Process for the manufacture of peptide compounds

Abstract

Provided is a method for producing a peptide containing an N-alkyl amino acid, comprising the following steps (1) to (3). Step (1): a step of mixing an N-terminal protected amino acid or an N-terminal protected peptide with a carboxylic acid halide or an alkyl haloformate; Step (2): a step of mixing an amino acid or a peptide that is not protected at the N- and C-termini with a trialkylsilylating agent; and a step of mixing the product obtained in Step (1) with the product obtained in Step (2).

Description

Technical Field

[0001] This invention relates to a method for manufacturing peptides having N-alkyl amino acids. Background Technology

[0002] In recent years, there has been a growing expectation for specialized peptides with low molecular weights (1,000-3,000) and containing non-natural amino acids as next-generation pharmaceutical alternatives to protein formulations. Examples of specialized peptides include peptides containing N-alkyl amino acids other than conventional amino acids. Compared to conventional peptides, peptides containing N-alkyl amino acids possess the following characteristics: low structural freedom, strong binding to targets; membrane permeability; low immunogenicity; and stability in vivo. Therefore, there is a strong desire to develop efficient methods for manufacturing peptides containing N-alkyl amino acids (see, for example, Non-Patent Literature 1).

[0003] Regarding methods for manufacturing peptides containing N-alkyl amino acids, the following methods are known, for example.

[0004] (1) The C-terminus of the product is protected peptide

[0005] - A method for activating the C-terminus of an N-terminal protected amino acid using neopentanoyl chloride and reacting it with a benzyl ester of an N-methyl amino acid (see, for example, Patent Document 1).

[0006] (2) The C-terminal unprotected peptide of the product

[0007] - A method for activating the C-terminus of an N-terminal protected amino acid using isobutyl chloroformate and reacting it with silylated N-methylglycine (sarcosine) and proline (see, for example, Non-Patent Literature 2).

[0008] - A method for activating the C-terminus of an N-terminal protected peptide using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and reacting it with N-methylalanine (see, for example, Patent Document 2).

[0009] In addition, as a method for manufacturing peptides composed of N-methyl amino acids in sequence, the following method is known: the C-terminus of N-Boc-N-methylleucine is activated by neopentanoyl chloride and reacted with N-methylphenylalanine methyl ester (for example, see Non-Patent Literature 3).

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: US Patent No. 5739104

[0013] Patent Document 2: International Publication No. 2009 / 134405

[0014] Non-patent literature

[0015] Non-patent literature 1: Pharmacia 2014, Vol. 50, pp. 751-755

[0016] Non-patent literature 2: Indian Journal of Chemistry, 2004, Vol. 43B, p. 1282

[0017] Non-patent literature 3: Tetrahedron, 2012, Vol. 68, pp. 7070 Summary of the Invention

[0018] The problem that the invention aims to solve

[0019] The inventors of this application have confirmed that when using the method described in Non-Patent Document 2 to introduce an N-terminal alkyl amino acid to the C-terminus of an N-terminal protected amino acid, the reaction does not necessarily proceed with sufficient conversion, depending on the type of amino acid introduced. This is particularly true for acyclic N-alkyl amino acids such as N-methylglycine and peptides, where a large amount of raw material remains, making it impossible to obtain the target product in a satisfactory yield. This residue of raw material contributes to the formation of peptides lacking several amino acids, which are difficult to remove due to their similar physical properties to the target peptide, thus causing quality problems. Furthermore, the method described in Patent Document 2 uses HATU, which contains an explosive triazole structure, as a condensing agent, which may not be suitable for the industrial production of peptides.

[0020] On the other hand, in the methods described in Patent Document 1 and Non-Patent Document 3, the C-terminus of the generated peptide is protected. In order to obtain a peptide with an unprotected C-terminus, a deprotection process is required. Therefore, in addition to the condensation process, a deprotection process must also occur, making it unsuitable for efficient peptide manufacturing methods.

[0021] This invention provides a method for manufacturing a peptide containing an N-alkyl amino acid and having an unprotected C-terminus. Additionally, this invention provides a method for manufacturing a peptide in which an amino acid or peptide containing an N-alkyl group and having unprotected N-terminus and C-terminus, and an N-terminal protected amino acid or peptide are used as materials.

[0022] Methods for solving problems

[0023] The inventors of this application conducted in-depth research and discovered that by mixing an unprotected amino acid or peptide containing an N-alkyl group and unprotected at both the N-terminus and C-terminus with a silylating agent, and then mixing the N-terminal protected amino acid or peptide with a carboxylic acid activator having a specific structure, the aforementioned problems can be solved, thus completing this invention. Specifically, the features of this invention are as follows.

[0024] [1] A method for manufacturing peptides, comprising the following steps (1) to (3),

[0025] Step (1) involves mixing the N-terminal protected amino acid or N-terminal protected peptide represented by formula (I) with an activator selected from the group consisting of a carboxylic acid halide represented by formula (II) and a alkyl haloformate represented by formula (III).

[0026] Formula (I): PA 1 -OH

[0027] (In the formula, P is the N-terminal protecting group, A) 1 Indicates groups derived from amino acids, derived from NC 1-6 alkyl amino acid groups (C 1-6 Alkyl groups (which may have substituents) or groups derived from peptides.

[0028] Equation (II):

[0029] [Chemical Formula 1]

[0030]

[0031] (In the formula, X represents a halogen atom, R...) 1 This refers to a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and may have substituents, or a primary aliphatic hydrocarbon group having 4 or more carbon atoms and may have substituents (wherein, the substituents of the primary aliphatic hydrocarbon group are located on the carbon atom bonded to the carbonyl carbon).

[0032] [Chemical Formula 2]

[0033]

[0034] (In the formula, X represents a halogen atom, R...) 2 (Indicates a secondary aliphatic hydrocarbon group with 5 or more carbon atoms and which may have substituents);

[0035] Step (2) involves mixing the amino acid or peptide represented by formula (IV) with a silylating agent.

[0036] Formula (IV): HA 2 -OH

[0037] (where A is in the formula) 2 Indicates originating from NC 1-6 alkyl amino acid groups (C 1-6 Alkyl groups may have substituents), or groups derived from 4-6 membered cyclic secondary amino acids (the 4-6 membered ring may be selected from C). 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8Cyclic compounds in the group consisting of cycloalkyl rings (fused together) or groups derived from peptides, wherein the N-terminal residue of the peptide is NC. 1-6 Alkyl amino acids (C 1-6 Alkyl groups (which may have substituents) or 4-6 membered cyclic secondary amino acids (the 4-6 membered ring can be selected from C... 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 (fusion of cyclic compounds in the group consisting of cycloalkyl rings);

[0038] Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

[0039] [2] A method for manufacturing peptides, comprising the following steps (1) to (3),

[0040] Step (1) involves mixing the N-terminal protected amino acid represented by formula (I) with an activator selected from the group consisting of a carboxylic acid halide represented by formula (II) and a alkyl haloformate represented by formula (III).

[0041] Formula (I): PA 1 -OH

[0042] (In the formula, P is the N-terminal protecting group, A) 1 Indicates a group derived from an amino acid or from NC. 1-6 alkyl amino acid groups (C 1-6 Alkyl groups may have substituents.

[0043] Equation (II):

[0044] [Chemical Formula 3]

[0045]

[0046] (In the formula, X represents a halogen atom, R...) 1 This refers to a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and may have substituents, or a primary aliphatic hydrocarbon group having 4 or more carbon atoms and may have substituents (wherein, the substituents of the primary aliphatic hydrocarbon group are located on the carbon atom bonded to the carbonyl carbon).

[0047] Equation (III):

[0048] [Chemical Formula 4]

[0049]

[0050] (In the formula, X represents a halogen atom, R...) 2 (Indicates a secondary aliphatic hydrocarbon group with 5 or more carbon atoms and which may have substituents);

[0051] Step (2) involves mixing the amino acid or peptide represented by formula (IV) with a silylating agent.

[0052] Formula (IV): HA 2 -OH

[0053] (where A is in the formula) 2 Indicates a group derived from N-methyl amino acids, or from NC 1-6 The group of alkylglycine (C 1-6 The alkyl group may have substituents, or a group derived from a 4-6 membered cyclic secondary amino acid, or a group derived from a peptide, wherein the N-terminal residue is an N-methyl amino acid, NC... 1-6 Alkyl glycine (C 1-6 Alkyl groups may have substituents, or 4-6 membered cyclic secondary amino acids;

[0054] Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

[0055] [3] A method for manufacturing peptides, comprising the following steps (1) to (3),

[0056] Step (1) involves mixing the N-terminal protective peptide represented by formula (V) with the carboxylic acid halide represented by formula (II).

[0057] Formula (V): PA 3 -OH

[0058] (In the formula, P is the N-terminal protecting group, A) 3 (Indicates a group derived from a peptide)

[0059] Equation (II):

[0060] [Chemical Formula 5]

[0061]

[0062] (In the formula, X represents a halogen atom, R...) 1 It represents a secondary or tertiary aliphatic hydrocarbon group with 5 or more carbon atoms and may have substituents, or a primary aliphatic hydrocarbon group with 4 or more carbon atoms and has substituents (wherein, the substituents of the primary aliphatic hydrocarbon group are present on the carbon atom bonded to the carbonyl carbon).

[0063] Step (2) involves mixing the amino acid represented by formula (IV') with a silylating agent.

[0064] Formula (IV'): HA 2’ -OH

[0065] (where A is in the formula) 2’ Indicates a group derived from N-methyl amino acids, or from NC1-6 The group of alkylglycine (C 1-6 Alkyl groups may have substituents (or groups derived from 4-6 cyclic secondary amino acids);

[0066] Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

[0067] [4] A method for manufacturing a peptide as described in any one of [1] to [3] above, comprising a step of removing the N-terminal protecting group of the peptide obtained in step (3).

[0068] [5] The method for manufacturing the peptide as described in any one of [1] to [3] above further includes repeating the following steps (4) and (5) more than once.

[0069] Step (4) removes the protecting group at the N-terminus of the peptide obtained in step (3) or (5);

[0070] Step (5) involves reacting the N-terminal protected amino acid or N-terminal protected peptide with the N-terminus of the peptide obtained in step (4).

[0071] [6] The method for manufacturing peptides as described in [1] or [3] above, wherein, located in formula (I)PA 1 -OH or formula (V)PA 3 The -OH group represents the C-terminal amino acid in the N-terminal protective peptide, excluding N and C. 1-6 Alkyl amino acids (C 1-6 Alkyl groups (which may have substituents) or 4-6 membered cyclic secondary amino acids (the 4-6 membered ring can be selected from C... 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 Amino acids other than those in the group consisting of cycloalkyl rings (fused cyclic compounds).

[0072] In equations (I) and (V), P is an N-terminal protecting group, and A 1 and A 3 Each represents a group derived from the peptide.

[0073] [7] The method for manufacturing peptides as described in [1] or [2] above, wherein A 1 It is a group derived from amino acids.

[0074] [8] The method for manufacturing a peptide as described in [1] or [2] above, wherein the N-terminal protected amino acid represented by formula (I) or the C-terminal amino acid located in the N-terminal protected peptide represented by formula (I) is an α-amino acid, a β-amino acid or a γ-amino acid.

[0075] [9] The method for manufacturing peptides as described in [8] above, wherein the N-terminal protected amino acid represented by formula (I) or the C-terminal amino acid located in the N-terminal protected peptide represented by formula (I) is an α-amino acid.

[0076]

[10] The method for manufacturing a peptide as described in [1] above, wherein the amino acid represented by formula (IV) or the amino acid located at the N-terminus of the peptide represented by formula (IV) is NC. 1-6 Alkyl-α-amino acids (C 1-6 Alkyl groups may have substituents) or 4-6 cyclic secondary-α-amino acids.

[0077]

[11] The method for manufacturing peptides as described in [1] above, wherein the amino acid represented by formula (IV) or the amino acid located at the N-terminus of the peptide represented by formula (IV) is an N-methyl-α-amino acid or an N-ethyl-α-amino acid (N-methyl and N-ethyl may each have substituents) or a 4-6 member cyclic secondary-α-amino acid.

[0078]

[12] A method for manufacturing a peptide as described in any one of [1] to

[11] above, wherein the activator is a carboxylic acid halide represented by formula (II), R 1 The number of carbon atoms ranges from 5 to 20, and X is a chlorine atom.

[0079]

[13] The method for manufacturing a peptide as described in any one of [1] to

[12] above, wherein the activator is a carboxylic acid halide represented by formula (II) selected from the group of compounds below.

[0080] [Chemical Formula 6]

[0081]

[0082]

[14] A method for manufacturing a peptide as described in any one of [1] to

[12] above, wherein the activator is a carboxylic acid halide represented by formula (II) selected from the group of compounds below.

[0083] [Chemical Formula 7]

[0084]

[0085]

[15] The method for manufacturing peptides as described in

[13] or

[14] above, wherein the activator is a compound described below.

[0086] [Chemical Formula 8]

[0087]

[0088]

[16] The method for manufacturing peptides as described in [1] or [2] above, wherein the activator is an alkyl haloformate represented by formula (III), where X is a chlorine atom.

[0089]

[17] The method for manufacturing peptides as described in [1] or [2] above, wherein the activator is an alkyl haloformate represented by formula (III) selected from the group of compounds below.

[0090] [Chemical Formula 9]

[0091]

[0092]

[18] The method for manufacturing a peptide as described in [1] or [2] above, wherein the activator is an alkyl haloformate represented by formula (III) selected from the group of compounds below.

[0093] [Chemical Formula 10]

[0094]

[0095]

[19] The method for manufacturing a peptide as described in any one of [1] to

[18] above, wherein the silylating agent is a trimethylsilylating agent.

[0096]

[20] The method for manufacturing a peptide as described in any one of [1] to

[19] above, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea or N,O-bis(trimethylsilyl)trifluoroacetamide.

[0097]

[21] The method for manufacturing a peptide as described in any one of [1] to

[20] above, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide.

[0098]

[22] The method for manufacturing peptides as described in [1] above, wherein the amino acid or peptide represented by formula (IV) is an amino acid other than proline or a peptide whose N-terminal residue is an amino acid residue other than proline.

[0099]

[23] The method for manufacturing peptides as described in [3] above, wherein the amino acid represented by formula (IV') is an amino acid other than proline.

[0100]

[24] A compound represented by the following formula:

[0101] [Chemical Formula 11]

[0102]

[0103] The effects of the invention

[0104] This invention provides a novel method for manufacturing C-terminal unprotected peptides, using N-alkyl-containing amino acids or peptides with unprotected N-terminus and C-terminus, and N-terminal protected amino acids or peptides as materials. According to the manufacturing method of this invention, regardless of the type of the introduced N-alkyl amino acid or the type of N-alkyl amino acid in the N-terminal residues of the introduced peptide, the target peptide can be obtained in a satisfactory yield using industrially applicable reagents and with few steps. Detailed Implementation

[0105] The present invention will now be described in detail.

[0106] In this specification, “n-” indicates normal, “s-” indicates secondary, “t-” and “tert-” indicate tert-, “Me” indicates methyl, “Et” indicates ethyl, “Pr” indicates propyl, “Bu” indicates butyl, “Ph” indicates phenyl, “Bn” indicates benzyl, “Boc” indicates tert-butoxycarbonyl, “Cbz” indicates benzyloxycarbonyl, “Fmoc” indicates 9-fluorenylmethoxycarbonyl, “Trt” indicates triphenylmethyl, “TMS” indicates trimethylsilyl, and “TFA” indicates trifluoroacetic acid.

[0107] "Halogen atom" refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom.

[0108] In this invention, "alkyl" refers to a monovalent group of a straight-chain or branched saturated aliphatic hydrocarbon. "C 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 3,3-dimethylbutane-2-yl, etc.

[0109] "Zhong or Shu C" 5-40 "Alkyl" refers to a monovalent group obtained by removing a hydrogen atom from a saturated aliphatic hydrocarbon having 5 to 40 carbon atoms and containing at least one secondary or tertiary carbon atom. Specific examples include 2-methylbutane-2-yl, 3-methylbutane-2-yl, 3,3-dimethylbutane-2-yl, 3-pentyl, 2,2,4-trimethylpentane-3-yl, 2,4-dimethylpentane-3-yl, 4-ethyl-2,2-dimethylhexane-3-yl, 3-heptyl, and 2,2,4,8,10,10-hexamethylundecane-5-yl. Additionally, "secondary or tertiary C..." 5-20 "Alkyl" refers to secondary or tertiary alkyl groups with 5 to 20 carbon atoms.

[0110] "Ber C" 4-40"Alkyl" refers to a monovalent group obtained by removing hydrogen from a primary carbon atom of a straight-chain or branched saturated aliphatic hydrocarbon having 4 to 40 carbon atoms. Examples include n-butyl, isobutyl, n-pentyl, 2-methylbutyl or 3-methylbutyl, or n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-eicosyl, n-trianealkyl, n-tetraalkyl, or primary alkyl groups as their isomers. Additionally, "primary C..." 4-20 "Alkyl" refers to primary alkyl groups with 4 to 20 carbon atoms.

[0111] In this invention, "alkenyl" refers to a monovalent group in a straight-chain or branched unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond. "Secondary or tertiary alkenyl" refers to a monovalent group obtained by removing the hydrogen atom from the secondary or tertiary carbon atom of an unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom and at least one carbon-carbon double bond; specific examples include isopropenyl and 1-methyl-1-propenyl. Additionally, "secondary or tertiary alkenyl" refers to... 5-40 "Alkenyl" refers to secondary or tertiary alkenyl groups with 5 to 40 carbon atoms. 5-20 "Alkenyl" refers to secondary or tertiary alkenyl groups with 5 to 20 carbon atoms.

[0112] "Ber C" 4-40 "Alkenyl" refers to a monovalent group obtained by removing a hydrogen atom from a primary carbon atom of a straight-chain or branched unsaturated aliphatic hydrocarbon with 4 to 40 carbon atoms and containing at least one carbon-carbon double bond. Examples include 2-butenyl, 3-butenyl, and 2-pentenyl. Additionally, "primary carbon..." 4-20 "Alkenyl" refers to primary alkenyl groups with 4 to 20 carbon atoms.

[0113] “C 6-14 "Aryl" refers to an aromatic hydrocarbon group with 6 to 14 carbon atoms. Specific examples include phenyl, 1-naphthyl, 2-naphthyl, 1-anthrayl, 2-anthrayl, 9-anthrayl, and biphenyl. Additionally, "C..." 6-14 "Aromatic ring" refers to an aromatic hydrocarbon ring with 6 to 14 carbon atoms.

[0114] “C 6-14 "Halogenated aryl" refers to an aromatic hydrocarbon group with 6 to 14 carbon atoms substituted by one or more halogen atoms. Specific examples include 4-chlorophenyl, 2,4-dichlorophenyl, 5-fluoro-1-naphthyl, 6-bromo-2-naphthyl, 6,7-diiodo-1-anthrayl, 10-bromo-9-anthrayl, and 4'-chloro-(1,1'-biphenyl)-2-yl. 6-14 "Halogenated aromatic rings" refer to aromatic hydrocarbon rings with 6 to 14 carbon atoms that are replaced by one or more halogen atoms.

[0115] “C 6-14"Aryloxy group" refers to aryloxy groups with 6 to 14 carbon atoms. Specific examples include phenoxy, 1-naphthoxy, 2-naphthoxy, 1-anthraoxy, 2-anthraoxy, 9-anthraoxy, biphenyloxy, etc.

[0116] "5-10 membered heterocyclic group" refers to a heterocyclic group of monocyclic or fused ring systems in which the number of atoms constituting the ring is 5 to 10, and the atoms constituting the ring contain 1 to 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur atoms. The heterocyclic group can be any of the following: saturated, partially unsaturated, or unsaturated. Specific examples include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyridazinyl, azaheptanyl, oxaheptanyl, thioheptanyl, acrylonitrileyl, oxaheptenyl, thioheptenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolinyl, pyrazinyl, morpholinyl, thiazolinyl, indolyl, isoindolyl, benzimidazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalolinyl, boryl, pteridinyl, chromenyl, isochromenyl, etc.

[0117] “C 1-6 "Alkoxy" refers to a straight-chain or branched alkoxy group with 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, and n-hexoxy.

[0118] “C 3-6 "Cycloalkyl" refers to a monovalent group in a cyclic saturated aliphatic hydrocarbon with 3 to 6 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0119] “C 3-8 "Cycloalkyl" refers to cycloalkyl groups with 3 to 8 carbon atoms. As a specific example, besides the "C" mentioned above, other cycloalkyl groups include those with 3 to 8 carbon atoms. 3-6 Besides examples of "cycloalkyl", other examples include cycloheptyl and cyclooctyl. Additionally, "C" 5-8 "Cycloalkyl" refers to cycloalkyl groups with 5 to 8 carbon atoms. 5-6 "Cycloalkyl" refers to cycloalkyl groups with 5 to 6 carbon atoms. 3-8 "Cycloalkyl ring" refers to a cycloalkyl ring with 3 to 8 carbon atoms.

[0120] “C 3-6 "Cycloalkoxy" refers to cycloalkoxy groups with 3 to 6 carbon atoms. Specific examples include cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0121] "Single C" 1-6 "alkylamino" refers to one of the above "C" groups. 1-6Alkyl groups are groups formed by bonding an amino group. Specific examples include monomethylamino, monoethylamino, monon-n-propylamino, monoisopropylamino, monon-n-butylamino, monoisobutylamino, monotert-butylamino, monon-n-pentylamino, and monon-n-hexylamino.

[0122] "2C" 1-6 "alkylamino" refers to two identical or different C atoms mentioned above. 1-6 An alkyl group is a group formed by bonding an amino group. Specific examples include dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-tert-butylamino, di-n-pentylamino, di-n-hexylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-methylamino, N-n-butyl-N-methylamino, N-isobutyl-N-methylamino, N-tert-butyl-N-methylamino, N-methyl-N-pentylamino, N-n-hexyl-N-methylamino, N-ethyl-N-n-propylamino, N-ethyl-N-isopropylamino, N-n-butyl-N-ethylamino, N-ethyl-N-isobutylamino, N-tert-butyl-N-ethylamino, N-ethyl-N-pentylamino, N-ethyl-N-hexylamino, etc.

[0123] “C 1-6 "Alkoxycarbonyl" refers to a straight-chain or branched alkoxycarbonyl group with 1 to 6 carbon atoms. Specific examples include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, n-pentoxycarbonyl, and n-hexyloxycarbonyl.

[0124] "Three Cs" 1-6 "alkylsilyl" refers to three identical or different C's mentioned above. 1-6 Alkyl groups are formed by bonding alkyl groups to silyl groups. Specific examples include trimethylsilane (TMS) group, triethylsilane, triisopropylsilane, tert-butyldimethylsilane, and di-tert-butylisobutylsilane.

[0125] "Three Cs" 1-6 "alkylsilyloxy" refers to three identical or different "C" groups. 1-6 "alkyl" refers to a group formed by bonding an alkyl group to a silyloxy group. Specific examples include trimethylsilyloxy, triethylsilyloxy, triisopropylsilyloxy, tert-butyldimethylsilyloxy, and di-tert-butylisobutylsilyloxy.

[0126] "Bicycloalkyl" refers to a monovalent group in a saturated aliphatic hydrocarbon containing two bridgehead carbons and having two rings. Specific examples include octahydroinden-3-yl, octahydronaphth-4-yl, bicyclo[2.2.1]heptane-1-yl, or bicyclo[2.2.1]heptane-2-yl. Additionally, "C..." 5-10 "Bicycloalkyl" refers to a bicycloalkyl group with 5 to 10 carbon atoms. 7-10 "Bicycloalkyl" refers to bicycloalkyl groups with 7 to 10 carbon atoms.

[0127] "Tricycloalkyl" refers to a monovalent group in a saturated aliphatic hydrocarbon containing at least three bridgehead carbons and having three rings. A specific example is a tricycloalkyl group [3.3.1.1]. 3,7 ] Decane-1-yl (adamantane-1-yl) or tricyclic [3.3.1.1] 3,7 Decane-2-yl (adamantane-2-yl) group, etc. Additionally, the so-called "C..." 5-15 "Tricycloalkyl" refers to tricycloalkyl groups with 5 to 15 carbon atoms. 7-15 "Tricyclic alkyl" refers to tricyclic alkyl groups with 7 to 15 carbon atoms.

[0128] "Secondary or tertiary aliphatic hydrocarbon group" refers to a monovalent group obtained by removing the hydrogen atom from a branched or cyclic saturated or unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom in the hydrocarbon chain. Examples include secondary or tertiary alkyl groups, bicycloalkyl groups, tricycloalkyl groups, and secondary or tertiary alkenyl groups. Specific examples include secondary or tertiary alkyl groups, bicycloalkyl groups, tricycloalkyl groups, and secondary or tertiary alkenyl groups with 5 or more carbon atoms. Preferably, secondary or tertiary C groups are also mentioned. 5-40 Alkyl, C 5-10 Bicycloalkyl, C 5-15 Tricycloalkyl, secondary or tert-C 5-40 Alkenyl groups, etc., more preferably secondary or tertiary C, can be mentioned. 5-20 Alkyl, C 7-10 Bicycloalkyl, C 7-15 Tricycloalkyl, secondary or tert-C 5-20 Alkenyl groups, etc.

[0129] "Second aliphatic hydrocarbon group" refers to a monovalent group obtained by removing the hydrogen atom from a branched or cyclic saturated or unsaturated aliphatic hydrocarbon containing at least one secondary carbon atom in the hydrocarbon chain. Examples include secondary alkyl, cycloalkyl, and secondary alkenyl groups. Specific examples include secondary alkyl, cycloalkyl, and secondary alkenyl groups with 5 or more carbon atoms. Preferably, secondary C... 5-40 Alkyl, C 3-8 cycloalkyl, secondary C 5-40 Alkenyl groups, etc., and more preferably secondary C can be cited as examples. 5-20 Alkyl, C 3-6 cycloalkyl, secondary C 5-20Alkenyl groups, etc.

[0130] "Primary aliphatic hydrocarbon group" refers to a monovalent group obtained by removing hydrogen from a primary carbon atom of a straight-chain or branched saturated or unsaturated aliphatic hydrocarbon. Examples include primary alkyl groups and primary alkenyl groups. Specific examples include primary alkyl groups and primary alkenyl groups with 4 or more carbon atoms. Preferred examples include primary C4 groups. 4-40 Alkyl, primary C 4-40 Alkenyl groups, etc., and more preferably primary C can be cited as examples. 4-20 Alkyl, primary C 4-20 Alkenyl groups, etc.

[0131] "May have substituents" means unsubstituted or substituted by any number of substituents.

[0132] "Having substituents" means being replaced by any number of any substituents.

[0133] The term "any substituent" can refer to any substituent that does not adversely affect the reaction intended for use in this invention, and there is no particular limitation on the type of substituent.

[0134] As "C" 1-6 The term "substituent" in "alkyl groups may have substituents" can be exemplified by C14. 6-14 Aryl, C 6-14 Halogenated aryl, C 6-14 aryloxy group, 5-10 membered heterocyclic group, hydroxyl group, C 1-6 Alkoxy, C 3-6 Cycloalkoxy, acetoxy, benzoyloxy, mono-C 1-6 Alkylamino, N-acetylamino, di-C 1-6 Alkylamino, halogen atom, C 1-6 Alkoxycarbonyl, phenoxycarbonyl, N-methylcarbamoyl, N-phenylcarbamoyl, tri-C 1-6 Alkyl silyl, tri-C 1-6 Alkylsilyloxy, C 3-8 Cycloalkyl, cyano, nitro, etc., preferably C 6-14 Aryl, C 6-14 Halogenated aryl, C 1-6 Alkoxy, diC 1-6 Alkylamino, tri-C 1-6 Alkyl silyl, tri-C 1-6 Alkyl silyloxy group, C 3-8 Cycloalkyl, more preferably C 6-14 Aryl, C 6-14 Halogenated aryl, C 1-6 Alkoxy, tri-C 1-6 Alkyl silyl, C 3-8 Cycloalkyl, more preferably C 6-14 Aryl or C3-8 Cycloalkyl, particularly phenyl or cyclohexyl.

[0135] As a "substituent" in "secondary or tertiary aliphatic hydrocarbon group that may have substituents" or "primary aliphatic hydrocarbon group that has substituents," for example, C 6-14 Aryl, C 6-14 Halogenated aryl, C 6-14 aryloxy group, 5-10 membered heterocyclic group, hydroxyl group, C 1-6 Alkoxy, C 3-6 Cycloalkoxy, acetoxy, benzoyloxy, mono-C 1-6 Alkylamino, N-acetylamino, di-C 1-6 Alkylamino, halogen atom, C 1-6 Alkoxycarbonyl, phenoxycarbonyl, N-methylcarbamoyl, N-phenylcarbamoyl, tri-C 1-6 Alkyl silyl, tri-C 1-6 Alkyl silyloxy group, C 3-8 Cycloalkyl, cyano, nitro, etc., preferably C 6-14 Aryl, C 6-14 Halogenated aryl, C 1-6 Alkoxy, diC 1-6 Alkylamino, tri-C 1-6 Alkyl silyl, tri-C 1-6 Alkyl silyloxy group, C 3-8 Cycloalkyl, more preferably C 6-14 Aryl, C 6-14 Halogenated aryl, C 1-6 Alkoxy, tri-C 1-6 Alkyl silyl, C 3-8 Cycloalkyl, more preferably C 6-14 Aryl or C 3-8 Cycloalkyl. It should be noted that the carboxylic acid halides represented by formula (II) are R... 1 In the phrase “a primary aliphatic hydrocarbon group with substituents”, the “substituent” can be replaced by a hydrogen atom bonded to the primary carbon atom of the carbonyl carbon.

[0136] The terms "N-terminal protected amino acid" and "N-terminal protected peptide" refer to amino acids and peptides where the N-terminal amino group is protected and the C-terminal carboxyl group is unprotected, respectively. Similarly, "C-terminal protected amino acid" and "C-terminal protected peptide" refer to amino acids and peptides where the C-terminal carboxyl group is protected and the N-terminal amino group is unprotected, respectively.

[0137] The terms "unprotected amino acids at the N-terminus and C-terminus" and "unprotected peptides at the N-terminus and C-terminus" refer to amino acids and peptides whose N-terminal amino and C-terminal carboxyl groups are unprotected, respectively. It should be noted that in unprotected amino acids at the N-terminus and C-terminus, if reactive functional groups such as side chains that do not participate in peptide formation are present, these functional groups may or may not be protected. Side-chain protected amino acids are preferred.

[0138] The amino acids used in this invention are organic compounds having both amino and carboxyl functional groups, referring to both natural and non-natural amino acids. Preferably, they are α-, β-, or γ-amino acids, or homoamino acids, with α-amino acids being more preferred. Furthermore, in cases where these amino acids contain two or more amino groups (e.g., arginine, lysine, 2,3-diaminopropionic acid (Dap), etc.), two or more carboxyl groups (e.g., glutamic acid, aspartic acid, etc.), or reactive functional groups (e.g., cysteine, serine, tyrosine, glutamine, histidine, tryptophan, etc.), the amino acids used in this invention also include amino acids whose amino, carboxyl, and / or reactive functional groups that do not participate in peptide formation are protected and / or modified.

[0139] The term "4-6 membered cyclic secondary amino acid" used in this invention refers to an amino acid in which the nitrogen atom of the amino group, together with two alkyl groups bonded to the amino group, forms a 4-6 membered ring. Proline is a specific example. The 4-6 membered ring is selected from C... 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 When cyclic compounds in the group consisting of cycloalkyl rings are fused, the preferred cyclic compound is C. 6-14 Aromatic rings, more preferably benzene. Therefore, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) is a specific example of the fusion of a 4- to 6-membered cyclic secondary amino acid with a cyclic compound.

[0140] The NC used in this invention 1-6 Alkyl amino acids are amino acids whose amino group is replaced by a C-shaped group that can have substituents. 1-6 Alkyl-substituted amino acids, preferably amino groups of amino acids that may have a C10 group. 6-14 Aryl, C 6-14 Halogenated aryl, C 1-6 Alkoxy, diC 1-6 Alkylamino, tri-C 1-6 Alkyl silyl, tri-C 1-6 alkylsilyloxy, or C 3-8 C of cycloalkyl 1-6 Alkyl-substituted amino acids, more preferably amino groups of amino acids, may have a C10 group. 6-14 Aryl, C 6-14 Halogenated aryl, C1-6 Alkoxy, tri-C 1-6 alkylsilyl, or C 3-8 C of cycloalkyl 1-6 Alkyl-substituted amino acids, more preferably amino acids in which the amino group of the amino acid is substituted by methyl, ethyl, propyl, butyl, benzyl or cyclohexylmethyl, even more preferably amino acids in which the amino group of the amino acid is substituted by methyl or ethyl, and particularly preferably amino acids in which the amino group of the amino acid is substituted by methyl.

[0141] The terms "groups derived from amino acids" and "groups derived from NC" used in this invention 1-6 "alkyl amino acid group" refers to the group derived from the amino or NC group of an amino acid. 1-6 NC of alkyl amino acids 1-6 A divalent group formed by removing a hydrogen atom from the nitrogen atom of an alkylamino group and removing a hydroxyl group from the carboxyl group. Similarly, "a group derived from an N-methylamino acid" refers to a divalent group formed by removing a hydrogen atom from the nitrogen atom of the N-methylamino group of an N-methylamino acid and removing a hydroxyl group from the carboxyl group; "a group derived from an N-methylamino acid" refers to a divalent group formed by removing a hydrogen atom from the nitrogen atom of the N-methylamino group of an N-methylamino acid and removing a hydroxyl group from the carboxyl group. 1-6 "alkylglycine group" refers to the group derived from NC 1-6 NC of alkylglycine 1-6 A divalent group is formed by removing a hydrogen atom from the nitrogen atom of an alkylamino group and removing a hydroxyl group from the carboxyl group. A "group derived from a cyclic secondary amino acid" refers to a divalent group formed by removing a hydrogen atom from the nitrogen atom of the secondary amino group of a cyclic secondary amino acid and removing a hydroxyl group from the carboxyl group. A "group derived from a 4-6 member cyclic secondary amino acid" refers to a divalent group formed by removing a hydrogen atom from the nitrogen atom of the secondary amino group of a 4-6 member cyclic secondary amino acid and removing a hydroxyl group from the carboxyl group.

[0142] The amino acids constituting the peptides used in this invention are the amino acids described above.

[0143] The term "group derived from peptide" as used in this invention refers to a divalent group formed by removing hydrogen atoms from the primary or secondary amino groups of various amino acids constituting the N-terminus and removing hydroxyl groups from the carboxyl groups of amino acids constituting the C-terminus.

[0144] The stereostructure of amino acids is not particularly limited, but the L-type is preferred.

[0145] In this invention, the "N-terminal protecting group" represented by P in formulas (I) and (V), for example, refers to the protecting group on the N-terminal side during peptide elongation (amidation) and known protecting groups can be used. Specific examples include urethane-based protecting groups (9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(p-biphenyl)isopropoxycarbonyl, etc.), amide-based protecting groups (acetyl, trifluoroacetyl, etc.), imide-based protecting groups (phthaloyl, etc.), sulfonamide-based protecting groups (p-toluenesulfonyl, 2-nitrobenzenesulfonyl, etc.), benzyl, etc., with 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, and benzyloxycarbonyl being preferred examples.

[0146] All technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Any methods and materials identical or equivalent to those described in this specification may be used in the implementation or testing of this invention, but preferred methods and materials are described below. All publications and patents mentioned in this specification are incorporated herein by reference for the purpose of describing and disclosing, for example, constructs and methodologies described in publications that are applicable in connection with the described invention.

[0147] (Detailed description of the method for manufacturing the peptides of the present invention)

[0148] The steps (1) to (5) of the peptide manufacturing method of the present invention will be described below.

[0149] In one manner, the manufacture of the peptide of the present invention consists of each unit process described as the following steps (1) to (5).

[0150] In one manner, the manufacture of the peptides of the present invention can be carried out by performing all or a suitable combination of the unit steps described as steps (1) to (5) below.

[0151] It should be noted that this specific explanation is based on the following content.

[0152] (a) R in the records of processes (1) to (5) 1 and R 2 Same meaning as above.

[0153] (b) As for the specific reaction conditions, there are no particular limitations as long as the production of the peptides of the present invention can be achieved. Preferred conditions for each reaction will be described in detail as appropriate.

[0154] (c) The solvents described in each reaction may be used alone or in combination of two or more.

[0155] Process (1)

[0156] This step involves mixing an N-terminal protected amino acid or N-terminal protected peptide with a carboxylic acid halide or an alkyl haloformate. This step also involves activating the C-terminus of the N-terminal protected amino acid or N-terminal protected peptide using a carboxylic acid halide or an alkyl haloformate. In one embodiment of the invention, formula (I) PA... 1 -OH (where P is the N-terminal protecting group, A 1 Indicates groups derived from amino acids, derived from NC 1-6 alkyl amino acid groups (C 1-6 The alkyl group may have substituents or groups derived from peptides. The process involves mixing an N-terminal protected amino acid or N-terminal protected peptide (represented by ) with a carboxylic acid halide or an alkyl ester of a haloformate. Alternatively, in another embodiment of the invention, formula (V)PA is used. 3 -OH (where P is the N-terminal protecting group, A 3 The group indicates a group derived from the peptide. The ) indicates the process of mixing the N-terminal protected peptide with a carboxylic acid halide.

[0157] The N-terminal protected amino acid or N-terminal protected peptide is the aforementioned amino acid or peptide that has been protected at the N-terminus. Specifically, it is an N-terminal protected amino acid or an N-terminal protected N-terminal peptide. 1-6 Alkyl amino acids (C 1-6 The alkyl group may have substituents) or the peptide may be N-terminally protected. It should be noted that the N-terminal protected peptide in this process preferably has an N-terminal amino acid of type N-C. 1-6 Alkyl amino acids (C 1-6 Alkyl groups (which may have substituents) or 4-6 membered cyclic secondary amino acids (the 4-6 membered ring can be selected from C... 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 N-terminal protected peptides of amino acids other than those in the group consisting of cycloalkyl rings (fused cyclic compounds).

[0158] Carboxylic acid halides are represented by the following formula (II).

[0159] [Chemical Formula 12]

[0160]

[0161] (In the formula, X represents a halogen atom, R...) 1 This indicates a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and may have substituents, or a primary aliphatic hydrocarbon group having 4 or more carbon atoms and may have substituents (wherein the substituents of the primary aliphatic hydrocarbon group are located on the carbon atom bonded to the carbonyl carbon).

[0162] The carboxylic acid halide represented by formula (II) is preferably R.1 For secondary or tertiary C that may have substituents 5-40 Alkyl groups, C groups that may have substituents 5-10 Bicycloalkyl, C-type compounds that may have substituents 5-15 Tricyclic alkyl groups, secondary or tertiary C groups that may have substituents. 5-40 alkenyl groups, or primary C with substituents 4-40 Alkyl or primary C with substituents 4-40 Alkenyl (of which, primary C) 4-40 Alkyl or primary C 4-40 A carboxylic acid halide (where the alkenyl substituent is located on a carbon atom bonded to the carbonyl carbon), more preferably R 1 For secondary or tertiary C that may have substituents 5-20 Alkyl groups, C groups that may have substituents 7-10 Bicycloalkyl, C that may have substituents 7-15 Tricyclic alkyl groups, secondary or tertiary C groups that may have substituents. 5-20 alkenyl groups, or primary C with substituents 4-20 Alkyl or primary C with substituents 4-20 Alkenyl (of which, primary C) 4-20 Alkyl or primary C 4-20 Carboxylic acid halides in which the alkenyl substituent is located on a carbon atom bonded to the carbonyl carbon are preferred, R. 1 For secondary or tertiary C that may have substituents 5-20 Alkyl groups, C groups that may have substituents 7-10 Bicycloalkyl, C-type compounds that may have substituents 7-15 Tricyclic alkyl groups, secondary or tertiary C groups that may have substituents. 5-20 Alkenyl or isobutyl acyl chlorides with substituents, further preferably selected from the group consisting of the following compounds.

[0163] [Chemical Formula 13]

[0164]

[0165] The compounds are particularly preferred from the group consisting of the following compounds.

[0166] [Chemical Formula 14]

[0167]

[0168] Alkyl haloformates are represented by the following formula (III).

[0169] [Chemical Formula 15]

[0170]

[0171] (In the formula, X represents a halogen atom, R...) 2This refers to a secondary aliphatic hydrocarbon group with 5 or more carbon atoms and which may have substituents.

[0172] The alkyl haloformate represented by formula (III) is preferably R. 2 For secondary C that can have substituents 5-40 Alkyl groups, C groups that may have substituents 5-8 cycloalkyl, secondary C that may have substituents 5-40 Alkenyl haloformate esters, more preferably R 2 For secondary C that can have substituents 5-20 Alkyl groups, C groups that may have substituents 5-6 cycloalkyl, secondary C that may have substituents 5-20 Alkenyl haloformate esters, more preferably R 2 For secondary C that can have substituents 5-20 Alkyl groups, C groups that may have substituents 5-6 cycloalkyl, secondary C that may have substituents 5-20 Alkyl chloroformate of alkenyl group, particularly preferably selected from the group of compounds listed below.

[0173] [Chemical Formula 16]

[0174]

[0175] R 1 or R 2 The number of carbon atoms in it is R 1 or R 2 The total number of carbon atoms in each, R 1 or R 2 In the case of substituents, the number of carbon atoms in the substituents is also included.

[0176] The mixture of carboxylic acid halides or alkyl halogenated carboxylic acid esters with N-terminal protected amino acids or N-terminal protected peptides (activation reaction) can be carried out in the presence of base and / or solvent as needed.

[0177] There are no particular limitations on the alkali used in this process. Examples include aliphatic amines (e.g., triethylamine, N,N-diisopropylethylamine, N-methylmorpholine), aromatic amines (e.g., pyridine, imidazole, N,N-dimethyl-4-aminopyridine), amidines (e.g., diazabicycloundecene), and alkali metal salts (e.g., sodium bicarbonate, potassium carbonate). Aliphatic amines are preferred, and N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine are more preferred.

[0178] The amount of carboxylic acid halide or alkyl haloformate used in this process is preferably 0.2 to 50 equivalents relative to the N-terminal protected amino acid or N-terminal protected peptide, more preferably 0.5 to 20 equivalents, and even more preferably 0.8 to 5 equivalents.

[0179] The amount of alkali used in this process is preferably 0.2 to 50 equivalents relative to the carboxylic acid halide or alkyl haloformate, more preferably 0.5 to 20 equivalents, and even more preferably 0.8 to 5 equivalents.

[0180] The solvent used in this process is not particularly limited as long as it does not hinder the activation reaction. Examples include halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl tert-butyl ether), amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide), nitrile solvents (e.g., acetonitrile), ketone solvents (e.g., acetone, methyl ethyl ketone), aliphatic hydrocarbon solvents (e.g., hexane, heptane, cyclohexane), and ester solvents (e.g., ethyl acetate). Nitrile solvents, amide solvents, or ether solvents are preferred, and acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide are more preferred.

[0181] The amount of solvent used in this process is preferably less than 100 times the mass of the carboxylic acid halide or alkyl haloformate, more preferably 1 to 50 times the mass, and even more preferably 3 to 20 times the mass.

[0182] Thus, as needed, the N-terminal protected amino acid or N-terminal protected peptide is mixed with a carboxylic acid halide or a haloformate alkyl ester in the presence of a solvent and / or a base. For the resulting mixture, the temperature is controlled using an oil bath or a cooling bath as needed. The temperature of the mixture is not particularly limited, but is preferably from -40°C to the reflux temperature of the mixture, more preferably from -20°C to 50°C, and even more preferably from -10°C to 30°C.

[0183] This process forms C-terminal activated N-terminal protected amino acids or N-terminal protected peptides. Therefore, the product obtained from this process refers to C-terminal activated N-terminal protected amino acids or N-terminal protected peptides, or mixtures containing either of them. C-terminal activated N-terminal protected amino acids or N-terminal protected peptides obtained in this manner can be used in process (3) in the form of a reaction solution or as a (crude) purified product without undergoing a purification process.

[0184] Process (2)

[0185] This step involves mixing an N-alkyl amino acid or a peptide having an N-terminal N-alkyl amino acid with a silylating agent. It should be noted that in this step, "N-alkyl amino acid" refers to an N-alkyl amino acid whose N-terminal amino group and C-terminal carboxyl group are not protected, and "peptide having an N-terminal N-alkyl amino acid" refers to a peptide having an N-alkyl amino acid at the N-terminus and whose N-terminal amino group and C-terminal carboxyl group are not protected. This step involves reacting an N-alkyl amino acid or a peptide having an N-terminal N-alkyl amino acid with a silylating agent to obtain an N-alkyl amino acid or a peptide having an N-terminal N-alkyl amino acid (hereinafter also referred to as "trialkyl-silylated amino acid or peptide") with at least a portion of the functional groups such as the C-terminus, N-terminus, and / or (if present) hydroxyl groups of the amino acid or peptide being trialkyl-silylated. In one embodiment of the invention, the amino acid is reacted with a silylating agent to obtain an N-alkyl amino acid or peptide having an N-terminal N-alkyl amino acid (hereinafter also referred to as "trialkyl-silylated amino acid or peptide") having at least a portion of the functional groups such as the C-terminus, N-terminus, and / or (if present) hydroxyl groups of the amino acid or peptide being trialkyl-silylated. 2 -OH[where A] 2 Indicates originating from NC 1-6 alkyl amino acid groups (C 1-6 Alkyl groups may have substituents), or groups derived from 4-6 membered cyclic secondary amino acids (the 4-6 membered ring may be selected from C). 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 Cyclic compounds in the group consisting of cycloalkyl rings (fused together) or groups derived from peptides, wherein the N-terminal residue of the peptide is NC. 1-6 Alkyl amino acids (C 1-6 Alkyl groups (which may have substituents) or 4-6 membered cyclic secondary amino acids (the 4-6 membered ring can be selected from C... 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 The process of mixing an amino acid or a peptide, represented by the group consisting of cycloalkyl rings and fused cyclic compounds, with a silylating agent, where the N-terminal amino group and C-terminal carboxyl group are unprotected, is described in [IV'](represented by fusion of cyclic compounds). Alternatively, in another aspect of the invention, the process involves mixing an amino acid with an N-terminal amino group and a C-terminal carboxyl group unprotected, represented by formula (IV')HA. 2’ -OH[where A] 2’ Indicates a group derived from N-methyl amino acids, or from NC 1-6 The group of alkylglycine (C 1-6 The process of mixing an amino acid with an unprotected N-terminal amino group and a C-terminal carboxyl group, represented by an alkyl group (which may have substituents) or a group derived from a 4-6 member cyclic secondary amino acid, with a silylating agent.

[0186] Furthermore, the N-alkyl amino acid in this process, or the N-terminal amino acid in a peptide having an N-alkyl amino acid at the N-terminus, is preferably NC. 1-6 Alkyl amino acids (C 1-6The alkyl group may be substituted with cyclohexyl or phenyl, more preferably N-methyl amino acid, N-ethyl amino acid, N-propyl amino acid, N-butyl amino acid, N-pentyl amino acid, N-cyclohexylmethyl amino acid or N-benzyl amino acid, further preferably N-methyl amino acid or N-ethyl amino acid, and particularly preferably N-methyl amino acid.

[0187] There are no particular limitations on the silylating agent used in this process. Examples include trimethylsilyl chloride, trimethylsilyl cyanide, 1,1,1,3,3,3-hexamethyldisilazane, N-trimethylsilylacetamide, N,N'-bis(trimethylsilyl)urea, N-methyl-N-trimethylsilyltrifluoroacetamide, N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Trimethylsilyl chloride, N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea, or N,O-bis(trimethylsilyl)trifluoroacetamide are preferred, and N,O-bis(trimethylsilyl)acetamide is more preferred.

[0188] The amount of silylating agent used is preferably 0.01 to 50 equivalents relative to the N-alkyl amino acid or the peptide having an N-terminus of an N-alkyl amino acid, more preferably 0.1 to 20 equivalents, and even more preferably 0.2 to 5 equivalents.

[0189] N-alkyl amino acids or peptides having an N-terminus of an N-alkyl amino acid, mixed with a silylating agent (silylation reaction), can be carried out in the presence of a base and / or solvent as needed.

[0190] There are no particular limitations on the base used; examples include aliphatic amines (e.g., dicyclohexylamine, piperidine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine), aromatic amines (e.g., pyridine, imidazole, N,N-dimethyl-4-aminopyridine), and alkali metal salts (e.g., sodium bicarbonate, potassium carbonate). Aliphatic amines are preferred, and triethylamine and N,N-diisopropylethylamine are more preferred.

[0191] The amount of base used is preferably 0.01 to 50 equivalents relative to the N-alkyl amino acid or the peptide having an N-alkyl amino acid at the N-terminus, more preferably 0.1 to 20 equivalents, and even more preferably 0.2 to 5 equivalents.

[0192] The solvent used in this process is not particularly limited as long as it does not hinder the silylation reaction. Examples include halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl tert-butyl ether), amide solvents (e.g., N,N-dimethylformamide), and nitrile solvents (e.g., acetonitrile). Nitrile solvents, amide solvents, or ether solvents are preferred, and acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide are more preferred.

[0193] The amount of solvent used in this process is preferably less than 100 times by mass relative to the N-alkyl amino acid or the peptide having an N-alkyl amino acid at the N-terminus, more preferably 1 to 50 times by mass, and even more preferably 3 to 20 times by mass.

[0194] Thus, as needed, N-alkyl amino acids or peptides having N-alkyl amino acids at the N-terminus are mixed with a silylating agent in the presence of a solvent and / or a base. The resulting mixture is then subjected to temperature control using an oil bath or a cooling bath, as needed. The temperature of the mixture is not particularly limited, but is preferably from 0°C to the reflux temperature of the mixture, more preferably from 10°C to 100°C, and even more preferably from 20°C to 80°C. Additionally, the mixture may be subjected to microwave irradiation.

[0195] This process forms a trialkylsilylated N-alkyl amino acid or a peptide with an N-terminus of an N-alkyl amino acid. Therefore, the product obtained from this process is a mixture containing a trialkylsilylated N-alkyl amino acid or a peptide with an N-terminus of an N-alkyl amino acid. The trialkylsilylated amino acid or peptide obtained in this manner can be used in process (3) without undergoing a purification process, either as a reaction solution or as a (crude) purified product. Furthermore, the trialkylsilylated N-alkyl amino acid or the peptide with an N-terminus of an N-alkyl amino acid can also be analyzed using analytical equipment such as NMR.

[0196] Process (3)

[0197] This step involves mixing the product obtained in step (1) with the product obtained in step (2). This step is a peptide extension step in which the C-terminally activated N-terminal protected amino acid or N-terminal protected peptide obtained in step (1) reacts with the trialkylsilylated amino acid or peptide obtained in step (2). Preferably, this is carried out by mixing and stirring the reaction solution obtained in step (1) and the reaction solution obtained in step (2). In one embodiment of the invention, the peptide extension step involves reacting the C-terminally activated N-terminal protected amino acid obtained in step (1) with the trialkylsilylated amino acid or peptide obtained in step (2). In another embodiment of the invention, the peptide extension step involves reacting the C-terminally activated N-terminal protected peptide obtained in step (1) with the trialkylsilylated amino acid obtained in step (2).

[0198] Regarding the resulting mixture, the temperature may be controlled using an oil bath or a cooling bath as needed. The temperature of the mixture is not particularly limited, but is preferably from -40°C to the reflux temperature of the reaction mixture, more preferably from -20°C to 50°C, and even more preferably from -10°C to 30°C.

[0199] Furthermore, in the peptide manufacturing method of the present invention, the peptide chain can be further extended by repeating the peptide obtained in step (3) to step (4) to (5) as desired.

[0200] Step (4) removes the protecting group at the N-terminus of the peptide obtained in step (3) or (5).

[0201] Step (5) involves reacting the N-terminal protected amino acid or N-terminal protected peptide with the N-terminus of the peptide obtained in step (4).

[0202] Step (5) can be carried out by the same operation as steps (1), (2) and (3) above, or by the usual peptide synthesis reaction.

[0203] In the peptide manufacturing method of the present invention, the purification steps of steps (1) to (5) may be appropriately omitted within the range that does not affect the reaction of subsequent steps.

[0204] Process (4): Deprotection process of N-terminus

[0205] This process involves removing the N-terminal protecting group from the peptide obtained in process (3) or (5) above to obtain an unprotected peptide with both N-terminus and C-terminus.

[0206] The deprotecting agent used in this process is selected appropriately based on the protecting group used. Examples include acids (e.g., trifluoroacetic acid, hydrochloric acid, Lewis acids), secondary or tertiary amines (e.g., pyrrolidine, piperidine, morpholine, triethylamine), and hydrogenolysis (e.g., palladium catalyst / hydrogenation).

[0207] The deprotection conditions used in this process are appropriately selected according to the type of N-terminal protecting group. For example, in the case of 9-fluorenylmethoxycarbonyl, it is carried out by treatment with a base; in the case of tert-butoxycarbonyl, it is carried out by treatment with an acid; and in the case of benzyloxycarbonyl or allyloxycarbonyl, it is carried out by hydrogenation in a neutral environment, in the presence of, for example, a metal catalyst.

[0208] In each reaction, if the reaction substrate has hydroxyl, thiol, amino, carboxyl, or carbonyl groups (especially if the side chain of an amino acid or peptide has a functional group), a protecting group commonly used in peptide chemistry can be introduced into these groups, and the protecting group can be removed as needed after the reaction to obtain the target compound.

[0209] Protection and deprotection can be carried out by using commonly known protecting groups and performing protection-deprotection reactions (e.g., Protective Group in Organic Synthesis, Fourth edition, T.W. Greene, John Wiley & Sons Inc. (2006)).

[0210] There are no particular limitations on the combination of the N-terminal protected amino acid or the N-terminal protected peptide used in step (1) (hereinafter referred to as amino acid A) and the N-terminal amino acid or peptide used in step (2) (hereinafter referred to as amino acid B). It is preferred that amino acid A and B are each α-, β- or γ-amino acids. More preferably, either amino acid A or amino acid B is an α-amino acid. It is even more preferred that amino acid A is an α-amino acid and amino acid B is an α-amino acid, β-amino acid or γ-amino acid, or amino acid A is an α-amino acid, β-amino acid or γ-amino acid and amino acid B is an α-amino acid. It is particularly preferred that amino acid A is an α-amino acid and amino acid B is an α-amino acid, β-amino acid or γ-amino acid.

[0211] In addition, the N-terminal amino acid in the peptide used in step (2) is preferably an α-amino acid.

[0212] Example

[0213] The present invention will be described in more detail below as examples of synthesis, comparative examples and embodiments, but the present invention is not limited to these embodiments.

[0214] In this specification, when abbreviations are used to refer to amino acids, etc., each expression is based on the abbreviation of IUPAC-IUB Commission on Biochemical Nomenclature or based on commonly used abbreviations in the field.

[0215] It should be noted that in the synthesis example, "(v / v)" refers to (volume / volume), and "M" refers to mol / L.

[0216] In the following synthesis examples, the microwave reaction apparatus used is an initiator (Biotage).

[0217] Unless otherwise specified, the proton nuclear magnetic resonance of the synthetic example ( 1 H-NMR) uses JNM-ECP300 or JNM-ECX300 manufactured by JEOL Corporation, or Ascend manufactured by Bruker Corporation. TM 500, determined in deuterated chloroform or deuterated dimethyl sulfoxide solvent, the chemical shift is expressed as δ value (ppm) with tetramethylsilane as internal standard (0.0 ppm).

[0218] In NMR spectroscopy, "s" indicates a singlet, "d" indicates a doublet, "t" indicates a triplet, "q" indicates a quartet, "dd" indicates a double doublet, "dt" indicates a double triplet, "sept" indicates a septet, "m" indicates a multiplet, "br" indicates a broad peak, "J" indicates the coupling constant, "Hz" indicates Hertz, "CDCl3" indicates deuterated chloroform, and "DMSO-d6" indicates deuterated dimethyl sulfoxide.

[0219] For high performance liquid chromatography / mass spectrometry, unless otherwise specified, any of the following instruments shall be used for determination: Waters ACQUITY UPLC H-Class / QDa, Waters ACQUITY UPLC H-Class / SQD2, or Shimadzu LC-20AD / Triple Tof 5600.

[0220] In high performance liquid chromatography / mass spectrometry (HPLC / MS) records, ESI+ refers to the positive ion mode of electrospray ionization, M+H refers to the proton adduct, and M+Na refers to the sodium adduct.

[0221] In high performance liquid chromatography / mass spectrometry (HPLC / MS) records, ESI- refers to the negative ion mode of electrospray ionization, and MH refers to deprotonated ions.

[0222] In terms of the ratio of raw materials to products, in synthesis examples 8 to 50, the ratio was calculated by using high performance liquid chromatography analysis under any of the following conditions: <Analytical Condition 1> to <Analytical Condition 3>.

[0223] <Analysis Condition 1>

[0224] High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2

[0225] Column: Phenomenex Kinetex EVO C18 (1.7 μm, 2.1 × 50 mm)

[0226] Column oven temperature: 60℃

[0227] Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution

[0228] 5:95 (0-2.1 minutes), 95:5 (2.1-2.84 minutes) (v / v)

[0229] Elution rate: 0.6 mL / min

[0230] Detection wavelength: 220nm

[0231] <Analysis Condition 2>

[0232] High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2

[0233] Chromatographic column: Waters ACQUITY BEH C18 (1.7μm, 2.1×100mm)

[0234] Column oven temperature: 60℃

[0235] Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution

[0236] 5:95 (0-3.7 minutes), 95:5 (3.7-4.81 minutes) (v / v)

[0237] Elution rate: 0.6 mL / min

[0238] Detection wavelength: 220nm

[0239] <Analysis Condition 3>

[0240] High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2

[0241] Chromatographic column: Waters ACQUITY BEH C18 (1.7μm, 2.1×100mm)

[0242] Column oven temperature: 40℃

[0243] Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution

[0244] 5:95 (0-5.56 minutes), 95:5 (5.56-7.22 minutes) (v / v)

[0245] Elution rate: 0.4 mL / min

[0246] Detection wavelength: 220nm

[0247] For purification using silica column chromatography, unless otherwise specified, any one of the following shall be used: Yamazen Hi-Flash column, Biotage SNAP Ultra Silica Cartridge, Merck silica gel 60, or Fuji SILYSIA Chemical PSQ60B.

[0248] Unless otherwise specified, N,O-bis(trimethylsilyl)acetamide is used in the following commercially available product with a purity of 98%.

[0249] It should be noted that in the following embodiments, the yield or quantitative yield sometimes exceeds 100%. These are all due to measurement errors, the influence of the purity of raw materials or products, or factors based on other common technical knowledge. Although the reasons for the yield exceeding 100% are not mentioned separately in the following embodiments, those skilled in the art will fully understand the scientific rationale of these embodiments.

[0250] Synthetic Example 1: Synthesis of 4-ethyl-2,2-dimethylhexane-3-ol

[0251] [Chemical Formula 17]

[0252]

[0253] 2-Ethylbutyraldehyde (3.69 g, 36.8 mmol) was mixed with cyclopentyl methyl ether (40 mL), and tert-butyllithium pentane solution (1.53 M, 26.5 mL, 40.5 mmol) was added at -78 °C, followed by stirring for 5 minutes. The resulting reaction solution was heated to 25 °C, and ethanol (1.0 mL) was added. The solution was then washed successively with 20% ammonium chloride aqueous solution (25 mL) and saturated sodium chloride aqueous solution (25 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain 4-ethyl-2,2-dimethylhexane-3-ol (5.22 g, 90% yield) as a colorless, transparent liquid.

[0254] 1 H-NMR (CDCl3)

[0255] δppm: 0.88-0.94(9H+4H,m), 1.06-1.16(1H,m), 1.27(1H, d, J=6.0Hz), 1.29 -1.38(2H,m), 1.40-1.48(1H,m), 1.52-1.58(1H,m), 3.24(1H,d,J=6.0Hz).

[0256] Synthetic Example 2: Synthesis of 4-ethyl-2,2-dimethylhexane-3-yl chloroformate

[0257] [Chemical Formula 18]

[0258]

[0259] 4-Ethyl-2,2-dimethylhexane-3-ol (5.21 g, 32.9 mmol), pyridine (2.99 g, 37.9 mmol), and carbon tetrachloride (50 mL) were mixed and cooled to 0 °C. A solution containing triphosgene (4.00 g, 13.5 mmol) and carbon tetrachloride (10 mL) was added to this solution, and the mixture was heated to 60 °C and stirred for 8 hours. The resulting reaction solution was washed twice with water (50 mL) and then with a saturated aqueous sodium chloride solution (50 mL). The resulting organic layer was concentrated to give 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (6.56 g, 90% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0260] 1 H-NMR (CDCl3)

[0261] δppm: 0.90-0.94(6H,m), 0.97(9H,s), 1.10-1.20(1H,m), 1.25-1.35(1H,m), 1.45-1.53(2H,m), 1.59-1.67(1H,m), 4.72(1H,d,J=2.0Hz).

[0262] Synthetic Example 3: Synthesis of 2,2,4-trimethylpentan-3-ol

[0263] [Chemical Formula 19]

[0264]

[0265] Isobutyraldehyde (3.61 g, 50.0 mmol) was mixed with cyclopentyl methyl ether (50 mL), and tert-butyllithium pentane solution (1.52 M, 36.2 mL, 55.0 mmol) was added at -78 °C, followed by stirring for 5 minutes. The resulting reaction solution was heated to 25 °C, and ethanol (1.0 mL) was added. The solution was then washed successively with 20% ammonium chloride aqueous solution (25 mL) and saturated sodium chloride aqueous solution (25 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain 2,2,4-trimethylpentan-3-ol (5.25 g, yield 81%) as a colorless, transparent liquid.

[0266] 1 H-NMR (CDCl3)

[0267] δppm: 0.91 (3H, d, J = 7.0Hz), 0.94 (9H, s), 1.01 (3H, d, J = 7.0Hz), 1.32 (1H, d, J = 6.5Hz), 1.91-1.99 (1H, m), 3.11 (1H, dd, J = 6.5Hz, 2.0Hz).

[0268] Synthetic Example 4: Synthesis of 2,2,4-trimethylpentane-3-yl chloroformate

[0269] [Chemical Formula 20]

[0270]

[0271] 2,2,4-Trimethylpentane-3-ol (5.25 g, 40.3 mmol), pyridine (3.67 g, 46.3 mmol), and carbon tetrachloride (40 mL) were mixed and cooled to 0 °C. A solution containing triphosgene (4.90 g, 16.5 mmol) and carbon tetrachloride (20 mL) was added to this solution, and the mixture was heated to 60 °C and stirred for 8 hours. The resulting reaction solution was washed twice with water (50 mL) and then with a saturated aqueous sodium chloride solution (50 mL). The resulting organic layer was concentrated to give 2,2,4-trimethylpentane-3-yl chloroformate (6.06 g, 78% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0272] 1 H-NMR (CDCl3)

[0273] δppm: 0.97 (3H, d, J = 7.0Hz), 0.99 (9H, s), 1.02 (3H, d, J = 7.0Hz), 2.04-2.13 (1H, m), 4.58 (1H, d, J = 3.0Hz).

[0274] Synthetic Example 5: Synthesis of 3,3-dimethylbutane-2-yl chloroformate

[0275] [Chemical Formula 21]

[0276]

[0277] 3,3-Dimethylbutane-2-ol (3.36 g, 32.9 mmol), pyridine (2.99 g, 37.8 mmol), and carbon tetrachloride (40 mL) were mixed and cooled to 0 °C. A solution containing triphosgene (4.00 g, 13.5 mmol) and carbon tetrachloride (15 mL) was added to this solution, and the mixture was heated to 60 °C and stirred for 8 hours. The resulting reaction solution was washed twice with water (50 mL) and then with a saturated sodium chloride aqueous solution (50 mL). The resulting organic layer was concentrated to give 3,3-dimethylbutane-2-yl chloroformate (4.50 g, 83% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0278] 1 H-NMR (CDCl3)

[0279] δppm: 0.96 (9H, s), 1.29 (3H, d, J=6.5Hz), 4.74 (1H, q, J=6.5Hz).

[0280] Synthetic Example 6: Synthesis of 2,4-dimethylpentane-3-yl chloroformic acid ester

[0281] [Chemical Formula 22]

[0282]

[0283] 2,4-Dimethylpentane-3-ol (3.49 g, 30.0 mmol), pyridine (2.73 g, 34.5 mmol), and carbon tetrachloride (40 mL) were mixed and cooled to 0 °C. A solution containing triphosgene (3.65 g, 12.3 mmol) and carbon tetrachloride (15 mL) was added to this solution, and the mixture was heated to 60 °C and stirred for 8 hours. The resulting reaction solution was washed twice with water (50 mL) and then with a saturated aqueous sodium chloride solution (50 mL). The resulting organic layer was concentrated to give 2,4-dimethylpentane-3-yl chloroformate (5.10 g, 95% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0284] 1 H-NMR (CDCl3)

[0285] δppm: 0.94 (6H, d, J = 2.5Hz), 0.95 (6H, d, J = 2.5Hz), 1.94-2.04 (2H, m) 4.60 (1H, t, J = 6.0Hz).

[0286] Synthetic Example 7: Synthesis of 3-methylbutane-2-yl chloroformate

[0287] [Chemical Formula 23]

[0288]

[0289] 3-Methylbutane-2-ol (3.97 g, 45.0 mmol), pyridine (4.09 g, 51.8 mmol), and carbon tetrachloride (40 mL) were mixed and cooled to 0 °C. A solution containing triphosgene (5.47 g, 18.5 mmol) and carbon tetrachloride (20 mL) was added to this solution, and the mixture was heated to 60 °C and stirred for 8 hours. The resulting reaction solution was washed twice with water (50 mL) and then with a saturated aqueous sodium chloride solution (50 mL). The resulting organic layer was concentrated to give 3-methylbutane-2-yl chloroformate (5.10 g, 95% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0290] 1 H-NMR (CDCl3)

[0291] δppm: 0.95 (3H, d, J = 4.0Hz), 0.97 (3H, d, J = 4.0Hz), 1.31 (3H, d, J = 6.5Hz), 1.87-1.94 (1H, m), 4.80 (1H, sept, J = 6.5Hz).

[0292] Synthetic Example 8: Synthesis of Boc-Phe-MePhe-OH

[0293] [Chemical Formula 24]

[0294]

[0295] Boc-Phe-OH (0.066 g, 0.25 mmol), triethylamine (0.033 g, 0.32 mmol), and tetrahydrofuran (5 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.040 g, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.067 g, 0.38 mmol), N,O-bis(trimethylsilyl)acetamide (0.161 g, 0.750 mmol), and acetonitrile (4 mL) and stirring at 75 °C for 3 minutes under microwave irradiation was added, and the mixture was stirred at 0 °C for 2 hours, and then further stirred at 25 °C for 16 hours (starting material: target substance = 1:70 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL). It was then washed twice with 10% citric acid aqueous solution (10 mL), followed by washing with 5% sodium chloride aqueous solution (10 mL) and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was concentrated to obtain Boc-Phe-MePhe-OH (0.125 g, yield 117%) as a white solid.

[0296] MASS(ESI+)m / z;(M+H)+427.3

[0297] Synthesis Example 9: Synthesis of Fmoc-Phe-MePhe-OH

[0298] [Chemical Formula 25]

[0299]

[0300] Fmoc-Phe-OH (0.097 g, 0.25 mmol), N-methylmorpholine (0.033 g, 0.33 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (0.066 g, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.058 g, 0.33 mmol), N,O-bis(trimethylsilyl)acetamide (0.141 g, 0.67 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 20 minutes was added, and the mixture was further stirred at 25 °C for 5 hours (starting material: target analyte = 1:13 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (20 mL) and washed successively with 10% citric acid aqueous solution (20 mL), 10% sodium chloride aqueous solution (20 mL), and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-Phe-MePhe-OH (0.137 g, 100% yield) as a white solid.

[0301] MASS(ESI+)m / z;(M+H)+549.3

[0302] Synthetic Example 10: Synthesis of Fmoc-Phe-MePhe-OH

[0303] [Chemical Formula 26]

[0304]

[0305] Fmoc-Phe-OH (0.097 g, 0.25 mmol), N-methylmorpholine (0.033 g, 0.33 mmol), and N,N-dimethylacetamide (5.0 mL) were mixed, and 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (0.066 g, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.058 g, 0.33 mmol), N,O-bis(trimethylsilyl)acetamide (0.141 g, 0.67 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 20 minutes was added, and the mixture was further stirred at 25 °C for 5 hours (starting material: target analyte = 1:31 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (30 mL) and washed successively with 10% citric acid aqueous solution (50 mL), 10% sodium chloride aqueous solution (20 mL), and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-Phe-MePhe-OH (0.137 g, 100% yield) as a white solid.

[0306] MASS(ESI+)m / z;(M+H)+549.3

[0307] Synthetic Example 11: Synthesis of Fmoc-Phe-MePhe-OH

[0308] [Chemical Formula 27]

[0309]

[0310] Fmoc-Phe-OH (0.194 g, 0.500 mmol), triethylamine (0.0607 g, 0.60 mmol), and tetrahydrofuran (10 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.074 g, 0.550 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.108 g, 0.600 mmol), N,N'-bis(trimethylsilyl)urea (0.250 g, 1.20 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 60 minutes under microwave irradiation was added, and the mixture was further stirred at 25 °C for 67 hours (starting material: target analyte = 1:20 (analytical condition 2)). The resulting reaction solution was concentrated and diluted with ethyl acetate (80 mL), and then washed successively with 10% citric acid aqueous solution (50 mL), 5% sodium chloride aqueous solution (50 mL), and saturated sodium chloride aqueous solution (50 mL). The resulting organic layer was concentrated to obtain Fmoc-Phe-MePhe-OH (0.322 g, yield 117%) as a white solid.

[0311] MASS(ESI+)m / z;(M+H)+549.4

[0312] Synthetic Example 12: Synthesis of Fmoc-Phe-MePhe-OH

[0313] [Chemical Formula 28]

[0314]

[0315] Fmoc-Phe-OH (0.097 g, 0.250 mmol), triethylamine (0.0304 g, 0.300 mmol), and tetrahydrofuran (5 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.0371 g, 0.275 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.0538 g, 0.300 mmol), N,O-bis(trimethylsilyl)trifluoroacetamide (0.155 g, 0.601 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 30 minutes was added, and the mixture was further stirred at 25 °C for 16 hours (starting material: target analyte = 4.4:95.6 (analytical condition 1; at the 4-hour stirring time)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), then washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was then washed successively with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated to obtain Fmoc-Phe-MePhe-OH (0.135 g, 98% yield) as a white solid.

[0316] MASS(ESI+)m / z;(M+H)+549.4

[0317] Synthetic Example 13: Synthesis of Boc-MePhe-MePhe-OH

[0318] [Chemical Formula 29]

[0319]

[0320] Boc-MePhe-OH (0.070 g, 0.250 mmol), triethylamine (0.033 g, 0.32 mmol), and tetrahydrofuran (5 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.040 g, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.067 g, 0.38 mmol), N,O-bis(trimethylsilyl)acetamide (0.161 g, 0.774 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 3 minutes was added, and the mixture was further stirred at 25 °C for 16 hours (reactant:target substance = 7:93 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), and washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was washed successively with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). After concentration, the organic layer was purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-OH (0.127 g, yield 116%) as a colorless oil.

[0321] MASS(ESI+)m / z;(M+H)+441.4

[0322] Synthetic Example 14: Synthesis of Boc-MePhe-Phe-OH

[0323] [Chemical Formula 30]

[0324]

[0325] Boc-MePhe-OH (1.40 g, 5.00 mmol), N-methylmorpholine (0.556 g, 5.50 mmol), and tetrahydrofuran (50 mL) were mixed, and isopropyl chloroformate (0.643 g, 5.25 mmol) was added at 0 °C, followed by stirring for 15 minutes. To this solution, a solution prepared by separately mixing H-Phe-OH (0.991 g, 6.00 mmol), N,O-bis(trimethylsilyl)acetamide (2.57 g, 12.4 mmol), and acetonitrile (15 mL) and stirring at 75 °C for 60 minutes under microwave irradiation was added. The mixture was stirred at 0 °C for 30 minutes, and then further stirred at 25 °C for 1.5 hours (starting material: target substance = 0:100 (analytical condition 1)). The resulting reaction solution was diluted with ethyl acetate (200 mL) and washed successively with 10% citric acid aqueous solution (75 mL), 5% sodium chloride aqueous solution (75 mL), and saturated sodium chloride aqueous solution (75 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-Phe-OH (2.20 g, yield 103%) as a white solid.

[0326] MASS(ESI+)m / z;(M+H)+427.3

[0327] Synthetic Example 15: Synthesis of Fmoc-MePhe-Phe-OH

[0328] [Chemical Formula 31]

[0329]

[0330] Fmoc-MePhe-OH (2.00 g, 5.00 mmol), N-methylmorpholine (0.556 g, 5.50 mmol), and tetrahydrofuran (30 mL) were mixed, and isopropyl chloroformate (0.663 g, 5.25 mmol) was added at 0 °C, and the mixture was stirred for 30 minutes. To this solution, a solution prepared by separately mixing H-Phe-OH (0.991 g, 6.00 mmol), N,O-bis(trimethylsilyl)acetamide (2.68 g, 12.9 mmol), and acetonitrile (15 mL) and stirring at 75 °C for 30 minutes was added. The mixture was stirred at 0 °C for 50 minutes, and then further stirred at 25 °C for 1 hour (starting material: target analyte = 3:97 (analytical condition 1)). The resulting reaction solution was diluted with ethyl acetate (75 mL) and washed successively with 10% citric acid aqueous solution (50 mL), 5% sodium chloride aqueous solution (50 mL), and saturated sodium chloride aqueous solution (50 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-MePhe-Phe-OH (2.85 g, yield 98%) as a white solid.

[0331] MASS(ESI+)m / z;(M+H)+549.4

[0332] Synthesis Example 16: Synthesis of H-MePhe-Phe-OH

[0333] [Chemical Formula 32]

[0334]

[0335] Fmoc-MePhe-Phe-OH (0.137 g, 0.250 mmol), triethylamine (0.505 g, 4.99 mmol), and acetonitrile (4.4 mL) were mixed and stirred at 80 °C for 60 minutes. The resulting reaction solution was concentrated, and tetrahydrofuran (1.0 mL) and diisopropyl ether (3.0 mL) were added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with diisopropyl ether (5 mL) and dried to obtain H-MePhe-Phe-OH (0.071 g, 87% yield) as a white solid.

[0336] MASS(ESI+)m / z;(M+H)+327.3

[0337] Synthetic Example 17: Synthesis of H-MePhe-Phe-OH·HCl

[0338] [Chemical Formula 33]

[0339]

[0340] Boc-MePhe-Phe-OH (0.213 g, 0.500 mmol) was mixed with 4 M HCl / ethyl acetate (10 mL) and stirred at 25 °C for 1 hour. The resulting reaction solution was concentrated, and diisopropyl ether was added to suspend it. The solid was filtered through a Kiriyama funnel and dried to obtain H-MePhe-Phe-OH·HCl (0.164 g, 91% yield) as a white solid.

[0341] MASS(ESI+)m / z;(M+H)+327.3

[0342] Synthetic Example 18: Synthesis of Boc-MePhe-MePhe-Phe-OH

[0343] [Chemical Formula 34]

[0344]

[0345] Solution A

[0346] Boc-MePhe-OH (0.280 g, 1.00 mmol), N-methylmorpholine (0.112 g, 1.10 mmol), and tetrahydrofuran (10 mL) were mixed, and 2,2,4-trimethylpentane-3-yl chloroformate (0.203 g, 1.05 mmol) was added at 0 °C and stirred for 1 hour.

[0347] Solution B

[0348] Boc-MePhe-Phe-OH (0.449 g, 1.05 mmol) was mixed with 4 M HCl / cyclopentyl methyl ether (5 mL) and stirred at 25 °C for 30 min. The resulting reaction solution was concentrated, ethyl acetate (20 mL) was added and concentrated, N,N-diisopropylethylamine (1.30 g, 10.0 mmol) and acetonitrile (8 mL) were added and concentrated. Acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.644 g, 3.11 mmol) were mixed in the residue and stirred at 25 °C for 20 min to obtain a colorless and transparent solution.

[0349] Condensation process

[0350] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 18 hours (raw material: target analyte = 1:21 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain a white solid. Boc-MePhe-MePhe-Phe-OH (0.532g, 90% yield). MASS(ESI+)m / z; (M+H)+588.4

[0351] Synthetic Example 19: Synthesis of Boc-Phe-MePhe-MePhe-Phe-OH

[0352] [Chemical Formula 35]

[0353]

[0354] Solution A

[0355] Boc-Phe-OH (0.177 g, 0.669 mmol), N-methylmorpholine (0.744 g, 0.736 mmol), and tetrahydrofuran (10 mL) were mixed, and 2,2,4-trimethylpentane-3-yl chloroformate (0.135 g, 0.702 mmol) was added at 0 °C and stirred for 1 hour.

[0356] Solution B

[0357] Boc-MePhe-MePhe-Phe-OH (0.413 g, 0.702 mmol) was mixed with 4M HCl / cyclopentyl methyl ether (10 mL) and stirred at 25 °C for 1 hour. The resulting reaction solution was concentrated, ethyl acetate (20 mL) was added and concentrated, N,N-diisopropylethylamine (0.865 g, 6.69 mmol) and acetonitrile (8 mL) were added and concentrated. Acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.430 g, 2.07 mmol) were mixed in the residue and stirred at 25 °C for 20 minutes to obtain a colorless and transparent solution.

[0358] Condensation process

[0359] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 18 hours (raw material: target substance = 0:100 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (20 mL) and washed successively with 10% citric acid aqueous solution (20 mL), 10% sodium chloride aqueous solution (20 mL), and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Phe-MePhe-MePhe-Phe-OH (0.453 g, yield 92%) as a white solid.

[0360] MASS(ESI+)m / z;(M+H)+735.5

[0361] Synthetic Example 20: Synthesis of Boc-MePhe-MePhe-Phe-OH

[0362] [Chemical Formula 36]

[0363]

[0364] Solution A

[0365] Boc-MePhe-OH (0.324 g, 1.16 mmol), triethylamine (0.141 g, 1.39 mmol), and tetrahydrofuran (10 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.172 g, 1.28 mmol) was added at 0 °C. The mixture was stirred for 1 hour.

[0366] Solution B

[0367] Boc-MePhe-Phe-OH (0.544 g, 1.28 mmol) was mixed with 4 M HCl / cyclopentyl methyl ether (20 mL) and stirred at 25 °C for 1 hour. The resulting reaction solution was concentrated, cyclopentyl methyl ether (20 mL) was added and the mixture was concentrated, then N,N-diisopropylethylamine (1.50 g, 11.6 mmol) and acetonitrile (8 mL) were added and the mixture was concentrated. Acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.621 g, 2.99 mmol) were mixed in the residue and stirred at 25 °C for 20 minutes to obtain a colorless and transparent solution.

[0368] Condensation process

[0369] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 48 hours (raw material: target analyte = 1:37 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-Phe-OH (0.701 g, yield 103%) as a brown solid.

[0370] MASS(ESI+)m / z;(M+H)+588.4

[0371] Synthetic Example 21: Synthesis of Boc-MePhe-Pro-OH

[0372] [Chemical Formula 37]

[0373]

[0374] Boc-MePhe-OH (1.40 g, 5.00 mmol), N-methylmorpholine (0.556 g, 5.50 mmol), and tetrahydrofuran (30 mL) were mixed, and 3,3-dimethylbutane-2-yl chloroformate (0.864 g, 5.25 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-Pro-OH (0.691 g, 6.00 mmol), N,O-bis(trimethylsilyl)acetamide (3.85 g, 18.6 mmol), and acetonitrile (12 mL) and stirring at 70 °C for 10 minutes was added, and the mixture was further stirred at 0 °C for 15 minutes (starting material: target substance = 0:100 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to give Boc-MePhe-Pro-OH (1.94 g, yield 94%) as a white solid.

[0375] MASS(ESI+)m / z;(M+H)+377.3

[0376] Synthetic Example 22: Synthesis of Boc-MePhe-MePhe-Pro-OH

[0377] [Chemical Formula 38]

[0378]

[0379] Solution A

[0380] Mix Boc-MePhe-OH (0.838 g, 3.00 mmol), triethylamine (0.364 g, 3.60 mmol), and tetrahydrofuran (60 mL). Add 2,2-dimethylbutyryl chloride (0.444 g, 3.30 mmol) at 0 °C and stir for 30 minutes.

[0381] Solution B

[0382] Boc-MePhe-Pro-OH (1.24 g, 3.30 mmol) was mixed with 4M HCl / cyclopentyl methyl ether (20 mL) and stirred at 25 °C for 1 hour. The resulting reaction solution was concentrated, acetonitrile (20 mL) was added and concentrated, N,N-diisopropylethylamine (0.865 g, 6.69 mmol) and acetonitrile (20 mL) were added and concentrated. Acetonitrile (20 mL) and N,O-bis(trimethylsilyl)acetamide (2.31 g, 11.1 mmol) were mixed in the residue and stirred at 25 °C for 20 minutes to obtain a colorless and transparent solution.

[0383] Condensation process

[0384] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 48 hours (raw material: target analyte = 1:24 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-Pro-OH (1.71 g, yield 90%) as a white solid.

[0385] MASS(ESI+)m / z;(M+H)+538.5

[0386] Synthetic Example 23: Synthesis of H-MePhe-MePhe-Pro-OH·HCl

[0387] [Chemical Formula 39]

[0388]

[0389] Boc-MePhe-MePhe-Pro-OH (1.61 g, 3.00 mmol) was mixed with 4 M HCl / ethyl acetate (15 mL) and stirred at 25 °C for 90 min. The resulting reaction solution was concentrated, and ethyl acetate (5 mL) and diisopropyl ether (20 mL) were added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with diisopropyl ether (10 mL) and dried to obtain H-MePhe-MePhe-Pro-OH·HCl (1.30 g, 91% yield) as a white solid. The obtained solid was used in the next step.

[0390] Synthetic Example 24: Synthesis of Boc-Tyr-MePhe-MePhe-Pro-OH

[0391] [Chemical Formula 40]

[0392]

[0393] Solution A

[0394] Mix Boc-Tyr-OH (0.282 g, 1.00 mmol), triethylamine (0.122 g, 1.20 mmol), and tetrahydrofuran (10 mL). Add 2,2-dimethylbutyryl chloride (0.148 g, 1.10 mmol) at 0 °C and stir for 1 hour.

[0395] Solution B

[0396] H-MePhe-MePhe-Pro-OH·HCl (0.482 g, 1.02 mmol) was mixed with N,N-diisopropylethylamine (2 mL) and stirred at 25 °C for 2 minutes. The resulting reaction solution was concentrated. Acetonitrile (6 mL) and N,O-bis(trimethylsilyl)acetamide (0.773 g, 3.74 mmol) were mixed into the residue and stirred at 25 °C for 20 minutes to obtain a colorless and transparent solution.

[0397] Condensation process

[0398] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 1 hour. The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Tyr-MePhe-MePhe-Pro-OH (0.698 g, 99% yield) as a white solid.

[0399] MASS(ESI+)m / z;(M+H)+701.5

[0400] Synthetic Example 25: Synthesis of Boc-MePhe-MeAla-Tyr-OH

[0401] [Chemical Formula 41]

[0402]

[0403] Solution A

[0404] Mix Boc-MePhe-OH (0.894 g, 3.20 mmol), triethylamine (0.389 g, 3.84 mmol), and tetrahydrofuran (25 mL). Add 2,2-dimethylbutyryl chloride (0.474 g, 3.52 mmol) at 0 °C and stir for 45 minutes.

[0405] Solution B

[0406] Acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (2.28 g, 10.9 mmol) were mixed in H-MeAla-Tyr-OH (0.937 g, 3.52 mmol) and stirred at 25 °C for 15 minutes to obtain a colorless and transparent solution.

[0407] Condensation process

[0408] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 2 hours (raw material: target substance = 0:100 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (30 mL), and washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was washed successively with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-MeAla-Tyr-OH (1.67 g, yield 94%) as a white solid.

[0409] MASS(ESI+)m / z;(M+H)+528.3

[0410] Synthetic Example 26: Synthesis of H-MePhe-MeAla-Tyr-OH

[0411] [Chemical Formula 42]

[0412]

[0413] Boc-MePhe-MeAla-Tyr-OH (0.474 g, 0.898 mmol) was mixed with 4 M HCl / ethyl acetate (5 mL) and stirred at 25 °C for 60 min. The resulting reaction solution was concentrated, and acetonitrile (5 mL) and N,N-diisopropylethylamine (1.74 g, 13.5 mmol) were added to suspend it. The resulting solid was collected by filtration using a Kiriyama funnel. The solution was washed with a solution of N,N-diisopropylethylamine (1.74 g, 13.5 mmol) and diisopropyl ether (5 mL) and dried to obtain H-MePhe-MeAla-Tyr-OH (0.370 g, 96% yield) as a white solid.

[0414] MASS(ESI+)m / z;(M+H)+428.8

[0415] Synthetic Example 27: Synthesis of Boc-Phe-MePhe-MeAla-Tyr-OH

[0416] [Chemical Formula 43]

[0417]

[0418] Boc-Phe-OH (0.125 g, 0.470 mmol), triethylamine (0.057 g, 0.564 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.070 g, 0.52 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-MePhe-MeAla-Tyr-OH (0.221 g, 0.517 mmol), N,O-bis(trimethylsilyl)acetamide (0.332 g, 1.60 mmol), and acetonitrile (5.0 mL) and stirring at 25 °C for 15 minutes was added, followed by stirring at 25 °C for 2 hours (reactant:target substance = 1:80 (analytical condition 2)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), followed by washing with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Phe-MePhe-MeAla-Tyr-OH (0.295 g, yield 93%) as a white solid.

[0419] MASS(ESI+)m / z;(M+H)+675.5

[0420] Synthesis Example 28: Synthesis of H-Phe-MePhe-MeAla-Tyr-OH

[0421] [Chemical Formula 44]

[0422]

[0423] Boc-Phe-MePhe-MeAla-Tyr-OH (0.317 g, 0.470 mmol) was mixed with trifluoroacetic acid (1.45 mL) and stirred at 25 °C for 15 minutes. The resulting reaction solution was concentrated, and acetonitrile (7 mL) and triethylamine (0.476 g, 4.70 mmol) were added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with ethyl acetate (2 mL) and diisopropyl ether (8 mL) and dried to obtain H-Phe-MePhe-MeAla-Tyr-OH (0.268 g, 99% yield) as a white solid.

[0424] MASS(ESI+)m / z;(M+H)+575.4

[0425] Synthetic Example 29: Synthesis of Boc-MePhe-Phe-MePhe-MeAla-Tyr-OH

[0426] [Chemical Formula 45]

[0427]

[0428] Boc-MePhe-OH (0.092 g, 0.33 mmol), triethylamine (0.040 g, 0.396 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.0489 g, 0.363 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-Phe-MePhe-MeAla-Tyr-OH (0.209 g, 0.363 mmol), N,O-bis(trimethylsilyl)acetamide (0.233 g, 1.12 mmol), and acetonitrile (5.0 mL) and stirring at 25 °C for 15 minutes was added, followed by stirring at 25 °C for 1 hour (raw material: target substance = 0:100 (analytical condition 2)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), followed by washing with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-Phe-MePhe-MeAla-Tyr-OH (0.282 g, yield 87%) as a white solid.

[0429] MASS(ESI+)m / z;(M+H)+836.5

[0430] Synthetic Example 30: Synthesis of Cbz-MeAla-Phe-OH

[0431] [Chemical Formula 46]

[0432]

[0433] Cbz-MeAla-OH (1.19 g, 5.00 mmol), triethylamine (0.607 g, 6.00 mmol), and tetrahydrofuran (50 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.740 g, 5.50 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-Phe-OH (0.991 g, 6.00 mmol), N,O-bis(trimethylsilyl)acetamide (2.57 g, 12.4 mmol), and acetonitrile (17 mL) and stirring at 75 °C for 60 minutes under microwave irradiation was added, followed by stirring at 0 °C for 1 hour (reactant:target substance = 1:62 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), followed by washing with saturated sodium bicarbonate aqueous solution (60 mL), water (60 mL), and saturated sodium chloride aqueous solution (30 mL). The obtained organic layer was washed sequentially with 10% citric acid aqueous solution (30 mL) and saturated sodium chloride aqueous solution (30 mL). The obtained organic layer was concentrated to obtain Cbz-MeAla-Phe-OH (1.76 g, yield 91%) as a colorless and transparent slurry.

[0434] MASS(ESI+)m / z;(M+H)+385.3

[0435] Synthetic Example 31: Synthesis of H-MeAla-Phe-OH

[0436] [Chemical Formula 47]

[0437]

[0438] Cbz-MeAla-Phe-OH (1.76 g, 4.57 mmol), triethylamine (0.023 g, 0.229 mmol), ammonium formate (1.44 g, 22.9 mmol), and 10% palladium-carbon (0.486 g by mass) were mixed with methanol (50 mL) and stirred at 60 °C for 1 hour. The reaction solution was filtered through diatomaceous earth and washed three times with methanol (20 mL). The filtrate was concentrated to give H-MeAla-Phe-OH (1.03 g, 90%) as a white solid.

[0439] MASS(ESI+)m / z;(M+H)+251.2

[0440] Synthetic Example 32: Synthesis of Boc-MePhe-MeAla-Phe-OH

[0441] [Chemical Formula 48]

[0442]

[0443] Boc-MePhe-OH (0.559 g, 2.00 mmol), triethylamine (0.243 g, 2.40 mmol), and tetrahydrofuran (30 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.296 g, 2.20 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-MeAla-Phe-OH (0.601 g, 2.40 mmol), N,O-bis(trimethylsilyl)acetamide (1.03 g, 4.95 mmol), and acetonitrile (20 mL) and stirring at 25 °C for 40 minutes was added, followed by stirring at 0 °C for 2 hours (reactant:target substance = 1:55 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), and washed twice each with saturated sodium bicarbonate aqueous solution (30 mL), water (30 mL), and saturated sodium chloride aqueous solution (15 mL). The obtained organic layer was washed successively with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). The obtained organic layer was concentrated to obtain Boc-MePhe-MeAla-Phe-OH (1.08 g, yield 105%) as a white solid.

[0444] MASS(ESI+)m / z;(M+H)+512.3

[0445] Synthesis Example 33: Synthesis of H-MePhe-MeAla-Phe-OH·HCl

[0446] [Chemical Formula 49]

[0447]

[0448] Boc-MePhe-MeAla-Phe-OH (1.06 g, 2.08 mmol) was mixed with 4 M HCl / ethyl acetate (20 mL) and stirred at 25 °C for 60 min. The resulting reaction solution was concentrated, and diisopropyl ether (20 mL) was added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solution was washed with diisopropyl ether (10 mL) and dried to obtain H-MePhe-MeAla-Phe-OH·HCl (0.886 g, 95% yield) as a white solid.

[0449] MASS(ESI+)m / z;(M+H)+411.5

[0450] Synthetic Example 34: Synthesis of Fmoc-Trp(Boc)-MePhe-MeAla-Phe-OH

[0451] [Chemical Formula 50]

[0452]

[0453] Fmoc-Trp(Boc)-OH (0.439 g, 0.833 mmol), triethylamine (0.101 g, 1.00 mmol), and tetrahydrofuran (30 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.123 g, 0.917 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-MePhe-MeAla-Phe-OH·HCl (0.448 g, 1.00 mmol), N,O-bis(trimethylsilyl)acetamide (0.428 g, 2.06 mmol), N,N-diisopropylethylamine (1.08 g, 8.33 mmol), and acetonitrile (20 mL) and stirring at 25 °C for 40 minutes was added, and then stirred at 25 °C for 3 hours (raw material: target substance = 1:12 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), then washed twice each with saturated sodium bicarbonate aqueous solution (30 mL), water (30 mL), and saturated sodium chloride aqueous solution (12 mL). The resulting organic layer was washed sequentially with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated, and the residue was dissolved in ethyl acetate (5.0 mL) and injected into hexane (95 mL). The precipitated solid was filtered off, yielding Fmoc-Trp(Boc)-MePhe-MeAla-Phe-OH (0.657 g, yield 86%) as a white solid.

[0454] MASS(ESI+)m / z;(M+H)+920.5

[0455] Synthetic Example 35: Synthesis of Fmoc-Gln(Trt)-MePhe-MeAla-Tyr-OH

[0456] [Chemical Formula 51]

[0457]

[0458] Solution A

[0459] Mix Fmoc-Gln(Trt)-OH (0.204 g, 0.333 mmol), triethylamine (0.040 g, 0.40 mmol), and tetrahydrofuran (20 mL). Add 2,2-dimethylbutyryl chloride (0.049 g, 0.37 mmol) at 0 °C and stir for 45 minutes.

[0460] Solution B

[0461] H-MePhe-MeAla-Tyr-OH (0.171 g, 0.400 mmol), N,N-diisopropylethylamine (0.431 g, 3.33 mmol), and acetonitrile (20 mL) were mixed, and the solution was concentrated to remove the solvent. Acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.343 g, 1.65 mmol) were mixed into the residue, and the mixture was stirred at 25 °C for 40 minutes to obtain a colorless and transparent solution.

[0462] Condensation process

[0463] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 23 hours (raw material: target substance = 1:5 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), and washed twice each with saturated sodium bicarbonate aqueous solution (15 mL), water (15 mL), and saturated sodium chloride aqueous solution (7.5 mL). The resulting organic layer was washed sequentially with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). The resulting organic layer was concentrated to obtain Fmoc-Gln(Trt)-MePhe-MeAla-Tyr-OH (0.209 g, yield 62%) as a white solid.

[0464] MASS(ESI+)m / z;(M+H)+1020.6

[0465] Synthetic Example 36: Synthesis of Fmoc-BnGly-Phe-OH

[0466] [Chemical Formula 52]

[0467]

[0468] Fmoc-BnGly-OH (0.387 g, 1.00 mmol), N-methylmorpholine (0.111 g, 1.10 mmol), and tetrahydrofuran (20 mL) were mixed, and isopropyl chloroformate (0.129 g, 1.05 mmol) was added at 0 °C, followed by stirring for 5 minutes. To this solution, a solution prepared by separately mixing H-Phe-OH (0.198 g, 1.20 mmol), N,O-bis(trimethylsilyl)acetamide (0.519 g, 2.48 mmol), and acetonitrile (5 mL) and stirring at 75 °C for 60 minutes under microwave irradiation was added. The mixture was then stirred at 0 °C for 15 minutes, followed by stirring at 25 °C for 20 hours (raw material: target analyte = 1:55 (analytical condition 1)). The obtained reaction solution was concentrated and diluted with ethyl acetate (80 mL), and then washed successively with 10% citric acid aqueous solution (50 mL), 10% sodium chloride aqueous solution (50 mL), and saturated sodium chloride aqueous solution (50 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-BnGly-Phe-OH (0.570 g, yield 107%) as a white solid.

[0469] MASS(ESI+)m / z;(M+H)+535.3

[0470] Synthesis Example 37: Synthesis of H-BnGly-Phe-OH

[0471] [Chemical Formula 53]

[0472]

[0473] Fmoc-BnGly-Phe-OH (0.535 g, 1.00 mmol), triethylamine (2.02 g, 20.0 mmol), and acetonitrile (20 mL) were mixed and stirred at 60 °C for 60 minutes. The resulting reaction solution was concentrated, and diisopropyl ether (50 mL) was added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with diisopropyl ether and dried to obtain H-BnGly-Phe-OH (0.293 g, 94% yield) as a white solid.

[0474] MASS(ESI+)m / z;(M+H)+313.2

[0475] Synthetic Example 38: Synthesis of Boc-Phe-BnGly-Phe-OH

[0476] [Chemical Formula 54]

[0477]

[0478] Boc-Phe-OH (0.066 g, 0.25 mmol), triethylamine (0.030 g, 0.30 mmol), and tetrahydrofuran (5 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.037 g, 0.28 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-BnGly-Phe-OH (0.093 g, 0.30 mmol), N,O-bis(trimethylsilyl)acetamide (0.129 g, 0.616 mmol), and acetonitrile (4 mL) and stirring at 25 °C for 20 minutes was added, followed by stirring at 25 °C for 14 hours (raw material: target substance = 1:93 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), followed by washing with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was washed successively with 10% citric acid aqueous solution and 20 mL of saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Phe-BnGly-Phe-OH (0.139 g, 100% yield) as a white solid.

[0479] MASS(ESI+)m / z;(M+H)+560.4

[0480] Synthetic Example 39: Synthesis of Fmoc-n-PrGly-Phe-OH

[0481] [Chemical Formula 55]

[0482]

[0483] Fmoc-n-PrGly-OH (0.339 g, 1.00 mmol), triethylamine (0.121 g, 1.20 mmol), and tetrahydrofuran (15 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.148 g, 1.10 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing H-Phe-OH (0.198 g, 1.20 mmol), N,O-bis(trimethylsilyl)acetamide (0.519 g, 2.48 mmol), and acetonitrile (4.5 mL) and stirring at 75 °C for 60 minutes under microwave irradiation was added, followed by stirring at 0 °C for 1 hour (raw material: target substance = 1:32 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (20 mL), followed by washing with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was washed twice each with 10% citric acid aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL). The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-n-PrGly-Phe-OH (0.538 g, yield 111%) as a white solid.

[0484] MASS(ESI+)m / z;(M+H)+487.3

[0485] Synthetic Example 40: Synthesis of Hn-PrGly-Phe-OH

[0486] [Chemical Formula 56]

[0487]

[0488] Fmoc-n-PrGly-Phe-OH (0.487 g, 1.00 mmol), triethylamine (2.02 g, 20.0 mmol), and acetonitrile (20 mL) were mixed and stirred at 60 °C for 60 minutes. The resulting reaction solution was concentrated, and diisopropyl ether (50 mL) was added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with diisopropyl ether and dried to obtain Hn-PrGly-Phe-OH (0.245 g, 93% yield) as a white solid.

[0489] MASS(ESI+)m / z;(M+H)+265.2

[0490] Synthetic Example 41: Synthesis of Boc-Phe-n-PrGly-Phe-OH

[0491] [Chemical Formula 57]

[0492]

[0493] Boc-Phe-OH (0.066 g, 0.25 mmol), triethylamine (0.030 g, 0.300 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.037 g, 0.27 mmol) was added at 0 °C, followed by stirring for 45 minutes. To this solution, a solution prepared by separately mixing Hn-PrGly-Phe-OH (0.066 g, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (0.118 g, 0.564 mmol), and acetonitrile (4.0 mL) and stirring at 25 °C for 30 minutes was added, and then stirred at 25 °C for at least 1 hour (raw material: target substance = 1:85 (analytical condition 3)). The obtained reaction solution was concentrated and diluted with ethyl acetate (40 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Phe-n-PrGly-Phe-OH (0.125 g, yield 98%) as a white solid.

[0494] MASS(ESI+)m / z;(M+H)+512.4

[0495] Synthetic Example 42: Synthesis of Fmoc-MePhe-n-PrGly-Phe-OH

[0496] [Chemical Formula 58]

[0497]

[0498] Fmoc-MePhe-OH (0.080 g, 0.20 mmol), triethylamine (0.024 g, 0.24 mmol), and tetrahydrofuran (5 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.030 g, 0.22 mmol) was added at 0 °C, and the mixture was stirred for 45 minutes. To this solution, a solution prepared by separately mixing Hn-PrGly-Phe-OH (0.063 g, 0.24 mmol), N,O-bis(trimethylsilyl)acetamide (0.104 g, 0.50 mmol), and acetonitrile (2 mL) and stirring at 0 °C for 30 minutes was added, and then the mixture was stirred at 25 °C for 2 hours (raw material: target substance = 1:18 (analytical condition 1)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), and then washed successively with 10% citric acid aqueous solution (30 mL), 5% sodium chloride aqueous solution (30 mL), and saturated sodium chloride aqueous solution (30 mL). The resulting organic layer was concentrated to obtain Fmoc-MePhe-n-PrGly-Phe-OH (0.153 g, yield 118%) as a white solid.

[0499] MASS(ESI+)m / z;(M+H)+648.4

[0500] Synthetic Example 43: Synthesis of Boc-MePhe-MePhe-MeAla-Tyr-OH

[0501] [Chemical Formula 59]

[0502]

[0503] Boc-MePhe-OH (0.084 g, 0.30 mmol), triethylamine (0.036 g, 0.36 mmol), and tetrahydrofuran (6.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.044 g, 0.331 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-MeAla-Tyr-OH (0.141 g, 0.305 mmol), N,O-bis(trimethylsilyl)acetamide (0.215 g, 1.023 mmol), N,N-diisopropylethylamine (0.086 g, 0.662 mmol), and acetonitrile (5.0 mL) and stirring at 0 °C for 60 minutes was added, and then stirred at 25 °C for 16 hours (raw material: target substance = 1:59 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), then washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-MeAla-Tyr-OH (0.177 g, yield 85%) as a white solid.

[0504] MASS(ESI+)m / z;(M+H)+689.5

[0505] Synthetic Example 44: Synthesis of Fmoc-Glu(tBu)-MePhe-MePhe-MeAla-Tyr-OH

[0506] [Chemical Formula 60]

[0507]

[0508] Solution A

[0509] Mix Fmoc-Glu(tBu)-OH (0.091 g, 0.21 mmol), triethylamine (0.026 g, 0.257 mmol), and tetrahydrofuran (5.0 mL). Add 2,2-dimethylbutyryl chloride (0.032 g, 0.24 mmol) at 0 °C and stir for 1 hour.

[0510] Solution B

[0511] Mix Boc-MePhe-MePhe-MeAla-Tyr-OH (0.177 g, 0.257 mmol) and trifluoroacetic acid (3.0 mL), and stir at 25 °C for 30 minutes. Concentrate the solution to remove the trifluoroacetic acid, then mix with acetonitrile (20 mL) and concentrate. Add acetonitrile (8.0 mL) and triethylamine (0.026 g, 0.257 mmol) to the residue, and concentrate the solution to remove the solvent and triethylamine. Mix acetonitrile (5.0 mL) and N,O-bis(trimethylsilyl)acetamide (0.167 g, 0.80 mmol) to the residue, and stir at 25 °C for 5 minutes to obtain a colorless and transparent solution.

[0512] Condensation process

[0513] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 25°C for 17 hours (raw material: target substance = 1:5 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (30 mL), and washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was concentrated to obtain Fmoc-Glu(tBu)-MePhe-MePhe-MeAla-Tyr-OH (0.192 g, yield 90%) as a white solid.

[0514] MASS(ESI+)m / z; (M+H)+730.4, +996.5

[0515] Synthetic Example 45: Synthesis of Fmoc-Cys(Trt)-MePhe-OH

[0516] [Chemical Formula 61]

[0517]

[0518] Fmoc-Cys(Trt)-OH (0.147 g, 0.251 mmol), triethylamine (0.031 g, 0.30 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.037 g, 0.28 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.054 g, 0.30 mmol), N,O-bis(trimethylsilyl)acetamide (0.130 g, 0.62 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 20 minutes was added, and the mixture was then stirred at 0 °C for 1 hour (reactant:target substance = 1:28 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), and washed successively with 10% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-Cys(Trt)-MePhe-OH (0.187 g, 100% yield) as a white solid.

[0519] MASS(ESI+) m / z; (M+H) +243.2 (triphenylmethyl cation), +747.2 Synthesis Example 46: Boc-Arg Synthesis of (Cbz)2-MePhe-OH

[0520] [Chemical Formula 62]

[0521]

[0522] Boc-Arg(Cbz)2-OH (0.136 g, 0.250 mmol), triethylamine (0.030 g, 0.30 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.037 g, 0.28 mmol) was added at 0 °C, and the mixture was stirred for 45 minutes. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.054 g, 0.30 mmol), N,O-bis(trimethylsilyl)acetamide (0.128 g, 0.62 mmol), and acetonitrile (4.0 mL) and stirring at 75 °C for 10 minutes was added, and then stirred at 25 °C for at least 1 hour (raw material: target substance = 1:25 (analytical condition 2)). The obtained reaction solution was concentrated and diluted with ethyl acetate (40 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Arg(Cbz)2-MePhe-OH (0.175 g, yield 99%) as a white solid.

[0523] MASS(ESI+)m / z;(M+H)+704.5

[0524] Synthetic Example 47: Synthesis of Boc-Arg(Cbz)2-MeAla-Phe-OH

[0525] [Chemical Formula 63]

[0526]

[0527] Boc-Arg(Cbz)2-OH (0.136 g, 0.251 mmol), triethylamine (0.030 g, 0.30 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.037 g, 0.28 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeAla-Phe-OH (0.075 g, 0.30 mmol), N,O-bis(trimethylsilyl)acetamide (0.130 g, 0.621 mmol), and acetonitrile (4.0 mL) and stirring at 25 °C for 20 minutes was added, and the mixture was then stirred at 25 °C for 14 hours (raw material: target substance = 1:13 (analytical condition 2)). The obtained reaction solution was concentrated and diluted with ethyl acetate (40 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (20 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Arg(Cbz)2-MeAla-Phe-OH (0.192 g, yield 99%) as a white solid.

[0528] MASS(ESI+)m / z;(M+H)+775.5

[0529] Synthetic Example 48: Synthesis of Fmoc-His(Boc)-MePhe-OH

[0530] [Chemical Formula 64]

[0531]

[0532] Fmoc-His(Boc)-OH (0.120 g, 0.251 mmol), N-methylmorpholine (0.033 g, 0.33 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (0.067 g, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.058 g, 0.33 mmol), N,O-bis(trimethylsilyl)acetamide (0.141 g, 0.679 mmol), and acetonitrile (4 mL) and stirring at 75 °C for 20 minutes was added, and the mixture was further stirred at 25 °C for 13 hours (starting material: target substance = 1:6 (analytical condition 2)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-His(Boc)-MePhe-OH (0.131 g, yield 82%) as a white solid.

[0533] MASS(ESI+)m / z; (M+H)+539.6, +639.4

[0534] Synthetic Example 49: Synthesis of Fmoc-MeHis(Trt)-Leu-OH

[0535] [Chemical Formula 65]

[0536]

[0537] Solution A

[0538] Fmoc-MeHis(Trt)-OH (1.27 g, 2.00 mmol), N,N-dimethylformamide (0.015 g, 0.20 mmol), and tetrahydrofuran (30 mL) were mixed. Thionyl chloride (1.19 g, 10.0 mmol) was added at 0 °C, and the mixture was stirred at 25 °C for 1 hour. The solution was concentrated to remove thionyl chloride and solvent. Tetrahydrofuran (10 mL) was then mixed and concentrated. Tetrahydrofuran (10 mL) was added to the residue to give a pale yellow, transparent acyl chloride solution.

[0539] Solution B

[0540] H-Leu-OH (0.315 g, 2.40 mmol), N,O-bis(trimethylsilyl)acetamide (1.30 g, 6.26 mmol), and acetonitrile (5 mL) were mixed and stirred at 80 °C for 1 hour to obtain a colorless and transparent solution.

[0541] Condensation process

[0542] Solution A was cooled to 0°C and then directly mixed with solution B, followed by stirring at 0°C for 1 hour (raw material: target substance = 1:25 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), and washed successively with 10% citric acid aqueous solution and saturated sodium chloride aqueous solution. The resulting organic layer was concentrated and purified by silica gel column chromatography to obtain Fmoc-MeHis(Trt)-Leu-OH (1.37 g, yield 92%) as a pale yellow solid.

[0543] MASS(ESI+)m / z;(M+H)+747.4

[0544] Synthetic Example 50: Synthesis of H-MeHis(Trt)-Leu-OH

[0545] [Chemical Formula 66]

[0546]

[0547] Fmoc-His(Trt)-Leu-OH (0.202 g, 0.270 mmol), triethylamine (0.564 g, 5.40 mmol), acetonitrile (4.0 mL), and tetrahydrofuran (4.0 mL) were mixed and stirred at 80 °C for 60 minutes. The resulting reaction solution was concentrated, and tetrahydrofuran (3.0 mL) and diisopropyl ether (9.0 mL) were added to suspend it. The solid was collected by filtration using a Kiriyama funnel. The solid was washed with diisopropyl ether (10 mL) and dried to obtain H-MeHis(Trt)-Leu-OH (0.130 g, 92% yield) as a white solid.

[0548] MASS(ESI+)m / z;(M+H)+525.8

[0549] Synthetic Example 51: Synthesis of Fmoc-Phe-MeHis(Trt)-Leu-OH

[0550] [Chemical Formula 67]

[0551]

[0552] Fmoc-Phe-OH (0.078 g, 0.20 mmol), triethylamine (0.024 g, 0.24 mmol), and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutyryl chloride (0.030 g, 0.22 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MeHis(Trt)-Leu-OH (0.116 g, 0.221 mmol), N,O-bis(trimethylsilyl)acetamide (0.096 g, 0.46 mmol), and acetonitrile (4.0 mL) and stirring at 25 °C for 30 minutes was added, followed by stirring at 25 °C for 5 hours (reactant:target substance = 1:5 (analytical condition 2)). The resulting reaction solution was concentrated and diluted with ethyl acetate (40 mL), and washed successively with 10% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution. The obtained organic layer was concentrated to give Fmoc-Phe-MeHis(Trt)-Leu-OH (0.154 g, yield 86%) as a white solid.

[0553] MASS(ESI+)m / z;(M+H)+894.5

[0554] Synthetic Example 52: Synthesis of Fmoc-Val-MePhe-OH

[0555] [Chemical Formula 68]

[0556]

[0557] Fmoc-Val-OH (0.101 g, 0.298 mmol), triethylamine (0.053 mL, 0.383 mmol), and tetrahydrofuran (1.0 g) were mixed, and neopentanoyl chloride (0.043 mL, 0.354 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.080 g, 0.446 mmol), trimethylchlorosilane (0.070 mL, 0.554 mmol), triethylamine (0.082 mL, 0.592 mmol), and acetonitrile (0.80 g) and stirring at 50 °C for 1.5 hours was added, followed by stirring at 0 °C for 70 hours (reactant:target substance = 3.5:1). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (3.0 g) and saturated saline solution (2.0 g). The obtained organic layer was analyzed by HPLC, and the quantitative yield was 11%.

[0558] Unless otherwise specified, the ratio of raw material Fmoc-Val-OH to product Fmoc-Val-MePhe-OH was calculated using high performance liquid chromatography (HPLC) analysis <Analysis Condition 4>.

[0559] <Analysis Condition 4>

[0560] High performance liquid chromatography: SHIMADZU HPLC LC-20A

[0561] Column: Agilent Poroshell 120EC-C18 (2.7μm, 3.0×100mm)

[0562] Column oven temperature: 40℃

[0563] Eluent: Acetonitrile: 0.05 vol% phosphoric acid aqueous solution

[0564] 50:50 (0-15 minutes), 50:50-95:5 (15-18 minutes), 95:5 (18-22 minutes) (v / v)

[0565] Elution rate: 0.7 mL / min

[0566] Detection wavelength: 210nm

[0567] Unless otherwise specified, the quantitative yields of Fmoc-Val-MePhe-OH were calculated using a quantitative analytical method based on <Analytical Condition 4>.

[0568] Standard substance: The Fmoc-Val-MePhe-OH obtained by the method described in Synthesis Example 57 was purified by silica gel chromatography and used as a standard substance.

[0569] MASS(ESI+)m / z;(M+H)+501.22

[0570] Quantitative method: Absolute standard curve method

[0571] Synthetic Example 53: Synthesis of Fmoc-Val-MePhe-OH

[0572] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.38 mmol), and tetrahydrofuran (1.0 g) were mixed, and neopentanoyl chloride (0.043 mL, 0.35 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.81 g) and stirring at 50 °C for 1.5 hours was added, followed by stirring at 0 °C for 70 hours (starting material: target substance = 1:7). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 79%.

[0573] Synthetic Example 54: Synthesis of Fmoc-Val-MePhe-OH

[0574] Fmoc-Val-OH (0.100 g, 0.295 mmol), N-methylmorpholine (0.042 mL, 0.383 mmol), and tetrahydrofuran (1.0 g) were mixed, and isobutyl chloroformate (0.046 mL, 0.35 mmol) was added at 0 °C, followed by stirring for 5 minutes. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, followed by stirring at 0 °C for 21 hours (starting material: target substance = 3.5:1). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 18%.

[0575] Synthetic Example 55: Synthesis of Fmoc-Val-MePhe-OH

[0576] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.38 mmol), and tetrahydrofuran (1.0 g) were mixed, and 2-ethylbutyryl chloride (0.048 mL, 0.35 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, followed by stirring at 0 °C for 110 hours (starting material: target substance = 1:13). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 87%.

[0577] Synthetic Example 56: Synthesis of Fmoc-Val-MePhe-OH

[0578] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.38 mmol), and tetrahydrofuran (1.0 g) were mixed, and 2,2-dimethylbutyryl chloride (0.049 mL, 0.35 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, followed by stirring at 0 °C for 95 hours (starting material: target substance = 1:18). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 89%.

[0579] Synthetic Example 57: Synthesis of Fmoc-Val-MePhe-OH

[0580] Fmoc-Val-OH (0.100 g, 0.295 mmol), N,N-diisopropylethylamine (0.058 mL, 0.32 mmol), and acetonitrile (1.0 g) were mixed, and a 50% by mass toluene solution (0.107 g, 0.354 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.442 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, and the mixture was then stirred at 0 °C for 67 hours and at 20 °C for 7 hours (starting material: target substance = 1:35). The resulting reaction solution was quenched with methanol (0.5 mL) and N,N-diisopropylethylamine (0.05 mL), diluted with ethyl acetate (5.0 g), and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer, Fmoc-Val-MePhe-OH, was 96%.

[0581] 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was synthesized with reference to Japanese Patent No. 3406093.

[0582] Synthesis Example 58: Synthesis of Fmoc-Val-MePhe-OH

[0583] Fmoc-Val-OH (0.100 g, 0.295 mmol), N-methylmorpholine (0.071 mL, 0.648 mmol), and N,N-dimethylacetamide (5.9 mL) were mixed, and 2,4-dimethylpentane-3-yl chloroformate (0.105 g, 0.589 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.106 g, 0.589 mmol), N,O-bis(trimethylsilyl)acetamide (0.288 mL, 1.179 mmol), and acetonitrile (7.9 mL) and stirring at 75 °C for 15 minutes was added, and the mixture was then stirred at 0 °C for 114 hours and then at 20 °C for 4 hours (raw material: target substance = 1:11). The resulting reaction solution was quenched with methanol (5.0 mL) and N,N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and washed twice with 10% citric acid aqueous solution (18.0 g) and saturated saline solution (6.0 g). The quantitative yield of the collected organic layer, Fmoc-Val-MePhe-OH, was 85%.

[0584] Synthetic Example 59: Synthesis of Fmoc-Val-MePhe-OH

[0585] Fmoc-Val-OH (0.100 g, 0.295 mmol), N-methylmorpholine (0.071 mL, 0.648 mmol), and N,N-dimethylacetamide (5.9 mL) were mixed, and 2,2,4-triphenylmethylpentane-3-yl chloroformate (0.114 g, 0.589 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.106 g, 0.589 mmol), N,O-bis(trimethylsilyl)acetamide (0.288 mL, 1.179 mmol), and acetonitrile (7.9 mL) and stirring at 75 °C for 15 minutes was added, and the mixture was then stirred at 0 °C for 91 hours and then at 20 °C for 7 hours (raw material: target substance = 1:10). The resulting reaction solution was quenched with methanol (5.0 mL) and N,N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and washed twice with 10% citric acid aqueous solution (18.0 g) and saturated saline solution (6.0 g). The quantitative yield of the collected organic layer, Fmoc-Val-MePhe-OH, was 88%.

[0586] Synthetic Example 60: Synthesis of Fmoc-Val-MePhe-OH

[0587] Fmoc-Val-OH (0.100 g, 0.295 mmol), N,N-diisopropylethylamine (0.065 mL, 0.38 mmol), and tetrahydrofuran (1.0 g) were mixed, and 1-adamantaneformyl chloride (0.070 g, 0.35 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.44 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, and then stirred at 0 °C for 96 hours (starting material: target substance = 1:10). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 90%.

[0588] Synthetic Example 61: Synthesis of Fmoc-Val-MePhe-OH

[0589] Fmoc-Val-OH (0.10 g, 0.30 mmol), N-methylmorpholine (0.071 mL, 0.65 mmol), and N,N-dimethylacetamide (5.9 mL) were mixed, and 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (0.13 g, 0.59 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.106 g, 0.59 mmol), N,O-bis(trimethylsilyl)acetamide (0.29 mL, 1.18 mmol), and acetonitrile (7.9 mL) and stirring at 75 °C for 15 minutes was added, and the mixture was then stirred at 0 °C for 164 hours (raw material: target substance = 1:9). The resulting reaction solution was quenched with methanol (5.0 mL) and N,N-diisopropylethylamine (0.50 mL), diluted with ethyl acetate (30.0 g), and washed twice with 10% citric acid aqueous solution (18.0 g), saturated saline solution (10.0 g), and saturated saline solution (6.0 g). The quantitative yield of the collected organic layer, Fmoc-Val-MePhe-OH, was 83%.

[0590] Synthetic Example 62: Synthesis of Fmoc-Val-MePhe-OH

[0591] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.383 mmol), and tetrahydrofuran (1.0 g) were mixed, and 2-ethylhexanoyl chloride (0.061 mL, 0.354 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.442 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, followed by stirring at 0 °C for 111 hours (starting material: target substance = 1:11). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 90%.

[0592] Synthetic Example 63: Synthesis of Fmoc-Val-MePhe-OH

[0593] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.383 mmol), and tetrahydrofuran (1.0 g) were mixed, and 2-(4-chlorophenyl)-3-methylbutyryl chloride (0.070 mL, 0.354 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.442 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, and then stirred at 0 °C for 66 hours (starting material: target substance = 1:10). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% citric acid aqueous solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of the collected organic layer Fmoc-Val-MePhe-OH was 84%.

[0594] Synthetic Example 64: Synthesis of Fmoc-Val-MePhe-OH

[0595] [Chemical Formula 69]

[0596]

[0597] Fmoc-Val-OH (0.100 g, 0.295 mmol), triethylamine (0.053 mL, 0.383 mmol), and tetrahydrofuran (1.0 g) were mixed, and 3,5,5-trimethylhexanoyl chloride (0.067 mL, 0.354 mmol) was added at 0 °C, followed by stirring for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.079 g, 0.442 mmol), N,O-bis(trimethylsilyl)acetamide (0.135 mL, 0.552 mmol), and acetonitrile (0.79 g) and stirring at 50 °C for 1 hour was added, and then stirred at 0 °C for 68 hours (raw material: target substance = 16:1).

[0598] Synthetic Example 65: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0599] [Chemical Formula 70]

[0600]

[0601] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) was prepared, and neopentanoyl chloride (24 mg, 0.20 mmol) was added at 0 °C, followed by stirring for 2 hours. To this solution, a solution prepared by separately reacting H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) was added and stirred at 50 °C for 1 hour. The mixture was then heated to 25 °C and stirred for 3 hours (reactant:target compound = 1:10 (analytical condition 5)). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 77% (analytical condition 5).

[0602] MASS(ESI+)m / z;(M+H)+608.44

[0603] Unless otherwise specified, the ratio of the raw material Cbz-Phe-OH to the product Cbz-Phe-MePhe-Phe-OH was calculated by high performance liquid chromatography (HPLC) analysis <Analysis Condition 5>.

[0604] <Analysis Condition 5>

[0605] High performance liquid chromatography: SHIMADZU HPLC-20A

[0606] Column: Agilent Poroshell 120EC-C18 (2.7μm, 3.0×100mm)

[0607] Column oven temperature: 50℃

[0608] Eluent: 0.2 vol% acetonitrile phosphate solution; 0.2 vol% aqueous phosphoric acid solution

[0609] 12:88-95:5 (0-15 minutes), 95:5 (15-19 minutes), (v / v)

[0610] Elution rate: 0.7 mL / min

[0611] Detection wavelength: 214nm

[0612] Unless otherwise specified, the quantitative yields of Cbz-Phe-MePhe-Phe-OH were calculated using a quantitative analytical method based on <Analytical Condition 5>.

[0613] Standard substance: Cbz-Phe-MePhe-Phe-OH synthesized by the method described in Synthesis Example 71 was purified by silica gel chromatography and used as a standard substance.

[0614] The NMR and MASS of the standard substance are shown.

[0615] 1 H NMR (300MHz, DMSO-d6):

[0616] δppm: 2.64 (3H, s), 2.67-3.33 (6H, m), 4.27-4.45 (3H, m), 4.89 (2H, s), 5.21 (1H, m), 7.04-7.33 (20H, m)

[0617] MASS(ESI+)m / z;(M+H)+608.44

[0618] Quantitative method: Absolute standard curve method

[0619] Synthetic Example 66: Synthesis of H-MePhe-Phe-OH

[0620] [Chemical Formula 71]

[0621]

[0622] Cbz-MePhe-OH (2.0 g, 6.38 mmol), dichloromethane (20.0 g), N,N-diisopropylethylamine (2.9 g, 22.3 mmol), and H-Phe-OBn (2.2 g, 7.66 mmol) were mixed, and N-[1-(cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino(morpholino)]urea hexafluorophosphate (3.28 g, 7.66 mmol) was added at 22 °C, and the mixture was stirred for 1 hour and 30 minutes. 10% hydrochloric acid (16 g) and water (16 g) were added to the resulting reaction mixture, and the organic layer was washed twice with water after separation. Then, 10% potassium bicarbonate aqueous solution (16 g) and water (16 g) were added, and the mixture was separated twice, followed by washing with water. The resulting organic layer was concentrated under reduced pressure, purified by silica gel column chromatography, and diluted with trifluoroethanol (40 g). 10% Pd-C (0.4 g) was added to the obtained solution, and the mixture was stirred at room temperature for 14 hours and 30 minutes under a hydrogen atmosphere. The reaction solution was concentrated under reduced pressure, diluted with methanol (300 g), filtered, and the filtrate was diluted again with methanol (300 g) and filtered. This operation was repeated 3 times. All the filtrates were concentrated, diluted with acetonitrile (100 g), and concentrated again to obtain H-MePhe-Phe-OH (1.35 g, yield 65%) as a white solid.

[0623] MASS(ESI+)m / z;(M+H)+327.2

[0624] Synthetic Example 67: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0625] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and N-methylmorpholine (20 mg, 0.20 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 3 hours (reactant:target compound = 1:1.6). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 43%.

[0626] Synthetic Example 68: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0627] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 21 hours (starting material: target substance = 1:24). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 95%.

[0628] Synthetic Example 69: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0629] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 27 hours (starting material: target substance = 1:30). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 102%.

[0630] Synthetic Example 70: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0631] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) was added to a 50% by mass toluene solution (124 mg, 0.20 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride at 25 °C, and the mixture was stirred for 9 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added, and the mixture was heated to 25 °C and stirred for 14 hours (raw material: target substance = 1:80). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 100%.

[0632] Synthetic Example 71: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0633] A mixture of Cbz-Phe-OH (99 mg, 0.33 mmol), acetonitrile (1.0 g), and N,N-diisopropylethylamine (52 mg, 0.40 mmol) was added to a 50% by mass toluene solution (250 mg, 0.41 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride at room temperature, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (164 mg, 0.50 mmol), N,O-bis(trimethylsilyl)acetamide (195 mg, 0.96 mmol), and acetonitrile (1.6 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 25 °C for 21 hours (starting material: target substance = 1:335). The resulting reaction solution was diluted with ethyl acetate (5.0 g), separated using a 10% citric acid aqueous solution (3.0 g), and then washed twice with a saturated sodium chloride aqueous solution (2.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 100%.

[0634] Synthetic Example 72: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0635] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 3 hours (starting material: target substance = 1:32). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 99%.

[0636] Synthetic Example 73: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0637] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) was added at 0 °C, and the mixture was stirred for 1.5 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 29 hours (starting material: target substance = 1:15). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% citric acid aqueous solution (1.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 83%.

[0638] Synthetic Example 74: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0639] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and N,N-diisopropylethylamine (30 mg, 0.23 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (110 mg, 1.0 mmol), N,O-bis(trimethylsilyl)acetamide (129 mg, 0.63 mmol), and acetonitrile (1.1 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 28 hours (starting material: target substance = 1:58). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated using a 10% citric acid aqueous solution (3.0 g), and then washed twice with a saturated sodium chloride aqueous solution (1.0 g). The obtained organic layer was quantified, and Cbz-Phe-MePhe-Phe-OH was obtained with a quantitative yield of 98%.

[0640] Synthetic Example 75: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0641] A mixture of Cbz-Phe-OH (0.050 g, 0.17 mmol), N,N-dimethylacetamide (3.0 mL), and N-methylmorpholine (0.040 mL, 0.37 mmol) was prepared, and 4-ethyl-2,2-dimethylhexane-3-yl chloroformate (0.074 g, 0.33 mmol) was added at 0 °C. The mixture was stirred for 1 hour and 30 minutes. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (0.109 g, 0.334 mmol), N,O-bis(trimethylsilyl)acetamide (0.16 mL, 0.67 mmol), and acetonitrile (4.0 mL) and stirring at 50 °C for 1 hour was added. The mixture was then heated to room temperature and stirred for 21 hours (starting material: target substance = 1:43). The obtained reaction solution was diluted with ethyl acetate (15 g), separated with 10% citric acid aqueous solution (9.0 g) and saturated saline solution (5.0 g), and then washed twice with saturated saline solution (3.0 g). The quantitative yield of the obtained organic layer Cbz-Phe-MePhe-Phe-OH was 92%.

[0642] Synthetic Example 76: Synthesis of Cbz-Phe-MePhe-Phe-OH

[0643] A mixture of Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) was added at 0 °C, and the mixture was stirred for 1.5 hours. To this solution, a solution prepared by separately mixing H-MePhe-Phe-OH (82 mg, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (97 mg, 0.48 mmol), and acetonitrile (0.82 g) and stirring at 50 °C for 1 hour was added. The mixture was then heated to 25 °C and stirred for 16 hours (raw material: target substance = 1:1.2).

[0644] Synthetic Example 77: Synthesis of Boc-Val-Pro-OH

[0645] [Chemical Formula 72]

[0646]

[0647] Boc-Val-OH (128 mg, 0.59 mmol), tetrahydrofuran (1.3 g), and N-methylmorpholine (60 mg, 0.59 mmol) were mixed, and isobutyl chloroformate (72 mg, 0.59 mol) was added at 0 °C, followed by stirring for 2.5 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (71 mg, 0.62 mmol), N,N-diisopropylethylamine (0.16 g, 1.2 mmol), trimethylchlorosilane (0.13 g, 1.2 mmol), and dichloromethane (3 mL) and stirring at 40 °C for 2 hours was added. The mixture was then stirred at 0 °C for 15 hours to obtain Boc-Val-Pro-OH (starting material: target compound = 1:15).

[0648] MASS(ESI+)m / z;(M+H)+315.20

[0649] Unless otherwise specified, the ratio of raw material Boc-Val-OH to product Boc-Val-Pro-OH was calculated using high performance liquid chromatography (HPLC) analysis <Analysis Condition 5>.

[0650] Synthetic Example 78: Synthesis of Boc-Val-Pro-OH

[0651] A mixture of Boc-Val-OH (128 mg, 0.59 mmol), acetonitrile (1.3 g), and N,N-diisopropylethylamine (91 mg, 0.71 mmol) was added to a 50% by mass toluene solution (0.43 g, 0.71 mol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride at 25 °C, and the mixture was stirred for 4 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (71 mg, 0.62 mmol), N,O-bis(trimethylsilyl)acetamide (0.22 g, 1.1 mmol), and acetonitrile (1.3 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 25 °C for 15 hours (starting material: target substance = 1:731). The obtained reaction solution was diluted with ethyl acetate (10 mL), separated by water (2 mL) and 10% citric acid aqueous solution (2 mL), and then washed successively with 5% sodium chloride aqueous solution (5 mL) and water (5 mL). The quantitative yield of the obtained organic layer Boc-Val-Pro-OH was 94%.

[0652] Unless otherwise specified, the quantitative yield of Boc-Val-Pro-OH was calculated using the quantitative analysis method based on <Analytical Condition 5>.

[0653] Standard reference material: Boc-Val-Pro-OH, which was separately isolated and purified with reference to Indian Journal of Chemistry, 2004, Vol. 43B, p. 1282, will be used as the standard reference material.

[0654] The MASS of the standard substance is shown.

[0655] MASS(ESI+)m / z;(M+H)+315.20

[0656] Quantitative method: Absolute standard curve method

[0657] Synthetic Example 79: Synthesis of Boc-Val-Pro-OH

[0658] A mixture of Boc-Val-OH (50 mg, 0.23 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (30 mg, 0.30 mmol) was added at 0 °C, and the mixture was stirred for 2.5 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (34 mg, 0.77 mmol), N,O-bis(trimethylsilyl)acetamide (0.80 g, 1.0 mmol), and acetonitrile (0.50 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 15 hours (starting material: target substance = 1:99). The resulting reaction solution was diluted with ethyl acetate (5 mL), separated with 10% citric acid aqueous solution (2 mL), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of Boc-Val-Pro-OH in the organic and aqueous layers was 90%.

[0659] Synthetic Example 80: Synthesis of Boc-Val-Pro-OH

[0660] A mixture of Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (77 mg, 0.60 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (64 mg, 0.55 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 g, 0.87 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 21 hours (starting material: target substance = 1:520). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated by water (2 mL) and 10% citric acid aqueous solution (2 mL), and then washed twice with 10% sodium chloride aqueous solution (2 mL). The quantitative yield of the obtained organic layer, Boc-Val-Pro-OH, was 99%.

[0661] Synthetic Example 81: Synthesis of Boc-Val-Pro-OH

[0662] A mixture of Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (77 mg, 0.60 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (64 mg, 0.55 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 g, 0.87 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 21 hours (starting material: target compound = 1:248). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated by water (2 mL), 10% citric acid aqueous solution (2 mL), and then washed twice with 10% sodium chloride aqueous solution (2 mL). The quantitative yield of the obtained organic layer of Boc-Val-Pro-OH was 99%.

[0663] Synthetic Example 82: Synthesis of Boc-Val-Pro-OH

[0664] A mixture of Boc-Val-OH (100 mg, 0.46 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (61 mg, 0.60 mmol) was added at 0 °C, followed by the addition of 2,4-dimethylpentane-3-yl chloroformate (99 mg, 0.55 mol), and the mixture was stirred for 2.5 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (64 mg, 0.55 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 g, 0.88 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 21 hours (starting material: target substance = 1:179). The resulting reaction solution was diluted with ethyl acetate (5 mL), separated with 10% citric acid aqueous solution (2 mL), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of Boc-Val-Pro-OH in the organic and aqueous layers was 89%.

[0665] Synthetic Example 83: Synthesis of Fmoc-Val-MeGly-OH

[0666] [Chemical Formula 73]

[0667]

[0668] Fmoc-Val-OH (200 mg, 0.59 mmol), tetrahydrofuran (2.0 g), and N-methylmorpholine (60 mg, 0.59 mmol) were mixed, and isobutyl chloroformate (72 mg, 0.59 mmol) was added at 0 °C, followed by stirring for 2.5 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (55 mg, 0.62 mmol), N,N-diisopropylethylamine (0.16 g, 1.2 mmol), trimethylchlorosilane (0.13 g, 1.2 mmol), and dichloromethane (3 mL) and stirring at 40 °C for 2 hours was added. The mixture was then stirred at 0 °C for 15 hours to obtain Fmoc-Val-MeGly-OH (starting material: target compound = 1:1.9).

[0669] MASS(ESI+)m / z;(M+H)+411.28

[0670] Unless otherwise specified, the ratio of raw material Fmoc-Val-OH to product Fmoc-Val-MeGly-OH was calculated by high performance liquid chromatography analysis <Analysis Condition 5>.

[0671] Synthetic Example 84: Synthesis of Fmoc-Val-MeGly-OH

[0672] Fmoc-Val-OH (200 mg, 0.59 mmol), acetonitrile (2.0 g), and N,N-diisopropylethylamine (91 mg, 0.71 mmol) were mixed, and a 50% by mass toluene solution (0.43 g, 0.71 mol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at 25 °C, and the mixture was stirred for 4 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (79 mg, 0.88 mmol), N,O-bis(trimethylsilyl)acetamide (0.22 g, 1.1 mmol), and acetonitrile (2.0 g) and stirring at 75 °C for 1 hour was added, and the mixture was stirred at 25 °C for 15 hours (starting material: target substance = 1:61). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% citric acid aqueous solution (2.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The quantitative yield of the obtained organic layer, Fmoc-Val-MeGly-OH, was 97%.

[0673] Unless otherwise specified, the quantitative yield of Fmoc-Val-MeGly-OH was calculated using the quantitative analysis method based on <Analytical Condition 5>.

[0674] Standard reference material: Fmoc-Val-MeGly-OH, which was separately isolated and purified with reference to Indian Journal of Chemistry, 2004, Vol. 43B, p. 1282, will be used as the standard reference material.

[0675] The MASS of the standard substance is shown.

[0676] MASS(ESI+)m / z;(M+H)+411.28

[0677] Quantitative method: Absolute standard curve method

[0678] Synthetic Example 85: Synthesis of Fmoc-Val-MeGly-OH

[0679] Fmoc-Val-OH (200 mg, 0.59 mmol), N,N-dimethylacetamide (2.0 g), and N-methylmorpholine (78 mg, 0.77 mmol) were mixed, and 2,2,4-trimethylpentane-3-yl chloroformate (0.14 g, 0.71 mol) was added at 0 °C, and the mixture was stirred for 2.5 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (79 mg, 0.88 mmol), N,O-bis(trimethylsilyl)acetamide (0.22 g, 1.1 mmol), and acetonitrile (2.0 g) and stirring at 75 °C for 1 hour was added, and the mixture was stirred at 0 °C for 15 hours (starting material: target substance = 1:64). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated by water (5 mL), 10% citric acid aqueous solution (5 mL), and then the aqueous layer was extracted with ethyl acetate (10 mL). The organic layers were mixed and washed with a 10% (w / w) sodium chloride aqueous solution. The quantitative yield of the obtained organic layer, Fmoc-Val-MeGly-OH, was 97%.

[0680] Synthetic Example 86: Synthesis of Fmoc-Val-MeGly-OH

[0681] Fmoc-Val-OH (100 mg, 0.29 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (50 mg, 0.38 mmol) were mixed, and 2,2-dimethylbutyryl chloride (48 mg, 0.35 mol) was added at 0 °C, followed by stirring for 2 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (32 mg, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.11 g, 0.55 mmol), and acetonitrile (1.0 g) and stirring at 75 °C for 1 hour was added, and the mixture was stirred at 0 °C for 20 hours (starting material: target substance = 1:48). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 mL). The quantitative yield of the obtained organic layer, Fmoc-Val-MeGly-OH, was 95%.

[0682] Synthetic Example 87: Synthesis of Fmoc-Val-MeGly-OH

[0683] Fmoc-Val-OH (100 mg, 0.29 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (50 mg, 0.38 mmol) were mixed, and 2-ethylbutyryl chloride (48 mg, 0.35 mol) was added at 0 °C, followed by stirring for 2 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (32 mg, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.11 g, 0.55 mmol), and acetonitrile (1.0 g) and stirring at 75 °C for 1 hour was added, and the mixture was stirred at 0 °C for 20 hours (starting material: target substance = 1:20). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 mL). The quantitative yield of the obtained organic layer, Fmoc-Val-MeGly-OH, was 92%.

[0684] Synthetic Example 88: Synthesis of Fmoc-Val-MeGly-OH

[0685] Fmoc-Val-OH (100 mg, 0.29 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (39 mg, 0.38 mmol) were mixed, and 2,4-dimethylpentane-3-yl chloroformate (63 mg, 0.35 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeGly-OH (32 mg, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.11 g, 0.55 mmol), and acetonitrile (1.0 g) and stirring at 75 °C for 1 hour was added, and the mixture was stirred at 0 °C for 20 hours (starting material: target substance = 1:33). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 mL). The quantitative yield of Fmoc-Val-MeGly-OH in the organic and aqueous layers was 88%.

[0686] Synthetic Example 89: Synthesis of Boc-Cys(Bn)-Pro-OH

[0687] [Chemical Formula 74]

[0688]

[0689] A mixture of Boc-Cys(Bn)-OH (200 mg, 0.64 mmol), chloroform (1.3 mL), and triethylamine (67 mg, 0.66 mmol) was added at -20 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-Pro-OH (96 mg, 0.84 mmol), triethylamine (0.16 g, 1.6 mmol), trimethylchlorosilane (0.11 g, 1.0 mmol), dichloromethane (1.6 mL), and N,N-dimethylformamide (0.32 mL) and stirring at 40 °C for 2 hours was added. The mixture was then stirred at -20 °C for 3 hours to obtain Boc-Cys(Bn)-Pro-OH (starting material: target compound = 1:2.2).

[0690] MASS(ESI+)m / z;(M+H)+409.26

[0691] Unless otherwise specified, the ratio of the raw material Boc-Cys(Bn)-OH to the product Boc-Cys(Bn)-Pro-OH was calculated using high performance liquid chromatography (HPLC) analysis <Analytical Condition 6>.

[0692] <Analysis Condition 6>

[0693] High performance liquid chromatography: SHIMADZU HPLC-20A

[0694] Column: Agilent Poroshell 120EC-C18 (2.7μm, 3.0×100mm)

[0695] Column oven temperature: 50℃

[0696] Eluent: 0.2 vol% acetonitrile phosphate solution; 0.2 vol% aqueous phosphoric acid solution

[0697] 35:65 (7 minutes), 35:65-95:5 (7-14 minutes), 95:5 (14-18 minutes), (v / v)

[0698] Elution rate: 0.7 mL / min

[0699] Detection wavelength: 214nm

[0700] Synthetic Example 90: Synthesis of Boc-Cys(Bn)-Pro-OH

[0701] A mixture of Boc-Cys(Bn)-OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), and N,N-diisopropylethylamine (50 mg, 0.39 mmol) was added at 25 °C, followed by the addition of a 50% by mass toluene solution (231 mg, 0.39 mol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (48 mg, 0.42 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 g, 0.59 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 25 °C for 23 hours (starting material: target substance = 1:265). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% citric acid aqueous solution (2.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The organic and aqueous layers were quantified to obtain Boc-Cys(Bn)-Pro-OH in a quantitative yield of 97%.

[0702] Unless otherwise specified, the quantitative yield of Boc-Cys(Bn)-Pro-OH was calculated using the quantitative analysis method based on <Analytical Condition 6>.

[0703] Standard reference material: Boc-Cys(Bn)-Pro-OH, which was separately isolated and purified with reference to Chemistry of Nature Compounds, 1992, Vol. 28, p. 344, will be used as the standard reference material.

[0704] The MASS of the standard substance is shown.

[0705] MASS(ESI+)m / z;(M+H)+409.26

[0706] Quantitative method: Absolute standard curve method

[0707] Synthetic Example 91: Synthesis of Boc-Cys(Bn)-Pro-OH

[0708] A mixture of Boc-Cys(Bn)-OH (100 mg, 0.32 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (42 mg, 0.42 mmol) was prepared, and 2,2,4-trimethylpentane-3-yl chloroformate (74 mg, 0.39 mol) was added at 0 °C, followed by stirring for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (48 mg, 0.42 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 g, 0.59 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 15 hours (starting material: target substance = 1:461). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% citric acid aqueous solution (2.0 g), and then washed twice with 10% sodium chloride aqueous solution (1.0 g). The obtained organic and aqueous layers were quantified to obtain Boc-Cys(Bn)-Pro-OH with a quantitative yield of 96%.

[0709] Synthetic Example 92: Synthesis of Boc-Cys(Bn)-Pro-OH

[0710] A mixture of Boc-Cys(Bn)-OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), and N,N-diisopropylethylamine (54 mg, 0.41 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (44 mg, 0.38 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 g, 0.61 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 19 hours (starting material: target substance = 1:31). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 mL). The obtained organic layer was quantified to obtain Boc-Cys(Bn)-Pro-OH with a quantitative yield of 97%.

[0711] Synthetic Example 93: Synthesis of Boc-Cys(Bn)-Pro-OH

[0712] A mixture of Boc-Cys(Bn)-OH (100 mg, 0.32 mmol), acetonitrile (1.0 g), and N,N-diisopropylethylamine (54 mg, 0.41 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (44 mg, 0.38 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 g, 0.61 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 19 hours (starting material: target substance = 1:30). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 mL). The obtained organic layer was quantified to obtain Boc-Cys(Bn)-Pro-OH with a quantitative yield of 97%.

[0713] Synthetic Example 94: Synthesis of Boc-Cys(Bn)-Pro-OH

[0714] A mixture of Boc-Cys(Bn)-OH (100 mg, 0.32 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (42 mg, 0.42 mmol) was added at 0 °C, followed by the addition of 2,4-dimethylpentane-3-yl chloroformate (69 mg, 0.38 mol), and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-Pro-OH (48 mg, 0.42 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 g, 0.59 mmol), and acetonitrile (1.0 g) and stirring at 50 °C for 1 hour was added, and the mixture was stirred at 0 °C for 19 hours (starting material: target substance = 1:60). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% citric acid aqueous solution (2.0 mL), and then washed twice with 10% sodium chloride aqueous solution (2.0 g). The obtained organic layer was quantified to obtain Boc-Cys(Bn)-Pro-OH with a quantitative yield of 98%.

[0715] Synthetic Example 95: Synthesis of Cbz-Phe-Phe-MePhe-OH

[0716] [Chemical Formula 75]

[0717]

[0718] Cbz-Phe-Phe-OH (0.100 g, 0.224 mmol), N,N-diisopropylethylamine (0.050 mL, 0.291 mmol), and tetrahydrofuran (1.0 g) were mixed, and 1-adamantaneformyl chloride (0.0534 g, 0.269 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.060 g, 0.336 mmol), N,O-bis(trimethylsilyl)acetamide (0.103 mL, 0.420 mmol), and acetonitrile (0.60 g) and stirring at 50 °C for 1 hour was added, and then stirred at 0 °C for 22 hours (raw material: target substance = 1:32). The resulting reaction solution was quenched with a 2% n-propylamine / acetonitrile solution, diluted with ethyl acetate (5.0 g), and washed twice with a 10% citric acid aqueous solution (2.0 g) and a saturated saline aqueous solution (1.0 g). The quantitative yield of the collected organic layer Cbz-Phe-Phe-MePhe-OH was 89%.

[0719] The ratio of the raw material Cbz-Phe-Phe-OH to the product Cbz-Phe-Phe-MePhe-OH was calculated by high performance liquid chromatography analysis <analysis condition 7>.

[0720] <Analysis Condition 7>

[0721] High performance liquid chromatography: SHIMADZU HPLC LC-20A

[0722] Column: Agilent Poroshell 120EC-C18 (2.7μm, 3.0×100mm)

[0723] Column oven temperature: 50℃

[0724] Eluent: 0.2 vol% TFA / acetonitrile:methanol = 1:1

[0725] 0.2 vol% TFA aqueous solution

[0726] 55:45 (0-20 minutes), 55:45-95:5 (20-20.1 minutes), 95:5 (20.1-24 minutes), Post time 5 minutes (v / v)

[0727] Elution rate: 0.7 mL / min

[0728] Detection wavelength: 214nm

[0729] The quantitative yield of Cbz-Phe-Phe-MePhe-OH was calculated using a quantitative analytical method based on <Analytical Condition 7>.

[0730] Standard substance: Cbz-Phe-Phe-MePhe-OH synthesized in Synthesis Example 96 was used as the standard substance.

[0731] The MASS of the standard substance is shown.

[0732] MASS(ESI+)m / z;(M+H)+608.4

[0733] Quantitative method: Absolute standard curve method

[0734] Synthetic Example 96: Synthesis of Cbz-Phe-Phe-MePhe-OH

[0735] Add dichloromethane (1.0 mL), Fmoc-MePhe-OH (0.062 g, 0.15 mmol), and N,N-diisopropylethylamine (0.11 mL, 0.61 mmol) to 2-chlorotriphenylmethyl chloride resin (200-400 mesh) (0.10 g, 0.13 mmol) and stir overnight. Add the following solutions sequentially to the resulting suspension, filtering each time: (1) a mixed solution of (dichloromethane / methanol / N,N-diisopropylethylamine = 17 / 2 / 1) 3 times, (2) dichloromethane 3 times, (3) N-methylpyrrolidone 2 times, (4) dichloromethane 2 times, and (5) methanol 5 times. Add N-methylpyrrolidone to the filtered resin to fully impregnate it, and stir for 15 minutes. Then filter, add a 20% piperidine / N-methylpyrrolidone solution to the resin to fully impregnate it, and stir for 20 minutes. Filtration was continued, and the resin was washed 10 times with N-methylpyrrolidone. To the obtained resin (0.10 g, 0.050 mmol), 0.80 mL of 0.5 M cyano(hydroxyimino)ethyl acetate / N-methylpyrrolidone solution, Fmoc-Phe-OH (0.078 mg, 0.20 mmol), N-[1-(cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino(morpholino)]urea hexafluorophosphate (0.086 g, 0.20 mmol), and N,N-diisopropylethylamine (0.085 mL, 0.50 mmol) were added, and the mixture was stirred for 2 hours. The reaction solution was filtered, and the resin was washed 10 times with N-methylpyrrolidone. Then, the above condensation-washing operation was repeated, with the addition of 20% piperidine / N-methylpyrrolidone followed by 20 minutes of stirring and washing with N-methylpyrrolidone, repeated 10 times. To the obtained resin, add 0.400 mL of 0.5 M cyano(hydroxyimino)ethyl acetate / N-methylpyrrolidone solution, Cbz-Phe-OH (0.060 mg, 0.20 mmol), and N,N-diisopropylcarbodiimide (0.032 mL, 0.20 mmol), and stir overnight. Filter the reaction solution, wash 10 times with N-methylpyrrolidone, wash 10 times with methanol, and then add 30% hexafluoroisopropanol to thoroughly impregnate the resin. Stir for 10 minutes and filter. Repeat this process 5 times. Concentrate the collected solution under reduced pressure to obtain Cbz-Phe-Phe-MePhe-OH (0.031 g, 100%).

[0736] MASS(ESI+)m / z;(M+H)+608.4

[0737] In the above synthesis examples, Synthesis Examples 1 to 7, 66 and 96 are reference examples related to the synthesis of the raw materials used in the embodiments, and Synthesis Examples 1 to 2 and Synthesis Examples 3 to 4 are also embodiments of the invention related to the compounds of this application. In addition, Synthesis Examples 8 to 13, 18 to 28, 32 to 35, 38, 41 to 48, 51, 55 to 63, 68 to 76, 78 to 82, 84 to 88, 90 to 95 are embodiments of the invention related to the peptide manufacturing method of this application, and Synthesis Examples 14 to 17, 29 to 31, 36 to 37, 39 to 40, 49 to 50, 52 to 54, 64 to 65, 67, 77, 83, 89 and 96 are comparative examples.

[0738] Synthetic Example 97: Synthesis of Fmoc-Val-MeTyr(tBu)-OH

[0739] [Chemical Formula 76]

[0740]

[0741] Fmoc-Val-OH (0.102 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.050 mL, 0.36 mmol) and neopentanoyl chloride (0.041 mL, 0.33 mmol) were added at 0 °C, and the mixture was stirred at 0 °C for 2 hours. To this solution, a solution prepared by separately mixing H-MeTyr(tBu)-OH (0.090 g, 0.36 mmol), N,O-bis(trimethylsilyl)acetamide (0.19 mL, 0.72 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 20 hours (reactant:target compound = 1:7 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (20 mL) and washed successively with saturated sodium bicarbonate aqueous solution (20 mL), water (5 mL), and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated to give Fmoc-Val-MeTyr(tBu)-OH (0.18 g, yield 106%) as a light yellow solid.

[0742] Synthetic Example 98: Synthesis of Fmoc-Val-MeTyr(tBu)-OH

[0743] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeTyr(tBu)-OH (0.090 g, 0.36 mmol), N,O-bis(trimethylsilyl)acetamide (0.185 mL, 0.72 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 24 hours (starting material: target analyte = 1:20 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeTyr(tBu)-OH (0.170 g, yield 99%) as a white solid.

[0744] Synthetic Example 99: Synthesis of Fmoc-Val-MeVal-OH

[0745] [Chemical Formula 77]

[0746]

[0747] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.050 mL, 0.36 mmol) and neopentanoyl chloride (0.041 mL, 0.33 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeVal-OH (0.047 g, 0.36 mmol), N,O-bis(trimethylsilyl)acetamide (0.19 mL, 0.72 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 15 hours (reactant:target compound = 2.4:1 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL) and washed successively with saturated sodium bicarbonate aqueous solution (5 mL), 10% citric acid aqueous solution (5 mL), water (5 mL), and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated to obtain Fmoc-Val-MeVal-OH (0.16 g, yield 116%) as a light yellow solid.

[0748] Synthetic Example 100: Synthesis of Fmoc-Val-MeVal-OH

[0749] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeVal-OH (0.032 g, 0.24 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 mL, 0.48 mmol), and acetonitrile (1.0 mL) and stirring at 50 °C for 1 hour was added, and the mixture was further stirred at 0 °C for 2 hours, and then at 25 °C for 36 hours (starting material: target substance = 1:16 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeVal-OH (0.092 g, yield 92%) as a white solid.

[0750] Synthetic Example 101: Synthesis of Fmoc-Val-MeSer(tBu)-OH

[0751] [Chemical Formula 78]

[0752]

[0753] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.050 mL, 0.36 mmol) and neopentanoyl chloride (0.041 mL, 0.33 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeSer(tBu)-OH (0.063 g, 0.36 mmol), N,O-bis(trimethylsilyl)acetamide (0.19 mL, 0.72 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added, and the mixture was further stirred at 0 °C for 15 hours. (Reagent:Target = 1:19 (Analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL) and washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated to give Fmoc-Val-MeSer(tBu)-OH (0.14 g, yield 94%) as a light yellow solid.

[0754] Synthetic Example 102: Synthesis of Fmoc-Val-MeSer(tBu)-OH

[0755] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeSer(tBu)-OH (0.045 g, 0.26 mmol), N,O-bis(trimethylsilyl)acetamide (0.13 mL, 0.52 mmol), and acetonitrile (1.0 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 15 hours (starting material: target substance = 1:25 (analytical condition 3)). The obtained reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeSer(tBu)-OH (0.095 g, yield 96%) as a white solid.

[0756] Synthetic Example 103: Synthesis of Fmoc-Val-MeDap(Boc)-OH

[0757] [Chemical Formula 79]

[0758]

[0759] Fmoc-Val-OH (0.034 g, 0.1 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.017 mL, 0.12 mmol) and neopentanoyl chloride (0.014 mL, 0.11 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing Nα-Boc-Nβ-methyl-2,3-diaminopropionic acid (H-MeDap(Boc)-OH) (0.026 g, 0.12 mmol), N,O-bis(trimethylsilyl)acetamide (0.62 mL, 0.24 mmol), and acetonitrile (1.0 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 24 hours. (Reagent:Target analyte = 1:2.4 (Analytical condition 3)).

[0760] Synthetic Example 104: Synthesis of Fmoc-Val-MeDap(Boc)-OH

[0761] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeDap(Boc)-OH (0.052 g, 0.24 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 mL, 0.48 mmol), and acetonitrile (1.0 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 36 hours (starting material: target analyte = 1:20 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeDap(Boc)-OH (0.107 g, yield 99%) as a white solid.

[0762] Synthetic Example 105: Synthesis of Fmoc-Val-MeGln(Trt)-OH

[0763] [Chemical Formula 80]

[0764]

[0765] Fmoc-Val-OH (0.33 g, 0.10 mmol) was mixed with tetrahydrofuran (0.5 mL), and triethylamine (0.017 mL, 0.12 mmol) and neopentanoyl chloride (0.014 mL, 0.11 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGln(Trt)-OH (0.048 g, 0.12 mmol), N,O-bis(trimethylsilyl)acetamide (0.062 mL, 0.24 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 30 minutes, and then at 25 °C for 24 hours (reactant:target substance = 1:8 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated to obtain Fmoc-Val-MeGln(Trt)-OH (0.75 g, yield 105%) as a light yellow solid.

[0766] Synthetic Example 106: Synthesis of Fmoc-Val-MeGln(Trt)-OH

[0767] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeGln(Trt)-OH (0.097 g, 0.24 mmol), N,O-bis(trimethylsilyl)acetamide (0.12 mL, 0.48 mmol), and acetonitrile (1.0 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 44 hours (starting material: target substance = 1:21 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGln(Trt)-OH (0.151 g, yield 104%) as a white solid.

[0768] Synthetic Example 107: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH

[0769] [Chemical Formula 81]

[0770]

[0771] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.050 mL, 0.36 mmol) and neopentanoyl chloride (0.041 mL, 0.33 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGlu(OtBu)-OH (0.098 g, 0.45 mmol), N,O-bis(trimethylsilyl)acetamide (0.23 mL, 0.90 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 30 minutes, and then at 25 °C for 18 hours (reactant:target substance = 1:18 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The obtained organic layer was concentrated to obtain Fmoc-Val-MeGlu(OtBu)-OH (0.16 g, 100% yield) as a light yellow solid.

[0772] Synthetic Example 108: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH

[0773] Fmoc-Val-OH (0.068 g, 0.20 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.033 mL, 0.24 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66 g, 0.22 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeGlu(OtBu)-OH (0.078 g, 0.36 mmol), N,O-bis(trimethylsilyl)acetamide (0.185 mL, 0.72 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 1 hour was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 24 hours (starting material: target substance = 1:25 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL), and saturated sodium bicarbonate aqueous solution (5 mL) was added. After stirring for 1 hour, the solution was washed successively with water (5 mL) and saturated sodium chloride aqueous solution (5 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGlu(OtBu)-OH (0.176 g, yield 109%) as a white solid.

[0774] Synthetic Example 109: Synthesis of Fmoc-Val-EtAla-OH

[0775] [Chemical Formula 82]

[0776]

[0777] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.11 g, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-EtAla-OH (0.041 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 22 hours (starting material: target substance = 1:19 (analytical condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (2.0 mL), and washed with hexane (2.0 mL). The acetonitrile solution was diluted with ethyl acetate (6.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (5.0 mL). The resulting organic layer was concentrated to obtain Fmoc-Val-EtAla-OH (0.13 g, yield 104%) as a pale yellow solid.

[0778] Synthetic Example 110: Synthesis of Fmoc-Ala-BnAla-OH

[0779] [Chemical Formula 83]

[0780]

[0781] Fmoc-Ala-OH (0.10 g, 0.32 mmol) was mixed with tetrahydrofuran (1.6 mL), and triethylamine (0.054 mL, 0.39 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.13 g, 0.35 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-BnAla-OH (0.069 g, 0.39 mmol), N,O-bis(trimethylsilyl)acetamide (0.19 mL, 0.77 mmol), and acetonitrile (1.6 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 12 hours (starting material: target analyte = 1:1 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (8.0 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (6.0 mL), water (6.0 mL), and saturated sodium chloride aqueous solution (6.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Ala-BnAla-OH (0.13 g, yield 82%) as a white solid.

[0782] Synthetic Example 111: Synthesis of Fmoc-Ala-BnAla-OH

[0783] [Chemical Formula 84]

[0784]

[0785] Fmoc-Ala-OH (0.10 g, 0.32 mmol) was mixed with tetrahydrofuran (1.6 mL), and triethylamine (0.054 mL, 0.39 mmol) and neopentanoyl chloride (0.44 mL, 0.35 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-BnAla-OH (0.069 g, 0.39 mmol), N,O-bis(trimethylsilyl)acetamide (0.19 mL, 0.77 mmol), and acetonitrile (1.6 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 12 hours (reactant:target substance = 3:1 (analytical condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (8.0 mL), and washed successively with saturated sodium bicarbonate aqueous solution (6.0 mL), water (6.0 mL), and saturated sodium chloride aqueous solution (6.0 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Ala-BnAla-OH (0.12 g, 80%) as a white solid.

[0786] Synthetic Example 112: Synthesis of Fmoc-Gly-EtAla-Phe-OH

[0787] [Chemical Formula 85]

[0788]

[0789] Fmoc-Gly-OH (0.020 g, 0.067 mmol) was mixed with tetrahydrofuran (0.42 mL), and triethylamine (0.011 mL, 0.080 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.026 g, 0.074 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-EtAla-Phe-OH (0.022 g, 0.080 mmol), N,O-bis(trimethylsilyl)acetamide (0.039 mL, 0.16 mmol), and acetonitrile (0.84 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 6 hours (starting material: target substance = 1:33 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (5.0 mL), water (5.0 mL), and saturated sodium chloride aqueous solution (5.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.040 g, yield 109%) as a white solid.

[0790] Synthetic Example 113: Synthesis of Fmoc-Gly-EtAla-Phe-OH

[0791] [Chemical Formula 86]

[0792]

[0793] Fmoc-Gly-OH (0.025 g, 0.084 mmol) was mixed with tetrahydrofuran (0.42 mL), and triethylamine (0.014 mL, 0.10 mmol) and neopentanoyl chloride (0.011 mL, 0.092 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-EtAla-Phe-OH (0.027 g, 0.10 mmol), N,O-bis(trimethylsilyl)acetamide (0.049 mL, 0.20 mmol), and acetonitrile (0.42 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 6 hours (reactant:target substance = 1:9 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (5.0 mL), water (5.0 mL), and saturated sodium chloride aqueous solution (5.0 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.050 g, yield 109%) as a white solid.

[0794] Synthetic Example 114: Synthesis of Fmoc-Val-cHexmGly-OH

[0795] [Chemical Formula 87]

[0796]

[0797] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.098 g, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing N-(cyclohexylmethyl)glycine (H-cHexmGly-OH) (0.061 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 15 hours (starting material: target substance = 1:32 (analytical condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (6.0 mL), and washed with hexane (11 mL). The acetonitrile solution was diluted with ethyl acetate (10 mL) and washed successively with saturated sodium bicarbonate aqueous solution (10 mL), 1M hydrochloric acid (10 mL), water (10 mL), and saturated sodium chloride aqueous solution (12 mL). The resulting organic layer was concentrated to obtain Fmoc-Val-cHexmGly-OH (0.15 g, yield 104%) as a white solid.

[0798] Synthetic Example 115: Synthesis of Fmoc-Val-cHexmGly-OH

[0799] [Chemical Formula 88]

[0800]

[0801] Fmoc-Val-OH (0.030 g, 0.088 mmol) was mixed with tetrahydrofuran (0.44 mL), and triethylamine (0.015 mL, 0.11 mmol) and neopentanoyl chloride (0.012 mL, 0.097 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-cHexmGly-OH (0.018 g, 0.11 mmol), N,O-bis(trimethylsilyl)acetamide (0.052 mL, 0.21 mmol), and acetonitrile (0.44 mL) and stirring at 50 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 15 hours (starting material: target analyte = 1:3 (analytical condition 3)). The resulting reaction solution was concentrated and diluted with ethyl acetate (5.0 mL), and then washed successively with saturated sodium bicarbonate aqueous solution (5.0 mL), 1M hydrochloric acid (5.0 mL), water (6.0 mL), and saturated sodium chloride aqueous solution (6.0 mL). The resulting organic layer was concentrated to obtain Fmoc-Val-cHexmGly-OH (0.048 g, yield 109%) as a white solid.

[0802] Synthetic Example 116: Synthesis of Fmoc-Val-Tic-OH

[0803] [Chemical Formula 89]

[0804]

[0805] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2,2-dimethylbutyryl chloride (0.045 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (H-Tic-OH) (0.063 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 4 hours (starting material: target substance = 1:99 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 10% citric acid aqueous solution (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The collected organic layer was concentrated to obtain Fmoc-Val-Tic-OH (0.15 g, 100% yield) as a white solid.

[0806] Synthetic Example 117: Synthesis of Fmoc-Val-Tic-OH

[0807] [Chemical Formula 90]

[0808]

[0809] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-Tic-OH (0.063 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 4 hours (reactant:target substance = 1:32 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 10% citric acid aqueous solution (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The collected organic layer was concentrated to obtain Fmoc-Val-Tic-OH (0.14 g, yield 97%) as a white solid.

[0810] Synthetic Example 118: Synthesis of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide

[0811] [Chemical Formula 91]

[0812]

[0813] 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoic acid (3.0 g, 10.6 mmol) was mixed with phosphorus tribromide (0.94 g, 3.48 mmol) and stirred at 50 °C for 24 hours. The resulting mixture was diluted with hexane (30 mL) and washed with water (20 mL). After concentrating the organic layer, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide (3.7 g, 10.6 mmol) was obtained as a colorless liquid.

[0814] Synthetic Example 119: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH

[0815] [Chemical Formula 92]

[0816]

[0817] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide (0.13 g, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGlu(OtBu)-OH (0.077 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 50 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 20 hours (starting material: target substance = < 1:99 (analytical condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (8.0 mL), and washed twice with hexane (8.0 mL). The acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (7.0 mL), 1M hydrochloric acid (7.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was concentrated to give Fmoc-Val-MeGlu(OtBu)-OH (0.16 g, yield 98%) as a white solid.

[0818] Synthetic Example 120: Synthesis of Fmoc-Val-(Me)βAla-OH

[0819] [Chemical Formula 93]

[0820]

[0821] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.058 mL, 0.41 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 g, 0.38 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.058 g, 0.41 mmol), N,O-bis(trimethylsilyl)acetamide (0.40 mL, 1.6 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 7 hours (starting material: target substance = 1:24 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) was added, and the mixture was stirred at 25 °C for 1 hour. The obtained organic layer was washed sequentially with 10% citric acid aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-OH (0.125 g, yield 94%) as a white solid.

[0822] Synthetic Example 121: Synthesis of Fmoc-Val-(Me)βAla-OH

[0823] [Chemical Formula 94]

[0824]

[0825] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.049 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.35 mL, 1.4 mmol), and acetonitrile (1.4 mL) and stirring at 25 °C for 60 minutes was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 1 hour (reactant:target substance = 1:1 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (3.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) was added. The mixture was stirred at 25 °C for 1 hour. The obtained organic layer was washed sequentially with 10% citric acid aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-OH (0.13 g, yield 102%) as a white solid.

[0826] Synthetic Example 122: Synthesis of Fmoc-Val-MeGABA-OH

[0827] [Chemical Formula 95]

[0828]

[0829] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.058 mL, 0.41 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 mL, 0.38 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-MeGABA-OH hydrochloride (0.063 g, 0.41 mmol), N,O-bis(trimethylsilyl)acetamide (0.38 mL, 1.5 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 7 hours, followed by stirring at 10 °C for 12 hours (starting material: target substance = 1:19 (analytical condition 3)). The reaction solution was diluted with ethyl acetate (4.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) was added. The mixture was stirred at 25 °C for 1 hour. The resulting organic layer was washed successively with 10% citric acid aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-OH (0.135 g, yield 104%) as a white solid.

[0830] Synthetic Example 123: Synthesis of Fmoc-Val-MeGABA-OH

[0831] [Chemical Formula 96]

[0832]

[0833] Fmoc-Val-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGABA-OH hydrochloride (0.054 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.35 mL, 1.4 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added. The mixture was further stirred at 0 °C for 1 hour, and then at 25 °C for 1 hour (reactant:target substance = 1:4 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) was added. The mixture was stirred at 25 °C for 1 hour. The obtained organic layer was washed successively with 10% citric acid aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-OH (0.125 g, yield 97%) as a white solid.

[0834] Synthetic Example 124: Synthesis of Fmoc-Val-(Me)βAla-MePhe-OH

[0835] [Chemical Formula 97]

[0836]

[0837] Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 g, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-MePhe-OH (0.093 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was stirred at 0 °C for 2 hours (starting material: target substance = 1:48 (analytical condition 3)). The reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-MePhe-OH (0.18 g, yield 107%) as a white solid.

[0838] Synthetic Example 125: Synthesis of Fmoc-Val-(Me)βAla-MePhe-OH

[0839] [Chemical Formula 98]

[0840]

[0841] Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-MePhe-OH (0.093 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was stirred at 0 °C for 2 hours (reactant:target substance = 1:24 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-MePhe-OH (0.19 g, yield 109%) as a white solid.

[0842] Synthetic Example 126: Synthesis of Fmoc-Val-MeGABA-MePhe-OH

[0843] [Chemical Formula 99]

[0844]

[0845] Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 g, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGABA-MePhe-OH (0.098 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was stirred at 0 °C for 2 hours (starting material: target analyte = 1:16 (analytical condition 3)). The reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-MePhe-OH (0.18 g, yield 107%) as a white solid.

[0846] Synthetic Example 127: Synthesis of Fmoc-Val-MeGABA-MePhe-OH

[0847] [Chemical Formula 100]

[0848]

[0849] Fmoc-Val-OH (0.100 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MeGABA-MePhe-OH (0.098 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was stirred at 0 °C for 2 hours (reactant:target substance = 1:3 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-MePhe-OH (0.19 g, yield 108%) as a white solid.

[0850] Synthetic Example 128: Synthesis of Fmoc-Val-(Me)βhomoTrp(1-Me)-OH

[0851] [Chemical Formula 101]

[0852]

[0853] Fmoc-Val-OH (0.030 g, 0.11 mmol) was mixed with tetrahydrofuran (0.44 mL), and triethylamine (0.015 mL, 0.11 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.035 g, 0.097 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-(Me)βhomoTrp(1-Me)-OH hydrochloride (0.030 g, 0.11 mmol), N,O-bis(trimethylsilyl)acetamide (0.10 mL, 0.42 mmol), and acetonitrile (0.44 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was stirred at 25 °C for 15 hours (starting material: target substance = 1:24 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βhomoTrp(1-Me)-OH (0.056 g, yield 111%) as a white solid.

[0854] Synthetic Example 129: Synthesis of Fmoc-Val-(Me)βhomoTrp(1-Me)-OH

[0855] [Chemical Formula 102]

[0856]

[0857] Fmoc-Val-OH (0.070 g, 0.21 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.035 mL, 0.11 mmol) and neopentanoyl chloride (0.028 mL, 0.23 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βhomoTrp(1-Me)-OH hydrochloride (0.070 g, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (0.24 mL, 0.99 mmol), and acetonitrile (1.0 mL) and stirring at 25 °C for 60 minutes was added. After stirring at 0 °C for 15 hours, the mixture was further stirred at 25 °C for 4 hours (starting material: target substance = 1:8 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) was added. The mixture was stirred at 25 °C for 1 hour. The obtained organic layer was washed sequentially with 10% citric acid aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βhomoTrp(1-Me)-OH (0.090 g, yield 77%) as a white solid.

[0858] Synthetic Example 130: Synthesis of Fmoc-Val-βhomoTrp(1-Me)-OH

[0859] [Chemical Formula 103]

[0860]

[0861] Fmoc-Val-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.74 mL), and triethylamine (0.025 mL, 0.18 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.049 g, 0.16 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-βhomoTrp(1-Me)-OH hydrochloride (0.048 g, 0.18 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (0.74 mL) and stirring at 50 °C for 60 minutes was added, and the mixture was stirred at 25 °C for 15 hours (starting material: target analyte = 1:99 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-βhomoTrp(1-Me)-OH (0.11 g, yield 130%) as a white solid.

[0862] Synthetic Example 131: Synthesis of Fmoc-Val-βhomoTrp(1-Me)-OH

[0863] [Chemical Formula 104]

[0864]

[0865] Fmoc-Val-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.74 mL), and triethylamine (0.025 mL, 0.18 mmol) and neopentanoyl chloride (0.020 g, 0.16 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-βhomoTrp(1-Me)-OH hydrochloride (0.048 g, 0.18 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (0.74 mL) and stirring at 50 °C for 60 minutes was added, and the mixture was stirred at 25 °C for 15 hours (reactant:target substance = 1:19 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The obtained organic layer was concentrated and washed with hexane to obtain Fmoc-Val-βhomoTrp(1-Me)-OH (0.079 g, 97% yield) as a white solid.

[0866] Synthetic Example 132: Synthesis of Fmoc-Val-(Me)βhomoLeu-OH

[0867] [Chemical Formula 105]

[0868]

[0869] Fmoc-Val-OH (0.030 g, 0.088 mmol) was mixed with tetrahydrofuran (0.44 mL), and triethylamine (0.015 mL, 0.11 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.035 g, 0.097 mmol) were added at 0 °C, and the mixture was stirred for 2 hours. To this solution, a solution prepared by separately mixing H-(Me)βhomoLeu-OH hydrochloride (0.021 g, 0.11 mmol), N,O-bis(trimethylsilyl)acetamide (0.10 mL, 0.42 mmol), and acetonitrile (0.44 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 15 hours (starting material: target substance = 1:24 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (4.0 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The resulting organic layer was concentrated and washed with a mixed solution of hexane and 10% isopropyl ether / hexane to obtain Fmoc-Val-(Me)βhomoLeu-OH (0.045 g, yield 106%) as a white solid.

[0870] Synthetic Example 133: Synthesis of Fmoc-Val-(Me)βhomoLeu-OH

[0871] [Chemical Formula 106]

[0872]

[0873] Fmoc-Val-OH (0.070 g, 0.21 mmol) was mixed with tetrahydrofuran (1.0 mL), and triethylamine (0.035 mL, 0.25 mmol) and neopentanoyl chloride (0.028 mL, 0.23 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βhomoLeu-OH hydrochloride (0.048 g, 0.25 mmol), N,O-bis(trimethylsilyl)acetamide (0.24 mL, 0.99 mmol), and acetonitrile (1.0 mL) and stirring at 25 °C for 60 minutes was added. The mixture was stirred at 0 °C for 15 hours, and then further stirred at 25 °C for 2 hours (starting material: target substance = 1:5 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), and saturated sodium bicarbonate aqueous solution (4.0 mL) and water (3.0 mL) were added. The mixture was stirred at 25 °C for 1 hour. The obtained organic layer was washed sequentially with 10% citric acid aqueous solution (4 mL), water (3.0 mL), and saturated sodium chloride aqueous solution (3.0 mL). The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βhomoLeu-OH (0.095 g, yield 96%) as a white solid.

[0874] Synthetic Example 134: Synthesis of Fmoc-(Me)βAla-MePhe-OH

[0875] [Chemical Formula 107]

[0876]

[0877] Fmoc-(Me)βAla-OH (1.0 g, 3.1 mmol) was mixed with tetrahydrofuran (15 mL), and triethylamine (0.51 mL, 3.7 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (1.2 g, 3.4 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.66 g, 3.7 mmol), N,O-bis(trimethylsilyl)acetamide (1.8 mL, 7.4 mmol), and acetonitrile (15 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 3 hours (starting material: target substance = 1:48 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with water (15 mL) and saturated sodium chloride aqueous solution (15 mL). The collected organic layer was concentrated and purified by column chromatography to obtain Fmoc-(Me)βAla-MePhe-OH (1.3 g, yield 85%) as a white solid.

[0878] Synthetic Example 135: Synthesis of Fmoc-(Me)βAla-MePhe-OH

[0879] [Chemical Formula 108]

[0880]

[0881] Fmoc-(Me)βAla-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.051 mL, 0.37 mmol) and neopentanoyl chloride (0.041 mL, 0.34 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.066 g, 0.37 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 mL, 0.74 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 6 hours (reactant:target substance = 1:33 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 1M hydrochloric acid (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The collected organic layer was concentrated to obtain Fmoc-(Me)βAla-MePhe-OH (0.16 g, yield 107%) as a white solid.

[0882] Synthetic Example 136: Synthesis of Fmoc-MeGABA-MePhe-OH

[0883] [Chemical Formula 109]

[0884]

[0885] Fmoc-MeGABA-OH (1.0 g, 3.0 mmol) was mixed with tetrahydrofuran (15 mL), and triethylamine (0.50 mL, 3.5 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (1.2 g, 3.2 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.63 g, 3.5 mmol), N,O-bis(trimethylsilyl)acetamide (1.7 mL, 7.1 mmol), and acetonitrile (15 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 3 hours (starting material: target substance = 1:48 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (40 mL) and washed successively with water (15 mL) and saturated sodium chloride aqueous solution (15 mL). The collected organic layer was concentrated and purified by column chromatography to obtain Fmoc-MeGABA-MePhe-OH (1.3 g, yield 88%) as a white solid.

[0886] Synthetic Example 137: Synthesis of Fmoc-MeGABA-MePhe-OH

[0887] [Chemical Formula 110]

[0888]

[0889] Fmoc-MeGABA-OH (0.10 g, 0.30 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.049 mL, 0.35 mmol) and neopentanoyl chloride (0.040 mL, 0.32 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-MePhe-OH (0.063 g, 0.35 mmol), N,O-bis(trimethylsilyl)acetamide (0.17 mL, 0.71 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 6 hours (reactant:target substance = 1:12 (analytical condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 1M hydrochloric acid (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The collected organic layer was concentrated to obtain Fmoc-MeGABA-MePhe-OH (0.15 g, yield 105%) as a white solid.

[0890] Synthetic Example 138: Synthesis of Fmoc-(2SMe)βAla-(Me)βAla-OH

[0891] [Chemical Formula 111]

[0892]

[0893] Fmoc-(2SMe)βAla-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.42 mL), and triethylamine (0.026 mL, 0.18 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.051 g, 0.17 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.026 g, 0.18 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 mL, 0.74 mmol), and acetonitrile (0.84 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 2 hours (starting material: target analyte = 1:7 (analytical condition 3)). The resulting reaction solution was diluted with acetonitrile (6.0 mL) and washed with hexane (8.0 mL). The resulting acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 10% citric acid aqueous solution (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The resulting organic layer was concentrated to obtain Fmoc-(2SMe)βAla-(Me)βAla-OH (0.069 g, yield 109%) as a colorless oil.

[0894] Synthetic Example 139: Synthesis of Fmoc-(2SMe)βAla-(Me)βAla-OH

[0895] [Chemical Formula 112]

[0896]

[0897] Fmoc-(2SMe)βAla-OH (0.050 g, 0.15 mmol) was mixed with tetrahydrofuran (0.42 mL), and triethylamine (0.026 mL, 0.18 mmol) and neopentanoyl chloride (0.021 mL, 0.17 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.026 g, 0.18 mmol), N,O-bis(trimethylsilyl)acetamide (0.18 mL, 0.74 mmol), and acetonitrile (0.84 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 25 °C for 2 hours (starting material: target substance = 1:3 (analytical condition 3)). The resulting reaction solution was diluted with acetonitrile (6.0 mL) and washed with hexane (8.0 mL). The obtained acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated sodium bicarbonate aqueous solution (8.0 mL), 10% citric acid aqueous solution (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was concentrated to obtain Fmoc-(2SMe)βAla-(Me)βAla-OH (0.070 g, yield 110%) as a colorless oil.

[0898] Synthetic Example 140: Synthesis of Fmoc-GABA-(Me)βAla-OH

[0899] [Chemical Formula 113]

[0900]

[0901] Fmoc-GABA-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.051 mL, 0.37 mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 g, 0.34 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.052 g, 0.37 mmol), N,O-bis(trimethylsilyl)acetamide (0.36 mL, 1.5 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 15 hours (starting material: target substance = 1:99 (analytical condition 3)). The resulting reaction solution was diluted with acetonitrile (6.0 mL) and washed with hexane (10 mL). The obtained acetonitrile solution was diluted with ethyl acetate (10 mL) and washed successively with 1 M hydrochloric acid (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was concentrated to obtain Fmoc-GABA-(Me)βAla-OH (0.14 g, yield 108%) as a colorless oil.

[0902] Synthetic Example 141: Synthesis of Fmoc-GABA-(Me)βAla-OH

[0903] [Chemical Formula 114]

[0904]

[0905] Fmoc-GABA-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.051 mL, 0.37 mmol) and neopentanoyl chloride (0.042 mL, 0.34 mmol) were added at 0 °C, and the mixture was stirred for 1 hour. To this solution, a solution prepared by separately mixing H-(Me)βAla-OH hydrochloride (0.052 g, 0.37 mmol), N,O-bis(trimethylsilyl)acetamide (0.36 mL, 1.5 mmol), and acetonitrile (1.5 mL) and stirring at 25 °C for 60 minutes was added, and the mixture was further stirred at 0 °C for 15 hours (starting material: target substance = 1:49 (analytical condition 3)). The resulting reaction solution was diluted with acetonitrile (8.0 mL) and washed with hexane (15 mL). The obtained acetonitrile solution was diluted with ethyl acetate (10 mL) and washed successively with 1 M hydrochloric acid (8.0 mL), water (10 mL), and saturated sodium chloride aqueous solution (10 mL). The obtained organic layer was concentrated to obtain Fmoc-GABA-(Me)βAla-OH (0.13 g, yield 104%) as a colorless oil.

[0906] In the above synthesis examples, synthesis example 118 is a reference example related to the synthesis of the raw materials used in the examples. Additionally, synthesis examples 98, 100, 102, 104, 106, 108 to 110, 112, 114, 116, 119, 120, 122, 124, 126, 128, 132, 134, 136, 138, and 140 are embodiments of the invention related to the peptide manufacturing method of this application; synthesis examples 97, 99, 101, 103, 105, 107, 111, 113, 115, 117, 121, 123, 125, 127, 129, 133, 135, 137, 139, and 141 are comparative examples; and synthesis examples 130 and 131 are reference examples.

Claims

1. A method for manufacturing peptides, comprising the following steps (1) to (3). Step (1) involves mixing the N-terminal protected amino acid or N-terminal protected peptide represented by formula (I) with a carboxylic acid halide. Formula (I): PA 1 -OH In formula (I), P is the N-terminal protecting group, and A 1 Indicates groups derived from amino acids, derived from NC 1-6 The alkyl amino acid group or the group derived from the peptide, wherein, C 1-6 The alkyl group may optionally have substituents. The carboxylic acid halide is selected from the group consisting of: ； Step (2) involves mixing the amino acid or peptide represented by formula (IV) with a silylating agent, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide. Formula (IV): HA 2 -OH In equation (IV), A 2 express: From NC 1-6 The alkyl amino acid group, wherein, C 1-6 The alkyl group may optionally have substituents, or A group derived from a 4-6 membered cyclic secondary amino acid, wherein the 4-6 membered ring is optionally associated with a C-membered ring. 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 Cyclic compounds in the group consisting of cycloalkyl rings are fused together, or A group derived from a peptide, wherein the N-terminal residue is NC. 1-6 Alkyl amino acids or 4-6 cyclic secondary amino acids, wherein C 1-6 The alkyl group optionally has substituents, and the 4-6 membered ring optionally has a C-shaped ring. 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 Cyclic compounds in the group consisting of cycloalkyl rings are fused together; Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

2. A method for manufacturing peptides, comprising the following steps (1) to (3). Step (1) involves mixing the N-terminal protected amino acid represented by formula (I) with a carboxylic acid halide. Formula (I): PA 1 -OH In formula (I), P is the N-terminal protecting group, and A 1 Indicates a group derived from an amino acid or from NC. 1-6 The alkyl amino acid group, wherein, C 1-6 The alkyl group may optionally have substituents. The carboxylic acid halide is selected from the group consisting of: ； Step (2) involves mixing the amino acid or peptide represented by formula (IV) with a silylating agent, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide. Formula (IV): HA 2 -OH In equation (IV), A 2 express: Groups derived from N-methyl amino acids, From NC 1-6 The alkylglycine group, wherein C 1-6 The alkyl group may optionally have substituents, or Derived from a cyclic secondary amino acid group of 4-6 members, or, A group derived from a peptide, wherein the N-terminal residue is an N-methyl amino acid, NC 1-6 Alkylglycine, or a 4-6 membered cyclic secondary amino acid, wherein C 1-6 The alkyl group may optionally have substituents; Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

3. A method for manufacturing peptides, comprising the following steps (1) to (3). Step (1) involves mixing the N-terminal protective peptide represented by formula (V) with a carboxylic acid halide. Formula (V): PA 3 -OH In formula (V), P is the N-terminal protecting group, and A 3 Indicates a group derived from a peptide. The carboxylic acid halide is selected from the group consisting of: ； Step (2) involves mixing the amino acid represented by formula (IV') with a silylating agent, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide. Formula (IV'): HA 2’ -OH In formula (IV'), A 2’ Indicates a group derived from N-methyl amino acids, or from NC 1-6 The group is an alkylglycine group, or a group derived from a 4-6 membered cyclic secondary amino acid, wherein... C 1-6 The alkyl group may optionally have substituents; Step (3) involves mixing the product obtained in step (1) with the product obtained in step (2).

4. A method for manufacturing a peptide according to any one of claims 1 to 3, comprising a step of removing the N-terminal protecting group of the peptide obtained in step (3).

5. The method for manufacturing a peptide as described in any one of claims 1 to 3, further comprising repeating the following steps (4) and (5) more than once. Step (4) removes the N-terminal protecting group of the peptide obtained in step (3) or (5); Step (5) involves reacting the N-terminal protected amino acid or N-terminal protected peptide with the N-terminus of the peptide obtained in step (4).

6. The method for manufacturing the peptide as described in claim 1 or 3, wherein, Located in formula (I)PA 1 -OH or formula (V)PA 3 The -OH group represents the C-terminal amino acid in the N-terminal protective peptide, excluding N and C. 1-6 Amino acids other than alkyl amino acids or 4-6 cyclic secondary amino acids, wherein C 1-6 The alkyl group optionally has substituents, and the 4-6 membered ring optionally has a C-shaped ring. 6-14 Aromatic rings, C 6-14 Halogenated aromatic rings and C 3-8 Cyclic compounds in the group consisting of cycloalkyl rings fuse together. In equations (I) and (V), P is an N-terminal protecting group, and A 1 and A 3 Each represents a group derived from the peptide.

7. The method for manufacturing the peptide as described in claim 1 or 2, wherein, A 1 It is a group derived from amino acids.

8. The method for manufacturing the peptide as described in claim 1 or 2, wherein, The N-terminal protective amino acid represented by formula (I) or the C-terminal amino acid in the N-terminal protective peptide represented by formula (I) is an α-amino acid, β-amino acid or γ-amino acid.

9. The method for manufacturing the peptide as described in claim 8, wherein, The N-terminal protective amino acid represented by formula (I) or the C-terminal amino acid in the N-terminal protective peptide represented by formula (I) is an α-amino acid.

10. The method for manufacturing the peptide as described in claim 1, wherein, The amino acid represented by formula (IV) or the amino acid located at the N-terminus of the peptide represented by formula (IV) is NC. 1-6 Alkyl-α-amino acids or 4-6 cyclic secondary-α-amino acids, wherein C 1-6 The alkyl group may optionally have substituents.

11. The method for manufacturing the peptide as described in claim 1, wherein, The amino acid represented by formula (IV) or the amino acid at the N-terminus of the peptide represented by formula (IV) is an N-methyl-α-amino acid or an N-ethyl-α-amino acid or a 4-6 membered cyclic secondary-α-amino acid, wherein the N-methyl and N-ethyl members optionally have substituents.

12. The method for manufacturing the peptide according to any one of claims 1 to 3, wherein, Carboxylic acid halides are the following compounds: 。 13. The method for manufacturing the peptide as described in claim 1, wherein, The amino acids or peptides represented by formula (IV) are amino acids other than proline or peptides with an N-terminal residue other than proline.

14. The method for manufacturing the peptide as described in claim 3, wherein, The amino acid represented by formula (IV') is an amino acid other than proline.

15. Uses of compounds represented by the following formula as activators of carboxylic acids: 。 16. Uses of compounds represented by the following formula as activators of carboxylic acids: 。

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