, relative to the cyclic peptide as the purification target. In step (1) or step (1)', the metal atom or metal ion is preferably 0.2 to 12 molar equivalents, more preferably 0.5 to 4 molar equivalents, and most preferably 0.8 to 1.2 molar equivalents.

[0415] In step (1) or step (1)', the liquid temperature of the first solvent may be -20°C or higher, -10°C or higher, 0°C or higher, 4°C or higher, 8°C or higher, 12°C or higher, 16°C or higher, or 20°C or higher, and may be 100°C or lower, 80°C or lower, 50°C or lower, 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower. In step (1) or step (1)', the liquid temperature of the first solvent may be -20°C to 100°C, 4°C to 30°C, 20°C to 40°C, or 20°C to 30°C. In step (1) or step (1)', the liquid temperature of the first solvent is preferably -20°C to 100°C, more preferably 20°C to 40°C, and most preferably 20°C to 30°C.

[0416] In step (1) or step (1)', the time for mixing a mixture containing a cyclic peptide as a purification target or a mixture containing a cyclic peptide as a purification target and a peptide as an impurity with a metal atom or a metal ion in a first solvent can be 0.5 hours or more, 1 hour or more, 1.5 hours or more, or 2 hours or more, and can be 72 hours or less, 48 hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, or 5 hours or less. In step (1) or step (1)', the time for mixing a mixture containing a cyclic peptide as a purification target or a mixture containing a cyclic peptide as a purification target and a peptide as an impurity with a metal atom or a metal ion in a first solvent can be 0.5 to 72 hours, 0.5 to 48 hours, 0.5 to 5 hours, 2 to 48 hours, or 2 to 5 hours. In step (1) or step (1)', the time for mixing a mixture containing a cyclic peptide as a purification target or a mixture containing a cyclic peptide as a purification target and a peptide as an impurity with a metal atom or a metal ion in a first solvent is preferably 0.5 to 72 hours, more preferably 0.5 to 48 hours, and most preferably 2 to 5 hours.

[0417] In step (1) or step (1)', the concentration of the cyclic peptide as a purification target in the first solvent can be 0.1 mg / mL or more, 0.5 mg / mL or more, 1.0 mg / mL or more, 2.0 mg / mL or more, or 2.7 mg / mL or more, and can be 1000 mg / mL or less, 800 mg / mL or less, 500 mg / mL or less, 300 mg / mL or less, or 150 mg / mL or less. In step (1) or step (1)', the concentration of the cyclic peptide as a purification target in the first solvent can be 0.1 mg / mL to 1000 mg / mL, 0.5 mg / mL to 800 mg / mL, 1.0 mg / mL to 500 mg / mL, 2.0 mg / mL to 300 mg / mL, or 2.7 mg / mL to 150 mg / mL. In step (1) or step (1)', the concentration of the cyclic peptide as a purification target 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 2.7 mg / mL to 150 mg / mL.

[0418] In step (2), when mixing the mixture obtained in step (1) or step (1)' with the second solvent, the liquid temperature can be -20°C or higher, -10°C or higher, 0°C or higher, 4°C or higher, 8°C or higher, 12°C or higher, 16°C or higher, or 20°C or higher, and can be 100°C or lower, 80°C or lower, 50°C or lower, 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower. In step (2), when mixing the residue with the second solvent, the liquid temperature can be -20°C to 100°C, 4°C to 30°C, 20°C to 40°C, or 20°C to 30°C. In step (2), when mixing the mixture obtained in step (1) or step (1)' with the second solvent, the liquid temperature is preferably -20°C to 100°C, more preferably 20°C to 40°C, and most preferably 20°C to 30°C.

[0419] In step (2), the time for mixing the mixture obtained in step (1) or step (1)' with the second solvent can be 0.5 hours or more, 1 hour or more, 1.5 hours or more, or 2 hours or more, and can be 72 hours or less, 48 hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, or 5 hours or less. In step (2), the time for mixing the residue with the second solvent can be 0.5 to 72 hours, 0.5 to 48 hours, 0.5 to 5 hours, 2 to 48 hours, or 2 to 5 hours. In step (2), the time for mixing the mixture obtained in step (1) or step (1)' with the second solvent is preferably 0.5 to 72 hours, more preferably 0.5 to 48 hours, and most preferably 1 to 2 hours.

[0420] In step (2), the concentration of the complex in the second solvent can be 0.1 mg / mL or more, 0.3 mg / mL or more, 0.5 mg / mL or more, 0.8 mg / mL or more, or 1.0 mg / mL or more, and can be 200 mg / mL or less, 100 mg / mL or less, 50 mg / mL or less, 45 mg / mL or less, or 30 mg / mL or less. In step (2), the concentration of the complex in the second solvent can be 0.1 mg / mL to 200 mg / mL, 0.3 mg / mL to 100 mg / mL, 0.5 mg / mL to 50 mg / mL, 0.8 mg / mL to 45 mg / mL, or 1.0 mg / mL to 30 mg / mL. In step (2), the concentration of the complex in the second solvent is preferably 0.1 mg / mL to 200 mg / mL, more preferably 0.5 mg / mL to 50 mg / mL, and most preferably 1.0 mg / mL to 30 mg / mL.

[0421] The purification method according to an embodiment of the present invention may include step (3): separating the complex from the mixed solution of the mixture obtained in step (1) or step (1)' and a second solvent.

[0422] The purification method according to still another embodiment of the present invention relates to a purification method of a cyclic peptide, wherein the purification method may include step (4): mixing a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or a metal ion and a second solvent, wherein the metal atom may be at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom, and the metal ion may be at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion, and a rare earth metal ion.

[0423] The purification method according to still another embodiment of the present invention may further include step (5): separating the complex from the mixed solution of the complex and the second solvent.

[0424] As used herein, the separation of the complex is carried out by solid-liquid separation or liquid-liquid partitioning. Examples of the solid-liquid separation method include centrifugation and filtration. Examples of the liquid-liquid partitioning method include centrifugation and decantation.

[0425] As used herein, the purification method of the cyclic peptide may include step (6): removing the metal atom or metal ion from the separated complex. Step (6) may include the following steps: mixing the separated complex, a third solvent, and water and separating them into an aqueous phase and an organic phase to remove the metal atom or metal ion present in the aqueous phase. Step (6) may further include the following steps: removing the third solvent from the organic phase and obtaining the cyclic peptide as the purification target. Specific examples of the method for removing the third solvent from the organic phase include distillation at atmospheric pressure, distillation under heating, and distillation under reduced pressure. Removing the third solvent from the organic phase may be carried out by distillation under reduced pressure.

[0426] Step (6) may further include the following steps: mixing the separated complex, a fourth solvent, and a ligand or a compound that generates a ligand or an anion or a compound that generates an anion, and the metal atom or metal ion forms a first complex or a first metal salt with the ligand or the anion. Step (6) may further include the step of removing the first metal complex or the first metal salt. The first complex may mean a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or a metal ion (complex), and a complex different from the second complex, and a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or a metal ion and the same complex as the second complex may be used.

[0427] As used herein, the method for purifying a cyclic peptide may further include step (7): confirming that the cyclic peptide as the purification target or the peptide as the impurity forms a complex with a metal atom or a metal ion. Step (7) may be carried out by: comparing the NMR peaks of the cyclic peptide as the purification target or the peptide as the impurity with the NMR peaks of a mixture obtained by mixing the cyclic peptide as the purification target or the peptide as the impurity with a metal atom or a metal ion. The measurement of NMR may be carried out in a manner well known to those skilled in the art. For example, NMR may be measured by: adding a solution of the compound to be measured dissolved in a solvent suitable for measuring NMR into a sample tube for NMR measurement, and setting the sample tube in the measuring device. The solvent suitable for measuring NMR is preferably a solvent capable of dissolving the compound to be measured, and may be a commercially available deuterated solvent. The measurement conditions of NMR may be the conditions well known to those skilled in the art and the conditions described in the measuring device manual. The measurement conditions are not particularly limited as long as the target peak components can be measured. For example, the measurement temperature may be in the range of 273K to 320K, the integration time may be in the range of 1 second to 7 days, and the rotation speed of the sample tube may be in the range of 0 to 20Hz. Specifically, in step (7), the following items are measured at 278K and 298K respectively 1 1H-NMR: (A) a solution obtained by dissolving the cyclic peptide or the peptide as the impurity in acetonitrile-d3 (deuterated acetonitrile, CD3CN, CAS: 2206-26-0) (concentration of the cyclic peptide or the peptide as the impurity: 1.4 mM); and (B) a solution obtained by mixing a metal atom or a metal ion with the cyclic peptide or the peptide as the impurity in acetonitrile-d3 (deuterated acetonitrile, CD3CN, CAS: 2206-26-0) such that the metal atom or the metal ion is 1 to 6 molar equivalents relative to the cyclic peptide or the peptide as the impurity (concentration of the cyclic peptide or the peptide as the impurity: 1.4 mM). When a new peak of the cyclic peptide as the purification target or the peptide as the impurity is observed in the peak components of (B) compared with the peak components of (A), when a change in the peak intensity ratio is observed in the peak components of (B) compared with the peak components of (A), or when peak sharpening or broadening is observed in the peak components of (B) compared with the peak components of (A), it can be determined that the cyclic peptide as the purification target or the peptide as the impurity forms a complex with a metal atom or a metal ion.

[0428] As used herein, the complex may be a complex of the cyclic peptide as the purification target with a metal atom or a metal ion, and may be a complex of the peptide as the impurity with a metal atom or a metal ion. The complex may be a complex obtained by bringing the metal into contact with the cyclic peptide or the peptide as the impurity. As used herein, a complex is a compound in which the cyclic peptide as the purification target or the peptide as the impurity interacts with a metal atom or a metal ion. The complex may include a solid, a crystal, a liquid or an amorphous form.

[0429] The complex may be a coordination complex. As used herein, a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or metal ion is a compound in such a state that the metal atom or metal ion coordinates with the cyclic peptide as a purification target or the peptide as an impurity. For example, the state of coordination of the metal atom or metal ion with the cyclic peptide as a purification target or the peptide as an impurity is determined by X-ray crystal structure analysis and changes in absorption spectra (see "Complex Chemistry - Fundamentals and Recent Topics" edited by society of basic coordination chemistry, KODANSHA SCIENTIFIC LTD., 1994, pages 39-49). The complex may include solids, crystals, liquids, or amorphous forms.

[0430] As used herein, a mixture containing a cyclic peptide as a purification target or a mixture containing a cyclic peptide as a purification target and a peptide as an impurity is a crude product obtained by producing the cyclic peptide as a purification target.

[0431] As used herein, the number of amino acid residues of the cyclic peptide as a purification target 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 of the cyclic peptide as a purification target may be 5 to 20, 5 to 14, 10 to 20, or 10 to 14.

[0432] As used herein, the number of amino acid residues in the cyclic portion of the cyclic peptide as a purification target 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 as a purification target may be 5 to 15, 5 to 14, 10 to 15, or 8 to 14, and may be 8, 11, 13, or 14.

[0433] As used herein, the term "amino acid" includes natural amino acids and unnatural amino acids. As used herein, the term "amino acid" may mean an amino acid residue. As used herein, the term "natural amino acid" refers 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. The "unnatural amino acids" are not particularly limited, and examples thereof include β-amino acids, γ-amino acids, D-type amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids in which the side chain is different from the side chain of a natural amino acid. As the amino acids used herein, any spatial configuration is acceptable. The selection of the amino acid side chain is not particularly limited, and in addition to a hydrogen atom, the side chain can be freely selected from, 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, and a cycloalkyl group bonded in a spiro manner. Substituents can be further added to each of these substituents. Such substituents are not limited and can be one or two or more substituents each independently freely selected from any substituents including the following: a halogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a boron atom, a silicon atom, or a phosphorus atom. That is, examples of the side chain include an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or an optionally substituted cycloalkyl group, or an oxo group, an aminocarbonyl group, and a halogen atom.

[0434] As used herein, the "side chain of an amino acid", in the case of an α-amino acid, means the atomic group bonded to the carbon (α-carbon) to which an amino group and a carboxyl 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 attached to the α-carbon and / or β-carbon can be the side chain of the amino acid, and in the case of a γ-amino acid, the atomic group attached to the α-carbon, β-carbon, and / or γ-carbon can be the side chain of the amino acid.

[0435] In the present specification, the term "main chain of an amino acid" means, in the case of an α-amino acid, the chain portion formed by an amino group, an α-carbon, and a carboxyl group, in the case of a β-amino acid, the chain portion formed by an amino group, a β-carbon, an α-carbon, and a carboxyl group, and in the case of a γ-amino acid, the chain portion formed by an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group.

[0436] The amino group of the amino acid backbone can be unsubstituted (-NH2) or substituted (i.e., -NHR). R represents, for example, an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group or cycloalkyl group, or a carbon chain attached to the N atom, and the carbon atom at position α can form a ring, such as proline.

[0437] As used herein, examples of "substituents containing halogen atoms" include fluorine (-F), chlorine (-Cl), bromine (-Br) and iodine (-I).

[0438] As used herein, examples of "substituents containing oxygen atoms" include hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxy (-CO2H), oxycarbonyl (-C=O-OR), carbonyloxy (-O-C=O-R), thiocarbonyl (-C(=O)-SR), carbonylthio group (-S-C(=O)-R), aminocarbonyl (-C(=O)-NHR), carbamoyl (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), aminosulfonylamino (-NH-SO2-NHR), thiocarboxy (-C(=O)-SH) and carboxycarbonyl (-C(=O)-CO2H).

[0439] As used herein, examples of "substituents containing nitrogen atoms" include azido (-N3, also referred to as "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R") and aminocarbonylamino (-NR-CO-NR'R").

[0440] As used herein, examples of "substituents containing sulfur atoms" include mercapto (-SH), thio (-S-R), sulfinyl (-S=O-R), sulfonyl (-S(O)2-R), sulfo (-SO3H) and pentafluorosulfonyl (-SF5).

[0441] As used herein, examples of "substituents containing boron atoms" include boryl (-BR(R')), dioxaborolyl (-B(OR)(OR')) and trifluoroborate (-BF3 -)。Specific examples include such "substituents derived from boron atoms" where the two substituents R and R' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl; or such "substituents derived from boron atoms" where the two substituents R and R' together with the atoms to which they are each attached form a ring, i.e., a borocyclic group.

[0442] As used herein, examples of "substituents containing a phosphorus atom" include phosphoryl (-P(O)-R1R2), phosphinyl (-O-P(O)-R1R2), and phosphoric acid (-PO3H2).

[0443] As used herein, an amino acid in which the amino group of the main chain is substituted is called an "N-substituted amino acid". Examples of N-substituted amino acids include N-C1-C6 alkyl amino acids substituted with an alkyl group having 1 to 6 carbon atoms (C1-C6 alkyl group), preferably N-C1-C4 alkyl amino acids. Specific examples of N-substituted amino acids include N-methyl amino acid, N-ethyl amino acid, and N-propyl amino acid. N-propyl amino acids include N-isopropyl amino acid and N-n-propyl amino acid. As used herein, a cyclic peptide as a purification target may contain at least one or more unnatural amino acids. As used herein, a cyclic peptide as a purification target may contain N-substituted amino acids. A cyclic peptide as a purification target may contain 3 or more, 4 or more, 5 or more, or 6 or more N-substituted amino acids, and may include 15 or fewer, 13 or fewer, or 10 or fewer N-substituted amino acids. The number of N-substituted amino acid residues in a cyclic peptide as a purification target may be 45% or more, 50% or more, 55% or more, or 60% or more relative to the number of amino acid residues in the cyclic part of the cyclic peptide, may be 80% or less, 75% or less, 70% or less, or 65% or less, and is preferably 45% to 80%.

[0444] As used herein, an N-substituted amino acid may be at least one selected from the group consisting of N-methyl amino acid, N-ethyl amino acid, and N-propyl amino acid, and may be N-methyl amino acid.

[0445] As used herein, an amino acid in which the amino group of the main chain is unsubstituted is referred to as an "N-unsubstituted amino acid". As used herein, a cyclic peptide as a purification target may contain an N-unsubstituted amino acid. The N-unsubstituted amino acid may be an unnatural amino acid. The proportion of the number of N-unsubstituted amino acids in the cyclic peptide as a purification target may be 55% or less, 50% or less, 45% or less, or 40% or less relative to the total number of amino acid residues of the cyclic peptide as a purification target, and may be 20% or more, 25% or more, 30% or more, or 35% or more, and is preferably 20% to 55%.

[0446] The compounds described herein may contain isotope atoms in unnatural ratios in one or more atoms constituting the compound. The present invention also includes such compounds in which any atom in the compound is replaced by another isotope atom having the same atomic number (number of protons) and a different mass number (sum of the number of protons and neutrons), whereby the abundance ratio of the isotope is different from the abundance ratio in nature, i.e., a compound labeled with an isotope atom. Examples of the isotope elements contained in the compounds in the present text include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, and they include respectively 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 can be used as therapeutic agents or prophylactic agents, research reagents (e.g., assay reagents), or diagnostic agents (e.g., in vivo imaging diagnostic agents). For the compounds as used herein, all compounds containing radioactive or non-radioactive isotope elements in any proportion are covered within the scope of the present invention. Compounds labeled with isotope atoms can be produced in a manner similar to the method of producing unlabeled compounds by using reagents or solvents containing the corresponding isotope atoms.

[0447] As used herein, the ClogP / number of amino acid residues of the cyclic peptide as the purification target can be 1.0 or more, 1.1 or more, 1.2 or more, or 1.3 or more, and can be 1.8 or less, 1.7 or less, 1.6 or less, or 1.5 or less. The ClogP / number of amino acid residues of the cyclic peptide as the purification target can be 1.0 or more and 1.8 or less. ClogP is the computer-calculated partition coefficient and can be determined according to the rules described in the following document: "CLOGP Reference Manual Daylight version 4.9 (release date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / ) [access date: December 25, 2023]". Examples of the method for calculating ClogP include using Daylight version 4.95 of Daylight Chemical Information Systems, Inc (release date: August 1, 2011, ClogP algorithm version 5.4, database version 28, https: / / www.daylight.com / dayhtml / doc / release_notes / index.html) for calculation.

[0448] As used herein, the peptide as an impurity can be a peptide generated during the synthesis of the cyclic peptide as the purification target. The peptide as an impurity can be a cyclic peptide different from the cyclic peptide as the purification target. The peptide as an impurity can be a cyclic peptide having twice the number of amino acids of the cyclic peptide as the purification target, a cyclic peptide having three times the number of amino acids of the cyclic peptide as the purification target, or an isomer of the cyclic peptide as the purification target. Examples of isomers preferably include diastereoisomers.

[0449] As used herein, the metal atom or metal ion can be an atom or ion of an alkali metal atom, alkaline earth metal atom, transition metal atom, rare earth metal atom, or poor metal atom.

[0450] The alkali metal is preferably at least one selected from the group consisting of: The alkali metal is preferably selected from lithium, sodium, potassium, rubidium, and cesium, and more preferably lithium or potassium.

[0451] The alkaline earth metal is preferably at least one selected from the group consisting of: magnesium, calcium, strontium, and barium, and more preferably at least one selected from the group consisting of: magnesium, calcium, and barium.

[0452] The transition metal is preferably at least one selected from the group consisting of: scandium, manganese, iron, zinc, and tungsten, and more preferably at least one selected from the group consisting of: scandium, manganese, and zinc.

[0453] The rare earth metal is preferably at least one selected from the group consisting of cerium, samarium, and ytterbium.

[0454] The poor metal is preferably at least one selected from the group consisting of bismuth and indium.

[0455] The metal atom is preferably at least one selected from the group consisting of a magnesium atom, a scandium atom, and a samarium atom, and more preferably a magnesium atom. The metal ion is preferably at least one selected from the group consisting of a magnesium ion, a scandium ion, and a samarium ion, and more preferably a magnesium ion.

[0456] The metal atom or metal ion can be an atom or ion derived from a metal salt or its solvate or a second complex, and can be an atom or ion derived from a metal salt or its solvate. The second complex can mean a complex (coordination compound) of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or metal ion, and a complex different from the first complex, and a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or metal ion and the same complex as the first complex can be used. The metal salt can mean a metal salt different from the first metal salt, and a metal salt the same as the first metal salt can be used.

[0457] The metal salt is preferably at least one selected from the group consisting of iodide salts, bromide salts, chloride salts, perchlorates, oxide salts, trifluoromethanesulfonates, toluenesulfonates, isopropylsulfonates, methanesulfonates, carbonates, acetates, bis(trifluoromethanesulfonyl)imide salts, ethylmalonates, nitrites, and sulfates, and more preferably at least one selected from the group consisting of iodide salts, bromide salts, perchlorates, and trifluoromethanesulfonates, and most preferably perchlorates and trifluoromethanesulfonates.

[0458] The iodide salt can 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.

[0459] The bromide salt can be at least one selected from the group consisting of lithium bromide, magnesium bromide, calcium bromide, samarium(III) bromide, zinc bromide, and indium bromide.

[0460] The chloride salt can be samarium chloride, cerium chloride, or magnesium chloride, and can be samarium chloride or cerium chloride.

[0461] The perchlorate can be at least one selected from the group consisting of lithium perchlorate, barium perchlorate, magnesium perchlorate, calcium perchlorate, and zinc perchlorate.

[0462] The oxide salt can be magnesium oxide.

[0463] The trifluoromethanesulfonate may be at least one selected from the group consisting of magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, scandium trifluoromethanesulfonate, samarium(III) trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, iron(III) trifluoromethanesulfonate, and copper trifluoromethanesulfonate.

[0464] The toluenesulfonate may be zinc(II) toluenesulfonate.

[0465] The isopropylsulfonate may be zinc(II) isopropylsulfonate.

[0466] The methanesulfonate may be at least one selected from the group consisting of cerium(III) methanesulfonate and zinc difluoromethanesulfonate.

[0467] The carbonate may be at least one selected from the group consisting of potassium carbonate, calcium carbonate, and zinc carbonate.

[0468] The acetate may be at least one selected from the group consisting of potassium acetate and magnesium acetate.

[0469] The bis(trifluoromethanesulfonyl)imide salt may be at least one selected from the group consisting of magnesium bis(trifluoromethanesulfonyl)imide, zinc bis(trifluoromethanesulfonyl)imide, and iron(II) bis(trifluoromethanesulfonyl)imide.

[0470] The ethylmalonate may be magnesium ethylmalonate.

[0471] The nitrite may be potassium nitrite.

[0472] The sulfate may be magnesium sulfate.

[0473] The metal atom or metal ion may be an atom or ion derived from at least one selected from the group consisting of lithium iodide, lithium perchlorate, lithium tetrafluoroborate, lithium bromide, lithium chloride, potassium iodide, lithium fluoride, potassium carbonate, potassium nitrite, potassium acetate, potassium tetrafluoroborate, potassium hexafluorophosphate, barium iodide, barium perchlorate, magnesium ethylmalonate, magnesium bis(trifluoromethanesulfonate)imide, magnesium oxide, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium bromide, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, scandium trifluoromethanesulfonate, scandium chloride, samarium(III) trifluoromethanesulfonate, samarium(III) iodide, samarium(III) bromide, samarium(III) chloride, cerium(III) methanesulfonate, cerium(III) chloride, cerium(III) chloride heptahydrate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, zinc(II) tetrafluoroborate, zinc perchlorate, zinc difluoromethanesulfonate, zinc(II) isopropylsulfonate, zinc(II) toluenesulfonate, zinc bis(trifluoromethanesulfonate)imide, zinc bromide, zinc chloride, zinc carbonate, zinc fluoride, zinc iodide, manganese trifluoromethanesulfonate, manganese chloride, indium trifluoromethanesulfonate, indium bromide, indium chloride, indium iodide, iron(III) trifluoromethanesulfonate, iron(II) bis(trifluoromethanesulfonate)imide, tungsten(VI) fluoride, tungsten(VI) chlorofluoride, and copper trifluoromethanesulfonate.

[0474] The metal atom or metal ion is preferably an atom or ion derived from at least one metal salt or its solvate selected from the group consisting of lithium iodide, lithium perchlorate, potassium iodide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, scandium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, and magnesium chloride.

[0475] The metal atom or metal ion may be an atom or ion derived from at least one metal salt or its solvate selected from the group consisting of lithium perchlorate, potassium iodide, magnesium bromide, magnesium perchlorate, magnesium iodide, and manganese trifluoromethanesulfonate. When the metal atom or metal ion is an atom or ion derived from the metal salt or its solvate described in this paragraph, the purity of the purified cyclic peptide can be further improved.

[0476] The ligand of the second complex may be at least one selected from the group consisting of olefinic compounds, carbonyl-based compounds, phosphine-based compounds, and carbon monoxide, and may be at least one selected from the group consisting of ethylene, dibenzylideneacetone, acetylacetone, triphenylphosphine, and carbon monoxide.

[0477] The second complex may be at least one selected from the group consisting of iron(III) tris(acetylacetonate), ferrocene, zinc(II) acetylacetonate, and tungsten hexacarbonyl.

[0478] The first solvent can be a solvent capable of forming a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or metal ion, and can include at least one selected from the group consisting of alcohol-based solvents, nitrile-based solvents, benzene-based solvents, ether-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, sulfoxide-based solvents, and amide-based solvents. When the first solvent is the solvent described in this paragraph, it is easier to form a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or metal ion.

[0479] Specific examples of the alcohol-based solvents herein may include methanol, ethanol, 1-propanol, 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, and ethylene glycol. Specific examples of the nitrile-based solvents include chain-like nitriles such as acetonitrile, propionitrile, and acrylonitrile; and cyclic nitriles such as benzonitrile. Specific examples of the benzene-based solvents include toluene, o-dichlorobenzene, 1,2,4-trichlorobenzene, and xylene. Specific examples of the ether-based solvents may include diethyl ether, DME (dimethyl ether), tetrahydrofuran, ethylene glycol dimethyl ether, 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. Specific examples of the ketone-based solvents may include acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone. Specific examples of the halogen-based solvents may include haloalkanes such as dichloromethane and dichloroethane, and haloarenes such as chlorobenzene. Specific examples of the ester-based solvents may 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.

[0480] The alcohol-based solvent as the first solvent is preferably at least one selected from the group consisting of: 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, ethanol, and methanol. The nitrile-based solvent as the first solvent is preferably acetonitrile. The benzene-based solvent as the first solvent is preferably toluene or xylene. The ether-based solvent as the first solvent is preferably at least one selected from the group consisting of: tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, MTBE (tert-butyl methyl ether), DME (dimethyl ether), and CPME (cyclopentyl methyl ether). The ketone-based solvent as the first solvent is preferably acetone or methyl ethyl ketone. The halogen-based solvent as the first solvent is preferably dichloromethane. The sulfoxide-based solvent as the first solvent is preferably dimethyl sulfoxide. The amide-based solvent as the first solvent is preferably dimethylformamide or dimethylacetamide.

[0481] The first solvent is preferably at least one selected from the group consisting of: 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, acetonitrile, tetrahydrofuran, toluene, 1,4-dioxane, acetone, dichloromethane, ethanol, methanol, dimethyl sulfoxide, dimethylformamide, and dimethylacetamide, and more preferably at least one selected from the group consisting of: 2,2,2-trifluoroethanol, acetonitrile, tetrahydrofuran, and acetone.

[0482] The second solvent may include at least one selected from the group consisting of: ether-based solvents, benzene-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, nitrile-based solvents, hydrocarbon-based solvents, alcohol-based solvents, sulfoxide-based solvents, and amide-based solvents.

[0483] The ether-based solvent as the second solvent is preferably at least one selected from the group consisting of: 2-methyltetrahydrofuran, tetrahydrofuran, 1,4-dioxane, MTBE (tert-butyl methyl ether), DME (dimethyl ether), and CPME (cyclopentyl methyl ether). The benzene-based solvent as the second solvent is preferably toluene or xylene. The ketone-based solvent as the second solvent is preferably acetone or methyl ethyl ketone. The halogen-based solvent as the second solvent is preferably dichloromethane. The hydrocarbon-based solvent as the second solvent is preferably at least one selected from the group consisting of: hexane, heptane, cyclohexane, methylcyclohexane, or isooctane. The ester-based solvent as the second solvent is preferably isopropyl acetate or ethyl acetate. The nitrile-based solvent as the second solvent is preferably acetonitrile. The alcohol-based solvent as the second solvent is preferably at least one selected from the group consisting of: methanol, ethanol, butanol, or benzyl alcohol. The sulfoxide-based solvent as the second solvent is preferably dimethyl sulfoxide. The amide-based solvent as the second solvent is preferably dimethylformamide.

[0484] The second solvent is preferably a combination of isopropyl acetate or methyl ethyl ketone and heptane, a combination of dichloromethane and hexane, or a combination of acetonitrile and MTBE.

[0485] The third solvent can be a solvent that is immiscible with water (e.g., a solvent having low solubility in water, a solvent having a high octanol / water partition coefficient (log Kow), or a solvent having a predicted high octanol / water partition coefficient).

[0486] The octanol / water partition coefficient (Log Kow) can be determined by any method known in the art or described herein. The predicted value of the octanol / water partition coefficient (Log Kow) can be determined in a separate definitive measurement by known means, for example but not particularly limited to database search or literature search. Examples of methods for measuring the octanol / water partition coefficient include but are not limited to the method according to Japanese Industrial Standard JIS 7260-107:2000 Measurement of Partition coefficient (1-octanol / water) Shake flask method (https: / / kikakurui.com / z7 / Z7260-107-2000-01.html) [Access date: December 25, 2023].

[0487] In the examples, organic solvents immiscible with water include but are not particularly limited to organic solvents having low water solubility (e.g., a solubility in water of 200 g / L or less, preferably 150 g / L or less). The organic solvent immiscible with water may contain trace amounts (e.g., 0.01 wt% or less) of other organic solvents miscible with water. The solubility in water can be determined by any method known in the art or described herein. Examples of methods for determining solubility include but are not intended to be particularly limited to gas chromatography, and the solubility can be determined by measuring the concentration of the organic solvent in water prepared by mixing the organic solvent with an equal volume of water at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C).

[0488] In the examples, to determine the miscibility of an organic solvent with water, the miscibility can also be shown by the fact that when the same volume of the solvent and water are mixed in a container, for example, at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C), the solvent and water separate into two layers. It can be determined whether the solvent and water separate into two layers, for example, by visual inspection or by collecting and examining the liquids in the upper and lower layers of the container. When it is confirmed that the solvent and water separate into two layers in this way, the solvent can be referred to as a solvent immiscible with water. However, even for a solvent miscible with water, depending on the solute in the solvent and the salt concentration in water, the solvent may still form an interface with water and separate into two layers.

[0489] In an embodiment, the water-immiscible solvent can be characterized as an ester having 3 or more and 10 or fewer carbon atoms, and specific examples thereof include ethyl acetate, isopropyl acetate, n-propyl acetate, tert-butyl acetate, methyl propionate, or ethyl propionate.

[0490] In an embodiment, the water-immiscible solvent can be characterized as a cyclic ether having 4 or more and 10 or fewer carbon atoms, and specific examples thereof include 2-methyltetrahydrofuran, tetrahydrofuran, 4-methyltetrahydropyran, or 1,4-dioxane. In an embodiment, the water-immiscible solvent can be characterized as an acyclic ether having 4 or more and 10 or fewer carbon atoms, and specific examples thereof include MTBE (tert-butyl methyl ether), diisopropyl ether, or diethyl ether.

[0491] In an embodiment, the water-immiscible solvent can be characterized as an ether having both a cyclic alkyl group and an acyclic alkyl group having 6 or more and 10 or fewer carbon atoms, and a specific example thereof is CPME.

[0492] In an embodiment, the water-immiscible solvent can be characterized as a carbonate having 3 or more and 10 or fewer carbon atoms, and specific examples thereof include dimethyl carbonate, diethyl carbonate, or diisopropyl carbonate.

[0493] In an embodiment, the water-immiscible solvent can be characterized as a hydrocarbon having 5 or more and 10 or fewer carbon atoms, and specific examples thereof include pentane, hexane, or heptane.

[0494] In an embodiment, the water-immiscible solvent can be characterized as an aromatic hydrocarbon ring having 6 or more and 10 or fewer carbon atoms, and specific examples thereof include toluene, xylene, or benzene.

[0495] In an embodiment, the water-immiscible solvent can be characterized as having a low boiling point at ambient pressure (about 1 atm). In an embodiment, examples of low boiling points at atmospheric pressure (close to 1 atm) include 35 °C or higher and lower than 140 °C.

[0496] 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.

[0497] The fourth solvent of the present invention may be at least one selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-butanol, ethylene glycol, cyclohexane, methylcyclohexane, heptane, isooctane, toluene, xylene, dimethyl sulfoxide, acetone, methyl ethyl ketone, tetrahydrofuran, tetrahydropyran, 4-methyltetrahydropyran, 2-methyltetrahydrofuran, 1,4-dioxane, MTBE (tert-butyl methyl ether), DME (dimethyl ether), CPME (cyclopentyl methyl ether), methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methyl isobutyl ketone, cyclohexanone, and acetonitrile.

[0498] Specific examples of ligands or ligand-generating compounds include olefinic compounds, carbonyl-based compounds, phosphine-based compounds, and carbon monoxide. Specific examples of anions or anion-generating compounds include iodide salts, bromide salts, chloride salts, perchlorates, oxide salts, trifluoromethanesulfonates, toluenesulfonates, isopropylsulfonates, methanesulfonates, carbonates, acetates, bis(trifluoromethanesulfonic acid) imide salts, ethyl malonates, 1,5-cyclooctadiene, dibenzylideneacetone, triphenylphosphine, and nitrites.

[0499] The method for purifying the cyclic peptide in this article can be a method for purifying the cyclic peptide as the purification target to have a purity of 85% or higher, 90% or higher, or 95% or higher, and can be a method for purifying the cyclic peptide as the purification target to have a purity of 100%, 98% or higher, or 97% or higher. The method for purifying the cyclic peptide in this article can increase the purity of the cyclic peptide as the purification target through the purification operations described herein. For example, the purity of the cyclic peptide as the purification target with 80% purity is 83%, 86%, 89%, 92%, 96% or 100%, or the purity of the cyclic peptide as the purification target is 1.03 times, 1.08 times, 1.11 times, 1.15 times or 1.20 times the purity before the purification operation until 100% purity. The method for purifying the cyclic peptide in this article can also be referred to as a method for reducing the impurity content in a mixture containing the cyclic peptide as the purification target and impurities, a method for reducing the ratio of impurities to the cyclic peptide as the purification target through the purification operations described herein, or a method in which the purification efficiency of impurities is a positive value, and this purification efficiency can be an index of the removal of impurities before and after the purification operations described herein. The purity (%) of the cyclic peptide as the purification target can be determined, for example, from the ratio of the peak area of the cyclic peptide as the purification target at 220 nm or 225 nm to the sum of each peak area of the entire mixture containing the cyclic peptide as the purification target, where the UV spectrum of the mixture containing the cyclic peptide as the purification target is measured using HPLC and PDA (photodiode array detector). The mixing ratio (%) of the peptide as an impurity (e.g., an isomer of the cyclic peptide as the purification target) can be determined, for example, from the ratio of the peak area of the peptide as an impurity at 220 nm or 225 nm to the sum of each peak area of the entire mixture containing the cyclic peptide as the purification target, where the UV spectrum of the mixture containing the cyclic peptide as the purification target is measured using HPLC and PDA (photodiode array detector).

[0500] The purification efficiency of impurities can be determined, for example, by the following: measuring the UV spectrum of the mixture containing the cyclic peptide as the purification target using HPLC and PDA (photodiode array detector), calculating the ratio of the peak area of the impurities (e.g., isomers) at 220 nm or 225 nm to the peak area of the cyclic peptide as the purification target, and comparing the ratios before and after the purification operations described herein. For example, the purification efficiency (%) of impurities can be determined by the following expression.

[0501] Purification efficiency of impurities (%) = {1 - (ratio of the cyclic peptide as the purification target to impurities after the purification operation) / (ratio of the cyclic peptide as the purification target to impurities before the purification operation)} × 100{1 - (ratio of the cyclic peptide as the purification target to impurities before the purification operation) / (ratio of the cyclic peptide as the purification target to impurities before the purification operation)} × 100

[0502] Yet another embodiment of the present invention relates to a method for producing a cyclic peptide, which production method includes the purification method of the cyclic peptide according to the present invention. The production method of the cyclic peptide may further include the step of obtaining the cyclic peptide as the purification target by a liquid-phase synthesis method, may further include the step of obtaining the cyclic peptide as the purification target by a solid-phase synthesis method, and may further include the step of obtaining the cyclic peptide as the purification target by a cultivation method. The step of obtaining the cyclic peptide as the purification target by the liquid-phase synthesis method means that the liquid-phase synthesis includes the step of obtaining the cyclic peptide as the purification target. The step of obtaining the cyclic peptide as the purification target by the solid-phase synthesis method means that the solid-phase synthesis includes the step of obtaining the cyclic peptide as the purification target. The liquid-phase synthesis method herein includes a method for synthesizing a peptide using an organic tag (also referred to as a hydrophobic tag). The method for synthesizing a peptide using an organic tag is not particularly limited, but examples thereof include those described in WO 2012 / 029794, WO 2007 / 122847, and WO2019 / 009317.

[0503] This application claims the priority of Japanese Patent Application No. 2022-212468 filed on December 28, 2022, the content of which is incorporated herein by reference in its entirety. All references cited herein, including the following references, including patent applications and publications, are incorporated herein by reference in their entirety: International Publication No. WO 2013 / 100132; International Publication No. WO2018 / 225851; International Publication No. WO 2018 / 225864; International Publication No. WO 2019 / 117274; International Publication No. WO2020 / 111238; International Publication No. WO 2020 / 122182; International Publication No. WO 2021 / 075478; International Publication No. WO2021 / 090856; International Publication No. WO 2021 / 132545; International Publication No. WO 202 / 1246471; International Publication No. WO2022 / 097540; International Publication No. WO 2022 / 138891; International Publication No. WO 2022 / 145444; International Publication No. WO2022 / 234864; and International Publication No. WO 2023 / 127869.

[0504] [Examples]

[0505] The present invention is further described in detail by way of examples and reference examples, but the present invention is not limited thereto. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods. The analysis conditions for LC / MS are shown in Table 1.

[0506]

[0507]

[0508] In the examples and reference examples, the following abbreviations are used.

[0509] AA: Ammonium acetate

[0510] COMU: (1-Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate

[0511] DBU: 1,8-Diazabicyclo[5.4.0]-7-undecene

[0512] DCM: Dichloromethane

[0513] DCE: 1,2-Dichloroethane

[0514] DMF: N,N-Dimethylformamide

[0515] DIC: N,N'-Diisopropylcarbodiimide

[0516] DIPEA: N,N-Diisopropylethylamine

[0517] DVB: Divinylbenzene

[0518] t-Bu group: Tert-butyl group

[0519] FA: Formic acid

[0520] Fmoc group: 9-Fluorenylmethyloxycarbonyl group

[0521] NMP: N-Methyl-2-pyrrolidone

[0522] NMR: Nuclear magnetic resonance spectroscopy

[0523] mM: mmol / L

[0524] PDA: Photodiode array detector

[0525] TFA: Trifluoroacetic acid

[0526] TFE: 2,2,2-Trifluoroethanol

[0527] THF: Tetrahydrofuran

[0528] THP: Tetrahydropyranyl

[0529] MTBE: Methyl tert-butyl ether

[0530] HOAt: 1-Hydroxy-7-azabenzotriazole

[0531] HOBt: 1-Hydroxybenzotriazole

[0532] IPAC: Isopropyl acetate

[0533] MEK: Methyl ethyl ketone

[0534] HPLC: High performance liquid chromatography

[0535] oxyma: Ethyl cyano(hydroxyimino)acetate

[0536] EDCI·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0537] DMSO: Dimethyl sulfoxide

[0538] MeCN: Acetonitrile, CH3CN

[0539] 1 H: Proton

[0540] v / v: Volume / volume

[0541] NMR measurements were carried out using an AVANCE III 600 Cryo-TCI, Avance III HD 600 SMART-BBFO, AVANCE NEO 600 iProbeTBO, or AVANCE NEO 600 Cryo-TCI-H&F manufactured by Bruker at an absolute temperature of 298 K or 278 K.

[0542] [Reference Example 1: Synthesis of Cyclic Peptide]

[0543] <General Synthesis Method of Amino Acids>

[0544] The Fmoc group-protected amino acids (Fmoc-amino acids) used in the examples and reference examples can be produced by general amino acid synthesis methods, such as those described in WO 2021 / 090855. Fmoc group-protected amino acids can also be obtained from commercial suppliers. In the examples and reference examples, amino acids in which the Fmoc group is introduced at the N-terminus of commercially available amino acids or amino acids in which the C-terminus of commercially available amino acids is deprotected can also be used in the extension reaction of the peptide of interest. The reactions for attachment and removal of the protecting group can be carried out, for example, by the methods described in "Greene's, Protective Groups in Organic Synthesis" (5th Edition, John Wiley & Sons 2014). Amino acids having side chains (functional groups on the α-carbon of the amino acid) different from those of naturally occurring amino acids can be produced by appropriately converting the side chains of amino acids (naturally occurring amino acids) available from commercial suppliers into the side chains of interest. The conversion of the side chain can be carried out, for example, by the methods described in the following literature: R.C. Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition; M.B. March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition; etc. N-Terminally alkylated amino acids (N-alkyl amino acids) can be produced by reacting the N-terminally unalkylated amino acid with an aldehyde according to Freidinger et al. (J. Org. Chem., 1983, 48(1), 77-81) to produce an oxazolidinone compound in which a cyclic protecting group is introduced, and carrying out a ring-opening reaction of the cyclic protecting group.

[0545] The Fmoc-amino acids used in the examples and reference examples are shown in Tables 2 to 4. The Fmoc-amino acids described in Tables 2 to 4 were purchased from commercial suppliers or synthesized with reference to the methods described in WO 2021 / 090855.

[0546] [Table 2]

[0547]

[0548] [Table 3]

[0549]

[0550] [Table 4]

[0551]

[0552] <Synthesis Method of Fmoc-Amino Acid-Loaded Resin>

[0553] Synthesize compound aa01, compound aa02, and compound aa03 (which are Fmoc-amino acids) as shown in the following formula (1). Use the obtained compound aa03 to synthesize compound aa03-resin ((3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutyric acid 2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro), where compound aa03 is loaded on the resin. Perform peptide synthesis using compound aa03-resin with a peptide synthesizer. The loading reaction of the Fmoc-amino acid onto the resin is carried out by the method described in WO 2013 / 100132 or WO 2018 / 225864. 2-Chlorotrityl chloride resin (100-200 mesh, 1% DVB) was purchased from WATANABE CHEMICAL INDUSTRIES, LTD. and Chem-Impex International, Inc. Note that pyrro means pyrrolidine. For example, in compound aa03-resin, the C-terminal carboxyl group forms an amide bond with pyrrolidine.

[0554] [Formula 1]

[0555]

[0556] In this article, in the chemical formula of a compound obtained by binding a polymer or resin to an amino acid or peptide compound, a part or all of the polymer or resin can be represented as a circle (○). The chemical structure representing the binding site of the resin to the amino acid or peptide compound can be shown to clarify the structure of the binding site. For example, in compound aa03-resin of the above formula (1), a part of the resin is indicated by ○, and it is shown that the 2-chlorotrityl group of the resin and the side-chain carboxyl group of Asp are bound via an ester bond.

[0557] In formula (1), compound aa01 was purchased from a commercial supplier. The synthesis method of compound aa02 will be described in detail. EDCI·HCl (67.1 g, 350 mmol), HOBt (43.4 g, 321 mmol), and Fmoc-Asp(OtBu)-OH (compound aa01, CAS: 71989-14-5) (120 g, 292 mmol) were successively mixed into DMF (600 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 0 °C for 1 hour. To obtain the reaction solution, pyrrolidine (26.3 mL, 321 mmol) was slowly added, and the mixture was stirred at 0 °C for 1 hour and 30 minutes. Ethyl acetate (10 v / w) and 0.5 mol / L hydrochloric acid aqueous solution (2 v / w) were added to this reaction solution at 0 °C, and the organic phase was separated. The obtained organic phase was successively washed with 0.5 mol / L hydrochloric acid aqueous solution, water, saturated sodium bicarbonate aqueous solution / water (1 / 1 (v / v)), and saturated sodium chloride aqueous solution / 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 aa02 (137.1 g, quantitative) as a crude product.

[0558] LCMS(ESI) m / z = 465 (M + H) +

[0559] Retention time: 1.05 minutes (synthesis conditions: SQDAA05)

[0560] The synthesis method of compound aa03 will be described in detail. TFA (271 mL) was slowly added to a solution of compound aa02 (137 g, 395 mmol) in DCM (137 mL) in such a way that the internal temperature did not exceed 10 °C with ice cooling. The resulting mixed solution was stirred at room temperature for 1 hour, and diisopropyl ether (3.4 L) was added in four portions. The precipitated solid was taken out by filtration and dried to obtain compound aa03 ((3s)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutyric acid, Fmoc-Asp-pyrro) (108.4 g, 90%).

[0561] LCMS(ESI) m / z = 409 (M + H) +

[0562] Retention time: 0.83 minutes (synthesis conditions: SQDAA05)

[0563] The synthesis method of compound aa03-resin will be described in detail. Charge 2-chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, 48.7 g) and dehydrated dichloromethane (500 mL) into a reaction vessel equipped with a filter, and shake the reaction vessel at room temperature for 20 minutes. After applying nitrogen pressure to the reaction vessel with a filter to remove the dehydrated dichloromethane, a mixed solution of compound aa03 (15.91 g), dehydrated dichloromethane (350 mL), dehydrated methanol (12.63 mL), and diisopropylethylamine (DlPEA) (32.6 mL) is added to the reaction vessel, and the reaction vessel is shaken for 60 minutes. After applying nitrogen pressure to the reaction vessel to remove the liquid components, a mixed solution of dehydrated dichloromethane (350 mL), dehydrated methanol (97.3 mL), and diisopropylethylamine (DIPEA) (32.6 mL) is added to the reaction vessel, and the reaction vessel is shaken for 1 hour and 30 minutes. After applying nitrogen pressure to remove the liquid components, dichloromethane (350 mL) is added, and the reaction vessel is shaken for 5 minutes. After applying nitrogen pressure to the reaction vessel to remove the liquid components, dichloromethane (350 mL) is added, and the reaction vessel is shaken for 5 minutes. Thus, washing the resin with dichloromethane is repeated five times. The washed resin is dried under reduced pressure overnight to obtain (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutyric acid 2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound aa03-resin, 59.79 g).

[0564] To determine the loading rate, the obtained aa03-resin (12.6 mg) was placed in a reaction vessel, DMF (2 mL) was added, and the reaction vessel was shaken at room temperature for 1 hour. DBU (40 μL) was added to the reaction vessel, and the reaction vessel was shaken at 30 °C for 30 minutes. After that, DMF (8 mL) was mixed in the vessel, and 1 mL of the reaction solution was collected from the reaction vessel. The 1 mL of the collected reaction solution was diluted with DMF (11.5 mL). The absorbance (294 nm) of the obtained diluted solution was measured (SHIMADZU CORPORATION, UV-1600PC (cell length: 1.0 cm)). By measuring the absorbance of dibenzofluorene of the Fmoc group of the Fmoc-amino acid loaded on the resin, the loading amount of compound aa03 was calculated to be 0.464 mmol / g. Another batch of different amounts that had been similarly synthesized and loaded was also used for peptide synthesis, research, etc.

[0565] Synthesize compound aa04-resin according to the description of WO 2013 / 100132.

[0566] [Formula 2]

[0567]

[0568] <Synthesis Method of Fmoc-Peptide-Loaded Resin>

[0569] [Formula 3]

[0570]

[0571] In the above formula (3), the compounds aa05 (Fmoc-Ile-OH, CAS: 71989-23-6), Fmoc-Pro-OH (CAS: 71989-31-6) and Fmoc-Asp(OAl)-OH (CAS: 146982-24-3) were purchased from commercial suppliers. The synthesis method of the compound aa06-resin (Fmoc-Ile-pip) will be described in detail. Under a nitrogen atmosphere, EDCI·HCl (3.25 g, 17.0 mmol) was mixed with DMF (30 mL), and the mixture was stirred at room temperature for 10 minutes and then cooled to 0 °C. HOBt (2.10 g, 3.25 mmol) and the compound aa05 (Fmoc-Ile-OH, CAS: 71989-23-6) (15.00 g, 14.15 mmol) were successively mixed, and the mixture was stirred at 0 °C for 1 hour. To obtain the reaction solution, piperidine (1.27 g, 14.9 mmol) was slowly added, and the mixture was stirred at 0 °C for 1 hour. Ethyl acetate (10 v / w) and 0.5 mol / L hydrochloric acid aqueous solution (10 v / w) were added to this reaction solution, and the organic phase was separated. The obtained organic phase was successively washed with 0.5 mol / L hydrochloric acid aqueous solution, water, 5% sodium carbonate aqueous solution and saturated sodium chloride aqueous solution / 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 the compound aa06 (Fmoc-Ile-pip) (6.11 g) as a crude product.

[0572] LCMS (ESI) m / z = 421 (M+H) +

[0573] Retention time: 0.98 minutes (synthesis conditions: SQDFA05)

[0574] The synthesis method of compound aa07 (Fmoc-Pro-Ile-pip) will be described in detail. 1,8-Diazabicyclo[5.4.0]-7-undecene (DBU, 2.21 g, 14.53 mmol) was added to a solution of compound aa06 (Fmoc-Ile-pip) in DMF (50 mL), and then the mixture was stirred at room temperature for 10 minutes. Triethylamine hydrochloride (2.00 g, 14.53 mmol), N,N-diisopropylethylamine (DIPEA, 2.54 mL, 14.53 mmol), Fmoc-Pro-OH (CAS: 71989-31-6) (4.90, 14.53 mmol), EDCI·HCl (3.90 g, 20.35 mmol) and HOAt (2.37 g, 17.43 mmol) were successively added to this reaction solution, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate (10 v / w) and 0.5 mol / L hydrochloric acid aqueous solution (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 hydrochloric acid aqueous solution, water, 5% sodium carbonate aqueous solution and saturated sodium chloride aqueous solution / 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 aa07 as a crude product. DMSO (5 mL) and ethyl acetate (20 mL) were added to the obtained crude product, and the obtained solution was filtered, and then ethyl acetate was distilled off to some extent under reduced pressure. The obtained residue was purified by reverse-phase medium-pressure column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain compound aa07 (Fmoc-Pro-Ile-pip) (2.41 g, 32%, 97% purity [peak area percentage]).

[0575] LCMS (ESI) m / z = 518 (M+H) +

[0576] Retention time: 0.94 minutes (synthesis conditions: SQDFA05)

[0577] The synthesis method of compound aa08 (Fmoc-Asp(OAI)-Pro-Ile-pip) will be described in detail. 1,8-Diazabicyclo[5.4.0]-7-undecene (DBU, 0.702 mL, 4.66 mmol) was added to a solution of compound aa07 (Fmoc-Pro-Ile-pip) (2.41 g, 4.66 mmol) in DMF (23 mL), and then the mixture was stirred at room temperature for 10 minutes. Triethylamine hydrochloride (0.641 g, 4.66 mmol), N,N-diisopropylethylamine (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 successively added to this reaction solution, and the mixture was stirred at room temperature for 1 hour and 30 minutes. Ethyl acetate (10 v / w) and 0.5 mol / L hydrochloric acid aqueous solution (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 hydrochloric acid aqueous solution, water, 5% sodium carbonate aqueous solution, and saturated sodium chloride aqueous solution / 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 aa08 as a crude product. The obtained crude product was dissolved in DMSO and purified by medium-pressure column chromatography in the reverse phase (acetonitrile / water containing 0.1% formic acid) to obtain compound aa08 (Fmoc-Asp(OAl)-Pro-Ile-pip) (1.71 g, 55%, 84% purity [peak area percentage]).

[0578] LCMS (ESI) m / z = 674 (M+H) +

[0579] Retention time: 2.94 minutes (synthesis conditions: SQDA05 long)

[0580] The synthesis method of compound aa09 will be described in detail. To a solution of compound aa08 (Fmoc-Asp(OAl)-Pro-Ile-pip) (1.71 g, 84% purity) in dichloromethane (5.08 mL) was added tetrakis(triphenylphosphine)palladium(0) (CAS: 14221-01-3, tetrakis(triphenylphosphine)palladium(0), Pd(PPhe3)4, 0.294 g, 0.254 mmol) and phenylsilane (CAS: 694-53-1, phenylsilane, 0.313 mL, 2.54 mmol), and the mixture was stirred at 0 °C for 15 minutes. The solvent in the obtained reaction solution was distilled off under reduced pressure, and purification was carried out by medium-pressure column chromatography on a reversed phase (acetonitrile / water containing 0.1% formic acid) to obtain compound aa09 (Fmoc-Asp-Pro-Ile-pip) (1.11 g, 69%, 99% purity [peak area percentage]).

[0581] LCMS(ESI) m / z = 633 (M+H) +

[0582] Retention time: 0.80 minutes (synthesis conditions: SQDFA05)

[0583] Compound aa09-resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-Ile-pip) was synthesized according to the description in WO 2013 / 100132.

[0584] <General Synthesis Method of Peptides>

[0585] The peptide compounds used in the examples and reference examples were synthesized by solid-phase synthesis. Examples of solid-phase synthesis methods can include Amino Acids, 2018, 50, 39-68 or Solid-Phase Peptide Synthesis (published by Bachem Holding AG) [search date: December 20, 2022], Internet <URL: https: / / www.bachem.com / wpfd_file / solid-phase-peptide-synthesis / >. In the examples and reference examples, the peptide synthesis method by the Fmoc method described in WO 2013 / 100132 or WO 2018 / 225864 was referred to. In the examples and reference examples, peptide elongation was carried out by Figure 1 the basic synthesis method of cyclic peptides shown in Figure 1 The basic synthesis method of cyclic peptides shown in includes the following five-step method:

[0586] 1) Peptide elongation reaction is carried out from the N-terminus of an amino acid by the Fmoc method using a substance in which the carboxyl group of the Asp side chain or the carboxyl group of the peptide main chain is loaded on 2-chlorotrityl resin (peptide chain elongation reaction is carried out using Fmoc-group protected amino acids as raw materials).

[0587] 2) Method for cleaving a peptide from 2-chlorotrityl resin;

[0588] 3) Amide cyclization is carried out by condensation of the carboxyl group of the Asp side chain or the carboxyl group of the peptide main chain generated by the method of cleaving from 2-chlorotrityl resin with the amino group (triangular unit) at the N-terminus of the peptide chain

[0589] 4) Optionally, deprotect the protecting groups of the side chain functional groups contained in the peptide chain, and

[0590] 5) Purify the compound by preparative HPLC. In the examples and reference examples, unless otherwise specified, the synthesis of cyclic peptides is carried out by Figure 1 the basic synthesis method of cyclic peptides shown in

[0591] <Synthesis Method of Peptide Compounds Containing N-Alkyl Amino Acids>

[0592] Peptide compounds containing N-alkyl amino acids can be synthesized using N-alkyl amino acids protected by Fmoc groups shown in Tables 2 to 4 as raw materials according to the "General Synthesis Method of Peptides" described in the examples and reference examples.

[0593] <1. Solid-Phase Synthesis of Peptides by Automated Synthesizer>

[0594] Compound 1 is synthesized by the method described in International Publication No. WO 2013 / 100132 or WO 2018 / 225864. The detailed operating procedures of the peptide synthesizer (Multipep RS; CEM Corporation (formerly Intavis AG)) comply with the manual attached to the synthesizer. The abbreviations, amino acid structural formulas and official names of each amino acid residue constituting Compound 1 (cyclic peptide) and Compound 6 (cyclic peptide) are shown in Table 5 below.

[0595] [Table 5]

[0596]

[0597]

[0598] <1-1. Peptide Elongation Reaction from the N-Terminus of Amino Acids by Fmoc Method>

[0599] Dissolve the Fmoc-amino acids (concentration in Solution 1: 0.3 to 0.6 mol / L) that constitute the cyclic peptide as the purification target and HOAt, oxyma, or HOOBt (concentration in Solution 1: 0.375 mol / L), which is an activator of the carboxyl group, in NMP or NMP / DMF (1 / 1) to prepare Solution 1. Mix N,N'-diisopropylcarbodiimide (DIC) (10% v / v) with N,N-dimethylformamide (DMF) to prepare Solution 2.

[0600] When using Fmoc-Ser(THP)-OH or Fmoc-Thr(THP)-OH as the Fmoc-amino acids having a THP group in the side chain, use oxyma as the activator of the carboxyl group to prepare Solution 1, and add Molecular Sieves 4A 1 / 8 (FUJIFILM Wako Pure Chemical Corporation) or Molecular Sieves 4A 1 / 16 (FUJIFILM Wako Pure Chemical Corporation) for peptide synthesis.

[0601] Place 2-chlorotrityl resin (100 mg) bound to the side-chain carboxyl group of aspartic acid whose N-terminus is protected with an Fmoc group into a reaction vessel with a filter and set it in a peptide synthesizer. Add dichloromethane (DCM) (0.8 mL) to this resin (100 mg), and allow the mixture to stand for about 1 hour to swell the resin. Then drain the solution from the reaction vessel through the filter. Set Solution 1 and Solution 2 in the peptide synthesizer and start the automatic synthesis with the peptide synthesizer.

[0602] <Deprotection of Fmoc Step>

[0603] Add a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF (2% v / v, 0.7 mL) to the reaction vessel with a filter containing the swollen resin at room temperature to deprotect the Fmoc group at the N-terminus. Carry out the reaction for 4.5 minutes in the deprotection of the first residue and for 10 minutes in the deprotection of the second and subsequent residues, and then drain the solution from the reaction vessel through the filter. Add DMF (0.7 mL) thereto to wash the resin and allow it to stand for 5 minutes, and drain the solution from the reaction vessel through the filter. Repeat this resin washing step three more times to obtain a resin in which the N-terminal Fmoc group of the amino acid or peptide attached to the resin has been removed to become an amino group.

[0604] <Elongation Step>

[0605] Subsequently, solution 1 (0.3 mL / reaction vessel) and solution 2 (0.36 mL / reaction vessel) were mixed with the mixing vial of the synthesizer, and the mixture was then added to the deprotected resin, and the filtered reaction vessel was heated to 40°C. The condensation reaction between the amino group on the resin and the Fmoc-amino acid was carried out for 2.5 hours. After the reaction, the solution was then discharged from the reaction vessel with a filter. Subsequently, the reacted resin was washed 3 times with DMF (0.7 mL). This condensation reaction of the Fmoc-amino acid after the deprotection reaction of the Fmoc group was set as one cycle, and this cycle was repeated to extend the peptide on the resin surface. After the last amino acid extension, no de-Fmoc step was performed, and the resin was washed four times with DCM (1.0 mL / reaction vessel).

[0606] <Synthesis of Fmoc-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal- Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1)>

[0607] [Formula 4]

[0608]

[0609] Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound aa03-resin, 0.497 mmol / g, 100 mg, 48 reaction vessels with filters, each containing 0.0497 mmol of the compound), Fmoc-MeVal-OH, Fmoc-MePhe(4-F)-OH, Fmoc-Ala(cPent)-OH, Fmoc-MePhe(34-F2)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Cha-OH, Fmoc-MeAla-OH, and Fmoc-Pro-OH were used as starting materials. Compound 1 was prepared by peptide extension reaction using the Fmoc method described above.

[0610] <1-2. Synthesis of Compound 6 (3S,6S,9S,12S,18S,22S,25S,28S,31S,34S,37S)-6-(Cyclohexylmethyl)-31- (Cyclopentylmethyl)-34-[(3,4-Difluorophenyl)methyl]-28-[(4-Fluorophenyl)methyl]-3-Isobutyl-25- Isopropyl-9,10,18,19,26,29,35-Heptamethyl-22-(Pyrrolidine-1-Carbonyl)-1,4,7,10,16,19,23,26,29, 32,35-Undecaazatricyclo[35.3.0.012,16]tetratetracontane-2,5,8,11,17,20,24,27,30,33,36-Undecaone> <Synthesis of Compound 6>

[0611] [Formula 5]

[0612]

[0613] <Deprotection of Fmoc Step>

[0614] Fmoc-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1) was washed by adding toluene (1 mL per reaction vessel), and allowed to stand for 5 minutes, and then the solution was drained from the reaction vessel through a filter. This washing step of the resin was repeated two more times to obtain the resin swollen with toluene.

[0615] A solution of diazabicycloundecene (DBU) in toluene (2% v / v, 0.7 mL per reaction vessel) was added to the solid-phase reaction vessel containing the resin swollen with toluene at room temperature to deprotect the N-terminal Fmoc group. After reacting for 10 minutes, the solution was drained from the reaction vessel through a filter. Toluene (0.7 mL per reaction vessel) was added thereto to wash the resin and allowed to stand for 5 minutes, and the solution was drained from the reaction vessel through a filter. The step of washing the resin with toluene was repeated once more, and then DCM (0.7 mL per reaction vessel) was added thereto to wash the resin and allowed to stand for 5 minutes, and the solution was drained from the reaction vessel through a filter. The step of washing the resin with DCM was repeated once more to obtain H-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2), in which the N-terminal Fmoc group attached to the peptide on the resin was removed to become an amino group.

[0616] <Elongation Step>

[0617] To the resin obtained as described above was added a solution prepared by adding Fmoc-MeAla-OH (4 molar equivalents) to 2)A solution of 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim, 153433-21-7, 4 molar equivalents) in DCM (0.25 mL) and DIPEA (6 molar equivalents) and a solution obtained by allowing the mixture to stand for about 1 to 2 minutes, and the mixture was shaken at room temperature for 3 hours. The number of molar equivalents was calculated based on the value obtained by multiplying the ratio (mmol / g) of the amino acid loaded on the resin used as the raw material by the amount of the resin used (usually 100 mg). The solution was drained from the reaction vessel through a filter, and then the resin was washed 4 times with DMF (0.7 mL per reaction vessel) and 4 times with DCM (0.7 mL per reaction vessel), and dried to obtain Fmoc-MeAla-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 3). After completion of the peptide elongation, a solution of diazabicycloundecene (DBU) in DMF (2% v / v, 0.7 mL per reaction vessel) was added to the resin, and the reaction was carried out for 15 minutes to carry out the deprotection reaction of the Fmoc group, and then the solution was drained from the reaction vessel through a filter. The obtained resin was washed 4 times with DMF (0.7 mL) and 4 times with DCM (0.7 mL) to obtain H-MeAla-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 4).

[0618] <Step of Cleaving the Elongated Peptide from the Resin>

[0619] To the resin (Compound 4) obtained by the above method, 2,2,2-trifluoroethanol (TFE) / DCM (1 / 1, v / v, 2 mL) containing 0.75% (v / v) DIPEA was added, and the reaction was carried out at room temperature for 2 hours to cleave the peptide chain from the resin. After the reaction, the solution in the tube was recovered from the reaction vessel using a filter. The operation of adding 2,2,2-trifluoroethanol (TFE) / DCM (1 / 1, v / v, 1 mL) to the remaining resin and recovering the solution from the reaction vessel with a filter was carried out twice. After mixing all the obtained cleavage solutions and mixing DMF (4 mL) or 1,2-dichloroethane (4 mL), the solvent was distilled off under reduced pressure using a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac Ltd. to obtain H-MeAla-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp-pyro (Compound 5).

[0620] <Cyclization Method of the Cleaved Peptide>

[0621] The residue obtained by the above method was dissolved in a mixed solution of DMF (8 mL) and DCM (8 mL), and a DMF solution (0.5 M, 1.5 molar equivalents) of (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) and DIPEA (1.8 molar equivalents) were added, and the mixture was stirred at room temperature for 30 minutes to carry out a condensation cyclization reaction between the amino group at the N-terminus and the carboxyl group at the C-terminus. The number of molar equivalents was calculated based on the value obtained by multiplying the ratio (mmol / g) of amino acid loading on the resin used as the raw material by the amount of resin used (usually 100 mg). The generation of the cyclic peptide of interest was carried out by LC / MS measurement (SQ detector 2 manufactured by Waters Corporation), and then the reaction solution was subjected to a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac Ltd. to distill off the solvent under reduced pressure.

[0622] <Purification Method of Cyclic Peptide>

[0623] DMSO was added to the residue obtained by the above method (48 filtered reaction vessels), and the mixture was purified in one batch by medium-pressure reverse-phase column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain compound 6 ((3S,6S,9S,12S,18S,22S,25S,28S,31S,34S,37S)-6-(cyclohexylmethyl)-31-(cyclopentylmethyl)-34-[(3,4-difluorophenyl)methyl]-28-[(4-fluorophenyl)methyl]-3-isobutyl-25-isopropyl-9,10,18,19,26,29,35-heptamethyl-22-(pyrrolidin-1-carbonyl)-1,4,7,10,16,19,23,26,29,32,35-undecaazatricyclo[35.3.0.012,16]tetracontane-2,5,8,11,17,20,24,27,30,33,36-undecanone) (2.3 g, 68%, 84% purity [peak area percentage]). Under the conditions of LC for purity analysis (purification analysis condition A), isomer 1 and isomer 2 (which are isomers of compound 6) were confirmed from the results of precision mass spectrometry before and after the retention time of compound 6 (about 11.1 minutes), and their retention times were about 10.8 minutes and about 11.4 minutes, respectively. From the analysis of the peak areas of the LC, it was also confirmed that the peak area percentage of isomer 1 of compound 6 observed near the retention time of 10.8 minutes (relative retention time: 0.972) was 2.0%, and the peak area percentage of isomer 2 of compound 6 observed near the retention time of 11.4 minutes (relative retention time: 1.03) was 6.8%. Based on the results of the synthesis method and precision mass spectrometry, it was assumed that isomer 1 and / or isomer 2 were diastereomers of compound 6. The relative retention time refers to the retention time of each peak divided by the retention time of the target (in this case, compound 6).

[0624] [Formula 6]

[0625]

[0626] Relative amount, yield: 2.3 g, 68%

[0627] Purity: 84.0%

[0628] LCMS(ESI) m / z = 1426 (M-H) -

[0629] Retention time: 0.85 minutes (analysis condition SQDAA50)

[0630] HPLC Conditions for Purification Analysis (Purification Analysis Condition A)

[0631] Column: ACQUITY UPLC Peptide CSH C18, 2.1x 150mm, 1.7μm

[0632] Solvents: A) 0.1% FA in H2O, B) 0.1% FA in CH3CN

[0633] Gradient program: 50% → 100% B (15 min) → 100% B (20 min) → 50% B (20.1 min)

[0634] Column temperature: 50 °C

[0635] Flow rate: 0.3 mL / min

[0636] Autosampler temperature: 20 °C

[0637] Sample concentration: Approximately 0.3 mg / mL in CH3CN

[0638] Injection volume: 5 μL

[0639] UV detection: 220 nm

[0640] System: Nexera X2 (SHIMADZU CORPORATION)

[0641] <1-3. Synthesis of Compound 9 (S)-1-((2S,5S,8S,11S,14S,17S,20S,23S)-14,20-Dibenzyl-8- ((S)-sec-Butyl)-5,17-Diisobutyl-11-Isopropyl-2,7,13,19-Tetramethyl-3,6,9,12,15,18,21,25-Octa Oxy-1,4,7,10,13,16,19,22-Octaazacyclopentacosane-23-Carbonyl)-N-((2S,3S)-3-Methyl-1-Oxo- 1-(Piperidin-1-yl)Pent-2-yl)Pyrrolidine-2Carboxamide)>

[0642] Compound aa09-resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-Ile-pip) was synthesized and purified in the same manner as compound 6 shown in the following formula (7) to obtain compound 9 (246 mg, 47%, 93.0% purity [peak area percentage]).

[0643] [Formula 7]

[0644]

[0645] Relative amount, yield: 246 mg, 47%

[0646] Purity: 93.0%

[0647] LCMS (ESI) m / z = 1237 (M-H) -

[0648] Retention time: 0.81 min (analysis condition SQDFA50)

[0649] HPLC Conditions for Purity Analysis: Purification Analysis Condition C

[0650] <1-4. Synthesis of Compound 11 ((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-(Piper ((3S,6S,9S,12S,16S,19S,25S,28S,31S,36aS)-25,28-Benzyl-19- ((R)-1-Hydroxyethyl)-3,31-Diisobutyl-9-Isopropyl-2,6,8,15,16,21,24,27,30-Nonamethyl-12-(Piperidin-1-Carbonyl)Tetrahydro-Pyrrolo[2,1-l][1,4,7,10,13,16,19,22,25,28,31]Undecaazacyclotetratriacontane-1,4,7,10,14,17,20,23,26,29,32(11H)-Undecanone) Synthesis>

[0651] The compound 10 ((3S,6S,9S,12S,18S,21S,24S,27S,30R,34S)-6,27-dibenzyl-3,12,21,24-tetraisobutyl-30-isopropyl-4,7,13,16,22,28-hexamethyl-34-(piperidine-1-carbonyl)-9-(propoxymethyl)-18-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotetratriacontane-2,5,8,11,14,17,20,23,26,29,32-undecanone) was synthesized according to the description of WO 2021 / 090855 using the compound aa04-resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip).

[0652] ((3S,6S,9S,12S,15S,18S,21S,27S,30S,33S,41S)-6,9-Dibenzyl-

[0653] In the sequence synthesized using Fmoc-protected amino acids having a THP-protected hydroxyl group in the side chain (e.g., Fmoc-Thr(THP)-OH and Fmoc-Ser(THP)-OH), a solution of 4 mL of tetramethylammonium hydrogensulfate (0.05 M) in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (containing 2% (v / v) triisopropylsilane (TIPS) and 1% (v / v) 1,2-dichloroethane) was added to the residue obtained above (14 reaction vessels). The residue was dissolved and allowed to stand at room temperature for 4 hours to deprotect the THP group. After confirming the completion of the reaction by LC / MS (SQ detector 2 manufactured by Waters Corporation), diisopropylethylamine (DIPEA) (70 μL) was added to the reaction solution (14 reaction vessels), and the solvent was distilled off under reduced pressure using a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac Ltd. to obtain compound 11 (372 mg, 44%, 92.3% purity [peak area percentage]).

[0654] [Formula 8]

[0655]

[0656] Relative amount, yield: 372 mg, 44%

[0657] Purity: 92.3%

[0658] LCMS(ESI) m / z = 1398 (M-H) -

[0659] Retention time: 0.82 minutes (analysis condition SQDFA50)

[0660] ((S)-sec-Butyl)-27-((R)-1-Hydroxyethyl)-12,18,21,30,33-Pentaisobutyl-3,7,13,19,22,

[0661] To the cyclic peptide compounds synthesized according to the production method of Compound 6 (14 reaction vessels / compound), add a solution of 4 mL of tetramethylammonium hydrogensulfate (0.05 M) in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (containing 2% (v / v) triisopropylsilane (TIPS), 1% (v / v) 1,2-dichloroethane) (14 reaction vessels / compound). Dissolve the residue and allow it to stand at room temperature for 4 hours to deprotect the THP group. After confirming the completion of the reaction by LC / MS (SQ detector 2 manufactured by Waters Corporation), add diisopropylethylamine (DIPEA) (70 μL) to the reaction solution, and distill off the solvent under reduced pressure using a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac Ltd. Collect the obtained residue of each compound in a container, and purify each compound by medium-pressure reverse-phase column chromatography (acetonitrile / water containing 0.1% formic acid) to obtain Compounds 12 to 15.

[0662] ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,41S)-6,24-Dibenzyl- ((S)-sec-Butyl)-21-((R)-1-Hydroxymethyl)-3,9,12,18,30-Pentaisobutyl-4,10,13,15,16, ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39R,43S)- ((3S,6S,9S,12S,16S,19S,25S,28S,31S,36aS)-25,28-Benzyl-19-

[0663] [Formula 9]

[0664]

[0665] Relative amount, yield: 233 mg, 30%

[0666] Purity: 85.7%

[0667] LCMS(ESI) m / z = 1296 (M-H) -

[0668] Retention time: 0.54 minutes (analysis condition SQDFA50)

[0669] ((R)-1-Hydroxyethyl)-3,31-Diisobutyl-9-Isopropyl-2,6,8,15,16,21,24,27,30-Nonamethyl-12-(Piperidin-1-Carbonyl)Tetrahydro-Pyrrolo[2,1-l][1,4,7,10,13,16,19,22,25,28,31]Undecaazacyclotetratriacontane-1,4,7,10,14,17,20,23,26,29,32(11H)-Undecanone) Synthesis>

[0670] ((3S,6S,9S,12S,15S,18S,21S,27S,30S,33S,41S)-6,9-Dibenzyl- ((S)-sec-Butyl)-27-((R)-1-Hydroxyethyl)-12,18,21,30,33-Pentaisobutyl-3,7,13,19,22, ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,41S)-6,24-Dibenzyl- ((S)-sec-Butyl)-21-((R)-1-Hydroxymethyl)-3,9,12,18,30-Pentaisobutyl-4,10,13,15,16,

[0671] [Formula 10]

[0672]

[0673] Relative quantity, yield: 388 mg, 42%

[0674] Purity: 92.3%

[0675] LCMS(ESI) m / z = 1552 (M-H) -

[0676] Retention time: 0.74 minutes (analysis condition SQDFA50)

[0677] ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39R,43S)-

[0678] ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,41S)-6,24-Dibenzyl- ((S)-sec-Butyl)-21-((R)-1-Hydroxymethyl)-3,9,12,18,30-Pentaisobutyl-4,10,13,15,16, ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39R,43S)- ((3S,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,41S)-6,24-Dibenzyl-

[0679] [Formula 11]

[0680]

[0681] Relative quantity, yield: 203 mg, 21%

[0682] Purity: 84.3%

[0683] LCMS(ESI) m / z = 1594 (M-H) -

[0684] Retention time: 0.81 minutes (analysis condition SQDFA50)

[0685] ((S)-sec-Butyl)-21-((R)-1-Hydroxymethyl)-3,9,12,18,30-Pentaisobutyl-4,10,13,15,16,

[0686] ((S)-sec-Butyl)-21-((R)-1-Hydroxymethyl)-3,9,12,18,30-Pentaisobutyl-4,10,13,15,16, Synthesis of 21,36-dibenzyl-27-((S)-sec-butyl)-6-((R)-1-hydroxyethyl)-9-(hydroxymethyl)-3,12,18,24,30-penta isobutyl-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-tetradecaazacyclotetratetracontane-2,5,8,11,14,17,20,23,26,29,32, 35,38,41-tetradecanone)>

[0687] [Formula 12]

[0688]

[0689] Relative quantity, yield: 388 mg, 42%

[0690] Purity: 89.5%

[0691] LCMS(ESI) m / z = 1667 (M-H) -

[0692] Retention time: 0.74 minutes (analysis condition SQDFA50)

[0693] HPLC conditions for purity analysis: Purification analysis condition C

[0694] [Reference Example 2: Search for metal salts capable of forming complexes]

[0695] <Basic operation>

[0696] Measure each of the following solutions at 278 K or 298 K for 1 1H-NMR: a solution obtained by dissolving Compound 6 in acetonitrile-d3 (deuterated acetonitrile, CD3CN, CAS: 2206-26-0) (concentration of cyclic peptide: 1.4 mM, experiment without addition of metal salt), and a solution obtained by mixing the metal salts shown in Table 6 below with the cyclic peptide such that the metal salts are 1, 3, or 6 molar equivalents with respect to the cyclic peptide (concentration of cyclic peptide: 1.4 mM, experiment with addition of metal salt). Summarize the results of the 1 1H-NMR spectra obtained for comparison in Table 6.

[0697] [Table 6]

[0698]

[0699] 1 The characteristics of the 1H-NMR spectra reflect the interaction between the compound and the metal and the change in the conformational state accompanying the interaction. By comparing the peak components observed in the 1 1H-NMR spectrum obtained without addition of metal salt with those observed in the 1 1H-NMR spectrum obtained when a metal salt is added, the formation of the complex is determined. In other words, the peak components obtained without addition of metal salt are used as the initial peak components to compare those observed in the 1 1H-NMR spectrum when a metal salt is added, and a complex is determined to be formed when any of the following occurs: when new peaks of the cyclic peptide other than the initial peak components appear, when a change in the peak intensity ratio occurs, or when the peak components observed upon addition of the metal salt are sharpened and / or broadened compared to the initial peak components ( Figures 2 to 7 ). The official names and CAS numbers of the metal salts used herein are shown in Table 7.

[0700] [Table 7]

[0701]

[0702] [Reference Example 3: Confirmation of solvents capable of forming complexes]

[0703] <Basic operation>

[0704] Measure each of the following solutions at 298 K for1 1H-NMR: Solutions obtained by dissolving Compound 6 in various deuterated solvents shown in Table 8 (concentration of cyclic peptide: 1.4 mM, experiment without addition of metal salts), and solutions obtained by adding approximately 1 molar equivalent of magnesium perchlorate to the cyclic peptide and dissolving it in various deuterated solvents shown in Table 8 (concentration of cyclic peptide: 1.4 mM, experiment without addition of metal salts), to obtain 1 1H-NMR spectra.

[0705] In Table 8, compare the 1 1H-NMR spectrum of Compound 6 without addition of magnesium perchlorate with the 1 1H-NMR spectrum of Compound 6 when approximately 1 molar equivalent of magnesium perchlorate is added to Compound 6, and when it is demonstrated that a complex is formed in the solution, such as when the peak components of the cyclic peptide disappear and new peak components of the cyclic peptide appear without addition of magnesium perchlorate, when there is a change in the peak intensity ratio, or when the peak components with addition of magnesium perchlorate are sharpened, broadened, etc. compared to the peak components without addition of magnesium perchlorate, it is described as "formed". In particular, 2,2,2-trifluoroethanol-d3, acetonitrile-d3, and acetone-d6 showed significant changes in peak components. Compare the 1 1H-NMR spectrum of Compound 6 without addition of magnesium perchlorate with the 1 1H-NMR spectrum of Compound 6 when approximately 1 molar equivalent of magnesium perchlorate is added to Compound 6, and when it is not demonstrated that a complex is formed, such as when no peak components of the cyclic peptide other than the peak components appear without addition of magnesium perchlorate, when there is no change in the peak intensity ratio, or when the peak components with addition of magnesium perchlorate are neither sharpened nor broadened compared to the peak components without addition of magnesium perchlorate, it is described as "not formed".

[0706] [Table 8]

[0707] Run CAS number Solvent name Solvent Complex formation 1 811-98-3 Methanol-d4 <![CDATA[CD3OD]]> Not formed 2 August 1, 1516 Ethanol-d6 <![CDATA[CD3CD2OD]]> Not formed 3 22739-76-0 2-Propanol-d8 <![CDATA[(D3C)2CDOD]]> Formed 4 53001-22-2 tert-Butanol-d10 <![CDATA[(D3C)3COD]]> Formed 5 77253-67-9 2,2,2-Trifluoroethanol-d3 <![CDATA[CF3CD2OD]]> Formed 6 2206-27-1 Dimethyl sulfoxide-d6 DMSO-d6 Not formed 7 2206-26-0 Acetonitrile-d3 <![CDATA[CD3CN]]> Formed 8 4472-41-7 N,N-Dimethylformamide-d4 DMF-d7 Not formed 9 1693-74-9 Tetrahydrofuran-d8 THF-d8 Formed 10 2037-26-5 Toluene-d8 Toluene-dB Formed 11 17647-74-4 1,4-Dioxane-d8 1,4-Dioxane-d8 Formed 12 666-52-4 Acetone-d6 Acetone-d6 Formed 13 1665-00-5 Dichloromethane-d2 DCM-d2 Formed

[0708] Runs 2, 6, 8 to 13 of Table 8

[0709] A solution of 0.333 mL (Compound 6, 1.01 mg, 0.705 μmol) of Compound 6 (30.24 mg) in acetonitrile (CH3CN, CAS: 75-05-8, 10 mL) was dispensed into a container and concentrated to dryness. Then, a solution of 0.100 mL (Mg(ClO4)2, 0.188 mg, 0.840 μmol) of magnesium perchlorate (Mg(ClO4)2, 4.52 mg, 83 w%) in acetonitrile (2.0 mL) was added to each container and concentrated to dryness. The resulting material was dissolved in 0.5 mL of the deuterated solvents shown in Table 8 (toluene-d8, ethanol-d6, tetrahydrofuran-d8, acetone-d6, dichloromethane-d2, N,N-dimethylformamide-d4, dimethyl sulfoxide-d6, 1,4-dioxane-d8), and measured at 298 K 1 1H-NMR.

[0710] Run 1 of Table 8

[0711] A solution of 0.028 mL (Compound 6, 0.999 mg, 0.700 μmol) of Compound 6 (2.78 mg) in methanol-d4 (0.0779 mL) was mixed with a solution of 0.0135 mL (Mg(ClO4)2, 0129 mg, 0.580 μmol) of magnesium perchlorate (Mg(ClO4)2, 2.31 mg, 83 w%) in methanol-d4 (0.2 mL). Then methanol-d4 (0.4585 mL) was added, and measured at 298 K 1 1H-NMR.

[0712] Runs 3 to 5 of Table 8

[0713] A solution of 0.010 mL (Compound 6, 1.00 mg, 0.700 μmol) of Compound 6 (6.69 mg) in acetonitrile (CH3CN, 0.0669 mL) and a solution of 0.0355 mL (Mg(ClO4)2, 0.160 mg, 0.717 μmol) of magnesium perchlorate (Mg(ClO4)2, 10.9 mg, 83 w%) in CH3CN (2.0 mL) were added to each container and concentrated to dryness. The resulting material was dissolved in 0.5 mL of the deuterated solvents shown in Table 8 (2-propanol-d8, tert-butanol-d10, 2,2,2-trifluoroethanol-d3), and measured at 298 K 1 1H-NMR.

[0714] Run 7 of Table 8

[0715] A solution of 0.028 mL (Compound 6, 1.00 mg, 0.700 μmol) of Compound 6 (13.04 mg) in acetonitrile-d3 (0.365 mL), a solution of 0.0253 mL (Mg(ClO4)2, 0.129 mg, 0.580 μmol) of magnesium perchlorate (Mg(ClO4)2, 9.25 mg, 83 w%) in acetonitrile-d3 (1.5 mL), and 0.4467 mL of acetonitrile-d3 were mixed and measured at 298 K. 1 1H-NMR.

[0716] [Example 1: Solvent range confirmation]

[0717] [Example 1-1: Confirmation of the solvent range available for complex formation]

[0718] <Basic operation>

[0719] Compound 6 (about 10 mg) was mixed with about 1 molar equivalent of magnesium perchlorate and dissolved in various solvents shown in Table 9 below. Hexane was added directly to the solution, or the solvent was distilled off under reduced pressure or lyophilized, and dichloromethane and hexane were added, and then the mixture was stirred at room temperature as described below. The resulting solid and supernatant were centrifuged, the solvent was distilled off under reduced pressure or purged with nitrogen, the supernatant and solid after vacuum drying were redissolved in acetonitrile, and their respective HPLCs were measured. Table 9 shows the percentage of peak area of Compound 6, the purity calculated based on the peak area value, the recovery rate, the ratio of isomer 1 and isomer 2 to Compound 6, and the purification efficiency.

[0720] Based on the initial concentration, the sample for HPLC measurement in Example 1-1 was prepared to be about 0.3 mg / mL. In Example 1-1, the HPLC of the prepared sample was measured under the same purity confirmation conditions (the same condition purity analysis condition A) as that of Compound 6 in Reference Example 1-2. Unless otherwise mentioned, the preparation accurately prepared for about 0.3 mg / mL calculates the purity and recovery rate based on the percentage of peak area and the peak area (measured at 220 nm) measured by HPLC under the above conditions. The ratio of isomer 1 and isomer 2 to Compound 6 and the purification efficiency were also calculated. In Reference Example 1-2, Compound 6 used as a raw material was synthesized in large quantities, and its purity was 84% based on the percentage of peak area under the conditions of HPLC used in the purity analysis. Therefore, when the purity of Compound 6 is improved relative to 84% after this operation, it is determined that there is a purification effect. In addition, even when the purity of Compound 6 is not improved relative to 84% after this operation, it is determined that there is a purification effect for those compounds in which the purification efficiency of the isomers of Compound 6 is positive.

[0721] Recovery calculation method 1

[0722] For example, assume that an experiment is conducted using approximately a mg of raw material. The solid and the solution are centrifuged, the solvent is distilled off under reduced pressure or purged with nitrogen, and (150b / a) μL of the solution obtained by adding b mL of acetonitrile to the supernatant and the solid dried under vacuum is sampled, and then a sample is prepared such that the entire solution is a 50% v / v aqueous acetonitrile solution of 0.5 mL. When HPLC is measured for each of the prepared sample and a precisely prepared 0.30 mg / mL preparation, and the peak areas (at 220 nm) of the target are set as c and d respectively, the recovery rate is expressed by the following expression. Considering the case where the actual weighed value (e mg) does not match the assumed weight of a mg, correction is made using the weighed value.

[0723] [Expression 1]

[0724]

[0725] Purification efficiency calculation method

[0726] The ratio of isomer 1 or isomer 2 to compound 6 and the purification efficiency are calculated by the following expressions to confirm how much of isomer 1 and isomer 2 are separated from compound 6 with respect to compound 6.

[0727] - Ratio of isomer 1 to compound 6 x 100:

[0728] (Peak area of isomer 1) / (Peak area of compound 6) x 100

[0729] - Ratio of isomer 2 to compound 6 x 100:

[0730] (Peak area of isomer 2) / (Peak area of compound 6) x 100

[0731] - Purification efficiency of isomer 1 (%):

[0732] {1 - (Ratio of compound 6 to isomer 1) / (Ratio of compound 6 to isomer 1 in the preparation)} x 100

[0733] - Purification efficiency of isomer 2 (%):

[0734] {1 - (Ratio of compound 6 to isomer 2) / (Ratio of compound 6 to isomer 2 in the preparation)} x 100

[0735] In Example 1-1, Labsolution manufactured by SHIMADZU CORPORATION was used as the HPLC analysis software, and the recovery rate was calculated by the above-described recovery rate calculation method 1 (a = 10). The purification efficiency of the isomers was calculated by the above-described calculation method of the purification efficiency.

[0736]

[0737] Run 1 of Table 9: Dimethylformamide

[0738] To Compound 6 (10.1 mg), a 35 mM dimethylformamide solution (0.200 mL) prepared by diluting a dimethylformamide solution of Mg(ClO4)2 (6.5 mg) (0.325 mL) was added, and the mixture was freeze-dried. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 3 days. Hexane (0.33 mL) was added and the mixture was stirred for another two days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained supernatant and acetonitrile solution (3.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, the purity and recovery rate of the solid were found to be 94% and 75% respectively, and the purity and recovery rate of the supernatant were 72% and 32% respectively. Through these operations, it was confirmed that the precipitated solid was purified. The purification efficiencies of Isomers 1 and 2 relative to Compound 6 in the supernatant were -124% and -123% respectively, and the purification efficiencies in the solid were 72% and 64% respectively. It is considered that the solid exhibits good purification because Compound 6 precipitates preferentially to Isomers 1 and 2 under the condition of metal addition.

[0739] Calculation of the recovery rate of Run 1

[0740] After measuring the HPLC of the preparation of the 0.30 mg / mL solution, the peak area of the target (2950511, 220 nm) was measured. The HPLC of the prepared 50% v / v aqueous acetonitrile solution was measured, and the peak area of the target (supernatant: 962751, solid: 2240371) was measured, and the recovery rate was calculated according to the following method.

[0741] Supernatant: (962751 x 10) / (2950511 x 10.1) x 100 ≈ 32%

[0742] Solid: (2240371 x 10) / (2950511 x 10.1) x 100 ≈ 75%

[0743] Calculation of the purification efficiency of Run 1

[0744] After HPLC of the preparation of the 0.30 mg / mL solution was measured, the peak area of the target (2,950,511, 220 nm) was measured. The peak areas of the isomers were measured in the same manner (Isomer 1: 77,125, Isomer 2: 225,732). HPLC of the prepared 50% v / v aqueous acetonitrile solution was measured, and the peak area of the target (supernatant: 962,751, solid: 2,240,371), the peak area of Isomer 1 (supernatant: 56,402, solid: 16,355), and the peak area of Isomer 2 (supernatant: 164,256, solid: 61,383) were measured, and the purification efficiency was calculated according to the following method.

[0745] Ratio of Isomer Ratio 1 in supernatant: 56,402 / 962,751 x 100 ≈ 5.9%

[0746] Ratio of Isomer Ratio 2 in supernatant: 164,256 / 962,751 x 100 ≈ 17.1%

[0747] Ratio of Isomer Ratio 1 in solid: 16,355 / 2,240,371 x 100 ≈ 0.7%

[0748] Ratio of Isomer Ratio 2 in solid: 61,383 / 2,240,371 x 100 ≈ 2.7%

[0749] Purification efficiency of Isomer Ratio 1 in supernatant:

[0750] {1 - (56,402 / 962,751) / (77,125 / 2,950,511)} x 100 ≈ -124%

[0751] Purification efficiency of Isomer Ratio 2 in supernatant:

[0752] {1 - (164,256 / 962,751) / (225,732 / 2,950,511)} x 100 ≈ -123%

[0753] Purification efficiency of Isomer Ratio 1 in solid:

[0754] {1 - (16,355 / 2,240,371) / (77,125 / 2,950,511)} x 100 ≈ 72%

[0755] Purification efficiency of Isomer Ratio 2 in solid:

[0756] {1 - (61,383 / 2,240,371) / (225,732 / 2,950,511)} x 100 ≈ 64%

[0757] Run 2 of Table 9: Dimethylacetamide

[0758] To compound 6 (10.0 mg), a 35 mM dimethylacetamide solution (0.200 mL) prepared by diluting a dimethylacetamide solution of Mg(ClO4)2 (5.3 mg) (0.265 mL) was added, and the mixture was freeze-dried. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 3 days. Hexane (0.33 mL) was added and the mixture was stirred for another two days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained supernatant and the acetonitrile solution (3.0 mL) of the solid, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0759] Run 3 of Table 9: Dimethyl sulfoxide

[0760] To compound 6 (10.0 mg), a 35 mM dimethyl sulfoxide solution (0.200 mL) prepared by diluting a dimethyl sulfoxide solution of Mg(ClO4)2 (5.2 mg) (0.260 mL) was added, and the mixture was freeze-dried. Dichloromethane (0.66 mL) and hexane (0.33 mL) were successively added to the obtained solid, and the mixture was stirred for 4 days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained supernatant and the acetonitrile solution (3.0 mL) of the solid, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0761] Run 4 of Table 9: Methanol

[0762] To compound 6 (10.0 mg), a 35 mM methanol solution (0.200 mL) prepared by diluting a methanol solution of Mg(ClO4)2 (6.1 mg) (0.305 mL) was added, and the mixture was concentrated and vacuum-dried. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 3 days. Hexane (0.33 mL) was added and the mixture was stirred for another two days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0763] Run 5 of Table 9: Ethanol

[0764] To Compound 6 (9.9 mg), a 35 mM ethanol solution (0.200 mL) prepared by diluting an ethanol solution of Mg(ClO4)2 (8.2 mg) (0.410 mL) was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 3 days. Hexane (0.33 mL) was added and the mixture was stirred for another two days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the supernatant was purified.

[0765] Run 6 of Table 9: Dichloromethane

[0766] To a mixture of Compound 6 (10.1 mg) and Mg(ClO4)2 (2.14 mg, 83 w%) was added dichloromethane (0.66 mL), and the mixture was stirred for 3 days. Hexane (0.33 mL) was added and the mixture was stirred for another two days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0767] From the above results, it was found that in the formation of the complex of magnesium perchlorate and cyclic peptide, solvents in which complex formation was not observed, in addition to the solvents in which complex formation was observed in Reference Example 3, could also be used for the purification method of cyclic peptide.

[0768] [Example 1-2: Confirmation of the solvent range capable of purification]

[0769] <Basic operation>

[0770] Compound 6 (about 15 mg or about 10 mg) was mixed with about 1 molar equivalent of magnesium perchlorate and dissolved in acetonitrile. The solvent was distilled off under reduced pressure, various solvents shown in Table 10 below were added, and the mixture was stirred at room temperature as described below. The supernatant and the resulting solid were centrifuged, and HPLC of each of the supernatant and the solid dried under vacuum was measured. The purity and recovery rate were determined based on the peak area percentage and peak area value of Compound 6, and are shown in Table 10.

[0771] [Table 10]

[0772]

[0773] Based on the initial concentration, samples for HPLC measurement in Examples 1-2 and 2-1 were prepared to be approximately 0.3 mg / mL. In Examples 1-2 and 2-1, the HPLC of the prepared samples was measured under the same conditions (purity analysis condition A) as the purity confirmation conditions of Compound 6 in Reference Example 1-2. Unless otherwise mentioned, the formulations precisely prepared for approximately 0.3 mg / mL were corrected based on the peak areas measured by HPLC under the above conditions (measured at 220 nm) to calculate the recovery rate. In Reference Example 1-2, Compound 6 used as a raw material was synthesized in large quantities, and its purity was 84% based on the peak area percentage under the HPLC conditions used in the purity analysis. Therefore, when the purity was improved relative to 84%, purification was determined to exist.

[0774] For Runs 7 to 18, HPLC was measured under purity analysis condition A. The purity and recovery rate of the formulations precisely prepared for approximately 0.3 mg / mL were calculated based on the peak area percentage and peak area (measured at 220 nm) measured by HPLC under purity analysis condition A. The ratios of Isomer 1 and Isomer 2 to Compound 6 and the purification efficiency were also calculated. For Runs 7 to 18, even when the purity of Compound 6 did not improve relative to 84% after this operation, purification was determined to exist for those compounds in which the purification efficiency of the isomers of Compound 6 was positive.

[0775] Recovery calculation method 2

[0776] For example, assuming that a mg of raw material was used for the experiment and the amount of the solution used was b mL, when measuring the HPLC of the supernatant, first (b / 100) mL of the supernatant was sampled, and then the sample was prepared by adding acetonitrile so that the entire solution was 0.5 mL. Separately, d mg of raw material was weighed, and a formulation was prepared using a 10-mL volumetric flask. When measuring the HPLC of each of the prepared sample and formulation and setting the peak areas (220 nm) of the target to c and e, respectively, the recovery rate is expressed by the following formula.

[0777] [Expression 2]

[0778]

[0779] Recovery calculation method 3

[0780] For example, assume that an experiment is conducted using a mg of raw material. The supernatant and the solid are centrifuged, the solvent contained in the supernatant or the solid is distilled off under reduced pressure and dried, and then 0.5 mL (or 1.0 mL) of acetonitrile is added. 5 μL (or 10 μL) of the obtained solution is mixed with 495 μL (or 490 μL) of acetonitrile to prepare a sample. Separately, d mg of the raw material is weighed and a preparation is made using a 10 mL volumetric flask. When measuring the HPLC of each of the prepared sample and the preparation and setting the peak areas (at 220 nm) of the target to f and e, respectively, the recovery rate is expressed by the following expression.

[0781] [Expression 3]

[0782]

[0783] In Runs 2 to 6, ACD / Spectrus manufactured by Advanced Chemistry Development, Inc. was used as the HPLC analysis software, and the recovery rate was calculated by the above-described recovery rate calculation method 3. In Runs 7 to 18, Labsolution manufactured by SHIMADZU CORPORATION was used as the HPLC analysis software, and the recovery rate was calculated by the above-described recovery rate calculation method 1 (a = 10). The purification efficiency of the isomers was calculated by the above-described purification efficiency calculation method.

[0784] Run 1 of Table 10: Dichloromethane / hexane

[0785] Dichloromethane (1 mL) and hexane (0.5 mL) were added to the solid obtained after concentrating a solution of Compound 6 (15.00 mg) and Mg(ClO4)2 (3.03 mg, 83 w%) in CH3CN (1.0 mL), and the mixture was stirred overnight. The resulting solid and solution were centrifuged. After concentration, the solution was dried for 6 hours, acetonitrile (0.5 mL) was added, and the purity and recovery rate were confirmed by HPLC. As a result, it was found that based on the percentage of the peak area, the purity was 68% and the recovery rate was 14%. The obtained solid was washed with a mixed solution of dichloromethane (1 mL) and hexane (0.5 mL), then centrifuged, and the obtained solid was vacuum dried overnight to obtain 13.64 mg of the Compound 6-Mg(ClO4)2 complex in solid form. As a result of confirming the purity and recovery rate of the obtained complex by HPLC, it was found that the purity was 90% and the recovery rate was 82%. Through these operations, it was confirmed that the precipitated solid was purified.

[0786] Calculation of the recovery rate of the solid based on dichloromethane / hexane

[0787] Measure the HPLC of the preparation of the 0.297 mg / mL solution, and then measure the peak area of the target (51555, 220 nm).

[0788] Dissolve 3.00 mg of the 13.64 mg of the obtained solid in 10 mL of acetonitrile. Then measure the HPLC, measure the peak area of the target (46818), and calculate the recovery rate according to the following method.

[0789] (13.64 / 0.300) x 46818 / {(15 / 0.297) x 51555} x 100 ≈ 82%

[0790] Run 2 of Table 10: Acetone / hexane

[0791] Add acetone (1 mL) and hexane (1 mL) to the solid obtained after concentrating a solution of compound 6 (15.00 mg) and Mg(ClO4)2 (3.01 mg, 83 w%) in CH3CN (1.0 mL), and stir the mixture overnight. Centrifuge the resulting solid and solution. Dry the solution for 6 hours after concentration, add acetonitrile (0.5 mL), and confirm the purity and recovery rate by HPLC. As a result, it was found that based on the peak area percentage, the purity was 69% and the recovery rate was 22%. Dry the obtained solid for 6 hours and dissolve it in acetonitrile (0.5 mL), and confirm the purity and recovery rate by HPLC, and it was found that the purity was 92% and the recovery rate was 68%. Through these operations, it was confirmed that the precipitated solid was purified.

[0792] Run 3 of Table 10: 1,4-Dioxane

[0793] Add 1,4-dioxane (1 mL) to the solid obtained after concentrating a solution of compound 6 (14.98 mg) and Mg(ClO4)2 (2.96 mg, 83 w%) in CH3CN (1.0 mL), and stir the mixture overnight. Centrifuge the resulting solid and solution. Dry the solution for 6 hours after concentration, add acetonitrile (0.5 mL), and confirm the purity and recovery rate by HPLC. As a result, it was found that based on the peak area percentage, the purity was 72% and the recovery rate was 15%. Dry the obtained solid for 6 hours and dissolve it in acetonitrile (0.5 mL), and confirm the purity and recovery rate by HPLC, and it was found that the purity was 90% and the recovery rate was 76%. Through these operations, it was confirmed that the precipitated solid was purified.

[0794] Runs 4, 5 and 6 of Table 10

[0795] The solution (5.0 mL) of compound 6 (75.12 mg) and Mg(ClO4)2 (14.68 mg, 83 w%) in CH3CN was aliquoted in 1-mL portions and each portion was concentrated. Each of the solvents shown in Runs 4 to 6 below was added to the obtained solid and the mixture was stirred over the weekend. The resulting solid and solution were centrifuged. After concentration, the solution was dried under vacuum, acetonitrile (0.5 mL) was added, and the purity and recovery were confirmed by HPLC.

[0796] After drying under vacuum, the obtained solid was dissolved in acetonitrile (0.5 mL) and the purity and recovery were confirmed by HPLC. Note that samples for purity and recovery measurement were prepared by diluting 10 μL of each of the solutions of the solid and the solution prepared after concentration and drying under vacuum with 990 μL of acetonitrile. As a result, it was confirmed that both solid phases were purified.

[0797] Run 7 of Table 10: Ethyl acetate

[0798] To compound 6 (9.9 mg) was added a 35 mM acetonitrile solution (0.200 mL) prepared by diluting an acetonitrile solution of Mg(ClO4)2 (20.7 mg) (0.414 mL), and the mixture was concentrated and dried under vacuum. Ethyl acetate (0.66 mL) was added to the obtained solid and the mixture was stirred for 3 days. The resulting solid and solution were centrifuged. After distilling off the solvent, the supernatant dried under vacuum and the solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery by HPLC, it was confirmed that the solid was purified.

[0799] Calculation of the recovery rate of Run 7

[0800] After HPLC measurement of the preparation of the 0.30 mg / mL solution, the peak area of the target (2950511, 220 nm) was measured. HPLC of the prepared 50% v / v aqueous acetonitrile solution was measured, and the peak area of the target (supernatant: 255084, solid: 2369897) was measured, and the recovery was calculated according to the following method.

[0801] Supernatant: (255084 x 10) / (2950511 x 9.9) x 100 ≈ 9%

[0802] Solid: (2369897 x 10) / (2950511 x 9.9) x 100 ≈ 81%

[0803] Run 8 of Table 10: Ethanol / heptane

[0804] To Compound 6 (10.0 mg), a 35 mM acetonitrile solution (0.200 mL) prepared by diluting a solution of Mg(ClO4)2 (20.7 mg) in acetonitrile (0.414 mL) was added, and the mixture was concentrated and dried under vacuum. Ethanol (0.66 mL) was added to the obtained solid, and the mixture was stirred for 3 days. Heptane (1.0 mL) was added and the mixture was stirred overnight, and then further heptane (1.0 mL) was added and the mixture was stirred for 2 days, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0805] Run 9 of Table 10: Butanol / heptane

[0806] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting a solution of 0.100 mL of Compound 6 (92.5 mg) in acetonitrile (0.925 mL) and a solution of 0.038 mL of Mg(ClO4)2 (25.0 mg) in acetonitrile (0.500 mL) was concentrated and dried under vacuum. Butanol (0.5 mL) and heptane (0.5 mL) were added to the obtained solid, and the mixture was stirred for 2 days. Further heptane (0.25 mL) was added and the mixture was stirred for 6 hours, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0807] Runs 10 and 12 of Table 10

[0808] A 35 mM acetonitrile solution (0.200 mL each) prepared by dispensing and diluting 0.100 mL of an acetonitrile solution (0.925 mL) of Compound 6 (92.5 mg) and 0.038 mL of an acetonitrile solution (0.500 mL) of Mg(ClO4)2 (25.0 mg) was concentrated and dried under vacuum. Each solvent shown in Runs 10 and 12 of Table 10 was added to the obtained solid, and the mixture was stirred for 3 days. The resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0809] Run 11 of Table 10: Acetonitrile / tert-butyl methyl ether

[0810] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting 0.100 mL of an acetonitrile solution (0.925 mL) of Compound 6 (92.5 mg) and 0.038 mL of an acetonitrile solution (0.500 mL) of Mg(ClO)42 (25.0 mg) was concentrated and dried under vacuum. Acetonitrile (0.13 mL) and tert-butyl methyl ether (0.87 mL) were added to the obtained solid, and the mixture was stirred for 2 days. Further, acetonitrile (0.13 mL) was added, and the mixture was stirred for 6 hours, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0811] Run 13 of Table 10: Isopropyl acetate / heptane

[0812] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting a solution of 0.100 mL of Compound 6 (92.5 mg) in acetonitrile (0.925 mL) and 0.038 mL of Mg(ClO4)2 (25.0 mg) in acetonitrile (0.500 mL) was concentrated and dried under vacuum. Isopropyl acetate (0.66 mL) and heptane (0.33 mL) were added to the obtained solid, and the mixture was stirred for 2 days. Further, isopropyl acetate (0.66 mL) was added and the mixture was stirred for 6 hours, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0813] Run 14 of Table 10: Methyl ethyl ketone / heptane

[0814] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting a solution of 0.100 mL of Compound 6 (92.5 mg) in acetonitrile (0.925 mL) and 0.038 mL of Mg(ClO4)2 (25.0 mg) in acetonitrile (0.500 mL) was concentrated and dried under vacuum. Methyl ethyl ketone (0.66 mL) and heptane (0.66 mL) were added to the obtained solid, and the mixture was stirred for 2 days. Further, methyl ethyl ketone (0.33 mL) was added and the mixture was stirred for 6 hours, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0815] Run 15 of Table 10: Methanol / toluene

[0816] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting a 0.100 mL acetonitrile solution (0.925 mL) of Compound 6 (92.5 mg) and a 0.038 mL acetonitrile solution (0.500 mL) of Mg(ClO4)2 (25.0 mg) was concentrated and dried under vacuum. Methanol (33.3 μL) and toluene (966.7 μL) were added to the obtained solid and the mixture was stirred for 3 days, and then the resulting oily precipitate and solution were centrifuged. After distilling off the solvent, the supernatant and precipitate dried under vacuum were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained acetonitrile solution (1.0 mL) of the supernatant and precipitate, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the supernatant was purified.

[0817] Run 16 of Table 10: Dimethyl sulfoxide / tert-butyl methyl ether

[0818] A 35 mM acetonitrile solution (0.200 mL) prepared by dispensing and diluting a 0.100 mL acetonitrile solution (0.485 mL) of Compound 6 (48.5 mg) and a 0.094 mL acetonitrile solution (0.265 mL) of Mg(ClO4)2 (5.3 mg) was concentrated and dried under vacuum. Dimethyl sulfoxide (90 μL) and tert-butyl methyl ether (910 μL) were added to the obtained solid and the mixture was stirred for 3 days, and then the resulting solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained acetonitrile solution (3.0 mL) of the supernatant and solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0819] Run 17 of Table 10: Dimethylformamide / tert-butyl methyl ether

[0820] To Compound 6 (10.1 mg), a 35 mM acetonitrile solution (0.200 mL) prepared by diluting an acetonitrile solution (0.475 mL) of Mg(ClO4)2 (9.5 mg) was added, and the mixture was concentrated and dried under vacuum. To the obtained solid, dimethylformamide (0.1 mL) and tert-butyl methyl ether (0.4 mL) were added and the mixture was stirred for 3 days, then further tert-butyl methyl ether (0.4 mL) was added and the mixture was stirred overnight, and then the obtained solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained supernatant and the acetonitrile solution (3.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, there was no significant improvement in the purity, but with respect to Compound 6 in the solid, the purification efficiency of Isomer 1 was 40% and that of Isomer 2 was 13%, and thus it was determined that the solid was purified.

[0821] Run 18 of Table 10: Benzyl alcohol / tert-butyl methyl ether

[0822] To Compound 6 (10.1 mg), a 35 mM acetonitrile solution (0.200 mL) prepared by diluting an acetonitrile solution (0.475 mL) of Mg(ClO4)2 (9.5 mg) was added, and the mixture was concentrated and dried under vacuum. To the obtained solid, benzyl alcohol (0.3 mL) and tert-butyl methyl ether (0.7 mL) were added, and the mixture was stirred for 4 days, and then the obtained solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in acetonitrile (3.0 mL). 45 μL was sampled from the obtained supernatant and the acetonitrile solution (3.0 mL) of the solid, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the solid was purified.

[0823] From the above results, it was found that in the formation of the complex of magnesium perchlorate and the cyclic peptide, solvents in which complex formation has not been observed, in addition to the solvents in which complex formation was observed in Reference Example 3, can also be used for the purification of the cyclic peptide.

[0824] [Example 2-1: Confirmation of the preparation and purification effect of complexes]

[0825] <Basic Operations>

[0826] Compound 6 (about 15 mg or about 10 mg) is mixed with each of the various metal salts shown in the following table 11 of about 1 molar equivalent and dissolved in acetonitrile, ethanol or aqueous ethanol.Then the solvent is distilled out under reduced pressure, and the various solvents shown in the following table 11 are added, and the mixture is stirred at room temperature as described below, and then the supernatant and the resulting solid are centrifuged. Each of the HPLC in the supernatant and the vacuum-dried solid is measured under purity analysis condition A, and purity and recovery are determined based on the peak area percentage and peak area value of compound 6, as shown in Table 12. HPLC analysis is performed using ACD / Spectrus (runs 1 to 26) manufactured by Advanced Chemistry Development, Inc. or Labsolution (runs 27 to 31) manufactured by SHIMADZUCORPORATION. Recovery is calculated by the recovery calculation method 1, 2 or 3 of Example 1. In Reference Example 1-2, a large amount of compound 6 used as raw material is synthesized, and its purity is 84% based on the peak area percentage under the condition (purification analysis condition A) of the HPLC used in the purity analysis. Therefore, based on HPLC analysis measured under the same conditions, when the purity improved from 84%, a purification effect was confirmed. Furthermore, even when the purity of Compound 6 did not improve from 84% after this operation, a purification effect was confirmed for compounds in which the purification efficiency for the isomers of Compound 6 was positive. The mass and molar equivalent of the metal salt actually used, as well as the mass of Compound 6, are shown in Table 13.

[0827] [Table 11]

[0828]

[0829]

[0830]

[0831]

[0832] [Table 13]

[0833]

[0834] The mass of the inorganic salts for runs 27 to 31 was calculated from the volume of the solution obtained by preparing an acetonitrile solution of each inorganic salt, dispensing, and mixing with compound 6.

[0835] The following experiments were performed on the metal salts shown in Run 10 of Table 11.

[0836] To the solid obtained after concentrating a solution of compound 6 (15.00 mg) and Mg(ClO4)2 (3.03 mg, 83 w%) in CH3CN (1.0 mL), dichloromethane (1 mL) and hexane (0.5 mL) were added, and the mixture was stirred overnight. The resulting solid and solution were centrifuged. After concentration, the solution was dried for 6 hours, acetonitrile (0.5 mL) was added, and the purity and recovery were confirmed by HPLC. As a result, it was found that based on the peak area percentage, the purity was 68% and the recovery was 14%. The obtained solid was washed with a mixed solution of dichloromethane (1 mL) and hexane (0.5 mL), then centrifuged, and the obtained solid was dried under vacuum overnight to obtain 13.64 mg of the compound 6-Mg(ClO4)2 complex as a solid. As a result of confirming the purity and recovery of the obtained complex by HPLC, it was found that the purity was 90% and the recovery was 82%. By these operations, it was confirmed that the precipitated solid was purified.

[0837] Calculation of Recovery Rate

[0838] After measuring the HPLC of the formulation of the 0.297 mg / mL solution, the peak area of the target (51555, 220 nm) was measured.

[0839] 3.00 mg of the 13.64 mg of the obtained solid was dissolved in 10 mL of acetonitrile. Then HPLC was measured, the peak area of the target (46818) was measured, and the recovery was calculated according to the following method.

[0840] (13.64 / 0.300) x 46818 / {(15 / 0.297) x 51555} x 100 ≈ 82%

[0841] The following experiment was conducted on the metal salt shown in Run 26 of Table 11.

[0842] To the solid obtained by vacuum drying a solution of compound 6 (15.0 mg) and AgOTf (2.65 mg, 0.98 molar equivalent) in CH3CN (0.5 mL), dichloromethane (1 mL) and hexane (0.6 mL, divided into a total of six portions, 0.1 mL each, every 2 to 5 hours) were added. No precipitation occurred, so the purity and recovery of the solution (supernatant) were confirmed by HPLC (calculation method 2).

[0843] Calculation of Recovery Rate

[0844] HPLC was measured before and after adding hexane during the above experiment, the peak area of the target (before adding hexane: 55176, after adding hexane: 49150) was measured, and the recovery was calculated according to the following method.

[0845] (49150 / 55176) x 100 ≈ 89%

[0846] The following experiments were conducted on the metal salts shown in Runs 11, 12, 13, 18, 19, and 23 of Table 11.

[0847] Dichloromethane (1 mL) and hexane (1 mL) were added to the solid obtained after the CH3CN solution (0.2 mL) or suspension (0.2 mL) of concentrated Compound 6 (approx. 15 mg) and each of the various metal salts (approx. 1 molar equivalent), and the mixture was stirred for 4.5 hours, and the resulting solid and solution were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 0.5 mL of CH3CN, and the purity and recovery were confirmed by HPLC (Calculation Method 3).

[0848] The following experiments were conducted on the metal salts shown in Runs 9, 14, 15, and 16 of Table 11.

[0849] Dichloromethane (1 mL) and hexane (1 mL) were added to the solid obtained after leaving the CH3CN solution (0.4 mL) or suspension (0.4 mL) of Compound 6 (approx. 15 mg) and each of the various metal salts (approx. 1 molar equivalent) in an open state overnight and then vacuum drying, and the mixture was stirred for 5 hours, and the resulting solid and solution were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC.

[0850] The following experiments were conducted on the metal salts shown in Runs 2, 3, 7, 8, and 17 of Table 11.

[0851] Dichloromethane (1 mL) and hexane (1 mL) were added to the solid obtained after leaving the CH3CN solution (0.4 mL) of Compound 6 (approx. 15 mg) and each of the various metal salts (approx. 1 molar equivalent) in an open state overnight and then vacuum drying, and the mixture was stirred for 5 hours, then an additional 1 mL of hexane was added and the mixture was stirred for 19 hours, and the resulting solid and solution were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC (Calculation Method 3).

[0852] The following experiments were conducted on the metal salts shown in Runs 21, 24, and 25 of Table 11.

[0853] To the solid obtained after vacuum drying a CH3CN solution (0.2 mL) of compound 6 (about 15 mg) and each of various metal salts (about 1 molar equivalent) was added dichloromethane (1 mL) and hexane (1 mL), and the mixture was stirred overnight. Then, hexane (1 mL) was further added and the mixture was stirred for 5 hours, and the resulting solid and solution were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC (calculation method 3).

[0854] The following experiments were conducted on the metal salts shown in Runs 5, 6, 20, and 22 of Table 11.

[0855] To the solid obtained after vacuum drying a CH3CN solution (0.1 mL) of compound 6 (about 15 mg) and each of various metal salts (about 1 molar equivalent) was added dichloromethane (1 mL) and hexane (1 mL), and the mixture was stirred for 17 hours. Then, hexane (1 mL) was further added and the mixture was stirred for 6 hours, and the resulting solid and solution were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC (calculation method 3).

[0856] The following experiment was conducted on the metal salt shown in Run 4 of Table 11.

[0857] A solution (0.2 mL) of compound 6 (15.02 mg) and KI (1.82 mg, 1.04 molar equivalent) in CH3CN was stirred ultrasonically, and the resulting solid and liquid were centrifuged. After decanting the supernatant, the solid obtained after vacuum drying was dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC. For the supernatant, the solvent was distilled off under reduced pressure, and then the supernatant was vacuum dried and dissolved in 1.0 mL of CH3CN, and the purity and recovery were confirmed by HPLC (calculation method 3).

[0858] The following experiment was conducted on the metal salts shown in Run 27 of Table 11. A solution (0.200 mL) prepared by dispensing 0.087 mL of a solution of MgSO4·7H2O (8.9 mg) in 60% ethanol (0.445 mL) and diluting with 0.113 mL of 60% ethanol was added to Compound 6 (10.0 mg), and the mixture was concentrated and dried under vacuum. Tetrahydrofuran (0.2 mL) and heptane (0.8 mL) were added to the obtained solid, and the mixture was stirred for 3 days, and then the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in 60% aqueous ethanol (1.0 mL). 15 μL of the 60% aqueous ethanol solution (1.0 mL) of the obtained supernatant and solid was sampled, the solvent was distilled off, and a sample was prepared such that the entire solution was 0.5 mL of a 50% v / v aqueous acetonitrile solution. The purity and recovery were confirmed by HPLC.

[0859] The following experiment was conducted on the metal salts shown in Run 28 of Table 11. A solution (0.200 mL) prepared by dispensing 0.100 mL of an ethanol solution of Compound 6 (48.5 mg) (0.485 mL) and 0.075 mL of an ethanol solution of Mg(OAc)2·4H2O (10.3 mg) (0.515 mL) and then diluting with 0.025 mL of ethanol was concentrated and dried under vacuum. Tetrahydrofuran (0.2 mL) and heptane (0.8 mL) were added to the obtained solid, and the mixture was stirred for 3 days, and then the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in ethanol (1.0 mL). 15 μL of the ethanol solution (1.0 mL) of the obtained supernatant and solid was sampled, the solvent was distilled off, and a sample was prepared such that the entire solution was 0.5 mL of a 50% v / v aqueous acetonitrile solution. The purity and recovery were confirmed by HPLC.

[0860] The following experiments were conducted on the metal salts shown in Run 29 of Table 11. A solution (0.200 mL) prepared by dispensing 0.100 mL of an ethanol solution (0.485 mL) of Compound 6 (48.5 mg) and 0.071 mL of an ethanol solution (0.330 mL) of MgCl2·6H2O (6.6 mg) and then diluting with 0.029 mL of ethanol was concentrated and dried under vacuum. Tetrahydrofuran (0.4 mL) and heptane (0.4 mL) were added to the obtained solid, and the mixture was stirred for 3 days, and then the resulting solid and solution were centrifuged. After distilling off the solvent, the supernatant and solid dried under vacuum were redissolved in ethanol (1.0 mL). 15 μL of the obtained supernatant and the ethanol solution (1.0 mL) of the solid were sampled, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. The purity and recovery were confirmed by HPLC.

[0861] The following experiments were conducted on the metal salts shown in Run 30 of Table 11. A solution (0.200 mL) prepared by dispensing 0.050 mL of a solution of Mg(OAc)2·4H2O (13.5 mg) in an aqueous ethanol solution (0.450 mL) and diluting with 0.150 mL of ethanol was added to Compound 6 (10.0 mg), and the mixture was concentrated and dried under vacuum. Acetonitrile (0.3 mL) and water (0.6 mL) were added to the obtained solid, and the mixture was stirred for 4 days, and then the resulting oily precipitate and solution were centrifuged. After distilling off the solvent, the supernatant and precipitate dried under vacuum were redissolved in ethanol (1.0 mL). 15 μL of the obtained supernatant and the ethanol solution (1.0 mL) of the precipitate were sampled, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. The purity and recovery were confirmed by HPLC.

[0862] The following experiments were conducted on the metal salts shown in Run 31 of Table 11. A 35 mM aqueous ethanol solution (0.200 mL) prepared by diluting an aqueous ethanol solution (0.475 mL) of MgCl2·6H2O (9.5 mg) was added to Compound 6 (9.9 mg), and the mixture was centrifuged and dried under vacuum. Acetonitrile (0.3 mL) and water (0.6 mL) were added to the obtained solid, and the mixture was stirred for 4 days, and then the resulting oily precipitate and solution were centrifuged. After distilling off the solvent, the supernatant and precipitate dried under vacuum were redissolved in ethanol (1.0 mL). 15 μL of the obtained supernatant and the ethanol solution (1.0 mL) of the precipitate were sampled, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. The purity and recovery were confirmed by HPLC.

[0863] Table 11 shows the purity and recovery rates of the supernatant and solid, the ratios of isomer 1 or isomer 2 to compound 6, and the purification efficiency calculated by the following expressions to confirm how much isomer 1 and isomer 2 are separated from compound 6 relative to compound 6.

[0864] - Ratio of isomer 1 to compound 6 x 100:

[0865] (Peak area of isomer 1) / (Peak area of compound 6) x 100

[0866] - Ratio of isomer 2 to compound 6 x 100:

[0867] (Peak area of isomer 2) / (Peak area of compound 6) x 100

[0868] - Purification efficiency of isomer 1 (%):

[0869] {1 - (Ratio of isomer 1 to compound 6) / (Ratio of isomer 1 to compound 6 in the preparation)} x 100

[0870] - Purification efficiency of isomer 2 (%):

[0871] {1 - (Ratio of isomer 2 to compound 6) / (Ratio of isomer 2 to compound 6 in the preparation)} x 100

[0872] For Run 29, the ratios and purification efficiencies of individual impurities were calculated. The ratio and purification efficiency of an impurity (relative retention time 0.71, molecular weight 1797.06, estimated to be a linear analog by-product peptide from LC-MS / MS) to compound 6 were calculated by the following expressions to confirm how much impurity was separated from compound 6 relative to compound 6.

[0873] - Ratio of impurity to compound 6 x 100:

[0874] (Peak area of impurity) / (Peak area of compound 6) x 100

[0875] - Purification efficiency of impurity (%):

[0876] {1 - (Ratio of impurity to compound 6) / (Ratio of impurity 1 to reference compound 6)} x 100

[0877] It was found that when using lithium iodide (LiI, Run 2, purity of supernatant 88%, recovery rate 78%), lithium perchlorate (LiClO4, Run 3, purity of supernatant 91%, recovery rate 60%), or ytterbium(III) trifluoromethanesulfonate (Yb(OTf)3, Run 21, purity of supernatant 87%, recovery rate 32%), the purity of the supernatant was 3% or higher than the purity of the preparation, which was 84%. In particular, when using lithium iodide and lithium perchlorate, the purification efficiencies of isomer 1 were 60% and 73% respectively, and the purification efficiencies of isomer 2 were 23% and 43% respectively, and a certain amount of both isomers was successfully removed. On the other hand, when using ytterbium(III) trifluoromethanesulfonate, the purification efficiency of isomer 2 decreased to -10%, but the purification efficiency of isomer 1 was higher, at 58%, resulting in the purity being improved to 87%.

[0878] It was found that when using indium(III) trifluoromethanesulfonate (In(OTf)3, Run 24, purity of supernatant 89%, recovery rate 37%), it was found that the purity of the supernatant was 5% higher than the purity of the preparation, which was 84%, but compound 6, isomer 1, and isomer 2 were each isomerized, and the proportions of each in the preparation and their respective proportions after the purification operation changed.

[0879] It was found that when using potassium iodide (KI, Run 4, solid purity 90%, recovery rate 57%), magnesium bromide (MgBr2, Run 9, solid purity 98%, recovery rate 43%), magnesium perchlorate (Mg(ClO4)2, Run 10, solid purity 90%, recovery rate 82%), magnesium iodide (MgI2, Run 11, solid purity 90%, recovery rate 73%), magnesium trifluoromethanesulfonate (Mg(OTf)2, Run 12, solid purity 89%, recovery rate 41%), scandium(III) trifluoromethanesulfonate (Sc(OTf)3, Run 18, solid purity 87%, recovery rate 40%), samarium(III) trifluoromethanesulfonate (Sm(OTf)3, Run 19, solid purity 87%, recovery rate 33%), cerium trifluoromethanesulfonate (Ce(OTf)3, Run 20, solid purity 88%, recovery rate 63%), or manganese bis(trifluoromethanesulfonate) (Mn(OTf)2, Run 23, solid purity 90%, recovery rate 50%), the purity of the precipitated solid was 3% or higher than the purity of the preparation, which was 84%. In particular, when using magnesium bromide, the purification efficiencies of isomer 1 and isomer 2 were relatively high, 88% and 82% respectively, and the purity was 98%. On the other hand, when using magnesium trifluoromethanesulfonate and samarium(III) trifluoromethanesulfonate, the purification efficiency of isomer 1 was relatively low, 2% and 12% respectively in each case, but the purification efficiency of isomer 2 was 59% and 53% respectively, resulting in the purity being improved to 89% or 87% respectively. For other metal salts, it was found that the purification efficiency of isomer 1 was 19% or higher, and the purification efficiency of isomer 2 was 34% or higher, indicating that both isomers could be removed in a balanced manner (Runs 4, 10, 11, 18, 20, and 23).

[0880] When using potassium tetrafluoroborate (KBF4, Run 5) or potassium hexafluorophosphate (KPF6, Run 6), the metal salts used precipitated when dichloromethane and hexane were added, and no solid containing Compound 6 was obtained. In addition, the purity of the supernatant and the purity of the preparation did not change. It is considered that this is because the metal salts precipitated during the experiment, and Compound 6 could not form a complex with the metal.

[0881] For magnesium sulfate (MgSO4, Run 13), magnesium acetate tetrahydrate (Mg(OAc)2·4H2O, Run 14), magnesium chloride (MgCl2, Run 15), and magnesium fluoride (MgF2, Run 16), when Compound 6 was mixed with each metal salt and then acetonitrile was added, the metal salts did not dissolve and a suspension was produced, and as a result of continuing the experiment as it was, no solid containing Compound 6 was obtained. In addition, the purity of the supernatant and the purity of the preparation did not change. It is considered that this is because the metal salts are insoluble in acetonitrile, and Compound 6 could not form a complex with the metal.

[0882] On the other hand, as a result of using ethanol (or 60% ethanol-water) as a solvent for complex formation in Runs 27 to 31, purification can be confirmed in the supernatants of Runs 27 and 28 and in the solids of Runs 30 and 31. Although the purity was not significantly improved in Run 29, purification was confirmed by focusing on the peak at a relative retention time of 0.71 as shown in Figure 8 and calculating the purification efficiency, such that the purification efficiency was -131% in the solid and 100% (not observed) in the supernatant. It is considered that purification was observed in Runs 27 to 31 because both the metal salt and Compound 6 (or impurities) were dissolved in ethanol (or 60% ethanol-water) and mixed to enable complex formation.

[0883] It was found that when using barium iodide (BaI2, Run 7, purity of solid 85%, recovery rate 54%), barium perchlorate (Ba(ClO4)2, Run 8, purity of solid 86%, recovery rate 65%), or zinc(II) trifluoromethanesulfonate (Zn(OTf)2, Run 22, purity of solid 86%, recovery rate 58%), the purity of the precipitated solid was improved by 1% to 2% compared to the purity of the preparation, but the purification efficiency of Isomer 2 was 23% to 27%. On the other hand, the purification efficiency of all of Isomer 1 was 10% or lower. From these results, it was confirmed that when using these metal salts, Isomer 2 can be removed to some extent.

[0884] It was found that when using calcium bromide (CaBr2, Run 17, purity of supernatant 86%, recovery rate 45%), the purity of the supernatant was improved by 2% compared to the purity of the preparation, but the purification efficiency of Isomer 1 was 59%. On the other hand, the purification efficiency of Isomer 2 was -19%, which hindered the improvement of purity. From these results, it was confirmed that when using these metal salts, Isomer 1 can be removed to some extent.

[0885] When using bismuth(III) trifluoromethanesulfonate (Bi(OTf)3, Run 25), Compound 6 was decomposed, so purification could not be confirmed.

[0886] When using silver trifluoromethanesulfonate (AgOTf, Run 26), no solid precipitate was observed, and the purity of the supernatant and the purity of the preparation did not change. Based on the 1H-NMR spectroscopic analysis in Reference Example 2 1 it was considered that the purity of the supernatant did not increase because Compound 6 did not form a complex with silver trifluoromethanesulfonate. It was also considered that the same results could be obtained when conducting the experiment in the same manner as Run 26 but without adding the metal because Compound 6 does not form a complex with silver trifluoromethanesulfonate.

[0887] Calculation Based on the Peak Area of the Preparation (Recovery Rate Calculation Method 3)

[0888] Calculation of solid recovery rates for runs 11, 12, 13, 18, 19, and 23 (examples)

[0889] After HPLC of the preparation of the 0.306 mg / mL solution, the peak area of the target (60127) was measured. 4 μL of the solid acetonitrile solution obtained in the above experiment was diluted with 396 μL of acetonitrile and measured by HPLC, and then the peak area of the target was measured, and the recovery rate of the target in the supernatant or solid was calculated based on the area of the preparation.

[0890] MgI2: (42893 / 15.05 x 5) / (60127 / 3.06) x 100 ≈ 73%

[0891] Mg(OTf)2: (24183 / 15.07 x 5) / (60127 / 3.06) x 100 ≈ 41%

[0892] MgSO4: (257 / 15.04 x 5) / (60127 / 3.06) x 100 ≈ 0%

[0893] Sc(OTf)3: (23815 / 14.99 x 5) / (60127 / 3.06) x 100 ≈ 40%

[0894] Sm(OTf)3: (19507 / 15.05 × 5) / (60127 / 3.06) x 100 ≈ 33%

[0895] Mn(OTf)2: (29747 / 15.00 x 5) / (60127 / 3.06) x 100 ≈ 50%

[0896] Calculation of the Recovery Rate of Run 27

[0897] In runs 27 to 31, the recovery rates of the supernatant and solid were calculated by the above recovery rate calculation method 1. The calculation of the supernatant and solid recovery rates for run 27 will be described as an example.

[0898] After HPLC of the preparation of the 0.30 mg / mL solution, the peak area of the target (2950511, 220 nm) was measured. The HPLC of the prepared 50% v / v acetonitrile aqueous solution was measured, and the peak area of the target (supernatant: 2704577, solid: 234383) was measured, and the recovery rate was calculated according to the following method.

[0899] Supernatant: (2704577 x 10) / (2950511 x 10.0) x 100 ≈ 92%

[0900] Solid: (234383x 10) / (2950511x 10.0)x 100≈8%

[0901] From the above results, it was confirmed that depending on the type of metal salt forming a complex with the cyclic peptide, there are cases where purification of the cyclic peptide as a purification target is caused or not caused.

[0902] It was confirmed that the relative solubility of the complex and impurities other than the complex can be changed by the formation of a complex of the cyclic peptide as a purification target with a metal ion, and it was confirmed that the cyclic peptide as a target can be purified by taking advantage of the difference in its solubility. It was also found that this method can particularly remove a certain amount of isomers of the cyclic peptide as a purification target that are generally considered difficult to separate.

[0903] [Example 2-2: Confirmation of Experiment Condition Dependence]

[0904] <Basic Operations>

[0905] Compound 6 was mixed with about 1 molar equivalent of magnesium perchlorate and dissolved in acetonitrile. The solvent was distilled off under reduced pressure, dichloromethane and hexane were added, and the mixture was stirred at room temperature as described below. The resulting solid and liquid were centrifuged and the supernatant was sampled for HPLC measurement. The purity and recovery rate calculated based on the peak area percentage and peak area value of each compound are shown in Tables 14 and 15.

[0906] Based on the initial concentration, the sample for HPLC measurement was prepared to be about 0.3 mg / mL. In Example 2-2, HPLC was measured under purity analysis condition B using ACD / Spectrus manufactured by Advanced Chemistry Development, Inc. as HPLC analysis software, and the purity and recovery rate were calculated based on the peak area percentage and peak area at the stage where only dichloromethane was added in each experimental operation. It should be noted that since it was confirmed in a similar experiment in Run 1 of Table 8 that the total recovery rate of the solid and the supernatant was close to 100% and the purity of the solid was improved, the solid recovery experiment was omitted in this experiment, and it was determined that there was a purification effect when the purity of the supernatant decreased relative to the reference value.

[0907] Recovery Rate Calculation Method 4

[0908] For example, assume that an experiment is conducted using a mg of raw material, and the amount of the solution used is b mL. When measuring the HPLC of the supernatant, the solid and the solution are centrifuged, and after sampling (150b / a) μL of the supernatant, the solvent is distilled off, and a sample is prepared such that the entire solution is a 50% v / v aqueous acetonitrile solution of 0.5 mL. When measuring the HPLC of each of the prepared samples, and setting the peak areas (at 220 nm) of the target after adding the reference sample and the solvent to c and d, respectively, the recovery rate is expressed by the following expression.

[0909] [Expression 4]

[0910]

[0911] [Example 2-2-1: Confirmation of Time Dependence]

[0912] [Table 14]

[0913]

[0914] Runs 1 to 3 of Table 14

[0915] To Compound 6 (Run 1: 15.0 mg, Run 2: 15.1 mg, Run 3: 15.1 mg), add a 53 mM acetonitrile solution (0.200 mL each) prepared by dispensing an acetonitrile solution (0.423 mL) of Mg(ClO4)2 (12.7 mg) (Run 1: 0.094 mL, Run 2: 0.095 mL, Run 3: 0.095 mL) and dilute with acetonitrile, and centrifuge and vacuum-dry the mixture. Add dichloromethane (1.0 mL) to the obtained solid, and stir the mixture for 15 minutes, then sample 10 μL from the solution. Add hexane (0.5 mL) to each solution and stir the mixture for 1 minute, then quickly centrifuge the resulting solid and liquid, and sample 15 μL from the supernatant. After stirring again and 10 minutes after adding hexane, centrifuge the solution again and sample the supernatant (15 μL). Similarly, at the stages of 30 minutes, 1 hour, 2 hours, 6 hours, and 2 days after adding hexane, centrifuge each solution and sample the supernatant (15 μL). For the supernatant sampled at each stage, distill off the solvent, and prepare a sample such that the entire solution is a 50% v / v aqueous acetonitrile solution of 50 μL. As a result of confirming the purity and recovery rate by HPLC, the purity of the supernatant decreased at any time, and purification was observed. In addition, since the purity and recovery rate hardly changed 1 hour after adding hexane, it is considered that the approximate value of the equilibrium is about 1 hour.

[0916] Calculation Example of the Supernatant Recovery Rate

[0917] In Run 1, the peak area of the target (3152623360, 225 nm) in the stage of adding only dichloromethane was measured, and the peak area of the target (523598976, 225 nm) 1 hour after adding hexane was measured. The recovery rate was calculated according to the following method.

[0918] 5253598976 / 3125623360×100≈17%

[0919] From the above results, it was confirmed that in any case, from immediately after the addition of the second solvent to a sufficient time after the addition of the second solvent, there was a purification effect on the cyclic peptide in the complex of magnesium perchlorate and the cyclic peptide. It was also confirmed that the purification effect became constant after a certain period of time.

[0920] [Example 2-2-2: Confirmation of Concentration Dependence]

[0921] [Table 15]

[0922]

[0923] Run 1 of Table 15

[0924] To Compound 6 (Run 1: 1.5 mg), a 5.3 mM acetonitrile solution (0.200 mL) prepared by diluting a 0.550 mL acetonitrile solution of 0.0084 mL of Mg(ClO4)2 (16.7 mg) with 0.1916 mL of acetonitrile was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (1.0 mL) was added to the obtained solid and the mixture was stirred for 15 minutes, then hexane (0.5 mL) was added and the mixture was stirred for 7 hours, but no precipitation occurred.

[0925] Run 2 of Table 15

[0926] To Compound 6 (Run 2: 1.5 mg), a 5.3 mM acetonitrile solution (0.200 mL) prepared by diluting 0.0084 mL of an acetonitrile solution (0.550 mL) of Mg(ClO4)2 (16.7 mg) with 0.1916 mL of acetonitrile was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (1.0 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled from the solution. After distilling off the solvent and drying the resultant under vacuum, dichloromethane (0.75 mL) was added to redissolve, and hexane (0.75 mL) was added, and the mixture was stirred overnight, and then the resultant solid and liquid were centrifuged, and 15 μL was sampled from the supernatant. For the supernatant sampled at each stage, the solvent was distilled off, and a sample was prepared such that the entire solution was 50 μL of a 50% v / v aqueous acetonitrile solution. As a result of confirming the purity and recovery by HPLC, the purity of the supernatant decreased, and purification was observed.

[0927] Run 3 of Table 15

[0928] To Compound 6 (Run 3: 7.5 mg), a 26 mM acetonitrile solution (0.200 mL) prepared by diluting 0.047 mL of an acetonitrile solution (0.550 mL) of Mg(ClO4)2 (16.7 mg) with 0.153 mL of acetonitrile was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (1.0 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled from each solution. Hexane (0.5 mL) was added to each solution and the mixture was stirred for 7 hours, and then centrifuged, and 15 μL was sampled from the supernatant of each solution. For the supernatant sampled at each stage, the solvent was distilled off, and a sample was prepared such that the entire solution was 250 μL, 1.5 mL of a 50% v / v aqueous acetonitrile solution to match the concentration. As a result of confirming the purity and recovery by HPLC, the purity of the supernatant decreased in any case, and purification was observed.

[0929] Run 4 of Table 15

[0930] To Compound 6 (15.1 mg), a 53 mM acetonitrile solution (0.200 mL) prepared by diluting 0.143 mL of an acetonitrile solution of Mg(ClO4)2 (7.7 mg) (0.387 mL) with 0.057 mL of acetonitrile was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (1.0 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, then 10 μL was sampled from the solution. Hexane (0.5 mL) was added and the mixture was stirred overnight, then the resulting solid and liquid were centrifuged, and 15 μL was sampled from the supernatant. For the supernatant sampled at each stage, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate of the supernatant by HPLC, the purity of the reference was found to be 80%, while the purity of the supernatant was 62% and the recovery rate was 21%, indicating that purification occurred.

[0931] Run 5 of Table 15

[0932] To Compound 6 (Run ⑤: 45.0 mg), a 63 mM acetonitrile solution (0.500 mL) prepared by diluting 0.283 mL of an acetonitrile solution of Mg(ClO4)2 (16.7 mg) (0.550 mL) with 0.217 mL of acetonitrile was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (1.0 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, then 10 μL was sampled from each solution. Hexane (0.5 mL) was added to each solution and the mixture was stirred for 7 hours, then centrifuged, and 15 μL was sampled from the supernatant of each solution. For the supernatant sampled at each stage, the solvent was distilled off, and a sample was prepared such that the whole solution was 250 μL, a 50% v / v aqueous acetonitrile solution of 1.5 mL to match the concentration. As a result of confirming the purity and recovery rate by HPLC, the purity of the supernatant decreased in any case, and purification was observed.

[0933] It is considered that precipitation did not occur in Run ① because the amount of the cyclic peptide was less than that in Runs ③ to ⑤, and the amount of the complex of magnesium perchlorate with the produced cyclic peptide was small. On the other hand, it is considered that precipitation occurred in Run ② because although the amount and concentration of the cyclic peptide were the same as those in Run ① (concentration: 1 mg / mL), the solubility of the complex of magnesium perchlorate with the produced cyclic peptide was lower than that in Run ① because the ratio of hexane as a poor solvent was higher than that in Run ①.

[0934] [Example 2-3: Example of Using Cyclic Peptides with Different Numbers of Amino Acid Residues in the Ring Portion]

[0935] [Example 2-3-1: Confirmation of Substrate Generalization for Purification Using Mg(ClO4)2 and Dichloromethane / Hexane]<Basic Operations>

[0936] Compounds 9, 11, 12 to 15 (each about 10 mg), each having a different number of amino acid residues in the cyclic portion of the cyclic peptide, were mixed with about 1 molar equivalent of magnesium perchlorate and dissolved in acetonitrile. The solvent was distilled off under reduced pressure, dichloromethane and hexane were added, and the mixture was stirred at room temperature as described below. The resulting solid and liquid were centrifuged, the solvent was distilled off, and the vacuum-dried supernatant and solid were redissolved in acetonitrile, and the respective HPLCs were measured. HPLC analysis was performed using ACD / Spectrus manufactured by Advanced Chemistry Development, Inc. The purity and recovery rate calculated based on the peak area percentage and the value of the peak area (measured at 225 nm) of each compound are shown in Tables 16 and 17. The compounds used as raw materials were the compounds synthesized in Reference Examples 1 to 3 to 8. Based on the analysis of HPLC measured under the same conditions (purity analysis condition C) as the HPLC used in the above purity analysis, it was determined that there was a purification effect when the purity was improved. In addition, even if the purity was not improved relative to the value before the operation, individual impurities were focused on, and it was determined that there was a purification effect on the impurities for which the purification efficiency was positive.

[0937] Based on the initial concentration, the samples for HPLC measurement were prepared to be about 0.3 mg / mL. In Examples 2-3, HPLC was measured under purification analysis condition C. In this experiment, the purity and recovery rate were calculated based on the peak area percentage and the peak area at the stage of adding dichloromethane (0.66 mL) in each experimental operation. The recovery rate was calculated by the above recovery rate calculation method 4. For some, individual impurities were focused on, and the ratio and purification efficiency were calculated based on the impurities.

[0938]

[0939] For Compound 9, the ratio and purification efficiency of two individual impurities were calculated.

[0940] The ratio and purification efficiency of Impurity 1 (relative retention time 0.88, molecular weight 2604.69, estimated to be a linear dimer analog by-product peptide from LC-MS / MS) or Impurity 2 (relative retention time 0.89, molecular weight 2618.71, estimated to be a linear dimer analog by-product peptide from LC-MS / MS) to Compound 9 were calculated by the following expressions to confirm how much Impurity 1 and Impurity 2 were separated from Compound 9 relative to Compound 9.

[0941] - Ratio of Impurity 1 to Compound 9 x 100:

[0942] (Peak area of Impurity 1) / (Peak area of Compound 9) x 100

[0943] - Ratio of impurity 2 to compound 9 x 100:

[0944] (Peak area of impurity 2) / (Peak area of compound 9) x 100

[0945] - Purification efficiency of impurity 1 (%):

[0946] {1 - (Ratio of impurity 1 to compound 9) / (Ratio of impurity 1 to reference compound 9)} x 100

[0947] - Purification efficiency of impurity 2 (%):

[0948] {1 - (Ratio of impurity 2 to compound 9) / (Ratio of impurity 2 to reference compound 9)} x 100 Run 1 of Table 16

[0949] Acetonitrile (0.2 mL) was added to compound 9 (10.0 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, after which 10 μL was sampled. Hexane (0.33 mL) was added and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours, and then centrifuged, but no precipitation occurred. 20 μL was sampled from the supernatant. A sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0950] Run 2 of Table 16

[0951] Acetonitrile solution (0.99 mL) of separately prepared Mg(ClO4)2 (9.9 mg) (0.217 mL) was added to compound 9 (10.2 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, after which 10 μL was sampled. Hexane (0.33 mL) was added, and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours. After centrifugation, the solid precipitated, and 20 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, then the vacuum-dried solid was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from the solution. A sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was 92%, and there was no improvement compared to the initial purity of 92%. On the other hand, when as Figure 9When calculating the purification efficiency of impurity 1 (relative retention time 0.88) and impurity 2 (0.89), it was shown that the purification efficiencies of impurity 1 and impurity 2 in the supernatant relative to compound 9 were 67% and 62% respectively. Therefore, it was determined that the supernatant was purified.

[0952] [Table 17]

[0953]

[0954] Run 1 of Table 17

[0955] Acetonitrile (0.2 mL) was added to compound 12 (9.8 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (0.33 mL) was added and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours, and then centrifuged, but no precipitation occurred. 20 μL was sampled from the supernatant. A sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0956] Run 2 of Table 17

[0957] An acetonitrile solution (0.99 mL) of separately prepared Mg(ClO4)2 (9.9 mg) (0.207 mL) was added to compound 12 (10.1 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (0.33 mL) was added, and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours. After centrifugation, the solid precipitated, and 20 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, and then the vacuum-dried solid was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from each solution. A sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that any supernatant was purified.

[0958] Run 3 of Table 17

[0959] Acetonitrile (0.2 mL) was added to Compound 14 (10.0 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, after which 10 μL was sampled. Hexane (0.33 mL) was added and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours, and then centrifuged, but no precipitation occurred. 20 μL was sampled from the supernatant. Samples were prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0960] Run 4 of Table 17

[0961] An acetonitrile solution (0.99 mL) of separately prepared Mg(ClO4)2 (9.9 mg) (0.169 mL) was added to Compound 14 (10.0 mg), and the resulting solution was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, after which 10 μL was sampled. Hexane (0.33 mL) was added, and the mixture was stirred overnight, then further hexane (0.33 mL) was added and the mixture was stirred for 3.5 hours. After centrifugation, the solid precipitated, and 20 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, then the solid dried under vacuum was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from each solution. Samples were prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming purity and recovery by HPLC, it was confirmed that any supernatant was purified.

[0962] Run 5 of Table 17

[0963] An acetonitrile solution (0.200 mL) of Compound 11 (9.9 mg) was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, after which 10 μL was sampled. Hexane (0.66 mL) was added and the mixture was stirred for 4.5 hours, and then centrifuged, but no precipitation occurred. 20 μL was sampled from the supernatant. For each sampled solution, the solvent was distilled off, and samples were prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0964] Run 6 of Table 17

[0965] To Compound 11 (10.1 mg), a 36 mM acetonitrile solution (0.200 mL) prepared by diluting a solution of Mg(ClO4)2 (3.8 mg) in acetonitrile (0.380 mL) was added, and the mixture was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (0.66 mL) was added and the mixture was stirred for 4.5 hours. After centrifugation, the solid precipitated, and 20 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, and the solid dried under vacuum was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from each solution. For each sampled solution, the solvent was distilled off, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery by HPLC, it was confirmed that the supernatant was purified.

[0966] Run 7 of Table 17

[0967] The acetonitrile solution (0.200 mL) of Compound 13 (9.9 mg) was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (0.66 mL) was added and the mixture was stirred for 4.5 hours, and then centrifugation was carried out, but no precipitation occurred. 20 μL was sampled from the supernatant. For each sampled solution, the solvent was distilled off, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0968] Run 8 of Table 17

[0969] To Compound 13 (9.9 mg), a 32 mM acetonitrile solution (0.200 mL) prepared by diluting a solution of Mg(ClO4)2 (4.1 mg) in acetonitrile (0.410 mL) was added, and the mixture was concentrated and dried under vacuum. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (0.66 mL) was added and the mixture was stirred for 4.5 hours. After centrifugation, the solid precipitated, and 20 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, and the solid dried under vacuum was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from each solution. For each sampled solution, the solvent was distilled off, and a sample was prepared such that the entire solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery by HPLC, it was confirmed that the supernatant was purified.

[0970] Run 9 of Table 17

[0971] The acetonitrile solution (0.200 mL) of Compound 15 (9.9 mg) was concentrated and dried under vacuum. Then, dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (1.32 mL) was added and the mixture was stirred for 4.5 hours, and then centrifuged, but no precipitation occurred. 30 μL was sampled from the supernatant. For each sampled solution, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL.

[0972] Run 10 of Table 17

[0973] A 30 mM acetonitrile solution (0.200 mL) prepared by diluting an acetonitrile solution of Mg(ClO4)2 (4.7 mg) (0.470 mL) was added to Compound 15 (9.9 mg), and the mixture was concentrated and dried under vacuum. Dichloromethane (0.66 mL) was added to the obtained solid, and the mixture was stirred for 15 minutes, and then 10 μL was sampled. Hexane (1.32 mL) was added and the mixture was stirred for 4.5 hours. After centrifugation, the solid precipitated, and 30 μL was sampled from the supernatant. The solid and the liquid were centrifuged, the solvent was distilled off, and then the solid dried under vacuum was redissolved in acetonitrile (1.0 mL), and 15 μL was sampled from each solution. For each sampled solution, the solvent was distilled off, and a sample was prepared such that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. As a result of confirming the purity and recovery rate by HPLC, it was confirmed that the supernatant was purified.

[0974] From the above findings, it was found that the purification method of the cyclic peptide of the present invention can also be applied to cyclic peptides having different numbers of amino acid residues in the cyclic moiety and cyclic peptides having branched chains.

[0975] [Example 2-3-2: Confirmation of Substrate Generalization Using MgI2 and Multiple Solvents]

[0976] <Basic Operations>

[0977] Each compound shown in Table 18 below (about 10 mg), which is a cyclic peptide having different numbers of amino acid residues in the cyclic portion, was mixed with about 1 molar equivalent of magnesium iodide and dissolved in acetonitrile. The solvent was distilled off under reduced pressure, various solvents shown in Table 18 below were added, and the mixture was stirred at room temperature as described below. The resulting solid and liquid were centrifuged, the solvent was distilled off, and the vacuum-dried supernatant and solid were redissolved in acetonitrile, and the respective HPLCs were measured under purification analysis conditions C. HPLC analysis was performed using ACD / Spectrus manufactured by Advanced Chemistry Development, Inc. The purity and recovery rate calculated based on the peak area percentage and peak area value (measured at 225 nm) of each compound are shown in Table 18.

[0978] Based on the initial concentration, the sample for HPLC measurement was prepared to be about 0.3 mg / mL. In Examples 2-3, HPLC was measured under purification analysis conditions C. In this experiment, the purity and recovery rate were calculated based on the peak area percentage and peak area at the stage of adding dichloromethane (0.66 mL) in Example 2-3-1. The recovery rate was calculated by the above Recovery Rate Calculation Method 4 to calculate the recovery rate.

[0979] [Table 18]

[0980]

[0981]

[0982] Runs 1 to 6 of Table 18

[0983] To each about 10 mg of the compound, an acetonitrile solution (2.62 mL) of 1 molar equivalent of separately prepared MgI2 (26.2 mg) was added, and the resulting solution was concentrated and vacuum-dried. Then, each solvent shown in Runs 1 to 6 of Table 18 was added to the obtained solid, and the mixture was stirred overnight. The resulting solid and solution were centrifuged, the solvent was distilled off, and then the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). 15 μL was sampled from the obtained supernatant and the acetonitrile solution (1.0 mL) of the solid, and the sample was prepared so that the whole solution was a 50% v / v aqueous acetonitrile solution of 0.5 mL. The purity and recovery rate were confirmed by HPLC. In Runs 1, 2, 3, 4, and 6, it was confirmed by the supernatant that the solid was purified. In Run 5, it was confirmed that the solid was purified.

[0984] From the above findings, the purification method of the cyclic peptides of the present invention can be carried out on cyclic peptides having different numbers of amino acid residues in the cyclic portion and cyclic peptides having branched chains using various metal salts and solvents.

[0985] [Example 3: Extraction of Cyclic Peptides]

[0986] [Example 3-1: Study on the Separation of Cyclic Peptides and Metal Salts]

[0987] <Experimental Operation 1>

[0988] The solution of compound 6 (30.0 mg, 84% purity [peak area percentage]) and Mg(ClO4)2 (5.88 mg) in CH3CN (1.0 mL) was concentrated under reduced pressure, and the resulting solid was dissolved in 2 mL of dichloromethane. Further, 1 mL of hexane was added, and the mixture was stirred for 3 hours. The obtained suspension was centrifuged and separated into a solid and a liquid. Then, the solid was added to a mixed solution of dichloromethane - hexane (2 mL / 1 mL), and the mixture was stirred for another 3 hours. The obtained suspension was centrifuged and dried to obtain a solid (43.07 mg, 93% purity [peak area percentage]).

[0989] <Experimental Operation 2>

[0990] Deuterated acetonitrile (CD3CN, 0.6 mL) was added to 1.13 mg of the solid obtained in Experimental Procedure 1, and 0.5 mL of the resulting solution was used for measurement 1 1H-NMR( Figure 10 -a).

[0991] <Experimental Operation 3>

[0992] 1 mL of dichloromethane was added to 1.02 mg of the solid obtained in Experimental Procedure 1, and the mixture was allowed to stand and dry overnight. Deuterated acetonitrile (CD3CN, 0.5 mL) was added, and measurement was carried out 1 1H-NMR( Figure 10 -b).

[0993] <Experimental Operation 4>

[0994] 1 mL of dichloromethane was added to 1.14 mg of the solid obtained in Experimental Procedure 1, and then 1 mL of water was added for liquid separation operation. The obtained organic phase and aqueous phase were separated, 1 mL of water was added to the organic phase, and the liquid separation operation was carried out twice more. As a result of drying the obtained organic phase, 0.72 mg of a solid was obtained. Deuterated acetonitrile (CD3CN, 0.5 mL) was added to the obtained solid, and measurement was carried out 1 1H-NMR( Figure 10 -c). For comparison, the 1 1H-NMR spectrum of the raw material compound 6 (84% purity [peak area percentage]) is shown in Figure 10 -d.

[0995] For comparison Figure 10 -a and Figure 10 as shown in -d, 1 the 1H-NMR spectrum of the obtained solid 1 1H-NMR spectrum ( Figure 10 -a) is different from that of the raw material 1 1H-NMR spectrum ( Figure 10 -d), so it is confirmed that the obtained solid is a complex.

[0996] For comparison Figure 10 -a and Figure 10 as shown in -b, 1 the results of the 1H-NMR spectrum show the same spectrum for both, so it is confirmed that even when dichloromethane is added, the complex obtained in Experimental Operation 1 is not decomposed.

[0997] For comparison Figure 10 -a, Figure 10 -c and Figure 10 as shown in -d, 1 the results of the 1H-NMR spectrum, the NMR spectrum obtained after the liquid separation operation ( Figure 10 -c) is different from that of the solid obtained in Experimental Operation 1 1 1H-NMR spectrum ( Figure 10 -a), and is consistent with the main peak of the 1H-NMR spectrum of the raw material 1 1H-NMR spectrum ( Figure 10 -d). So it is confirmed that the complex is decomposed by the liquid separation operation without metal addition to obtain Compound 6.

[0998] From the above findings, Mg(ClO4)2 can be removed by subjecting the complex obtained in Experimental Operation 1 to a liquid separation operation using water and dichloromethane.

[0999] [Example 3-2: Extraction of Cyclic Peptides]

[1000] <Experimental Operation 1 (Extraction and Purification of Complex)>

[1001] A solution of Compound 6 (15.17 mg, 84% purity [peak area percentage]) and Mg(ClO4)2 (2.95 mg) in CH3CN (0.5 mL) was concentrated under reduced pressure, and the resulting solid was dissolved in 1 mL of dichloromethane. Then 0.6 mL of hexane was further added, and the mixture was stirred for 2 hours. The obtained suspension was centrifuged and separated into a solid and a liquid. Then the solid was added to a mixed solution of dichloromethane - hexane (1 mL / 0.6 mL), and the mixture was stirred for another 30 minutes. The obtained suspension was centrifuged and dried to obtain a solid (13.16 mg, 88% purity).

[1002] <Experimental Operation 2 (Extraction of Compound 6 from the Complex)>

[1003] To the solid obtained in Experimental Operation 1, add dichloromethane (1 mL) and water (1 mL) for liquid separation operation. Separate the aqueous phase and the organic phase, and add water (2 mL) to the organic phase for liquid separation operation. Repeat this operation once more. Separate the aqueous phase and the organic phase, and then add water (2 mL) and acetonitrile (0.05 mL) to the organic phase for liquid separation operation. Separate the aqueous phase and the organic phase, and add water (2 mL) to the organic phase for liquid separation operation. Separate the aqueous phase and the organic phase, and add dichloromethane (0.5 mL) and water (1 mL) to the organic phase for liquid separation operation. Separate the aqueous phase and the organic phase, add dichloromethane (1 mL) to the aqueous phase for liquid separation operation, and separate the organic phase and the aqueous phase. Conduct the same operation two more times. Collect the obtained organic phase and distill off the solvent under reduced pressure to obtain a solid (10.57 mg, purity 88%, recovery rate 73%).

[1004] The recovery rate is calculated as follows, and 1 1H-NMR is measured to confirm that the obtained solid is Compound 6 rather than a complex.

[1005] Calculation of the recovery rate: 10.57 x 0.88 / (15.17 x 0.84) x 100 = 73%

[1006] Hereinafter, it will be described in detail Figures 2 to 10 . Figure 2 is measured at 298 K by measuring the 1 1H-NMR of each of the following solutions 1 Schematic diagram of the 1H-NMR spectrum: a solution obtained by dissolving Compound 6 in acetonitrile-d3 (CD3CN) (concentration of the cyclic peptide: 1.4 mM) and solutions obtained by adding magnesium perchlorate (Mg(ClO4)2) to the cyclic peptide such that the magnesium perchlorate is 1, 3, or 6 molar equivalents relative to the cyclic peptide (concentration of the cyclic peptide: 1.4 mM, metal salt addition experiment).

[1007] Figure 2 -a) is a schematic diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution without the addition of magnesium perchlorate (Mg(ClO4)2). 1 1H-NMR spectrum.

[1008] Figure 2 -b) is the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of magnesium perchlorate (Mg(ClO4)2) is added to Compound 6. 1Schematic diagram of the ¹H-NMR spectrum.

[1009] Figure 2 -c) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of magnesium perchlorate (Mg(ClO4)2) are added to Compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1010] Figure 2 -d) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of magnesium perchlorate (Mg(ClO4)2) are added to Compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1011] Figure 3 is measured at 298 K by measuring the 1 ¹H-NMR obtained 1 Schematic diagram of the ¹H-NMR spectrum: a solution obtained by dissolving Compound 6 in acetonitrile-d3 (CD3CN) (concentration of the cyclic peptide: 1.4 mM) and solutions obtained by adding magnesium iodide (MgI2) to the cyclic peptide such that the magnesium iodide is 1, 3, or 6 molar equivalents relative to the cyclic peptide (concentration of the cyclic peptide: 1.4 mM, metal salt addition experiment).

[1012] Figure 3 -a) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution without the addition of magnesium iodide (MgI2). 1 Schematic diagram of the ¹H-NMR spectrum.

[1013] Figure 3 -b) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of magnesium iodide (MgI2) is added to Compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1014] Figure 3 -c) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of magnesium iodide (MgI2) are added to Compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1015] Figure 3 -d) is of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of magnesium iodide (MgI2) are added to Compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1016] Figure 4The following solutions were measured at 298K: 1 H-NMR 1 Schematic diagram of the H-NMR spectrum: a solution obtained by dissolving compound 6 in acetonitrile-d3(CD3CN) (concentration of cyclic peptide: 1.4 mM) and a solution obtained by adding magnesium trifluorosulfonate (Mg(OTf)2) to the cyclic peptide so that the amount of magnesium trifluorosulfonate is 1, 3, or 6 molar equivalents relative to the cyclic peptide (concentration of cyclic peptide: 1.4 mM, metal salt addition experiment).

[1017] Figure 4 -a) is the reaction of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution without the addition of magnesium trifluorosulfonate (Mg(OTf)2) 1 A simplified diagram of the H-NMR spectrum.

[1018] Figure 4 -b) is the reaction of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of magnesium trifluorosulfonate (Mg(OTf)2) is added to compound 6. 1 A simplified diagram of the H-NMR spectrum.

[1019] Figure 4 -c) is the reaction of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of magnesium trifluorosulfonate (Mg(OTf)2) are added to compound 6. 1 A simplified diagram of the H-NMR spectrum.

[1020] Figure 4 -d) is the reaction of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of magnesium trifluorosulfonate (Mg(OTf)2) are added to compound 6. 1 A simplified diagram of the H-NMR spectrum.

[1021] Figure 5 The following solutions were measured at 298K: 1 H-NMR 1 Schematic diagram of H-NMR spectra: solutions obtained by dissolving compound 6 in acetonitrile-d3 (CD3CN) (concentration of cyclic peptide: 1.4 mM) and solutions obtained by adding calcium iodide (CaI2) to the cyclic peptide so that the amount of calcium iodide is 1, 3 or 6 molar equivalents relative to the cyclic peptide (concentration of cyclic peptide: 1.4 mM, metal salt addition experiment).

[1022] Figure 5 -a) is the reaction of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution without calcium iodide (CaI2) addition 1Schematic diagram of the ¹H-NMR spectrum.

[1023] Figure 5 -b) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of calcium iodide (CaI2) is added to compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1024] Figure 5 -c) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of calcium iodide (CaI2) are added to compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1025] Figure 5 -d) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of calcium iodide (CaI2) are added to compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1026] Figure 6 It was measured at 298 K by measuring the 1 ¹H-NMR obtained 1 Schematic diagram of the ¹H-NMR spectrum: the solution obtained by dissolving compound 6 in acetonitrile-d3 (CD3CN) (concentration of cyclic peptide: 1.4 mM) and the solution obtained by adding scandium(III) triflate (Sc(OTf)3) to the cyclic peptide such that the amount of scandium(III) triflate relative to the cyclic peptide is 1, 3, or 6 molar equivalents (concentration of cyclic peptide: 1.4 mM, metal salt addition experiment).

[1027] Figure 6 -a) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution without the addition of scandium(III) triflate (Sc(OTf)3). 1 Schematic diagram of the ¹H-NMR spectrum.

[1028] Figure 6 -b) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of scandium(III) triflate (Sc(OTf)3) is added to compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1029] Figure 6 -c) is the ¹H-NMR spectrum of compound 6 (1.4 mM) in acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of scandium(III) triflate (Sc(OTf)3) are added to compound 6. 1 Schematic diagram of the ¹H-NMR spectrum.

[1030] Figure 6 -d) is a simplified diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of scandium(III) trifluoromethanesulfonate (Sc(OTf)3) are added to Compound 6. 1 Simplified 1H-NMR spectrum.

[1031] Figure 7 It was measured at 298 K by measuring the 1H-NMR of each of the following solutions. 1 Obtained by 1H-NMR 1 Simplified 1H-NMR spectra: a solution obtained by dissolving Compound 6 in acetonitrile-d3 (CD3CN) (concentration of the cyclic peptide: 1.4 mM) and solutions obtained by adding silver trifluoromethanesulfonate (AgOTf) to the cyclic peptide such that the silver trifluoromethanesulfonate is 1, 3, or 6 molar equivalents relative to the cyclic peptide (concentration of the cyclic peptide: 1.4 mM, metal salt addition experiment).

[1032] Figure 7 -a) is a simplified diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution without the addition of silver trifluoromethanesulfonate (AgOTf). 1 Simplified 1H-NMR spectrum

[1033] Figure 7 -b) is a simplified diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 1 molar equivalent of silver trifluoromethanesulfonate (AgOTf) is added to Compound 6. 1 Simplified 1H-NMR spectrum

[1034] Figure 7 -c) is a simplified diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 3 molar equivalents of silver trifluoromethanesulfonate (AgOTf) are added to Compound 6. 1 Simplified 1H-NMR spectrum

[1035] Figure 7 -d) is a simplified diagram of the 1H-NMR spectrum of Compound 6 (1.4 mM) in an acetonitrile-d3 (CD3CN) solution when 6 molar equivalents of silver trifluoromethanesulfonate (AgOTf) are added to Compound 6. 1 Simplified 1H-NMR spectrum

[1036] Figure 8 It is a simplified diagram showing the LC chromatogram measured in Run No. 29 of Example 2-1.

[1037] Figure 9 It is a simplified diagram showing the LC chromatogram measured in Run No. 2 of Example 2-3-1.

[1038] Figure 10 by dissolving each of the solids obtained in each of the experimental operations (experimental operations 1 to 4) in Example 3-1 in CD3CN and measuring 1 1H-NMR spectrum and by dissolving compound 6 (84% purity [peak area percentage]) in CD3CN and measuring 1 1H-NMR spectrum and by dissolving compound 6 (84% purity [peak area percentage]) in CD3CN and measuring 1 1H-NMR spectrum and by dissolving compound 6 (84% purity [peak area percentage]) in CD3CN and measuring 1 Sketches of the 1H-NMR spectra.

[1039] Figure 10 -a) is a sketch of the 1H-NMR spectrum obtained by adding deuterated acetonitrile (CD3CN, 0.6 mL) to 1.13 mg of the solid obtained in experimental operation 1 and measuring 1H-NMR using 0.5 mL of the resulting solution 1 1H-NMR spectrum and by dissolving compound 6 (84% purity [peak area percentage]) in CD3CN and measuring 1 Sketches of the 1H-NMR spectra.

[1040] Figure 10 -b) is a sketch of the 1H-NMR spectrum obtained by adding 1 mL of dichloromethane to 1.02 mg of the solid obtained in experimental operation 1, allowing the mixture to stand and dry overnight, adding deuterated acetonitrile (CD3CN, 0.5 mL) and measuring 1H-NMR 1 Sketches of the 1H-NMR spectra.

[1041] Figure 10 -c) is a sketch of the 1H-NMR spectrum obtained by adding 1 mL of dichloromethane to 1.14 mg of the solid obtained in experimental operation 1, then subjecting it to a liquid separation operation according to experimental operation 3, adding deuterated acetonitrile (CD3CN, 0.5 mL) to the obtained solid and measuring 1H-NMR 1 Sketches of the 1H-NMR spectra.

[1042] Figure 10 -d) is a sketch of the 1H-NMR spectrum of compound 6 (84% purity [peak area percentage]) in deuterated acetonitrile (CD3CN) without the addition of metal salts of cancer 1 Sketches of the 1H-NMR spectra.

Claims

1. A method for purifying a cyclic peptide, The purification method includes the following separation step (i) or (ii): (i) Separating the cyclic peptide as the purification target in the form of a complex with a metal atom or metal ion from a mixture containing the cyclic peptide as the purification target, or (ii) Separating the cyclic peptide as the purification target or the peptide as an impurity in the form of a complex with a metal atom or metal ion from a mixture containing the cyclic peptide as the purification target and the peptide as an impurity, wherein The metal atom is at least one selected from the group consisting of: alkali metal atoms, alkaline earth metal atoms, transition metal atoms, poor metal atoms, and rare earth metal atoms, and The metal ion is at least one selected from the group consisting of: alkali metal ions, alkaline earth metal ions, transition metal ions, poor metal ions, and rare earth metal ions.

2. The purification method according to claim 1, which includes step (1): Mixing the mixture containing the cyclic peptide as the purification target or the mixture containing the cyclic peptide as the purification target and the peptide as an impurity with the metal atom or the metal ion, as a preliminary step of the separation step.

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

4. A method for purifying a cyclic peptide, the purification method includes Step (1)': Mixing a mixture containing the cyclic peptide as the purification target or a mixture containing the cyclic peptide as the purification target and the peptide as an impurity with a metal atom or a metal ion in a first solvent, wherein The metal atom is at least one selected from the group consisting of: alkali metal atoms, alkaline earth metal atoms, transition metal atoms, poor metal atoms, and rare earth metal atoms, and The metal ion is at least one selected from the group consisting of: alkali metal ions, alkaline earth metal ions, transition metal ions, poor metal ions, and rare earth metal ions.

5. The purification method according to claim 4, wherein in the step (1)', a complex of the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion is formed in the first solvent.

6. The purification method according to any one of claims 3 to 5, which further includes step (2): Mixing the mixture obtained in the step (1) or the step (1)' with a second solvent, wherein The first solvent and the second solvent are different solvents from each other.

7. The purification method according to claim 6, which includes step (2)': Removing at least a part of the first solvent, as a preliminary step of the step (2).

8. The purification method according to claim 6 or 7, which includes step (3): Separating the complex of the cyclic peptide as the purification target or the peptide as an impurity with the metal atom or the metal ion from the mixed solution of the mixture obtained in the step (1) or the step (1)' and the second solvent.

9. The purification method according to any one of claims 3 to 8, wherein the first solvent is at least one selected from the group consisting of alcohol-based solvents, nitrile-based solvents, benzene-based solvents, ether-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, sulfoxide-based solvents, and amide-based solvents.

10. A method for purifying a cyclic peptide, the purification method comprising step (4): mixing a complex of a cyclic peptide as a purification target or a peptide as an impurity with a metal atom or a metal ion with a second solvent, wherein the metal atom is at least one selected from the group consisting of alkali metal atoms, alkaline earth metal atoms, transition metal atoms, poor metal atoms, and rare earth metal atoms, and the metal ion is at least one selected from the group consisting of alkali metal ions, alkaline earth metal ions, transition metal ions, poor metal ions, and rare earth metal ions.

11. The purification method according to claim 10, further comprising step (5): separating the complex from the mixed solution of the complex and the second solvent.

12. The purification method according to any one of claims 6 to 8, 10, and 11, wherein the second solvent is at least one selected from the group consisting of ether-based solvents, benzene-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, nitrile-based solvents, hydrocarbon-based solvents, alcohol-based solvents, sulfoxide-based solvents, and amide-based solvents.

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

14. The production method according to claim 13, further comprising the step of obtaining the cyclic peptide as a purification target by a liquid phase synthesis method.

15. The production method according to claim 13, further comprising the step of obtaining the cyclic peptide as a purification target by a solid phase synthesis method.

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