Method for synthesizing peptides containing N-substituted amino acids

Abstract

The object of the present invention is to provide a novel method for producing peptides containing N-substituted amino acids or N-substituted amino acid analogs with high purity and high efficiency. [Solution] The method for producing a peptide containing an N-substituted amino acid or an N-substituted amino acid analog according to the present invention is: A step of preparing Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or Fmoc-protected peptides. A step of deprotecting a protecting group having an Fmoc skeleton, such as an Fmoc-protected amino acid, with a base, and The process includes adding a new Fmoc-protecting amino acid, etc., to form an amide bond. When the peptide is produced by a solid-phase method, the obtained peptide is characterized by being cleaved from the solid phase under conditions that are weaker than TFA. Furthermore, at least one side chain of the obtained peptide is characterized by having a protecting group that is not deprotected under basic conditions but is deprotected under conditions that are weaker than TFA.

Description

[Technical Field]

[0001] The present invention relates to a novel method for synthesizing peptides containing N-substituted amino acids, which enables high purity and high synthesis efficiency. [Background technology]

[0002] Peptides are highly valuable chemical species, with over 40 types already marketed as pharmaceuticals (Non-Patent Document 1). Among these, cyclic peptides and N-methylated (or N-alkylated) non-natural peptides are expected to offer improved membrane permeability due to enhanced lipophilicity and improved metabolic stability by acquiring resistance to hydrolytic enzymes (Non-Patent Document 2). Recently, research has been progressing on drug-like cyclic peptides (preferably exhibiting both membrane permeability and metabolic stability) that are key to intracellular translocation and oral formulation (Non-Patent Documents 3 and 4). Furthermore, a patent document clarifying the conditions required for drug-like cyclic peptides has been published (Patent Document 1), and their importance in drug discovery and the level of awareness surrounding them are increasing.

[0003] On the other hand, progress in developing peptide synthesis methods for many unnatural amino acids, such as N-alkyl amino acids, is relatively limited. Many methods are simply applying techniques established for natural peptides to unnatural peptides.

[0004] The Fmoc and Boc methods are widely known as peptide synthesis methods, and much of the knowledge gained from these methods was derived from the development of synthesis methods for natural peptides. Since the Fmoc group is stable to acids, when the N-terminal amino group is protected with an Fmoc group, the deprotection reaction is carried out using a base such as DBU or piperidine. Therefore, as a protecting group for the peptide side chain functional group, one that can be deprotected with an acid, for example, is used to selectively deprotect the N-terminal amino group and extend the peptide chain. Commonly used protecting groups in the Fmoc method include t-butyl (tBu) and trityl (Trt) groups, which can be deprotected with acids such as trifluoroacetic acid (TFA), allowing for peptide cleavage from resin and deprotection of the protecting group of the side chain functional group under milder conditions compared to the Boc method.

[0005] However, even in solid-phase synthesis using the Fmoc method, which allows for cleavage from resin and deprotection of side-chain functional groups under relatively mild conditions, it has become clear that N-alkylated peptide synthesis has the following problems in the cleavage from resin using TFA or in the deprotection of side-chain functional groups.

[0006] Typically, when peptide synthesis is performed using the Fmoc method, TFA is commonly used for the cleavage step from the resin and the deprotection of the protecting group of the side chain functional group. In most cases, the cleavage reaction from the resin and the deprotection reaction of the side chain functional group are carried out simultaneously using a 90% TFA aqueous solution. However, in the case of N-methylated peptides, especially those with sequences of consecutive N-methyl amino acids, a side reaction is known to occur in which acid hydrolysis via oxazolonium proceeds, causing the peptide chain to be cleaved (Non-Patent Documents 5, 6). Furthermore, in the case of peptides containing amino acids with β-hydroxyl groups, such as serine and threonine, it is known that in these steps using TFA, not only acid hydrolysis but also an N→O acyl shift reaction may proceed as a side reaction, potentially leading to depsipeptidization (Non-Patent Documents 7, 8).

[0007] Attempts have been made to avoid the hydrolysis problem in the cleavage and deprotection steps using this acid by using a low concentration of TFA solution and controlling the reaction time to be short. For example, according to a report by Albericio et al., when the Boc group on the amino group in an N-methylated cyclic hexapeptide was deprotected using a TFA-DCM (1:1) solution during the solid-phase synthesis of a peptide named NMe-IB-01212, degradation of the peptide at the N-Me site was observed. Attempts have been made to improve the situation by using even lower concentrations of TFA or minimizing the reaction time to avoid degradation, but sufficient improvement has not been achieved (Non-Patent Literature 9). In the first place, with protecting groups commonly used in peptide synthesis, in the deprotection step using a low concentration of TFA solution, the cleavage reaction from the resin proceeds at a satisfactory rate, but the side chain deprotection reaction does not proceed or proceeds extremely slowly.

[0008] Furthermore, to prevent the cleavage of the N-terminal Ac-MePhe, which proceeds via the same reaction mechanism as the hydrolysis of highly N-methylated peptides, Fang et al. used TFA to lower the reaction temperature to 4°C and deprotected the Pbf group, which is a protecting group of the Arg side chain (Non-Patent Literature 10). However, even with this method of lowering the temperature, it is difficult to completely prevent the cleavage of Ac-MePhe, and the reaction is only stopped at the time when the target product is maximized.

[0009] Furthermore, in addition to the problems during deprotection, there is also a known issue of low reactivity during the extension process. When the N-terminus of the newly formed amide bond is an N-methylamino acid, the bulkiness of the secondary amine may prevent the subsequent amide formation reaction (extension reaction) with the amino acid from proceeding sufficiently (Non-Patent Documents 2, 5).

[0010] To address the problems in this extension process, efforts have been made to reduce unreacted sites by repeating the exact same reaction conditions two or more times (the method of repeating twice is called double coupling). Furthermore, efforts have been made to improve condensation efficiency by, for example, replacing the activation of the amino acids to be condensed with more active acid halides (Non-Patent Literature 11). However, repeating the same reaction conditions as in double coupling doubles or more the time and reagent costs, and when condensation is performed with acid halides, preparation is required each time, and there are concerns about whether the generated acid halides can remain stable during the peptide synthesis process. In addition, the generation of HCl and HF by the reaction may raise concerns about the progression of deprotection reactions.

[0011] Other measures to improve low reactivity in the extension process include reducing the amount of resin loaded to decrease the density of peptide chains on the solid phase and thereby increase condensation efficiency, and increasing the concentration of the reaction solution (Non-Patent Document 9). More recently, efforts have also been made to improve condensation efficiency by increasing the reaction temperature through microwave irradiation (Non-Patent Documents 12, 13).

[0012] However, no fundamental solutions have been reported for concerns regarding the decrease in purity and yield of the synthesized peptides, or in some cases, the inability to obtain the desired product at all, in the synthesis of N-methylated peptides. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication Number WO 2013 / 100132 A1 [Non-patent literature]

[0014] [Non-Patent Document 1] S. R. Gracia, et al. Synthesis of chemically modified bioactive peptides: recent advances, challenges and developments for medicinal chemistry. Future Med. Chem., 2009, 1, 1289. [Non-Patent Document 2] J. Chatterjee, et al. N-Methylation of peptides: A new perspective in medicinal chemistry. Acc. Chem. Res., 2008, 41, 1331. [Non-Patent Document 3] J. E. Bock, et al. Getting in Shape: Controlling Peptide Bioactivity and Bioavailability Using Conformational Constraints. ACS Chem. Biol., 2013, 8, 488. [Non-Patent Document 4] K. Jpsephson, et al. mRNA display: from basic principles to macrocycle drug discovery. Drug Discovery Today, DOI:10.1016 / j.drudis.2013.10.011 [Non-Patent Document 5] M. Teixido, et al. Solid-phase synthesis and characterization of N-methyl-rich peptides. J. Peptide Res., 2005, 65, 153. [Non-Patent Document 6] J. Urban, et al. Lability of N-alkylated peptides towards TFA cleavage. Int. J. Pept. Prot. Res., 1996, 47, 182. [Non-Patent Document 7] LA Carpino, et al. Dramatically enhanced N→O acyl migration during the trifluoroacetic acid-based deprotectionstep in solid phase peptide synthesis. Tetrahedron Lett., 2005, 46, 1361. [Non-Patent Document 8] H. Eberhard, et al. N→O-Acyl shiftin Fmoc-based synthesis of phosphopeptides. Org. Biomol. Chem., 2008, 6, 1349. [Non-Patent Document 9] E. Marcucci, et al. Solid-PhaseSynthesis of NMe-IB-01212, a Highly N-Methylated Cyclic Peptide. Org. Lett.,2012, 14, 612. [Non-Patent Document 10] W.-J. Fang, et al. Deletion ofAc-NMePhe1 From [NMePhe1]arodyn Under Acidic Conditions,Part 1: Effects of Cleavage Conditions and N-Terminal Functionality. PeptideScience Vol. 96, 97 [Non-Patent Document 11] LA Carpino, et al. StepwiseAutomated Solid Phase Synthesis of Naturally Occurring Peptaibols Using FMOCAmino Acid Fluorides. J. Org. Chem., 1995, 60, 405. [Non-Patent Document 12] H. Rodriguez, et al. A convenient microwave-enhanced solid-phase synthesis of short chain N-methyl-rich peptides.J. Pept. Sci., 2010, 16, 136. [Non-Patent Document 13] R. Roodbeen, et al. MicrowaveHeating in the Solid-Phase Synthesis of N-Methylated Peptides: When Is RoomTemperature Better? Eur. J. Org. Chem., 2012, 7106. [Overview of the project] [Problems that the invention aims to solve]

[0015] The inventors focused on cyclic peptides containing N-alkylated amino acids that can act as drug-like peptides and investigated a method for parallel synthesis of peptide compounds with such characteristics. As a result, they found that with cyclic peptides containing N-alkylated amino acids that can act as drug-like peptides, the problems found in the compounds described in the above-mentioned known literature become more pronounced with conventional TFA synthesis methods, making it impossible to isolate the cyclic peptides. Specifically, in the case of peptides containing N-alkylated amino acids, the inventors found that in reactions under acidic conditions using TFA (cleavage from the solid phase or deprotection of the protecting group of the side chain functional group), a side reaction in which the peptide chain is cleaved becomes the main reaction, making it difficult to obtain the target peptide. Furthermore, they found that when an amino acid with a β-hydroxyl group is included in the peptide, an N→O acyl shift reaction proceeds in reactions under acidic conditions using TFA, making it difficult to obtain the target peptide. These problems were found in the compounds described in the above-mentioned known literature, but the inventors further found that these problems are similarly observed in many other peptides as well. In addition to these problems, we also discovered that when peptides containing amino acids with hydroxyl groups in their backbone, not just at the β-position, are reacted under acidic conditions using TFA, the hydroxyl groups become TFA-esterified.

[0016] Furthermore, the inventors have found that, when considering the industrialization of peptide synthesis containing N-alkylated amino acids that can become drug-like peptides, the conventional deprotection method using TFA is extremely difficult to industrialize, not only in terms of the deprotection and extension reactions themselves, but also in terms of post-processing steps and large-scale synthesis. For example, when removing the solvent from a TFA / DCM solution by concentration, the TFA concentration increases as concentration progresses, and problems such as hydrolysis and N→O-acyl shift occur simultaneously with concentration, resulting in the failure to obtain the target compound or a significant decrease in yield. It also becomes necessary to carry out the concentration step at low temperatures. Even if the TFA concentration is low, it is present in a large excess relative to the target product, so if one tries to neutralize it to stop the reaction, the amount of base to be added will also be in a large excess, and the excess salt will remain along with the target peptide, adding complexity to the purification step. Moreover, although TFA itself is a solvent that effectively dissolves peptides, if the concentration of the TFA solution is reduced, it leads to a decrease in the solubility of the peptide. Regarding solubility, not only when considering industrialization, but also in parallel synthesis where many different peptide compounds are handled simultaneously, it is necessary to select a solvent that has high solubility for a group of peptides.

[0017] In addition, the inventors focused on improving reactivity by reducing the steric size of the protecting group of Fmoc-amino acids having a functional group with a protecting group attached to the side chain, an area that has not been actively addressed until now. For example, since threonine (Thr) has a hydroxyl group, a protecting group for the hydroxyl group is necessary to selectively allow the reaction to proceed with the amino group during subsequent acylation. However, because it has a secondary alcohol branched at the β-position as a side chain functional group, the protected form of Thr has relatively low condensation efficiency due to its bulkiness. Commonly used protecting groups for throcytes in peptide synthesis include acetyl (Ac) group, tBu group, Trt group, benzyl (Bn) group, and t-butyldimethylsilyl (TBS) group (Albert Isidro-Llobet, et al. Amino Acid-Protecting Groups. Chem.Rev., 2009, 109, 2455., Watanabe Chemical Reagent Catalog Amino acids & Chiral building blocks to new medicine 2012-14). However, the bulkiness of the Trt group and TBS group reduces condensation efficiency. Furthermore, even with the tBu group, which can be deprotected with acid, high concentrations of TFA are required for deprotection, thus exacerbating the deprotection problems already mentioned. Other protecting groups cannot be easily removed using acid. In other words, there is a need to find a protecting group that is sterically small, does not reduce condensation efficiency, and can be easily deprotected with an acid that avoids the problems of acid hydrolysis and N→O-acyl shift mentioned above. The same thing applies broadly to N-methylserine (MeSer), which has increased bulk due to N-substitution, even though it does not have a branching site at the β-position, and to other amino acids that have a hydroxyl group as a functional group.

[0018] In other words, the present invention aims to find a novel reaction process that can mitigate the problems of side reactions such as acid hydrolysis of peptides, N→O-acyl shift, and TFA esterification of hydroxyl groups in the deprotection step using TFA, which have been found to become prominent during the parallel synthesis of peptides containing N-substituted amino acids, while ensuring the solubility of the peptide. Furthermore, the present invention aims to provide a method for obtaining peptides containing N-substituted amino acids with high purity and high synthetic efficiency by using an appropriate protecting group on the side chain functional group (an appropriate protecting group from the viewpoint of reducing the bulkiness of the protecting group in order to improve low reactivity during extension, and from the viewpoint of being able to deprotect under the deprotection conditions according to the present invention).

[0019] In other words, when parallel synthesizing peptide compounds containing N-substituted amino acids with various sequences, (1) To find the reaction conditions necessary to suppress hydrolysis during acid addition (during the cleavage reaction from the solid phase and the side chain deprotection reaction), particularly hydrolysis originating from N-substituted amino groups. (2) To find reaction conditions that enable practical post-treatment during acid addition, (3) Find reaction conditions that include a solvent that takes into account the unique solubility of non-natural peptide compounds. (4) When a non-natural peptide compound contains a functional group such as a hydroxyl group, it is necessary to suppress side reactions after deprotection (such as N→O acyl shift or side reactions between the hydroxyl group and the reaction reagent (for example, the TFA acylation reaction when TFA is used as the reagent)). This is the challenge. In addition, the challenge is to find protecting groups that satisfy the above four conditions for each functional group in the amino acid side chain. Furthermore, considering the industrial production of peptide compounds containing N-substituted amino acids, another challenge is to find manufacturing methods that can be applied to optimization for specific sequences. [Means for solving the problem]

[0020] The inventors have discovered a novel method to efficiently synthesize cyclic peptides containing N-substituted amino acids. This method addresses numerous challenges encountered when using conventional peptide synthesis methods with TFA to synthesize compounds described in known literature. These challenges could not be adequately addressed by commonly implemented improvement methods, such as reducing the TFA concentration or lowering the reaction temperature. The novel method addresses issues such as suppressing hydrolysis and N→O-acyl shift, establishing a practical work-up method, suppressing TFA ester formation in the presence of hydroxyl groups, and selecting a solvent that ensures peptide solubility. This novel method does not use TFA, which is used in conventional peptide synthesis, and successfully obtains the target product with high selectivity.

[0021] In one aspect of the present invention, TFA is not used in the cleavage step from the solid phase, and a weaker acid, such as 2,2,2-trifluoroethanol (TFE) or hexafluoro-2-propanol (HFIP), is used instead. In addition, in another aspect of the present invention, a protecting group for side-chain functional groups that are not deprotected is used in the cleavage step. In the cleavage step using an acid weaker than TFA, such as TFE or HFIP, unlike when TFA is used, the rate of side reactions, such as hydrolysis of amide bonds, is sufficiently small even after concentration following the reaction. In particular, when an acid weaker than TFA, such as TFE or HFIP, is used, the rate of side reactions is small even with peptides containing highly N-substituted amino acids or cyclized peptides that are more prone to side reactions. Therefore, the target compound can be obtained as the main product. In another aspect of the present invention, the cleavage step uses a reagent that satisfies the following conditions: (1) the cleavage reaction from the solid phase proceeds smoothly while suppressing side reactions of the peptide (such as hydrolysis); (2) the rate of side reactions is sufficiently slow even after post-treatment such as concentration; (3) it exhibits high solubility even for non-natural peptides with high lipophilicity; and (4) it is possible to cleave while retaining the protecting group of the side chain functional group. By using a reagent that satisfies these conditions, it becomes possible to synthesize peptides containing many N-substituted amino acids, in particular drug-like peptides containing many N-alkyl groups. Reagents that satisfy these conditions can be used not only in parallel synthesis but also in the industrial synthesis of specific peptides.

[0022] In one aspect of the present invention, a method for synthesizing peptides is provided that can deprotect the protecting group of the side chain in order to suppress hydrolysis and N→O-acyl shift and promote the deprotection reaction, which is the main reaction of the present invention. For hydrolysis and N→O-acyl shift to proceed, only the acid strength (proton concentration) may be important. Therefore, we have found that by using a weak acid with reduced acidity instead of a strong acid such as TFA, the progress of hydrolysis and N→O-acyl shift can be suppressed. Furthermore, for the desired deprotection to proceed, in addition to the acid strength (proton concentration), the step in which the protecting group is eliminated from the protected functional group as a cationic species (carbocation or oxonium cation) may also be important. Therefore, we have found that by using a solvent with ionizing ability as a solvent that promotes the step in which the protecting group is eliminated as a cationic species, deprotection with the weak acid described above can be promoted.

[0023] In addition, in order to establish an efficient synthesis method for drug-like peptides as described in Patent Document 1, we have found protecting groups for amino acid side-chain functional groups having a low degree of ionization under neutral conditions, such as hydroxyl groups, which are amino acid side-chain functional groups such as Ser and Thr; alkyl alcohol groups having a hydroxyl group in the side chain; phenol groups, which are amino acid side-chain functional groups such as Tyr; imidazole groups, which are amino acid side-chain functional groups such as His; side-chain carboxylic acids, which are amino acid side-chain functional groups such as Asp and Glu; and carboxylic acids of the main chain of peptides or amino acids. These protecting groups are not deprotected under weak acid conditions when cleaved from resin, but can be deprotected under the aforementioned weak acid conditions.

[0024] Furthermore, we have discovered a protecting group that allows for deprotection under the weak acid conditions described above, and that can improve the low reactivity during the extension reaction, even when amino acids such as β-hydroxy-α-amino acids (e.g., Thr, Ser, and their derivatives), which are suspected to have low reactivity during the extension reaction, have a protecting group.

[0025] In other words, the present invention is as follows: [1] The following steps: 1) A step of preparing an amino acid having at least one of the following functional groups i) and ii) (Fmoc-protected amino acid), an amino acid analog having at least one of the following i) and ii) (Fmoc-protected amino acid analog), or a peptide containing both or either the Fmoc-protected amino acid and the Fmoc-protected amino acid analog (Fmoc-protected peptide); i) The amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group, 2) A step of supporting the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide prepared in step 1) onto a solid phase. 3) A step of deprotecting the protecting group having the Fmoc skeleton of an Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide supported on a solid phase with a base to expose the amino group. 4) A step of adding a new Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide to form an amide bond, and 5) A step in which the peptide obtained in step 4) is cleaved from the solid phase under conditions that are weaker than TFA, A method for producing a peptide comprising at least one N-substituted amino acid or an N-substituted amino acid analog. [2] At least one side chain of an amino acid or amino acid analog constituting the peptide obtained in step 4) is protected by a protecting group that is not deprotected under basic conditions but is deprotected by a first acid, and further comprises a step of deprotecting the protecting group with the first acid before or after step 5), In step 5) above, the peptide is cleaved using a second acid, as described in [1], A manufacturing method in which both the first and second acids are weaker acids than TFA, and the acidity of the first acid is higher than the acidity of the second acid. [3] The following steps: 1) A step of preparing an amino acid having at least one of the functional groups i) and ii) below (Fmoc-protected amino acid), an amino acid analog having at least one of the functional groups i) and ii) below (Fmoc-protected amino acid analog), or a peptide containing both or either the Fmoc-protected amino acid and the Fmoc-protected amino acid analog (Fmoc-protected peptide); i) The amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group, 2) A step of deprotecting the protecting group having the Fmoc skeleton of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide with a base to expose the amino group. 3) A step of adding a new Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide to form an amide bond, wherein at least one side chain of the amino acid or amino acid analog constituting the peptide obtained in this step has a protecting group that is not deprotected under basic conditions but is deprotected under conditions weaker than TFA, and 4) A step of deprotecting the protecting group of the side chain under conditions that are weaker than TFA, A method for producing a peptide comprising at least one N-substituted amino acid or an N-substituted amino acid analog. [4] The manufacturing method described in [3], wherein the peptide is produced by a solid-phase method. [5] The method for producing the peptide obtained in step 3) from the solid phase, further comprising the step of cleaving the peptide obtained in step 4) from the solid phase under conditions that are even weaker than the weak acid conditions used in step 4) before or after step 4). [6] The manufacturing method described in [3], wherein the peptide is produced by a liquid-phase method. [7] Step 4) of [1] or step 3) of [3] A step of deprotecting the protecting group having the Fmoc skeleton of a newly added Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide with a base to expose the amino group, and The process further includes the step of adding a new Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide to form an amide bond. A manufacturing method according to any one of [1] to [6], wherein these steps are repeated once or multiple times. [8] The method for producing a peptide according to any one of [1] to [7], wherein the produced peptide contains an amino acid residue or amino acid analog residue having one reaction site at the C-terminus and an amino acid residue, amino acid analog residue or carboxylic acid analog having another reaction site at the N-terminus. [9] The method for producing the peptide according to [8], further comprising the step of connecting one reaction site with the other reaction site to cyclize the peptide.

[10] The method for producing the product according to [9], wherein the amino acid residue, amino acid analog residue, or carboxylic acid analog having the other reaction site is at the N-terminus, and the bond is an amide bond.

[11] The method for producing the product according to [9], wherein the amino acid residue, amino acid analog residue, or carboxylic acid analog having the other reaction site is at the N-terminus, and the bond is a carbon-carbon bond.

[12] A process carried out under conditions that result in a weaker acid than TFA, in which a weak acid with a pKa value of 0-9 in water is ionized by Y OTs A manufacturing method according to any one of [1] to

[11] , wherein the method is carried out using a weak acid solution contained in a solvent having a positive value and a pKa of 5 to 14 in water.

[13] The manufacturing method according to

[12] , wherein the solvent is a fluoroalcohol.

[14] The method for producing the product according to

[13] , wherein the fluoroalcohol is TFE or HFIP.

[15] The manufacturing method according to any one of [2] to

[14] , wherein the protecting group of the side chain is a protecting group that is deprotected in the range of pH 1 to pH 7, or a protecting group that is deprotected in TFA of 10% or less.

[16] The manufacturing method according to any one of [2] to

[15] , wherein the protecting group of the side chain is selected from a) to d) below: a) When the protecting group of the side chain is a protecting group for the hydroxyl group of the side chain of Ser, Thr, Hyp, and their derivatives, any protecting group selected from the MOM skeleton group, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, and Boc skeleton represented by the following general formula; b) When the protecting group of the side chain is a protecting group for the hydroxyl group of the side chain of Tyr and its derivatives, any protecting group selected from the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, Boc skeleton, and tBu skeleton represented by the following general formula; c) When the protecting group of the side chain is a protecting group for the imidazole ring of the side chain of His and its derivatives, any protecting group selected from the MOM skeleton, Bn skeleton, and Trt skeleton represented by the following general formula; d) When the protecting group of the side chain is a protecting group for the carboxylic acid group of the side chain of Asp, Glu, and their derivatives, any protecting group selected from the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, phenyl-EDOTn skeleton, and orthoester skeleton obtained by converting the carbon atom of the carboxylic acid group to be protected into a skeleton substituted with three alkoxy groups; <Protecting group having MOM skeleton> [Chemical formula] (In the formula, R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or 2-trimethylsilylethyl, or R1 is methyl, R2 is H, and X is ethyl, or R1, R2, and R3 are all methyl, or R1 and X together form -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R2 is H Here, when any of R1, R2, and X is methyl or ethyl, these groups may be further substituted with alkyl, benzyl, or aryl.) <Protecting group having Bn skeleton> [Chemical formula] (wherein, R1 to R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are alkyl, or R1, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, R3 is methoxy, and R6 and R7 are H, or R1 and R3 are methoxy, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are H, or R1, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R2 and R3 together form -O-CH2-O-.) ><Protecting group having a Dpm skeleton> [Chemical formula] (wherein, R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen, or R1 to R4 and R7 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form -O- or -CH2-CH2-.) ><Protecting group having a Trt skeleton> [Chemical formula] (wherein, R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, or R1, R2, and R4 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R3 is methyl or methoxy, or R1 is Cl, and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, or R1 to R4, and R7 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form -O-. ) <Protecting group having a silyl skeleton> [Chemical formula] (In the formula, R1 to R3 are each independently alkyl or aryl. ) <Protecting group having a Boc skeleton> [Chemical formula] (In the formula, R1 to R9 are each independently H, alkyl, or aryl. ) <Protecting group having a tBu skeleton> [Chemical formula] (In the formula, R1 to R9 are each independently H, alkyl, or aryl. ) <Protecting group having a phenyl-EDOTn skeleton> [Chemical formula] (In the formula, R1 to R3 are each independently H or methoxy). [Advantages of the Invention]

[0026] According to the present invention, a peptide containing an N-substituted amino acid can be obtained with high purity at high synthesis efficiency.

[0027] For example, in the case of a peptide sequence containing an amino acid having a protecting group on the side chain, (1) The combination of an acid weaker than TFA and a solvent exhibiting ionizing ability discovered by the present invention minimizes acid hydrolysis of peptide chains and N→O-acyl shifts and TFA esterification that may occur in sequences containing β-hydroxy-α-amino acids (e.g., Ser, Thr, and their derivatives), thereby enabling deprotection. (2) When extending the amino acid in an amide bond formation reaction, the reaction rate and reaction efficiency can be improved compared to when the amino acid has a protecting group commonly used in peptide synthesis. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 shows the basic synthetic route of a cyclic peptide containing an N-methylamino acid in its sequence. [Figure 2] Figure 2 shows the results of LC-MS analysis of the target peptide (compound 131), its hydrolyzed product (TM+H2O), and its HFIP-mediated solvolysis product (TM+HFIP) under deprotection conditions in a 0.1M tetramethylammonium bisulfate / HFIP solution (2% TIPS). [Figure 3] Figure 3 shows the results of LC-MS analysis under deprotection conditions using a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) to detect the target peptide (compound 131), its hydrolyzed product (TM+H2O), and its HFIP-mediated solvolysis product (TM+HFIP). [Figure 4] Figure 4 shows the results of LCMS analysis under deprotection conditions using a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS), illustrating the detection of the target peptide (compound 133) and its N→O-acyl shift variant. [Figure 5] Figure 5 shows the results of LC-MS analysis of the target peptide (compound 131), its hydrolyzed product (TM+H2O), and its HFIP-mediated solvolysis product (TM+HFIP) under deprotection conditions in a 0.05 M oxalic acid / HFIP solution (2% TIPS). [Figure 6] Figure 6 shows the results of LC-MS analysis under deprotection conditions using a 0.05 M maleic acid / HFIP solution (2% TIPS) to detect the target peptide (compound 131), its hydrolyzed product (TM+H2O), and its HFIP-mediated solvolysis product (TM+HFIP). [Figure 7] Figure 7 shows the results of LC-MS analysis under deprotection conditions using a 0.05 M oxalic acid / HFIP solution (2% TIPS), illustrating the detection of the target peptide (compound 133) and its N→O-acyl shift variant. [Figure 8] Figure 8 shows the results of LC-MS analysis under deprotection conditions using a 0.05 M maleic acid / HFIP solution (2% TIPS), illustrating the detection of the target peptide (compound 133) and its N→O-acyl shift variant. [Figure 9] Figure 9 shows the results of LC-MS analysis under deprotection conditions of 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS), illustrating the detection of the target peptide (compound 137) and its HFIP-mediated solvolysis product (in which one of the amide bonds has been solvoly digested by HFIP). [Figure 10] Figure 10 shows the results of LC-MS analysis under deprotection conditions of 0.05 M tetramethylammonium bisulfate / TFE (2% TIPS), illustrating the detection of the target peptide (compound 137) and its solvolysis product by TFE (in which one of the amide bonds has been solvoly digested by TFE). [Figure 11] Figure 11 shows the results of LC-MS analysis indicating the detection of the target peptide (compound 135) when a 0.1M tetramethylammonium bisulfate / HFIP solution (2% TIPS) was used as a deprotection condition, and the reaction was stopped by adding a base (DIPEA) to the solution. [Figure 12]Figure 12 shows the results of LC-MS analysis indicating the detection of the target peptide (compound 133) when a 0.1M tetramethylammonium bisulfate / HFIP solution (2% TIPS) was used as a deprotection condition, and the reaction was stopped by adding a base (DIPEA) to the solution. [Figure 13] Figure 13 shows the results of LC-MS analysis when Fmoc-Thr(Trt)-OH was added, indicating the detection of the target peptide (compound 112) and the target peptide with the Thr group removed (compound 113). [Figure 14] Figure 14 shows the results of LC-MS analysis demonstrating the detection of the target peptide (compound 114) when Fmoc-Thr(THP)-OH was added. No peptide with the Thr group removed (compound 113) was detected. [Figure 15] Figure 15 shows the results of LC-MS analysis, which detects the target peptide (compound 115) and the target peptide with the MeSer removed (compound 116) when synthesized using Fmoc-MeSer(DMT)-OH·0.75DIPEA. [Figure 16] Figure 16 shows the results of LC-MS analysis, which detects the target peptide (compound 115) and the target peptide with the MeSer removed (compound 116) when synthesized using Fmoc-MeSer(THP)-OH (compound 6). [Figure 17] Figure 17 shows the results of LC-MS analysis under deprotection conditions of 5% TFA / DCE (5% TIPS), illustrating the detection of the target peptide (compound 131) and its hydrolyzed product (TM+H2O). [Figure 18] Figure 18 shows the results of LCMS analysis under deprotection conditions of 5% TFA / DCE (5% TIPS), showing the detection of the target peptide (compound 133), the N→O-acyl shift derivative of the target, a compound in which one hydroxyl group of the target is esterified with TFA, and a compound in which both hydroxyl groups of the target are esterified with TFA. [Figure 19] Figure 19 shows the results of LCMS analysis under deprotection conditions of 5% TFA / DCE (5% TIPS) (0°C), indicating the detection of the target peptide (compound 133), the N→O-acyl shift derivative of the target, a compound in which one hydroxyl group of the target is esterified with TFA, and a compound in which both hydroxyl groups of the target are esterified with TFA. [Figure 20] Figure 20 shows the results of LCMS analysis under deprotection conditions of 5% TFA / DCE (5% TIPS) (25°C), illustrating the detection of the target peptide (compound 133), the N→O-acyl shift derivative of the target, a compound in which one hydroxyl group of the target is esterified with TFA, and a compound in which both hydroxyl groups of the target are esterified with TFA. [Figure 21] Figure 21 shows a synthesis method that includes an extension reaction in the liquid phase. [Modes for carrying out the invention]

[0029] In one embodiment, the present invention relates to a method for producing a peptide comprising at least one N-substituted amino acid or an N-substituted amino acid analog, comprising the following steps. 1) A step of preparing an amino acid having at least one of the following functional groups i) and ii) (Fmoc-protected amino acid), an amino acid analog having at least one of the following i) and ii) (Fmoc-protected amino acid analog), or a peptide containing both or either the Fmoc-protected amino acid and the Fmoc-protected amino acid analog (Fmoc-protected peptide); i) The amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group, 2) A step of supporting the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide prepared in step 1) onto a solid phase. 3) A step of deprotecting the protecting group having the Fmoc skeleton of an Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide supported on a solid phase with a base to expose the amino group. 4) A step of adding a new Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide to form an amide bond, and 5) A step in which the peptide obtained in step 4) is cleaved from the solid phase under conditions that are weaker than TFA.

[0030] In another embodiment, the present invention relates to a method for producing a peptide comprising at least one N-substituted amino acid or an N-substituted amino acid analog, comprising the following steps. 1) A step of preparing an amino acid having at least one of the functional groups i) and ii) below (Fmoc-protected amino acid), an amino acid analog having at least one of the functional groups i) and ii) below (Fmoc-protected amino acid analog), or a peptide containing both or either the Fmoc-protected amino acid and the Fmoc-protected amino acid analog (Fmoc-protected peptide); i) The amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group, 2) A step of deprotecting the protecting group having the Fmoc skeleton of an Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide with a base to expose the amino group. 3) A step of adding a new Fmoc-protected amino acid, an Fmoc-protected amino acid analog, or an Fmoc-protected peptide to form an amide bond, wherein at least one side chain of the amino acid or amino acid analog constituting the peptide obtained in this step has a protecting group that is not deprotected under basic conditions but is deprotected under conditions weaker than TFA, and 4) A step of deprotecting the protecting group of the side chain under conditions that are weaker than TFA. The above peptide may be manufactured by a solid-phase method or a liquid-phase method.

[0031] The term "peptide" in this invention is not particularly limited as long as it is a peptide formed by amide or ester bonds between amino acids and / or amino acid analogs, but is preferably a peptide of 5 to 30 residues, more preferably 7 to 15 residues, and even more preferably 9 to 13 residues. The peptide synthesized in this invention contains at least one N-substituted amino acid or amino acid analog (also called an N-substituted amino acid), preferably two or more, more preferably three or more, and even more preferably five or more N-substituted amino acids. These N-substituted amino acids may be present continuously or discontinuously in the peptide. The peptide in this invention may be a linear peptide or a cyclic peptide, but a cyclic peptide is preferred.

[0032] The "cyclic peptide" in this invention can be obtained by synthesizing a linear peptide according to the method of this invention and then cyclizing it. The cyclization can take any form, such as cyclization via a carbon-nitrogen bond like an amide bond, cyclization via a carbon-oxygen bond like an ester bond or an ether bond, cyclization via a carbon-sulfur bond like a thioether bond, cyclization via a carbon-carbon bond, or cyclization by constructing a heterocycle. There are no particular limitations, but cyclization via a covalent bond such as an amide bond or a carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxylic acid group on the side chain and an amino group on the N-terminal main chain is particularly preferred. The position of the carboxylic acid group, amino group, etc. used in cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position where cyclization is possible.

[0033] In the present invention, "N-substituted amino acid" refers to an amino acid or amino acid analog in which the main chain amino group is N-substituted, among the "amino acids" or "amino acid analogs" described later, and amino acids or amino acid analogs that are N-alkylated, such as by N-methylation, are preferred. Specifically as N-substituted amino acids, the main chain amino group of the amino acid or amino acid analog is an NHR group, where R is an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group, or a group such as proline in which a carbon atom bonded to the N atom and a carbon atom from the α position form a ring. The substituents of each optionally substituted group are not particularly limited and include, for example, halogen groups, ether groups, hydroxyl groups, etc. Specifically, alkyl groups, aralkyl groups, and cycloalkyl groups are preferred as such N-substituted amino acids.

[0034] In this invention, "amino acids" refer to α, β, and γ amino acids, and are not limited to natural amino acids (in this application, natural amino acids refer to the 20 types of amino acids contained in proteins, specifically Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro), but may also be non-natural amino acids. In the case of α-amino acids, they may be L-type amino acids, D-type amino acids, or α,α-dialkyl amino acids. The selection of amino acid side chains is not particularly limited, but examples include hydrogen atoms, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. Each amino acid side chain may have substituents attached, which can be freely selected from any functional group including, for example, N atoms, O atoms, S atoms, B atoms, Si atoms, and P atoms. The number of substituents is not particularly limited and may be one or two or more.

[0035] The "amino acid analog" in the present invention preferably means an α-hydroxycarboxylic acid. The side chain of the α-hydroxycarboxylic acid is not particularly limited, similar to that of an amino acid, and examples include a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group. The steric structure of the α-hydroxycarboxylic acid may correspond to the L-form or the D-form of an amino acid. The side chain is not particularly limited and is freely selected from any functional groups containing, for example, N atom, O atom, S atom, B atom, Si atom, and P atom. The number of substituents is not particularly limited and may be one or two or more. For example, it may have an S atom and further have functional groups such as an amino group or a halogen group. In the case of β- or γ-amino acids, any configuration is allowed, similar to the case of α-amino acids, and the selection of the side chain is also the same as that of α-amino acids without particular restriction.

[0036] The "amino acids" and "amino acid analogs" constituting the peptides synthesized in the present invention include all corresponding isotopes. The isotopes of "amino acids" and "amino acid analogs" are those in which at least one atom is replaced by an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of the isotopes included in the "amino acids" and "amino acid analogs" constituting the peptide compounds of the present invention include, for example, hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, and chlorine atom. Specifically, for example, 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl are included.

[0037] Amino acids or amino acid analogs may have one or more substituents. Examples of such substituents include those derived from oxygen, nitrogen, sulfur, boron, phosphorus, silicon, and halogen atoms.

[0038] Examples of halogen-derived substituents include fluoro(-F), chloro(-Cl), bromo(-Br), and iod(-I).

[0039] Examples of substituents derived from the oxygen atom include hydroxyl (-OH), oxy (-OR), carbonyl (-C=OR), carboxyl (-CO2H), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio group (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), and carboxylcarbonyl (-C(=O)-CO2H).

[0040] Examples of oxy (-OR) compounds include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy.

[0041] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, and aralkylcarbonyl.

[0042] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl. (-C=O-OR)

[0043] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

[0044] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl.

[0045] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.

[0046] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, and aralkylaminocarbonyl. In addition to these, compounds in which the hydrogen atom bonded to the nitrogen atom in -C=O-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0047] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0048] Examples of oxycarbonylaminos (-NH-C=O-OR) include alkoxycarbonylaminos, cycloalkoxycarbonylaminos, alkenyloxycarbonylaminos, alkynyloxycarbonylaminos, aryloxycarbonylaminos, heteroaryloxycarbonylaminos, and aralkyloxycarbonylaminos. In addition to these, compounds in which the hydrogen atom bonded to the nitrogen atom in the -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl groups are also included.

[0049] Examples of sulfonylaminos (-NH-SO2-R) include alkylsulfonylaminos, cycloalkylsulfonylaminos, alkenylsulfonylaminos, alkynylsulfonylaminos, arylsulfonylaminos, heteroarylsulfonylaminos, and aralkylsulfonylaminos. In addition to these, compounds in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0050] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl. In addition to these, compounds in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0051] Examples of sulfamoylamino (-NH-SO2-NHR) include alkyl sulfamoylamino, cycloalkyl sulfamoylamino, alkenyl sulfamoylamino, alkynyl sulfamoylamino, aryl sulfamoylamino, heteroaryl sulfamoylamino, and aralkyl sulfamoylamino. 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.

[0052] Examples of substituents derived from the sulfur atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), and sulfo (-SO3H).

[0053] Examples of thio(-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio.

[0054] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, and aralkylsulfinyl.

[0055] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, and aralkylsulfonyl.

[0056] Substituents derived from the N atom include azide (-N3, also called the "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'').

[0057] Examples of secondary amino acids (-NH-R) include alkylaminos, cycloalkylaminos, alkenylaminos, alkynylaminos, arylaminos, heteroarylaminos, and aralkylaminos.

[0058] Examples of tertiary aminos (-NR(R')) include alkyl(aralkyl)aminos, which are amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, and these two substituents may form a ring.

[0059] Examples of substituted amidinos (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, such as alkyl(aralkyl)(aryl)amidinos.

[0060] Examples of substituted guanidinos (-NR-C(=NR''')-NR'R'') include groups where R, R', R'', and R''' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, or groups in which these groups form a ring.

[0061] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are independently selected from hydrogen atoms, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or groups that form a ring.

[0062] Examples of substituents derived from the B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' can be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, or they may form a ring.

[0063] Thus, the amino acids or amino acid analogs of the present invention may have one or more substituents, including oxygen atoms, nitrogen atoms, sulfur atoms, boron atoms, phosphorus atoms, silicon atoms, and halogen atoms, which are commonly used in low-molecular-weight compounds. These substituents may be further substituted with other substituents.

[0064] In this specification, the "amino acids" and "amino acid analogs" that constitute the peptides synthesized in this invention may also be referred to as "amino acid residues" and "amino acid analog residues," respectively.

[0065] In the present invention, "Fmoc-protected amino acid" and "Fmoc-protected amino acid analog" are amino acids and amino acid analogs, respectively, having at least one of the following functional groups i) and ii): i) The amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group.

[0066] In the present invention, "protecting group having an Fmoc skeleton" means an Fmoc group or a group in which any substituent is introduced at any position on the constituent skeleton of an Fmoc group. Specifically, examples of protecting groups having an Fmoc skeleton include 9-fluorenylmethyloxycarbonyl (Fmoc) group, 2,7-di-tert-butyl-Fmoc (Fmoc*) group, 2-fluoro-Fmoc (Fmoc(2F)) group, 2-monoisooctyl-Fmoc (mio-Fmoc) group, and 2,7-diisooctyl-Fmoc (dio-Fmoc) group. In the present invention, instead of protecting groups having an Fmoc skeleton, protecting groups that can be deprotected under basic conditions or by a nucleophile exhibiting basicity (e.g., piperidine or hydrazine) can also be used. Examples of such protecting groups include, for instance, 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group, (1,1-dioxobenzo[b]thiophene-2-yl)methyloxycarbonyl (Bsmoc) group, (1,1-dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc) group, 1-(4,4-dimethyl-2,6-dioxocyclohexy-1-ylidene)-3-methylbutyl (ivDde) ​​group, tetrachlorophthaloyl (TCP) group, 2-[phenyl(methyl)sulfonio]ethyloxycarbonyltetrafluoroborate (Pms) group, ethanesulfonylethoxycarbonyl (Esc) group, and 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps) group. In addition, protecting groups other than acids and bases that can be used for deprotection can also be utilized.Examples of such protecting groups include the benzyloxycarbonyl (Z) group, which can be deprotected by hydrogenation in the presence of a transition metal catalyst such as palladium; the allyloxycarbonyl (Alloc) group, which can be deprotected by a combination of a palladium catalyst and a scavenger (for example, a combination of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and phenylsilane); the o-nitrobenzenesulfonyl (oNBS, Ns), 2,4-dinitrobenzenesulfonyl (dNBS), and dithiasuccinoyl (Dts) groups, which can be deprotected by a combination of an alkylthiol or arylthiol and a base; and the p-nitrobenzyloxycarbonyl (pNZ) group, which can be deprotected by hydrogenation in the presence of a transition metal catalyst or by reductive deprotection with a reducing agent such as sodium dithionite (Na2S2O4) (Reference: Amino Acid-Protecting Groups, Chem. Rev. 2009, 109, 2455-2504).

[0067] In the present invention using the Fmoc method, for example, an Fmoc-protected amino acid or Fmoc-protected amino acid analog can be preferably used in which the amino group of the main chain is protected by an Fmoc group, the functional group of the side chain is optionally protected by a protecting group that is not cleaved by basics such as piperidine or DBU, and the carboxylic acid group of the main chain is not protected. An Fmoc-protected amino acid or Fmoc-protected amino acid analog having an amino group protected by a protecting group having an Fmoc skeleton and a carboxylic acid group without a protecting group can also be preferably used.

[0068] In the present invention, if an Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide has a functional group in its side chain, it is preferable that the functional group is protected by a protecting group. When the functional group in the side chain is protected by a protecting group, any well-known protecting group that can be deprotected under any conditions can be used. Preferred protecting groups are those that are not cleaved under basic conditions and can be deprotected under conditions that are weaker than TFA. Examples of protecting groups that can be deprotected in acidic conditions include those that can be deprotected in the range of pH 1 to pH 7, preferably in the range of pH 2 to pH 6. Alternatively, a protecting group that can be deprotected in TFA of 10% or less, or a protecting group having the structure described later, can be used. In the present invention, any well-known protecting group can be used as the protecting group for the side chain. For example, a protecting group that satisfies the above conditions can be selected from among the protecting groups described in the following references i) and ii) as the protecting group for the side chain. Non-patent literature i) Greene's Protective Groups inOrganic Synthesis, Fourth Edition, Non-patent literature ii)Chemical Reviews, 2009, 109(6),2455-2504.

[0069] The method of the present invention can be used for peptide synthesis by parallel synthesis. In this case, a protecting group is not necessarily required on the side chain of the amino acid, but if a protecting group is required on the side chain, it is preferable that the protecting group used is rapidly deprotected under the deprotection conditions of the present invention. It is preferable that 50% of the protecting group on the side chain is deprotected within 24 hours, and particularly preferable that 90% is deprotected within 4 hours. As protecting groups that satisfy these conditions, protecting groups having the Trt skeleton, THP skeleton, THF skeleton, and TBS skeleton described later are preferred. Furthermore, in order to be easily deprotected by acid and to have high reactivity during extension, it is preferable that the atom on the protecting group side directly attached to the functional group has at least one hydrogen atom substituted (sterically less bulky than the protecting group of the Trt skeleton). Among these, protecting groups in which substituents other than hydrogen form a ring are more preferred, and THP and THF are particularly preferred.

[0070] The method of the present invention can also be used for industrial peptide synthesis. In this case, as with parallel synthesis, it is not necessarily required that the side chains of the amino acids have protecting groups, but if protecting groups are present on the side chains, it is preferable that they be the same type of protecting groups as in parallel synthesis. The sequence of the synthesized peptide does not have problems with hydrolysis and N→O-acyl shift during deprotection, but if there are problems with the elongation reaction due to the bulkiness of the protecting group, a strong acid such as TFA, which is commonly used during deprotection, may be used. Also, if there are no problems with the elongation reaction of the synthesized peptide, a bulky protecting group can be used.

[0071] In this invention, the "conditions for being a weaker acid than TFA" are preferably a weak acid having a pKa value of 0 to 9 in water, and an ionization capacity of Y OTs One example of a condition is the use of a weak acid solution containing a solvent with a positive value and a pKa of 5-14 in water.

[0072] A "weak acid with a pKa value of 0 to 9 in water" is preferred, and more preferably a weak acid with a pKa of 1 to 5 in water. Specific examples of such weak acids include tetramethylammonium bisulfate (pKa = 2.0 in water), oxalic acid (pKa = 1.23 in water), and maleic acid (pKa = 1.92 in water). The concentration of the weak acid dissolved in the solvent can be arbitrary, as long as the conditions for being weaker than TFA are met.

[0073] "Ionization capacity Y OTs Preferably, a solvent is a fluoroalcohol, which has a positive value and a pKa of 5 to 14 in water. A fluoroalcohol is a general term for a compound in which a fluorine atom is bonded to a carbon atom other than the carbon atom to which a hydroxyl group is bonded among the carbon atoms that make up the alcohol. In the present invention, compounds in which a hydroxyl group is bonded to an aromatic ring, such as 2,3,4,5,6-pentafluorophenol, are also included as fluoroalcohols. Preferred fluoroalcohols include 2,2,2-trifluoroethanol (TFE) and hexafluoro-2-propanol (HFIP).

[0074] In the present invention, if the conditions for the solution to be weaker than TFA are met, other organic solvents (e.g., dichloromethane or 1,2-dichloroethane) or cation scavengers (e.g., triisopropylsilane) can also be added to the weak acid solution.

[0075] In the present invention, when an Fmoc-protected amino acid or an Fmoc-protected amino acid analog has a protecting group in its side chain, the following are preferably used as the protecting group in the side chain.

[0076] When the side chain protecting group is a protecting group for the hydroxyl group of Ser, Thr, Hyp, and their derivatives, a protecting group having a MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, or Boc skeleton represented by the following general formula is preferred.

[0077] [ka] Representative examples of protecting groups with an MOM skeleton include MOM (R1=H, R2=H, X=Me), EE (R1=Me, R2=H, X=Et), MIP (R1=Me, R2=Me, X=Me), THP (R2=H, with a ring structure of four carbon atoms between R1 and X), THF (R2=H, with a ring structure of three carbon atoms between R1 and X), and SEM (R1=H, R2=H, X=2-trimethylsilylethyl). The Me and Et substituents on the skeleton can also be substituted with other alkyl groups, benzyl groups, aryl groups, etc.

[0078] [ka] Representative examples of protecting groups with a Bn skeleton include Pis (R6=Me, R7=Me, other R=H), PMB (R3=OMe, other R=H), and DMB (R1=OMe, R3=OMe, other R=H). Other alkyl groups may be used instead of the Me substituent. Alkyl groups, aryl groups, halogen groups, etc., may also be substituted on the benzene ring.

[0079] [ka] A typical example of a protecting group having a Dpm skeleton is Dpm (all R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc. Alternatively, a cross-linked structure between R5 and R6 may be used, such as a Xan group cross-linked via an oxygen atom, or a dibenzosberyl group cross-linked via two carbon atoms.

[0080] [ka] Representative examples of protecting groups having a Trt skeleton include Trt (all R=H), Mmt (R3=Me, other R=H), Mtt (R3=OMe, other R=H), Dmt (R3=OMe, R8=OMe, other R=H), and Clt (R1=Cl, other R=H). Alkyl groups, aryl groups, alkoxy groups, halogen groups, etc., may be substituted on the aromatic ring. Furthermore, a Pixyl group can be used that is bridged between R5 and R6, for example, a Pixyl group bridged via an oxygen atom.

[0081] [ka] Typical examples of protecting groups with a silyl skeleton include TBS (R1=Me, R2=Me, R3=tBu). Other alkyl groups, aryl groups, etc., may be substituted for Me and tBu.

[0082] [ka] Typical examples of protecting groups with a Boc skeleton include Boc (all R=H), but other alkyl groups, aryl groups, etc., may also be substituted.

[0083] In addition, the following protecting groups can also be used. [ka]

[0084] Among these protecting groups, THP and Trt are particularly preferred. Furthermore, when the amino acid residue is Ser, THP and Trt are particularly preferred as the side chain protecting group, and when the amino acid residue is Thr, THP is particularly preferred as the side chain protecting group.

[0085] When the side chain protecting group is a protecting group for an amino acid having a hydroxyl group substituted with an aryl group, such as Tyr, D-Tyr, or Tyr(3-F), a protecting group having a MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, Boc skeleton, or tBu skeleton, represented by the following general formula, is preferred.

[0086] [ka] Representative examples of protecting groups with an MOM skeleton include MOM (R1=H, R2=H, X=Me), BOM (R1=H, R2=H, X=Bn), EE (R1=Me, R2=H, X=Et), THP (R2=H, with a ring structure of four carbon atoms between R1 and X), THF (R2=H, with a ring structure of three carbon atoms between R1 and X), and SEM (R1=H, R2=H, X=2-trimethylsilylethyl). The Me and Et substituents on the skeleton can also be substituted with other alkyl groups, benzyl groups, aryl groups, etc.

[0087] [ka] Representative examples of protecting groups with a Bn skeleton include Pis (R6=Me, R7=Me, other R=H), PMB (R3=OMe, other R=H), and DMB (R1=OMe, R3=OMe, other R=H). Other alkyl groups may be used instead of the Me substituent. Alkyl groups, aryl groups, halogen groups, etc., may also be substituted on the benzene ring.

[0088] [ka] A typical example of a protecting group having a Dpm skeleton is Dpm (all R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc. Alternatively, a cross-linked structure between R5 and R6 may be used, such as a Xan group cross-linked via an oxygen atom, or a dibenzosberyl group cross-linked via two carbon atoms.

[0089] [ka] Representative examples of protecting groups having a Trt skeleton include Trt (all R=H), Mmt (R3=Me, other R=H), Mtt (R3=OMe, other R=H), and Clt (R1=Cl, other R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc. Furthermore, a Pixyl group can be used that is bridged between R5 and R6, for example, a Pixyl group bridged via an oxygen atom.

[0090] [ka] Typical examples of protecting groups with a silyl skeleton include TBS (R1=Me, R2=Me, R3=tBu). Other alkyl groups, aryl groups, etc., may be substituted for Me and tBu.

[0091] [ka] Typical examples of protecting groups with a Boc skeleton include Boc (all R=H), but other alkyl groups, aryl groups, etc., may also be substituted.

[0092] [ka] A typical example of a protecting group having a tBu skeleton is tBu (all R=H). Other alkyl groups, aryl groups, etc. may be substituted.

[0093] Among these protecting groups, tBu, Pis, Trt, Clt, THP, and THF are particularly preferred. Furthermore, when the amino acid residue is Tyr or D-Tyr, tBu, Trt, Clt, and THP are particularly preferred as the side chain protecting group, and when the amino acid residue is Tyr(3-F), tBu and Pis are particularly preferred as the side chain protecting group.

[0094] When the side chain protecting group is a protecting group for an amino acid having an imidazole in its side chain, such as His or MeHis, it is preferable to use a protecting group having a MOM skeleton, Bn skeleton, or Trt skeleton represented by the following general formula.

[0095] [ka] Representative examples of protecting groups with an MOM skeleton include MBom (R1=H, R2=H, X=4-methoxybenzyl), 2,4-DMBom (R1=H, R2=H, X=2,4-dimethoxybenzyl), 3,4-DMBom (R1=H, R2=H, X=3,4-dimethoxybenzyl), EE (R1=Me, R2=H, X=Et), THP (R2=H, with a ring structure of four carbon atoms between R1 and X), and THF (R2=H, with a ring structure of three carbon atoms between R1 and X). Substituents such as Me and Et on the skeleton can also be substituted with other alkyl groups, benzyl groups, or aryl groups.

[0096] [ka] Representative examples of protecting groups with a Bn skeleton include Pis (R6=Me, R7=Me, other R=H), PMB (R3=OMe, other R=H), and DMB (R1=OMe, R3=OMe, other R=H). Other alkyl groups may be used instead of the Me substituent. Alkyl groups, aryl groups, halogen groups, etc., may also be substituted on the benzene ring.

[0097] [ka] Representative examples of protecting groups having a Trt skeleton include Trt (all R=H), Mmt (R3=Me, other R=H), Mtt (R3=OMe, other R=H), and Clt (R1=Cl, other R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc.

[0098] Among these, Trt is particularly preferred. Furthermore, when the amino acid residues are His or MeHis, Trt is particularly preferred as the side chain protecting group.

[0099] Furthermore, for example, when the carboxylic acid group of the main chain is used as a "free or activated esterified carboxylic acid group," protecting groups having the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, or phenyl-EDOTn skeleton, represented by the following general formulas, can be used as protecting groups for the carboxylic acid group of the side chain of Asp, Glu, and their derivatives, or when the carboxylic acid group of the side chain of Asp, Glu, and their derivatives is used as a "free or activated esterified carboxylic acid group," protecting groups having the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, or phenyl-EDOTn skeleton, represented by the following general formulas, can be used as protecting groups for the carboxylic acid group. In addition, protecting groups having an orthoester skeleton in which three alkoxy groups are bonded to a carbon atom derived from the carboxylic acid group can also be used as protecting groups for carboxylic acids. The carbon atoms forming these protecting groups may be substituted.

[0100] [ka] Representative examples of protecting groups with an MOM skeleton include BOM (R1=H, R2=H, X=Bn), THP (R2=H, with a ring structure of four carbon atoms between R1 and X), and THF (R2=H, with a ring structure of three carbon atoms between R1 and X). Substituents on the skeleton can also be other alkyl groups, benzyl groups, aryl groups, etc.

[0101] [ka] Representative examples of protecting groups with a Bn skeleton include Pis (R6=Me, R7=Me, other R=H), PMB (R3=OMe, other R=H), DMB (R1=OMe, R3=OMe, other R=H), and piperonyl (a structure in which oxygen atoms are substituted on R2 and R3, and these oxygen atoms are bridged by one carbon atom, with other R=H). Other alkyl groups may be used instead of the substituted Me. Alkyl groups, aryl groups, halogen groups, etc., may also be substituted on the benzene ring.

[0102] [ka] A typical example of a protecting group having a Dpm skeleton is Dpm (all R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc. Alternatively, a cross-linked structure between R5 and R6, such as a dibenzosveryl group cross-linked via two carbon atoms, may also be used.

[0103] [ka] Representative examples of protecting groups having a Trt skeleton include Trt (all R=H), Mmt (R3=Me, other R=H), Mtt (R3=OMe, other R=H), and Clt (R1=Cl, other R=H). The aromatic ring may be substituted with alkyl groups, aryl groups, alkoxy groups, halogen groups, etc. Furthermore, a Pixyl group can be used that is bridged between R5 and R6, for example, a Pixyl group bridged via an oxygen atom.

[0104] [ka] Typical examples of protecting groups with a tBu skeleton include tBu (all R=H) and Mpe (R1=Me, R4=Me, other R=H). Other alkyl groups, aryl groups, etc., may also be substituted.

[0105] [ka] Phenyl-EDOTn can be a combination of substituents such as (i) R1=R2=R3=OMe, (ii) R1=R2=OMe,R3=H, (iii) R1=R2=H,R3=OMe, or (iv) R1=R2=R3=H.

[0106] [ka] A dicyclopropylmethyl group can also be used.

[0107] Among these, tBu, Pis, and Trt are particularly preferred.

[0108] In the present invention, "Fmoc-protected peptide" means a peptide containing both or either of the "Fmoc-protected amino acid" and the "Fmoc-protected amino acid analog." Examples of such peptides include dipeptides and oligopeptides having two or more molecules containing either or both of the above-mentioned Fmoc-protected amino acid and Fmoc-protected amino acid analog.

[0109] In the solid-phase peptide synthesis of the present invention, Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or Fmoc-protected peptides (sometimes referred to as Fmoc-protected amino acids, etc.) can be supported on a solid phase using a resin. The group (resin binding group) used for binding to the Fmoc-protected amino acids, etc. of the resin is not particularly limited as long as the peptide can be cleaved by acid. The amount and loading rate of the Fmoc-protected amino acids, etc. are also not particularly limited. In the present invention, for example, trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (Clt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt) can be used. The resin is preferably one that has a resin binding group that is judged to be "H (<5% TFAin DCM)" as an acid-sensitive group as described in the Solid-Phase Synthesis Handbook (published by Merck KGaA, May 1, 2002), and can be appropriately selected according to the functional group of the amino acid used. For example, when using a carboxylic acid (main-chain carboxylic acid, or a side-chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (a phenol group represented by Tyr) as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (Clt resin) as the resin. When using an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (Clt resin), or 4-methyltrityl chloride resin (Mtt resin) as the resin. The type of polymer constituting the resin is not particularly limited. In the case of a resin composed of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The crosslinking ratio is also not particularly limited, but 1% DVB (divinylbenzene) crosslinking is preferred.

[0110] Fmoc-protected amino acids, Fmoc-protected amino acid analogs, or Fmoc-protected peptides are supported on a resin by a chemical reaction between a resin-binding group and a free carboxylic acid group or an activated esterified carboxylic acid group of the C-terminal amino acid of the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide. In this case, the free carboxylic acid may be the main chain carboxylic acid of the amino acid or amino acid analog, or it may be a side chain carboxylic acid (such as Asp). Instead of a carboxylic acid group, a free OH group or free SH group of the main chain or side chain of the C-terminal amino acid of the Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide can also be used for support to the solid phase.

[0111] The protecting group having the Fmoc skeleton of an Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide supported on a solid phase is deprotected with a base to expose the amino group. The base used here is not particularly limited, but deprotecting agents commonly used in peptide synthesis can be used (e.g., Amino Acid-Protecting Groups (Chem. Rev. 2009, 109, 2455-2504)). Preferred deprotecting agents include, for example, secondary amines, bases having an amidine skeleton, and bases having a guanidine skeleton. Specific secondary amines include, for example, piperidine, morpholine, pyrrolidine, and piperazine. Specific bases having an amidine skeleton include, for example, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU) and 1,5-diazabicyclo[4.3.0]-5-nonene (DBN). Specific examples of bases having a guanidine skeleton include, for instance, 1,1,3,3-tetramethylguanidine.

[0112] The exposed amino group is condensed with the newly added Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide free or activated esterified carboxylic acid group to form a peptide bond.

[0113] The coupling agent used when condensing an amino group and a carboxylic acid group is not particularly limited as long as it can form an amide bond, and coupling agents commonly used in peptide synthesis are preferred (e.g., Peptide Coupling Reagents, More than a Letter Soup (Chem. Rev. 2011, 111, 6557-6602)). Specific examples of such coupling agents include those having a carbodiimide skeleton. For example, a coupling agent having a carbodiimide skeleton can be used in a condensation reaction in combination with a hydroxy compound that can form an active ester. Examples of coupling agents having a carbodiimide skeleton include N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI·HCl) (see, for example, Watanabe Chemical catalog, Amino acids and chiral building blocks to new medicine). Examples of hydroxy compounds that can form active esters include 1-hydroxy-1H-benzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 2-cyano-2-(hydroxyimino)ethyl acetate (oxyma), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt or HODhbt), N-hydroxy-5-norbornene-2,3-dicarboxymide (HONB), 2,3,4,5,Examples include 6-pentafluorophenol (HOPfp), N-hydroxysuccinimide (HOSu), and 6-chloro-1-hydroxy-1H-benzotriazole (Cl-HOBt) (see, for example, Watanabe Chemical's catalog, Amino acids and chiral building blocks to new medicine). Salts having these skeletons, such as K-oxyma, the potassium salt of oxyma, can also be used. Among these, HOBt, HOAt, oxyma, and HOOBt are particularly preferred. In particular, it is preferable to use a combination of DIC and HOAt, or a combination of DIC and oxyma. Other phosphonium-based and uronium-based condensing agents include O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethyl Tylamino(morpholino)uronium hexafluorophosphate (COMU), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-Tetramethyluronium tetrafluoroborate (TATU), 1H-benzotriazole-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 1H-benzotriazole-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBroP), chlorotri(pyrrolidino)phosphonium Muhexafluorophosphate (PyCloP), (7-azabenzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotris(dimethylamino)phosphonium hexafluorophosphate (Brop), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one (DEPBT), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetramethyl Trafluoroboric acid (TSTU), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), tetramethylthiuronium S-(1-oxide-2-pyridyl)-N,N,N',N'-tetrafluoroboric acid The following can be used in condensation reactions: a salt (TOTT), O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TPTU), and a base (N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), 2,4,6-trimethylpyridine (2,4,6-collidine), or 2,6-dimethylpyridine (2,6-lutidine). In particular, it is preferable to use HATU in combination with DIPEA, or COMU in combination with DIPEA. In addition, N,N'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), 4-(4,6-dimethoxy-1,3,Condensing agents such as 5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and propylphosphonic anhydride (T3P) can also be used.

[0114] The present invention's production method involves a step of deprotecting the protecting group having an Fmoc skeleton of a newly added Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide with a base to expose the amino group, and The method further includes the step of adding a new Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide to form an amide bond. These steps may be repeated once or multiple times. The method of the present invention makes it possible to obtain a desired peptide sequence by repeatedly deprotecting a protecting group having an Fmoc skeleton and condensing it with the next new Fmoc-protected amino acid, Fmoc-protected amino acid analog, or Fmoc-protected peptide.

[0115] When the present invention is carried out by a solid-phase method, after the target peptide is obtained, it is cleaved from the solid phase (cleavage step). It is also possible to perform structural transformation or cyclization of the peptide before the cleavage step. In the present invention, at the time of cleavage, the side chain functional groups protected by protecting groups may or may not be deprotected, and only some of the protecting groups may be deprotected. It is preferable that the cleavage step is performed while the side chain functional groups remain protected.

[0116] Specifically, the reaction conditions for the cleavage step of the present invention are preferably weakly acidic, and more preferably weaker than TFA. Specifically, such weak acids are those that have a pKa value higher than TFA in water. More specifically, those with a pKa value in the range of 0 to 15 are preferred, and those with a pKa value in the range of 6 to 15 in water are more preferred. Examples of acids that are weaker than TFA used in this step include TFE and HFIP. Two or more weak acids may be combined in any proportion, such as TFE / acetic acid. Any solvent such as DCM, DCE, and water may also be mixed in any proportion. Among such combinations of weak acids and solvents, the combination of TFE and DCM is particularly preferred. Other organic solvents, reagents (e.g., DIPEA), and cation scavengers (e.g., triisopropylsilane) may be added to the solution used for cleavage.

[0117] When a cleavage step is performed before deprotecting the protecting group of the side chain of the synthesized peptide, it is preferable that the weak acid used for cleavage is weaker than the acid used in the deprotection reaction. In this case, two acids with different acidities that are weaker than TFA are prepared in advance, and the weaker acid is used for cleavage. When a cleavage step is performed after deprotecting the protecting group of the side chain of the synthesized peptide, the weak acid used for cleavage is not particularly limited as long as it is weaker than TFA.

[0118] In the side-chain protecting group deprotection step of the present invention, it is possible to selectively carry out the desired deprotection reaction by reducing side reactions such as hydrolysis and N→O-acyl shift. Deprotection of the side-chain protecting group is preferably carried out under conditions that are weaker than TFA. The reaction can be carried out at any temperature, but is preferably carried out between 0 and 40°C. When the deprotection is completed or when the reaction is stopped during deprotection, a base such as ammonia or primary to tertiary amines can be used. Basic heterocyclic compounds (e.g., pyridine, imidazole, and their analogs) can also be used.

[0119] If further modifications or alterations are to be made to the peptide synthesized by the manufacturing method of the present invention, these steps can be carried out either before or after the cleavage step.

[0120] Peptides produced by the production method of the present invention may be peptides that contain an amino acid residue or amino acid analog residue having one reaction site in the side chain at the C-terminus and an amino acid residue, amino acid analog residue, or carboxylic acid analog having another reaction site at the N-terminus. Such peptides can be produced, for example, by selecting Fmoc-protected amino acids, Fmoc-protected amino acid analogs, and Fmoc-protected peptides as raw materials such that the C-terminal side chain contains an amino acid residue or amino acid analog residue having one reaction site and the N-terminus contains an amino acid residue, amino acid analog residue, or carboxylic acid analog having another reaction site.

[0121] This peptide can be cyclized by joining one reaction site to another. The production method of the present invention may include such a cyclization step. Specifically, the cyclization step can be carried out according to the description in WO2013 / 100132.

[0122] If the cyclization process is carried out after the excision process, the reaction solution obtained in the excision process (excision solution) may be concentrated under reduced pressure and used in the cyclization process of the residue, or the excision solution may be used directly in the cyclization process.

[0123] In the present invention, "carboxylic acid analogues" include compounds that simultaneously possess an amino group and a carboxyl group with three or more atoms between them, various carboxylic acid derivatives that do not possess an amino group, peptides formed from 2 to 4 residues, and amino acids in which the main chain amino group is chemically modified by an amide bond with a carboxylic acid. Furthermore, "carboxylic acid analogues" may have boric acid or boric acid ester moieties that can be used for cyclization. In addition, "carboxylic acid analogues" may be carboxylic acids having double or triple bond moieties, or carboxylic acids having ketones or halides. It should be noted that the parts of these compounds other than the defined functional groups may be substituted, for example, selected (free substituents) from alkyl groups, aralkyl groups, aryl groups, cycloalkyl groups, heteroaryl groups, alkenyl groups, alkynyl groups, etc.

[0124] The cyclization step includes, but is not limited to, the step of cyclizing the two reaction sites by, for example, an amide bond, a disulfide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, or a carbon-carbon bond.

[0125] Cyclization by amide bond formation is a cyclization process in which an amide bond is formed between the reaction site (an amino group on the main chain or an amino group on the side chain) of an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog, and the reaction site of an amino acid residue or amino acid analog having one carboxylic acid on the side chain. In these reactions, the same coupling agents used in peptide bonding described above can be used. Specifically, for example, a combination of HATU and DIPEA, or a combination of COMU and DIPEA can be used to condense a side-chain carboxylic acid with an amino group on the N-terminal main chain, or a side-chain amino group with a carboxylic acid on the C-terminal main chain. In this case, it is preferable to select the protecting group of the carboxylic acid on the C-terminal side and the protecting group of the carboxylic acid on the side chain to be cyclized, or the protecting group of the amino group on the N-terminal main chain and the protecting group of the amino group on the side chain to be cyclized, taking into consideration their orthogonality. The preferred protecting groups in this series of peptide synthesis are as described above.

[0126] Cyclization by carbon-carbon bond formation is a cyclization process in which a carbon-carbon bond is formed between, for example, the reaction site of an N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog and the reaction site of an amino acid residue or amino acid analog having one reaction site on its side chain. Specifically, for example, an alkenyl group can be selected as the reaction site of the N-terminal amino acid residue, an N-terminal amino acid analog residue, or an N-terminal carboxylic acid analog, and an alkenyl group can be selected as the reaction site of an amino acid residue or amino acid analog residue having one reaction site on its side chain, and the cyclization reaction can be carried out by a carbon-carbon bond reaction catalyzed by a transition metal. Examples of transition metals that can be used as catalysts include ruthenium, molybdenum, titanium, and tungsten. For example, when ruthenium is used, the cyclization reaction can be carried out by a metathesis reaction. Furthermore, for example, a combination of an aryl halide and a boronic acid or boronic acid analog can be used as the reaction site for the N-terminal amino acid residue, the N-terminal amino acid analog residue, or the N-terminal carboxylic acid analog, and for the reaction site for an amino acid residue or amino acid analog residue having one reaction site on the side chain, and a cyclization reaction can be carried out by a carbon-carbon bond reaction catalyzed by a transition metal. In this case, palladium, nickel, and iron can be used as transition metals to carry out the cyclization reaction. For example, when palladium is used, the cyclization reaction can be carried out by the Suzuki reaction. Furthermore, for example, a combination of an alkenyl group and an aryl halide or alkenyl halide can be used as the reaction site for the N-terminal amino acid residue, the N-terminal amino acid analog residue, or the N-terminal carboxylic acid analog, and for the reaction site for an amino acid residue or amino acid analog residue having one reaction site on the side chain, and a cyclization reaction can be carried out by a carbon-carbon bond reaction catalyzed by a transition metal. In this case, palladium and nickel can be used as transition metals to carry out the cyclization reaction. For example, when palladium is used, the cyclization reaction can be carried out by a Heck-type chemical reaction.Furthermore, for example, a combination of an acetylene group and an aryl halide or alkenyl halide can be selected as the reaction site for the N-terminal amino acid residue, the N-terminal amino acid analog residue, or the N-terminal carboxylic acid analog, and as the reaction site for an amino acid residue or amino acid analog residue having one reaction site in its side chain, and a cyclization reaction can be carried out by a carbon-carbon bond reaction catalyzed by a transition metal. Examples of transition metals that can be used as catalysts include palladium, copper, gold, and iron. For example, when a combination of palladium and copper is used, the cyclization reaction can be carried out by the Sonogashira reaction.

[0127] In this invention, the obtained product can be purified as needed. For example, common peptide purification methods such as reversed-phase column chromatography or molecular sieve column chromatography can be used. Purification can also be performed by crystallization or solidification using an appropriate solvent. Concentration under reduced pressure is also possible before purification. Furthermore, all prior art documents cited herein are incorporated herein by reference. [Examples]

[0128] The present invention is further illustrated by the following embodiments, but is not limited to those embodiments.

[0129] The following abbreviations were used in the examples. DCM Dichloromethane DCE 1,2-Dichloroethane DMF (N,N-dimethylformamide) DIC N,N'-Diisopropylcarbodiimide DIPEA N,N-diisopropylethylamine DBU 1,8-diazabicyclo[5.4.0]-7-undecé hmm NMP N-methyl-2-pyrrolidone FA Formic Acid TFA (Trifluoroacetic Acid) TFE 2,2,2-trifluoroethanol HFIP 1,1,1,3,3,3-Hexafluoroisopropyl Alcohol HOAt 1-Hydroxy-7-azabenzotriazole HOBt 1-Hydroxybenzotriazole WSCI·HCl 1-Ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride TBME t-Butyl methyl ether TIPS Triisopropylsilane HATU O-(7-Aza-1H-benzotriazol-1-yl )-N,N,N’,N’-Tetramethyluronium hexa Fluorophosphate

[0130] Also, for the reaction solvents used in peptide synthesis and solid-phase synthesis, those for peptide synthesis (purchased from Watanabe Chemical Industries, Ltd. and Wako Pure Chemical Industries, Ltd.) were used. For example, DCM, DMF, NMP, 2% DBU in DMF, 20% piperidine in DMF, etc. Also, for reactions where water was not added as a solvent, dehydrated solvents, ultra-dehydrated solvents, and anhydrous solvents (purchased from Kanto Chemical Co., Inc. and Wako Pure Chemical Industries, Ltd.) were used.

[0131] The analysis conditions for LCMS are as shown in Table 1. [Table 1]

[0132] Example 1. Basic synthetic route for cyclic peptides containing N-methylamino acids in their sequence The synthesis of cyclic peptides containing N-methyl amino acids in the sequence adopted solid-phase synthesis by the Fmoc method and was carried out according to the synthesis route described in Figure 1 in the following five steps. A) Step of extending the peptide from the N-terminus of Asp whose carboxylic acid on the side chain was supported on 2-chlorotrityl resin by the Fmoc method using a peptide synthesizer B) Step of cleaving the peptide from 2-chlorotrityl resin C) A step in which the carboxylic acid (white circle unit) of the side chain of the excised peptide Asp is condensed with the amino group of the N-terminus (triangular unit) of the peptide chain, and then cyclized by an amide bond. D) A step of deprotecting the protecting group of the side chain functional group contained in the peptide chain. E) A step in which the compound is purified by preparative HPLC.

[0133] In this embodiment, unless otherwise specified, cyclic peptides were synthesized based on this basic synthetic route.

[0134] Fmoc-amino acids used in peptide synthesis by peptide synthesizers In the peptide synthesis described in this embodiment, the following Fmoc-amino acids were used for synthesis using a peptide synthesizer (step A above).

[0135] Fmoc-Pro-OH, Fmoc-Thr(Trt)-OH, Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-D-Tyr(C lt)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-MeP he-OH, Fmoc-MeAla-OH, Fmoc-MeGly-OH, Fmoc-MeLeu-OH, Fmoc-Phe(4-CF3)-OH, Fmoc-b-Ala-OH, Fmo c-b-MeAla-OH, Fmoc-Nle-OH, Fmoc-Met(O2)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-MeVal, and Fmoc-Val-OH. These were purchased from companies such as Watanabe Chemical, Chempep, or Chem-Impex.

[0136] Fmoc-MeSer(DMT)-OH, Fmoc-MePhe(3-Cl)-OH, Fmoc-MeAla(4-Thz)-OH, Fmoc-Hyp(Et)-OH, and Fmoc-γEtAbu-OH, Fmoc-nPrGly-OH. These were synthesized using the method described in the literature (Literature: International Publication Number WO2013 / 100132 A1).

[0137] Fmoc-Ser(THP)-OH(compound 1), Fmoc-Thr(THP)-OH(compound 2), Fmoc-MeSer(THP)-OH(compound 6), Fmoc-MeHis(Trt)-OH(compound 7), Fmoc-D-Tyr(THP)-OH(compound Compound 8), Fmoc-D-Tyr(Pis)-OH (Compound 11), Fmoc-Tyr(3-F,tBu)-OH (Compound 13), Fmoc-MePhe(4-Cl)-OH (Compound 16), and Fmoc-Tyr(3-F,Pis)-OH (Compound 22). These were synthesized as follows. These synthesized Fmoc-amino acids were used not only for peptide synthesis but also for investigating the deprotection of side-chain functional groups or C-terminal carboxylic acid groups.

[0138] Example 1-1: Synthesis of (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (compound 1, Fmoc-Ser(THP)-OH) [ka] A mixture of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxypropanoic acid (Fmoc-Ser-OH, purchased from Watanabe Chemical, 1.0 g, 3.06 mmol) and pyridinium p-toluenesulfonate (PPTS, 0.038 g, 0.153 mmol) was mixed with toluene (10 mL), and the water was removed by azeotropy by distillation under reduced pressure. To the resulting residue, super-dehydrated tetrahydrofuran (THF, 6.1 mL) and 3,4-dihydro-2H-pyran (1.9 mL, 21.3 mmol) were added, and the mixture was stirred at 50°C for 4 hours under a nitrogen atmosphere. After confirming the disappearance of the starting materials by LCMS (SQDFA05), the mixture was cooled to 25°C, and ethyl acetate (6 mL) was added. Subsequently, saturated sodium chloride aqueous solution (6 mL) was added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (6 mL). All the resulting organic layers were mixed and washed twice with saturated sodium chloride aqueous solution (6 mL). The organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The obtained residue was dissolved in tetrahydrofuran (THF, 12.2 mL), and then 1.0 M phosphate buffer (12.2 mL) prepared to pH 8.0 was added. This mixture was stirred at 50°C for 3 hours. After cooling to 25°C, ethyl acetate (12.2 mL) was added to separate the organic layer from the aqueous layer. After extraction by adding ethyl acetate (12.2 mL) to the aqueous layer, all the resulting organic layers were mixed and washed twice with saturated sodium chloride aqueous solution (12.2 mL). The organic layers were dried over sodium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure at 25°C for 30 minutes using a pump. The obtained residue was dissolved in dichloromethane (7 mL), and then heptane (16.6 mL) was added. Under controlled reduced pressure (~100 hPa), only the dichloromethane was removed by distillation, and the resulting mixture was filtered to obtain a solid. This washing operation with heptane was repeated twice. The obtained solid was dried under reduced pressure using a pump at 25°C for 2 hours to obtain 1.40 g of residue. To the obtained residue, t-butyl methyl ether (TBME, 25 mL) and a 0.05 M aqueous phosphoric acid solution with pH 2.1 (70 mL) were added and stirred at 25°C for 5 minutes, after which the organic layer and aqueous layer were separated. After extraction by adding t-butyl methyl ether (TBME, 25 mL) to the aqueous layer, all the obtained organic layers were mixed and washed twice with saturated aqueous sodium chloride solution (25 mL). The organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was dried under reduced pressure with a pump at 25°C for 2 hours to obtain (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (compound 1, Fmoc-Ser(THP)-OH, 1.22 g, with 30 mol% t-butyl methyl ether (TBME) remaining). The obtained Fmoc-Ser(THP)-OH was stored in a freezer at -25 degrees Celsius. LCMS(ESI)m / z=410.2(MH) - Retention time: 0.81 minutes (Analysis conditions SQDFA05)

[0139] Examples 1-2: Synthesis of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (compound 2, Fmoc-Thr(THP)-OH) [ka] A mixture of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid monohydrate (Fmoc-Thr-OH monohydrate, purchased from Tokyo Chemical Industry Co., Ltd., 5.0 g, 13.9 mmol) and pyridinium p-toluenesulfonate (PPTS, 0.175 g, 0.70 mmol) was mixed with toluene (50 mL), and the water was removed by azeotropy by distillation under reduced pressure. To the resulting residue, super-dehydrated tetrahydrofuran (THF, 28 mL) and 3,4-dihydro-2H-pyran (8.8 mL, 97 mmol) were added, and the mixture was stirred at 50°C for 4 hours under a nitrogen atmosphere. After confirming the disappearance of the starting materials by LCMS (SQDFA05), the mixture was cooled to 25°C, and ethyl acetate (30 mL) was added. Next, saturated sodium chloride aqueous solution (30 mL) was added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (30 mL). All the obtained organic layers were mixed and washed twice more with saturated sodium chloride aqueous solution (30 mL). The organic layers were dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain 9.3 g of crude product. Of the obtained crude product, 4.65 g was dissolved in tetrahydrofuran (THF, 30 mL), and then 1.0 M phosphate buffer (30 mL) prepared to pH 8.0 was added. This mixture was stirred at 50°C for 4 hours. After cooling to 25°C, ethyl acetate (30 mL) was added to separate the organic layer from the aqueous layer. After extraction by adding ethyl acetate (30 mL) to the aqueous layer, all the obtained organic layers were mixed and washed twice with saturated sodium chloride aqueous solution (30 mL). The organic layers were dried over sodium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure at 25°C for 30 minutes using a pump. The obtained residue was dissolved in diethyl ether (50 mL), and then heptane (50 mL) was added. Under controlled reduced pressure (~100 hPa), only the diethyl ether was removed by distillation, and the resulting mixture was filtered to obtain a solid. This washing operation with heptane was repeated twice. The obtained solid was dried under reduced pressure using a pump at 25°C for 2 hours to obtain the sodium salt of Fmoc-Thr(THP)-OH (2.80 g, 6.26 mmol). Ethyl acetate (50 mL) and 0.05 M aqueous phosphoric acid solution (140 mL) at pH 2.1 were added to the sodium salt of the total amount of the obtained Fmoc-Thr(THP)-OH, and after stirring at 25 °C for 5 minutes, the organic layer and the aqueous layer were separated. The aqueous layer was extracted by adding ethyl acetate (50 mL), and all the obtained organic layers were mixed and washed twice with a saturated aqueous sodium chloride solution (50 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure with a pump at 25 °C for 2 hours, and then the obtained solid was dissolved in t-butyl methyl ether (TBME, 50 mL), and the solvent was distilled off under reduced pressure. Further drying under reduced pressure with a pump at 25 °C for 1 hour gave (2S,3R)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (Compound 2, Fmoc-Thr(THP)-OH, 2.70 g, with 30 mol% of t-butyl methyl ether (TBME) remaining) as a diastereomer derived from the asymmetric carbon on the THP protection. The obtained Fmoc-Thr(THP)-OH was stored in a freezer at -25 °C. LCMS(ESI) m / z = 424.2 (M - H) - Retention time: 0.84 min, 0.85 min (analysis conditions SQDFA05)

[0140] Examples 1 - 3: Synthesis of (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (compound 6, Fmoc-MeSer(THP)-OH)

Chemical Structure

[0141] Examples 1-4: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazole-4-yl)propanoic acid (compound 7, Fmoc-MeHis(Trt)-OH) [ka] In a 3000 mL flask, a solution of (S)-3-(1H-imidazole-4-yl)-2-(methylamino)propano hydrochloride (75 g, 364.71 mmol) in dichloromethane (1000 mL), dichlorodimethylsilane (51 g, 395.16 mmol), and triethylamine (40 g, 395.30 mmol) were added. Subsequently, a solution of (chloromethanetriyl)tribenzene (Trt-Cl, 111 g, 398.17 mmol) in dichloromethane (500 mL) and triethylamine (40 g, 395.30 mmol) were added. The resulting reaction solution was heated under reflux and stirred for 4 hours, and then stirred at 20°C for another 2 hours. Methanol was added to the reaction solution to stop the reaction, and the solvent was then removed under reduced pressure. The pH was adjusted to 8-8.5 with triethylamine, and 125 g of solid was obtained. To the obtained solid, 1,4-dioxane (1000 mL), potassium carbonate (84 g, 603.39 mmol), and water (1000 mL) were added. Further, (2,5-dioxopyrrolidine-1-yl)(9H-fluoren-9-yl)methyl carbonate (Fmoc-OSu, 102 g, 302.38 mmol) was added, and the mixture was stirred at 0°C for 2 hours. The resulting reaction solution was washed with diethyl ether (2000 mL), and the pH of the solution was adjusted to 6-7 using acetic acid. The obtained solid was filtered to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazole-4-yl)propanoic acid (compound 7, Fmoc-MeHis(Trt)-OH, 155 g). LCMS(ESI)m / z=634.4(M+H) + Retention time: 1.07 minutes (Analysis conditions SQDAA05)

[0142] Examples 1-5: Synthesis of (2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid (compound 8, Fmoc-D-Tyr(THP)-OH) [ka] (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoic acid (Fmoc-D-Tyr-OH, purchased from Watanabe Chemical, 500 mg, 1.24 mmol) and a catalytic amount of pyridinium p-toluenesulfonate (PPTS, 15.6 mg, 0.062 mmol) were mixed with toluene (5.0 mL), and the water was removed by azeotropy by distillation of the toluene under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (THF) (2.5 mL), and 3,4-dihydro-2H-pyran (785 μL, 8.68 mmol) was added. The mixture was stirred at 50°C for 4 hours under a nitrogen atmosphere. The reaction mixture was cooled to 25°C, and ethyl acetate (3 mL) was added. Subsequently, saturated saline solution (3 mL) was added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (3 mL). All the obtained organic layers were mixed and washed twice with saturated brine (3 mL). The organic layers were dried over sodium sulfate, the solvent was removed under reduced pressure, and the layers were further dried under reduced pressure using a pump to obtain a residue of 596 mg. The obtained residue (300 mg) was dissolved in tetrahydrofuran (THF) (2.5 mL), and 1.0 M aqueous phosphoric acid solution (pH 8.0, 2.5 mL) was added and the mixture was stirred at 50°C for 3 hours. Ethyl acetate (3 mL) was added to the reaction mixture to separate the organic layer from the aqueous layer, and the aqueous layer was extracted with ethyl acetate (3 mL). All the obtained organic layers were mixed and washed twice with saturated brine (3 mL). The organic layers were dried over sodium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure with a pump for 30 minutes. The obtained residue was dissolved in dichloromethane (DCM) (2 mL) and heptane (5 mL) was added. Only the dichloromethane (DCM) was removed using an evaporator, and the resulting white solid was collected by filtration. The same procedure was repeated twice with the obtained white solid. The white solid thus obtained was dried under reduced pressure using a pump for 2 hours. To the above white solid, t-butyl methyl ether (TBME) (4.6 mL) and 0.05 M aqueous phosphoric acid solution (pH 2.1, 13 mL) were added and the mixture was stirred at 25°C for 5 minutes. After separating the organic layer, the aqueous layer was extracted with t-butyl methyl ether (TBME) (4.6 mL). The obtained organic layer was collected and washed twice with saturated brine (4.6 mL). The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The resulting residue was purified by reverse-phase chromatography (Wakosil 25C18 10 g, water / acetonitrile) to obtain (2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid (compound 8, Fmoc-D-Tyr(THP)-OH, 173 mg) in 57% yield as a diastereomer derived from the chiral carbon of the THP protection. LCMS(ESI)m / z=488.4(M+H) + Retention time: 0.92 minutes (Analysis conditions SQDFA05)

[0143] Examples 1-6: Synthesis of 2,2,2-trichloroacetimide 2-phenylpropan-2-yl (compound 9) [ka] To a 4.8 mL solution of 2-phenylpropan-2-ol (purchased from Wako, 2.0 g, 14.7 mmol) in diethyl ether (Et2O), 850 μL (1.62 mmol) of 1.9 M NaHMDS in tetrahydrofuran (THF) was added dropwise at 22°C. The reaction mixture was stirred at the same temperature for 20 minutes, then cooled to 0°C, and 2,2,2-trichloroacetonitrile (1.47 mL, 14.7 mmol) was added dropwise. The reaction mixture was stirred at 0°C for 10 minutes, then heated to 15°C and stirred for a further 1 hour. The reaction mixture was concentrated using an evaporator, and hexane (1.8 mL) and methanol (65 μL) were added to the resulting residue and stirred at 15°C for 15 minutes. The resulting solid was filtered and washed three times with hexane (2.0 mL) to obtain 4.19 g of 2,2,2-trichloroacetimide 2-phenylpropan-2-yl (compound 9). This compound was used directly in the reaction without further purification. 1 H NMR(Varian 400-MR, 400 MHz, CDCl3) δ 1.89 (6H, s), 7.28 (1H, m), 7.36(2H, m), 7.43 (2H, m), 8.20 (1H, brs)

[0144] Examples 1-7: Synthesis of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoic acid (R)-methyl (compound 10, Fmoc-D-Tyr-OMe) [ka] Under a nitrogen atmosphere, thionyl chloride (1.59 mL, 21.76 mmol) was added dropwise at 0°C to a mixture of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanoic acid (Fmoc-D-Tyr(tBu)-OH, purchased from Watanabe Chemical, 5.0 g, 10.88 mmol) and methanol (8.80 mL, 218 mmol). The resulting reaction solution was stirred at 25°C for 3 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in ethyl acetate, and the solution was washed twice with saturated sodium chloride aqueous solution. The organic layer was dried over sodium sulfate, the solid was removed by filtration, and the solvent was removed under reduced pressure to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoic acid (R)-methyl (compound 10, Fmoc-D-Tyr-OMe, 4.50 g). LCMS(ESI)m / z=418.3(M+H) + Retention time: 0.81 minutes (Analysis conditions SQDFA05)

[0145] Examples 1-8: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (Compound 11, Fmoc-D-Tyr(Pis)-OH) [ka] To a tetrahydrofuran (THF) solution (240 μL) of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoic acid (R)-methyl (compound 10, Fmoc-D-Tyr-OMe, 100 mg, 0.24 mmol), a separately prepared cyclohexane solution (60 μL) of 2,2,2-trichloroacetimic acid 2-phenylpropan-2-yl (compound 9) and a catalytic amount of borontrifluoride-ethyl ether complex (BF3-OEt, 4.55 μL, 0.036 mmol) were added dropwise at 0°C. After stirring the reaction mixture at 25°C for 1 hour, the same amount of cyclohexane solution (60 μL) of 10M 2,2,2-trichloroacetimide 2-phenylpropan-2-yl (compound 9) and boron trifluoride-ethyl ether complex (BF3-OEt, 4.55 μL, 0.036 mmol) were added again, and the reaction mixture was stirred at 25°C for a further 30 minutes. The reaction mixture was diluted with dichloromethane (DCM), and saturated sodium bicarbonate aqueous solution was added. After extraction with dichloromethane, the organic layer was washed with saturated brine. The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and the layer was further dried with a pump. A dichloromethane (DCM) / hexane = 1 / 1 solution was added to the resulting residue, and the precipitate was removed by filtration. The filtrate was concentrated using an evaporator, and the resulting residue was purified by flash column chromatography (purif pack® SIZE 20, hexane / ethyl acetate) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (R)-methyl(Fmoc-D-Tyr(Pis)-OMe) as a mixture. The mixture obtained above was dissolved in dichloroethane (DCE) (535 μL), and trimethyltin(IV) hydroxide (Me3SnOH, 58.1 mg, 0.321 mmol) was added, and the mixture was stirred at 60°C for 7 hours. Further trimethyltin(IV) hydroxide (Me3SnOH, 29.1 mg, 0.161 mmol) was added to the reaction mixture, and the mixture was stirred at 60°C for 15 hours. The reaction mixture was concentrated using an evaporator, and t-butyl methyl ether (TBME, 1 mL) and 0.05 M aqueous phosphoric acid solution (pH 2.1, 2 mL) were added, and the mixture was stirred at 25°C for 5 minutes. After separating the organic layer, the aqueous layer was extracted twice with t-butyl methyl ether (TBME, 1 mL). The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried using a pump. The obtained residue was purified by column chromatography (purif pack® SIZE 20, dichloromethane / methanol) to obtain (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (compound 11, Fmoc-D-Tyr(Pis)-OH, 33 mg) in a two-step yield of 39%. LCMS(ESI)m / z=522.4(M+H) + Retention time: 1.00 minutes (Analysis conditions SQDFA05)

[0146] Examples 1-9: Synthesis of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (S)-methyl (compound 12, Fmoc-Tyr(3-F)-OMe) [ka] (S)-2-amino-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (H2N-Tyr(3-F)-OH, purchased from Astatech, 2.0 g, 10.0 mmol) was dissolved in a 10% aqueous sodium carbonate solution. Then, using a dropping funnel, a solution of (2,5-dioxopyrrolidine-1-yl)(9H-fluoren-9-yl)methyl carbonate (Fmoc-OSu, 3.39 g, 10.0 mmol) in 1,4-dioxane (35 mL) was added at 0°C. The reaction mixture was stirred at 25°C for 40 minutes, then water (35 mL) and diethyl ether (70 mL) were added, and the mixture was washed three times with diethyl ether. The pH of the aqueous layer was adjusted to 2-3 with 5N aqueous hydrochloric acid solution, and then extracted three times with ethyl acetate (100 mL x 3). The organic layer was dried over magnesium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried with a pump. The resulting residue (4.08 g) was used directly in the next reaction without further purification. The above residue (1.04 g) was dissolved in methanol (10 mL), and thionyl chloride (SOCl2, 539 μL, 7.38 mmol) was added dropwise at 0°C. The reaction mixture was stirred at 60°C for 1 hour, then cooled to room temperature, and the solvent was removed using an evaporator. Ethyl acetate and water were added to the obtained residue, and it was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate, the solvent was removed under reduced pressure, and it was further dried with a pump. The obtained residue was purified by flash column chromatography (purif pack® SIZE 200, hexane / ethyl acetate) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (S)-methyl (compound 12, Fmoc-Tyr(3-F)-OMe, 900 mg, 2.07 mmol) in a two-step yield of 84%. LCMS(ESI)m / z=436.4(M+H) + Retention time: 0.82 minutes (Analysis conditions SQDFA05)

[0147] Examples 1-10: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid (compound 13, Fmoc-Tyr(3-F,tBu)-OH) [ka] To a tetrahydrofuran (THF) solution (690 μL) of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (S)-methyl (compound 12, Fmoc-Tyr(3-F)-OMe, 300 mg, 0.689 mmol), 2,2,2-trichloroacetimide tert-butyl (308 μL, 1.72 mmol) and a catalytic amount of boron trifluoride-ethyl ether complex (BF3-OEt, 13.1 μL, 0.103 mmol) were added dropwise at 0°C. After stirring the reaction mixture at 25°C for 1 hour, the same amount of tert-butyl 2,2,2-trichloroacetimate (308 μL, 1.72 mmol) and boron trifluoridoethyl ether complex (BF3-OEt, 13.1 μL, 0.103 mmol) were added again, and the reaction mixture was stirred at 25°C for another 1 hour. The reaction mixture was diluted with dichloromethane (DCM), and saturated sodium bicarbonate aqueous solution was added. After extraction with dichloromethane, the organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and the layer was further dried with a pump. The obtained residue was purified by flash column chromatography (purif pack® SIZE 60, hexane / ethyl acetate) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoate(S)-methyl (Fmoc-Tyr(3-F,tBu)-OMe) as a mixture. The mixture obtained above (40 mg) was dissolved in dichloroethane (DCE) (810 μL), trimethyltin(IV) hydroxide (Me3SnOH, 29.4 mg, 0.163 mmol) was added, and the mixture was stirred at 60°C for 1 hour. Formic acid (15.35 μL, 0.407 mmol) was added to the reaction mixture, and the mixture was purified by reverse-phase chromatography (Wakosil 25C18 10 g, 0.1% aqueous formic acid solution / 0.1% acetonitrile formic acid solution) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid (compound 13, Fmoc-Tyr(3-F,tBu)-OH, 27 mg, 56.5 μmol) in a two-step yield of 93%. LCMS(ESI)m / z=478.3(M+H) + Retention time: 0.94 minutes (Analysis conditions SQDFA05)

[0148] Examples 1-11: Synthesis of pyrrolidine-1,2-dicarboxylic acid 2-(2-phenylpropan-2-yl) (S)-1-((9H-fluoren-9-yl)methyl) (Compound 14, Fmoc-Pro-OPis) [ka] A mixture of 2-phenyl-2-propanol (14.2 g, 104 mmol) and anhydrous diethyl ether (35 mL) was mixed under a nitrogen atmosphere at room temperature, to which 1.9 M NaHMDS (tetrahydrofuran solution, 0.85 mL, 1.62 mmol) was added dropwise over 3 minutes or more, and the mixture was then stirred at room temperature for 30 minutes. Next, the reaction mixture was cooled to 0°C on ice, and trichloroacetonitrile (11.5 mL, 115 mmol) was added dropwise over 5 minutes or more. The mixture was stirred at 0°C for 10 minutes, then removed from the ice bath and stirred at room temperature for a further 1 hour. The resulting mixture was cooled to 0°C on ice, and a mixture of (S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)pyrrolidine-2-carboxylic acid (Fmoc-Pro-OH, 42.3 g, 125 mmol) and dichloromethane (100 mL) was added over 15 minutes. After stirring at 0°C for 30 minutes, the mixture was filtered, washed with hexane-dichloromethane (5 / 1) solution, and then the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain pyrrolidine-1,2-dicarboxylic acid 2-(2-phenylpropan-2-yl) (S)-1-((9H-fluoren-9-yl)methyl) (Compound 14, Fmoc-Pro-OPis, 26.3 g, 57.7 mmol). LCMS(ESI)m / z=456.4(M+H) + Retention time: 0.76 minutes (Analysis conditions SQDAA50)

[0149] Examples 1-12: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid (compound 16, Fmoc-MePhe(4-Cl)-OH) [ka] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-chlorophenyl)propanoic acid (Fmoc-Phe(4-Cl)-OH, 170 g, 402.96 mmol) was dissolved in toluene (2.5 L), to which paraformaldehyde (48 g, 1.60 mol) and 10-camphasulfonic acid (CSA, 4.6 g, 19.83 mmol) were added, and the mixture was stirred at 110°C for 16 hours. Subsequently, the reaction solution was washed twice with saturated sodium bicarbonate aqueous solution (1 L) and twice with saturated sodium chloride aqueous solution (1 L). The organic layer was dried over sodium sulfate, the solid was removed by filtration, and the solvent was removed under reduced pressure to obtain 160 g of 4-(4-chlorobenzyl)-5-oxoxazolidine-3-carboxylic acid (S)-(9H-fluoren-9-yl)methyl. A solution of 4-(4-chlorobenzyl)-5-oxoxazolidine-3-carboxylic acid (S)-(9H-fluoren-9-yl)methyl (230 g, 530.10 mmol) in dichloromethane (2.5 L) was mixed with triethylsilane (881 g, 7.58 mol) and trifluoroacetic acid (TFA, 2518 g, 22.28 mol), and the mixture was stirred at 30°C for 12 hours. Subsequently, the solvent was removed under reduced pressure, and the resulting residue was recrystallized in dichloromethane / hexane (1 / 10, v / v) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid (compound 16, Fmoc-MePhe(4-Cl)-OH, 205 g). LCMS(ESI)m / z=436.3(M+H) + Retention time: 0.99 minutes (Analysis conditions SQDAA05)

[0150] Examples 1-13: Synthesis of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (S)-methyl (compound 21, Fmoc-Tyr(3-F,Pis)-OMe) [ka] 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (S)-methyl (compound 12, Fmoc-Tyr(3-F)-OMe, 200 mg, 0.459 mmol) was dissolved in THF (460 μL), and then separately prepared 2,2,2-trichloroacetimide 2-phenylpropan-2-yl (compound 9, 322 mg, 1.15 mmol) and a catalytic amount of boron trifluoride-ethyl ether complex (BF3-OEt, 8.73 μL, 0.069 mmol) were added dropwise at 0°C. The reaction mixture was stirred at room temperature for 30 minutes, and then an equal amount of 2,2,2-trichloroacetimide 2-phenylpropan-2-yl (322 mg, 1.15 mmol) and a catalytic amount of borontrifluoride ethyl ether complex (BF3-OEt, 8.73 μL, 0.069 mmol) were added dropwise at 0°C. The reaction mixture was stirred further at room temperature for 30 minutes, then diluted with dichloromethane, and saturated sodium bicarbonate aqueous solution was added under ice cooling. After extraction with dichloromethane, the organic layer was washed with saturated brine. The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and then dried with a pump. The resulting residue was washed twice with dichloromethane / hexane = 1 / 1 (20 mL, 10 mL), and the white solid was removed by filtration. The obtained filtrate was concentrated, and the residue was purified by flash column chromatography (purif pack® SIZE 20, hexane / ethyl acetate, 0.1% diisopropylethylamine (DIPEA)) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (S)-methyl (compound 21, Fmoc-Tyr(3-F,Pis)-OMe, 210 mg, 0.379 mmol) in 83% yield. LCMS(ESI)m / z=554.4(M+H) + Retention time: 1.09 minutes (Analysis conditions SQDFA05)

[0151] Examples 1-14: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (compound 22, Fmoc-Tyr(3-F,Pis)-OH) [ka] 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (S)-methyl (compound 21, Fmoc-Tyr(3-F, Pis)-OMe, 210 mg, 0.379 mmol) was dissolved in dichloroethane (DCE) (1.26 mL), trimethyltin(IV) hydroxide (Me3SnOH, 137 mg, 0.379 mmol) was added, and the mixture was stirred at 60°C for 3 hours. The reaction mixture was concentrated using an evaporator, and t-butyl methyl ether (TBME, 2.0 mL) and 0.05 M aqueous phosphoric acid solution (pH 2.1, 4.0 mL) were added, and the mixture was stirred at 25°C for 15 minutes. After separating the organic layer, the aqueous layer was extracted twice with t-butyl methyl ether (TBME, 1 mL). The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and the layer was further dried using a pump. The resulting residue was dissolved in a 0.1% acetonitrile formate solution, stirred for 15 minutes, and the resulting solution was purified by reverse-phase chromatography (Wakosil 25C18 30 g, 0.1% aqueous formic acid solution / 0.1% acetonitrile formate) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (compound 22, Fmoc-Tyr(3-F,Pis)-OH, 190 mg, 0.352 mmol) in 93% yield. LCMS(ESI)m / z=538.2(MH) - Retention time: 1.00 minutes (Analysis conditions SQDFA05)

[0152] Synthesis of a compound of resin and Fmoc-amino acid used in peptide synthesis by a peptide synthesizer. The resin-Fmoc-amino acid conjugate used for peptide synthesis in a peptide synthesizer was synthesized as follows.

[0153] Examples 1-15: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidine-1-yl)butanoic acid-2-chlorotrityllresin (compound 50, Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) [ka] (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidine-1-yl)butanoic acid-2-chlorotrityllesin (compound 50, Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) was synthesized by the method described in the literature (Literature: International Publication No. WO 2013 / 100132 A1).

[0154] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin portion may be indicated by a circle (○). Furthermore, to clearly indicate the reaction site of the resin portion, the chemical structure of the reaction site may be shown connected to the circle. For example, in the above structure (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip(compound 50)), the 2-chlorotrityl group of the resin is bonded to the side-chain carboxylic acid of Asp via an ester bond. Note that "pip" refers to piperidine, and in the above structure, the C-terminal carboxylic acid group forms an amide bond with piperidine.

[0155] Examples 1-16 Synthesis of compound 52, obtained by bonding Fmoc-Asp-piptBu (compound 51) to a resin using its side-chain carboxylic acid.

[0156] Example 1-16-1: Synthesis of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (S)-tert-butyl (compound 53, Fmoc-Asp(OtBu)-piptBu) (Note that piptBu refers to 4-(tert-butyl)piperidine, and here it indicates that the C-terminal carboxylic acid group forms an amide bond with 4-(tert-butyl)piperidine.) [ka] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (10 g, 24.30 mmol), 4-(tert-butyl)piperidine hydrochloride (4.10 g, 23.09 mmol), and 1-hydroxybenzotriazole monohydrate (HOBt, 3.61 g) were dissolved in DMF (80 mL), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI·HCl, 5.59 g) was added at 0°C and the mixture was stirred at 0°C for 30 minutes. Subsequently, 4-methylmorpholine (2.54 mL) was added and the mixture was stirred at room temperature for 1 hour. Hexane-ethyl acetate (1 / 1, v / v, 500 mL) was added to the reaction solution, and the organic layer was washed twice with saturated ammonium chloride aqueous solution, twice with saturated sodium bicarbonate aqueous solution, and once with saturated sodium chloride aqueous solution. The resulting organic layer was dried over sodium sulfate, the solid was removed by filtration, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (mobilization phase: hexane-ethyl acetate) to obtain 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (S)-tert-butyl (compound 53, Fmoc-Asp(OtBu)-piptBu, 11.5 g, 21.51 mmol). LCMS(ESI)m / z=535.4(M+H) + Retention time: 1.17 minutes (Analysis conditions SQDAA05)

[0157] Example 1-16-2: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (compound 51, Fmoc-Asp-piptBu) [ka] 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (S)-tert-butyl (compound 53, Fmoc-Asp(OtBu)-piptBu, 2.0 g, 3.74 mmol) was mixed with toluene, and the solvent was removed under reduced pressure by azeotropy, thereby removing the water. The resulting residue was dissolved in dichloromethane (1.66 mL), and the water content was confirmed to be 110 ppm by Karl Fischer assay. Subsequently, the mixture was stirred at 0°C for 5 minutes, and trifluoroacetic acid (TFA, 1.66 mL) was added dropwise at 0°C, and the mixture was stirred for 5 minutes. The reaction solution was allowed to return to room temperature, and stirring was continued for 4 hours. The mixture was cooled to 0°C, and triethylamine (3.1 mL) was added dropwise. The mixture was diluted with dichloromethane (30 mL) and washed with a 5% sodium dihydrogen phosphate aqueous solution (5% NaH2PO4aq, pH 4.4). The organic layer was dried over sodium sulfate, the solid was removed by filtration, and the solvent was removed under reduced pressure at 20°C. The resulting residue was then collected. 19 FNMR measurement (DMSO-d6) confirmed the presence of TFA, so the residue was diluted again with dichloromethane (30 mL) and washed with 5% sodium dihydrogen phosphate aqueous solution (5% NaH2PO4aq, pH 4.4). The organic layer was dried over sodium sulfate, and after removing the solid by filtration, the solvent was removed by distillation at 20°C under reduced pressure to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (compound 51, Fmoc-Asp-piptBu, 1.73 g). 19 FNMR confirmed that TFA residue was below the detection limit. LCMS(ESI)m / z=479.4(M+H) + Retention time: 1.00 minutes (Analysis conditions SQDAA05)

[0158] Example 1-16-3: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid-2-chlorotrityllesin (compound 52, Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu) [ka] 2-chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, purchased from Watanabe Chemical, 4.52 g, 7.23 mmol) and anhydrous dichloromethane (72 mL) were placed in a filter-equipped reaction vessel and shaken at 25°C for 10 minutes. After removing the dichloromethane by applying nitrogen pressure, a mixture of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid (compound 51, Fmoc-Asp-piptBu, 1.73 g) and anhydrous methanol (1.17 mL) and diisopropylethylamine (DIPEA, 3.02 mL) was added to the reaction vessel and shaken for 15 minutes. After removing the reaction mixture under nitrogen pressure, a mixture of anhydrous dichloromethane (72 mL), anhydrous methanol (9.0 mL), and diisopropylethylamine (DIPEA, 3.02 mL) was added to the reaction vessel and shaken for 90 minutes. After removing the reaction mixture under nitrogen pressure, dichloromethane was added and shaken for 5 minutes. The reaction mixture was removed under nitrogen pressure. The resin was washed with this dichloromethane five times, and the resulting resin was dried overnight under reduced pressure to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(4-(tert-butyl)piperidine-1-yl)-4-oxobutanoic acid-2-chlorotrityllesin (compound 52, Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu, 5.23 g). The obtained Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu (compound 52, 16.5 mg) was placed in a reaction vessel, 1 mL of 20% piperidine / DMF solution was added, and the mixture was shaken at 25°C for 30 minutes. 30 μL was taken from the reaction mixture, diluted with DMF (2.97 mL), and its absorbance (301.2 nm) was measured (using a Shimadzu, UV-1600PC (cell length 1.0 cm)). The loading amount of Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu (compound 52) was calculated to be 0.356 mmol / g. Furthermore, a different batch with a different loading amount, synthesized in the same way, was also used for peptide synthesis.

[0159] Examples 1-17: Synthesis of compound (compound 55) obtained by bonding Fmoc-Asp-MeOctyl (compound 54) to a resin using its side-chain carboxylic acid.

[0160] Example 1-17-1: Synthesis of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (S)-tert-butyl (compound 56, Fmoc-Asp(OtBu)-MeOctyl) (Note that MeOctyl refers to N-methyloctane-1-amine, and here it indicates that the C-terminal carboxylic acid group forms an amide bond with N-methyloctane-1-amine.) [ka] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (Fmoc-Asp(OtBu)-OH, 8.00 g, 19.44 mmol) and DMF (65 mL) were added to a 300 mL flask and stirred at room temperature for 5 minutes. Subsequently, 4-methylmorpholine (2.57 mL) and O-(7-aza-1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 8.87 g, 23.33 mmol) were added and stirred at 0°C for 5 minutes. Furthermore, N-methyloctane-1-amine (3.35 mL, 18.47 mmol) was added dropwise over 2 minutes, and the resulting reaction solution was stirred at 0°C for 30 minutes. To this reaction solution, hexane-ethyl acetate (1 / 1, v / v, 400 mL) was added, and the mixture was washed with water (400 mL), saturated ammonium chloride aqueous solution (400 mL), 50% sodium bicarbonate aqueous solution (400 mL), water (400 mL × 2), and saturated sodium chloride aqueous solution (400 mL). The resulting organic layer was dried over sodium sulfate, the solid was removed, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (S)-tert-butyl (compound 56, Fmoc-Asp(OtBu)-MeOctyl, 10.2 g, 19.00 mmol). LCMS(ESI)m / z=537.5(M+H) + Retention time: 0.84 minutes (Analysis conditions SQDFA50)

[0161] Example 1-17-2: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (compound 54, Fmoc-Asp-MeOctyl) [ka] 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (S)-tert-butyl (compound 56, Fmoc-Asp(OtBu)-MeOctyl, 8.1 g, 15.09 mmol) was mixed with toluene, and the water was removed by azeotropy by distillation under reduced pressure. The resulting residue was dissolved in dichloromethane (anhydrous, 6.7 mL), and the water content was confirmed to be 380 ppm by Karl Fischer assay. Subsequently, the mixture was stirred at 0°C for 5 minutes, and trifluoroacetic acid (TFA, 6.7 mL) was added dropwise over 5 minutes at 0°C, followed by stirring for another 5 minutes. The reaction solution was allowed to return to room temperature, and stirring was continued for 4 hours. The mixture was cooled to 0°C, and triethylamine (12.62 mL) was added dropwise. The mixture was diluted with dichloromethane (100 mL) and washed with a 5% sodium dihydrogen phosphate aqueous solution (5% NaH2PO4aq). The organic layer was dried over sodium sulfate, and the solid was removed by filtration. The solvent was then removed under reduced pressure at 20°C. The resulting residue was then collected. 19 FNMR measurement (DMSO-d6) confirmed the presence of TFA, so the residue was dissolved again in dichloromethane and washed with 5% sodium dihydrogen phosphate aqueous solution (5% NaH2PO4aq). The organic layer was dried over sodium sulfate, and after removing the solid by filtration, the solvent was removed by distillation under reduced pressure at 20°C to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (compound 54, Fmoc-Asp-MeOctyl, 6.61 g, 13.75 mmol). 19 FNMR confirmed that TFA residue was below the detection limit. LCMS(ESI)m / z=481.4(M+H) + Retention time: 0.65 minutes (Analysis conditions SQDAA50)

[0162] Example 1-17-3: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid-2-chlorotrityllesin (compound 55, Fmoc-Asp(O-Trt(2-Cl)-resin)-MeOctyl) [ka] 2-chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, purchased from Watanabe Chemical, 16.3 g, 26.1 mmol) and anhydrous dichloromethane (261 mL) were placed in a filter-equipped reaction vessel and shaken at 25°C for 10 minutes. After removing the dichloromethane by applying nitrogen pressure, a mixture of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid (compound 54, Fmoc-Asp-MeOctyl, 6.28 g, 13.07 mmol) and anhydrous methanol (4.23 mL) and diisopropylethylamine (DIPEA, 10.9 mL) was added to the reaction vessel and shaken for 15 minutes. After removing the reaction mixture under nitrogen pressure, a mixture of anhydrous dichloromethane (261 mL), anhydrous methanol (32.4 mL), and diisopropylethylamine (DIPEA, 10.9 mL) was added to the reaction vessel and shaken for 90 minutes. After removing the reaction mixture under nitrogen pressure, dichloromethane (261 mL) was added and shaken for 5 minutes. The reaction mixture was removed under nitrogen pressure. The resin was washed with this dichloromethane twice, and the resulting resin was dried overnight under reduced pressure to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(methyl(octyl)amino)-4-oxobutanoic acid-2-chlorotrityllresin (compound 55, Fmoc-Asp(O-Trt(2-Cl)-resin)-MeOctyl, 18.2 g). Loading amount: 0.366 mmol / g Furthermore, a different batch with a different loading amount, synthesized in the same way, was also used for peptide synthesis.

[0163] Examples 1-18: Synthesis of compound (compound 58) obtained by bonding Fmoc-Asp-Pro-OPis (compound 57) to a resin using its side-chain carboxylic acid.

[0164] Example 1-18-1: Synthesis of 1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)pyrrolidine-2-carboxylic acid (S)-2-phenylpropan-2-yl (compound 59, Fmoc-Asp(OAll)-Pro-OPis) [ka] A solution of pyrrolidine-1,2-dicarboxylic acid 2-(2-phenylpropan-2-yl)(S)-1-((9H-fluoren-9-yl)methyl) (compound 14, Fmoc-Pro-OPis, 20.0 g, 43.9 mmol) prepared by the previously described method was cooled to 20°C in an anhydrous DMF (40 mL) bath. 1,8-diazabicyclo[5.4.0]-7-undecene (DBU, 6.57 mL, 43.9 mmol) was added dropwise over 7 minutes, and the mixture was stirred at room temperature for 5 minutes. Subsequently, the reaction mixture was cooled to 0°C, pyridine hydrochloride (5.07 g, 43.9 mmol) was added, and the mixture was stirred at 0°C for 10 minutes. Subsequently, a mixture of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoic acid (Fmoc-Asp(OAll)-OH, 17.35 g, 43.9 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI·HCl, 11.8 g, 61.4 mmol), and 1-hydroxy-7-azabenzotriazole (HOAt, 7.17 g, 52.7 mmol) was added, and then diisopropylethylamine (DIPEA, 7.6 mL, 43.9 mmol) was added dropwise over 7 minutes at 0°C. The reaction mixture was stirred at room temperature for 20 minutes. To the resulting reaction mixture, hexane (50 mL), diethyl ether (50 mL), saturated sodium bicarbonate aqueous solution (10 mL), and saturated sodium chloride aqueous solution (50 mL) were added, and the aqueous layer was extracted twice with diethyl ether. All of the resulting organic layers were combined and washed three times with saturated sodium chloride aqueous solution (50 mL), and then dried over sodium sulfate. After removing the solvent under reduced pressure, the resulting residue was purified by silica gel column chromatography (mobile phase: hexane-ethyl acetate) to obtain 1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)pyrrolidine-2-carboxylic acid (S)-2-phenylpropan-2-yl (compound 59, Fmoc-Asp(OAll)-Pro-OPis, 24.5 g, 40.1 mmol). LCMS(ESI)m / z=611.4(M+H)+ Retention time: 1.03 minutes (Analysis conditions SQDFA05)

[0165] Example 1-18-2: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phenylpropane-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic acid (compound 57, Fmoc-Asp-Pro-OPis) [ka] 1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoyl)pyrrolidine-2-carboxylic acid (S)-2-phenylpropan-2-yl (compound 59, Fmoc-Asp(OAll)-Pro-OPis, 24.16 g, 39.6 mmol) and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 0.114 g, 0.099 mmol) were placed in a 300 mL two-necked flask, and the flask was purged with nitrogen. Then, dichloromethane (40 mL) was added, and the mixture was stirred at room temperature, after which it was cooled to 14°C in a water bath. Phenylsilane (3.30 mL, 26.7 mmol) was added dropwise over 5 minutes, and the reaction mixture was stirred under a nitrogen atmosphere at 14-17°C for 35 minutes. Next, SH silica (Fuji Silysia, 5 g) and methanol (32.1 mL) were added, followed by Kieselgel 60 (15 g). Further methanol (30 mL), SH silica (Fuji Silysia, 5 g), and Kieselgel 60 (25 g) were added, and the mixture was stirred at 17-24 degrees Celsius until the liquid phase became colorless. The resulting mixture was filtered through Celite, washed with dichloromethane-methanol (10 / 1, v / v), and the solvent was removed under reduced pressure to obtain the crude product (25.38 g) of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phenylpropane-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic acid (compound 57, Fmoc-Asp-Pro-OPis). The resulting crude product was used for subsequent loading onto resin without purification. LCMS(ESI)m / z=571.3(M+H) + Retention time: 0.88 minutes (Analysis conditions SQDFA05)

[0166] Example 1-18-3: Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phenylpropane-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic acid-2-chlorotrityllresin (Compound 58, Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis) [ka] 2-chlorotrityl chloride resin (1.60 mmol / g, 100-200 mesh, 1% DVB, purchased from Watanabe Chemical, 47.8 g, 76.48 mmol) and anhydrous dichloromethane (150 mL) were placed in a reaction vessel with a filter and shaken at 25°C for 35 minutes. After removing the dichloromethane by applying nitrogen pressure, a mixture of anhydrous dichloromethane (115 mL) of the prepared (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phenylpropane-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic acid (compound 57, Fmoc-Asp-Pro-OPis, 21.94 g, 38.4 mmol) with anhydrous methanol (3.11 mL) and diisopropylethylamine (DIPEA, 32.1 mL) was added to the reaction vessel and shaken for 45 minutes. After removing the reaction mixture by applying nitrogen pressure, a mixture of anhydrous dichloromethane (100 mL) with anhydrous methanol (55 mL) and diisopropylethylamine (DIPEA, 25 mL) was added to the reaction vessel and shaken for 90 minutes. After removing the reaction solution by applying nitrogen pressure, 100 mL of dichloromethane was added and shaken for 5 minutes. The reaction solution was removed again by applying nitrogen pressure. The resin was washed with 100 mL of dichloromethane four times, and the resulting resin was dried under reduced pressure for 15 and a half hours to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-((S)-2-(((2-phenylpropane-2-yl)oxy)carbonyl)pyrrolidine-1-yl)butanoic acid-2-chlorotrityllresin (compound 58, Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis, 61.55 g). The obtained Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (compound 58, 12.3 mg) was placed in a reaction vessel, and DMF (0.2 mL) and piperidine (0.2 mL) were added. The mixture was shaken at 25°C for 30 minutes. After adding DMF (1.6 mL) to the reaction vessel, 0.4 mL was taken from the reaction mixture, diluted with DMF (9.6 mL), and its absorbance (301.2 nm) was measured (measured using Shimadzu, UV-1600PC (cell length 1.0 cm)). The loading amount of Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (compound 58) was calculated to be 0.3736 mmol / g using the following formula. (Absorbance (301.2 nm) × 1000 × 50) / (Resin weight (mg) × 7800) = (0.717 × 1000 × 50) / (12.3 × 7800) = 0.3736 mmol / g Furthermore, a different batch with a different loading amount, synthesized in the same way, was also used for peptide synthesis.

[0167] Example 2. Chemical synthesis of peptides using a peptide synthesizer (Steps A to C) Unless otherwise specified, peptide synthesis using the basic synthesis route described above was performed in the following manner.

[0168] Example 2-1: Solid-phase peptide synthesis using an automated synthesizer (Process A) Peptides were synthesized using the Fmoc method with a peptide synthesizer (Multipep RS; Intavis). Detailed procedures were followed according to the synthesizer's manual.

[0169] A 2-chlorotrityl resin (100 mg per column) bonded to the carboxylic acid moiety of the side chain of aspartic acid, whose N-terminus is protected with Fmoc, along with an NMP solution of various Fmoc-amino acids (0.6 mol / L, 0.5 mol / L for Fmoc-MeHis(Trt)-OH) and 1-hydroxy-7-azabenzotriazole (HOAt) or oxyma (0.375 mol / L), and a 10% v / v solution of diisopropylcarbodiimide (DIC) in N,N-dimethylformamide (DMF), were set in the synthesizer. Furthermore, if the Fmoc-amino acid is Fmoc-Ser(THP)-OH (compound 1), Fmoc-Thr(THP)-OH (compound 2), or Fmoc-MeSer(THP)-OH (compound 6), these Fmoc-amino acids were kept in the NMP solution with oxyma, and then molecular sieves 4A 1 / 8 (Wako Pure Chemical Industries) or molecular sieves 4A 1 / 16 (Wako Pure Chemical Industries) were added before setting in the synthesizer.

[0170] A 2% v / v solution of diazabicycloundecene (DBU) in DMF was used as the Fmoc deprotection solution. After washing the resin with DMF, the Fmoc group was deprotected, and then a condensation reaction with a new Fmoc amino acid was carried out. This constituted one cycle, and the peptide was extended onto the resin surface by repeating this cycle multiple times.

[0171] Example 2-2: Excavation of extended peptide from resin (Process B) After removing the N-terminal Fmoc group of the peptide extended by the method described above on a peptide synthesizer, the resin was washed with DMF. Subsequently, the resin was re-swelled with DCM, and then TFE / DCM (1 / 1, v / v, 2 mL) was added to the resin and shaken at room temperature for 2 hours. The solution in the tube was then filtered through a synthesis column to remove the resin, and the remaining resin was washed twice more with TFE / DCM (1 / 1, v / v, 1 mL). All the resulting cleaved solutions were mixed and concentrated under reduced pressure.

[0172] Examples 2-3: Cyclization of the excised peptide (Process C) The residue, concentrated under reduced pressure after cleavage, was dissolved in DMF / DCM (1 / 1, v / v, 8 mL). A 0.5 M O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) / DMF solution (in a volume equivalent to 1.5 times the number of moles on the resin used (calculated by multiplying the loading volume (mmol / g) by the amount of resin used (usually 0.10 g))) and DIPEA (1.8 times the number of moles on the resin used) were added, and the mixture was shaken at room temperature for 2 hours. The solvent was then removed under reduced pressure. The formation of the target cyclic peptide was confirmed by LC-MS measurement.

[0173] Using the method described above, peptides Pep1-Pep7, which will be used in the deprotection reaction study described later, were synthesized. The sequences of Pep1-Pep7 are shown in Table 2-1, their structures in Table 2-2, and their LCMS data in Table 2-3. The deprotection conditions studied later (the degree of hydrolysis or N→O-acyl shift malfunctions observed during deprotection, or the peptides to be present in the solution under consideration) were evaluated using the residue containing the cyclic peptides obtained in this process.

[0174] [Table 2-1]

[0175] [Table 2-2] TIFF2026088137000060.tif189149TIFF2026088137000061.tif189149TIFF2026088137000062.tif96149

[0176] [Table 2-3]

[0177] Example 3. A weak acid (pKa of 0-9 in water) and a solvent (Y OTs Deprotection of the protecting group of the side chain functional group of a peptide using a weak acid solution (a solvent with a positive value, weak acidity (pKa of 5-14 in water, and low nucleophilicity) (Process D)

[0178] Example 3-1. Deprotection of the protecting group of the side chain functional group of Fmoc-amino acid by the above weak acid solution. The protecting group of the side chain functional group of the Fmoc-amino acid used in peptide synthesis is a weaker acid than TFA, i.e., a weak acid with a pKa of 0-9 in water, Y OTs We investigated whether deprotection was possible in a solution where the value was positive, the solution was weakly acidic (pKa of 5-14 in water), and the solvent had low nucleophilicity.

[0179] Specifically, tetramethylammonium hydrogen sulfate (pKa 2.0) was used as the weak acid, and HFIP (Y) was used as the solvent. OTs A value of 3.82 (literature value: Prog. Phys. Org. Chem. 1990, 17,121-158, pKa 9.30) was used. More specifically, either a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) or a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) was used.

[0180] Example 3-1-1: Preparation of 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) A 0.1 M tetrabutylammonium bisulfate / HFIP solution (2% TIPS) was prepared by taking 4 mL from a mixture of HFIP (11.66 mL), TIPS (0.24 mL), and DCE (0.10 mL), and dissolving 68.5 mg of tetrabutylammonium bisulfate in it.

[0181] Example 3-1-2: Preparation of 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) A 0.05 M tetrabutylammonium bisulfate / HFIP solution (2% TIPS) was prepared by taking 4 mL from a mixture of HFIP (11.66 mL), TIPS (0.24 mL), and DCE (0.10 mL), and dissolving 34.3 mg of tetrabutylammonium bisulfate in it.

[0182] Deprotection of Fmoc-amino acids with a protecting group on the side chain, or peptides containing amino acid residues with a protecting group on the side chain, was performed using either method A or method B below.

[0183] Example 3-1-3: Method A A mixture of Fmoc-amino acid (4.0 umol) with a protecting group attached to the side chain and a peptide (one of the previously synthesized cyclic peptides Pep 1 to Pep 6 (residue after cyclization); maximum 3.66 umol) was mixed with either 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (0.20 mL) or 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (0.40 mL), shaken for 3 minutes, then allowed to stand at 25°C, and LCMS (FAO5) was measured after a certain period of time. The progress of deprotection was calculated from the UV area ratio of the deprotected and protected compounds.

[0184] Example 3-1-4: Method B Peptides containing amino acid residues with protecting groups attached to the side chain (any of the already synthesized cyclic peptides Pep 1 to Pep 6 (residue after cyclization); maximum 3.66 umol) were mixed with 0.1 M tetramethylammonium bisulfate / HFIP (2% TIPS) (0.20 mL) or 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS) (0.40 mL), shaken for 3 minutes, then allowed to stand at 25°C, and LCMS (FAO5) was measured after a certain period of time. The progress of deprotection was calculated from the UV area ratio of the deprotected and protected molecules.

[0185] The peptides used in methods A and B were prepared by extending and cleaving them from the resin using the previously described method, and then, after the cyclization reaction using the previously described method, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and then concentrated again under reduced pressure.

[0186] The evaluation results are shown in Table 3 below. [Table 3]

[0187] The LCMS measurement results for Fmoc-amino acids after deprotection are as follows: Fmoc-Tyr(3-F)-OH (deprotection product of run3, run4, and run15) LCMS(ESI)m / z=422.3(M+H) + Retention time: 0.73 minutes (Analysis conditions SQDFA05) Fmoc-MeHis-OH (deprotection product of run5) LCMS(ESI)m / z=392.3(M+H) + Retention time: 0.47 minutes (Analysis conditions SQDFA05) Fmoc-MeSer-OH (deprotection product of run8) LCMS(ESI)m / z=342.3(M+H) + Retention time: 0.67 minutes (Analysis conditions SQDFA05) Fmoc-Ser-OH (deprotection product of run10) LCMS(ESI)m / z=328.2(M+H) + Retention time: 0.64 minutes (Analysis conditions SQDFA05) Fmoc-Pro-OH (deprotection product of run11) LCMS(ESI)m / z=338.3(M+H) + Retention time: 0.75 minutes (Analysis conditions SQDFA05) Fmoc-D-Tyr-OH (deprotection product of runs 12-14) LCMS(ESI)m / z=404.3(M+H) + Retention time: 0.72 minutes (Analysis conditions SQDFA05)

[0188] [ka] Deprotection product of Pep 1 (compound 101) (deprotection product of run 1 and run 2, compound 131) LCMS(ESI)m / z=1424.0(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05)

[0189] [ka] Deprotection product of Pep 2 (compound 102) (deprotection product of run 6, compound 132) LCMS(ESI)m / z=1526.3(M+H) + Retention time: 0.89 minutes (Analysis conditions SQDFA05)

[0190] [ka] Deprotection product of Pep 3 (compound 103) (deprotection product of run 7, compound 133) LCMS(ESI)m / z=1474.1(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05)

[0191] [ka] Deprotection product of Pep 5 (compound 105) (deprotection product of run 9, compound 135) LCMS(ESI)m / z=1331.7(M+H) + Retention time: 0.74 minutes (Analysis conditions SQDFA05)

[0192] These results confirm that these protecting groups undergo deprotection under conditions of 0.1 M tetramethylammonium bisulfate / HFIP (2% TIPS) or 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS).

[0193] Furthermore, the protecting groups of the side chains are not affected by peptide cleavage conditions using TFE-DCM (1 / 1, v / v) solution or TFE-DCM (1 / 1, v / v) / DIPEA (1.8 equiv. added to the theoretical amount of moles obtained by multiplying the loading amount of resin used by the amount of resin used). Therefore, in the intramolecular cyclization at the N-terminus of the peptide backbone and the carboxylic acid moiety of the Asp side chain that follows the cleavage step, the amino acid side chain functional groups remain protected. This suppresses undesirable cyclization reactions in which the functional groups of the amino acid side chains act as nucleophiles.

[0194] 3-2. Possibility of suppressing hydrolysis and N→O-acyl shift compound formation when the protecting group of the side chain functional group of Fmoc-amino acids is deprotected with the above weak acid solution.

[0195] Example 3-2-1: Deprotection of a cyclic compound (compound 101, Pep1) formed by an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) as the deprotection condition. Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, resin load: 0.342 mmol / g, 100 mg) as the resin, a cyclic compound (compound 101) was synthesized by forming an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp, according to the previously described method. After cyclization, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and then the solvent was concentrated again under reduced pressure. To one of the ten divided test tubes, 0.20 mL of a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL was withdrawn, and 68.5 mg of tetramethylammonium bisulfate was dissolved in it) was added. The test tubes were stoppered with rubber septums, shaken for 3 minutes, and then allowed to stand at 25°C for 24 hours. The reaction was confirmed by LCMS (FAO5), and the completion of side chain deprotection (deprotection of the tBu group of D-Tyr(tBu)) was confirmed. Furthermore, the ratio of the target deprotected peptide (compound 131), the solvolysis product (a compound showing the mass spectrum of a peptide whose amide bond is solvoly hydrolyzed with HFIP as the solvent), and the hydrolysate (a compound showing the mass spectrum of a peptide whose amide bond is solvoly hydrolyzed with water) was 72:10:18 (Figure 2). In this example, "TM+H2O" refers to a compound in which one of the amide bonds of the target product has undergone hydrolysis, and "TM+HFIP" refers to a compound in which one of the amide bonds of the target product has undergone solvolysis by HFIP.

[0196] The data for Figure 2 is shown below. Target peptide (compound 131) LCMS(ESI)m / z=1424.0(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) Hydrolysis (TM+H2O) LCMS(ESI)m / z=1442.0(M+H) + Retention time: 0.61 minutes (Analysis conditions SQDFA05) HFIP-mediated solvolysis product (TM+HFIP) LCMS(ESI)m / z=1592.0(M+H) + Retention time: 0.69 minutes, 0.71 minutes (Analysis conditions SQDFA05)

[0197] Example 3-2-2: Deprotection of a cyclic compound (compound 101, Pep1) formed by an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) as the deprotection condition. After synthesizing compound 101 (Pep1), 0.40 mL of 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (4 mL was withdrawn from a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, DCE: 0.10 mL, and then 34.3 mg of tetramethylammonium bisulfate was dissolved in it) was added to one of the 10 equal portions obtained in the above procedure. The test tube was stoppered with a rubber septum, shaken for 3 minutes, and then allowed to stand at 25°C for 24 hours. The reaction was confirmed by LCMS (FA05). As a result, 81% of the side chain deprotection (deprotection of the tBu group of D-Tyr(tBu)) proceeded, and at this time, the ratio of the target deprotected peptide (compound 131), the solvolysis product (a compound representing the mass of the peptide after solvolysis of any amide bond by HFIP with solvent) and the hydrolysis product (a compound representing the mass of the peptide after hydrolysis of any amide bond by water) was 93:3:4 (Figure 3). Note that "TM+H2O" represents a compound in which one of the amide bonds of the target product has undergone hydrolysis. Similarly, "TM+HFIP" represents a compound in which one of the amide bonds of the target product has undergone solvolysis by HFIP.

[0198] The data for Figure 3 is shown below. Target peptide (compound 131) LCMS(ESI)m / z=1424.1(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) hydrolysis LCMS(ESI)m / z=1442.0(M+H) + Retention time: 0.61 minutes (Analysis conditions SQDFA05) HFIP-mediated solvolysis products LCMS(ESI)m / z=1592.0(M+H) + Retention time: 0.69 minutes, 0.71 minutes (Analysis conditions SQDFA05)

[0199] As shown in the comparative example below, the cyclic compounds (compounds 101 and Pep1) in which an amide bond is formed between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp underwent 87% hydrolysis after 2.5 hours at 25 degrees Celsius when deprotected under 5% TFA / DCE conditions. In contrast, using 0.1 M or 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS) instead of 5% TFA for the same peptide sequence significantly reduced the formation of hydrolysates (and solvolysis products). This result also suggests the possibility of arbitrarily adjusting the concentration of the weak acid if conditions for a weaker acid than TFA are met.

[0200] Example 3-2-3: Deprotection of a cyclic compound (compound 103, Pep3) formed by an amide bond between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) as the deprotection condition. Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, loading: 0.316 mmol / g, 100 mg) as the resin, a cyclic compound (compound 103, Pep3) was synthesized by forming an amide bond between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp, according to the previously described method. After cyclization, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and then the solvent was concentrated again under reduced pressure. To one of the ten divided test tubes, 0.40 mL of a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL was withdrawn, and 34.3 mg of tetramethylammonium bisulfate was dissolved in it) was added. The test tubes were stoppered with rubber septums, shaken for 3 minutes, and then allowed to stand at 25 degrees Celsius. The reaction was confirmed by LC-MS (SQDFA05) after 4 hours. As a result, the completion of side chain deprotection (deprotection of the DMT group of MeSer(DMT) and deprotection of the Trt group of Ser(Trt)) was confirmed. Furthermore, at this time, the UV area ratio in LC between the deprotected target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) and the N→O-acyl shift product (depshipeptide) of the target peptide was 96:4. After 22 hours from the start of the reaction and standing at 25 degrees Celsius, LC-MS (SQDFA05) measurements were performed. The LC UV area ratio between the deprotected target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) and the N→O-acyl shift product (depshipeptide) of the target peptide was 83:17 (Figure 4).

[0201] The data for Figure 4 is shown below. The target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) LCMS(ESI)m / z=1474.1(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05) N→O-Acylshift compound LCMS(ESI)m / z=1474.1(M+H) + Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0202] As described below, the cyclic compound (compound 103, Pep3) in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp underwent deprotection under 5% TFA / DCE conditions, and after 2 hours at 25 degrees Celsius, 70% of the N→O acyl shift proceeded. In contrast, using 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS) instead of 5% TFA for the same peptide sequence significantly reduced the formation of the N→O-acyl shift compound.

[0203] Example 3-2-4: Deprotection of a cyclic compound (compound 101, Pep1) formed by an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M oxalic acid / HFIP solution (2% TIPS) as the deprotection condition. After synthesizing compound 101 (Pep1), 0.40 mL of a 0.05 M oxalic acid / HFIP solution (2% TIPS) (prepared by taking 4 mL of a solution made by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL, and dissolving 18.0 mg of oxalic acid in it) was added to one of the 10 equal portions obtained in the above procedure. The test tube was stoppered with a rubber septum, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LCMS (FA05). As a result, the deprotection of the side chain (deprotection of the tBu group of D-Tyr(tBu)) was completed, and at this time, the ratio of the target deprotected peptide (compound 131), the solvolysis product (a compound representing the mass of the peptide after solvolysis of any amide bond with HFIP, which is the solvent), and the hydrolysis product (a compound representing the mass of the peptide after hydrolysis of any amide bond with water) was 79:17:4 (Figure 5). Note that "TM+H2O" represents a compound in which one of the amide bonds of the target product has undergone hydrolysis. Similarly, "TM+HFIP" represents a compound in which one of the amide bonds of the target product has undergone solvolysis by HFIP.

[0204] The data for Figure 5 is shown below. Target peptide (compound 131) LCMS(ESI)m / z=1423.5(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) hydrolysis LCMS(ESI)m / z=1441.5(M+H) + Retention time: 0.61 minutes (Analysis conditions SQDFA05) HFIP-mediated solvolysis products LCMS(ESI)m / z=1591.5(M+H) + Retention time: 0.68 minutes, 0.71 minutes (Analysis conditions SQDFA05)

[0205] Example 3-2-5: Deprotection of a cyclic compound (compound 101, Pep1) formed by an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M maleic acid / HFIP solution (2% TIPS) as the deprotection condition. After synthesizing compound 101 (Pep1), 0.40 mL of a 0.05 M maleic acid / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, DCE: 0.10 mL, and then dissolving 23.2 mg of oxalic acid in it) was added to one of the 10 equal portions obtained in the above procedure. The test tube was stoppered with a rubber septum, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LCMS (FA05). As a result, the deprotection of the side chain (deprotection of the tBu group of D-Tyr(tBu)) was completed, and at this time, the ratio of the target deprotected peptide (compound 131), the solvolysis product (a compound representing the mass of the peptide after solvolysis of any amide bond with HFIP, which is the solvent), and the hydrolysis product (a compound representing the mass of the peptide after hydrolysis of any amide bond with water) was 81:12:7 (Figure 6). Note that "TM+H2O" represents a compound in which one of the amide bonds of the target product has undergone hydrolysis. Similarly, "TM+HFIP" represents a compound in which one of the amide bonds of the target product has undergone solvolysis by HFIP.

[0206] The data for Figure 6 is shown below. Target peptide (compound 131) LCMS(ESI)m / z=1423.5(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) hydrolysis LCMS(ESI)m / z=1441.5(M+H) + Retention time: 0.61 minutes (Analysis conditions SQDFA05) HFIP-mediated solvolysis products LCMS(ESI)m / z=1591.4(M+H) + Retention time: 0.68 minutes, 0.71 minutes (Analysis conditions SQDFA05)

[0207] Example 3-2-6: Deprotection of a cyclic compound (compound 103, Pep3) formed by an amide bond between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M oxalic acid / HFIP solution (2% TIPS) as the deprotection condition. After synthesizing compound 103 (Pep3), 0.40 mL of a 0.05 M oxalic acid / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, DCE: 0.10 mL, and then dissolving 18.0 mg of oxalic acid in it) was added to one of the 10 equal portions obtained in the above procedure. The test tube was stoppered with a rubber septum, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LC-MS (SQDFA05). As a result, the deprotection of the side chains (deprotection of the DMT group of MeSer(DMT) and deprotection of the Trt group of Ser(Trt)) was confirmed. Furthermore, at this time, the UV area ratio in LC between the deprotected target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) and the N→O-acyl shift product (depshipeptide) of the target peptide was 86:14 (Figure 7).

[0208] The data for Figure 7 is shown below. The target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) LCMS(ESI)m / z=1473.5(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05) N→O-Acylshift compound LCMS(ESI)m / z=1473.5(M+H) + Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0209] Example 3-2-7: Deprotection of a cyclic compound (compound 103, Pep3) formed by an amide bond between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp, using a 0.05 M maleic acid / HFIP solution (2% TIPS) as the deprotection condition. After synthesizing compound 103 (Pep3), 0.40 mL of a 0.05 M maleic acid / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, DCE: 0.10 mL, and then dissolving 23.2 mg of oxalic acid in it) was added to one of the 10 equal portions obtained in the above procedure. The test tube was stoppered with a rubber septum, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LC-MS (SQDFA05). As a result, the deprotection of the side chains (deprotection of the DMT group of MeSer(DMT) and deprotection of the Trt group of Ser(Trt)) was confirmed. Furthermore, at this time, the UV area ratio in LC between the deprotected target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) and the N→O-acyl shift product (depshipeptide) of the target peptide was 86:14 (Figure 8).

[0210] The data for Figure 8 is shown below. The target peptide (compound 133, a cyclic compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp) LCMS(ESI)m / z=1473.5(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) N→O-Acylshift compound LCMS(ESI)m / z=1473.5(M+H) + Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0211] These results indicate that even when oxalic acid (pKa 1.23) or maleic acid (pKa 1.92) were used as the weak acid instead of tetramethylammonium bisulfate (pKa 2.0), deprotection could be carried out while suppressing hydrolysis, solvolysis, and N→O-acyl shift.

[0212] Example 3-2-8: Comparison of deprotection of a cyclic compound (compound 107, Pep7) formed by an amide bond between the N-terminal amino group of H-Ala-Phe(4-CF3)-Trp-Trp-MeLeu-MeGly-MeGly-Pro-Hyp(Et)-Ser(Trt)-Asp-pip(tBu) and the side-chain carboxylic acid of Asp, using 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS) as the deprotection condition and using 0.05 M tetramethylammonium bisulfate / TFE (2% TIPS) as the deprotection condition. Using Fmoc-Asp(O-Trt(2-Cl)-resin)-piptBu (compound 52, loading: 0.356 mmol / g, 100 mg) as the resin, a cyclic compound (compound 107) was synthesized by forming an amide bond between the N-terminal amino group of H-Ala-Phe(4-CF3)-Trp-Trp-MeLeu-MeGly-MeGly-Pro-Hyp(Et)-Ser(Trt)-Asp-pip(tBu) and the side-chain carboxylic acid of Asp, according to the previously described method. After cyclization, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and the solvent was removed again under reduced pressure. To one of the ten divided test tubes, 0.40 mL of a 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (prepared by dissolving 205.8 mg of tetramethylammonium bisulfate in a solution made by mixing HFIP: 23.32 mL, TIPS: 0.48 mL, and DCE: 0.20 mL) was added. The test tubes were stoppered with rubber septums and allowed to stand at 25°C for 4 hours, after which the reaction was confirmed by LCMS (FA05). As a result, deprotection of the side chain (deprotection of the Trt group of Ser(Trt)) was completed, and at this time, the UV area ratio of the deprotected target peptide (compound 137) to the solvolysis product (a compound indicating the mass of the peptide after solvolysis of one of its amide bonds with HFIP, the solvent) was 53:47 (Figure 9). Note that "TM+HFIP" represents a compound in which one of the target amide bonds has undergone solvolysis by HFIP.

[0213] [ka]

[0214] The data for Figure 9 is shown below. Target peptide (compound 137) LCMS(ESI)m / z=1492.1(M+H) + Retention time: 0.90 minutes (Analysis conditions SQDFA05) HFIP-mediated solvolysis product (a product in which one of the amide bonds has been solvoly analyzed by HFIP) LCMS(ESI)m / z=1660.1(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05)

[0215] To another test tube, which was divided into 10 equal parts, 0.40 mL of 0.05 M tetramethylammonium bisulfate / TFE solution (2% TIPS) (205.8 mg of tetramethylammonium bisulfate dissolved in a solution of TFE: 23.32 mL, TIPS: 0.48 mL, DCE: 0.20 mL) was added. The test tube was stoppered with a rubber septum and allowed to stand at 25°C, and the reaction was confirmed by LC-MS (FA05). As a result, after 4 hours, 96% of the side chain deprotection (deprotection of the Trt group of Ser(Trt)) had proceeded, and at this time, the solvolysis product (a compound indicating the mass of the product obtained by solvolysis of one of the amide bonds of the peptide with TFE as the solvent) was below the detection limit by LC-MS. When the reaction was observed after 20 hours, the deprotection of the side chain (deprotection of the Trt group of Ser(Trt)) was complete. At this time, the UV area ratio of the deprotected target peptide (compound 137) to the solvolysis product (a compound indicating the mass of which one of the amide bonds of the peptide has been solvoly decomposed by TFE, which is the solvent) was 97:3 (Figure 10). In this example, "TM+TFE" refers to a compound in which the target product (TM) has been solvated by TFE (a compound in which one of any amide bonds has been solvoly decomposed by TFE).

[0216] The data for Figure 10 is shown below. Target peptide (compound 137) LCMS(ESI)m / z=1492.2(M+H) + Retention time: 0.90 minutes (Analysis conditions SQDFA05) TFE-mediated solvolysis product (a product in which one of the amide bonds has been solvoly decomposed by TFE). LCMS(ESI)m / z=1592.0(M+H) + Retention time: 0.75 minutes (Analysis conditions SQDFA05)

[0217] The above results demonstrate that TFE can be used as a solvent instead of HFIP for dissolving weak acids. Furthermore, this result indicates that Y OTs The possibility of using any solvent is suggested if it satisfies the conditions that the value is positive, the solvent itself is weakly acidic (pKa of 5-14 in water), and it has low nucleophilicity.

[0218] Example 3-2-9: Deprotection was performed using a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) as the deprotection condition, and the reaction was stopped by adding a base (DIPEA) to the solution. Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, loading: 0.342 mmol / g, 100 mg) as the resin, a cyclic compound (compound 105, Pep5) was synthesized by forming an amide bond between the N-terminal amino group of H-Ala-Trp-Nle-Trp-Ser(Trt)-Gly-MeAla-MePhe(3-Cl)-MeGly-Pro-Asp-pip and the side-chain carboxylic acid of Asp, according to the previously described method. After cyclization, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and then the solvent was concentrated again under reduced pressure. To each of the two test tubes, which were divided into 10 equal parts, 0.40 mL of a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL was withdrawn, and 68.5 mg of tetrabutylammonium bisulfate was dissolved in it) was added. The test tubes were stoppered with rubber septums, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LCMS (SQDFA05). As a result, the deprotection of the side chain (deprotection of the Trt group of Ser(Trt)) was completed, and at this time, no peaks indicating the mass of solvodegraded products (compounds indicating the mass of products in which any amide bond of the peptide is solvograded with HFIP, the solvent) or hydrolyzed products (compounds indicating the mass of products in which any amide bond of the peptide is solvograded with water) other than the target deprotected peptide (compound 135) were detected (Figure 11). Diisopropylethylamine (DIPEA, 14 μL, 2 equivalents relative to tetramethylammonium bisulfate) was added to each of the reaction mixtures. One test tube was allowed to stand at 25°C for 18 hours, while the other test tube was concentrated under reduced pressure. When these were measured using LCMS (SQDFA05), no peaks indicating mass of solvolysis or hydrolysis products other than the deprotected target peptide were detected at this stage (Figure 11).

[0219] [ka]

[0220] The data for Figure 11 is shown below. Target peptide (compound 135) LCMS(ESI)m / z=1331.9(M+H) + Retention time: 0.74 minutes (Analysis conditions SQDFA05)

[0221] Example 3-2-10: Deprotection was performed using a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) as the deprotection condition, and the reaction was stopped by adding a base (DIPEA) to the solution. Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, loading: 0.316 mmol / g, 100 mg) as the resin, a cyclic compound (compound 103, Pep3) was synthesized by forming an amide bond between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the side-chain carboxylic acid of Asp, according to the previously described method. After cyclization, the residue was concentrated under reduced pressure, dissolved in dichloromethane, divided into 10 equal parts in test tubes, and then the solvent was concentrated again under reduced pressure. To each of the two test tubes, which were divided into 10 equal parts, 0.40 mL of a 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) (4 mL of a solution prepared by mixing HFIP: 11.66 mL, TIPS: 0.24 mL, and DCE: 0.10 mL was withdrawn, and 68.5 mg of tetramethylammonium bisulfate was dissolved in it) was added. The test tubes were stoppered with rubber septums, shaken for 3 minutes, and then allowed to stand at 25°C for 4 hours. The reaction was confirmed by LC-MS (FA05). As a result, the deprotection of the side chains (deprotection of the DMT group of MeSer(DMT) and deprotection of the Trt group of Ser(Trt)) was completed, and the UV area ratio on LC between the deprotected target peptide (compound 133) and the N→O-acyl shift derivative (depshipeptide) of the target peptide was 93:7. Diisopropylethylamine (DIPEA, 14 μL, 2 equivalents to tetramethylammonium bisulfate) was added to each of the reaction mixtures. One test tube was left to stand at 25°C for 18 hours, while the other test tube was concentrated under reduced pressure immediately after adding DIPEA. When these LCMS(FAO5) samples were measured, the UV area ratio of the target peptide and its N→O-acylshift compound remained unchanged in the sample left to stand for 18 hours, while the UV area ratio of the target peptide and its N→O-acylshift compound became 98:2 in the sample concentrated under reduced pressure (Figure 12).

[0222] The data shown in Figure 12 is presented below. Target peptide (compound 133) LCMS(ESI)m / z=1474.1(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05) N→O-Acylshift compound LCMS(ESI)m / z=1474.1(M+H) + Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0223] The above results demonstrate that by adding DIPEA after the deprotection reaction is complete (or when you want to stop the reaction), workup can be performed while suppressing problems such as hydrolysis (solvolysis) and N→O-acyl shift.

[0224] Example 4. Reactivity of Thr and MeSer when a THP group is used as a protecting group for the side-chain hydroxyl group. The following experiments were conducted to investigate the reactivity when using a THP group as a protecting group for the hydroxyl side chains of Thr and MeSer, which are suspected to have low reactivity during the extension reaction.

[0225] Example 4-1: Comparative evaluation of the extension reactivity of Fmoc-Thr(Trt)-OH and Fmoc-Thr(THP)-OH (compound 2) to a peptide with an N-methylamino group at its N-terminus (H-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-2-Cl-Trt-resin)-pip) extended on resin. The comparative evaluation of extension reactivity was performed using sequences with MePhe(3-Cl) at the N-terminus, which has low reactivity of the amino group due to steric hindrance. Fmoc-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-Trt(2-Cl)-resin)-pip (compound 108) was synthesized using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, loading: 0.329 mmol / g, 100 mg), which was prepared by the previously described method, as the resin, and by the previously described method. Dichloromethane (600uL) was added to the obtained Fmoc-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp(O-Trt(2-Cl)-resin)-pip (compound 108), and the mixture was allowed to stand for 30 minutes to swell the resin. After removing the liquid phase, the resin was washed three times with DMF (600uL). 2% DBU / DMF (v / v, 600uL) was added to the obtained resin and shaken for 20 minutes to remove the liquid phase. The resin was washed three times with DMF (600uL). To this resin, a solution prepared by mixing 0.60 M Fmoc-Thr(Trt)-OH / 0.375 Moxyma NMP solution (300 uL) with 10% (v / v) DIC / DMF (300 uL), or a solution prepared by mixing 0.60 M Fmoc-Thr(THP)-OH(compound 2) / 0.375 M oxyma NMP solution (300 uL) with 10% (v / v) DIC / DMF (300 uL), was added and shaken. During shaking, approximately 10 mg of the resin was removed at 1 hour, 2 hours, and 4 hours. The removed resin was washed three times with DMF (600 uL), and then 2% DBU / DMF (v / v, 600 uL) was added and shaken for 20 minutes to remove the liquid phase. The resin was washed three times with DMF (600uL) and then three times with dichloromethane (600uL). The obtained resin was mixed with TFE / DCM (1 / 1, v / v, 1 mL) and shaken for 10 minutes. After removing the resin by filtration, the liquid phase was concentrated under reduced pressure. The residue was analyzed by LC-MS (analytical conditions SQDFA05). The results are shown in Table 4.

[0226] [Table 4]

[0227] The ratio of unreacted compound to extended compound in the table represents the UV area ratio of the LC. The unreacted compound in the table is H-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip (compound 109), and the extended compound is H-Thr(Trt)-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip (compound 110: when Fmoc-Thr(Trt)-OH is added) or H-Thr(THP)-MePhe(3-Cl)-D-Tyr(tBu)-Trp-MePhe-Trp-MePhe-Ile-Asp-pip (compound 111: when Fmoc-Thr(THP)-OH is added).

[0228] [ka] Unreacted compound (compound 109) LCMS(ESI)m / z=1422.9(M+H) + Retention time: 0.77 minutes (Analysis conditions SQDFA05)

[0229] [ka] Elongated form (Thr(Trt)) (Compound 110) LCMS(ESI)m / z=1766.2(M+H) + Retention time: 0.92 minutes (Analysis conditions SQDFA05)

[0230] [ka] Elongated form (Thr(THP)) (compound 111) LCMS(ESI)m / z=1608.1(M+H) + Retention time: 0.80 minutes (Analysis conditions SQDFA05)

[0231] Example 4-2: The elongation reactivity of Fmoc-Thr(Trt)-OH or Fmoc-Thr(THP)-OH (compound 2) when Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr(PG)-MePhe-MeAla-MeLeu-MePhe-Asp(O-Trt(2-Cl)-resin)-pip is synthesized using a synthesizer. We investigated the extension reactivity by extending Thr in sequences having MePhe at the N-terminus, which has low reactivity due to steric hindrance, and in sequences having MeLeu at the N-terminus, which is sterically bulkier relative to the amino group of Thr. Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr(PG)-MePhe-MeAla-MeLeu-MePhe-Asp(O-Trt(2-Cl)-resin)-pip (where PG in the Thr side chain represents a protecting group, in this experiment representing either Trt protection or THP protection) was prepared using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, 100 mg) prepared by the previously described method as the resin, and peptide elongation was carried out according to the peptide synthesis method by Fmoc already described in the examples. In this process, Fmoc-Thr(Trt)-OH or Fmoc-Thr(THP)-OH (compound 2) was used for Thr elongation. After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and the resin was washed with DMF and DCM. After re-swelling the resin with DCM, TFE / DCM (1 / 1, v / v, 2 mL) was added to the resin and shaken at room temperature for 2 hours to excavate the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with TFE / DCM (1 / 1, v / v, 1 mL). After cleavage, when using Fmoc-Thr(Trt)-OH, the Trt group of Thr(Trt) was deprotected by shaking at 25°C for 5 minutes with a solution of 4N HCl / 1,4-dioxane (19.5 uL), TIPS (0.25 mL), and DCM (0.73 mL). Subsequently, the acid was neutralized by adding DIPEA (24 uL), and the extension reactivity was examined by LC-MS.

[0232] The LCMS results of this product are shown in Figure 13. The target peptide (compound 112, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr-MePhe-MeAla-MeLeu-MePhe-Asp-pip) and the target peptide with the Thr group removed (compound 113, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-MePhe-MeAla-MeLeu-MePhe-Asp-pip) were observed at the same retention time of 0.69 minutes. Furthermore, MS (negative mode) revealed that approximately 30% of the peptide contained the Thr group removed.

[0233] [ka]

[0234] The data for Figure 13 is shown below. Target compound (compound 112, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr-MePhe-MeAla-MeLeu-MePhe-Asp-pip) LCMS(ESI)m / z=1373.6(M+H) + Retention time: 0.69 minutes (Analysis conditions SQDAA50)

[0235] [ka] Target - Thr (Compound 113, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-MePhe-MeAla-MeLeu-MePhe-Asp-pip) LCMS(ESI)m / z=1272.6(M+H) + Retention time: 0.69 minutes (Analysis conditions SQDAA50)

[0236] The LCMS results of this product are shown in Figure 14. In contrast to the case where Fmoc-Thr(Trt)-OH was added, when Fmoc-Thr(THP)-OH was added, the peptide with the Thr group removed from the target peptide (compound 114, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-Thr(THP)-MePhe-MeAla-MeLeu-MePhe-Asp-pip) (compound 113, Hb-MeAla-Ile-MeLeu-MeAla-MeLeu-MePhe-MeAla-MeLeu-MePhe-Asp-pip) was not detected. Note that when Fmoc-Thr(THP)-OH (compound 2) was used, the cleaved solution was measured directly by LCMS without deprotection after cleavage from the resin, so the THP protection of the Thr side chain remained.

[0237] [ka]

[0238] The data for Figure 14 is shown below. Target compound (compound 114) LCMS(ESI)m / z=1458.1(M+H) + Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0239] The results above demonstrate that Fmoc-Thr(THP)-OH (compound 2) exhibits higher reactivity compared to Fmoc-Thr(Trt)-OH, which is commonly used to extend Thr in peptide synthesis. In particular, it was shown that high condensation efficiency can be achieved when extending bulky amino groups with N-alkylated N-terminuses. Furthermore, it was confirmed that the extension of bulky Fmoc-amino acids (in this case, Fmoc-MeLeu-OH) to the N-terminal amino group of Thr(THP) proceeds without any problems in the subsequent extension reaction.

[0240] Example 4-2: Confirmation of MeSer extension reactivity when using Fmoc-MeSer(DMT)-OH or Fmoc-MeSer(THP)-OH (compound 6) when synthesizing H-MeSer(PG)-MeVal-MeHis(Trt)-Tyr(3-F,tBu)-Pro-MeHis(Trt)-Pro-Trp-MePhe(4-Cl)-Asp(O-Trt(2-Cl)-resin)-Pro-OPis using a synthesizer. H-MeSer(PG)-MeVal-MeHis(Trt)-Tyr(3-F,tBu)-Pro-MeHis(Trt)-Pro-Trp-MePhe(4-Cl)-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (where PG in the MeSer side chain represents a protecting group, in this experiment representing either DMT protection or THP protection) was prepared using Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (compound 58, loading amount 0.3736 mmol / g, 100 mg), which was prepared by the previously described method, as the resin, and peptide extension was carried out according to the peptide synthesis method by Fmoc already described in the examples. In this process, Fmoc-MeSer(DMT)-OH or Fmoc-MeSer(THP)-OH (compound 6) was used for MeSer extension. After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and the resin was washed with DMF and DCM. After drying the obtained resins under reduced pressure, 30 mg of each resin was extracted. Each 30 mg of resin sample was re-swelled with DCM, then TFE / DCM (1 / 1, v / v, 2 mL) was added to the resin and shaken at room temperature for 2 hours to cleave the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with TFE / DCM (1 / 1, v / v, 1 mL). All the resulting cleaved solutions were mixed and concentrated under reduced pressure. To the obtained residue, 1.3 mL of 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) prepared by the previously described method was added to dissolve the residue. After standing at room temperature for 1 hour, the side chain protecting groups other than the tBu protection of the Tyr(3-F) side chain (DMT or THP protection of the MeSer side chain, and Trt protection of the MeHis side chain) and the Pis protection of the main chain C-terminus were deprotected, and the reaction was confirmed by measuring LCMS.

[0241] Figure 15 shows the LCMS results when Fmoc-MeSer(DMT)-OH·0.75 was synthesized using DIPEA. The target peptide (compound 115, H-MeSer-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH) and a compound with MeSer removed from the target peptide (compound 116, H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH) were observed with the same retention time of 0.50 minutes. Furthermore, MS (negative mode) revealed that 50% of the compound (compound 116, H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH) contained a peptide lacking MeSer.

[0242] [ka]

[0243] The data for Figure 15 is shown below. Target compound (compound 115) LCMS(ESI)m / z=1559.7(M+H) + Retention time: 0.50 minutes (Analysis conditions SQDFA50) Target substance - MeSer (compound 116) LCMS(ESI)m / z=1458.8(M+H) + Retention time: 0.50 minutes (Analysis conditions SQDFA50)

[0244] [ka]

[0245] In contrast, Figure 16 shows the LCMS results when synthesis was performed using Fmoc-MeSer(THP)-OH (compound 6). The target peptide (compound 115, H-MeSer-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH) and a compound with MeSer removed from the target peptide (compound 116) were observed at the same retention time of 0.50 minutes. However, MS (negative mode) confirmed that the peptide with MeSer removed (compound 116, H-MeVal-MeHis-Tyr(3-F,tBu)-Pro-MeHis-Pro-Trp-MePhe(4-Cl)-Asp-Pro-OH) accounted for less than 10%.

[0246] The data for Figure 16 is shown below. Target compound (compound 115) LCMS(ESI)m / z=1559.7(M+H) + Retention time: 0.50 minutes (Analysis conditions SQDFA50) Target substance - MeSer (compound 116) LCMS(ESI)m / z=1458.7(M+H) + Retention time: 0.50 minutes (Analysis conditions SQDFA50)

[0247] The results above demonstrate that Fmoc-MeSer(THP)-OH (compound 6) exhibits higher reactivity compared to Fmoc-MeSer(DMT)-OH, and that high condensation efficiency can be achieved when extending the N-terminus to an N-methylated amino group.

[0248] Deprotection of cyclized peptides in 5.5% TFA (comparative example)

[0249] Comparative Example 1: Deprotection of the side chain protecting group of a compound (compound 101, Pep-1) in which an amide bond is formed between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the Asp side-chain carboxylic acid (deprotection of the tBu protection of D-Tyr(tBu)) (when using 5% TFA / DCE (5% TIPS)). Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, resin load 0.373 mmol / g, 100 mg) synthesized by the previously described method, peptide extension was performed on a synthesizer to obtain H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp(O-Trt(2-Cl)-resin)-pip. Next, the resin was re-swollen with DCM, and then TFE / DCM (1 / 1, v / v, 2 mL) was added to the resin and shaken at room temperature for 2 hours to cleave the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with TFE / DCM (1 / 1, v / v, 1 mL). All the resulting cleaved solutions were mixed and concentrated under reduced pressure. The obtained residue was dissolved in DMF / DCM (1 / 1, v / v, 8 mL), and O-(7-aza-1Hbenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU, 21 mg) / DMF solution (0.5 M) and DIPEA (12 μL) / DMF (88 μL) solution were added, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by LCMS measurement (analytical conditions SQDFA05), and the formation of a compound (compound 101, Pep-1) was confirmed, in which an amide bond was formed between the N-terminal amino group of H-Ala-Trp-Nle-Trp-D-Tyr(tBu)-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and the Asp side-chain carboxylic acid. LCMS(ESI)m / z=1480.0(M+H) + Retention time: 0.93 minutes (Analysis conditions SQDFA05) Thereafter, the solvent was distilled off under reduced pressure, and 5% TFA / DCE (5% TIPS) (8 mL, the water content was confirmed by Karl Fischer measurement to be <200 ppm) was added to the obtained residue, followed by stirring for 2 hours and 30 minutes. The solvent was distilled off under reduced pressure, and LCMS (FA05) measurement of the residue was performed. As a result, the MS of the compound (Compound 131) in which an amide bond was formed between the N-terminal amino group and the Asp side-chain carboxylic acid of the target product H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip and its hydrolyzate (in which any amide bond was hydrolyzed) was confirmed, and the ratio of the corresponding UV areas was 13:87 (Figure 17). The measurement results of LC are shown in Figure 17. Note that "TM + H2O" described in this example represents a compound in which one of the amide bonds of the target product has undergone hydrolysis.

[0250] [Chemical formula]

[0251] The data in Figure 17 are shown below. Target product (Compound 131, a compound in which an amide bond is formed between the N-terminal amino group and the Asp side-chain carboxylic acid of H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip) LCMS (ESI) m / z = 1424.0 (M + H) + Retention time: 0.79 minutes (analysis condition SQDFA05) Hydrolyzate (a compound in which any amide bond of the compound in which an amide bond is formed between the N-terminal amino group and the Asp side-chain carboxylic acid of H-Ala-Trp-Nle-Trp-D-Tyr-MeGly-MeAla-MePhe(3-Cl)-MeGly-nPrGly-Asp-pip has been hydrolyzed) LCMS (ESI) m / z = 1442.0 (M + H) + Retention time: 0.61 minutes (analysis condition SQDFA05)

[0252] These results confirm that in deprotection using 5% TFA / DCE (5% TIPS), in the case of cyclic peptides that are highly N-methylated, especially those with a sequence of consecutive N-methyl amino acids, approximately 90% of the target product is hydrolyzed, making it difficult to obtain the target product.

[0253] Comparative Example 2: Deprotection of the side chain protecting groups (DMT protection of the MeSer side chain and Trt protection of the Ser side chain) of a compound (compound 103, Pep3) in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side chain carboxylic acid (when using 5% TFA / DCE (5% TIPS)). Using Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 50, resin load 0.316 mmol / g, 100 mg) synthesized by the previously described method, peptide extension was performed on a synthesizer to obtain Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp(O-Trt(2-Cl)-resin)-pip. Next, the resin was re-swollen with DCM, and then TFE / DCM (1 / 1, v / v, 2 mL) was added to the resin and shaken at room temperature for 2 hours to excavate the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice more with TFE / DCM (1 / 1, v / v, 1 mL). The peptide was excised from the resin twice, and all the resulting excised solutions were mixed and concentrated under reduced pressure. The obtained residue was dissolved in DMF / DCM (1 / 1, v / v, 8 mL), and O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU, 18 mg) / DMF solution (0.5 M) and DIPEA (9.9 μL) / DMF (39.6 μL) solution were added, and the mixture was stirred at room temperature for 2 hours. Confirmation of the reaction by LC-MS measurement (SQDFA05) revealed that 70% of the compounds (compound 103, Pep3) in which the DMT protection of the MeSer side chain was removed from the compound in which an amide bond was formed between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3-Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid were formed had the DMT protection of the MeSer side chain removed (LC-MS (ESI) m / z = 1716.2 (M+H)). + (Retention time: 1.06 mins), 30% of the samples showed loss of both DMT protection on the MeSer side chain and TRT protection on the Ser side chain (LCMS(ESI) m / z = 1474.1 (M+H)). + (Retention time: 0.78 minutes) was confirmed. The ratio was calculated by the UV area ratio of LC. Subsequently, the solvent was removed under reduced pressure, the residue was dissolved in dichloromethane, and divided into 10 equal parts in test tubes. These were concentrated under reduced pressure to remove the dichloromethane. Note that the "residue after cyclization" used in the above example refers to the residue obtained by concentrating under reduced pressure after dividing into 10 equal parts. 5% TFA / DCE (5% TIPS) (0.8 mL, water content 32.5 ppm, measured by Karl Fischer) was added to one of the test tubes and shaken for 3 minutes, then allowed to stand at 25°C for 2 hours. The solvent was removed under reduced pressure, and the residue was measured by LCMS (FA05). It was confirmed that the UV area ratios of the compound (compound 133) in which an amide bond was formed between the N-terminal amino group of the target product Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid, the N→O-acyl shift derivative of the target product, the product in which one hydroxyl group of the target product was esterified with TFA, and the product in which both hydroxyl groups of the target product were esterified with TFA were 17:46:32:5. The LC measurement results are shown in Figure 18.

[0254] [ka]

[0255] The data for Figure 18 is shown below. The target peptide (compound 133, a compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid) LCMS(ESI)m / z=1473.8(M+H) + Retention time: 0.79 minutes (Analysis conditions SQDFA05) The N→O-acyl shift compound of the target product (a compound in which an N→O-acyl shift has occurred in one or both of the two hydroxyl groups of the target product). LCMS(ESI)m / z=1473.8(M+H) + Retention time: 0.62 minutes (Analysis conditions SQDFA05) A compound in which one of the hydroxyl groups of the target product is esterified with TFA. LCMS(ESI)m / z=1569.8(M+H) + Retention time: 0.89 min (analysis condition SQDFA05) Compound in which two hydroxyl groups of the target substance are esterified with TFA LCMS (ESI) m / z = 1665.9 (M+H) + Retention time: 0.99 min (analysis condition SQDFA05)

[0256] From these results, it was confirmed that when an amino acid having a β-hydroxyl group such as MeSer or Ser is included in the sequence, in the deprotection using 5% TFA / DCE (5% TIPS), the N→O-acyl shift proceeds. Also, in the deprotection under these conditions, it was confirmed that either one or both of the two side-chain hydroxyl groups are esterified with TFA. It was confirmed that these unwanted reactions make it difficult to obtain the target substance.

[0257] Tests were conducted to lower the reaction temperature to 0°C and to lower the TFA concentration, aiming to suppress the formation of the N→O-acyl shift product and the TFA esterification of the hydroxyl group.

[0258] Comparative Example 3: Deprotection of the side chain protecting groups (DMT protection of the MeSer side chain and Trt protection of the Ser side chain) of a compound (compound 103, Pep3) in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side chain carboxylic acid (when performed at 0°C using 5% TFA / DCE (5% TIPS)). After the above cyclization, 5% TFA / DCE (5% TIPS) (0.8 mL, water content measured by Karl Fischer was 36.6 ppm) was added to one of the ten test tubes at 0°C and shaken for 1 minute, and then left standing at 0°C for 4 hours. When the reaction solution was measured by LCMS (FA05), the compound (Compound 133) in which an amide bond was formed between the N-terminal amino group of the target substance H-g-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid, the N→O-acyl shift product of the target substance, the one in which one hydroxyl group of the target substance was esterified with TFA, and the one in which one hydroxyl group of the N→O-acyl shift product of the target substance was esterified with TFA had a UV area ratio of 56:12:21:11. The measurement results of LC are shown in Fig. 19.

[0259] The data for Figure 19 is shown below. The target peptide (compound 133, a compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid) LCMS(ESI)m / z=1473.7(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05) The N→O-acyl shift compound of the target product (a compound in which an N→O-acyl shift has occurred in one or both of the two hydroxyl groups of the target product). LCMS(ESI)m / z=1473.7(M+H) + Retention time: 0.65 minutes (Analysis conditions SQDFA05) A compound in which one of the hydroxyl groups of the target product is esterified with TFA. LCMS(ESI)m / z=1569.7(M+H) + Retention time: 0.89 minutes (Analysis conditions SQDFA05) A compound in which one hydroxyl group of the N→O-acyl shift product of the target product is esterified with TFA. LCMS(ESI)m / z=1569.7(M+H) + Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0260] Comparative Example 4: Deprotection of the side chain protecting groups (DMT protection of the MeSer side chain and Trt protection of the Ser side chain) of a compound (compound 103, Pep3) in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer(DMT)-Hyp(Et)-Ile-MePhe(3Cl)-Ser(Trt)-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side chain carboxylic acid (when carried out at 25°C using 2% TFA / DCE (5% TIPS)). After the cyclization described above, 2% TFA / DCE (5% TIPS) (0.8 mL, water content 30.1 ppm, measured by Karl Fischer) was added at 25°C to one of the 10 divided test tubes, shaken for 1 minute, and then allowed to stand at room temperature for 4 hours. LC-MS (FA05) measurements of the reaction solution revealed that the UV area ratios for the compound (compound 133) formed by the amide bond between the N-terminal amino group of the target product Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid, the N→O-acyl shift derivative of the target product, the product with one hydroxyl group esterified with TFA, the product with both hydroxyl groups esterified with TFA, and the N→O-acyl shift derivative of the target product with one hydroxyl group esterified with TFA were 30:34:24:3:9. The LC measurement results are shown in Figure 20.

[0261] The data for Figure 20 is shown below. The target peptide (compound 133, a compound in which an amide bond is formed between the N-terminal amino group of Hg-EtAbu-MeSer-Hyp(Et)-Ile-MePhe(3-Cl)-Ser-Trp-Trp-Pro-MeGly-Asp-pip and the Asp side-chain carboxylic acid) LCMS(ESI)m / z=1473.8(M+H) + Retention time: 0.78 minutes (Analysis conditions SQDFA05) The N→O-acyl shift compound of the target product (a compound in which an N→O-acyl shift has occurred in one or both of the two hydroxyl groups of the target product). LCMS(ESI)m / z=1473.8(M+H) + Retention time: 0.65 minutes (Analysis conditions SQDFA05) A compound in which one of the hydroxyl groups of the target product is esterified with TFA. LCMS(ESI)m / z=1569.8(M+H) + Retention time: 0.89 minutes (Analysis conditions SQDFA05) Compounds in which the two hydroxyl groups of the target product are esterified with TFA. LCMS(ESI)m / z=1665.7(M+H) + Retention time: 0.99 minutes (Analysis conditions SQDFA05) A compound in which one hydroxyl group of the N→O-acyl shift product of the target product is esterified with TFA. LCMS(ESI)m / z=1569.8(M+H) + Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0262] Based on these results, problems such as the N→O-acyl shift and the TFA esterification of hydroxyl groups in the compound could not be completely solved simply by lowering the reaction temperature or using a lower concentration of TFA solution.

[0263] Example 6. Application of the present invention to parallel synthesis (solid-phase method)

[0264] Example 6-1: Synthesis of peptides cyclized with an N-terminal amino group and a side-chain carboxylic acid group of aspartic acid. We synthesized a group of peptides in which the carboxylic acid group of the side chain of aspartic acid, which is either amidated at the C-terminus (amidated with piperidine, 4-(tert-butyl)piperidine, or N-methyloctane-1-amine) or has a proline bonded to the C-terminus, is cyclized by an amide bond to the amino group of the main chain at the N-terminus.

[0265] Using 100 mg of any of the following compounds: compound 50 (2-chlorotrityllase resin supported with compound 48 (Fmoc-Asp-pip)), compound 52 (2-chlorotrityllase resin supported with compound 51 (Fmoc-Asp-piptBu)), compound 55 (2-chlorotrityllase resin supported with compound 54 (Fmoc-Asp-MeOctyl)), or compound 58 (2-chlorotrityllase resin supported with compound 57 (Fmoc-Asp-Pro-OPis)), and as Fmoc amino acids, Fmoc-MeVal-OH, Fmoc-MePhe(3-Cl)-OH (compound 15), Fmoc-MePhe(4-Cl)-OH (compound 16), Fmoc-MeHis(Trt)-OH (compound 7), Fmoc-MePhe-OH, Fmoc-Me Ser(DMT)-OH (compound 5), Fmoc-MeSer(THP)-OH (compound 6), Fmoc-MeAla-OH, Fmoc-nPrGly-OH (compound 20), Fmoc-M eGly-OH, Fmoc-Hyp(Et)-OH (compound 18), Fmoc-Pro-OH, Fmoc-Thr(THP)-OH (compound 2), Fmoc-Ile-OH, Fmoc-Va Peptide elongation was performed using l-OH, Fmoc-Trp-OH, Fmoc-Tyr(3-F,tBu)-OH (compound 13), Fmoc-Phe(4-CF3)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Met(O2)-OH, Fmoc-b-Ala-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, etc. (For MeSer elongation, Fmoc-MeSer(DMT)-OH (compound 5) was used for PS-53 and PS-54 (Table 5-1), and Fmoc-MeSer(THP)-OH (compound 6) was used for all others.) Peptide elongation was performed according to the peptide synthesis method using the Fmoc method already described in the examples. After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and then the resin was washed with DMF.

[0266] Next, the resin was re-swollen with DCM, and then TFE / DCM (1 / 1, v / v, 2 mL) and diisopropylethylamine (DIPEA, in a volume equivalent to 1.8 times the number of moles on the resin used (loading volume (mmol / g) multiplied by the amount of resin used (usually 0.10 g))) were added to the resin and shaken at room temperature for 2 hours to cleave the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with TFE / DCM (1 / 1, v / v, 1 mL). All the resulting cleaved solutions were mixed and concentrated under reduced pressure.

[0267] The obtained residue was dissolved in DMF / DCM (1 / 1, v / v, 8 mL), and 0.5 M O-(7-aza-1H-benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU) / DMF solution (in a volume equivalent to 1.5 times the number of moles of resin used (resin loading amount (mmol / g) multiplied by the amount of resin used (0.1 g))) and DIPEA (1.8 times the number of moles of resin used) were added, and the mixture was stirred at room temperature for 2 hours. After that, the solvent was removed under reduced pressure.

[0268] The obtained residue was deprotected as follows. If the sequence contained Tyr(3-F,tBu), 2 mL of 0.1 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) prepared by the previously described method was added to dissolve the residue, and the mixture was allowed to stand at room temperature or 30°C for 24 hours. If the sequence did not contain Tyr(3-F), 4 mL of 0.05 M tetramethylammonium bisulfate / HFIP solution (2% TIPS) prepared by the previously described method was added to dissolve the residue, and the mixture was allowed to stand at room temperature for 4 hours. After standing for a certain period of time, diisopropylethylamine (DIPEA, 70 μL) was added, and the solvent was removed under reduced pressure.

[0269] After removing the solvent under reduced pressure, the peptides were dissolved in DMF. Insoluble matter was removed by filter filtration, and the peptides were purified by preparative-HPLC to obtain the amide-cyclized cyclic peptides (PS-1 to PS-54) mentioned in the title. The sequences of PS-1 to PS-54 are shown in Table 5-1, their structures in Table 5-2, and the mass spectral values, liquid chromatography retention times, purity, and yield of the obtained peptides are shown in Table 5-3, respectively.

[0270] [Table 5-1] TIFF2026088137000083.tif216149

[0271] [Table 5-2] TIFF2026088137000085.tif198149TIFF2026088137000086.tif198149TIFF2026088137000087.tif198149TIFF202 6088137000088.tif198149TIFF2026088137000089.tif198149TIFF2026088137000090.tif198149TIFF20260881370 00091.tif198149TIFF2026088137000092.tif198149TIFF2026088137000093.tif198149TIFF2026088137000094.t if198149TIFF2026088137000095.tif198149TIFF2026088137000096.tif198149TIFF2026088137000097.tif198149 TIFF2026088137000098.tif198149TIFF2026088137000099.tif198149TIFF2026088137000100.tif198149TIFF202 6088137000101.tif198149TIFF2026088137000102.tif198149TIFF2026088137000103.tif198149TIFF20260881370 00104.tif198149TIFF2026088137000105.tif198149TIFF2026088137000106.tif198149TIFF2026088137000107.t if198149TIFF2026088137000108.tif198149TIFF2026088137000109.tif198149TIFF2026088137000110.tif198149

[0272] [Table 5-3]

[0273] Example 7. Application of the present invention to a liquid-phase method The synthesis, including the extension reaction using the liquid phase method, is shown below.

[0274] Application to partial liquid phase methods Synthesis of a cyclic peptide (compound 154, a cyclic peptide in which an amide bond is formed between the amino group at the N-terminus of the main chain of H-Ala-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 151)) in the liquid phase. The peptide was synthesized using the synthetic route shown in Figure 21, which includes an extension reaction in the liquid phase.

[0275] Example 7-1: Synthesis of H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 151) [ka] Using Fmoc-Asp(O-Trt(2-Cl)-resin)-Pro-OPis (compound 58, loading amount 0.3736 mmol / g, 200 mg) synthesized by the previously described method, peptide elongation was performed according to the peptide synthesis method using the Fmoc method described in the examples. After peptide elongation, the N-terminal Fmoc group was removed on the peptide synthesizer, and the resin was washed with DMF. Next, the resin was re-swollen with DCM, and then TFE / DCM (1 / 1, v / v, 4 mL) and diisopropylethylamine (24 μL) were added to the resin and shaken at room temperature for 2 hours to cleave the peptide from the resin. Subsequently, the resin was removed by filtering the solution in the tube through a synthesis column, and the remaining resin was washed twice with TFE / DCM (1 / 1, v / v, 2 mL). All the resulting cleaved solutions were mixed and concentrated under reduced pressure to obtain the title compound (Compound 151, H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis, 113.8 mg). This compound was used in the next step without purification. LCMS(ESI)m / z=1860.9(M+H) + Retention time: 0.72 minutes (Analysis conditions SQDFA05)

[0276] Example 7-2: Synthesis of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (S)-2,5-dioxopyrrolidine-1-yl (compound 152, Fmoc-Ala-OSu) [ka] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Ala-OH, 1.00 g, 3.21 mmol), 1-hydroxypyrrolidine-2,5-dione (HOSu, 0.554 g, 4.82 mmol), and dichloromethane (6.4 mL) were mixed under a nitrogen atmosphere. This mixture was cooled to 0°C on ice, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI·HCl, 0.924 g, 4.82 mmol) was added. The resulting reaction solution was stirred at 0°C for 1 hour and at room temperature for 15 hours. Next, the solvent was removed under reduced pressure, and the resulting residue was purified by reverse-phase silica gel chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (S)-2,5-dioxopyrrolidine-1-yl (compound 152, Fmoc-Ala-OSu, 1.05 g). LCMS(ESI)m / z=409.3(M+H) + Retention time: 0.80 minutes (Analysis conditions SQDFA05)

[0277] Example 7-3: Coupling of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (S)-2,5-dioxopyrrolidine-1-yl (compound 152, Fmoc-Ala-OSu) and H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 151), followed by the de-Fmoc reaction. [ka] To a solution of the obtained H-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 151, 113.8 mg) in dichloromethane (245 μL), 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (S)-2,5-dioxopyrrolidine-1-yl (compound 152, Fmoc-Ala-OSu, 26.2 mg) and diisopropylethylamine (DIPEA, 12.8 μL) were added, and the mixture was stirred at 25°C for 1 hour. Subsequently, methylamine (40% methanol solution, 11.9 μL) was added to the reaction solution and stirred for 30 minutes, after which DBU (11.1 μL) was added and stirred for another 30 minutes. The resulting reaction solution was purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution), and the resulting fraction was freeze-dried. The resulting residue was dissolved in dichloromethane and washed with saturated sodium bicarbonate aqueous solution and saturated sodium chloride aqueous solution to obtain H-Ala-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 153, 79.3 mg, 0.041 mmol). LCMS(ESI)m / z=1931.8(M+H) + Retention time: 0.73 minutes (Analysis conditions SQDFA05)

[0278] Example 7-4: Synthesis of compound 154, in which the N-terminal amino group of H-Ala-Trp-Nle-Trp-Ser-nPrGly-MePhe(3-Cl)-MeHis-MeGly-Pro-Asp-Pro-OH and the carboxylic acid group of the Asp side chain are amide-cyclized (cyclization reaction of compound 153 followed by deprotection reaction). [ka] The obtained H-Ala-Trp-Nle-Trp-Ser(THP)-nPrGly-MePhe(3-Cl)-MeHis(Trt)-MeGly-Pro-Asp-Pro-OPis (compound 153, 79.3 mg, 0.041 mmol) was dissolved in DMF (20 mL) and dichloromethane (20 mL), and HATU (17.2 mg, 0.045 mmol) and diisopropylethylamine (10.8 μL, 0.062 mmol) were added, and the mixture was stirred at 25°C for 2 hours. Subsequently, the solvent was removed under reduced pressure, and a 0.05 M tetramethylammonium bisulfate / HFIP (2% TIPS) solution (prepared by the method previously described in this example, 8 mL) was added, and the mixture was allowed to stand at 25°C for 1 hour. Diisopropylethylamine (140 μL) was added to the resulting reaction solution, and the solvent was removed under reduced pressure. The obtained residue was purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution), and the resulting fraction was freeze-dried to obtain a compound (compound 154, 59 mg, 0.040 mmol, 98%) in which the N-terminal amino group of H-Ala-Trp-Nle-Trp-Ser-nPrGly-MePhe(3-Cl)-MeHis-MeGly-Pro-Asp-Pro-OH and the carboxylic acid group of the Asp side chain were amide-cyclized. LCMS(ESI)m / z=1469.7(M+H) + Retention time: 0.61 minutes (Analysis conditions SQDFA05)

[0279] As demonstrated by the peptide synthesis described above, including segment coupling in the liquid phase, the synthesis method of the present invention is also applicable to liquid-phase methods. [Industrial applicability]

[0280] According to the present invention, peptides containing N-substituted amino acids, which are expected to be useful as pharmaceuticals, can be synthesized with high purity and high synthesis efficiency. The present invention is useful in fields such as the industrial production of peptides containing N-substituted amino acids that can be used as raw materials for pharmaceuticals.

Claims

1. (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid, (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid, (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazole-4-yl)propanoic acid, (2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropane-2-yl)oxy)phenyl)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid, or The compound is (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropane-2-yl)oxy)phenyl)propanoic acid.

2. An amino acid (Fmoc-protected amino acid) having at least one of the following functional groups i) and ii), or an amino acid analog (Fmoc-protected amino acid analog) having at least one of the following i) and ii); i) an amino group of the main chain protected by at least one protecting group having an Fmoc skeleton, ii) At least one free or activated esterified carboxylic acid group, A raw material composition containing, The protecting group of the side chain of the Fmoc-protected amino acid or the Fmoc-protected amino acid analog is a protecting group that is deprotected in the pH range of pH 1 to pH 7, or a protecting group that is deprotected in 10% or less trifluoroacetic acid. A raw material composition for use in a method for producing a peptide containing an N-substituted amino acid or an N-substituted amino acid analog, which has a deprotection step of deprotecting the protecting group of the side chain of the Fmoc-protected amino acid or the Fmoc-protected amino acid analog that constitutes the peptide, under conditions using a weak acid solution containing a weak acid with a pKa value of 1 to 5 in water and a fluoroalcohol.

3. The raw material composition according to claim 2, wherein the protecting group of the side chain is selected from a) to d) below: a) When the protecting group of the side chain is a protecting group of the hydroxyl group of the side chain of Ser, Thr, Hyp, and their derivatives, it is any protecting group selected from the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton and Boc skeleton represented by the following general formula; b) If the protecting group of the side chain is a protecting group of the hydroxyl group of the side chain of Tyr and its derivatives, it is any protecting group selected from the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, silyl skeleton, Boc skeleton and tBu skeleton represented by the following general formulas; c) When the protecting group of the side chain is a protecting group of the imidazole ring of the side chain of His and its derivatives, it is any protecting group selected from the MOM skeleton, Bn skeleton and Trt skeleton represented by the following general formula; d) When the protecting group of the side chain is a protecting group of the carboxylic acid group of the side chain of Asp, Glu, and their derivatives, it is one of the protecting groups selected from the MOM skeleton, Bn skeleton, Dpm skeleton, Trt skeleton, tBu skeleton, phenyl-EDOTn skeleton, and orthoester skeleton obtained by converting the carbon atom of the carboxylic acid group to be protected to a skeleton in which three alkoxy groups are substituted, as represented by the following general formula; <Protective group with MOM framework> 【Chemistry 1】 (In the formula, R1 is H, R2 is H, and X is methyl, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, or 2-trimethylsilylethyl. R1 is methyl, R2 is H, and X is ethyl. R1, R2, and R3 are all methyl, or R1 and X together form -CH2-CH2-CH2- or -CH2-CH2-CH2-CH2-, and R2 is H. Here, if any of R1, R2, and X are methyl or ethyl, then these The group may be further substituted with alkyl, benzyl, or aryl groups. <Protecting group with a Bn skeleton> 【Chemistry 2】 (In the formula, R1 to R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are alkyl. R1, R2, R4, and R5 are each independently H, alkyl, aryl, or halogen, R3 is methoxy, and R6 and R7 are H. R1 and R3 are methoxy, R2, R4 and R5 are each independently H, alkyl, aryl, or halogen, and R6 and R7 are H, or R1, R4, and R5 are each independently H, alkyl, aryl, or halogen, and R2 and R3 together form -O-CH2-O-. <Protecting group with a Dpm skeleton> 【Transformation 3】 (In the formula, R1 to R10 are each independently H, alkyl, aryl, alkoxy, or halogen, or R1-R4 and R7-R10 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form -O- or -CH2-CH2-. <Protecting group with a Trt skeleton> 【Chemistry 4】 (In the formula, R1 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen. R1, R2, and R4-R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R3 is methyl or methoxy. R1 is Cl, and R2 to R15 are each independently H, alkyl, aryl, alkoxy, or halogen, or R1-R4 and R7-R15 are each independently H, alkyl, aryl, alkoxy, or halogen, and R5 and R6 together form -O-. <Protecting groups with a silyl skeleton> 【Transformation 5】 (In the formula, R1 to R3 are each independently alkyl or aryl. <Protecting group with a Boc skeleton> 【Transformation 6】 (In the formula, R1 to R9 are each independently H, alkyl, or aryl. <Protecting group with tBu skeleton> 【Transformation 7】 (In the formula, R1 to R9 are each independently H, alkyl, or aryl. <Protecting group with a phenyl-EDOTn skeleton> 【Transformation 8】 (In the formula, R1 to R3 are each independently either H or methoxy.

4. The Fmoc-protecting amino acid or the Fmoc-protecting amino acid analog is (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid, (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid, (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazole-4-yl)propanoic acid, (2R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-phenylpropane-2-yl)oxy)phenyl)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropane-2-yl)oxy)phenyl)propanoic acid, (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(trityloxy)butanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-chlorophenyl)diphenylmethoxy)phenyl)propanoic acid, N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-trityl-L-serine, N α-(((9H-fluoren-9-yl)methoxy)carbonyl)-N τ-trityl-L-histidine, or The raw material composition according to claim 2 or 3, wherein the raw material is N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(bis(4-methoxyphenyl)(phenyl)methyl)-N-methyl-L-serine.

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