Synthesis method of peptide compounds

Abstract

The present inventors have found that in the synthesis of a peptide compound including the condensation of the C-terminal active form of an acid component with an amine component, the C-terminal active form can be removed by mixing a solution containing the C-terminal active form remaining after the condensation reaction with a tertiary amine and water or an aqueous solution.

Description

Technical Field

[0001] The present invention relates to a method for efficiently removing unnecessary C-terminal active substances generated during the synthesis of a peptide compound, thereby efficiently producing a target peptide compound. Background Art

[0002] One form of peptide synthesis involves using a compound that activates the C-terminal carboxyl group of an amino acid or peptide so that it can react with an amine, such as an amino acid or peptide, to form an amide bond. In this case, if the compound with the activated carboxyl group remains in the reaction solution after the reaction is completed, the quality of the resulting peptide may be reduced.

[0003] Such C-terminally activated compounds include not only compounds with activated carboxyl groups used in peptide synthesis reactions, but also compounds that are activated during the reaction, such as azlactones, NCA (N-carboxyanhydride), etc., and are thus capable of reacting with amines (hereinafter, these compounds may be referred to as "C-terminally activated forms"). In addition, the C-terminally activated forms used in peptide synthesis reactions are not limited to active esters, mixed acid anhydrides, and acylisoureas synthesized using peptide condensing agents, such as those described in Non-Patent Documents 1 or 2, but include any compounds as long as they are activated so as to react with amines.

[0004] As examples of the degradation of the quality of the produced peptide, it is known that impurity peptides are produced as by-products due to the residual C-terminal active form and peptides with inserted sequences are mixed as impurities in the final product (Patent Documents 1 and 2).

[0005] As a method for eliminating this problem of residual C-terminal active forms, a method is known in which the active ester is hydrolyzed with alkaline water and removed as an aqueous alkaline solution of the corresponding amino acid (Patent Document 1). However, this method requires multiple alkaline water hydrolysis treatments, making the operation complex. Furthermore, it is speculated that increasing the number of alkaline water treatments and the length of the treatment time may lead to side reactions such as epimerization of the product, which may impair stability.

[0006] Furthermore, a method is known in which the residual C-terminal active form is captured with a polyamine having a primary amino group, such as N,N-dimethylpropane-1,3-diamine, and converted into a basic compound. The amide compound derived from the residual C-terminal active form is then removed by aqueous washing with an acidic aqueous solution to transfer it to the aqueous layer (Patent Document 3, Non-Patent Document 3). However, when highly nucleophilic primary amines are used, it is presumed that the highly electrophilic sites of the target peptide react with the primary amine, producing impurities that form covalent bonds. Therefore, this method is not suitable for the synthesis of high-purity peptides.

[0007] A method is also known in which the residual C-terminal active form is reacted with a trapping agent, such as an amine containing a potential anion with a protecting group, to convert it into an amide compound for removal (Patent Document 2). However, this method requires an aqueous extraction step after the amide compound is formed, followed by hydrocracking, and then a further aqueous extraction step, making the operation complex.

[0008] Furthermore, if the C-terminal active form remains, deprotection of the N-terminal protecting group of the resulting peptide may also occur simultaneously with deprotection of the N-terminal protecting group of the C-terminal active form. The deprotected form of the remaining C-terminal active form is an impurity, but if its absorption coefficient is low, it is difficult to detect using general HPLC, making it undesirable for quality control.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent No. 5171613

[0012] Patent Document 2: Japanese Patent No. 4142907

[0013] Patent Document 3: Japanese Patent No. 5212371

[0014] Non-patent literature

[0015] Non-patent document 1: Chem. Rev., 2011, 111, 6557.

[0016] Non-patent document 2: Organic Process Research & Development, 2016, 20, 140.

[0017] Non-patent document 3: Tetrahedron Lett., 1974, 15, 1785. Summary of the Invention

[0018] Problems to be solved by the invention

[0019] The present invention has been made in view of such circumstances, and in one aspect, an object of the present invention is to efficiently remove the residual C-terminal active form during the synthesis of a peptide compound.

[0020] Means for solving problems

[0021] The present inventors have discovered that, in the synthesis of a peptide compound comprising condensing a C-terminal active form of an acid component with an amine component, the C-terminal active form remaining in the reaction mixture can be removed by reacting it with a tertiary amine.

[0022] The present invention includes the following in a non-limiting specific embodiment.

[0023] [1] A method for producing a peptide compound, comprising:

[0024] Step A: a step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent; and

[0025] Step B: A step of mixing the reaction mixture, a tertiary amine, and water or an aqueous solution to remove the C-terminal active form.

[0026] [2] A method for producing a peptide compound, comprising:

[0027] Step A: a step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent; and

[0028] Step B: A step of mixing the reaction mixture, a tertiary amine, and water or an aqueous solution, and allowing the tertiary amine to act on the unreacted C-terminal active form to remove the C-terminal active form.

[0029] [3] The method according to [1] or [2], wherein the acid component is a first amino acid whose amino group is protected by a protecting group, or a first peptide whose N-terminal amino group is protected by a protecting group.

[0030] [4] The method according to any one of [1] to [3], wherein the amine component is a second amino acid whose carboxyl group is protected by a protecting group, or a second peptide whose C-terminal carboxyl group is protected by a protecting group.

[0031] [5] The method according to any one of [1] to [4], wherein step A is performed in the presence of a condensing agent.

[0032] [6] The method according to any one of [1] to [5], wherein the tertiary amine has nucleophilic reactivity with the C-terminal active form.

[0033] [7] The method according to any one of [1] to [6], wherein the tertiary amine is an amine having little steric hindrance near nitrogen.

[0034] [8] The method according to any one of [1] to [7], wherein the tertiary amine is represented by the following formula (A), (B), or (C):

[0035]

[0036] Where,

[0037] Among R1 to R3, (i) R1 and R2 together with the nitrogen atom to which they are bonded form a 5- to 6-membered non-aromatic heterocyclic ring, and R3 is a C1-C2 alkyl group or a C2 hydroxyalkyl group, or (ii) each independently is a C1-C2 alkyl group or a C2 hydroxyalkyl group,

[0038] X is N or O,

[0039] R4 and R5 are each independently a C1-C2 alkyl group or a C2 hydroxyalkyl group, or together with the nitrogen atom to which they are bonded, form a 5- to 6-membered non-aromatic heterocyclic ring, wherein when X is O, R5 is absent,

[0040] R6 and R7 are each independently H, C1-C2 alkyl, or methoxy,

[0041] R8 and R9 are each independently H, C1-C2 alkyl, or C2 hydroxyalkyl, or form a 5- to 6-membered non-aromatic heterocyclic ring together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded.

[0042] [9] The method according to [8], wherein R1 to R3 are each independently a C1-C2 alkyl group.

[0043]

[10] The method according to [8], wherein X is N, R4 and R5 are each independently a C1-C2 alkyl group, and R6 and R7 are H.

[0044]

[11] The method according to [8], wherein R8 and R9 are each independently H or C1-C2 alkyl.

[0045]

[12] The method according to any one of [1] to

[11] , wherein the tertiary amine is NMI, DMAP or trimethylamine.

[0046]

[13] The method according to any one of [1] to

[12] , wherein the peptide compound contains one or more unnatural amino acids.

[0047]

[14] The method according to any one of [1] to

[13] , wherein the temperature at which the tertiary amine is allowed to react with the C-terminal active form is 25°C to 60°C.

[0048]

[15] The method according to any one of [1] to

[14] , wherein 0.5 equivalents or more of the tertiary amine is added relative to the amine component.

[0049]

[16] The method according to any one of [1] to

[15] , wherein the residual rate of the C-terminal active form is 3% or less.

[0050]

[17] The method according to any one of [1] to

[16] , wherein in step B, the step further comprises separating the reaction mixture into an organic layer and an aqueous layer, and then washing the organic layer, wherein the residual amount of the C-terminal active form after washing is 1.0% or less.

[0051]

[18] The method according to any one of [1] to

[17] , wherein the solvent in the above step A is toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl acetate, ethyl acetate, methyl tert-butyl ether, cyclopentyl methyl ether, or N,N-dimethylformamide, or a mixed solvent thereof.

[0052]

[19] The method according to any one of [1] to

[18] , wherein in the step B, the aqueous solution is an alkaline aqueous solution.

[0053]

[20] The method according to any one of [1] to

[19] , wherein the side chain of the first amino acid contains one or more carbon atoms.

[0054]

[21] The method according to

[20] , wherein the side chain is an alkyl group which may be substituted, an alkenyl group which may be substituted, an alkynyl group which may be substituted, a cycloalkyl group which may be substituted, an alkoxyalkyl group which may be substituted, a cycloalkylalkyl group which may be substituted, an aralkyl group which may be substituted, or a heteroarylalkyl group which may be substituted.

[0055]

[22] The method according to any one of [1] to

[21] , wherein the time for allowing the tertiary amine to act on the C-terminal active form is 2 hours or less.

[0056]

[23] The method according to any one of [1] to

[22] , wherein the time for allowing the tertiary amine to react with the C-terminal active form is 2 minutes to 2 hours.

[0057]

[24] The method according to any one of [1] to

[23] , wherein the time for allowing the tertiary amine to react with the C-terminal active form is 5 to 60 minutes.

[0058]

[25] The method according to any one of [1] to

[24] , wherein the time for allowing the tertiary amine to react with the C-terminal active form is 5 to 50 minutes.

[0059]

[26] The method according to any one of [1] to

[25] , wherein the C-terminal active form is formed in the presence of a condensing agent comprising T3P, HATU, BEP, DMT-MM, a combination of EDC and PfpOH, a combination of EDC and HOOBt, or a combination of EDC and HOBt.

[0060]

[27] The method according to any one of [1] to

[26] , further comprising a step C: a step of deprotecting the protecting group at the N-terminus of the peptide compound.

[0061]

[28] The method according to [1] to

[27] , wherein the C-terminal active form is allowed to react with the tertiary amine to be hydrolyzed and removed.

[0062]

[29] A method for promoting the hydrolysis of a C-terminal active body, comprising the step of adding a tertiary amine and water or an aqueous solution to a solution containing residual C-terminal active bodies, and allowing the C-terminal active bodies to react with the tertiary amine.

[0063]

[30] A method for removing a hydrolyzate, comprising the step of aqueous washing a solution of the hydrolyzate containing the residual C-terminal active form.

[0064] Effects of the Invention

[0065] By using the method of the present invention, the C-terminal active form remaining after the condensation reaction can be removed efficiently and simply in a short time by a single hydrolysis treatment followed by aqueous washing, thereby enabling the synthesis of a peptide compound with high purity without column purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 It is a graph showing the relative values ​​of the residual amount of the C-terminal active form.

[0067] Figure 2 It is a graph showing the relative values ​​of the residual amount of the C-terminal active form.

[0068] Figure 3 It is a graph showing the relative values ​​of the residual amount of the C-terminal active form.

[0069] Figure 4 It is a graph showing the transition of the residual rate of the C-terminal active form. DETAILED DESCRIPTION

[0070] Hereinafter, preferred non-limiting embodiments of the present invention will be described.

[0071] All elements described in the embodiment described later are not subject to any patent practices, customs, laws, etc. in the country where the present patent application is intended to be granted, which may provide a restrictive interpretation of the contents described in the embodiment, and are intended to be regarded as being described equivalently in this "Specific Implementation Method".

[0072] As long as there is no technical contradiction based on the technical common sense of those skilled in the art, any combination of part or all of any one or more elements described in the present invention is intended to be included in the present invention and is described as should be understood by those skilled in the art.

[0073] (abbreviation)

[0074] The abbreviations used in this specification are as follows.

[0075] Abbreviation for amino acids

[0076] Aib: alpha-methylalanine

[0077] Ala: alanine

[0078] Arg: Arginine

[0079] Asn: Asparagine

[0080] Asp: Aspartic acid

[0081] Asp(tBu): O-tert-butylaspartic acid

[0082] Aze: Azetidine-2-carboxylic acid

[0083] Cys: Cysteine

[0084] Glu: glutamate

[0085] Gln: glutamine

[0086] Gly: glycine

[0087] His:histidine

[0088] Hph: Homophenylalanine

[0089] Ile: Isoleucine

[0090] Leu: leucine

[0091] Lys: lysine

[0092] MeAla: N-methylalanine

[0093] MeAsp(tBu): N-methyl O-tert-butyl aspartate

[0094] MeGly: N-methylglycine

[0095] MeIle: N-methylisoleucine

[0096] MeLeu: N-methylleucine

[0097] MePhe: N-methylphenylalanine

[0098] MeVal: N-methylvaline

[0099] Met: methionine

[0100] Phe: Phenylalanine

[0101] Phe-OtBu: O-tert-butylphenylalanine

[0102] Phe(3-F): 3-fluorophenylalanine

[0103] Pro: Proline

[0104] Ser: serine

[0105] Ser(tBu): O-tert-butylserine

[0106] Thr:Threonine

[0107] Thr(tBu): O-tert-butyl-threonine

[0108] Trp: Tryptophan

[0109] Tyr: tyrosine

[0110] Val: valine

[0111] Abbreviation of reagent / solvent

[0112] BEP: 2-bromo-1-ethylpyridinium tetrafluoroborate

[0113] DABCO: 1,4-diazabicyclo[2.2.2]octane

[0114] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0115] DCM: dichloromethane

[0116] DIPEA: diisopropyldiethylamine

[0117] DMAP: dimethylaminopyridine

[0118] DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

[0119] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

[0120] HATU: O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0121] HOAt: 1-azahydroxybenzotriazole

[0122] HOBt: 1-hydroxybenzotriazole

[0123] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

[0124] HOSu: N-hydroxysuccinimide

[0125] MTBE: Methyl tert-butyl ether

[0126] NMI: N-methylimidazole

[0127] NMM: N-methylmorpholine

[0128] T3P: Propylphosphonic anhydride (cyclic trimer)

[0129] TBAF: Tetrabutylammonium fluoride

[0130] TsOH: p-toluenesulfonic acid

[0131] Functional group abbreviation

[0132] Bn: benzyl

[0133] Boc: tert-Butoxycarbonyl

[0134] Cbz: benzyloxycarbonyl

[0135] Pfp: pentafluorophenyl

[0136] Teoc: 2-(trimethylsilyl)ethoxycarbonyl

[0137] (Definition of functional groups, etc.)

[0138] As the "halogen atom" in this specification, F, Cl, Br or I can be exemplified.

[0139] In this specification, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing one arbitrary hydrogen atom, does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and includes a hydrocarbon group or a subset of hydrocarbon groups having hydrogen and carbon atoms. Alkyl groups include not only straight-chain alkyl groups but also branched-chain alkyl groups. Specifically, alkyl groups include groups having 1 to 20 carbon atoms (C1-C 20 , below, “C p -C q " refers to an alkyl group with p to q carbon atoms, preferably C1-C 10The alkyl group is more preferably a C1-C6 alkyl group, and further preferably a C1-C2 alkyl group. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl (2-methylpropyl), n-pentyl, sec-pentyl (1-methylbutyl), tert-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl.

[0140] In this specification, "alkenyl" refers to a group having at least one double bond (two adjacent SP 2 A monovalent group containing 1-carbon atoms. Depending on the configuration of the double bond and the substituent (if any), the double bond geometry may be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl groups include not only straight-chain alkenyl groups but also branched alkenyl groups. Preferred alkenyl groups include C2-C 10 Alkenyl groups, more preferably C2-C6 alkenyl groups, specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl and the like.

[0141] In this specification, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight-chain alkynyl but also branched-chain alkynyl. As alkynyl, preferably C2-C 10 Alkynyl, more preferably C2-C6 alkynyl, specifically, for example, ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2′-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, etc.

[0142] In this specification, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic groups. Examples of the cycloalkyl group include preferably C3-C8 cycloalkyl groups, and specifically include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl.

[0143] In this specification, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Aryl group. Specific examples of the aryl group include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

[0144] In this specification, "heterocyclic group" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclic group may have a double bond and / or a triple bond in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl group, and may be a monocyclic or condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4 to 10-membered heterocyclic group), more preferably 4 to 7 (4 to 7-membered heterocyclic group). As the heterocyclic group, specifically, for example, azetidinyl, oxirane, oxetane, azetidinyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, nitrogen heterocyclobutane, oxazolidinone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 3-oxa-8-azabicyclo[3.2.1]octyl, sultam, 2-oxaspiro[3.3]heptyl, and the like.

[0145] In this specification, "heteroaryl" refers to an aromatic, cyclic, monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring, a fused ring with other rings, or a partially saturated ring. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of the heteroaryl group include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl groups.

[0146] In this specification, "alkoxy" refers to an oxy group bonded to the "alkyl" group defined above, and preferably includes a C1-C6 alkoxy group. Specific examples of the alkoxy group include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, and 3-methylbutoxy.

[0147] In this specification, "alkenyloxy" refers to an oxy group bonded to the "alkenyl" defined above, and preferably a C2-C6 alkenyloxy group is mentioned. Specific examples of alkenyloxy groups include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans forms), 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0148] In the present specification, "cycloalkoxy" refers to an oxy group bonded to the above-defined "cycloalkyl" group, and preferably a C3-C8 cycloalkoxy group is mentioned. Specific examples of the cycloalkoxy group include cyclopropyloxy, cyclobutyloxy, and cyclopentyloxy.

[0149] In this specification, "aryloxy" refers to an oxy group bonded to the above-defined "aryl", and preferably C6-C 10 Aryloxy group. Specific examples of the aryloxy group include phenoxy group, 1-naphthyloxy group, and 2-naphthyloxy group.

[0150] In this specification, "amino" refers to -NH2 in a narrow sense and -NRR' in a broad sense, where R and R' are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl, or R and R' together with the nitrogen atom to which they are bonded form a ring. Preferred examples of the amino group include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and 4- to 8-membered cyclic amino groups.

[0151] In this specification, "monoalkylamino" refers to an "amino" group defined above in which R is hydrogen and R' is an "alkyl" group defined above. Preferred examples include mono-C1-C6 alkylamino groups. Specific examples of monoalkylamino groups include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, sec-butylamino, and tert-butylamino groups.

[0152] In this specification, "dialkylamino" refers to an "amino" group defined above in which R and R' are independently an "alkyl" group defined above, preferably a diC1-C6 alkylamino group. Specific examples of dialkylamino groups include dimethylamino and diethylamino groups.

[0153] In this specification, a "cyclic amino group" refers to an "amino group" defined above in which R and R', together with the nitrogen atom to which they are bonded, form a ring. Preferred examples include 4- to 8-membered cyclic amino groups. Specific examples of cyclic amino groups include 1-azetidinyl, 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, 4-morpholinyl, 3-oxazolidinyl, 1,1-dioxidothiomorpholinyl-4-yl, and 3-oxa-8-azabicyclo[3.2.1]octan-8-yl.

[0154] In this specification, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by a hydroxy group. A C1-C6 hydroxyalkyl group is preferred, and a C2 hydroxyalkyl group is more preferred. Specific examples of the hydroxyalkyl group include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0155] As used herein, a "haloalkyl" group is an "alkyl" group defined above in which one or more hydrogen atoms are replaced by a halogen. A C1-C6 haloalkyl group is preferred, and a C1-C6 fluoroalkyl group is more preferred. Specific examples of the haloalkyl group include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, and 5,5-difluoropentyl.

[0156] In this specification, "cyanoalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by a cyano group, preferably a C1-C6 cyanoalkyl. Specific examples of the cyanoalkyl group include cyanomethyl and 2-cyanoethyl.

[0157] In this specification, "aminoalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by an "amino" group defined above, and is preferably a C1-C6 aminoalkyl group. Specific examples of the aminoalkyl group include 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, and 4-aminobutyl.

[0158] In this specification, "carboxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by a carboxyl group, and is preferably a C2-C6 carboxyalkyl. Specific examples of the carboxyalkyl group include carboxymethyl.

[0159] In this specification, "alkenyloxycarbonylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by an "alkenyloxycarbonyl" group defined above. Preferably, it is a C2-C6 alkenyloxycarbonylC1-C6 alkyl group, and more preferably a C2-C6 alkenyloxycarbonylC1-C2 alkyl group. Specific examples of the alkenyloxycarbonylalkyl group include allyloxycarbonylmethyl and 2-(allyloxycarbonyl)ethyl.

[0160] In this specification, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by an "alkoxy" group defined above. Preferably, the group comprises a C1-C6 alkoxy C1-C6 alkyl group, and more preferably a C1-C6 alkoxy C1-C2 alkyl group. Specific examples of the alkoxyalkyl group include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, isobutoxymethyl, sec-butoxymethyl, tert-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0161] In this specification, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by a "cycloalkyl" group defined above. Preferably, it is a C3-C8 cycloalkylC1-C6 alkyl group, and more preferably a C3-C6 cycloalkylC1-C2 alkyl group. Specific examples of the cycloalkylalkyl group include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0162] In this specification, a "cycloalkoxyalkyl" group refers to an "alkyl" group defined above in which one or more hydrogen atoms are replaced by a "cycloalkoxy" group defined above. Preferably, the group comprises a C3-C8 cycloalkoxy C1-C6 alkyl group, and more preferably a C3-C6 cycloalkoxy C1-C2 alkyl group. Specific examples of the cycloalkoxyalkyl group include cyclopropyloxymethyl and cyclobutyloxymethyl.

[0163] In this specification, "heterocyclylalkyl" refers to an "alkyl" group defined above in which one or more hydrogen atoms are replaced by a "heterocyclyl" group defined above. A 4- to 7-membered heterocyclyl C1-C6 alkyl group is preferred, and a 4- to 7-membered heterocyclyl C1-C2 alkyl group is more preferred. Specific examples of heterocyclylalkyl groups include 2-(tetrahydro-2H-pyran-4-yl)ethyl and 2-(azetidin-3-yl)ethyl.

[0164] In this specification, "alkylsulfonylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" group defined above are replaced by an "alkylsulfonyl" group defined above. Preferably, it is a C1-C6 alkylsulfonylC1-C6 alkyl group, and more preferably a C1-C6 alkylsulfonylC1-C2 alkyl group. Specific examples of the alkylsulfonylalkyl group include methylsulfonylmethyl and 2-(methylsulfonyl)ethyl.

[0165] In this specification, "aminocarbonylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "aminocarbonyl" defined above, preferably an aminocarbonyl C1-C6 alkyl group, more preferably an aminocarbonyl C1-C4 alkyl group. Specific examples of the aminocarbonylalkyl group include methylaminocarbonylmethyl, dimethylaminocarbonylmethyl, tert-butylaminocarbonylmethyl, 1-azetidinylcarbonylmethyl, 1-pyrrolidinylcarbonylmethyl, 1-piperidinylcarbonylmethyl, 4-morpholinylcarbonylmethyl, 2-(methylaminocarbonyl)ethyl, 2-(dimethylaminocarbonyl)ethyl, 2-(1-azetidinylcarbonyl)ethyl, 2-(1-pyrrolidinylcarbonyl)ethyl, 2-(4-morpholinylcarbonyl)ethyl, 3-(dimethylaminocarbonyl)propyl, and 4-(dimethylaminocarbonyl)butyl.

[0166] The "aryloxyalkyl" in this specification refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "aryloxy" defined above, preferably a C6-C 10 Aryloxy C1-C6 alkyl, more preferably C6-C 10 Aryloxy C1-C2 alkyl group. Specific examples of the aryloxyalkyl group include phenoxymethyl and 2-phenoxyethyl.

[0167] In this specification, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of the "alkyl" defined above is replaced by the "aryl" defined above, preferably C7-C 14 Arylalkyl, more preferably C7-C 10 Aralkyl group: Specific examples of the aralkyl group include benzyl, phenethyl, and 3-phenylpropyl.

[0168] In the present specification, "heteroarylalkyl" refers to a group in which at least one hydrogen atom of the "alkyl" group defined above is substituted by a "heteroaryl" group defined above. It is preferably a 5- to 10-membered heteroaryl C1-C6 alkyl group, and more preferably a 5- to 10-membered heteroaryl C1-C2 alkyl group. Specific examples of the heteroarylalkyl group include 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.

[0169] As used herein, a "non-aromatic heterocycle" refers to a non-aromatic heterocycle containing 1 to 5 heteroatoms among the atoms constituting the ring. The non-aromatic heterocycle may have double and / or triple bonds in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl group. Furthermore, the non-aromatic heterocycle may be a monocyclic ring, a condensed ring, or a spirocyclic ring. The number of atoms constituting the ring is not limited, but is preferably 5 to 6 (5- to 6-membered non-aromatic heterocycle). Specific examples of the non-aromatic heterocycle include azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, piperidine, tetrahydropyran, thiolane, piperazine, morpholine, thiomorpholine, dioxane, dithiane, azepane, oxepane, thiepane, and diazepane.

[0170] In this specification, the term "peptide chain" refers to a peptide chain composed of 1, 2, 3, 4 or more natural amino acids and / or non-natural amino acids linked by amide bonds and / or ester bonds.

[0171] In the present specification, "may be substituted" means that a certain group may be substituted with any substituent.

[0172] In this specification, "one or more" means one or more. When used in the context of a substituent of a group, the term refers to a number from one to the maximum number of substituents permitted in the group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0173] In this specification, "C-terminal active forms" include not only compounds whose carboxyl groups are activated (e.g., active esters related to the production of the target peptide compound) used in the peptide synthesis reaction, but also compounds that are activated during the reaction by, for example, converting to azlactones, NCA (N-carboxyanhydride), etc., and thereby reacting with amines (e.g., amine components) to provide the target peptide compound. Furthermore, the C-terminal active forms of compounds whose carboxyl groups are activated used in the peptide synthesis reaction are not limited to active esters, mixed anhydrides, and acylisoureas synthesized using peptide condensing agents, such as those described in, for example, Chem. Rev., 2011, 111, 6557. or Organic Process Research & Development, 2016, 20(2), 140., but include any compounds activated in a manner capable of reacting with amines.

[0174] In this specification, "active ester" refers to a compound containing a carbonyl group that reacts with an amino group to form an amide bond, and is a compound in which, for example, OBt, OAt, OSu, OPfp, etc. are bonded to the carbonyl group, and is a compound that promotes reaction with amines.

[0175] The term "amino acid" as used herein includes both natural and unnatural amino acids. "Natural amino acids" as used herein refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Unnatural amino acids are not particularly limited, and examples thereof include β-amino acids, γ-amino acids, D-type amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with side chains different from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids in any stereoconfiguration are permitted. The choice of amino acid side chains is not particularly limited, and in addition to hydrogen atoms, they can be freely selected from, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl groups. One or two non-adjacent methylene groups in these groups may be substituted with oxygen atoms, carbonyl (-CO-), or sulfonyl (-SO2-), phosphoryl, or phosphonyl groups. Substituents may be added separately, and these substituents are also not limited. For example, one or more substituents may be independently selected from any substituent containing a halogen atom, an O atom, an S atom, a N atom, a B atom, a Si atom, or a P atom. That is, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, etc., which may be substituted, may be exemplified. In a non-limiting embodiment, the amino acid in this specification may be a compound having a carboxyl group and an amino group in the same molecule.

[0176] The main chain amino group of an amino acid may be unsubstituted (NH2 group) or substituted (i.e., -NHR group: R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group which may have a substituent, and one or two non-adjacent methylene groups in these groups may be substituted with an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO2-). In addition, the carbon chain bonded to the N atom and the carbon atom at the α position may form a ring as in proline. Amino acids in which the main chain amino group is substituted are referred to as "N-substituted amino acids" in this specification. As the "N-substituted amino acids" in this specification, preferred examples include N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, and N-methyl amino acids, but are not limited thereto.

[0177] The "amino acids" constituting the peptide compounds in this specification include all corresponding isotopes. An isotope of an "amino acid" is a substance in which at least one atom is replaced by an atom having the same atomic number (number of protons) but a different mass number (the sum of the number of protons and neutrons). Examples of isotopes contained in the "amino acids" constituting the peptide compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., including 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 Cl et al.

[0178] Examples of the substituent containing a halogen atom in the present specification include alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups each having a halogen atom. More specific examples include fluoroalkyl, difluoroalkyl, and trifluoroalkyl groups.

[0179] Examples of the substituent containing an O atom include a hydroxyl group (-OH), an oxy group (-OR), a carbonyl group (-C(=O)-R), a carboxyl group (-CO2H), an oxycarbonyl group (-C(=O)-OR), a carbonyloxy group (-OC(=O)-R), a thiocarbonyl group (-C(=O)-SR), a carbonylthio group (-SC(=O)-R), an aminocarbonyl group (-C(=O)-NHR), a carbonylamino group (-NH-C(=O)-R), an oxycarbonylamino group (-NH-C(=O)-OR), a sulfonylamino group (-NH-SO2-R), an aminosulfonyl group (-SO2-NHR), a sulfamoylamino group (-NH-SO2-NHR), a thiocarboxy group (-C(=O)-SH), and a carboxycarbonyl group (-C(=O)-CO2H).

[0180] Examples of the oxy group (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy. The alkoxy group is preferably a C1-C4 alkoxy group or a C1-C2 alkoxy group, and among these, a methoxy group or an ethoxy group is preferred.

[0181] Examples of the carbonyl group (—C(═O)—R) include formyl (—C(═O)—H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, and aralkylcarbonyl.

[0182] Examples of the oxycarbonyl group (—C(═O)—OR) include an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, and an aralkyloxycarbonyl group.

[0183] Examples of the carbonyloxy group (—OC(═O)—R) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group, and an aralkylcarbonyloxy group.

[0184] Examples of the thiocarbonyl group (—C(═O)—SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, and an aralkylthiocarbonyl group.

[0185] Examples of the carbonylthio group (—SC(═O)—R) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group, and an aralkylcarbonylthio group.

[0186] Examples of the aminocarbonyl group (-C(=O)-NHR) include alkylaminocarbonyl groups (e.g., C1-C6 or C1-C4 alkylaminocarbonyl groups, particularly ethylaminocarbonyl and methylaminocarbonyl groups), cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. In addition, examples include groups in which the H atom bonded to the nitrogen atom in -C(=O)-NHR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0187] Examples of the carbonylamino group (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino. In addition, examples include groups in which the H atom bonded to the nitrogen atom in -NH-C(=O)-R is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0188] Examples of the oxycarbonylamino group (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, and aralkyloxycarbonylamino. In addition, examples include groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0189] Examples of the sulfonylamino group (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, and aralkylsulfonylamino groups. Examples include groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0190] Examples of the aminosulfonyl group (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl. In addition, examples include groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0191] Examples of the sulfamoylamino group (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, and aralkylsulfamoylamino. Furthermore, the two H atoms bonded to the nitrogen atom in -NH-SO2-NHR may be substituted with a substituent independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, and two of these substituents may form a ring.

[0192] Examples of the substituent containing an S atom include thiol (—SH), sulfenyl (—SR), sulfinyl (—S(═O)—R), sulfonyl (—SO 2 —R), and sulfo (—SO 3 H).

[0193] Examples of the thio group (—SR) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio groups.

[0194] Examples of the sulfonyl group (-SO2-R) include an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, and an aralkylsulfonyl group.

[0195] Examples of substituents containing a nitrogen atom include azide (-N3, also referred to as "azido"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also referred to as a monosubstituted amino group), tertiary amino (-NR(R'); also referred to as a disubstituted amino group), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidine (-NH-C(=NH)-NH2), substituted guanidine (-NR-C(=NR"')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridyl, piperidinyl, morpholino, and azetidinyl groups.

[0196] Examples of the secondary amino group (—NH—R; monosubstituted amino group) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, and aralkylamino groups.

[0197] Examples of tertiary amino groups (-NR(R'); disubstituted amino groups) include amino groups having two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these two substituents may form a ring. Specifically, dialkylamino groups, particularly C1-C6 dialkylamino groups, C1-C4 dialkylamino groups, dimethylamino groups, diethylamino groups, etc., may be mentioned. In this specification, "C p -C q "Dialkylamino" refers to an amino group substituted with 2 C p -C q Alkyl group, two C p -C q The alkyl groups may be the same or different.

[0198] As examples of substituted amidino groups (-C(=NR)-NR′R″), there can be mentioned groups in which the three substituents R, R′, and R″ on the N atom are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, for example, an alkyl (aralkyl) (aryl) amidino group, etc.

[0199] Examples of substituted guanidine groups (-NR-C(=NR"')-NR'R") include groups in which R, R', R", and R"' are each independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or a group in which these groups form a ring.

[0200] Examples of aminocarbonylamino groups (-NR-CO-NR'R") include groups in which R, R', and R" are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or a group in which these groups form a ring.

[0201] In this specification, the "amino acid residue" constituting a peptide compound may be simply referred to as "amino acid".

[0202] (Method for producing peptide compound)

[0203] In one embodiment, the present invention relates to a method for producing a peptide compound, comprising the following steps.

[0204] Step A: a step of obtaining a reaction mixture comprising a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent, and

[0205] Step B: A step of mixing the reaction mixture, a tertiary amine, and water or an aqueous solution to remove the C-terminal active form.

[0206] Step A is a step in which an acid component and an amine component react in a solvent using a condensing agent to obtain a reaction mixture containing a peptide compound. While not being limited to a particular theory, in step A, the acid component reacts with the condensing agent to form a C-terminal active form of the acid component. Subsequently, the amine component nucleophilically attacks the C-terminal active form, thereby reacting to produce the peptide compound.

[0207] As the acid component, an amino acid whose amino group is protected by a protecting group or a peptide whose N-terminal amino group is protected by a protecting group can be used. In this specification, the amino acid used as the acid component is sometimes referred to as the "first amino acid" and the peptide used as the acid component is sometimes referred to as the "first peptide".

[0208] The first amino acid is not particularly limited, and any natural amino acid or non-natural amino acid can be used. In addition, the first peptide is also not particularly limited, and a peptide formed by linking two or more arbitrary natural amino acids and / or non-natural amino acids can be used.

[0209] As the first amino acid, preferably, an amino acid containing one or more carbon atoms in its side chain can be cited. As such amino acids, specifically, amino acids having substituted alkyl, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted alkoxyalkyl, substituted cycloalkylalkyl, substituted aralkyl, substituted heteroarylalkyl, etc. in the side chain can be exemplified. In addition, when the above-mentioned side chain has a functional group such as an amino group, a carboxyl group, or a hydroxyl group that can affect the formation reaction of the peptide bond, it is preferably protected with an appropriate protecting group. Without being bound by a particular theory, when an amino acid has a bulky group in its side chain, due to its steric hindrance, the hydrolysis of the residual C-terminal active form of the amino acid is sometimes not fully carried out using previous methods. Even in such cases, by using the method of the present invention, the residual C-terminal active form can be quickly and effectively hydrolyzed.

[0210] The side chain of the C-terminal amino acid contained in the first peptide may have the same side chain as the first amino acid described above.

[0211] As the protecting group for the amino group of the first amino acid and the protecting group for the amino group at the N-terminus of the first peptide, any amino protecting group commonly used in the art can be used. Specific examples of such protecting groups include Cbz, Boc, Teoc, Fmoc, Tfa, Alloc, nonyl, dinitrononyl, t-Bu, trityl, and cumyl.

[0212] In one embodiment, the acid component is preferably used in an amount at least equal to that of the amine component, preferably in excess relative to the amine component. Specifically, for example, 1 to 1.1 equivalents, 1 to 1.2 equivalents, 1 to 1.3 equivalents, 1 to 1.4 equivalents, 1 to 1.5 equivalents, 1 to 2.0 equivalents, or 1 to 3.0 equivalents of the acid component can be used relative to the amine component.

[0213] In one embodiment, the C-terminal active form of the acid component in the present invention can be formed by reacting the acid component with a condensing agent in a solvent. The condensing agent is not particularly limited as long as it can introduce a group having a leaving ability into the hydroxyl portion of the carboxyl group of the acid component in order to increase the electrophilicity of the carbonyl carbon of the acid component. Specifically, for example, T3P, HATU, BEP, carbodiimides (DIC, EDC, etc.), combinations of carbodiimides and additives (oxyma, HOOBt, HOBt, etc.), DMT-MM, CDI, etc. can be mentioned.

[0214] The step of condensing the C-terminal active form with the amine component to obtain the peptide compound (step A) can be carried out by stirring the reaction mixture at a temperature from -20°C to around the boiling point of the solvent, preferably at a temperature from 0°C to 60°C, for 1 minute to 48 hours, preferably 15 minutes to 4 hours.

[0215] In step A, the condensation reaction between the acid component and the amine component can proceed quantitatively.

[0216] As the amine component, an amino acid whose carboxyl group is protected by a protecting group or a peptide whose C-terminal carboxyl group is protected by a protecting group can be used. In this specification, an amino acid used as an amine component is sometimes referred to as a "second amino acid" and a peptide used as an amine component is sometimes referred to as a "second peptide."

[0217] The second amino acid is not particularly limited, and any natural amino acid or any non-natural amino acid can be used. In addition, the second peptide is not particularly limited either, and a peptide formed by connecting two or more arbitrary natural amino acids and / or non-natural amino acids can be used.

[0218] As the protecting group for the carboxyl group of the second amino acid and the protecting group for the carboxyl group at the C-terminus of the second peptide, any carboxyl protecting group commonly used in the art can be used. Specific examples of such protecting groups include methyl, allyl, tert-butyl, trityl, cumyl, benzyl, methoxytrityl, and 1-piperidinyl.

[0219] In one embodiment, any solvent can be used as the solvent in the present invention as long as the condensation reaction can proceed to obtain the peptide compound. Specific examples of such solvents include toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl acetate, ethyl acetate, methyl tert-butyl ether, cyclopentyl methyl ether, N,N-dimethylformamide, and mixtures of two or more selected from these solvents.

[0220] In one embodiment, the "peptide compound" of the present invention, which is obtained by condensing the C-terminal active form of the acid component with the amine component, includes a linear or cyclic peptide compound formed by linking two or more amino acids. In addition, the meaning of a cyclic peptide compound is the same as that of a "peptide compound having a cyclic portion".

[0221] The "linear peptide compound" of the present invention is not particularly limited as long as it is a compound formed by natural amino acids and / or non-natural amino acids linked by amide bonds or ester bonds and does not have a cyclic portion. The total number of natural amino acids or non-natural amino acids constituting the linear peptide compound may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, with preferred ranges being 6-20, 7-19, 7-18, 7-17, 7-16, 7-15, 8-14, or 9-13.

[0222] The "cyclic peptide compound" in the present invention is formed by natural amino acids and / or non-natural amino acids linked by amide bonds or ester bonds, and is not particularly limited as long as it is a compound with a cyclic portion. The cyclic peptide compound may have one or more linear portions. The total number of natural amino acids or non-natural amino acids constituting the cyclic peptide compound may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, and the preferred range is 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, or 9 to 13.

[0223] The number of amino acids constituting the cyclic portion of the cyclic peptide compound is not limited, and examples thereof include 4 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 20 or less, 18 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. The number of amino acids constituting the cyclic portion is preferably 5 to 15, more preferably 5 to 14, 7 to 14, or 8 to 14, further preferably 8 to 13, 9 to 13, 8 to 12, 8 to 11, or 9 to 12, and particularly preferably 9 to 11.

[0224] The number of amino acids in the linear portion of the cyclic peptide is preferably 0-8, more preferably 0-5, and even more preferably 0-3.

[0225] The peptide compound may contain one or more, two or more, three or more, four or more, five or more, or six or more unnatural amino acids. Furthermore, the peptide compound may contain 20 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 10 or fewer, or 9 or fewer unnatural amino acids. When the peptide compound contains unnatural amino acids, the proportion of the unnatural amino acids can be, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the total number of amino acids constituting the peptide compound.

[0226] In addition to the above-mentioned conditions regarding the total number of natural and non-natural amino acids, the peptide compound may be a linear or cyclic peptide containing at least two (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, particularly preferably 5, 6, or 7, preferably in the range of 2 to 30, 3 to 30, 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, or 9 to 13) N-substituted amino acids and at least one amino acid that is not N-substituted. Examples of "N-substituted" include, but are not limited to, substitution of a hydrogen atom bonded to the N atom with a methyl, ethyl, propyl, butyl, or hexyl group. Preferred examples of N-substituted amino acids include those in which the amino group contained in natural amino acids is N-methylated, N-ethylated, N-propylated, N-butylated, or N-pentylated. These are referred to as N-methyl amino acids, N-ethyl amino acids, N-propyl amino acids, N-butyl amino acids, and N-pentyl amino acids. The conversion of an N-unsubstituted amino acid to an N-substituted amino acid is referred to as N-substitution, sometimes also referred to as N-alkylation, N-methylation, or N-ethylation. The proportion of N-substituted amino acids contained in the peptide compound of the present invention may be, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the total number of amino acids constituting the peptide compound.

[0227] The peptide compound may include a salt thereof or a solvate thereof.

[0228] In this specification, the term "side chain" is used in the context of a side chain of an amino acid or a side chain of a cyclic portion of a cyclic peptide compound and refers to a portion not included in the main chain structure.

[0229] In this specification, the "number of amino acids" refers to the number of amino acid residues constituting a peptide compound, and refers to the number of amino acid units produced when amide bonds, ester bonds, and bonds in cyclized portions connecting amino acids are cleaved.

[0230] Step B is a step of removing the unreacted C-terminal active form contained in the reaction mixture obtained in step A. In one embodiment, the removal of the unreacted C-terminal active form is carried out by allowing the unreacted C-terminal active form to react with a tertiary amine. In this specification, the unreacted C-terminal active form, specifically, the C-terminal active form in the reaction mixture that has not reacted with the amine component and remains during the condensation process, is sometimes referred to as the "residual C-terminal active form". In step B, the reaction mixture obtained in step A, the tertiary amine and water or an aqueous solution are mixed. In the case where an excess of an acid component relative to the amine component is used in step A, or when the condensation reaction in step A is not sufficiently carried out, the C-terminal active form of the acid component that has not reacted with the amine component and remains remains in the reaction solvent as an impurity. If the residual C-terminal active form is not fully decomposed and exists in the system, it will have an adverse effect on the subsequent deprotection step of the peptide compound and the further extension reaction of the peptide chain, so it is important to remove it reliably. Conventional liquid-phase synthesis methods employ methods such as hydrolysis of residual active esters using aqueous alkaline solutions. However, the present inventors have confirmed that, particularly in the case of residual C-terminal active forms of amino acids with bulky side chains, or when the leaving group of the residual C-terminal active form is not sufficiently active to readily react with water, decomposition is sometimes insufficient. In contrast, the present method of hydrolyzing the C-terminal active form by mixing a reaction mixture containing the unreacted C-terminal active form with a tertiary amine and water or an aqueous solution, or by contacting the residual C-terminal active form with a tertiary amine, can solve the aforementioned problems.

[0231] As the tertiary amine, a tertiary amine having nucleophilic reactivity toward the residual C-terminal active form of the acid component can be preferably used. Such tertiary amines are preferably those with minimal steric hindrance near the nitrogen. Examples of such tertiary amines include those represented by the following formulas (A), (B), or (C).

[0232]

[0233] In one embodiment, in formula (A), among R1 to R3, R1 and R2, together with the nitrogen atom to which they are bonded, form a 5- to 6-membered non-aromatic heterocyclic ring, and R3 is a C1-C2 alkyl group (i.e., methyl or ethyl) or a C2 hydroxyalkyl group. The 5- to 6-membered non-aromatic heterocyclic ring is preferably pyrrolidine, piperidine, or morpholine, and the C2 hydroxyalkyl group is preferably a 2-hydroxyethyl group.

[0234] In another embodiment, in formula (A), R1 to R3 are each independently a C1-C2 alkyl group or a C2 hydroxyalkyl group. The C2 hydroxyalkyl group is preferably a 2-hydroxyethyl group.

[0235] Preferred examples of the tertiary amine represented by formula (A) include tertiary amines in which R1 to R3 are each independently a C1-C2 alkyl group.

[0236] Specific examples of the tertiary amine represented by formula (A) include trimethylamine, N,N-dimethylethylamine, N,N-diethylmethylamine, triethylamine, and triethanolamine. Among them, trimethylamine is particularly preferred.

[0237] In one embodiment, in formula (B), X is N or O. When X is N, R4 and R5 are each independently a C1-C2 alkyl group or a C2 hydroxyalkyl group, or form a 5- to 6-membered non-aromatic heterocyclic ring together with the nitrogen atom to which they are bonded. When X is O, R4 is a C1-C2 alkyl group or a C2 hydroxyalkyl group, and R5 is absent. As the 5- to 6-membered non-aromatic heterocyclic ring, pyrrolidine, piperidine, or morpholine is preferred, and as the C2 hydroxyalkyl group, 2-hydroxyethyl is preferred. In addition, in formula (B), R6 and R7 are each independently H, a C1-C2 alkyl group, or a methoxy group.

[0238] Preferred examples of the tertiary amine represented by formula (B) include those in which X is N, R4 and R5 are each independently a C1-C2 alkyl group, and R6 and R7 are H.

[0239] Specific examples of the tertiary amine represented by formula (B) include DMAP, 4-piperidylpyridine, and 4-morpholinopyridine. Among them, DMAP is particularly preferred.

[0240] In one embodiment, in formula (C), R8 and R9 are each independently H, a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded, form a 5- to 6-membered non-aromatic heterocyclic ring. The 5- to 6-membered non-aromatic heterocyclic ring is preferably pyrrolidine, piperidine, or morpholine, and the C2 hydroxyalkyl group is preferably a 2-hydroxyethyl group.

[0241] As the tertiary amine represented by formula (C), preferably, R8 and R9 are each independently H or a C1-C2 alkyl group, and more preferably, R8 is a C1-C2 alkyl group and R9 is H.

[0242] Specific examples of the tertiary amine represented by formula (C) include NMI, imidazole-1-ethanol, and 5,6,7,8-tetrahydroimidazo[1,5-α]pyridine. Among them, NMI is particularly preferred.

[0243] Without being bound by a particular theory, the tertiary amines of the present invention can promote the hydrolysis of residual C-terminal active forms by performing a nucleophilic attack on them. Tertiary amines like DIPEA have bulky substituents and therefore have low nucleophilicity, which is undesirable. The hydrolyzate of the residual C-terminal active form can be removed by migrating to the aqueous layer, allowing the resulting peptide compound to be used in the subsequent condensation reaction without undergoing a separate purification step such as column purification. By using the method of the present invention, the residual C-terminal active form can be effectively removed quickly (e.g., within 5 minutes) and with a small number of hydrolysis treatments (e.g., only one). In one embodiment, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the residual C-terminal active form can be removed. In other words, according to the present invention, the residual C-terminal active form can be reduced to 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0244] The tertiary amine can be used in a catalytic amount relative to the amine component, or in an amount greater than a stoichiometric amount. Specifically, for example, 0.1 to 10 equivalents, preferably 0.5 to 3 equivalents, of the tertiary amine can be added to the reaction mixture relative to the amine component.

[0245] When the tertiary amine is allowed to react with the residual C-terminal active form, the reaction mixture can be stirred at a temperature from -20°C to near the boiling point of the solvent, preferably at a temperature of 25°C to 60°C, for 1 minute to 48 hours, preferably for 2 hours or less, for example, 2 minutes to 2 hours, 5 minutes to 60 minutes, 5 minutes to 50 minutes, or 5 minutes to 30 minutes.

[0246] In one embodiment, water or an aqueous solution may be added during the step of treating the remaining C-terminal active form with a tertiary amine. As the aqueous solution, an alkaline aqueous solution is preferably used. Such an alkaline aqueous solution is not particularly limited. Specific examples include aqueous potassium carbonate solution, aqueous lithium hydroxide solution, aqueous sodium carbonate solution, sodium hydroxide, aqueous potassium hydroxide solution, aqueous sodium hydroxide solution, or aqueous cesium carbonate solution. Among these, aqueous potassium carbonate solution and aqueous sodium carbonate solution, which have mild alkalinity, are preferred.

[0247] In one embodiment, the present invention further comprises a step of allowing the tertiary amine to react with the residual C-terminal active form, separating the reaction mixture into an organic layer and an aqueous layer, collecting the organic layer, and then washing the organic layer. For example, this step includes washing the organic layer with an acidic aqueous solution and an alkaline aqueous solution. In one embodiment, this step can reduce the residual amount of the residual C-terminal active form to 1.0% or less, 0.5% or less, and preferably 0.1% or less.

[0248] In one embodiment, the present invention further comprises a step of deprotecting the N-terminal protecting group of the peptide compound (step C). Deprotection of the protecting group can be performed, for example, by conventional methods described in "Greene's, 'Protective Groups in Organic Synthesis' (5th edition, John Wiley & Sons 2014)." Conventional methods sometimes prevent the deprotection reaction from proceeding sufficiently due to residual C-terminal active forms. However, the method of the present invention allows the deprotected form of the peptide compound to be obtained in high yield.

[0249] In one embodiment, the present invention comprises repeating step A and step B multiple times. In another embodiment, the present invention comprises repeating step A, step B, and step C multiple times. By such repetition, the peptide chain is extended, and a peptide compound can be obtained.

[0250] In one embodiment, the present invention relates to a method for promoting the hydrolysis of residual C-terminal active forms, comprising the step of adding a tertiary amine and water or an aqueous solution to a solution containing residual C-terminal active forms, thereby allowing the C-terminal active forms to react with the tertiary amine. In this embodiment, the residual C-terminal active forms and / or tertiary amine can be the aforementioned substances. When the aqueous solution is added to the solution containing residual C-terminal active forms, the aqueous solution is preferably the aforementioned alkaline water.

[0251] In one embodiment, the present invention relates to a method for removing a hydrolyzate of residual C-terminal active bodies, comprising the step of aqueous washing a solution containing the hydrolyzate. In this embodiment, as an aqueous wash, in addition to water, washing with an alkaline aqueous solution can also be implemented. The alkaline aqueous solution is not particularly limited, and preferably an aqueous potassium carbonate solution or an aqueous sodium carbonate solution. In addition, in another embodiment, when the base used forms a salt with the hydrolyzate and becomes difficult to transfer to the aqueous layer, the base can be washed with an acidic aqueous solution after being removed. The acidic aqueous solution is not particularly limited, and preferably an aqueous potassium hydrogen sulfate solution or an aqueous sodium hydrogen sulfate solution. The alkaline aqueous solution is preferably an aqueous potassium carbonate solution or an aqueous sodium carbonate solution.

[0252] It should be noted that all prior art documents cited in this specification are incorporated into this specification as reference.

[0253] Example

[0254] The present invention is further illustrated by the following examples, but is not limited to the following examples.

[0255] The purity of the peptide compound (the target product in peptide synthesis) and the amount of the C-terminal active form remaining were measured using LCMS equipped with QDA and PDA detectors (column: Ascentis Express C18, 5 cm × 4.6 mm, 2.7 μm, mobile phase: 0.5% trifluoroacetic acid aqueous solution / 0.5% trifluoroacetic acid acetonitrile solution = 95 / 5-0 / 100, 1.0 mL / min, detector: UV 210 nm).

[0256] The residual amount of the C-terminal active form was evaluated by converting the residual C-terminal active form into propionamide, as the residual C-terminal active form may be hydrolyzed under analytical conditions (LCMS).

[0257] The purity of the peptide compound (the target compound in peptide synthesis) is reported as a percentage of the peak area by LCMS. The C-terminal active form residual rate and the relative amount of C-terminal active form residual were calculated using the formula described in each example. It should be noted that the total peak area was corrected by subtracting the blank peak and solvent peak area values.

[0258] The "nd" in the table means "not detected".

[0259] (Example 1) Effect of Adding Amines in the Hydrolysis of Residual C-Terminal Active Forms

[0260] (Preparation of Mixed Acid Anhydride)

[0261] 463 mg (1.7 mmol) of Cbz-Ile-OH and 31 mg of pentamethylbenzene (internal standard: 0.21 mmol) were dissolved in 3.0 mL of 2-methyltetrahydrofuran. 1.1 mL (6.2 mmol) of diisopropylethylamine and 1.9 mL (3.2 mmol) of a 50% T3P / THF solution were added at room temperature, and the mixture was stirred at 40°C for 1 hour to prepare a mixed anhydride solution (C-terminal active form). 5 μL of the prepared mixed anhydride solution was reacted with 100 μL (1.2 mmol) of n-propylamine, then diluted with 0.9 mL of methanol. The reaction conversion to the mixed anhydride was determined based on the peak area of ​​LC / MS (conversion: 97%). Cbz-Ile-NHPr / MS (ESI): m / z 307.1 [M+H]+.

[0262] Conversion rate (%) = {Cbz-Ile-NHPr (area %) / [Cbz-Ile-OH (area %) + Cbz-Ile-NHPr (area %)]} × 100

[0263] (Hydrolysis treatment - no amine added)

[0264] Take 1.0 mL from the total amount of the prepared mixed anhydride solution (6 mL), add 0.5 mL of alkaline water (5% lithium hydroxide aqueous solution, 5% sodium carbonate aqueous solution, 5% potassium carbonate aqueous solution, 5% potassium hydroxide aqueous solution, or 5% cesium carbonate aqueous solution), and stir with a stirrer at 25°C (1200 rpm). After stopping stirring, let it stand to allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the residual C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Perform LC / MS analysis on this solution and calculate the peak area ratio [propionamide: pentamethylbenzene (internal standard)].

[0265] (Hydrolysis treatment-amine addition)

[0266] Take 1.0 mL of the prepared mixed anhydride solution (6 mL), add an amine additive (0.19 mmol) and 0.5 mL of a 5% potassium carbonate aqueous solution, and stir at 25°C with a stirrer (1200 rpm). Stop stirring, let the mixture stand, and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. Then dilute with 0.9 mL of methanol. Analyze this solution by LC / MS, and calculate the peak area ratio [propionamide: pentamethylbenzene (internal standard)].

[0267] (Evaluation of the residual amount of C-terminal active form)

[0268] Peak area ratio [propionamide / pentamethylbenzene (internal standard)] using LC / MS. The relative values ​​of the C-terminal active form residue in the following table are relative values ​​when the peak area ratio [propionamide / pentamethylbenzene] value of 3.5 when treated with 5% potassium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 100 (1 5-minute column injection).

[0269] Relative value of the residual amount of C-terminal active form (%) = {[propionamide (area %) / pentamethylbenzene (area %)] / 3.5 ([propionamide (area %) / pentamethylbenzene (area %) at 1.5 min after injection])}×100

[0270]

Table 1

[0271]

[0272] 1) Not applicable.

[0273] The smaller the relative value of the residual C-terminal active form in Table 1, the more hydrolyzed the residual C-terminal active form. When using alkaline water alone, the hydrolysis rate remained largely unchanged even when the countercation of the base was changed, resulting in slower hydrolysis compared to when amines were added. Furthermore, among the amines added, the addition of DMAP and NMI significantly accelerated the hydrolysis of the residual C-terminal active form.

[0274] (Example 2) Effect of Adding Amines in Hydrolysis of Residual C-Terminal Active Forms

[0275] (Preparation of Active Ester)

[0276] 701 mg (2.64 mmol) of Cbz-Ile-OH and 46 mg (0.31 mmol) of pentamethylbenzene were dissolved in 7.0 mL of 2-methyltetrahydrofuran. 1.0 g (2.64 mmol) of HATU and 1.5 mL (8.79 mmol) of diisopropylethylamine were added at room temperature, and the mixture was stirred at 60°C for 4 hours to prepare an active ester solution (C-terminal active form). 5 μL of the prepared active ester solution was reacted with 100 μL (1.2 mmol) of n-propylamine, then diluted with 0.9 mL of methanol. The reaction conversion to the active ester was determined from the peak area of ​​LC / MS (conversion: 94%). Cbz-Ile-NHPr / MS (ESI): m / z 307.1 [M+H]+.

[0277] Conversion rate (%) = {Cbz-Ile-NHPr (area %) / [Cbz-Ile-OH (area %) + Cbz-Ile-NHPr (area %)]} × 100

[0278] (Hydrolysis treatment using alkaline water alone)

[0279] Take 1.5 mL of the prepared active ester solution (9 mL) and add 0.75 mL of alkaline water (5% potassium carbonate aqueous solution). Stir at 25°C with a stirrer (1200 rpm). After stopping stirring, let the mixture stand to allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. Then dilute with 0.9 mL of methanol. Analyze this solution by LC / MS, and calculate the peak area ratio [propionamide: pentamethylbenzene (internal standard)].

[0280] (Hydrolysis treatment with amine addition)

[0281] Take 1.5 mL of the prepared active ester solution (9 mL), add the amine additive (0.44 mmol) and 0.75 mL of a 5% aqueous potassium carbonate solution, and stir at 25°C with a stirrer (1200 rpm). Stop stirring, let the mixture stand, and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. Then, dilute with 0.9 mL of methanol. LC / MS analysis of this solution was performed, and the peak area ratio (propionamide: pentamethylbenzene (internal standard)) was calculated.

[0282] (Evaluation of the residual amount of C-terminal active form)

[0283] Peak area ratio [propionamide / pentamethylbenzene (internal standard)] using LC / MS. The relative values ​​of the C-terminal active form residue in the following table are relative values ​​when the peak area ratio [propionamide / pentamethylbenzene] value of 3.0 when treated with 5% potassium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 100 (1 5-minute column injection).

[0284] Relative value of the residual amount of C-terminal active form (%) = {[propionamide (area %) / pentamethylbenzene (area %)] / 3.0 ([propionamide (area %) / pentamethylbenzene (area %) at 1.5 min after injection])}×100

[0285]

Table 2

[0286]

[0287] The smaller the relative value of the residual C-terminal active form in Table 2, the more hydrolyzed the residual C-terminal active form was. It was found that the addition of amines promoted the hydrolysis of the residual C-terminal active form compared to the case of using only alkaline water. Specifically, it was found that the addition of DBU, MeN, NMI, and DMAP had an effect, with NMI and DMAP showing a particularly significant effect.

[0288] (Example 3) Effect of Adding Amines in Hydrolysis of Residual C-Terminal Active Forms

[0289] To a solution of 617 mg (2.6 mmol) of Cbz-MeAla-OH, 46 mg (0.31 mmol) of pentamethylbenzene, and 4.5 mL of 2-methyltetrahydrofuran, 1.5 mL (8.6 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution were added at room temperature. The mixture was stirred at 40°C for 1 hour to prepare a mixed anhydride solution (C-terminal active form). 5 μL of the prepared mixed anhydride solution was reacted with 100 μL (1.2 mmol) of n-propylamine, then diluted with 0.9 mL of methanol. The reaction conversion to the mixed anhydride was determined from the peak area of ​​LC / MS (conversion: 90%). Cbz-MeAla-NHPr / MS (ESI): m / z 279.1 [M+H]+.

[0290] Conversion rate (%) = {Cbz-MeAla-NHPr (area %) / [Cbz-MeAla-OH (area %) + Cbz-MeAla-NHPr (area %)]} × 100

[0291] (Hydrolysis treatment using alkaline water alone)

[0292] Take 1.5 mL of the total amount of the prepared mixed anhydride solution (9 mL), add 0.75 mL of alkaline water (5% sodium carbonate aqueous solution or 5% potassium carbonate aqueous solution), and stir at 25°C with a stirrer (1200 rpm). After stopping stirring, let it stand to allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Analyze this solution by LC / MS and calculate the peak area ratio [propionamide: pentamethylbenzene (internal standard)].

[0293] (Hydrolysis treatment with amine addition)

[0294] From the total amount of the prepared mixed anhydride solution (9 mL), 1.5 mL was added to an amine additive (0.43 mmol, 0.67 equivalents) and 0.75 mL of a 5% aqueous potassium carbonate solution. The mixture was stirred at 25°C (1200 rpm) with a stirrer. Stirring was stopped and the mixture was allowed to stand for separation of the organic and aqueous layers. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the peak area ratio [propionamide: pentamethylbenzene (internal standard)] was calculated.

[0295] (Evaluation of the residual amount of C-terminal active form)

[0296] Peak area ratio [propionamide / pentamethylbenzene (internal standard)] using LC / MS. The relative values ​​of the C-terminal active form residue in the following table are relative values ​​when the peak area ratio [propionamide / pentamethylbenzene] value of 1.1 when treated with 5% sodium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 100 (1 5-minute column injection).

[0297] Relative value of the residual amount of C-terminal active form (%) = {[propionamide (area %) / pentamethylbenzene (area %)] / 1.1 ([propionamide (area %) / pentamethylbenzene (area %) at 1.5 min of injection]}×100

[0298]

Table 3

[0299]

[0300] The smaller the relative value of the residual C-terminal active form in Table 3, the more hydrolyzed the residual C-terminal active form. When using alkaline water alone, the hydrolysis rate remained almost unchanged even when the counter cation of the base was changed. Furthermore, it was found that the addition of DMAP and NMI promoted the hydrolysis of the residual C-terminal active form compared to the case of using alkaline water alone. The addition of DMAP and NMI was found to be sufficiently effective within 5 minutes, with the addition of DMAP particularly effective in completely hydrolyzing the residual C-terminal active form.

[0301] (Example 4) Synthesis of Cbz-Ile-Phe-OtBu

[0302] (Condensation reaction)

[0303] To a solution of 458 mg (1.8 mmol) of H-Phe-OtBu hydrochloride, 699 mg (2.7 mmol) of Cbz-Ile-OH, and 4.5 mL of 2-methyltetrahydrofuran, 1.6 mL (8.9 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution were added at room temperature, and the mixture was stirred at 40°C for 1 hour to allow a peptide bond formation reaction to proceed. 5 μL of the reaction solution was reacted with 100 μL (1.2 mmol) of n-propylamine, then diluted with 0.9 mL of methanol. The reaction conversion was determined from the peak area of ​​LC / MS (conversion: 100%).

[0304] Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area %) / [H-Phe-OtBu (area %) + Cbz-Ile-Phe-OtBu (area %)]} × 100

[0305] (Hydrolysis treatment using alkaline water alone)

[0306] From the total amount of the dipeptide solution prepared above (9 mL), 1.5 mL was taken and added to 0.75 mL of a 5% aqueous potassium carbonate solution. The mixture was stirred at 25°C (1200 rpm) with a stirrer. After stopping stirring, the mixture was allowed to stand and the organic and aqueous layers separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS. The peak areas of propionamide and the target peptide were determined, and the residual C-terminal active form ratio (%) was calculated. The remaining aqueous layer of the reaction solution was removed, and the organic layer was washed sequentially with 0.5 mL of a 5% aqueous potassium bisulfate solution and 0.5 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form).

[0307] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0308] (Hydrolysis treatment with amine addition)

[0309] From the total amount of the prepared dipeptide solution (9 mL), 1.5 mL was taken and added with an amine (0.15 mmol, 0.5 equivalents; 0.30 mmol, 1.0 equivalents; or 0.89 mmol, 3 equivalents: equivalents are relative to H-Phe-OtBu hydrochloride) and 0.75 mL of a 5% aqueous potassium carbonate solution. The mixture was stirred at 25°C using a stirrer (1200 rpm). After stopping stirring, the mixture was allowed to stand and separate into the organic and aqueous layers. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS. The peak areas of propionamide and the target peptide were determined, and the residual C-terminal active form ratio (%) was calculated. The remaining aqueous layer of the reaction solution was removed, and the organic layer was washed sequentially with 0.75 mL of a 5% aqueous potassium bisulfate solution and 0.75 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The resulting solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed to determine the peak areas of the target peptide and the remaining C-terminal active form (propionamide conversion). MS (ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+.

[0310] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0311]

Table 4

[0312]

[0313] 1) Equivalent to N-terminal amino acid derivative (H-Phe-OtBu hydrochloride)

[0314] 2) LCMS peak area ratio

[0315] It was found that the use of the added amine NMI in an amount of 0.5 to 3.0 equivalents relative to the N-terminal amino acid derivative significantly promoted hydrolysis compared to hydrolysis using alkaline water alone. Furthermore, it was found that the use of the added amine DMAP in an amount of 0.5 to 3.0 equivalents relative to the N-terminal amino acid derivative significantly promoted hydrolysis compared to hydrolysis using alkaline water alone.

[0316] It was discovered that after a primary hydrolysis treatment with amine, washing the organic layer with 5% KHSO₄ and 5% K₂CO₃ completely removed the remaining C-terminal active form. In this case, the target peptide was obtained at a high purity. On the other hand, treatment with alkaline water alone left the C-terminal active form and the dipeptide had a low purity.

[0317] (Example 5) Synthesis of Cbz-Ile-Phe-OtBu

[0318] (Condensation reaction)

[0319] To a solution of 452 mg (1.8 mmol) of H-Phe-OtBu hydrochloride, 702 mg (2.6 mmol) of Cbz-Ile-OH, and 4.5 mL of 2-methyltetrahydrofuran, 1.5 mL (8.8 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution were added at room temperature, and the mixture was stirred at 40°C for 1 hour to allow a peptide bond formation reaction to proceed. 5 μL of the reaction solution was reacted with 100 μL (1.2 mmol) of n-propylamine, then diluted with 0.9 mL of methanol. The reaction conversion was determined from the peak area of ​​LC / MS (conversion: 100%).

[0320] Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area %) / [H-Phe-OtBu (area %) + Cbz-Ile-Phe-OtBu (area %)]} × 100

[0321] (Hydrolysis treatment using alkaline water alone)

[0322] From the total amount of the dipeptide solution prepared above (9 mL), 1.5 mL was taken and added to 0.75 mL of a 5% aqueous potassium carbonate solution. The mixture was stirred at 60°C (1200 rpm) with a stirrer. After stopping stirring, the mixture was allowed to stand and the organic and aqueous layers separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS. The peak areas of propionamide and the target peptide were determined, and the residual C-terminal active form ratio (%) was calculated. The remaining aqueous layer of the reaction solution was removed, and the organic layer was washed sequentially with 0.75 mL of a 5% aqueous potassium bisulfate solution and 0.75 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form).

[0323] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0324] (Hydrolysis treatment with amine addition)

[0325] From the total amount of the prepared dipeptide solution (9 mL), 1.5 mL was taken, and an amine (0.29 mmol) and 0.75 mL of a 5% aqueous potassium carbonate solution were added. The mixture was stirred at 60°C (1200 rpm) with a stirrer. After stopping stirring, the mixture was allowed to stand and separate into the organic and aqueous layers. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS. The peak areas of propionamide and the target peptide were determined, and the residual C-terminal active form ratio (%) was calculated. The remaining aqueous layer of the reaction solution was removed, and the organic layer was washed sequentially with 0.75 mL of a 5% aqueous potassium bisulfate solution and 0.75 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). MS (ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+.

[0326] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0327]

Table 5

[0328]

[0329] 1) LCMS peak area ratio

[0330] Even when hydrolysis of the residual C-terminal active form was performed at 60°C with the addition of an amine, the target peptide was obtained at a high purity comparable to that obtained at 25°C. In particular, when the added amines were DMAP and NMI, hydrolysis proceeded more rapidly than with alkaline water alone. Furthermore, it was found that when the added amines were DMAP and NMI, hydrolysis proceeded efficiently within 5 minutes after a single hydrolysis treatment, and that the residual C-terminal active form could be completely removed from the organic layer by subsequent separation (5% KHSO4, 5% K2CO3 washing).

[0331] (Example 6) Synthesis of Cbz-MeIle-MePhe-OMe

[0332] (Condensation reaction)

[0333] 300 mg (1.3 mmol) of MePhe-OMe hydrochloride and 442 mg (1.6 mmol) of Cbz-MeIle-OH were suspended in 3.0 mL of acetonitrile, and 683 μL (3.9 mmol) of diisopropylethylamine was added. Subsequently, 594 mg (1.6 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 30 minutes, then at 40°C for 3 hours, and further at 60°C for 3 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide, and then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined from the peak area value of the LC / MS (conversion rate: >99%).

[0334] Conversion rate (%) = {Cbz-MeIle-MePhe-OMe (area %) / [MePhe-OMe (area %) + Cbz-MeIle-MePhe-OMe (area %)]} × 100

[0335] (Hydrolysis treatment)

[0336] (1) When no amine is added

[0337] Add 3.0 mL of MTBE and 3.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above, and stir with a stirrer at 25°C for 30 minutes. Stop stirring and allow the organic layer and aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0338] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0339] (2) When amine is added

[0340] To the peptide-containing reaction solution prepared above, add 3.0 mL of MTBE, 103 μL (1.3 mmol) of N-methylimidazole, and 3.0 mL of a 5% aqueous potassium carbonate solution, and stir at 25°C for 30 minutes with a stirring bar. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Based on the peak area value of LC / MS, calculate the residual rate of the C-terminal active form according to the following calculation formula.

[0341] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0342] (Post-processing)

[0343] After stopping stirring, the mixture was allowed to stand for separation of the organic and aqueous layers, and the aqueous layer was removed. Next, the organic layer was washed with 3 mL of 10% potassium hydrogen sulfate aqueous solution x 2, 3 mL of 5% potassium carbonate aqueous solution x 3, and 1 mL of water x 5, in that order. Stirring was stopped and the organic and aqueous layers were separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine was 671.8 mg (yield 113%: although the concentrate contained impurities (residual C-terminal active form), it was calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 563.7 mg (yield 95%). MS(ESI): m / z455.2[M+H]+, 477.2[M+Na]+.

[0344]

Table 6

[0345]

[0346] 1) LCMS peak area ratio

[0347] The researchers found that during hydrolysis using alkaline water alone, the residual C-terminal active form was not completely hydrolyzed, and subsequent aqueous washing could not remove the residual C-terminal active form. However, when hydrolysis was performed with the addition of NMI, the residual C-terminal active form was completely hydrolyzed and removed. Furthermore, the target dipeptide was obtained with 100% purity (95% yield).

[0348] (Example 7) Synthesis of Cbz-MeVal-MeAsp(tBu)-piperidine

[0349] (Condensation reaction)

[0350] 303 mg (1.1 mmol) of MeAsp(tBu)-piperidine and 448 mg (1.7 mmol) of Cbz-MeVal-OH were suspended in a mixed solvent of 0.6 mL of acetonitrile and 2.4 mL of cyclopentyl methyl ether. 586 μL (3.4 mmol) of diisopropylethylamine was added. Subsequently, 642 mg (1.7 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 6.5 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide. The solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined from the peak area value (conversion rate: 100%).

[0351] Conversion rate (%) = {Cbz-MeVal-MeAsp(tBu)-piperidine (area %) / [MeAsp(tBu)-piperidine (area %) + Cbz-MeVal-MeAsp(tBu)-piperidine (area %)]} × 100

[0352] (Hydrolysis treatment)

[0353] (1) When no amine is added

[0354] Add 3.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0355] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0356] (2) When amine is added

[0357] To the peptide-containing reaction solution prepared above, add 136 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% potassium carbonate aqueous solution, and stir at 25°C with a stirrer for 5 minutes. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Based on the peak area value of LC / MS, calculate the residual rate of the C-terminal active form according to the following calculation formula.

[0358] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0359] (Post-processing)

[0360] After stopping stirring, the mixture was allowed to stand for separation of the organic and aqueous layers, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% potassium hydrogen sulfate aqueous solution x 2, 3 mL of 5% potassium carbonate aqueous solution x 2, and 1.5 mL of water x 3. Stirring was stopped and the organic and aqueous layers were separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine was 782.0 mg (yield 136%: although the concentrate contained impurities (remaining C-terminal active form), it was calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 530.6 mg (yield 92%). MS(ESI): m / z518.4[M+H]+, 540.4[M+Na]+.

[0361]

Table 7

[0362]

[0363] 1) LCMS peak area ratio

[0364] The researchers found that hydrolysis with alkaline water alone did not completely hydrolyze the remaining C-terminal active form, and subsequent aqueous washing could not remove the remaining C-terminal active form. However, hydrolysis with the addition of DMAP completely hydrolyzed the remaining C-terminal active form, allowing for complete removal. Furthermore, the target dipeptide was obtained with 100% purity (92% yield).

[0365] (Example 8) Synthesis of Cbz-MeVal-MeAsp(tBu)-piperidine

[0366] (Condensation reaction)

[0367] 299 mg (1.1 mmol) of MeAsp(tBu)-piperidine and 458 mg (1.7 mmol) of Cbz-MeVal-OH were suspended in 4.5 mL of 2-MeTHF solvent, and 775 μL (4.4 mmol) of diisopropylethylamine was added. Next, 1.6 mL (2.8 mmol) of a 50% T3P / THF solution was added at 25°C, and the mixture was stirred at 25°C for 15 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form to propionamide, and then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the conversion rate was determined from the peak area value (conversion rate: 100%).

[0368] Conversion rate (%) = {Cbz-MeVal-MeAsp(tBu)-piperidine (area %) / [MeAsp(tBu)-piperidine (area %) + Cbz-MeVal-MeAsp(tBu)-piperidine (area %)]} × 100

[0369] (Hydrolysis treatment)

[0370] (1) When no amine is added

[0371] Add 3.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0372] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0373] (2) When amine is added

[0374] To the peptide-containing reaction solution prepared above, 141 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred at 25°C for 5 minutes using a stirring bar. Stirring was stopped, and the organic and aqueous layers were separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active form into propionamide, and then diluted with 0.9 mL of methanol. Based on the peak area value of LC / MS, the residual rate of the C-terminal active form was calculated according to the following calculation formula.

[0375] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0376] (Post-processing)

[0377] After stopping stirring, let it stand to separate the organic layer and the aqueous layer, and remove the aqueous layer. Next, wash the organic layer with 3 mL of 10% potassium hydrogen sulfate aqueous solution and 3 mL of 5% potassium carbonate aqueous solution in sequence. Stop stirring and separate the organic layer and the aqueous layer. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of n-propylamine to convert the residual C-terminal active form into propionamide. Then dilute it with 0.9 mL of methanol and perform LC / MS analysis to determine the peak area values ​​of the target peptide and the residual C-terminal active form (propionamide conversion form). The remaining organic layer is concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without adding amine is 561.6 mg (yield 98%: although the concentrate contains impurities (residual C-terminal active form), it is calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine is 501.1 mg (yield 87%). MS(ESI): m / z 518.4[M+H]+, 540.4[M+Na]+.

[0378]

Table 8

[0379]

[0380] 1) LCMS peak area ratio

[0381] The researchers found that hydrolysis with alkaline water alone did not completely hydrolyze the residual C-terminal active form, and subsequent aqueous washing could not remove the residual C-terminal active form. However, hydrolysis with the addition of DMAP completely hydrolyzed the residual C-terminal active form, allowing for complete removal. Furthermore, the target dipeptide was obtained with 100% purity (yield 87%).

[0382] (Example 9) Synthesis of Cbz-Ile-MeVal-MeAsp(tBu)-piperidine

[0383] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis without adding an amine)

[0384] 782 mg of Cbz-MeVal-MeAsp(tBu)-piperidine (containing 17.6 area % of residual C-terminal active form), synthesized in Example 7 without the addition of an amine, was dissolved in 4.2 mL of cyclopentyl methyl ether. Hydrogenolysis was performed using 115 mg of 5% Pd / C (50% wet) and hydrogen. Since the reaction barely progressed, the Pd / C was removed using a filter, the mixture was concentrated to dryness, and the mixture was redissolved in 4.2 mL of cyclopentyl methyl ether. 105 mg of 5% Pd / C (50% wet) was added, and hydrogenolysis was performed again. However, even after a total reaction time of 3 hours, the reaction barely progressed (reaction conversion: 1.6%). The reaction conversion was determined by diluting 5 μL of the reaction solution with 1.0 mL of acetonitrile. The filtered solution was then analyzed by LC / MS, and the peak area values ​​were determined based on the LC / MS results.

[0385] Conversion rate (%) = {MeVal-MeAsp(tBu)-piperidine (area %) / [MeVal-MeAsp(tBu)-piperidine (area %) + Cbz-MeVal-MeAsp(tBu)-piperidine (area %)]} × 100

[0386] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis treatment with amine addition)

[0387] 543 mg (1.0 mmol) of Cbz-MeVal-MeAsp(tBu)-piperidine, synthesized under the amine addition conditions in Example 7, was dissolved in 4.3 mL of cyclopentyl methyl ether. Hydrogenolysis was carried out using 124 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring at room temperature for 2 hours yielded MeVal-MeAsp(tBu)-piperidine as a de-Cbz product (conversion rate 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of acetonitrile, filtering the solution, and analyzing it by LC / MS. The LC / MS peak area was used for the analysis. MS (ESI): m / z 384.3 [M+H]+.

[0388] Conversion rate (%) = {MeVal-MeAsp(tBu)-piperidine (area %) / [MeVal-MeAsp(tBu)-piperidine (area %) + Cbz-MeVal-MeAsp(tBu)-piperidine (area %)]} × 100

[0389] (Condensation reaction)

[0390] The reaction solution was filtered to remove Pd / C, then concentrated to dryness. The solid was dissolved in 4.3 mL of 2-methyltetrahydrofuran, and 362 mg (1.3 mmol) of Cbz-Ile-OH and 715 μL (4.1 mmol) of diisopropylethylamine were added. Next, 1.4 mL (2.4 mmol) of a 50% T3P / THF solution was added at 25°C, and the mixture was stirred at 40°C for 7 hours and further at room temperature for 14 hours to allow peptide bond formation to proceed (conversion rate: 100%). To the prepared reaction solution were added 81 μL (1.0 mmol) of N-methylimidazole and 2.6 mL of a 20% aqueous potassium carbonate solution, and the mixture was stirred at 25°C for 45 minutes using a stirrer. After stopping stirring, the mixture was allowed to stand, and the organic and aqueous layers were allowed to separate, and the aqueous layer was removed. The organic layer was then washed with 5.2 mL of a 10% aqueous potassium hydrogen sulfate solution and then 5.2 mL of a 5% aqueous potassium carbonate solution twice. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. LC / MS analysis was performed on the solution to determine the peak area percentages of the target peptide and the residual C-terminal active form. The target peptide Cbz-Ile-MeVal-MeAsp(tBu)-piperidine was 95.1%, and no Cbz-Ile-NHPr from the residual C-terminal active form was detected. The remaining organic layer was concentrated to obtain 542.7 mg of concentrate (yield 82%). MS (ESI): m / z 631.5 [M+H] + ,653.4[M+Na] + .

[0391] It has been shown that when a peptide solution containing residual C-terminal active groups treated with alkaline water alone is used, the Cbz deprotection reaction hardly proceeds. On the other hand, it has been found that when a peptide solution in which the residual C-terminal active groups have been completely removed by adding DMAP is used, the Cbz deprotection reaction proceeds smoothly, enabling the subsequent peptide synthesis reaction. In other words, the method of the present invention allows the reductive removal reaction of the N-terminal protecting group of the generated peptide compound to proceed without stagnation. This allows for the efficient production of high-purity peptide compounds having the desired amino acid sequence.

[0392] (Example 10) Synthesis of Cbz-Phe(3-F)-Phe-OtBu

[0393] 200 mg (0.8 mmol) of Phe-OtBu hydrochloride and 297 mg (0.9 mmol) of Cbz-Phe(3-F)-OH were suspended in 3.0 mL of toluene, and 407 μL (2.3 mmol) of diisopropylethylamine was added. Subsequently, 0.9 mL (1.6 mmol) of a 50% T3P / THF solution was added at 25°C, and the mixture was stirred at room temperature for 30 minutes to allow a peptide bond formation reaction to proceed (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, adding it to 100 μL of n-propylamine, and diluting the resulting solution with 0.9 mL of methanol. LC / MS analysis was performed, and the peak area value obtained by LC / MS was used to determine the conversion rate.

[0394] Conversion rate (%) = {Cbz-Phe(3-F)-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-Phe(3-F)-Phe-OtBu (area %)]} × 100

[0395] To the above reaction solution, 95 mg (0.8 mmol) of DMAP and 2.0 mL of 5% aqueous potassium carbonate solution were added, and the mixture was stirred at 25°C for 5 minutes using a stirrer. After stopping stirring, the mixture was allowed to stand to allow the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 1 mL of 10% aqueous potassium bisulfate solution, 1 mL of 5% aqueous potassium carbonate solution, and 1 mL of water. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution to determine the peak area percentages of the target peptide and the residual C-terminal active form. The purity of the target peptide, Cbz-Phe(3-F)-Phe-OtBu, was 100%, and no Cbz-Phe(3-F)-NHPr from the residual C-terminal active form was detected. The remaining organic layer was concentrated to obtain 387.2 mg of concentrate (96% yield). MS(ESI): m / z 465.2[M-tBu+H]+, 521.1[M+H]+, 543.2[M+Na]+.

[0396] When DMAP was added for hydrolysis, the residual C-terminal active form was completely removed, and the target dipeptide was obtained with 100% purity (yield 96%).

[0397] (Example 11) Synthesis of Cbz-Ser(OtBu)-Phe-OtBu

[0398] 300 mg (1.2 mmol) of Phe-OtBu hydrochloride and 450 mg (1.5 mmol) of Cbz-Ser(OtBu)-OH were suspended in 3.6 mL of 2-methyltetrahydrofuran, and 610 μL (3.5 mmol) of diisopropylethylamine was added. Subsequently, 1.4 mL (2.3 mmol) of a 50% T3P / THF solution was added at 25°C, and the mixture was stirred at room temperature for 1 hour to allow a peptide bond formation reaction to proceed (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, adding it to 100 μL of n-propylamine, and diluting the resulting solution with 0.9 mL of methanol. LC / MS analysis was performed, and the peak area value obtained by LC / MS was used to determine the conversion rate.

[0399] Conversion rate (%) = {Cbz-Ser(tBu)-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-Ser(tBu)-Phe-OtBu (area %)]} × 100

[0400] To the reaction solution, 143 mg (1.2 mmol) of DMAP and 1.5 mL of 20% aqueous potassium carbonate solution were added, and the mixture was stirred at 25°C for 5 minutes using a stirring bar. After stopping stirring, the mixture was allowed to stand for separation of the organic and aqueous layers, and the aqueous layer was removed. The organic layer was then washed sequentially with 3.0 mL of 10% aqueous potassium bisulfate solution (2 times), 3.0 mL of 5% aqueous potassium carbonate solution, and 3.0 mL of water. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution to determine the peak area percentages of the target peptide and the residual C-terminal active form. The purity of the target peptide, Cbz-Ser(OtBu)-Phe-OtBu, was 100%, and no Cbz-Ser(OtBu)-NHPr from the residual C-terminal active form was detected. The remaining organic layer was concentrated to obtain 556.4 mg of concentrate (96% yield). MS(ESI): m / z 387.1[M-2tBu+H]+, 499.3[M+H]+, 521.2[M+Na]+.

[0401] When DMAP was added for hydrolysis, the residual C-terminal active form was completely removed, and the target dipeptide was obtained with 100% purity (yield 96%).

[0402] (Example 12) Synthesis of Boc-MeVal-Phe-piperidine

[0403] (Boc deprotection reaction)

[0404] Dissolve 471 mg (1.4 mmol) of Boc-Phe-piperidine in 4.7 mL of dichloromethane, and add 180 μL (2.8 mmol) of methanesulfonic acid. Stir at 35°C for 2 hours to carry out a Boc removal reaction (conversion rate 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and analyzing the resulting solution by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0405] Conversion rate (%) = {Phe-piperidine (area %) / [Boc-Phe-piperidine (area %) + Phe-piperidine (area %)]} × 100

[0406] (Condensation reaction)

[0407] After adding 742 μL (4.3 mmol) of diisopropylethylamine to the reaction solution, the solvent was distilled off. Subsequently, 1.4 mL of acetonitrile, 3.3 mL of 2-methyltetrahydrofuran, 742 μL (4.3 mmol) of diisopropylethylamine, and 492 mg (2.1 mmol) of Boc-MeVal-OH were added. 804 mg (2.2 mmol) of HATU was added at 25°C, and the mixture was stirred at room temperature for 1 hour to allow a peptide bond formation reaction to proceed (conversion rate: 100%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was added to 100 μL of n-propylamine, and the resulting solution was diluted with 0.9 mL of methanol. LC / MS analysis was performed, and the peak area value of the LC / MS analysis was used to determine the conversion rate.

[0408] Conversion rate (%) = {Boc-MeVal-Phe-piperidine (area %) / [Phe-piperidine (area %) + Boc-MeVal-Phe-piperidine (area %)]} × 100

[0409] 168 mg (1.4 mmol) of DMAP and 4.6 mL of 5% potassium carbonate aqueous solution were added to the reaction solution prepared above, and the mixture was stirred at 25°C for 5 minutes with a stirrer. After stopping the stirring, the mixture was allowed to stand to separate the organic layer and the aqueous layer, and the aqueous layer was removed. Next, the organic layer was washed with 4.6 mL of 10% potassium hydrogen sulfate aqueous solution, 4.6 mL of 5% potassium carbonate aqueous solution, and 1.5 mL of water x 6. 5 μL of the obtained organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. The solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the residual C-terminal active form. The purity of the target peptide Boc-MeVal-Phe-OtBu was 99.7%, and no Boc-MeVal-NHPr from the residual C-terminal active form was detected. The remaining organic layer was concentrated to obtain 542.3 mg of concentrate (yield 86%). MS(ESI): m / z 346.2[M-Boc+H]+, 446.3[M+H]+, 468.3[M+Na]+.

[0410] When DMAP was added for hydrolysis, the residual C-terminal active form was completely removed, and the target dipeptide was obtained with a purity of 99.7% (yield 86%) even though the N-terminal protecting group was Boc.

[0411] (Example 13) Synthesis of Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 1 (5mer)

[0412] (Synthesis of Cbz-MePhe-MeGly-OtBu)

[0413] (Condensation reaction)

[0414] 2.0 g (11.0 mmol) of MeGly-OtBu hydrochloride was suspended in 16 mL of isopropyl acetate and 4 mL of acetonitrile, and 7.7 mL (44.0 mmol) of diisopropyldiethylamine and 3.6 g (11.5 mmol) of Cbz-MePhe-OH were added. The reaction solution was cooled to 0°C, and 9.7 mL (16.5 mmol) of a T3P / ethyl acetate solution was added. The mixture was stirred at room temperature for 30 minutes to allow for peptide bond formation (conversion: 100%). The reaction conversion rate was determined by diluting 3 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The peak area value was determined by LC / MS.

[0415] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0416] Next, 1.7 mL (22.0 mmol) of NMI and 20 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with a 5% aqueous potassium sulfate solution and a 5% aqueous potassium carbonate solution × 2. The obtained organic layer was concentrated to obtain 5.0 g of a concentrate (yield quant.). This concentrate was analyzed by LC / MS to determine the peak area percentage (100 area %) of the target Cbz-MePhe-MeGly-OtBu. MS (ESI): m / z 441.2 [M+H] + ,463.2[M+Na] + .

[0417] (Synthesis of Cbz-Aze-MePhe-MeGly-OtBu)

[0418] (Cbz deprotection reaction)

[0419] The total amount of Cbz-MePhe-MeGly-OtBu obtained by the above method was dissolved in 75 mL of isopropyl acetate and hydrogenolyzed with 0.98 g of 10% Pd / C (3% wet) and hydrogen gas. Stirring at room temperature for 2 hours yielded the de-Cbz product (conversion: 100%). The reaction conversion rate was determined by LC / MS analysis of 3 μL of the reaction solution diluted with 1.0 mL of methanol, and the peak area value was determined by LC / MS.

[0420] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0421] (Condensation reaction)

[0422] The reaction solution was filtered and azeotropically dehydrated by adding toluene. The concentrate was dissolved in 39 mL of isopropyl acetate and 9.7 mL of acetonitrile and cooled to 0°C. After adding 2.6 g (11.0 mmol) of Cbz-Aze-OH, 13.0 mL (22.0 mmol) of a 50% T3P / ethyl acetate solution, and 7.7 mL (44.0 mmol) of diisopropylethylamine, the mixture was stirred at room temperature for 30 minutes to allow a peptide bond formation reaction to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 3 μL of the reaction solution was diluted with 1.0 mL of methanol and analyzed by LC / MS. The conversion rate was determined based on the peak area value of the LC / MS.

[0423] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0424] Next, 1.7 mL (22.0 mmol) of NMI and 34 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 34 mL of a 5% aqueous potassium sulfate solution and 34 mL of a 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 5.5 g of a concentrate (yield 96%). This concentrate was analyzed by LC / MS to determine the peak area percentage of the target Cbz-Aze-MePhe-MeGly-OtBu (99.8 area %). MS (ESI): m / z 546.2 [M+Na] + .

[0425] (Synthesis of Cbz-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 2)

[0426] (Cbz deprotection reaction)

[0427] 5.5 g (10.6 mmol) of Cbz-Aze-MePhe-MeGly-OtBu obtained by the above method was dissolved in 75 mL of isopropyl acetate and subjected to hydrogenolysis with 0.95 g of 10% Pd / C (3% wet) and hydrogen gas. The mixture was stirred at 50°C for 2 hours to obtain the de-Cbz product (conversion: 100%). The reaction conversion rate was determined by LC / MS analysis of 3 μL of the reaction solution diluted with 1.0 mL of methanol, and the peak area value was determined by LC / MS.

[0428] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0429] (Condensation reaction)

[0430] The reaction mixture was filtered and azeotropically dehydrated twice with toluene. The concentrate was dissolved in 32.8 mL of isopropyl acetate and 8.2 mL of acetonitrile. 2.7 g (11.1 mmol) of Cbz-MeAla-OH and 7.4 mL (42.3 mmol) of diisopropylethylamine were added, followed by 12.5 mL (21.1 mmol) of a 50% T3P / ethyl acetate solution and 7.4 mL (42.3 mmol) of diisopropylethylamine. After stirring at room temperature for 2 hours, 0.39 g (1.7 mmol) of Cbz-MeAla-OH, 1.9 mL (3.2 mmol) of a T3P / ethyl acetate solution, and 1.1 mL (6.3 mmol) of diisopropylethylamine were added, and the mixture was stirred at room temperature for a further 2 hours to allow peptide bond formation to proceed (conversion: 97%). The reaction conversion was determined by diluting 3 μL of the reaction mixture with 1.0 mL of methanol and analyzing it by LC / MS. The peak area value was determined by LC / MS.

[0431] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0432] Next, 1.7 mL (21.1 mmol) of NMI and 41 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 41 mL of a 5% aqueous potassium sulfate solution and 41 mL of a 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 5.8 g of a concentrate (yield 91%). This concentrate was analyzed by LC / MS, and the peak area percentage of the target Cbz-MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 2) was determined (99.5 area %). MS (ESI): m / z 609.3 [M+H] + 631.3[M+Na] + .

[0433] (Synthesis of Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 1)

[0434] (Cbz deprotection reaction)

[0435] 5.8 g (9.6 mmol) of Cbz-MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 2) obtained by the above method was dissolved in 88 mL of isopropyl acetate and subjected to hydrogenolysis using 0.93 g of 10% Pd / C (3% wet) and hydrogen gas. After stirring at room temperature for 5 hours, the reaction solution was filtered (conversion: 100%). The reaction conversion rate was determined by LC / MS analysis of 3 μL of the reaction solution diluted with 1.0 mL of methanol. The peak area value was determined by LC / MS.

[0436] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0437] The filtrate was concentrated to obtain 4.4 g of concentrate (yield 97%). LC / MS analysis of this concentrate revealed the peak area percentage of the target MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 3) (99.7 area %). MS (ESI): m / z 475.3 [M+H] + .

[0438] (Condensation reaction)

[0439] 1.5 g (3.2 mmol) of the above concentrate and 1.3 g (4.7 mmol) of Cbz-Ile-OH were dissolved in 18 mL of isopropyl acetate and 4.5 mL of acetonitrile. 2.2 mL (12.6 mmol) of diisopropylethylamine and 2.4 g (6.3 mmol) of HATU were added, and the mixture was stirred at room temperature for 30 minutes to allow for peptide bond formation (conversion rate: >99%). The reaction conversion rate was determined by diluting 3 μL of the reaction solution with 1.0 mL of methanol, analyzing it by LC / MS, and determining the conversion rate based on the peak area value obtained by LC / MS.

[0440] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0441] Next, 0.75 mL (9.5 mmol) of NMI and 22.5 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 20 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 22.5 mL of a 5% aqueous potassium sulfate solution twice and 22.5 mL of a 5% aqueous potassium carbonate solution three times. The resulting organic layer was concentrated to obtain 2.4 g of a concentrate (yield, quant.). This concentrate was analyzed by LC / MS, and the peak area percentage of the target Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 1) was determined (99.1 area %). MS (ESI): m / z 744.3 [M+Na] + .

[0442] By adding NMI for a primary hydrolysis followed by aqueous washing, the remaining C-terminal active form was completely removed, yielding the target pentapeptide with a purity of 99.1%. The total yield from the initial amino acid was 87%. These results demonstrate that the use of an amine additive in continuous liquid-phase peptide synthesis completely removes the remaining C-terminal active form, enabling the synthesis of high-purity pentapeptides with high yield.

[0443] (Example 14)

[0444] Synthesis of Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 4 (11mer)

[0445] (Synthesis of Cbz-MeVal-Asp(tBu)-piperidine)

[0446] (Condensation reaction)

[0447] 8.6 g (33.5 mmol) of Asp(tBu)-piperidine was dissolved in 108 mL of cyclopentyl methyl ether. 9.79 g (36.9 mmol) of Cbz-MeVal-OH and 17.6 mL (101 mmol) of diisopropylethylamine were added. 13.8 g (50.3 mmol) of BEP was dissolved in 21.5 mL of acetonitrile and added to the reaction mixture. The mixture was stirred at room temperature for 3 minutes to allow peptide bond formation (conversion rate: >99%). The reaction conversion rate was determined by diluting 5 μL of the reaction mixture with 1.0 mL of methanol and analyzing it by LC / MS. The peak area value was determined by LC / MS.

[0448] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0449] The reaction mixture was washed with 150 mL of a 10% aqueous potassium bisulfate solution, and then 150 mL of a 5% aqueous potassium carbonate solution and 9.52 g (101 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40°C for 90 minutes. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the remaining organic layer was washed with 150 mL of a 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 17 g of a concentrate (yield: quant.). LC / MS analysis of this concentrate revealed the peak area percentage of the target Cbz-MeVal-Asp(tBu)-piperidine (99.7 area %).

[0450] (Synthesis of Cbz-MePhe-MeVal-Asp(tBu)-piperidine)

[0451] (Cbz deprotection reaction)

[0452] 9.5 g (9.6 mmol) of Cbz-MeVal-Asp(tBu)-piperidine obtained by the above method was dissolved in 50 mL of cyclopentyl methyl ether and subjected to hydrogenolysis with 1.9 g of 10% Pd / C (3% wet) and hydrogen gas. The mixture was stirred at 35°C for 2 hours (conversion: 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0453] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0454] The same operation was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to obtain 14.0 g of a concentrate (yield: quant.). This concentrate was analyzed by LC / MS to determine the peak area percentage of the target MeVal-Asp(tBu)-piperidine (99.5 area %).

[0455] (Condensation reaction)

[0456] The above concentrate was dissolved in 126 mL of cyclopentyl methyl ether and 14 mL of acetonitrile. 13.0 g (41.7 mmol) of Cbz-MePhe-OH and 52.9 mL (303 mmol) of diisopropylethylamine were added. 67.0 mL (114 mmol) of a 50% T3P / ethyl acetate solution was added and stirred at room temperature for 1 hour to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol and analyzed by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0457] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0458] The reaction mixture was washed with 140 mL of a 5% aqueous potassium bisulfate solution, and then 140 mL of a 5% aqueous potassium carbonate solution and 10.9 g (114 mmol) of trimethylamine hydrochloride were added, followed by stirring at room temperature for 30 minutes. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the remaining organic layer was washed with 140 mL of a 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 24.1 g of a concentrate (yield 96%). LC / MS analysis of this concentrate revealed the peak area percentage of the target Cbz-MePhe-MeVal-Asp(tBu)-piperidine (99.6 area %).

[0459] (Synthesis of Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 5)

[0460] (Cbz deprotection reaction)

[0461] 11.5 g (9.6 mmol) of Cbz-MePhe-MeVal-Asp(tBu)-piperidine obtained by the above method was dissolved in 58 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 2.3 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35°C for 2 hours (conversion: 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol, analyzing it by LC / MS, and determining the conversion rate based on the peak area value obtained by LC / MS.

[0462] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0463] The same operation was repeated, and the combined reaction solution was filtered through a filter, and the filtrate was concentrated to obtain 18.1 g of a concentrate (yield 99%).

[0464] (Condensation reaction)

[0465] 17.3 g (32.6 mmol) of the above concentrate was dissolved in 153 mL of cyclopentyl methyl ether and 17 mL of acetonitrile. 10.6 g (35.9 mmol) of Cbz-Ser(tBu)-OH and 45.5 mL (261 mmol) of diisopropylethylamine were added. 57.6 mL (98.0 mmol) of a 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 15 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol, and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0466] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0467] The reaction mixture was washed with 170 mL of 5% potassium bisulfate aqueous solution, and then 170 mL of 5% potassium carbonate aqueous solution and 9.4 g (98.0 mmol) of trimethylamine hydrochloride were added, followed by stirring at room temperature for 2 hours. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the remaining organic layer was washed with 170 mL of 5% potassium carbonate aqueous solution. The resulting organic layer was concentrated to obtain 26.5 g of a concentrate (yield: quant.). LC / MS analysis of this concentrate revealed the peak area percentage of the target Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 5) (98.9 area %). MS (ESI): 830.4 [M+Na] + .

[0468] (Synthesis of Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 6)

[0469] (Cbz deprotection reaction)

[0470] 12.0 g (14.9 mmol) of Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 5) was dissolved in 60 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 2.4 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35°C for 2 hours (conversion rate: >98%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0471] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0472] The same operation was repeated, and the combined reaction solution was filtered through a filter, and the filtrate was concentrated to obtain 19.5 g of a concentrate (yield 97%).

[0473] (Condensation reaction)

[0474] 16.0 g (23.7 mmol) of the above concentrate was dissolved in 200 mL of cyclopentyl methyl ether. 7.3 g (26.1 mmol) of Cbz-MeIle-OH and 12.4 mL (71.2 mmol) of diisopropylethylamine were added. 9.8 g (35.6 mmol) of BEP was dissolved in 40 mL of acetonitrile and added to the reaction solution. The mixture was stirred at room temperature for 5 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The peak area value was determined by LC / MS.

[0475] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0476] The reaction mixture was washed with 240 mL of a 10% aqueous sodium bisulfate solution, and then 240 mL of a 5% aqueous potassium carbonate solution and 6.7 g (71.2 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40°C for 1.5 hours. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the remaining organic layer was washed with 240 mL of a 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 22.2 g of a concentrate (yield: quant.). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 6), which was 99.4 area %.

[0477] (Synthesis of Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 7)

[0478] (Cbz deprotection reaction)

[0479] 9.5 g (10.2 mmol) of Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 6) was dissolved in 48 mL of cyclopentyl methyl ether and hydrogenolysis was performed using 1.9 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35°C for 2 hours. The same procedure was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to obtain 15.6 g of a concentrate (yield 96%).

[0480] (Condensation reaction)

[0481] 15.3 g (19.1 mmol) of the above concentrate was dissolved in 138 mL of cyclopentyl methyl ether and 15 mL of acetonitrile. 4.7 g (21.0 mmol) of Cbz-MeGly-OH and 26.7 mL (153 mmol) of diisopropylethylamine were added. 33.8 mL (57.3 mmol) of a 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 15 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol, analyzing it by LC / MS, and determining the conversion rate based on the peak area value obtained by LC / MS.

[0482] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0483] The reaction mixture was washed with 153 mL of 5% potassium bisulfate aqueous solution, followed by the addition of 153 mL of 5% potassium carbonate aqueous solution and stirring at room temperature for 5 minutes. Stirring was stopped, and the organic and aqueous layers were separated. After the aqueous layer was removed, 153 mL of 5% potassium carbonate aqueous solution was added and stirred at room temperature for 1 hour. After the aqueous layer was removed, the resulting organic layer was concentrated to obtain 19.5 g of a concentrate (yield: quant.). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 7), which was 99.6 area %.

[0484] (Synthesis of Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 8)

[0485] (Cbz deprotection reaction)

[0486] 9.5 g (10.2 mmol) of Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 7) was dissolved in 48 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 1.9 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35°C for 3 hours (conversion: 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0487] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0488] The same operation was repeated, and the combined reaction solution was filtered through a filter, and the filtrate was concentrated to obtain 16.3 g of a concentrate (yield 99%).

[0489] (Condensation reaction)

[0490] 16.0 g (18.4 mmol) of the above concentrate was dissolved in 144 mL of cyclopentyl methyl ether and 16 mL of acetonitrile. 5.1 g (20.2 mmol) of Cbz-Val-OH and 25.6 mL (147 mmol) of diisopropylethylamine were added. 32.4 mL (55.0 mmol) of a 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 30 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol, and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0491] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0492] The reaction mixture was washed with 160 mL of 5% aqueous potassium bisulfate solution, and then 153 mL of 5% aqueous potassium carbonate solution and 5.3 g (55.0 mmol) of trimethylamine hydrochloride were added, followed by stirring at 60°C for 1 hour. Stirring was stopped, and the organic and aqueous layers were separated. After the aqueous layer was removed, the resulting organic layer was washed with 160 mL of 5% aqueous potassium carbonate solution and concentrated to obtain 20.0 g of a concentrate (yield 99%). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 8), which was 99.6 area %.

[0493] (Synthesis of Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 9)

[0494] (Cbz deprotection reaction)

[0495] 9.2 g (8.3 mmol) of Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 8) was dissolved in 46 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 1.8 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35°C for 6 hours and further at 45°C for 4 hours (conversion rate: 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0496] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0497] The same procedure was repeated, and the combined reaction mixture was filtered. The filtrate was concentrated to obtain 15.9 g of a concentrate (yield 98%). LC / MS analysis of this concentrate revealed the peak area percentage of the target Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 10) (97.9 area %).

[0498] (Condensation reaction)

[0499] 14.5 g (14.9 mmol) of the above concentrate was dissolved in 181 mL of cyclopentyl methyl ether. 4.6 g (16.4 mmol) of Cbz-MeLeu-OH and 7.8 mL (44.8 mmol) of diisopropylethylamine were added. 4.9 g (17.9 mmol) of BEP was dissolved in 36 mL of acetonitrile, and the resulting BEP solution was added to the reaction solution. The mixture was stirred at 40°C for 1 minute to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol, analyzing it by LC / MS, and determining the conversion rate based on the peak area value obtained by LC / MS.

[0500] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0501] The reaction mixture was washed with 128 mL of a 10% aqueous sodium bisulfate solution, and then 128 mL of a 5% aqueous potassium carbonate solution and 4.2 g (44.8 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40°C for 30 minutes. Stirring was stopped, and the organic and aqueous layers were separated. After the aqueous layer was removed, the resulting organic layer was washed with 128 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 18.0 g of a concentrate (yield 98%). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 9), which was 96.0 area %.

[0502] (Synthesis of Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 11)

[0503] (Cbz deprotection reaction)

[0504] 8.0 g (6.5 mmol) of Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 9) was dissolved in 40 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 1.6 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 45°C for 4 hours (conversion: 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0505] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0506] The same operation was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to obtain 14.3 g of a concentrate (quantitative yield). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12), which was 95.8 area %. MS (ESI): m / z 1098.6 [M+H] + .

[0507] (Condensation reaction)

[0508] 13.0 g (11.8 mmol) of the above concentrate was dissolved in 117 mL of cyclopentyl methyl ether and 13 mL of acetonitrile. 3.5 g (13.0 mmol) of Cbz-Leu-OH and 16.5 mL (95.0 mmol) of diisopropylethylamine were added. 20.9 mL (35.5 mmol) of a 50% T3P / ethyl acetate solution was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol, and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0509] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0510] The reaction mixture was washed with 130 mL of a 5% aqueous potassium bisulfate solution, and then 130 mL of a 5% aqueous potassium carbonate solution and 3.4 g (35.5 mmol) of trimethylamine hydrochloride were added, followed by stirring at 60°C for 45 minutes. Stirring was stopped, and the organic and aqueous layers were separated. After the aqueous layer was removed, the resulting organic layer was washed with 130 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 15.6 g of a concentrate (yield 98%). LC / MS analysis of this concentrate revealed a peak area percentage of 97.2 area % for the target compound Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 11).

[0511] (Synthesis of Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 12)

[0512] (Cbz deprotection reaction)

[0513] 10.0 g (7.4 mmol) of Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 11) was dissolved in 50 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 2.0 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 45°C for 4 hours (conversion rate: 100%). The reaction solution was filtered and the filtrate was concentrated to obtain 8.9 g of a concentrate (yield: 99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol and analyzed by LC / MS. The conversion rate was determined based on the peak area value of the LC / MS. MS (ESI): m / z 1211.7 [M+H] +,1233.7[M+Na] + .

[0514] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0515] (Condensation reaction)

[0516] 7.0 g (5.8 mmol) of the above concentrate was dissolved in 87.5 mL of cyclopentyl methyl ether. 2.0 g (6.4 mmol) of Cbz-MePhe-OH and 3.0 mL (17.3 mmol) of diisopropylethylamine were added. 1.9 g (17.9 mmol) of BEP was dissolved in 17.5 mL of acetonitrile, and the resulting BEP solution was added to the reaction solution. The mixture was stirred at room temperature for 3 minutes to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol, analyzing it by LC / MS, and determining the conversion rate based on the peak area value obtained by LC / MS.

[0517] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0518] The reaction mixture was washed with 105 mL of a 10% aqueous sodium bisulfate solution, and then 105 mL of a 5% aqueous potassium carbonate solution and 1.7 g (17.3 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40°C for 30 minutes. Stirring was stopped, and the organic and aqueous layers were separated. After the aqueous layer was removed, the resulting organic layer was washed with 105 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 8.6 g of a concentrate (yield 99%). LC / MS analysis of this concentrate revealed the peak area percentage of the target compound, Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12), which was 97.0 area %.

[0519] (Synthesis of Cbz-MeAla-MePhe-Leu-MeLeu-ValMeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 4)

[0520] (Cbz deprotection reaction)

[0521] 7.6 g (5.0 mmol) of Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIe-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12) was dissolved in 38 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 2.0 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 45°C for 4 hours (conversion rate: 100%). The reaction solution was filtered and the filtrate was concentrated to obtain 6.8 g of a concentrate (yield: 98%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of methanol and analyzing it by LC / MS. The peak area value was determined by LC / MS.

[0522] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0523] (Condensation reaction)

[0524] 500 mg (0.4 mmol) of the above concentrate was dissolved in 4.5 mL of cyclopentyl methyl ether and 0.5 mL of acetonitrile. 95.0 mg (0.4 mmol) of Cbz-MeAla-OH and 509 μL (2.9 mmol) of diisopropylethylamine were added. 644 μL (1.1 mmol) of a 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 2 hours to allow peptide bond formation to proceed (conversion rate: >99%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of methanol, and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0525] Conversion rate (%) = {target compound (area %) / [raw material (area %) + target compound (area %)]} × 100

[0526] The reaction mixture was washed with 5.0 mL of a 10% aqueous sodium bisulfate solution, and then 5.0 mL of a 5% aqueous potassium carbonate solution and 104 mg (1.1 mmol) of trimethylamine hydrochloride were added, followed by stirring at room temperature for 1 hour. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the resulting organic layer was washed with 5.0 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 555 mg of a concentrate (yield 96%, Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 4) with a 95.3 area % yield). MS (ESI): m / z 1591.9 [M+H] + ,1613.9[M+Na] + .

[0527] A single hydrolysis with the addition of trimethylamine, followed by aqueous washing, completely removed any remaining C-terminal active forms, yielding a peptide consisting of 11 amino acids with a high purity of 95.3%. The total yield from the initial amino acid was 75.3%. These results demonstrate that the use of an amine additive in continuous liquid-phase peptide synthesis to remove any remaining C-terminal active forms enables the synthesis of high-purity peptides.

[0528] (Example 15) Synthesis of Teoc-MeLeu-Phe-OtBu

[0529] (Synthesis of Teoc-MeLeu-Opfp)

[0530] 2.35 g (16.2 mmol) of MeLeu-OH was dissolved in 23.5 mL of 1,4-dioxane, and 4.61 g (17.8 mmol) of Teoc-OSu, 23.5 mL of water, and 4.5 mL (32.4 mmol) of triethylamine were added. The mixture was stirred at room temperature for 1 hour to allow the Teoc reaction to proceed. A 5% aqueous potassium bisulfate solution was added to acidify the reaction solution, followed by extraction with 50 mL of ethyl acetate and washing the organic layer with saturated brine. The resulting organic layer was concentrated to dryness, and the concentrate was dissolved in 30 mL of dichloromethane. 3.10 g (16.2 mmol) of Pfp-OH and 4.53 g (24.3 mmol) of EDC hydrochloride were added, and the mixture was stirred at room temperature for 30 minutes to allow the Pfp reaction to proceed. The reaction solution was washed with saturated brine, and the aqueous layer was extracted with 50 mL of ethyl acetate. The combined organic layers were concentrated, and the obtained concentrate was purified by column chromatography (ethyl acetate / heptane) to obtain 6.63 g of Teoc-MeLeu-OPfp (yield: 90%).

[0531] (Condensation reaction)

[0532] 201 mg (0.8 mmol) of Phe-OtBu hydrochloride and 536 mg (1.2 mmol) of Teoc-MeLeu-OPfp were suspended in 3.0 mL of isopropyl acetate, and 257 μL (2.3 mmol) of 4-methylmorpholine was added. The mixture was stirred at 25°C for 3 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined from the peak area value obtained by LC / MS (conversion rate: 100%).

[0533] Conversion rate (%) = {Teoc-MeLeu-Phe-OtBu (area %) / [Phe-OtBu (area %) + Teoc-MeLeu-Phe-OtBu (area %)]} × 100

[0534] (Hydrolysis treatment)

[0535] (1) When no amine is added

[0536] Add 2.0 mL of 5% aqueous sodium carbonate solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 20 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0537] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0538] (2) When amine is added

[0539] To the peptide-containing reaction solution prepared above, add 95 mg (0.8 mmol) of DMAP and 2.0 mL of a 5% aqueous sodium carbonate solution, and stir at 25°C for 20 minutes with a stirrer. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the C-terminal active form residual rate based on the peak area value of LC / MS according to the following calculation formula.

[0540] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0541] (Post-processing)

[0542] After stopping stirring, the mixture was allowed to stand for separation of the organic and aqueous layers, and the aqueous layer was removed. The organic layer was then washed with 2 mL of 10% potassium bisulfate aqueous solution twice and 2 mL of 5% sodium carbonate aqueous solution. The mixture was further washed three times with 1 mL of 5% potassium carbonate aqueous solution and 1 mL of water twice. 5 μL of the resulting organic layer was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS to determine the peak areas of the target peptide and the remaining C-terminal active form (propionamide conversion form). The concentrate (peptide) obtained by hydrolysis without the addition of amine was 576.6 mg (yield 150%: although the concentrate contained impurities (residual C-terminal active form), the calculation was performed as if the concentrate contained only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 369.6 mg (yield 96%). MS(ESI): m / z 437.3[M-tBu+H]+, 493.3[M+H]+, 515.3[M+Na]+.

[0543]

Table 9

[0544]

[0545] 1) LCMS peak area ratio

[0546] In hydrolysis of residual C-terminal active forms with a Teoc protecting group and a Pfp C-terminal active site, alkaline water alone did not completely hydrolyze the residual C-terminal active form, and even continued aqueous washing could not remove the residual C-terminal active form. On the other hand, the addition of DMAP to the hydrolysis process resulted in complete hydrolysis and removal of the residual C-terminal active form. The target dipeptide was obtained with a purity of 96.3% (yield 96%).

[0547] (Example 16) Synthesis of Cbz-Aib-MeLeu-Phe-OtBu

[0548] (Teoc deprotection reaction using a dipeptide obtained by hydrolysis without adding an amine)

[0549] 576.6 mg of Teoc-MeLeu-Phe-OtBu (containing 8.9 area % of the residual C-terminal active form), synthesized in Example 15 without the addition of amine, was dissolved in 2.0 mL of 2-methyltetrahydrofuran. 1.5 mL (1.5 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added, and the mixture was stirred at 50°C for 2.5 hours. Since the reaction was not complete, 0.75 mL (0.75 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added, and the mixture was stirred for 2.5 hours. Further 0.75 mL (0.75 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added, and the mixture was stirred for 30 minutes to obtain MeLeu-Phe-OtBu as a de-Teoc form (conversion rate 100%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. The conversion rate was determined based on the peak area value of the LC / MS.

[0550] Conversion rate (%) = {MeLeu-Phe-OtBu (area %) / [Teoc-MeLeu-Phe-OtBu (area %) + MeLeu-Phe-OtBu (area %)]} × 100

[0551] (Condensation reaction)

[0552] After concentrating the solution to approximately 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This procedure was repeated two more times, and 0.5 mL of acetonitrile, 276 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine were added to the resulting 2-methyltetrahydrofuran solution. Subsequently, 441 mg (1.1 mmol) of HATU was added at 25°C, and the mixture was stirred at room temperature for 14 hours. 579 mg (1.5 mmol) of HATU was added, and the mixture was stirred at 40°C for 1 hour. Then, 684 mg (1.8 mmol) of HATU was added, the temperature was raised to 60°C, and the mixture was stirred for 4.5 hours. Furthermore, 455 mg (1.1 mmol) of HATU was added, and the mixture was stirred at 60°C for 2 hours, at room temperature for 12 hours, and at 60°C for 2 hours. However, no condensation reaction was observed (conversion rate 0%). The reaction conversion rate was determined by adding 5 μL of the reaction solution to 100 μL of propylamine, diluting with 0.9 mL of methanol, and performing LC / MS analysis to determine the reaction conversion rate based on the peak area value of LC / MS.

[0553] Conversion rate (%) = {Cbz-Aib-MeLeu-Phe-OtBu (area %) / [MeLeu-Phe-OtBu (area %) + Cbz-Aib-MeLeu-Phe-OtBu (area %)]} × 100

[0554] (Teoc deprotection reaction using a dipeptide obtained by hydrolysis treatment with an amine)

[0555] 369.6 mg (0.75 mmol) of Teoc-MeLeu-Phe-OtBu, synthesized under amine addition conditions in Example 15, was dissolved in 2.0 mL of 2-methyltetrahydrofuran. 1.5 mL (1.5 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added, and the mixture was stirred at 50°C for 2.5 hours to obtain MeLeu-Phe-OtBu as a de-Teoc compound (conversion rate 100%). The reaction conversion rate was determined by diluting 5 μL of the reaction solution with 1.0 mL of acetonitrile and analyzing it by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0556] Conversion rate (%) = {MeLeu-Phe-OtBu (area %) / [Teoc-MeLeu-Phe-OtBu (area %) + MeLeu-Phe-OtBu (area %)]} × 100

[0557] (Condensation reaction)

[0558] After concentrating the solution to approximately 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This operation was repeated two more times, and 0.5 mL of acetonitrile, 273 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine were added to the resulting 2-methyltetrahydrofuran solution. Next, 439 mg (1.1 mmol) of HATU was added at 25°C, and the mixture was stirred at room temperature for 14 hours. 576 mg (1.5 mmol) of HATU was added, and the mixture was stirred at 40°C for 5.5 hours. 452 mg (1.1 mmol) of HATU was further added, and the mixture was stirred at 60°C for 2 hours, at room temperature for 12 hours, and at 60°C for 2 hours (conversion rate 86%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was added to 100 μL of propylamine, diluted with 0.9 mL of methanol, and analyzed by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS. Conversion rate (%) = {Cbz-Aib-MeLeu-Phe-OtBu (area %) / [MeLeu-Phe-OtBu (area %) + Cbz-Aib-MeLeu-Phe-OtBu (area %)]} × 100

[0559] To the prepared reaction solution, 92 mg (0.75 mmol) of DMAP and 4.0 mL of a 10% aqueous potassium carbonate solution were added, and the mixture was stirred at 25°C for 1 hour using a stirrer. After stopping stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. LC / MS analysis of this solution was performed to determine the peak area percentages of the target peptide and the residual C-terminal active form. The peak area percentage for the target peptide, Cbz-Aib-MeLeu-Phe-OtBu, was 86.7%, while that for the starting material, MeLeu-Phe-OtBu, was 13.3%. No Cbz-Aib-NHPr derived from the residual C-terminal active form was detected. The remaining organic layer was concentrated to yield 295.6 mg of the concentrate with the above composition. MS (ESI): m / z 568.4 [M+H]+, 590.4 [M+Na]+.

[0560] It was found that if C-terminal active species remain in a solution of an N-terminally protected peptide, deprotection of the peptide's N-terminal protecting group (Teoc) using an aqueous fluorinated reagent requires a large excess of the reagent. This is presumably because the deprotection reagent also reacts with the remaining C-terminal active species.

[0561] Furthermore, it was determined that the condensation reaction (peptide bond formation reaction) with other C-terminal active forms in the next step after deprotection did not proceed at all. It is speculated that the excess reagent used in the previous step (N-terminal deprotection) may have degraded the C-terminal active form. It was found that if the C-terminal active form remains, a large excess of reagent is required for N-terminal deprotection, which would hinder the subsequent condensation reaction. On the other hand, it was found that if a peptide solution in which the residual C-terminal active form has been completely removed is used as the starting material, the deprotection reaction can be completed with an appropriate amount of deprotection reagent, and the condensation reaction in the next step can also be carried out.

[0562] (Example 17) Synthesis of Cbz-MeAla-Phe-OtBu

[0563] (Condensation reaction)

[0564] 302 mg (1.2 mmol) of Phe-OtBu hydrochloride, 417 mg (1.7 mmol) of Cbz-MeAla-OH, and 290 mg (1.8 mmol) of HOOBt were suspended in 0.9 mL of acetonitrile and 3.6 mL of MTBE. 1.0 mL (5.8 mmol) of diisopropylethylamine was added. Next, 443 mg (2.3 mmol) of EDC hydrochloride was added, and the mixture was stirred at 25°C for 30 minutes to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined from the peak area value obtained by LC / MS (conversion rate: 100%).

[0565] Conversion rate (%) = {Cbz-MeAla-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-MeALa-Phe-OtBu (area %)]} × 100

[0566] (Hydrolysis treatment)

[0567] (1) When no amine is added

[0568] Add 3.0 mL of 5% aqueous sodium carbonate solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0569] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0570] (2) When amine is added

[0571] To the peptide-containing reaction solution prepared above, add 147 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% aqueous sodium carbonate solution, and stir at 25°C with a stirrer for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the C-terminal active form residual rate based on the peak area value of LC / MS according to the following calculation formula.

[0572] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0573] (Post-processing)

[0574] After stopping stirring, let the mixture stand to allow the organic layer and aqueous layer to separate, and remove the aqueous layer. Next, wash the organic layer with 3 mL of 10% sodium bisulfate aqueous solution x 2 and 3 mL of 5% sodium carbonate aqueous solution, respectively. Add 5 μL of the obtained organic layer to 100 μL of propylamine and dilute with 0.9 mL of methanol. Analyze the solution by LC / MS to determine the peak area percentage of the target peptide and the residual C-terminal active form. MS (ESI): m / z 385.2 [M-tBu+H]+, 441.3 [M+H]+, 463.2 [M+Na]+.

[0575]

Table 10

[0576]

[0577] 1) LCMS peak area ratio

[0578] Even with alanine, an amino acid with a small substituent, the residual C-terminal active form was not completely hydrolyzed during hydrolysis using alkaline water alone, and the subsequent liquid separation procedure (aqueous washing) could not fully remove the residual C-terminal active form. On the other hand, it was discovered that the addition of DMAP to the hydrolysis completely hydrolyzed the residual C-terminal active form and completely removed the residual C-terminal active form. Furthermore, in this case, the target dipeptide was obtained with a purity of 98.4%.

[0579] (Example 18) Synthesis of Cbz-Hph-MeAla-Phe-OtBu

[0580] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis without adding an amine)

[0581] The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized in Example 17 without the addition of amine was replaced with 2-methyltetrahydrofuran and concentrated. Hydrogenolysis was carried out using 101 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was performed at 25°C for 6 hours, but the reaction was not complete (conversion rate 35%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of acetonitrile and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0582] Conversion (%) = {MeAla-Phe-OtBu (area %) / [Cbz-MeAla-Phe-OtBu (area %) + MeALa-Phe-OtBu (area %)]} × 100

[0583] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis treatment with amine addition)

[0584] The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized under amine addition conditions in Example 17 was replaced with 2-methyltetrahydrofuran and concentrated. A hydrogenolysis reaction was carried out using 102 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was performed at 25°C for 3 hours to obtain MeAla-Phe-OtBu as a de-Cbz product (conversion rate 100%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of acetonitrile and analyzed by LC / MS. The conversion rate was determined based on the peak area value of the LC / MS. MS (ESI): m / z 307.2 [M+H]+.

[0585] Conversion (%) = {MeAla-Phe-OtBu (area %) / [Cbz-MeAla-Phe-OtBu (area %) + MeALa-Phe-OtBu (area %)]} × 100

[0586] (Condensation reaction)

[0587] The Cbz-deprotected dipeptide reaction solution obtained by hydrolysis with amine was filtered through a filter to remove Pd / C, and then concentrated to dryness. The dried product was dissolved in 2.5 mL of 2-methyltetrahydrofuran, and 474 mg (1.5 mmol) of Cbz-Hph-OH and 610 μL (3.5 mmol) of diisopropylethylamine were added. Next, 1.37 mL (2.33 mmol) of a T3P / 2-methyltetrahydrofuran solution was added, and the mixture was stirred at 25°C for 1.5 hours to allow the peptide bond to form (conversion rate: 100%). The reaction conversion rate was determined by adding 5 μL of the reaction solution to 100 μL of propylamine, diluting with 0.9 mL of methanol, and analyzing by LC / MS. The peak area value was determined by LC / MS.

[0588] Conversion rate (%) = {Cbz-Hph-MeAla-Phe-OtBu (area %) / [MeAla-Phe-OtBu (area %) + Cbz-Hph-MeAla-Phe-OtBu (area %)]} × 100

[0589] To the prepared reaction solution, 74 mg (0.6 mmol) of DMAP and 3.0 mL of 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 25°C for 15 minutes. Stirring was stopped, and after standing, the organic and aqueous layers were separated and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous sodium bisulfate solution, 3 mL of 5% aqueous sodium carbonate solution, and 3 mL of water. 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution to determine the peak area percentages of the target peptide and the residual C-terminal active form. The target peptide, Cbz-Hph-MeAla-Phe-OtBu, was 99.0%, with no Cbz-Hph-NHPr derived from the residual C-terminal active form detected. The remaining organic layer was concentrated to yield 618.4 mg of concentrate (88% yield from Phe-OtBu in Example 17). MS(ESI): m / z 602.4[M+H]+, 624.4[M+Na]+.

[0590] It was clarified that if C-terminal active forms derived from EDC and HOOBt remain, the Cbz deprotection reaction after peptide elongation will hardly proceed. On the other hand, it was found that if a peptide solution from which the residual C-terminal active forms have been completely removed is used as the starting material, the Cbz deprotection reaction proceeds smoothly, enabling the peptide synthesis reaction to proceed. In other words, it was found that, similar to the case of Example 9, the use of the method of the present invention allows the reductive removal reaction of the N-terminal protecting group of the generated peptide compound to proceed without stagnation.

[0591] (Example 19) Synthesis of Cbz-Aib-D-Val-OBn

[0592] (Condensation reaction)

[0593] 502 mg (1.3 mmol) of D-Val-OBn TsOH salt and 478 mg (2.0 mmol) of Cbz-Aib-OH were suspended in 6.0 mL of 2-MeTHF, and 1.2 mL (6.9 mmol) of diisopropylethylamine was added. Next, 1.9 mL (3.3 mmol) of a 50% T3P / 2-methyltetrahydrofuran solution was added at 25°C, and the mixture was stirred at 25°C for 15 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide, and the solution was then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the conversion rate was determined from the peak area of ​​the LC / MS results (conversion rate: 100%).

[0594] Conversion rate (%) = {Cbz-Aib-D-Val-OBn (area %) / [D-Val-OBn (area %) + Cbz-Aib-D-Val-OBn (area %)]} × 100

[0595] (Hydrolysis treatment)

[0596] (1) When no amine is added

[0597] Add 5.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 30 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0598] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0599] (2) When amine is added

[0600] To the peptide-containing reaction solution prepared above, add 484 mg (4.0 mmol) of DMAP and 5.0 mL of a 5% potassium carbonate aqueous solution, and stir at 25°C for 30 minutes with a stirring bar. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Based on the peak area value of LC / MS, calculate the residual rate of the C-terminal active form according to the following calculation formula.

[0601] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0602] (Post-processing)

[0603] After stopping the stirring, the mixture was allowed to stand for separation of the organic layer and the aqueous layer, and the aqueous layer was removed. Next, the organic layer was washed sequentially with 5 mL of a 10% aqueous potassium hydrogen sulfate solution and 2.5 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine was 653.8 mg (yield 116%: although the concentrate contained impurities (residual C-terminal active form), it was calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 549.4 mg (yield 97%). MS (ESI): m / z 427.3 [M+H] +,449.2[M+Na] + .

[0604]

Table 11

[0605]

[0606] 1) LCMS peak area ratio

[0607] It was found that during hydrolysis using alkaline water alone, the residual C-terminal active form was not completely hydrolyzed, and the subsequent aqueous wash could not fully remove the residual C-terminal active form. However, when DMAP was added to the hydrolysis, the residual C-terminal active form was completely hydrolyzed and completely removed. In this case, the target dipeptide was obtained with a purity of 98.6% (yield of 97%).

[0608] (Example 20) Synthesis of Cbz-Thr(tBu)-Phe-OtBu

[0609] (Condensation reaction)

[0610] 300 mg (1.2 mmol) of Phe-OtBu hydrochloride, 855 mg (1.7 mmol) of Cbz-Thr(tBu)-OH dicyclohexylamine salt, and 237 mg (1.8 mmol) of HOBt were suspended in 4.2 mL of 2-MeTHF and 0.9 mL of acetonitrile. 813 μL (4.6 mmol) of diisopropylethylamine was added. Next, 447 mg (2.3 mmol) of EDC hydrochloride was added at 25°C, and the mixture was stirred at 25°C for 3 hours to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined based on the peak area of ​​the LC / MS results (conversion rate: 100%).

[0611] Conversion rate (%) = {Cbz-Thr(tBu)-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-Thr(tBu)-Phe-OtBu (area %)]} × 100

[0612] (Hydrolysis treatment)

[0613] (1) When no amine is added

[0614] Add 3.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0615] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0616] (2) When amine is added

[0617] Add 142 mg (1.2 mmol) of DMAP and 3.0 mL of 5% potassium carbonate aqueous solution to the reaction solution containing the peptide prepared above, and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the residual C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Based on the peak area value of LC / MS, calculate the residual C-terminal active form according to the following calculation formula.

[0618] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0619] (Post-processing)

[0620] After stopping stirring, let it stand to allow the organic layer and the aqueous layer to separate, and remove the aqueous layer. Next, wash the organic layer with 3 mL of 10% potassium bisulfate aqueous solution, 3 mL of 5% potassium carbonate aqueous solution, and 1.5 mL of water in sequence. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the residual C-terminal active form into propionamide. Then dilute it with 0.9 mL of methanol and perform LC / MS analysis to determine the peak area values ​​of the target peptide and the residual C-terminal active form (propionamide conversion form). Concentrate the remaining organic layer to obtain the peptide. MS (ESI): m / z 401.2 [M-2tBu+H] + , 457.2[M-tBu+H] + , 513.3[M+H] + ,535.3[M+Na] + .

[0621]

Table 12

[0622]

[0623] 1) LCMS peak area ratio

[0624] It was found that during hydrolysis using alkaline water alone, the remaining C-terminal active form was not completely hydrolyzed, and the subsequent aqueous wash could not fully remove the remaining C-terminal active form. However, when hydrolysis was performed with the addition of DMAP, the remaining C-terminal active form was completely hydrolyzed and removed. In this case, the target dipeptide was obtained with a purity of 98.4%.

[0625] (Example 21) Synthesis of Cbz-Leu-Thr(tBu)-Phe-OtBu

[0626] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis without adding an amine)

[0627] The MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu synthesized in Example 20 without the addition of amine was replaced with 2-methyltetrahydrofuran and concentrated. Hydrogenolysis was carried out using 99 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was performed at 25°C for 1 hour, but the reaction was not completed (conversion rate 53%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of acetonitrile and analyzed by LC / MS. The peak area value was determined by LC / MS.

[0628] Conversion rate (%) = {Thr(tBu)-Phe-OtBu (area %) / [Cbz-Thr(tBu)-Phe-OtBu (area %) + Thr(tBu)-Phe-OtBu (area %)]} × 100

[0629] (Cbz deprotection reaction using a dipeptide obtained by hydrolysis treatment with amine addition)

[0630] The MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu synthesized under the conditions of amine addition in Example 20 was replaced with 2-methyltetrahydrofuran and concentrated. A hydrogenolysis reaction was carried out using 104 mg of 5% Pd / C (50% wet) and hydrogen. Stirring at 25°C for 1 hour gave Thr(tBu)-Phe-OtBu as a de-Cbz product (conversion rate 100%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was diluted with 1.0 mL of acetonitrile, and then analyzed by LC / MS. The conversion rate was determined based on the peak area value of the LC / MS.

[0631] Conversion rate (%) = {Thr(tBu)-Phe-OtBu (area %) / [Cbz-Thr(tBu)-Phe-OtBu (area %) + Thr(tBu)-Phe-OtBu (area %)]} × 100

[0632] (Condensation reaction)

[0633] The Cbz-deprotected dipeptide reaction solution obtained by hydrolysis with amine was filtered through a filter to remove Pd / C, and then concentrated to dryness. The dried product was dissolved in 5.0 mL of 2-methyltetrahydrofuran, and 382 mg (1.4 mmol) of Cbz-Leu-OH and 814 μL (4.7 mmol) of diisopropylethylamine were added. Next, 1.37 mL (2.3 mmol) of a 50% T3P / 2-methyltetrahydrofuran solution was added, and the mixture was stirred at 25°C for 30 minutes to allow for peptide bond formation (conversion rate: 100%). The reaction conversion rate was determined as follows: 5 μL of the reaction solution was added to 100 μL of propylamine, diluted with 0.9 mL of methanol, and analyzed by LC / MS. The conversion rate was determined based on the peak area value obtained by LC / MS.

[0634] Conversion rate (%) = {Cbz-Leu-Thr(tBu)-Phe-OtBu (area %) / [Thr(tBu)-Phe-OtBu (area %) + Cbz-Leu-Thr(tBu)-Phe-OtBu (area %)]} × 100. 139 mg (1.1 mmol) of DMAP and 3.0 mL of 10% aqueous sodium carbonate solution were added to the prepared reaction solution, and the mixture was stirred at 25°C for 5 minutes. Stirring was stopped, and after standing, the organic layer and the aqueous layer were separated and the aqueous layer was removed. Next, the organic layer was washed with 3 mL of 10% aqueous sodium bisulfate solution × 2, 3 mL of 5% aqueous sodium carbonate solution, and 3 mL of water in that order. 5 μL of the obtained organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. The solution was analyzed by LC / MS to determine the peak area percentages of the target peptide and the residual C-terminal active form. The target peptide, Cbz-Leu-Thr(tBu)-Phe-OtBu, was 98.3% in concentration, with no Cbz-Leu-NHPr from the residual C-terminal active form detected. The remaining organic layer was concentrated to yield 638.0 mg of concentrate (88% yield from Phe-OtBu in Example 20). MS (ESI): m / z 514.3 [M-2tBu+H] + , 570.3[M-tBu+H] + ,626.5[M+H] + ,648.4[M+Na] + .

[0635] It was found that when C-terminal active forms derived from EDC and HOBt remained, the Cbz deprotection reaction proceeded more slowly than when the remaining C-terminal active forms were completely removed. It was discovered that using a peptide solution from which the remaining C-terminal active forms had been completely removed as a starting material allowed the Cbz deprotection reaction to proceed smoothly, enabling peptide synthesis. Specifically, by using the method of the present invention, as in Examples 9 and 18, the reductive removal reaction of the N-terminal protecting group of the produced peptide compound could proceed without stagnation. This allows for the efficient production of high-purity peptide compounds having the desired amino acid sequence.

[0636] (Example 22) Synthesis of Cbz-Ile-Phe-OtBu

[0637] (Condensation reaction)

[0638] 301 mg (1.2 mmol) of Phe-OtBu hydrochloride and 465 mg (1.8 mmol) of Cbz-Ile-OH were suspended in 3.6 mL of MTBE and 0.9 mL of acetonitrile, and 610 μL (3.5 mmol) of diisopropylethylamine was added. Next, 479 mg (1.8 mmol) of BEP was added at 25°C, and the mixture was stirred at 25°C for 45 minutes to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide, and the solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined based on the peak area of ​​the LC / MS results (conversion rate: 100%).

[0639] Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-Ile-Phe-OtBu (area %)]} × 100

[0640] (Hydrolysis treatment)

[0641] (1) When no amine is added

[0642] Add 3.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 3 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0643] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0644] (2) When amine is added

[0645] To the peptide-containing reaction solution prepared above, 139 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% potassium carbonate aqueous solution were added, and the mixture was stirred at 25°C for 3 minutes using a stirring bar. Stirring was stopped, and the organic and aqueous layers were separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, and then diluted with 0.9 mL of methanol. Based on the peak area value of LC / MS, the residual rate of the C-terminal active form was calculated according to the following calculation formula.

[0646] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0647] (Post-processing)

[0648] After stopping stirring, let it stand to allow the organic layer and the aqueous layer to separate, and remove the aqueous layer. Next, wash the organic layer with 3 mL of 10% potassium bisulfate aqueous solution and 3 mL of 5% potassium carbonate aqueous solution in sequence. After adding 2 mL of 2-MeTHF, wash with 1.5 mL of water. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the residual C-terminal active form into propionamide. Then dilute it with 0.9 mL of methanol and perform LC / MS analysis to determine the peak area values ​​of the target peptide and the residual C-terminal active form (propionamide conversion form). Concentrate the remaining organic layer to obtain the peptide. MS (ESI): m / z 413.3 [M-tBu+H] + , 469.3[M+H] + ,491.3[M+Na] + .

[0649]

Table 13

[0650]

[0651] 1) LCMS peak area ratio

[0652] It was found that during hydrolysis using alkaline water alone, the remaining C-terminal active form was not completely hydrolyzed, and the subsequent aqueous wash could not fully remove the remaining C-terminal active form. However, when DMAP was added to the hydrolysis, the remaining C-terminal active form was completely hydrolyzed and completely removed. In this case, the target dipeptide was obtained with a purity of 96.3%.

[0653] (Example 23) Cbz-Phe-MeGly-Phe-piperidine

[0654] (Boc deprotection reaction)

[0655] Dissolve 334 mg (1.0 mmol) of Boc-Phe-piperidine in 3.4 mL of dichloromethane, and add 131 μL (2.0 mmol) of methanesulfonic acid. Stir at 35°C for 3 hours to carry out a Boc removal reaction (conversion rate: 100%). The reaction conversion rate was determined by LCMS analysis of 5 μL of the reaction solution diluted with 1.0 mL of acetonitrile. The peak area value was determined by LC / MS.

[0656] Conversion rate (%) = {Phe-piperidine (area %) / [Boc-Phe-piperidine (area %) + Phe-piperidine (area %)]} × 100

[0657] (Condensation reaction)

[0658] After adding 528 μL (3.0 mmol) of diisopropylethylamine to the above reaction solution, the solvent was distilled off. Next, 1.0 mL of acetonitrile, 3.4 mL of 2-methyltetrahydrofuran, 528 μL (3.0 mmol) of diisopropylethylamine, 505 mg (1.5 mmol) of Cbz-Phe-MeGly-OH, and 256 mg (1.6 mmol) of HOOBt were added. 388 mg (2.0 mmol) of EDC hydrochloride was added at 25°C, and the mixture was stirred at 25°C for 1 hour to perform a peptide bond formation reaction. 5 μL of the reaction solution was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, and then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the conversion rate was calculated from the peak area of ​​LC / MS (conversion rate: 100%).

[0659] Conversion rate (%) = {Cbz-Phe-MeGly-Phe-piperidine (area %) / [Phe-piperidine (area %) + Cbz-Phe-MeGly-Phe-piperidine (area %)]} × 100

[0660] 128 mg (1.0 mmol) of DMAP and 3.5 mL of 5% potassium carbonate aqueous solution were added to the reaction solution prepared above, and stirred at 25°C for 3 minutes with a stirrer. After stopping stirring, the mixture was allowed to stand to separate the organic layer and the aqueous layer, and the aqueous layer was removed. Next, the organic layer was diluted with 3.5 mL of 10% potassium sulfate aqueous solution × 2 and 3.5 mL of 5% potassium carbonate aqueous solution. The solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the residual C-terminal active form. The purity of the target peptide Cbz-Phe-MeGly-Phe-piperidine was 94.6%, and Cbz-Phe-MeGly-NHPr from the residual C-terminal active form was not detected. The remaining organic layer was concentrated to obtain 520.4 mg of concentrate (yield 94%).

[0661] When DMAP was added for hydrolysis, the residual C-terminal active form from the peptide fragment was completely decomposed and removed, and the target tripeptide was obtained with a purity of 94.6%. (Yield 94%)

[0662] 1) J. Org. Chem., 2003, 68, 7505-7508.

[0663] 2) Bull.Chem.Soc.Jpn., 2004, 77, 1187-1193.

[0664] (Example 24) Synthesis of Cbz-Val-Phe-OtBu

[0665] (Condensation reaction)

[0666] 200 mg (0.8 mmol) of Phe-OtBu hydrochloride and 294 mg (1.2 mmol) of Cbz-Val-OH were suspended in 2.4 mL of 2-methyltetrahydrofuran and 0.6 mL of acetonitrile, and 294 μL (2.3 mmol) of N-ethylmorpholine was added. Next, 447 mg (1.2 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 2.5 hours to allow the peptide bond to form. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide, and then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). MS (ESI): m / z 399.3 [M-tBu+H] + , 455.3[M+H] +

[0667] Conversion rate (%) = {Cbz-Val-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-Val-Phe-OtBu (area %)]} × 100

[0668] (Hydrolysis treatment)

[0669] (1) When no amine is added

[0670] Add 2.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above, and stir with a stirrer at 25°C. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the residual C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Perform LC / MS analysis on this solution, and calculate the C-terminal active form residual rate according to the peak area value of LC / MS according to the following calculation formula (table below).

[0671] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0672] (2) When amine is added

[0673] To the peptide-containing reaction solution prepared above, add the amine additive (0.8 mmol) shown in the table below and 2.0 mL of a 5% potassium carbonate aqueous solution, and stir with a stirrer at 25°C. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the residual C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Perform LC / MS analysis on this solution, and calculate the C-terminal active form residual rate according to the peak area value of LC / MS according to the following calculation formula (table below).

[0674] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0675]

Table 14

[0676] Changes in the residual rate of C-terminal active form

[0677]

[0678] The addition of DIPEA slightly promoted the hydrolysis of the residual C-terminal active form compared to alkaline water alone without the addition of an amine, but no significant effect was observed. On the other hand, the addition of DMAP and NMI significantly promoted the hydrolysis of the residual C-terminal active form compared to DIPEA. In particular, the use of DMAP completely hydrolyzed the residual C-terminal active form within 5 minutes. This demonstrates that amines with less steric hindrance near the nitrogen nitrogen, such as DMAP, are more effective in promoting the hydrolysis of the residual C-terminal active form than amines with steric hindrance near the nitrogen nitrogen, such as DIPEA.

[0679] (Example 25) Synthesis of Cbz-Ile-Val-OBn

[0680] (Condensation reaction)

[0681] 300 mg (1.2 mmol) of Val-OBn hydrochloride and 495 mg (1.9 mmol) of Cbz-Ile-OH were suspended in 3.0 mL of cyclopentyl methyl ether and 0.9 mL of acetonitrile, and 859 μL (4.9 mmol) of diisopropylethylamine was added. Subsequently, 705 mg (1.9 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 1 hour to allow peptide bond formation. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide, and the solution was then diluted with 0.9 mL of methanol. LC / MS analysis was performed on this solution, and the conversion rate was determined from the peak area of ​​the LC / MS results (conversion rate: 100%).

[0682] Conversion rate (%) = {Cbz-Ile-Val-OBn (area %) / [Val-OBn (area %) + Cbz-Ile-Val-OBn (area %)]} × 100

[0683] (Hydrolysis treatment)

[0684] (1) When no amine is added

[0685] Add 3.0 mL of neutral water to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0686] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0687] (2) When amine is added

[0688] To the peptide-containing reaction solution prepared above, add 91 mg (0.7 mmol) of DMAP and 3.0 mL of neutral water, and stir at 25°C with a stirrer for 5 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0689] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0690] (Post-processing)

[0691] After stopping stirring, the mixture was allowed to stand for separation of the organic layer and the aqueous layer, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of a 10% aqueous sodium bisulfate solution, 3 mL of a 5% aqueous sodium carbonate solution, and 1.5 mL of water. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and analyzed by LC / MS to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine was 744 mg (yield 132%: although the concentrate contained impurities (residual C-terminal active form), it was calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 528 mg (yield 94%). MS (ESI): m / z 455.3 [M+H] + ,477.3[M+Na] + .

[0692]

Table 15

[0693]

[0694] 1) LCMS peak area ratio

[0695] It was found that during hydrolysis using neutral water alone, the residual C-terminal active form was not completely hydrolyzed, and subsequent aqueous washing could not remove the residual C-terminal active form. However, when DMAP was added to the hydrolysis, the residual C-terminal active form was completely hydrolyzed and removed. In this case, the target dipeptide was obtained with a purity of 99.5% (yield of 94%).

[0696] (Example 26) Synthesis of Cbz-MeAla-Phe-OtBu

[0697] (Condensation reaction)

[0698] 200 mg (0.8 mmol) of Phe-OtBu hydrochloride and 260 mg (1.2 mmol) of Cbz-MeAla-OH were suspended in 2.4 mL of 2-methyltetrahydrofuran and 0.6 mL of acetonitrile, and 271 μL (1.6 mmol) of diisopropylethylamine was added. Subsequently, 265 mg (0.8 mmol, 13 wt% water content) of DMT-MM-n-hydrate (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate) was added at 25°C. After stirring at 25°C for 1 hour, 119 mg (0.4 mmol, 13 wt% water content) of DMT-MM-n-hydrate was added, and the mixture was stirred at 25°C for 1 hour to carry out a peptide bond formation reaction. 5 μL of the reaction solution was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form to propionamide. The resulting solution was then diluted with 0.9 mL of methanol. This solution was analyzed by LC / MS, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%).

[0699] Conversion rate (%) = {Cbz-MeAla-Phe-OtBu (area %) / [Phe-OtBu (area %) + Cbz-MeAla-Phe-OtBu (area %)]} × 100

[0700] (Hydrolysis treatment)

[0701] (1) When no amine is added

[0702] Add 2.0 mL of 5% potassium carbonate aqueous solution to the peptide-containing reaction solution prepared above and stir with a stirrer at 25°C for 10 minutes. Stop stirring and allow the organic and aqueous layers to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, then dilute with 0.9 mL of methanol. Calculate the residual C-terminal active form rate based on the peak area value of LC / MS according to the following calculation formula.

[0703] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0704] (2) When amine is added

[0705] Add 96 mg (0.8 mmol) of DMAP and 2.0 mL of a 5% aqueous potassium carbonate solution to the reaction solution containing the peptide prepared above, and stir with a stirrer at 25°C for 10 minutes. Stop stirring and allow the organic layer and the aqueous layer to separate. Take 5 μL of the organic layer and add it to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide, and then dilute with 0.9 mL of methanol. Calculate the C-terminal active form residual rate according to the following calculation formula based on the peak area value of LC / MS.

[0706] Residual rate of C-terminal active form (%) = {propionamide (area %) / [propionamide (area %) + dipeptide (area %)]} × 100

[0707] (Post-processing)

[0708] After stopping the stirring, the mixture was allowed to stand for separation of the organic layer and the aqueous layer, and the aqueous layer was removed. Next, the organic layer was washed with 2 mL of a 10% aqueous potassium hydrogen sulfate solution, 2 mL of a 5% aqueous potassium carbonate solution, and 1 mL of water twice. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propionamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak area values ​​of the target peptide and the remaining C-terminal active form (propionamide conversion form). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine was 387 mg (yield 114%: although the concentrate contained impurities (residual C-terminal active form), it was calculated as a concentrate containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine was 336 mg (yield 98%). MS (ESI): m / z 385.2 [M-tBu+H] + , 441.3[M+H] + ,463.3[M+Na] + .

[0709] Table 16

[0710]

[0711] 1) LCMS peak area ratio

[0712] It was found that when DMT-MM was used as a condensing agent, the residual C-terminal active form was not completely hydrolyzed during hydrolysis using alkaline water alone, and the residual C-terminal active form could not be removed by the subsequent aqueous wash. However, when DMAP was added to the hydrolysis, the residual C-terminal active form was completely hydrolyzed and removed completely. In this case, the target dipeptide was obtained with a purity of 98.6% (yield 98%). The C-terminal active form generated by DMT-MM, a condensing agent that can also be used in aqueous solvents, is known to be relatively resistant to hydrolysis. However, the use of an amine additive allowed the residual C-terminal active form, even when prepared using DMT-MM, to be completely hydrolyzed in a short time and a single treatment, and completely removed in the subsequent aqueous wash.

[0713] Reference Example 1: Synthesis of MeAsp(tBu)-piperidine

[0714] (Condensation reaction)

[0715] 10.2 g (19.6 mmol) of Cbz-MeAsp(tBu)-OH dicyclohexylamine salt was suspended in 100 mL of ethyl acetate, and 20.6 mL (118 mmol) of diisopropylethylamine and 9.7 mL (98.0 mmol) of piperidine were added. 35.0 mL (58.9 mmol) of a 50% T3P / ethyl acetate solution was added dropwise at 3-10°C over 45 minutes. After the addition, 5 μL of the reaction solution was diluted with 1.0 mL of methanol and analyzed by LCMS. The reaction conversion rate was determined from the peak area of ​​the LC / MS results (conversion rate: 100%).

[0716] Conversion rate (%) = {Cbz-MeAsp(tBu)-piperidine (area %) / [Cbz-MeAsp(tBu)-OH (area %) + Cbz-MeAsp(tBu)-piperidine (area %)]} × 100

[0717] The reaction solution was washed with 100 mL of 10% potassium hydrogen sulfate aqueous solution and 100 mL of 10% potassium carbonate, and the obtained organic layer was dried over magnesium sulfate, filtered, and concentrated. MS (ESI) m / z 349.1 [M-tBu+H] + , 405.2[M+H] + ,427.3[M+Na] + .

[0718] (Cbz deprotection reaction)

[0719] The concentrated solution was dissolved in 100 mL of cyclopentyl methyl ether and subjected to hydrogenolysis using 2.0 g of 5% Pd / C (50% wet) and hydrogen gas. Stirring at room temperature for 5 hours yielded the target product, MeAsp(tBu)-piperidine (conversion rate 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and analyzing it by LCMS. The reaction conversion rate was determined from the peak area value obtained by LC / MS.

[0720] Conversion rate (%) = {MeAsp(tBu)-piperidine (area %) / [Cbz-MeAsp(tBu)-piperidine (area %) + MeAsp(tBu)-piperidine (area %)]} × 100

[0721] The reaction mixture was filtered and the filtrate was concentrated to obtain 5.69 g of concentrate (quantitative yield). LC / MS analysis of this concentrate revealed the peak area percentage of the target MeAsp(tBu)-piperidine (99.2 area %). MS (ESI): m / z 215.1 [M-tBu+H] + , 271.1[M+H] + .

[0722] Industrial applicability

[0723] The present invention can produce a high-purity peptide compound without column purification by effectively removing the C-terminal active form remaining after the condensation reaction during the production of the peptide compound.

Claims

1. A method for manufacturing a peptide compound, comprising: Step A: a step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent; and Step B: a step of removing the remaining C-terminal active form by contacting it with a tertiary amine, wherein the tertiary amine is represented by the following formula (B) or (C): In the formula, X is N or O, R4 and R5 are each independently a C1-C2 alkyl group or a C2 hydroxyalkyl group, or together with the nitrogen atom to which they are bonded form a 5- to 6-membered non-aromatic heterocycle, provided that when X is O, R5 does not exist, R6 and R7 are each independently H, a C1-C2 alkyl group, or a methoxy group, R8 and R9 are each independently H, a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded form a 5- to 6-membered non-aromatic heterocycle.

2. A method for manufacturing a peptide compound, comprising: Step A: a step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent; and Step B: a step of mixing the above reaction mixture, a tertiary amine, and water or an aqueous solution, and removing the unreacted C-terminal active form by allowing the tertiary amine to act on it, wherein the tertiary amine is represented by the following formula (B) or (C): In the formula, X is N or O, R4 and R5 are each independently a C1-C2 alkyl group or a C2 hydroxyalkyl group, or together with the nitrogen atom to which they are bonded form a 5- to 6-membered non-aromatic heterocycle, provided that when X is O, R5 does not exist, R6 and R7 are each independently H, a C1-C2 alkyl group, or a methoxy group, R8 and R9 are each independently H, a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded form a 5- to 6-membered non-aromatic heterocycle.

3. The method according to claim 1 or 2, wherein, The acid component is a first amino acid whose amino group is protected by a protecting group, or a first peptide whose N-terminal amino group is protected by a protecting group.

4. The method according to claim 1 or 2, wherein The amine component is a second amino acid whose carboxyl group is protected by a protecting group, or a second peptide whose C-terminal carboxyl group is protected by a protecting group.

5. The method according to claim 1 or 2, wherein Step A is carried out in the presence of a condensing agent.

6. The method according to claim 1 or 2, wherein The above tertiary amine has a nucleophilic reactivity with respect to the above C-terminal active form.

7. The method according to claim 1 or 2, wherein The above tertiary amine is an amine with little steric hindrance near the nitrogen.

8. The method according to claim 1 or 2, wherein X is N, R4 and R5 are each independently a C1-C2 alkyl group, and R6 and R7 are H.

9. The method according to claim 1 or 2, wherein R8 and R9 are each independently H or a C1-C2 alkyl group.

10. The method according to claim 1 or 2, wherein The above tertiary amine is NMI or DMAP.

11. The method according to claim 1 or 2, wherein The above peptide compound contains one or more non-natural amino acids.

12. The method according to claim 1 or 2, wherein The temperature when allowing the above tertiary amine to act on the above C-terminal active form is 25°C to 60°C.

13. The method according to claim 1 or 2, wherein 0.5 equivalent or more of the above tertiary amine is added relative to the above amine component.

14. The method according to claim 1 or 2, wherein The residual rate of the C-terminal active form is 3% or less.

15. The method according to claim 1 or 2, wherein In Step B, it further includes a step of separating the above reaction mixture into an organic layer and an aqueous layer, and then washing the organic layer, and the residual amount of the C-terminal active form after this washing is 1.0% or less.

16. The method according to claim 1 or 2, wherein The solvent in the above-mentioned process A is toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl acetate, ethyl acetate, methyl tert-butyl ether, cyclopentyl methyl ether, or N,N-dimethylformamide, or a mixed solvent thereof.

17. The method according to claim 2, wherein In the above-mentioned process B, the above-mentioned aqueous solution is an aqueous alkali solution.

18. The method according to claim 3, wherein The side chain of the first amino acid contains one or more carbon atoms.

19. The method according to claim 18, wherein, The above-mentioned side chain is an alkyl group that can be substituted, an alkenyl group that can be substituted, an alkynyl group that can be substituted, a cycloalkyl group that can be substituted, an alkoxyalkyl group that can be substituted, a cycloalkylalkyl group that can be substituted, an aralkyl group that can be substituted, or a heteroarylalkyl group that can be substituted.

20. The method according to any one of claims 1, 2, and 19, wherein The time when the above-mentioned tertiary amine acts on the above-mentioned C-terminal active body is 2 hours or less.

21. The method according to any one of claims 1, 2, and 19, wherein, The time when the above-mentioned tertiary amine acts on the above-mentioned C-terminal active body is 2 minutes to 2 hours.

22. The method according to any one of claims 1, 2, and 19, wherein The time when the above-mentioned tertiary amine acts on the above-mentioned C-terminal active body is 5 minutes to 60 minutes.

23. The method according to any one of claims 1, 2, and 19, wherein, The time when the above-mentioned tertiary amine acts on the above-mentioned C-terminal active body is 5 minutes to 50 minutes.

24. The method according to any one of claims 1, 2, and 19, wherein The above-mentioned C-terminal active body is formed in the presence of a condensing agent, and the condensing agent includes a combination of T3P, HATU, BEP, DMT-MM, EDC and PfpOH, a combination of EDC and HOOBt, or a combination of EDC and HOBt.

25. The method according to any one of claims 1, 2, and 19, further comprising a process C: a process of deprotecting the protecting group at the N-terminal of the above-mentioned peptide compound.

26. The method according to any one of claims 1, 2, and 19, wherein, The above-mentioned C-terminal active body is hydrolyzed and removed by acting on the above-mentioned tertiary amine.

27. A method for promoting the hydrolysis of a C-terminal active body, which includes a process of adding a tertiary amine and water or an aqueous solution to a solution containing a residual C-terminal active body and making the C-terminal active body act on the tertiary amine. The above-mentioned tertiary amine is represented by the following formula (B) or (C): In the formula, X is N or O. R4 and R5 are each independently a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom to which they are bonded form a 5- to 6-membered non-aromatic heterocycle, wherein, When X is O, R5 does not exist. R6 and R7 are each independently H, a C1-C2 alkyl group, or a methoxy group. R8 and R9 are each independently H, a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom bonded to R8 and the carbon atom bonded to R9 form a 5- to 6-membered non-aromatic heterocycle.

28. A method for removing a C-terminal active body, which includes a process of hydrolyzing by bringing a residual C-terminal active body into contact with a tertiary amine and performing an aqueous washing on a solution containing a hydrolyzate of the residual C-terminal active body. The above-mentioned tertiary amine is represented by the following formula (B) or (C): In the formula, X is N or O. R4 and R5 are each independently a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom to which they are bonded form a 5- to 6-membered non-aromatic heterocycle, wherein, When X is O, R5 does not exist. R6 and R7 are each independently H, a C1-C2 alkyl group, or a methoxy group. R8 and R9 are each independently H, a C1-C2 alkyl group, or a C2 hydroxyalkyl group, or together with the nitrogen atom bonded to R8 and the carbon atom bonded to R9 form a 5- to 6-membered non-aromatic heterocycle.

Images