Method for preparing a peptide compound containing an amino acid with large steric hindrance

Abstract

It was discovered that a peptide compound having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue is linked to the N-substituted amino acid or the N-substituted amino acid residue at the N-terminus can be efficiently prepared by linking an N-unsubstituted α,α-disubstituted amino acid, in which the amino group is protected by an electron-containing protecting group, to a peptide compound having an N-substituted amino acid or an N-substituted amino acid residue at the N-terminus, and then selectively introducing a substituent to the N-terminus amino group by applying a substituent introducing agent in the presence of a specific base.

Description

Technology Field

[0001] The present invention relates to a method for preparing a peptide compound comprising an amino acid with a large steric hindrance. Background Technology

[0002] Examples of so-called tough targets that have not been used as drug targets in conventional medicine include drugs that target molecules within cells, or drugs that inhibit protein-protein interactions. The reason these have not been used as drug targets is that the drug molecule cannot reach the target molecule of the disease, or that the shape of the site of action of the target molecule is difficult for conventional medicine to act upon (Non-patent Literature 1).

[0003] Recently, cyclic peptides have attracted attention as a method for accessing tough targets that were difficult to access with conventional technology (Non-patent document 2). For the application of cyclic peptides in pharmaceuticals, not only have the ability to bind to targets been improved, but drug-like cyclic peptides (drug-likeness: preferably exhibiting compatibility of membrane permeability and metabolic stability) and efficient screening methods for cyclic peptides have also been considered (Non-patent documents 3, 4). Furthermore, conditions required for non-natural type cyclic peptides containing drug-like non-natural amino acids have been identified, and their importance and recognition in drug discovery are increasing (Patent document 1). Since non-natural amino acids are an important component of this non-natural type peptide, it is widely recognized that the synthesis of non-natural amino acids, for example, peptides containing N-methyl (or N-alkyl) amino acids, or in some cases, peptides containing a series of N-methyl (or N-alkyl) amino acid units, is more difficult than that of natural type peptides due to their bulkiness (Non-patent literature 5, 6).

[0004] When shifting the focus to non-natural amino acids in which the side chains of the amino acids differ from those of natural amino acids, it has been reported that α,α-disubstituted amino acids, in which the α position of the amino acid is substituted with two substituents, contribute to increased lipid solubility, restriction of the conformational freedom of peptides containing α,α-disubstituted amino acids due to steric hindrance, and stabilization in vivo (Non-patent Literature 7), and it can be expected that they will have a significantly positive effect in terms of both pharmacological efficacy and drug-like properties.

[0005] In the preparation of peptides containing these non-natural amino acids, it is known that the Fmoc method, which is the most common method for peptide synthesis, is not suitable due to the structural bulkiness of these non-natural amino acids (Non-patent document 8).

[0006] From these, it is easily inferred that peptide synthesis by the condensation reaction of the carboxyl group of an α,α-disubstituted amino acid to the amino group of an amino acid, particularly the condensation reaction of the carboxyl group of an N-alkylated α,α-disubstituted amino acid to the amino group of an N-alkylated amino acid, will be difficult due to the stereochemical bulkiness of one or both amino acids.

[0007] As one method for continuously introducing N-alkyl amino acids, a method is known in which a peptide is synthesized by condensing an NH amino acid with less steric hindrance than the N-alkyl amino acid, and among the multiple NH groups in the obtained peptide, a target NH group is selectively N-alkylated (Non-patent literature 9, 10). However, these methods are methods for combining α-monosubstituted amino acids with N-alkyl amino acids, and no practical method for synthesizing a peptide having an arrangement in which an N-alkyl-α,α-disubstituted amino acid is combined with an N-alkyl amino acid is known. Prior art literature

[0008] International Publication No. 2018 / 225864

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

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

[0011] Non-patent document 6 illustrates a method for preparing N-alkylamino acids and a method for preparing peptides containing N-alkylamino acids. While this document illustrates a method focused on amino acid condensation agents, it does not describe the condensation reaction of bulkier α,α-disubstituted amino acids.

[0012] Non-patent document 7 describes a method for preparing α,α-disubstituted amino acids, the utility of peptides containing α,α-disubstituted amino acids, examples of α,α-disubstituted amino acids, and methods for preparing them, but there is no description of the preparation of peptides containing stereobulky amino acids.

[0013] Non-patent document 8 provides examples of methods for preparing peptides that are difficult to synthesize. This document describes the preparation of peptides that have low solubility or are prone to aggregation, but it does not describe a method for solving the problem using a condensation reaction.

[0014] Non-patent literature 9 describes a method for forming peptide bonds using N-alkylamino acids with a trifluoroacetyl group that can be removed under mild conditions, and a method for preparing peptides containing N-alkylamino acids. However, there is no mention of a condensation reaction with a bulkier α,α-disubstituted amino acid. Furthermore, it is known that alkyl groups cannot be selectively introduced only to the nitrogen atom to which the trifluoroacetyl group is attached, and that by-products of isomers are formed in which alkyl groups are introduced to the oxygen atom of the trifluoroacetyl group as well.

[0015] Non-patent document 10 describes a method for preparing a peptide containing an N-alkylamino acid by introducing an alkyl group to the nitrogen atom of an amino acid protected by a nosyl group. However, it is known that there are problems in preparing the target product because side reactions occur during the deprotection process of the nosyl group (Non-patent document 9).

[0016] As described above, the reaction for forming a bond 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-alkyl amino acids, is difficult, but no effective means of solving this is known. The present invention was made in light of this situation and aims to provide a method for preparing a peptide compound comprising an N-substituted-α,α-disubstituted amino acid residue and / or an N-substituted amino acid residue. More specifically, the present invention aims to provide a method for introducing an N-substituted-α,α-disubstituted amino acid into an N-substituted amino acid. Furthermore, the present invention aims to provide a method for highly selectively converting the amino group of an N-unsubstituted-α,α-disubstituted amino acid residue into an N-functional group. means of solving the problem

[0017] The inventors have discovered a method for linking N-substituted amino acids with N-substituted-α,α-disubstituted amino acids. Specifically, they discovered that a target amino acid can be introduced efficiently by using an N-unsubstituted-α,α-disubstituted amino acid, which has a lower steric bulk compared to N-substituted amino acids and enhances the reactivity of the carboxyl group by protecting the amino group with an electron-containing protecting group. Furthermore, regarding the functionalization of the amino group that follows, they discovered that N-functionalization reactions, such as N-alkylation reactions, proceed selectively on the NH group, whose acidity has increased due to the electron-containing protecting group. Additionally, noting that the acidity of the NH group increases due to the electron-containing protecting group, they discovered a specific base used in the N-functionalization reaction and completed the present invention.

[0018] That is, the present invention includes the following in a specific, non-limiting aspect.

[0019] [1] A method for preparing a peptide compound, its salt, or their solvates, comprising having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked, comprising the following steps:

[0020] Process A: a process of reacting an N-substituted amino acid, its salt, or their solvate, or a peptide compound having an N-substituted amino acid residue at the N-terminus, its salt, or their solvate, with an N-unsubstituted α,α-disubstituted amino acid having an amino group protected by an electron-receiving protecting group, its salt, its dehydrate, or their solvate in the presence or absence of a condensation reagent to obtain a peptide compound, its salt, or their solvate, comprising a dipeptide residue having an N-unsubstituted α,α-disubstituted amino acid residue having an amino group protected by an electron-receiving protecting group at the N-terminus, wherein the N-unsubstituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked; and

[0021] Process B: A process of introducing a substituent into the amino group of an N-unsubstituted α,α-disubstituted amino acid residue in the presence of a base and a substituent introducing agent, so as to have an N-substituted α,α-disubstituted amino acid residue in which the amino group is protected by an electron-receiving protecting group at the N-terminus, and comprising a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked, to obtain a peptide compound, its salt, or their solvates.

[0022] [2] The method described in [1], wherein the electron-receptive protecting group is a protecting group to which the NH group to which the protecting group is bonded has a pKa (in water) of 6 to 11.

[0023] [3] A method described in [1] or [2] in which the pKa (in acetonitrile) of the conjugate acid of the base is 18 to 31.

[0024] [4] A method described in any one of [1] to [3], wherein an 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 synthesis.

[0025] [5] An N-substituted amino acid, or a peptide compound having an N-substituted amino acid residue at the N-terminus, formula (2):

[0026]

[0027] [Among the formula,

[0028] P2 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0029] R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonyl C1-C6 alkyl, C2-C6 alkynyl, a C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is an aralkyl, and

[0030] R3 is a hydroxyl group, O-PG2, any amino acid residue, or any peptide residue, and

[0031] PG2 is a protector for the carboxyl group.

[0032] A method described in any one of [1] to [4], indicated by [1].

[0033] [6] An N-unsubstituted-α,α-disubstituted amino acid in which the amino group is protected by an electron-retaining protecting group, Formula (3):

[0034]

[0035] [Among the formula,

[0036] PG1 is a protector of electronic incubation, and

[0037] R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C7-C6 that may be substituted. 14 Independently selected from aralkyls, or

[0038] R1 and Q1 combine with the carbon atoms to which they are bonded to form a 3–8-membered alicyclic ring or a 4–7-membered saturated heterocyclic ring.

[0039] A method described in any one of [1] to [5], indicated by

[0040] [7] The peptide compound obtained in process A is formula (4):

[0041]

[0042] [Among the formula,

[0043] PG1, R1, and Q1 are respectively equivalent to PG1, R1, and Q1 of Equation (3), and

[0044] P2, R2, and R3 are respectively identical to P2, R2, and R3 of Equation (2)

[0045] A method described in any one of [1] to [6], indicated by .

[0046] [8] The substituent introducing agent in process B is P1X (wherein P1 is identical to P1 of formula (1) and X is a degreasing agent), and the peptide compound obtained in process B is formula (1):

[0047]

[0048] [Among the formula,

[0049] P1 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0050] PG1, R1, and Q1 are respectively equivalent to PG1, R1, and Q1 of Equation (3), and

[0051] P2, R2, and R3 are respectively identical to P2, R2, and R3 of Equation (2)

[0052] A method described in any one of [1] to [7], indicated by .

[0053] [9] Formula (1), including the following processes:

[0054]

[0055] [Among the formula,

[0056] PG1 is a protecting group of the amino group, and

[0057] P1 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0058] R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C7-C6 that may be substituted. 14 Independently selected from aralkyls, or

[0059] R1 and Q1 combine with the carbon atom to which they are bonded to form a 3–8 member alicyclic ring or a 4–7 member saturated heterocyclic ring, and

[0060] P2 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0061] R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonyl C1-C6 alkyl, C2-C6 alkynyl, a C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is an aralkyl, and

[0062] R3 is a hydroxyl group, O-PG2, any amino acid residue, or any peptide residue, and

[0063] PG2 is a protector for the carboxyl group.

[0064] A method for preparing a peptide compound, its salt, or their solvate comprising a structure in which two amino acid residues are linked, denoted by:

[0065] Process A: Formula (2):

[0066]

[0067] [In the formula, P2, R2, and R3 are respectively identical to P2, R2, and R3 of formula (1)]

[0068] Compounds represented by, their salts, or their solvates, and formula (3):

[0069]

[0070] [In the formula, PG1, Q1, and R1 are respectively identical to PG1, Q1, and R1 of formula (1)]

[0071] A compound represented by, its salt, its dehydrate, or their solvate is reacted with a condensation reagent, or a compound represented by the corresponding formula (2), its salt, or their solvate is reacted with a dehydrate of a compound represented by the corresponding formula (3), its salt, or their solvate, by formula (4):

[0072]

[0073] [In the formula, PG1, P2, Q1, and R1 to R3 are each identical to PG1, P2, Q1, and R1 to R3 of formula (1)]

[0074] A process of obtaining a compound represented by, its salt, or their solvate, and

[0075] Process B: A process of reacting a compound represented by Formula (4), its salt, or their solvate with a P1-introducing reagent to obtain a peptide compound represented by Formula (1), its salt, or their solvate.

[0076]

[10] R1 and Q1 may unite with the carbon atom to which they are bonded to form a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, or a tetrahydropyran ring, or

[0077] R1 and Q1 are independently selected from methyl, ethyl, 2-methylpropyl, allyl, methoxymethyl, cyclohexylmethyl, benzyl which may be substituted, or phenethyl which may be substituted,

[0078] A method described in any one of [6] to [9].

[0079]

[11] A method described in any one of [6] to

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

[0080]

[12] A method described in any one of [6] to

[11] in which PG1 is a C2-C6 haloacyl.

[0081]

[13] The method described in

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

[0082]

[14] Dehydrated bodies, the following formula:

[0083]

[0084] [In the formula, Q1 and R1 are respectively identical to Q1 and R1 of formula (1), and R4 is a C1-C5 haloalkyl.]

[0085] A method described in any one of [1] to

[13] , indicated by .

[0086]

[15] The method described in

[14] in which R1 and Q1 become one with the carbon atom to which they are bonded to form a 3- to 8-membered ring.

[0087]

[16] The method described in

[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.

[0088]

[17] P1 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl, a method described in any one of [8] to

[16] .

[0089]

[18] P2 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl, a method described in any one of [5] to

[17] .

[0090]

[19] A method described in any one of [5] to

[18] in which R3 is any amino acid residue or any peptide residue supported on a resin for solid synthesis.

[0091]

[20] A method described in any one of [4] to [8] and

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

[0092]

[21] A method described in any one of [1] to

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

[0093]

[22] A method described in any one of [9] to

[21] in which the P1-introducing reagent is a combination of P1X (wherein P1 is synonymous with P1 of formula (1) and X is a degreasing agent) and a base.

[0094]

[23] The method described in

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

[0095]

[24] The base,

[0096]

[0097] [Among the formula,

[0098] RB1 and RB4 are each independently C1-C4 alkyl, or RB1 and RB4 are combined with the nitrogen atom to which RB1 is bonded and the carbon atom to which RB4 is bonded to form a 5- to 8-membered ring, and

[0099] RB2 and RB3 are each independently C1-C4 alkyl, or RB2 and RB3 are combined with the nitrogen atom to which RB2 is bonded, the nitrogen atom to which RB3 is bonded, and the carbon atom to which the corresponding nitrogen atom is bonded to form a 5- to 8-membered ring.

[0100]

[0101] [Among the formula,

[0102] RB6 is a C1-C4 alkyl, and

[0103] RB5 and RB7 are each independently C1-C4 alkyl, or they form a 5- to 8-membered ring by combining each nitrogen atom to which they are bonded with the carbon atom to which that nitrogen atom is bonded, and

[0104] RB8 is a C1-C4 alkyl, and RB9 is a C1-C4 alkyl or phenyl; RB8 and RB9 are combined with each nitrogen atom to which they are bonded and the carbon atom to which that nitrogen atom is bonded to form a 5- to 8-membered ring, and

[0105] Here, if RB9 is phenyl, the two B2s may form naphthalene by condensing the two benzene rings of the corresponding phenyl group.

[0106]

[0107] [Among the formula,

[0108] RB 10 Silver, C1-C4 alkyl, or RB 10 and RB 11 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0109] RB 11 Silver, RB 10 and RB 11 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 11 and RB 12 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0110] RB 12 neun, RB 11 and RB 12 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB12 and RB 13 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0111] RB 13 Silver, RB 12 and RB 13 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 13 and RB 14 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0112] RB 14 neun, RB 13 and RB 14 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 14 and RB 15 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0113] RB 15 neun, RB 14 and RB 15 Except for cases where it forms a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0114] RB 16 Silver hydrogen, C1-C8 alkyl, or C6-C 10 It is Aril.], and

[0115]

[0116] [Among the formula,

[0117] RB 17 silver, independently C1-C4 alkyl, or RB 17 and RB 18 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0118] RB 18 Silver, RB 17 and RB 18 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB18 and RB 19 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0119] RB 19 neun, RB 18 and RB 19 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 19 and RB 20 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0120] RB 20 Silver, RB 19 and RB 20 Except for cases forming a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0121] RB 21 Silver, C1-C4 alkyl, or RB 21 and RB 22 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0122] RB 22 neun, RB 21 and RB 22 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 22 and RB 23 Silver, each nitrogen atom to which they are bonded, and the phosphorus atom to which that respective nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0123] RB 23 Silver, RB 22 and RB 23 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 23 and RB 24 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0124] RB 24 neun, RB 23 and RB24 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 24 and RB 25 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0125] RB 25 neun, RB 24 and RB 25 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 25 and RB 26 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0126] RB 26 Silver, RB 25 and RB 26 Except for cases forming a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0127] RB 27 silver, C1-C4 alkyl, or C6-C 10 It is Aril.

[0128] A method described in any one of [3] to [8] and

[22] to

[23] selected from the group consisting of

[0129]

[25] Bases, 1,8-diazabicyclo[5.4.0]undeca-7-en (DBU), 1,5-diazabicyclo[4.3.0]non-5-en (DBN), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-en (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]deca-5-en (TBD), tert-butylimino-tris(dimethylamino)phosphoran (P1-tBu), tert-butylimino-tri(pyrrolidino)phosphoran (P1-t-Bu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP), tert-octylimino-tris(dimethylamino)phosphoran (P1-t-Oct), imino-tris(dimethylamino)phosphoran (HP1(dma)), 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 -Catenadi(phosphazene)(P2-t-Bu), and 1-ethyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 A method described in any one of [3] to [8] and

[22] to

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

[0130]

[26] A method described in any one of [1] to

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

[0131]

[27] A method for preparing a peptide compound, its salt, or their solvates, comprising a dipeptide residue in which an N-substituted α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked, comprising a method described in any one of [1] to

[26] .

[0132]

[28] A process for deprotecting an N-terminal protecting group from a peptide compound, its salt, or their solvate prepared by a method described in any one of [1] to

[27] ,

[0133] A process for arbitrarily extending a peptide chain, and

[0134] A method for preparing a cyclic peptide compound, its salt, or their solvate, comprising a process of cyclically converting a C-terminal group and an N-terminal group to form a cyclic portion, wherein

[0135] The method wherein the cyclic peptide compound comprises 8 to 15 amino acid residues, comprises at least 3 N-substituted amino acid residues, and also comprises at least 1 N-unsubstituted amino acid residue, and the cyclic portion comprises at least 8 amino acid residues. Effects of the invention

[0136] According to the present invention, a peptide compound comprising a dipeptide residue connected to an N-substituted α,α-disubstituted amino acid residue, which is useful for peptide drugs, the search for peptide drugs, and / or the supply of raw materials for drugs, can be efficiently prepared. In addition, since it is possible to prepare peptide compounds combined with various non-natural amino acid residues, peptide compounds with diverse structures can be provided. Brief explanation of the drawing

[0137] [Fig. 1] Fig. 1 is a diagram showing the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Examples 2-4 detected at the maximum absorption wavelength using a photodiode array detector. [Fig. 2] Fig. 2 is a diagram showing the results of the LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 1, detected at the maximum absorption wavelength using a photodiode array detector. [Fig. 3] Fig. 3 is a diagram showing the results of the LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Examples 2-4, detected at the maximum absorption wavelength using a photodiode array detector. [Fig. 4] Fig. 4 is a diagram showing the results of the LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Examples 2-5, detected at the maximum absorption wavelength using a photodiode array detector. Specific details for implementing the invention

[0138] The abbreviations used in the present invention are listed below.

[0139] AA: Ammonium acetate

[0140] CSA: (+)-10-campasulfonic acid

[0141] DBU: 1,8-diazabicyclo[5.4.0]-7-undecen

[0142] DCC: N,N'-Dicyclohexylcarbodiimide

[0143] DCM: Dichloromethane

[0144] DCE: 1,2-Dichloroethane

[0145] DEAD: Diethyl azodicarboxylate

[0146] DMA: Dimethylacetamide

[0147] DMF: N,N-dimethylformamide

[0148] DIAD: Diisopropyl azodicarboxylic acid

[0149] DIC: N,N'-Diisopropylcarbodiimide

[0150] DIPEA: N,N-Diisopropylethylamine

[0151] DMAP: N,N-dimethyl-4-aminopyridine

[0152] dtbbpy: 4,4'-di-tert-butyl-2,2'-bipyridyl

[0153] EDTA: Ethylenediamine tetraacetic acid

[0154] FA: Pomsan

[0155] Fmoc: 9-fluorenylmethyloxycarbonyl group

[0156] NMP: N-methyl-2-pyrrolidone

[0157] TBME: t-butyl methyl ether

[0158] TES: Triethylsilane

[0159] TFA: Trifluoroacetic acid

[0160] TFE: 2,2,2-trifluoroethanol

[0161] THF: Tetrahydrofuran

[0162] THP: Tetrahydropyran Diary

[0163] TMSCl: Chlorotrimethylsilane

[0164] HFIP: 1,1,1,3,3,3-hexafluoroisopropyl alcohol

[0165] HOAt: 1-hydroxy-7-azabenzotriazole

[0166] HOBt: 1-hydroxybenzotriazole

[0167] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

[0168] IPAC: Isopropyl acetate

[0169] oxyma: ethyl cyano(hydroxyimino)acetate

[0170] PPTS: Pyridinium p-toluenesulfonate

[0171] EDCI·HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0172] TIPS: Triisopropylsilane

[0173] TfOH: Trifluoromethanesulfonic acid

[0174] HATU: O-(7-aza-1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluroronium hexafluorophosphate

[0175] DMSO: Dimethyl sulfoxide

[0176] Fmoc-Cl: Carbonochloride (9H-fluorene-9-yl)methyl carbonochloride

[0177] Fmoc-OSu: N-succinimidyl 9-fluorenylmethyl carbonate

[0178] Ns: o-Nitrobenzenesulfone diary

[0179] Trt: triphenylmethyl group, or trityl group

[0180] Tfa: Trifluoroacetyl group

[0181] MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-en

[0182] TMGN: 1,8-Bis(tetramethylguanidino)naphthalene

[0183] P1-tBu: tert-butylimino-tris(dimethylamino)phosphorane

[0184] (Definition of functional groups, etc.)

[0185] Examples of “halogen atoms” in this specification include F, Cl, Br, or I.

[0186] In this specification, "alkyl" refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in its backbone, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyls include not only straight-chain types but also branched-chain types. Specifically as alkyls, they include those with 1 to 20 carbon atoms (C1-C 20 , below 「C p -C q "" means having p to q carbon atoms) and is an alkyl group, preferably C1-C10 Examples include alkyl, more preferably C1-C6 alkyl. Specifically as alkyl, 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, Examples include 1-ethylbutyl, 2-ethylbutyl, etc.

[0187] In this specification, "alkenyl" refers to at least one double bond (two adjacent sp 2 It is a monovalent group having a carbon atom. Depending on the arrangement of the double bond and substituents (if present), the geometric form of the double bond can take an entgegen (E) or zuzanmen (Z), cis, or trans configuration. Alkenyls include not only straight-chain types but also branched-chain types. Preferably as an alkenyl, C2-C 10 Examples include alkenyls, more preferably C2-C6 alkenyls, and specifically, examples include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, etc.

[0188] In this specification, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkyns include not only straight chains but also branched chains. Preferably as an alkynyl, C2-C 10Examples include alkynal, more preferably C2-C6 alkynal, specifically, ethynyl, 1-propinyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propinyl, 3-(2'-fluorophenyl)-2-propinyl, 2-hydroxy-2-propinyl, 3-(3-fluorophenyl)-2-propinyl, 3-methyl-(5-phenyl)-4-pentynyl, etc.

[0189] In this specification, "cycloalkyl" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group and includes a monocyclic, bicyclocyclic, and spirocyclic group. Preferably, C3-C8 cycloalkyls may be used as cycloalkyls, and specifically, examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, etc.

[0190] In this specification, "aryl" means a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Examples of aryls include phenyl, naphthyl (e.g., 1-naphthyl, 2-naphthyl).

[0191] In this specification, "heterocyclile" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to a carbon atom. The heterocyclile may have double and / or triple bonds within the ring, the carbon atoms within the ring may be oxidized to form carbonyls, and it 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 heterocyclile), and more preferably 4 to 7 (4 to 7-membered heterocyclile). Specifically as heterocyclils, for example, azetidine yl, oxetan yl, dihydrofuryl, tetrahydrofuryl, dihydropyran yl, tetrahydropyran yl, tetrahydropyridyl, tetrahydropyrimidyl, morpholine yl, thiomopoline yl, pyrrolidine yl, piperidine yl, piperazine yl, pyrazolidin yl, imidazoline yl, imidazolidin yl, oxazolidin yl, isooxazolidin yl, thiazolidin yl, isothiazolidin yl, 1,2-thiazineine, thiadiazolidin yl, azetidine yl, oxazolidone, benzodioxane yl, benzoxazolyl, dioxolan yl, dioxane yl, tetrahydropyrrolo[1,2-c]imidazole, thiethane yl, Examples include 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sulfam, 2-oxaspyro[3.3]heptyl, etc.

[0192] In this specification, "heteroaryl" means an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to a carbon atom. The ring may be a single ring, a condensed ring with another ring, or partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5 to 10-membered heteroaryl) and more preferably 5 to 7 (5 to 7-membered heteroaryl). Specifically, heteroaryls include, for example, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolineyl, quinazolinyl, quinoxalineyl, benzodioxolyl, indolizinyl, imidazopyridyl, etc.

[0193] In this specification, "alkoxy" means an oxy group to which an "alkyl" of the above definition is bonded, and preferably, C1-C6 alkoxy may be used. Specifically, examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, 3-methylbutoxy, etc.

[0194] In this specification, "acyl (alkanoyl)" means a group in which a carbonyl group is bonded to hydrogen or the "alkyl" group, preferably a C1-C6 acyl, more preferably a C2-C4 acyl. Specifically, examples of acyl include formyl, acetyl, propionyl, butanoyl, etc.

[0195] In this specification, "cycloalkoxy" means an oxy group to which the "cycloalkyl" defined above is bonded, and preferably, a C3-C8 cycloalkoxy may be used. Specifically, examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0196] In this specification, "alkylsulfonyl" means a sulfonyl group to which the "alkyl" defined above is bonded, and preferably, C1-C6 alkylsulfonyl may be used. Specifically, examples of alkylsulfonyl include, for instance, methylsulfonyl.

[0197] In this specification, "hydroxyalkyl" refers to a group in which one or more hydrogens of the "alkyl" defined above are substituted with hydroxyl groups, and C1-C6 hydroxyalkyl is preferred. Specifically, examples of hydroxyalkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, 5-hydroxypentyl, etc.

[0198] In this specification, "haloalkyl" means a group in which one or more hydrogens of the "alkyl" defined above are substituted with a halogen, and C1-C6 haloalkyl is preferred, and C1-C6 fluoroalkyl is more preferred. Specifically, examples of haloalkyls include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, etc.

[0199] In this specification, "haloalkoxy" refers to a group in which one or more hydrogens of the "alkoxy" defined above are substituted with a halogen, and a C1-C6 haloalkoxy is preferred. Specifically, examples of haloalkoxy include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, etc.

[0200] In this specification, "haloacyl (haloalkanoyl)" means a group in which a carbonyl group is bonded to the "haloalkyl" group, preferably a C2-C6 haloacyl, more preferably a C2-C4 haloacyl. Specifically, examples of haloacyl include trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, 3,3,3-trifluoro-2-(trifluoromethyl)propionyl, etc.

[0201] In this specification, "alkoxyalkyl" means a group in which one or more hydrogens of the "alkyl" defined above are substituted with the "alkoxy" defined above, and C1-C6 alkoxyC1-C6 alkyl is preferred, and C1-C6 alkoxyC1-C2 alkyl is more preferred. Specifically, examples of alkoxyalkyls include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, 2-ethoxyethyl, etc.

[0202] In this specification, "cycloalkylalkyl" means a group in which one or more hydrogens of the "alkyl" defined above are substituted with the "cycloalkyl" defined above, and C3-C8 cycloalkyl C1-C6 alkyl is preferred, and C3-C6 cycloalkyl C1-C2 alkyl is more preferred. Specifically, examples of cycloalkylalkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc.

[0203] In this specification, "cycloalkoxyalkyl" means a group in which one or more hydrogens of the "alkyl" defined above are substituted with the "cycloalkoxy" defined above, and C3-C8 cycloalkoxy C1-C6 alkyl is preferred, and C3-C6 cycloalkoxy C1-C2 alkyl is more preferred. Specifically, examples of cycloalkoxyalkyl include cyclopropoxymethyl, cyclobutoxymethyl, etc.

[0204] In this specification, "alkylsulfonylalkyl" means a group in which one or more hydrogens of the "alkyl" defined above are substituted with the "alkylsulfonyl" defined above, and C1-C6 alkylsulfonyl C1-C6 alkyl is preferred, and C1-C6 alkylsulfonyl C1-C2 alkyl is more preferred. Specifically, examples of alkylsulfonylalkyl include methylsulfonylmethyl, 2-(methylsulfonyl)ethyl, etc.

[0205] In this specification, "aralkyl (arylalkyl)" means a group in which at least one hydrogen atom of the "alkyl" defined above is substituted with the "aryl" defined above, and C7-C 14 Aralkyl is preferred, and C7-C 10 Aralkyl is more preferable. Specifically, examples of aralkyl include benzyl, phenethyl, 3-phenylpropyl, etc.

[0206] In this specification, "heteroaralkyl (heteroarylalkyl)" means a group in which at least one hydrogen atom of the "alkyl" defined above is substituted with the "heteroaryl" defined above, and a 5- to 10-membered heteroaryl C1-C6 alkyl is preferred, and a 5- to 10-membered heteroaryl C1-C2 alkyl is more preferred. Specifically, examples of heteroarylkyl 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, 2-(5-benzofuranyl)ethyl, etc.

[0207] In this specification, "protecting group of a carboxyl group" may include an alkyl ester type protecting group, a benzyl ester type protecting group, a substituted alkyl ester type protecting group, etc. Specifically, examples of protecting groups of a carboxyl group include a methyl group, an ethyl group, a t-Bu group, a benzyl group, a trityl group, a cumyl group, a methoxymethyl group, a 2-(trimethylsilyl)ethyl group, a 2,2,2-trichloroethyl group, an allyl group, etc.

[0208] In this specification, "protecting groups of amino groups" may include carbamate-type protecting groups, amide-type protecting groups, imide-type protecting groups, sulfonamide-type protecting groups, etc. Specifically, examples of protecting groups of amino groups include Fmoc, Boc, Cbz, Alloc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, tosyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, etc.

[0209] In this specification, "allicyclic ring" means a non-aromatic hydrocarbon ring. An alicyclic ring may have unsaturated bonds within the ring, or it may be a polycyclic ring having two or more rings. Additionally, carbon atoms constituting the ring may be oxidized to form carbonyls. Preferably, a 3 to 8-membered alicyclic ring may be used as an alicyclic ring, and specifically, examples include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, a bicyclo[2.2.1]heptane ring, etc.

[0210] In this specification, "heterocyclic ring" refers to a non-aromatic heterocyclic ring containing 1 to 5 heteroatoms in addition to a carbon atom. The heterocyclic ring may have double and / or triple bonds within the ring, the carbon atoms within the ring may be oxidized to form carbonyls, and may be a single ring, a condensed ring, or a spirocyclic ring. The number of atoms constituting the ring is not limited, but is preferably 3 to 12 (3 to 12-membered heterocyclic ring), and more preferably 4 to 7 (4 to 7-membered heterocyclic ring). Examples of heterocyclic compounds include, specifically, piperazine, pyrrolidine, piperidine, morpholine, homomorpholine, hexahydropyrazine, 3-oxopiperazine, 2-oxopyrrolidine, azetidine, 2-oxomidazolidine, oxetane, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, tetrahydropyridine, thiomopoline, pyrazolidine, imidazoline, oxazolidine, isooxazolidine, thiazolidine, imidazolidine, isothiazolidine, thiadiazolidine, oxazolidone, benzodioxane, dioxolein, dioxane, tetrahydrothiopyran, etc.

[0211] In this specification, "saturated heterocyclic ring" means a non-aromatic heterocyclic ring containing 1 to 5 heteroatoms in addition to carbon atoms and not containing double bonds and / or triple bonds in the ring. A saturated heterocyclic ring may be a single ring, or it may form a condensation ring with another ring, for example, an aromatic ring such as a benzene ring. When a saturated heterocyclic ring forms a condensed ring, preferably a 4- to 7-membered saturated heterocyclic ring can be used as the saturated heterocyclic ring, and specifically, examples include the azetidine ring, oxetane ring, tetrahydrofuran ring, tetrahydropyran ring, morpholine ring, thiomopoline ring, pyrrolidine ring, 4-oxopyrrolidine ring, piperidine ring, 4-oxopiperidine ring, piperazine ring, pyrazolidin ring, imidazolidin ring, oxazolidin ring, isooxazolidin ring, thiazolidin ring, isothiazolidin ring, thiadiazolidin ring, oxazolidone ring, dioxolein ring, dioxane ring, thiethane ring, octahydroindole ring, indoline ring, etc.

[0212] In this specification, "peptide chain" refers to a peptide chain in which one or more natural amino acids and / or non-natural amino acids are connected by amide bonds and / or ester bonds. Preferably, the peptide chain is a peptide chain comprising 1 to 15 amino acid residues, and more preferably, a peptide chain composed of 5 to 12 amino acid residues.

[0213] In the present invention, the "peptide compound" is not particularly limited as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are connected by amide bonds or ester bonds, but preferably has 5 to 30 amino acid residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. A peptide compound supported on a resin for solid-state synthesis is also included in the peptide compound. In the present invention, the peptide compound synthesized preferably contains at least 3 N-substituted amino acids in one peptide, and more preferably contains at least 5 N-substituted amino acids. These N-substituted amino acids may exist continuously or discontinuously in the peptide compound. In this specification, "amino acids" constituting the peptide compound may be referred to as "amino acid residues," and "peptides" constituting all or part of the peptide compound may be referred to as "peptide residues." The peptide compound in the present invention may be linear or cyclic, and a cyclic peptide compound is preferred.

[0214] The "cyclic peptide compound" in the present invention is a cyclic peptide compound obtained by cyclizing the N-terminal group and the C-terminal group of a straight-chain peptide compound. The cyclization may take any form, such as cyclization by carbon-nitrogen bonds like amide bonds, cyclization by carbon-oxygen bonds like ester bonds or ether bonds, cyclization by carbon-sulfur bonds like thioether bonds, cyclization by carbon-carbon bonds, or cyclization by heterocyclic formation. Among these, cyclization involving covalent bonds such as amide bonds or carbon-carbon bonds is preferred, and cyclization involving amide bonds between the carboxylic acid group of the side chain and the amino group of the N-terminal main chain is more preferred. The position of the carboxylic acid group or amino group used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position where cyclization is possible.

[0215] In this specification, “one or more” means one or two or more numbers. When “one or more” is used in a context relating to a substituent of a group, this term means a number from one up to the maximum number of substituents allowed by that group. Specifically, “one or more” may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or larger numbers.

[0216] In this specification, "resin for solid-state synthesis" is not particularly limited as long as it can be used for the synthesis of peptide compounds by a solid-state method. Specifically, examples of such resins for solid-state synthesis include those that can be removed under acidic conditions, such as CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt). The resin can be appropriately selected according to the functional group on the amino acid side used. For example, when using a carboxylic acid (main chain carboxylic acid, or a side chain carboxylic acid represented by Asp or Glu) or an aromatic ring hydroxyl group (phenol group represented by Tyr) as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin) as the resin. When using an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) 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. Meanwhile, in this specification, the resin may also be described as resin. The resin for solid-state synthesis can be connected to an amino acid at any position, not limited to the C-terminal amino acid in the peptide. It is preferable that the carboxyl group of the C-terminal amino acid be connected to the resin for solid-state synthesis, and the carboxyl group may be a carboxyl group of the main chain or a carboxyl group of the side chain.

[0217] The type of polymer constituting the resin is not particularly limited. In the case of a resin composed of polystyrene, either 100-200 mesh or 200-400 mesh may be used. In addition, the crosslinking rate is not particularly limited, but it is preferable to have 1% DVB (divinylbenzene) crosslinked. Furthermore, Tentagel or Chemmatrix may be used as the type of polymer constituting the resin.

[0218] In the preparation of the compounds described in this specification, if a defined group undergoes an undesirable chemical transformation under the conditions of the method, the said compound may be prepared by using means such as protecting or deprotecting the functional group. Here, the selection and desorption operations of the protecting group may be, for example, the methods described in “Greene’s, “Protective Groups in Organic Synthesis” (5th ed., John Wiley & Sons 2014), and these may be appropriately utilized depending on the reaction conditions. Additionally, the order of reaction processes, such as the introduction of substituents, may be changed as necessary.

[0219] In the present specification, where the modifier “may be substituted” is applied, examples of the substituents include, for instance, alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclil, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, etc.

[0220] Additionally, substituents may be attached to each of these, and such substituents are not limited, and one or more may be freely selected independently from any substituents including, for example, halogen atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms, or phosphorus atoms. That is, examples of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, etc. that may be substituted are given.

[0221] The compounds described in the present invention may be their salts or their solvates. Salts of the compounds described in the present invention include, for example, hydrochloride; hydrobromide; hydroiodide; phosphate; phosphonate; sulfate; sulfonate such as methanesulfonate, p-toluenesulfonate, etc.; carboxylates such as acetate, citrate, malate, tartrate, succinate, salicylate, etc.; or alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as magnesium salt, calcium salt, etc.; ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, tetraalkylammonium salt, etc. These salts are prepared, for example, by contacting the said compounds with an acid or a base. A solvate of a compound described in the present invention refers to a phenomenon in which solute molecules in a solution strongly attract solvent molecules to form a single molecular group, and if the solvent is water, it is called a hydrate. The compound described in the present invention may be a solvate with a single solvent selected from alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), organic solvents such as dimethylformamide or diglyme, or water, or a solvate with multiple solvents.

[0222] In this specification, "amino acid" includes natural amino acids and non-natural amino acids (which may be referred to as amino acid derivatives). In this specification, "natural amino acid" 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 include β-amino acids, D-type amino acids, N-substituted amino acids, α,α-di-substituted amino acids, amino acids whose side chains differ from natural amino acids, and hydroxycarboxylic acids. As for amino acids in this specification, any stereochemical configuration is permitted, but preferably, they are L-type amino acids. There are no specific restrictions on the selection of the side chain of the amino acid, but in addition to hydrogen atoms, it is freely selected from, for example, alkyl groups, alkenyl groups, alkyneyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-linked cycloalkyl groups. Each may be provided with a substituent, and such substituents are not limited and may be freely selected independently from any substituents including, for example, halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms, or P atoms. That is, examples include alkyl groups, alkoxy groups, alkenyl groups, alkyneyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., or oxo, aminocarbonyl, halogen atoms, etc., which may be substituted. In one non-limiting aspect, the amino acid in this specification may be a compound having a carboxyl group and an amino group within the same molecule (in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0223] Examples of substituents containing a halogen atom in this specification include alkyl groups, cycloalkyl groups, alkenyl groups, alkyneyl groups, aryl groups, heteroaryl groups, aralkyl groups, etc., having a halogen as a substituent, and more specifically, fluoroalkyl, difluoroalkyl, trifluoroalkyl, etc. are examples.

[0224] Examples of substituents containing an O atom include hydroxy (-OH), oxy (-OR), carbonyl (-C=OR), carboxy (-CO2H), 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).

[0225] Examples of oxy(-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As for alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among these, methoxy or ethoxy is preferred.

[0226] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkyl carbonyl, cycloalkyl carbonyl, alkenyl carbonyl, alkenyl carbonyl, aryl carbonyl, heteroaryl carbonyl, aralkyl carbonyl, etc.

[0227] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkenyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, etc.

[0228] Examples of carbonyloxy (-OC=OR) include alkyl carbonyloxy, cycloalkyl carbonyloxy, alkenyl carbonyloxy, alkenyl carbonyloxy, aryl carbonyloxy, heteroaryl carbonyloxy, aralkyl carbonyloxy, etc.

[0229] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkenylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, etc.

[0230] Examples of carbonylthio (-SC=OR) include alkyl carbonylthio, cycloalkyl carbonylthio, alkenyl carbonylthio, alkenyl carbonylthio, aryl carbonylthio, heteroaryl carbonylthio, aralkyl carbonylthio, etc.

[0231] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl (e.g., C1-C6 or C1-C4 alkylaminocarbonyl, among which ethylaminocarbonyl, methylaminocarbonyl, etc.), cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, a group 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 group may be used.

[0232] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, groups may be further substituted in which the H atom bonded to the N atom in -NH-C=OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0233] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, there are groups in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0234] Examples of sulfonylamin (-NH-SO2-R) include alkylsulfonylamin, cycloalkylsulfonylamin, alkenylsulfonylamin, alkynylsulfonylamin, arylsulfonylamin, heteroarylsulfonylamin, aralkylsulfonylamin, etc. In addition to these, there are groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0235] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups may be provided in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0236] 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.

[0237] Examples of substituents containing an S atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-SO2-R), sulfo (-SO3H), and pentafluorosulfanyl (-SF5).

[0238] Examples of thio(-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, etc.

[0239] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkenylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, etc.

[0240] Examples of sulfonyls (-SO2-R) include alkylsulfonyls, cycloalkylsulfonyls, alkenylsulfonyls, alkenylsulfonyls, arylsulfonyls, heteroarylsulfonyls, aralkylsulfonyls, etc.

[0241] Substituents containing an N atom include azaido (-N3, also called "azido group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also called monosubstituted amino), tertiary amino (-NR(R'); also called disubstituted amino), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridyl, piperidino, morpholino, azetidinyl, etc.

[0242] Examples of secondary aminos (-NH-R) include alkylaminos, cycloalkylaminos, alkenylaminos, alkynylaminos, arylaminos, heteroarylaminos, aralkylaminos, etc.

[0243] Examples of tertiary aminos (-NR(R'; disubstituted aminos) include, for instance, alkyl(aralkyl)aminos, amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and any two of these substituents may form a ring. Specifically, examples include dialkylaminos, among which C1-C6 dialkylamino, C1-C4 dialkylamino, dimethylamino, diethylamino, etc. are exemplified. In this specification, "C p -C q "Dialkylamino group" refers to C in the amino group p -C q It refers to a group substituted with two alkyl groups, and both C p -C q The alkyl group may be the same or different.

[0244] Examples of substituted amidinos (-C(=NR)-NR'R'') include three substituents R, R', and R'' on the N atom, each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, such as alkyl(aralkyl)(aryl)amidino.

[0245] Examples of substituted guanidino (-NR-C(=NR''')-NR'R'') include R, R', R'', and R''', each being a group independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or a group in which they form a ring.

[0246] Examples of aminocarbonylamino(-NR-CO-NR'R'') include R, R', and R'' being groups independently selected from hydrogen atoms, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups that form a ring.

[0247] Examples of substituents containing a B atom include boryl (-BR(R')) or dioxyboryl (-B(OR)(OR')). These two substituents R and R' may each be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or they may form a ring. Specifically, examples include cyclic boryl groups, and more specifically, examples include pinacoletoboryl groups, neopentanediolatetoboryl groups, catecholetoboryl groups.

[0248] Specifically, as substituents on the nitrogen atom of N-substituted amino acids in this specification, alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, C7-C 14 Examples include aralkyl, benzyl, and phenethyl.

[0249] The main chain amino group of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR. Here, R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group that may have a substituent, and may also form a ring with the carbon chain bonded to the N atom and the carbon atom on the α-position, such as proline). Amino acids in which a hydrogen atom of such a main chain amino group is substituted may be referred to as "N-substituted amino acids" in this specification. Preferably, "N-substituted amino acids" in this specification include 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, and N-C7-C 14 Aralkylamino acids, N-benzylamino acids, and N-phenethylamino acids are examples, but are not limited to these.

[0250] In this specification, "amino acid" includes all corresponding isotopes. An isotope of "amino acid" is one in which at least one atom is substituted with an atom having the same atomic number (number of protons) but different mass numbers (sum of the number of protons and neutrons). Examples of isotopes included in "amino acid" in this specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., and each, 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.

[0251] (Manufacturing method)

[0252] In one aspect, the present invention relates to a method for preparing a peptide compound, its salt, or their solvates, comprising the N-substituted α,α-disubstituted amino acid residue having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue is connected to the N-substituted amino acid residue, comprising the processes A and B described below.

[0253] (Process A)

[0254] Process A is a process of reacting an N-substituted amino acid, its salt, or their solvate, or a peptide compound having an N-substituted amino acid residue at the N-terminus, its salt, or their solvate, with an N-unsubstituted-α,α-disubstituted amino acid, its salt, its dehydrate, or their solvate in which an amino group is protected by an electron-receiving protecting group, in the presence or absence of a condensation reagent, to obtain a peptide compound, its salt, or their solvate, having an N-unsubstituted-α,α-disubstituted amino acid residue at the N-terminus and comprising a dipeptide residue in which the N-unsubstituted-α,α-disubstituted amino acid residue is linked to the N-substituted amino acid residue. In this specification, the statement that a peptide compound comprises a dipeptide means that the amino acid sequence constituting the peptide compound comprises the corresponding dipeptide.

[0255] In one embodiment, the “N-substituted amino acid” used in process A is any natural or non-natural amino acid in which the amino group of the main chain is -NHR, wherein R is any group other than hydrogen. Specifically, R may be, for example, an alkyl that may be substituted, an alkenyl that may be substituted, an alkyneyl that may be substituted, an aryl that may be substituted, a heteroaryl that may be substituted, an aralkyl that may be substituted, a cycloalkyl that may be substituted, etc. Additionally, R may form a ring with the carbon chain bonded to the N atom and the carbon atom on the α-position, such as proline, and the ring may be further substituted with any substituent. Furthermore, the N-substituted amino acid may be in the form of a salt or a solvate.

[0256] In any embodiment, the "peptide compound having an N-substituted amino acid residue at the N-terminus" used in process A is not limited in the type and number of other amino acids included in the peptide compound, provided that it has the N-substituted amino acid residue at the N-terminus. Additionally, the peptide compound may be in the form of a salt or a solvate.

[0257] The N-substituted amino acid used in Process A, or the peptide compound having an N-substituted amino acid residue at the N-terminus, may be purchased from a commercial supplier or prepared by modifying one purchased from a commercial supplier.

[0258] As such, an N-substituted amino acid, or a peptide compound having an N-substituted amino acid residue at the N-terminus, specifically, the following formula (2):

[0259]

[0260] [Among the formula,

[0261] P2 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0262] R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonyl C1-C6 alkyl, C2-C6 alkynyl, a C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl, or C7-C 14 It is an aralkyl, and

[0263] R3 is a hydroxyl group, O-PG2, any amino acid residue, or any peptide residue, and

[0264] PG2 is a protector for the carboxyl group.

[0265] Examples include compounds represented by, their salts, or their solvates.

[0266] In one embodiment, the “N-unsubstituted-α,α-disubstituted amino acid in which an amino group is protected by an electron-attracting protecting group” used in process A means an amino acid having two optional substituents other than hydrogen on the α-carbon of the amino acid, in which the amino group of the main chain of the amino acid is unsubstituted, and in which the amino group is protected by an electron-attracting 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. Specifically, examples of the substituents include, for instance, alkyl that may be substituted, alkoxyalkyl that may be substituted, alkenyl that may be substituted, alkynyl that may be substituted, aryl that may be substituted, heteroaryl that may be substituted, aralkyl that may be substituted, heteroaralkyl that may be substituted, cycloalkyl that may be substituted, cycloalkylalkyl that may be substituted, etc. In addition, the two substituents bonded to the α-carbon may form a cycloaliphatic ring or a heterocyclic ring that may be substituted with the carbon atom to which they are bonded.

[0267] The N-unsubstituted-α,α-disubstituted amino acid in which the amino group is protected by an electron-receiving protecting group used in process A may be prepared by purchasing it from a commercial supplier or by modifying one purchased from a commercial supplier.

[0268] In any given sun, process A may be carried out in the presence of a condensation reagent. On the other hand, process A may be carried out in the absence of a condensation reagent if the condensation reaction proceeds, such as when using a dehydrate of an N-unsubstituted α,α-disubstituted amino acid.

[0269] As an N-unsubstituted-α,α-disubstituted amino acid in which the amino group is protected by such an electron-retaining protecting group, specifically, the following formula (3):

[0270]

[0271] [Among the formula,

[0272] PG1 is a protector of electronic incubation, and

[0273] R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C7-C6 that may be substituted. 14 Independently selected from aralkyls, or

[0274] R1 and Q1 combine with the carbon atoms to which they are bonded to form a 3–8-membered alicyclic ring or a 4–7-membered saturated heterocyclic ring.

[0275] Examples include compounds represented by, their salts, or their solvates.

[0276] In one aspect, the electron-receptive protecting group attached to the N-unsubstituted-α,α-disubstituted amino acid is a protecting group to which the NH group is attached, wherein the pKa (in water) of the NH group is 6 to 11, and a protecting group to which the pKa (in water) of the NH group is 8 to 11 is preferred. Specifically, examples of such protecting groups include C2-C6 haloacyl, and more specifically, trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, or 3,3,3-trifluoro-2-(trifluoromethyl)propionyl, etc.

[0277] In any sun, a peptide compound having an N-unsubstituted α,α-disubstituted amino acid residue at the N-terminus obtained by process A, and comprising a dipeptide residue in which the N-unsubstituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are connected, specifically, the following formula (4):

[0278]

[0279] [Among the formula,

[0280] PG1, R1, and Q1 are respectively equivalent to PG1, R1, and Q1 of Equation (3), and

[0281] P2, R2, and R3 are respectively identical to P2, R2, and R3 of Equation (2)

[0282] Examples include compounds represented by, their salts, or their solvates.

[0283] (Process B)

[0284] Process B is a process of introducing a substituent into the amino group of an N-unsubstituted α,α-disubstituted amino acid residue, in the presence of a base and a substituent introducing agent, so as to have an N-substituted α,α-disubstituted amino acid residue at the N-terminus, in which the amino group is protected by an electron-convex protecting group at the N-terminus of the peptide compound obtained by Process A, and also comprising a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked.

[0285] Specifically, substituents introduced in this process include alkyl, alkenyl, alkynyl, aralkyl, and cycloalkyl.

[0286] In one aspect, the base used in process B is preferably one in which the pKa (in acetonitrile) of the conjugate acid is 23 to 30. Specifically, such a base may be a base having an amidine backbone, a base having a guanidine backbone, or a base having a phosphazene backbone, as described later.

[0287] In one aspect, the substituent introduced in process B is used to introduce a substituent to 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 N-terminal electron-receiving protecting group. As the substituent introduced, an electron reagent may be used. Specifically, a compound in which the substituent to be introduced is combined with a detaching group (e.g., a halogen, a trifluoromethanesulfonyl group, a methanesulfonyl group, a sulfonic acid group such as a tosyl group, or a phosphate group) may be used.

[0288] In one aspect, a peptide compound comprising a dipeptide residue having an N-substituted α,α-disubstituted amino acid residue having an amino group protected by an electron-receptive protecting group obtained by process B at the N-terminus, and wherein the N-substituted α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked, specifically, the following formula (1):

[0289]

[0290] [Among the formula,

[0291] P1 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl, and

[0292] PG1, R1, and Q1 are respectively equivalent to PG1, R1, and Q1 of Equation (3), and

[0293] P2, R2, and R3 are respectively identical to P2, R2, and R3 of Equation (2)

[0294] Examples include compounds represented by, their salts, or their solvates.

[0295] A “peptide compound having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and comprising a dipeptide residue in which the N-substituted α,α-disubstituted amino acid residue is connected to the N-substituted amino acid residue” produced by the method of the present invention may have the amino group at the N-terminus protected by a protecting group, or the protecting group removed to become a free amino group (NHR-). If the amino group at the N-terminus is protected by a protecting group, the protecting group may be an electron-fixing protecting group derived from the “N-unsubstituted α,α-disubstituted amino acid in which the amino group is protected by an electron-fixing protecting group” used in process A, or another protecting group (e.g., an Fmoc group) introduced after the electron-fixing protecting group is deprotected. In addition to process A and process B, the present invention may include a process for removing the electron-fixing protecting group and a process for introducing any protecting group different from the protecting group. For removing the protector, for example, the method described in “Greene’s, “Protective Groups in Organic Synthesis” (5th edition, John Wiley & Sons 2014)” can be used.

[0296] In one aspect, the present invention relates to a method for preparing a peptide compound, its salt, or their solvate, comprising a structure in which two amino acid residues are connected as represented by formula (1), comprising process A and process B as shown in the scheme below.

[0297]

[0298] Among the above formulas, PG1 is a protecting group of the amino group, and in formula (4), a protecting group is preferably used such that the pKa of the NH group to which PG1 is bound is 11 or less. When the pKa of the NH group to which PG1 is bound is 11 or less, preferably 6 to 11, and more preferably 8 to 11, it is possible to selectively introduce a P1 group to the NH group of formula (4) to which PG1 is bound. The pKa can be calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C) 1994-2019 ACD / Labs). For example, the pka of the NH group of tert-butyl(2,2,2-trifluoroacetyl)alaninate, in which a trifluoroacetyl group 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-trifluoroacetamide)propanoate is 9.21. Also, the pka of the NH group of methyl-2-(2,2,3,3,3-pentafluoropropaneamide)propanoate, in which a pentafluoropropionyl group is bonded to a nitrogen atom, is 9.27, and the pka of the NH group of methyl-2-(2,2,2-trichloroacetamide)propanoate, in which a trichloroacetyl group is bonded to a nitrogen atom, is 9.72. On the other hand, the pKa of the NH group of 2-acetamide-2-methylpropanoate, in which an acetyl group with weaker electron-attracting power than these haloacyl groups is bonded to a 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-attracting protecting group in which the acidity of the proton of the NH group increases, and C2-C6 haloacyl can be cited as such a protecting group. As C2-C6 haloacyl, trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, or 3,3,3-trifluoro-2-(trifluoromethyl)propionyl are preferred.

[0299] In formula (1), P1 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is an aralkyl. If P1 is a C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, n-propyl, or i-propyl, and if P1 is a C2-C6 alkenyl, the C2-C6 alkenyl is preferably allyl, and P1 is a C7-C 14 In the case of aralkyl, C7-C 14 As an aralkyl, it is preferably benzyl or phenethyl.

[0300] 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 C7-C6 that may be substituted. 14 Independently selected from aralkyls, or

[0301] R1 and Q1 combine with the carbon atom they are bonded to to form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring.

[0302] If R1 and / or Q1 is a C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, i-propyl, or 2-methylpropyl. If R1 and / or Q1 is a C2-C6 alkenyl, the C2-C6 alkenyl is preferably allyl. If R1 and / or Q1 is a C1-C6 alkoxyC1-C6 alkyl, the C1-C6 alkoxyC1-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. Where R1 and / or Q1 is a 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. C7-C where R1 and / or Q1 may be substituted 14 In the case of aralkyl, C7-C 14 Preferably benzyl or phenethyl as aralkyl, and C7-C 14 Preferably, as a substituent of the aryl of the aralkyl group, it is 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.

[0303] When R1 and Q1 are bonded to the carbon atom to 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.

[0304] In each of the above formulas, P2 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14It is an aralkyl. If P2 is a C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, n-propyl, or i-propyl, and if P2 is a C2-C6 alkenyl, the C2-C6 alkenyl is preferably allyl, and P2 is a C7-C 14 In the case of aralkyl, C7-C 14 As an aralkyl, it is preferably benzyl or phenethyl.

[0305] In each of the above formulas, R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonyl C1-C6 alkyl, C2-C6 alkyneyl, a C1-C6 alkoxyC1-C6 alkyl that may be substituted by one or more halogens, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C3-C8 cycloalkoxyC1-C6 alkyl, or a C7-C 14 It is an aralkyl.

[0306] Preferably as R2, it is C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C4 hydroxyalkyl, methylsulfonyl C1-C2 alkyl, C2-C3 alkynyl, C1-C4 alkoxy C1-C2 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkoxy C1-C2 alkyl, C3-C6 cycloalkoxy C1-C2 alkyl, benzyl, and phenethyl, which may be substituted by one or more fluorines.

[0307] Specifically as R2, examples 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, phenethyl, etc.

[0308] In each of the above formulas, R3 is a hydroxyl group, O-PG2, any amino acid residue, or any peptide residue, wherein PG2 is a protecting group of a carboxyl group. When R3 is O-PG2, specific examples of PG2 include, for instance, alkyl groups such as t-butyl, trityl, cumyl, allyl, benzyl, etc. 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-state synthesis. When a peptide residue is supported on a resin for solid-state synthesis, the resin may be supported on the amino acid residue at the C-terminal end of the peptide residue, or on an amino acid residue at any other position. Preferably, the resin for solid-state synthesis is a CTC resin, a Wang resin, or a SASRIN resin, and more preferably, a CTC resin. Furthermore, 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, and more preferably 2 to 9.

[0309] Among the equations (1), the following equations:

[0310]

[0311] Specifically, amino acid residues represented as such include, for example, 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.

[0312] Among the equations (1), the following equations:

[0313]

[0314] Specifically, amino acid residues represented by can be, for example, 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.

[0315] In formula (1), where R3 is any amino acid residue, the corresponding amino acid residue specifically includes, for example, 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-NMe2, 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(iPen)-(C#CH2), D-Ser(NtBu-Aca)-(C#CH2), EtAsp-pip, MeAsp-aze, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-mor(SO2), MeAsp-NMe2, MeAsp-oxz, MeAsp-pip, MeAsp-pip(345-F6), MeAsp-pip(3-F2), MeAsp-pip(4-F2), MeAsp-pip(4-Me), Examples include MeAsp-piz(oxe), MeAsp-pyrro, MeAsp-pyrro(34-F4), MeAsp-pyrro(3-Me2), and nPrAsp-pip.

[0316] Process A is a process of reacting a compound represented by Formula (2), its salt, or their solvate with a compound represented by Formula (3), its salt, its dehydrate, or their solvate with a condensation reagent to obtain a compound represented by Formula (4), its salt, or their solvate, or a process of reacting a compound represented by Formula (2), its salt, or their solvate, and a dehydrate of a compound represented by Formula (3) (i.e., a compound represented by Formula (3'), its salt, or their solvate in the absence of a condensation reagent to obtain a compound represented by Formula (4), its salt, or their solvate.

[0317] The compound represented by the following formula (2) can be purchased from a commercial supplier or, if necessary, modified from one purchased from a commercial supplier. Specifically, for example, the compound represented by formula (2) can be manufactured by introducing P2 into one purchased from a commercial supplier.

[0318]

[0319] P2, R2, and R3 of Equation (2) are each equivalent to P2, R2, and R3 of Equation (1).

[0320] The compound represented by the following formula (3) can be purchased from a commercial supplier or, if necessary, modified from one purchased from a commercial supplier. Specifically, for example, the compound represented by formula (3) can be prepared by introducing PG1 into one purchased from a commercial supplier using a base and a PG1 introduction reagent in a solvent. Specific examples of the PG1 introduction reagent include, for example, ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate, trifluoroacetic anhydride, pentafluoropropionate anhydride, trichloroacetic anhydride, etc. Specific examples of the base include, for example, N,N-diisopropylethylamine, triethylamine, sodium methoxide, sodium ethoxide, etc. Specifically, as solvents used when introducing PG1, examples include methanol and ethanol when ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate is used as an introduction reagent. In addition, examples include dichloromethane, tetrahydrofuran, and pyridine when trifluoroacetic anhydride, pentafluoropropionate, or trichloroacetic anhydride is used as an introduction reagent.

[0321]

[0322] Among the formulas, PG1, Q1, and R1 are equivalent to PG1, Q1, and R1 of formula (1), respectively.

[0323] Process A can be carried out by applying reaction conditions known in literature. For example, methods described in the Solid State Synthesis Handbook published by Merck & Co., Ltd. on May 1, 2002, etc., are exemplified, and these may be appropriately used according to reaction conditions. As condensation reagents used in Process A, carbodiimide-based condensation agents represented by DCC (N,N'-dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), and EDCI·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), combinations of carbodiimide-based condensation agents and additives represented by HOAt, HOBt, and oxyma, HATU (O-(7-az-1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluroronium hexafluorophosphate), and HBTU (O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluroronium hexafluorophosphate), Euronium salt-based condensates represented by HCTU (O-(6-chloro-1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluroronium hexafluorophosphate) and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomopolinocarbenium hexafluorophosphate), PyAOP ((7-azabenzotriazole-1-yloxy)trispirolidinophosphonium hexafluorophosphate), PyBOP (1H-benzotriazole-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate), and PyOxim ([ethylcyano(hydroxyimino)acetate-O 2Phosphonium salt-based condensing agents represented by ]tri-1-pyrrolidinylphosphonium hexafluorophosphate, 1-chloro-N,N-2-trimethyl-1-propenylamine (Ghosez reagent), formamidinium salt-based condensing agents represented by 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), and TFFH (fluoro-N,N,N',N'-tetramethylamidinium hexafluorophosphate) may be used. Preferably, either DIC or EDCI·HCl, or a combination of DIC and Oxyma.

[0324] In addition, if PG1 is a C2-C6 haloacyl, an oxazolone represented by formula (3'), which is a dehydrated form of the compound prepared from the compound represented by formula (3), may be used in process A. The oxygen atom constituting the oxazolone ring and the carbon atom between the oxygen atom and the nitrogen atom 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 the oxazolone include, for instance, N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, thionyl chloride, etc.

[0325]

[0326] In the compound represented by formula (3'), R1 and Q1 are identical to R1 and Q1 of formula (3). R1 ​​and Q1 may form a 3- to 8-membered alicyclic ring with the carbon atom to which they are bonded, and specific examples of such 3- to 8-membered alicyclic rings include the cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, etc.

[0327] In the compound represented by formula (3'), R4 is a C1-C5 haloalkyl, and specifically, examples of C1-C5 haloalkyls include trifluoromethyl, trichloromethyl, pentafluoroethyl, 1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl, or 2,2,2-trifluoro-1-(trifluoromethyl)ethyl. Among these, trifluoromethyl is preferred.

[0328] Process B is a process of reacting a compound represented by formula (4), its salt, or their solvate with a P1-introducing reagent to obtain a peptide compound represented by formula (1), its salt, or their solvate.

[0329] In the present invention, a combination of P1X (wherein P1 is synonymous with P1 of formula (1) and X is a degreasing agent) and a base can be used as a P1 introduction reagent. In process B, P1 can be selectively introduced to the nitrogen atom to which PG1 is bound by acting P1X on the compound represented by formula (4) in the presence of a base of suitable pKa.

[0330] Specifically, as P1X, examples 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-introducing reagent is a methylation reagent, specific examples as the methylation reagent include, for instance, methyl iodide, dimethyl sulfate, methyl trifluoromethanesulfonate, methyl p-toluenesulfonate, methyl methanesulfonate, etc. When the P1-introductory reagent is an ethylation reagent, specific examples of ethylation reagents include, for instance, ethyl iodide, ethyl bromide, diethyl sulfate, ethyl trifluoromethanesulfonate, ethyl p-toluenesulfonate, ethyl methanesulfonate, etc. When the P1-introductory reagent is an allylation reagent, specific examples of allylation reagents include, for instance, allyl chloride, allyl bromide, etc. When the P1-introductory reagent is a benzylation reagent, specific examples of benzylation reagents include, for instance, benzyl chloride, benzyl bromide, etc. When the P1-introductory reagent is a phenethylation reagent, specific examples of phenethylation reagents include, for instance, (2-iodoethyl)benzene, (2-bromoethyl)benzene, etc.

[0331] When using a combination of P1X and a base as a P1-introduction reagent, the base may be one that possesses a basicity suitable for introducing P1 into the target nitrogen atom. The basicity of a base is expressed as the pKa of its conjugate acid. The pKa of the base's conjugate acid is sometimes referred to as the base's pKa.

[0332] Specifically, a base having a pKa sufficient to dehydrogenate the hydrogen of the NH group to which P1 is bound can be used.

[0333] The pKa of the conjugate acid of a base can be appropriately referenced from values ​​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 values ​​listed in the catalog of Sigma-Aldrich.

[0334] The pKa varies depending on the solvent. The pKas 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)))

[0335] Meanwhile, the pKa of the conjugated 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, respectively (Chem. Eur. J. 2002, 8, 1682-1693, values ​​listed in the Sigma-Aldrich catalog). The pKa of the conjugated acid of DBN in acetonitrile is 23.89 (Eur. J. Org. Chem., 2019, 40, 6735-6748).

[0336]

[0337] The pKa value of the conjugate acid of a base in water (calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C) 1994-2019 ACD / Labs)) and the pKa value in acetonitrile are generally 10 to 14 greater in acetonitrile.

[0338] In the present invention, a protecting group is preferably used in which the pKa (in water) of the NH group to which PG1 is bound is 11 or less. The pKa (in water) of the NH group to which PG1 is bound is preferably 6 to 11, and more preferably 8 to 11.

[0339] The pKa of the conjugate acid of the base required to deprotonate the proton of the NH group needs to be at least 2, preferably 2 to 3, more preferably 2 to 6, apart from the pKa of the NH group.

[0340] Accordingly, when the pKa of the NH group to which PG1 is bound is 6 to 11, the pKa of the conjugate acid of the base used is preferred to be (1) greater than the pKa of the NH group, (2) furthermore, the pKa is separated by at least 2, preferably 6 or more, and (3) furthermore, the base has a pKa value greater than the converted value (10 to 14) of the pKa in water to the pKa in acetonitrile. Specifically, if the pKa value (in acetonitrile) of the conjugate acid of the base is 18 to 31, 22 to 29, 22 to 30, 22 to 31, 23 to 29, 23 to 30, or 23 to 31, it can be used as the base for this reaction. The range of the pKa (in acetonitrile) of the conjugate acid of the base used is preferably 22 to 31.

[0341] In addition, when the pKa of the NH group to which PG1 is bound (in acetonitrile) is 8 to 11, the base used can be used as the base for this reaction if the pKa of the conjugate acid of the base used (in acetonitrile) 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. Preferably, the range of the pKa of the conjugate acid of the base used (in acetonitrile) is 23 to 30.

[0342] In any sun, the above base is represented by the following formula B1 having an amidine skeleton.

[0343]

[0344] [Among the formula,

[0345] RB1 and RB4 are each independently C1-C4 alkyl, or RB1 and RB4 are combined with the nitrogen atom to which RB1 is bonded and the carbon atom to which RB4 is bonded to form a 5- to 8-membered ring, and

[0346] RB2 and RB3 are each independently C1-C4 alkyl, or RB2 and RB3 are combined with the nitrogen atom to which RB2 is bonded, the nitrogen atom to which RB3 is bonded, and the carbon atom to which the corresponding nitrogen atom is bonded to form a 5- to 8-membered ring.

[0347] When RB1 to RB4 are C1-C4 alkyls, preferably methyl and ethyl can be used as the corresponding C1-C4 alkyls.

[0348] When RB1 and RB4 form a 5- to 8-membered ring, the 5- to 8-membered ring preferably includes a pyrrolidine ring, a piperidine ring, an azepan ring, etc.

[0349] When RB2 and RB3 form a 5- to 8-membered ring, preferably, the 1,4,5,6-tetrahydropyrimidine ring can be used as the 5- to 8-membered ring.

[0350] Specifically, bases represented by formula B1 include, for example, 1,8-diazabicyclo[5.4.0]undeca-7-en (DBU), 1,5-diazabicyclo[4.3.0]non-5-en (DBN).

[0351]

[0352] In a given sun, the above base is represented by the following formula B2 having a guanidine skeleton.

[0353]

[0354] [Among the formula,

[0355] RB6 is hydrogen or C1-C4 alkyl, and

[0356] RB5 and RB7 are each independently C1-C4 alkyl, or they form a 5- to 8-membered ring by combining each nitrogen atom to which they are bonded with the carbon atom to which that nitrogen atom is bonded, and

[0357] RB8 is a C1-C4 alkyl, and RB9 is a C1-C4 alkyl or phenyl; RB8 and RB9 are combined with each nitrogen atom to which they are bonded and the carbon atom to which that nitrogen atom is bonded to form a 5- to 8-membered ring, and

[0358] Here, if RB9 is phenyl, the two B2s may form naphthalene by condensing the two benzene rings of the corresponding phenyl group.

[0359] When RB5 to RB8 are C1-C4 alkyls, the corresponding C1-C4 alkyl is preferably methyl, and when RB9 is C1-C4 alkyl, the corresponding C1-C4 alkyl is preferably t-butyl.

[0360] When RB5 and RB7 form a 5- to 8-membered ring, the 5- to 8-membered ring preferably includes an imidazolidine ring, a hexahydropyrimidine ring, a 1,3-diazepan ring, etc.

[0361] When RB8 and RB9 form a 5- to 8-membered ring, preferably, the 1,4,5,6-tetrahydropyrimidine ring can be used as the 5- to 8-membered ring.

[0362] Specifically, bases represented by formula B2 include, for example, 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-en (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]deca-5-en (TBD), etc.

[0363]

[0364] In a certain sun, the above base is represented by the following formula B3 having a phosphazene backbone.

[0365]

[0366] [Among the formula,

[0367] RB 10 Silver, C1-C4 alkyl, or RB 10 and RB 11 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0368] RB 11 Silver, RB 10 and RB 11 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 11 and RB 12 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0369] RB 12 neun, RB 11 and RB 12 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 12 and RB 13 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0370] RB 13 Silver, RB 12 and RB 13 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 13 and RB 14 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0371] RB 14 neun, RB 13 and RB 14 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 14and RB 15 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0372] RB 15 neun, RB 14 and RB 15 Except for cases where it forms a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0373] RB 16 Silver hydrogen, C1-C8 alkyl, or C6-C 10 It is Aril.

[0374] RB 10 ~RB 15 a. In the case of a C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and RB 16 In the case of this, if it is a C1-C8 alkyl, the corresponding C1-C8 alkyl is preferably t-butyl or t-octyl.

[0375] RB 10 and RB 11 , RB 12 and RB 13 , and / or RB 14 and RB 15 When a 5- to 8-membered ring is formed, preferably, the 5- to 8-membered ring can be a pyrrolidine ring, a piperidine ring, an azepan ring, etc.

[0376] RB 11 and RB 12 , and / or RB 13 and RB 14 If ga forms a 5–8 member ring, the said 5–8 member ring is RB 11 , RB 12 , RB 13 , and RB 14 It is preferable that the ring be a saturated ring of 5 to 8, which does not contain heteroatoms other than each nitrogen atom bonded to it and the phosphorus atom bonded to each nitrogen atom.

[0377] Specifically, bases represented by formula B3 include, for example, tert-butylimino-tris(dimethylamino)phosphoran (P1-tBu), tert-octylimino-tris(dimethylamino)phosphoran (P1-t-Oct), tert-butylimino-tri(pyrrolidino)phosphoran (P1-t-Bu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorin (BEMP), imino-tris(dimethylamino)phosphoran (HP1(dma)), etc.

[0378]

[0379] In a certain sun, the above base is represented by the following B4, which has a phosphazene backbone comprising two phosphorus atoms interposed with a nitrogen atom.

[0380]

[0381] [Among the formula,

[0382] RB 17 silver, independently C1-C4 alkyl, or RB 17 and RB 18 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0383] RB 18 Silver, RB 17 and RB 18 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 18 and RB 19 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0384] RB 19 neun, RB 18 and RB 19 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 19 and RB 20Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0385] RB 20 Silver, RB 19 and RB 20 Except for cases forming a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0386] RB 21 Silver, C1-C4 alkyl, or RB 21 and RB 22 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0387] RB 22 neun, RB 21 and RB 22 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 22 and RB 23 Silver, each nitrogen atom to which they are bonded, and the phosphorus atom to which that respective nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0388] RB 23 Silver, RB 22 and RB 23 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 23 and RB 24 They unite with the nitrogen atoms they are bonded to to form a 5- to 8-membered ring, and

[0389] RB 24 neun, RB 23 and RB 24 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 24 and RB 25 Each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5- to 8-membered ring, and

[0390] RB 25 neun, RB 24 and RB 25Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 25 and RB 26 Silver, combined with the nitrogen atoms to which they are bonded, forms a 5–8 member ring, and

[0391] RB 26 Silver, RB 25 and RB 26 Except for cases forming a 5- to 8-membered ring, it is a C1-C4 alkyl, and

[0392] RB 27 silver, C1-C4 alkyl, or C6-C 10 It is Aril.

[0393] RB 17 ~RB 26 In the case where it is a C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and RB 27 a. In the case of a C1-C4 alkyl, the corresponding C1-C4 alkyl is preferably t-butyl.

[0394] RB 17 and RB 18 , RB 19 and RB 20 , RB 21 and RB 22 , RB 23 and RB 24 , RB 25 and RB 26 When forming this 5-8 member ring, preferably, the 5-8 member ring can be a pyrrolidine ring, a piperidine ring, an azepan ring, etc.

[0395] RB 17 and RB 18 In the case where all of these are C1-C4 alkyls, RB 19 and RB 20 It is preferable that all are C1-C4 alkyl, and RB 17 and RB 18 RB forming this 5–8-membered ring 19 and RB 20 It is preferable to form a ring of 5 to 8.

[0396] RB 21and RB 22 In the case where all are C1-C4 alkyls, RB 23 and RB 24 and RB 25 and RB 26 It is preferable that all are C1-C4 alkyl, and RB 21 and RB 22 RB, which forms a 5–8-membered ring 23 and RB 24 and RB 25 and RB 26 It is preferable to form a ring of 5 to 8.

[0397] RB 18 and RB 19 , and / or RB 22 and RB 23 In the case where this 5–8 member ring is formed, the corresponding 5–8 member ring is RB 11 , RB 12 , RB 13 , and RB 14 It is preferable that the ring be a saturated ring of 5 to 8, which does not contain heteroatoms other than each nitrogen atom bonded to it and the phosphorus atom bonded to each nitrogen atom.

[0398] Specifically, as a base represented by formula B4, for example, 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 -Catenadi(phosphazene)(P2-t-Bu), 1-ethyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 Examples include catenady (phosphazene) (P2-Et).

[0399]

[0400] In the present invention, when a combination of P1X and a base is used as a P1-introducing reagent, the solvent used in the reaction may be an amide-based solvent represented by DMF or NMP, a urea-based solvent represented by DMI, an ether-based solvent represented by tetrahydrofuran or 2-methyltetrahydrofuran, and acetonitrile, among which an amide-based solvent is preferred.

[0401] When using P1X and a base as P1 introduction reagents, a combination of PG1 and a base is preferred such that the pKa of the amino group protected by PG1 (in water) is 6 to 11 and the pKa of the conjugate acid of the base (in acetonitrile) is 23 to 30. Furthermore, a combination of PG1 and a base is more preferred such that the pKa of the amino group protected by PG1 (in water) is 8 to 11 and the pKa of the conjugate acid of the base (in acetonitrile) is 23 to 27. As for the 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, or benzyl bromide, and the base is P1-tBu, TMGN, or MTBD. Specific combinations of PG1 and bases include trifluoroacetyl and TMGN, trifluoroacetyl and P1-tBu, and trifluoroacetyl and MTBD.

[0402] In one aspect, the present invention relates to a method for preparing a peptide compound, its salt, or their solvates, comprising a dipeptide residue having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and a dipeptide residue having the N-substituted α,α-disubstituted amino acid residue linked thereto. The method may further include a step of condensing one or more amino acid residues and / or peptide residues to the N-terminus and / or C-terminus of a peptide compound having an N-substituted α,α-disubstituted amino acid residue at the N-terminus and a dipeptide residue having the N-substituted α,α-disubstituted amino acid residue linked thereto, prepared by the method described herein. The peptide compound prepared by this method is any peptide compound comprising a dipeptide residue to which an N-substituted α,α-disubstituted amino acid residue is linked, and includes a peptide compound in which any number and type of amino acids are linked to the N-terminal and C-terminal sides of the said dipeptide residue.

[0403] In one aspect, the present invention also relates to a method for producing a cyclic peptide compound, further comprising the steps of: deprotecting an N-terminal protecting group (e.g., PG1) from a peptide compound represented by formula (1) produced by the method of the present invention, its salt, or their solvate; optionally extending a peptide chain; and cyclizing a C-terminal group and an N-terminal group to form a cyclic portion.

[0404] The cyclic peptide compound comprises 8 to 15 amino acid residues, preferably 10 to 13 amino acid residues, at least 3, preferably at least 3 to (number of amino acid residues constituting the cyclic peptide compound minus 1) N-substituted amino acid residues, and also comprises at least 1, preferably at least 3 N-unsubstituted amino acid residues, and the cyclic portion comprises at least 8 amino acid residues, preferably at least 10 amino acid residues.

[0405] For the process of deprotecting PG1 from the peptide compound represented by formula (1), for example, the method described in “Greene’s, “Protective Groups in Organic Synthesis” (5th edition, John Wiley & Sons 2014) can be used.

[0406] For the process of extending the peptide chain and the process of forming the annular portion, known methods, such as the methods described in WO2013 / 100132 or WO2018 / 225864, may be used. When the peptide chain is extended by solid-state synthesis, a process of cutting out from the resin may be included prior to the extension process and the process of forming the annular portion.

[0407] Meanwhile, all prior art literature cited in this specification is incorporated herein by reference.

[0408] Examples

[0409] The contents of the present invention are further explained in the following examples, comparative examples, and reference examples, but the present invention 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.

[0410]

[0411] Example 1: Preparation of amino acids or peptides supported on resin used in this example

[0412] Example 1-1: Fmoc-amino acid used in peptide synthesis by a peptide synthesizer

[0413] In the peptide synthesis described in this specification, Fmoc-amino acids listed in Tables 3 to 5 were used for synthesis by the peptide synthesizer.

[0414] The Fmoc-amino acids listed in Tables 3 and 5 were purchased from commercial suppliers.

[0415] The Fmoc-amino acids listed in Table 4 were synthesized according to the scheme shown below.

[0416]

[0417]

[0418]

[0419]

[0420] Example 1-1-1: Synthesis of Compound AA2-001, (2S)-4-[3-chloro-4-(trifluoromethyl)phenyl]-2-(9H-fluorene-9-ylmethoxycarbonylamino)butanoic acid, (Fmoc-Hph(4-CF3-3-Cl)-OH).

[0421]

[0422] DIC (138 mL, 1.54 equivalents) was added dropwise under a nitrogen atmosphere at 0°C to a THF (2 L) solution containing (4S)-4-[(2-methylpropane-2-yl)oxycarbonylamino]-5-oxo-5-phenylmethoxypentanoic acid (Boc-Glu-OBn, CAS No. 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). The reaction mixture was stirred at 25°C for 16 hours, the solids were removed by filtration, and the filtrate was removed by solvent distillation under reduced pressure. The residue was diluted with toluene, the resulting solids were removed by filtration, and the filtrate was removed by solvent distillation under reduced pressure. The residue was purified by recrystallization (acetone / heptane) to obtain compound AA2-001-a(1-O-benzyl5-O-(1,3-dioxoisoindole-2-yl)(2S)-2-[(2-methylpropane-2-yl)oxycarbonylamino]pentanedioate). (230 g, 80%)

[0423] LCMS(ESI) m / z=505.2(M+Na)+

[0424] Retention time: 0.992 min (Analysis condition SMDmethod_16)

[0425] Nickel bromide trihydrate (NiBr2·3H2O) (4 g, 0.07 equivalent) and 4,4'-di-tert-butyl-2,2'-bipyridyl (dtbbpy) (3.9 g, 14.55 mmol, 0.07 equivalent) were added to DMA (500 mL) and stirred at 50°C for 2 hours under a nitrogen atmosphere to prepare a Ni solution.

[0426] To a DMA (500 mL) mixture of compound AA2-001-a (100 g, 207.3 mmol), zinc powder (70 g, 5 equal parts), and 4-bromo-2-chloro-1-(trifluoromethyl)benzene (160 g, 617 mmol, 3 equal parts), a previously adjusted Ni solution was added and stirred at 25°C for 16 hours. An aqueous solution of EDTA·2Na (10%) was added to the reaction mixture and extracted with ethyl acetate. The combined organic layer was washed with a saturated aqueous sodium chloride solution, dried with anhydrous sodium sulfate, and removed by solvent distillation 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%).

[0427] LCMS(ESI) m / z=494(M+Na)+

[0428] Retention time: 2.863 min (Analysis condition SMDmethod_17)

[0429] 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 equal parts) 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 with anhydrous sodium sulfate, and then removed by solvent distillation 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 equivalent weight) was added to this solution, and the mixture was stirred at room temperature for 16 hours while maintaining a pH of 7.8 to 8.0. The reaction mixture was filtered, and the pH of the filtrate was adjusted to 2 with 6 mol / L hydrochloric acid water. The precipitated solid was collected and dried at 50°C to obtain compound AA2-001 ((2S)-4-[3-chloro-4-(trifluoromethyl)phenyl]-2-(9H-fluorene-9-ylmethoxycarbonylamino)butanoic acid, Fmoc-Hph(4-CF3-3-Cl)-OH). (70 g, 87%)

[0430] LCMS(ESI) m / z=525.8(M+Na)+

[0431] Retention time: 2.180 min (Analysis condition SMDmethod_21)

[0432] 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)

[0433] Example 1-1-2: Synthesis of Compound AA2-002, (2S)-3-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]propanoic acid (Fmoc-MeAla(cBu)-OH)

[0434]

[0435] To a 46 mL solution of compound AA2-002-a ((2S)-3-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonylamino]propanoic acid, Fmoc-Ala(cBu)-OH) (3.36 g, 9.19 mmol) in DCM, 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 stirred at room temperature for 2 hours. An aqueous solution of saturated sodium chloride diluted with water to half its concentration was added to the reaction mixture, and additional DCM was added to dilute it. The separated organic layer was washed with a saturated sodium chloride aqueous solution and filtered. The obtained organic layer was removed by solvent distillation under reduced pressure to obtain compound AA2-002-b as a crude product (3.63 g).

[0436] LCMS(ESI) m / z=378(M+H)+

[0437] Retention time: 1.01 min (Analysis condition SQDFA05)

[0438] To a DCM (30.6 mL) solution of the obtained compound AA2-002-b (3.47 g), 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) were added under a nitrogen atmosphere and stirred for 2 hours. An aqueous solution of saturated sodium chloride diluted with water to half its concentration was added to the reaction mixture and stirred at room temperature for 15 minutes. The organic layer separated from the obtained mixture was removed by solvent distillation under reduced pressure. Compound AA2-002 ((2S)-3-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]propanoic acid, Fmoc-MeAla(cBu)-OH) was obtained by purifying the obtained residue by reverse-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). (3.18 g, Process 2 91%)

[0439] LCMS(ESI) m / z=380(M+H)+

[0440] Retention time: 0.94 min (Analysis condition SQDFA05)

[0441] Example 1-1-3: Synthesis of Compound AA2-003, (2S)-2-cyclopentyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]acetic acid (Fmoc-MeGly(cPent)-OH)

[0442]

[0443] Trifluoroacetic acid (TFA) (9.0 mL) was added to a mixture of compound AA2-003-a ((2S)-2-cyclopentyl-2-[9H-fluorene-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), and the mixture was stirred at 60°C for 4 hours. After cooling the reaction mixture to room temperature, the solid was removed by filtration. The filtrate was diluted with ethyl acetate (220 mL) and then washed sequentially with a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound AA2-003-b as a crude product. No further purification was performed, and the following reaction was carried out.

[0444] LCMS(ESI) m / z=378(M+H)+

[0445] Retention time: 1.01 min (Analysis condition SQDFA05)

[0446] The obtained compound AA2-003-b (31 g, 82 mmol) and triethylsilane (TES) (65.5 mL, 410 mmol) were mixed with dichloroethane (DCE) (90 mL) and trifluoroacetic acid (TFA) (76 mL, 984 mmol) was added and stirred at 60°C for 16 hours. After cooling the reaction mixture to room temperature, it was concentrated under reduced pressure, and 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-fluorene-9-ylmethoxycarbonyl(methyl)amino]acetic acid, Fmoc-MeGly(cPent)-OH) (29.1 g, 93%).

[0447] LCMS(ESI) m / z=380(M+H)+

[0448] Retention time: 0.92 min (Analysis condition SQDFA05)

[0449] Example 1-1-4: Synthesis of Compound AA2-004, (2S)-2-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]acetic acid (Fmoc-MeGly(cBu)-OH)

[0450]

[0451] Compound AA2-004-a(2S)-2-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonylamino]acetic acid, Fmoc-Gly(cBu)-OH)(2.5g, 7.11mmol) was used as a starting material, and compound AA2-004-b was obtained as a crude product by the same method as the synthesis of compound AA2-002-b.

[0452] LCMS(ESI) m / z=364(M+H)+

[0453] Retention time: 0.97 min (Analysis condition SQDFA05)

[0454] Using the entire amount of compound AA2-004-b obtained above, compound AA2-004 ((2S)-2-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]acetic acid, Fmoc-MeGly(cBu)-OH) was obtained by reacting in the same manner as the synthesis of compound AA2-002 and purifying by reverse-phase column chromatography (0.1% aqueous formic acid / 0.1% acetonitrile formic acid). (2.32 g, Process 2 89%)

[0455] LCMS(ESI) m / z=366(M+H)+

[0456] Retention time: 0.88 min (Analysis condition SQDFA05)

[0457] Example 1-1-5: Synthesis of Compound AA2-005, (2S)-3-cyclopentyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]propanoic acid (Fmoc-MeAla(cPent)-OH)

[0458]

[0459] Compound AA2-005-a ((2S)-3-cyclopentyl-2-[9H-fluorene-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 by the same method as the synthesis of compound AA2-002-b.

[0460] LCMS(ESI) m / z=392(M+H)+

[0461] Retention time: 1.05 min (Analysis condition SQDFA05)

[0462] Using the obtained compound AA2-005-b (10.5 g), compound AA2-005 ((2S)-3-cyclopentyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]propanoic acid, Fmoc-MeAla(cPent)-OH) was obtained by reacting in the same manner as the synthesis of compound AA2-002 and purifying by reverse-phase column chromatography (0.1% aqueous formic acid / 0.1% acetonitrile formic acid solution). (10.11 g, Process 2 96%)

[0463] LCMS(ESI) m / z=394(M+H)+

[0464] Retention time: 0.98 min (Analysis condition SQDFA05)

[0465] Examples 1-2: Preparation of amino acids and peptides, etc. supported on the resin used in these examples

[0466] Example 1-2-1: Synthesis of Compound 1-2-1, (3 S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidine-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro)

[0467]

[0468] In this specification, when a polymer or resin is combined with a compound, the polymer or resin portion may be indicated by ○. Additionally, for the purpose of clarifying the reaction site of the resin portion, the chemical structure of the reaction site may be indicated by connecting it to ○. 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. Meanwhile, pyrro means pyrrolidine, and in the above structure, the C-terminal carboxylic acid group forms an amide bond with pyrrolidine.

[0469]

[0470] Under a nitrogen atmosphere at 0°C, EDCI·HCl (67.1g, 350mmol), HOBt (43.4g, 321mmol), and Fmoc-Asp(OtBu)-OH (120g, 292mmol) were sequentially added to DMF (600mL) and stirred at 0°C for 1 hour. Pyrrolidine (26.3mL, 321mmol) was slowly added to the reaction mixture and stirred at 0°C for 1.5 hours. Ethyl acetate (10v) and 0.5 mol / L hydrochloric acid water (2v) were added to the reaction mixture at 0°C and the organic layer was separated. The obtained organic layer was washed sequentially with 0.5 mol / L hydrochloric acid solution, water, saturated sodium bicarbonate aqueous solution / water (1 / 1 (v / v)), and saturated sodium chloride aqueous solution / water (1 / 1 (v / v)); dried with anhydrous sodium sulfate; and removed by solvent distillation under reduced pressure to obtain compound 1-2-1-a as the crude product. (137.1 g, quant.)

[0471] LCMS(ESI) m / z=465(M+H)+

[0472] Retention time: 1.05 min (Analysis condition SQD compound AA05)

[0473] Under ice cooling, TFA (271 mL) was slowly added to a DCM (137 mL) solution of compound 1-2-1-a (137 g, 395 mmol) while keeping the temperature below 10 °C. After stirring at room temperature for 1 hour, diisopropyl ether (3.4 L) was added in four installments, the precipitated solid was filtered, taken, and dried to obtain compound 1-2-1-b ((3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidine-1-ylbutanoic acid, Fmoc-Asp-pyrro). (108.4 g, 90%)

[0474] LCMS(ESI) m / z=409(M+H)+

[0475] Retention time: 0.83 min (Analysis condition SQD compound AA05)

[0476] The loading reaction of Fmoc amino acids onto a 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 filtered reaction vessel and shaken at room temperature for 20 minutes. After removing the 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. The resin was washed with dichloromethane five times, and the obtained resin was dried overnight under reduced pressure to obtain (3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidine-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1, 59.79 g).

[0477] 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. After that, 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 (measured using Shimadzu, UV-1600 PC (cell length 1.0 cm)). By measuring the dibenzofulben derived from Fmoc of the Fmoc amino acid loaded on the resin, the loading amount of compound 1-2-1 was calculated to be 0.464 mmol / g.

[0478] Meanwhile, separate lots with different loading amounts synthesized in the same way were also used for peptide synthesis or examination.

[0479] Example 1-2-2: Preparation of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 1-2-2)

[0480]

[0481] 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 procedure of operation was followed according to the manual attached to the synthesizer.

[0482] Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) prepared in Example 1-2-1 (100 mg per column), 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%) were set in a synthesizer.

[0483] At the time of starting the synthesis, 1 mL of dichloromethane (DCM) was added per column to the set 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.

[0484] Post-Fmoc process

[0485] 0.7 mL of a 2% DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added per column and left to stand for 5 to 10 minutes to perform Fmoc removal. Subsequently, the resin was washed with DMF (0.7 mL per column, repeated 4 times).

[0486] Xinjiang process

[0487] For the resin that underwent the Fmoc removal process, a solution mixed with the set Fmoc-amino acid solution (0.30 mL per column) and DIC / DMF solution (0.36 mL per column) was added, and the mixture was left to stand at 40°C. After the reaction was complete, the resin was washed with DMF (0.7 mL per column, repeated 4 times).

[0488] Fmoc-MeVal was stretched using the above process. After stretching, the Fmoc removal process was not performed; instead, it was additionally washed with DCM, dried, and then used for subsequent examination.

[0489] Meanwhile, for the purpose of confirming that compound 1-2-2 was obtained, peptide excision was performed on a portion of the obtained resin using a TFE / DCM solution (1 / 1 (v / v)). When the excision solution was analyzed by LCMS, the formation of the target peptide Fmoc-MeVal-Asp-pyrro (compound 1-2-2*) was confirmed. Meanwhile, in this example, when * is attached to the compound number, it indicates a compound identified by excising a peptide from the resin to confirm the reaction. Compound 1-2-2* represents 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.

[0490]

[0491] LCMS(ESI) m / z=522.32(M+H)+

[0492] Retention time: 0.76 min (Analysis condition SQDFA05)

[0493] Example 1-2-3: Preparation of Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-3)

[0494]

[0495] As in Example 1-2-2, it was prepared by extending Fmoc-MePhe-OH to Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-1, 0.464 mmol / g).

[0496] Meanwhile, to confirm that compound 1-2-3 was obtained, peptide excision was performed on a portion of the obtained resin using a TFE / DCM solution (1 / 1 (v / v). The excision solution was analyzed by LCMS, and the formation of the target peptide Fmoc-MePhe-Asp-pyrro (compound 1-2-3*) was confirmed.

[0497]

[0498] LCMS(ESI) m / z=570.31(M+H)+

[0499] Retention time: 0.80 min (Analysis condition SQDFA05)

[0500] Example 1-2-4: Synthesis of (3R)-3-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, Compound 1-2-4)

[0501]

[0502] Using (3R)-3-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]butanoic acid (Fmoc-D-3-MeAbu-OH) (11.5 g, 33.9 mmol) and 2-chlorotrityl chloride resin (1.69 mmol / g, 100-200 mesh, 1% DVB, 50 g, 84.5 mmol) purchased from a commercial supplier, (3R)-3-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, Compound 1-2-4) was obtained by the same method as in the synthesis of Compound 1-2-1. (58.95 g, loading 0.343 mmol / g)

[0503] Meanwhile, in a separate lot with a different loading amount synthesized in the same way, it was also used for the peptide synthesis in this example.

[0504] Examples 1-3: Amino acids protected by protecting groups other than Fmoc for peptide synthesis, and their dehydrates

[0505] The amino acid protected by a protecting group other than Fmoc and its dehydrate, used in the peptide synthesis described in this specification, were synthesized as follows.

[0506] Example 1-3-1: Preparation of 2-methyl-2-[(2,2,2-trifluoroacetyl)amino]propanoic acid (Tfa-Aib-OH) (Compound 1-3-1)

[0507]

[0508] To 2-amino-2-methylpropanoic acid (25.0 g), methanol (242 mL), DIPEA (63.5 mL, 1.5 equal parts), and ethyl trifluoroacetate (CAS No. 383-63-1), 37.6 mL, 1.3 equal parts were added, and the mixture was stirred at 50°C for 18 hours. Afterward, the solvent was removed by vacuum distillation, and a 1N aqueous hydrochloric acid solution and ethyl acetate were added to the resulting residue to separate the organic layer and the aqueous layer. The obtained organic layer was washed with a saturated aqueous sodium chloride solution, dried with anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain 18.2 g of crude product.

[0509] After dissolving the crude product (16.0 g) in TBME (80 mL), heptane (320 mL) was added dropwise over a period of more than 1 hour while stirring. The mixture was ice-cooled, stirred for an additional 1 hour, and then filtered. The obtained 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).

[0510] LCMS(ESI) m / z=197.93(MH)-

[0511] Retention time: 0.40 min (Analysis condition SQDFA05)

[0512] Example 1-3-2. Synthesis of Tfa-(Me) Abu-OH((S)-2-methyl-2-(2,2,2-trifluoroacetamide)butanoic acid, Compound 1-3-2-b).

[0513]

[0514] Diisopropylethylamine (82.7 g, 640 mmol) and ethyl trifluoroacetate (54.6 g, 384 mmol) were added to a methanol (150 mL) solution of compound 1-3-2-a ((S)-2-amino-2-methylbutanoic acid, isovalin, H-(Me)Abu-OH) (15.0 g, 128 mmol), and the mixture was stirred at 50°C for 16 hours. After cooling the reaction mixture to room temperature, it was concentrated under reduced pressure, and the resulting residue was dissolved in TBME and washed twice with a 1N aqueous hydrochloric acid solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. By recrystallizing the obtained crude product from TBME / hexane (1:7), compound 1-3-2-b ((S)-2-methyl-2-(2,2,2-trifluoroacetamide)butanoic acid) (12 g, 44%) was obtained.

[0515] LCMS(ESI) m / z=214.0(M+H)+

[0516] Retention time: 0.32 min (Analysis condition SQDFA05)

[0517] Example 1-3-3. Synthesis of Tfa-(Me)Leu-OH((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamide)pentanoic acid, Compound 1-3-3-b).

[0518]

[0519] Diisopropylethylamine (40.1 g, 310 mmol) and ethyl trifluoroacetate (44.0 g, 310 mmol) were added to a methanol (50 mL) solution of compound 1-3-3-a (2-methylleucine, (S)-2-amino-2,4-dimethylpentanoic acid, H-(Me)Leu-OH) (15.0 g, 103 mmol), and the mixture was stirred at 50°C for 16 hours. After cooling the reaction mixture to room temperature, it was concentrated under reduced pressure, and the resulting residue was dissolved in TBME and washed twice with a 1N aqueous hydrochloric acid solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Compound 1-3-3-b ((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamide)pentanoic acid) (10 g, 40%) was obtained by recrystallizing the obtained crude product from TBME / hexane (1:7).

[0520] LCMS(ESI) m / z=242.1(M+H)+

[0521] Retention time: 0.66 min (Analysis condition SQDFA05)

[0522] Example 1-3-4. Synthesis of Tfa-(Me)Ser(Me)-OH((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid, Compound 1-3-4-b).

[0523]

[0524] Diisopropylethylamine (5.9 mL, 34 mmol) and ethyl trifluoroacetate (4.0 mL) were added to a methanol (19 mL) solution of 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), and the mixture was stirred at 50°C for 21 hours. After cooling the reaction mixture to room temperature, it was concentrated under reduced pressure, and the resulting residue was dissolved in TBME (45 mL). The mixture was then washed twice with a 1N aqueous hydrochloric acid solution (45 mL) and once with a saturated aqueous sodium chloride solution (45 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the crude product. Compound 1-3-4-b ((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid) (2.07 g, 72%) was obtained by purifying the obtained crude product by reverse-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile).

[0525] LCMS(ESI) m / z=228.2(MH)-

[0526] Retention time: 0.41 min (Analysis condition SQDFA05)

[0527] Example 1-3-5. Synthesis of Tfa-(Me)Phe-OH((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamide)propanoic acid, Compound 1-3-5-b).

[0528]

[0529] Diisopropylethylamine (21.63 g, 167.4 mmol) and ethyl trifluoroacetate (23.78 g, 167.4 mmol) were added to a methanol (500 mL) solution of compound 1-3-5-a ((2S)-2-amino-2-methyl-3-phenylpropanoic acid, H-(Me)Phe-OH) (10.0 g, 55.8 mmol), and the mixture was stirred at 50°C for 16 hours. After cooling the reaction mixture to room temperature, it was concentrated under reduced pressure, and the resulting residue was dissolved in TBME and washed twice with a 1N aqueous hydrochloric acid solution and once with a saturated aqueous sodium chloride solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. By recrystallizing the obtained crude product from TBME / hexane (1:15), compound 1-3-5-b ((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamide)propanoic acid) (8 g, 52%) was obtained.

[0530] LCMS(ESI) m / z=274.0(MH)-

[0531] Retention time: 0.68 min (Analysis condition SQDFA05)

[0532] Example 1-3-6. Synthesis of Tfa-(Me)Cha-OH((S)-3-cyclohexyl-2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid, Compound 1-3-6-c).

[0533]

[0534] 4-(3-phenylpropyl)piperidine (4.7 mL, 22 mmol) was added to a dichloromethane (18.4 mL) solution of compound 1-3-6a (2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-cyclohexyl-2-methylpropanoic acid, Fmoc-(Me)Cha-OH) and stirred for 16 hours at room temperature under a nitrogen atmosphere. Water (8 mL) was added to the reaction mixture to extract the product, and the aqueous layer was purified by reverse-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). Additionally, water (5 mL) and 2N hydrochloric acid (5 mL) were added to the organic phase, and the remaining crude product was extracted into the aqueous layer, after which the aqueous layer was purified by reverse-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). The column purified product was combined to form compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropanoic acid, H-(Me)Cha-OH) (1.1 g, 81%) and used in the following reaction.

[0535] LCMS(ESI) m / z=186.1(M+H)+

[0536] Retention time: 0.32 min (Analysis condition SQDFA05)

[0537] Diisopropylethylamine (3.1 mL, 18 mmol) and ethyl trifluoroacetate (2.1 mL) were added to a methanol (20 mL) solution of compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropanoic acid, H-(Me)Cha-OH) (1.1 g, 6.0 mmol), and the mixture was stirred at 50°C for 2 hours. After cooling the reaction mixture to room temperature, diisopropylethylamine (3.1 mL, 18 mmol) and ethyl trifluoroacetate (2.1 mL) were added, and the mixture was stirred at 50°C for 20 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in TBME (30 mL), then washed twice with a 1N aqueous hydrochloric acid solution (30 mL) and once with a saturated aqueous sodium chloride solution (40 mL). The organic layer was dried with 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-trifluoroacetamide)propanoic acid) (1.22 g, 72%).

[0538] LCMS(ESI) m / z=280.2(MH)-

[0539] Retention time: 0.75 min (Analysis condition SQDFA05)

[0540] Example 1-3-7. Synthesis of Tfa-(Me)Val-OH((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamide)butanoic acid, Compound 1-3-7-b).

[0541]

[0542] Diisopropylethylamine (8.0 mL, 46 mmol) and ethyl trifluoroacetate (5.5 mL) were added to a methanol (25 mL) solution of compound 1-3-7-a ((S)-2-amino-2,3-dimethylbutanoic acid, H-(Me)Val-OH) (2.0 g, 15 mmol), and the mixture was stirred at 50°C for 3 hours. After cooling the reaction mixture to room temperature, diisopropylethylamine (4.0 mL, 23 mmol) and ethyl trifluoroacetate (2.7 mL) were added, and the mixture was stirred at 50°C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in TBME (40 mL) and then washed sequentially with a 1N aqueous hydrochloric acid solution (40 mL) and a saturated aqueous sodium chloride solution (40 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the crude product. Compound 1-3-7-b ((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamide)butanoic acid) (1.17 g, 34%) was obtained by purifying the obtained crude product by reverse-phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile).

[0543] LCMS(ESI) m / z=226.1(MH)-

[0544] Retention time: 0.54 min (Analysis condition SQDFA05)

[0545] Example 1-3-8. Synthesis of Tfa-cLeu-OH(1-(2,2,2-trifluoroacetamide)cyclopentane-1-carboxylic acid, Compound 1-3-8-b)

[0546]

[0547] Diisopropylethylamine (37.5 g, 290 mmol) and ethyl trifluoroacetate (41.3 g, 290 mmol) were added to a 100 mL methanol solution of compound 1-3-8-a (1-aminocyclopentane carboxylic acid, H-cLeu-OH) (25 g, 194 mmol), and the mixture was stirred at 50°C for 2 days. After cooling the reaction mixture to room temperature, diisopropylethylamine (4.0 mL, 23 mmol) and ethyl trifluoroacetate (2.7 mL) were added, and the mixture was stirred at 50°C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in TBME and then washed sequentially with a 1 N aqueous hydrochloric acid solution and a saturated aqueous sodium chloride solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the crude product. By recrystallizing the obtained crude product from TBME / hexane (3:20), compound 1-3-8-b (1-(2,2,2-trifluoroacetamide)cyclopentane-1-carboxylic acid) (20 g, 46%) was obtained.

[0548] LCMS(ESI) m / z=224.0(MH)-

[0549] Retention time: 0.49 min (Analysis condition SQDFA05)

[0550] Example 1-3-9. Synthesis of 2-(trifluoromethyl)-3-oxa-1-azaspiro[4,4]nona-1-en-4-one (Compound 1-3-9)

[0551]

[0552] 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-trifluoroacetamide)cyclopentane-1-carboxylic acid) (25 g, 111 mmol) and stirred at room temperature for 2 days. After the reaction, the reaction mixture 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]nona-1-en-4-one) (11.9 g, 52%).

[0553] LCMS(ESI) m / z=208.1(M+H)+

[0554] Retention time: 0.86 min (Analysis condition SQDAA05)

[0555] Example 2: An experiment in which, regarding an N-terminally N-substituted amino acid residue in a peptide during solid-phase synthesis, an N-unsubstituted α,α-disubstituted amino acid with a Tfa-protected N-terminus was extended, and the introduction of an N-substituted α,α-disubstituted amino acid residue was attempted through N-functionalization in the solid phase.

[0556]

[0557] Example 2-1: Extension of Tfa-Aib-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0558] Example 2-1-1: Extension of Tfa-Aib-OH using DIC after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0559] 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 added to a reaction vessel 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 5 minutes to de-Fmoc. After removing the de-Fmoc solution, the resin was washed four times with DMF (0.7 mL).

[0560] For the obtained resin, an extension reaction of Tfa-Aib-OH was performed.

[0561] The elongation reaction was carried out by adding a solution of 0.6M Tfa-Aib-OH / NMP solution (0.3mL) and 10% DIC / DMF solution (0.36mL) to the resin and shaking at 40 degrees for 20 hours.

[0562] After removing the liquid phase of the elongation 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 Lot 1 of compound 2-1 (Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) (hereinafter referred to as compound 2-1-1).

[0563] To confirm the progress of the reaction, the obtained resin (Compound 2-1-1) was subjected to peptide excision with a TFE / DCM solution (1 / 1 (v / v)), and the excision solution was analyzed by LCMS to confirm the formation 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.

[0564]

[0565] LCMS(ESI) m / z=481.21(M+H)+

[0566] Retention time: 0.53 min (Analysis condition SQDFA05)

[0567] Example 2-1-2: Extension of Tfa-Aib-OH with oxyma added as an additive using DIC after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0568] 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 added to a reaction vessel with a filter, and Tfa-Aib was extended in the same manner as in Example 2-1-1, except for the extension reagent. The extension reagent used was a solution mixed with 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, a peptide was extruded from the resin and analyzed by LCMS. The results showed that the target peptide Tfa-Aib-MeVal-Asp-pyrro (Compound 2-1*) accounted for 87.50% (UV area), in addition to 2.2% (UV area) of unreacted H-MeVal-Asp-pyrro and two unidentified peaks (8.85% and 1.43%, respectively (UV area)). This resin (Lot 2 of Compound 2-1, hereinafter referred to as Compound 2-1-2) was used in Comparative Example 1.

[0569] Example 2-1-3: Extension of Tfa-Aib-OH using EDCI·HCl after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0570] 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 added to a reaction vessel with a filter, and Tfa-Aib was extended in the same manner as in Example 2-1-1, except for the extension reagent. The extension reagent used was a solution prepared by mixing 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, a peptide was extruded from the resin and analyzed by LCMS. The results showed that in addition to the target peptide Tfa-Aib-MeVal-Asp-pyrro (Compound 2-1*) at 93.1% (UV area), unreacted H-MeVal-Asp-pyrro at 3.0% (UV area) and an unidentified peak at 3.9% (UV area) were detected. 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.

[0571] Example 2-2: N-methylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) by nucleus substitution reaction (methyl iodide as methylating agent, DBU as base)

[0572] 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 in a reaction vessel with a filter, 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).

[0573] To the obtained resin, 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. After removing the liquid phase with a filter, the resin was washed four times with NMP (0.7 mL) and four times with dichloromethane. A small sample of the obtained resin was taken, and peptides were extracted using a TFE / DCM solution (1 / 1 (v / v)), and the extracted solution was analyzed by LCMS.

[0574] For the resin that underwent the first methylation, the same operation was performed again for the purpose of improving the reaction conversion rate. The second methylation was carried out by shaking at 40°C for 20 hours. Compound 2-2 was obtained by washing the resin. A small amount of the obtained resin was sampled, and peptide excision was performed using a TFE / DCM solution (1 / 1 (v / v)). The excision solution was analyzed by LCMS to identify the target product (Compound 2-2*) and unreacted product (Compound 2-1*). The results are as shown in Table 6.

[0575]

[0576]

[0577] LCMS(ESI) m / z=495.23(M+H)+

[0578] Retention time: 0.57 min (Analysis condition SQDFA05)

[0579] From these results, it was found that in a nucleus substitution reaction using methyl iodide as a methylating agent and DBU (pKa=24.34 in conjugated acetonitrile (J. Org. Chem. 2005, 70, 1019-1028)) as a base, Tfa-protected N-terminal selective N-methylation proceeded. It was shown that the conversion rate of the reaction could be improved by repeating the reaction with different reagents.

[0580] Example 2-3: N-methylation of the Tfa amide site of Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) by nucleus substitution reaction (methyl iodide and various bases as methylating agents)

[0581] 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 in a reaction vessel with a filter, 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).

[0582] To the obtained resin, a base (amount added is listed in Table 7) / DMF (0.35 mL) solution was added, followed by the addition of a 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 in DMF (0.7 mL) and four times in dichloromethane to obtain Compound 2-2. A small amount of the obtained resin was sampled, and the peptide was excised using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS.

[0583] The results of confirming the reaction by cutting were as shown in Table 7. When P1-tBu was used as the base, the formation of an excess methylated product (compound 2-3*) was also observed.

[0584]

[0585]

[0586] LCMS(ESI) m / z=509.25(M+H)+

[0587] Retention time: 0.59 min (Analysis condition SQDFA05)

[0588] From these results, it was shown that the desired N-methylation proceeded in a nucleus substitution reaction using MTBD (pKa=25.43 in conjugated acetonitrile) (Chem. Eur. J. 2002, 8, 1682-1693)), TMGN (pKa=25.1 in conjugated acetonitrile) and P1-tBu (pKa=26.9 in conjugated acetonitrile) (Aldrich website on phosphazenes bases https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html (accessed on October 10, 2019)) which have stronger basicity than DBU as bases. On the other hand, when P1-tBu was used, a small amount (3.8%) of excess methylated product was found at a secondary amide site (an amide site composed of the amino group of Asp and the carboxyl group of MeVal) separate from the Tfa amide site in question. From this result, it is inferred that in order to selectively N-methylate at the Tfa amide site, it is more desirable to use a base with a pKa value of 27 or less of the conjugated acid.

[0589] Example 2-4: Deprotection of Tfa protection by Tfa-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-2) after N-alkylation

[0590] Under a nitrogen atmosphere, 0.5 g of sodium borohydride was mixed with 6.6 mL of triglaim (triethylene glycol dimethyl ether) and stirred at room temperature for 10 minutes to obtain a 2.0 M sodium borohydride / triglaim solution.

[0591] In a reaction vessel with a filter, N-methylated resin (Compound 2-2) prepared in run 3 of Table 7, Example 2-3, using TMGN as the base was added, 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).

[0592] THF (125 μL), methanol (63 μL), and the previously prepared 2.0 M sodium borohydride / triglaim solution (63 μL) were added to the obtained resin and shaken at room temperature for 30 minutes. After removing the liquid phase with a filter, the resin was washed four times with methanol (0.7 mL) (each wash time was 1 minute), followed by four washes with dichloromethane (0.7 mL) to obtain Compound 2-4. A small sample of the obtained resin was taken, and the peptide was excised using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS.

[0593] The Tfa-protected source 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 as shown in Figure 1, and it was confirmed that synthesis with high purity is possible.

[0594]

[0595] Purpose peptide H-MeAib-MeVal-Asp-pyrro(compound 2-4*)

[0596] LCMS(ESI) m / z=399.23(M+H)+

[0597] Retention time: 0.35 min (Analysis condition SQDFA05)

[0598] As described above, the present invention has shown that N-methyl-α,α-dialkylamino acid can be introduced with high purity following bulky N-alkylamino acid. Furthermore, it has been confirmed that the subsequent Tfa removal process proceeds well, and it is possible to continue performing conventional peptide extensions from the N-terminus.

[0599] Examples 2-5. Experiments in which various N-methyl-α,α-dialkylamino acids were subsequently introduced into bulky N-methylamino acid (MeVal) in a solid phase.

[0600] 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 general formula below.

[0601]

[0602]

[0603]

[0604] Example 2-5-1. Synthesis of Tfa-Me(Me) Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-1-2)

[0605] Example 2-5-1-1. Extension of Tfa-(Me) Abu-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0606] 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 added to a reaction vessel 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 5 minutes to de-Fmoc. After removing the de-Fmoc solution, the resin was washed four times with DMF (0.7 mL).

[0607] For the obtained resin, an extension reaction of Tfa-(Me) Abu-OH (compound 1-3-2-b) was performed.

[0608] The elongation reaction was carried out by adding a solution of 0.6M Tfa-(Me) Abu-OH (compound 1-3-2-b) / NMP solution (0.3mL) and 10% DIC / DMF solution (0.36mL) to the resin and shaking at 60 degrees for 48 hours.

[0609] 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).

[0610] To confirm the progress of the reaction, a portion of the obtained resin (Compound 2-5-1-1) was extracted, 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 formation of the target peptide Tfa-(Me) Abu-MeVal-Asp-pyrro (Compound 2-5-1-1*). No other peptide components were detected. After elongation, the material was washed with DCM, dried, and used for subsequent examination.

[0611]

[0612] LCMS(ESI) m / z=495.4(M+H)+

[0613] Retention time: 0.56 min (Analysis condition SQDFA05)

[0614] Example 2-5-1-2. N-methylation of the Tfa amide site of Tfa-(Me) Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-1-1) by nucleus substitution reaction

[0615] 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 added to a reaction vessel 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 four times with DMF (0.7 mL).

[0616] To the obtained resin, a TMGN (27 mg) / DMF (0.175 mL) solution was added, followed by a methyl iodide (31 μL) / DMF (0.175 mL) solution, and the mixture was shaken at 40°C for 1 hour. After removing the liquid phase with a filter, the mixture was washed twice with DMF (0.7 mL). A small sample of the obtained resin was taken, and peptides were extracted using a TFE / DCM / DIPEA solution (1:1:0.015), and the extracted solution was analyzed by LCMS.

[0617] For the resin that underwent the first methylation, the same operation was performed three additional times for the purpose of improving the reaction conversion rate. The second methylation was carried out by shaking at 40°C for 1.5 hours. The third and fourth methylations were carried out by shaking at 40°C for 1 hour. After four methylations, the resin was washed four times with DMF and additionally four times with DCM to obtain compound 2-5-1-2. A portion of the obtained resin was extracted, and the peptide was excised using a TFE / DCM / DIPEA solution (1:1:0.015). The excised solution was analyzed by LCMS to confirm 94.0% (UV area) of the target peptide Tfa-Me(Me) Abu-MeVal-Asp-pyrro (compound 2-5-1-2*) and 6.0% (UV area) of the O-methylated form of the Tfa amide site (compound 2-5-1-2a*).

[0618]

[0619] LCMS(ESI) m / z=509.5(M+H)+

[0620] Retention time: 0.60 min (Analysis condition SQDFA05)

[0621]

[0622] LCMS(ESI) m / z=509.5(M+H)+

[0623] Retention time: 0.69 min (Analysis condition SQDFA05)

[0624] Example 2-5-2. Synthesis of Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-2-2)

[0625] Example 2-5-2-1. Extension of Tfa-(Me)Leu-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0626] Compound 2-5-2-1 (Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same way 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) according to the method shown in Example 2-5-1-1. A portion of the obtained resin was extracted, 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-(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-1*) (84.5% UV area), an excess elongator of MeVal-Asp-pyrro (compound 2-5-2-1a*) 6.8% (UV area) was identified as the main impurity (conversion efficiency after de-Fmoc was >99%).

[0627]

[0628] LCMS(ESI) m / z=523.5(M+H)+

[0629] Retention time: 0.67 min (Analysis condition SQDFA05)

[0630]

[0631] LCMS(ESI) m / z=804.7(M+H)+

[0632] Retention time: 0.73 min (Analysis condition SQDFA05)

[0633] Example 2-5-2-2. N-methylation of the Tfa amide site of Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-2-1) by nucleus substitution reaction

[0634] Compound 2-5-2-2 (Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same way using compound 2-5-2-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the obtained resin was extracted, and the peptide was cut using a TFE / DCM / DIPEA solution (1:1:0.015). The cut solution was analyzed by LCMS to confirm the target peptide Tfa-Me(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-2*) (68.5% UV area), in addition to 17.0% (UV area) of the O-methylated form of the Tfa amide site (compound 2-5-2-2a*) and the starting material compound 2-5-2-1* (14.5% UV area).

[0635]

[0636] LCMS(ESI) m / z=537.5(M+H)+

[0637] Retention time: 0.70 min (Analysis condition SQDFA05)

[0638]

[0639] LCMS(ESI) m / z=537.5(M+H)+

[0640] Retention time: 0.81 min (Analysis condition SQDFA05)

[0641] Example 2-5-3. Synthesis of Tfa-Me(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-3-2)

[0642] Example 2-5-3-1. Extension of Tfa-(Me)Ser(Me)-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0643] 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) according to the method shown in Example 2-5-1-1. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-(Me)Ser(Me)-MeVal-Asp-pyrro (compound 2-5-3-1*). No other peptide components were detected.

[0644]

[0645] LCMS(ESI) m / z=511.4(M+H)+

[0646] Retention time: 0.55 min (Analysis condition SQDFA05)

[0647] Example 2-5-3-2. N-methylation of the Tfa amide site of Tfa by nucleus substitution reaction

[0648] 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 obtained resin was extracted, 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 formation of the target peptide Tfa-Me(Me)Ser(Me)-MeVal-Asp-pyrro (compound 2-5-3-2*) (94.0% UV area).

[0649]

[0650] LCMS(ESI) m / z=525.5(M+H)+

[0651] Retention time: 0.61 min (Analysis condition SQDFA05)

[0652] Example 2-5-4. Synthesis of Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-4-2)

[0653] Example 2-5-4-1. Extension of Tfa-(Me)Phe-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0654] 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 using compound 1-2-2 (0.552 mmol / g, 100 mg) and a 0.6 M Tfa-(Me)Phe-OH (compound 1-3-5-b) / DMF solution (0.3 mL) with a reaction time of 72 hours. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-1*) (81.4% UV area). The conversion efficiency after de-Fmoc is >99%, and in addition to the excess elongator of MeVal-Asp-pyrro (compound 2-5-4-1a*) 4.1% (UV area) as the main impurity, a peak of unknown structure was detected.

[0655]

[0656] LCMS(ESI) m / z=557.5(M+H)+

[0657] Retention time: 0.68 min (Analysis condition SQDFA05)

[0658]

[0659] LCMS(ESI) m / z=838.7(M+H)+

[0660] Retention time: 0.73 min (Analysis condition SQDFA05)

[0661] Example 2-5-4-2. N-methylation of the Tfa amide site of Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-4-1) by nucleus substitution reaction

[0662] Compound 2-5-4-2 (Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-5-4-1 (45 mg) by the method shown in Example 2-5-1-2. At this time, the second N-methylation was also carried out for 1 hour. A portion of the obtained resin was extracted, and the peptide was cut using a TFE / DCM / DIPEA solution (1:1:0.015). The cut solution was analyzed by LCMS to confirm the formation of the target peptide Tfa-Me(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-2*) (79.7% UV area), and in addition, an excess elongated 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%).

[0663]

[0664] LCMS(ESI) m / z=571.5(M+H)+

[0665] Retention time: 0.74 min (Analysis condition SQDFA05)

[0666]

[0667] LCMS(ESI) m / z=852.7(M+H)+

[0668] Retention time: 0.79 min (Analysis condition SQDFA05)

[0669]

[0670] LCMS(ESI) m / z=571.5(M+H)+

[0671] Retention time: 0.79 min (Analysis condition SQDFA05)

[0672] Example 2-5-5. Synthesis of Tfa-Me(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-5-2)

[0673] Example 2-5-5-1. Extension of Tfa-(Me)Cha-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0674] 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 using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-(Me)Cha-OH (compound 1-3-6-c) / DMF solution (0.3 mL) with a reaction time of 72 hours. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-(Me)Cha-MeVal-Asp-pyrro (compound 2-5-5-1*) (81.1% UV area). Multiple peaks of unknown structure were detected, including an excess elongator of MeVal-Asp-pyrro (compound 2-5-5-1a*) as the main impurity (conversion efficiency after de-Fmoc was 100%).

[0675]

[0676] LCMS(ESI) m / z=563.6(M+H)+

[0677] Retention time: 0.76 min (Analysis condition SQDFA05)

[0678]

[0679] LCMS(ESI) m / z=844.8(M+H)+

[0680] Retention time: 0.80 min (Analysis condition SQDFA05)

[0681] Example 2-5-5-2. N-methylation of the Tfa amide site of Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-5-1) by nucleus substitution reaction

[0682] 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 for 1 hour. A portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to confirm the target peptide Tfa-Me(Me)Cha-MeVal-Asp-pyrro (compound 2-5-5-2*) (74.4% UV area), in addition to the O-methylated form of the Tfa amide site (compound 2-5-5-2a*) at 12.1% (UV area) and the starting material compound 2-5-5-1* (13.5% UV area).

[0683]

[0684] LCMS(ESI) m / z=577.5(M+H)+

[0685] Retention time: 0.80 min (Analysis condition SQDFA05)

[0686]

[0687] LCMS(ESI) m / z=577.5(M+H)+

[0688] Retention time: 0.92 min (Analysis condition SQDFA05)

[0689] Example 2-5-6. Synthesis of Tfa-Me(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-6-2)

[0690] Example 2-5-6-1. Extension of Tfa-(Me)Val-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0691] 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 using compound 1-2-2 (0.552 mmol / g, 100 mg) and a 0.6 M Tfa-(Me)Val-OH (compound 1-3-7-b) / DMF solution (0.3 mL) with a reaction time of 72 hours. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-(Me)Val-MeVal-Asp-pyrro (compound 2-5-6-1*) (66.6% UV area). Multiple peaks of unknown structure containing an excess elongator of MeVal-Asp-pyrro (compound 2-5-6-1a*) as an impurity were detected (conversion efficiency after de-Fmoc was 98%).

[0692]

[0693] LCMS(ESI) m / z=509.5(M+H)+

[0694] Retention time: 0.59 min (Analysis condition SQDFA05)

[0695]

[0696] LCMS(ESI) m / z=790.7(M+H)+

[0697] Retention time: 0.67 min (Analysis condition SQDFA05)

[0698] Example 2-5-6-2. N-methylation of the Tfa amide site of Tfa-(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-6-1) by nucleus substitution reaction

[0699] 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 way using compound 2-5-6-1 (40 mg). At this time, the second N-methylation was also carried out for 1 hour. A portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded 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), 17.1% of the O-methylated form of the Tfa amide site (compound 2-5-6-2a*) (UV area) and the starting material compound 2-5-6-1* (39.6% UV area) were identified. Additionally, multiple peaks of unknown structure were detected.

[0700]

[0701] LCMS(ESI) m / z=523.5(M+H)+

[0702] Retention time: 0.65 min (Analysis condition SQDFA05)

[0703]

[0704] LCMS(ESI) m / z=523.5(M+H)+

[0705] Retention time: 0.77 min (Analysis condition SQDFA05)

[0706] Example 2-5-7. Synthesis of Tfa-MecLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 2-5-7-2)

[0707] Example 2-5-7-1. Extension of Tfa-cLeu-OH after deFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2).

[0708] Compound 2-5-7-1 (Tfa-cLeu-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-cLeu-OH (compound 1-3-8-b) / DMF solution (0.3 mL) according to the method shown in Example 2-5-1-1. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-cLeu-MeVal-Asp-pyrro (compound 2-5-7-1*). No other peptide components were detected.

[0709]

[0710] LCMS(ESI) m / z=507.4(M+H)+

[0711] Retention time: 0.56 min (Analysis condition SQDFA05)

[0712] Example 2-5-7-2. N-methylation of the Tfa amide site of Tfa-cLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-5-7-1) by nucleus substitution reaction

[0713] 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 extracted, and the peptide was cut using a TFE / DCM / DIPEA solution (1:1:0.015). The cut solution was analyzed by LCMS to confirm the formation of the target peptide Tfa-MecLeu-MeVal-Asp-pyrro (compound 2-5-7-2*) (92.1% UV area), and 7.9% (UV area) of O-methylated bodies (compound 2-5-7-2a*) of the Tfa amide site were detected as impurities (conversion efficiency of the starting material was 100%).

[0714]

[0715] LCMS(ESI) m / z=521.4(M+H)+

[0716] Retention time: 0.61 min (Analysis condition SQDFA05)

[0717]

[0718] LCMS(ESI) m / z=521.4(M+H)+

[0719] Retention time: 0.70 min (Analysis condition SQDFA05)

[0720] From the results of Examples 2-5 above, it has been shown that, by the method of the present invention, it is possible to introduce various N-methyl-α,α-dialkylamino acids other than MeAib into bulky N-methylamino acids in solid-phase synthesis at a practical level.

[0721] Examples 2-6. Experiments in which various N-substituted α,α-dialkylamino acids were subsequently introduced into bulky N-methylamino acid (MeVal) in a solid phase.

[0722] Compounds 2-6-1 to 2-6-4 were synthesized using various Tfa-amino acids according to the general formula below.

[0723]

[0724]

[0725] 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)

[0726] Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) (0.552 mmol / g, 50 mg), prepared by the same method as in Example 2-1-1, was added to a reaction vessel 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 four times with DMF (0.7 mL).

[0727] To the obtained resin, a TMGN (29 mg) / DMF (0.175 mL) solution was added, followed by the addition of an ethyl iodide (44 μL) / DMF (0.175 mL) solution, and the mixture was shaken at 60°C for 1 hour. After removing the liquid phase with a filter, the mixture was washed twice with DMF (0.7 mL). A small sample of the obtained resin was taken, and peptides were extracted using a TFE / DCM / DIPEA solution (1:1:0.015), and the extracted solution was analyzed by LCMS.

[0728] For the resin that underwent the first ethylation, the same operation was performed four additional times for the purpose of improving the reaction conversion rate. After the fifth ethylation, the resin was washed four times with DMF and additionally four times with DCM to obtain compound 2-6-1(Tfa-EtAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to confirm the target peptide Tfa-EtAib-MeVal-Asp-pyrro (compound 2-6-1*) (26.1% UV area), in addition to the O-ethylated form of the Tfa amide site (compound 2-6-1a*) at 15.4% (UV area), and the starting material compound 2-1* (54.8% UV area).

[0729]

[0730] LCMS(ESI) m / z=509.4(M+H)+

[0731] Retention time: 0.60 min (Analysis condition SQDFA05)

[0732]

[0733] LCMS(ESI) m / z=509.4(M+H)+

[0734] Retention time: 0.67 min (Analysis condition SQDFA05)

[0735] 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)

[0736] Compound 2-6-2 (Tfa-nPrAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same way 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 portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to confirm the target peptide Tfa-nPrAib-MeVal-Asp-pyrro (compound 2-6-2*) (10.0% UV area), in addition to the On-propylated form of the Tfa amide site (compound 2-6-2a*) at 6.3% (UV area), and the starting material compound 2-1* (82.7% UV area).

[0737]

[0738] LCMS(ESI) m / z=523.5(M+H)+

[0739] Retention time: 0.66 min (Analysis condition SQDFA05)

[0740]

[0741] LCMS(ESI) m / z=523.5(M+H)+

[0742] Retention time: 0.73 min (Analysis condition SQDFA05)

[0743] 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)

[0744] 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 portion of the obtained resin was extracted, and the peptide was cut using a TFE / DCM / DIPEA solution (1:1:0.015). The cut solution was analyzed by LCMS to confirm the target peptide Tfa-AllylAib-MeVal-Asp-pyrro (compound 2-6-3*) (20.3% UV area), in addition to 6.9% of the O-allylated compound of the Tfa amide site (compound 2-6-3a*) (UV area) and the starting material compound 2-1* (72.8% UV area).

[0745]

[0746] LCMS(ESI) m / z=521.4(M+H)+

[0747] Retention time: 0.64 min (Analysis condition SQDFA05)

[0748]

[0749] LCMS(ESI) m / z=521.5(M+H)+

[0750] Retention time: 0.71 min (Analysis condition SQDFA05)

[0751] 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)

[0752] 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 portion of the obtained resin was extracted, and the peptide was cut using a TFE / DCM / DIPEA solution (1:1:0.015). The cut solution was analyzed by LCMS to confirm the target peptide Tfa-BnAib-MeVal-Asp-pyrro (compound 2-6-4*) (6.2% UV area), in addition to 7.7% of the O-benzyl derivative of the Tfa amide site (compound 2-6-4a*) (UV area) and the starting material compound 2-1* (79.5% UV area).

[0753]

[0754] LCMS(ESI) m / z=571.5(M+H)+

[0755] Retention time: 0.72 min (Analysis condition SQDFA05)

[0756]

[0757] LCMS(ESI) m / z=571.5(M+H)+

[0758] Retention time: 0.79 min (Analysis condition SQDFA05)

[0759] From the results of Examples 2-6 above, it has been shown that, according to the method of the present invention, in solid-phase synthesis, the introduction of N-substituted α,α-dialkylamino acids into bulky N-methylamino acids is possible at a practical level, not limited to N-methyl-α,α-dialkylamino acids.

[0760] Examples 2-7. Experiment in which N-methyl-α,α-dialkylamino acid (MecLeu) was subsequently introduced into bulky N-alkylamino acid (EtVal / nPrVal) in the solid phase.

[0761] 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 formulas.

[0762]

[0763]

[0764]

[0765] Example 2-7-1. Preparation of Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-1)

[0766] Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 1-2-1) (0.552 mmol / g, 100 mg), prepared by the same method as in Example 1-2-1, was added to a reaction vessel 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 5 minutes to de-Fmoc. After removing the de-Fmoc solution, the resin was washed four times with DMF (0.7 mL).

[0767] For the obtained resin, an extension reaction of Fmoc-Val-OH was performed.

[0768] The elongation reaction was carried out by adding a solution of 0.3 mL of an NMP solution of Fmoc-Val-OH (0.6 mol / L) and 1-hydroxy-7-azabenotriazole (HOAt, 0.375 mol / L) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 3 hours.

[0769] 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).

[0770] To confirm the progress of the reaction, a portion (∼5 mg) of the obtained resin (compound 2-7-1) was extracted, 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 formation of the target peptide Fmoc-Val-Asp-pyrro (compound 2-7-1*) (97.3% UV area). Additionally, an excess stretch of Val (compound 2-7-1a*) at 2.7% (UV area) was simultaneously detected. After stretching, the sample was washed with DCM, dried, and used for subsequent examination.

[0771]

[0772] LCMS(ESI) m / z=508.4(M+H)+

[0773] Retention time: 0.72 min (Analysis condition SQDFA05)

[0774]

[0775] LCMS(ESI) m / z=607.5(M+H)+

[0776] Retention time: 0.74 min (Analysis condition SQDFA05)

[0777] Example 2-7-2. De-Fmoc of Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-1) and Nsification of the N-terminus

[0778] 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 added to a reaction vessel 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 10 minutes to de-Fmoc. After removing the Fmoc solution, the resin was washed sequentially with DMF (0.7 mL), a DMF solution (0.7 mL) of 1-hydroxy-7-azabenotriazole (HOAt, 0.157 mol / L) and DIPEA (0.157 mol / L), and DMF (0.7 mL), and then washed three times with THF (0.7 mL).

[0779] 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 degrees for 3 hours.

[0780] After removing the liquid phase with a filter, the resin was washed 5 times with THF (1 mL) and 5 times with dichloromethane (1 mL) to obtain compound 2-7-2(Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0781] To confirm the progression of elongation, a portion (∼5 mg) of the obtained resin was extracted, 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 Nsification, the resin was washed with DCM, dried, and used for subsequent examination.

[0782]

[0783] LCMS(ESI) m / z=471.3(M+H)+

[0784] Retention time: 0.55 min (Analysis condition SQDFA05)

[0785] Example 2-7-3-1. N-ethylation of the Ns-amide site of Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-2) by photoextension reaction

[0786] 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 added to a reaction vessel 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 THF (1 mL).

[0787] Separately, 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) were added to a 1.5 mL vial, mixed by gentle shaking, and left to stand at room temperature for 15 minutes. Then, ethanol (32 μL, 0.552 mmol) was added and mixed, followed by leaving to stand for 5 minutes. The resulting solution was added to the swollen resin and shaken at 35°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) to obtain compound 2-7-3-1 (Ns-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0788] A portion (∼5 mg) of the obtained resin was excised from the peptide using a TFE / DCM / DIPEA solution (1:1:0.015), and the excised solution was analyzed by LCMS to confirm the formation 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 subsequent examination.

[0789]

[0790] LCMS(ESI) m / z=499.4(M+H)+

[0791] Retention time: 0.64 min (Analysis condition SQDFA05)

[0792] Example 2-7-3-2. De-Nsification of Ns-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 2-7-3-1)

[0793] 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 added to a reaction vessel 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 NMP (0.7 mL).

[0794] 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 the mixture was shaken at 60°C for 4 hours. After removing the liquid phase with a filter, the resin was washed twice with NMP (0.7 mL). A portion of the obtained resin was extracted, and peptides were extracted using a TFE / DCM / DIPEA solution (1:1:0.015), and the extracted solution was analyzed by LCMS.

[0795] For the resin that underwent the first de-Nsification, the same operation was performed again for the purpose of improving 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 additionally four times with DCM to obtain compound 2-7-3-2(H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to detect 84.6% (UV area) of the target peptide H-EtVal-Asp-pyrro (compound 2-7-3-2*), in addition to 1-dodecanethiol being ipso-substituted for the Ns-protected nitro group (compound 2-7-3-2a*) (12.6% UV area).

[0796]

[0797] LCMS(ESI) m / z=314.3(M+H)+

[0798] Retention time: 0.26 min (Analysis condition SQDFA05)

[0799]

[0800] LCMS(ESI) m / z=654.5(M+H)+

[0801] Retention time: 1.25 min (Analysis condition SQDFA05)

[0802] Example 2-7-3-3. The stretching reaction of Tfa-cLeu-OH on H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-3-2) is the stretching of Tfa-cLeu using 2-(trifluoromethyl)-3-oxa-1-azapyrro[4.4]nona-1-en-4-one (Compound 1-3-9) on resin.

[0803] 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 added to a reaction vessel 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 2-(trifluoromethyl)-3-oxa-1-azaspiro[4,4]nona-1-en-4-one (compound 1-3-9) (0.582 g, 2.81 mmol) to the resin and shaking at 60°C for 48 hours. To confirm the progress of the reaction, a small amount of the obtained resin was sampled after 24 hours, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to confirm the formation 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).

[0804] To confirm the progress of the reaction, a portion of the obtained resin (compound 2-7-3-3) was extracted, 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 formation of the target peptide Tfa-cLeu-EtVal-Asp-pyrro (compound 2-7-3-3*) (55.5% UV area), as well as the detection of an excess elongator of EtVal-Asp-pyrro (compound 2-7-3-3a*) (18.0% UV area), and an impurity (compound 2-7-3-2a*) (11.8% UV area) presumed to be an inverted substitution of 1-dodecanethiol for the Ns-protected nitro group. Additionally, a peak indicating that Tfa-cLeu-OH was supported on the resin was also detected. After stretching, it was washed with DCM, dried, and then used for subsequent review.

[0805]

[0806] LCMS(ESI) m / z=521.5(M+H)+

[0807] Retention time: 0.60 min (Analysis condition SQDFA05)

[0808]

[0809] LCMS(ESI) m / z=816.7(M+H)+

[0810] Retention time: 0.69 min (Analysis condition SQDFA05)

[0811] Example 2-7-3-4. N-methylation of the Tfa amide site of Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-3-3) by nucleus substitution reaction

[0812] 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 added to a reaction vessel 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 four times with DMF (0.7 mL).

[0813] To the obtained resin, a TMGN (59 mg) / DMF (0.35 mL) solution was added, followed by a methyl iodide (69 μL) / DMF (0.35 mL) solution, and the mixture was shaken at 40°C for 1 hour. After removing the liquid phase with a filter, the mixture was washed twice with DMF (0.7 mL). A portion of the obtained resin was extracted, and peptides were extracted using a TFE / DCM / DIPEA solution (1:1:0.015), and the extracted solution was analyzed by LCMS.

[0814] For the resin that underwent the first methylation, the same operation was performed two additional times for the purpose of improving the reaction conversion rate. After three methylations, the resin was washed four times with DMF and additionally 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 obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to identify the target peptide Tfa-MecLeu-EtVal-Asp-pyrro (compound 2-7-3-4*) (53.2% UV area), a compound that was Me-ized after EtVal-Asp-pyrro was over-elongated (compound 2-7-3-4a*) (29.8% UV area), and an impurity (compound 2-7-3-2a*) (11.9% UV area) presumed to have been substituted with 1-dodecanethiol for the Ns-protected nitro group. Additionally, a peak was detected in which Tfa-cLeu-OH was supported on the resin and Me-ized.

[0815]

[0816] LCMS(ESI) m / z=535.4(M+H)+

[0817] Retention time: 0.66 min (Analysis condition SQDFA05)

[0818]

[0819] LCMS(ESI) m / z=830.7(M+H)+

[0820] Retention time: 0.75 min (Analysis condition SQDFA05)

[0821] Example 2-7-4-1. Nn-propylation of the Ns-amide site of Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-2) by photoreaction

[0822] Compound 2-7-4-1 (Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-7-2 (0.552 mmol / g, 100 mg) and 1-propanol (41 μL, 0.552 mmol) by the method shown in Example 2-7-3-1. A portion of the obtained resin was extracted, 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 formation of 94.7% (UV area) of the target peptide Ns-nPrVal-Asp-pyrro (compound 2-7-4-1*) (conversion rate from compound 2-7-2 was 100%).

[0823]

[0824] LCMS(ESI) m / z=513.4(M+H)+

[0825] Retention time: 0.69 min (Analysis condition SQDFA05)

[0826] Example 2-7-4-2. De-Nsification of Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 2-7-4-1)

[0827] 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 obtained resin was extracted, 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-nPrVal-Asp-pyrro (compound 2-7-4-2*) at 81.1% (UV area), an impurity (compound 2-7-4-2a*) (15.1% UV area) presumed to be an upside substitution of 1-dodecanethiol for the Ns-protected nitro group was detected.

[0828]

[0829] LCMS(ESI) m / z=328.3(M+H)+

[0830] Retention time: 0.28 min (Analysis condition SQDFA05)

[0831]

[0832] LCMS(ESI) m / z=668.6(M+H)+

[0833] Retention time: 1.28 min (Analysis condition SQDFA05)

[0834] Example 2-7-4-3. Extension of Tfa-cLeu using 2-(trifluoromethyl)-3-oxa-1-azaspyrro[4.4]nona-1-en-4-one (Compound 1-3-9) against H-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-4-2).

[0835] Compound 2-7-4-3 (Tfa-cLeu-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same way using compound 2-7-4-2 (0.552 mmol / g, 100 mg) by the method shown in Example 2-7-3-3. A portion of the obtained resin was extracted, 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 formation of the target peptide Tfa-cLeu-nPrVal-Asp-pyrro (compound 2-7-4-3*) (53.1% UV area), and an excess elongator of nPrVal-Asp-pyrro (compound 2-7-4-3a*) (31.0% UV area), and an impurity presumed to be an inverted substitution of 1-dodecanethiol for the Ns-protected nitro group (compound 2-7-4-2a*) (13.3% UV area) were detected. Additionally, a peak indicating that Tfa-cLeu-OH was supported on the resin was also detected.

[0836]

[0837] LCMS(ESI) m / z=535.5(M+H)+

[0838] Retention time: 0.65 min (Analysis condition SQDFA05)

[0839]

[0840] LCMS(ESI) m / z=844.8(M+H)+

[0841] Retention time: 0.77 min (Analysis condition SQDFA05)

[0842] Example 2-7-4-4. N-methylation of the Tfa amide site of Tfa-cLeu-nPrVal-Asp(OTrt(2-Cl)-resin)-pyrro (Compound 2-7-4-3) by nucleus substitution reaction

[0843] Compound 2-7-4-4 (Tfa-MecLeu-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same way using compound 2-7-4-3 (0.552 mmol / g, 60 mg) by the method shown in Example 2-7-3-4. A portion of the obtained resin was extracted, and the peptide was extruded using a TFE / DCM / DIPEA solution (1:1:0.015). The extruded solution was analyzed by LCMS to identify the target peptide Tfa-MecLeu-nPrVal-Asp-pyrro (compound 2-7-4-4*) (46.1% UV area), a compound that was Me-ized after nPrVal-Asp-pyrro was over-elongated (compound 2-7-4-4a*) (38.3% UV area), and an impurity (compound 2-7-4-2a*) (12.6% UV area) presumed to have been substituted with 1-dodecanethiol for the Ns-protected nitro group. Additionally, a peak was detected in which Tfa-cLeu-OH was supported on the resin and Me-ized.

[0844]

[0845] LCMS(ESI) m / z=549.5(M+H)+

[0846] Retention time: 0.71 min (Analysis condition SQDFA05)

[0847]

[0848] LCMS(ESI) m / z=858.7(M+H)+

[0849] Retention time: 0.83 min (Analysis condition SQDFA05)

[0850] From the results of Examples 2-7 above, it has been shown that, according to the method of the present invention, the introduction of N-methyl-α,α-dialkylamino acid into bulky N-alkylamino acid in solid-phase synthesis is possible at a practical level.

[0851] Example 3: An example of peptide synthesis performed by introducing MeAib using the method of the present invention

[0852] In accordance with the peptide synthesis method by Fmoc described in WO2013 / 100132 or WO2018 / 225864, peptide extension was performed via the following basic route. That is,

[0853] 1) A peptide extension reaction by the Fmoc method from the N-terminus of an amino acid, wherein the carboxylic acid of the Asp side chain or the carboxylic acid of the peptide main chain is supported on 2-chlorotrityl resin,

[0854] 2) Process of peptide excision from 2-chlorotrityl resin,

[0855] 3) Amide cyclization resulting from the condensation of the amino group at the N-terminus (triangular unit) of the peptide chain with the carboxylic acid of the Asp side chain or peptide main chain carboxylic acid formed by detachment from the 2-chlorotrityl resin during the excision process,

[0856] 4) Deprotection of the protecting group of the side chain functional group included in the peptide chain as needed,

[0857] 5) This is a five-step process for purifying compounds by preparative HPLC. In this example, unless otherwise specifically described, the synthesis of peptide compounds was performed based on this basic route.

[0858]

[0859] Example 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-isopropyl-5,6,12,15,16,19,21,21,22,3 Synthesis of 3,36-undecamethyltetracosahydro-2H-azet[2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16,19,22,25,28,31]undecazacyclotetratriacontin-4,7,10,13,17,20,23,28,31,34,37(14H)-undecaone (Compound 3-1)

[0860]

[0861] The synthesis of compound 3-1 was carried out from compound 1-2-4 according to the following scheme.

[0862]

[0863] (3R)-3-[9H-fluorene-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 Fmoc-MeVal-OH was extended in a filtered reaction vessel using the peptide extension method described in Example 1-2-2, followed by the extension of Tfa-Aib-OH (2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid) (compound 1-3-1) by the same operation as in Example 2-1-1 to obtain compound 3-1-a.

[0864] 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 sealed and shaken at 40°C for 30 minutes. After removing the reaction solution, the resin was washed four times with DMF (1 mL) and additionally washed four times with DCM (1 mL) to obtain compound 3-1-b. A portion of the obtained resin was extracted with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS to confirm the formation of compound 3-1-b*.

[0865]

[0866] LCMS(ESI) m / z=424(MH)-

[0867] Retention time: 0.57 min (Analysis condition SQDFA05)

[0868] Sodium borohydride (NaBH4) (758 mg, 20 mmol) was placed in a flask, pumped up, and placed under a nitrogen atmosphere. It was dissolved in triglaim (10 mL) to obtain Solution A. The 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 shaken at room temperature in an open system for 40 minutes. After removing the reaction solution, methanol (0.7 mL) was added, and the washing operation of discarding the liquid after 1 minute was repeated four times. Additionally, the solution 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 excised with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS to confirm the formation of compound 3-1-c*.

[0869]

[0870] LCMS(ESI) m / z=330(M+H)+

[0871] Retention time: 0.33 min (Analysis condition SQDFA05)

[0872] The processes of peptide extension, cyclization, and purification after preparing compound 3-1-c were carried out according to the following synthesis method.

[0873] 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), an NMP solution (Solution 1) of HOAt or oxyma or HOOBt (0.375 mol / L), and an N,N-dimethylformamide (DMF) solution of diisopropyl carbodiimide (DIC) (10%, Solution 2) were set in a peptide synthesizer.

[0874] Solution 1 and Solution 2 were mixed in a mixing vial of a synthesizer and then added to the resin to perform a condensation reaction between the amino groups on the resin and the Fmoc amino acid.

[0875] Synthesis was performed using a 2% DMF solution of diazobicyclodecene (DBU) as the Fmoc deprotection solution. After washing the resin with DMF, the condensation reaction of Fmoc amino acids was performed as one cycle following Fmoc deprotection, and the peptide was extended on the resin surface by repeating this cycle. After the peptide extension was completed, the N-terminal Fmoc group of the resin was removed on the peptide synthesizer, and then the resin was washed with DMF.

[0876] For the chain peptide supported on the obtained solid phase, DCM was added to reswell the resin, and then 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. Subsequently, the resin was 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 obtained extraction solutions were mixed and concentrated under reduced pressure.

[0877] After cutting, the residue concentrated under reduced pressure was dissolved in DMF / DCM (1 / 1 (v / v), 8 mL). 0.5 M O-(7-aza-1H-benzotriazole-1-yl)-N,N,N,N-tetramethyluroronium hexafluorophosphate (HATU) / DMF solution (a volume equivalent to 1.5 times the amount of resin used (the amount of resin used (usually 0.10 g) multiplied by the amount of loading (mmol / g))) and DIPEA (a volume equivalent to 1.8 times the amount of resin used) were added, and the mixture was shaken at room temperature for 2 hours. Afterward, the solvent was removed by distillation under reduced pressure. The formation of the target cyclic peptide was confirmed by LCMS measurement.

[0878] Thereafter, under reduced pressure, the solvent was removed by distillation, DMF or DMSO was added, and insoluble matter was removed by filter filtration. After purification by preparative HPLC, 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-isopropyl-5,6,12,15,16,19,21,21,22,3 3,36-undecamethyltetracosahydro-2H-azet [2,1-u]pyrrolo [2,1-i] [1,4,7,10,13,16,19,22,25,28,31]undecazacyclotetratriacontin-4,7,10,13,17,20,23,28,31,34,37(14H)-undecaone)(4.1 mg, 9%) was obtained.

[0879] The analysis results of the LCMS are listed in Table 12.

[0880] Example 3-2: An example in which peptide synthesis was performed in the same manner as in Example 3-1

[0881] Compounds 3-2 to 3-9 were synthesized in the same manner by the method shown in Example 3-1. Meanwhile, the relationship between the formal names, structures, and abbreviations of each amino acid residue constituting the cyclic peptides described in Compounds 3-1 to 3-9 (structural formulas are listed in Table 13) is determined from the aforementioned Tables 3 to 5 and Table 11 below.

[0882] The analysis results of the LCMS are listed in Table 12.

[0883]

[0884]

[0885]

[0886]

[0887]

[0888]

[0889]

[0890]

[0891]

[0892]

[0893]

[0894]

[0895]

[0896]

[0897]

[0898]

[0899]

[0900]

[0901]

[0902]

[0903]

[0904] 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 photoreaction

[0905] As a comparative example with the present invention, a known method (Org. Lett. 2013, 15, 5012-5015) of performing a photo-ignition reaction as a selective N-methylation method at a trifluoroacetamide site was attempted.

[0906] 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 in a reaction vessel with a filter, 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).

[0907] To the obtained resin, a solution of triphenylphosphine (66.0 mg) / THF (0.7 mL), methanol (20 μL), and DIAD (49 μL) were added, and the mixture was shaken at 40 degrees for 30 minutes. After removing the liquid phase with a filter, the solution of triphenylphosphine (66.0 mg) / THF (0.7 mL), methanol (20 μL), and DIAD (49 μL) were added again, and the mixture was shaken at 40 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).

[0908] The obtained resin was subjected to peptide excision with a TFE / DCM solution (1 / 1 (v / v)), and the excision solution was analyzed by LCMS. In addition to the formation of the target peptide Tfa-MeAib-MeVal-Asp-pyrro (Compound 2-2*), an O-methylated product of the Tfa amide site (Compound C1-1) and H-Aib-MeVal-Asp-pyrro (Compound C1-2), which was hydrolyzed therefrom, were detected. The LC chart is shown in Figure 2.

[0909] Purpose peptide Tfa-MeAib-MeVal-Asp-pyrro(compound 2-2*)

[0910]

[0911] LCMS(ESI) m / z=495.26(M+H)+

[0912] Retention time: 0.58 min (Analysis condition SQDFA05)

[0913] O-methylated product at the Tfa amide site (compound C1-1)

[0914]

[0915] LCMS(ESI) m / z=495.26(M+H)+

[0916] Retention time: 0.64 min (Analysis condition SQDFA05)

[0917] H-Aib-MeVal-Asp-pyrro (compound C1-2) hydrolyzed from compound C1-1

[0918]

[0919] LCMS(ESI) m / z=385.26(M+H)+

[0920] Retention time: 0.35 min (Analysis condition SQDFA05)

[0921] From these results, it was confirmed that, unlike in the literature (Org. Lett. 2013, 15, 5012-5015), when the N-terminus is an α,α-dialkylamino acid, O-methylation proceeds significantly simultaneously with N-methylation, resulting in a decrease in yield and purity. This result is in contrast to the N-selective methylation results shown in Examples 2-2 and 2-3.

[0922] Comparative Example 2: An experiment attempting the introduction of MeAib by performing Fmoc-Aib-OH extension on N-methylamino acid via a conventional solid-state synthesis method, followed by an exchange from Fmoc protection to Ns protection, N-methylation of the N-terminus on the resin, and deNs.

[0923] As a comparative example with the present invention, an attempt was made to introduce MeAib by performing the same method as described in the literature (Nature Protocols 2012, 7, 3, 432-444), by performing Fmoc-Aib-OH extension following N-methylamino acid in a solid-phase synthesis method, then exchanging from Fmoc protection to Ns protection, N-methylation on the N-terminal resin, and deNs.

[0924]

[0925] Comparative Example 2-1: Solid-phase stretching reaction of Fmoc-Aib-OH against Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0926] In a reaction vessel with a filter, 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, 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 10 minutes to de-Fmoc. After removing the de-Fmoc solution, the resin was washed four times with DMF (0.7 mL).

[0927] For the obtained resin, an extension reaction of Fmoc-Aib-OH was performed.

[0928] The elongation reaction was carried out by adding a solution of 0.6M Fmoc-Aib-OH / 0.375M oxyma / NMP solution (0.3mL) and 10% DIC / DMF solution (0.36mL) to the resin and shaking at 50 degrees for 15 hours.

[0929] This renal response was repeated two additional times. (2nd renal condition: 50°C for 24 hours, 3rd renal condition: 50°C for 20 hours)

[0930] After removing the liquid phase of the renal reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL).

[0931] To confirm the progression of the renal process, a portion (∼5 mg) of the obtained resin was extracted, and the unreacted points were capped with Fmoc-Gly-OH.

[0932] Capping was performed by adding a solution of 0.6M Fmoc-Gly-OH / 0.375MHOAt / NMP solution (0.3mL) and 10% DIC / DMF solution (0.36mL) to the resin and shaking at 40 degrees for 45 minutes.

[0933] After removing the liquid phase of the renal reaction with a filter, the resin was washed four times with DMF (0.7 mL) and four times with dichloromethane (0.7 mL).

[0934] Peptide excision was performed using TFE / DCM solution (1 / 1 (v / v)), and the excision solution was analyzed by LCMS, confirming that the target peptide Fmoc-Aib-MeVal-Asp-pyrro (compound C2-1*) was produced at 60.4%.

[0935]

[0936] LCMS(ESI) m / z=605.52(MH)-

[0937] Retention time: 2.16 min (Analysis condition SQDFA05long)

[0938] Meanwhile, the unreacted points of the obtained resin were capped with Z-Gly-OH (N-α-carbozoxyglycine, CAS: 1138-80-3) purchased from a commercial supplier.

[0939] Capping was performed by adding a solution of 0.6 MZ-Gly-OH / 0.375 MOH OAt / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 2 hours.

[0940] After removing the liquid phase of the capping 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 C2-1.

[0941] Comparative Example 2-2: De-Fmoc and Ns-conversion of the N-terminus of Fmoc-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound C2-1)

[0942] 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 added to a reaction vessel 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). Subsequently, a 2% DBU / DMF solution (de-Fmoc solution: 0.7 mL) was added to the resin and the mixture was shaken at room temperature for 10 minutes to de-Fmoc. After removing the de-Fmoc solution, the resin was washed three times with DMF (1 mL) and then four times with THF (1 mL).

[0943] To the obtained 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 degrees for 2 hours.

[0944] After removing the liquid with a filter, the resin was washed three times with THF (1 mL) and four times with dichloromethane (1 mL).

[0945] The above Ns conversion by 2-nitrobenzenesulfonyl chloride was repeated two additional times (2nd time: 40°C, shaking for 16 hours; 3rd time: 40°C, shaking for 21 hours).

[0946] To confirm the progression of the renal process, a portion (∼5 mg) of the obtained resin was extracted, and the peptide was excised using a TFE / DCM solution (1 / 1 (v / v)). When the excised solution was analyzed by LCMS, it was confirmed that the target peptide Ns-Aib-MeVal-Asp-pyrro (compound C2-2*) was produced in 64.9%.

[0947] The unreacted points of the obtained resin were capped with Z-Gly-OH.

[0948] Capping was performed by adding a solution of 0.6 MZ-Gly-OH / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 2 hours.

[0949] 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).

[0950]

[0951] LCMS(ESI) m / z=568.45(MH)-

[0952] Retention time: 0.59 min (Analysis condition SQDFA05)

[0953] 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 photoreaction

[0954] In a reaction vessel 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).

[0955] To the obtained resin, a THF (0.7 mL) solution of triphenylphosphine (61.0 mg, 0.232 mmol) and methanol (19 μL, 0.464 mmol) was added, followed by the addition of DIAD (45 μL, 0.232 mmol), and the mixture was shaken at 40 degrees 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).

[0956] A portion (∼5 mg) of the obtained resin was excised from the peptide using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS to confirm the formation 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 following process.

[0957]

[0958] LCMS(ESI) m / z=582.47(MH)-

[0959] Retention time: 0.63 min (Analysis condition SQDFA05)

[0960] Comparative Example 2-4: De-Nsification of Ns-MeAib-MeVal-Asp-pyrro resin (Compound C2-3)

[0961] 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 in a reaction vessel with a filter, 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).

[0962] To the obtained resin, DBU (17 μL, 0.115 mmol) / NMP solution (0.35 mL) and 2-mercaptoethanol (16 μL, 0.230 mmol) / NMP solution (0.30 mL) were added, and the mixture was shaken at room temperature for 1 hour. After removing the liquid phase with a filter, the resin was washed 4 times with NMP (0.5 mL) and 4 times with dichloromethane (0.5 mL).

[0963] A portion (∼5 mg) of the obtained resin was excised from the resin using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS. As shown in Figure 3, the formation of the target peptide H-MeAib-MeVal-Asp-pyrro (compound C2-4*) with deNsification was confirmed.

[0964]

[0965] LCMS(ESI) m / z=399.29(M+H)+

[0966] Retention time: 0.34 min (Analysis condition SQDFA05)

[0967] Comparative Example 2-5: De-Nsification of Ns-MeAib-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro

[0968]

[0969] In a reaction vessel with a filter, dichloromethane (0.5 mL) was added to Ns-MeAib-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-5-1) (0.464 mmol / g, 50 mg), prepared by the same procedure as Comparative Examples 2-1 to 2-3, for Compound 1-2-3 (100 mg) prepared in Example 1-2-3, 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).

[0970] For the obtained resin, DBU (17 μL, 0.115 mmol) / NMP solution (0.35 mL) and 2-mercaptoethanol (16 μL, 0.230 mmol) / NMP solution (0.30 mL) were added and the mixture was shaken at room temperature for 1 hour. After removing the liquid phase with a filter, the resin was washed 4 times with NMP (0.5 mL) and 4 times with dichloromethane (0.5 mL).

[0971] A portion (∼5 mg) of the obtained resin was excised from the resin using a TFE / DCM solution (1 / 1 (v / v)), and the excised solution was analyzed by LCMS. As shown in Figure 4, in addition to the formation of the target peptide H-MeAib-MePhe-Asp-pyrro (compound C2-5-2*) in which Ns-protection was performed, an impurity (compound C2-5-3*) presumed to have been substituted with 2-mercaptoethanol for the Ns-protecting nitro group was detected.

[0972]

[0973] LCMS(ESI) m / z=447.31(M+H)+

[0974] Retention time: 0.39 min (Analysis condition SQDFA05)

[0975]

[0976] LCMS(ESI) m / z=661.74(MH)-

[0977] Retention time: 0.65 min (Analysis condition SQDFA05)

[0978] From the results of Comparative Example 2, it was confirmed that while bulky N-methyl-α,α-dialkylamino acid (in this example, MeAib) can be introduced to the N-terminus of bulky N-methylamino acid, a series of processes including the extension of Fmoc-Aib and the exchange of the protecting group to the Ns group resulted in low purity and low yield. Furthermore, it was found that a decrease in purity was caused by side reactions on the Ns protecting group during the Ns removal step. Under known conditions described in the literature, it was confirmed that it is difficult to introduce bulky N-methyl-α,α-dialkylamino acid to the N-terminus of bulky N-methylamino acid with high purity and high yield.

[0979] Reference Example: Attempt to extend Fmoc-MeAib-OH to N-methylamino acid using a conventional solid-phase synthesis method

[0980] Reference Example 1: Solid-phase stretching reaction of Fmoc-MeAib-OH for Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0981]

[0982] 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 added to a reaction vessel with a filter, and the extension of Fmoc-MeAib-OH was attempted by the same operation as in Comparative Example 2-1.

[0983] For the elongation reaction, a solution mixed with 0.6M Fmoc-MeAib-OH / 0.375M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) was applied to the resin and shaken at 40°C for 21 hours. After draining the reaction solution, the same operation was repeated once more (21.5 hours at 40°C).

[0984] After the extension reaction, the resin was properly cleaned, the unreacted sites were capped with Fmoc-Gly-OH, and the peptide was extruded from the resin by the same operation as in Comparative Example 2-1, and the extruded solution was analyzed by LCMS, but the target peptide Fmoc-MeAib-MeVal-Asp-pyrro (compound R1*) was not detected.

[0985]

[0986] Reference Example 2: Solid-phase stretching reaction of Fmoc-MeAib-OH for Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-3)

[0987]

[0988] 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 added to a reaction vessel with a filter, and the extension of Fmoc-MeAib was attempted by the same procedure as in Reference Example 1. The extension reaction was carried out at 40°C for 15 hours.

[0989] Afterwards, the resin was properly cleaned, the unreacted sites were capped with Fmoc-Gly-OH, and the peptide was extruded from the resin by the same operation as in Reference Example 1. When the extruded solution was analyzed by LCMS, the production of the target peptide Fmoc-MeAib-MePhe-Asp-pyrro (compound R2*) remained at 3.1%.

[0990]

[0991] LCMS(ESI) m / z=669.43(M+H)+

[0992] Retention time: 0.88 min (Analysis condition SQDFA05)

[0993] From the results of the reference example above, it was confirmed that the extension of Fmoc-MeAib-OH (i.e., N-substituted-α,α-disubstituted amino acid) following N-methylamino acid (i.e., N-substituted amino acid) in the conventional solid-phase synthesis method (Fmoc method) is very difficult, and there are cases where the target peptide is not obtained. Industrial applicability

[0994] According to the present invention, it has been discovered that in the preparation of peptide compounds using a solid-state method, a 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 can be efficiently prepared. The present invention is useful in the field of peptide synthesis.

Claims

Claim 1 Formula (1), including the following process: [In the formula, PG1 is a protecting group of the amino group, and P1 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 It is aralkyl, and R1 and Q1 are C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C7-C alkyl that may be substituted. 14 They are independently selected from aralkyls, or R1 and Q1 are combined with the carbon atom to which they are bonded to form a 3–8-membered alicyclic ring or a 4–7-membered saturated heterocyclic ring, and P2 is a C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 R2 is an aralkyl, and R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonyl C1-C6 alkyl, C2-C6 alkyneyl, a C1-C6 alkoxyC1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkoxyC1-C6 alkyl that may be substituted by one or more halogens, or a C7-C 14 A method for preparing a peptide compound, its salt, or their solvates comprising a structure in which two amino acid residues are connected, represented by [aralkyl, where R3 is a hydroxyl, O-PG2, any amino acid residue, or any peptide residue, and PG2 is a protecting group of a carboxyl group]: Process A: Formula (2): [In the formula, P2, R2, and R3 are respectively equivalent to P2, R2, and R3 of formula (1)] a compound represented by, its salt, or their solvate, and formula (3): [In the formula, PG1, Q1, and R1 are each equivalent to PG1, Q1, and R1 of formula (1)] react a compound represented by, its salt, its dehydrate, or their solvate with a condensation reagent, or react a compound represented by the corresponding formula (2), its salt, or their solvate with a dehydrate of a compound represented by the corresponding formula (3), its salt, or their solvate, so as to formula (4): [In the formula, PG1, P2, Q1, and R1 to R3 are each synonymous with PG1, P2, Q1, and R1 to R3 of formula (1)] a process for obtaining a compound represented by formula (4), its salt, or their solvate, and process B: a process for reacting a compound represented by formula (4), its salt, or their solvate with a P1-introducing reagent to obtain a peptide compound represented by formula (1), its salt, or their solvate (provided, in formula (3) and / or formula (4), the pKa (in water) of the NH group to which PG1 is bound is 6 to 11, and the P1-introducing reagent is a combination of P1X (in the formula, P1 is synonymous with P1 of formula (1) and X is a degreasing group) and a base, and the pKa (in acetonitrile) of the conjugate acid of the base is 22 to 31). Claim 2 A method according to claim 1, wherein R1 and Q1 are combined with the carbon atom to which they are bonded to 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, benzyl which may be substituted, or phenethyl which may be substituted. Claim 3 The method according to claim 1 or 2, wherein PG1 is a C2-C6 haloacyl. Claim 4 The method of claim 3, 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. Claim 5 In claim 1 or 2, the dehydrate is of the following formula: A method represented by [wherein Q1 and R1 are respectively identical to Q1 and R1 of Formula (1), and R4 is a C1-C5 haloalkyl]. Claim 6 A method according to claim 5, wherein R1 and Q1 become one with the carbon atom to which they are bonded to form a 3- to 8-membered alicyclic ring. Claim 7 Method of claim 5, wherein R4 is trifluoromethyl, trichloromethyl, pentafluoroethyl, 1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl, or 2,2,2-trifluoro-1-(trifluoromethyl)ethyl. Claim 8 Method according to claim 1 or 2, wherein P1 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl. Claim 9 Method according to claim 1 or 2, wherein P2 is methyl, ethyl, n-propyl, i-propyl, allyl, benzyl, or phenethyl. Claim 10 The method of claim 1 or 2, wherein R3 is any amino acid residue or any peptide residue supported on a resin for solid synthesis. Claim 11 In claim 10, the method wherein the resin for solid synthesis is a CTC resin, Wang resin, or SASRIN resin. Claim 12 A method according to claim 1 or 2, wherein the condensation reagent is either DIC or EDCI·HCl, or a combination of DIC and Oxyma. Claim 13 In claim 1 or 2, the base, [In the formula, RB1 and RB4 are each independently C1-C4 alkyl, or RB1 and RB4 are combined with the nitrogen atom to which RB1 is bonded and the carbon atom to which RB4 is bonded to form a 5- to 8-membered ring, and RB2 and RB3 are each independently C1-C4 alkyl, or RB2 and RB3 are combined with the nitrogen atom to which RB2 is bonded, the nitrogen atom to which RB3 is bonded, and the carbon atom to which the corresponding nitrogen atom is bonded to form a 5- to 8-membered ring], [In the formula, RB6 is hydrogen or a C1-C4 alkyl, RB5 and RB7 are each independently a C1-C4 alkyl, or they form a 5- to 8-membered ring by combining each nitrogen atom to which they are bonded with the carbon atom to which that nitrogen atom is bonded, RB8 is a C1-C4 alkyl, and RB9 is a C1-C4 alkyl or phenyl, or RB8 and RB9 form a 5- to 8-membered ring by combining each nitrogen atom to which they are bonded with the carbon atom to which that nitrogen atom is bonded, wherein if RB9 is phenyl, the two B2s may form naphthalene by condensing the two benzene rings of the corresponding phenyl group], [Among the formula, RB 10 Silver, C1-C4 alkyl, or RB 10 and RB 11 Silver, uniting with the nitrogen atoms to which they are bonded to form a 5–8 member ring, and RB 11 Silver, RB 10 and RB 11 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 11 and RB 12 They unite with each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded to form a 5–8 member ring, and RB 12 neun, RB 11 and RB 12 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 12 and RB 13 Silver, uniting with the nitrogen atoms to which they are bonded to form a 5–8 member ring, and RB 13 Silver, RB 12 and RB 13 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 13 and RB 14 They unite with each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded to form a 5–8 member ring, and RB 14 neun, RB 13 and RB 14 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 14 and RB 15 They unite with the nitrogen atoms they are bonded to to form a 5–8 member ring, and RB 15 neun, RB 14 and RB 15 Except for cases where it forms a 5- to 8-membered ring, it is a C1-C4 alkyl, and RB 16 Silver hydrogen, C1-C8 alkyl, or C6-C 10 It is Aril.], and [Among the formula, RB 17 silver, independently C1-C4 alkyl, or RB 17 and RB 18 Silver, uniting with the nitrogen atoms to which they are bonded to form a 5–8 member ring, and RB 18 Silver, RB 17 and RB 18 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 18 and RB 19 They unite with each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded to form a 5–8 member ring, and RB 19 neun, RB 18 and RB 19 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 19 and RB 20 Silver, uniting with the nitrogen atoms to which they are bonded to form a 5–8 member ring, and RB 20 Silver, RB 19 and RB 20 Except for the case where this 5–8-membered ring is formed, it is a C1-C4 alkyl, and RB 21 Silver, C1-C4 alkyl, or RB 21 and RB 22 They unite with the nitrogen atoms they are bonded to to form a 5–8 member ring, and RB 22 neun, RB 21 and RB 22 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 22 and RB 23 Silver, each nitrogen atom to which they are bonded, and the phosphorus atom to which that nitrogen atom is bonded become one to form a 5–8 member ring, and RB 23 Silver, RB 22 and RB 23 Except for cases forming this 5–8-membered ring, it is C1-C4 alkyl, or RB 23 and RB 24 They unite with the nitrogen atoms they are bonded to to form a 5–8 member ring, and RB 24 neun, RB 23 and RB 24 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 24 and RB 25 They unite with each nitrogen atom to which they are bonded and the phosphorus atom to which that nitrogen atom is bonded to form a 5–8 member ring, and RB 25 neun, RB 24 and RB 25 Except where it forms a 5–8-membered ring, it is a C1-C4 alkyl, or RB 25 and RB 26 Silver, uniting with the nitrogen atoms to which they are bonded to form a 5–8 member ring, and RB 26 Silver, RB 25 and RB 26 Except for the case where this 5–8-membered ring is formed, it is a C1-C4 alkyl, and RB 27 silver, C1-C4 alkyl, or C6-C 10 A method selected from a group consisting of [Aryl.] Claim 14 In claim 1 or 2, the base is 1,8-diazabicyclo[5.4.0]undeca-7-en (DBU), 1,5-diazabicyclo[4.3.0]non-5-en (DBN), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-en (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]deca-5-en (TBD), tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu), tert-butylimino-tri(pyrrolidino)phosphorane (P1-t-Bu-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 -Catenadi(phosphazene)(P2-t-Bu), and 1-ethyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 - A method selected from the group consisting of catenadi(phosphazene)(P2-Et). Claim 15 A method according to claim 1 or 2, wherein process B is carried out in a solvent selected from the group consisting of DMF, NMP, DMI, tetrahydrofuran, 2-methyltetrahydrofuran, and acetonitrile. Claim 16 A method for preparing a peptide compound, its salt, or their solvates comprising a dipeptide residue in which an N-substituted α,α-disubstituted amino acid residue and an N-substituted amino acid residue are linked, comprising the method described in claim 1 or 2. Claim 17 A method for preparing a cyclic peptide compound, its salt, or its solvate, comprising the steps of: deprotecting an N-terminal protecting group from a peptide compound, its salt, or their solvate prepared by the method described in claim 1 or 2; optionally extending a peptide chain; and cyclizing a C-terminal group and an N-terminal group to form a cyclic portion, wherein the cyclic peptide compound comprises 8 to 15 amino acid residues, comprises at least 3 N-substituted amino acid residues, and also comprises at least 1 N-unsubstituted amino acid residue, and the cyclic portion comprises at least 8 amino acid residues. Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete

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