Peptide synthesis

Abstract

To provide novel peptide synthesis which can avoid the use of a main chain-protecting group and a condensation agent in a peptide supply process, can inhibit epimerization, and can enhance solubility of an unprotected amino acid element to accelerate the reaction.SOLUTION: A method comprises reacting a compound represented by the specified formula (I) with an amino acid represented by the specified formula (II) in the presence of a compound represented by the specified formula (III) and a compound represented by the specified formula (IV) thereby preparing a compound of the formula (V).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel method for peptide synthesis, more specifically, for elongating a peptide chain from the N-terminus to the C-terminus via a thiocarboxylic acid intermediate. [Background technology]

[0002] Medium-sized peptide compounds are attracting attention as next-generation drug candidates. In the exploration stage, they are supplied by solid-phase synthesis, but when it comes to large-scale trials and commercialization, the supply is insufficient, so a quantitative supply by liquid-phase synthesis is required.

[0003] The conventional mainstream chemical synthesis method that can freely incorporate various amino acids, including unnatural amino acids, is the method of extending from the C-terminus to the N-terminus (C→N-terminus extension method), but it is necessary to use an equivalent or more of condensation agent and a protected amino acid element that is expensive and has low atom efficiency. This has led to the problem of producing a large amount of waste from the main chain amino group protecting group and the condensation agent, which has led to an increase in the cost and environmental burden of the peptide supply process (see Figure 1).

[0004] On the other hand, a method of extending from the N-terminus to the C-terminus in the opposite direction (N→C-terminus extension method) has the potential to solve this problem, but because epimerization occurs at the same time, it has been difficult to realize a synthesis method that produces products with excellent purity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Holanders, K.; Mars, BUW; Ballet, S. Synthesis 2019, 51, 2261. [Non-Patent Document 2] de Figueiredo, RM; Suppo, J.-S.; Campagne, J.-M. Chem. Rev. [Non-Patent Document 3] Crich, D.; Sana, K.; Guo, S. Org. Lett. 2007, 9, 4423. [Non-Patent Document 4] Crich, D.; Sharma, I. Angew. Chem. Int. Ed. 2009, 48, 7591. [Non-Patent Document 5] Wu, W.; Zhang, Z.; Liebeskind, LSJ Am. Chem. Soc 2011, 133, 14256-14259. [Non-Patent Document 6] Mali, SM; Jadhav, SV; Gopi, HN Chem. Commun. 2012, 48, 7085. [Non-Patent Document 7] Wang, P.; Danishefsky, SJJ Am. Chem. Soc. 2015, 137, 13167. [Non-Patent Document 8] Review: Narenda N, Thimmalapura VM, Hosamani B, Prabhu G, Kumar LR, Sureshbabu VV. Org. Biomol. Chem. 2018, 16, 3524. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel peptide synthesis method that can avoid the use of condensation agents and main chain protecting groups in the peptide supply process, suppress epimerization, and further improve the solubility of unprotected amino acid elements and accelerate the reaction. [Means for solving the problem]

[0007] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by a novel peptide synthesis method comprising the following three elements, and have thus completed the present invention. (1) Peptide chain elongation from the N to C terminus via peptide thiocarboxylic acid. (2) A newly discovered epimerization inhibitor. (3) A solubilizer for unprotected amino acid elements.

[0008] That is, the present invention provides: [1] A method for preparing a compound of formula (V) by reacting a compound of formula (I) below with an amino acid of formula (II) below in the presence of a compound of formula (III) below and a compound of formula (IV) below. TIFF0007640994000001.tif42161 (in the formula, TIFF0007640994000002.tif15142 is represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group, R1 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group. TIFF0007640994000003.tif44145 (wherein R2 is a side chain of an α-amino acid, and the side chain may be protected with a protecting group.) TIFF0007640994000004.tif52159 (in the formula, L is an ester group (-COR; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R''; R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' represents an alkyl group substituted with an amino group), and an acyl group (-COR a '; R a ' is an alkyl group having 1 to 4 carbon atoms), provided that at least one of R3 and R4 is an ester group (-CO2R a ;R a is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H), R3 and R4 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a halogen atom, or an ester group (-COR a ;R a represents an alkyl group having 1 to 4 carbon atoms. TIFF0007640994000005.tif45155 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded.) TIFF0007640994000006.tif50163 (in the formula, TIFF0007640994000007.tif15142, R1 and R2 are as defined above.) [2] The method according to [1], wherein a mixed solvent of DMSO and toluene is used as the reaction solvent. [3] The method according to [1] or [2], comprising, prior to the coupling reaction, a step of reacting a compound represented by the following formula (VI) with a compound represented by the following formula (1) to prepare a compound represented by formula (I). TIFF0007640994000008.tif47168 (wherein R1 is as defined in formula (I)). TIFF0007640994000009.tif28158 (wherein R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.) [4] PG-(AA) n -SH: (PG represents an N-terminal protecting group, AA represents any amino acid residue, which may be the same or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups; n is an integer from 1 to 4. A compound represented by the formula: H-(AA') m -OH: (AA represents any amino acid residue, which may be the same or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with a protecting group; m is an integer from 1 to 4. An amino acid or peptide represented by In the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV), PG-(AA) n (AA') m A method for preparing a compound represented by -OH. TIFF0007640994000010.tif54163 (in the formula, L is an ester group (-COR; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R''; R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' represents an alkyl group substituted with an amino group), and an acyl group (-COR a '; R a ' is an alkyl group having 1 to 4 carbon atoms), provided that at least one of R3 and R4 is an ester group (-CO2R a ;R a is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H), R3 and R4 each independently represent an alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group (-COR a ;R a represents an alkyl group having 1 to 4 carbon atoms. TIFF0007640994000011.tif43147 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded.) [5] Following the method according to any one of [1] to [3], (i) preparing a compound represented by formula (VII) by reacting a compound represented by formula (V) with a compound represented by formula (1): TIFF0007640994000012.tif43169 (in the formula, TIFF0007640994000013.tif15142, R1 and R2 are as defined in formulas (I) and (II). TIFF0007640994000014.tif38152 (wherein R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.) (ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV): A process for preparing a compound of formula (VIIII), comprising: TIFF0007640994000016.tif48158 (in the formula, R 3a is the side chain of an α-amino acid, which may be protected with a protecting group. TIFF0007640994000017.tif61156 (in the formula, L is an ester group (-COR; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R''; R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' represents an alkyl group substituted with an amino group), and an acyl group (-COR a '; R a ' is an alkyl group having 1 to 4 carbon atoms), provided that at least one of R3 and R4 is an ester group (-CO2R a ;R a is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H), R3 and R4 each independently represent an alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group (-COR a ;R a represents an alkyl group having 1 to 4 carbon atoms. TIFF0007640994000018.tif44151 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded.) TIFF0007640994000019.tif36131 (in the formula, TIFF0007640994000020.tif15142, R1~R2, R 3a is as defined above.) [6] A method of using a compound represented by the following formula (IV) as a solubilizing agent in peptide synthesis. TIFF0007640994000021.tif40150 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded.) This provides: Effect of the Invention

[0009] The present invention provides a novel peptide synthesis method that can avoid the use of condensation agents and main chain protecting groups in the peptide supply process, suppress epimerization, and further improve the solubility of unprotected amino acid elements and accelerate the reaction. [Brief description of the drawings]

[0010] [Figure 1] Schematic diagram of the conventional C→N-terminal elongation method using coupling agents. [Diagram 2] Basic reaction scheme of the peptide synthesis method of the present invention. [Diagram 3] Summary of reaction schemes for non-limiting methods to obtain compounds of formula (I). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Peptide synthesis One embodiment of the present invention is a method for preparing a compound of formula (V) by coupling reaction of a compound of formula (I) with an amino acid of formula (II) in the presence of a compound of formula (III) and a compound of formula (IV) (hereinafter also referred to as the "peptide synthesis method of the present invention").

[0012] The basic reaction scheme of the peptide synthesis method of the present invention is shown in Figure 2. In the present invention, it is important to have three elements: (1) a peptide chain elongation method in the N→C-terminal direction via peptide thiocarboxylic acid, (2) a newly discovered epimerization inhibitor, and (3) a solubilizer for main chain unprotected amino acid elements. The peptide synthesis method of the present invention will be explained below, including the details of these three elements.

[0013] (1) Peptide chain elongation from N to C terminal via thiocarboxylic acid compounds The peptide synthesis method of the present invention employs a peptide chain elongation method in the N→C-terminal direction via a peptide thiocarboxylic acid, which is a compound represented by formula (I). TIFF0007640994000022.tif49156

[0014] In formula (I), TIFF0007640994000023.tif15142 is It represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group.

[0015] Examples of the protecting group include protecting groups commonly used as protecting groups for the N-terminus of amino acids, such as tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 9-fluorenylmethoxycarbonyl group (Fmoc), acetyl group (Ac), and benzoyl group (Bz).

[0016] An amino acid whose N-terminus is protected with a protecting group means an amino acid whose N-terminus is protected with the above-mentioned protecting group. The amino acid can be used without any particular limitation. Examples include α-amino acids and β-amino acids. Examples of α-amino acids include, but are not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline, ornithine, citrulline, N-methylglycine (sarcosine), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, and α-aminoisobutyric acid. Examples of β-amino acids include, but are not limited to, β-alanine. The above amino acids may be either L- or D-optical isomers. When the side chains of these amino acids have functional groups, R1 may be protected with a protecting group as described below.

[0017] A peptide whose N-terminus is protected with a protecting group means a peptide whose N-terminus is protected with the above-mentioned protecting group. Here, the peptide has a structure in which 2 to 9, preferably 2 to 6, and more preferably 2 to 4 of the above-mentioned amino acid residues are bonded.

[0018] R1 is the side chain of the α-amino acid. R1 is determined by the type of α-amino acid residue that is bonded to SH. For example, if the amino acid residue is glycine, R1 is hydrogen, and if it is alanine, R1 is a methyl group. That is, the α-amino acid residue bound to SH in formula (I); NH-CH(R1)-C(=O) may be any α-amino acid residue other than phenylglycine. Here, examples of α-amino acids include, but are not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline, ornithine, citrulline, N-methylglycine (sarcosine), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, and α-aminoisobutyric acid. R1 is the side chain of an amino acid selected from these. The above α-amino acids may be either L- or D-optical isomers.

[0019] The side chain of the above α-amino acid residue represented by R1 may be protected with a protecting group. For example, when R1 is a lysine side chain (having an amino group), the protecting group can be a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), or a 9-fluorenylmethoxycarbonyl group (Fmoc). When R1 is a glutamic acid or aspartic acid side chain having a carboxy group, a benzyl ester (Bzl) or a tert-butyl ester (t-Bu) can be used as a protecting group. Also, when R1 is a serine or threonine side chain (containing a hydroxy group), a benzyl group or a tert-butyl group can be used as a protecting group. When R1 is a tyrosine side chain (having a phenolic hydroxy group), 2-bromobenzyloxycarbonyl (Z(2Br)) or a tert-butyl group can be used as a protecting group. When R1 is a cysteine ​​side chain (having a sulfhydryl group), the protecting group can be 4-methylbenzyl (Bzl(4Me)), trityl (Trt), tert-butyl, or N-(acetyl)aminomethyl (Acm). When R1 is an arginine side chain (having a guanidino group), a protecting group such as p-toluenesulfonyl group (p-Ts) can be used. In addition, when R1 is a histidine side chain (containing an imidazole ring), the π-nitrogen can be protected with a benzyloxymethyl group (Bom) or a tert-butoxymethyl group (Bum), and the τ-nitrogen can be protected with a 2,4-dinitrophenyl group (Dnp), a trityl group, or the like.

[0020] The thiocarboxylic acid compound represented by formula (I) can be obtained using the one-step conversion conditions reported by the present inventors' group (Chem. Commun. 2018, 54, 1222) or any existing method.

[0021] In one preferred aspect of the present invention, the compound represented by formula (I) can be obtained by reacting a compound represented by the following formula (VI) with a compound represented by the following formula (1). TIFF0007640994000024.tif77153 In formula (1), R7 is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

[0022] In formula (VI), TIFF0007640994000025.tif15142 is represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group, and R1 is the side chain of an α-amino acid, and the details thereof are the same as those explained in relation to formula (I).

[0023] The outline of the reaction scheme for obtaining the compound represented by the above formula (I) is shown in Figure 3. In this reaction, acetyl sulfide is used as a catalyst. This reaction is carried out usually using DMF as a solvent at a temperature of 0° C. to room temperature.

[0024] In the peptide synthesis method of the present invention, a thiocarboxylic acid compound represented by formula (I) is reacted with a compound represented by the following formula (II). TIFF0007640994000026.tif44158

[0025] The compound represented by formula (II) is an unprotected α-amino acid, and in addition to natural amino acids, any α-amino acid can be used as the α-amino acid, such as, but not limited to, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, ornithine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, tert-leucine, phenylglycine, and α-aminoisobutyric acid. The above α-amino acids may be either L- or D-optical isomers.

[0026] R2 in formula (II) is the side chain of the α-amino acid and is determined by the type of α-amino acid. For example, if the amino acid is glycine, R2 is hydrogen, and if the amino acid is alanine, R2 is a methyl group.

[0027] The side chain of the α-amino acid represented by R2 may be protected with a protecting group, which may be the same as that described for R1 in formula (I).

[0028] In the peptide synthesis method of the present invention, by converting a peptide or amino acid to be elongated into a thiocarboxylic acid as represented by formula (I), it becomes possible to distinguish, to the minimum extent possible, between the C-terminus of the starting compound of formula (I) and the C-terminus of the unprotected amino acid element of formula (II), and it is possible to elongate the peptide one amino acid at a time by selectively activating the C-terminus of the starting compound. In addition, by using a peptide chain elongation method in the N→C-terminal direction via thiocarboxylic acid, an equivalent amount or more of a condensation agent is not required, and waste generated during the elongation process can in principle be reduced to only solvent and elemental sulfur. In addition, inexpensive unprotected amino acids can be used as synthetic building blocks. Therefore, a significant reduction in waste and costs can be expected compared to conventional peptide elongation methods. In addition, since the product is a peptide chain with no C-terminal protection, it is possible to rapidly elongate the peptide chain by repeating the thiocarboxylic acid conversion → peptide chain linkage.

[0029] (2) Epimerization inhibitors In the peptide synthesis method of the present invention, a novel epimerization inhibitor, which is a compound represented by the following formula (III), is used.

[0030] When N→C-terminal elongation is performed according to conventional thinking, epimerization (loss of stereochemistry) occurs via a cyclization intermediate, making it impossible to supply a product with excellent purity. As a result of synthesizing and screening various additives expected to suppress epimerization, the present inventors found that N-hydroxypyridone-type additives solve the problem of epimerization in the peptide synthesis method of the present invention.

[0031] TIFF0007640994000027.tif52153

[0032] In formula (III), L is an ester group (-COR; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R''; R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' represents an alkyl group substituted with an amino group), or an acyl group (-COR a '; R a ' is selected from the group consisting of alkyl groups having 1 to 4 carbon atoms.

[0033] The ester group is represented by -CO2R, where R is an alkyl group having a carbon number of 1 to 30. The alkyl group may be unsubstituted or may have a substituent. The alkyl group may be either a linear alkyl group or a branched alkyl group. In the case of a branched alkyl group, even if the total number of carbon atoms becomes large, such as about 30, it is advantageous in that the additive can be easily recovered from the product by recrystallization. Branched alkyl groups include -(CH2CH2CH(CH3)CH2) s A branched alkyl group of -H (s is 1 to 4) is preferred from the viewpoint of increasing the recovery rate of the additive from the product by recrystallization. In the case of a linear alkyl group, the yield and degree of epimerization are good even if the carbon number is large, but the degree of crystallization increases, and recovery from the product may become difficult. Among linear alkyl groups, an alkyl group having 1 to 4 carbon atoms is preferred from the viewpoints of the yield, degree of epimerization, and ease of recovery of the additive from the product, and a methyl group (i.e., as an ester group, a methyl ester group (-CO2CH3)) is particularly preferred.

[0034] The amido group which may be substituted with an amino group is preferably a dimethylaminoethylamido group (-CONHC2H4N(CH3)2).

[0035] The aryl group may be a phenyl group, which may be substituted or unsubstituted. The substituent of the aryl group may be an alkyl group, an ester group, etc., and is preferably an ester group (e.g., a methyl ester group (-CO2CH3)).

[0036] The substituent of the benzoyl group may be an alkyl group, an amino group, or the like, and is preferably an amino group (eg, a dimethylamino group).

[0037] The acyl group is -COR a '(R a ' is an alkyl group having 1 to 4 carbon atoms, and is preferably an acetyl group.

[0038] L is preferably selected from the groups listed above, but at least one of R3 and R4 is an ester group (-COR a ;R a When L is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H). In this case, the epimerization degree tends to be slightly higher than when L is an ester group or the like, but it is possible to obtain the product in high yield.

[0039] In formula (III), R3 and R4 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a halogen atom, or an ester group (-COR a ;R a represents an alkyl group having 1 to 4 carbon atoms. Chlorine is preferred as the halogen. A methyl ester group (-CO2CH3) is preferred as the ester group.

[0040] In one preferred aspect of the invention, both R3 and R4 are hydrogen atoms. In another preferred aspect of the present invention, one of R3 and R4 is chlorine and the other is a hydrogen atom. In another preferred aspect of the present invention, one of R3 and R4 is a methyl ester group, and the other is a hydrogen atom.

[0041] (3) Solubilizer for unprotected amino acids The peptide synthesis method of the present invention employs a solubilizing agent which is a compound represented by formula (IV).

[0042] Since unprotected amino acid elements (raw materials) generally do not require organic solvents, peptide elongation methods using them have poor reaction efficiency and require a long time to complete one step. The present inventors searched for additives that solubilize unprotected amino acid elements and found that phosphite-based additives contribute to improving the solubility of unprotected amino acid elements, improving the reaction efficiency in the liquid phase and making it possible to shorten the reaction time.

[0043] TIFF0007640994000028.tif42148

[0044] In formula (IV), R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group, provided that both R5 and R6 are not hydroxy groups. The alkyl group is preferably an n-butyl group, and the alkoxy group is preferably a methoxy group, an ethoxy group, an i-propoxy group, or an n-butoxy group.

[0045] In one preferred aspect of the present invention, the compound of formula (IV) is represented by the following formula (IVa): TIFF0007640994000029.tif43147

[0046] In formula (IVa), R5' and R6' each independently represent an alkyl group having 1 to 4 carbon atoms, a benzyl group which may have a substituent, or hydrogen, provided that both R5' and R6' are not hydrogen.

[0047] In one preferred aspect of the invention, both R5' and R6' are methyl groups. In addition, in one preferred aspect of the present invention, both R5' and R6' are ethyl groups. Also, in a preferred aspect of the present invention, both R5' and R6' are i-propyl groups. In addition, in one preferred aspect of the present invention, both R5' and R6' are n-butyl groups. Also, in one preferred aspect of the present invention, both R5' and R6' are benzyl groups.

[0048] In formula (IV), R5 and R6 may join together to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded. Such a heterocyclyl is preferably a 5- or 6-membered ring structure, more preferably a 6-membered ring structure. The substituents include alkyl groups having 1 to 4 substituents, and the number of the substituents may be 1 or 2 or more. The alkyl group is preferably a methyl group or an ethyl group.

[0049] Non-limiting examples of formula (IV) that may be used in the method of the present invention are shown below.

[0050] TIFF0007640994000030.tif54152

[0051] The peptide synthesis method of the present invention can provide a compound represented by the following formula (V). TIFF0007640994000031.tif39128

[0052] In formula (V), TIFF0007640994000032.tif15142, R1 and R2 are as described for formulas (I) and (II).

[0053] In the peptide synthesis method of the present invention, the molar amount of the compound of formula (I) is usually reacted with 1 to 2 equivalents of the compound of formula (II), 1 equivalent of the compound of formula (III), and 1 equivalent of the compound of formula (IV).

[0054] In the peptide synthesis method of the present invention, a solvent usually used in liquid phase peptide synthesis can be used, but a DMSO-based solvent is preferred because it can efficiently activate the thioacid. Furthermore, a mixed solvent such as DMSO and toluene is preferred because it can reduce polarity and effectively suppress epimerization. In one preferred aspect of the method of the present invention, a mixed solvent of DMSO:toluene=1:1 (volume ratio) is used.

[0055] The peptide synthesis method of the present invention is usually carried out by reacting at about room temperature for 3 to 6 hours.

[0056] As explained above, the peptide synthesis method of the present invention is based on the peptide chain elongation method in the N→C-terminal direction via a thiocarboxylic acid compound of a peptide or amino acid, but the method of the present invention can also be applied to peptide fragment coupling and repeated coupling.

[0057] 2. Fragment Coupling Another embodiment of the present invention is PG-(AA) n -SH: (PG represents an N-terminal protecting group, AA represents any amino acid residue, which may be the same or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups; n is an integer from 1 to 4. A compound represented by the formula: H-(AA') m -OH: (AA' represents any amino acid residue, which may be the same or different in each occurrence, and some or all of the side chains of the amino acid residue may be protected with protecting groups; m is an integer from 1 to 4. The peptide represented by In the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV), PG-(AA) n (AA') m This is a method for preparing a compound represented by —OH (hereinafter also referred to as the “fragment coupling of the present invention”). TIFF0007640994000033.tif65155(in the formula, L is an ester group (-COR; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted aryl group, a substituted or unsubstituted benzoyl group, an amide group which may be substituted with an amino group (-CONR'R''; R' and R'' each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least one of R' and R'' represents an alkyl group substituted with an amino group), an acyl group (-COR a '; R a' is an alkyl group having 1 to 4 carbon atoms), provided that at least one of R3 and R4 is an ester group (-CO2R a ;R a is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H), R3 and R4 each independently represent an alkyl group having 1 to 4 carbon atoms, a halogen, or an ester group (-COR a ;R a represents an alkyl group having 1 to 4 carbon atoms. TIFF0007640994000034.tif42157 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may be joined together to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are attached.

[0058] As the N-terminal protecting group of PG, the same N-terminal protecting group as explained in the peptide synthesis method of the present invention can be used. The arbitrary amino acid residues of AA and AA' include α-amino acid and β-amino acid residues, and the α-amino acid residues include leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, ornithine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, and the like. The β-amino acid includes β-alanine and the like. However, the amino acid residues bound to SH among the amino acid residues of AA are arbitrary amino acid residues other than phenylglycine, and the amino acid residue at the N-terminus among the amino acid residues of AA' are amino acid residues other than proline and N-methylamino acid. When the side chains of these amino acid residues are protected, the same protecting groups as those explained in the peptide synthesis method of the present invention can be used. Any amino acid residue of AA and AA' may be either the L- or D-optical isomer.

[0059] In the fragment coupling of the present invention, n and m are preferably the same. n and m are preferably 2 or 3.

[0060] In the fragment coupling of the present invention, the reaction conditions such as solvent, temperature and time explained in the peptide synthesis method of the present invention can be used similarly.

[0061] 3. Iterative Coupling Another embodiment of the present invention comprises carrying out the peptide synthesis method of the present invention as described above, followed by: (i) preparing a compound represented by formula (VII) by reacting a compound represented by formula (V) with a compound represented by formula (1): TIFF0007640994000035.tif36133(in the formula, TIFF0007640994000036.tif15142, R1 and R2 are as defined in formulas (I) and (II). TIFF0007640994000037.tif73161(ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV): (hereinafter also referred to as "the iterative coupling of the present invention") TIFF0007640994000038.tif49153 (in the formula, R 3a is the side chain of an α-amino acid, which may be protected with a protecting group. TIFF0007640994000039.tif56159 (wherein L, R3 to R4 are as defined above.) TIFF0007640994000040.tif47160 (wherein R5 to R6 are as defined above.) TIFF0007640994000041.tif56168 (in the formula, TIFF0007640994000042.tif15142, R1~R2, R 3a is as defined above.)

[0062] R in formula (VIII) 3a is the side chain of the α-amino acid, and the α-amino acid is the same as that explained for the amino acid of formula (II). The side chain may be protected with a protecting group, and the protecting group is the same as that explained for formula (II).

[0063] In the repetitive coupling of the present invention, the above-described method can be repeatedly used to elongate a peptide chain. The peptide chain elongation can be carried out until a peptide having usually 2 to 5 amino acid residues is obtained.

[0064] In the iterative coupling of the present invention, the reaction conditions such as solvent, temperature and time described in the peptide synthesis method of the present invention can be used similarly.

[0065] The above-mentioned peptide synthesis method of the present invention, the fragment coupling of the present invention, and the iterative coupling of the present invention can be applied to liquid phase synthesis of peptides.

[0066] Another aspect of the present invention is a method of using a compound represented by the following formula (IV) as a solubilizing agent in peptide synthesis. TIFF0007640994000043.tif44141 (wherein R5 and R6 each independently represent an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a benzyloxy group which may have a substituent, or a hydroxy group (however, R5 and R6 cannot both be hydroxy groups), R5 and R6 may join together to form a 4-7 membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R5 and R6 are bonded.) EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0068] 1.General method (1) General theory Electrospray ionization (ESI) mass spectra were measured on a Shimadzu LCMS-2020 spectrometer (for LRMS). Analytical HPLC charts were obtained using a JASCO HPLC system equipped with a UV-2075 spectrometer, a PU-2080 pump, a DG-2080-54 degasser, and an MX-2080-32 mixer.

[0069] (2) Analytical HPLC Peptide composition was assessed by analytical reverse-phase HPLC using a gradient of acetonitrile vs. 0.1% trifluoroacetic acid (TFA) in water. Reverse phase analytical HPLC was performed as follows: YMC-Triart-C 18 A column (inner diameter 4.6 mm I.D. × 150 mm) was used, and a linear gradient of 0 to 100% acetonitrile in 0.1% TFA aqueous solution was used at room temperature for 40 minutes or more at a flow rate of 1 mL / min. -1 The eluent was monitored by absorbance at 230 nm.

[0070] (3) Preparative HPLC Longer peptides were purified by preparative reversed-phase HPLC using a gradient of acetonitrile vs. 0.1% TFA in water. Preparative HPLC was performed as follows: 40 °C, flow rate 10.0 mL min -1 The column was YMC-Triart C18 (20 nm inner diameter × 250 mm) at 100° C. The gradient conditions were modified for each peptide. The eluent was monitored by absorbance at 230 nm.

[0071] (4) Preparation of a standard curve for peptides Authentic peptides (5 μmol) were dissolved in DMSO (250 μL) to make a 20 mM peptide solution. This solution was diluted to 15 mM, 10 mM, 8 mM, 5 mM, and 2 mM. For peptides containing Trt or Pbf, authentic peptides (1 μmol) were dissolved in DMSO (500 μL) to make a 2 mM peptide solution, which was then diluted to 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM, and these solutions were then analyzed by analytical HPLC by absorbance at 230 nm. For each solution, the peak area corresponding to the peptide was measured, and a calibration curve was constructed from these values.

[0072] (5) Calculation method for epimerization level of thioacid The peptide thioacids were converted to p-methoxybenzyl thioesters with p-methoxybenzyl chloride, and the reaction mixtures containing the thioesters were analyzed by normal-phase chiral HPLC to measure the peak areas corresponding to the thioester isomers, and the epimerization rates were calculated using the following equation:

number

[0073] (6) Method for calculating the epimerization level of peptides after coupling reaction After the peptide coupling reaction, the reaction mixture was analyzed by reversed-phase HPLC. The peak areas corresponding to LLL isomers and LDL isomers were measured, and the apparent epimerization level (AEL) was calculated based on the following formula:

number

number

[0074] <Synthesis Examples> 1. Preparation of Peptide Thioacids The peptide thioacids were obtained by the method described in Chem. Commun., 2018, 54, 12222. The preparation method of each peptide thioacid is described below.

[0075] (1) Synthesis of Cbz-Phe-Val-SH To a frame-dried flask, Cbz-Phe-Val-OH (1.19 g, 3.0 mmol) and DMF (20 mM) were added under argon atmosphere. AcSK (3.43 g, 30 mmol) and Ac2S (161 μL, 1.5 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Val-SH (870 mg, 70%) as a yellow solid. MS (ESI): m / z 415.53 (calcd [M+H] + =415.10). Save time: 29.75 min.

[0076] (2) Synthesis of Cbz-Phe-Phe-SH To a frame-dried flask, Cbz-Phe-Phe-OH (446 mg, 1.0 mmol) and DMF (10 mM) were added under argon atmosphere. AcSK (342 mg, 3.0 mmol) and Ac2S (21 μL, 0.2 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Phe-SH (254 mg, 55%) as a white solid. MS (ESI): m / z 485.15 (calcd [M+Na] + =485.55). Save time: 31.67 min.

[0077] (3) Synthesis of Boc-Phe-Val-SH To a frame-dried flask, Boc-Phe-Val-OH (109 mg, 0.3 mmol) and DMF (3 mM) were added under argon atmosphere. AcSK (171 mg, 1.5 mmol) and Ac2S (18 μL, 0.15 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Boc-Phe-Val-SH (57 mg, 50%) as a white solid. MS (ESI): m / z 403.15 (calcd [M+Na] + =403.49). Save time: 30.16 min.

[0078] (4) Synthesis of Cbz-Phe-Cys(Trt)-SH To a frame-dried test tube, Cbz-Phe-Cys(Trt)-OH (129 mg, 0.2 mmol) and DMF (2 mM) were added under argon atmosphere. AcSK (114 mg, 1 mmol) and Ac2S (10 μL, 0.1 mmol) were then added to the solution. After the resulting mixture was stirred at 0° C. for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Cys(Trt)-SH (25 mg, 19%) as a yellow solid. MS(ESI): m / z 683.15(calcd [M+Na] + =683.84). Save time: 37.89 min.

[0079] (5) Synthesis of Cbz-Phe-Lys(Boc)-SH To a frame-dried test tube, Cbz-Phe-Lys(Boc)-OH (105 mg, 0.2 mmol) and DMF (2 mM) were added under argon atmosphere. AcSK (68 mg, 0.6 mmol) and Ac2S (5 μL, 40 μmol) were then added to the solution. After the resulting mixture was stirred at 0 °C for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automated RP-column chromatography to give Cbz-Phe-Lys(Boc)-SH (30 mg, 28%) as a yellow solid. MS (ESI): m / z 566.10 (calcd [M+Na] + =566.67). Save time: 31.80 min.

[0080] (6) Synthesis of Cbz-Phe-Ala-Val-SH To a frame-dried test tube, Cbz-Phe-Ala-Val-OH (47 mg, 0.1 mmol) and DMF (1 mM) were added under argon atmosphere. AcSK (57 mg, 0.5 mmol) and Ac2S (5 μL, 50 μmol) were then added to the solution. After the resulting mixture was stirred at 0 °C for 3 h, the reaction mixture was extracted with ethyl acetate and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Cbz-Phe-Ala-Val-SH (11 mg, 25%) as a yellow solid. MS (ESI): m / z 508.15 (calcd [M+Na] + =508.59). Save time: 29.34 min.

[0081] 2. Preparation of N-hydroxy-pyridone additives (1) Methyl 1-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylate (Compound 1) Compound 1 was synthesized according to the following synthetic scheme. TIFF0007640994000047.tif35148

[0082] This compound was synthesized as described in the literature (Ando, ​​M.; Sato, N.; Nagase, T.; Nagai, K.; Ishikawa, S.; Takahashi, H.; Ohtake, N.; Ito, J.; Hirayama, M.; Mitobe, Y.; et al. Bioorganic Med. Chem. 2009, 17, 6106-6122.). To a stirred solution of S1 (10.2 g, 60 mmol) and DCM (80 mL), urea hydrogen peroxide (12.0 g, 122 mmol) and TFAA (17.1 mL, 120 mmol) were added dropwise successively at 0 °C, and the reaction mixture was warmed to room temperature and stirred for 3 h. After cooling to 0 °C, the reaction mixture was added with aqueous Na2S2O3 and concentrated. The mixture was extracted with DCM, washed with aqueous NaHCO3 and brine, and dried over Na2SO4. The organic layer was concentrated to give crude S2. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane=3:1) to give S2 (7.8 g, 70%) as a yellow solid.

[0083] To a stirred solution of S2 (7.8 g, 41.7 mmol) and MeCN (3 mL) was added TFAA (7 ml, 50 mmol) at room temperature, and the mixture was stirred for 3 h. The reaction mixture was concentrated under reduced pressure. To the residue was added solid NaHCO3 and methanol. After filtration, the filtrate was concentrated to give crude product 1. The crude product was purified by flash column chromatography (DCM / MeOH=19:1) to give 1 (3.0 g, 43%) as a pale yellow solid. 1 H NMR (500 MHz, CD3OD): δ 3.86 (3H, s), 6.62 (1H. d, J = 9.2 Hz), 7.92 (1H, dd, J = 2.4, 9.2 Hz), 8.56 (1H, d, J = 2.4 Hz); MS (ESI): m / z 170.10 (calcd [M+H] + = 170.14).

[0084] (2) 1-Hydroxy-5-phenylpyridin-2(1H)-one (Compound 2) Compound 2 was synthesized according to the following synthetic scheme. TIFF0007640994000048.tif55152

[0085] To a stirred solution of S3 (241 μL, 2 mmol) and DCM (10 mL) was added mCPBA (986 mg, 4 mmol, containing 30% H2O) at 0 °C, and the mixture was stirred for 2 h. 1 M aqueous Na2S2O3 was added to the reaction mixture, and the mixture was extracted with DCM. The combined organic layers were washed with aqueous NaHCO3 and brine, and dried over Na2SO4. After filtration, the organic layers were concentrated to give crude product S4. Crude product S4 was purified by flash column chromatography (DCM / MeOH=19:1) to give S4 (320 mg, 79%) as a white solid.

[0086] To a stirred solution of S4 (50 mg, 0.25 mmol) and DMF (500 μL), phenylboronic acid (45 mg, 0.375 mmol), Pd(PPh3)4 (14 mg, 2.5 μmol) and K2CO3 (69 mg, 0.5 mmol) were added and stirred at 100 °C overnight. The reaction mixture was extracted with DCM and the combined organic layers were washed with brine, dried over Na2SO4 and concentrated to give crude product S5. The crude mixture was purified by flash column chromatography (DCM / MeOH=19:1) to give S5 (34 mg, 67%) as a white solid.

[0087] To a stirred solution of S5 (20 mg, 0.1 mmol) and dry DCM (500 μL) was added BCl3 (1 M in heptane, 150 μL, 0.15 mmol) dropwise under an argon atmosphere at −10° C., and the reaction mixture was allowed to warm to room temperature and stirred overnight. After addition of MeOH (1 mL), the reaction mixture was concentrated to give crude product 2. The crude product was purified by flash column chromatography (ethyl acetate / hexanes=1:3) to give 2 (5 mg, 27%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 6.84 (1H, d, J = 9.2), 7.36 (1H, m), 7.44 (4H, m), 7.67 (1H, d, J = 10.3), 8.02 (1H, s); MS (ESI): m / z 188.10 (calcd [M+H] + =188.21).

[0088] (3) Methyl 1-hydroxyl-2-oxo-5-phenyl-1,2-dihydropyridine-3-carboxylate (Compound 3) Compound 3 was synthesized according to the following synthetic scheme. TIFF0007640994000049.tif65151

[0089] To a frame-dried 50 mL flask, Na (340 mg, 14 mmol) and MeOH (10 mL) were added at 0° C. under argon atmosphere, and S6 (1.0 g, 4 mmol) was added to the solution, and the mixture was stirred for 3 h. Acetic acid was added to the reaction mixture until neutralization, and the mixture was concentrated under reduced pressure. Ethyl acetate and H2O were added to the residue, and the product was extracted with ethyl acetate. The organic layer was washed with H2O and brine, dried over Na2SO4, and concentrated to give crude product S7. The crude mixture was purified by flash column chromatography (ethyl acetate / hexanes=1:3) to give S7 (800 mg, 77%) as a colorless oil.

[0090] In a 30 mL flask, S7 (390 mg, 1.5 mmol), phenylboronic acid (272 mg, 2.25 mmol), Pd(PPh3)4 (86 mg, 75 μmol), K2CO3 (414 mg, 3 mmol), and DMF (8 mL) were added under argon atmosphere, and the mixture was stirred at 100 °C overnight. Ethyl acetate and H2O were added to the reaction mixture, and the product was extracted with ethyl acetate. The combined organic layers were washed with H2O, dried over Na2SO4 with brine, and concentrated to the crude product S8. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1:3) to give S8 as a white solid (280 mg, 77%).

[0091] A test tube was charged with S8 (120 mg, 0.5 mmol), urea hydrogen peroxide (294 mg, 6.1 mmol), and MeCN (2 mL). TFAA (450 μL, 6 mmol) was added dropwise to the 0°C solution, the reaction was allowed to warm to room temperature, and the mixture was stirred overnight. To the reaction mixture was added aqueous Na2S2O3, and the mixture was concentrated. Ethyl acetate and H2O were added to the residue, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, and concentrated to give the crude product S9. The crude mixture was purified by flash column chromatography (ethyl acetate / hexanes=3:1) to give S9 (50 mg, 38%) as a white solid.

[0092] To a test tube was added S9 (25 mg, 0.1 mmol) and DCM (500 μL) under argon atmosphere. AcCl (72 μL, 1 mmol) was added to the solution and the mixture was heated to reflux for 1 h. The reaction mixture was concentrated. Methanol was added and the mixture was stirred at room temperature overnight. The mixture was concentrated to give crude product 3. The crude mixture was purified by flash column chromatography (DCM / MeOH=19:1) to give 3 (11 mg, 45%) as a white solid. 1 H NMR (500 MHz, CDCl3): δ3.97 (3H, s), 7.39 (2H, m), 7.46 (3H, m), 8.26 (1H, s), 8.47 (1H, s); MS (ESI): m / z 246 (calcd [M+H] + = 246.24).

[0093] (4) Methyl 2-(1-hydroxy-6-oxo-1,6-dihydropyridin-3-yl)benzoate (Compound 4) Compound 4 was synthesized according to the following synthetic scheme. TIFF0007640994000050.tif72153

[0094] In a 100 mL flask, S3 (482 mg, 4 mmol), 2-(methoxycarbonyl)-phenylboronic acid (1074 mg, 6 mmol), Pd(PPh3)4 (231 mg, 0.2 mmol), K2CO3 (1100 mg, 12 mmol) and DMF (20 mL) were added under argon atmosphere and the mixture was stirred at 100 °C overnight. Ethyl acetate and H2O were added to the reaction mixture and the product was extracted with ethyl acetate. The combined organic layers were washed with H2O and brine, dried over Na2SO4 and concentrated to give crude product S10. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1:3) to give S10 (800 mg, 82%) as a white solid.

[0095] To S10 (600 mg, 2.5 mmol) in DCM (10 mL) in a 50 mL flask was added mCPBA (1.85 g, 7.5 mmol, containing 30% H2O) at 0° C., and the mixture was allowed to warm to room temperature and stirred overnight. 1M Na2S2O3 aqueous solution was added to the reaction mixture, and the mixture was extracted with DCM. The combined organic layers were washed with NaHCO3 aqueous solution and brine, and dried over Na2SO4. The organic layers were concentrated to give the crude product S11. The crude mixture was purified by flash column chromatography (DCM / MeOH=19:1) to give S11 (580 mg, 90%) as a white solid.

[0096] The reaction mixture was concentrated, methanol was added and stirred at room temperature overnight. The mixture was concentrated to give crude product 3. The crude mixture was purified by flash column chromatography (DCM / MeOH=19:1) to give 4 (125 mg, 51%) as a white solid. 1 H NMR (500 MHz, CDCl3): δ 3.65 (3H, s), 6.79 (1H, s), 7.41 (4H, m), 7.84 (1H, d, J = 8.0), 8.10 (1H, s); MS (ESI): m / z 246 (calcd [M+H] + = 246.24).

[0097] (5) Methyl 1-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylate (Compound 5) Compound 5 was synthesized according to the following synthetic scheme. TIFF0007640994000051.tif38154

[0098] This compound was prepared using the procedure described for compound 1 as a yellow solid (72 mg, 85%). (500 MHz, (CD3)2SO): δ 3.74 (3H, s), 6.25 (1H, dd, J = 6.7, 7.4), 7.96 (1H, d, J = 7.4), 8.23 ​​(1H, d, J = 6.7); MS (ESI): m / z 170.10 (calcd [M+H] + = 170.14).

[0099] (6) N-(2-(dimethylamino)ethyl)-1-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxamide (compound 6) Compound 6 was synthesized according to the following synthetic scheme. TIFF0007640994000052.tif73149

[0100] To a stirred solution of compound 1 (3.0 g, 17.75 mmol), K2CO3 (7.3 g, 53.25 mmol), and DMF (40 mL), BnBr (2.5 mL, 21.3 mmol) was added and stirred at 80 °C for 3 h, and the reaction mixture was concentrated. Ethyl acetate and H2O were added to the resulting mixture, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give crude product S13. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1:3) to give S13 (2.0 g, 44%) as a white solid.

[0101] To a stirred solution of S13 (2.0 g, 8 mmol) and MeOH (8 mL) was added 3M aqueous NaOH (8 mL, 24 mmol) and the mixture was stirred overnight. To the reaction mixture was added 1M aqueous HCl until neutralization. The product was concentrated. To the resulting residue was added ethyl acetate and 1M aqueous HCl and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give S14 (1.7 g, 87%) as a white solid.

[0102] To a stirred solution of S14 (49 mg, 0.2 mmol), N,N-dimethylethylenediamine (43 μL, 0.4 mmol), and DCM (1 mL) were added WSCI (76 mg, 0.4 mmol), and HOBt (54 mg, 0.4 mmol) at 0° C., and the reaction mixture was warmed to room temperature and stirred for 3 h. To the resulting mixture were added DCM and aqueous NaHCO3. The product was extracted with DCM, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give the crude product S15. This crude mixture was used in the next reaction without further purification.

[0103] To a stirred solution of crude S15 and DCM (1 mL), BCl3 (240 μL of a 1 M solution in heptane, 0.24 mmol) was added and the mixture was stirred overnight. The reaction mixture was concentrated to give crude 6. The crude mixture was purified by preparative HPLC to give 6 (11 mg, 24%, 2 steps) as a white solid. 1 H NMR (500 MHz, CDCl3): δ 2.25 (6H, s), 2.44 (2H, t, J = 5.7), 3.39 (2H, t, J = 5.7), 6.67 (1H, d, J = 9.7), 7.58 (1H, d, J = 9.7), 7.88 (1H, s); MS (ESI): m / z 259 (calcd [M+H] + = 259.28).

[0104] (7) 5-(3-(dimethylamino)benzyl)-1-hydroxypyridin-2(1H)-one (compound 7) Compound 7 was synthesized according to the following synthetic scheme. TIFF0007640994000053.tif66149

[0105] To a stirred solution of S14 (490 mg, 2.4 mmol) and p-toluenethiol (298 mg, 2.4 mmol) in DCM (10 mL) was added WSCI (458 mg, 2.4 mmol) and HOBt (270 mg, 2 mmol) at 0° C., and the reaction mixture was warmed to room temperature and stirred for 3 h. The resulting mixture was concentrated. Ethyl acetate and aqueous NaHCO3 were added, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give crude product S16. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane=1:3) to give S16 (480 mg, 68%) as a white solid.

[0106] To a stirred solution of S16 (70 mg, 0.2 mmol) and THF (2 mL) was added 2-dimethylaminophenylboronic acid (99 mg, 0.6 mmol), Pd2(dba)3·CHCl3 (5 mg, 4 μmol), tri(2-furyl)phosphine (3.5 mg, 15 μmol), and CuTC (115 mg, 0.6 mmol). The reaction mixture was heated to reflux and stirred overnight. After filtration through Celite, the filtrate was concentrated to give S17. This crude mixture was used in the next reaction without further purification.

[0107] To a stirred solution of S17 and DCM (1 mL) was added BCl3 (690 μL, 0.7 mmol) and the mixture was stirred overnight. The reaction solution was concentrated to give the crude product 7. The crude mixture was purified by preparative HPLC to give 7 (6 mg, 12%, 2 steps) as a colorless oil. 1 H NMR (500 MHz, CDCl3): δ 3.28 (6H, s), 6.73 (1H, s), 7.28-7.43 (4H, m), 7.75 (1H, d, J = 8.0), 7.99 (1H, d, J = 8.0); MS (ESI): m / z 259 (calcd [M+H] += 259.28).

[0108] (8) Synthesis of compound 8 Compound 7 was synthesized according to the following synthetic scheme. TIFF0007640994000054.tif46149

[0109] To a mixture of S18 (4 g, 25.2 mmol), WSCI (7.2 g, 37.8 mmol), DMAP (4.6 g, 37.8 mmol) and DCM (80 mL), 3,7,11,15-tetramethylhexadecan-1-ol (10.8 mL, 30.2 mmol) and triethylamine (7.2 mL, 37.8 mmol) were added at room temperature, and the mixture was stirred at room temperature under argon for 25 h, and the solvent was removed under reduced pressure. Ethyl acetate and 1M aqueous HCl were added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium bicarbonate, washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to give crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 20:80 → 40:60) to give S19 (8.85 g, 80%).

[0110] Trifluoroacetic anhydride (5.7mL, 40.4mmol) was added dropwise to a mixture of S19 (8.85g, 20.2mmol), urea hydrogen peroxide (3.95g, 42.4mmol) and DCM (50mL) at 0℃, and the mixture was stirred at room temperature under argon for 16 hours. After cooling to 0℃, saturated aqueous sodium bisulfite solution (5.5mL) was added to the reaction solution, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium bicarbonate, washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to give the crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 10:90 → 100:0) to give S20 (7.51g, 82%).

[0111] To a stirred solution of S20 (7.51 g, 16.5 mmol) and acetonitrile (15 mL) was added TFAA (30 mL) at room temperature and stirred under argon atmosphere at room temperature for 14 h. The solvent was removed under reduced pressure, and solid NaHCO3 and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to give the crude product 8. The crude product was purified by neutral silica gel column chromatography (ethyl acetate / hexane = 40:60 → 100:0) to give a yellow liquid 8 (5.82 g, 81%). 1 H NMR (500 MHz, CD3OD): δ 0.83-0.89 (12H, m), 0.96 (3H, d, J = 6.3 Hz), 1.02-1.44 (20H, m), 1.45-1.66 (3H, m), 1.70-1.82 (1H, m), 4.24-4.40 (2H. m), 6.63 (1H, d, J = 9.2 Hz),7.92 (1H, dd, J = 2.3, 9.7 Hz), 8.52 (1H, d, J =2.9 Hz); MS (ESI): m / z 436.30 (calcd [M+H] + = 436.34).

[0112] (9) Synthesis of Compound 9 Compound 9 was synthesized according to the following synthetic scheme. TIFF0007640994000055.tif74144

[0113] This compound was prepared using the procedure described for compound 8 to give a yellow liquid 9 (1.53 g, 96%). 1H NMR (500 MHz, CDCl3): δ0.87 (6H, d, J = 6.3 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.08-1.20 (3H, m), 1.23-1.38 (3H, m), 1.44-1.64 (3H, m), 1.70-1.82 (1H, m), 4.28-4.38 (1H, m), 6.73 (1H, d, J =9.7 Hz), 7.96 (1H, dd, J =2.9, 9.7 Hz), 8.55 (1H, d, J =2.3 Hz); MS (ESI): m / z 296.20 (calcd [M+H] + = 296.19).

[0114] (10) Synthesis of Compound 10 Compound 10 was synthesized according to the following synthetic scheme. TIFF0007640994000056.tif74152

[0115] This compound was prepared using the procedure described for compound 8 to give a yellow solid 10 (205 mg, 92%). 1 H NMR (500 MHz, CD3OD): δ0.91 (3H, t, J =7.4 Hz), 1.30-1.38 (4H, m), 1.40-1.46 (2H, m),1.70-1.76 (2H, m), 4.26 (3H, t, J =6.9 Hz), 6.61 (1H, d, J = 9.2 Hz), 7.90-7.92 (1H, m), 8.48-8.54 (1H, m); MS (ESI): m / z 240.15 (calcd [M+H] + = 240.12).

[0116] (11) Synthesis of Compound 11 Compound 11 was synthesized according to the following synthetic scheme. TIFF0007640994000057.tif92145

[0117] This compound was prepared using the procedure described for compound 8 to give yellow liquid 11 (131 mg, 48%). 1 H NMR (500 MHz, CD3OD): δ0.93 (6H, t, J = 7.4 Hz), 1.60-1.75 (4H, m), 4.90-4.95 (1H. m), 6.63 (1H, d, J = 9.2 Hz), 7.93 (1H, dd, J = 2.3 Hz, 9.2 Hz), 8.53 (1H, d, J = 2.3 Hz); MS (ESI): m / z 226.40 (calcd [M+H] + =226.11).

[0118] (12) Synthesis of compound 12 Compound 12 was synthesized according to the following synthetic scheme. TIFF0007640994000058.tif33150

[0119] A mixture of S18 (632 mg, 4 mmol), Boc2O (1.31 g, 6 mmol), DMAP (48.9 mg, 0.4 mmol) and tetrahydrofuran (10 mL) was stirred under argon atmosphere with heating under reflux for 3 hours, and the solvent was removed under reduced pressure. Diethyl ether and water were added to the residue, and the mixture was extracted with diethyl ether. The organic layer was washed with saturated sodium bicarbonate water and saturated brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain crude product S27. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 5:95 → 30:70) to obtain S27 (622 mg, 73%).

[0120] Trifluoroacetic anhydride (374 μL, 2.66 mmol) was added dropwise to a mixture of S27 (285 mg, 1.33 mmol), urea hydrogen peroxide (263 mg, 2.80 mmol) and DCM (13.3 mL) at 0° C., and the mixture was stirred at room temperature under argon for 7 hours. The mixture was cooled to 0° C., and saturated aqueous sodium bisulfite and water were added to the reaction solution, and the reaction mixture was extracted with chloroform. The organic layer was washed twice with saturated aqueous sodium bicarbonate, washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the crude product S28. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane=20:90 → 100:0) to obtain S28 (280 mg, 92%).

[0121] To a stirred solution of S28 (280 mg, 1.22 mmol) and acetonitrile (12.2 mL), trifluoroacetic anhydride (344 μL, 2.44 mmol) was added at room temperature, and the reaction solution was stirred for 1 h 20 min under argon atmosphere. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the reaction solution was stirred for 14 h 30 min at room temperature. The mixture was stirred for 1 h 20 min. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the reaction solution was stirred for 2 h at room temperature. The solvent was removed under reduced pressure, and solid NaHCO3 and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to give crude product 12. The crude product was purified by neutral silica gel column chromatography (ethyl acetate / methanol = 100:0 → 85:15) to give white solid 12 (119 mg, 46%). 1 H NMR (500 MHz, CD3OD): δ1.56 (9H,s), 6.60 (1H, d, J = 9.2 Hz), 7.84--7.90 (1H, m), 8.43 (1H, d, J =2.3 Hz); MS (ESI): m / z 240.12 (calcd [M+H] + = 240.15).

[0122] [Synthesis Example 1] Condensation of dipeptide thioacids with various amino acids. Condensation of dipeptide thioacids with various amino acids was carried out according to the following reaction scheme. TIFF0007640994000059.tif60144H-AA-OH represents any amino acid.

[0123] N-hydroxypyridone additive, secondary phosphorous acid, and peptide thioacid were used as stock solutions. N-hydroxypyridone additive, secondary phosphorous acid, and amino acid were added to a 1.5 mL microtube. Then, a solution of peptide thioacid was added and stirred at 30°C for a given reaction time. The reaction mixture was diluted with 1% TFA in DMSO and analyzed by HPLC. The yield was determined by a calibration curve.

[0124] The results obtained (yield, epimerization degree) are shown in Table 1 below. [Table 1]

[0125] The characteristics of the peptide obtained above were examined, and the results are described below. Cbz-Phe-Val-Gly-OH MS (ESI): m / z 478.15 (calcd [M+Na] + = 478.50) Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.10 min Cbz-Phe-Val-Ala-OH MS (ESI): m / z 470.10 (calcd [M+H] + = 470.23) Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.69 min Cbz-Phe-Val-Val-OH MS (ESI): m / z 520.10 (calcd [M+Na] + = 520.24) Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.72 min Cbz-Phe-Val-Ile-OH MS (ESI): m / z 512.20 (calcd [M+H] + = 512.28) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.56 min Cbz-Phe-Val-Phe-OH MS (ESI): m / z 568.15 (calcd [M+Na] + = 568.24) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.21 min Cbz-Phe-Val-Met-OH MS (ESI): m / z 552.10 (calcd [M+Na] + = 552.21) Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.59 min Cbz-Phe-Val-Pro-OH MS (ESI): m / z 518.10 (calcd [M+Na] + = 518.23) Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.52 min Cbz-Phe-Val-Trp(Boc)-OH MS (ESI): m / z 707.20 (calcd [M+Na] + = 707.31) Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.68 min Cbz-Phe-Val-Lys(Boc)-OH MS (ESI): m / z 649.25 (calcd [M+Na] + = 649.32) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.58 min Cbz-Phe-Val-Tyr(O t Bu)-OH MS (ESI): m / z 584.40 (calcd [M+Na] + = 584.24) Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.46 min Cbz-Phe-Val-Cys(Trt)-OH MS (ESI): m / z 766.20 (calcd [M+Na] + = 766.29) Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.59 min Cbz-Phe-Val-Asp( t Bu)-OH MS (ESI): m / z 592.10 (calcd [M+Na] + = 592.64) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.25 min Cbz-Phe-Val-Asn(Trt)-OH MS (ESI): m / z 777.25 (calcd [M+Na] + = 777.87) Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.25 min Cbz-Phe-Val-Ser(O t Bu)-OH MS (ESI): m / z 564.20 (calcd [M+Na] + = 564.27) Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.08 min Cbz-Phe-Val-Thr(O tBu)-OH MS (ESI): m / z 500.10 (calcd [M+Na] + = 500.24) Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.47 min Cbz-Phe-Val-Arg(Pbf)-OH MS (ESI): m / z 807.05 (calcd [M+H] + = 807.37) Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.87 min Cbz-Phe-Val-His(Trt)-OH MS (ESI): m / z 778.20 (calcd [M+H] + = 778.93) Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.87 min Cbz-Phe-Val-tertLeu-OH MS (ESI): m / z 512.10 (calcd [M+H] + = 512.63) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.53 min Cbz-Phe-Val-Aib-OH MS (ESI): m / z 484.15 (calcd [M+H] + = 484.57) Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.31 min Cbz-Phe-Val-Pg-OH MS (ESI): m / z 532.15 (calcd [M+H] + = 532.62)

[0126] [Synthesis Example 2] Consideration of Protecting Groups The study was carried out using terminal Val dipeptides having Fmoc and Boc, which are commonly used in peptide synthesis. The results are shown below.

[0127] [Table 2]

[0128] The characteristics of the peptide obtained above were examined, and the results are described below. Fmoc-Phe-Val-Ala-OH MS (ESI): m / z 580.15 (calcd [M+Na] + = 580.64) Purity: >95% (HPLC analysis at 230 nm) Retention time 29.83 min Boc-Phe-Val-Ala-OH MS (ESI): m / z 458.15 (calcd [M+Na] + = 458.51) Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.62 min

[0129] As shown in Table 2, the reaction proceeded with high yields and low epimerization rates, similar to the case with Cbz.

[0130] [Synthesis Example 3] Examination of C-terminal residues An investigation was carried out using dipeptide thioacids whose C-terminal residue was other than Val. The results are shown below.

[0131] [Table 3]

[0132] The characteristics of the peptide obtained above were examined, and the results are described below. Cbz-Phe-Phe-Ala-OH MS (ESI): m / z 540.05 (calcd [M+Na] += 540.21) Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.56 min Cbz-Phe-Phe-Val-OH MS (ESI): m / z 568.10 (calcd [M+Na] + = 568.24) Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.28 min Cbz-Phe-Lys(Boc)-Ala-OH MS (ESI): m / z 621.20 (calcd [M+Na] + = 621.69) Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.95 min Cbz-Phe-Lys(Boc)-Val-OH MS (ESI): m / z 649.25 (calcd [M+Na] + = 649.74) Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.53 min Cbz-Phe-Cys(Trt)-Ala-OH MS (ESI): m / z 738.15 (calcd [M+Na] + = 738.85) Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.15 min Cbz-Phe-Cys(Trt)-Val-OH MS (ESI): m / z 766.10 (calcd [M+Na] + = 766.91) Purity: >95% (HPLC analysis at 230 nm) Retention time: 35.33 min

[0133] In both cases, when the hydrophobic amino acid Phe was used as the C-terminus, and when the hydrophilic amino acid Lys(Boc) was used, the reaction proceeded in high yields, and the epimerization rate was suppressed to less than 1%. On the other hand, when Cys(Trt), which is known to be prone to epimerization, was used, epimerization was observed at about 4% in coupling with Ala and about 2% in coupling with valine.

[0134] [Synthesis Example 4] Study of additives (epimerization inhibitors) (1) In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of additive was changed, and the results are summarized below.

[0135] TIFF0007640994000063.tif30148

[0136] TIFF0007640994000064.tif37151

[0137] The methyl ester moiety was replaced by an amide structure, and the basic pyridine moiety was used, probably because deprotonation was promoted at the active ester stage, and the reactivity of the active ester was also lower than that of the ester type, leading to poor results in both yield and epimerization rate. The chlorine-substituted product had a low nucleophilicity of the hydroxyl group, and the active ester formation did not occur at an appropriate rate, leading to a tendency for the yield to decrease.

[0138] [Synthesis Example 5] Study of additives (epimerization inhibitors) (2) In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of additive was changed, and the results are summarized below.

[0139] TIFF0007640994000065.tif32149

[0140] The results (yield, degree of epimerization) when the reaction time was set to 22 hours and various additives (epimerization inhibitors) were used are shown below. TIFF0007640994000066.tif50153

[0141] In the general formula (III), compound 5, in which R4 is an ester group and L is hydrogen, has a slightly higher epimerization degree than compound 1 and the like, but the product could be obtained in high yield.

[0142] TIFF0007640994000067.tif53131

[0143] The results (yield, epimerization rate) when the reaction time was set to 6 hours and various additives were used are shown below. TIFF0007640994000068.tif66160

[0144] The results (yield, epimerization degree) when H-Al-OH was 2 equivalents and the additive was 2 equivalents were used with various additives are shown below. TIFF0007640994000069.tif53139

[0145] TIFF0007640994000070.tif72162

[0146] The compounds in which the alkyl chain of the methyl ester portion was extended showed good yields and epimerization rates. However, compound 11, which has an alkyl chain of 6 carbon atoms in the ester portion, had a high degree of crystallinity and was difficult to separate from product 2ba. In contrast, compounds 12 and 13, which have alkyl chains with a larger number of carbon atoms than compound 11 but have branched alkyl chains, were able to recover 76% and 73%, respectively, of the additive by recrystallization.

[0147] [Synthesis Example 6] Condensation of dipeptide thioacids with various amino acids. Condensation of dipeptide thioacids with various amino acids was carried out according to the following reaction scheme. TIFF0007640994000071.tif42150

[0148] N-hydroxypyridone (78.4 mg, 0.18 mmol), dipeptide thioacid (50 mg, 0.12 mmol), and L-valine (28.1 mg, 0.24 mmol) were added to a test tube. Toluene (600 μL) and dimethyl sulfoxide (600 μL) were then added, and the reaction solution was stirred at 30° C. for 6 hours. The reaction solution (12 μL) was diluted with 1% trifluoroacetic acid in dimethyl sulfoxide (68 μL) and analyzed by HPLC. The yield and epimerism were determined by a calibration curve (yield <99%, epimerism <1%). The solvent of the remaining reaction solution was removed under reduced pressure, and ethyl acetate and 1M HCl aqueous solution were added to the residue, followed by extraction with ethyl acetate. The mixture was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain the crude tripeptide product. The crude product was purified by silica gel column chromatography (ethyl acetate / hexane=30:70→100:0) to give the tripeptide (50 mg, 84%).

[0149] [Synthesis Example 7] Examination of solubilizers In the following condensation reaction of Cbz-Phe-Val-SH with alanine, the type of solubilizer was changed, and the results are summarized below. TIFF0007640994000072.tif108132

[0150] Dialkyl phosphites gave good results, and the highest reaction acceleration effect was observed when diethyl phosphite was used. The yield increased from 59% without the addition to 84% at 6 hours from the start of the reaction. When dialkyl phosphites with functional groups more bulky than the ethoxy group were used, the yield decreased, suggesting that the steric properties of the phosphite contribute to the reactivity. In addition, the reactivity decreased when a phosphite with an alkyl group directly introduced on the phosphorus was used, suggesting that in addition to the steric effect, the electronic state also contributes to the reactivity. When diethyl thiophosphite was used, the system became complicated, probably due to the direct reaction of the thioacid and thiophosphite, and the yield of the tripeptide also decreased to 34%.

[0151] [Synthesis Example 8] General Method for Fragment Coupling Reactions N-hydroxypyridone additive 1 (12 μmol), diethyl phosphite (12 μmol), DIPEA (24 μmol) and peptide thioacid (12 μmmol) were used as stock solutions, respectively. To a 1.5 mL microtube, the peptide fragment (14.4 μmol) was added. Then, stock solutions of N-hydroxypyridone additive 1 (12 μmol in DMSO, 12 μL), diethyl phosphite (12 μmol in DMSO, 12 μL), and DIPEA (12 μmol in DMSO, 12 μL) were added to the microtube. Finally, a solution of peptide thioacid (12 μmol in DMSO, 48 μL) was added and stirred at 30 °C for the indicated reaction time. The reaction mixture was diluted with 1% TFA solution in DMSO (680 μL) and analyzed by HPLC. The yield was determined by HPLC based on a calibration curve.

[0152] [Synthesis Example 9] Sequential peptide synthesis (1) Cbz-Tyr(O t Bu)-OH -> Cbz-Tyr(O t Bu)-SH In a flame-dried test tube, Cbz-Tyr(O t Bu)-OH (74 mg, 0.2 mmol) and DMF (2 mL) were added under argon atmosphere. Then AcSK (114 mg, 1.0 mmol) and Ac2S (12 μL, 0.1 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layers were washed with 1 M aqueous HCl and brine, then dried over Na2SO4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

[0153] (2) Cbz-Tyr(O t Bu)-SH -> Cbz-Tyr(O t Bu)-Gly-OH In a test tube, Cbz-Tyr(O tBu)-Gly-SH and a mixed solvent of DMSO:toluene = 1:1 (2mL) were added. N-Hydroxypyridone additive 1 (34mg, 0.2mmol), Gly (18mg, 0.24mmol), and diethyl phosphite (26μL) were then added and stirred at 30°C for 6 hours. The reaction mixture was diluted to 1.5mM and analyzed by analytical HPLC, and the HPLC yield was 89%. Insoluble materials were then removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. Cbz-Tyr(O t The yield was 66 mg (77%) of (Bu)-Gly-OH. Cbz-Tyr(O t The properties of Bu)-Gly-OH were investigated and the results are as follows. MS (ESI): m / z 451.15 (calcd [M+Na] + = 451.47) Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min

[0154] (3) Cbz-Tyr(O t Bu)-Gly-OH -> Cbz-Tyr(O t Bu)-Gly-SH In a flame-dried test tube, Cbz-Tyr(O t Bu)-Gly-OH (85 mg, 0.2 mmol) and DMF (2 mL) were added under argon atmosphere. Then AcSK (114 mg, 1.0 mmol) and Ac2S (12 μL, 0.1 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layers were washed with 1 M aqueous HCl and brine, then dried over Na2SO4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

[0155] (4) Cbz-Tyr(O t Bu)-Gly-SH -> Cbz-Tyr(O t Bu)-Gly-Gly-OH In a test tube, Cbz-Tyr(Ot Bu)-Gly-SH and a mixed solvent of DMSO:toluene = 1:1 (2mL) were added. N-hydroxypyridone additive 1 (34mg, 0.2mmol), Gly (18mg, 0.24mmol), and diethyl phosphite (26μL) were then added and stirred at 30°C for 6 hours. The reaction mixture was diluted to 1.5mM and analyzed by analytical HPLC, and the HPLC yield was 94%. Insoluble materials were then removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. Cbz-Tyr(O t 71 mg (73%) of Bu)-Gly-Gly-OH was obtained. Cbz-Tyr(O t The properties of Bu)-Gly-Gly-OH were investigated and the results are as follows. MS (ESI): m / z 508.15 (calcd [M+Na] + = 508.21) Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min

[0156] (5) Cbz-Tyr(O t Bu)-Gly-Gly-OH -> Cbz-Tyr(O t Bu)-Gly-Gly-SH In a frame-dried test tube, Cbz-Tyr(O t Bu)-Gly-Gly-OH (49 mg, 0.1 mmol) and DMF (1 mL) were added under argon atmosphere. Then AcSK (34 mg, 0.3 mmol) and Ac2S (2.1 μL, 0.02 mmol) were added to the solution. The reaction mixture was stirred at 0° C. for 3 h, the mixture was extracted with ethyl acetate, and the combined organic layers were washed with 1 M aqueous HCl and brine, then dried over Na2SO4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

[0157] (6) Cbz-Tyr(O t Bu)-Gly-Gly-SH -> Cbz-Tyr(O t Bu)-Gly-Gly-Phe-OH In a test tube, Cbz-Tyr(O t Bu)-Gly-Gly-SH and a mixed solvent of DMSO:toluene = 1:1 (1mL) were added. N-Hydroxypyridone additive 1 (17mg, 0.1mmol), Phe (20mg, 0.12mmol), and diethyl phosphite (13μL) were then added and stirred at 30°C for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 87%. Insoluble materials were then removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. Cbz-Tyr(O t The compound was synthesized by the method of the present invention and synthesized by the method of the present invention. Cbz-Tyr(O t The properties of Bu)-Gly-Gly-Phe-OH were examined and the results are as follows. MS (ESI): m / z 655.25 (calcd [M+Na] + = 655.27) Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.39 min

[0158] (7) Cbz-Tyr(O t Bu)-Gly-Gly-Phe-OH -> Cbz-Tyr(O t Bu)-Gly-Gly-Phe-SH In a frame-dried test tube, Cbz-Tyr(O t Bu)-Gly-Gly-Phe-OH (13 mg, 20 μmol) and DMF (200 μL) were added under an argon atmosphere. Then AcSK (11 mg, 100 μmol) and Ac2S (1 μL, 10 μmol) were added to the solution. The reaction mixture was stirred at 0°C for 5 h, the mixture was extracted with ethyl acetate, and the combined organic layers were washed with 1M aqueous HCl and brine, then dried over Na2SO4. After filtering off the drying agent, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.

[0159] (8) Cbz-Tyr(O tBu)-Gly-Gly-Phe-SH -> Cbz-Tyr(O t Bu)-Gly-Gly-Phe-Leu-OH In a test tube, Cbz-Tyr(O t Bu)-Gly-Gly-Phe-SH and a mixed solvent of DMSO:toluene = 1:1 (200 μL) were added. N-hydroxypyridone additive 1 (4 mg, 20 μmol), Leu (4 mg, 24 μmol), and diethyl phosphite (13 μL) were then added and stirred at 30 °C for 6 hours. The reaction mixture was diluted to 1.5 mM and analyzed by analytical HPLC, and the HPLC yield was 54%. Insoluble materials were then removed with a syringe filter, and the reaction mixture was purified by preparative HPLC. Cbz-Tyr(O t The compound (7 mg, 47%) was obtained as a 3-aminopropyl phenylalanine salt of 1,2-dihydropyridine-2,4-trimethylphenylalanine (III)-Gly-Gly-Phe-Leu-OH. Cbz-Tyr(O t The properties of Bu)-Gly-Gly-Phe-LeuOH were investigated and the results are as follows. MS (ESI): m / z 768.25 (calcd [M+Na] + = 768.36) Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.00 min

Claims

1. A method for preparing a compound of formula (V) by reacting a compound of formula (I) below with an amino acid of formula (II) below in the presence of a compound of formula (III) below and a compound of formula (IV) below. (In the formula, teeth, represents a protecting group, or represents an amino acid or peptide whose N-terminus is protected with a protecting group; wherein the peptide has a structure in which 2 to 9 amino acid residues derived from the same or different amino acids selected from the group consisting of α-amino acids and β-amino acids are bound together; R １ is a side chain of an α-amino acid, and the side chain may be protected with a protecting group. (In the formula, R ２ is a side chain of an α-amino acid, and the side chain may be protected with a protecting group. (In the formula, L is an ester group (-CO ２ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH 2 CH 2 CH(CH 3 )CH 2 ) s -H (s is 1 to 4), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group), a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group), a dimethylaminoethylamide group (-CONHC 2 H 4 N(CH 3 ) 2 ), and an acyl group (-COR ａ '; R ａ R ′ is an alkyl group having 1 to 4 carbon atoms, ３ and R ４ At least one of the groups is an ester group (—CO ２ R ａ ;R ａ is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H); R ３ and R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a chlorine atom, or an ester group (—CO ２ R ａ ;R ａ represents an alkyl group having 1 to 4 carbon atoms. (In the formula, R ５ and R ６ each independently represents an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group, a benzyloxy group which may have a substituent, or a hydroxy group (provided that R ５ and R ６ and (b) cannot both be hydroxy groups. R ５ and R ６ Let's get together and ５ and R ６ may form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which is bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms). (In the formula, , R １ and R ２ is as defined above.)

2. The method according to claim 1, wherein a mixed solvent of DMSO and toluene is used as a reaction solvent.

3. 3. The method according to claim 1 or 2, comprising, prior to the coupling reaction, a step of preparing a compound represented by formula (I) by reacting a compound represented by formula (VI) below with a compound represented by formula (1) below. (In the formula, R １ is as defined in formula (I). (In the formula, R ７ is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group.

4. PG-(AA) ｎ -SH: (PG represents an N-terminal protecting group, AA represents any amino acid residue, which may be the same or different in each occurrence, some or all of the side chains of the amino acid residue may be protected with protecting groups; n is an integer from 1 to 4. A compound represented by the formula: H-(AA’) ｍ -OH: (AA' represents any amino acid residue, which may be the same or different in each occurrence; Some or all of the side chains of the amino acid residues may be protected with protecting groups; m is an integer from 1 to 4. An amino acid or peptide represented by In the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV), PG-(AA) ｎ (AA') ｍ A method for preparing a compound represented by -OH. (In the formula, L is an ester group (-CO ２ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH 2 CH 2 CH(CH 3 )CH 2 ) s -H (s is 1 to 4), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group), a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group), a dimethylaminoethylamide group (-CONHC 2 H 4 N(CH 3 ) 2 ), and an acyl group (-COR ａ '; R ａ R ′ is an alkyl group having 1 to 4 carbon atoms, ３ and R ４ At least one of the groups is an ester group (—CO ２ R ａ ;R ａ is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H); R ３ and R ４ each independently represents an alkyl group having 1 to 4 carbon atoms, or a chlorine or ester group (—CO ２ R ａ ;R ａ represents an alkyl group having 1 to 4 carbon atoms. (In the formula, R ５ and R ６ each independently represents an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group, a benzyloxy group which may have a substituent, or a hydroxy group (provided that R ５ and R ６ and (b) cannot both be hydroxy groups. R ５ and R ６ Let's get together and ５ and R ６ may form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which is bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms).

5. The method according to any one of claims 1 to 3, followed by (i) reacting a compound represented by the following formula (V) with a compound represented by the following formula (1) to prepare a compound represented by formula (VII): (In the formula, , R １ , R ２ is as defined in formulas (I) and (II). (In the formula, R ７ is an alkyl group having 1 to 4 carbon atoms or a substituted or unsubstituted aryl group. (ii) reacting a compound represented by formula (VII) with an amino acid represented by the following formula (VIII) in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV): A method for preparing a compound of formula (VIIII), comprising: (In the formula, R ３ａ is a side chain of an α-amino acid, and the side chain may be protected with a protecting group. (In the formula, L is an ester group (-CO ２ R; R is a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group of -(CH 2 CH 2 CH(CH 3 )CH 2 ) s -H (s is 1 to 4), a substituted or unsubstituted phenyl group (the substituent of the phenyl group is an alkyl group or an ester group), a substituted or unsubstituted benzoyl group (the substituent of the benzoyl group is an alkyl group or an amino group), a dimethylaminoethylamide group (-CONHC 2 H 4 N(CH 3 ) 2 ), and an acyl group (-COR ａ '; R ａ R ′ is an alkyl group having 1 to 4 carbon atoms, ３ and R ４ At least one of the groups is an ester group (—CO ２ R ａ ;R ａ is an alkyl group having 1 to 4 carbon atoms, L may be hydrogen (H); R ３ and R ４ each independently represents an alkyl group having 1 to 4 carbon atoms, chlorine, or an ester group (—CO ２ R ａ ;R ａ represents an alkyl group having 1 to 4 carbon atoms. (In the formula, R ５ and R ６ each independently represents an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group, a benzyloxy group which may have a substituent, or a hydroxy group (provided that R ５ and R ６ and (b) cannot both be hydroxy groups. R ５ and R ６ Let's get together and ５ and R ６ may form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which is bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms). (In the formula, , R １ ~R ２ , R ３ａ is as defined above.)

6. A method for preparing a compound of formula (V) as defined in claim 1, comprising using a compound of formula (IV) below as a solubilizing agent: (In the formula, R ５ and R ６ each independently represents an n-butyl group, a methoxy group, an ethoxy group, an i-propoxy group, an n-butoxy group, a benzyloxy group which may have a substituent, or a hydroxy group (provided that R ５ and R ６ and (b) cannot both be hydroxy groups. R ５ and R ６ Let's get together and ５ and R ６ may form a 5- or 6-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which is bonded (the substituent of the heterocyclyl is an alkyl group having 1 to 4 carbon atoms).

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