Synthesis method of peptide compounds

Abstract

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

Description

Technical Field

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

Background Art

[0002] Peptide synthesis, in one form thereof, is carried out using a compound in which the C-terminal carboxyl group of an amino acid or a peptide is activated so as to react with amines such as amino acids and peptides to enable the formation of an amide bond. In this case, if a compound having an activated carboxyl group remains in the reaction solution after the reaction, it causes a problem of deterioration in the quality of the produced peptide.

[0003] Such a compound having an activated C-terminus includes not only a compound in which the carboxyl group used in the peptide synthesis reaction is activated, but also a compound that can react with amines by changing to an activated state during the reaction, for example, azlactone, NCA (N-carboxyanhydride), etc. (hereinafter, these compounds may be referred to as "C-terminal active substances"). In addition, the C-terminal active substances used in the peptide synthesis reaction are not limited to active esters, mixed acid anhydrides, acylisoureas, etc. synthesized using a peptide condensing agent as described in Non-Patent Document 1 or Non-Patent Document 2, and any compound is included as long as it is activated so as to be able to react with amines. Examples of the deterioration in the quality of the produced peptide include the by-production of impurity peptides due to the remaining C-terminal active substances and the mixing of peptides of the inserted sequence as impurities in the final product (Patent Documents 1 and 2).

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

[0005] Further, a method is known in which the residual C-terminal active substance is captured with a polyamine having a primary amino group such as N,N-dimethylpropane-1,3-diamine and converted into a basic compound, and then the amide compound derived from the residual C-terminal active substance is transferred to the aqueous layer by aqueous washing with an acidic aqueous solution and removed (Patent Document 3, Non-Patent Document 3). However, when using primary amines with high nucleophilicity, it is presumed that the primary amine reacts with the electrophilic site of the target peptide to form impurities with a covalent bond, so it is not suitable for the synthesis of high-purity peptides.

[0006] Also, a method is known in which the residual C-terminal active substance is reacted with a scavenger which is an amine containing a latent anion having a protecting group to convert it into an amide compound and remove it (Patent Document 2). However, in this method, after forming the amide compound, it is necessary to go through an aqueous extraction step, perform hydrogenolysis, and then go through another aqueous extraction step, so the operation is complicated.

[0007] In addition, when the C-terminal active substance remains, when deprotecting the protecting group at the N-terminus of the produced peptide, deprotection of the protecting group at the N-terminus of the C-terminal active substance may also occur simultaneously. The deprotected form of the residual C-terminal active substance is an impurity, but when the extinction coefficient is small, it is difficult to detect by general-purpose HPLC, which is not preferable for quality control.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

[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] [Problems to be Solved by the Invention]

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

[0011] The present inventors have found a method capable of removing a remaining C-terminal active form in a reaction mixture by reacting it with a tertiary amine in the synthesis of a peptide compound including condensing a C-terminal active form of an acid component with an amine component.

[0012] The present invention includes the following in a non-limiting specific aspect. [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 active 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 substance The method as described above, including this step. 〔2〕A method for producing a peptide compound, comprising: Step A: A step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active substance 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, and removing the C-terminal active substance by allowing the tertiary amine to act on the unreacted C-terminal active substance The method as described above, including these steps. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the acid component is a first amino acid whose amino group is protected by a protecting group, or a first peptide whose N-terminal amino group is protected by a protecting group. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein the amine component is a second amino acid whose carboxyl group is protected by a protecting group, or a second peptide whose C-terminal carboxyl group is protected by a protecting group. 〔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 with respect to the C-terminal active substance. 〔7〕The method according to any one of 〔1〕 to 〔6〕, wherein the tertiary amine is an amine having a small steric hindrance near nitrogen. 〔8〕The tertiary amine is represented by the following formula (A), (B), or (C): TIFF0007709920000001.tif56158 wherein, R1 to R3 are such that (i) R1 and R2 together with the nitrogen atom to which they are attached form a 5- to 6-membered non-aromatic heterocyclic ring, 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, R8 and R9 are each independently H, C1-C2 alkyl, or C2 hydroxyalkyl, or together with the nitrogen atom to which R8 is attached and the carbon atom to which R9 is attached form a 5- to 6-membered non-aromatic heterocycle, the method according to any one of [1] to [7]. [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 contains one or more non-natural amino acids.

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

[13] , wherein the temperature when the tertiary amine acts on the C-terminal active body is 25°C to 60°C.

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

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

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

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

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

[16] , further comprising, in step B, separating the reaction mixture into an organic layer and an aqueous layer, and then washing the organic layer, and the remaining amount of the C-terminal active body after the washing is 1.0% or less. 〔18〕The method according to any one of 〔1〕~〔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, or N,N-dimethylformamide, or a mixed solvent thereof. 〔19〕The method according to any one of 〔1〕~〔18〕, wherein in the step B, the aqueous solution is an alkaline aqueous solution. 〔20〕The method according to any one of 〔1〕~〔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〕~〔21〕, wherein the time for reacting the tertiary amine with the C-terminal active body is 2 hours or less. 〔23〕The method according to any one of 〔1〕~〔22〕, wherein the time for reacting the tertiary amine with the C-terminal active body is 2 minutes to 2 hours. 〔24〕The method according to any one of 〔1〕~〔23〕, wherein the time for reacting the tertiary amine with the C-terminal active body is 5 minutes to 60 minutes. 〔25〕The method according to any one of 〔1〕~〔24〕, wherein the time for reacting the tertiary amine with the C-terminal active body is 5 minutes to 50 minutes. 〔26〕The method according to any one of 〔1〕~〔25〕, wherein the C-terminal active body 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 HOBt, or a combination of EDC and HOBt. 〔27〕The method according to any one of 〔1〕~〔26〕, further including step C: a step of deprotecting the protecting group at the N-terminus of the peptide compound. The method according to any one of [1] to

[27] , wherein the C-terminal active substance is hydrolyzed and removed by reacting with the tertiary amine. 〔29〕A method for promoting the hydrolysis of a 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 cause the C-terminal active substance to react with the tertiary amine. 〔30〕A method for removing the hydrolyzate, comprising the step of aqueous-washing a solution containing the hydrolyzate of the residual C-terminal active substance.

Advantages of the Invention

[0013] By using the method of the present invention, the C-terminal active substance remaining after the condensation reaction can be easily, efficiently, and in a short time removed by a single hydrolysis treatment followed by aqueous washing. Therefore, a peptide compound can be synthesized with high purity without column purification.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0015] Hereinafter, preferred non-limiting embodiments of the present disclosure will be described.

[0016] All elements described in the following examples are described in this "Embodiments for Carrying Out the Invention" equally, without being bound by any patent practice, custom, law, etc. that may attempt to restrictively interpret the content described in the examples in the country where the patent application is intended to be patented.

[0017] Any partial or complete combination of one or more of the elements described in this disclosure is also intended to be included in this disclosure and is described as being naturally understood by those skilled in the art, as long as it is not technically inconsistent based on the common general knowledge of those skilled in the art.

[0018] (Abbreviations) The abbreviations used in this specification are listed below. Abbreviations of Amino Acids Aib: α-methylalanine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Asp(tBu): O-t-butylaspartic 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 O-t-butylaspartic acid MeGly: N-methylglycine MeIle: N-methylisoleucine MeLeu: N-methyleucine MePhe: N-methylphenylalanine MeVal: N-methylvaline Met: Methionine Phe: Phenylalanine Phe-OtBu: O-t-butylphenylalanine Phe(3-F): 3-fluorophenylalanine Pro: Proline Ser: Serine Ser(tBu): O-tert-butylserine Thr: Threonine Thr(tBu): O-tert-butyl-threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine

[0019] Abbreviations of Reagents / Solvents 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: Diisopropylethylamine 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 tert-butyl ether NMI: N-Methylimidazole NMM: N-Methylmorpholine T3P: Propylphosphonic anhydride (cyclic trimer) TBAF: Tetrabutylammonium fluoride TsOH: p-Toluenesulfonic acid

[0020] Abbreviations of Functional Groups Bn: Benzyl Boc: t-Butoxycarbonyl Cbz: Benzyloxycarbonyl Pfp: Pentafluorophenyl Teoc: 2-(Trimethylsilyl)ethoxycarbonyl

[0021] (Definition of functional groups, etc.) Examples of the "halogen atom" in this specification include F, Cl, Br, or I.

[0022] In this specification, "alkyl" is a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, and does not contain a heteroatom (an atom other than a carbon and hydrogen atom) or an unsaturated carbon-carbon bond in its skeleton, and has a subset of a hydrocarbyl or hydrocarbon group structure containing hydrogen and carbon atoms. Alkyl includes not only linear ones but also branched ones. Specifically, alkyl is an alkyl having 1 to 20 carbon atoms (C1-C 20 , hereinafter "C p -C q " means having p to q carbon atoms), preferably C1-C 10 alkyl, more preferably C1-C6 alkyl, and even more preferably C1-C2 alkyl. Specifically, 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, 2-ethylbutyl, etc.

[0023] As used herein, "alkenyl" refers to a monovalent group having at least one double bond (two adjacent sp 2 carbon atoms). Depending on the arrangement of the double bond and the substituents (if any), the geometric form of the double bond can take an entgegen (E) or zusammen (Z), cis or trans configuration. Alkenyl includes not only linear but also branched-chain ones. Preferably, alkenyl is C2-C 10 alkenyl, more preferably C2-C6 alkenyl, and specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, 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 linear but also branched-chain ones. Preferably, alkynyl is C2-C 10 alkynyl, more preferably C2-C6 alkynyl, and specifically, for example, ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, etc.

[0025] As used herein, "cycloalkyl" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spiro rings. Preferably, cycloalkyl is C3-C8 cycloalkyl, and specifically, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, etc.

[0026] As used herein, "aryl" means a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Examples of aryl include phenyl, naphthyl (e.g., 1-naphthyl, 2-naphthyl), etc.

[0027] As used herein, "heterocyclyl" means a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. Heterocyclyl may have double and / or triple bonds in the ring, the carbon atoms in the ring may be oxidized to form carbonyls, and it may be a monocyclic or fused 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, for example, azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiadinane, 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, 2-oxaspiro[3.3]heptyl, and the like.

[0028] As used herein, "heteroaryl" means an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring, a fused ring with other rings, or may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include, for example, 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, imidazopyridyl, and the like.

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

[0030] As used herein, "alkenyloxy" means an oxy group to which the "alkenyl" as defined above is bonded, and preferably includes C2-C6 alkenyloxy. Specific examples of alkenyloxy include, for example, vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentyloxy, hexyloxy, and the like.

[0031] As used herein, "cycloalkoxy" means an oxy group to which the "cycloalkyl" as defined above is attached, and preferably includes C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include, for example, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and the like.

[0032] As used herein, "aryloxy" means an oxy group to which the "aryl" as defined above is attached, and preferably includes C6-C 10 aryloxy. Specific examples of aryloxy include, for example, phenoxy, 1-naphthyloxy, 2-naphthyloxy, and the like.

[0033] As used herein, "amino" means -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 examples of amino include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, 4- to 8-membered cyclic amino, and the like.

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

[0035] As used herein, "dialkylamino" means a group in which R and R' are independently the "alkyl" as defined above among the "amino" as defined above, and preferably includes di-C1-C6 alkylamino. Specific examples of dialkylamino include, for example, dimethylamino, diethylamino, and the like.

[0036] As used herein, "cyclic amino" means, among the "amino" as defined above, a group in which R and R' together with the nitrogen atom to which they are attached form a ring, preferably a 4- to 8-membered cyclic amino. Specific examples of the cyclic amino include, for example, 1-azetidyl, 1-pyrrolidyl, 1-piperidyl, 1-piperazinyl, 4-morpholinyl, 3-oxazolidyl, 1,1-dioxidothiomorpholinyl-4-yl, 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, and the like.

[0037] As used herein, "hydroxyalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are substituted with a hydroxyl group, preferably C1-C6 hydroxyalkyl, more preferably C2 hydroxyalkyl. Specific examples of the hydroxyalkyl include, for example, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, 5-hydroxypentyl, and the like.

[0038] As used herein, "haloalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are substituted with a halogen, preferably C1-C6 haloalkyl, more preferably C1-C6 fluoroalkyl. Specific examples of the haloalkyl include, for example, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, and the like.

[0039] As used herein, "cyanoalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are substituted with a cyano group, preferably C1-C6 cyanoalkyl. Specific examples of the cyanoalkyl include, for example, cyanomethyl, 2-cyanoethyl, and the like.

[0040] As used herein, "aminoalkyl" means a group in which one or more hydrogens of "alkyl" as defined above are replaced by "amino" as defined above, and C1-C6 aminoalkyl is preferred. Specific examples of aminoalkyl include, for example, 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, 4-aminobutyl, and the like.

[0041] As used herein, "carboxyalkyl" means a group in which one or more hydrogens of "alkyl" as defined above are replaced by carboxy, and C2-C6 carboxyalkyl is preferred. Specific examples of carboxyalkyl include, for example, carboxymethyl, and the like.

[0042] As used herein, "alkenyloxycarbonylalkyl" means a group in which one or more hydrogens of "alkyl" as defined above are replaced by "alkenyloxycarbonyl" as defined above, and C2-C6 alkenyloxycarbonyl C1-C6 alkyl is preferred, and C2-C6 alkenyloxycarbonyl C1-C2 alkyl is more preferred. Specific examples of alkenyloxycarbonylalkyl include, for example, allyloxycarbonylmethyl, 2-(allyloxycarbonyl)ethyl, and the like.

[0043] As used herein, "alkoxyalkyl" means a group in which one or more hydrogens of "alkyl" as defined above are replaced by "alkoxy" as defined above, and C1-C6 alkoxy C1-C6 alkyl is preferred, and C1-C6 alkoxy C1-C2 alkyl is more preferred. Specific examples of alkoxyalkyl include, for example, methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, 2-ethoxyethyl, and the like.

[0044] As used herein, "cycloalkylalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are replaced by the "cycloalkyl" as defined above, and C3-C8 cycloalkyl C1-C6 alkyl is preferred, and C3-C6 cycloalkyl C1-C2 alkyl is more preferred. Specific examples of cycloalkylalkyl include, for example, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl and the like.

[0045] As used herein, "cycloalkoxyalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are replaced by the "cycloalkoxy" as defined above, and C3-C8 cycloalkoxy C1-C6 alkyl is preferred, and C3-C6 cycloalkoxy C1-C2 alkyl is more preferred. Specific examples of cycloalkoxyalkyl include, for example, cyclopropoxymethyl, cyclobutoxymethyl and the like.

[0046] As used herein, "heterocyclylalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are replaced by the "heterocyclyl" as defined above, and 4- to 7-membered heterocyclyl C1-C6 alkyl is preferred, and 4- to 7-membered heterocyclyl C1-C2 alkyl is more preferred. Specific examples of heterocyclylalkyl include, for example, 2-(tetrahydro-2H-pyran-4-yl)ethyl, 2-(azetidin-3-yl)ethyl and the like.

[0047] As used herein, "alkylsulfonylalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are replaced by the "alkylsulfonyl" as defined above, and C1-C6 alkylsulfonyl C1-C6 alkyl is preferred, and C1-C6 alkylsulfonyl C1-C2 alkyl is more preferred. Specific examples of alkylsulfonylalkyl include, for example, methylsulfonylmethyl, 2-(methylsulfonyl)ethyl and the like.

[0048] As used herein, "aminocarbonylalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are substituted by the "aminocarbonyl" as defined above, aminocarbonyl C1-C6 alkyl is preferred, and aminocarbonyl C1-C4 alkyl is more preferred. Specific examples of aminocarbonylalkyl include, for example, 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, 4-(dimethylaminocarbonyl)butyl, and the like.

[0049] As used herein, "aryloxyalkyl" means a group in which one or more hydrogens of the "alkyl" as defined above are substituted by the "aryloxy" as defined above, C6-C 10 aryloxy C1-C6 alkyl is preferred, C6-C 10 aryloxy C1-C2 alkyl is more preferred. Specific examples of aryloxyalkyl include, for example, phenoxymethyl, 2-phenoxyethyl, and the like.

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

[0051] As used herein, "heteroarylalkyl" means a group in which at least one hydrogen atom of the "alkyl" as defined above is substituted with the "heteroaryl" as defined above, and 5- to 10-membered heteroaryl C1-C6 alkyl is preferred, and 5- to 10-membered heteroaryl C1-C2 alkyl is more preferred. Specific examples of heteroarylalkyl include, for example, 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, 2-(5-benzofuranyl)ethyl, and the like.

[0052] As used herein, "non-aromatic heterocyclic ring" means a non-aromatic heterocyclic ring containing 1 to 5 heteroatoms among the atoms constituting the ring. The non-aromatic heterocyclic ring may have double and / or triple bonds in the ring, and the carbon atoms in the ring may be oxidized to form carbonyls. The non-aromatic heterocyclic ring may be a monocyclic ring, a condensed ring, or a spiro ring. The number of atoms constituting the ring is not limited, but is preferably 5 to 6 (5- to 6-membered non-aromatic heterocyclic ring). Specific examples of non-aromatic heterocyclic rings include, for example, 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, diazepane, and the like.

[0053] As used herein, "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, "optionally substituted" means that a group may be optionally substituted by any substituent.

[0055] As used herein, "one or more" means one or a number of two or more. When "one or more" is used in the context related to the substituents of a group, this term means a number from one to the maximum number of substituents allowed for the group. Specifically, examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or a number greater than that.

[0056] As used herein, the "C-terminal active form" includes not only a compound in which the carboxyl group used in the peptide synthesis reaction is activated (e.g., an active ester leading to the production of the target peptide compound), but also a compound that can react with amines (e.g., an amine component) to give the target peptide compound by changing to an activated state during the reaction, such as azlactone, NCA (N-carboxyanhydride), etc. Further, the C-terminal active form, which is a compound in which the carboxyl group used in the peptide synthesis reaction is activated, is not limited to active esters, mixed acid anhydrides, and acylisoureas synthesized using peptide condensing agents as described in, for example, Chem. Rev., 2011, 111, 6557. or Organic Process Research & Development, 2016, 20 (2), 140., and includes any compound that is activated to be able to react with amines.

[0057] As used herein, an "active ester" is a compound containing a carbonyl group that reacts with an amino group to form an amide bond, and a compound in which, for example, OBt, OAt, OSu, OPfp, etc. are bonded to the carbonyl group, and is a compound that promotes the reaction with an amine.

[0058] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. As used herein, "natural amino acid" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, Pro. Unnatural amino acids are not particularly limited, and examples include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with side chains different from natural ones, hydroxycarboxylic acids, and the like. Any configuration of the amino acids is acceptable as used herein. The selection of the side chain of the amino acid is not particularly limited, and in addition to a hydrogen atom, it can be freely selected, for example, from an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group, and one or two non-adjacent methylene groups in these groups may be substituted with an oxygen atom, a carbonyl group (-CO-), a sulfonyl group (-SO2-), a phosphoryl group, or a phosphonyl group. Each may be provided with a substituent, and these substituents are also not limited, and may be independently selected freely from one or more of any substituents containing, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. That is, examples include an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, cycloalkyl group, and the like. In one non-limiting aspect, the amino acid as used herein may be a compound having a carboxy group and an amino group within the same molecule.

[0059] The amino group of the main chain of the amino acid may be unsubstituted (NH2 group) or may be substituted (i.e., -NHR group: R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl 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-), and the carbon chain bonded to the N atom and the α-position carbon atom may form a ring like proline. The amino acid with such a substituted main chain amino group is referred to as "N-substituted amino acid" in this specification. Examples of the "N-substituted amino acid" in this specification include, but are not limited to, N-alkylamino acid, N-C1-C6 alkylamino acid, N-C1-C4 alkylamino acid, N-methylamino acid.

[0060] The "amino acids" constituting the peptide compound in this specification include all corresponding isotopes. The isotope of an "amino acid" is one in which at least one atom is substituted with an atom having the same atomic number (number of protons) and a different mass number (sum of the number of protons and neutrons). Examples of the isotopes included in the "amino acids" constituting the peptide compound of the present invention include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, chlorine atom, etc., respectively, 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, etc. are included.

[0061] Examples of the substituent containing a halogen atom in this specification include alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl having a halogen as a substituent, and more specifically, fluoroalkyl, difluoroalkyl, trifluoroalkyl, etc. are exemplified.

[0062] Examples of substituents containing an O atom include groups such as hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxy (-CO2H), oxycarbonyl (-C(=O)-OR), carbonyloxy (-O-C(=O)-R), thiocarbonyl (-C(=O)-SR), carbonylthio (-S-C(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), carboxylcarbonyl (-C(=O)-CO2H), and the like.

[0063] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, and the like. As alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among them, 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 (-O-C(=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 (-S-C(=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, especially ethylaminocarbonyl, methylaminocarbonyl, etc. are exemplified.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, and the like. In addition to these, groups in which the H atom bonded to the N atom in -C(=O)-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl are also included.

[0070] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and the like. 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, and the like. 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, and the like. 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, and the like. 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-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, and the like. Further, the two H atoms bonded to the N atom in -NH-SO2-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 groups such as thiol (-SH), thio (-S-R), sulfinyl (-S(=O)-R), sulfonyl (-SO2-R), and sulfo (-SO3H).

[0076] Examples of thio (-S-R) 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 "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also referred to as a mono-substituted amino), tertiary amino (-NR(R'); also referred to as a di-substituted 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, azetidinyl, and other groups.

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

[0080] Examples of tertiary amino (-NR(R'); di-substituted amino) include, for example, alkyl(aralkyl)amino and the like, and amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and any two of these substituents may form a ring. Specifically, dialkylamino, especially C1-C6 dialkylamino, C1-C4 dialkylamino, dimethylamino, diethylamino, etc. are exemplified. In this specification, "C p -C q The "dialkylamino group" refers to a group in which two C p -C q alkyl groups are substituted on the amino group, and the two C p -C q alkyl groups may be the same or different.

[0081] Examples of the 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, aralkyl, such as alkyl(aralkyl)(aryl)amidino and the like.

[0082] Examples of the 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, aralkyl, or groups formed by these groups forming a ring and the like.

[0083] Examples of the 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, aralkyl, or groups formed by these groups forming a ring and the like.

[0084] In this specification, the "amino acid residue" constituting the peptide compound may sometimes be simply referred to as "amino acid".

[0085] (Method for producing peptide compound) In one aspect, the present invention relates to a method for producing a peptide compound, the method comprising the following steps. Step A: A step of obtaining a reaction mixture containing a peptide compound obtained by condensing a C-terminal active form of an acid component with an amine component in a solvent, and Step B: A step of mixing the reaction mixture with a tertiary amine and water or an aqueous solution to remove the C-terminal active form.

[0086] Step A is a step of reacting an acid component and an amine component in a solvent using a condensing agent to obtain a reaction mixture containing a peptide compound. Without being bound by a specific theory, in Step A, the acid component reacts with the condensing agent to form a C-terminal active form of the acid component, and then the reaction proceeds by nucleophilic attack of the amine component on the C-terminal active form, resulting in the production of a peptide compound.

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

[0088] The first amino acid is not particularly limited, and any natural amino acid or unnatural amino acid can be used. Also, the first peptide is not particularly limited, and a peptide in which two or more arbitrary natural amino acids and / or unnatural amino acids are linked can be used.

[0089] Preferably, the first amino acid includes those having one or more carbon atoms in its side chain. Specific examples of such amino acids include those having, in their side chains, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted alkoxyalkyl, optionally substituted cycloalkylalkyl, optionally substituted aralkyl, optionally substituted heteroarylalkyl, etc. Also, when the side chain has a functional group that can affect the peptide bond formation reaction, such as an amino group, a carboxyl group, or a hydroxyl group, it is preferable to protect these groups with appropriate protecting groups. Without being bound by a specific theory, when an amino acid has a bulky group in its side chain, due to the steric hindrance, the hydrolysis of the remaining C-terminal active form of the amino acid may not proceed sufficiently by conventional methods. Even in such cases, by using the method of the present invention, the remaining C-terminal active form can be hydrolyzed rapidly and efficiently.

[0090] The side chain of the C-terminal amino acid contained in the first peptide can also have a side chain similar to that of the first amino acid.

[0091] As the protecting group for the amino group of the first amino acid and the N-terminal amino group of the first peptide, a protecting group for amino groups common in the art can be used. Specific examples of such protecting groups include, 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, and 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 can be used relative to the amine component.

[0093] In one embodiment, the C-terminal active form of the acid component in the present invention can be formed by reacting the acid component with a condensing agent in a solvent. The condensing agent is not particularly limited as long as it can introduce a group having a leaving ability to the hydroxy portion of the carboxyl group of the acid component in order to enhance the electrophilicity of the carbonyl carbon of the acid component. Specifically, for example, T3P, HATU, BEP, carbodiimides (such as DIC, EDC, etc.), combinations of carbodiimides and additives (such as oxyma, HOBt, HOBt, etc.), DMT-MM, CDI, etc. can be mentioned.

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

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

[0096] As the amine component, an amino acid in which the carboxyl group is protected by a protecting group, or a peptide in which the carboxyl group at the C-terminus is protected by a protecting group can be used. In the present specification, the amino acid used as the amine component may be referred to as the "second amino acid", and the peptide used as the amine component may be referred to as the "second peptide".

[0097] The second amino acid is not particularly limited, and any natural amino acid or any unnatural amino acid can be used. Also, the second peptide is not particularly limited, and one in which two or more arbitrary natural amino acids and / or unnatural amino acids are linked can be used.

[0098] As the protecting group for the carboxyl group of the second amino acid and the protecting group for the carboxyl group at the C-terminus of the second peptide, a commonly used carboxyl group protecting group in the technical field can be used. Specific examples of such protecting groups include, for example, methyl, allyl, t-butyl, trityl, cumyl, benzyl, methoxytrityl, 1-piperidinyl, and the like.

[0099] In one aspect, as the solvent in the present invention, any solvent can be used as long as the condensation reaction proceeds and a peptide compound can be obtained. Specific examples of such solvents include, for example, toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl acetate, ethyl acetate, methyl tert-butyl ether, cyclopentyl methyl ether, N,N-dimethylformamide, and a solvent obtained by mixing two or more solvents selected from these.

[0100] In one aspect, the "peptide compound" obtained by condensing the C-terminal active form of the acid component with the amine component in the present invention includes linear or cyclic peptide compounds in which two or more amino acids are linked. The cyclic peptide compound is synonymous with the "peptide compound having a cyclic part".

[0101] The "linear peptide compound" in the present invention is formed by linking natural amino acids and / or unnatural amino acids through amide bonds or ester bonds, and is not particularly limited as long as it does not have a cyclic moiety. The total number of natural amino acids 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, 30, and the preferred ranges are 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, 9 to 13.

[0102] The "cyclic peptide compound" in the present invention is formed by linking natural amino acids and / or unnatural amino acids through amide bonds or ester bonds, and is not particularly limited as long as it has a cyclic moiety. The cyclic peptide compound may have one or more linear moieties. The total number of natural amino acids 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, 30, and the preferred ranges are 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, 9 to 13.

[0103] The number of amino acids constituting the cyclic moiety 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 moiety is preferably 5 to 15, more preferably 5 to 14, 7 to 14, or 8 to 14, still 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 moiety of the cyclic peptide is preferably 0 to 8, more preferably 0 to 5, and still more preferably 0 to 3.

[0105] The peptide compound can contain one or more, two or more, three or more, four or more, five or more, or six or more non-natural amino acids. Also, the peptide compound can contain 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, 9 or less non-natural amino acids. When the peptide compound contains non-natural amino acids, examples of the ratio of the number of non-natural amino acids include 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more of the total number of amino acids constituting the peptide compound.

[0106] In addition to, or independently of, the conditions regarding the total number of the above-described natural and non-natural amino acids, the peptide compound can be a linear or cyclic peptide containing at least two (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, particularly preferably 5, 6 or 7, and the preferred range is 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, 9 to 13) N-substituted amino acids and at least one non-N-substituted amino acid. Examples of "N-substitution" include, but are not limited to, substitution of the hydrogen atom bonded to the N atom with a methyl group, an ethyl group, a propyl group, a butyl group, a hexyl group, etc. Preferred N-substituted amino acids include amino acids in which the amino group contained in natural amino acids is N-methylated, N-ethylated, N-propylated, N-butylated, N-pentylated, and these are referred to as N-methyl amino acids, N-ethyl amino acids, N-propyl amino acids, N-butyl amino acids, N-pentyl amino acids. Converting a non-N-substituted amino acid to an N-substituted amino acid is referred to as N-substitution, and may also be referred to as N-alkylation, N-methylation, or N-ethylation. Examples of the ratio of the number of N-substituted amino acids contained in the peptide compound in the present invention include 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more of the total number of amino acids constituting the peptide compound.

[0107] The peptide compound may contain its salt 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 moiety of a cyclic peptide compound, and means a portion not included in each main chain structure.

[0109] As used herein, the "number of amino acids" refers to the number of amino acid residues constituting the peptide compound, and means the number of amino acid units generated when the amide bond, ester bond, and bond of the cyclized portion that link the amino acids are cleaved.

[0110] Step B is a step of removing the unreacted C-terminal active species contained in the reaction mixture obtained in Step A. In one embodiment, the removal of the unreacted C-terminal active species is carried out by reacting the unreacted C-terminal active species with a tertiary amine. In the present specification, the unreacted C-terminal active species, specifically, for example, the C-terminal active species in the reaction mixture remaining without reacting with the amine component during the condensation process may be referred to as "residual C-terminal active species". In Step B, the reaction mixture obtained in Step A, a tertiary amine, and water or an aqueous solution are mixed. When an excessive amount of acid component is used with respect to the amine component in Step A, or when the condensation reaction does not proceed sufficiently in Step A, the C-terminal active species of the acid component remaining without reacting with the amine component remains as an impurity in the reaction solvent. If this remaining C-terminal active species is present in the system without being sufficiently decomposed, it will have an adverse effect on the subsequent deprotection step of the peptide compound and the further peptide chain elongation reaction, so it is important to remove it reliably. In the conventional liquid-phase synthesis method, a method of hydrolyzing the residual active ester using an aqueous alkali solution has been known, but in particular, in the case of the residual C-terminal active species of an amino acid having a bulky functional group in its side chain, or when the leaving ability of the leaving group of the residual C-terminal active species is not high enough to easily react with water, it has been confirmed by the present inventors that the decomposition may be insufficient. On the other hand, by using the method of the present invention in which the C-terminal active species is hydrolyzed by mixing the reaction mixture containing the unreacted C-terminal active species with a tertiary amine and water or an aqueous solution, or by bringing the residual C-terminal active species into contact with a tertiary amine, such problems can be solved.

[0111] As the tertiary amine, those having a nucleophilic reactivity with respect to the remaining C-terminal active form of the acid component can preferably be used. As such a tertiary amine, an amine with a small 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). TIFF0007709920000002.tif56158

[0112] In one embodiment, in formula (A), R1 to R3 are such that R1 and R2 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. Preferably, the 5- to 6-membered non-aromatic heterocycle is pyrrolidine, piperidine, or morpholine, and preferably, the C2 hydroxyalkyl is 2-hydroxyethyl.

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

[0114] Preferably, as the tertiary amine represented by formula (A), those in which R1 to R3 are each independently C1-C2 alkyl are exemplified.

[0115] Specific examples of the tertiary amine represented by formula (A) include trimethylamine, N,N-dimethylethylamine, N,N-diethylmethylamine, triethylamine, triethanolamine, etc. Among these, trimethylamine is particularly preferred.

[0116] In one aspect, 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. Also, in formula (B), R6 and R7 are each independently H, C1-C2 alkyl, or methoxy.

[0117] Preferred as the tertiary amine represented by formula (B) are 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, 4-morpholinopyridine, etc. Among these, DMAP is particularly preferred.

[0119] In one aspect, in formula (C), R8 and R9 are each independently H, C1-C2 alkyl, or C2 hydroxyalkyl, or together with the nitrogen atom to which R8 is attached and the carbon atom to which R9 is attached 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] Preferred as the tertiary amine represented by formula (C) are those in which R8 and R9 are each independently H or C1-C2 alkyl, and more preferably those in which R8 is C1-C2 alkyl and R9 is H.

[0121] As the tertiary amine represented by formula (C), specifically, NMI, imidazole-1-ethanol, 5,6,7,8-tetrahydroimidazo[1,5-α]pyridine, etc. can be mentioned. Among these, NMI is particularly preferable.

[0122] Although not bound by a specific theory, the tertiary amine of the present invention can promote the hydrolysis of the residual C-terminal active substance by performing a nucleophilic attack on the residual C-terminal active substance. Since a tertiary amine such as DIPEA has a bulky substituent, its nucleophilicity is low, which is not desirable. Since the hydrolysis product of the residual C-terminal active substance can be removed by transferring it to the aqueous layer, the produced peptide compound can be subjected to the next condensation reaction without going through a separate purification step such as column purification. By using the method of the present invention, the residual C-terminal active substance can be efficiently removed by a hydrolysis treatment that is rapid (for example, within 5 minutes) and with a small number of times (for example, only 1 time). In certain embodiments, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the residual C-terminal active substance can be removed. In other words, according to the present invention, the residual rate of the C-terminal active substance can be made 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, 1% or less.

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

[0124] When allowing the tertiary amine to act on the residual C-terminal active substance, the reaction mixture can be stirred at a temperature of -20°C to near the boiling point of the solvent, preferably at a temperature of 25°C to 60°C, for 1 minute to 48 hours, preferably 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 aspect, in the step of treating the residual C-terminal active species with a tertiary amine, water or an aqueous solution can be added, and an alkaline aqueous solution can preferably be used as the aqueous solution. Such an alkaline aqueous solution is not particularly limited, and specifically, for example, an aqueous solution of potassium carbonate, an aqueous solution of lithium hydroxide, an aqueous solution of sodium carbonate, sodium hydroxide, an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, or an aqueous solution of cesium carbonate can be mentioned. Among these, an aqueous solution of potassium carbonate or an aqueous solution of sodium carbonate having mild basicity is preferred.

[0126] In one aspect, the present invention further includes a step of, after allowing a tertiary amine to act on the residual C-terminal active species, separating the reaction mixture into an organic layer and an aqueous layer, taking the organic layer, and then washing the organic layer. As an example, it includes washing the organic layer with an acidic aqueous solution and a basic aqueous solution. In one aspect, when this step is carried out, the remaining amount of the residual C-terminal active species can be made 1.0% or less, 0.5% or less, preferably 0.1% or less.

[0127] In one aspect, the present invention further includes a step (step C) of deprotecting the protecting group at the N-terminus of the peptide compound. The deprotection of the protecting group can be carried out, for example, by a conventional method described in "Greene’s, “Protective Groups in Organic Synthesis″ (5th Edition, John Wiley & Sons 2014)". In the conventional method, the deprotection reaction may not proceed sufficiently due to the remaining C-terminal active species, but by using the method of the present invention, a deprotected form of the produced peptide compound can be obtained in a high yield.

[0128] In one aspect, the present invention includes repeating steps A and B a plurality of times. Also, in one aspect, the present invention includes repeating steps A, B, and C a plurality of times. By such repetition, the peptide chain can be extended to obtain a peptide compound.

[0129] In one aspect, the present invention relates to a method for promoting the hydrolysis of a residual C-terminal active substance, which includes the step of adding a tertiary amine and water or an aqueous solution to a solution containing the residual C-terminal active substance to cause the C-terminal active substance to act on the tertiary amine. In this aspect, the residual C-terminal active substance and / or the tertiary amine can be those described above. When adding an aqueous solution to the solution containing the residual C-terminal active substance, the alkaline water is preferably used as the aqueous solution.

[0130] In one aspect, the present invention relates to a method for removing a hydrolyzate of a residual C-terminal active substance, which includes the step of aqueous-washing a solution containing the hydrolyzate of the residual C-terminal active substance. In this aspect, as the aqueous washing, in addition to water, washing with an alkaline aqueous solution can be carried out. The alkaline aqueous solution is not particularly limited, but an aqueous potassium carbonate solution or an aqueous sodium carbonate solution is preferred. Further, in another aspect, when the base used forms a salt with the hydrolyzate and is difficult to transfer to the aqueous layer, after washing and removing the base with an acidic aqueous solution, it can also be washed with an alkaline aqueous solution. The acidic aqueous solution is not particularly limited, but an aqueous potassium hydrogen sulfate solution or an aqueous sodium hydrogen sulfate solution is preferred. The alkaline aqueous solution is preferably an aqueous potassium carbonate solution or an aqueous sodium carbonate solution.

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

Examples

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

[0133] The purity of the peptide compound (the target product in peptide synthesis) and the residual amount of the C-terminal active substance were measured using LCMS equipped with QDA and PDA detectors (column: Ascentis Express C18, 5 cm x 4.6 mm, 2.7 μm, mobile phase: 0.5% aqueous trifluoroacetic acid solution / 0.5% trifluoroacetic acid acetonitrile solution = 95 / 5 - 0 / 100, 1.0 mL / min, detector: UV210 nm). The remaining amount of the C-terminal active substance was evaluated by converting the remaining C-terminal active substance into propylamide because the remaining C-terminal active substance might be hydrolyzed under the analysis conditions (LCMS). The purity of the peptide compound (the target substance in peptide synthesis) was described as the peak area percentage of LCMS. The remaining rate of the C-terminal active substance and the relative value of the remaining amount of the C-terminal active substance were calculated according to the calculation formulas described in each example. The total peak area was corrected by subtracting the area values of the blank peak and the solvent peak. In the table, nd represents not detected.

[0134] (Example 1) Effect of added amine in hydrolysis of remaining C-terminal active substance (Preparation of mixed acid anhydride) 463 mg (1.7 mmol) of Cbz-Ile-OH and 31 mg (internal standard substance: 0.21 mmol) of pentamethylbenzene were dissolved in 3.0 mL of 2-methyltetrahydrofuran. 1.1 mL (6.2 mmol) of diisopropylethylamine and 1.9 mL (3.2 mmol) of 50% T3P / THF solution were added at room temperature, and the mixture was stirred at 40 °C for 1 hour to prepare a mixed acid anhydride (C-terminal active substance) solution. 5 μL was taken from the prepared mixed acid anhydride solution, reacted with 100 μL (1.2 mmol) of normal propylamine, and then diluted with 0.9 mL of methanol. The reaction conversion rate to the mixed acid anhydride was determined from the peak area of LC / MS (conversion rate: 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 - without added amine) Take 1.0 mL from the total amount (6 mL) of the prepared mixed acid anhydride solution, add 0.5 mL of alkaline water (5% lithium hydroxide aqueous solution, 5% sodium carbonate aqueous solution, 5% potassium carbonate aqueous solution, 5% potassium hydroxide aqueous solution, or 5% cesium carbonate aqueous solution), and stir (1200 rpm) with a stir bar at 25 °C. After stopping the stirring, let it stand still to separate the organic layer and the aqueous layer. Take 5 μL of the organic layer, add it to 100 μL (1.2 mmol) of normal propylamine, convert the remaining C-terminal active substance to propylamide, and then dilute it with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard substance)] was calculated.

[0136] (Hydrolysis treatment - amine addition) Take 1.0 mL from the total amount (6 mL) of the prepared mixed acid anhydride solution, add 0.19 mmol of amine additive and 0.5 mL of 5% potassium carbonate aqueous solution, and stir (1200 rpm) with a stir bar at 25 °C. Stop the stirring, let it stand still, and separate the organic layer and the aqueous layer. Take 5 μL of the organic layer, add it to 100 μL (1.2 mmol) of normal propylamine, convert the remaining C-terminal active substance to propylamide, and then dilute it with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the peak area ratio [propylamide:pentamethylbenzene (internal standard substance)] was calculated.

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

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

[0139] The relative value of the remaining amount of the C-terminal active substance in Table 1 indicates that the smaller the value, the more the remaining C-terminal active substance is hydrolyzed. When using only alkaline water, even if the counter cation of the alkali is changed, there is almost no change in the hydrolysis rate, and the hydrolysis is slower than when an amine is added. Among the added amines, it was found that the addition of DMAP and NMI dramatically promoted the hydrolysis of the remaining C-terminal active substance.

[0140] (Example 2) Effect of added amine on hydrolysis of remaining C-terminal active substance (Adjustment 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. At room temperature, 1.0 g (2.64 mmol) of HATU and 1.5 mL (8.79 mmol) of diisopropylethylamine were added, and the mixture was stirred at 60 °C for 4 hours to prepare an active ester (C-terminal active substance) solution. 5 μL was taken from the prepared active ester solution, reacted with 100 μL (1.2 mmol) of normal propylamine, and then diluted with 0.9 mL of methanol. The reaction conversion rate to the active ester was determined from the peak area of LC / MS (conversion rate: 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 with only alkaline water) 1.5 mL was taken from the total amount (9 mL) of the prepared active ester solution, 0.75 mL of alkaline water (5% potassium carbonate aqueous solution) was added, and stirring (1200 rpm) was carried out with a magnetic stirrer at 25 °C. After stopping the stirring, it was allowed to stand still to separate the organic layer and the aqueous layer. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal 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 peak area ratio [propylamide:pentamethylbenzene (internal standard substance)] was calculated.

[0142] (Hydrolysis treatment with amine addition) 1.5 mL was taken from the total amount (9 mL) of the prepared active ester solution, an amine additive (0.44 mmol), and 0.75 mL of 5% potassium carbonate aqueous solution were added, and stirring (1200 rpm) was carried out with a magnetic stirrer at 25 °C. After stopping the stirring, it was allowed to stand still to separate the organic layer and the aqueous layer. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal 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 peak area ratio [propylamide:pentamethylbenzene (internal standard substance)] was calculated.

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

[0144]

Table 2

[0145] The relative value of the remaining amount of the C-terminal active substance in Table 2 indicates that the smaller the value, the more the remaining C-terminal active substance is hydrolyzed. It was found that the addition of an amine promotes the hydrolysis of the remaining C-terminal active substance more than when only alkaline water is used. That is, it was confirmed that the addition of DBU, Me3N, NMI, and DMAP is effective, and in particular, it was found that the addition of NMI and DMAP has a dramatic effect.

[0146] (Example 3) Effect of added amine in hydrolysis of remaining C-terminal active substance 617 mg (2.6 mmol) of Cbz-MeAla-OH and 46 mg (0.31 mmol) of pentamethylbenzene were added to a solution consisting of 4.5 mL of 2-methyltetrahydrofuran at room temperature with 1.5 mL (8.6 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution, and stirred at 40 °C for 1 hour to prepare a mixed acid anhydride solution (C-terminal active substance). 5 μL was taken from the prepared mixed acid anhydride solution, reacted with 100 μL (1.2 mmol) of normal propylamine, and then diluted with 0.9 mL of methanol, and the reaction conversion rate to the mixed acid anhydride was determined from the peak area of LC / MS (conversion rate: 90%). 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 with only alkaline water) Take 1.5 mL from the total amount (9 mL) of the prepared mixed acid anhydride solution, add 0.75 mL of alkaline water (5% sodium carbonate aqueous solution or 5% potassium carbonate aqueous solution), and stir (1200 rpm) with a stir bar at 25 °C. After stopping the stirring, let it stand to separate the organic layer and the aqueous layer. Take 5 μL of the organic layer, add it to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then dilute it with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to calculate the peak area ratio [propylamide:pentamethylbenzene (internal standard substance)].

[0148] (Hydrolysis treatment with amine addition) Take 1.5 mL from the total amount (9 mL) of the prepared mixed acid anhydride solution, add an amine additive (0.43 mmol, 0.67 equivalent), and 0.75 mL of 5% potassium carbonate aqueous solution, and stir (1200 rpm) with a stir bar at 25 °C. Stop the stirring, let it stand to separate the organic layer and the aqueous layer, take 5 μL of the organic layer, add it to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then dilute it with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to calculate the peak area ratio [propylamide:pentamethylbenzene (internal standard substance)].

[0149] (Evaluation of the remaining amount of C-terminal active form) The peak area ratio [propylamide / pentamethylbenzene (internal standard substance)] of LC / MS was used. The relative value of the remaining amount of the C-terminal active form in the following table is the relative value when the value of the peak area ratio [propylamide / pentamethylbenzene] when treated with 5% sodium carbonate aqueous solution for 5 minutes without adding an amine additive is set to 1.1 (the column of 5 min in entry 1) as 100. Relative value of the remaining amount of C-terminal active form (%) = {[propylamide (area %) / pentamethylbenzene (area %)] / 1.1 (propylamide (area %) / pentamethylbenzene (area %) at entry 1, 5 min)} x 100

[0150]

Table 3

[0151] The relative value of the remaining amount of the C-terminal active substance in Table 3 indicates that the smaller the value, the more the remaining C-terminal active substance is hydrolyzed. When using only alkaline water, even when changing the counter cation of the alkali, there was almost no change in the hydrolysis rate. In addition, it was found that when DMAP and NMI were added, the hydrolysis of the remaining C-terminal active substance was promoted compared to the case of using only alkaline water. The addition of DMAP and NMI was sufficiently effective even within 5 minutes. In particular, it was found that the addition of DMAP completely hydrolyzed 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 a peptide bond formation reaction. 5 μL was taken from the reaction solution, reacted with 100 μL (1.2 mmol) of normal propylamine, and then diluted with 0.9 mL of methanol, and the reaction conversion rate was determined from the peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area%) / [H-Phe-OtBu (area%) + Cbz-Ile-Phe-OtBu (area%)]} × 100

[0153] (Hydrolysis treatment with only alkaline water) 1.5 mL was taken from the total amount (9 mL) of the prepared dipeptide solution, 0.75 mL of 5% aqueous potassium carbonate solution was added, and stirring (1200 rpm) was carried out with a stir bar at 25 °C. After stopping the stirring, it was allowed to stand, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of propylamide and the target peptide, and the remaining rate (%) of the C-terminal active form was calculated. The aqueous layer of the remaining reaction solution was removed, and the organic layer was washed successively with 0.5 mL of 5% aqueous potassium bisulfate solution and 0.5 mL of 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active form (converted to propylamide). Remaining rate of C-terminal active form (%) = {[Area % of propylamide] / ([Area % of propylamide] + [Area % of dipeptide])} × 100

[0154] (Hydrolysis treatment with amine addition) 1.5 mL was taken from the total amount (9 mL) of the prepared dipeptide solution, and amine (0.15 mmol, 0.5 equivalent; 0.30 mmol, 1.0 equivalent; or 0.89 mmol, 3 equivalents: the equivalent is based on H-Phe-OtBu hydrochloride), and 0.75 mL of 5% aqueous potassium carbonate solution were added, and stirring (1200 rpm) was carried out at 25 °C with a stir bar. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active substance was converted to propylamide, then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of propylamide and the target peptide, and the residual rate (%) of the C-terminal active substance was calculated. The aqueous layer of the remaining reaction solution was removed, and the organic layer was washed successively with 0.75 mL of 5% aqueous potassium bisulfate solution and 0.75 mL of 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and the remaining C-terminal active substance was converted to propylamide, then diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active substance (converted to propylamide). MS(ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+. Residual rate of C-terminal active substance (%) = {[Area percentage of propylamide] / ([Area percentage of propylamide] + [Area percentage of dipeptide])} × 100

[0155]

Table 4

[0156] It was found that by using 0.5 to 3.0 equivalents of added amine NMI with respect to the N-terminal amino acid derivative, hydrolysis was promoted more advantageously than hydrolysis by treatment with alkaline water alone. Also, it was found that by using 0.5 to 3.0 equivalents of added amine DMAP with respect to the N-terminal amino acid derivative, hydrolysis was promoted more advantageously than hydrolysis by treatment with alkaline water alone. It was found that after performing hydrolysis treatment once with the addition of an amine, the residual C-terminal active substance in the organic layer could be completely removed by washing the organic layer with 5% KHSO4 and 5% K2CO3. At this time, the target peptide was obtained with high purity. On the other hand, in the case of treatment with alkaline water alone, the C-terminal active substance remained and the purity of the dipeptide was also low.

[0157] (Example 5) Synthesis of Cbz-Ile-Phe-OtBu (Condensation reaction) To a solution consisting of 452 mg (1.8 mmol) of H-Phe-OtBu hydrochloride, 702 mg (2.6 mmol) of Cbz-Ile-OH, and 4.5 mL of 2-methyltetrahydrofuran, 1.5 mL (8.8 mmol) of diisopropylethylamine and 2.6 mL (4.4 mmol) of a 50% T3P / THF solution were added at room temperature, and the mixture was stirred at 40 °C for 1 hour to carry out a peptide bond formation reaction. 5 μL was taken from the reaction solution, reacted with 100 μL (1.2 mmol) of normal propylamine, and then diluted with 0.9 mL of methanol, and the reaction conversion rate was determined from the peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {Cbz-Ile-Phe-OtBu (area%) / [H-Phe-OtBu (area%) + Cbz-Ile-Phe-OtBu (area%)]} × 100

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

[0159] (Hydrolysis treatment with amine addition) 1.5 mL was taken from the total amount (9 mL) of the prepared dipeptide solution, 0.29 mmol of amine and 0.75 mL of 5% aqueous potassium carbonate solution were added, and stirring (1200 rpm) was carried out with a stir bar at 60 °C. After stopping the stirring, it was allowed to stand, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active substance was converted to propylamide. Then, it was diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of propylamide and the target peptide, and the residual rate (%) of the C-terminal active substance was calculated. The aqueous layer of the remaining reaction solution was removed, and the organic layer was washed successively with 0.75 mL of 5% aqueous potassium hydrogen sulfate solution and 0.75 mL of 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and the remaining C-terminal active substance was converted to propylamide. Then, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active substance (converted to propylamide). MS(ESI): m / z 413.3 [M-tBu+H]+, 469.3 [M+H]+, 491.3 [M+Na]+. Residual rate of C-terminal active substance (%) = {[Area % of propylamide] / ([Area % of propylamide] + [Area % of dipeptide])} × 100

[0160]

Table 5

[0161] Even when the hydrolysis of the remaining C-terminal active substance was carried out at 60 °C by adding amine, the target peptide was obtained with a purity as high as that when carried out at 25 °C. In particular, when the added amine was DMAP and NMI, the hydrolysis proceeded faster than in the case of using only alkaline water. Also, when the added amine was DMAP and NMI, it was found that the hydrolysis effectively proceeded within 5 minutes in a single hydrolysis treatment, and the remaining C-terminal active substance could be completely removed from the organic layer by subsequent liquid separation operations (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. Then, 594 mg (1.6 mmol) of HATU was added at 25 °C, and the mixture was stirred at 25 °C for 30 minutes, then at 40 °C for 3 hours, and further at 60 °C for 3 hours to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active species 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 peak area values of LC / MS (conversion rate: >99%). Conversion rate (%) = {Area% of Cbz-MeIle-MePhe-OMe / [Area% of MePhe-OMe + Area% of Cbz-MeIle-MePhe-OMe]} × 100

[0163] (Hydrolysis treatment) (1) In the case of no amine addition 3.0 mL of MTBE and 3.0 mL of 5% aqueous potassium carbonate solution were added to the reaction solution containing the above-prepared peptide, and the mixture was stirred at 25 °C with a stir bar for 30 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active species to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active species was calculated from the peak area values of LC / MS according to the following formula. Residual rate of C-terminal active species (%) = {Area% of propylamide / [Area% of propylamide + Area% of dipeptide]} × 100

[0164] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 3.0 mL of MTBE, 103 μL (1.3 mmol) of N-methylimidazole, and 3.0 mL of 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a magnetic stirrer at 25 °C for 30 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and the remaining C-terminal active species was converted to propylamide, followed by dilution with 0.9 mL of methanol. The residual rate of the C-terminal active species was calculated from the peak area values of LC / MS according to the following formula. Residual rate of C-terminal active species (%) = {[Area % of propylamide] / ([Area % of propylamide] + [Area % of dipeptide])} × 100

[0165] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL × 2 of 10% aqueous potassium hydrogen sulfate solution, 3 mL of 5% aqueous potassium carbonate solution, and 1 mL × 5 of deionized water. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine, and the remaining C-terminal active species was converted to propylamide, followed by dilution with 0.9 mL of methanol and subjected to LC / MS analysis to determine the peak area values of the target peptide and the residual C-terminal active species (converted to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without amine addition was 671.8 mg (yield 113%: calculated as containing only the peptide, although the concentrate contains impurities (residual C-terminal active species)). The concentrate obtained by hydrolysis with amine addition was 563.7 mg (yield 95%). MS (ESI): m / z 455.2 [M+H]+, 477.2 [M+Na]+.

[0166]

Table 6

[0167] In the hydrolysis with alkaline water alone, the residual C-terminal active substance was not completely hydrolyzed, and the residual C-terminal active substance could not be removed even by subsequent aqueous washing. However, when hydrolysis was carried out by adding NMI, it was found that the hydrolysis of the residual C-terminal active substance was completely achieved and the removal of the residual C-terminal active substance was also completely possible. Moreover, at this time, the target 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 mixed solvent of 0.6 mL of acetonitrile and 2.4 mL of cyclopentyl methyl ether, and 586 μL (3.4 mmol) of diisopropylethylamine was added. Then, 642 mg (1.7 mmol) of HATU was added at 25 °C, and the mixture was stirred at 25 °C for 6.5 hours to carry out a peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of normal propylamine, and after converting the remaining C-terminal active substance to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the peak area value of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-MeVal-MeAsp(tBu)-piperidine] / [Area% of MeAsp(tBu)-piperidine + Area% of Cbz-MeVal-MeAsp(tBu)-piperidine]} × 100

[0169] (Hydrolysis treatment) (1) In the case of no amine addition 3.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a magnetic stirrer at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated from the peak area value of LC / MS according to the following calculation formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0170] (2) In the case of adding amine 136 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a magnetic stirrer at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated from the peak area value of LC / MS according to the following calculation formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0171] (Work-up) After stopping the stirring, the mixture was allowed to stand still to separate the organic layer and the aqueous layer, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL × 2 of 10% potassium hydrogen sulfate aqueous solution, 3 mL × 2 of 5% potassium carbonate aqueous solution, and 1.5 mL × 3 of normal water. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken and added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active substance to propylamide, and then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active substance (the converted product to propylamide). The remaining organic layer was concentrated to obtain a peptide. The concentrate (peptide) obtained by amine-free hydrolysis was 782.0 mg (yield 136%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active substance)). The concentrate obtained by hydrolysis with amine addition was 530.6 mg (yield 92%). MS(ESI): m / z 518.4 [M+H]+, 540.4 [M+Na]+.

[0172]

Table 7

[0173] In the hydrolysis with treatment by alkaline water alone, the remaining C-terminal active substance was not completely hydrolyzed, and the remaining C-terminal active substance could not be removed even by subsequent aqueous washing. However, it was found that when hydrolysis was carried out by adding DMAP, the hydrolysis of the remaining C-terminal active substance was completely achieved and the removal of the remaining C-terminal active substance was also completely possible. Moreover, at this time, the target dipeptide was obtained with a purity of 100% (yield 92%).

[0174] (Example 8) Synthesis of Cbz-MeVal-MeAsp(tBu)-piperidine (Condensation reaction) MeAsp(tBu)-piperidine 299 mg (1.1 mmol), Cbz-MeVal-OH 458 mg (1.7 mmol) were suspended in 4.5 mL of 2-MeTHF as a solvent, and 775 μL (4.4 mmol) of diisopropylethylamine was added. Then, 1.6 mL (2.8 mmol) of 50% T3P / THF solution was added at 25 °C, and the mixture was stirred at 25 °C for 15 hours to conduct the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active species 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 peak area values of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-MeVal-MeAsp(tBu)-piperidine] / [Area% of MeAsp(tBu)-piperidine + Area% of Cbz-MeVal-MeAsp(tBu)-piperidine]} × 100

[0175] (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 above-prepared peptide, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active species to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active species was calculated from the peak area values of LC / MS according to the following formula. Residual rate of C-terminal active species (%) = {[Area% of propylamide] / [Area% of propylamide + Area% of dipeptide]} × 100

[0176] (2) When amine is added To the reaction solution containing the above-prepared peptide, 141 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated according to the following formula from the peak area values of LC / MS. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0177] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then the organic layer was washed successively with 3 mL of a 10% aqueous potassium hydrogen sulfate solution and 3 mL of a 5% aqueous potassium carbonate solution. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of normal propylamine to convert the remaining C-terminal active form to propylamide, and then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active form (the converted form to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without amine addition was 561.6 mg (yield 98%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active form)). The concentrate obtained by hydrolysis with amine addition was 501.1 mg (yield 87%). MS (ESI): m / z 518.4 [M+H]+, 540.4 [M+Na]+.

[0178]

Table 8

[0179] In the hydrolysis with aqueous alkali alone, the residual C-terminal active species was not completely hydrolyzed, and the residual C-terminal active species could not be removed even by subsequent aqueous washing. However, when hydrolysis was carried out with the addition of DMAP, it was found that the hydrolysis of the residual C-terminal active species was completely achieved and the removal of the residual C-terminal active species was also completely possible. Moreover, at this time, the target dipeptide was obtained with a purity of 100% (yield 87%).

[0180] (Example 9) Synthesis of Cbz-Ile-MeVal-MeAsp(tBu)-piperidine (Cbz deprotection reaction using the dipeptide obtained by hydrolysis treatment without adding amine) 782 mg of Cbz-MeVal-MeAsp(tBu)-piperidine (containing 17.6 area% of residual C-terminal active species) synthesized under amine-free conditions in Example 7 was dissolved in 4.2 mL of cyclopentyl methyl ether. It was subjected to a hydrogenolysis reaction with 115 mg of 5% Pd / C (50% wet) and hydrogen gas. Since the reaction hardly proceeded, after filtering off Pd / C using a filter, it was concentrated to dryness, dissolved again in 4.2 mL of cyclopentyl methyl ether, 105 mg of 5% Pd / C (50% wet) was added, and the hydrogenolysis reaction was carried out again. However, even after reacting for a total of 3 hours, the reaction hardly proceeded (reaction conversion rate: 1.6%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting the filtered solution to LC / MS analysis. Conversion rate (%) = {MeVal-MeAsp(tBu)-piperidine (area%) / [MeVal-MeAsp(tBu)-piperidine (area%) + Cbz-MeVal-MeAsp(tBu)-piperidine (area%)]} × 100

[0181] (Cbz deprotection reaction using the dipeptide obtained by hydrolysis treatment with the addition of amine) 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. It was subjected to a hydrogenolysis reaction with 124 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirred at room temperature for 2 hours to obtain MeVal-MeAsp(tBu)-piperidine which is the Cbz-removed product (conversion rate 100%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, filtering the solution with a filter, and subjecting it to LC / MS analysis. 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 solution was filtered through a filter to remove Pd / C, and then concentrated to dryness. The dried solid was dissolved in 4.3 mL of 2-methyltetrahydrofuran, and 362 mg (1.3 mmol) of Cbz-Ile-OH and 715 μL (4.1 mmol) of diisopropylethylamine were added. Then, 1.4 mL (2.4 mmol) of a 50% T3P / THF solution was added at 25 °C, and the mixture was stirred at 40 °C for 7 hours and then at room temperature for 14 hours to carry out a peptide bond formation reaction (conversion rate: 100%). To the prepared reaction solution, 81 μL (1.0 mmol) of N-methylimidazole and 2.6 mL of a 20% aqueous potassium carbonate solution were added, and the mixture was stirred at 25 °C with a stir bar for 45 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then, the organic layer was successively washed with 5.2 mL of a 10% aqueous potassium hydrogen sulfate solution and 5.2 mL × 2 of a 5% aqueous potassium carbonate solution. 5 μL of the obtained organic layer was added to 100 μL of normal 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 residual C-terminal active species. The target peptide Cbz-Ile-MeVal-MeAsp(tBu)-piperidine was 95.1%, and Cbz-Ile-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 542.7 mg of a concentrate (yield 82%). MS(ESI): m / z 631.5 [M+H] + , 653.4 [M+Na] + .

[0183] It was found that when a peptide solution containing a residual C-terminal active species treated with only alkaline water was used, the Cbz deprotection reaction hardly proceeded. On the other hand, it was found that when a peptide solution obtained by adding DMAP and treating it, in which the residual C-terminal active species was completely removed, was used, the Cbz deprotection reaction proceeded smoothly and the subsequent peptide synthesis reaction became possible. That is, it was found that by using the method of the present invention, the reductive removal reaction of the protecting group at the N-terminus of the produced peptide compound can proceed without stagnation. As a result, a high-purity peptide compound having a desired amino acid sequence could be efficiently produced.

[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. Then, 0.9 mL (1.6 mmol) of a 50% T3P / THF solution was added at 25 °C, and the mixture was stirred at room temperature for 30 minutes to carry out the peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, adding it to 100 μL of normal propylamine, diluting the solution with 0.9 mL of methanol, and subjecting it to LC / MS analysis, and calculating from the peak area values of LC / MS. Conversion rate (%) = {[Area% of Cbz-Phe(3-F)-Phe-OtBu] / [Area% of Phe-OtBu + Area% of Cbz-Phe(3-F)-Phe-OtBu]} × 100

[0185] To the above reaction solution, 95 mg (0.8 mmol) of DMAP and 2.0 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred at 25 °C with a stir bar for 5 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then, the organic layer was washed successively with 1 mL of a 10% aqueous potassium hydrogen sulfate solution, 1 mL of a 5% aqueous potassium carbonate solution, and 1 mL of normal water. 5 μL of the obtained organic layer was added to 100 μL of normal 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 residual C-terminal active species. The target peptide Cbz-Phe(3-F)-Phe-OtBu had a purity of 100%, and Cbz-Phe(3-F)-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 387.2 mg of the concentrate (yield 96%). MS(ESI): m / z 465.2 [M-tBu+H]+, 521.1 [M+H]+, 543.2 [M+Na]+.

[0186] When hydrolysis was carried out by adding DMAP, the removal of the residual C-terminal active form was completely achieved, and the target dipeptide could be 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. Then, 1.4 mL (2.3 mmol) of a 50% T3P / THF solution was added at 25 °C, and the mixture was stirred at room temperature for 1 hour to carry out the peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, adding it to 100 μL of normal propylamine, diluting the solution with 0.9 mL of methanol, and subjecting the resulting solution to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {[Area% of Cbz-Ser(tBu)-Phe-OtBu] / [Area% of Phe-OtBu + Area% of Cbz-Ser(tBu)-Phe-OtBu]} × 100

[0188] To the above reaction solution, 143 mg (1.2 mmol) of DMAP and 1.5 mL of 20% aqueous potassium carbonate solution were added, and the mixture was stirred with a magnetic stirrer at 25 °C for 5 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3.0 mL×2 of 10% aqueous potassium hydrogen sulfate solution, 3.0 mL of 5% aqueous potassium carbonate solution, and 3.0 mL of deionized water. 5 μL of the obtained organic layer was added to 100 μL of normal 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 residual C-terminal active species. The target peptide Cbz-Ser(OtBu)-Phe-OtBu had a purity of 100%, and Cbz-Ser(OtBu)-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 556.4 mg of the concentrate (yield 96%). MS(ESI): m / z 387.1 [M-2tBu+H]+, 499.3 [M+H]+, 521.2 [M+Na]+.

[0189] When hydrolysis was carried out by adding DMAP, the removal of the residual C-terminal active species was completely achieved, and the target dipeptide could be obtained with a purity of 100% (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 deprotection reaction (conversion rate 100%). The reaction conversion rate was determined from the peak area values of LC / MS by subjecting 5 μL of the reaction solution to LC / MS analysis after diluting it with 1.0 mL of acetonitrile. Conversion rate (%) = {Phe-piperidine (area%) / [Boc-Phe-piperidine (area%) + Phe-piperidine (area%)]} × 100

[0191] (Coupling Reaction) To the above reaction solution, 742 μL (4.3 mmol) of diisopropylethylamine was added, and then the solvent was distilled off. Subsequently, 1.4 mL of acetonitrile, 3.3 mL of 2-methyltetrahydrofuran, 742 μL (4.3 mmol) of diisopropylethylamine, and 492 mg (2.1 mmol) of Boc-MeVal-OH were added. At 25 °C, 804 mg (2.2 mmol) of HATU was added, and the mixture was stirred at room temperature for 1 hour to conduct a peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, adding it to 100 μL of normal propylamine, diluting the solution with 0.9 mL of methanol, and subjecting the resulting solution to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {[Area % of Boc-MeVal-Phe-piperidine] / ([Area % of Phe-piperidine] + [Area % of Boc-MeVal-Phe-piperidine])} × 100

[0192] To the above-prepared reaction solution, 168 mg (1.4 mmol) of DMAP and 4.6 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 4.6 mL of a 10% aqueous potassium hydrogen sulfate solution, 4.6 mL of a 5% aqueous potassium carbonate solution, and 1.5 mL × 6 of normal water. 5 μL of the obtained organic layer was added to 100 μL of normal 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 residual C-terminal active species. The target peptide Boc-MeVal-Phe-OtBu had a purity of 99.7%, and Boc-MeVal-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 542.3 mg of a concentrate (yield 86%). MS (ESI): m / z 346.2 [M - Boc + H]+, 446.3 [M + H]+, 468.3 [M + Na]+.

[0193] When hydrolysis was carried out with the addition of DMAP, the removal of the residual C-terminal active form was completely achieved, and even when the N-terminal protecting group was Boc, the target dipeptide could be obtained with a purity of 99.7% (yield 86%).

[0194] (Synthesis example of Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu / SEQ ID NO: 1 (5 mer)) (Synthesis of Cbz-MePhe-MeGly-OtBu) (Coupling reaction) 2.0 g (11.0 mmol) of MeGly-OtBu hydrochloride was suspended in 16 mL of isopropyl acetate and 4 mL of acetonitrile, and 7.7 mL (44.0 mmol) of diisopropyldiethylamine and 3.6 g (11.5 mmol) of Cbz-MePhe-OH were added. The reaction solution was cooled to 0 °C, 9.7 mL (16.5 mmol) of a T3P / ethyl acetate solution was added, and then the mixture was stirred at room temperature for 30 minutes to carry out a peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 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 layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 5% aqueous potassium sulfate solution and 5% aqueous potassium carbonate solution × 2, and then the obtained organic layer was concentrated to obtain 5.0 g of a concentrate (quantitative yield). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MePhe-MeGly-OtBu (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 total amount of Cbz-MePhe-MeGly-OtBu obtained by the above method was dissolved in 75 mL of isopropyl acetate, and subjected to a hydrogenolysis reaction with 0.98 g of 10% Pd / C (3% wet) and hydrogen gas. It was stirred at room temperature for 2 hours to obtain a Cbz-depleted product (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 3 μL of the reaction solution and diluting it with 1.0 mL of methanol for LC / MS analysis. Conversion rate (%) = {[Area % of target compound] / ([Area % of raw material] + [Area % of target compound])} × 100

[0197] (Condensation Reaction) The reaction solution was filtered through a filter, 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. After adding 2.6 g (11.0 mmol) of Cbz-Aze-OH, 13.0 mL (22.0 mmol) of 50% T3P / ethyl acetate solution, and 7.7 mL (44.0 mmol) of diisopropylethylamine, it was stirred at room temperature for 30 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis. Conversion rate (%) = {[Area % of target compound] / ([Area % of raw material] + [Area % of target compound])} × 100

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

[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 a hydrogenolysis reaction with 0.95 g of 10% Pd / C (3% wet) and hydrogen gas. The mixture was stirred at 50 °C for 2 hours to obtain a de-Cbz product (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting the diluted solution to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0200] (Condensation Reaction) The reaction solution was filtered through a filter, and toluene was added for azeotropic dehydration twice. The concentrate was dissolved in 32.8 mL of isopropyl acetate and 8.2 mL of acetonitrile. After adding 2.7 g (11.1 mmol) of Cbz-MeAla-OH and 7.4 mL (42.3 mmol) of diisopropylethylamine, 12.5 mL (21.1 mmol) of 50% T3P / ethyl acetate solution and 7.4 mL (42.3 mmol) of diisopropylethylamine were added. 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 T3P / ethyl acetate solution, and 1.1 mL (6.3 mmol) of diisopropylethylamine were added, and the mixture was further stirred at room temperature for 2 hours to perform a peptide bond formation reaction (conversion rate: 97%). The reaction conversion rate was determined from the peak area value of LC / MS by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting the diluted solution to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 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 layer and the aqueous layer 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. Then, the obtained organic layer was concentrated to obtain 5.8 g of a concentrate (yield 91%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 2) (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 a hydrogenation reaction with 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 through a filter (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS by taking 3 μL of the reaction solution and diluting it with 1.0 mL of methanol for LC / MS analysis. Conversion rate (%) = {Target compound (area%) / [Starting material (area%) + Target compound (area%)]} × 100

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

[0204] (Condensation Reaction) 1.5 g (3.2 mmol) of the above concentrate and 1.3 g (4.7 mmol) of Cbz-Ile-OH were dissolved in 18 mL of isopropyl acetate and 4.5 mL of acetonitrile. 2.2 mL (12.6 mmol) of diisopropylethylamine and 2.4 g (6.3 mmol) of HATU were added, and the mixture was stirred at room temperature for 30 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 3 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 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 layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 22.5 mL × 2 of 5% aqueous potassium sulfate solution and 22.5 mL × 3 of 5% aqueous potassium carbonate solution, and then the obtained organic layer was concentrated to obtain 2.4 g of a concentrate (yield quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-Ile-MeAla-Aze-MePhe-MeGly-OtBu (SEQ ID NO: 1) (99.1 area %). MS (ESI): m / z 744.3 [M+Na] + 。

[0206] By adding NMI and performing hydrolysis once, followed by aqueous washing, complete removal of the residual C-terminal active species was achieved, and the target pentapeptide was obtained with a purity of 99.1%. Its yield was 87% in total from the initial amino acid. This result shows that in continuous liquid-phase peptide synthesis, high-purity pentapeptide synthesis was achieved in high yield by completely removing the residual C-terminal active species using an amine additive.

[0207] (Example 14) Synthesis of Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine / Sequence No.: 4 (11 mer) (Synthesis of Cbz-MeVal-Asp(tBu)-piperidine) (Condensation reaction) 8.6 g (33.5 mmol) of Asp(tBu)-piperidine was dissolved in 108 mL of cyclopentyl methyl ether. 9.79 g (36.9 mmol) of Cbz-MeVal-OH and 17.6 mL (101 mmol) of diisopropylethylamine were added. 13.8 g (50.3 mmol) of BEP was dissolved in 21.5 mL of acetonitrile and then added to the reaction solution, followed by stirring at room temperature for 3 minutes to conduct the peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0208] The reaction solution was washed with 150 mL of 10% potassium hydrogen sulfate aqueous solution, then 150 mL of 5% potassium carbonate aqueous solution and 9.52 g (101 mmol) of trimethylamine hydrochloride were added, and the mixture was stirred at 40 °C for 90 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 150 mL of 5% potassium carbonate aqueous solution, and then the obtained organic layer was concentrated to obtain 17 g of a concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MeVal-Asp(tBu)-piperidine (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 a hydrogenolysis reaction with 1.9 g of 10% Pd / C (3% wet) and hydrogen gas, and stirred at 35 °C for 2 hours (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0210] The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 14.0 g of a concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target MeVal-Asp(tBu)-piperidine (99.5 area %).

[0211] (Condensation reaction) The above concentrate was dissolved in 126 mL of cyclopentyl methyl ether and 14 mL of acetonitrile. 13.0 g (41.7 mmol) of Cbz-MePhe-OH and 52.9 mL (303 mmol) of diisopropylethylamine were added. 67.0 mL (114 mmol) of 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 1 hour to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0212] The reaction solution was washed with 140 mL of a 5% aqueous potassium hydrogen sulfate solution, and then 140 mL of a 5% aqueous potassium carbonate solution and 10.9 g (114 mmol) of trimethylamine hydrochloride were added, followed by stirring at room temperature for 30 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 140 mL of a 5% aqueous potassium carbonate solution. Then, the obtained organic layer was concentrated to obtain 24.1 g of a concentrate (yield: 96%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MePhe-MeVal-Asp(tBu)-piperidine (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 a hydrogenolysis reaction with 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 by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {Area percentage of target compound / [Area percentage of raw material + Area percentage of target compound]} × 100

[0214] The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 18.1 g of a concentrate (yield: 99%).

[0215] (Condensation Reaction) 17.3 g (32.6 mmol) of the above concentrate was dissolved in 153 mL of cyclopentyl methyl ether and 17 mL of acetonitrile. 10.6 g (35.9 mmol) of Cbz-Ser(tBu)-OH and 45.5 mL (261 mmol) of diisopropylethylamine were added. 57.6 mL (98.0 mmol) of 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 15 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0216] The reaction solution was washed with 170 mL of 5% potassium bisulfate aqueous solution, then 170 mL of 5% potassium carbonate aqueous solution and 9.4 g (98.0 mmol) of trimethylamine hydrochloride were added, and the mixture was stirred at room temperature for 2 hours. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 170 mL of 5% potassium carbonate aqueous solution, and then the obtained organic layer was concentrated to obtain 26.5 g of a concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 5) (98.9 area %). MS (ESI): 830.4 [M+Na] + 。

[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 a hydrogenolysis reaction with 2.4 g of 10% Pd / C and hydrogen gas, and stirred at 35 °C for 2 hours (conversion rate: >98%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0218] The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 19.5 g of the concentrate (yield 97%).

[0219] (Condensation reaction) 16.0 g (23.7 mmol) of the above concentrate was dissolved in 200 mL of cyclopentyl methyl ether. 7.3 g (26.1 mmol) of Cbz-MeIle-OH and 12.4 mL (71.2 mmol) of diisopropylethylamine were added. 9.8 g (35.6 mmol) of BEP was dissolved in 40 mL of acetonitrile, then added to the reaction solution, and stirred at room temperature for 5 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0220] The reaction solution was washed with 240 mL of a 10% aqueous sodium bisulfate solution, and then 240 mL of a 5% aqueous potassium carbonate solution and 6.7 g (71.2 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40 °C for 1.5 h. Stirring was stopped, and the organic layer and the aqueous layer were separated. The aqueous layer was removed, and the remaining organic layer was washed with 240 mL of a 5% aqueous potassium carbonate solution. Then, the obtained organic layer was concentrated to obtain 22.2 g of a concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 6) (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 a hydrogenolysis reaction with 1.9 g of 10% Pd / C and hydrogen gas, followed by stirring at 35 °C for 2 h. The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 15.6 g of a concentrate (yield 96%).

[0222] (Coupling Reaction) 15.3 g (19.1 mmol) of the above concentrate was dissolved in 138 mL of cyclopentyl methyl ether and 15 mL of acetonitrile. 4.7 g (21.0 mmol) of Cbz-MeGly-OH and 26.7 mL (153 mmol) of diisopropylethylamine were added. 33.8 mL (57.3 mmol) of a 50% T3P / ethyl acetate solution was added, followed by stirring at room temperature for 15 min to conduct a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0223] The reaction solution was washed with 153 mL of 5% potassium bisulfate aqueous solution, then 153 mL of 5% potassium carbonate aqueous solution was added, and the mixture was stirred at room temperature for 5 minutes. Stirring was stopped, the organic layer and the aqueous layer were separated, and after removing the aqueous layer, 153 mL of 5% potassium carbonate aqueous solution was added, and the mixture was stirred at room temperature for 1 hour. After removing the aqueous layer, the obtained organic layer was concentrated to obtain 19.5 g of a concentrate (yield: quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 7) (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 a hydrogenolysis reaction with 1.9 g of 10% Pd / C and hydrogen gas, and stirred at 35 °C for 3 hours (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0225] The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 16.3 g of a concentrate (yield 99%).

[0226] (Condensation Reaction) 16.0 g (18.4 mmol) of the above concentrate was dissolved in 144 mL of cyclopentyl methyl ether and 16 mL of acetonitrile. 5.1 g (20.2 mmol) of Cbz-Val-OH and 25.6 mL (147 mmol) of diisopropylethylamine were added. 32.4 mL (55.0 mmol) of 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 30 minutes to conduct a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area% of target compound / [Area% of raw material + Area% of target compound]} × 100

[0227] The reaction solution was washed with 160 mL of 5% potassium bisulfate aqueous solution, then 153 mL of 5% potassium carbonate aqueous solution and 5.3 g (55.0 mmol) of trimethylamine hydrochloride were added, and the mixture was stirred at 60 °C for 1 hour. Stirring was stopped, the organic layer and the aqueous layer were separated, the aqueous layer was removed, and the obtained organic layer was washed with 160 mL of 5% potassium carbonate aqueous solution and concentrated to obtain 20.0 g of a concentrate (yield 99%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 8) (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 a hydrogenolysis reaction with 1.8 g of 10% Pd / C and hydrogen gas. The mixture was stirred at 35 °C for 6 hours and further at 45 °C for 4 hours (conversion rate: 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = { (Area % of target compound) / [(Area % of raw material) + (Area % of target compound)]} × 100

[0229] The same operation was performed again, and the combined reaction solution was filtered through a filter. The filtrate was concentrated to obtain 15.9 g of a concentrate (yield 98%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 10) (97.9 area %).

[0230] (Condensation reaction) 14.5 g (14.9 mmol) of the above concentrate was dissolved in 181 mL of cyclopentyl methyl ether. 4.6 g (16.4 mmol) of Cbz-MeLeu-OH and 7.8 mL (44.8 mmol) of diisopropylethylamine were added. 4.9 g (17.9 mmol) of BEP was dissolved in 36 mL of acetonitrile, and the resulting BEP solution was added to the reaction solution, followed by stirring at 40 °C for 1 minute to conduct a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = { (Area % of target compound) / [(Area % of raw material) + (Area % of target compound)]} × 100

[0231] The reaction solution was washed with 128 mL of a 10% aqueous sodium bisulfate solution, and then 128 mL of a 5% aqueous potassium carbonate solution and 4.2 g (44.8 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40 °C for 30 minutes. Stirring was stopped, the organic layer and the aqueous layer were separated, and after removing the aqueous layer, the obtained organic layer was washed with 128 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 18.0 g of a concentrate (yield 98%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 9) (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 a hydrogenolysis reaction with 1.6 g of 10% Pd / C and hydrogen gas, followed by stirring at 45 °C for 4 hours (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area percentage of target compound / [Area percentage of starting material + Area percentage of target compound]} × 100

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

[0234] (Condensation reaction) 13.0 g (11.8 mmol) of the above concentrate was dissolved in 117 mL of cyclopentyl methyl ether and 13 mL of acetonitrile. 3.5 g (13.0 mmol) of Cbz-Leu-OH and 16.5 mL (95.0 mmol) of diisopropylethylamine were added. 20.9 mL (35.5 mmol) of 50% T3P / ethyl acetate solution was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and obtaining it from the peak area value of LC / MS. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0235] The reaction solution was washed with 130 mL of 5% potassium bisulfate aqueous solution, then 130 mL of 5% potassium carbonate aqueous solution and 3.4 g (35.5 mmol) of trimethylamine hydrochloride were added, and the mixture was stirred at 60 °C for 45 minutes. Stirring was stopped, the organic layer and the aqueous layer were separated, the aqueous layer was removed, and the obtained organic layer was washed with 130 mL of 5% potassium carbonate aqueous solution and concentrated to obtain 15.6 g of a concentrate (yield 98%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 11) (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 a hydrogenolysis reaction with 2.0 g of 10% Pd / C and hydrogen gas, and stirred at 45 °C for 4 hours (conversion rate: 100%). After the reaction solution was filtered through a filter, the filtrate was concentrated to obtain 8.9 g of a concentrate (yield 99%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. MS (ESI): m / z 1211.7 [M+H] + , 1233.7 [M+Na] + . Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0237] (Condensation reaction) 7.0 g (5.8 mmol) of the above concentrate was dissolved in 87.5 mL of cyclopentyl methyl ether. 2.0 g (6.4 mmol) of Cbz-MePhe-OH and 3.0 mL (17.3 mmol) of diisopropylethylamine were added. 1.9 g (17.9 mmol) of BEP was dissolved in 17.5 mL of acetonitrile, and the resulting BEP solution was added to the reaction solution, and stirred at room temperature for 3 minutes to carry out a peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0238] The reaction solution was washed with 105 mL of a 10% aqueous sodium bisulfate solution, and then 105 mL of a 5% aqueous potassium carbonate solution and 1.7 g (17.3 mmol) of trimethylamine hydrochloride were added, followed by stirring at 40 °C for 30 minutes. Stirring was stopped, the organic layer and the aqueous layer were separated, and after removing the aqueous layer, the obtained organic layer was washed with 105 mL of a 5% aqueous potassium carbonate solution and concentrated to obtain 8.6 g of a concentrate (yield 99%). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target Cbz-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 12) (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 a hydrogenolysis reaction with 2.0 g of 10% Pd / C and hydrogen gas, followed by stirring at 45 °C for 4 hours (conversion rate: 100%). After filtering the reaction solution through a filter, the filtrate was concentrated to obtain 6.8 g of a concentrate (yield 98%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and then subjecting it to LC / MS analysis and calculating from the peak area values of LC / MS. Conversion rate (%) = {Area percentage of target compound / [Area percentage of raw material + Area percentage of target compound]} × 100

[0240] (Condensation Reaction) The above 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 50% T3P / ethyl acetate solution was added, and the mixture was stirred at room temperature for 2 hours to carry out the peptide bond formation reaction (conversion rate: >99%). The reaction conversion rate was determined from the peak area value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of target compound / [Area % of raw material + Area % of target compound]} × 100

[0241] The reaction solution was washed with 5.0 mL of 10% aqueous sodium bisulfate solution, then 5.0 mL of 5% aqueous potassium carbonate solution and 104 mg (1.1 mmol) of trimethylamine hydrochloride were added, and the mixture was stirred at room temperature for 1 hour. Stirring was stopped, the organic layer and the aqueous layer were separated, the aqueous layer was removed, and the obtained organic layer was washed with 5.0 mL of 5% aqueous potassium carbonate solution and concentrated to obtain 555 mg of a concentrate (yield 96%, Cbz-MeAla-MePhe-Leu-MeLeu-Val-MeGly-MeIle-Ser(tBu)-MePhe-MeVal-Asp(tBu)-piperidine (SEQ ID NO: 4) was 95.3 area %). MS (ESI): m / z 1591.9 [M+H] + , 1613.9 [M+Na] + 。

[0242] By adding trimethylamine and performing hydrolysis once, followed by aqueous washing, complete removal of the residual C-terminal active species was achieved, and a peptide consisting of 11 amino acids was obtained with a high purity of 95.3%. The yield was 75.3% in total from the first amino acid. This result indicates that in continuous liquid-phase peptide synthesis, high-purity polypeptide synthesis can be achieved by removing the residual C-terminal active species using an amine additive.

[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 Teocylation reaction. A 5% aqueous potassium hydrogen sulfate solution was added to make the reaction solution acidic, and then it was extracted with 50 mL of ethyl acetate. The organic layer was washed with saturated brine. The obtained 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 Pfpylation reaction. After the reaction solution was washed with saturated brine, the aqueous layer was extracted with 50 mL of ethyl acetate. The combined organic layers were concentrated, and the obtained concentrate was purified by column chromatography (ethyl acetate / heptane) to obtain 6.63 g of Teoc-MeLeu-OPfp (yield: 90%).

[0244] (Condensation reaction) 201 mg (0.8 mmol) of Phe-OtBu hydrochloride and 536 mg (1.2 mmol) of Teoc-MeLeu-OPfp were suspended in 3.0 mL of isopropyl acetate, 257 μL (2.3 mmol) of 4-methylmorpholine was added, and the mixture was stirred at 25 °C for 3 hours to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, the remaining C-terminal active species was converted 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 peak area value of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Teoc-MeLeu-Phe-OtBu] / [Area% of Phe-OtBu + Area% of Teoc-MeLeu-Phe-OtBu]} × 100

[0245] (Hydrolysis treatment) (1) In the case of no amine addition To the reaction solution containing the prepared peptide, 2.0 mL of a 5% aqueous sodium carbonate solution was added, and the mixture was stirred with a magnetic stirrer at 25 °C for 20 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated from the peak area values of LC / MS according to the following calculation formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0246] (2) In the case of amine addition To the reaction solution containing the prepared peptide, 95 mg (0.8 mmol) of DMAP and 2.0 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred with a magnetic stirrer at 25 °C for 20 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated from the peak area values of LC / MS according to the following calculation formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0247] (Post-treatment) After stopping the stirring, the mixture was allowed to stand still to separate the organic layer and the aqueous layer, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 2 mL of 10% potassium hydrogen sulfate aqueous solution twice and 2 mL of 5% sodium carbonate aqueous solution. Further, washing with 1 mL of 5% potassium carbonate aqueous solution and 1 mL of normal water twice was repeated three times. 5 μL of the obtained organic layer was taken and added to 100 μL (1.2 mmol) of propylamine to convert the remaining C-terminal active form into propylamide, and 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 (the converted form to propylamide). The concentrate (peptide) obtained by hydrolysis without adding amine was 576.6 mg (yield 150%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active form)). The concentrate obtained by hydrolysis with amine addition was 369.6 mg (yield 96%). MS(ESI): m / z 437.3 [M-tBu+H]+, 493.3 [M+H]+, 515.3 [M+Na]+.

[0248]

Table 9

[0249] In the hydrolysis of the remaining C-terminal active form with only alkali water treatment, where the protecting group was Teoc and the C-terminal active form site was Pfp, the remaining C-terminal active form was not completely hydrolyzed, and the remaining C-terminal active form could not be removed even by subsequent aqueous washing. On the other hand, it was found that when hydrolysis was carried out by adding DMAP, the hydrolysis of the remaining C-terminal active form was completely achieved and the removal of the remaining C-terminal active form was also complete. The target dipeptide was obtained with a purity of 96.3% (yield 96%).

[0250] (Example 16) Synthesis of Cbz-Aib-MeLeu-Phe-OtBu (Teoc deprotection reaction using the dipeptide obtained by hydrolysis without adding amine) 576.6 mg of Teoc-MeLeu-Phe-OtBu (including 8.9 area% of residual C-terminal active form) synthesized under amine-free conditions in Example 15 was dissolved in 2.0 mL of 2-methyltetrahydrofuran. After adding 1.5 mL (1.5 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF, the mixture was stirred at 50 °C for 2.5 hours. Since the reaction was not completed, 0.75 mL (0.75 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added and stirred for 2.5 hours. Further, 0.75 mL (0.75 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF was added and stirred for 30 minutes to obtain MeLeu-Phe-OtBu which is a Teoc-removed product (conversion rate 100%). The reaction conversion rate was determined from the peak area values of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area% of MeLeu-Phe-OtBu / [Area% of Teoc-MeLeu-Phe-OtBu + Area% of MeLeu-Phe-OtBu]} × 100

[0251] (Condensation reaction) After concentrating until the internal volume became about 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This operation was repeated 2 more times, and 0.5 mL of acetonitrile, 276 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine were added to the obtained 2-methyltetrahydrofuran solution. Then, 441 mg (1.1 mmol) of HATU was added at 25 °C, and the mixture was stirred at room temperature for 14 hours. 579 mg (1.5 mmol) of HATU was added, and after stirring at 40 °C for 1 hour, 684 mg (1.8 mmol) of HATU was added and the temperature was raised to 60 °C, and the mixture was stirred for 4.5 hours. Further, 455 mg (1.1 mmol) of HATU was added, and the mixture was stirred at 60 °C for 2 hours, at room temperature for 12 hours, and at 60 °C for 2 hours, but no progress of the condensation reaction was observed (conversion rate 0%). The reaction conversion rate was determined from the peak area values of LC / MS by adding 5 μL of the reaction solution to 100 μL of propylamine, diluting it with 0.9 mL of methanol, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area percentage of Cbz-Aib-MeLeu-Phe-OtBu / [Area percentage of MeLeu-Phe-OtBu + Area percentage of Cbz-Aib-MeLeu-Phe-OtBu]} × 100

[0252] (Teoc Deprotection Reaction Using the Dipeptide Obtained by Hydrolysis Treatment with Amine Addition) 369.6 mg (0.75 mmol) of Teoc-MeLeu-Phe-OtBu synthesized under the amine addition conditions in Example 15 was dissolved in 2.0 mL of 2-methyltetrahydrofuran. After adding 1.5 mL (1.5 mmol) of an 8.4% aqueous tetrahydrofuran solution of TBAF, the mixture was stirred at 50 °C for 2.5 hours to obtain MeLeu-Phe-OtBu, the Teoc-deprotected product (conversion rate 100%). The reaction conversion rate was determined from the peak area values of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting it to LC / MS analysis. Conversion rate (%) = {Area percentage of MeLeu-Phe-OtBu / [Area percentage of Teoc-MeLeu-Phe-OtBu + Area percentage of MeLeu-Phe-OtBu]} × 100

[0253] (Condensation Reaction) After concentration until the content volume reached approximately 1 mL, 2 mL of 2-methyltetrahydrofuran was added. This operation was repeated two more times. Then, 0.5 mL of acetonitrile, 273 mg (1.1 mmol) of Cbz-Aib-OH, and 0.66 mL (3.8 mmol) of diisopropylethylamine were added to the resulting 2-methyltetrahydrofuran solution. Next, 439 mg (1.1 mmol) of HATU was added at 25 °C, and the mixture was stirred at room temperature for 14 hours. 576 mg (1.5 mmol) of HATU was added, and the mixture was stirred at 40 °C for 5.5 hours. Further, 452 mg (1.1 mmol) of HATU was added, and the mixture was stirred at 60 °C for 2 hours, at room temperature for 12 hours, and at 60 °C for 2 hours (conversion rate 86%). The reaction conversion rate was determined from the peak area values 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 (%) = {[Area% of Cbz-Aib-MeLeu-Phe-OtBu] / [Area% of MeLeu-Phe-OtBu + Area% of Cbz-Aib-MeLeu-Phe-OtBu]} × 100

[0254] 92 mg (0.75 mmol) of DMAP and 4.0 mL of 10% aqueous potassium carbonate solution were added to the prepared reaction solution, and the mixture was stirred with a stir bar at 25 °C for 1 hour. After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. 5 μL of the obtained 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 residual C-terminal active species. The target peptide Cbz-Aib-MeLeu-Phe-OtBu was 86.7%, the starting material MeLeu-Phe-OtBu was 13.3%, and Cbz-Aib-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 295.6 mg of a concentrate with the above composition. MS(ESI): m / z 568.4 [M+H]+, 590.4 [M+Na]+.

[0255] When a C-terminal activator remains in a solution of a peptide with a protected N-terminus, it has been found that a large excess of reagent is required when deprotecting the N-terminal protecting group (Teoc) of the peptide with a hydrofluoric acid-containing reagent. This is presumably because the deprotection reagent also reacts with the remaining C-terminal activator. Also, it has been clarified that the subsequent condensation reaction (peptide bond formation reaction) with another C-terminal activator in the next step following deprotection does not proceed at all. Presumably, the excess reagent used in the previous step (deprotection of the N-terminus) decomposed the C-terminal activator. Thus, it has been found that when a C-terminal activator remains, a large excess of reagent is required for deprotection of the N-terminus, and this leads to hindrance of the progress of the subsequent condensation reaction. On the other hand, it has been found that if a peptide solution from which the remaining C-terminal activator has been completely removed is used as a raw material, the deprotection reaction can be completed with an appropriate amount of deprotection reagent, and the condensation reaction in the next step can also be carried out.

[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 HOBt were suspended in 0.9 mL of acetonitrile and 3.6 mL of MTBE, and 1.0 mL (5.8 mmol) of diisopropylethylamine was added. Then, 443 mg (2.3 mmol) of EDC hydrochloride was added, and the mixture was stirred at 25 °C for 30 minutes to carry out a peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal activator to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the peak area value of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-MeAla-Phe-OtBu] / [Area% of Phe-OtBu + Area% of Cbz-MeALa-Phe-OtBu]} × 100

[0257] (Hydrolysis treatment) (1) In the case of no amine addition To the reaction solution containing the above-prepared peptide, 3.0 mL of a 5% aqueous sodium carbonate solution was added, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0258] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 147 mg (1.1 mmol) of DMAP and 3.0 mL of a 5% aqueous sodium carbonate solution were added, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0259] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then, the organic layer was washed successively with 3 mL × 2 of a 10% aqueous sodium hydrogen sulfate solution and 3 mL of a 5% aqueous sodium carbonate solution. 5 μL of the obtained 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 form. MS (ESI): m / z 385.2 [M-tBu+H]+, 441.3 [M+H]+, 463.2 [M+Na]+.

[0260]

Table 10

[0261] Even for alanine, which is a small amino acid with a substituent, in hydrolysis with only alkaline water, the residual C-terminal active form was not completely hydrolyzed, and the residual C-terminal active form could not be sufficiently removed even by subsequent liquid separation operation (aqueous washing). On the other hand, when hydrolysis was carried out by adding DMAP, it was found that the hydrolysis of the residual C-terminal active form was completely achieved and the removal of the residual C-terminal active form could also be completed. Moreover, at this time, the target dipeptide was obtained with a purity of 98.4%.

[0262] (Example 18) Synthesis of Cbz-Hph-MeAla-Phe-OtBu (Cbz deprotection reaction using the dipeptide obtained by hydrolysis without adding amine) The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized under the condition of no amine addition in Example 17 was replaced and concentrated with 2-methyltetrahydrofuran. It was subjected to a hydrogenolysis reaction with 101 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was carried out 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 value of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and then subjecting it to LC / MS analysis. Conversion rate (%) = {MeAla-Phe-OtBu (area %) / [Cbz-MeAla-Phe-OtBu (area %) + MeALa-Phe-OtBu (area %)]} × 100

[0263] (Cbz deprotection reaction using the dipeptide obtained by hydrolysis with amine addition) The MTBE solution of Cbz-MeAla-Phe-OtBu synthesized under the amine addition conditions in Example 17 was replaced with 2-methyltetrahydrofuran and concentrated. It was subjected to a hydrogenolysis reaction with 102 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was carried out at 25 °C for 3 hours to obtain MeAla-Phe-OtBu, which is the de-Cbz product (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and then subjecting it to LC / MS analysis. 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 reaction solution of the Cbz-deprotected product of the dipeptide obtained by hydrolysis treatment with amine addition was filtered through a filter, and after filtering off Pd / C, it 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. Then, 1.37 mL (2.33 mmol) of a T3P / 2-methyltetrahydrofuran solution was added, and stirring was carried out at 25 °C for 1.5 hours to perform a peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS by adding 5 μL of the reaction solution to 100 μL of propylamine, diluting it with 0.9 mL of methanol, and then subjecting it to LC / MS analysis. 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 solution were added, and the mixture was stirred at 25 °C for 15 minutes. After stopping the stirring and allowing it to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL of 10% aqueous sodium bisulfate solution, 3 mL of 5% aqueous sodium carbonate solution, and 3 mL of normal water. 5 μL of the obtained 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 residual C-terminal active species. The target peptide Cbz-Hph-MeAla-Phe-OtBu was 99.0%, and Cbz-Hph-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 618.4 mg of the concentrate (yield 88% from Phe-OtBu of Example 17). MS(ESI): m / z 602.4 [M+H]+, 624.4 [M+Na]+.

[0266] It was revealed that when the C-terminal active species derived from EDC and HOOBt remained, the Cbz deprotection reaction after peptide elongation hardly proceeded. On the other hand, it was found that if a peptide solution in which the residual C-terminal active species was completely removed was used as a raw material, the Cbz deprotection reaction proceeded smoothly and peptide synthesis reaction became possible. That is, similar to the case of Example 9, it was found that by using the method of the present invention, the reductive removal reaction of the protecting group at the N-terminus of the produced peptide compound can proceed without stagnation.

[0267] (Example 19) Synthesis of Cbz-Aib-D-Val-OBn (Coupling 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. Then, 1.9 mL (3.3 mmol) of 50% T3P / 2-methyltetrahydrofuran solution was added at 25 °C, and the mixture was stirred at 25 °C for 15 hours to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active species was converted 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 peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area % of Cbz-Aib-D-Val-OBn] / ([Area % of D-Val-OBn] + [Area % of Cbz-Aib-D-Val-OBn])} × 100

[0268] (Hydrolysis treatment) (1) In the case of no amine addition 5.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred at 25 °C with a stir bar for 30 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active species was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area value of LC / MS, the residual rate of the C-terminal active species was calculated according to the following formula. Residual rate of C-terminal active species (%) = {[Area % of propylamide] / ([Area % of propylamide] + [Area % of dipeptide])} × 100

[0269] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 484 mg (4.0 mmol) of DMAP and 5.0 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a magnetic stirrer at 25 °C for 30 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, followed by dilution with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0270] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 5 mL of a 10% aqueous potassium hydrogen sulfate solution and 2.5 mL of a 5% aqueous potassium carbonate solution. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, followed by dilution with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active form (the converted form to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without adding amine was 653.8 mg (yield 116%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active form)). The concentrate obtained by hydrolysis with amine addition was 549.4 mg (yield 97%). MS (ESI): m / z 427.3 [M+H] + , 449.2 [M+Na] + 。

[0271]

Table 11

[0272] In the hydrolysis with alkaline water alone, the residual C-terminal active substance was not completely hydrolyzed, and the residual C-terminal active substance could not be sufficiently removed even by subsequent aqueous washing. However, when hydrolysis was carried out by adding DMAP, it was found that the hydrolysis of the residual C-terminal active substance was completely achieved and the residual C-terminal active substance could be completely removed. At this time, 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. Then, 447 mg (2.3 mmol) of EDC hydrochloride was added at 25°C, and the mixture was stirred at 25°C for 3 hours to carry out a peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active substance was converted 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 peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-Thr(tBu)-Phe-OtBu] / [Area% of Phe-OtBu + Area% of Cbz-Thr(tBu)-Phe-OtBu]} × 100

[0274] (Hydrolysis treatment) (1) In the case of no amine addition To the reaction solution containing the above-prepared peptide, 3.0 mL of a 5% aqueous potassium carbonate solution was added, and the mixture was stirred with a magnetic stirrer at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0275] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 142 mg (1.2 mmol) of DMAP and 3.0 mL of a 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a magnetic stirrer at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0276] (Work-up) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL of a 10% aqueous potassium hydrogen sulfate solution, 3 mL of a 5% aqueous potassium carbonate solution, and 1.5 mL of water. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, and then diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain the peak area values of the target peptide and the remaining C-terminal active form (the converted form 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) Peak area ratio of LCMS

[0278] In the hydrolysis with aqueous alkali alone, the remaining C-terminal active form was not completely hydrolyzed, and the remaining C-terminal active form could not be sufficiently removed even by subsequent aqueous washing. However, when hydrolysis was carried out with the addition of DMAP, it was found that the hydrolysis of the remaining C-terminal active form was completely achieved and the remaining C-terminal active form could be completely removed. At this time, 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 the dipeptide obtained by hydrolysis without adding amine) The MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu synthesized under the amine-free condition in Example 20 was concentrated by substituting it with 2-methyltetrahydrofuran. It was subjected to a hydrogenolysis reaction with 99 mg of 5% Pd / C (50% wet) and hydrogen gas. Stirring was carried out at 25 °C for 1 hour, but the reaction was not completed (conversion rate 53%). The reaction conversion rate was determined from the peak area values of LC / MS after taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting it to LC / MS analysis. Conversion rate (%) = {Thr(tBu)-Phe-OtBu (area%) / [Cbz-Thr(tBu)-Phe-OtBu (area%) + Thr(tBu)-Phe-OtBu (area%)]} × 100

[0280] (Cbz deprotection reaction using the dipeptide obtained by hydrolysis with the addition of amine) The MTBE / 2-MeTHF solution of Cbz-Thr(tBu)-Phe-OtBu synthesized under the amine addition conditions in Example 20 was replaced with 2-methyltetrahydrofuran and concentrated. It was subjected to a hydrogenolysis reaction with 104 mg of 5% Pd / C (50% wet) and hydrogen gas. It was stirred at 25 °C for 1 hour to obtain Thr(tBu)-Phe-OtBu, which is the de-Cbz product (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and then subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of Thr(tBu)-Phe-OtBu / [Area % of Cbz-Thr(tBu)-Phe-OtBu + Area % of Thr(tBu)-Phe-OtBu]} × 100

[0281] (Condensation reaction) The reaction solution of the Cbz-deprotected product of the dipeptide obtained by hydrolysis treatment with amine addition was filtered through a filter, the Pd / C was filtered off, and then concentrated to dryness. The dried product was dissolved in 5.0 mL of 2-methyltetrahydrofuran, and 382 mg (1.4 mmol) of Cbz-Leu-OH and 814 μL (4.7 mmol) of diisopropylethylamine were added. Then, 1.37 mL (2.3 mmol) of a 50% T3P / 2-methyltetrahydrofuran solution was added and stirred at 25 °C for 30 minutes to carry out a peptide bond formation reaction (conversion rate: 100%). The reaction conversion rate was determined from the peak area values of LC / MS by adding 5 μL of the reaction solution to 100 μL of propylamine, diluting it with 0.9 mL of methanol, and then subjecting it to LC / MS analysis. Conversion rate (%) = {Area % of Cbz-Leu-Thr(tBu)-Phe-OtBu / [Area % of Thr(tBu)-Phe-OtBu + Area % of Cbz-Leu-Thr(tBu)-Phe-OtBu]} × 100

[0282] To the prepared reaction solution, 139 mg (1.1 mmol) of DMAP and 3.0 mL of 10% aqueous sodium carbonate solution were added, and the mixture was stirred at 25 °C for 5 minutes. After stopping the stirring and allowing it to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL × 2 of 10% aqueous sodium bisulfate solution, 3 mL of 5% aqueous sodium carbonate solution, and 3 mL of normal water. 5 μL of the obtained 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 residual C-terminal active species. The target peptide Cbz-Leu-Thr(tBu)-Phe-OtBu was 98.3%, and Cbz-Leu-NHPr derived from the residual C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 638.0 mg of the concentrate (yield 88% from Phe-OtBu in Example 20). MS (ESI): m / z 514.3 [M - 2tBu + H] + , 570.3 [M - tBu + H] + , 626.5 [M + H] + , 648.4 [M + Na] + .

[0283] It was found that when the C-terminal active species derived from EDC and HOBt remained, the progress of the Cbz deprotection reaction was slower compared to the case where the residual C-terminal active species was completely removed. It was found that if a peptide solution in which the residual C-terminal active species was completely removed was used as a raw material, the Cbz deprotection reaction proceeded smoothly and peptide synthesis reaction became possible. That is, by using the method of the present invention, it was found that, as in the case of Example 9 and Example 18, the reductive removal reaction of the protecting group at the N-terminus of the produced peptide compound could proceed without stagnation. As a result, a high-purity peptide compound having a desired amino acid sequence could be efficiently produced.

[0284] (Example 22) Synthesis of Cbz-Ile-Phe-OtBu (Coupling 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. Then, 479 mg (1.8 mmol) of BEP was added at 25°C, and the mixture was stirred at 25°C for 45 minutes to conduct a peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active substance was converted 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 peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-Ile-Phe-OtBu] / ([Area% of Phe-OtBu] + [Area% of Cbz-Ile-Phe-OtBu])} × 100

[0285] (Hydrolysis treatment) (1) In the case of no amine addition 3.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a stir bar at 25°C for 3 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active substance was converted to propylamide, and then diluted with 0.9 mL of methanol. From the peak area value of LC / MS, the residual rate of the C-terminal active substance was calculated according to the following formula. Residual rate of C-terminal active substance (%) = {[Area% of propylamide] / ([Area% of propylamide] + [Area% of dipeptide])} × 100

[0286] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 139 mg (1.1 mmol) of DMAP and 3.0 mL of 5% aqueous potassium carbonate solution were added, and the mixture was stirred with a stir bar at 25 °C for 3 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated according to the following formula from the peak area values of LC / MS. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0287] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then the organic layer was washed successively with 3 mL of 10% aqueous potassium hydrogen sulfate solution and 3 mL of 5% aqueous potassium carbonate solution. After adding 2 mL of 2-MeTHF, it was washed with 1.5 mL of water. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol and subjected to LC / MS analysis to obtain 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

[0289] In the hydrolysis with alkaline water alone, the residual C-terminal active substance was not completely hydrolyzed, and the residual C-terminal active substance could not be sufficiently removed even by subsequent aqueous washing. However, when hydrolysis was carried out by adding DMAP, it was found that the hydrolysis of the residual C-terminal active substance was completely achieved and the residual C-terminal active substance could be completely removed. At this time, 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) 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 de-Boc reaction (conversion rate: 100%). The reaction conversion rate was determined from the peak area value of LC / MS by subjecting a 5 μL aliquot of the reaction solution diluted with 1.0 mL of acetonitrile to LCMS analysis. Conversion rate (%) = {Phe-piperidine (area %) / [Boc-Phe-piperidine (area %) + Phe-piperidine (area %)]} × 100

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

[0292] 128 mg (1.0 mmol) of DMAP and 3.5 mL of 5% aqueous potassium carbonate solution were added to the above-prepared reaction solution, and the mixture was stirred with a stir bar at 25 °C for 3 minutes. After stopping the stirring, the mixture was allowed to stand, and the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Then, the organic layer was diluted with 3.5 mL × 2 of 10% aqueous potassium sulfate solution and 3.5 mL of 5% aqueous potassium carbonate solution. This solution was subjected to LC / MS analysis to determine the peak area percentages of the target peptide and the remaining C-terminal active species. The target peptide Cbz-Phe-MeGly-Phe-piperidine had a purity of 94.6%, and Cbz-Phe-MeGly-NHPr derived from the remaining C-terminal active species was not detected. The remaining organic layer was concentrated to obtain 520.4 mg of the concentrate (yield 94%).

[0293] When hydrolysis was carried out by adding DMAP, the decomposition and removal of the remaining C-terminal active species derived from the peptide fragment were completely achieved, and the target tripeptide could be 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. Then, 447 mg (1.2 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 2.5 hours to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active species to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the peak area of LC / MS (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) In the case of no amine addition 2.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a stir bar at 25°C. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active species to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the residual rate of the C-terminal active species was calculated from the peak area value of LC / MS according to the following calculation formula (the following table). Residual rate of C-terminal active species (%) = {propylamide (area%) / [propylamide (area%) + dipeptide (area%)]} × 100

[0297] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 0.8 mmol of the amine additive shown in the following table and 2.0 mL of a 5% aqueous potassium carbonate solution were added, and stirring was carried out with a stir bar at 25°C. Stirring was stopped, and the organic layer and the aqueous layer 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 residual rate of the C-terminal active form was calculated according to the following formula from the peak area value of LC / MS (the following table). Residual rate of C-terminal active form (%) = {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 form compared to the case of using only alkaline water without adding an amine, 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 form more than the addition of DIPEA. In particular, it was found that the remaining C-terminal active form was completely hydrolyzed within 5 minutes when DMAP was used. Thus, it was found that amines with less steric hindrance near nitrogen, such as DMAP, promoted the hydrolysis of the remaining C-terminal active form more than amines with steric hindrance near nitrogen, such as DIPEA.

[0300] (Example 25) Synthesis of Cbz-Ile-Val-OBn (Coupling reaction) 300 mg (1.2 mmol) of Val-OBn hydrochloride and 495 mg (1.9 mmol) of Cbz-Ile-OH were suspended in 3.0 mL of cyclopentyl methyl ether and 0.9 mL of acetonitrile, and 859 μL (4.9 mmol) of diisopropylethylamine was added. Then, 705 mg (1.9 mmol) of HATU was added at 25°C, and the mixture was stirred at 25°C for 1 hour to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol. This solution was subjected to LC / MS analysis, and the conversion rate was determined from the peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {[Area% of Cbz-Ile-Val-OBn] / ([Area% of Val-OBn] + [Area% of Cbz-Ile-Val-OBn])} × 100

[0301] (Hydrolysis treatment) (1) In the case of no amine addition 3.0 mL of neutral water was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a stir bar at 25°C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and after converting the remaining C-terminal active form to propylamide, it was diluted with 0.9 mL of methanol. From the peak area values of LC / MS, the residual rate of the C-terminal active form was calculated according to the following formula. Residual rate of C-terminal active form (%) = {[Area% of propylamide] / ([Area% of propylamide] + [Area% of dipeptide])} × 100

[0302] (2) In the case of amine addition To the reaction solution containing the above-prepared peptide, 91 mg (0.7 mmol) of DMAP and 3.0 mL of neutral water were added, and the mixture was stirred with a stir bar at 25 °C for 5 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, followed by dilution with 0.9 mL of methanol. The residual rate of the C-terminal active form was calculated according to the following formula from the peak area values of LC / MS. Residual rate of C-terminal active form (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0303] (Workup) After stopping the stirring, the mixture was allowed to stand, the organic layer and the aqueous layer were separated, and the aqueous layer was removed. Subsequently, the organic layer was washed successively with 3 mL of 10% aqueous sodium bisulfate solution, 3 mL × 2 of 5% aqueous sodium carbonate solution, and 1.5 mL × 3 of normal water. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active form was converted to propylamide, followed by dilution 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. The concentrate (peptide) obtained by hydrolysis without amine addition was 744 mg (yield 132%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active form)). The concentrate obtained by hydrolysis with amine addition was 528 mg (yield 94%). MS (ESI): m / z 455.3 [M+H] + , 477.3 [M+Na] + .

[0304]

Table 15

[0305] In the hydrolysis with neutral water alone, the residual C-terminal active substance was not completely hydrolyzed, and the residual C-terminal active substance could not be removed even by subsequent aqueous washing. However, when hydrolysis was carried out by adding DMAP, it was found that the hydrolysis of the residual C-terminal active substance was completely achieved and the removal of the residual C-terminal active substance could also be completed. At this time, the target dipeptide was obtained with a purity of 99.5% (yield 94%).

[0306] (Example 26) Synthesis of Cbz-MeAla-Phe-OtBu (Condensation reaction) 200 mg (0.8 mmol) of Phe-OtBu hydrochloride and 260 mg (1.2 mmol) of Cbz-MeAla-OH were suspended in 2.4 mL of 2-methyltetrahydrofuran and 0.6 mL of acetonitrile, and 271 μL (1.6 mmol) of diisopropylethylamine was added. Then, 265 mg (0.8 mmol, 13 w% water-containing) of DMT-MM-n hydrate (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n hydrate) was added at 25 °C, and the mixture was stirred at 25 °C for 1 hour. Then, 119 mg (0.4 mmol, 13 w% water-containing) of DMT-MM-n hydrate was added, and the mixture was stirred at 25 °C for 1 hour to carry out the peptide bond formation reaction. 5 μL of the reaction solution was taken, added to 100 μL (1.2 mmol) of propylamine, and the residual C-terminal active substance was converted 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 peak area of LC / MS (conversion rate: 100%). Conversion rate (%) = {Cbz-MeAla-Phe-OtBu (area%) / [Phe-OtBu (area%) + Cbz-MeAla-Phe-OtBu (area%)]} × 100

[0307] (Hydrolysis treatment) (1) In the case of no amine addition 2.0 mL of 5% aqueous potassium carbonate solution was added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a stir bar at 25 °C for 10 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active species was converted to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active species was calculated from the peak area values of LC / MS according to the following calculation formula. Residual rate of C-terminal active species (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0308] (2) In the case of amine addition 96 mg (0.8 mmol) of DMAP and 2.0 mL of 5% aqueous potassium carbonate solution were added to the reaction solution containing the above-prepared peptide, and the mixture was stirred with a stir bar at 25 °C for 10 minutes. Stirring was stopped, and the organic layer and the aqueous layer were separated. 5 μL of the organic layer was taken, added to 100 μL (1.2 mmol) of propylamine, and the remaining C-terminal active species was converted to propylamide, and then diluted with 0.9 mL of methanol. The residual rate of the C-terminal active species was calculated from the peak area values of LC / MS according to the following calculation formula. Residual rate of C-terminal active species (%) = {propylamide (area %) / [propylamide (area %) + dipeptide (area %)]} × 100

[0309] (Post-treatment) After stopping the stirring, the mixture was allowed to stand still to separate the organic layer and the aqueous layer, and the aqueous layer was removed. Subsequently, the organic layer was successively washed with 2 mL of 10% potassium hydrogen sulfate aqueous solution, 2 mL of 5% potassium carbonate aqueous solution, and 1 mL×2 of normal 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 substance into propylamide. Then, it was 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 substance (the converted product to propylamide). The remaining organic layer was concentrated to obtain the peptide. The concentrate (peptide) obtained by hydrolysis without adding amine was 387 mg (yield 114%: calculated as containing only the peptide although the concentrate contains impurities (remaining C-terminal active substance)). The concentrate obtained by hydrolysis with amine addition 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

[0311] When DMT-MM was used as the condensing agent, in the hydrolysis with only alkaline water treatment, the remaining C-terminal active substance was not completely hydrolyzed, and the remaining C-terminal active substance could not be removed even by subsequent aqueous washing. However, when hydrolysis was carried out by adding DMAP, it was found that the hydrolysis of the remaining C-terminal active substance was completely achieved and the removal of the remaining C-terminal active substance was also complete. At this time, the target dipeptide was obtained with a purity of 98.6% (yield 98%). The C-terminal active substance generated from DMT-MM, which is a condensing agent that can also be used in a water-containing solvent, is known to be relatively resistant to hydrolysis. However, it was found that by using an amine additive, even the remaining C-terminal active substance prepared using DMT-MM can be completely hydrolyzed in a short time and with a single treatment, and can be completely removed by subsequent aqueous washing.

[0312] Reference Example 1: Synthesis method 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. A 35.0 mL (58.9 mmol) solution of 50% T3P / ethyl acetate was added dropwise over 45 minutes at 3 - 10 °C. After the addition was complete, 5 μL of the reaction solution was taken, diluted with 1.0 mL of methanol, and subjected to LCMS analysis. The reaction conversion rate was determined from the peak area values of LC / MS (conversion rate: 100%). Conversion rate (%) = {Cbz-MeAsp(tBu)-piperidine (area %) / [Cbz-MeAsp(tBu)-OH (area %) + Cbz-MeAsp(tBu)-piperidine (area %)]} × 100

[0313] The reaction solution was washed with 100 mL of 10% potassium bisulfate aqueous solution and 100 mL of 10% potassium carbonate, and the obtained organic layer was dried over magnesium sulfate, filtered, and concentrated. MS (ESI) m / z 349.1 [M-tBu+H] + 、405.2 [M+H] + 、427.3 [M+Na] + 。

[0314] (Cbz deprotection reaction) The above concentrated solution was dissolved in 100 mL of cyclopentyl methyl ether, and subjected to a hydrogenolysis reaction with 2.0 g of 5% Pd / C (50% wet) and hydrogen gas. After stirring at room temperature for 5 hours, the target product MeAsp(tBu)-piperidine was obtained (conversion rate 100%). The reaction conversion rate was determined by taking 5 μL of the reaction solution, diluting it with 1.0 mL of acetonitrile, and subjecting it to LCMS analysis. The reaction conversion rate was determined from the peak area values of LC / MS. Conversion rate (%) = {[Area % of MeAsp(tBu)-piperidine] / [[Area % of Cbz-MeAsp(tBu)-piperidine] + [Area % of MeAsp(tBu)-piperidine]]} × 100

[0315] After filtering the reaction solution through a filter, the filtrate was concentrated to obtain 5.69 g of a concentrate (yield quant.). This concentrate was subjected to LC / MS analysis to determine the peak area percentage of the target MeAsp(tBu)-piperidine (99.2 area %). MS (ESI): m / z 215.1 [M-tBu+H] + , 271.1 [M+H] + .

Industrial Applicability

[0316] When producing a peptide compound, the present invention can produce a high-purity peptide compound without column purification by efficiently removing the C-terminal active substance remaining after the condensation reaction.

Claims

1. A method for producing a peptide compound, comprising: Step A: obtaining a reaction mixture containing a peptide compound obtained by condensing the C-terminal active 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 alkali solution to remove the C-terminal active form wherein the tertiary amine is represented by the following formula (B) or (C), said method. 【Chemical 1】 [In the formula, 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 heterocyclic ring, provided that when X is O, R ５ is absent. 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 together with the nitrogen atom to which R is attached and the carbon atom to which R ９ is attached form a 5- to 6-membered non-aromatic heterocyclic ring.]

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

3. The method according to claim 1 or 2, wherein the peptide compound contains one or more unnatural amino acids.

4. The method according to any one of claims 1 to 3, wherein the temperature at which the tertiary amine acts on the C-terminal active form is 25°C to 60°C.

5. The method according to any one of claims 1 to 4, wherein 0.5 equivalent or more of the tertiary amine is added to the amine component.

6. The method according to any one of claims 1 to 5, wherein the C-terminal active form is hydrolyzed and removed by reacting with the tertiary amine.

7. The method according to any one of claims 1 to 6, further comprising, in Step B, separating the reaction mixture into an organic layer and an aqueous layer, and then washing the organic layer, and the remaining amount of the C-terminal active form after the washing is 1.0% or less.

8. The method according to any one of claims 1 to 7, wherein the solvent in Step A is toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl acetate, ethyl acetate, methyl tert-butyl ether, or cyclopentyl methyl ether, N,N-dimethylformamide or a mixed solvent thereof.

9. The method according to any one of claims 1 to 8, wherein the aqueous alkali solution in Step B is an aqueous potassium carbonate solution or an aqueous sodium carbonate solution.

10. The method according to any one of claims 1 to 9, wherein the acid component is a first amino acid having an amino group protected by a protecting group, and the side chain of the first amino acid contains one or more carbon atoms.

11. The method according to claim 10, 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.

12. The method according to any one of claims 1 to 11, wherein the time for reacting the tertiary amine with the C-terminal active substance is 2 hours or less.

13. The method according to any one of claims 1 to 12, 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.

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