Method for producing peptide compound comprising highly sterically hindered amino acid

Abstract

To provide a method for efficiently producing a peptide compound having a structure in which an N-substituted-α,α-disubstituted amino acid and an N-substituted amino acid are linked together.SOLUTION: It was found that the peptide compound can be efficiently produced by linking an NH-α,α-disubstituted amino acid whose amino group is protected with an electron-withdrawing protecting group to an N-substituted amino acid, and then allowing a reagent to act in the presence of a specific base to selectively introduce a substituent to the NH group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a peptide compound containing a highly sterically hindered amino acid. [Background technology]

[0002] Examples of so-called tough targets that could not be used as drug discovery targets with conventional medicines include drug discovery that targets intracellular molecules, or drug discovery that inhibits protein-protein interactions. The reason these have not been used as targets for drug discovery is that the drug molecules cannot reach the target molecules of the disease, or the shape of the site of action of the target molecule is such that conventional medicines have difficulty acting on them (Non-Patent Document 1).

[0003] Recently, cyclic peptides have been attracting attention as a method for accessing tough targets that were difficult to access using conventional techniques (Non-Patent Document 2). For the application of cyclic peptides to medicines, consideration has been given not only to improving the binding ability to the target, but also to the fact that the cyclic peptide is drug-like (preferably exhibiting both membrane permeability and metabolic stability) and to efficient screening methods for cyclic peptides (Non-Patent Documents 3 and 4). In addition, the conditions required for non-natural cyclic peptides containing drug-like non-natural amino acids have been clarified, and their importance in drug discovery and their recognition have been increasing (Patent Document 1). Non-natural amino acids are an important element of these non-natural peptides, and it is widely recognized that the synthesis of non-natural amino acids, such as peptides containing N-methyl (or N-alkyl) amino acids, and in some cases peptides containing units in which N-methyl (or N-alkyl) amino acids are consecutive, is more difficult than that of natural peptides due to their bulkiness (Non-Patent Documents 5 and 6).

[0004] Turning our attention to unnatural amino acids, in which the side chain of an amino acid differs from that of natural amino acids, it has been reported that α,α-disubstituted amino acids, in which the α position of an amino acid is substituted with two substituents, increase lipid solubility, restrict the conformational freedom of peptides containing α,α-disubstituted amino acids due to steric hindrance, and contribute to stabilization in the body (Non-Patent Document 7), and are expected to have a significant positive impact on both pharmacological efficacy and drug-like properties.

[0005] In producing peptides containing these unnatural amino acids, the Fmoc method, which is the most widely used method for peptide synthesis, is known to be unsuitable due to the structural bulkiness of these unnatural amino acids (Non-Patent Document 8).

[0006] From these facts, it is easily inferred that peptide synthesis by condensation reaction of the carboxyl group of an α,α-disubstituted amino acid with the amino group of an amino acid, in particular, synthesis of a peptide by condensation reaction of the carboxyl group of an N-alkylated α,α-disubstituted amino acid with the amino group of an N-alkylated amino acid, is difficult due to the steric bulkiness of one or both of the amino acids.

[0007] As a method for continuously introducing N-alkylamino acids, a method is known in which a peptide is synthesized by condensing an NH amino acid that has a smaller steric hindrance than the N-alkylamino acid, and then the desired NH group among the multiple NH groups in the resulting peptide is selectively N-alkylated (Non-Patent Documents 9 and 10). However, these methods are methods for binding an α-monosubstituted amino acid to an N-alkylamino acid, and no practical method for synthesizing a peptide having a sequence in which an N-alkyl-α,α-disubstituted amino acid is bound to an N-alkylamino acid is known. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2018 / 225864 [Non-patent literature]

[0009] [Non-Patent Document 1] Annu. Rev. Pharmacol. Toxicol.,2016, 56, 23-40. [Non-Patent Document 2] Future Med. Chem. 2009, 1,1289-1310. [Non-Patent Document 3] ACS Chem. Biol., 2013, 8, 488-499. [Non-Patent Document 4] Drug Discovery Today, 2014, 19,388-399. [Non-Patent Document 5] J. Peptide Res., 2005, 65,153-166. [Non-Patent Document 6] Biopolymers, 109; e23110(DOI:10.1002 / bip.23110) [Non-Patent Document 7] Journal of the Society of Organic Synthetic Chemistry 2002, vol.60, No.2,125-136. [Non-Patent Document 8] Chem. Soc. Rev., 2016, 45,631-654. [Non-Patent Document 9] Org. Lett., 2013, 15, 5012-5015. [Non-Patent Document 10] J. Am. Chem. Soc., 1997, 119,2301-2302. Summary of the Invention [Problem to be solved by the invention]

[0010] Non-Patent Document 5 exemplifies a method for producing a peptide containing an N-alkylamino acid as a method for producing a peptide containing a bulky amino acid, but this document only describes the mechanism of side reactions in the condensation reaction of amino acids and condensation reagents, and does not describe the condensation reaction of bulky α,α-disubstituted amino acids.

[0011] Non-Patent Document 6 illustrates a method for producing an N-alkylamino acid and a method for producing a peptide containing an N-alkylamino acid. This document illustrates a production method focusing on a condensing agent for amino acids, but does not describe a condensation reaction of a bulkier α,α-disubstituted amino acid.

[0012] Non-Patent Document 7 describes a method for producing α,α-disubstituted amino acids, the usefulness of peptides containing α,α-disubstituted amino acids, examples of α,α-disubstituted amino acids, and methods for producing them, but does not describe the production of peptides containing sterically bulky amino acids.

[0013] Non-Patent Document 8 illustrates a method for producing a peptide that is difficult to synthesize. This document describes the production of peptides that have low solubility or are prone to aggregation, but does not describe a method for solving the problems using a condensation reaction.

[0014] Non-Patent Document 9 exemplifies a method for forming a peptide bond with an N-alkylamino acid using a trifluoroacetyl group that can be removed under mild conditions, and a method for producing a peptide containing an N-alkylamino acid. However, there is no mention of a condensation reaction with a bulkier α,α-disubstituted amino acid. In addition, it is known that an alkyl group cannot be selectively introduced only to the nitrogen atom to which the trifluoroacetyl group is bonded, and an isomeric by-product in which an alkyl group is also introduced to the oxygen atom of the trifluoroacetyl group is also generated.

[0015] Non-Patent Document 10 describes a method for producing a peptide containing an N-alkylamino acid by introducing an alkyl group to the nitrogen atom of an amino acid protected with a nosyl group. However, it is known that there is a problem in producing the target product because a side reaction occurs in the deprotection step of the nosyl group (Non-Patent Document 9).

[0016] As described above, the bond-forming reaction between N-substituted α,α-disubstituted amino acids such as N-alkyl-α,α-disubstituted amino acids and N-substituted amino acids such as N-alkylamino acids is difficult, but no effective means for solving this problem is known. The present invention has been made in view of this situation, and an objective of the present invention is to provide a method for producing N-substituted α,α-disubstituted amino acid residues and / or peptide compounds containing N-substituted amino acid residues. More specifically, an objective of the present invention is to provide a method for introducing N-substituted α,α-disubstituted amino acids into N-substituted amino acids. Furthermore, an objective of the present invention is to provide a method for highly selectively N-functionalizing the amino groups of N-unsubstituted α,α-disubstituted amino acid residues. [Means for solving the problem]

[0017] The present inventors have found a method for linking an N-substituted amino acid with an N-substituted-α,α-disubstituted amino acid. Specifically, they have found that the target amino acid can be efficiently introduced by using an N-unsubstituted-α,α-disubstituted amino acid that is sterically less bulky than an N-substituted amino acid and has improved reactivity of the carboxyl group by protecting the amino group with an electron-withdrawing protecting group. In addition, they have found that in the subsequent functionalization of the amino group, an N-functionalization reaction such as an N-alkylation reaction proceeds selectively on the NH group whose acidity has increased due to the electron-withdrawing protecting group. Furthermore, they have focused on the fact that the acidity of the NH group has increased due to the electron-withdrawing protecting group, and have found a specific base to be used in the N-functionalization reaction, thereby completing the present invention.

[0018] That is, in one non-limiting specific embodiment, the present invention includes the following. [1] A method for producing a peptide compound, a salt thereof, or a solvate thereof, which has an N-substituted α,α-disubstituted amino acid residue at the N-terminus and contains a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together, the method comprising the steps of: Step A: reacting an N-substituted amino acid, a salt thereof, or a solvate thereof, or a peptide compound having an N-substituted amino acid residue at the N-terminus, a salt thereof, or a solvate thereof with an N-non-substituted-α,α-disubstituted amino acid in which the amino group is protected with an electron-withdrawing protecting group, a salt thereof, a dehydrated form thereof, or a solvate thereof in the presence or absence of a condensation reagent to obtain a peptide compound, a salt thereof, or a solvate thereof, which has an N-non-substituted-α,α-disubstituted amino acid residue in which the amino group is protected with an electron-withdrawing protecting group at the N-terminus and contains a dipeptide residue in which the N-non-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together; and Step B: A step of introducing a substituent into the amino group of an N-unsubstituted-α,α-disubstituted amino acid residue, the amino group of which is protected by an electron-withdrawing protecting group at the N-terminus, in the presence of a base and a substituent-introducing agent, to obtain a peptide compound, a salt thereof, or a solvate thereof, which has an N-substituted-α,α-disubstituted amino acid residue, the amino group of which is protected by an electron-withdrawing protecting group at the N-terminus, and which contains a dipeptide residue in which the N-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together. [2] The method according to [1], wherein the electron-withdrawing protecting group is a protecting group having a pKa (in water) of 6 to 11 for the NH group to which the protecting group is bonded. [3] The method according to [1] or [2], wherein the pKa (in acetonitrile) of the conjugate acid of the base is 18 to 31. [4] The method according to any one of [1] to [3], wherein the N-substituted amino acid or a peptide compound having an N-substituted amino acid residue at the N-terminus is supported on a resin for solid phase synthesis. [5] An N-substituted amino acid or a peptide compound having an N-substituted amino acid residue at the N-terminus, represented by the formula (2): [ka] [In the formula, P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is aralkyl, R3 is hydroxy, O-PG2, any amino acid residue, or any peptide residue; PG2 is a protecting group for the carboxyl group. The method according to any one of [1] to [4], [6] An N-unsubstituted α,α-disubstituted amino acid having an amino group protected by an electron-withdrawing protecting group is represented by the formula (3): [ka] [In the formula, PG1 is an electron-withdrawing protecting group, R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or optionally substituted C7-C 14 aralkyl; R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring. The method according to any one of [1] to [5], [7] The peptide compound obtained in step A is represented by the formula (4): [ka] [In the formula, PG1, R1, and Q1 are defined the same as PG1, R1, and Q1 in formula (3), respectively. P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2), respectively. The method according to any one of [1] to [6], [8] The substituent-introducing agent in step B is P1X (wherein P1 has the same meaning as P1 in formula (1), and X is a leaving group), and the peptide compound obtained in step B is represented by formula (1): [ka] [In the formula, P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, PG1, R1, and Q1 are defined the same as PG1, R1, and Q1 in formula (3), respectively. P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2), respectively. The method according to any one of [1] to [7], [9] A method comprising the steps of: [ka] [In the formula, PG1 is a protecting group for an amino group, P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or optionally substituted C7-C 14 aralkyl; R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring; P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is aralkyl, R3 is hydroxy, O-PG2, any amino acid residue, or any peptide residue; PG2 is a protecting group for the carboxyl group. A method for producing a peptide compound, a salt thereof, or a solvate thereof, comprising a structure in which two amino acid residues represented by the following formula (1) are linked: Process A: Formula (2): [ka] [In the formula, P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (1), respectively.] A compound represented by the formula (3): [ka] [In the formula, PG1, Q1, and R1 have the same meanings as PG1, Q1, and R1 in formula (1), respectively.] or a solvate thereof with a condensation reagent, or by reacting the compound represented by formula (2), a salt thereof, or a solvate thereof with the dehydrate of the compound represented by formula (3), a salt thereof, or a solvate thereof to obtain a compound represented by formula (4): [ka] [In the formula, PG1, P2, Q1, and R1 to R3 are defined the same as PG1, P2, Q1, and R1 to R3 in formula (1), respectively.] A step of obtaining a compound represented by the formula: Step B: A step of reacting a compound represented by formula (4), a salt thereof, or a solvate thereof with a P1-introducing reagent to obtain a peptide compound represented by formula (1), a salt thereof, or a solvate thereof.

[10] R1 and Q1 together with the carbon atom to which they are attached form a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, or a tetrahydropyran ring, or R1 and Q1 are independently selected from methyl, ethyl, 2-methylpropyl, allyl, methoxymethyl, cyclohexylmethyl, optionally substituted benzyl, or optionally substituted phenethyl; The method according to any one of [6] to [9].

[11] The method according to any one of [6] to

[10] , wherein in the formula (3) and / or the formula (4), the pKa (in water) of the NH group to which PG1 is bonded is 6 to 11.

[12] The method according to any one of [6] to

[11] , wherein PG1 is C2-C6 haloacyl.

[13] The method according to

[12] , wherein the C2-C6 haloacyl is trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, or 3,3,3-trifluoro-2-(trifluoromethyl)propionyl.

[14] The dehydrated product is represented by the following formula: [ka] [In the formula, Q1 and R1 are defined as Q1 and R1 in formula (1), respectively, and R4 is a C1-C5 haloalkyl.] The method according to any one of [1] to

[13] ,

[15] The method according to

[14] , wherein R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring.

[16] The method according to

[14] or

[15] , wherein R4 is trifluoromethyl, trichloromethyl, pentafluoroethyl, 1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl, or 2,2,2-trifluoro-1-(trifluoromethyl)ethyl.

[17] The method according to any one of [8] to

[16] , wherein P1 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl.

[18] The method according to any one of [5] to

[17] , wherein P2 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl.

[19] The method according to any one of [5] to

[18] , wherein R3 is any amino acid residue or any peptide residue supported on a resin for solid phase synthesis.

[20] The method according to any one of [4] to [8] and

[19] , wherein the resin for solid phase synthesis is a CTC resin, a Wang resin, or a SASRIN resin.

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

[20] , wherein the condensation reagent is either DIC or EDCI·HCl, or a combination of DIC and Oxyma.

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

[21] , wherein the P1-introducing reagent is a combination of P1X (wherein P1 has the same meaning as P1 in formula (1), and X is a leaving group) and a base.

[23] The method according to

[22] , wherein the pKa (in acetonitrile) of the conjugate acid of the base is 18 to 31.

[24] The base is [ka] [In the formula, R and R are each independently a C-C alkyl, or R and R together with the nitrogen atom to which R is bonded and the carbon atom to which R is bonded form a 5- to 8-membered ring; RB2 and RB3 are each independently a C1-C4 alkyl, or RB2 and RB3 together with the nitrogen atom to which RB2 is bonded and the nitrogen atom to which RB3 is bonded and the carbon atom to which the nitrogen atoms are bonded form a 5- to 8-membered ring. [ka] [In the formula, RB6 is C1-C4 alkyl; RB5 and RB7 are each independently C1-C4 alkyl or form a 5-8 membered ring together with the nitrogen atom to which they are bonded and the carbon atom to which each nitrogen atom is bonded; RB8 is C1-C4 alkyl and RB9 is C1-C4 alkyl or phenyl, or RB8 and RB9 together with the nitrogen atoms to which they are bonded and the carbon atoms to which they are bonded form a 5- to 8-membered ring; When RB9 is phenyl, two B2's may be fused together at two benzene rings of the phenyl group to form a naphthalene. [ka] [In the formula, R.B. 10 is C1-C4 alkyl, or RB 10 and R.B. 11 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 11 RB 10 and R.B. 11 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 12 RB 11 and R.B. 12 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R12 and R.B. 13 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 13 RB 12 and R.B. 13 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 14 RB 13 and R.B. 14 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 14 and R.B. 15 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 15 RB 14 and R.B. 15 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 16 is hydrogen, C1-C8 alkyl, or C6-C 10 aryl; and [ka] [In the formula, R.B. 17 are independently C1-C4 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, R.B. 18 RB 17 and R.B. 18 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 19 RB18 and R.B. 19 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 19 and R.B. 20 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 20 RB 19 and R.B. 20 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 21 is C1-C4 alkyl, or RB 21 and R.B. 22 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 22 RB 21 and R.B. 22 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 23 RB 22 and R.B. 23 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 23 and R.B. 24 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 24 RB 23 and R.B. 24 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 24 and R.B. 25 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atom to which they are bonded, R.B. 25 RB 24 and R.B. 25 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 25 and R.B. 26form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 26 RB 25 and R.B. 26 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 27 is C1-C4 alkyl, or C6-C 10 It is aryl. The method according to any one of [3] to [8] and

[22] to

[23] , wherein the method is selected from the group consisting of:

[25] The base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu), tert-butylimino-tris(pyrrolidino)phosphorane (P1-tBu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP), tert-octylimino-tris(dimethylamino)phosphorane (P1-t-Oct), imino-tris(dimethylamino)phosphorane (HP1(dma)), 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 -Catenazi(phosphazene)(P2-t-Bu), and 1-ethyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 The method according to any one of [3] to [8] and

[22] to

[24] , wherein the compound is selected from the group consisting of -catenadi(phosphazene) (P2-Et).

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

[25] , wherein step B is carried out in a solvent selected from the group consisting of DMF, NMP, DMI, tetrahydrofuran, 2-methyltetrahydrofuran, and acetonitrile.

[27] A method for producing a peptide compound containing a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked together, a salt thereof, or a solvate thereof, the method comprising any one of the methods described in [1] to

[26] .

[28] A step of deprotecting the N-terminal protecting group from a peptide compound, a salt thereof, or a solvate thereof produced by the method according to any one of [1] to

[27] ; Optionally, extending the peptide chain; and A method for producing a cyclic peptide compound, a salt thereof, or a solvate thereof, comprising a step of cyclizing a group on the C-terminus side and a group on the N-terminus side to form a cyclic moiety, The above method, wherein the cyclic peptide compound contains 8 to 15 amino acid residues, at least three N-substituted amino acid residues and at least one non-N-substituted amino acid residue, and the cyclic portion contains at least 8 amino acid residues. Effect of the Invention

[0019] According to the present invention, it is possible to efficiently produce peptide compounds containing a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked, which are useful for peptide drugs, the discovery of peptide drugs, and / or the supply of drug substances. In addition, it is also possible to produce peptide compounds in which various non-natural amino acid residues are linked, so that it is possible to provide peptide compounds with diverse structures. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Example 2-4, detected at the maximum absorption wavelength using a photodiode array detector. [Diagram 2]FIG. 2 shows the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 1, detected at the maximum absorption wavelength using a photodiode array detector. [Diagram 3] FIG. 3 shows the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 2-4, detected at the maximum absorption wavelength using a photodiode array detector. [Figure 4] FIG. 4 shows the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 2-5, detected at the maximum absorption wavelength using a photodiode array detector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The abbreviations used in the present invention are listed below. AA: Ammonium acetate CSA: (+)-10-Camphorsulfonic acid DBU: 1,8-diazabicyclo[5.4.0]-7-undecene DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane DCE: 1,2-dichloroethane DEAD: Diethyl azodicarboxylate DMA: Dimethylacetamide DMF: N,N-dimethylformamide DIAD: Diisopropyl azodicarboxylate DIC: N,N'-diisopropylcarbodiimide DIPEA: N,N-diisopropylethylamine DMAP: N,N-dimethyl-4-aminopyridine dtbbpy: 4,4'-di-tert-butyl-2,2'-bipyridyl EDTA: Ethylenediaminetetraacetic acid FA: Formic acid Fmoc: 9-fluorenylmethyloxycarbonyl group NMP: N-methyl-2-pyrrolidone TBME: t-Butyl methyl ether TES: Triethylsilane TFA: Trifluoroacetic acid TFE: 2,2,2-trifluoroethanol THF: tetrahydrofuran THP: tetrahydropyranyl group TMSCl: chlorotrimethylsilane HFIP: 1,1,1,3,3,3-hexafluoroisopropyl alcohol HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxybenzotriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine IPAC: Isopropyl acetate oxyma: ethyl cyano(hydroxyimino)acetate PPTS: Pyridinium p-toluenesulfonate EDCI·HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride TIPS: Triisopropylsilane TfOH: Trifluoromethanesulfonic acid HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate DMSO: Dimethyl sulfoxide Fmoc-Cl: 9H-fluoren-9-ylmethyl carbonochloridate Fmoc-OSu: N-succinimidyl 9-fluorenylmethyl carbonate Ns: o-nitrobenzenesulfonyl group Trt: triphenylmethyl or trityl Tfa: Trifluoroacetyl group MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene TMGN: 1,8-bis(tetramethylguanidino)naphthalene P1-tBu: tert-butylimino-tris(dimethylamino)phosphorane

[0022] (Definition of functional groups, etc.) As used herein, the term "halogen atom" includes, for example, F, Cl, Br or I.

[0023] In this specification, "alkyl" refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only linear ones but also branched ones. Specific examples of alkyl include those having 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q " means that the number of carbon atoms is p to q), and preferably C1-C 10 Examples of alkyl include alkyl, more preferably C1-C6 alkyl. Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc.

[0024] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent sp 2Depending on the arrangement of the double bond and the substituents (if any), the geometry of the double bond can be in the Entgegen (E) or Zusammen (Z) configuration, in the cis or trans configuration. Alkenyl includes not only straight chains but also branched chains. Alkenyl is preferably C2-C 10 Alkenyl, more preferably C2-C6 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, etc.

[0025] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Alkynyl is preferably C2-C 10 Alkynyl, more preferably C2-C6 alkynyl, specifically, for example, ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, and the like.

[0026] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferred examples of cycloalkyl include C3-C8 cycloalkyl, specifically, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl.

[0027] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably a C6-C 10Specific examples of the aryl include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

[0028] In the present specification, 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 the carbon atom in the ring may be oxidized to form a carbonyl, and may be a single ring or a condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), and more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxetanyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2- Examples include thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, and 2-oxaspiro[3.3]heptyl.

[0029] In the present specification, "heteroaryl" refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a single ring or a condensed ring with other rings, and may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), 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.

[0030] In the present specification, "alkoxy" refers to an oxy group to which the above-defined "alkyl" is bonded, and preferably includes C1-C6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0031] In the present specification, "acyl (alkanoyl)" means a group in which a carbonyl group is bonded to hydrogen or the above-mentioned "alkyl", and preferably includes C1-C6 acyl, more preferably C2-C4 acyl. Specific examples of acyl include formyl, acetyl, propionyl, butanoyl, etc.

[0032] As used herein, "cycloalkoxy" refers to an oxy group bonded to the above-defined "cycloalkyl", and preferably includes C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, and cyclopentyloxy.

[0033] As used herein, the term "alkylsulfonyl" refers to a sulfonyl group having an "alkyl" bonded thereto, preferably C1-C6 alkylsulfonyl. Specific examples of alkylsulfonyl include methylsulfonyl.

[0034] In the present specification, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with hydroxyl groups, and C1-C6 hydroxyalkyl is preferred. Specific examples of hydroxyalkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0035] In the present specification, "haloalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with halogen, and C1-C6 haloalkyl is preferred, and C1-C6 fluoroalkyl is more preferred. Specific examples of haloalkyl include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, and 5,5-difluoropentyl.

[0036] In the present specification, "haloalkoxy" refers to a group in which one or more hydrogen atoms of the "alkoxy" defined above are replaced with halogen, and C1-C6 haloalkoxy is preferred. Specific examples of haloalkoxy include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, and 2,2,2-trifluoroethoxy.

[0037] In the present specification, "haloacyl (haloalkanoyl)" means a group in which a carbonyl group is bonded to the "haloalkyl", and preferably includes C2-C6 haloacyl, more preferably C2-C4 haloacyl. Specific examples of haloacyl include trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, and 3,3,3-trifluoro-2-(trifluoromethyl)propionyl.

[0038] In the present specification, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "alkoxy" defined above, and C1-C6 alkoxyC1-C6 alkyl is preferred, and C1-C6 alkoxyC1-C2 alkyl is more preferred. Specific examples of alkoxyalkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0039] In the present specification, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "cycloalkyl" defined above, and C3-C8 cycloalkylC1-C6 alkyl is preferred, and C3-C6 cycloalkylC1-C2 alkyl is more preferred. Specific examples of cycloalkylalkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0040] In the present specification, "cycloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "cycloalkoxy" defined above, and C3-C8 cycloalkoxyC1-C6 alkyl is preferred, and C3-C6 cycloalkoxyC1-C2 alkyl is more preferred. Specific examples of cycloalkoxyalkyl include cyclopropoxymethyl and cyclobutoxymethyl.

[0041] In the present specification, "alkylsulfonylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "alkylsulfonyl" defined above, and C1-C6 alkylsulfonylC1-C6 alkyl is preferred, and C1-C6 alkylsulfonylC1-C2 alkyl is more preferred. Specific examples of alkylsulfonylalkyl include methylsulfonylmethyl and 2-(methylsulfonyl)ethyl.

[0042] As used herein, the term "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is replaced by an "aryl" as defined above, and is a C7-C 14 Aralkyl is preferred, C7-C 10 Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0043] In the present specification, the term "heteroaralkyl (heteroarylalkyl)" refers to a group in which at least one hydrogen atom of the "alkyl" defined above is replaced by the "heteroaryl" defined above, and is preferably a 5- to 10-membered heteroaryl C1-C6 alkyl, and more preferably a 5- to 10-membered heteroaryl C1-C2 alkyl. Specific examples of heteroarylalkyl include 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.

[0044] In the present specification, the term "protecting group for a carboxyl group" includes protecting groups of an alkyl ester type, protecting groups of a benzyl ester type, protecting groups of a substituted alkyl ester type, etc. Specific examples of protecting groups for a carboxyl group include a methyl group, an ethyl group, a t-Bu group, a benzyl group, a trityl group, a cumyl group, a methoxytrityl group, a 2-(trimethylsilyl)ethyl group, a 2,2,2-trichloroethyl group, an allyl group, etc.

[0045] In the present specification, the "protecting group for amino group" includes a carbamate type protecting group, an amide type protecting group, an imide type protecting group, a sulfonamide type protecting group, etc. Specific examples of the protecting group for amino group include Fmoc, Boc, Cbz, Alloc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, tosyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, etc.

[0046] In the present specification, the term "alicyclic ring" refers to a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond in the ring, or may be a polycyclic ring having two or more rings. The carbon atoms constituting the ring may be oxidized to form a carbonyl. Preferred examples of the alicyclic ring include 3- to 8-membered alicyclic rings, and specific examples thereof include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, and a bicyclo[2.2.1]heptane ring.

[0047] In the present specification, the term "heterocycle" refers to a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocycle may have a double and / or triple bond in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl, and may be a monocycle, a condensed ring, or a spiro ring. The number of atoms constituting the ring is not limited, but is preferably 3 to 12 (3- to 12-membered heterocycle), and more preferably 4 to 7 (4- to 7-membered heterocycle). Specific examples of the heterocycle include piperazine, pyrrolidine, piperidine, morpholine, homomorpholine, hexahydropyrazine, 3-oxopiperazine, 2-oxopyrrolidine, azetidine, 2-oxoimidazolidine, oxetane, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyridine, thiomorpholine, pyrazolidine, imidazoline, oxazolidine, isoxazolidine, thiazolidine, imidazolidine, isothiazolidine, thiadiazolidine, oxazolidone, benzodioxane, dioxolane, dioxane, and tetrahydrothiopyran.

[0048] In the present specification, the term "saturated heterocycle" refers to a non-aromatic heterocycle that contains 1 to 5 heteroatoms in addition to carbon atoms and does not contain a double bond and / or a triple bond in the ring. The saturated heterocycle may be a monocycle or may form a condensed ring with another ring, for example, an aromatic ring such as a benzene ring. When a saturated heterocycle forms a condensed ring, the saturated heterocycle is preferably a 4- to 7-membered saturated heterocycle. Specific examples thereof include an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, a sazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, and an indoline ring.

[0049] As used herein, the term "peptide chain" refers to a peptide chain in which one or more natural amino acids and / or non-natural amino acids are linked by amide bonds and / or ester bonds. The peptide chain is preferably a peptide chain containing 1 to 15 amino acid residues, more preferably a peptide chain consisting of 5 to 12 amino acid residues.

[0050] The "peptide compound" in the present invention is not particularly limited as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are linked by amide bonds or ester bonds, but is preferably a peptide compound with 5 to 30 amino acid residues, more preferably 8 to 15 amino acid residues, and even more preferably 9 to 13 amino acid residues. Peptide compounds supported on a resin for solid phase synthesis are also included. The peptide compound synthesized in the present invention preferably contains at least three N-substituted amino acids in one peptide, and more preferably contains at least five or more N-substituted amino acids. These N-substituted amino acids may be present consecutively or discontinuously in the peptide compound. In the present specification, the "amino acid" constituting the peptide compound may be referred to as the "amino acid residue", and the "peptide" constituting all or a part of the peptide compound may be referred to as the "peptide residue". The peptide compound in the present invention may be linear or cyclic, and a cyclic peptide compound is preferred.

[0051] The "cyclic peptide compound" in the present invention is a cyclic peptide compound that can be obtained by cyclizing a group on the N-terminus side and a group on the C-terminus 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 via a heterocyclic ring construction. 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 in a side chain and an amino group in the main chain of the N-terminus 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.

[0052] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of a substituent of a group, the term means a number from one to the maximum number of substituents permitted by the group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0053] In the present specification, 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 the solid-phase method. 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), 4-methoxytrityl chloride resin (Mmt), and other resins that can be removed under acidic conditions. The resin can be appropriately selected according to the functional group of the amino acid used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid represented by Asp or Glu) or a hydroxy group on an aromatic ring (phenol group represented 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 (aliphatic alcohol group represented 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 be described as resin. The solid-phase synthesis resin can be linked to an amino acid at any position in a peptide, not limited to the C-terminal amino acid. It is preferable that the carboxyl group of the C-terminal amino acid is linked to the solid-phase synthesis resin, and the carboxyl group may be a main chain carboxyl group or a side chain carboxyl group.

[0054] The type of polymer constituting the resin is not particularly limited. In the case of a resin made of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The cross-linking rate is also not particularly limited, but 1% DVB (divinylbenzene) cross-linking is preferable. The type of polymer constituting the resin includes Tentagel and Chemmatrix.

[0055] In the production of the compounds described herein, when the defined groups undergo undesired chemical conversion under the conditions of the method, the compounds can be produced by using, for example, means for protecting and deprotecting functional groups. The selection and deprotection of the protecting groups can be carried out, for example, by the methods described in "Greene's, "Protective Groups in Organic Synthesis" (5th Edition, John Wiley & Sons 2014), which can be used appropriately depending on the reaction conditions. In addition, the order of reaction steps such as introducing substituents can be changed as necessary.

[0056] 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.

[0057] Furthermore, each of these may have 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. That is, examples of the substituents include optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, and the like.

[0058] The compounds described in the present invention can be their salts or solvates. The salts of the compounds described in the present invention include, for example, hydrochloride, hydrobromide, hydroiodide, phosphate, phosphonate, sulfate, sulfonate such as methanesulfonate and p-toluenesulfonate, carboxylate such as acetate, citrate, malate, tartrate, succinate, salicylate, or alkali metal salt such as sodium salt and potassium salt, alkaline earth metal salt such as magnesium salt and calcium salt, ammonium salt such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, tetraalkylammonium salt, and the like. These salts are produced, for example, by contacting the compound with an acid or base. The solvate of the compound described in the present invention refers to a phenomenon in which solute molecules strongly attract solvent molecules in a solution to form a molecular group, and if the solvent is water, it is called a hydrate. The compounds according to the invention may be solvated with a single or multiple solvents selected from organic solvents such as alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), dimethylformamide, or diglyme, or water.

[0059] The term "amino acid" as used herein includes natural amino acids and non-natural amino acids (sometimes referred to as amino acid derivatives). The term "natural amino acid" as used herein refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Non-natural amino acids (amino acid derivatives) are not particularly limited, but examples thereof include β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains are different from those of natural amino acids, and hydroxycarboxylic acids. As used herein, any configuration is acceptable for the amino acid, but L-amino acids are preferred. There are no particular limitations on the selection of the side chain of the amino acid, and the side chain 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 them may have a substituent, and the substituents are not limited, and may be independently selected from any substituents including, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. That is, examples of the substituents include an optionally substituted alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, and the like, or an oxo, an aminocarbonyl, a halogen atom, and the like. In a non-limiting embodiment, the amino acid in the present specification 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).

[0060] 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 as a substituent. More specific examples of the substituent include a fluoroalkyl, a difluoroalkyl, a trifluoroalkyl, and the like.

[0061] Examples of the substituent containing an O atom include hydroxyl (-OH), oxy (-OR), carbonyl (-C=OR), carboxy (-COH), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C=O-SH), and carboxylcarbonyl (-C=O-CO2H).

[0062] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As the alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among them, methoxy and ethoxy are preferred.

[0063] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0064] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0065] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0066] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0067] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0068] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl (e.g., C1-C6 or C1-C4 alkylaminocarbonyl, particularly ethylaminocarbonyl, methylaminocarbonyl, etc.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, examples include groups in which the H atom bonded to the N atom in -C=O-NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0069] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-C=OR may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0070] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, there are also 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.

[0071] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-SO2-R may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0072] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, the H atom bonded to the N atom in -SO2-NHR may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0073] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0074] Substituents containing an S atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-SO2-R), sulfo (-SO3H), and pentafluorosulfanyl (-SF5).

[0075] Examples of thio (-SR) are selected from among alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0076] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

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

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

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

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

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

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

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

[0084] Examples of the substituent containing a B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or may form a ring. Specific examples include cyclic boryl groups, and more specific examples include pinacolatoboryl, neopentanediolateboryl, and catecholateboryl groups.

[0085] Specific examples of the substituent on the nitrogen atom of the N-substituted amino acid in the present specification include alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, C7-C 14 Examples include aralkyl, benzyl, and phenethyl.

[0086] The main chain amino group of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR, where R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group that may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline). Such amino acids in which the hydrogen atom of the main chain amino group is substituted may be referred to as "N-substituted amino acids" in the present specification. As used herein, the "N-substituted amino acids" are preferably N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, N-C2-C6 alkenyl amino acids, N-allyl amino acids, N-C7-C8 alkyl amino acids, N-methyl amino acids, N-C8-C9 alkenyl amino acids, N-allyl amino acids, N-C9-C10 alkyl amino acids, N-methyl amino acids, N-C1-C20 alkyl amino acids, N-methyl amino acids, N-C1-C3 alkyl amino acids, N-methyl amino acids, N-C2-C3 alkenyl amino acids, N-aryl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, N-C1-C20 alkenyl amino acids, N-aryl amino acids, N-C1-C3 ...3 alkyl amino acids, N-methyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, N-C1-C20 alkenyl amino acids, N-aryl amino acids, N-C1-C3 alkyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids, N-methyl amino acids 14 Examples include, but are not limited to, aralkyl amino acids, N-benzyl amino acids, and N-phenethyl amino acids.

[0087] In this specification, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is an isotope in which at least one atom is 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 the "amino acid" in this specification include a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, a sulfur atom, a fluorine atom, and a chlorine atom, each of which is represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Cl etc. are included.

[0088] (Manufacturing method) In one aspect, the present invention relates to a method for producing a peptide compound, a salt thereof, or a solvate thereof, which has an N-substituted-α,α-disubstituted amino acid residue at the N-terminus and contains a dipeptide residue in which the N-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together, the method comprising steps A and B described below.

[0089] (Process A) Step A is a step of reacting an N-substituted amino acid, a salt thereof, or a solvate thereof, or a peptide compound having an N-substituted amino acid residue at the N-terminus, a salt thereof, or a solvate thereof with an N-non-substituted-α,α-disubstituted amino acid in which the amino group is protected with an electron-withdrawing protecting group, a salt thereof, a dehydrate thereof, or a solvate thereof, in the presence or absence of a condensation reagent, to obtain a peptide compound, a salt thereof, or a solvate thereof, which has an N-non-substituted-α,α-disubstituted amino acid residue at the N-terminus and contains a dipeptide residue in which the N-non-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together. In this specification, "a peptide compound contains a dipeptide" means that the dipeptide is contained in the amino acid sequence constituting the peptide compound.

[0090] In one embodiment, the "N-substituted amino acid" used in step A is any natural or unnatural amino acid in which the amino group in the main chain is -NHR, where R is any group other than hydrogen. Specific examples of R include optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, and optionally substituted cycloalkyl, and R may form a ring with the carbon chain bonded to the N atom and the carbon atom at the α-position, as in proline, and the ring may be further substituted with any substituent. The N-substituted amino acid may also be in the form of a salt or a solvate.

[0091] In one embodiment, the "peptide compound having an N-substituted amino acid residue at its N-terminus" used in step A is not limited in type or number of other amino acids contained in the peptide compound, so long as it has the N-substituted amino acid residue at its N-terminus. The peptide compound may be in the form of a salt or a solvate.

[0092] The N-substituted amino acid or peptide compound having an N-substituted amino acid residue at the N-terminus used in step A may be purchased from a commercial supplier or may be prepared by modifying a product purchased from a commercial supplier.

[0093] Specific examples of such N-substituted amino acids or peptide compounds having an N-substituted amino acid residue at the N-terminus include those represented by the following formula (2): [ka] [In the formula, P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is aralkyl, R3 is hydroxy, O-PG2, any amino acid residue, or any peptide residue; PG2 is a protecting group for the carboxyl group. or a salt thereof, or a solvate thereof.

[0094] In one embodiment, the "N-unsubstituted-α,α-disubstituted amino acid whose amino group is protected by an electron-withdrawing protecting group" used in step A means an amino acid having two optional substituents other than hydrogen on the α-carbon of the amino acid, an amino group in the main chain of the amino acid being unsubstituted, and the amino group being protected by an electron-withdrawing protecting group (i.e., "protecting group -NH-"). The amino acid may be in the form of a salt or a solvate. The two substituents bonded to the α-carbon may be the same or different. Specific examples of the substituent include optionally substituted alkyl, optionally substituted alkoxyalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted cycloalkyl, and optionally substituted cycloalkylalkyl. In addition, the two substituents bonded to the α-carbon may form an optionally substituted alicyclic ring or an optionally substituted heterocyclic ring together with the carbon atom to which they are bonded.

[0095] The N-unsubstituted-α,α-disubstituted amino acids in which the amino group is protected with an electron-withdrawing protecting group used in step A may be purchased from commercial suppliers or may be prepared by modification of those purchased from commercial suppliers.

[0096] In one embodiment, the reaction in step A can be carried out in the presence of a condensation reagent. On the other hand, in step A, for example, when a dehydrate of an N-unsubstituted-α,α-disubstituted amino acid is used, the reaction may be carried out in the absence of a condensation reagent as long as the condensation reaction proceeds.

[0097] Specific examples of N-unsubstituted α,α-disubstituted amino acids in which the amino group is protected with such an electron-withdrawing protecting group include those represented by the following formula (3): [ka] [In the formula, PG1 is an electron-withdrawing protecting group, R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or optionally substituted C7-C 14 aralkyl; R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring. or a salt thereof, or a solvate thereof.

[0098] In one embodiment, the electron-withdrawing protecting group bonded to the N-unsubstituted-α,α-disubstituted amino acid is a protecting group that provides a pKa (in water) of 6 to 11 for the NH group to which the protecting group is bonded, and preferably a protecting group that provides a pKa (in water) of 8 to 11 for the NH group. Specific examples of such protecting groups include C2-C6 haloacyls, more specifically, trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, and 3,3,3-trifluoro-2-(trifluoromethyl)propionyl.

[0099] In one embodiment, the peptide compound obtained by step A has an N-unsubstituted α,α-disubstituted amino acid residue at its N-terminus and contains a dipeptide residue in which the N-unsubstituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together, and specifically has a structure represented by the following formula (4): [ka] [In the formula, PG1, R1, and Q1 are defined the same as PG1, R1, and Q1 in formula (3), respectively. P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2), respectively. or a salt thereof, or a solvate thereof.

[0100] (Process B) Step B is a step of introducing a substituent into the amino group of the N-unsubstituted-α,α-disubstituted amino acid residue, the amino group of which is protected with an electron-withdrawing protecting group at the N-terminus of the peptide compound obtained by step A, in the presence of a base and a substituent-introducing agent, to obtain a peptide compound, a salt thereof, or a solvate thereof, which has an N-substituted-α,α-disubstituted amino acid residue, the amino group of which is protected with an electron-withdrawing protecting group at the N-terminus, and which contains a dipeptide residue in which the N-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together.

[0101] Specific examples of the substituent introduced in this step include optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aralkyl, and optionally substituted cycloalkyl.

[0102] In one embodiment, the base used in step B is preferably one whose conjugate acid has a pKa (in acetonitrile) of 23 to 30. Specific examples of such bases include bases having an amidine skeleton, bases having a guanidine skeleton, and bases having a phosphazene skeleton, which will be described later.

[0103] In one embodiment, the substituent-introducing agent used in step B is used to introduce a substituent into the amino group (i.e., "protecting group -NH-") of an N-unsubstituted-α,α-disubstituted amino acid residue in which the amino group is protected by an electron-withdrawing protecting group at the N-terminus. An electrophilic reagent can be used as the substituent-introducing agent. Specifically, a compound in which the substituent to be introduced is bonded to a leaving group (e.g., a halogen, a sulfonic acid group such as a trifluoromethanesulfonyl group, a methanesulfonyl group, or a tosyl group, or a phosphate group) can be used.

[0104] In one embodiment, the peptide compound has an N-substituted α,α-disubstituted amino acid residue at its N-terminus, the amino group of which is protected with an electron-withdrawing protecting group obtained by step B, and contains a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked together, specifically, a peptide compound represented by the following formula (1): [ka] [In the formula, P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, PG1, R1, and Q1 are defined the same as PG1, R1, and Q1 in formula (3), respectively. P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2), respectively. or a salt thereof, or a solvate thereof.

[0105] The "peptide compound having an N-substituted-α,α-disubstituted amino acid residue at the N-terminus and containing a dipeptide residue in which the N-substituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked" produced by the method of the present invention may have an N-terminal amino group protected with a protecting group, or may have a free amino group (NHR-) after the protecting group has been removed. When the N-terminal amino group is protected with a protecting group, the protecting group may be an electron-withdrawing protecting group originating from the "N-unsubstituted-α,α-disubstituted amino acid whose amino group is protected with an electron-withdrawing protecting group" used in step A, or may be another protecting group (e.g., Fmoc group) introduced after the electron-withdrawing protecting group is removed. In addition to step A and step B, the present invention can include a step of removing the electron-withdrawing protecting group and a step of introducing any protecting group other than the protecting group. The protective group can be removed, for example, by the method described in Greene's, "Protective Groups in Organic Synthesis" (5th Edition, John Wiley & Sons 2014).

[0106] In one aspect, the present invention relates to a method for producing a peptide compound, a salt thereof, or a solvate thereof, which comprises a structure in which two amino acid residues represented by formula (1) are linked, the method comprising steps A and B as shown in the following scheme. [ka]

[0107] In each of the above formulas, PG1 is a protecting group for an amino group, and a protecting group that makes the pKa of the NH group to which PG1 is bonded in formula (4) 11 or less is preferably used. When the pKa of the NH group to which PG1 is bonded is 11 or less, preferably 6 to 11, more preferably 8 to 11, it is possible to selectively introduce a P1 group into the NH group of formula (4) to which PG1 is bonded. For pKa, a calculated value using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C)1994-2019 ACD / Labs) can be used. For example, the pka of the NH group of tert-butyl (2,2,2-trifluoroacetyl) alaninate in which trifluoroacetyl is bonded to a nitrogen atom is 9.71, and the pka of the NH group of tert-butyl 2-methyl-2-(2,2,2-trifluoroacetamido)propanoate is 9.21. In addition, the pka of the NH group of methyl 2-methyl-2-(2,2,3,3,3-pentafluoropropanamido)propanoate in which pentafluoropropionyl is bonded to the nitrogen atom is 9.27, and the pka of the NH group of methyl 2-methyl-2-(2,2,2-trichloroacetamido)propanoate in which trichloroacetyl is bonded to the nitrogen atom is 9.72. On the other hand, the pka of the NH group of methyl 2-acetamido-2-methylpropanoate in which an acetyl group, which has a weaker electron-withdrawing power than these haloacyl groups, is bonded to the nitrogen atom is 14.36, and the acidity of the NH group is weaker than that of the haloacyl group. In the present invention, PG1 is preferably an electron-withdrawing protecting group that increases the acidity of the proton of the NH group, and an example of such a protecting group is C2-C6 haloacyl. Preferred C2-C6 haloacyl includes trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, 3,3,3-trifluoro-2-(trifluoromethyl)propionyl, and the like.

[0108] In formula (1), P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14When P1 is a C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, n-propyl, or i-propyl; when P1 is a C1-C6 alkenyl, the C1-C6 alkenyl is preferably allyl; when P1 is a C7-C 14 If aralkyl, C7-C 14 The aralkyl is preferably benzyl or phenethyl.

[0109] In each of the above formulas, R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or optionally substituted C7-C 14 aralkyl; R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring.

[0110] When R1 and / or Q1 are C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, i-propyl, or 2-methylpropyl. When R1 and / or Q1 are C2-C6 alkenyl, the C2-C6 alkenyl is preferably allyl. When R1 and / or Q1 are C1-C6 alkoxy C1-C6 alkyl, the C1-C6 alkoxy C1-C6 alkyl is preferably methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, or 2-ethoxyethyl. When R1 and / or Q1 are C3-C8 cycloalkyl C1-C6 alkyl, the C3-C8 cycloalkyl C1-C6 alkyl is preferably cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl. 14 If aralkyl, C7-C 14 The aralkyl is preferably benzyl or phenethyl, and C7-C 14 Preferred substituents for the aryl of the aralkyl are one or more groups selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, and cyano.

[0111] When R1 and Q1, together with the carbon atom to which they are bonded, form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring, the 3- to 8-membered alicyclic ring is preferably a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, or a cyclohexane ring, and the 4- to 7-membered saturated heterocyclic ring is preferably a tetrahydropyran ring.

[0112] In each of the above formulas, P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14When P2 is a C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, n-propyl, or i-propyl; when P2 is a C1-C6 alkenyl, the C1-C6 alkenyl is preferably allyl; when P2 is a C7-C 14 If aralkyl, C7-C 14 The aralkyl is preferably benzyl or phenethyl.

[0113] In each of the above, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is aralkyl.

[0114] Preferred examples of R2 include C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C4 hydroxyalkyl, methylsulfonylC1-C2 alkyl, C2-C3 alkynyl, C1-C4 alkoxyC1-C2 alkyl optionally substituted by one or more fluorines, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C2 alkyl, C3-C6 cycloalkoxyC1-C2 alkyl, benzyl, and phenethyl.

[0115] Specific examples of R2 include methyl, ethyl, n-propyl, i-propyl, 1-methylpropyl, 2-methylpropyl, n-butyl, 2-methylbutyl, 3-methylbutyl, n-pentyl, propargyl, 3,3-difluorobutyl, 5,5-difluoropentyl, methoxymethyl, 1-methoxyethyl, 2-methoxyethyl, n-propoxymethyl, 1-hydroxyethyl, cyclopropoxymethyl, cyclobutoxymethyl, (2,2,2-trifluoroethoxy)methyl, 2-methylsulfonylethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, benzyl, and phenethyl.

[0116] In each of the above formulas, R3 is hydroxy, O-PG2, any amino acid residue, or any peptide residue, where PG2 is a protecting group for a carboxyl group. When R3 is O-PG2, specific examples of PG2 include alkyl such as t-butyl, trityl, cumyl, allyl, and benzyl. When R3 is any amino acid residue or any peptide residue, the amino acid residue or peptide residue may be supported on a resin for solid-phase synthesis. When a peptide residue is supported on a resin for solid-phase synthesis, the resin may be supported on the amino acid residue at the C-terminus of the peptide residue, or on an amino acid residue at any other position. As the resin for solid-phase synthesis, CTC resin, Wang resin, or SASRIN resin is preferable, and CTC resin is more preferable. When R3 is any peptide residue, the peptide residue is composed of any type and number of amino acid residues. The number of amino acid residues constituting the peptide residue is preferably 2 to 13, more preferably 2 to 9.

[0117] In formula (1), the following formula: [ka] Specific examples of amino acid residues represented by the formula (I) include MeAib, MecLeu, Me(Me)Phe, Me(Me)Abu, Me(Me)Leu, Me(Me)Ser(Me), Me(Me)Phe, Me(Me)Cha, Me(Me)Val, EtAib, nPrAib, AllylAib, and BnAib.

[0118] In formula (1), the following formula: [ka] Specific examples of amino acid residues represented by the formula (I) include MeAla, MeLeu, MeCha, MeVal, MeAla(cPent), MeAla(cBu), MeAla(cPr), MeChg, MeGly(cPent), MeGly(cBu), MeGly(cPr), MeAbu, MeNva, MeNle, MeNva(5-F2), MeHle, MeIle, MeSer(nPr), MeSer(cPr), MeHnl, MeHnl(7-F2), MePRA, MeSer(Me), MeThr, MeSer(cBu), MeSer(Tfe), MeThr(Me), MeHse(Me), MeMet(O2), EtVal, and nPrVal.

[0119] In the formula (1), when R3 is any amino acid residue, specific examples of the amino acid residue include MeSer(tBuOH), bAla, bMeAla, MeGly, MePhe, MePhe(3-F), MePhe(4-F), D-MePhe, 2-ACHxC, 2-ACPnC, 3-CF3-bAla, Asp-mor, Asp-mor(26-bicyc), Asp-mor(SO2), Asp-NMe 2, Asp-oxz, Asp-pip, Asp-pip(345-F6), Asp-pip(4-Me), Asp-pip-tBu, Asp-piz(oxe), Asp-pyrro, Asp-pyrro( 34-F4), Asp-pyrro(3-Me2), D-(Propargyl)Gly-(C#CH2), D-3-Abu, D-3-MeAbu, D-Gly(Allyl)-(C#CH2), D-Hph -(C#CH2), D-Leu-(C#CH2), D-MeAsp-pyrro, D-MeLeu-(C#CH2), D-Pic(2)-(C#CH2), D-Pro-(C#CH2), D-Ser(iPe n)-(C#CH2), D-Ser(NtBu-Aca)-(C#CH2), EtAsp-pip, MeAsp-aze, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-mo r(SO2), MeAsp-NMe2, MeAsp-oxz, MeAsp-pip, MeAsp-pip(345-F6), MeAsp-pip(3-F2), MeAsp-pip(4-F2), MeAsp -pip(4-Me), MeAsp-piz(oxe), MeAsp-pyrro, MeAsp-pyrro(34-F4), MeAsp-pyrro(3-Me2), and nPrAsp-pip.

[0120] Step A is a step of reacting a compound represented by formula (2), a salt thereof, or a solvate thereof with a compound represented by formula (3), a salt thereof, a dehydrated product thereof, or a solvate thereof with a condensing reagent to obtain a compound represented by formula (4), a salt thereof, or a solvate thereof; or a step of reacting a compound represented by formula (2), a salt thereof, or a solvate thereof with a dehydrated product of a compound represented by formula (3) (i.e., a compound represented by formula (3')), a salt thereof, or a solvate thereof in the absence of a condensing reagent to obtain a compound represented by formula (4), a salt thereof, or a solvate thereof.

[0121] The compound represented by the following formula (2) can be purchased from a commercial supplier, or can be modified as necessary after purchase from a commercial supplier. Specifically, for example, the compound represented by formula (2) can be produced by introducing P2 into the compound purchased from a commercial supplier. [ka] P2, R2, and R3 in formula (2) have the same definitions as P2, R2, and R3 in formula (1), respectively.

[0122] The compound represented by the following formula (3) can be purchased from a commercial supplier, or can be used by modifying the compound purchased from a commercial supplier as necessary. Specifically, for example, the compound represented by formula (3) can be produced by introducing PG1 into the compound purchased from a commercial supplier in a solvent using a base and a PG1-introducing reagent. Specific examples of the PG1-introducing reagent include ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate, trifluoroacetic anhydride, pentafluoropropionic anhydride, and trichloroacetic anhydride, and specific examples of the base include N,N-diisopropylethylamine, triethylamine, sodium methoxide, and sodium ethoxide. Specific examples of the solvent used when introducing PG1 include, for example, methanol and ethanol when ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate is used as the introduction reagent. In addition, for example, dichloromethane, tetrahydrofuran, and pyridine can be used when trifluoroacetic anhydride, pentafluoropropionic anhydride, and trichloroacetic anhydride are used as the introduction reagent. [ka] In the formula, PG1, Q1, and R1 have the same meanings as PG1, Q1, and R1 in formula (1), respectively.

[0123] Step A can be carried out by applying reaction conditions known in the literature. For example, methods described in the Solid Phase Synthesis Handbook published by Merck Ltd. on May 1, 2002 can be exemplified, and these can be used appropriately depending on the reaction conditions. Condensation reagents used in step A include carbodiimide condensing agents such as DCC (N,N'-dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), and EDCI·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), combinations of carbodiimide condensing agents and additives such as HOAt, HOBt, and oxyma, 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), and the like. Uronium salt-based condensing agents represented by HCTU (O-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate), PyBOP (1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate), PyOxim ([ethylcyano(hydroxyimino)acetato-O 2]tri-1-pyrrolidinylphosphonium hexafluorophosphate), phosphonium salt-based condensation agents, 1-chloro-N,N-2-trimethyl-1-propenylamine (Ghosez reagent), TCFH (chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate), PyCIU (N,N,N',N'-bis(tetramethylene)chloroformamidinium hexafluorophosphate), BTFFH (fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate), TFFH (fluoro-N,N,N',N'-tetramethylamidinium hexafluorophosphate), and the like can be used. Preferably, either DIC or EDCI·HCl, or a combination of DIC and Oxyma.

[0124] In addition, when PG1 is a C2-C6 haloacyl, oxazolone represented by formula (3'), which is a dehydrate of the compound represented by formula (3), can also be used in step A. The oxygen atom and the carbon atom between the oxygen atom and the nitrogen atom constituting the oxazolone ring are derived from the carbonyl group of the C2-C6 haloacyl of PG1, and R4 is a C1-C5 haloalkyl derived from the haloalkyl group of the C2-C6 haloacyl of PG1. Specific examples of reactants for preparing oxazolone include N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and thionyl chloride. [ka]

[0125] In the compound represented by formula (3'), R1 and Q1 have the same meaning as R1 and Q1 in formula (3). R1 ​​and Q1 may form a 3- to 8-membered alicyclic ring together with the carbon atom to which they are bonded, and specific examples of such a 3- to 8-membered alicyclic ring include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, and a cyclohexane ring.

[0126] In the compound represented by formula (3'), R4 is C1-C5 haloalkyl, and specific examples of C1-C5 haloalkyl include trifluoromethyl, trichloromethyl, pentafluoroethyl, 1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl, and 2,2,2-trifluoro-1-(trifluoromethyl)ethyl. Among these, trifluoromethyl is preferred.

[0127] Step B is a step of reacting a compound represented by formula (4), a salt thereof, or a solvate thereof with a P1-introducing reagent to obtain a peptide compound represented by formula (1), a salt thereof, or a solvate thereof.

[0128] In the present invention, a combination of P1X (wherein P1 has the same meaning as P1 in formula (1), and X is a leaving group) and a base can be used as a P1-introducing reagent. In step B, P1X is allowed to act on the compound represented by formula (4) in the presence of a base having a suitable pKa, thereby selectively introducing P1 into the nitrogen atom to which PG1 is bonded.

[0129] Specific examples of P1X include alkyl iodide, alkyl bromide, alkyl trifluoromethanesulfonate, alkyl p-toluenesulfonate, alkenyl iodide, alkenyl bromide, alkenyl trifluoromethanesulfonate, alkenyl p-toluenesulfonate, aralkyl iodide, aralkyl bromide, aralkyl trifluoromethanesulfonate, aralkyl p-toluenesulfonate, etc. When the P1 introduction reagent is a methylation reagent, specific examples of the methylation reagent include methyl iodide, dimethyl sulfate, methyl trifluoromethanesulfonate, methyl p-toluenesulfonate, methyl methanesulfonate, etc. When the P1 introduction reagent is an ethylation reagent, specific examples of the ethylation reagent include ethyl iodide, ethyl bromide, diethyl sulfate, ethyl trifluoromethanesulfonate, ethyl p-toluenesulfonate, ethyl methanesulfonate, etc. When the P1 introduction reagent is an allylation reagent, specific examples of the allylation reagent include allyl chloride, allyl bromide, etc. When the P1 introduction reagent is a benzylation reagent, specific examples of the benzylation reagent include benzyl chloride, benzyl bromide, etc. When the P1 introduction reagent is a phenethylation reagent, specific examples of the phenethylation reagent include (2-iodoethyl)benzene, (2-bromoethyl)benzene, etc.

[0130] When using a combination of P1X and a base as a P1-introducing reagent, the base can have a basicity suitable for introducing P1 to the target nitrogen atom. The basicity of a base is expressed by the pKa of the base's conjugate acid. The pKa of the base's conjugate acid is sometimes called the pKa of the base. Specifically, a base having a pKa sufficient to dehydrogenate the hydrogen of the NH group to which P1 is bonded can be used.

[0131] The pKa of the conjugate acid of the base can be calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((c) 1994-2019 ACD / Labs), Chem. Eur. J. 2002, 8, 1682-1693, J. Org.Chem.2005, 70, 3, 1019-1028, Eur. J. Org. Chem., 2019,40, 6735-6748, or the values ​​listed in the Sigma-Aldrich catalog, etc., as appropriate.

[0132] The pKa varies depending on the solvent. The pKa of the conjugate acids of DBU, DBN, TMGN, MTBD, and BTMG in water are 13.28, 13.42, 12.26, 14.37, and 13.81, respectively. (Calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((c)1994-2019 ACD / Labs))

[0133] On the other hand, the pKa of the conjugate acids of DBU, TMGN, MTBD, P1-tBu, BTPP, and BEMP in acetonitrile is 24.32, 25.1, 25.43, 26.9, 28.4, and 27.6 (Chem. Eur. J. 2002, 8, 1682-1693, values ​​listed in the Sigma-Aldrich catalog). The pKa of the conjugate acid of DBN in acetonitrile is 23.89 (Eur. J. Org. Chem., 2019, 40, 6735-6748).

[0134] [Table 1]

[0135] The pKa value of the conjugate acid of the base in water (calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C) 1994-2019 ACD / Labs)) is approximately 10-14 higher than the pKa value in acetonitrile.

[0136] In the present invention, a protecting group is preferably used such that the pKa (in water) of the NH group to which PG1 is bonded is not more than 11. The pKa (in water) of the NH group to which PG1 is bonded is preferably 6 to 11, and more preferably 8 to 11.

[0137] The pKa of the conjugate acid of the base required to deprotonate the proton of the NH group must be at least 2 or more, preferably 2 to 3, and more preferably 2 to 6, away from the pKa of the NH group.

[0138] Therefore, when the pKa of the NH group to which PG1 is bonded is 6 to 11, the pKa of the conjugate acid of the base used is preferably (1) higher than the pKa of the NH group, (2) furthermore, the pKa is at least 2 or more, preferably 6 or more, and (3) a base having a pKa value that is larger by the converted value of the pKa in acetonitrile from the pKa in water (10 to 14). Specific pKa values ​​(in acetonitrile) of the conjugate acid of the base can be used as the base for this reaction if they are 18 to 31, 22 to 29, 22 to 30, 22 to 31, 23 to 29, 23 to 30, or 23 to 31. The pKa range (in acetonitrile) of the conjugate acid of the base used is preferably 22 to 31.

[0139] In addition, when the pKa (in acetonitrile) of the NH group to which PG1 is bonded is 8 to 11, the base can be used as the base in this reaction if the pKa (in acetonitrile) of the conjugate acid of the base used is 20 to 31, 20 to 30, 20 to 29, 21 to 31, 21 to 30, 21 to 29, 22 to 31, 22 to 30, 22 to 29, 23 to 31, 23 to 30, or 23 to 29. The pKa (in acetonitrile) of the conjugate acid of the base used is preferably in the range of 23 to 30.

[0140] In one embodiment, the base is represented by the following formula B1 having an amidine skeleton. [ka] [In the formula, R and R are each independently a C-C alkyl, or R and R together with the nitrogen atom to which R is bonded and the carbon atom to which R is bonded form a 5- to 8-membered ring; RB2 and RB3 are each independently a C1-C4 alkyl, or RB2 and RB3 together with the nitrogen atom to which RB2 is bonded and the nitrogen atom to which RB3 is bonded and the carbon atom to which the nitrogen atoms are bonded form a 5- to 8-membered ring.

[0141] When RB1 to RB4 are C1-C4 alkyl, preferred examples of the C1-C4 alkyl include methyl and ethyl. When RB1 and RB4 form 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. When RB2 and RB3 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0142] Specific examples of the base represented by formula B1 include 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN). [ka]

[0143] In one embodiment, the base is represented by the following formula B2 having a guanidine skeleton. [ka] [In the formula, RB6 is hydrogen or C1-C4 alkyl; RB5 and RB7 are each independently C1-C4 alkyl or form a 5-8 membered ring together with the nitrogen atom to which they are bonded and the carbon atom to which each nitrogen atom is bonded; RB8 is C1-C4 alkyl and RB9 is C1-C4 alkyl or phenyl, or RB8 and RB9 together with the nitrogen atoms to which they are bonded and the carbon atoms to which they are bonded form a 5- to 8-membered ring; When RB9 is phenyl, two B2 may be fused with two benzene rings of the phenyl group to form a naphthalene.

[0144] When RB5 to RB8 are C1-C4 alkyl, the C1-C4 alkyl is preferably methyl, and when RB9 is C1-C4 alkyl, the C1-C4 alkyl is preferably t-butyl. When RB5 and RB7 form a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include an imidazolidine ring, a hexahydropyrimidine ring, and a 1,3-diazepane ring. When RB8 and RB9 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring.

[0145] Specific examples of the base represented by formula B2 include 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD). [ka]

[0146] In one embodiment, the base is represented by the following formula B3 having a phosphazene skeleton. [ka] [In the formula, R.B. 10 is C1-C4 alkyl, or RB 10 and R.B. 11 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 11 RB 10 and R.B. 11 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 12 RB 11 and R.B. 12 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 12 and R.B. 13 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 13 RB 12 and R.B. 13 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 14 RB 13 and R.B. 14 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 14 and R.B. 15 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 15 RB 14 and R.B. 15 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 16is hydrogen, C1-C8 alkyl, or C6-C 10 It is aryl.

[0147] R.B. 10 ~RB 15 When R is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl. 16 When is C1-C8 alkyl, the C1-C8 alkyl is preferably t-butyl or t-octyl. R.B. 10 and R.B. 11、 R.B. 12 and R.B. 13 , and / or R.B. 14 and R.B. 15 When forms a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include a pyrrolidine ring, a piperidine ring, and an azepane ring. R.B. 11 and R.B. 12 , and / or R.B. 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.

[0148] Specific examples of the base represented by formula B3 include tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu), tert-octylimino-tris(dimethylamino)phosphorane (P1-t-Oct), tert-butylimino-tris(pyrrolidino)phosphorane (P1-t-Bu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP), and imino-tris(dimethylamino)phosphorane (HP1(dma)). [ka]

[0149] In one embodiment, the base is represented by the following B4 having a phosphazene skeleton containing two phosphorus atoms connected via a nitrogen atom. [ka] [In the formula, R.B. 17 are independently C1-C4 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, R.B. 18 RB 17 and R.B. 18 is C1-C4 alkyl, except when R forms a 5-8 membered ring, 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 atom to which they are bonded, R.B. 19 RB 18 and R.B. 19 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 19 and R.B. 20 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 20 RB 19 and R.B. 20 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 21 is C1-C4 alkyl, or RB 21 and R.B. 22 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 22 RB 21 and R.B. 22 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 22 and R.B. 23form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atom to which they are bonded, R.B. 23 RB 22 and R.B. 23 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 23 and R.B. 24 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 24 RB 23 and R.B. 24 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 24 and R.B. 25 form a 5- to 8-membered ring together with the nitrogen atoms to which they are bonded and the phosphorus atom to which they are bonded, R.B. 25 RB 24 and R.B. 25 is C1-C4 alkyl, except when R forms a 5-8 membered ring, or R 25 and R.B. 26 form a 5- to 8-membered ring together with the nitrogen atom to which they are attached, R.B. 26 RB 25 and R.B. 26 is C1-C4 alkyl, except when it forms a 5-8 membered ring; R.B. 27 is C1-C4 alkyl, or C6-C 10 It is aryl.

[0150] R.B. 17 ~RB 26 When R is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl. 27 When is C1-C4 alkyl, the C1-C4 alkyl is preferably t-butyl. R.B. 17 and R.B. 18、 R.B. 19 and R.B. 20 , R.B. 21 and R.B.22、 R.B. 23 and R.B. 24、 R.B. 25 and R.B. 26 When forms a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include a pyrrolidine ring, a piperidine ring, and an azepane ring. R.B. 17 and R.B. 18 When both are C1-C4 alkyl, R 19 and R.B. 20 Preferably, both are C1-C4 alkyl, and R 17 and R.B. 18 forms a 5-8 membered ring, RB 19 and R.B. 20 It is preferable that the ring also forms a 5- to 8-membered ring. R.B. 21 and R.B. 22 When both are C1-C4 alkyl, R 23 and R.B. 24 and R.B. 25 and R.B. 26 Preferably, both are C1-C4 alkyl, and R 21 and R.B. 22 forms a 5-8 membered ring, RB 23 and R.B. 24 and R.B. 25 and R.B. 26 It is preferable that the ring also forms a 5- to 8-membered ring. R.B. 18 and R.B. 19 , and / or R.B. 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.

[0151] Specific examples of the base represented by formula B4 include 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5-Catenazi(phosphazene)(P2-t-Bu), 1-ethyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 , 4λ 5 -catenazi(phosphazene) (P2-Et) and the like. [ka]

[0152] In the present invention, when a combination of P1X and a base is used as a P1-introducing reagent, examples of the solvent used in the reaction include amide-based solvents such as DMF and NMP, urea-based solvents such as DMI, ether-based solvents such as tetrahydrofuran and 2-methyltetrahydrofuran, and acetonitrile, and among these, amide-based solvents are preferred.

[0153] When P1X and a base are used as the P1 introduction reagent, a combination of PG1 and a base in which the pKa (in water) of the amino group protected by PG1 is 6 to 11 and the pKa (in acetonitrile) of the conjugate acid of the base is 23 to 30 is preferred. A combination of PG1 and a base in which the pKa (in water) of the amino group protected by PG1 is 8 to 11 and the pKa (in acetonitrile) of the conjugate acid of the base is 23 to 27 is more preferred. As a combination of PG1, P1X, and a base, it is preferred that PG1 is trifluoroacetyl, P1X is methyl iodide, dimethyl sulfate, ethyl iodide, allyl bromide, n-propyl iodide, benzyl bromide, and the base is P1-tBu, TMGN, or MTBD. Specific combinations of PG1 and a base include trifluoroacetyl and TMGN, trifluoroacetyl and P1-tBu, and trifluoroacetyl and MTBD.

[0154] In one aspect, the present invention relates to a method for producing a peptide compound, a salt thereof, or a solvate thereof comprising a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue is linked to an N-substituted amino acid residue, comprising the above-mentioned method for producing a peptide compound, a salt thereof, or a solvate thereof, comprising a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue is linked to an N-substituted amino acid residue, the method comprising the step of further condensing one or more amino acid residues and / or peptide residues to the N-terminus and / or C-terminus of the peptide compound comprising a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue is linked to an N-substituted amino acid residue, the dipeptide residue being produced by the method described herein. The peptide compound produced by this method is any peptide compound containing a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked, and this includes peptide compounds in which any number and type of amino acids are linked to the N-terminal and C-terminal sides of the dipeptide residue.

[0155] In one embodiment, the present invention also relates to a method for producing a cyclic peptide compound, which further comprises the steps of deprotecting the N-terminal protecting group (e.g., PG1) from the peptide compound represented by formula (1), a salt thereof, or a solvate thereof produced by the method of the present invention, optionally extending the peptide chain, and cyclizing the group on the C-terminus and the group on the N-terminus to form a cyclic moiety. The cyclic peptide compound contains 8 to 15 amino acid residues, preferably 10 to 13 amino acid residues, at least 3, preferably at least 3 to (the number of amino acid residues constituting the cyclic peptide compound minus 1) N-substituted amino acid residues, and at least 1, preferably at least 3, non-N-substituted amino acid residues, and the cyclic portion contains at least 8 amino acid residues, preferably at least 10 amino acid residues.

[0156] The step of deprotecting PG1 from the peptide compound represented by formula (1) can be carried out, for example, by the method described in Greene's, "Protective Groups in Organic Synthesis" (5th ed., John Wiley & Sons 2014).

[0157] The step of extending the peptide chain and the step of forming the cyclic portion can be carried out by known methods such as those described in WO2013 / 100132 or WO2018 / 225864. When the peptide chain is extended by solid-phase synthesis, a step of cleavage from the resin may be included before the step of extending the peptide chain and the step of forming the cyclic portion.

[0158] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0159] The present invention will be further described in the following examples, comparative examples and reference examples, but is not limited thereto. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods. The LCMS analysis conditions are listed in Table 2.

[0160] [Table 2]

[0161] Example 1: Preparation of amino acids and resin-supported peptides used in this example Example 1-1: Fmoc-amino acids used in peptide synthesis by a peptide synthesizer In the peptide synthesis described herein, the Fmoc-amino acids shown in Tables 3 to 5 were used in the synthesis by a peptide synthesizer. The Fmoc-amino acids listed in Tables 3 and 5 were purchased from commercial suppliers. The Fmoc-amino acids shown in Table 4 were synthesized according to the scheme shown below.

[0162] [Table 3] JPEG2025061589000035.jpg197149

[0163] [Table 4]

[0164] [Table 5]

[0165] Example 1-1-1: Synthesis of compound AA2-001, (2S)-4-[3-chloro-4-(trifluoromethyl)phenyl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid, (Fmoc-Hph(4-CF3-3-Cl)-OH) [ka]

[0166] To a solution of (4S)-4-[(2-methylpropan-2-yl)oxycarbonylamino]-5-oxo-5-phenylmethoxypentanoic acid (Boc-Glu-OBn, CAS number 30924-93-7) (200 g, 592.82 mmol), N-hydroxyphthalimide (106 g, 649.78 mmol, 1.10 equivalents), and DMAP (3.6 g, 29.47 mmol, 0.05 equivalents) in THF (2 L), DIC (138 mL, 1.54 equivalents) was added dropwise at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 25°C for 16 hours, the solid matter was removed by filtration, and the filtrate was evaporated under reduced pressure. The residue was diluted with toluene, the resulting solid matter was removed by filtration, and the filtrate was evaporated under reduced pressure. The residue was purified by recrystallization (acetone / heptane) to obtain compound AA2-001-a (1-O-benzyl 5-O-(1,3-dioxoisoindol-2-yl) (2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanedioate). (230g, 80%) LCMS(ESI)m / z=505.2(M+Na)+ Retention time: 0.992 minutes (Analysis conditions SMDmethod_16)

[0167] Nickel bromide trihydrate (NiBr2·3H2O) (4 g, 0.07 equiv.) and 4,4'-di-tert-butyl-2,2'-bipyridyl (dtbbpy) (3.9 g, 14.55 mmol, 0.07 equiv.) were added to DMA (500 mL) and stirred at 50 °C for 2 h under a nitrogen atmosphere to prepare a Ni solution.

[0168] The Ni solution prepared above was added to a mixture of compound AA2-001-a (100 g, 207.3 mmol), zinc powder (70 g, 5 equivalents), and 4-bromo-2-chloro-1-(trifluoromethyl)benzene (160 g, 617 mmol, 3 equivalents) in DMA (500 mL), and the mixture was stirred at 25°C for 16 hours. An aqueous solution of EDTA·2Na (10%) was added to the reaction solution, and the mixture was extracted with ethyl acetate. The combined organic layer was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain compound AA2-001-b. (75 g, 77%) LCMS(ESI)m / z=494(M+Na)+ Retention time: 2.863 minutes (Analysis conditions SMDmethod_17)

[0169] A toluene solution (900 mL) of compound AA2-001-b (75 g, 158.93 mmol) was cooled to 0° C., and trifluoromethanesulfonic acid (TfOH) (42 mL, 3.00 equivalents) was added dropwise. After stirring at room temperature for 1 hour, water (75 mL) was added. This mixture was extracted with water, and the combined aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Acetonitrile / water (900 / 900 mL) was added to the residue, and the pH was adjusted to 7 with an aqueous sodium hydroxide solution (48%). Fmoc-OSu (51.2 g, 151.93 mmol, 0.95 equivalents) was added to this solution, and the mixture was stirred at room temperature for 16 hours while maintaining the pH at 7.8 to 8.0. The reaction solution was filtered, and the pH of the filtrate was adjusted to 2 with 6 mol / L aqueous hydrochloric acid. The precipitated solid was collected and dried at 50° C. to obtain compound AA2-001 ((2S)-4-[3-chloro-4-(trifluoromethyl)phenyl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid, Fmoc-Hph(4-CF3-3-Cl)-OH). (70 g, 87%) LCMS(ESI)m / z=525.8(M+Na)+ Retention time: 2.180 minutes (Analysis conditions SMDmethod_21) 1H-NMR(300MHz,DMSO-d6)δ12.70(s,1H),7.91(d,J=7.5Hz,2H),7.79-7.59(m,5H),7.45 -7.28(m,5H),4.40-4.19(m,3H),3.96-3.88(m,1H),2.82-2.60(m,2H),2.11-1.77(m,2H)

[0170] Example 1-1-2: Synthesis of compound AA2-002, (2S)-3-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid (Fmoc-MeAla(cBu)-OH) [ka]

[0171] To a solution of compound AA2-002-a ((2S)-3-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]propanoic acid, Fmoc-Ala(cBu)-OH) (3.36 g, 9.19 mmol) in DCM (46 mL), paraformaldehyde (0.828 g, 27.6 mmol), anhydrous magnesium sulfate (2.77 g, 22.99 mmol), and boron trifluoride diethyl ether complex (BF3·OEt2) (1.398 mL, 11.03 mmol) were added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous solution of sodium chloride diluted with water to half its concentration was added to the reaction solution, and DCM was further added for dilution. The separated organic layer was washed with a saturated aqueous solution of sodium chloride and filtered. The solvent was removed from the obtained organic layer under reduced pressure to obtain compound AA2-002-b as a crude product (3.63 g). LCMS(ESI)m / z=378(M+H)+ Retention time: 1.01 minutes (Analysis conditions SQDFA05)

[0172] To a solution of the obtained compound AA2-002-b (3.47 g) in DCM (30.6 mL) was added triethylsilane (4.39 mL, 27.6 mmol), water (0.166 g, 9.19 mmol), and boron trifluoride diethyl ether complex (BF3·OEt2) (3.50 mL, 27.6 mmol) under a nitrogen atmosphere, and the mixture was stirred for 2 hours. A saturated aqueous solution of sodium chloride diluted with water to half its concentration was added to the reaction solution, and the mixture was stirred at room temperature for 15 minutes. The organic layer separated from the mixture was distilled off under reduced pressure. The residue obtained was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound AA2-002 ((2S)-3-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid, Fmoc-MeAla(cBu)-OH). (3.18g, 2 steps 91%) LCMS(ESI)m / z=380(M+H)+ Retention time: 0.94 minutes (Analysis conditions SQDFA05)

[0173] Example 1-1-3: Synthesis of compound AA2-003, (2S)-2-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]acetic acid (Fmoc-MeGly(cPent)-OH) [ka]

[0174] Compound AA2-003-a ((2S)-2-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]acetic acid, Fmoc-Gly(cPent)-OH) (30.0 g, 82 mmol), paraformaldehyde (7.39 g, 246 mmol) and CSA (0.954 g, 4.10 mmol) in toluene (160 mL) were mixed, and trifluoroacetic acid (TFA) (9.0 mL) was added, followed by stirring at 60° C. for 4 hours. The reaction solution was cooled to room temperature, and the solid was removed by filtration. The filtrate was diluted with ethyl acetate (220 mL), and then washed with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride in that order. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound AA2-003-b as a crude product. The next reaction was carried out without further purification. LCMS(ESI)m / z=378(M+H)+ Retention time: 1.01 minutes (Analysis conditions SQDFA05)

[0175] The obtained compound AA2-003-b (31 g, 82 mmol) and triethylsilane (TES) (65.5 mL, 410 mmol) were added to a mixture of dichloroethane (DCE) (90 mL) and trifluoroacetic acid (TFA) (76 mL, 984 mmol) and stirred at 60 ° C. for 16 hours. The reaction solution was cooled to room temperature and then concentrated under reduced pressure. The obtained solid was washed with n-hexane / ethyl acetate (95 / 5) and dried under reduced pressure to obtain compound AA2-003 ((2S)-2-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl (methyl) amino] acetic acid, Fmoc-MeGly (cPent)-OH) (29.1 g, 93%). LCMS(ESI)m / z=380(M+H)+ Retention time: 0.92 minutes (Analysis conditions SQDFA05)

[0176] Example 1-1-4: Synthesis of compound AA2-004, (2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]acetic acid (Fmoc-MeGly(cBu)-OH) [ka]

[0177] Compound AA2-004-a (2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]acetic acid, Fmoc-Gly(cBu)-OH) (2.5 g, 7.11 mmol) was used as a starting material, and compound AA2-004-b was obtained as a crude product in the same manner as in the synthesis of compound AA2-002-b. LCMS(ESI)m / z=364(M+H)+ Retention time: 0.97 minutes (Analysis conditions SQDFA05)

[0178] The entire amount of compound AA2-004-b obtained above was reacted in the same manner as in the synthesis of compound AA2-002, and then purified by reverse phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain compound AA2-004 ((2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]acetic acid, Fmoc-MeGly(cBu)-OH). (2.32 g, 89% for 2 steps) LCMS(ESI)m / z=366(M+H)+ Retention time: 0.88 minutes (Analysis conditions SQDFA05)

[0179] Example 1-1-5: Synthesis of compound AA2-005, (2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid (Fmoc-MeAla(cPent)-OH) [ka]

[0180] Compound AA2-005-a ((2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]propanoic acid, Fmoc-Ala(cPent)-OH) (10 g, 26.4 mmol) was used as a starting material, and compound AA2-005-b (10.5 g) was obtained as a crude product in the same manner as in the synthesis of compound AA2-002-b. LCMS(ESI)m / z=392(M+H)+ Retention time: 1.05 minutes (Analysis conditions SQDFA05)

[0181] The obtained compound AA2-005-b (10.5 g) was reacted in the same manner as in the synthesis of compound AA2-002, and then purified by reverse phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain compound AA2-005 ((2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propanoic acid, Fmoc-MeAla(cPent)-OH). (10.11 g, 96% for 2 steps) LCMS(ESI)m / z=394(M+H)+ Retention time: 0.98 minutes (Analysis conditions SQDFA05)

[0182] Example 1-2: Preparation of resin-supported amino acids and peptides used in this example Example 1-2-1: Synthesis of compound 1-2-1, (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro) [ka]

[0183] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin site may be represented by a circle. In addition, in order to clarify the reaction site of the resin site, the chemical structure of the reaction 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 side chain carboxylic acid 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 pyrrolidine. [ka]

[0184] Under a nitrogen atmosphere, EDCI·HCl (67.1 g, 350 mmol), HOBt (43.4 g, 321 mmol), and Fmoc-Asp(OtBu)-OH (120 g, 292 mmol) were added in order to DMF (600 mL) at 0 °C, and the mixture was stirred at 0 °C for 1 hour. Pyrrolidine (26.3 mL, 321 mmol) was slowly added to the reaction solution, and the mixture was stirred at 0 °C for 1.5 hours. Ethyl acetate (10 v) and 0.5 mol / L hydrochloric acid (2 v) were added to the reaction solution at 0 °C, and the organic layer was separated. The obtained organic layer was washed with 0.5 mol / L hydrochloric acid, water, saturated aqueous sodium bicarbonate solution / water (1 / 1 (v / v)), and saturated aqueous sodium chloride solution / water (1 / 1 (v / v)) in that order, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain compound 1-2-1-a as a crude product. (137.1g, quant.) LCMS(ESI)m / z=465(M+H)+ Retention time: 1.05 minutes (Analysis conditions SQD compound AA05)

[0185] 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 conditions SQD compound AA05)

[0186] The reaction of supporting Fmoc amino acid on resin 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 reaction vessel equipped with a filter and shaken at room temperature for 20 minutes. After removing dichloromethane by applying 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 by applying 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 by applying nitrogen pressure, dichloromethane (350 mL) was added and shaken for 5 minutes, and then the reaction solution was removed by applying nitrogen pressure. This washing of the resin with dichloromethane was repeated five times, and the obtained resin was dried overnight under reduced pressure to obtain (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1, 59.79 g).

[0187] 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 solution was diluted with DMF (11.5 mL). The absorbance (294 nm) of the obtained diluted solution was measured (Shimadzu, UV-1600PC (cell length 1.0 cm)). By measuring the dibenzofulvene derived from the Fmoc of the Fmoc amino acid supported on the resin, the loading amount of compound 1-2-1 was calculated to be 0.464 mmol / g. In addition, other lots synthesized in the same manner but with different loading amounts were also used for peptide synthesis, investigations, etc.

[0188] Example 1-2-2: Preparation of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2) [ka]

[0189] The preparation of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) used in this example was carried out by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by Intavis). The detailed operation procedure was as described in the manual attached to the synthesizer.

[0190] The Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) (100 mg per column) prepared in Example 1-2-1, an NMP solution of Fmoc-MeVal-OH (0.6 mol / L) and 1-hydroxy-7-azabenzotriazole (HOAt, 0.375 mol / L), and an N,N-dimethylformamide (DMF) solution of diisopropylcarbodiimide (DIC) (10% v / v) were set in a synthesizer.

[0191] To start the synthesis, 1 mL of dichloromethane (DCM) was added to the Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) (100 mg per column) and left to stand for about 30 minutes to swell the resin. The resin was then washed with DMF.

[0192] Fmoc removal process 0.7 mL of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in DMF (2% v / v) was added to each column and left for 5-10 minutes to remove Fmoc. The resin was then washed with DMF (0.7 mL per column, repeated 4 times).

[0193] Extension process A mixture of the Fmoc-amino acid solution (0.30 mL per column) and DIC / DMF solution (0.36 mL per column) was added to the resin that had undergone the Fmoc removal process, and the resin was left to stand at 40°C. After the reaction was completed, the resin was washed with DMF (0.7 mL per column, repeated four times).

[0194] Fmoc-MeVal was elongated by the above process. After elongation, the product was washed with DCM and dried without carrying out the Fmoc removal step, and then used for the following study. In order to confirm that compound 1-2-2 was obtained, a part of the obtained resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v)). The cleaved solution was analyzed by LCMS, and the production of the target peptide Fmoc-MeVal-Asp-pyrro (compound 1-2-2*) was confirmed. In this example, when an * is attached to the compound number, it indicates a compound that was confirmed by cleaving the peptide from the resin to confirm the reaction. Compound 1-2-2* indicates a peptide compound obtained by cleaving the bond between the carboxylic acid of the peptide contained in compound 1-2-2 and the 2-chlorotrityl group of the resin.

[0195] [ka] LCMS (ESI) m / z = 522.32 (M+H)+ Retention time: 0.76 minutes (Analysis conditions SQDFA05)

[0196] Example 1-2-3: Preparation of Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-3) [ka]

[0197] Similarly to Example 1-2-2, Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) was prepared by extending Fmoc-MePhe-OH.

[0198] In order to confirm that compound 1-2-3 was obtained, a part of the obtained resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v)). The cleaved solution was analyzed by LCMS, and the production of the target peptide Fmoc-MePhe-Asp-pyrro (compound 1-2-3*) was confirmed.

[0199] [ka] LCMS (ESI) m / z = 570.31 (M+H)+ Retention time: 0.80 minutes (Analysis conditions SQDFA05)

[0200] Example 1-2-4: Synthesis of (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, compound 1-2-4) [ka]

[0201] (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butanoic acid (Fmoc-D-3-MeAbu-OH) (11.5 g, 33.9 mmol) purchased from a commercial supplier and 2-chlorotrityl chloride resin (1.69 mmol / g, 100-200 mesh, 1% DVB, 50 g, 84.5 mmol) were used to obtain (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, compound 1-2-4) in the same manner as in the synthesis of compound 1-2-1. (58.95 g, loading 0.343 mmol / g) In addition, other lots synthesized in the same manner but with different loading amounts were also used for the peptide synthesis in this example.

[0202] Example 1-3: Amino acids protected with protecting groups other than Fmoc for use in peptide synthesis, and their dehydrates Amino acids protected with protecting groups other than Fmoc and their dehydrated derivatives used in the peptide synthesis described herein were synthesized as follows.

[0203] Example 1-3-1: Preparation of 2-methyl-2-[(2,2,2-trifluoroacetyl)amino]propanoic acid (Tfa-Aib-OH) (compound 1-3-1) [ka]

[0204] Methanol (242 mL), DIPEA (63.5 mL, 1.5 equivalents), and ethyl trifluoroacetate ((CAS number 383-63-1), 37.6 mL, 1.3 equivalents) were added to 2-amino-2-methylpropanoic acid (25.0 g), and the mixture was stirred at 50°C for 18 hours. The solvent was then removed under reduced pressure, and 1N aqueous hydrochloric acid and ethyl acetate were added to the resulting residue, and the organic layer and the aqueous layer were separated. The resulting organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 18.2 g of crude product.

[0205] The crude product (16.0 g) was dissolved in TBME (80 mL), and heptane (320 mL) was added dropwise over 1 hour while stirring. The mixture was stirred for another hour under ice cooling, and then filtered. The resulting powder was washed with a TBME / heptane solution (1 / 4, 32 mL) and dried under reduced pressure to obtain 13.5 g of 2-methyl-2-[(2,2,2-trifluoroacetyl)amino]propanoic acid (Tfa-Aib-OH) (compound 1-3-1). LCMS (ESI) m / z = 197.93 (MH)- Retention time: 0.40 minutes (Analysis conditions SQDFA05)

[0206] Example 1-3-2. Synthesis of Tfa-(Me)Abu-OH ((S)-2-methyl-2-(2,2,2-trifluoroacetamido)butanoic acid, compound 1-3-2-b) [ka]

[0207] Compound 1-3-2-a ((S)-2-amino-2-methylbutanoic acid, isovaline, H-(Me)Abu-OH) (15.0 g, 128 mmol) was dissolved in methanol (150 mL) and diisopropylethylamine (82.7 g, 640 mmol) and ethyl trifluoroacetate (54.6 g, 384 mmol) were added, and the mixture was stirred at 50 ° C. for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the resulting residue was dissolved in TBME and washed twice with 1N aqueous hydrochloric acid. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (1:7) to obtain compound 1-3-2-b ((S)-2-methyl-2-(2,2,2-trifluoroacetamido)butanoic acid) (12 g, 44%). LCMS(ESI)m / z=214.0(M+H)+ Retention time: 0.32 minutes (Analysis conditions SQDFA05)

[0208] Example 1-3-3. Synthesis of Tfa-(Me)Leu-OH ((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamido)pentanoic acid, compound 1-3-3-b) [ka]

[0209] Compound 1-3-3-a (2-methylleucine, (S)-2-amino-2,4-dimethylpentanoic acid, H-(Me)Leu-OH) (15.0 g, 103 mmol) was added to a methanol (50 mL) solution of diisopropylethylamine (40.1 g, 310 mmol) and ethyl trifluoroacetate (44.0 g, 310 mmol), and then stirred at 50 ° C. for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was dissolved in TBME and washed twice with 1N aqueous hydrochloric acid. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (1:7) to obtain compound 1-3-3-b ((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamido)pentanoic acid) (10 g, 40%). LCMS(ESI)m / z=242.1(M+H)+ Retention time: 0.66 minutes (Analysis conditions SQDFA05)

[0210] Example 1-3-4. Synthesis of Tfa-(Me)Ser(Me)-OH ((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetamido)propanoic acid, compound 1-3-4-b) [ka]

[0211] Compound 1-3-4-a (3-methoxy-2-methyl-L-alanine, (S)-2-amino-3-methoxy-2-methylpropanoic acid, H-(Me)Ser(Me)-OH) (1.5 g, 11 mmol) was added to a methanol (19 mL) solution of diisopropylethylamine (5.9 mL, 34 mmol) and ethyl trifluoroacetate (4.0 mL), and then stirred at 50 ° C. for 21 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was dissolved in TBME (45 mL), and then washed twice with 1N aqueous hydrochloric acid (45 mL) and once with saturated aqueous sodium chloride (45 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-4-b ((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetamido)propanoic acid) (2.07 g, 72%). LCMS(ESI)m / z=228.2(MH)- Retention time: 0.41 minutes (Analysis conditions SQDFA05)

[0212] Example 1-3-5. Synthesis of Tfa-(Me)Phe-OH ((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamido)propanoic acid, compound 1-3-5-b) [ka]

[0213] Compound 1-3-5-a ((2S)-2-amino-2-methyl-3-phenylpropanoic acid, H-(Me)Phe-OH) (10.0 g, 55.8 mmol) was added to a methanol (500 mL) solution of diisopropylethylamine (21.63 g, 167.4 mmol) and ethyl trifluoroacetate (23.78 g, 167.4 mmol), and then stirred at 50 ° C. for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was dissolved in TBME, and then washed twice with 1N aqueous hydrochloric acid and once with saturated aqueous sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (1:15) to obtain compound 1-3-5-b ((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamido)propanoic acid) (8 g, 52%). LCMS(ESI)m / z=274.0(MH)- Retention time: 0.68 minutes (Analysis conditions SQDFA05)

[0214] Example 1-3-6. Synthesis of Tfa-(Me)Cha-OH ((S)-3-cyclohexyl-2-methyl-2-(2,2,2-trifluoroacetamido)propanoic acid, compound 1-3-6-c) [ka]

[0215] 4-(3-phenylpropyl)piperidine (4.7mL, 22mmol) was added to a dichloromethane (18.4mL) solution of compound 1-3-6a (2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-cyclohexyl-2-methylpropanoic acid, Fmoc-(Me)Cha-OH) and stirred at room temperature under a nitrogen atmosphere for 16 hours. Water (8mL) was added to the reaction solution to extract the product, and the aqueous layer was purified by reversed-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). Water (5mL) and 2N hydrochloric acid (5mL) were added to the organic phase, and the remaining crude product was extracted into the aqueous layer, and the aqueous layer was purified by reversed-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). The column purified products were combined to obtain compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropanoic acid, H-(Me)Cha-OH) (1.1 g, 81%), which was used in the next reaction. LCMS(ESI)m / z=186.1(M+H)+ Retention time: 0.32 minutes (Analysis conditions SQDFA05)

[0216] Compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropanoic acid, H-(Me)Cha-OH) (1.1 g, 6.0 mmol) was added to a methanol (20 mL) solution of diisopropylethylamine (3.1 mL, 18 mmol) and ethyl trifluoroacetate (2.1 mL), and then stirred at 50 ° C for 2 hours. After cooling the reaction solution to room temperature, diisopropylethylamine (3.1 mL, 18 mmol) and ethyl trifluoroacetate (2.1 mL) were added, and then stirred at 50 ° C for 20 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in TBME (30 mL), and then washed twice with 1N hydrochloric acid aqueous solution (30 mL) and once with saturated sodium chloride aqueous solution (40 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-6-c ((S)-3-cyclohexyl-2-methyl-2-(2,2,2-trifluoroacetamido)propanoic acid) (1.22 g, 72%). LCMS(ESI)m / z=280.2(MH)- Retention time: 0.75 minutes (Analysis conditions SQDFA05)

[0217] Example 1-3-7. Synthesis of Tfa-(Me)Val-OH ((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid, compound 1-3-7-b) [ka]

[0218] Compound 1-3-7-a ((S)-2-amino-2,3-dimethylbutanoic acid, H-(Me)Val-OH) (2.0 g, 15 mmol) was added to a methanol (25 mL) solution of diisopropylethylamine (8.0 mL, 46 mmol) and ethyl trifluoroacetate (5.5 mL), and then stirred at 50 degrees for 3 hours. After cooling the reaction solution to room temperature, diisopropylethylamine (4.0 mL, 23 mmol) and ethyl trifluoroacetate (2.7 mL) were added, and then stirred at 50 degrees for 16 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in TBME (40 mL), and then washed with 1N aqueous hydrochloric acid solution (40 mL) and saturated aqueous sodium chloride solution (40 mL) in that order. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-7-b ((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid) (1.17 g, 34%). LCMS(ESI)m / z=226.1(MH)- Retention time: 0.54 minutes (Analysis conditions SQDFA05)

[0219] Example 1-3-8. Synthesis of Tfa-cLeu-OH (1-(2,2,2-trifluoroacetamido)cyclopentane-1-carboxylic acid, compound 1-3-8-b) [ka]

[0220] Compound 1-3-8-a (1-aminocyclopentanecarboxylic acid, H-cLeu-OH) (25 g, 194 mmol) was dissolved in methanol (100 mL) and diisopropylethylamine (37.5 g, 290 mmol) and ethyl trifluoroacetate (41.3 g, 290 mmol) were added, and the mixture was stirred at 50 degrees for 2 days. The reaction solution was cooled to room temperature, and diisopropylethylamine (4.0 mL, 23 mmol) and ethyl trifluoroacetate (2.7 mL) were added, and the mixture was stirred at 50 degrees for 16 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in TBME, and then washed with 1N aqueous hydrochloric acid and saturated aqueous sodium chloride in that order. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (3:20) to obtain compound 1-3-8-b (1-(2,2,2-trifluoroacetamido)cyclopentane-1-carboxylic acid) (20 g, 46%). LCMS(ESI)m / z=224.0(MH)- Retention time: 0.49 minutes (Analysis conditions SQDFA05)

[0221] Example 1-3-9. Synthesis of 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (compound 1-3-9) [ka]

[0222] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (27.7 g, 144 mmol) was added to a dichloromethane (225 mL) solution of compound 1-3-8-b (Tfa-cLeu-OH, 1-(2,2,2-trifluoroacetamido)cyclopentane-1-carboxylic acid) (25 g, 111 mmol), and the mixture was stirred at room temperature for 2 days. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain compound 1-3-9 (2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one) (11.9 g, 52%). LCMS(ESI)m / z=208.1(M+H)+ Retention time: 0.86 minutes (Analysis conditions SQDAA05)

[0223] Example 2: An experiment in which an N-substituted α,α-disubstituted amino acid residue was introduced by extending an N-unsubstituted α,α-disubstituted amino acid whose N-terminus was protected by Tfa to an amino acid residue whose N-terminus was N-substituted in a peptide during solid-phase synthesis, and then performing N-functionalization on the solid phase. [ka]

[0224] Example 2-1: Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) was de-Fmoc-enhanced to Tfa-Aib-OH Example 2-1-1: Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) was de-Fmoc-enhanced, followed by elongation of Tfa-Aib-OH using DIC Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 1 hour to swell the resin. After removing the dichloromethane with a filter, the resin was washed three times with DMF (0.7 mL). Next, 2% DBU / DMF solution (Fmoc-removal solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc-removal solution, the resin was washed four times with DMF (0.7 mL). The resulting resin was subjected to an extension reaction of Tfa-Aib-OH.

[0225] The extension reaction was carried out by adding a mixture of 0.6 M Tfa-Aib-OH / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40° C. for 20 hours.

[0226] After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain compound 2-1 (Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) lot 1 (hereinafter referred to as compound 2-1-1).

[0227] To confirm the progress of the reaction, the resulting resin (compound 2-1-1) was subjected to peptide cleavage in a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS to confirm the production of the target peptide Tfa-Aib-MeVal-Asp-pyrro (compound 2-1*). No other peptide components were detected. This resin (compound 2-1-1) was used in Example 2-3.

[0228] [ka] LCMS (ESI) m / z = 481.21 (M+H)+ Retention time: 0.53 minutes (Analysis conditions SQDFA05)

[0229] Example 2-1-2: After removing Fmoc from Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2), elongation of Tfa-Aib-OH with oxyma as an additive using DIC In a reaction vessel equipped with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed, and Tfa-Aib was elongated in the same manner as in Example 2-1-1 except for the elongation reagent. The elongation reagent used was a mixture of 0.6 M Tfa-Aib-OH / 0.375 M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL). As in Example 2-1-1, peptides were excised from the resin and analyzed by LCMS. As a result, the target peptide Tfa-Aib-MeVal-Asp-pyrro (compound 2-1*) was 87.50% (UV area), and unreacted H-MeVal-Asp-pyrro was 2.2% (UV area), and two unidentified peaks (8.85, 1.43% (UV area) respectively) were confirmed. This resin (lot 2 of compound 2-1, hereinafter referred to as compound 2-1-2) was used in Comparative Example 1.

[0230] Example 2-1-3: Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) was de-Fmoc-enhanced to Tfa-Aib-OH using EDCI·HCl Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed in a reaction vessel equipped with a filter, and Tfa-Aib was elongated in the same manner as in Example 2-1-1 except for the elongation reagent. The elongation reagent used was a mixture of 0.6 M Tfa-Aib-OH / NMP solution (0.3 mL) and EDCI·HCl (48 mg, 0.250 mmol) / DMF solution (0.36 mL). As in Example 2-1-1, the peptide was excised from the resin and analyzed by LCMS. As a result, the target peptide Tfa-Aib-MeVal-Asp-pyrro (compound 2-1*) was 93.1% (UV area), and the peaks of unreacted H-MeVal-Asp-pyrro were 3.0% (UV area) and unidentified 3.9% (UV area). This resin (lot 3 of compound 2-1, hereinafter referred to as compound 2-1-3) was used in Examples 2-2 and 2-3.

[0231] Example 2-2: N-methylation by nucleophilic substitution reaction (methyl iodide as methylating agent, DBU as base) at the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1) In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-3) (25 mg) prepared in Example 2-1, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with NMP (0.7 mL).

[0232] To the resin obtained, a DBU (23 μL) / NMP (0.35 mL) solution was added, followed by a methyl iodide (63 μL) / NMP (0.35 mL) solution, and the mixture was shaken at 40°C for 30 minutes. The liquid phase was removed with a filter, and the mixture was washed four times with NMP (0.7 mL) and four times with dichloromethane. A small amount of the resin obtained was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS.

[0233] The same procedure was carried out again on the resin that had been methylated the first time in order to improve the reaction conversion rate. The second methylation was carried out by shaking at 40 degrees for 20 hours. Compound 2-2 was obtained by washing the resin. A small amount of the obtained resin was sampled and the peptide was cleaved using a TFE / DCM solution (1 / 1 (v / v)). The cleaved solution was analyzed by LCMS to confirm the target product (compound 2-2*) and unreacted product (compound 2-1*). The results are shown in Table 6.

[0234] [Table 6]

[0235] [ka] LCMS (ESI) m / z = 495.23 (M+H)+ Retention time: 0.57 minutes (Analysis conditions SQDFA05)

[0236] From these results, it was found that the N-methylation of the Tfa-protected N-terminus was selectively carried out in a nucleophilic substitution reaction using methyl iodide as a methylating agent and DBU (pKa of the conjugate acid in acetonitrile = 24.34 (J. Org. Chem. 2005, 70, 1019-1028)) as a base. It was shown that the conversion rate of the reaction could be improved by replacing the reagents and repeating the reaction.

[0237] Example 2-3: N-methylation by nucleophilic substitution reaction (methyl iodide as methylating agent, various bases) at the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1) In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-1 or 2-1-3) (25 mg) prepared in Example 2-1, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with DMF (0.7 mL).

[0238] To the obtained resin, a base (addition amount is shown in Table 7) / DMF (0.35 mL) solution was added, followed by methyl iodide (63 μL) / DMF (0.35 mL) solution, and the mixture was shaken at 40°C for 15 hours. After removing the liquid phase with a filter, the mixture was washed four times with DMF (0.7 mL) and four times with dichloromethane to obtain compound 2-2. A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS. The results of cutting out and confirming the reaction are shown in Table 7. When P1-tBu was used as the base, a small amount of overmethylated product (compound 2-3*) was also observed.

[0239] [Table 7]

[0240] [ka] LCMS (ESI) m / z = 509.25 (M+H)+ Retention time: 0.59 minutes (Analysis conditions SQDFA05)

[0241] These results indicate that the desired N-methylation proceeds in a nucleophilic substitution reaction using MTBD (pKa of the conjugate acid in acetonitrile = 25.43 (Chem. Eur. J. 2002, 8, 1682-1693)), TMGN (pKa of the conjugate acid in acetonitrile = 25.1 (Chem. Eur. J. 2002, 8, 1682-1693)), and P1-tBu (pKa of the conjugate acid in acetonitrile = 26.9 (Aldrich phosphazene base website https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html (accessed October 10, 2019))), which have stronger basicity than DBU. When P1-tBu was used, a small amount (3.8%) of a product was confirmed in which the secondary amide site (the amide site consisting of the amino group of Asp and the carboxyl group of MeVal) other than the corresponding Tfa amide site was overmethylated. From this result, it is inferred that it is more preferable to use a base with a conjugate acid pKa value of 27 or less in order to selectively N-methylate the Tfa amide site.

[0242] Example 2-4: Deprotection of Tfa-protected Tfa-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-2) after N-alkylation Triglyme (triethylene glycol dimethyl ether) (6.6 mL) was added to sodium borohydride (0.5 g) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 10 minutes to obtain a 2.0 M sodium borohydride / triglyme solution.

[0243] The resin (compound 2-2) N-methylated with TMGN as a base, prepared in run 3 of Example 2-3 and Table 7, was added to a reaction vessel equipped with a filter, and dichloromethane (1 mL) was added and the mixture was shaken at room temperature for 30 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with THF (0.7 mL).

[0244] To the resin obtained, THF (125 μL), methanol (63 μL), and the previously prepared 2.0 M sodium borohydride / triglyme solution (63 μL) were added, and the mixture was shaken at room temperature for 30 minutes. After removing the liquid phase with a filter, the mixture was washed four times with methanol (0.7 mL) (each washing time was 1 minute), followed by washing four times with dichloromethane (0.7 mL) to obtain compound 2-4. A small amount of the resin obtained was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS.

[0245] The Tfa-protected starting peptide Tfa-MeAib-MeVal-Asp-pyrro (compound 2-2*) was completely consumed, and the target peptide H-MeAib-MeVal-Asp-pyrro (compound 2-4*) was observed. The LC chart is shown in Figure 1, and it was confirmed that the synthesis of high purity was possible.

[0246] [ka] Target peptide H-MeAib-MeVal-Asp-pyrro (compound 2-4*) LCMS (ESI) m / z = 399.23 (M+H)+ Retention time: 0.35 minutes (Analysis conditions SQDFA05)

[0247] As described above, it has been demonstrated that the present invention enables the introduction of bulky N-alkylamino acids followed by N-methyl-α,α-dialkylamino acids with high purity. It has also been confirmed that the subsequent Tfa removal step proceeds smoothly, making it possible to carry out conventional peptide elongation from the N-terminus.

[0248] Example 2-5. Experiments on the introduction of bulky N-methyl amino acid (MeVal) followed by various N-methyl-α,α-dialkyl amino acids on a solid phase Compounds 2-5-1-1 to 2-5-7-1 and compounds 2-5-1-2 to 2-5-7-2 were synthesized using various Tfa-amino acids according to the following general scheme. [ka]

[0249] [Table 8] JPEG2025061589000068.jpg204149

[0250] Example 2-5-1. Synthesis of Tfa-Me(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-1-2) Example 2-5-1-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) was de-Fmoc-treated, followed by elongation of Tfa-(Me)Abu-OH. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.552 mmol / g, 100 mg) prepared by the same method as in Example 1-2-2 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed three times with DMF (0.7 mL). Next, 2% DBU / DMF solution (Fmoc-free solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc-free solution, the resin was washed four times with DMF (0.7 mL).

[0251] The obtained resin was subjected to an extension reaction of Tfa-(Me)Abu-OH (compound 1-3-2-b).

[0252] The extension reaction was carried out by adding a mixture of 0.6 M Tfa-(Me)Abu-OH (compound 1-3-2-b) / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 60 degrees for 48 hours.

[0253] After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain compound 2-5-1-1 (Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0254] To confirm the progress of the reaction, a portion of the resin (compound 2-5-1-1) was taken out and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS to confirm the production of the target peptide Tfa-(Me)Abu-MeVal-Asp-pyrro (compound 2-5-1-1*). No other peptide components were detected. After elongation, the resin was washed with DCM, dried, and then used for further investigation. [ka] LCMS(ESI)m / z=495.4(M+H)+ Retention time: 0.56 minutes (Analysis conditions SQDFA05)

[0255] Example 2-5-1-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-1-1) In a reaction vessel equipped with a filter, Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-1-1) (45 mg) prepared in Example 2-5-1-1 was placed, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with DMF (0.7 mL).

[0256] To the obtained resin, TMGN (27 mg) / DMF (0.175 mL) solution was added, followed by methyl iodide (31 μL) / DMF (0.175 mL) solution, and the mixture was shaken at 40°C for 1 hour. The liquid phase was removed with a filter, and then washed twice with DMF (0.7 mL). A small amount of the obtained resin was sampled, and peptides were cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0257] The resin that had been methylated the first time was subjected to the same procedure three more times in order to improve the reaction conversion rate. The second methylation was performed by shaking at 40°C for 1.5 hours. The third and fourth methylations were performed by shaking at 40°C for 1 hour. After the four methylations, the resin was washed four times with DMF and four times with DCM to obtain compound 2-5-1-2. A portion of the resin was taken out and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the target peptide Tfa-Me(Me)Abu-MeVal-Asp-pyrro (compound 2-5-1-2*) was 94.0% (UV area) and the O-methylated form of the Tfa amide site (compound 2-5-1-2a*) was 6.0% (UV area).

[0258] [ka] LCMS(ESI)m / z=509.5(M+H)+ Retention time: 0.60 minutes (Analysis conditions SQDFA05)

[0259] [ka] LCMS(ESI)m / z=509.5(M+H)+ Retention time: 0.69 minutes (Analysis conditions SQDFA05)

[0260] Example 2-5-2. Synthesis of Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-2-2) Example 2-5-2-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) followed by Fmoc removal and elongation of Tfa-(Me)Leu-OH Compound 2-5-2-1 (Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1 using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-(Me)Leu-OH (compound 1-3-3-b) / DMF solution (0.3 mL). A portion of the resin was removed and the peptide was extracted using a TFE / DCM / DIPEA solution (1:1:0.015). The extracted solution was analyzed by LCMS, and the main impurities were identified as the target peptide Tfa-(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-1*) (84.5% UV area) and an over-extended form of MeVal-Asp-pyrro (compound 2-5-2-1a*) 6.8% (UV area) (conversion efficiency after Fmoc removal was >99%).

[0261] [ka] LCMS(ESI)m / z=523.5(M+H)+ Retention time: 0.67 minutes (Analysis conditions SQDFA05)

[0262] [ka] LCMS(ESI)m / z=804.7(M+H)+ Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0263] Example 2-5-2-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-2-1) Compound 2-5-2-2 (Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-2-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the resin obtained was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-Me(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-2*) (68.5% UV area), the O-methylated form of the Tfa amide site (compound 2-5-2-2a*) 17.0% (UV area), and the starting material compound 2-5-2-1* (14.5% UV area) were confirmed.

[0264] [ka] LCMS(ESI)m / z=537.5(M+H)+ Retention time: 0.70 minutes (Analysis conditions SQDFA05)

[0265] [ka] LCMS(ESI)m / z=537.5(M+H)+ Retention time: 0.81 minutes (Analysis conditions SQDFA05)

[0266] Example 2-5-3. Synthesis of Tfa-Me(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-3-2) Example 2-5-3-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) followed by Fmoc removal and elongation of Tfa-(Me)Ser(Me)-OH Compound 2-5-3-1 (Tfa-(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-(Me)Ser(Me)-OH (compound 1-3-4-b) / DMF solution (0.3 mL) by the method shown in Example 2-5-1-1. A part of the obtained resin was taken out, and the peptide was excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-(Me)Ser(Me)-MeVal-Asp-pyrro (compound 2-5-3-1*) was confirmed. No other peptide components were detected.

[0267] [ka] LCMS(ESI)m / z=511.4(M+H)+ Retention time: 0.55 minutes (Analysis conditions SQDFA05)

[0268] Example 2-5-3-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-3-1) Compound 2-5-3-2 (Tfa-Me(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-3-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the resulting resin was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-Me(Me)Ser(Me)-MeVal-Asp-pyrro (compound 2-5-3-2*) was confirmed (94.0% UV area).

[0269] [ka] LCMS(ESI)m / z=525.5(M+H)+ Retention time: 0.61 minutes (Analysis conditions SQDFA05)

[0270] Example 2-5-4. Synthesis of Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-4-2) Example 2-5-4-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) followed by Fmoc removal and elongation of Tfa-(Me)Phe-OH Compound 2-5-4-1 (Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1, by setting the reaction time to 72 hours, using compound 1-2-2 (0.552 mmol / g, 100 mg), 0.6 M Tfa-(Me)Phe-OH (compound 1-3-5-b) / DMF solution (0.3 mL). A portion of the resin obtained was taken out, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-1*) was confirmed (81.4% UV area). The conversion efficiency after Fmoc removal was >99%, and the main impurities detected were an overextended MeVal-Asp-pyrro (compound 2-5-4-1a*) at 4.1% (UV area) and a peak of unknown structure.

[0271] [ka] LCMS(ESI)m / z=557.5(M+H)+ Retention time: 0.68 minutes (Analysis conditions SQDFA05)

[0272] [ka] LCMS(ESI)m / z=838.7(M+H)+ Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0273] Example 2-5-4-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-4-1) Compound 2-5-4-2 (Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was similarly synthesized using compound 2-5-4-1 (45 mg) by the method shown in Example 2-5-1-2. The second N-methylation was also carried out in 1 hour. A portion of the resin was removed and the peptide was extracted using a TFE / DCM / DIPEA solution (1:1:0.015). The extracted solution was analyzed by LCMS, confirming the production of the target peptide Tfa-Me(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-2*) (79.7% UV area). In addition, an over-extended form of MeVal-Asp-pyrro (compound 2-5-4-2a*) and an O-methylated form of the Tfa amide site (compound 2-5-4-2b*) (total 11.8% UV area) were detected as impurities (conversion efficiency of the starting material was 100%).

[0274] [ka] LCMS(ESI)m / z=571.5(M+H)+ Retention time: 0.74 minutes (Analysis conditions SQDFA05)

[0275] [ka] LCMS(ESI)m / z=852.7(M+H)+ Retention time: 0.79 minutes (Analysis conditions SQDFA05)

[0276] [ka] LCMS(ESI)m / z=571.5(M+H)+ Retention time: 0.79 minutes (Analysis conditions SQDFA05)

[0277] Example 2-5-5. Synthesis of Tfa-Me(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-5-2) Example 2-5-5-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) followed by Fmoc removal and elongation of Tfa-(Me)Cha-OH Compound 2-5-5-1 (Tfa-(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1, by setting the reaction time to 72 hours, using compound 1-2-2 (0.552 mmol / g, 100 mg), 0.6 M Tfa-(Me)Cha-OH (compound 1-3-6-c) / DMF solution (0.3 mL). A portion of the resin obtained was taken out, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-(Me)Cha-MeVal-Asp-pyrro (compound 2-5-5-1*) was confirmed (81.1% UV area). As the main impurity, multiple peaks of unknown structure were detected, including an overextended form of MeVal-Asp-pyrro (compound 2-5-5-1a*) (conversion efficiency after Fmoc removal was 100%).

[0278] [ka] LCMS(ESI)m / z=563.6(M+H)+ Retention time: 0.76 minutes (Analysis conditions SQDFA05)

[0279] [ka] LCMS(ESI)m / z=844.8(M+H)+ Retention time: 0.80 minutes (Analysis conditions SQDFA05)

[0280] Example 2-5-5-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-5-1) Compound 2-5-5-2 (Tfa-Me(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-5-1 (45 mg) by the method shown in Example 2-5-1-2. At this time, the second N-methylation was also carried out in 1 hour. A part of the obtained resin was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-Me(Me)Cha-MeVal-Asp-pyrro (compound 2-5-5-2*) (74.4% UV area), the O-methylated form of the Tfa amide site (compound 2-5-5-2a*) 12.1% (UV area) and the starting material compound 2-5-5-1* (13.5% UV area) were confirmed.

[0281] [ka] LCMS(ESI)m / z=577.5(M+H)+ Retention time: 0.80 minutes (Analysis conditions SQDFA05)

[0282] [ka] LCMS(ESI)m / z=577.5(M+H)+ Retention time: 0.92 minutes (Analysis conditions SQDFA05)

[0283] Example 2-5-6. Synthesis of Tfa-Me(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-6-2) Example 2-5-6-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) followed by Fmoc removal and elongation of Tfa-(Me)Val-OH Compound 2-5-6-1 (Tfa-(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1, by setting the reaction time to 72 hours, using compound 1-2-2 (0.552 mmol / g, 100 mg), 0.6 M Tfa-(Me)Val-OH (compound 1-3-7-b) / DMF solution (0.3 mL). A portion of the resin obtained was taken out, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-(Me)Val-MeVal-Asp-pyrro (compound 2-5-6-1*) was confirmed (66.6% UVarea). Several peaks of unknown structure were detected as impurities, including an overextended MeVal-Asp-pyrro (compound 2-5-6-1a*) (conversion efficiency after Fmoc removal was 98%).

[0284] [ka] LCMS(ESI)m / z=509.5(M+H)+ Retention time: 0.59 minutes (Analysis conditions SQDFA05)

[0285] [ka] LCMS(ESI)m / z=790.7(M+H)+ Retention time: 0.67 minutes (Analysis conditions SQDFA05)

[0286] Example 2-5-6-2. N-Methylation by nucleophilic substitution reaction at the amide site of Tfa in Tfa-(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-6-1) Based on the method shown in Example 2-5-1-2, the reaction temperature was set to 60 degrees, and compound 2-5-6-2 (Tfa-Me(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-6-1 (40 mg). At this time, the second N-methylation was also carried out in 1 hour. A part of the obtained resin was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-Me(Me)Val-MeVal-Asp-pyrro (compound 2-5-6-2*) (21.2% UV area), the O-methylated form of the Tfa amide site (compound 2-5-6-2a*) 17.1% (UV area) and the starting material compound 2-5-6-1* (39.6% UV area) were confirmed. In addition, several peaks of unknown structure were detected.

[0287] [ka] LCMS(ESI)m / z=523.5(M+H)+ Retention time: 0.65 minutes (Analysis conditions SQDFA05)

[0288] [ka] LCMS(ESI)m / z=523.5(M+H)+ Retention time: 0.77 minutes (Analysis conditions SQDFA05)

[0289] Example 2-5-7. Synthesis of Tfa-MecLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-7-2) Example 2-5-7-1. Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) was de-Fmoc-enhanced to Tfa-cLeu-OH Compound 2-5-7-1 (Tfa-cLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1 using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-cLeu-OH (compound 1-3-8-b) / DMF solution (0.3 mL). A portion of the resulting resin was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-cLeu-MeVal-Asp-pyrro (compound 2-5-7-1*) was confirmed. No other peptide components were detected.

[0290] [ka] LCMS(ESI)m / z=507.4(M+H)+ Retention time: 0.56 minutes (Analysis conditions SQDFA05)

[0291] Example 2-5-7-2. N-Methylation by nucleophilic substitution reaction at the Tfa amide site of Tfa-cLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-7-1) Compound 2-5-7-2 (Tfa-MecLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-7-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the obtained resin was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-MecLeu-MeVal-Asp-pyrro (compound 2-5-7-2*) (92.1% UV area) was confirmed, and an O-methylated form of the Tfa amide site (compound 2-5-7-2a*) 7.9% (UV area) was detected as an impurity (the conversion efficiency of the starting material was 100%).

[0292] [ka] LCMS(ESI)m / z=521.4(M+H)+ Retention time: 0.61 minutes (Analysis conditions SQDFA05)

[0293] [ka] LCMS(ESI)m / z=521.4(M+H)+ Retention time: 0.70 minutes (Analysis conditions SQDFA05)

[0294] The results of Examples 2-5 above demonstrate that the method of the present invention makes it possible to practically introduce various N-methyl-α,α-dialkylamino acids other than MeAib following bulky N-methylamino acids in solid-phase synthesis.

[0295] Example 2-6. Experiments on the introduction of bulky N-methylamino acid (MeVal) followed by various N-substituted α,α-dialkylamino acids on a solid phase Compounds 2-6-1 to 2-6-4 were synthesized using various Tfa-amino acids according to the following general scheme. [ka]

[0296] [Table 9]

[0297] Example 2-6-1. N-Ethylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1) (0.552 mmol / g, 50 mg) prepared in the same manner as in Example 2-1-1 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to allow the resin to swell. After removing the dichloromethane with a filter, the resin was washed four times with DMF (0.7 mL).

[0298] To the obtained resin, TMGN (29 mg) / DMF (0.175 mL) solution was added, followed by ethyl iodide (44 μL) / DMF (0.175 mL) solution, and the mixture was shaken at 60°C for 1 hour. The liquid phase was removed with a filter, and then washed twice with DMF (0.7 mL). A small amount of the obtained resin was sampled, and peptides were cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0299] The resin that had been ethylated the first time was subjected to the same procedure four more times in order to improve the reaction conversion rate. After the fifth ethylation, the resin was washed four times with DMF and four times with DCM to obtain compound 2-6-1 (Tfa-EtAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the resin was taken out and the peptide was excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-EtAib-MeVal-Asp-pyrro (compound 2-6-1*) (26.1% UV area), the O-ethylated form of the Tfa amide moiety (compound 2-6-1a*) 15.4% (UV area), the starting material compound 2-1* (54.8% UV area), etc. were confirmed.

[0300] [ka] LCMS(ESI)m / z=509.4(M+H)+ Retention time: 0.60 minutes (Analysis conditions SQDFA05)

[0301] [ka] LCMS(ESI)m / z=509.4(M+H)+ Retention time: 0.67 minutes (Analysis conditions SQDFA05)

[0302] Example 2-6-2. Nn-propylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Compound 2-6-2 (Tfa-nPrAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-1 (0.552 mmol / g, 50 mg) and n-propyl iodide (54 μL × 5) by the method shown in Example 2-6-1. A part of the obtained resin was taken out, and peptides were excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-nPrAib-MeVal-Asp-pyrro (compound 2-6-2*) (10.0% UV area), the On-propylated form of the Tfa amide site (compound 2-6-2a*) 6.3% (UV area), the starting material compound 2-1* (82.7% UV area), etc. were confirmed.

[0303] [ka] LCMS(ESI)m / z=523.5(M+H)+ Retention time: 0.66 minutes (Analysis conditions SQDFA05)

[0304] [ka] LCMS(ESI)m / z=523.5(M+H)+ Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0305] Example 2-6-3. N-allylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Compound 2-6-3 (Tfa-AllylAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-1 (0.552 mmol / g, 50 mg) and allyl bromide (48 μL × 5) by the method shown in Example 2-6-1. A part of the obtained resin was taken out, and peptides were excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-AllylAib-MeVal-Asp-pyrro (compound 2-6-3*) (20.3% UV area), the O-allylated form of the Tfa amide site (compound 2-6-3a*) 6.9% (UV area) and the starting material compound 2-1* (72.8% UV area) were confirmed.

[0306] [ka] LCMS(ESI)m / z=521.4(M+H)+ Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0307] [ka] LCMS(ESI)m / z=521.5(M+H)+ Retention time: 0.71 minutes (Analysis conditions SQDFA05)

[0308] Example 2-6-4. N-benzylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Compound 2-6-4 (Tfa-BnAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-1 (0.552 mmol / g, 50 mg) and benzyl bromide (66 μL × 5) by the method shown in Example 2-6-1. A part of the obtained resin was taken out, and peptides were excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-BnAib-MeVal-Asp-pyrro (compound 2-6-4*) (6.2% UV area), the O-benzylated form of the Tfa amide site (compound 2-6-4a*) 7.7% (UV area) and the starting material compound 2-1* (79.5% UV area) were confirmed.

[0309] [ka] LCMS(ESI)m / z=571.5(M+H)+ Retention time: 0.72 minutes (Analysis conditions SQDFA05)

[0310] [ka] LCMS(ESI)m / z=571.5(M+H)+ Retention time: 0.79 minutes (Analysis conditions SQDFA05)

[0311] The results of Examples 2-6 show that the method of the present invention makes it possible to practically introduce N-substituted α,α-dialkylamino acids, not limited to N-methyl-α,α-dialkylamino acids, following bulky N-methylamino acids in solid-phase synthesis.

[0312] Example 2-7. Experiment in which bulky N-alkylamino acid (EtVal / nPrVal) was introduced followed by N-methyl-α,α-dialkylamino acid (MecLeu) on the solid phase Compounds 2-7-1 to 2-7-2, compounds 2-7-3-1 to 2-7-3-4, and compounds 2-7-4-1 to 2-7-4-4 were synthesized according to the following general formula. [ka]

[0313] [Table 10] JPEG2025061589000106.jpg119149

[0314] Example 2-7-1. Preparation of Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-1) Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1) (0.552 mmol / g, 100 mg) prepared in the same manner as in Example 1-2-1 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed three times with DMF (0.7 mL). Next, 2% DBU / DMF solution (Fmoc-free solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc-free solution, the resin was washed four times with DMF (0.7 mL).

[0315] The resulting resin was subjected to an extension reaction of Fmoc-Val-OH. The extension reaction was carried out by adding a mixture of Fmoc-Val-OH (0.6 mol / L), 1-hydroxy-7-azabenzotriazole (HOAt, 0.375 mol / L) in NMP (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40°C for 3 hours.

[0316] After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain compound 2-7-1 (Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0317] To confirm the progress of the reaction, a portion (~5 mg) of the obtained resin (compound 2-7-1) was taken out and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Fmoc-Val-Asp-pyrro (compound 2-7-1*) was confirmed (97.3% UV area). Val overextension (compound 2-7-1a*) 2.7% (UV area) was also detected at the same time. After the extension, the resin was washed with DCM, dried, and used for further investigation.

[0318] [ka] LCMS(ESI)m / z=508.4(M+H)+ Retention time: 0.72 minutes (Analysis conditions SQDFA05)

[0319] [ka] LCMS(ESI)m / z=607.5(M+H)+ Retention time: 0.74 minutes (Analysis conditions SQDFA05)

[0320] Example 2-7-2. Fmoc removal from Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-1) and Ns-conjugation of the N-terminus Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-1) (0.552 mmol / g, 100 mg per column) prepared in Example 2-7-1 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed twice with DMF (0.7 mL). Next, 2% DBU / DMF solution (Fmoc-removal solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 10 minutes to remove Fmoc. After removing the Fmoc deprotection solution, the resin was washed successively with DMF (0.7 mL), a solution of 1-hydroxy-7-azabenzotriazole (HOAt, 0.157 mol / L) and DIPEA (0.157 mol / L) in DMF (0.7 mL), DMF (0.7 mL), and then three times with THF (0.7 mL).

[0321] To the obtained resin, a THF solution (0.35 mL) of 2,4,6-trimethylpyridine (0.074 mL, 0.552 mmol) and a THF solution (0.35 mL) of 2-nitrobenzenesulfonyl chloride (0.049 g, 0.221 mmol) were added, and the mixture was shaken at 40° C. for 3 hours.

[0322] After removing the liquid phase by filtration, the resin was washed five times with THF (1 mL) and five times with dichloromethane (1 mL) to obtain compound 2-7-2 (Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0323] To confirm the progress of elongation, a portion of the resin (~5 mg) was taken out and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and it was confirmed that the target peptide Ns-Val-Asp-pyrro (compound 2-7-2*) was produced in 94.3% (UV area). After Ns conversion, the resin was washed with DCM, dried, and used for further investigation.

[0324] [ka] LCMS(ESI)m / z=471.3(M+H)+ Retention time: 0.55 minutes (Analysis conditions SQDFA05)

[0325] Example 2-7-3-1. N-Ethylation of the Ns-amide moiety of Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-2) by Mitsunobu reaction Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-2) (0.552 mmol / g, 100 mg) prepared in Example 2-7-2 was placed in a reaction vessel equipped with a filter, and dichloromethane (1 mL) was added and the mixture was shaken at room temperature for 45 minutes to allow the resin to swell. After removing the dichloromethane with a filter, the resin was washed twice with THF (1 mL).

[0326] Separately, a 1.5 mL vial was charged with a solution of triphenylphosphine (72.0 mg, 0.276 mmol) in THF (0.35 mL) and a solution of DIAD (54 μL, 0.276 mmol) in THF (0.35 mL), and the mixture was gently shaken and left to stand at room temperature for 15 minutes. After that, ethanol (32 μL, 0.552 mmol) was added and mixed, and the mixture was left to stand for 5 minutes. The resulting solution was added to the swollen resin, and the mixture was shaken at 35 degrees for 1 hour. After removing the liquid phase with a filter, the resin was washed four times with THF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain compound 2-7-3-1 (Ns-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0327] A portion of the obtained resin (~5 mg) was cleaved from the peptide with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS, confirming the production of the target peptide Ns-EtVal-Asp-pyrro (compound 2-7-3-1*) (conversion rate from compound 2-7-2 was 100%). The obtained resin was dried and used for further investigation.

[0328] [ka] LCMS(ESI)m / z=499.4(M+H)+ Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0329] Example 2-7-3-2. De-Nsification of Ns-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-3-1) Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-1) (0.552 mmol / g, 100 mg) prepared in Example 2-7-3-1 was placed in a reaction vessel equipped with a filter, and dichloromethane (1 mL) was added and the mixture was shaken at room temperature for 45 minutes to allow the resin to swell. After removing the dichloromethane with a filter, the resin was washed twice with NMP (0.7 mL).

[0330] To the obtained resin, DBU (42 μL, 0.276 mmol) / NMP solution (0.35 mL) and 1-dodecanethiol (126 μL, 0.552 mmol) / NMP solution (0.35 mL) were added and shaken at 60°C for 4 hours. The liquid phase was removed with a filter and washed twice with NMP (0.7 mL). A portion of the obtained resin was taken out and peptides were cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0331] The resin that had been de-Nsified the first time was subjected to the same procedure again in order to improve the reaction conversion rate. The second de-Nsification was performed by shaking at 60 degrees for 12 hours. After the second de-Nsification, the resin was washed four times with NMP and four times with DCM to obtain compound 2-7-3-2 (H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the resulting resin was taken out and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide H-EtVal-Asp-pyrro (compound 2-7-3-2*) 84.6% (UVarea), an impurity (compound 2-7-3-2a*) (12.6% UVarea) estimated to be ipso-substituted 1-dodecanethiol for the nitro group of the Ns protection was detected.

[0332] [ka] LCMS(ESI)m / z=314.3(M+H)+ Retention time: 0.26 minutes (Analysis conditions SQDFA05)

[0333] [ka] LCMS(ESI)m / z=654.5(M+H)+ Retention time: 1.25 minutes (Analysis conditions SQDFA05)

[0334] Example 2-7-3-3. The extension reaction of Tfa-cLeu-OH to H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-2) was carried out by the same method as that of Tfa-cLeu extension using 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (compound 1-3-9) as the resin. H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-2) (0.552 mmol / g, 100 mg) prepared in Example 2-7-3-2 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 45 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed three times with DMF (0.7 mL). The extension reaction of Tfa-cLeu-OH was carried out by adding neat 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (compound 1-3-9) (0.582 g, 2.81 mmol) to the resin and shaking at 60 degrees for 48 hours. To confirm the progress of the reaction, a small amount of the resin was sampled after 24 hours, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS to confirm the production of the target peptide. After removing the liquid phase after the extension reaction with a filter, the resin was washed four times with DMF (1 mL) and four times with dichloromethane (1 mL) to obtain compound 2-7-3-3 (Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0335] To confirm the progress of the reaction, a part of the obtained resin (compound 2-7-3-3) was taken out, and the peptide was excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-cLeu-EtVal-Asp-pyrro (compound 2-7-3-3*) (55.5% UV area) was confirmed. In addition, an over-extension of EtVal-Asp-pyrro (compound 2-7-3-3a*) (18.0% UV area) and an impurity estimated to be ipso-substituted 1-dodecanethiol for the nitro group of the Ns protection (compound 2-7-3-2a*) (11.8% UV area) were detected. In addition, a peak like Tfa-cLeu-OH supported on the resin was also detected. After the extension, it was washed with DCM, dried, and used for further investigation.

[0336] [ka] LCMS(ESI)m / z=521.5(M+H)+ Retention time: 0.60 minutes (Analysis conditions SQDFA05)

[0337] [ka] LCMS(ESI)m / z=816.7(M+H)+ Retention time: 0.69 minutes (Analysis conditions SQDFA05)

[0338] Example 2-7-3-4. N-Methylation by nucleophilic substitution reaction at the Tfa amide site of Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-3) In a reaction vessel equipped with a filter, Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-3) (66 mg) prepared in Example 2-7-3-3 was placed, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 1 hour to allow the resin to swell. After removing the dichloromethane with a filter, the resin was washed four times with DMF (0.7 mL).

[0339] To the obtained resin, TMGN (59 mg) / DMF (0.35 mL) solution was added, followed by methyl iodide (69 μL) / DMF (0.35 mL) solution, and the mixture was shaken at 40°C for 1 hour. The liquid phase was removed with a filter, and then washed twice with DMF (0.7 mL). A portion of the obtained resin was taken out, and peptides were cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0340] The resin that had been methylated the first time was subjected to the same procedure twice more to improve the conversion rate. After the third methylation, the resin was washed four times with DMF and four times with DCM to obtain compound 2-7-3-4 (Tfa-MecLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the resin was taken out, and the peptide was excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-MecLeu-EtVal-Asp-pyrro (compound 2-7-3-4*) (53.2% UV area), a compound that was Me-converted after EtVal-Asp-pyrro was over-extended (compound 2-7-3-4a*) (29.8% UV area), an impurity that is estimated to be ipso-substituted by 1-dodecanethiol for the nitro group of the Ns protection (compound 2-7-3-2a*) (11.9% UV area), etc. were confirmed. In addition, a peak that resembled Tfa-cLeu-OH supported on the resin and Me-converted was also detected.

[0341] [ka] LCMS(ESI)m / z=535.4(M+H)+ Retention time: 0.66 minutes (Analysis conditions SQDFA05)

[0342] [ka] LCMS(ESI)m / z=830.7(M+H)+ Retention time: 0.75 minutes (Analysis conditions SQDFA05)

[0343] Example 2-7-4-1. Nn-Propylation of the Ns-amide moiety of Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-2) by Mitsunobu reaction Compound 2-7-4-1 (Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-7-3-1 using compound 2-7-2 (0.552mmol / g, 100mg) and 1-propanol (41μL, 0.552mmol). A portion of the resin obtained was taken out, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Ns-nPrVal-Asp-pyrro (compound 2-7-4-1*) was confirmed at 94.7% (UV area) (the conversion rate from compound 2-7-2 was 100%).

[0344] [ka] LCMS(ESI)m / z=513.4(M+H)+ Retention time: 0.69 minutes (Analysis conditions SQDFA05)

[0345] Example 2-7-4-2. De-Nsification of Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-4-1) Compound 2-7-4-2 (H-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-7-4-1 (0.552 mmol / g, 100 mg) by the method shown in Example 2-7-3-2. A portion of the resin obtained was taken out, and peptides were excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide H-nPrVal-Asp-pyrro (compound 2-7-4-2*) 81.1% (UVarea), an impurity (compound 2-7-4-2a*) (15.1% UVarea) estimated to be ipso-substituted 1-dodecanethiol for the nitro group of the Ns protection was detected.

[0346] [ka] LCMS(ESI)m / z=328.3(M+H)+ Retention time: 0.28 minutes (Analysis conditions SQDFA05)

[0347] [ka] LCMS(ESI)m / z=668.6(M+H)+ Retention time: 1.28 minutes (Analysis conditions SQDFA05)

[0348] Example 2-7-4-3. Extension of Tfa-cLeu to H-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-4-2) using 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (Compound 1-3-9) Compound 2-7-4-3 (Tfa-cLeu-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-7-3-3 using compound 2-7-4-2 (0.552 mmol / g, 100 mg). A portion of the resin was taken out, and the peptide was excised with a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and the production of the target peptide Tfa-cLeu-nPrVal-Asp-pyrro (compound 2-7-4-3*) (53.1% UV area) was confirmed. In addition, an over-extension of nPrVal-Asp-pyrro (compound 2-7-4-3a*) (31.0% UV area) and an impurity estimated to be ipso-substituted 1-dodecanethiol for the nitro group of the Ns protection (compound 2-7-4-2a*) (13.3% UV area) were detected. In addition, a peak that resembled Tfa-cLeu-OH supported on the resin was also detected.

[0349] [ka] LCMS(ESI)m / z=535.5(M+H)+ Retention time: 0.65 minutes (Analysis conditions SQDFA05)

[0350] [ka] LCMS(ESI)m / z=844.8(M+H)+ Retention time: 0.77 minutes (Analysis conditions SQDFA05)

[0351] Example 2-7-4-4. N-Methylation by nucleophilic substitution reaction at the Tfa amide site of Tfa-cLeu-nPrVal-Asp(OTrt(2-Cl)-resin)-pyrro (compound 2-7-4-3) Compound 2-7-4-4 (Tfa-MecLeu-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-7-3-4 using compound 2-7-4-3 (0.552 mmol / g, 60 mg). A portion of the resin was removed, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS, and in addition to the target peptide Tfa-MecLeu-nPrVal-Asp-pyrro (compound 2-7-4-4*) (46.1% UV area), a compound in which nPrVal-Asp-pyrro was over-elongated and then converted to Me (compound 2-7-4-4a*) (38.3% UV area), an impurity estimated to be ipso-substituted 1-dodecanethiol for the Ns-protected nitro group (compound 2-7-4-2a*) (12.6% UV area), etc. were confirmed. In addition, a peak was detected that suggested that Tfa-cLeu-OH was supported on the resin and converted to Me.

[0352] [ka] LCMS(ESI)m / z=549.5(M+H)+ Retention time: 0.71 minutes (Analysis conditions SQDFA05)

[0353] [ka] LCMS(ESI)m / z=858.7(M+H)+ Retention time: 0.83 minutes (Analysis conditions SQDFA05)

[0354] The results of Examples 2 to 7 show that the method of the present invention makes it possible to practically introduce bulky N-alkylamino acids and then N-methyl-α,α-dialkylamino acids in solid-phase synthesis.

[0355] Example 3: An example of peptide synthesis using MeAib introduced by the method of the present invention According to the peptide synthesis method by the Fmoc method described in WO2013 / 100132 or WO2018 / 225864, the peptide was elongated by the following basic route. 1) The carboxylic acid of the Asp side chain or the carboxylic acid of the peptide main chain is supported on 2-chlorotrityl resin, and the peptide is elongated from the N-terminus of the amino acid by the Fmoc method. 2) The process of peptide cleavage from 2-chlorotrityl resin, 3) Amide cyclization by condensation of the carboxylic acid of the Asp side chain or the carboxylic acid of the peptide main chain generated by the cleavage process from the 2-chlorotrityl resin with the amino group of the N-terminus of the peptide chain (triangle unit). 4) Deprotection of the protecting groups of the side chain functional groups contained in the peptide chain, if necessary; 5) Purification of the compound by preparative HPLC. In this example, unless otherwise specified, the peptide compound was synthesized based on this basic route. [ka]

[0356] Example 3-1: Synthesis of (5S,8S,11S,15R,18S,23aS,29S,35S,37aS)-8,11-di((S)-sec-butyl)-29-(3-chloro-4-(trifluoromethyl)phenethyl)-35-(cyclohexylmethyl)-18-isopropyl-5,6,12,15,16,19,21,21,22,33,36-undecamethyltetracosahydro-2H-azeto[2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16,19,22,25,28,31]undecaazacyclotetratriacontin-4,7,10,13,17,20,23,28,31,34,37(14H)-undecaone (compound 3-1) [ka]

[0357] Compound 3-1 was synthesized from compound 1-2-4 according to the following scheme. [ka]

[0358] (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin) (compound 1-2-4, 100 mg, 0.343 mmol / g, 0.0343 mmol) was used as a raw material, and in a reaction vessel equipped with a filter, Fmoc-MeVal-OH was elongated by the peptide elongation method described in Example 1-2-2, followed by elongation of Tfa-Aib-OH (2-methyl-2-(2,2,2-trifluoroacetamido)propanoic acid) (compound 1-3-1) in the same manner as in Example 2-1-1, to obtain compound 3-1-a.

[0359] The obtained compound 3-1-a was swollen with DCM (1 mL) and then washed four times with DMF (1 mL). A DMF solution (180 μL) of phosphazene base P1-tBu (38 μL, 0.150 mmol) and a DMF solution (180 μL) of methyl iodide (62 μL, 1 mmol) were added, and the mixture was shaken for 30 minutes at 40°C under a sealed condition. After removing the reaction solution, the resin was washed four times with DMF (1 mL) and then four times with DCM (1 mL) to obtain compound 3-1-b. A portion of the obtained resin was cut out with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS, confirming the formation of compound 3-1-b*.

[0360] [ka] LCMS(ESI)m / z=424(MH)- Retention time: 0.57 minutes (Analysis conditions SQDFA05)

[0361] Sodium borohydride (NaBH4) (758 mg, 20 mmol) was put into the flask, pumped up, and then placed under a nitrogen atmosphere. The solution was dissolved in triglyme (10 mL) to obtain solution A. Compound 3-1-b obtained above was swollen with DCM (1 mL) and then washed four times with THF (0.7 mL). THF (0.5 mL), methanol (0.25 mL), and solution A (0.25 mL) were added to the resin, and the mixture was shaken at room temperature for 40 minutes in an open system. After removing the reaction solution, methanol (0.7 mL) was added and the washing operation of discharging the liquid after 1 minute was repeated four times, and then the mixture was washed four times with DCM (0.7 mL) in the same manner to obtain compound 3-1-c. A portion of the obtained resin was cut out with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS, and the production of compound 3-1-c* was confirmed.

[0362] [ka] LCMS(ESI)m / z=330(M+H)+ Retention time: 0.33 minutes (Analysis conditions SQDFA05)

[0363] After preparing compound 3-1-c, the peptide elongation, cyclization and purification steps were carried out according to the following synthesis method.

[0364] As in Example 1-2-2, compound 3-1-c (100 mg per column), various Fmoc-amino acids (Fmoc-Pro-OH, Fmoc-Hph(4-CF3-3-Cl)-OH (compound AA2-001), Fmoc-MeGly-OH, Fmoc-MeCha-OH, Fmoc-Aze(2)-OH, Fmoc-MeAla-OH, Fmoc-Ile-OH, Fmoc-MeLeu-OH) (0.3-0.6 mol / L), HOAt or oxyma or HOOBt (0.375 mol / L) in NMP solution (solution 1), and diisopropylcarbodiimide (DIC) in N,N-dimethylformamide (DMF) solution (10% v / v, solution 2) were set in a peptide synthesizer.

[0365] Solutions 1 and 2 were mixed in the mixing vial of the synthesizer and then added to the resin to carry out a condensation reaction between the amino group on the resin and the Fmoc amino acid.

[0366] Synthesis was performed using a DMF solution (2% v / v) of diazabicycloundecene (DBU) as the Fmoc deprotection solution. After washing the resin with DMF, one cycle of Fmoc deprotection followed by Fmoc amino acid condensation reaction was repeated to elongate the peptide on the resin surface. After peptide elongation was completed, the Fmoc group at the N-terminus of the resin was removed on the peptide synthesizer, and the resin was washed with DMF.

[0367] DCM was added to the resulting linear peptide supported on the solid phase to reswell the resin, and 2,2,2-trifluoroethanol (TFE) / DCM (1 / 1 (v / v), 2 mL) was added to the resin and shaken at room temperature for 2 hours. The resin was then removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with 2,2,2-trifluoroethanol (TFE) / DCM (1 / 1 (v / v), 1 mL). All of the resulting cleavage solutions were mixed and concentrated under reduced pressure.

[0368] The residue concentrated under reduced pressure after cleavage was dissolved in DMF / DCM (1 / 1 (v / v), 8 mL). 0.5 M O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) / DMF solution (volume equivalent to 1.5 equivalents relative to the number of moles on the resin used (loading amount (mmol / g) multiplied by the amount of resin used (usually 0.10 g))) and DIPEA (1.8 equivalents relative to the number of moles on the resin used) were added and shaken at room temperature for 2 hours. The solvent was then distilled off under reduced pressure. The production of the desired cyclic peptide was confirmed by LCMS measurement.

[0369] After that, the solvent was distilled off under reduced pressure, DMF or DMSO was added, and the insoluble matter was removed by filtration. The mixture was purified by preparative-HPLC to obtain compound 3-1 ((5S,8S,11S,15R,18S,23aS,29S,35S,37aS)-8,11-di((S)-sec-butyl)-29-(3-chloro-4-(trifluoromethyl)phenethyl)-35-(cyclohexylmethyl)-18-(cyclohexylmethyl)-2-(1 ... The resulting mixture was 1,3,4-isopropyl-5,6,12,15,16,19,21,21,22,33,36-undecamethyltetracosahydro-2H-azeto[2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16,19,22,25,28,31]undecaazacyclotetratriacontin-4,7,10,13,17,20,23,28,31,34,37(14H)-undecaone) (4.1 mg, 9%). The LCMS analysis results are shown in Table 12.

[0370] Example 3-2: Peptide synthesis carried out in the same manner as in Example 3-1 Compounds 3-2 to 3-9 were similarly synthesized by the method shown in Example 3-1. The relationship between the formal names, structures, and abbreviations of the amino acid residues constituting the cyclic peptides shown in Compounds 3-1 to 3-9 (the structural formulas are shown in Table 13) can be understood from Tables 3 to 5 above and Table 11 below. The LCMS analysis results are shown in Table 12.

[0371] [Table 11] JPEG2025061589000130.jpg197149 JPEG2025061589000131.jpg194149 JPEG2025061589000132.jpg194149 JPEG2025061589000133.jpg197149 JPEG2025061589000134.jpg194149 JPEG2025061589000135.jpg196149 JPEG2025061589000136.jpg194149 JPEG2025061589000137.jpg175149 JPEG2025061589000138.jpg181149 JPEG2025061589000139.jpg197149 JPEG2025061589000140.jpg194149 JPEG2025061589000141.jpg202149 JPEG2025061589000142.jpg186149 JPEG2025061589000143.jpg60149

[0372] [Table 12]

[0373] [Table 13] JPEG2025061589000146.jpg224149 JPEG2025061589000147.jpg219149 JPEG2025061589000148.jpg212149 JPEG2025061589000149.jpg224149

[0374] Comparative Example 1: N-methylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1) by Mitsunobu reaction As a comparative example to the present invention, a known method of performing Mitsunobu reaction was attempted as a selective N-methylation method at the trifluoroacetamide site (Org. Lett. 2013, 15, 5012-5015).

[0375] In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-2) (0.473 mmol / g, 100 mg) prepared in Example 2-1-2, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with THF (0.7 mL).

[0376] To the obtained resin, triphenylphosphine (66.0 mg) / THF (0.7 mL) solution, methanol (20 μL), and DIAD (49 μL) were added, and the mixture was shaken at 40°C for 30 minutes. After removing the liquid phase with a filter, triphenylphosphine (66.0 mg) / THF (0.7 mL) solution, methanol (20 μL), and DIAD (49 μL) were added again, and the mixture was shaken at 40°C for 1 hour. After removing the liquid phase with a filter, the resin was washed four times with THF (0.7 mL) and four times with dichloromethane (0.7 mL).

[0377] The resulting resin was used to cleave peptides in a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS. In addition to the production of the target peptide Tfa-MeAib-MeVal-Asp-pyrro (compound 2-2*), the O-methylated product of the Tfa amide moiety (compound C1-1) and the hydrolysis product H-Aib-MeVal-Asp-pyrro (compound C1-2) were detected. The LC chart is shown in Figure 2.

[0378] Target peptide Tfa-MeAib-MeVal-Asp-pyrro (compound 2-2*) [ka] LCMS (ESI) m / z = 495.26 (M+H)+ Retention time: 0.58 minutes (Analysis conditions SQDFA05)

[0379] O-methylated product at the Tfa amide site (compound C1-1) [ka] LCMS (ESI) m / z = 495.26 (M+H)+ Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0380] H-Aib-MeVal-Asp-pyrro (compound C1-2) hydrolyzed from compound C1-1 [ka] LCMS (ESI) m / z = 385.26 (M+H)+ Retention time: 0.35 minutes (Analysis conditions SQDFA05)

[0381] From this result, it was confirmed that, unlike the literature (Org. Lett. 2013, 15, 5012-5015), when the N-terminus is an α,α-dialkylamino acid, O-methylation proceeds significantly in addition to N-methylation, resulting in a decrease in yield and purity. This result is in contrast to the results of N-selective methylation shown in Examples 2-2 and 2-3.

[0382] Comparative Example 2: An experiment in which MeAib was introduced by changing Fmoc protection to Ns protection, N-methylating the N-terminus on the resin, and removing Ns after Fmoc-Aib-OH extension of N-methyl amino acid in the conventional solid phase synthesis method. As a comparative example to the present invention, we attempted to introduce MeAib by performing Fmoc-Aib-OH elongation following N-methylamino acid synthesis in a solid-phase synthesis method, switching from Fmoc protection to Ns protection, N-methylation of the N-terminus on a resin, and removal of Ns, in a manner similar to that described in the literature (Nature Protocols 2012, 7, 3, 432-444). [ka]

[0383] Comparative Example 2-1: Solid-phase extension reaction of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) with Fmoc-Aib-OH Dichloromethane (1 mL) was added to Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.464 mmol / g, 100 mg) prepared in Example 1-2-2 in a reaction vessel equipped with a filter, and the mixture was shaken at room temperature for 30 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed twice with DMF (1 mL). Next, 2% DBU / DMF solution (Fmoc-removal solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 10 minutes to remove Fmoc. After removing the Fmoc-removal solution, the resin was washed four times with DMF (0.7 mL).

[0384] The resulting resin was subjected to an extension reaction with Fmoc-Aib-OH. The extension reaction was carried out by adding a mixture of 0.6 M Fmoc-Aib-OH / 0.375 M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 50 degrees for 15 hours. This extension reaction was repeated two more times (2nd extension condition: 50°C for 24 hours, 3rd extension condition: 50°C for 20 hours).

[0385] After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL).

[0386] To confirm the progress of elongation, a portion (~5 mg) of the resulting resin was taken out and the unreacted sites were capped with Fmoc-Gly-OH. Capping was performed by adding a mixture of 0.6 M Fmoc-Gly-OH / 0.375 M HOAt / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40° C. for 45 minutes. After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL).

[0387] The peptide was excised using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS, confirming that the target peptide Fmoc-Aib-MeVal-Asp-pyrro (compound C2-1*) was produced in 60.4% yield.

[0388] [ka] LCMS (ESI) m / z = 605.52 (MH)- Retention time: 2.16 minutes (Analysis conditions SQDFA05long)

[0389] Unreacted sites of the resulting resin were capped with Z-Gly-OH (N-α-carbobenzoxyglycine, CAS: 1138-80-3) purchased from a commercial supplier.

[0390] Capping was performed by adding a mixture of 0.6 M Z-Gly-OH / 0.375 M HOAt / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 2 hours.

[0391] After removing the liquid phase of the capping reaction by filtration, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain compound C2-1.

[0392] Comparative Example 2-2: Fmoc removal and Ns-conjugation of the N-terminus of Fmoc-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-1) Fmoc-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-1) (0.464 mmol / g, 100 mg) prepared in Comparative Example 2-1 was placed in a reaction vessel equipped with a filter, dichloromethane (1 mL) was added, and the mixture was shaken at room temperature for 30 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed twice with DMF (1 mL). Next, 2% DBU / DMF solution (Fmoc-removal solution: 0.7 mL) was added to the resin, and the mixture was shaken at room temperature for 10 minutes to remove Fmoc. After removing the Fmoc-removal solution, the resin was washed three times with DMF (1 mL), followed by four times with THF (1 mL).

[0393] To the resulting resin, a THF solution (0.35 mL) of 2,4,6-trimethylpyridine (0.062 mL, 0.464 mmol) and a THF solution (0.35 mL) of 2-nitrobenzenesulfonyl chloride (0.041 g, 0.186 mmol) were added, and the mixture was shaken at 40° C. for 2 hours.

[0394] After removing the liquid phase by filtration, the resin was washed three times with THF (1 mL) and four times with dichloromethane (1 mL).

[0395] The above Ns conversion using 2-nitrobenzenesulfonyl chloride was repeated two more times (second time: 40°C, shaking for 16 hours, third time: 40°C, shaking for 21 hours).

[0396] To confirm the progress of elongation, a portion of the resin (~5 mg) was taken and the peptide was excised using a TFE / DCM solution (1 / 1 (v / v)). The excised solution was analyzed by LCMS, and it was confirmed that the desired peptide Ns-Aib-MeVal-Asp-pyrro (compound C2-2*) was produced in 64.9% yield.

[0397] Unreacted sites of the resulting resin were capped with Z-Gly-OH. Capping was performed by adding a mixture of 0.6 M Z-Gly-OH / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40° C. for 2 hours.

[0398] After removing the liquid phase of the extension reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL) to obtain Ns-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-2).

[0399] [ka] LCMS (ESI) m / z = 568.45 (MH)- Retention time: 0.59 minutes (Analysis conditions SQDFA05)

[0400] Comparative Example 2-3: N-Methylation of the Ns amide site of Ns-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound C2-2) by Mitsunobu reaction In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Ns-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-2) (0.464 mmol / g, 100 mg) prepared in Comparative Example 2-2, and the mixture was shaken at room temperature for 20 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with THF (1 mL).

[0401] A solution of triphenylphosphine (61.0 mg, 0.232 mmol) and methanol (19 μL, 0.464 mmol) in THF (0.7 mL) was added to the obtained resin, followed by addition of DIAD (45 μL, 0.232 mmol) and shaking at 40°C for 30 minutes. After removing the liquid phase with a filter, the resin was washed four times with THF (1 mL) and four times with dichloromethane (1 mL).

[0402] A portion of the resulting resin (~5 mg) was cleaved from the peptide in a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS, confirming the production of the target peptide Ns-MeAib-MeVal-Asp-pyrro (compound C2-3*) (conversion rate from compound C2-2 was 96%). The remaining Ns-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-3) was used in the next step. [ka] LCMS (ESI) m / z = 582.47 (MH)- Retention time: 0.63 minutes (Analysis conditions SQDFA05)

[0403] Comparative Example 2-4: De-Nsification of Ns-MeAib-MeVal-Asp-pyrro resin (compound C2-3) In a reaction vessel equipped with a filter, dichloromethane (0.5 mL) was added to Ns-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-3) (0.464 mmol / g, 50 mg) prepared in Comparative Example 2-3, and the mixture was shaken at room temperature for 20 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with NMP (0.5 mL).

[0404] To the resin obtained, DBU (17μL, 0.115mmol) / NMP solution (0.35mL) and 2-mercaptoethanol (16μL, 0.230mmol) / NMP solution (0.30mL) were added and shaken at room temperature for 1 hour. After removing the liquid phase with a filter, the resin was washed four times with NMP (0.5mL) and four times with dichloromethane (0.5mL).

[0405] A portion of the obtained resin (~5 mg) was cleaved from the resin using a TFE / DCM solution (1 / 1 (v / v)). The cleaved solution was analyzed by LCMS, and the production of the desired peptide H-MeAib-MeVal-Asp-pyrro (compound C2-4*) that had been de-Nsified was confirmed, as shown in Figure 3.

[0406] [ka] LCMS (ESI) m / z = 399.29 (M+H)+ Retention time: 0.34 minutes (Analysis conditions SQDFA05)

[0407] Comparative Example 2-5: De-Nsification of Ns-MeAib-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro [ka]

[0408] In a reaction vessel equipped with a filter, compound 1-2-3 (100 mg) prepared in Example 1-2-3 was added to Ns-MeAib-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-5-1) (0.464 mmol / g, 50 mg) prepared in the same manner as in Comparative Examples 2-1 to 2-3, and dichloromethane (0.5 mL) was added and the mixture was shaken at room temperature for 20 minutes to swell the resin. After removing the dichloromethane with a filter, the resin was washed four times with NMP (0.5 mL).

[0409] To the resin obtained, DBU (17μL, 0.115mmol) / NMP solution (0.35mL) and 2-mercaptoethanol (16μL, 0.230mmol) / NMP solution (0.30mL) were added and shaken at room temperature for 1 hour. After removing the liquid phase with a filter, the resin was washed four times with NMP (0.5mL) and four times with dichloromethane (0.5mL).

[0410] A portion of the obtained resin (~5 mg) was cleaved from the resin using a TFE / DCM solution (1 / 1 (v / v)). The cleaved solution was analyzed by LCMS. As shown in Figure 4, in addition to the production of the target peptide H-MeAib-MePhe-Asp-pyrro (compound C2-5-2*) in which Ns-deprotection had progressed, an impurity (compound C2-5-3*) presumed to be the ipso substitution of 2-mercaptoethanol for the Ns-protected nitro group was detected.

[0411] [ka] LCMS (ESI) m / z = 447.31 (M+H)+ Retention time: 0.39 minutes (Analysis conditions SQDFA05)

[0412] [ka] LCMS (ESI) m / z = 661.74 (MH)- Retention time: 0.65 minutes (Analysis conditions SQDFA05)

[0413] From the results of Comparative Example 2, it was confirmed that while a bulky N-methyl-α,α-dialkylamino acid (MeAib in this example) can be introduced to the N-terminus of a bulky N-methylamino acid, the result is low purity and low yield through a series of steps including the extension of Fmoc-Aib and the replacement of the protecting group with the Ns group. It was also found that a side reaction on the Ns protecting group at the stage of Ns removal causes a decrease in purity. It was confirmed that it is difficult to introduce a bulky N-methyl-α,α-dialkylamino acid with high purity and high yield to the N-terminus of a bulky N-methylamino acid under known conditions described in the literature.

[0414] Reference example: Attempted Fmoc-MeAib-OH elongation following N-methyl amino acid synthesis using conventional solid phase synthesis Reference Example 1: Solid-phase extension reaction of Fmoc-MeAib-OH to Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) [ka]

[0415] Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.464 mmol / g, 100 mg) prepared in Example 1-2-2 was placed in a reaction vessel equipped with a filter, and the elongation of Fmoc-MeAib-OH was attempted by the same procedure as in Comparative Example 2-1.

[0416] For the extension reaction, a mixture of 0.6 M Fmoc-MeAib-OH / 0.375 M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) was added to the resin, shaken at 40 degrees for 21 hours, the reaction solution was discharged, and the same procedure was repeated once more (21.5 hours at 40 degrees).

[0417] After the extension reaction, the resin was washed appropriately, unreacted sites were capped with Fmoc-Gly-OH, and the peptide was cleaved from the resin in the same manner as in Comparative Example 2-1. The cleaved solution was analyzed by LCMS, but the target peptide Fmoc-MeAib-MeVal-Asp-pyrro (compound R1*) was not detected. [ka]

[0418] Reference Example 2: Solid-phase extension reaction of Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-3) with Fmoc-MeAib-OH [ka]

[0419] Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-3) (0.464 mmol / g, 100 mg) prepared in Example 1-2-3 was placed in a reaction vessel equipped with a filter, and elongation of Fmoc-MeAib was attempted in the same manner as in Reference Example 1. The elongation reaction was carried out at 40° C. for 15 hours.

[0420] Thereafter, the resin was appropriately washed, unreacted sites were capped with Fmoc-Gly-OH, and the peptide was cleaved from the resin using the same procedures as in Reference Example 1. The cleaved solution was analyzed by LCMS, and the production of the target peptide Fmoc-MeAib-MePhe-Asp-pyrro (compound R2*) was found to be only 3.1%. [ka] LCMS (ESI) m / z = 669.43 (M+H)+ Retention time: 0.88 minutes (Analysis conditions SQDFA05)

[0421] From the results of the Reference Example described above, it was confirmed that the extension of N-methylamino acid (i.e., N-substituted amino acid) followed by Fmoc-MeAib-OH (i.e., N-substituted-α,α-disubstituted amino acid) using the conventional solid-phase synthesis method (Fmoc method) is extremely difficult, and there are cases in which the desired peptide cannot be obtained. [Industrial Applicability]

[0422] According to the present invention, it has been found that a peptide compound containing a dipeptide residue in which an N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked can be efficiently produced in the production of a peptide compound using a solid phase method. The present invention is useful in the field of peptide synthesis.

Claims

[Claim 1] The invention described in the specification.

Images