Method for preparing peptide compounds containing sterically hindered amino acids

Abstract

It was found that by linking an N-unsubstituted-α,α-disubstituted amino acid whose amino group is protected with an electron-withdrawing protecting group to an N-substituted amino acid or a peptide compound having an N-substituted amino acid residue at the N-terminal, and then allowing a substituent-introducing agent to act in the presence of a specific base to selectively introduce a substituent into the N-terminal amino group, a peptide compound having an N-substituted-α,α-disubstituted amino acid residue at the N-terminal and comprising a dipeptide residue linked to the N-substituted-α,α-disubstituted amino acid residue can be efficiently prepared.

Description

Technical Field

[0001] The present invention relates to a method for preparing a peptide compound containing sterically hindered amino acids. Background Art

[0002] Examples of so-called tough targets that are not used as drug development targets in conventional drugs include drug development targeting intracellular molecules, drug development by inhibiting protein-protein interactions, etc. These targets that are not used as drug development are because drug molecules cannot reach the target molecules of the disease, or the shape of the site of action of the target molecule is a shape that is difficult for conventional drugs to act on (Non-Patent Document 1).

[0003] Recently, as a method for approaching difficult targets that are difficult to approach in conventional techniques, cyclic peptides have received attention (non-patent literature 2). In the application of cyclic peptides to drugs, it is not only considered to improve the ability to bind to the target, but also to consider the screening methods of drug-like (drug-like: preferably showing both membrane permeability and metabolic stability) cyclic peptides and efficient cyclic peptides (non-patent literature 3, 4). In addition, the conditions required for non-natural cyclic peptides containing drug-like non-natural amino acids become clear, and their importance in drug development and their recognition increase (patent literature 1). Non-natural amino acids are important elements of the non-natural peptides, and it is generally believed that the synthesis of peptides containing non-natural amino acids such as N-methyl (or N-alkyl) amino acids, and peptides containing N-methyl (or N-alkyl) amino acids continuous units according to the situation is due to its hugeness and is more difficult than natural peptides (non-patent literature 5, 6).

[0004] When shifting the viewpoint to non-natural amino acids whose side chains are different from those of natural amino acids, it is reported that α,α-disubstituted amino acids in which the α-position of the amino acid is substituted by two substituents impart increased lipid solubility, restriction of conformational freedom of peptides containing α,α-disubstituted amino acids due to steric hindrance, and stabilization in vivo (Non-Patent Document 7), and can be expected to exert a large positive influence on both drug efficacy and drug-likeness.

[0005] In the preparation method of peptides containing these unnatural amino acids, it is known that the Fmoc method most commonly used for peptide synthesis is not suitable due to the bulky structure of these unnatural amino acids (Non-Patent Document 8).

[0006] Based on these, it is easy to infer that the synthesis of peptides by condensation reaction of the carboxyl group of an α,α-disubstituted amino acid with the amino group of an amino acid, especially the synthesis of peptides by condensation reaction of the carboxyl group of an N-alkylated α,α-disubstituted amino acid with the amino group of an N-alkylated amino acid, is difficult due to the large space of one or both amino acids.

[0007] As one of the methods for continuously introducing N-alkyl amino acids, there is known a method of synthesizing a peptide by condensing an NH amino acid having less steric hindrance than an N-alkyl amino acid, and selectively N-alkylating the target NH group among a plurality of NH groups in the obtained peptide (Non-Patent Documents 9 and 10). However, these methods are methods for combining an α-monosubstituted amino acid and an N-alkyl amino acid, and a practical method for synthesizing a peptide having a sequence in which an N-alkyl-α,α-disubstituted amino acid and an N-alkyl amino acid are combined is unknown.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. 2018 / 225864

[0011] Non-patent literature

[0012] Non-patent literature 1: Annu. Rev. Pharmacol. Toxicol., 2016, 56, 23-40.

[0013] Non-patent document 2: Future Med. Chem. 2009, 1, 1289-1310.

[0014] Non-patent document 3: ACS Chem. Biol., 2013, 8, 488-499.

[0015] Non-patent document 4: Drug Discovery Today, 2014, 19, 388-399.

[0016] Non-patent document 5: J. Peptide Res., 2005, 65, 153-166.

[0017] Non-patent document 6: Biopolymers, 109; e23110 (DOI: 10.1002 / bip.23110)

[0018] Non-patent literature 7: Journal of the Society of Synthetic Organic Chemistry 2002, vol.60, No.2, 125-136.

[0019] Non-patent document 8: Chem. Soc. Rev., 2016, 45, 631-654.

[0020] Non-patent document 9: Org. Lett., 2013, 15, 5012-5015.

[0021] Non-patent document 10: J. Am. Chem. Soc., 1997, 119, 2301-2302. SUMMARY OF THE INVENTION

[0023] Problems to be solved by the invention

[0024] In Non-Patent Document 5, a method for producing a peptide containing N-alkyl amino acids is exemplified as a method for producing a peptide containing bulky amino acids. However, this document only describes the study of the mechanism of side reactions in the condensation reaction of amino acids and condensation reagents, and does not describe the condensation reaction of a larger α,α-disubstituted amino acid.

[0025] Non-patent document 6 exemplifies a method for producing an N-alkyl amino acid and a method for producing a peptide containing an N-alkyl amino acid. This document exemplifies a method for producing a condensing agent focused on amino acids, but does not describe a more extensive condensation reaction of α,α-disubstituted amino acids.

[0026] Non-patent document 7 describes methods for preparing α,α-disubstituted amino acids, the usefulness of peptides containing α,α-disubstituted amino acids, examples of α,α-disubstituted amino acids, and methods for preparing them, but does not describe the preparation of peptides containing sterically bulky amino acids.

[0027] A method for preparing a peptide that is difficult to synthesize is exemplified in Non-Patent Document 8. This document describes the preparation of a peptide that has low solubility or is prone to aggregation, but does not describe a method for solving the problem by applying a condensation reaction.

[0028] In Non-Patent Document 9, a method for forming a peptide bond by using a trifluoroacetyl group that can be removed under mild conditions through an N-alkyl amino acid and a method for preparing a peptide containing an N-alkyl amino acid are exemplified. However, there is no mention of a condensation reaction with a more bulky α,α-disubstituted amino acid. In addition, it is known that an alkyl group is not selectively introduced only to the nitrogen atom to which the trifluoroacetyl group is bonded, but an isomeric byproduct in which an alkyl group is also introduced to the oxygen atom of the trifluoroacetyl group is produced.

[0029] Non-patent document 10 describes a method for preparing a peptide containing an N-alkyl amino acid by introducing an alkyl group into the nitrogen atom of an amino acid protected with a p-nitrobenzenesulfonyl group (nosyl). However, it is known that there are problems in preparing the target product due to side reactions occurring in the step of deprotecting the p-nitrobenzenesulfonyl group (Non-patent document 9).

[0030] As mentioned above, the bond-forming reaction between N-substituted-α,α-disubstituted amino acids such as N-alkyl-α,α-disubstituted amino acids and N-substituted amino acids such as N-alkyl amino acids is difficult, and an effective solution is unknown. The present invention is completed in view of such a situation, with the aim of providing 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 aim is to provide a method for introducing an N-substituted-α,α-disubstituted amino acid into an N-substituted amino acid. Furthermore, the aim is to provide a method for highly selectively N-functionalizing the amino group of an N-unsubstituted-α,α-disubstituted amino acid residue.

[0031] Means for solving problems

[0032] The present inventors have discovered a method for connecting N-substituted amino acids and N-substituted-α, α-disubstituted amino acids. Specifically, it has been found that by using N-unsubstituted-α, α-disubstituted amino acids that have a smaller steric volume than N-substituted amino acids and that improve the reactivity of the carboxyl group by protecting the amino group with an electron-withdrawing protective group, the target amino acid can be efficiently introduced. In addition, it has been found that in the subsequent functionalization of the amino group, the NH group whose acidity increases due to the electron-withdrawing protective group is selectively subjected to N-functionalization reactions such as N-alkylation reactions. Further, focusing on the increase in acidity of the NH group due to the electron-withdrawing protective group, a specific base used in the N-functionalization reaction was discovered, thereby completing the present invention.

[0033] That is, the present invention includes the following inventions in a non-limiting specific embodiment.

[0034] [1] A method for preparing a peptide compound having an N-substituted-α,α-disubstituted amino acid residue at the N-terminus, a dipeptide residue comprising the N-substituted-α,α-disubstituted amino acid residue linked to an N-substituted amino acid residue, a salt thereof, or a solvate thereof, comprising the following steps:

[0035] Step A: reacting an N-substituted amino acid, a salt thereof, or a solvate thereof, or a peptide compound having an N-substituted amino acid residue at the N-terminus, a salt thereof, or a solvate thereof, with an N-unsubstituted-α,α-disubstituted amino acid, a salt thereof, a dehydrate thereof, or a solvate thereof in which the amino group is protected with an electron-withdrawing protecting group, in the presence or absence of a condensation reagent, to obtain an N-unsubstituted-α,α-disubstituted amino acid residue having an amino group protected with an electron-withdrawing protecting group at the N-terminus, a peptide compound comprising a dipeptide residue linked to the N-unsubstituted-α,α-disubstituted amino acid residue, a salt thereof, or a solvate thereof; and

[0036] Step B: In the presence of a base and a substituent-introducing agent, a substituent is introduced into the amino group of an N-unsubstituted-α,α-disubstituted amino acid residue whose N-terminus is protected with an electron-withdrawing protecting group, to obtain a peptide compound having an N-substituted-α,α-disubstituted amino acid residue whose N-terminus is protected with an electron-withdrawing protecting group, a dipeptide residue comprising the N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue connected, a salt thereof, or a solvate thereof.

[0037] [2] The method according to [1], wherein the electron withdrawing protecting group is a protecting group having a pKa (in water) of 6 to 11 for the NH group to which the protecting group binds.

[0038] [3] The method according to [1] or [2], wherein the pKa (in acetonitrile) of the conjugate acid of the base is 18 to 31.

[0039] [4] The method according to any one of [1] to [3], wherein the N-substituted amino acid or a peptide compound having an N-substituted amino acid residue at the N-terminus is supported on a resin for solid phase synthesis.

[0040] [5] The method according to any one of [1] to [4], wherein the N-substituted amino acid or the peptide compound having an N-substituted amino acid residue at the N-terminus is represented by formula (2):

[0041]

[0042] [Wherein,

[0043] P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0044] R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkyloxyC1-C6 alkyl, or C7-C 14 Aralkyl,

[0045] R3 is hydroxyl, O-PG2, any amino acid residue, or any peptide residue,

[0046] PG2 is a protecting group for the carboxyl group].

[0047] [6] The method according to any one of [1] to [5], wherein the N-unsubstituted-α,α-disubstituted amino acid whose amino group is protected with an electron-withdrawing protecting group is represented by the formula (3):

[0048]

[0049] [Wherein,

[0050] PG1 is an electron-withdrawing protecting group.

[0051] R1 and Q1 are independently selected from C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, or optionally substituted C7-C 14 Aralkyl, or

[0052] R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring]. [7] The method according to any one of [1] to [6], wherein the peptide compound obtained in step A is represented by formula (4):

[0053]

[0054] [Wherein,

[0055] PG1, R1, and Q1 have the same meanings as PG1, R1, and Q1 in formula (3), respectively.

[0056] P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2).

[0057] [8] The method according to any one of [1] to [7], wherein the substituent-introducing agent in step B is P1X (wherein P1 has the same meaning as P1 in formula (1) and X is a leaving group), and the peptide compound obtained in step B is represented by formula (1):

[0058]

[0059] [Wherein,

[0060] P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0061] PG1, R1, and Q1 have the same meanings as PG1, R1, and Q1 in formula (3), respectively.

[0062] P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2).

[0063] [9] A method for preparing a peptide compound having a structure in which two amino acid residues are linked represented by formula (1), a salt thereof, or a solvate thereof:

[0064]

[0065] [Wherein,

[0066] PG1 is a protecting group of the amino group,

[0067] P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0068] R1 and Q1 are independently selected from C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, or optionally substituted C7-C 14 Aralkyl, or

[0069] R1 and Q1 together with the carbon atoms to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring.

[0070] P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0071] R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkyloxyC1-C6 alkyl, or C7-C 14 Aralkyl,

[0072] R3 is hydroxyl, O-PG2, any amino acid residue, or any peptide residue,

[0073] PG2 is a protecting group for the carboxyl group],

[0074] The method comprises the following steps:

[0075] Step A: a compound represented by formula (2), a salt thereof, or a solvate thereof:

[0076]

[0077] [wherein, P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (1)]

[0078] and a compound represented by formula (3), a salt thereof, a dehydrate thereof, or a solvate thereof:

[0079]

[0080] [wherein, PG1, Q1, and R1 have the same meanings as PG1, Q1, and R1 in formula (1)]

[0081] A step of reacting with a condensation reagent, or reacting the compound represented by the formula (2), its salt, or their solvate with the dehydrated product of the compound represented by the formula (3), its salt, or their solvate to obtain the compound represented by the formula (4), its salt, or their solvate:

[0082]

[0083] [wherein, PG1, P2, Q1, and R1 to R3 have the same meanings as PG1, P2, Q1, and R1 to R3 in formula (1), respectively]

[0084] and

[0085] Step B: A step of reacting the compound represented by formula (4), a salt thereof, or a solvate thereof with a P1-introducing reagent to obtain a peptide compound represented by formula (1), a salt thereof, or a solvate thereof.

[0086]

[10] The method according to any one of [6] to [9], wherein R1 and Q1 together with the carbon atoms to which they are attached form a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, or a tetrahydropyran ring, or

[0087] R1 and Q1 are independently selected from methyl, ethyl, 2-methylpropyl, allyl, methoxymethyl, cyclohexylmethyl, optionally substituted benzyl, or optionally substituted phenethyl.

[0088]

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

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

[0089]

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

[11] , wherein PG1 is a C2-C6 acyl halide.

[0090]

[13] The method described in

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

[0091]

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

[13] , wherein the dehydrated product is represented by the following formula:

[0092]

[0093] [wherein, Q1 and R1 have the same meanings as Q1 and R1 in formula (1), and R4 is a C1-C5 haloalkyl group].

[0094]

[15] The method according to

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

[0095]

[16] The method according to

[14] or

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

[0096]

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

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

[0097]

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

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

[0098]

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

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

[0099]

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

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

[0100]

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

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

[0101]

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

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

[0102]

[23] The method according to

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

[0103]

[24] The method according to any one of [3] to [8] and

[22] to

[23] , wherein the base is selected from:

[0104]

[0105] [Wherein,

[0106] RB1 and RB4 are independently C1-C4 alkyl, or RB1 and RB4 together with the nitrogen atom to which RB1 is bound and the carbon atom to which RB4 is bound form a 5- to 8-membered ring,

[0107] RB2 and RB3 are independently C1-C4 alkyl, or RB2 and RB3, together with the nitrogen atom to which RB2 is bound, the nitrogen atom to which RB3 is bound, and the carbon atom to which the nitrogen atom is bound, form a 5- to 8-membered ring],

[0108]

[0109] [Wherein,

[0110] RB6 is a C1-C4 alkyl group,

[0111] RB5 and RB7 are each independently a C1-C4 alkyl group, or together with each nitrogen atom to which they are bound and the carbon atom to which the nitrogen atom is bound, form a 5- to 8-membered ring,

[0112] RB8 is a C1-C4 alkyl group, and RB9 is a C1-C4 alkyl group or a phenyl group, or RB8 and RB9 together with each nitrogen atom to which they are bound and the carbon atom to which each nitrogen atom is bound form a 5- to 8-membered ring,

[0113] Here, when RB9 is a phenyl group, for two B2 groups, the two benzene rings of the phenyl group are optionally condensed to form naphthalene],

[0114]

[0115] [Wherein,

[0116] RB 10 is C1-C4 alkyl, or RB 10 and RB 11 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0117] RB 11 In RB 10 and RB 11 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 11 and RB 12 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0118] RB 12 In RB 11 and RB 12 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 12 and RB 13 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0119] RB 13 In RB 12 and RB 13 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 13and RB 14 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0120] RB 14 In RB 13 and RB 14 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 14 and RB 15 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0121] RB 15 In RB 14 and RB 15 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0122] RB 16 is hydrogen, C1-C8 alkyl, or C6-C 10 aryl], and

[0123]

[0124] [Wherein,

[0125] RB 17 are independently C1-C4 alkyl, or RB 17 and RB 18 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0126] RB 18 In RB 17 and RB 18 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 18 and RB 19 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0127] RB 19 In RB 18 and RB 19 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 19 and RB 20 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0128] RB 20 In RB 19 and RB 20 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0129] RB 21 is C1-C4 alkyl, or RB 21and RB 22 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0130] RB 22 In RB 21 and RB 22 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 22 and RB 23 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0131] RB 23 In RB 22 and RB 23 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 23 and RB 24 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0132] RB 24 In RB 23 and RB 24 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 24 and RB 25 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0133] RB 25 In RB 24 and RB 25 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 25 and RB 26 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0134] RB 26 In RB 25 and RB 26 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0135] RB 27 is a C1-C4 alkyl group, or a C6-C 10 Aryl].

[0136]

[25] The method according to any one of [3] to [8] and

[22] to

[24] , wherein the base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (TBD), tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu), tert-butylimino-tris(pyrrolidinyl)phosphorane (P1-t-Bu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphanphenyl (BEMP), tert-octylimino-tris(dimethylamino)phosphorane (P1-t-Oct), imino-tris(dimethylamino)phosphorane (HP1(dma)), 1-tert-butyl-2,2,4,4,4-penta(dimethylamino)-2λ 5 , 4λ 5 -bis(phosphazene) (P2-t-Bu), and 1-ethyl-2,2,4,4,4-penta(dimethylamino)-2λ 5 , 4λ 5 -bis(phosphazene) (P2-Et).

[0137]

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

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

[0138]

[27] A method for preparing 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, a salt thereof, or a solvate thereof, which comprises the method described in any one of [1] to

[26] .

[0139]

[28] A method for producing a cyclic peptide compound, a salt thereof, or a solvate thereof, comprising: deprotecting the N-terminal protecting group from the peptide compound, a salt thereof, or a solvate thereof prepared by the method described in any one of [1] to

[27] ,

[0140] optionally a step of extending the peptide chain, and

[0141] a step of cyclizing the group on the C-terminal side and the group on the N-terminal side to form a cyclic portion,

[0142] The cyclic peptide compound contains 8 to 15 amino acid residues, at least 3 N-substituted amino acid residues, and at least one amino acid residue not substituted by N, and the cyclic part contains at least 8 amino acid residues.

[0143] Effects of the Invention

[0144] According to the present invention, a peptide compound containing a dipeptide residue linked to an N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue can be efficiently prepared, which is useful in peptide drugs, research on peptide drugs, and / or the supply of raw materials for drugs. In addition, since peptide compounds combined with various non-natural amino acid residues can also be prepared, peptide compounds with diverse structures can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] [ Figure 1 ] Figure 1 This is a diagram showing the results of LCMS analysis of the reaction mixture of Example 2-4 using a photodiode array detector and detecting at the maximum absorption wavelength (analysis conditions: SQDFA05).

[0147] [ Figure 2 ] Figure 2 This is a diagram showing the results of LCMS analysis of the reaction mixture of Comparative Example 1 using a photodiode array detector and detecting at the maximum absorption wavelength (analysis conditions: SQDFA05).

[0148] [ Figure 3 ] Figure 3 This is a diagram showing the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 2-4 using a photodiode array detector and detecting at the maximum absorption wavelength.

[0149] [ Figure 4 ] Figure 4 This is a graph showing the results of LCMS analysis (analysis conditions: SQDFA05) of the reaction mixture of Comparative Example 2-5 using a photodiode array detector and detecting at the maximum absorption wavelength.

[0150] Modes for carrying out the invention

[0151] Abbreviations used in the present invention are described below.

[0152] AA: Ammonium acetate

[0153] CSA: (+)-10-Camphorsulfonic acid

[0154] DBU: 1,8-diazabicyclo[5.4.0]-7-undecene

[0155] DCC: N, N'-dicyclohexylcarbodiimide

[0156] DCM: dichloromethane

[0157] DCE: 1,2-dichloroethane

[0158] DEAD: Diethyl azodicarboxylate

[0159] DMA: dimethylacetamide

[0160] DMF: N, N-dimethylformamide

[0161] DIAD: diisopropyl azodicarboxylate

[0162] DIC: N, N'-diisopropylcarbodiimide

[0163] DIPEA: N, N-diisopropylethylamine

[0164] DMAP: N, N-dimethyl-4-aminopyridine

[0165] dtbbpy: 4,4'-di-tert-butyl-2,2'-bipyridine

[0166] EDTA: Ethylenediaminetetraacetic acid

[0167] FA: Formic acid

[0168] Fmoc: 9-fluorenylmethoxycarbonyl

[0169] NMP: N-methyl-2-pyrrolidone

[0170] TBME: tert-butyl methyl ether

[0171] TES: Triethylsilane

[0172] TFA: trifluoroacetic acid

[0173] TFE: 2,2,2-trifluoroethanol

[0174] THF: Tetrahydrofuran

[0175] THP: Tetrahydropyranyl

[0176] TMSCl: trimethylchlorosilane

[0177] HFIP: 1,1,1,3,3,3-hexafluoroisopropanol

[0178] HOAt: 1-Hydroxy-7-azabenzotriazole

[0179] HOBt: 1-Hydroxybenzotriazole

[0180] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

[0181] IPAC: Isopropyl Acetate

[0182] oxyma: ethyl cyano(hydroxyimino)acetate

[0183] PPTS: Pyridinium p-toluenesulfonate

[0184] EDCI·HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0185] TIPS: Triisopropylsilane

[0186] TfOH: trifluoromethanesulfonic acid

[0187] HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0188] DMSO: dimethyl sulfoxide

[0189] Fmoc-Cl: (9H-fluoren-9-yl)methylcarbonyl chloride

[0190] Fmoc-OSu: 9-fluorenylmethyl N-succinimidyl carbonate

[0191] Ns: o-nitrobenzenesulfonyl

[0192] Trt: triphenylmethyl or trityl

[0193] Tfa: trifluoroacetyl

[0194] MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene

[0195] TMGN: 1,8-bis(tetramethylguanidinyl)naphthalene

[0196] P1-tBu: tert-butylimino-tris(dimethylamino)phosphorane

[0197] (Definition of functional group, etc.)

[0198] As the "halogen atom" in this specification, F, Cl, Br or I is exemplified.

[0199] The "alkyl group" in this specification is a monovalent group derived from an aliphatic hydrocarbon by removing any hydrogen atom, and has a hydrocarbon group or a subset of hydrocarbon group structures that does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton and contains hydrogen and carbon atoms. Alkyl groups include not only straight-chain ones but also branched ones. Specifically, alkyl groups include those with 1 to 20 carbon atoms (C1-C 20 , below “C p-C q " means an alkyl group having p to q carbon atoms, preferably C1-C 10 The alkyl group is preferably a C1-C6 alkyl group. Specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl (2-methylpropyl), n-pentyl, sec-pentyl (1-methylbutyl), tert-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc. are listed.

[0200] In this specification, an "alkenyl group" is a group having at least one double bond (two adjacent sp 2 The double bond geometry may be entgegen (E) or zusammen (Z), cis or trans, depending on the configuration of the double bond and the substituent (when present). Alkenyl groups include not only straight-chain ones but also branched ones. Preferred alkenyl groups include C2-C 10 Alkenyl, more preferably C2-C6 alkenyl, specifically, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl and the like.

[0201] In this specification, "alkynyl" is a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkyl groups include not only straight-chain ones but also branched ones. 10 Alkynyl, more preferably C2-C6 alkynyl, specifically, for example, ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2′-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl and the like.

[0202] "Cycloalkyl" in this specification means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. As the cycloalkyl group, preferably C3-C8 cycloalkyl is listed, and specifically, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, etc. are listed.

[0203] The term "aryl" as used herein means a monovalent aromatic hydrocarbon ring, and preferably C6-C 10 Aryl group. Specific examples of the aryl group include phenyl and naphthyl (eg, 1-naphthyl and 2-naphthyl).

[0204] The "heterocyclic group" in this specification means a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclic group may have a double bond and / or a triple bond in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl group, and may be a monocyclic or condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4 to 10-membered heterocyclic group), and more preferably 4 to 7 (4 to 7-membered heterocyclic group). As the heterocyclic group, specifically, for example, azetidinyl, oxetanyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, Azetidyl, oxazolidinone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, 2-oxaspiro[3.3]heptyl, and the like.

[0205] In this specification, "heteroaryl" means an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a single ring or a condensed ring with other rings, and may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of the heteroaryl group include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridinyl.

[0206] In this specification, "alkoxy" means an oxy group bonded to the "alkyl" defined above, and preferably C1-C6 alkoxy groups are listed. As alkoxy groups, specifically, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, 3-methylbutoxy, etc. are listed.

[0207] In the present specification, "acyl (alkanoyl)" means a group in which a carbonyl group is bonded to hydrogen or the aforementioned "alkyl", preferably C1-C6 acyl, more preferably C2-C4 acyl. Specific examples of acyl include formyl, acetyl, propionyl, butyryl, and the like.

[0208] In the present specification, "cycloalkoxy" means an oxy group to which the above-defined "cycloalkyl" is bonded, and preferably C3-C8 cycloalkoxy is mentioned. Specific examples of the cycloalkoxy include cyclopropyloxy, cyclobutyloxy, and cyclopentyloxy.

[0209] In the present specification, the "alkylsulfonyl group" means a sulfonyl group to which the "alkyl group" defined above is bonded, and preferably a C1-C6 alkylsulfonyl group is mentioned. Specific examples of the alkylsulfonyl group include a methylsulfonyl group and the like.

[0210] The "hydroxyalkyl" in this specification means a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by hydroxyl groups, preferably a C1-C6 hydroxyalkyl group. As the hydroxyalkyl group, specifically, for example, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, 5-hydroxypentyl, etc. are listed.

[0211] In this specification, "haloalkyl" means a group in which one or more hydrogen atoms of the aforementioned "alkyl" are replaced by halogen, preferably C1-C6 haloalkyl, more preferably C1-C6 fluoroalkyl. As haloalkyl, specifically, for example, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, etc. are listed.

[0212] In the present specification, "haloalkoxy" means a group in which one or more hydrogen atoms of the "alkoxy" defined above are replaced by halogen, preferably C1-C6 haloalkoxy. Specifically, for example, difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, etc. are listed as haloalkoxy.

[0213] In the present specification, "haloacyl (haloalkanoyl)" means a group in which a carbonyl group is bonded to the aforementioned "haloalkyl" group, preferably a C2-C6 haloacyl group, more preferably a C2-C4 haloacyl group. As haloacyl groups, specifically, trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, 3,3,3-trifluoro-2-(trifluoromethyl)propionyl, etc. are exemplified.

[0214] In the present specification, "alkoxyalkyl" means a group in which one or more hydrogen atoms of the aforementioned "alkyl" are replaced by the aforementioned "alkoxy" group, preferably a C1-C6 alkoxy C1-C6 alkyl group, and more preferably a C1-C6 alkoxy C1-C2 alkyl group. As the alkoxyalkyl group, specifically, for example, methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, isobutoxymethyl, sec-butoxymethyl, tert-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, 2-ethoxyethyl, etc. are listed.

[0215] In the present specification, "cycloalkylalkyl" means a group in which one or more hydrogen atoms of the aforementioned "alkyl" are replaced by the aforementioned "cycloalkyl", preferably C3-C8cycloalkylC1-C6alkyl, more preferably C3-C6cycloalkylC1-C2alkyl. As cycloalkylalkyl, specifically, for example, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. are listed.

[0216] In the present specification, "cycloalkoxyalkyl" means a group in which one or more hydrogen atoms of the aforementioned "alkyl" are replaced by the aforementioned "cycloalkoxy" group, preferably a C3-C8 cycloalkoxy C1-C6 alkyl group, more preferably a C3-C6 cycloalkoxy C1-C2 alkyl group. Specifically, for example, cycloalkoxyalkyl groups include cyclopropyloxymethyl and cyclobutyloxymethyl groups.

[0217] In the present specification, "alkylsulfonylalkyl" means a group in which one or more hydrogen atoms of the aforementioned "alkyl" are replaced by the aforementioned "alkylsulfonyl" group, preferably C1-C6 alkylsulfonylC1-C6 alkyl, more preferably C1-C6 alkylsulfonylC1-C2 alkyl. Specifically, for example, methylsulfonylmethyl, 2-(methylsulfonyl)ethyl, etc. are cited as examples of the alkylsulfonylalkyl group.

[0218] The term "aralkyl" in this specification means a group in which at least one hydrogen atom of the aforementioned "alkyl" is replaced by an "aryl" as defined above, preferably a C7-C 14 Arylalkyl, more preferably C7-C 10 Aralkyl group. Specific examples of the aralkyl group include benzyl, phenethyl, and 3-phenylpropyl.

[0219] In the present specification, "heteroaralkyl (heteroarylalkyl)" means a group in which at least one hydrogen atom of the "alkyl" defined above is substituted by the "heteroaryl" defined above, preferably a 5- to 10-membered heteroaryl C1-C6 alkyl, and more preferably a 5- to 10-membered heteroaryl C1-C2 alkyl. As the heteroarylalkyl, specifically, for example, 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, 2-(5-benzofuranyl)ethyl and the like are listed.

[0220] In the present specification, the "protecting group of carboxyl group" includes alkyl ester type protecting groups, benzyl ester type protecting groups, substituted alkyl ester type protecting groups, etc. As the protecting group of carboxyl group, specifically, methyl, ethyl, t-Bu group, benzyl, trityl, cumyl, methoxytrityl, 2-(trimethylsilyl)ethyl, 2,2,2-trichloroethyl, allyl group, etc. are exemplified.

[0221] In the present specification, the "amino group protecting group" includes carbamate type protecting groups, amide type protecting groups, imide type protecting groups, sulfonamide type protecting groups, etc. As the amino group protecting group, specifically, Fmoc, Boc, Cbz, Alloc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, toluenesulfonyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, etc. are exemplified.

[0222] In the present specification, "alicyclic ring" means a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond in the ring, or may be a polycyclic ring having two or more rings. In addition, the carbon atoms constituting the ring may be oxidized to form a carbonyl group. As the alicyclic ring, preferably a 3- to 8-membered alicyclic ring is listed, and specifically, for example, 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. are listed.

[0223] In this specification, "heterocycle" means a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocycle may have double bonds and / or triple bonds in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl group. The heterocycle may be a monocyclic ring, a condensed ring, or a spirocyclic ring. The number of atoms constituting the ring is not limited, but preferably 3 to 12 (3 to 12-membered heterocycle), and more preferably 4 to 7 (4 to 7-membered heterocycle). Specific examples of the heterocycle include piperazine, pyrrolidine, piperidine, morpholine, homomorpholine, hexahydropyrazine, 3-oxopiperazine, 2-oxopyrrolidine, azetidine, 2-oxoimidazolidine, oxetane, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, tetrahydropyridine, thiomorpholine, pyrazolidine, imidazoline, oxazolidine, isoxazolidine, thiazolidine, imidazolidine, isothiazolidine, thiadiazolidine, oxazolidinone, benzodioxane, dioxolane, dioxane, and tetrahydrothiopyran.

[0224] In this specification, "saturated heterocycle" means a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms and containing no double bonds and / or triple bonds in the ring. The saturated heterocycle may be a monocyclic ring or may form a condensed ring with other rings, such as aromatic rings such as a benzene ring. When the saturated heterocycle forms a condensed ring, a 4- to 7-membered saturated heterocycle is preferably listed as a saturated heterocycle, specifically, for example, an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, an oxazolidinone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, a dihydroindole ring, and the like.

[0225] In this specification, "peptide chain" refers to a peptide chain of one or more natural amino acids and / or non-natural amino acids connected by amide bonds and / or ester bonds. As a peptide chain, a peptide chain containing 1 to 15 amino acid residues is preferred, and a peptide chain consisting of 5 to 12 amino acid residues is more preferred.

[0226] The "peptide compound" in the present invention is not particularly limited as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are linked by amide bonds and / or ester bonds. The number of amino acid residues is preferably 5 to 30 residues, more preferably 8 to 15 residues, and further preferably 9 to 13 residues. The peptide compound loaded on a resin for solid phase synthesis is also included in the peptide compound. The peptide compound synthesized in the present invention preferably contains at least 3 N-substituted amino acids in one peptide, and more preferably contains at least 5 or more N-substituted amino acids. These N-substituted amino acids may exist continuously or discontinuously in the peptide compound. In this specification, the "amino acids" constituting the peptide compound are sometimes referred to as "amino acid residues", and the "peptide" constituting all or part of the peptide compound is sometimes referred to as "peptide residues". The peptide compound in the present invention may be linear or cyclic, preferably a cyclic peptide compound.

[0227] The "cyclic peptide compound" in the present invention is a cyclic peptide compound that can be obtained by cyclizing the group on the N-terminal side of a linear peptide compound with the group on the C-terminal side. The cyclization can be any form such as cyclization through a carbon-nitrogen bond such as an amide bond, cyclization through a carbon-oxygen bond such as an ester bond and an ether bond, cyclization through a carbon-sulfur bond such as a thioether bond, cyclization through a carbon-carbon bond, or cyclization through heterocyclic construction. Among them, cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxylic acid group on the side chain and an amino group on the main chain of the N-terminal is more preferred. The position of the carboxylic acid group and the amino group used in the cyclization can be on the main chain or on the side chain, and there is no particular limitation as long as it is a cyclizable position.

[0228] In this specification, "one or more" means one or more than two. When "one or more" is used in the context of a substituent of a group, the term means from one to the maximum number of substituents allowed by the group. As "one or more", specifically, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or a number greater than these are listed.

[0229] The "resin for solid phase synthesis" in this specification is not particularly limited as long as it can be used in the synthesis of peptide compounds by the solid phase method. As such a resin for solid phase synthesis, specifically, for example, CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), 4-methoxytrityl chloride resin (Mmt) and the like that can be removed under acidic conditions are listed. The resin can be appropriately selected according to the functional group on the amino acid side to be used. For example, when a carboxylic acid (main chain carboxylic acid, or a side chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (a phenolic group represented by Tyr) is used as the functional group on the amino acid side, it is preferred to use a trityl chloride resin (Trt resin) or a 2-chlorotrityl chloride resin (CTC resin) as the resin. When an aliphatic hydroxyl group (aliphatic alcohol group represented by Ser or Thr) is used as the functional group on the amino acid side, it is preferred to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin) or 4-methyltrityl chloride resin (Mtt resin) as the resin. In addition, in this specification, the resin is sometimes described as resin. The resin for solid phase synthesis can be connected to an amino acid at any position not limited to the C-terminal amino acid in the peptide. It is preferred that the carboxyl group of the C-terminal amino acid is connected to the resin for solid phase synthesis, and the carboxyl group may be a main chain carboxyl group or a side chain carboxyl group.

[0230] There is no particular limitation on the type of polymer constituting the resin. In the case of a resin composed of polystyrene, either 100-200 mesh or 200-400 mesh can be used. In addition, there is no particular limitation on the crosslinking rate, but 1% DVB (divinylbenzene) crosslinking is preferred. In addition, as the type of polymer constituting the resin, Tentagel or Chemmatrix is ​​listed.

[0231] In the preparation of the compounds described in this specification, when the defined groups are subjected to undesirable chemical transformation under the conditions of the implementation method, for example, the compounds can be prepared by applying means such as protection and deprotection of functional groups. Here, the selection of protecting groups and the introduction and removal operations can be cited, for example, as described in "Greene's, "Protective Groups in Organic Synthesis" (5th edition, John Wiley & Sons 2014), which can be appropriately applied according to the reaction conditions. In addition, the order of the reaction steps such as the introduction of substituents can also be changed as needed.

[0232] In the present specification, when the modifier "optionally substituted" is given, examples of the substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclic group, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl and the like.

[0233] Further, they can be given substituents respectively, and these substituents are also not limited, for example, one or more can be freely selected independently from any substituents comprising halogen atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms, or phosphorus atoms. That is, examples include alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, etc., which are optionally substituted.

[0234] The compounds described in the present invention may be salts thereof or solvates thereof. The salts of the compounds described in the present invention include, for example: hydrochloride; hydrobromide; hydroiodide; phosphate; phosphonate; sulfate; sulfonates such as methanesulfonate and p-toluenesulfonate; carboxylates such as acetate, citrate, malate, tartrate, succinate, and salicylate; or alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, and tetraalkylammonium salt. These salts are prepared, for example, by contacting the compound with an acid or a base. The solvate of the compound described in the present invention refers to the phenomenon that the solute molecules in the solution strongly attract the solvent molecules to form a molecular group. If the solvent is water, it is called a hydrate. The compound described in the present invention may be a solvate with an organic solvent selected from alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), dimethylformamide, diglyme, or water, or may be a solvate with multiple solvents.

[0235] The "amino acids" in this specification include natural amino acids and non-natural amino acids (sometimes referred to as amino acid derivatives). The "natural amino acids" in this specification refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, Pro. Non-natural amino acids (amino acid derivatives) are not particularly limited, and examples include β-amino acids, D-type amino acids, N-substituted amino acids, α, α-disubstituted amino acids, amino acids with side chains different from natural amino acids, hydroxycarboxylic acids, etc. As amino acids in this specification, any spatial configuration is allowed, but L-type amino acids are preferred. The selection of the side chain of the amino acid is not particularly limited, and in addition to hydrogen atoms, it is also freely selected from, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and spiro-bound cycloalkyl. Substituents may be given separately, and these substituents are also not limited. For example, one or more substituents may be independently selected from any substituents containing halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms, or P atoms. That is, examples include optionally substituted alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, or oxo, aminocarbonyl, halogen atoms, etc. In a non-limiting manner, the amino acid in this specification may be a compound having a carboxyl group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acid).

[0236] As the substituent containing a halogen atom in the present specification, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl and the like in which a halogen is present in the substituent are exemplified, and more specifically, fluoroalkyl, difluoroalkyl, trifluoroalkyl and the like are exemplified.

[0237] As substituents containing an O atom, there are listed groups such as hydroxyl (-OH), oxyl (-OR), carbonyl (-C=OR), carboxyl (-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), aminosulfonylamino (-NH-SO2-NHR), thiocarboxy (-C=O-SH), and carboxycarbonyl (-C=O-CO2H).

[0238] Examples of the oxy group (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As the alkoxy group, a C1-C4 alkoxy group or a C1-C2 alkoxy group is preferred, and a methoxy group or an ethoxy group is particularly preferred.

[0239] Examples of the carbonyl group (—C═OR) include formyl (—C═OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl and the like.

[0240] Examples of the oxycarbonyl group (—C═O—OR) include an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, an aralkyloxycarbonyl group and the like.

[0241] Examples of the carbonyloxy group (—OC═OR) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group and an aralkylcarbonyloxy group.

[0242] Examples of the thiocarbonyl group (—C═O—SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, an aralkylthiocarbonyl group and the like.

[0243] Examples of the carbonylthio group (-SC=OR) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group and an aralkylcarbonylthio group.

[0244] Examples of aminocarbonyl groups (-C=O-NHR) include alkylaminocarbonyl groups (e.g., C1-C6 or C1-C4 alkylaminocarbonyl groups, particularly ethylaminocarbonyl and methylaminocarbonyl groups), cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. In addition to these, groups in which the H atom bonded to the N atom in -C=O-NHR is further substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0245] Examples of the carbonylamino group (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, there are also groups in which the H atom bonded to the N atom in -NH-C=OR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0246] Examples of the oxycarbonylamino group (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkyloxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, there are also groups in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0247] Examples of the sulfonylamino group (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0248] Examples of aminosulfonyl groups (-SO2-NHR) include alkylaminosulfonyl groups, cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups, aralkylaminosulfonyl groups, etc. In addition to these, groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group are also included.

[0249] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Further, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with a substituent independently selected from an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and an aralkyl group, and the two substituents may form a ring.

[0250] Examples of the substituent containing a S atom include a thiol group (—SH), a sulfenyl group (—SR), a sulfinyl group (—S═OR), a sulfonyl group (—SO 2 —R), a sulfo group (—SO 3 H), and a pentafluorosulfanyl group (—SF 5 ).

[0251] Examples of the thio group (—SR) include alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio and the like.

[0252] Examples of the sulfinyl group (—S═OR) include an alkylsulfinyl group, a cycloalkylsulfinyl group, an alkenylsulfinyl group, an alkynylsulfinyl group, an arylsulfinyl group, a heteroarylsulfinyl group, and an aralkylsulfinyl group.

[0253] Examples of the sulfonyl group (-SO2-R) include an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, an aralkylsulfonyl group and the like.

[0254] As substituents containing a N atom, there are listed azide (-N3, also called "azido"), 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″), guanidine (-NH-C(=NH)-NH2), substituted guanidine (-NR-C(=NR″′)-NR′R″), aminocarbonylamino (-NR-CO-NR′R″), pyridyl, piperidino, morpholino, azetidinyl and the like groups.

[0255] Examples of the secondary amino group (—NH—R) include an alkylamino group, a cycloalkylamino group, an alkenylamino group, an alkynylamino group, an arylamino group, a heteroarylamino group, and an aralkylamino group.

[0256] Examples of tertiary amino groups (-NR(R'); disubstituted amino groups) include amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these two substituents may form a ring. Specifically, dialkylamino groups, particularly C1-C6 dialkylamino groups, C1-C4 dialkylamino groups, dimethylamino groups, diethylamino groups, etc., are exemplified. In this specification, "C p -C q "Dialkylamino" refers to an amino group substituted with 2 C p -C q Alkyl group, two C p -C q The alkyl groups may be the same or different.

[0257] As examples of substituted amidino groups (-C(=NR)-NR′R″), the three substituents R, R′, and R″ on the N atom are listed as groups independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, for example, alkyl (aralkyl) (aryl) amidino groups, etc.

[0258] As examples of substituted guanidine groups (-NR-C(=NR"')-NR'R"), R, R', R", and R"' are listed as groups independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or groups forming a ring thereof, etc.

[0259] Examples of aminocarbonylamino (-NR-CO-NR'R") include groups wherein R, R', and R" are independently selected from hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or groups forming a ring thereof.

[0260] As the substituent containing a B atom, a boron group (-BR(R')) and a dioxyboryl group (-B(OR)(OR')) are listed. These two substituents R and R' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or they can form a ring. Specifically, a cyclic boron group is listed, and more specifically, a pinacholatoboryl group, a neopentanediolatoboryl group, a catechol boron group, etc. are listed.

[0261] As the substituent on the nitrogen atom of the N-substituted amino acid in the present specification, specifically, alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, C7-C 14 Aralkyl, benzyl, phenethyl, etc.

[0262] The amino group in the main chain of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR. Here, R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group which may optionally have a substituent. In addition, as in proline, the carbon chain bound to the N atom and the carbon atom at the α position may form a ring.). Amino acids in which the hydrogen atoms of the main chain amino group are substituted are sometimes referred to as "N-substituted amino acids" in this specification. Preferred examples of "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, N-C7-C 14 Aralkyl amino acids, N-benzyl amino acids, N-phenylethyl amino acids, but not limited to these.

[0263] The "amino acid" in this specification includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom is replaced by an atom with the same atomic number (number of protons) but a different mass number (the sum of the number of protons and neutrons). Examples of isotopes included in the "amino acid" in this specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., respectively. 2 H. 3 H. 13 C. 14 C. 15 N. 17 O.18 O. 32 P. 35 S. 18 F. 36 Cl et al.

[0264] (Preparation method)

[0265] In a certain embodiment, the present invention relates to a method for preparing a peptide compound having an N-substituted-α,α disubstituted amino acid residue at the N-terminus, a dipeptide residue comprising the N-substituted-α,α disubstituted amino acid residue and an N-substituted amino acid residue connected thereto, a salt thereof, or a solvate thereof, the method comprising steps A and B described later.

[0266] (Step A)

[0267] Step A is a step of reacting an N-substituted amino acid, a salt thereof, or a solvate thereof, or a peptide compound having an N-substituted amino acid residue at the N-terminus, a salt thereof, or a solvate thereof, with an N-unsubstituted-α,α-disubstituted amino acid, a salt thereof, a dehydrate thereof, or a solvate thereof in which the amino group is protected with an electron-withdrawing protecting group, in the presence or absence of a condensation reagent, to obtain a peptide compound having an N-unsubstituted-α,α-disubstituted amino acid residue at the N-terminus, or a dipeptide residue comprising the N-unsubstituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue, a salt thereof, or a solvate thereof. In the present specification, the peptide compound comprising a dipeptide means that the dipeptide is contained in the amino acid sequence constituting the peptide compound.

[0268] In a certain embodiment, the "N-substituted amino acid" used in step A is any natural or non-natural amino acid whose main chain amino group is -NHR, where R is any group other than hydrogen. As R, specifically, for example, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, etc. are listed. In addition, R is such as proline, and the carbon chain bonded to the N atom and the carbon atom at the α position can form a ring, and the ring can be further substituted by any substituent. In addition, the N-substituted amino acid can be in the form of a salt or a solvate.

[0269] In one embodiment, the "peptide compound having an N-substituted amino acid residue at the N-terminus" used in step A is not limited to the type and number of other amino acids contained in the peptide compound as long as it has the aforementioned N-substituted amino acid residue at the N-terminus. In addition, the peptide compound may be in the form of a salt or a solvate.

[0270] The N-substituted amino acid or the peptide compound having an N-substituted amino acid residue at the N-terminus used in step A can be purchased from a supplier, or the one purchased from a supplier can be formulated with modifications.

[0271] Specific examples of such N-substituted amino acids or peptide compounds having an N-substituted amino acid residue at the N-terminus include compounds represented by the following formula (2), or salts thereof, or solvates thereof:

[0272]

[0273] [Wherein,

[0274] P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0275] R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkyloxyC1-C6 alkyl, or C7-C 14 Aralkyl,

[0276] R3 is hydroxyl, O-PG2, any amino acid residue, or any peptide residue,

[0277] PG2 is a protecting group for the carboxyl group].

[0278] In a certain embodiment, the "N-unsubstituted-α, α-disubstituted amino acid in which the amino group is protected with an electron-withdrawing protecting group" used in step A means an amino acid in which the α carbon of the amino acid has two arbitrary substituents other than hydrogen, the amino group of the main chain of the amino acid is unsubstituted, and the amino group is protected with an electron-withdrawing protecting group (i.e., "protecting group -NH-"). The amino acid may be in the form of a salt or a solvate. The two substituents bound to the α carbon may be the same or different. As the substituent, specifically, for example, there are listed optionally substituted alkyl, optionally substituted alkoxyalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, etc. In addition, the two substituents bound to the α carbon may form an optionally substituted alicyclic ring or an optionally substituted heterocyclic ring together with the carbon atom to which they are bound.

[0279] The N-unsubstituted-α,α-disubstituted amino acid in which the amino group is protected with an electron withdrawing protecting group used in step A is purchased from a supplier or can be prepared by modifying the one purchased from the supplier.

[0280] In one embodiment, step A can be carried out in the presence of a condensation reagent. On the other hand, step A can be carried out in the absence of a condensation reagent when, for example, a dehydrate of an N-unsubstituted-α,α-disubstituted amino acid is used for the condensation reaction.

[0281] As such an N-unsubstituted-α,α-disubstituted amino acid in which the amino group is protected with an electron-withdrawing protecting group, specifically, there are exemplified compounds represented by the following formula (3), or salts thereof, or solvates thereof:

[0282]

[0283] [Wherein,

[0284] PG1 is an electron-withdrawing protecting group.

[0285] R1 and Q1 are independently selected from C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, or optionally substituted C7-C 14 Aralkyl, or

[0286] R1 and Q1 together with the carbon atom to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring].

[0287] In a certain embodiment, the electron-withdrawing protecting group bound to the N-unsubstituted-α,α-disubstituted amino acid is a protecting group having a pKa (in water) of 6 to 11 for the NH group to which the protecting group is bound, preferably a protecting group having a pKa (in water) of 8 to 11 for the NH group. As such a protecting group, specifically, C2-C6 halogenated acyl groups are listed, and more specifically, trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, or 3,3,3-trifluoro-2-(trifluoromethyl)propionyl and the like are listed.

[0288] In a certain embodiment, as the peptide compound obtained by step A, which has an N-unsubstituted-α,α-disubstituted amino acid residue at the N-terminus, and comprises a dipeptide residue to which the N-unsubstituted-α,α-disubstituted amino acid residue and the N-substituted amino acid residue are linked, specifically, a compound represented by the following formula (4), or a salt thereof, or a solvate thereof is exemplified:

[0289]

[0290] [Wherein,

[0291] PG1, R1, and Q1 are synonymous with PG1, R1, and Q1 in formula (3), respectively.

[0292] P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2).

[0293] (Step B)

[0294] Step B is a step of introducing a substituent into the amino group of the N-unsubstituted-α,α-disubstituted amino acid residue whose amino group is protected with an electron-withdrawing protective group at the N-terminus of the peptide compound obtained in step A in the presence of a base and a substituent-introducing agent, thereby obtaining a peptide compound having an N-substituted-α,α-disubstituted amino acid residue whose amino group is protected with an electron-withdrawing protective group at the N-terminus and comprising a dipeptide residue connected to the N-substituted amino acid residue, a salt thereof, or a solvate thereof.

[0295] Specific examples of the substituent introduced in this step include an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aralkyl group, an optionally substituted cycloalkyl group, and the like.

[0296] In one embodiment, the base used in step B is preferably one whose conjugate acid has a pKa (in acetonitrile) of 23 to 30. Specific examples of such a base include bases having an amidine skeleton, bases having a guanidine skeleton, or bases having a phosphazene skeleton, which will be described later.

[0297] In a certain embodiment, the substituent-introducing agent used in step B is used to introduce a substituent into the amino group of the N-unsubstituted-α,α-disubstituted amino acid residue whose amino group is protected with an electron-withdrawing protecting group at the N-terminus (i.e., "protecting group -NH-"). As the substituent-introducing agent, an electrophilic reagent can be used. Specifically, a compound combining an introduced substituent and a leaving group (e.g., a sulfonic acid group such as a halogen, a trifluoromethanesulfonyl group, a mesyl group, or a tosyl group, or a phosphate group) can be used.

[0298] In a certain embodiment, as the peptide compound obtained by step B having an N-substituted-α,α-disubstituted amino acid residue having an amino group protected by an electron-withdrawing protecting group at the N-terminus, or a dipeptide residue comprising the N-substituted-α,α-disubstituted amino acid residue and an N-substituted amino acid residue, specifically, a compound represented by the following formula (1), or a salt thereof, or a solvate thereof:

[0299]

[0300] [Wherein,

[0301] P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 Aralkyl,

[0302] PG1, R1, and Q1 have the same meanings as PG1, R1, and Q1 in formula (3), respectively.

[0303] P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (2).

[0304] The "peptide compound having an N-substituted-α,α disubstituted amino acid residue at the N-terminus and a dipeptide residue comprising the N-substituted-α,α disubstituted amino acid residue and the N-substituted amino acid residue connected" prepared by the method of the present invention may be a peptide compound having an N-terminal amino group protected by a protecting group or a free amino group (NHR-) with the protecting group removed. When the amino group at the N-terminus is protected by a protecting group, the protecting group may be an electron-withdrawing protecting group resulting from the "N-unsubstituted-α,α disubstituted amino acid in which the amino group is protected by an electron-withdrawing protecting group" applied in step A, or may be another protecting group (e.g., Fmoc group) introduced after the electron-withdrawing protecting group is deprotected. In addition to the aforementioned steps A and B, the present invention may also include a step of removing the electron-withdrawing protecting group and a step of introducing an arbitrary protecting group different from the protecting group. In the introduction and removal of the protecting group, for example, the method described in "Greene's, "Protective Groups in Organic Synthesis" (5th edition, John Wiley & Sons 2014)" may be applied.

[0305] In one embodiment, the present invention relates to a method for preparing a peptide compound having a structure in which two amino acid residues are linked represented by formula (1), a salt thereof, or a solvate thereof, the method comprising step A and step B as shown in the following scheme.

[0306]

[0307] In each of the above formulas, PG1 is a protecting group for an amino group. In formula (4), it is preferred that a protecting group whose pKa is 11 or less is used for the NH group to which PG1 is bound. 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, the P1 group can be selectively introduced into the NH group of formula (4) to which PG1 is bound. The pKa can be a value calculated using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C) 1994-2019 ACD / Labs). For example, the pKa of the NH group of (2,2,2-trifluoroacetyl)alanine tert-butyl ester in which a trifluoroacetyl group is bound to a nitrogen atom is 9.71, and the pKa of the NH group of 2-methyl-2-(2,2,2-trifluoroacetamide)propionic acid tert-butyl ester is 9.21. In addition, the pka of the NH group of methyl 2-methyl-2-(2,2,3,3,3-pentafluoropropionamide) propionate in which a pentafluoropropionyl group is bonded to a nitrogen atom is 9.27, and the pka of the NH group of methyl 2-methyl-2-(2,2,2-trichloroacetamide) propionate 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 methyl 2-acetamide-2-methylpropionate in which an acetyl group having a weaker electron-withdrawing force than these halogenated acyl groups is bonded to a nitrogen atom is 14.36, and the acidity of the NH group is weaker than that of the halogenated acyl groups. In the present invention, PG1 preferably has an electron-withdrawing protective group that increases the acidity of the proton of the NH group, and as such a protective group, a C2-C6 halogenated acyl group is listed. As the C2-C6 haloacyl group, a trifluoroacetyl group, a trichloroacetyl group, a pentafluoropropionyl group, a 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl group, or a 3,3,3-trifluoro-2-(trifluoromethyl)propionyl group is preferred.

[0308] In formula (1), P1 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 When P1 is a C1-C6 alkyl group, the C1-C6 alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. When P1 is a C2-C6 alkenyl group, the C2-C6 alkenyl group is preferably an allyl group. When P1 is a C7-C 14 When aralkyl is C7-C 14 The aralkyl group is preferably a benzyl group or a phenethyl group.

[0309] In the above formulae, R1 and Q1 are independently selected from C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, or optionally substituted C7-C 14 Aralkyl, or

[0310] R1 and Q1 together with the carbon atoms to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring.

[0311] When R1 and / or Q1 are C1-C6 alkyl, the C1-C6 alkyl is preferably methyl, ethyl, isopropyl, or 2-methylpropyl. When R1 and / or C2-C6 alkenyl, the C2-C6 alkenyl is preferably allyl. When R1 and / or Q1 are C1-C6 alkoxy C1-C6 alkyl, the C1-C6 alkoxy C1-C6 alkyl is preferably methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, isobutoxymethyl, sec-butoxymethyl, tert-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, or 2-ethoxyethyl. When R1 and / or Q1 are C3-C8 cycloalkyl C1-C6 alkyl, the C3-C8 cycloalkyl C1-C6 alkyl is preferably cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, 2-cyclohexylethyl. 14 When aralkyl is C7-C 14 Arylalkyl, preferably benzyl or phenethyl, as C7-C 14 The substituent of the aryl group of the aralkyl group is preferably one or more groups selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, and cyano.

[0312] When R1 and Q1 together with the carbon atoms to which they are attached 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.

[0313] In the above formulas, P2 is C1-C6 alkyl, C2-C6 alkenyl, or C7-C 14 When P2 is a C1-C6 alkyl group, the C1-C6 alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. When P2 is a C2-C6 alkenyl group, the C2-C6 alkenyl group is preferably an allyl group. 14 When aralkyl is C7-C 14 The aralkyl group is preferably a benzyl group or a phenethyl group.

[0314] In the above formulas, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkyloxyC1-C6 alkyl, or C7-C 14 Aralkyl.

[0315] As R2, preferred are C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C4 hydroxyalkyl, methylsulfonyl C1-C2 alkyl, C2-C3 alkynyl, C1-C4 alkoxy C1-C2 alkyl optionally substituted with one or more fluorine, C3-C6 cycloalkyl, C3-C6 cycloalkyl C1-C2 alkyl, C3-C6 cycloalkyloxy C1-C2 alkyl, benzyl, phenethyl.

[0316] As R2, specifically, for example, methyl, ethyl, n-propyl, isopropyl, 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, cyclopropyloxymethyl, cyclobutyloxymethyl, (2,2,2-trifluoroethoxy)methyl, 2-methylsulfonylethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, benzyl, phenethyl and the like are listed.

[0317] In the above formulas, R3 is a hydroxyl group, O-PG2, an arbitrary amino acid residue, or an arbitrary peptide residue, and PG2 is a protecting group for a carboxyl group. When R3 is O-PG2, as PG2, specifically, for example, alkyl groups such as tert-butyl and trityl, cumyl, allyl, benzyl, etc. are listed. When R3 is an arbitrary amino acid residue or an arbitrary peptide residue, the amino acid residue or peptide residue can be loaded onto a resin for solid phase synthesis. When a peptide residue is loaded onto a resin for solid phase synthesis, the resin can be loaded onto an amino acid residue at the C-terminal end of the peptide residue, or onto an amino acid residue at any position other than the C-terminal end. As a resin for solid phase synthesis, CTC resin, Wang resin, or SASRIN resin are preferably listed, and CTC resin is further preferred. In addition, when R3 is an arbitrary peptide residue, the peptide residue is composed of an arbitrary type and number of amino acid residues. As the number of amino acid residues constituting the peptide residue, 2 to 13 are preferred, and 2 to 9 are more preferred.

[0318] In formula (1), as the following formula:

[0319]

[0320] Specific examples of the amino acid residue represented by include MeAib, MecLeu, Me(Me)Phe, Me(Me)Abu, Me(Me)Leu, Me(Me)Ser(Me), Me(Me)Phe, Me(Me)Cha, Me(Me)Val, EtAib, nPrAib, AllylAib, and BnAib.

[0321] In formula (1), as the following formula:

[0322]

[0323] Specific examples of the amino acid residues represented include MeAla, MeLeu, MeCha, MeVal, MeAla (cPent), MeAla (cBu), MeAla (cPr), MeChg, MeGly (cPent), MeGly (cBu), MeGly (cPr), MeAbu, MeNva, MeNle, MeNva (5-F2), MeHle, MeIle, MeSer (nPr), MeSer (cPr), MeHnl, MeHnl (7-F2), MePRA, MeSer (Me), MeThr, MeSer (cBu), MeSer (Tfe), MeThr (Me), MeHse (Me), MeMet (O2), EtVal, and nPrVal.

[0324] In the formula (1), when R3 is an arbitrary amino acid residue, specific examples of the amino acid residue include MeSer(tBuOH), bAla, bMeAla, MeGly, MePhe, MePhe(3-F), MePhe(4-F), D-MePhe, 2-ACHxC, 2-ACPnC, 3-CF3-bAla, Asp-mor, Asp-mor(26-bicyc), Asp-mor(SO2), Asp-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(iP en)-(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), MeAsp-piz(oxe), MeAsp-pyrro, MeAsp-pyrro(34-F4), MeAsp-pyrro(3-Me2), nPrAsp-pip.

[0325] Step A is a step of reacting a compound represented by formula (2), a salt thereof, or a solvate thereof, and a compound represented by formula (3), a salt thereof, a dehydrate thereof, or a solvate thereof with a condensation agent to obtain a compound represented by formula (4), a salt thereof, or a solvate thereof, or a step of reacting a compound represented by formula (2), a salt thereof, or a solvate thereof, and a dehydrate of a compound represented by formula (3) (i.e., a compound represented by formula (3')), a salt thereof, or a solvate thereof in the absence of a condensation agent to obtain a compound represented by formula (4), a salt thereof, or a solvate thereof.

[0326] The compound represented by the following formula (2) can be purchased from a supplier, or the one purchased from a supplier can be modified and used as needed. Specifically, for example, the compound represented by formula (2) can be prepared by introducing P2 into the one purchased from a supplier.

[0327]

[0328] P2, R2, and R3 in formula (2) have the same meanings as P2, R2, and R3 in formula (1).

[0329] The compound represented by the following formula (3) can be purchased from a supplier, or the one purchased from the supplier can be changed and applied as needed. Specifically, for example, the compound represented by formula (3) can be prepared by introducing PG1 into the one purchased from the supplier using a base and a PG1 introduction reagent in a solvent. As the PG1 introduction reagent, specifically, for example, ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate, trifluoroacetic anhydride, pentafluoropropionic anhydride, trichloroacetic anhydride, etc. are listed, and as the base, specifically, for example, N, N-diisopropylethylamine, triethylamine, sodium methoxide, sodium ethoxide, etc. are listed. As the solvent used when PG1 is introduced, specifically, when ethyl trifluoroacetate, ethyl pentafluoropropionate, or ethyl trichloroacetate is used as an introduction reagent, for example, methanol, ethanol, etc. are listed. In addition, when trifluoroacetic anhydride, pentafluoropropionic anhydride, trichloroacetic anhydride are used as introduction reagents, for example, dichloromethane, tetrahydrofuran, pyridine, etc. are listed.

[0330]

[0331] In the formula, PG1, Q1, and R1 have the same meanings as PG1, Q1, and R1 in formula (1), respectively.

[0332] Step A can be carried out under reaction conditions known in the literature. For example, methods described in the Solid Phase Synthesis Handbook published by Merck on May 1, 2002, etc. can be used appropriately according to the reaction conditions. As the condensation reagent used in step A, carbodiimide condensation agents represented by DCC (N, N'-dicyclohexylcarbodiimide), DIC (N, N'-diisopropylcarbodiimide), EDCI·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), combinations of carbodiimide condensation agents and additives represented by HOAt, HOBt, and oxyma, HATU (O-(7-aza-1H-benzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate), HBTU (O-(1H-benzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate), can be used. Urea salt condensing agents represented by HCTU (O-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbonium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyBOP (1H-benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate), PyOxim ([ethylcyano(hydroxyimino)acetic acid-O 2

[0043] Phosphonium salt condensing agents represented by tris-1-pyrrolidinylphosphonium hexafluorophosphate), 1-chloro-N, N-2-trimethyl-1-propyleneamine (Ghosez reagent), TCFH (chloro-N, N, N', N'-tetramethylformamidinium hexafluorophosphate), PyCIU (N, N, N', N'-bis(tetramethylene)chloroformamidinium hexafluorophosphate), BTFFH (fluoro-N, N, N', N'-bis(tetramethylene)formamidinium hexafluorophosphate), TFFH (fluoro-N, N, N', N'-tetramethylamidinium hexafluorophosphate), etc. Preferably, any one of DIC and EDCI·HCl, or a combination of DIC and Oxyma is used.

[0333] In addition, when PG1 is a C2-C6 acyl halide, an oxazolone represented by formula (3') prepared from a compound represented by formula (3) as a dehydrate of the compound can also be used in step A. The oxygen atom and the carbon atom between the oxygen atom and the nitrogen atom constituting the oxazolone ring are derived from the carbonyl group of the C2-C6 acyl halide of PG1, and R4 is a C1-C5 haloalkyl group derived from the haloalkyl group of the C2-C6 acyl halide of PG1. As the reactant for preparing oxazolone, specifically, for example, N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, thionyl chloride, etc. are listed.

[0334]

[0335] In the compound represented by formula (3'), R1 and Q1 have the same meanings as R1 and Q1 in formula (3). R1 ​​and Q1 may form a 3- to 8-membered alicyclic ring together with the carbon atom to which they are bound. Specific examples of such a 3- to 8-membered alicyclic ring include cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, and the like.

[0336] In the compound represented by formula (3'), R4 is a C1-C5 haloalkyl group, and specific examples of the C1-C5 haloalkyl group include trifluoromethyl, trichloromethyl, pentafluoroethyl, 1,2,2,2-tetrafluoro-1-(trifluoromethyl)ethyl, and 2,2,2-trifluoro-1-(trifluoromethyl)ethyl. Among them, trifluoromethyl is preferred.

[0337] Step B is a step of reacting the compound represented by formula (4), a salt thereof, or a solvate thereof with a P1-introducing reagent to obtain a peptide compound represented by formula (1), a salt thereof, or a solvate thereof.

[0338] In the present invention, as a P1-introducing reagent, a combination of P1X (wherein P1 is synonymous with P1 in formula (1) and X is a leaving group) and a base can be used. In step B, by reacting a compound represented by formula (4) with P1X in the presence of a base having an appropriate pKa, P1 can be selectively introduced into the nitrogen atom to which PG1 is bound.

[0339] As P1X, specifically, 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. are listed. When the P1-introducing reagent is a methylating reagent, specifically, for example, methyl iodide, dimethyl sulfate, methyl trifluoromethanesulfonate, methyl p-toluenesulfonate, methyl methanesulfonate, etc. are listed. When the P1-introducing reagent is an ethylating reagent, specifically, for example, ethyl iodide, ethyl bromide, diethyl sulfate, ethyl trifluoromethanesulfonate, ethyl p-toluenesulfonate, ethyl methanesulfonate, etc. are listed. When the P1-introducing reagent is an allylation reagent, specifically, for example, allyl chloride, allyl bromide, etc. are listed. When the P1-introducing reagent is a benzylation reagent, specific examples of the benzylation reagent include benzyl chloride and benzyl bromide. When the P1-introducing reagent is a phenethylation reagent, specific examples of the phenethylation reagent include (2-iodoethyl)benzene and (2-bromoethyl)benzene.

[0340] When a combination of P1X and a base is used as a P1-introducing reagent, the base can be one having a basicity suitable for introducing P1 into the target nitrogen atom. The basicity of the base is represented by the pKa of the conjugate acid of the base. The pKa of the conjugate acid of the base is sometimes referred to as the pKa of the base.

[0341] Specifically, a base having a pKa sufficient to dehydrogenate the hydrogen of the NH group to which P1 is bound may be used.

[0342] The pKa of the conjugate acid of the base can be appropriately referred to the calculated value using Advanced Chemistry Development (ACD / Labs) Software V11.02 ((c) 1994-2019 ACD / Labs), Chem. Eur. J. 2002, 8, 1682-1693, J. Org. Chem. 2005, 70, 3, 1019-1028, Eur. J. Org. Chem., 2019, 40, 6735-6748, or the value described in the catalog of Sigma-Aldrich Company.

[0343] The pKa varies depending on the solvent. The pKa of the conjugate acid 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))).

[0344] On the other hand, the pKa of the conjugate acid of DBU, TMGN, MTBD, P1-tBu, BTPP, and BEMP in acetonitrile is 24.32, 25.1, 25.43, 26.9, 28.4, 27.6 (Chem. Eur. J. 2002, 8, 1682-1693, catalog value of Sigma-Aldrich). The pKa of the conjugate acid of DBN in acetonitrile is 23.89 (Eur. J. Org. Chem., 2019, 40, 6735-6748).

[0345] [Table 1]

[0346]

[0347] With respect to the pKa value of the conjugate acid of the base in water (calculated by Advanced Chemistry Development (ACD / Labs) Software V11.02 ((C) 1994-2019 ACD / Labs)) and the pKa value in acetonitrile, the pKa value in acetonitrile is approximately 10 to 14 higher.

[0348] In the present invention, it is preferred to use a protecting group having a pKa (in water) of 11 or less for the NH group to which PG1 is bonded. The pKa (in water) of the NH group to which PG1 is bonded is preferably 6-11, and more preferably 8-11.

[0349] The pKa of the conjugate acid of the base required for deprotonating the proton of the NH group needs to be different from the pKa of the NH group by at least 2, preferably 2 to 3, and more preferably 2 to 6.

[0350] Therefore, 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 to be used is (1) larger than the pKa of the NH group, (2) further, the pKa differs by at least 2, preferably by 6, and (3) further, preferably a base with a pKa greater than the pKa in water by (10 to 14) in terms of the converted pKa in acetonitrile. As a specific base, the pKa value of the conjugate acid (in acetonitrile) of 18 to 31, 22 to 29, 22 to 30, 22 to 31, 23 to 29, 23 to 30, 23 to 31 can be used as the base in this reaction. The range of the pKa of the conjugate acid (in acetonitrile) of the base to be used is preferably 22 to 31.

[0351] In addition, when the pKa (in acetonitrile) of the NH group to which PG1 is bonded is 8 to 11, the base used can be used as the base in this reaction as long as the pKa (in acetonitrile) of the conjugate acid of the base used is 20 to 31, 20 to 30, 20 to 29, 21 to 31, 21 to 30, 21 to 29, 22 to 31, 22 to 30, 22 to 29, 23 to 31, 23 to 30, or 23 to 29. The range of the pKa (in acetonitrile) of the conjugate acid of the base used is preferably 23 to 30.

[0352] In one embodiment, the base is represented by the following formula B1 having an amidine skeleton.

[0353]

[0354] [Wherein,

[0355] RB1 and RB4 are independently C1-C4 alkyl, or RB1 and RB4 together with the nitrogen atom to which RB1 is bound and the carbon atom to which RB4 is bound form a 5- to 8-membered ring,

[0356] RB2 and RB3 are each independently a C1-C4 alkyl group, or RB2 and RB3 together with the nitrogen atom to which RB2 is bound, the nitrogen atom to which RB3 is bound, and the carbon atom to which the nitrogen atom is bound form a 5- to 8-membered ring].

[0357] When RB1 to RB4 are C1-C4 alkyl groups, preferably, the C1-C4 alkyl group includes a methyl group and an ethyl group.

[0358] When RB1 and RB4 form a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include a pyrrolidine ring, a piperidine ring, and an azepane ring.

[0359] When RB2 and RB3 form a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include 1,4,5,6-tetrahydropyrimidine ring and the like.

[0360] As the base represented by the formula B1, specifically, for example, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and the like are cited.

[0361]

[0362] In one embodiment, the base is represented by the following formula B2 having a guanidine skeleton.

[0363]

[0364] [Wherein,

[0365] RB6 is hydrogen or C1-C4 alkyl,

[0366] RB5 and RB7 are each independently a C1-C4 alkyl group, or together with each nitrogen atom to which they are bound and the carbon atom to which the nitrogen atom is bound, form a 5- to 8-membered ring,

[0367] RB8 is a C1-C4 alkyl group, and RB9 is a C1-C4 alkyl group or a phenyl group, or RB8 and RB9 together with each nitrogen atom to which they are bound and the carbon atom to which each nitrogen atom is bound form a 5- to 8-membered ring,

[0368] Here, when RB9 is a phenyl group, the two benzene rings of the phenyl group may be condensed to form naphthalene for two B2 groups].

[0369] When RB5 to RB8 are C1-C4 alkyl groups, the C1-C4 alkyl group is preferably a methyl group, and when RB9 is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably a tert-butyl group.

[0370] When RB5 and RB7 form a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include an imidazolidine ring, a hexahydropyrimidine ring, and a 1,3-diazepane ring.

[0371] When RB8 and RB9 form a 5- to 8-membered ring, preferred examples of the 5- to 8-membered ring include 1,4,5,6-tetrahydropyrimidine ring.

[0372] As the base represented by formula B2, specifically, for example, 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), 2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and the like are listed.

[0373]

[0374] In one embodiment, the base is represented by the following formula B3 having a phosphazene skeleton.

[0375]

[0376] [Wherein,

[0377] RB 10 is C1-C4 alkyl, or RB 10 and RB 11 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0378] RB 11 In RB 10 and RB 11 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 11 and RB 12 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0379] RB 12 In RB 11 and RB 12 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 12 and RB 13 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0380] RB 13 In RB 12 and RB 13 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 13 and RB 14 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0381] RB 14 In RB13 and RB 14 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 14 and RB 15 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0382] RB 15 In RB 14 and RB 15 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0383] RB 16 is hydrogen, C1-C8 alkyl, or C6-C 10 Aryl].

[0384] RB 10 ~RB 15 When it is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably a methyl group or an ethyl group. 16 In the case of a C1-C8 alkyl group, the C1-C8 alkyl group is preferably a tert-butyl group or a tert-octyl group.

[0385] 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, preferred examples of the 5- to 8-membered ring include a pyrrolidine ring, a piperidine ring, and an azepane ring.

[0386] RB 11 and RB 12 , and / or RB 13 and RB 14 When a 5- to 8-membered ring is formed, the 5- to 8-membered ring preferably does not contain RB 11 , RB 12 , RB 13 , and RB 14 A 5- to 8-membered saturated ring consisting of each nitrogen atom bonded to the ring and a heteroatom other than the phosphorus atom bonded to the nitrogen atom.

[0387] As the base represented by formula B3, specifically, for example, tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu), tert-octylimino-tris(dimethylamino)phosphorane (P1-t-Oct), tert-butylimino-tris(pyrrolidinyl)phosphorane (P1-t-Bu-tris(tetramethylene), BTPP), 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphinane (BEMP), imino-tris(dimethylamino)phosphorane (HP1(dma)), and the like are listed.

[0388]

[0389] In one embodiment, the base is represented by the following B4 having a phosphazene skeleton containing two phosphorus atoms via a nitrogen atom.

[0390]

[0391] [Wherein,

[0392] RB 17 are independently C1-C4 alkyl, or RB 17 and RB 18 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0393] RB 18 In RB 17 and RB 18 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 18 and RB 19 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0394] RB 19 In RB 18 and RB 19 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 19 and RB 20 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0395] RB 20 In RB 19 and RB 20 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0396] RB 21 is C1-C4 alkyl, or RB 21 and RB 22 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0397] RB 22 In RB 21 and RB 22 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 22 and RB 23 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0398] RB 23 In RB 22 and RB 23When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 23 and RB 24 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0399] RB 24 In RB 23 and RB 24 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 24 and RB 25 Together with the nitrogen atoms to which they are bonded and the phosphorus atoms to which the nitrogen atoms are bonded, they form a 5- to 8-membered ring.

[0400] RB 25 In RB 24 and RB 25 When forming a 5- to 8-membered ring, it is C1-C4 alkyl, or RB 25 and RB 26 Together with the nitrogen atoms they are bound to, they form a 5- to 8-membered ring.

[0401] RB 26 In RB 25 and RB 26 When forming a 5- to 8-membered ring, it is a C1-C4 alkyl group.

[0402] RB 27 is a C1-C4 alkyl group, or a C6-C 10 Aryl].

[0403] RB 17 ~RB 26 When it is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably a methyl group or an ethyl group. 27 When it is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably a tert-butyl group.

[0404] 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 a 5- to 8-membered ring is formed, preferred examples of the 5- to 8-membered ring include a pyrrolidine ring, a piperidine ring, and an azepane ring.

[0405] RB 17 and RB 18 When all are C1-C4 alkyl, RB is preferred 19 and RB 20They are all C1-C4 alkyl, preferably RB 17 and RB 18 Forming 5- to 8-membered rings, RB 19 and RB 20 It also forms 5- to 8-membered rings.

[0406] RB 21 and RB 22 When all are C1-C4 alkyl, RB is preferred 23 and RB 24 and RB 25 and RB 26 They are all C1-C4 alkyl, preferably RB 21 and RB 22 Forming 5- to 8-membered rings, RB 23 and RB 24 and RB 25 and RB 26 It also forms a 5- to 8-membered ring.

[0407] RB 18 and RB 19 , and / or RB 22 and RB 23 When a 5- to 8-membered ring is formed, it is preferred that the 5- to 8-membered ring does not contain RB 11 , RB 12 , RB 13 , and RB 14 A 5- to 8-membered saturated ring consisting of each nitrogen atom bonded to the ring and a heteroatom other than the phosphorus atom bonded to the nitrogen atom.

[0408] As the base represented by the formula B4, specifically, for example, 1-tert-butyl-2,2,4,4,4-penta(dimethylamino)-2λ is exemplified. 5 , 4λ 5 -bis(phosphazene) (P2-t-Bu), 1-ethyl-2,2,4,4,4-penta(dimethylamino)-2λ 5 , 4λ 5 -bis(phosphazene) (P2-Et), etc.

[0409]

[0410] In the present invention, when a combination of P1X and a base is used as a P1-introducing reagent, examples of solvents used in the reaction include amide solvents represented by DMF and NMP, urea solvents represented by DMI, ether solvents represented by tetrahydrofuran and 2-methyltetrahydrofuran, and acetonitrile, among which amide solvents are preferred.

[0411] When P1X and a base are used as the P1 introduction reagent, a combination of PG1 and a base in which the pKa (in water) of the amino group protected with PG1 is 6 to 11 and the pKa (in acetonitrile) of the conjugate acid of the base is 23 to 30 is preferred. In addition, a combination of PG1 and a base in which the pKa (in water) of the amino group protected with PG1 is 8 to 11 and the pKa (in acetonitrile) of the conjugate acid of the base is 23 to 27 is more preferred. As a combination of PG1, P1X, and a base, it is preferred that PG1 is trifluoroacetyl, P1X is methyl iodide, dimethyl sulfate, ethyl iodide, allyl bromide, n-propyl iodide, benzyl bromide, and the base is P1-tBu, TMGN, or MTBD. Specific combinations of PG1 and a base include trifluoroacetyl and TMGN, trifluoroacetyl and P1-tBu, trifluoroacetyl and MTBD, and the like.

[0412] In a certain embodiment, the present invention relates to a method for preparing a peptide compound comprising an N-substituted-α,α disubstituted amino acid residue and a dipeptide residue to which an N-substituted amino acid residue is linked, a salt thereof, or a solvate thereof, comprising the aforementioned method for preparing a peptide compound having an N-substituted-α,α disubstituted amino acid residue at the N-terminus and comprising a dipeptide residue to which the N-substituted-α,α disubstituted amino acid residue is linked, a salt thereof, or a solvate thereof. The method may include the step of further condensing one or more amino acid residues and / or peptide residues at the N-terminus and / or the C-terminus of a peptide compound having an N-substituted-α,α disubstituted amino acid residue at the N-terminus and comprising a dipeptide residue to which the N-substituted-α,α disubstituted amino acid residue is linked, prepared by the method described in the present specification. The peptide compound prepared by the method is an arbitrary peptide compound comprising an N-substituted-α,α disubstituted amino acid residue and a dipeptide residue to which an N-substituted amino acid residue is linked, wherein the peptide compound comprises an arbitrary number and type of amino acids to which the N-terminus and the C-terminus of the dipeptide residue are linked.

[0413] In a certain embodiment, the present invention also relates to a method for preparing a cyclic peptide compound, which further comprises: a step of deprotecting the N-terminal protecting group (e.g., PG1) from a peptide compound represented by formula (1), a salt thereof, or a solvate thereof prepared by the method of the present invention, a step of optionally extending the peptide chain, and a step of cyclizing the group on the C-terminal side and the group on the N-terminal side to form a cyclic portion.

[0414] The cyclic peptide compound contains 8 to 15 amino acid residues, preferably 10 to 13 amino acid residues, contains at least 3, preferably at least 3 to (the number of amino acid residues constituting the cyclic peptide compound - 1) N-substituted amino acid residues, and contains at least 1, preferably at least 3 amino acid residues that are not substituted by N, and the cyclic portion contains at least 8 amino acid residues, preferably at least 10 amino acid residues.

[0415] In the step 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 applied.

[0416] In the step of extending the peptide chain and the step of forming the cyclic portion, known methods such as the method described in WO2013 / 100132 or WO2018 / 225864 can be applied. When the peptide chain is extended by solid phase synthesis, a step of cutting from the resin can be included before the step of extending and forming the cyclic portion.

[0417] In addition, all prior art documents cited in this specification are incorporated into this specification as reference. Example

[0418] The following examples, comparative examples and reference examples are used to further illustrate the present invention, but the present invention is not limited to these examples. All raw materials and reagents were obtained from suppliers or synthesized using known methods. The LCMS analysis conditions are listed in Table 2.

[0419] [Table 2]

[0420]

[0421] Example 1: Preparation of amino acids used in this example and peptides loaded onto resin

[0422] Example 1-1: Fmoc-amino acid used in peptide synthesis using a peptide synthesizer

[0423] In the peptide synthesis described in the present specification, the Fmoc-amino acids described in Tables 3 to 5 were used for synthesis using a peptide synthesizer.

[0424] The Fmoc-amino acids listed in Tables 3 and 5 were purchased from suppliers.

[0425] The Fmoc-amino acids listed in Table 4 were synthesized according to the scheme shown below.

[0426] [Table 3]

[0427]

[0428]

[0429] [Table 4]

[0430]

[0431] [Table 5]

[0432]

[0433] Example 1-1-1: Compound AA2-001, (2S)-4-[3-chloro-4-(trifluoromethyl)phenyl]-2-(9H-fluorene-9- Synthesis of (4-(4-methoxycarbonylamino)butyric acid, (Fmoc-Hph(4-CF3-3-Cl)-OH)

[0434]

[0435] DIC (138 mL, 1.54 eq.) was added dropwise to a solution of (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 eq.), and DMAP (3.6 g, 29.47 mmol, 0.05 eq.) in THF (2 L) under a nitrogen atmosphere at 0°C. The reaction solution was stirred at 25°C for 16 hours, the solid was removed by filtration, and the solvent was removed from the filtrate under reduced pressure. The residue was diluted with toluene, the generated solid was removed by filtration, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by recrystallization (acetone / heptane) to give compound AA2-001-a (1-O-benzyl 5-O-(1,3-dioxoisoindol-2-yl)(2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanedioate) (230 g, 80%).

[0436] LCMS (ESI) m / z = 505.2 (M + Na) +

[0437] Retention time: 0.992 minutes (analysis conditions SMDmethod_16)

[0438] Nickel bromide trihydrate (NiBr2·3H2O) (4 g, 0.07 eq.) and 4,4'-di-tert-butyl-2,2'-bipyridine (dtbbpy) (3.9 g, 14.55 mmol, 0.07 eq.) were added to DMA (500 mL) and stirred at 50°C for 2 hours under a nitrogen atmosphere to prepare a Ni solution.

[0439] The Ni solution prepared previously was added to a mixture of compound AA2-001-a (100 g, 207.3 mmol), zinc powder (70 g, 5 equivalents) and 4-bromo-2-chloro-1-(trifluoromethyl)benzene (160 g, 617 mmol, 3 equivalents) in DMA (500 mL), and stirred at 25 ° C for 16 hours. EDTA·2Na aqueous solution (10%) was added to the reaction solution and extracted with ethyl acetate. After washing the combined organic layer with saturated sodium chloride aqueous solution, it was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain compound AA2-001-b. (75 g, 77%)

[0440] LCMS (ESI) m / z = 494 (M + Na) +

[0441] Retention time: 2.863 minutes (analysis conditions SMDmethod_17)

[0442] A toluene solution (900 mL) of compound AA2-001-b (75 g, 158.93 mmol) was cooled at 0°C, and trifluoromethanesulfonic acid (TfOH) (42 mL, 3.00 equiv) was added dropwise. After stirring at room temperature for 1 hour, water (75 mL) was added. The mixed solution was extracted with water, and the combined aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Acetonitrile / water (900 / 900 mL) was added to the residue, and the pH was adjusted with aqueous sodium hydroxide solution (48%). Fmoc-OSu (51.2 g, 151.93 mmol, 0.95 equiv) was added to the solution, and stirred at room temperature for 16 hours while maintaining the pH at 7.8 to 8.0. The reaction solution was filtered, and the pH of the filtrate was adjusted to 2 with 6 mol / L 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-fluoren-9-ylmethoxycarbonylamino)butyric acid, Fmoc-Hph(4-CF3-3-Cl)-OH) (70 g, 87%).

[0443] LCMS (ESI) m / z = 525.8 (M + Na) +

[0444] Retention time: 2.180 minutes (analysis conditions SMDmethod_21)

[0445] 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)

[0446] Example 1-1-2: Compound AA2-002, (2S)-3-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl) Synthesis of amino]propionic acid (Fmoc-MeAla(cBu)-OH)

[0447]

[0448] To a DCM (46 mL) solution of compound AA2-002-a ((2S)-3-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonylamino]propionic acid, Fmoc-Ala(cBu)-OH) (3.36 g, 9.19 mmol), paraformaldehyde (0.828 g, 27.6 mmol), anhydrous magnesium sulfate (2.77 g, 22.99 mmol), and boron trifluoride ether complex (BF3·OEt2) (1.398 mL, 11.03 mmol) were added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. An aqueous solution prepared by diluting a saturated sodium chloride aqueous solution with water to half the concentration was added to the reaction solution, and DCM was further added for dilution. The separated organic layer was washed with a saturated sodium chloride aqueous solution and filtered. The obtained organic layer was freed from the solvent under reduced pressure to obtain compound AA2-002-b (3.63 g) as a crude product.

[0449] LCMS (ESI) m / z = 378 (M+H) +

[0450] Retention time: 1.01 minutes (analysis conditions SQDFA05)

[0451] To the obtained DCM (30.6 mL) solution of the compound AA2-002-b (3.47 g), triethylsilane (4.39 mL, 27.6 mmol), water (0.166 g, 9.19 mmol), and boron trifluoride 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 aqueous solution diluted with water to half the concentration was added to the reaction solution and stirred at room temperature for 15 minutes. The organic layer separated from the obtained mixture was freed from the solvent under reduced pressure. The obtained residue was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound AA2-002 ((2S)-3-cyclobutyl-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]propionic acid, Fmoc-MeAla(cBu)-OH). (3.18 g, 91% in 2 steps)

[0452] LCMS (ESI) m / z = 380 (M + H) +

[0453] Retention time: 0.94 minutes (analysis conditions SQDFA05)

[0454] Example 1-1-3: Compound AA2-003, (2S)-2-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl) Synthesis of amino]acetic acid (Fmoc-MeGly(cPent)-OH)

[0455]

[0456] After adding trifluoroacetic acid (TFA) (9.0 mL) 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), the mixture was stirred at 60°C for 4 hours. After cooling the reaction solution to room temperature, the solid was removed by filtration. After diluting the filtrate with ethyl acetate (220 mL), it was washed with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution in sequence. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound AA2-003-b as a crude product. The subsequent reaction was carried out without the above purification.

[0457] LCMS (ESI) m / z = 378 (M+H) +

[0458] Retention time: 1.01 minutes (analysis conditions SQDFA05)

[0459] Trifluoroacetic acid (TFA) (76 mL, 984 mmol) was added to a mixture of the obtained compound AA2-003-b (31 g, 82 mmol), triethylsilane (TES) (65.5 mL, 410 mmol) and dichloroethane (DCE) (90 mL), and the mixture was stirred at 60° C. for 16 hours. The reaction solution was cooled to room temperature and 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-fluoren-9-ylmethoxycarbonyl(methyl)amino]acetic acid, Fmoc-MeGly(cPent)-OH) (29.1 g, 93%).

[0460] LCMS (ESI) m / z = 380 (M + H) +

[0461] Retention time: 0.92 minutes (analysis conditions SQDFA05)

[0462] Example 1-1-4: Compound AA2-004, (2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl) Synthesis of amino]acetic acid (Fmoc-MeGly(cBu)-OH)

[0463]

[0464] Using compound AA2-004-a (2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]acetic acid, Fmoc-Gly(cBu)-OH) (2.5 g, 7.11 mmol) as starting materials, compound AA2-004-b was obtained as a crude product by the same method as the synthesis of compound AA2-002-b.

[0465] LCMS (ESI) m / z = 364 (M + H) +

[0466] Retention time: 0.97 minutes (analysis conditions SQDFA05)

[0467] The total amount of compound AA2-004-b obtained above was reacted in the same manner as in the synthesis of compound AA2-002, and then purified by reverse phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain compound AA2-004 ((2S)-2-cyclobutyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]acetic acid, Fmoc-MeGly(cBu)-OH) (2.32 g, 89% in 2 steps)

[0468] LCMS (ESI) m / z = 366 (M + H) +

[0469] Retention time: 0.88 minutes (analysis conditions SQDFA05)

[0470] Example 1-1-5: Compound AA2-005, (2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl) Synthesis of amino]propionic acid (Fmoc-MeAla(cPent)-OH)

[0471]

[0472] Using compound AA2-005-a ((2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonylamino]propionic acid, Fmoc-Ala(cPent)-OH) (10 g, 26.4 mmol) as the starting material, the same method as the synthesis of compound AA2-002-b was used to obtain compound AA2-005-b (10.5 g) as a crude product.

[0473] LCMS (ESI) m / z = 392 (M+H) +

[0474] Retention time: 1.05 minutes (analysis conditions SQDFA05)

[0475] The obtained compound AA2-005-b (10.5 g) was reacted in the same manner as in the synthesis of compound AA2-002, and then purified by reverse phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain compound AA2-005 ((2S)-3-cyclopentyl-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]propionic acid, Fmoc-MeAla(cPent)-OH) (10.11 g, 96% in 2 steps)

[0476] LCMS (ESI) m / z = 394 (M+H) +

[0477] Retention time: 0.98 minutes (analysis conditions SQDFA05)

[0478] Example 1-2: Preparation of amino acids and peptides loaded onto resin used in this example

[0479] Example 1-2-1: Compound 1-2-1, (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyridine Synthesis of 2-chlorotrityl-1-pyrrolidinobutyric acid resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro

[0480]

[0481] In this specification, when a compound is bound to a polymer or resin, the polymer or resin site is sometimes represented by ○. In addition, for the purpose of clarifying the reaction point of the resin site, the chemical structure of the reaction site connected to ○ is sometimes represented. 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 bound to the side chain carboxylic acid of Asp via an ester bond. In addition, pyrro means pyrrolidine, and in the above structure, the carboxylic acid group at the C-terminus forms an amide bond with pyrrolidine.

[0482]

[0483] Under nitrogen atmosphere, EDCI·HCl (67.1 g, 350 mmol), HOBt (43.4 g, 321 mmol), and Fmoc-Asp(OtBu)-OH (120 g, 292 mmol) were added to DMF (600 mL) at 0°C in order, and stirred at 0°C for 1 hour. Pyrrolidine (26.3 mL, 321 mmol) was slowly added to the reaction solution, and stirred at 0°C for 1.5 hours. Ethyl acetate (10 v) and 0.5 mol / L hydrochloric acid water (2 v) were added to the reaction solution at 0°C, and the organic layer was separated. The obtained organic layer was washed with 0.5 mol / L hydrochloric acid water, water, saturated sodium bicarbonate aqueous solution / water (1 / 1 (v / v)), and saturated sodium chloride aqueous solution / water (1 / 1 (v / v)) in order, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain compound 1-2-1-a as a crude product. (137.1 g, quant.)

[0484] LCMS (ESI) m / z = 465 (M + H) +

[0485] Retention time: 1.05 minutes (analysis conditions SQD compound AA05)

[0486] 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) so that the internal temperature did not exceed 10°C. After stirring at room temperature for 1 hour, diisopropyl ether (3.4 L) was added in 4 portions, and the precipitated solid was collected by filtration and dried to obtain compound 1-2-1-b ((3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid, Fmoc-Asp-pyrro). (108.4 g, 90%)

[0487] LCMS (ESI) m / z = 409 (M + H) +

[0488] Retention time: 0.83 minutes (analysis conditions SQD compound AA05)

[0489] The loading reaction of Fmoc amino acid to resin was carried out according to the method described in WO2013 / 100132 or WO2018 / 225864. 2-Chlorotrityl chloride resin (1.60mmol / g, 100-200 mesh, 1% DVB, 48.7g) and dehydrated dichloromethane (500mL) were added to a reaction vessel with a filter and shaken at room temperature for 20 minutes. After applying nitrogen pressure to remove dichloromethane, a mixture of dehydrated methanol (12.63mL) and diisopropylethylamine (DIPEA) (32.6mL) was added to compound 1-2-1-b (15.91g) and dehydrated dichloromethane (350mL) in the reaction vessel and shaken for 60 minutes. After the reaction solution was removed by nitrogen pressure, a mixed solution of dehydrated methanol (97.3 mL) and diisopropylethylamine (DIPEA) (32.6 mL) was added to dehydrated dichloromethane (350 mL) in a reaction vessel and shaken for 1 hour and 30 minutes. After the reaction solution was removed by nitrogen pressure, dichloromethane (350 mL) was added and shaken for 5 minutes, and then the reaction solution was removed by nitrogen pressure. The washing of the resin with dichloromethane was repeated 5 times, and the obtained resin was dried overnight under reduced pressure to obtain (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutyric acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro, compound 1-2-1, 59.79 g).

[0490] In order to confirm the loading rate, the obtained compound 1-2-1 (12.6 mg) was added to the reaction vessel, DMF (2 mL) was added, and the mixture was shaken at room temperature for 1 hour. Thereafter, DBU (40 μL) was added and shaken at 30°C for 30 minutes. Thereafter, DMF (8 mL) was added to the reaction mixture, and the solution was diluted to 1 mL with DMF (11.5 mL). The absorbance (294 nm) of the obtained diluted solution was measured (using Shimadzu, UV-1600PC (cell length 1.0 cm)). By measuring the dibenzofulvene of Fmoc from the Fmoc amino acid loaded on the resin, the loading amount of compound 1-2-1 was calculated to be 0.464 mmol / g.

[0491] In addition, other batches of the same synthesis with different loading amounts are also used in peptide synthesis and research.

[0492] Example 1-2-2: Preparation of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2)

[0493]

[0494] Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2) used in this Example was prepared by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by Intavis). The detailed procedure of the operation was in accordance with the manual attached to the synthesizer.

[0495] In the synthesizer, Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) (100 mg per column) prepared in Example 1-2-1, Fmoc-MeVal-OH (0.6 mol / L) and 1-hydroxy-7-azabenzotriazole (HOAt, 0.375 mol / L) in NMP, and diisopropylcarbodiimide (DIC) in N,N-dimethylformamide (DMF) solution (10% v / v) were placed.

[0496] At the beginning of the synthesis, 1 ml of dichloromethane (DCM) was added to the placed Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) (100 mg per column) and the mixture was allowed to stand for about 30 minutes to swell the resin. The resin was then washed with DMF.

[0497] De-Fmoc step

[0498] 0.7 mL of a DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v) was added per column and allowed to stand for 5 to 10 minutes to remove Fmoc. The resin was then washed with DMF (0.7 mL per column, repeated 4 times).

[0499] Extension steps

[0500] To the resin after the Fmoc removal step, add the mixed solution of the Fmoc-amino acid solution (0.30 mL per column) and the DIC / DMF solution (0.36 mL per column) and let it stand at 40 degrees. After the reaction is completed, wash the resin with DMF (0.7 mL per column, repeated 4 times).

[0501] Fmoc-MeVal was extended by the above steps. After extension, no Fmoc removal step was performed, and the product was further washed with DCM and dried for subsequent studies.

[0502] In addition, for the purpose of confirming the acquisition of compound 1-2-2, a portion of the obtained resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, the generation of the target peptide Fmoc-MeVal-Asp-pyrro (compound 1-2-2*) was confirmed. In addition, in this example, when a * is assigned to the compound number, it indicates a compound that was confirmed by cleaving the peptide from the resin in order to confirm the reaction. Compound 1-2-2* represents a peptide compound that cleaves the bond between the carboxylic acid of the peptide contained in compound 1-2-2 and the 2-chlorotrityl group of the resin.

[0503]

[0504] LCMS (ESI) m / z = 522.32 (M+H) +

[0505] Retention time: 0.76 minutes (analysis conditions SQDFA05)

[0506] Example 1-2-3: Preparation of Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-3)

[0507]

[0508] In the same manner as in Example 1-2-2, Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-1, 0.464 mmol / g) was extended to prepare Fmoc-MePhe-OH.

[0509] In order to confirm the acquisition of compound 1-2-3, a portion of the obtained resin was subjected to peptide cleavage using a TFE / DCM solution (1 / 1 (v / v). When the cleaved solution was analyzed by LCMS, the generation of the target peptide Fmoc-MePhe-Asp-pyrro (compound 1-2-3*) was confirmed.

[0510]

[0511] LCMS (ESI) m / z = 570.31 (M+H) +

[0512] Retention time: 0.80 minutes (analysis conditions SQDFA05)

[0513] Example 1-2-4: (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butyric acid-2-chlorotrityl Synthesis of resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, compound 1-2-4)

[0514]

[0515] Using (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butyric 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 supplier, the same method as the synthesis of compound 1-2-1 was used to obtain (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butyric acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin, compound 1-2-4). (58.95 g, loading amount 0.343 mmol / g)

[0516] In addition, other batches of the same synthesis with different loading amounts were also used in the peptide synthesis in this example.

[0517] Example 1-3: Amino acids protected with protecting groups other than Fmoc and their dehydrates used in peptide synthesis

[0518] Amino acids protected with a protecting group other than Fmoc and their dehydrates used in the peptide synthesis described in the present specification were synthesized as follows.

[0519] Example 1-3-1: 2-methyl-2-[(2,2,2-trifluoroacetyl)amino]propionic acid (Tfa-Aib-OH) (compound 1-3-1) Preparation

[0520]

[0521] Methanol (242 mL), DIPEA (63.5 mL, 1.5 equivalents), and ethyl trifluoroacetate ((CAS No. 383-63-1), 37.6 mL, 1.3 equivalents) were added to 2-amino-2-methylpropionic acid (25.0 g), and the mixture was stirred at 50 degrees for 18 hours. Thereafter, the solvent was removed under reduced pressure, and a 1N hydrochloric acid aqueous solution and ethyl acetate were added to the obtained residue, and the organic layer and the aqueous layer were separated. The obtained organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 18.2 g of a crude product.

[0522] After the crude product (16.0 g) was dissolved in TBME (80 mL), heptane (320 mL) was added dropwise with stirring for more than 1 hour. The mixture was further stirred for 1 hour under ice-cooling and filtered. The obtained powder was washed with 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]propionic acid (Tfa-Aib-OH) (Compound 1-3-1).

[0523] LCMS (ESI) m / z = 197.93 (MH) -

[0524] Retention time: 0.40 minutes (analysis conditions SQDFA05)

[0525] Example 1-3-2. Tfa-(Me)Abu-OH ((S)-2-methyl-2-(2,2,2-trifluoroacetamide)butyric acid, compound 1-3-2-b) Synthesis

[0526]

[0527] To a solution of compound 1-3-2-a ((S)-2-amino-2-methylbutyric acid, isovaline, H-(Me)Abu-OH) (15.0 g, 128 mmol) in methanol (150 mL) were added diisopropylethylamine (82.7 g, 640 mmol) and ethyl trifluoroacetate (54.6 g, 384 mmol), followed by stirring at 50°C for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The obtained residue was dissolved in TBME and washed twice with 1N aqueous hydrochloric acid solution. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (1:7) to obtain compound 1-3-2-b ((S)-2-methyl-2-(2,2,2-trifluoroacetamide)butyric acid) (12 g, 44%).

[0528] LCMS (ESI) m / z = 214.0 (M + H) +

[0529] Retention time: 0.32 minutes (analysis conditions SQDFA05)

[0530] Example 1-3-3. Tfa-(Me)Leu-OH ((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamide)pentanoic acid, Synthesis of Compound 1-3-3-b)

[0531]

[0532] 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) were added diisopropylethylamine (40.1 g, 310 mmol) and ethyl trifluoroacetate (44.0 g, 310 mmol), followed by stirring at 50°C for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The obtained residue was dissolved in TBME and washed twice with 1N aqueous hydrochloric acid solution. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (1:7) to obtain compound 1-3-3-b ((S)-2,4-dimethyl-2-(2,2,2-trifluoroacetamide)pentanoic acid) (10 g, 40%).

[0533] LCMS (ESI) m / z = 242.1 (M+H) +

[0534] Retention time: 0.66 minutes (analysis conditions SQDFA05)

[0535] Example 1-3-4. Tfa-(Me)Ser(Me)-OH((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetyl) Amine) propionic acid, synthesis of compound 1-3-4-b)

[0536]

[0537] To a solution of compound 1-3-4-a (3-methoxy-2-methyl-L-alanine, (S)-2-amino-3-methoxy-2-methylpropionic acid, H-(Me)Ser(Me)-OH) (1.5 g, 11 mmol) in methanol (19 mL) were added diisopropylethylamine (5.9 mL, 34 mmol) and ethyl trifluoroacetate (4.0 mL), followed by stirring at 50°C for 21 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in TBME (45 mL), washed twice with 1N aqueous hydrochloric acid solution (45 mL) and once with saturated aqueous sodium chloride solution (45 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-4-b ((S)-3-methoxy-2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid) (2.07 g, 72%).

[0538] LCMS (ESI) m / z = 228.2 (MH) -

[0539] Retention time: 0.41 minutes (analysis conditions SQDFA05)

[0540] Example 1-3-5. Tfa-(Me)Phe-OH((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamide)propane Acid, Synthesis of Compound 1-3-5-b)

[0541]

[0542] To a solution of compound 1-3-5-a ((2S)-2-amino-2-methyl-3-phenylpropionic acid, H-(Me)Phe-OH) (10.0 g, 55.8 mmol) in methanol (500 mL) were added diisopropylethylamine (21.63 g, 167.4 mmol) and ethyl trifluoroacetate (23.78 g, 167.4 mmol), followed by stirring at 50°C for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in TBME, washed twice with a 1N aqueous hydrochloric acid solution and once with a saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The resulting crude product was recrystallized from TBME / hexane (1:15) to obtain compound 1-3-5-b ((S)-2-methyl-3-phenyl-2-(2,2,2-trifluoroacetamide)propionic acid) (8 g, 52%).

[0543] LCMS (ESI) m / z = 274.0 (MH) -

[0544] Retention time: 0.68 minutes (analysis conditions SQDFA05)

[0545] Example 1-3-6. Tfa-(Me)Cha-OH((S)-3-cyclohexyl-2-methyl-2-(2,2,2-trifluoroacetamide)propane Acid, Synthesis of Compound 1-3-6-c)

[0546]

[0547] 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-methylpropionic acid, Fmoc-(Me)Cha-OH), and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. Water (8 mL) was added to the reaction solution 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). In addition, water (5 mL) and 2N hydrochloric acid (5 mL) were added to the organic phase, and after extracting the residual crude product in the aqueous layer, the aqueous layer was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile). Compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropionic acid, H-(Me)Cha-OH) (1.1 g, 81%) was obtained by combining the column purified products and used for the subsequent reaction.

[0548] LCMS (ESI) m / z = 186.1 (M+H) +

[0549] Retention time: 0.32 minutes (analysis conditions SQDFA05)

[0550] To a methanol (20 mL) solution of compound 1-3-6-b ((S)-2-amino-3-cyclohexyl-2-methylpropionic acid, H-(Me)Cha-OH) (1.1 g, 6.0 mmol) was added diisopropylethylamine (3.1 mL, 18 mmol) and ethyl trifluoroacetate (2.1 mL), and the mixture was stirred at 50°C for 2 hours. After the reaction solution was cooled 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 solution was concentrated under reduced pressure, and the obtained residue was dissolved in TBME (30 mL), washed twice with 1N aqueous hydrochloric acid solution (30 mL), and once with saturated aqueous sodium chloride solution (40 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-6-c ((S)-3-cyclohexyl-2-methyl-2-(2,2,2-trifluoroacetamide)propanoic acid) (1.22 g, 72%).

[0551] LCMS (ESI) m / z = 280.2 (MH) -

[0552] Retention time: 0.75 minutes (analysis conditions SQDFA05)

[0553] Example 1-3-7. Tfa-(Me)Val-OH((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamide)butyric acid, Synthesis of Compound 1-3-7-b)

[0554]

[0555] After adding diisopropylethylamine (8.0mL, 46mmol) and ethyl trifluoroacetate (5.5mL) to a methanol (25mL) solution of compound 1-3-7-a ((S)-2-amino-2,3-dimethylbutyric acid, H-(Me)Val-OH) (2.0g, 15mmol), the mixture was stirred at 50 degrees for 3 hours. After the reaction solution was cooled to room temperature, diisopropylethylamine (4.0mL, 23mmol) and ethyl trifluoroacetate (2.7mL) were added, and the mixture was stirred at 50 degrees for 16 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in TBME (40mL), and then washed with 1N aqueous hydrochloric acid solution (40mL) and saturated aqueous sodium chloride solution (40mL) in that order. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by reverse phase column chromatography (0.1% formic acid-water / 0.1% formic acid-acetonitrile) to obtain compound 1-3-7-b ((S)-2,3-dimethyl-2-(2,2,2-trifluoroacetamide)butanoic acid) (1.17 g, 34%).

[0556] LCMS (ESI) m / z = 226.1 (MH) -

[0557] Retention time: 0.54 minutes (analysis conditions SQDFA05)

[0558] Example 1-3-8. Tfa-cLeu-OH (1-(2,2,2-trifluoroacetamide) cyclopentane-1-carboxylic acid, compound 1-3- 8-b) Synthesis

[0559]

[0560] After adding diisopropylethylamine (37.5g, 290mmol) and ethyl trifluoroacetate (41.3g, 290mmol) to a methanol (100mL) solution of compound 1-3-8-a (1-aminocyclopentanecarboxylic acid, H-cLeu-OH) (25g, 194mmol), stir at 50 degrees for 2 days. After cooling the reaction solution to room temperature, add diisopropylethylamine (4.0mL, 23mmol) and ethyl trifluoroacetate (2.7mL), and stir at 50 degrees for 16 hours. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in TBME, and then washed with 1N hydrochloric acid aqueous solution and saturated sodium chloride aqueous solution in order. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The obtained crude product was recrystallized from TBME / hexane (3:20) to give compound 1-3-8-b (1-(2,2,2-trifluoroacetamide)cyclopentane-1-carboxylic acid) (20 g, 46%).

[0561] LCMS (ESI) m / z = 224.0 (MH) -

[0562] Retention time: 0.49 minutes (analysis conditions SQDFA05)

[0563] Example 1-3-9. 2-(Trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (Compound 1-3- 9) Synthesis

[0564]

[0565] 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 the mixture was stirred at room temperature for 2 days. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether) to obtain compound 1-3-9 (2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one) (11.9 g, 52%).

[0566] LCMS (ESI) m / z = 208.1 (M+H) +

[0567] Retention time: 0.86 minutes (analysis conditions SQDAA05)

[0568] Example 2: Extension of the N-terminus of a peptide in solid phase synthesis by N-substituted amino acid residues Protected N-unsubstituted-α,α-disubstituted amino acids, N-functionalization on solid phase, attempt to introduce N-substituted-α,α-disubstituted amino acids Experiments on amino acid residues

[0569]

[0570] Example 2-1: DeFmoc of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) Afterwards, the extension of Tfa-Aib-OH

[0571] Example 2-1-1: Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, Tfa-Aib-OH extension using DIC

[0572] In a reaction vessel with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 1 hour to swell the resin. After removing dichloromethane with a filter, the resin was washed 3 times with DMF (0.7 mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7 mL) was added to the resin and shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc removal solution, the resin was washed 4 times with DMF (0.7 mL).

[0573] The obtained resin was subjected to an extension reaction of Tfa-Aib-OH.

[0574] The extension reaction was carried out by adding a mixed solution of 0.6 M Tfa-Aib-OH / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 20 hours.

[0575] After removing the liquid phase of the extension reaction with a filter, the resin was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain batch number 1 of compound 2-1 (Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) (hereinafter referred to as compound 2-1-1).

[0576] In order to confirm the progress of the reaction, the obtained resin (compound 2-1-1) was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS to confirm the generation of the target peptide Tfa-Aib-MeVal-Asp-pyrro (compound 2-1*). Other peptide components were not detected. The resin (compound 2-1-1) was used in Example 2-3.

[0577]

[0578] LCMS (ESI) m / z = 481.21 (M+H) +

[0579] Retention time: 0.53 minutes (analysis conditions SQDFA05)

[0580] Example 2-1-2: Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) Extension of Tfa-Aib-OH using DIC and oxyma as an additive after Fmoc

[0581] In a reaction vessel with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed, and the extension of Tfa-Aib was carried out in the same manner as in Example 2-1-1 except for the extension reagent. The extension reagent used was a mixed solution of 0.6 M Tfa-Aib-OH / 0.375 M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL). The peptide was cleaved from the resin in the same manner as in Example 2-1-1, and the result of LCMS analysis showed that in addition to the target peptide Tfa-Aib-MeVal-Asp-pyrro (Compound 2-1*) 87.50% (UVarea), unreacted H-MeVal-Asp-pyrro was also confirmed at 2.2% (UVarea) and two unidentified peaks (8.85 and 1.43% (UVarea) respectively). This resin (Batch No. 2 of Compound 2-1, hereinafter referred to as Compound 2-1-2) was used in Comparative Example 1.

[0582] Example 2-1-3: Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) Elongation of Tfa-Aib-OH using EDCI·HCl after Fmoc

[0583] In a reaction vessel with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.473 mmol / g, 100 mg) prepared in Example 1-2-2 was placed, and the extension of Tfa-Aib was carried out in the same manner as in Example 2-1-1 except for the extension reagent. The extension reagent used was a mixed solution of 0.6 M Tfa-Aib-OH / NMP solution (0.3 mL) and EDCI·HCl (48 mg, 0.250 mmol) / DMF solution (0.36 mL). The peptide was cleaved from the resin in the same manner as in Example 2-1-1, and the result of LCMS analysis showed that in addition to the target peptide Tfa-Aib-MeVal-Asp-pyrro (Compound 2-1*) 93.1% (UVarea), unreacted H-MeVal-Asp-pyrro was also confirmed at 3.0% (UVarea) and unidentified 3.9% (UVarea). This resin (Batch No. 3 of Compound 2-1, hereinafter referred to as Compound 2-1-3) was used in Examples 2-2 and 2-3.

[0584] Example 2-2: Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Nucleophilic substitution reaction of the Tfa amide site (methyl iodide as methylating agent, DBU as base) to N-methylate

[0585] In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-3) (25 mg) prepared in Example 2-1, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with NMP (0.7 mL).

[0586] DBU (23 μL) / NMP (0.35 mL) solution was added to the obtained resin, and then methyl iodide (63 μL) / NMP (0.35 mL) solution was added, and the mixture was shaken at 40 degrees for 30 minutes. After removing the liquid phase with a filter, the mixture was washed 4 times with NMP (0.7 mL) and 4 times with dichloromethane. A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS.

[0587] The same operation was performed again on the resin that had been methylated for the first time in order to increase the reaction conversion rate. The second methylation was performed at 40 degrees with shaking for 20 hours. Compound 2-2 was obtained by washing the resin. A small amount of the obtained resin was sampled and the peptide was cut with a TFE / DCM solution (1 / 1 (v / v)). The cut solution was analyzed by LCMS to confirm the target (compound 2-2*) and the unreacted product (compound 2-1*). The results are shown in Table 6.

[0588] [Table 6]

[0589]

[0590]

[0591] LCMS (ESI) m / z = 495.23 (M+H) +

[0592] Retention time: 0.57 minutes (analysis conditions SQDFA05)

[0593] According to the results, in the nucleophilic substitution reaction using methyl iodide as the methylating agent and DBU as the base (pKa of the conjugate acid in acetonitrile = 24.34 (J. Org. Chem. 2005, 70, 1019-1028)), the Tfa-protected N-terminus was selectively N-methylated. Repeating the reaction by replacing the reagents showed that the conversion rate of the reaction was improved.

[0594] Example 2-3: Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) Nucleophilic substitution reaction of the amide site of Tfa (methyl iodide as methylating agent, various bases) to N-methylate

[0595] In a reaction vessel equipped with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-1 or 2-1-3) (25 mg) prepared in Example 2-1, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with DMF (0.7 mL).

[0596] To the obtained resin, a base (the amount added is described in Table 7) / DMF (0.35 mL) solution was added, and then a methyl iodide (63 μL) / DMF (0.35 mL) solution was added, and the mixture was shaken at 40 degrees for 15 hours. After removing the liquid phase with a filter, the mixture was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane to obtain compound 2-2. A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS.

[0597] The results of confirming the cleavage reaction are shown in Table 7. When P1-tBu was used as a base, a small amount of overmethylated product (Compound 2-3*) was also observed.

[0598] [Table 7]

[0599]

[0600]

[0601] LCMS (ESI) m / z = 509.25 (M+H) +

[0602] Retention time: 0.59 minutes (analysis conditions SQDFA05)

[0603] According to the results, in the nucleophilic substitution reaction using MTBD (pKa of the conjugate acid in acetonitrile = 25.43 (Chem. Eur. J. 2002, 8, 1682-1693)), TMGN (pKa of the conjugate acid in acetonitrile = 25.1 (Chem. Eur. J. 2002, 8, 1682-1693)), and P1-tBu (pKa of the conjugate acid in acetonitrile = 26.9 (Aldrich's website for phosphazene bases: https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html (available on October 10, 2019))) which have stronger basicity than DBU as bases, the target N-methylation was shown to occur. In addition, when P1-tBu was used, a small amount (3.8%) of overmethylated products were confirmed in the secondary amide site (amide site composed of the amino group of Asp and the carboxyl group of MeVal) different from the Tfa amide site. Based on this result, it is considered more preferable to use a base with a conjugate acid pKa value of 27 or less in order to selectively N-methylate the Tfa amide site.

[0604] Example 2-4: N-alkylated Tfa-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( Deprotection of Tfa protection of compound 2-2)

[0605] Under a nitrogen atmosphere, triglyme (6.6 mL) was added to sodium borohydride (0.5 g), and the mixture was stirred at room temperature for 10 minutes to obtain a 2.0 M sodium borohydride / triglyme solution.

[0606] In a reaction container equipped with a filter, the N-methylated resin (Compound 2-2) prepared in Example 2-3 and the third batch of Table 7 using TMGN as a base was added, and dichloromethane (1 mL) was added and shaken at room temperature for 30 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with THF (0.7 mL).

[0607] THF (125 μL), methanol (63 μL), and a previously prepared 2.0 M sodium borohydride / triethylene glycol dimethyl ether solution (63 μL) were added to the obtained resin, and the mixture was shaken at room temperature for 30 minutes. After removing the liquid phase with a filter, the mixture was washed 4 times with methanol (0.7 mL) (each washing time was 1 minute), and then washed 4 times with dichloromethane (0.7 mL) to obtain compound 2-4. A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and the cleaved solution was analyzed by LCMS.

[0608] The Tfa-protected raw material 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. Figure 1 , confirming that it can be synthesized with high purity.

[0609]

[0610] Target peptide H-MeAib-MeVal-Asp-pyrro (Compound 2-4*)

[0611] LCMS (ESI) m / z = 399.23 (M+H) +

[0612] Retention time: 0.35 minutes (analysis conditions SQDFA05)

[0613] As described above, the present invention shows that N-methyl-α,α-dialkyl amino acids can be introduced at high purity after bulky N-alkyl amino acids. In addition, it can be confirmed that the subsequent Tfa removal step also proceeds well, and conventional peptide extension from the N-terminus can be performed.

[0614] Example 2-5. Introduction of various N-methyl-α,α-diamino acids after bulky N-methyl amino acids (MeVal) on a solid phase Experiments with alkyl amino acids

[0615] According to the following general formula, 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.

[0616]

[0617] [Table 8]

[0618]

[0619]

[0620] Example 2-5-1. Tfa-Me(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2- 5-1-2) Synthesis

[0621] Example 2-5-1-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Abu-OH

[0622] In a reaction container with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.552 mmol / g, 100 mg) prepared by the same method as in Example 1-2-2 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 3 times with DMF (0.7 mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7 mL) was added to the resin and shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc removal solution, the resin was washed 4 times with DMF (0.7 mL).

[0623] The obtained resin was subjected to an extension reaction with Tfa-(Me)Abu-OH (Compound 1-3-2-b).

[0624] The extension reaction was carried out by adding a mixed solution of 0.6M Tfa-(Me)Abu-OH (Compound 1-3-2-b) / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 60 degrees for 48 hours.

[0625] After removing the liquid phase of the extension reaction with a filter, the resin was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain compound 2-5-1-1 (Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0626] In order to confirm the progress of the reaction, a portion of the obtained resin (compound 2-5-1-1) was taken out and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the formation of the target peptide Tfa-(Me)Abu-MeVal-Asp-pyrro (compound 2-5-1-1*). Other peptide components were not detected. After extension, it was washed with DCM and dried for subsequent research.

[0627]

[0628] LCMS (ESI) m / z = 495.4 (M+H) +

[0629] Retention time: 0.56 minutes (analysis conditions SQDFA05)

[0630] Example 2-5-1-2. By Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( N-methylation of the nucleophilic substitution reaction of the Tfa amide site of compound 2-5-1-1)

[0631] In a reaction container with a filter, Tfa-(Me)Abu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-5-1-1) (45 mg) prepared in Example 2-5-1-1 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with DMF (0.7 mL).

[0632] To the obtained resin, a TMGN (27 mg) / DMF (0.175 mL) solution was added, and then a methyl iodide (31 μL) / DMF (0.175 mL) solution was added, and the mixture was shaken at 40 degrees for 1 hour. After removing the liquid phase with a filter, the mixture was washed twice with DMF (0.7 mL). A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0633] The resin subjected to the first methylation was subjected to the same operation for the purpose of increasing the reaction conversion rate three more times. The second methylation was carried out at 40 degrees with shaking for 1.5 hours. The third and fourth methylations were carried out at 40 degrees with shaking for 1 hour. After the fourth methylation, the resin was washed with DMF four times and further washed with DCM four times to obtain compound 2-5-1-2. A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-Me(Me)Abu-MeVal-Asp-pyrro (compound 2-5-1-2*) 94.0% (UVarea), the O-methylated product of the Tfa amide site (compound 2-5-1-2a*) 6.0% (UVarea) was also confirmed.

[0634]

[0635] LCMS (ESI) m / z = 509.5 (M+H) +

[0636] Retention time: 0.60 minutes (analysis conditions SQDFA05)

[0637]

[0638] LCMS (ESI) m / z = 509.5 (M+H) +

[0639] Retention time: 0.69 minutes (analysis conditions SQDFA05)

[0640] Example 2-5-2. Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2- 5-2-2) Synthesis

[0641] Example 2-5-2-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Leu-OH

[0642] Compound 2-5-2-1 (Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-5-1-1 using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6M Tfa-(Me)Leu-OH (compound 1-3-3-b) / DMF solution (0.3 mL). A portion of the obtained resin was taken out and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-1*) (84.5% UVarea), the over-extension product of MeVal-Asp-pyrro (compound 2-5-2-1a*) 6.8% (UVarea) was also confirmed as the main impurity (the conversion efficiency after removal of Fmoc was >99%).

[0643]

[0644] LCMS (ESI) m / z = 523.5 (M + H) +

[0645] Retention time: 0.67 minutes (analysis conditions SQDFA05)

[0646]

[0647] LCMS (ESI) m / z = 804.7 (M+H) +

[0648] Retention time: 0.73 minutes (analysis conditions SQDFA05)

[0649] Example 2-5-2-2. By Tfa-(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( N-methylation of the nucleophilic substitution reaction of the Tfa amide site of compound 2-5-2-1)

[0650] Compound 2-5-2-2 (Tfa-Me(Me)Leu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as compound 2-5-2-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-Me(Me)Leu-MeVal-Asp-pyrro (compound 2-5-2-2*) (68.5% UVarea), the O-methylated product (compound 2-5-2-2a*) at the amide site of Tfa was confirmed at 17.0% (UVarea), and the compound 2-5-2-1* (14.5% UVarea) as the starting material was confirmed.

[0651]

[0652] LCMS (ESI) m / z = 537.5 (M + H) +

[0653] Retention time: 0.70 minutes (analysis conditions SQDFA05)

[0654]

[0655] LCMS (ESI) m / z = 537.5 (M + H) +

[0656] Retention time: 0.81 minutes (analysis conditions SQDFA05)

[0657] Example 2-5-3. Tfa-Me(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound Synthesis of Compound 2-5-3-2)

[0658] Example 2-5-3-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Ser(Me)-OH

[0659] Compound 2-5-3-1 (Tfa-(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized by the method shown in Example 2-5-1-1 using compound 1-2-2 (0.552mmol / g, 100mg) and 0.6M Tfa-(Me)Ser(Me)-OH (compound 1-3-4-b) / DMF solution (0.3mL). A portion of the obtained resin was taken out and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved 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*). Other peptide components were not detected.

[0660]

[0661] LCMS (ESI) m / z = 511.4 (M+H) +

[0662] Retention time: 0.55 minutes (analysis conditions SQDFA05)

[0663] Example 2-5-3-2. Tfa-(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro N-methylation of the nucleophilic substitution reaction of the Tfa amide site of (Compound 2-5-3-1)

[0664] Compound 2-5-3-2 (Tfa-Me(Me)Ser(Me)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as compound 2-5-3-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved 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% UVarea).

[0665]

[0666] LCMS (ESI) m / z = 525.5 (M + H) +

[0667] Retention time: 0.61 minutes (analysis conditions SQDFA05)

[0668] Example 2-5-4. Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2- 5-4-2) Synthesis

[0669] Example 2-5-4-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Phe-OH

[0670] Compound 2-5-4-1 (Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1 by setting the reaction time to 72 hours and using compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-(Me)Phe-OH (compound 1-3-5-b) / DMF solution (0.3 mL). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the generation of the target peptide Tfa-(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-1*) (81.4% UVarea). The conversion efficiency after Fmoc removal was >99%. In addition to the over-extension product of MeVal-Asp-pyrro (compound 2-5-4-1a*) 4.1% (UVarea) as the main impurity, a peak of unknown structure was also detected.

[0671]

[0672] LCMS (ESI) m / z = 557.5 (M+H) +

[0673] Retention time: 0.68 minutes (analysis conditions SQDFA05)

[0674]

[0675] LCMS (ESI) m / z = 838.7 (M + H) +

[0676] Retention time: 0.73 minutes (analysis conditions SQDFA05)

[0677] Example 2-5-4-2. By Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( N-methylation of the nucleophilic substitution reaction of the Tfa amide site of compound 2-5-4-1)

[0678] Compound 2-5-4-2 (Tfa-Me(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as compound 2-5-4-1 (45 mg) by the method described in Example 2-5-1-2. In this case, the second N-methylation was also carried out for 1 hour. A portion of the obtained resin was taken out and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to confirming the production of the target peptide Tfa-Me(Me)Phe-MeVal-Asp-pyrro (compound 2-5-4-2*) (79.7% UVarea), the over-extension product of MeVal-Asp-pyrro (compound 2-5-4-2a*) and the O-methylated product of the amide site of Tfa (compound 2-5-4-2b*) (a total of 11.8% UVarea) were also detected as impurities (the conversion efficiency of the starting material was 100%).

[0679]

[0680] LCMS (ESI) m / z = 571.5 (M+H) +

[0681] Retention time: 0.74 minutes (analysis conditions SQDFA05)

[0682]

[0683] LCMS (ESI) m / z = 852.7 (M + H) +

[0684] Retention time: 0.79 minutes (analysis conditions SQDFA05)

[0685]

[0686] LCMS (ESI) m / z = 571.5 (M+H) +

[0687] Retention time: 0.79 minutes (analysis conditions SQDFA05)

[0688] Example 2-5-5. Tfa-Me(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2- 5-5-2) Synthesis

[0689] Example 2-5-5-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Cha-OH

[0690] Compound 2-5-5-1 (Tfa-(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as in Example 2-5-1-1 by setting the reaction time to 72 hours and 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). A portion of the obtained resin was taken out and the peptide was cleaved using a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved 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% UVarea). As a major impurity, multiple peaks of unknown structure including an over-extended product of MeVal-Asp-pyrro (compound 2-5-5-1a*) were detected (the conversion efficiency after Fmoc removal was 100%).

[0691]

[0692] LCMS (ESI) m / z = 563.6 (M + H) +

[0693] Retention time: 0.76 minutes (analysis conditions SQDFA05)

[0694]

[0695] LCMS (ESI) m / z = 844.8 (M+H) +

[0696] Retention time: 0.80 minutes (analysis conditions SQDFA05)

[0697] Example 2-5-5-2. By Tfa-(Me)Phe-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( N-methylation of the nucleophilic substitution reaction of the Tfa amide site of compound 2-5-5-1)

[0698] Compound 2-5-5-2 (Tfa-Me(Me)Cha-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as compound 2-5-5-1 (45 mg) by the method shown in Example 2-5-1-2. The second N-methylation was also performed for 1 hour. A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-Me(Me)Cha-MeVal-Asp-pyrro (compound 2-5-5-2*) (74.4% UVarea), O-methylated product (compound 2-5-5-2a*) at the Tfa amide site was confirmed at 12.1% (UVarea), and compound 2-5-5-1* (13.5% UVarea) as the starting material was confirmed.

[0699]

[0700] LCMS (ESI) m / z = 577.5 (M + H) +

[0701] Retention time: 0.80 minutes (analysis conditions SQDFA05)

[0702]

[0703] LCMS (ESI) m / z = 577.5 (M + H) +

[0704] Retention time: 0.92 minutes (analysis conditions SQDFA05)

[0705] Example 2-5-6. Tfa-Me(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2- 5-6-2) Synthesis

[0706] Example 2-5-6-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-(Me)Val-OH

[0707] By the same method as Example 2-5-1-1, the reaction time was set to 72 hours, and compound 1-2-2 (0.552 mmol / g, 100 mg) and 0.6 M Tfa-(Me)Val-OH (compound 1-3-7-b) / DMF solution (0.3 mL) were used to synthesize compound 2-5-6-1 (Tfa-(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the obtained resin was taken out, and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the generation of the target peptide Tfa-(Me)Val-MeVal-Asp-pyrro (compound 2-5-6-1*) (66.6% UVarea). Multiple peaks of unknown structure including an over-extended product of MeVal-Asp-pyrro (compound 2-5-6-1a*) were detected as impurities (the conversion efficiency after Fmoc removal was 98%).

[0708]

[0709] LCMS (ESI) m / z = 509.5 (M+H) +

[0710] Retention time: 0.59 minutes (analysis conditions SQDFA05)

[0711]

[0712] LCMS (ESI) m / z = 790.7 (M+H) +

[0713] Retention time: 0.67 minutes (analysis conditions SQDFA05)

[0714] Example 2-5-6-2. By Tfa-(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro( N-methylation of the nucleophilic substitution reaction of the Tfa amide site of compound 2-5-6-1)

[0715] Based on the method shown in Example 2-5-1-2, the reaction temperature was set to 60 degrees, and compound 2-5-6-1 (40 mg) was used to synthesize compound 2-5-6-2 (Tfa-Me(Me)Val-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro). In this case, the second N-methylation was also carried out for 1 hour. A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-Me(Me)Val-MeVal-Asp-pyrro (compound 2-5-6-2*) (21.2% UVarea), O-methylated product (compound 2-5-6-2a*) at the Tfa amide site was confirmed at 17.1% (UVarea), and compound 2-5-6-1* (39.6% UVarea) as the starting material was also confirmed. In addition, multiple peaks with unknown structures were detected.

[0716]

[0717] LCMS (ESI) m / z = 523.5 (M + H) +

[0718] Retention time: 0.65 minutes (analysis conditions SQDFA05)

[0719]

[0720] LCMS (ESI) m / z = 523.5 (M + H) +

[0721] Retention time: 0.77 minutes (analysis conditions SQDFA05)

[0722] Example 2-5-7. Tfa-MecLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(Compound 2-5-7- 2) Synthesis

[0723] Example 2-5-7-1. Desorption of Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) After Fmoc, extension of Tfa-cLeu-OH

[0724] Compound 2-5-7-1 (Tfa-cLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-5-1-1 using compound 1-2-2 (0.552mmol / g, 100mg) and 0.6MTfa-cLeu-OH (compound 1-3-8-b) / DMF solution (0.3mL). A portion of the obtained resin was taken out and the peptide was cleaved with TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the formation of the target peptide Tfa-cLeu-MeVal-Asp-pyrro (compound 2-5-7-1*). Other peptide components were not detected.

[0725]

[0726] LCMS (ESI) m / z = 507.4 (M+H) +

[0727] Retention time: 0.56 minutes (analysis conditions SQDFA05)

[0728] Example 2-5-7-2. By Tfa-cLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2-5-7-1) N-methylation of the nucleophilic substitution reaction of the Tfa amide site

[0729] Compound 2-5-7-2 (Tfa-MecLeu-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as compound 2-5-7-1 (45 mg) by the method shown in Example 2-5-1-2. A portion of the obtained resin was taken out and the peptide was cleaved using a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to confirming the formation of the target peptide Tfa-MecLeu-MeVal-Asp-pyrro (compound 2-5-7-2*) (92.1% UVarea), 7.9% (UVarea) of the O-methylated product (compound 2-5-7-2a*) at the amide site of Tfa was detected as an impurity (the conversion efficiency of the starting material was 100%).

[0730]

[0731] LCMS (ESI) m / z = 521.4 (M + H) +

[0732] Retention time: 0.61 minutes (analysis conditions SQDFA05)

[0733]

[0734] LCMS (ESI) m / z = 521.4 (M + H) +

[0735] Retention time: 0.70 minutes (analysis conditions SQDFA05)

[0736] As described above, the results of Examples 2 to 5 show that the method of the present invention can introduce various N-methyl-α,α-dialkyl amino acids other than MeAib into a practical level after the bulky N-methyl amino acid in solid phase synthesis.

[0737] Example 2-6. Introduction of various N-substituted-α,α- Experiments with dialkyl amino acids

[0738] According to the following general formula, various Tfa-amino acids were used to synthesize compounds 2-6-1 to 2-6-4.

[0739]

[0740] [Table 9]

[0741]

[0742] Example 2-6-1. Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) N-ethylation of the amide site

[0743] In a reaction container with a filter, Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1) (0.552 mmol / g, 50 mg) prepared in the same manner as in Example 2-1-1 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with DMF (0.7 mL).

[0744] To the obtained resin, a TMGN (29 mg) / DMF (0.175 mL) solution was added, followed by an ethyl iodide (44 μL) / DMF (0.175 mL) solution, and the mixture was shaken at 60 degrees for 1 hour. The liquid phase was removed with a filter, and then washed twice with DMF (0.7 mL). A small amount of the obtained resin was sampled, and the peptide was cleaved with a TFE / DCM / DIPEA solution (1: 1: 0.015), and the cleaved solution was analyzed by LCMS.

[0745] The resin subjected to the first ethylation was subjected to the same operation for the purpose of increasing the reaction conversion rate. After the fifth ethylation, the resin was washed with DMF 4 times and further washed with DCM 4 times to obtain compound 2-6-1 (Tfa-EtAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-EtAib-MeVal-Asp-pyrro (compound 2-6-1*) (26.1% UVarea), O-ethylated product (compound 2-6-1a*) at the amide site of Tfa (15.4% UVarea) and starting material compound 2-1* (54.8% UVarea) were also confirmed.

[0746]

[0747] LCMS (ESI) m / z = 509.4 (M+H) +

[0748] Retention time: 0.60 minutes (analysis conditions SQDFA05)

[0749]

[0750] LCMS (ESI) m / z = 509.4 (M+H) +

[0751] Retention time: 0.67 minutes (analysis conditions SQDFA05)

[0752] Example 2-6-2. Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) N-propylation of the amide site

[0753] Compound 2-6-2 (Tfa-nPrAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-6-1 using compound 2-1 (0.552mmol / g, 50mg) and n-propyl iodide (54μL×5). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-nPrAib-MeVal-Asp-pyrro (compound 2-6-2*) (10.0% UVarea), O-n-propylated product (compound 2-6-2a*) at the amide site of Tfa was confirmed at 6.3% (UVarea), starting material compound 2-1* (82.7% UVarea), etc.

[0754]

[0755] LCMS (ESI) m / z = 523.5 (M + H) +

[0756] Retention time: 0.66 minutes (analysis conditions SQDFA05)

[0757]

[0758] LCMS (ESI) m / z = 523.5 (M + H) +

[0759] Retention time: 0.73 minutes (analysis conditions SQDFA05)

[0760] Example 2-6-3. Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) N-allylation of the amide site

[0761] Compound 2-6-3 (Tfa-AllylAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-6-1 using compound 2-1 (0.552 mmol / g, 50 mg) and allyl bromide (48 μL×5). A portion of the obtained resin was taken out and the peptide was cleaved using a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-AllylAib-MeVal-Asp-pyrro (compound 2-6-3*) (20.3% UVarea), O-allylated product (compound 2-6-3a*) at the Tfa amide site was confirmed at 6.9% (UVarea), and starting material compound 2-1* (72.8% UVarea).

[0762]

[0763] LCMS (ESI) m / z = 521.4 (M + H) +

[0764] Retention time: 0.64 minutes (analysis conditions SQDFA05)

[0765]

[0766] LCMS (ESI) m / z = 521.5 (M+H) +

[0767] Retention time: 0.71 minutes (analysis conditions SQDFA05)

[0768] Example 2-6-4. Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) N-benzylation of the amide site

[0769] Compound 2-6-4 (Tfa-BnAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-6-1 using compound 2-1 (0.552 mmol / g, 50 mg) and benzyl bromide (66 μL×5). A portion of the obtained resin was taken out and the peptide was cleaved using a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-BnAib-MeVal-Asp-pyrro (compound 2-6-4*) (6.2% UVarea), O-benzylation of the Tfa amide site (compound 2-6-4a*) 7.7% (UVarea) and starting material compound 2-1* (79.5% UVarea) were also confirmed.

[0770]

[0771] LCMS (ESI) m / z = 571.5 (M+H) +

[0772] Retention time: 0.72 minutes (analysis conditions SQDFA05)

[0773]

[0774] LCMS (ESI) m / z = 571.5 (M+H) +

[0775] Retention time: 0.79 minutes (analysis conditions SQDFA05)

[0776] As described above, the results of Examples 2-6 show that the method of the present invention can be used to introduce N-substituted-α,α-dialkyl amino acids, not limited to N-methyl-α,α-dialkyl amino acids, after the bulky N-methyl amino acids in solid phase synthesis, at a practical level.

[0777] Example 2-7. On a solid phase, after bulky N-alkyl amino acids (EtVal / nPrVal), N-methyl-α- Experiments with α-dialkylamino acids (MecLeu)

[0778] According to the following general formula, 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.

[0779]

[0780] [Table 10]

[0781]

[0782]

[0783] Example 2-7-1. Preparation of Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-7-1)

[0784] In a reaction container with a filter, 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 placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 3 times with DMF (0.7 mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7 mL) was added to the resin and shaken at room temperature for 5 minutes to remove Fmoc. After removing the Fmoc removal solution, the resin was washed 4 times with DMF (0.7 mL).

[0785] The obtained resin was subjected to an extension reaction of Fmoc-Val-OH.

[0786] The extension reaction was carried out by adding a mixed solution of Fmoc-Val-OH (0.6 mol / L), 1-hydroxy-7-azabenzotriazole (HOAt, 0.375 mol / L) in NMP (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 3 hours.

[0787] After removing the liquid phase of the extension reaction with a filter, the resin was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain compound 2-7-1 (Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0788] In order to confirm the progress of the reaction, a portion (~5 mg) of the obtained resin (compound 2-7-1) was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the generation of the target peptide Fmoc-Val-Asp-pyrro (compound 2-7-1*) (97.3% UVarea). In addition, 2.7% (UVarea) of Val over-extension (compound 2-7-1a*) was also detected at the same time. After extension, it was washed with DCM and dried for subsequent research.

[0789]

[0790] LCMS (ESI) m / z = 508.4 (M+H) +

[0791] Retention time: 0.72 minutes (analysis conditions SQDFA05)

[0792]

[0793] LCMS (ESI) m / z = 607.5 (M+H) +

[0794] Retention time: 0.74 minutes (analysis conditions SQDFA05)

[0795] Example 2-7-2. DeFmoc of Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-1) and N-terminal Ns

[0796] In a reaction vessel with a filter, Fmoc-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-1) (0.552 mmol / g, 100 mg per column) prepared in Example 2-7-1 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed twice with DMF (0.7 mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7 mL) was added to the resin and shaken at room temperature for 10 minutes to remove Fmoc. After removing the de-Fmoc solution, the resin was washed sequentially with DMF (0.7 mL), 1-hydroxy-7-azabenzotriazole (HOAt, 0.157 mol / L) and DIPEA (0.157 mol / L) in DMF (0.7 mL), DMF (0.7 mL), and then washed three times with THF (0.7 mL).

[0797] To the obtained resin were added a THF solution (0.35 mL) of 2,4,6-collidine (0.074 mL, 0.552 mmol) and a THF solution (0.35 mL) of 2-nitrobenzenesulfonyl chloride (0.049 g, 0.221 mmol), and the mixture was shaken at 40 degrees for 3 hours.

[0798] 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).

[0799] In order to confirm the progress of extension, a portion of the obtained resin (~5 mg) was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). When the cleaved solution was analyzed by LCMS, it was confirmed that the target peptide Ns-Val-Asp-pyrro (compound 2-7-2*) was generated at 94.3% (UVarea). After Ns conversion, it was washed with DCM and dried for subsequent research.

[0800]

[0801] LCMS (ESI) m / z = 471.3 (M + H) +

[0802] Retention time: 0.55 minutes (analysis conditions SQDFA05)

[0803] Example 2-7-3-1. By Ns-Val-Asp (O-Trt (2-Cl) -resin) -pyrro (compound 2-7-2) N-Ethylation of the Ns-Amide Site by Mitsunobu Reaction

[0804] In a reaction container with a filter, Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-2) (0.552mmol / g, 100mg) prepared in Example 2-7-2 was placed, and dichloromethane (1mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed twice with THF (1mL).

[0805] 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, and the mixture was gently shaken and allowed to stand at room temperature for 15 minutes. Then, ethanol (32 μL, 0.552 mmol) was added, mixed, and allowed to stand for 5 minutes. The resulting solution was added to the swollen resin and shaken at 35 degrees for 1 hour. After removing the liquid phase with a filter, the resin was washed 4 times with THF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain compound 2-7-3-1 (Ns-EtVal-Asp (O-Trt (2-Cl) -resin) -pyrro).

[0806] A portion of the obtained resin (~5 mg) was cleaved from the peptide using a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS to confirm the formation of the target peptide Ns-EtVal-Asp-pyrro (compound 2-7-3-1*) (the conversion rate from compound 2-7-2 was 100%). The obtained resin was dried and used for subsequent studies.

[0807]

[0808] LCMS (ESI) m / z = 499.4 (M+H) +

[0809] Retention time: 0.64 minutes (analysis conditions SQDFA05)

[0810] Example 2-7-3-2. Desorption of Ns-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-1) Ns

[0811] In a reaction container with a filter, Ns-Val-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-1) (0.552mmol / g, 100mg) prepared in Example 2-7-3-1 was placed, and dichloromethane (1mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed twice with NMP (0.7mL).

[0812] 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 to the obtained resin, and the mixture was shaken at 60 degrees for 4 hours. The liquid phase was removed with a filter, and then washed twice with NMP (0.7 mL). A portion of the obtained resin was taken out, and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015), and the cleaved solution was analyzed by LCMS.

[0813] The resin subjected to the first de-Ns treatment was subjected to the same operation again for the purpose of improving the reaction conversion rate. The second de-Ns treatment was performed by shaking at 60 degrees for 12 hours. After the second de-Ns treatment, the resin was washed 4 times with NMP and further 4 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 taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide H-EtVal-Asp-pyrro (compound 2-7-3-2*) 84.6% (UVarea), an impurity (compound 2-7-3-2a*) (12.6% UVarea) in which the nitro 1-dodecanethiol protected by Ns was presumably substituted at the present position was detected.

[0814]

[0815] LCMS (ESI) m / z = 314.3 (M+H) +

[0816] Retention time: 0.26 minutes (analysis conditions SQDFA05)

[0817]

[0818] LCMS (ESI) m / z = 654.5 (M+H) +

[0819] Retention time: 1.25 minutes (analysis conditions SQDFA05)

[0820] Example 2-7-3-3. H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-2) The elongation reaction of Tfa-cLeu-OH is a resin-based 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one Extension of Tfa-cLeu of (Compound 1-3-9)

[0821] In a reaction vessel with a filter, H-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-2) (0.552 mmol / g, 100 mg) prepared in Example 2-7-3-2 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 45 minutes to swell the resin. After removing 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 pure 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (compound 1-3-9) (0.582 g, 2.81 mmol) to the resin and shaking at 60 degrees for 48 hours. In order to confirm the progress of the reaction, a small amount of the obtained resin was sampled after 24 hours, and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved 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 4 times with DMF (1 mL) and 4 times with dichloromethane (1 mL) to obtain compound 2-7-3-3 (Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro).

[0822] In order to confirm the progress of the reaction, a portion of the obtained resin (compound 2-7-3-3) was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to confirming the formation of the target peptide Tfa-cLeu-EtVal-Asp-pyrro (compound 2-7-3-3*) (55.5% UVarea), an over-extension product of EtVal-Asp-pyrro (compound 2-7-3-3a*) (18.0% UVarea) and an impurity in which the Ns-protected nitro 1-dodecanethiol was presumably substituted at the same position (compound 2-7-3-2a*) (11.8% UVarea) were also detected. In addition, a peak such as Tfa-cLeu-OH loaded on the resin was also detected. After extension, the solution was washed with DCM and dried, and used for subsequent studies.

[0823]

[0824] LCMS (ESI) m / z = 521.5 (M+H) +

[0825] Retention time: 0.60 minutes (analysis conditions SQDFA05)

[0826]

[0827] LCMS (ESI) m / z = 816.7 (M+H) +

[0828] Retention time: 0.69 minutes (analysis conditions SQDFA05)

[0829] Example 2-7-3-4. By Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro(compound 2-7-3-3) N-methylation of the nucleophilic substitution reaction of the Tfa amide site

[0830] In a reaction container with a filter, Tfa-cLeu-EtVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-3-3) (66 mg) prepared in Example 2-7-3-3 was placed, and dichloromethane (1 mL) was added and shaken at room temperature for 1 hour to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with DMF (0.7 mL).

[0831] 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 degrees for 1 hour. The liquid phase was removed with a filter, and then washed twice with DMF (0.7 mL). A portion of the obtained resin was taken out, and the peptide was cleaved with a TFE / DCM / DIPEA solution (1: 1: 0.015), and the cleaved solution was analyzed by LCMS.

[0832] The resin subjected to the first methylation was subjected to the same operation twice for the purpose of increasing the reaction conversion rate. After the third methylation, the resin was washed 4 times with DMF and 4 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 taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-MecLeu-EtVal-Asp-pyrro (compound 2-7-3-4*) (53.2% UVarea), a compound Me-ylated after excessive extension of EtVal-Asp-pyrro (compound 2-7-3-4a*) (29.8% UVarea), and an impurity in which the Ns-protected nitro 1-dodecanethiol was presumably substituted at the same position (compound 2-7-3-2a*) (11.9% UVarea) were also confirmed. In addition, a Me-ylated peak such as Tfa-cLeu-OH loaded on the resin was also detected.

[0833]

[0834] LCMS (ESI) m / z = 535.4 (M+H) +

[0835] Retention time: 0.66 minutes (analysis conditions SQDFA05)

[0836]

[0837] LCMS (ESI) m / z = 830.7 (M+H) +

[0838] Retention time: 0.75 minutes (analysis conditions SQDFA05)

[0839] Example 2-7-4-1. By Ns-Val-Asp (O-Trt (2-Cl) -resin) -pyrro (compound 2-7-2) N-propylation of the Ns-amide site by Mitsunobu reaction

[0840] Compound 2-7-4-1 (Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner as described in Example 2-7-3-1 using compound 2-7-2 (0.552 mmol / g, 100 mg) and 1-propanol (41 μL, 0.552 mmol). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS to confirm the generation of 94.7% (UVarea) of the target peptide Ns-nPrVal-Asp-pyrro (compound 2-7-4-1*) (the conversion rate from compound 2-7-2 was 100%).

[0841]

[0842] LCMS (ESI) m / z = 513.4 (M+H) +

[0843] Retention time: 0.69 minutes (analysis conditions SQDFA05)

[0844] Example 2-7-4-2. Ns-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-4-1) De-Ns

[0845] 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 taken out and the peptide was cleaved using a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide H-nPrVal-Asp-pyrro (compound 2-7-4-2*) 81.1% (UVarea), an impurity (compound 2-7-4-2a*) (15.1% UVarea) in which the Ns-protected nitro 1-dodecanethiol was presumably substituted at the present position was also detected.

[0846]

[0847] LCMS (ESI) m / z = 328.3 (M+H) +

[0848] Retention time: 0.28 minutes (analysis conditions SQDFA05)

[0849]

[0850] LCMS (ESI) m / z = 668.6 (M+H) +

[0851] Retention time: 1.28 minutes (analysis conditions SQDFA05)

[0852] Example 2-7-4-3. H-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-7-4-2) Extension of Tfa-cLeu with 2-(trifluoromethyl)-3-oxa-1-azaspiro[4.4]non-1-en-4-one (Compound 1-3-9)

[0853] Compound 2-7-4-3 (Tfa-cLeu-nPrVal-Asp(O-Trt(2-Cl)-resin)-pyrro) was synthesized in the same manner using compound 2-7-4-2 (0.552 mmol / g, 100 mg). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to confirming the formation of the target peptide Tfa-cLeu-nPrVal-Asp-pyrro (compound 2-7-4-3*) (53.1% UVarea), an over-extended product of nPrVal-Asp-pyrro (compound 2-7-4-3a*) (31.0% UVarea), an impurity in which the Ns-protected nitro 1-dodecanethiol was presumably substituted at the original position (compound 2-7-4-2a*) (13.3% UVarea), etc. were also detected. In addition, a peak such as Tfa-cLeu-OH loaded on the resin was also detected.

[0854]

[0855] LCMS (ESI) m / z = 535.5 (M+H) +

[0856] Retention time: 0.65 minutes (analysis conditions SQDFA05)

[0857]

[0858] LCMS (ESI) m / z = 844.8 (M+H) +

[0859] Retention time: 0.77 minutes (analysis conditions SQDFA05)

[0860] Example 2-7-4-4. By Tfa-cLeu-nPrVal-Asp(OTrt(2-Cl)-resin)-pyrro(compound 2-7-4-3) N-methylation of the nucleophilic substitution reaction of the Tfa amide site

[0861] Compound 2-7-4-4 (Tfa-MecLeu-nPrVal-Asp(O-Trt(2-Gl)-resin)-pyrro) was synthesized in the same manner using compound 2-7-4-3 (0.552 mmol / g, 60 mg). A portion of the obtained resin was taken out and the peptide was cleaved with a TFE / DCM / DIPEA solution (1:1:0.015). The cleaved solution was analyzed by LCMS. In addition to the target peptide Tfa-MecLeu-nPrVal-Asp-pyrro (compound 2-7-4-4*) (46.1% UVarea), a compound that was Me-ylated after over-extension of nPrVal-Asp-pyrro (compound 2-7-4-4a*) (38.3% UVarea), and an impurity that was presumed to be substituted at the same position for the Ns-protected nitro 1-dodecanethiol (compound 2-7-4-2a*) (12.6% UVarea) were also confirmed. In addition, a Me-ylated peak such as Tfa-cLeu-OH loaded on the resin was also detected.

[0862]

[0863] LCMS (ESI) m / z = 549.5 (M + H) +

[0864] Retention time: 0.71 minutes (analysis conditions SQDFA05)

[0865]

[0866] LCMS (ESI) m / z = 858.7 (M + H) +

[0867] Retention time: 0.83 minutes (analysis conditions SQDFA05)

[0868] As described above, the results of Examples 2-7 show that the method of the present invention can introduce N-methyl-α,α-dialkyl amino acids at a practical level after the bulky N-alkyl amino acids in solid phase synthesis.

[0869] Example 3: Example of using the method of the present invention to introduce MeAib and perform peptide synthesis

[0870] According to the peptide synthesis method using the Fmoc method described in WO2013 / 100132 or WO2018 / 225864, peptide extension is performed through the following basic pathway. That is,

[0871] 1) Loading the carboxylic acid of the Asp side chain or the peptide backbone carboxylic acid onto 2-chlorotrityl resin, and performing peptide extension reaction from the N-terminus of the amino acid by the Fmoc method,

[0872] 2) Cleavage of peptides from 2-chlorotrityl resin,

[0873] 3) amide cyclization by condensation of the carboxylic acid of the Asp side chain or the peptide backbone carboxylic acid generated by the cleavage process released from the 2-chlorotrityl resin with the amino group at the N-terminus (triangular unit) of the peptide chain,

[0874] 4) deprotection of the protective groups of the side chain functional groups contained in the peptide chain as required,

[0875] 5) 5-step procedure for purification of compounds by preparative HPLC In the present examples, unless otherwise stated, the synthesis of peptide compounds was carried out based on this basic route.

[0876]

[0877] 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, 22,33,36-Undecyltetrahydro-2H-azetidin-2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16, 19,22,25,28,31]undecane-4,7,10,13,17,20,23,28,31,34,37(14H)-undecane Synthesis of ketone (compound 3-1)

[0878]

[0879] The synthesis of compound 3-1 was carried out from compound 1-2-4 according to the following scheme.

[0880]

[0881] Using (3R)-3-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]butyric 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) as a raw material, Fmoc-MeVal-OH was extended by the peptide extension method described in Example 1-2-2 in a reaction container with a filter, and then Tfa-Aib-OH (2-methyl-2-(2,2,2-trifluoroacetamide)propionic acid) (compound 1-3-1) was extended by the same operation as in Example 2-1-1 to obtain compound 3-1-a.

[0882] The obtained compound 3-1-a was swelled with DCM (1 mL) and washed 4 times with DMF (1 mL). A DMF solution (180 μL) of phosphazene base P1-tBu (38 μL, 0.150 mmol) and a DMF solution (180 μL) of methyl iodide (62 μL, 1 mmol) were added, and the mixture was shaken at 40°C for 30 minutes in a sealed state. After removing the reaction solution, the resin was washed 4 times with DMF (1 mL) and further washed 4 times with DCM (1 mL) to obtain compound 3-1-b. A portion of the obtained resin was cut with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS to confirm the formation of compound 3-1-b*.

[0883]

[0884] LCMS (ESI) m / z = 424 (MH) -

[0885] Retention time: 0.57 minutes (analysis conditions SQDFA05)

[0886] Sodium borohydride (NaBH4) (758 mg, 20 mmol) was placed in a flask, evacuated and placed under a nitrogen atmosphere, and dissolved in triethylene glycol dimethyl ether (10 mL) to obtain solution A. Compound 3-1-b obtained above was swelled with DCM (1 mL) and then washed 4 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 for 40 minutes in an open system. After removing the reaction solution, methanol (0.7 mL) was added, and the liquid was discarded after 1 minute. The washing operation was repeated 4 times, and then washed 4 times with DCM (0.7 mL) to obtain compound 3-1-c. A portion of the obtained resin was cut with TFE / DCM (1 / 1 (v / v)) and analyzed by LCMS to confirm the generation of compound 3-1-c*.

[0887]

[0888] LCMS (ESI) m / z = 330 (M + H) +

[0889] Retention time: 0.33 minutes (analysis conditions SQDFA05)

[0890] After preparing compound 3-1-c, the peptide extension, cyclization and purification steps were performed according to the following synthetic method.

[0891] 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) and HOAt or oxyma or HOOBt (0.375 mol / L) in NMP (solution 1), and diisopropylcarbodiimide (DIC) in N,N-dimethylformamide (DMF) (10% v / v, solution 2) were placed in a peptide synthesizer.

[0892] Solution 1 and solution 2 were mixed in a mixing bottle of a synthesizer and added to the resin to carry out a condensation reaction between the amino groups on the resin and the Fmoc amino acid.

[0893] The synthesis was performed using a DMF solution (2% v / v) of diazabicycloundecene (DBU) as an Fmoc deprotection solution. After washing the resin with DMF, the Fmoc deprotection followed by the condensation reaction of the Fmoc amino acid was repeated as one cycle to extend the peptide on the resin surface. After the peptide extension was completed, the Fmoc group at the N-terminus of the resin was removed on a peptide synthesizer, and the resin was washed with DMF.

[0894] After adding DCM to the chain peptide loaded on the obtained solid phase to swell the resin again, 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. Then, the resin was removed by filtering the solution in the tube with a synthesis column, and the remaining resin was further washed twice with 2,2,2-trifluoroethanol (TFE) / DCM (1 / 1 (v / v), 1 mL). All the cleavage solutions obtained were mixed and concentrated under reduced pressure.

[0895] The residue concentrated under reduced pressure after cleavage was dissolved in DMF / DCM (1 / 1 (v / v), 8 mL). 0.5 M O-(7-aza-1H-benzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) / DMF solution (1.5 equivalents of capacity for the molar number on the applied resin (loaded amount (mmol / g) multiplied by the amount of resin used (usually 0.10 g))) and DIPEA (1.8 equivalents for the molar number on the applied resin) were added and shaken at room temperature for 2 hours. Thereafter, the solvent was removed under reduced pressure. The formation of the target cyclic peptide was confirmed by LCMS measurement.

[0896] Thereafter, the solvent was removed under reduced pressure, DMF or DMSO was added, the insoluble matter was removed by filtration, and then purified by preparative HPLC to obtain compound 3-1 ((5S, 8S, 11S, 15R, 18S, 23aS, 29S, 35S, 37aS)-8,11-di((S)-sec-butyl)-29-(3-chloro-4-(trifluoromethyl)phenethyl)-35-(cyclohexylmethyl)-18-isopropyl-5,6- 12,15,16,19,21,21,22,33,36-Undecyltetrahydro-2H-azetidinol[2,1-u]pyrrolo[2,1-i][1,4,7,10,13,16,19,22,25,28,31]undecaneazacyclotetratriacontane-4,7,10,13,17,20,23,28,31,34,37(14H)-undecanone) (4.1 mg, 9%).

[0897] The analysis results of LCMS are shown in Table 12.

[0898] Example 3-2: Example of peptide synthesis in the same manner as in Example 3-1

[0899] Compounds 3-2 to 3-9 were synthesized in the same manner as described in Example 3-1. In addition, the relationship between the formal name, structure, and abbreviation of each amino acid residue constituting the cyclic peptide described in Compounds 3-1 to 3-9 (structural formulas are described in Table 13) can be understood from Tables 3 to 5 above and Table 11 below.

[0900] The analysis results of LCMS are shown in Table 12.

[0901] [Table 11]

[0902]

[0903]

[0904]

[0905]

[0906]

[0907]

[0908]

[0909]

[0910]

[0911]

[0912]

[0913]

[0914]

[0915]

[0916]

[0917] [Table 12]

[0918] Compound No. LCMS conditions Retention time (minutes) LCMS (ESI) m / z MS polarity Compound 3-1 SSC-A-AF-01 7.513 1318.6 (M+H)+ Compound 3-2 SSC-A-FA-01 5.337 1302.6 (M+H)+ Compound 3-3 SSC-A-AF-01 7.408 1288.7 (MH)- Compound 3-4 SSC-A-FA-01 4.595 1262.5 (M+H)+ Compound 3-5 SSC-A-AF-01 7.603 1314.9 (MH)- Compound 3-6 SSC-A-FA-01 5.383 1300.7 (MH)- Compound 3-7 SSC-A-AF-01 7.243 1286.9 (MH)- Compound 3-8 SSC-A-FA-01 5.564 1316.6 (M+H)+ Compound 3-9 SSC-A-FA-01 5.204 1290.6 (M+H)+

[0919] [Table 13]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925] Comparative Example 1: Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 2-1) N-methylation of the amide site of Tfa by Mitsunobu reaction

[0926] As a comparative example with the present invention, a known method of Mitsunobu reaction (Org. Lett. 2013, 15, 5012-5015) was tried as a method for selective N-methylation of the trifluoroacetamide site.

[0927] In a reaction vessel with a filter, dichloromethane (1 mL) was added to Tfa-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 2-1-2) (0.473 mmol / g, 100 mg) prepared in Example 2-1-2, and the mixture was shaken at room temperature for 15 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with THF (0.7 mL).

[0928] To the obtained resin, triphenylphosphine (66.0 mg) / THF (0.7 mL) solution, methanol (20 μL), and DIAD (49 μL) were added, and the mixture was shaken at 40 degrees for 30 minutes. After the liquid phase was removed by a filter, triphenylphosphine (66.0 mg) / THF (0.7 mL) solution, methanol (20 μL), and DIAD (49 μL) were added again, and the mixture was shaken at 40 degrees for 1 hour. After the liquid phase was removed by a filter, the resin was washed 4 times with THF (0.7 mL) and 4 times with dichloromethane (0.7 mL).

[0929] The obtained resin was cleaved with a TFE / DCM solution (1 / 1 (v / v)), and when the cleaved solution was analyzed by LCMS, in addition to the formation of the target peptide Tfa-MeAib-MeVal-Asp-pyrro (Compound 2-2*), the O-methylated product of the Tfa amide site (Compound C1-1) and H-Aib-MeVal-Asp-pyrro (Compound C1-2) hydrolyzed therefrom were also detected. The LC graph is shown in FIG. Figure 2 .

[0930] Target peptide Tfa-MeAib-MeVal-Asp-pyrro (Compound 2-2*)

[0931]

[0932] LCMS (ESI) m / z = 495.26 (M+H) +

[0933] Retention time: 0.58 minutes (analysis conditions SQDFA05)

[0934] The product of O-methylation at the amide site of Tfa (Compound C1-1)

[0935]

[0936] LCMS (ESI) m / z = 495.26 (M+H) +

[0937] Retention time: 0.64 minutes (analysis conditions SQDFA05)

[0938] H-Aib-MeVal-Asp-pyrro (Compound C1-2) hydrolyzed from Compound C1-1

[0939]

[0940] LCMS (ESI) m / z = 385.26 (M+H) +

[0941] Retention time: 0.35 minutes (analysis conditions SQDFA05)

[0942] According to this result, unlike the literature (Org. Lett. 2013, 15, 5012-5015), when the N-terminus is an α,α-dialkyl amino acid, in addition to N-methylation, O-methylation is also greatly performed, which is confirmed to cause a decrease in yield and purity. This result is in contrast to the results of N-selective methylation shown in Examples 2-2 and 2-3.

[0943] Comparative Example 2: After the N-methylamino acid Fmoc-Aib-OH extension in the conventional solid phase synthesis method, Experiments to change Fmoc protection to Ns protection, N-methylation and Ns removal on the N-terminal resin, and to try the introduction of MeAib

[0944] As a comparative example of the present invention, the introduction of MeAib was attempted by the same method as described in the literature (Nature Protocols 2012, 7, 3, 432-444), after the N-methyl amino acid was extended with Fmoc-Aib-OH in the solid phase synthesis method, and then the protection was changed from Fmoc to Ns, and the N-methylation and Ns removal on the N-terminal resin were carried out.

[0945]

[0946] Comparative Example 2-1: Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound 1-2-2), Fmoc- Extension reaction of Aib-OH in the solid phase

[0947] 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 resin was swollen by shaking at room temperature for 30 minutes. After removing dichloromethane with a filter, the resin was washed twice with DMF (1 mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7 mL) was added to the resin, and Fmoc removal was carried out by shaking at room temperature for 10 minutes. After removing the Fmoc removal solution, the resin was washed 4 times with DMF (0.7 mL).

[0948] The obtained resin was subjected to an extension reaction of Fmoc-Aib-OH.

[0949] The extension reaction was carried out by adding a mixed solution of 0.6 M Fmoc-Aib-OH / 0.375 M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 50 degrees for 15 hours.

[0950] This extension reaction was further repeated 2 times. (Second extension condition: 50°C for 24 hours, third extension condition: 50°C for 20 hours)

[0951] 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).

[0952] In order to confirm the progress of the extension, a portion (~5 mg) of the obtained resin was taken out, and the unreacted sites were capped with Fmoc-Gly-OH.

[0953] Covering was performed by adding a mixed solution of 0.6 M Fmoc-Gly-OH / 0.375 M HOAt / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 45 minutes.

[0954] 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).

[0955] The peptide was cleaved using a TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, it was confirmed that the target peptide Fmoc-Aib-MeVal-Asp-pyrro (compound C2-1*) was produced in 60.4%.

[0956]

[0957] LCMS (ESI) m / z = 605.52 (MH) -

[0958] Retention time: 2.16 minutes (analysis conditions SQDFA05long)

[0959] In addition, the unreacted sites of the obtained resin were capped with Z-Gly-OH (N-α-benzyloxycarbonylglycine, CAS: 1138-80-3) purchased from a supplier.

[0960] Covering was performed by adding a mixed solution of 0.6 M Z-Gly-OH / 0.375 M HOAt / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 2 hours.

[0961] After removing the liquid phase covering the reaction with a filter, the resin was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain compound C2-1.

[0962] Comparative Example 2-2: Desorption of Fmoc-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound C2-1) Fmoc and N-terminal Nsylation

[0963] In a reaction container with a filter, Fmoc-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (Compound C2-1) (0.464mmol / g, 100mg) prepared in Comparative Example 2-1 was placed, and dichloromethane (1mL) was added and shaken at room temperature for 30 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed twice with DMF (1mL). Then, 2% DBU / DMF solution (Fmoc removal solution: 0.7mL) was added to the resin and shaken at room temperature for 10 minutes to remove Fmoc. After removing the Fmoc removal solution, the resin was washed 3 times with DMF (1mL) and then 4 times with THF (1mL).

[0964] To the obtained resin were added a THF solution (0.35 mL) of 2,4,6-collidine (0.062 mL, 0.464 mmol) and a THF solution (0.35 mL) of 2-nitrobenzenesulfonyl chloride (0.041 g, 0.186 mmol), and the mixture was shaken at 40 degrees for 2 hours.

[0965] After removing the liquid phase with a filter, the resin was washed once with THF (1 mL) and four times with dichloromethane (1 mL).

[0966] The above-mentioned Ns-reduction by 2-nitrobenzenesulfonyl chloride was further repeated twice (the second time: shaking at 40 degrees for 16 hours, the third time: shaking at 40 degrees for 21 hours).

[0967] In order to confirm the progress of extension, a portion of the obtained resin (~5 mg) was taken out and the peptide was cleaved with TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, 64.9% of the target peptide Ns-Aib-MeVal-Asp-pyrro (compound C2-2*) was confirmed.

[0968] The unreacted sites of the obtained resin were capped with Z-Gly-OH.

[0969] Covering was performed by adding a mixed solution of 0.6 M Z-Gly-OH / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) to the resin and shaking at 40 degrees for 2 hours.

[0970] After removing the liquid phase of the extension reaction with a filter, the resin was washed 4 times with DMF (0.7 mL) and 4 times with dichloromethane (0.7 mL) to obtain Ns-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-2).

[0971]

[0972] LCMS (ESI) m / z = 568.45 (MH) -

[0973] Retention time: 0.59 minutes (analysis conditions SQDFA05)

[0974] Comparative Example 2-3: By Ns-Aib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-2) N-methylation of the Ns amide site by Mitsunobu reaction

[0975] In a reaction container 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 dichloromethane with a filter, the resin was washed 4 times with THF (1 mL).

[0976] To the obtained resin, a solution of triphenylphosphine (61.0 mg, 0.232 mmol) and methanol (19 μL, 0.464 mmol) in THF (0.7 mL) was added, followed by 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 4 times with THF (1 mL) and 4 times with dichloromethane (1 mL).

[0977] A portion of the obtained resin (~5 mg) was cleaved from the peptide using a TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, the formation of the target peptide Ns-MeAib-MeVal-Asp-pyrro (compound C2-3*) was confirmed (the conversion rate from compound C2-2 was 96%). The residual Ns-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-3) was used in the next step.

[0978]

[0979] LCMS (ESI) m / z = 582.47 (MH) -

[0980] Retention time: 0.63 minutes (analysis conditions SQDFA05)

[0981] Comparative Example 2-4: De-Nsification of Ns-MeAib-MeVal-Asp-pyrro Resin (Compound C2-3)

[0982] In a reaction container with a filter, dichloromethane (0.5 mL) was added to Ns-MeAib-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound C2-3) (0.464 mmol / g, 50 mg) prepared in Comparative Example 2-3, and the mixture was shaken at room temperature for 20 minutes to swell the resin. After removing dichloromethane with a filter, the resin was washed 4 times with NMP (0.5 mL).

[0983] 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 to the obtained resin, and 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).

[0984] A portion (~5 mg) of the obtained resin was cleaved from the resin using a TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, it was found that Figure 3 , the production of the target peptide H-MeAib-MeVal-Asp-pyrro (compound C2-4*) which had undergone Ns depolymerization was confirmed.

[0985]

[0986] LCMS (ESI) m / z = 399.29 (M+H) +

[0987] Retention time: 0.34 minutes (analysis conditions SQDFA05)

[0988] Comparative Example 2-5: De-Nsification of Ns-MeAib-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro

[0989]

[0990] In a reaction container 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 in the same operation as in Comparative Examples 2-1 to 2-3, relative to 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 dichloromethane with a filter, the resin was washed 4 times with NMP (0.5 mL).

[0991] 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 to the obtained resin, and 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).

[0992] A portion (~5 mg) of the obtained resin was cleaved from the resin using a TFE / DCM solution (1 / 1 (v / v)). When the cleaved solution was analyzed by LCMS, it was found that Figure 4 In addition to the production of the target peptide H-MeAib-MePhe-Asp-pyrro (compound C2-5-2*) after de-Nsification, an impurity (compound C2-5-3*) in which the nitro-2-mercaptoethanol protected by Ns was presumably substituted at the present position was also detected.

[0993]

[0994] LCMS (ESI) m / z = 447.31 (M+H) +

[0995] Retention time: 0.39 minutes (analysis conditions SQDFA05)

[0996]

[0997] LCMS (ESI) m / z = 661.74 (MH) -

[0998] Retention time: 0.65 minutes (analysis conditions SQDFA05)

[0999] According to the results of these comparative examples 2, a bulky N-methyl-α, α-dialkyl amino acid (in this example, MeAib) can be introduced into the N-terminus of a bulky N-methyl amino acid. On the other hand, a series of steps including extension of Fmoc-Aib and change of the protecting group to the Ns group confirmed that the purity was low and the yield was low. In addition, it was clear that the purity was reduced by the side reaction on the Ns protecting group in the Ns removal stage. It was confirmed that it was difficult to introduce a bulky N-methyl-α, α-dialkyl amino acid into the N-terminus of a bulky N-methyl amino acid with high purity and high yield under the known conditions recorded in the literature.

[1000] Reference example: Attempt to extend N-methylamino acid with Fmoc-MeAib-OH in conventional solid phase synthesis

[1001] Reference Example 1: Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2), Fmoc- Extension reaction of MeAib-OH in the solid phase

[1002]

[1003] In a reaction container with a filter, Fmoc-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-2) (0.464 mmol / g, 100 mg) prepared in Example 1-2-2 was placed, and the extension of Fmoc-MeAib-OH was attempted by the same operation as in Comparative Example 2-1.

[1004] For the extension reaction, a mixed solution of 0.6M Fmoc-MeAib-OH / 0.375M oxyma / NMP solution (0.3 mL) and 10% DIC / DMF solution (0.36 mL) was added to the resin, shaken at 40 degrees for 21 hours, and after discarding the reaction solution, the same operation was repeated again (at 40 degrees for 21.5 hours).

[1005] After the extension reaction, the same operations as in Comparative Example 2-1 were performed, including appropriate washing of the resin, covering of unreacted sites with Fmoc-Gly-OH, and cleavage of the peptide from the resin. The cleaved solution was analyzed by LCMS, and the target peptide Fmoc-MeAib-MeVal-Asp-pyrro (compound R1*) was not detected.

[1006]

[1007] Reference Example 2: Fmoc-MePhe-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 1-2-3), Fmoc- Extension reaction of MeAib-OH in the solid phase

[1008]

[1009] In a reaction vessel with a filter, 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, and the extension of Fmoc-MeAib was attempted by the same operation as in Reference Example 1. The extension reaction was carried out at 40 degrees for 15 hours.

[1010] Thereafter, the same operations as in Reference Example 1 were performed, including appropriate washing of the resin, covering of unreacted sites with Fmoc-Gly-OH, and cleavage of the peptide from the resin. When the cleaved solution was analyzed by LCMS, the production of the target peptide Fmoc-MeAib-MePhe-Asp-pyrro (compound R2*) was maintained at 3.1%.

[1011]

[1012] LCMS (ESI) m / z = 669.43 (M+H) +

[1013] Retention time: 0.88 minutes (analysis conditions SQDFA05)

[1014] As described above, based on the results of the reference examples, it was confirmed that the extension of N-methyl amino acid (i.e., N-substituted amino acid) followed by Fmoc-MeAib-OH (i.e., N-substituted-α, α disubstituted amino acid) in the conventional solid phase synthesis method (Fmoc method) is extremely difficult, and there are cases where the target peptide cannot be obtained.

[1015] Industrial Applicability

[1016] According to the present invention, it has been found that in the preparation of a peptide compound using a solid phase 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

1. A method for preparing a peptide compound or a salt thereof comprising a structure in which two amino acid residues are linked represented by formula (1), In the formula, PG1 is trifluoroacetyl, P1 is a methyl group, R1 and Q1 are independently selected from methyl, ethyl, 2-methylpropyl, C1-C3 alkoxy C1-C3 alkyl, cyclohexyl C1-C3 alkyl and benzyl, or R1 and Q1 together with the carbon atom to which they are attached form a cyclopentane ring, P2 is a C1-C3 alkyl group, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 alkylsulfonylC1-C6 alkyl, C2-C6 alkynyl, C1-C6 alkoxyC1-C6 alkyl optionally substituted with one or more halogens, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C3-C8 cycloalkyloxyC1-C6 alkyl, or C7-C 14 Aralkyl, R3 is hydroxyl, O-PG2, any amino acid residue, or any peptide residue, PG2 is a protecting group for the carboxyl group. The method comprises the following steps: Step A: a compound represented by formula (2) or a salt thereof: In the formula, P2, R2, and R3 have the same meanings as P2, R2, and R3 in formula (1), respectively. and a compound represented by formula (3) or a salt thereof: In the formula, PG1, Q1, and R1 have the same meanings as PG1, Q1, and R1 in formula (1), respectively. The step of reacting with a condensation reagent to obtain a compound represented by formula (4) or a salt thereof: In the formula, PG1, P2, Q1, and R1 to R3 have the same meanings as PG1, P2, Q1, and R1 to R3 in formula (1), respectively. and Step B: a step of reacting a compound represented by formula (4) or a salt thereof with a P1-introducing reagent to obtain a peptide compound represented by formula (1) or a salt thereof, Wherein, the P1 introducing reagent is a combination of P1X and a base, wherein P1 is synonymous with P1 in formula (1), X is a leaving group, and The base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), and tert-butylimino-tris(dimethylamino)phosphorane (P1-tBu).

2. The method of claim 1, wherein: R1 and Q1 together with the carbon atom to which they are attached form a cyclopentane ring, or R1 and Q1 are independently selected from methyl, ethyl, 2-methylpropyl, methoxymethyl, cyclohexylmethyl and benzyl.

3. The method of claim 1, wherein: P2 is methyl, ethyl, n-propyl or isopropyl.

4. The method of claim 1, wherein: R3 is any amino acid residue or any peptide residue loaded on the resin for solid phase synthesis.

5. The method of claim 4, wherein: The resin used for solid phase synthesis is CTC resin, Wang resin, or SASRIN resin.

6. The method of claim 1, wherein: The condensation reagent is either DIC or EDCI·HCl, or a combination of DIC and Oxyma.

7. The method of claim 1, wherein: Step B is carried out in a solvent selected from the group consisting of DMF, NMP, DMI, tetrahydrofuran, 2-methyltetrahydrofuran, and acetonitrile.

8. A method for preparing a peptide compound comprising an N-substituted-α,α disubstituted amino acid residue and a dipeptide residue to which an N-substituted amino acid residue is linked, or a salt thereof, which comprises the method according to any one of claims 1 to 7.

9. A method for preparing a cyclic peptide compound or a salt thereof, comprising: a step of deprotecting the protective group at the N-terminus from the peptide compound or a salt thereof prepared by the method according to any one of claims 1 to 8, and a step of cyclizing the group on the C-terminal side and the group on the N-terminal side to form a cyclic portion, The cyclic peptide compound contains 8 to 15 amino acid residues, at least 3 N-substituted amino acid residues, and at least one amino acid residue not substituted by N, and the cyclic part contains at least 8 amino acid residues.

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