Method for producing peptide compound using mixed anhydride procedure

Abstract

Provided is a method for synthesizing a peptide compound with high diastereoselectivity at high yield even in fragment coupling, said method producing a peptide compound or a salt thereof and including a step (linking step) in which an amino group of a first amino acid or peptide and a carboxy group of a second amino acid or peptide are linked by an amide bond in a two-layer solvent containing water and one or more types of organic solvent immiscible with water.

Description

Method for producing peptide compounds using the mixed acid anhydride method

[0001] The present invention relates to a method for producing a peptide compound using a mixed acid anhydride method.

[0002] In recent years, it has been found that the metabolic stability and membrane permeability of peptides can be improved by using unnatural amino acids, such as cyclic peptides (Non-Patent Documents 1 and 2). Among these, cyclic peptide compounds containing N-substituted amino acids have been suggested to be particularly useful for the development of inhibitors of protein-protein interactions due to their drug-like properties, such as metabolic stability and membrane permeability (Patent Document 1 and Non-Patent Document 3).

[0003] As peptide compounds have come to be of interest as pharmaceuticals, methods for producing them have also become important (Non-Patent Document 4). Peptide compound synthesis is generally carried out by repeatedly linking the C-terminal carboxyl group of an amino acid to the N-terminal amino group of another amino acid or a peptide, thereby elongating the peptide chain. One known method for elongating a peptide chain is to use EDC as a condensation agent in a two-phase solvent system of isopropyl acetate and water (Non-Patent Document 5). Other known methods include using HATU as a condensation agent, T3P as a condensation agent, acid chlorides, and mixed acid anhydrides using acyl halides such as pivaloyl chloride.

[0004] Among these, peptide chain elongation via mixed acid anhydrides is carried out by reacting the mixed acid anhydride obtained by activating the C-terminal carboxy group of a reaction substrate with an acyl halide with the N-terminal amine of another reaction substrate. Examples of such methods for peptide chain elongation via mixed acid anhydrides include a method in which an unprotected amino acid is reacted with a substrate temporarily protected by silylation (Non-Patent Document 6), and a method in which a two-residue peptide is synthesized from amino acids via a mixed acid anhydride using pivaloyl chloride in a two-phase solvent system of water and ethyl acetate (Non-Patent Document 7). Both of these methods involve sequentially subjecting amino acids, one residue at a time, to an amide bond-forming reaction.

[0005] Meanwhile, a method for peptide chain elongation, called fragment coupling, in which peptide fragments consisting of multiple amino acid residues are subjected to an amide bond formation reaction is also known. Fragment coupling has the advantage of increasing the overall yield of the final product, and this advantage becomes even more pronounced as the number of amino acid residues in the desired peptide compound increases. However, under conventional amide bond formation conditions, impurities increase due to factors such as racemization at the α-position of amino acid residues, resulting in a significant decrease in the yield and purity of the target product, making purification more difficult and resulting in increased production time and effort. Furthermore, when an N-substituted amino acid serves as the reaction site for the amide bond formation reaction in fragment coupling, reduced reactivity due to steric hindrance of the substituent on the nitrogen atom, which is the reaction site, has been an issue (Non-Patent Document 8). Therefore, when synthesizing peptide compounds containing N-substituted amino acids, synthetic methods have been limited to either sequentially subjecting amino acids one by one to an amide bond formation reaction or fragment coupling at residues that do not undergo epimerization.

[0006] International Publication No. 2013 / 100132

[0007] Acc. Chem. Res., 2008, 41, 1331-1342.Angew. Chem. Int. Ed., 2013, 52, 254-269.Chem. Rev., 2019, 119, 10360-10391.Amino Acids, Peptides and Proteins in Organic Chemistry: Building Blocks, Catalysis and Coupling Chemistry., 2011, 3.J. Org. Chem., 1995, 60, 3569-3570.Org. Lett., 2020, 22, 8039-8043.ACS Sustainable Chem. Eng., 2022, 10, 5307-5314.J. Peptide Res., 2005, 65, 153-166.

[0008] The present invention has been made in view of these circumstances. In one aspect, an object is to establish a novel synthesis method for peptide compounds. In one aspect, an object is to establish a novel synthesis method suitable for fragment coupling of peptide compounds. In one aspect, an object is to establish a method for synthesizing peptide compounds with high diastereoselectivity and high yield even in fragment coupling. In one aspect, an object is to establish a synthesis method suitable for fragment coupling of peptide compounds containing N-substituted amino acids. In one aspect, an object is to establish a synthesis method suitable for fragment coupling of peptide compounds having bulky side chains near the reaction site of an amide bond.

[0009] As a result of extensive research to solve the above problems, the present inventors have discovered a method for synthesizing peptide compounds with high diastereoselectivity and high yield even in fragment coupling by performing an amide bond formation reaction in a mixture containing water and one or more water-immiscible solvents. The present inventors have also discovered a method for solving at least one of the above problems by activating the carboxyl groups of amino acids or peptides using a specific acyl halide in the above mixture. The present inventors have also discovered reaction conditions that can suppress the formation of by-products in the condensation reaction.

[0010] In one non-limiting specific aspect, the present invention includes the following: [A1] A method for producing a peptide compound or a salt thereof, comprising a step of linking the amino group of a first amino acid or peptide to the carboxy group of a second amino acid or peptide via an amide bond (linking step) in a two-phase solvent system containing one or more water-immiscible organic solvents and water. [A2] The method of [A1], wherein the linking step is carried out in the presence of an activating agent. [A3] The method of [A1] or [A2], wherein the activating agent is an acyl halide. [A4] The method of [A1], wherein the linking step comprises: (1) a step of contacting the second amino acid or peptide with the acyl halide in an organic solvent to prepare a mixed acid anhydride, and (2) a step of contacting the first amino acid or peptide with the mixed acid anhydride obtained in step (1) in the presence of a base in a two-phase solvent system containing one or more water-immiscible organic solvents and water. [A5] The method according to [A4], wherein the step of contacting the first amino acid or peptide with the mixed acid anhydride obtained in step (1) in (2) is a step of adding the first amino acid or peptide to the mixed acid anhydride obtained in step (1). [A6] The method according to any one of [A1] to [A5], wherein the second amino acid or peptide is a peptide containing two or more amino acid residues. [A7] The method according to any one of [A1] to [A6], wherein the α-carbon of the carboxy group of the second amino acid or peptide is optionally substituted. [A8] The method according to any one of [A1] to [A6], wherein the α-carbon of the carboxy group of the second amino acid or peptide is optionally substituted. [A9] The method according to any one of [A1] to [A9], wherein the α-carbon of the carboxy group of the second amino acid or peptide is a group represented by the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ６ Alkyl, optionally substituted branched C ３ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl C １ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl, optionally substituted phenyl C １ -C ２ Alkyl, optionally substituted C１ -C ６ Alkoxy C １ -C ２ Alkyl, optionally substituted 5- to 6-membered heteroaryl C １ -C ２ Alkyl or optionally substituted phenyl C １ -C ２ Alkylthio C １ -C ２ alkyl or R １ and R ２ together with the carbon atoms to which they are attached, form C ３ -C ８ [A9] The method according to any one of [A1] to [A6], wherein the α-carbon of the carboxy group of the second amino acid or peptide is represented by the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ４ Alkyl, branched C ３ -C ６ Alkyl, C ３ -C ６ Cycloalkyl C １ -C ６ Alkyl, C １ -C ６ Alkoxymethyl, 1-(C １ -C ６ alkoxy)ethyl, optionally substituted benzyl, optionally substituted phenethyl, or phenyl C １ -C ２ [A10] The method according to any one of [A1] to [A6], wherein R is an alkylthiomethyl group. １ [A11] The method according to [A8] or [A9], wherein the α-carbon of the carboxy group of the second amino acid or peptide is a group represented by the formula: -CR １ R ２ - (wherein, R 1 is a hydrogen atom, and R ２is a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, t-butoxymethyl, 1-(t-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl, or benzylthiomethyl. [A12] The method according to any one of [A1] to [A6], wherein the nitrogen atom at the β-position of the carboxy group of the second amino acid or peptide is represented by the formula: -NR ３ - (wherein, R ３ is a hydrogen atom). [A13] The method according to any one of [A1] to [A12], wherein the first amino acid or peptide is a peptide containing two or more amino acid residues. [A14] The method according to any one of [A1] to [A13], wherein the carboxy group of the first amino acid or peptide is protected with a protecting group. [A15] The method according to any one of [A1] to [A14], wherein the amino group of the second amino acid or peptide is protected with a protecting group. [A16] The method according to any one of [A1] to [A14], wherein the acyl halide is a linear C １ -C １８ Alkylcarbonyl chloride, branched C ３ -C １８ Alkylcarbonyl chloride, linear C １ -C １８ Alkylcarbonyl bromide, branched C ３ -C １８ Alkylcarbonyl bromide and monocyclic or condensed ring C ３ -C １５ Cycloalkylcarbonyl chloride (C ３ -C １５ Cycloalkylcarbonyl chloride is C １ -C ６[A17] The method according to any one of [A3] to [A16], wherein the acyl halide is at least one selected from the group consisting of pivaloyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexanecarbonyl chloride, 1-adamantanecarbonyl chloride, and 2-ethylbutyryl chloride. [A18] The method according to any one of [A3] to [A17], wherein the acyl halide is pivaloyl chloride or 2,2-dimethylbutyryl chloride. [A19] The method according to any one of [A3] to [A18], wherein the acyl halide is used in an amount of 0.6 to 1.0 molar equivalents relative to the second amino acid or peptide. [A20] The method according to any one of [A4] to [A19], wherein step (1) is carried out in the presence of a base. [A21] The method according to [A20], wherein the pKa of the conjugate acid of the base used in step (1) is 15.0 or less, more preferably 11.5 or less. [A22] The method according to [A20] or [A21], wherein the base used in step (1) is an organic base, preferably at least one selected from the group consisting of amines and pyridines. [A23] The method according to any one of [A20] to [A22], wherein the base used in step (1) is at least one selected from the group consisting of diisopropylethylamine, triethylamine, 2,6-lutidine, and 2,4,6-collidine. [A24] The method according to any one of [A4] to [A23], wherein the base used in step (2) is at least one selected from the group consisting of sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium hydrogencarbonate, sodium hydrogenphosphate, triethylamine, and pyridine. [A24-0] The method according to any one of [A1] to [A24], wherein the linking step is carried out in the presence of an additive. [A24-1] The method according to any one of [A4] to [A24], wherein step (1) is carried out in the presence of an additive. [A24-2] The method according to [A24-0] or [A24-1], wherein the additive is at least one selected from the group consisting of bromide salts, iodide salts, and trifluoromethanesulfonate salts.[A24-3] The method according to [A24-0] or [A24-1], wherein the additive is at least one selected from the group consisting of sodium bromide, sodium iodide, sodium trifluoromethanesulfonate, potassium bromide, potassium iodide, potassium trifluoromethanesulfonate, magnesium bromide, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, calcium iodide, calcium trifluoromethanesulfonate, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, barium bromide, barium iodide, and barium trifluoromethanesulfonate. [A24-4] The method according to [A24-0] or [A24-1], wherein the additive is at least one selected from the group consisting of sodium bromide, sodium iodide, sodium trifluoromethanesulfonate, and potassium trifluoromethanesulfonate. [A24-5] The method according to any one of [A24-0] to [A24-4], wherein the additive is used in an amount ranging from 0.2 to 3.0 molar equivalents relative to the second amino acid or peptide. [A24-6] The method according to any one of [A24-0] to [A24-4], wherein the additive is used in an amount ranging from 0.3 to 1.5 molar equivalents relative to the second amino acid or peptide. [A25] The method according to any one of [A4] to [A24], wherein the organic solvent used in step (1) is one or more water-immiscible organic solvents. [A26] The method according to any one of [A1] to [A25], wherein the water-immiscible organic solvent is a solvent having an octanol / water partition coefficient (Log Kow) of 5 or more, or a solvent having a predicted octanol / water partition coefficient (Log Kow) of 5 or more. [A27] The method according to any one of [A1] to [A26], wherein the water-immiscible organic solvent is at least one selected from the group consisting of isopropyl acetate, ethyl acetate, butyl acetate, methyl t-butyl ether, diethyl ether, dichloromethane, carbon tetrachloride, 2-methyltetrahydrofuran, toluene, and hexane. [A28] The method according to any one of [A1] to [A27], wherein the water-immiscible organic solvent is at least one selected from the group consisting of isopropyl acetate, methyl t-butyl ether, dichloromethane, 2-methyltetrahydrofuran, and toluene.[A29] The amino group of the first amino acid or peptide has the formula: -NR. ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom, linear C １ -C ６ Alkyl, branched C ３ -C ６ Alkyl, or C ３ -C ８ [A30] The method according to any one of [A1] to [A28], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom or a linear C １ -C ４ [A31] The method according to any one of [A1] to [A29], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ [A32] The method according to any one of [A1] to [A30], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ [A33] The method according to any one of [A1] to [A31], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５[A35] The method according to any one of [A1] to [A34], wherein the peptide compound or a salt thereof produced contains 11 to 14 amino acid residues. [A36] The method according to any one of [A1] to [A35], wherein the peptide compound or a salt thereof produced contains 11 to 14 amino acid residues. [A37] The method according to any one of [A1] to [A36], wherein the peptide compound produced contains at least one unnatural amino acid residue. [A38] The method according to any one of [A1] to [A37], wherein the peptide compound produced contains at least four unnatural amino acid residues. [A39] The method according to any one of [A36] to [A38], wherein the peptide compound produced contains at least five unnatural amino acid residues. [A40] The method according to any one of [A1] to [A39], wherein the peptide compound or a salt thereof produced comprises a cyclic portion consisting of 4 to 14 amino acid residues, and the cyclic portion comprises 1 to 7 amide bonds linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide. [A41] The method according to any one of [A1] to [A40], wherein the cyclic portion comprises 1 to 7 amide bonds linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide. [A42] The method according to any one of [A1] to [A41], wherein the peptide compound or a salt thereof is produced by any one of the methods [A1] to [A41]. [A43] A pharmaceutical composition comprising the peptide compound or a salt thereof produced by any one of the methods [A1] to [A41]. [B1] A method for producing a peptide compound or a salt thereof, comprising a step of linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide via an amide bond (linking step) in a two-phase solvent system comprising one or more water-immiscible organic solvents and water, wherein the amino group of the first amino acid or peptide is a group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom, linear C １ -C ６ Alkyl, branched C ３ -C ６Alkyl, or C ３ -C ８ [B2] The method of [B1], wherein the linking step comprises: (1) a step of contacting a second amino acid or peptide with an acyl halide in an organic solvent to prepare a mixed acid anhydride, and (2) a step of contacting the first amino acid or peptide with the mixed acid anhydride obtained in step (1) in the presence of a base in a two-phase solvent containing one or more water-immiscible organic solvents and water. [B3] The method of [B2], wherein the step of contacting the first amino acid or peptide with the mixed acid anhydride obtained in step (1) in (2) is a step of adding the first amino acid or peptide to the mixed acid anhydride obtained in step (1). [B4] The method of [B2], wherein the amino group of the first amino acid or peptide is represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom or a linear C １ -C ４ [B5] The method according to any one of [B1] to [B3], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ [B6] The method according to any one of [B1] to [B4], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ [B7] The method according to any one of [B1] to [B5], wherein the amino group of the first amino acid or peptide is an amino group represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５[B8] The method according to any one of [B1] to [B7], wherein the second amino acid or peptide is a peptide containing two or more amino acid residues. [B9] The method according to any one of [B1] to [B8], wherein the carboxy group of the first amino acid or peptide is protected. [B10] The method according to any one of [B1] to [B9], wherein the amino group of the second amino acid or peptide is protected. [B11] The method according to any one of [B1] to [B10], wherein the α-carbon of the carboxy group of the second amino acid or peptide is optionally substituted. [B12] The method according to any one of [B1] to [B10], wherein the α-carbon of the carboxy group of the second amino acid or peptide is optionally substituted. [B13] The method according to any one of [B1] to [B14], wherein the α-carbon of the carboxy group of the second amino acid or peptide is a group represented by the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ６ Alkyl, optionally substituted branched C ３ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl C １ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl, optionally substituted phenyl C １ -C ２ Alkyl, optionally substituted C １ -C ６ Alkoxy C １ -C ２ Alkyl, optionally substituted 5- to 6-membered heteroaryl C １ -C ２ Alkyl or optionally substituted phenyl C １ -C ２ Alkylthio C １ -C ２ alkyl or R １ and R ２ together with the carbon atoms to which they are attached, form C ３ -C ８[B13] The method according to any one of [B1] to [B10], wherein the α-carbon of the carboxy group of the second amino acid or peptide is represented by the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ４ Alkyl, branched C ３ -C ６ Alkyl, C ３ -C ６ Cycloalkyl C １ -C ６ Alkyl, C １ -C ６ Alkoxymethyl, 1-(C １ -C ６ alkoxy)ethyl, optionally substituted benzyl, optionally substituted phenethyl, or phenyl C １ -C ２ [B14] The method according to any one of [B1] to [B10], wherein R is alkylthiomethyl. １ [B15] The method according to [B12] or [B13], wherein the α-carbon of the carboxy group of the second amino acid or peptide is a group represented by the formula: -CR １ R ２ - (wherein, R 1 is a hydrogen atom, and R ２ is a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, t-butoxymethyl, 1-(t-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl, or benzylthiomethyl. [B16] The method according to any one of [B1] to [B10], wherein the nitrogen atom at the β-position of the carboxy group of the second amino acid or peptide is represented by the formula: -NR ３ - (wherein, R ３is a hydrogen atom). [B17] The method according to any one of [B1] to [B16], wherein the first amino acid or peptide is a peptide containing two or more amino acid residues. [B18] The method according to any one of [B1] and [B4] to [B17], wherein the linking step is carried out in the presence of an activating agent. [B19] The method according to [B18], wherein the activating agent is an acyl halide. [B20] The method according to [B21], wherein the acyl halide is a linear C １ -C １８ Alkylcarbonyl chloride, branched C ３ -C １８ Alkylcarbonyl chloride, linear C １ -C １８ Alkylcarbonyl bromide, branched C ３ -C １８ Alkylcarbonyl bromide and monocyclic or condensed ring C ３ -C １５ Cycloalkylcarbonyl chloride (C ３ -C １５ Cycloalkylcarbonyl chloride is C １ -C ６[B21] The method according to any one of [B2] to [B3] and [B19], wherein the acyl halide is at least one selected from the group consisting of pivaloyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexanecarbonyl chloride, 1-adamantanecarbonyl chloride, and 2-ethylbutyryl chloride. [B22] The method according to any one of [B2] to [B3] and [B19] to [B21], wherein the acyl halide is pivaloyl chloride or 2,2-dimethylbutyryl chloride. [B23] The method according to any one of [B2] to [B3] and [B19] to [B22], wherein the acyl halide is used in an amount of 0.6 to 1.0 molar equivalent relative to the second amino acid or peptide. [B24] The method according to any one of [B2] to [B17] and [B20] to [B23], wherein step (1) is carried out in the presence of a base. [B25] The method according to [B24], wherein the pKa of the conjugate acid of the base used in step (1) is 15.0 or less, more preferably 11.5 or less. [B26] The method according to [B24] or [B25], wherein the base used in step (1) is an organic base, preferably at least one selected from the group consisting of amines and pyridines. [B27] The method according to any one of [B23] to [B26], wherein the base used in step (1) is at least one selected from the group consisting of diisopropylethylamine, triethylamine, 2,6-lutidine, and 2,4,6-collidine. [B28] The method according to any one of [B2] to [B27], wherein the base used in step (2) is at least one selected from the group consisting of sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium hydrogen carbonate, sodium hydrogen phosphate, triethylamine, and pyridine. [B28-0] The method according to any one of [B1] to [B28], wherein the linking step is carried out in the presence of an additive. [B28-1] The method according to any one of [B2] to [B28], wherein step (1) is carried out in the presence of an additive.[B28-2] The method according to [B28-0] or [B28-1], wherein the additive is at least one selected from the group consisting of bromide salts, iodide salts, and trifluoromethanesulfonate salts. [B28-3] The method according to [B28-0] or [B28-1], wherein the additive is at least one selected from the group consisting of sodium bromide, sodium iodide, sodium trifluoromethanesulfonate, potassium bromide, potassium iodide, potassium trifluoromethanesulfonate, magnesium bromide, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, calcium iodide, calcium trifluoromethanesulfonate, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, barium bromide, barium iodide, and barium trifluoromethanesulfonate. [B28-4] The method according to [B28-0] or [B28-1], wherein the additive is at least one selected from the group consisting of sodium bromide, sodium iodide, sodium trifluoromethanesulfonate, and potassium trifluoromethanesulfonate. [B28-5] The method according to any one of [B28-0] to [B28-4], wherein the additive is used in an amount ranging from 0.2 to 3.0 molar equivalents relative to the second amino acid or peptide. [B28-6] The method according to any one of [B28-0] to [B28-4], wherein the additive is used in an amount ranging from 0.3 to 1.5 molar equivalents relative to the second amino acid or peptide. [B29] The method according to any one of [B2] to [B17] and [B20] to [B28], wherein the organic solvent used in step (1) is one or more water-immiscible organic solvents. [B30] The method according to any one of [B1] to [B29], wherein the water-immiscible organic solvent is a solvent having an octanol / water partition coefficient (Log Kow) of at least 5, or a solvent having a predicted octanol / water partition coefficient (Log Kow) of at least 5. [B31] The method according to any one of [B1] to [B30], wherein the water-immiscible organic solvent is at least one selected from the group consisting of isopropyl acetate, ethyl acetate, butyl acetate, methyl t-butyl ether, diethyl ether, dichloromethane, carbon tetrachloride, 2-methyltetrahydrofuran, toluene, and hexane.[B32] The method according to any one of [B1] to [B31], wherein the water-immiscible organic solvent is at least one selected from the group consisting of isopropyl acetate, methyl t-butyl ether, dichloromethane, 2-methyltetrahydrofuran, and toluene. [B33] The method according to any one of [B1] to [B32], wherein the peptide compound or a salt thereof produced contains 8 to 20 amino acid residues. [B34] The method according to any one of [B1] to [B33], wherein the peptide compound or a salt thereof produced contains 11 to 14 amino acid residues. [B35] The method according to any one of [B1] to [B34], wherein the peptide compound produced contains at least one unnatural amino acid residue. [B36] The method according to any one of [B1] to [B35], wherein the peptide compound produced contains at least four unnatural amino acid residues. [B37] The method according to any one of [B1] to [B36], wherein the peptide compound produced contains at least five unnatural amino acid residues. [B38] The method of any of [B34] to [B37], wherein the unnatural amino acid residue is an N-methyl amino acid residue. [B39] The method of any of [B1] to [B38], wherein the produced peptide compound or a salt thereof comprises a cyclic portion composed of 4 to 14 amino acid residues, and the cyclic portion comprises 1 to 7 amide bonds linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide. [B40] The method of any of [B1] to [B39], wherein the method is carried out using a flow reactor. [B41] A peptide compound or a salt thereof produced by any of the methods of [B1] to [B40]. [B42] A pharmaceutical composition comprising a peptide compound or a salt thereof produced by any of the methods of [B1] to [B40].

[0011] According to the present invention, peptide compounds can be synthesized with high diastereoselectivity and high yield even in fragment coupling. Furthermore, according to the present invention, a synthesis method suitable for fragment coupling of peptide compounds containing N-substituted amino acids and / or peptide compounds having bulky side chains near the reaction site of an amide bond can be provided.

[0012] The abbreviations used in this specification are as follows: 2-MeTHF: 2-methyltetrahydrofuran IPAC: isopropyl acetate EtOAc: ethyl acetate MeCN: acetonitrile THF: tetrahydrofuran MeOH: methanol MTBE: methyl tert-butyl ether CPME: cyclopentyl methyl ether DCM: dichloromethane DIPEA: N,N-diisopropylethylamine TEA: triethylamine NMM: N-methylmorpholine NMI: N-methylimidazole T3P: propylphosphonic anhydride HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate PivCl: pivaloyl chloride Me2BtrCl: 2,2-Dimethylbutyryl chloride EDC・HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HOPO: 2-hydroxypyridine-N-oxide H3PO4: Phosphoric acid NaCl: Sodium chloride Na2CO3: Sodium carbonate K2CO3: Potassium carbonate Cs2CO3: Cesium carbonate NaHCO3: Sodium bicarbonate K3PO4: Tripotassium phosphate Na2HPO4: Disodium hydrogen phosphate K2HPO4: Dipotassium hydrogen phosphate NaOH: Sodium hydroxide KOH: Potassium hydroxide LiOH: Lithium hydroxide NaHSO4: Sodium hydrogen sulfate Na2SO4: Sodium sulfate H2O: Water TFA: Trifluoroacetic acid HMDS: Hexamethyldisilazane TMSOTf: Trimethylsilyl trifluoromethanesulfonate Pd / C: Palladium on carbon Alloc: Allyloxycarbonyl Boc: t-butoxycarbonyl Cbz: Benzyloxycarbonyl Teoc: 2-(trimethylsilyl)ethoxycarbonyl Fmoc: 9-fluorenylmethyloxycarbonyl, TBAF: tetrabutylammonium fluoride, LiBH4: lithium borohydride, EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, BEP: 2-bromo-1-ethylpyridinium tetrafluoroborate, PyBOP: 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, PyOxim: (ethylcyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate, NaBr: sodium bromide, NaI: sodium iodide, NaOTf: sodium trifluoromethanesulfonate, KOTf: potassium trifluoromethanesulfonate.

[0013] Definitions of functional groups, etc. (The terms exemplified below are merely examples and are not intended to be particularly limiting and are commonly understood by those skilled in the art.)

[0014] As used herein, "halogen atom" is exemplified by fluorine, chlorine, bromine, and iodine. In addition, in the specification, F represents fluorine, Cl represents chlorine, Br represents bromine, and I represents iodine. Examples of halogen include fluorine, chlorine, and bromine, and preferred examples are fluorine and chlorine.

[0015] In this specification, "alkyl" refers to a linear or branched monovalent saturated hydrocarbon group derived from an aliphatic saturated hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a hydrocarbyl or hydrocarbon group structure subset containing hydrogen atoms and carbon atoms. Alkyl includes not only linear ones but also branched ones. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C １ -C ２０ , hereinafter referred to as “C ｐ -C ｑ " means that the number of carbon atoms is p to q), and preferably C １ -C １０ Alkyl, more preferably C １ -C ６ Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl. １ -C ６ Specific examples of alkyl include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. １-C ３ Specific examples of alkyl include methyl, ethyl, and n-propyl. ３ -C ６ Specific examples of alkyl include i-propyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, and the like.

[0016] As used herein, "alkenyl" refers to a straight-chain or branched-chain monovalent unsaturated hydrocarbon group having one or more carbon-carbon double bonds (bonds between two adjacent sp2 carbon atoms). Depending on the configuration of the atom or atomic group bonded to the sp2 carbon atom, the geometry of the double bond can be entgegen (E) or zusammen (Z), cis or trans. Examples of alkenyl include C ２ -C １０ alkenyl, C ２ -C ８ Alkenyl is preferred, C ２ -C ７ Alkenyl is more preferred, C ２ -C ６ Alkenyl is most preferred, and examples thereof include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl.

[0017] As used herein, the term "alkynyl" refers to a linear or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon triple bonds (bonds between two adjacent sp carbon atoms). Examples of alkynyl include C ２ -C １０alkynyl, C ２ -C ８ Alkynyl is preferred, C ２ -C ７ Alkynyl is more preferred, C ２ -C ６ Alkynyl is most preferred. Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl (2-propynyl), 1-butynyl, 2-butynyl, 3-butynyl, pentynyl, and hexynyl.

[0018] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent non-aromatic hydrocarbon ring group (alicyclic ring group). The carbon atoms constituting the ring may be oxidized to include a carbonyl. The cycloalkyl may be selected from the group consisting of a monocyclic ring, a fused ring, and a spiro ring. As used herein, a cycloalkyl containing a monocyclic ring is referred to as a monocyclic cycloalkyl, i.e., a monocyclic alicyclic ring group; a cycloalkyl containing a fused ring is referred to as a fused ring cycloalkyl, i.e., a fused ring alicyclic ring group; and a cycloalkyl containing a spiro ring is referred to as a spirocyclic cycloalkyl, i.e., a spirocyclic alicyclic ring group. A cycloalkyl may form a fused ring with a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, an unsaturated alicyclic ring such as a cyclopentene ring or a cyclohexene ring, or an aromatic hydrocarbon ring such as a benzene ring or a naphthalene ring. A cycloalkyl may form a spiro ring with a saturated alicyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, or a cyclohexane ring. Examples of cycloalkyl include C ３ -C １０ is cycloalkyl, and C ３ -C ８ Cycloalkyl is preferred, C ３ -C ７ Cycloalkyl is more preferred, C ３ -C ６ Cycloalkyl is most preferred. Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, cyclohexenyl, and the like.

[0019] In the present specification, the term "aryl" refers to a monovalent aromatic hydrocarbon ring group consisting of a single ring or fused rings that exhibits monovalent aromaticity. In the present specification, an aryl consisting of a single ring is referred to as a monocyclic aryl, and an aryl consisting of a fused ring is referred to as a fused ring aryl. Examples of aryl include C ６ -C １４ aryl, C ６ Aryl, C １０ Aryl and C １４ Aryl is preferred, C ６ Aryl and C １０ Aryl is more preferred, C ６ Aryl is most preferred. Specific examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, tolyl, and xylyl.

[0020] As used herein, the term "heterocyclyl" refers to a heterocyclic group containing, in addition to carbon atoms, preferably 1 to 5, and more preferably 1 to 3, heteroatoms selected from the group consisting of nitrogen atoms, oxygen atoms, and sulfur atoms as ring-constituting atoms, and which may have a double and / or triple bond within the ring. A carbon atom within the heterocyclyl ring may be oxidized to form a carbonyl. As used herein, a heterocyclyl containing a single ring is referred to as a monocyclic heterocyclyl, a heterocyclyl containing a fused ring is referred to as a fused-ring heterocyclyl, and a heterocyclyl containing a spiro ring is referred to as a spirocyclic heterocyclyl. A heterocyclyl may form a fused ring or a spiro ring with, for example, a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, or a saturated heterocycle such as a tetrahydropyran ring, a dioxane ring, or a pyrrolidine ring. The number of atoms constituting the heterocyclyl ring is, for example, 3 to 14 (3- to 14-membered heterocyclyl), preferably 3 to 12 (3- to 12-membered heterocyclyl), more preferably 3 to 10 (3- to 10-membered heterocyclyl), and most preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxiranyl, oxetanyl, thietanyl, tetrahydrofuranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, thiadiazolidinyl, oxazolidonyl, dioxolanyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, 4-oxopyrrolidinyl, piperidinyl, 4-oxopiperidinyl, piperazinyl, dioxanyl, and rings in which one or more single bonds in these saturated heterocycles are replaced with double bonds or triple bonds.

[0021] As used herein, the term "heteroaryl" refers to a monovalent aromatic heterocyclic group that contains at least one heteroatom in addition to carbon atoms and is composed of a monocyclic or fused ring that exhibits aromaticity. In this specification, a heteroaryl composed of a single ring is referred to as a monocyclic heteroaryl, and a heteroaryl composed of a fused ring is referred to as a fused-ring heteroaryl. The number of atoms constituting the heteroaryl ring is, for example, 5 to 14 (5- to 14-membered heteroaryl), preferably 5 to 13 (5- to 13-membered heteroaryl), more preferably 5 to 10 (5- to 10-membered heteroaryl), and most preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include 5-membered heteroaryls such as furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, and tetrazolyl; 6-membered heteroaryls such as pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, and triazinyl; 9-membered heteroaryls such as benzofuranyl, benzothienyl, benzothiazolyl, benzimidazolyl, benzotriazolyl, indolyl, indazolyl, and pyrazolopyridyl; and 10-membered heteroaryls such as quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, and quinoxalinyl.

[0022] As used herein, "alkoxy" refers to a group in which an "alkyl" as defined herein is bonded to an oxygen atom (-OR (R is alkyl)). Examples of alkoxy include C １ -C ２０ Alkoxy, C １ -C １０ Alkoxy is preferred, C １ -C ８ Alkoxy is more preferred, C １ -C ６ Alkoxy is most preferred, and specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0023] As used herein, "alkenyloxy" refers to a group in which an "alkenyl" as defined herein is bonded to an oxygen atom (-OR (R is alkenyl)). Examples of alkenyloxy include C２ -C １０ alkenyloxy, C ２ -C ８ Alkenyloxy is preferred, C ２ -C ７ Alkenyloxy is more preferred, C ２ -C ６ Alkenyloxy is most preferred. Specific examples of alkenyloxy include ethenyl(vinyl)oxy, 1-propenyloxy, 2-propenyl(allyl)oxy, isopropenyloxy, 1-butenyloxy, 2-cis-butenyloxy, 2-trans-butenyloxy, 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0024] As used herein, "cycloalkoxy" refers to a group in which a "cycloalkyl" as defined herein is bonded to an oxygen atom (-OR (R is cycloalkyl)). Examples of cycloalkoxy include C ３ -C １０ cycloalkoxy, C ３ -C ８ Cycloalkoxy is preferred, C ３ -C ７ Cycloalkoxy is more preferred, C ３ -C ６ Cycloalkoxy is most preferred. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, and the like.

[0025] As used herein, "aryloxy" refers to a group in which an "aryl" as defined herein is bonded to an oxygen atom (-OAr (Ar is aryl)). Examples of aryloxy include C ６ -C １４ aryloxy, C ６ Aryloxy, C １０ Aryloxy and C １４ Aryloxy is preferred, C ６ Aryloxy and C １０ Aryloxy is more preferred, C ６Aryloxy is most preferred. Specific examples of aryloxy include phenoxy, 1-naphthyloxy, 2-naphthyloxy, tolyloxy, and xylyloxy.

[0026] As used herein, "amino" refers to -NRR', where N represents a nitrogen atom, and R and R' are each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or R and R' are groups that form a ring together with the nitrogen atom to which they are attached. ２ , Mono C １ -C ６ Alkylamino, DiC １ -C ６ Examples thereof include alkylamino and 4- to 8-membered cyclic amino.

[0027] As used herein, "monoalkylamino" refers to a group in which R is a hydrogen atom and R' is an "alkyl" in the "amino (-NRR')" defined herein. Examples of monoalkylamino include monoC １ -C ２０ alkylamino, mono C １ -C １５ Alkylamino is preferred, mono C １ -C １０ Alkylamino is more preferred, and mono C １ -C ６ Alkylamino is most preferred. Specific examples of monoalkylamino include methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, and t-butylamino.

[0028] As used herein, "dialkylamino" refers to a group in which R and R' are each independently "alkyl" within the "amino (-NRR')" defined herein. Examples of dialkylamino include diC １ -C ２０ alkylamino, diC １ -C １５ Alkylamino is preferred, diC １ -C１０ Alkylamino is more preferred, diC １ -C ６ Alkylamino is most preferred. Specific examples of dialkylamino include dimethylamino, diethylamino, and methylethylamino.

[0029] As used herein, "cyclic amino" refers to the "amino (-NRR')" defined herein, in which R and R' form a ring together with the nitrogen atom to which they are bonded. Examples of cyclic amino include 3- to 14-membered cyclic amino, preferably 3- to 12-membered cyclic amino, more preferably 3- to 10-membered cyclic amino, and most preferably 4- to 7-membered cyclic amino. Specific examples of cyclic amino include 1-azetidyl, 1-pyrrolidyl, 1-piperidyl, 1-piperazyl, 4-morpholinyl, 3-oxazolidyl, 1,1-dioxidethiomorpholinyl-4-yl, 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, and the like.

[0030] As used herein, the term "protected amino" refers to an amino group protected with any protecting group. Specific examples of the protected amino include amino protected with a protecting group such as Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), Cbz (benzyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Alloc (allyloxycarbonyl), Teoc (2-(trimethylsilyl)ethoxycarbonyl), or trifluoroacetyl.

[0031] As used herein, "aminocarbonyl" refers to a group in which an "amino" as defined herein is bonded to the carbon atom of a carbonyl. It may also be called an amide. Examples of aminocarbonyl include -CONH ２ , Mono C １ -C ６ Alkylaminocarbonyl, diC １ -C ６ alkylaminocarbonyl, and 4- to 8-membered cyclic aminocarbonyl. Specific examples of aminocarbonyl include -CONH ２methylaminocarbonyl, ethylaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl, 1-azetidinylcarbonyl, 1-pyrrolidinylcarbonyl, 1-piperidinylcarbonyl, 1-piperazinylcarbonyl, 4-morpholinylcarbonyl, 3-oxazolidinylcarbonyl and the like.

[0032] As used herein, "alkenyloxycarbonyl" refers to a carbonyl group to which "alkenyloxy" as defined herein is bonded. Examples of alkenyloxycarbonyl include C ２ -C １０ alkenyloxycarbonyl, C ２ -C ８ Alkenyloxycarbonyl is preferred, C ２ -C ７ Alkenyloxycarbonyl is more preferred, C ２ -C ６ Alkenyloxycarbonyl is most preferred. Specific examples of alkenyloxycarbonyl include ethenyl(vinyl)oxycarbonyl, 1-propenyloxycarbonyl, 2-propenyl(allyl)oxycarbonyl, isopropenyloxycarbonyl, 1-butenyloxycarbonyl, 2-cis-butenyloxycarbonyl, 2-trans-butenyloxycarbonyl, 3-butenyloxycarbonyl, pentenyloxycarbonyl, and hexenyloxycarbonyl.

[0033] As used herein, "alkylsulfonyl" refers to a sulfonyl group to which an "alkyl" as defined herein is bonded. Examples of alkylsulfonyl include C １ -C ２０ alkylsulfonyl, C １ -C １０ Alkylsulfonyl is preferred, C １ -C ８ Alkylsulfonyl is more preferred, C １ -C ６Specific examples of alkylsulfonyl include methylsulfonyl, ethylsulfonyl, 1-propylsulfonyl, 2-propylsulfonyl, n-butylsulfonyl, i-butylsulfonyl, s-butylsulfonyl, t-butylsulfonyl, pentylsulfonyl, and 3-methylbutylsulfonyl.

[0034] As used herein, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with a hydroxy group. Preferred hydroxyalkyl groups are those in which one hydrogen atom of an alkyl is substituted with a hydroxy group. Examples of hydroxyalkyl include hydroxy C １ -C ２０ alkyl, and hydroxy C １ -C １０ Alkyl is preferred, hydroxy C １ -C ８ Alkyl is more preferred, and hydroxy C １ -C ６ Specific examples of hydroxyalkyl include hydroxyethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0035] As used herein, "haloalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with halogen. As the haloalkyl, a group in which 1 to 6 hydrogen atoms, which are allowed to be substituted, are substituted with halogen is preferred. Examples of haloalkyl include haloC １ -C ２０ alkyl and haloC １ -C １０ Alkyl is preferred, haloC １ -C ８ Alkyl is more preferred, haloC １ -C ６ Alkyl is most preferred. HaloC １ -C ６The alkyl is, for example, a group in which 1 to 6 hydrogen atoms, which are permissible for substitution, are substituted with fluorine, preferably a group in which 1 to 5 hydrogen atoms are substituted with fluorine, more preferably a group in which 1 to 4 hydrogen atoms are substituted with fluorine, and most preferably a group in which 1 to 3 hydrogen atoms are substituted with fluorine. Specific examples of haloalkyl include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, etc.

[0036] As used herein, "cyanoalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with cyano. Preferred cyanoalkyl groups include alkyl groups in which one hydrogen atom is substituted with cyano. Examples of cyanoalkyl include cyanoC １ -C ２０ alkyl and cyano C １ -C １０ Alkyl is preferred, and cyano C １ -C ８ Alkyl is more preferred, and cyano C １ -C ６ Alkyl is most preferred. Specific examples of cyanoalkyl include cyanomethyl and 2-cyanoethyl.

[0037] As used herein, "aminoalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "amino". As the aminoalkyl, a group in which one hydrogen atom of an alkyl is substituted with amino is preferred. Examples of aminoalkyl include amino C １ -C ２０ alkyl and amino C １ -C １０ Alkyl is preferred, and amino C １ -C ８ Alkyl is more preferred, and amino C １ -C ６Alkyl is most preferred. Specific examples of aminoalkyl include aminomethyl, aminoethyl, 4-aminobutyl, methylaminomethyl, dimethylaminomethyl, methylaminoethyl, and dimethylaminoethyl.

[0038] As used herein, "carboxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "carboxy". Preferred carboxyalkyl groups are those in which one hydrogen atom of an alkyl is substituted with carboxy. Examples of carboxyalkyl include carboxyC １ -C ２０ alkyl and carboxy C １ -C １５ Alkyl is preferred, and carboxy C １ -C １０ Alkyl is more preferred, and carboxy C １ -C ６ Alkyl is most preferred. Specific examples of carboxyalkyl include carboxymethyl and carboxyethyl.

[0039] As used herein, "alkenyloxycarbonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "alkenyloxycarbonyl". As the alkenyloxycarbonylalkyl, a group in which one hydrogen atom of an alkyl is substituted with alkenyloxycarbonyl is preferred. Examples of alkenyloxycarbonylalkyl include C ２ -C １０ Alkenyloxycarbonyl C １ -C ６ alkyl, C ２ -C ８ Alkenyloxycarbonyl C １ -C ６ Alkyl is preferred, C ２ -C ７ Alkenyloxycarbonyl C １ -C ６ Alkyl is more preferred, C ２ -C ６ Alkenyloxycarbonyl C １ -C ６Specific examples of alkenyloxycarbonylalkyl include ethenyl(vinyl)oxycarbonylmethyl, 1-propenyloxycarbonylethyl, 2-propenyl(allyl)oxycarbonylmethyl, isopropenyloxycarbonylmethyl, 1-butenyloxycarbonylethyl, 2-cis-butenyloxycarbonylmethyl (including cis and trans), 2-trans-butenyloxycarbonylmethyl, 3-butenyloxycarbonylethyl, pentenyloxycarbonylmethyl, and hexenyloxycarbonylethyl.

[0040] As used herein, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "alkoxy". Preferred alkoxyalkyl groups are those in which one hydrogen atom of an alkyl is substituted with alkoxy. Examples of alkoxyalkyl include C １ -C ６ Alkoxy C １ -C ２０ alkyl, C １ -C ６ Alkoxy C １ -C １５ Alkyl is preferred, C １ -C ６ Alkoxy C １ -C １０ Alkyl is more preferred, C １ -C ６ Alkoxy C １ -C ６ Specific examples of alkoxyalkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0041] As used herein, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with a "cycloalkyl". As the cycloalkylalkyl, a group in which one hydrogen atom of an alkyl is substituted with a cycloalkyl is preferred. Examples of cycloalkylalkyl include C ３ -C １０ Cycloalkyl C １ -C ２０ alkyl, C ３ -C １０ Cycloalkyl C １ -C ６ Alkyl is preferred, C ３ -C ８ Cycloalkyl C １ -C ６ Alkyl is more preferred, C ３ -C ６ Cycloalkyl C １ -C ２ Alkyl is most preferred. Specific examples of cycloalkylalkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl.

[0042] As used herein, "cycloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "cycloalkoxy". As the cycloalkoxyalkyl, a group in which one hydrogen atom of an alkyl is substituted with cycloalkoxy is preferred. Examples of cycloalkoxyalkyl include C ３ -C １０ Cycloalkoxy C １ -C ６ alkyl, C ３ -C ８ Cycloalkoxy C １ -C ６ Alkyl is preferred, C ３ -C ７ Cycloalkoxy C １ -C ６ Alkyl is more preferred, C ３ -C ６ Cycloalkoxy C １ -C ６Alkyl is most preferred. Specific examples of cycloalkoxyalkyl include cyclopropoxymethyl, cyclobutoxymethyl, cyclopentyloxymethyl, and the like.

[0043] As used herein, "heterocyclylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with a "heterocyclyl". Preferred heterocyclylalkyl groups are those in which one hydrogen atom of an alkyl is substituted with a heterocyclyl. Examples of heterocyclylalkyl include 3- to 14-membered heterocyclyl C １ -C ６ alkyl and 3- to 12-membered heterocyclyl C １ -C ６ Alkyl is preferred, and 3- to 10-membered heterocyclyl C １ -C ４ Alkyl is more preferred, and 4- to 7-membered heterocyclyl C １ -C ３ Specific examples of heterocyclylalkyl include azetidin-1-ylmethyl, oxetan-3-ylmethyl, 2-(tetrahydrofuran-3-yl)ethyl, (1-methylpyrrolidin-3-yl)methyl, 2-morpholinoethyl, 3-(1-piperidinyl)propyl, and 3-(4-methylpiperazin-1-yl)propyl.

[0044] As used herein, "alkylsulfonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with "alkylsulfonyl". As the alkylsulfonylalkyl, a group in which one hydrogen atom of an alkyl is substituted with alkylsulfonyl is preferred. For example, C １ -C ２０ Alkylsulfonyl C １ -C ４ alkyl, C １ -C １０ Alkylsulfonyl C １ -C ４ Alkyl is preferred, C １ -C ８ Alkylsulfonyl C １ -C ４ Alkyl is more preferred, C １ -C６ Alkylsulfonyl C １ -C ４ Specific examples of alkylsulfonylalkyl include methylsulfonylmethyl, methylsulfonylpropyl, methylsulfonylbutyl, ethylsulfonylethyl, 1-propylsulfonylmethyl, 2-propylsulfonylethyl, n-butylsulfonylpropyl, i-butylsulfonylmethyl, s-butylsulfonylmethyl, t-butylsulfonylmethyl, pentylsulfonylmethyl, and 3-methylbutylsulfonylmethyl.

[0045] As used herein, "aminocarbonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with an "aminocarbonyl" as defined herein. As the aminocarbonylalkyl, a group in which one hydrogen atom of an alkyl is substituted with an aminocarbonyl is preferred. As the aminocarbonylalkyl, for example, aminocarbonyl C １ -C １０ alkyl, and aminocarbonyl C １ -C ６ Alkyl is preferred, and aminocarbonyl C １ -C ４ Alkyl is more preferred, and aminocarbonyl C １ -C ２ Alkyl is most preferred. Specific examples of aminocarbonylalkyl include -CH ２ CONH ２ , methylaminocarbonylmethyl, ethylaminocarbonylethyl, dimethylaminocarbonylpropyl, diethylaminocarbonylmethyl, 1-azetidinylcarbonylmethyl, 1-pyrrolidinylcarbonylethyl, 1-piperidinylcarbonylpropyl, 1-piperazinylcarbonylbutyl, 4-morpholinylcarbonylmethyl, 3-oxazolidinylcarbonylethyl and the like.

[0046] As used herein, "aryloxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with an "aryloxy" as defined herein. ６ -C１０ Aryloxy C １ -C ６ Alkyl is preferred, C ６ -C １０ Aryloxy C １ -C ２ Alkyl is more preferred. Specific examples of aryloxyalkyl include phenoxymethyl and 2-phenoxyethyl.

[0047] As used herein, "aralkyl (arylalkyl)" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with an "aryl" as defined herein. Examples of aralkyl include C ７ -C ２０ aralkyl, C ７ -C １８ Aralkyl is preferred, C ７ -C １６ Aralkyl is more preferred, C ７ -C １４ Aralkyl is most preferred. ７ -C ２０ Examples of aralkyl include C ６ -C １０ Aryl C １ -C １０ alkyl, C ６ -C １０ Aryl C １ -C ８ Alkyl is preferred, C ６ Aryl C １ -C ８ Alkyl or C １０ Aryl C １ -C ８ Alkyl is more preferred, C ６ Aryl C １ -C ８ Alkyl is most preferred. ７ -C １８ Examples of aralkyl include C ６ -C １０ Aryl C １ -C ８ alkyl, C ６ -C １０ Aryl C １ -C ６Alkyl is preferred, C ６ Aryl C １ -C ６ Alkyl or C １０ Aryl C １ -C ６ Alkyl is more preferred, C ６ Aryl C １ -C ６ Alkyl is most preferred. ７ -C １６ As the aralkyl, for example, C ６ -C １０ Aryl C １ -C ６ alkyl, C ６ -C １０ Aryl C １ -C ４ Alkyl is preferred, C ６ Aryl C １ -C ４ Alkyl or C １０ Aryl C １ -C ４ Alkyl is more preferred, C ６ Aryl C １ -C ４ Alkyl is most preferred. ７ -C １４ As the aralkyl, for example, C ６ -C １０ Aryl C １ -C ４ alkyl, C ６ -C １０ Aryl C １ -C ３ Alkyl is preferred, C ６ Aryl C １ -C ３ Alkyl or C １０ Aryl C １ -C ３ Alkyl is more preferred, C ６ Aryl C １ -C ３ Alkyl is most preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0048] As used herein, "aralkoxy" refers to a group in which the alkyl moiety of an "aralkyl" defined herein is bonded to an oxygen atom (-O-R-Ar (R is alkylene)). Examples of aralkoxy include C ７ -C ２０ Aralkoxy, C ７ -C １８ Aralkoxy is preferred, C ７ -C １６ Aralkoxy is more preferred, C ７ -C １４ Aralkoxy is most preferred. ７ -C ２０ Examples of aralkoxy include C ６ -C １０ Aryl C １ -C １０ Alkoxy, C ６ -C １０ Aryl C １ -C ８ Alkoxy is preferred, C ６ Aryl C １ -C ８ Alkoxy or C １０ Aryl C １ -C ８ Alkoxy is more preferred, C ６ Aryl C １ -C ８ Alkoxy is most preferred. ７ -C １８ Examples of aralkoxy include C ６ -C １０ Aryl C １ -C ８ Alkoxy, C ６ -C １０ Aryl C １ -C ６ Alkoxy is preferred, C ６ Aryl C １ -C ６ Alkoxy or C １０ Aryl C １ -C ６ Alkoxy is more preferred, C ６ Aryl C １ -C ６ Alkoxy is most preferred. ７ -C１６ As the aralkoxy, for example, C ６ -C １０ Aryl C １ -C ６ Alkoxy, C ６ -C １０ Aryl C １ -C ４ Alkoxy is preferred, C ６ Aryl C １ -C ４ Alkoxy or C １０ Aryl C １ -C ４ Alkoxy is more preferred, C ６ Aryl C １ -C ４ Alkoxy is most preferred. ７ -C １４ As the aralkoxy, for example, C ６ -C １０ Aryl C １ -C ４ Alkoxy, C ６ -C １０ Aryl C １ -C ３ Alkoxy is preferred, C ６ Aryl C １ -C ３ Alkoxy or C １０ Aryl C １ -C ３ Alkoxy is more preferred, C ６ Aryl C １ -C ３ Alkoxy is most preferred. Specific examples of aralkoxy include benzyloxy, phenethyloxy, and 3-phenylpropoxy.

[0049] As used herein, "aralkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with an "aralkoxy" as defined herein. Aralkoxyalkyl includes C ７ -C １４ Aralkoxy C １ -C ６ Alkyl is preferred, C ７ -C １４ Aralkoxy C １ -C２ Alkyl is more preferred. Specific examples of aralkoxyalkyl include benzyloxymethyl and 1-(benzyloxy)ethyl.

[0050] As used herein, "heteroarylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with a "heteroaryl". As the heteroarylalkyl, a group in which one hydrogen atom of an alkyl is substituted with a heteroaryl is preferred. Examples of heteroarylalkyl include 5- to 10-membered heteroaryl C １ -C ６ alkyl and 5-10 membered heteroaryl C １ -C ４ Alkyl is preferred, and 5- to 10-membered heteroaryl C １ -C ３ Alkyl is more preferred, and 5- to 10-membered heteroaryl C １ -C ２ Specific examples of heteroarylalkyl include 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-furanylmethyl, 2-thienylmethyl, 3-thienylmethyl, and 4-thiazolylmethyl.

[0051] As used herein, "heteroarylalkoxy" refers to a group in which the alkyl moiety of "heteroarylalkyl" as defined herein is bonded to an oxygen atom. Examples of heteroarylalkoxy include 5- to 10-membered heteroaryl C １ -C ６ alkoxy and 5-10 membered heteroaryl C １ -C ４ Alkoxy is preferred, and 5- to 10-membered heteroaryl C １ -C ３ Alkoxy is more preferred, and 5- to 10-membered heteroaryl C １ -C ２ Alkoxy is most preferred. Specific examples of heteroarylalkoxy include 2-pyridylmethoxy, 3-pyridylmethoxy, 4-pyridylmethoxy, 2-furanylmethoxy, 2-thienylmethoxy, 3-thienylmethoxy, and 4-thiazolylmethoxy.

[0052] As used herein, "heteroarylalkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are substituted with a "heteroarylalkoxy" as defined herein. As the heteroarylalkoxyalkyl, a group in which one hydrogen atom of an alkyl is substituted with a heteroarylalkoxy is preferred. As the heteroarylalkoxyalkyl, for example, a 5- to 10-membered heteroaryl C １ -C ６ Alkoxy C １ -C ６ alkyl and 5-10 membered heteroaryl C １ -C ４ Alkoxy C １ -C ６ Alkyl is preferred, and 5- to 10-membered heteroaryl C １ -C ３ Alkoxy C １ -C ６ Alkyl is more preferred, and 5- to 10-membered heteroaryl C １ -C ２ Alkoxy C １ -C ６ Specific examples of heteroarylalkoxyalkyl include 2-pyridylmethoxymethyl, 3-pyridylmethoxymethyl, 4-pyridylmethoxymethyl, 2-furanylmethoxymethyl, 2-thienylmethoxymethyl, 3-thienylmethoxymethyl, and 4-thiazolylmethoxymethyl.

[0053] As used herein, "heterocycloalkylidenealkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined herein are replaced with a "heterocycloalkylidene" as defined herein. Heterocycloalkylidenealkyl includes 4- to 7-membered heterocycloalkylidene C １ -C ６ Alkyl is preferred, and 4- to 7-membered heterocycloalkylidene C １ -C ２ Specific examples of heterocycloalkylidenealkyl include tetrahydro-4H-pyran-4-ylidenemethyl, azetidin-3-ylidenemethyl, and the like.

[0054] As used herein, "alkoxyalkenyl" refers to a group in which one or more hydrogen atoms of an "alkenyl" as defined herein are substituted with "alkoxy". As the alkoxyalkenyl, a group in which one hydrogen atom of an alkenyl is substituted with alkoxy is preferred. Examples of the alkoxyalkenyl include C １ -C ６ Alkoxy C ２ -C １０ alkenyl, C １ -C ６ Alkoxy C ２ -C ８ Alkenyl is preferred, C １ -C ６ Alkoxy C ２ -C ７ Alkenyl is more preferred, C １ -C ６ Alkoxy C ２ -C ６ Alkenyl is most preferred. Specific examples of alkoxyalkenyl include (E)-4-methoxybut-2-en-1-yl.

[0055] As used herein, "aminocarbonylalkenyl" refers to a group in which one or more hydrogen atoms of an "alkenyl" as defined herein are substituted with "aminocarbonyl". As the aminocarbonylalkenyl, a group in which one hydrogen atom of an alkenyl is substituted with aminocarbonyl is preferred. As the aminocarbonylalkenyl, for example, aminocarbonyl C ２ -C １０ alkenyl, and aminocarbonyl C ２ -C ８ Alkenyl is preferred, and aminocarbonyl C ２ -C ６ Alkenyl is more preferred, and aminocarbonyl C ２ -C ４ Alkenyl is most preferred. Specific examples of aminocarbonylalkenyl include (E)-3-(dimethylaminocarbonyl)-prop-2-en-1-yl.

[0056] The term "haloalkoxy" as used herein refers to a group in which one or more hydrogen atoms of an "alkoxy" as defined herein are substituted with halogen. The haloalkoxy is preferably a group in which 1 to 6 hydrogen atoms of an alkoxy that are allowed to be substituted are substituted with halogen. For example, haloC １ -C ２０ Alkoxy and haloC １ -C １０ Alkoxy is preferred, haloC １ -C ８ Alkoxy is more preferred, haloC １ -C ６ Alkoxy is most preferred. HaloC １ -C ６ The alkoxy is, for example, a group in which 1 to 6 hydrogen atoms of an alkoxy, which are permissible for substitution, are substituted with fluorine, preferably a group in which 1 to 5 hydrogen atoms are substituted with fluorine, more preferably a group in which 1 to 4 hydrogen atoms are substituted with fluorine, and most preferably a group in which 1 to 3 hydrogen atoms are substituted with fluorine. Specific examples of haloalkoxy include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 3-fluoropropoxy, 2,2-difluoropropoxy, etc.

[0057] As used herein, "alkylene" refers to a divalent group derived from an "alkyl" as defined herein by further removing one optional hydrogen atom. １ -C ８ Alkylene is preferred, C ４ -C ８ Alkylene is more preferred. Specific examples of alkylene include —CH ２ -, -(CH ２ ) ２ -, -(CH ２ ) ３ -, -CH(CH ３ ) CH ２ -, -C(CH ３ ) ２ -, -(CH ２ ) ４ -, -CH(CH ３ ) CH ２CH ２ -, -C(CH ３ ) ２ CH ２ -, -CH ２ CH (CH ３ ) CH ２ -, -CH ２ C(CH ３ ) ２ -, -CH ２ CH ２ CH (CH ３ ) -, -CH ２ CH (CH ２ CH ３ ) -, -(CH ２ ) ５ -, -CH(CH ３ ) CH(CH ２ CH ３ ) -, -(CH ２ ) ６ -, -(CH ２ ) ７ -, -(CH ２ ) ８ - and others.

[0058] As used herein, an "alicyclic ring" refers to a saturated or partially saturated non-aromatic hydrocarbon ring. The carbon atoms constituting the ring may be oxidized to include a carbonyl. The alicyclic ring may be selected from the group consisting of a monocyclic ring, a fused ring, and a spiro ring. As used herein, an alicyclic ring containing a monocyclic ring is referred to as a monocyclic alicyclic ring, an alicyclic ring containing a fused ring is referred to as a fused alicyclic ring, and an alicyclic ring containing a spiro ring is referred to as a spirocyclic alicyclic ring. The alicyclic ring may form a fused ring with a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, an unsaturated alicyclic ring such as a cycloheptene ring or a cyclohexene ring, or an aromatic hydrocarbon ring such as a benzene ring or a naphthalene ring. The alicyclic ring may form a spiro ring with a saturated alicyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, or a cyclohexane ring. Examples of alicyclic rings include C ３ -C １０ is an alicyclic ring, ３ -C ８ An alicyclic ring is preferred, and C ３ -C ７ An alicyclic ring is more preferred, and C ３ -C６ Alicyclic rings are most preferred. Specific examples of alicyclic rings include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a bicyclo[2.2.1]heptane ring, and a cyclohexene ring.

[0059] The term "saturated heterocycle" as used herein refers to a "heterocycle" that does not have an unsaturated bond within the ring. The number of atoms constituting the ring of the saturated heterocycle is 3 to 14 (3- to 14-membered heterocycle), preferably 3 to 12 (3- to 12-membered heterocycle), more preferably 3 to 10 (3- to 10-membered heterocycle), and most preferably 4 to 7 (4- to 7-membered heterocycle). Specific examples of saturated heterocycles include an azetidine ring, an epoxy ring, an oxetane ring, a thietane ring, a tetrahydrofuran ring, a pyrrolidine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidone ring, a dioxolane ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, and a dioxane ring.

[0060] As used herein, "peptide compound" refers to a compound in which two or more amino acids are linked by amide bonds. As long as two or more amino acids are linked by amide bonds, the peptide chain may contain other bonds (e.g., ester bonds, thioester bonds, etc.). The number of amino acid residues contained in the peptide is, for example, 5 to 30 residues, preferably 7 to 20 residues, more preferably 8 to 18 residues, and most preferably 9 to 15 residues. The peptide may be linear, branched, or cyclic.

[0061] As used herein, the term "peptide chain" refers to a chain-like portion of a peptide in which two or more amino acids are linked by amide bonds. As long as two or more amino acids are linked by amide bonds, the peptide chain may contain other bonds (e.g., ester bonds, thioester bonds, etc.). The number of amino acid residues contained in the peptide chain is, for example, 5 to 30 residues, preferably 7 to 20 residues, more preferably 8 to 18 residues, and most preferably 9 to 15 residues.

[0062] As used herein, the term "optionally substituted" means that a certain group and / or a certain atom may be substituted with any substituent and / or any atom. That is, either a state in which the certain group and the certain atom are not substituted with any substituent or any atom, or a state in which the certain group and / or the certain atom are substituted with any substituent and / or any atom, can be selected. Each of the certain group and / or the certain atom may be further substituted with any substituent and / or any atom. That is, either a state in which the certain group and the certain atom are not substituted with any substituent or any atom, or a state in which the certain group and / or the certain atom are substituted with any substituent and / or any atom, can be selected. The certain group and the certain substituent are not limited and may be freely selected, for example, from groups derived from atoms selected from the group consisting of hydrogen atoms, halogen atoms, carbon atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms, and phosphorus atoms. The certain atom and the optional atom are not limited and may be freely selected from, for example, a halogen atom, a carbon atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom. Examples of the optional substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, etc.

[0063] As used herein, the term "optionally protected" means that a group may be protected by any protecting group.

[0064] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0065] The compounds or salts thereof described herein may be solvates thereof. Examples of salts of the compounds include hydrochlorides, hydrobromides, hydroiodides, phosphates, phosphonates, sulfates, sulfonates such as methanesulfonates and p-toluenesulfonates, carboxylates such as acetates, citrates, malates, tartrates, succinates, and salicylates, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and ammonium salts such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts. These salts can be produced, for example, by contacting the compound with an acid or a base. In this specification, the term "solvate" refers to a compound that forms a molecular group together with a solvent, and is not particularly limited as long as it is a solvate formed with a solvent that is acceptable for ingestion accompanying the administration of a drug. Specific examples of solvates include solvates with a single solvent such as water, alcohol (ethanol, methanol, 1-propanol, 2-propanol, etc.), dimethyl sulfoxide, etc., as well as solvates formed with multiple solvents per molecule of the compound, or solvates formed with multiple types of solvents per molecule of the compound. For example, a solvate formed with water of a compound is called a hydrate. These solvates can be produced, for example, by contacting the compound with a solvent. As the solvate of the compound of the present invention, hydrates are preferred. Specific examples of such hydrates include mono- to xodecahydrates, more preferably mono- to decahydrates, even more preferably mono- to pentahydrates, and most preferably mono- to trihydrates. Solvates of these salts can be produced, for example, by contacting the compound with an acid or base and a solvent.

[0066] As used herein, "amino acid" includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives). Furthermore, as used herein, "amino acid" may refer to an amino acid residue. As used herein, "natural amino acid" refers to any L-amino acid selected from the group consisting of Gly (glycine), L-Ala (alanine), L-Ser (serine), L-Thr (threonine), L-Val (valine), L-Leu (leucine), L-Ile (isoleucine), L-Phe (phenylalanine), L-Tyr (tyrosine), L-Trp (tryptophan), L-His (histidine), L-Glu (glutamic acid), L-Asp (aspartic acid), L-Gln (glutamine), L-Asn (asparagine), L-Cys (cysteine), L-Met (methionine), L-Lys (lysine), L-Arg (arginine), and L-Pro (proline). As used herein, "unnatural amino acid" refers to an amino acid other than natural amino acids. Examples of unnatural amino acids include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids other than Pro, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids. The amino acids referred to herein may have any configuration. The side chains of the amino acids are not particularly limited and may be freely selected from groups such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and spiro-linked cycloalkyl, and / or atoms such as hydrogen atoms. Each of these groups and / or atoms may be further substituted. In a non-limiting embodiment, the amino acids referred to herein may be compounds having a carboxy group and an amino group in the same molecule. Even in this case, amino acids also include proline, hydroxyproline, azetidine-2-carboxylic acid, and the like, in which the nitrogen atom of the amino group and any atom in the side chain of the amino acid form a ring.

[0067] The main chain amino group of the amino acid is unsubstituted (NH ２group), and may be substituted (i.e., an —NHR group: R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group which may have a substituent, and one or two non-adjacent methylene groups in these groups are replaced by an oxygen atom, a carbonyl group (—CO—), or a sulfonyl group (—SO ２ -), or, as in proline, the carbon chain bonded to the N atom may form a ring with the carbon atom at the α-position.) The substituent of R is selected in the same manner as the substituent in the amino acid side chain described above. When the main chain amino group is substituted, R is included in the "amino acid side chain" in this specification. An amino acid in which such a main chain amino group is substituted is referred to as an "N-substituted amino acid" in this specification. As used herein, the "N-substituted amino acid" is preferably an N-alkyl amino acid, an N-C １ ~C ６ Alkyl amino acids, N-C １ ~C ４ Examples include, but are not limited to, alkyl amino acids and N-methyl amino acids.

[0068] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom is replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons) in an abundance ratio different from the natural abundance ratio. Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Cl, etc. Compounds herein containing all proportions of radioactive or non-radioactive isotopes are within the scope of the present invention.

[0069] As used herein, the term "substituent derived from halogen" includes fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), and the like.

[0070] As used herein, examples of an "oxygen atom-derived substituent" include hydroxy (-OH), oxy (-OR), oxo (=O), carbonyl (-C(=O)-R), carboxy (-COH), oxycarbonyl (-C(=O)-OR), carbonyloxy (-OC(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio group (-SC(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO-R), aminosulfonyl (-SO-NHR), sulfamoylamino (-NH-SO-NHR), thiocarboxy (-C(=O)-SH), and carboxycarbonyl (-C(=O)-COH).

[0071] Examples of oxy (—OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. Alkoxy includes C １ ~C ４ Alkoxy, C １ ~C ２ Alkoxy is preferred, and methoxy or ethoxy is particularly preferred.

[0072] Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0073] Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0074] Examples of carbonyloxy (-OC(=O)-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

[0075] Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0076] Examples of carbonylthio (-SC(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.

[0077] Examples of aminocarbonyl (-C(=O)-NHR) include alkylaminocarbonyl (e.g., C １ ~C ６ or C １ ~C ４ Examples thereof include alkylaminocarbonyl, specifically ethylaminocarbonyl, methylaminocarbonyl, etc.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, examples thereof include groups in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0078] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0079] Examples of oxycarbonylamino (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0080] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0081] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0082] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0083] As used herein, examples of a "sulfur atom-derived substituent" include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)-R), sulfo (-SOH), pentafluorosulfanyl (-SF), and disulfanyl (-SSR).

[0084] Examples of thio (-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0085] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0086] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0087] As used herein, examples of a "nitrogen atom-derived substituent" include azide (-N3, also referred to as an "azido 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'').

[0088] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, and aralkylamino.

[0089] Examples of tertiary amino (—NR(R′)) include an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring.

[0090] Examples of substituted amidino (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0091] Examples of substituted guanidino (-NR-C(=NR''')-NR'R'') include groups in which R, R', R'', and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0092] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0093] In this specification, the "amino acid residues" that constitute a peptide compound may be simply referred to as "amino acids".

[0094] In this specification, the term "to" indicating a numerical range includes both ends of the range. For example, "A to B" means a numerical range that is equal to or greater than A and equal to or less than B.

[0095] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus or minus 10% of that numerical value.

[0096] In this specification, the meaning of the term "and / or" includes any combination of "and" and "or." Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0097] Method for Producing a Peptide Compound In one aspect, the present invention relates to a method for producing a peptide compound or a salt thereof, the method comprising a step of linking an amino group of a first amino acid or peptide to a carboxy group of a second amino acid or peptide via an amide bond in a two-phase solvent system containing one or more water-immiscible organic solvents and water (linking step).

[0098] In one aspect, the present invention relates to a method for producing a peptide compound or a salt thereof, the method comprising a step of linking an amino group of a first amino acid or peptide with a carboxy group of a second amino acid or peptide via an amide bond in a two-phase solvent system containing one or more water-immiscible organic solvents and water (linking step). In this aspect, the amino group of the first amino acid or peptide is selected from the group consisting of a hydrogen atom, a linear C １-C ６ Alkyl, branched C ３ -C ６ Alkyl, or C ３ -C ８ It is a cycloalkyl-substituted amino group.

[0099] In one embodiment, the linking step is carried out in the presence of an activating agent. Examples of the activating agent include acyl halides, phosphoric acid halides, phosphinic acid halides, carbodiimides, uronium, phosphonium, etc. The acyl halides are preferably linear C １ -C １８ Alkylcarbonyl chloride, branched C ３ -C １８ Alkylcarbonyl chloride, linear C １ -C １８ Alkylcarbonyl bromide, branched C ３ -C １８ Alkylcarbonyl bromide or monocyclic or condensed cyclic C ３ -C １５ Cycloalkylcarbonyl chloride (C ３ -C １５ Cycloalkylcarbonyl chloride is C １ -C ６ Examples of suitable phosphate halides include diethyl chlorophosphate and diphenyl chlorophosphate. Examples of suitable phosphinic acid halides include diphenylphosphinic acid chloride. Examples of suitable carbodiimides include N,N-diisopropylalbodiimide, N,N-dicyclohexylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Examples of suitable uronium halides include HATU and COMU. Examples of suitable phosphonium halides include PyFOP.

[0100] In one embodiment, the linking step is carried out in the presence of an additive. Preferably, step (1) of the linking step is carried out in the presence of an additive. Examples of the additive include bromide salts, iodide salts, and trifluoromethanesulfonate salts. Examples of bromide salts include sodium bromide, potassium bromide, magnesium bromide, calcium bromide, and barium bromide. Examples of iodide salts include sodium iodide, potassium iodide, magnesium iodide, calcium iodide, and barium iodide. Examples of trifluoromethanesulfonate salts include sodium trifluoromethanesulfonate, potassium trifluoromethanesulfonate, magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, and barium trifluoromethanesulfonate. A preferred additive is at least one selected from the group consisting of sodium bromide, sodium iodide, sodium trifluoromethanesulfonate, and potassium trifluoromethanesulfonate. The molar equivalent of the additive used relative to the second amino acid or peptide is, for example, in the range of 0.2 to 3.0 molar equivalents relative to the second amino acid or peptide, and preferably in the range of 0.3 to 1.5 molar equivalents.

[0101] In one embodiment, the coupling step can be carried out by stirring the reaction mixture for 10 minutes to 24 hours at a reaction temperature of, for example, −50° C. to 30° C., preferably −20° C. to 10° C., more preferably −18° C. to 0° C., and most preferably −18° C. to −5° C.

[0102] In one embodiment, the linking step comprises: (1) contacting a second amino acid or peptide with an acyl halide in an organic solvent to prepare a mixed acid anhydride, and (2) contacting a first amino acid or peptide with the mixed acid anhydride obtained in step (1) in the presence of a base in a biphasic solvent system containing one or more water-immiscible organic solvents and water. Preferably, (2) of the linking step comprises adding the first amino acid or peptide dropwise to the mixed acid anhydride obtained in step (1) in the presence of a base.

[0103] In one embodiment, step (1) in the linking step can be carried out by stirring the reaction mixture for 75 minutes to 7 hours at a reaction temperature of, for example, −50° C. to 30° C., preferably −20° C. to 10° C., more preferably −18° C. to 0° C., and most preferably −18° C. to −5° C.

[0104] In one embodiment, step (2) in the linking step can be carried out by stirring the reaction mixture for 10 minutes to 24 hours at a reaction temperature of, for example, −50° C. to 30° C., preferably −20° C. to 10° C., more preferably −18° C. to 0° C., and most preferably −18° C. to −5° C.

[0105] As used herein, the term "two-phase solvent" refers to a solvent in which a water-immiscible organic solvent and water form an interface that is visible. Depending on the water-immiscible organic solvent, the organic solvent may be the lower layer, or the water may be the lower layer.

[0106] In certain embodiments, the water-immiscible organic solvent includes, but is not limited to, an organic solvent having low water solubility (e.g., solubility in water of 200 g / L or less, preferably 150 g / L or less). The water-immiscible organic solvent may contain a trace amount of another water-miscible organic solvent, for example, 0.01 wt % or less. The water solubility can be determined by any method known in the art or described herein. Exemplary methods for determining solubility include, but are not limited to, gas chromatography, which can be determined by measuring the concentration of the organic solvent in water prepared by mixing equal volumes of the organic solvent and water at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C).

[0107] Furthermore, the miscibility of an organic solvent with water can be determined, for example, by the separation of the solvent and water into two layers when equal volumes of the solvent and water are mixed in a container at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C). Whether the solvent and water separate into two layers can be determined, for example, by visual inspection or by sampling the upper and lower liquids in the container. When it is confirmed by such a method that the solvent and water have separated into two layers, the solvent may be referred to as a water-immiscible solvent. However, even a solvent that is miscible with water may form an interface with water and separate into two layers depending on the solute in the solvent and the salt concentration in the water.

[0108] In certain embodiments, the water-immiscible organic solvent is a solvent having an octanol / water partition coefficient (Log Kow) of 5 or greater, or a solvent having a predicted octanol / water partition coefficient (Log Kow) of 5 or greater. The octanol / water partition coefficient (Log Kow) can be determined by any method known in the art or described herein. The predicted octanol / water partition coefficient (Log Kow) can be determined by known means in a separate explicit measurement, for example, but not limited to, a database search or literature search. The octanol / water partition coefficient can be measured by, for example, but not limited to, a method in accordance with Japanese Industrial Standard JIS 7260-107:2000, Determination of Partition Coefficient (1-Octanol / Water) - Shake Flask Method (https: / / kikakurui.com / z7 / Z7260-107-2000-01.html [Accessed September 9, 2024]).

[0109] In one embodiment, examples of the water-immiscible organic solvent include ester solvents, ether solvents, halogenated hydrocarbon solvents, and aromatic hydrocarbon solvents. Examples of the ester solvent include isopropyl acetate, ethyl acetate, and butyl acetate. Examples of the ether solvent include methyl t-butyl ether and diethyl ether. Examples of the halogenated hydrocarbon solvent include dichloromethane and carbon tetrachloride. Examples of the aromatic hydrocarbon solvent include toluene and hexane. Among these, isopropyl acetate, methyl t-butyl ether, dichloromethane, 2-methyltetrahydrofuran, and toluene are preferred.

[0110] In one embodiment, the ratio of water to one or more water-immiscible organic solvents in the linking step is, for example, 0.001 to 10,000 by weight, preferably 0.01 to 1,000, more preferably 0.1 to 100, and most preferably 1 to 10.

[0111] In one embodiment, the organic solvent used in step (1) in the linking step is not particularly limited as long as it does not inhibit the production of a mixed acid anhydride, but is preferably an organic solvent that is immiscible with water. Examples of solvents used in step (1) include ester solvents, ether solvents, amide solvents, and nitrile solvents. Examples of ester solvents include isopropyl acetate, ethyl acetate, and butyl acetate. Examples of ether solvents include tetrahydrofuran, 2-methyltetrahydrofuran, and diethyl ether. Examples of amide solvents include N,N-dimethylformamide, N,N-dimethylacetamide, and N,N-diethylformamide. Examples of nitrile solvents include acetonitrile and propionitrile. Among these, isopropyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, and acetonitrile are preferred.

[0112] In one embodiment, the organic solvent used in step (1) of the linking step is used at a concentration that dissolves the second amino acid or peptide. The second amino acid or peptide is used at a concentration of, for example, 0.1 to 300 mg / mL, preferably 1 to 200 mg / mL, more preferably 10 to 200 mg / mL, and most preferably 20 to 200 mg / mL.

[0113] In certain embodiments, organic solvents that may react with the mixed acid anhydride or peptide compound may be excluded from the water-immiscible organic solvents of the present invention. Examples of organic solvents that are inappropriate as water-immiscible organic solvents include amine solvents (e.g., n-propylamine or diisopropylamine) and alcohol solvents (e.g., methanol, ethanol, n-propanol, phenol).

[0114] In one embodiment, in step (2) of the linking step, it is preferable to use a mixture of the mixed acid anhydride obtained in step (1) and an organic solvent.

[0115] As used herein, the term "contact" refers to contacting a solid phase with a liquid phase in a solid-liquid phase synthesis method, or mixing, adding, dropping, etc. in a liquid-liquid phase synthesis method.

[0116] As described herein, step (2) in the linking step of the method of the present invention is carried out in a two-phase system containing one or more water-immiscible solvents and water, which can be useful in the following respects: (a) the water-immiscible solvent can be utilized as an extraction solvent in post-treatment of the reaction mixture; and (b) the extracted solution containing the peptide compound can be used as a starting material for a subsequent step / reaction (i.e., a solution containing the starting compound of the subsequent step). In certain embodiments, this method allows the entire reaction to be carried out and completed without isolating intermediates from the solvent of the starting reaction.

[0117] In some embodiments, the acyl halide is a linear C １ -C １８ Alkylcarbonyl chloride, branched C ３ -C１８ Alkylcarbonyl chloride, linear C １ -C １８ Alkylcarbonyl bromide, branched C ３ -C １８ Alkylcarbonyl bromide or monocyclic or condensed cyclic C ３ -C １５ Cycloalkylcarbonyl chloride (C ３ -C １５ Cycloalkylcarbonyl chloride is C １ -C ６ Among these, pivaloyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexanecarbonyl chloride, 1-adamantanecarbonyl chloride, or 2-ethylbutyryl chloride is preferred, pivaloyl chloride or 2,2-dimethylbutyryl chloride is more preferred, pivaloyl chloride or 2,2-dimethylbutyryl chloride is even more preferred, and pivaloyl chloride is most preferred.

[0118] In one embodiment, the acyl halide is used in an amount of, for example, 0.01 to 100 molar equivalents relative to the second amino acid or peptide, preferably 0.1 to 10 molar equivalents, more preferably 0.5 to 1.0 molar equivalents, and most preferably 0.8 to 1.0 molar equivalents.

[0119] In one embodiment, step (1) in the linking step is carried out in the presence of a base.

[0120] In an embodiment, the base used in step (1) in the linking step has a conjugate acid having a pKa of 15.0 or less, preferably 13.0 or less, and more preferably 11.5 or less. In an embodiment, the base used in step (1) in the linking step has a conjugate acid having a pKa of 0 or more, preferably 3 or more, and more preferably 5 or more. In an embodiment, the base used in step (1) in the linking step has a conjugate acid having a pKa of 0 to 15.0, preferably 0 to 11.5, more preferably 3 to 11.5, even more preferably 5 to 11.5, and most preferably 6 to 11.5. The "pKa" herein can be measured using water as a solvent. As the pKa herein, an actual measured value that has already been reported as the pKa when water is used as a solvent may be used. If the measured value of pKa is not available, it may be calculated using ADMETPredictor (Simulations Plus Inc., ver. 8.0), and the calculated value may be used as the pKa in this specification.The pKa of representative reagents is listed below: conjugate acid of DBU (pKa=11.9; R. Srivastava, J. Mol. Catal. A: Chem. 264 (2007) 146-152); conjugate acid of piperidine (pKa=11.22; Hall, HK, Jr. JA Am Chem. Soc. 1957, 79, 5441); conjugate acid of triethylamine (pKa=10.65; Hall, HK, Jr. JA Am Chem. Soc. 1957, 79, 5441); conjugate acid of diisopropylethylamine (pKa=11.44; Chemical and Pharmaceutical Bulletin, 1995, 43, 1872-1877); The conjugate acid of 2,4,6-collidine (pKa = 7.48; Clarke, K., Rothwell, K.J. Chem. Soc. 1960, 1885); the conjugate acid of 2,6-lutidine (pKa = 6.77; Clarke, K., Rothwell, K.J. Chem. Soc. 1960, 1885); the conjugate acid of pyridine (pKa = 5.21; D.H. Ripin, D.A. Evans, pKa's of Nitrogen Acids, [online], [Retrieved September 22, 2023], Internet<URL:http: / / evans.rc.fas.harvard.edu / pdf / evans_pKa_table.pdf> ).

[0121] In one embodiment, the base used in step (1) in the linking step is an organic base, and is preferably at least one selected from the group consisting of amines and pyridines. Examples of amines include diisopropylethylamine and triethylamine. Examples of pyridines include 2,6-lutidine and 2,4,6-collidine. The base used in step (1) in the linking step is preferably one selected from the group consisting of diisopropylethylamine, triethylamine, 2,6-lutidine, and 2,4,6-collidine, more preferably one selected from the group consisting of triethylamine, 2,6-lutidine, and 2,4,6-collidine, and most preferably 2,6-lutidine.

[0122] In one embodiment, the base used in step (1) in the linking step is used in an amount of, for example, 0.5 to 100 molar equivalents, preferably 0.8 to 10 molar equivalents, more preferably 0.9 to 5 molar equivalents, and most preferably 1.0 to 1.3 molar equivalents relative to the second amino acid or peptide.

[0123] In an embodiment, the base used in step (2) in the linking step is not particularly limited, but is preferably an organic base or an inorganic base, more preferably at least one selected from the group consisting of sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium hydrogencarbonate, sodium hydrogenphosphate, triethylamine, and pyridine, even more preferably one selected from the group consisting of sodium carbonate, potassium carbonate, cesium carbonate, and potassium phosphate, and most preferably one selected from the group consisting of sodium carbonate and potassium carbonate.

[0124] As used herein, the terms "first amino acid" and "second amino acid" are used solely to distinguish the reactive sites between amino acids that are substrates in the ligation step. Specifically, a "first amino acid" refers to an amino acid whose reactive site is an amino group, and a "second amino acid" refers to an amino acid whose reactive site is a carboxy group.

[0125] In some embodiments, the first amino acid or peptide is a peptide comprising two or more amino acid residues.

[0126] In some embodiments, the amino group of the first amino acid or peptide has the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a linear C １ -C ６ Alkyl, branched C ３ -C ６ Alkyl, or C ３ -C ８ In certain embodiments, the amino group of the first amino acid or peptide is represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom or a linear C １ -C ４ In certain embodiments, the amino group of the first amino acid or peptide is represented by the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom, methyl, or ethyl. In certain embodiments, the amino group of the first amino acid or peptide is an amino group of the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is a hydrogen atom or methyl. In certain embodiments, the amino group of the first amino acid or peptide is an amino group of the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, and R ５ is methyl).

[0127] In some embodiments, the carboxyl group of the first amino acid or peptide may be protected with a protecting group. Any protecting group known in the art may be used as the protecting group for the carboxyl group, as long as it does not reduce the solubility of the peptide in a solvent. Specific examples of such a protecting group for the carboxyl group include a methyl group, an ethyl group, a t-butyl group, a trityl group, and a cumyl group, with a t-butyl group being preferred.

[0128] In some embodiments, the second amino acid or peptide is a peptide comprising two or more amino acid residues.

[0129] In some embodiments, the α-carbon of the carboxy group of the second amino acid or peptide is optionally substituted. In some embodiments, the α-carbon of the carboxy group of the second amino acid or peptide is substituted with a group of the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ６ Alkyl, optionally substituted branched C ３ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl C １ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl, optionally substituted phenyl C １ -C ２ Alkyl, optionally substituted C １ -C ６ Alkoxy C １ -C ２ Alkyl, optionally substituted 5- to 6-membered heteroaryl C １ -C ２ Alkyl or optionally substituted phenyl C １ -C ２ Alkylthio C １ -C ２ alkyl or R 1 and R ２together with the carbon atoms to which they are attached, form C ３ -C ８ R １ or R ２ is branched chain C １ -C ６ When the branched chain C １ -C ６ The alkyl may be substituted with one or more halogens. 1 or R ２ is C ３ -C ８ Cycloalkyl C １ -C ６ When it is alkyl, the C ３ -C ８ Cycloalkyl C １ -C ６ The alkyl may be substituted with one or more selected from the group consisting of methyl, ethyl, propyl, halogen, methoxy, ethoxy, and trifluoromethyl. 1 or R ２ is C ３ -C ８ When it is cycloalkyl, the C ３ -C ８ The cycloalkyl may be substituted with one or more selected from the group consisting of methyl, ethyl, propyl, halogen, methoxy, ethoxy, and trifluoromethyl. 1 or R ２ Phenyl C １ -C ２ When the phenyl C is alkyl, １ -C ２ The alkyl may be substituted with one or more selected from the group consisting of methyl, ethyl, propyl, halogen, methoxy, ethoxy, and trifluoromethyl. １ or R ２ is C １ -C ６ Alkoxy C １ -C ２ When it is alkyl, the C １ -C ６ Alkoxy C １ -C ２The alkyl may be substituted with one or more selected from the group consisting of methyl, ethyl, propyl, halogen, methoxy, ethoxy, and trifluoromethyl. １ or R ２ is a 5- to 6-membered heteroaryl C １ -C ２ When it is alkyl, the 5- to 6-membered heteroaryl C １ -C ２ The alkyl may be substituted with one or more selected from the group consisting of methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0130] In certain embodiments, the alpha carbon of the carboxy group of the second amino acid or peptide has the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and represent a hydrogen atom, a linear C １ -C ３ Alkyl, branched C ３ -C ６ Alkyl, C ３ -C ６ Cycloalkyl C １ -C ６ Alkyl, C １ -C ６ Alkoxymethyl, 1-(C １ -C ６ alkoxy)ethyl, optionally substituted benzyl, optionally substituted phenethyl, or phenyl C １ -C ２ alkylthiomethyl). 1 or R ２ When is benzyl or phenethyl, the benzyl or phenethyl may be substituted with one or more selected from the group consisting of fluorine, methyl, ethyl, methoxy, ethoxy and trifluoromethyl.

[0131] In certain embodiments, the alpha carbon of the carboxy group of the second amino acid or peptide has the formula: -CR １ R ２ - (wherein, R 1 is a hydrogen atom, and R ２is a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, t-butoxymethyl, 1-(t-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl, or benzylthiomethyl).

[0132] In some embodiments, the nitrogen at the β-position of the carboxy group of the second amino acid or peptide has the formula: -NR ３ - (wherein, R ３ is a hydrogen atom).

[0133] In some embodiments, the amino group of the second amino acid or peptide may be protected with a protecting group. Any protecting group known in the art may be used as the amino group protecting group as long as it does not reduce the solubility of the peptide in a solvent. Specific examples of such amino group protecting groups include Cbz, p-nitrobenzyloxycarbonyl, 2-naphthylmethyloxycarbonyl, diphenylmethyloxycarbonyl, 9-anthrylmethyloxycarbonyl, Teoc, Fmoc, Boc, Alloc, and trifluoroacetyl. Of these, Cbz, Teoc, and trifluoroacetyl are preferred.

[0134] In one embodiment, the peptide compound or salt thereof produced by the method of the present invention comprises 8 to 20 amino acid residues, preferably 11 to 14 amino acid residues, more preferably 11 to 13 amino acid residues, and most preferably 11 amino acid residues.

[0135] In certain aspects, the peptide compound or salt thereof produced by the methods of the invention can comprise at least 1, at least 2, at least 3, at least 4, or at least 5 unnatural amino acid residues. In certain embodiments, the unnatural amino acid residue contained in the peptide compound or salt thereof produced by the methods of the invention is an N-methyl amino acid residue.

[0136] In one embodiment, the peptide compound produced by the method of the present invention can be a linear peptide compound. In another embodiment, the peptide compound produced by the method of the present invention can be a cyclic peptide compound. In one embodiment, the linear or cyclic peptide compound may contain a cyclic structure (cyclic portion) as a partial structure. Specific examples of cyclic structures include a structure in which the side chain of one amino acid residue is linked to the side chain of another amino acid residue, a structure in which the N-substituent of one amino acid residue is linked to the side chain of another amino acid residue, or a structure in which the N-substituent of one amino acid residue is linked to the N-substituent of another amino acid residue. The two amino acid residues involved in the linkage for the cyclic structure may be adjacent, or any number of amino acid residues, for example, any amino acid residue selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19, may be present between them. The size of the ring formed by the cyclic structure is not particularly limited, and examples include a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, a 12-membered ring, a 13-membered ring, a 14-membered ring, a 15-membered ring, a 16-membered ring, a 17-membered ring, an 18-membered ring, a 19-membered ring, a 20-membered ring, a 21-membered ring, a 22-membered ring, a 23-membered ring, a 24-membered ring, a 25-membered ring, a 26-membered ring, a 27-membered ring, a 28-membered ring, a 29-membered ring, a 30-membered ring, a 31-membered ring, a 32-membered ring, a 33-membered ring, a 34-membered ring, or a 35-membered ring. Preferably, the ring is a 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-membered ring, more preferably a 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, or 18-membered ring, and most preferably an 11-, 12-, 13-, or 14-membered ring. When a peptide compound has a cyclic structure, the number of cyclic structures is not limited, but it is preferred that one, two, three, four, or five cyclic structures be present.

[0137] In certain embodiments, the peptide compound or salt thereof produced by the method of the present invention can comprise a cyclic portion composed of 4 or more, 6 or more, 8 or more, or 11 or more amino acid residues. In certain embodiments, the peptide compound or salt thereof produced by the method of the present invention is a cyclic peptide compound or salt thereof, and comprises a cyclic portion composed of 4 to 14 amino acid residues, preferably 6 to 14 amino acid residues, more preferably 8 to 14 amino acid residues, and most preferably 11 to 14 amino acid residues. In certain embodiments, the peptide compound or salt thereof produced by the method of the present invention is a cyclic peptide compound or salt thereof, and comprises a cyclic portion composed of 11 amino acid residues. In these embodiments, the cyclic portion comprises one, two, three, four, five, six, or seven amide bonds linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide.

[0138] In certain embodiments, the method of the present invention may further comprise a step of removing / deprotecting the protecting groups of the amino and carboxy groups derived from the second amino acid or peptide and / or the protecting groups of the amino and carboxy groups derived from the first amino acid or peptide in the peptide compound obtained in the linking step (deprotection step) immediately before, immediately after, and / or during the linking step. The deprotection step can be carried out using a method known in the art, for example, using reagents and conditions described in "Greene's Protective Groups in Organic Synthesis, Fifth Edition, 2014."

[0139] In one embodiment, the deprotection step of the amino-protecting group can be carried out by catalytic hydrogenation, for example, when the protecting group is Cbz, p-nitrobenzyloxycarbonyl, 2-naphthylmethyloxycarbonyl, diphenylmethyloxycarbonyl, or 9-anthrylmethyloxycarbonyl. Any catalyst known in the art can be used for catalytic hydrogenation. Specific examples of the catalyst include Pd / C, Pd(OH) / C, and PtO, with Pd / C being preferred.

[0140] In some embodiments, the deprotection step of the amino protecting group can be carried out using a reagent such as TBAF, LiBH4, piperidine, trifluoroacetic acid, or methanesulfonic acid when the protecting group is Teoc, Fmoc, Boc, Alloc, or trifluoroacetyl.

[0141] In one embodiment, the deprotection step of the carboxy protecting group can be carried out under acidic conditions in the presence of a deprotecting reagent, for example, when the protecting group is a methyl group, an ethyl group, a t-butyl group, a trityl group, or a cumyl group.

[0142] In some embodiments, the method of the present invention may further comprise a step of linking / condensing a third amino acid or peptide to the peptide compound obtained in the linking step or deprotection step (linking step A).

[0143] In one embodiment, the method of the present invention may further comprise a step of linking / condensing the amino group and the carboxy group in the peptide compound obtained in the linking step or the deprotection step (linking step B). In this case, the peptide compound obtained in linking step B is a cyclic peptide compound.

[0144] In some embodiments, the ligation steps A and B can be carried out in the presence or absence of, for example, a condensation reagent, such as T3P, EDCI, HATU, COMU, BEP, PyBOP, DMT-MM, or PyOxim.

[0145] In one embodiment, the method of the present invention is carried out using a flow reactor. As used herein, the term "flow reactor" refers to a device that continuously supplies raw materials to a tubular reactor, mixes and reacts them to produce a target compound. The flow reactor may be a commercially available flow reactor (e.g., the Africa flow chemistry system or the Asia flow chemistry system manufactured by Syrris), or may be a device that combines a tubular reactor with a syringe pump.

[0146] Peptide Compound In one aspect, the present invention relates to a peptide compound or a salt thereof produced by the above-mentioned "method for producing a peptide compound." In one aspect, the present invention relates to a pharmaceutical composition comprising a peptide compound or a salt thereof produced by the above-mentioned "method for producing a peptide compound."

[0147] All prior art documents cited herein are incorporated herein by reference. This application claims priority from Japanese Patent Application No. 2023-168751, filed September 28, 2023, the contents of which are incorporated herein by reference in their entirety. Furthermore, all references cited herein, including patent applications and publications, including the following documents, are incorporated herein by reference in their entirety: WO 2013 / 100132, WO 2018 / 225851, WO 2018 / 225864, WO 2019 / 117274, WO 2020 / 111238, WO 2020 / 122182, WO 2021 / 075478, WO 202 ... Publication No. 2021 / 090856, International Publication No. 2021 / 132545, International Publication No. 2021 / 246471, International Publication No. 2022 / 097540, International Publication No. 2022 / 138891, International Publication No. 2022 / 145444, International Publication No. 2022 / 234864, International Publication No. 2023 / 127869, International Publication No. 2023 / 219152, International Publication No. 2024 / 096023, International Publication No. 2024 / 143514.

[0148] The present invention is further illustrated, but not limited, by the following examples. Unless otherwise noted, starting materials, starting materials, solvents, and reagents were obtained from commercial suppliers or synthesized using known methods.

[0149] 1H-NMR spectra were measured using a JNM-ECZ500 nuclear magnetic resonance spectrometer (JEOL Ltd.). The chemical shift of tetramethylsilane, used as an internal standard, was set at 0 ppm, and the deuterium lock signal from the sample solvent was referenced. The chemical shifts of the analyte signals were expressed in ppm. Signal splitting was abbreviated as s: singlet, brs: broad singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, and m: multiplet, and the signal split width was expressed as a J value (Hz). The signal integral was calculated based on the ratio of the area intensities of each signal.

[0150] HPLC analysis was performed using a Waters H-Class system with a PDA detector at a wavelength of 210 nm. The reactivity, selectivity, and purity of each substrate used in the examples were evaluated using the analytical methods shown in Table 1 below. LCMS analysis was performed using an SQD2 or QDa detector.

[0151]

[0152] Unless otherwise specified in the experimental section, HPLC samples were prepared in the following manner: Activation step: 2–3 μL of the reaction mixture was dissolved in MeCN (1.0 mL) and azetidine (0.1 mL). Condensation step: 5 μL of the reaction mixture was dissolved in MeCN (1.0 mL) and azetidine (0.1 mL).

[0153] The conversion rate and diastereomeric ratio of the condensation reaction were calculated using the HPLC area values ​​of each component in the reaction mixture according to the following formula. In the formula, "substrate" refers to the substrate with the smaller molar amount of the two substrates to be condensed. Conversion rate = [(desired product + epimer) / (substrate + desired product + epimer)] x 100 Diastereomeric ratio = desired product / epimer

[0154] (Example 1) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: organic solvent and water bilayer system conditions

[0155] Activation and condensation reaction step: A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration: 157 mg / mL, 2.00 mL, 314.0 mg, 0.800 mmol) and 2,6-lutidine (104.0 μL, 0.895 mmol) were added to a test tube containing a stir bar (hereafter referred to as "test tube A"), and the reaction mixture was cooled to -10 °C while stirring. PivCl (91.8 μL, 0.746 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. HPLC analysis of the reaction mixture confirmed the progress of activation. H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (300.0 mg, 0.597 mmol), toluene (2.00 mL), Na2CO3 (190.0 mg, 1.790 mmol), and water (1.90 mL) were added to another test tube (hereinafter referred to as "test tube B") containing a stir bar, and the reaction mixture was cooled to 0 °C with stirring. The mixture in test tube A was added dropwise to test tube B, and stirring was continued at 0 °C for 22 hours. Reaction conversion: 98.8% Diastereomer ratio: 99.6 / 0.4 Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 1.8%

[0156] Post-treatment step: The aqueous layer of the reaction mixture was removed, and N,N-dimethylethylenediamine (65.0 μL, 0.597 mmol) was added to the organic layer. The organic layer was heated to 25°C with stirring. 2.5% aqueous ammonia (2.00 mL) was added to the organic layer and stirred, and after standing, the aqueous layer was removed. 5% aqueous sulfuric acid (2.00 mL) was added to the organic layer and stirred, and after standing, the aqueous layer was removed. 5% aqueous Na2CO3 was added to the organic layer and stirred, and after standing, the aqueous layer was removed. The resulting organic layer was concentrated to dryness to obtain 513.4 mg of the desired product. Yield: 98% Purity: 96.8% Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.0 min, Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 13.1 min, desired product: 16.4 min, epimer: 16.8 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.68 ([M+H] + ), Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 587.81 ([M+H] + ), desired product: m / z 732.98 ([M-Sar-OtBu] + )

[0157] (Example 2) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A to D.

[0158] Condition A: A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.240 mL, 44.4 mg, 0.113 mmol) and DIPEA (22.4 μL, 0.128 mmol), and the mixture was cooled to -20 °C with stirring. PivCl (13.2 μL, 0.107 mmol) was added to the reaction mixture, which was then stirred for 1 hour and 25 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (156.0 mg / mL, 0.280 mL, 43.7 mg, 0.087 mmol) was added to the reaction mixture, which was then stirred for 15 minutes while warming to 0 °C. An aqueous solution of Na2CO3 (27.2 mg Na2CO3, 0.270 mL HO) was then added, and the mixture was stirred for 8 hours. Condition B: A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.240 mL, 44.4 mg, 0.113 mmol) and DIPEA (22.4 μL, 0.128 mmol), followed by stirring and cooling to -20 °C. PivCl (13.2 μL, 0.107 mmol) was added to the reaction mixture, which was then stirred for 1 hour and 25 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (156.0 mg / mL, 0.280 mL, 43.7 mg, 0.087 mmol) was added to the reaction mixture, which was then stirred for 15 minutes while warming to 0 °C. DIPEA (44.7 μL, 0.257 mmol) was then added, and the mixture was stirred for 8 hours. Condition C: HATU (76.0 mg, 0.199 mmol), a MeCN solution of Cbz-MeLeu-Ile-OH (concentration: 200.0 mg / mL, 0.210 mL, 42.0 mg, 0.107 mmol), a MeCN solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration: 142.9 mg / mL, 0.270 mL, 38.6 mg, 0.077 mmol), and DIPEA (34.7 μL, 0.199 mmol) were sequentially added to a vial containing a stir bar and stirred.Condition D: HOPO (9.5 mg, 0.086 mmol), an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.180 mL, 33.6 mg, 0.086 mmol), an IPAC solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (156.0 mg / mL, 0.280 mL, 43.0 mg, 0.086 mmol), and water (0.46 mL) were added sequentially to a test tube containing a stir bar and cooled to 0 °C. EDC·HCl (18.0 mg, 0.094 mmol) was added to the reaction mixture, and the mixture was stirred at 0 °C for 14 h. Synthesis of the epimer: The epimer was synthesized according to condition A, except that Cbz-MeLeu-D-allo-Ile-OH was used instead of Cbz-MeLeu-Ile-OH. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 7.8 min, desired product: 16.2 min, epimer: 16.6 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.79 ([M+H]. + ), desired product: m / z 732.87 ([M-Sar-OtBu] + ), epimer: m / z 732.87 ([M-Sar-OtBu] + )

[0159]

[0160] As shown in Table 2, both the conversion rate and the diastereomeric ratio were high under the two-layer system condition of an organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system condition of an organic solvent only (condition B), the condition using HATU as a condensing agent (condition C), and the known two-layer system condition (condition D).

[0161] (Example 3) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A to C.

[0162] Condition A: A solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.220 mL, 40.7 mg, 0.100 mmol) and DIPEA (18.8 μL, 0.108 mmol) were added to a test tube containing a stir bar and cooled to -20°C with stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture and stirred for 1 hour and 30 minutes. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) was added to the reaction mixture, which was then stirred for 15 min while warming to 0 °C. An aqueous solution of Na2CO3 (21.0 mg Na2CO3, 0.210 mL HO) was then added, and stirring was continued for 22 h. Condition B: A solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.220 mL, 40.7 mg, 0.100 mmol) and DIPEA (18.8 μL, 0.108 mmol) were added to a test tube containing a stir bar, and the mixture was cooled to -20 °C while stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture, which was then stirred for 1 hour and 30 minutes. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) was added to the reaction mixture, which was then stirred for 15 minutes while warming to 0 °C. DIPEA (35.2 μL, 0.202 mmol) was then added, and the mixture was stirred for 22 hours. Condition C: HATU (51.2 mg, 0.135 mmol), a MeCN solution of Cbz-MeLeu-Ile-OH (concentration: 200.0 mg / mL, 0.150 mL, 30.0 mg, 0.076 mmol), a MeCN solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration: 170.0 mg / mL, 0.300 mL, 51.0 mg, 0.069 mmol), and DIPEA (23.4 μL, 0.134 mmol) were added sequentially to a vial containing a stir bar and stirred for 3 h.Synthesis of epimers. The epimers were synthesized according to condition A, except that Cbz-MeLeu-D-allo-Ile-OH was used instead of Cbz-MeLeu-Ile-OH. Measurement method: HPLC Method A. Retention times: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min. Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.92 ([M+H]). + ), desired product: m / z 1118.26 ([M+H] + ), epimer: m / z 1118.32 ([M+H] + )

[0163]

[0164] As shown in Table 3, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system of organic solvent only (condition B) and the condition using HATU as a condensing agent (condition C).

[0165] (Example 4) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A to C.

[0166] Condition A: A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.230 mL, 42.6 mg, 0.108 mmol) and DIPEA (21.2 μL, 0.122 mmol), and the mixture was cooled to -20 °C with stirring. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, which was then stirred for 1 hour and 15 minutes. A toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) was added to the reaction mixture, which was then heated to 0 °C with stirring for 15 minutes. An aqueous solution of Na2CO3 (26.0 mg Na2CO3, 0.260 mL HO) was then added, and the mixture was stirred for 9 hours. Condition B: A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 0.230 mL, 42.6 mg, 0.108 mmol) and DIPEA (21.2 μL, 0.122 mmol), followed by stirring and cooling to -20 °C. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, which was then stirred for 1 hour and 15 minutes. A toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) was added to the reaction mixture, which was then stirred for 15 minutes while warming to 0 °C. DIPEA (42.4 μL, 0.244 mmol) was then added, and the mixture was stirred for 9 hours. Condition C: HATU (51.5 mg, 0.135 mmol), a MeCN solution of Cbz-MeLeu-Ile-OH (200.0 mg / mL, 0.150 mL, 30.0 mg, 0.076 mmol), a MeCN solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (75.8 mg / mL, 0.330 mL, 25.0 mg, 0.068 mmol), and DIPEA (23.4 μL, 0.134 mmol) were added sequentially to a vial containing a stir bar and stirred for 2 hours. Synthesis of the epimer: The epimer was synthesized according to condition A, except that Cbz-MeLeu-D-allo-Ile-OH was used instead of Cbz-MeLeu-Ile-OH.Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 16.7 min, epimer: 17.5 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.64 ([M+H]. + ), desired product: m / z 766.78 ([M+Na] + ), epimer: m / z 767.01 ([M+Na] + )

[0167]

[0168] As shown in Table 4, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system of organic solvent only (condition B) and the condition using HATU as a condensing agent (condition C).

[0169] (Example 5) Condensation reaction of Cbz-MeLeu-Ala-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under conditions A to C shown in the table below.

[0170] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ala-OH (156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol) and 2,6-lutidine (13.5 μL, 0.115 mmol), and cooled to −10°C with stirring. PivCl (12.0 μL, 0.098 mmol) was added to the reaction mixture, which was then stirred for 3 hours. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.330 mL, 66.0 mg, 0.089 mmol) was added to the reaction mixture, which was then stirred for 10 min while warming to 0 °C. An aqueous solution of Na2CO3 (28.0 mg Na2CO3, 0.280 mL HO) was then added, and stirring was continued for 6 h. Condition B: A solution of Cbz-MeLeu-Ala-OH in IPAC (156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol) and 2,6-lutidine (13.5 μL, 0.115 mmol) were added to a test tube containing a stir bar, and the mixture was cooled to -10 °C while stirring. PivCl (12.0 μL, 0.098 mmol) was added to the reaction mixture, which was then stirred for 3 h. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.330 mL, 66.0 mg, 0.089 mmol) was added to the reaction mixture, which was then stirred for 10 min while warming to 0 °C. DIPEA (46.0 μL, 0.266 mmol) was then added, and the mixture was stirred for 6 h. Condition C: HATU (67.6 mg, 0.178 mmol), a solution of Cbz-MeLeu-Ala-OH in IPAC (concentration: 156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol), a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration: 170 mg / mL, 0.390 mL, 66.0 mg, 0.089 mmol), and DIPEA (31.0 μL, 0.178 mmol) were sequentially added to a vial containing a stir bar and stirred.Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.4 min, desired product: 17.4 min, epimer: 17.7 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.15 ([M+H]. + ), desired product: m / z 931.09 ([M-Sar-OtBu] + ), epimer: m / z 931.27 ([M-Sar-OtBu] + )

[0171]

[0172] As shown in Table 5, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were lower than those under condition A under the one-layer system of organic solvent only (condition B) and the condition using HATU as a condensing agent (condition C).

[0173] (Example 6) Condensation reaction of Cbz-MeLeu-Phe-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A to C.

[0174] Condition A: A solution of Cbz-MeLeu-Phe-OH in IPAC (concentration: 159.0 mg / mL, 0.270 mL, 42.9 mg, 0.101 mmol) and 2,6-lutidine (12.6 μL, 0.108 mmol) were added to a test tube containing a stir bar and cooled to -10°C with stirring. PivCl (11.3 μL, 0.092 mmol) was added to the reaction mixture and stirred for 3 hours and 30 minutes. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.310 mL, 62.0 mg, 0.083 mmol) was added to the reaction mixture, which was then stirred for 10 min while warming to 0 °C. An aqueous solution of Na2CO3 (26.0 mg Na2CO3, 0.260 mL HO) was then added, and stirring was continued for 3 h. Condition B: A solution of Cbz-MeLeu-Phe-OH in IPAC (159.0 mg / mL, 0.270 mL, 42.9 mg, 0.101 mmol) and 2,6-lutidine (12.6 μL, 0.108 mmol) were added to a test tube containing a stir bar, and the mixture was cooled to -10 °C while stirring. PivCl (11.3 μL, 0.092 mmol) was added to the reaction mixture, which was then stirred for 3 hours and 30 minutes. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.310 mL, 62.0 mg, 0.083 mmol) was added to the reaction mixture, which was then stirred for 10 minutes while warming to 0 °C. DIPEA (43.6 μL, 0.250 mmol) was then added, and the mixture was stirred for 3 hours. Condition C: HATU (63.5 mg, 0.167 mmol), a MeCN solution of Cbz-MeLeu-Phe-OH (concentration: 195.0 mg / mL, 0.200 mL, 39.0 mg, 0.091 mmol), a MeCN solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration: 170.0 mg / mL, 0.360 mL, 61.2 mg, 0.082 mmol), and DIPEA (29.0 μL, 0.167 mmol) were sequentially added to a vial containing a stir bar and stirred.Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.4 min, desired product: 18.2 min, epimer: 18.7 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.04 ([M+H]. + ), desired product: m / z 1007.35 ([M-Sar-OtBu] + ), epimer: m / z 1007.30 ([M-Sar-OtBu] + )

[0175]

[0176] As shown in Table 6, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system of organic solvent only (condition B) and the condition using HATU as a condensing agent (condition C).

[0177] (Example 7) Condensation reaction of Cbz-MeLeu-Ser(OtBu)-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A to C.

[0178] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ser(OtBu)-OH (155.0 mg / mL, 0.260 mL, 40.3 mg, 0.096 mmol) and 2,6-lutidine (12.0 μL, 0.103 mmol), followed by stirring and cooling to -20 °C. PivCl (10.8 μL, 0.088 mmol) was added to the reaction mixture, followed by stirring for 3 h. A toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol) was added to the reaction mixture, followed by stirring for 15 min. The mixture was then warmed to 0 °C, followed by addition of an aqueous solution of Na2CO3 (25.0 mg Na2CO3, 0.250 mL HO), followed by stirring for 3 h. Condition B: A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Ser(OtBu)-OH in IPAC (155.0 mg / mL, 0.260 mL, 40.3 mg, 0.096 mmol) and 2,6-lutidine (12.0 μL, 0.103 mmol), followed by stirring and cooling to -20 °C. PivCl (10.8 μL, 0.088 mmol) was added to the reaction mixture, followed by stirring for 3 h. A toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol) was added to the reaction mixture, followed by stirring for 15 min. The mixture was then warmed to 0 °C, followed by DIPEA (41.6 μL, 0.239 mmol), followed by stirring for 3 h. Condition C: HATU (71.1 mg, 0.187 mmol), a solution of Cbz-MeLeu-Ser(OtBu)-OH in IPAC (concentration: 155.0 mg / mL, 0.280 mL, 43.4 mg, 0.103 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration: 142.9 mg / mL, 0.330 mL, 47.1 mg, 0.094 mmol), and DIPEA (32.6 μL, 0.187 mmol) were sequentially added to a vial containing a stir bar and stirred.Synthesis of epimers. The epimers were synthesized according to condition A, except that Cbz-MeLeu-D-Ser(OtBu)-OH was used instead of Cbz-MeLeu-Ser(OtBu)-OH. Measurement method: HPLC Method A. Retention times: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.5 min, desired product: 17.1 min, epimer: 17.3 min. Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.74 ([M+H]). + ), desired product: m / z 762.96 ([M-Sar-OtBu] + ), epimer: m / z 763.02 ([M-Sar-OtBu] + )

[0179]

[0180] As shown in Table 7, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were lower than those under condition A under the one-layer system of organic solvent (condition B) and the condition using HATU as a condensing agent (condition C).

[0181] (Example 8) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeLeu-Ile-OtBu The condensation reaction was carried out under the following conditions A or B.

[0182] Condition A: A 100 mL flask was purged with nitrogen, and a solution of Cbz-MeLeu-Ile-OH in IPAC (185.0 mg / mL, 12.14 mL, 2.247 g, 5.72 mmol) and 2,6-lutidine (756 μL, 6.49 mmol) were added. The mixture was cooled to -10 °C with stirring. PivCl (657 μL, 5.34 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. A solution of H-MeLeu-Ile-OtBu in IPAC (137.6 mg / mL, 8.72 mL, 1.20 g, 3.82 mmol) and an aqueous solution of NaCO (1.215 g NaCO, 12.13 mL HO) were added to the reaction mixture, and the mixture was heated to 0 °C and stirred for 5 h. Condition B: COMU (109.0 mg, 0.254 mmol), an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.364 mL, 67.4 mg, 0.172 mmol), an IPAC solution of H-MeLeu-Ile-OtBu (137.6 mg / mL, 0.291 mL, 40.0 mg, 0.127 mmol), and DIPEA (44.3 μL, 0.254 mmol) were added sequentially to a test tube containing a stir bar and stirred. Synthesis of the epimer: The epimer was synthesized according to condition A, except that Cbz-MeLeu-D-allo-Ile-OH was used instead of Cbz-MeLeu-Ile-OH. Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-OtBu: 8.0 min, desired product: 18.3 min, epimer: 18.8 min Mass spectrometry: H-MeLeu-Ile-OtBu: m / z 315.37 ([M+H] + ), desired product: m / z 689.65 ([M+H] + ), epimer: m / z 689.56 ([M+H] + )

[0183]

[0184] As shown in Table 8, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using COMU as a condensing agent (condition B).

[0185] (Example 9) Condensation reaction of Cbz-MeLeu-Ile-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A to C.

[0186] Condition A: A solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration: 116.2 mg / mL, 0.731 mL, 85.0 mg, 0.134 mmol) and 2,6-lutidine (17.4 μL, 0.149 mmol) were added to a test tube containing a stir bar and cooled to -10°C with stirring. PivCl (15.3 μL, 0.124 mmol) was added to the reaction mixture and stirred for 5 hours and 30 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (148.7 mg / mL, 0.336 mL, 50.0 mg, 0.099 mmol) and an aqueous solution of Na2CO3 (31.9 mg Na2CO3, 0.316 mL HO) were added to the reaction mixture, which was then heated to 0 °C and stirred for 16 h. Condition B: A solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (116.2 mg / mL, 0.585 mL, 68.0 mg, 0.107 mmol) and 2,6-lutidine (13.8 μL, 0.119 mmol) were added to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (12.3 μL, 0.100 mmol) was added to the reaction mixture, which was then stirred for 5 hours and 30 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (148.7 mg / mL, 0.269 mL, 40.0 mg, 0.080 mmol) and DIPEA (41.6 μL, 0.239 mmol) were then added to the reaction mixture, which was then warmed to 0°C and stirred for 16 hours. Condition C: COMU (42.6 mg, 0.099 mmol), a solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration: 116.2 mg / mL, 0.366 mL, 42.5 mg, 0.067 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration: 148.7 mg / mL, 0.168 mL, 25.0 mg, 0.050 mmol), and DIPEA (17.3 μL, 0.099 mmol) were added sequentially to a test tube containing a stir bar and stirred.Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.0 min, desired product: 17.8 min, epimer: 18.5 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.36 ([M+H]. + ), desired product: m / z 1117.83 ([M+H] + ), epimer: m / z 1117.88 ([M+H] + )

[0187]

[0188] As shown in Table 9, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system of organic solvent only (condition B) and the condition using COMU as a condensing agent (condition C).

[0189] (Example 10) Condensation reaction of Cbz-MeLeu-Ile-MeLeu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A to C.

[0190] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH (116.2 mg / mL, 0.597 mL, 69.4 mg, 0.110 mmol) and 2,6-lutidine (14.2 μL, 0.122 mmol), followed by stirring and cooling to -10 °C. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, followed by stirring for 7 h. A toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (140.2 mg / mL, 0.214 mL, 30.0 mg, 0.081 mmol) and an aqueous solution of Na2CO3 (25.8 mg Na2CO3, 0.258 mL HO) were added to the reaction mixture, which was then warmed to 0 °C and stirred for 24 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH (116.2 mg / mL, 0.597 mL, 69.4 mg, 0.110 mmol) and 2,6-lutidine (14.1 μL, 0.122 mmol), followed by stirring and cooling to -10 °C. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, followed by stirring for 7 h. A toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (140.2 mg / mL, 0.214 mL, 30.0 mg, 0.081 mmol) and DIPEA (42.4 μL, 0.244 mmol) were added to the reaction mixture, which was then heated to 0 °C and stirred for 24 h. Condition C: COMU (34.8 mg, 0.081 mmol), an IPAC solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH (concentration: 116.2 mg / mL, 0.298 mL, 34.7 mg, 0.055 mmol), a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (concentration: 140.2 mg / mL, 0.107 mL, 15.0 mg, 0.041 mmol), and DIPEA (14.1 μL, 0.081 mmol) were added sequentially to a test tube containing a stir bar and stirred.Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 18.3 min, epimer: 19.4 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.39 ([M+H]. + ), desired product: m / z 1007.04 ([M+Na] + ), epimer: m / z 1006.86 ([M+Na] + )

[0191]

[0192] As shown in Table 10, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were significantly low under the one-layer system of organic solvent only (condition B) and the condition using COMU as a condensing agent (condition C).

[0193] (Example 11) Condensation reaction of Cbz-MeLeu-Ile-OH and H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A to C.

[0194] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.298 mL, 55.1 mg, 0.140 mmol) and 2,6-lutidine (18.2 μL, 0.156 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (16.0 μL, 0.130 mmol) was added to the reaction mixture, and the mixture was stirred for 3.5 hours. A toluene solution of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 (128.7 mg / mL, 0.389 mL, 50.0 mg, 0.104 mmol) and an aqueous solution of Na2CO3 (33.1 mg Na2CO3, 0.331 mL HO) were added to the reaction mixture, and the mixture was heated to 0 °C and stirred for 19 hours. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.298 mL, 55.1 mg, 0.140 mmol) and 2,6-lutidine (18.1 μL, 0.156 mmol), followed by stirring and cooling to -10 °C. PivCl (16.0 μL, 0.130 mmol) was added to the reaction mixture, followed by stirring for 3.5 hours. A toluene solution of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 (128.7 mg / mL, 0.389 mL, 50.0 mg, 0.104 mmol) and DIPEA (54.4 μL, 0.312 mmol) were added to the reaction mixture, which was then heated to 0 °C and stirred for 19 hours. Condition C: COMU (44.7 mg, 0.104 mmol), an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.149 mL, 27.6 mg, 0.070 mmol), a toluene solution of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 (128.7 mg / mL, 0.194 mL, 25.0 mg, 0.052 mmol), and DIPEA (18.1 μL, 0.104 mmol) were added sequentially to a test tube containing a stir bar and stirred. Synthesis of the epimer: The epimer was synthesized according to condition A, except that Cbz-MeLeu-D-allo-Ile-OH was used instead of Cbz-MeLeu-Ile-OH.Measurement method: HPLC Method A Retention time: H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2: 7.6 min, desired product: 16.4 min, epimer: 16.9 min Mass spectrometry: H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 481.43 ([M+H]. + ), desired product: m / z 877.81 ([M+Na] + ), epimer: m / z 877.79 ([M+Na] + )

[0195]

[0196] As shown in Table 11, both the conversion rate and the diastereomeric ratio were high under the two-layer system of organic solvent and water (condition A), whereas either or both of the conversion rate and the diastereomeric ratio were lower than those under condition A under the one-layer system of organic solvent only (condition B) and the condition using COMU as a condensing agent (condition C).

[0197] (Example 12) Evaluation of the influence of base and solvent on mixed acid anhydride formation reaction A condensation reaction was carried out under the conditions shown in Table 12 below.

[0198] A test tube containing a stir bar was charged with a solution of Cbz-MeLeu-Phe-OH in each solvent (concentration: 198.0 mg / mL, 0.300 mL, 59.4 mg, 0.139 mmol) and each base (0.153 mmol), and the mixture was cooled to -20°C while stirring. PivCl (16.5 μL, 0.134 mmol) was added to the reaction mixture, and the mixture was stirred. 5.0 μL of the reaction mixture was sampled over time, dissolved in MeCN (1.0 mL) and azetidine (0.1 mL), and analyzed by HPLC. Measurement method: HPLC Method A. Retention times: Cbz-MeLeu-Phe-OH: 13.1 min, Cbz-MeLeu-Phe-Aze: 13.35 min, epimer: 13.44 min. Mass spectrometry: Cbz-MeLeu-Phe-OH: m / z 427.88 ([M+H] +), Cbz-MeLeu-Phe-Aze: m / z 466.87 ([M+H] + ), epimer: m / z 466.87 ([M+H] + )

[0199]

[0200] As shown in Table 12, it was found that the conversion rate and diastereomeric ratio were high when 2,6-lutidine or diisopropylethylamine was used as the base.

[0201] (Example 12-1) Effect of Solvent on Activation Reaction of Cbz-MeLeu-Ile-OH The activation reaction was carried out under the following conditions.

[0202] Cbz-MeLeu-Ile-OH (53.4 mg, 0.136 mmol), solvent (0.400 mL), and 2,6-lutidine (17.6 μL, 0.151 mmol) were added sequentially to a test tube containing a stir bar and cooled to 0 °C with stirring. PivCl (15.5 μL, 0.126 mmol) was added to the reaction mixture and stirred. 3 μL of the reaction mixture was sampled over time, dissolved in MeCN (1.0 mL) and azetidine (0.1 mL), and analyzed by HPLC for the conversion and diastereomeric ratio of the activation reaction. Measurement method: HPLC Method A Retention time: Cbz-MeLeu-Ile-OH: 13.2 min, Cbz-MeLeu-Ile-Aze: 13.4 min, epimer: 13.8 min Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.79 ([M+H] + ), Cbz-MeLeu-Ile-Aze: m / z 432.90 ([M+H] + ), epimer: m / z 432.84 ([M+H] + ) Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100 Diastereomer ratio = Cbz-MeLeu-Ile-aze / epimer

[0203]

[0204] As shown in Table 12-1, high reactivity and diastereomeric ratio were obtained under the conditions of using ester solvents such as IPAC and aromatic solvents such as toluene.

[0205] (Example 12-2) Effect of base on activation reaction of Cbz-MeLeu-Ile-OH The activation reaction was carried out under the following conditions.

[0206] Cbz-MeLeu-Ile-OH (53.4 mg, 0.136 mmol), toluene (0.400 mL), and each base (0.151 mmol) were sequentially added to a test tube containing a stir bar, and the mixture was cooled to 0°C while stirring. PivCl (15.5 μL, 0.126 mmol) was added to the reaction mixture and stirred. 3 μL of the reaction mixture was sampled over time and dissolved in MeCN (1.0 mL) and azetidine (0.1 mL). The conversion rate and diastereomeric ratio of the activation reaction were analyzed by HPLC. Measurement method: HPLC Method A. Retention times: Cbz-MeLeu-Ile-OH: 13.2 min, Cbz-MeLeu-Ile-Aze: 13.4 min, epimer: 13.8 min. Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.79 ([M+H] + ), Cbz-MeLeu-Ile-Aze: m / z 432.90 ([M+H] + ), epimer: m / z 432.84 ([M+H] + ) Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100 Diastereomer ratio = Cbz-MeLeu-Ile-aze / epimer

[0207]

[0208] As shown in Table 12-2, high reactivity and diastereomeric ratios were obtained under conditions using weak bases such as pyridine and 2,6-lutidine. (Conditions using 2,6-di-tert-butylpyridine resulted in significantly lower reaction conversion, so the diastereomeric ratio is not shown (NA: Not Analyzed).)

[0209] (Example 13) Evaluation of the effect of temperature on mixed acid anhydride formation reaction A condensation reaction was carried out under the conditions shown in Table 13 below.

[0210] A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (concentration: 200.0 mg / mL, 0.200 mL, 40.0 mg, 0.102 mmol) and 2,6-lutidine (11.8 μL, 0.101 mmol), and the mixture was cooled to the temperatures listed in Table 13 while stirring. PivCl (10.4 μL, 0.084 mmol) was added to the reaction mixture, and the mixture was stirred. 5.0 μL of the reaction mixture was sampled over time, dissolved in MeCN (1.0 mL) and azetidine (0.1 mL), and analyzed by HPLC. Measurement method: HPLC Method A Retention time: Cbz-MeLeu-Ile-OH: 12.9 min, Cbz-MeLeu-Ile-Aze: 13.1 min, epimer: 13.5 min Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.74 ([M+H] + ), Cbz-MeLeu-Ile-Aze: m / z 432.95 ([M+H] + ), epimer: m / z 432.90 ([M+H] + )

[0211]

[0212] As shown in Table 13, it was found that the conversion rate and diastereomeric ratio were high at both temperatures of -18°C and -5°C.

[0213] Example 13-1 Reaction Acceleration Effect of Additives on Activation Reaction of Cbz-MeLeu-Ile-OH The activation reaction was carried out under the following conditions.

[0214] A test tube containing a stir bar was charged with an additive, an IPAC solution of Cbz-MeLeu-Ile-OH (concentration: 157.0 mg / mL, 0.200 mL, 31.4 mg, 0.081 mmol), and 2,6-lutidine (10.4 μL, 0.090 mmol), and the mixture was cooled to -10°C while stirring. 2,2-dimethylbutyryl chloride (10.2 μL, 0.075 mmol) was added to the reaction mixture, and the mixture was stirred. 2-3 μL of the reaction mixture was sampled over time and dissolved in MeCN (1.0 mL) and azetidine (0.1 mL). The conversion and diastereomeric ratio of the activation reaction were analyzed by HPLC. Measurement method: HPLC Method A Retention time: Cbz-MeLeu-Ile-OH: 12.8 min, Cbz-MeLeu-Ile-Aze: 13.1 min, epimer: 13.4 min Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.74 ([M+H] + ), Cbz-MeLeu-Ile-Aze: m / z 432.95 ([M+H] + ), epimer: m / z 432.90 ([M+H] + ) Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100 Diastereomer ratio = Cbz-MeLeu-Ile-aze / epimer

[0215]

[0216] As shown in Table 13-1, it was found that the addition of NaOTf, KOTf, and NaI had a significant effect on accelerating the reaction compared to the condition without any additives.

[0217] (Example 14) Evaluation of the effect of solvent on fragment condensation reaction Condensation reactions were carried out under the conditions shown in Table 14 below.

[0218] A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (185.0 mg / mL, 0.231 mL, 42.8 mg, 0.109 mmol) and 2,6-lutidine (14.0 μL, 0.121 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (12.4 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in each solvent (170.0 mg / mL, 0.353 mL, 60.0 mg, 0.081 mmol) and an aqueous solution of Na2CO3 (25.7 mg Na2CO3, 0.257 mL HO) were added to the reaction mixture, and the mixture was heated to 0 °C and stirred for 5 h. Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.60 ([M+H] + ), desired product: m / z 1117.78 ([M+H] + ), epimer: m / z 1117.85 ([M+H] + )

[0219]

[0220] It was found that under the conditions using the solvents shown in Table 14, the conversion rate and diastereomeric ratio were high.

[0221] (Example 15) Evaluation of the scope of acyl halide reagents in fragment condensation reaction Condensation reactions were carried out under the conditions shown in Table 15 below.

[0222] A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (concentration: 185.0 mg / mL, 0.231 mL, 42.8 mg, 0.109 mmol) and 2,6-lutidine (14.0 μL, 0.121 mmol), and cooled to −10°C with stirring. The reaction mixture was added with each acyl halide reagent (pivaloyl chloride (12.4 μL, 0.101 mmol), 1-methylcyclohexanecarbonyl chloride (16.1 μL, 0.101 mmol), 1-adamantanecarbonyl chloride (21.1 mg, 0.101 mmol), and 2-ethylbutyryl chloride (14.0 μL, 0.101 mmol), and stirred for 3 hours. A toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration: 170.0 mg / mL, 0.353 mL, 60.0 mg, 0.081 mmol) and an aqueous solution of Na2CO3 (25.7 mg Na2CO3, 0.257 mL HO) were added to the reaction mixture, and the mixture was heated to 0 °C and stirred for 5 hours. 2,2-Dimethylbutyryl The example using Cbz-MeLeu-Ile-OH was carried out as follows. A test tube containing a stir bar was charged with an IPAC solution (180.0 mg / mL, 0.225 mL, 40.5 mg, 0.103 mmol) and DIPEA (19.9 μL, 0.114 mmol), and the mixture was cooled to -20°C while stirring (hereafter referred to as test tube A). 2,2-Dimethylbutyryl chloride (13.4 μL, 0.098 mmol) was added to the reaction mixture, and the mixture was stirred for 1 hour and 15 minutes. Another test tube was charged with a toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) and an aqueous solution of Na2CO3 (21.4 mg Na2CO3, 0.210 mL). HO) was added and cooled to 0°C (hereinafter referred to as test tube B). The contents of test tube A was added to test tube B, and stirring was continued at 0°C for 6 hours.Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.60 ([M+H]. + ), desired product: m / z 1117.78 ([M+H] + ), epimer: m / z 1117.85 ([M+H] + )

[0223]

[0224] It was found that under the conditions using the acyl halide reagents shown in Table 15, the conversion rate and diastereomeric ratio were high.

[0225] (Example 16) Evaluation of the scope of bases in fragment condensation reaction Condensation reactions were carried out under the conditions shown in Table 16 below.

[0226] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration: 185.0 mg / mL, 0.285 mL, 52.7 mg, 0.134 mmol) and 2,6-lutidine (17.3 μL, 0.149 mmol) were added to a test tube containing a stir bar and cooled to -10°C with stirring. PivCl (15.3 μL, 0.124 mmol) was added to the reaction mixture and stirred for 5 hours. A toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (144.2 mg / mL, 0.347 mL, 50.0 mg, 0.100 mmol), bases (Na2CO3 (31.7 mg), K2CO3 (41.9 mg), Cs2CO3 (97.6 mg), K3PO4 (64.1 mg), NaHCO3 (25.7 mg), Na2HPO4 (42.5 mg), TEA (41.6 μL), pyridine (24.1 μL), 0.298 mmol), and water (0.316 mL) were added to the reaction mixture, which was then heated to 0 °C and stirred for 4 h. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 7.9 min, desired product: 16.3 min, epimer: 16.8 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.36 ([M+H] + ), desired product: m / z 877.71 ([M+H] + ), epimer: m / z 877.73 ([M+H] + )

[0227]

[0228] It was found that under the conditions using the bases shown in Table 16, the conversion rate and diastereomeric ratio were high, and that the conversion rate and diastereomeric ratio were further increased when a strong base was used as the base.

[0229] (Example 17) Condensation reaction of Cbz-Leu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0230] Condition A: Cbz-Leu-Ile-OH (50.8 mg, 0.134 mmol), IPAC (0.3 mL), 2-MeTHF (0.05 mL), and 2,6-lutidine (17.4 μL, 0.149 mmol) were added to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (15.3 μL, 0.124 mmol) was added to the reaction mixture and stirred for 2 h. A 10% aqueous solution of K2CO3 (0.382 mL, 41.2 mg K2CO3) and a toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.250 mL, 50.0 mg, 0.100 mmol) were added sequentially to the reaction mixture. The mixture was then heated to 0 °C and stirred for 6 h 30 min. Condition B: Cbz-Leu-Ile-OH (41.5 mg, 0.110 mmol), MeCN (0.25 mL), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (200.0 mg / mL, 0.250 mL, 50.0 mg, 0.100 mmol), and DIPEA (34.7 μL, 0.199 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (75.6 mg, 0.199 mmol) was added to the reaction mixture, and stirring was continued for 4 h. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.7 min, desired product: 16.3 min, epimer: 16.7 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.79 ([M+H] + ), desired product: m / z 864.05 ([M+H] + ), epimer: m / z 864.22 ([M+H] + )

[0231]

[0232] As shown in Table 17, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0233] (Example 18) Condensation reaction of Cbz-Leu-Ile-OH with H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0234] Condition A: Cbz-Leu-Ile-OH (37.0 mg, 0.098 mmol), IPAC (0.3 mL), and 2,6-lutidine (13.2 μL, 0.114 mmol) were added sequentially to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture and stirred for 2 h 55 min. A 10% aqueous solution of K2CO3 (0.315 mL, 33.9 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture, which was then heated to 0 °C and stirred for 20 h. Condition B: Cbz-Leu-Ile-OH (36.9 mg, 0.097 mmol), MeCN (0.3 mL), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (75.8 mg / mL, 0.400 mL, 30.3 mg, 0.081 mmol), and DIPEA (28.3 μL, 0.162 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and stirring was continued for 3 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 15.5 min, epimer: 16.5 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] +), desired product: m / z 752.96 ([M+Na] + ), epimer: m / z 753.24 ([M+Na] + )

[0235]

[0236] As shown in Table 18, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0237] (Example 19) Condensation reaction of Cbz-Leu-D-allo-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0238] Cbz-Leu-D-allo-Ile-OH (36.9 mg, 0.097 mmol), IPAC (0.3 mL), and 2,6-lutidine (13.2 μL, 0.114 mmol) were added sequentially to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture and stirred for 3.5 hours. 10% aqueous K2CO3 (0.315 mL, 33.9 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture, which was then warmed to 0 °C and stirred for 8 hours. Condition B: Cbz-Leu-D-allo-Ile-OH (36.9 mg, 0.097 mmol), MeCN (0.3 mL), a MeCN solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (75.8 mg / mL, 0.400 mL, 30.3 mg, 0.081 mmol), and DIPEA (28.3 μL, 0.162 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and stirring was continued for 3 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 16.5 min, epimer: 15.5 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), desired product: m / z 685.98 ([M-NMe2] + ), epimer: m / z 685.98 ([M-NMe2] + )

[0239]

[0240] As shown in Table 19, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0241] (Example 20) Condensation reaction of Cbz-D-Leu-Ile-OH with H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0242] Condition A: Cbz-D-Leu-Ile-OH (36.9 mg, 0.097 mmol), IPAC (0.3 mL), and 2,6-lutidine (13.2 μL, 0.114 mmol) were added sequentially to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture and stirred for 3.5 hours. 10% aqueous K2CO3 (0.315 mL, 33.9 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture, and the mixture was heated to 0 °C and stirred for 8 hours. Condition B: Cbz-D-Leu-Ile-OH (36.9 mg, 0.097 mmol), MeCN (0.3 mL), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (75.8 mg / mL, 0.400 mL, 30.3 mg, 0.081 mmol), and DIPEA (28.3 μL, 0.162 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and stirring was continued for 4 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 15.7 min, epimer: 16.2 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] +), desired product: m / z 686.04 ([M-NMe2] + ), epimer: m / z 686.10 ([M-NMe2] + )

[0243]

[0244] As shown in Table 20, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0245] (Example 21) Condensation reaction of Cbz-MeLeu-Thr(OtBu)-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0246] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Thr(OtBu)-OH (147.0 mg / mL, 0.300 mL, 44.1 mg, 0.101 mmol) and 2,6-lutidine (13.2 μL, 0.114 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (11.7 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 3.5 hours. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.280 mL, 28.0 mg, 0.076 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 20 hours. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Thr(OtBu)-OH (147.0 mg / mL, 0.200 mL, 29.4 mg, 0.067 mmol), an MeCN solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (75.8 mg / mL, 0.250 mL, 19.0 mg, 0.050 mmol), and DIPEA (17.5 μL, 0.100 mmol), followed by stirring and cooling to 10 °C. HATU (38.3 mg, 0.100 mmol) was added to the reaction mixture, which was then stirred for 3 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 6.7 min, desired product: 18.7 min, epimer: 19.0 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.77 ([M+H] + ), desired product: m / z 811.10 ([M+Na] + ), epimer: m / z 811.61 ([M+Na] + )

[0247]

[0248] As shown in Table 21, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0249] (Example 22) Condensation reaction of Cbz-MeLeu-Thr(OtBu)-OH and H-MeGly(nPr)-Ile-Pro-OtBu Condensation reaction was carried out under the following conditions A and B.

[0250] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Thr(OtBu)-OH (147.0 mg / mL, 0.300 mL, 44.1 mg, 0.101 mmol) and 2,6-lutidine (13.2 μL, 0.114 mmol), and the mixture was cooled to -10 °C with stirring. 2,2-dimethylbutyryl chloride (13.0 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(nPr)-Ile-Pro-OtBu (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 6 h. Condition B: H-MeGly(nPr)-Ile-Pro-OtBu (31.0 mg, 0.076 mmol), MeCN (0.3 mL), an IPAC solution of Cbz-MeLeu-Thr(OtBu)-OH (147.0 mg / mL, 0.300 mL, 44.1 mg, 0.101 mmol), and DIPEA (26.3 μL, 0.151 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (57.4 mg, 0.151 mmol) was added to the reaction mixture, and the mixture was stirred for 5 h. Measurement method: HPLC Method A Retention time: H-MeGly(nPr)-Ile-Pro-OtBu: 8.6 min, desired product: 19.3 min, epimer: 19.8 min Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.81 ([M+H] + ), desired product: m / z 817.37 ([M+H] + ), epimer: m / z 817.26 ([M+H] + )

[0251]

[0252] As shown in Table 22, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0253] (Example 23) Condensation reaction of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0254] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol) and 2,6-lutidine (15.4 μL, 0.132 mmol), and cooled to −10°C with stirring. PivCl (13.4 μL, 0.109 mmol) was added to the reaction mixture, which was then stirred for 3 hours. To the reaction mixture, 10% aqueous K2CO3 solution (0.370 mL, 39.9 mg K2CO3) and a toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration 200.0 mg / mL, 0.350 mL, 70.0 mg, 0.095 mmol) were added sequentially, and the mixture was heated to 0 °C and stirred for 6 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol), a MeCN solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (170.0 mg / mL, 0.410 mL, 69.7 mg, 0.095 mmol), and DIPEA (33.0 μL, 0.189 mmol), followed by stirring and cooling to 10 °C. HATU (72.0 mg, 0.189 mmol) was added to the reaction mixture, which was then stirred for 3 h. Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.9 min, desired product: 18.9 min, epimer: 19.2 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.18 ([M+H] + ), desired product: m / z 1125.35 ([M-Sar-OtBu] + ), epimer: m / z 1125.29 ([M-Sar-OtBu] + )

[0255]

[0256] As shown in Table 23, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0257] (Example 24) Condensation reaction of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0258] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol) and 2,6-lutidine (15.4 μL, 0.133 mmol), and cooled to −10°C with stirring. PivCl (13.4 μL, 0.109 mmol) was added to the reaction mixture, which was then stirred for 3 hours. To the reaction mixture, 10% aqueous K2CO3 solution (0.370 mL, 39.9 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (concentration: 100.0 mg / mL, 0.350 mL, 35.0 mg, 0.095 mmol) were added sequentially, and the mixture was then heated to 0 °C and stirred for 6 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol), a MeCN solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (75.8 mg / mL, 0.460 mL, 34.9 mg, 0.095 mmol), and DIPEA (33.0 μL, 0.189 mmol), followed by stirring and cooling to 10 °C. HATU (72.0 mg, 0.189 mmol) was added to the reaction mixture, which was then stirred for 3 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 18.2 min, epimer: 18.5 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), desired product: m / z 852.14 ([M-NMe2] + ), epimer: m / z 852.03 ([M-NMe2] + )

[0259]

[0260] As shown in Table 24, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0261] (Example 25) Condensation reaction of Cbz-MeLeu-Cys(SBn)-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0262] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Cys(SBn)-OH (116.0 mg / mL, 0.475 mL, 55.1 mg, 0.107 mmol) and 2,6-lutidine (13.9 μL, 0.119 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (12.2 μL, 0.100 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 6 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-Cys(SBn)-OH (116.0 mg / mL, 0.475 mL, 55.1 mg, 0.107 mmol), a MeCN solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol), and DIPEA (27.7 μL, 0.159 mmol), and the mixture was cooled to 10 °C with stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture and stirred for 6 h. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.5 min, desired product: 18.7 min, epimer: 18.9 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.92 ([M+H] + ), desired product: m / z 958.28 ([M+H] + ), epimer: m / z 958.39 ([M+H] + )

[0263]

[0264] As shown in Table 25, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0265] (Example 26) Condensation reaction of Cbz-MeAla-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0266] Condition A: Cbz-MeAla-Ile-OH (37.5 mg, 0.102 mmol), IPAC (0.300 mL), and 2,6-lutidine (13.2 μL, 0.113 mmol) were added sequentially to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture and stirred for 3 h. 10% aqueous K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.280 mL, 56.0 mg, 0.075 mmol) were added sequentially to the reaction mixture, which was then warmed to 0 °C and stirred for 20 h. Condition B: Cbz-MeAla-Ile-OH (37.5 mg, 0.102 mmol), H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (56.0 mg, 0.075 mmol), MeCN (0.500 mL), and DIPEA (26.3 μL, 0.151 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (57.3 mg, 0.151 mmol) was added to the reaction mixture, and the mixture was stirred for 6 h. Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.9 min, desired product: 17.8 min, epimer: 18.4 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.12 ([M+H] + ), desired product: m / z 931.43 ([M-Sar-OtBu] + ), epimer: m / z 931.49 ([M-Sar-OtBu] + )

[0267]

[0268] As shown in Table 26, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0269] (Example 27) Condensation reaction of Cbz-MeAla-Ile-OH and H-MeGly(nPr)-Ile-Pro-OtBu Condensation reaction was carried out under the following conditions A and B.

[0270] Condition A: Cbz-MeAla-Ile-OH (37.5 mg, 0.102 mmol), IPAC (0.300 mL), and 2,6-lutidine (13.2 μL, 0.113 mmol) were added sequentially to a test tube containing a stir bar and cooled to -10 °C with stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture and stirred for 3 h. 10% aqueous K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(nPr)-Ile-Pro-OtBu (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture, which was then warmed to 0 °C and stirred for 20 h. Condition B: Cbz-MeAla-Ile-OH (37.6 mg, 0.102 mmol), H-MeGly(nPr)-Ile-Pro-OtBu (30.0 mg, 0.075 mmol), MeCN (0.500 mL), and DIPEA (26.3 μL, 0.151 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10°C with stirring. HATU (57.4 mg, 0.151 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours. Measurement method: HPLC Method A. Retention times: H-MeGly(nPr)-Ile-Pro-OtBu: 8.6 min, desired product: 17.1 min, epimer: 17.6 min. Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.75 ([M+H] + ), desired product: m / z 731.24 ([M+H] +), epimer: m / z 731.18 ([M+H] + )

[0271]

[0272] As shown in Table 27, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0273] (Example 28) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0274] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-MeAla-Ile-OH (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-lutidine (13.2 μL, 0.114 mmol), and cooled to −10°C with stirring. PivCl (11.6 μL, 0.095 mmol) was added to the reaction mixture, which was then stirred for 8 hours. To the reaction mixture, 10% aqueous K2CO3 solution (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration 200.0 mg / mL, 0.280 mL, 56.0 mg, 0.076 mmol) were added sequentially, and the mixture was warmed to 0 °C and stirred for 18 h. Condition B: A test tube containing a stir bar was charged with a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (170.0 mg / mL, 0.330 mL, 56.1 mg, 0.076 mmol), a solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol), and DIPEA (26.3 μL, 0.152 mmol), followed by stirring and cooling to 10 °C. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture, which was then stirred for 14 h. Measurement method: HPLC Method A Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.9 min, desired product: 19.1 min, epimer: 19.8 min Mass spectrometry: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.07 ([M+H] + ), desired product: m / z 1058.43 ([M-Sar-OtBu] + ), epimer: m / z 1059.45 ([M-Sar-OtBu] + )

[0275]

[0276] As shown in Table 28, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0277] (Example 29) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeGly(nPr)-Ile-Pro-OtBu Condensation reaction was carried out under the following conditions A and B.

[0278] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-MeAla-Ile-OH (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-lutidine (13.2 μL, 0.114 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (11.6 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(nPr)-Ile-Pro-OtBu (100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 18 h. Condition B: H-MeGly(nPr)-Ile-Pro-OtBu (30.0 mg, 0.076 mmol), an IPAC solution of Cbz-MeLeu-MeAla-Ile-OH (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol), and DIPEA (26.3 μL, 0.152 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture, and the mixture was stirred for 5 h. Measurement method: HPLC Method A Retention time: H-MeGly(nPr)-Ile-Pro-OtBu: 8.5 min, desired product: 18.7 min, epimer: 19.2 min Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.87 ([M+H] +), desired product: m / z 858.24 ([M+H] + ), epimer: m / z 858.18 ([M+H] + )

[0279]

[0280] As shown in Table 29, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0281] (Example 30) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2 Condensation reaction was carried out under the following conditions A and B.

[0282] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-MeAla-Ile-OH (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-lutidine (13.2 μL, 0.114 mmol), and the mixture was cooled to -10 °C with stirring. PivCl (11.6 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (100.0 mg / mL, 0.280 mL, 28.0 mg, 0.076 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 18 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Cbz-MeLeu-MeAla-Ile-OH (146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol), a 2-MeTHF solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 (129.6 mg / mL, 0.220 mL, 28.5 mg, 0.077 mmol), and DIPEA (26.3 μL, 0.152 mmol) in that order. The mixture was cooled to 10 °C with stirring. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture and stirred for 5 h. Measurement method: HPLC Method A Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 6.6 min, desired product: 17.7 min, epimer: 18.9 min Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), desired product: m / z 785.22 ([M-NMe2] + ), epimer: m / z 785.11 ([M-NMe2] + )

[0283]

[0284] As shown in Table 30, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0285] (Example 31) Condensation reaction of Boc-MeLeu-Phe-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0286] Condition A: A test tube containing a stir bar was charged with an IPAC solution of Boc-MeLeu-Phe-OH (134.0 mg / mL, 0.280 mL, 37.5 mg, 0.096 mmol) and 2,6-lutidine (13.0 μL, 0.111 mmol), and the mixture was cooled to -8 °C with stirring. PivCl (10.8 μL, 0.088 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol) were added sequentially to the reaction mixture, and the mixture was then heated to 0 °C and stirred for 3 h. Condition B: A test tube containing a stir bar was charged with an IPAC solution of Boc-MeLeu-Phe-OH (134.0 mg / mL, 0.280 mL, 37.5 mg, 0.096 mmol), a MeCN solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol), and DIPEA (27.7 μL, 0.159 mmol), and the mixture was cooled to 10 °C with stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture and stirred for 5 h. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.5 min, desired product: 18.4 min, epimer: 18.5 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.92 ([M+H] + ), desired product: m / z 878.13 ([M+H] + ), epimer: m / z 878.08 ([M+H] + )

[0287]

[0288] As shown in Table 31, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition using HATU as a condensing agent (condition B).

[0289] (Example 32) Condensation reaction of Fmoc-MeAlGly-Val-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Condensation reaction was carried out under the following conditions A and B.

[0290] Condition A: Fmoc-MeAlGly-Val-OH (52.6 mg, 91.7% content, net 48.4 mg, 0.107 mmol), IPAC (0.300 mL), and 2,6-lutidine (13.9 μL, 0.119 mmol) were added sequentially to a test tube containing a stir bar and cooled to -8 °C with stirring. PivCl (12.2 μL, 0.100 mmol) was added to the reaction mixture and stirred for 3 h. A 10% aqueous solution of K2CO3 (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (concentration 169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol) were added sequentially to the reaction mixture, which was then warmed to 0 °C and stirred for 5 h. Condition B: Fmoc-MeAlGly-Val-OH (52.6 mg, 91.7% content, net 48.4 mg, 0.107 mmol), MeCN (0.300 mL), a MeCN solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol), and DIPEA (27.7 μL, 0.159 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture, and the mixture was stirred for 8 h. Measurement method: HPLC Method A Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.6 min, desired product: 18.6 min, epimer: 18.7 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.81 ([M+H] + ), desired product: m / z 936.10 ([M+H] + ), epimer: m / z 936.39 ([M+H] + )

[0291]

[0292] As shown in Table 32, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0293] (Example 33) Condensation reaction of Fmoc-MeAlGly-Val-OH and H-MeGly(nPr)-Ile-Pro-OtBu Condensation reaction was carried out under the following conditions A and B.

[0294] Condition A: Fmoc-MeAlGly-Val-OH (52.6 mg, 91.7% content, net 48.4 mg, 0.107 mmol), IPAC (0.300 mL), and 2,6-lutidine (13.9 μL, 0.119 mmol) were added sequentially to a test tube containing a stir bar and cooled to -8 °C with stirring. PivCl (12.2 μL, 0.100 mmol) was added to the reaction mixture and stirred for 3 h. 10% aqueous K2CO3 solution (0.300 mL, 32.3 mg K2CO3) and a toluene solution of H-MeGly(nPr)-Ile-Pro-OtBu (concentration 100.0 mg / mL, 0.316 mL, 31.6 mg, 0.080 mmol) were added sequentially to the reaction mixture, which was then warmed to 0 °C and stirred for 5 h. Condition B: Fmoc-MeAlGly-Val-OH (52.6 mg, 91.7% content, net 48.4 mg, 0.107 mmol), H-MeGly(nPr)-Ile-Pro-OtBu (31.6 mg, 0.080 mmol), MeCN (0.500 mL), and DIPEA (27.7 μL, 0.159 mmol) were added sequentially to a test tube containing a stir bar, and the mixture was cooled to 10 °C with stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture, and the mixture was stirred for 8 hours. Measurement method: HPLC Method A Retention time: H-MeGly(nPr)-Ile-Pro-OtBu: 9.2 min, desired product: 19.5 min, epimer: 19.9 min Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.81 ([M+H] +), desired product: m / z 831.51 ([M+H] + ), epimer: m / z 831.16 ([M+H] + )

[0295]

[0296] As shown in Table 33, both the conversion rate and the diastereomeric ratio were high under the two-phase system of organic solvent and water (condition A), whereas the diastereomeric ratio was significantly low under the condition in which HATU was used as a condensing agent (condition B).

[0297] (Example 34) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu The condensation reaction was carried out according to the following procedure.

[0298] A test tube containing a stir bar (hereafter referred to as "test tube A") was charged with an IPAC solution of Cbz-MeLeu-Ile-OH (concentration: 157 mg / mL, 0.400 mL, 62.8 mg, 0.160 mmol) and 2,6-lutidine (20.8 μL, 0.179 mmol), and the reaction mixture was cooled to -10 °C with stirring. PivCl (18.4 μL, 0.149 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. Activation was confirmed by HPLC analysis. 10% aqueous Na2CO3 (0.380 mL) was added, and the reaction mixture was stirred at -5 °C for 2 h and then warmed to 0 °C. A toluene solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (200 mg / mL, 0.300 mL, 60.0 mg, 0.119 mmol) was added to test tube A over 1 hour and 30 minutes using a syringe pump. The reaction mixture was stirred at 0 °C for 17 hours. Conversion: 98.8% Diastereomer ratio: 99.4 / 0.6 Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 0.9% Measurement method: HPLC Method A Retention times: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.6 min, Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 13.7 min, desired product: 17.0 min, epimer: 17.3 min Mass spectrometry: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.35 ([M+H] + ), Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 587.45 ([M+H] + ), desired product: m / z 899.54 ([M+Na] + ), epimer: m / z 899.59 ([M+Na] + )

[0299] (Raw material synthesis 1) Synthesis of Cbz-MeLeu-Ile-OH

[0300] H-Ile-OtBu·HCl (6.04 g, 27.0 mmol) and 2-MeTHF (120 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (60 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ile-OtBu (4.8 g, 25.6 mmol). Cbz-MeLeu-OH (7.39 g, 26.4 mmol), MeCN (50 mL), and NMM (5.6 mL, 51.3 mmol) were added to the concentrate, and the reaction mixture was cooled with stirring. HATU (12.07 g, 31.74 mmol) was added while maintaining the internal temperature below 17 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 1 hour and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. CPME (100 mL), 5% aqueous K2CO3 (50 mL), and NMI (2.0 mL, 25.6 mmol) were added to the reaction mixture and stirred for 1 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed with 2.5% aqueous ammonia three times (50 mL x 3), 5% aqueous NaHSO4 twice (50 mL x 2), and 5% aqueous Na2CO3 once (50 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Ile-OtBu (11.28 g, 25.14 mmol). 2-MeTHF (110 mL) and HMDS (21.0 mL, 100.30 mmol) were added to the resulting concentrate, followed by the dropwise addition of TMSOTf (14.0 mL, 75.23 mmol). The reaction mixture was stirred for 3 hours, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (100 mL) was added dropwise while maintaining the reaction mixture at 28 °C or below. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (250 mL) was added to the resulting aqueous layer, followed by 85% H3PO4 (10 mL). The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (50 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Ile-OH (8.20 g).Yield: 77% (over 2 steps) Purity: 100% Measurement method: HPLC Method A, retention time: 12.7 min Mass spectrometry: m / z 393.55 ([M+H]. + )

[0301] (Synthesis of analytical preparation 1) Synthesis of Cbz-MeLeu-D-allo-Ile-OH

[0302] Cbz-MeLeu-OH (1.00 g, 3.58 mmol) and THF (70 mL) were added to a 300 mL three-neck flask equipped with a stir bar and cooled to 0 °C with stirring. TEA (1.50 mL, 10.74 mmol) and isobutyl chloroformate (0.44 mL, 3.40 mmol) were added sequentially to the reaction mixture and stirred for 30 min. A solution of HD-allo-Ile-OH (0.71 g, 5.37 mmol) in water (70 mL) was added dropwise, and the reaction mixture was stirred for 4.5 h. The reaction mixture was concentrated under reduced pressure, and the resulting concentrate was transferred to a separatory funnel with the addition of 2-MeTHF (150 mL) and 5% aqueous NaHSO4 (50 mL). The aqueous layer was removed, and the resulting organic layer was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-D-allo-Ile-OH (0.71 g). Yield: 51%. HPLC purity: 95.3%. Measurement method: HPLC Method A. Retention time: 13.1 min. Mass spectrometry: m / z 393.55 ([M+H] + )

[0303] (Raw Material Synthesis 2) Synthesis of Cbz-MeLeu-Ala-OH

[0304] H-Ala-OtBu·HCl (3.01 g, 16.57 mmol) and 2-MeTHF (60 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (30 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ala-OtBu (1.22 g, 8.40 mmol). Cbz-MeLeu-OH (2.54 g, 9.09 mmol), MeCN (25 mL), and NMM (1.8 mL, 16.80 mmol) were added to the concentrate and cooled with stirring. HATU (3.89 g, 10.23 mmol) was added to the reaction mixture and stirring continued at room temperature. Stirring was continued for 1 h, and completion of the reaction was confirmed by HPLC analysis. CPME (60 mL), 5% aqueous K2CO3 (20 mL), and NMI (0.65 mL, 8.23 ​​mmol) were added to the reaction mixture and stirring continued for 1 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed three times with 2.5% aqueous ammonia (25 mL x 3), twice with 5% aqueous NaHSO (25 mL x 2), and once with 5% aqueous NaCO (5 mL x 1). The resulting organic layer was dried over anhydrous NaSO. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Ala-OtBu (3.27 g, 8.04 mmol). 2-MeTHF (33 mL) and HMDS (5.1 mL, 24.1 mmol) were added to the resulting concentrate, and TMSOTf (2.2 mL, 12.1 mmol) was added dropwise. Stirring was continued for 2 hours and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO (35 mL) was added dropwise while maintaining the reaction mixture below 19 °C. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (70 mL) was added to the aqueous layer, and 85% H3PO4 (5 mL) was slowly added. The aqueous layer was removed, and the organic layer was washed with 5% NaCl aqueous solution (25 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Ala-OH (2.32 g). Yield: 79% (over 2 steps). Purity: 100%. Measurement method: HPLC Method A. Retention time: 11.2 min. Mass spectrometry: m / z 351.48 ([M+H]+ )

[0305] (Raw Material Synthesis 3) Synthesis of Cbz-MeLeu-Phe-OH

[0306] H-Phe-OtBu·HCl (5.00 g, 19.40 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (50 mL x 2), the organic layer was concentrated under reduced pressure to give H-Phe-OtBu (4.09 g, 18.48 mmol). Cbz-MeLeu-OH (5.71 g, 20.33 mmol), MeCN (30 mL), and NMM (6.0 mL, 55.40 mmol) were added to the concentrate. HATU (9.14 g, 24.03 mmol) was added to the reaction mixture, which was then stirred at room temperature. Stirring was continued for 2 h, and completion of the reaction was confirmed by HPLC analysis. CPME (80 mL), 5% aqueous K2CO3 (40 mL), and NMI (1.47 mL, 18.48 mmol) were added to the reaction mixture, which was then stirred for 30 min. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed three times with 3% aqueous ammonia (40 mL x 3), twice with 5% aqueous NaHSO (40 mL x 2), and once with 5% aqueous NaCO (40 mL x 2). The resulting organic layer was dried over anhydrous NaSO. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Phe-OtBu (9.10 g, 18.86 mmol). 2-MeTHF (91 mL) and HMDS (20.0 mL, 95.42 mmol) were added to the concentrate, followed by the dropwise addition of TMSOTf (14.0 mL, 77.35 mmol). Stirring was continued for 1 hour and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO (50 mL) was added dropwise while maintaining the reaction mixture below 25 °C. The entire contents of the flask were transferred to a separatory funnel, and 2-MeTHF (100 mL) was added. 85% H3PO4 (6 mL) was then slowly added. The aqueous layer was removed, and the organic layer was washed with 5% NaCl aqueous solution (50 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Phe-OH (6.93 g). Yield: 84% (over 2 steps). Purity: 100%. Measurement method: HPLC Method A. Retention time: 13.1 min. Mass spectrometry: m / z 427.82 ([M+H] + )

[0307] (Raw Material Synthesis 4) Synthesis of Cbz-MeLeu-Ser(OtBu)-OH

[0308] H-Ser(OtBu)-OtBu·HCl (3.76 g, 14.82 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (50 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ser(OtBu)-OtBu (2.71 g, 12.47 mmol). Cbz-MeLeu-OH (3.66 g, 13.10 mmol), MeCN (30 mL), and NMM (4.0 mL, 36.38 mmol) were added to the concentrate and cooled with stirring. HATU (6.14 g, 16.15 mmol) was added while maintaining the reaction temperature below 17 °C, and stirring was continued at room temperature. Stirring was continued for 1 h, and completion of the reaction was confirmed by HPLC analysis. CPME (100 mL), 5% aqueous K2CO3 (50 mL), and NMI (1.0 mL, 12.54 mmol) were added to the reaction mixture, and stirring was continued for 2 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed with 2.5% aqueous ammonia three times (50 mL x 3), 5% aqueous NaHSO4 (50 mL x 2), and 5% aqueous Na2CO3 (50 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Ser(OtBu)-OtBu (5.90 g, 12.54 mmol). 2-MeTHF (60 mL) and HMDS (5.3 mL, 25.07 mmol) were added to the resulting concentrate, and TMSOTf (2.5 mL, 13.79 mmol) was added dropwise. Stirring was continued for 4 hours, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (50 mL) was added dropwise while maintaining the reaction mixture below 18 °C. The entire contents of the flask were transferred to a separatory funnel, and 85% H3PO4 (6 mL) was slowly added. The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (50 mL). The resulting organic layer was concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Ser(OtBu)-OH (4.30 g).Yield: 69% (over 2 steps) Purity: 100% Assay: HPLC Method A, Retention time: 12.8 min Mass Spectrometry: m / z 423.59 ([M+H]. + )

[0309] (Synthesis of analytical standard 2) Synthesis of Cbz-MeLeu-D-Ser(OtBu)-OH

[0310] HD-Ser(OtBu)-OtBu·HCl (380 mg, 1.50 mmol) and 2-MeTHF (25 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (10 mL x 2), the organic layer was concentrated under reduced pressure to give HD-Ser(OtBu)-OtBu (325 mg, 1.50 mmol). Cbz-MeLeu-OH (463 mg, 1.66 mmol), MeCN (5.0 mL), and NMM (0.33 mL, 2.99 mmol) were added to the concentrate. HATU (854 mg, 2.25 mmol) was added to the reaction mixture, which was then stirred at room temperature. Stirring was continued for 2 hours and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. CPME (20 mL), 5% aqueous K2CO3 (10 mL), and NMI (120 μL, 1.50 mmol) were added to the reaction mixture, which was then stirred for 1 hour. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed three times with 2.5% aqueous ammonia (10 mL x 3), twice with 5% aqueous NaHSO (10 mL x 2), and once with 5% aqueous NaCO (10 mL x 1). The resulting organic layer was dried over anhydrous NaSO. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-d-Ser(OtBu)-OtBu (0.690 g, 1.44 mmol). 2-MeTHF (7.0 mL) and HMDS (0.6 mL, 2.88 mmol) were added to the concentrate, followed by the dropwise addition of TMSOTf (300 μL, 1.66 mmol). Stirring was continued for 4 hours, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO (6 mL) was added dropwise while maintaining the reaction mixture below 14 °C. The entire contents of the flask were transferred to a separatory funnel, and 85% H3PO4 (5 mL) was slowly added. The aqueous layer was removed, and the organic layer was washed with 5% NaCl aqueous solution and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-D-Ser(OtBu)-OH (470 mg). Yield: 74% (over 2 steps). Purity: 100%. Measurement method: HPLC Method A. Retention time: 13.6 min. Mass spectrometry: m / z 423.59 ([M+H] + )

[0311] (Raw Material Synthesis 5) Synthesis of Cbz-MeLeu-Ile-MeLeu-Ile-OH

[0312] Cbz-MeLeu-Ile-MeLeu-Ile-OtBu (2.22 g, 3.22 mmol), synthesized by the method described in Example 8, was added to 22.2 mL of 2-MeTHF and HMDS (3.38 mL, 16.11 mmol). TMSOTf (2.33 mL, 12.89 mmol) was added dropwise to the reaction mixture, and after stirring for 5 hours, the reaction was confirmed to be complete by HPLC analysis. The flask was cooled, and 5% aqueous Na2CO3 solution (20 mL) was added dropwise while maintaining the reaction temperature below 53 °C. The entire contents of the flask were transferred to a separatory funnel. The aqueous layer was drained, and the organic layer was washed once with 5% aqueous Na2CO3 solution (20 mL). The combined aqueous layer was slowly added to 2-MeTHF (30 mL) and 85% H3PO4 (2 mL). The aqueous layer was removed, and the combined organic layer was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Ile-MeLeu-Ile-OH (708 mg). Yield: 35%. HPLC purity: 99.6%. Measurement method: HPLC Method A. Retention time: 15.2 min. Mass spectrometry: m / z 655.52 ([M+Na] + )

[0313] (Raw Material Synthesis 6) Synthesis of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0314] It was synthesized according to the method described in WO 2023 / 127869.

[0315] (Raw Material Synthesis 7) Synthesis of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0316] The L-tartrate of the title compound obtained by the method described in WO 2023 / 127869 was desalted and synthesized.

[0317] (Raw Material Synthesis 8) Synthesis of H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0318] The hydrochloride salt of the title compound obtained by the method described in WO 2023 / 127869 was desalted and synthesized.

[0319] (Raw Material Synthesis 9) Synthesis of H-MeLeu-Ile-OtBu

[0320] Cbz-MeLeu-Ile-OtBu (4.00 g, 8.92 mmol), synthesized by the method described in Raw Material Synthesis 1, was dissolved in 2-MeTHF (24 mL) and added to a pressure reactor. 10% Pd / C (0.522 g, 0.223 mmol Pd metal basis) was added, and the mixture was purged with nitrogen and hydrogen. The mixture was stirred under hydrogen pressure (0.3 atm) for 1 hour and 30 minutes. The reaction mixture was filtered, and the Pd / C was washed with 2-MeTHF (20 mL). The filtrate was concentrated under reduced pressure, and the resulting concentrate was purified by silica gel column chromatography to give H-MeLeu-Ile-OtBu (1.66 g). Yield: 59%. HPLC Purity: 98.8%. Measurement Method: HPLC Method A. Retention Time: 8.0 min. Mass Spectrometry: m / z 315.38 ([M+H] + )

[0321] (Raw Material Synthesis 10) Synthesis of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0322] It was synthesized according to the method described in WO 2023 / 127869.

[0323] (Raw Material Synthesis 11) Synthesis of Cbz-Leu-Ile-OH

[0324] H-Ile-OtBu·HCl (2.00 g, 8.94 mmol) and 2-MeTHF (50 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (25 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ile-OtBu (1.58 g, 8.44 mmol). Cbz-Leu-OH (2.48 g, 9.35 mmol), MeCN (16 mL), and NMM (1.9 mL, 16.87 mmol) were added to the resulting concentrate, and the reaction mixture was cooled with stirring. HATU (3.87 g, 10.18 mmol) was added while maintaining the internal temperature of the reaction mixture below 11 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 2 h, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (25 mL), 5% aqueous K2CO3 (20 mL), and NMI (0.67 mL, 8.44 mmol) were added to the reaction mixture and stirred for 2 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed with 2.5% aqueous ammonia three times (25 mL x 3), 5% aqueous NaHSO4 twice (25 mL x 2), and 5% aqueous Na2CO3 once (25 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-Leu-Ile-OtBu (3.55 g, 8.17 mmol). 2-MeTHF (36 mL) and HMDS (7.0 mL, 32.7 mmol) were added to the resulting concentrate, followed by the dropwise addition of TMSOTf (4.4 mL, 24.5 mmol). The reaction mixture was stirred for 2 hours and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (36 mL) was added dropwise while maintaining the reaction mixture at 25°C or below. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (100 mL) was added to the resulting aqueous layer, followed by 85% H3PO4 (4.5 mL). The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (36 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-Leu-Ile-OH (2.00 g).Yield: 59% (over 2 steps) Purity: 98.6% Measurement method: HPLC Method A, retention time: 11.8 min Mass spectrometry: m / z 379.77 ([M+H]. + )

[0325] (Raw Material Synthesis 12) Synthesis of Cbz-Leu-D-allo-Ile-OH

[0326] Cbz-Leu-OH (1.50 g, 5.65 mmol) and THF (105 mL) were added to a 300 mL three-neck flask equipped with a stir bar and cooled to -3 °C with stirring. TEA (2.4 mL, 17.0 mmol) and isobutyl chloroformate (0.70 mL, 5.37 mmol) were added sequentially to the reaction mixture and stirred for 30 min. A solution of HD-allo-Ile-OH (1.11 g, 8.48 mmol) in water (105 mL) was added dropwise, and the reaction mixture was stirred for 2 h. The reaction mixture was concentrated under reduced pressure, and the resulting concentrate was transferred to a separatory funnel with the addition of 2-MeTHF (225 mL) and 5% aqueous NaHSO4 (75 mL). The aqueous layer was removed, and the resulting organic layer was concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-Leu-D-allo-Ile-OH (0.645 g). Yield: 30% HPLC purity: 100.0% Measurement method: HPLC Method A, retention time: 12.2 min Mass spectrometry: m / z 379.72 ([M+H] + )

[0327] (Raw Material Synthesis 13) Synthesis of Cbz-D-Leu-Ile-OH

[0328] H-Ile-OtBu·HCl (0.65 g, 2.91 mmol) and 2-MeTHF (50 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (12 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ile-OtBu (0.55 g, 2.90 mmol). Cbz-D-Leu-OH (0.86 g, 3.2 mmol), MeCN (5.5 mL), and NMM (0.65 mL, 5.9 mmol) were added to the resulting concentrate, and the reaction mixture was cooled with stirring. COMU (1.5 g, 3.5 mmol) was added while maintaining the internal temperature below 11 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 2 h, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (10 mL), 5% aqueous K2CO3 (10 mL), and NMI (0.23 mL, 2.9 mmol) were added to the reaction mixture and stirred for 1 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed once with 5% aqueous Na2CO3 (12 mL x 1), three times with 2.5% aqueous ammonia (12 mL x 3), two times with 5% aqueous NaHSO4 (12 mL x 2), and once with 5% aqueous Na2CO3 (12 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-D-Leu-Ile-OtBu (1.17 g, 2.69 mmol). To the resulting concentrate, 2-MeTHF (12 mL) and HMDS (2.3 mL, 10.75 mmol) were added, and TMSOTf (1.5 mL, 8.06 mmol) was added dropwise. The reaction mixture was stirred for 3 h, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (12 mL) was added dropwise while maintaining the reaction temperature below 24 °C. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (20 mL) and 85% H3PO4 (1.3 mL) were added. The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (12 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-D-Leu-Ile-OH (0.57 g, 1.51 mmol).Yield: 52% (over 2 steps) Purity: 99.3% Assay: HPLC Method A, Hold time: 12.0 min Mass Spectrometry: m / z 379.66 ([M+H]. + )

[0329] (Synthesis of raw materials 14) Synthesis of Cbz-MeLeu-Thr(OtBu)-OH

[0330] A flask containing a stir bar was charged with Fmoc-Thr(OtBu)-OH (2.00 g, 5.03 mmol), cyclohexane (16 mL), and DCM (6 mL). tert-Butyl 2,2,2-trichloroacetimidate (1.8 mL, 10.10 mmol) was added to the flask. While cooling the flask in an ice bath, boron trifluoride diethyl etherate (0.06 mL, 0.50 mmol) was added dropwise, and the mixture was stirred at room temperature for 1 hour and 15 minutes. After confirming the completion of the reaction by HPLC analysis, the reaction mixture was filtered, and the filtered solid was washed with cyclohexane. The filtrate and washings were combined and washed five times with 10% aqueous citric acid (16 mL x 5). The resulting organic layer was washed twice with 5% aqueous Na2CO3 (16 mL x 2). The resulting organic layer was concentrated under reduced pressure to give Fmoc-Thr(OtBu)-OtBu. MeCN (18 mL) and DBU (0.75 mL, 5.03 mmol) were added to the flask and stirred for 30 minutes. After confirming the completion of the reaction by HPLC analysis, TEA (2.8 mL, 20.10 mmol) and water (0.91 mL, 50.3 mmol) were added to the reaction mixture. Sodium bisulfite (1.31 g, 12.60 mmol) was added to the flask while cooling it in an ice bath, and the mixture was stirred at room temperature for 1 hour. MTBE (57 mL) and 10% aqueous ammonia (32 mL) were added to the reaction mixture, and the entire contents of the flask were transferred to a separatory funnel. The aqueous layer was removed, and the resulting organic layer was washed three times with 10% aqueous ammonia (32 mL x 3) and once with 5% aqueous NaCl (32 mL x 1). The resulting organic layer was concentrated under reduced pressure to give H-Thr(OtBu)-OtBu. To the flask were added MeCN (14 mL), Cbz-MeLeu-OH (1.54 g, 5.53 mmol), and NMM (1.7 mL, 15.1 mmol). While cooling the flask in an ice bath, COMU (2.80 g, 6.53 mmol) was added to the reaction mixture, and stirring was continued at room temperature for 2 hours. After confirming the completion of the reaction by HPLC analysis, 2-MeTHF (34 mL), 5% aqueous K2CO3 (17 mL), and NMI (0.40 mL, 5.03 mmol) were added to the reaction mixture, and stirring was continued for 30 minutes. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed.The resulting organic layer was washed once with 5% K2CO3 aqueous solution (27 mL x 1), three times with 2.5% ammonia solution (27 mL x 3), twice with 5% NaHSO4 aqueous solution (27 mL x 2), once with 5% K2CO3 aqueous solution (27 mL x 1), and once with 5% NaCl aqueous solution (27 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Thr(OtBu)-OtBu (2.35 g, 4.76 mmol). 2-MeTHF (23 mL) and HMDS (4.0 mL, 19.00 mmol) were added to the flask, followed by the dropwise addition of TMSOTf (2.6 mL, 14.20 mmol). After stirring for 4 h, the flask was cooled and 5% NaHCO3 aqueous solution (29 mL) was added dropwise while maintaining the reaction mixture below 24 °C. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (56 mL) was added to the resulting aqueous layer, and 85% H3PO4 (3.5 mL) was slowly added. The aqueous layer was removed, and the resulting organic layer was washed with 5% aqueous NaCl (29 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Thr(OtBu)-OH (0.53 g, 1.20 mmol). Yield: 24% (over 4 steps starting from Fmoc-Thr(OtBu)-OH). Purity: 99.5%. Measurement method: HPLC Method A. Retention time: 14.7 min. Mass spectrometry: m / z 437.74 ([M+H]). + )

[0331] (Raw Material Synthesis 15) Synthesis of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH

[0332] Cbz-Hph(3,5-F2-4-CF3)-OH·Cy2NH (5.00 g, 8.35 mmol) and 2-MeTHF (75 mL) were added to a separatory funnel. After washing twice with 5% aqueous H2SO4 (25 mL x 2), the organic layer was concentrated under reduced pressure to give Cbz-Hph(3,5-F2-4-CF3)-OH. Cyclohexane (40 mL) and DCM (15 mL) were added to the concentrate. tert-Butyl 2,2,2-trichloroacetimidate (3.0 mL, 16.7 mmol) was added dropwise to the flask while cooling in an ice bath. The mixture was then stirred at room temperature for 2.5 hours. After confirming the completion of the reaction by HPLC analysis, the reaction mixture was filtered, and the filtered solid was washed with cyclohexane. The filtrate and washings were combined and washed five times with 10% aqueous citric acid (20 mL x 5). The resulting organic layer was washed twice with 5% aqueous Na2CO3 (20 mL x 2). The resulting organic layer was concentrated under reduced pressure to give Cbz-Hph(3,5-F2-4-CF3)-OtBu (3.93 g, 8.30 mmol). 5% Pd / C (50% wet, 1.77 g, 0.42 mmol on Pd metal basis) was added to the flask, and the atmosphere was purged with nitrogen and hydrogen. The mixture was stirred under a hydrogen atmosphere (1 atm) for 2 hours and 30 minutes. The reaction mixture was filtered, and the Pd / C was washed with 2-MeTHF. The filtrate and washings were combined and concentrated under reduced pressure to give H-Hph(3,5-F2-4-CF3)-OtBu (2.60 g, 7.66 mmol). Cbz-MeLeu-OH (2.35 g, 8.43 mmol), MeCN (26 mL), and NMM (1.7 mL, 15.3 mmol) were added to the resulting concentrate, and the reaction mixture was cooled with stirring. COMU (3.94 g, 9.20 mmol) was added while maintaining the internal temperature of the reaction mixture below 7°C, and stirring was continued at room temperature. The reaction mixture was stirred for 2 hours, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (40 mL), 5% aqueous K2CO3 solution (20 mL), and NMM (0.61 mL, 7.66 mmol) were added to the reaction mixture, and stirring was continued for 30 minutes. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed.The resulting organic layer was washed once with 5% aqueous Na2CO3 (20 mL x 1), three times with 2.5% aqueous ammonia (20 mL x 3), twice with 5% aqueous NaHSO4 (20 mL x 2), and once with 5% aqueous Na2CO3 (20 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OtBu (4.26 g, 7.09 mmol). 2-MeTHF (44 mL) and HMDS (6.0 mL, 28.4 mmol) were added to the flask, followed by the dropwise addition of TMSOTf (3.8 mL, 21.3 mmol). After stirring for 2 h, the flask was cooled and 5% aqueous NaHCO3 (40 mL) was added dropwise while maintaining the reaction mixture below 22 °C. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The resulting organic layer was washed with 5% aqueous NaHSO4 (40 mL x 1), and the aqueous layer was removed. The resulting organic layer was washed with 5% aqueous NaCl (30 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (2.64 g, 4.85 mmol). Yield: 58% (over 4 steps starting from Cbz-Hph(3,5-F2-4-CF3)-OH·Cy2NH Purity: 100.0% Measurement method: HPLC Method A, Retention time: 15.2 min Mass spectrometry: m / z 545.70 ([M+H]). + )

[0333] (Raw Material Synthesis 16) Synthesis of Cbz-MeLeu-Cys(SBn)-OH

[0334] H-Cys(SBn)-OMe·HCl (1.97 g, 7.53 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (40 mL x 2), the organic layer was concentrated under reduced pressure to give H-Cys(SBn)-OMe (1.49 g, 6.61 mmol). Cbz-MeLeu-OH (2.04 g, 7.27 mmol), MeCN (15 mL), and NMM (1.5 mL, 13.2 mmol) were added to the resulting concentrate, and the reaction mixture was cooled with stirring. COMU (3.40 g, 7.94 mmol) was added while maintaining the internal temperature of the reaction mixture below 11 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 2 h, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (45 mL), 5% aqueous K2CO3 (23 mL), and NMI (0.52 mL, 6.61 mmol) were added to the reaction mixture and stirred for 1 h. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed once with 5% aqueous K2CO3 (38 mL x 1), three times with 2.5% aqueous ammonia (38 mL x 3), twice with 5% aqueous NaHSO4 (38 mL x 2), once with 5% aqueous K2CO3 (38 mL x 1), and once with 5% aqueous NaCl (38 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeLeu-Cys(SBn)-OMe (3.30 g, 6.78 mmol). THF (30 mL) was added to the concentrate, and the resulting solution was cooled in an ice bath. While maintaining the internal temperature of the mixture below 2°C, 2M aqueous NaOH (5.0 mL) was added dropwise. The reaction mixture was stirred for 2 hours, and completion of the reaction was confirmed by HPLC analysis. The contents of the flask were concentrated under reduced pressure, and 2-MeTHF (60 mL) was added to the concentrate. 5% aqueous NaHSO4 (30 mL) was added to the resulting solution, which was then transferred to a separatory funnel and the aqueous layer was removed. The resulting organic layer was washed with 5% aqueous NaCl (45 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-Cys(SBn)-OH (0.693 g, 1.467 mmol).Yield: 22% (over 2 steps) Purity: 99.2% Assay: HPLC Method A, Hold time: 15.5 min Mass Spectrometry: m / z 473.71 ([M+H]. + )

[0335] (Synthesis of raw materials 17) Synthesis of Cbz-MeAla-Ile-OH

[0336] H-Ile-OtBu·HCl (5.05 g, 22.6 mmol) and 2-MeTHF (250 mL) were added to a separatory funnel. After washing twice with 5% aqueous Na2CO3 (100 mL x 2), the organic layer was concentrated under reduced pressure to give H-Ile-OtBu (4.13 g, 22.1 mmol). Cbz-MeAla-OH (5.76 g, 24.3 mmol), MeCN (41 mL), and NMM (4.8 mL, 44.1 mmol) were added to the concentrate, and the reaction mixture was cooled with stirring. COMU (11.3 g, 26.5 mmol) was added while maintaining the internal temperature below 11 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 3 h, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (83 mL), 5% aqueous K2CO3 (83 mL), and NMI (1.7 mL, 22.1 mmol) were added to the reaction mixture and stirred for 30 min. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed once with 5% aqueous K2CO3 (83 mL x 1), three times with 2.5% aqueous ammonia (83 mL x 3), two times with 5% aqueous NaHSO4 (83 mL x 2), and once with 5% aqueous K2CO3 (83 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Cbz-MeAla-Ile-OtBu (8.82 g, 21.7 mmol). The resulting concentrate was dissolved in 2-MeTHF (80 mL), and the resulting solution was divided into three portions. To one of the three aliquots was added HMDS (6.0 mL, 28.9 mmol), followed by the dropwise addition of TMSOTf (4.0 mL, 21.7 mmol). The reaction mixture was stirred for 2 hours, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (36 mL) was added dropwise while maintaining the reaction mixture below 24 °C. The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (72 mL) was added to the resulting aqueous layer, followed by the addition of 85% H3PO4 (5.2 mL). The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (36 mL) and concentrated under reduced pressure.The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeAla-Ile-OH (2.20 g, 6.28 mmol). Yield: 85% (over 2 steps). Purity: 99.8%. Measurement method: HPLC Method A. Retention time: 10.7 min. Mass spectrometry: m / z 351.73 ([M+H]). + )

[0337] (Raw Material Synthesis 18) Synthesis of Cbz-MeLeu-MeAla-Ile-OH

[0338] To one of the three aliquots of Cbz-MeAla-Ile-OtBu in 2-MeTHF (Synthesis 17), 5% Pd / C (50% wet, 1.53 g, 0.36 mmol on Pd metal basis) was added. The mixture was purged with nitrogen and hydrogen and stirred under a hydrogen atmosphere (1 atm) for 2 hours. The reaction mixture was filtered, and the Pd / C was washed with 2-MeTHF. The filtrate and washings were combined and concentrated under reduced pressure to give H-MeAla-Ile-OtBu (1.95 g, 7.17 mmol). To the resulting concentrate, Cbz-MeLeu-OH (2.20 g, 7.87 mmol), MeCN (20 mL), and NMM (1.6 mL, 14.3 mmol) were added, and the reaction mixture was cooled with stirring. COMU (3.69 g, 8.59 mmol) was added while maintaining the internal temperature of the reaction mixture below 5 °C, and stirring was continued at room temperature. The reaction mixture was stirred for 1 hour and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. 2.5% aqueous ammonia (60 mL), 2-MeTHF (60 mL), 5% aqueous K2CO3 (30 mL), and NMI (0.57 mL, 7.16 mmol) were added to the reaction mixture, and stirring was continued for 30 minutes. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed once with 5% aqueous K2CO3 (50 mL x 1), three times with 2.5% aqueous ammonia (50 mL x 3), two times with 5% aqueous NaHSO4 (50 mL x 2), one time with 5% aqueous K2CO3 (50 mL x 1), and one time with 5% aqueous NaCl (50 mL x 1). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting concentrate was purified by silica gel chromatography to give Cbz-MeLeu-MeAla-Ile-OtBu (3.45 g, 6.46 mmol). To the resulting Cbz-MeLeu-MeAla-Ile-OtBu, MeTHF (35 mL) and HMDS (5.4 mL, 25.9 mmol) were added, and TMSOTf (3.6 mL, 19.4 mmol) was added dropwise. The reaction mixture was stirred for 3 hours and 30 minutes, and completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and 5% aqueous NaHCO3 (41 mL) was added dropwise while maintaining the reaction temperature below 24 °C.The entire contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (83 mL) and 85% H3PO4 (4.8 mL) were added to the resulting aqueous layer. The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl (41 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Cbz-MeLeu-MeAla-Ile-OH (1.79 g, 3.75 mmol). Yield: 51% (over 4 steps). Purity: 100.0%. Measurement method: HPLC Method A. Retention time: 14.03 min. Mass spectrometry: m / z 479.07 ([M+H]). + )

[0339] (Raw Material Synthesis 19) Synthesis of Boc-MeLeu-Phe-OH

[0340] H-Phe-OMe·HCl (2.00 g, 9.27 mmol), Boc-MeLeu-OH (2.50 g, 10.2 mmol), MeCN (20 mL), and NMM (4.1 mL, 37.1 mmol) were added to the flask sequentially, and the reaction mixture was cooled with stirring. COMU (4.77 g, 11.1 mmol) was added while maintaining the internal temperature of the reaction mixture below 5°C, and stirring was continued at room temperature. The reaction mixture was stirred for 2 hours, and completion of the reaction was confirmed by HPLC analysis. 2-MeTHF (60 mL), 5% aqueous K2CO3 solution (30 mL), and NMM (0.73 mL, 9.27 mmol) were added to the reaction mixture, and stirring was continued for 1 hour. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The organic layer was washed once with 5% aqueous K2CO3 (50 mL x 1), three times with 2.5% aqueous ammonia (50 mL x 3), twice with 5% aqueous NaHSO4 (50 mL x 2), once with 5% aqueous K2CO3 (50 mL x 1), and once with 5% aqueous NaCl (50 mL). The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Boc-MeLeu-Phe-OMe (3.93 g, 9.67 mmol). THF (38 mL) was added to the concentrate, and the resulting solution was cooled in an ice bath. 2M aqueous NaOH (7.0 mL) was added dropwise while maintaining the internal temperature below 2 °C. The reaction mixture was stirred for 3 h, and completion of the reaction was confirmed by HPLC analysis. The contents of the flask were concentrated under reduced pressure, and 2-MeTHF (75 mL) was added to the concentrate. The resulting solution was added with 5% aqueous NaHSO4 (38 mL), transferred to a separatory funnel, and the aqueous layer was removed. The resulting organic layer was washed with 5% aqueous NaCl (57 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Boc-MeLeu-Phe-OH (3.13 g, 7.97 mmol). Yield: 86% (over 2 steps). Purity: 99.6%. Measurement method: HPLC Method A. Retention time: 14.5 min. Mass spectrometry: m / z 393.74 ([M+H] + )

[0341] (Material Synthesis 20) Synthesis of Fmoc-MeAlGly-Val-OH

[0342] A flask containing a stir bar was charged with Cbz-Val-OH (3.02 g, 11.9 mmol), cyclohexane (24 mL), and DCM (9 mL). tert-Butyl 2,2,2-trichloroacetimidate (4.3 mL, 23.9 mmol) was added to the flask. Boron trifluoride diethyl etherate (0.15 mL, 1.2 mmol) was added dropwise while the flask was cooled in an ice bath, and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction by HPLC analysis, the reaction mixture was filtered, and the filtered solid was washed with cyclohexane. The filtrate and washings were combined and washed five times with 10% aqueous citric acid (24 mL x 5). The resulting organic layer was washed twice with 5% aqueous Na2CO3 (24 mL x 2). The resulting organic layer was concentrated under reduced pressure to give Cbz-Val-OtBu (3.67 g, 11.9 mmol). The resulting concentrate was added with 2-MeTHF (37 mL) and 5% Pd / C (50% wet, 2.54 g, 0.60 mmol on Pd metal basis). The mixture was purged with nitrogen and hydrogen and stirred under a hydrogen atmosphere (1 atm) for 2 hours. The reaction mixture was filtered, and the Pd / C was washed with 2-MeTHF. The filtrate and washings were combined and concentrated under reduced pressure to give H-Val-OtBu (2.06 g, 11.9 mmol). To the same flask were added MeCN (21 mL), Fmoc-MeAlgly-OH (4.63 g, 13.1 mmol), and NMM (3.9 mL, 35.8 mmol). While cooling the flask in an ice bath, COMU (6.14 g, 14.3 mmol) was added to the reaction mixture, and stirring was continued at room temperature for 2 hours and 30 minutes. After confirming the completion of the reaction by HPLC analysis, 2-MeTHF (60 mL) and 5% aqueous K2CO3 (30 mL) were added to the reaction mixture and stirred for 30 minutes. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was removed. The resulting organic layer was washed four times with 5% aqueous K2CO3 (50 mL x 4), two times with 5% aqueous NaHSO4 (50 mL x 2), one time with 5% aqueous K2CO3 (50 mL x 1), and one time with 5% aqueous NaCl (50 mL x 1).The resulting organic layer was dried over anhydrous Na2SO4. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to give Fmoc-MeAlGly-Val-OtBu (5.69 g, 11.2 mmol). To the flask, 2-MeTHF (57 mL) and HMDS (9.4 mL, 44.9 mmol) were added, followed by the dropwise addition of TMSOTf (6.2 mL, 33.7 mmol). After stirring for 2 h, the flask was cooled, and 2-MeTHF (57 mL) was added, followed by the dropwise addition of 5% aqueous NaHCO3 (68 mL) while maintaining the reaction mixture below 24 °C. The entire contents of the flask were transferred to a separatory funnel, and the aqueous layer was separated. The resulting organic layer was washed with 5% aqueous NaHCO3 (20 mL), and the aqueous layer was separated. The two aqueous layers were combined, 2-MeTHF (114 mL) was added, and 85% H3PO4 (8.2 mL) was slowly added. The aqueous layer was removed, and the resulting organic layer was washed with 5% aqueous NaCl (68 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to give Fmoc-MeAlGly-Val-OH (4.37 g, 9.70 mmol). Yield: 82% (over 4 steps). Purity: 99.6%. Measurement method: HPLC Method A. Retention time: 15.0 min. Mass spectrometry: m / z 451.76 ([M+H]). + )

[0343] (Material Synthesis 21) Synthesis of H-MeGly(nPr)-Ile-Pro-OtBu

[0344] Step 21-1: Synthesis of Compound 21-1: tert-butyl (2S)-1-[(2S,3S)-2-(benzyloxycarbonylamino)-3-methyl-pentanoyl]pyrrolidine-2-carboxylic acid

[0345] In a nitrogen-purged reactor, tert-butyl(2S)-pyrrolidine-2-carboxylate (18.8 g), (2S,3S)-2-(benzyloxycarbonylamino)-3-methyl-pentanoic acid (20.0 g), and DMF (140 mL) were added at room temperature and stirred. After confirming complete dissolution, the mixture was cooled to 0°C and DIPEA (52.7 mL) was added. A 50 wt.% propylphosphonic anhydride ethyl acetate solution (58.3 mL) was added to the mixture over 20 minutes at 0°C and stirred at 0°C for 1.5 hours. Water (100 mL) and ethyl acetate (200 mL) were added to the mixture in that order. Aqueous layer 1 and organic layer 1 were separated by liquid-liquid separation, and aqueous layer 1 was set aside. Organic layer 1 was washed with 5% aqueous potassium hydrogen sulfate (100 mL), 5% aqueous sodium carbonate (100 mL), and 10% brine (100 mL). Water (100 mL) and ethyl acetate (200 mL) were added to the separated aqueous layer 1, and the mixture was stirred. Then, the organic layer 2 was obtained by liquid-liquid separation. The organic layers 1 and 2 were combined and concentrated under reduced pressure to obtain compound 21-1 (33.8 g). LCMS (ESI) of compound 21-1: retention time: 2.75 minutes, m / z=419 [M+H] ＋

[0346] Step 21-2: Synthesis of Compound 21-2: tert-butyl (2S)-1-[(2S,3S)-2-amino-3-methyl-pentanoyl]pyrrolidine-2-carboxylic acid

[0347] Compound 21-1 (31.6 g) obtained in step 21-1 and 2-MeTHF (221 mL) were added to a reaction vessel purged with nitrogen and cooled to 10°C. 5% Pd / C (6.32 g, 50% water content) was added to the mixed solution, followed by triethylsilane (60.3 mL) over 20 minutes. After stirring at an internal temperature of 15°C for 6 hours, the mixture was stirred at room temperature for an additional 17 hours. The mixed solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 21-2. Compound 21-2 was used in step 21-3 without further purification. LCMS (ESI) of compound 21-2: retention time: 1.14 minutes, m / z = 285 [M+H] ＋

[0348] Step 21-3: Synthesis of Compound 21-3: tert-butyl(2S)-1-[(2S,3S)-2-[[(2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]pentanoyl]amino]-3-methyl-pentanoyl]pyrrolidine-2-carboxylic acid

[0349] Acetonitrile (221 mL) was added to compound 21-2 obtained in step 21-2 at 5°C, followed by (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]pentanoic acid (28.0 g) and DIPEA (39.6 mL). HATU (34.5 g) was slowly added to the mixed solution at 2.5°C. The mixture was stirred at 2.5°C for 1 hour and then at room temperature for 2.5 hours. 5% aqueous sodium carbonate solution (189 mL) and water (150 mL) were added to the mixed solution in this order. Toluene (80 mL) and 2-MeTHF (140 mL) were added, stirred, and then the liquids were separated. The organic layer was washed with 5% aqueous potassium hydrogen sulfate solution (190 mL x 2) and 10% brine (190 mL x 2). The resulting organic layer was concentrated under reduced pressure to obtain compound 21-3. Compound 21-3 was used in step 21-4 without further purification. LCMS (ESI) of compound 21-3: Retention time: 3.43 minutes, m / z = 620 [M+H] ＋

[0350] Step 21-4 Compound 21-4: Synthesis of tert-butyl(2S)-1-[(2S,3S)-3-methyl-2-[[(2S)-2-(methylamino)pentanoyl]amino]pentanoyl]pyrrolidine-2-acetate (Synthesis of H-MeGly(nPr)-Ile-Pro-OtBu)

[0351] Compound 21-3 (296 mg), synthesized by the same method as in step 21-3, and toluene (2.07 mL) were added to a reaction vessel purged with nitrogen and stirred. DBU (0.072 mL) was added to the mixed solution and stirred for 30 minutes. Acetonitrile (1.00 mL) was added to the mixed solution, followed by stirring for 30 minutes. DBU (0.072 mL) was added and stirred for an additional 30 minutes. 1N hydrochloric acid (2.00 mL) and n-heptane (1.00 mL) were added and stirred, after which aqueous layer 1 and organic layer 1 were separated. Organic layer 1 was extracted with 1N hydrochloric acid (1.00 mL) to obtain aqueous layer 2 containing compound 21-4. Aqueous layer 1 and aqueous layer 2 were combined and extracted with 5% aqueous potassium carbonate (2.00 mL) and toluene (4.00 mL), and the organic layer containing compound 21-4 was separated. The obtained organic layer was washed with 10% brine (2.00 mL) and then concentrated under reduced pressure to give compound 21-4 (166 mg). LCMS (ESI) of compound 21-4: retention time: 1.36 minutes, m / z = 398 [M+H] ＋

[0352] The analytical conditions for compounds 21-1 to 21-4 by LCMS are shown below. Apparatus: Waters UPLC / SQD Column: Ascentis Express RP 90A amide, 2.1 mm ID x 50 mm, 2.7 μm Mobile phase: 0.1% FA / water (A), 0.1% FA / MeCN (B) Elution method: B) 5% (0 min) → 100% (4.5 min) → 100% (5.0 min) → 5% (5.01 min) → 5% (7 min) Flow rate: 0.5 mL / min Column temperature: 40°C Detection wavelength: 210-400 nm (PDA)

[0353] The present invention enables the synthesis of peptide compounds with high diastereoselectivity and high yield even in fragment coupling. Furthermore, the present invention provides a synthesis method suitable for fragment coupling of peptide compounds containing N-substituted amino acids and / or peptide compounds having bulky side chains near the reaction site of an amide bond.

Claims

1. A method for producing a peptide compound or a salt thereof, comprising a step of linking an amino group of a first amino acid or peptide to a carboxy group of a second amino acid or peptide via an amide bond (linking step) in a two-phase solvent system containing water and one or more water-immiscible organic solvents.

2. The method of claim 1, wherein the linking step is carried out in the presence of an activating agent.

3. The method of claim 1 or 2, wherein the activating agent is an acyl halide.

4. The method according to claim 1, wherein the linking step comprises the steps of: (1) contacting a second amino acid or peptide with an acyl halide in an organic solvent to prepare a mixed acid anhydride; and (2) contacting a first amino acid or peptide with the mixed acid anhydride obtained in step (1) in the presence of a base in a mixture containing one or more water-immiscible organic solvents and water.

5. The method according to any one of claims 1 to 4, wherein the second amino acid or peptide is a peptide containing two or more amino acid residues.

6. The α carbon of the carboxy group of the second amino acid or peptide has the formula: -CR １ R ２ - (wherein, R １ and R ２ are the same or different and each represents a hydrogen atom, a linear C １ -C ６ Alkyl, optionally substituted branched C ３ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl C １ -C ６ Alkyl, optionally substituted C ３ -C ８ Cycloalkyl, optionally substituted phenyl C １ -C ２ Alkyl, optionally substituted C １ -C ６ Alkoxy C １ -C ２ Alkyl, optionally substituted 5- to 6-membered heteroaryl C １ -C ２ Alkyl or optionally substituted phenyl C １ -C ２ Alkylthio C １ -C ２ alkyl or R １ and R ２ together with the carbon atom to which they are attached, ３ -C ８ The method according to any one of claims 1 to 5, wherein the aryl group is a cyclic group that forms a saturated alicyclic ring.

7. The method according to any one of claims 1 to 6, wherein the first amino acid or peptide is a peptide comprising two or more amino acid residues.

8. The method according to any one of claims 3 to 7, wherein the acyl halide is pivaloyl chloride or 2,2-dimethylbutyryl chloride.

9. The method according to any one of claims 4 to 8, wherein step (1) is carried out in the presence of a base.

10. The method according to claim 9, wherein the base used in step (1) is at least one selected from the group consisting of diisopropylethylamine, triethylamine, 2,6-lutidine, and 2,4,6-collidine.

11. The amino group of a first amino acid or peptide has the formula: -NR ４ R ５ (In the formula, R ４ is a hydrogen atom, R ５ is a hydrogen atom, linear C １ -C ６ Alkyl, branched C ３ -C ６ Alkyl, or C ３ -C ８ The method according to any one of claims 1 to 10, wherein the amino group is represented by the formula (I) (cycloalkyl).

12. The method according to any one of claims 1 to 11, wherein the peptide compound or a salt thereof produced contains 8 to 20 amino acid residues.

13. The method of any one of claims 1 to 12, wherein the peptide compound or a salt thereof produced comprises an unnatural amino acid residue, and the unnatural amino acid is an N-methyl amino acid residue.

14. The method according to any one of claims 1 to 13, wherein the produced peptide compound or a salt thereof comprises a cyclic portion composed of 4 to 14 amino acid residues, and the cyclic portion comprises 1 to 7 amide bonds linking the amino group of a first amino acid or peptide with the carboxy group of a second amino acid or peptide.

15. A peptide compound or a salt thereof produced by the method according to any one of claims 1 to 14.

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