Method for producing peptide compounds using mixed anhydride procedure

Abstract

Provided is a method for synthesizing a peptide compound with high diastereoselectivity in high yield even in fragment coupling, the method producing a peptide compound or a salt thereof, and comprising a step of linking the amino group of the first amino acid or peptide to the carboxyl group of the second amino acid or peptide through an amide bond in a bilayer solvent containing water and one or more types of water-immiscible organic solvents (linking step).

Description

Technical Field

[0001] This invention relates to a method for producing peptide compounds using a mixed anhydride method. Background Technology

[0002] Recently, it has been found that the metabolic stability and membrane permeability of peptides can be improved by using non-natural amino acids such as cyclic peptides (Non-Patent Literature 1 and 2). In particular, cyclic peptide compounds containing N-substituted amino acids are considered to have pharmaceutical similarities (such as metabolic stability and membrane permeability) and can be used to generate inhibitors of protein-protein interactions (Patent Literature 1, Non-Patent Literature 3).

[0003] Because peptide compounds have attracted attention as pharmaceuticals, methods for producing them are also considered important (Non-Patent Literature 4). The synthesis of peptide compounds is generally carried out by extending the peptide chain through repeated steps of linking the C-terminal carboxyl group of an amino acid to another amino acid or the N-terminal amino group of a peptide. An example of a known method for extending peptide chains is the use of EDC as a condensing agent in a bilayer solvent of isopropyl acetate and water (Non-Patent Literature 5). Other known examples of methods for extending peptide chains include methods using HATU as a condensing agent, methods using T3P as a condensing agent, methods using acyl chlorides, and methods using acyl halides (such as neopentyl chloride) through the extension by mixing acid anhydrides.

[0004] In the mixed anhydride method, peptide chain elongation is achieved by reacting the mixed anhydride (obtained by activating the carboxyl group at the C-terminus of the substrate with an acyl halide) with an amine at the N-terminus of another substrate. Examples of known methods for extending peptide chains via mixed anhydrides include reacting an unprotected amino acid with a substrate temporarily protected by silanization (Non-Patent Document 6), and synthesizing a diretin peptide from an amino acid using a mixed anhydride of neopentanoyl chloride in a water and ethyl acetate bilayer solvent (Non-Patent Document 7). All these methods involve subjecting the amino acid to amide bond formation reactions one residue at a time.

[0005] Another known method for extending peptide chains is called fragment coupling, which involves subjecting peptide fragments, each composed of multiple amino acid residues, to amide bond formation reactions. Fragment coupling offers the advantage of high overall yield of the final product. The more amino acid residues in the desired peptide compound, the greater the advantage. However, under conventional amide bond formation conditions, the increased impurities due to racemization at the α-position of amino acid residues, and the resulting decrease in the yield and purity of the target product, significantly increase the difficulty of purification, thus increasing the time and effort required for production. Furthermore, when N-substituted amino acids become the reaction sites for amide bond formation reactions in fragment coupling, the reduced reactivity due to steric hindrance of the substituent at the nitrogen atom (i.e., the reaction site) has always been a problem (Non-Patent Literature 8). Therefore, when synthesizing peptide compounds containing N-substituted amino acids, this synthetic method has been limited to methods that subject amino acids to amide bond formation reactions one residue at a time, or to fragment coupling with residues that do not cause epimerization.

[0006] [List of Citations]

[0007] [Patent Literature]

[0008] [Patent Document 1] International Publication No. WO 2013 / 100132

[0009] [Non-patent literature]

[0010] [Non-patent literature 1] Acc. Chem. Res. 2008, 41, 1331-1342.

[0011] [Non-Patent Literature 2] Angew. Chem. Int. (ed.), 2013, 52, 254-269.

[0012] [Non-Patent Literature 3] Chem. Rev., 2019, 119, 10360-10391.

[0013] [Non-Patent Literature 4] Amino Acids, Peptides and Proteins in Organic Chemistry: Building Blocks, Catalysis and Coupling Chemistry, Vol. 3, 2011.

[0014] [Non-Patent Literature 5] J. Org.Chem., 1995, 60, 3569-3570.

[0015] [Non-Patent Literature 6] Org. Lett. 2020, 22, 8039-8043.

[0016] [Non-patent literature 7] ACS Sustainable Chem. Eng., 2022, 10, 5307-5314.

[0017] [Non-Patent Literature 8] J. Peptide Res., 2005, 65, 153-166. Summary of the Invention

[0018] [Problem to be solved through invention]

[0019] The present invention was made in view of these circumstances. In one aspect, the problem to be solved is to establish a novel synthetic method for peptide compounds. In another aspect, the problem to be solved is to establish a novel synthetic method suitable for fragment coupling of peptide compounds. In another aspect, the problem to be solved is to establish a method for synthesizing peptide compounds with high diastereomeric selectivity and high yield even through fragment coupling. In another aspect, the problem to be solved is to establish a synthetic method suitable for fragment coupling of peptide compounds containing N-substituted amino acids. In another aspect, the problem to be solved is to establish a synthetic method suitable for fragment coupling of peptide compounds with large side chains near the reaction site of the amide bond.

[0020] [Methods used to solve problems]

[0021] The inventors have conducted in-depth research to address the above-mentioned problems. Therefore, the inventors have discovered a method for synthesizing peptide compounds with high diastereomeric selectivity and high yield, even via fragment coupling, by performing an amide bond formation reaction in a mixture containing water and one or more water-immiscible solvents. Furthermore, the inventors have discovered a method for solving at least one of the above problems by activating the carboxyl group of an amino acid or peptide with a specific acyl halide from the aforementioned mixture. In addition, the inventors have discovered reaction conditions that can suppress the formation of byproducts in condensation reactions.

[0022] In one specific non-limiting aspect, the invention covers the following.

[0023] [A1] A method for producing a peptide compound or a salt thereof, the method comprising the step of linking an amino group of a first amino acid or peptide to a carboxyl group of a second amino acid or peptide by an amide bond in a bilayer solvent containing water and one or more organic solvents immiscible with water (linking step).

[0024] [A2] The method according to [A1], wherein the connection step is performed in the presence of an activator.

[0025] [A3] The method according to [A1] or [A2], wherein the activator is an acyl halide.

[0026] [A4] According to the method described in [A1], the connection step includes the following steps:

[0027] (1) Preparing a mixed anhydride by contacting the second amino acid or peptide with an acyl halide in an organic solvent; and

[0028] (2) In the presence of a base, the mixed anhydride obtained in step (1) is contacted with the first amino acid or peptide in the presence of a base in the bilayer solvent containing water and one or more organic solvents that are immiscible with water.

[0029] [A5] According to the method of [A4], wherein step (2) of contacting the mixed anhydride obtained in step (1) with the first amino acid or peptide is a step of adding the first amino acid or peptide to the mixed anhydride obtained in step (1).

[0030] [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.

[0031] [A7] The method according to any one of [A1] to [A6], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is optionally substituted.

[0032] [A8] According to any one of [A1] to [A6], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C6 alkyl, an optionally substituted branched C3-C6 alkyl, an optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, an optionally substituted C3-C8 cycloalkyl, an optionally substituted phenyl-C1-C2 alkyl, an optionally substituted C1-C6 alkoxy-C1-C2 alkyl, an optionally substituted 5- to 6-membered heteroaryl-C1-C2 alkyl, or an optionally substituted phenyl-C1-C2 alkyl thio-C1-C2 alkyl, or R 1 and R 2 Together with the carbon atoms they are attached to, they form C3-C8 saturated alicyclic rings.

[0033] [A9] According to any one of [A1] to [A6], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C4 alkyl, a branched C3-C6 alkyl, a C3-C6 cycloalkyl-C1-C6 alkyl, a C1-C6 alkoxymethyl, a 1-(C1-C6 alkoxy)ethyl, an optionally substituted benzyl, an optionally substituted phenethyl or phenyl C1-C2 alkyl thiomethyl.

[0034] [A10] According to the method described in [A8] or [A9], where R 1 It is a hydrogen atom.

[0035] [A11] According to any one of [A1] to [A6], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 It is a hydrogen atom, and R 2 It can be a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, tert-butoxymethyl, 1-(tert-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl or benzylthiomethyl.

[0036] [A12] According to any one of [A1] to [A11], wherein the β-nitrogen at the carboxyl group of the second amino acid or peptide is represented by the following formula: -NR 3 -, where R 3 It is a hydrogen atom.

[0037] [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.

[0038] [A14] The method according to any one of [A1] to [A13], wherein the carboxyl group of the first amino acid or peptide is protected by a protecting group.

[0039] [A15] The method according to any one of [A1] to [A14], wherein the amino group of the second amino acid or peptide is protected by a protecting group.

[0040] [A16] The method according to any one of [A3] to [A15], wherein the acyl halide is at least one selected from the group consisting of: straight-chain C1-C 18 Alkyl carboxyl chloride, branched C3-C 18 Alkyl carbamate chloride, straight-chain C1-C 18 Alkyl carboyl bromide, branched C3-C 18 Alkyl carbamate bromide and monocyclic or condensed ring C3-C 15 cycloalkyl carbonyl chloride, wherein the C3-C 15 The cycloalkyl carbonyl chloride is optionally substituted with C1-C6 alkyl groups.

[0041] [A17] The method according to any one of [A3] to [A16], wherein the acyl halogen is at least one selected from the group consisting of: neopentanoyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexanecarbonyl chloride, 1-adamantanecarbonyl chloride and 2-ethylbutyryl chloride.

[0042] [A18] The method according to any one of [A3] to [A17], wherein the acyl halide is neopentanoyl chloride or 2,2-dimethylbutyryl chloride.

[0043] [A19] The method according to any one of [A3] to [A18], wherein the acyl halide is used in a molar equivalent of 0.6 to 1.0 relative to the second amino acid or peptide.

[0044] [A20] The method according to any one of [A4] to [A19], wherein step (1) is carried out in the presence of a base.

[0045] [A21] According to the method of [A20], wherein the pKa of the conjugated acid of the base used in step (1) is 15.0 or lower, preferably 11.5 or lower.

[0046] [A22] According to the method of [A20] or [A21], wherein the base used in step (1) is an organic base, preferably at least one of the group consisting of free amines and pyridines.

[0047] [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-dimethylpyridine and 2,4,6-trimethylpyridine.

[0048] [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 bicarbonate, sodium biphosphate, triethylamine and pyridine.

[0049] [A24-0] The method according to any one of [A1] to [A24], wherein the connection step is carried out in the presence of a base.

[0050] [A24-1] The method according to any one of [A4] to [A24], wherein step (1) is carried out in the presence of an additive.

[0051] [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 trifluoromethanesulfonates.

[0052] [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.

[0053] [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.

[0054] [A24-5] The method according to any one of [A24-0] to [A24-4], wherein the additive is used in a range of 0.2 molar equivalents to 3.0 molar equivalents relative to the second amino acid or peptide.

[0055] [A24-6] The method according to any one of [A24-0] to [A24-4], wherein the additive is used in the range of 0.3 molar equivalents to 1.5 molar equivalents relative to the second amino acid or peptide.

[0056] [A25] The method according to any one of [A4] to [A24], wherein the organic solvent used in step (1) is one or more organic solvents that are immiscible with water.

[0057] [A26] The method according to any one of [A1] to [A25], wherein the water-immiscible organic solvent is a solvent with an octanol / water partition coefficient (Log Kow) of 5 or higher, or a solvent with a predicted octanol / water partition coefficient (Log Kow) of 5 or higher.

[0058] [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 tert-butyl ether, diethyl ether, dichloromethane, carbon tetrachloride, 2-methyltetrahydrofuran, toluene, and hexane.

[0059] [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 tert-butyl ether, dichloromethane, 2-methyltetrahydrofuran, and toluene.

[0060] [A29] 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 following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, a straight-chain C1-C6 alkyl group, a branched C3-C6 alkyl group, or a C3-C8 cycloalkyl group.

[0061] [A30] 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 following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It consists of hydrogen atoms or straight-chain C1-C4 alkyl groups.

[0062] [A31] 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 following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, a methyl group, or an ethyl group.

[0063] [A32] 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 following formula: -NR4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom or a methyl group.

[0064] [A33] The method according to any one of [A1] to [A32], wherein the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It is a methyl group.

[0065] [A34] The method according to any one of [A1] to [A33], wherein the peptide compound to be produced or a salt thereof contains 8 to 20 amino acid residues.

[0066] [A35] The method according to any one of [A1] to [A34], wherein the peptide compound to be produced or its salt contains 11 to 14 amino acid residues.

[0067] [A36] The method according to any one of [A1] to [A35], wherein the peptide compound to be produced contains at least one non-natural amino acid residue.

[0068] [A37] The method according to any one of [A1] to [A36], wherein the peptide compound to be produced contains at least four non-natural amino acid residues.

[0069] [A38] The method according to any one of [A1] to [A37], wherein the peptide compound to be produced contains at least five non-natural amino acid residues.

[0070] [A39] The method according to any one of [A36] to [A38], wherein the non-natural amino acid is an N-methyl amino acid residue.

[0071] [A40] The method according to any one of [A1] to [A39], wherein the peptide compound to be produced or a salt thereof contains a cyclic moiety consisting of 4 to 14 amino acid residues, and wherein an amide bond connecting the amino group of the first amino acid or peptide to the carboxyl group of the second amino acid or peptide is contained at 1 to 7 positions of the cyclic moiety.

[0072] [A41] The method according to any one of [A1] to [A40] is carried out using a flow reaction apparatus.

[0073] [A42] A peptide compound or a salt thereof, which is produced according to any one of [A1] to [A41].

[0074] [A43] A pharmaceutical composition comprising a peptide compound or a salt thereof produced by the method according to any one of [A1] to [A41].

[0075] [B1] A method for producing a peptide compound or a salt thereof, the method comprising the step of linking an amino group of a first amino acid or peptide to a carboxyl group of a second amino acid or peptide by an amide bond in a bilayer solvent containing water and one or more organic solvents immiscible with water (linking step), wherein the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, a straight-chain C1-C6 alkyl group, a branched C3-C6 alkyl group, or a C3-C8 cycloalkyl group.

[0076] [B2] According to the method described in [B1], the connection step includes the following steps:

[0077] (1) Preparing a mixed anhydride by contacting the second amino acid or peptide with an acyl halide in an organic solvent; and

[0078] (2) In the presence of a base, the mixed anhydride obtained in step (1) is contacted with the first amino acid or peptide in the presence of a base in the bilayer solvent containing water and one or more organic solvents that are immiscible with water.

[0079] [B3] According to the method described in [B2], step (2) of contacting the mixed anhydride obtained in step (1) with the first amino acid or peptide is a step of adding the first amino acid or peptide to the mixed anhydride obtained in step (1).

[0080] [B4] 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 following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It consists of hydrogen atoms or straight-chain C1-C4 alkyl groups.

[0081] [B5] 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 following formula: -NR 4 R5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, a methyl group, or an ethyl group.

[0082] [B6] 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 following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom or a methyl group.

[0083] [B7] The method according to any one of [B1] to [B6], wherein the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom and R 5 It is a methyl group.

[0084] [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.

[0085] [B9] The method according to any one of [B1] to [B8], wherein the carboxyl group of the first amino acid or peptide is protected.

[0086] [B10] The method according to any one of [B1] to [B9], wherein the amino group of the second amino acid or peptide is protected.

[0087] [B11] The method according to any one of [B1] to [B10], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is optionally substituted.

[0088] [B12] According to any one of [B1] to [B10], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2They are the same or different and each is a hydrogen atom, a straight-chain C1-C6 alkyl, an optionally substituted branched C3-C6 alkyl, an optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, an optionally substituted C3-C8 cycloalkyl, an optionally substituted phenyl-C1-C2 alkyl, an optionally substituted C1-C6 alkoxy-C1-C2 alkyl, an optionally substituted 5- to 6-membered heteroaryl-C1-C2 alkyl, or an optionally substituted phenyl-C1-C2 alkyl thio-C1-C2 alkyl, or R 1 and R 2 Together with the carbon atoms they are attached to, they form C3-C8 saturated alicyclic rings.

[0089] [B13] According to any one of [B1] to [B10], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C4 alkyl, a branched C3-C6 alkyl, a C3-C6 cycloalkyl-C1-C6 alkyl, a C1-C6 alkoxymethyl, a 1-(C1-C6 alkoxy)ethyl, an optionally substituted benzyl, an optionally substituted phenethyl or phenyl C1-C2 alkyl thiomethyl.

[0090] [B14] According to the method described in [B12] or [B13], wherein R 1 It is a hydrogen atom.

[0091] [B15] According to any one of [B1] to [B10], wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 It is a hydrogen atom, and R 2 It can be a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, tert-butoxymethyl, 1-(tert-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl or benzylthiomethyl.

[0092] [B16] According to any one of [B1] to [B15], wherein the β-nitrogen at the carboxyl group of the second amino acid or peptide is represented by the following formula: -NR 3 -, where R 3 It is a hydrogen atom.

[0093] [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.

[0094] [B18] The method according to any one of [B1] and [B4] to [B17], wherein the connection step is performed in the presence of an activator.

[0095] [B19] The method according to [B18], wherein the activator is an acyl halide.

[0096] [B20] The method according to any one of [B2], [B3] and [B19], wherein the acyl halogen is at least one selected from the group consisting of: linear C1-C 18 Alkyl carboxyl chloride, branched C3-C 18 Alkyl carbamate chloride, straight-chain C1-C 18 Alkyl carboyl bromide, branched C3-C 18 Alkyl carbamate bromide and monocyclic or condensed ring C3-C 15 cycloalkyl carbonyl chloride, wherein the C3-C 15 The cycloalkyl carbonyl chloride is optionally substituted with C1-C6 alkyl groups.

[0097] [B21] The method according to any one of [B2] to [B3] and [B19] to [B20], wherein the acyl halogen is at least one selected from the group consisting of: neopentanoyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexanecarbonyl chloride, 1-adamantanecarbonyl chloride and 2-ethylbutyryl chloride.

[0098] [B22] The method according to any one of [B2] to [B3] and [B19] to [B21], wherein the acyl halide is neopentanoyl chloride or 2,2-dimethylbutyryl chloride.

[0099] [B23] The method according to any one of [B2] to [B3] and [B19] to [B22], wherein the acyl halide is used in a molar equivalent of 0.6 to 1.0 relative to the second amino acid or peptide.

[0100] [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.

[0101] [B25] According to the method of [B24], wherein the pKa of the conjugated acid of the base used in step (1) is 15.0 or lower, preferably 11.5 or lower.

[0102] [B26] According to the method of [B24] or [B25], wherein the base used in step (1) is an organic base, preferably at least one of the group consisting of free amines and pyridines.

[0103] [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-dimethylpyridine and 2,4,6-trimethylpyridine.

[0104] [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 bicarbonate, sodium biphosphate, triethylamine and pyridine.

[0105] [B28-0] The method according to any one of [B1] to [B28], wherein the connection step is carried out in the presence of a base.

[0106] [B28-1] The method according to any one of [B2] to [B28], wherein step (1) is carried out in the presence of an additive.

[0107] [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 trifluoromethanesulfonates.

[0108] [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.

[0109] [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.

[0110] [B28-5] The method according to any one of [B28-0] to [B28-4], wherein the additive is used in a range of 0.2 molar equivalents to 3.0 molar equivalents relative to the second amino acid or peptide.

[0111] [B28-6] The method according to any one of [B28-0] to [B28-4], wherein the additive is used in the range of 0.3 molar equivalents to 1.5 molar equivalents relative to the second amino acid or peptide.

[0112] [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 organic solvents that are immiscible with water.

[0113] [B30] The method according to any one of [B1] to [B29], wherein the water-immiscible organic solvent is a solvent with an octanol / water partition coefficient (Log Kow) of 5 or higher, or a solvent with a predicted octanol / water partition coefficient (Log Kow) of 5 or higher.

[0114] [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 tert-butyl ether, diethyl ether, dichloromethane, carbon tetrachloride, 2-methyltetrahydrofuran, toluene, and hexane.

[0115] [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 tert-butyl ether, dichloromethane, 2-methyltetrahydrofuran and toluene.

[0116] [B33] The method according to any one of [B1] to [B32], wherein the peptide compound to be produced or its salt contains 8 to 20 amino acid residues.

[0117] [B34] The method according to any one of [B1] to [B33], wherein the peptide compound to be produced or a salt thereof contains 11 to 14 amino acid residues.

[0118] [B35] The method according to any one of [B1] to [B34], wherein the peptide compound to be produced contains at least one non-natural amino acid residue.

[0119] [B36] The method according to any one of [B1] to [B35], wherein the peptide compound to be produced contains at least four non-natural amino acid residues.

[0120] [B37] The method according to any one of [B1] to [B36], wherein the peptide compound to be produced contains at least five non-natural amino acid residues.

[0121] [B38] The method according to any one of [B34] to [B37], wherein the non-natural amino acid is an N-methyl amino acid residue.

[0122] [B39] The method according to any one of [B1] to [B38], wherein the peptide compound to be produced or its salt contains a cyclic moiety consisting of 4 to 14 amino acid residues, and wherein the amide bond connecting the amino group of the first amino acid or peptide to the carboxyl group of the second amino acid or peptide is contained in 1 to 7 positions of the cyclic moiety.

[0123] [B40] The method according to any one of [B1] to [B39] is carried out using a flow reaction apparatus.

[0124] [B41] A peptide compound or a salt thereof, which is produced according to any one of [B1] to [B40].

[0125] [B42] A pharmaceutical composition comprising a peptide compound or a salt thereof produced by the method according to any one of [B1] to [B40].

[0126] [Beneficial effects of the invention]

[0127] According to the present invention, peptide compounds with high diastereomeric selectivity and high yield can be synthesized even through fragment coupling. Furthermore, according to the present invention, a synthetic method suitable for fragment coupling of peptide compounds containing N-substituted amino acids and / or peptide compounds having large side chains near the reaction site of the amide bond can be provided.

[0128] [Mode for carrying out the invention]

[0129] The abbreviations used in this article are listed below.

[0130] 2-MeTHF: 2-Methyltetrahydrofuran

[0131] IPAC: Isopropyl acetate

[0132] EtOAc: Ethyl acetate

[0133] MeCN: Acetonitrile

[0134] THF: Tetrahydrofuran

[0135] MeOH: Methanol

[0136] MTBE: Methyl tert-butyl ether

[0137] CPME: Cyclopentylmethyl ether

[0138] DCM: Dichloromethane

[0139] DIPEA: N,N-Diisopropylethylamine

[0140] TEA: Triethylamine

[0141] NMM: N-methylmorpholine

[0142] NMI: N-methylimidazole

[0143] T3P: n-propylphosphonic anhydride

[0144] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate

[0145] COMU: (1-Cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinocarbomon hexafluorophosphate

[0146] PivCl: Neopentanoyl chloride

[0147] Me2BtrCl: 2,2-Dimethylbutyryl chloride

[0148] EDC·HCl: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0149] HOPO: 2-hydroxypyridine-N-oxide

[0150] H3PO4: Phosphoric acid

[0151] NaCl: Sodium chloride

[0152] Na2CO3: Sodium carbonate

[0153] K2CO3: Potassium carbonate

[0154] Cs2CO3: Cesium carbonate

[0155] NaHCO3: Sodium bicarbonate

[0156] K3PO4: Tripotassium phosphate

[0157] Na2HPO4: Disodium hydrogen phosphate

[0158] K2HPO4: dipotassium hydrogen phosphate

[0159] NaOH: Sodium hydroxide

[0160] KOH: Potassium hydroxide

[0161] LiOH: Lithium hydroxide

[0162] NaHSO4: Sodium bisulfate

[0163] Na2SO4: Sodium sulfate

[0164] H2O: water

[0165] TFA: Trifluoroacetic acid

[0166] HMDS: Hexamethyldisilazane

[0167] TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0168] Pd / C: Palladium on carbon

[0169] Alloc: allyloxycarbonyl

[0170] Boc: tert-Butoxycarbonyl

[0171] Cbz: Benzyloxycarbonyl

[0172] Teoc: 2-(trimethylsilyl)ethoxycarbonyl

[0173] Fmoc: 9-fluorenylmethyloxycarbonyl

[0174] TBAF: Tetrabutylammonium fluoride

[0175] LiBH4: Lithium borohydride

[0176] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0177] BEP: 2-Bromo-1-ethylpyridine tetrafluoroborate

[0178] PyBOP: 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate

[0179] DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride

[0180] PyOxim: (ethyl cyano(hydroxyimino)ethyl acetate-O2)-tris(1-pyrrolidinyl)-phosphorus hexafluorophosphate

[0181] NaBr: Sodium bromide

[0182] NaI: Sodium iodide

[0183] NaOTf: Sodium trifluoromethanesulfonate

[0184] KOTf: Potassium trifluoromethanesulfonate

[0185] Definitions of functional groups, etc. (The terms explained below are illustrative and not intended to be particularly limiting, and are terms that are generally understood by those skilled in the art).

[0186] As used herein, "halogen atom" includes fluorine, chlorine, bromine, and iodine. As used herein, F refers to fluorine, Cl refers to chlorine, Br refers to bromine, and I refers to iodine. Examples of halogens include fluorine, chlorine, and bromine, with fluorine and chlorine being preferred.

[0187] As used herein, "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group derived from saturated aliphatic hydrocarbons by removing any one of the hydrogen atoms. This group has a hydrocarbon group or subset of hydrocarbon group structures that do not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in their main chain, but contain hydrogen and carbon atoms. Alkyl groups include not only straight-chain alkyl groups but also branched alkyl groups. Specifically, the alkyl group is one having 1 to 20 carbon atoms (C1-C2). 20 Alkyl groups, preferably C1-C 10 Alkyl, more preferably C1-C6 alkyl. In the following, "C" refers to... p -C q"This means that it has p to q" One carbon atom. Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl (2-methylpropyl), n-pentyl, sec-pentyl (1-methylbutyl), tert-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. Specific examples of straight-chain C1-C6 alkyl groups include methyl, ethyl, ... n-Propyl, n-Butyl, n-Pentyl, and n-Hexyl. Specific examples of straight-chain C1-C3 alkyl groups include methyl, ethyl, and n-propyl. Specific examples of branched C3-C6 alkyl groups include isopropyl, sec-butyl, tert-butyl, isobutyl (2-methylpropyl), sec-pentyl (1-methylbutyl), tert-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, and 2-ethylbutyl.

[0188] As used herein, "alkenyl" is a straight-chain or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon double bonds (bonded by two adjacent sp2 carbon atoms). Depending on the conformation of the atom or group of atoms attached to the sp2 carbon atom, the geometry of the double bond can be entgegen (E) or zusammen (Z) and in cis or trans conformations. Alkenyl groups are, for example, C2-C 10 Alkenyl, preferably C2-C8 alkenyl, more preferably C2-C7 alkenyl, and most preferably C2-C6 alkenyl. Specific examples of alkenyl include ethenyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl.

[0189] As used herein, "alkynyl" is a straight-chain or branched monovalent unsaturated hydrocarbon group having one or more carbon-carbon triple bonds (composed of two adjacent sp carbon atoms). The alkynyl group is, for example, C2-C. 10The alkynyl group is preferably C2-C8 alkynyl, more preferably C2-C7 alkynyl, and most preferably C2-C6 alkynyl. Specific examples of alkynyl groups include ethynyl, 1-propynyl, propynyl (2-propynyl), 1-butynyl, 2-butynyl, 3-butynyl, pentynyl, and hexynyl.

[0190] As used herein, “cycloalkyl” is a saturated or partially saturated cyclic monovalent non-aromatic hydrocarbon cyclic group (alicyclic cyclic group). The carbon atom constituting the ring can be oxidized to form a carbonyl group. Cycloalkyl can be selected from the group consisting of monocyclic, condensed, and spirocyclic. As used herein, a cycloalkyl containing a monocyclic ring is called a monocyclic cycloalkyl or monocyclic alicyclic cyclic group; a cycloalkyl containing a condensed ring is called a condensed cycloalkyl or condensed cycloalicyclic cyclic group; and a cycloalkyl containing a spirocyclic ring is called a spirocyclic cycloalkyl or spirocyclic alicyclic cyclic group. Cycloalkyl can form condensed rings with saturated alicyclic rings (such as cyclopentane or cyclohexane), unsaturated alicyclic rings (such as cycloheptene or cyclohexene), or aromatic hydrocarbon rings (such as benzene or naphthalene). Cycloalkyl can form spirocyclic rings with saturated alicyclic rings (such as cyclopropane, cyclobutane, cyclopentane, or cyclohexane). Cycloalkyl is, for example, C3-C 10 Cycloalkyl, preferably C3-C8 cycloalkyl, more preferably C3-C7 cycloalkyl, and most preferably C3-C6 cycloalkyl. Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and cyclohexenyl.

[0191] As used herein, "aryl" is a monovalent aromatic hydrocarbon cyclic group consisting of a monovalent, monocyclic, or condensed ring that exhibits aromaticity. As used herein, aryl groups consisting of a monocyclic ring are called monocyclic aryl groups, and aryl groups consisting of a condensed ring are called condensed aryl groups. Aryl groups are, for example, C6-C... 14 Aryl, preferably C6 aryl, C 10 Aryl and C 14 Aryl, more preferably C6 aryl and C 10 Aryl, most preferably C6 aryl. Specific examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, tolyl, and xylyl.

[0192] As used herein, a "heterocyclic group" is a heterocyclic group whose constituent atoms of the ring contain heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur atoms, and a carbon atom, preferably 1 to 5 heteroatoms, more preferably 1 to 3 heteroatoms, and may have double and / or triple bonds in the ring. The carbon atom of the ring of the heterocyclic group can be oxidized to form a carbonyl group. As used herein, a heterocyclic group containing a monocyclic ring is called a monocyclic heterocyclic group; a heterocyclic group containing a condensed ring is called a condensed ring heterocyclic group; and a heterocyclic group containing a spirocyclic ring is called a spirocyclic heterocyclic group. Heterocyclic groups can form condensed or spirocyclic rings with saturated alicyclic rings (such as cyclopentane or cyclohexane rings) or saturated heterocyclic rings (such as tetrahydropyran, dioxane, or pyrrolidine rings). The number of atoms in the ring constituting the heterocyclic group is, for example, 3 to 14 (3-membered to 14-membered heterocyclic group), preferably 3 to 12 (3-membered to 12-membered heterocyclic group), more preferably 3 to 10 (3-membered to 10-membered heterocyclic group), and particularly preferably 4 to 7 (4-membered to 7-membered heterocyclic group). Specific examples of heterocyclic groups include azocyclobutane, ethylene oxide, oxocyclobutane, thiocyclobutane, tetrahydrofuranyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazoyl, isothiazolyl, thiadiazoyl, oxazolyl, dioxopentane, tetrahydropyranyl, morpholinyl, thiomorpholinyl, 4-oxopyrrolyl, piperidinyl, 4-oxopyridinyl, piperazine, and dioxyl, and rings in which one or more single bonds in these saturated heterocycles are replaced by double or triple bonds.

[0193] As used herein, a "heteroaryl" is a monovalent aromatic heterocyclic group that contains at least one heteroatom in addition to a carbon atom and is composed of a monocyclic or fused ring exhibiting aromaticity. As used herein, a heteroaryl composed of a monocyclic ring is called a monocyclic heteroaryl, and a heteroaryl composed of a condensed ring is called a condensed-ring heteroaryl. The number of atoms in the ring constituting the heteroaryl is, for example, 5 to 14 (5-membered to 14-membered heteroaryl), preferably 5 to 13 (5-membered to 13-membered heteroaryl), more preferably 5 to 10 (5-membered to 10-membered heterocyclic group), and most preferably 5 to 7 (5-membered to 7-membered heteroaryl). Specific examples of heteroaryl groups include 5-membered heteroaryl groups, such as furanyl, thiopheneyl, pyrroleyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, or tetrazolyl; 6-membered heteroaryl groups, such as pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, or triazinyl; 9-membered heteroaryl groups, such as benzofuranyl, benzothiopheneyl, benzothiazolyl, benzoimidazolyl, benzotriazolyl, indolyl, inazolyl, or pyrazolopyridyl; and 10-membered heteroaryl groups, such as quinolinyl, isoquinolinyl, cyclolinyl, quinazolinyl, or quinoxalinyl.

[0194] As used herein, “alkoxy” is a group in which an alkyl group (-OR, where R is an alkyl group) is attached to an oxygen atom as defined herein. Alkoxy groups are, for example, C1-C 20 Alkoxy, preferably C1-C 10 Alkoxy, more preferably C1-C8 alkoxy, and most preferably C1-C6 alkoxy. Specific examples of alkoxy groups include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and 3-methylbutoxy.

[0195] As used herein, “alkenyloxy group” is a group in which an “alkenyl” as defined herein is attached to an oxygen atom (-OR, where R is an alkenyl group). An alkenyloxy group is, for example, C2-C… 10 Alkenyloxy, preferably C2-C8 alkenyloxy, more preferably C2-C7 alkenyloxy, and most preferably C2-C6 alkenyloxy. Specific examples of alkenyloxy include ethenyloxy, 1-propenyloxy, 2-propenyloxy, isopropenyloxy, 1-butenyloxy, 2-cis-butenyloxy, 2-trans-butenyloxy, 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0196] As used herein, “cycloalkoxy” is a group in which a “cycloalkyl” as defined herein is attached to an oxygen atom (-OR, where R is a cycloalkyl group). Cycloalkoxy groups are, for example, C3-C… 10 Cycloalkoxy, preferably C3-C8 cycloalkoxy, more preferably C3-C7 cycloalkoxy, and most preferably C3-C6 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, and cyclopentoxy.

[0197] As used herein, "aryloxy group" is a group in which an aryl group (-OAr, where Ar is an aryl group) is attached to an oxygen atom as defined herein. Aryloxy groups are, for example, C6-C. 14 Aryloxy group, preferably C6 aryloxy group, C 10 aryloxy groups and C 14 aryloxy, more preferably C6 aryloxy and C 10 Aryloxy group, most preferably C6 aryloxy group. Specific examples of aryloxy groups include phenoxy, 1-naphthoxy, 2-naphthoxy, tolyloxy, and xyleneoxy.

[0198] As used herein, “amino” means -NRR’, where N represents a nitrogen atom and R and R’ are each independently selected from the group consisting of: hydrogen atom, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or R and R’ form a ring together with the nitrogen atom to which they are attached. Examples of amino groups include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and 4- to 8-membered cyclic amino groups.

[0199] As used herein, “monoalkylamino” means an amino (-NRR’) group as defined above, where R is a hydrogen atom and R’ is an alkyl group. Monoalkylamino groups are, for example, C1-C 20 Alkylamino, preferably mono-C1-C 15 Alkylamino, more preferably mono-C1-C 10 Alkylamino, most preferably mono-C1-C6 alkylamino. Specific examples of mono-alkylamino include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, sec-butylamino, and tert-butylamino.

[0200] As used herein, “dialkylamino” means an amino (-NRR') group as defined above, where R and R' are each independently an alkyl group. Dialkylamino groups are, for example, C1-C 20 Alkylamino, preferably di-C1-C 15 Alkylamino, more preferably di-C1-C 10 Alkylamino, most preferably di-C1-C6 alkylamino. Specific examples of dialkylamino include dimethylamino, diethylamino, and methylethylamino.

[0201] As used herein, “cyclic amino” is a group of “amino (-NRR’)” as defined herein, wherein R and R’ together with the nitrogen atom to which they are attached form a ring. Cyclic amino groups are, for example, 3- to 14-membered cyclic amino groups, preferably 3- to 12-membered cyclic amino groups, more preferably 3- to 10-membered cyclic amino groups, and most preferably 4- to 7-membered cyclic amino groups. Specific examples of cyclic amino groups include 1-azacyclobutane, 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, 4-morpholinyl, 3-oxazolidinyl, 1,1-thiomorpholinyl-4-yl, and 3-oxa-8-azabicyclo[3.2.1]octane-8-yl.

[0202] As used herein, “protected amino” means an amino group protected by an optional protecting group. Specific examples of protected amino groups include amino groups protected by protecting groups such as Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), Cbz (benzyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Alloc (allyloxycarbonyl), Teoc (2-(trimethylsilyl)ethoxycarbonyl), or trifluoroacetyl.

[0203] As used herein, “aminocarbonyl” is a group in which an “amino” group, as defined herein, is attached to a carbon atom of a carbonyl group. It is sometimes also referred to as an amide. Examples of aminocarbonyl groups include -CONH2, mono-C1-C6 alkylaminocarbonyl, di-C1-C6 alkylaminocarbonyl, and 4- to 8-membered cyclic aminocarbonyl groups. Specific examples of aminocarbonyl groups include -CONH2, methylaminocarbonyl, ethylaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl, 1-azacyclobutylcarbonyl, 1-pyrrolidinylcarbonyl, 1-piperidinylcarbonyl, 1-piperazinylcarbonyl, 4-morpholinylcarbonyl, and 3-oxazolylalkylcarbonyl.

[0204] As used herein, "alkenyloxycarbonyl" is a carbonyl group attached to an "alkoxy" group as defined above. An alkenyloxycarbonyl group is, for example, C2-C... 10 Alkenyloxycarbonyl, preferably C2-C8 alkenyloxycarbonyl, more preferably C2-C7 alkenyloxycarbonyl, and most preferably C2-C6 alkenyloxycarbonyl. Specific examples of alkenyloxycarbonyl include ethenyloxycarbonyl, 1-propenyloxycarbonyl, 2-propenyloxycarbonyl, isopropenyloxycarbonyl, 1-butenyloxycarbonyl, 2-cis-butenyloxycarbonyl, 2-trans-butenyloxycarbonyl, 3-butenyloxycarbonyl, pentenyloxycarbonyl, and hexenyloxycarbonyl.

[0205] As used herein, “alkylsulfonyl” is a sulfonyl group attached to an alkyl group as defined herein. Alkylsulfonyl groups are, for example, C1-C2. 20 Alkyl sulfonyl, preferably C1-C 10 Alkylsulfonyl, more preferably C1-C8 alkylsulfonyl, and most preferably C1-C6 alkylsulfonyl. Specific examples of alkylsulfonyl include methylsulfonyl, ethylsulfonyl, 1-propylsulfonyl, 2-propylsulfonyl, n-butylsulfonyl, isobutylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl, pentylsulfonyl, and 3-methylbutylsulfonyl.

[0206] As used herein, "hydroxyalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by a hydroxyl group. Preferably, a hydroxyalkyl group is a group in which one hydrogen atom of an alkyl group is replaced by a hydroxyl group. Hydroxyalkyl is, for example, a hydroxyl C1-C1 group. 20 Alkyl, preferably hydroxyl C1-C 10 Alkyl, more preferably hydroxy C1-C8 alkyl, most preferably hydroxy C1-C6 alkyl. Specific examples of hydroxyalkyl include hydroxyethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0207] As used herein, “haloalkyl” is a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with a halogen. Haloalkyl is preferably a group in which one or more and six or fewer hydrogen atoms of the alkyl group are substituted with a halogen. Haloalkyl is, for example, a halogenated-C1-C1 group. 20 Alkyl, preferably halogenated -C1-C 10 Alkyl, more preferably halogenated-C1-C8 alkyl, and most preferably halogenated-C1-C6 alkyl. Halogenated-C1-C6 alkyl is, for example, a group in which one or more and six or fewer hydrogen atoms of the alkyl group are substituted with fluorine, preferably a group in which one or more and five or fewer hydrogen atoms are substituted with fluorine, more preferably a group in which one or more and four or fewer hydrogen atoms are substituted with fluorine, and most preferably a group in which one or more and three or fewer hydrogen atoms are substituted with fluorine. Specific examples of halogenated alkyl groups include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, and 5,5-difluoropentyl.

[0208] As used herein, "cyanoalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by a cyano group. Cyanoalkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by a cyano group. Cyanoalkyl is, for example, a cyano C1-C... 20 Alkyl, preferably cyano C1-C 10 Alkyl, more preferably cyanoC1-C8 alkyl, and most preferably cyanoC1-C6 alkyl. Specific examples of cyanoalkyl include cyanomethyl and 2-cyanoethyl.

[0209] As used herein, "aminoalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by an amino group. Aminoalkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by an amino group. Aminoalkyl is, for example, an amino C1-C group. 20 Alkyl, preferably amino C1-C 10Alkyl, more preferably amino C1-C8 alkyl, most preferably amino C1-C6 alkyl. Specific examples of aminoalkyl include aminomethyl, aminoethyl, 4-aminobutyl, methylaminomethyl, dimethylaminomethyl, methylaminoethyl, and dimethylaminoethyl.

[0210] As used herein, “carboxyalkyl” is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by a carboxyl group. Carboxyalkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by a carboxyl group. Carboxyalkyl is, for example, a carboxyl C1-C… 20 Alkyl, preferably carboxyl C1-C 15 Alkyl, more preferably carboxyl C1-C 10 Alkyl, most preferably carboxyl C1-C6 alkyl. Specific examples of carboxyl alkyl include carboxymethyl and carboxyethyl.

[0211] As used herein, "alkenyloxycarbonylalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by an "alkenyloxycarbonyl" group. Preferably, an alkenyloxycarbonylalkyl group is a group in which one hydrogen atom of an alkyl group is replaced by an alkenyloxycarbonyl group. The alkenyloxycarbonylalkyl group is, for example, C2-C 10 Alkenyloxycarbonyl-C1-C6 alkyl, preferably C2-C8 alkenyloxycarbonyl-C1-C6 alkyl, more preferably C2-C7 alkenyloxycarbonyl-C1-C6 alkyl, and most preferably C2-C6 alkenyloxycarbonyl-C1-C6 alkyl. Specific examples of alkenyloxycarbonyl alkyl 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.

[0212] As used herein, "alkoxyalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with an alkoxy group. Alkoxyalkyl is preferably a group in which one hydrogen atom of an alkyl group is substituted with an alkoxy group. Alkoxyalkyl is, for example, C1-C6 alkoxy-C1-C 20 Alkyl, preferably C1-C6 alkoxy-C1-C 15 Alkyl, more preferably C1-C6 alkoxy-C1-C 10Alkyl, most preferably C1-C6 alkoxy-C1-C6 alkyl. Specific examples of alkoxyalkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, isobutoxymethyl, sec-butoxymethyl, tert-butoxymethyl, pentoxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0213] As used herein, "cycloalkylalkyl" is a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted by a cycloalkyl group. Preferably, a cycloalkylalkyl group is a group in which one hydrogen atom of an alkyl group is substituted by a cycloalkyl group. A cycloalkylalkyl group is, for example, C3-C4. 10 cycloalkyl-C1-C 20 Alkyl, preferably C3-C 10 Cycloalkyl-C1-C6 alkyl, more preferably C3-C8 cycloalkyl-C1-C6 alkyl, and most preferably C3-C6 cycloalkyl-C1-C2 alkyl. Specific examples of cycloalkyl alkyl groups include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl.

[0214] As used herein, “cycloalkoxyalkyl” is a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with a cycloalkoxy group. Cycloalkoxyalkyl is preferably a group in which one hydrogen atom of an alkyl group is substituted with a cycloalkoxy group. Cycloalkoxyalkyl is, for example, C3-C4. 10 Cycloalkoxy-C1-C6 alkyl, preferably C3-C8 cycloalkoxy-C1-C6 alkyl, more preferably C3-C7 cycloalkoxy-C1-C6 alkyl, and most preferably C3-C6 cycloalkoxy-C1-C6 alkyl. Specific examples of cycloalkoxyalkyl include cyclopropoxymethyl, cyclobutoxymethyl, and cyclopentyloxymethyl.

[0215] As used herein, “heterocyclic alkyl” is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by a “heterocyclic group”. Heterocyclic alkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by a heterocyclic group. Heterocyclic alkyl is, for example, a 3- to 14-membered heterocyclic-C1-C6 alkyl, preferably a 3- to 12-membered heterocyclic-C1-C6 alkyl, more preferably a 3- to 10-membered heterocyclic-C1-C4 alkyl, and most preferably a 4- to 7-membered heterocyclic-C1-C3 alkyl. Specific examples of heterocyclic alkyl include azirmonobutane-1-ylmethyl, oxadiazonobutane-3-ylmethyl, 2-(tetrahydrofuran-3-yl)ethyl, (1-methylpyrrolidine-3-yl)methyl, 2-morpholinoethyl, 3-(1-piperidinyl)propyl, and 3-(4-methylpiperazin-1-yl)propyl.

[0216] As used herein, "alkylsulfonylalkyl" is a group in which one or more hydrogen atoms of an alkyl group, as defined herein, are replaced by an alkylsulfonyl group. Alkylsulfonylalkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by an alkylsulfonyl group. Alkylsulfonylalkyl is, for example, C1-C1. 20 alkylsulfonyl-C1-C4 alkyl, preferably C1-C 10 Alkylsulfonyl-C1-C4 alkyl, more preferably C1-C8 alkylsulfonyl-C1-C4 alkyl, and most preferably C1-C6 alkylsulfonyl-C1-C4 alkyl. Specific examples of alkylsulfonyl alkyl include methylsulfonylmethyl, methylsulfonylpropyl, methylsulfonylbutyl, ethylsulfonylethyl, 1-propylsulfonylmethyl, 2-propylsulfonylethyl, n-butylsulfonylpropyl, isobutylsulfonylmethyl, sec-butylsulfonylmethyl, tert-butylsulfonylmethyl, pentylsulfonylmethyl, and 3-methylbutylsulfonylmethyl.

[0217] As used herein, “aminocarbonylalkyl” means a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by an aminocarbonyl group as defined herein. Preferably, an aminocarbonylalkyl group is a group in which one hydrogen atom of an alkyl group is replaced by an aminocarbonyl group. An aminocarbonylalkyl group is, for example, aminocarbonyl-C1-C1. 10 Alkyl, preferably aminocarbonyl-C1-C6 alkyl, more preferably aminocarbonyl-C1-C4 alkyl, and most preferably aminocarbonyl-C1-C2 alkyl. Specific examples of aminocarbonyl alkyl include -CH2CONH2, methylaminocarbonylmethyl, ethylaminocarbonylethyl, dimethylaminocarbonylpropyl, diethylaminocarbonylmethyl, 1-azacyclobutylcarbonylmethyl, 1-pyrrolidinylcarbonylethyl, 1-piperidinylcarbonylpropyl, 1-piperazinylcarbonylbutyl, 4-morpholinylcarbonylmethyl, and 3-oxazolylcarbonylethyl.

[0218] As used herein, "aryloxyalkyl" means a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted by an aryloxy group as defined herein. The aryloxyalkyl group is preferably C6-C. 10 aryloxy-C1-C6 alkyl, more preferably C6-C 10 Aryloxy-C1-C2 alkyl. Specific examples of aryloxyalkyl groups include phenoxymethyl and 2-phenoxyethyl.

[0219] As used herein, "aralkyl (arylalkyl)" is a group in which one or more hydrogen atoms of an "alkyl" as defined herein are replaced by an "aryl" as defined herein. Aralkyl groups are, for example, C7-C64 groups. 20 Aryl alkyl group, preferably C7-C 18 Aryl alkyl, more preferably C7-C 16Aryl group, most preferably C7-C 14 Aryl group. C7-C 20 Aryl groups are, for example, C6-C 10 Aryl-C1-C 10 Alkyl, preferably C6-C 10 aryl-C1-C8 alkyl, more preferably C6 aryl-C1-C8 alkyl or C 10 Aryl-C1-C8 alkyl, most preferably C6 aryl-C1-C8 alkyl. C7-C 18 Aryl groups are, for example, C6-C 10 aryl-C1-C8 alkyl, preferably C6-C 10 aryl-C1-C6 alkyl, more preferably C6 aryl-C1-C6 alkyl or C 10 Aryl-C1-C6 alkyl, most preferably C6 aryl-C1-C6 alkyl. C7-C 16 Aryl groups are, for example, C6-C 10 aryl-C1-C6 alkyl, preferably C6-C 10 aryl-C1-C4 alkyl, more preferably C6 aryl-C1-C4 alkyl or C 10 Aryl-C1-C4 alkyl, most preferably C6 aryl-C1-C4 alkyl. C7-C 14 Aryl groups are, for example, C6-C 10 aryl-C1-C4 alkyl, preferably C6-C 10 aryl-C1-C3 alkyl, more preferably C6 aryl-C1-C3 alkyl or C 10 Aryl-C1-C3 alkyl, most preferably C6 aryl-C1-C3 alkyl. Specific examples of aryl alkyl groups include benzyl, phenethyl, and 3-phenylpropyl.

[0220] As used herein, “aranalkoxy” is a group in which the alkyl portion of “aranyl” as defined herein is attached to an oxygen atom (-OR-Ar, where R is an alkylene group). Aranalkoxy groups are, for example, C7-C 20 Arylalkoxy, preferably C7-C 18 arylalkoxy, more preferably C7-C 16 Arylalkoxy, most preferably C7-C 14 Arylalkoxy group. C7-C 20 Aryl alkoxy groups are, for example, C6-C. 10 Aryl-C1-C 10 Alkoxy, preferably C6-C 10 aryl-C1-C8 alkoxy, more preferably C6 aryl-C1-C8 alkoxy or C 10Aryl-C1-C8 alkoxy, most preferably C6 aryl-C1-C8 alkoxy. C7-C 18 Aryl alkoxy groups are, for example, C6-C. 10 aryl-C1-C8 alkoxy, preferably C6-C 10 aryl-C1-C6 alkoxy, more preferably C6 aryl-C1-C6 alkoxy or C 10 Aryl-C1-C6 alkoxy, most preferably C6 aryl-C1-C6 alkoxy. C7-C 16 Aryl alkoxy groups are, for example, C6-C. 10 aryl-C1-C6 alkoxy, preferably C6-C 10 aryl-C1-C4 alkoxy, more preferably C6 aryl-C1-C4 alkoxy or C 10 Aryl-C1-C4 alkoxy, most preferably C6 aryl-C1-C4 alkoxy. C7-C 14 Aryl alkoxy groups are, for example, C6-C. 10 aryl-C1-C4 alkoxy, preferably C6-C 10 aryl-C1-C3 alkoxy, more preferably C6 aryl-C1-C3 alkoxy or C 10 Aryl-C1-C3 alkoxy, most preferably C6 aryl-C1-C3 alkoxy. Specific examples of arylalkoxy compounds include benzyloxy, phenethoxy, and 3-phenylpropoxy.

[0221] As used herein, "aranyloxyalkyl" means a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with an aranyloxy group as defined herein. Aranyloxyalkyl is preferably C7-C6. 14 Arylalkoxy-C1-C6 alkyl, more preferably C7-C 14 Aranealkyl-C1-C2 alkyl. Specific examples of aranealkyl-C1 include benzyloxymethyl and 1-(benzyloxy)ethyl.

[0222] As used herein, “heteroarylalkyl” is a group in which one or more hydrogen atoms of an alkyl group as defined herein are replaced by a “heteroaryl”. Heteroarylalkyl is preferably a group in which one hydrogen atom of an alkyl group is replaced by a heteroaryl. Heteroarylalkyl is, for example, a 5- to 10-membered heteroaryl-C1-C6 alkyl, preferably a 5- to 10-membered heteroaryl-C1-C4 alkyl, more preferably a 5- to 10-membered heteroaryl-C1-C3 alkyl, and most preferably a 5- to 10-membered heteroaryl-C1-C2 alkyl. Specific examples of heteroarylalkyl include 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-furanylmethyl, 2-thienylmethyl, 3-thienylmethyl, and 4-thiazolylmethyl.

[0223] As used herein, “heteroarylalkoxy” is a group in which the alkyl portion of “heteroarylalkyl” as defined herein is attached to an oxygen atom. Heteroarylalkoxy groups are, for example, 5- to 10-membered heteroaryl-C1-C6 alkoxy groups, preferably 5- to 10-membered heteroaryl-C1-C4 alkoxy groups, more preferably 5- to 10-membered heteroaryl-C1-C3 alkoxy groups, and most preferably 5- to 10-membered heteroaryl-C1-C2 alkoxy groups. Specific examples of heteroarylalkoxy groups include 2-pyridylmethoxy, 3-pyridylmethoxy, 4-pyridylmethoxy, 2-furanylmethoxy, 2-thienylmethoxy, 3-thienylmethoxy, and 4-thiazolylmethoxy.

[0224] As used herein, “heteroarylalkoxyalkyl” means a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with a “heteroarylalkoxy” as defined herein. A heteroarylalkoxyalkyl group is preferably a group in which one hydrogen atom of an alkyl group is substituted with a heteroarylalkoxy. A heteroarylalkoxyalkyl group is, for example, a 5- to 10-membered heteroaryl-C1-C6 alkoxy-C1-C6 alkyl group, preferably a 5- to 10-membered heteroaryl-C1-C4 alkoxy-C1-C6 alkyl group, more preferably a 5- to 10-membered heteroaryl-C1-C3 alkoxy-C1-C6 alkyl group, and most preferably a 5- to 10-membered heteroaryl-C1-C2 alkoxy-C1-C6 alkyl group. Specific examples of heteroarylalkoxyalkyl groups include 2-pyridylmethoxymethyl, 3-pyridylmethoxymethyl, 4-pyridylmethoxymethyl, 2-furanylmethoxymethyl, 2-thienylmethoxymethyl, 3-thienylmethoxymethyl, and 4-thiazolylmethoxymethyl.

[0225] As used herein, “heterocyclic alkylalkyl” means a group in which one or more hydrogen atoms of an alkyl group as defined herein are substituted with a “heterocyclic alkyl” as defined herein. Heterocyclic alkylalkyl groups are preferably 4- to 7-membered heterocyclic alkyl-C1-C6 alkyl groups, more preferably 4- to 7-membered heterocyclic alkyl-C1-C2 alkyl groups. Specific examples of heterocyclic alkylalkyl groups include tetrahydro-4H-pyran-4-ylemethylene and azirmonobutane-3-ylemethylene.

[0226] As used herein, "alkoxyalkenyl" is a group in which one or more hydrogen atoms of an "alkenyl" group, as defined herein, are substituted with an "alkoxy" group. Preferably, an alkoxyalkenyl group is a group in which one hydrogen atom of an alkenyl group is substituted with an alkoxy group. Alkoxyalkenyl groups are, for example, C1-C6 alkoxy-C2-C... 10Alkenyl, preferably C1-C6 alkoxy-C2-C8 alkenyl, more preferably C1-C6 alkoxy-C2-C7 alkenyl, and most preferably C1-C6 alkoxy-C2-C6 alkenyl. Specific examples of alkoxyalkenyl include (E)-4-methoxybut-2-en-1-yl.

[0227] As used herein, "aminocarbonylalkenyl" is a group in which one or more hydrogen atoms of an "alkenyl" group, as defined herein, are replaced by an "aminocarbonyl" group. Preferably, an aminocarbonylalkenyl group is a group in which one hydrogen atom of an alkenyl group is replaced by an aminocarbonyl group. An aminocarbonylalkenyl group is, for example, aminocarbonyl-C2-C... 10 Alkenyl, preferably aminocarbonyl-C2-C8 alkenyl, more preferably aminocarbonyl-C2-C6 alkenyl, and most preferably aminocarbonyl-C2-C4 alkenyl. Specific examples of aminocarbonyl alkenyl include (E)-3-(dimethylaminocarbonyl)-prop-2-en-1-yl.

[0228] As used herein, “haloalkoxy” is a group in which one or more hydrogen atoms of an alkoxy group, as defined herein, are substituted with a halogen. Haloalkoxy is preferably a group in which one or more and six or fewer hydrogen atoms of the alkoxy group are substituted with a halogen. Haloalkoxy groups are, for example, halogenated-C1-C. 20 Alkoxy, preferably halogenated-C1-C 10 Alkoxy, more preferably halogenated-C1-C8 alkoxy, and most preferably halogenated-C1-C6 alkoxy. Halogenated-C1-C6 alkoxy is, for example, a group in which one or more and six or fewer hydrogen atoms of the alkoxy group are substituted with fluorine, preferably a group in which one or more and five or fewer hydrogen atoms are substituted with fluorine, more preferably a group in which one or more and four or fewer hydrogen atoms are substituted with fluorine, and most preferably a group in which one or more and three or fewer hydrogen atoms are substituted with fluorine. Specific examples of halogenated alkoxy groups include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 3-fluoropropoxy, and 2,2-difluoropropoxy.

[0229] As used herein, "alkylene" means a divalent group induced by further removing any hydrogen atom from an "alkyl" as described herein. Alkylenes are preferably C1-C8 alkylenes, more preferably C4-C8 alkylenes. Examples of alkylenes specifically include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)CH2-, -C(CH3)2-, -(CH2)4-, -CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, -CH2CH(CH2CH3)-, -(CH2)5-, -CH(CH3)CH(CH2CH3)-, -(CH2)6-, -(CH2)7-, and -(CH2)8-.

[0230] The term "alicyclic ring" in this article refers to a saturated or partially saturated non-aromatic hydrocarbon ring. The carbon atoms constituting the ring can be oxidized to form a carbonyl group. Alicyclic rings can be selected from the group consisting of monocyclic, condensed, and spirocyclic rings. As used herein, an alicyclic ring containing a monocyclic ring is called a monocyclic alicyclic ring; an alicyclic ring containing a condensed ring is called a condensed alicyclic ring; and an alicyclic ring containing a spirocyclic ring is called a spirocyclic alicyclic ring. Alicyclic rings can form condensed rings with saturated alicyclic rings (such as cyclopentane or cyclohexane); with unsaturated alicyclic rings (such as cycloheptene or cyclohexene); or with aromatic hydrocarbon rings (such as benzene or naphthalene). Alicyclic rings can form spirocyclic rings with saturated alicyclic rings (such as cyclopropane, cyclobutane, cyclopentane, or cyclohexane). Alicyclic rings are, for example, C3-C 10 Alicyclic rings, preferably C3-C8 alicyclic rings, more preferably C3-C7 alicyclic rings, and most preferably C3-C6 alicyclic rings. Specific examples of alicyclic rings include cyclopropane rings, cyclobutane rings, cyclopentane rings, cyclohexane rings, cycloheptane rings, cyclooctane rings, bicyclic [2.2.1]heptane rings, and cyclohexene rings.

[0231] As used herein, a “saturated heterocycle” is a ring that does not have unsaturated bonds in its ring. The number of atoms constituting a saturated heterocycle is 3 to 14 (3-membered to 14-membered heterocycles), preferably 3 to 12 (3-membered to 12-membered heterocycles), more preferably 3 to 10 (3-membered to 10-membered heterocycles), and most preferably 4 to 7 (4-membered to 7-membered heterocycles). Specific examples of saturated heterocycles include nitrogen-containing butane rings, epoxy-containing rings, oxo-hetero-butane rings, thio-hetero-butane rings, tetrahydrofuran rings, pyrrolidine rings, pyrazolidine rings, imidazoline rings, oxazolidine rings, isoxazolidine rings, thiazoline rings, isothiazolidine rings, thiadiazolidine rings, oxazolidine ketone rings, dioxopentane rings, tetrahydropyran rings, morpholine rings, thiomorpholine rings, 4-oxopyrrolidine rings, piperidine rings, 4-oxopyridine rings, piperazine rings, and dioxane rings.

[0232] As used herein, "peptide compound" means a compound in which two or more amino acids are linked by an amide bond. As long as two or more amino acids are linked by an amide bond, the peptide chain may contain another bond, such as an ester bond or a thioester bond. The number of amino acid residues contained in a 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. Peptides can be linear, branched, or cyclic.

[0233] As used herein, "peptide chain" means the 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, a peptide chain may contain another bond, such as an ester bond or a thioester bond. The number of amino acid residues contained in a 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.

[0234] As used herein, "optionally substituted" means that a group and / or an atom may be substituted by optional substituents and / or optional atoms. That is, it is possible to select either a state in which neither a group nor an atom is substituted by optional substituents or optional atoms, or a state in which one group and / or an atom is substituted by optional substituents and / or optional atoms. Each of the group and / or an atom may be further substituted by optional substituents and / or optional atoms. That is, it is possible to select either a state in which neither a group nor an atom is substituted by optional substituents or optional atoms, or a state in which one group and / or an atom is substituted by optional substituents and / or optional atoms. The group and optional substituents are not limited and can be freely selected, for example, from groups derived from atoms, which are 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 atom and optional atoms are not limited and can be freely selected, for example, from halogen atoms, carbon atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms, and phosphorus atoms. Examples of optional substituents include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclic, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, and formyl.

[0235] As used herein, “optionally protected” means that the group may be protected by an optional protecting group.

[0236] As used herein, “one or more” means a quantity of one, two or more. When “one or more” is used in the context of substituents of a group, this term means a number from 1 to the maximum number of substituents allowed for that group. Specific examples of “one or more” include numbers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and / or greater.

[0237] The compounds described herein, or their salts, may be solvates thereof. Examples of salts of the compounds include hydrochlorides; hydrobroms; hydroiodates; 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 and potassium salts; alkaline earth metal salts, such as magnesium and calcium salts; and ammonium salts, such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts. These salts may be generated, for example, by contacting the compound with an acid or a base. As used herein, a solvate means a solvate in which the compound and the solvent together form a molecular aggregate, and there is no particular limitation as long as it is a solvate formed by a solvent acceptable for uptake when administered with a drug. Specific examples of solvates include not only solvates formed with a single solvent such as water, alcohol (ethanol, methanol, 1-propanol, 2-propanol, etc.) or dimethyl sulfoxide, but also solvates formed by multiple solvents per molecule of the compound, or solvates formed by multiple types of solvents per molecule of the compound. For example, a solvate formed by the compound and water is called a hydrate. These solvates can be generated, for example, by contacting the compound with a solvate. Hydrates are preferred as solvates of the compounds of the present invention. Specifically, hydrates are preferably 1 to 20 hydrates, more preferably 1 to 10 hydrates, further preferably 1 to 5 hydrates, and most preferably 1 to 3 hydrates. Solvates of these salts can be generated, for example, by contacting the compound with an acid or base and a solvent.

[0238] As used herein, “amino acid” includes both natural and non-natural amino acids (sometimes referred to as amino acid derivatives). As used herein, “amino acid” can refer to an amino acid residue. As used herein, “natural amino acid” is any L-amino acid selected from the following: Glycine, L-Ala, L-Ser, L-Thr, L-Val, L-Leu, L-Ile, L-Phe, L-Tyr, L-Trp, L-His, L-Glutamic acid, L-Asp, L-Gln, L-Asn, L-Cys, L-Met, L-Lys, L-Arg, or L-Pro. As used herein, “non-natural amino acid” is an amino acid other than a natural amino acid. Examples of non-natural amino acids include β-amino acids, -Amino acids, D-type amino acids, N-substituted amino acids (except Pro), α,α-disubstituted amino acids, and amino acids having side chains different from those of natural amino acids. As amino acids herein, any conformation of amino acids is acceptable. There are no particular limitations on the side chains of amino acids, and each side chain is freely selected from groups such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaryl, cycloalkyl, or spiro-bonded cycloalkyl, and / or atoms such as hydrogen atoms. Each group and / or atom may further optionally be substituted. In a non-limiting aspect, amino acids as used herein can be compounds having a carboxyl group and an amino group in the same molecule. Even in this case, compounds in which the nitrogen atom of the amino group of the amino acid and any atom of the side chain together form a ring (such as proline, hydroxyproline, and azircyclobutane-2-carboxylic acid) are included in amino acids.

[0239] The main chain amino group of an amino acid can be unsubstituted (i.e., NH2 group) or substituted (i.e., -NHR group, where R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group optionally having a substituent, one or both non-adjacent methylene groups of any such group can be substituted by an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO2-); and the carbon chain bonded to the N atom and the carbon atom at position α can form a ring, as in proline). The substituents of R are selected in the same manner as those for the substituents of the side chains of amino acids described above. In the case of a substituted main chain amino group, R is present in what is referred to herein as the “side chain of the amino acid.” Such amino acids having a substituted main chain amino group are referred to herein as “N-substituted amino acids.” Examples of “N-substituted amino acids,” as used herein, preferably include, but are not limited to, N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, and N-methyl amino acids.

[0240] As used herein, “amino acid” includes all isotopes corresponding to each amino acid. An isotope of an amino acid is one in which at least one atom is replaced by atoms having the same atomic number (number of protons) but different mass numbers (total number of protons and neutrons) at an abundance ratio different from that of the natural amino acid. Examples of isotopes contained in “amino acid” herein include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine atoms, and they each include… 2 H, 3 H, 13 C 14 C 15 N、 17 O、 18 O、 32 P, 35 S, 18 F, 36 Cl, etc. For compounds as used herein, all compounds containing radioactive or non-radioactive isotopic elements in any proportion are covered within the scope of this invention.

[0241] As used herein, examples of "halogen-derived substituents" include fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0242] Examples of "substituents derived from oxygen atoms" include hydroxyl (-OH), oxy group (-OR), oxo (=O), carbonyl (-C(=O)-R), carboxyl (-CO2H), oxycarbonyl (-C(=O)-OR), carbonyloxy group (-OC(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio (-SC(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), aminosulfonylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), and carboxylcarbonyl (-C(=O)-CO2H).

[0243] Examples of alkoxy groups (-OR) include alkoxy, cycloalkoxy, alkenoxy, alkynoxy, aryloxy, heteroaryloxy, and arylalkoxy. Alkoxy groups are preferably C1-C4 alkoxy, C1-C2 alkoxy, and particularly preferably methoxy or ethoxy.

[0244] Examples of carbonyl groups (-C(=O)-R) include formyl (-C(=O)-H), alkyl carbonyl, cycloalkyl carbonyl, alkenyl carbonyl, alkynyl carbonyl, aryl carbonyl, heteroaryl carbonyl, and aralkyl carbonyl.

[0245] Examples of oxycarbonyl groups (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl.

[0246] Examples of carbonyloxy groups (-OC(=O)-R) include alkyl carbonyloxy groups, cycloalkyl carbonyloxy groups, alkenyl carbonyloxy groups, alkynyl carbonyloxy groups, aryl carbonyloxy groups, heteroaryl carbonyloxy groups, and aralkyl carbonyloxy groups.

[0247] Examples of thiocarbonyl (-C(=O)-SR) include alkyl thiocarbonyl, cycloalkyl thiocarbonyl, alkenyl thiocarbonyl, alkynyl thiocarbonyl, aryl thiocarbonyl, heteroaryl thiocarbonyl, and aralkyl thiocarbonyl.

[0248] Examples of carbonyl thiolation (-SC(=O)-R) include alkyl carbonyl thiolation, cycloalkyl carbonyl thiolation, alkenyl carbonyl thiolation, alkynyl carbonyl thiolation, aryl carbonyl thiolation, heteroaryl carbonyl thiolation, and aralkyl carbonyl thiolation.

[0249] Examples of aminocarbonyl groups (-C(=O)-NHR) include alkylaminocarbonyl groups (e.g., C1-C6 or C1-C4 alkylaminocarbonyl groups, specifically ethylaminocarbonyl or methylaminocarbonyl groups), cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. Further examples include groups in which the H atom bonded to the N atom in -C(=O)-NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl group.

[0250] Examples of carbonylamino groups (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino. Additional examples include groups in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl group.

[0251] Examples of oxycarbonylamino groups (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenoxycarbonylamino, alkynoxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, and arylalkoxycarbonylamino. Additional examples include groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl group.

[0252] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, and aralkylsulfonylamino. Additional examples include groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl group.

[0253] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl. Additional examples include groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl group.

[0254] Examples of aminosulfonylamino (-NH-SO2-NHR) include alkylaminosulfonylamino, cycloalkylaminosulfonylamino, alkenylaminosulfonylamino, alkynylaminosulfonylamino, arylaminosulfonylamino, heteroarylaminosulfonylamino, and aralkylaminosulfonylamino. Furthermore, the two H atoms bonded to the N atom in -NH-SO2-NHR are optionally substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and the two substituents may form a ring.

[0255] As used herein, examples of "sulfur-derived substituents" include thiols (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), sulfonyl (-SO3H), pentafluorosulfonyl (-SF5), and dithioalkyl (-SSR).

[0256] Examples of thiolated (-SR) groups include alkyl thiolated, cycloalkyl thiolated, alkenyl thiolated, alkynyl thiolated, aryl thiolated, heteroaryl thiolated, and aralkyl thiolated.

[0257] Examples of sulfinyl groups (-S=OR) include alkyl sulfinyl groups, cycloalkyl sulfinyl groups, alkenyl sulfinyl groups, alkynyl sulfinyl groups, aryl sulfinyl groups, heteroaryl sulfinyl groups, and aralkyl sulfinyl groups.

[0258] Examples of sulfonyl groups (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, and aralkylsulfonyl.

[0259] As used herein, examples of "nitrogen-derived substituents" include azide (-N3, also known as "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amido (-C(=NH)-NH2), substituted amido (-C(=NR)-NR'R''), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R''), and aminocarbonylamino (-NR-CO-NR'R'').

[0260] Examples of secondary amino groups (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, and aralkylamino.

[0261] Examples of tertiary amino groups (-NR(R')) include an amino group having two optional substituents, each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl (aralkyl)amino, and the optional two substituents may form a ring.

[0262] Examples of substituted amidoyl groups (C(=NR)-NR'R'') include those 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 groups, such as alkyl(aralkyl)(aryl)amidinyl.

[0263] Examples of substituted guanidino groups (-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 groups, and groups in which these substituents form a ring.

[0264] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are each independently selected from hydrogen atoms, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, and groups in which these substituents form a ring.

[0265] As used in this article, the "amino acid residues" that form peptide compounds are sometimes simply referred to as "amino acids".

[0266] As used in this article, "to" indicates a range of values ​​that includes both ends of the range. For example, "A to B" means a range of values ​​where A is greater than or equal to B.

[0267] As used in this article, when used in conjunction with a numerical value, the term “approximately” means a range of values ​​between +10% and -10% of that value.

[0268] As used herein, the term “and / or” is intended to include every appropriate combination of the terms “and” and “or”. Specifically, for example, the terms “A, B and / or C” include 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.

[0269] Methods for producing peptide compounds

[0270] In one aspect, the present invention relates to a method for producing a peptide compound or a salt thereof, the method comprising the step of linking an amino group of a first amino acid or peptide to a carboxyl group of a second amino acid or peptide by an amide bond in a bilayer solvent containing water and one or more organic solvents immiscible with water (linking step).

[0271] In one aspect, the present invention relates to a method for producing a peptide compound or a salt thereof, the method comprising the step of linking an amino group of a first amino acid or peptide to a carboxyl group of a second amino acid or peptide by an amide bond in a bilayer solvent containing water and one or more water-immiscible organic solvents (the linking step). In this aspect, the amino group of the first amino acid or peptide is an amino group substituted with a hydrogen atom, a straight-chain C1-C6 alkyl group, a branched C3-C6 alkyl group, or a C3-C8 cycloalkyl group.

[0272] In the embodiments, the linking step is performed in the presence of an activator. Examples of activators include acyl halides, phosphate halides, phosphonate halides, carbodiimides, ureonium, and phosphonium. The acyl halides are preferably straight-chain C1-C. 18 Alkyl carboxyl chloride, branched C3-C 18 Alkyl carbamate chloride, straight-chain C1-C 18 Alkyl carboyl bromide, branched C3-C 18 Alkyl carbohydride, or monocyclic or condensed ring C3-C 15 cycloalkyl carbonyl chloride, wherein C3-C 15 Cycloalkyl carboacyl chlorides are optionally substituted with C1-C6 alkyl groups, more preferably neopentanoyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexane carboacyl chloride, 1-adamantane carboacyl chloride, 2-ethylbutyryl chloride, further preferably neopentanoyl chloride or 2,2-dimethylbutyryl chloride, and most preferably neopentanoyl chloride. Examples of phosphate halides include diethylphosphoryl chloride and diphenylphosphoryl chloride. Examples of phosphonic acid halides include diphenylphosphine chloride. Examples of carbodiimides include N,N-diisopropylcarbodiimide, N,N-dicyclohexylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Examples of ureonium include HATU and COMU. Examples of phosphonium include PyFOP.

[0273] In the embodiments, the linking step is performed in the presence of a solvent. Preferably, step (1) of the linking step is performed in the presence of an additive. Examples of additives include bromide salts, iodide salts, and trifluoromethanesulfonates. 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 trifluoromethanesulfonates include sodium trifluoromethanesulfonate, potassium trifluoromethanesulfonate, magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, and barium trifluoromethanesulfonate. The additive is preferably selected from at least one of 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 in the range of 0.2 molar equivalents to 3.0 molar equivalents, preferably 0.3 molar equivalents to 1.5 molar equivalents relative to the second amino acid or peptide.

[0274] On one hand, the connection step can be carried out, for example, by stirring the reaction mixture for 10 minutes to 24 hours at a reaction temperature of -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.

[0275] In an embodiment, the linking step includes the following steps: (1) contacting a second amino acid or peptide with an acyl halide in an organic solvent to prepare a mixed anhydride; and (2) contacting the mixed anhydride obtained in step (1) with a first amino acid or peptide in the presence of a base in a bilayer solvent containing water and one or more water-immiscible organic solvents. Preferably, linking step (2) includes adding the first amino acid or peptide dropwise to the mixed anhydride obtained in step (1) in the presence of a base.

[0276] On one hand, step (1) in the linking step can be performed, for example, by stirring the reaction mixture for 75 minutes to 7 hours at a reaction temperature of -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.

[0277] On one hand, step (2) in the linking step can be carried out, for example, by stirring the reaction mixture for 10 minutes to 24 hours at a reaction temperature of -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.

[0278] As used herein, "bilayer solvent" refers to a solvent in which water and an immiscible organic solvent form an interface that is visible. Depending on the immiscible organic solvent, the organic solvent may be the bottom layer, or water may be the bottom layer.

[0279] In the embodiments, water-immiscible organic solvents include, but are not particularly limited to, organic solvents having low water solubility (e.g., a solubility in water of 200 g / L or less, preferably 150 g / L or less). Water-immiscible organic solvents may contain trace amounts (e.g., 0.01 wt% or less) of other water-miscible organic solvents. Solubility in water can be determined by any method known in the art or described herein. Examples of methods for determining solubility include, but are not intended to be particularly limited to, gas chromatography, and solubility can be determined by measuring the concentration of the organic solvent in water prepared by mixing the organic solvent with an equal volume of water at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C).

[0280] To determine the miscibility of an organic solvent with water, miscibility can also be demonstrated by the following: when equal volumes of solvent and water are mixed in a container, for example at room temperature (e.g., 15°C to 40°C, preferably 20°C to 30°C), the solvent and water separate into two layers. Whether the solvent and water separate into two layers can be determined, for example, by visual inspection or by collecting and examining the upper and lower layers of liquid in the container. When it is confirmed that the solvent and water separate into two layers in this manner, the solvent can be described as an immiscible solvent. However, even solvents that are miscible with water can still form an interface with water and separate into two layers, depending on the solute in the solvent and the salt concentration in the water.

[0281] In the embodiments, the water-immiscible organic solvent is a solvent with an octanol / water partition coefficient (Log Kow) of 5 or higher, or a predicted octanol / water partition coefficient (Log Kow) of 5 or higher. 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 methods in independent, explicit measurements, for example, but not specifically limited to, database searches or literature searches. Examples of methods for measuring the octanol / water partition coefficient include, but are not limited to, the method according to Japanese Industrial Standard JIS 7260-107: 2000 Measurement of Partition coefficient (1-octanol / water) Shake flask method (https: / / kikakurui.com / z7 / Z7260-107-2000-01.html [Accessed September 9, 2024]).

[0282] In the embodiments, water-immiscible organic solvents include ester solvents, ether solvents, haloalkyl solvents, aromatic hydrocarbon solvents, etc. Examples of ester solvents include isopropyl acetate, ethyl acetate, and butyl acetate. Examples of ether solvents include methyl tert-butyl ether and diethyl ether. Examples of haloalkyl solvents include dichloromethane and carbon tetrachloride. Examples of aromatic hydrocarbon solvents include toluene and hexane. Among these solvents, isopropyl acetate, methyl tert-butyl ether, dichloromethane, 2-methyltetrahydrofuran, and toluene are preferred.

[0283] In the embodiments, the ratio of water to one or more water-immiscible organic solvents in the bonding 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.

[0284] In the embodiments, the organic solvent used in step (1) of the connecting step is not particularly limited unless it inhibits the formation of mixed anhydrides, but is preferably an organic solvent immiscible with water. The solvent used in step (1) includes ester-based solvents, ether-based solvents, amide-based solvents, and nitrile-based solvents. Examples of ester-based solvents include isopropyl acetate, ethyl acetate, and butyl acetate. Examples of ether-based solvents include tetrahydrofuran, 2-methyltetrahydrofuran, and diethyl ether. Examples of amide-based solvents include N,N-dimethylformamide, N,N-dimethylacetamide, and N,N-diethylformamide. Examples of nitrile-based solvents include acetonitrile and propionitrile. Among these solvents, isopropyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, and acetonitrile are preferred.

[0285] In the embodiments, the organic solvent used in step (1) of the linking step is used at a concentration sufficient to dissolve the second amino acid or peptide. The concentration of the second amino acid or peptide used is, 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.

[0286] In some embodiments, organic solvents that may react with the mixed acid anhydrides or peptide compounds may be excluded from the list of water-immiscible organic solvents. Examples of organic solvents that are not suitable for use with water-immiscible organic solvents include amine solvents (e.g., n-propylamine or diisopropylamine) and alcohol solvents (e.g., methanol, ethanol, n-propanol, or phenol).

[0287] In the embodiment, in step (2) of the connection step, the mixture of mixed anhydride and organic solvent obtained in step (1) is preferably used.

[0288] As used in this article, "contact" refers to bringing the solid phase into contact with the liquid phase in a solid-liquid synthesis method, or mixing, adding, or dropping in a liquid-liquid synthesis method.

[0289] As described herein, step (2) of the connecting steps in the method of the present invention is carried out in a bilayer system containing water and one or more solvents immiscible with water. Therefore, it may be useful in the following ways: (a) the water-immiscible solvent can be used as an extraction solvent in the post-treatment of the reaction mixture, and (b) the extraction solution containing the peptide compound can be used as a starting material for subsequent steps / reactions (i.e., a solution containing a starting compound for subsequent steps), etc. In the embodiments, the method of the present invention enables the entire reaction to be carried out and completed without separating the intermediate from the solvent of the initial reaction.

[0290] In the embodiments, the acyl halide is a straight-chain C1-C halide. 18 Alkyl carboxyl chloride, branched C3-C18 Alkyl carbamate chloride, straight-chain C1-C 18 Alkyl carboyl bromide, branched C3-C 18 Alkyl carbohydride or monocyclic or condensed ring C3-C 15 cycloalkyl carbonyl chloride, wherein C3-C 15 The cycloalkyl carboacyl chloride is optionally substituted with a C1-C6 alkyl group. Among these groups, the acyl halide is preferably neopentanoyl chloride, 2,2-dimethylbutyryl chloride, 1-methylcyclohexane carboacyl chloride, 1-adamantane carboacyl chloride or 2-ethylbutyryl chloride, more preferably neopentanoyl chloride or 2,2-dimethylbutyryl chloride, more preferably neopentanoyl chloride or 2,2-dimethylbutyryl chloride, and most preferably neopentanoyl chloride.

[0291] In the embodiments, the acyl halide is used, for example, in amounts of 0.01 to 100 molar equivalents, 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, relative to the second amino acid or peptide.

[0292] In the embodiment, step (1) of the connection step is performed in the presence of an alkali.

[0293] In the embodiments, the base used in step (1) of the connecting step has a pKa of 15.0 or less, preferably 13.0 or less, and more preferably 11.5 or less for the conjugate acid. In the embodiments, the base used in step (1) of the connecting step has a pKa of 0 or more, preferably 3 or more, and more preferably 5 or more for the conjugate acid. In the embodiments, the base used in step (1) of the connecting step has a pKa of 0 to 15.0, preferably 0 to 11.5, more preferably 3 to 11.5, more preferably 5 to 11.5, and most preferably 6 to 11.5 for the conjugate acid. As used herein, “pKa” can be measured using water as a solvent. As used herein, the pKa can be a measurement already reported as pKa when using water as a solvent. When a pKa measurement is not available, the pKa can be calculated using ADMETPredictor (Simulations Plus Inc., version 8.0) and is used herein as pKa. The following are the pKas of representative reagents:

[0294] The conjugate acid of DBU (pKa = 11.9; R. Srivastava, J. Mol. Catal. A: Chem. 264(2007) 146-152);

[0295] The conjugate acid of piperidine (pKa = 11.22; Hall, HK, Jr. JAmChem. Soc. 1957, 79, 5441);

[0296] The conjugate acid of triethylamine (pKa = 10.65; Hall, HK, Jr. JAmChem. Soc. 1957, 79, 5441);

[0297] The conjugate acid of diisopropylethylamine (pKa = 11.44; Chemical and Pharmaceutical Bulletin, 1995, 43, 1872-1877).

[0298] The conjugate acid of 2,4,6-trimethylpyridine (pKa = 7.48; Clarke, K., Rothwell, KJ Chem.Soc. 1960, 1885);

[0299] The conjugate acid of 2,6-dimethylpyridine (pKa = 6.77; Clarke, K., Rothwell, KJ Chem.Soc. 1960, 1885);

[0300] The conjugate acid of pyridine (pKa = 5.21; DH Ripin, DA Evans, pKa's of NitrogenAcids, [online], [searched September 22, 2023], internet)<URL:http: / / evans.rc.fas.harvard.edu / pdf / evans_pKa_table.pdf> ).

[0301] In the embodiments, the base used in step (1) of the linking step is an organic base, 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-dimethylpyridine and 2,4,6-trimethylpyridine. The base used in step (1) of the linking step is preferably one selected from the group consisting of diisopropylethylamine, triethylamine, 2,6-dimethylpyridine, and 2,4,6-trimethylpyridine; more preferably one selected from the group consisting of triethylamine, 2,6-dimethylpyridine, and 2,4,6-trimethylpyridine; most preferably 2,6-dimethylpyridine.

[0302] In the embodiments, the base used in step (1) of the linking step is used, for example, in amounts of 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.

[0303] In the embodiments, the base used in step (2) of the connection 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 bicarbonate, sodium biphosphate, triethylamine and pyridine; further preferably one selected from the group consisting of: sodium carbonate, potassium carbonate, cesium carbonate and potassium phosphate; most preferably one selected from the group consisting of sodium carbonate and potassium carbonate.

[0304] As used herein, "first amino acid" and "second amino acid" are terms used only to distinguish the reaction sites between amino acids, which are substrates in the linking step. Specifically, "first amino acid" refers to an amino acid in which the amino group is the reaction site, and "second amino acid" refers to an amino acid in which the carboxyl group is the reaction site.

[0305] In the embodiments, the first amino acid or peptide is a peptide containing two or more amino acid residues.

[0306] In the embodiments, the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It is a straight-chain C1-C6 alkyl, a branched C3-C6 alkyl, or a C3-C8 cycloalkyl. In some embodiments, the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It is a hydrogen atom or a straight-chain C1-C4 alkyl group. In some embodiments, the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, methyl, or ethyl. In some embodiments, the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5It can be a hydrogen atom or a methyl group. In some embodiments, the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It is a methyl group.

[0307] In the embodiments, the carboxyl group of the first amino acid or peptide is optionally protected by a protecting group. Any protecting group known in the art can be used as a protecting group for the carboxyl group, as long as it does not reduce the solubility of the peptide in the solvent. Specific examples of such protecting groups for the carboxyl group include methyl groups, ethyl groups, tert-butyl groups, triphenylmethyl groups, and cumyl groups, and among these groups, tert-butyl groups are preferred.

[0308] In the embodiments, the second amino acid or peptide is a peptide containing two or more amino acid residues.

[0309] In the embodiments, the α-carbon of the carboxyl group of the second amino acid or peptide is optionally substituted. In the embodiments, the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C6 alkyl, an optionally substituted branched C3-C6 alkyl, an optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, an optionally substituted C3-C8 cycloalkyl, an optionally substituted phenyl-C1-C2 alkyl, an optionally substituted C1-C6 alkoxy-C1-C2 alkyl, an optionally substituted 5- to 6-membered heteroaryl-C1-C2 alkyl, or an optionally substituted phenyl-C1-C2 alkyl thio-C1-C2 alkyl, or R 1 and R 2 Together with the carbon atoms they are attached to, they form C3-C8 saturated alicyclic rings.

[0310] When R 1 Or R 2 When the alkyl group is branched C1-C6, the branched C1-C6 alkyl group may optionally be substituted with one or more halogens.

[0311] When R 1 Or R 2 When it is a C3-C8 cycloalkyl-C1-C6 alkyl, the C3-C8 cycloalkyl-C1-C6 alkyl may optionally be substituted by one or more of the following groups: methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0312] When R 1 Or R 2 When it is a C3-C8 cycloalkyl group, the C3-C8 cycloalkyl group may optionally be substituted by one or more of the following groups: methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0313] When R 1 Or R 2 When it is phenyl-C1-C2 alkyl, the phenyl-C1-C2 alkyl may optionally be substituted by one or more of the group consisting of: methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0314] When R 1 Or R 2 It is a C1-C6 alkoxy-C1-C2 alkyl group, which may optionally be substituted by one or more of the group consisting of: methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0315] When R 1 Or R 2 It is a 5- to 6-membered heteroaryl-C1-C2 alkyl group, which is optionally substituted by one or more of the group consisting of: methyl, ethyl, propyl, halogen, methoxy, ethoxy and trifluoromethyl.

[0316] In some embodiments, the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C3 alkyl, a branched C3-C6 alkyl, a C3-C6 cycloalkyl-C1-C6 alkyl, a C1-C6 alkoxymethyl, a 1-(C1-C6 alkoxy)ethyl, an optionally substituted benzyl, an optionally substituted phenethyl or phenyl-C1-C2 alkylthiomethyl.

[0317] When R 1 Or R 2 When benzyl or phenethyl, benzyl or phenethyl may optionally be substituted by one or more of the group consisting of: fluorine, methyl, ethyl, methoxy, ethoxy, and trifluoromethyl.

[0318] In some embodiments, the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R1 It is a hydrogen atom, and R 2 It can be a hydrogen atom, methyl, 1-methylethyl, 1-methylpropyl, tert-butoxymethyl, 1-(tert-butoxy)ethyl, benzyl, 2-(4-trifluoromethyl-3,5-difluorophenyl)ethyl or benzylthiomethyl.

[0319] In the embodiments, the β-nitrogen at the carboxyl group of the second amino acid or peptide is represented by the following formula: -NR 3 -, where R 3 It is a hydrogen atom.

[0320] In the embodiments, the amino group of the second amino acid or peptide is optionally protected by a protecting group. Any protecting group known in the art can be used as a protecting group for the amino group, as long as it does not reduce the solubility of the peptide in the solvent. Specific examples of such protecting groups for the amino group include Cbz, p-nitrobenzyloxycarbonyl, 2-naphthylmethyloxycarbonyl, diphenylmethyloxycarbonyl, 9-anthraylmethyloxycarbonyl, Teoc, Fmoc, Boc, Alloc, and trifluoroacetyl, and of these groups, Cbz, Teoc, and trifluoroacetyl are preferred.

[0321] In the embodiments, the peptide compound or its salt produced by the method of the present invention contains 8 to 20 residues, preferably 11 to 14 residues, more preferably 11 to 13 residues, and most preferably 11 residues of amino acid residues.

[0322] In one aspect, the peptide compound or its salt produced by the method of the present invention may contain at least one, at least two, at least three, at least four, or at least five non-natural amino acid residues. In some embodiments, the non-natural amino acid residues contained in the peptide compound or its salt produced by the method of the present invention are N-methyl amino acid residues.

[0323] In one aspect, the peptide compound produced by the method of the present invention can be a linear peptide compound. In another aspect, the peptide compound produced by the method of the present invention can be a cyclic peptide compound. In one aspect, a linear or cyclic peptide compound may contain a cyclic structure (cyclic portion) as a part of its structure. Specific examples of said cyclic structure include those cyclic structures in which the side chain of one amino acid residue is linked to the side chain of another amino acid residue, those cyclic structures in which the N-substituent of one amino acid residue is linked to the side chain of another amino acid residue, and those cyclic structures 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 linking of the cyclic structure can be adjacent, or any number of amino acid residues can be present between them, for example, any amino acid residues 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 amino acid residues. Examples of ring sizes formed by ring structures include, but are not intended to be particularly limited to, 4-membered rings, 5-membered rings, 6-membered rings, 7-membered rings, 8-membered rings, 9-membered rings, 10-membered rings, 11-membered rings, 12-membered rings, 13-membered rings, 14-membered rings, 15-membered rings, 16-membered rings, 17-membered rings, 18-membered rings, 19-membered rings, 20-membered rings, 21-membered rings, 22-membered rings, 23-membered rings, 24-membered rings, 25-membered rings, 26-membered rings, 27-membered rings, 28-membered rings, 29-membered rings, 30-membered rings, 31-membered rings, 32-membered rings, 33-membered rings, 34-membered rings, and 35-membered rings. Preferably, the rings formed by the cyclic structure are 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered, 13-membered, 14-membered, 15-membered, 16-membered, 17-membered, 18-membered, 19-membered, or 20-membered rings; more preferably, 10-membered, 11-membered, 12-membered, 13-membered, 14-membered, 15-membered, 16-membered, 17-membered, or 18-membered rings; most preferably, 11-membered, 12-membered, 13-membered, or 14-membered rings. When a cyclic structure is present in the peptide compound, the number of cyclic structures is not limited, but preferably one, two, three, four, or five cyclic structures are present.

[0324] In embodiments, the peptide compound or its salt produced by the method of the present invention may contain a cyclic moiety consisting of 4 or more, 6 or more, 8 or more, or 11 or more amino acid residues. In embodiments, the peptide compound or its salt produced by the method of the present invention is a cyclic peptide compound or its salt consisting of a cyclic moiety consisting of 4 to 14, preferably 6 to 14, more preferably 8 to 14, and most preferably 11 to 14 amino acid residues. In embodiments, the peptide compound or its salt produced by the method of the present invention is a cyclic peptide compound or its salt consisting of a cyclic moiety consisting of 11 amino acid residues. In these respects, the amide bond connecting the amino group of the first amino acid or peptide to the carboxyl group of the second amino acid or peptide is contained at positions 1, 2, 3, 4, 5, 6, or 7 in the cyclic moiety.

[0325] In embodiments, the method of the present invention may further include the following step (deprotection step): removing / deprotecting the protecting groups of the amino or carboxyl groups of the second amino acid or peptide and / or the amino or carboxyl groups of the first amino acid or peptide in the peptide compound obtained in the ligation step, immediately before or after and / or during the ligation step. The deprotection step may be performed using methods known in the art (e.g., using the reagents and conditions described in Greene's Protective Groups in Organic Synthesis, Fifth Edition, 2014).

[0326] On one hand, the deprotection step of the protecting group of the amino group can be carried out by contact hydrogenation, for example, when the protecting group is Cbz, p-nitrobenzyloxycarbonyl, 2-naphthylmethyloxycarbonyl, diphenylmethyloxycarbonyl, or 9-anthraylmethyloxycarbonyl. For contact hydrogenation, any catalyst known in the art can be used. Specific examples of catalysts include Pd / C, Pd(OH)2 / C, and PtO2, with Pd / C being preferred.

[0327] On the one hand, the deprotection step of the protecting group of the amino group can be carried out using reagents (such as TBAF, LiBH4, piperidine, trifluoroacetic acid or methanesulfonic acid), for example when the protecting group is Teoc, Fmoc, Boc, Alloc or trifluoroacetyl.

[0328] On the one hand, the deprotection step of the carboxyl group can be carried out under acidic conditions in the presence of a deprotecting agent, for example, when the protecting group is a methyl group, an ethyl group, a tert-butyl group, a triphenylmethyl group, or a cumyl group.

[0329] In an embodiment, the method of the present invention may further include a step of linking / condensing a third amino acid or peptide with a peptide compound obtained in the linking step or deprotection step (linking step A).

[0330] In embodiments, the method of the present invention may further include a step of linking / condensing amino and carboxyl groups in the peptide compound obtained in the linking or deprotection step (linking step B). In this case, the peptide compound obtained in linking step B is a cyclic peptide compound.

[0331] On one hand, steps A and B can be performed, for example, in the presence or absence of a condensing agent. Examples of condensing agents include T3P, EDCI, HATU, COMU, BEP, PyBOP, DMT-MM, and PyOxim.

[0332] In the embodiments, the method of the present invention is carried out using a flow reaction apparatus. As used herein, a “flow reaction apparatus” refers to an apparatus that continuously feeds raw materials into a tubular reaction vessel and mixes and reacts the raw materials to produce a target compound. A flow reaction apparatus may be a commercially available flow reaction apparatus (e.g., an African or Asian flow chemistry system manufactured by Syrris), or it may be an apparatus combining a tubular reaction vessel and a syringe pump.

[0333] peptide compounds

[0334] In one aspect, the present invention relates to a peptide compound or a salt thereof produced by the above-described "method for producing a peptide compound". In another aspect, the present invention relates to a pharmaceutical composition containing a peptide compound or a salt thereof produced by the above-described "method for producing a peptide compound".

[0335] All prior art referenced herein is incorporated herein by reference. This application claims priority to Japanese Patent Application No. 2023-168751, filed on September 28, 2023, the contents of which are hereby incorporated by reference in their entirety. All references cited in this document (including patent applications and publications) are incorporated herein by reference in their entirety, including the following references: International Publication No. WO 2013 / 100132, International Publication No. WO 2018 / 225851, International Publication No. WO2018 / 225864, International Publication No. WO 2019 / 117274; International Publication No. WO 2020 / 111238; International Publication No. WO 2020 / 122182; International Publication No. WO 2021 / 075478; International Publication No. WO 2021 / 090856; International Publication No. WO 2021 / 132545; International Publication No. WO 2021 / 246471; International Publication No. WO 2022 / 097540; International Publication No. WO 2022 / 138891; International Publication No. WO 2022 / 145444; International Publication No. WO 2022 / 234864; International Publication No. WO 2023 / 127869; International Publication No. WO 2023 / 219152; International Publication No. 2024 / 096023; and International Publication No. 2024 / 143514.

[0336] [Example]

[0337] The invention will be further described through the following examples, but the invention is not limited thereto. Except as specifically described, the starting materials, starting ingredients, solvents, and reagents are obtained from commercial suppliers or synthesized using known methods.

[0338] 1 H-NMR spectra were measured using a JNM-ECZ 500 nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.). The chemical shift of tetramethylsilane, used as an internal standard, was set to 0 ppm, and the signals followed the deuterium-locked signal from the sample solvent. The chemical shifts of the signals from the analytes are expressed in ppm. Signal splits are abbreviated as follows: s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quadruplet, dd = doublet, m = multiplet. The width of the signal split is expressed as the J value (Hz). The integral value of the signal is calculated based on the ratio of the area intensity of each signal.

[0339] HPLC analysis was performed using a Class H system manufactured by Waters Corporation, with measurements taken at 210 nm using a PDA detector. 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 either an SQD2 or QDa detector.

[0340] [Table 1]

[0341]

[0342] Unless otherwise specified in the experimental section, HPLC samples should be prepared as follows.

[0343] Activation step: Dissolve 2 to 3 μL of the reaction mixture in MeCN (1.0 mL) and aziridine (0.1 mL).

[0344] Condensation step: Dissolve 5 μL of the reaction mixture in MeCN (1.0 mL) and aziridine (0.1 mL).

[0345] The conversion 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. The substrate in the formula refers to the substrate with the smaller molar equivalent between the two substrates.

[0346] Condensation = [(Desired Product + Epimer) / (Substrate + Desired Product + Epimer)] x 100

[0347] Diastereomer ratio = Desired product / Epimer

[0348] (Example 1) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: Two-layer conditions with organic solvent and water

[0349] [Formula 1]

[0350]

[0351] Activation and condensation reaction steps

[0352] 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-dimethylpyridine (104.0 μL, 0.895 mmol) were added to a test tube equipped with a stirrer (hereinafter 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. The reaction mixture was analyzed by HPLC to confirm that activation had progressed. 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 equipped with a stirrer (hereinafter referred to as "test tube B"), and the reaction mixture was cooled to 0°C while stirring. The mixture in test tube A was added dropwise to test tube B, and the resulting mixture was continuously stirred at 0°C for 22 hours.

[0353] Reaction conversion rate: 98.8%

[0354] Diastereomer ratio: 99.6 / 0.4

[0355] Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 1.8%

[0356] Post-processing steps

[0357] 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 while the temperature was raised to 25 °C with stirring. A 2.5% aqueous ammonia solution (2.00 mL) was added to the organic layer. The mixture was stirred and allowed to stand, then the aqueous layer was removed. A 5% aqueous sulfuric acid solution (2.00 mL) was added to the organic layer. The mixture was stirred and allowed to stand, then the aqueous layer was removed. A 5% aqueous Na₂CO₃ solution was added to the organic layer. The mixture was stirred and allowed to stand, then the aqueous layer was removed. The resulting organic layer was concentrated to dryness to obtain 513.4 mg of the target substance.

[0358] Yield: 98%

[0359] Purity: 96.8%

[0360] Measurement method: HPLC method A

[0361] Retention times: 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

[0362] Mass spectrum: 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] + ), Required product: m / z 732.98 ([M-Sar-OtBu] + )

[0363] (Example 2) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0364] The condensation reaction was carried out under the following conditions A through D.

[0365] [Equation 2]

[0366]

[0367] Condition A

[0368] Add a solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.240 mL, 44.4 mg, 0.113 mmol) and DIPEA (22.4 μL, 0.128 mmol) to a test tube equipped with a stirrer, and cool the mixture to -20°C while stirring. Add PivCl (13.2 μL, 0.107 mmol) to the reaction mixture and stir the mixture for 1 hour and 25 minutes. Add a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (concentration 156.0 mg / mL, 0.280 mL, 43.7 mg, 0.087 mmol) to the reaction mixture, and stir the mixture for 15 minutes while heating to 0°C. Then add an aqueous Na2CO3 solution (27.2 mg Na2CO3, 0.270 mL H2O) and stir the mixture continuously for 8 hours.

[0369] Condition B

[0370] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.240 mL, 44.4 mg, 0.113 mmol) and DIPEA (22.4 μL, 0.128 mmol) was added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring. PivCl (13.2 μL, 0.107 mmol) was added to the reaction mixture, and the mixture was stirred for 1 hour and 25 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (concentration 156.0 mg / mL, 0.280 mL, 43.7 mg, 0.087 mmol) was added to the reaction mixture, and the mixture was stirred for 15 minutes while heating to 0°C. Then DIPEA (44.7 μL, 0.257 mmol) was added, and the mixture was continuously stirred for 8 hours.

[0371] Condition C

[0372] Add HATU (76.0 mg, 0.199 mmol), a solution of Cbz-MeLeu-Ile-OH in MeCN (concentration 200.0 mg / mL, 0.210 mL, 42.0 mg, 0.107 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 142.9 mg / mL, 0.270 mL, 38.6 mg, 0.077 mmol), and DIPEA (34.7 μL, 0.199 mmol) to a vial equipped with a stirrer in sequence, and stir the mixture.

[0373] Condition D

[0374] HOPO (9.5 mg, 0.086 mmol), a solution of Cbz-MeLeu-Ile-OH in IPAC (concentrations 185.0 mg / mL, 0.180 mL, 33.6 mg, 0.086 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (concentrations 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 equipped with a stirrer, and the mixture was 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 hours.

[0375] Synthesis of epimers

[0376] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-allo-Ile-OH is used instead of Cbz-MeLeu-Ile-OH.

[0377] Measurement method: HPLC method A

[0378] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 7.8 min, desired product: 16.2 min, epimer: 16.6 min

[0379] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.79 ([M+H] + ), Required product: m / z 732.87 ([M-Sar-OtBu] + Epimer: m / z 732.87 ([M-Sar-OtBu]) + )

[0380] [Table 2]

[0381]

[0382] As shown in Table 2, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion rate and the diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B), the conditions using HATU as a condensing agent (condition C), and the known bilayer conditions (condition D), one or both of the conversion rate and the diastereomeric ratio were significantly lower.

[0383] (Example 3) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0384] The condensation reaction was carried out under the following conditions A to C.

[0385] [Formula 3]

[0386]

[0387] Condition A

[0388] Add a solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.220 mL, 40.7 mg, 0.100 mmol) and DIPEA (18.8 μL, 0.108 mmol) to a test tube equipped with a stirrer, and cool the mixture to -20°C while stirring. Add PivCl (11.6 μL, 0.094 mmol) to the reaction mixture and stir the mixture for 1 hour and 30 minutes. Add a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) to the reaction mixture, and stir the mixture for 15 minutes while heating to 0°C. Then add an aqueous Na2CO3 solution (21.0 mg Na2CO3, 0.210 mL H2O) and stir the mixture continuously for 22 hours.

[0389] Condition B

[0390] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.220 mL, 40.7 mg, 0.100 mmol) and DIPEA (18.8 μL, 0.108 mmol) was added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture, and the mixture was stirred for 1 hour and 30 minutes. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) was added to the reaction mixture, and the mixture was stirred for 15 minutes while heating to 0°C. Then DIPEA (35.2 μL, 0.202 mmol) was added, and the mixture was continuously stirred for 22 hours.

[0391] Condition C

[0392] Add HATU (51.2 mg, 0.135 mmol), Cbz-MeLeu-Ile-OH in MeCN (concentrations 200.0 mg / mL, 0.150 mL, 30.0 mg, 0.076 mmol), H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentrations 170.0 mg / mL, 0.300 mL, 51.0 mg, 0.069 mmol), and DIPEA (23.4 μL, 0.134 mmol) sequentially to a vial equipped with a stirrer, and stir the mixture for 3 hours.

[0393] Synthesis of epimers

[0394] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-allo-Ile-OH is used instead of Cbz-MeLeu-Ile-OH.

[0395] Measurement method: HPLC method A

[0396] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min

[0397] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.92 ([M+H] + ), Required product: m / z 1118.26 ([M+H] + Epimer: m / z 1118.32 ([M+H]) + )

[0398] [Table 3]

[0399]

[0400] As shown in Table 3, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion rate and the diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B) and the conditions using HATU as a condensing agent (condition C), one or both of the conversion rate and the diastereomeric ratio were significantly lower.

[0401] (Example 4) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0402] The condensation reaction was carried out under the conditions A to C shown below.

[0403] [Formula 4]

[0404]

[0405] Condition A

[0406] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.230 mL, 42.6 mg, 0.108 mmol) and DIPEA (21.2 μL, 0.122 mmol) was added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 1 hour and 15 minutes. A solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) was added to the reaction mixture, and the mixture was stirred for 15 minutes while heating to 0°C. Then, an aqueous Na2CO3 solution (26.0 mg Na2CO3, 0.260 mL H2O) was added, and the mixture was continuously stirred for 9 hours.

[0407] Condition B

[0408] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.230 mL, 42.6 mg, 0.108 mmol) and DIPEA (21.2 μL, 0.122 mmol) was added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 1 hour and 15 minutes. A solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) was added to the reaction mixture, and the mixture was stirred for 15 minutes while heating to 0°C. Then DIPEA (42.4 μL, 0.224 mmol) was added, and the mixture was continuously stirred for 9 hours.

[0409] Condition C

[0410] Add HATU (51.5 mg, 0.135 mmol), a solution of Cbz-MeLeu-Ile-OH in MeCN (concentrations of 200.0 mg / mL, 0.150 mL, 30.0 mg, 0.076 mmol), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (concentrations of 75.8 mg / mL, 0.330 mL, 25.0 mg, 0.068 mmol), and DIPEA (23.4 μL, 0.134 mmol) to a vial equipped with a stirrer in sequence, and stir the mixture for 2 hours.

[0411] Synthesis of epimers

[0412] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-allo-Ile-OH is used instead of Cbz-MeLeu-Ile-OH.

[0413] Measurement method: HPLC method A

[0414] Retention times: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 16.7 min, epimer: 17.5 min

[0415] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.64 ([M+H] + ), Required product: m / z 766.78 ([M+Na] + Epimer: m / z 767.01 ([M+Na]) + )

[0416] [Table 4]

[0417]

[0418] As shown in Table 4, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion rate and the diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B) and the conditions using HATU as a condensing agent (condition C), one or both of the conversion rate and the diastereomeric ratio were significantly lower.

[0419] (Example 5) Condensation reaction of Cbz-MeLeu-Ala-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0420] The condensation reaction was carried out under conditions A to C shown in the table below.

[0421] [Formula 5]

[0422]

[0423] Condition A

[0424] Add a solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol) and 2,6-dimethylpyridine (13.5 μL, 0.115 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (12.0 μL, 0.098 mmol) to the reaction mixture, and stir the mixture for 3 hours. Add a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.330 mL, 66.0 mg, 0.089 mmol) to the reaction mixture, and stir the mixture for 10 minutes while heating to 0 °C. Then add an aqueous Na2CO3 solution (28.0 mg Na2CO3, 0.280 mL H2O) and stir the mixture continuously for 6 hours.

[0425] Condition B

[0426] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol) and 2,6-dimethylpyridine (13.5 μL, 0.115 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (12.0 μL, 0.098 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.330 mL, 66.0 mg, 0.089 mmol) was added to the reaction mixture, and the mixture was stirred for 10 minutes while heating to 0 °C. Then DIPEA (46.0 μL, 0.266 mmol) was added, and the mixture was continuously stirred for 6 hours.

[0427] Condition C

[0428] Add HATU (67.6 mg, 0.178 mmol), Cbz-MeLeu-Ala-OH in IPAC (concentration 156.0 mg / mL, 0.240 mL, 37.4 mg, 0.107 mmol), 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) to a vial equipped with a stirrer in sequence, and stir the mixture.

[0429] Measurement method: HPLC method A

[0430] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.4 min, desired product: 17.4 min, epimer: 17.7 min

[0431] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.15 ([M+H] + ), Required product: m / z 931.09 ([M-Sar-OtBu] + Epimer: m / z 931.27 ([M-Sar-OtBu]) + )

[0432] [Table 5]

[0433]

[0434] As shown in Table 5, it was found that both the conversion and diastereomeric ratio were higher under the bilayer conditions of organic solvent and water (condition A), while one or both of the conversion and diastereomeric ratio were lower under the monolayer conditions of organic solvent only (condition B) and the conditions using HATU as a condensing agent (condition C) compared to condition A.

[0435] (Example 6) Condensation reaction of Cbz-MeLeu-Phe-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0436] The condensation reaction was carried out under the following conditions A to C.

[0437] [Formula 6]

[0438]

[0439] Condition A

[0440] Add 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-dimethylpyridine (12.6 μL, 0.108 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (11.3 μL, 0.092 mmol) to the reaction mixture, and stir the mixture for 3 hours and 30 minutes. Add a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.310 mL, 62.0 mg, 0.083 mmol) to the reaction mixture, and stir the mixture for 10 minutes while heating to 0 °C. Then add an aqueous Na2CO3 solution (26.0 mg Na2CO3, 0.260 mL H2O) and stir the mixture continuously for 3 hours.

[0441] Condition B

[0442] 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-dimethylpyridine (12.6 μL, 0.108 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.3 μL, 0.092 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours and 30 minutes. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.310 mL, 62.0 mg, 0.083 mmol) was added to the reaction mixture, and the mixture was stirred for 10 minutes while heating to 0 °C. Then DIPEA (43.6 μL, 0.250 mmol) was added, and the mixture was continuously stirred for 3 hours.

[0443] Condition C

[0444] Add HATU (63.5 mg, 0.167 mmol), Cbz-MeLeu-Phe-OH in MeCN (concentration 195.0 mg / mL, 0.200 mL, 39.0 mg, 0.091 mmol), H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 170.0 mg / mL, 0.360 mL, 61.2 mg, 0.082 mmol), and DIPEA (29.0 μL, 0.167 mmol) to a vial equipped with a stirrer in sequence, and stir the mixture.

[0445] Measurement method: HPLC method A

[0446] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 10.4 min, desired product: 18.2 min, epimer: 18.7 min

[0447] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.04 ([M+H] + ), Required product: m / z 1007.35 ([M-Sar-OtBu] + Epimer: m / z 1007.30 ([M-Sar-OtBu]) + )

[0448] [Table 6]

[0449]

[0450] As shown in Table 6, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion and diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B) and the conditions using HATU as a condensing agent (condition C), one or both of the conversion and diastereomeric ratio were significantly lower.

[0451] (Example 7) Condensation reaction of Cbz-MeLeu-Ser(OtBu)-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0452] The condensation reaction was carried out under the following conditions A to C.

[0453] [Formula 7]

[0454]

[0455] Condition A

[0456] Add a solution of Cbz-MeLeu-Ser(OtBu)-OH in IPAC (concentration 155.0 mg / mL, 0.260 mL, 40.3 mg, 0.096 mmol) and 2,6-dimethylpyridine (12.0 μL, 0.103 mmol) to a test tube equipped with a stirrer, and cool the mixture to -20°C while stirring. Add PivCl (10.8 μL, 0.088 mmol) to the reaction mixture, and stir the mixture for 3 hours. Add a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol) to the reaction mixture, and stir the mixture for 15 minutes while heating to 0°C. Then add an aqueous Na2CO3 solution (25.0 mg Na2CO3, 0.250 mL H2O) and stir the mixture continuously for 3 hours.

[0457] Condition B

[0458] A solution of Cbz-MeLeu-Ser(OtBu)-OH in IPAC (concentration 155.0 mg / mL, 0.260 mL, 40.3 mg, 0.096 mmol) and 2,6-dimethylpyridine (12.0 μL, 0.103 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring. PivCl (10.8 μL, 0.088 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.200 mL, 40.0 mg, 0.080 mmol) was added to the reaction mixture, and the mixture was stirred for 15 minutes while heating to 0°C. Then DIPEA (41.6 μL, 0.239 mmol) was added, and the mixture was continuously stirred for 3 hours.

[0459] Condition C

[0460] Add HATU (71.1 mg, 0.187 mmol), Cbz-MeLeu-Ser(OtBu)-OH in IPAC (concentration 155.0 mg / mL, 0.280 mL, 43.4 mg, 0.103 mmol), 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) to a vial equipped with a stirrer in sequence, and stir the mixture.

[0461] Synthesis of epimers

[0462] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-Ser(OtBu)-OH is used instead of Cbz-MeLeu-Ser(OtBu)-OH.

[0463] Measurement method: HPLC method A

[0464] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.5 min, desired product: 17.1 min, epimer: 17.3 min

[0465] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.74 ([M+H] + ), Required product: m / z 762.96 ([M-Sar-OtBu] + Epimer: m / z 763.02 ([M-Sar-OtBu]) + )

[0466] [Table 7]

[0467]

[0468] As shown in Table 7, it was found that both the conversion and diastereomeric ratio were higher under the bilayer conditions of organic solvent and water (condition A), while one or both of the conversion and diastereomeric ratio were lower under the monolayer conditions of organic solvent (condition B) and the conditions using HATU as a condensing agent (condition C) compared to condition A.

[0469] (Example 8) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeLeu-Ile-OtBu

[0470] The condensation reaction is carried out under either condition A or B.

[0471] [Formula 8]

[0472]

[0473] Condition A

[0474] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 12.14 mL, 2.247 g, 5.72 mmol) and 2,6-dimethylpyridine (756 μL, 6.49 mmol) were added to a nitrogen-replaced 100 mL flask, and 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 hours. A solution of H-MeLeu-Ile-OtBu in IPAC (concentration 137.6 mg / mL, 8.72 mL, 1.20 g, 3.82 mmol) and an aqueous Na₂CO₃ solution (1.215 g Na₂CO₃, 12.13 mL H₂O) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 5 hours.

[0475] Condition B

[0476] Add COMU (109.0 mg, 0.254 mmol), Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.364 mL, 67.4 mg, 0.172 mmol), H-MeLeu-Ile-OtBu in IPAC (concentration 137.6 mg / mL, 0.291 mL, 40.0 mg, 0.127 mmol), and DIPEA (44.3 μL, 0.254 mmol) to a test tube equipped with a stirrer in sequence, and stir the mixture.

[0477] Synthesis of epimers

[0478] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-allo-Ile-OH is used instead of Cbz-MeLeu-Ile-OH.

[0479] Measurement method: HPLC method A

[0480] Retention time: H-MeLeu-Ile-OtBu: 8.0 min, desired product: 18.3 min, epimer: 18.8 min

[0481] Mass spectrometry: H-MeLeu-Ile-OtBu: m / z 315.37 ([M+H] + ), Required product: m / z 689.65 ([M+H] + Epimer: m / z 689.56 ([M+H]) + )

[0482] [Table 8]

[0483]

[0484] As shown in Table 8, under the bilayer conditions of organic solvent and water (condition A), both the conversion and diastereomeric ratios are high, while under the condition of using COMU as a condensing agent (condition B), the diastereomeric ratio is significantly lower.

[0485] (Example 9) Condensation reaction of Cbz-MeLeu-Ile-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0486] The condensation reaction was carried out under the conditions A to C shown below.

[0487] [Formula 9]

[0488]

[0489] Condition A

[0490] Add 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-dimethylpyridine (17.4 μL, 0.149 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (15.3 μL, 0.124 mmol) to the reaction mixture, and stir the mixture for 5 hours and 30 minutes. Add a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (concentration 148.7 mg / mL, 0.336 mL, 50.0 mg, 0.099 mmol) and an aqueous Na2CO3 solution (31.9 mg Na2CO3, 0.316 mL H2O) to the reaction mixture. After raising the temperature to 0°C, the mixture was stirred continuously for 16 hours.

[0491] Condition B

[0492] A solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration 116.2 mg / mL, 0.585 mL, 68.0 mg, 0.107 mmol) and 2,6-dimethylpyridine (13.8 μL, 0.119 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (12.3 μL, 0.100 mmol) was added to the reaction mixture, and the mixture was stirred for 5 hours and 30 minutes. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in IPAC (concentration 148.7 mg / mL, 0.269 mL, 40.0 mg, 0.080 mmol) and DIPEA (41.6 μL, 0.239 mmol) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 16 hours.

[0493] Condition C

[0494] Add COMU (42.6 mg, 0.099 mmol), Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration 116.2 mg / mL, 0.366 mL, 42.5 mg, 0.067 mmol), 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) to a test tube equipped with a stirrer in sequence, and stir the mixture.

[0495] Measurement method: HPLC method A

[0496] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.0 min, desired product: 17.8 min, epimer: 18.5 min

[0497] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.36 ([M+H] + ), Required product: m / z 1117.83 ([M+H] + Epimer: m / z 1117.88 ([M+H]) + )

[0498] [Table 9]

[0499]

[0500] As shown in Table 9, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion and diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B) and the conditions using COMU as a condensing agent (condition C), one or both of the conversion and diastereomeric ratio were significantly lower.

[0501] (Example 10) Condensation reaction of Cbz-MeLeu-Ile-MeLeu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0502] The condensation reaction was carried out under the following conditions A to C.

[0503] [Formula 10]

[0504]

[0505] Condition A

[0506] A solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration 116.2 mg / mL, 0.597 mL, 69.4 mg, 0.110 mmol) and 2,6-dimethylpyridine (14.2 μL, 0.122 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 7 hours. A solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 140.2 mg / mL, 0.214 mL, 30.0 mg, 0.081 mmol) and an aqueous Na2CO3 solution (25.8 mg Na2CO3, 0.258 mL H2O) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 24 hours.

[0507] Condition B

[0508] A solution of Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration 116.2 mg / mL, 0.597 mL, 69.4 mg, 0.110 mmol) and 2,6-dimethylpyridine (14.1 μL, 0.122 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (12.5 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 7 hours. A solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 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. After raising the temperature to 0 °C, the mixture was continuously stirred for 24 hours.

[0509] Condition C

[0510] Add COMU (34.8 mg, 0.081 mmol), Cbz-MeLeu-Ile-MeLeu-Ile-OH in IPAC (concentration 116.2 mg / mL, 0.298 mL, 34.7 mg, 0.055 mmol), H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 140.2 mg / mL, 0.107 mL, 15.0 mg, 0.041 mmol), and DIPEA (14.1 μL, 0.081 mmol) to a test tube equipped with a stirrer in sequence, and stir the mixture.

[0511] Measurement method: HPLC method A

[0512] Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 18.3 min, epimer: 19.4 min

[0513] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.39 ([M+H] + ), Required product: m / z 1007.04 ([M+Na] + Epimer: m / z 1006.86 ([M+Na]) + )

[0514] [Table 10]

[0515]

[0516] As shown in Table 10, it was found that under the bilayer conditions of organic solvent and water (condition A), both the conversion and diastereomeric ratio were high, while under the monolayer conditions of organic solvent only (condition B) and the conditions using COMU as a condensing agent (condition C), one or both of the conversion and diastereomeric ratio were significantly lower.

[0517] (Example 11) Condensation reaction of Cbz-MeLeu-Ile-OH and H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0518] The condensation reaction was carried out under the following conditions A to C.

[0519] [Equation 11]

[0520]

[0521] Condition A

[0522] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.298 mL, 55.1 mg, 0.140 mmol) and 2,6-dimethylpyridine (18.2 μL, 0.156 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (16.0 μL, 0.130 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours and 30 minutes. A solution of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 128.7 mg / mL, 0.389 mL, 50.0 mg, 0.104 mmol) and an aqueous Na2CO3 solution (33.1 mg Na2CO3, 0.331 mL H2O) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 19 hours.

[0523] Condition B

[0524] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.298 mL, 55.1 mg, 0.140 mmol) and 2,6-dimethylpyridine (18.1 μL, 0.156 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (16.0 μL, 0.130 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours and 30 minutes. A solution of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 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. After raising the temperature to 0 °C, the mixture was continuously stirred for 19 hours.

[0525] Condition C

[0526] Add COMU (44.7 mg, 0.104 mmol), Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.149 mL, 27.6 mg, 0.070 mmol), H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 128.7 mg / mL, 0.194 mL, 25.0 mg, 0.052 mmol), and DIPEA (18.1 μL, 0.104 mmol) to a test tube equipped with a stirrer in sequence, and stir the mixture.

[0527] Synthesis of epimers

[0528] Synthesize the diastereomer according to condition A, the difference being that Cbz-MeLeu-D-allo-Ile-OH is used instead of Cbz-MeLeu-Ile-OH.

[0529] Measurement method: HPLC method A

[0530] Retention time: H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2: 7.6 min, desired product: 16.4 min, epimer: 16.9 min

[0531] Mass spectrum: H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 481.43 ([M+H] + ), Required product: m / z 877.81 ([M+Na] + Epimer: m / z 877.79 ([M+Na]) + )

[0532] [Table 11]

[0533]

[0534] As shown in Table 11, it was found that both the conversion and diastereomeric ratio were higher under the bilayer conditions of organic solvent and water (condition A), while one or both of the conversion and diastereomeric ratio were lower under the monolayer conditions of organic solvent only (condition B) and the conditions using COMU as a condensing agent (condition C) compared to condition A.

[0535] (Example 12) Evaluation of the effects of base and solvent on the formation of mixed acid anhydrides

[0536] The condensation reaction was carried out under the conditions shown in Table 12 below.

[0537] [Equation 12]

[0538]

[0539] Solutions of Cbz-MeLeu-Phe-OH in each solvent (concentrations of 198.0 mg / mL, 0.300 mL, 59.4 mg, 0.139 mmol) and each base (0.153 mmol) were added to test tubes equipped with stirrers, 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 collected over time and dissolved in MeCN (1.0 mL) and aziridine (0.1 mL), and then analyzed by HPLC.

[0540] Measurement method: HPLC method A

[0541] Retention times: Cbz-MeLeu-Phe-OH: 13.1 min, Cbz-MeLeu-Phe-Aze: 13.35 min, epimer: 13.44 min

[0542] Mass spectrometry: Cbz-MeLeu-Phe-OH: m / z 427.88 ([M+H]) + ), Cbz-MeLeu-Phe-Aze: m / z466.87 ([M+H] + Epimer: m / z 466.87 ([M+H]) + )

[0543] [Table 12]

[0544]

[0545] As shown in Table 12, higher conversion rates and diastereomeric ratios were found when using 2,6-dimethylpyridine or diisopropylethylamine as bases.

[0546] (Example 12-1) The role of solvent in the Cbz-MeLeu-Ile-OH activation reaction

[0547] The activation reaction is carried out under the following conditions.

[0548] [Equation 13]

[0549]

[0550] Cbz-MeLeu-Ile-OH (53.4 mg, 0.136 mmol), solvent (0.400 mL), and 2,6-dimethylpyridine (17.6 μL, 0.151 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 0 °C while stirring. PivCl (15.5 μL, 0.126 mmol) was added to the reaction mixture, and the mixture was stirred. 3 μL of the reaction mixture was collected over time and dissolved in MeCN (1.0 mL) and aziridine (0.1 mL). The conversion rate and diastereomeric ratio of the activation reaction were then analyzed by HPLC.

[0551] Measurement method: HPLC method A

[0552] Retention times: Cbz-MeLeu-Ile-OH: 13.2 min, Cbz-MeLeu-Ile-Aze: 13.4 min, epimer: 13.8 min

[0553] Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.79 ([M+H]) + ), Cbz-MeLeu-Ile-Aze: m / z432.90 ([M+H] + Epimer: m / z 432.84 ([M+H]) + )

[0554] Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100

[0555] Diastereomer ratio = Cbz - MeLeu - Ile - aze / diastereomer

[0556] [Table 12-1]

[0557]

[0558] As shown in Table 12-1, under conditions using ester solvents (such as IPAC) and aromatic cyclic solvents (such as toluene), both the reactivity and diastereomeric ratios are high.

[0559] (Example 12-2) The role of base in the activation reaction of Cbz-MeLeu-Ile-OH

[0560] The activation reaction is carried out under the following conditions.

[0561] [Formula 14]

[0562]

[0563] Cbz-MeLeu-Ile-OH (53.4 mg, 0.136 mmol), toluene (0.400 mL), and each base (0.151 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 0 °C while stirring. PivCl (15.5 μL, 0.126 mmol) was added to the reaction mixture, and the mixture was stirred. 3 μL of the reaction mixture was collected over time and dissolved in MeCN (1.0 mL) and aziridine (0.1 mL). The conversion rate and diastereomeric ratio of the activation reaction were then analyzed by HPLC.

[0564] Measurement method: HPLC method A

[0565] Retention times: Cbz-MeLeu-Ile-OH: 13.2 min, Cbz-MeLeu-Ile-Aze: 13.4 min, epimer: 13.8 min

[0566] Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.79 ([M+H]) + ), Cbz-MeLeu-Ile-Aze: m / z432.90 ([M+H] + Epimer: m / z 432.84 ([M+H]) + )

[0567] Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100

[0568] Diastereomer ratio = Cbz - MeLeu - Ile - aze / diastereomer

[0569] [Table 12-2]

[0570]

[0571] As shown in Table 12-2, both reactivity and diastereomeric ratios were high under conditions using weak bases (such as pyridine and 2,6-dimethylpyridine). (The diastereomeric ratios under conditions using 2,6-di-tert-butylpyridine are not shown because the reaction conversion was significantly lower (NA: not analyzed)).

[0572] (Example 13) Evaluation of the effect of temperature on the formation of mixed acid anhydrides

[0573] The condensation reaction was carried out under the conditions shown in Table 13 below.

[0574] [Formula 15]

[0575]

[0576] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentrations of 200.0 mg / mL, 0.200 mL, 40.0 mg, 0.102 mmol) and 2,6-dimethylpyridine (11.8 μL, 0.101 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to each temperature described 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 collected over time and dissolved in MeCN (1.0 mL) and aziridine (0.1 mL), and then analyzed by HPLC.

[0577] Measurement method: HPLC method A

[0578] Retention times: Cbz-MeLeu-Ile-OH: 12.9 min, Cbz-MeLeu-Ile-Aze: 13.1 min, epimer: 13.5 min

[0579] Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.74 ([M+H]) + ), Cbz-MeLeu-Ile-Aze: m / z432.95 ([M+H] + Epimer: m / z 432.90 ([M+H]) + )

[0580] [Table 13]

[0581]

[0582] As shown in Table 13, the conversion rate and diastereomeric ratio were both high at temperatures of -18℃ and -5℃.

[0583] (Example 13-1) Accelerating effect of additives in the Cbz-MeLeu-Ile-OH activation reaction

[0584] The activation reaction is carried out under the following conditions.

[0585] [Formula 16]

[0586]

[0587] Add the additive, a solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 157.0 mg / mL, 0.200 mL, 31.4 mg, 0.081 mmol), and 2,6-dimethylpyridine (10.4 μL, 0.090 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add 2,2-dimethylbutyryl chloride (10.2 μL, 0.075 mmol) to the reaction mixture and stir. Collect 2 to 3 μL of the reaction mixture over time and dissolve it in MeCN (1.0 mL) and aziridine (0.1 mL), then analyze the conversion and diastereomeric ratio of the activation reaction by HPLC.

[0588] Measurement method: HPLC method A

[0589] Retention times: Cbz-MeLeu-Ile-OH: 12.8 min, Cbz-MeLeu-Ile-Aze: 13.1 min, epimer: 13.4 min

[0590] Mass spectrometry: Cbz-MeLeu-Ile-OH: m / z 393.74 ([M+H]) + ), Cbz-MeLeu-Ile-Aze: m / z432.95 ([M+H] + Epimer: m / z 432.90 ([M+H]) + )

[0591] Conversion rate = [(Cbz-MeLeu-Ile-aze + epimer) / (Cbz-MeLeu-Ile-OH + Cbz-MeLeu-Ile-aze + epimer)] x 100

[0592] Diastereomer ratio = Cbz - MeLeu - Ile - aze / diastereomer

[0593] [Table 13-1]

[0594]

[0595] As shown in Table 13-1, a significant reaction acceleration effect was found when NaOTf, KOTf, or NaI was added compared to the condition without additives.

[0596] (Example 14) Evaluation of the effect of solvent on fragment condensation reaction

[0597] The condensation reaction was carried out under the conditions shown in Table 14 below.

[0598] [Equation 17]

[0599]

[0600] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentrations 185.0 mg / mL, 0.231 mL, 42.8 mg, 0.109 mmol) and 2,6-dimethylpyridine (14.0 μL, 0.121 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (12.4 μL, 0.101 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in each solvent (concentrations 170.0 mg / mL, 0.353 mL, 60.0 mg, 0.081 mmol) and an aqueous Na₂CO₃ solution (25.7 mg Na₂CO₃, 0.257 mL H₂O) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 5 hours.

[0601] Measurement method: HPLC method A

[0602] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min

[0603] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.60 ([M+H] +), Required product: m / z 1117.78 ([M+H] + Epimer: m / z 1117.85 ([M+H]) + )

[0604] [Table 14]

[0605]

[0606] It was found that the conversion rate and diastereomer ratio were high when using the solvents shown in Table 14.

[0607] (Example 15) Assessment of the scope of acyl halide reagents in fragment condensation reactions

[0608] The condensation reaction was carried out under the conditions shown in Table 15 below.

[0609] [Formula 18]

[0610]

[0611] Add a solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 185.0 mg / mL, 0.231 mL, 42.8 mg, 0.109 mmol) and 2,6-dimethylpyridine (14.0 μL, 0.121 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Each acyl halide reagent (neopentyl 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), 2-ethylbutyryl chloride (14.0 μL, 0.101 mmol)) was added to the reaction mixture, and the mixture was stirred for 3 hours. A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 170.0 mg / mL, 0.353 mL, 60.0 mg, 0.081 mmol) and an aqueous Na2CO3 solution (25.7 mg Na2CO3, 0.257 mL H2O) were added to the reaction mixture. After raising the temperature to 0 °C, the mixture was stirred continuously for 5 hours.

[0612] Examples of using 2,2-dimethylbutyryl chloride were performed using the following procedure. A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 180.0 mg / mL, 0.225 mL, 40.5 mg, 0.103 mmol) and DIPEA (19.9 μL, 0.114 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -20°C while stirring (hereinafter 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. Add a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.250 mL, 50.0 mg, 0.067 mmol) and an aqueous Na2CO3 solution (21.4 mg Na2CO3, 0.210 mL H2O) to another test tube, and cool the mixture to 0°C (hereinafter referred to as test tube B). Add the contents of test tube A to test tube B, and stir the mixture at 0°C for 6 hours.

[0613] Measurement method: HPLC method A

[0614] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.8 min, desired product: 17.9 min, epimer: 18.5 min

[0615] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 743.60 ([M+H] + ), Required product: m / z 1117.78 ([M+H] + Epimer: m / z 1117.85 ([M+H]) + )

[0616] [Table 15]

[0617]

[0618] It was found that the conversion rate and diastereomer ratio were high when using the acyl halide reagents shown in Table 15.

[0619] (Example 16) Assessment of the range of bases in fragment condensation reactions

[0620] The condensation reaction was carried out under the conditions shown in Table 16 below.

[0621] [Formula 19]

[0622]

[0623] 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-dimethylpyridine (17.3 μL, 0.149 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (15.3 μL, 0.124 mmol) was added to the reaction mixture, and the mixture was stirred for 5 hours. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentrations 144.2 mg / mL, 0.347 mL, 50.0 mg, 0.100 mmol), each base (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. After raising the temperature to 0 °C, the mixture was stirred continuously for 4 hours.

[0624] Measurement method: HPLC method A

[0625] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 7.9 min, desired product: 16.3 min, epimer: 16.8 min

[0626] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.36 ([M+H] + ), Required product: m / z 877.71 ([M+H] + Epimer: m / z 877.73 ([M+H]) + )

[0627] [Table 16]

[0628]

[0629] It was found that the conversion and diastereomer ratio were high when using the bases shown in Table 16, and the conversion and diastereomer ratio were further increased when using stronger bases.

[0630] (Example 17) Condensation reaction of Cbz-Leu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0631] The condensation reaction is carried out under the following conditions A and B.

[0632] [Formula 20]

[0633]

[0634] Condition A

[0635] Add Cbz-Leu-Ile-OH (50.8 mg, 0.134 mmol), IPAC (0.3 mL), 2-MeTHF (0.05 mL), and 2,6-dimethylpyridine (17.4 μL, 0.149 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (15.3 μL, 0.124 mmol) to the reaction mixture, and stir the mixture for 2 hours. Add sequentially a 10% aqueous K₂CO₃ solution (0.382 mL, 41.2 mg K₂CO₃) and a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentrations: 200.0 mg / mL, 0.250 mL, 50.0 mg, 0.100 mmol). After the temperature reaches 0 °C, stir the mixture continuously for 6 hours and 30 minutes.

[0636] Condition B

[0637] 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 (concentrations of 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (75.6 mg, 0.199 mmol) was added to the reaction mixture, and the mixture was stirred continuously for 4 hours.

[0638] Measurement method: HPLC method A

[0639] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 8.7 min, desired product: 16.3 min, epimer: 16.7 min

[0640] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.79 ([M+H] + ), Required product: m / z 864.05 ([M+H] + Epimer: m / z 864.22 ([M+H]) + )

[0641] [Table 17]

[0642]

[0643] As shown in Table 17, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0644] (Example 18) Condensation reaction of Cbz-Leu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0645] The condensation reaction is carried out under the following conditions A and B.

[0646] [Equation 21]

[0647]

[0648] Condition A

[0649] Cbz-Leu-Ile-OH (37.0 mg, 0.098 mmol), IPAC (0.3 mL), and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture, and the mixture was stirred for 2 hours and 55 minutes. A 10% aqueous K₂CO₃ solution (0.315 mL, 33.9 mg K₂CO₃) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe₂ in toluene (concentrations 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 20 hours.

[0650] Condition B

[0651] 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 (concentrations 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and the mixture was stirred continuously for 3 hours.

[0652] Measurement method: HPLC method A

[0653] Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 15.5 min, epimer: 16.5 min

[0654] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), Required product: m / z 752.96 ([M+Na] + Epimer: m / z 753.24 ([M+Na]) + )

[0655] [Table 18]

[0656]

[0657] As shown in Table 18, both the conversion rate and the diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0658] (Example 19) Condensation reaction of Cbz-Leu-D-allo-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0659] The condensation reaction is carried out under the following conditions A and B.

[0660] [Equation 22]

[0661]

[0662] Cbz-Leu-D-allo-Ile-OH (36.9 mg, 0.097 mmol), IPAC (0.3 mL), and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours and 30 minutes. A 10% aqueous K₂CO₃ solution (0.315 mL, 33.9 mg K₂CO₃) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe₂ in toluene (concentrations 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 8 hours.

[0663] Condition B

[0664] Cbz-Leu-D-allo-Ile-OH (36.9 mg, 0.097 mmol), MeCN (0.3 mL), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (concentrations 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 equipped with a stirrer, and the mixture was cooled to 10 °C while stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and the mixture was stirred continuously for 3 hours.

[0665] Measurement method: HPLC method A

[0666] Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 16.5 min, epimer: 15.5 min

[0667] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), Required product: m / z 685.98 ([M-NMe2] + Epimer: m / z 685.98 ([M-NMe2]) + )

[0668] [Table 19]

[0669]

[0670] As shown in Table 19, both the conversion rate and the diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0671] (Example 20) Condensation reaction of Cbz-D-Leu-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0672] The condensation reaction is carried out under the following conditions A and B.

[0673] [Equation 23]

[0674]

[0675] Condition A

[0676] Cbz-D-Leu-Ile-OH (36.9 mg, 0.097 mmol), IPAC (0.3 mL), and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.5 μL, 0.093 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours and 30 minutes. A 10% aqueous K₂CO₃ solution (0.315 mL, 33.9 mg K₂CO₃) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe₂ in toluene (concentrations 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.081 mmol) were added sequentially to the reaction mixture. After raising the temperature to 0 °C, the mixture was continuously stirred for 8 hours.

[0677] Condition B

[0678] 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 (concentrations 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (61.7 mg, 0.162 mmol) was added to the reaction mixture, and the mixture was stirred continuously for 4 hours.

[0679] Measurement method: HPLC method A

[0680] Retention times: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 15.7 min, epimer: 16.2 min

[0681] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), Required product: m / z 686.04 ([M-NMe2] + Epimer: m / z 686.10 ([M-NMe2]) + )

[0682] [Table 20]

[0683]

[0684] As shown in Table 20, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0685] (Example 21) Condensation reaction of Cbz-MeLeu-Thr(OtBu)-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0686] The condensation reaction is carried out under the following conditions A and B.

[0687] [Equation 24]

[0688]

[0689] Condition A

[0690] Add a solution of Cbz-MeLeu-Thr(OtBu)-OH in IPAC (concentration 147.0 mg / mL, 0.300 mL, 44.1 mg, 0.101 mmol) and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (11.7 μL, 0.095 mmol) to the reaction mixture, and stir the mixture for 3 hours and 30 minutes. Add sequentially a 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe₂ in toluene (concentration 100.0 mg / mL, 0.280 mL, 28.0 mg, 0.076 mmol). The temperature was then raised to 0°C, and the mixture was stirred continuously for 20 hours.

[0691] Condition B

[0692] A solution of Cbz-MeLeu-Thr(OtBu)-OH in IPAC (concentration 147.0 mg / mL, 0.200 mL, 29.4 mg, 0.067 mmol), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (concentration 75.8 mg / mL, 0.250 mL, 19.0 mg, 0.050 mmol), and DIPEA (17.5 μL, 0.100 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (38.3 mg, 0.100 mmol) was added to the reaction mixture and the mixture was stirred for 3 hours.

[0693] Measurement method: HPLC method A

[0694] Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 6.7 min, desired product: 18.7 min, epimer: 19.0 min

[0695] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.77 ([M+H] + ), Required product: m / z 811.10 ([M+Na] + Epimer: m / z 811.61 ([M+Na]) + )

[0696] [Table 21]

[0697]

[0698] As shown in Table 21, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0699] (Example 22) Condensation reaction of Cbz-MeLeu-Thr(OtBu)-OH and H-MeGly(nPr)-Ile-Pro-OtBu

[0700] The condensation reaction is carried out under the following conditions A and B.

[0701] [Equation 25]

[0702]

[0703] Condition A

[0704] A solution of Cbz-MeLeu-Thr(OtBu)-OH in IPAC (concentration 147.0 mg / mL, 0.300 mL, 44.1 mg, 0.101 mmol) and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. 2,2-Dimethylbutyryl chloride (13.0 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(nPr)-Ile-Pro-OtBu in toluene (concentration 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0 °C, and the mixture was continuously stirred for 6 hours.

[0705] Condition B

[0706] H-MeGly(nPr)-Ile-Pro-OtBu (31.0 mg, 0.076 mmol), MeCN (0.3 mL), a solution of Cbz-MeLeu-Thr(OtBu)-OH in IPAC (concentrations 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 equipped with a stirrer, and the mixture was cooled to 10 °C while stirring. HATU (57.4 mg, 0.151 mmol) was added to the reaction mixture and the mixture was stirred for 5 hours.

[0707] Measurement method: HPLC method A

[0708] Retention times: H-MeGly(nPr)-Ile-Pro-OtBu: 8.6 min, desired product: 19.3 min, epimer: 19.8 min

[0709] Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.81 ([M+H] + ), Required product: m / z 817.37 ([M+H] + Epimer: m / z 817.26 ([M+H]) + )

[0710] [Table 22]

[0711]

[0712] As shown in Table 22, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0713] (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

[0714] The condensation reaction is carried out under the following conditions A and B.

[0715] [Equation 26]

[0716]

[0717] Condition A

[0718] Add a solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH in IPAC (concentration 145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol) and 2,6-dimethylpyridine (15.4 μL, 0.132 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10°C while stirring. Add PivCl (13.4 μL, 0.109 mmol) to the reaction mixture, and stir the mixture for 3 hours. Then, sequentially add a 10% aqueous K2CO3 solution (0.370 mL, 39.9 mg K2CO3), a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.350 mL, 70.0 mg, 0.095 mmol), and the mixture into a toluene solution. The temperature was then raised to 0°C, and the mixture was stirred continuously for 6 hours.

[0719] Condition B

[0720] A solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH in IPAC (concentration 145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol), a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 170.0 mg / mL, 0.410 mL, 69.7 mg, 0.095 mmol), and DIPEA (33.0 μL, 0.189 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (72.0 mg, 0.189 mmol) was added to the reaction mixture and the mixture was stirred for 3 hours.

[0721] Measurement method: HPLC method A

[0722] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu 9.9 min, desired product: 18.9 min, epimer: 19.2 min

[0723] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.18 ([M+H] + ), Required product: m / z 1125.35 ([M-Sar-OtBu] + Epimer: m / z 1125.29 ([M-Sar-OtBu]) + )

[0724] [Table 23]

[0725]

[0726] As shown in Table 23, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0727] (Example 24) Condensation reaction of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0728] The condensation reaction is carried out under the following conditions A and B.

[0729] [Equation 27]

[0730]

[0731] Condition A

[0732] Add a solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH in IPAC (concentration 145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol) and 2,6-dimethylpyridine (15.4 μL, 0.133 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10°C while stirring. Add PivCl (13.4 μL, 0.109 mmol) to the reaction mixture, and stir the mixture for 3 hours. Add sequentially a 10% aqueous K2CO3 solution (0.370 mL, 39.9 mg K2CO3) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in toluene (concentration 100.0 mg / mL, 0.350 mL, 35.0 mg, 0.095 mmol). The temperature was then raised to 0°C, and the mixture was stirred continuously for 6 hours.

[0733] Condition B

[0734] A solution of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH in IPAC (concentration 145.0 mg / mL, 0.430 mL, 62.3 mg, 0.115 mmol), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in MeCN (concentration 75.8 mg / mL, 0.460 mL, 34.9 mg, 0.095 mmol), and DIPEA (33.0 μL, 0.189 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (72.0 mg, 0.189 mmol) was added to the reaction mixture and the mixture was stirred for 3 hours.

[0735] Measurement method: HPLC method A

[0736] Retention time: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 5.6 min, desired product: 18.2 min, epimer: 18.5 min

[0737] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), Required product: m / z 852.14 ([M-NMe2] + Epimer: m / z 852.03 ([M-NMe2]) + )

[0738] [Table 24]

[0739]

[0740] As shown in Table 24, both the conversion and diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0741] (Example 25) Condensation reaction of Cbz-MeLeu-Cys(SBn)-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0742] The condensation reaction is carried out under the following conditions A and B.

[0743] [Equation 28]

[0744]

[0745] Condition A

[0746] Add a solution of Cbz-MeLeu-Cys(SBn)-OH in IPAC (concentration 116.0 mg / mL, 0.475 mL, 55.1 mg, 0.107 mmol) and 2,6-dimethylpyridine (13.9 μL, 0.119 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (12.2 μL, 0.100 mmol) to the reaction mixture, and stir the mixture for 3 hours. Add sequentially a 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol). After raising the temperature to 0°C, the mixture was stirred continuously for 6 hours.

[0747] Condition B

[0748] A solution of Cbz-MeLeu-Cys(SBn)-OH in IPAC (concentration 116.0 mg / mL, 0.475 mL, 55.1 mg, 0.107 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture and the mixture was stirred for 6 hours.

[0749] Measurement method: HPLC method A

[0750] Retention times: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.5 min, desired product: 18.7 min, epimer: 18.9 min

[0751] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.92 ([M+H] + ), Required product: m / z 958.28 ([M+H] + Epimer: m / z 958.39 ([M+H]) + )

[0752] [Table 25]

[0753]

[0754] As shown in Table 25, both the conversion and diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0755] (Example 26) Condensation reaction of Cbz-MeAla-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0756] The condensation reaction is carried out under the following conditions A and B.

[0757] [Equation 29]

[0758]

[0759] Condition A

[0760] Cbz-MeAla-Ile-OH (37.5 mg, 0.102 mmol), IPAC (0.300 mL), and 2,6-dimethylpyridine (13.2 μL, 0.113 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -10°C while stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentrations 200.0 mg / mL, 0.280 mL, 56.0 mg, 0.075 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0°C, and the mixture was continuously stirred for 20 hours.

[0761] Condition B

[0762] 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (57.3 mg, 0.151 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours.

[0763] Measurement method: HPLC method A

[0764] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu 10.9 min, desired product: 17.8 min, epimer: 18.4 min

[0765] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.12 ([M+H] + ), Required product: m / z 931.43 ([M-Sar-OtBu]+ Epimer: m / z 931.49 ([M-Sar-OtBu]) + )

[0766] [Table 26]

[0767]

[0768] As shown in Table 26, both the conversion rate and diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0769] (Example 27) Condensation reaction of Cbz-MeAla-Ile-OH and H-MeGly(nPr)-Ile-Pro-OtBu

[0770] The condensation reaction is carried out under the following conditions A and B.

[0771] [Formula 30]

[0772]

[0773] Condition A

[0774] Cbz-MeAla-Ile-OH (37.5 mg, 0.102 mmol), IPAC (0.300 mL), and 2,6-dimethylpyridine (13.2 μL, 0.113 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.6 μL, 0.094 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(nPr)-Ile-Pro-OtBu in toluene (concentrations 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0 °C, and the mixture was continuously stirred for 20 hours.

[0775] Condition B

[0776] 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (57.4 mg, 0.151 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours.

[0777] Measurement method: HPLC method A

[0778] Retention time: H-MeGly(nPr)-Ile-Pro-OtBu: 8.6 min, desired product: 17.1 min, epimer: 17.6 min

[0779] Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.75 ([M+H] + ), Required product: m / z 731.24 ([M+H] + Epimer: m / z 731.18 ([M+H]) + )

[0780] [Table 27]

[0781]

[0782] As shown in Table 27, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0783] (Example 28) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0784] The condensation reaction is carried out under the following conditions A and B.

[0785] [Equation 31]

[0786]

[0787] Condition A

[0788] Add a solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) to a test tube equipped with a stirrer, and cool the mixture to -10 °C while stirring. Add PivCl (11.6 μL, 0.095 mmol) to the reaction mixture, and stir the mixture for 8 hours. Add sequentially a 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 200.0 mg / mL, 0.280 mL, 56.0 mg, 0.076 mmol). Then raise the temperature to 0°C and stir the mixture continuously for 18 hours.

[0789] Condition B

[0790] A solution of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 170.0 mg / mL, 0.330 mL, 56.1 mg, 0.076 mmol), a solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture and the mixture was stirred for 14 hours.

[0791] Measurement method: HPLC method A

[0792] Retention time: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu 10.9 min, desired product: 19.1 min, epimer: 19.8 min

[0793] Mass spectrum: H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 744.07 ([M+H] +), Required product: m / z 1058.43 ([M-Sar-OtBu] + Epimer: m / z 1059.45 ([M-Sar-OtBu]) + )

[0794] [Table 28]

[0795]

[0796] As shown in Table 28, both the conversion and diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0797] (Example 29) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeGly(nPr)-Ile-Pro-OtBu

[0798] The condensation reaction is carried out under the following conditions A and B.

[0799] [Equation 32]

[0800]

[0801] Condition A

[0802] A solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.6 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(nPr)-Ile-Pro-OtBu in toluene (concentration 100.0 mg / mL, 0.300 mL, 30.0 mg, 0.076 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0 °C, and the mixture was continuously stirred for 18 hours.

[0803] Condition B

[0804] H-MeGly(nPr)-Ile-Pro-OtBu (30.0 mg, 0.076 mmol), a solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 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 equipped with a stirrer, and the mixture was cooled to 10 °C while stirring. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture and the mixture was stirred for 5 hours.

[0805] Measurement method: HPLC method A

[0806] Retention times: H-MeGly(nPr)-Ile-Pro-OtBu: 8.5 min, desired product: 18.7 min, epimer: 19.2 min

[0807] Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.87 ([M+H] + ), Required product: m / z 858.24 ([M+H] + Epimer: m / z 858.18 ([M+H]) + )

[0808] [Table 29]

[0809]

[0810] As shown in Table 29, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0811] (Example 30) Condensation reaction of Cbz-MeLeu-MeAla-Ile-OH and H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0812] The condensation reaction is carried out under the following conditions A and B.

[0813] [Equation 33]

[0814]

[0815] Condition A

[0816] A solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol) and 2,6-dimethylpyridine (13.2 μL, 0.114 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -10 °C while stirring. PivCl (11.6 μL, 0.095 mmol) was added to the reaction mixture, and the mixture was stirred for 6 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe₂ in toluene (concentration 100.0 mg / mL, 0.280 mL, 28.0 mg, 0.076 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0 °C, and the mixture was continuously stirred for 18 hours.

[0817] Condition B

[0818] A solution of Cbz-MeLeu-MeAla-Ile-OH in IPAC (concentration 146.0 mg / mL, 0.330 mL, 48.2 mg, 0.102 mmol), a solution of H-MeGly(cPent)-MeAsp(OtBu)-NMe2 in 2-MeTHF (concentration 129.6 mg / mL, 0.220 mL, 28.5 mg, 0.077 mmol), and DIPEA (26.3 μL, 0.152 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (57.4 mg, 0.152 mmol) was added to the reaction mixture and the mixture was stirred for 5 hours.

[0819] Measurement method: HPLC method A

[0820] Retention times: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: 6.6 min, desired product: 17.7 min, epimer: 18.9 min

[0821] Mass spectrometry: H-MeGly(cPent)-MeAsp(OtBu)-NMe2: m / z 370.71 ([M+H] + ), Required product: m / z 785.22 ([M-NMe2] + Epimer: m / z 785.11 ([M-NMe2])+ )

[0822] [Table 30]

[0823]

[0824] As shown in Table 30, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0825] (Example 31) Condensation reaction of Boc-MeLeu-Phe-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0826] The condensation reaction is carried out under the following conditions A and B.

[0827] [Equation 34]

[0828]

[0829] Condition A

[0830] A solution of Boc-MeLeu-Phe-OH in IPAC (concentration 134.0 mg / mL, 0.280 mL, 37.5 mg, 0.096 mmol) and 2,6-dimethylpyridine (13.0 μL, 0.111 mmol) were added to a test tube equipped with a stirrer, and the mixture was cooled to -8°C while stirring. PivCl (10.8 μL, 0.088 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol) were added sequentially to the reaction mixture. After raising the temperature to 0°C, the mixture was continuously stirred for 3 hours.

[0831] Condition B

[0832] A solution of Boc-MeLeu-Phe-OH in IPAC (concentration 134.0 mg / mL, 0.280 mL, 37.5 mg, 0.096 mmol), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 200 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture and the mixture was stirred for 5 hours.

[0833] Measurement method: HPLC method A

[0834] Retention time: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.5 min, desired product: 18.4 min, epimer: 18.5 min

[0835] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.92 ([M+H] + ), Required product: m / z 878.13 ([M+H] + Epimer: m / z 878.08 ([M+H]) + )

[0836] [Table 31]

[0837]

[0838] As shown in Table 31, both the conversion rate and the diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0839] (Example 32) Condensation reaction of Fmoc-MeAlGly-Val-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0840] The condensation reaction is carried out under the following conditions A and B.

[0841] [Formula 35]

[0842]

[0843] Condition A

[0844] Fmoc-MeAlGly-Val-OH (52.6 mg, purity 91.7%, net content 48.4 mg, 0.107 mmol), IPAC (0.300 mL), and 2,6-dimethylpyridine (13.9 μL, 0.119 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -8°C while stirring. PivCl (12.2 μL, 0.100 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 169.0 mg / mL, 0.240 mL, 40.6 mg, 0.080 mmol) were added sequentially to the reaction mixture. After raising the temperature to 0°C, the mixture was stirred continuously for 5 hours.

[0845] Condition B

[0846] Fmoc-MeAlGly-Val-OH (52.6 mg, purity 91.7%, net content 48.4 mg, 0.107 mmol), MeCN (0.300 mL), a solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in MeCN (concentration 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture and the mixture was stirred for 8 hours.

[0847] Measurement method: HPLC method A

[0848] Retention times: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 9.6 min, desired product: 18.6 min, epimer: 18.7 min

[0849] Mass spectrum: H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: m / z 503.81 ([M+H] + ), Required product: m / z 936.10 ([M+H]+ Epimer: m / z 936.39 ([M+H]) + )

[0850] [Table 32]

[0851]

[0852] As shown in Table 32, both the conversion rate and diastereomer ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomer ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0853] (Example 33) Condensation reaction of Fmoc-MeAlGly-Val-OH and H-MeGly(nPr)-Ile-Pro-OtBu

[0854] The condensation reaction is carried out under the following conditions A and B.

[0855] [Formula 36]

[0856]

[0857] Condition A

[0858] Fmoc-MeAlGly-Val-OH (52.6 mg, purity 91.7%, net content 48.4 mg, 0.107 mmol), IPAC (0.300 mL), and 2,6-dimethylpyridine (13.9 μL, 0.119 mmol) were added sequentially to a test tube equipped with a stirrer, and the mixture was cooled to -8°C while stirring. PivCl (12.2 μL, 0.100 mmol) was added to the reaction mixture, and the mixture was stirred for 3 hours. A 10% aqueous K₂CO₃ solution (0.300 mL, 32.3 mg K₂CO₃) and a solution of H-MeGly(nPr)-Ile-Pro-OtBu in toluene (concentration 100.0 mg / mL, 0.316 mL, 31.6 mg, 0.080 mmol) were added sequentially to the reaction mixture. The temperature was then raised to 0°C, and the mixture was continuously stirred for 5 hours.

[0859] Condition B

[0860] Fmoc-MeAlGly-Val-OH (52.6 mg, purity 91.7%, net content 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 equipped with a stirrer, and the mixture was cooled to 10°C while stirring. HATU (60.5 mg, 0.159 mmol) was added to the reaction mixture, and the mixture was stirred for 8 hours.

[0861] Measurement method: HPLC method A

[0862] Retention times: H-MeGly(nPr)-Ile-Pro-OtBu: 9.2 min, desired product: 19.5 min, epimer: 19.9 min

[0863] Mass spectrometry: H-MeGly(nPr)-Ile-Pro-OtBu: m / z 398.81 ([M+H] + ), Required product: m / z 831.51 ([M+H] + Epimer: m / z 831.16 ([M+H]) + )

[0864] [Table 33]

[0865]

[0866] As shown in Table 33, both the conversion rate and the diastereomeric ratio were high under the bilayer conditions of organic solvent and water (condition A), while the diastereomeric ratio was significantly lower under the condition of using HATU as a condensing agent (condition B).

[0867] (Example 34) Condensation reaction of Cbz-MeLeu-Ile-OH and H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0868] The condensation reaction is carried out in the following procedure.

[0869] [Formula 37]

[0870]

[0871] A solution of Cbz-MeLeu-Ile-OH in IPAC (concentration 157 mg / mL, 0.400 mL, 62.8 mg, 0.160 mmol) and 2,6-dimethylpyridine (20.8 μL, 0.179 mmol) were added to a test tube equipped with a stirrer (hereinafter referred to as "test tube A"), and the reaction mixture was cooled to -10 °C while stirring. PivCl (18.4 μL, 0.149 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. The progress of activation was confirmed by HPLC analysis. A 10% aqueous Na₂CO₃ solution (0.380 mL) was added, and the reaction mixture was stirred at -5 °C for 2 h, and then the temperature was raised to 0 °C. A solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu in toluene (concentration 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.

[0872] Reaction conversion rate: 98.8%

[0873] Diastereomer ratio: 99.4 / 0.6

[0874] Piv-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 0.9%

[0875] Measurement method: HPLC method A

[0876] 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

[0877] Mass spectrum: 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] + ), Required product: m / z 899.54 ([M+Na] + Epimer: m / z 899.59 ([M+Na]) + )

[0878] (Synthesis of raw material 1) Synthesis of Cbz-MeLeu-Ile-OH

[0879] [Formula 38]

[0880]

[0881] H-Ile-OtBu·HCl (6.04 g, 27.0 mmol) and 2-MeTHF (120 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (60 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate, and the reaction mixture was cooled while stirring. HATU (12.07 g, 31.74 mmol) was added to the reaction mixture, while maintaining the internal temperature of the reaction mixture at 17 °C or lower, and the mixture was continuously stirred at room temperature. Stirring of the reaction solution was continued for 1 hour and 30 minutes, and the completion of the reaction was confirmed by HPLC analysis. CPME (100 mL), 5% aqueous K₂CO₃ solution (50 mL), and NMI (2.0 mL, 25.6 mmol) were added to the reaction mixture, and the mixture was stirred continuously for 1 h. All 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 solution (50 mL x 3), twice with 5% aqueous NaHSO₄ solution (50 mL x 2), and once with 5% aqueous Na₂CO₃ solution (50 mL x 1). The resulting organic layer was dried over anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was then concentrated under reduced pressure to obtain Cbz-MeLeu-Ile-OtBu (11.28 g, 25.14 mmol). Add 2-MeTHF (110 mL) and HMDS (21.0 mL, 100.30 mmol) to the obtained concentrate, and add TMSOTf (14.0 mL, 75.23 mmol) dropwise. Continue stirring the reaction mixture for 3 hours, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 100 mL of 5% aqueous NaHCO3 solution dropwise to the reaction mixture while maintaining the reaction mixture at 28°C or lower. Transfer all contents of the flask to a separatory funnel and remove the organic layer. Add 2-MeTHF (250 mL) to the resulting aqueous layer, and add 10 mL of 85% H3PO4. Remove the aqueous layer, and wash the organic layer with 50 mL of 5% aqueous NaCl solution and concentrate under reduced pressure.The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-Ile-OH (8.20 g).

[0882] Yield: 77% (after 2 steps)

[0883] Purity: 100%

[0884] Measurement method: HPLC method A, retention time: 12.7 min

[0885] Mass spectrometry: m / z 393.55 ([M+H]) + )

[0886] (Analysis of the synthesis of formulation 1) Synthesis of Cbz-MeLeu-D-allo-Ile-OH

[0887] [Formula 39]

[0888]

[0889] Add Cbz-MeLeu-OH (1.00 g, 3.58 mmol) and THF (70 mL) to a 300 mL three-necked flask equipped with a stirrer, and stir the mixture and cool to an internal temperature of 0 °C. Add TEA (1.50 mL, 10.74 mmol) and isobutyl chloroformate (0.44 mL, 3.40 mmol) sequentially to the reaction mixture, and stir for 30 min. Add dropwise an aqueous solution prepared by dissolving HD-allo-Ile-OH (0.71 g, 5.37 mmol) in water (70 mL), and stir the reaction mixture for 4 h 30 min. Concentrate the reaction mixture under reduced pressure, and transfer the concentrate to a separatory funnel, add 2-MeTHF (150 mL) and 5% aqueous NaHSO4 solution (50 mL). Remove the aqueous layer and concentrate the resulting organic layer under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-D-allo-Ile-OH (0.71 g).

[0890] Yield: 51%

[0891] HPLC purity: 95.3%

[0892] Measurement method: HPLC method A, retention time: 13.1 min

[0893] Mass spectrometry: m / z 393.55 ([M+H]) + )

[0894] (Synthesis of raw material 2) Synthesis of Cbz-MeLeu-Ala-OH

[0895] [Formula 40]

[0896]

[0897] H-Ala-OtBu·HCl (3.01 g, 16.57 mmol) and 2-MeTHF (60 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (30 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate, and the mixture was cooled while stirring. HATU (3.89 g, 10.23 mmol) was added to the reaction mixture, and the mixture was continuously stirred at room temperature. Stirring was continued for 1 hour, and the reaction was confirmed to be complete by HPLC analysis. Add CPME (60 mL), 5% aqueous K₂CO₃ solution (20 mL), and NMI (0.65 mL, 8.23 ​​mmol) to the reaction mixture, and stir continuously for 1 hour. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer three times with 2.5% aqueous ammonia solution (25 mL x 3), twice with 5% aqueous NaHSO₄ solution (25 mL x 2), and once with 5% aqueous Na₂CO₃ solution (5 mL x 1). Dry the obtained organic layer with anhydrous Na₂SO₄. Filter off the desiccant, and then concentrate the filtrate under reduced pressure to obtain Cbz-MeLeu-Ala-OtBu (3.27 g, 8.04 mmol). Add 2-MeTHF (33 mL) and HMDS (5.1 mL, 24.1 mmol) to the obtained concentrate, and add TMSOTf (2.2 mL, 12.1 mmol) dropwise. Continue stirring for 2 hours and 30 minutes, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 35 mL of 5% aqueous NaHCO3 solution dropwise to the reaction mixture while maintaining the reaction mixture at 19°C or lower. Transfer all contents of the flask to a separatory funnel and remove the organic layer. Add 70 mL of 2-MeTHF to the aqueous layer, and gradually add 5 mL of 85% H3PO4. Remove the aqueous layer, and wash the organic layer with 25 mL of 5% aqueous NaCl solution and concentrate under reduced pressure. Purify the resulting concentrate by silica gel column chromatography to obtain Cbz-MeLeu-Ala-OH (2.32 g).

[0898] Yield: 79% (after 2 steps)

[0899] Purity: 100%

[0900] Measurement method: HPLC method A, retention time: 11.2 min

[0901] Mass spectrometry: m / z 351.48 ([M+H]) + )

[0902] (Synthesis of raw material 3) Synthesis of Cbz-MeLeu-Phe-OH

[0903] [Formula 41]

[0904]

[0905] H-Phe-OtBu·HCl (5.00 g, 19.40 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (50 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate. HATU (9.14 g, 24.03 mmol) was added to the reaction mixture, and the mixture was continuously stirred at room temperature. Stirring was continued for 2 hours, and the completion of the reaction was confirmed by HPLC analysis. Add CPME (80 mL), 5% aqueous K₂CO₃ solution (40 mL), and NMI (1.47 mL, 18.48 mmol) to the reaction mixture and stir continuously for 30 minutes. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer three times with 3% aqueous ammonia solution (40 mL x 3), twice with 5% aqueous NaHSO₄ solution (40 mL x 2), and once with 5% aqueous Na₂CO₃ solution (40 mL x 2). Dry the obtained organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain Cbz-MeLeu-Phe-OtBu (9.10 g, 18.86 mmol). Add 2-MeTHF (91 mL) and HMDS (20.0 mL, 95.42 mmol) to the obtained concentrate, and add TMSOTf (14.0 mL, 77.35 mmol) dropwise. Continue stirring for 1 hour and 30 minutes, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 5% aqueous NaHCO3 solution (50 mL) dropwise to the reaction mixture while maintaining the reaction mixture at 25°C or lower. Transfer all contents of the flask to a separatory funnel, then add 2-MeTHF (100 mL), and gradually add 85% H3PO4 (6 mL). Remove the aqueous layer, and wash the organic layer with 5% aqueous NaCl solution (50 mL) and concentrate under reduced pressure. Purify the resulting concentrate by silica gel column chromatography to obtain Cbz-MeLeu-Phe-OH (6.93 g).

[0906] Yield: 84% (after 2 steps)

[0907] Purity: 100%

[0908] Measurement method: HPLC method A, retention time: 13.1 min

[0909] Mass spectrometry: m / z 427.82 ([M+H]) + )

[0910] (Synthesis of raw material 4) Synthesis of Cbz-MeLeu-Ser(OtBu)-OH

[0911] [Equation 42]

[0912]

[0913] H-Ser(OtBu)-OtBu·HCl (3.76 g, 14.82 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (50 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate, and the mixture was cooled while stirring. HATU (6.14 g, 16.15 mmol) was added to the reaction mixture, and the reaction mixture was kept at 17 °C or lower while the mixture was continuously stirred at room temperature. Stirring was continued for 1 hour, and the reaction was confirmed to be complete by HPLC analysis. Add CPME (100 mL), 5% aqueous K₂CO₃ solution (50 mL), and NMI (1.0 mL, 12.54 mmol) to the reaction mixture and stir continuously for 2 hours. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer three times with 2.5% aqueous ammonia solution (50 mL x 3), twice with 5% aqueous NaHSO₄ solution (50 mL x 2), and once with 5% aqueous Na₂CO₃ solution (50 mL x 1). Dry the obtained organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain Cbz-MeLeu-Ser(OtBu)-OtBu (5.90 g, 12.54 mmol). Add 2-MeTHF (60 mL) and HMDS (5.3 mL, 25.07 mmol) to the obtained concentrate, and add TMSOTf (2.5 mL, 13.79 mmol) dropwise. Continue stirring for 4 hours, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 5% aqueous NaHCO3 solution (50 mL) dropwise to the reaction mixture while maintaining the reaction mixture at 18°C ​​or lower. Transfer all contents of the flask to a separatory funnel, and then gradually add 85% H3PO4 (6 mL). Remove the aqueous layer, and wash the organic layer with 5% aqueous NaCl solution (50 mL) and concentrate the organic layer under reduced pressure. Purify the resulting concentrate by silica gel column chromatography to obtain Cbz-MeLeu-Ser(OtBu)-OH (4.30 g).

[0914] Yield: 69% (after 2 steps)

[0915] Purity: 100%

[0916] Measurement method: HPLC method A, retention time: 12.8 min

[0917] Mass spectrometry: m / z 423.59 ([M+H]) + )

[0918] (Analysis of the synthesis of formulation 2) Synthesis of Cbz-MeLeu-D-Ser(OtBu)-OH

[0919] [Formula 43]

[0920]

[0921] HD-Ser(OtBu)-OtBu·HCl (380 mg, 1.50 mmol) and 2-MeTHF (25 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (10 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate. HATU (854 mg, 2.25 mmol) was added to the reaction mixture, and the mixture was continuously stirred at room temperature. Stirring was continued for 2 hours and 30 minutes, and the reaction was confirmed to be complete by HPLC analysis. Add CPME (20 mL), 5% aqueous K₂CO₃ solution (10 mL), and NMI (120 μL, 1.50 mmol) to the reaction mixture and stir continuously for 1 hour. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer three times with 2.5% aqueous ammonia solution (10 mL x 3), twice with 5% aqueous NaHSO₄ solution (10 mL x 2), and once with 5% aqueous Na₂CO₃ solution (10 mL x 1). Dry the obtained organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain Cbz-MeLeu-D-Ser(OtBu)-OtBu (0.690 g, 1.44 mmol). Add 2-MeTHF (7.0 mL) and HMDS (0.6 mL, 2.88 mmol) to the obtained concentrate, and add TMSOTf (300 μL, 1.66 mmol) dropwise. Continue stirring for 4 hours, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 5% aqueous NaHCO3 solution (6 mL) dropwise to the reaction mixture while maintaining the reaction mixture at 14°C or lower. Transfer all contents of the flask to a separatory funnel, and then gradually add 85% H3PO4 (5 mL). Remove the aqueous layer, and wash the organic layer with 5% aqueous NaCl solution and concentrate under reduced pressure. Purify the resulting concentrate by silica gel column chromatography to obtain Cbz-MeLeu-D-Ser(OtBu)-OH (470 mg).

[0922] Yield: 74% (after 2 steps)

[0923] Purity: 100%

[0924] Measurement method: HPLC method A, retention time: 13.6 min

[0925] Mass spectrometry: m / z 423.59 ([M+H]) + )

[0926] (Synthesis of raw material 5) Synthesis of Cbz-MeLeu-Ile-MeLeu-Ile-OH

[0927] [Formula 44]

[0928]

[0929] Add 2-MeTHF (22.2 mL) and HMDS (3.38 mL, 16.11 mmol) to Cbz-MeLeu-Ile-MeLeu-Ile-OtBu (2.22 g, 3.22 mmol) synthesized by the method described in Example 8. Add TMSOTf (2.33 mL, 12.89 mmol) dropwise to the reaction mixture. Stir the mixture for 5 hours, and then confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 20 mL of 5% aqueous Na₂CO₃ solution dropwise to the reaction mixture while maintaining the reaction mixture at 53°C or lower. Transfer all contents of the flask to a separatory funnel. Discard the aqueous layer, and then wash the organic layer once with 20 mL of 5% aqueous Na₂CO₃ solution. Combine the aqueous layers, and then gradually add 30 mL of 2-MeTHF and 2 mL of 85% H₃PO₄. The aqueous layer was removed, and the organic layers were then combined and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-Ile-MeLeu-Ile-OH (708 mg).

[0930] Yield: 35%

[0931] HPLC purity: 99.6%

[0932] Measurement method: HPLC method A, retention time: 15.2 min

[0933] Mass spectrometry: m / z 655.52 ([M+Na]) + )

[0934] Synthesis of (raw material 6) H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0935] [Formula 45]

[0936]

[0937] The compound was synthesized according to the method described in International Publication No. WO 2023 / 127869.

[0938] Synthesis of (raw material 7) H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu

[0939] [Formula 46]

[0940]

[0941] The compound was synthesized by desalting the L-tartrate of the title compound obtained by the method described in International Publication No. WO 2023 / 127869.

[0942] Synthesis of (raw material 8) H-MeGly(cPent)-MeAsp(OtBu)-NMe2

[0943] [Formula 47]

[0944]

[0945] The compound was synthesized by desalting the hydrochloride salt of the title compound obtained by the method described in International Publication No. WO 2023 / 127869.

[0946] Synthesis of H-MeLeu-Ile-OtBu (Synthesis of Raw Material 9)

[0947] [Formula 48]

[0948]

[0949] Cbz-MeLeu-Ile-OtBu (4.00 g, 8.92 mmol), synthesized by the method described in the synthesis of starting material 1, was dissolved in 2-MeTHF (24 mL) and added to a pressurized reaction vessel. 10% Pd / C (0.522 g, 0.223 mmol, on a Pd metal base) was added, and nitrogen and hydrogen purgings were performed. The mixture was then stirred at a 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 obtain H-MeLeu-Ile-OtBu (1.66 g).

[0950] Yield: 59%

[0951] HPLC purity: 98.8%

[0952] Measurement method: HPLC method A, retention time: 8.0 min

[0953] Mass spectrometry: m / z 315.38 ([M+H]) + )

[0954] Synthesis of H-cLeu-MeGly(cPent)-MeAsp(OtBu)-NMe2 (from raw material 10)

[0955] [Formula 49]

[0956]

[0957] The compound was synthesized according to the method described in International Publication No. WO 2023 / 127869.

[0958] (Synthesis of raw material 11) Synthesis of Cbz-Leu-Ile-OH

[0959] [Formula 50]

[0960]

[0961] H-Ile-OtBu·HCl (2.00 g, 8.94 mmol) and 2-MeTHF (50 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (25 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 while stirring. HATU (3.87 g, 10.18 mmol) was added to the reaction mixture, while maintaining the internal temperature of the reaction mixture at 11 °C or lower, and the mixture was continuously stirred at room temperature. Stirring of the reaction solution was continued for 2 hours, and the completion of the reaction was confirmed by HPLC analysis. Add 2-MeTHF (25 mL), 5% aqueous K₂CO₃ solution (20 mL), and NMI (0.67 mL, 8.44 mmol) to the reaction mixture, and stir continuously for 2 hours. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer three times with 2.5% aqueous ammonia solution (25 mL x 3), twice with 5% aqueous NaHSO₄ solution (25 mL x 2), and once with 5% aqueous Na₂CO₃ solution (25 mL x 1). Dry the resulting organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain Cbz-Leu-Ile-OtBu (3.55 g, 8.17 mmol). Add 2-MeTHF (36 mL) and HMDS (7.0 mL, 32.7 mmol) to the obtained concentrate, and add TMSOTf (4.4 mL, 24.5 mmol) dropwise. Continue stirring the reaction mixture for 2 hours and 30 minutes, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 36 mL of 5% aqueous NaHCO3 solution dropwise to the reaction mixture while maintaining the reaction mixture at 25°C or lower. Transfer all contents of the flask to a separatory funnel and remove the organic layer. Add 100 mL of 2-MeTHF to the resulting aqueous layer, and add 4.5 mL of 85% H3PO4. Remove the aqueous layer, and wash the organic layer with 36 mL of 5% aqueous NaCl solution and concentrate under reduced pressure.The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-Leu-Ile-OH (2.00 g).

[0962] Yield: 59% (after 2 steps)

[0963] Purity: 98.6%

[0964] Measurement method: HPLC method A, retention time: 11.8 min

[0965] Mass spectrometry: m / z 379.77 ([M+H]) + )

[0966] Synthesis of Cbz-Leu-D-allo-Ile-OH (from raw material 12)

[0967] [Equation 51]

[0968]

[0969] Add Cbz-Leu-OH (1.50 g, 5.65 mmol) and THF (105 mL) to a 300 mL three-necked flask equipped with a stirrer, and cool the mixture to an internal temperature of -3 °C while stirring. Add TEA (2.4 mL, 17.0 mmol) and isobutyl chloroformate (0.70 mL, 5.37 mmol) sequentially to the reaction mixture, and stir for 30 min. Add dropwise an aqueous solution prepared by dissolving HD-allo-Ile-OH (1.11 g, 8.48 mmol) in water (105 mL), and stir the reaction mixture for 2 h. Concentrate the reaction mixture under reduced pressure, and transfer the concentrate to a separatory funnel, adding 2-MeTHF (225 mL) and a 5% aqueous NaHSO4 solution (75 mL). Remove the aqueous layer and concentrate the resulting organic layer under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-Leu-D-allo-Ile-OH (0.645 g).

[0970] Yield: 30%

[0971] HPLC purity: 100.0%

[0972] Measurement method: HPLC method A, retention time: 12.2 min

[0973] Mass spectrometry: m / z 379.72 ([M+H]) + )

[0974] Synthesis of Cbz-D-Leu-Ile-OH (from raw material 13)

[0975] [Equation 52]

[0976]

[0977] Add H-Ile-OtBu·HCl (0.65 g, 2.91 mmol) and 2-MeTHF (50 mL) to a separatory funnel. Wash the mixture twice with 5% aqueous Na₂CO₃ solution (12 mL x 2), and concentrate the organic layer under reduced pressure to obtain H-Ile-OtBu (0.55 g, 2.90 mmol). Add Cbz-D-Leu-OH (0.86 g, 3.2 mmol), MeCN (5.5 mL), and NMM (0.65 mL, 5.9 mmol) to the resulting concentrate, and cool the reaction mixture while stirring. Add COMU (1.5 g, 3.5 mmol) to the reaction mixture, maintaining the internal temperature of the reaction mixture at 11 °C or lower, and continuously stir the mixture at room temperature. Continue stirring the reaction solution for 2 hours, and confirm the completion of the reaction by HPLC analysis. Add 10 mL of 2-MeTHF, 10 mL of 5% aqueous K₂CO₃ solution, and 0.23 mL of NMI (2.9 mmol) to the reaction mixture, and stir continuously for 1 hour. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer once with 12 mL of 5% aqueous Na₂CO₃ solution, three times with 3 x 12% aqueous ammonia solution, twice with 2 x 12% aqueous NaHSO₄ solution, and once with 1 x 12% aqueous Na₂CO₃ solution. Dry the resulting organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain Cbz-D-Leu-Ile-OtBu (1.17 g, 2.69 mmol). Add 12 mL of 2-MeTHF and 2.3 mL of HMDS (10.75 mmol) to the obtained concentrate, and add TMSOTf (1.5 mL, 8.06 mmol) dropwise. Continue stirring the reaction mixture for 3 hours, and confirm the completion of the reaction by HPLC analysis. Cool the flask, and then add 12 mL of 5% aqueous NaHCO3 solution dropwise to the reaction mixture while maintaining the reaction mixture at 24 °C or lower. Transfer all contents of the flask to a separatory funnel and remove the organic layer. Add 20 mL of 2-MeTHF and 1.3 mL of 85% H3PO4 to the resulting aqueous layer. Remove the aqueous layer, and wash the organic layer with 12 mL of 5% aqueous NaCl solution and concentrate under reduced pressure.The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-D-Leu-Ile-OH (0.57 g, 1.51 mmol).

[0978] Yield: 52% (after 2 steps)

[0979] Purity: 99.3%

[0980] Measurement method: HPLC method A, retention time: 12.0 min

[0981] Mass spectrometry: m / z 379.66 ([M+H]) + )

[0982] Synthesis of Cbz-MeLeu-Thr(OtBu)-OH (Synthesis of raw material 14)

[0983] [Formula 53]

[0984]

[0985] Add Fmoc-Thr(OtBu)-OH (2.00 g, 5.03 mmol), cyclohexane (16 mL), and DCM (6 mL) to a flask equipped with a stirrer. Add 2,2,2-trichloroacetylimine tert-butyl ester (1.8 mL, 10.10 mmol) to the flask. While cooling the flask in an ice bath, add boron trifluoride diethyl ether complex (0.06 mL, 0.50 mmol) dropwise, and then continuously stir the mixture at room temperature for 1 hour and 15 minutes. Confirm the completion of the reaction by HPLC analysis, filter the reaction mixture, and wash the filtered solid with cyclohexane. Combine the filtrate and wash solution, and wash 5 times with 10% aqueous citric acid solution (16 mL x 5). Wash the resulting organic layer twice with 5% aqueous Na₂CO₃ solution (16 mL x 2). Concentrate the resulting organic layer under reduced pressure to obtain Fmoc-Thr(OtBu)-OtBu. MeCN (18 mL) and DBU (0.75 mL, 5.03 mmol) were added to the flask, and the mixture was stirred for 30 min. The reaction mixture was confirmed by HPLC analysis, and 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 while the flask was cooled in an ice bath, and the mixture was stirred at room temperature for 1 h. MTBE (57 mL) and 10% aqueous ammonia solution (32 mL) were added to the reaction mixture, and all contents of the flask were transferred to a separatory funnel. The organic layer obtained by removing the aqueous layer was washed three times with 10% aqueous ammonia solution (32 mL x 3) and once with 5% aqueous NaCl solution (32 mL x 1). The resulting organic layer was concentrated under reduced pressure to obtain H-Thr(OtBu)-OtBu. Add MeCN (14 mL), Cbz-MeLeu-OH (1.54 g, 5.53 mmol), and NMM (1.7 mL, 15.1 mmol) to the flask. While cooling the flask in an ice bath, add COMU (2.80 g, 6.53 mmol) to the reaction mixture and stir continuously at room temperature for 2 hours. Confirm the completion of the reaction by HPLC analysis, and then add 2-MeTHF (34 mL), 5% aqueous K2CO3 solution (17 mL), and NMI (0.40 mL, 5.03 mmol) to the reaction mixture and stir continuously for 30 minutes. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer.The resulting organic layer was washed once with 5% aqueous K₂CO₃ solution (27 mL x 1), three times with 2.5% aqueous ammonia solution (27 mL x 3), twice with 5% aqueous NaHSO₄ solution (27 mL x 2), once with 5% aqueous K₂CO₃ solution (27 mL x 1), and once with 5% aqueous NaCl solution (27 mL x 1). The resulting organic layer was dried over anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain 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, and then TMSOTf (2.6 mL, 14.20 mmol) was added dropwise. After stirring continuously for 4 hours, the flask was cooled, and then 29 mL of 5% aqueous NaHCO3 solution was added dropwise to the reaction mixture while maintaining the reaction mixture at 24°C or lower. All contents of the flask were transferred to a separatory funnel and the organic layer was removed. 56 mL of 2-MeTHF was added to the resulting aqueous layer, followed by 3.5 mL of 85% H3PO4. The aqueous layer was removed, and the resulting organic layer was washed with 29 mL of 5% aqueous NaCl solution and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-Thr(OtBu)-OH (0.53 g, 1.20 mmol).

[0986] Yield: 24% (starting from Fmoc-Thr(OtBu)-OH, through 4 steps)

[0987] Purity: 99.5%

[0988] Measurement method: HPLC method A, retention time: 14.7 min

[0989] Mass spectrometry: m / z 437.74 ([M+H]) + )

[0990] Synthesis of Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (Synthesis of raw material 15)

[0991] [Formula 54]

[0992]

[0993] Add Cbz-Hph(3,5-F2-4-CF3)-OH·Cy2NH (5.00 g, 8.35 mmol) and 2-MeTHF (75 mL) to a separatory funnel. Wash the mixture twice with 5% aqueous H2SO4 solution (25 mL x 2), and concentrate the organic layer under reduced pressure to obtain Cbz-Hph(3,5-F2-4-CF3)-OH. Add cyclohexane (40 mL) and DCM (15 mL) to the obtained concentrate. Add 2,2,2-trichloroacetylimine tert-butyl ester (3.0 mL, 16.7 mmol) to a flask. While cooling the flask in an ice bath, add boron trifluoride diethyl ether complex (0.11 mL, 0.84 mmol) dropwise, and then continuously stir the mixture at room temperature for 2.5 h. Confirm the completion of the reaction by HPLC analysis, filter the reaction mixture, and wash the filtered solid with cyclohexane. The filtrate and washing solution were combined and washed five times with 10% aqueous citric acid solution (20 mL x 5). The resulting organic layer was washed twice with 5% aqueous Na₂CO₃ solution (20 mL x 2). The resulting organic layer was concentrated under reduced pressure to obtain 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, Pd metal-based) was added to the flask, and nitrogen and hydrogen purging were performed. The mixture was then stirred under hydrogen pressure (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 washing solution were combined and concentrated under reduced pressure to obtain H-Hph(3,5-F₂-4-CF₃)-OtBu (2.60 g, 7.66 mmol). Add Cbz-MeLeu-OH (2.35 g, 8.43 mmol), MeCN (26 mL), and NMM (1.7 mL, 15.3 mmol) to the resulting concentrate, and cool the reaction mixture while stirring. Add COMU (3.94 g, 9.20 mmol) to the reaction mixture, maintaining the internal temperature of the reaction mixture at 7 °C or lower, and continuously stir the mixture at room temperature. Continue stirring the reaction solution for 2 hours, and confirm the completion of the reaction by HPLC analysis. Add 2-MeTHF (40 mL), 5% aqueous K₂CO₃ solution (20 mL), and NMI (0.61 mL, 7.66 mmol) to the reaction mixture, and continuously stir the mixture for 30 minutes. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer.The resulting organic layer was washed once with 5% aqueous Na₂CO₃ solution (20 mL x 1), three times with 2.5% aqueous ammonia solution (20 mL x 3), twice with 5% aqueous NaHSO₄ solution (20 mL x 2), and once with 5% aqueous Na₂CO₃ solution (20 mL x 1). The resulting organic layer was dried over anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain 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, and then TMSOTf (3.8 mL, 21.3 mmol) was added dropwise. After stirring continuously for 2 hours, the flask was cooled, and then 40 mL of 5% aqueous NaHCO3 solution was added dropwise to the reaction mixture while maintaining the reaction mixture at 22°C or lower. All contents of the flask were transferred to a separatory funnel and the aqueous layer was removed. The resulting organic layer was washed with 1 x 40 mL of 5% aqueous NaHSO4 solution and the aqueous layer was removed. The resulting organic layer was washed with 30 mL of 5% aqueous NaCl solution and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-Hph(3,5-F2-4-CF3)-OH (2.64 g, 4.85 mmol).

[0994] Yield: 58% (through 4 steps, starting from Cbz-Hph(3,5-F2-4-CF3)-OH·Cy2NH)

[0995] Purity: 100.0%

[0996] Measurement method: HPLC method A, retention time: 15.2 min

[0997] Mass spectrometry: m / z 545.70 ([M+H]) + )

[0998] Synthesis of Cbz-MeLeu-Cys(SBn)-OH (Synthesis of raw material 16)

[0999] [Formula 55]

[1000]

[1001] H-Cys(SBn)-OMe·HCl (1.97 g, 7.53 mmol) and 2-MeTHF (100 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (40 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 while stirring. COMU (3.40 g, 7.94 mmol) was added to the reaction mixture, while maintaining the internal temperature of the reaction mixture at 11 °C or lower, and the mixture was continuously stirred at room temperature. Stirring of the reaction solution was continued for 2 hours, and the completion of the reaction was confirmed by HPLC analysis. Add 45 mL of 2-MeTHF, 23 mL of 5% aqueous K₂CO₃ solution, and 0.52 mL of NMI (6.61 mmol) to the reaction mixture, and stir continuously for 1 hour. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer once with 38 mL of 5% aqueous K₂CO₃ solution, three times with 38 mL of 2.5% aqueous ammonia solution, twice with 28 mL of 5% aqueous NaHSO₄ solution, once with 38 mL of 5% aqueous K₂CO₃ solution, and once with 38 mL of 5% aqueous NaCl solution. Dry the resulting organic layer with anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain Cbz-MeLeu-Cys(SBn)-OMe (3.30 g, 6.78 mmol). THF (30 mL) was added to the resulting concentrate, and the solution was cooled in an ice bath. 2M aqueous NaOH solution (5.0 mL) was added dropwise while maintaining the intermediate temperature of the mixture at 2°C or lower. Stirring of the reaction mixture was continued for 2 hours, and the 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 solution (30 mL) was added to the resulting solution, and the mixture was transferred to a separatory funnel, and the aqueous layer was removed. The resulting organic layer was washed with 5% aqueous NaCl solution (45 mL) and concentrated under reduced pressure.The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-Cys(SBn)-OH (0.693 g, 1.467 mmol).

[1002] Yield: 22% (after 2 steps)

[1003] Purity: 99.2%

[1004] Measurement method: HPLC method A, retention time: 15.5 min

[1005] Mass spectrometry: m / z 473.71 ([M+H]) + )

[1006] Synthesis of Cbz-MeAla-Ile-OH (from raw material 17)

[1007] [Formula 56]

[1008]

[1009] H-Ile-OtBu·HCl (5.05 g, 22.6 mmol) and 2-MeTHF (250 mL) were added to a separatory funnel. The mixture was washed twice with 5% aqueous Na₂CO₃ solution (100 mL x 2), and the organic layer was concentrated under reduced pressure to obtain 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 resulting concentrate, and the reaction mixture was cooled while stirring. COMU (11.3 g, 26.5 mmol) was added to the reaction mixture, while maintaining the internal temperature of the reaction mixture at 11 °C or lower, and the mixture was continuously stirred at room temperature. Stirring of the reaction solution was continued for 3 hours, and the completion of the reaction was confirmed by HPLC analysis. Add 2-MeTHF (83 mL), 5% aqueous K₂CO₃ solution (83 mL), and NMI (1.7 mL, 22.1 mmol) to the reaction mixture, and stir continuously for 30 minutes. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. Wash the organic layer once with 5% aqueous K₂CO₃ solution (83 mL x 1), three times with 2.5% aqueous ammonia solution (83 mL x 3), twice with 5% aqueous NaHSO₄ solution (83 mL x 2), and once with 5% aqueous K₂CO₃ solution (83 mL x 1). Dry the resulting organic layer with anhydrous Na₂SO₄. Filter off the desiccant and then concentrate the filtrate under reduced pressure to obtain 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 fractions. HMDS (6.0 mL, 28.9 mmol) was added to one of the three fractions, followed by dropwise addition of TMSOTf (4.0 mL, 21.7 mmol). The reaction mixture was stirred for 2 hours, and the completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and then 5% aqueous NaHCO3 solution (36 mL) was added dropwise to the reaction mixture while maintaining the reaction mixture at 24°C or lower. All contents of the flask were transferred to a separatory funnel, and the organic layer was removed. 2-MeTHF (72 mL) and 85% H3PO4 (5.2 mL) were added to the resulting aqueous layer.The aqueous layer was removed, and the organic layer was washed with 5% aqueous NaCl solution (36 mL) and concentrated under reduced pressure. The resulting concentrate was purified by silica gel column chromatography to obtain Cbz-MeAla-Ile-OH (2.20 g, 6.28 mmol).

[1010] Yield: 85% (after 2 steps)

[1011] Purity: 99.8%

[1012] Measurement method: HPLC method A, retention time: 10.7 min

[1013] Mass spectrometry: m / z 351.73 ([M+H]) + )

[1014] Synthesis of Cbz-MeLeu-MeAla-Ile-OH (from raw material 18)

[1015] [Formula 57]

[1016]

[1017] Add Cbz-MeAla-Ile-OtBu, 5% Pd / C (50% wet, 1.53 g, 0.36 mmol, on a Pd metal base), in 2-MeTHF to one of three separate solutions, and perform nitrogen and hydrogen purging. The mixture is then stirred for 2 hours under a hydrogen atmosphere (1 atm). Filter the reaction mixture and wash the Pd / C with 2-MeTHF. Combine the filtrate and wash solution and concentrate under reduced pressure to obtain H-MeAla-Ile-OtBu (1.95 g, 7.17 mmol). Add Cbz-MeLeu-OH (2.20 g, 7.87 mmol), MeCN (20 mL), and NMM (1.6 mL, 14.3 mmol) to the resulting concentrate, and cool the reaction mixture while stirring. Add COMU (3.69 g, 8.59 mmol) to the reaction mixture while maintaining the internal temperature of the reaction mixture at 5°C or lower, and continuously stir the mixture at room temperature. Continue stirring the reaction solution for 1 hour and 30 minutes, and confirm the completion of the reaction by HPLC analysis. Add 2.5% aqueous ammonia solution (60 mL), 2-MeTHF (60 mL), 5% aqueous K2CO3 solution (30 mL), and NMI (0.57 mL, 7.16 mmol) to the reaction mixture, and continuously stir the mixture for 30 minutes. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. The organic layer was washed once with 5% aqueous K₂CO₃ solution (50 mL x 1), three times with 2.5% aqueous ammonia solution (50 mL x 3), twice with 5% aqueous NaHSO₄ solution (50 mL x 2), once with 5% aqueous K₂CO₃ solution (50 mL x 1), and once with 5% aqueous NaCl solution (50 mL x 1). The resulting organic layer was dried over anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel chromatography to obtain Cbz-MeLeu-MeAla-Ile-OtBu (3.45 g, 6.46 mmol). MeTHF (35 mL) and HMDS (5.4 mL, 25.9 mmol) were added to the obtained Cbz-MeLeu-MeAla-Ile-OtBu, and TMSOTf (3.6 mL, 19.4 mmol) was added dropwise.The reaction mixture was stirred for 3 hours and 30 minutes, and the completion of the reaction was confirmed by HPLC analysis. The flask was cooled, and then 41 mL of 5% aqueous NaHCO3 solution was added dropwise to the reaction mixture while maintaining the reaction mixture at 24°C or lower. All contents of the flask were transferred to a separatory funnel and the organic layer was removed. 83 mL of 2-MeTHF and 4.8 mL of 85% H3PO4 were added to the resulting aqueous layer. The aqueous layer was removed, and the organic layer was washed with 41 mL of 5% aqueous NaCl solution and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to obtain Cbz-MeLeu-MeAla-Ile-OH (1.79 g, 3.75 mmol).

[1018] Yield: 51% (after 4 steps)

[1019] Purity: 100.0%

[1020] Measurement method: HPLC method A, retention time: 14.03 min

[1021] Mass spectrometry: m / z 479.07 ([M+H]) + )

[1022] Synthesis of Boc-MeLeu-Phe-OH (from raw material 19)

[1023] [Formula 58]

[1024]

[1025] Add 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) sequentially to the flask, cooling the reaction mixture while stirring. Add COMU (4.77 g, 11.1 mmol) to the reaction mixture, maintaining the internal temperature of the reaction mixture at 5 °C or lower, and continuously stir the mixture at room temperature. Continue stirring the reaction solution for 2 hours, and confirm the completion of the reaction by HPLC analysis. Add 2-MeTHF (60 mL), 5% aqueous K2CO3 solution (30 mL), and NMI (0.73 mL, 9.27 mmol) to the reaction mixture, and continuously stir the mixture for 1 hour. Transfer all contents of the flask to a separatory funnel and remove the aqueous layer. The organic layer was washed once with 5% aqueous K₂CO₃ solution (50 mL x 1), three times with 2.5% aqueous ammonia solution (50 mL x 3), twice with 5% aqueous NaHSO₄ solution (50 mL x 2), once with 5% aqueous K₂CO₃ solution (50 mL x 1), and once with 5% aqueous NaCl solution (50 mL). The resulting organic layer was dried over anhydrous Na₂SO₄. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure to obtain Boc-MeLeu-Phe-OMe (3.93 g, 9.67 mmol). THF (38 mL) was added to the resulting concentrate, and the solution was cooled in an ice bath. 2M aqueous NaOH solution (7.0 mL) was added dropwise while maintaining the intermediate temperature of the reaction mixture at 2°C or lower. The reaction mixture was stirred for another 3 hours, and the 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. 5% aqueous NaHSO4 solution (38 mL) was added to the resulting solution, and the mixture was transferred to a separatory funnel to remove the aqueous layer. The resulting organic layer was washed with 5% aqueous NaCl solution (57 mL) and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to obtain Boc-MeLeu-Phe-OH (3.13 g, 7.97 mmol).

[1026] Yield: 86% (after 2 steps)

[1027] Purity: 99.6%

[1028] Measurement method: HPLC method A, retention time: 14.5 min

[1029] Mass spectrometry: m / z 393.74 ([M+H]) + )

[1030] Synthesis of Fmoc-MeAlGly-Val-OH (from raw material 20)

[1031] [Formula 59]

[1032]

[1033] Cbz-Val-OH (3.02 g, 11.9 mmol), cyclohexane (24 mL), and DCM (9 mL) were added to a flask equipped with a stirrer. 2,2,2-Trichloroacetylimine tert-butyl ester (4.3 mL, 23.9 mmol) was added to the flask. While cooling the flask in an ice bath, boron trifluoride diethyl ether complex (0.15 mL, 1.2 mmol) was added dropwise, and the mixture was continuously stirred at room temperature for 1 hour. The completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the filtered solid was washed with cyclohexane. The filtrate and wash solution were combined and washed five times with 10% aqueous citric acid solution (24 mL x 5). The resulting organic layer was washed twice with 5% aqueous Na₂CO₃ solution (24 mL x 2). The resulting organic layer was concentrated under reduced pressure to obtain Cbz-Val-OtBu (3.67 g, 11.9 mmol). 2-MeTHF (37 mL) and 5% Pd / C (50% wet, 2.54 g, 0.60 mmol, Pd metal-based) were added to the resulting concentrate, followed by nitrogen and hydrogen purging. The mixture was then stirred for 2 hours under a hydrogen atmosphere (1 atm). The reaction mixture was filtered, and the Pd / C was washed with 2-MeTHF. The filtrate and wash solution were combined and concentrated under reduced pressure to obtain H-Val-OtBu (2.06 g, 11.9 mmol). MeCN (21 mL), Fmoc-MeAlgly-OH (4.63 g, 13.1 mmol), and NMM (3.9 mL, 35.8 mmol) were added to the flask. While cooling the flask in an ice bath, COMU (6.14 g, 14.3 mmol) was added to the reaction mixture, and the mixture was continuously stirred at room temperature for 2 hours and 30 minutes. The completion of the reaction was confirmed by HPLC analysis. Then, 60 mL of 2-MeTHF and 30 mL of 5% aqueous K₂CO₃ solution were added to the reaction mixture, and the mixture was stirred continuously for 30 minutes. All 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 solution (50 mL x 4), twice with 5% aqueous NaHSO₄ solution (50 mL x 2), once with 5% aqueous K₂CO₃ solution (50 mL x 1), and once with 5% aqueous NaCl solution (50 mL x 1). The resulting organic layer was dried over anhydrous Na₂SO₄.The desiccant was filtered off, and the filtrate was then concentrated under reduced pressure to obtain Fmoc-MeAlGly-Val-OtBu (5.69 g, 11.2 mmol). 2-MeTHF (57 mL) and HMDS (9.4 mL, 44.9 mmol) were added to the flask, followed by dropwise addition of TMSOTf (6.2 mL, 33.7 mmol). After stirring continuously for 2 hours, the flask was cooled, and 2-MeTHF (57 mL) was added while maintaining the reaction mixture at 24°C or lower, followed by dropwise addition of 5% aqueous NaHCO3 solution (68 mL). All 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 solution (20 mL), and the aqueous layer was separated. The two aqueous layers were combined, and 2-MeTHF (114 mL) was added, followed by gradual addition of 85% H3PO4 (8.2 mL). The aqueous layer was removed, and the resulting organic layer was washed with 5% aqueous NaCl solution (68 mL) and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography to obtain Fmoc-MeAlGly-Val-OH (4.37 g, 9.70 mmol).

[1034] Yield: 82% (after 4 steps)

[1035] Purity: 99.6%

[1036] Measurement method: HPLC method A, retention time: 15.0 min

[1037] Mass spectrometry: m / z 451.76 ([M+H]) + )

[1038] Synthesis of H-MeGly(nPr)-Ile-Pro-OtBu (Synthesis of raw material 21)

[1039] [Formula 60]

[1040]

[1041] Step 21-1

[1042] Compound 21-1: (2S)-1-[(2S,3S)-2-(benzyloxycarbonylamino)-3-methyl-pentanoyl]pyrrolidine- Synthesis of tert-butyl 2-carboxylate

[1043] [Formula 61]

[1044]

[1045] (2S)-pyrrolidine-2-carboxylic acid tert-butyl ester (18.8 g), (2S,3S)-2-(benzyloxycarbonylamino)-3-methylvaleric acid (20.0 g), and DMF (140 mL) were added to the nitrogen-replaced reactor at room temperature, and the mixture was stirred. After confirming complete dissolution, the mixture was cooled to 0°C, and DIPEA (52.7 mL) was added. A 50 wt.% solution of propylphosphonic anhydride in ethyl acetate (58.3 mL) was added to the mixture over 20 minutes at 0°C, and the mixture was stirred at 0°C for 1.5 hours. Water (100 mL) and ethyl acetate (200 mL) were added to the mixture in this order. Aqueous layer 1 and organic layer 1 were separated by liquid-liquid separation, and aqueous layer 1 was removed. Organic layer 1 was washed with 100 mL of 5% aqueous potassium bisulfate solution, 100 mL of 5% aqueous sodium carbonate solution, and 100 mL of 10% brine. Water (100 mL) and 200 mL of ethyl acetate were added to the extracted aqueous layer 1, and the mixture was stirred. Organic layer 2 was then obtained by liquid-liquid separation. Organic layers 1 and 2 were combined and concentrated under reduced pressure to obtain compound 21-1 (33.8 g).

[1046] LCMS (ESI) of compound 21-1: Retention time: 2.75 min, m / z = 419 [M+H] +

[1047] Step 21-2

[1048] Compound 21-2: (2S)-1-[(2S,3S)-2-amino-3-methyl-pentanoyl]pyrrolidine-2-carboxylic acid tert-butyl ester Synthesis

[1049] [Formula 62]

[1050]

[1051] Compound 21-1 (31.6 g) and 2-MeTHF (221 mL) obtained in step 21-1 were added to the nitrogen-replaced reactor, and the mixture was cooled to 10 °C. After adding 5% Pd / C (6.32 g, 50% aqueous product) to the mixture, triethylsilane (60.3 mL) was added over 20 minutes. The mixture was stirred at an intermediate temperature of 15 °C for 6 hours, and then stirred again at room temperature for 17 hours. The mixture 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.

[1052] LCMS (ESI) of compound 21-2: Retention time: 1.14 min, m / z = 285 [M+H] +

[1053] Steps 21-3

[1054] Compound 21-3: (2S)-1-[(2S,3S)-2-[[(2S)-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino] Synthesis of tert-butyl pyrrolidine-2-carboxylate [amino]-3-methyl-pentanoyl]pyrrolidine-2-carboxylate

[1055] [Formula 63]

[1056]

[1057] Acetonitrile (221 mL) was added to compound 21-2 obtained in step 21-2 at 5 °C, followed by (2S)-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]valeric acid (28.0 g) and DIPEA (39.6 mL). HATU (34.5 g) was slowly added to the mixture at 2.5 °C. After stirring at 2.5 °C for 1 hour, the mixture was stirred at room temperature for 2.5 hours. 5% aqueous sodium carbonate solution (189 mL) and water (150 mL) were added to the mixture in this order. Toluene (80 mL) and 2-MeTHF (140 mL) were added, and the mixture was stirred, followed by liquid-liquid separation. The organic layer was washed with 5% aqueous potassium bisulfate solution (190 mL x 2) and 10% brine (190 mL x 2). The obtained organic layer was concentrated under reduced pressure to obtain compound 21-3. Compound 21-3 was used in step 21-4 without further purification.

[1058] LCMS (ESI) of compound 21-3: Retention time: 3.43 min, m / z = 620 [M+H] +

[1059] Steps 21-4

[1060] Compound 21-4: (2S)-1-[(2S,3S)-3-methyl-2-[[(2S)-2-(methylamino)pentanoyl]amino] Synthesis of tert-butyl 2-acetate [pentanoyl]pyrrolidine (Synthesis of H-MeGly(nPr)-Ile-Pro-OtBu)

[1061] [Formula 64]

[1062]

[1063] Compound 21-3 (296 mg), synthesized using the same method as in step 21-3, and toluene (2.07 mL) were added to the nitrogen-replaced reactor, and the mixture was stirred. DBU (0.072 mL) was added to the mixture, and the mixture was stirred for 30 minutes. After adding acetonitrile (1.00 mL), the mixture was stirred for 30 minutes, then DBU (0.072 mL) was added, and the mixture was stirred for another 30 minutes. 1N hydrochloric acid (2.00 mL) and n-heptane (1.00 mL) were added, and the mixture was stirred, then 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 solution (2.00 mL) and toluene (4.00 mL) to separate the organic layer containing compound 21-4. The obtained organic layer was washed with 10% brine (2.00 mL) and then concentrated under reduced pressure to obtain compound 21-4 (166 mg).

[1064] LCMS (ESI) of compound 21-4: Retention time: 1.36 min, m / z = 398 [M+H] +

[1065] The analytical conditions for compounds 21-1 to 21-4 by LCMS are shown below.

[1066] Device: Waters UPLC / SQD

[1067] Column: Ascentis Express RP 90A amide, 2.1 mm ID x 50 mm, 2.7 μm

[1068] Mobile phase: 0.1% FA / water (A), 0.1% FA / MeCN (B)

[1069] Elution method: B) 5% (0 min) 100% (4.5 min) 100% (5.0 min) 5% (5.01 min) 5% (7 min)

[1070] Flow rate: 0.5 mL / min

[1071] Column temperature: 40°C

[1072] Detection wavelength: 210 to 400 nm (PDA)

[1073] [Industrial Applicability]

[1074] According to the present invention, even in the case of peptide fragment coupling, peptide compounds can be synthesized with high diastereoselectivity and high yield. Furthermore, according to the present invention, a synthetic method suitable for fragment coupling of peptide compounds containing N-substituted amino acids and / or peptide compounds having large side chains near the reaction site of the amide bond can be provided.

Claims

1. A method for producing a peptide compound or a salt thereof, the method comprising the step of linking an amino group of a first amino acid or peptide to a carboxyl group of a second amino acid or peptide by an amide bond in a bilayer solvent system containing water and one or more organic solvents immiscible with water (linking step).

2. The method according to claim 1, wherein the connection step is performed in the presence of an activator.

3. The method according to claim 1 or 2, wherein the activator is an acyl halide.

4. The method according to claim 1, wherein the connection step comprises the following steps: (1) To prepare a mixed anhydride, the second amino acid or peptide is contacted with an acyl halide in an organic solvent; as well as (2) In the presence of a base, the mixed anhydride obtained in step (1) is contacted with the first amino acid or peptide in a mixed solution containing water and one or more organic solvents that are immiscible with 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 method according to any one of claims 1 to 5, wherein the α-carbon of the carboxyl group of the second amino acid or peptide is represented by the following formula: -CR 1 R 2 -, where R 1 and R 2 They are the same or different and each is a hydrogen atom, a straight-chain C1-C6 alkyl, an optionally substituted branched C3-C6 alkyl, an optionally substituted C3-C8 cycloalkyl-C1-C6 alkyl, an optionally substituted C3-C8 cycloalkyl, an optionally substituted phenyl-C1-C2 alkyl, an optionally substituted C1-C6 alkoxy-C1-C2 alkyl, an optionally substituted 5- to 6-membered heteroaryl-C1-C2 alkyl, or an optionally substituted phenyl-C1-C2 alkylthio-C1-C2 alkyl, or R 1 and R 2 Together with the carbon atoms they are attached to, they form C3-C8 saturated alicyclic rings.

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

8. The method according to any one of claims 3 to 7, wherein the acyl halide is neopentanoyl 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-dimethylpyridine and 2,4,6-trimethylpyridine.

11. The method according to any one of claims 1 to 10, wherein the amino group of the first amino acid or peptide is an amino group represented by the following formula: -NR 4 R 5 , where R 4 It is a hydrogen atom, and R 5 It can be a hydrogen atom, a straight-chain C1-C6 alkyl group, a branched C3-C6 alkyl group, or a C3-C8 cycloalkyl group.

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

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

14. The method according to any one of claims 1 to 13, wherein the peptide compound to be produced or a salt thereof contains a cyclic moiety consisting of 4 to 14 amino acid residues, and wherein the amide bond connecting the amino group of the first amino acid or peptide to the carboxyl group of the second amino acid or peptide is contained at 1 to 7 positions of the cyclic moiety.

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

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