Peptide synthesis method for suppressing defect caused by diketopiperazine formation

Abstract

The present invention addresses the problem of a desired elongation reaction not progressing as a result of a 6-membered cyclic amidine skeleton structure and a diketopiperazine formed when removing an N-terminated protective group during peptide synthesis. The inventors have discovered that it is possible to solve said problem during peptide production by treating a peptide protected by an N-terminated amino group in a protective group having an Fmoc skeleton by using a base which has a pKa of 23 or higher in an acetonitrile of a conjugate acid in a specific solvent, and next, performing peptide chain elongation.

Description

Peptide synthesis method that suppresses defects caused by diketopiperazine formation

[0001] The present invention relates to a novel method for synthesizing peptides, which enables the synthesis of peptides with high purity and high efficiency.

[0002] The Fmoc method is a widely used peptide synthesis method due to its reliability and mild deprotection conditions (Fmoc deprotection conditions). Recently, there has been active research into the application of peptides containing many N-alkylamino acids, such as N-methylamino acids, to pharmaceuticals. However, the acid lability of such molecular species is well known (Non-Patent Document 1, Patent Document 1). Therefore, these documents employ the Fmoc method, which allows deprotection under basic conditions, rather than the Boc method, which requires acidic conditions for N-terminal deprotection.

[0003] Diketopiperazine (DKP) formation has long been recognized as a problem in peptide synthesis. Diketopiperazine formation can occur when the N-terminal protecting group of a dipeptide supported on a solid phase via an ester bond is removed, exposing a free amino group. Several approaches have been reported to address this diketopiperazine formation problem in peptide synthesis using the Fmoc method.

[0004] Specifically, a method is known in which diketopiperazine formation is suppressed by using an Alloc group, which undergoes a deprotection reaction under neutral conditions without using a base, as a protecting group at a site where diketopiperazine formation is likely to occur (Non-Patent Document 2).

[0005] Furthermore, in order to avoid the N-terminus becoming a free amino group at a site where diketopiperazine formation is likely to proceed, a method is known in which a dipeptide containing such a sequence is synthesized in advance and used to elongate the peptide chain (Non-Patent Document 3).

[0006] Furthermore, a method is known in which, instead of piperidine, which is commonly used in the deprotection of Fmoc, stronger bases such as DBU or TBAF are used to shorten the time required for deprotection, thereby suppressing the formation of diketopiperazine (Non-Patent Document 4).

[0007] All of these methods address the diketopiperazine formation of dipeptides supported on a solid phase via an ester bond.

[0008] International Publication No. WO 2018 / 225851 A1

[0009] J. Peptide Res., 2005, 65, 153-166.Org. Lett., 2012, 14, 612-615.Bachem Publishing Solid Phase Peptide Synthesis (https: / / www.bachem.com / fileadmin / user_upload / pdf / Catalogs_Brochures / Solid_Phase_Peptide_Synthesis.pdf)Org. Lett., 2008, 10, 3857-3860.

[0010] As described above, it is known that a dipeptide supported on a solid phase via an ester bond forms a diketopiperazine upon cleavage of the ester bond. However, when an N-substituted amino acid is contained in the peptide sequence, a diketopiperazine is formed by cleavage of an amide bond, which is stronger than an ester bond, and the dipeptide containing the N-substituted amino acid may be removed from the peptide sequence. This removal can occur at any location in the peptide sequence where the N-substituted amino acid is present, and therefore it has been found that it can occur not only in dipeptides supported on a solid phase.

[0011] Furthermore, the present inventors have revealed that when an N-substituted amino acid is contained in a peptide sequence, in addition to the problem of diketopiperazine formation, there is also the problem that the desired elongation reaction does not proceed, and instead of the target product, an impurity having a six-membered cyclic amidine skeleton at the N-terminus (six-membered cyclic amidine skeleton structure) is formed. No method for solving these problems has been reported so far.

[0012] In order to suppress not only the diketopiperazine elimination that occurs at the ester bond of a dipeptide supported on a solid phase but also the diketopiperazine elimination that occurs at the amide bond and the formation of the six-membered cyclic amidine skeleton structure, it is conceivable to apply the above-mentioned conventional techniques, but the scope of application is limited, and these techniques cannot be said to provide a sufficient solution.

[0013] For example, the method described in Non-Patent Document 2 requires Alloc amino acids, which are less common than Fmoc amino acids in terms of availability. Furthermore, the peptide chain elongation method using dipeptides described in Non-Patent Document 3 raises concerns about racemization of the C-terminal amino acid of the dipeptide. Therefore, it is difficult to apply the method described in this document except when the C-terminal amino acid of the dipeptide fragment is achiral (e.g., glycine) or when racemization is difficult to proceed (e.g., proline). Furthermore, the method described in Non-Patent Document 4 is characterized by an extremely short time required for the Fmoc removal step, requiring the Fmoc removal step and solid-phase washing operation within one minute, for example, 10 to 20 seconds. It is impossible to apply such extremely short operations to scale-up synthesis, including industrialization.

[0014] Furthermore, the present inventors have revealed that in addition to the problem of diketopiperazine formation and / or the problem of the formation of a 6-membered cyclic amidine skeleton structure, there is also the problem of the formation of a dimer urea impurity, and no method for solving this problem of the formation of a dimer urea has been reported so far.

[0015] As described above, no practical methodology for suppressing diketopiperazine formation has yet been presented. The present invention has been made in light of these circumstances, and in one aspect, the present invention aims to provide a peptide synthesis method capable of suppressing the formation of diketopiperazines, which (i) uses a protecting group containing an Fmoc skeleton as an N-terminal protecting group, (ii) is not subject to sequence application restrictions from the standpoint of racemization, and (iii) allows for scale-up synthesis, such as industrialization. In another aspect, the present invention aims to provide a peptide synthesis method capable of suppressing the formation of a 6-membered cyclic amidine skeleton structure. In yet another aspect, the present invention aims to provide a peptide synthesis method capable of suppressing the formation of a dimeric urea compound.

[0016] The present inventors have discovered that in peptide production, the formation of diketopiperazine, a 6-membered cyclic amidine skeleton structure, and / or a dimeric urea compound can be suppressed from the removal of the protecting group to the elongation of the peptide chain by treating a peptide having a protecting group containing an Fmoc skeleton with a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a specific solvent, and then reacting the peptide with an active elongation species such as an activated ester generated using an acid chloride or a condensing agent, thereby completing the present invention.

[0017] That is, the present invention includes the following: [0-1] A method for producing a peptide, comprising: (1) a step of providing a first peptide having a protecting group containing an Fmoc backbone, (2) after the step (1), a step of treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent, and (3) after the step (2), a step of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. [0-2] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in the production of a peptide, the method comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent. [0-3] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeletal structure impurities, and dimer urea impurities, in the production of a peptide, the method comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent. [0-4] (3) The method according to [0-2] or [0-3], further comprising, after step (2), a step of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. [0-5] The method according to any one of [0-1] to [0-4], which is carried out by a solid phase method. [0-6] The method according to any one of [0-1] to [0-4], wherein the first peptide is a first peptide having a protecting group containing an Fmoc skeleton supported on a solid phase. [0-7] The method according to any one of [0-1] to [0-4], which is carried out by a liquid phase method.[1] A method for producing a peptide by a solid phase method, comprising: (1) providing a first peptide having a protecting group including an Fmoc skeleton supported on a solid phase; (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent; and (3) after the step (2), condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. [2] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in peptide production by a solid-phase method, the method comprising: (1) providing a first peptide having a protecting group containing an Fmoc skeleton supported on a solid phase; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent. [2-1] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine backbone impurities, and dimeric urea impurities in peptide production by a solid-phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or higher, in a solvent containing a sulfoxide solvent. [3] The method of [2], further comprising: (3) after step (2), condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. [4] The method according to any one of [0-1] to [3], wherein the step (2) is a step of removing a protecting group containing an Fmoc skeleton in the first peptide and / or a step of converting a protecting group containing an Fmoc skeleton in the first peptide into a carbamate form.[5] The method according to any one of [0-1] to [3], wherein the step of removing a protecting group containing an Fmoc skeleton in the first peptide is carried out substantially only in the step (2). [6] The method according to any one of [0-1] to [5], wherein the step of treating the first peptide with piperidine as a single base prior to the step (2). [7] The method according to any one of [0-1] to [5], wherein the step of treating the first peptide with a single base whose conjugate acid has a pKa in acetonitrile of less than 23 prior to the step (2). [8] The method according to any one of [0-1] to [7], wherein the solvent in the step (2) is a solvent having a swelling capacity of the solid phase resin of 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more. [9] The method according to any one of [0-1] to [8], wherein at least a portion of the first peptide obtained in step (2) is in the form of a carbamate.

[10] The method according to [9], wherein the carbamate is a salt with a base whose conjugate acid has a pKa in acetonitrile of 23 or higher.

[11] The method according to [9] or

[10] , wherein the carbamate is a salt with at least one base selected from the group consisting of amidines, guanidines, and phosphazenes.

[12] The method according to any one of [9] to

[11] , wherein the carbamate is a DBU salt, TMG salt, HP1(dma) salt, MTBD salt, P1-tBu salt, or P2-Et salt.

[13] The method according to any one of [9] to

[12] , wherein in the step (2), the molar ratio of the carbamate to the amine formed by further decarboxylation (carbamate / amine) is 0.6 or more, 0.8 or more, 1.0 or more, 2.0 or more, 3.0 or more, 4.0 or more, 4.6 or more, 5.0 or more, 6.0 or more, 8.0 or more, or 10.0 or more. [13-1] In the step (2), １The method of any one of [9] to

[12] , wherein the molar ratio of the carbamate to the amine formed by further decarboxylation (carbamate / amine), determined from the proton integral ratio in H-NMR, is 0.6 or more, 0.8 or more, 1.0 or more, 2.0 or more, 3.0 or more, 4.0 or more, 4.6 or more, 5.0 or more, 6.0 or more, 8.0 or more, or 10.0 or more.

[14] The method of any one of [0-1] to

[13] , wherein the solvent in step (2) contains a sulfoxide solvent at 50 v / v% or more.

[15] The method of any one of [0-1] to

[14] , wherein the solvent in step (2) contains a sulfoxide solvent at 70 v / v% or more.

[16] The method of any one of [0-1] to

[15] , wherein the solvent in step (2) contains a sulfoxide solvent at 90 v / v% or more.

[17] The method according to any one of [0-1] to

[16] , wherein the solvent in step (2) further comprises one or more solvents selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, phosphate ester-based solvents, amide-based solvents, urea-based solvents, and sulfone-based solvents.

[18] The method according to any one of [0-1] to

[16] , wherein the solvent in step (2) further comprises one or more solvents selected from the group consisting of amide-based solvents, urea-based solvents, and sulfone-based solvents.

[19] The method according to any one of [0-1] to

[13] , wherein the solvent in step (2) consists of a sulfoxide-based solvent.

[20] The method according to any one of [0-1] to

[19] , wherein the sulfoxide-based solvent is one or more solvents selected from the group consisting of DMSO, diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide.

[21] The method according to any one of [0-1] to

[19] , wherein the sulfoxide solvent is DMSO.

[22] The method according to any one of [0-1] to

[21] , wherein the base in the step (2) is at least one selected from the group consisting of amidines, guanidines, and phosphazenes.

[23] The method according to any one of [0-1] to

[22] , wherein the base in step (2) is at least one selected from the group consisting of DBU, MTBD, TMG, P1tBu, P2Et, and HP1(dma).

[24] The method according to any one of [0-1] to

[23] , wherein the base in step (2) is DBU, MTBD, TMG, P1tBu, P2Et, or HP1(dma).

[25] The method according to any one of [0-1] to

[23] , wherein the base in step (2) is (i) a combination of DBU and piperidine, or (ii) a combination of DBU and MTBD, HP1(dma), P1tBu, or P2Et. [25-1] The method according to any one of [0-1] to

[25] , wherein the solvent in step (2) is DMSO, and the base is DBU. [25-2] The method according to any one of [0-1] to

[25] , wherein the solvent in step (2) is DMSO and the base is TMG. [25-3] The method according to any one of [0-1] to

[25] , wherein the solvent in step (2) is DMSO and the base is a combination of DBU and piperidine.

[26] The method according to any one of [0-1] to

[25] , wherein the base in step (2) is contained in the solvent at a concentration of 1 to 8 v / v %.

[27] The method according to any one of [0-1] to

[25] , wherein the solvent in step (2) is CO. ２

[28] The method according to any one of [0-1] to

[26] , further comprising a step of contacting the solvent with CO ２

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

[27] , further comprising a step of bubbling the solvent in the step (2). ２

[30] The method according to any one of [0-1] to

[26] , wherein the solvent in the step (2) is a solvent that has been previously contacted with CO ２

[31] The method according to any one of [0-1] to

[30] , wherein step (2) is repeated multiple times.

[32] The method according to any one of [0-1] to

[30] , wherein step (2) is repeated multiple times, and the same solvent is used each time.

[33] The method according to

[31] , wherein step (2) is repeated multiple times, and a different solvent is used each time.

[34] The method according to any one of

[31] to

[33] , wherein step (2) is repeated multiple times, and the same base is used each time.

[35] The method according to any one of

[31] to

[33] , wherein step (2) is repeated multiple times, and a different base is used each time.

[36] The method according to any one of

[31] to

[35] , wherein step (2) is repeated multiple times, and a different base is used each time.

[37] The carboxylic acid or carboxylic acid analog is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkylcarboxylic acid, or a C6-C 10 an aryl carboxylic acid, or a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 an activated ester of an aryl carboxylic acid, or a protected amino acid, a protected second peptide, a C1-C8 alkyl carboxylic acid, or a C6-C 10 The acid halide of an arylcarboxylic acid is a C1-C8 alkylcarboxylic acid and a C6-C 10The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[36] , wherein the arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl, and dialkylaminocarbonyl.

[38] The method according to

[37] , wherein the protecting group is a carbamate protecting group, a sulfonyl protecting group, or an acyl protecting group.

[39] The method according to

[38] , wherein the carbamate protecting group is a protecting group containing an Fmoc skeleton, Alloc, Teoc, Boc, or Cbz, the sulfonyl protecting group is Ns, and the acyl protecting group is Tfa.

[40] The method according to

[41] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkylcarboxylic acid or a C6-C8 alkylcarboxylic acid. 10 aryl carboxylic acid or its activated ester or acid halide, and the C1-C8 alkyl carboxylic acid and C6-C 10The method of

[37] , wherein the arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, and halogen.

[41] The method of

[37] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkylcarboxylic acid, or an activated ester or acid halide thereof, optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, and halogen.

[42] The method of

[37] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkylcarboxylic acid, or an activated ester or acid halide thereof.

[43] The method of any of [0-1] to

[42] , wherein the first peptide contains 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues.

[44] The method of any of [0-1] to

[43] , wherein the first peptide is a dipeptide.

[45] The method of any of [0-1], [0-4] to [0-6], and [3] to

[44] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group, or an activated ester of an amino acid or a second peptide having a protecting group, or an acid halide of an amino acid or a second peptide having a protecting group, and the first peptide and / or the second peptide having a protecting group contains one or more N-substituted amino acids, and / or the amino acid having a protecting group is an N-substituted amino acid.

[46] The method of any one of [0-1], [0-4] to [0-6], and [3] to

[45] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group containing an Fmoc backbone, or an activated ester of an amino acid or a second peptide having a protecting group containing an Fmoc backbone, or an acid halide of an amino acid or a second peptide having a protecting group containing an Fmoc backbone, and the first peptide and / or the second peptide having a protecting group containing an Fmoc backbone contains one or more N-substituted amino acids, and / or the amino acid having a protecting group containing an Fmoc backbone is an N-substituted amino acid.

[47] The method of any one of [0-1] to

[46] , wherein the second amino acid residue from the N-terminus of the first peptide is an N-substituted amino acid.

[48] The method of

[46] or

[47] , wherein the N-substituted amino acid is an N-alkylamino acid.

[49] The method of

[46] or

[47] , wherein the N-substituted amino acid is an N-C1-C6 alkylamino acid.

[50] The method according to

[46] or

[47] , wherein the N-substituted amino acid is an N-methyl amino acid.

[51] The condensing agent in the step (3) is in the form of a salt, the counter anion of which is PF6. - or BF4 -

[52] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[50] , wherein the condensing agent in the step (3) is in the form of a salt, and the counter anion thereof is PF6 - or BF4 -

[53] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[51] , wherein the condensing agent in step (3) comprises at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, and TOTU.

[54] The method according to

[52] or

[53] , wherein the condensing agent in step (3) is PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, or TOTU.

[55] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[54] , wherein step (3) is carried out in the presence of a base.

[56] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[55] , wherein the step (3) is carried out in the presence of a base having a pKa in water of 5 to 12.

[57] The method according to

[55] or

[56] , wherein the base in the step (3) is DIPEA.

[58] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[51] , wherein the condensing agent in the step (3) is a carbodiimide condensing agent.

[59] The method according to

[58] , wherein the carbodiimide condensing agent is at least one selected from the group consisting of diisopropylcarbodiimide (DIC), di-sec-butylcarbodiimide (DsBC), 1-tert-butyl-3-ethylcarbodiimide (tBEC), di-tert-butylcarbodiimide (DtBC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[60] The method according to

[58] or

[59] , wherein the carbodiimide condensing agent is diisopropylcarbodiimide (DIC) or di-sec-butylcarbodiimide (DsBC).

[61] The method according to any one of [1],

[51] , and

[58] to

[60] , wherein step (3) is carried out in the presence of an additive.

[62] The method according to

[61] , wherein the additive is at least one selected from the group consisting of HOAt, HOBt, HOOBt, Oxyma, and Oxyma-B.

[63] The method according to

[62] , wherein the additive is HOAt or Oxyma.

[64] The method according to any one of [0-1] to

[63] , wherein the protecting group containing an Fmoc skeleton is represented by the following formula (1): wherein R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; and R9-R 10are independently hydrogen or methyl.

[65] The method according to

[64] , wherein the protecting group containing an Fmoc skeleton is an Fmoc group, an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br) group, a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, an sm-Fmoc group, or an rm-Fmoc group.

[66] The method according to

[65] , wherein the protecting group containing an Fmoc skeleton is an Fmoc group.

[67] The method according to any one of [0-1], [0-3] to [0-6], and [3] to

[66] , further comprising a step of treating the mixture in step (2) between step (2) and step (3) (treating step). [67-1] The method according to

[67] , wherein the treating step is a step of contacting the mixture with an acid or a salt. [67-2] The method according to

[67] , wherein the treating step is a step of adding a solution of a sulfoxide-based solvent containing an acid or a salt to the mixture. [67-3] The method according to

[67] , wherein the treating step is a step of adding a DMSO solution containing an acid or a salt to the mixture.

[68] The method according to

[67] , wherein the treating step is a step of washing a resin for solid phase synthesis. [68-1] The method according to

[68] , wherein the washing step comprises washing with a solvent containing a sulfoxide-based solvent. [68-2] The method according to

[68] , wherein the washing step comprises washing with a solvent containing DMSO. [68-3] The method according to

[68] , wherein the washing step comprises washing with DMSO. [68-4] The method according to

[68] , wherein the washing step comprises washing with a solution of a sulfoxide-based solvent containing a base. [68-5] The method according to

[68] , wherein the washing step comprises washing with a DMSO solution containing a base. [68-6] The method according to either [68-4] or [68-5], wherein the washing step comprises washing with a DMSO solution containing at least one base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, and 2,4,6-trimethylpyridine. [68-7] The method according to any one of [68-1] to [68-6], wherein the washing step is carried out two, three, four, five, or six times.[68-8] The method according to any one of

[68] to [68-7], wherein one or more washings in the washing step comprise a step of washing with a solution of a sulfoxide-based solvent containing an acid or a salt. [68-8-1] The method according to [68-8], wherein one or more washings in the washing step comprise a step of washing with a solution of a sulfoxide-based solvent containing an acid. [68-8-2] The method according to [68-8], wherein one or more washings in the washing step comprise a step of washing with a DMSO solution containing an acid. [68-8-3] The method according to [68-8], wherein one or more washings in the washing step comprise a step of washing with a solution of a sulfoxide-based solvent containing a salt. [68-8-4] The method according to [68-8], wherein one or more washings in the washing step comprise a step of washing with a DMSO solution containing a salt. [68-9] The method of any of

[68] to [68-7], wherein one or more washings in the washing step comprise a step of washing with a sulfoxide-based solvent solution containing an acid and a base. [68-10] The method of any of

[68] to [68-7], wherein one or more washings in the washing step comprise a step of washing with a DMSO solution containing an acid and a base. [68-11] The method of any of [68-8] to [68-11], wherein the one or more washings in the washing step are a single wash.

[69] The method of any of [67-1] to [68-3] and [68-8] to [68-11], wherein the acid is at least one selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid. [69-1] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the acid is at least one selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, and HOOBt.[69-2] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the salt is a salt consisting of an acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid, and a base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine. [69-3] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the salt is pyridine hydrochloride. [69-4] The method according to [68-9] or [68-10], wherein the base is at least one selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine.

[70] The washing step comprises: i) pre-cleaning CO. ２ and / or ii) washing the resin for solid phase synthesis with a solvent that has been previously contacted with CO ２

[71] The method according to

[68] , comprising a step of washing the resin for solid phase synthesis with a solvent that has not been in contact with CO.

[72] The method according to

[68] , wherein the washing step comprises: i) washing the resin with a solvent that has not been in contact with CO ２ and / or ii) washing the resin for solid phase synthesis with a solvent into which CO ２

[72] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[71] , wherein the solvent in step (3) is the same as the solvent in step (2).

[73] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[72] , wherein the solvent in step (3) is a solvent in which the condensing agent is soluble.

[74] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[73] , wherein the solvent in step (3) comprises at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[75] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[73] , wherein the solvent in step (3) contains 50 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate-based solvents.

[76] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[73] , wherein the solvent in step (3) contains 70 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate-based solvents.

[77] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[73] , wherein the solvent in step (3) contains 90 v / v % or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents.

[78] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[77] , wherein the solvent in step (3) is at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate-based solvents.

[79] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[78] , wherein the solvent in step (3) is aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, or phosphate-based solvents.

[80] The aromatic hydrocarbon solvent is one or more selected from the group consisting of benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzeneanisole, ethylbenzene, nitrobenzene, and cumene; the halogen-based solvent is one or more selected from the group consisting of dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride; the ether-based solvent is one or more selected from the group consisting of diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,3-dioxolane, 1,4-dioxane, 1,2-dimethoxyethane, diisopropyl ether, t-butyl methyl ether, diglyme, triglyme, and tetraglyme; and the amide-based solvent is one or more selected from the group consisting of DMF, NMP, DMA, NEP, NBP, and formamide. the sulfoxide solvent is one or more selected from the group consisting of DMSO and methylphenyl sulfoxide, the sulfone solvent is one or more selected from the group consisting of diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone, the urea solvent is one or more selected from the group consisting of DMI and DMPU,The method according to any one of

[74] to

[79] , wherein the ester solvent is one or more selected from the group consisting of methyl acetate, ethyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone; the ketone solvent is one or more selected from the group consisting of acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, diethyl ketone, and cyclopentanone; the carbonate solvent is one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate, and dibutyl carbonate; and the phosphate ester solvent is one or more selected from the group consisting of trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[81] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[80] , wherein the carboxylic acid used in the step (3) is an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, and the protecting group containing an Fmoc skeleton possessed by the amino acid or the second peptide is the same as or different from the protecting group containing an Fmoc skeleton possessed by the first peptide used in the step (1).

[82] The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[80] , wherein the carboxylic acid analog used in the step (3) is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkylcarboxylic acid, or a C6-C 10 the carboxyl group of an amino acid having a protecting group containing an Fmoc skeleton, the C-terminal carboxyl group of a second peptide having a protecting group containing an Fmoc skeleton, the carboxyl group of the C1-C8 alkylcarboxylic acid, or the C6-C 10 The method according to any one of [0-1], [0-4] to [0-6], and [3] to

[81] , wherein a carboxyl group of the arylcarboxylic acid is converted to an acid halide group.

[0018] Although [1] to

[82] are methods for producing peptides by a solid phase method, these methods can also be applied to methods for producing peptides by a liquid phase method. That is, one aspect of the present invention provides the following

[83] to [84-1].

[83] to [84-1] may include features other than those specific to the production methods by a solid phase method among [3] to

[82] .

[83] A method for producing a peptide by a liquid phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc skeleton; (2) after step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile, in a solvent containing a sulfoxide solvent; and (3) after step (2), condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[84] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton impurities produced in peptide production by a liquid phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc skeleton supported on a solid phase; and (2) after step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent. [84-1] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine backbone impurities, and dimer urea impurities in peptide production by a liquid phase method, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or higher, in a solvent containing a sulfoxide solvent. This method enables the production of a target peptide with high efficiency.

[0019] According to the present invention, even in peptide synthesis containing an amino acid sequence for which the desired elongation reaction does not proceed sufficiently under conventional conditions due to the formation of a diketopiperazine and / or a 6-membered cyclic amidine backbone structure, the formation of such impurities can be significantly reduced, thereby enabling efficient peptide chain elongation to obtain a peptide having the desired amino acid sequence. The method of the present invention does not require the use of a special protecting group such as less common Alloc, and is not subject to operational restrictions such as extremely short reaction times, making it a highly versatile and practical synthesis method that can be scaled up.

[0020] FIG. 1 shows the synthesis of Fmoc-Phe-NMe in N,N-dimethylformamide-d7 (DMF-d7). ２ Between 3.6-6.8 ppm before and after the action of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) on (Compound 1-4-1) １ The H-NMR spectrum shows the characteristic H-NMR spectrum of Compound 1-4-1, Compound 1-4-2, Compound 1-4-3a, and dibenzofulvene observed in this range. １ In FIG. 1, a) shows the H signal of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ 1 shows the H-NMR spectrum of a solution of compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ 2 shows a H-NMR spectrum. In N,N-dimethylformamide-d7 (DMF-d7), compound 1-4-2, which is produced by reacting compound 1-4-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), and compound 1-4-3a undergo chemical exchange, and the ratio of the two compounds is 82:18. １ In FIG. 2, a)-1 represents a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １2 shows the H-NMR spectrum. In FIG. 2, a)-2 shows the H-NMR spectrum of 4.82 ppm of the solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ 2 shows a 1D-ROESY spectrum obtained by selectively exciting the H signal. In FIG. 2, a)-3 shows the 3.95 ppm peak of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ 2 shows a 1D-ROESY spectrum obtained by selectively exciting the H signal. In FIG. 2, b) shows the 4.85 ppm peak of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ H signal and 3.95 ppm １ The integral ratio of the H signal is shown. １ 3 shows a H-NMR spectrum of Compound 1-4-2 produced by reacting Compound 1-4-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). １ 3 shows the H-NMR spectrum. In FIG. 3, a)-1 shows the H-NMR spectrum of the authentic sample of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １ 3 shows the H-NMR spectrum. In FIG. 3, a)-2 shows the H-NMR spectrum of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ In FIG. 3, a)-3 represents a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-4-2. １In FIG. 3, a)-4 represents the 1H-NMR spectrum of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-4-2. １ 3 shows a 1D-NOESY spectrum obtained by selectively exciting the H signal. In FIG. 3, a)-5 shows the 3.95 ppm peak of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-4-2. １ 3 shows the 1D-NOESY spectrum obtained by selectively exciting the H signal. In FIG. 3, b) shows the spectrum of a solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then adding 3 μL of a sample of compound 1-4-2. １ The integral values ​​of the proton signal at 4.84 ppm and the signal at 3.95 ppm in the H-NMR spectrum were measured, and the abundance ratio of Compound 1-4-3 to Compound 1-4-2 in the solution was calculated based on the integral values. １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was reacted with the resulting solution, and the concentration of the compound 1-4-3a was around 5.1-3.7 ppm. １ 4 shows H-NMR and 1D-NOESY spectra. In FIG. 4, a) shows the H-NMR spectrum of a standard sample of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １ 4 shows the H-NMR spectrum. In FIG. 4, b) shows the result of 1-4-2 immersed in an N,N-dimethylformamide-d7 (DMF-d7) solution for 1 minute. １３ CO ２ The solution obtained by bubbling １4 shows the H-NMR spectrum. In FIG. 4, c) shows the result of 1-4-2 immersed in an N,N-dimethylformamide-d7 (DMF-d7) solution for 1 minute. １３ CO ２ The solution obtained by bubbling １ 4 shows the 1D-NOESY spectrum obtained by selectively exciting the H signal. In FIG. 4, d) shows the 1D-NOESY spectrum obtained by selectively exciting the H signal in an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-4-2 for 1 minute. １３ CO ２ The solution obtained by bubbling １ 4 shows the 1D-NOESY spectrum obtained by selectively exciting the H signal. In FIG. 4, e) shows the result of 1D-NOESY of a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute. １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was allowed to act on the resulting solution. １ 4 shows the H-NMR spectrum. In FIG. 4, f) shows the result of 1 minute incubation of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １３ CO ２ After bubbling, １ H signal and 3.96 ppm １ The integral ratio of the H signal is shown. １ In FIG. 4, e) shows the H-NMR spectrum of compound 1-4-2 in an N,N-dimethylformamide-d7 (DMF-d7) solution for 1 minute. １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was allowed to act on the resulting solution. １ H-NMR spectrum at 4.83 ppm １ This is an enlarged view of the H signal. To improve the resolution, this view was converted using a Sin-bell window function. Figure 5 shows the results of the analysis of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １３ CO ２After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was reacted with the resulting solution, and the peaks at around 164-124 ppm indicated that compound 1-4-3a was produced. １３ 5 shows the C-NMR spectrum of Compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １３ CO ２ The solution obtained by bubbling １３ 5 shows the C-NMR spectrum of Compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was allowed to react with the resulting solution. １３ 6 shows a C-NMR spectrum of Compound 1-4-3a in a solution (Solution B, Sol B) obtained by reacting Compound 1-4-1 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7). １ 6 shows the H-NMR spectrum of a solution (solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). １ 6 shows H-NMR spectra. In FIG. 6, b) shows a solution (solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7), and a solution (solution C) obtained by reacting an N,N-dimethylformamide-d7 (DMF-d7) solution of compound 1-4-2 with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) for 1 minute. １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was reacted with the resulting solution (solution A) to obtain a solution of １ 6 shows the H-NMR spectrum. In FIG. 6, c) shows the result of 1-4-2 immersed in an N,N-dimethylformamide-d7 (DMF-d7) solution for 1 minute.１３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was allowed to act on the resulting solution (Solution A). １ 7 shows H-NMR spectra of a solution (solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7), and a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) after 1 minute. １３ CO ２ After bubbling, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added to the resulting solution (Solution A). The resulting solution was analyzed by NH-HSQC ( １５ N[F1]: around 97-85 ppm, １ H[F2]: around 5.7-4.7ppm), NH-HMBC ( １５ N[F1]: around 101-83 ppm, １ H[F2]: around 3.04-2.64ppm), CH-HMBC ( １３ C[F1]: around 165-158 ppm, １ 7A shows the spectra of a solution (solution B) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7), and a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) after 1 minute. １３ CO ２ 7 shows the NH-HSQC spectrum of a solution obtained by bubbling Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then mixing it with a solution (Solution A) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). In FIG. 7, b) shows a solution obtained by reacting Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (Solution B), and a solution of Compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute.１３ CO ２ 7 shows the NH-HMBC spectrum of a solution obtained by bubbling Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then mixing it with a solution (Solution A) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). In FIG. 7, c) shows a solution obtained by reacting Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (Solution B), and a solution of Compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) for 1 minute. １３ CO ２ 8 shows the CH-HMBC spectrum of a solution obtained by bubbling Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and then mixing it with a solution (Solution A) obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). 9 shows the CH-HMBC spectrum of a solution obtained by reacting Compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) with Compound 1-4-2 for 1 minute. １３ CO ２ After bubbling, the solution obtained by reacting 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) with the compound (solution A) was mixed, and the carbamate moiety of the compound 1-4-3a present in the solution obtained was １ H. １３ C. １５ Figure 1 shows that all N signals were identified.

[0021] (Definition of Terms) In this specification, examples of the "halogen atom" include F, Cl, Br and I.

[0022] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a hydrocarbyl or hydrocarbon group structure subset containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C １-C ２０ , hereinafter referred to as “C ｐ -C ｑ " means that the number of carbon atoms is p to q), and preferably C １ -C １０ Alkyl, more preferably C １ -C ６ Specific examples of the alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl.

[0023] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP ２ It is a monovalent group having 1 carbon atom. Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be in the Entgegen (E) or Zusammen (Z), cis or trans configuration. Alkenyl includes not only straight chains but also branched chains. C is preferred as alkenyl. ２ -C １０ alkenyl, more preferably C ２ -C ６ Examples include alkenyl, specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, and the like.

[0024] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. C ２ -C １０ Alkynyl, more preferably C ２ -C ６ Specific examples include alkynyl, such as 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, and 3-methyl-(5-phenyl)-4-pentynyl.

[0025] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. ３ -C ８ Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like.

[0026] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C ６ -C １０ Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

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

[0028] As used herein, "heteroaryl" refers to an aromatic, cyclic, monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a monocyclic ring or a condensed ring with another ring, and may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0029] As used herein, "alkoxy" refers to an oxy group to which the above-defined "alkyl" is bonded, and preferably C １ -C ６ Specific examples of the alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0030] As used herein, "alkenyloxy" refers to an oxy group having the above-defined "alkenyl" bonded thereto, and preferably C ２ -C ６ Specific examples of alkenyloxy include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentenyloxy, and hexenyloxy.

[0031] As used herein, "cycloalkoxy" refers to an oxy group to which the above-defined "cycloalkyl" is bonded, and preferably C ３ -C ８ Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0032] As used herein, "aryloxy" refers to an oxy group to which the above-defined "aryl" is bonded, and preferably C ６ -C １０ Specific examples of the aryloxy include phenoxy, 1-naphthyloxy, and 2-naphthyloxy.

[0033] As used herein, the term "heteroaryloxy" refers to an oxy group having the above-defined "heteroaryl" bonded thereto, and preferably includes 5- to 10-membered heteroaryloxy.

[0034] In the present specification, "amino" means, in a narrow sense, -NH ２ and broadly, -NRR', 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. ２ , Mono C １ -C ６ Alkylamino, DiC １ -C ６ Examples thereof include alkylamino and 4- to 8-membered cyclic amino.

[0035] As used herein, the term "monoalkylamino" refers to a group in which R is hydrogen and R' is an "alkyl" as defined above, among the "amino" groups defined above, and preferably monoC １ -C ６ Specific examples of monoalkylamino include methylamino, ethylamino, n-propylamino, i-propylamino, n-butylamino, s-butylamino, and t-butylamino.

[0036] As used herein, "dialkylamino" refers to a group in which R and R' are independently "alkyl" as defined above, among the "amino" groups defined above, and preferably diC １ -C ６ Specific examples of dialkylamino include dimethylamino and diethylamino.

[0037] As used herein, "cyclic amino" refers to the above-defined "amino" in which R and R' form a ring together with the nitrogen atom to which they are attached, and preferably includes 4- to 8-membered cyclic amino. Specific examples of cyclic amino include 1-azetidyl, 1-pyrrolidyl, 1-piperidyl, 1-piperazyl, 4-morpholinyl, 3-oxazolidyl, 1,1-dioxidethiomorpholinyl-4-yl, 3-oxa-8-azabicyclo[3.2.1]octan-8-yl, and the like.

[0038] As used herein, "haloalkyl" refers to an "alkyl" defined above in which one or more hydrogen atoms are substituted with halogen. １ -C ８ Haloalkyl is preferred, C １ -C ６ Haloalkyl is more preferred.

[0039] As used herein, "fluoroalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are substituted with fluorine atoms, and C １ -C ８ Fluoroalkyl is preferred. Specific examples of haloalkyl include monofluoromethyl, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl.

[0040] As used herein, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" defined above is substituted with an "aryl" defined above, and C ７ -C １４ Aralkyl is preferred, C ７ -C １０ Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0041] As used herein, "aralkyloxy" refers to an oxy group to which the above-defined "aralkyl" is bonded, and ７ -C １４ Aralkyloxy is preferred, C ７ -C １０ Aralkyloxy is more preferred. Specific examples of aralkyloxy include benzyloxy, phenethyloxy, and 3-phenylpropoxy.

[0042] As used herein, the term "peptide chain" refers to a peptide chain in which one or more natural amino acids and / or unnatural amino acids are linked by amide bonds and / or ester bonds. The peptide chain preferably contains 1 to 15 amino acid residues, and more preferably contains 5 to 12 amino acid residues.

[0043] The "peptide compound" of the present invention is not particularly limited as long as it is a peptide compound in which natural amino acids and / or unnatural amino acids are linked by amide bonds or ester bonds, but is preferably a peptide compound of 5 to 30 residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. The peptide compound synthesized in the present invention preferably contains at least three N-substituted amino acids, and more preferably at least five or more N-substituted amino acids, in one peptide. These N-substituted amino acids may be present consecutively or discontinuously in the peptide compound. The peptide compound of the present invention may be linear or cyclic, with cyclic peptide compounds being preferred. In this specification, "peptide compound" may also be referred to as "peptide."

[0044] The "cyclic peptide compound" of the present invention is a cyclic peptide compound obtainable by cyclizing a group on the N-terminal side and a group on the C-terminal side of a linear peptide compound. The cyclization may be in any form, such as cyclization via a carbon-nitrogen bond such as an amide bond, cyclization via a carbon-oxygen bond such as an ester bond or an ether bond, cyclization via a carbon-sulfur bond such as a thioether bond, cyclization via a carbon-carbon bond, or cyclization by constructing a heterocyclic ring. Among these, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxylic acid group on a side chain and an amino group on the N-terminal main chain is more preferred. The position of the carboxylic acid group or amino group used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position that allows cyclization.

[0045] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0046] As used herein, the term "resin for solid-phase synthesis" is not particularly limited as long as it can be used in the synthesis of peptide compounds by solid-phase synthesis. Specific examples of such resins for solid-phase synthesis include CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt), which are removable under acidic conditions. The resin can be appropriately selected depending on the functional group of the amino acid used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid typified by Asp or Glu) or a hydroxy group on an aromatic ring (phenol group typified by Tyr) is used as the functional group of the amino acid, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin). When an aliphatic hydroxy group (an aliphatic alcohol group typified by Ser or Thr) is used as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin), or 4-methyltrityl chloride resin (Mtt resin) as the resin. In this specification, resin may also be referred to as "resin." Cellulose may also be used as the solid phase. In this specification, the term "solid phase" may also be used synonymously with "resin for solid-phase synthesis."

[0047] There are no particular limitations on the type of polymer that constitutes the resin. In the case of a resin made of polystyrene, either 100-200 mesh or 200-400 mesh may be used. There are also no particular limitations on the cross-linking rate, but 1% DVB (divinylbenzene) cross-linking is preferred. Examples of the type of polymer that constitutes the resin include Tentagel and Chemmatrix.

[0048] In the production of the compounds described herein, if a defined group undergoes an undesired chemical transformation under the conditions of the method, the compound can be produced by, for example, using means such as protection and deprotection of the functional group. Here, the selection and deprotection of the protecting group can be performed using, for example, the method described in "Greene's, 'Protective Groups in Organic Synthesis' (5th ed., John Wiley & Sons 2014)," which can be used appropriately depending on the reaction conditions. Furthermore, the order of reaction steps such as the introduction of substituents can also be changed as necessary.

[0049] In the present specification, when the modifier "optionally substituted" is used, examples of the substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, and the like.

[0050] Furthermore, each of these may be given a substituent, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom, such as optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl.

[0051] The compounds described herein may be in the form of salts or solvates thereof. Examples of the salts described herein include hydrochlorides; hydrobromides; hydroiodides; phosphates; phosphonates; sulfates; sulfonates such as methanesulfonate and p-toluenesulfonate; carboxylates such as acetate, citrate, malate, tartrate, succinate, and salicylate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; and ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, and tetraalkylammonium salt. These salts are produced, for example, by contacting the compound with an acid or a base. A solvate of a compound described herein refers to a compound that forms a molecular group together with a solvent, and is not particularly limited as long as it is a solvate formed with a solvent. Examples of solvates include hydrates, alcoholates (ethanol solvates, methanol solvates, 1-propanol solvates, 2-propanol solvates, etc.), and solvates with a single solvent such as dimethyl sulfoxide, as well as solvates formed with multiple solvents per molecule of the compound, or solvates formed with multiple types of solvents per molecule of the compound. When the solvent is water, the solvate is called a hydrate. As the solvate of the compound of the present invention, hydrates are preferred, and specific examples of such hydrates include mono- to decahydrates, preferably mono- to pentahydrates, and more preferably mono- to trihydrates.

[0052] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Examples of unnatural amino acids include, but are not limited to, β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains differ from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids may have any configuration. The side chain of an amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, and a spiro-linked cycloalkyl group. Each of these may be substituted, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom. Examples of such substituents include optionally substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., as well as oxo, aminocarbonyl, and halogen atoms. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0053] In this specification, examples of the substituent containing a halogen atom include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, each of which has a halogen atom as a substituent, and more specific examples thereof include a fluoroalkyl, a difluoroalkyl, a trifluoroalkyl, and the like.

[0054] Substituents containing an O atom include hydroxy (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxy (-CO ２H), 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-SO ２ -R), aminosulfonyl (-SO ２ -NHR), sulfamoylamino (-NH-SO ２ -NHR), thiocarboxyl (-C(=O)-SH), carboxylcarbonyl (-C(=O)-CO ２ H).

[0055] Examples of oxy (—OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. Alkoxy includes C １ -C ４ Alkoxy, C １ -C ２ Alkoxy is preferred, and methoxy or ethoxy is particularly preferred.

[0056] Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0057] Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0058] Examples of carbonyloxy (—O—C(═O)—R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

[0059] Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0060] Examples of carbonylthio (-S-C(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0061] Examples of aminocarbonyl (—C(═O)—NHR) include alkylaminocarbonyl (e.g., C １ -C ６ or C １ -C ４ Examples include alkylaminocarbonyl, especially ethylaminocarbonyl and methylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0062] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0063] Examples of oxycarbonylamino (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0064] Sulfonylamino (-NH-SO ２ Examples of —R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, —NH—SO ２ Examples include groups in which the H atom bonded to the N atom in —R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0065] Aminosulfonyl (-SO ２ Examples of —NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, and the like. ２Examples include groups in which the H atom bonded to the N atom in —NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0066] Sulfamoylamino (-NH-SO ２ Examples of —NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, and aralkylsulfamoylamino. ２ The two H atoms bonded to the N atom in —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.

[0067] Substituents containing an S atom include thiol (-SH), thio (-S-R), sulfinyl (-S(=O)-R), sulfonyl (-SO ２ -R), sulfo (-SO ３ H).

[0068] Examples of thio (-S-R) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0069] Sulfonyl (-SO ２ Examples of —R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0070] As a substituent containing a nitrogen atom, azide (-N ３ , also called "azido group"), cyano (-CN), primary amino (-NH ２ ), secondary amino (-NH-R; also called monosubstituted amino), tertiary amino (-NR(R'); also called disubstituted amino), amidino (-C(=NH)-NH ２), substituted amidino (—C(═NR)—NR′R″), guanidino (—NH—C(═NH)—NH ２ ), substituted guanidino (—NR—C(═NR′″)—NR′R″), aminocarbonylamino (—NR—CO—NR′R″), pyridyl, piperidino, morpholino, azetidinyl, and the like.

[0071] Examples of secondary amino (-NH-R; monosubstituted amino) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, and aralkylamino.

[0072] Examples of tertiary amino (—NR(R′); disubstituted amino) include an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring. Specifically, dialkylamino, especially C １ -C ６ Dialkylamino, C １ -C ４ Examples include dialkylamino, dimethylamino, and diethylamino. ｐ -C ｑ "Dialkylamino group" means an amino group having C ｐ -C ｑ It means a group substituted with two alkyl groups, and both C ｐ -C ｑ The alkyl groups may be the same or different.

[0073] Examples of substituted amidino (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0074] Examples of substituted guanidino (-NR-C(=NR'")-NR'R") include groups in which R, R', R", and R'" are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0075] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0076] In this specification, the "amino acid residues" that constitute a peptide compound may be simply referred to as "amino acids".

[0077] In the present invention, the term "N-substituted amino acid" refers to an amino acid defined above in which the main chain amino group is N-substituted. Specific examples of N-substituted amino acids include those in which the main chain amino group of the amino acid is an NHR group, where R is any group other than hydrogen, such as an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group, and cyclic amino acids such as proline in which the carbon atom bonded to the N atom and the carbon atom from the α-position form a ring. Examples include prolines that form a five-membered ring, azetidines that form a four-membered ring, and piperidines that form a six-membered ring. The substituent of each optionally substituted group is not particularly limited, and examples include a halogen group, an ether group, and a hydroxyl group.

[0078] In the present invention, the term "N-alkylamino acid" refers to an amino acid in which the main chain amino group is substituted with an alkyl group, among the N-substituted amino acids defined above. １ -C １０ Alkyl amino acids, N-C １ -C ６Specific examples of N-alkyl amino acids include N-methyl amino acids, N-ethyl amino acids, N-(n-propyl) amino acids, and N-(i-propyl) amino acids.

[0079] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is ２ H. ３ H. １３ C. １４ C. １５ N. １７ O. １８ O. ３２ P. ３５ S. １８ F. ３６ Cl and the like are included.

[0080] As used herein, the term "prolines" refers to a group of compounds comprising proline and one or more optional substituents bonded to any carbon atom forming the five-membered ring of the proline. When multiple substituents are present on the five-membered ring, these substituents may be combined to form a ring, which may be any aromatic ring. Specific examples of "prolines" include proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-4-fluoro-L-proline, cis-4-fluoro-L-proline, and 2-methyl-L-proline. When a proline contains a hydroxy group, the hydroxy group may be protected by an optional protecting group, or an ether or ester bond may be formed between the optional substituent and the oxygen atom of the hydroxy group. Furthermore, any one of the carbon atoms forming the five-membered ring may be replaced with an oxygen atom or a sulfur atom. Specific examples of such compounds include L-thioproline. Furthermore, prolines may contain an unsaturated bond within the five-membered ring they form. A specific example of such a compound is 3,4-dehydro-L-proline, which may be in the D-form as well as the L-form.

[0081] As used herein, "azetidines" refers to cyclic amino acids based on a compound in which one nitrogen atom and three carbon atoms form a four-membered ring, with a carboxyl group bonded to the carbon atom adjacent to the nitrogen atom. Any carbon atom forming the four-membered ring may be bonded with one or more optional substituents. When multiple substituents are present on the four-membered ring, these substituents may be joined together to form a ring. Specific examples of "azetidines" include (S)-azetidine-2-carboxylic acid and (R)-azetidine-2-carboxylic acid. Compounds in which a carboxyl group is bonded to the second carbon atom, counting from the nitrogen atom forming the four-membered ring, are also included in the "azetidines." Examples of such compounds include azetidine-3-carboxylic acid.

[0082] As used herein, "piperidines" refers to cyclic amino acids based on a compound in which one nitrogen atom and five carbon atoms form a six-membered ring, with a carboxyl group bonded to the carbon atom adjacent to the nitrogen atom. Any carbon atom forming the six-membered ring may be bonded to one or more optional substituents. When multiple substituents are present on the six-membered ring, these substituents may together form a ring, which may be any aromatic ring. Specific examples of "piperidines" include (R)-piperidine-2-carboxylic acid, (S)-piperidine-2-carboxylic acid, (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0083] In this specification, examples of aromatic hydrocarbon solvents include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene.

[0084] In this specification, examples of halogen-based solvents include dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride.

[0085] In this specification, examples of ether solvents include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme.

[0086] In this specification, examples of amide solvents include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide.

[0087] In this specification, examples of sulfoxide solvents include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide.

[0088] In this specification, examples of sulfone solvents include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0089] In this specification, examples of urea-based solvents include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0090] In this specification, examples of the ester solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone.

[0091] In this specification, examples of ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone.

[0092] In this specification, examples of carbonate solvents include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate.

[0093] In this specification, examples of phosphate ester solvents include trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[0094] As used herein, "amidines" refer to bases represented by the following formula B1: [In the formula, RB １ and R.B. ４ are each independently C １ -C ４ alkyl or R １ and R.B. ４ is RB １and the nitrogen atom to which R ４ forms a 5- to 8-membered ring together with the carbon atom to which R B is attached, ２ and R.B. ３ are each independently C １ -C ４ alkyl or R ２ and R.B. ３ is RB ２ and the nitrogen atom to which R ３ is bonded to the nitrogen atom and the carbon atom to which the nitrogen atom is bonded to form a 5- to 8-membered ring.

[0095] RB １ ~RB ４ But C １ -C ４ When it is alkyl, the C １ -C ４ Preferred examples of alkyl include methyl and ethyl. １ and R.B. ４ When R B forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. ２ and R.B. ３ When forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0096] Specific examples of amidines include DBU: 1,8-diazabicyclo[5.4.0]-7-undecene and DBN: 1,5-diazabicyclo[4.3.0]-5-nonene.

[0097] As used herein, the term "guanidines" refers to bases represented by the following formula B2: [In the formula, RB ６ is hydrogen or C １ -C ４ is alkyl, and R B ５ and R.B. ７ are each independently C １ -C ４ alkyl or form a 5- to 8-membered ring together with the nitrogen atoms to which they are attached and the carbon atoms to which they are attached;８ is C １ -C ４ alkyl, and RB ９ is hydrogen, C １ -C ４ alkyl or phenyl, or R ８ and R.B. ９ form a 5- to 8-membered ring together with the nitrogen atoms to which they are attached and the carbon atoms to which they are attached, wherein R, ９ is phenyl, two benzene rings of the phenyl groups of the two formulae B2 may be fused to form a naphthalene.

[0098] RB ５ ~RB ８ But C １ -C ４ When it is alkyl, the C １ -C ４ The alkyl is preferably methyl, and R ９ But C １ -C ４ When it is alkyl, the C １ -C ４ The alkyl is preferably t-butyl. ５ and R.B. ７ When R B forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably an imidazolidine ring, a hexahydropyrimidine ring, a 1,3-diazepane ring, or the like. ８ and R.B. ９ When forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0099] Specific examples of guanidines include TMG: 1,1,3,3-tetramethylguanidine, TBD: 1,5,7-triazabicyclo[4.4.0]dec-5-ene, and MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene.

[0100] As used herein, the term "phosphazenes" refers to bases represented by the following formula B3 or B4:

[0101] [In the formula, RB１０ is C １ -C ４ alkyl or R １０ and R.B. １１ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B １１ is RB １０ and R.B. １１ forms a 5- to 8-membered ring, １ -C ４ alkyl or R １１ and R.B. １２ form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, and RB １２ is RB １１ and R.B. １２ forms a 5- to 8-membered ring, １ -C ４ alkyl or R １２ and R.B. １３ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B １３ is RB １２ and R.B. １３ forms a 5- to 8-membered ring, １ -C ４ alkyl or R １３ and R.B. １４ form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, and RB １４ is RB １３ and R.B. １４ forms a 5- to 8-membered ring, １ -C ４ alkyl or R １４ and R.B. １５ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B １５ is RB １４ and R.B. １５ forms a 5- to 8-membered ring, １ -C ４ is alkyl, and R B １６ is hydrogen, C１ -C ８ Alkyl, or C ６ -C １０ It is aryl.

[0102] RB １０ ~RB １５ But C １ -C ４ When it is alkyl, the C １ -C ４ The alkyl is preferably methyl or ethyl, and R １６ But C １ -C ８ When it is alkyl, the C １ -C ８ The alkyl is preferably t-butyl or t-octyl. １０ and R.B. １１、 RB １２ and R.B. １３ , and / or RB １４ and R.B. １５ When R B forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. １１ and R.B. １２ , and / or RB １３ and R.B. １４ When R forms a 5- to 8-membered ring, the 5- to 8-membered ring is １１ , R.B. １２ , R.B. １３ , and R.B. １４ is preferably a 5- to 8-membered saturated ring that does not contain any heteroatoms other than the nitrogen atoms to which each is bonded and the phosphorus atom to which each nitrogen atom is bonded.

[0103] [In the formula, RB １７ is independently C １ -C ４ alkyl or R １７ and R.B. １８ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B １８ is RB １７ and R.B. １８ forms a 5- to 8-membered ring, １ -C ４alkyl or R １８ and R.B. １９ form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, and RB １９ is RB １８ and R.B. １９ forms a 5- to 8-membered ring, １ -C ４ alkyl or R １９ and R.B. ２０ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B ２０ is RB １９ and R.B. ２０ forms a 5- to 8-membered ring, １ -C ４ is alkyl, and R B ２１ is C １ -C ４ alkyl or R ２１ and R.B. ２２ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B ２２ is RB ２１ and R.B. ２２ forms a 5- to 8-membered ring, １ -C ４ alkyl or R ２２ and R.B. ２３ form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, and RB ２３ is RB ２２ and R.B. ２３ forms a 5- to 8-membered ring, １ -C ４ alkyl or R ２３ and R.B. ２４ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B ２４ is RB ２３ and R.B. ２４ forms a 5- to 8-membered ring, １ -C ４ alkyl or R２４ and R.B. ２５ form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which they are bonded, and RB ２５ is RB ２４ and R.B. ２５ forms a 5- to 8-membered ring, １ -C ４ alkyl or R ２５ and R.B. ２６ form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, and R B ２６ is RB ２５ and R.B. ２６ forms a 5- to 8-membered ring, １ -C ４ is alkyl, and R B ２７ is C １ -C ４ Alkyl, or C ６ -C １０ It is aryl.

[0104] RB １７ ~RB ２６ But C １ -C ４ When it is alkyl, the C １ -C ４ The alkyl is preferably methyl or ethyl, and R ２７ But C １ -C ４ When it is alkyl, the C １ -C ４ The alkyl is preferably ethyl or t-butyl. １７ and R.B. １８、 RB １９ and R.B. ２０ , R.B. ２１ and R.B. ２２、 RB ２３ and R.B. ２４、 RB ２５ and R.B. ２６ When R B forms a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring, or the like. １７ and R.B. １８ Both are C １ -C ４When R is alkyl, １９ and R.B. ２０ Also C １ -C ４ Preferably, R is alkyl; １７ and R.B. １８ form a 5- to 8-membered ring, R B １９ and R.B. ２０ It is preferable that R B also forms a 5- to 8-membered ring. ２１ and R.B. ２２ Both are C １ -C ４ When R is alkyl, ２３ and R.B. ２４ and R.B. ２５ and R.B. ２６ Also C １ -C ４ Preferably, R is alkyl; ２１ and R.B. ２２ form a 5- to 8-membered ring, R B ２３ and R.B. ２４ and R.B. ２５ and R.B. ２６ It is preferable that R B also forms a 5- to 8-membered ring. １８ and R.B. １９ , and / or RB ２２ and R.B. ２３ When R forms a 5- to 8-membered ring, the 5- to 8-membered ring is １１ , R.B. １２ , R.B. １３ , and R.B. １４ is preferably a 5- to 8-membered saturated ring that does not contain any heteroatoms other than the nitrogen atoms to which each is bonded and the phosphorus atom to which each nitrogen atom is bonded.

[0105] Specific examples of phosphazenes include P2tBu: 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ ５ , 4λ ５-catenadi(phosphazene), P2Et: tetramethyl(tris(dimethylamino)phosphoranylidene)phosphoric acid triamide-ethylimine, HP1(dma): imino-tris(dimethylamino)phosphorane, BTPP: tert-butylimino-tri(pyrrolidino)phosphorane, P1tBu: tert-butylimino-tris(dimethylamino)phosphorane, BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine, and the like.

[0106] As used herein, the term "and / or" includes any combination of "and" and "or." Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0107] (Production Method) In one aspect, the present invention relates to a method for producing a peptide by a solid phase method, comprising the following steps (1) to (3): (1) providing a first peptide supported on a solid phase and having a protecting group containing an Fmoc backbone; (2) after step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or higher, in a solvent containing a sulfoxide solvent; and (3) after step (2), condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. Note that the "first peptide" in step (2) refers to the first peptide having a protecting group containing an Fmoc backbone.

[0108] Step (1) Step (1) of the present invention is a step of providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase.

[0109] The "solid-phase-supported first peptide having a protecting group containing an Fmoc backbone" in step (1) is usually supported on a resin for solid-phase synthesis via a functional group (e.g., a carboxyl group) in the C-terminal amino acid residue, but may also be supported on the solid phase via a functional group (e.g., a carboxyl group) of an amino acid residue other than the C-terminus. The "solid-phase-supported first peptide having a protecting group containing an Fmoc backbone" has the amino group of its N-terminal amino acid residue protected by a protecting group containing an Fmoc backbone.

[0110] The solid phase supporting the first peptide may be any known in the art, and is generally linked to a predetermined amino acid residue (e.g., the C-terminal amino acid residue) of the first peptide via an ester bond. The resin preferably has a resin-binding group whose acid sensitivity is determined to be "H (<5% TFA in DCM)" as described in the Solid Phase Synthesis Handbook (published by Merck Ltd., May 1, 2002), and can be appropriately selected depending on the functional group of the amino acid used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid such as Asp or Glu) or a hydroxy group on an aromatic ring (phenol group such as Tyr) is used as the functional group of the amino acid, trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (Clt resin) is preferably used. When an aliphatic hydroxy group (an aliphatic alcohol group typified by Ser or Thr) is used as the functional group on the amino acid side, it is preferable to use a trityl chloride resin (Trt resin), a 2-chlorotrityl chloride resin (Clt resin), or a 4-methyltrityl chloride resin (Mtt resin) as the resin.

[0111] In the present invention, the term "protecting group containing an Fmoc skeleton" refers to an Fmoc group or a group in which an arbitrary substituent has been introduced at an arbitrary position of the structural skeleton of the Fmoc group. Specific examples of such a protecting group containing an Fmoc skeleton include a protecting group represented by the following formula (1): (In the formula, R １ ~R ８ are independently hydrogen, C １ -C８ Alkyl, C １ -C ８ R is selected from the group consisting of fluoroalkyl, halogen, sulfo, and trimethylsilyl; ９ ~R １０ are independently hydrogen or methyl).

[0112] More specific examples of the protecting group containing an Fmoc skeleton include a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, a 1-methyl-Fmoc (Fmoc(1Me)) group, a 2-fluoro-Fmoc (Fmoc(2F)) group, a 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, a 2-monoisooctyl-Fmoc (mio-Fmoc) group, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, a 2,7-(3,3,4 ,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc (tdf-Fmoc) group, 2,7-bis(trimethylsilyl)-Fmoc (Fmoc(2TMS)) group, (2-sulfo-9H-fluoren-9-yl)methoxycarbonyl group (Fmoc(2so3h)), [(1S)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (sm-Fmoc), [(1R)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (rm-Fmoc), and the like. The Fmoc group is preferred as a protecting group containing the Fmoc skeleton. These protecting groups containing the Fmoc skeleton can be introduced by known methods using commercially available reagents, etc.

[0113] In the present invention, the type and number of amino acid residues constituting the first peptide are not limited, and any peptide having an amino acid sequence containing two or more amino acid residues, for example, 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, can be used as the first peptide. The amino acids can be natural amino acids and / or unnatural amino acids. The first peptide preferably contains one or more N-substituted amino acids.

[0114] The first peptide having a protecting group containing an Fmoc backbone provided in step (1) (e.g., a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase) can be obtained from a commercial supplier or can be produced by applying a method known in the art, for example, a method described in WO2013 / 100132 or WO2018 / 225864.

[0115] In one embodiment, the first peptide is a dipeptide. In this case, the C-terminal amino acid residue of the dipeptide is supported on a solid phase, for example, by an ester bond. As shown in the following scheme, the free amino group resulting from removal of the protecting group of the N-terminal amino acid residue may react with the carbonyl group in the ester bond to produce undesired DKP. By using the method of the present invention, a decrease in the yield of the target peptide due to DKP elimination can be suppressed. (In the formula, R １ ~R ４ represents any atom or group, and * represents the bonding site with the resin for solid phase synthesis.)

[0116] In one embodiment, the first peptide is a peptide in which the second amino acid residue from the N-terminus is an "N-substituted amino acid." In this case, as shown in the following scheme, the free amino group resulting from removal of the protecting group of the N-terminal amino acid residue may react with a carbonyl group in the peptide bond between the second and third amino acids from the N-terminus, resulting in the formation of undesired DKPs, DKP elimination products, and impurities having an amidine skeleton (6-membered cyclic amidine skeleton structures). By using the method of the present invention, the formation of such DKPs, DKP elimination products, and 6-membered cyclic amidine skeleton structures can be suppressed, thereby preventing a decrease in the yield of the target peptide. (In the formula, R ４ means any atom or group other than hydrogen, and R １ ~R ３ and R ５ ~R ８ represents any atom or group, and * represents the bonding site with the adjacent amino acid residue or solid phase. ６When R is hydrogen, either Route A or Route B in the above scheme can be taken, ６ When is other than hydrogen, only Route A in the above scheme can be taken.

[0117] Preferred examples of the "N-substituted amino acid" in the present invention include N-alkylamino acids that are N-alkylated, such as N-methylated, and cyclic amino acids such as prolines and azetidines.

[0118] Step (2) of the present invention is a step of treating the first peptide provided in step (1) with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent.

[0119] In one embodiment, at least a portion of the first peptide obtained in step (2) is in the form of a carbamate. Without being bound by a particular theory, in step (2), when the protecting group containing the Fmoc skeleton is removed, dibenzofulvene is generated in at least a portion of the first peptide without decarboxylation, and the N-terminal amino group is converted to C(═O)O derived from the protecting group. － together with the carbamate ion (-NRC(=O)O － , where R is hydrogen or any substituent of an amino group), which forms a carbamate with a protonated base in the system (below is a scheme showing the formation of a carbamate when Fmoc is used as a protecting group containing an Fmoc skeleton and DBU is used as a base). The formation of a carbamate salt can suppress the intramolecular nucleophilic reaction of the terminal amino group, and it is presumed that this suppresses the formation of a six-membered ring intermediate even under basic deprotection conditions, thereby reducing the by-production of DKP elimination products and six-membered cyclic amidine skeleton structures.

[0120] In one embodiment, at least a portion of the first peptide obtained in step (2) is in the form of a carbamate salt. Without being bound by any particular theory, it is believed that when the carbamate salt is stable until step (3), a dimeric urea is produced by nucleophilic attack of the amino group at the adjacent N-terminus on an isocyanate formed by the reaction of a condensing agent with a carbamate anion, resulting in an intermolecular reaction (here, this is referred to as intermolecular even when the peptides are supported on the same solid phase) to produce a dimeric urea. Therefore, it is presumed that the production of dimer urea compounds in step (3) can be reduced by decomposing the carbamate salt and converting it to an amine in the step of treating the mixture in step (2) (for example, the step of washing the resin for solid phase synthesis).

[0121] In the method of the present invention, in which a base having a pKa of 23 or higher in acetonitrile is used in step (2), the first peptide can be present as a carbamate in an amount sufficient to significantly reduce the formation of DKP elimination products and 6-membered cyclic amidine skeletal structures. For example, in the method of the present invention, the carbamate can be formed in an amount such that the molar ratio of the carbamate to the amine formed by further decarboxylation (carbamate / amine) is 0.6 or higher, 0.8 or higher, 1.0 or higher, 2.0 or higher, 3.0 or higher, 4.0 or higher, 4.6 or higher, 5.0 or higher, 6.0 or higher, 8.0 or higher, or 10.0 or higher. Such a molar ratio can be determined, for example, by using an amide (e.g., dimethylamide) of the N-terminal amino acid in the first peptide having a protecting group containing an Fmoc skeleton, and treating the solution with the base. １ It can be determined from the proton integral ratio in H-NMR.

[0122] The carbamate may be a salt with any base whose conjugate acid has a pKa in acetonitrile of 23 or higher. Specific preferred examples of such salts include DBU salt, TMG salt, HP1(dma) salt, MTBD salt, P1-tBu salt, and P2-Et salt.

[0123] In one embodiment, the solvent used in step (2) may be a single solvent or a mixed solvent as long as it contains a sulfoxide solvent. In the case of a mixed solvent, it is preferable that the mixed solvent contains 50 v / v % or more, 70 v / v % or more, or 90 v / v % or more of a sulfoxide solvent.

[0124] Specific examples of the sulfoxide solvent used in step (2) include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide, with DMSO being preferred.

[0125] When the solvent used in step (2) is a mixed solvent, the mixed solvent may contain, in addition to a sulfoxide-based solvent, one or more solvents selected from aromatic hydrocarbon-based solvents, halogen-based solvents, ether-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, phosphate ester-based solvents, amide-based solvents, urea-based solvents, and sulfone-based solvents.

[0126] In an embodiment, when the solvent used in step (2) is a mixed solvent, the mixed solvent may contain, in addition to a sulfoxide-based solvent, one or more solvents selected from an amide-based solvent, a urea-based solvent, and a sulfone-based solvent.

[0127] When an aromatic hydrocarbon solvent is used in step (2), specific examples of the solvent include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene, and preferred are toluene and cumene.

[0128] When a halogen-based solvent is used in step (2), specific examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride, with dichloromethane being preferred.

[0129] When an ether solvent is used in step (2), specific examples of the solvent include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane (DME), diisopropyl ether, cyclopentyl methyl ether (CPME), t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme, and preferred are tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, and 1,3-dioxolane.

[0130] When an ester solvent is used in step (2), specific examples of the solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone, and preferred are butyl acetate and methyl propionate.

[0131] When a ketone solvent is used in step (2), specific examples of the solvent include acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone (3-pentanone), and preferred are methyl ethyl ketone and diethyl ketone.

[0132] When a carbonate solvent is used in step (2), specific examples of the solvent include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, with dimethyl carbonate being preferred.

[0133] When a phosphate ester solvent is used in step (2), specific examples of the solvent include trimethyl phosphate, triethyl phosphate, and tributyl phosphate, with tributyl phosphate being preferred.

[0134] When an amide solvent is used in step (2), specific examples of the solvent include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide.

[0135] When a urea-based solvent is used in step (2), specific examples of the solvent include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0136] When a sulfone solvent is used in step (2), specific examples of the solvent include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0137] In one embodiment, the solvent used in step (2) preferably has a swelling capacity of the solid phase resin of 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more. As an index of the swelling capacity of the solid phase resin of each solvent, values ​​described in "Tetrahedron Lett., 1998, 39, 8951-8954", "Org. Process Res Dev. 2018, 22, 494-503", and "RSC Adv., 2020, 10, 42457-42492" can be used. Specific examples of the swelling capacity (mL / g) of the solid phase resin include DMI: 8.5, DMPU: 8.0, NMP: 6.4, THF: 6.0, DMA: 5.8, and CHCl 3. ３ :5.6,CPME:5.6,CH ２ Cl ２ : 5.4, 2-methyltetrahydrofuran: 5.4, DMF: 5.2, butyl acetate: 5.2, DME: 4.8, MEK: 4.4, toluene: 4.0, AcOEt: 4.2, xylene: 3.0, Et ２ O: 2.8, dimethyl carbonate: 2.8, MTBE: 2.4, CH ３ CN: 2.0, Tributyl Phosphate: 1.6, MeOH: 1.6, H ２ O: 1.6. In addition, the judgment in the table described in "Green Chem., 2017, 19, 952-962" or table 2 in "Green Chem., 2020, 22, 996-1018" can also be used.

[0138] In step (2), one or more bases are used, including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile. When only one base is used, that base is a base whose conjugate acid has a pKa of 23 or more in acetonitrile. On the other hand, when multiple bases are used, at least one of the bases is a base whose conjugate acid has a pKa of 23 or more in acetonitrile, and the other bases may be bases whose conjugate acid has a pKa of 23 or more in acetonitrile, or bases whose pKa is less than 23.

[0139] The base having a pKa of 23 or more in acetonitrile of the conjugate acid used in step (2) may be a base having a pKa of 34 or less, preferably 30 or less, more preferably 28 or less.

[0140] pKa is an index that indicates the degree of basicity, and the larger the pKa, the higher the basicity. In the present invention, the pKa (CH ３ CN) as "New J.Chem.2009,33,588" １) or "Sigma-Aldrich, Phosphazene Bases: https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html" ２) Alternatively, values ​​such as those shown in "Eur. J. Org. Chem. 2019, 6735-6748" may be adopted.

[0141] Examples of bases whose conjugate acids have a pKa of 23 or more in acetonitrile include amidines, guanidines, and phosphazenes. For example, the pKa of the bases listed below is 23 or more. １） =24.3 DBN: pKa １） =23.8 TMG: pKa １） =23.3 TBD: pKa １） =26.0 MTBD: pKa １） =25.5 P2tBu: pKa １） =33.5 P2Et:pKa １）=32.9 HP1(dma): pKa １） =25.9 BTPP: pKa ２） =28.4 P1tBu:pKa １） =27.0 BEMP:pKa ２） = 27.6

[0142] In this step, when multiple bases having a pKa of 23 or more in acetonitrile of the conjugate acid are used, multiple bases, for example, two bases selected from the amidines, guanidines, or phosphazenes described above can be used in combination. Specific preferred combinations of such bases include DBU and MTBD, DBU and HP1(dma), DBU and P1tBu, and DBU and P2Et.

[0143] In this step, when a base having a pKa of 23 or more in acetonitrile and a base having a pKa of less than 23 are used in combination, the base having a pKa of 23 or more in acetonitrile can be selected from the amidines, guanidines, or phosphazenes described above. Furthermore, the base having a pKa of less than 23 in acetonitrile is preferably selected from piperidine, piperazine, and morpholine. A preferred example of a specific combination of such bases is DBU and piperidine.

[0144] In one embodiment, step (2) can be repeated multiple times before step (3). In this case, the same solvent and / or the same base may be used each time step (2), or different solvents and / or different bases may be used. In the present invention, the term "same solvent" means that the type of solvent and the mixing ratio of the solvents are the same, and the term "different solvents" means that either or both of the type of solvent and the mixing ratio of the solvents are different. Furthermore, in the present invention, the term "same base" means that the type of base and the concentration of the base in the solvent are the same, and the term "different bases" means that either or both of the type of base and the concentration of the base in the solvent are different.

[0145] In one embodiment, when step (2) is included multiple times, the method of the present invention may include a step of discharging the solution between each step (2), thereby replacing the solvent and / or base used in the previous step (2) with the solvent and / or base used in the next step (2).

[0146] In one embodiment, step (2) is a step of removing a protecting group containing an Fmoc backbone and / or a step of converting a protecting group containing an Fmoc backbone in the first peptide into a carbamate form.

[0147] In one embodiment, the step of removing the protecting group including the Fmoc backbone in the first peptide is carried out substantially only in step (2).

[0148] In one embodiment, the method of the present invention does not include a step of treating the first peptide with a single base, such as piperidine, whose conjugate acid has a pKa of less than 23 in acetonitrile, prior to step (2). Specifically, the present invention excludes an embodiment in which the first peptide provided in step (1) having a protecting group containing an Fmoc skeleton is treated, for example, in any solvent (e.g., DMF) with a base (e.g., piperidine) whose conjugate acid has a pKa of less than 23 in acetonitrile, and then step (2) of the present invention is performed (e.g., treatment with DBU or a combination of DBU and piperidine in a solvent containing DMSO).

[0149] In one embodiment, the base used in step (2) can be contained in the solvent at any concentration that allows the reaction of this step to proceed. Specific examples of such a concentration range include 0.25 to 20 v / v%, 0.5 to 20 v / v%, 1 to 20 v / v%, 1 to 15 v / v%, 1 to 10 v / v%, 1 to 9 v / v%, 1 to 8 v / v%, 1 to 7 v / v%, 1 to 6 v / v%, 1 to 5 v / v%, 1 to 4 v / v%, 1 to 3 v / v%, and 1 to 2 v / v%, with 1 to 8 v / v% being preferred.

[0150] In one embodiment, step (2) comprises adding CO to a solvent. ２In one embodiment, the solvent used in step (2) may be a solvent containing CO 2 prior to the step (2). ２ The solvent and CO ２ The contact with the solvent may be carried out, for example, by adding CO ２ This can be done by bubbling CO with the solvent during or before step (2). ２ By contacting with ２ Without being bound by any particular theory, in step (2), the N-terminal amino group of at least a part of the first peptide is converted to C(═O)O derived from the protecting group. － and a protonated base to form a carbamate, and CO ２ By adding CO to the reaction system, the amine compound in the reaction system can be more easily converted into a carbamate. ２ By including the above, it is possible to further reduce the amount of amine compounds during the reaction and further suppress the intramolecular nucleophilic reaction of the terminal amino group, and it is presumed that even under basic deprotection conditions, the formation of six-membered ring intermediates is further suppressed, and the by-production of DKP elimination products and six-membered cyclic amidine skeleton structures is further reduced.

[0151] In one embodiment, the reaction in step (2) can be carried out at a temperature of from −100° C. to about the boiling point of the solvent, preferably from 0 to 50° C. The reaction in step (2) can be carried out for a reaction time of from 1 minute to 1 week, preferably from 1 minute to 3 hours, 3 minutes to 3 hours, or 5 minutes to 3 hours, and more preferably from 1 minute to 20 minutes, 3 minutes to 20 minutes, or 5 minutes to 20 minutes.

[0152] Step (3) of the present invention is a step, which follows step (2), of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide. In this step, the N-terminal amino group of the first peptide is condensed with the carboxyl group of the carboxylic acid to synthesize the third peptide.

[0153] In some embodiments, the carboxylic acid used in step (3) is selected from the group consisting of an amino acid having a protecting group, a second peptide having a protecting group, C １ -C ８ Alkyl carboxylic acid, or C ６ -C １０ It can be an aryl carboxylic acid.

[0154] In some embodiments, the carboxylic acid analog used in step (3) can be an activated ester of the carboxylic acid or an acid halide of the carboxylic acid.

[0155] an amino acid having a protecting group, a second peptide having a protecting group, C １ -C ８ Alkyl carboxylic acid, or C ６ -C １０ By using the respective activated esters of arylcarboxylic acids, the condensation reaction of step (3) can be carried out without a condensing agent. Specific examples of activated esters include those having a group capable of promoting the reaction with an amine, such as an O-(benzotriazol-1-yl) group (OBt), an O-(7-azabenzotriazol-1-yl) group (OAt), an N-hydroxysuccinimide group (OSu), a pentafluorophenoxy group (OPfp), or an S-R group, at the carbonyl group in these compounds, particularly at the carbonyl group contained in the C-terminal amino acid residue in the case of the second peptide. １１ (R １１ represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, an optionally substituted heteroaryl group, an optionally substituted alkenyl group, an optionally substituted alkylene group, etc.

[0156] an amino acid having a protecting group, a second peptide having a protecting group, C １ -C ８ Alkyl carboxylic acid, or C ６ -C １０By using an acid halide of each arylcarboxylic acid, the condensation reaction of step (3) can be carried out without a condensing agent. Specific examples of the acid halide include those in which the carboxyl group in these compounds, particularly the carboxyl group contained in the C-terminal amino acid residue in the case of the second peptide, has been converted to a chlorocarbonyl group, a fluorocarbonyl group, a bromocarbonyl group, an iodocarbonyl group, or the like.

[0157] When the carboxylic acid or carboxylic acid analog used in step (3), such as an amino acid or a second peptide, has a protecting group, the protecting group can be a carbamate-based protecting group, a sulfonyl-based protecting group, or an acyl-based protecting group.

[0158] As used herein, the term "carbamate protecting group" refers to a protecting group that comprises a carbamate structure, and includes, for example, a protecting group having an Fmoc skeleton, Alloc, Teoc, Boc, Cbz, and a 2,2,2-trichloroethoxycarbonyl group (Troc). The term "sulfonyl protecting group" refers to a protecting group that contains a sulfonyl structure, and includes, for example, a methanesulfonyl (Ms) group, a p-toluenesulfonyl (Ts) group, a 2-nitrobenzenesulfonyl (Ns) group, a trifluoromethanesulfonyl (Tf) group, and a (2-trimethylsilyl)-ethanesulfonyl (SES) group. The term "acyl protecting group" refers to a protecting group that contains an acyl structure, and includes, for example, an acetyl group, a pivaloyl group, a benzoyl group, and a trifluoroacetyl (Tfa) group.

[0159] In one embodiment, when the carboxylic acid or carboxylic acid analog used in step (3) is an amino acid having a protecting group containing an Fmoc backbone, the amino acid having a protecting group containing an Fmoc backbone can be any natural or unnatural amino acid protected with a protecting group containing an Fmoc backbone.

[0160] In one embodiment, when the carboxylic acid or carboxylic acid analog used in step (3) is a second peptide having a protecting group containing an Fmoc backbone, the second peptide having a protecting group containing an Fmoc backbone is not limited in type or number of amino acid residues constituting the peptide, as long as its N-terminal amino acid is protected with a protecting group containing an Fmoc backbone, and a peptide having any amino acid sequence containing two or more natural and / or unnatural amino acid residues can be used. Preferably, the second peptide contains one or more N-substituted amino acids.

[0161] In one embodiment, the carboxylic acid or carboxylic acid analog used in step (3) is C １ -C ８ Alkyl carboxylic acid, C ６ -C １０ When it is an aryl carboxylic acid, or an activated ester or acid halide thereof, it may be substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl, and dialkylaminocarbonyl. １ -C ８ The alkylcarboxylic acid, or its active ester or acid halide is preferably C １ -C ４ Alkylcarboxylic acids, or their activated esters or acid halides, and specific examples thereof include acetic acid, propanoic acid, butanoic acid, or pentanoic acid, or their activated esters or acid halides. ６ -C １０ Specific examples of the arylcarboxylic acid, or its active ester, or its acid halide include benzoic acid, naphthoic acid, or its active ester, or its acid halide.

[0162] In step (3), 1 to 20 equivalents, preferably 2 to 10 equivalents, of an amino acid having a protecting group containing an Fmoc backbone or a second peptide having a protecting group containing an Fmoc backbone can be used relative to the first peptide.

[0163] In one embodiment, the first peptide and / or the second peptide having a protecting group containing an Fmoc backbone preferably contains at least one N-substituted amino acid. Furthermore, the amino acid having a protecting group containing an Fmoc backbone is preferably an N-substituted amino acid.

[0164] In one embodiment, the protecting group containing an Fmoc backbone carried by the amino acid or second peptide used in step (3) may be the same as or different from the protecting group containing an Fmoc backbone carried by the first peptide used in step (1).

[0165] In one embodiment, the solvent used in step (3) may be a single solvent or a mixed solvent as long as it contains at least one of an aromatic hydrocarbon solvent, a halogen-based solvent, an ether-based solvent, an amide-based solvent, a sulfoxide-based solvent, a sulfone-based solvent, a urea-based solvent, an ester-based solvent, a ketone-based solvent, a carbonate-based solvent, or a phosphate-based solvent. In the case of a mixed solvent, the solvent used in step (3) preferably contains at least one of an aromatic hydrocarbon solvent, a halogen-based solvent, an ether-based solvent, an amide-based solvent, a sulfoxide-based solvent, a sulfone-based solvent, a urea-based solvent, an ester-based solvent, a ketone-based solvent, a carbonate-based solvent, or a phosphate-based solvent in an amount of 25 v / v % or more, 50 v / v % or more, or 75 v / v % or more.

[0166] When an aromatic hydrocarbon solvent is used in step (3), specific examples of the solvent include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene, and preferred are toluene and cumene.

[0167] When a halogen-based solvent is used in step (3), specific examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride, with dichloromethane being preferred.

[0168] When an ether solvent is used in step (3), specific examples of the solvent include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxolane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, t-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme, and tetraglyme, and preferred are tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane, and 1,3-dioxolane.

[0169] When an amide solvent is used in step (3), specific examples of the solvent include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), N-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP), and formamide, and preferred are DMF and NMP.

[0170] When a sulfoxide solvent is used in step (3), specific examples of the solvent include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide, and preferably DMSO.

[0171] When a sulfone solvent is used in step (3), specific examples of the solvent include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone, and ethyl isopropyl sulfone.

[0172] When a urea-based solvent is used in step (3), specific examples of the solvent include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), and preferably DMI.

[0173] When an ester solvent is used in step (3), specific examples of the solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate, and γ-valerolactone, and preferred are ethyl acetate, butyl acetate, and methyl propionate.

[0174] When a ketone solvent is used in step (3), specific examples of the solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone, and preferred are methyl ethyl ketone and diethyl ketone.

[0175] When a carbonate solvent is used in step (3), specific examples of the solvent include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate, with dimethyl carbonate being preferred.

[0176] When a phosphate ester solvent is used in step (3), specific examples of the solvent include trimethyl phosphate, triethyl phosphate, and tributyl phosphate, with tributyl phosphate being preferred.

[0177] When the solvent used in step (3) is a mixed solvent, the mixed solvent may consist of two or more solvents selected from aromatic hydrocarbon solvents, halogen-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, sulfone-based solvents, urea-based solvents, ester-based solvents, ketone-based solvents, carbonate-based solvents, and phosphate ester-based solvents, or may contain any other solvent.

[0178] When the solvent used in step (3) is a single solvent, the solvent is preferably toluene, cumene, 1,2-dichlorobenzene, benzene, anisole, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxolane, DMF, NMP, DSMO, DMI, DMPU, ethyl acetate, isopropyl acetate, butyl acetate, methyl propionate, acetone, methyl ethyl ketone, diethyl ketone, dimethyl carbonate, diethyl carbonate, trimethyl phosphate, tributyl phosphate, sulfolane, or 3-methylsulfolane.

[0179] In some embodiments, the solvent used in step (3) can be the same as the solvent used in step (2).

[0180] The solvent used in step (3) is preferably one in which the condensing agent can be dissolved. In the present invention, the base used in step (2) may remain, and this remaining base may cause by-products in which amino acids or peptides are excessively elongated. When a solvent in which the condensing agent can be dissolved is used in step (3), the formation of such over-elongated products can be suppressed.

[0181] In one embodiment, the condensing agent used in step (3) is in the form of a salt, the counter anion of which is PF ６ － or BF ４ － Specific examples of such condensing agents include PyOxim ([ethylcyano(hydroxyimino)acetato-O ２]tri-1-pyrrolidinylphosphonium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate) salt), HATU (O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate), HCTU (O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), TDBTU (O-(3,4 -dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate), TATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate ), TBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate), TCTU (O-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), and TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate).

[0182] In one embodiment, the condensing agent used in step (3) preferably includes at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU, and TOTU, and it is more preferable to use any one of these.

[0183] In one embodiment, the condensation reaction in step (3) can be carried out in the presence of a base. While the type of base is not limited as long as it contributes to the progress of the condensation reaction, a base whose conjugate acid has a pKa of 5 to 12 in water is preferred. Specific examples of such bases include DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine. In step (3), 1 to 20 equivalents, preferably 2 to 10 equivalents, of base can be used relative to the first peptide. The pKa of the conjugate acid of the base in water can be calculated, for example, using the value calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((c)1994-2020 ACD / Labs).

[0184] In one embodiment, the condensing agent used in step (3) is a carbodiimide-based condensing agent. Specific examples of such condensing agents include diisopropylcarbodiimide (DIC), di-sec-butylcarbodiimide (DsBC), 1-tert-butyl-3-ethylcarbodiimide (tBEC), di-tert-butylcarbodiimide (DtBC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and preferably diisopropylcarbodiimide (DIC) or di-sec-butylcarbodiimide (DsBC).

[0185] When the condensing agent used in step (3) is a carbodiimide-based condensing agent, the condensation reaction in step (3) can be carried out in the presence of an additive. The type of additive is not limited as long as it contributes to the progress of the condensation reaction, and specific examples of such additives include HOAt, HOBt, HOOBt, Oxyma, and Oxyma-B, with HOAt and Oxyma being preferred.

[0186] In step (3), the condensing agent can be used in an amount of 1 to 20 equivalents, preferably 2 to 10 equivalents, relative to the first peptide.

[0187] In one embodiment, the present invention may further include a step of treating the mixture in step (2) (treating step) between steps (2) and (3). In one embodiment, the treating step is a step of contacting the mixture with an acid or a salt. In the step of contacting the mixture with an acid or a base, the acid or base may be added directly to the mixture, or a solution containing the acid or base may be added to the mixture. Any solvent can be used for the solution; for example, it is preferable to use the solvent used in step (2), more preferably to use a sulfoxide solvent, and even more preferably to use DMSO. The treating step may also be a step of decomposing the carbamate salt produced in step (2).

[0188] Examples of acids used in the treatment step include HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid. Among these, HOAt, Oxyma, 4-nitrophenol, and HOOBt are preferred. Examples of salts used in the treatment step include salts made from at least one acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid, and a base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine, with pyridine hydrochloride being a preferred example.

[0189] In one embodiment, the step of treating the mixture in step (2) may be a step of washing the resin for solid phase synthesis. Any solvent can be used to wash the resin. For example, the solvent used in step (2) is preferably used, a sulfoxide solvent is more preferably used, and DMSO is even more preferably used. In another embodiment, the solvent used in the step of washing the resin for solid phase synthesis may be a solution containing a base. Examples of such bases include DIPEA, triethylamine, 2,6-lutidine, and 2,4,6-trimethylpyridine. The resin can be washed multiple times (e.g., two, three, four, five, or six times) with the same solvent or different solvents.

[0190] In one embodiment, the step of washing the resin for solid phase synthesis is a step of washing with a solution of a sulfoxide-based solvent containing an acid or a salt. Examples of acids used in washing the resin include HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid. Among these, HOAt, Oxyma, 4-nitrophenol, and HOOBt are preferred. Examples of salts used in washing the resin include salts made from at least one acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt, and hydrochloric acid, and a base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, and pyridine, with pyridine hydrochloride being a preferred example.

[0191] In one embodiment, the step of washing the resin for solid phase synthesis may be preceded by CO ２ A solvent (e.g., CO ２ A solvent bubbled with CO ２ A solvent that has not been contacted with CO may be used. ２ A solvent that has been contacted with CO may be used in combination with a solvent that has not been contacted with CO. ２ The resin is washed one or more times with a solvent that has been contacted with CO ２ Preferably, the resin is washed one or more times with a solvent that has not been in contact with the resin.

[0192] In one embodiment, the reaction in step (3) can be carried out at a temperature of from −100° C. to about the boiling point of the solvent, preferably from 0 to 60° C. The reaction in step (3) can be carried out for a reaction time of from 1 minute to 1 week, preferably from 30 minutes to 20 hours.

[0193] In one embodiment, the peptide produced by the method of the present invention comprises a third peptide produced via steps (1) to (3) above. In this case, the peptide produced by the method of the present invention can further comprise any number of any amino acids in addition to the third peptide.

[0194] In one embodiment, the peptide produced by the method of the present invention may be the third peptide produced through the steps (1) to (3) above.

[0195] In one embodiment, the peptide produced by the method of the present invention can contain at least one, or two or more, three or more, four or more, or five or more N-substituted amino acids. The N-substituted amino acids can be N-alkyl amino acids, preferably N-methyl amino acids.

[0196] In one embodiment, the method of the present invention may include a step of cleaving the peptide from the solid-phase synthesis resin after the peptide chain elongation step. For the cleavage step, a method known in the art or the method described in this Example can be used.

[0197] In one aspect, the present invention also relates to a method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeletal structure impurities produced in peptide production (e.g., peptide production by a solid-phase method), comprising the steps (1) and (2). The method may further comprise, after the step (2), a step (3) of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0198] In one aspect, the present invention also relates to a method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine framework impurities, and dimeric urea impurities in peptide production (e.g., peptide production by a solid-phase method), comprising steps (1) and (2). The method may further comprise, after step (2), step (3) of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0199] All prior art documents cited in this specification are hereby incorporated by reference.

[0200] The present invention is further illustrated, but not limited, by the following examples, comparative examples, and reference examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0201] The following abbreviations are used in the examples: AA: ammonium acetate, Ac: acetyl group, Alloc: allyloxycarbonyl group, Boc: tert-butoxycarbonyl group, Cbz: benzyloxycarbonyl group, COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate, DBU: 1,8-diazabicyclo[5.4.0]-7-undecene, DCM: dichloromethane, DCE: 1,2-dichloroethane, DMF: N,N-dimethylformamide, DKP: diketopiperazine, DIC: N,N'-diisopropylcarbodiimide, DIPEA: N,N-diisopropylethylamine, DMSO: dimethyl sulfoxide, DsBC: N,N'-di-sec-butylcarbodiimide EDCI·HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc: ethyl acetate FA: formic acid Fmoc: 9-fluorenylmethyloxycarbonyl group HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxybenzotriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU: O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HCTU: O-(1H-6-chloro-1-yl)-1,1,3,3 -Tetramethyluronium hexafluorophosphate HOTU: O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate HP1(dma): Imino-tris(dimethylamino)phosphorane MTBD: 7-methyl-1,5,7-Triazabicyclo[4.4.0]dec-5-ene NMP: N-methyl-2-pyrrolidone Ns: 2-nitrobenzenesulfonyl group Oxyma: ethyl cyano(hydroxyimino)acetate pip: piperidine P1tBu: t-butylimino-tris(dimethylamino)phosphorane P2Et: tetramethyl(tris(dimethylamino)phosphoranylidene)phosphoric triamide-ethylimine PyAOP: (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate PyBOP: benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate PyOxim: [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate Teoc: 2-trimethylsilylethoxycarbonyl group TES: Triethylsilane Tfa: Trifluoroacetyl group TFA: Trifluoroacetic acid TFE: 2,2,2-trifluoroethanol THF: Tetrahydrofuran TMG: 1,1,3,3-tetramethylguanidine TATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate TBTU: 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate TCTU: O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TDBTU: O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, TOTU: O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate,

[0202] The LCMS analysis conditions are shown in Table 1.

[0203]

[0204] Example 1: Preparation of amino acids, resin-supported peptides, etc. used in this example Example 1-1: Fmoc-amino acids used in peptide synthesis by peptide synthesizer In the peptide synthesis described herein, the Fmoc-amino acids listed in Tables 2 to 4 were used for synthesis by a peptide synthesizer. The Fmoc-amino acids listed in Table 2 were synthesized according to the methods described in WO2018 / 225864 and WO2021 / 090856. The Fmoc-amino acids listed in Table 3 were purchased from a commercial supplier. The Fmoc-amino acids listed in Table 4 were synthesized according to the scheme shown below.

[0205]

[0206]

[0207]

[0208] Synthesis of compound aa3-1, (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid (Fmoc-MeAla(3-Pyr)-OH)

[0209] Under a nitrogen atmosphere, paraformaldehyde (696 mg, 23.17 mmol) and trifluoroacetic acid (TFA) (5.36 mL, 69.5 mmol) were added to a toluene solution (10.5 mL) of commercially available (2S)-2-[9H-fluoren-9-ylmethoxycarbonylamino]-3-pyridin-3-ylpropanoic acid (Fmoc-Ala(3-Pyr)-OH, compound aa3-1-a) (3 g, 7.72 mmol), and the mixture was stirred at 40°C for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with dichloromethane (DCM), washed with saturated aqueous sodium bicarbonate, dried over anhydrous magnesium sulfate, and then filtered. The resulting solution was concentrated under reduced pressure to give compound aa3-1-b (2.95 g, 95%) as a crude product. This was used in the next reaction without further purification. LCMS (ESI) m / z = 401 (M+H) + Retention time: 0.64 min (Analysis conditions SQDFA05)

[0210] Under a nitrogen atmosphere, triethylsilane (TES) (10.59 mL, 66.3 mmol) and trifluoroacetic acid (TFA) (15.32 mL, 199 mmol) were added to a solution of the crude product compound aa3-1-b (2.95 g) in 1,2-dichloroethane (DCE) (15.5 mL) at room temperature, and the mixture was stirred at 70°C for 5 hours. The reaction solution was cooled to room temperature, and the solvent was distilled off under reduced pressure to obtain a crude product, which was dissolved in ethyl acetate, and t-butyl methyl ether / n-hexane = 9 / 1 (40 mL) was added and stirred. This mixture was cooled in a refrigerator for 30 minutes, and the resulting solid was filtered, washed three times with t-butyl methyl ether / n-hexane = 9 / 1 (30 mL), and then dried under reduced pressure. The resulting crude product was suspended in DCM (50 mL), and 3 mol / L aqueous sodium dihydrogen phosphate (45 mL, containing 1.5 mol / L sodium chloride) was added and stirred. After removing the aqueous layer, the organic layer was washed twice with 3 mol / L aqueous sodium dihydrogen phosphate (45 mL, containing 1.5 mol / L sodium chloride). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give compound aa3-1, (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-ylpropanoic acid, Fmoc-MeAla(3-Pyr)-OH (2.67 g, 90%). LCMS (ESI) m / z = 403 (M+H) + retention time: 0.53 min (analysis condition SQDFA05).

[0211] Synthesis of compound aa3-2, (2S)-3-[4-(trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxycarbonylamino)propanoic acid (Teoc-Phe(4-CF3)-OH)

[0212] Under a nitrogen atmosphere, commercially available (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoic acid (H-Phe(4-CF3)-OH, compound aa3-2-a) (2.00 g, 8.58 mmol) was dissolved in water (12.0 mL), and then a mixed solution of triethylamine (2.38 mL, 17.15 mmol) and 1,4-dioxane (10.0 mL) was added, followed by stirring at room temperature for 10 minutes. Thereafter, 2,5-dioxopyrrolidin-1-yl(2-(trimethylsilyl)ethyl)carbonate (2.45 g, 9.43 mmol) was added, followed by stirring at room temperature for 1 hour. 2-Methyltetrahydrofuran (5 mL) was added to the reaction solution, and the mixture was washed twice with 10% aqueous sodium carbonate solution (10 mL). The resulting organic layer was washed with saturated aqueous potassium hydrogen sulfate solution (10 mL), then with 10% aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and then filtered. The resulting solution was concentrated under reduced pressure to give compound aa3-2, 2S)-3-[4-(trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxycarbonylamino)propanoic acid (Teoc-Phe(4-CF3)-OH) (2.88 g, 89%). LCMS (ESI) m / z = 376 (M-H)-. Retention time: 0.87 minutes (analysis condition SQDFA05).

[0213] Synthesis of compound aa3-3, 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propanoic acid (Ns-Aib-OH)

[0214] A DCM solution (30.0 mL) of 2-nitrobenzenesulfonyl chloride (6.93 g, 31.2 mmol) was added to a DCM solution (22.1 mL) of commercially available 2-amino-2-methylpropanoic acid methyl ester hydrochloride (4.0 g, 26.0 mmol) and DIPEA (10.92 mL, 62.5 mmol) while cooling in an ice bath. The mixture was stirred at 0°C for 5 minutes and then at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give the crude product aa3-3-b. LCMS (ESI) m / z = 301 (M-H) - retention time: 0.64 minutes (analysis condition SQDFA05).

[0215] To a THF / methanol solution (1 / 1, 52 mL) of the obtained crude product aa3-3-b (3.63 g), 5N aqueous sodium hydroxide solution (26 mL) was added while cooling in an ice bath, and the mixture was stirred at 0°C for 5 minutes and then at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in 4N hydrochloric acid (30 mL), and the solvent was distilled off. The mixture was extracted twice with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and filtered, and the solution was concentrated under reduced pressure. The obtained crude product was purified twice by reverse-phase column chromatography (acetonitrile containing 0.1% formic acid / distilled water containing 0.1% formic acid) to obtain compound aa3-3, 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propanoic acid (Ns-Aib-OH) (2.46 g, 71%). LCMS (ESI) m / z = 287 (MH) - Retention time: 0.53 minutes (Analysis conditions SQDFA05)

[0216] Example 1-2: Preparation of resin-supported amino acids and peptides used in this example Example 1-2-1: Synthesis of (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoate-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1)

[0217] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin site may be represented by a circle. Furthermore, to clarify the reactive site of the resin site, the chemical structure of the reactive site may be represented by connecting it to a circle. For example, in the above structure (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1)), the 2-chlorotrityl group of the resin is bonded to the carboxylic acid on the side chain of Asp via an ester bond. Note that pyrro means pyrrolidine, and in the above structure, the carboxylic acid group at the C-terminus forms an amide bond with the pyrrolidine.

[0218] Under a nitrogen atmosphere, EDCI·HCl (67.1 g, 350 mmol), HOBt (43.3 g, 321 mmol), and Fmoc-Asp(OtBu)-OH (120 g, 2929 mmol) were added in that order to DMF (600 mL) at 0°C, and the mixture was stirred at 0°C for 1 hour. Ethyl acetate (10 v) and 0.5 mol / L aqueous hydrochloric acid (2 v) were added to the reaction solution at 0°C, and the organic layer was separated. The resulting organic layer was washed with 0.5 mol / L aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate / water (1 / 1 (v / v)), and saturated saline / water (1 / 1 (v / v)), successively, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain Compound 1-2-1-a as a crude product. (137.1 g, quant.) LCMS (ESI) m / z = 465 (M+H) + Retention time: 1.05 minutes (Analysis conditions SQDAA05)

[0219] Under ice-cooling, TFA (271 mL) was slowly added to a solution of compound 1-2-1-a (137 g, 395 mmol) in DCM (137 mL) so that the internal temperature did not exceed 10°C. After stirring at room temperature for 1 hour, diisopropyl ether (3.4 L) was added in four portions, and the precipitated solid was collected by filtration and dried to obtain compound 1-2-1-b ((3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid, Fmoc-Asp-pyrro). (108.4 g, 90%) LCMS (ESI) m / z = 409 (M + H) + Retention time: 0.83 minutes (Analysis condition SQDAA05).

[0220] The Fmoc amino acid resin loading reaction was carried out according to the method described in WO2013 / 100132 or WO2018 / 225864. 2-Chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, 48.7 g) and dehydrated dichloromethane (500 mL) were placed in a filter-equipped reaction vessel and shaken at room temperature for 20 minutes. After removing the dichloromethane under nitrogen pressure, a mixture of compound 1-2-1-b (15.91 g), dehydrated dichloromethane (350 mL), dehydrated methanol (12.63 mL), and diisopropylethylamine (DIPEA) (32.6 mL) was added to the reaction vessel and shaken for 60 minutes. After removing the reaction solution under nitrogen pressure, a mixture of dehydrated dichloromethane (350 mL), dehydrated methanol (97.3 mL), and diisopropylethylamine (DIPEA) (32.6 mL) was added to the reaction vessel and shaken for 1 hour and 30 minutes. After removing the reaction solution under nitrogen pressure, dichloromethane (350 mL) was added and the vessel was shaken for 5 minutes, after which the reaction solution was removed under nitrogen pressure. The resin was washed five times with dichloromethane, and the resulting resin was dried overnight under reduced pressure to give (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoate-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1, 59.79 g).

[0221] To confirm the loading rate, the obtained compound 1-2-1 (12.6 mg) was placed in a reaction vessel, DMF (2 mL) was added, and the mixture was shaken at room temperature for 1 hour. Then, DBU (40 μL) was added and the mixture was shaken at 30°C for 30 minutes. Then, DMF (8 mL) was added to the reaction mixture, and 1 mL of the resulting solution was diluted with DMF (11.5 mL). The absorbance (294 nm) of the resulting diluted solution was measured (measured using a Shimadzu UV-1600PC (cell length 1.0 cm)), and the loading amount of compound 1-2-1 was calculated to be 0.464 mmol / g. Note that a different lot synthesized in the same manner but with a different loading amount was also used for peptide synthesis, investigations, etc.

[0222] Example 1-2-2: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip, compound 1-2-2)

[0223] (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoate-2-chlorotrityl resin (compound 1-2-2, Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) was synthesized by a method described in a literature (literature: International Publication No. WO2013 / 100132 A1). Note that pip means piperidine, and in the above structure, the carboxylic acid group at the C-terminus forms an amide bond with the piperidine.

[0224] Example 1-2-3: Synthesis of (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin, compound 1-2-3)

[0225] Compound 1-2-3, (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-OH) (7.1 g, 21.82 mmol) purchased from a commercial supplier and 2-chlorotrityl chloride resin (1.6 mmol / g, 100-200 mesh, 1% DVB, 27.25 g, 43.6 mmol) were used in the same manner as in the synthesis of compound 1-2-1 to obtain compound 1-2-3, (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin). (33.44 g, loading: 0.598 mmol / g) Note that a different lot synthesized in the same manner but with a different loading was also used in the peptide synthesis in this example.

[0226] Example 1-2-4: Preparation of solid-phase supported peptide compounds (compounds 1-2-4 to 1-2-12) used in this example Compounds 1-2-4 to 1-2-12 used in this example were prepared by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by Intavis). Detailed operating procedures were performed in accordance with the manual provided with the synthesizer.

[0227] Solution 1 was prepared by dissolving the Fmoc-protected amino acid (0.6 mol / L) constituting the target peptide and HOAt, Oxyma, or HOOBt (0.375 mol / L) as a carboxylic acid activator in NMP. When the Fmoc-protected amino acid or HOOBt was poorly soluble, DMSO was added to prepare a 20% solution. Solution 2 was prepared by mixing N,N'-diisopropylcarbodiimide (DIC) (10 v / v%) and N,N-dimethylformamide (DMF).

[0228] The 2-chlorotrityl resin (Compound 1-2-1, Compound 1-2-2, or Compound 1-2-3) (100 mg per column) prepared in Examples 1-2-1, 1-2-2, or 1-2-3, to which the side chain carboxylic acid moiety of aspartic acid, N-terminally protected with an Fmoc group, or the main chain carboxylic acid moiety of a β-amino acid, N-terminally protected with an Fmoc group, was bound, was added to a solid-phase reaction vessel and set in a peptide synthesizer. Dichloromethane (DCM) (1.0 mL) was added to this resin (100 mg) and the mixture was allowed to stand for approximately 30 minutes to allow the resin to swell. Solution 1 and Solution 2 were set in the peptide synthesizer, and automated synthesis using the peptide synthesizer was initiated. The solution was discharged from the frit, and the resin was then washed twice with DMF (0.7 mL per column).

[0229] Fmoc removal step: A DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the Fmoc group was deprotected at room temperature. The reaction was allowed to proceed for 4.5 minutes for deprotection of the first residue, and for 10 minutes for deprotection of the second and subsequent residues, after which the solution was drained through the frit. The column was then washed twice with DMF (0.7 mL per column), once with a solution of DIPEA (0.14 mol / L) and HOAt (0.14 mol / L) dissolved in DMF (0.7 mL per column), and twice with DMF (0.7 mL per column).

[0230] Elongation Step: Solution 1 (0.3 mL per column) and Solution 2 (0.36 mL per column) were mixed in the synthesizer's mixing vial and then added to the resin. The solid-phase reaction vessel was heated from 40°C to 60°C for difficult-to-elongate sequences, and the reaction time was 2.5 hours to 14 hours for difficult-to-elongate sequences, resulting in a condensation reaction of the amino groups on the resin with the Fmoc-protected amino acids. The solution was then discharged through the frit. If the elongation efficiency was low, this condensation reaction of the Fmoc-protected amino acids was repeated one or two more times. The resin was then washed three times with DMF (0.7 mL per column).

[0231] This Fmoc group deprotection reaction followed by the Fmoc amino acid condensation reaction constituted one cycle, and this cycle was repeated to elongate the peptide on the resin surface. After elongation of the final amino acid, the column was washed four times with DCM (1.0 mL per column) without the Fmoc removal step, dried, and then used for further investigation. Using this method as the standard conditions, the following solid-phase-supported peptide compounds (compounds 1-2-4 to 1-2-12) were prepared. As described above, 100 mg of compound 1-2-1, compound 1-2-2, or compound 1-2-3 was used per column for preparation; however, multiple columns per sequence were similarly prepared.

[0232] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-4) It was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.552 mmol / g).

[0233] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-5) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0234] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(O-Trt(2-Cl)-resin)-pip (Compound 1-2-6) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0235] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (compound 1-2-7) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0236] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (compound 1-2-8) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0237] Fmoc-Ser(nPr)-MeAla-Phe-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-9) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0238] Fmoc-1-ACPrC-MeAla-Tyr(tBu)-Asp-(O-Trt(2-Cl)-resin)-pip (compound 1-2-10) Prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0239] Fmoc-Aib-Pro-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-11) Prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.558 mmol / g).

[0240] Fmoc-cLeu-MeVal-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-12) Prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.558 mmol / g).

[0241] As an example, to confirm that compound 1-2-4 had been obtained, a portion of the obtained resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). Analysis of the cleaved solution by LCMS confirmed the production of the target peptide (compound 1-2-4*). For compounds 1-2-5 to 1-2-12, the production of the target peptides (compounds 1-2-5* to 1-2-12*) was confirmed using the same method as for compound 1-2-4. In this example, when an * is attached to a compound number, it indicates a compound that was confirmed by cleaving the peptide from the resin to confirm the reaction. Compound 1-2-4* indicates a peptide compound obtained by cleaving the bond between the carboxylic acid of the peptide contained in compound 1-2-4 and the 2-chlorotrityl group of the resin.

[0242] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (compound 1-2-4*) LCMS (ESI) m / z=928.69 (M+H)+ Retention time: 0.85 min (Analysis conditions SQDFA05)

[0243] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip (Compound 1-2-5*) LCMS (ESI) m / z = 928.80 (M + H) + Holding time: 0.87 minutes (Analysis conditions SQDFA05)

[0244] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip (Compound 1-2-6*) LCMS (ESI) m / z = 1003.80 (M + H) + Holding time: 1.06 minutes (Analysis conditions SQDFA05)

[0245] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip(Compound 1-2-7*) LCMS (ESI) m / z = 855.73 (M + H) + Holding time: 0.76 minutes (Analysis conditions SQDFA05)

[0246] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-pip(Compound 1-2-8*) LCMS (ESI) m / z = 888.67 (M + H) + Holding time: 1.05 minutes (Analysis conditions SQDFA05)

[0247] Fmoc-Ser(nPr)-MeAla-Phe-Asp-pip (Compound 1-2-9*) LCMS (ESI) m / z = 784.66 (M + H) + Holding time: 0.86 minutes (Analysis conditions SQDFA05)

[0248] Fmoc-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Compound 1-2-10*) LCMS (ESI) m / z = 810.44 (M + H) + Holding time: 2.60 minutes (Analysis conditions SQDFA05long)

[0249] Fmoc-Aib-Pro-D-3-Abu-OH (Compound 1-2-11*) LCMS (ESI) m / z = 508.47 (M + H) + Holding time: 0.66 minutes (Analysis conditions SQDFA05)

[0250] Fmoc-cLeu-MeVal-D-3-Abu-OH (compound 1-2-12*) LCMS (ESI) m / z = 326.41 (M-H)- Detected as MS of the Fmoc-deprotected fragment ((M-H-Fmoc)-) Retention time: 0.79 minutes (analysis condition SQDFA05)

[0251] Example 1-3: Preparation of linear peptides used in this example Example 1-3-1: Synthesis of Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(OMe)-pip, compound 1-3-1

[0252] 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-5 prepared in Example 1-2-4 from 0.452 mmol / g of Compound 1-2-2) already prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added and the vessel was left to stand for 10 minutes to allow the resin to swell. The solution was then discharged through the frit, followed by the addition of a TFE / DCM solution (1 / 1 (v / v), 2.0 mL) and shaking at room temperature for 2 hours. The solution was then discharged through the frit into a recovery flask, and the resin was washed twice with a TFE / DCM solution (1 / 1 (v / v), 1.0 mL), and the discharged solution was recovered in a recovery flask. The same procedure was performed for seven solid-phase reaction vessels, and the discharged solutions were combined and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in DCE (5 mL), and the solvent was distilled off under reduced pressure twice to give the crude product Compound 1-2-5* (209.2 mg, 71%). This was used in the next reaction without further purification. LCMS (ESI) m / z = 928.78 (M + H) + Retention time: 0.88 min (Analysis condition SQDFA05).

[0253] Under a nitrogen atmosphere, the crude product, Compound 1-2-5* (209.2 mg, 0.23 mmol), was dissolved in toluene (0.75 mL) and methanol (1.50 mL) to which a hexane solution of trimethylsilyldiazomethane (0.6 M, 0.75 mL, 0.45 mmol) was added dropwise at room temperature. After stirring at room temperature for 5 minutes, acetic acid (0.013 mL, 0.23 mmol) was added, and the solvent was evaporated under reduced pressure to obtain a residue. The residue was purified by normal phase column chromatography (DCM / methanol) to obtain Compound 1-3-1, Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(OMe)-pip (175.9 mg, 83%). LCMS (ESI) m / z = 942.63 (M + H) + Retention time: 0.94 minutes (Analysis condition SQDFA05).

[0254] Example 1-3-3: Synthesis of Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip, Compound 1-3-2

[0255] 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-6 prepared in Example 1-2-4 from 0.452 mmol / g of Compound 1-2-2) already prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added and the vessel was left to stand for 10 minutes to allow the resin to swell. The solution was then discharged through the frit, followed by the addition of a TFE / DCM solution (1 / 1 (v / v), 2.0 mL) and shaking at room temperature for 2 hours. The solution was then discharged through the frit into a recovery flask, and the resin was washed twice with a TFE / DCM solution (1 / 1 (v / v), 1.0 mL), and the discharged solution was recovered in a recovery flask. The same procedure was performed for seven solid-phase reaction vessels, and the discharged solutions were combined and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in DCE (5 mL), and the solvent was distilled off under reduced pressure twice to obtain the crude product Compound 1-2-6* (202.8 mg, 64%). This was used in the next reaction without further purification. LCMS (ESI) m / z = 1003.70 (M + H) + Retention time: 1.06 min (Analysis condition SQDFA05).

[0256] Under a nitrogen atmosphere, the crude product, Compound 1-2-6* (202.8 mg, 0.20 mmol), was dissolved in toluene (0.67 mL) and methanol (1.35 mL). A hexane solution of trimethylsilyldiazomethane (0.6 M, 0.67 mL, 0.40 mmol) was added dropwise at room temperature. After stirring for 5 minutes at room temperature, acetic acid (0.012 mL, 0.20 mmol) was added, and the solvent was evaporated under reduced pressure. The resulting residue was purified by normal phase column chromatography (ethyl acetate / hexane) to give Compound 1-3-2, Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip (168.1 mg, 82%). LCMS (ESI) m / z = 1017.61 (M+H) + retention time: 1.12 minutes (analysis condition SQDFA05).

[0257] Example 2: Experiment to confirm the effects of reagents and solvents on the Fmoc removal step, resin washing step, and elongation step in solid-phase peptide synthesis. Solid-phase peptide synthesis involves Fmoc removal and resin washing, followed by amino acid elongation. In this example, compound 1-2-4 was used as the solid-phase-supported peptide sequence, and the reagents and solvents in each step were changed to compare the degree of reduction in impurities (diketopiperazine elimination products, 6-membered cyclic amidine framework structures, and over-elongation products) generated by peptide synthesis, thereby identifying the range of appropriate conditions.

[0258] Basic Procedure A (Table 5, runs 1-7, 14-15, 29-30) 100 mg of the peptide-loaded solid-phase resin (compound 1-2-4) previously prepared in Example 1-2-4 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-purged glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc removal (0.7 mL per column). The Fmoc removal solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc removal solution was prepared by dissolving the base listed in Table 5 in the Fmoc removal solvent at the volume percent (v / v%) listed in Table 5. After draining the solution through the frit, the resin was washed six times with the washing solvent (0.7 mL per column).

[0259] To the resin obtained above, a solution (0.3 mL per column) of Fmoc-MeLeu-OH (0.6 mol / L) and Oxyma or HOAt (0.375 mol / L) dissolved in an elongation solvent and a solution (0.36 mL per column) of DIC (10 v / v%) or DsBC (12 v / v%) dissolved in an elongation solvent were mixed, and the mixture was shaken for 2.5 hours at 40°C. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and then dried.

[0260] For example, run 5 in Table 5 was carried out as follows based on General Procedure A. 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-4, prepared in Example 1-2-4 from 0.552 mmol / g of Compound 1-2-1) previously prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added in a nitrogen-substituted glove box. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0261] To the resin obtained above, a mixed solution of Fmoc-MeLeu-OH (0.6 mol / L), Oxyma (0.375 mol / L) in DMSO (0.3 mL), and DIC (10 v / v%) in DMSO (0.36 mL) was added, and the mixture was shaken at 40° C. for 2.5 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0262] To confirm the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A 2% (v / v) solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in DMF was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was then drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a 1 / 1 (v / v) TFE / DCM solution containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDAA05long) to confirm the formation of the target peptide compound (TM) (2-1*), the diketopiperazine (DKP) elimination product (compound 2-2*), the six-membered cyclic amidine backbone structure (compound 2-3*), and the MeLeu-excess-extension product (compound 2-4*).

[0263] Target peptide compound (TM), H-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (compound 2-1*) LCMS (ESI) m / z=833.58 (M+H)+ Retention time: 2.72 minutes (Analysis conditions SQDAA05long)

[0264] Diketopiperazine elimination product, H-MeLeu-Aze(2)-MeCha-MeGly-Asp-pyrro (compound 2-2*) LCMS (ESI) m / z=635.45 (M+H)+ Retention time: 2.24 minutes (Analysis conditions SQDAA05long)

[0265] Six-membered cyclic amidine skeleton structure (compound 2-3*) LCMS (ESI) m / z=688.47 (M+H)+ Retention time: 2.52 minutes (Analysis conditions SQDAA05long)

[0266] MeLeu-extended complex, H-MeLeu-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrro (compound 2-4*) LCMS (ESI) m / z=960.66 (M+H)+ Retention time: 3.19 minutes (Analysis conditions SQDAA05long)

[0267] Basic Procedure B (Table 5, runs 8-13, 16-23, 25-28) 100 mg of the peptide-loaded solid-phase resin (compound 1-2-4) previously prepared in Example 1-2-4 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-substituted glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc removal (0.7 mL per column). The Fmoc removal solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc removal solution was prepared by dissolving the base listed in Table 5 in the Fmoc removal solvent at the volume percent (v / v%) listed in Table 5. After draining the solution through the frit, the resin was washed six times with the washing solvent (0.7 mL per column).

[0268] To the resin obtained above, a solution (0.25 mL) of Fmoc-MeLeu-OH (4 equivalents) in an elongation solvent, a solution (0.25 mL) of a uronium or phosphonium condensing agent (4 equivalents) in an elongation solvent, and DIPEA (6 equivalents) were mixed and left to stand for 1-2 minutes, and then the mixture was added and shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0269] For example, run 8 in Table 5 was carried out as follows based on Basic Procedure B. 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-4, prepared in Example 1-2-4 from 0.552 mmol / g of Compound 1-2-1) previously prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added in a nitrogen-substituted glove box. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0270] To the resin obtained above, a solution of Fmoc-MeLeu-OH (0.221 mmol, 4 equiv.) in DMSO (0.25 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equiv.) in DMSO (0.25 mL), and DIPEA (0.057 mL, 0.331 mmol, 6 equiv.) was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0271] To confirm the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was then added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was then drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDAA05long).

[0272] Basic Procedure C (Table 5, run 24): 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-4, prepared in Example 1-2-4 from 0.552 mmol / g of Compound 1-2-1) previously prepared in Example 1-2-4 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-purged glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0273] To the resin obtained above, a mixture of an NMP solution (0.7 mL) of Fmoc-MeLeu-Cl (described below) (0.221 mmol, 4 equivalents) and 2,4,6-trimethylpyridine (0.55 mmol, 10 equivalents) was added, and the mixture was shaken at 40°C for 1.5 hours. After the solution was discharged through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried. Fmoc-MeLeu-Cl was prepared as follows. (2S)-2-[9H-Fluoren-9-ylmethoxycarbonyl(methyl)amino]-4-methylpentanoic acid (Fmoc-MeLeu-OH) (0.081 g, 0.22 mmol) was dissolved in dichloromethane (0.44 mL). DMF (0.017 mL, 0.22 mmol) was added dropwise to the solution while cooling in an ice bath, followed by the addition of thionyl chloride (0.024 mL, 0.33 mmol). The resulting reaction mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. Toluene (0.81 mL, 10 v / w) was added to the resulting residue, and this process of concentration under reduced pressure was repeated twice. The resulting crude product was dissolved in NMP (0.7 mL) and used in the above reaction.

[0274] To confirm the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was then added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was then drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDAA05long).

[0275] The formation of diketopiperazine elimination products is a commonly known synthetic problem in peptide synthesis. The formation of six-membered cyclic amidine skeletal structures is unprecedented and represents a new challenge in peptide synthesis discovered by the present inventors. Without being limited to a particular theory, the formation of diketopiperazine elimination products proceeds by nucleophilic attack of the N-terminal amino group on the carbonyl group forming the second amide bond from the N-terminus to form a six-membered ring, followed by elimination of the amino group forming the original amide bond. On the other hand, the formation of six-membered cyclic amidine skeletal structures is thought to similarly proceed by the formation of a six-membered ring followed by elimination of a hydroxyl group derived from the carbonyl group generated by nucleophilic attack. In other words, the formation of diketopiperazine elimination products and six-membered cyclic amidine skeletal structures both share the same peptide synthesis challenge resulting from the formation of a six-membered ring.

[0276] The results obtained (relative ratios of UV areas in LCMS of Compound 2-1*, Compound 2-2*, Compound 2-3*, and Compound 2-4* under each condition, expressed as percentages) are shown in Table 5 below.

[0277]

[0278] Runs 1 and 2 represent the Fmoc removal step, resin washing step, and elongation step, which are typically performed in peptide synthesis (comparative experiment). For example, under the conditions of run 2, DBU was used as the base in the Fmoc removal step, and DMF was used as the solvent in the Fmoc removal step, resin washing step, and elongation step. Under these conditions, the production of a diketopiperazine (DKP) elimination product lacking two residues was confirmed at approximately 2%, and the production of a 6-membered cyclic amidine skeletal structure was confirmed at approximately 5%. In contrast, as shown in run 5, it was confirmed that using DMSO as the solvent in the Fmoc removal step, resin washing step, and elongation step not only produced the diketopiperazine elimination product and the 6-membered cyclic amidine skeletal structure, but also suppressed the production of MeLeu-excess elongation products. Furthermore, as shown in the results in run 3, when piperidine (pip) (pKa 19.35 (in acetonitrile), reference Eur. J. Org. Chem. 2019, 6735-6748), which is most commonly used as a base for Fmoc removal, was used and DMSO was used as the solvent for the Fmoc removal step, resin washing step, and elongation step, the production of diketopiperazine elimination products and the production of a 6-membered cyclic amidine skeletal structure were confirmed in approximately 2% each. In contrast, as shown in runs 4 to 7, it was confirmed that the production of diketopiperazine elimination products and the 6-membered cyclic amidine skeletal structure could be suppressed when a stronger base with a conjugate acid pKa of 23 or higher was used.

[0279] Furthermore, as shown in the results of run 8, run 10 to run 13, and run 16 to run 23, it was confirmed that the production of diketopiperazine elimination products and 6-membered cyclic amidine skeleton structures could be suppressed even when various phosphonium-based and uronium-based condensing agents were used in the elongation reaction under conditions in which DMSO was used as the solvent in the Fmoc removal step, resin washing step, and elongation step.

[0280] As shown in the results of run 9, it was confirmed that the production of the diketopiperazine elimination product and the 6-membered cyclic amidine skeleton structure could be suppressed even when DMSO solvent was used in the Fmoc removal step and the resin washing step, and PyOxim was used as the condensing agent and a solvent other than DMSO was used in the elongation step.

[0281] Runs 14 and 15 show the results of using DMSO solvent in the Fmoc removal step, DMSO solvent in either the resin washing step or the elongation step, and DMF solvent in the remaining step. Compared to run 5, in which DMSO solvent was used in all three steps, using DMF solvent only in the elongation step resulted in a slight increase in the production of 6-membered cyclic amidine skeletal structures when DIC / Oxyma was used as the condensing agent. On the other hand, when DMF solvent was used only in the resin washing step, it was confirmed that the production of the diketopiperazine elimination product and 6-membered cyclic amidine skeletal structures could be suppressed to the same extent as when DMSO solvent was used in all three steps.

[0282] In the present invention, an acid chloride may be used in the elongation step. For example, as in run 24, it was confirmed that the formation of the diketopiperazine elimination product and the 6-membered cyclic amidine skeleton structure can be suppressed even when a separately prepared acid chloride is reacted in NMP in the presence of 2,4,6-trimethylpyridine under conditions in which DMSO is used in the Fmoc removal step and the resin washing step.

[0283] In the present invention, two or more types of bases may be present at the same time in the Fmoc removal step. For example, in the case of two types of bases being present, as long as one of them is an optimal base (here, DBU) as in run 25, the formation of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures can be suppressed even in the presence of piperidine, which is widely known to give an adduct to dibenzofulvene generated in the Fmoc removal step.

[0284] Furthermore, it was confirmed that the formation of diketopiperazine elimination products and six-membered cyclic amidine structures could be suppressed even in the presence of HP1(dma), P1tBu, and P2Et, which are bases stronger than DBU (runs 26 to 28).

[0285] As shown in the results of run 29 and run 30, it was confirmed that the formation of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures could be suppressed under conditions in which DMSO was used as the solvent in the Fmoc removal step, resin washing step, and elongation step, even when DsBC or HOAt was used in the elongation reaction.

[0286] As described above, the outline of the scope of each step (Fmoc removal step, resin washing step, and elongation step) capable of suppressing the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures was identified from the results of Example 2. Note that the suppression of the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures is not limited to the combination of reaction conditions shown in Example 2.

[0287] Example 3 Experiment to confirm the generality of the peptide sequence for the effect of reducing diketopiperazine elimination during peptide synthesis under the discovered conditions In this example, for various peptide sequences prepared in Example 1-2-4 (peptide sequences extended up to Core 2 in Table 6, Compounds 1-2-5 to 1-2-9), the amino acids listed in Core 1 were extended by a general conventional method (Condition-1) and the method of the present invention (Condition-2), and the amounts of diketopiperazine elimination products produced were compared.

[0288] Condition-1 (Conventional Method) 100 mg of the peptide-loaded solid-phase resin (Compounds 1-2-5 to 1-2-9 (corresponding to runs 1 to 5 in Table 6, respectively)) already prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was subsequently washed twice with DMF (0.7 mL per column). A piperidine DMF solution (20 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After the solution was drained through the frit, the resin was washed eight times with DMF (0.7 mL per column).

[0289] To the resin obtained above, a mixed solution of Fmoc-protected amino acid (0.6 mol / L), HOAt (0.375 mol / L) in DMF (0.3 mL), and DIC (10 v / v%) in DMF (0.36 mL) was added, and the mixture was shaken for 4 hours at 40° C. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0290] Condition 2 (Method of the Present Invention) 100 mg of the peptide-loaded solid-phase resin (Compounds 1-2-5 to 1-2-9 (corresponding to runs 1 to 5 in Table 6, respectively)) already prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After the solution was drained through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0291] To the resin obtained above, a solution of 0.25 mL of a DMSO solution of Fmoc-protected amino acid (4 equiv.), 0.25 mL of a DMSO solution of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (4 equiv.), and 6 equiv. of DIPEA was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0292] To confirm the progress of the reaction, a portion of each resin was removed and swollen with DCM, after which the peptide was cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long), and the production of the target peptide (TM) (compounds 3-1-1* to 3-1-5*) and diketopiperazine elimination products (compounds 3-2-1* to 3-2-5*) was confirmed.

[0293] The amount of diketopiperazine (DKP) elimination product produced for each sequence and each condition is shown in Table 6. The amount of diketopiperazine (DKP) elimination product shown in Table 6 is the UV area percentage of the diketopiperazine (DKP) elimination product when the total UV area percentage of the target peptide (TM) and the diketopiperazine (DKP) elimination product by LCMS is taken as 100%.

[0294] Target peptide compound (TM), Fmoc-MeLeu-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip(run1) (compound 3-1-1*) LCMS (ESI) m / z=1055.68 (M+H)+ Retention time: 3.10 minutes (Analysis conditions SQDFA05long)

[0295] Diketopiperazine elimination product, Fmoc-MeLeu-Aze(2)-MeCha-MeGly-Asp-pip(run1) (compound 3-2-1*) LCMS (ESI) m / z=871.61 (M+H)+ Retention time: 2.97 minutes (Analysis conditions SQDFA05long)

[0296] Target peptide compound (TM), Fmoc-Thr(tBu)-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip(run2) (Compound 3-1-2*) LCMS (ESI) m / z=1160.61 (M+H)+ Retention time: 3.81 minutes (Analysis conditions SQDFA05long)

[0297] Diketopiperazine eliminator, Fmoc-Thr(tBu)-Cys(StBu)-Phe-Asp-pip(run 2) (compound 3-2-2*) LCMS (ESI) m / z=918.44 (M+H)+ Retention time: 3.49 minutes (Analysis conditions SQDFA05long)

[0298] Target peptide compound (TM), Fmoc-Nle-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip(run3) (compound 3-1-3*) LCMS (ESI) m / z = 968.59 (M + H) + Holding time: 2.50 minutes (Analysis conditions SQDFA05long)

[0299] Separate body, Fmoc-Nle-Ser (iP en)-Asp-pip (run3) (compound 3-2-3*) LCMS (ESI) m / z = 693.72 (M + H) + Holding time: 2.88 minutes (Analysis conditions SQDFA05long)

[0300] Target ペプチド compound (TM), Fmoc-Nle-Nle-MePhe (3-C l)-Ser(iPen)-Asp-pip(run4)(Compound 3-1-4*) LCMS (ESI) m / z = 1001.54 (M + H) + Holding time: 3.55 minutes (Analysis conditions SQDFA05long)

[0301] Separate body, Fmoc-Nle-Ser (iP en)-Asp-pip (run4) (compound 3-2-4*) LCMS (ESI) m / z = 693.42 (M + H) + Holding time: 2.88 minutes (Analysis conditions SQDFA05long)

[0302] Objective Peptide compound (TM), Fmoc-Pro-Ser (nPr)-MeAla-Phe-Asp-pip (run5) (Compound 3-1-5*) LCMS (ESI) m / z = 881.47 (M + H) + Holding time: 2.60 minutes (Analysis conditions SQDFA05long)

[0303] Separate body, Fmoc-Pro-Phe-Asp-pip (run5) (Compound 3-2-5*) LCMS (ESI) m / z = 667.38 (M + H) + Holding time: 2.44 minutes (Analysis conditions SQDFA05long)

[0304]

[0305] As described above, the results of Example 3 confirmed that, in the synthesis of various peptides, not limited to specific peptide sequences, the method of the present invention is more effective in suppressing the production of diketopiperazine elimination products than general conventional methods when elongation is performed using the method of the present invention.

[0306] Example 4: Experiment to confirm the effect of the neutralization step and acid washing in a sequence in which dimer urea compounds are produced under the conditions of WO 2022 / 097540, in addition to the problem of diketopiperazine elimination. Compound 1-2-10 has a sequence in which diketopiperazine elimination is likely to proceed, and applying the method of WO 2022 / 097540 makes it possible to significantly suppress diketopiperazine elimination. However, it has been confirmed that a dimer urea compound derived from carbamate is produced as a new by-product. Without being bound by a particular theory, it is believed that when a carbamate salt is present stably, a nucleophilic attack is caused by the amino group at the adjacent N-terminus on an isocyanate produced by the reaction of a condensing agent with a carbamate anion, resulting in an intermolecular reaction to produce a dimer urea compound. In this example, Compound 1-2-10 was used as the solid-phase-supported peptide sequence, and the extent to which impurities (diketopiperazine elimination products and dimer urea products) generated during peptide synthesis were reduced by adding a neutralization step and by adding an acid or base to the resin washing step was compared, and the range of appropriate conditions capable of reducing both problems was identified.

[0307] Basic Procedure D (Table 7, runs 1 and 4): 100 mg of the peptide-loaded solid-phase resin (compound 1-2-10) prepared in Example 1-2-4 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-substituted glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc removal (0.7 mL per column). The Fmoc removal solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc removal solution was prepared by dissolving the base listed in Table 7 in the Fmoc removal solvent at the volume percent (v / v%) listed in Table 7. After draining the solution through the frit, the resin was washed eight times with DMF in run 1 and six times with DMSO in run 4, each time using 0.7 mL per column.

[0308] To the resin obtained above, a solution (0.3 mL per column) of Fmoc-MeAla-OH (0.6 mol / L) and HOAt or Oxyma (0.375 mol / L) dissolved in an elongation solvent and a solution (0.36 mL per column) of DIC (10 v / v%) dissolved in an elongation solvent were mixed, and the mixture was shaken for 2.5 hours at 40°C. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and then dried.

[0309] For example, run 4 in Table 7 was carried out as follows based on General Procedure D. 100 mg of the peptide-loaded solid-phase resin (compound 1-2-10 prepared in Example 1-2-4 from 0.452 mmol / g of compound 1-2-2) was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added in a nitrogen-substituted glove box. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0310] To the resin obtained above, a mixed solution of Fmoc-MeAla-OH (0.6 mol / L), Oxyma (0.375 mol / L) in DMSO (0.3 mL), and DIC (10 v / v%) in DMSO (0.36 mL) was added, and the mixture was shaken for 2.5 hours at 40° C. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0311] To confirm the progress of the reaction, a portion of the resulting resin was removed, swollen with DCM, and the solution was discharged through the frit. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long), confirming the production of the target peptide compound (TM) (4-1*), a diketopiperazine (DKP) elimination product (compound 4-2*), and a dimer urea product (compound 4-3*).

[0312] Target peptide compound (TM), Fmoc-MeAla-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (compound 4-1*) LCMS (ESI) m / z=895.61 (M+H)+ Retention time: 2.66 minutes (Analysis conditions SQDFA05long)

[0313] Diketopiperazine eliminator, Fmoc-MeAla-Tyr(tBu)-Asp-pip (Compound 4-2*) LCMS (ESI) m / z=727.52 (M+H)+ Retention time: 2.81 minutes (Analysis conditions SQDFA05long)

[0314] Dimer urea (compound 4-3*) LCMS (ESI) m / z=1201.77 (M+H)+ Retention time: 2.18 minutes (Analysis conditions SQDFA05long)

[0315] Basic Procedure E (Table 7, runs 2, 3, 6-25) 100 mg of the peptide-loaded solid-phase resin (compound 1-2-10) prepared in Example 1-2-4 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-substituted glove box, and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc removal (0.7 mL per column). The Fmoc removal solution (0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc removal solution was prepared by dissolving the base listed in Table 7 in the Fmoc removal solvent at the volume percent (v / v%) listed in Table 7. After draining the solution through the frit, the resin was washed with the washing solvent listed in Table 7 (0.7 mL per column). The multiple washing operations were carried out with the same or different solvents, or in combination with acid washing, and the order and number of washings are shown in Table 7.

[0316] To the resin obtained above, a solution (0.25 mL) of Fmoc-MeAla-OH (4 equivalents) in an elongation solvent, a solution (0.25 mL) of a phosphonium condensing agent (4 equivalents) in an elongation solvent, and DIPEA (6 equivalents) were mixed and left to stand for 1-2 minutes, and then the mixture was added and shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0317] For example, run 17 in Table 7 was carried out as follows based on General Procedure E. 100 mg of the peptide-loaded solid-phase resin (compound 1-2-10 prepared in Example 1-2-4 from 0.452 mmol / g of compound 1-2-2) was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-substituted glove box and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed twice with a DMSO solution of DIPEA (2 equivalents) (0.7 mL per column). Subsequently, the column was washed once with a DMSO solution (0.7 mL per column) of HOAt (2 equivalents) and DIPEA (2 equivalents), and then three times with a DMSO solution (0.7 mL per column) of DIPEA (2 equivalents).

[0318] To the resin obtained above, a solution of Fmoc-MeAla-OH (0.181 mmol, 4 equiv.) in DMSO (0.25 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equiv.) in DMSO (0.25 mL), and DIPEA (0.047 mL, 0.271 mmol, 6 equiv.) was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0319] To confirm the progress of the reaction, a portion of the resulting resin was taken, swollen with DCM, and the solution was drained through a frit. The peptide was then cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long).

[0320] Basic Procedure F (Table 7, run 5) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-10 prepared in Example 1-2-4 from 0.452 mmol / g of Compound 1-2-2) was placed in a solid-phase reaction vessel. In a nitrogen-purged glove box, DCM (1.0 mL) was added and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with the solvent used for Fmoc removal (0.7 mL per column) listed in Table 7. The Fmoc removal solution (0.7 mL per column) was added, and the mixture was allowed to stand at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc removal solution was prepared by dissolving the base listed in Table 7 in the Fmoc removal solvent at the volume percent (v / v%) listed in Table 7. Next, for the neutralization step, 0.4 mL of a DMSO solution of HOAt (0.25 mol / L) (approximately 2 equivalents relative to the peptide supported on the solid phase) was added and allowed to stand for an additional 5 minutes. After the solution was drained through the frit, the resin was washed six times with the wash solvent (0.7 mL per column) listed in Table 7. To the resin obtained above, a solution of a DMSO solution (0.3 mL) of Fmoc-MeAla-OH (0.181 mmol, 4 equivalents), a DMSO solution (0.3 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equivalents), and DIPEA (0.047 mL, 0.271 mmol, 6 equivalents) was added and allowed to stand for approximately 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and dried.

[0321] For example, run 5 in Table 7 was performed as follows based on Basic Procedure F. 100 mg of the peptide-supported solid-phase resin (compound 1-2-10 prepared in Example 1-2-4 from 0.452 mmol / g of compound 1-2-2) was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added in a nitrogen-substituted glove box. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and a DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added to the resin and allowed to stand at room temperature for 10 minutes to remove Fmoc. Subsequently, as a neutralization step, 0.4 mL of a DMSO solution of HOAt (0.5 mol / L) (equivalent to approximately 4 equivalents of the peptide supported on the solid phase) was added, and the mixture was allowed to stand for an additional 5 minutes. After the solution was drained through the frit, the resin was washed six times with DMSO (0.7 mL per column).

[0322] To the resin obtained above, a solution of Fmoc-MeAla-OH (0.181 mmol, 4 equiv.) in DMSO (0.3 mL), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equiv.) in DMSO (0.3 mL), and DIPEA (0.047 mL, 0.271 mmol, 6 equiv.) was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried. To confirm the progress of the reaction, a portion of the resulting resin was taken, swollen with DCM, and the solution was drained through a frit. The peptide was then cleaved in a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long).

[0323] The results obtained (relative ratios of UV areas in LCMS of Compound 4-1*, Compound 4-2*, and Compound 4-3* under each condition, expressed as percentages) are shown in Table 7 below.

[0324]

[0325] Run 1 shows the Fmoc removal step, resin washing step, and elongation step (comparative experiment) that are typically performed in peptide synthesis. Piperidine (pip) was used as the base in the Fmoc removal step, and DMF was used as the solvent in the Fmoc removal step, resin washing step, and elongation step. Under these conditions, the production of a diketopiperazine (DKP) elimination product lacking two residues was confirmed at approximately 75%, while the production of the target product was approximately 25%. In contrast, as shown in the results in run 2, when the method of WO 2022 / 097540 was used, the production of the diketopiperazine elimination product could be suppressed to approximately 4%, but a dimer urea product was confirmed as a new by-product at approximately 30%, resulting in approximately 68% of the target product.

[0326] In contrast, as shown in run 3, when DMSO was used as the solvent in all of the Fmoc removal step, resin washing step, and elongation step, the production of the diketopiperazine elimination product was reduced to about 10%, the production of the dimer urea product was reduced to about 7%, and the yield of the target product was improved to about 83%.

[0327] In order to suppress the formation of both the diketopiperazine elimination product and the dimer urea product, the condensing agent was immobilized on PyOxim, and the conditions for the resin washing step after the Fmoc removal step were investigated.

[0328] First, as shown in run 5, a neutralization step in which 2 equivalents of HOAt were added after the Fmoc removal step was performed, and the production of diketopiperazine elimination products was suppressed to approximately 8% and the production of dimer urea products to approximately 4%. Without being bound by any particular theory, this result in which the dimer urea products were reduced is thought to be due to the decomposition of carbamates promoted by the neutralization step. Furthermore, as shown in run 6, when washing with a DMSO solution containing 2 equivalents of HOAt was performed only in the sixth of six resin washing steps for the same purpose as the neutralization step, the production of diketopiperazine elimination products and dimer urea products was further suppressed compared to when the neutralization step was used.

[0329] On the other hand, as shown in runs 6 to 8, although the production of diketopiperazine elimination products increased with increasing exposure time to the DMSO solution containing 2 equivalents of HOAt, it was found that the method still showed a superior effect compared to run 1, which is a general method.

[0330] As shown in runs 9 to 12, when DIPEA was added to an acid-containing washing solvent, the production of both the diketopiperazine elimination product and the dimer urea product could be suppressed, even when the washing solvent was exposed for a long time, just as it was when the washing solvent was exposed for a short time. This indicates that the method can be applied to scale-up, when the time required for each step, such as washing time, is extended.

[0331] As shown in runs 13 and 14, when washing with a DMSO solution containing HOAt and DIPEA was performed in the first of six resin washing steps, the production of dimer urea products increased slightly compared to when washing was performed in the sixth step. However, when washing was performed in the third step, as shown in runs 15 and 16, it was confirmed that the production of diketopiperazine elimination products and dimer urea products could be suppressed to the same extent as when washing was performed in the sixth step.

[0332] Furthermore, as shown in run 17 and run 18, when the remaining five of the six resin washing steps, excluding the washing with a DMSO solution containing HOAt and DIPEA, were washed with a DMSO solution containing 2 equivalents of DIPEA, it was confirmed that the production of diketopiperazine elimination products could be further suppressed compared to when the remaining five washing steps were done with DMSO alone.

[0333] As shown in run 19 to run 25, it was confirmed that the formation of both diketopiperazine elimination products and dimer urea products could be suppressed even when Oxyma, 4-nitrophenol, HOOBt, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, or pyridine hydrochloride was used as the acid or base in the washing with the DMSO solution containing an acid and a base, and in the remaining five washings with the DMSO solution containing a base.

[0334] As described above, the results of Example 4 identified the ranges of each step (Fmoc removal step, resin washing step, and elongation step) that can suppress the production of both diketopiperazine elimination products and dimer urea products for peptide sequences for which diketopiperazine elimination products are a problem and the production of dimer urea products becomes a new problem under the conditions of WO 2022 / 097540. Note that, with regard to the suppression of the production of diketopiperazine elimination products and dimer urea products, this Example is only a part of the present invention, and is not limited to the combination of reaction conditions shown in Example 4.

[0335] Example 5: Experiment to confirm the peptide sequence generality of the effect of reducing diketopiperazine elimination during peptide synthesis under the conditions discovered in Example 4 In this example, for various peptide sequences prepared in Example 1-2-4 (peptide sequences extended up to Core 2 in Table 8, Compounds 1-2-4 to 1-2-9, and Compounds 1-2-11 to 1-2-12), the amino acids listed in Core 1 were extended using a general conventional method (Condition-1), the method of the present invention described in Example 3 (Condition-2), and the method discovered in Example 4 of the present invention (Condition-3), and the amounts of diketopiperazine elimination products and by-products derived from carbamates were compared. Note that in run 1, the amino acids listed in Core 1 were extended using the method described in Example 2, Table 5, run 1, which is a general conventional method.

[0336] Condition 3 (Method Discovered in Example 4 of the Present Invention) 100 mg of the peptide-loaded solid-phase resin (Compounds 1-2-4 to 1-2-9 and Compounds 1-2-11 to 1-2-12 (corresponding to Runs 1 to 8 in Table 6, respectively)) already prepared in Example 1-2-4 was placed in a solid-phase reaction vessel, and DCM (1.0 mL) was added in a nitrogen-substituted glove box. The resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v %, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed twice with a solution of DIPEA (2 equiv.) in DMSO (0.7 mL per column), once with a solution of HOAt (2 equiv.) and DIPEA (2 equiv.) in DMSO (0.7 mL per column), and then three times with a solution of DIPEA (2 equiv.) in DMSO (0.7 mL per column).

[0337] To the resin obtained above, a solution of 0.25 mL of a DMSO solution of Fmoc-protected amino acid (4 equiv.), 0.25 mL of a DMSO solution of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (4 equiv.), and 6 equiv. of DIPEA was added and allowed to stand for 1-2 minutes. The mixture was then shaken at room temperature for 4 hours. After the solution was drained through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and then dried.

[0338] To confirm the progress of the reaction, a portion of each resin was removed and swollen with DCM. The peptide was then excised using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The excised solution was analyzed by LCMS (SQDFA05long), and the production of the target peptides (TM) (compounds 3-1-1* to 3-1-5* and compounds 5-1-1* to 5-1-2*) and diketopiperazine elimination products (compounds 3-2-1* to 3-2-5* and compounds 5-2-1* to 5-2-2*) was confirmed.

[0339] In Run 1, to confirm the progress of the reaction, a portion of the resulting resin was removed, swollen in DCM, and washed twice with DMF. A DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was then added and allowed to react at room temperature for 10 minutes to deprotect the Fmoc group. The solution was then drained through the frit, and the resin was washed four times with DMF and four times with DCM. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDAA05long) to confirm the formation of the target peptide (TM) (Compound 2-1*), the diketopiperazine elimination product (Compound 2-2*), and the 6-membered cyclic amidine framework (Compound 2-3*).

[0340] The amount of diketopiperazine (DKP) elimination product produced for each sequence and each condition is shown in Table 8. The amount of diketopiperazine (DKP) elimination product produced in Table 8 is the UV area percentage of the diketopiperazine (DKP) elimination product when the total UV area percentage of the target peptide (TM), diketopiperazine (DKP) elimination product, and carbamate-derived by-products by LCMS is taken as 100%. Note that the amount of diketopiperazine (DKP) elimination product produced in run 1 shown in Table 8 also includes the amount of the 6-membered cyclic amidine skeleton structure produced.

[0341] Target peptide compound (TM), Fmoc-MeIle-Aib-Pro-D-3-Abu-OH (run7) (compound 5-1-1*) LCMS (ESI) m / z=633.42 (MH) - Retention time: 2.41 minutes (Analysis conditions SQDFA05long)

[0342] Diketopiperazine eliminator, Fmoc-MeIle-D-3-Abu-OH (run 7) (compound 5-2-1*) LCMS (ESI) m / z=453.55 (M+H)+ Retention time: 2.46 minutes (Analysis conditions SQDFA05long)

[0343] Carbamate-derived by-product (run 7) (compound 5-3-1*) LCMS (ESI) m / z=412.23 (M+H)+ Retention time: 1.03 minutes (Analysis conditions SQDFA05long)

[0344] Target peptide compound (TM), Fmoc-Pro-cLeu-MeVal-D-3-Abu-OH (run8) (compound 5-1-2*) LCMS (ESI) m / z=645.41 (MH) - Retention time: 2.39 minutes (Analysis conditions SQDFA05long)

[0345] Diketopiperazine eliminator, Fmoc-Pro-D-3-Abu-OH (run 8) (compound 5-2-2*) LCMS (ESI) m / z=423.53 (M+H)+ Retention time: 1.87 minutes (Analysis conditions SQDFA05long)

[0346] Carbamate-derived by-product (run 8) (compound 5-3-2*) LCMS (ESI) m / z=452.24 (MH) - Retention time: 1.58 minutes (Analysis conditions SQDFA05long)

[0347]

[0348] The results of runs 1 to 6 confirmed that, in various peptide syntheses, not limited to the sequences described in Example 4, the method (Condition-3) discovered in Example 4 of the present invention is more effective in suppressing the production of diketopiperazine elimination products than general conventional methods when elongation is performed by the method.

[0349] In run 7 and run 8, when the method of Example 3 of the present invention (Condition-2) was applied to these sequences, it was confirmed that while the production of diketopiperazine elimination products could be suppressed compared to conventional methods, by-products derived from carbamates (compounds 5-3-1* and 5-3-2*) were produced. Without being bound by a particular theory, it is thought that when carbamates are present in a stable state, the condensing agent reacts with carbamate anions to produce isocyanates, which are then produced by nucleophilic attack by TFE, which is used during cleavage.

[0350] It was confirmed that when these sequences were elongated by the method (Condition-3) discovered in Example 4 of the present invention, the production of both carbamate-derived by-products and diketopiperazine elimination products could be suppressed (Runs 7 and 8). Without being bound by any particular theory, it is believed that the result of being able to reduce carbamate-derived by-products is due to the accelerated decomposition of carbamates by washing with acid.

[0351] As described above, the results of Example 5 confirmed that, not only for specific peptide sequences but also for various peptide syntheses, when elongation is performed by the method discovered in Example 4 of the present invention, the production of both diketopiperazine elimination products and by-products derived from carbamates can be generally suppressed compared to general conventional methods.

[0352] Example 6: Experiment to confirm the substrate generality of carboxylic acids that elongate the diketopiperazine elimination reducing effect during peptide synthesis under the conditions discovered in Example 4 In this example, various carboxylic acids described in Core 1 were elongated using the peptide sequence (compound 1-2-10) prepared in Example 1-2-4 by the method (condition-4) discovered in Example 4 of the present invention, and the amounts of diketopiperazine elimination products and dimer urea products produced were confirmed.

[0353] Condition 4 (Method Found in Example 4 of the Present Invention) 100 mg of the peptide-loaded solid-phase resin (Compound 1-2-10 prepared in Example 1-2-4 from 0.452 mmol / g of Compound 1-2-2) previously prepared in Example 1-2-2 was placed in a solid-phase reaction vessel. DCM (1.0 mL) was added in a nitrogen-substituted glove box and the resin was allowed to stand for 10 minutes to swell. The solution was then drained through the frit, and the resin was washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the reaction was allowed to proceed at room temperature for 10 minutes to deprotect the Fmoc group. After draining the solution through the frit, the resin was washed twice with a DMSO solution of DIPEA (2 equivalents) (0.7 mL per column). Subsequently, the column was washed once with a DMSO solution (0.7 mL per column) of HOAt (2 equivalents) and DIPEA (2 equivalents), and then three times with a DMSO solution (0.7 mL per column) of DIPEA (2 equivalents).

[0354] To the resin obtained above, a solution of a DMSO solution (0.25 mL) of the elongating carboxylic acid (0.181 mmol, 4 equiv.) listed in Table 9, a DMSO solution (0.25 mL) of [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equiv.) and DIPEA (0.047 mL, 0.271 mmol, 6 equiv.) was added and allowed to stand for 1-2 minutes, followed by shaking at room temperature for 4 hours. After draining the solution through the frit, the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column) and then dried.

[0355] To confirm the progress of the reaction, a portion of each resin was removed and swollen in DCM. The peptide was then cleaved using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The cleaved solution was analyzed by LCMS (SQDFA05long or SQDAA05long), and the production of the target peptide (TM) (Compounds 6-1-1* to 3-1-6*), diketopiperazine elimination products (Compounds 6-2-1* to 6-2-6*), and dimer urea (Compound 4-3*) was confirmed.

[0356] The amounts of diketopiperazine elimination product (TM-DKP) and dimer urea product produced for each sequence are shown in Table 9. The amounts of diketopiperazine (DKP) elimination product and dimer urea product produced shown in Table 9 are the UV area percentages of the diketopiperazine (DKP) elimination product and dimer urea product when the total UV area percentages of the target peptide (TM), diketopiperazine (DKP) elimination product, and dimer urea product by LCMS are taken as 100%.

[0357] Target peptide compound (TM), Teoc-Phe(4-CF3)-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip(run1) (compound 6-1-1*) LCMS (ESI) m / z=947.60 (M+H)+ Retention time: 2.99 minutes (Analysis conditions SQDFA05long)

[0358] Diketopiperazine eliminator, Teoc-Phe(4-CF3)-Tyr(tBu)-Asp-pip(run1) (compound 6-2-1*) LCMS (ESI) m / z=779.53 (M+H)+ Retention time: 3.11 minutes (Analysis conditions SQDFA05long)

[0359] Target peptide compound (TM), Boc-MeAla-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip(run2) (compound 6-1-2*) LCMS (ESI) m / z=773.62 (M+H)+ Retention time: 2.18 minutes (Analysis conditions SQDFA05long)

[0360] ジケトピペラジンbreakaway body, Boc-MeAla-Tyr(t Bu)-Asp-pip (run2) (compound 6-2-2*) LCMS (ESI) m / z = 603.58 (M-H) - Holding time: 2.29 minutes (Analysis conditions SQDFA05long)

[0361] Target compound (TM), Cbz-Pro-1-ACPrC-MeAl a-Tyr(tBu)-Asp-pip(run3)(Compound 6-1-3*) LCMS (ESI) m / z = 819.58 (M + H) + Holding time: 2.16 minutes (Analysis conditions SQDFA05long)

[0362] Separate body, Cbz-Pro-Tyr (tBu)-Asp-pip (run3) (compound 6-2-3*) LCMS (ESI) m / z = 649.61 (M-H) - Holding time: 2.27 minutes (Analysis conditions SQDFA05long)

[0363] Target compound (TM), Ac-1-ACPrC-MeAla-T yr(tBu)-Asp-pip(run4)(compound 6-1-4*) LCMS (ESI) m / z = 630.50 (M + H) + Holding time: 2.21 minutes (Analysis conditions SQDAA05long)

[0364] ジケトピペラジン detachment, Ac-Tyr (tBu)-Asp-pip (run4) (compound 6-2-4*) LCMS (ESI) m / z = 462.42 (M + H) + Holding time: 2.17 minutes (Analysis conditions SQDAA05long)

[0365] Target compound (TM), Tfa-Aib-1-ACPrC-MeAl a-Tyr(tBu)-Asp-pip(run5)(Compound 6-1-5*) LCMS (ESI) m / z = 769.56 (M + H) + Holding time: 1.90 minutes (Analysis conditions SQDFA05long)

[0366] Diketopiperazine eliminator, Tfa-Aib-Tyr(tBu)-Asp-pip(run5) (Compound 6-2-5*) LCMS (ESI) m / z=601.54 (M+H)+ Retention time: 1.98 minutes (Analysis conditions SQDFA05long)

[0367] Target peptide compound (TM), Ns-Aib-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip(run6) (compound 6-1-6*) LCMS (ESI) m / z=858.54 (M+H)+ Retention time: 2.09 minutes (Analysis conditions SQDFA05long)

[0368] Diketopiperazine elimination product, Ns-Aib-Tyr(tBu)-Asp-pip(run6) (compound 6-2-6*) LCMS (ESI) m / z=690.41 (M+H)+ Retention time: 2.17 minutes (Analysis conditions SQDFA05long)

[0369]

[0370] As described above, the results of Example 6 confirmed that, in the elongation of various carboxylic acids, not limited to specific Fmoc amino acids, the production of diketopiperazine elimination products and dimer urea products can be suppressed by performing the elongation using the method of the present invention, thereby enabling the synthesis of target peptides.

[0371] Example 7: Confirmation of the presence of carbamate in the Fmoc removal step and confirmation of its effect on diketopiperazine elimination The compounds described in the examples are shown in the table below.

[0372]

[0373] The following abbreviations are used in the examples: NMR: Nuclear magnetic resonance spectroscopy; ROESY: Rotating frame Overhauser effect spectroscopy; NOESY: Nuclear Overhauser effect spectroscopy; HSQC: Heteronuclear single quantum correlation; HMBC: Heteronuclear multiple bond correlation; cpd.: Compound

[0374] NMR measurements were carried out at 298K using AVANCE III 600 Cryo-TCI or AVANCE III HD 600 BBFO manufactured by Bruker.

[0375] When a base containing multiple nitrogen atoms is protonated to form a cation and forms a salt with an anion in the system, any of the protonated nitrogen atoms can serve as the cation source. For example, in the case of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), the following three are possible cation sources. However, in this specification, we will refer to any cation that forms a salt with the corresponding anion as a DBU salt (Figure 1). Other bases, such as 1,1,3,3-tetramethylguanidine (TMG) and iminotris(dimethylamino)phosphouran (HP1(dma)), will also be referred to as TMG salts, HP1(dma) salts, or MTBD salts.

[0376] Compound 1-4-1, 9H-fluoren-9-ylmethyl N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl]carbamate (Fmoc-Phe-NMe ２Synthesis of N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-Phe-OH, CAS: 35661-40-6, compound aa2-12) (2.0 g, 5.16 mmol) commercially available N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-Phe-OH, CAS: 35661-40-6, compound aa2-12) was dissolved in a mixture of N,N-dimethylformamide (DMF) (14 mL) and dichloromethane (DCM) (4 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl) (1.19 g, 6.19 mmol) and 1-hydroxybenzotriazole (HOBt) (0.767 g, 5.68 mmol) were then added to the mixture at room temperature. A solution of dimethylamine in tetrahydrofuran (THF) (2.0 M, 2.84 mL, 5.68 mmol) was then added under ice-cooling, and the mixture was stirred for 15 minutes. Isopropyl acetate (20 mL) was added to the solution, and the organic phase was washed twice with 1 M hydrochloric acid (20 mL), once with water (50 mL), twice with half-saturated aqueous sodium bicarbonate (20 mL), and twice with half-saturated aqueous sodium chloride (20 mL). The resulting organic phase was dried over sodium sulfate, filtered, and the solvent in the resulting mother liquor was evaporated under reduced pressure. The resulting crude product was azeotroped twice with toluene and then allowed to cool at 5°C, yielding white crystals of compound 1-4-1 (1.88 g, 88%). １ H-NMR (600MHz, toluene-d8, 298K) δ7.51 (d, 2H, J = 7.2Hz), 7.43 (d, 2H, J = 7.3Hz), 7.21-7.16 (m, 2 H), 7.14-7.07 (m, 2H), 7.05-7.00 (m, 4H), 7.00-6.95 (m, 1H), 6.00 (d, 1H, J = 8.4Hz, NH), 4.88 (ddd , 1H, J = 8.4, 8.4, 6.0Hz), 4.32 (dd, 1H, J = 10.5, 7.4Hz), 4.23 (dd, 1H, J = 10.5, 7.4Hz), 4.03 (t, 1H, J=7.4Hz), 2.93 (dd, 1H, J=13.0, 8.4Hz), 2.86 (dd, 1H, J=13.0, 6.0Hz), 2.49 (s, 3H), 2.02 (s, 3H). １３ ​C-NMR (151MHz, toluene-d8, 298K) δ171.1, 155.9, 144.6, 141.8, 129.9, 128.5, 127.8 (overlapped with solvent peak, detected by CH-HSQC), 127.3, 127.0, 125.6, 125.4, 120.2, 67.2, 52.3, 47.7, 40.2, 36.0, 35.0. LCMS (ESI) m / z=415.44 (M+H)+ Retention time: 0.89 min (Analysis conditions SQDFA05)

[0377] Example 7-1: DBU·HOCO-Phe-NMe obtained by treating compound 1-4-1 with DBU ２ Example 7-1-1: Product when compound 1-4-1 is treated with DBU in N,N-dimethylformamide-d7 (DMF-d7) solution Compound 1-4-1 (12.9 mg, 0.031 mmol) in N,N-dimethylformamide-d7 (DMF-d7) solution (0.5 mL) １ After measuring H-NMR (FIG. 1-b), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (5.11 μL, 0.034 mmol) was added to the solution and stirred vigorously. １ H-NMR (Figure 1-a) and 1D-ROESY (Figures 2-a-2 and 2-a-3) were measured. As a result, it was confirmed from the NMR spectrum that three compounds were mainly produced: dibenzofulvene and compound 1-4-2 (H-Phe-NMe ２ ), N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl]carbamate DBU salt (compound 1-4-3a, DBU·HOCO-Phe-NMe ２ ) were present in the solution, and it was confirmed that chemical exchange had occurred between compound 1-4-2 and compound 1-4-3a. ２ The identification of compound 1-4-3a was described in detail in Example 7-1-3.

[0378] In DMF-d7, compound 1-4-1 was reacted with DBU to give compound 1-4-2 (H-Phe-NMe ２ ) and compound 1-4-3a undergo chemical exchange and their ratio were confirmed. The 1D-ROESY of the solution was measured (4.84 ppm and 3.95 ppm). １ 2-a)-2, 2-a)-3), and the H signal at 4.84 ppm of compound 1-4-3a. １ H signal and the signal at 3.95 ppm corresponding to the α-methine proton of compound 1-4-2 １ It was confirmed that the H signal was undergoing chemical exchange in the system (Fig. 2-a-2, Fig. 2-a-3). １ When the H signal was selectively excited, the １ From the H signal (Fig. 2-a)-2), 3.95 ppm １ When the H signal was selectively excited, the １ From the H signal (Fig. 2-a)-3), ROESY was observed in phase with the selectively excited signal, confirming that the respective signals were chemically exchanged (Fig. 2-a). １ H-NMR １ From the integral ratio of the H signal, it was confirmed that the abundance ratio of compound 1-4-3a to compound 1-4-2 was 82:18 (FIG. 2-b).

[0379] Compound 1-4-2 (H-Phe-NMe) in the solution ２ Confirmation of the presence of ) 3 μL of a separately prepared sample of compound 1-4-2 was mixed with the solution. １ H-NMR (Fig. 3-a)-3, Fig. 3-b)) and 1D-NOSEY (Fig. 3-a)-4, Fig. 3-a)-5) were measured. 1) 3 μL of a sample of compound 1-4-2 was added to the solution. １ As a result of measuring H-NMR, １ 3.95 ppm observed by H-NMR １ H signal (Figure 3-a)-2) and the signal at 3.94 ppm observed in the authentic sample of compound 1-4-2 １ The H signal (Fig. 3-a)-1) was observed at 3.95 ppm as a triplet without separation.１ 2) 1D-NOESY of the solution obtained by adding 3 μL of the compound 1-4-2 preparation to the solution was measured (4.84 ppm and 3.95 ppm). １ H signal), 4.84 ppm １ When the H signal was selectively excited, the １ From the H signal (Figure 3-a)-4), 3.95 ppm １ When the H signal was selectively excited, the １ From the H signal (Figure 3-a)-5), NOESY (4.85 ppm: double triplet, 3.95 ppm: triplet) was observed, which was in phase with the selectively excited signal and was the same shape as the original signal. 3) When 3 μL of the sample of compound 1-4-2 was added to the solution, the 4.84 ppm of compound 1-4-3a １ H signal and 3.95 ppm of compound 1-4-2 １ The integral ratio of the H signals changed from 82:18 (integral ratio before the addition of compound 1-4-2 [Figure 2-b]) to 61:39 (Figure 3-b). The presence of compound 1-4-2 in the solution was confirmed from the above three points 1) to 3).

[0380] Example 7-1-2: Compound 1-4-2 １３ CO ２ All of the following experimental procedures in Example 7-1-2 were carried out in a glove box (under a nitrogen atmosphere and dehydrated conditions). NMR was measured using N,N-dimethylformamide-d7 (DMF-d7) as a solvent, which had been dried for 12 hours or more with MS4A that had been activated by heating to a high temperature using microwaves and then cooling under vacuum (x2). The sample was then placed in a valved, airtight NMR tube in a glove box and completely isolated from the outside air.

[0381] Preparation of Solution A (SolA) To a solution of compound 1-4-2 (4.64 mg, 0.024 mmol) in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO２ After bubbling for 1 minute, １ H-NMR (Figure 4-b)), １３ C-NMR (Figure 5-a), 1D-NOESY (4.77 ppm and 3.96 ppm) １ The H signals were selectively excited and measured (Figure 4-c, Figure 4-d). Then, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (3.97 μL, 0.027 mmol) was added to obtain compound 1-4-3a (DBU·HOCO-Phe-NMe ２ Prepare a solution A containing １ H-NMR (Figure 4-e)), １３ C-NMR (FIG. 5-b)) was measured.

[0382] Identification of Compound 1-4-3a in Solution A The structure of compound 1-4-3a is: １ H-NMR, １３ This can be explained without contradiction from the results of C-NMR and 1D-NOESY measurements. 4) For a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２ of the solution was bubbled for 1 minute. １ As a result of measuring H-NMR, the raw material compound 1-4-2 １ A new peak at 4.76 ppm was observed that was not observed in H-NMR (Fig. 4-a). １ 1H signal was confirmed (Figure 4-b) (production of carbamate anion or carbamic acid, see Figure 5-a)). 5) To a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２ of the solution was bubbled for 1 minute. １ H-NMR observed at 4.76 ppm １ H signal and the α-methylene signal at 3.96 ppm of compound 1-4-2. １ The integral value of the H signal was measured, and it was confirmed that the ratio was 52:48 (FIG. 4-f). 6) To a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２The 1D-NOESY of the solution in which the compound was bubbled for 1 minute was measured, and the result was 4.76 ppm. １ 3.96 ppm when the H signal is selectively excited １ From the H signal (FIG. 4-c), and from the １ When the H signal was selectively excited, the １ The NOESY of the same phase was observed from the H signal (Figure 4-d), confirming that the two compounds were undergoing chemical exchange. 7) To a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２ After bubbling for 1 minute, DBU was further added to the solution. １ As a result of measuring H-NMR, the peak at 4.76 ppm observed before adding DBU was １ It was confirmed that the H signal shifted upfield to 4.83 ppm when DBU was added (FIG. 4-e) (confirmation of the production of compound 1-4-3a). 8) To a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２ After bubbling for 1 minute, DBU was further added to the solution, and the 1H-NMR of the solution was measured. １ H signal and １３ C-labeled carbonyl at 161.5 ppm １３ C signal (Fig. 5-b)) ３ J ＣＨ Coupling was confirmed (Figure 4-g).

[0383] 9) To a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL), １３ CO ２ of the solution obtained by bubbling for 1 minute １３ C-NMR was measured (Figure 5-a), and then DBU was added to the resulting solution. １３ As a result of measuring C-NMR (Figure 5-b) and comparing, １３ Corresponding to C-labeled carbonyl １３It was confirmed that the C signal shifted upfield from 157.1 ppm to 161.5 ppm by adding DBU (confirming the production of compound 1-4-3a).

[0384] By preparing Solution A (SolA) from the six points 4) to 9), the primary amine of compound 1-4-2 and １３ CO ２ is reacted to form a carbamate or carbamic acid, which is then reacted with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to give compound 1-4-3a (DBU·HOCO-Phe-NMe ２ It was confirmed that compound 1-4-3a (DBU·HOCO-Phe-NMe) was produced. ２ This structure was identified in more detail in Example 7-1-3.

[0385] Example 7-1-3: Proof of Structure of Compound 1-4-3a Present in Solution A + Solution B (SolA + SolB) All of the following experimental procedures described in Example 7-1-3 were carried out in a glove box (under a nitrogen atmosphere and dehydrated conditions). NMR was measured using N,N-dimethylformamide-d7 (DMF-d7) as a solvent, which had been dried for 12 hours or more with MS4A that had been activated by heating to a high temperature using a microwave and then cooling under vacuum (x2). The sample was then placed in a valved, airtight NMR tube in a glove box and completely isolated from the outside air.

[0386] Preparation of Solution B (SolB) Solution B was prepared by adding 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (4.56 μL, 0.031 mmol) to a solution (0.5 mL) of compound 1-4-1 (11.5 mg, 0.028 mmol) in N,N-dimethylformamide-d7 (DMF-d7). １ H-NMR was measured (SolB, FIG. 6-a).

[0387] Preparation of Solution A + Solution B (SolA + SolB) The above solution B (SolB) (0.15 mL, Figure 6-a), 0.0084 mmol) and solution A (SolA) (0.35 mL, SolA, Figure 6-c), 0.0168 mmol) were mixed. １H-NMR (Figure 6-b)), １３ C-NMR, CH-HMBC (FIG. 7-c), CH-HSQC, NH-HMBC (FIG. 7-b), and NH-HSQC (FIG. 7-a) were measured.

[0388] Confirmation of the presence of compound 1-4-3a in solution B (SolB) １ H-NMR observed at 4.83 ppm, which corresponds to the α-methine of compound 1-4-3a. １ H signal (Fig. 6-c)) and solution B １ H-NMR observed at 4.85 ppm １ The H signal (Fig. 6-a) was 4.84 ppm. １ It was confirmed that the H signal was completely consistent with the 4.85 ppm observed in solution B, while shifting to the H signal (Figure 6-b). １ The peak at 4.83 ppm observed in solution A (the peak surrounded by the dotted line in Figure 6-a) is in the low magnetic field without coupling in the H signal. １ H signal １３ It was confirmed that the peak on the low magnetic field side (circled peak in Figure 6-b) where coupling with C (2.7 Hz) can be recognized shifts relative to the peak when solution B and solution A are mixed, and is converted into an intermediate broad peak (circled peak in Figure 6-b). From these results, １ The H signal confirmed the presence of compound 1-4-3a in (SolB).

[0389] Proof of structure of compound 1-4-3a in solution A + solution B (SolA + SolB) The structure of compound 1-4-3a in solution A + solution B (SolA + SolB) is １ H-NMR (Figure 6-b)), １３ By using C-NMR, CH-HMBC (Figure 7-c), CH-HSQC, NH-HMBC (Figure 7-b), and NH-HSQC (Figure 7-a), it can be consistently explained that this is a carbamic acid DBU salt. 10) When the CH-HMBC of the mixed solution of Solution A (SolA) and Solution B (SolB) was measured, the α-methine at 4.84 ppm was detected. １ H signal and solvent １３ Higher than C signal １３C labeled 161.5 ppm １３ It was confirmed that there is a CH-HMBC correlation between the C signal and the CH-HMBC signal (Fig. 7-c). １３ C signal value, experimentally １３ The origin of C bubbled up １３ CO ２ Therefore, 161.5 ppm １３ The C signal can be identified as the carbonyl of carbamate, which is consistent with the above. 11) The NH-HSQC measurement of the mixed solution of Solution A (SolA) and Solution B (SolB) showed that the NH １ H signals at 5.30 ppm and 91.4 ppm １５ Since NH-HSQC correlation was observed in the N signal, the １５ The N signal was identified as the nitrogen atom of the carbamate (Figure 7-a). １ H signal and 91.4 ppm corresponding to the nitrogen atom of the carbamate １５ The NH-HMBC correlation was also confirmed between the N signal (Figure 7-b). １５ The N signal value is 91.4 ppm. １５ This can be explained without contradiction by the N signal being the nitrogen atom of the carbamate.

[0390] When Solution A + Solution B (SolA + SolB) was prepared from the two points 10) and 11), the presence of Compound 1-4-3a was confirmed in the system. Furthermore, as shown below, all NMR signals of the carbamate moiety of Compound 1-4-3a could be assigned (Figure 8), and the values ​​of all NMR signals can consistently explain the structure of Compound 1-4-3a. １ H-NMR (600MHz, DMF-d7, 298K) δ7.29-7.23 (m, 4H), 7.22-7.11 (m, 1H), 5.30 (brs, 1H, NH), 4.90 (ddd, 1H, J = 6.8, 6.2Hz, ３ J ＣＨ = 2.7Hz), 2.91 (dd, 1H, J = 13.3, 6.8Hz), 2.87 (s, 3H), 2.83 (dd, 1H, J = 13.3, 6.2Hz), 2.78 (s, 3H).​１３ C-NMR (151MHz, DMF-d7, 298K) δ173.6 (qC), 161.5 (qC), 139.4 (qC), 129.7 (CH x2), 128.3 (CH x2), 126.3 (CH), 53.0 (CH), 40.0 (CH2), 36.4 (CH3), 34.8 (CH3). １５ N-NMR (61 MHz, DMF-d7, 298K, assigned by NH-HSQC and NH-HMBC) δ96,91.

[0391] From the above results, it was found that when the Fmoc-protected amino group was treated with DBU, the amino group was not completely exposed through the elimination of dibenzofulvene followed by decarboxylation, but was mostly exposed by DBU·HOCO-Phe-NMe. ２ It was confirmed that it exists as a １ This was confirmed by H-NMR.

[0392] Example 7-2: Confirmation of the presence of carbamate when compound 1-4-1 is treated with DBU or piperidine in various solvents. Basic procedure A solution of compound 1-4-1 (approximately 25 mg) in a heavy solvent (1.25 mL, tetrahydrofuran-d8 (THF-d8) [Entry 1], toluene-d8 [Entry 2], dichloromethane-d2 (DCM-d2) [Entry 3], N,N-dimethylformamide-d7 (DMF-d7) [Entry 4], dimethyl sulfoxide-d6 (DMSO-d6) [Entry 5]) was prepared, and 1.0 mL of this solution was divided into two (0.5 mL). One was treated with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL) and the other with piperidine (67.5 μL). NMR of each was measured, and the ratio of compound 1-4-3a to compound 1-4-2 was determined. １ The amount of compound 1-4-1 used in this experiment was 25.1 mg in entry 1, 24.8 mg in entry 2, 25.0 mg in entry 3, 25.3 mg in entry 4, and 25.2 mg in entry 5.

[0393]

[0394] From the above results, it was confirmed that when the Fmoc-protected amino group was treated with DBU, carbamate was present in a majority ratio in all solvents, whereas when the Fmoc-protected amino group was treated with piperidine, no carbamate was detected in any solvent, confirming that decarboxylation had proceeded to the complete exposure of the amino group.

[0395] When the results of solid-phase experiments are considered based on this finding, and without being bound by any particular theory, it can be explained that the reduction effect is due to the fact that the presence of a carbamate prevents the formation of a six-membered ring that causes diketopiperazine elimination, etc., as shown in the following formula.

[0396] For example, deprotection with DBU, in which the formation of a carbamate was confirmed by NMR, was compared with deprotection with piperidine, in which the formation of a carbamate was not confirmed, in a solid-phase experiment to confirm the effects. Reactions were carried out using the reagents and solvents listed in Table 12, runs 1 and 2, in a manner similar to that of Basic Procedure B described in Example 2, and the results obtained (relative ratios of the UV areas in LCMS of Compound 2-1*, Compound 2-2*, Compound 2-3*, and Compound 2-4* under each condition, expressed as percentages) are shown in Table 12 below.

[0397]

[0398] Without being bound by any particular theory, when run 1 (2% DBU) and run 2 (20% piperidine) are compared in Table 12, the fact that the production of diketopiperazine elimination products and 6-membered cyclic amidine skeletal structures can be significantly suppressed when treated with DBU can be explained without contradiction by the fact that the presence of a carbamate prevents the formation of a 6-membered ring that causes diketopiperazine elimination, etc.

[0399] Example 7-3: Confirmation of the presence of Compound 1-4-3a when Compound 1-4-1 was treated with various bases in DMSO. Basic procedure. Five solutions (0.5 mL) of Compound 1-4-1 (approximately 10 mg, 0.24 mmol) in dimethyl sulfoxide-d6 (DMSO-d6) were prepared, and then piperidine (67.5 μL, Entry 1), 1,1,3,3-tetramethylguanidine (TMG) (13.5 μL, Entry 2), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL, Entry 3), and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL, Entry 4) were added. 1-4-3a, 1-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) (13.5 μL, Entry 3), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) (13.5 μL, Entry 4), and iminotris(dimethylamino)phosphouran (HP1(dma)) (13.5 μL, Entry 5) were reacted with each other, and NMR was measured for each. The ratio of Compound 1-4-3a and Compound 1-4-2 was determined. １ The amount of compound 1-4-1 used in this experiment was 10.1 mg in entry 1, 10.0 mg in entry 2, 10.1 mg in entry 3, 10.1 mg in entry 4, and 10.1 mg in entry 5.

[0400]

[0401] From the above results, it was confirmed that carbamate salts were present at a ratio of 90% when an Fmoc-protected amino group was treated with a base whose conjugate acid had a pKa of at least 23. When the solid-phase experimental results were considered based on this finding, and without being limited to a particular theory, it was found that the reduction in diketopiperazine elimination and the like under the conditions of runs 4 to 7 compared to run 3 in Example 2 was due to the fact that the presence of carbamate salts prevented the formation of a six-membered ring that would cause diketopiperazine elimination and the like, as described above.

[0402] As described above, without being limited to a particular theory, when Fmoc protection is treated with a base, the N-terminal amino group exists as a carbamate, which can reduce diketopiperazine elimination, etc. Furthermore, the results of Example 7-3 identified the range of bases that can exist as carbamates.

[0403] Example 8: Experiment to confirm the effect of discovered conditions on reducing diketopiperazine elimination during peptide synthesis by the liquid phase method In this example, the linear peptide prepared in Example 1-3 was elongated with the Fmoc-protected amino acids listed in Table 14 by the method of the present invention (Condition-X), and the amounts of diketopiperazine elimination products and dimer urea products produced by the liquid phase method were confirmed.

[0404] Condition-X: DBU (0.0091 mL, 0.061 mmol, 1 equivalent) was added to a DMSO (0.20 mL) solution of the linear peptides (Compounds 1-3-1 to 1-3-2) (0.061 mmol) already prepared in Example 1-3, and the mixture was stirred at room temperature for 5 minutes. A DMSO (0.1 mL) solution of HOAt (16.52 mg, 0.121 mmol, 2 equivalents) and DIPEA (0.021 mL, 0.121 mmol, 2 equivalents) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 minutes. This was used as solution A.

[0405] In a separate reaction vessel, DIPEA (0.019 mL, 0.109 mmol, 1.8 equivalents) was added to a DMSO (0.20 mL) solution of an Fmoc-protected amino acid (0.073 mmol, 1.2 equivalents) and [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (38.4 mg, 0.073 mmol, 1.2 equivalents) listed in Table 14, and the mixture was stirred at room temperature for 3 minutes, followed by the dropwise addition of separately prepared solution A. After stirring at room temperature for 1 hour, a portion of the reaction solution was analyzed by LCMS (SQDFA05long), and the production of the target peptides (TM) (compounds 8-1-1 to 8-1-2) and diketopiperazine elimination products (compounds 8-2-1 to 8-2-2) was confirmed.

[0406] The amount of diketopiperazine elimination product (TM-DKP) produced for each peptide sequence is shown in Table 14. The amount of diketopiperazine elimination product (TM-DKP) produced shown in Table 14 is the UV area percentage of the diketopiperazine elimination product (TM-DKP) when the total UV area percentage of the target peptide (TM) and the diketopiperazine elimination product (TM-DKP) by LCMS is taken as 100%.

[0407] Target peptide compound (TM), Fmoc-MeLeu-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(OMe)-pip(run1) (compound 8-1-1) LCMS (ESI) m / z=1069.97 (M+H)+ Retention time: 3.35 minutes (Analysis conditions SQDFA05long)

[0408] Diketopiperazine eliminator, Fmoc-MeLeu-Aze(2)-MeCha-MeGly-Asp(OMe)-pip(run1) (compound 8-2-1) LCMS (ESI) m / z=885.78 (M+H)+ Retention time: 3.26 minutes (Analysis conditions SQDFA05long)

[0409] Target peptide compound (TM), Fmoc-Thr(tBu)-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip(run2) (Compound 8-1-2) LCMS (ESI) m / z=1074.90 (M+H)+ Retention time: 4.05 minutes (Analysis conditions SQDFA05long)

[0410] Diketopiperazine eliminator, Fmoc-Thr(tBu)-Cys(StBu)-Phe-Asp(OMe)-pip(run2) (compound 8-2-2) LCMS (ESI) m / z=932.69 (M+H)+ Retention time: 3.76 minutes (Analysis conditions SQDFA05long)

[0411]

[0412] From the results of Example 8, it was confirmed that the method of the present invention can suppress the production of diketopiperazine elimination products and synthesize the target peptide even in liquid phase peptide synthesis.

[0413] According to the present invention, it has been found that in peptide production, the formation of by-products such as diketopiperazine and 6-membered cyclic amidine structures can be suppressed, and peptide chains can be efficiently elongated. The present invention is useful in the field of peptide synthesis.

Claims

1. A method for producing a peptide, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent; and (3) The production method as described above, which comprises, after the step (2), a step of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

2. 1. A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine backbone impurities produced in the production of a peptide, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent; The method comprising:

3. 1. A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeletal structure impurities, and dimer urea impurities in peptide production, comprising: (1) providing a first peptide having a protecting group containing an Fmoc backbone supported on a solid phase; and (2) after the step (1), treating the first peptide with one or more bases, including at least a base whose conjugate acid in acetonitrile has a pKa of 23 or more, in a solvent containing a sulfoxide solvent; The method comprising:

4. (3) The method according to claim 2 or 3, further comprising, after step (2), a step of condensing the first peptide with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

5. The method according to claim 1 or 2, which is carried out by a solid phase method.

6. The method according to claim 1 or 2, wherein the first peptide is a first peptide supported on a solid phase and having a protecting group containing an Fmoc backbone.

7. 3. The method according to claim 1 or 2, which is carried out by a liquid phase method.

8. 3. The method according to claim 1 or 2, wherein step (2) is a step of removing a protecting group containing an Fmoc backbone in the first peptide and / or a step of converting a protecting group containing an Fmoc backbone in the first peptide into a carbamate form.

9. 3. The method of claim 1 or 2, which does not include a step of treating the first peptide with piperidine as a sole base prior to step (2).

10. 3. The method according to claim 1 or 2, which does not include a step of treating the first peptide with a single base having a conjugate acid with a pKa in acetonitrile of less than 23 prior to step (2).

11. 3. The method according to claim 1 or 2, wherein at least a portion of the first peptide obtained in step (2) is in the form of a carbamate.

12. 3. The method according to claim 1, wherein the solvent in step (2) contains a sulfoxide solvent in an amount of 50 v / v or more.

13. 3. The method according to claim 1, wherein the sulfoxide solvent is one or more selected from the group consisting of DMSO, diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide.

14. 3. The method according to claim 1, wherein the base in step (2) is at least one selected from the group consisting of amidines, guanidines, and phosphazenes.

15. 3. The method according to claim 1, wherein the base in step (2) is at least one selected from the group consisting of DBU, MTBD, TMG, P1tBu, P2Et, and HP1(dma).

16. 3. The method of claim 1 or 2, wherein the first peptide comprises 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues.

17. The method of claim 1, wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group, or an active ester of an amino acid or a second peptide having a protecting group, or an acid halide of an amino acid or a second peptide having a protecting group, and the first peptide and / or the second peptide having a protecting group contains one or more N-substituted amino acids, and / or the amino acid having a protecting group is an N-substituted amino acid.

18. The method according to claim 1 or 2, wherein the second amino acid residue from the N-terminus of the first peptide is an N-substituted amino acid.

19. The condensing agent in the step (3) is in the form of a salt, and its counter anion is PF ６ － or BF ４ － or a carbodiimide-based condensing agent.