N-alkyl amino acid and method for producing peptides containing n-alkyl amino acid

Abstract

To provide a method for producing an N-monoalkyl amino acid or an ester thereof, or a peptide containing the N-monoalkyl amino acid, or an ester thereof.SOLUTION: Provided is a method for producing an N-monoalkyl amino acid or an ester thereof, or a peptide containing the N-monoalkyl amino acid, or an ester thereof, the method comprising an alkylation step of mixing in a solvent, in the presence of hydrogen, a starting amino acid or an ester thereof or a peptide comprising the starting amino acid or, an ester thereof; C1-C6 primary alkylating agent or substituted methyl halide, and a catalyst, the alkylation step being performed under a pressure of 1 atmosphere or more, and a primary alkyl group corresponding to the C1-C6 primary alkylating agent or substituted methyl halide being bonded to the amino group of the starting amino acid or an ester thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing N-alkylamino acids and peptides containing N-alkylamino acids. [Background technology]

[0002] In recent years, it has become known that compounds with a molecular weight of more than 500 can contribute to interactions on the surface of target proteins, which are difficult to interact with using conventional low molecular weight compounds, i.e., inhibition of protein-protein interactions. These molecules are called medium molecular weight compounds (molecular weight of 500 to 2000) and are distinguished from low molecular weight compounds (molecular weight of 500 or less) that have been used as oral drugs and high molecular weight compounds (molecular weight of more than 100,000) such as antibody drugs. Medium molecular weight compounds are attracting attention as a new modality that can realize drug discovery for tough targets. Among medium molecular weight compounds, more than 40 types of peptide drugs have already been marketed, and for example, cyclosporine is used as an immunosuppressant (Non-Patent Document 1).

[0003] It is known that many of the peptides that are the active ingredients of peptide drugs contain unnatural amino acids such as N-alkylamino acids in the molecule. In particular, peptides that contain N-alkylamino acids as unnatural amino acids are known to have improved metabolic stability and membrane permeability, which are necessary for active ingredients of drugs (Patent Documents 1 and 2).

[0004] In order to supply them as pharmaceuticals, it is essential to establish a chemical synthesis method that is efficient and suitable for large-scale synthesis. In the production of peptides containing unnatural amino acids in their sequences, particularly peptides containing N-alkylamino acids, the yield of the target product is reduced due to the low reactivity of the condensation reaction caused by the steric hindrance of the alkyl group on the nitrogen atom of the amino group, and the racemization of the α-position of the amino acid residue (Non-Patent Document 2). In addition, in the production of N-alkylamino acids or peptides containing N-alkylamino acid residues by alkylation of amino groups, a method is known in which an alkylaldehyde or an alkylnitrile is used as an alkylating agent to alkylate aliphatic primary amino groups in amino acids or peptide structures under a hydrogen atmosphere (Patent Document 3, Non-Patent Documents 3 and 4). In addition, a method is known in which an alkylnitrile is used as an alkylating agent to convert aliphatic primary amino groups to secondary amino groups by selective alkylation under a hydrogen atmosphere (Non-Patent Documents 5 and 6). As an N-alkylation method for peptides, for example, a method is known in which a primary amine protected with a 2-nitrobenzenesulfonyl group is N-alkylated with an alkylating agent such as dialkyl sulfate, and then deprotected (Non-Patent Document 7). A synthetic method in which a peptide is N-alkylated using a borohydride reagent as a reducing agent is also known (Non-Patent Document 8). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 225864 [Patent Document 2] International Publication No. 2020 / 122182 [Patent Document 3] International Publication No. 2000 / 015656 [Non-patent literature]

[0006] [Non-Patent Document 1] Future Med. Chem., 2009, 1,1289-1310. [Non-Patent Document 2] J. Peptide Res., 2005, 65,153-166. [Non-Patent Document 3] J. Med. Chem. 1992, 35,4195-4204. [Non-Patent Document 4] J. Org. Chem. 1984, 49,5269-5271. [Non-Patent Document 5] Org. Lett. 2004, 6, 4977-4980. [Non-Patent Document 6] Org. Biomol. Chem. 2012, 10,293-304. [Non-Patent Document 7] Acc. Chem. Res. 2008, 41,1331-1342. [Non-Patent Document 8] Chem. Sci. 2017, 8, 2717-2722. Summary of the Invention [Problem to be solved by the invention]

[0007] In order to efficiently N-alkylate the amino group at the N-terminus of amino acids and peptides, a synthetic method capable of mono-alkylating aliphatic primary amino groups contained in the structures of amino acids and peptides with high selectivity and converting them to secondary amino groups is required. In addition, the production of peptides containing N-alkylamino acids requires multiple steps, including deprotection of the amino group at the N-terminus, N-alkylation of the amino group at the N-terminus, and then elongation of the amino acid. Therefore, an object of the present invention is to provide an efficient method for producing N-monoalkylamino acids or peptides containing N-monoalkylamino acid residues.

[0008] In the conventional N-alkylation method, not only secondary amines in which the primary amino group of the substrate is monoalkylated, but also many dialkylated tertiary amines are produced as by-products, which may be difficult to separate. In general, when the bulkiness of the substrate amino group and the alkyl group to be introduced are both small, it is difficult to increase the selectivity of monoalkylation over dialkylation. Non-Patent Documents 3-6 and Patent Document 3 refer to a method of introducing a sterically hindered alkyl group to the amino group of a substrate with a steric hindrance, but do not refer to highly selective monoalkylation. In fact, when a method of selectively converting an aliphatic primary amine to a secondary amine under a hydrogen atmosphere using an alkylnitrile as an alkylating agent according to the disclosures of Non-Patent Documents 5 and 6 was applied to the production of peptides, the reactivity was low due to the influence of the steric hindrance of the amino group, and it was difficult to efficiently obtain the target product.

[0009] On the other hand, in the liquid-phase peptide production, amino acids whose N-terminal amino group is protected with a benzyloxycarbonyl group (also called a Cbz group) are widely used. When such protected amino acids are used to perform an elongation reaction of a peptide chain having a sequence containing an N-alkylamino acid, the reaction conditions such as the solvent and the liquidity of the reaction solution may differ in each of the steps of removing the Cbz group, alkylating the N-terminal amino group, and elongating the peptide chain. Furthermore, multiple work steps are required, including post-treatment operations in each step and isolation of the target product, and the process is complicated. The protecting group used in Non-Patent Document 7 and the alkylation reaction in the next step are not practical methods because they require three steps, namely, introduction of a specific protecting group into the N-terminal amino group, alkylation, and deprotection. In the method described in Non-Patent Document 8, which uses a boron hydride reagent as a reducing agent, only the production of an N-substituted peptide using benzaldehyde, which has a large steric hindrance, is exemplified. In addition, hydride reagents, such as boron hydride reagents, require an equal or greater amount of reagent to the substrate compound, and the post-treatment process for the reaction solution containing the excess reagent is complicated. In addition, when these methods are applied to peptide synthesis, the reaction for removing the protecting group at the N-terminus of the peptide and the reaction for alkylating the amino group at the N-terminus are different, and therefore must be carried out as separate steps, and it is difficult to carry out the deprotection reaction and the N-alkylation reaction in one pot. [Means for solving the problem]

[0010] The present invention relates to a method for producing N-alkylamino acids and peptides containing N-alkylamino acids. The inventors aimed to establish an efficient method for producing N-alkylated amino acids or peptides that does not depend on the bulkiness of the substrate or the alkyl group to be introduced, and investigated the conditions for the N-alkylation reaction, focusing on the alkylating agent, reducing agent, catalyst, and additive. They investigated alkylation using an alkylnitrile or alkylaldehyde as an alkylating agent, under a hydrogen atmosphere at normal pressure (1 atm) or higher, in the presence of a transition metal catalyst, and adding an organic base as an additive, and found that the method is applicable to the N-alkylation reaction of peptides in addition to the N-alkylation reaction of amino acids. When an amino acid or peptide having a protecting group at the N-terminus that can be removed under hydrogenolysis conditions was subjected to the conditions of the present invention, they found that the deprotection reaction and the N-alkylation reaction could be carried out in one pot. They found that when the reaction is carried out in one pot, the desired N-alkylated product can be obtained efficiently by adding an acid as an additive.

[0011] For example, the present invention provides the following (1) to (35).

[0012] (1) A method for producing an N-monoalkylamino acid or an ester thereof, or a peptide containing an N-monoalkylamino acid or an ester thereof, comprising the steps of: The method includes an alkylation step of mixing a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, and a catalyst in a solvent in the presence of hydrogen; A method in which the alkylation step is carried out under a pressure of 1 atmosphere or more, and an N-monoalkylamino acid or an ester thereof, or a peptide or an ester thereof containing said N-monoalkylamino acid, in which a primary alkyl group corresponding to a C1-C6 primary alkylating agent or a substituted methyl halide is bound to the amino group of the starting amino acid or an ester thereof, is produced. (1.1) A method for producing an N-monoalkylamino acid or an ester thereof, or a peptide containing an N-monoalkylamino acid or an ester thereof, comprising the steps of: The alkylation step comprises mixing a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, a hydride reducing agent and a catalyst in a solvent; A method in which the alkylation step produces an N-monoalkylamino acid or an ester thereof in which a primary alkyl group corresponding to a C1-C6 primary alkylating agent or a substituted methyl halide is bonded to the amino group of the starting amino acid or an ester thereof, or a peptide containing said N-monoalkylamino acid or an ester thereof. (1.2) The method according to (1) or (1.1), wherein the C1-C6 primary alkylating agent or substituted methyl halide is a C1-C6 primary alkylating agent. (2) The method according to any one of (1) to (1.2), wherein the C1-C6 primary alkylating agent is a C1-C5 alkylnitrile or a C1-C5 alkylaldehyde. (2.1) The method according to (2), wherein the substituted methyl halide is one selected from the group consisting of methoxymethyl chloride (MOM-Cl), ethoxymethyl chloride (EOM-Cl), 2-methoxyethoxymethyl chloride (MEM-Cl), and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl). (2.2) The method according to any one of (1.1) to (2.1), wherein the hydride reducing agent is a trialkylsilane. (2.3) The method according to (2.2), wherein the trialkylsilane is triethylsilane. (3) The method according to any one of (1) to (2.2), wherein the catalyst is a heterogeneous hydrogenation catalyst containing a transition metal. (4) The method according to (3), wherein the heterogeneous hydrogenation catalyst is a catalyst containing a transition metal selected from the group consisting of Pd, Rh and Pt. (5) The method according to (3) or (4), wherein the heterogeneous hydrogenation catalyst is a catalyst selected from the group consisting of Pd-C, Pd(OH)2-C, Rh-C, and Adams' catalyst. (6) The method according to any one of (1) to (5), wherein the solvent comprises at least one solvent selected from the group consisting of ether-based solvents, alcohol-based solvents, and ester-based solvents. (7) The method according to any one of (1) to (6), wherein the solvent is selected from the group consisting of ether solvents, alcohol solvents, ester solvents, and combinations thereof. (8) The method according to any one of (1) to (7), wherein the solvent is an ether solvent selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, dimethyl ether, methyl t-butyl ether, cyclopentyl methyl ether, diisopropyl ether, 4-methyltetrahydropyran, dioxane, and diethyl ether. (9) The method according to any one of (1) to (7), wherein the solvent is an alcohol solvent selected from the group consisting of methanol, ethanol, propanol, butanol and pentanol. (10) The method according to any one of (1) to (7), wherein the solvent is an ester solvent selected from the group consisting of ethyl acetate, propyl acetate, and butyl acetate. (11) The method according to any one of (1) to (10), further comprising a step of contacting the reaction mixture in the alkylation step with additional hydrogen. (12) The method according to any one of (1) to (11), wherein the amino group of the starting amino acid or an ester thereof, or the peptide containing the starting amino acid or an ester thereof is bound to a protecting group removable under hydrogenolysis conditions. (12.1) The method according to (12), comprising a step of removing the protecting group (removal of the protecting group). (13) The method according to (12) or (12.1), wherein the protecting group is an arylmethyloxycarbonyl group. (14) The method according to (12) or (13), wherein the removal of the protecting group is carried out in the presence of an additive selected from the group consisting of p-toluenesulfonic acid, methanesulfonic acid, sodium hydrogen sulfate, triethylamine hydrochloride and propylphosphonic acid. (14.1) The method according to any one of (12) to (13), wherein the reaction mixture in the alkylation step further contains a base. (14.2) The method according to any one of (12) to (14.1), wherein the removal of the protecting group and the alkylation step are carried out in one pot. (15) The amino group of the starting amino acid or its ester is a primary amino group; The method according to any one of (1) to (11), wherein the reaction mixture in the alkylation step further contains a base. (16) The method according to (14.1) or (15), wherein the base is a tertiary amine. (17) The method according to (16), wherein the tertiary amine is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undecene-7, 1,5-diazabicyclo[4.3.0]nonene-5, N-methylmorpholine, 1,4-diazabicyclo[2.2.2]octane, triethylamine, N,N-diisopropylethylamine, pyridine and collidine. (18) A method for producing a peptide or an ester thereof, comprising the steps of: (a) obtaining an N-monoalkylamino acid or an ester thereof, or a peptide containing an N-monoalkylamino acid or an ester thereof according to any one of the methods described in (1) to (17); and (b) optionally extending one or more amino acids or peptides to the N-monoalkyl amino acid or its ester, or a peptide or its ester containing the N-monoalkyl amino acid, by a bond-forming reaction to obtain a peptide or its ester. (18.1) A method for producing the peptide or ester thereof according to (18), comprising repeating steps (a) and (b) multiple times until the desired peptide is obtained. (18.2) A method for producing a peptide having a cyclic portion composed of at least four amino acids or an ester thereof, comprising the steps of: (c) obtaining a peptide or an ester thereof according to the method of (18) or (18.1); and (d) cyclizing the C-terminal group and the N-terminal group of the peptide or ester thereof to form the cyclic moiety. (18.3) The method according to (18.2), wherein the peptide or its ester in step (d) is a linear peptide or its ester. (18.4) The linear peptide or ester thereof has the following formula: [ka] or a salt or solvate thereof. (19) The method according to (18.2) or (18.3), wherein the step of forming a cyclic portion is carried out by forming a bond between the C-terminal carboxyl group and the N-terminal amino group of the peptide or ester. (20) The cyclic portion is composed of at least 8 amino acids, The method according to any one of (18.2) to (19), wherein the peptide having a cyclic portion or an ester thereof is a peptide consisting of 8 to 15 amino acids or an ester thereof. (20.1) A method for producing a peptide or an ester thereof containing 5 or more, 6 or more, or 7 or more N-alkylamino acid residues, the method comprising the method according to any one of (18) to (20). (20.2) The peptide having a cyclic portion or an ester thereof has the following formula (1): [ka] or a salt thereof, or a solvate thereof. (20.3) The method according to (20.2), wherein the peptide having a cyclic portion, or a salt thereof, or a solvate thereof is a solvate of a peptide having a cyclic portion represented by formula (1). (20.4) The method according to (20.2), wherein the peptide having a cyclic portion, or a salt thereof, or a solvate thereof is a hydrate of a peptide having a cyclic portion represented by formula (1). (20.5) The method according to (20.2), wherein the peptide having a cyclic portion, or a salt thereof, or a solvate thereof, is a peptide having a cyclic portion represented by formula (1). (20.6) The method according to any one of (18) to (20.5), wherein column chromatography is not used for isolation and / or purification of the peptide having a cyclic portion, or a salt thereof, or a solvate thereof. (20.7) The method according to any one of (18) to (20.6), further comprising a step of isolating and / or purifying the peptide having a cyclic portion, or a salt thereof, or a solvate thereof by crystallization to obtain a crystal of the peptide having a cyclic portion, or a salt thereof, or a solvate thereof. (20.8) The crystal of the peptide having a cyclic portion, or a salt thereof, or a solvate thereof, is represented by the following formula (1): [ka] The method according to (20.7), wherein the crystal is a non-solvated or solvated crystal of a peptide having a cyclic portion represented by the formula: (20.9) The method according to (20.8), wherein the crystal of the peptide having the cyclic portion is a solvate crystal. (20.10) The method according to (20.9), wherein the peptide solvate crystal having a cyclic portion is a hydrate crystal. (21) A method for suppressing production of a compound in which an amino group of a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof is dialkylated in an N-monoalkylation reaction of a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, comprising the steps of: The method includes an alkylation step of mixing a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, and a catalyst in a solvent in the presence of hydrogen; The alkylation step is carried out under a pressure of 1 atmosphere or more, and produces an N-monoalkylamino acid or its ester, or a peptide containing an N-monoalkylamino acid or its ester, in which a primary alkyl group corresponding to a C1-C6 primary alkylating agent or a substituted methyl halide is bonded to the amino group of the starting amino acid or its ester. (21.1) A method for suppressing the production of a compound in which an amino group of a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof is dialkylated in an N-monoalkylation reaction of a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, comprising the steps of: The alkylation step comprises mixing a starting amino acid or an ester thereof, or a peptide containing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, a hydride reducing agent and a catalyst in a solvent; A method in which the alkylation step produces an N-monoalkylamino acid or an ester thereof in which a primary alkyl group corresponding to a C1-C6 primary alkylating agent or a substituted methyl halide is bonded to the amino group of the starting amino acid or an ester thereof, or a peptide containing said N-monoalkylamino acid or an ester thereof. (21.2) The method according to (21) or (21.1), wherein the C1-C6 primary alkylating agent or substituted methyl halide is a C1-C6 primary alkylating agent. (22) extending a peptide chain of an N-monoalkylamino acid or an ester thereof, or a peptide containing an N-monoalkylamino acid or an ester thereof, by a bond forming reaction; The method according to any one of (21) to (21.2), further comprising the step of treating the extended peptide with an acidic aqueous solution to remove dialkylated compounds. (23) The method according to any one of (1) to (22), wherein the N-monoalkylamino acid or its ester is a compound represented by formula C. [ka] [In the formula, R1 represents the side chain of an amino acid, and R2 represents a hydrogen atom or a C1-C6 alkyl group.] (24) The method according to any one of (1) to (11) and (15) to (23), wherein the starting amino acid or an ester thereof is a compound represented by formula A. [ka] [In the formula, R1 represents the side chain of an amino acid, and R2 represents a hydrogen atom or a C1-C6 alkyl group.] (25) The method according to any one of (1) to (14) and (18) to (23), wherein the starting amino acid or an ester thereof is a compound represented by formula B. [ka] [In the formula, PG1 is a protecting group for an amino group, R1 represents the side chain of an amino acid, and R2 represents a hydrogen atom or a C1-C6 alkyl group.] (26) The method according to any one of (1) to (22), wherein the peptide containing an N-monoalkylamino acid or an ester thereof is a compound represented by formula F. [ka] [In the formula, R3 represents a side chain of an amino acid residue, and R4 represents a peptide residue.] (27) The method according to any one of (1) to (11), (15) to (22), and (26), wherein the peptide containing the starting amino acid (hereinafter also referred to as the "starting peptide") or an ester thereof is a compound represented by formula D. [ka] [In the formula, R3 represents a side chain of an amino acid residue, and R4 represents a peptide residue.] (28) The method according to any one of (1) to (14), (18) to (22), (26) and (27), wherein the peptide containing the starting amino acid is a compound represented by formula E. [ka] [In the formula, PG2 is a protecting group for an amino group, R3 represents a side chain of an amino acid residue, and R4 represents a peptide residue.] (29) The method according to any one of (21) to (26), wherein the C1-C6 alkyl group for R2 is a group that is not removed under hydrogenolysis conditions. (30) The method according to any one of (23) to (25), wherein the C1-C6 alkyl group of R2 is selected from a t-butyl group, an n-butyl group, a 1-methylpropyl group, a 2-methylpropyl group, an n-propyl group, an isopropylmethyl group, and an ethyl group. (31) The method according to any one of (25) and (28) to (30), wherein PG1 and PG2 are protecting groups removable under hydrogenolysis conditions. (32) The method according to any one of (25) and (28) to (31), wherein PG1 and PG2 are protecting groups selected from the group consisting of a benzyloxycarbonyl group, a benzyloxymethyl group, and a benzyl group. (33) The method according to any one of (23) to (32), wherein R1 and R3 are not groups that may undergo unintended structural transformation depending on the conditions of the alkylation step. (34) R1 and R3 are each independently a hydrogen atom, a C1-C6 alkyl group, a halo C1-C6 alkyl group, a C3-C6 cycloalkyl group, a C3-C6 cycloalkyl C1-C6 alkyl group, a carboxy C1-C6 alkyl group, a C6-C alkyl group optionally having a substituent on the aryl group, 10The method according to any one of (21) to (31), wherein the alkyl group is selected from an aryl C1-C6 alkyl group, a 5- to 10-membered heteroaryl C1-C6 alkyl group optionally having a substituent on the heteroaryl group, a 5- to 10-membered heterocyclyl C1-C6 alkyl group optionally having a substituent on the heterocyclyl group, a C3-C6 cycloalkoxy C1-C6 alkyl group, a halo C1-C6 alkoxy C1-C6 alkyl group, a protected amino C3-C6 alkyl group, a protected hydroxy C1-C6 alkyl group, or a C1-C6 alkoxy C1-C6 alkyl group. (35) R1 and R3 are each independently a hydrogen atom, a C1-C6 alkyl group, or a C6-C aryl group optionally having a substituent on the aryl group. 10 The method according to any one of (23) to (34), wherein the alkyl group is selected from an aryl C1-C6 alkyl group. Effect of the Invention

[0013] The present invention makes it possible to produce N-alkylamino acids and peptides containing N-alkylamino acids by selective N-alkylation reaction. Furthermore, the production method according to one embodiment of the present invention can reduce costs by simplifying the reaction procedures including purification procedures and shortening the process by a one-pot reaction, compared with existing production methods, and can supply active ingredients as pharmaceuticals and their intermediates in large quantities at low cost.

[0014] In particular, it has been found that the N-alkylation reaction can be applied to the introduction reaction of a primary alkyl group. Furthermore, whereas the target product was conventionally obtained through two steps, namely, the removal reaction of the N-terminal protecting group and the subsequent alkylation reaction, according to one embodiment of the present invention, the desired N-monoalkylated product can be obtained in one pot. Furthermore, in the one-pot reaction of the present invention, the generation of diketopiperazine in the deprotection step can be further suppressed by adding an organic acid, and the target product can be obtained more efficiently. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0016] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of a substituent of a group, the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0017] In this specification, the term "to" indicating a range includes both ends of the range. For example, "A to B" means a range that is equal to or greater than A and equal to or less than B.

[0018] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus or minus 10% of that numerical value.

[0019] In this specification, the meaning of the term "and / or" includes any combination of "and" and "or" appropriately combined. Specifically, for example, "A, B and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B and C.

[0020] As used herein, the term "amino acid" includes natural amino acids and non-natural amino acids (sometimes referred to as amino acid derivatives). As used herein, the term "amino acid" may refer to amino acid residues. As used herein, the term "natural amino acid" refers to glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine ​​(Cys), methionine (Met), lysine (Lys), arginine (Arg), and proline (Pro). Examples of non-natural amino acids (amino acid derivatives) include, but are not limited to, β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains are different from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids may have any configuration. There is no particular restriction on the selection of the side chain of an amino acid, and in addition to a hydrogen atom, the side chain may be freely selected from, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, and a spiro-linked cycloalkyl group. Each of these may have a substituent, and the substituents are not limited, and may be independently selected from any substituents including, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. That is, examples of the side chain include an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, and the like, which may be substituted, or an oxo, an aminocarbonyl, a halogen atom, and the like. In a non-limiting embodiment, the amino acid herein 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 amino acids).

[0021] In this specification, the "amino acid residues" that constitute a peptide may be simply referred to as "amino acids".

[0022] As used herein, the term "side chain of an amino acid" refers to, in the case of an α-amino acid, an atomic group attached to the carbon (α-carbon) to which the amino group and the carboxyl group are attached. For example, the methyl group of Ala is the side chain of an amino acid. In the case of a β-amino acid, the atomic group attached to the α-carbon and / or the β-carbon can be the side chain of the amino acid, and in the case of a γ-amino acid, the atomic group attached to the α-carbon, the β-carbon, and / or the γ-carbon can be the side chain of the amino acid.

[0023] In the present specification, examples of the "halogen" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0024] In this specification, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is an atom in which at least one atom is replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons), in an abundance ratio different from the natural abundance ratio. Examples of isotopes included in the "amino acids" in this specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Cl, etc. Compounds herein containing all proportions of radioactive or non-radioactive isotopes are within the scope of the present invention.

[0025] The compounds described herein may contain unnatural proportions of isotope atoms in one or more atoms constituting such compounds. The present invention also includes compounds in which any atom in a compound is replaced with another isotope atom having the same atomic number (number of protons) and a different mass number (sum of the number of protons and neutrons), thereby replacing the isotopes with an abundance ratio different from that of natural isotopes, i.e., compounds labeled with isotope atoms. Examples of isotope elements contained in the compounds of the present specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Cl, etc. Compounds labeled with isotope atoms are useful as therapeutic or preventive agents, research reagents (e.g., assay reagents), and diagnostic agents (e.g., in vivo imaging diagnostic agents). Compounds herein containing radioactive or non-radioactive isotopes in any proportion are within the scope of the present invention. Compounds labeled with isotope atoms can be produced by using reagents and solvents containing the corresponding isotope atoms in the same manner as in the production method of non-labeled compounds.

[0026] As used herein, "N-protected amino acid" refers to a natural or non-natural amino acid whose amino group is protected, and "N-protected peptide" refers to a peptide whose N-terminal amino acid residue has its amino group protected. The peptide may be composed of only natural amino acid residues, only non-natural amino acid residues, or any combination of natural and non-natural amino acid residues.

[0027] The term "peptide" as used herein is not particularly limited as long as it is a compound in which two or more natural amino acids and / or non-natural amino acids are linked. The bonds between amino acid residues may be, for example, only amide bonds, or some of the bonds may be amide bonds and the rest may be bonds other than amide bonds, such as ester bonds, ether bonds, thioether bonds, sulfoxide bonds (-S(=O)-), sulfone bonds (-S(=O)2-), disulfide bonds, carbon-carbon bonds, or bonds formed by heterocyclic ring construction. Examples of groups involved in the bonds between amino acid residues include groups between the main chains of amino acids, groups between the main chains and side chains of amino acids, and groups between the side chains of amino acids, and the "peptide" as used herein also includes those groups bonded according to the examples of the bond modes of the amino acid residues. In the present specification, a chain-like "peptide" in which these amino acids are linked is also referred to as a "peptide chain". The number of amino acid residues contained in the "peptide" is preferably 5 to 30 residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. The peptide synthesized in the present invention preferably contains at least three N-substituted amino acids, more preferably at least five or more N-substituted amino acids in one peptide. These N-substituted amino acids may be present consecutively or discontinuously in the peptide. The peptide in the present invention may be linear or cyclic, and a cyclic peptide is preferred.

[0028] As used herein, the terms "peptide main chain" and "cyclic peptide main chain" refer to a structure formed by linking a plurality of the above-mentioned "amino acid main chains" via amide bonds.

[0029] The compounds described herein (including peptides) can be salts or solvates thereof. Examples of salts of compounds include hydrochloride, hydrobromide, hydroiodide, phosphate, phosphonate, sulfate, sulfonate such as methanesulfonate and p-toluenesulfonate, carboxylate such as acetate, citrate, malate, tartrate, succinate, salicylate, alkali metal salt such as sodium salt and potassium salt, alkaline earth metal salt such as magnesium salt and calcium salt, ammonium salt such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, tetraalkylammonium salt, and the like. These salts are produced, for example, by contacting the compound with an acid or base. In the present specification, the solvate refers to a compound that forms a molecular group together with a solvent, and is not particularly limited as long as it is a solvate formed with a solvent that can be ingested in association with the administration of a medicine. Examples of the solvate include solvates with a single solvent such as hydrates, alcohol solvates (ethanol solvates, methanol solvates, 1-propanol solvates, 2-propanol solvates, etc.), and dimethylsulfoxide, as well as solvates with multiple solvents per molecule of the compound, or solvates with multiple types of solvents per molecule of the compound. If the solvent is water, it is called a hydrate. As the solvate of the compound of the present invention, hydrates are preferred, and specific examples of such hydrates include monohydrates to 10 hydrates, preferably monohydrates to 5 hydrates, and more preferably monohydrates to 3 hydrates. The solvates of the compound of the present invention include solvates with a single solvent such as water, alcohol (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), and dimethylformamide, as well as solvates with multiple solvents.

[0030] When the compound according to the present invention is obtained as a free form, the compound can be converted into its hydrate or solvate according to a conventional method.In addition, when the compound according to the present invention is obtained as a free form, the compound can be converted into the salt that the compound may form, or into its hydrate or solvate according to a conventional method.For example, the compound represented by formula (1) or its salt hydrate, ethanol solvate, etc. can be mentioned. Specifically, the solvate may be a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nodahydrate, decahydrate, or monoethanolate of the compound represented by formula (1), or a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nodahydrate, decahydrate, or monoethanolate of the sodium salt of the compound represented by formula (1), or a hydrate or ethanolate of the hydrochloride of the compound represented by formula (1), but is not limited thereto. The hydrate or solvate may be produced in a crystalline or non-crystalline form, and in the case of a crystalline form, it may be a crystalline polymorph. As a method for producing a hydrate or solvate, for example, a solvent such as ethanol and / or water can be added to a compound represented by formula (1) or a peptide compound described in the present specification, and the hydrate or solvate can be obtained by a conventional method, such as stirring, cooling, concentrating, and / or drying. [ka]

[0031] Furthermore, when the compound according to the present invention is obtained as a salt, hydrate, or solvate of the compound, the compound can be converted into its free form in a conventional manner.

[0032] In the production of the compounds described herein, when the defined groups undergo undesired chemical conversion under the conditions of the method, the compounds can be produced by using, for example, means for protecting and deprotecting functional groups. The selection and deprotection of the protecting groups can be carried out, for example, by the methods described in "Greene's, "Protective Groups in Organic Synthesis" (5th Edition, John Wiley & Sons 2014), which can be used appropriately depending on the reaction conditions. In addition, the order of reaction steps such as introducing substituents can be changed as necessary.

[0033] In one aspect, the first embodiment of the present invention is a method for producing an N-monoalkylamino acid or an ester thereof, comprising an alkylation step of mixing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, and a catalyst in a solvent in the presence of hydrogen, the alkylation step being carried out under a pressure of 1 atmosphere or more, and producing an N-monoalkylamino acid or an ester thereof in which a primary alkyl group corresponding to the C1-C6 primary alkylating agent or substituted methyl halide is bonded to the amino group of the starting amino acid or ester thereof.

[0034] Another first embodiment of the present invention, in one aspect, is a method for producing an N-monoalkylamino acid or an ester thereof, comprising an alkylation step of mixing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, a hydride reducing agent and a catalyst in a solvent, and the alkylation step produces an N-monoalkylamino acid or an ester thereof in which a primary alkyl group corresponding to the C1-C6 primary alkylating agent or the substituted methyl halide is bonded to the amino group of the starting amino acid or ester thereof, or a peptide containing the N-monoalkylamino acid or an ester thereof.

[0035] According to the method of this embodiment, an N-monoalkylamino acid or an ester, salt or solvate thereof can be efficiently obtained.

[0036] The alkylation step according to this embodiment includes a method of obtaining an N-monoalkylamino acid represented by formula C or its ester by selectively N-monoalkylating an amino acid represented by formula A or its ester (also referred to as starting amino acid A), and a method of obtaining an N-monoalkylamino acid represented by formula C or its ester by carrying out a deprotection reaction and an N-alkylation reaction of an N-protected amino acid represented by formula B or its ester (also referred to as starting amino acid B) in one pot. An outline of the method according to this embodiment is shown below. [ka]

[0037] The starting amino acids A and B may be used in the form of their free forms or in the form of the corresponding salts or solvates.

[0038] In the above chemical formula, R1 represents the side chain of an amino acid, PG1 represents a protecting group of an amino group, and R2 represents a hydrogen atom or a protecting group of a carboxyl group. The protecting group of a carboxyl group is, for example, a C1-C6 alkyl group. For convenience, in the above reaction formula, the starting amino acids A and B are exemplified in the form of α-amino acids, but they may be β-amino acids or γ-amino acids. In addition, in the above reaction formula, the side chain R1 of the amino acid preferably does not have a functional group that can undergo unintended structural transformation due to an alkylation reaction or a reduction reaction depending on the conditions of the alkylation step. When R1 has a functional group that can undergo unintended structural transformation depending on the conditions of the alkylation step, the target product can be produced by introducing a protecting group into the functional group beforehand and then carrying out the alkylation step.

[0039] R1 is, for example, a hydrogen atom, a C1-C6 alkyl group, a halo C1-C6 alkyl group, a C3-C6 cycloalkyl group, a C3-C6 cycloalkyl C1-C6 alkyl group, a carboxy C1-C6 alkyl group, a C6-C alkyl group optionally having a substituent on the aryl. 10It is selected from an aryl C1-C6 alkyl group, a 5-10 membered heteroaryl C1-C6 alkyl group optionally having a substituent on the heteroaryl, a 5-10 membered heterocyclyl C1-C6 alkyl group optionally having a substituent on the heterocyclyl, a C3-C6 cycloalkoxy C1-C6 alkyl group, a halo C1-C6 alkoxy C1-C6 alkyl group, a protected amino C3-C6 alkyl group, a protected hydroxy C1-C6 alkyl group, or a C1-C6 alkoxy C1-C6 alkyl group.

[0040] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only straight-chain but also branched-chain alkyls. Specific examples of C1-C6 alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl.

[0041] Examples of amino acids whose side chain (R1 or R3) is a C1-C6 alkyl group include alanine (Ala), isoleucine (Ile), leucine (Leu), valine (Val), 2-aminobutanoic acid (Abu), norvaline (Nva), norleucine (Nle), and tert-leucine (Tle).

[0042] In the present specification, "haloalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with halogen. As the haloalkyl, haloC1-C6 alkyl is preferable, and fluoroC1-C6 alkyl is more preferable. Specific examples of haloC1-C6 alkyl include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, etc.

[0043] Examples of amino acids in which the side chain (R1 or R3) is a halo C1-C6 alkyl group include 5-difluoronorvaline (Nva(5-F2)) and 2-amino-4-trifluorobutanoic acid (Abu(4-F3)).

[0044] In the present specification, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including a monocyclic ring, a bicyclic ring, and a spiro ring. Preferred examples of cycloalkyl include C3-C6 cycloalkyl. Specific examples of C3-C6 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0045] Examples of amino acids whose side chain (R1 or R3) is a C3-C6 cycloalkyl group include α-cyclopropylglycine (Gly(cPr)), α-cyclobutylglycine (Gly(cBu)), α-cyclopentylglycine (Gly(cPent)), and α-cyclohexylglycine (Chg).

[0046] In the present specification, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "cycloalkyl" defined above. Specific examples of C3-C6 cycloalkylC1-C6 alkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0047] As used herein, the term "carboxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by carboxy. Specific examples of carboxyC1-C6 alkyl include carboxymethyl and the like.

[0048] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring or aromatic hydrocarbon ring group. Aryl is preferably a C6-C 10 Examples of the aryl include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

[0049] As used herein, the term "arylalkyl (aralkyl)" refers to a group in which at least one hydrogen atom of the "alkyl" defined above is replaced with the "aryl" defined above. The arylalkyl is a C6-C 10 Aryl C1-C6 alkyl is preferred. C6-C 10 Specific examples of aryl C1-C6 alkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0050] The amino acid side chain (R1 or R3) is C6-C, which may have a substituent on the aryl 10 Examples of amino acids that are aryl C1-C6 alkyl groups include phenylalanine (Phe), 4-methylphenylalanine (Phe(4-Me)), and 4-trifluoromethyl-3,5-difluorohomophenylalanine (Hph(4-CF3-35-F2)).

[0051] In the present specification, "heteroaryl" refers to an aromatic cyclic monovalent group and an aromatic heterocyclic 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 heteroaryl ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, benzotriazolyl, indolyl, isoindolyl, indazolyl, azaindolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, imidazopyridyl, pyrazolopyridyl, imidazopyridyl, triazolopyridyl, pyrrolopyrazinyl, and furopyridyl.

[0052] In the present specification, the term "heteroaryl alkyl" refers to a group in which at least one hydrogen atom of the "alkyl" defined above is substituted with the "heteroaryl" defined above. As the heteroaryl alkyl, a 5- to 10-membered heteroaryl C1-C6 alkyl is preferred, and a 5- to 10-membered heteroaryl C1-C2 alkyl is more preferred. Specific examples of the 5- to 10-membered heteroaryl C1-C6 alkyl include 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.

[0053] The side chain of the amino acid (R 1or R 3 Examples of amino acids in which A is a 5- to 10-membered heteroaryl C1-C6 alkyl group optionally having a substituent on the heteroaryl include 2-amino-4-(pyridin-2-yl)-butanoic acid (Abu(4-Pyr)) and 3-(6-trifluoromethylpyridin-3-yl)alanine (Ala(3-Pyr-4-CF3)).

[0054] In the present specification, the term "heterocyclyl" refers to a non-aromatic cyclic monovalent group and heterocyclic group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl may be a saturated heterocycle, may have a double and / or triple bond in the ring, may be oxidized to form a carbonyl, and may be a monocycle or a condensed ring. In the case of a condensed ring, it may form a condensed ring with an aromatic ring such as a benzene ring, a pyridine ring, or a pyrimidine ring. It may form a condensed ring with a saturated alicyclic ring such as a cyclopentane ring or a cyclohexane ring, or a saturated heterocycle such as a tetrahydropyran ring, a dioxane ring, or a pyrrolidine ring.

[0055] The number of atoms constituting the heterocyclyl ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), and more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxoazetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, oxopyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiazinane, thiadiazolidinyl, oxazolidone, benzyl, phenyl ... Examples of such aryl groups include benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, 2-oxaspiro[3.3]heptyl, 6,7-dihydro-pyrrolo[1,2-a]imidazolyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazinyl, azepanyl, dioxepanyl, and 5,9-dioxaspiro[3.5]nonanyl.

[0056] In the present specification, the term "heterocyclylalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "heterocyclyl" defined above. As the heterocyclylalkyl, 5- to 10-membered heterocyclylC1-C6 alkyl is preferred, 4- to 7-membered heterocyclylC1-C6 alkyl is more preferred, and 4- to 7-membered heterocyclylC1-C2 alkyl is even more preferred. Specific examples of the 5- to 10-membered heterocyclylC1-C6 alkyl include 2-(tetrahydro-2H-pyran-4-yl)ethyl, 2-(azetidin-3-yl)ethyl, 4-(oxolan-2-ylmethyl)piperazin-1-yl, 2-(1-piperidyl)ethyl, and 3-(1-piperidyl)propyl.

[0057] In the present specification, "cycloalkoxy" refers to an oxy group to which the above-defined "cycloalkyl" is bonded. Cycloalkoxy is preferably C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0058] In the present specification, "cycloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with the "cycloalkoxy" defined above. As the cycloalkoxyalkyl, C3-C8 cycloalkoxyC1-C6 alkyl is preferred, C3-C6 cycloalkoxyC1-C6 alkyl is more preferred, and C3-C6 cycloalkoxyC1-C2 alkyl is more preferred. Specific examples of the C3-C6 cycloalkoxyC1-C6 alkyl include cyclopropoxymethyl and cyclobutoxymethyl.

[0059] In the present specification, "alkoxy" refers to an oxy group to which the above-defined "alkyl" is bonded. Preferred examples of alkoxy include C1-C6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0060] In the present specification, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with the "alkoxy" defined above. As the alkoxyalkyl, C1-C6 alkoxyC1-C6 alkyl is preferred, and C1-C6 alkoxyC1-C2 alkyl is more preferred. Specific examples of the C1-C6 alkoxyC1-C6 alkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0061] As used herein, the term "haloalkoxy" refers to a group in which one or more hydrogen atoms of the "alkoxy" defined above are substituted with halogen. As the haloalkoxy, halo C1-C6 alkoxy is preferred, and fluoro C1-C6 alkoxy is more preferred.

[0062] In the present specification, the term "haloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with the "haloalkoxy" defined above. As the haloalkoxyalkyl, preferred examples include halo C1-C6 alkoxy C1-C6 alkyl. Specific examples of the halo C1-C6 alkoxy C1-C6 alkyl include difluoromethoxymethyl, trifluoromethoxymethyl, 2,2-difluoroethoxymethyl, 2,2,2-trifluoroethoxymethyl, 3,3-difluoropropoxymethyl, 4,4-difluorobutoxymethyl, 5,5-difluoropentoxymethyl, and the like.

[0063] C6-C above 10 The aryl in the arylC1-C6 alkyl, the heteroaryl in the above 5- to 10-membered heteroarylC1-C6 alkyl, and the heterocyclyl in the above 5- to 10-membered heterocyclylC1-C6 alkyl may be further substituted by a substituent.

[0064] In the present specification, "optionally substituted" means that a group may be substituted by any substituent.Furthermore, each of these may be given a substituent, and the substituents are not limited, and may be independently selected from any substituents including, for example, a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom.

[0065] Examples of the substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, and the like.

[0066] In the present specification, "aminoalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced by the "amino" defined above. As the aminoalkyl, aminoC3-C6 alkyl is preferable. Specific examples of the aminoalkyl include aminomethyl, aminoethyl, 4-aminobutyl, methylaminomethyl, dimethylaminomethyl, methylaminoethyl, dimethylaminoethyl, and the like.

[0067] In the present specification, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above are replaced with hydroxyl groups. Preferred examples of hydroxyalkyl include hydroxy C1-C6 alkyl. Specific examples of hydroxy C1-C6 alkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0068] As used herein, the term "substituent derived from halogen" includes fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) and the like.

[0069] In the present specification, examples of an "oxygen atom-derived substituent" include hydroxyl (-OH), oxy (-OR), carbonyl (-C(=O)-R), carboxyl (-CO2H), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C(=O)-SR), carbonylthio group (-SC(=O)-R), aminocarbonyl (-C(=O)-NHR), carbonylamino (-NH-C(=O)-R), oxycarbonylamino (-NH-C(=O)-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), and carboxylcarbonyl (-C(=O)-CO2H).

[0070] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, and the like.

[0071] Examples of carbonyl (-C(=O)-R) include formyl (-C(=O)-H), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0072] Examples of oxycarbonyl (-C(=O)-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0073] Examples of carbonyloxy (-OC(=O)-R) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0074] Examples of thiocarbonyl (-C(=O)-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0075] Examples of carbonylthio (-SC(=O)-R) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0076] Examples of aminocarbonyl (-C(=O)-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, the H atom bonded to the N atom in -C(=O)-NHR may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0077] Examples of carbonylamino (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-C(=O)-R may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0078] Examples of oxycarbonylamino (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-C(=O)-OR may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0079] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-SO2-R may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0080] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, the H atom bonded to the N atom in -SO2-NHR may be further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0081] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0082] As used herein, examples of a "nitrogen atom-derived substituent" include azide (-N3, also referred to as an "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R''), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R'') and aminocarbonylamino (-NR-CO-NR'R'').

[0083] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0084] Examples of tertiary amino (-NR(R')) include amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring.

[0085] Examples of substituted amidino (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0086] Examples of substituted guanidino (-NR-C(=NR''')-NR'R'') include groups in which R, R', R'', and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl, or groups in which these form a ring.

[0087] Examples of aminocarbonylamino (-NR-C(=O)-NR'R'') include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these form a ring.

[0088] As used herein, examples of a "sulfur atom-derived substituent" include thiol (-SH), thio (-SR), sulfinyl (-S(=O)-R), sulfonyl (-S(=O)2-R), sulfo (-SO3H), and pentafluorosulfanyl (-SF5).

[0089] Examples of thio (-SR) are selected from among alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0090] Examples of sulfinyl (-S(=O)-R) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0091] Examples of sulfonyl (-S(=O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0092] As used herein, the term "boron atom-derived substituent" refers to boryl (-BR(R')), dioxyboryl (-B(OR)(OR')), and trifluoroborate (-BF3 - ) and the like. Specifically, these two substituents R and R' are "boron atom-derived substituents" which are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and the like, or these two substituents R and R' form a ring together with the atoms to which R and R' are bonded, which is a "boron atom-derived substituent", i.e., a cyclic boryl group.

[0093] A preferred example of the "boron atom-derived substituent" is a cyclic boryl group.

[0094] More specific examples of the cyclic boryl group include a pinacolatoboryl group, a neopentanediolateboryl group, a catecholateboryl group, and a 9-borabicyclo[3.3.1]nonan-9-yl group.

[0095] In the present specification, the term "optionally protected" means that a certain group may be protected by any protecting group.

[0096] In the present specification, the "protecting group for amino group" includes a carbamate type protecting group, an amide type protecting group, an arylsulfonamide type protecting group, an alkylamine type protecting group, an imide type protecting group, etc. Specific examples include a 9-fluorenylmethoxycarbonyl (Fmoc) group, a t-butoxycarbonyl (Boc) group, an allyloxycarbonyl (Alloc) group, a benzyloxycarbonyl (Cbz) group, a triethylsilyloxycarbonyl (Teoc) group, a trifluoroacetyl group, a pentafluoropropionyl group, a phthaloyl group, a benzenesulfonyl group, a tosyl group, a nosyl group, a dinitronosyl group, a t-butyl group, a trityl group, a cumyl group, a benzylidene group, a 4-methoxybenzylidene group, a diphenylmethylidene group, etc.

[0097] In the present specification, the "hydroxy-protecting group" includes an alkyl ether-type protecting group, an aralkyl ether-type protecting group, a silyl ether-type protecting group, a carbonate-type protecting group, etc. Specific examples of the hydroxy-protecting group include a methoxymethyl group, a benzyloxymethyl group, a tetrahydropyranyl group, a t-butyl group, an allyl group, a 2,2,2-trichloroethyl group, a benzyl group, a 4-methoxybenzyl group, a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, a methoxycarbonyl group, a 9-fluorenylmethoxycarbonyl (Fmoc) group, a 2,2,2-trichloroethoxycarbonyl group, etc.

[0098] Those skilled in the art can appropriately select and remove the protecting group. The selecting and removing of the protecting group may be appropriately selected according to the reaction conditions, for example, by referring to the method described in "Greene's, "Protective Groups in Organic Synthesis" (5th Edition, John Wiley & Sons 2014).

[0099] The structural transformation reaction of a compound can be appropriately carried out by those skilled in the art. For example, the methods described in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (8th Edition, John Wiley & Sons, Inc., 2019) and Comprehensive Organic Transformations (3rd Edition, John Wiley & Sons, Inc., 2018) by RC Laroch may be referred to and appropriately adopted depending on the reaction conditions.

[0100] The C1-C6 primary alkylating agent may be any alkylating agent having 1 to 6 carbon atoms and a formyl group or a cyano group bonded to a C1-C5 alkyl group, and is preferably a C1-C5 aldehyde or a C1-C5 alkylnitrile. Specific examples of C1-C5 aldehydes include acetaldehyde, propanal, 1-butanal, 1-pentanal, 3-methylbutanal, 1-hexanal, 4-methylpentanal, and 3,3-dimethylbutanal. Specific examples of C1-C5 alkylnitriles include acetonitrile, propionitrile, n-butyronitrile, n-pentylnitrile (1-cyanopentane), 3-methylbutyronitrile, valeronitrile (1-cyanobutane), and isovaleronitrile (1-cyano-2-methylpropane).

[0101] In this specification, the term "substituted methyl halide" refers to a methyl halide in which one of the hydrogen atoms on the carbon of the methyl halide (methyl halide) is replaced with another atom or functional group. Examples of the substituted methyl halide include alkoxymethyl halides, alkoxyalkoxymethyl halides, and trialkylsilylalkoxymethyl halides. A preferred example is substituted methyl chloride. Specific examples of the substituted methyl chloride include MOM-Cl (methoxymethyl chloride), EOM-Cl (ethoxymethyl chloride), MEM-Cl (2-methoxyethoxymethyl chloride), and SEM-Cl (2-(trimethylsilyl)ethoxymethyl chloride).

[0102] The amount of the C1-C6 primary alkylating agent or substituted methyl halide used may be in the range of 1.0 to 20.0 mol, 1.5 to 15.0 mol, 2.0 to 10.0 mol, or 2.5 to 5.0 mol relative to 1 mol of the starting amino acid or ester thereof.

[0103] Hydride reducing agents react with substrates as hydrides (H - The hydride reducing agent may be any hydride reducing agent that provides a silyl group and a nitrile group, and reduces a carbonyl group, a nitrile group, etc. The hydride reducing agent is preferably a hydride reducing agent containing silicon, more preferably a trialkylsilane, and most preferably triethylsilane.

[0104] The amount of the hydride reducing agent used may be in the range of 1.0 to 20.0 mol, 1.0 to 15.0 mol, 1.0 to 10.0 mol, or 1.2 to 5.0 mol per mol of the starting amino acid or ester thereof, or the starting peptide or ester thereof.

[0105] The catalyst may be any catalyst that promotes the alkylation of the primary amino group and improves the reaction rate. The catalyst is preferably a heterogeneous hydrogenation catalyst containing a transition metal, more preferably a transition metal supported on a suitable carrier. The transition metal preferably contains at least one selected from the group consisting of Pd, Rh and Pt. Specific examples of catalysts containing a transition metal supported on a carrier include palladium carbon (Pd-C), palladium hydroxide carbon (Pd(OH)2-C), rhodium carbon (Rh-C) and Adams catalyst. When the catalyst contains a transition metal supported on a carrier, it is easy to handle and the reaction can be carried out under simpler conditions.

[0106] The amount of the catalyst used may be in the range of 0.001 to 0.5 mol, 0.005 to 0.4 mol, 0.01 to 0.4 mol, or 0.03 to 0.1 mol per 1 mol of the starting amino acid or ester thereof, or the starting peptide or ester thereof.

[0107] The above process is carried out in a sealed reaction vessel. In the above process, the gas phase in the reaction vessel may be composed of only hydrogen gas, or may be mixed with an inert gas within a range that does not inhibit the desired reaction. The pressure (partial pressure) of hydrogen gas in the reaction vessel may be 1 atmosphere or more, in the range of 1 atmosphere to 10 atmospheres, in the range of 1 atmosphere to 7 atmospheres, in the range of 1 atmosphere to 6 atmospheres, or in the range of 1 atmosphere to 5 atmospheres. When the reaction is carried out under a pressure of about 1 atmosphere, for example, a method in which a rubber or vinyl balloon is attached to the reaction vessel and the balloon is replaced with hydrogen gas is well known. In this process, if the reaction solution is vigorously stirred, the frequency of contact with the gaseous hydrogen gas increases, and the reaction rate can be improved.

[0108] Additional hydrogen gas may be contacted with the mixture obtained by contacting the starting amino acid or its ester, the C1-C6 primary alkylating agent or substituted methyl halide, the catalyst, and hydrogen in a solvent. Additional hydrogen can be added to the reaction mixture by, for example, feeding hydrogen into the reaction vessel, degassing the reaction vessel under reduced pressure and then adding hydrogen to the reaction vessel, degassing the reaction vessel under reduced pressure, replacing the reaction vessel with nitrogen, degassing again under reduced pressure, and then replacing the reaction vessel with hydrogen. The number of times additional hydrogen gas is contacted depends on the progress of the reaction, and can be in the range of 1 to 10 times, 1 to 5 times, or 1 to 3 times. The time from the start of the reaction to contacting additional hydrogen gas can be in the range of 10 minutes to 5 hours, 20 minutes to 3 hours, or 10 minutes to 2 hours. Additional reagents can also be added for the purpose of efficiently controlling the reaction when the reaction vessel is opened to contact additional hydrogen gas. Examples of the additional reagent include a C1-C6 primary alkylating agent or a substituted methyl halide, a catalyst, a solvent, an acid, and a base.

[0109] As an alternative to hydrogen gas, a hydride reducing agent can be used, and depending on the progress of the reaction, additional hydride reducing agent can be added to the reaction vessel.

[0110] The solvent includes at least one solvent selected from the group consisting of ether solvents, alcohol solvents, and ester solvents. The solvent is preferably selected from the group consisting of ether solvents, alcohol solvents, ester solvents, and mixed solvents thereof.

[0111] Examples of ether solvents include tetrahydrofuran (THF), 2-methyltetrahydrofuran, dimethoxyethane (DME), methyl t-butyl ether (MTBE), cyclopentyl methyl ether (CPME), diisopropyl ether (IPE), 4-methyltetrahydropyran, dioxane, diethyl ether, and combinations thereof. Examples of alcohol solvents include methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, 3-pentanol, and combinations thereof. Examples of ester solvents include ethyl acetate, n-propyl acetate, butyl acetate, and combinations thereof. In this process, the above solvents may be appropriately combined and mixed in any ratio.

[0112] The amount of the solvent used may be in the range of 1 to 100 mL, 3 to 75 mL, 5 to 50 mL, or 7 to 25 mL relative to 1 mole of the starting amino acid or its ester.

[0113] When starting amino acid A is used as a raw material in the alkylation step, it is preferable to add a base to the reaction solution. By adding a base to the reaction solution, the ratio of dialkylated products and by-products can be reduced, and the monoalkylation selectivity can be further improved. When starting amino acid B is used as a raw material, it is preferable to add a base to the reaction solution in the N-alkylation step after the deprotection reaction.

[0114] The base may be an organic base or an inorganic base. The base is preferably a tertiary amine, more preferably 1,8-diazabicyclo[5.4.0]undecene-7 (DBU), 1,5-diazabicyclo[4.3.0]nonene-5 (DBN), N-methylmorpholine (NMM), 1,4-diazabicyclo[2.2.2]octane (DABCO), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine and collidine.

[0115] The amount of the base used may be in the range of 0.01 to 20.0 mol, 0.03 to 15.0 mol, 0.05 to 10.0 mol, or 0.7 to 5.0 mol, relative to 1 mol of the starting amino acid or its ester. When the amount of the base used is in the above range, the effect of improving the monoalkylation selectivity becomes more remarkable.

[0116] When the starting amino acid B is used as a raw material in the alkylation step, it is preferable to add an acid to the reaction solution. By adding an acid to the reaction solution, the proportion of by-products is reduced, and the target N-monoalkylamino acid or its ester (C) can be obtained more efficiently.

[0117] The acid may be any acid capable of effectively removing the protecting group attached to the primary amino group. Examples of the acid include p-toluenesulfonic acid (TsOH), methanesulfonic acid (MsOH), sodium hydrogen sulfate, triethylamine hydrochloride and propylphosphonic acid. The acid may be used in the form of a hydrate or any solution.

[0118] The amount of the acid used may be 0.1 to 10.0 mol, 0.3 to 7.0 mol, 0.5 to 5.0 mol, or 0.7 to 3.0 mol relative to 1 mol of the starting amino acid or its ester.

[0119] The reaction can be performed at a temperature ranging from -40°C to near the boiling point of the solvent, and can be performed at a temperature ranging from -20°C to 50°C or from 0°C to 30°C.

[0120] The reaction time may be within a range of 5 minutes to 72 hours, or within a range of 10 minutes to 48 hours, or within a range of 10 minutes to 24 hours.

[0121] After the alkylation step, the resulting N-monoalkylamino acid or ester thereof can also be converted to the corresponding salt or solvate by methods well known to those skilled in the art.

[0122] A second embodiment of the present invention, in one aspect, is a method for producing a peptide containing an N-monoalkylamino acid residue or an ester thereof, comprising an alkylation step of mixing a peptide containing a starting amino acid residue or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, and a catalyst in a solvent in the presence of hydrogen, the alkylation step being carried out under a pressure of 1 atmosphere or more, and producing a peptide containing an N-monoalkylamino acid residue or an ester thereof in which a primary alkyl group corresponding to the C1-C6 primary alkylating agent or the substituted methyl halide is bonded to the amino group of the starting amino acid residue.

[0123] Another second embodiment of the present invention, in one aspect, is a method for producing a peptide containing an N-monoalkylamino acid or an ester thereof, comprising an alkylation step of mixing a peptide containing a starting amino acid or an ester thereof, a C1-C6 primary alkylating agent or a substituted methyl halide, a hydride reducing agent and a catalyst in a solvent, wherein the alkylation step produces a peptide containing an N-monoalkylamino acid or an ester thereof in which a primary alkyl group corresponding to the C1-C6 primary alkylating agent or the substituted methyl halide is bonded to the amino group of the starting amino acid residue.

[0124] The alkylation step according to this embodiment includes a method of selectively N-monoalkylating a peptide represented by formula D or its ester (also referred to as starting peptide D) to obtain a peptide represented by formula F or its ester containing an N-monoalkyl amino acid residue, and a method of performing a deprotection reaction and an N-alkylation reaction of an N-protected peptide represented by formula E or its ester (also referred to as starting peptide E) in one pot to obtain a peptide represented by formula F or its ester. The method according to this embodiment is outlined below. [ka]

[0125] In the above chemical formula, R3 represents the side chain of the amino acid residue at the N-terminus of the peptide, PG2 represents a protecting group for the amino group, and R4 represents the peptide chain bound to the N-terminus amino acid residue. For convenience, in the above reaction formula, starting peptides D and E are exemplified in the form of α-amino acids, but they may be β-amino acids or γ-amino acids. In addition, in the above reaction formula, the side chain R3 of the amino acid residue at the N-terminus and the peptide chain R4 preferably do not have a functional group that can be subjected to unintended structural transformation by alkylation reaction or reduction reaction depending on the conditions of the alkylation step. When R3 and / or R4 have a functional group that can be subjected to unintended structural transformation depending on the conditions of the alkylation step, the target product can be produced by introducing a protecting group to the functional group beforehand and then performing the alkylation step.

[0126] R3 is, for example, a hydrogen atom, a C1-C6 alkyl group, a halo C1-C6 alkyl group, a C3-C6 cycloalkyl group, a C3-C6 cycloalkyl C1-C6 alkyl group, a carboxy C1-C6 alkyl group, a C6-C alkyl group optionally having a substituent on the aryl. 10It is selected from an aryl C1-C6 alkyl group, a 5-10 membered heteroaryl C1-C6 alkyl group optionally having a substituent on the heteroaryl, a 5-10 membered heterocyclyl C1-C6 alkyl group optionally having a substituent on the heterocyclyl, a C3-C6 cycloalkoxy C1-C6 alkyl group, a halo C1-C6 alkoxy C1-C6 alkyl group, a protected amino C3-C6 alkyl group, a protected hydroxy C1-C6 alkyl group, or a C1-C6 alkoxy C1-C6 alkyl group.

[0127] R4 may be a peptide chain of two or more amino acid residues, which may contain an N-alkylamino acid residue. The side chain of the amino acid residue contained in R4 may be an amino acid having a side chain that does not have a functional group that may undergo unintended structural transformation by an alkylation reaction or a reduction reaction depending on the conditions of the alkylation step. In addition, when the side chain of the amino acid residue contained in R4 has a functional group that may undergo unintended structural transformation by an alkylation reaction or a reduction reaction depending on the conditions of the alkylation step, the side chain may be an amino acid having a side chain in which a protecting group has been introduced to the functional group in advance. Such a side chain may be, for example, a hydrogen atom, a C1-C6 alkyl group, a halo C1-C6 alkyl group, a C3-C6 cycloalkyl group, a C3-C6 cycloalkyl C1-C6 alkyl group, a carboxy C1-C6 alkyl group, a C6-C alkyl group optionally having a substituent on the aryl. 10 It is selected from an aryl C1-C6 alkyl group, a 5-10 membered heteroaryl C1-C6 alkyl group optionally having a substituent on the heteroaryl, a 5-10 membered heterocyclyl C1-C6 alkyl group optionally having a substituent on the heterocyclyl, a C3-C6 cycloalkoxy C1-C6 alkyl group, a halo C1-C6 alkoxy C1-C6 alkyl group, a protected amino C3-C6 alkyl group, a protected hydroxy C1-C6 alkyl group, or a C1-C6 alkoxy C1-C6 alkyl group.

[0128] The reaction conditions for the alkylation step in the second embodiment can be referenced by replacing the "starting amino acid" in the reaction conditions described in the first embodiment with "peptide containing the starting amino acid" (also referred to as "starting peptide" in this specification).

[0129] The third embodiment of the present invention is a method for producing a peptide or an ester thereof, comprising a step of using as a starting material the N-monoalkylamino acid or ester thereof (see formula C) obtained in the first embodiment, or a peptide having an N-monoalkylamino acid residue or an ester thereof (see formula F) obtained in the second embodiment, and optionally extending one or more amino acids by a bond formation reaction (e.g., a peptide bond formation reaction) to obtain a desired peptide or an ester thereof. This embodiment also includes a method for producing a peptide having a cyclic portion composed of at least four amino acids or an ester thereof, which additionally comprises a step of cyclizing the peptide (cyclization precursor peptide, i.e., linear peptide) obtained by the above production method (or an ester thereof) at the C-terminal group and the N-terminal group to form a cyclic portion.

[0130] In the step of extending the peptide main chain, when the N-monoalkylamino acid or its ester (see formula C) obtained in the first embodiment is used as the starting material, extension of the peptide main chain is essential to form a cyclic portion, but when the peptide having an N-monoalkylamino acid residue or its ester (see formula F) obtained in the second embodiment is used as the starting material, it is not necessarily required to extend the peptide main chain. Those skilled in the art can appropriately select whether or not to carry out this step depending on the chemical structure of the desired peptide having a cyclic portion.

[0131] According to the method of this embodiment, a peptide having a cyclic portion composed of at least 4 amino acids or an ester thereof can be produced. The method of this embodiment is more suitable for producing a peptide having a cyclic portion composed of at least 8 amino acids and composed of 8 to 15 amino acids or an ester thereof. The peptide or ester thereof obtained in this embodiment may contain 5 or more, 6 or more, or 7 or more N-alkyl amino acid residues.

[0132] The peptide chain elongation by bond formation reaction and the peptide bond formation reaction can be carried out by methods well known to those skilled in the art, for example, by referring to Biopolym. Pept. Sci. 2000, 55, 227-250, or W. M. Hussein et al., Peptide Synthesis Methods and Protocols (Humana Press, 2020).

[0133] The step of forming the cyclic portion is a step of reacting a group on the C-terminus side of the peptide main chain and a group on the N-terminus side of the cyclization precursor peptide obtained above or a peptide having an N-monoalkylamino acid residue or an ester thereof obtained in the second embodiment to form a cyclic portion. The combination of the group on the C-terminus side and the group on the N-terminus side may be any combination as long as they can form an organic bond with each other.

[0134] A preferred combination is one in which the group on the C-terminus side is a carboxy group and the group on the N-terminus side is an amino group. In this case, the step of forming the cyclic portion can be a condensation reaction (peptide bond forming reaction). The conditions for the condensation reaction can be those well known to those skilled in the art, and examples include stirring in a solvent in the presence of a condensing agent. The position of the carboxyl group, amino group, etc. used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position that allows cyclization.

[0135] The condensation agent may be any agent capable of binding a carboxy group and an amino group to form a peptide bond. Specific examples of the condensation agent include carbodiimide-based condensation agents such as N,N'-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), phosphoric acid azide-based condensation agents such as diphenyl phosphoric acid azide (DPPA), phosphonium-based condensation agents such as BOP reagent and PyBOP reagent, uronium-based condensation agents such as TBTU, HBTU, TATU, and HATU, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMT-MM), and T3P (propylphosphonic anhydride).

[0136] In the condensation reaction, an additive may be further added to the reaction mixture, such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HOOBt), ethyl (hydroxyimino)cyanoacetate (Oxyma), etc.

[0137] A fourth embodiment of the present invention is a method for producing an N-monoalkylamino acid or its ester (see formula C) or a peptide containing an N-monoalkylamino acid or its ester (see formula F), which comprises an alkylation step of mixing the starting amino acid A or B or the starting peptide D or E, a C1-C6 primary alkylating agent or a substituted methyl halide, and a catalyst in a solvent in the presence of hydrogen, wherein the alkylation step is carried out under a pressure of 1 atmosphere or more, and an N-monoalkylamino acid or its ester (see formula C) or a peptide containing an N-monoalkylamino acid or its ester (see formula F) is produced in which a primary alkyl group corresponding to the C1-C6 primary alkylating agent or substituted methyl halide is bonded to the amino group of the starting amino acid A or B, the method being a method for suppressing the production of a compound in which the amino group of the starting amino acid or its ester, or the peptide containing the starting amino acid or its ester, is dialkylated.

[0138] In this embodiment, reference can be made to the definitions described in the first or second embodiment. According to the alkylation step, the production of dialkylated products can be suppressed.

[0139] Furthermore, the N-monoalkylamino acid or its ester (see formula C) obtained in the alkylation step, or a peptide containing an N-monoalkylamino acid or its ester (see formula F) can be used as a starting material to carry out an additional bond formation reaction to extend the main chain of the peptide, and then the resulting peptide can be treated with an acidic aqueous solution, whereby the resulting dialkylated compound can be easily removed.

[0140] In one embodiment, the linear peptide comprises: [ka] or a salt or solvate thereof.

[0141] In one embodiment, the peptide having a cyclic portion produced by the method of the present invention has the following formula (1): [ka] The peptide having a cyclic portion represented by the formula (1) is preferably a solvate, more preferably a hydrate, DMSO-hydrate, acetone-hydrate, or DMSO solvate, and even more preferably a hydrate. As described in WO 2021 / 090855, the peptide having a cyclic portion represented by the formula (1) is useful as a KRAS inhibitor and can be used for various KRAS-related diseases, such as KRAS-related cancers.

[0142] In one embodiment, it is preferred not to use column chromatography for isolation and / or purification of a peptide having a cyclic portion produced by the method of the present invention, or a salt thereof, or a solvate thereof.

[0143] The peptide having a cyclic portion produced by the method of the present invention, or a salt thereof, or a solvate thereof can be isolated and / or purified by, for example, crystallization by crystallization instead of column chromatography. Specifically, for example, the reaction solution after the condensation reaction is subjected to a liquid separation operation, and the organic layer is concentrated and / or filtered as necessary, and then a solvent suitable for crystallization is added to the resulting residue, and seed crystals are optionally added, and the mixture is stirred as necessary to obtain crystals of the peptide having a cyclic portion, or a salt thereof, or a solvate thereof. The solvent added during crystallization is not particularly limited as long as it is a solvent that allows the peptide having a cyclic portion to form crystals, but a solvent that allows an operation to reduce the solubility of the peptide having a cyclic portion in the solution in which the peptide having a cyclic portion is dissolved is preferable. For example, when the solubility of the peptide having a cyclic portion can be reduced by adding a poor solvent or cooling the solution, examples of the solvent that allows such an operation are solvents that allow such an operation. In addition, when the crystals of the peptide having a cyclic portion can be obtained by keeping the crude crystals of the peptide having a cyclic portion in a suspension state for an arbitrary time, a solvent that allows such an operation can be used for crystallization. Specific examples of the solvent added during crystallization include acetone, water, DMSO, acetonitrile, or ethanol, and mixtures thereof. This crystallization can also be applied to peptides containing N-monoalkylamino acids or their esters.

[0144] In one embodiment, the crystal of a peptide having a cyclic portion, or a salt thereof, or a solvate thereof produced by the method of the present invention can be a nonsolvate crystal, a solvate crystal, a salt crystal, or a solvate crystal of a salt of a peptide having a cyclic portion represented by the above formula (1). In one embodiment, a nonsolvate crystal (nonsolvate crystal) may refer to a crystal that is not a solvate crystal or a hydrate crystal. The crystal of a peptide having a cyclic portion represented by the above formula (1), or a salt thereof, or a solvate thereof is preferably a solvate crystal, more preferably a hydrate crystal. EXAMPLES

[0145] The present invention will be described in more detail with reference to the following examples. The abbreviations used in the examples should be understood in the ordinary sense in the field of organic chemistry, and examples are shown below. Bn: Benzyl Boc: t-butoxycarbonyl Cbz: benzyloxycarbonyl CPME: Cyclopentyl methyl ether DABCO: 1,4-diazabicyclo[2.2.2]octane DBN: 1,5-diazabicyclo[4.3.0]nonene-5 DBU: 1,8-diazabicyclo[5.4.0]undecene-7 DCHA: dicyclohexylamine DIPEA: N,N-diisopropylethylamine DKP: Diketopiperazine DMA: Dimethylacetamide DMI: 1,3-Dimethyl-2-imidazolidinone DMSO: Dimethyl sulfoxide EOM-Cl: Ethoxymethyl chloride HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate 1 H-NMR spectrum: Proton nuclear magnetic resonance spectrum HPLC: High-performance liquid chromatography i-:iso LCMS: Liquid Chromatography and Mass Spectrum MEM-Cl: 2-Methoxyethoxymethyl chloride MOM-Cl: Methoxymethyl chloride MTBE: Methyl tert-butyl ether n-:Normal NMM: N-methylmorpholine PFP: Pentafluorophenyl s-: Secondary SEM-Cl: 2-(trimethylsilyl)ethoxymethyl chloride tBu: tertiary butyl TEA: Triethylamine Teoc: 2-(trimethylsilyl)ethoxycarbonyl t-: tertiary THF: tetrahydrofuran TMSCl: chlorotrimethylsilane TsOH·H2O: paratoluenesulfonic acid monohydrate Sar: Sarcosine

[0146] 1 H-NMR spectra were measured using a nuclear magnetic resonance spectrometer JNM-ECZ500 (manufactured by JEOL Ltd.), and the chemical shift of tetramethylsilane used as an internal standard was set to 0 ppm, and the deuterium lock signal from the sample solvent was used as a reference. The chemical shift of the signal of the analyte compound was expressed in ppm. The abbreviations for the signal splitting were s: singlet, brs: broad singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, and m: multiplet, and the signal splitting width was expressed in J value (Hz). The signal integral value was calculated based on the ratio of the area intensity of each signal.

[0147] For HPLC analysis, a Waters H-Class system was used, and a PDA detector was used at a wavelength of 220 nm for compound 4 and at a wavelength of 210 nm for other compounds. The reactivity, selectivity, and purity of each substrate used in the examples were evaluated by the analytical methods shown in Table 1 below. For LCMS analysis, an SQD2 was used to detect compounds 3, 4, 11, and 12, and a QDa detector was used to detect other compounds. [Table 1] JPEG2024052694000016.jpg116149

[0148] The melting point of the crystal was measured by thermal analysis carried out under the following conditions.

[0149] (Measurement condition 1) Measurement equipment: TGA / DSC3+ (Mettler Toledo) Heating rate: 10℃ / min Atmosphere: dry nitrogen

[0150] (Raw material synthesis 1) Preparation of H-Phe-OtBu [ka] In a separatory funnel, t-butyl L-phenylalaninate hydrochloride (15.05 g, 58.2 mmol) was suspended in 2-methyltetrahydrofuran (200 mL), and 5% aqueous sodium carbonate solution (150 mL) was added and mixed. The solid gradually dissolved. 5% saline (about 10 mL) was added to the mixture. The aqueous layer was drained, and the organic layer was washed again with 5% aqueous sodium carbonate solution (150 mL). The aqueous layer was drained, and anhydrous sodium sulfate was added to the obtained organic layer to dry it. After filtering off the sodium sulfate, the mixture was concentrated under reduced pressure until the weight did not change, and t-butyl L-phenylalaninate (hereinafter also referred to as compound 1) was obtained (12.43 g, yield 97%). HPLC purity: 100% Measurement method: HPLC Method A Retention time: 2.72 minutes 1 H-NMR(500MHz,DMSO-d6) δ:1.32(9H, s), 1.66(2H,bs),2.79(2H, m), 3.44(1H, t, J=6.9Hz), 7.19-7.21(3H, m), 7.25-7.28(2H, m) Mass spectrometry: m / z 166.17([M-tBu+H] + )

[0151] (Example 1) Evaluation of the effect of adding a base on the monomethylation reaction of H-Phe-OtBu [ka] A flask containing a stirrer was charged with 5% palladium on carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% Pd metal basis) and a methanol solution of compound 1 (20 mL / g substrate, 2.0 mL, 0.452 mmol). To the resulting mixture was added 37% formaldehyde aqueous solution (33.4 μL, 0.452 mmol, 1.0 eq.), followed by the addition of a base. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced again with hydrogen. Sampling was performed 3 and 6 hours after the hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The results are shown in Table 2. [Table 2] Measurement method: HPLC Method A Retention time: 2.82 minutes (compound 1), 2.93 minutes (compound 1-A), 3.05 minutes (compound 1-B) Mass spectrometry: m / z 236.31 (compound 1-A, [M+H] + ), 250.30 (compound 1-B, [M+H] + )

[0152] (Example 2) Monomethylation reaction of H-Phe-OtBu [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% Pd metal basis) and a methanol solution of compound 1 (20 mL / g substrate, 2.0 mL, 0.452 mmol). A 37% aqueous formaldehyde solution (67 μL, 0.904 mmol, 2.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced with hydrogen again. Sampling was performed 3 and 11 hours after the hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The reaction was carried out in the same manner, except that DBU (6.7 μL, 0.045 mmol, 0.1 eq.) was added before the nitrogen replacement. The results are shown in Table 3. [Table 3] Measurement method: HPLC Method A Retention time: 2.44 min (compound 1), 2.54 min (compound 1-A), 2.66 min (compound 1-B) Mass spectrometry: m / z 236.31 (compound 1-A, [M+H] + ), 250.32 (compound 1-B, [M+H] + )

[0153] (Example 3) Monoethylation reaction of H-Phe-OtBu with acetaldehyde [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% metal Pd basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). Acetaldehyde (51 μL, 0.904 mmol, 2.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced with hydrogen again. Three hours after the hydrogen replacement, a sample was taken and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. The reaction was carried out in the same manner, except that DBU (6.7 μL, 0.045 mmol, 0.1 eq.) was added before the nitrogen replacement. The results are shown in Table 4. [Table 4] Measurement method: HPLC Method A Retention time: 2.43 min (compound 1), 2.63 min (compound 1-C), 2.85 min (compound 1-D) Mass spectrometry: m / z 250.31 (compound 1-C, [M+H] + ), 278.33 (compound 1-D, [M+H] + )

[0154] (Example 4) Monoethylation reaction of H-Phe-OtBu using acetonitrile [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% metal Pd basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). Acetonitrile (236 μL, 4.52 mmol, 10.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, then replaced with hydrogen again, and stirred at 30°C. Sampling was performed 5 and 11 hours after the hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The reaction was performed in the same manner, except that DBU (6.7 μL, 0.045 mmol, 0.1 eq.) was added before replacing with nitrogen. The results are shown in Table 5. [Table 5] Measurement method: HPLC Method A Retention time: 2.61 min (compound 1), 2.83 min (compound 1-C), 3.06 min (compound 1-D) Mass spectrometry: m / z 250.09 (compound 1-C, [M+H] + ), 278.06 (compound 1-D, [M+H] + )

[0155] (Example 5) Monopropylation reaction of H-Phe-OtBu with propanal [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% metal Pd basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). Propanal (65 μL, 0.904 mmol, 2.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced with hydrogen again. Sampling was performed 3 and 6 hours after the hydrogen replacement, and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. The reaction was performed in the same manner, except that DBU (6.7 μL, 0.045 mmol, 0.1 eq.) was added before the nitrogen replacement. The results are shown in Table 6. [Table 6] Measurement method: HPLC Method A Retention time: 2.42 min (compound 1), 2.82 min (compound 1-E), 3.27 min (compound 1-F) Mass spectrometry: m / z 264.36 (compound 1-E, [M+H] + ), 306.38 (compound 1-F, [M+H] + )

[0156] (Example 6) Monopropylation reaction of H-Phe-OtBu using propionitrile [ka] A flask containing a stir bar was charged with 5% palladium on carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% Pd metal basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). To the resulting mixture was added propionitrile (322 μL, 4.52 mmol, 10.0 eq.). The gas phase in the flask was replaced with nitrogen while stirring, and then replaced again with hydrogen. Eight hours after the hydrogen replacement, a sample was taken and the ratio of each compound was confirmed by HPLC analysis. The results are shown in Table 7. [Table 7] Measurement method: HPLC Method A Retention time: 2.69 min (compound 1), 3.11 min (compound 1-E), 3.58 min (compound 1-F) Mass spectrometry: m / z 264.29 (compound 1-E, [M+H] + ), 306.31 (compound 1-F, [M+H] + )

[0157] (Example 7) Monobutylation reaction of H-Phe-OtBu with butanal [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% metal Pd basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). Butanal (81 μL, 0.904 mmol, 2.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced with hydrogen again. Sampling was performed 3 and 6 hours after the hydrogen replacement, and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. The reaction was performed in the same manner, except that DBU (6.7 μL, 0.045 mmol, 0.1 eq.) was added before the nitrogen replacement. The results are shown in Table 8. [Table 8] Measurement method: HPLC Method A Retention time: 2.42 min (compound 1), 3.04 min (compound 1-G), 3.68 min (compound 1-H) Mass spectrometry: m / z 278.35 (compound 1-G, [M+H] + ), 334.43 (compound 1-H, [M+H] + )

[0158] (Example 8) Monobutylation reaction of H-Phe-OtBu with butyronitrile [ka] A flask containing a stir bar was charged with 5% palladium on carbon (50% wet, 96 mg, 0.023 mmol, 5 mol% Pd metal basis) and an ethanol solution of compound 1 (15 mL / g substrate, 1.5 mL, 0.452 mmol). Butyronitrile (393 μL, 4.52 mmol, 10.0 eq.) was added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, and then replaced with hydrogen again. Five hours after the hydrogen replacement, a sample was taken and the ratio of each compound was confirmed by HPLC analysis. The results are shown in Table 9. [Table 9] Measurement method: HPLC Method A Retention time: 2.67 min (compound 1), 3.32 min (compound 1-G), 3.99 min (compound 1-H) Mass spectrometry: m / z 278.30 (compound 1-G, [M+H] + ), 334.39 (compound 1-H, [M+H] + )

[0159] Example 9 H-Val-MeAsp(OtBu)-NMe 2 Monopropylation reaction of [ka] A vial containing a stir bar was charged with 5% palladium carbon (50% wet, 48 mg, 0.011 mmol, 5 mol% metal Pd standard) and an ethanol solution of compound 2 (10 mL / g substrate, 0.75 mL, 0.228 mmol). Propanal (82 μL, 1.138 mmol, 5.0 eq.) was added to the resulting mixture. The vial was placed in a pressure-resistant container, and the gas phase in the pressure-resistant container was replaced with nitrogen while stirring the reaction solution in the vial, and then replaced with hydrogen again. Sampling was performed 6 hours after hydrogen replacement, and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. The reaction was performed in the same manner, except that TEA (63.5 μL, 0.455 mmol, 2.0 eq.) was added before replacing with nitrogen. The results are shown in Table 10. [Table 10] Measurement method: HPLC Method A Retention time: 2.06 min (compound 2), 2.41 min (compound 2-E), 2.77 min (compound 2-F) Mass spectrometry: m / z 372.68 (compound 2-E, [M+H] + ), 414.65 (compound 2-F, [M+H] + )

[0160] Example 10 H-Val-MeAsp(OtBu)-NMe 2 Monobutylation of butanal [ka] 5% palladium carbon (50% wet, 48 mg, 0.011 mmol, 5 mol% metal Pd standard) and an ethanol solution of compound 2 (10 mL / g substrate, 0.75 mL, 0.228 mmol) were added to a vial containing a stir bar. Butanal (103 μL, 1.138 mmol, 5.0 eq.) was added to the resulting mixture. The vial was then placed in a pressure-resistant container, and the gas phase in the pressure-resistant container was replaced with nitrogen while stirring in the pressure-resistant container, and then replaced with hydrogen again. Sampling was performed 6 hours after hydrogen replacement, and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. In addition, the reaction was performed in the same manner except that TEA (63.5 μL, 0.455 mmol, 2.0 eq.) was added before replacing with nitrogen. The results are shown in Table 11. [Table 11] Measurement method: HPLC Method A Retention time: 2.06 min (compound 2), 2.64 min (compound 2-G), 3.22 min (compound 2-H) Mass spectrometry: m / z 386.71 (compound 2-G, [M+H] + ), 442.87(compound-H, [M+H] + )

[0161] (Example 11) Deprotection and monoethylation of Cbz-Phe-OtBu in one pot [ka] 5% palladium carbon (50% wet, 120 mg, 0.028 mmol, 5 mol% metal Pd basis) and p-toluenesulfonic acid (107 mg, 0.563 mmol, 1.0 eq.) were added to a flask or vial containing a stirrer. Compound P1 in THF solution (10 mL / g substrate, 2.0 mL, 0.563 mmol) and acetonitrile (294 μL, 5.63 mmol, 10.0 eq.) were added to the resulting mixture in sequence. The gas phase in the flask was replaced with nitrogen while stirring, then replaced with hydrogen, and stirred at 30°C. When the hydrogen pressure was 1 atm, hydrogen was supplied using a balloon filled with hydrogen. When the hydrogen pressure was 3.5 or 5.5 atm, the vial containing the reaction solution was placed in a pressure-resistant container, and the pressure-resistant container was replaced with nitrogen, followed by hydrogen replacement. Sampling was performed 10 hours after hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The results are shown in Table 12. [Table 12] Measurement method: HPLC Method A Retention time: 2.61 min (compound 1), 2.83 min (compound 1-C), 3.06 min (compound 1-D) Mass spectrometry: m / z 250.09 (compound 1-C, [M+H] + ), 278.06 (compound 1-D, [M+H] + )

[0162] (Raw material synthesis 2) Cbz-Asp(OtBu)-NMe 2 Synthesis of [ka] Compound P4 (3.55 g, 10.4 mmol) and 2-methyltetrahydrofuran (32 mL, 30 eq.) were added to a 200 mL flask purged with nitrogen. While the flask was cooled in an ice bath, a THF solution of dimethylamine (2.0 M, 7.8 mL, 15.6 mmol, 1.5 eq.) and DIPEA (6.2 mL, 36.4 mmol, 3.5 eq.) were added successively to the resulting mixture. Then, a 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 16.3 mL, 26.0 mmol, 2.5 eq.) was added dropwise to the mixture. The internal temperature during the dropwise addition was 15.0 to 26.0 °C. After the dropwise addition, the mixture was stirred at room temperature for 1.5 hours, sampled, and the reaction completion was confirmed by HPLC analysis. While the flask was cooled in an ice bath, a 5% aqueous sodium hydrogen sulfate solution (20 mL) was slowly added. After stirring for 15 minutes, the entire reaction solution was transferred to a separatory funnel, and after standing, the aqueous layer was removed. 5% aqueous sodium hydrogen sulfate solution (20 mL) was added to the organic layer, and after shaking well, the mixture was allowed to stand, and the aqueous layer was removed. 5% aqueous potassium carbonate solution (20 mL) was added to the organic layer, and after shaking well, the mixture was allowed to stand, and the aqueous layer was removed. Separation washing with 5% aqueous potassium carbonate solution (20 mL) was repeated once more. The obtained organic layer was concentrated under reduced pressure to obtain 3.9 g of a crude product. The obtained crude product was purified by silica gel column chromatography to obtain compound P5 (3.48 g, yield 95%). HPLC purity: 100% Measurement method: HPLC Method A Retention time: 3.83 minutes Mass spectrometry: m / z 295.21([M-tBu+H] + )

[0163] Example 12 Cbz-Asp(OtBu)-NMe 2 One-pot deprotection and monopropylation of [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 60 mg, 0.014 mmol, 5 mol% metal Pd standard) and an ethanol solution of compound P5 (15 mL / g substrate, 1.5 mL, 0.285 mmol). The mixture was substituted with nitrogen and hydrogen while stirring to carry out the deprotection reaction. After 3 hours, the mixture was degassed under reduced pressure and propanal (41 μL, 0.571 mmol, 2.0 eq.) was added. The gas phase in the flask was substituted with nitrogen while stirring and then with hydrogen. After 4 hours from the hydrogen substitution, the mixture was sampled and the ratio of each compound and the amount of impurities were confirmed by HPLC analysis. The reaction was carried out in the same manner except that DBU (4.3 μL, 0.029 mmol, 0.1 eq.) was added immediately before the addition of propanal. The results are shown in Table 13. [Table 13] Measurement method: HPLC Method A Retention time: 3.57 min (compound P5), 1.69 min (compound 5), 2.04 min (compound 5-E), 2.49 min (compound 5-F) Mass spectrometry: m / z 295.23 (compound P5, [M-tBu+H] + ), 259.32 (compound 5-E, [M+H] + ), 301.35 (compound 5-F, [M+H] + )

[0164] (Raw Material Synthesis 3) Synthesis of Cbz-Phe(4-Me)-Sar-OtBu [ka] Compound P6 (30.36 g, 96 mmol) and t-butyl sarcosinate hydrochloride (20.87 g, 115 mmol, 1.2 eq.) were added to a 2 L flask, and the gas phase in the flask was replaced with nitrogen. 2-Methyltetrahydrofuran (290 mL, 30 eq.) was added, the external temperature was cooled to 15°C, and DIPEA (88 mL, 517 mmol, 5.4 eq.) was added dropwise from the dropping funnel. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 132 mL, 211 mmol, 2.2 eq.) was added dropwise from the dropping funnel over 1 hour and 20 minutes. The internal temperature during the dropping was 15.0 to 17.4°C. One hour after the end of the dropping, a sample was taken, and the reaction completion was confirmed by HPLC analysis. 5% aqueous sodium carbonate solution (180 mL) was slowly added from the dropping funnel. The internal temperature during the dropwise addition was maintained at 30.3°C or less. After completion of the dropwise addition, the external temperature was set to 23°C. After stirring for 15 minutes or more, the mixture was allowed to stand, and the aqueous layer was removed. A 5% aqueous sodium hydrogen sulfate solution (180mL) was added to the organic layer, and after stirring for 10 minutes or more, the mixture was allowed to stand, and the aqueous layer was removed. Separation washing with a 5% aqueous sodium hydrogen sulfate solution (180mL) was repeated once more. A 5% aqueous sodium carbonate solution (180mL) was added to the organic layer, and after stirring for 10 minutes or more, the mixture was allowed to stand, and the aqueous layer was removed. The obtained organic layer was concentrated under reduced pressure conditions to obtain 44.93g of a crude product. A portion of the obtained crude product was purified by silica gel column chromatography to obtain compound P7. HPLC purity: 100% Measurement method: HPLC Method B Holding time: 3.80 minutes Mass spectrometry: m / z 385.34([M-tBu+H] + )

[0165] (Example 13) One-pot deprotection and monoethylation of Cbz-Phe(4-Me)-Sar-OtBu: Inhibitory effect of DKP formation by addition of TsOH [ka] 5% palladium carbon (50% wet, 48 mg, 0.011 mmol, 5 mol% metal Pd basis) and TsOH·H2O (43 mg, 0.227 mmol, 1.0 eq.) were added to a flask containing a stirrer. Compound P7 in 2-methyltetrahydrofuran solution (10 mL / g substrate, 1.0 mL, 0.227 mmol) and acetonitrile (119 μL, 2.27 mmol, 10.0 eq.) were added to the resulting mixture. The gas phase in the flask was replaced with nitrogen while stirring, then replaced with hydrogen, and stirred at 25 °C. Sampling was performed 6 hours or 10.5 hours after hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The reaction was performed in the same manner except that TsOH·H2O (43 mg, 0.227 mmol, 1.0 eq.) was added before replacing with nitrogen. The results are shown in Table 14. [Table 14] Measurement method: HPLC Method B Retention time: 1.66 min (compound 7-DKP), 2.23 min (compound 7), 2.32 min (compound 7-C), 2.46 min (compound 7-D) Mass spectrometry: m / z 233.26 (compound 7-DKP, [M+H] + ), 307.26 (compound 7, [M+H] + ), 335.34 (compound 7-C, [M+H] + ), 363.38 (compound 7-D, [M+H] + )

[0166] (Example 14) One-pot deprotection and monoethylation of Cbz-Phe(4-Me)-Sar-OtBu [ka] 5% palladium carbon (50% wet, 97 mg, 0.023 mmol, 5 mol% metal Pd standard) and p-toluenesulfonic acid (86 mg, 0.454 mmol, 1.0 eq.) were added to a flask or pressure-resistant reaction vessel containing a stirrer. A 2-methyltetrahydrofuran solution of compound P7 (10 mL / g substrate, 2.0 mL, 0.454 mmol) and acetonitrile (237 μL, 4.54 mmol, 10.0 eq.) were added in sequence. The gas phase in the flask was replaced with nitrogen while stirring, then replaced with hydrogen, and the reaction was carried out at 30 ° C. When the hydrogen pressure was 1 atm, hydrogen was supplied using a balloon filled with hydrogen. When the hydrogen pressure was 5.5 atm, the vial containing the reaction solution was placed in a pressure-resistant vessel, and the inside of the pressure-resistant vessel was replaced with nitrogen, followed by hydrogen replacement. Sampling was carried out 10 hours after hydrogen replacement, and the ratio of each compound was confirmed by HPLC analysis. The results are shown in Table 15. [Table 15] Measurement method: HPLC Method B Retention time: 2.23 min (compound 7), 2.32 min (compound 7-C), 2.46 min (compound 7-D) Mass spectrometry: m / z 307.26 (compound 7, [M+H] + ), 335.34 (compound 7-C, [M+H] + ), 363.38 (compound 7-D, [M+H] + )

[0167] (Example 15) Synthesis of Cbz-Aze-EtPhe(4-Me)-Sar-OtBu [ka] Compound 7-C (3.00 g, 8.97 mmol, containing 1.6% of compound 7-D) was dissolved in 2-methyltetrahydrofuran (27.0 mL, 30 eq.) in a 200 mL flask, compound 8 (3.17 g, 13.45 mmol, 1.5 eq.) was added, and the atmosphere was replaced with nitrogen. The external temperature was cooled to 15°C, and DIPEA (9.0 mL, 53.8 mmol, 6.0 eq.) was added by syringe. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 20 mL, 31.4 mmol, 3.5 eq.) was added dropwise from the dropping funnel over 17 minutes. The internal temperature during the dropping was 15.0 to 18.0°C. Five hours after the end of the dropping, the mixture was sampled, and the reaction completion was confirmed by HPLC analysis. 5% aqueous sodium carbonate solution (27 mL) was slowly added from the dropping funnel. The internal temperature during the dropwise addition was maintained at 28.0°C or less. After completion of the dropwise addition, the external temperature was set to 25°C. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous layer was removed. Cyclohexane (15mL) and 5% aqueous sodium hydrogen sulfate solution (30mL) were added, and the mixture was stirred for 10 minutes or more, allowed to stand and the aqueous layer was removed. Separation washing with 5% aqueous sodium hydrogen sulfate solution (30mL) was repeated once more. A 5% aqueous sodium carbonate solution (30mL) was added, and the mixture was stirred for 10 minutes or more, allowed to stand and the aqueous layer was removed. It was confirmed by HPLC analysis that compound 7-D contained in the raw material was completely removed by the separation washing. The analytical results before the reaction and after the separation washing are shown in Table 16. [Table 16] Measurement method: HPLC Method B Retention time: 2.47 min (compound 7-C), 2.61 min (compound 7-D), 3.84 min (compound 9) Mass spectrometry: m / z 407.36 (compound 9, [M-Sar+H] + )

[0168] (Raw Material Synthesis 4) 50 gram scale synthesis of Cbz-Phe(4-Me)-Sar-OtBu [ka] Compound P6 (50.03 g, 160 mmol) and t-butyl sarcosinate hydrochloride (34.50 g, 191 mmol, 1.2 eq.) were added to a 2 L flask and replaced with nitrogen. 2-Methyltetrahydrofuran (485 mL, 30 eq.) was added, cooled to an external temperature of 15°C, and DIPEA (147 mL, 862 mmol, 5.4 eq.) was added dropwise from the dropping funnel. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 219 mL, 2.2 eq.) was added dropwise from the dropping funnel over 1 hour and 20 minutes. The internal temperature during the dropwise addition was 15.5 to 18.5°C. One hour after the end of the dropwise addition, a sample was taken, and the reaction completion was confirmed by HPLC analysis. A 5% aqueous sodium carbonate solution (300 mL) was slowly added from the dropping funnel. The internal temperature during the dropwise addition was kept below 22.8°C. After the dropwise addition, the external temperature was set to 23°C. After stirring for 15 minutes or more, the mixture was allowed to stand, and the aqueous layer was removed. 5% aqueous sodium hydrogen sulfate solution (300mL) was added, and the mixture was stirred for 10 minutes or more, and the aqueous layer was removed. Separation washing with 5% aqueous sodium hydrogen sulfate solution (300mL) was repeated two more times. 5% aqueous sodium carbonate solution (300mL) was added, and the mixture was stirred for 10 minutes or more, and the aqueous layer was removed. 5% aqueous sodium chloride solution (300mL) was added, and the mixture was stirred for 10 minutes or more, and the aqueous layer was removed. Separation washing with 5% aqueous sodium chloride solution (300mL) was repeated once more. The obtained organic layer was concentrated under reduced pressure conditions. 2-Methyltetrahydrofuran (229mL) was added to the obtained solution of compound P7 (175.41g) to prepare a solution with a concentration of 0.189g / g. HPLC purity: 99.37% Measurement method: HPLC Method B Retention time: 3.78 minutes Mass spectrometry: m / z 385.32([M-tBu+H] + )

[0169] Example 16: One-pot deprotection and monoethylation of Cbz-Phe(4-Me)-Sar-OtBu on a 30 gram scale [ka] The inside of a 1L pressure-resistant reaction vessel with a stirring blade was replaced with nitrogen, and 5% palladium carbon (50% wet, 20.30g, 4.77mmol, 7mol% metal Pd basis) and TsOH·H2O (12.95g, 68.1mmol, 1.0eq.) were added. A 2-methyltetrahydrofuran solution of compound P7 (concentration 0.189g / g, 159.0g solution, 30.0g substrate, 68.1mmol) was added, and 2-methyltetrahydrofuran (150mL) and acetonitrile (35.6mL, 681mmol, 10.0eq.) were added in sequence. The atmosphere was replaced with nitrogen and hydrogen while stirring, and the reaction was carried out at 30℃. Hydrogen was supplied so that the pressure inside the vessel during the reaction was maintained at 2-4atm. Degassing was carried out for 1 minute at a reduced pressure of 150-300torr at 1 hour, 2 hours, and 3 hours after the start of the reaction. At the point when the reaction time was 5 hours, 5% palladium carbon (50% wet, 8.70 g, 2.04 mmol, 3 mol% metal Pd basis) was added. At the point when the reaction time was 11 hours, the mixture was replaced with nitrogen and left to stand under a nitrogen atmosphere for 12 hours. After storage, the mixture was replaced with hydrogen and the reaction was resumed, and 7 hours after the restart, the reaction was terminated by replacing with nitrogen. The palladium carbon was filtered off by vacuum suction, and the filtered palladium carbon was washed three times with 2-methyltetrahydrofuran (90 mL). The filtrate and the washings were mixed, and the mixture was washed twice with a 5% aqueous sodium carbonate solution (150 mL) using a separatory funnel. The organic layer after washing was concentrated under reduced pressure to obtain the target compound 7-C. The yield was 93% through the two steps from compound P6. The analytical results during and after the reaction are shown in Table 17. [Table 17] Measurement method: HPLC Method B Retention time: 2.23 min (compound 7), 2.32 min (compound 7-C), 2.46 min (compound 7-D) Mass spectrometry: m / z 307.26 (compound 7, [M+H] + ), 335.34 (compound 7-C, [M+H] + ), 363.38 (compound 7-D, [M+H] + )

[0170] (Example 17) Evaluation of methylating reagents in the monomethylation reaction of H-Phe-OtBu [ka] A flask containing a stirrer was charged with 5% palladium on carbon (50% wet, 72 mg, 0.017 mmol, 5 mol% on Pd metal basis) and a solution of H-Phe-OtBu in tetrahydrofuran (20 mL / g substrate, 1.5 mL, 0.339 mmol). TEA (71 μL, 0.508 mmol, 1.5 eq.) and a methylation reagent (0.407 mmol, 1.2 eq.) were added in that order. The atmosphere was replaced with nitrogen and then with hydrogen while stirring, and the reaction was carried out. The ratio of the raw material, monomethylated product, and dimethylated product was confirmed by HPLC analysis. [Table 18] Measurement method: HPLC Method A Retention time: H-Phe-OtBu: 2.75 min, mono-Me: 2.84 min, di-Me: 2.98 min Mass spectrometry: H-Phe-OtBu:m / z 166.47 ([M-tBu+H] + ), mono-Me: m / z 236.59 ([M+H] + ),di-Me: m / z 250.61 ([M+H] + )

[0171] (Example 18) Monomethylation of H-Phe-OtBu using SEM-Cl as a methylating agent: 1 gram scale [ka] A flask containing a stir bar was charged with 5% palladium on carbon (50% wet, 1.154 g, 0.271 mmol, 6 mol% on Pd metal basis) and a 2-methyltetrahydrofuran solution of H-Phe-OtBu (10 mL / g substrate, 10.0 mL, 4.52 mmol). TEA (0.945 mL, 6.78 mmol, 1.5 eq.) and DBU (68 μL, 0.452 mmol, 0.1 eq.) were added in sequence, and finally SEM-Cl (0.96 mL, 5.42 mmol, 1.2 eq.) was added. The atmosphere was replaced with nitrogen and then with hydrogen while stirring, and the reaction was carried out at 30°C. After 8 hours, a sample was taken and the ratio of the raw material, monomethylated product, and dimethylated product was confirmed by HPLC analysis. [Table 19] Measurement method: HPLC Method A Retention time: H-Phe-OtBu: 2.82 min, mono-Me: 2.90 min, di-Me: 3.06 min Mass spectrometry: H-Phe-OtBu:m / z 166.42 ([M-tBu+H] + ), mono-Me: m / z 236.53 ([M+H] + ),di-Me: m / z 250.55 ([M+H] + )

[0172] (Example 19) Monomethylation reaction of H-Phe-OtBu: Comparative experiment using aqueous formaldehyde solution as a methylation reagent [ka] H-Phe-OtBu (75 mg, 0.339 mmol) was added to a flask containing a stirrer, followed by tetrahydrofuran (1.5 mL). 5% palladium carbon (50% wet, 72 mg, 0.017 mmol, 5 mol% on Pd metal basis) and 37% formaldehyde aqueous solution (25.1 μL, 0.339 mmol, 1.0 eq.) were added in that order. The atmosphere was replaced with nitrogen and then with hydrogen while stirring, and the reaction was carried out at 25°C. After 6 hours, the mixture was sampled, and the ratio of the raw material, monomethylated product, and dimethylated product was confirmed by HPLC analysis. [Table 20] Measurement method: HPLC Method A Retention time: H-Phe-OtBu: 2.75 min, mono-Me: 2.85 min, di-Me: 2.98 min Mass spectrometry: H-Phe-OtBu: m / z 166.47 ([M-tBu+H] + ), mono-Me: m / z 236.59 ([M+H] + ),di-Me: m / z 250.61 ([M+H] + )

[0173] Example 20: Evaluation of reducing agents other than hydrogen gas in the monomethylation reaction of H-Phe-OtBu [ka] A flask containing a stirrer was charged with 5% palladium on carbon (50% wet, 231 mg, 0.054 mmol, 6 mol% on Pd metal basis) and a solution of H-Phe-OtBu in tetrahydrofuran (10 mL / g substrate, 2.0 mL, 0.904 mmol). TEA (189 μL, 1.356 mmol, 1.5 eq.) was added. SEM-Cl (192 μL, 1.085 mmol, 1.2 eq.) and a reducing agent (1.356 mmol, 1.5 eq.) were added in sequence. The atmosphere was replaced with nitrogen while stirring and the reaction was carried out at 30°C. After 5 hours, a sample was taken and the ratio of the raw material, monomethylated product, and dimethylated product, as well as the amount of impurities, were confirmed by HPLC analysis. [Table 21] Measurement method: HPLC Method A Retention time: H-Phe-OtBu: 2.80 min, mono-Me: 2.88 min, di-Me: 3.03 min Mass spectrometry: H-Phe-OtBu: m / z 166.36 ([M-tBu+H] + ), mono-Me: m / z 236.53 ([M+H] + ),di-Me: m / z 250.55 ([M+H] + )

[0174] (Example 21) One-pot deprotection and monomethylation of Cbz-Val-MeAsp(OtBu)-NMe2 [ka] A flask containing a stir bar was charged with 5% palladium carbon (50% wet, 46 mg, 0.004 mmol, 2 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Val-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 1.5 mL, 0.216 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and 5% palladium carbon (50% wet, 46 mg, 0.011 mmol, 5 mol% on Pd metal basis), TEA (45 μL, 0.324 mmol, 1.5 eq.), and SEM-Cl (46 μL, 0.259 mmol, 1.2 eq.) were added in that order. The mixture was purged with nitrogen and hydrogen while stirring, and the methylation reaction was carried out at 30°C. Sampling was performed after 8.5 and 10 hours, and the ratios of the raw material, NH2, monomethylated and dimethylated compounds were confirmed by HPLC analysis. [Table 22] Measurement method: HPLC Method A Retention time: NH2: 2.34 min, mono-Me: 2.39 min, di-Me: 2.48 min Mass spectrometry: NH2: m / z 330.75 ([M+H] + ), mono-Me: m / z 344.94 ([M+H] + ), di-Me: m / z 358.80 ([M+H] + )

[0175] (Example 22) One-pot deprotection and monomethylation of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 21 mg, 0.005 mmol, 2 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu (15 mL / g substrate, 2.0 mL, 0.250 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and 5% palladium carbon (50% wet, 85 mg, 0.020 mmol, 8 mol% on Pd metal basis), TEA (70 μL, 0.500 mmol, 2.0 eq.), and SEM-Cl (58 μL, 0.325 mmol, 1.3 eq.) were added in that order. The mixture was purged with nitrogen and hydrogen while stirring, and the methylation reaction was carried out at 30°C. Sampling was performed after 8 and 10 hours, and the ratios of the raw material, NH2, monomethylated, and dimethylated compounds were confirmed by HPLC analysis. [Table 23] Measurement method: HPLC Method A Retention time: mono-Me: 3.33 min, di-Me: 3.40 min Mass spectrometry: mono-Me: m / z 420.93 ([M+H] + ),di-Me: m / z 434.89([M+H] + )

[0176] (Example 23) One-pot deprotection and monomethylation of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu: Comparative experiment using aqueous formaldehyde as a methylation reagent [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 53 mg, 0.013 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu (15 mL / g substrate, 2.0 mL, 0.250 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and a 37% aqueous formaldehyde solution (22.2 μL, 0.300 mmol, 1.2 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring, and the methylation reaction was carried out at 25°C. After 7 hours, the mixture was sampled, and the ratios of the raw material, NH2, monomethylated, and dimethylated products were confirmed by HPLC analysis. [Table 24] Measurement method: HPLC Method A Retention time: mono-Me: 3.34 min, di-Me: 3.41 min Mass spectrometry: mono-Me: m / z 420.93 ([M+H] + ),di-Me: m / z 434.84([M+H] + )

[0177] (Example 24) One-pot deprotection and monomethylation of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A flask containing a stir bar was charged with 5% palladium carbon (50% wet, 12 mg, 0.003 mmol, 2 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.138 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and 5% palladium carbon (50% wet, 47 mg, 0.011 mmol, 8 mol% on Pd metal basis), TEA (39 μL, 0.276 mmol, 2.0 eq.), and SEM-Cl (39 μL, 0.276 mmol, 1.5 eq.) were added in that order. The mixture was purged with nitrogen and hydrogen while stirring, and the methylation reaction was carried out at 35°C. After 8 hours, a sample was taken and the ratios of the raw material, NH2, monomethylated and dimethylated compounds were confirmed by HPLC analysis. [Table 25] Measurement method: HPLC Method A Retention time: NH2: 4.22 min, mono-Me: 4.28 min, di-Me: 4.40 min Mass spectrometry: NH2: m / z 843.93 ([M+H] + ), mono-Me: m / z 858.35 ([M+H] + ), di-Me: m / z 872.20 ([M+H] + )

[0178] (Example 25) One-pot deprotection and monomethylation of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2: Comparative experiment using aqueous formaldehyde as a methylation reagent [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 29 mg, 0.007 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.138 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and a 37% aqueous formaldehyde solution (12.3 μL, 0.166 mmol, 1.2 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring, and the methylation reaction was carried out at 25°C. After 7 hours, the mixture was sampled, and the ratios of the raw material, NH2, monomethylated, and dimethylated products were confirmed by HPLC analysis. [Table 26] Measurement method: HPLC Method A Retention time: Cbz-NHR: 6.00 min, NH2: 4.21 min, mono-Me: 4.29 min, di-Me: 4.38 min Mass spectrometry: Cbz-NHR: m / z 1000.23 ([M+Na] + ),NH2: m / z 844.16 ([M+H] + ), mono-Me: m / z 858.35 ([M+H] + ),di-Me: m / z 872.43 ([M+H] + )

[0179] (Example 26) Monopropylation of H-Phe-OtBu: Example using triethylsilane as a reducing agent [ka] H-Phe-OtBu (84 mg, 0.380 mmol) was added to a flask containing a stirrer, followed by tetrahydrofuran (2.0 mL). 5% palladium carbon (50% wet, 82 mg, 0.015 mmol, 5 mol% on Pd metal basis) was added. n-Propanal (36 μL, 0.494 mmol, 1.3 eq.) and triethylsilane (303 μL, 1.899 mmol, 5.0 eq.) were added in sequence, and the reaction was carried out at 25°C while stirring and replacing with nitrogen. After 2.5 hours, a sample was taken, and the ratio of the raw material, NH2, monopropylated product, and dipropylated product was confirmed by HPLC analysis. [Table 27] Measurement method: HPLC Method A Retention time: H-Phe-OtBu: 2.70 min, mono-Pr: 3.12 min, di-Pr: 3.61 min Mass spectrometry: H-Phe-OtBu: m / z 166.47 ([M-tBu+H] + ), mono-Pr: m / z 264.74 ([M+H] + ),di-Pr: m / z 306.81 ([M+H] + )

[0180] (Example 27) One-pot deprotection and monopropylation of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 53 mg, 0.013 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu (15 mL / g substrate, 2.0 mL, 0.250 mmol). The mixture was purged with nitrogen and hydrogen while stirring to carry out the de-Cbz reaction. After 1 hour, the mixture was degassed under reduced pressure and n-propanal (27 μL, 0.375 mmol, 1.5 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring to carry out the propylation reaction. After 5 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of the raw material, NH2, monopropylated, and dipropylated products, as well as the amount of impurities. Two experiments were carried out, one with and one without the addition of DBU (3.7 μL, 0.025 mmol, 0.1 eq.) just before the addition of n-propanal, and the results were compared. [Table 28] Measurement method: HPLC Method A Retention time: NH2: 3.30 min, mono-Pr: 3.59 min, di-Pr: 3.95 min Mass spectrometry: NH2: m / z 406.85 ([M+H] + ), mono-Pr: m / z 449.03 ([M+H] + ), di-Pr: m / z 490.92 ([M+H] + )

[0181] (Example 28) One-pot deprotection and monopropylation of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 29 mg, 0.014 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.138 mmol). The mixture was purged with nitrogen and hydrogen while stirring to carry out the de-Cbz reaction. After 1 hour, the mixture was degassed under reduced pressure and n-propanal (13 μL, 0.180 mmol, 1.3 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring to carry out the propylation reaction. After 7 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of the raw material, NH2, monopropylated, and dipropylated products, as well as the amount of impurities. Two experiments were carried out, one with and one without adding DBU (2.1 μL, 0.014 mmol, 0.1 eq.) immediately before the addition of n-propanal, and the results were compared. [Table 29] Measurement method: HPLC Method A Retention time: mono-Pr: 4.47 min, di-Pr: 4.79 min Mass spectrometry: mono-Pr: m / z 886.34 ([M+H] + ),di-Pr: m / z 928.41 ([M+H] + )

[0182] (Example 29) One-pot deprotection and monobutylation of Cbz-Val-MeAsp(OtBu)-NMe2 [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 124 mg, 0.029 mmol, 10 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Val-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.291 mmol). Butyronitrile (253 μL, 2.91 mmol, 10.0 eq.) was added, and the atmosphere was replaced with nitrogen and then hydrogen while stirring, and the reaction was carried out at 30°C. After 8 hours, a sample was taken, and the ratio of the raw material, NH2, monobutylated product, and dibutylated product was confirmed by HPLC analysis. [Table 30] Measurement method: HPLC Method A Retention time: NH2: 2.28 min, mono-Bu: 2.86 min, di-Bu: 3.49 min Mass spectrometry:NH2:m / z 330.75 ([M+H] + ), mono-Bu: m / z 386.90 ([M+H] + ), di-Bu: m / z 442.99 ([M+H] + )

[0183] (Example 30) One-pot deprotection and monobutylation of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 53 mg, 0.013 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu (15 mL / g substrate, 2.0 mL, 0.250 mmol). The mixture was purged with nitrogen and hydrogen while stirring to carry out the Cbz removal reaction. After 1 hour, the mixture was degassed under reduced pressure and n-butanal (33 μL, 0.375 mmol, 1.5 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring to start the butylation reaction. After 5 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of the raw material, NH2, monobutylated, and dibutylated products, as well as the amount of impurities. Two experiments were carried out, one with and one without the addition of DBU (3.7 μL, 0.025 mmol, 0.1 eq.) just before the addition of n-butanal, and the results were compared. [Table 31] Measurement method: HPLC Method A Holding time: NH2: 3.30 min, mono-Bu: 3.80 min, di-Bu: 4.35 min Mass spectrometry: NH2: m / z 406.85 ([M+H] + ), mono-Bu: m / z 463.11 ([M+H] + ), di-Bu: m / z 519.03 ([M+H] + )

[0184] (Example 31) One-pot deprotection and monobutylation of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 12 mg, 0.003 mmol, 2 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.138 mmol). The mixture was purged with nitrogen and hydrogen while stirring, and the de-Cbz reaction was carried out at 25°C. After 1 hour, the mixture was degassed under reduced pressure, and 5% palladium carbon (50% wet, 47 mg, 0.011 mmol, 8 mol% on Pd metal basis) and butyronitrile (120 μL, 1.382 mmol, 10.0 eq.) were added in that order. The mixture was purged with nitrogen and hydrogen while stirring, and the butylation reaction was carried out at 30°C. After 10 hours, a sample was taken and the ratio of the raw material, NH2, monobutylated and dibutylated compounds was confirmed by HPLC analysis. [Table 32] Measurement method: HPLC Method A Retention time: mono-Bu: 4.56 min Mass spectrometry: mono-Bu: m / z 900.31 ([M+H] + )

[0185] (Example 32) One-pot deprotection and monohexylation of Cbz-Val-MeAsp(OtBu)-NMe2 [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 62 mg, 0.015 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Val-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.291 mmol). The mixture was purged with nitrogen and hydrogen while stirring to carry out the de-Cbz reaction. After 1 hour, the mixture was degassed under reduced pressure, and n-hexanal (46 μL, 0.379 mmol, 1.3 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring to carry out the hexylation reaction at 25°C. After 6 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of the raw material, NH2, monohexyl, and dihexyl, as well as the amount of impurities. Two experiments were carried out, one with and one without the addition of DBU (4.4 μL, 0.029 mmol, 0.1 eq.) immediately before the addition of n-hexanal, and the results were compared. [Table 33] Measurement method: HPLC Method A Retention time: NH2: 2.32 min, mono-Hex: 3.42 min, di-Hex: 4.47 min Mass spectrometry: NH2: m / z ([M+H] + ), mono-Hex: m / z 415.11 ([M+H] + ), di-Hex: m / z499.02 ([M+H] + )

[0186] (Example 33) One-pot deprotection and monohexylation of Cbz-Val-MeAsp(OtBu)-NMe2: Example using triethylsilane as a reducing agent [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 62 mg, 0.015 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Val-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.291 mmol). n-Hexanal (46 μL, 0.379 mmol, 1.3 eq.) and triethylsilane (233 μL, 1.456 mmol, 5.0 eq.) were added in sequence, and the reaction was carried out at 25°C under nitrogen replacement with stirring. After 7 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of raw material, NH2, monohexyl, and dihexyl, as well as the amount of impurities. [Table 34] Measurement method: HPLC Method A Retention time: mono-Hex: 3.44 min, di-Hex: 4.47 min Mass spectrometry: mono-Hex: m / z 415.00 ([M+H] + ),di-Hex: m / z 499.02 ([M+H] + )

[0187] (Example 34) One-pot deprotection and monohexylation of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A flask containing a stirrer was charged with 5% palladium carbon (50% wet, 29 mg, 0.014 mmol, 5 mol% on Pd metal basis) and a tetrahydrofuran solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (15 mL / g substrate, 2.0 mL, 0.138 mmol). The mixture was purged with nitrogen and hydrogen while stirring to carry out the de-Cbz reaction. After 1 hour, the mixture was degassed under reduced pressure and n-hexanal (22 μL, 0.180 mmol, 1.3 eq.) was added. The mixture was purged with nitrogen and hydrogen while stirring to carry out the hexylation reaction at 25°C. After 6 hours, the mixture was sampled and analyzed by HPLC to confirm the ratio of the raw material, NH2, monohexyl, and dihexyl, as well as the amount of impurities. Two experiments were carried out, one with and one without the addition of DBU (2.1 μL, 0.014 mmol, 0.1 eq.) immediately before the addition of n-hexanal, and the results were compared. [Table 35] Measurement method: HPLC Method A Retention time: mono-Hex: 4.87 min, di-Hex: 5.54 min Mass spectrometry: mono-Hex: m / z 928.41([M+H] + ),di-Hex: m / z 1012.54 ([M+H] + )

[0188] (Example 35) Monoethylation of H-Phe(4-Me)-OH [ka] H-Phe(4-Me)-OH (100mg, 0.558mmol) was added to a flask containing a stirrer, followed by ethanol (2.0mL). 2M NaOH aqueous solution (0.265mL, 0.530mmol, 0.95eq.) was added, and the substrate was dissolved in the solution. 5% palladium carbon (50% wet, 119mg, 0.028mmol, 5mol% on Pd metal basis) and acetonitrile (0.291mL, 5.58mmol, 10.0eq.) were added. The atmosphere was replaced with nitrogen and then with hydrogen while stirring, and the ethylation reaction was carried out at 30°C. After 7 hours, a sample was taken, and the ratio of the raw material, NH2, monoethylated product, and diethylated product was confirmed by HPLC analysis. [Table 36] Measurement method: HPLC Method A Retention time: mono-Et: 2.00 min, di-Et: 2.29 min Mass spectrometry: mono-Et: m / z 208.77 ([M+H] + ), di-Et: m / z 236.76([M+H] + )

[0189] (Example 36) Monopropylation reaction of H-Phe(4-Me)-OH [ka] H-Phe(4-Me)-OH (100 mg, 0.558 mmol) was added to a flask containing a stirrer, followed by the addition of tetrahydrofuran (2.0 mL). 2M NaOH aqueous solution (x eq., see Table 37) was added, and the substrate was dissolved in the solution, to which 5% palladium carbon (50% wet, 119 mg, 0.028 mmol, 5 mol% on Pd metal basis) was added. A basic additive (y eq., see Table 37) and n-propanal (52 μL, 0.725 mmol, 1.3 eq.) were added. The atmosphere was replaced with nitrogen and then with hydrogen while stirring, and the propylation reaction was carried out at 25°C. The ratio of the raw material, NH2 form, monoethylated form, and diethylated form was confirmed by HPLC analysis. The effect of adding an equivalent amount of NaOH and Et3N (78 μL, 1.0 eq., 0.558 mmol) or DBU (8.3 μL, 0.056 mmol, 0.1 eq.) was evaluated. [Table 37] Measurement method: HPLC Method A Retention time: H-Phe(4-Me)-OH: 1.88 min, mono-Pr: 2.23 min, di-Pr: 3.00 min Mass spectrometry: H-Phe(4-Me)-OH: m / z 180.50 ([M+H] + ), mono-Pr: m / z 222.68 ([M+H] + ), di-Pr: m / z 264.63 ([M+H] + )

[0190] (Raw Material Synthesis 5) Synthesis of Cbz-Val-MeAsp(OtBu)-NMe2 [ka] The solution of H-MeAsp(OtBu)-NMe2 obtained by the synthesis method described in Example 49 was concentrated under reduced pressure to remove the solvent, and 4.26 g of oil was obtained. 2-Methyltetrahydrofuran (43 mL) was added to make a solution, and Cbz-Val-OH (5.11 g, 20.35 mmol, 1.1 eq.) was added. The flask was cooled in an ice bath, and DIPEA (12.9 mL, 74.0 mmol, 4.0 eq.) was added. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 23 mL, 37.0 mmol, 2.0 eq.) was added dropwise with a syringe over 10 minutes. The internal temperature during the dropwise addition was maintained at 8.0 to 19.0 °C. One hour after the end of the dropwise addition, the mixture was sampled, and the reaction completion was confirmed by HPLC analysis. 5% aqueous sodium carbonate solution (34 mL) was added dropwise and stirred, then transferred to a separatory funnel and allowed to stand, and the aqueous layer was removed. A 5% aqueous solution of sodium hydrogen sulfate (34 mL) was added, the mixture was shaken well, and then allowed to stand, and the aqueous layer was removed. Separation washing with a 5% aqueous solution of sodium hydrogen sulfate (34 mL) was repeated once more. A 5% aqueous solution of sodium carbonate (34 mL) was added, the mixture was shaken well, and then allowed to stand, and the aqueous layer was removed. The resulting organic layer was concentrated under reduced pressure to obtain 9.0 g of a crude product. The resulting crude product was purified by silica gel column chromatography to obtain 7.0 g of Cbz-Val-MeAsp(OtBu)-NMe2. Yield: 82% HPLC purity: 100% Measurement method: HPLC Method A Holding time: 4.45 min Mass spectrometry: m / z 486.76 ([M+Na] + )

[0191] (Raw Material Synthesis 6) Synthesis of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu [ka] H-EtPhe(4-Me)-Sar-OtBu hydrochloride (6.0 g, 16.18 mmol) obtained by the synthesis method described in Example 38 was suspended in 2-methyltetrahydrofuran (100 mL) in a separatory funnel, washed with 5% aqueous sodium carbonate solution (100 mL), and the aqueous layer was removed. Separation and washing with 5% aqueous sodium carbonate solution (100 mL) was repeated once more. The obtained organic layer was concentrated under reduced pressure to obtain 4.81 g of H-EtPhe(4-Me)-Sar-OtBu. 2-Methyltetrahydrofuran (48 mL) was added to make a solution, and Cbz-Ala-OH (3.52 g, 15.79 mmol, 1.1 eq.) was added. DIPEA (10.0 mL, 57.4 mmol, 4.0 eq.) was added, and a solution of propylphosphonic anhydride in 2-methyltetrahydrofuran (1.6 M, 18 mL, 28.7 mmol, 2.0 eq.) was added dropwise over 10 minutes using a syringe. After the dropwise addition, the mixture was stirred at room temperature for 3 hours. After 5% aqueous sodium carbonate solution (40 mL) was added dropwise and stirred, the mixture was transferred to a separatory funnel and allowed to stand, and the aqueous layer was removed. After separation and washing with 5% aqueous sodium hydrogen sulfate solution (40 mL) was performed four times, separation and washing with 5% aqueous sodium carbonate solution (40 mL) was performed twice. The obtained organic layer was concentrated under reduced pressure to obtain 5.26 g of a crude product. The obtained crude product was purified by silica gel column chromatography to obtain 3.14 g of Cbz-Ala-EtPhe(4-Me)-Sar-OtBu. Yield: 40% HPLC purity: 99.5% Measurement method: HPLC Method A Holding time: 5.50 min Mass spectrometry: m / z 562.86 ([M+Na] + )

[0192] (Raw material synthesis 7)Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] The solution of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 obtained by the synthesis method described in Example 59 was concentrated under reduced pressure to remove the solvent, and 6.1 g of oil was obtained. The obtained crude product was purified by silica gel column chromatography to obtain 4.4 g of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2. Yield: 88% HPLC purity: 100% Measurement method: HPLC Method A Holding time: 6.00 min Mass spectrometry: m / z 1000.23 ([M+Na] + )

[0193] (Example 37) Synthesis of Cbz-Phe(4-Me)-Sar-OtBu [ka] Cbz-Phe(4-Me)-OH (17.22g, 55.0mmol), H-Sar-OtBu hydrochloride (11.06g, 59.7mmol), 2-methyltetrahydrofuran (141g) and DIPEA (37.91g, 293mmol) were added to a reaction vessel at 25°C. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (50.4wt%, 75.31g, 119mmol) was added dropwise over 1 hour and 30 minutes. After stirring for 2 hours after the dropwise addition, the mixture was sampled and the reaction completion was confirmed by HPLC analysis. A 5% aqueous sodium carbonate solution (102g) was added dropwise over 40 minutes. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous layer was removed. The obtained organic layer was separated and washed with a 5% aqueous solution of sodium hydrogen sulfate monohydrate (102 g, twice), a 5% aqueous solution of sodium carbonate (102 g), and a 5% aqueous solution of sodium chloride (102 g, twice), and then concentrated under reduced pressure to obtain a solution containing Cbz-Phe(4-Me)-Sar-OtBu (38.80 g). HPLC purity: 99.87% Measurement method: HPLC Method B Retention time: 4.11 min Mass spectrometry: m / z 441([M+H] + )

[0194] (Example 38) Synthesis of H-EtPhe(4-Me)-Sar-OtBu hydrochloride [ka] A reaction vessel was charged with 5% palladium carbon (55.31% wet, 2.60 g, 0.546 mmol, 1 mol% on Pd metal basis), the solution of Cbz-Phe(4-Me)-Sar-OtBu obtained in Example 37 (38.80 g), 2-methyltetrahydrofuran (179 g), acetonitrile (22.29 g, 543 mmol, 10.0 eq.), TsOH·H2O (10.85 g, 57.0 mmol, 1.0 eq.) and water (1.03 g, 56.9 mmol). The atmosphere was replaced with nitrogen at 25°C, replaced with hydrogen, and stirred for 2 hours under a hydrogen atmosphere (0.20 MPaG), after which a sample was taken and it was confirmed by HPLC analysis that Cbz-Phe(4-Me)-Sar-OtBu had disappeared. The reaction vessel was replaced with nitrogen, and 5% palladium carbon (55.31% wet, 15.50 g, 3.26 mmol, 6 mol% on Pd metal basis) was added. The reaction vessel was replaced with hydrogen, and then the temperature was raised to 33°C. After stirring for 7 hours at 33°C under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and HPLC analysis confirmed that the reaction rate was 99%. The reaction mixture was filtered, and the cake was washed three times in total with 2-methyltetrahydrofuran (twice with 68 g and once with 51 g). The mixed solution of the filtrate and the washings was washed twice with a 5% aqueous sodium carbonate solution (119 g), and the resulting organic layer was concentrated under reduced pressure. 2-Methyltetrahydrofuran (27 g) was added, and the mixture was concentrated to 34 mL under reduced pressure, and then 2-methyltetrahydrofuran (12 g) was added. To the resulting solution, pyridine hydrochloride (6.27 g, 54.3 mmol, 1.0 eq.) dissolved in acetonitrile (19 g) was added dropwise over 70 min to precipitate crystals of H-EtPhe(4-Me)-Sar-OtBu hydrochloride. Acetonitrile (6.8 g) was added, followed by H-EtPhe(4-Me)-Sar-OtBu hydrochloride crystals (17.04 mg) obtained in a separate experiment performed in the same manner as above. After stirring for 30 min, MTBE (73 g) was added dropwise over 60 min. After stirring for 60 min, MTBE (122 g) was added dropwise over 60 min. After stirring for 14 h, the slurry was filtered.The solid obtained was washed with 2-methyltetrahydrofuran (68 g), and then washed with a mixed solution of 2-methyltetrahydrofuran (34 g) and MTBE (34 g). The solid obtained was dried under reduced pressure to obtain H-EtPhe(4-Me)-Sar-OtBu hydrochloride (16.62 g). Melting point of H-EtPhe(4-Me)-Sar-OtBu hydrochloride: 196°C Yield: 82% (yield in two steps from Cbz-Phe(4-Me)-Sar-OtBu) HPLC purity: 100% Measurement method: HPLC Method B Holding time: 2.51 min Mass spectrometry: m / z 335([M+H] + )

[0195] Example 39: Synthesis of Cbz-Aze-EtPhe(4-Me)-Sar-OtBu [ka] Cbz-Aze-OH (12.94 g, 55.0 mmol), 2-methyltetrahydrofuran (132 g), H-EtPhe(4-Me)-Sar-OtBu hydrochloride (17.01 g, 45.9 mmol) synthesized by the same method as in Example 38, and DIPEA (47.41 g, 367 mmol) were added to a reaction vessel at room temperature. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (50.4 wt%, 87.00 g, 137 mmol) was added at 25 ° C. over 1 hour and 30 minutes. After the addition was completed, the mixture was stirred for 2 hours, then sampled, and the reaction completion was confirmed by HPLC analysis. A 5% aqueous sodium carbonate solution (155 g) was added, stirred for 20 minutes, and then allowed to stand, and the aqueous layer was removed. The obtained organic layer was separated and washed with 4% aqueous sulfuric acid solution (156 g), 10% aqueous potassium hydrogen sulfate solution (155 g), and 5% aqueous sodium carbonate solution (155 g), and then 2-methyltetrahydrofuran (43 g) was added and concentrated under reduced pressure three times. 2-Methyltetrahydrofuran (29 g) was added to the obtained residue to obtain a solution containing Cbz-Aze-EtPhe(4-Me)-Sar-OtBu (78.24 g). HPLC purity: 98.22% Measurement method: HPLC Method B Holding time: 4.14 min Mass spectrometry: m / z 552([M+H] + )

[0196] (Example 40) Synthesis of H-Aze-EtPhe(4-Me)-Sar-OtBu [ka] A reaction vessel was charged with 10% palladium carbon (55.65% wet, 7.76 g, 3.21 mmol, 7 mol% on Pd metal basis) and 2-methyltetrahydrofuran (39 g). The atmosphere was replaced with nitrogen and hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. The solution of Cbz-Aze-EtPhe(4-Me)-Sar-OtBu obtained in Example 39 (78.24 g), 2-methyltetrahydrofuran (29 g) and water (1.71 g, 94.9 mmol) were added. After stirring for 3 hours under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with 2-methyltetrahydrofuran (61 g). The mixed solution of the filtrate and washings was concentrated under reduced pressure, and then 2-methyltetrahydrofuran (34 g) was added and concentrated under reduced pressure, and this operation was repeated twice. To the resulting residue, 2-methyltetrahydrofuran (34 g) was added to obtain a solution (70.59 g) containing H-Aze-EtPhe(4-Me)-Sar-OtBu. HPLC purity: 96.53% Measurement method: HPLC Method B Holding time: 2.67 min Mass spectrometry: m / z 418([M+H] + )

[0197] (Example 41) Synthesis of Cbz-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu [ka] The solution of H-Aze-EtPhe(4-Me)-Sar-OtBu obtained in Example 40 (70.59 g), Cbz-MeAla-OH (13.13 g, 55.0 mmol), 2-methyltetrahydrofuran (68 g), and N-methylmorpholine (11.60 g, 115 mmol) were added to the reaction vessel at 25 ° C. A solution of propylphosphonic anhydride in 2-methyltetrahydrofuran (50.4 wt%, 69.40 g, 110 mmol) was added at 25 ° C. over 1 hour and 30 minutes. After the addition was completed, the mixture was stirred for 5 hours, and then sampled, and the reaction completion was confirmed by HPLC analysis. After adding a 5% aqueous sodium carbonate solution (177 g), 1-methylimidazole (3.77 g, 45.9 mmol) was immediately added, and the mixture was stirred for 2 hours, then allowed to stand, and the aqueous layer was removed. The obtained organic layer was separated and washed with 4% aqueous sulfuric acid solution (138 g), 10% aqueous potassium hydrogen sulfate solution (138 g), and 5% aqueous sodium carbonate solution (138 g), and then 2-methyltetrahydrofuran (51 g) was added and concentrated under reduced pressure three times. 2-Methyltetrahydrofuran (27 g) was added to the obtained residue to obtain a solution containing Cbz-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (83.89 g). HPLC purity: 97.28% Measurement method: HPLC Method B Holding time: 4.07 min Mass spectrometry: m / z 637([M+H] + )

[0198] Example 42 Synthesis of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu [ka] A reaction vessel was charged with 10% palladium carbon (55.65% wet, 5.54 g, 2.29 mmol, 5 mol% on Pd metal basis) and 2-methyltetrahydrofuran (39 g). The atmosphere was replaced with nitrogen and hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. The solution of Cbz-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu obtained in Example 41 (83.89 g), 2-methyltetrahydrofuran (27 g) and water (2.10 g, 117 mmol) were added. After stirring for 3 hours under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with 2-methyltetrahydrofuran (59 g). The mixture of the filtrate and the washings was concentrated under reduced pressure, and then filtered. The filtrate obtained was added with CPME (85 g) and concentrated to 68 mL under reduced pressure conditions three times. CPME (15 g), MTBE (26 g) and n-heptane (27 g) were added to the residue obtained while stirring at 40° C., and then crystals of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (16.66 mg) obtained by the same method as in Example 66 were added. After stirring at 40° C. for 1 hour, the mixture was cooled to 20° C. over 2 hours. After stirring at 20° C. for 16 hours, n-heptane (241 g) was added over 1 hour. After stirring at 20° C. for 4 hours, the mixture was cooled to 8° C. over 2 hours. After stirring at 8° C. for 16 hours, the slurry was filtered. The obtained solid was washed with n-heptane (66 g) and then dried under reduced pressure to obtain crystals of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (18.28 g). Melting point of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 95°C Yield: 79% (4-step yield from H-EtPhe(4-Me)-Sar-OtBu) HPLC purity: 99.76% Measurement method: HPLC Method B Holding time: 2.62 min Mass spectrometry: m / z 503([M+H] + )

[0199] Example 43 Synthesis of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu [ka] Toluene (92 g), 5% sodium hydrogen sulfate monohydrate aqueous solution (100 g) and Cbz-Ile-OH dicyclohexylamine salt (21.43 g, 47.7 mmol) were added to a reaction vessel with stirring at 25°C. 5% sodium hydrogen sulfate monohydrate aqueous solution (220 g) was added, stirred for 10 minutes, allowed to stand, and the aqueous layer was removed. The obtained organic layer was washed three times with 5% sodium hydrogen sulfate monohydrate aqueous solution (320 g), and then washed twice with 5% sodium chloride aqueous solution (220 g). The obtained organic layer was concentrated to 26 mL under reduced pressure conditions. To the resulting residue, H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (20.2 g, 40.2 mmol), 2-methyltetrahydrofuran (72 g), toluene (62 g), acetonitrile (22 g) and DIPEA (22.63 g, 175 mmol) obtained in the same manner as in Example 42 were added with stirring at 25 ° C., and then HATU (22.69 g, 59.7 mmol) was added with stirring at 22 ° C. After the addition was completed, the mixture was stirred for 2 hours, sampled, and the reaction completion was confirmed by HPLC analysis. 5% aqueous sodium carbonate solution (172 g) and 1-methylimidazole (3.27 g, 39.8 mmol) were added. After stirring for 2 hours at 22 ° C., the temperature was raised to 25 ° C., and 2.5% aqueous ammonia solution (172 g) was added. After stirring for 10 minutes, the mixture was left to stand, and the aqueous layer was removed. The obtained organic layer was washed with 2.5% aqueous ammonia solution (172 g), 4% aqueous sulfuric acid solution (172 g), 10% aqueous sodium hydrogen sulfate monohydrate solution (172 g), and 3% aqueous dipotassium hydrogen phosphate solution (172 g), and then concentrated to 60 mL under reduced pressure. Toluene (52 g) was added, and the operation of concentrating to 60 mL under reduced pressure was repeated twice, and the obtained residue was filtered. Toluene (66 g) was added to the filtrate, and n-heptane (102 g) was added over 10 minutes while stirring at 22 ° C. Crystals of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (285 mg) obtained by the same method as in Example 67 were added, and the mixture was cooled to 18 ° C. over 4 hours, and then cooled to 10 ° C. over another 4 hours. After stirring at 10 ° C. for 18 hours, n-heptane (102 g) was added over 3 hours. After addition was complete, the mixture was stirred at 10° C. for a further 18 hours before filtering the slurry.The obtained solid was washed twice with n-heptane (92 g) and then dried under reduced pressure to obtain crystals of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (28.08 g). Melting point of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu: 70 °C Yield: 93% HPLC purity: 99.84% Measurement method: HPLC Method B Holding time: 4.21 min Mass spectrometry: m / z 750([M+H] + )

[0200] (Example 44) Synthesis of Teoc-MeLeu-OPFP [ka] Teoc-MeLeu-OH (19.34 g, 66.8 mmol), 1,3-dimethyl-2-imidazolidinone (132 g) and pentafluorophenol (15.36 g, 83.4 mmol) were added to a reaction vessel at 25°C. After cooling to 0°C with stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.34 g, 83.7 mmol) was added. The temperature was raised to 25°C over 1 hour, and after stirring at 25°C for another 1 hour, a sample was taken and the completion of the reaction was confirmed by HPLC analysis. Isopropyl acetate (110 g) and 0.5 M aqueous hydrochloric acid solution (126 g) were added in sequence. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous layer was removed. The obtained organic layer was washed with 0.5 M aqueous hydrochloric acid solution (126 g) and then 1,3-dimethyl-2-imidazolidinone (22 g) was added. The obtained organic layer was separated and washed with 5% potassium carbonate aqueous solution (126 g, twice) and 10% sodium chloride aqueous solution (126 g), and then concentrated under reduced pressure to 44.4 mL. Isopropyl acetate (19 g) was added to the obtained residue to obtain a solution containing Teoc-MeLeu-OPFP (67 mL). HPLC purity: 97.95% Measurement method: HPLC Method B Holding time: 5.58 min Mass spectrometry: m / z 428([M-CH2=CH2+H] + )

[0201] Example 45 Synthesis of Teoc-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu [ka] Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (25.00 g, 33.3 mmol) obtained by the same method as in Example 43, a solution containing Teoc-MeLeu-OPFP obtained in Example 44 (67 mL), acetone (70 g), N-methylmorpholine (20.22 g, 200 mmol) and 10% palladium carbon (54.33% wet, 7.84 g, 3.34 mmol, 10 mol% on Pd metal basis) were added to the reaction vessel in sequence. The atmosphere was replaced with nitrogen and then with hydrogen while stirring at 25°C. After stirring for 2 hours under a hydrogen atmosphere (0.18 MPaG), a sample was taken and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed three times with acetone (29 g). The mixed solution of the filtrate and washings was concentrated to 120 mL under reduced pressure conditions. Toluene (87 g), 5% potassium carbonate aqueous solution (110 g), and 4-dimethylaminopyridine (4.07 g, 33.3 mmol) were added to the obtained residue in this order while stirring at 25°C. After stirring for 5 hours, the mixture was allowed to stand and the aqueous layer was removed. The obtained organic layer was separated and washed with 4% sulfuric acid aqueous solution (110 g), 10% potassium hydrogen sulfate aqueous solution (110 g), and 5% potassium carbonate aqueous solution (110 g, twice), and then concentrated under reduced pressure to obtain a solution (47.5 mL) containing Teoc-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu. HPLC purity: 98.61% Measurement method: HPLC Method B Holding time: 5.28 min Mass spectrometry: m / z 888([M+H] + )

[0202] Example 46 Synthesis of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu [ka] A toluene solution (61.47 w / w%, 4.88 g, 3.38 mmol) containing Teoc-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu obtained by the same method as in Example 45 and 2-methyltetrahydrofuran (8.9 mL) were added to the reaction vessel. The temperature was raised to 45°C, and a tetrabutylammonium fluoride tetrahydrofuran solution (1.2 M, 7.04 mL, 8.45 mmol) was added over 20 minutes. After stirring for 1 hour after the addition, the mixture was sampled and the completion of the reaction was confirmed by HPLC analysis. The mixture was cooled to 25°C, and isopropyl acetate (9.0 mL) was added. The reaction mixture was separated and washed with a 5% aqueous sodium carbonate solution (9.13 g, 3 times) and a 5% aqueous sodium chloride solution (9.13 g), and the obtained organic layer was concentrated under reduced pressure. The resulting residue was added with ethanol (12.5 mL) and concentrated twice to give a solution (6.9 mL) containing H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu. HPLC purity: 98.17% Measurement method: HPLC Method B Holding time: 3.04 min Mass spectrometry: m / z 743.4([M+H] + )

[0203] Example 47 Synthesis of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBuL-tartrate [ka] A solution containing H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (38.46 w / w%, 56.16 g, 29.1 mmol) obtained by the same method as in Example 46 and ethanol (8.0 mL) were added to a reaction vessel. L-tartaric acid (4.80 g, 32.0 mmol) was added at 22°C, and MTBE (207 mL) was added over 2 minutes. A slurry of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu L-tartrate crystals (108.0 mg) obtained by the same method as in Example 68 suspended in MTBE (1.62 mL) was added, and then MTBE (77 g) was added over 1 hour. After stirring for 18 hours, n-heptane (106 g) was added dropwise over 1 hour. The internal temperature was cooled to 10°C over 1 hour. After stirring at 10°C for an additional 27 hours, the slurry was filtered. The resulting solid was washed with MTBE (130 mL) and then dried under reduced pressure to obtain H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu L-tartrate (26.13 g, content 92.2 w / w%). The content and yield were calculated by HPLC analysis using a standard sample. Melting point of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBuL-tartrate: 94°C Yield: 88% (3-step yield from H-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu) HPLC purity: 99.38% Measurement method: HPLC Method B Retention time: 3.01 min Mass spectrometry: m / z 743.6([M+H] + )

[0204] (Example 48) Synthesis of Cbz-MeAsp(OtBu)-NMe2 [ka] Cbz-MeAsp(OtBu)-OH dicyclohexylamine salt (25.00 g, 48.2 mmol) and 2-methyltetrahydrofuran (126 g) were added to a reaction vessel at 25° C. After separation and washing with 10% sodium hydrogen sulfate monohydrate aqueous solution (150 g) was repeated twice, washing was performed with 5% sodium chloride aqueous solution (150 g). The obtained organic layer was concentrated under reduced pressure. 2-Methyltetrahydrofuran (95 g) was added to the obtained residue, and the operation of concentrating under reduced pressure was repeated twice. 2-Methyltetrahydrofuran (95 g), acetonitrile (75 g), DIPEA (35.46 g, 274 mmol), and dimethylamine hydrochloride (7.88 g, 96.6 mmol) were added to the obtained residue (47.91 g) at 25° C. A solution of propylphosphonic anhydride in 2-methyltetrahydrofuran (50.4 wt%, 61.33 g, 97.1 mmol) was added dropwise over 1 hour and 30 minutes. After stirring for 1 hour after the end of the dropwise addition, the mixture was sampled and the completion of the reaction was confirmed by HPLC analysis. 2M aqueous sodium hydroxide solution (150 g) was added. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous layer was removed. The obtained organic layer was washed with 2M aqueous sodium hydroxide solution (150 g), 13% aqueous sulfuric acid solution (150 g), 10% aqueous sodium hydrogen sulfate monohydrate solution (150 g), and 5% aqueous sodium carbonate solution (150 g), and then concentrated under reduced pressure. The operation of adding 2-methyltetrahydrofuran (125 g) and concentrating under reduced pressure was repeated twice to obtain a solution containing Cbz-MeAsp(OtBu)-NMe2 (42.39 g). HPLC purity: 99.85% Measurement method: HPLC Method C Holding time: 3.37 min Mass spectrometry: m / z 387([M+Na] + )

[0205] (Example 49) Synthesis of H-MeAsp(OtBu)-NMe2 [ka] A reaction vessel was charged with 10% palladium carbon (54.33% wet, 3.39 g, 1.45 mmol, 3 mol% on Pd metal basis) and 2-methyltetrahydrofuran (75 g). The atmosphere was replaced with nitrogen and hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. The solution of Cbz-MeAsp(OtBu)-NMe2 obtained in Example 48 (42.39 g) and 2-methyltetrahydrofuran (22 g) were added. After stirring for 1 hour and 30 minutes under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with 2-methyltetrahydrofuran (75 g). The mixture of the filtrate and washings was concentrated under reduced pressure to obtain a solution containing H-MeAsp(OtBu)-NMe2 (30.76 g). HPLC purity: 98.64% Measurement method: HPLC Method C Holding time: 1.44 min Mass spectrometry: m / z 231([M+H] + )

[0206] (Example 50) Synthesis of Cbz-MeGcp-MeAsp(OtBu)-NMe2 [ka] The solution of H-MeAsp(OtBu)-NMe2 obtained in Example 49 (30.76 g), Cbz-MeGcp-OH (16.96 g, 58.2 mmol), 2-methyltetrahydrofuran (40 g), acetonitrile (17 g), and DIPEA (27.74 g, 215 mmol) were added to the reaction vessel at 25°C. HATU (27.49 g, 72.3 mmol) was added over 10 minutes. After stirring for 3 hours after the addition, the mixture was sampled and the reaction completion was confirmed by HPLC analysis. Toluene (30 g), 5% aqueous potassium carbonate solution (23 g), and 1-methylimidazole (3.97 g, 48.4 mmol) were added and stirred for 30 minutes. 2.5% aqueous ammonia solution (88 g) and 2-methyltetrahydrofuran (25 g) were added. After stirring for 10 minutes, the mixture was left to stand and the aqueous layer was removed. The organic layer was washed with 2.5% aqueous ammonia (113 g), 10% aqueous sodium hydrogen sulfate monohydrate (113 g, twice), and 5% aqueous potassium carbonate (113 g), then concentrated under reduced pressure. 2-Methyltetrahydrofuran (42 g) was added and concentrated under reduced pressure to obtain a solution containing Cbz-MeGcp-MeAsp(OtBu)-NMe2 (52.78 g). HPLC purity: 98.59% Measurement method: HPLC Method C Holding time: 4.10 min Mass spectrometry: m / z 526([M+Na] + )

[0207] (Example 51) Synthesis of H-MeGcp-MeAsp(OtBu)-NMe2 [ka] A reaction vessel was charged with 10% palladium carbon (54.33% wet, 3.39 g, 1.45 mmol, 3 mol% on Pd metal basis) and 2-methyltetrahydrofuran (75 g). The atmosphere was replaced with nitrogen at 25°C, then replaced with hydrogen, and stirred for 2 hours under a hydrogen atmosphere (0.40 MPaG). A solution of Cbz-MeGcp-MeAsp(OtBu)-NMe2 obtained in Example 50 (52.78 g) and 2-methyltetrahydrofuran (15 g) were added, and the temperature was raised to 30°C. After stirring for 2 hours under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with 2-methyltetrahydrofuran (75 g). The mixture of the filtrate and washings was concentrated under reduced pressure, and then acetonitrile (75 g) was added and concentrated under reduced pressure twice to obtain a solution (42.5 mL) containing H-MeGcp-MeAsp(OtBu)-NMe2. HPLC purity: 96.86% Measurement method: HPLC Method D Holding time: 2.96 min Mass spectrometry: m / z 370([M+H] + )

[0208] (Example 52) Synthesis of H-MeGcp-MeAsp(OtBu)-NMe2 hydrochloride [ka] The solution (42.5 mL) of H-MeGcp-MeAsp(OtBu)-NMe2 obtained in Example 51 and acetonitrile (8.0 g) were added to a reaction vessel. After adding MTBE (65 g) at 40° C., a solution of pyridine hydrochloride in acetonitrile (16.94 w / w%, 4.50 g) was added dropwise over 30 minutes. After stirring for 1 hour, a solution of pyridine hydrochloride in acetonitrile (16.94 w / w%, 31.34 g) was added dropwise over 3 hours and 30 minutes, and acetonitrile (14 g) was added. After stirring for 1 hour, the mixture was cooled to 10° C. over 6 hours. After stirring for an additional 11 hours at 10° C., the slurry was filtered. The obtained solid was washed twice with MTBE (38 g) and then dried under reduced pressure to obtain H-MeGcp-MeAsp(OtBu)-NMe2 hydrochloride (15.68 g). Melting point of H-MeGcp-MeAsp(OtBu)-NMe2 hydrochloride: 227℃ Yield: 80% (yield in 5 steps from Cbz-MeAsp(OtBu)-OH dicyclohexylamine salt) HPLC purity: 99.62% Measurement method: HPLC Method D Holding time: 2.92 min Mass spectrometry: m / z 370([M+H] + )

[0209] (Example 53) Synthesis of Cbz-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] Into a reaction vessel, H-MeGcp-MeAsp(OtBu)-NMe2 hydrochloride (1.00 g, 2.46 mmol) synthesized by the same method as in Example 52 and acetonitrile (10 mL) were added. Then, DIPEA (2.72 mL, 15.6 mmol), Cbz-cLeu-OH (1.74 g, 6.61 mmol) and HATU (2.75 g, 7.23 mmol) were added in sequence while stirring, and the temperature was raised to 50 ° C. After stirring at 50 ° C for 6 hours, a sample was taken and the reaction completion was confirmed by HPLC analysis. 1-Methylimidazole (0.78 mL, 9.78 mmol) and water (3.99 mL) were added. After stirring at 50 ° C for 1 hour, it was cooled to 25 ° C. After stirring at 25 ° C for 14 hours, the slurry was filtered. The obtained solid was washed with a mixed solvent of acetonitrile / water (8:3 (v / v), 5.33 mL) and then dried under reduced pressure to obtain Cbz-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (1.27 g). Melting point of Cbz-cLeu-MeGcp-MeAsp(OtBu)-NMe2: 202°C Yield: 84% HPLC purity: 99.86% Measurement method: HPLC Method D Holding time: 6.70 min Mass spectrometry: m / z 637([M+Na] + )

[0210] (Example 54) Synthesis of H-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A reaction vessel was charged with 5% palladium carbon (50% wet, 0.85 g, 0.20 mmol, 3.5 mol% on Pd metal basis) and tetrahydrofuran (14.0 mL). The atmosphere was replaced with nitrogen and hydrogen at 25°C, and the mixture was stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. A solution of Cbz-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (3.51 g, 5.71 mmol) synthesized by the same method as in Example 53 dissolved in tetrahydrofuran (42 mL) was added. After stirring for 2 hours under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. After filtering the reaction mixture, the cake was washed twice with tetrahydrofuran (14 mL). The mixture of the filtrate and washings was concentrated under reduced pressure to obtain a solution (7.37 g) containing H-cLeu-MeGcp-MeAsp(OtBu)-NMe2. HPLC purity: 99.98% Measurement method: HPLC Method C Holding time: 2.42 min Mass spectrometry: m / z 503([M+Na] + )

[0211] (Example 55) Synthesis of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A tetrahydrofuran solution (9.63 g) containing H-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (5.00 g, 10.4 mmol) obtained in the same manner as in Example 54, acetonitrile (25 mL) and Cbz-Pro-OH (3.37 g, 13.5 mmol) were added to a reaction vessel at room temperature. N-Methylmorpholine (3.12 g, 30.8 mmol) and HATU (5.93 g, 15.6 mmol) were added with stirring at 25 ° C. After stirring for 1 hour after the addition, a sample was taken and the completion of the reaction was confirmed by HPLC analysis. Toluene (40 mL) and 1-methylimidazole (0.90 g, 10.9 mmol) were added, and then a 5% aqueous potassium carbonate solution (10 mL) was added at 10 ° C. The mixture was heated to 25 ° C. and stirred for 30 minutes, then cooled to 10 ° C. and 2.5% aqueous ammonia solution (20 mL) was added. The temperature was raised to 25°C, and the mixture was stirred for another 10 minutes, then allowed to stand, and the aqueous layer was removed. The resulting organic layer was washed with 2.5% aqueous ammonia solution (30mL), 3% aqueous sulfuric acid solution (30mL), 10% aqueous potassium hydrogen sulfate solution (30mL), and 5% aqueous sodium carbonate solution (30mL, twice), and then concentrated to 25mL under reduced pressure. Toluene (25mL) was added, and the mixture was concentrated to 25mL under reduced pressure. Tetrahydrofuran (15mL) was added to the resulting residue, and the mixture was stirred at 40-60°C to prepare a homogeneous solution. After cooling to 25°C, a slurry of crystals (49.9 mg) of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 obtained by the same method as in Example 69 suspended in a mixed solution of n-heptane (0.16 mL) and tetrahydrofuran (0.04 mL) was added, followed by a mixed solution of n-heptane (0.32 mL) and tetrahydrofuran (0.08 mL). After stirring at 25°C for 13 hours, n-heptane (5 mL) was added over 17 minutes. After stirring for 1 hour after the addition, n-heptane (5 mL) was added over 15 minutes. After stirring for another hour, n-heptane (25 mL) was added over 16 minutes. After stirring for another 3 hours, the slurry was filtered. The obtained solid was washed twice with a mixed solution of n-heptane (10 mL) and tetrahydrofuran (2.5 mL) and then dried under reduced pressure to obtain crystals of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (6.51 g). Melting point of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2: 178°C Yield: 88% HPLC purity: 100% Measurement method: HPLC Method D Holding time: 6.54 min Mass spectrometry: m / z 734([M+Na] + )

[0212] (Example 56) Synthesis of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] A reaction vessel was charged with 10% palladium carbon (54.33% wet, 1.36 g, 0.568 mmol, 2.2 mol% on Pd metal basis) and tetrahydrofuran (27 mL). The atmosphere was replaced with nitrogen at 25°C, then replaced with hydrogen, and stirred under a hydrogen atmosphere (0.40 MPaG) for 2 hours. Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (18.03 g, 25.3 mmol) synthesized by the same method as in Example 55 and tetrahydrofuran (52 mL) were added. After stirring for 1 hour under a hydrogen atmosphere (0.18 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. After filtering the reaction mixture, the cake was washed three times with 2-methyltetrahydrofuran (38 mL). The mixed solution of the filtrate and washings was concentrated to 81 mL under reduced pressure, and then 2-methyltetrahydrofuran (68 mL) was added and concentrated to 81 mL, and this operation was repeated three times. To the resulting residue, n-heptane (37 mL) was added while stirring at 45° C., and then a slurry of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 crystals (40.60 mg) obtained by the same method as in Example 70 suspended in n-heptane (0.75 mL) and n-heptane (0.75 mL) were added in sequence. After stirring at 45° C. for 2 hours, n-heptane (22 g) was added over 15 minutes. After stirring at 45° C. for an additional 18 hours, n-heptane (109 g) was added over 75 minutes. After stirring at 45° C. for an additional 2 hours, the mixture was cooled to 22° C. over 2 hours and then cooled to 10° C. over 1 hour. After stirring at 10° C. for 16 hours, the slurry was filtered. The obtained solid was washed successively with a mixed solvent of 2-methyltetrahydrofuran / heptane (1:9 (v / w), 47 g) and then with 2-methyltetrahydrofuran (68 mL), and then dried under reduced pressure to obtain crystals of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (12.96 g). Melting point of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2: 147°C Yield: 95.8% HPLC purity: 100% Measurement method: HPLC Method C Holding time: 2.73 min Mass spectrometry: m / z 578.5([M+H] + )

[0213] (Example 57-1) Synthesis of Boc-Hph(3,5-F2-4-CF3)-OBn [ka] A slurry of nickel(II) bromide trihydrate (1.58 g, 5.80 mmol) suspended in 1,3-dimethyl-2-imidazolidinone (160 mL) and 4,4'-di-tert-butyl-2,2'-bipyridine (1.56 g, 5.80 mmol) were added to a nitrogen-substituted reaction vessel. Boc-Glu(NHPI)-OBn (40.0 g, 83.0 mmol) synthesized by the method described in International Publication No. 2020 / 189540 was added with stirring, and then 1,3-dimethyl-2-imidazolidinone (40 mL), 5-bromo-1,3-difluoro-2-(trifluoromethyl)-benzene (26.0 g, 99 mmol) and N-methylmorpholine (22.8 mL, 207 mmol) were added in sequence. After cooling to 10°C, activated zinc (16.26g, 249mmol) was added. TMSCl (21.0mL, 166mmol) was added dropwise over 1 hour while the temperature was raised to 25°C. Immediately after the addition was completed, a sample was taken and the reaction was confirmed to be complete by HPLC analysis. 15% ammonium chloride aqueous solution (416g) was added at 0°C, and the temperature was raised to 25°C and stirred for 50 minutes. Toluene (200mL) was added, the slurry was filtered using Celite, and the cake was washed with toluene (200mL). The resulting solution was stirred for 20 minutes, then allowed to stand, and the aqueous layer was removed. A solution of disodium dihydrogen ethylenediaminetetraacetate dihydrate (31.4g, 84.0mmol) dissolved in 0.1M aqueous potassium hydroxide solution (600mL) was added to the resulting organic layer while stirring. After stirring for 3 hours, the solution was allowed to stand, and the aqueous layer was removed. The obtained organic layer was separated and washed with a 10% aqueous sodium chloride solution (400 mL), and then concentrated under reduced pressure to obtain a solution (90.81 g) containing Boc-Hph(3,5-F2-4-CF3)-OBn. HPLC purity: 78.47% Measurement method: HPLC Method E Holding time: 7.40 min Mass spectrometry: m / z 374([M-Boc+2H] + )

[0214] (Examples 57-1 to 57-3) Synthesis of Boc-Hph(3,5-F2-4-CF3)-OBn: Evaluation of the effect of adding a basic compound A slurry of nickel (II) bromide trihydrate (0.07 eq.) suspended in a solvent (4.0 v / w of Boc-Glu(NHPI)-OBn) and 4,4'-di-tert-butyl-2,2'-bipyridine (0.07 eq.) were added to a nitrogen-substituted reaction vessel. Boc-Glu(NHPI)-OBn (X g (see Table 38), 1.0 eq.) synthesized by the method described in International Publication No. 2020 / 189540 was added with stirring, and then solvent (1.0 v / w of Boc-Glu(NHPI)-OBn), 5-bromo-1,3-difluoro-2-(trifluoromethyl)-benzene (1.2 eq.) and NMM (2.5 eq.) were added in sequence only in Example 57-1. After cooling to 10 ° C., activated zinc (3.0 eq.) was added. The temperature was raised to 25°C while TMSCl (Y eq. (see Table 38)) was added dropwise over 1 hour. After Z hours from the end of the addition, a sample was taken to confirm the reaction rate. The reaction rate was calculated according to the following formula using the area values ​​of Boc-Glu(NHPI)-OBn and Boc-Hph(3,5-F2-4-CF3)-OBn calculated by HPLC analysis. Reaction rate (%) = area value of Boc-Hph(3,5-F2-4-CF3)-OBn / (area value of Boc-Glu(NHPI)-OBn+area value of Boc-Hph(3,5-F2-4-CF3)-OBn) × 100 [Table 38]

[0215] (Example 58) Synthesis of Cbz-Hph(3,5-F2-4-CF3)-OH dicyclohexylamine salt [ka] The solution (90.81 g) of Boc-Hph(3,5-F2-4-CF3)-OBn obtained in Example 57-1 and toluene (135 mL) were added to a nitrogen-substituted reaction vessel, and then cooled to 0°C. Trifluoromethanesulfonic acid (22.0 mL, 249 mmol) was added over 17 minutes, and the temperature was raised to 25°C. After the addition was completed, the mixture was stirred for 1 hour, sampled, and the reaction completion was confirmed by HPLC analysis. Water (40 mL) was added, stirred for 15 minutes, and then water (160 mL) was added. After stirring for another 1 hour, the mixture was left to stand, and the organic layer was removed to obtain a solution containing H-Hph(3,5-F2-4-CF3)-OH. The obtained H-Hph(3,5-F2-4-CF3)-OH was used in the next step without purification.

[0216] To the solution containing H-Hph(3,5-F2-4-CF3)-OH obtained as described above, 40% potassium phosphate aqueous solution (60 mL) was added with stirring. Acetonitrile (100 mL) and 40% potassium phosphate aqueous solution (18 mL) were added, and then N-carbobenzoxyoxysuccinimide (16.53 g, 66.3 mmol) was added. After stirring for 2 hours after the addition, the mixture was sampled and the reaction completion was confirmed by HPLC analysis. A mixed solution consisting of n-heptane (80 mL) and MTBE (80 mL) was added, stirred for 10 minutes, allowed to stand, and the organic layer was removed. MTBE (200 mL), 0.2 M sodium hydroxide aqueous solution (80 mL), and 20% sodium chloride aqueous solution (80 mL) were added to the obtained aqueous layer, stirred for 13 minutes, allowed to stand, and the aqueous layer was removed. The obtained organic layer was washed twice with a mixed solution of 0.2M sodium hydroxide aqueous solution (80mL) and 20% sodium chloride aqueous solution (80mL), and then further washed with 0.2M sodium hydroxide aqueous solution (80mL) and 1M hydrochloric acid aqueous solution (600mL). MTBE (80mL) and 10% sodium chloride aqueous solution (214mL) were added to the obtained organic layer, stirred for 10 minutes, left to stand, and the aqueous layer was removed. The obtained organic layer was concentrated under reduced pressure. Toluene (120mL) was added to the obtained residue and concentrated to 80mL to obtain a solution (116.07g) containing Cbz-Hph(3,5-F2-4-CF3)-OH. The obtained Cbz-Hph(3,5-F2-4-CF3)-OH was used in the next step without purification.

[0217] In a nitrogen-purged reaction vessel, the toluene solution (116.07 g) of Cbz-Hph(3,5-F2-4-CF3)-OH prepared as above and toluene (188 mL) were added and the temperature was raised to 50°C. Dicyclohexylamine (22.4 mL, 112 mmol) and MTBE (94 mL) were added, followed by Cbz-Hph(3,5-F2-4-CF3)-OH dicyclohexylamine salt (117 mg). After stirring for 3 hours after the addition was completed, n-heptane (94 mL) was added over 2 hours with stirring. After stirring for an additional hour after the addition was completed, n-heptane (188 mL) was added over 3 hours with stirring. After stirring for an additional hour after the addition was completed, the mixture was cooled to 20°C. After stirring for 13 hours at 20°C, the slurry was filtered. The cake was washed with a mixed solvent of MTBE / n-heptane (1:1 (v / v), 94 mL), and the obtained wet powder was dried under reduced pressure to obtain Cbz-Hph(3,5-F2-4-CF3)-OH dicyclohexylamine salt (31.7 g). Melting point of Cbz-Hph(3,5-F2-4-CF3)-OH dicyclohexylamine salt: 154℃ Yield: 64% (4-step yield from Boc-Glu(NHPI)-OBn) HPLC purity: 99.70% Measurement method: HPLC Method F Holding time: 9.73 min Mass spectrometry: m / z 418([M+H] + )

[0218] (Example 59) Synthesis of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 [ka] Into a reaction vessel, Cbz-Hph(3,5-F2-4-CF3)-OH dicyclohexylamine salt (42.0 g, 70.2 mmol) synthesized by the same method as in Example 58 and 2-methyltetrahydrofuran (169 mL) were added. After repeating separation washing with 10% sodium hydrogen sulfate monohydrate aqueous solution (170 mL) twice, it was washed with 5% sodium chloride aqueous solution (170 mL). The obtained organic layer was concentrated under reduced pressure. After filtering the obtained residue, 2-methyltetrahydrofuran (240 mL) was added and concentrated under reduced pressure to obtain a solution containing Cbz-Hph(3,5-F2-4-CF3)-OH (29.3 g, 70.2 mmol) (70.63 g, 41.5 w / w%).

[0219] In a separate reaction vessel, the Cbz-Hph(3,5-F2-4-CF3)-OH solution (41.5 w / w%, 62.7 g, 62.3 mmol) prepared as above, H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (30.0 g, 51.9 mmol) synthesized by the same method as in Example 56, 2-methyltetrahydrofuran (167 mL) and DIPEA (39.9 mL, 228 mmol) were added. A 2-methyltetrahydrofuran solution of propylphosphonic anhydride (1.6 M, 78 mL, 125 mmol) was added with stirring at 10 ° C., and then the temperature was raised to 25 ° C. After stirring for 1 hour after the completion of the addition, the mixture was sampled and the completion of the reaction was confirmed by HPLC analysis. 5% potassium carbonate aqueous solution (180mL) and 1-methylimidazole (4.1mL, 51.9mmol) were added with stirring at 15°C, and then the temperature was raised to 25°C. After stirring for 50 minutes, the mixture was left to stand, and the aqueous layer was removed. The obtained organic layer was washed with 4% sulfuric acid aqueous solution (180mL) and 10% sodium hydrogen sulfate monohydrate aqueous solution (180mL) in sequence, and then n-heptane (108mL), MTBE (72mL), and acetonitrile (69mL) were added. After washing with 2.5% potassium carbonate aqueous solution (171mL), 2-methyltetrahydrofuran (60mL) and acetonitrile (102mL) were added, and the mixture was washed with 2.5% potassium carbonate aqueous solution (171mL). 2-Methyltetrahydrofuran (60mL) and acetonitrile (102mL) were added to the obtained organic layer, and the mixture was washed with 2.5% potassium carbonate aqueous solution (171mL), and then concentrated under reduced pressure. The residue was added with isopropyl acetate (210 mL) and concentrated under reduced pressure twice to obtain a solution (84.49 g, 60.0 w / w%) containing Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2. HPLC purity: 97.90% Measurement method: HPLC Method C Holding time: 4.63 min Mass spectrometry: m / z 999([M+Na] + )

[0220] (Example 60) Synthesis of Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 diethylamine salt [ka] A solution containing Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (60.0 w / w%, 42.25 g, 26.0 mmol), isopropyl acetate (108 mL) and 1,1,1,3,3,3-hexamethyldisilazane (13.6 mL, 65.0 mmol) were added to a reaction vessel, and then cooled to 0 ° C. Trimethylsilyl trifluoromethanesulfonate (4.7 mL, 26.0 mmol) was added, and the temperature was raised to 20 ° C. After stirring for 3 hours after the addition, sampling was performed, and the completion of the reaction was confirmed by HPLC analysis. 2-Methyltetrahydrofuran (128 mL) was added, and the mixture was cooled to 0 ° C., and 5% dipotassium hydrogen phosphate aqueous solution (254 mL) was added, and the temperature was raised to 25 ° C. After stirring for 20 minutes, the mixture was left to stand, and the aqueous layer was removed. The obtained organic layer was washed with 5% aqueous sodium dihydrogen phosphate solution (254 mL), and then diethylamine (10.8 mL, 104 mmol) was added and concentrated under reduced pressure to 144 mL. The residue obtained was concentrated under reduced pressure to 144 mL by adding isopropyl acetate (120 mL) and diethylamine (2.69 mL, 26.0 mmol) three times to form a slurry. Further, isopropyl acetate (120 mL) and diethylamine (2.69 mL, 26.0 mmol) were added and concentrated under reduced pressure to 108 mL, and then isopropyl acetate (38.2 mL) and diethylamine (2.18 mL, 21.1 mmol) were added at 25° C. with stirring. After stirring for 4.5 hours after the completion of the addition, MTBE (192 mL) was added over 80 minutes. After stirring for 2 hours after the completion of the addition, the slurry was filtered. The obtained solid was washed twice with a mixed solution of isopropyl acetate / MTBE / diethylamine (1:5:0.06 (v / v), 72 mL) and then dried under reduced pressure to obtain Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 diethylamine salt (23.23 g, content 92.0 w / w%). Yield: 83% (2 steps from H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2) HPLC purity: 99.85% Measurement method: HPLC Method C Holding time: 4.18 min Mass spectrometry: m / z 921([M+H] + )

[0221] (Example 61) Synthesis of Compound 11 [ka] The Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 diethylamine salt (92.0 w / w%, 20.78 g, 19.2 mmol) obtained in Example 60, acetonitrile (177 mL), dicyclohexylmethylamine (8.2 mL, 38.6 mmol) and DIPEA (10.1 mL, 58.0 mmol) were added to a reaction vessel and concentrated to 42.5 mL under reduced pressure. The residue was concentrated under reduced pressure to 42.5 mL with acetonitrile (177 mL) and DIPEA (3.4 mL, 19.5 mmol) three times to give a solution (46.76 g, 37.9 w / w%) containing Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 (17.70 g).

[0222] In a separate reaction vessel, H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBuL-tartrate (93.8 w / w%, 18.66 g, 19.6 mmol) synthesized by the same method as in Example 47 and 2-methyltetrahydrofuran (87 mL) were added. 5% aqueous sodium carbonate solution (87 mL) was added while stirring at 25 ° C. After stirring for 10 minutes, the mixture was left to stand and the aqueous layer was removed. The obtained organic layer was separated and washed with 5% aqueous sodium carbonate solution (87 mL) and 5% aqueous sodium chloride solution (87 mL), and then concentrated under reduced pressure to obtain a solution containing H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (14.55 g) (29.71 g, 49.0 w / w%).

[0223] In a separate reaction vessel, an acetonitrile solution (37.9 w / w%, 39.0 g, 16.0 mmol) containing Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 prepared as above, a 2-methyltetrahydrofuran solution (49.0 w / w%, 26.7 g, 17.6 mmol) containing H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu prepared as above, 2-methyltetrahydrofuran (118 mL) and acetonitrile (13.5 mL) were added. After cooling to 10 °C, DIPEA (5.5 mL, 31.6 mmol) and HATU (9.14 g, 24.1 mmol) were added successively with stirring, and the temperature was raised to 25 °C. After stirring for 3 hours after the completion of the addition, a sample was taken and the completion of the reaction was confirmed by HPLC analysis. 2-Methyltetrahydrofuran (44 mL) was added, cooled to 10° C., and then 10% aqueous ammonia (100 mL) was added. The mixture was heated to 25° C., stirred for 30 minutes, and then allowed to stand, and the aqueous layer was removed. The obtained organic layer was separated and washed with 10% aqueous ammonia (100 mL), 10% aqueous citric acid (100 mL), 5% aqueous sodium carbonate (100 mL), and 20% aqueous sodium chloride (100 mL), and then concentrated to 79 mL under reduced pressure. 2-Methyltetrahydrofuran (45 mL) was added to the obtained residue and concentrated under reduced pressure to obtain a solution containing compound 11 (69.84 g, content 37.8 w / w%). HPLC purity: 96.49% Measurement method: HPLC Method G Holding time: 10.27 min Mass spectrometry: m / z 1668([M+Na] + )

[0224] (Example 62) Synthesis of Compound 12 [ka] A solution containing compound 11 obtained in Example 61 (37.8 w / w%, 69.14 g, 15.9 mmol) and 2-methyltetrahydrofuran (247 mL) were added to a reaction vessel, and then cooled to 0°C. 1,1,1,3,3,3-hexamethyldisilazane (22.6 mL, 108 mmol) and trimethylsilyl trifluoromethanesulfonate (12.0 mL, 66.4 mmol) were added, and then the temperature was raised to 24°C. After stirring for 3 hours after the addition, sampling was performed, and the completion of the reaction was confirmed by HPLC analysis. The mixture was cooled to 0°C, and a 5% dipotassium hydrogen phosphate aqueous solution (120 mL) was added, and then the temperature was raised to 24°C. After stirring for 50 minutes, the mixture was left to stand, and the aqueous layer was removed. The obtained organic layer was separated and washed with a 5% aqueous ammonium chloride solution (131 mL, four times) and a 5% aqueous sodium carbonate solution (131 mL), and then concentrated under reduced pressure to obtain a solution containing compound 12 (54.99 g, content 45.8 w / w%). HPLC purity: 95.41% Measurement method: HPLC Method G Holding time: 9.22 min Mass spectrometry: m / z 1612([M+Na] + )

[0225] (Example 63) Synthesis of Compound 3 [ka] A reaction vessel was charged with 10% palladium carbon (54.13% wet, 3.97 g, 1.71 mmol, 11 mol% on Pd metal basis) and tetrahydrofuran (84 mL). The atmosphere was replaced with nitrogen at 25°C, then replaced with hydrogen, and stirred under a hydrogen atmosphere (0.35 MPaG) for 2 hours. A solution containing compound 12 obtained in Example 62 (45.8 w / w%, 53.95 g, 15.5 mmol) and tetrahydrofuran (84 mL) were added. After stirring for 1 hour under a hydrogen atmosphere (0.20 MPaG), a sample was taken, and the completion of the reaction was confirmed by HPLC analysis. The reaction mixture was filtered, and the cake was washed twice with tetrahydrofuran (57 mL). The mixed solution of the filtrate and washings was concentrated under reduced pressure, and then acetonitrile (226 mL) was added and concentrated to 90 mL under reduced pressure, and this operation was repeated three times. The obtained residue was filtered, and then tetrahydrofuran (47 mL) and toluene (110 mL) were added and concentrated under reduced pressure. Tetrahydrofuran (224 mL) was added and the mixture was concentrated under reduced pressure to 134 mL to obtain a concentrated liquid (120.80 g).

[0226] Tetrahydrofuran (20 mL) was added to a portion (107.90 g) of the obtained concentrated solution to prepare a solution containing compound 3. n-Heptane (240 mL) was added to another reaction vessel, and the solution containing compound 3 prepared above was added dropwise over 1 hour while stirring at 25°C. Tetrahydrofuran (10 mL) was added, and the mixture was stirred at 25°C for 1 hour, after which the slurry was filtered. The obtained solid was washed with n-heptane (100 mL) and then dried under reduced pressure to obtain compound 3 (19.07 g, content 97.06 w / w%). The content and yield were calculated by HPLC analysis using a standard sample. Yield: 91.8% (3-step yield from Cbz-Hph(3,5-F2-4-CF3)-Pro-cLeu-MeGcp-MeAsp(OH)-NMe2 diethylamine salt) HPLC purity: 97.56% Measurement method: HPLC Method G Holding time: 6.57 min Mass spectrometry: m / z 1456([M+H] + )

[0227] (Example 64) Synthesis of Compound 4 [ka] HATU (2.36 g, 6.21 mmol) and acetonitrile (76 mL) were added to the reaction vessel. Compound 3 (9.50 g, 6.53 mmol), DIPEA (2.62 mL, 15.0 mmol) and acetonitrile (152 mL) were added to another reaction vessel in the same manner as in Example 63 to prepare a solution, and half of the solution was added to the previously prepared acetonitrile solution of HATU at 25 ° C. over 6 hours while stirring. After the addition was completed and the mixture was left to stand for 13 hours, a solution in which HATU (2.36 g, 6.21 mmol) was dissolved in acetonitrile (11.8 mL) and acetonitrile (3.8 mL) were added in that order. The remaining half of the previously prepared acetonitrile solution containing compound 3 and DIPEA was added over 6 hours. After the addition was completed and the mixture was stirred for 30 minutes, the mixture was sampled, and the completion of the reaction was confirmed by HPLC analysis. MTBE (124 mL), heptane (9.5 mL) and 2.5% aqueous ammonia solution (95 g) were added. After stirring for 10 minutes, the mixture was allowed to stand and the aqueous layer was removed. The obtained organic layer was washed with 4% aqueous sulfuric acid solution (133 g), 5% aqueous dipotassium hydrogen phosphate solution (95 g), and 0.5% aqueous sodium chloride solution (95 g, twice), and then filtered to remove dust. The obtained filtrate was concentrated under reduced pressure. After adding acetonitrile (66.5 mL) and concentrating under reduced pressure twice, the operation of adding acetone (66.5 mL) and concentrating under reduced pressure was repeated six times. Acetone (38 mL) was added to the obtained residue to obtain a solution containing compound 4 (54.3 g). HPLC purity: 90.22% Measurement method: HPLC Method H Holding time: 17.99 min Mass spectrometry: m / z 1439([M+H] + )

[0228] (Example 65) Synthesis of hydrate crystals of compound 4 (C type) A reaction vessel was charged with an acetone solution (33.33 g) containing compound 4 (6.00 g, 4.17 mmol) synthesized by the same method as in Example 64 and acetone (12.53 g). The temperature was raised to 40° C., and water (19.2 mL) was added over 10 minutes while stirring. Compound 4 hydrate crystals (C type) (18 mg) were added to a glass vial and suspended in a mixed solution of acetone / water (5:4 (v / v), 0.24 mL), and the suspension was added to the crystallization solution. Further, a mixed solution of acetone / water (5:4 (v / v), 0.24 mL) was added to the glass vial, and the resulting suspension was added to the crystallization solution. After stirring for 2 hours, water (4.8 mL) was added over 10 minutes. After stirring for another 3 hours, water (4.8 mL) was added over 10 minutes. After stirring for another hour, the mixture was cooled to 25° C. over 1 hour. After stirring at 25°C for 1 hour, the suspension was left to stand for 13 hours. After stirring at 25°C for another 2 hours, the suspension was filtered. The obtained wet powder was washed with a mixed solution of acetone (16.8mL) and water (13.2mL), and then washed with a mixed solution of methanol (15mL) and water (15mL). The obtained wet powder was further suspended in a mixed solution of methanol (15mL) and water (15mL), left to stand for 14 hours, and then the suspension was filtered. The obtained wet powder was suspended in water (30mL), left to stand for 2 hours, and then filtered. After the above-mentioned suspension washing with water was performed again, the obtained wet powder was dried under reduced pressure to obtain hydrate crystals of compound 4 (C type) (4.97g). HPLC purity: 99.74% Measurement method: HPLC Method H Holding time: 17.91 min Mass spectrometry: m / z 1439([M+H] + )

[0229] (Example 66) Crystallization of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Amorphous H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (14.82 g) was mixed with CPME (21.7 g) to prepare a CPME solution of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (content 40.6 w / w%). The solution was left to stand at 5°C for one day to obtain crystals of H-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu.

[0230] Example 67 Crystallization of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu Amorphous Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (10 mg) and a mixed solvent of toluene / heptane (1:2 (v / v), 0.1 mL) were added to a vial, and the resulting solution was shaken at room temperature for 2 days to obtain crystals of Cbz-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu.

[0231] Example 68 Crystallization of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBuL-tartrate A solution was prepared by adding amorphous H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu (600 mg, 0.808 mmol), L-tartaric acid (121 mg, 0.808 mmol) and methanol (6 mL) to a glass vial. 0.1 mL of the prepared solution was added to another glass vial, and the solvent was removed by concentrating to dryness under reduced pressure. n-Butyl acetate (0.02 mL) and glass beads were added to the vial, and the mixture was shaken at 25 °C for 7 days to obtain crystals of H-MeLeu-Ile-MeAla-Aze-EtPhe(4-Me)-Sar-OtBu L-tartrate.

[0232] (Example 69) Crystallization of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 Amorphous Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (1.19 g) was added to CPME (5.94 mL) and stirred at room temperature for 4 hours, after which the slurry was filtered. The solid obtained was washed twice with CPME (2.38 mL) and then dried under reduced pressure to obtain crystals of Cbz-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2.

[0233] (Example 70) Crystallization of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 Amorphous H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2 (10 mg) and tetrahydrofuran (0.02 mL) were added to a vial and shaken at room temperature for 6 days. Heptane (0.04 mL) was added to the resulting solution and shaken at room temperature for another 6 days to obtain crystals of H-Pro-cLeu-MeGcp-MeAsp(OtBu)-NMe2. [Industrial Applicability]

[0234] INDUSTRIAL APPLICABILITY The present invention provides pharmaceuticals and intermediates containing N-monoalkylamino acids and peptides containing N-monoalkylamino acids, as well as methods for producing the same.

Claims

1. A method for producing a peptide containing an N-monoalkylamino acid or an ester thereof, comprising: In the presence of hydrogen, a peptide containing the starting amino acid or an ester thereof, C １ -C ６ an alkylation step of mixing a primary alkylating agent or a substituted methyl halide and a catalyst in a solvent; the C 1 -C 6 primary alkylating agent is a C 1 -C 5 alkyl nitrile or a C 1 -C 5 alkyl aldehyde; the substituted methyl halide is one selected from the group consisting of methoxymethyl chloride (MOM-Cl), ethoxymethyl chloride (EOM-Cl), 2-methoxyethoxymethyl chloride (MEM-Cl), and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl); the catalyst is a heterogeneous hydrogenation catalyst containing a transition metal, the solvent includes at least one solvent selected from the group consisting of an ether-based solvent, an alcohol-based solvent, and an ester-based solvent; The alkylation step is carried out under a pressure of 1 atmosphere or more, and the amino group of the peptide containing the starting amino acid or an ester thereof is substituted with the C １ -C ６ A method for producing a peptide or ester thereof comprising an N-monoalkylamino acid having attached thereto a primary alkyl group corresponding to a primary alkylating agent or a substituted methyl halide.

2. A method for producing a peptide containing an N-monoalkylamino acid or an ester thereof, comprising: A peptide containing the starting amino acid or an ester thereof, C １ -C ６ an alkylation step of combining a primary alkylating agent or a substituted methyl halide, a hydride reducing agent, and a catalyst in a solvent; the C 1 -C 6 primary alkylating agent is a C 1 -C 5 alkyl nitrile or a C 1 -C 5 alkyl aldehyde; the substituted methyl halide is one selected from the group consisting of methoxymethyl chloride (MOM-Cl), ethoxymethyl chloride (EOM-Cl), 2-methoxyethoxymethyl chloride (MEM-Cl), and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl); the catalyst is a heterogeneous hydrogenation catalyst containing a transition metal, the solvent includes at least one solvent selected from the group consisting of an ether-based solvent, an alcohol-based solvent, and an ester-based solvent; The alkylation step is carried out by adding the C alkyl group to the amino group of the peptide containing the starting amino acid or an ester thereof. １ -C ６ A method for producing a peptide or an ester thereof comprising the N-monoalkylamino acid having attached thereto a primary alkyl group corresponding to a primary alkylating agent or a substituted methyl halide.

3. The method of claim 2 wherein the hydride reducing agent is a trialkylsilane.

4. 2. The method of claim 1, wherein the heterogeneous hydrogenation catalyst is a catalyst containing a transition metal selected from the group consisting of Pd, Rh, and Pt.

5. The method of claim 2, wherein the heterogeneous hydrogenation catalyst is a catalyst containing a transition metal selected from the group consisting of Pd, Rh and Pt.

6. 2. The method of claim 1, wherein the solvent is selected from the group consisting of ether solvents, alcohol solvents, ester solvents, and combinations thereof.

7. The method of claim 6, wherein the ether solvent is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, dimethoxyethane, methyl t-butyl ether, cyclopentyl methyl ether, diisopropyl ether, 4-methyltetrahydropyran, dioxane, and diethyl ether.

8. The method of claim 6, wherein the alcohol-based solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.

9. The method of claim 6, wherein the ester solvent is selected from the group consisting of ethyl acetate, propyl acetate, and butyl acetate.

10. The method of claim 2, wherein the solvent is selected from the group consisting of ether-based solvents, alcohol-based solvents, ester-based solvents, and combinations thereof.

11. The method of claim 10, wherein the ether solvent is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, dimethoxyethane, methyl t-butyl ether, cyclopentyl methyl ether, diisopropyl ether, 4-methyltetrahydropyran, dioxane, and diethyl ether.

12. The method of claim 10, wherein the alcohol-based solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.

13. The method of claim 10, wherein the ester solvent is selected from the group consisting of ethyl acetate, propyl acetate, and butyl acetate.

14. 10. The method of claim 1, further comprising contacting the reaction mixture in said alkylating step with additional hydrogen.

15. The method of claim 1, wherein the amino group of the peptide or ester thereof containing the starting amino acid is bound to a protecting group that can be removed under hydrogenolysis conditions.

16. The method described in claim 15, wherein the peptide or its ester containing the starting amino acid is a compound represented by formula E. 【Chemistry 1】 [In the formula, PG 3 is a protecting group for an amino group, R 3 represents the side chain of an amino acid residue, and R 4 represents a peptide residue.]

17. 17. The method of claim 16, wherein the protecting group is an arylmethyloxycarbonyl group.

18. 16. The method of claim 15, wherein the removal of the protecting group is carried out in the presence of an additive selected from the group consisting of p-toluenesulfonic acid, methanesulfonic acid, sodium hydrogen sulfate, triethylamine hydrochloride, and propylphosphonic acid.

19. 16. The method of claim 15, wherein the reaction mixture in the alkylation step further comprises a base.

20. 16. The method of claim 15, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

21. The method of claim 18, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

22. The method of claim 19, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

23. The method of claim 2, wherein the amino group of the peptide or ester thereof containing the starting amino acid is bound to a protecting group that can be removed under hydrogenolysis conditions.

24. The method described in claim 23, wherein the peptide or its ester containing the starting amino acid is a compound represented by formula E. 【Chemistry 2】 [In the formula, PG 3 is a protecting group for an amino group, R 3 represents the side chain of an amino acid residue, and R 4 represents a peptide residue.] 25. The method of claim 24, wherein the protecting group is an arylmethyloxycarbonyl group.

26. The method of claim 23, wherein the removal of the protecting group is carried out in the presence of an additive selected from the group consisting of p-toluenesulfonic acid, methanesulfonic acid, sodium hydrogen sulfate, triethylamine hydrochloride and propylphosphonic acid.

27. The method of claim 23, wherein the reaction mixture in the alkylation step further comprises a base.

28. The method of claim 23, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

29. The method of claim 26, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

30. The method of claim 27, wherein the removal of the protecting group and the alkylation step are carried out in one pot.

31. The amino group of the peptide containing the starting amino acid or its ester is a primary amino group, The method of claim 1 , wherein the reaction mixture in the alkylation step further comprises a base.

32. The method described in claim 31, wherein the peptide or its ester containing the starting amino acid is a compound represented by formula D. 【Transformation 3】 [wherein R 3 represents the side chain of an amino acid residue, and R 4 represents a peptide residue.] 33. The method of claim 19, wherein the base is a tertiary amine.

34. The method of claim 27, wherein the base is a tertiary amine.

35. The method of claim 31, wherein the base is a tertiary amine.

36. The method of claim 33, wherein the tertiary amine is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undecene-7, 1,5-diazabicyclo[4.3.0]nonene-5, N-methylmorpholine, 1,4-diazabicyclo[2.2.2]octane, triethylamine, N,N-diisopropylethylamine, pyridine, and collidine.

37. The method of claim 34, wherein the tertiary amine is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undecene-7, 1,5-diazabicyclo[4.3.0]nonene-5, N-methylmorpholine, 1,4-diazabicyclo[2.2.2]octane, triethylamine, N,N-diisopropylethylamine, pyridine, and collidine.

38. The method of claim 35, wherein the tertiary amine is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undecene-7, 1,5-diazabicyclo[4.3.0]nonene-5, N-methylmorpholine, 1,4-diazabicyclo[2.2.2]octane, triethylamine, N,N-diisopropylethylamine, pyridine, and collidine.

39. A method for producing a peptide having a cyclic portion composed of at least four amino acids or an ester thereof, comprising the steps of: (a) obtaining a peptide containing the N-monoalkylamino acid or an ester thereof according to the method of any one of claims 1 to 38; (b) optionally extending the N-monoalkylamino acid-containing peptide or its ester with one or more amino acids by a bond-forming reaction to obtain a peptide or its ester; and (c) a step of cyclizing the C-terminal group and the N-terminal group of the peptide or ester thereof obtained in step (a) or (b) to form the cyclic moiety.

40. A method for suppressing the production of a compound in which an amino group of a peptide containing a starting amino acid or an ester thereof is dialkylated in an N-monoalkylation reaction of the peptide containing the starting amino acid or an ester thereof, comprising: In the presence of hydrogen, a peptide containing the starting amino acid or an ester thereof, C １ -C ６ an alkylation step of mixing a primary alkylating agent or a substituted methyl halide and a catalyst in a solvent; the C 1 -C 6 primary alkylating agent is a C 1 -C 5 alkyl nitrile or a C 1 -C 5 alkyl aldehyde; the substituted methyl halide is one selected from the group consisting of methoxymethyl chloride (MOM-Cl), ethoxymethyl chloride (EOM-Cl), 2-methoxyethoxymethyl chloride (MEM-Cl), and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl); the catalyst is a heterogeneous hydrogenation catalyst containing a transition metal, the solvent includes at least one solvent selected from the group consisting of an ether-based solvent, an alcohol-based solvent, and an ester-based solvent; The alkylation step is carried out under a pressure of 1 atmosphere or more, and the amino group of the peptide containing the starting amino acid or an ester thereof is substituted with the C １ -C ６ A method for producing a peptide or ester thereof comprising an N-monoalkylamino acid having attached thereto a primary alkyl group corresponding to a primary alkylating agent or a substituted methyl halide.

41. A method for suppressing the production of a compound in which an amino group of a peptide containing a starting amino acid or an ester thereof is dialkylated in an N-monoalkylation reaction of a peptide containing a starting amino acid or an ester thereof, comprising: A peptide containing the starting amino acid or an ester thereof, C １ -C ６ an alkylation step of combining a primary alkylating agent or a substituted methyl halide, a hydride reducing agent, and a catalyst in a solvent; the C 1 -C 6 primary alkylating agent is a C 1 -C 5 alkyl nitrile or a C 1 -C 5 alkyl aldehyde; the substituted methyl halide is one selected from the group consisting of methoxymethyl chloride (MOM-Cl), ethoxymethyl chloride (EOM-Cl), 2-methoxyethoxymethyl chloride (MEM-Cl), and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl); the catalyst is a heterogeneous hydrogenation catalyst containing a transition metal, the solvent includes at least one solvent selected from the group consisting of an ether-based solvent, an alcohol-based solvent, and an ester-based solvent; The alkylation step involves adding a C to the amino group of the peptide containing the starting amino acid or its ester. １ -C ６ A method for producing a peptide or ester thereof comprising an N-monoalkylamino acid having attached thereto a primary alkyl group corresponding to a primary alkylating agent or a substituted methyl halide.

42. A method for producing a peptide comprising the steps of: extending a peptide chain of a peptide containing an N-monoalkylamino acid or an ester thereof by a bond-forming reaction; 42. The method of claim 40 or 41, further comprising treating the extended peptide with aqueous acid to remove the dialkylated compound.

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