Process for the manufacture of peptide compounds

Abstract

The objective of this invention is to provide an efficient method for manufacturing peptides, comprising the following steps (1) and (2): step (1) mixing an N-protected amino acid or an N-protected peptide with a carboxylic acid halide represented by formula (I); and step (2) mixing the product obtained in step (1) with a C-protected amino acid or a C-protected peptide. In formula (I), X represents a halogen atom, R... 1 R 2 and R 3 Each of the above can independently represent an aliphatic hydrocarbon group that may have substituents, R 1 R 2 and R 3 The total number of carbon atoms in it is between 3 and 40.

Description

Technical Field

[0001] This invention relates to a method for manufacturing peptide compounds. Background Technology

[0002] Condensation reagents used to form amide bonds are widely used in the manufacture of physiologically active substances in pharmaceuticals, pesticides, and other fields. Especially in the manufacture of peptide compounds, where multiple amide bonds need to be formed, there is a strong desire for condensation reagents that can obtain the target compound in high yield (Non-Patent Literature 1).

[0003] In the manufacture of peptide compounds, industrially usable condensing agents include (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbomony hexafluorophosphate (COMU), isobutyl chloroformate, and tervapotranolol chloride (Patent Documents 1 and 2, Non-Patent Documents 2 and 3). On the other hand, COMU and isobutyl chloroformate are known to have exothermic properties due to self-decomposition (Non-Patent Document 4).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2017 / 129796

[0007] Patent Document 2: International Publication No. 2012 / 004554

[0008] Non-Patent Literature 1: Chemical Review, 2011, Vol. 111, pp. 6557-6602

[0009] Non-patent document 2: Report of the Bulgarian Academy of Sciences (Bulgarian Academy of Sciences) 2004, Volume 57, Pages 53-58

[0010] Non-patent document 3: Russian Journal of Bioorganic Chemistry (Russian Journal of Bioorganic Chemistry), 2009, Volume 35, Pages 150-156

[0011] Non-patent Document 4: Organic Process Research and Development (オーガニックプロセスリサーチアンドディベロップメント) 2018, Volume 22, Pages 1262-1275 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The inventors of this application, through verification, discovered that when pivaloyl chloride is used as a condensing agent to react the N-terminus of a C-protected amino acid or C-protected peptide with the C-terminus of an N-protected amino acid or N-protected peptide, a byproduct is generated, resulting in the introduction of a neopentanoyl group from the condensing agent into the N-terminus of the C-protected amino acid or C-protected peptide, thus reducing the yield of the target peptide compound. In the manufacture of peptide compounds that repeatedly form amide bonds, these byproducts accumulate as impurities. Therefore, it is envisioned that the above method presents challenges in terms of efficiency and economy as an industrial manufacturing method.

[0014] This invention provides a method for producing peptide compounds in high yield using a safe condensation reagent that does not generate heat due to self-decomposition.

[0015] Methods for solving problems

[0016] The inventors of this application conducted in-depth research and discovered that the aforementioned problems could be solved by using carboxylic acid halides with specific structures, thereby completing this invention. Specifically, the features of this invention are as follows. [1]

[0018] A method for manufacturing peptides, comprising the following steps (1) and (2),

[0019] Process (1):

[0020] Mix N-protected amino acids or N-protected peptides with carboxylic acid halides represented by formula (I),

[0021] [Chemical Formula 1]

[0022]

[0023] (In the formula, X represents a halogen atom,

[0024] R 1 R 2 and R 3 Each of the above can independently represent an aliphatic hydrocarbon group that may have substituents, R 1 R 2 and R 3 The total number of carbon atoms in it is 3 to 40; and

[0025] Process (2):

[0026] The product obtained in step (1) is mixed with C-protected amino acids or C-protected peptides. [2]

[0028] The method for manufacturing peptides as described in [1] further includes repeating the following steps (3) to (5) more than once.

[0029] Process (3):

[0030] Remove the protecting group at the N-terminus of the peptide obtained in step (2) or (5);

[0031] Process (4):

[0032] Mixing an N-protected amino acid or N-protected peptide with a carboxylic acid halide represented by formula (I); and

[0033] Process (5):

[0034] The product obtained in step (4) is mixed with the product obtained in step (3). [3]

[0036] The method for manufacturing peptides as described in [1] or [2], wherein R 1 R 2 and R 3 Two or more of them are aliphatic hydrocarbon groups that contain one or more tertiary or quaternary carbon atoms, and are independent of each other. [4]

[0038] The method for manufacturing peptides as described in [1] or [2], wherein R 1 It is methyl, R 2 and R 3 Each is an aliphatic hydrocarbon group containing one or more tertiary or quaternary carbon atoms, and each group is independent of the others. [5]

[0040] The method for manufacturing peptides as described in [4], wherein R 2 and R 3 Each of the following is an independent carbon atom containing more than one tertiary or quaternary carbon atom: 3-10 alkyl. [6]

[0042] The method for manufacturing peptides as described in [1], wherein the carboxylic acid halide is a compound represented by formula (II).

[0043] [Chemical Formula 2]

[0044]

[0045] (In the formula, X represents a halogen atom) [7]

[0047] The method for manufacturing a peptide as described in any one of [1] to [6], wherein X is a chlorine atom or a bromine atom. [8]

[0049] The method for manufacturing a peptide as described in any one of [1] to [6], wherein X is a chlorine atom. [9]

[0051] The method for manufacturing a peptide as described in any one of [1] to [8], wherein the amino acid in the N-protected amino acid is an α-amino acid other than glycine.

[10]

[0053] The method for manufacturing peptides as described in [9], wherein the amino acid in the N-protected amino acid is an α-amino acid other than glycine, and the reactive functional groups of its amino acid side chain are protected.

[11]

[0055] The method for manufacturing peptides as described in [9] or

[10] , wherein the α-amino acid other than glycine is valine, phenylalanine, threonine, leucine, tryptophan, serine, cysteine, aspartic acid or tyrosine.

[12]

[0057] In any of the methods for manufacturing peptides as described in [1] to

[11] , the amino acid in the C-protected amino acid or the amino acid in the N-terminal residue of the C-protected peptide is an α-amino acid other than an N-substituted amino acid.

[13]

[0059] A method for manufacturing a peptide as described in any one of [1] to

[12] , wherein step (2) is a step of mixing the product obtained in step (1) with a C-protective peptide.

[14]

[0061] The method for manufacturing a peptide as described in any one of [1] to

[13] , wherein the N-terminal protecting group of the N-protected amino acid or the N-protected peptide is a carbamate-based protecting group.

[15]

[0063] The method for manufacturing peptides as described in

[14] , wherein the urethane ester protecting group is 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl.

[0064] Invention Effects

[0065] This invention enables the use of industrially applicable reagents to obtain the target peptide in high yield. Detailed Implementation

[0066] The present invention will now be described in detail.

[0067] In this specification, "n-" refers to normal, "s-" refers to secondary, "t-" and "tert-" refer to tertiary, "Me" refers to methyl, "Et" refers to ethyl, "Bu" refers to butyl, "Bn" refers to benzyl, "Boc" refers to tert-butoxycarbonyl, "Cbz" refers to benzyloxycarbonyl, "Fmoc" refers to 9-fluorenylmethoxycarbonyl, "Trt" refers to triphenylmethyl, and "NMP" refers to N-methylpyrrolidone.

[0068] The term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0069] The term "alkyl" refers to a monovalent group in a straight-chain or branched saturated aliphatic hydrocarbon. The term "C" refers to a monovalent group in a hydrocarbon. 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 3,3-dimethylbutane-2-yl, etc.

[0070] The so-called "C" 1-40 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 40 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, dodecyl, triacontyl, tetradecyl, 3,7,11,15-tetramethylhexadecyl (hereinafter sometimes also called 2,3-dihydrochlorophyllin), etc.

[0071] The so-called "C" 3-10 "Alkyl" refers to a straight-chain or branched alkyl group with 3 to 10 carbon atoms. Specific examples include n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, octyl, decyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl, 2,2,4-trimethylpentyl, 2,2,4-trimethylhexyl, 2,2,3,4-tetramethylhexyl, 4-ethyl-2,2-dimethylhexyl, etc.

[0072] The term "cycloalkyl" refers to a monovalent group in a cyclic saturated aliphatic hydrocarbon. The term "C" refers to a monovalent group in a cyclic saturated aliphatic hydrocarbon. 3-6 "Cycloalkyl" refers to cycloalkyl groups with 3 to 6 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0073] The term "alkenyl" refers to a monovalent group in a straight-chain or branched unsaturated aliphatic hydrocarbon containing a double bond. The term "C" refers to...2-6 "Alkenyl" refers to alkenyl groups with 2 to 6 carbon atoms. Specific examples include vinyl, 1-propenyl, allyl, isopropenyl, 2-butenyl, and 3-butenyl.

[0074] The term "alkynyl group" refers to a monovalent group in a straight-chain or branched unsaturated aliphatic hydrocarbon containing a triple bond. The term "C" refers to... 2-6 "Alynyl" refers to an alkynyl group with 2 to 6 carbon atoms. Specific examples include ethynyl and 1-propynyl.

[0075] The term "aralkyl group" refers to an alkyl group that has aromatic hydrocarbons as substituents. The term "C" refers to... 7-14 "Aryl group" refers to aryl groups with 7 to 14 carbon atoms. Specific examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, and 1-naphthylpropyl.

[0076] The so-called "C" 6-14 "Aryl" refers to an aromatic hydrocarbon group with 6 to 14 carbon atoms. Specific examples include phenyl, 1-naphthyl, 2-naphthyl, 1-anthrayl, 2-anthrayl, 9-anthrayl, biphenyl, etc.

[0077] The so-called "C" 6-14 "Halogenated aryl" refers to an aromatic hydrocarbon group with 6 to 14 carbon atoms replaced by one or more halogen atoms. Specific examples include 4-chlorophenyl, 2,4-dichlorophenyl, 5-fluoro-1-naphthyl, 6-bromo-2-naphthyl, 6,7-diiodo-1-anthrayl, 10-bromo-9-anthrayl, 4'-chloro-(1,1'-biphenyl)-2-yl, etc.

[0078] The so-called "C" 6-14 "Aryloxy" refers to aryloxy groups with 6 to 14 carbon atoms. Specific examples include phenoxy, 1-naphthyloxy, 2-naphthyloxy, 1-anthrayloxy, 2-anthrayloxy, 9-anthrayloxy, and biphenyloxy.

[0079] The term "5-10 membered heterocyclic group" refers to a monocyclic or fused-ring heterocyclic group consisting of 5 to 10 atoms forming the ring, with 1 to 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur atoms. This heterocyclic group can be saturated, partially unsaturated, or unsaturated. Specific examples include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyridazinyl, azaheptanyl, oxoheptanyl, thioheptanyl, and azaheptanyl. The compounds include: oxoheptenyl, thioheptenyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolinyl, pyrazinyl, morpholinyl, thiazolinyl, indolyl, isoindolyl, benzimidazolyl, purine, quinolinyl, isoquinolinyl, quinoxalinyl, porphyrinyl, pteridinyl, benzopyranyl, isobenzopyranyl, etc.

[0080] The so-called "C" 1-6 "Alkoxy" refers to a straight-chain or branched alkoxy group with 1 to 6 carbon atoms. Specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, and n-hexyloxy.

[0081] The so-called "C" 1-40 "Alkoxy" refers to a straight-chain or branched alkoxy group with 1 to 40 carbon atoms. Specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, n-hexyloxy, octyloxy, decyloxy, dodecyloxy, hexadecyloxy, octadecyloxy, dodecyloxy, triadecyloxy, triadecyloxy, tetradecyloxy, and 3,7,11,15-tetramethylhexadecyloxy (hereinafter sometimes also called 2,3-dihydrochlorophylloxyloxy).

[0082] The so-called "C" 3-6 "Cycloalkoxy" refers to cycloalkyloxy groups with 3 to 6 carbon atoms. Specific examples include cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.

[0083] The so-called "single C" 1-6 "alkylamino" refers to one of the aforementioned "C" groups. 1-6 Alkyl groups are groups formed by bonding an amino group. Specific examples include monomethylamino, monoethylamino, monon-n-propylamino, monoisopropylamino, monon-n-butylamino, monoisobutylamino, monotert-butylamino, monon-n-pentylamino, and monon-n-hexylamino.

[0084] The so-called "two C" 1-6 "alkylamino" refers to two identical or different C atoms mentioned above. 1-6An alkyl group is a group formed by bonding an amino group. Specific examples include dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-tert-butylamino, di-n-pentylamino, di-n-hexylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-methylamino, N-n-butyl-N-methylamino, N-isobutyl-N-methylamino, N-tert-butyl-N-methylamino, N-methyl-N-pentylamino, N-n-hexyl-N-methylamino, N-ethyl-N-n-propylamino, N-ethyl-N-isopropylamino, N-n-butyl-N-ethylamino, N-ethyl-N-isobutylamino, N-tert-butyl-N-ethylamino, N-ethyl-N-pentylamino, N-ethyl-N-hexylamino, etc.

[0085] The so-called "C" 1-6 "Alkoxycarbonyl" refers to a straight-chain or branched alkoxycarbonyl group with 1 to 6 carbon atoms. Specific examples include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, n-pentyloxycarbonyl, and n-hexyloxycarbonyl.

[0086] The so-called "three Cs" 1-6 "alkylsilyl" refers to three identical or different C atoms. 1-6 "alkyl" refers to a group formed by bonding an alkyl group to a silyl group. Specific examples include trimethylsilyl (TMS) group, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, di-tert-butylisobutylsilyl, etc.

[0087] The so-called "three Cs" 1-6 "alkylsilyloxy" refers to three identical or different C atoms mentioned above. 1-6 "alkyl" refers to a group formed by bonding an alkyl group to a silyloxy group. Specific examples include trimethylsilyloxy, triethylsilyloxy, triisopropylsilyloxy, tert-butyldimethylsilyloxy, and di-tert-butylisobutylsilyloxy.

[0088] The term "bicycloalkyl" refers to a monovalent group in a saturated aliphatic hydrocarbon containing two bridgehead carbons and having two rings. Specific examples include octahydroinden-3-yl, octahydronaphth-4-yl, bicyclo[2.2.1]heptane-1-yl, or bicyclo[2.2.1]heptane-2-yl. Additionally, the term "C..." 5-10 "Bicycloalkyl" refers to bicycloalkyl groups with 5 to 10 carbon atoms. 5-7 "Bicycloalkyl" refers to bicycloalkyl groups with 5 to 7 carbon atoms.

[0089] The term "tricyclic alkyl" refers to a monovalent group in a saturated aliphatic hydrocarbon containing at least three bridgehead carbons and having three rings. A specific example is a tricyclic alkyl group [3.3.1.1]. 3,7 [Decan-1-yl (adamant-1-yl) group or tricyclic [3.3.1.1] 3,7 Decane-2-yl (adamantane-2-yl) group, etc. Additionally, the so-called "C..." 5-15 "Tricycloalkyl" refers to tricycloalkyl groups with 5 to 15 carbon atoms. 7-15 "Tricyclic alkyl" refers to tricyclic alkyl groups with 7 to 15 carbon atoms.

[0090] The term "aliphatic hydrocarbon group" refers to a straight-chain, branched, or cyclic aliphatic hydrocarbon group, which can be saturated or unsaturated. Examples include alkyl, cycloalkyl, alkenyl, alkynyl, and aralkyl groups. For specific examples, C1... 1-40 Alkyl, C 3-6 cycloalkyl, C 2-6 Alkenyl, C 2-6 alkynyl group, C 7-14 Aryl groups, etc.

[0091] The term "aliphatic hydrocarbon group containing one or more tertiary or quaternary carbon atoms" refers to a group in which one or more of the carbon atoms constituting the aliphatic hydrocarbon group are tertiary or quaternary carbon atoms. Specific examples include 2-methylbutane-2-yl, 3-methylbutane-2-yl, 3,3-dimethylbutane-2-yl, tert-butyl, 3-pentyl, 2,2,4-trimethylpentane-3-yl, 2,4,4-trimethylpentyl, 2,4-dimethylpentane-3-yl, 4-ethyl-2,2-dimethylhexane-3-yl, 3-heptyl, 2,2,4,8,10,10-hexamethylundecane-5-yl, 3-methylcyclobutyl, 2-methylcyclopentyl, and 4-isopropylcyclohexyl.

[0092] The term "C containing one or more tertiary or quaternary carbon atoms" 3-10 "alkyl" refers to the C-shaped structure. 3-10 A group in which one or more carbon atoms of an alkyl group are tertiary or quaternary carbon atoms. Specific examples include isobutyl, tert-butyl, isopentyl, neopentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2,4-trimethylpentyl, 2,4,4-trimethylpentyl, 2,2,4-trimethylhexyl, 2,2,3,4-tetramethylhexyl, 2,2,4,4-tetramethylpentyl, 4-ethyl-2,2-dimethylhexyl, etc.

[0093] The term "reactive functional group of amino acid side chain" refers to a group present in the side chain of an amino acid that can react with other groups to form a covalent bond. Specific examples include hydroxyl, carboxyl, amino, amide, and thiol groups. Examples of amino acids containing these functional groups in their side chains include arginine, asparagine, aspartic acid, lysine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan, and histidine.

[0094] In addition, the so-called "protection of reactive functional groups of amino acid side chains" means that a protecting group commonly used in peptide chemistry is introduced onto the aforementioned reactive functional groups.

[0095] The term "may have substituents" means that the substance is either unsubstituted or substituted with any number of chemically permissible substituents.

[0096] The term "having substituents" means being replaced by any number of chemically permissible substituents.

[0097] The term "any substituent" as used above refers to any substituent that does not adversely affect the reaction that is the subject of this invention, and there is no particular limitation on the type.

[0098] As a "substituent" in "aliphatic hydrocarbon groups that may have substituents", for example, C 6-14 Aryl, C 6-14 aryloxy group, 5-10 membered heterocyclic group, hydroxyl group, C 1-40 Alkoxy, C 3-6 Cycloalkoxy, acetoxy, benzoyloxy, amino, mono-C 1-6 Alkylamino, N-acetylamino, di-C 1-6 Alkylamino, halogen atom, C 1-6 Alkoxycarbonyl, phenoxycarbonyl, N-methylcarbamoyl, N-phenylcarbamoyl, tri-C 1-6 Alkyl silyl, tri-C 1-6 Alkyl, silyl, alkyl, cyano, nitro, carboxyl, etc.

[0099] The terms "N-protected amino acid" and "N-protected peptide" refer to amino acids or peptides in which the amino group at the amino end of the amino acid backbone or the amino group at the N-terminus of the peptide is protected, while the carboxyl group at the carboxyl end of the amino acid backbone or the carboxyl group at the C-terminus of the peptide is unprotected.

[0100] The terms "C-protected amino acid" and "C-protected peptide" refer to amino acids or peptides in which the carboxyl group at the C-terminus of the amino acid backbone is protected, or the amino group at the N-terminus of the peptide is unprotected.

[0101] The amino acids used in this invention also include N-substituted amino acids.

[0102] The term "N-substituted amino acid" refers to an amino acid obtained by introducing a substituent onto the amino group of the amino group in the amino chain. A specific example is NC... 1-6 Alkyl amino acids, NC 2-6 Alkenyl amino acids, NC 2-6 Alkyne amino acids, NC 6-14 Aromatic amino acids, NC 1-6 Alkoxy amino acids (the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 alkynyl group, C 6-14 Aryl and C 1-6 Alkoxy groups can have substituents, and examples of such substituents are the same as those in the "substituents" section above under "aliphatic hydrocarbon groups that can have substituents".

[0103] The above NC 1-6 Alkyl amino acids are preferably those with a C atom introduced onto the amino group. 6-14 Aryl, C 1-6 Alkoxy, C 1-6 Alkoxycarbonyl, diC 1-6 Alkylamino, 5-10 membered heterocyclic or tricyclic 1-6 C of alkylsilyl 1-6 The amino acid obtained by alkylation is more preferably an amino group having a C3 group introduced onto the amino group. 6-14 Aryl, C 1-6 alkoxy, or C 1-6 C of alkoxycarbonyl 1-6 The amino acid obtained by alkylation is more preferably an amino acid obtained by introducing a methyl, ethyl, n-propyl, n-butyl or benzyl group onto an amino group.

[0104] The term "group derived from amino acids" as used in this invention refers to a divalent group obtained by removing a hydrogen atom from the nitrogen atom of the primary or secondary amino group present in the main chain of an amino acid and removing a hydroxyl group from the carboxyl group present in the main chain.

[0105] The term "peptide-derived group" as used in this invention refers to a divalent group obtained by removing a hydrogen atom from the nitrogen atom of the primary or secondary amino group constituting the N-terminus of the amino acid and removing a hydroxyl group from the carboxyl group constituting the C-terminus of the amino acid.

[0106] The stereostructure of α-amino acids is not particularly limited, but the L-form is preferred.

[0107] All technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Any methods and materials identical or equivalent to those described in this specification may be used in the implementation or testing of this invention, but preferred methods and materials are described below. All publications and patents mentioned in this specification are incorporated herein by reference for the purpose of describing and disclosing, for example, constructs and methodologies described in publications that are applicable in connection with the described invention.

[0108] (Detailed description of the method for manufacturing the peptides of the present invention)

[0109] Hereinafter, the peptide manufacturing method of the present invention can be carried out by performing all or appropriately combining the unit steps described as each step (1) to (5).

[0110] It should be noted that this specific explanation is based on the following content.

[0111] (a) R in the records of processes (1) to (5) 1 R 2 and R 3 Same meaning as above.

[0112] (b) The specific reaction conditions are not particularly limited as long as they enable the production of the peptides of the present invention. Preferred conditions for each reaction are described in detail as appropriate.

[0113] (c) Each reaction may be carried out in a solvent as needed, preferably in a solvent. The solvents described in each reaction may be used alone or in combination of two or more.

[0114] Process (1)

[0115] This step involves mixing an N-protected amino acid or N-protected peptide with a carboxylic acid halide. This step also involves activating the C-terminus of the N-protected amino acid or N-protected peptide using the carboxylic acid halide. In one embodiment of the invention, formula (III)P... 1 -A 1 -OH(where P is in the formula) 1 As an N-terminal protecting group, A 1 This indicates a group derived from an amino acid or a peptide. (This refers to the process of mixing an N-protected amino acid or N-protected peptide with a carboxylic acid halide.)

[0116] Carboxylic acid halides are represented by the following formula (I).

[0117] [Chemical Formula 3]

[0118]

[0119] (In the formula, X represents a halogen atom, R) 1 R 2 and R 3 Each of the above can independently represent an aliphatic hydrocarbon group that may have substituents, R 1 R 2 and R 3 The total number of carbon atoms in it ranges from 3 to 40.

[0120] The carboxylic acid halide represented by formula (I) is preferably R. 1 R 2 and R 3 The carboxylic acid halide in which two or more of the groups are independently aliphatic hydrocarbon groups containing one or more tertiary or quaternary carbon atoms, more preferably R 1 It is methyl, and R 2 and R 3 Carboxylic acid halides that are independently composed of aliphatic hydrocarbon groups containing one or more tertiary or quaternary carbon atoms, and are further preferably R 1 It is methyl, and R 2 and R 3 Each of the following is an independent carbon atom containing more than one tertiary or quaternary carbon atom: 3-10 Alkyl carboxylic acid halides, particularly preferably compounds represented by formula (II) below.

[0121] [Chemical Formula 4]

[0122]

[0123] The halogen atom represented by X in formulas (I) and (II) can be any halogen atom without any particular restriction, but is preferably a chlorine atom or a bromine atom, and more preferably a chlorine atom.

[0124] The amount of carboxylic acid halide used relative to N-protected amino acids or N-protected peptides is preferably 0.2 to 50 equivalents, more preferably 0.5 to 20 equivalents, and even more preferably 0.8 to 5 equivalents.

[0125] P of equation (III) 1 The N-terminal protecting group is not particularly limited, but specific examples include urethane-based protecting groups (9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(p-biphenyl)isopropyloxycarbonyl, etc.), amide-based protecting groups (acetyl, trifluoroacetyl, etc.), imide-based protecting groups (phthaloyl, etc.), sulfonamide-based protecting groups (p-toluenesulfonyl, 2-nitrobenzenesulfonyl, etc.), benzyl, etc., preferably 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl or benzyloxycarbonyl, more preferably 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl.

[0126] The amino acids in the N-protected amino acid represented by formula (III) and the amino acids in the C-terminal residues of the N-protected peptide are not particularly limited, but are preferably α-amino acids, β-amino acids, γ-amino acids, or δ-amino acids, more preferably α-amino acids or β-amino acids, further preferably α-amino acids, and even more preferably α-amino acids other than glycine, particularly preferably valine, phenylalanine, threonine, leucine, tryptophan, serine, cysteine, aspartic acid, or tyrosine. It should be noted that in the presence of two or more amino groups (e.g., arginine, lysine, etc.), two or more carboxyl groups (e.g., glutamic acid, aspartic acid, etc.), or reactive functional groups (e.g., cysteine, serine, etc.), the reactive functional groups present in the amino acid side chains that do not participate in peptide formation can be protected.

[0127] In equation (III), A 1 When the group is derived from a peptide, there is no particular limitation on the number of amino acid residues contained in the group, but it is preferably 2 to 40, more preferably 2 to 20.

[0128] This process can be carried out by adding alkali as needed.

[0129] There are no particular limitations on the alkali used in this process. Examples include aliphatic amines (e.g., triethylamine, N,N-diisopropylethylamine, N-methylmorpholine), aromatic amines (e.g., pyridine, imidazole, N,N-dimethyl-4-aminopyridine), amidines (e.g., diazabicycloundecene), and alkali metal salts (e.g., sodium bicarbonate, potassium carbonate). Aliphatic amines are preferred, and N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine are more preferred.

[0130] The amount of alkali used in this process is preferably 0.2 to 50 equivalents relative to the carboxylic acid halide, more preferably 0.5 to 20 equivalents, and even more preferably 0.8 to 5 equivalents.

[0131] The solvent used in this process is not particularly limited as long as it does not hinder the activation reaction. Examples include halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl tert-butyl ether), amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide), nitrile solvents (e.g., acetonitrile), ketone solvents (e.g., acetone, methyl ethyl ketone), aliphatic hydrocarbon solvents (e.g., hexane, heptane, cyclohexane), and ester solvents (e.g., ethyl acetate). Nitrile solvents, amide solvents, or ether solvents are preferred, and acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide are more preferred.

[0132] The amount of solvent used relative to the carboxylic acid halide is preferably less than 100 times by mass, more preferably 1 to 50 times by mass, and even more preferably 3 to 20 times by mass.

[0133] This process can use an oil bath or a cooling bath to control the temperature as needed. There are no particular limitations on the temperature, but it is preferably -40°C to the reflux temperature of the mixture, more preferably -20°C to 50°C, and even more preferably -10°C to 30°C.

[0134] This process generates C-terminal activated N-protected amino acids or N-protected peptides. Therefore, the product obtained through this process refers to C-terminal activated N-protected amino acids or N-protected peptides, or mixtures containing either. The C-terminal activated N-protected amino acids or N-protected peptides thus obtained can be directly mixed with the C-protected peptide in the form of a reaction solution without purification, or they can be separated as a (crude) purified product and then mixed with the C-protected peptide.

[0135] Process (2)

[0136] This step involves mixing the product obtained in step (1) with a C-protected amino acid or a C-protected peptide. This step also involves mixing the C-terminally activated N-protected amino acid or N-protected peptide obtained in step (1) with a C-protected amino acid or C-protected peptide. In one embodiment of the invention, the C-terminally activated N-protected amino acid or N-protected peptide obtained in step (1) is mixed with formula (IV)HA. 2 -OP 2 (A 2 P indicates a group derived from an amino acid or a peptide. 2 The process of mixing C-protected amino acids or C-protected peptides, represented by a C-terminal protecting group.

[0137] The amino acids in the C-protected amino acid represented by formula (IV) and the amino acids in the N-terminal residues of the C-protected peptide are not particularly limited, but are preferably α-amino acids, β-amino acids, γ-amino acids, or δ-amino acids, more preferably α-amino acids or β-amino acids, further preferably α-amino acids, and even more preferably phenylalanine, glycine, valine, proline, leucine, or ornithine. It should be noted that in the presence of two or more amino groups (e.g., arginine, lysine, etc.), two or more carboxyl groups (e.g., glutamic acid, aspartic acid, etc.), or reactive functional groups (e.g., cysteine, serine, etc.), the reactive functional groups of the amino acid side chains that do not participate in peptide formation can be protected.

[0138] In equation (IV), A2 When the group is derived from a peptide, there is no particular limitation on the number of amino acid residues contained in the group, but it is preferably 2 to 40, more preferably 2 to 20.

[0139] P of equation (IV) 2 The C-terminal protecting group can be any protecting group commonly used in the synthesis of amino acids or peptides, and there are no particular restrictions. Examples include methyl, ethyl, tert-butyl, benzyl, allyl, silyl, etc.

[0140] In equation (IV), P 2 The groups can originate from a solid support, and the method of bonding is not particularly limited; they can be directly bonded or bonded through a linker. The solid support is not particularly limited; examples include synthetic resins such as nitrocellulose, agarose beads, modified cellulose fibers, polypropylene, polyethylene glycol, polystyrene, or polyacrylamide. The linker is also not particularly limited; examples include 2-chlorotriphenylmethyl (2-ClTrt), 4-(hydroxymethyl)benzoic acid, 3,4-dihydro-2H-pyran-2-ylmethanol, 4-(hydroxymethyl)phenoxyacetic acid, 3-hydroxyxanthan-9-one, N-methoxy-3-aminopropionic acid, and 3-methoxy-2-nitropyridine.

[0141] The solvents used in this process are not particularly limited. Examples include halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform), aromatic hydrocarbon solvents (e.g., toluene, xylene), ether solvents (e.g., tetrahydrofuran, 1,4-dioxane, cyclopentylmethyl ether, methyl tert-butyl ether), amide solvents (e.g., N,N-dimethylformamide), and nitrile solvents (e.g., acetonitrile). Nitrile solvents, amide solvents, or ether solvents are preferred, and acetonitrile, tetrahydrofuran, or N,N-dimethylacetamide are more preferred.

[0142] The amount of solvent used is preferably less than 100 times by mass relative to the C-protected amino acid or C-protected peptide, more preferably 1 to 50 times by mass, and even more preferably 3 to 20 times by mass.

[0143] For the resulting mixture, the temperature is controlled using an oil bath or a cooling bath as needed. There are no particular limitations on the temperature of the mixture, but it is preferably -40°C to the reflux temperature of the reaction mixture, more preferably -20°C to 50°C, and even more preferably -10°C to 30°C.

[0144] If the C-protected amino acids or C-protected peptides used in this process form salts, they can be converted into free bodies by adding organic amines.

[0145] There are no particular limitations on the organic amines used in converting the salts of C-protected amino acids or C-protected peptides into their free forms. Examples include aliphatic amines (e.g., dicyclohexylamine, piperidine, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine) and aromatic amines (e.g., pyridine, imidazole, N,N-dimethyl-4-aminopyridine). Aliphatic amines are preferred, and triethylamine or N,N-diisopropylethylamine are more preferred.

[0146] Regarding the amount of organic amine used in converting the salt of C-protected amino acids or C-protected peptides into free bodies, it is preferably 0.01 equivalents to 50 equivalents relative to the C-protected amino acids or C-protected peptides, more preferably 0.1 equivalents to 20 equivalents, and even more preferably 0.2 equivalents to 5 equivalents.

[0147] In addition, in the peptide manufacturing method of the present invention, the following steps (3) to (5) can be repeated a desired number of times for the peptide obtained in step (2), thereby further extending the peptide chain.

[0148] Step (3) removes the protecting group at the N-terminus of the peptide obtained in step (2) or (5).

[0149] Step (4) involves mixing an N-protected amino acid or N-protected peptide with a carboxylic acid halide represented by formula (I).

[0150] Step (5) involves mixing the product obtained in step (4) with the product obtained in step (3).

[0151] Steps (4) and (5) can be carried out by the same operation as steps (1) and (2) above, or by the usual peptide synthesis reaction.

[0152] The “N-protected amino acid or N-protected peptide” used in step (4) may be the same as or different from the substance used in step (1). Similarly, the “carboxylic acid halide represented by formula (I)” used in step (4) may be the same as or different from the carboxylic acid halide used in step (1).

[0153] In the peptide manufacturing method of the present invention, the purification of the peptides obtained in steps (1) to (5) can be appropriately omitted to the extent that it does not affect the reaction of subsequent steps.

[0154] Process (3)

[0155] This step is to remove the N-terminal protecting group from the peptide obtained from step (2) or step (5).

[0156] The deprotection conditions used in this process can be appropriately selected according to the type of N-terminal protecting group. For example, in the case of 9-fluorenylmethoxycarbonyl, it is carried out by treatment with a secondary or tertiary amine (e.g., pyrrolidine, piperidine, morpholine, triethylamine); in the case of tert-butoxycarbonyl, it is carried out by treatment with an acid (e.g., trifluoroacetic acid, hydrochloric acid, Lewis acid); and in the case of benzyloxycarbonyl or allyloxycarbonyl, it is carried out by hydrogenation under neutral conditions, for example, in the presence of a metal catalyst.

[0157] In each reaction, when the reaction matrix has hydroxyl, thiol, amino, carboxyl or carbonyl groups (especially when the side chain of an amino acid or peptide has a functional group), a protecting group, as commonly used in peptide chemistry, can be introduced onto these groups. The target compound can be obtained by removing the protecting group as needed after the reaction.

[0158] Protection and deprotection can be carried out by using protection-deprotection reactions with commonly known protecting groups (e.g., see Protective Groups in Organic Synthesis, Fourth Edition, T.W. Greene, John Wiley & Sons Inc. (2006)).

[0159] Example

[0160] The following examples, which are provided as reference examples, comparative examples and embodiments, illustrate the present invention in more detail, but the present invention is not limited to these embodiments.

[0161] In this specification, when abbreviations are used to designate amino acids, etc., each designation is based on the abbreviations prescribed by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the field.

[0162] In this specification, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl is sometimes referred to as ISTA, and neopentanoyl is referred to as Piv.

[0163] It should be noted that in the synthesis example, "(v / v)" refers to (volume / volume), and "M" refers to mol / L.

[0164] The safety evaluation of ISTA-Cl and ISTA-Br was conducted using a differential scanning calorimeter manufactured by METTLER TOLED, under the conditions described below.

[0165] <Measurement Conditions>

[0166] Heating range: 30-300℃

[0167] Heating rate: 10℃ / min

[0168] The dish used: Swiss Institute Au dish M20

[0169] For high performance liquid chromatography / mass spectrometry, unless otherwise specified, use any of the following: Waters ACQUITY UPLC H-Class / QDa, Waters ACQUITY UPLC H-Class / SQD2, or Shimadzu LC-20AD / Triple Tof5600.

[0170] In high performance liquid chromatography / mass spectrometry (HPLC / MS) records, ESI+ represents the positive mode of electrospray ionization, and M+H refers to proton adducts.

[0171] Unless otherwise specified, the quantitative yields of each product and byproduct were calculated using high performance liquid chromatography (HPLC) analysis under analytical conditions 1.

[0172] <Analysis Condition 1>

[0173] High performance liquid chromatography: SHIMADZU LC-20

[0174] Column: Agilent Poroshell 120EC-C18 (2.7μm, 3.0×100mm)

[0175] Column oven temperature: 40℃

[0176] Eluent: 0.025 vol% trifluoroacetic acid; Acetonitrile: 0.025 vol% trifluoroacetic acid aqueous solution

[0177] 95:5 (0-12 minutes), 95:5 (12-18 minutes), 10:90 (18.1-23 minutes) (v / v)

[0178] Elution rate: 0.7 mL / min

[0179] Detection wavelength: 210nm

[0180] Unless otherwise specified, purification based on silica column chromatography used any of the following: Yamazen Hi-Flash column, Biotage SNAP Ultra Silica Cartridge, Merck Silica 60, or Fuji Silysia Chemical Ltd. PSQ60B.

[0181] In the following reference examples and embodiments, the yield or quantitative yield sometimes exceeds 100%. These exceed 100% due to measurement errors, the influence of the purity of the raw materials or products, or other important factors based on common technical knowledge. In the following embodiments, the reasons for yields exceeding 100% are not specifically mentioned, but those skilled in the art will fully understand the scientific soundness of these embodiments.

[0182] In the following reference examples and synthesis examples, quantitative yields of the target substance and byproducts are sometimes shown, which are the result of the reaction. Furthermore, when quantitative yields are shown, unless otherwise specified regarding the yield of byproducts, it means that no byproducts were produced.

[0183] Reference Example 1: Safety evaluation of ISTA-Cl based on differential scanning calorimetry

[0184] ISTA-Cl (16.8 mg) was placed in an Au dish, sealed, and its calorific value was measured. No calorific peak was detected. This confirms that ISTA-Cl does not exhibit calorific value due to self-decomposition. It should be noted that the calorific values ​​of COMU and isobutyl chloroformate are 773.17 J / g and 467.31 J / g, respectively (Non-Patent Literature 4).

[0185] Reference Example 2: Safety evaluation of ISTA-Br based on differential scanning calorimetry

[0186] ISTA-Br (14.9 mg) was placed in an Au dish, sealed, and calorimetry was performed. No calorimetric peak was detected. Therefore, it is confirmed that ISTA-Br does not exhibit calorimetry due to self-decomposition.

[0187] Reference Example 3: Synthesis of ISTA-Br

[0188] [Chemical Formula 5]

[0189]

[0190] 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoic acid (4.0 g, 14.06 mmol), N,N-dimethylformamide (0.031 g, 0.422 mmol), and xylene (4.0 mL) were mixed, and thionyl bromide (4.8 g, 21.15 mmol) was added at 0 °C. The mixture was heated to room temperature and stirred for 1 hour. The resulting reaction solution was distilled under reduced pressure to obtain 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoic acid bromide (4.31 g, yield 88%) as a clear liquid.

[0191] Reference Example 4: Synthesis of Fmoc-Val-Phe-OEt

[0192] [Chemical Formula 6]

[0193]

[0194] Fmoc-Val-OH (0.177 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.8 mL) were mixed, and tervapotranol chloride (0.058 g, 0.479 mmol) was added at 0 °C, followed by stirring for 1 hour. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Val-Phe-OEt (target analyte) in the organic layer was 79%, and the quantitative yield of Piv-Phe-OEt (byproduct) was 21%.

[0195] Piv-Phe-OEt MASS(ESI+)m / z; 278.0(M+H)+

[0196] Reference Example 5: Synthesis of Cbz-Val-Phe-OEt

[0197] [Chemical Formula 7]

[0198]

[0199] Cbz-Val-OH (0.131 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.3 mL) were mixed, and tervapotranol chloride (0.058 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Cbz-Val-Phe-OEt (target analyte) in the organic layer was 77%, and the quantitative yield of Piv-Phe-OEt (byproduct) was 22%.

[0200] See Example 6: Synthesis of Boc-Val-Phe-OEt

[0201] [Chemical Formula 8]

[0202]

[0203] Boc-Val-OH (0.114 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.1 mL) were mixed, and tervapotranol chloride (0.058 g, 0.479 mmol) was added at 0 °C, followed by stirring for 1 hour. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Boc-Val-Phe-OEt (target analyte) was 79%, and the quantitative yield of Piv-Phe-OEt (byproduct) was 18%.

[0204] See Example 7: Synthesis of Fmoc-Trp(Boc)-Ala-OBn

[0205] [Chemical Formula 9]

[0206]

[0207] Fmoc-Trp(Boc)-OH (0.293 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (2.9 mL) were mixed, and tervapotranol chloride (0.061 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Trp(Boc)-Ala-OBn (target analyte) in the organic layer was 84%, and the quantitative yield of Piv-Ala-OBn (byproduct) was 10%.

[0208] Piv-Ala-OBn MASS(ESI+)m / z;264.9(M+H)+

[0209] Refer to Example 8: Synthesis of Fmoc-Val-Phe-Phe-OEt

[0210] [Chemical Formula 10]

[0211]

[0212] Fmoc-Val-OH (0.118 g, 0.348 mmol), N,N-diisopropylethylamine (0.048 g, 0.377 mmol), and acetonitrile (1.2 mL) were mixed, and tervapotranol chloride (0.039 g, 0.32 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. A solution obtained by mixing HCl·H-Phe-Phe-OEt (0.1 g, 0.29 mmol), N,N-diisopropylethylamine (0.045 g, 0.348 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (3.0 g), the quantitative yield of Fmoc-Val-Phe-Phe-OEt (target analyte) in the organic layer was 85%, and the yield of Piv-Phe-Phe-OEt (byproduct) was 18%.

[0213] Piv-Phe-Phe-OEt MASS(ESI+)m / z; 425.2(M+H)+

[0214] See Example 9: Synthesis of Fmoc-Trp(Boc)-Ala-Phe-OEt

[0215] [Chemical Formula 11]

[0216]

[0217] Fmoc-Trp(Boc)-OH (0.209 g, 0.40 mmol), N,N-diisopropylethylamine (0.055 g, 0.43 mmol), and acetonitrile (2.1 mL) were mixed, and tervapotranol chloride (0.044 g, 0.36 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. A solution obtained by mixing HCl·H-Ala-Phe-OEt (0.1 g, 0.33 mmol), N,N-diisopropylethylamine (0.051 g, 0.40 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (3.0 g), the quantitative yield of Fmoc-Trp(Boc)-Ala-Phe-OEt (target analyte) in the organic layer was 69%, and the quantitative yield of Piv-Ala-Phe-OEt (byproduct) was 26%.

[0218] Piv-Ala-Phe-OEt MASS(ESI+)m / z; 349.1(M+H)+

[0219] Synthesis Example 1: Synthesis of Fmoc-Val-Phe-OEt

[0220] [Chemical Formula 12]

[0221]

[0222] Fmoc-Val-OH (0.177 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.8 mL) were mixed. A 50% by mass toluene solution (0.29 g, 0.479 mmol) of 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl chloride (ISTA-Cl) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by separately mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The obtained reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After separating the aqueous layer again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Val-Phe-OEt (target analyte) in the organic layer was 95%, and the quantitative yield of ISTA-Phe-OEt (byproduct) was 1%.

[0223] Fmoc-Val-Phe-OEt MASS(ESI+)m / z; 515.7(M+H)+

[0224] ISTA-Phe-OEt MASS(ESI+)m / z; 461.1(M+H)+

[0225] Synthesis Example 2: Synthesis of Fmoc-Val-Phe-OEt

[0226] [Chemical Formula 13]

[0227]

[0228] Mix Fmoc-Val-OH (0.207 g, 0.610 mmol), N,N-diisopropylethylamine (0.084 g, 0.650 mmol), and acetonitrile (1 mL). Add a solution obtained by mixing 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctanoyl bromide (ISTA-Br) (0.197 g, 0.566 g) and toluene (0.197 g) at 0 °C and stir for 1 hour. Add another solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.526 mmol), and acetonitrile (1.0 mL) to this reaction solution and stir for 1 hour. The obtained reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (6.0 g), and then 10% potassium hydrogen sulfate aqueous solution (1.0 g) and water (2.0 g) were added. The mixture was then separated. After further separation of the aqueous layer with ethyl acetate (3.0 g), the quantitative yield of Fmoc-Val-Phe-OEt (the target analyte) in the organic layer was 94%, and the quantitative yield of ISTA-Phe-OEt (the byproduct) was 1%.

[0229] Fmoc-Val-Phe-OEt MASS(ESI+)m / z; 515.7(M+H)+

[0230] ISTA-Phe-OEt MASS(ESI+)m / z; 461.1(M+H)+

[0231] Synthesis Example 3: Synthesis of Cbz-Val-Phe-OEt

[0232] [Chemical Formula 14]

[0233]

[0234] Cbz-Val-OH (0.131 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.3 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Cbz-Val-Phe-OEt (target analyte) in the organic layer was 95%, and the quantitative yield of ISTA-Phe-OEt (byproduct) was 1%.

[0235] Cbz-Val-Phe-OEt MASS(ESI+)m / z; 427.6(M+H)+

[0236] Synthesis Example 4: Synthesis of Boc-Val-Phe-OEt

[0237] [Chemical Formula 15]

[0238]

[0239] Boc-Val-OH (0.114 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.1 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Boc-Val-Phe-OEt (target analyte) was 96%, and the quantitative yield of ISTA-Phe-OEt (byproduct) was 1%.

[0240] Boc-Val-Phe-OEt MASS(ESI+)m / z; 393.5(M+H)+

[0241] Synthesis Example 5: Synthesis of Fmoc-Phe-Phe-OEt

[0242] [Chemical Formula 16]

[0243]

[0244] Fmoc-Phe-OH (0.202 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (2.0 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Phe-Phe-OEt (target analyte) was 101%.

[0245] Fmoc-Phe-Phe-OEt MASS(ESI+)m / z; 563.6(M+H)+

[0246] Synthesis Example 6: Synthesis of Cbz-Phe-Phe-OEt

[0247] [Chemical Formula 17]

[0248]

[0249] Cbz-Phe-OH (0.156 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (1.6 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Cbz-Phe-Phe-OEt (target analyte) was 99%.

[0250] Cbz-Phe-Phe-OEt MASS(ESI+)m / z; 563.6(M+H)+

[0251] Synthesis Example 7: Synthesis of Fmoc-Phe-MePhe-OMe

[0252] [Chemical Formula 18]

[0253]

[0254] Fmoc-Phe-OH (0.202 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (2.0 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-MePhe-OMe (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Phe-MePhe-OMe (target analyte) was 91%.

[0255] Fmoc-Phe-MePhe-OMe MASS(ESI+)m / z; 563.7(M+H)+

[0256] Synthesis Example 8: Synthesis of Fmoc-Leu-Gly-OBn

[0257] [Chemical Formula 19]

[0258]

[0259] Fmoc-Leu-OH (0.210 g, 0.60 mmol), N,N-diisopropylethylamine (0.083 g, 0.65 mmol), and acetonitrile (2.1 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.330 g, 0.55 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Gly-OBn (0.1 g, 0.50 mmol), N,N-diisopropylethylamine (0.077 g, 0.60 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Leu-Gly-OBn (target analyte) in the organic layer was 98%, and the quantitative yield of ISTA-Gly-OBn (byproduct) was 1%.

[0260] Fmoc-Leu-Gly-OBn MASS(ESI+)m / z; 501.6(M+H)+

[0261] ISTA-Gly-OBn MASS(ESI+)m / z;432.7(M+H)+

[0262] Synthesis Example 9: Synthesis of Fmoc-Trp(Boc)-Gly-OBn

[0263] [Chemical Formula 20]

[0264]

[0265] Fmoc-Trp(Boc)-OH (0.313 g, 0.60 mmol), N,N-diisopropylethylamine (0.083 g, 0.65 mmol), and acetonitrile (3.1 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.330 g, 0.55 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Gly-OBn (0.1 g, 0.50 mmol), N,N-diisopropylethylamine (0.077 g, 0.60 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Trp(Boc)-Gly-OBn (target analyte) in the organic layer was 93%, and the quantitative yield of ISTA-Gly-OBn (byproduct) was 2%.

[0266] Fmoc-Trp-Gly-OBn MASS(ESI+) m / z; 574.3(M+H)+ (detected as a de-Boc body)

[0267] Synthesis Example 10: Synthesis of Fmoc-Leu-Ala-OBn

[0268] [Chemical Formula 21]

[0269]

[0270] Fmoc-Leu-OH (0.197 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (2.0 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.309 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Leu-Ala-OBn (target analyte) was 98%.

[0271] Fmoc-Leu-Ala-OBn MASS(ESI+)m / z; 515.7(M+H)+

[0272] Synthesis Example 11: Synthesis of Fmoc-Trp(Boc)-Ala-OBn

[0273] [Chemical Formula 22]

[0274]

[0275] Fmoc-Trp(Boc)-OH (0.293 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (2.9 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.309 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Trp(Boc)-Ala-OBn (target analyte) in the organic layer was 96%, and the quantitative yield of ISTA-Ala-OBn (byproduct) was 1%.

[0276] Fmoc-Trp(Boc)-Ala-OBn MASS(ESI+)m / z; 688.4(M+H)+

[0277] ISTA-Ala-OBn MASS(ESI+)m / z;446.3(M+H)+

[0278] Synthesis Example 12: Synthesis of Fmoc-Ser(tBu)-Ala-OBn

[0279] [Chemical Formula 23]

[0280]

[0281] Fmoc-Ser(tBu)-OH (0.213 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (2.1 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.309 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Ser(tBu)-Ala-OBn (target analyte) was 97%.

[0282] Fmoc-Ser(tBu)-Ala-OBn MASS(ESI+)m / z; 545.3(M+H)+

[0283] Synthesis Example 13: Synthesis of Fmoc-Cys(Trt)-Ala-OBn

[0284] [Chemical Formula 24]

[0285]

[0286] Fmoc-Cys(Trt)-OH (0.326 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (3.3 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.309 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Cys(Trt)-Ala-OBn (target analyte) was 96%.

[0287] Synthesis Example 14: Synthesis of Fmoc-Thr(tBu)-Ala-OBn

[0288] [Chemical Formula 25]

[0289]

[0290] Fmoc-Thr(tBu)-OH (0.221 g, 0.556 mmol), N,N-diisopropylethylamine (0.078 g, 0.602 mmol), and acetonitrile (2.2 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.309 g, 0.509 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Ala-OBn (0.1 g, 0.463 mmol), N,N-diisopropylethylamine (0.072 g, 0.556 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Fmoc-Thr(tBu)-Ala-OBn (target analyte) was 92%.

[0291] Fmoc-Thr(tBu)-Ala-OBn MASS(ESI+)m / z; 559.4(M+H)+

[0292] Synthesis Example 15: Synthesis of Fmoc-Asp(tBu)-Phe-OEt

[0293] [Chemical Formula 26]

[0294]

[0295] Fmoc-Asp(tBu)-OH (0.215 g, 0.522 mmol), N,N-diisopropylethylamine (0.073 g, 0.566 mmol), and acetonitrile (2.2 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.29 g, 0.479 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.1 g, 0.435 mmol), N,N-diisopropylethylamine (0.068 g, 0.522 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of Fmoc-Asp(tBu)-Phe-OEt (target analyte) in the organic layer was 99%, and the quantitative yield of ISTA-Phe-OEt (byproduct) was 1%.

[0296] Fmoc-Asp(tBu)-Phe-OEt MASS(ESI+)m / z; 587.7(M+H)+

[0297] Synthesis Example 16: Synthesis of Boc-Gly-Pro-OBn

[0298] [Chemical Formula 27]

[0299]

[0300] Boc-Gly-OH (0.086 g, 0.50 mmol), N,N-diisopropylethylamine (0.070 g, 0.538 mmol), and acetonitrile (0.9 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.27 g, 0.456 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Pro-OBn (0.1 g, 0.414 mmol), N,N-diisopropylethylamine (0.053 g, 0.50 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After separating the aqueous layer again with ethyl acetate (2.0 g), the yield of the organic layer containing Boc-Gly-Pro-OBn (the target compound) was 91%.

[0301] Boc-Gly-Pro-OBn MASS(ESI+)m / z; 363.1(M+H)+

[0302] Synthesis Example 17: Synthesis of Boc-Asp(tBu)-Leu-OMe

[0303] [Chemical Formula 28]

[0304]

[0305] Boc-Asp(tBu)-OH (0.191 g, 0.66 mmol), N,N-diisopropylethylamine (0.092 g, 0.715 mmol), and acetonitrile (2.0 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.367 g, 0.61 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Leu-OMe (0.1 g, 0.55 mmol), N,N-diisopropylethylamine (0.085 g, 0.66 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Boc-Asp(tBu)-Leu-OMe (target analyte) was 99%.

[0306] Boc-Asp(tBu)-Leu-OMe MASS(ESI+)m / z; 417.1(M+H)+

[0307] Synthesis Example 18: Synthesis of Boc-Tyr(tBu)-Orn(Boc)-OMe

[0308] [Chemical Formula 29]

[0309]

[0310] Boc-Tyr(tBu)-OH (0.142 g, 0.42 mmol), N,N-diisopropylethylamine (0.059 g, 0.46 mmol), and acetonitrile (1.4 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.233 g, 0.39 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. A solution obtained by mixing HCl·H-Orn(Boc)-OMe (0.1 g, 0.35 mmol), N,N-diisopropylethylamine (0.054 g, 0.42 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (2.0 g), the quantitative yield of the organic layer Boc-Tyr(tBu)-Orn(Boc)-OMe (target analyte) was 99%.

[0311] Boc-Tyr(tBu)-Orn(Boc)-OMe MASS(ESI+)m / z; 566.4(M+H)+

[0312] Synthesis Example 19: Synthesis of Fmoc-Val-Phe-Phe-OEt

[0313] [Chemical Formula 30]

[0314]

[0315] Fmoc-Val-OH (0.118 g, 0.348 mmol), N,N-diisopropylethylamine (0.048 g, 0.377 mmol), and acetonitrile (1.2 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.193 g, 0.32 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Phe-Phe-OEt (0.1 g, 0.29 mmol), N,N-diisopropylethylamine (0.045 g, 0.348 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (3.0 g), the quantitative yield of Fmoc-Val-Phe-Phe-OEt (target analyte) in the organic layer was 96%, and the yield of ISTA-Phe-Phe-OEt (byproduct) was 1%.

[0316] Fmoc-Val-Phe-Phe-OEt MASS(ESI+)m / z; 662.4(M+H)+

[0317] ISTA-Phe-Phe-OEt MASS(ESI+)m / z; 607.4(M+H)+

[0318] Synthesis Example 20: Synthesis of Fmoc-Asp(tBu)-Phe-Phe-OEt

[0319] [Chemical Formula 31]

[0320]

[0321] Fmoc-Asp(tBu)-OH (0.143 g, 0.348 mmol), N,N-diisopropylethylamine (0.048 g, 0.377 mmol), and acetonitrile (1.4 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.193 g, 0.32 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Phe-Phe-OEt (0.1 g, 0.29 mmol), N,N-diisopropylethylamine (0.045 g, 0.348 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (3.0 g), the quantitative yield of the organic layer Fmoc-Asp(tBu)-Phe-Phe-OEt (target analyte) was 94%.

[0322] Fmoc-Asp(tBu)-Phe-Phe-OEt MASS(ESI+)m / z; 734.4(M+H)+

[0323] Synthesis Example 21: Synthesis of Fmoc-Trp(Boc)-Ala-Phe-OEt

[0324] [Chemical Formula 32]

[0325]

[0326] Fmoc-Trp(Boc)-OH (0.209 g, 0.40 mmol), N,N-diisopropylethylamine (0.055 g, 0.43 mmol), and acetonitrile (2.1 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.220 g, 0.36 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Ala-Phe-OEt (0.1 g, 0.33 mmol), N,N-diisopropylethylamine (0.051 g, 0.40 mmol), and acetonitrile (1.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with tetrahydrofuran (3.0 g) and ethyl acetate (3.0 g), and then a saturated ammonium chloride aqueous solution (1.0 g) and water (1.0 g) were added, followed by separation. After the aqueous layer was separated again with ethyl acetate (3.0 g), the quantitative yield of Fmoc-Trp(Boc)-Ala-Phe-OEt (target analyte) in the organic layer was 97%, and the quantitative yield of ISTA-Ala-Phe-OEt (byproduct) was 1%.

[0327] Fmoc-Trp(Boc)-Ala-Phe-OEt MASS(ESI+)m / z; 773.4(M+H)+

[0328] ISTA-Ala-Phe-OEt MASS(ESI+)m / z; 531.3(M+H)+

[0329] Synthesis Example 22: Synthesis of Boc-Val-Phe-OEt

[0330] [Chemical Formula 33]

[0331]

[0332] Boc-Val-OH (0.340 g, 1.56 mmol), N,N-diisopropylethylamine (0.219 g, 1.69 mmol), and acetonitrile (3.4 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.870 g, 1.43 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Phe-OEt (0.3 g, 1.3 mmol), N,N-diisopropylethylamine (0.203 g, 1.32 mmol), and acetonitrile (3.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with ethyl acetate (6.0 g), and then a saturated ammonium chloride aqueous solution (3.0 g) and water (2.0 g) were added, followed by separation. The aqueous layer was separated again with ethyl acetate (3.0 g), and the resulting organic layer was concentrated and purified by silica gel column chromatography to give Boc-Val-Phe-OEt (0.49 g, yield 95%) as a white solid.

[0333] Boc-Val-Phe-OEt MASS(ESI+)m / z; 393.2(M+H)+

[0334] Synthesis Example 23: Synthesis of Cbz-Val-Val-Phe-OEt

[0335] [Chemical Formula 34]

[0336]

[0337] Boc-Val-Phe-OEt (0.20 g, 0.51 mmol) was mixed with 4 M HCl / 1,4-dioxane (1.0 mL) and stirred at 25 °C for 2 hours. The resulting reaction solution was concentrated, and the resulting white solid HCl·H-Val-Phe-OEt was used in subsequent processes.

[0338] Cbz-Val-OH (0.154 g, 0.612 mmol), N,N-diisopropylethylamine (0.086 g, 0.663 mmol), and acetonitrile (3.0 mL) were mixed, and a 50% (w / w) toluene solution of ISTA-Cl (0.340 g, 0.561 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing HCl·H-Val-Phe-OEt (0.51 mmol), N,N-diisopropylethylamine (0.079 g, 0.612 mmol), and acetonitrile (2.0 mL) was added to this solution, and the mixture was stirred for 1 hour. The resulting reaction solution was diluted with acetonitrile (4.0 g) and ethyl acetate (8.0 g), and then a saturated ammonium chloride aqueous solution (2.0 g) and water (2.0 g) were added, followed by separation. The aqueous layer was separated again with ethyl acetate (5.0 g), dried with sodium sulfate, and the resulting organic layer was concentrated to obtain a white solid. Ethyl acetate (4.0 g) was added to the obtained white solid, and the precipitated solid was filtered off to obtain Cbz-Val-Val-Phe-OEt (0.26 g, yield 97%) as a white solid.

[0339] Cbz-Val-Val-Phe-OEt MASS(ESI+)m / z; 526.7(M+H)+

[0340] Synthesis Example 24: Synthesis of Fmoc-Val-Val-Val-Phe-OEt

[0341] [Chemical Formula 35]

[0342]

[0343] Cbz-Val-Val-Phe-OEt (0.10 g, 0.19 mmol) was dissolved in 2,2,2-trifluoroethanol (4 mL), and 10% Pd-C (20 mg by mass) was added. The mixture was stirred at 25 °C for 1 hour under a hydrogen atmosphere. The reaction solution was filtered and concentrated, and the resulting H-Val-Val-Phe-OEt was used in subsequent processes.

[0344] Fmoc-Val-OH (0.077 g, 0.228 mmol), N,N-diisopropylethylamine (0.032 g, 0.247 mmol), and acetonitrile (0.77 mL) were mixed, and a 50% by mass toluene solution of ISTA-Cl (0.127 g, 0.21 mmol) was added at 0 °C, and the mixture was stirred for 3 hours. A solution obtained by mixing H-Val-Val-Phe-OEt (0.19 mmol), acetonitrile (1.0 mL), and NMP (0.2 mL) obtained in the previous step was added to this solution, and the mixture was stirred for 1 hour. Diisopropyl ether (10 mL) was added to the resulting reaction solution, and the precipitated solid was filtered off, yielding Fmoc-Val-Val-Val-Phe-OEt (0.14 g, yield 103%) as a white solid.

[0345] Fmoc-Val-Val-Val-Phe-OEt MASS(ESI+)m / z; 713.4(M+H)+

[0346] Synthetic Example 25: Synthesis of Fmoc-Phe-Phe-OH

[0347] (1) Add NMP (1.0 mL) to H-Phe-2-ClTrt resin (109.9 mg, 0.1 mmol, made by Merck, the resin is polystyrene), stir for 15 minutes, and then remove the solvent.

[0348] (2) Fmoc-Phe-OH (155.0 mg, 0.4 mmol) and N,N-diisopropylethylamine (0.07 mL, 0.4 mmol) were dissolved in NMP (0.8 mL), and then 50% toluene solution of ISTA-Cl (0.24 mL, 0.4 mmol) was added at room temperature and stirred for 3 hours.

[0349] (3) Add the solid obtained in (1) to the solution obtained in (2) above, and stir for 1 hour. After removing the reaction solvent, wash the obtained solid with NMP and methanol in sequence, and then dry it to obtain Fmoc-Phe-Phe-2-ClTrt resin.

[0350] (4) Add acetic acid / 2,2,2-trifluoroethanol / dichloromethane (volume ratio 1 / 2 / 7) (2 mL) to the total amount of Fmoc-Phe-Phe-2-ClTrt resin obtained in (3) above, and stir for 2 hours. After removing the detached resin by filtration, concentrate the reaction solution under reduced pressure, add diisopropyl ether to the residue, and collect the precipitated solid by filtration to obtain Fmoc-Phe-Phe-OH (51.4 mg, yield 96.1%) as a white solid.

[0351] Fmoc-Phe-Phe-OH MASS(ESI+)m / z; 535.3(M+H)+

[0352] Industrial availability

[0353] This invention provides an efficient method for manufacturing peptides.

Claims

1. A method for manufacturing peptides, comprising the following steps (1) and (2). Process (1): N-protected amino acids or N-protected peptides, and carboxylic acid halides represented by formula (II), are mixed in the presence of a base at temperatures ranging from -10°C to 30°C. In formula (II), X represents a halogen atom; and Process (2): The product obtained in step (1) is mixed with C-protected amino acids or C-protected peptides in the presence of a base.

2. The method for manufacturing the peptide as described in claim 1, further comprising repeating the following steps (3) to (5) one or more times. Process (3): Remove the protecting group at the N-terminus of the peptide obtained in step (2) or (5); Process (4): Mixing an N-protected amino acid or N-protected peptide with a carboxylic acid halide represented by formula (II); and Process (5): The product obtained in step (4) is mixed with the product obtained in step (3).

3. The method of producing a peptide according to claim 1 or 2, wherein, X is a chlorine atom or a bromine atom.

4. The method of producing a peptide according to claim 1 or 2, wherein, X is a chlorine atom.

5. The method of producing a peptide according to claim 1 or 2, wherein, The amino acids in the N-protected amino acid group are α-amino acids other than glycine.

6. The method for manufacturing the peptide as described in claim 5, wherein, The amino acids in N-protected amino acids are α-amino acids other than glycine, and the reactive functional groups of their amino acid side chains are protected.

7. The method for manufacturing the peptide as described in claim 5, wherein, The α-amino acids other than glycine are valine, phenylalanine, threonine, leucine, tryptophan, serine, cysteine, aspartic acid, or tyrosine.

8. The method for manufacturing the peptide as described in claim 1 or 2, wherein, The amino acid in the C-protected amino acid or the amino acid in the N-terminal residue of the C-protected peptide is an α-amino acid other than an N-substituted amino acid.

9. The method for manufacturing the peptide as described in claim 1 or 2, wherein, Step (2) is a process of mixing the product obtained in step (1) with the C-protective peptide.

10. The method for manufacturing the peptide as described in claim 1 or 2, wherein, The N-terminal protecting group of an N-protected amino acid or N-protected peptide is a carbamate-based protecting group.

11. The method for manufacturing the peptide as described in claim 10, wherein, The protecting group of the urethane series is 9-fluorenylmethoxycarbonyl or benzyloxycarbonyl.

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