Method for synthesizing peptide compound

Abstract

To provide a method for efficiently removing a residual C-terminal activator in the synthesis of a peptide compound.SOLUTION: The present invention provides a method for the synthesis of a peptide compound involving the condensation of a C-terminal activator of an acid component with an amine component, wherein a solution containing the C-terminal activator remaining after the condensation is mixed with a tertiary amine and water or an aqueous solution to promote hydrolysis of the C-terminal activator.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently producing a desired peptide compound by efficiently removing unnecessary C-terminal active compounds generated during the synthesis of the peptide compound. [Background technology]

[0002] In one form of peptide synthesis, a compound in which the C-terminal carboxyl group of an amino acid or peptide is activated is used so that it can react with amines in the amino acid, peptide, etc. to form an amide bond. In this case, if the compound with an activated carboxyl group remains in the reaction solution after completion of the reaction, it can cause a problem of a decrease in the quality of the produced peptide.

[0003] Such compounds with activated C-terminus include not only compounds with activated carboxyl groups used in peptide synthesis reactions, but also compounds that are converted to, for example, azlactones, NCAs (N-carboxyanhydrides), etc. during the reaction, and are in an activated state that allows them to react with amines (hereinafter, these compounds may be referred to as "activated C-terminus.") Furthermore, the activated C-terminus used in peptide synthesis reactions is not limited to, for example, activated esters, mixed acid anhydrides, and acylisourea synthesized using peptide condensing agents as described in Non-Patent Document 1 or Non-Patent Document 2, but includes any compound that is activated so as to be capable of reacting with amines. Examples of known degradation of the quality of the produced peptides include the by-production of impurity peptides due to the residual C-terminal active form, and the contamination of the final product with peptides of inserted sequences as impurities (Patent Documents 1 and 2).

[0004] As a method for solving the problem of such residual C-terminal active compounds, a method is known in which the active ester is hydrolyzed by treatment with alkaline water and then removed as an alkaline aqueous solution of the corresponding amino acid (Patent Document 1). However, this method requires multiple hydrolysis treatments with alkaline water, making the operation complicated. In addition, if the number of treatments with alkaline water increases and the treatment time becomes longer, it is thought that side reactions such as epimerization (isomerization) of the product may occur, which may impair robustness.

[0005] Another known method involves capturing the remaining C-terminal active species with a polyamine having a primary amino group, such as N,N-dimethylpropane-1,3-diamine, converting it to a basic compound, and then removing the amide compound derived from the remaining C-terminal active species by transferring it to the aqueous layer through aqueous washing with an acidic aqueous solution (Patent Document 3, Non-Patent Document 3). However, when highly nucleophilic primary amines are used, it is thought that the primary amine will react with the highly electrophilic site of the target peptide, generating impurities through the formation of covalent bonds, making this method unsuitable for synthesizing highly pure peptides.

[0006] Another known method involves reacting the remaining C-terminal active compound with a scavenger, which is an amine containing a latent anion having a protecting group, to convert it into an amide compound and then remove it (Patent Document 2). However, this method requires a complicated procedure, since it requires an aqueous extraction step after forming the amide compound, hydrogenolysis, and then another aqueous extraction step.

[0007] Furthermore, if the C-terminal active form remains, deprotection of the N-terminal protecting group of the resulting peptide may also occur at the same time. The remaining deprotected C-terminal active form is an impurity, but if its extinction coefficient is small, it is difficult to detect by general-purpose HPLC, making it undesirable from the standpoint of quality control. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5171613 [Patent Document 2] Patent No. 4142907 [Patent Document 3] Patent No. 5212371 [Non-patent literature]

[0009] [Non-Patent Document 1] Chem. Rev., 2011, 111, 6557. [Non-patent document 2] Organic Process Research &Development, 2016, 20, 140. [Non-patent document 3] Tetrahedron Lett., 1974, 15,1785. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made in view of the above circumstances, and in one aspect, an object of the present invention is to efficiently remove the remaining C-terminal active form in the synthesis of a peptide compound. [Means for solving the problem]

[0011] The present inventors have discovered a method for removing residual C-terminal active forms in a reaction mixture in the synthesis of peptide compounds, which involves condensing a C-terminal active form of an acid component with an amine component, by reacting the C-terminal active form with a tertiary amine.

[0012] In one non-limiting specific embodiment, the present invention includes the following. [1] A method for producing a peptide compound, comprising: Step A: obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal activated form of an acid component with an amine component in a solvent; and Step B: mixing the reaction mixture with a tertiary amine and water or an aqueous solution to remove the C-terminal active compound The method comprising: [2] A method for producing a peptide compound, comprising: Step A: obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal activated form of an acid component with an amine component in a solvent; and Step B: Mixing the reaction mixture with a tertiary amine and water or an aqueous solution, and allowing the tertiary amine to react with the unreacted C-terminal active substance to remove the C-terminal active substance. The method comprising: [3] The method according to [1] or [2], wherein the acid component is a first amino acid whose amino group is protected with a protecting group, or a first peptide whose N-terminal amino group is protected with a protecting group. [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 with a protecting group, or a second peptide whose C-terminal carboxyl group is protected with a protecting group. [5] The method according to any one of [1] to [4], wherein step A is carried out in the presence of a condensing agent. [6] The method according to any one of [1] to [5], wherein the tertiary amine has nucleophilic reactivity toward the C-terminal activated form. [7] The method according to any one of [1] to [6], wherein the tertiary amine is an amine having little steric hindrance near the nitrogen. [8] The tertiary amine is represented by the following formula (A), (B), or (C): [ka] During the ceremony, R1 to R3 are (i) R1 and R2 taken together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocycle, and R3 is C1-C2 alkyl or C2 hydroxyalkyl, or (ii) each independently is C1-C2 alkyl or C2 hydroxyalkyl; X is N or O; R4 and R5 are each independently C1-C2 alkyl or C2 hydroxyalkyl, or together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocycle, provided that when X is O, R5 is absent; R6 and R7 are each independently H, C1-C2 alkyl, or methoxy; The method according to any one of [1] to [7], wherein R8 and R9 are each independently H, C1-C2 alkyl, or C2 hydroxyalkyl, or form a 5- to 6-membered non-aromatic heterocycle together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded. [9] The method according to [8], wherein R1 to R3 are each independently C1-C2 alkyl.

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

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

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

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

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

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

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

[13] , wherein the temperature when the tertiary amine is reacted with the C-terminal activated form is 25°C to 60°C.

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

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

[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.

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

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

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

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

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

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

[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.

[21] The method according to

[20] , wherein the side chain is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkylalkyl, optionally substituted aralkyl, or optionally substituted heteroarylalkyl.

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

[21] , wherein the time for reacting the tertiary amine with the C-terminal activated form is 2 hours or less.

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

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

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

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

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

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

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

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

[27] Step C: The method according to any one of [1] to

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

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

[27] , wherein the C-terminal activated form is hydrolyzed by reacting it with the tertiary amine and removed.

[29] A method for promoting the hydrolysis of the C-terminal active substance, comprising the step of adding a tertiary amine and water or an aqueous solution to a solution containing the remaining C-terminal active substance, thereby reacting the C-terminal active substance with the tertiary amine.

[30] A method for removing the hydrolysate of the remaining C-terminal active compound, comprising the step of subjecting a solution containing the hydrolysate to aqueous washing. [Effects of the Invention]

[0013] By using the method of the present invention, the C-terminal active form remaining after the condensation reaction can be easily and efficiently removed in a short time by a single hydrolysis treatment followed by aqueous washing, thereby enabling the synthesis of peptide compounds with high purity without column purification. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the relative remaining amounts of C-terminal active substances. [Figure 2] FIG. 1 shows the relative remaining amounts of C-terminal active substances. [Figure 3] FIG. 1 shows the relative remaining amounts of C-terminal active substances. [Figure 4] FIG. 1 shows the transition of the residual rate of C-terminal active form. DETAILED DESCRIPTION OF THE INVENTION

[0015] Preferred, non-limiting embodiments of the present disclosure are described below.

[0016] All elements described in the following examples are intended to be considered to be equivalently described in this "Form for Carrying Out the Invention" without being bound by any patent practice, custom, laws, regulations, etc. in any country in which the patent application is intended to be granted that may attempt to restrictively interpret the contents described in the examples.

[0017] Any combination of part or all of one or more elements described anywhere in this disclosure is intended to be included in this disclosure, unless there is a technical contradiction based on the common technical knowledge of a person skilled in the art, and is described as being naturally understood by a person skilled in the art.

[0018] (abbreviation) The abbreviations used in this specification are listed below. Amino Acid Abbreviations Aib: α-methylalanine Ala: Alanine Arg: arginine Asn: asparagine Asp: aspartic acid Asp(tBu): 0t-butyl aspartic acid Aze: Azetidine-2-carboxylic acid Cys: cysteine Glu: glutamic acid Gln: glutamine Gly: glycine His: histidine Hph: homophenylalanine Ile: Isoleucine Leu: Leucine Lys: Lysine MeAla: N-methylalanine MeAsp(tBu): N-methyl t-butyl aspartic acid MeGly: N-methylglycine MeIle: N-methylisoleucine MeLeu: N-methylleucine MePhe: N-methylphenylalanine MeVal: N-methylvaline Met: methionine Phe: phenylalanine Phe-OtBu: Ot-butylphenylalanine Phe(3-F): 3-fluorophenylalanine Pro: Proline Ser: Serine Ser(tBu): Ot-butylserine Thr: Threonine Thr(tBu): Ot-butyl-threonine Trp: tryptophan Tyr: Tyrosine Val: Valin

[0019] Reagent / Solvent Abbreviations BEP: 2-bromo-1-ethylpyridinium tetrafluoroborate DABCO: 1,4-diazabicyclo[2.2.2]octane DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene DCM: dichloromethane DIPEA: Diisopropyldiethylamine DMAP: dimethylaminopyridine DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HOAt: 1-azahydroxybenzotriazole HOBt: 1-hydroxybenzotriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HOSu: N-hydroxysuccinimide MTBE: Methyl t-butyl ether NMI: N-methylimidazole NMM: N-methylmorpholine T3P: Propylphosphonic anhydride (cyclic trimer) TBAF: Tetrabutylammonium fluoride TsOH: p-toluenesulfonic acid

[0020] Functional Group Abbreviations Bn: Benzyl Boc: t-butoxycarbonyl Cbz: benzyloxycarbonyl Pfp: pentafluorophenyl Teoc: 2-(trimethylsilyl)ethoxycarbonyl

[0021] (Definition of functional groups, etc.) As used herein, the term "halogen atom" includes, for example, F, Cl, Br, or I.

[0022] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q " means that the number of carbon atoms is p to q), and preferably C1-C 10 Alkyl is preferably C1-C6 alkyl, and more preferably C1-C2 alkyl. Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-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.

[0023] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP 2 Alkenyl is a monovalent group having 2-4 carbon atoms. Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl includes not only straight chains but also branched chains. Alkenyl is preferably C2-C 10 Alkenyl, more preferably C2-C6 alkenyl, is exemplified, and specific examples include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, etc.

[0024] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Alkynyl is preferably C2-C 10 Alkynyl, more preferably C2-C6 alkynyl, is included, and specific examples include 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, and the like.

[0025] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferred examples of cycloalkyl include C3-C8 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl.

[0026] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

[0027] As used herein, the term "heterocyclyl" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl, and may be a single ring or a condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, and isothiazolidinyl. Examples include thiadiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, and 2-oxaspiro[3.3]heptyl.

[0028] As used herein, "heteroaryl" refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a single ring or a condensed ring with other rings, and may be partially saturated. 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 heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0029] As used herein, "alkoxy" refers to an oxy group bonded to an "alkyl" as defined above, and preferably includes C1-C6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0030] As used herein, "alkenyloxy" refers to an oxy group bonded to the above-defined "alkenyl," and preferably includes C2-C6 alkenyloxy. Specific examples of alkenyloxy include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0031] As used herein, "cycloalkoxy" refers to an oxy group bonded to a "cycloalkyl" as defined above, and preferably includes C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0032] As used herein, "aryloxy" refers to an oxy group to which the above-defined "aryl" is bonded, and preferably has a C6-C 10 Specific examples of the aryloxy include phenoxy, 1-naphthyloxy, and 2-naphthyloxy.

[0033] As used herein, "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, heterocyclyl, aryl, or heteroaryl, or R and R' together with the nitrogen atom to which they are attached form a ring. Preferred amino groups include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and 4- to 8-membered cyclic amino.

[0034] As used herein, "monoalkylamino" refers to a group in which R is hydrogen and R' is an "alkyl" as defined above, among the "amino" groups defined above, and preferably includes mono-C1-C6 alkylamino. Specific examples of monoalkylamino include methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, and t-butylamino.

[0035] As used herein, "dialkylamino" refers to a group in which R and R' are independently "alkyl" as defined above, among the "amino" groups defined above, and preferably includes diC1-C6 alkylamino. Specific examples of dialkylamino include dimethylamino and diethylamino.

[0036] As used herein, "cyclic amino" refers to the above-defined "amino" in which R and R' form a ring together with the nitrogen atom to which they are attached, and preferably includes 4- to 8-membered cyclic amino. Specific examples of cyclic amino include 1-azetidyl, 1-pyrrolidyl, 1-piperidyl, 1-piperazyl, 4-morpholinyl, 3-oxazolidyl, 1,1-dioxidethiomorpholinyl-4-yl, and 3-oxa-8-azabicyclo[3.2.1]octan-8-yl.

[0037] As used herein, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been replaced with hydroxyl groups, preferably C1-C6 hydroxyalkyl, and more preferably C2 hydroxyalkyl. Specific examples of hydroxyalkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0038] As used herein, "haloalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been substituted with halogen atoms, preferably C1-C6 haloalkyl, more preferably C1-C6 fluoroalkyl. Specific examples of haloalkyl include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, and 5,5-difluoropentyl.

[0039] As used herein, "cyanoalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been replaced with cyano, and C1-C6 cyanoalkyl is preferred. Specific examples of cyanoalkyl include cyanomethyl and 2-cyanoethyl.

[0040] As used herein, "aminoalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "amino" as defined above, and C1-C6 aminoalkyl is preferred. Specific examples of aminoalkyl include 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, and 4-aminobutyl.

[0041] As used herein, "carboxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are substituted with carboxy, and C2-C6 carboxyalkyl is preferred. Specific examples of carboxyalkyl include carboxymethyl.

[0042] As used herein, "alkenyloxycarbonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkenyloxycarbonyl" as defined above, with C2-C6 alkenyloxycarbonylC1-C6 alkyl being preferred, and C2-C6 alkenyloxycarbonylC1-C2 alkyl being more preferred. Specific examples of alkenyloxycarbonylalkyl include allyloxycarbonylmethyl and 2-(allyloxycarbonyl)ethyl.

[0043] As used herein, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkoxy" as defined above, with C1-C6 alkoxyC1-C6 alkyl being preferred, and C1-C6 alkoxyC1-C2 alkyl being more preferred. Specific examples of alkoxyalkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0044] As used herein, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with a "cycloalkyl" as defined above, with C3-C8 cycloalkylC1-C6 alkyl being preferred, and C3-C6 cycloalkylC1-C2 alkyl being more preferred. Specific examples of cycloalkylalkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0045] As used herein, "cycloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with a "cycloalkoxy" as defined above, with C3-C8 cycloalkoxyC1-C6 alkyl being preferred, and C3-C6 cycloalkoxyC1-C2 alkyl being more preferred. Specific examples of cycloalkoxyalkyl include cyclopropoxymethyl and cyclobutoxymethyl.

[0046] As used herein, "heterocyclylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with a "heterocyclyl" as defined above, and is preferably a 4- to 7-membered heterocyclylC1-C6 alkyl, more preferably a 4- to 7-membered heterocyclylC1-C2 alkyl. Specific examples of heterocyclylalkyl include 2-(tetrahydro-2H-pyran-4-yl)ethyl and 2-(azetidin-3-yl)ethyl.

[0047] As used herein, "alkylsulfonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkylsulfonyl" as defined above, with C1-C6 alkylsulfonylC1-C6 alkyl being preferred, and C1-C6 alkylsulfonylC1-C2 alkyl being more preferred. Specific examples of alkylsulfonylalkyl include methylsulfonylmethyl and 2-(methylsulfonyl)ethyl.

[0048] As used herein, "aminocarbonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "aminocarbonyl" as defined above, preferably an aminocarbonyl C1-C6 alkyl, and more preferably an aminocarbonyl C1-C4 alkyl. Specific examples of aminocarbonylalkyl include methylaminocarbonylmethyl, dimethylaminocarbonylmethyl, t-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.

[0049] As used herein, "aryloxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" defined above are substituted with an "aryloxy" defined above, and is a C6-C 10 Aryloxy C1-C6 alkyl is preferred, C6-C 10 Aryloxy C1-C2 alkyl is more preferred. Specific examples of aryloxy alkyl include phenoxymethyl and 2-phenoxyethyl.

[0050] As used herein, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is substituted with an "aryl" as defined above, and is a C7-C 14 Aralkyl is preferred, C7-C 10 Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0051] As used herein, the term "heteroarylalkyl" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is substituted with a "heteroaryl" as defined above, preferably a 5- to 10-membered heteroaryl C1-C6 alkyl, and more preferably a 5- to 10-membered heteroaryl C1-C2 alkyl. Specific examples of heteroarylalkyl 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.

[0052] As used herein, the term "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 a double and / or triple bond within the ring, and a carbon atom within the ring may be oxidized to form a carbonyl. The non-aromatic heterocycle may be a monocycle, a fused ring, or a spirocycle. The number of atoms constituting the ring is not limited, but is preferably 5 to 6 (5- or 6-membered non-aromatic heterocycle). Specific examples of non-aromatic heterocycles include azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, piperidine, tetrahydropyran, thiane, piperazine, morpholine, thiomorpholine, dioxane, dithiane, azepane, oxepane, thiepane, and diazepane.

[0053] As used herein, the term "peptide chain" refers to a peptide chain in which one, two, three, four, or more natural amino acids and / or unnatural amino acids are linked by amide bonds and / or ester bonds.

[0054] As used herein, the term "optionally substituted" means that a group may be substituted with any substituent.

[0055] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0056] As used herein, the term "C-terminal activated form" refers not only to compounds with activated carboxyl groups used in peptide synthesis reactions (e.g., activated esters that lead to the production of target peptide compounds), but also to compounds that are activated during the reaction, e.g., converted to azlactones, NCAs (N-carboxyanhydrides), etc., and can react with amines (e.g., amine components) to give target peptide compounds. Furthermore, C-terminal activated forms, which are compounds with activated carboxyl groups used in peptide synthesis reactions, are not limited to, for example, activated esters, mixed acid anhydrides, and acylisourea synthesized using peptide condensing agents as described in Chem. Rev., 2011, 111, 6557, or Organic Process Research & Development, 2016, 20 (2), 140, but also include any compound that is activated to be reactive with amines.

[0057] As used herein, the term "activated 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, or the like is bound to the carbonyl group, and the reaction with an amine is promoted.

[0058] As used herein, the term "amino acid" includes natural and unnatural amino acids. As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Examples of unnatural amino acids include, but are not limited to, β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with unnatural side chains, and hydroxycarboxylic acids. As used herein, amino acids may have any configuration. The side chain of an amino acid is not particularly limited and may be freely selected from, in addition to a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group. In these groups, one or two non-adjacent methylene groups may be substituted with an oxygen atom, a carbonyl group (-CO-), a sulfonyl group (-SO-), a phosphoryl group, or a phosphonyl group. Each of these may have a substituent, and the substituents are not limited and may be independently selected from any substituents including, for example, a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. Examples of such substituents include optionally substituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxy group and an amino group in the same molecule.

[0059] The main chain amino group of an amino acid may be unsubstituted (NH group) or substituted (i.e., an -NHR group: R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, 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-), or the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline. An amino acid in which such a main chain amino group is substituted is referred to herein as an "N-substituted amino acid." Preferred examples of the "N-substituted amino acid" herein include, but are not limited to, N-alkylamino acid, N-C1-C6 alkylamino acid, N-C1-C4 alkylamino acid, and N-methylamino acid.

[0060] The "amino acids" constituting the peptide compounds herein include all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (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, and chlorine atoms, each of which is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Includes Cl etc.

[0061] In this specification, examples of the substituent containing a halogen atom include alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and the like, each of which has a halogen atom as a substituent, and more specific examples thereof include fluoroalkyl, difluoroalkyl, trifluoroalkyl, and the like.

[0062] Examples of the substituent containing an O atom include hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxy (-COH), oxycarbonyl (-C(=O)-OR), carbonyloxy (-OC(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio (-SC(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO-R), aminosulfonyl (-SO-NHR), sulfamoylamino (-NH-SO-NHR), thiocarboxyl (-C(=O)-SH), and carboxylcarbonyl (-C(=O)-COH).

[0063] Examples of oxy (—OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among these, methoxy or ethoxy is preferred.

[0064] Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0065] Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0066] Examples of carbonyloxy (-OC(=O)-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0067] Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0068] Examples of carbonylthio (-SC(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0069] Examples of aminocarbonyl (-C(=O)-NHR) include alkylaminocarbonyl (e.g., C1-C6 or C1-C4 alkylaminocarbonyl, particularly ethylaminocarbonyl, methylaminocarbonyl, etc.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in -C(=O)-NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0070] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0071] Examples of oxycarbonylamino (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0072] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0073] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0074] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0075] Examples of substituents containing an S atom include thiol (-SH), thio (-SR), sulfinyl (-S(=O)-R), sulfonyl (-SO2-R), and sulfo (-SO3H).

[0076] Examples of thio (-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0077] Examples of sulfonyl (-SO2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0078] Examples of substituents containing an N atom include azide (-N3, also referred to as an "azido group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also referred to as monosubstituted amino), tertiary amino (-NR(R'; also referred to as disubstituted amino), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridyl, piperidino, morpholino, and azetidinyl.

[0079] Examples of secondary amino (-NH-R; monosubstituted amino) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0080] Examples of tertiary amino (-NR(R'); disubstituted amino) include alkyl(aralkyl)amino and other amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these two substituents may form a ring. Specific examples include dialkylamino, particularly C1-C6 dialkylamino, C1-C4 dialkylamino, dimethylamino, diethylamino, etc. In the present specification, "C p -C q "Dialkylamino group" means an amino group with C p -C q A group substituted with two alkyl groups, both C p -C q The alkyl groups may be the same or different.

[0081] Examples of substituted amidino (-C(=NR)-NR'R") include groups in which the three substituents R, R', and R" on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0082] Examples of substituted guanidino (-NR-C(=NR''')-NR'R") include groups in which R, R', R", and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0083] Examples of aminocarbonylamino (-NR-CO-NR'R") include groups in which R, R', and R" are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0084] In this specification, the "amino acid residues" that constitute a peptide compound may be simply referred to as "amino acids".

[0085] (Method for producing peptide compounds) In one aspect, the present invention relates to a method for producing a peptide compound, the method comprising the steps of: Step A: obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal activated form of an acid component with an amine component in a solvent; and 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.

[0086] In step A, an acid component and an amine component are reacted in a solvent using a condensing agent to obtain a reaction mixture containing a peptide compound. Without being bound by any particular theory, in step A, the acid component reacts with the condensing agent to form a C-terminal activated form of the acid component, and then the amine component undergoes nucleophilic attack on the C-terminal activated form, thereby proceeding with the reaction to produce a peptide compound.

[0087] The acid component can be an amino acid whose amino group is protected with a protecting group, or a peptide whose N-terminal amino group is protected with a protecting group. In this specification, an amino acid used as the acid component may be referred to as a "first amino acid," and a peptide used as the acid component may be referred to as a "first peptide."

[0088] The first amino acid is not particularly limited and can be any natural or unnatural amino acid. The first peptide is also not particularly limited and can be any combination of two or more natural and / or unnatural amino acids.

[0089] The first amino acid is preferably one containing one or more carbon atoms in its side chain. Specific examples of such amino acids include those having an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted cycloalkyl, an optionally substituted alkoxyalkyl, an optionally substituted cycloalkylalkyl, an optionally substituted aralkyl, an optionally substituted heteroarylalkyl, or the like, in the side chain. Furthermore, if the side chain contains functional groups such as an amino group, a carboxyl group, or a hydroxyl group that may affect the peptide bond formation reaction, it is preferable to protect these groups with an appropriate protecting group. Without being bound by any particular theory, when an amino acid has a bulky group in its side chain, the steric hindrance may prevent the hydrolysis of the remaining C-terminal active form of the amino acid from proceeding sufficiently using conventional methods. Even in such cases, the method of the present invention can rapidly and efficiently hydrolyze the remaining C-terminal active form.

[0090] The side chain of the C-terminal amino acid contained in the first peptide may also have the same side chain as that of the first amino acid.

[0091] The amino-protecting group of the first amino acid and the N-terminal amino-protecting group of the first peptide can be any amino-protecting group commonly used in the art, including, for example, Cbz, Boc, Teoc, Fmoc, Tfa, Alloc, nosyl, dinitronosyl, t-Bu, trityl, and cumyl.

[0092] In one embodiment, the acid component is preferably used in at least the same equivalent amount as the amine component, preferably in an excess amount 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 relative to the amine component can be used.

[0093] In one embodiment, the C-terminal activated form of the acid component of 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 leaving group into the hydroxy moiety of the carboxyl group of the acid component to increase the electrophilicity of the carbonyl carbon of the acid component. Specific examples of the condensing agent include T3P, HATU, BEP, carbodiimides (DIC, EDC, etc.), combinations of carbodiimides and additives (oxyma, HOOBt, HOBt, etc.), DMT-MM, and CDI.

[0094] The step (Step A) of obtaining a peptide compound by condensing the C-terminal activated moiety with an amine component can be carried out by stirring the reaction mixture at a temperature of from -20°C to near the boiling point of the solvent, preferably from 0°C to 60°C, for 1 minute to 48 hours, preferably from 15 minutes to 4 hours.

[0095] In step A, the condensation reaction between the acid component and the amine component can proceed quantitatively.

[0096] The amine component can be an amino acid whose carboxyl group is protected with a protecting group, or a peptide whose C-terminal carboxyl group is protected with a protecting group. In this specification, an amino acid used as an amine component may be referred to as a "second amino acid," and a peptide used as an amine component may be referred to as a "second peptide."

[0097] The second amino acid is not particularly limited and can be any natural or unnatural amino acid. The second peptide is also not particularly limited and can be any combination of two or more natural and / or unnatural amino acids.

[0098] The protecting group for the carboxyl group of the second amino acid and the protecting group for the C-terminus of the second peptide can be any protecting group for a carboxyl group commonly used in the art, including, for example, methyl, allyl, t-butyl, trityl, cumyl, benzyl, methoxytrityl, 1-piperidinyl, etc.

[0099] In one embodiment, any solvent can be used in the present invention as long as it allows the condensation reaction to proceed and a peptide compound can be obtained. 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 solvents selected from these.

[0100] In one embodiment, the "peptide compound" of the present invention obtained by condensing the C-terminal activated form of an acid component with an amine component includes a linear or cyclic peptide compound in which two or more amino acids are linked. Note that a cyclic peptide compound is synonymous with a "peptide compound having a cyclic portion."

[0101] The "linear peptide compound" of the present invention is formed by linking natural amino acids and / or unnatural amino acids via amide bonds or ester bonds, and is not particularly limited as long as it is a compound that does not have a cyclic moiety. The total number of natural or unnatural amino acids constituting the linear peptide compound can 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 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, and 9 to 13.

[0102] The "cyclic peptide compound" of the present invention is formed by linking natural amino acids and / or unnatural amino acids via amide bonds or ester bonds, and is not particularly limited as long as it is a compound having a cyclic portion. The cyclic peptide compound may have one or more linear portions. The total number of natural or unnatural amino acids constituting the cyclic peptide compound can 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 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, and 9 to 13.

[0103] The number of amino acids constituting the cyclic portion of the cyclic peptide compound is not limited, and examples 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, even more preferably 8 to 13, 9 to 13, 8 to 12, 8 to 11, or 9 to 12, and particularly preferably 9 to 11.

[0104] 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.

[0105] A peptide compound can contain one or more, two or more, three or more, four or more, five or more, or six or more unnatural amino acids. Furthermore, a peptide compound can 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 a peptide compound contains unnatural amino acids, the proportion of the number of 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.

[0106] The peptide compound may be a linear or cyclic peptide that contains at least two N-substituted amino acids (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, with preferred ranges being 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), in addition to or solely within the above-mentioned total number of natural and unnatural amino acids, and at least one non-N-substituted amino acid. Examples of "N-substitution" include, but are not limited to, substitution of a hydrogen atom bonded to an N atom with a methyl group, ethyl group, propyl group, butyl group, or hexyl group. N-substituted amino acids preferably include amino acids in which the amino group contained in a natural amino acid has been N-methylated, N-ethylated, N-propylated, N-butylated, or N-pentylated, and are referred to as N-methylamino acids, N-ethylamino acids, N-propylamino acids, N-butylamino acids, and N-pentylamino acids. Converting an N-unsubstituted amino acid into an N-substituted amino acid is referred to as N-substitution, and is sometimes 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 is, 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.

[0107] The peptide compound may include a salt thereof or a solvate thereof.

[0108] As used herein, the term "side chain" is used in the context of the side chain of an amino acid or the side chain of the cyclic portion of a cyclic peptide compound, and refers to a portion that is not included in the main chain structure of each.

[0109] As used herein, the term "number of amino acids" refers to the number of amino acid residues constituting a peptide compound, and means the number of amino acid units generated upon cleavage of the amide bond, ester bond, and bond at the cyclization site that link the amino acids.

[0110] Step B is a step of removing unreacted C-terminal active form contained in the reaction mixture obtained in Step A. In one embodiment, removal of unreacted C-terminal active form is achieved by reacting the unreacted C-terminal active form with a tertiary amine. Herein, unreacted C-terminal active form, specifically, for example, C-terminal active form remaining in the reaction mixture without reacting with the amine component during the condensation process, may be referred to as "residual C-terminal active form." In Step B, the reaction mixture obtained in Step A is mixed with a tertiary amine and water or an aqueous solution. If an excess amount of acid component relative to the amine component is used in Step A, or if the condensation reaction does not proceed sufficiently in Step A, the C-terminal active form of the acid component remaining without reacting with the amine component will remain as an impurity in the reaction solvent. If this remaining C-terminal active form remains in the system without being sufficiently decomposed, it will adversely affect the subsequent deprotection step of the peptide compound and further peptide chain elongation reactions, and therefore it is important to ensure its removal. In conventional liquid phase synthesis, methods such as hydrolyzing the remaining activated ester using an alkaline aqueous solution have been known, but the present inventors have confirmed that decomposition may be insufficient, particularly in the case of the remaining C-terminal activated form of an amino acid having a bulky functional group in its side chain, or when the leaving ability of the leaving group of the remaining C-terminal activated form is not high enough to easily react with water.In contrast, these problems can be solved by using the method of the present invention, which hydrolyzes the C-terminal activated form by mixing a reaction mixture containing unreacted C-terminal activated form with a tertiary amine and water or an aqueous solution, or by contacting the remaining C-terminal activated form with a tertiary amine.

[0111] As the tertiary amine, one having nucleophilic reactivity with the remaining C-terminal activated form of the acid component can be preferably used. As such a tertiary amine, an amine with little steric hindrance near the nitrogen is preferred. Examples of such a tertiary amine include tertiary amines represented by the following formula (A), (B), or (C). [ka]

[0112] In one embodiment, in formula (A), R1 to R3, together with the nitrogen atom to which they are attached, form a 5- to 6-membered non-aromatic heterocycle, and R3 is C1-C2 alkyl (i.e., methyl or ethyl) or C2 hydroxyalkyl. The 5- to 6-membered non-aromatic heterocycle is preferably pyrrolidine, piperidine, or morpholine, and the C2 hydroxyalkyl is preferably 2-hydroxyethyl.

[0113] In another embodiment, in formula (A), R1 to R3 are each independently C1-C2 alkyl or C2 hydroxyalkyl. C2 hydroxyalkyl is preferably 2-hydroxyethyl.

[0114] The tertiary amine represented by formula (A) is preferably one in which R1 to R3 are each independently C1-C2 alkyl.

[0115] Specific examples of the tertiary amine represented by formula (A) include trimethylamine, N,N-dimethylethylamine, N,N-diethylmethylamine, triethylamine, and triethanolamine, with trimethylamine being particularly preferred.

[0116] In one embodiment, in formula (B), X is N or O. When X is N, R4 and R5 are each independently C1-C2 alkyl or C2 hydroxyalkyl, or together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocycle. When X is O, R4 is C1-C2 alkyl or C2 hydroxyalkyl, and R5 is absent. The 5- to 6-membered non-aromatic heterocycle is preferably pyrrolidine, piperidine, or morpholine, and the C2 hydroxyalkyl is preferably 2-hydroxyethyl. Furthermore, in formula (B), R6 and R7 are each independently H, C1-C2 alkyl, or methoxy.

[0117] Preferred tertiary amines represented by formula (B) include those in which X is N, R4 and R5 are each independently C1-C2 alkyl, and R6 and R7 are H.

[0118] Specific examples of the tertiary amine represented by formula (B) include DMAP, 4-piperidinopyridine, and 4-morpholinopyridine, and among these, DMAP is particularly preferred.

[0119] In one embodiment, in formula (C), R8 and R9 are each independently H, C1-C2 alkyl, or C2 hydroxyalkyl, or are joined together with the nitrogen atom to which R8 is bonded and the carbon atom to which R9 is bonded to form a 5- to 6-membered non-aromatic heterocycle. The 5- to 6-membered non-aromatic heterocycle is preferably pyrrolidine, piperidine, or morpholine, and the C2 hydroxyalkyl is preferably 2-hydroxyethyl.

[0120] The tertiary amine represented by formula (C) is preferably one in which R8 and R9 are each independently H or C1-C2 alkyl, and more preferably one in which R8 is C1-C2 alkyl and R9 is H.

[0121] 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, and among these, NMI is particularly preferred.

[0122] Without being bound by any particular theory, the tertiary amines of the present invention can promote the hydrolysis of the remaining C-terminal active substance by nucleophilic attack on the remaining C-terminal active substance. Tertiary amines such as DIPEA have bulky substituents, making them less nucleophilic and undesirable. The hydrolyzate of the remaining C-terminal active substance can be removed by transferring it to the aqueous phase, allowing the resulting peptide compound to be subjected to the subsequent condensation reaction without a separate purification step such as column purification. Using the method of the present invention, the remaining C-terminal active substance can be efficiently removed quickly (e.g., within 5 minutes) and with a small number of hydrolysis treatments (e.g., only one). In some embodiments, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the remaining C-terminal active substance can be removed. In other words, according to the present invention, the residual rate of C-terminal active substance 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.

[0123] The tertiary amine may be used in a catalytic amount relative to the amine component, or in an amount equal to or greater than the stoichiometric amount. Specifically, for example, 0.1 to 10 equivalents of the tertiary amine relative to the amine component can be added to the reaction mixture, and it is preferable to add 0.5 to 3 equivalents of the tertiary amine.

[0124] When reacting the tertiary amine with the remaining C-terminal activated form, the reaction mixture can be stirred at a temperature of from -20°C to a temperature near the boiling point of the solvent, preferably from 25°C to 60°C, for 1 minute to 48 hours, preferably 2 hours or less, for example, 2 minutes to 2 hours, 5 minutes to 60 minutes, 5 minutes to 50 minutes, or 5 to 30 minutes.

[0125] In one embodiment, water or an aqueous solution can be added to the step of treating the remaining C-terminal active form with a tertiary amine, and an alkaline aqueous solution can be preferably used as the aqueous solution. While such an alkaline aqueous solution is not particularly limited, specific examples include an aqueous potassium carbonate solution, an aqueous lithium hydroxide solution, an aqueous sodium carbonate solution, sodium hydroxide, an aqueous potassium hydroxide solution, an aqueous sodium hydroxide solution, and an aqueous cesium carbonate solution. Among these, an aqueous potassium carbonate solution or an aqueous sodium carbonate solution, which have mild basicity, are preferred.

[0126] In one embodiment, the present invention further comprises the step of separating the reaction mixture into an organic layer and an aqueous layer after reacting the tertiary amine with the remaining C-terminal active substance, separating the organic layer, and then washing the organic layer. For example, this step includes washing the organic layer with an acidic aqueous solution and a basic aqueous solution. In one embodiment, after this step, the amount of remaining C-terminal active substance can be reduced to 1.0% or less, 0.5% or less, and preferably 0.1% or less.

[0127] In one embodiment, the present invention further comprises a step (Step C) of deprotecting the protecting group at the N-terminus of the peptide compound. Deprotection of the protecting group can be carried out, for example, by the standard method described in "Greene's, 'Protective Groups in Organic Synthesis' (5th ed., John Wiley & Sons 2014)." In conventional methods, the deprotection reaction may not proceed sufficiently due to residual C-terminal active species. However, by using the method of the present invention, the deprotected form of the resulting peptide compound can be obtained in high yield.

[0128] In one embodiment, the present invention includes repeating step A and step B multiple times. Also, in another embodiment, the present invention includes repeating step A, step B, and step C multiple times. By repeating in this manner, the peptide chain can be elongated to obtain a peptide compound.

[0129] In one embodiment, the present invention relates to a method for promoting the hydrolysis of a residual C-terminal active substance, comprising the step of adding a tertiary amine and water or an aqueous solution to a solution containing the residual C-terminal active substance to react with the tertiary amine. In this embodiment, the residual C-terminal active substance and / or the tertiary amine may be those described above. When an aqueous solution is added to a solution containing the residual C-terminal active substance, the aqueous solution is preferably alkaline water.

[0130] In one aspect, the present invention relates to a method for removing a hydrolysate of a residual C-terminal active form, comprising a step of subjecting a solution containing the hydrolysate to aqueous washing. In this aspect, in addition to water, washing with an alkaline aqueous solution can be performed as the aqueous washing. The alkaline aqueous solution is not particularly limited, but a potassium carbonate aqueous solution or a sodium carbonate aqueous solution is preferred. In another aspect, when the base used forms a salt with the hydrolysate and makes it difficult to migrate to the aqueous layer, the base can be removed by washing with an acidic aqueous solution, and then washed with an alkaline aqueous solution. The acidic aqueous solution is not particularly limited, but a potassium hydrogen sulfate aqueous solution or a sodium hydrogen sulfate aqueous solution is preferred. The alkaline aqueous solution is preferably a potassium carbonate aqueous solution or a sodium carbonate aqueous solution.

[0131] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0132] The present invention is further illustrated by, but not limited to, the following examples.

[0133] The purity of the peptide compound (the target product in peptide synthesis) and the amount of remaining C-terminal active compound were measured using an LCMS equipped with a QDA and PDA detector (column: Ascentis Express C18, 5 cm x 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). The remaining amount of C-terminal active substance was evaluated by converting the remaining C-terminal active substance into propylamide, since there was a possibility that the remaining C-terminal active substance would be hydrolyzed under analytical conditions (LCMS). The purity of the peptide compound (the target product in peptide synthesis) was reported as a peak area percentage by LCMS. The C-terminal active form remaining rate and the relative amount of C-terminal active form remaining were calculated using the formulas described in each example. The total peak area was corrected by subtracting the areas of the blank peak and solvent peak. In the table, nd means not detected.

[0134] (Example 1) Effect of added amine on hydrolysis of remaining C-terminal active substance (Preparation of mixed acid anhydride) Cbz-Ile-OH (463 mg, 1.7 mmol) and pentamethylbenzene (31 mg, internal standard: 0.21 mmol) were dissolved in 3.0 mL of 2-methyltetrahydrofuran. Diisopropylethylamine (1.1 mL, 6.2 mmol) and 50% T3P / THF (1.9 mL, 3.2 mmol) were added at room temperature and stirred at 40 °C for 1 hour to prepare a mixed acid anhydride (C-terminal activated form). A 5 μL aliquot of the mixed acid anhydride solution was reacted with n-propylamine (100 μL, 1.2 mmol) and then diluted with methanol (0.9 mL). The conversion to the mixed acid anhydride was determined by LC / MS peak area (97%). Cbz-Ile-NHPr / MS (ESI): m / z 307.1 [M+H]+. Conversion rate (%) = {Cbz-Ile-NHPr (area%) / [Cbz-Ile-OH (area%) + Cbz-Ile-NHPr (area%)]} × 100

[0135] (Hydrolysis treatment - no amine added) A 1.0 mL aliquot of the total mixed acid anhydride solution (6 mL) was added to 0.5 mL of alkaline water (5% lithium hydroxide, 5% sodium carbonate, 5% potassium carbonate, 5% potassium hydroxide, or 5% cesium carbonate) and stirred at 25 °C with a stirrer (1200 rpm). After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active moiety to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0136] (hydrolysis treatment - amine addition) A 1.0 mL aliquot of the total mixed acid anhydride solution (6 mL) was added to an amine additive (0.19 mmol) and 0.5 mL of 5% aqueous potassium carbonate solution, and the mixture was stirred at 25 °C with a stir bar at 1200 rpm. The stirring was stopped, the mixture was allowed to stand, and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active moiety to propylamide, and the mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0137] (Evaluation of remaining amount of C-terminal active substance) The LC / MS peak area ratio [propylamide / pentamethylbenzene (internal standard)] was used. The relative values ​​of the remaining amount of C-terminal active substance in the table below are relative values ​​when the peak area ratio [propylamide / pentamethylbenzene] of 3.5 obtained when treated with 5% potassium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 100 (5-minute column for entry 1). Relative value of remaining amount of C-terminal active substance (%) = {[propylamide (area%) / pentamethylbenzene (area%)] / 3.5 (entry 1, 5 min [propylamide (area%) / pentamethylbenzene (area%)])} x 100

[0138] [Table 1] 1) Not applicable.

[0139] The relative values ​​of the remaining C-terminal active species in Table 1 indicate that the smaller the value, the more hydrolysis of the remaining C-terminal active species. When alkaline water alone was used, changing the counter cation of the alkali hardly affected the hydrolysis rate, and hydrolysis was slower than when an amine was added. Furthermore, we found that the addition of DMAP and NMI, among the amines added, dramatically accelerated the hydrolysis of the remaining C-terminal active species.

[0140] (Example 2) Effect of added amine on hydrolysis of remaining C-terminal active substance (Preparation of active ester) 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 stirred at 60 °C for 4 hours to prepare an activated ester (C-terminal activated form). 5 μL of the activated ester solution was reacted with 100 μL (1.2 mmol) of n-propylamine and then diluted with 0.9 mL of methanol. The conversion rate to the activated ester was determined from the peak area of ​​LC / MS (94%). Cbz-Ile-NHPr / MS (ESI): m / z 307.1 [M+H]+. Conversion rate (%) = {Cbz-Ile-NHPr(area%) / [Cbz-Ile-OH(area%)+Cbz-Ile-NHPr(area%)]}×100

[0141] (Hydrolysis treatment using alkaline water alone) A 1.5 mL aliquot of the total volume (9 mL) of the prepared activated ester solution was added to 0.75 mL of alkaline water (5% aqueous potassium carbonate solution) and stirred at 25 °C with a stirrer (1200 rpm). After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal activated moiety to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0142] (Amine-added hydrolysis treatment) A 1.5 mL aliquot of the total activated ester solution (9 mL) was added to an amine additive (0.44 mmol) and 0.75 mL of 5% aqueous potassium carbonate solution, followed by stirring at 25 °C with a stir bar at 1200 rpm. The stirring was stopped, the mixture was allowed to stand, and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal activated moiety to propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0143] (Evaluation of remaining amount of C-terminal active substance) The LC / MS peak area ratio [propylamide / pentamethylbenzene (internal standard)] was used. The relative values ​​of the remaining amount of C-terminal active substance in the table below are relative values ​​when the peak area ratio [propylamide / pentamethylbenzene] value of 3.0 obtained when treated with 5% potassium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 100 (5-minute column for entry 1). Relative value of remaining amount of C-terminal active substance (%) = {[propylamide (area%) / pentamethylbenzene (area%)] / 3.0 (entry 1, [propylamide (area%) / pentamethylbenzene (area%)] at 5 min)} x 100

[0144] [Table 2]

[0145] The relative values ​​for the remaining C-terminal active substance in Table 2 indicate that the smaller the value, the more hydrolysis of the remaining C-terminal active substance has been achieved. We found that the addition of amines promotes the hydrolysis of the remaining C-terminal active substance more than the use of alkaline water alone. Specifically, we found that the addition of DBU, Me3N, NMI, and DMAP was effective, with the addition of NMI and DMAP being particularly dramatic.

[0146] (Example 3) Effect of added amine on hydrolysis of remaining C-terminal active substance A solution of 617 mg (2.6 mmol) of Cbz-MeAla-OH and 46 mg (0.31 mmol) of pentamethylbenzene in 4.5 mL of 2-methyltetrahydrofuran was added to 1.5 mL (8.6 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution at room temperature and stirred at 40 °C for 1 hour to prepare a mixed acid anhydride solution (C-terminal activated form). A 5 μL aliquot of the mixed acid anhydride solution was reacted with 100 μL (1.2 mmol) of n-propylamine and then diluted with 0.9 mL of methanol. The conversion to the mixed acid anhydride was determined from the peak area of ​​LC / MS (90% conversion). Cbz-MeAla-NHPr / MS (ESI): m / z 279.1 [M+H]+. Conversion rate (%) = {Cbz-MeAla-NHPr(area%) / [Cbz-MeAla-OH(area%)+Cbz-MeAla-NHPr(area%)]} × 100

[0147] (Hydrolysis treatment using alkaline water alone) A 1.5 mL aliquot of the total mixed acid anhydride solution (9 mL) was added to 0.75 mL of alkaline water (5% aqueous sodium carbonate or 5% aqueous potassium carbonate), and the mixture was stirred at 25 °C with a stirrer at 1200 rpm. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active moiety to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0148] (Amine-added hydrolysis treatment) A 1.5 mL aliquot of the total mixed anhydride solution (9 mL) was added to an amine additive (0.43 mmol, 0.67 equivalents) and 0.75 mL of 5% aqueous potassium carbonate solution. The mixture was stirred at 25 °C with a stir bar at 1200 rpm. The stirring was stopped, the mixture was allowed to stand, and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active moiety to propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard)] was calculated.

[0149] (Evaluation of remaining amount of C-terminal active substance) The LC / MS peak area ratio [propylamide / pentamethylbenzene (internal standard)] was used. The relative values ​​of the remaining amount of C-terminal active substance in the table below are relative values ​​when the peak area ratio [propylamide / 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 (5-minute column for entry 1). Relative value of remaining amount of C-terminal active substance (%) = {[propylamide (area%) / pentamethylbenzene (area%)] / 1.1 (entry 1, 5 min [propylamide (area%) / pentamethylbenzene (area%)]} x 100

[0150] [Table 3]

[0151] The relative values ​​for the remaining amount of C-terminal active substance in Table 3 indicate that the smaller the value, the more hydrolysis of the remaining C-terminal active substance. When alkaline water alone was used, there was almost no change in the hydrolysis rate even when the counter cation of the alkali was changed. We also found that the addition of DMAP or NMI accelerated the hydrolysis of the remaining C-terminal active substance more than when alkaline water alone was used. The addition of DMAP or NMI was sufficiently effective even within 5 minutes, and in particular, the addition of DMAP was found to completely hydrolyze the remaining C-terminal active substance.

[0152] Example 4: Synthesis of Cbz-Ile-Phe-OtBu (Condensation reaction) To a solution consisting 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 carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken and 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%). Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area%) / [H-Phe-OtBu (area%) + Cbz-Ile-Phe-OtBu (area%)]} × 100

[0153] (Hydrolysis treatment using alkaline water alone) A 1.5 mL portion of the total dipeptide solution (9 mL) prepared above was taken, and 0.75 mL of 5% aqueous potassium carbonate was added. The mixture was stirred at 25 °C with a stir bar (1200 rpm). After stopping the stirring, the mixture was allowed to stand 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 compound to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis. The peak areas of the propylamide and the target peptide were determined, and the remaining C-terminal active compound (%) was calculated. The remaining aqueous layer of the reaction solution was removed, and the organic layer was washed sequentially with 0.5 mL of 5% aqueous potassium hydrogen sulfate and 0.5 mL of 5% aqueous potassium carbonate. 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 compound to propylamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area values ​​of the target peptide and the remaining C-terminal active substance (converted to propylamide) were determined. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0154] (Amine-added hydrolysis treatment) A 1.5 mL aliquot of the total dipeptide solution (9 mL) was added to 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 5% aqueous potassium carbonate. The mixture was stirred at 25 °C with a stir bar at 1200 rpm. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active compound to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis. The peak areas of the propylamide and the target peptide were determined, and the percentage of remaining C-terminal active compound (%) was calculated. The remaining aqueous layer of the reaction mixture was removed, and the organic layer was washed sequentially with 0.75 mL of 5% aqueous potassium hydrogen sulfate and 0.75 mL of 5% aqueous potassium carbonate. 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 propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). MS (ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0155] [Table 4] 1) Equivalent to N-terminal amino acid derivative (H-Phe-OtBu hydrochloride) 2) LCMS peak area ratio

[0156] We found that the addition of the amine NMI, when used in an amount of 0.5 to 3.0 equivalents relative to the N-terminal amino acid derivative, significantly accelerated hydrolysis compared to alkaline water treatment alone. We also found that the addition of the amine DMAP, when used in an amount of 0.5 to 3.0 equivalents relative to the N-terminal amino acid derivative, significantly accelerated hydrolysis compared to alkaline water treatment alone. After performing a single hydrolysis treatment with an amine, we found that the remaining C-terminal active form in the organic layer could be completely removed by washing the organic layer with 5% KHSO4 and 5% K2CO3. In this case, the target peptide was obtained in high purity. On the other hand, when treated with alkaline water alone, the C-terminal active form remained and the purity of the dipeptide was low.

[0157] Example 5: Synthesis of Cbz-Ile-Phe-OtBu (Condensation reaction) 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 was added with 1.5 mL (8.8 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution at room temperature, and the mixture was stirred at 40 °C for 1 hour to form a peptide bond. 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%). Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area%) / [H-Phe-OtBu (area%) + Cbz-Ile-Phe-OtBu (area%)]} × 100

[0158] (Hydrolysis treatment using alkaline water alone) A 1.5 mL portion of the dipeptide solution (9 mL) was taken, and 0.75 mL of 5% aqueous potassium carbonate was added. The mixture was stirred at 60 °C with a stir bar (1200 rpm). After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active compound to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis. The peak areas of the propylamide and the target peptide were determined, and the remaining C-terminal active compound (%) was calculated. The aqueous layer of the reaction mixture was removed, and the organic layer was washed sequentially with 0.75 mL of 5% aqueous potassium hydrogen sulfate and 0.75 mL of 5% aqueous potassium carbonate. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active compound to propylamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area values ​​of the target peptide and the remaining C-terminal active substance (converted to propylamide) were determined. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0159] (Amine-added hydrolysis treatment) A 1.5 mL aliquot of the total dipeptide solution (9 mL) was added to amine (0.29 mmol) and 0.75 mL of 5% aqueous potassium carbonate solution. The mixture was stirred at 60 °C with a stir bar at 1200 rpm. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active compound to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis. The peak areas of the propylamide and the target peptide were determined, and the remaining C-terminal active compound (%) was calculated. The aqueous layer of the reaction mixture was removed, and the organic layer was washed sequentially with 0.75 mL of 5% aqueous potassium hydrogen sulfate and 0.75 mL of 5% aqueous potassium carbonate. 5 μL of the organic layer was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal active compound to propylamide. The mixture was then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak areas of the target peptide and the remaining C-terminal active compound (converted to propylamide) were determined. MS(ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0160] [Table 5] 1) LCMS peak area ratio

[0161] When hydrolysis of the remaining C-terminal active compound was performed at 60°C with the addition of amines, the target peptide was obtained with a high purity equivalent to that obtained when hydrolysis was performed at 25°C. In particular, when the added amines were DMAP and NMI, hydrolysis proceeded more rapidly than when alkaline water was used alone. Furthermore, when the added amines were DMAP and NMI, hydrolysis proceeded effectively within 5 minutes in a single hydrolysis treatment, and the remaining C-terminal active compound could be completely removed from the organic layer by subsequent separation (washing with 5% KHSO4 and 5% K2CO3).

[0162] Example 6: Synthesis of Cbz-MeIle-MePhe-OMe (Condensation reaction) 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. 594 mg (1.6 mmol) of HATU was then added at 25 °C. The mixture was stirred at 25 °C for 30 minutes, then at 40 °C for 3 hours, and then at 60 °C for another 3 hours to carry out the peptide bond formation reaction. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: >99%). Conversion rate (%) = {Cbz-MeIle-MePhe-OMe (area%) / [MePhe-OMe (area%) + Cbz-MeIle-MePhe-OMe (area%)]} × 100

[0163] (hydrolysis treatment) (1) When no amine is added To the reaction solution containing the peptide prepared above, 3.0 mL of MTBE and 3.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25 °C for 30 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0164] (2) When amine is added To the reaction solution containing the peptide prepared above, 3.0 mL of MTBE, 103 μL (1.3 mmol) of N-methylimidazole, and 3.0 mL of 5% aqueous potassium carbonate were added and stirred at 25 °C for 30 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula: C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0165] (Post-processing) After stopping the stirring, the mixture was allowed to stand, 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 potassium hydrogen sulfate solution twice, 3 mL of 5% aqueous potassium carbonate solution, and 1 mL of tap water five times. The stirring was stopped, and the organic and aqueous layers were separated. 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 propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained from hydrolysis without the addition of amine weighed 671.8 mg (yield 113%; the concentrate contained impurities (residual C-terminal active form), but was calculated as containing only the peptide). The concentrate obtained from hydrolysis with the addition of amine weighed 563.7 mg (yield 95%). MS(ESI): m / z 455.2 [M+H]+, 477.2 [M+Na]+.

[0166] [Table 6] 1) LCMS peak area ratio

[0167] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to remove it. However, when hydrolysis was performed with the addition of NMI, the residual C-terminal active compound was completely hydrolyzed and could also be completely removed. Moreover, the desired dipeptide was obtained with 100% purity (yield 95%).

[0168] (Example 7) Synthesis of Cbz-MeVal-MeAsp(tBu)-piperidine (Condensation reaction) 303 mg (1.1 mmol) of MeAsp(tBu)-piperidine and 448 mg (1.7 mmol) of Cbz-MeVal-OH were suspended in a mixture of 0.6 mL of acetonitrile and 2.4 mL of cyclopentyl methyl ether, and 586 μL (3.4 mmol) of diisopropylethylamine was added. 642 mg (1.7 mmol) of HATU was then added at 25 °C, and the mixture was stirred for 6.5 hours at 25 °C to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-MeVal-MeAsp(tBu)-piperidine(area%) / [MeAsp(tBu)-piperidine(area%)+Cbz-MeVal-MeAsp(tBu)-piperidine(area%)]}×100

[0169] (hydrolysis treatment) (1) When no amine is added To the reaction solution containing the peptide prepared above, 3.0 mL of 5% aqueous potassium carbonate was added and stirred at 25 °C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0170] (2) When amine is added To the reaction solution containing the peptide prepared above, 136 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25 °C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0171] (Post-processing) After stopping the stirring, the mixture was allowed to stand, 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 potassium hydrogen sulfate solution (2 x 3 mL), 3 mL of 5% aqueous potassium carbonate solution (2 x 3 mL), and 1.5 mL of tap water (3 x 3 mL). The stirring was stopped, and the organic and aqueous layers were separated. 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 propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained from hydrolysis without the addition of amine weighed 782.0 mg (yield 136%; the concentrate contained impurities (residual C-terminal active form), but was calculated as containing only the peptide). The concentrate obtained from hydrolysis with the addition of amine weighed 530.6 mg (yield 92%). MS(ESI): m / z 518.4 [M+H]+, 540.4 [M+Na]+.

[0172] [Table 7] 1) LCMS peak area ratio

[0173] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to remove it. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and could also be completely removed. Moreover, the desired dipeptide was obtained with 100% purity (yield 92%).

[0174] (Example 8) Synthesis of Cbz-MeVal-MeAsp(tBu)-piperidine (Condensation reaction) 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 and 775 μL (4.4 mmol) of diisopropylethylamine was added. 1.6 mL (2.8 mmol) of a 50% T3P / THF solution was then added at 25 °C and the mixture was stirred for 15 hours at 25 °C to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of n-propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-MeVal-MeAsp(tBu)-piperidine(area%) / [MeAsp(tBu)-piperidine(area%)+Cbz-MeVal-MeAsp(tBu)-piperidine(area%)]}×100

[0175] (hydrolysis treatment) (1) When no amine is added To the reaction solution containing the peptide prepared above, 3.0 mL of 5% aqueous potassium carbonate was added and stirred at 25 °C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0176] (2) When amine is added To the reaction solution containing the peptide prepared above, 141 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25 °C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0177] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous potassium hydrogen sulfate and 3 mL of 5% aqueous potassium carbonate. The stirring was stopped, and the organic and aqueous layers were separated. 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 propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained from hydrolysis without the addition of amine weighed 561.6 mg (yield 98%; the concentrate contained impurities (residual C-terminal active form), but was calculated as containing only the peptide). The concentrate obtained from hydrolysis with the addition of amine weighed 501.1 mg (yield 87%). MS(ESI): m / z 518.4 [M+H]+, 540.4 [M+Na]+.

[0178] [Table 8] 1) LCMS peak area ratio

[0179] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to remove it. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and could also be completely removed. Moreover, the desired dipeptide was obtained with 100% purity (87% yield).

[0180] (Example 9) Synthesis of Cbz-Ile-MeVal-MeAsp(tBu)-piperidine (Cbz deprotection reaction using dipeptides obtained by hydrolysis without the addition of amines) 782 mg of Cbz-MeVal-MeAsp(tBu)-piperidine (782 mg, containing 17.6 area % of the residual C-terminal active form) synthesized under the amine-free conditions of Example 7 was dissolved in 4.2 mL of cyclopentyl methyl ether. This was subjected to hydrogenolysis using 115 mg of 5% Pd / C (50% wet) and hydrogen gas. Since the reaction proceeded poorly, the Pd / C was removed by filtration, the solution was concentrated to dryness, and the solution was redissolved in 4.2 mL of cyclopentyl methyl ether. 105 mg of 5% Pd / C (50% wet) was added, and the solution was subjected to hydrogenolysis again. However, even after a total of 3 hours of reaction, the reaction proceeded very little (reaction conversion rate: 1.6%). The reaction conversion rate was determined by LC / MS analysis of 5 μL of the reaction mixture, diluting it with 1.0 mL of acetonitrile, and filtering the solution. Conversion rate (%)={MeVal-MeAsp(tBu)-piperidine(area %) / [MeVal-MeAsp(tBu)-piperidine(area %)+Cbz-MeVal-MeAsp(tBu)-piperidine(area %)]}×100

[0181] (Cbz deprotection reaction using dipeptides obtained by hydrolysis with amine addition) 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. The mixture was subjected to hydrogenolysis using 124 mg of 5% Pd / C (50% wet) and hydrogen gas. After stirring at room temperature for 2 hours, the Cbz-free form, MeVal-MeAsp(tBu)-piperidine, was obtained (100% conversion). The reaction conversion was determined by LC / MS analysis of the peak area obtained by aliquoting 5 μL of the reaction mixture, diluting it with 1.0 mL of acetonitrile, and filtering the resulting solution. MS (ESI): m / z 384.3 [M+H]+ Conversion rate (%)={MeVal-MeAsp(tBu)-piperidine(area %) / [MeVal-MeAsp(tBu)-piperidine(area %)+Cbz-MeVal-MeAsp(tBu)-piperidine(area %)]}×100

[0182] (Condensation reaction) The reaction mixture was filtered, the Pd / C removed, and then concentrated to dryness. The dried product 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. 1.4 mL (2.4 mmol) of a 50% T3P / THF solution was then added at 25 °C. The mixture was stirred at 40 °C for 7 hours and then at room temperature for 14 hours to form a peptide bond (100% conversion). 81 μL (1.0 mmol) of N-methylimidazole and 2.6 mL of 20% aqueous potassium carbonate were added to the reaction mixture, and the mixture was stirred at 25 °C for 45 minutes with a stir bar. After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 5.2 mL of 10% aqueous potassium hydrogen sulfate and 5.2 mL of 5% aqueous potassium carbonate. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Ile-MeVal-MeAsp(tBu)-piperidine, was 95.1%, and no Cbz-Ile-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 542.7 mg of concentrate (82% yield). MS (ESI): m / z 631.5 [M+H] + , 653.4[M+Na] + .

[0183] It was found that the Cbz deprotection reaction hardly proceeded when a peptide solution containing residual C-terminal active isomers treated with alkaline water alone was used. On the other hand, it was found that the Cbz deprotection reaction proceeded smoothly when a peptide solution from which residual C-terminal active isomers had been completely removed, obtained by treatment with DMAP, was used, enabling the subsequent peptide synthesis reaction. In other words, it was found that the reductive removal reaction of the N-terminal protecting group of the resulting peptide compound could proceed without stagnation by using the method of the present invention. This enabled the efficient production of highly pure peptide compounds having the desired amino acid sequence.

[0184] Example 10: Synthesis of Cbz-Phe(3-F)-Phe-OtBu 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. 0.9 mL (1.6 mmol) of a 50% T3P / THF solution was then added at 25 °C and stirred at room temperature for 30 minutes to carry out the peptide bond formation reaction (conversion: 100%). 5 μL of the reaction mixture was added to 100 μL of n-propylamine, diluted with 0.9 mL of methanol, and the resulting solution was analyzed by LC / MS. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {Cbz-Phe(3-F)-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-Phe(3-F)-Phe-OtBu (area%)]} × 100

[0185] To the reaction mixture, 95 mg (0.8 mmol) of DMAP and 2.0 mL of 5% aqueous potassium carbonate were added and stirred at 25 °C for 5 minutes using a stir bar. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. The aqueous layer was then removed. The organic layer was then washed sequentially with 1 mL of 10% aqueous potassium hydrogen sulfate, 1 mL of 5% aqueous potassium carbonate, and 1 mL of tap water. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Phe(3-F)-Phe-OtBu, was 100% pure, and no Cbz-Phe(3-F)-NHPr derived from the remaining C-terminal active compound 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]+.

[0186] By adding DMAP and carrying out hydrolysis, the remaining C-terminal active substance was completely removed, and the desired dipeptide was obtained with 100% purity (yield 96%).

[0187] Example 11: Synthesis of Cbz-Ser(OtBu)-Phe-OtBu 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. 1.4 mL (2.3 mmol) of a 50% T3P / THF solution was then added at 25 °C and stirred at room temperature for 1 hour to carry out the peptide bond formation reaction (conversion: 100%). 5 μL of the reaction mixture was added to 100 μL of n-propylamine, diluted with 0.9 mL of methanol, and the resulting solution was analyzed by LC / MS. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {Cbz-Ser(tBu)-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-Ser(tBu)-Phe-OtBu (area%)]} × 100

[0188] To the reaction mixture, 143 mg (1.2 mmol) of DMAP and 1.5 mL of 20% aqueous potassium carbonate were added and stirred at 25 °C for 5 minutes using a stir bar. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. The aqueous layer was then removed. The organic layer was then washed sequentially with 3.0 mL x 2 of 10% aqueous potassium hydrogen sulfate, 3.0 mL of 5% aqueous potassium carbonate, and 3.0 mL of tap water. 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Ser(OtBu)-Phe-OtBu, was 100% pure, and no Cbz-Ser(OtBu)-NHPr derived from the remaining C-terminal active compound 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]+.

[0189] By adding DMAP and carrying out hydrolysis, the remaining C-terminal active substance was completely removed, and the desired dipeptide was obtained with 100% purity (yield 96%).

[0190] (Example 12) Synthesis of Boc-MeVal-Phe-piperidine (Boc deprotection reaction) 471 mg (1.4 mmol) of Boc-Phe-piperidine was dissolved in 4.7 mL of dichloromethane, and 180 μL (2.8 mmol) of methanesulfonic acid was added. The mixture was stirred at 35 °C for 2 hours to carry out the Boc removal reaction (100% conversion). 5 μL of the reaction mixture was diluted with 1.0 mL of acetonitrile, and the resulting solution was subjected to LC / MS analysis to determine the reaction conversion rate from the LC / MS peak area. Conversion rate (%)={Phe-piperidine(area%) / [Boc-Phe-piperidine(area%)+Phe-piperidine(area%)]}×100

[0191] (Condensation reaction) To the reaction solution, 742 μL (4.3 mmol) of diisopropylethylamine was added, and the solvent was evaporated. Next, 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 form a peptide bond (conversion rate: 100%). The reaction conversion rate was determined by adding 5 μL of the reaction mixture to 100 μL of n-propylamine, diluting the solution with 0.9 mL of methanol, and analyzing the peak area of ​​the LC / MS. Conversion rate (%) = {Boc-MeVal-Phe-piperidine(area%) / [Phe-piperidine(area%)+Boc-MeVal-Phe-piperidine(area%)]}×100

[0192] To the reaction mixture prepared above, 168 mg (1.4 mmol) of DMAP and 4.6 mL of 5% aqueous potassium carbonate were added and stirred at 25 °C for 5 minutes using a stir bar. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. The aqueous layer was then removed. The organic layer was then washed sequentially with 4.6 mL of 10% aqueous potassium hydrogen sulfate, 4.6 mL of 5% aqueous potassium carbonate, and 1.5 mL of tap water (6 times). 5 μL of the resulting organic layer was added to 100 μL of n-propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Boc-MeVal-Phe-OtBu, was 99.7% pure, and no Boc-MeVal-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 542.3 mg of concentrate (86% yield). MS(ESI): m / z 346.2 [M-Boc+H]+, 446.3 [M+H]+,468.3 [M+Na]+.

[0193] By adding DMAP and carrying out hydrolysis, the remaining C-terminal active compound was completely removed, and the desired dipeptide was obtained with a purity of 99.7% (yield 86%), even though the N-terminal protecting group was Boc.

[0194] (Example 13) Synthesis example of Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 1 (5 mer) (Synthesis of Cbz-MePhe-MeGly-OtBu) (Condensation reaction) 2.0 g (11.0 mmol) of MeGly-OtBu hydrochloride was suspended in 16 mL of isopropyl acetate and 4 mL of acetonitrile, followed by the addition of 7.7 mL (44.0 mmol) of diisopropyldiethylamine and 3.6 g (11.5 mmol) of Cbz-MePhe-OH. The reaction mixture 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 form a peptide bond (100% conversion). 3 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0195] Next, 1.7 mL (22.0 mmol) of NMI and 20 mL of 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 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 5% aqueous potassium sulfate and 5% aqueous potassium carbonate solutions twice. The resulting organic layer was then concentrated to obtain 5.0 g of concentrate (yield quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target Cbz-MePhe-MeGly-OtBu was determined (100 area%). MS (ESI): m / z 441.2 [M+H] + , 463.2 [M+Na] + .

[0196] (Synthesis of Cbz-Aze-MePhe-MeGly-OtBu) (Cbz deprotection reaction) The entire amount of Cbz-MePhe-MeGly-OtBu obtained by the above method was dissolved in 75 mL of isopropyl acetate and subjected to hydrogenolysis using 0.98 g of 10% Pd / C (3% wet) and hydrogen gas. After stirring at room temperature for 2 hours, the de-Cbz product was obtained (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS analysis by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and analyzing it by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0197] (Condensation reaction) The reaction mixture was filtered, and toluene was added for azeotropic dehydration. The concentrate was dissolved in 39 mL of isopropyl acetate and 9.7 mL of acetonitrile and cooled to 0°C. 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 were added and stirred at room temperature for 30 minutes to form a peptide bond (conversion rate: >99%). The conversion rate was determined from the peak area of ​​the LC / MS analysis of 3 μL of the reaction mixture, diluted with 1.0 mL of methanol, and analyzed by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0198] Next, 1.7 mL (22.0 mmol) of NMI and 34 mL of 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 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 34 mL of 5% aqueous potassium sulfate solution and 34 mL of 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to obtain 5.5 g of concentrate (96% yield). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target Cbz-Aze-MePhe-MeGly-OtBu was determined (99.8% area). MS (ESI): m / z 546.2 [M+Na] + .

[0199] (Synthesis of Cbz-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 2) (Cbz deprotection reaction) 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 using 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 rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS analysis by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and analyzing it. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0200] (Condensation reaction) The reaction mixture was filtered and azeotropically evaporated 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 the T3P / ethyl acetate solution, and 1.1 mL (6.3 mmol) of diisopropylethylamine were added. The mixture was stirred at room temperature for an additional 2 hours to carry out the peptide bond formation reaction (conversion: 97%). The reaction conversion rate was calculated from the peak area value of LC / MS after 3 μL of the reaction solution was taken and diluted with 1.0 mL of methanol. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0201] Next, 1.7 mL (21.1 mmol) of NMI and 41 mL of 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 5 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 41 mL of 5% aqueous potassium sulfate solution and 41 mL of 5% aqueous potassium carbonate solution. The resulting organic layer was concentrated to give 5.8 g of concentrate (91% yield). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the desired 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] + .

[0202] (Synthesis of Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 1) (Cbz deprotection reaction) 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 rate: 100%). 3 μL of the reaction solution was taken, diluted with 1.0 mL of methanol, and the solution was subjected to LC / MS analysis, and the reaction conversion rate was determined from the LC / MS peak area value. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0203] The filtrate was concentrated to obtain 4.4 g of concentrate (97% yield). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 3) was determined (99.7% area). MS (ESI): m / z 475.3 [M+H] + .

[0204] (Condensation reaction) 1.5 g (3.2 mmol) of the 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 stirred at room temperature for 30 minutes to form a peptide bond (conversion rate: >99%). The conversion rate was determined from the peak area of ​​the LC / MS analysis of 3 μL of the reaction mixture, diluted with 1.0 mL of methanol, and analyzed by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0205] Next, 0.75 mL (9.5 mmol) of NMI and 22.5 mL of 5% aqueous sodium carbonate solution were added, and the mixture was stirred at 50°C for 20 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 22.5 mL of 5% aqueous potassium sulfate solution twice and 22.5 mL of 5% aqueous potassium carbonate solution three times. The resulting organic layer was concentrated to obtain 2.4 g of concentrate (yield quant.). This concentrate was subjected to LC / MS analysis, 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] + .

[0206] A single hydrolysis using NMI followed by aqueous washing achieved complete removal of residual C-terminal active compounds, yielding the desired pentapeptide with 99.1% purity. The yield, calculated from the starting amino acid, was 87%. These results demonstrate that complete removal of residual C-terminal active compounds using an amine additive in continuous solution-phase peptide synthesis can lead to the synthesis of a highly pure pentapeptide in high yield.

[0207] Example 14 Synthesis of Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 4 (11 mer) (Synthesis of Cbz-MeVal-Asp(tBu)-piperidine) (Condensation reaction) Asp(tBu)-piperidine (8.6 g, 33.5 mmol) was dissolved in 108 mL of cyclopentyl methyl ether. Cbz-MeVal-OH (9.79 g, 36.9 mmol) and diisopropylethylamine (17.6 mL, 101 mmol) were added. BEP (13.8 g, 50.3 mmol) 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 form a peptide bond (conversion rate: >99%). The conversion rate was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of methanol, and analyzed by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0208] The reaction mixture was washed with 150 mL of 10% aqueous potassium hydrogen sulfate, followed by the addition of 150 mL of 5% aqueous potassium carbonate and 9.52 g (101 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 40°C for 90 minutes. The 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 5% aqueous potassium carbonate. The resulting organic layer was concentrated to give 17 g of concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target Cbz-MeVal-Asp(tBu)-piperidine was determined (99.7 area%).

[0209] (Synthesis of Cbz-MePhe-MeVal-Asp(tBu)-piperidine) (Cbz deprotection reaction) 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 reaction with 1.9 g of 10% Pd / C (3% wet) and hydrogen gas, followed by stirring at 35 °C for 2 hours (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS analysis by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then analyzing it by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0210] The same procedure was repeated, and the combined reaction mixture was filtered. The filtrate was concentrated to obtain 14.0 g of concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target MeVal-Asp(tBu)-piperidine was determined (99.5 area%).

[0211] (Condensation reaction) The concentrate was dissolved in 126 mL of chloropentyl 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 carry out the peptide bond formation reaction (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the LC / MS peak area. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0212] The reaction mixture was washed with 140 mL of 5% aqueous potassium hydrogen sulfate, followed by the addition of 140 mL of 5% aqueous potassium carbonate and 10.9 g (114 mmol) of trimethylamine hydrochloride, and the mixture was stirred at room temperature for 30 minutes. The 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 5% aqueous potassium carbonate. The resulting organic layer was concentrated to give 24.1 g of concentrate (96% yield). This concentrate was analyzed by LC / MS, and the peak area percentage of the desired Cbz-MePhe-MeVal-Asp(tBu)-piperidine was determined (99.6% area).

[0213] (Synthesis of Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 5) (Cbz deprotection reaction) 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, followed by stirring at 35°C for 2 hours (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS analysis by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then analyzing it by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0214] The same procedure was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to give 18.1 g of a concentrate (yield 99%).

[0215] (Condensation reaction) 17.3 g (32.6 mmol) of the concentrate was dissolved in 153 mL of chloropentyl 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 stirred at room temperature for 15 minutes to carry out the peptide bond formation reaction (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0216] The reaction mixture was washed with 170 mL of 5% aqueous potassium hydrogen sulfate, followed by the addition of 170 mL of 5% aqueous potassium carbonate and 9.4 g (98.0 mmol) of trimethylamine hydrochloride, and the mixture was stirred at room temperature for 2 hours. The 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% aqueous potassium carbonate. The resulting organic layer was concentrated to obtain 26.5 g of concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the desired Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 5) was determined (98.9 area%). MS (ESI): 830.4 [M+Na] + .

[0217] (Synthesis of Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 6) (Cbz deprotection reaction) 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, followed by stirring at 35°C for 2 hours (conversion rate: >98%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis, and the conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0218] The same procedure was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to give 19.5 g of a concentrate (yield 97%).

[0219] (Condensation reaction) 16.0 g (23.7 mmol) of the 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 diisopropyl ethylamine were added. 9.8 g (35.6 mmol) of BEP was dissolved in 40 mL of acetonitrile and added to the reaction mixture. The mixture was stirred at room temperature for 5 minutes to form a peptide bond (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0220] The reaction mixture was washed with 240 mL of 10% aqueous sodium hydrogen sulfate, followed by the addition of 240 mL of 5% aqueous potassium carbonate and 6.7 g (71.2 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 40°C for 1.5 hours. The 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 5% aqueous potassium carbonate. The resulting organic layer was concentrated to give 22.2 g of concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the desired Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 6) was determined (99.4 area%).

[0221] (Synthesis of Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 7) (Cbz deprotection reaction) 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 subjected to hydrogenolysis using 1.9 g of 10% Pd / C and hydrogen gas, followed by stirring at 35°C for 2 hours. The same procedure was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to give 15.6 g of concentrate (yield 96%).

[0222] (Condensation reaction) 15.3 g (19.1 mmol) of the 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 stirred at room temperature for 15 minutes to carry out the peptide bond formation reaction (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0223] The reaction mixture was washed with 153 mL of 5% aqueous potassium hydrogen sulfate, followed by addition of 153 mL of 5% aqueous potassium carbonate and stirring at room temperature for 5 minutes. Stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, followed by addition of 153 mL of 5% aqueous potassium carbonate and stirring at room temperature for 1 hour. After removing the aqueous layer, the resulting organic layer was concentrated to obtain 19.5 g of concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the desired Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 7) was determined (99.6 area%).

[0224] (Synthesis of Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 8) (Cbz deprotection reaction) 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, followed by stirring at 35°C for 3 hours (conversion: 100%). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction solution, which was diluted with 1.0 mL of methanol. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0225] The same procedure was repeated, and the combined reaction solution was filtered. The filtrate was concentrated to give 16.3 g of a concentrate (yield 99%).

[0226] (Condensation reaction) 16.0 g (18.4 mmol) of the 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 stirred at room temperature for 30 minutes to carry out the peptide bond formation reaction (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0227] The reaction mixture was washed with 160 mL of 5% aqueous potassium hydrogen sulfate, followed by the addition of 153 mL of 5% aqueous potassium carbonate and 5.3 g (55.0 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 60°C for 1 hour. The stirring was stopped, the organic and aqueous layers were separated, and the aqueous layer was removed. The resulting organic layer was washed with 160 mL of 5% aqueous potassium carbonate and concentrated to give 20.0 g of concentrate (99% yield). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the desired Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 8) was determined (99.6% area).

[0228] (Synthesis of Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 9) (Cbz deprotection reaction) 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, followed by stirring at 35°C for 6 hours and then at 45°C for 4 hours (conversion: 100%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis, and the reaction conversion was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0229] 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%). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 10) was determined (97.9 area%).

[0230] (Condensation reaction) 14.5 g (14.9 mmol) of the 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 mixture. The mixture was stirred at 40°C for 1 minute to form a peptide bond (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0231] The reaction mixture was washed with 128 mL of 10% aqueous sodium hydrogen sulfate, followed by the addition of 128 mL of 5% aqueous potassium carbonate and 4.2 g (44.8 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 40°C for 30 minutes. The stirring was stopped, the organic and aqueous layers were separated, and the aqueous layer was removed. The resulting organic layer was washed with 128 mL of 5% aqueous potassium carbonate and concentrated to give 18.0 g of concentrate (98% yield). This concentrate was analyzed by LC / MS, and the peak area percentage of the desired Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 9) was determined (96.0 area%).

[0232] (Synthesis of Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 11) (Cbz deprotection reaction) 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, followed by stirring at 45°C for 4 hours (conversion: 100%). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction solution, diluted with 1.0 mL of methanol. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0233] The same procedure was repeated, and the combined reaction mixture was filtered. The filtrate was concentrated to obtain 14.3 g of concentrate (yield quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target compound MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12) was determined (95.8 area%). MS (ESI): m / z 1098.6 [M+H]+ .

[0234] (Condensation reaction) 13.0 g (11.8 mmol) of the 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 mixture, which was stirred at room temperature for 30 minutes to form a peptide bond (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0235] The reaction mixture was washed with 130 mL of 5% aqueous potassium hydrogen sulfate, followed by the addition of 130 mL of 5% aqueous potassium carbonate and 3.4 g (35.5 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 60°C for 45 minutes. The stirring was stopped, and the organic and aqueous layers were separated. The aqueous layer was removed, and the resulting organic layer was washed with 130 mL of 5% aqueous potassium carbonate and concentrated to give 15.6 g of concentrate (98% yield). This concentrate was analyzed by LC / MS, and the peak area percentage of the desired Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 11) was determined (97.2% area).

[0236] (Synthesis of Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 12) (Cbz deprotection reaction) 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, followed by stirring at 45°C for 4 hours (conversion: 100%). The reaction mixture was filtered and the filtrate was concentrated to give 8.9 g of concentrate (99% yield). The reaction conversion was determined from the peak area of ​​LC / MS analysis by aliquoting 5 μL of the reaction mixture, diluting it with 1.0 mL of methanol, and analyzing it. MS (ESI): m / z 1211.7 [M+H] + , 1233.7 [M+Na] + . Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0237] (Condensation reaction) 7.0 g (5.8 mmol) of the 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 mixture. The mixture was stirred at room temperature for 3 minutes to form a peptide bond (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0238] The reaction mixture was washed with 105 mL of 10% aqueous sodium hydrogen sulfate, followed by the addition of 105 mL of 5% aqueous potassium carbonate and 1.7 g (17.3 mmol) of trimethylamine hydrochloride, and the mixture was stirred at 40°C for 30 minutes. The stirring was stopped, the organic and aqueous layers were separated, and the aqueous layer was removed. The resulting organic layer was washed with 105 mL of 5% aqueous potassium carbonate and concentrated to give 8.6 g of concentrate (99% yield). This concentrate was analyzed by LC / MS, and the peak area percentage of the desired Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12) was determined (97.0% area).

[0239] (Synthesis of Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / SEQ ID NO: 4) (Cbz deprotection reaction) 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, followed by stirring at 45 °C for 4 hours (conversion: 100%). The reaction mixture was filtered and the filtrate was concentrated to give 6.8 g of concentrate (98% yield). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of methanol, and analyzed by LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0240] (Condensation reaction) 500 mg (0.4 mmol) of the 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 stirred at room temperature for 2 hours to carry out the peptide bond formation reaction (conversion rate: >99%). 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and subjected to LC / MS analysis. The conversion rate was determined from the peak area of ​​the LC / MS. Conversion rate (%) = {target compound (area%) / [raw material (area%) + target compound (area%)]} × 100

[0241] The reaction mixture was washed with 5.0 mL of 10% aqueous sodium hydrogen sulfate, followed by the addition of 5.0 mL of 5% aqueous potassium carbonate and 104 mg (1.1 mmol) of trimethylamine hydrochloride, and the mixture was stirred at room temperature for 1 hour. The 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 5% aqueous potassium carbonate and concentrated to give 555 mg of concentrate (96% yield, 95.3 area % of Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 4)). MS (ESI): m / z 1591.9 [M+H] + , 1613.9[M+Na] + .

[0242] A single hydrolysis using trimethylamine followed by aqueous washing completely removed the residual C-terminal active compound, affording a highly purified 11-amino acid peptide with a purity of 95.3%. The yield was 75.3% based on the starting amino acid. These results demonstrate that the use of an amine additive in continuous solution-phase peptide synthesis can be used to remove residual C-terminal active compounds, resulting in highly purified polypeptides.

[0243] (Example 15) Synthesis of Teoc-MeLeu-Phe-OtBu (Synthesis of Teoc-MeLeu-Opfp) 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 carry out the Teoc-conjugation reaction. The reaction solution was acidified by adding 5% aqueous potassium hydrogen sulfate, extracted with 50 mL of ethyl acetate, and the organic layer was washed with saturated saline. 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 carry out the Pfp-conjugation reaction. The reaction solution was washed with saturated saline, and the aqueous layer was extracted with 50 mL of ethyl acetate. The combined organic layers were concentrated, and the resulting concentrate was purified by column chromatography (ethyl acetate / heptane) to give 6.63 g of Teoc-MeLeu-OPfp (yield: 90%).

[0244] (Condensation reaction) Phe-OtBu hydrochloride (201 mg, 0.8 mmol) and Teoc-MeLeu-OPfp (536 mg, 1.2 mmol) 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 form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was calculated from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Teoc-MeLeu-Phe-OtBu (area%) / [Phe-OtBu (area%) + Teoc-MeLeu-Phe-OtBu (area%)]} × 100

[0245] (hydrolysis treatment) (1) When no amine is added 2.0 mL of 5% aqueous sodium carbonate was added to the reaction solution containing the peptide prepared above, and the mixture was stirred at 25°C for 20 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0246] (2) When amine is added To the reaction solution containing the peptide prepared above, 95 mg (0.8 mmol) of DMAP and 2.0 mL of 5% aqueous sodium carbonate were added and stirred at 25 °C for 20 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula: C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0247] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 2 mL of 10% aqueous potassium hydrogen sulfate solution (2 mL each) and 2 mL of 5% aqueous sodium carbonate solution. This was followed by three washes with 1 mL of 5% aqueous potassium carbonate solution and 1 mL of tap water (2 mL each). 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 to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). The yield of the concentrate (peptide) obtained by hydrolysis without the addition of amine was 576.6 mg (150% yield; the concentrate contained impurities (residual C-terminal active form), but was calculated assuming only peptide). The yield of the concentrate obtained by hydrolysis with the addition of amine was 369.6 mg (96% yield). MS(ESI): m / z437.3 [M-tBu+H]+, 493.3 [M+H]+, 515.3 [M+Na]+.

[0248] [Table 9] 1) LCMS peak area ratio

[0249] When the residual C-terminal activator, which had Teoc as the protecting group and Pfp as the C-terminal activator site, was hydrolyzed by alkaline water alone, the residual C-terminal activator was not completely hydrolyzed, and subsequent aqueous washing was unable to remove it. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal activator was completely hydrolyzed and could also be completely removed. The desired dipeptide was obtained with a purity of 96.3% (yield: 96%).

[0250] Example 16: Synthesis of Cbz-Aib-MeLeu-Phe-OtBu (TeO deprotection reaction using dipeptides obtained by hydrolysis without the addition of amines) 576.6 mg of Teoc-MeLeu-Phe-OtBu (containing 8.9 area% of residual C-terminal active form) synthesized under the amine-free 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. 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. Another 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 the Teoc-free form, MeLeu-Phe-OtBu (100% conversion). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. Conversion rate (%) = {MeLeu-Phe-OtBu (area%) / [Teoc-MeLeu-Phe-OtBu (area%) + MeLeu-Phe-OtBu (area%)]} × 100

[0251] (Condensation reaction) After concentrating the mixture to approximately 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This procedure was repeated twice more, and the resulting 2-methyltetrahydrofuran solution was added with 0.5 mL of acetonitrile, 276 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine. 441 mg (1.1 mmol) of HATU was then added at 25 °C and stirred at room temperature for 14 hours. 579 mg (1.5 mmol) of HATU was added and stirred at 40 °C for 1 hour. 684 mg (1.8 mmol) of HATU was then added, and the mixture was heated to 60 °C and stirred for 4.5 hours. 455 mg (1.1 mmol) of HATU was then added and stirred at 60 °C for 2 hours, room temperature for 12 hours, and 60 °C for 2 hours, but no progress of the condensation reaction was observed (0% conversion). The reaction conversion rate was determined from the peak area value of LC / MS after adding 5 μL of the reaction solution to 100 μL of propylamine and diluting with 0.9 mL of methanol. Conversion rate (%) = {Cbz-Aib-MeLeu-Phe-OtBu (area%) / [MeLeu-Phe-OtBu (area%) + Cbz-Aib-MeLeu-Phe-OtBu (area%)]} × 100

[0252] (TeO deprotection reaction using a dipeptide obtained by hydrolysis with the addition of an amine) 369.6 mg (0.75 mmol) of Teoc-MeLeu-Phe-OtBu synthesized under the amine addition conditions of 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 the Teoc-free form, MeLeu-Phe-OtBu (100% conversion). The reaction conversion rate was determined from the peak area value of the LC / MS analysis obtained by aliquoting 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting it to LC / MS analysis. Conversion rate (%) = {MeLeu-Phe-OtBu (area%) / [Teoc-MeLeu-Phe-OtBu (area%) + MeLeu-Phe-OtBu (area%)]} × 100

[0253] (Condensation reaction) After concentrating the mixture to approximately 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This procedure was repeated twice more, and the resulting 2-methyltetrahydrofuran solution was added with 0.5 mL of acetonitrile, 273 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine. 439 mg (1.1 mmol) of HATU was then added at 25 °C and stirred at room temperature for 14 hours. 576 mg (1.5 mmol) of HATU was added and stirred at 40 °C for 5.5 hours. 452 mg (1.1 mmol) of HATU was then added and stirred at 60 °C for 2 hours, room temperature for 12 hours, and 60 °C for 2 hours (86% conversion). The reaction conversion was determined by adding 5 μL of the reaction mixture to 100 μL of propylamine, diluting with 0.9 mL of methanol, and analyzing the peak area of ​​the LC / MS. Conversion rate (%) = {Cbz-Aib-MeLeu-Phe-OtBu (area%) / [MeLeu-Phe-OtBu (area%) + Cbz-Aib-MeLeu-Phe-OtBu (area%)]} × 100

[0254] To the prepared reaction mixture, 92 mg (0.75 mmol) of DMAP and 4.0 mL of 10% aqueous potassium carbonate solution were added and stirred at 25 °C for 1 hour using a stir bar. After stopping the stirring, the mixture was allowed to stand and the organic and aqueous layers were separated. The aqueous layer was then removed. 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide Cbz-Aib-MeLeu-Phe-OtBu was 86.7%, the starting material MeLeu-Phe-OtBu was 13.3%, and no Cbz-Aib-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 295.6 mg of the concentrate with the above composition. MS (ESI): m / z 568.4 [M+H]+, 590.4 [M+Na]+.

[0255] It was found that if the C-terminal active species remained in a solution of an N-terminal protected peptide, a large excess of the reagent was required when deprotecting the N-terminal protecting group (Teoc) of the peptide with a hydrofluoric acid reagent. This is presumably because the deprotecting reagent also reacts with the remaining C-terminal active species. Furthermore, it was found that the condensation reaction (peptide bond formation reaction) with another C-terminal activated form in the next step following deprotection did not proceed at all. It is presumed that the excess reagent used in the previous step (N-terminal deprotection) decomposed the C-terminal activated form. Thus, we found that if the C-terminal activated form remains, a large excess of reagent is required for N-terminal deprotection, which leads to the impediment of the subsequent condensation reaction. On the other hand, we found that if a peptide solution from which the remaining C-terminal activated form had been completely removed was used as the starting material, the deprotection reaction could be completed with an appropriate amount of deprotection reagent, and the condensation reaction in the next step could also be carried out.

[0256] Example 17: Synthesis of Cbz-MeAla-Phe-OtBu (Condensation reaction) 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, followed by the addition of 1.0 mL (5.8 mmol) of diisopropylethylamine. 443 mg (2.3 mmol) of EDC hydrochloride was then added, and the mixture was stirred at 25 °C for 30 minutes to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-MeAla-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-MeALa-Phe-OtBu (area%)]} × 100

[0257] (hydrolysis treatment) (1) When no amine is added 3.0 mL of 5% aqueous sodium carbonate was added to the reaction solution containing the peptide prepared above, and the mixture was stirred at 25°C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0258] (2) When amine is added To the reaction solution containing the peptide prepared above, 147 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous sodium carbonate were added and stirred at 25 °C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula: C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0259] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous sodium hydrogen sulfate (3 mL x 2) and 3 mL of 5% aqueous sodium carbonate (3 mL). 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. MS (ESI): m / z 385.2 [M-tBu+H]+, 441.3 [M+H]+, 463.2 [M+Na]+.

[0260] [Table 10] 1) LCMS peak area ratio

[0261] Even with alanine, an amino acid with a small substituent, the residual C-terminal active compound was not completely hydrolyzed when hydrolyzed with alkaline water alone, and the subsequent separation procedure (aqueous washing) did not sufficiently remove the residual C-terminal active compound. On the other hand, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and could also be completely removed. Moreover, the desired dipeptide was obtained with a purity of 98.4%.

[0262] Example 18: Synthesis of Cbz-Hph-MeAla-Phe-OtBu (Cbz deprotection reaction using dipeptides obtained by hydrolysis without the addition of amines) The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized under the amine-free conditions in Example 17 was substituted with 2-methyltetrahydrofuran and concentrated. The resulting mixture was subjected to hydrogenolysis using 101 mg of 5% Pd / C (50% wet) and hydrogen gas. The mixture was stirred at 25°C for 6 hours, but the reaction was not completed (conversion rate: 35%). The reaction conversion rate was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. Conversion rate (%) = {MeAla-Phe-OtBu (area%) / [Cbz-MeAla-Phe-OtBu (area%) + MeALa-Phe-OtBu (area%)]} × 100

[0263] (Cbz deprotection reaction using dipeptides obtained by hydrolysis with amine addition) The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized under the amine addition conditions in Example 17 was substituted with 2-methyltetrahydrofuran and concentrated. The resulting mixture was subjected to hydrogenolysis using 102 mg of 5% Pd / C (50% wet) and hydrogen gas. The mixture was stirred at 25°C for 3 hours to obtain the de-Cbzed form, MeAla-Phe-OtBu (100% conversion). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. MS (ESI): m / z 307.2 [M+H]+ Conversion rate (%) = {MeAla-Phe-OtBu (area%) / [Cbz-MeAla-Phe-OtBu (area%) + MeALa-Phe-OtBu (area%)]} × 100

[0264] (Condensation reaction) The Cbz-deprotected dipeptide reaction mixture obtained by hydrolysis with amine addition was filtered, the Pd / C was removed, and the mixture was 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. 1.37 mL (2.33 mmol) of T3P / 2-methyltetrahydrofuran solution was then added and stirred at 25°C for 1.5 hours to carry out the peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by adding 5 μL of the reaction mixture to 100 μL of propylamine, diluting with 0.9 mL of methanol, and analyzing the peak area of ​​the LC / MS. Conversion rate (%) = {Cbz-Hph-MeAla-Phe-OtBu (area%) / [MeAla-Phe-OtBu (area%) + Cbz-Hph-MeAla-Phe-OtBu (area%)]} × 100

[0265] To the prepared reaction solution, 74 mg (0.6 mmol) of DMAP and 3.0 mL of 5% aqueous sodium carbonate were added and stirred at 25°C for 15 minutes. After stopping the stirring and allowing the mixture to stand, 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 hydrogen sulfate, 3 mL of 5% aqueous sodium carbonate, and 3 mL of tap water. 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Hph-MeAla-Phe-OtBu, was 99.0%, and no Cbz-Hph-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 618.4 mg of concentrate (88% yield based on Phe-OtBu in Example 17). MS(ESI): m / z602.4 [M+H]+, 624.4 [M+Na]+.

[0266] It was found that if the C-terminal active forms derived from EDC and HOOBt remain, the Cbz deprotection reaction after peptide elongation hardly proceeds. On the other hand, it was found that if a peptide solution from which the remaining C-terminal active forms have been completely removed is used as a starting material, the Cbz deprotection reaction proceeds smoothly, making peptide synthesis possible. That is, as in Example 9, it was found that by using the method of the present invention, the reductive removal reaction of the N-terminal protecting group of the produced peptide compound can proceed without stagnation.

[0267] Example 19: Synthesis of Cbz-Aib-D-Val-OBn (Condensation reaction) 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. 1.9 mL (3.3 mmol) of a 50% T3P / 2-methyltetrahydrofuran solution was then added at 25 °C and the mixture was stirred for 15 hours to carry out the peptide bond formation reaction. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%)={Cbz-Aib-D-Val-OBn(area %) / [D-Val-OBn(area %)+Cbz-Aib-D-Val-OBn(area %)]}×100

[0268] (hydrolysis treatment) (1) When no amine is added 5.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C for 30 minutes. 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0269] (2) When amine is added To the reaction solution containing the peptide prepared above, 484 mg (4.0 mmol) of DMAP and 5.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25°C for 30 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0270] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 5 mL of 10% aqueous potassium hydrogen sulfate and 2.5 mL of 5% aqueous potassium carbonate. 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 propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without the addition of amine weighed 653.8 mg (yield 116%; the concentrate contained impurities (residual C-terminal active form), but was calculated as containing only the peptide). The concentrate obtained by hydrolysis with the addition of amine weighed 549.4 mg (yield 97%). MS (ESI): m / z 427.3 [M+H] + , 449.2 [M+Na] + .

[0271] [Table 11] 1) LCMS peak area ratio

[0272] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to sufficiently remove the residual C-terminal active compound. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and completely removed. In this case, the target dipeptide was obtained with a purity of 98.6% (yield 97%).

[0273] Example 20: Synthesis of Cbz-Thr(tBu)-Phe-OtBu (Condensation reaction) 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, and 813 μL (4.6 mmol) of diisopropylethylamine was added. 447 mg (2.3 mmol) of EDC hydrochloride was then added at 25°C, and the mixture was stirred for 3 hours to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to the propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-Thr(tBu)-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-Thr(tBu)-Phe-OtBu (area%)]} × 100

[0274] (hydrolysis treatment) (1) When no amine is added 3.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C for 5 minutes. 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0275] (2) When amine is added To the reaction solution containing the peptide prepared above, 142 mg (1.2 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25°C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0276] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous potassium hydrogen sulfate, 3 mL of 5% aqueous potassium carbonate, and 1.5 mL of water. 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 propylamide. 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 (converted to propylamide). The remaining organic layer was concentrated 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] + .

[0277] [Table 12] 1) LCMS peak area ratio

[0278] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to sufficiently remove the residual C-terminal active compound. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and could be completely removed. In this case, the target dipeptide was obtained with a purity of 98.4%.

[0279] Example 21: Synthesis of Cbz-Leu-Thr(tBu)-Phe-OtBu (Cbz deprotection reaction using dipeptides obtained by hydrolysis without the addition of amines) A MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu, synthesized under the amine-free conditions in Example 20, was substituted with 2-methyltetrahydrofuran and concentrated. The resulting mixture was subjected to hydrogenolysis using 99 mg of 5% Pd / C (50% wet) and hydrogen gas. The mixture was stirred at 25°C for 1 hour, but the reaction was not completed (53% conversion). The reaction conversion was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. Conversion rate (%) = {Thr(tBu)-Phe-OtBu (area%) / [Cbz-Thr(tBu)-Phe-OtBu (area%) + Thr(tBu)-Phe-OtBu (area%)]} × 100

[0280] (Cbz deprotection reaction using dipeptides obtained by hydrolysis with amine addition) A MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu, synthesized under the amine addition conditions of Example 20, was substituted with 2-methyltetrahydrofuran and concentrated. The resulting mixture was subjected to hydrogenolysis using 104 mg of 5% Pd / C (50% wet) and hydrogen gas. After stirring at 25°C for 1 hour, the de-Cbz product, Thr(tBu)-Phe-OtBu, was obtained (100% conversion). The reaction conversion rate was determined from the peak area of ​​the LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and analyzed by LC / MS. Conversion rate (%) = {Thr(tBu)-Phe-OtBu (area%) / [Cbz-Thr(tBu)-Phe-OtBu (area%) + Thr(tBu)-Phe-OtBu (area%)]} × 100

[0281] (Condensation reaction) The Cbz-deprotected dipeptide reaction mixture obtained by hydrolysis with amine addition was filtered, the Pd / C was removed, and the mixture was 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. 1.37 mL (2.3 mmol) of a 50% T3P / 2-methyltetrahydrofuran solution was then added and stirred at 25°C for 30 minutes to carry out the peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by adding 5 μL of the reaction mixture to 100 μL of propylamine, diluting with 0.9 mL of methanol, and analyzing the peak area of ​​the LC / MS. Conversion rate (%) = {Cbz-Leu-Thr(tBu)-Phe-OtBu (area%) / [Thr(tBu)-Phe-OtBu (area%) + Cbz-Leu-Thr(tBu)-Phe-OtBu (area%)]} × 100

[0282] To the prepared reaction solution, 139 mg (1.1 mmol) of DMAP and 3.0 mL of 10% aqueous sodium carbonate were added and stirred at 25°C for 5 minutes. After stopping the stirring and allowing the mixture to stand, 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 hydrogen sulfate (2×3 mL), 3 mL of 5% aqueous sodium carbonate, and 3 mL of tap water. 5 μL of the resulting organic layer was added to 100 μL of propylamine and diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Leu-Thr(tBu)-Phe-OtBu, was 98.3%, and no Cbz-Leu-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 638.0 mg of concentrate (88% yield based on 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] + .

[0283] It was found that when the C-terminal active isomer derived from EDC and HOBt remained, the Cbz deprotection reaction proceeded more slowly than when the remaining C-terminal active isomer was completely removed. It was also found that when a peptide solution from which the remaining C-terminal active isomer had been completely removed was used as a starting material, the Cbz deprotection reaction proceeded smoothly, enabling peptide synthesis. That is, it was found that, by using the method of the present invention, the reductive removal reaction of the N-terminal protecting group of the produced peptide compound could proceed without stagnation, as in Examples 9 and 18. This enabled the efficient production of highly pure peptide compounds having the desired amino acid sequence.

[0284] Example 22: Synthesis of Cbz-Ile-Phe-OtBu (Condensation reaction) 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. 479 mg (1.8 mmol) of BEP was then added at 25°C, and the mixture was stirred for 45 minutes at 25°C to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to a propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-Ile-Phe-OtBu (area%)]} × 100

[0285] (hydrolysis treatment) (1) When no amine is added 3.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C for 3 minutes. 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0286] (2) When amine is added To the reaction solution containing the peptide prepared above, 139 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25°C for 3 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0287] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous potassium hydrogen sulfate and 3 mL of 5% aqueous potassium carbonate. 2 mL of 2-MeTHF was added, followed by washing with 1.5 mL of water. 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 propylamide. 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 (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. MS (ESI): m / z 413.3 [M-tBu+H] + ,469.3 [M+H] + , 491.3 [M+Na] + .

[0288] [Table 13] 1) LCMS peak area ratio

[0289] When hydrolysis was performed using alkaline water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to sufficiently remove the residual C-terminal active compound. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and completely removed. In this case, the target dipeptide was obtained with a purity of 96.3%.

[0290] Example 23 Cbz-Phe-MeGly-Phe-piperidine (Boc deprotection reaction) Boc-Phe-piperidine 1 334 mg (1.0 mmol) of this product was dissolved in 3.4 mL of dichloromethane, and 131 μL (2.0 mmol) of methanesulfonic acid was added. The mixture was stirred at 35 °C for 3 hours to carry out the Boc removal reaction (conversion rate: 100%). 5 μL of the reaction mixture was taken, diluted with 1.0 mL of acetonitrile, and the resulting solution was subjected to LC / MS analysis to determine the reaction conversion rate from the LC / MS peak area value. Conversion rate (%)={Phe-piperidine(area %) / [Boc-Phe-piperidine(area %)+Phe-piperidine(area %)]}×100

[0291] (Condensation reaction) To the reaction solution, 528 μL (3.0 mmol) of diisopropylethylamine was added, and the solvent was evaporated. Then, 1.0 mL of acetonitrile, 3.4 mL of 2-methyltetrahydrofuran, 528 μL (3.0 mmol) of diisopropylethylamine, and Cbz-Phe-MeGly-OH were added. 2505 mg (1.5 mmol) of HOOBt and 256 mg (1.6 mmol) of HOOBt were added. 388 mg (2.0 mmol) of EDC hydrochloride was added at 25°C and stirred for 1 hour at 25°C to carry out the peptide bond formation reaction. 5 μL of the reaction mixture was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active group to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was calculated from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-Phe-MeGly-Phe-piperidine(area%) / [Phe-piperidine(area%)+Cbz-Phe-MeGly-Phe-piperidine(area%)]}×100

[0292] To the reaction solution prepared above, 128 mg (1.0 mmol) of DMAP and 3.5 mL of 5% aqueous potassium carbonate were added and stirred at 25 °C for 3 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic and aqueous layers were separated. The aqueous layer was then removed. The organic layer was then diluted with 3.5 mL of 10% aqueous potassium sulfate (3.5 mL x 2) and 3.5 mL of 5% aqueous potassium carbonate. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active compound. The target peptide, Cbz-Phe-MeGly-Phe-piperidine, was 94.6% pure, and no Cbz-Phe-MeGly-NHPr derived from the remaining C-terminal active compound was detected. The remaining organic layer was concentrated to obtain 520.4 mg of concentrate (94% yield).

[0293] When DMAP was added and hydrolysis was performed, the residual C-terminal active substance derived from the peptide fragment was completely decomposed and removed, and the desired tripeptide was obtained with a purity of 94.6% (yield 94%).

[0294] 1) J. Org. Chem., 2003, 68, 7505-7508. 2) Bull. Chem. Soc. Jpn., 2004, 77,1187-1193.

[0295] Example 24: Synthesis of Cbz-Val-Phe-OtBu (Condensation reaction) 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. 447 mg (1.2 mmol) of HATU was then added at 25°C, and the mixture was stirred for 2.5 hours at 25°C to carry out the peptide bond formation reaction. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, 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] + Conversion rate (%) = {Cbz-Val-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-Val-Phe-OtBu (area%)]} × 100

[0296] (hydrolysis treatment) (1) When no amine is added 2.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C. 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 to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the remaining C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula (see table below). C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0297] (2) When amine is added The amine additive (0.8 mmol) shown in the table below and 2.0 mL of 5% aqueous potassium carbonate solution were added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C. 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 to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula (table below). C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0298] [Table 14]

[0299] The addition of DIPEA slightly promoted the hydrolysis of the remaining C-terminal active substance compared with alkaline water alone without the addition of amines, but no significant effect was observed. On the other hand, the addition of DMAP and NMI significantly promoted the hydrolysis of the remaining C-terminal active substance more than the addition of DIPEA, and it was found that the remaining C-terminal active substance was completely hydrolyzed within 5 minutes when DMAP was used in particular. This indicates that amines with less steric hindrance near the nitrogen, such as DMAP, promote the hydrolysis of the remaining C-terminal active substance more than amines with steric hindrance near the nitrogen, such as DIPEA.

[0300] Example 25: Synthesis of Cbz-Ile-Val-OBn (Condensation reaction) Val-OBn hydrochloride (300 mg, 1.2 mmol) and Cbz-Ile-OH (495 mg, 1.9 mmol) were suspended in 3.0 mL of cyclopentyl methyl ether and 0.9 mL of acetonitrile, followed by the addition of 859 μL (4.9 mmol) of diisopropylethylamine. HATU (705 mg, 1.9 mmol) was then added at 25°C, and the mixture was stirred for 1 hour at 25°C to form a peptide bond. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal activated moiety to a propylamide, followed by dilution with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-Ile-Val-OBn (area%) / [Val-OBn (area%) + Cbz-Ile-Val-OBn (area%)]} × 100

[0301] (hydrolysis treatment) (1) When no amine is added 3.0 mL of neutral water was added to the reaction solution containing the peptide prepared above, and the mixture was stirred with a stir bar at 25°C for 5 minutes. 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0302] (2) When amine is added To the reaction solution containing the peptide prepared above, 91 mg (0.7 mmol) of DMAP and 3.0 mL of neutral water were added and stirred at 25°C for 5 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0303] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 3 mL of 10% aqueous sodium hydrogen sulfate, 2 x 3 mL of 5% aqueous sodium carbonate, and 3 x 1.5 mL of tap water. 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 propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). 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%; the concentrate contained impurities (residual C-terminal active form), but was calculated as 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] + .

[0304] [Table 15] 1) LCMS peak area ratio

[0305] When hydrolysis was performed using neutral water alone, the residual C-terminal active compound was not completely hydrolyzed, and subsequent aqueous washing was also unable to remove it. However, when hydrolysis was performed with the addition of DMAP, the residual C-terminal active compound was completely hydrolyzed and could also be completely removed. In this case, the desired dipeptide was obtained with a purity of 99.5% (yield 94%).

[0306] Example 26: Synthesis of Cbz-MeAla-Phe-OtBu (Condensation reaction) Phe-OtBu hydrochloride (200 mg, 0.8 mmol) and Cbz-MeAla-OH (260 mg, 1.2 mmol) were suspended in 2.4 mL of 2-methyltetrahydrofuran and 0.6 mL of acetonitrile, and diisopropylethylamine (271 μL, 1.6 mmol) was added. DMT-MM-n-hydrate (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate) (265 mg, 0.8 mmol, 13 wt% water content) was added at 25 °C. The mixture was stirred at 25 °C for 1 hour, followed by the addition of DMT-MM-n-hydrate (119 mg, 0.4 mmol, 13 wt% water content) and stirring at 25 °C for 1 hour to carry out the peptide bond formation reaction. 5 μL of the reaction mixture was added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active compound to propylamide, and then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was calculated from the LC / MS peak area (conversion rate: 100%). Conversion rate (%) = {Cbz-MeAla-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-MeAla-Phe-OtBu (area%)]} × 100

[0307] (hydrolysis treatment) (1) When no amine is added 2.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the peptide prepared above, and the mixture was stirred at 25°C for 10 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining percentage of C-terminal active form was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0308] (2) When amine is added To the reaction solution containing the peptide prepared above, 96 mg (0.8 mmol) of DMAP and 2.0 mL of 5% aqueous potassium carbonate solution were added and stirred at 25°C for 10 minutes with a stir 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 to propylamide, and then diluted with 0.9 mL of methanol. The remaining C-terminal active form percentage was calculated from the LC / MS peak area values ​​using the following formula. C-terminal active substance remaining rate (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0309] (Post-processing) After stopping the stirring, the mixture was allowed to stand, allowing the organic and aqueous layers to separate, and the aqueous layer was removed. The organic layer was then washed sequentially with 2 mL of 10% aqueous potassium hydrogen sulfate, 2 mL of 5% aqueous potassium carbonate, and 1 mL of tap 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 to propylamide. The mixture was then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak areas of the target peptide and the remaining C-terminal active form (converted to propylamide). 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%; the concentrate contained impurities (residual C-terminal active form), but was calculated as 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] + .

[0310] [Table 16] 1) LCMS peak area ratio

[0311] When DMT-MM was used as a condensation agent, the residual C-terminal active compound was not completely hydrolyzed by alkaline water alone, and subsequent aqueous washings were unable to remove it. However, we found that adding DMAP to the hydrolysis process completely hydrolyzed the residual C-terminal active compound and also removed it. The target dipeptide was obtained with a purity of 98.6% (98% yield). The C-terminal active compound produced from DMT-MM, a condensation agent that can be used in aqueous solvents, is known to be relatively resistant to hydrolysis. However, we found that the addition of an amine additive allowed complete hydrolysis of the residual C-terminal active compound, even when prepared using DMT-MM, in a short time and in a single treatment, and subsequent aqueous washings allowed complete removal.

[0312] Reference Example 1: Synthesis of MeAsp(tBu)-piperidine (Condensation reaction) 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 over 45 minutes at 3-10°C. After the addition was complete, 5 μL of the reaction mixture was diluted with 1.0 mL of methanol and analyzed by LC / MS. The reaction conversion was determined from the LC / MS peak area (conversion: 100%). Conversion rate (%) = {Cbz-MeAsp(tBu)-piperidine(area%) / [Cbz-MeAsp(tBu)-OH(area%)+Cbz-MeAsp(tBu)-piperidine(area%)]}×100

[0313] The reaction mixture was washed with 100 mL of 10% aqueous potassium hydrogen sulfate and 100 mL of 10% potassium carbonate, and the resulting 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] + .

[0314] (Cbz deprotection reaction) The concentrate 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. The mixture was stirred at room temperature for 5 hours to obtain the target product, MeAsp(tBu)-piperidine (100% conversion). The reaction conversion rate was determined by LC / MS analysis of 5 μL of the reaction mixture, diluted with 1.0 mL of acetonitrile, and the resulting solution. Conversion rate (%)={MeAsp(tBu)-piperidine(area%) / [Cbz-MeAsp(tBu)-piperidine(area%)+MeAsp(tBu)-piperidine(area%)]}×100

[0315] The reaction mixture was filtered, and the filtrate was concentrated to give 5.69 g of concentrate (yield quant.). This concentrate was subjected to LC / MS analysis, and the peak area percentage of the target MeAsp(tBu)-piperidine was determined (99.2 area%). MS (ESI): m / z 215.1 [M-tBu+H] + , 271.1 [M+H] + . [Industrial Applicability]

[0316] The present invention enables the production of highly pure peptide compounds without column purification by efficiently removing the C-terminal active form remaining after the condensation reaction when producing peptide compounds.

Claims

1. A method for promoting the hydrolysis of the C-terminal active substance, comprising the step of adding a tertiary amine and water or an aqueous solution to a solution containing the remaining C-terminal active substance, thereby reacting the C-terminal active substance with the tertiary amine.

2. The method of claim 1 , wherein the tertiary amine has nucleophilic reactivity toward the C-terminal activated form.

3. 2. The method of claim 1, wherein the tertiary amine is an amine with low steric hindrance near the nitrogen.

4. The tertiary amine is represented by the following formula (A), (B), or (C): 【Chemical 1】 During the ceremony, R １ ~R ３ is (i) R １ and R ２ together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocycle, and R ３ is C １ -C ２ Alkyl or C ２ hydroxyalkyl; or (ii) each independently, C １ -C ２ Alkyl or C ２ is a hydroxyalkyl, X is N or O; R ４ and R ５ are each independently C １ -C ２ Alkyl or C ２ hydroxyalkyl, or together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocycle, provided that when X is O, R ５ does not exist, R ６ and R ７ are each independently H, C １ -C ２ alkyl, or methoxy; R ８ and R ９ are each independently H, C １ -C ２ Alkyl or C ２ hydroxyalkyl or R ８ and the nitrogen atom to which R ９ The method of claim 1, wherein together with the carbon atom to which it is attached, forms a 5- to 6-membered non-aromatic heterocycle.

5. R １ ~R ３ However, each independently, C １ -C ２ The method of claim 4, wherein the alkyl is alkyl.

6. X is N and R ４ and R ５ However, each independently, C １ -C ２ alkyl, and R ６ and R ７ The method of claim 4 , wherein

7. R ８ and R ９ are each independently H or C １ -C ２ The method of claim 4, wherein the alkyl is alkyl.

8. A method for removing the residual C-terminal active hydrolysate, comprising the step of subjecting a solution containing the hydrolysate to aqueous washing.

9. 9. The method according to claim 8, wherein water or an aqueous solution is used as the aqueous wash.

10. 9. The method according to claim 8, wherein the aqueous wash is water or an aqueous alkaline solution.

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