Method for efficient peptide condensation of high difficulty sequences

Abstract

To provide a method for producing a high-purity peptide compound in a high yield under a condensation condition applicable even to a highly rare non-natural amino acid by using an easily available condensing agent and additive capable of suppressing prematurecleavage and also suppressing a side reaction especially in a condensation reaction process of a solid phase synthesis method of a peptide.SOLUTION: Provided is a method for producing a peptide compound, including a step of condensing a first amino acid or peptide and a second amino acid or peptide in the presence of an additive and a condensing agent to obtain a condensate, wherein the number of moles of the additive is smaller than the number of moles of the second amino acid or peptide. By using a small amount of the additive with respect to the amino acid or peptide to be added to the N-terminus, the condensation reaction can be allowed to proceed efficiently even when an amino acid having large steric hindrance is contained, and the target peptide compound can be obtained with high yield and high purity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a peptide compound using a condensation reaction. [Background technology]

[0002] Peptides are molecules composed of many linked amino acids and play an essential role in the life activities of living organisms. As our understanding of peptides deepens with advances in biology and chemistry, there has been active research and development into functional peptides, including the utilization of natural peptides aimed at the creation of new pharmaceuticals and the artificial design of peptides (Non-Patent Document 1). In particular, it has been reported that peptide cyclization and N-alkylation, particularly N-methylation, of constituent amino acids contribute to improved membrane permeability and metabolic stability (Non-Patent Documents 2 and 3). Furthermore, findings and considerations regarding drug-like cyclic peptide structures, which are key to intracellular transport and the development of oral agents, have been reported, and the importance of such structures in peptide drug discovery is becoming increasingly recognized (Patent Document 1).

[0003] Peptide synthesis is achieved by extending the desired sequence through the formation of an amide bond. More specific methods include liquid-phase and solid-phase methods (Non-Patent Document 4).

[0004] Among these, the solid phase method uses an atomic group linked to a polymer resin (solid phase synthesis resin) as a linker, 1) Formation of a covalent bond with the carboxyl group at the C-terminus of an amino acid or peptide (loading step); 2) deprotection of the N-terminal amino group of the supported amino acid or peptide, followed by condensation with the amino acid of the next sequence, and repeating these deprotection and condensation reactions until the desired sequence is reached (elongation step); 3) cleavage of the peptide from the resin for solid-phase synthesis (resin removal step) (Non-Patent Documents 5 and 6). The solid-phase method is carried out in a two-layer reaction system consisting of a solid and a liquid, and involves contacting a solid solid-phase synthesis resin to which the target peptide is bound with a liquid reaction solution containing dissolved reagents for deprotection and condensation reactions. Because the target peptide is bound to the solid-phase synthesis resin, excess reagents and impurities derived from the reagents can be separated from the solid-phase synthesis resin to which the target peptide is bound simply by washing the resin between each step, allowing for easy sequential elongation.

[0005] In the solid-phase method, the Fmoc group or Boc group is commonly used as a protecting group for the amino group at the N-terminus of the amino acid main chain.

[0006] Resins for solid-phase synthesis are broadly classified by the linker atomic group attached to the polymer used in the resin, and solid-phase synthesis resins with linker atomic groups containing trityl or benzyl groups are commonly used. More specifically, CTC resin, Wang resin, SASRIN resin, and Rink Amide resin are representative examples. The depolymerization step is primarily performed under acidic conditions, but the ease of depolymerization depends on the acid stability of the linker atomic group. For example, depolymerization of peptides from CTC resin, which can be bound to peptides using a trityl group as a linker, can be performed using weakly acidic reagents. On the other hand, depolymerization of peptides from Wang resin, which can be bound to peptides using a benzyl group as a linker, requires strong acidic conditions (Non-Patent Document 6).

[0007] As mentioned above, the peptide elongation process, whether by solid-phase or liquid-phase methods, consists of a repetition of a condensation step in which the amino group of an amino acid or peptide is dehydrated and condensed with the carboxyl group of a newly introduced amino acid whose N-terminal amino group has been protected, and a deprotection step in which the protecting group of the newly introduced N-terminal amino group is removed. If the condensation reaction is not completed and / or competing side reactions accumulate during this repetition process, impurities resulting from the deletion or addition of amino acids may be produced from the peptide having the desired amino acid sequence, reducing the yield and / or purity of the peptide having the desired sequence.

[0008] Side reactions that are likely to occur during solid-phase condensation reactions include epimerization of the α-position of the carboxyl group due to activation of the carboxylic acid or the properties of the reaction solution, guanylation of the N-terminus, or N-Fmoc removal, as well as the simultaneous occurrence of these reactions (Non-Patent Document 6). One report suggests that a countermeasure for incomplete condensation reactions is to repeat the reaction under the same conditions rather than prolonging the reaction time (Non-Patent Document 6). Furthermore, a deletion product may be generated when an unreacted N-terminal amino group, for which the desired condensation reaction has not been achieved, reacts with a newly introduced amino acid (also referred to as the next amino acid in the sequence) whose N-terminal amino group has been protected. To prevent the formation of such deletion products, an acylating agent, such as acetic anhydride or benzoyl chloride, is reacted in the presence of pyridine to cap the unreacted N-terminal amino group, for which the desired condensation reaction has not been achieved, thereby preventing the elongation of the undesired sequence (Non-Patent Document 6).

[0009] As previously mentioned, the use of CTC resin allows peptide deprotection under mild, weakly acidic conditions. In peptide production using CTC resin, peptides with protecting groups that are easily removed under acidic conditions can be selectively deprotected without deprotecting the protecting groups, making it useful for producing peptides protected with the desired protecting groups (Non-Patent Document 7). On the other hand, in solid-phase peptide synthesis using CTC resin, peptides can be deprotected from CTC resin under mild conditions. Therefore, it has been reported that under condensation reaction conditions, the covalent bond between the amino acid or peptide supported on the CTC resin and the resin linker is cleaved, resulting in a decrease in the yield of the desired peptide (also known as premature cleavage, premature peptide release, or premature acidolytic cleavage) (Non-Patent Document 8). This document lists Gly, Pro, and Leu as examples of amino acids supported on CTC resin, and investigates the stability of the amino acid-supported CTC resin during condensation reactions with Fmoc-Gly-OH. As a result, it has been confirmed that when a condensation reaction is carried out using N,N'-diisopropylcarbodiimide as a condensing agent in combination with HOAt, Oxyma, or HOBt as an additive, the yield decreases, although to varying degrees, regardless of the type of supported amino acid or additive, and that the decrease in yield increases over time.

[0010] Regarding solid-phase synthesis using CTC resin, it has been reported that the condensation conditions of DIC / K-Oxyma significantly improved the yield compared to DIC / Oxyma, and provided a partial sequence of myelin basic protein (MBP) with equal or higher purity (Non-Patent Document 9).

[0011] However, it has been reported that in condensation reactions using DIC / K-Oxyma, epimerization at the α-position of the C-terminal carbonyl group progressed significantly even if the pretreatment time to generate the activated ester obtained from the amino acid and condensation agent was only a few minutes different. Therefore, in the solid-phase peptide synthesis method using K-Oxyma, considering the longer reagent addition time that accompanies scale-up, there are concerns that not only will the epimerization rate increase, but the purity may also decrease.

[0012] TOTU is used as a substitute reagent for DIC (Non-Patent Document 10), but because it requires basic conditions for the reaction, there is a risk of epimerization. Furthermore, TOTU is an expensive reagent, which increases production costs and is therefore undesirable. This document also describes cases of reduced yield due to premature cleavage and reduced purity due to the by-production of over-extensions. Specifically, it describes the production of a by-product with the sequence Gly-Gly-OCTC when Fmoc amino acid elongation was performed using DIC / HOBt with Gly-OCTC, a starting material in which Gly was supported on a solid-phase synthesis resin. This by-product is thought to be the result of depolymerization of the Gly residue supported on the CTC resin during the elongation reaction of the second amino acid, followed by condensation with unreacted Gly-OCTC.

[0013] Furthermore, it has been reported that the acid stability of peptides on the CTC resin increases when the second amino acid residue is extended from a Gly bound to the CTC resin. However, this contradicts the statement in Non-Patent Document 8 that the yield decreases with increasing time. In other words, an effective solution to the premature cleavage problem is still unknown. From the above, the first extension using the CTC resin, i.e., dipeptide synthesis, faces the challenges of reduced yield and reduced purity due to the by-production of an over-extended product in which excess supported amino acid residues are incorporated.

[0014] A method using Oxyma as an additive in the condensation reaction of amino acids is known. It has been described that the use of Oxyma as an additive can suppress epimerization and improve the acylation rate (Non-Patent Document 11).

[0015] In Patent Documents 1, 2, 3, 4, 5, and 6, and Non-Patent Document 12, which will be described later, various condensing agents and additives are used in the production of peptides by condensation reactions. However, these documents do not describe problems in peptide condensation reactions, such as premature cleavage.

[0016] Patent Document 1 relates to a novel peptide cyclization method and novel peptides and libraries containing them, which provide an effective drug discovery method for tough targets that have traditionally been difficult to develop drugs for. This document includes examples in which reagents are used in specific ratios, but there is no mention of the reaction conditions being set with the aim of resolving issues specific to peptide condensation reactions, such as premature cleavage.

[0017] Patent Document 2 describes a solid-phase peptide synthesis method using CTC resin. The document only describes that previous production methods resulted in side reactions due to the atypical conformation that occurs during peptide chain elongation, and that the production method was improved to resolve this issue. Furthermore, because the amino acids that make up peptides do not include unnatural amino acids such as N-substituted amino acids, there is no mention of problems such as incomplete condensation reactions due to special amino acid structures, or of solutions to problems related to condensation reaction problems.

[0018] In Patent Document 3, solid-phase synthesis of peptides using Rink'samide resin is carried out by first carrying out a pretreatment step (also called preactivation) in which the C-terminal amino acid is converted into an activated ester, and then adding the solid-phase synthesis resin to the reaction solution that has undergone the pretreatment step. However, Patent Document 3 only describes the production of a peptide with a specific sequence (AMG416 or a salt thereof), and does not disclose any improvement in the elongation reaction of low-reactivity amino acids, such as the condensation reaction between amino acids with large steric hindrance.

[0019] Patent Document 4 describes an improvement in the Fmoc removal reaction. However, it does not describe the problems specific to peptide condensation reactions, such as premature cleavage. Furthermore, this document requires a large excess of amino acids, making it unsuitable for producing peptides containing unnatural amino acids, such as N-substituted amino acids, which are more difficult to obtain than natural amino acids.

[0020] Patent Document 5 describes a gentle de-resination step that avoids damage to peptides containing acid-labile N-substituted amino acids in solid-phase synthesis, and a protecting group for a side chain functional group that is not removed in this de-resination step. However, it does not describe issues specific to peptide condensation reactions, such as premature cleavage.

[0021] Patent Document 6 describes a method for treating a deprotecting agent in the deprotection step of solid-phase synthesis, but does not describe problems specific to peptide condensation reactions, such as premature cleavage.

[0022] Non-Patent Document 12 describes a solid-phase method that uses an environmentally friendly alternative solvent instead of DMF or DCM, which are commonly used in solid-phase methods. However, it does not describe the condensation reaction of highly sterically hindered amino acids, such as N-substituted amino acids, α,α-disubstituted amino acids, or β-branched amino acids.

[0023] As described above, in peptide production, the yield of condensation reactions and the decrease in peptide purity due to side reactions have been known, but recently, issues specific to peptide condensation reactions, such as premature cleavage, have become known. Previously, it was only known that when Gly, Pro, and Leu are supported on a solid-phase synthesis resin, the bond between the amino acid and the solid-phase synthesis resin is cleaved during the condensation reaction, and further, when a natural amino acid into which a protecting group has been introduced is condensed with Gly-OCTC supported on a solid-phase synthesis resin, an over-extended product with a sequence different from the target amino acid sequence is obtained. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] WO2013100132 [Patent Document 2] WO2011006644 [Patent Document 3] WO2015154031 [Patent Document 4] WO2017070512 [Patent Document 5] WO2018225851 [Patent Document 6] WO2019117274 [Non-patent literature]

[0025] [Non-Patent Document 1] Future Med. Chem., 2009, 1,1289-1310. [Non-patent document 2] Acc Chem. Res., 2008, 41,1331-1342. [Non-patent document 3] Angew. Chem. Int. Ed., 2013, 52,254-269. [Non-patent document 4] Amino Acids, Peptides and Proteins in Organic Chemistry: Building Blocks, Catalysis and CouplingChemistry, Volume 3, 2011. [Non-Patent Document 5] Amino Acids, 2018, 50, 39-68. [Non-patent document 6] Solid phase peptide synthesis (published by Bachem) [Retrieved May 28, 2020], Internet <URL:https: / / www.bachem.com / fileadmin / user_upload / pdf / Catalogs_Brochures / Solid_Phase_Peptide_Synthesis.pdf> [Non-Patent Document 7] QSAR Comb. Sci., 2007, 26,1027-1035. [Non-patent document 8] ACS Comb. Sci., 2013, 15,229-234. [Non-Patent Document 9] Eur. J. Org. Chem. 2013,6372-6378. [Non-Patent Document 10] Side reactions in PeptideSynthesis, 2015, 1-31, Academic Press. [Non-Patent Document 11] Chem. Eur. J. 2009, 15,9394-9403. [Non-Patent Document 12] Green. Chem., 2019, 21,2594-2600. Summary of the Invention [Problem to be solved by the invention]

[0026] The present inventors applied the condensation reaction conditions of the solid-phase method commonly used in conventional peptide synthesis to the production of peptide compounds containing unnatural amino acids using CTC resin, but encountered problems such as incomplete condensation and a significant decrease in yield and purity due to premature cleavage. They found that such problems during condensation reactions are not limited to the condensation reactions between natural α-amino acids described in Non-Patent Documents 8, 9, and 10, but are also significant in sequences with significant steric hindrance and poor condensation reactivity, such as N-substituted amino acids and amino acids with a branch at the β-position of the side chain (also known as β-branched amino acids). The present invention aims to provide a method for producing high-purity peptide compounds in high yields using condensation conditions that suppress premature cleavage, particularly in the condensation reaction step, and also suppress side reactions such as incomplete condensation and competitive epimerization. Furthermore, the present invention aims to provide a method for producing high-purity peptide compounds in high yields using readily available condensing agents and additives under condensation conditions that are applicable to unnatural amino acids, the amounts of which are preferably reduced due to their scarcity. [Means for solving the problem]

[0027] To solve the above-mentioned problems, the present inventors have intensively investigated 1+1 residue synthesis, in which a two-residue peptide is obtained by condensing a second amino acid with a first amino acid, primarily an N-substituted amino acid, supported on a solid-phase synthesis resin. In condensation reactions using a carbodiimide compound, such as DIC, a condensing agent commonly used in peptide synthesis, and an N-hydroxy compound, such as Oxyma, an additive, the commonly used conditions are that the amino acid to be introduced, the condensing agent, and the additive be used in the same equivalent ratio. However, these commonly used conditions did not solve the problems specific to condensation reactions of peptides, such as premature cleavage. After extensive research to solve these problems, the present inventors investigated changing the equivalent ratios of the condensing agent and additive to the first amino acid or peptide and / or the second amino acid or peptide. As a result, they discovered a method that solves the problems specific to condensation reactions of peptides, such as premature cleavage.

[0028] Specifically, we have discovered a method for condensing a sequence that is difficult to amidate due to significant steric hindrance, specifically an N-substituted amino acid, an α,α-disubstituted amino acid, a β-branched amino acid, or an amino acid with a bulky side chain, to elongate the amino acid, using a condensing agent and additive in a molar ratio calculated based on the first amino acid or peptide and / or the second amino acid or peptide. In one aspect, we have found that the method of the present invention solves problems specific to peptide condensation reactions, such as premature cleavage. Furthermore, in one aspect, we have found that the method of the present invention can produce a peptide compound having a desired sequence. Furthermore, we have found that the method of the present invention can perform the desired condensation reaction in high yield. Furthermore, we have found that the method of the present invention can suppress side reactions, such as epimerization. Furthermore, in one aspect, we have found that the method of the present invention can produce the desired peptide compound in high yield and with high purity, since the amidation reaction proceeds with a sufficient conversion rate.

[0029] Specifically, we found that changing the molar ratio of the additive to the amino acid or peptide (i.e., the second amino acid or peptide) added to the N-terminus is effective, and that using a small amount of additive relative to the amino acid or peptide added to the N-terminus is particularly effective. We also found that changing the molar ratio of the condensing agent is also effective, and that using an excess of the condensing agent relative to the amino acid or peptide added to the N-terminus is particularly effective. We also found that changing the molar ratio of both the condensing agent and the additive is even more effective. We found that the method of the present invention allows the amidation reaction to proceed with sufficient conversion, and allows the desired peptide compound to be obtained in high yield and high purity, not only in the condensation reaction of amino acids or peptides with large steric hindrance, but also in the condensation reaction of amino acids or peptides with small steric hindrance.

[0030] That is, the present invention includes the following in one non-limiting specific embodiment. [1] A method for producing a peptide compound, comprising a step of condensing a first amino acid or peptide with a second amino acid or peptide in the presence of an additive and a condensing agent to obtain a condensate, The method, wherein the number of moles of the additive is less than the number of moles of the second amino acid or peptide. [2] The method according to [1], wherein the molar ratio of the additive to the second amino acid or peptide is 0.8 or less. [3] The method according to [1] or [2], wherein the molar ratio of the condensing agent to the second amino acid or peptide is 1.0 or more. [4] The method according to any one of [1] to [3], wherein the molar ratio of the condensing agent to the second amino acid or peptide is 1.2 to 4.0. [5] The method according to any one of [1] to [4], wherein the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 10 or less. [6] The method according to any one of [1] to [5], wherein the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 1 to 2, and the molar ratio of the additive to the second amino acid or peptide is 0.7 or less. [7] The method according to any one of [1] to [6], wherein the molar ratio (first molar ratio) of the second amino acid or peptide to the first amino acid or peptide is 2 or more, and the molar ratio of the additive to the second amino acid or peptide is the first molar ratio minus 1 or less. [8] The method according to any one of [1] to [7], wherein the molar ratio of the additive to the first amino acid or peptide is 2.0 or less. [9] The method according to any one of [1] to [8], wherein the molar ratio of the condensing agent to the first amino acid or peptide is 1.3 or more.

[10] The method according to [1], wherein the molar ratio of the second amino acid or peptide, the condensing agent, and the additive is second amino acid or peptide:condensing agent:additive=about 2:about 4-6:about 1.

[11] The method according to [1], wherein the molar ratio of the first amino acid or peptide, the second amino acid or peptide, the condensing agent, and the additive is first amino acid or peptide:second amino acid or peptide:condensing agent:additive = about 1:about 2:about 4:about 1, about 1:about 2.4:about 7.2:about 1.2, or about 1:about 3:about 6:about 1.5.

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

[11] , wherein the additive is Oxyma, HOBt, HOOBt, or HOAt.

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

[12] , wherein the condensing agent is DIC, DCC, EDCI, or EDCI·HCl.

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

[13] , wherein the additive is Oxyma.

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

[14] , wherein the peptide compound is the condensate or contains the condensate in its structure.

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

[15] , which is carried out by a solid phase method.

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

[16] , wherein the first amino acid or peptide is supported on a resin for solid phase synthesis.

[18] The method according to

[17] , wherein the resin for solid phase synthesis is a trityl-based resin.

[19] The method according to

[18] , wherein the trityl-based resin is a CTC resin, an Mmt resin, or an Mtt resin.

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

[19] , wherein the amino group of the second amino acid or peptide is protected with a protecting group.

[21] The method according to

[20] , wherein the protecting group is a protecting group having an Fmoc skeleton.

[22] The production method according to any one of [1] to

[21] , wherein the step is carried out in a solvent selected from the group consisting of DMF, NMP, DMI, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, dimethyl carbonate, and acetonitrile.

[23] The method according to any one of

[17] to

[22] , further comprising a step of removing the resin for solid phase synthesis.

[24] The method according to any one of

[20] to

[23] , further comprising a step of removing a protecting group.

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

[24] , wherein the first amino acid, or the N-terminal amino acid of the first peptide, and / or the C-terminal amino acid of the first peptide is an N-alkyl amino acid.

[26] The method according to

[24] , wherein the first amino acid or the C-terminal amino acid of the first peptide is an N-alkyl β-amino acid.

[27] The method according to

[25] , wherein the first amino acid, or the N-terminal amino acid of the first peptide, and / or the C-terminal amino acid of the first peptide is represented by the following formula: [ka] During the ceremony, When the first amino acid or the N-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 11 is hydrogen, and when the C-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 11 means the bond point with the adjacent amino acid, P 11 is hydrogen or C1-C6 alkyl, and R 12 is hydrogen, CONR 12A R 12B , COOR 12c , C1-C6 alkyl, C3-C8 cycloalkyl, C7-C 14 aralkyl, 5-10 membered heteroaryl C1-C6 alkyl, protected 5-10 membered heteroaryl C1-C6 alkyl, C1-C6 hydroxyalkyl, protected C1-C6 hydroxyalkyl, C1-C6 carboxyalkyl, protected C1-C6 carboxyalkyl, C1-C6 aminoalkyl, protected C1-C6 aminoalkyl, C1-C6 alkylthio C1-C6 alkyl, or P 11 and R 12 is P 11 and the nitrogen atom to which R is attached. 12 forms a 4- to 7-membered saturated heterocyclic ring together with the carbon atom to which it is attached, Q 12 is hydrogen or C1-C6 alkyl R 12A and R 12B are independently C1-C4 alkyl; or R 12A and R 12B together with the nitrogen atom to which they are attached form a 4- to 8-membered ring which may contain one or more additional heteroatoms; L 11 is a single bond or -CH2-, When the first amino acid or the C-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 13 means a binding point with a resin for solid phase synthesis, and when the N-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 13 indicates the point of attachment to the adjacent amino acid.

[28] The method according to

[27] , wherein the first amino acid, or the N-terminal amino acid of the first peptide, and / or the C-terminal amino acid of the first peptide is MeAsp-pip, MeAsp-aze, MeAsp-pyrro, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-OtBu, MeAsp-NMe2, MeVal, MeGly, MeAla, MeLeu, D-3-MeAbu, bMeAla, MeIle, MeGly(cPent), MeChg, MePhe, MeTrp(Boc), MeThr(Bzl), MeGlu(OtBu), MeLys(Boc), MeMet, Aze(2), or Aib.

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

[28] , wherein the second amino acid or the C-terminal amino acid of the second peptide is an α,α-disubstituted amino acid, a β-branched amino acid, an N-alkyl amino acid, or any other amino acid having a side chain with two or more carbon atoms.

[30] The method according to

[29] , wherein the β-branched amino acid is represented by the following formula: [ka] During the ceremony, P21 is hydrogen or C1-C6 alkyl, R 21 and R 22 are each independently C1-C4 alkyl, C1-C6 alkoxy, or C1-C6 alkoxyC1-C6 alkyl, or R 21 and R 22 together with the carbon to which they are attached form a 3- to 8-membered alicyclic ring, When the second amino acid is an amino acid represented by formula (2A), R 23 means the point of attachment of the amino group to the protecting group, and when the C-terminal amino acid of the second peptide is an amino acid represented by formula (2A), R 23 indicates the point of attachment to the adjacent amino acid.

[31] The method according to

[30] , wherein the β-branched amino acid is MeVal, D-MeVal, Val, Ile, MeIle, MeChg, Chg, MeGly(cPent), Gly(cPent), MeGly(cBu), Gly(cBu), MeGly(cPr), Gly(cPr), MeThr(tBu), or Thr(tBu).

[32] The method according to

[29] , wherein the α,α-disubstituted amino acid is represented by the following formula: [ka] During the ceremony, P 22 is hydrogen or C1-C6 alkyl, R 23 and R 24 is C1-C6 alkyl, C2-C6 alkenyl, or optionally substituted C7-C 14 aralkyl; R 23 and R 24 form, together with the carbon atoms to which they are attached, a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring; When the second amino acid is an amino acid represented by formula (2B), R 25means the point of attachment of the amino group to the protecting group, and when the C-terminal amino acid of the second peptide is an amino acid represented by formula (2B), R 25 indicates the point of attachment to the adjacent amino acid.

[33] The method according to

[32] , wherein the α,α-disubstituted amino acid is Aib, (Me)Abu, (Me)Leu, (Me)Algly, (Me)Phe, (Me)Phe(3-I), 1-ACPrC, cVal, cLeu, cHex, or Athpc.

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

[33] , wherein the second amino acid or the C-terminal amino acid of the second peptide is an N-alkyl amino acid.

[35] The method according to

[34] , wherein the N-alkylamino acid is MeAsp-pip, MeAsp-aze, MeAsp-pyrro, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-OtBu, D-3-MeAbu, bMeAla, MeGly, MeAla, MeLeu, MePhe, Aze(2), Pro, MeAsp-NMe2, MeVal, MeIle, MeChg, MeGly(cPent), MeGly(cBu), MeGly(cPr), MeThr(tBu), D-MeVal, MeTrp(Boc), MeThr(Bzl), MeGlu(OtBu), MeLys(Boc), or MeMet.

[36] The method according to

[29] , wherein the other amino acid having a side chain with two or more carbon atoms is Lys (Z), Glu (OBzl), or Ser (tBu).

[37] A step of obtaining a peptide compound by the method according to any one of [1] to

[36] ; and cyclizing the peptide compound. A method for producing a cyclic peptide compound, comprising: [Effects of the Invention]

[0031] The present invention enables efficient production of peptide compounds containing sequences with poor reactivity in condensation reactions. Specifically, it enables improved productivity, including improved yield and purity of peptide compounds. In other words, in one aspect, the method of the present invention not only inhibits premature cleavage, but also enables production of peptide compounds containing amino acid sequences for which condensation reactions are incomplete when conventional methods are applied, or peptide compounds containing amino acid sequences for which epimerization is antagonized, thereby improving the yield and / or purity of peptide compounds. Furthermore, in one aspect, the method of the present invention minimizes side reactions even when the condensation reaction is prolonged, and / or allows the reaction to be completed using a minimal amount of amino acid or peptide added to the N-terminus, thereby reducing the amount of the amino acid or peptide used. The method of the present invention completes the reaction even with reduced amounts of reagents, eliminating the need for excessive amounts of reagents or repeated double coupling under the same conditions, as in conventional methods. As a result, the amounts of reagents can be minimized even when using amino acids or peptides that are difficult to amidate. This method is particularly suitable for synthesizing peptide compounds containing expensive and rare unnatural amino acid residues. In one aspect, the method of the present invention reduces the amount of solvent used during condensation compared to conventional methods, has a significant effect of promoting the reaction when the reaction is carried out at a high concentration, and can also reduce the amount of amino acid used, thereby reducing the amount of solvent required not only during condensation but also for washing reagents after condensation.

[0032] Furthermore, when amino acids are loaded onto various resins for solid-phase synthesis in amounts exceeding those optimized in conventional methods, it is known that the condensation reaction does not go to completion or side reactions become significant. However, even when such amounts are used, the reaction can be completed by applying one embodiment of the present invention. Therefore, the amount of peptide that can be produced in a single production run using the solid-phase method can be increased.

[0033] The present invention enables the condensation reaction between an amino acid or peptide supported on a trityl resin (e.g., CTC resin) and an amino acid or peptide to be elongated to be carried out while sufficiently consuming the amino acid or peptide supported on the resin, while solving problems specific to condensation reactions of peptides, such as premature cleavage, and therefore enables the starting material to be efficiently converted into the target product. DETAILED DESCRIPTION OF THE INVENTION

[0034] The abbreviations used in this specification are listed below. A%:area% Alloc: Allyloxycarbonyl Aze: Azetidino Boc: tert-butoxycarbonyl Cbz: benzyloxycarbonyl COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate CTC: 2-chlorotrityl CTC resin: Cl-Trt(2-Cl) resin DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane DIC: N,N'-diisopropylcarbodiimide DMA: N,N-dimethylacetamide DMF: N,N-dimethylformamide DMI: 1,3-dimethyl-2-imidazolidinone EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Fmoc: 9-fluorenylmethyloxycarbonyl HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HMDS: 1,1,1,3,3,3-hexamethyldisilazane HOAt: 1-hydroxy-7-azabenzotriazole HOBt: 1-hydroxybenzotriazole HPLC: High-performance liquid chromatography iPr2NEt: N,N-diisopropylethylamine K-Oxyma: Ethyl (hydroxyimino)cyanoacetate, potassium salt MeCN: acetonitrile MeOH: Methanol 2-MeTHF: 2-methyltetrahydrofuran Mmt resin: 4-methoxytrityl chloride resin Mor: Morpholino MTBE: Methyl tert-butyl ether Mtt resin: 4-methyltrityl chloride resin NMP: N-methyl-2-pyrrolidone Oxyma: Ethyl (hydroxyimino)cyanoacetate PDA: Photodiode array detector Pip: Piperidino Pyrro: Pyrrolidino t-Bu: t-butyl Teoc: 2-(trimethylsilyl)ethoxycarbonyl TFA: Trifluoroacetic acid TMSOTf: Trimethylsilyl trifluoromethanesulfonate TMU: Tetramethylurea TOTU:O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0035] The abbreviations for β-branched amino acids, α,α-disubstituted amino acids, and N-alkyl amino acids used herein and their structure relationships are shown below. In the table below, each amino acid is listed in a form in which the amino group is protected with an Fmoc group. However, the relationship between the abbreviations for each amino acid and its residue with a free amino group after removal of the Fmoc group and their structure can also be understood from the table below. Specifically, it is obvious to those skilled in the art that, for example, MeAsp(OH)-pip is an amino acid having the following structure obtained by removing the Fmoc group from Fmoc-MeAsp(OH)-pip in the table below, and the structure of the amino acid residue, MeAsp-pip, is also obvious to those skilled in the art. [ka] [Table 1-1] [Table 1-2]

[0036] (Definition of functional groups, etc.) As used herein, the term "halogen atom" includes, for example, F, Cl, Br, or I.

[0037] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Specific examples of alkyl include alkyls having 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q " means that the number of carbon atoms is p to q), and preferably C1-C 10Alkyl is preferably C1-C6 alkyl. Specific examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc.

[0038] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP 2 Alkenyl is a monovalent group having 2-4 carbon atoms. Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be Entgegen (E) or Zusammen (Z), cis or trans. Alkenyl includes not only straight chains but also branched chains. Alkenyl is preferably C2-C 10 Alkenyl, more preferably C2-C6 alkenyl, is exemplified, and specific examples include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, etc.

[0039] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Alkynyl is preferably C2-C 10Alkynyl, more preferably C2-C6 alkynyl, is included, and specific examples include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, 3-methyl-(5-phenyl)-4-pentynyl, and the like.

[0040] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferred examples of cycloalkyl include C3-C8 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, and spiro[3.3]heptyl.

[0041] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C 10 Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

[0042] As used herein, the term "heterocyclyl" refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The heterocyclyl may have a double and / or triple bond in the ring, and a carbon atom in the ring may be oxidized to form a carbonyl. The heterocyclyl may be a monocyclic or condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), more preferably 4 to 7 (4- to 7-membered heterocyclyl). Specific examples of heterocyclyl include azetidinyl, oxetanyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2- Examples include thiazinane, thiadiazolidinyl, azetidinyl, oxazolidone, benzodioxanyl, benzoxazolyl, dioxolanyl, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 3-oxa-8-azabicyclo[3.2.1]octanyl, sultam, and 2-oxaspiro[3.3]heptyl.

[0043] As used herein, "heteroaryl" refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. The ring may be a single ring or a condensed ring with other rings, and may be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0044] As used herein, "alkoxy" refers to an oxy group bonded to an "alkyl" as defined above, and preferably includes C1-C6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0045] As used herein, "alkylthio" refers to a thio group bonded to an "alkyl" as defined above, and preferably includes C1-C6 alkylthio. Specific examples of alkylthio include methylthio, ethylthio, 1-propylthio, 2-propylthio, n-butylthio, i-butylthio, s-butylthio, and t-butylthio.

[0046] As used herein, "amino" refers to -NH2 in a narrow sense and -NRR' in a broad sense, where R and R' are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R and R' together with the nitrogen atom to which they are attached form a ring. Preferred amino groups include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, and 4- to 8-membered cyclic amino.

[0047] As used herein, "acyl (alkanoyl)" refers to a group in which a carbonyl group is bonded to hydrogen or the aforementioned "alkyl," and is preferably C1-C6 acyl, more preferably C2-C4 acyl. Specific examples of acyl include formyl, acetyl, propionyl, and butanoyl.

[0048] As used herein, "cycloalkoxy" refers to an oxy group bonded to a "cycloalkyl" as defined above, and preferably includes C3-C8 cycloalkoxy. Specific examples of cycloalkoxy include cyclopropoxy, cyclobutoxy, cyclopentyloxy, etc.

[0049] As used herein, "alkylsulfonyl" refers to a sulfonyl group having an "alkyl" bonded thereto, as defined above, and preferably includes C1-C6 alkylsulfonyl. Specific examples of alkylsulfonyl include methylsulfonyl.

[0050] As used herein, "hydroxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been replaced with hydroxyl groups, and C1-C6 hydroxyalkyl is preferred. Specific examples of hydroxyalkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 2-hydroxy-2-methylpropyl, and 5-hydroxypentyl.

[0051] As used herein, "carboxyalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been replaced with a carboxyl group, and C1-C6 carboxyalkyl is preferred. Specific examples of carboxyalkyl include carboxymethyl, 1-carboxyethyl, and 2-carboxyethyl.

[0052] As used herein, the term "protected carboxyalkyl" refers to a group in which the carboxyl group included in the above-defined "carboxyalkyl" is protected with any protecting group. Specific examples of the protecting group for the carboxyl group include a methyl group, an ethyl group, a t-Bu group, a benzyl group, a trityl group, a cumyl group, a methoxytrityl group, a 2-(trimethylsilyl)ethyl group, a 2,2,2-trichloroethyl group, and an allyl group.

[0053] As used herein, "aminoalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "amino" as defined above, and C1-C6 aminoalkyl is preferred. Specific examples of aminoalkyl include 1-pyridylmethyl, 2-(1-piperidyl)ethyl, 3-(1-piperidyl)propyl, and 4-aminobutyl.

[0054] As used herein, the term "protected aminoalkyl" refers to a group in which the amino group included in the above-defined "aminoalkyl" is protected with any protecting group. Specific examples of the amino protecting group include Fmoc, Boc, Cbz, Alloc, Teoc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, tosyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, and 2,4-dinitrobenzenesulfonyl.

[0055] As used herein, "haloalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" defined above have been substituted with halogen atoms, preferably C1-C6 haloalkyl, more preferably C1-C6 fluoroalkyl. Specific examples of haloalkyl include difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, and 5,5-difluoropentyl.

[0056] As used herein, "alkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkoxy" as defined above, with C1-C6 alkoxyC1-C6 alkyl being preferred, and C1-C6 alkoxyC1-C2 alkyl being more preferred. Specific examples of alkoxyalkyl include methoxymethyl, ethoxymethyl, 1-propoxymethyl, 2-propoxymethyl, n-butoxymethyl, i-butoxymethyl, s-butoxymethyl, t-butoxymethyl, pentyloxymethyl, 3-methylbutoxymethyl, 1-methoxyethyl, 2-methoxyethyl, and 2-ethoxyethyl.

[0057] As used herein, "alkylthioalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkylthio" as defined above, with C1-C6 alkylthioC1-C6 alkyl being preferred, and C1-C6 alkylthioC1-C2 alkyl being more preferred. Specific examples of alkylthioalkyl include methylthiomethyl, ethylthiomethyl, 1-propylthiomethyl, 2-propylthiomethyl, n-butylthiomethyl, i-butylthiomethyl, s-butylthiomethyl, t-butylthiomethyl, 1-methylthioethyl, and 2-ethylthioethyl.

[0058] As used herein, "haloalkoxy" refers to a group in which one or more hydrogen atoms of the "alkoxy" defined above have been substituted with halogen atoms, and C1-C6 haloalkoxy is preferred. Specific examples of haloalkoxy include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, and 2,2,2-trifluoroethoxy.

[0059] As used herein, "haloacyl (haloalkanoyl)" refers to a group in which a carbonyl group is bonded to the "haloalkyl," and preferably includes C2-C6 haloacyl, more preferably C2-C4 haloacyl. Specific examples of haloacyl include trifluoroacetyl, trichloroacetyl, pentafluoropropionyl, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)propionyl, and 3,3,3-trifluoro-2-(trifluoromethyl)propionyl.

[0060] As used herein, "cycloalkylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with a "cycloalkyl" as defined above, with C3-C8 cycloalkylC1-C6 alkyl being preferred, and C3-C6 cycloalkylC1-C2 alkyl being more preferred. Specific examples of cycloalkylalkyl include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0061] As used herein, "cycloalkoxyalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with a "cycloalkoxy" as defined above, with C3-C8 cycloalkoxyC1-C6 alkyl being preferred, and C3-C6 cycloalkoxyC1-C2 alkyl being more preferred. Specific examples of cycloalkoxyalkyl include cyclopropoxymethyl and cyclobutoxymethyl.

[0062] As used herein, "alkylsulfonylalkyl" refers to a group in which one or more hydrogen atoms of an "alkyl" as defined above are substituted with an "alkylsulfonyl" as defined above, with C1-C6 alkylsulfonylC1-C6 alkyl being preferred, and C1-C6 alkylsulfonylC1-C2 alkyl being more preferred. Specific examples of alkylsulfonylalkyl include methylsulfonylmethyl and 2-(methylsulfonyl)ethyl.

[0063] As used herein, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is substituted with an "aryl" as defined above, and is a C7-C 14 Aralkyl is preferred, C7-C 10 Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0064] As used herein, the term "heteroarylalkyl" refers to a group in which at least one hydrogen atom of an "alkyl" as defined above is substituted with a "heteroaryl" as defined above, preferably a 5- to 10-membered heteroaryl C1-C6 alkyl, and more preferably a 5- to 10-membered heteroaryl C1-C2 alkyl. Specific examples of heteroarylalkyl include 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.

[0065] As used herein, the term "protected heteroarylalkyl" refers to a group in which one or more functional groups included in the above-defined "heteroarylalkyl," such as an amino group, are protected with any protecting group. Specific examples of the protecting group include Fmoc, Boc, Cbz, Alloc, Teoc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, tosyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, and 2,4-dinitrobenzenesulfonyl.

[0066] As used herein, the term "protecting group for a carboxyl group" includes alkyl ester type protecting groups, benzyl ester type protecting groups, substituted alkyl ester type protecting groups, etc. Specific examples of the protecting group for a carboxyl group include a methyl group, an ethyl group, a t-Bu group, a benzyl group, a trityl group, a cumyl group, a methoxytrityl group, a 2-(trimethylsilyl)ethyl group, a 2,2,2-trichloroethyl group, an allyl group, etc.

[0067] As used herein, the term "amino group-protecting group" includes carbamate-type protecting groups, amide-type protecting groups, imide-type protecting groups, sulfonamide-type protecting groups, etc. Specific examples of amino group-protecting groups include Fmoc, Boc, Cbz, Alloc, Teoc, trifluoroacetyl, pentafluoropropionyl, phthaloyl, tosyl, 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, etc.

[0068] As used herein, "alicyclic ring" refers to a non-aromatic hydrocarbon ring. The alicyclic ring may have an unsaturated bond within the ring, or may be a polycyclic ring having two or more rings. Furthermore, the carbon atoms constituting the ring may be oxidized to form a carbonyl. Preferred examples of the alicyclic ring include 3- to 8-membered alicyclic rings, and specific examples include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a cyclooctane ring, and a bicyclo[2.2.1]heptane ring.

[0069] As used herein, the term "saturated heterocycle" refers to a non-aromatic heterocycle containing 1 to 5 heteroatoms in addition to carbon atoms and no double and / or triple bonds within the ring. The saturated heterocycle may be a monocycle or may form a condensed ring with another ring, for example, an aromatic ring such as a benzene ring. Preferred examples of the saturated heterocycle include 4- to 7-membered saturated heterocycles, such as an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydropyran ring, a morpholine ring, a thiomorpholine ring, a pyrrolidine ring, a 4-oxopyrrolidine ring, a piperidine ring, a 4-oxopiperidine ring, a piperazine ring, a pyrazolidine ring, an imidazolidine ring, an oxazolidine ring, an isoxazolidine ring, a thiazolidine ring, an isothiazolidine ring, a thiadiazolidine ring, a sazolidone ring, a dioxolane ring, a dioxane ring, a thietane ring, an octahydroindole ring, and an indoline ring.

[0070] As used herein, the term "peptide" refers to a peptide in which one or more natural amino acids and / or unnatural amino acids are linked via amide bonds and / or ester bonds. The peptide preferably contains 1 to 15 amino acid residues, and more preferably 5 to 12 amino acid residues.

[0071] The "peptide compound" used herein is not particularly limited as long as it is a peptide compound in which natural amino acids and / or unnatural amino acids are linked by amide bonds or ester bonds, but is preferably a peptide compound having 5 to 30 residues, more preferably 8 to 15 residues, and even more preferably 9 to 13 residues. The peptide compound synthesized in the present invention preferably contains at least three N-substituted amino acids, and more preferably at least five or more N-substituted amino acids, in one peptide. These N-substituted amino acids may be present consecutively or discontinuously in the peptide compound. The peptide compound of the present invention may be linear or cyclic, with cyclic peptide compounds being preferred.

[0072] As used herein, a "cyclic peptide compound" refers to a cyclic peptide compound obtainable by cyclizing a group at the N-terminus and a group at the C-terminus of a linear peptide compound. The cyclization may be via any form, such as cyclization via a carbon-nitrogen bond such as an amide bond, cyclization via a carbon-oxygen bond such as an ester bond or an ether bond, cyclization via a carbon-sulfur bond such as a thioether bond, cyclization via a carbon-carbon bond, or cyclization via a heterocyclic ring structure. Among these, 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 carboxyl group in a side chain and an amino group in the N-terminal main chain is more preferred. The position of the carboxyl group or amino group used for cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is located in a position that allows cyclization.

[0073] "Cyclization" of a peptide compound refers to the formation of a cyclic moiety containing four or more amino acid residues. The number of amino acids contained in the cyclic moiety of a cyclic peptide compound herein is not particularly limited, but examples include 4 to 20 residues, 5 to 15 residues, and 6 to 13 residues. A linear peptide compound can be converted into a cyclic peptide compound by carrying out an intramolecular bond formation reaction using a method described in, for example, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (by R.C. Larock) or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition (by M.B. Smith and J. March). After the bond formation reaction, a functional group transformation reaction can also be carried out. Examples of bond-forming reactions include C(O)-N bonds formed between carboxylic acids and amines, COC bonds, C(O)-O bonds, C(S)-O bonds using oxygen atoms, C(O)-S bonds, C(S)-S bonds, CSSC bonds, CSC bonds, CS(O)-C bonds, CS(O2)-C bonds, and CNC bonds, C=NC bonds, NC(O)-N bonds, NC(S)N bonds, and C(S)-N bonds using nitrogen atoms. Further examples include C-C bond formation reactions using transition metals, such as the Suzuki reaction, the Heck reaction, and the Sonogashira reaction. Examples of functional group transformation reactions that can be performed after a bond-forming reaction include oxidation reactions and reduction reactions. Specifically, a reaction that oxidizes a sulfur atom to convert it to a sulfoxide group or a sulfone group is an example. Another example is a reduction reaction that reduces a triple or double bond in a carbon-carbon bond to convert it to a double or single bond. When two amino acids are bonded together at the backbone of the amino acid, a closed ring structure is formed by a peptide bond, but a covalent bond between two amino acids may also be formed by bonding between the side chains of the two amino acids, or between the side chain and the backbone, etc.

[0074] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means from one to the maximum number of substituents permitted by that group. Specific examples of "one or more" include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or more.

[0075] As used herein, the term "solid-phase synthesis resin" is not particularly limited as long as it can be used in the synthesis of peptide compounds by solid-phase synthesis. Specific examples of such solid-phase synthesis resins include CTC resin, NovaSyn TGT resin (TGT resin), Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt resin), which are removable under acidic conditions. The resin can be selected appropriately depending on the functional group of the amino acid used. For example, when a carboxyl group (main chain carboxyl group or side chain carboxyl group, such as Asp or Glu) or a hydroxy group on an aromatic ring (phenol group, such as Tyr) is used as the functional group of the amino acid, trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin) is preferably used as the resin. When an aliphatic hydroxy group (an aliphatic alcohol group such as Ser or Thr) is used as the functional group of the amino acid, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin), or 4-methyltrityl chloride resin (Mtt resin). Note that in this specification, resin may also be referred to as resin.

[0076] The type of polymer constituting the resin is not particularly limited. In the case of a resin made of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The cross-linking rate is also not particularly limited, but 1% DVB (divinylbenzene) cross-linking is preferred. Examples of the type of polymer constituting the resin include TentaGel (registered trademark) and ChemMatrix (registered trademark).

[0077] In the preparation of the compounds described herein, if a defined group undergoes an undesired chemical transformation under the conditions of the method, the compound can be prepared by, for example, using means such as protection and deprotection of the functional group. Here, the selection and deprotection of the protecting group can be performed using, for example, the methods described in "Greene's, 'Protective Groups in Organic Synthesis' (5th ed., John Wiley & Sons 2014)," which can be used appropriately depending on the reaction conditions. Furthermore, the order of reaction steps such as the introduction of substituents can also be changed as necessary.

[0078] In the present specification, when the modifier "optionally substituted" is used, examples of the substituent include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, alkoxycarbonyl, formyl, and the like.

[0079] Furthermore, each of these may be given a substituent, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom. Examples of such substituents include optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl.

[0080] The compounds described herein can be their salts or solvates. Examples of compound salts include hydrochlorides, hydrobromides, hydroiodides, phosphates, phosphonates, sulfates, sulfonates such as methanesulfonates and p-toluenesulfonates, carboxylates such as acetates, citrates, malates, tartrates, succinates, and salicylates, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, and ammonium salts such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts. These salts can be produced, for example, by contacting the compound with an acid or a base. A solvate of a compound refers to a phenomenon in which solute molecules attract solvent molecules in a solution to form a molecular cluster, and when the solvent is water, it is called a hydrate. The compounds described herein may be solvated with a single solvent or multiple solvents selected from organic solvents such as alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), dimethylformamide, or diglyme, or water.

[0081] As used herein, "amino acid" includes natural amino acids and unnatural amino acids (sometimes referred to as amino acid derivatives). Furthermore, as used herein, "amino acid" may refer to amino acid residues. As used herein, "natural amino acids" refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Examples of unnatural amino acids (amino acid derivatives) include, but are not limited to, β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids with side chains different from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids may have any stereoconfiguration. The side chain of an amino acid is not particularly limited and may be freely selected from, in addition to a hydrogen atom, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-linked cycloalkyl groups. Each of these may have a substituent, and the substituents are not limited, and may be independently selected from any substituents containing, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. Examples of such substituents include optionally substituted alkyl groups, alkoxy groups, alkoxyalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., as well as oxo, aminocarbonyl, and halogen atoms. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxyl group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the definition of amino acids).

[0082] Halogen-derived substituents include fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), and the like.

[0083] Substituents derived from O atoms include hydroxy (-OH), oxy (-OR), carbonyl (-C=OR), carboxyl (-COH), 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)-COH).

[0084] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, and the like.

[0085] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0086] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0087] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0088] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0089] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0090] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, groups in which the H atom bonded to the N atom in -C=O-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0091] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, the H atom bonded to the N atom in -NH-C=OR may be further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0092] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0093] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0094] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, groups 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 are also included.

[0095] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO-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.

[0096] Substituents derived from S atoms include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), sulfo (-SO3H), and pentafluorosulfanyl (-SF5).

[0097] Examples of thio (-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0098] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0099] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0100] Substituents derived from the N atom include azido (-N3, also referred to as "azido 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'').

[0101] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0102] Examples of tertiary amino (—NR(R′)) include an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring.

[0103] Examples of substituted amidino (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0104] Examples of substituted guanidino (-NR-C(=NR''')-NR'R'') include groups in which R, R', R'', and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0105] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0106] Examples of substituents derived from the B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents, R and R', are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or may form a ring. Specific examples include cyclic boryl groups, and more specific examples include pinacolatoboryl, neopentanediolateboryl, and catecholateboryl groups.

[0107] Specific examples of the substituent on the nitrogen atom of the N-substituted amino acid herein include alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, C7-C 14 Examples include aralkyl, benzyl, and phenethyl.

[0108] The main chain amino group of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR, where R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline). Such amino acids in which the main chain amino group is substituted may be referred to as "N-substituted amino acids" in the present specification. As used herein, the "N-substituted amino acids" preferably include N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, N-C7-C8 alkyl amino acids, N-methyl amino acids, N-C8-C9 alkyl amino acids, N-methyl amino acids, N-C9-C10 alkyl amino acids, N-methyl amino acids, N-C1-C12 alkyl amino acids, N-methyl amino acids, N-C1-C14 alkyl amino acids, N-methyl ... 14 Examples include, but are not limited to, aralkyl amino acids, N-benzyl amino acids, and N-phenethyl amino acids.

[0109] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes included in "amino acids" as used herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Includes Cl etc.

[0110] In this specification, the use of "to" indicating a numerical range includes both ends of the range. For example, "A to B" means a numerical range that is equal to or greater than A and equal to or less than B.

[0111] As used herein, the term "about" when used in conjunction with a numerical value means a range of values ​​of plus and minus 10% of that numerical value.

[0112] As used herein, the term "and / or" includes any combination of "and" and "or." Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, (vii) A, B, and C.

[0113] (Manufacturing method) The present invention relates to a method for producing a peptide compound, which comprises a step of condensing a first amino acid or peptide with a second amino acid or peptide in the presence of an additive and a condensing agent to obtain a condensate, wherein the number of moles of the additive is less than the number of moles of the second amino acid or peptide.

[0114] In one embodiment, the peptide compound obtained by the production method of the present invention may be an intermediate or final product of an amino acid or peptide elongation step by a solid-phase or liquid-phase method. The elongation step can be performed multiple times depending on the length of the amino acid sequence of the desired peptide compound, and the condensation step according to the present invention can be included at least once during such an elongation step, and may be included multiple times. During the elongation step, condensation steps other than the condensation step according to the present invention can be performed using methods known in the art.

[0115] In one embodiment, when the condensation step of the present invention is used as the final step of the elongation step, the condensate obtained by the condensation step of the present invention can be a peptide compound having a desired sequence. On the other hand, when a peptide compound having a desired sequence is obtained by further elongation by a known method in addition to the condensation step of the present invention, the condensate obtained by the condensation step of the present invention is included in the peptide compound as a partial structure thereof.

[0116] In the present invention, the "first amino acid" may be a natural amino acid or an unnatural amino acid.

[0117] In the present invention, a "first peptide" may be composed of only natural amino acids, only unnatural amino acids, or any combination of natural and unnatural amino acids.

[0118] In some embodiments, the first amino acid or first peptide is preferably an amino acid, more preferably an unnatural amino acid.

[0119] In some embodiments, the first amino acid or first peptide may be an amino acid or peptide in which the main chain amino group is not protected, preferably at the N-terminus of the first amino acid or first peptide.

[0120] When a solid-phase synthesis method is used for the condensation reaction of the present invention, the first amino acid or the C-terminal amino acid of the first peptide is supported on a resin for solid-phase synthesis. In this case, if the N-terminal and / or C-terminal amino acids of the first amino acid or the first peptide are unnatural amino acids, particularly N-substituted amino acids such as N-alkylamino acids, the desired condensation reaction may not proceed sufficiently using conventional methods due to premature cleavage. This is particularly noticeable when a trityl-based atomic group is used in the resin linker that connects the solid-phase synthesis resin and the amino acid. Even in such cases, the condensation reaction can proceed efficiently using the method of the present invention.

[0121] In one embodiment, when the first amino acid or the amino acid at the N-terminus and / or C-terminus of the first peptide is an N-alkyl amino acid, examples of the N-alkyl amino acid include those having a side chain different from that of a natural amino acid, and α, β, or γ amino acids, with N-alkyl β amino acids being preferred examples.

[0122] Specific examples of the first amino acid, or the N-terminal amino acid of the first peptide and / or the C-terminal amino acid of the first peptide include those having the following formula (1): [ka]

[0123] When the first amino acid or the N-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 11 On the other hand, when the C-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 11 means a bonding point with an adjacent amino acid, and usually, the amino acid of formula (1) forms an amide bond with the adjacent amino acid at this site.

[0124] In one embodiment, in formula (1), P 11 is hydrogen or C1-C6 alkyl, and R 12 is hydrogen, CONR12A R 12B , COOR 12c , C1-C6 alkyl, C3-C8 cycloalkyl, C7-C 14 aralkyl, 5-10 membered heteroaryl C1-C6 alkyl, protected 5-10 membered heteroaryl C1-C6 alkyl, C1-C6 hydroxyalkyl, protected C1-C6 hydroxyalkyl, C1-C6 carboxyalkyl, protected C1-C6 carboxyalkyl, C1-C6 aminoalkyl, protected C1-C6 aminoalkyl, C1-C6 alkylthio C1-C6 alkyl.

[0125] P 11 is preferably hydrogen or methyl, more preferably methyl.

[0126] R 12 CONR 12A R 12B If R 12A and R 12B are independently C1-C4 alkyl (preferably methyl, ethyl), or R 12A and R 12B together with the nitrogen atom to which they are attached to form a 4- to 8-membered ring (preferably a piperidine ring, an azetidine ring, a pyrrolidine ring, a morpholine ring, or a 3-oxa-8-azabicyclo[3.2.1]octane ring) which may contain one or more additional heteroatoms.

[0127] R 12 COOR 12C If R 12C is allyl, t-butyl, or benzyl.

[0128] R 12 is C1-C6 alkyl, preferably R 12 is methyl, isopropyl, or isobutyl.

[0129] R 12 When is C3-C8 cycloalkyl, preferably R 12 is cyclopentyl or cyclohexyl.

[0130] R 12 C7-C 14 When R is aralkyl, it is preferably 12 is benzyl or phenethyl.

[0131] R 12 is 5-10 membered heteroaryl C1-C6 alkyl, preferably R 12 is a 5- to 10-membered heteroarylmethyl or a 5- to 10-membered heteroarylethyl. 12 is a protected 5-10 membered heteroarylC1-C6 alkyl, R 12 is preferably one in which a functional group contained in the above group, for example, an amino group, is protected with a Boc group.

[0132] R 12 is C1-C6 hydroxyalkyl, preferably R 12 is hydroxymethyl, 1-hydroxyethyl, or 2-hydroxyethyl, and 1-hydroxyethyl is more preferred. 12 is a protected C1-C6 hydroxyalkyl, R 12 is preferably one in which the above group is protected with a hydroxyl-protecting group, such as a Bzl group or a tBu group.

[0133] R 12 is C1-C6 carboxyalkyl, preferably R 12 is carboxymethyl, 1-carboxyethyl, or 2-carboxyethyl, and 2-carboxyethyl is more preferred. 12 is a protected C-C carboxyalkyl, R 12 is preferably one in which the above group is protected with a carboxyl-protecting group, such as a Bzl group or a tBu group.

[0134] R 12 is C1-C6 aminoalkyl, preferably R 12 is 4-aminobutyl. 12 is a protected C1-C6 aminoalkyl, R 12is preferably one in which the above group is protected with an amino-protecting group, for example, a Boc group.

[0135] R 12 is C1-C6 alkylthioC1-C6 alkyl, preferably R 12 is 2-methylthioethyl.

[0136] In one embodiment, P 11 and R 12 is P 11 and the nitrogen atom to which R is attached. 12 forms a 4- to 7-membered saturated heterocyclic ring together with the carbon atom to which it is bonded. The 4- to 7-membered saturated heterocyclic ring is preferably an azetidine ring, a pyrrolidine ring, a piperidine ring, a piperazine ring, or a morpholine ring.

[0137] In formula (1), Q 12 is hydrogen or C1-C6 alkyl, preferably hydrogen or methyl.

[0138] In formula (1), L 11 is a single bond or -CH2-.

[0139] When the first amino acid or the C-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 13 On the other hand, when the N-terminal amino acid of the first peptide is an amino acid represented by formula (1), R 13 means a bonding point with an adjacent amino acid, and usually, the amino acid of formula (1) forms an amide bond with the adjacent amino acid at this site.

[0140] More specific examples of the first amino acid or the amino acid at the N-terminus and / or C-terminus of the first peptide include MeAsp-pip, MeAsp-aze, MeAsp-pyrro, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-OtBu, MeAsp-NMe2, MeVal, MeGly, MeAla, MeLeu, D-3-MeAbu, bMeAla, MeIle, MeGly(cPent), MeChg, MePhe, MeTrp(Boc), MeThr(Bzl), MeGlu(OtBu), MeLys(Boc), MeMet, Aze(2), and Aib.

[0141] According to the condensation step of the present invention, even when a first amino acid or peptide is supported on a solid-phase synthesis resin at such a high loading (for example, 0.5 mmol / g or more based on Fmoc quantification) that conventional methods would cause the peptide to fall off from the resin, a condensate can be obtained while maintaining the loading. Therefore, in the method of the present invention, the amount of the first amino acid or peptide supported on the resin for solid-phase synthesis can be any amount, for example, an amount of 0.2 mmol / g or more and 0.8 mmol / g or less based on the Fmoc quantification method, and the reaction can proceed efficiently even with a high supported amount of 0.3 mmol / g or more, or even 0.5 mmol / g or more.

[0142] In the present invention, the "second amino acid" may be a natural amino acid or an unnatural amino acid.

[0143] In some embodiments, the second amino acid or peptide may be an amino acid or peptide in which the main chain carboxyl group is not protected, preferably at the C-terminus of the second amino acid or peptide.

[0144] In the present invention, the "second peptide" may be composed of only natural amino acids, only unnatural amino acids, or any combination of natural and unnatural amino acids.

[0145] In some embodiments, when the second amino acid or the C-terminal amino acid of the second peptide is an unnatural amino acid, particularly an N-substituted amino acid such as an N-alkylamino acid, or an amino acid with two or more carbon atoms in its side chain (e.g., an α,α-disubstituted amino acid or a β-branched amino acid), the condensation reaction may not proceed sufficiently using conventional methods. Even in such cases, efficient condensation is possible using the method of the present invention.

[0146] In the present invention, the amino group of the second amino acid or peptide is preferably protected with a protecting group, such as those described above as "amino group-protecting groups," specifically, protecting groups having an Fmoc skeleton, Cbz, Boc, Teoc, and Alloc.

[0147] In the present invention, the term "protecting group having an Fmoc skeleton" refers to an Fmoc group or a group in which an arbitrary substituent has been introduced at an arbitrary position of the structural skeleton of the Fmoc group. Specific examples of such a protecting group having an Fmoc skeleton include protecting groups represented by the following formula: [ka] (In the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl).

[0148] More specific examples of the protecting group having an Fmoc skeleton include a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, a 1-methyl-Fmoc (Fmoc(1Me)) group, a 2-fluoro-Fmoc (Fmoc(2F)) group, a 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, a 2-monoisooctyl-Fmoc (mio-Fmoc) group, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, and a 2,7-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc Examples include a (tdf-Fmoc) group, a 2,7-bis(trimethylsilyl)-Fmoc (Fmoc(2TMS)) group, a (2-sulfo-9H-fluoren-9-yl)methoxycarbonyl group (Fmoc(2so3h)), a [(1S)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (sm-Fmoc), and a [(1R)-1-(9H-fluoren-9-yl)ethoxy]carbonyl group (rm-Fmoc).

[0149] When the second amino acid or the C-terminal amino acid of the second peptide is a β-branched amino acid, specific examples of the β-branched amino acid include those having the following formula (2A): [ka]

[0150] In formula (2A), is hydrogen or C1-C6 alkyl. 21 When is C1-C6 alkyl, it is preferably methyl.

[0151] In formula (2A), R 21 and R 22 are each independently C1-C4 alkyl, C1-C6 alkoxy, or C1-C6 alkoxyC1-C6 alkyl, or R 21 and R 22 R together with the carbon to which they are attached form a 3- to 8-membered alicyclic ring. 21 and R 22is C1-C4 alkyl, C1-C6 alkoxy, or C1-C6 alkoxyC1-C6 alkyl, R 21 and R 22 is preferably methyl, ethyl, or tert-butoxy, and R 21 and R 22 Preferred combinations include methyl and methyl, methyl and ethyl, and methyl and tert-butoxy. R 21 and R 22 When these are taken together to form a 3- to 8-membered alicyclic ring, the 3- to 8-membered alicyclic ring is preferably a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, or a cyclohexane ring.

[0152] When the second amino acid is an amino acid represented by formula (2A), R 23 On the other hand, when the C-terminal amino acid of the second peptide is an amino acid represented by formula (2A), R 23 means a bonding point with an adjacent amino acid, and usually, the amino acid of formula (2A) forms an amide bond with the adjacent amino acid at this site.

[0153] More specific examples of such a second amino acid or a β-branched amino acid at the C-terminus of the second peptide include MeVal, D-MeVal, Val, Ile, MeIle, MeChg, Chg, MeGly(cPent), Gly(cPent), MeGly(cBu), Gly(cBu), MeGly(cPr), Gly(cPr), MeThr(tBu), and Thr(tBu).

[0154] When the second amino acid or the C-terminal amino acid of the second peptide is an α,α-disubstituted amino acid, specific examples of the α,α-disubstituted amino acid include those having the following formula (2B): [ka]

[0155] In formula (2B), P22 is hydrogen or C1-C6 alkyl.

[0156] In formula (2B), R 23 and R 24 is C1-C6 alkyl, C2-C6 alkenyl, or optionally substituted C7-C 14 aralkyl, or R 23 and R 24 together with the carbon atoms to which they are attached form a 3- to 8-membered alicyclic ring or a 4- to 7-membered saturated heterocyclic ring. R 23 and R 24 When R is C1-C6 alkyl, it is preferably methyl, ethyl, or isopropyl; 23 and R 24 is C-C alkenyl, it is preferably allyl, and R 23 and R 24 C7-C 14 When R is aralkyl, it is preferably benzyl optionally substituted with halogen. 23 and R 24 Preferred combinations are methyl and methyl, methyl and ethyl, methyl and isopropyl, methyl and isobutyl, methyl and allyl, and methyl and benzyl which may be substituted with halogen. Also R 23 and R 24 When R form a 3- to 8-membered alicyclic ring together with the carbon atom to which they are attached, the alicyclic ring is preferably a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, or a cyclohexane ring, and R 23 and R 24 When these are taken together with the carbon atom to which they are attached to form a 4- to 7-membered saturated heterocycle, the saturated heterocycle is preferably a tetrahydropyran ring.

[0157] When the second amino acid is an amino acid represented by formula (2B), R 25 On the other hand, when the C-terminal amino acid of the second peptide is an amino acid represented by formula (2B), R 25means a bonding point with an adjacent amino acid, and usually, the amino acid of formula (2B) forms an amide bond with the adjacent amino acid at this site.

[0158] More specific examples of such a second amino acid or an α,α-disubstituted amino acid at the C-terminus of the second peptide include Aib, (Me)Abu, (Me)Leu, (Me)Algly, (Me)Phe, (Me)Phe(3-I), 1-ACPrC, cVal, cLeu, cHex, and Athpc.

[0159] In certain embodiments, the second amino acid or the C-terminal amino acid of the second peptide is an N-alkyl amino acid, specifically, MeAsp-pip, MeAsp-aze, MeAsp-pyrro, MeAsp-mor, MeAsp-mor(26-bicyc), MeAsp-OtBu, D-3-MeAbu, bMeAla, MeGly, MeAla, MeLeu, MePhe, Aze(2), Pro, MeAsp-NMe2, MeVal, MeIle, MeChg, MeGly(cPent), MeGly(cBu), MeGly(cPr), MeThr(tBu), D-MeVal, MeTrp(Boc), MeThr(Bzl), MeGlu(OtBu), MeLys(Boc), or MeMet, and preferably MeVal or MeIle.

[0160] In some embodiments, the second amino acid or the C-terminal amino acid of the second peptide may be an amino acid having two or more carbon atoms in its side chain that is not a β-branched amino acid or an α,α-disubstituted amino acid, such as Lys (Z), Glu (OBzl), or Ser (tBu).

[0161] In the present invention, the second amino acid or peptide is used in an equal or excess amount relative to the first amino acid or peptide. Specifically, the molar ratio of the second amino acid or peptide to the first amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the group consisting of 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 with an upper limit selected from the group consisting of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. As used herein, the term "lower limit" encompasses both "greater than" and "greater than," while the term "upper limit" encompasses both "less than" and "less than." The molar ratio of the second amino acid or peptide to the first amino acid or peptide is preferably 1.5 or greater, more preferably 2 or greater. The molar ratio of the second amino acid or peptide to the first amino acid or peptide is preferably 8 or less, more preferably 4 or less. Most preferably, the molar ratio of the second amino acid or peptide to the first amino acid or peptide is about 2.

[0162] The additive used in the condensation reaction of the present invention includes Oxyma, HOBt, HOOBt, HOAt, and the like.

[0163] In the present invention, the additive is used in a molar amount less than that of the second amino acid or peptide. In other words, in the present invention, the molar ratio of the additive to the second amino acid or peptide is less than 1. Preferably, the molar ratio is 0.8 or less, for example, 0.1 to 0.8. In this case, the molar ratio of the additive to the second amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 with an upper limit selected from the group consisting of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8. More preferably, the molar ratio of the additive to the second amino acid or peptide is 0.3 to 0.7.

[0164] In one embodiment, the molar ratio of the additive to the second amino acid or peptide is preferably 0.7 or less when the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 1 to 2. Specifically, when the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 1 to 2, the molar ratio of the additive to the second amino acid or peptide can be in the range of 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less. Alternatively, in the present invention, when the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 1 to 2, the molar ratio of the additive to the second amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 with an upper limit selected from the group consisting of 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7.

[0165] In another embodiment, when the molar ratio of the second amino acid or peptide to the first amino acid or peptide (this molar ratio may be referred to as the "first molar ratio") is 2 or more, the molar ratio of the additive to the second amino acid or peptide is preferably "(first molar ratio)-1" or less. Specifically, when the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 2 or more, the molar ratio of the additive to the second amino acid or peptide can be in the range of (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -1 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -2 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -3 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -4 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -5 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -6 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -7 or less, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -8 or less, or (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -9 or less. Alternatively, in the present invention, when the molar ratio of the second amino acid or peptide to the first amino acid or peptide is 2 or more, the molar ratio of the additive to the second amino acid or peptide is (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -9, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -8, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -7, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -6, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -5, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -4, (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -3, and (the molar ratio of the second amino acid or peptide to the first amino acid or peptide) -1. and an upper limit selected from the group consisting of (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -8, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -7, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -6, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -5, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -4, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -3, (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -2, and (molar ratio of the second amino acid or peptide to the first amino acid or peptide) -1.

[0166] In some embodiments, the molar ratio of the additive to the first amino acid or peptide can be 0.5 to 2.0. Specifically, the molar ratio of the additive to the first amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the group consisting of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9 with an upper limit selected from the group consisting of 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0. A molar ratio of about 1 is particularly preferred for the additive to the first amino acid or peptide.

[0167] Condensing agents used in the condensation reaction of the present invention include DIC, DCC, EDCI, EDCI·HCl, and the like.

[0168] In the present invention, the condensing agent is used in an amount equal to or greater than the number of moles of the second amino acid or peptide. Specifically, in the present invention, the molar ratio of the condensing agent to the second amino acid or peptide can be in the range of 1 or more, 2 or more, 3 or more, or 4 or more. More specifically, the molar ratio of the condensing agent to the second amino acid or peptide can be in the range of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0, with the lower limit selected from the group consisting of 1.1, 1.2, 1.3, 1.4 , 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0. Preferred ranges for the molar ratio of the condensing agent to the second amino acid or peptide include 1.0 to 5.0, 1.2 to 4.0, 1.2 to 3.0, and 2.0 to 3.0.

[0169] In some embodiments, the condensing agent is used in an equal or excess amount relative to the first amino acid or peptide. Specifically, for example, the molar ratio of the condensing agent to the first amino acid or peptide may be in the range of 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 2.1 or more, 2.2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, 2.7 or more, 2.8 or more, or 2.9 or more. Range, 3.0 or more, 3.1 or more, 3.2 or more, 3.3 or more, 3.4 or more, 3.5 or more, 3.6 or more, 3.7 or more, 3.8 or more, 3.9 or more, 4.0 or more, 4.1 or more, 4.2 or more, 4.3 or more, 4.4 or more, 4.5 or more, 4.6 or more, 4.7 or more, 4.8 or more, 4.9 or more, 5.0 or more, 5.1 or more, 5.2 or more, 5.3 or more Range, 5.4 or higher, 5.5 or higher, 5.6 or higher, 5.7 or higher, 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, 6.5 or higher, 6.6 or higher, 6.7 or higher, 6.8 or higher, 6.9 or higher, 7.0 or higher, 7.1 or higher, 7.2 or higher, 7.3 or higher, 7.4 or higher, 7.5 or higher, 7.6 or higher, 7.7 or higher and above, 7.8 and above, 7.9 and above, 8.0 and above, 8.1 and above, 8.2 and above, 8.3 and above, 8.4 and above, 8.5 and above, 8.6 and above, 8.7 and above, 8.8 and above, 8.9 and above, 9.0 and above, 9.1 and above, 9.2 and above, 9.3 and above, 9.4 and above, 9.5 and above, 9.6 and above, 9.7 and above, 9.8 and above, or 9.9 and above. Alternatively, in the present invention, the molar ratio of the condensing agent to the first amino acid or peptide is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0 The lower limit and the following are the values: 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 10.10, 10.11, 10.12, 10.13, 10.14, 10.15, 10.16, 10.17, 10.18 The range can be specified by combining with an upper limit selected from the group consisting of 0.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. The molar ratio of the condensing agent to the first amino acid or peptide is preferably 1.3 or more, more preferably 2.0 or more. The molar ratio of the condensing agent to the first amino acid or peptide is preferably 10 or less, more preferably 8.0 or less. The molar ratio of the condensing agent to the first amino acid or peptide is most preferably about 4.

[0170] In the present invention, the molar ratio of the condensing agent and the additive to the second amino acid or peptide is the molar ratio of the condensing agent to the second amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the values ​​consisting of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 with an upper limit selected from the values ​​consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0; The molar ratio of the additive to the second amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the values ​​consisting of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, and 0.5 with an upper limit selected from the values ​​consisting of 0.6, 0.7, 0.8, 0.9, and 1.0. The molar ratio of the condensing agent and additive to the second amino acid or peptide is preferably second amino acid or peptide:condensing agent:additive=about 2:about 4-6:about 1.

[0171] In the present invention, the molar ratio of the second amino acid or peptide, the condensing agent, and the additive to the first amino acid or peptide is the molar ratio of the second amino acid or peptide to the first amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the values ​​consisting of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 with an upper limit selected from the values ​​consisting of 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0; the molar ratio of the condensing agent to the first amino acid or peptide can be within a range that can be specified by a combination of a lower limit selected from the group consisting of 1.0, 2.0, 3.0, and 4.0 and an upper limit selected from the group consisting of 5.0, 6.0, 7.0, 8.0, and 9.0; The molar ratio of the additive to the first amino acid or peptide can be within a range that can be specified by combining a lower limit selected from the values ​​consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 with an upper limit selected from the values ​​consisting of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The molar ratio of the first amino acid or peptide, the second amino acid or peptide, the condensing agent, and the additive is preferably first amino acid or peptide:second amino acid or peptide:condensing agent:additive=about 1:about 2:about 4:about 1, first amino acid or peptide:second amino acid or peptide:condensing agent:additive=about 1:about 2.4:about 7.2:about 1.2, or first amino acid or peptide:second amino acid or peptide:condensing agent:additive=about 1:about 3:about 6:about 1.5, and more preferably first amino acid or peptide:second amino acid or peptide:condensing agent:additive=about 1:about 2:about 4:about 1.

[0172] The condensation reaction of the present invention can be carried out at a reaction temperature of 0 to 100°C, preferably 5 to 60°C, and more preferably 10 to 40°C.

[0173] The condensation reaction of the present invention can be carried out for a reaction time of 10 minutes to 1 week, preferably 10 minutes to 3 days, and more preferably 1 hour to 2 days.

[0174] In the present invention, it is preferred that the additive is Oxyma and the condensing agent is DIC, DCC, EDCI, or EDCI·HCl.

[0175] In the present invention, it is more preferred that the additive is Oxyma and the condensing agent is DIC.

[0176] The condensation reaction of the present invention can be carried out in a suitable solvent. The solvent can be an aprotic solvent, and examples thereof include amide solvents, ester solvents, ether solvents, alkylnitrile solvents, and urea solvents. Examples of amide solvents include DMF, DMA, and NMP. Examples of ester solvents include ethyl acetate and dimethyl carbonate. Examples of ether solvents include tetrahydrofuran and 2-methyltetrahydrofuran. Examples of alkylnitrile solvents include acetonitrile. Examples of urea solvents include DMI and TMU.

[0177] The method of the present invention can be applied to both solid phase and liquid phase methods.

[0178] When the solid phase method is used, the method of the present invention can be carried out by contacting a first amino acid or peptide bound to a resin with a second amino acid or peptide, a condensing agent, and an additive. The order in which the first amino acid or peptide is contacted with the second amino acid or peptide, the condensing agent, and the additive is arbitrary, and the first amino acid or peptide may be contacted with the second amino acid or peptide, the condensing agent, and the additive simultaneously or sequentially. The first amino acid or peptide may be contacted with a premix of all or any of the second amino acid or peptide, the condensing agent, and the additive. When the first amino acid or peptide is contacted with the second amino acid or peptide, the condensing agent, and the additive, a mixture of these with an appropriate solvent may be used.

[0179] The method of the present invention may be carried out using a solid-phase synthesis apparatus. In some embodiments, the method of the present invention can be carried out by mixing a first amino acid or peptide bound to a resin with a second amino acid or peptide, a condensation agent, and an additive in an appropriate solvent. When mixing the solid-phase synthesis resin with these reagents, the resin can be swollen by contacting it with an appropriate solvent as a pretreatment, thereby efficiently promoting the desired condensation reaction. The amount of solvent used in this pretreatment can be any amount as long as the swollen resin is immersed in the solvent. For example, when DMF is used as the solvent, an amount of 3 v / w to 15 v / w, preferably 4 v / w to 10 v / w, and more preferably 4 v / w to 8 v / w can be used. A solvent amount of 4 v / w indicates that the amount of solvent is 4 ml per gram of resin.

[0180] After the reaction is complete, the reaction solution is discharged from the solid-phase synthesis apparatus, and the remaining solid-phase synthesis resin is washed with an appropriate solvent to remove excess reagents and by-products, thereby obtaining the target peptide compound bound to the solid-phase synthesis resin. Examples of solvents suitable for washing and / or swelling the solid-phase synthesis resin include amide-based and alcohol-based solvents, with DMF or 2-propanol being preferred. These solvents may be used multiple times or alternately. The swollen solid-phase synthesis resin can be shrunk as needed by washing with an alcohol-based or ether-based solvent. Methanol is a preferred alcohol-based solvent, and MTBE is a preferred ether-based solvent.

[0181] The present invention can improve the yield of the condensation reaction and the purity of the condensation product compared to conventional methods, even when premature cleavage does not occur, and therefore allows the use of any resin. Resin linkers that may undergo premature cleavage include those classified as acid-sensitive "H (<5% TFA in CHCl)" in the Novabiochem Handbook of Solid-Phase Synthesis. Examples of resins equipped with such resin linkers include solid-phase synthesis resins that use trityl-based atomic groups in the resin linker, such as 2-chlorotrityl chloride resin (CTC resin), TGT resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), and 4-methoxytrityl chloride resin (Mmt resin) (these resins are sometimes referred to as "trityl-based resins"). The present invention is particularly useful when using such resins.

[0182] The type of polymer constituting the solid-phase synthesis resin used in the present invention is not particularly limited, but a resin composed of polystyrene is preferred. Furthermore, the particle size of the solid-phase synthesis resin is preferably about 100-200 mesh or 200-400 mesh. The cross-linking rate of the resin is also not particularly limited, but a resin with 1% DVB (divinylbenzene) cross-linking is preferred. Examples of polymers constituting the resin include TentaGel (registered trademark) and ChemMatrix (registered trademark).

[0183] In one embodiment, a mixture of the second amino acid or peptide, the condensing agent, and the additive may be prepared in advance in a solvent before the condensation reaction. The mixing time is not particularly limited, but is preferably 0 minutes to 2 hours, more preferably 0 minutes to 1 hour, and even more preferably 30 minutes.

[0184] Stirring or shaking the resin using an automated synthesizer can be important for ensuring that the resin is fully permeated into the reaction solution and that the reaction proceeds as desired. The stirring speed, shaking rate, and frequency are not particularly limited. However, excessive stirring can cause physical damage to the resin, so stirring at 60 rpm for approximately 2 minutes every hour is recommended. Furthermore, stirring or shaking is not necessarily required if permeation is sufficient.

[0185] In some embodiments, the method of the present invention may further comprise a step of removing the resin for solid phase synthesis, which can be accomplished by a method known in the art. The peptide compound elongated to the desired sequence can be cleaved from the resin and isolated.

[0186] In some embodiments, the method of the present invention may further include a step of removing a protecting group, which can be achieved by methods known in the art. For example, the method described in "Greene's, 'Protective Groups in Organic Synthesis' (5th ed., John Wiley & Sons 2014)" can be used, and these methods may be used appropriately depending on the reaction conditions. Specifically, when the amino group of the second amino acid or the amino group of the N-terminal amino acid of the second peptide is protected with a protecting group, the protecting group can be removed to prepare for the next condensation reaction. The protecting group may be removed simultaneously with the condensation reaction or separately from the condensation reaction.

[0187] In one embodiment, the present invention also relates to a method for producing a cyclic peptide compound by obtaining a linear peptide compound using the method of the present invention and then cyclizing the N-terminal group and the C-terminal group of the linear peptide compound by known techniques such as those described in Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd Edition (by R.C. Larock) or March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th Edition (by M.B. Smith and J. March).

[0188] In one embodiment, the present invention also relates to a method for inhibiting premature cleavage using the method of the present invention.

[0189] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0190] The following examples were analyzed by one or more of Waters' HPLC methods AD.

[0191] Analysis: Waters HPLC (reaction conversion rate, purity) HPLC method A Instrument: ACQUITY UPLC H-Class Column: Ascentis Express C18 (2.7 μm, 4.6mm x 50 mm), Supelco Column temperature: 35 deg. Eluent: A) 0.05% TFA / water, B) 0.05% TFA / MeCN Gradient (B): 5% (0 min.) → 100% (4 min.) →100% (4.5 min.) → 5% (4.6 min.) → 5% (6 min.) Flow rate: 1.0 mL / min Detection: PDA 210 nm (200-400 nm PDAtotal) Injection volume: A-a) 0.30 μL, A-b) 1.0μL, A-c) 2.0 μL Sample preparation: A-a) 5 μL of supernatant / 995 μL MeCN forloading evaluation A-b) 5 μL of supernatant / 995 μL MeCN forloading evaluation A-c) 40 μL of supernatant / 960 μL MeCN forGly-cap evaluation

[0192] HPLC method B Instrument: ACQUITY UPLC H-Class Column: CAPCELL Core ADME (2.7 μm, 2.1 mm x50 mm) Column temperature: 50 deg. Eluent: A) 0.05% TFA / water, B) 0.05% TFA / MeCN Gradient (B): 30% (0 min.) → 70% (20.0min.) → 100% (20.1 min.) → 100% (22.0 min.) → 30% (22.1 min.) → 30% (24.0 min.) Flow rate: 0.3 mL / min Detection: PDA 210 nm (200-400 nm PDAtotal) Injection volume: 1.0 μL Sample preparation: 40 μL of supernatant / 960 μL MeCN

[0193] HPLC method C Instrument: ACQUITY UPLC H-Class Column: ACQUITY UPLC CSH Fluoro-Phenyl (1.7μm, 2.1 mm x 150 mm) Column temperature: 35 deg. Eluent: A) 0.1% formic acid / water, B) 0.1%formic acid / MeCN Gradient (B): 5% (0 min.) → 100% (15.0min.) → 100% (18.0 min.) → 5% (18.01 min.) → 5% (20.0 min.) Flow rate: 0.3 mL / min Detection: PDA 254 nm (200-380 nm PDAtotal) Injection volume: 2.0 μL Sample preparation: 0.37 mg / ml

[0194] HPLC method D Instrument: ACQUITY UPLC H-Class Column: Bioshell A160 peptide (2.7 μm, 2.1mm x 150 mm) Column temperature: 40 deg. Eluent: A) 0.05% TFA / water, B) 0.05%TFA / MeCN Gradient (B): 20% (0 min.) → 60% (20 min.)→ 100% (20.1 min.) → 100% (22.0 min.) → 100% (22.1 min.) → 20% (24 min.) Flow rate: 0.5 mL / min Detection: PDA 254 nm Injection volume: 2.0 μL Sample preparation: 5 μL of supernatant / 1000 μL MeCN

[0195] HPLC method E Instrument: Acquity UPLC / SQD2 Column: Ascentis Express C18 Column temperature: 35 deg. Eluent: A) 0.1% FA / water, B) 0.1%FA / acetonitrile Gradient (B): 5% (0 min.) → 100% (1.0 min.)→ 100% (1.4 min.) Flow rate: 1.0 mL / min Detection: PDA 210-400 nm(total)

[0196] HPLC method F Instrument: Acquity UPLC / SQD2 Column: Ascentis Express C18 Column temperature: 35 deg. Eluent: A) 0.1% FA / water, B) 0.1%FA / acetonitrile Gradient (B): 5% (0 min.) → 100% (4.5 min.)→ 100% (5.0 min.) Flow rate: 1.0 mL / min Detection: PDA 210-400 nm(total)

[0197] HPLC method G Instrument: Acquity UPLC / SQD2 Column: Ascentis Express C18 Column temperature: 35 deg. Eluent: A) 10mM AA / water, B) methanol Gradient (B): 50% (0 min.) → 100% (4.5min.) → 100% (5.0 min.) Flow rate: 1.0 mL / min Detection: PDA 210-400 nm(total)

[0198] HPLC method H Instrument: ACQUITY UPLC H-Class Column: Ascentis Express RP-amide (2.7 μm,2.1 mm x 50 mm), Supelco Column temperature: 35 deg. Eluent: A) 0.05% TFA / water, B) 0.05%TFA / MeCN Gradient (B): 5% (0 min.) → 100% (4 min.) →100% (4.5 min.) → 5% (4.6 min.) → 5% (6 min.) Flow rate: 0.5 mL / min Detection: PDA 210 nm (200-400 nm PDAtotal) Injection volume: E-a) 0.50 μL, E-b) 0.50μL, E-c) 1.0 μL Sample preparation: E-a) 5 μL of supernatant / 960 μL MeCN forGly-cap evaluation or purity evaluation

[0199] HPLC method I Instrument: ACQUITY UPLC H-Class Column: Ascentis Express RP-amide (2.7 μm,2.1 mm x 50 mm), Supelco Column temperature: 50 deg. Eluent: A) 0.05% TFA / water, B) 0.05%TFA / MeCN Gradient (B): 5% (0 min.) → 70% (20 min.) →70% (21 min.) → 100% (22 min.) → 100% (22.1 min.) → 5% (22.6 min.) → 5% (24.0min.) Flow rate: 0.5 mL / min Detection: PDA 210 nm (200-400 nm PDAtotal) Injection volume: 0.50 or 1.0 μL Sample preparation: 40 μL of supernatant / 960 μL MeCN

[0200] HPLC method J Instrument: ACQUITY UPLC H-Class Column: ACQUITY UPLC CSH Fluoro-Phenyl (1.7μm, 2.1 mm x 150 mm) Column temperature: 40 deg. Eluent: A) 0.05% TFA / water, B) 0.05%TFA / MeCN Gradient (B): 20% (0 min.) → 100% (20 min.) → 20% (20.1 min.) → 12% (24.0 min.) Flow rate: 0.3 mL / min Detection: PDA 254 nm (200-400 nm PDAtotal) Injection volume: 1.0 μL Sample preparation: 40 μL of supernatant / 960 μL MeCN

[0201] The following Fmoc amino acids were purchased commercially. [Table 1-3] [Table 1-4]

[0202] [Liquid phase synthesis experiment] [Starting material synthesis] (Starting material synthesis 1) Synthesis of Fmoc-MeAsp(OtBu)-pip (Compound 2) [ka] A 500 mL three-neck flask was charged with 26.9 g (63.2 mmol, 1.0 eq) of Fmoc-MeAsp(OtBu)-OH (compound 1), 9.23 g (75.9 mmol, 1.2 eq) of piperidine hydrochloride, 10.8 g (69.5 mmol, 1.1 eq) of Oxyma, and 135 mL of N,N-dimethylformamide, and the mixture was stirred. Using a dropping funnel, 12.2 mL (69.5 mmol, 1.1 eq) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added dropwise over 10 min at 0 °C. After the addition, the dropping funnel was rinsed twice with 1.25 mL of N,N-dimethylformamide. Five hours after the start of the reaction, 269 mL of 2-MeTHF was added, followed by 135 mL of 0.5 mol / L aqueous hydrochloric acid over 10 min. After the aqueous layer was discarded, 135 mL of 5% aqueous sodium carbonate solution was added to the organic layer. After discarding the aqueous layer, 135 mL of 10% aqueous sodium chloride solution was added to the organic layer. After discarding the aqueous layer, the organic layer was stored at room temperature overnight. Approximately 60 mL of organic solvent was removed by distillation under reduced pressure, and 81 mL of ethyl acetate was added to the concentrate at room temperature. 323 mL of heptane was added with stirring. The external temperature was set to 0°C, and after 1 hour and 20 minutes, 161 mL of heptane was added. The crystals were filtered and washed using a Kiriyama funnel, and then dried under reduced pressure at an external temperature of 40°C to obtain 28.2 g of a white solid (Compound 2).

[0203] [Table 2]

[0204] [Table 3]

[0205] (Starting material synthesis 2) Synthesis of Fmoc-MeAsp(OH)-pip (Compound 3) [ka] 1.58 g (3.20 mmol, 1.0 eq) of Fmoc-MeAsp(OtBu)-pip (compound 2) was weighed into a 100 mL three-neck flask. Under a nitrogen atmosphere, 2-MeTHF was added via a 9.5 mL syringe and stirred. 1.0 mL (4.8 mmol, 1.5 eq) of HMDS was then added via a syringe. The flask was immersed in an ice bath, and when the internal temperature reached 1°C, 0.79 mL (4.4 mmol, 1.4 eq) of TMSOTf was added via a syringe. Five minutes after the completion of the dropwise addition, the temperature was raised to room temperature. After one hour, 100% conversion was confirmed by HPLC (method Aa). The reaction vessel was again immersed in the ice bath, and when the internal temperature reached 5°C, 15 mL of 5% aqueous sodium carbonate solution was added. After the addition of the aqueous sodium carbonate solution, the reaction solution was stirred for several minutes, after which the stirring was stopped. The reaction solution was transferred to a 50 mL separatory funnel and separated, after which the aqueous layer was separated. The obtained aqueous layer was washed with 15 mL of MTBE. 15 mL of MTBE was added to the separated aqueous layer, followed by 1.1 mL of phosphoric acid (mass fraction 85% or more). The solution was shaken in the separatory funnel, followed by separation and the organic layer was separated. Magnesium sulfate was added to the obtained organic layer, and the magnesium sulfate was filtered. 1.5 mL of heptane was added to the filtrate, which was then concentrated under reduced pressure to obtain 1.42 g of a white solid (compound 3) (content not determined).

[0206] [Table 4]

[0207] [Table 5]

[0208] (Starting material synthesis 3) Synthesis of Fmoc-MeAsp(OH)-NMe2 (Compound 21) [ka]

[0209] Under a nitrogen stream, EDCI (16.9 g, 88.0 mmol) and DMF (144 mL) were added to a reaction vessel and cooled to 0 °C. HOBt (10.9 g, 80.6 mmol) and a solution of compound 19 (30.0 g, 73.3 mmol) synthesized according to the method described in WO2018 / 225864 in DCM / DMF (60 mL / 60 mL) were added sequentially at 0 °C and stirred for 30 minutes at 0 °C. Dimethylamine (2 mol / L THF solution, 40.5 mL, 80.6 mmol) was added dropwise at 0 °C, followed by stirring at 0 °C for 30 minutes. Ethyl acetate (300 mL) was added to the reaction mixture, and the organic layer was washed twice with 1 mol / L aqueous hydrochloric acid (240 mL), twice with water (300 mL), twice with saturated aqueous sodium bicarbonate / water (1 / 1, 300 mL), and once with saturated aqueous sodium chloride / water (1 / 1, 300 mL). The organic layer was then dried over sodium sulfate. After filtering the desiccant, the filtrate was concentrated under reduced pressure to give compound 20 (32.7 g, 102% yield) as pale brown amorphous crystals. LCMS(ESI) m / z = 437.2 (M+H) + Retention time: 0.86 minutes (Analysis conditions HPLC METHOD E)

[0210] Compound 20 (32.0 g, 73.3 mmol) was used as the starting material, and tetrakistriphenylphosphine palladium and DCM were mixed. Phenylsilane was then added dropwise, and the reaction mixture was stirred at room temperature for 30 minutes. The target product was extracted, and the solution containing the target product was concentrated to dryness, yielding 25.1 g (86% yield) of compound 21 as pale brown amorphous crystals. LCMS(ESI) m / z = 397.2 (M+H) + Retention time: 0.68 minutes (Analysis conditions HPLC METHOD E)

[0211] Cl-Trt(2-Cl) resin (1.25-1.60 mmol / g, 100-200 mesh, 1% DVB) was purchased from Watanabe Chemical Industry Co., Ltd. or SUNRESIN.

[0212] [Solid-phase synthesis experiments] In this specification, when a compound is bonded to a solid support, the polymer or resin portion may be represented by a ●. Furthermore, to clarify the reaction site with the solid support portion, the chemical structure of the linker, which is the reaction site, may be represented by connecting it to the ●. For example, in the following structure (Fmoc-MeAsp(O-Trt(2-Cl)-resin)pip (compound 3), the 2-chlorotrityl group of the solid support forms an ester bond with the side chain carboxy group of MeAsp. Note that pip refers to a piperidino group, and in the above structure, the C-terminal carboxy group forms an amide bond with piperidine. [ka]

[0213] [1 residue + 1 residue condensation experiment] In this specification, the amount of solvent used during solid-phase reactions is sometimes expressed as a double volume (v / w). The double volume is based on the mass of the Cl-Trt(2-Cl) resin used in the loading step. For example, in solid-phase raw material synthesis 1, if 40 mL of dichloromethane was used for 3.97 g of Cl-Trt(2-Cl) resin, then 40 / 3.97 ≒ 10 v / w. Furthermore, in the 1+1 residue condensation experiment, the resin intermediate obtained in the loading step was used in small aliquots. The solvent volume during the 1+1 residue condensation experiment was 4 v / w, which, when converted to volume, corresponds to 4 (mL / g) x aliquot resin (g) ÷ loaded resin (g) x loading step raw resin (g) = 4 (mL / g) x aliquot resin (g) / 5.79 x 3.97 mL.

[0214] In this specification, the purity of the solid-phase condensation reaction was evaluated by measuring the reaction solution after the de-resination reaction using the dried resin obtained after elongation by LC to confirm the purity of the compound supported on the solid-phase synthesis resin. The specific procedure is as follows: Approximately 20 mg of the dried resin after elongation was placed in a 5 mL disposable syringe with a filter, and dichloromethane was aspirated using a 1 mL disposable syringe and allowed to stand at room temperature for 15 minutes. The dichloromethane was then drained, and 0.20 mL of dichloromethane containing 1% TFA was added. The mixture was then shaken at room temperature for 30 seconds. The solution was then drained into a 1 mL vial. 40 μL of the drained solution was diluted with 960 μL of acetonitrile to prepare an LC sample solution.

[0215] In all examples other than Examples 2-2 to 2-11 (Example 2-1, Examples 2-12 to 2-31) herein, the amount of substance (mmol) of the target compound was calculated from the product of the Fmoc quantitative value (mmol / g), the dry resin weight (g), and the LC purity (area %), and the yield of the solid-phase condensation reaction was evaluated. The specific procedure for Fmoc quantification is as follows: Approximately 20 mg of dry resin was weighed into a 100 mL volumetric flask, and then a 20% piperidine solution in N,N-dimethylformamide was added to the flask to make up the volume. This solution was shaken at room temperature for 45 minutes. The absorbance of the solution (wavelength 289.80 nm, coefficient ε = 6089) was measured using a spectrophotometer (UV3900-01), and the amount of substance per unit weight of the Fmoc-containing compound supported on the resin was calculated using the following formula (Fmoc quantification): Molar concentration of the measurement solution (mol / L) = absorbance ÷ ε Fmoc quantitative value (mmol / g) = molar concentration of measurement solution (mol / L) ÷ measurement resin (g) × 100 (mL)

[0216] In Examples 2-2 to 2-11 herein, the following Fmoc quantification method was used: Fmoc Quantitation Method (Examples 2-2 to 2-11) Approximately 10 mg of commercially available Fmoc-Gly-OH was weighed into a 10 mL volumetric flask and then 4 mL of DMF was added. After shaking for 30 minutes at room temperature, 40 μL of DBU was added, and the solution was shaken for 15 minutes at room temperature. DMF was then added to the flask to bring the total volume to 10 mL. A sample solution was prepared from 30 μL of the supernatant and 4 mL of the DMF solution, and LC data was acquired (5 μL injection). Calibration curves were created at wavelengths of 294 nm and 304 nm from the acquired data.

[0217] After the reaction, approximately 10 mg of resin was weighed into a 10 mL volumetric flask and DMF (4 mL) was added. After shaking at room temperature for 30 minutes, DBU (40 μL) was added, and the solution was shaken at room temperature for 15 minutes. DMF was then added to make up to 10 mL. A sample solution was prepared from 80 μL of the supernatant and 920 μL of DMF solution, and LC data was acquired (5 μL injection). The amount of substance per unit weight of the compound bearing the Fmoc group supported on the resin was calculated from the area of ​​the acquired data at each wavelength and the calibration curve created, and the average value was used as the Fmoc quantitative value.

[0218] [Starting material synthesis] (Solid phase raw material synthesis 1) Immobilization of Fmoc-MeAsp(OH)-pip (compound 3) on Trt(2-Cl) resin [ka] 3.97 g (1.36 mmol / g, 5.40 mmol, 1.7 eq) of Cl-Trt(2-Cl) resin was weighed into a solid-phase reaction column, and 40 mL (10 v / w) of dichloromethane was added. The column was then left to stand at room temperature for 30 min. After the dichloromethane was drained, a solution of 1.40 g (3.20 mmol, 1.0 eq) of Fmoc-MeAsp(OH)-pip (compound 3) and 1.6 mL (9.0 mmol, 2.8 eq) of N,N-diisopropylethylamine in 30 mL (7.5 v / w) of dichloromethane was added to the reaction column at room temperature and shaken for 2 h. After draining the reaction solution, a solution of 3.2 mL (0.8 v / w) of methanol and 1.6 mL (0.4 v / w) of N,N-diisopropylethylamine in 27 mL (6.8 v / w) of N,N-dimethylformamide was added to the reaction column at room temperature. After shaking at room temperature for 2 hours, the reaction solution was drained. The resin was washed four times with 40 mL (10 v / w) of N,N-dimethylformamide, then twice with 40 mL (10 v / w) of 2-propanol, and four times with 40 mL (10 v / w) of methanol. It was then dried overnight under vacuum at room temperature to obtain 5.79 g of dried resin (compound 4). 19.4 mg of the resulting resin was weighed into a 100 mL volumetric flask, made up to volume with 20% piperidine in N,N-dimethylformamide, and shaken for 45 minutes at room temperature. Fmoc quantification was carried out from the absorbance of the solution using a spectrophotometer, and the yield was calculated from the obtained Fmoc quantification value, the mass of the dry resin, and LC A%.

[0219] [Table 6] * Two-step yield including t-Bu removal step ((Compound 2) is calculated as 100% content)

[0220] [Table 7] * De-resined compound 3 was analyzed to confirm the change in purity before and after loading.

[0221] (Solid phase raw material synthesis 2) Immobilization of Fmoc-MeAsp(OH)-NMe2 (compound 21) on Trt(2-Cl) resin [ka]

[0222] A reaction vessel equipped with a filter was charged with Cl-Trt(2-Cl) resin (1.60 mmol / g, 8.83 g, 14.1 mmol) and DCM (90 mL) and shaken at room temperature for 20 min. After removing the DCM under nitrogen pressure, a mixture of compound 21 (2.80 g, 7.06 mmol), methanol (2.29 mL, 56.5 mmol), DIPEA (5.91 mL, 33.9 mmol), and DCM (64 mL) was added to the reaction vessel and shaken at room temperature for 60 min. After removing the reaction mixture under nitrogen pressure, a mixture of methanol (9.17 mL, 226 mmol), DIPEA (5.91 mL, 33.9 mmol), and DCM (64 mL) was added to the reaction vessel and shaken at room temperature for 90 min. After removing the reaction mixture under nitrogen pressure, DCM (90 mL) was added and the mixture was shaken for 5 minutes, after which the reaction mixture was removed under nitrogen pressure. The resin was washed twice with DCM, and the resulting resin was dried overnight under reduced pressure to obtain 10.4 g of compound 22. The loading rate was calculated using the dried resin (11.04 mg) by Fmoc quantification to be 0.442 mmol / g (UVarea value at 294 nm: 4990.63, UVarea value at 304 nm: 4516.89).

[0223] (Solid phase raw material synthesis 3) Immobilization of Fmoc-D-3-MeAbu-OH (compound 23) on Trt(2-Cl) resin [ka]

[0224] A reaction vessel equipped with a filter was charged with Cl-Trt(2-Cl) resin (1.60 mmol / g, 25.0 g, 40.0 mmol) and DCM (125 mL) and shaken at room temperature for 20 min. After removing the DCM under nitrogen pressure, a mixture of Fmoc-D-3-MeAbu-OH (compound 23, 3.60 g, 10.6 mmol), methanol (0.859 mL, 21.2 mmol), and DIPEA (12.3 mL, 70.7 mmol) with DCM (total volume: 145 mL) was added to the reaction vessel and shaken at room temperature for 30 min. After removing the reaction mixture under nitrogen pressure, a mixture of methanol (12.5 mL, 143 mmol), DIPEA (12.5 mL, 71.8 mmol), and DCM (total volume: 250 mL) was added to the reaction vessel and shaken at room temperature for 90 min. After removing the reaction mixture under nitrogen pressure, DCM (180 mL) was added and the mixture was shaken for 5 minutes, after which the reaction mixture was removed under nitrogen pressure. The resin was washed with DCM three times, and the resulting resin was dried overnight under reduced pressure to obtain 28.3 g of compound 24. The loading rate was calculated using the dried resin (10.36 mg) by Fmoc quantification to be 0.369 mmol / g (UVarea value at 294 nm: 3920.38, UVarea value at 304 nm: 3530.84).

[0225] (Solid phase raw material synthesis 4) Immobilization of Fmoc-MeGly-OH (compound 25) on Trt(2-Cl) resin [ka]

[0226] A filter-equipped reaction vessel was charged with Cl-Trt(2-Cl) resin (1.60 mmol / g, 12.3 g, 19.7 mmol) and DCM (125 mL) and shaken at room temperature for 20 min. After removing the DCM under nitrogen pressure, a mixture of Fmoc-MeGly-OH (3.07 g, 9.87 mmol), DIPEA (8.25 mL, 47.4 mmol), and DCM (110 mL) purchased from a commercial supplier was added to the reaction vessel and shaken at room temperature for 60 min. After removing the reaction mixture under nitrogen pressure, a mixture of methanol (12.8 mL, 316 mmol), DIPEA (8.25 mL, 47.4 mmol), and DCM (110 mL) was added to the reaction vessel and shaken at room temperature for 90 min. After removing the reaction mixture under nitrogen pressure, DCM (180 mL) was added and the mixture was shaken for 5 minutes, after which the reaction mixture was removed under nitrogen pressure. The resin was washed twice with DCM, and the resulting resin was dried overnight under reduced pressure to obtain 22.2 g of compound 26. The loading rate was calculated using the dried resin (10.00 mg) by Fmoc quantification to be 0.573 mmol / g (UVarea value at 294 nm: 5879.66, UVarea value at 304 nm: 5289.40).

[0227] (Solid phase raw material synthesis 5) Immobilization of Fmoc-MeLeu-OH (compound 37) on Trt(2-Cl) resin [ka] A solid-phase column was loaded with 1.01 g (1.13 mmol / g, 1.14 mmol, 1.4 eq) of Cl-Trt(2-Cl) resin, and 10 mL (10 v / w) of dichloromethane was added. The column was then left to stand at room temperature for 30 minutes. After the dichloromethane was drained, a solution of 0.29 g (0.79 mmol, 1.0 eq) of Fmoc-MeLeu-OH (compound 1) and 0.40 mL (2.3 mmol, 2.9 eq) of N,N-diisopropylethylamine in 8.0 mL (8 v / w) of dichloromethane was added to the column at room temperature and shaken for 2 hours. After draining the reaction solution, a solution of 0.80 mL (0.8 v / w) of methanol and 0.40 mL (0.4 v / w) of N,N-diisopropylethylamine in 6.8 mL (6.8 v / w) of N,N-dimethylformamide was added to the reaction column at room temperature. After shaking at room temperature for 2 hours, the reaction solution was drained. The resin was washed four times with 10 mL (10 v / w) of N,N-dimethylformamide, then twice with 10 mL (10 v / w) of 2-propanol, and four times with 10 mL (10 v / w) of methanol. It was then dried overnight under vacuum at room temperature to yield 1.27 g of dried resin (compound 38). 17.7 mg of the resulting resin was weighed into a 100 mL volumetric flask, made up to volume with 20% piperidine in N,N-dimethylformamide, and shaken for 45 minutes at room temperature. Fmoc quantification was carried out from the absorbance of the solution using a spectrophotometer, and the yield was calculated from the obtained Fmoc quantification value, the mass of the dry resin, and LC A%.

[0228] [Table 8]

[0229] [Table 9]

[0230] The results of immobilizing the following commercially available amino acids on CTC resin under the same reaction conditions as above are shown below.

[0231] [Table 10]

[0232] In Examples 1, 2-1, and 2-12 to 2-31, the amino acids were elongated by using 2 equivalents, 4 equivalents, and 1 equivalent of Fmoc amino acid, DIC, and Oxyma, respectively, relative to the amino acid on the resin for solid phase synthesis (Example Condition A). [Example 1] (1 residue + 1 residue condensation experiment) Synthesis of Fmoc-MeVal-MeAsp(OCTC)-pip (Compound 5) (Reaction conditions: Fmoc amino acid:DIC:additive (Oxyma) = 2:4:1, relative to the amino acid of the resin; hereinafter referred to as Example Condition A) [ka] 3.01 g (0.525 mmol / g, 1.57 mmol, 1.0 eq) of dried resin (compound 4) was weighed into a solid-phase reaction column, and 21 mL (10 v / w) of N,N-dimethylformamide was added and the column was left to stand at room temperature for 30 minutes. After discharging the N,N-dimethylformamide, 17 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the column was shaken at room temperature for 15 minutes. After discharging the reaction solution, 17 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the column was shaken at room temperature for 15 minutes. After discharging the reaction solution, the column was washed eight times with 21 mL (10 v / w) of N,N-dimethylformamide. Next, 1.12 g (3.16 mmol, 2.0 eq) of Fmoc-MeVal-OH and 0.226 g (1.58 mmol, 1.0 eq) of Oxyma were weighed into a vial and dissolved in 8.2 mL (4 v / w) of N,N-dimethylformamide. This solution was then loaded onto a solid-phase synthesis column. 0.99 mL (6.3 mmol, 4.0 eq) of DIC was then added, sealed, and shaken at room temperature for 6 hours. After draining the reaction solution, the column was washed four times with 21 mL (10 v / w) of N,N-dimethylformamide, two times with 21 mL (10 v / w) of 2-propanol, and four times with 21 mL (10 v / w) of methanol. The resin was then dried overnight at room temperature under reduced pressure to yield 2.78 g of dried resin (compound 5). The resulting resin (23.2 mg) was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0233] [Table 11]

[0234] [ka]

[0235] [Table 12]

[0236] The target product (compound 7) was obtained in high yield. The yields of the overstretched product (compound 8) and epimer (compound 6) were 2.3 A% and 0.4 A%, respectively.

[0237] [Example 2-1] Synthesis of Fmoc-D-MeVal-MeAsp(OCTC)-pip (Compound 10) (Synthesis under Example Condition A) [ka] 0.195 g (0.525 mmol / g, 0.101 mmol, 1.0 eq) of dried resin (compound 4) was weighed into a 5 mL disposable syringe with a filter, and 1.3 mL (10 v / w) of N,N-dimethylformamide was added and the mixture was left to stand at room temperature for 30 minutes. After discharging the N,N-dimethylformamide, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, the mixture was washed eight times with 1.3 mL (10 v / w) of N,N-dimethylformamide. Next, 72 mg (0.21 mmol, 2.0 eq) of Fmoc-D-MeVal-OH and 15 mg (0.11 mmol, 1.0 eq) of Oxyma were weighed into a vial and dissolved in 0.54 mL (4 v / w) of N,N-dimethylformamide. The solution was then aspirated using a disposable syringe. 66 μL (0.42 mmol, 4 eq) of DIC was then added, sealed, and shaken at room temperature for 6 hours. After draining the reaction solution, the resin was washed four times with 1.3 mL (10 v / w) of N,N-dimethylformamide, then twice with 1.3 mL (10 v / w) of 2-propanol, and four times with 1.3 mL (10 v / w) of methanol. The resin was then dried under vacuum overnight at room temperature to yield 0.188 g of dried resin (compound 10). The resulting resin (22.6 mg) was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0238] [Table 13]

[0239] [ka]

[0240] [Table 14]

[0241] The target product (compound 6) was obtained in high yield. The yields of the overstretched product (compound 11) and the epimer (compound 7) were 1.9 A% and 0.1 A%, respectively.

[0242] The solid-phase peptide synthesis described in Examples 2-2 to 2-11 was carried out by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by Intavis). Detailed procedures were described in the manual attached to the synthesizer.

[0243] Detailed synthesis conditions in Examples 2-2 to 2-11 are shown in Synthesis Method 1 below.

[0244] Synthesis method 1 Solution 1 was prepared by dissolving the Fmoc amino acid (second amino acid) constituting the target peptide, HOAt or Oxyma as an additive, and NMP to a concentration of 0.6 mol / L for the Fmoc amino acid and 0.375 mol / L for the additive. For poorly soluble Fmoc amino acids, DMSO was added to prepare Solution 1 at a concentration of 20–30% (v / v). Solution 2 was prepared by mixing the Fmoc amino acid or peptide-loaded resin (100 mg) prepared in solid-phase synthesis steps 1–4 with a filter (frit) in a solid-phase reaction vessel and loading it into a peptide synthesizer. After adding DCM (1.2 mL) and allowing it to stand for 30 minutes to swell the resin, the solution was then drained from the frit. Solutions 1 and 2 were loaded into the peptide synthesizer, and automated synthesis using the peptide synthesizer was initiated.

[0245] A DBU / DMF solution (2% v / v, 0.7 mL) was added to the solid-phase reaction vessel containing the resin, and the Fmoc group removal reaction was carried out at room temperature. The reaction was allowed to proceed for 4.5 minutes for the first deprotection and 10 minutes for the second and subsequent deprotection, after which the solution was drained through the frit. DMF (0.7 mL) was added, and the mixture was left standing for 5 minutes before draining through the frit. This resin washing process was repeated three more times. Next, Solution 1 (0.3 mL) and Solution 2 (0.36 mL) were mixed in the synthesizer's mixing vial (Fmoc amino acid: condensing agent: additive ratio approximately 1.6:2:1) and added to the resin. The solid-phase reaction vessel was heated to 40°C or 50°C, and the reaction was allowed to proceed for 2.5 hours or 10 hours, allowing the condensation reaction of the amino groups on the resin with the Fmoc amino acid. The solution was then drained through the frit. The resin was then washed three times with DMF (0.7 mL). This Fmoc group removal reaction followed by the condensation reaction of an Fmoc amino acid constituted one cycle, and this cycle was repeated to elongate the peptide.

[0246] After peptide elongation was complete, the resulting resin was washed four times with DMF (0.7 mL), followed by four times with DCM (0.7 mL), and air-dried at room temperature for 48 hours. A portion of the resulting resin (approximately 10 mg) was placed in a reaction vessel, and the peptide loading on the resin was calculated using the Fmoc quantification method described above. A portion of the resulting resin (approximately 20 mg) was also placed in a reaction vessel, and a TFE / DCM solution (1 / 1, 1 mL) with or without 0.75% (v / v) DIPEA was added. The mixture was shaken at room temperature for 2 hours to carry out the peptide cleavage reaction. After the reaction, the cleaved solution was analyzed by LCMS.

[0247] The tripeptides shown in [Example 2-2] to [Example 2-11] were synthesized by solid-phase reaction using a peptide synthesizer under the following two conditions, and the recovery rate and purity were calculated.

[0248] [Table 15]

[0249] The definition and calculation method of the recovery rate in Examples 2-1 to 2-30 are shown in the following recovery rate calculation method.

[0250] Recovery rate calculation method In Example 2, the recovery rate was defined as follows: The higher the recovery rate, the more premature cleavage was suppressed. Recovery rate = Actual Fmoc quantitative value after elongation reaction (mmol / g) ÷ Theoretical Fmoc quantitative value after elongation reaction (mmol / g) (Equation 1)

[0251] The theoretical Fmoc quantitative value (mmol / g) after the elongation reaction is calculated as follows: Theoretical Fmoc quantitative value after elongation reaction (mmol / g) = Fmoc quantitative value of raw resin (mmol / g) × weight of raw resin (g) ÷ weight of resin when 100% of the target product is produced (g) (Equation 2)

[0252] The weight (g) of resin when 100% of the desired product is produced is calculated as follows: Weight of resin when 100% of the target substance is produced (g) = Weight of raw resin (g) - Weight of amino acids or peptide components on raw resin (g) + Weight of peptide components on resin when 100% of the target substance is produced (g) (Equation 3)

[0253] The weight (g) of amino acid or peptide components on the raw resin is calculated as follows: Weight of amino acid or peptide component on raw resin (g) = Fmoc quantitative value of raw resin (mmol / g) × weight of raw resin (g) × molecular weight of amino acid or peptide component on raw resin (g / mol) × 0.001 (mol / mmol) (Equation 4)

[0254] When 100% of the target product is produced, the weight (g) of the peptide component on the resin is calculated as follows: Weight of peptide component on resin when 100% of target product is produced (g) = Fmoc quantitative value of raw resin (mmol / g) × weight of raw resin (g) × molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol) (Equation 5)

[0255] Substituting Equation 3, Equation 4, and Equation 5 into Equation 2 gives Theoretical Fmoc quantitative value after elongation reaction (mmol / g) = Fmoc quantitative value of starting resin (mmol / g) ÷ (1 - Fmoc quantitative value of starting resin (mmol / g) × molecular weight of amino acid or peptide component on starting resin (g / mol) × 0.001 (mol / mmol) + Fmoc quantitative value of starting resin (mmol / g) × molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol)) = 1 ÷ (1 ÷ Fmoc quantitative value of starting resin (mmol / g) - molecular weight of amino acid or peptide component on starting resin (g / mol) × 0.001 (mol / mmol) + molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol)) (Equation 6)

[0256] Substituting Equation 6 into Equation 1, the recovery rate is calculated using the following formula: Recovery rate = Actual Fmoc quantitative value after elongation reaction (mmol / g) × (1 ÷ Fmoc quantitative value of starting resin (mmol / g) - Molecular weight of amino acid or peptide component on starting resin (g / mol) × 0.001 (mol / mmol) + Molecular weight of target peptide component (g / mol) × 0.001 (mol / mmol))

[0257] [Example 2-2] Synthesis of Fmoc-Ile-MeVal-MeAsp(O-Trt(2-Cl)resin)-pip (Compound 27) [ka]

[0258] Synthesis of Compound 27 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 27 was synthesized by Fmoc-MeVal-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours). The loading rate was calculated using dried resin (10.45 mg) by Fmoc quantification to be 0.292 mmol / g (UVarea value at 294 nm: 3046.82, UVarea value at 304 nm: 2746.87).

[0259] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.292 × (1 ÷ 0.455 - 436.51 × 0.001 + 662.83 × 0.001) = 70.8% [ka]

[0260] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 27 was 61.6 area%, with the epimer form (compound 27a) being 6.5 area%, the over-elongated form (compound 27b) being 2.4 area%, and the truncated form (compound 27c) being 29.5 area%.

[0261] Analysis conditions: HPLC method G [Table 16]

[0262] Synthesis of compound 27 under condition 2 (synthesis method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 27 was synthesized by Fmoc-MeVal-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours). The loading rate was calculated using dried resin (10.89 mg) by Fmoc quantification to be 0.332 mmol / g (UVarea value at 294 nm: 3612.81, UVarea value at 304 nm: 3267.35).

[0263] The recovery rate is calculated using the following formula in accordance with the recovery rate calculation method. Recovery rate = 0.332 × (1 ÷ 0.455 - 436.51 × 0.001 + 662.83 × 0.001) = 80.5%

[0264] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 27 was 93.7 area%, with 2.6 area% of the epimer (compound 27a) and 3.7 area% of the over-elongated form (compound 27b) observed.

[0265] Analysis conditions: HPLC method G [Table 17]

[0266] The above results are summarized in the table below. [Table 18]

[0267] [Example 2-3] Synthesis of Fmoc-Ile-MeIle-MeAsp(O-Trt(2-Cl)resin)-pip (Compound 28) [ka]

[0268] Synthesis of compound 28 under condition 1 (synthesis method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 28 was synthesized by Fmoc-MeIle-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours). The loading rate was calculated using dried resin (10.81 mg) by Fmoc quantitation, yielding 0.326 mmol / g (UVarea value at 294 nm: 3524.40, UVarea value at 304 nm: 3171.55).

[0269] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.326 × (1 ÷ 0.455 - 436.51 × 0.001 + 676.86 × 0.001) = 79.5% [ka]

[0270] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 28 was 58.0 area%, with the epimer form (compound 28a) being 1.4 area%, the over-elongated form (compound 28b) being 1.8 area%, and the truncated form (compound 27c) being 38.8 area%.

[0271] Analysis conditions: HPLC method G [Table 19]

[0272] Synthesis of compound 28 under condition 2 (synthesis method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 28 was synthesized by Fmoc-MeIle-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours). The loading rate was calculated using dried resin (10.41 mg) by Fmoc quantification to be 0.330 mmol / g (UVarea value at 294 nm: 3437.60, UVarea value at 304 nm: 3100.91).

[0273] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.330 × (1 ÷ 0.455 - 436.51 × 0.001 + 676.86 × 0.001) = 80.5%

[0274] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 28 was 95.2 area%, with 1.4 area% of the epimer (compound 28a) and 3.4 area% of the over-elongated form (compound 28b) observed.

[0275] Analysis conditions: HPLC method G [Table 20]

[0276] The above results are summarized in the table below. [Table 21]

[0277] [Example 2-4] Synthesis of Fmoc-Ile-MeGly(cPent)-MeAsp(O-Trt(2-Cl)resin)-pip (Compound 29) [ka]

[0278] Synthesis of Compound 29 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 29 was synthesized by elongation of Fmoc-MeGly(cPent)-OH (HOAt, 40°C, 2.5 h) followed by elongation of Fmoc-Ile-OH (HOAt, 40°C, 2.5 h). The loading rate was calculated using the dried resin (10.05 mg) by Fmoc quantification to be 0.320 mmol / g (UVarea value at 294 nm: 3216.77, UVarea value at 304 nm: 2905.18).

[0279] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.320 × (1 ÷ 0.455 - 436.51 × 0.001 + 688.87 × 0.001) = 78.4% [ka]

[0280] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 29 was 83.9 area%, with the epimer form (compound 29a) being 1.2 area%, the over-elongated form (compound 29b) being 3.9 area%, and the truncated form (compound 27c) being 11.0 area%.

[0281] Analysis conditions: HPLC method G [Table 22]

[0282] Synthesis of compound 29 under condition 2 (synthesis method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 29 was synthesized by elongation of Fmoc-MeGly(cPent)-OH (Oxyma, 50 °C, 10 h) followed by elongation of Fmoc-Ile-OH (HOAt, 40 °C, 2.5 h). The loading rate was calculated using the dried resin (9.45 mg) by Fmoc quantification to be 0.232 mmol / g (UVarea value at 294 nm: 2194.45, UVarea value at 304 nm: 1975.12).

[0283] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.232 × (1 ÷ 0.455 - 436.51 × 0.001 + 688.87 × 0.001) = 56.8%

[0284] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 29 was 91.4 area%, the epimer form (compound 29a) was 0.1 area%, and the over-elongated form (compound 29b) was 8.5 area%.

[0285] Analysis conditions: HPLC method G [Table 23]

[0286] The above results are summarized in the table below. [Table 24]

[0287] [Example 2-5] Synthesis of Fmoc-Ile-MeChg-MeAsp(O-Trt(2-Cl)resin)-pip (Compound 30) [ka]

[0288] Synthesis of Compound 30 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 30 was synthesized by Fmoc-MeChg-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours). The loading rate was calculated using dried resin (9.68 mg) by Fmoc quantification to be 0.314 mmol / g (UVarea value at 294 nm: 3037.14, UVarea value at 304 nm: 2743.09).

[0289] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.314 × (1 ÷ 0.455 - 436.51 × 0.001 + 702.89 × 0.001) = 77.4% [ka]

[0290] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 30 was 74.1 area%, with the epimer form (compound 30a) being 2.3 area%, the over-elongated form (compound 30b) being 4.0 area%, and the truncated form (compound 27c) being 19.5 area%.

[0291] Analysis conditions: HPLC method G [Table 25]

[0292] Synthesis of Compound 30 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 30 was synthesized by Fmoc-MeChg-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours). The loading rate was calculated using dried resin (9.91 mg) by Fmoc quantification to be 0.226 mmol / g (UVarea value at 294 nm: 2235.50, UVarea value at 304 nm: 2016.81).

[0293] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.226 × (1 ÷ 0.455 - 436.51 × 0.001 + 702.89 × 0.001) = 55.7%

[0294] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 30 was 91.7 area%, with the epimer (compound 30a) being 0.6 area% and the over-elongated form (compound 30b) being 7.7 area%.

[0295] Analysis conditions: HPLC method G [Table 26]

[0296] The above results are summarized in the table below. [Table 27]

[0297] [Example 2-6] Synthesis of Fmoc-Ile-MeLeu-MeAsp(O-Trt(2-Cl)resin)-pip (Compound 31) [ka]

[0298] Synthesis of Compound 31 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 31 was synthesized by Fmoc-MeLeu-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours). The loading rate was calculated using dried resin (10.74 mg) by Fmoc quantitation, yielding 0.369 mmol / g (UVarea value at 294 nm: 3953.19, UVarea value at 304 nm: 3570.96).

[0299] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.369 × (1 ÷ 0.455 - 436.51 × 0.001 + 676.86 × 0.001) = 90.0% [ka]

[0300] Furthermore, analysis of the cleavage reaction solution by LCMS revealed that the purity of the deresinized form of compound 31 was 98.3 area%, with 0.1 area% of the epimer (compound 31a) and 1.6 area% of the over-extended form (compound 31b). With this substrate, no deletion form (compound 27c) was observed even under condition 1.

[0301] Analysis conditions: HPLC method G [Table 28]

[0302] Synthesis of Compound 31 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 4) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip) (100 mg, 0.455 mmol / g) prepared in solid-phase synthesis 1, compound 31 was synthesized by Fmoc-MeLeu-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours). The loading rate was calculated using dried resin (10.73 mg) by Fmoc quantification to be 0.337 mmol / g (UVarea value at 294 nm: 3610.72, UVarea value at 304 nm: 3254.78).

[0303] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.337 × (1 ÷ 0.455 - 436.51 × 0.001 + 676.86 × 0.001) = 82.2%

[0304] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 31 was 97.7 area%, the epimer form (compound 31a) was 0.1 area%, and the over-elongated form (compound 31b) was 2.2 area%.

[0305] Analysis conditions: HPLC method G [Table 29]

[0306] The above results are summarized in the table below. [Table 30]

[0307] [Example 2-7] Synthesis of Fmoc-Ile-MeGly(cPent)-MeAsp(O-Trt(2-Cl)resin)-NMe2 (Compound 32) [ka]

[0308] Synthesis of Compound 32 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 22) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-NMe2) (100 mg, 0.442 mmol / g) prepared in solid-phase synthesis 2, compound 32 was synthesized by elongation of Fmoc-MeGly(cPent)-OH (HOAt, 40°C, 2.5 h) followed by elongation of Fmoc-Ile-OH (HOAt, 40°C, 2.5 h). The loading rate was calculated using dried resin (10.73 mg) by Fmoc quantitation, yielding 0.304 mmol / g (UVarea value at 294 nm: 3263.10, UVarea value at 304 nm: 2945.28).

[0309] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.304 × (1 ÷ 0.442 - 396.44 × 0.001 + 648.80 × 0.001) = 76.5% [ka]

[0310] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 32 was 76.3 area%, with the epimer form (compound 32a) being 1.2 area%, the over-elongated form (compound 32b) being 5.6 area%, and the truncated form (compound 32c) being 16.9 area%.

[0311] Analysis conditions: HPLC METHOD E [Table 31]

[0312] Synthesis of Compound 32 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 22) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-NMe2) (100 mg, 0.442 mmol / g) prepared in solid-phase synthesis 2, compound 32 was synthesized by elongation of Fmoc-MeGly(cPent)-OH (Oxyma, 50 °C, 10 h) followed by elongation of Fmoc-Ile-OH (HOAt, 40 °C, 2.5 h). The loading rate was calculated using the dried resin (10.60 mg) by Fmoc quantification to be 0.211 mmol / g (UVarea value at 294 nm: 2239.56, UVarea value at 304 nm: 2018.13).

[0313] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.211 × (1 ÷ 0.442 - 396.44 × 0.001 + 648.80 × 0.001) = 53.1%

[0314] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of Compound 32 was 85.9 area %, and the excess elongation product (Compound 32b) was observed at 14.1 area %.

[0315] Analysis conditions: HPLC METHOD E [Table 32]

[0316] The above results are summarized in the table below. [Table 33]

[0317] [Example 2-8] Synthesis of Fmoc-Ile-MeLeu-MeAsp(O-Trt(2-Cl)resin)-NMe2 (Compound 33) [ka]

[0318] Synthesis of Compound 33 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 22) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-NMe2) (100 mg, 0.442 mmol / g) prepared in solid-phase synthesis 2, compound 33 was synthesized by Fmoc-MeLeu-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.15 mg) by Fmoc quantification to be 0.371 mmol / g (UVarea value at 294 nm: 3770.57, UVarea value at 304 nm: 3391.62).

[0319] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.371 × (1 ÷ 0.442 - 396.44 × 0.001 + 636.79 × 0.001) = 92.9% [ka]

[0320] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresinized form of compound 33 was 97.9 area%, and the over-extended form (compound 33b) was observed at 2.1 area%. With this substrate, no deletion form (compound 32c) was observed even under condition 1.

[0321] Analysis conditions: HPLC METHOD E [Table 34]

[0322] Synthesis of Compound 33 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 22) (Fmoc-MeAsp(O-Trt(2-Cl)resin)-NMe2) (100 mg, 0.442 mmol / g) prepared in solid-phase synthesis 2, compound 33 was synthesized by Fmoc-MeLeu-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours). The loading rate was calculated using the dried resin (10.73 mg) by Fmoc quantification to be 0.334 mmol / g (UVarea value at 294 nm: 3587.19, UVarea value at 304 nm: 3234.07).

[0323] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.334 × (1 ÷ 0.442 - 396.44 × 0.001 + 636.79 × 0.001) = 83.6%

[0324] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of Compound 33 was 96.6 area %, and the excess elongation product (Compound 33b) was observed at 3.4 area %.

[0325] Analysis conditions: HPLC METHOD E [Table 35]

[0326] The above results are summarized in the table below. [Table 36]

[0327] [Example 2-9] Synthesis of Fmoc-Ile-MeVal-D-3-MeAbu-O-Trt(2-Cl) resin (compound 34) [ka]

[0328] Synthesis of Compound 34 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 24) (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin) (100 mg, 0.369 mmol / g) prepared in solid-phase synthesis 3, compound 34 was synthesized by Fmoc-MeVal-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.62 mg) by Fmoc quantification to be 0.295 mmol / g (UVarea value at 294 nm: 3138.26, UVarea value at 304 nm: 2821.05).

[0329] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.295 × (1 ÷ 0.369 - 339.39 × 0.001 + 565.71 × 0.001) = 86.6% [ka]

[0330] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresinized form of compound 34 was 99.1 area%, and the over-extended form (compound 34b) was observed at 1.0 area%. With this substrate, no deletion form (compound 34c) was observed even under condition 1.

[0331] Analysis conditions: HPLC method G [Table 37]

[0332] Synthesis of compound 34 under condition 2 (synthesis method 1) Starting from the amino acid-loaded resin (compound 24) (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin) (100 mg, 0.369 mmol / g) prepared in solid-phase synthesis 3, compound 34 was synthesized by Fmoc-MeVal-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.44 mg) by Fmoc quantification to be 0.284 mmol / g (UVarea value at 294 nm: 2964.86, UVarea value at 304 nm: 2667.78).

[0333] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.284 × (1 ÷ 0.369 - 339.39 × 0.001 + 565.71 × 0.001) = 83.4%

[0334] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of Compound 34 was 98.1 area %, and the excess elongation product (Compound 34b) was observed at 1.9 area %.

[0335] Analysis conditions: HPLC method G [Table 38]

[0336] The above results are summarized in the table below. [Table 39]

[0337] [Example 2-10] Synthesis of Fmoc-Ile-MeChg-D-3-MeAbu-O-Trt(2-Cl) resin (compound 35) [ka]

[0338] Synthesis of Compound 35 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 24) (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin) (100 mg, 0.369 mmol / g) prepared in solid-phase synthesis 3, compound 35 was synthesized by Fmoc-MeChg-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.20 mg) by Fmoc quantitation to be 0.293 mmol / g (UVarea value at 294 nm: 2987.04, UVarea value at 304 nm: 2692.68).

[0339] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.293 × (1 ÷ 0.369 - 339.39 × 0.001 + 605.78 × 0.001) = 87.2% [ka]

[0340] Furthermore, when the excision reaction solution was analyzed by LCMS, the purity of the deresined form of compound 35 was 92.8 area%, with an over-extended form (compound 35b) at 3.4 area% and a truncated form (compound 34c) at 3.8 area%.

[0341] Analysis conditions: HPLC method F [Table 40]

[0342] Synthesis of Compound 35 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 24) (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin) (100 mg, 0.369 mmol / g) prepared in solid-phase synthesis 3, compound 35 was synthesized by Fmoc-MeChg-OH extension (Oxyma, 50 °C, 10 h) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 h). The loading rate was calculated using the dried resin (9.56 mg) by Fmoc quantification to be 0.217 mmol / g (UVarea value at 294 nm: 2072.81, UVarea value at 304 nm: 1864.82).

[0343] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.217 × (1 ÷ 0.369 - 339.39 × 0.001 + 605.78 × 0.001) = 64.6%

[0344] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of Compound 35 was 94.5 area %, and the excess elongation product (Compound 35b) was observed at 5.5 area %.

[0345] Analysis conditions: HPLC method F [Table 41]

[0346] The above results are summarized in the table below. [Table 42]

[0347] [Example 2-11] Synthesis of Fmoc-Ile-MeVal-MeGly-O-Trt(2-Cl) resin (compound 36) [ka]

[0348] Synthesis of Compound 36 under Condition 1 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 26) (Fmoc-MeGly-O-Trt(2-Cl) resin) (100 mg, 0.573 mmol / g) prepared in solid-phase synthesis 4, compound 36 was synthesized by Fmoc-MeVal-OH extension (HOAt, 40°C, 2.5 hours) followed by Fmoc-Ile-OH extension (HOAt, 40°C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.20 mg) by Fmoc quantitation to be 0.326 mmol / g (UVarea value at 294 nm: 3322.12, UVarea value at 304 nm: 3002.34).

[0349] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.326 × (1 ÷ 0.573 - 311.34 × 0.001 + 537.66 × 0.001) = 64.3% [ka]

[0350] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresinized form of compound 36 was 95.6 area%, and the over-extended form (compound 36b) was observed at 4.4 area%. With this substrate, no deletion form (compound 36c) was observed even under condition 1.

[0351] Analysis conditions: HPLC METHOD E [Table 43]

[0352] Synthesis of Compound 36 under Condition 2 (Synthesis Method 1) Starting from the amino acid-loaded resin (compound 26) (Fmoc-MeGly-(O-Trt(2-Cl) resin)) (100 mg, 0.573 mmol / g) prepared in solid-phase synthesis 4, compound 36 was synthesized by Fmoc-MeVal-OH extension (Oxyma, 50 °C, 10 hours) followed by Fmoc-Ile-OH extension (HOAt, 40 °C, 2.5 hours) according to synthesis method 1. The loading rate was calculated using the dried resin (10.69 mg) by Fmoc quantification to be 0.331 mmol / g (UVarea value at 294 nm: 3542.66, UVarea value at 304 nm: 3189.92).

[0353] The recovery rate was calculated using the following formula according to the recovery rate calculation method. Recovery rate = 0.331 × (1 ÷ 0.573 - 311.34 × 0.001 + 537.66 × 0.001) = 65.3%

[0354] Furthermore, when the cleavage reaction solution was analyzed by LCMS, the purity of the deresined form of compound 36 was 93.9 area %, and the excess elongation product (compound 36b) was observed at 6.1 area %.

[0355] Analysis conditions: HPLC METHOD E [Table 44]

[0356] The above results are summarized in the table below. [Table 45]

[0357] Verification of premature cleavage suppression effect of Example Condition A with various arrangements [Example 2-12] Synthesis of Fmoc-MeVal-MeLeu-OCTC (Compound 39) (Synthesis under Example Condition A) [ka] 0.195 g (0.650 mmol / g, 0.127 mmol, 1.0 eq) of dried resin (compound 38) was weighed into a 5 mL disposable syringe with a filter, and 1.6 mL (10 v / w) of N,N-dimethylformamide was added and the mixture was left to stand at room temperature for 30 minutes. After discharging the N,N-dimethylformamide, 1.3 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, 1.3 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, the mixture was washed eight times with 1.6 mL (10 v / w) of N,N-dimethylformamide. Next, 90 mg (0.25 mmol, 2.0 eq) of Fmoc-MeVal-OH and 18 mg (0.13 mmol, 1.0 eq) of Oxyma were weighed into a vial and dissolved in 0.62 mL (4 v / w) of N,N-dimethylformamide. 80 μL (0.51 mmol, 4.0 eq) of DIC was added to the solution, which was then immediately aspirated using a disposable syringe. The vial was then sealed and shaken at room temperature for 18 h. After draining the reaction solution, the vial was washed four times with 1.6 mL (10 v / w) of N,N-dimethylformamide, then twice with 1.6 mL (10 v / w) of 2-propanol, and four times with 1.6 mL (10 v / w) of methanol. The resin was then dried overnight under vacuum at room temperature to yield 0.201 g of dried resin (compound 39). The resulting resin (16.7 mg) was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0358] [Table 46]

[0359] [ka]

[0360] [Table 47]

[0361] Glycine capping was also performed to confirm the remaining percentage of starting materials. Approximately 10 mg of the dried resin from the condensation reaction was weighed into a 5 mL disposable syringe with a filter, and 1 mL of N,N-dimethylformamide was added. The mixture was then left to stand at room temperature for 15 minutes. After the N,N-dimethylformamide was removed, the reaction cocktail (approximately 70 mg of Fmoc-Gly-OH and approximately 0.11 g of HATU were weighed into a vial and suspended in 0.45 mL of N,N-dimethylformamide. 45 μL of N,N-diisopropylethylamine was added and the mixture was shaken for 1 minute to obtain a pale yellow homogeneous solution) was aspirated into the disposable syringe and shaken for 1 hour at room temperature. After the reaction solution was removed, the mixture was washed six times with 1 mL of N,N-dimethylformamide and then four times with 1 mL of dichloromethane, after which the resin was removed. The residual rate of the starting material was calculated as 0% (0 ÷ (0 + 98.6 + 0 + 0)) based on the sum of the LC A% of the Gly-capped compound (compound 40), the target compound (compound 39), the epimer (compound 39a), and the over-elongated compound (compound 39b).

[0362] [Table 48]

[0363] [ka] The resin-free product (compound 39) was obtained in good yield. The formation of the overstretched product (compound 39b) and the epimer (compound 39a) was not confirmed.

[0364] [Reference Example 4] Synthesis of Fmoc-MeVal-MeLeu-OCTC (compound 39) under the reaction conditions described in Chem. Eur. J., 2009, 15, 9394-9403 (reaction conditions of Fmoc amino acid:DIC:additive (Oxyma)=2:2:2 relative to the amino acid of the resin; hereinafter referred to as Reference Example Condition A). [ka] 0.204 g (0.650 mmol / g, 0.133 mmol, 1.0 eq) of dried resin (compound 38) was weighed into a 5 mL disposable syringe with a filter, and 1.6 mL (10 v / w) of N,N-dimethylformamide was added and the mixture was left to stand at room temperature for 30 minutes. After the N,N-dimethylformamide was drained, 1.3 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the mixture was shaken at room temperature for 15 minutes. After the reaction solution was drained, 1.3 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the mixture was shaken at room temperature for 15 minutes. After the reaction solution was drained, the mixture was washed eight times with 1.6 mL (10 v / w) of N,N-dimethylformamide. Next, 96 mg (0.26 mmol, 2.0 eq) of Fmoc-MeVal-OH and 38 mg (0.26 mmol, 2.0 eq) of Oxyma were weighed into a vial and dissolved in 0.65 mL (4 v / w) of N,N-dimethylformamide. Next, 42 μL (0.26 mmol, 2.0 eq) of DIC was added to the vial at room temperature, and the container was sealed and shaken at room temperature for 2 minutes. The solution was aspirated using a disposable syringe, sealed, and shaken at room temperature for 18 hours. After draining the reaction solution, the resin was washed four times with 1.6 mL (10 v / w) of N,N-dimethylformamide, then twice with 1.6 mL (10 v / w) of 2-propanol, and four times with 1.6 mL (10 v / w) of methanol. It was then dried overnight at room temperature under reduced pressure to yield 0.181 g of dried resin (compound 3). The resulting resin (19.0 mg) was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0365] [Table 49]

[0366] Using the same method as in Example Condition A, it was confirmed that the residual rate of starting materials was 0% under Reference Example Condition A.

[0367] [Table 50] It was found that under the reaction conditions of Reference Example Condition A above, both the yield and purity of the target product decreased.

[0368] Based on the above 1 residue + 1 residue condensation experiment, the following sequences were synthesized under Example Condition A and Reference Example Condition A. [Example 2-13] Synthesis of Fmoc-D-MeVal-MeLeu-OCTC (Compound 41) (Synthesis under Example Condition A) [ka]

[0369] [Table 51]

[0370] [Table 52]

[0371] [ka]

[0372] [Example 2-14] Synthesis of Fmoc-Lys(Z)-MeVal-OCTC (Compound 43) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0373] [Table 53]

[0374] [Table 54]

[0375] [ka]

[0376] [Example 2-15] Synthesis of Fmoc-MeVal-MeAla-OCTC (Compound 45) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0377] [Table 55]

[0378] [Table 56] [ka]

[0379] [Example 2-16] Synthesis of Fmoc-MeVal-MeGly-OCTC (Compound 47) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0380] [Table 57]

[0381] [Table 58]

[0382] [ka]

[0383] [Example 2-17] Synthesis of Fmoc-MeVal-MePhe-OCTC (Compound 49) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0384] [Table 59]

[0385] [Table 60]

[0386] [ka]

[0387] [Example 2-18] Synthesis of Fmoc-Glu(OBzl)-MeTrp(Boc)-OCTC (Compound 51) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0388] [Table 61]

[0389] [Table 62]

[0390] [ka]

[0391] [Example 2-19] Synthesis of Fmoc-Aze(2)-MeThr(Bzl)-OCTC (Compound 53) (Synthesis under Example Condition A and Reference Example Condition A)

[0392] [Table 63]

[0393] [Table 64]

[0394] [ka]

[0395] [Example 2-20] Synthesis of Fmoc-Lys(Z)-MeGlu(OtBu)-OCTC (Compound 55) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0396] [Table 65]

[0397] [Table 66]

[0398] [ka]

[0399] [Example 2-21] Synthesis of Fmoc-Thr(tBu)-MeLys(Boc)-OCTC (Compound 57) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0400] [Table 67]

[0401] [Table 68]

[0402] [ka]

[0403] [Example 2-22] Synthesis of Fmoc-MePhe-MeMet-OCTC (Compound 59) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0404] [Table 69]

[0405] [Table 70]

[0406] [ka]

[0407] [Example 2-23] Synthesis of Fmoc-cVal-Aze(2)-OCTC (Compound 61) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0408] [Table 71]

[0409] [Table 72]

[0410] [ka]

[0411] [Example 2-24] Synthesis of Fmoc-Gly-Gly-OCTC (Compound 63) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0412] [Table 73]

[0413] [Table 74]

[0414] [ka]

[0415] [Table 75]

[0416] In Entries 2 and 3, the reaction time was shortened from 18 hours to 1 hour.

[0417] [Example 2-25] Synthesis of Fmoc-Gly-Leu-OCTC (Compound 65) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0418] [Table 76]

[0419] [Table 77]

[0420] [ka]

[0421] [Table 78] In Entries 2 and 3, the reaction time was shortened from 18 hours to 1 hour.

[0422] [Example 2-26] Synthesis of Fmoc-Pro-Aib-OCTC (Compound 67) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0423] [Table 79]

[0424] [Table 80]

[0425] [ka]

[0426] [Example 2-27] Synthesis of Fmoc-Gly-bAla-OCTC (Compound 69) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0427] [Table 81]

[0428] [Table 82]

[0429] [ka]

[0430] [Example 2-28] Synthesis of Fmoc-MeVal-bMeAla-OCTC (Compound 71) [ka]

[0431] [Table 83]

[0432] The extension of this sequence was carried out under Example Condition A with three changes.

[0433] [Table 84]

[0434] [ka]

[0435] [Table 85]

[0436] [ka]

[0437] [Example 2-29] Synthesis of Fmoc-MeVal-D-3-MeAbu-OCTC (Compound 73) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0438] [Table 86]

[0439] The extension of this sequence was carried out under Example Condition A with one change.

[0440] [Table 87]

[0441] [Table 88]

[0442] [ka]

[0443] [Example 2-30] Synthesis of Fmoc-Ser(tBu)-MeLeu-OCTC (Compound 75) (Synthesis under Example Condition A and Reference Example Condition A) [ka]

[0444] [Table 89]

[0445] [Table 90]

[0446] [ka]

[0447] The above results are summarized in the table below.

[0448] [Table 91]

[0449] For all sequences, Example Condition A suppressed premature cleavage more than Reference Example Condition A, giving two-residue peptides in good yield. The inhibitory effect was particularly remarkable in the case of N-Me α-amino acids.

[0450] [Example 2-31] Experiments to explore the range of reagent equivalents that suppress premature cleavage Based on the above [1 residue + 1 residue condensation experiment] (Example condition A), the same reaction was carried out by changing the equivalent amount of reagents, and the results are shown below. [ka]

[0451] [Table 92]

[0452] Under Reference Example Condition A, increasing the equivalent amount of Fmoc amino acid hardly improved the yield (Entries 1 and 3). On the other hand, decreasing the equivalent amount of Oxyma improved the purity and yield (Entries 4, 8, 13, 16). Furthermore, increasing the equivalent amount of DIC improved the purity and yield (Entry 5, 6). Furthermore, decreasing the equivalent amount of Oxyma and increasing the equivalent amount of DIC improved the purity and yield (Entries 2, 9, 10, 11, 12, 14, and 15). In particular, Entries 2, 9, 10, and 11 showed yields exceeding 75%.

[0453] In Examples 2-1 to 2-31, the target peptides were synthesized by elongating amino acids or peptides whose N-terminus was N-Me-modified under conditions in which 1 equivalent or more of DIC was used relative to the Fmoc amino acid and 1 equivalent or less of an additive was used. The recovery rates for elongation up to the third residue were all above 53%, and in some cases even as high as 90% or higher. The yields and purities of the comparative experiments with Reference Example Condition A were equal to or higher than those of Example Condition A.

[0454] [Reference Example 1] Chem. Eur. J., 2009, 15, 9394-9403) Synthesis of Fmoc-MeVal-MeAsp(OCTC)-pip (Compound 5) (Reaction conditions: Fmoc amino acid:DIC:additive (Oxyma) = 2:2:2 for the amino acid of the resin: Reference Example Condition A) [ka] 0.200 g (0.525 mmol / g, 0.104 mmol, 1.0 eq) of dried resin (compound 4) was weighed into a 5 mL disposable syringe with a filter, and 1.3 mL (10 v / w) of N,N-dimethylformamide was added and the mixture was left to stand at room temperature for 30 minutes. After discharging the N,N-dimethylformamide, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, the mixture was washed eight times with 1.3 mL (10 v / w) of N,N-dimethylformamide. Next, 74 mg (0.21 mmol, 2.0 eq) of Fmoc-MeVal-OH and 30 mg (0.21 mmol, 2.0 eq) of Oxyma were weighed into a vial and dissolved in 0.54 mL (4 v / w) of N,N-dimethylformamide. Next, 33 μL (0.21 mmol, 2.0 eq) of DIC was added to the vial at room temperature, and the container was sealed and shaken at room temperature for 2 minutes. The solution was aspirated using a disposable syringe, sealed, and shaken at room temperature for 6 hours. After draining the reaction solution, the resin was washed four times with 1.3 mL (10 v / w) of N,N-dimethylformamide, then twice with 1.3 mL (10 v / w) of 2-propanol, and four times with 1.3 mL (10 v / w) of methanol. It was then dried under reduced pressure overnight at room temperature to yield 0.178 g of dried resin (compound 5). 21.6 mg of the resulting resin was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0455] [Table 93]

[0456] [ka]

[0457] [Table 94] * Mass spectrometry analysis of the glycopapping experiment identified this as overstretched form B.

[0458] Glycine capping was also performed to confirm the remaining percentage of starting materials. Specifically, approximately 10 mg of resin obtained by filtering the reaction solution after the condensation reaction was transferred to a 5 mL disposable syringe with a filter as a suspension in N,N-dimethylformamide. After draining the N,N-dimethylformamide, the mixture was washed three times with 1 mL of N,N-dimethylformamide. The reaction cocktail (approximately 70 mg of Fmoc-Gly-OH and approximately 0.11 g of HATU were weighed into a vial and suspended in 0.45 mL of N,N-dimethylformamide. 45 μL of N,N-diisopropylethylamine was added and the mixture was shaken for 1 minute to obtain a pale yellow homogeneous solution) was aspirated into the disposable syringe and shaken for 1 hour at room temperature. After draining the reaction solution, the mixture was washed six times with 1 mL of N,N-dimethylformamide and then four times with 1 mL of dichloromethane, followed by the resin removal reaction. The residual rate of the starting material was calculated to be 1.3% (1.3 ÷ (1.3 + 79.9 + 13.4 + 2.3)) from the sum of the LC A% of the Gly-capped compound (compound 13), epimer (compound 6), target compound (compound 7), over-elongated compound (compound 8), and over-elongated compound (compound 9).

[0459] [Table 95]

[0460] [ka]

[0461] The above reaction conditions resulted in a low yield of the desired product. The incomplete reaction was confirmed by the formation of Gly-capped product (compound 13), while the formation of over-extended products (compounds 8 and 9) was also observed.

[0462] [Reference Example 2] Synthesis of Fmoc-MeVal-MeAsp(OCTC)-pip (compound 5) under the reaction conditions described in Eur. J. Org. Chem., 2013, 6372-6378 (reaction conditions: Fmoc amino acid:DIC:additive (K-Oxyma) = 2:2:2 for the amino acid of the resin). [ka] 0.202 g (0.525 mmol / g, 0.105 mmol, 1.0 eq) of dried resin (compound 4) was weighed into a 5 mL disposable syringe with a filter, and 1.3 mL (10 v / w) of N,N-dimethylformamide was added and the mixture was left to stand at room temperature for 30 minutes. After discharging the N,N-dimethylformamide, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, 1.1 mL (8 v / w) of a 20% N,N-dimethylformamide solution was added again and the mixture was shaken at room temperature for 15 minutes. After discharging the reaction solution, the mixture was washed eight times with 1.3 mL (10 v / w) of N,N-dimethylformamide. Next, 75 mg (0.21 mmol, 2.0 eq) of Fmoc-MeVal-OH and 40 mg (0.21 mmol, 2.0 eq) of K-Oxyma were weighed into a vial and dissolved in 0.54 mL (4 v / w) of N,N-dimethylformamide. 33 μL (0.21 mmol, 2.0 eq) of DIC was then added to the vial at room temperature. The solution was immediately aspirated using a disposable syringe, sealed, and shaken at room temperature for 6 h. After draining the reaction solution, the resin was washed four times with 1.3 mL (10 v / w) of N,N-dimethylformamide, then twice with 1.3 mL (10 v / w) of 2-propanol, and four times with 1.3 mL (10 v / w) of methanol. It was then dried overnight at room temperature under reduced pressure to yield 0.188 g of dried resin (compound 5). 21.0 mg of the resulting resin was weighed into a 100 mL volumetric flask, and the flask was filled up with 20% piperidine in N,N-dimethylformamide and shaken for 45 minutes at room temperature. Fmoc quantification was performed using a spectrophotometer based on the absorbance of the solution, and the yield was calculated from the Fmoc quantification value, the mass of the dried resin, and LC A%.

[0463] [Table 96]

[0464] [ka]

[0465] [Table 97]

[0466] The above reaction conditions were found to result in a decreased yield of the target product. Although the overstretched product (compound 8) was suppressed, the production of the epimer (compound 6) was increased.

[0467] [1-residue + 2-residue condensation experiment] [Starting material synthesis] The starting material for the condensation reaction, H-MeVal-MeAsp(OCTC)-pip (compound 14), was synthesized according to the loading, condensation, and Fmoc removal conditions described herein. Condensation of one residue + one residue was carried out in accordance with Example Condition A. The theoretical peptide loading rate of the starting material for the condensation reaction (compound 14) was 0.63 mmol / g, which was calculated as follows. 1. Fmoc quantification of the dried resin (compound 4) after Fmoc-MeAsp(OH)-pip (compound 3) was performed on the CTC resin, and the measured value was 0.59 mmol / g. 2. Considering that the molecular weight of compound 3 is 436.51 (g / mol), 1 g of this dry resin (compound 4) is composed of 0.258 g of compound 1 and 0.742 g of resin component according to the following formula. Compound 3: 0.59 (mmol / g) x 1 (g) x 436.51 (g / mol) = 0.258 (g). Resin component: 1 (g) - 0.258 (g) = 0.742 (g) 3. The theoretical yield of the raw material for the condensation reaction (compound 14) obtained from 1 g of dried resin (compound 4) can be calculated based on the mass of H-MeVal-MeAsp(OH)-pip (compound 15) and the molecular weight of the peptide. Assuming that the mass of the resin component remains unchanged before and after the reaction, Compound 14: 0.59(mmol / g) x 1 (g) x 327.43 (g / mol) + 0.742 = 0.935 (g) Therefore, the theoretical peptide loading rate of the raw material for the condensation reaction (compound 14) is 0.63 (mmol / g) (0.59 ÷ 0.935 = 0.63).

[0468] In this Reference Example, the amount of solvent used in the solid-phase reaction is sometimes expressed as a double volume (v / w). Unlike the 1+1 residue condensation experiment, the double volume in this Reference Example is the mass of the dry resin (compound 14). When 0.80 mL of N,N-dimethylformamide is used for 100 mg of dry resin, the double volume is 0.80 mL ÷ 0.100 (g) = 8 v / w.

[0469] [Example 3] Synthesis of Fmoc-cLeu-MeVal-MeAsp(OCTC)-pip (Compound 16) (Synthesis under Example Condition A) [ka] [Condensation experiment] 67 mg (0.19 mmol, 3.0 eq) of Fmoc-cLeu-OH and 13 mg (0.095 mmol, 1.5 eq) of Oxyma were weighed into a vial and dissolved in 0.80 mL (8 v / w) of N,N-dimethylformamide. 59 μL (0.38 mmol, 6.0 eq) of DIC was added to the solution and shaken at room temperature for 2 hours. 0.10 g (0.63 mmol / g, 0.063 mmol, 1.0 eq) of dried resin (compound 14) was added to the reaction solution and shaken at room temperature for 24 hours. [Conversion rate confirmation experiment] In another vial, 62 mg (0.21 mmol, 3.3 eq) of Fmoc-Gly-OH and 30 mg (0.21 mmol, 3.3 eq) of Oxyma were weighed and dissolved in 0.40 mL (4 v / w) of N,N-dimethylformamide. 65 μL (0.42 mmol, 6.6 eq) of DIC was added to this solution and shaken at room temperature for 1 hour. This solution was transferred to the Fmoc-cLeu-OH reaction solution and shaken at room temperature for 2 hours. The reaction solution was transferred to a disposable syringe with a filter, and the reaction solution and resin were filtered off. The resin was washed twice with 0.80 mL (8 v / w) of N,N-dimethylformamide and 0.80 mL (8 v / w) of isopropanol, then twice with 0.80 mL (8 v / w) of N,N-dimethylformamide and finally with 0.80 mL (8 v / w) of MTBE. The dried resin (compound 16) was then immersed in 1 mL of a 1% TFA solution in dichloromethane to remove the resin. From the LC A% of the Gly-capped compound (compound 17) and the target product (compound 18), the conversion rate was calculated to be 75% (68.9 ÷ (23.5 + 68.9)).

[0470] [Table 98]

[0471] [ka]

[0472] Based on the above [Condensation Experiment], the same reaction was carried out with some modifications to the [Condensation Experiment], and the results are shown below.

[0473] [Table 99]

[0474] [ka]

[0475] In the condensation reaction of Fmoc-cLeu-OH, a sterically hindered amino acid, it was found that the condensation reaction proceeded efficiently when DIC and Oxyma were used. Compared to Entry 1, when the additive was changed from Oxyma to HOAt, HOOBt, or TCNHPI (Entry 2 to Entry 4), the conversion rate significantly decreased. When Oxyma was not used (Entry 5), the conversion rate significantly decreased. When the solvent was changed from 8 v / w (Entry 1) to 4 v / w (Entry 6), the conversion rate improved. It was found that Oxyma was a superior additive. It was found that the conversion rate improved when Oxyma was used. It was found that the conversion rate improved when the reaction solvent was reduced.

[0476] [Reference Example 3] Synthesis of Fmoc-cLeu-MeVal-MeAsp(OCTC)-pip (Compound 15) under the reaction conditions described in Chem. Eur. J., 2009, 15, 9404-9416 (reaction conditions: Fmoc amino acid: condensing agent (COMU) = 3:3 for the amino acid of the resin) [ka] [Condensation experiment] 73 mg (0.21 mmol, 3.0 eq) of Fmoc-cLeu-OH and 89 mg (0.21 mmol, 3.0 eq) of COMU were weighed into a vial and dissolved in 0.80 mL (8 v / w) of N,N-dimethylformamide. 73 μL (0.42 mmol, 6.0 eq) of N,N-diisopropylethylamine was added to the solution and shaken at room temperature for 30 minutes. 0.10 g (0.63 mmol / g, 0.063 mmol, 1.0 eq) of dried resin (compound 14) was added to the reaction solution and shaken at room temperature for 16 hours. [Conversion rate confirmation experiment] In another vial, 62 mg (0.21 mmol, 3.3 eq) of Fmoc-Gly-OH and 30 mg (0.21 mmol, 3.3 eq) of Oxyma were weighed and dissolved in 0.40 mL (4 v / w) of N,N-dimethylformamide. 65 μL (0.42 mmol, 6.6 eq) of DIC was added to this solution and the mixture was shaken at room temperature for 1 hour. This solution was transferred to the Fmoc-cLeu-OH reaction solution and shaken at room temperature for 2 hours. The reaction solution was transferred to a disposable syringe with a filter, and the reaction solution and resin were filtered off. The resin was washed twice with 0.80 mL (8 v / w) of N,N-dimethylformamide and 0.80 mL (8 v / w) of isopropanol, then twice with 0.80 mL (8 v / w) of N,N-dimethylformamide and finally with 0.80 mL (8 v / w) of MTBE. The dried resin (compound 16) was then immersed in 1 mL of a 1% TFA solution in dichloromethane to remove the resin. From the LC A% of the Gly-capped compound (compound 17) and the target product (compound 18), the conversion rate was calculated to be 5.1% (4.7 ÷ (88.3 + 4.7)).

[0477] [Table 100]

[0478] [ka]

[0479] When COMU was used in the condensation reaction of Fmoc-cLeu-OH, a sterically hindered amino acid, the rate of the condensation reaction was 5%, and the unreacted Gly-capped form (compound 17) was the main product. [Industrial Applicability]

[0480] The present invention provides a method for efficiently producing a peptide compound containing a poorly reactive sequence that would result in an incomplete condensation reaction when conventional methods are applied.

Claims

[Claim 1] The invention described in the specification.

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