Method for producing peptide compounds

Abstract

The present invention provides a method for producing a peptide containing an N-alkyl amino acid, the method comprising the following steps (1) to (3). Step (1): Mix an N-terminus protected amino acid or an N-terminus protected peptide with a carboxylic acid halide or a halogenated alkyl formate; step (2): mix an amino acid or peptide, in which the N-terminus and C-terminus are not protected, with a trialkyl silylating agent; and step (3): mix the product obtained in step (1) with the product obtained in step (2).

Description

[Technical field]

[0001] The present invention relates to a method for producing a peptide having an N-alkylamino acid. [Background technology]

[0002] In recent years, expectations are growing for non-standard peptides with relatively low molecular weights (1,000-3,000) and containing non-natural amino acids as next-generation drugs to replace protein preparations. Examples of non-standard peptides include peptides containing N-alkylamino acids other than normal amino acids. Compared with normal peptides, peptides containing N-alkylamino acids have characteristics such as low structural freedom, strong target binding, membrane permeability, low immunogenicity, and stability in vivo. Therefore, there is a strong demand for the development of an efficient method for producing peptides containing N-alkylamino acids (see, for example, Non-Patent Document 1).

[0003] Methods for producing peptides containing N-alkylamino acids are known, for example, as follows. (1) C-terminally protected peptide product A method in which the C-terminus of an N-terminal protected amino acid is activated with pivaloyl chloride and reacted with a benzyl ester of an N-methylamino acid (see, for example, Patent Document 1). (2) The product is a peptide with an unprotected C-terminus A method in which the C-terminus of an N-terminal protected amino acid is activated with isobutyl chloroformate and reacted with silylated N-methylglycine (sarcosine) or proline (see, for example, Non-Patent Document 2). A method in which the C-terminus of an N-terminal protected peptide is activated with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and then reacted with N-methylalanine (see, for example, Patent Document 2).

[0004] In addition, a method for producing a peptide containing consecutive N-methylamino acids is known in which the C-terminus of N-Boc-N-methylleucine is activated with pivaloyl chloride and then reacted with N-methylphenylalanine methyl ester (see, for example, Non-Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 5,739,104 [Patent Document 2] International Publication No. 2009 / 134405 [Non-patent literature]

[0006] [Non-Patent Document 1] Pharmacia 2014, vol. 50, pp. 751-755 [Non-Patent Document 2] Indian Journal of Chemistry 2004, Vol. 43B, p. 1282 [Non-Patent Document 3] Tetrahedron 2012, Vol. 68, p. 7070 Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors have confirmed that when an amino acid having an alkyl group at the N-terminus is introduced to the C-terminus of an amino acid whose N-terminus is protected using the method described in Non-Patent Document 2, the reaction does not necessarily proceed with a sufficient conversion rate depending on the type of amino acid to be introduced, and in particular, in the case of acyclic N-alkylamino acids such as N-methylglycine and peptides, a large amount of raw material remains, making it impossible to obtain the target product with a satisfactory yield. The remaining raw material causes the production of peptides lacking some amino acids, and since the physical properties are similar to those of the target peptide, it is difficult to remove them, which is a quality issue. In addition, the method described in Patent Document 2 uses HATU containing an explosive triazole structure as a condensing agent, and is not necessarily applicable to industrial peptide production. On the other hand, in the methods described in Patent Document 1 and Non-Patent Document 3, the C-terminus of the resulting peptide is protected, and a deprotection step is further required to obtain a peptide with an unprotected C-terminus. Therefore, since a deprotection step is necessarily required in addition to the condensation step, the methods cannot be applied as efficient methods for producing peptides.

[0008] The present invention provides a method for producing a peptide containing an N-alkylamino acid and having an unprotected C-terminus.The present invention also provides a method for producing a peptide using an amino acid or peptide containing an N-alkyl group and having unprotected N- and C-terminuses, and an N-terminal protected amino acid or peptide as materials. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have found that the above-mentioned problems can be solved by mixing an unprotected amino acid or peptide containing an N-alkyl group and having neither N-terminal nor C-terminal protected with a silylating agent, and further mixing the N-terminal protected amino acid or peptide with a carboxylic acid activating agent having a specific structure, and have thus completed the present invention. That is, the present invention is characterized as follows.

[0010] [1] The following steps (1) to (3): (1) Formula (I)1 -OH (wherein P is an N-terminal protecting group, A 1 is a group derived from an amino acid, NC 1-6 Alkylamino acid derived groups (C 1-6 wherein alkyl may have a substituent) or represents a group derived from a peptide, Formula (II) [ka] (In the formula, X represents a halogen atom, R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and which may have a substituent, or represents a primary aliphatic hydrocarbon group having 4 or more carbon atoms and which has a substituent (wherein the substituent of the primary aliphatic hydrocarbon group is present on the carbon atom bonded to the carbonyl carbon), and [ka] (In the formula, X represents a halogen atom, R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms which may have a substituent; (2) Formula (IV)HA 2 -OH(in the formula, A 2 NC 1-6 Alkylamino acid derived groups (C 1-6 alkyl may have a substituent), or a group derived from a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 cycloalkyl rings), or the N-terminal residue is NC 1-6 Alkylamino acids (C 1-6 alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14Aryl ring, C 6-14 Haloaryl rings and C 3-8 a step of mixing an amino acid or peptide represented by the formula (I) (which may be condensed with a cyclic compound selected from the group consisting of cycloalkyl rings) with a silylating agent; (3) mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

[0011] [2] The following steps (1) to (3): (1) Formula (I) 1 -OH (wherein P is an N-terminal protecting group, A 1 is a group derived from an amino acid or NC 1-6 Alkylamino acid derived groups (C 1-6 wherein the alkyl group may have a substituent. Formula (II) [ka] (In the formula, X represents a halogen atom, R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and which may have a substituent, or represents a primary aliphatic hydrocarbon group having 4 or more carbon atoms and which has a substituent (wherein the substituent of the primary aliphatic hydrocarbon group is present on the carbon atom bonded to the carbonyl carbon), and Formula (III) [ka] (In the formula, X represents a halogen atom, R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms which may have a substituent; (2) Formula (IV)HA 2 -OH(in the formula, A 2is the group derived from N-methylamino acids, NC 1-6 Alkylglycine derived groups (C 1-6 The alkyl may be substituted), or a group derived from a 4-6 membered cyclic secondary amino acid, or the N-terminal residue is an N-methyl amino acid, NC 1-6 Alkylglycine (C 1-6 a step of mixing an amino acid or peptide represented by the formula (I) (wherein the alkyl group may have a substituent) or a group derived from a peptide which is a 4- to 6-membered cyclic secondary amino acid) with a silylating agent; (3) mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

[0012] [3] The following steps (1) to (3): (1) Formula (V)PA 3 -OH (wherein P is an N-terminal protecting group, A 3 represents a group derived from a peptide) Formula (II) [ka] (In the formula, X represents a halogen atom, R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and which may have a substituent, or represents a primary aliphatic hydrocarbon group having 4 or more carbon atoms and which has a substituent (wherein the substituent of the primary aliphatic hydrocarbon group is present on the carbon atom bonded to the carbonyl carbon); (2) Formula (IV')HA 2’ -OH(in the formula, A 2’ is the group derived from N-methylamino acids, NC 1-6 Alkylglycine derived groups (C 1-6 a step of mixing an amino acid represented by the formula (I) (wherein the alkyl group may have a substituent) or a group derived from a 4- to 6-membered cyclic secondary amino acid) with a silylating agent; (3) mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

[0013] [4] A method for producing the peptide according to any one of [1] to [3] above, which comprises a step of removing the protecting group at the N-terminus of the peptide obtained in step (3).

[0014] [5] Further comprising the steps (4) and (5): (4) a step of removing the protecting group at the N-terminus of the peptide obtained in step (3) or (5); (5) a step of reacting an N-terminal protected amino acid or an N-terminal protected peptide with the N-terminus of the peptide obtained in the step (4); The method for producing the peptide according to any one of [1] to [3] above, comprising one or more repeats of the formula:

[0015] [6] Formula (I)PA 1 -OH or formula (V)PA 3 -OH (wherein P is an N-terminal protecting group, A 1 and A 3 Each of the N-terminal protected peptides is represented by the formula (1), and the amino acid located at the C-terminus of the N-terminal protected peptide represented by the formula (1) is NC 1-6 Alkylamino acids (C 1-6 alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 The method for producing the peptide according to the above-mentioned [1] or [3], wherein the amino acid is an amino acid other than an amino acid which may be condensed with a cyclic compound selected from the group consisting of cycloalkyl rings.

[0016] [7] A 1 is a group derived from an amino acid.

[0017] [8] The method for producing a peptide according to the above [1] or [2], wherein the N-terminal protected amino acid represented by formula (I) or the amino acid located at the C-terminus of the N-terminal protected peptide represented by formula (I) is an α-amino acid, a β-amino acid or a γ-amino acid.

[0018] [9] The method for producing a peptide according to the above [8], wherein the N-terminal protected amino acid represented by formula (I) or the amino acid located at the C-terminus of the N-terminal protected peptide represented by formula (I) is an α-amino acid.

[0019]

[10] 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 The method for producing a peptide according to the above-mentioned [1], wherein the aryl group is a cyclic 4- to 6-membered secondary α-amino acid, wherein the alkyl group may have a substituent.

[0020]

[11] The method for producing the peptide according to the above [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 an N-methyl-α-amino acid or an N-ethyl-α-amino acid (N-methyl and N-ethyl may each have a substituent), or a 4- to 6-membered cyclic secondary-α-amino acid.

[0021]

[12] The activator is a carboxylic acid halide represented by formula (II), R 1 and X is a chlorine atom.

[0022]

[13] A method for producing a peptide according to any one of [1] to

[12] above, wherein the activating agent is a carboxylic acid halide represented by formula (II) and selected from the following group of compounds: [ka]

[0023]

[14] A method for producing a peptide according to any one of [1] to

[12] above, wherein the activating agent is a carboxylic acid halide represented by formula (II) and selected from the following group of compounds: [ka]

[0024]

[15] A method for producing the peptide according to

[13] or

[14] above, wherein the activator is the following compound: [ka]

[0025]

[16] The method for producing a peptide according to the above [1] or [2], wherein the activating agent is a halogenated alkyl formate represented by formula (III), and X is a chlorine atom:

[0026]

[17] The method for producing the peptide according to the above [1] or [2], wherein the activating agent is an alkyl formate halide represented by formula (III) and selected from the following compound group: [ka]

[0027]

[18] The method for producing the peptide according to the above [1] or [2], wherein the activating agent is an alkyl formate halide represented by formula (III) and selected from the following compound group: [ka]

[0028]

[19] The method for producing a peptide according to any one of [1] to

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

[0029]

[20] A method for producing a peptide according to 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.

[0030]

[21] The method for producing a peptide according to any one of [1] to

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

[0031]

[22] The method for producing the peptide according to the above [1], 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, respectively.

[0032]

[23] The method for producing the peptide according to the above [3], wherein the amino acid represented by the formula (IV') is an amino acid other than proline.

[0033]

[24] The following formula: [ka] A compound represented by the formula: Effect of the Invention

[0034] The present invention provides a novel method for producing a peptide with an unprotected C-terminus by using an amino acid or peptide containing an N-alkyl group and an N-terminally protected amino acid or peptide as materials. The production method of the present invention makes it possible to obtain a desired peptide in a satisfactory yield with fewer steps using industrially applicable reagents, regardless of the type of N-alkyl amino acid introduced, or the type of N-alkyl amino acid at the N-terminal residue of the peptide introduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention will be described in detail below. In this specification, "n-" means normal, "s-" means secondary, "t-" and "tert-" mean tertiary, "Me" means methyl, "Et" means ethyl, "Pr" means propyl, "Bu" means butyl, "Ph" means phenyl, "Bn" means benzyl, "Boc" means t-butoxycarbonyl, "Cbz" means benzyloxycarbonyl, "Fmoc" means 9-fluorenylmethoxycarbonyl, "Trt" means trityl, "TMS" means trimethylsilyl, and "TFA" means trifluoroacetic acid.

[0036] The term "halogen atom" refers to a fluorine atom, chlorine atom, bromine atom or iodine atom.

[0037] In the present invention, an "alkyl group" refers to a monovalent group of a linear or branched, saturated aliphatic hydrocarbon. 1-6 The term "alkyl group" means a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, and specific examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a s-butyl group, a t-butyl group, an n-pentyl group, a 3-pentyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, a 1-ethylpropyl group, an n-hexyl group, and a 3,3-dimethylbutan-2-yl group.

[0038] "2nd or 3rd grade C 5-40 The term "alkyl group" refers to a monovalent group having 5 to 40 carbon atoms and obtained by removing hydrogen on a secondary or tertiary carbon atom from a saturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom. Specific examples include 2-methylbutan-2-yl group, 3-methylbutan-2-yl group, 3,3-dimethylbutan-2-yl group, 3-pentyl group, 2,2,4-trimethylpentan-3-yl group, 2,4-dimethylpentan-3-yl group, 4-ethyl-2,2-dimethylhexan-3-yl group, 3-heptyl group, and 2,2,4,8,10,10-hexamethylundecan-5-yl group. 5-20The term "alkyl group" refers to a secondary or tertiary alkyl group having from 5 to 20 carbon atoms.

[0039] "1st grade C 4-40 The term "alkyl group" refers to a monovalent group obtained by removing hydrogen on a primary carbon atom from a linear or branched saturated aliphatic hydrocarbon having 4 to 40 carbon atoms, and examples of the primary alkyl groups include n-butyl, isobutyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-icosyl, n-triacontyl, n-tetracontyl, and isomers thereof. 4-20 The term "alkyl group" refers to a primary alkyl group having from 4 to 20 carbon atoms.

[0040] In the present invention, the term "alkenyl group" refers to a monovalent group of a straight-chain or branched unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond. The term "secondary or tertiary alkenyl group" refers to a monovalent group obtained by removing a hydrogen atom on a secondary or tertiary carbon atom from an unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom and at least one carbon-carbon double bond, and specific examples include an isopropenyl group and a 1-methyl-1-propenyl group. In addition, the term "secondary or tertiary C 5-40 An alkenyl group has 5 to 40 carbon atoms and is a secondary or tertiary C 5-20 The term "alkenyl group" refers to a secondary or tertiary alkenyl group having 5 to 20 carbon atoms.

[0041] "1st grade C 4-40 The term "alkenyl group" refers to a monovalent group formed by removing hydrogen on a primary carbon atom from an unsaturated aliphatic hydrocarbon having 4 to 40 carbon atoms, a straight or branched chain, and at least one carbon-carbon double bond, and examples of the group include a 2-butenyl group, a 3-butenyl group, and a 2-pentenyl group. 4-20 The term "alkenyl group" refers to a primary alkenyl group having 4 to 20 carbon atoms.

[0042] "C 6-14 The term "aryl group" refers to an aromatic hydrocarbon group having 6 to 14 carbon atoms, and specific examples thereof include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a 1-anthryl group, a 2-anthryl group, a 9-anthryl group, and a biphenyl group. 6-14 The term "aryl ring" refers to an aromatic hydrocarbon ring having 6 to 14 carbon atoms.

[0043] "C 6-14 The term "haloaryl group" refers to an aromatic hydrocarbon group having 6 to 14 carbon atoms substituted with one or more halogen atoms, and specific examples thereof include a 4-chlorophenyl group, a 2,4-dichlorophenyl group, a 5-fluoro-1-naphthyl group, a 6-bromo-2-naphthyl group, a 6,7-diiodo-1-anthryl group, a 10-bromo-9-anthryl group, and a 4'-chloro-(1,1'-biphenyl)-2-yl group. 6-14 The term "haloaryl ring" refers to an aromatic hydrocarbon ring having 6 to 14 carbon atoms substituted with one or more halogen atoms.

[0044] "C 6-14 The term "aryloxy group" means an aryloxy group having 6 to 14 carbon atoms, and specific examples include a phenoxy group, a 1-naphthyloxy group, a 2-naphthyloxy group, a 1-anthryloxy group, a 2-anthryoxy group, a 9-anthryoxy group, and a biphenyloxy group.

[0045] The term "5- to 10-membered heterocyclic group" refers to a monocyclic or condensed ring heterocyclic group having 5 to 10 ring-constituting atoms and containing, among the ring-constituting atoms, 1 to 4 heteroatoms independently selected from the group consisting of nitrogen atoms, oxygen atoms, and sulfur atoms. This heterocyclic group may be saturated, partially unsaturated, or unsaturated. Specific examples include a pyrrolidinyl group, a tetrahydrofuryl group, a tetrahydrothienyl group, a piperidyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a pyrrole group, a furyl group, a thienyl group, a pyridyl group, a pyrimidinyl group, a pyridazinyl group, an azepanyl group, an oxepanyl group, a thiepanyl group, an azepinyl group, an oxepinyl group, a thiepinyl group, an imidazolyl group, a pyrazolyl group, an oxazolyl group, a thiazolyl group, an imidazolinyl group, a pyrazinyl group, a morpholinyl group, a thiazinyl group, an indolyl group, an isoindolyl group, a benzimidazolyl group, a purinyl group, a quinolyl group, an isoquinolyl group, a quinoxalinyl group, a cinnolinyl group, a pteridinyl group, a chromenyl group, and an isochromenyl group.

[0046] "C 1-6 The term "alkoxy group" means a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms, and specific examples include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a t-butoxy group, an n-pentyloxy group, and an n-hexyloxy group.

[0047] "C 3-6 The term "cycloalkyl group" refers to a monovalent cyclic saturated aliphatic hydrocarbon group having 3 to 6 carbon atoms, and specific examples include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0048] "C 3-8 The term "cycloalkyl group" means a cycloalkyl group having 3 to 8 carbon atoms. Specific examples include the above-mentioned "C 3-6 Examples of "cycloalkyl groups" include cycloheptyl and cyclooctyl groups. 5-8 A cycloalkyl group has 5 to 8 carbon atoms.5-6 "Cycloalkyl group" means a cycloalkyl group having 5 to 6 carbon atoms. 3-8 The term "cycloalkyl ring" refers to a cycloalkyl ring having from 3 to 8 carbon atoms.

[0049] "C 3-6 The term "cycloalkoxy group" means a cycloalkyloxy group having 3 to 6 carbon atoms, and specific examples include a cyclopropoxy group, a cyclobutoxy group, a cyclopentyloxy group, and a cyclohexyloxy group.

[0050] "Mono C 1-6 The term "alkylamino group" refers to one of the above "C 1-6 "Alkyl group" means a group in which an "alkyl group" is bonded to an amino group, and specific examples include a monomethylamino group, a monoethylamino group, a mono-n-propylamino group, a monoisopropylamino group, a mono-n-butylamino group, a monoisobutylamino group, a mono-t-butylamino group, a mono-n-pentylamino group, and a mono-n-hexylamino group.

[0051] "JiC 1-6 The term "alkylamino group" refers to a group consisting of two identical or different "C 1-6Specific examples of the alkyl group include dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-t-butylamino, di-n-pentylamino, di-n-hexylamino, N-ethyl-N-methylamino, N-methyl-Nn-propylamino, N-isopropyl-N-methylamino, Nn-butyl-N-methylamino, Examples of such amino groups include N-isobutyl-N-methylamino group, Nt-butyl-N-methylamino group, N-methyl-Nn-pentylamino group, Nn-hexyl-N-methylamino group, N-ethyl-Nn-propylamino group, N-ethyl-N-isopropylamino group, Nn-butyl-N-ethylamino group, N-ethyl-N-isobutylamino group, Nt-butyl-N-ethylamino group, N-ethyl-Nn-pentylamino group, and N-ethyl-Nn-hexylamino group.

[0052] "C 1-6 The term "alkoxycarbonyl group" means a straight-chain or branched-chain alkoxycarbonyl group having 1 to 6 carbon atoms, and specific examples include a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, an isopropoxycarbonyl group, an n-butoxycarbonyl group, an isobutoxycarbonyl group, a t-butoxycarbonyl group, an n-pentyloxycarbonyl group, and an n-hexyloxycarbonyl group.

[0053] "Tori C 1-6 The term "alkylsilyl group" refers to a group having three identical or different "C 1-6 "Alkyl group" means a group in which the alkyl group is bonded to a silyl group, and specific examples include a trimethylsilyl (TMS) group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, and a di-t-butylisobutylsilyl group.

[0054] "Tori C 1-6 The term "alkylsilyloxy group" refers to a group having three identical or different "C 1-6Specific examples of the silyloxy group include a trimethylsilyloxy group, a triethylsilyloxy group, a triisopropylsilyloxy group, a t-butyldimethylsilyloxy group, and a di-t-butylisobutylsilyloxy group.

[0055] The term "bicycloalkyl group" refers to a monovalent group of a saturated aliphatic hydrocarbon containing two bridgehead carbons and having two rings. Specific examples include an octahydroinden-3-yl group, an octahydronaphthalene-4-yl group, a bicyclo[2.2.1]heptan-1-yl group, and a bicyclo[2.2.1]heptan-2-yl group. 5-10 A bicycloalkyl group has 5 to 10 carbon atoms and is defined as "C 7-10 The term "bicycloalkyl group" refers to a bicycloalkyl group having from 7 to 10 carbon atoms.

[0056] The term "tricycloalkyl group" refers to a monovalent group of a saturated aliphatic hydrocarbon having at least three bridgehead carbons and three rings. Specific examples include tricyclo[3.3.1.1 3,7 ]decan-1-yl (adamantan-1-yl) group or tricyclo[3.3.1.1 3,7 ]decan-2-yl (adamantan-2-yl) group. 5-15 A "tricycloalkyl group" has 5 to 15 carbon atoms and is defined as "C 7-15 The term "tricycloalkyl group" refers to a tricycloalkyl group having from 7 to 15 carbon atoms.

[0057] The term "secondary or tertiary aliphatic hydrocarbon group" refers to a monovalent group formed by removing a hydrogen atom on a secondary or tertiary carbon atom from a branched or cyclic, saturated or unsaturated aliphatic hydrocarbon containing at least one secondary or tertiary carbon atom in the hydrocarbon chain, and examples of the group include secondary or tertiary alkyl groups, bicycloalkyl groups, tricycloalkyl groups, and secondary or tertiary alkenyl groups, and specific examples of the group include secondary or tertiary alkyl groups, bicycloalkyl groups, tricycloalkyl groups, and secondary or tertiary alkenyl groups having 5 or more carbon atoms, and preferably secondary or tertiary C 5-40 Alkyl group, C 5-10 Bicycloalkyl group, C 5-15 Tricycloalkyl groups, secondary or tertiary C 5-40 alkenyl groups, and more preferably secondary or tertiary C 5-20 Alkyl group, C 7-10 Bicycloalkyl group, C 7-15 Tricycloalkyl groups, secondary or tertiary C 5-20 Alkenyl groups and the like.

[0058] The term "secondary aliphatic hydrocarbon group" refers to a monovalent group formed by removing a hydrogen atom on a secondary carbon atom from a branched or cyclic, saturated or unsaturated aliphatic hydrocarbon containing at least one secondary carbon atom in the hydrocarbon chain, and examples of the group include secondary alkyl groups, cycloalkyl groups, and secondary alkenyl groups. Specific examples of the group include secondary alkyl groups, cycloalkyl groups, and secondary alkenyl groups having 5 or more carbon atoms, and preferably secondary C 5-40 Alkyl group, C 3-8 Cycloalkyl groups, secondary C 5-40 alkenyl groups, and more preferably secondary C 5-20 Alkyl group, C 3-6 Cycloalkyl groups, secondary C 5-20 Alkenyl groups and the like.

[0059] The term "primary aliphatic hydrocarbon group" refers to a monovalent group formed by removing hydrogen on a primary carbon atom from a linear or branched, saturated or unsaturated aliphatic hydrocarbon, and examples of such a group include primary alkyl groups and primary alkenyl groups. Specific examples of such a group include primary alkyl groups and primary alkenyl groups having 4 or more carbon atoms, and preferably primary C 4-40 Alkyl group, primary C 4-40 alkenyl groups, and more preferably primary C 4-20 Alkyl group, primary C 4-20 Alkenyl groups and the like.

[0060] The term "optionally substituted" means unsubstituted or substituted with any number of optional substituents.

[0061] "Having a substituent" means substituted with any number of optional substituents.

[0062] The above-mentioned "optional substituent" is not particularly limited in type, so long as it is a substituent that does not adversely affect the reaction that is the subject of the present invention.

[0063] "C 1-6 In the case where the alkyl group may have a substituent, examples of the substituent include, for example, C 6-14 Aryl group, C 6-14 Haloaryl groups, C 6-14 Aryloxy group, 5-10 membered heterocyclic group, hydroxy group, C 1-6 Alkoxy group, C 3-6 Cycloalkoxy group, acetoxy group, benzoyloxy group, mono C 1-6 Alkylamino group, N-acetylamino group, diC 1-6 Alkylamino group, halogen atom, C 1-6 Alkoxycarbonyl group, phenoxycarbonyl group, N-methylcarbamoyl group, N-phenylcarbamoyl group, triC 1-6 Alkylsilyl group, triC 1-6 Alkylsilyloxy group, C 3-8 Examples of the alkyl group include a cycloalkyl group, a cyano group, and a nitro group. Preferably, the alkyl group is C6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, diC 1-6 Alkylamino group, TriC 1-6 Alkylsilyl group, triC 1-6 Alkylsilyloxy group, C 3-8 is a cycloalkyl group, more preferably C 6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, TriC 1-6 Alkylsilyl group, C 3-8 is a cycloalkyl group, more preferably C 6-14 Aryl group or C 3-8 A cycloalkyl group is preferred, and a phenyl group or a cyclohexyl group is particularly preferred.

[0064] Examples of the "substituent" in the "secondary or tertiary aliphatic hydrocarbon group which may have a substituent" or the "primary aliphatic hydrocarbon group which has a substituent" include, for example, C 6-14 Aryl group, C 6-14 Haloaryl groups, C 6-14 Aryloxy group, 5-10 membered heterocyclic group, hydroxy group, C 1-6 Alkoxy group, C 3-6 Cycloalkoxy group, acetoxy group, benzoyloxy group, mono C 1-6 Alkylamino group, N-acetylamino group, diC 1-6 Alkylamino group, halogen atom, C 1-6 Alkoxycarbonyl group, phenoxycarbonyl group, N-methylcarbamoyl group, N-phenylcarbamoyl group, triC 1-6 Alkylsilyl group, triC 1-6 Alkylsilyloxy group, C 3-8 Examples of the alkyl group include a cycloalkyl group, a cyano group, and a nitro group. Preferably, the alkyl group is C 6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, diC 1-6 Alkylamino group, TriC 1-6 Alkylsilyl group, triC 1-6 Alkylsilyloxy group, C3-8 is a cycloalkyl group, more preferably C 6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, TriC 1-6 Alkylsilyl group, C 3-8 is a cycloalkyl group, more preferably C 6-14 Aryl group or C 3-8 It is a cycloalkyl group. R of the carboxylic acid halide represented by formula (II) 1 The "substituent" in the "substituent-containing primary aliphatic hydrocarbon group" is replaced with a hydrogen atom on the primary carbon atom bonded to the carbonyl carbon.

[0065] An "N-terminal protected amino acid" and an "N-terminal protected peptide" respectively refer to an amino acid and a peptide in which the amino group at the N-terminus is protected and the carboxy group at the C-terminus is not protected. Also, a "C-terminal protected amino acid" and a "C-terminal protected peptide" respectively refer to an amino acid and a peptide in which the carboxy group at the C-terminus is protected and the amino group at the N-terminus is not protected.

[0066] "An amino acid whose N-terminus and C-terminus are not protected" and "a peptide whose N-terminus and C-terminus are not protected" respectively refer to an amino acid and a peptide whose N-terminus amino group and C-terminus carboxy group are not protected. In addition, in an amino acid whose N-terminus and C-terminus are not protected, if a reactive functional group that is not involved in the formation of a peptide, such as a side chain, is present, the functional group may be protected or not protected. Side-chain-protected amino acids are preferred.

[0067] The amino acids used in the present invention are organic compounds having both amino and carboxy functional groups, and refer to natural and non-natural amino acids. They are preferably α-, β- or γ-amino acids, or homoamino acids, and more preferably α-amino acids. In addition, when these amino acids have two or more amino groups (e.g., arginine, lysine, 2,3-diaminopropionic acid (Dap), etc.), when they have two or more carboxy groups (e.g., glutamic acid, aspartic acid, etc.), or when they have a reactive functional group (e.g., cysteine, serine, tyrosine, glutamine, histidine, tryptophan, etc.), the amino acids used in the present invention also include amino acids in which the amino groups, carboxy groups, and / or reactive functional groups that are not involved in the formation of a peptide are protected and / or modified.

[0068] The term "4-6 membered cyclic secondary amino acid" as used herein means an amino acid in which the nitrogen atom of the amino group and two alkyl groups bound to the amino group together form a 4-6 membered ring, and a specific example is proline. 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 When condensed with a cyclic compound selected from the group consisting of cycloalkyl rings, the preferred cyclic compound is C 6-14 An aryl ring is more preferably benzene. Thus, a specific example of a 4-6 membered cyclic secondary amino acid condensed with a cyclic compound is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic).

[0069] NC used in the present invention 1-6 Alkylamino acids are amino acids in which the amino group is optionally substituted. 1-6 An amino acid substituted with an alkyl group, preferably the amino group of the amino acid is 6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, diC 1-6 Alkylamino group, TriC 1-6 Alkylsilyl group, triC 1-6Alkylsilyloxy group, or C 3-8 C which may have a cycloalkyl group 1-6 More preferably, the amino acid is an amino acid substituted with an alkyl group, and more preferably, the amino group of the amino acid is 6-14 Aryl group, C 6-14 Haloaryl groups, C 1-6 Alkoxy group, TriC 1-6 Alkylsilyl group, or C 3-8 C which may have a cycloalkyl group 1-6 The amino acid is preferably an amino acid substituted with an alkyl group, more preferably an amino acid in which the amino group is substituted with a methyl group, an ethyl group, a propyl group, a butyl group, a benzyl group or a cyclohexylmethyl group, still more preferably an amino acid in which the amino group is substituted with a methyl group or an ethyl group, and particularly preferably an amino acid in which the amino group is substituted with a methyl group.

[0070] The "amino acid-derived group," "NC 1-6 The "alkylamino acid derived group" is the amino group of an amino acid or NC 1-6 Alkylamino acid NC 1-6 A divalent group formed by removing a hydrogen atom from the nitrogen atom of an alkylamino group and 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 a hydroxyl group from the carboxyl group, such as "NC 1-6 The alkylglycine-derived group is NC 1-6 Alkylglycine NC 1-6 A divalent group in which a hydrogen atom is removed from the nitrogen atom of an alkylamino group and a hydroxy group is removed from the carboxy group; a "group derived from a cyclic secondary amino acid" means a divalent group in which a hydrogen atom is removed from the nitrogen atom of a secondary amino group of a cyclic secondary amino acid and a hydroxy group is removed from the carboxy group; and a "group derived from a 4- to 6-membered cyclic secondary amino acid" means a divalent group in which a hydrogen atom is removed from the nitrogen atom of a secondary amino group of a 4- to 6-membered cyclic secondary amino acid and a hydroxy group is removed from the carboxy group.

[0071] The amino acids constituting the peptides used in the present invention are the amino acids described above.

[0072] The term "peptide-derived group" as used herein means a divalent group formed by removing a hydrogen atom from the primary or secondary amino group of various amino acids constituting the N-terminus and by removing a hydroxyl group from the carboxy group of the amino acid constituting the C-terminus.

[0073] The three-dimensional structure of the amino acid is not particularly limited, but is preferably the L-form.

[0074] The "N-terminal protecting group" used in the present invention and represented by, for example, P in formulas (I) and (V) is a protecting group on the N-terminal side when carrying out a peptide elongation reaction (amidation reaction), and a known protecting group can be used. Specific examples thereof include carbamate-based protecting groups (9-fluorenylmethoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, 2-(p-biphenyl)isopropyloxycarbonyl group, etc.), amide-based protecting groups (acetyl group, trifluoroacetyl group, etc.), imide-based protecting groups (phthaloyl group, etc.), sulfonamide-based protecting groups (p-toluenesulfonyl group, 2-nitrobenzenesulfonyl group, etc.), benzyl group, etc., and preferably include 9-fluorenylmethoxycarbonyl group, t-butoxycarbonyl group, and benzyloxycarbonyl group.

[0075] All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that can be used in connection with the described invention.

[0076] (Specific Description of the Method for Producing the Peptide of the Present Invention) Each step (1) to (5) of the method for producing the peptide of the present invention is explained below. In one embodiment, the production of the peptide of the present invention comprises the following unit steps (1) to (5). In one embodiment, the peptide of the present invention can be produced by all or a suitable combination of the unit steps described as steps (1) to (5) below. This specific description will be given based on the following. (a) R in the description of steps (1) to (5) 1 and R 2 is synonymous with the above. (b) The specific conditions for the reactions are not particularly limited as long as the production of the peptide of the present invention is achieved. Preferred conditions for each reaction will be described in detail as appropriate. (c) The solvents described in each reaction may be used alone or in combination of two or more.

[0077] Process (1) This step is a step of mixing an N-terminal protected amino acid or an N-terminal protected peptide with a carboxylic acid halide or an alkyl formate halide. This step is a step of activating the C-terminus of the N-terminal protected amino acid or the N-terminal protected peptide with a carboxylic acid halide or an alkyl formate halide. In one embodiment of the present invention, a compound of formula (I) PA 1 -OH (wherein P is an N-terminal protecting group, A 1 is a group derived from an amino acid, NC 1-6 Alkylamino acid derived groups (C 1-6 In another embodiment of the present invention, a compound represented by the formula (V) PA is mixed with an N-terminal protected amino acid or an N-terminal protected peptide represented by the formula (V) PA 3 -OH (wherein P is an N-terminal protecting group, A 3represents a group derived from a peptide.

[0078] The N-terminal protected amino acid or N-terminal protected peptide is the above-mentioned amino acid or peptide whose N-terminus is protected, specifically, an N-terminal protected amino acid, an N-terminal protected NC 1-6 Alkylamino acids (C 1-6 The N-terminal protected peptide in this step is preferably a peptide in which the C-terminal amino acid is NC 1-6 Alkylamino acids (C 1-6 alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 It is an N-terminal protected peptide, which is an amino acid other than an amino acid selected from the group consisting of cycloalkyl rings, which may be condensed with a cyclic compound selected from the group consisting of cycloalkyl rings.

[0079] The carboxylic acid halide is represented by the following formula (II).

[0080] [ka] (In the formula, X represents a halogen atom, R 1 represents a secondary or tertiary aliphatic hydrocarbon group having 5 or more carbon atoms and which may have a substituent, or represents a primary aliphatic hydrocarbon group having 4 or more carbon atoms and which has a substituent (wherein the substituent of the primary aliphatic hydrocarbon group is present on the carbon atom bonded to the carbonyl carbon).

[0081] The carboxylic acid halide of formula (II) is preferably 1 may be substituted with a secondary or tertiary C 5-40 Alkyl group, optionally substituted C 5-10 Bicycloalkyl group, optionally substituted C 5-15Tricycloalkyl group, optionally substituted secondary or tertiary C 5-40 Alkenyl group or primary C having a substituent 4-40 Primary C having an alkyl group or a substituent 4-40 Alkenyl group (wherein the primary C 4-40 Alkyl or primary C 4-40 The substituent of the alkenyl group is a carboxylic acid halide, which is present on the carbon atom bonded to the carbonyl carbon, and more preferably, R 1 may be substituted with a secondary or tertiary C 5-20 Alkyl group, optionally substituted C 7-10 Bicycloalkyl group, optionally substituted C 7-15 Tricycloalkyl group, optionally substituted secondary or tertiary C 5-20 Alkenyl group or primary C with a substituent 4-20 Primary C having an alkyl group or a substituent 4-20 Alkenyl group (wherein the primary C 4-20 Alkyl or primary C 4-20 The substituent of the alkenyl group is a carboxylic acid halide, where R 1 may be substituted with a secondary or tertiary C 5-20 Alkyl group, optionally substituted C 7-10 Bicycloalkyl group, optionally substituted C 7-15 Tricycloalkyl group, optionally substituted secondary or tertiary C 5-20 The carboxylic acid chloride is an alkenyl group or an isobutyl group having a substituent, and is more preferably selected from the following compound group. [ka]

[0082] Particularly preferably, the compound is selected from the following group of compounds: [ka]

[0083] The alkyl formate halide is represented by the following formula (III): [ka] (In the formula, X represents a halogen atom, R 2 represents a secondary aliphatic hydrocarbon group having 5 or more carbon atoms which may have a substituent.

[0084] The alkyl halide formate of formula (III) is preferably R 2 C optionally having a secondary substituent 5-40 Alkyl group, optionally substituted C 5-8 Cycloalkyl groups, optionally substituted secondary C 5-40 More preferably, R is an alkyl halide formate which is an alkenyl group. 2 C optionally having a secondary substituent 5-20 Alkyl group, optionally substituted C 5-6 Cycloalkyl groups, optionally substituted secondary C 5-20 Alkenyl halide alkyl formates, more preferably R 2 C optionally having a secondary substituent 5-20 Alkyl group, optionally substituted C 5-6 Cycloalkyl groups, optionally substituted secondary C 5-20 The alkenyl group is alkyl chloroformate, and is particularly preferably selected from the following compound group: [ka]

[0085] R 1 or R 2 The number of carbon atoms in R 1 or R 2 is the total number of carbon atoms that each of R 1 or R 2 When the group has a substituent, the number of carbon atoms in the substituent is also included.

[0086] The mixing (activation reaction) of the carboxylic acid halide or alkyl formate halide with the N-terminal protected amino acid or N-terminal protected peptide can be carried out, if necessary, in the presence of a base and / or a solvent.

[0087] The base used in this step is not particularly limited, but examples thereof 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), alkali metal salts (e.g., sodium hydrogen carbonate, potassium carbonate), etc. Preferred are aliphatic amines, and more preferred are N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine.

[0088] The amount of the carboxylic acid halide or alkyl formate halide used in this step is preferably 0.2 to 50 equivalents, more preferably 0.5 to 20 equivalents, and even more preferably 0.8 to 5 equivalents relative to the N-terminal protected amino acid or N-terminal protected peptide.

[0089] The amount of the base used in this step is preferably 0.2 to 50 equivalents, more preferably 0.5 to 20 equivalents, and further preferably 0.8 to 5 equivalents relative to the carboxylic acid halide or alkyl formate halide.

[0090] The solvent used in this step is not particularly limited as long as it does not interfere with the activation reaction, and examples thereof include halogen-containing hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, methyl-t-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), ester solvents (e.g., ethyl acetate), etc. Preferred are nitrile solvents, amide solvents, or ether solvents, more preferably acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide.

[0091] The amount of the solvent used in this step is preferably 100 times by mass or less, more preferably 1 to 50 times by mass, and even more preferably 3 to 20 times by mass, relative to the amount of the carboxylic acid halide or alkyl formate halide.

[0092] In this manner, the N-terminal protected amino acid or N-terminal protected peptide is mixed with the carboxylic acid halide or alkyl formate halide in the presence of a solvent and / or a base as necessary. The temperature of the resulting mixture is controlled using an oil bath or a cooling bath as necessary. 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.

[0093] This step forms an N-terminally protected amino acid or an N-terminally protected peptide with an activated C-terminus. Therefore, the product obtained by this step means an N-terminally protected amino acid or an N-terminally protected peptide with an activated C-terminus, or a mixture containing either of them. The N-terminally protected amino acid or N-terminally protected peptide with an activated C-terminus thus obtained may be used in the subsequent step (3) as the reaction solution or after being isolated as a (crude) purified product without being subjected to a purification step.

[0094] Process (2) This step is a step of mixing an N-alkylamino acid or a peptide having an N-alkylamino acid at the N-terminus with a silylating agent. In this step, "N-alkylamino acid" means an N-alkylamino acid whose N-terminal amino group and C-terminal carboxy group are not protected, and "peptide having an N-alkylamino acid at the N-terminus" means a peptide having an N-alkylamino acid at the N-terminus and whose N-terminal amino group and C-terminal carboxy group are not protected. This step is a step of reacting an N-alkylamino acid or a peptide having an N-alkylamino acid at the N-terminus with a silylating agent to obtain an N-alkylamino acid or a peptide having an N-alkylamino acid at the N-terminus in which at least a part of the functional groups such as the C-terminus, N-terminus and / or hydroxyl group (if present) of the amino acid or peptide is trialkylsilylated (hereinafter also referred to as "trialkylsilylated amino acid or peptide"). In one aspect of the present invention, a compound of formula (IV) HA 2 -OH[wherein, A 2 NC 1-6 Alkylamino acid derived groups (C 1-6 alkyl may have a substituent), or a group derived from a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 cycloalkyl rings), or the N-terminal residue is NC 1-6 Alkylamino acids (C 1-6alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is C 6-14 Aryl ring, C 6-14 Haloaryl rings and C 3-8 a cyclic compound selected from the group consisting of cycloalkyl rings) amino The carboxyl group at the C-terminus and the carboxyl group at the N-terminus are not protected. amino In another embodiment of the present invention, a peptide having an unprotected carboxyl group and a unprotected C-terminal carboxyl group is mixed with a silylating agent. 2’ -OH[wherein, A 2’ is the group derived from N-methylamino acids, NC 1-6 Alkylglycine derived groups (C 1-6 This is a process of mixing an amino acid in which the N-terminal amino group and the C-terminal carboxy group are not protected, represented by the formula (I) (alkyl may have a substituent), or a group derived from a 4- to 6-membered cyclic secondary amino acid), with a silylating agent.

[0095] In addition, the N-terminal amino acid in the N-alkyl amino acid or peptide having an N-alkyl amino acid at the N-terminus in this step is preferably NC 1-6 Alkylamino acids (C 1-6 The alkyl may be substituted with cyclohexyl or phenyl), more preferably an N-methyl amino acid, an N-ethyl amino acid, an N-propyl amino acid, an N-butyl amino acid, an N-pentyl amino acid, an N-cyclohexylmethyl amino acid or an N-benzyl amino acid, further preferably an N-methyl amino acid or an N-ethyl amino acid, and particularly preferably an N-methyl amino acid.

[0096] The silylating agent in this step is not particularly limited, but examples thereof include trimethylsilylating agents such as 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, and N,O-bis(trimethylsilyl)trifluoroacetamide, and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Preferred are trimethylsilyl chloride, N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea, or N,O-bis(trimethylsilyl)trifluoroacetamide, and more preferably N,O-bis(trimethylsilyl)acetamide.

[0097] The amount of the silylating agent used is preferably 0.01 to 50 equivalents, more preferably 0.1 to 20 equivalents, and even more preferably 0.2 to 5 equivalents relative to the N-alkylamino acid or peptide having an N-alkylamino acid at the N-terminus.

[0098] Mixing the N-alkylamino acid or the peptide having an N-alkylamino acid at the N-terminus with the silylating agent (silylation reaction) can be carried out in the presence of a base and / or a solvent, if necessary.

[0099] The base to be used is not particularly limited, and examples thereof include aliphatic amines (e.g., dicyclohexylamine, piperidine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine), aromatic amines (e.g., pyridine, imidazole, N,N-dimethyl-4-aminopyridine), alkali metal salts (e.g., sodium hydrogen carbonate, potassium carbonate), etc. Preferred are aliphatic amines, and more preferred are triethylamine and N,N-diisopropylethylamine.

[0100] The amount of the base used is preferably 0.01 to 50 equivalents, more preferably 0.1 to 20 equivalents, and even more preferably 0.2 to 5 equivalents relative to the N-alkylamino acid or peptide having an N-alkylamino acid at the N-terminus.

[0101] The solvent used in this step is not particularly limited as long as it does not interfere with the silylation reaction, and examples thereof include halogen-containing hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentyl methyl ether, methyl-t-butyl ether), amide solvents (e.g., N,N-dimethylformamide), nitrile solvents (e.g., acetonitrile), etc. Nitrile solvents, amide solvents, or ether solvents are preferred, and acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide are more preferred.

[0102] The amount of the solvent used in this step is preferably 100 times or less by mass, more preferably 1 to 50 times by mass, and even more preferably 3 to 20 times by mass, relative to the amount of the N-alkylamino acid or the peptide having an N-alkylamino acid at the N-terminus.

[0103] In this manner, the N-alkylamino acid or the peptide having an N-alkylamino acid at the N-terminus is mixed with the silylating agent in the presence of a solvent and / or a base as necessary. The temperature of the obtained mixture is controlled using an oil bath or a cooling bath as necessary. 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. The mixture may also be subjected to microwave irradiation.

[0104] This step forms a trialkylsilylated N-alkylamino acid or a peptide having an N-alkylamino acid at the N-terminus. Therefore, the product obtained by this step means a mixture containing a trialkylsilylated N-alkylamino acid or a peptide having an N-alkylamino acid at the N-terminus. The trialkylsilylated amino acid or peptide thus obtained may be used in the subsequent step (3) as the reaction solution or isolated as a (crude) purified product without a purification step. In addition, the trialkylsilylated N-alkylamino acid or the peptide having an N-alkylamino acid at the N-terminus can also be analyzed by an analytical instrument such as NMR.

[0105] Process (3) This step is a step of mixing the product obtained in step (1) with the product obtained in step (2). This step is a peptide elongation step in which the N-terminal protected amino acid or N-terminal protected peptide with an activated C-terminus obtained in step (1) is reacted with the trialkylsilylated amino acid or peptide obtained in step (2), and can be preferably carried out by mixing and stirring the reaction solution obtained in step (1) with the reaction solution obtained in step (2). In one aspect of the present invention, this is a peptide elongation step in which the N-terminal protected amino acid with an activated C-terminus obtained in step (1) is reacted with the trialkylsilylated amino acid or peptide obtained in step (2). In another aspect of the present invention, this is a peptide elongation step in which the N-terminal protected peptide with an activated C-terminus obtained in step (1) is reacted with the trialkylsilylated amino acid obtained in step (2).

[0106] The temperature of the obtained mixture is controlled using an oil bath or a cooling bath as necessary. 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 further preferably from -10°C to 30°C.

[0107] In the method for producing a peptide of the present invention, the peptide chain can be further elongated by repeating the following steps (4) and (5) for the peptide obtained in step (3) a desired number of times. (4) A step of removing the protecting group at the N-terminus of the peptide obtained in step (3) or (5). (5) A step of reacting the N-terminus of the peptide obtained in the step (4) with an N-terminal protected amino acid or an N-terminal protected peptide. Step (5) can be carried out by the same procedures as in steps (1), (2) and (3) above, or by a general peptide synthesis reaction.

[0108] In the method for producing the peptide of the present invention, the purification steps (1) to (5) can be omitted as appropriate to the extent that the reaction in the subsequent step is not affected.

[0109] Step (4): N-terminal deprotection step This step is a step in which the N-terminal protecting group is removed from the peptide obtained in step (3) or (5) above to obtain a peptide having unprotected N- and C-termini.

[0110] The deprotection reagent used in this step is appropriately selected depending on the protecting group used. Examples include acids (e.g., trifluoroacetic acid, hydrochloric acid, Lewis acid), secondary or tertiary amines (e.g., pyrrolidine, piperidine, morpholine, triethylamine), hydrogenolysis (e.g., palladium catalyst / hydrogenation), etc.

[0111] The deprotection conditions used in this step are appropriately selected depending on the type of N-terminal protecting group. For example, in the case of a 9-fluorenylmethoxycarbonyl group, deprotection is carried out by treatment with a base, in the case of a t-butoxycarbonyl group, deprotection is carried out by treatment with an acid, and in the case of a benzyloxycarbonyl group or an allyloxycarbonyl group, deprotection is carried out in neutral conditions, for example, by hydrogenation in the presence of a metal catalyst.

[0112] In each reaction, when the reaction substrate has a hydroxy group, a mercapto group, an amino group, a carboxy group, or a carbonyl group (particularly when the amino acid or peptide has a functional group in the side chain), a protecting group generally used in peptide chemistry or the like may be introduced into these groups, and the target compound can be obtained by removing the protecting group as necessary after the reaction.

[0113] The protection and deprotection can be carried out by carrying out a protection-deprotection reaction using a commonly known protecting group (see, for example, Protective Groups in Organic Synthesis, Fourth edition, TW Greene, John Wiley & Sons Inc. (2006)).

[0114] The combination of the N-terminal protected amino acid or N-terminal amino acid in the N-terminal protected peptide used in step (1) (hereinafter, amino acid A) and the N-terminal amino acid in the amino acid or peptide used in step (2) (hereinafter, amino acid B) is not particularly limited, but preferably, amino acids A and B are each an α-, β-, or γ-amino acid, more preferably, either amino acid A or amino acid B is an α-amino acid, even more preferably, amino acid A is an α-amino acid and amino acid B is an α-, β-, or γ-amino acid, or amino acid A is an α-, β-, or γ-amino acid and amino acid B is an α-amino acid, and particularly preferably, amino acid A is an α-amino acid and amino acid B is an α-, β-, or γ-amino acid. In addition, the N-terminal amino acid in the peptide used in step (2) is preferably an α-amino acid. EXAMPLES

[0115] The present invention will be described in more detail below with reference to Reference Examples, Comparative Examples and Synthesis Examples as Examples, but the present invention is not limited to these Examples.

[0116] In this specification, when amino acids and the like are represented by abbreviations, each representation is based on the abbreviations defined by the IUPAC-IUB Commission on Biochemical Nomenclature or the commonly used abbreviations in the relevant field.

[0117] In the synthesis examples, "(v / v)" means (volume / volume) and "M" means mol / L.

[0118] In the following synthesis examples, the microwave reaction apparatus used was Initiator+ (manufactured by Biotage).

[0119] Proton nuclear magnetic resonance of synthesis examples ( 1 Unless otherwise specified, H-NMR was performed using a JEOL JNM-ECP300, a JEOL JNM-ECX300, or a Bruker Ascend TM The measurements were made using a 500 spectrometer in a deuterated chloroform or deuterated dimethyl sulfoxide solvent, and the chemical shifts are shown as δ values ​​(ppm) relative to tetramethylsilane as the internal standard (0.0 ppm).

[0120] In describing NMR spectra, "s" stands for singlet, "d" for doublet, "t" for triplet, "q" for quartet, "dd" for doublet of doublet, "dt" for doublet of triplet, "sept" for septet, "m" for multiplet, "br" for broad, "J" for coupling constant, "Hz" for Hertz, "CDCl3" for deuterated chloroform, and "DMSO-d6" for deuterated dimethyl sulfoxide.

[0121] Unless otherwise specified, high performance liquid chromatography / mass spectrometry was measured using either Waters ACQUITY UPLC H-Class / QDa, Waters ACQUITY UPLC H-Class / SQD2, or Shimadzu LC-20AD / Triple Tof5600.

[0122] In the descriptions of high performance liquid chromatography / mass spectrometry, ESI+ is electrospray ionization positive mode, M+H means proton adduct, and M+Na means sodium adduct.

[0123] In the description of high performance liquid chromatography / mass spectrometry, ESI- is electrospray ionization negative mode, and MH means proton deficient.

[0124] In Synthesis Examples 8 to 50, the ratio of the raw material to the product was calculated by any one of the analyses <Analysis Condition 1> to <Analysis Condition 3> using high performance liquid chromatography. <Analysis conditions 1> High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2 Column: Phenomenex Kinetex EVO C18 (1.7μm, 2.1 x 50 mm) Column oven temperature: 60℃ Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution 5:95 (0-2.1 minutes), 95:5 (2.1-2.84 minutes) (v / v) Eluent rate: 0.6 mL / min Detection wavelength: 220 nm <Analysis conditions 2> High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2 Column: Waters ACQUITY BEH C18 (1.7μm, 2.1 x 100 mm) Column oven temperature: 60℃ Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution 5:95 (0-3.7 minutes), 95:5 (3.7-4.81 minutes) (v / v) Eluent rate: 0.6 mL / min Detection wavelength: 220 nm <Analysis conditions 3> High performance liquid chromatography: Waters ACQUITY UPLC H-Class / SQD2 Column: Waters ACQUITY BEH C18 (1.7μm, 2.1 x 100 mm) Column oven temperature: 40℃ Eluent: Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution 5:95 (0-5.56 minutes), 95:5 (5.56-7.22 minutes) (v / v) Eluent rate: 0.4 mL / min Detection wavelength: 220 nm

[0125] For purification by silica gel column chromatography, unless otherwise specified, either a Yamazen Hi-Flash column, a Biotage SNAP Ultra Silica Cartridge, Merck Silica Gel 60, or Fuji Silysia Chemical PSQ60B was used.

[0126] Unless otherwise specified below, a commercially available N,O-bis(trimethylsilyl)acetamide with a purity of 98% was used.

[0127] In the following examples, the yield or quantitative yield may exceed 100%. These are all due to measurement errors, the influence of the purity of the raw materials or products, or other factors based on common technical knowledge. In the following examples, the causes of the yield exceeding 100% are not mentioned individually, but a person skilled in the art would be able to fully understand the scientific validity of these examples.

[0128] Synthesis Example 1: Synthesis of 4-ethyl-2,2-dimethylhexan-3-ol [ka]

[0129] 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. and stirred for 5 minutes. The obtained reaction liquid was heated to 25 ° C., and then ethanol (1.0 mL) was added, and the mixture was washed with 20 mass % ammonium chloride aqueous solution (25 mL) and saturated sodium chloride aqueous solution (25 mL) in that order. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain 4-ethyl-2,2-dimethylhexane-3-ol (5.22 g, yield 90%) as a colorless transparent liquid.

[0130] 1 H-NMR (CDCl3) 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).

[0131] Synthesis Example 2: Synthesis of 4-ethyl-2,2-dimethylhexan-3-yl carbonochloridate [ka]

[0132] 4-Ethyl-2,2-dimethylhexan-3-ol (5.21 g, 32.9 mmol), pyridine (2.99 g, 37.9 mmol) were mixed with carbon tetrachloride (50 mL) and cooled to 0°C. A solution of 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 reaction solution was washed twice with water (50 mL) and with a saturated aqueous sodium chloride solution (50 mL) in that order. The organic layer was concentrated to obtain 4-ethyl-2,2-dimethylhexan-3-yl carbonochloridate (6.56 g, 90% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0133] 1 H-NMR (CDCl3) 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).

[0134] Synthesis Example 3: Synthesis of 2,2,4-trimethylpentan-3-ol [ka]

[0135] 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. and stirred for 5 minutes. The resulting reaction solution was heated to 25 ° C., and then ethanol (1.0 mL) was added, and the solution was washed with 20 mass % ammonium chloride aqueous solution (25 mL) and saturated sodium chloride aqueous solution (25 mL) in that order. 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.

[0136] 1 H-NMR (CDCl3) 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).

[0137] Synthesis Example 4: Synthesis of 2,2,4-trimethylpentan-3-yl carbonochloridate [ka]

[0138] 2,2,4-Trimethylpentan-3-ol (5.25 g, 40.3 mmol), pyridine (3.67 g, 46.3 mmol) were mixed with carbon tetrachloride (40 mL) and cooled to 0°C. A solution of 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 reaction solution was washed twice with water (50 mL) and with a saturated aqueous sodium chloride solution (50 mL) in that order. The organic layer was concentrated to obtain 2,2,4-trimethylpentan-3-yl carbonochloridate (6.06 g, 78% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0139] 1 H-NMR (CDCl3) 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).

[0140] Synthesis Example 5: Synthesis of 3,3-dimethylbutan-2-yl carbonochloridate [ka]

[0141] 3,3-Dimethylbutan-2-ol (3.36 g, 32.9 mmol), pyridine (2.99 g, 37.8 mmol) were mixed with carbon tetrachloride (40 mL) and cooled to 0°C. A solution of 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 reaction solution was washed twice with water (50 mL) and with a saturated aqueous sodium chloride solution (50 mL) in that order. The organic layer was concentrated to obtain 3,3-dimethylbutan-2-yl carbonochloridate (4.50 g, 83% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0142] 1 H-NMR (CDCl3) δppm:0.96(9H,s),1.29(3H,d,J=6.5Hz),4.74(1H,q,J=6.5Hz).

[0143] Synthesis Example 6: Synthesis of 2,4-dimethylpentan-3-yl carbonochloridate [ka]

[0144] 2,4-Dimethylpentan-3-ol (3.49 g, 30.0 mmol), pyridine (2.73 g, 34.5 mmol) were mixed with carbon tetrachloride (40 mL) and cooled to 0°C. A solution of 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 reaction solution was washed twice with water (50 mL) and with a saturated aqueous sodium chloride solution (50 mL) in that order. The organic layer was concentrated to obtain 2,4-dimethylpentan-3-yl carbonochloridate (5.10 g, 95% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0145] 1 H-NMR (CDCl3) 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).

[0146] Synthesis Example 7: Synthesis of 3-methylbutan-2-yl carbonochloridate [ka] 3-Methylbutan-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 of 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 reaction solution was washed twice with water (50 mL) and with a saturated aqueous sodium chloride solution (50 mL) in that order. The organic layer was concentrated to obtain 3-methylbutan-2-yl carbonochloridate (5.10 g, 95% yield) as a colorless, transparent liquid. This compound was used in the next step without further purification.

[0147] 1 H-NMR (CDCl3) 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).

[0148] Synthesis Example 8: Synthesis of Boc-Phe-MePhe-OH [ka]

[0149] Boc-Phe-OH (0.066g, 0.25mmol), triethylamine (0.033g, 0.32mmol) were mixed with tetrahydrofuran (5mL), and 2,2-dimethylbutanoyl chloride (0.040g, 0.30mmol) was added at 0°C and stirred for 1 hour. This solution was mixed with H-MePhe-OH (0.067g, 0.38mmol), N,O-bis(trimethylsilyl)acetamide (0.161g, 0.750mmol), and acetonitrile (4mL) separately, and stirred at 75°C for 3 minutes under microwave irradiation to prepare a solution, which was then added and stirred at 0°C for 2 hours, and further stirred at 25°C for 16 hours (raw material: target product = 1:70 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed with 10% by mass citric acid solution (10 mL) twice, 5% by mass sodium chloride solution (10 mL), and saturated sodium chloride solution (10 mL) in that order. The resulting organic layer was concentrated to obtain Boc-Phe-MePhe-OH (0.125 g, yield 117%) as a white solid. MASS(ESI+) m / z;(M+H)+427.3

[0150] Synthesis Example 9: Synthesis of Fmoc-Phe-MePhe-OH [ka]

[0151] Fmoc-Phe-OH (0.097g, 0.25mmol), N-methylmorpholine (0.033g, 0.33mmol) were mixed with tetrahydrofuran (5.0mL), and 4-ethyl-2,2-dimethylhexane-3-yl carbonochloridate (0.066g, 0.30mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.058g, 0.33mmol), N,O-bis(trimethylsilyl)acetamide (0.141g, 0.67mmol), and acetonitrile (4.0mL) separately and stirring at 75°C for 20 minutes, and further stirring was performed at 25°C for 5 hours (raw material: target product = 1:13 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (20 mL) and washed successively with 10% by mass aqueous citric acid solution (20 mL), 10% by mass aqueous sodium chloride solution (20 mL), and saturated aqueous sodium chloride 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. MASS(ESI+) m / z;(M+H)+549.3

[0152] Synthesis Example 10: Synthesis of Fmoc-Phe-MePhe-OH [ka]

[0153] Fmoc-Phe-OH (0.097g, 0.25mmol), N-methylmorpholine (0.033g, 0.33mmol) was mixed with N,N-dimethylacetamide (5.0mL), 4-ethyl-2,2-dimethylhexane-3-yl carbonochloridate (0.066g, 0.30mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.058g, 0.33mmol), N,O-bis(trimethylsilyl)acetamide (0.141g, 0.67mmol), and acetonitrile (4.0mL) separately and stirring at 75°C for 20 minutes, and further stirring was performed at 25°C for 5 hours (raw material: target product = 1:31 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (30 mL) and washed successively with 10% by mass aqueous citric acid solution (50 mL), 10% by mass aqueous sodium chloride solution (20 mL), and saturated aqueous sodium chloride 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. MASS(ESI+) m / z;(M+H)+549.3

[0154] Synthesis Example 11: Synthesis of Fmoc-Phe-MePhe-OH [ka]

[0155] Fmoc-Phe-OH (0.194g, 0.500mmol), triethylamine (0.0607g, 0.60mmol) were mixed with tetrahydrofuran (10mL), and 2,2-dimethylbutanoyl chloride (0.074g, 0.550mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.108g, 0.600mmol), N,N'-bis(trimethylsilyl)urea (0.250g, 1.20mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 75°C for 60 minutes under microwave irradiation to prepare a solution, which was then added and further stirred at 25°C for 67 hours (raw material: target product = 1:20 (analysis condition 2)). The resulting reaction solution was concentrated, diluted with ethyl acetate (80 mL), and washed successively with 10% by mass citric acid aqueous solution (50 mL), 5% by mass sodium chloride aqueous solution (50 mL), and saturated sodium chloride aqueous solution (50 mL). The resulting organic layer was concentrated to give Fmoc-Phe-MePhe-OH (0.322 g, yield 117%) as a white solid. MASS(ESI+)m / z;(M+H)+549.4

[0156] Synthesis Example 12: Synthesis of Fmoc-Phe-MePhe-OH [ka]

[0157] Fmoc-Phe-OH (0.097g, 0.250mmol), triethylamine (0.0304g, 0.300mmol) were mixed with tetrahydrofuran (5mL), and 2,2-dimethylbutanoyl chloride (0.0371g, 0.275mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.0538g, 0.300mmol), N,O-bis(trimethylsilyl)trifluoroacetamide (0.155g, 0.601mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 75°C for 30 minutes to prepare a solution, which was then added and stirred at 25°C for 16 hours (raw material: target product = 4.4:95.6 (analysis condition 1; but at 4 hours of stirring)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (20 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution (20 mL), and saturated aqueous sodium chloride solution (20 mL). The resulting organic layer was concentrated to obtain Fmoc-Phe-MePhe-OH (0.135 g, yield 98%) as a white solid. MASS(ESI+)m / z;(M+H)+549.4

[0158] Synthesis Example 13: Synthesis of Boc-MePhe-MePhe-OH [ka]

[0159] Boc-MePhe-OH (0.070g, 0.250mmol), triethylamine (0.033g, 0.32mmol) were mixed with tetrahydrofuran (5mL), and 2,2-dimethylbutanoyl chloride (0.040g, 0.30mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.067g, 0.38mmol), N,O-bis(trimethylsilyl)acetamide (0.161g, 0.774mmol), and acetonitrile (4.0mL) separately and stirring at 75°C for 3 minutes, and further stirring was performed at 25°C for 16 hours (raw material: target product = 7:93 (analysis condition 1)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (20 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution (20 mL), and saturated aqueous sodium chloride solution (20 mL). The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-OH (0.127 g, yield 116%) as a colorless oil. MASS(ESI+) m / z;(M+H)+441.4

[0160] Synthesis Example 14: Synthesis of Boc-MePhe-Phe-OH [ka]

[0161] Boc-MePhe-OH (1.40g, 5.00mmol), N-methylmorpholine (0.556g, 5.50mmol) were mixed with tetrahydrofuran (50mL), and isopropyl chloroformate (0.643g, 5.25mmol) was added at 0°C and stirred for 15 minutes. H-Phe-OH (0.991g, 6.00mmol), N,O-bis(trimethylsilyl)acetamide (2.57g, 12.4mmol), and acetonitrile (15mL) were added to this solution and stirred at 75°C for 60 minutes under microwave irradiation, and the prepared solution was added, stirred at 0°C for 30 minutes, and further stirred at 25°C for 1.5 hours (raw material: target product = 0:100 (analysis condition 1)). The resulting reaction solution was diluted with ethyl acetate (200 mL) and washed successively with 10% by mass aqueous citric acid solution (75 mL), 5% by mass aqueous sodium chloride solution (75 mL), and saturated aqueous sodium chloride 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. MASS(ESI+) m / z;(M+H)+427.3

[0162] Synthesis Example 15: Synthesis of Fmoc-MePhe-Phe-OH [ka]

[0163] Fmoc-MePhe-OH (2.00 g, 5.00 mmol), N-methylmorpholine (0.556 g, 5.50 mmol) were mixed with tetrahydrofuran (30 mL), and isopropyl chloroformate (0.663 g, 5.25 mmol) was added at 0° C. and stirred for 30 minutes. To this solution, a solution was added which was prepared by 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) separately and stirring at 75° C. for 30 minutes, and the mixture was stirred at 0° C. for 50 minutes and further stirred at 25° C. for 1 hour (raw material: target product = 3:97 (analysis condition 1)). The resulting reaction solution was diluted with ethyl acetate (75 mL) and washed successively with 10% by mass aqueous citric acid solution (50 mL), 5% by mass aqueous sodium chloride solution (50 mL), and saturated aqueous sodium chloride 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. MASS(ESI+)m / z;(M+H)+549.4

[0164] Synthesis Example 16: Synthesis of H-MePhe-Phe-OH [ka]

[0165] Fmoc-MePhe-Phe-OH (0.137 g, 0.250 mmol), triethylamine (0.505 g, 4.99 mmol) were mixed with acetonitrile (4.4 mL) 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 the mixture, and the resulting solid was filtered off using a Kiriyama funnel. It was washed with diisopropyl ether (5 mL) and dried to obtain H-MePhe-Phe-OH (0.071 g, 87% yield) as a white solid. MASS(ESI+) m / z;(M+H)+327.3

[0166] Synthesis Example 17: Synthesis of H-MePhe-Phe-OH·HCl [ka]

[0167] 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 suspended in diisopropyl ether. The resulting solid was filtered using a Kiriyama funnel and dried to obtain H-MePhe-Phe-OH·HCl (0.164 g, 91% yield) as a white solid. MASS(ESI+) m / z;(M+H)+327.3

[0168] Synthesis Example 18: Synthesis of Boc-MePhe-MePhe-Phe-OH [ka]

[0169] Solution A Boc-MePhe-OH (0.280 g, 1.00 mmol), N-methylmorpholine (0.112 g, 1.10 mmol) were mixed with tetrahydrofuran (10 mL), and 2,2,4-trimethylpentan-3-yl carbonochloridate (0.203 g, 1.05 mmol) was added at 0° C. and stirred for 1 hour.

[0170] Solution B Boc-MePhe-Phe-OH (0.449 g, 1.05 mmol) was mixed with 4M-HCl / cyclopentyl methyl ether (5 mL) and stirred at 25° C. for 30 minutes. The resulting reaction solution was concentrated, ethyl acetate (20 mL) was added and concentrated, and N,N-diisopropylethylamine (1.30 g, 10.0 mmol) and acetonitrile (8 mL) were added and concentrated. The resulting residue was mixed with acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.644 g, 3.11 mmol) and stirred at 25° C. for 20 minutes to obtain a colorless transparent solution.

[0171] condensation process While keeping solution A cooled to 0°C, solution B was mixed and further stirred at 25°C for 18 hours (raw material: target product = 1:21 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with a 10% by mass aqueous solution of citric acid and a saturated aqueous solution of sodium chloride. The obtained organic layer was concentrated and then purified by silica gel column chromatography. Boc-MePhe-MePhe-Phe-OH (0.532 g, 90% yield) was obtained as a white solid. MASS(ESI+)m / z;(M+H)+588.4

[0172] Synthesis Example 19: Synthesis of Boc-Phe-MePhe-MePhe-Phe-OH [ka]

[0173] Solution A 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-trimethylpentan-3-yl carbonochloridate (0.135 g, 0.702 mmol) was added at 0° C. and stirred for 1 hour.

[0174] Solution B 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, and N,N-diisopropylethylamine (0.865 g, 6.69 mmol) and acetonitrile (8 mL) were added and concentrated. The resulting residue was mixed with acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.430 g, 2.07 mmol) and stirred at 25° C. for 20 minutes to obtain a colorless transparent solution.

[0175] condensation process While cooling Solution A to 0°C, Solution B was mixed and the mixture was stirred at 25°C for 18 hours (raw material: target product = 0:100 (analysis condition 3)). The reaction solution obtained was diluted with ethyl acetate (20 mL) and washed successively with 10% by mass citric acid aqueous solution (20 mL), 10% by mass sodium chloride aqueous solution (20 mL), and saturated sodium chloride aqueous solution (20 mL). The organic layer obtained 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. MASS(ESI+) m / z;(M+H)+735.5

[0176] Synthesis Example 20: Synthesis of Boc-MePhe-MePhe-Phe-OH [ka]

[0177] Solution A 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-dimethylbutanoyl chloride (0.172 g, 1.28 mmol) was added at 0° C. and stirred for 1 hour.

[0178] Solution B Boc-MePhe-Phe-OH (0.544 g, 1.28 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, cyclopentyl methyl ether (20 mL) was added and concentrated, and N,N-diisopropylethylamine (1.50 g, 11.6 mmol) and acetonitrile (8 mL) were added and concentrated. The resulting residue was mixed with acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.621 g, 2.99 mmol) and stirred at 25° C. for 20 minutes to obtain a colorless transparent solution.

[0179] condensation process While keeping solution A cooled to 0°C, solution B was mixed and further stirred at 25°C for 48 hours (raw material: target product = 1:37 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The obtained 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. MASS(ESI+)m / z;(M+H)+588.4

[0180] Synthesis Example 21: Synthesis of Boc-MePhe-Pro-OH [ka]

[0181] Boc-MePhe-OH (1.40g, 5.00mmol), N-methylmorpholine (0.556g, 5.50mmol) were mixed with tetrahydrofuran (30mL), and 3,3-dimethylbutan-2-yl carbonochloridate (0.864g, 5.25mmol) was added at 0°C and stirred for 1 hour. To this solution, H-Pro-OH (0.691g, 6.00mmol), N,O-bis(trimethylsilyl)acetamide (3.85g, 18.6mmol), and acetonitrile (12mL) were mixed separately and stirred at 70°C for 10 minutes to prepare a solution, which was then added and stirred at 0°C for 15 minutes (raw material: target product = 0:100 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (40mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-MePhe-Pro-OH (1.94 g, yield 94%) as a white solid. MASS(ESI+) m / z;(M+H)+377.3

[0182] Synthesis Example 22: Synthesis of Boc-MePhe-MePhe-Pro-OH [ka]

[0183] Solution A Boc-MePhe-OH (0.838 g, 3.00 mmol), triethylamine (0.364 g, 3.60 mmol) and tetrahydrofuran (60 mL) were mixed, and 2,2-dimethylbutanoyl chloride (0.444 g, 3.30 mmol) was added at 0° C. and stirred for 30 minutes.

[0184] Solution B 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, and N,N-diisopropylethylamine (0.865 g, 6.69 mmol) and acetonitrile (20 mL) were added and concentrated. The resulting residue was mixed with acetonitrile (20 mL) and N,O-bis(trimethylsilyl)acetamide (2.31 g, 11.1 mmol) and stirred at 25° C. for 20 minutes to obtain a colorless transparent solution.

[0185] condensation process While cooling Solution A to 0°C, Solution B was mixed and the mixture was stirred at 25°C for 48 hours (raw material: target product = 1:24 (analysis condition 3)). The reaction solution obtained was diluted with ethyl acetate (40 mL) and washed with a 10% by mass aqueous solution of citric acid and a saturated aqueous solution of sodium chloride in that order. The organic layer obtained 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. MASS(ESI+) m / z;(M+H)+538.5

[0186] Synthesis Example 23: Synthesis of H-MePhe-MePhe-Pro-OH·HCl [ka]

[0187] Boc-MePhe-MePhe-Pro-OH (1.61 g, 3.00 mmol) was mixed with 4M-HCl / ethyl acetate (15 mL) and stirred at 25°C for 90 minutes. The resulting reaction solution was concentrated, and ethyl acetate (5 mL) and diisopropyl ether (20 mL) were added and suspended, and the resulting solid was filtered using a Kiriyama funnel. It 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 resulting solid was used in the next step.

[0188] Synthesis Example 24: Synthesis of Boc-Tyr-MePhe-MePhe-Pro-OH [ka]

[0189] Solution A Boc-Tyr-OH (0.282 g, 1.00 mmol), triethylamine (0.122 g, 1.20 mmol) and tetrahydrofuran (10 mL) were mixed, and 2,2-dimethylbutanoyl chloride (0.148 g, 1.10 mmol) was added at 0° C. and stirred for 1 hour.

[0190] Solution B 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. The resulting residue was mixed with acetonitrile (6 mL) and N,O-bis(trimethylsilyl)acetamide (0.773 g, 3.74 mmol) and stirred at 25°C for 20 minutes to obtain a colorless, transparent solution.

[0191] condensation process While keeping solution A cooled to 0°C, solution B was mixed and further stirred at 25°C for 1 hour. The obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The obtained organic layer was concentrated and purified by silica gel column chromatography to obtain Boc-Tyr-MePhe-MePhe-Pro-OH (0.698 g, yield 99%) as a white solid. MASS(ESI+) m / z;(M+H)+701.5

[0192] Synthesis Example 25: Synthesis of Boc-MePhe-MeAla-Tyr-OH [ka]

[0193] Solution A Boc-MePhe-OH (0.894 g, 3.20 mmol), triethylamine (0.389 g, 3.84 mmol) and tetrahydrofuran (25 mL) were mixed, and 2,2-dimethylbutanoyl chloride (0.474 g, 3.52 mmol) was added at 0° C. and stirred for 45 minutes.

[0194] Solution B H-MeAla-Tyr-OH (0.937 g, 3.52 mmol) was mixed with acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (2.28 g, 10.9 mmol), and the mixture was stirred at 25° C. for 15 minutes to obtain a colorless, transparent solution.

[0195] condensation process While cooling solution A to 0°C, solution B was mixed and the mixture was stirred at 25°C for 2 hours (raw material:target product=0:100 (analysis condition 1)). The reaction solution obtained was concentrated, then diluted with ethyl acetate (30 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The organic layer obtained was washed successively with 10% by mass aqueous citric acid solution (20 mL), and saturated aqueous sodium chloride solution. The organic layer obtained was concentrated, then purified by silica gel column chromatography to obtain Boc-MePhe-MeAla-Tyr-OH (1.67 g, yield 94%) as a white solid. MASS(ESI+) m / z;(M+H)+528.3

[0196] Synthesis Example 26: Synthesis of H-MePhe-MeAla-Tyr-OH [ka]

[0197] Boc-MePhe-MeAla-Tyr-OH (0.474 g, 0.898 mmol) was mixed with 4M-HCl / ethyl acetate (5 mL) and stirred at 25° C. for 60 minutes. The resulting reaction solution was concentrated, and acetonitrile (5 mL) and N,N-diisopropylethylamine (1.74 g, 13.5 mmol) were added and suspended, and the resulting solid was collected by filtration using a Kiriyama funnel. It 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. MASS(ESI+) m / z;(M+H)+428.8

[0198] Synthesis Example 27: Synthesis of Boc-Phe-MePhe-MeAla-Tyr-OH [ka]

[0199] Boc-Phe-OH (0.125g, 0.470mmol), triethylamine (0.057g, 0.564mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.070g, 0.52mmol) was added at 0°C and stirred for 45 minutes. To this solution, H-MePhe-MeAla-Tyr-OH (0.221g, 0.517mmol), N,O-bis(trimethylsilyl)acetamide (0.332g, 1.60mmol), and acetonitrile (5.0mL) were mixed separately and stirred at 25°C for 15 minutes to prepare a solution, which was then added and stirred for 2 hours at 25°C (raw material: target product = 1:80 (analysis condition 2)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40mL), and washed successively with 10% by mass 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. MASS(ESI+) m / z;(M+H)+675.5

[0200] Synthesis Example 28: Synthesis of H-Phe-MePhe-MeAla-Tyr-OH [ka]

[0201] 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 and suspended, and the resulting solid was collected by filtration using a Kiriyama funnel. It 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. MASS(ESI+) m / z;(M+H)+575.4

[0202] Synthesis Example 29: Synthesis of Boc-MePhe-Phe-MePhe-MeAla-Tyr-OH [ka]

[0203] Boc-MePhe-OH (0.092g, 0.33mmol), triethylamine (0.040g, 0.396mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.0489g, 0.363mmol) was added at 0°C and stirred for 45 minutes. To this solution, H-Phe-MePhe-MeAla-Tyr-OH (0.209g, 0.363mmol), N,O-bis(trimethylsilyl)acetamide (0.233g, 1.12mmol), and acetonitrile (5.0mL) were mixed separately and stirred at 25°C for 15 minutes to prepare a solution, which was then added and stirred for 1 hour at 25°C (raw material: target product = 0:100 (analysis condition 2)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10% by mass aqueous citric acid and saturated aqueous sodium chloride solution. The resulting 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. MASS(ESI+) m / z;(M+H)+836.5

[0204] Synthesis Example 30: Synthesis of Cbz-MeAla-Phe-OH [ka]

[0205] Cbz-MeAla-OH (1.19 g, 5.00 mmol), triethylamine (0.607 g, 6.00 mmol) were mixed with tetrahydrofuran (50 mL), and 2,2-dimethylbutanoyl chloride (0.740 g, 5.50 mmol) was added at 0 ° C. and stirred for 45 minutes. To this solution, H-Phe-OH (0.991 g, 6.00 mmol), N,O-bis(trimethylsilyl)acetamide (2.57 g, 12.4 mmol), and acetonitrile (17 mL) were mixed separately and stirred at 75 ° C. for 60 minutes under microwave irradiation to prepare a solution, and the mixture was further stirred at 0 ° C. for 1 hour (raw material: target product = 1:62 (analysis condition 1)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (60 mL), water (60 mL), and saturated aqueous sodium chloride solution (30 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution (30 mL), and saturated aqueous sodium chloride solution (30 mL). The resulting organic layer was concentrated to obtain Cbz-MeAla-Phe-OH (1.76 g, 91% yield) as a colorless transparent syrup. MASS(ESI+) m / z;(M+H)+385.3

[0206] Synthesis Example 31: Synthesis of H-MeAla-Phe-OH [ka]

[0207] 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% by mass palladium-carbon (0.486 g) were mixed with methanol (50 mL) and stirred at 60° C. for 1 hour. The reaction solution was filtered through Celite and washed three times with methanol (20 mL). The obtained filtrate was concentrated to obtain H-MeAla-Phe-OH (1.03 g, 90%) as a white solid. MASS(ESI+) m / z;(M+H)+251.2

[0208] Synthesis Example 32: Synthesis of Boc-MePhe-MeAla-Phe-OH [ka]

[0209] Boc-MePhe-OH (0.559g, 2.00mmol), triethylamine (0.243g, 2.40mmol) were mixed with tetrahydrofuran (30mL), and 2,2-dimethylbutanoyl chloride (0.296g, 2.20mmol) was added at 0°C and stirred for 45 minutes. To this solution, a solution was added which was prepared by mixing H-MeAla-Phe-OH (0.601g, 2.40mmol), N,O-bis(trimethylsilyl)acetamide (1.03g, 4.95mmol), and acetonitrile (20mL) separately and stirring at 25°C for 40 minutes, and further stirring was continued for 2 hours at 0°C (raw material: target product = 1:55 (analysis condition 1)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (20 mL), and washed twice with saturated aqueous sodium bicarbonate (30 mL), water (30 mL), and saturated aqueous sodium chloride (15 mL). The resulting organic layer was washed twice with 10% by mass aqueous citric acid (20 mL), and saturated aqueous sodium chloride (20 mL). The resulting organic layer was concentrated to give Boc-MePhe-MeAla-Phe-OH (1.08 g, 105% yield) as a white solid. MASS(ESI+) m / z;(M+H)+512.3

[0210] Synthesis Example 33: Synthesis of H-MePhe-MeAla-Phe-OH·HCl [ka]

[0211] Boc-MePhe-MeAla-Phe-OH (1.06 g, 2.08 mmol) was mixed with 4M-HCl / ethyl acetate (20 mL) and stirred at 25°C for 60 minutes. The resulting reaction solution was concentrated, suspended in diisopropyl ether (20 mL), and the resulting solid was filtered using a Kiriyama funnel. It 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. MASS(ESI+) m / z;(M+H)+411.5

[0212] Synthesis Example 34: Synthesis of Fmoc-Trp(Boc)-MePhe-MeAla-Phe-OH [ka]

[0213] Fmoc-Trp(Boc)-OH (0.439g, 0.833mmol), triethylamine (0.101g, 1.00mmol) were mixed with tetrahydrofuran (30mL), and 2,2-dimethylbutanoyl chloride (0.123g, 0.917mmol) was added at 0℃ and stirred for 45 minutes. To this solution, H-MePhe-MeAla-Phe-OH·HCl (0.448g, 1.00mmol), N,O-bis(trimethylsilyl)acetamide (0.428g, 2.06mmol), N,N-diisopropylethylamine (1.08g, 8.33mmol), and acetonitrile (20mL) were mixed separately and stirred at 25℃ for 40 minutes. The solution was added and further stirred at 25℃ for 3 hours (raw material: target product = 1:12 (analysis condition 1)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (20 mL), and washed twice with saturated aqueous sodium bicarbonate solution (30 mL), water (30 mL), and saturated aqueous sodium chloride solution (12 mL). The resulting organic layer was washed twice with 10% by mass aqueous citric acid solution (20 mL), and saturated aqueous sodium chloride solution (20 mL). The resulting organic layer was concentrated, and the residue was dissolved in ethyl acetate (5.0 mL) and poured into hexane (95 mL). The precipitated solid was collected by filtration to obtain Fmoc-Trp(Boc)-MePhe-MeAla-Phe-OH (0.657 g, 86% yield) as a white solid. MASS(ESI+) m / z;(M+H)+920.5

[0214] Synthesis Example 35: Synthesis of Fmoc-Gln(Trt)-MePhe-MeAla-Tyr-OH [ka]

[0215] Solution A Fmoc-Gln(Trt)-OH (0.204 g, 0.333 mmol), triethylamine (0.040 g, 0.40 mmol) were mixed with tetrahydrofuran (20 mL), and 2,2-dimethylbutanoyl chloride (0.049 g, 0.37 mmol) was added at 0° C. and stirred for 45 minutes.

[0216] Solution B 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. The resulting residue was mixed with acetonitrile (10 mL) and N,O-bis(trimethylsilyl)acetamide (0.343 g, 1.65 mmol), and stirred at 25° C. for 40 minutes to obtain a colorless, transparent solution.

[0217] condensation process While cooling solution A to 0°C, solution B was mixed and the mixture was stirred at 25°C for 23 hours (raw material: target product = 1:5 (analysis condition 1)). The resulting reaction solution was concentrated, diluted with ethyl acetate (20 mL), and washed twice with saturated aqueous sodium bicarbonate solution (15 mL), water (15 mL), and saturated aqueous sodium chloride solution (7.5 mL). The resulting organic layer was washed twice with 10% by mass aqueous citric acid solution (20 mL), and saturated aqueous sodium chloride 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. MASS(ESI+) m / z;(M+H)+1020.6

[0218] Synthesis Example 36: Synthesis of Fmoc-BnGly-Phe-OH [ka]

[0219] Fmoc-BnGly-OH (0.387g, 1.00mmol), N-methylmorpholine (0.111g, 1.10mmol) were mixed with tetrahydrofuran (20mL), and isopropyl chloroformate (0.129g, 1.05mmol) was added at 0°C and stirred for 5 minutes. A solution was added to this solution, which was prepared by mixing H-Phe-OH (0.198g, 1.20mmol), N,O-bis(trimethylsilyl)acetamide (0.519g, 2.48mmol), and acetonitrile (5mL) separately and stirring at 75°C for 60 minutes under microwave irradiation, and the mixture was stirred at 0°C for 15 minutes and further stirred at 25°C for 20 hours (raw material: target product = 1:55 (analysis condition 1)). The resulting reaction solution was concentrated, diluted with ethyl acetate (80 mL), and washed successively with 10% by mass citric acid aqueous solution (50 mL), 10% by mass 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-BnGly-Phe-OH (0.570 g, yield 107%) as a white solid. MASS(ESI+) m / z;(M+H)+535.3

[0220] Synthesis Example 37: Synthesis of H-BnGly-Phe-OH [ka]

[0221] Fmoc-BnGly-Phe-OH (0.535 g, 1.00 mmol) and triethylamine (2.02 g, 20.0 mmol) were mixed with acetonitrile (20 mL) and stirred at 60° C. for 60 minutes. The resulting reaction solution was concentrated, and diisopropyl ether (50 mL) was added and suspended, and the resulting solid was filtered using a Kiriyama funnel. It was washed with diisopropyl ether and dried to obtain H-BnGly-Phe-OH (0.293 g, 94% yield) as a white solid. MASS(ESI+) m / z;(M+H)+313.2

[0222] Synthesis Example 38: Synthesis of Boc-Phe-BnGly-Phe-OH [ka]

[0223] Boc-Phe-OH (0.066g, 0.25mmol), triethylamine (0.030g, 0.30mmol) were mixed with tetrahydrofuran (5mL), and 2,2-dimethylbutanoyl chloride (0.037g, 0.28mmol) was added at 0°C and stirred for 45 minutes. To this solution, H-BnGly-Phe-OH (0.093g, 0.30mmol), N,O-bis(trimethylsilyl)acetamide (0.129g, 0.616mmol), and acetonitrile (4mL) were mixed separately and stirred at 25°C for 20 minutes to prepare a solution, which was then added and stirred for 14 hours at 25°C (raw material: target product = 1:93 (analysis condition 3)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution (20 mL). The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-Phe-BnGly-Phe-OH (0.139 g, 100% yield) as a white solid. MASS(ESI+) m / z;(M+H)+560.4

[0224] Synthesis Example 39: Synthesis of Fmoc-n-PrGly-Phe-OH [ka]

[0225] Fmoc-n-PrGly-OH (0.339g, 1.00mmol), triethylamine (0.121g, 1.20mmol) were mixed with tetrahydrofuran (15mL), and 2,2-dimethylbutanoyl chloride (0.148g, 1.10mmol) was added at 0°C and stirred for 45 minutes. To this solution, H-Phe-OH (0.198g, 1.20mmol), N,O-bis(trimethylsilyl)acetamide (0.519g, 2.48mmol), and acetonitrile (4.5mL) were mixed separately and stirred at 75°C for 60 minutes under microwave irradiation to prepare a solution, and the mixture was stirred for 1 hour at 0°C (raw material: target product = 1:32 (analysis condition 1)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (20 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively twice each with 10% by mass aqueous citric acid solution (20 mL) and saturated aqueous sodium chloride solution (20 mL). The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Fmoc-n-PrGly-Phe-OH (0.538 g, 111% yield) as a white solid. MASS(ESI+) m / z;(M+H)+487.3

[0226] Synthesis Example 40: Synthesis of Hn-PrGly-Phe-OH [ka]

[0227] Fmoc-n-PrGly-Phe-OH (0.487 g, 1.00 mmol) and triethylamine (2.02 g, 20.0 mmol) were mixed with acetonitrile (20 mL) and stirred at 60° C. for 60 minutes. The resulting reaction solution was concentrated, and diisopropyl ether (50 mL) was added and suspended, and the resulting solid was filtered using a Kiriyama funnel. It was washed with diisopropyl ether and dried to obtain Hn-PrGly-Phe-OH (0.245 g, 93% yield) as a white solid. MASS(ESI+) m / z;(M+H)+265.2

[0228] Synthesis Example 41: Synthesis of Boc-Phe-n-PrGly-Phe-OH [ka]

[0229] Boc-Phe-OH (0.066g, 0.25mmol), triethylamine (0.030g, 0.300mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.037g, 0.27mmol) was added at 0°C and stirred for 45 minutes. To this solution, Hn-PrGly-Phe-OH (0.066g, 0.25mmol), N,O-bis(trimethylsilyl)acetamide (0.118g, 0.564mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 25°C for 30 minutes to prepare a solution, which was then added and stirred at 25°C for 1 hour or more (raw material: target product = 1:85 (analysis condition 3)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-Phe-n-PrGly-Phe-OH (0.125 g, yield 98%) as a white solid. MASS(ESI+) m / z;(M+H)+512.4

[0230] Synthesis Example 42: Synthesis of Fmoc-MePhe-n-PrGly-Phe-OH [ka]

[0231] Fmoc-MePhe-OH (0.080g, 0.20mmol), triethylamine (0.024g, 0.24mmol) were mixed with tetrahydrofuran (5mL), and 2,2-dimethylbutanoyl chloride (0.030g, 0.22mmol) was added at 0°C and stirred for 45 minutes. To this solution, Hn-PrGly-Phe-OH (0.063g, 0.24mmol), N,O-bis(trimethylsilyl)acetamide (0.104g, 0.50mmol), and acetonitrile (2mL) were mixed separately and stirred at 0°C for 30 minutes to prepare a solution, which was then added and stirred for 2 hours at 25°C (raw material: target product = 1:18 (analysis condition 1)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10% by mass citric acid aqueous solution (30 mL), 5% by mass 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. MASS(ESI+) m / z;(M+H)+648.4

[0232] Synthesis Example 43: Synthesis of Boc-MePhe-MePhe-MeAla-Tyr-OH [ka]

[0233] Boc-MePhe-OH (0.084g, 0.30mmol), triethylamine (0.036g, 0.36mmol) were mixed with tetrahydrofuran (6.0mL), and 2,2-dimethylbutanoyl chloride (0.044g, 0.331mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MePhe-MeAla-Tyr-OH (0.141g, 0.305mmol), N,O-bis(trimethylsilyl)acetamide (0.215g, 1.023mmol), N,N-diisopropylethylamine (0.086g, 0.662mmol), and acetonitrile (5.0mL) were mixed separately and stirred at 0°C for 60 minutes to prepare a solution, which was then added and stirred for 16 hours at 25°C (raw material: target product = 1:59 (analysis condition 3)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-MePhe-MePhe-MeAla-Tyr-OH (0.177 g, yield 85%) as a white solid. MASS(ESI+)m / z;(M+H)+689.5

[0234] Synthesis Example 44: Synthesis of Fmoc-Glu(tBu)-MePhe-MePhe-MeAla-Tyr-OH [ka]

[0235] Solution A Fmoc-Glu(tBu)-OH (0.091 g, 0.21 mmol), triethylamine (0.026 g, 0.257 mmol) and tetrahydrofuran (5.0 mL) were mixed, and 2,2-dimethylbutanoyl chloride (0.032 g, 0.24 mmol) was added at 0° C. and stirred for 1 hour.

[0236] Solution B Boc-MePhe-MePhe-MeAla-Tyr-OH (0.177 g, 0.257 mmol) and trifluoroacetic acid (3.0 mL) were mixed and stirred at 25°C for 30 minutes. The solution was concentrated to remove trifluoroacetic acid, and acetonitrile (20 mL) was mixed and concentrated. Acetonitrile (8.0 mL) and triethylamine (0.026 g, 0.257 mmol) were added to the obtained residue, and the solution was concentrated to remove the solvent and triethylamine. Acetonitrile (5.0 mL) and N,O-bis(trimethylsilyl)acetamide (0.167 g, 0.80 mmol) were mixed with the obtained residue and stirred at 25°C for 5 minutes to obtain a colorless transparent solution.

[0237] condensation process While cooling Solution A to 0°C, Solution B was mixed and the mixture was stirred at 25°C for 17 hours (raw material: target product = 1:5 (analysis condition 3)). The reaction solution obtained was concentrated, diluted with ethyl acetate (30 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The organic layer obtained was concentrated to obtain Fmoc-Glu(tBu)-MePhe-MePhe-MeAla-Tyr-OH (0.192 g, 90% yield) as a white solid. MASS(ESI+)m / z;(M+H)+730.4,+996.5

[0238] Synthesis Example 45: Synthesis of Fmoc-Cys(Trt)-MePhe-OH [ka]

[0239] Fmoc-Cys(Trt)-OH (0.147g, 0.251mmol), triethylamine (0.031g, 0.30mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.037g, 0.28mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.054g, 0.30mmol), N,O-bis(trimethylsilyl)acetamide (0.130g, 0.62mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 75°C for 20 minutes to prepare a solution, which was then added and stirred at 0°C for 1 hour (raw material: target product = 1:28 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40mL), and washed successively with 10% by mass citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution. The resulting 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. MASS(ESI+) m / z;(M+H)+243.2 (trityl cation), +747.2

[0240] Synthesis example 46: Boc-Arg(Cbz) 2 Synthesis of -MePhe-OH [ka]

[0241] Boc-Arg(Cbz)2-OH (0.136g, 0.250mmol), triethylamine (0.030g, 0.30mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.037g, 0.28mmol) was added at 0°C and stirred for 45 minutes. To this solution, H-MePhe-OH (0.054g, 0.30mmol), N,O-bis(trimethylsilyl)acetamide (0.128g, 0.62mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 75°C for 10 minutes to prepare a solution, which was then added and stirred at 25°C for 1 hour or more (raw material: target product = 1:25 (analysis condition 2)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-Arg(Cbz)2-MePhe-OH (0.175 g, yield 99%) as a white solid. MASS(ESI+) m / z;(M+H)+704.5

[0242] Synthesis example 47: Boc-Arg(Cbz) 2 Synthesis of -MeAla-Phe-OH [ka]

[0243] Boc-Arg(Cbz)2-OH (0.136g, 0.251mmol), triethylamine (0.030g, 0.30mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.037g, 0.28mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MeAla-Phe-OH (0.075g, 0.30mmol), N,O-bis(trimethylsilyl)acetamide (0.130g, 0.621mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 25°C for 20 minutes to prepare a solution, which was then added and stirred for 14 hours at 25°C (raw material: target product = 1:13 (analysis condition 2)). The resulting reaction solution was concentrated, then diluted with ethyl acetate (40 mL), and washed successively with saturated aqueous sodium bicarbonate solution (20 mL), water (20 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was washed successively with 10% by mass aqueous citric acid solution and saturated aqueous sodium chloride solution. The resulting organic layer was concentrated, then purified by silica gel column chromatography to obtain Boc-Arg(Cbz)2-MeAla-Phe-OH (0.192 g, yield 99%) as a white solid. MASS(ESI+) m / z;(M+H)+775.5

[0244] Synthesis Example 48: Synthesis of Fmoc-His(Boc)-MePhe-OH [ka]

[0245] Fmoc-His(Boc)-OH (0.120g, 0.251mmol), N-methylmorpholine (0.033g, 0.33mmol) were mixed with tetrahydrofuran (5.0mL), and 4-ethyl-2,2-dimethylhexane-3-yl carbonochloridate (0.067g, 0.30mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.058g, 0.33mmol), N,O-bis(trimethylsilyl)acetamide (0.141g, 0.679mmol), and acetonitrile (4mL) separately and stirring at 75°C for 20 minutes, and further stirring at 25°C for 13 hours (raw material: target product = 1:6 (analysis condition 2)). The obtained reaction solution was diluted with ethyl acetate (40mL) and washed successively with 10% by mass 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-His(Boc)-MePhe-OH (0.131 g, yield 82%) as a white solid. MASS(ESI+)m / z;(M+H)+539.6,+639.4

[0246] Synthesis Example 49: Synthesis of Fmoc-MeHis(Trt)-Leu-OH [ka]

[0247] Solution A Fmoc-MeHis(Trt)-OH (1.27 g, 2.00 mmol), N,N-dimethylformamide (0.015 g, 0.20 mmol) were mixed with tetrahydrofuran (30 mL), 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 the solvent, and tetrahydrofuran (10 mL) was mixed and concentrated. Tetrahydrofuran (10 mL) was added to the resulting residue to obtain a pale yellow, transparent acid chloride solution.

[0248] Solution B 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, transparent solution.

[0249] condensation process While keeping solution A cooled to 0°C, solution B was mixed and further stirred at 0°C for 1 hour (raw material: target product = 1:25 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with a 10% by mass aqueous solution of citric acid and a saturated aqueous solution of sodium chloride. 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. MASS(ESI+) m / z;(M+H)+747.4

[0250] Synthesis Example 50: Synthesis of H-MeHis(Trt)-Leu-OH [ka]

[0251] Fmoc-His(Trt)-Leu-OH (0.202 g, 0.270 mmol), triethylamine (0.564 g, 5.40 mmol) were mixed with acetonitrile (4.0 mL) and tetrahydrofuran (4.0 mL) 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 the mixture and suspended. The resulting solid was filtered using a Kiriyama funnel. It 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. MASS(ESI+) m / z;(M+H)+525.8

[0252] Synthesis Example 51: Synthesis of Fmoc-Phe-MeHis(Trt)-Leu-OH [ka]

[0253] Fmoc-Phe-OH (0.078g, 0.20mmol), triethylamine (0.024g, 0.24mmol) were mixed with tetrahydrofuran (5.0mL), and 2,2-dimethylbutanoyl chloride (0.030g, 0.22mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MeHis(Trt)-Leu-OH (0.116g, 0.221mmol), N,O-bis(trimethylsilyl)acetamide (0.096g, 0.46mmol), and acetonitrile (4.0mL) were mixed separately and stirred at 25°C for 30 minutes to prepare a solution, which was then added and stirred for 5 hours at 25°C (raw material: target product = 1:5 (analysis condition 2)). The resulting reaction solution was concentrated, diluted with ethyl acetate (40 mL), and washed successively with 10% by mass aqueous citric acid, saturated aqueous sodium bicarbonate, water, and saturated aqueous sodium chloride, and the resulting organic layer was concentrated to give Fmoc-Phe-MeHis(Trt)-Leu-OH (0.154 g, yield 86%) as a white solid. MASS(ESI+)m / z;(M+H)+894.5

[0254] Synthesis Example 52: Synthesis of Fmoc-Val-MePhe-OH [ka]

[0255] Fmoc-Val-OH (0.101g, 0.298mmol), triethylamine (0.053mL, 0.383mmol) were mixed with tetrahydrofuran (1.0g), and pivaloyl chloride (0.043mL, 0.354mmol) was added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.080g, 0.446mmol), trimethylsilyl chloride (0.070mL, 0.554mmol), triethylamine (0.082mL, 0.592mmol), and acetonitrile (0.80g) were mixed separately and stirred at 50°C for 1.5 hours to prepare a solution, which was then added and stirred for 70 hours at 0°C (raw material: target product = 3.5:1). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% by mass aqueous citric acid solution (3.0 g) and saturated saline solution (2.0 g). HPLC analysis of the resulting organic layer showed that the quantitative yield was 11%.

[0256] Hereinafter, unless otherwise specified, the ratio of the raw material Fmoc-Val-OH to the product Fmoc-Val-MePhe-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 4>.

[0257] <Analysis conditions 4> High performance liquid chromatography: SHIMADZU HPLC LC-20A Column: Agilent Poroshell 120EC-C18 (2.7 μm, 3.0 × 100 mm) Column oven temperature: 40℃ Eluent: Acetonitrile: 0.05 vol% phosphoric acid aqueous solution 50:50 (0-15 minutes), 50:50-95:5 (15-18 minutes), 95:5 (18-22 minutes) (v / v) Eluent rate: 0.7 mL / min Detection wavelength: 210 nm

[0258] Hereinafter, unless otherwise specified, the quantitative yield of Fmoc-Val-MePhe-OH was calculated by the quantitative analysis method under <Analysis Condition 4>. Standard substance: Fmoc-Val-MePhe-OH obtained by the method described in Synthesis Example 57 was purified by silica gel chromatography to prepare a standard substance. MASS(ESI+) m / z;(M+H)+501.22 Quantitative method: Absolute calibration method

[0259] Synthesis Example 53: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.38mmol) were mixed with tetrahydrofuran (1.0g), and pivaloyl chloride (0.043mL, 0.35mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.44mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.81g) separately and stirring at 50°C for 1.5 hours, and further stirring was continued for 70 hours at 0°C (raw material: target product = 1:7). The obtained reaction solution was diluted with ethyl acetate (5.0g) and washed twice with 10% by mass aqueous citric acid solution (2.0g) and saturated saline solution (1.0g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 79%.

[0260] Synthesis Example 54: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), N-methylmorpholine (0.042mL, 0.383mmol) were mixed with tetrahydrofuran (1.0g), and isobutyl carbonochloridate (0.046mL, 0.35mmol) was added at 0°C and stirred for 5 minutes. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.44mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and further stirring was continued for 21 hours at 0°C (raw material: target product = 3.5:1). The obtained reaction solution was diluted with ethyl acetate (5.0g) and washed twice with 10% by mass aqueous citric acid solution (2.0g) and saturated saline solution (1.0g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 18%.

[0261] Synthesis Example 55: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.38mmol) were mixed with tetrahydrofuran (1.0g), and 2-ethylbutanoyl chloride (0.048mL, 0.35mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.44mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and further stirring was continued for 110 hours at 0°C (raw material: target product = 1:13). The obtained reaction solution was diluted with ethyl acetate (5.0g) and washed twice with 10% by mass aqueous citric acid solution (2.0g) and saturated saline solution (1.0g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 87%.

[0262] Synthesis Example 56: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.38mmol) were mixed with tetrahydrofuran (1.0g), and 2,2-dimethylbutanoyl chloride (0.049mL, 0.35mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.44mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and further stirring was continued for 95 hours at 0°C (raw material: target product = 1:18). The obtained reaction solution was diluted with ethyl acetate (5.0g) and washed twice with 10% by mass citric acid aqueous solution (2.0g) and saturated saline solution (1.0g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 89%.

[0263] Synthesis Example 57: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), N,N-diisopropylethylamine (0.058mL, 0.32mmol) was mixed with acetonitrile (1.0g), and 50% by mass toluene solution (0.107g, 0.354mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at 0°C and stirred for 2 hours. To this solution, H-MePhe-OH (0.079g, 0.442mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) were mixed separately and stirred at 50°C for 1 hour to prepare a solution, and the mixture was stirred at 0°C for 67 hours and at 20°C for 7 hours (raw material: target product = 1:35). The reaction mixture 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% by mass aqueous citric acid (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 96%.

[0264] 2-(4,4-Dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was synthesized with reference to Japanese Patent No. 3406093.

[0265] Synthesis Example 58: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), N-methylmorpholine (0.071mL, 0.648mmol) was mixed with N,N-dimethylacetamide (5.9mL), and 2,4-dimethylpentan-3-yl carbonochloridate (0.105g, 0.589mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.106g, 0.589mmol), N,O-bis(trimethylsilyl)acetamide (0.288mL, 1.179mmol), and acetonitrile (7.9mL) separately and stirring at 75°C for 15 minutes, and the mixture was stirred at 0°C for 114 hours and at 20°C for 4 hours (raw material: target product = 1:11). The reaction mixture 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% by mass aqueous citric acid (18.0 g) and saturated saline solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 85%.

[0266] Synthesis Example 59: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), N-methylmorpholine (0.071mL, 0.648mmol) was mixed with N,N-dimethylacetamide (5.9mL), and 2,2,4-tritylpentan-3-yl carbonochloridate (0.114g, 0.589mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.106g, 0.589mmol), N,O-bis(trimethylsilyl)acetamide (0.288mL, 1.179mmol), and acetonitrile (7.9mL) separately and stirring at 75°C for 15 minutes, and the mixture was stirred at 0°C for 91 hours and at 20°C for 7 hours (raw material: target product = 1:10). The reaction mixture 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% by mass aqueous citric acid (18.0 g) and saturated saline solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 88%.

[0267] Synthesis Example 60: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), N,N-diisopropylethylamine (0.065mL, 0.38mmol) were mixed with tetrahydrofuran (1.0g), and 1-adamantanecarbonyl chloride (0.070g, 0.35mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.44mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and the mixture was further stirred at 0°C for 96 hours (raw material: target product = 1:10). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% by mass aqueous citric acid solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 90%.

[0268] Synthesis Example 61: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.10g, 0.30mmol), N-methylmorpholine (0.071mL, 0.65mmol) was mixed with N,N-dimethylacetamide (5.9mL), 4-ethyl-2,2-dimethylhexane-3-yl carbonochloridate (0.13g, 0.59mmol) was added at 0°C, and the mixture was stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.106g, 0.59mmol), N,O-bis(trimethylsilyl)acetamide (0.29mL, 1.18mmol), and acetonitrile (7.9mL) separately and stirring at 75°C for 15 minutes, and the mixture was stirred for 164 hours at 0°C (raw material: target product = 1:9). The reaction mixture 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% by mass citric acid solution (18.0 g), saturated saline solution (10.0 g), and saturated saline solution (6.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 83%.

[0269] Synthesis Example 62: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.383mmol) were mixed with tetrahydrofuran (1.0g), and 2-ethylhexanoyl chloride (0.061mL, 0.354mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.442mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and further stirring was continued for 111 hours at 0°C (raw material: target product = 1:11). The obtained reaction solution was diluted with ethyl acetate (5.0g) and washed twice with 10% by mass aqueous citric acid solution (2.0g) and saturated saline solution (1.0g). The quantitative yield of Fmoc-Val-MePhe-OH in the collected organic layer was 90%.

[0270] Synthesis Example 63: Synthesis of Fmoc-Val-MePhe-OH Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.383mmol) were mixed with tetrahydrofuran (1.0g), and 2-(4-chlorophenyl)-3-methylbutanoyl chloride (0.070mL, 0.354mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.442mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and the mixture was further stirred at 0°C for 66 hours (raw material: target product = 1:10). The resulting reaction solution was diluted with ethyl acetate (5.0 g) and washed twice with 10% by mass aqueous citric acid solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of Fmoc-Val-MePhe-OH from the collected organic layer was 84%.

[0271] Synthesis Example 64: Synthesis of Fmoc-Val-MePhe-OH [ka]

[0272] Fmoc-Val-OH (0.100g, 0.295mmol), triethylamine (0.053mL, 0.383mmol) were mixed with tetrahydrofuran (1.0g), and 3,5,5-trimethylhexanoyl chloride (0.067mL, 0.354mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.079g, 0.442mmol), N,O-bis(trimethylsilyl)acetamide (0.135mL, 0.552mmol), and acetonitrile (0.79g) separately and stirring at 50°C for 1 hour, and the mixture was further stirred at 0°C for 68 hours (raw material: target product = 16:1).

[0273] Synthesis Example 65: Synthesis of Cbz-Phe-MePhe-Phe-OH [ka]

[0274] Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) were mixed, and pivaloyl chloride (24 mg, 0.20 mmol) was added at 0 ° C. and stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the temperature was raised to 25 ° C. and stirring was continued for 3 hours (raw material: target product = 1:10 (analysis condition 5)). The obtained reaction solution was diluted with ethyl acetate (2.5 g), separated with 10 mass% citric acid aqueous solution (1.0 g), and washed twice with 10 mass% sodium chloride aqueous solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the obtained organic layer was 77% (analysis condition 5). MASS(ESI+)m / z;(M+H)+608.44

[0275] Hereinafter, unless otherwise specified, the ratio of the raw material Cbz-Phe-OH to the product Cbz-Phe-MePhe-Phe-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 5>.

[0276] <Analysis conditions 5> High performance liquid chromatography: SHIMADZU HPLC-20A Column: Agilent Poroshell 120EC-C18 (2.7 μm, 3.0 × 100 mm) Column oven temperature: 50℃ Eluent: 0.2 vol% phosphoric acid Acetonitrile solution: 0.2 vol% phosphoric acid aqueous solution 12:88-95:5(0-15 minutes), 95:5(15-19 minutes), (v / v) Eluent rate: 0.7 mL / min Detection wavelength: 214 nm

[0277] Hereinafter, unless otherwise specified, the quantitative yield of Cbz-Phe-MePhe-Phe-OH was calculated by the quantitative analysis method under <Analysis Condition 5>. Standard substance: Cbz-Phe-MePhe-Phe-OH synthesized by the method described in Synthesis Example 71 was purified by silica gel chromatography to prepare a standard substance. NMR and MASS of standard materials are shown. 1 H NMR (300MHz, DMSO-d6): 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) MASS(ESI+)m / z;(M+H)+608.44 Quantitative method: Absolute calibration method

[0278] Synthesis Example 66: Synthesis of H-MePhe-Phe-OH [ka]

[0279] Cbz-MePhe-OH (2.0 g, 6.38 mmol), methylene chloride (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-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (3.28 g, 7.66 mmol) was added at 22°C and stirred for 1.5 hours. 10% by mass hydrochloric acid (16 g) and water (16 g) were added to the resulting reaction solution, and the organic layer was washed with water twice. Then, 10% by mass potassium hydrogen carbonate aqueous solution (16 g) and water (16 g) were added and the organic layer was washed with water twice. The obtained organic layer was concentrated under reduced pressure, purified by silica gel column chromatography, and diluted with trifluoroethanol (40 g). 10% by mass Pd-C (0.4 g) was added to the obtained solution, and the mixture was stirred at room temperature for 14.5 hours under a hydrogen gas atmosphere. The reaction solution was concentrated under reduced pressure, diluted with methanol (300 g), and filtered. The filtrate obtained was diluted again with methanol (300 g) and filtered three times. The filtrate obtained was concentrated, diluted with acetonitrile (100 g), and concentrated again to obtain H-MePhe-Phe-OH (1.35 g, yield 65%) as a white solid. MASS(ESI+) m / z;(M+H)+327.2

[0280] Synthesis Example 67: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and N-methylmorpholine (20 mg, 0.20 mmol) were mixed, and isobutyl carbonochloridate (27 mg, 0.20 mmol) was added at 0 ° C. and stirred for 3 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the mixture was heated to 25 ° C. and stirred for 3 hours (raw material: target product = 1: 1.6). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10 mass% citric acid aqueous solution (1.0 g), and washed twice with 10 mass% sodium chloride aqueous solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 43%.

[0281] Synthesis Example 68: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) were mixed, and 2,2-dimethylbutanoyl chloride (27 mg, 0.20 mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50°C for 1 hour, and the mixture was heated to 25°C and stirred for 21 hours (raw material: target product = 1:24). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with a 10% by mass aqueous citric acid solution (1.0 g), and washed twice with a 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 95%.

[0282] Synthesis Example 69: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) were mixed, and 2-ethylbutanoyl chloride (27 mg, 0.20 mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50°C for 1 hour, and the mixture was heated to 25°C and stirred for 27 hours (raw material: target product = 1:30). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with a 10% by mass aqueous citric acid solution (1.0 g), and washed twice with a 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 102%.

[0283] Synthesis Example 70: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and triethylamine (20 mg, 0.20 mmol) were mixed, and 50% by mass toluene solution (124 mg, 0.20 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at 25 ° C. and stirred for 9 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the mixture was heated to 25 ° C. and stirred for 14 hours (raw material: target product = 1:80). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% by mass aqueous citric acid solution (1.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 100%.

[0284] Synthesis Example 71: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (99 mg, 0.33 mmol), acetonitrile (1.0 g), and N,N-diisopropylethylamine (52 mg, 0.40 mmol) were mixed, and 50% by mass toluene solution (250 mg, 0.41 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at room temperature and stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C for 1 hour, and the mixture was stirred at 25 ° C for 21 hours (raw material: target product = 1: 335). The resulting reaction solution was diluted with ethyl acetate (5.0 g), separated with 10% by mass aqueous citric acid solution (3.0 g), and washed twice with saturated aqueous sodium chloride solution (2.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 100%.

[0285] Synthesis Example 72: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) were mixed, and 2,2,4-trimethylpentan-3-yl carbonochloridate (39 mg, 0.20 mmol) was added at 0° C. and stirred for 2 hours. To this solution, a solution was added that was prepared by 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) separately and stirring at 50° C. for 1 hour, and the mixture was heated to 25° C. and stirred for 3 hours (raw material: target product = 1:32). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% by mass aqueous citric acid solution (1.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 99%.

[0286] Synthesis Example 73: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) were mixed, and 2,4-dimethylpentan-3-yl carbonochloridate (39 mg, 0.20 mmol) was added at 0° C. and stirred for 1.5 hours. To this solution, a solution was added that was prepared by 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) separately and stirring at 50° C. for 1 hour, and the mixture was heated to 25° C. and stirred for 29 hours (raw material: target product = 1:15). The resulting reaction solution was diluted with ethyl acetate (2.5 g), separated with 10% by mass aqueous citric acid solution (1.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 83%.

[0287] Synthesis Example 74: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), tetrahydrofuran (0.5 g), and N,N-diisopropylethylamine (30 mg, 0.23 mmol) were mixed, and 1-adamantanecarbonyl chloride (51 mg, 0.26 mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by 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) separately and stirring at 50°C for 1 hour, and stirred at 0°C for 28 hours (raw material: target product = 1:58). The obtained reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% by mass citric acid aqueous solution (3.0 g), and washed twice with saturated sodium chloride aqueous solution (1.0 g). The resulting organic layer was quantified to give Cbz-Phe-MePhe-Phe-OH in a quantitative yield of 98%.

[0288] Synthesis Example 75: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (0.050g, 0.17mmol), N,N-dimethylacetamide (3.0mL), and N-methylmorpholine (0.040mL, 0.37mmol) were mixed, and 4-ethyl-2,2-dimethylhexane-3-yl carbonochloridate (0.074g, 0.33mmol) was added at 0°C and stirred for 1.5 hours. To this solution, a solution was added which was prepared by mixing H-MePhe-Phe-OH (0.109g, 0.334mmol), N,O-bis(trimethylsilyl)acetamide (0.16mL, 0.67mmol), and acetonitrile (4.0mL) separately and stirring at 50°C for 1 hour, and the mixture was heated to room temperature and stirred for 21 hours (raw material: target product = 1:43). The resulting reaction solution was diluted with ethyl acetate (15 g), separated with 10% by mass aqueous citric acid 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 Cbz-Phe-MePhe-Phe-OH in the resulting organic layer was 92%.

[0289] Synthesis Example 76: Synthesis of Cbz-Phe-MePhe-Phe-OH Cbz-Phe-OH (50 mg, 0.17 mmol), N,N-dimethylacetamide (0.5 g), and N-methylmorpholine (22 mg, 0.22 mmol) were mixed, and 3,3-dimethylbutan-2-yl carbonochloridate (39 mg, 0.20 mmol) was added at 0 ° C. and stirred for 1.5 hours. To this solution, a solution was added that was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the mixture was heated to 25 ° C. and stirred for 16 hours (raw material: target product = 1: 1.2).

[0290] Synthesis Example 77: Synthesis of Boc-Val-Pro-OH [ka]

[0291] Boc-Val-OH (128 mg, 0.59 mmol), tetrahydrofuran (1.3 g), and N-methylmorpholine (60 mg, 0.59 mmol) were mixed, and isobutyl carbonochloridate (72 mg, 0.59 mmol) was added at 0° C. and stirred for 2.5 hours. To this solution, a solution was added which was prepared by mixing H-Pro-OH (71 mg, 0.62 mmol), N,N-diisopropylethylamine (0.16 g, 1.2 mmol), trimethylsilyl chloride (0.13 g, 1.2 mmol), and dichloromethane (3 mL) separately and stirring at 40° C. for 2 hours, and the mixture was stirred at 0° C. for 15 hours to obtain Boc-Val-Pro-OH (raw material: target product = 1:15). MASS(ESI+) m / z;(M+H)+315.20

[0292] Hereinafter, unless otherwise specified, the ratio of the raw material Boc-Val-OH to the product Boc-Val-Pro-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 5>.

[0293] Synthesis Example 78: Synthesis of Boc-Val-Pro-OH Boc-Val-OH (128 mg, 0.59 mmol), acetonitrile (1.3 g), and N,N-diisopropylethylamine (91 mg, 0.71 mmol) were mixed, and 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 stirred for 4 hours. A solution prepared by 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) separately and stirring at 50°C for 1 hour was added to this solution, and the mixture was stirred for 15 hours at 25°C (raw material: target product = 1:731). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated with water (2 mL) and 10% by mass aqueous citric acid solution (2 mL), and then washed with 5% by mass aqueous sodium chloride solution (5 mL) and water (5 mL) in that order. The quantitative yield of Boc-Val-Pro-OH in the resulting organic layer was 94%.

[0294] Hereinafter, unless otherwise specified, the quantitative yield of Boc-Val-Pro-OH was calculated by the quantitative analysis method under <Analysis Condition 5>. Standard substance: Boc-Val-Pro-OH, which was isolated and purified separately with reference to Indian Journal of Chemistry, 2004, Vol. 43B, p. 1282, was used as the standard substance. The mass of the standard material is shown. MASS(ESI+) m / z;(M+H)+315.20 Quantitative method: Absolute calibration method

[0295] Synthesis Example 79: Synthesis of Boc-Val-Pro-OH Boc-Val-OH (50 mg, 0.23 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (30 mg, 0.30 mmol) were mixed, and 2,2,4-trimethylpentan-3-yl carbonochloridate (53 mg, 0.28 mmol) was added at 0 ° C. and stirred for 2.5 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the mixture was stirred at 0 ° C. for 15 hours (raw material: target product = 1:99). The resulting reaction solution was diluted with ethyl acetate (5 mL), separated with 10 mass% citric acid aqueous solution (2 mL), and washed twice with 10 mass% sodium chloride aqueous solution (1.0 g). The quantitative yield of Boc-Val-Pro-OH in the resulting organic and aqueous layers was 90%.

[0296] Synthesis Example 80: Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (77 mg, 0.60 mmol) were mixed, and 2,2-dimethylbutanoyl chloride (74 mg, 0.55 mmol) was added at 0°C, and the mixture was stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50°C for 1 hour, and the mixture was stirred at 0°C for 21 hours (raw material: target product = 1:520). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated with water (2 mL) and 10% by mass aqueous citric acid solution (2 mL), and then washed twice with 10% by mass aqueous sodium chloride solution (2 mL). The quantitative yield of Boc-Val-Pro-OH in the obtained organic layer was 99%.

[0297] Synthesis Example 81: Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), tetrahydrofuran (1.0 g), and N,N-diisopropylethylamine (77 mg, 0.60 mmol) were mixed, and 2-ethylbutanoyl chloride (74 mg, 0.55 mmol) was added at 0°C, and the mixture was stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50°C for 1 hour, and the mixture was stirred at 0°C for 21 hours (raw material: target product = 1:248). The resulting reaction solution was diluted with ethyl acetate (10 mL), separated with water (2 mL) and 10% by mass aqueous citric acid solution (2 mL), and then washed twice with 10% by mass aqueous sodium chloride solution (2 mL). The quantitative yield of Boc-Val-Pro-OH in the obtained organic layer was 99%.

[0298] Synthesis Example 82: Synthesis of Boc-Val-Pro-OH Boc-Val-OH (100 mg, 0.46 mmol), N,N-dimethylacetamide (1.0 g), and N-methylmorpholine (61 mg, 0.60 mmol) were mixed, and 2,4-dimethylpentan-3-yl carbonochloridate (99 mg, 0.55 mmol) was added at 0 ° C. and stirred for 2.5 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 50 ° C. for 1 hour, and the mixture was stirred at 0 ° C. for 21 hours (raw material: target product = 1:179). The resulting reaction solution was diluted with ethyl acetate (5 mL), separated with 10% by mass citric acid aqueous solution (2 mL), and washed twice with 10% by mass sodium chloride aqueous solution (1.0 g). The quantitative yield of Boc-Val-Pro-OH in the resulting organic and aqueous layers was 89%.

[0299] Synthesis Example 83: Synthesis of Fmoc-Val-MeGly-OH [ka]

[0300] Fmoc-Val-OH (200 mg, 0.59 mmol), tetrahydrofuran (2.0 g), and N-methylmorpholine (60 mg, 0.59 mmol) were mixed, and isobutyl carbonochloridate (72 mg, 0.59 mmol) was added at 0° C. and stirred for 2.5 hours. To this solution, a solution was added which was prepared by mixing H-MeGly-OH (55 mg, 0.62 mmol), N,N-diisopropylethylamine (0.16 g, 1.2 mmol), trimethylsilyl chloride (0.13 g, 1.2 mmol), and dichloromethane (3 mL) separately and stirring at 40° C. for 2 hours, and the mixture was stirred at 0° C. for 15 hours to obtain Fmoc-Val-MeGly-OH (raw material: target product = 1: 1.9). MASS(ESI+) m / z;(M+H)+411.28

[0301] Hereinafter, unless otherwise specified, the ratio of the raw material Fmoc-Val-OH to the product Fmoc-Val-MeGly-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 5>.

[0302] Synthesis Example 84: Synthesis of Fmoc-Val-MeGly-OH Fmoc-Val-OH (200 mg, 0.59 mmol), acetonitrile (2.0 g), and N,N-diisopropylethylamine (91 mg, 0.71 mmol) were mixed, and 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 stirred for 4 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 75° C. for 1 hour, and the mixture was stirred for 15 hours at 25° C. (raw material: target product = 1:61). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% by mass aqueous citric acid solution (2.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The quantitative yield of Fmoc-Val-MeGly-OH in the resulting organic layer was 97%.

[0303] Hereinafter, unless otherwise specified, the quantitative yield of Fmoc-Val-MeGly-OH was calculated by the quantitative analysis method under <Analysis Condition 5>. Standard substance: Fmoc-Val-MeGly-OH, which was isolated and purified separately with reference to Indian Journal of Chemistry, Vol. 43B, p. 1282, 2004, was used as the standard substance. The mass of the standard material is shown. MASS(ESI+) m / z;(M+H)+411.28 Quantitative method: Absolute calibration method

[0304] Synthesis Example 85: Synthesis of Fmoc-Val-MeGly-OH 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-trimethylpentan-3-yl carbonochloridate (0.14 g, 0.71 mol) was added at 0° C. and stirred for 2.5 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 75° C. for 1 hour, and the mixture was stirred at 0° C. for 15 hours (raw material: target product = 1:64). The resulting reaction solution was diluted with ethyl acetate (10 mL), and separated with water (5 mL) and 10% by mass aqueous citric acid solution (5 mL), and the aqueous layer was extracted with ethyl acetate (10 mL). The organic layers were mixed and washed with 10% by mass aqueous sodium chloride solution. The quantitative yield of Fmoc-Val-MeGly-OH in the resulting organic layer was 97%.

[0305] Synthesis Example 86: Synthesis of Fmoc-Val-MeGly-OH 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-dimethylbutanoyl chloride (48 mg, 0.35 mmol) was added at 0°C, and the mixture was stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 75°C for 1 hour, and the mixture was stirred at 0°C for 20 hours (raw material: target product = 1:48). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% by mass aqueous citric acid solution (2.0 mL), and washed twice with 10% by mass aqueous sodium chloride solution (2.0 mL). The quantitative yield of Fmoc-Val-MeGly-OH in the obtained organic layer was 95%.

[0306] Synthesis Example 87: Synthesis of Fmoc-Val-MeGly-OH 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-ethylbutanoyl chloride (48 mg, 0.35 mmol) was added at 0°C, and the mixture was stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 75°C for 1 hour, and the mixture was stirred for 20 hours at 0°C (raw material: target product = 1:20). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% by mass aqueous citric acid solution (2.0 mL), and washed twice with 10% by mass aqueous sodium chloride solution (2.0 mL). The quantitative yield of Fmoc-Val-MeGly-OH in the obtained organic layer was 92%.

[0307] Synthesis Example 88: Synthesis of Fmoc-Val-MeGly-OH 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-dimethylpentan-3-yl carbonochloridate (63 mg, 0.35 mmol) was added at 0°C and stirred for 2 hours. To this solution, a solution was added which was prepared by 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) separately and stirring at 75°C for 1 hour, and the mixture was stirred at 0°C for 20 hours (raw material: target product = 1:33). The resulting reaction solution was diluted with ethyl acetate (5.0 mL), separated with 10% by mass aqueous citric acid solution (2.0 mL), and washed twice with 10% by mass aqueous sodium chloride solution (2.0 mL). The quantitative yield of Fmoc-Val-MeGly-OH in the resulting organic and aqueous layers was 88%.

[0308] Synthesis Example 89: Synthesis of Boc-Cys(Bn)-Pro-OH [ka]

[0309] Boc-Cys(Bn)-OH (200mg, 0.64mmol), chloroform (1.3mL), triethylamine (67mg, 0.66mmol) were mixed, and pivaloyl chloride (81mg, 0.67mol) was added at -20℃ and stirred for 1 hour. To this solution, H-Pro-OH (96mg, 0.84mmol), triethylamine (0.16g, 1.6mmol), trimethylsilyl chloride (0.11g, 1.0mmol), dichloromethane (1.6mL), N,N-dimethylformamide (0.32mL) were mixed separately and stirred at 40℃ for 2 hours to prepare a solution, which was then added and stirred at -20℃ for 3 hours to obtain Boc-Cys(Bn)-Pro-OH (raw material: target product = 1:2.2). MASS(ESI+) m / z;(M+H)+409.26

[0310] Hereinafter, unless otherwise specified, the ratio of the raw material Boc-Cys(Bn)-OH to the product Boc-Cys(Bn)-Pro-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 6>.

[0311] <Analysis conditions 6> High performance liquid chromatography: SHIMADZU HPLC-20A Column: Agilent Poroshell 120EC-C18 (2.7 μm, 3.0 × 100 mm) Column oven temperature: 50℃ Eluent: 0.2 vol% phosphoric acid Acetonitrile solution: 0.2 vol% phosphoric acid aqueous solution 35:65(7 minutes), 35:65-95:5(7-14 minutes), 95:5(14-18 minutes), (v / v) Eluent rate: 0.7mL / min Detection wavelength: 214 nm

[0312] Synthesis Example 90: Synthesis of Boc-Cys(Bn)-Pro-OH Boc-Cys(Bn)-OH (100mg, 0.32mmol), acetonitrile (1.0g), and N,N-diisopropylethylamine (50mg, 0.39mmol) were mixed, and 50% by mass toluene solution (231mg, 0.39mol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride was added at 25°C and stirred for 2 hours. A solution was added to this solution, which was prepared by mixing H-Pro-OH (48mg, 0.42mmol), N,O-bis(trimethylsilyl)acetamide (0.12g, 0.59mmol), and acetonitrile (1.0g) separately and stirring at 50°C for 1 hour, and the mixture was stirred for 23 hours at 25°C (raw material: target product = 1:265). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% by mass aqueous citric acid solution (2.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The resulting organic layer and aqueous layer were quantified to obtain Boc-Cys(Bn)-Pro-OH in a quantitative yield of 97%.

[0313] Hereinafter, unless otherwise specified, the quantitative yield of Boc-Cys(Bn)-Pro-OH was calculated by the quantitative analysis method under <Analysis Condition 6>. Standard substance: Boc-Cys(Bn)-Pro-OH, which was isolated and purified separately with reference to Chemistry of Nature Compounds, 1992, Vol. 28, p. 344, was used as the standard substance. The mass of the standard material is shown. MASS(ESI+) m / z;(M+H)+409.26 Quantitative method: Absolute calibration method

[0314] Synthesis Example 91: Synthesis of Boc-Cys(Bn)-Pro-OH Boc-Cys(Bn)-OH (100mg, 0.32mmol), N,N-dimethylacetamide (1.0g), and N-methylmorpholine (42mg, 0.42mmol) were mixed, and 2,2,4-trimethylpentan-3-yl carbonochloridate (74mg, 0.39mol) was added at 0℃ and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-Pro-OH (48mg, 0.42mmol), N,O-bis(trimethylsilyl)acetamide (0.12g, 0.59mmol), and acetonitrile (1.0g) separately and stirring at 50℃ for 1 hour, and the mixture was stirred at 0℃ for 15 hours (raw material: target product = 1:461). The resulting reaction solution was diluted with ethyl acetate (3.0 g), separated with 10% by mass aqueous citric acid solution (2.0 g), and washed twice with 10% by mass aqueous sodium chloride solution (1.0 g). The resulting organic layer and aqueous layer were quantified to obtain Boc-Cys(Bn)-Pro-OH in a quantitative yield of 96%.

[0315] Synthesis Example 92: Synthesis of Boc-Cys(Bn)-Pro-OH Boc-Cys(Bn)-OH (100mg, 0.32mmol), acetonitrile (1.0g), and N,N-diisopropylethylamine (54mg, 0.41mmol) were mixed, and 2,2-dimethylbutanoyl chloride (52mg, 0.38mol) was added at 0℃ and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-Pro-OH (44mg, 0.38mmol), N,O-bis(trimethylsilyl)acetamide (0.12g, 0.61mmol), and acetonitrile (1.0g) separately and stirring at 50℃ for 1 hour, and the mixture was stirred at 0℃ for 19 hours (raw material: target product = 1:31). The resulting reaction solution was diluted with ethyl acetate (5.0mL), separated with 10% by mass citric acid aqueous solution (2.0mL), and washed twice with 10% by mass sodium chloride aqueous solution (2.0mL). The resulting organic layer was quantified to give Boc-Cys(Bn)-Pro-OH in a quantitative yield of 97%.

[0316] Synthesis Example 93: Synthesis of Boc-Cys(Bn)-Pro-OH Boc-Cys(Bn)-OH (100mg, 0.32mmol), acetonitrile (1.0g), and N,N-diisopropylethylamine (54mg, 0.41mmol) were mixed, and 2-ethylbutanoyl chloride (52mg, 0.38mol) was added at 0℃ and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-Pro-OH (44mg, 0.38mmol), N,O-bis(trimethylsilyl)acetamide (0.12g, 0.61mmol), and acetonitrile (1.0g) separately and stirring at 50℃ for 1 hour, and the mixture was stirred at 0℃ for 19 hours (raw material: target product = 1:30). The obtained reaction solution was diluted with ethyl acetate (5.0mL), separated with 10% by mass citric acid aqueous solution (2.0mL), and washed twice with 10% by mass sodium chloride aqueous solution (2.0mL). The resulting organic layer was quantified to give Boc-Cys(Bn)-Pro-OH in a quantitative yield of 97%.

[0317] Synthesis Example 94: Synthesis of Boc-Cys(Bn)-Pro-OH Boc-Cys(Bn)-OH (100mg, 0.32mmol), N,N-dimethylacetamide (1.0g), and N-methylmorpholine (42mg, 0.42mmol) were mixed, and 2,4-dimethylpentan-3-yl carbonochloridate (69mg, 0.38mol) was added at 0℃ and stirred for 2 hours. To this solution, a solution was added which was prepared by mixing H-Pro-OH (48mg, 0.42mmol), N,O-bis(trimethylsilyl)acetamide (0.12g, 0.59mmol), and acetonitrile (1.0g) separately and stirring at 50℃ for 1 hour, and the mixture was stirred at 0℃ for 19 hours (raw material: target product = 1:60). The resulting reaction solution was diluted with ethyl acetate (5.0mL), separated with 10% by mass citric acid aqueous solution (2.0mL), and washed twice with 10% by mass sodium chloride aqueous solution (2.0g). The resulting organic layer was quantified to give Boc-Cys(Bn)-Pro-OH in a quantitative yield of 98%.

[0318] Synthesis Example 95: Synthesis of Cbz-Phe-Phe-MePhe-OH [ka]

[0319] Cbz-Phe-Phe-OH (0.100g, 0.224mmol), N,N-diisopropylethylamine (0.050mL, 0.291mmol) were mixed with tetrahydrofuran (1.0g), and 1-adamantanecarbonyl chloride (0.0534g, 0.269mmol) was added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MePhe-OH (0.060g, 0.336mmol), N,O-bis(trimethylsilyl)acetamide (0.103mL, 0.420mmol), and acetonitrile (0.60g) separately and stirring at 50°C for 1 hour, and the mixture was further stirred at 0°C for 22 hours (raw material: target product = 1:32). The reaction mixture was quenched with 2% n-propylamine / acetonitrile solution, diluted with ethyl acetate (5.0 g), and washed twice with 10% by mass aqueous citric acid solution (2.0 g) and saturated saline solution (1.0 g). The quantitative yield of Cbz-Phe-Phe-MePhe-OH in the collected organic layer was 89%.

[0320] The ratio of the raw material Cbz-Phe-Phe-OH to the product Cbz-Phe-Phe-MePhe-OH was calculated by analysis using high performance liquid chromatography <Analysis Condition 7>.

[0321] <Analysis conditions 7> High performance liquid chromatography: SHIMADZU HPLC LC-20A Column: Agilent Poroshell 120EC-C18 (2.7 μm, 3.0 × 100 mm) Column oven temperature: 50℃ Eluent: 0.2 vol% TFA / acetonitrile:methanol = 1:1 0.2vol% TFA aqueous solution 55:45 (0-20 minutes), 55:45-95:5 (20-20.1 minutes), 95:5 (20.1-24 minutes) Post time5 minutes (v / v) Eluent rate: 0.7mL / min Detection wavelength: 214 nm

[0322] The quantitative yield of Cbz-Phe-Phe-MePhe-OH was calculated by the quantitative analysis method under <Analysis Condition 7>. Standard substance: Cbz-Phe-Phe-MePhe-OH synthesized in Synthesis Example 96 was used as the standard substance. The mass of the standard material is shown. MASS(ESI+) m / z;(M+H)+608.4 Quantitative method: Absolute calibration method

[0323] Synthesis Example 96: Synthesis of Cbz-Phe-Phe-MePhe-OH 2-Chlorotrityl chloride resin (200-400 mesh) (0.10 g, 0.13 mmol) was added with dichloromethane (1.0 mL), Fmoc-MePhe-OH (0.062 g, 0.15 mmol), and N,N-diisopropylethylamine (0.11 mL, 0.61 mmol) and stirred overnight. The following solutions were added to the resulting suspension in order and filtered each time: (1) a mixed solution of (dichloromethane / methanol / N,N-diisopropylethylamine = 17 / 2 / 1) three times, (2) dichloromethane three times, (3) N-methylpyrrolidone twice, (4) dichloromethane twice, and (5) methanol five times. N-methylpyrrolidone was added so that the resin after filtration was fully immersed, and the mixture was stirred for 15 minutes. After that, filtration was performed, and 20% piperidine / N-methylpyrrolidone solution was added so that the resin was fully immersed, and the mixture was stirred for 20 minutes. Filtration was then performed, and the resin was washed 10 times with N-methylpyrrolidone. 0.80 mL of 0.5 M ethyl cyano(hydroxyimino)acetate / N-methylpyrrolidone solution, Fmoc-Phe-OH (0.078 mg, 0.20 mmol), N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (0.086 g, 0.20 mmol), and N,N-diisopropylethylamine (0.085 mL, 0.50 mmol) were added to the obtained resin (0.10 g, 0.050 mmol), and the mixture was stirred for 2 hours. The reaction solution was filtered, and the resin was washed 10 times with N-methylpyrrolidone. The above condensation and washing procedures were then repeated, and 20% piperidine / N-methylpyrrolidone was added, followed by stirring for 20 minutes and washing with N-methylpyrrolidone 10 times. 0.400 mL of 0.5 M ethylcyano(hydroxyimino)acetate / N-methylpyrrolidone solution, Cbz-Phe-OH (0.060 mg, 0.20 mmol), and N,N-diisopropylcarbodiimide (0.032 mL, 0.20 mmol) were added to the resin obtained, and the mixture was stirred overnight. After filtering the reaction solution, the mixture was washed 10 times with N-methylpyrrolidone and 10 times with methanol, and 30% hexafluoroisopropanol was added so that the resin was fully immersed, followed by stirring for 10 minutes and then filtering 5 times.The collected solution was concentrated under reduced pressure to give Cbz-Phe-Phe-MePhe-OH (0.031 g, 100%). MASS(ESI+) m / z;(M+H)+608.4

[0324] In the above synthesis examples, synthesis examples 1 to 7, 66, and 96 are reference examples relating to the synthesis of raw materials used in the examples, while synthesis examples 1 to 2 and synthesis examples 3 to 4 are examples of the invention relating to the compounds of the present application. 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, and 90 to 95 are examples of the invention relating to the method for producing peptides of the present 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.

[0325] Synthesis Example 97: Synthesis of Fmoc-Val-MeTyr(tBu)-OH [ka]

[0326] 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 pivaloyl chloride (0.041 mL, 0.33 mmol) were added at 0 ° C. and stirred for 2 hours at 0 ° C. To this solution, 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) were separately mixed, and the mixture was stirred at 50 ° C. for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0 ° C. for 1 hour, and then stirred at 25 ° C. for 20 hours (raw material: target product = 1: 7 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (20 mL) and washed with saturated aqueous hydrogen carbonate (20 mL), water (5 mL), and saturated aqueous sodium chloride (5 mL) in that order. The organic layer was concentrated to give Fmoc-Val-MeTyr(tBu)-OH (0.18 g, 106% yield) as a pale yellow solid.

[0327] Synthesis Example 98: Synthesis of Fmoc-Val-MeTyr(tBu)-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. H-MeTyr(tBu)-OH (0.090g, 0.36mmol), N,O-bis(trimethylsilyl)acetamide (0.185mL, 0.72mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0°C for 1 hour, and then stirred at 25°C for 24 hours (raw material: target product = 1:20 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous sodium bicarbonate (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride (5 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-MeTyr(tBu)-OH (0.170 g, 99% yield) as a white solid.

[0328] Synthesis Example 99: Synthesis of Fmoc-Val-MeVal-OH [ka]

[0329] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.050mL, 0.36mmol) and pivaloyl chloride (0.041mL, 0.33mmol) were added at 0°C and stirred for 2 hours. H-MeVal-OH (0.047g, 0.36mmol), N,O-bis(trimethylsilyl)acetamide (0.19mL, 0.72mmol), and acetonitrile (1.5mL) were mixed with this solution separately, and the mixture was stirred at 50°C for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0°C for 1 hour, and then stirred at 25°C for 15 hours (raw material: target product = 2.4:1 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (5 mL) and washed successively with saturated aqueous hydrogen carbonate solution (5 mL), 10% by mass aqueous citric acid solution (5 mL), water (5 mL), and saturated aqueous sodium chloride solution (5 mL). The resulting organic layer was concentrated to give Fmoc-Val-MeVal-OH (0.16 g, 116% yield) as a pale yellow solid.

[0330] Synthesis Example 100: Synthesis of Fmoc-Val-MeVal-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. H-MeVal-OH (0.032g, 0.24mmol), N,O-bis(trimethylsilyl)acetamide (0.12mL, 0.48mmol), and acetonitrile (1.0mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour. The prepared solution was added, and the mixture was stirred at 0°C for 2 hours, and then stirred at 25°C for 36 hours (raw material: target product = 1:16 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride solution (5 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-MeVal-OH (0.092 g, 92% yield) as a white solid.

[0331] Synthesis Example 101: Synthesis of Fmoc-Val-MeSer(tBu)-OH [ka]

[0332] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.050mL, 0.36mmol) and pivaloyl chloride (0.041mL, 0.33mmol) were added at 0°C and stirred for 1 hour. H-MeSer(tBu)-OH (0.063g, 0.36mmol), N,O-bis(trimethylsilyl)acetamide (0.19mL, 0.72mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour. The prepared solution was added and further stirred at 0°C for 15 hours. (Raw material: target product = 1:19 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (5mL) and washed with water (5mL) and saturated aqueous sodium chloride solution (5mL) in that order. After the obtained organic layer was concentrated, Fmoc-Val-MeSer(tBu)-OH (0.14 g, yield 94%) was obtained as a pale yellow solid.

[0333] Synthesis Example 102: Synthesis of Fmoc-Val-MeSer(tBu)-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. This solution was mixed with H-MeSer(tBu)-OH (0.045g, 0.26mmol), N,O-bis(trimethylsilyl)acetamide (0.13mL, 0.52mmol), and acetonitrile (1.0mL) separately, stirred at 50°C for 1 hour, added the prepared solution, stirred at 0°C for 1 hour, and then stirred at 25°C for 15 hours (raw material: target product = 1:25 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride solution (5 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-MeSer(tBu)-OH (0.095 g, 96% yield) as a white solid.

[0334] Synthesis Example 103: Synthesis of Fmoc-Val-MeDap(Boc)-OH [ka]

[0335] Fmoc-Val-OH (0.034g, 0.1mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.017mL, 0.12mmol) and pivaloyl chloride (0.014mL, 0.11mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with Nα-Boc-Nβ-methyl-2,3-diaminopropionic acid (H-MeDap(Boc)-OH) (0.026g, 0.12mmol), N,O-bis(trimethylsilyl)acetamide (0.62mL, 0.24mmol), and acetonitrile (1.0mL) separately, and stirred at 50°C for 1 hour. The prepared solution was added, and the mixture was stirred at 0°C for 1 hour, and then stirred at 25°C for 24 hours. (Raw material: target product = 1:2.4 (analysis condition 3)).

[0336] Synthesis Example 104: Synthesis of Fmoc-Val-MeDap(Boc)-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. H-MeDap(Boc)-OH (0.052g, 0.24mmol), N,O-bis(trimethylsilyl)acetamide (0.12mL, 0.48mmol), and acetonitrile (1.0mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0°C for 1 hour, and then stirred at 25°C for 36 hours (raw material: target product = 1:20 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride solution (5 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-MeDap(Boc)-OH (0.107 g, 99% yield) as a white solid.

[0337] Synthesis Example 105: Synthesis of Fmoc-Val-MeGln(Trt)-OH [ka]

[0338] Fmoc-Val-OH (0.33g, 0.10mmol) was mixed with tetrahydrofuran (0.5mL), and triethylamine (0.017mL, 0.12mmol) and pivaloyl chloride (0.014mL, 0.11mmol) were added at 0°C and stirred for 1 hour. H-MeGln(Trt)-OH (0.048g, 0.12mmol), N,O-bis(trimethylsilyl)acetamide (0.062mL, 0.24mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour. The prepared solution was added, and the mixture was stirred at 0°C for 30 minutes, and then stirred at 25°C for 24 hours (raw material: target product = 1:8 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), and saturated aqueous hydrogen carbonate solution (5 mL) was added and stirred for 1 hour. The mixture was then washed with water (5 mL) and saturated aqueous sodium chloride solution (5 mL) in that order. The organic layer was concentrated to give Fmoc-Val-MeGln(Trt)-OH (0.75 g, 105% yield) as a pale yellow solid.

[0339] Synthesis Example 106: Synthesis of Fmoc-Val-MeGln(Trt)-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. H-MeGln(Trt)-OH (0.097g, 0.24mmol), N,O-bis(trimethylsilyl)acetamide (0.12mL, 0.48mmol), and acetonitrile (1.0mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0°C for 1 hour, and then stirred at 25°C for 44 hours (raw material: target product = 1:21 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate solution (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride solution (5 mL) in that order. The organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGln(Trt)-OH (0.151 g, 104% yield) as a white solid.

[0340] Synthesis Example 107: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH [ka]

[0341] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.050mL, 0.36mmol) and pivaloyl chloride (0.041mL, 0.33mmol) were added at 0°C and stirred for 1 hour. H-MeGlu(OtBu)-OH (0.098g, 0.45mmol), N,O-bis(trimethylsilyl)acetamide (0.23mL, 0.90mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour. The prepared solution was added, and the mixture was stirred at 0°C for 30 minutes, and then stirred at 25°C for 18 hours (raw material: target product = 1:18 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous hydrogen carbonate (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride (5 mL) in that order. The organic layer was concentrated to give Fmoc-Val-MeGlu(OtBu)-OH (0.16 g, 100% yield) as a pale yellow solid.

[0342] Synthesis Example 108: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH Fmoc-Val-OH (0.068g, 0.20mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.033mL, 0.24mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.66g, 0.22mmol) were added at 0°C and stirred for 2 hours. H-MeGlu(OtBu)-OH (0.078g, 0.36mmol), N,O-bis(trimethylsilyl)acetamide (0.185mL, 0.72mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 50°C for 1 hour, and the prepared solution was added, and the mixture was further stirred at 0°C for 1 hour, and then stirred at 25°C for 24 hours (raw material: target product = 1:25 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (5 mL), saturated aqueous sodium bicarbonate (5 mL) was added, and the mixture was stirred for 1 hour, and then washed with water (5 mL) and saturated aqueous sodium chloride (5 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-MeGlu(OtBu)-OH (0.176 g, 109% yield) as a white solid.

[0343] Synthesis Example 109: Synthesis of Fmoc-Val-EtAla-OH [ka]

[0344] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.11g, 0.32mmol) were added at 0°C and stirred for 1 hour. H-EtAla-OH (0.041g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 25°C for 60 minutes, and the prepared solution was added, and the mixture was further stirred at 25°C for 22 hours (raw material: target product = 1:19 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (2.0 mL), and then washed with hexane (2.0 mL). The acetonitrile solution was diluted with ethyl acetate (6.0 mL) and washed with saturated aqueous sodium bicarbonate solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (5.0 mL) in that order. The resulting organic layer was concentrated to give Fmoc-Val-EtAla-OH (0.13 g, 104% yield) as a pale yellow solid.

[0345] Synthesis Example 110: Synthesis of Fmoc-Ala-BnAla-OH [ka]

[0346] Fmoc-Ala-OH (0.10g, 0.32mmol) was mixed with tetrahydrofuran (1.6mL), and triethylamine (0.054mL, 0.39mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.13g, 0.35mmol) were added at 0°C and stirred for 1 hour. To this solution, H-BnAla-OH (0.069g, 0.39mmol), N,O-bis(trimethylsilyl)acetamide (0.19mL, 0.77mmol), and acetonitrile (1.6mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and further stirred at 25°C for 12 hours (raw material: target product = 1:1 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (8.0 mL), and washed successively with saturated aqueous sodium bicarbonate solution (6.0 mL), water (6.0 mL), and saturated aqueous sodium chloride 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.

[0347] Synthesis Example 111: Synthesis of Fmoc-Ala-BnAla-OH [ka]

[0348] 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 pivaloyl chloride (0.44 mL, 0.35 mmol) were added at 0 ° C. and stirred for 1 hour. To this solution, 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) were mixed separately and stirred at 25 ° C. for 60 minutes to prepare a solution, which was then added and stirred at 25 ° C. for 12 hours (raw material: target product = 3:1 (analysis condition 3)). The obtained 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 resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Ala-BnAla-OH (0.12 g, 80%) as a white solid.

[0349] Synthesis Example 112: Synthesis of Fmoc-Gly-EtAla-Phe-OH [ka]

[0350] Fmoc-Gly-OH (0.020g, 0.067mmol) was mixed with tetrahydrofuran (0.42mL), and triethylamine (0.011mL, 0.080mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.026g, 0.074mmol) were added at 0°C and stirred for 1 hour. H-EtAla-Phe-OH (0.022g, 0.080mmol), N,O-bis(trimethylsilyl)acetamide (0.039mL, 0.16mmol), and acetonitrile (0.84mL) were mixed with this solution, and the mixture was stirred at 25°C for 60 minutes, and the prepared solution was added and further stirred at 25°C for 6 hours (raw material: target product = 1:33 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (5.0 mL), water (5.0 mL), and saturated aqueous sodium chloride solution (5.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.040 g, 109% yield) as a white solid.

[0351] Synthesis Example 113: Synthesis of Fmoc-Gly-EtAla-Phe-OH [ka]

[0352] Fmoc-Gly-OH (0.025g, 0.084mmol) was mixed with tetrahydrofuran (0.42mL), and triethylamine (0.014mL, 0.10mmol) and pivaloyl chloride (0.011mL, 0.092mmol) were added at 0°C and stirred for 1 hour. H-EtAla-Phe-OH (0.027g, 0.10mmol), N,O-bis(trimethylsilyl)acetamide (0.049mL, 0.20mmol), and acetonitrile (0.42mL) were mixed with this solution separately, and stirred at 25°C for 60 minutes. The prepared solution was added and further stirred at 25°C for 6 hours (raw material: target product = 1:9 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (5.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (5.0 mL), water (5.0 mL), and saturated aqueous sodium chloride solution (5.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Gly-EtAla-Phe-OH (0.050 g, 109% yield) as a white solid.

[0353] Synthesis Example 114: Synthesis of Fmoc-Val-cHexmGly-OH [ka]

[0354] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.098g, 0.32mmol) were added at 0°C and stirred for 2 hours. This solution was mixed with N-(cyclohexylmethyl)glycine (H-cHexmGly-OH) (0.061g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.18mL, 0.71mmol), and acetonitrile (1.5mL) separately, and stirred at 50°C for 60 minutes. The prepared solution was added and further stirred at 25°C for 15 hours (raw material: target product = 1:32 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (6.0 mL), and then washed with hexane (11 mL). The acetonitrile solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium bicarbonate solution (10 mL), 1M hydrochloric acid (10 mL), water (10 mL), and saturated aqueous sodium chloride solution (12 mL). The resulting organic layer was concentrated to give Fmoc-Val-cHexmGly-OH (0.15 g, 104% yield) as a white solid.

[0355] Synthesis Example 115: Synthesis of Fmoc-Val-cHexmGly-OH [ka]

[0356] Fmoc-Val-OH (0.030g, 0.088mmol) was mixed with tetrahydrofuran (0.44mL), and triethylamine (0.015mL, 0.11mmol) and pivaloyl chloride (0.012mL, 0.097mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-cHexmGly-OH (0.018g, 0.11mmol), N,O-bis(trimethylsilyl)acetamide (0.052mL, 0.21mmol), and acetonitrile (0.44mL) separately, and stirred at 50°C for 60 minutes. The prepared solution was added and further stirred at 0°C for 15 hours (raw material: target product = 1:3 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with ethyl acetate (5.0 mL), and washed successively with saturated aqueous sodium bicarbonate solution (5.0 mL), 1 M hydrochloric acid (5.0 mL), water (6.0 mL), and saturated aqueous sodium chloride solution (6.0 mL). The resulting organic layer was concentrated to give Fmoc-Val-cHexmGly-OH (0.048 g, 109% yield) as a white solid.

[0357] Synthesis Example 116: Synthesis of Fmoc-Val-Tic-OH [ka]

[0358] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and 2,2-dimethylbutanoyl chloride (0.045mL, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (H-Tic-OH) (0.063g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 4 hours (raw material: target product = 1:99 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium bicarbonate solution (8.0 mL), 10% by mass aqueous citric acid solution (8.0 mL), water (10 mL), and saturated aqueous sodium chloride solution (10 mL). The collected organic layer was concentrated to give Fmoc-Val-Tic-OH (0.15 g, 100% yield) as a white solid.

[0359] Synthesis Example 117: Synthesis of Fmoc-Val-Tic-OH [ka]

[0360] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0℃ and stirred for 1 hour. To this solution, H-Tic-OH (0.063g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25℃ for 60 minutes to prepare a solution, which was then added and stirred at 0℃ for 4 hours (raw material: target product = 1:32 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium bicarbonate solution (8.0 mL), 10% by mass aqueous citric acid solution (8.0 mL), water (10 mL), and saturated aqueous sodium chloride solution (10 mL). The collected organic layer was concentrated to give Fmoc-Val-Tic-OH (0.14 g, 97% yield) as a white solid.

[0361] Synthesis Example 118: Synthesis of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide [ka]

[0362] 2-(4,4-Dimethylpentan-2-yl)-5,7,7-trimethyloctanoic acid (3.0 g, 10.6 mmol) and phosphorus tribromide (0.94 g, 3.48 mmol) were mixed 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.

[0363] Synthesis Example 119: Synthesis of Fmoc-Val-MeGlu(OtBu)-OH [ka]

[0364] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide (0.13g, 0.32mmol) were added at 0°C and stirred for 1 hour. H-MeGlu(OtBu)-OH (0.077g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed with this solution and stirred at 50°C for 60 minutes, and the prepared solution was added and further stirred at 25°C for 20 hours (raw material: target product = <1:99 (analysis condition 3)). The resulting reaction solution was concentrated, diluted with acetonitrile (8.0 mL), and then washed twice with hexane (8.0 mL). The acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (7.0 mL), 1M hydrochloric acid (7.0 mL), water (10 mL), and saturated aqueous sodium chloride solution (10 mL). The resulting organic layer was concentrated to give Fmoc-Val-MeGlu(OtBu)-OH (0.16 g, 98% yield) as a white solid.

[0365] Synthesis Example 120: Synthesis of Fmoc-Val-(Me)βAla-OH [ka]

[0366] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.058mL, 0.41mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12g, 0.38mmol) were added at 0°C and stirred for 2 hours. To this solution, H-(Me)βAla-OH hydrochloride (0.058g, 0.41mmol), N,O-bis(trimethylsilyl)acetamide (0.40mL, 1.6mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 7 hours (raw material: target product = 1:24 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), saturated aqueous sodium bicarbonate solution (4.0 mL) was added, and the mixture was stirred at 25° C. for 1 hour. The resulting organic layer was washed with 10% by mass aqueous citric acid solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL) in that order. The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-OH (0.125 g, 94% yield) as a white solid.

[0367] Synthesis Example 121: Synthesis of Fmoc-Val-(Me)βAla-OH [ka]

[0368] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-(Me)βAla-OH hydrochloride (0.049g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.35mL, 1.4mmol), and acetonitrile (1.4mL) separately, stirred at 25°C for 60 minutes, added the prepared solution, stirred at 0°C for 1 hour, and then stirred at 25°C for 1 hour (raw material: target product = 1:1 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (3.0mL), and saturated aqueous sodium bicarbonate solution (4.0mL) was added and stirred at 25°C for 1 hour. The organic layer was washed with 10% by mass aqueous citric acid (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL), successively. The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βAla-OH (0.13 g, 102% yield) as a white solid.

[0369] Synthesis Example 122: Synthesis of Fmoc-Val-MeGABA-OH [ka]

[0370] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.058mL, 0.41mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12mL, 0.38mmol) were added at 0°C and stirred for 2 hours. H-MeGABA-OH hydrochloride (0.063g, 0.41mmol), N,O-bis(trimethylsilyl)acetamide (0.38mL, 1.5mmol), and acetonitrile (1.5mL) were mixed with this solution, and the mixture was stirred at 25°C for 60 minutes, and the prepared solution was added, and the mixture was further stirred at 0°C for 7 hours, and then stirred at 10°C for 12 hours (raw material: target product = 1:19 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), saturated aqueous sodium bicarbonate solution (4.0 mL) was added, and the mixture was stirred at 25° C. for 1 hour. The resulting organic layer was washed with 10% by mass aqueous citric acid solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL) in that order. The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-OH (0.135 g, 104% yield) as a white solid.

[0371] Synthesis Example 123: Synthesis of Fmoc-Val-MeGABA-OH [ka]

[0372] Fmoc-Val-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, H-MeGABA-OH hydrochloride (0.054g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.35mL, 1.4mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then stirred at 0°C for 1 hour and then stirred at 25°C for 1 hour (raw material: target product = 1:4 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0mL), and a saturated aqueous sodium bicarbonate solution (4.0mL) was added and stirred at 25°C for 1 hour. The organic layer was washed with 10% by mass aqueous citric acid (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL), successively. 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.

[0373] Synthesis Example 124: Synthesis of Fmoc-Val-(Me)βAla-MePhe-OH [ka]

[0374] Fmoc-Val-OH (0.100g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12g, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, H-(Me)βAla-MePhe-OH (0.093g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 2 hours (raw material: target product = 1:48 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (4.0 mL) and washed with saturated aqueous sodium bicarbonate (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-(Me)βAla-MePhe-OH (0.18 g, 107% yield) as a white solid.

[0375] Synthesis Example 125: Synthesis of Fmoc-Val-(Me)βAla-MePhe-OH [ka]

[0376] Fmoc-Val-OH (0.100g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-(Me)βAla-MePhe-OH (0.093g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) separately and stirring at 25°C for 60 minutes, and the mixture was stirred at 0°C for 2 hours (raw material: target product = 1:24 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to give Fmoc-Val-(Me)βAla-MePhe-OH (0.19 g, 109% yield) as a white solid.

[0377] Synthesis Example 126: Synthesis of Fmoc-Val-MeGABA-MePhe-OH [ka]

[0378] Fmoc-Val-OH (0.100g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12g, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MeGABA-MePhe-OH (0.098g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) separately and stirring at 25°C for 60 minutes, and the mixture was stirred at 0°C for 2 hours (raw material: target product = 1:16 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-MePhe-OH (0.18 g, 107% yield) as a white solid.

[0379] Synthesis Example 127: Synthesis of Fmoc-Val-MeGABA-MePhe-OH [ka]

[0380] Fmoc-Val-OH (0.100g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by mixing H-MeGABA-MePhe-OH (0.098g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) separately and stirring at 25°C for 60 minutes, and the mixture was stirred at 0°C for 2 hours (raw material: target product = 1:3 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (4.0mL) and washed successively with saturated aqueous sodium bicarbonate solution (4.0mL), water (3.0mL), and saturated aqueous sodium chloride solution (3.0mL). The resulting organic layer was concentrated and washed with hexane to obtain Fmoc-Val-MeGABA-MePhe-OH (0.19 g, yield 108%) as a white solid.

[0381] Synthesis Example 128: Synthesis of Fmoc-Val-(Me)βhomoTrp(1-Me)-OH [ka]

[0382] Fmoc-Val-OH (0.030g, 0.11mmol) was mixed with tetrahydrofuran (0.44mL), and triethylamine (0.015mL, 0.11mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.035g, 0.097mmol) were added at 0°C and stirred for 2 hours. This solution was mixed with H-(Me)βhomoTrp(1-Me)-OH hydrochloride (0.030g, 0.11mmol), N,O-bis(trimethylsilyl)acetamide (0.10mL, 0.42mmol), and acetonitrile (0.44mL) separately, and stirred at 25°C for 60 minutes. The prepared solution was added and stirred at 25°C for 15 hours (raw material: target product = 1:24 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (4.0 mL) and washed with saturated aqueous sodium bicarbonate (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-(Me)βhomoTrp(1-Me)-OH (0.056 g, 111% yield) as a white solid.

[0383] Synthesis Example 129: Synthesis of Fmoc-Val-(Me)βhomoTrp(1-Me)-OH [ka]

[0384] Fmoc-Val-OH (0.070g, 0.21mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.035mL, 0.11mmol) and pivaloyl chloride (0.028mL, 0.23mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-(Me)βhomoTrp(1-Me)-OH hydrochloride (0.070g, 0.25mmol), N,O-bis(trimethylsilyl)acetamide (0.24mL, 0.99mmol), and acetonitrile (1.0mL) separately, and stirred at 25°C for 60 minutes. The prepared solution was added, stirred at 0°C for 15 hours, and then stirred at 25°C for 4 hours (raw material: target product = 1:8 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), saturated aqueous sodium bicarbonate solution (4.0 mL) was added, and the mixture was stirred at 25° C. for 1 hour. The resulting organic layer was washed with 10% by mass aqueous citric acid solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL) in that order. The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βhomoTrp(1-Me)-OH (0.090 g, 77% yield) as a white solid.

[0385] Synthesis Example 130: Synthesis of Fmoc-Val-βhomoTrp(1-Me)-OH [ka]

[0386] Fmoc-Val-OH (0.050g, 0.15mmol) was mixed with tetrahydrofuran (0.74mL), and triethylamine (0.025mL, 0.18mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.049g, 0.16mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-βhomoTrp(1-Me)-OH hydrochloride (0.048g, 0.18mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (0.74mL) separately, and stirred at 50°C for 60 minutes. The prepared solution was added and stirred at 25°C for 15 hours (raw material: target product = 1:99 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (4.0 mL) and washed with saturated aqueous sodium bicarbonate (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-βhomoTrp(1-Me)-OH (0.11 g, 130% yield) as a white solid.

[0387] Synthesis Example 131: Synthesis of Fmoc-Val-βhomoTrp(1-Me)-OH [ka]

[0388] Fmoc-Val-OH (0.050g, 0.15mmol) was mixed with tetrahydrofuran (0.74mL), and triethylamine (0.025mL, 0.18mmol) and pivaloyl chloride (0.020g, 0.16mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-βhomoTrp(1-Me)-OH hydrochloride (0.048g, 0.18mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (0.74mL) separately, and stirred at 50°C for 60 minutes. The prepared solution was added and stirred at 25°C for 15 hours (raw material: target product = 1:19 (analysis condition 3)). The reaction mixture was diluted with ethyl acetate (4.0 mL) and washed with saturated aqueous sodium bicarbonate (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride (3.0 mL) in that order. The organic layer was concentrated and washed with hexane to give Fmoc-Val-βhomoTrp(1-Me)-OH (0.079 g, 97% yield) as a white solid.

[0389] Synthesis Example 132: Synthesis of Fmoc-Val-(Me)βhomoLeu-OH [ka]

[0390] Fmoc-Val-OH (0.030g, 0.088mmol) was mixed with tetrahydrofuran (0.44mL), and triethylamine (0.015mL, 0.11mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.035g, 0.097mmol) were added at 0°C and stirred for 2 hours. This solution was mixed with H-(Me)βhomoLeu-OH hydrochloride (0.021g, 0.11mmol), N,O-bis(trimethylsilyl)acetamide (0.10mL, 0.42mmol), and acetonitrile (0.44mL) separately, and stirred at 25°C for 60 minutes. The prepared solution was added and further stirred at 25°C for 15 hours (raw material: target product = 1:24 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL) and washed successively with saturated aqueous sodium bicarbonate solution (4.0 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL). The resulting organic layer was concentrated and washed with hexane and a mixed solution of 10% isopropyl ether / hexane to obtain Fmoc-Val-(Me)βhomoLeu-OH (0.045 g, 106% yield) as a white solid.

[0391] Synthesis Example 133: Synthesis of Fmoc-Val-(Me)βhomoLeu-OH [ka]

[0392] Fmoc-Val-OH (0.070g, 0.21mmol) was mixed with tetrahydrofuran (1.0mL), and triethylamine (0.035mL, 0.25mmol) and pivaloyl chloride (0.028mL, 0.23mmol) were added at 0°C and stirred for 1 hour. This solution was mixed with H-(Me)βhomoLeu-OH hydrochloride (0.048g, 0.25mmol), N,O-bis(trimethylsilyl)acetamide (0.24mL, 0.99mmol), and acetonitrile (1.0mL) separately, and stirred at 25°C for 60 minutes. The prepared solution was added, stirred at 0°C for 15 hours, and then stirred at 25°C for 2 hours (raw material: target product = 1:5 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (4.0 mL), and saturated aqueous sodium bicarbonate solution (4.0 mL) and water (3.0 mL) were added, followed by stirring at 25° C. for 1 hour. The resulting organic layer was washed with 10% by mass aqueous citric acid solution (4 mL), water (3.0 mL), and saturated aqueous sodium chloride solution (3.0 mL) in that order. The collected organic layer was concentrated and washed with hexane to obtain Fmoc-Val-(Me)βhomoLeu-OH (0.095 g, 96% yield) as a white solid.

[0393] Synthesis Example 134: Synthesis of Fmoc-(Me)βAla-MePhe-OH [ka]

[0394] Fmoc-(Me)βAla-OH (1.0 g, 3.1 mmol) was mixed with tetrahydrofuran (15 mL), and triethylamine (0.51 mL, 3.7 mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (1.2 g, 3.4 mmol) were added at 0°C and stirred for 1 hour. To this solution, a solution was added which was prepared by 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) separately and stirring at 25°C for 60 minutes, and further stirred at 0°C for 3 hours (raw material: target product = 1:48 (analysis condition 3)). The obtained reaction solution was diluted with ethyl acetate (40 mL) and washed successively with water (15 mL) and saturated aqueous sodium chloride solution (15 mL). The combined organic layer was concentrated and purified by column chromatography to obtain Fmoc-(Me)βAla-MePhe-OH (1.3 g, 85% yield) as a white solid.

[0395] Synthesis Example 135: Synthesis of Fmoc-(Me)βAla-MePhe-OH [ka]

[0396] Fmoc-(Me)βAla-OH (0.10g, 0.31mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.051mL, 0.37mmol) and pivaloyl chloride (0.041mL, 0.34mmol) were added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.066g, 0.37mmol), N,O-bis(trimethylsilyl)acetamide (0.18mL, 0.74mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 6 hours (raw material: target product = 1:33 (analysis condition 3)). The resulting reaction mixture was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium bicarbonate (8.0 mL), 1 M hydrochloric acid (8.0 mL), water (10 mL), and saturated aqueous sodium chloride (10 mL). The collected organic layer was concentrated to give Fmoc-(Me)βAla-MePhe-OH (0.16 g, 107% yield) as a white solid.

[0397] Synthesis Example 136: Synthesis of Fmoc-MeGABA-MePhe-OH [ka]

[0398] Fmoc-MeGABA-OH (1.0g, 3.0mmol) was mixed with tetrahydrofuran (15mL), and triethylamine (0.50mL, 3.5mmol) and 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (1.2g, 3.2mmol) were added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.63g, 3.5mmol), N,O-bis(trimethylsilyl)acetamide (1.7mL, 7.1mmol), and acetonitrile (15mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 3 hours (raw material: target product = 1:48 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (40mL) and washed successively with water (15mL) and saturated aqueous sodium chloride solution (15mL). 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.

[0399] Synthesis Example 137: Synthesis of Fmoc-MeGABA-MePhe-OH [ka]

[0400] Fmoc-MeGABA-OH (0.10g, 0.30mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.049mL, 0.35mmol) and pivaloyl chloride (0.040mL, 0.32mmol) were added at 0°C and stirred for 1 hour. To this solution, H-MePhe-OH (0.063g, 0.35mmol), N,O-bis(trimethylsilyl)acetamide (0.17mL, 0.71mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 6 hours (raw material: target product = 1:12 (analysis condition 3)). The resulting reaction solution was diluted with ethyl acetate (10 mL) and washed successively with saturated aqueous sodium bicarbonate solution (8.0 mL), 1 M hydrochloric acid (8.0 mL), water (10 mL), and saturated aqueous sodium chloride solution (10 mL). The collected organic layer was concentrated to give Fmoc-MeGABA-MePhe-OH (0.15 g, 105% yield) as a white solid.

[0401] Synthesis Example 138: Synthesis of Fmoc-(2SMe)βAla-(Me)βAla-OH Growth [ka]

[0402] Fmoc-(2SMe)βAla-OH (0.050g, 0.15mmol) was mixed with tetrahydrofuran (0.42mL), and triethylamine (0.026mL, 0.18mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.051g, 0.17mmol) were added at 0°C and stirred for 1 hour. To this solution, H-(Me)βAla-OH hydrochloride (0.026g, 0.18mmol), N,O-bis(trimethylsilyl)acetamide (0.18mL, 0.74mmol), and acetonitrile (0.84mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and further stirred at 25°C for 2 hours (raw material: target product = 1:7 (analysis condition 3)). The reaction mixture was diluted with acetonitrile (6.0 mL) and washed with hexane (8.0 mL). The acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed with saturated aqueous sodium bicarbonate (8.0 mL), 10% aqueous citric acid (8.0 mL), water (10 mL), and saturated aqueous sodium chloride (10 mL) in that order. The organic layer was concentrated to give Fmoc-(2SMe)βAla-(Me)βAla-OH (0.069 g, 109% yield) as a colorless oil.

[0403] Synthesis Example 139: Synthesis of Fmoc-(2SMe)βAla-(Me)βAla-OH [ka]

[0404] Fmoc-(2SMe)βAla-OH (0.050g, 0.15mmol) was mixed with tetrahydrofuran (0.42mL), and triethylamine (0.026mL, 0.18mmol) and pivaloyl chloride (0.021mL, 0.17mmol) were added at 0°C and stirred for 1 hour. To this solution, H-(Me)βAla-OH hydrochloride (0.026g, 0.18mmol), N,O-bis(trimethylsilyl)acetamide (0.18mL, 0.74mmol), and acetonitrile (0.84mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 25°C for 2 hours (raw material: target product = 1:3 (analysis condition 3)). The resulting reaction solution was diluted with acetonitrile (6.0mL) and washed with hexane (8.0mL). The resulting acetonitrile solution was diluted with ethyl acetate (8.0 mL) and washed successively with saturated aqueous sodium bicarbonate (8.0 mL), 10% aqueous citric acid (8.0 mL), water (10 mL), and saturated aqueous sodium chloride (10 mL). The resulting organic layer was concentrated to give Fmoc-(2SMe)βAla-(Me)βAla-OH (0.070 g, 110% yield) as a colorless oil.

[0405] Synthesis Example 140: Synthesis of Fmoc-GABA-(Me)βAla-OH [ka]

[0406] Fmoc-GABA-OH (0.10 g, 0.31 mmol) was mixed with tetrahydrofuran (1.5 mL), and triethylamine (0.051 mL, 0.37 mmol), 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (0.12 g, 0.34 mmol) were added at 0°C and stirred for 1 hour. To this solution, 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) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 15 hours (raw material: target product = 1:99 (analysis condition 3)). The resulting reaction solution was diluted with acetonitrile (6.0 mL) and washed with hexane (10 mL). The resulting 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 aqueous sodium chloride solution (10 mL). The resulting organic layer was concentrated to give Fmoc-GABA-(Me)βAla-OH (0.14 g, 108% yield) as a colorless oil.

[0407] Synthesis Example 141: Synthesis of Fmoc-GABA-(Me)βAla-OH [ka]

[0408] Fmoc-GABA-OH (0.10g, 0.31mmol) was mixed with tetrahydrofuran (1.5mL), and triethylamine (0.051mL, 0.37mmol) and pivaloyl chloride (0.042mL, 0.34mmol) were added at 0°C and stirred for 1 hour. To this solution, H-(Me)βAla-OH hydrochloride (0.052g, 0.37mmol), N,O-bis(trimethylsilyl)acetamide (0.36mL, 1.5mmol), and acetonitrile (1.5mL) were mixed separately and stirred at 25°C for 60 minutes to prepare a solution, which was then added and stirred at 0°C for 15 hours (raw material: target product = 1:49 (analysis condition 3)). The resulting reaction solution was diluted with acetonitrile (8.0mL) and washed with hexane (15mL). The resulting 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 aqueous sodium chloride solution (10 mL). The resulting organic layer was concentrated to give Fmoc-GABA-(Me)βAla-OH (0.13 g, 104% yield) as a colorless oil.

[0409] In the above synthesis examples, synthesis example 118 is a reference example relating to the synthesis of the raw material used in the examples, 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 examples of the invention relating to the method for producing the peptide of the present 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. The following steps (1) to (3): （１） Formula (I) P-A １ -OH (wherein P is an N-terminal protecting group, A １ is a group derived from an amino acid, N-C 1－６ A group derived from an alkylamino acid (C 1－６ wherein alkyl may have a substituent) or a group derived from a peptide, The following compound groups: 【Chemistry 1】 with an carboxylic acid halide selected from （２） Formula (IV) H-A ２ -OH (wherein, A ２ is N-C １－６ A group derived from an alkylamino acid (C 1－６ The alkyl may have a substituent), or a group derived from a 4- to 6-membered cyclic secondary amino acid (the 4- to 6-membered ring is ６－１４ Aryl ring, C ６－１４ Haloaryl Rings and C ３－８ cycloalkyl rings), or the N-terminal residue is N-C １－６ Alkylamino acid (C 1－６ alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is ６－１４ Aryl ring, C ６－１４ Haloaryl Rings and C ３－８ a step of mixing an amino acid or peptide represented by the formula (I) (which may be condensed with a cyclic compound selected from the group consisting of cycloalkyl rings) with a silylating agent selected from N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea, or N,O-bis(trimethylsilyl)trifluoroacetamide; （３） mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

2. The following steps (1) to (3): （１） Formula (I) P-A １ -OH (wherein P is an N-terminal protecting group, A １ is a group derived from an amino acid or N-C 1－６ A group derived from an alkylamino acid (C 1－６ wherein the alkyl group may have a substituent. The following compound groups: 【Chemistry 3】 with an carboxylic acid halide selected from （２） Formula (IV) H-A ２ -OH (wherein, A ２ is a group derived from an N-methylamino acid, N-C 1－６ Alkylglycine-derived group (C 1－６ alkyl may have a substituent), or a group derived from a 4-6 membered cyclic secondary amino acid, or the N-terminal residue is an N-methyl amino acid, N-C 1－６ Alkylglycine (C 1－６ a step of mixing an amino acid or peptide represented by the formula (I) (alkyl may have a substituent), or a group derived from a peptide which is a 4-6 membered cyclic secondary amino acid), with a silylating agent selected from N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea, or N,O-bis(trimethylsilyl)trifluoroacetamide; （３） mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

3. The following steps (1) to (3): （１） Formula (V) P-A ３ -OH (wherein P is an N-terminal protecting group, A ３ represents a group derived from a peptide) The following compound groups: 【Chemistry 5】 with an carboxylic acid halide selected from （２） Formula (IV') H-A ２’ -OH (wherein, A ２’ is a group derived from an N-methylamino acid, N-C １－６ Alkylglycine-derived group (C 1－６ a step of mixing an amino acid represented by the formula (I) (alkyl may have a substituent), or a group derived from a 4- to 6-membered cyclic secondary amino acid) with a silylating agent selected from N,O-bis(trimethylsilyl)acetamide, N,N'-bis(trimethylsilyl)urea, or N,O-bis(trimethylsilyl)trifluoroacetamide; （３） mixing the product obtained in step (1) with the product obtained in step (2); A method for producing a peptide comprising the steps of:

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

5. Further, the following steps (4) and (5): （４） A step of removing the protecting group at the N-terminus of the peptide obtained in step (3) or (5); （５） a step of reacting an N-terminal protected amino acid or an N-terminal protected peptide with the N-terminus of the peptide obtained in the step (4); A method for producing the peptide according to any one of claims 1 to 3, comprising one or more repeats of the following:

6. Formula (I) P-A １ -OH or formula (V)P-A ３ -OH (wherein P is an N-terminal protecting group, A １ and A ３ Each of the amino acids located at the C-terminus of the N-terminal protected peptide represented by N-C １－６ Alkylamino acid (C 1－６ alkyl may have a substituent) or a 4-6 membered cyclic secondary amino acid (the 4-6 membered ring is ６－１４ Aryl ring, C ６－１４ Haloaryl Rings and C ３－８ 4. The method for producing the peptide according to claim 1 or 3, wherein the amino acid is an amino acid other than the amino acid (which may be condensed with a cyclic compound selected from the group consisting of cycloalkyl rings).

7. A １ The method for producing a peptide according to claim 1 or 2, wherein is a group derived from an amino acid.

8. 3. The method for producing a peptide according to claim 1 or 2, wherein the N-terminal protected amino acid represented by formula (I) or the amino acid located at the C-terminus of the N-terminal protected peptide represented by formula (I) is an α-amino acid, a β-amino acid or a γ-amino acid.

9. The method for producing a peptide according to claim 8, wherein the N-terminal protected amino acid represented by formula (I) or the amino acid located at the C-terminus of the N-terminal protected peptide represented by formula (I) is an α-amino acid.

10. The amino acid represented by formula (IV) or the amino acid located at the N-terminus of the peptide represented by formula (IV) is N-C １－６ Alkyl-α-amino acids (C 1－６ 2. The method for producing a peptide according to claim 1, wherein the aryl group is a cyclic 4- to 6-membered secondary α-amino acid, wherein the alkyl group may have a substituent.

11. The method for producing a peptide according to 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 an N-methyl-α-amino acid or an N-ethyl-α-amino acid (N-methyl and N-ethyl each may have a substituent), or a 4- to 6-membered cyclic secondary-α-amino acid.

12. A method for producing a peptide described in any one of claims 1 to 11, wherein the carboxylic acid halide is selected from the following group of compounds: 【Chemistry 7】

13. The method for producing the peptide according to claim 12, wherein the activating agent is the following compound: 【Chemistry 8】

14. The method for producing a peptide according to any one of claims 1 to 13, wherein the silylating agent is N,O-bis(trimethylsilyl)acetamide.

15. 2. The method for producing a peptide according to claim 1, 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, respectively.

16. The method for producing a peptide according to claim 3, wherein the amino acid represented by formula (IV') is an amino acid other than proline.

17. The following formula: 【Chemistry 12】 A carboxylic acid activator comprising a compound represented by the formula:

18. The following formula: 【Chemistry 13】 A carboxylic acid activator comprising a compound represented by the formula:

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