Peptide synthesis method for inhibiting defects caused by diketopiperazine formation

Abstract

The synthesis of peptides has the problem that diketopiperazine and 6-membered diamine backbone compounds formed when the protecting group at the N-terminus is removed hinder the progress of the desired elongation reaction. The present inventors found that, in the production of a peptide, a peptide in which an amino group at the N-terminal is protected by a protecting group having an Fmoc skeleton is treated in a specific solvent with a base having a pKa of 23 or more as a conjugated acid in acetonitrile, and then the peptide chain is extended, the above-mentioned problem can be solved.

Description

Technical Field

[0001] The present invention relates to a novel peptide synthesis method, which can synthesize a peptide with high purity and high efficiency in peptide synthesis. Background Art

[0002] In peptide synthesis, the Fmoc method is widely used because of its reliability and mild deprotection conditions (Fmoc deprotection conditions). In recent years, research on the application of peptides containing abundant N-alkyl amino acids (such as N-methyl amino acids) in medicine has been actively carried out, and it is known that these molecular species have acid instability (Non-patent Document 1 and Patent Document 1). Therefore, in these documents, the Fmoc method that can be deprotected under alkaline conditions is adopted, rather than the Boc method that requires acidic conditions to carry out the deprotection of the N-terminus.

[0003] The formation of diketopiperazines (DKP) in peptide synthesis is a problem that has been recognized for a long time. If the protecting group at the N-terminus of a dipeptide is supported on a solid phase via an ester bond, thereby exposing a free amino group, diketopiperazines may be formed. Several methods have been reported to ameliorate the problem of diketopiperazine formation in peptide synthesis using the Fmoc method.

[0004] Specifically, a method is known in which an Alloc group that undergoes a deprotection reaction under neutral conditions is used as a protecting group at a position where dionepiperazinedione formation is likely to occur, instead of a base, to suppress the formation of dionepiperazinedione (Non-Patent Document 2).

[0005] In addition, a method for avoiding the situation where the N-terminus is formed by a free amino group at a position where diketopiperazine formation is possible is known, in which a dipeptide containing such a sequence is synthesized in advance and used to extend the peptide chain (Non-Patent Document 3).

[0006] In addition, there is known a method of using a stronger base such as DBU or TBAF instead of piperidine which is generally used for deprotection of Fmoc to extremely shorten the time required for deprotection and thereby suppress the formation of diketopiperazine (Non-Patent Document 4).

[0007] All of these approaches are strategies directed toward the formation of diketopiperazines in dipeptides supported on a solid phase via ester bonds.

[0008] Citation List

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. WO 2018 / 225851 A1

[0011] Non-patent literature

[0012] Non-patent document 1: J. Peptide Res., 2005, 65, 153-166.

[0013] Non-patent document 2: Org. Lett., 2012, 14, 612-615.

[0014] Non-patent document 3: Solid Phase Peptide Synthesis published by Bachem (https: / / www.bachem.com / fileadmin / user_upload / pdf / Catalogs_Brochures / Solid_Phase_Peptide_Synthesis.pdf)

[0015] Non-patent document 4: Org. Lett., 2008, 10, 3857-3860. Summary of the invention

[0016] Technical issues

[0017] It is known that in a dipeptide supported on a solid phase via an ester bond, as described above, the ester bond is cleaved to form a diketopiperazine, and when the peptide sequence includes an N-substituted amino acid, an amide bond that is stronger than an ester bond may be cleaved to form a diketopiperazine, resulting in a deletion of the dipeptide containing the N-substituted amino acid in the peptide sequence. It is known that since such a deletion may occur at any position where an N-substituted amino acid exists in the peptide sequence, the dipeptide that may be deleted is not limited to the dipeptide supported on a solid phase.

[0018] Furthermore, the present inventors have found that when the peptide sequence includes an N-substituted amino acid, there is not only a problem of diketopiperazine formation, but also a problem of failure to carry out the desired elongation reaction and formation of an impurity having a 6-membered cyclic amidine skeleton (6-membered cyclic amidine skeleton compound) at the N-terminus instead of the target product. No method for solving these problems has been reported so far.

[0019] The above conventional techniques can be used not only to inhibit the elimination of diketopiperazines carried out with the ester bond of a dipeptide loaded on a solid phase, but also to inhibit the elimination of diketopiperazines carried out with an amide bond and the formation of a 6-membered cyclic amidine skeleton compound. However, the scope of its application is limited, and these techniques cannot provide sufficient solutions.

[0020] For example, the method described in Non-Patent Document 2 requires Alloc amino acids which are less versatile in availability than Fmoc amino acids.

[0021] In the peptide chain elongation method using a dipeptide described in Non-Patent Document 3, racemization of the amino acid of the dipeptide on the C-terminal side may be performed. Therefore, unless the amino acid of the dipeptide fragment on the C-terminal side is achiral (such as in the case of glycine) or racemization is impossible (such as in the case of proline), the method described herein is difficult to apply.

[0022] In addition, the method described in Non-Patent Document 4 is characterized in that the time required for the Fmoc deprotection step is very short, and the method requires an Fmoc deprotection step and a solid washing operation in less than one minute (e.g., 10 to 20 seconds). Such an extremely short time operation cannot be applied to industrial scale-up synthesis.

[0023] Furthermore, the present inventors have found that there is not only the problem of diketopiperazine formation and / or 6-membered cyclic amidine skeleton compound formation, but also the problem of formation of diurea compound impurities. So far, no method for solving the problem of diurea compound formation has been reported.

[0024] As mentioned above, there has never been proposed a method for suppressing diketopiperazine. The present invention is completed in view of the above situation, and in one aspect, the object of the present invention is to provide a peptide synthesis method capable of suppressing the formation of diketopiperazine, wherein (i) a protecting group containing an Fmoc skeleton is used as a protecting group at the N-terminus, (ii) the application of the sequence is not limited by racemization, and (iii) it can be used for expanded synthesis such as industrialization. On the other hand, the object of the present invention is to provide a peptide synthesis method capable of suppressing the formation of a 6-membered cyclic amidine skeleton compound. In addition, on the other hand, the object of the present invention is to provide a peptide synthesis method capable of suppressing the formation of a diurea compound.

[0025] Solution to the problem

[0026] The present inventors have found that, in the production of a peptide, a peptide having a protecting group containing an Fmoc skeleton is treated with a base having a pKa of 23 or more as a conjugate acid in acetonitrile in a specific solvent, and then an extended active substance such as an acid chloride, an active ester generated by using a condensing agent is reacted with the peptide, and the formation of a diketopiperazine and a 6-membered ring amidine skeleton compound and / or a diurea compound can be suppressed until the peptide chain after the removal of the protecting group is extended. Thus, the present invention has been completed.

[0027] That is, the present invention includes the following contents.

[0028] [0-1] A method for producing a peptide, the method comprising the following steps:

[0029] (1) providing a first peptide having a protecting group containing an Fmoc backbone;

[0030] (2) after step (1), treating the first peptide with one or more bases including at least a base whose conjugate acid has a pKa of 23 or greater in acetonitrile in a solvent containing a sulfoxide solvent; and

[0031] (3) After step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0032] [0-2] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton compound impurities formed in peptide production, the method comprising the following steps:

[0033] (1) providing a first peptide having a protecting group containing an Fmoc backbone; and

[0034] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0035] [0-3] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeleton compound impurities and diurea compound impurities formed in peptide production, the method comprising the following steps:

[0036] (1) providing a first peptide having a protecting group containing an Fmoc backbone; and

[0037] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0038] [0-4] According to the method described in [0-2] or [0-3], the method further comprises step (3): after step (2), in a solvent, in the presence or absence of a condensing agent, condensing the first peptide with a carboxylic acid or a carboxylic acid analogue to obtain a third peptide.

[0039] [0-5] The method according to any one of [0-1] to [0-4], which is performed by a solid phase method.

[0040] [0-6] The method according to any one of [0-1] to [0-4], wherein the first peptide is a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase.

[0041] [0-7] The method according to any one of [0-1] to [0-4], which is carried out by a liquid phase method.

[0042] [1] A method for producing a peptide by a solid phase method, the method comprising the following steps:

[0043] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase;

[0044] (2) after step (1), treating the first peptide with one or more bases including at least a base whose conjugate acid has a pKa of 23 or greater in acetonitrile in a solvent containing a sulfoxide solvent; and

[0045] (3) After step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0046] [2] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton compound impurities formed in peptide production by a solid phase method, the method comprising the following steps:

[0047] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and

[0048] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0049] [2-1] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeleton compound impurities and diurea compound impurities formed in peptide production, the method comprising the following steps:

[0050] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and

[0051] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0052] [3] The method according to [2] further comprises step (3): after step (2), condensing the first peptide with carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0053] [4] The method according to any one of [0-1] to [3], wherein step (2) is a step of removing the protecting group containing the Fmoc skeleton in the first peptide, and / or a step of converting the protecting group containing the Fmoc skeleton in the first peptide into a carbamate form.

[0054] [5] The method according to any one of [0-1] to [3], wherein the step of removing the protecting group containing the Fmoc backbone in the first peptide is substantially performed only in step (2).

[0055] [6] The method according to any one of [0-1] to [5], which does not include the step of treating the first peptide with piperidine as a single base before step (2).

[0056] [7] The method according to any one of [0-1] to [5], which does not include the step of treating the first peptide with a single base having a conjugate acid with a pKa of less than 23 in acetonitrile before step (2).

[0057] [8] The method according to any one of [0-1] to [7], wherein the solvent in step (2) is a solvent having a solid phase resin swelling capacity of 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more.

[0058] [9] The method according to any one of [0-1] to [8], wherein at least a portion of the first peptide obtained in step (2) is in the form of a carbamate.

[0059]

[10] The method according to [9], wherein the carbamate is a salt of a base having a pKa of 23 or more as a conjugate acid in acetonitrile.

[0060]

[11] The method according to [9] or

[10] , wherein the carbamate is a salt with at least one base selected from the group consisting of amidines, guanidines and phosphazenes.

[0061]

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

[11] , wherein the carbamate is a DBU salt, a TMG salt, a HP1(dma) salt, a MTBD salt, a P1-tBu salt or a P2-Et salt.

[0062]

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

[12] , wherein in step (2), the molar ratio of carbamate to amine form additionally formed during decarboxylation (carbamate / amine form) is 0.6 or greater, 0.8 or greater, 1.0 or greater, 2.0 or greater, 3.0 or greater, 4.0 or greater, 4.6 or greater, 5.0 or greater, 6.0 or greater, 8.0 or greater or 10.0 or greater.

[0063] [13-1] The method according to any one of [9] to

[12] , wherein in step (2), the molar ratio of carbamate to amine form additionally formed during decarboxylation (carbamate / amine form) is 0.6 or greater, 0.8 or greater, 1.0 or greater, 2.0 or greater, 3.0 or greater, 4.0 or greater, 4.6 or greater, 5.0 or greater, 6.0 or greater, 8.0 or greater, or 10.0 or greater, which is 1 The proton integration ratio in H-NMR was determined.

[0064]

[14] The method according to any one of [0-1] to

[13] , wherein the solvent in step (2) contains 50 v / v% or more of a sulfoxide solvent.

[0065]

[15] The method according to any one of [0-1] to

[14] , wherein the solvent in step (2) contains 70 v / v% or more of a sulfoxide solvent.

[0066]

[16] The method according to any one of [0-1] to

[15] , wherein the solvent in step (2) contains 90 v / v% or more of a sulfoxide solvent.

[0067]

[17] The method according to any one of [0-1] to

[16] , wherein the solvent in step (2) further contains one or more selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, phosphate solvents, amide solvents, urea solvents and sulfone solvents.

[0068]

[18] The method according to any one of [0-1] to

[16] , wherein the solvent in step (2) further contains one or more selected from the group consisting of an amide solvent, a urea solvent, and a sulfone solvent.

[0069]

[19] The method according to any one of [0-1] to

[13] , wherein the solvent in step (2) consists of a sulfoxide solvent.

[0070]

[20] The method according to any one of [0-1] to

[19] , wherein the sulfoxide solvent is one or more selected from the group consisting of DMSO, diethyl sulfoxide, methyl ethyl sulfoxide and methyl phenyl sulfoxide.

[0071]

[21] The method according to any one of [0-1] to

[19] , wherein the sulfoxide solvent is DMSO.

[0072]

[22] The method according to any one of [0-1] to

[21] , wherein the base in step (2) is at least one selected from the group consisting of amidines, guanidines and phosphazenes.

[0073]

[23] The method according to any one of [0-1] to

[22] , wherein the base in step (2) is at least one selected from the group consisting of DBU, MTBD, TMG, P1tBu, P2Et and HP1(dma).

[0074]

[24] The method according to any one of [0-1] to

[23] , wherein the base in step (2) is DBU, MTBD, TMG, P1tBu, P2Et or HP1(dma).

[0075]

[25] The method according to any one of [0-1] to

[23] , wherein the base in step (2) is (i) a combination of DBU and piperidine or (ii) a combination of DBU and MTBD, HP1(dma), P1tBu or P2Et.

[0076] [25-1] The method according to any one of [0-1] to

[25] , wherein in step (2), the solvent is DMSO and the base is DBU.

[0077] [25-2] The method according to any one of [0-1] to

[25] , wherein in step (2), the solvent is DMSO and the base is TMG.

[0078] [25-3] The method according to any one of [0-1] to

[25] , wherein in step (2), the solvent is DMSO, and the base is a combination of DBU and piperidine.

[0079]

[26] The method according to any one of [0-1] to

[25] , wherein the base in step (2) is contained in the solvent at a concentration of 1 to 8 v / v%.

[0080]

[27] The method according to any one of [0-1] to

[26] , wherein step (2) further includes a step of contacting the solvent with CO2.

[0081]

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

[27] , wherein step (2) further comprises a step of bubbling the solvent with CO2.

[0082]

[29] The method according to any one of [0-1] to

[26] , wherein the solvent in step (2) is a solvent that has been contacted with CO2 in advance.

[0083]

[30] The method according to any one of [0-1] to

[26] , wherein the solvent in step (2) is a solvent that has been pre-bubbled with CO2.

[0084]

[31] The method according to any one of [0-1] to

[30] , wherein step (2) is repeated two or more times.

[0085]

[32] The method according to

[31] , wherein when step (2) is repeated two or more times, the same solvent is used in the repetition of step (2).

[0086]

[33] The method according to

[31] , wherein when step (2) is repeated two or more times, different solvents are used in the repetitions of step (2).

[0087]

[34] The method according to any one of

[31] to

[33] , wherein when step (2) is repeated two or more times, the same base is used in the repetition of step (2).

[0088]

[35] The method according to any one of

[31] to

[33] , wherein when step (2) is repeated two or more times, different bases are used in the repetition of step (2).

[0089]

[36] According to the method described in any one of

[31] to

[35] , when step (2) is repeated two or more times, the method further comprises a step of draining the solution between repetitions of step (2).

[0090]

[37] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[36] , wherein the carboxylic acid or carboxylic acid analog is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 Aryl carboxylic acid; or an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 An active ester of an aromatic carboxylic acid; or an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 Acid halides of aryl carboxylic acids; wherein C1-C8 alkyl carboxylic acids and C6-C 10 The arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl and dialkylaminocarbonyl.

[0091]

[38] The method according to

[37] , wherein the protecting group is a carbamate type protecting group, a sulfonyl type protecting group or an acyl type protecting group.

[0092]

[39] The method according to

[38] , wherein the carbamate type protecting group is a protecting group containing an Fmoc skeleton, Alloc, Teoc, Boc or Cbz, the sulfonyl type protecting group is Ns, and the acyl type protecting group is Tfa.

[0093]

[40] The method according to

[37] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkyl carboxylic acid or a C6-C 10 Aryl carboxylic acid, or its active ester or acyl halide, and C1-C8 alkyl carboxylic acid C6-C 10 The arylcarboxylic acid is optionally substituted with one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl and halogen.

[0094]

[41] The method according to

[37] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkyl carboxylic acid or an active ester or acid halide thereof optionally substituted by one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl and halogen.

[0095]

[42] The method according to

[37] , wherein the carboxylic acid or carboxylic acid analog is a C1-C8 alkyl carboxylic acid or an active ester or acid halide thereof.

[0096]

[43] The method according to any one of [0-1] to

[42] , wherein the first peptide contains 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues.

[0097]

[44] The method according to any one of [0-1] to

[43] , wherein the first peptide is a dipeptide.

[0098]

[45] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[44] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group; or an active ester of an amino acid or a second peptide having a protecting group; or an acyl halide of an amino acid or a second peptide having a protecting group; wherein the first peptide and / or the second peptide having a protecting group contains one or more N-substituted amino acids, and / or the amino acid having a protecting group is an N-substituted amino acid.

[0099]

[46] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[45] , wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide having a protecting group containing an Fmoc skeleton; or an active ester of an amino acid or a second peptide having a protecting group containing an Fmoc skeleton; or an acyl halide of an amino acid or a second peptide having a protecting group containing an Fmoc skeleton; wherein the first peptide and / or the second peptide having a protecting group containing an Fmoc skeleton contains one or more N-substituted amino acids and / or the amino acid having a protecting group containing an Fmoc skeleton is an N-substituted amino acid.

[0100]

[47] The method according to any one of [0-1] to

[46] , wherein the amino acid at the second residue from the N-terminus of the first peptide is an N-substituted amino acid.

[0101]

[48] ​​The method according to

[46] or

[47] , wherein the N-substituted amino acid is an N-alkyl amino acid.

[0102]

[49] The method according to

[46] or

[47] , wherein the N-substituted amino acid is an N-C1-C6 alkyl amino acid.

[0103]

[50] The method according to

[46] or

[47] , wherein the N-substituted amino acid is an N-methyl amino acid.

[0104]

[51] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[50] , wherein the condensing agent in step (3) is in the form of a salt and its counter anion is PF6 - or BF4 - , or the condensing agent is a carbodiimide condensing agent.

[0105]

[52] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[50] , wherein the condensing agent in step (3) is in the form of a salt and its counter anion is PF6 - or BF4 - .

[0106]

[53] The method according to

[52] , wherein the condensing agent in step (3) contains at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU and TOTU.

[0107]

[54] The method according to

[52] or

[53] , wherein the condensing agent in step (3) is PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU or TOTU.

[0108]

[55] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[54] , wherein step (3) is performed in the presence of a base.

[0109]

[56] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[55] , wherein step (3) is carried out in the presence of water as a base having a pKa of 5 to 12 in the conjugate acid.

[0110]

[57] The method according to

[55] or

[56] , wherein the base in step (3) is DIPEA.

[0111]

[58] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[51] , wherein the condensing agent in step (3) is a carbodiimide condensing agent.

[0112]

[59] The method according to

[58] , wherein the carbodiimide condensing agent is at least one selected from the group consisting of diisopropylcarbodiimide (DIC), di-sec-butylcarbodiimide (DsBC), 1-tert-butyl-3-ethylcarbodiimide (tBEC), di-tert-butylcarbodiimide (DtBC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0113]

[60] The method according to

[58] or

[59] , wherein the carbodiimide condensation agent is diisopropylcarbodiimide (DIC) or di-sec-butylcarbodiimide (DSBC).

[0114]

[61] The method according to any one of [1],

[51] and

[58] to

[60] , wherein step (3) is performed in the presence of an additive.

[0115]

[62] The method according to

[61] , wherein the additive is at least one selected from the group consisting of HOAt, HOBt, HOOBt, Oxyma and Oxyma-B.

[0116]

[63] The method according to

[62] , wherein the additive is HOAt or Oxyma.

[0117]

[64] The method according to any one of [0-1] to

[63] , wherein the protecting group containing an Fmoc skeleton is represented by the following formula (1):

[0118] [Chem.1]

[0119]

[0120] in

[0121] R1 to R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl, and trimethylsilyl; and

[0122] R9 to R 10 Each is hydrogen or methyl.

[0123]

[65] The method according to

[64] , wherein the protecting group containing the Fmoc skeleton is an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br), a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, a sm-Fmoc group or a rm-Fmoc group.

[0124]

[66] The method according to

[65] , wherein the protecting group containing an Fmoc skeleton is an Fmoc group.

[0125]

[67] According to the method described in [0-1], [0-3] to [0-6] and [3] to

[66] , the method further includes a step of treating the mixture in step (2) between step (2) and step (3) (treating step).

[0126] [67-1] The method according to

[67] , wherein the treating step is a step of contacting an acid or a salt with the mixture.

[0127] [67-2] The method according to

[67] , wherein the treating step is a step of adding a sulfoxide solvent solution containing an acid or a salt to the mixture.

[0128] [67-3] The method according to

[67] , wherein the treating step is a step of adding a DMSO solution containing an acid or a salt to the mixture.

[0129]

[68] The method according to

[67] , wherein the treatment step is a step of washing the solid phase synthetic resin.

[0130] [68-1] The method according to

[68] , wherein the washing step includes a step of washing with a solvent containing a sulfoxide solvent.

[0131] [68-2] The method according to

[68] , wherein the washing step includes a step of washing with a solvent containing DMSO.

[0132] [68-3] The method according to

[68] , wherein the washing step includes a washing step with DMSO.

[0133] [68-4] The method according to

[68] , wherein the washing step includes a step of washing with a sulfoxide solvent solution containing a base.

[0134] [68-5] The method according to

[68] , wherein the washing step includes a step of washing with a DMSO solution containing an alkali.

[0135] [68-6] The method according to [68-4] or [68-5], comprising the step of washing with a DMSO solution containing at least one base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine and 2,4,6-trimethylpyridine.

[0136] [68-7] The method according to any one of [68-1] to [68-6], wherein the washing step is performed two, three, four, five or six times.

[0137] [68-8] The method according to any one of

[68] to [68-7], comprising a step of washing with a sulfoxide solvent solution containing an acid or a salt in one or more washing steps in the washing step.

[0138] [68-8-1] The method according to [68-8], which includes a step of washing with a sulfoxide solvent solution containing an acid in one or more washing operations in the washing step.

[0139] [68-8-2] The method according to [68-8], which includes a step of washing with a DMSO solution containing an acid in one or more washing operations in the washing step.

[0140] [68-8-3] The method according to [68-8], which includes a step of washing with a sulfoxide solvent solution containing a salt in one or more washing operations in the washing step.

[0141] [68-8-4] The method according to [68-8], which includes a step of washing with a DMSO solution containing a salt in one or more washing operations in the washing step.

[0142] [68-9] The method according to any one of

[68] to [68-7], comprising a step of washing with a sulfoxide solvent solution containing an acid and a salt in one or more washing operations in the washing step.

[0143] [68-10] The method according to any one of

[68] to [68-7], comprising a step of washing with a DMSO solution containing an acid and a salt in one or more washing operations in the washing step.

[0144] [68-11] The method according to any one of [68-8] to [68-11], wherein the number of washing operations in the washing step is 1.

[0145]

[69] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the acid is at least one selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid.

[0146] [69-1] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the acid is at least one selected from the group consisting of HOAt, Oxyma, 4-nitrophenol and HOOBt.

[0147] [69-2] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the salt is a salt formed by an acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid and a base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine and pyridine.

[0148] [69-3] The method according to any one of [67-1] to [68-3] and [68-8] to [68-11], wherein the salt is pyridine hydrochloride.

[0149] [69-4] The method according to [68-9] or [68-10], wherein the base is at least one selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-lutidine and pyridine.

[0150]

[70] The method according to

[68] , wherein the washing step comprises the following steps:

[0151] i) washing the solid phase synthetic resin with a solvent previously contacted with CO2; and / or

[0152] ii) Washing the solid phase synthetic resin with a solvent that has not been previously contacted with CO2.

[0153]

[71] The method according to

[68] , wherein the washing step comprises the following steps:

[0154] i) washing the solid phase synthetic resin with a solvent previously bubbled with CO2; and / or

[0155] ii) Washing the solid phase synthesis resin with a solvent that has not been previously bubbled with CO2.

[0156]

[72] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[71] , wherein the solvent in step (3) is the same as the solvent in step (2).

[0157]

[73] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[72] , wherein the solvent in step (3) is a solvent in which the condensing agent is soluble.

[0158]

[74] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[73] , wherein the solvent in step (3) contains at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents.

[0159]

[75] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[73] , wherein the solvent in step (3) contains 50 v / v% or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents.

[0160]

[76] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[73] , wherein the solvent in step (3) contains 70 v / v% or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents.

[0161]

[77] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[73] , wherein the solvent in step (3) contains 90 v / v% or more of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents.

[0162]

[78] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[73] , wherein the solvent in step (3) is composed of at least one selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents.

[0163]

[79] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[78] , wherein the solvent in step (3) is an aromatic hydrocarbon solvent, a halogen solvent, an ether solvent, an amide solvent, a sulfoxide solvent, a sulfone solvent, a urea solvent, an ester solvent, a ketone solvent, a carbonate solvent or a phosphate solvent.

[0164]

[80] The method according to any one of

[74] to

[79] , wherein the aromatic hydrocarbon solvent is one or more selected from the group consisting of benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene and isopropylbenzene,

[0165] The halogen solvent is one or more selected from the group consisting of dichloromethane, chloroform, 1,2-dichloroethane and carbon tetrachloride,

[0166] The ether solvent is one or more selected from the group consisting of diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,3-dioxolane, 1,4-dioxane, 1,2-dimethoxyethane, diisopropyl ether, tert-butyl methyl ether, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether and tetraethylene glycol dimethyl ether,

[0167] The amide solvent is one or more selected from the group consisting of DMF, NMP, DMA, NEP, NBP and formamide,

[0168] The sulfoxide solvent is one or more selected from the group consisting of DMSO and methylphenyl sulfoxide,

[0169] The sulfone solvent is one or more selected from the group consisting of diphenyl sulfone, dimethyl sulfone, sulfolane, 3-methyl sulfolane, ethyl methyl sulfone and ethyl isopropyl sulfone,

[0170] The urea solvent is one or more selected from the group consisting of DMI and DMPU,

[0171] The ester solvent is one or more selected from the group consisting of methyl acetate, ethyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, amyl acetate and γ-valerolactone,

[0172] The ketone solvent is one or more selected from the group consisting of acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, diethyl ketone and cyclopentanone,

[0173] The carbonate solvent is one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate and dibutyl carbonate, and

[0174] The phosphoric acid ester solvent is one or more selected from the group consisting of trimethyl phosphate, triethyl phosphate and tributyl phosphate.

[0175]

[81] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[80] , wherein the carboxylic acid used in step (3) is an amino acid or a second peptide having a protecting group containing an Fmoc skeleton, and the protecting group containing an Fmoc skeleton in the amino acid or the second peptide is the same as or different from the protecting group containing an Fmoc skeleton in the first peptide used in step (1).

[0176]

[82] The method according to any one of [0-1], [0-4] to [0-6] and [3] to

[81] , wherein the carboxylic acid analog used in step (3) is an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 The acyl halide of an aryl carboxylic acid, and the carboxyl group of an amino acid having a protecting group containing an Fmoc skeleton, the carboxyl group of the C-terminus of a second peptide having a protecting group containing an Fmoc skeleton, the carboxyl group of a C1-C8 alkyl carboxylic acid, or the carboxyl group of a C6-C 10 The carboxyl group of the aryl carboxylic acid is converted into an acyl halide group.

[0177] Although the methods of [1] to

[82] are methods for producing peptides by a solid phase method, these methods can also be applied to methods for producing peptides by a liquid phase method. That is, another aspect of the present invention provides the following

[83] to [84-1]. The methods described in

[83] to [84-1] may be the methods described in [3] to

[82] , which include features other than those inherent in the production method by a solid phase method.

[0178]

[83] A method for producing a peptide by a liquid phase method, the method comprising the following steps:

[0179] (1) providing a first peptide having a protecting group containing an Fmoc backbone;

[0180] (2) after step (1), treating the first peptide with one or more bases including at least a base whose conjugate acid has a pKa of 23 or greater in acetonitrile in a solvent containing a sulfoxide solvent; and

[0181] (3) After step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0182]

[84] A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton compound impurities formed in peptide production by a liquid phase method, the method comprising the following steps:

[0183] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and

[0184] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0185] [84-1] A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeleton compound impurities and diurea compound impurities formed in peptide production by a liquid phase method, the method comprising the following steps:

[0186] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and

[0187] (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

[0188] This method can efficiently obtain the desired peptide.

[0189] Effects of the Invention

[0190] In peptide synthesis, the present invention ensures that even if there is an amino acid sequence in which the desired extension reaction cannot be fully carried out under conventional conditions due to the formation of diketopiperazine and / or the formation of a 6-membered cyclic amidine skeleton compound, the formation of such impurities can be significantly reduced, thereby effectively extending the peptide chain to obtain a peptide having a desired amino acid sequence. Since the method of the present invention does not require the use of a special protective group such as Alloc with low versatility and has no restrictions on reaction operation, thereby achieving an extremely short reaction time, the method is a practical synthetic method with strong versatility and scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0191] [ Figure 1 ] Figure 1 The concentration of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) before and after application of Fmoc-Phe-NMe2 (compound 1-4-1) in N,N-dimethylformamide-d7 (DMF-d7) was shown to be between 3.6 ppm and 6.8 ppm. 1 H-NMR spectra and the characteristics of compound 1-4-1, compound 1-4-2, compound 1-4-3a and dibenzofulvene observed in this range 1 H signal.

[0192] exist Figure 1 In the figure, a) shows the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7).1 H-NMR spectrum.

[0193] exist Figure 1 b) shows a solution of compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0194] [ Figure 2 ] Figure 2 It is around 5.4 to 3.5 ppm 1 H-NMR spectrum, which shows that compound 1-4-2 and compound 1-4-3a formed by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) are chemically exchanged at a ratio of 82:18.

[0195] exist Figure 2 a)-1 shows a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0196] exist Figure 2 In a)-2, the solution selectively excited at 4.82 ppm by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) is shown. 1 The 1D-ROESY spectrum was obtained using the H signal.

[0197] exist Figure 2 In a)-3, the solution selectively excited at 3.95 ppm by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) is shown. 1 The 1D-ROESY spectrum was obtained using the H signal.

[0198] exist Figure 2 In b), it is shown 1 H-NMR spectrum, which shows that the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) has a concentration of 4.85 ppm. 1 H signal and 3.95ppm 1 The integral ratio between H signals.

[0199] [ Figure 3 ] Figure 3 It shows that the 1 H-NMR spectrum showing compound 1-4-2 formed by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7).

[0200] exist Figure 3 a)-1 shows the standard of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0201] exist Figure 3 a)-2 shows a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0202] exist Figure 3 a)-3 shows the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and adding 3 μL of a standard of compound 1-4-2. 1 H-NMR spectrum.

[0203] exist Figure 3 In a)-4, a)-5 shows the selective excitation of the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and adding 3 μL of a standard of compound 1-4-2 at 4.84 ppm. 1 1D-NOESY spectrum obtained by using H signal.

[0204] exist Figure 3 In a)-5, a)-5 shows the selective excitation of the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and adding 3 μL of a standard of compound 1-4-2 at 3.95 ppm. 1 1D-NOESY spectrum obtained by using H signal.

[0205] exist Figure 3b) indicates determination of a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and adding 3 μL of a standard of compound 1-4-2. 1 A graph of the integrated values ​​of proton signals at 4.84 ppm and 3.95 ppm in the H-NMR spectrum and the abundance ratio between the measured compound 1-4-3 and compound 1-4-2 calculated based on the integrated values.

[0206] [ Figure 4 ] Figure 4 It shows a range of 5.1 to 3.7 ppm 1 H-NMR and 1D-NOESY spectra showed that compound 1-4-3a was prepared by 13 Compound 1-4-2 was sparged with CO2 gas and then formed using 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7).

[0207] exist Figure 4 In the figure, a) shows the standard of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0208] exist Figure 4 b) shows the reaction of a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas for 1 minute 1 H-NMR spectrum.

[0209] exist Figure 4 In the example, c) shows the reaction of a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas for 1 minute was selectively excited at 4.77 ppm. 1 1D-NOESY spectrum obtained by using H signal.

[0210] exist Figure 4 In the figure, d) shows the reaction of a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas for 1 minute was selectively excited at 3.96 ppm. 1 1D-NOESY spectrum obtained by using H signal.

[0211] exist Figure 4In e), a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) is prepared by 13 The solution obtained by bubbling CO2 gas for 1 minute and then applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) 1 H-NMR spectrum.

[0212] exist Figure 4 f) shows that a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) is treated with 13 After bubbling CO2 for 1 minute, the concentration of 4.77 ppm 1 H signal and 3.96ppm 1 The integral ratio between the H signals 1 Figure 1 H-NMR spectrum.

[0213] exist Figure 4 e) is obtained by mixing a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas for 1 minute and then applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) 1 Amplification of the H-NMR spectrum at 4.83 ppm 1 Figure 2. Plot of the H signal. For this plot only, a transformation using Sin-bell as the window function was applied to improve the resolution.

[0214] [ Figure 5 ] Figure 5 Shows a range of 164 to 124 ppm 13 C-NMR spectrum, which shows that compound 1-4-3a is prepared by 13 Compound 1-4-2 was sparged with CO2 gas and then formed using 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) in N,N-dimethylformamide-d7 (DMF-d7).

[0215] exist Figure 5 In the figure, a) shows the reaction of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas 13 C-NMR spectrum.

[0216] exist Figure 5 b) shows the reaction of a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13The solution obtained by bubbling CO2 gas and then applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) 13 C-NMR spectrum.

[0217] [ Figure 6 ] Figure 6 It shows a range of 6.9 to 3.6 ppm. 1 H-NMR spectrum, which indicates that compound 1-4-3a exists in a solution (solution B, Sol B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7).

[0218] exist Figure 6 In the figure, a) shows a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7). 1 H-NMR spectrum.

[0219] exist Figure 6 In the figure, b) shows a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO2 gas for 1 minute and then mixing with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A) was 1 H-NMR spectrum.

[0220] exist Figure 6 In c), compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) is reacted with 13 The solution (Solution A) obtained by bubbling CO2 gas for 1 minute and then applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) 1 H-NMR spectrum.

[0221] [ Figure 7 ] Figure 7 Shown is NH-HSQC ( 15 N[F1]: about 97 to 85 ppm and 1 H[F2]: about 5.7 to 4.7 ppm), NH-HMBC ( 15 N[F1]: about 101 to 83 ppm and1 H[F2]: about 3.04 to 2.64 ppm) and CH-HMBC ( 13 C[F1]: about 165 to 158 ppm and 1 H[F2]: about 5.7 to 4.5 ppm) spectrum, which shows that compound 1-4-3a is present in a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and a solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) with 13 The solution obtained by bubbling CO 2 gas for 1 minute and then mixing with a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A).

[0222] exist Figure 7 In the figure, a) shows a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) to compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) 13 NH-HSQC spectrum of a solution obtained by bubbling CO 2 gas for 1 minute and then mixing with a solution obtained with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A).

[0223] exist Figure 7 In the figure, b) shows a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) to compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) 13 NH-HMBC spectrum of a solution obtained by bubbling CO 2 gas for 1 minute and then mixing with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A).

[0224] exist Figure 7 In the figure, c) shows a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) and a solution (solution B) obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) to compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) 13CH-HMBC spectrum of a solution obtained by bubbling CO 2 gas for 1 minute and then mixing with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A).

[0225] [ Figure 8 ] Figure 8 is a graph showing all the carbamate moieties of compound 1-4-3a present in the solution obtained by 1 H. 13 C and 15 Figure 1 shows N signal: a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) (solution B) and a solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to compound 1-4-1 in N,N-dimethylformamide-d7 (DMF-d7) (solution B). 13 CO2 was bubbled for 1 minute and then the solution obtained by applying 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (solution A) was mixed. DETAILED DESCRIPTION

[0226] (the term)

[0227] Examples of the "halogen atom" in the present specification include F, Cl, Br, and I.

[0228] In this specification, "alkyl" is a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, and has a hydrocarbon group or a subset of hydrocarbon group structures containing hydrogen and carbon atoms but not containing heteroatoms (which refer to atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton. Alkyl groups include not only straight chain forms but also branched forms. Alkyl groups are specifically alkyl groups having 1 to 20 carbon atoms (C1-C 20 ; below, "C p -C q " means an alkyl group having carbon atoms of p to q), preferably C1-C 10Alkyl, more preferably C1-C6 alkyl. Examples of alkyl specifically include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl (2-methylpropyl), n-pentyl, sec-pentyl (1-methylbutyl), tert-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl and 2-ethylbutyl.

[0229] In this specification, "alkenyl" is a group having at least one double bond (two adjacent SP 2 Depending on the configuration of the double bond and the substituents (if present), the double bond geometry may present an entgegen (E) or zusammen (Z) and a cis or trans configuration. Alkenyl includes not only straight-chain forms but also branched forms. Alkenyl is preferably C2-C 10 Alkenyl, more preferably C2-C6 alkenyl. Examples thereof specifically include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl and hexenyl.

[0230] In the present specification, "alkynyl" is a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain forms but also branched chain forms. Alkynyl is preferably C2-C 10 Alkynyl, more preferably C2-C6 alkynyl. Examples thereof specifically include ethynyl, 1-propynyl, propynyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3'-fluorophenyl)-2-propynyl and 3-methyl-(5-phenyl)-4-pentynyl.

[0231] In this specification, "cycloalkyl" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, and includes monocyclic, bicyclic and spirocyclic rings. Cycloalkyl is preferably C3-C8 cycloalkyl. Examples thereof specifically include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl and spiro[3.3]heptyl.

[0232] In the present specification, "aryl" means a monovalent aromatic hydrocarbon ring, and is preferably a C6-C 10Aryl. Examples of the aryl group specifically include phenyl and naphthyl (eg, 1-naphthyl and 2-naphthyl).

[0233] In this specification, "heterocyclic group" means a non-aromatic ring monovalent group containing carbon atoms and 1 to 5 heteroatoms. The heterocyclic group may have double bonds and / or triple bonds in the ring. The carbon atoms in the ring can be oxidized to form a carbonyl group, and the ring may be a monocyclic or condensed ring. The number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclic groups), more preferably 4 to 7 (4- to 7-membered heterocyclic groups). Examples of heterocyclic groups specifically include azetidinyl, oxiranyl, oxetanyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,2-thiazinane, thiazolidinyl diazolidinyl, oxazolidinone, benzodioxanyl, benzoxazolyl, dioxolane, dioxanyl, tetrahydropyrrolo[1,2-c]imidazole, thietanyl, 3,6-diazabicyclo[3.1.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 3-oxa-8-azabicyclo[3.2.1]octyl, sultam, and 2-oxaspiro[3.3]heptyl.

[0234] In the present specification, "heteroaryl" means an aromatic ring monovalent group containing carbon atoms and 1 to 5 heteroatoms. The ring may be a single ring or a condensed ring with other rings, and may also be partially saturated. The number of atoms constituting the ring is preferably 5 to 10 (5-membered to 10-membered heteroaryl), more preferably 5 to 7 (5-membered to 7-membered heteroaryl). Specific examples of heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiophenyl, benzothiadiazole, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnoazinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl and imidazopyridinyl.

[0235] In the present specification, "alkoxy" means an oxy group bonded to the "alkyl" defined above, and is preferably a C1-C6 alkoxy group. Examples of alkoxy groups specifically include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and 3-methylbutoxy.

[0236] In the present specification, "alkenyloxy" means an oxy group bonded to the "alkenyl" defined above, and is preferably a C2-C6 alkenyloxy group. Examples of alkenyloxy groups specifically include vinyloxy, allyloxy, 1-propenyloxy, 2-propenyloxy, 1-butenyloxy, 2-butenyloxy (including cis and trans), 3-butenyloxy, pentenyloxy and hexenyloxy.

[0237] In the present specification, "cycloalkoxy" means an oxy group bonded to the above-defined "cycloalkyl" and is preferably a C3-C8 cycloalkoxy. Examples of the cycloalkoxy group specifically include cyclopropyloxy, cyclobutyloxy and cyclopentyloxy.

[0238] In the present specification, "aryloxy" means an oxy group bonded to the above-defined "aryl" and is preferably a C6-C 10 Aryloxy group. Examples of the aryloxy group specifically include a phenoxy group, a 1-naphthyloxy group and a 2-naphthyloxy group.

[0239] In the present specification, the "heteroaryloxy group" means an oxy group bonded to the "heteroaryl group" defined above, and is preferably a 5- to 10-membered heteroaryloxy group.

[0240] In this specification, "amino" means -NH2 in a narrow sense, and means -NRR' in a broad sense. Herein, R and R' are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or R and R' form a ring together with the nitrogen atom to which they are bonded. Examples of amino preferably include -NH2, mono-C1-C6 alkylamino, di-C1-C6 alkylamino and 4- to 8-membered cyclic amino.

[0241] In the present specification, "monoalkylamino" means an "amino" group as defined above, wherein R is hydrogen, and R' is an "alkyl" as defined above, and is preferably a mono-C1-C6 alkylamino group. Examples of monoalkylamino groups specifically include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, sec-butylamino, and tert-butylamino.

[0242] In the present specification, "dialkylamino" means an "amino" group as defined above, wherein R and R' are each independently an "alkyl" as defined above, and is preferably a di-C1-C6 alkylamino group. Examples of dialkylamino groups specifically include dimethylamino and diethylamino.

[0243] In the present specification, "cyclic amino" means an "amino" group as defined above, wherein R and R' together with the nitrogen atom to which they are bonded form a ring, and preferably a 4- to 8-membered cyclic amino group. Examples of cyclic amino groups specifically include 1-azetidinyl, 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, 4-morpholinyl, 3-oxazolidinyl, 1,1-dioxothiomorpholinyl-4-yl and 3-oxa-8-azabicyclo[3.2.1]octan-8-yl.

[0244] In the present specification, "haloalkyl" means a group in which one or more hydrogen atoms of the above-defined "alkyl" are substituted by halogen, and is preferably a C1-C8 haloalkyl, more preferably a C1-C6 haloalkyl.

[0245] In the present specification, "fluoroalkyl" means a group in which one or more fluorine atoms of the "alkyl" defined above are substituted by halogen, preferably by C1-C8 haloalkyl. Examples of haloalkyl specifically include monofluoromethyl, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl.

[0246] In the present specification, "aralkyl (arylalkyl)" means a group in which at least one hydrogen atom of the above-defined "alkyl" is substituted by the above-defined "aryl" group, and is preferably C7-C 14 Arylalkyl, more preferably C7-C 10 Aralkyl group. Examples of the aralkyl group specifically include benzyl, phenethyl and 3-phenylpropyl.

[0247] In the present specification, "aralkyloxy" means an oxy group bonded to the "aralkyl" defined above, and is preferably a C7-C 14 Aralkyloxy, more preferably C7-C 10 Aralkyloxy. Examples of the aralkyloxy group specifically include benzyloxy, phenethoxy and 3-phenylpropoxy.

[0248] In this specification, "peptide chain" refers to a peptide chain of one or more natural amino acids and / or non-natural amino acids connected by amide bonds and / or ester bonds. The peptide chain is preferably a peptide chain comprising 1 to 15 amino acid residues, more preferably a peptide chain consisting of 5 to 12 amino acid residues.

[0249] As used herein, the term "peptide compound" is not particularly limited, as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are linked by amide bonds or ester bonds, and the peptide compound is preferably a peptide compound having 5 to 30 residues, more preferably a peptide compound having 8 to 15 residues, and even more preferably a peptide compound having 9 to 13 residues. The peptide compound synthesized in the present invention preferably contains at least three N-substituted amino acids in one peptide, more preferably at least 5 N-substituted amino acids. N-substituted amino acids may be present continuously or discontinuously in N-substituted cyclic peptide compounds. The peptide compound according to the present invention may be linear or cyclic, and is preferably a cyclic peptide compound. In this specification, "peptide residues" are sometimes referred to as "peptides".

[0250] The "cyclic peptide compound" in the present invention refers to a cyclic peptide compound obtained by cyclizing the group on the N-terminal side and the group on the C-terminal side in a linear peptide compound. The cyclization can be in any form, such as cyclization through a carbon-nitrogen bond (such as an amide bond), cyclization through a carbon-oxygen bond (such as an ester bond or an ether bond), cyclization through a carbon-sulfur bond (such as a thioether bond), cyclization through a carbon-carbon bond, or cyclization through a heterocyclic structure. Among them, it is preferred to cyclize through a covalent bond such as an amide bond or a carbon-carbon bond, and it is more preferred to cyclize through an amide bond of a carboxylic acid group on the side chain and an amino group on the n-terminus of the main chain. The position of the carboxylic acid group, amino group, etc. used for cyclization can be on the main chain or on the side chain, and there is no specific restriction, as long as the position allows these groups to be cyclized.

[0251] In this specification, the term "one or more" means 1 or 2 or more. When the term "one or more" is used in the context of a substituent of a particular group, the term means a number from 1 to the maximum number of substituents that the group accepts. Examples of the term "one or more" specifically include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0252] In this specification, "solid phase synthetic resin" is not particularly limited as long as it can be used to synthesize peptide compounds by solid phase method. Examples of solid phase synthetic resins specifically include resins that can be removed under acidic conditions, such as CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin) and 4-methyltrityl chloride resin (Mtt resin). The resin can be appropriately selected according to the functional group on the amino acid side used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (phenolic group represented by Tyr) is used as the functional group on the amino acid side, it is preferred to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin) as the resin. When an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) is used as the functional group on the amino acid side, it is preferred to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin) or 4-methyltrityl chloride resin (Mtt resin) as the resin. In this specification, resin is referred to as resin. Cellulose can be used as a solid phase. In this specification, the term "solid phase" can be used interchangeably with "solid phase synthetic resin".

[0253] The type of polymer constituting the resin is not particularly limited. For a resin consisting of polystyrene, a resin of 100 to 200 mesh or a resin of 200 to 400 mesh can be used. The degree of crosslinking is not particularly limited, and a resin crosslinked with 1% DVB (divinylbenzene) is preferred. Examples of the type of polymer constituting the resin include Tentagel and Chemmatrix.

[0254] If in the production of the compounds described in this specification, the defined groups undergo undesirable chemical transformations under the conditions of the implementation method, the compounds can be produced using methods such as protection or deprotection of functional groups. Herein, for the selection of protecting groups and the operation of desorption, for example, the methods described in "Greene's, "Protective Groups in Organic Synthesis" (5th edition, John Wiley & Sons 2014)" can be mentioned, and can be used appropriately according to the reaction conditions. If necessary, the order of the reaction steps can also be changed, such as by introducing substituents.

[0255] When the modifier "optionally substituted" is given in the present specification, examples of relevant substituents include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxy, alkoxycarbonyl and formyl.

[0256] Substituents may be added to each group. Such substituents are not limited and may be independently and freely selected from one or two or more of any substituents comprising halogen atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms and phosphorus atoms. Specifically, examples thereof include optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl and cycloalkyl.

[0257] The compound described in this specification may be a salt thereof, or a solvate thereof. Examples of salts described in this specification include: hydrochloride; hydrobromide; hydroiodide; phosphate; phosphonate; sulfate; sulfonate, such as methanesulfonate and p-toluenesulfonate; carboxylates, such as acetate, citrate, malate, tartrate, succinate and salicylate; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as magnesium and calcium salts; and ammonium salts, such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts and tetraalkylammonium salts. These salts are produced, for example, by contacting the relevant compound with an acid or a base. The solvate of the compound described in this specification refers to a group of molecules formed by the compound and a solvent, and there is no restriction as long as it is a solvate formed by a solvent. Its examples include not only solvates with a single solvent, such as hydrates, alcohol adducts (ethanol adducts, methanol adducts, 1-propanol adducts, 2-propanol adducts, etc.) and dimethyl sulfoxide, but also solvates formed with multiple solvents per compound molecule, or solvates formed with multiple types of solvents per compound molecule. When the solvent is water, the solvate is a hydrate. The solvate of the compound of the present invention is preferably a hydrate, and examples of the hydrate specifically include monohydrates to decahydrates, preferably monohydrates to pentahydrates, and more preferably monohydrates to trihydrates.

[0258] The term "amino acid" in this specification includes natural amino acids and non-natural amino acids. In this specification, "natural amino acid" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg or Pro. Non-natural amino acids are not particularly limited, and examples thereof include β-amino acids, D-type 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 amino acids herein, amino acids with any conformation are acceptable. The selection of the side chain of amino acids is not particularly limited, and in addition to hydrogen atoms, the side chain can also be freely selected from alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, spiral bond cycloalkyl groups, etc. Substituents can be added to each group. Substituents are also not limited, and one or two or more substituents can be independently and freely selected from any substituent including, for example, halogen atoms, O atoms, S atoms, N atoms, B atoms, Si atoms or P atoms. That is, examples of side chains include alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, or substituted cycloalkyl groups, oxo, aminocarbonyl and halogen atoms. In non-limiting terms, the amino acid in this specification can be a compound having a carboxyl group and an amino group in the same molecule (even in this case, the amino acid also includes imino acids, such as proline and hydroxyproline).

[0259] In the present specification, examples of the substituent containing a halogen atom include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group and an aralkyl group having a halogen as a substituent, and more specifically include a fluoroalkyl group, a difluoroalkyl group and a trifluoroalkyl group.

[0260] Examples of substituents containing an oxygen atom include a hydroxyl group (-OH), an oxy group (-OR), a carbonyl group (-C(=O)-R), a carboxyl group (-CO2H), an oxycarbonyl group (-C(=O)-OR), a carbonyloxy group (-OC(=O)-R), a thiocarbonyl group (-C(=O)-SR), a carbonylthio group (-SC(=O)-R), an aminocarbonyl group (-C(=O)-NHR), a carbonylamino group (-NH-C(=O)-R), an oxycarbonylamino group (-NH-C(=O)-OR), a sulfonylamino group (-NH-SO2-R), an aminosulfonyl group (-SO2-NHR), an aminosulfonyl group (-NH-SO2-NHR), a sulfonylamino group (-NH-SO2-NHR), a thiocarboxy group (-C(=O)-SH) and a carboxycarbonyl group (-C(=O)-CO2H).

[0261] Examples of the oxy group (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy and aralkyloxy. The alkoxy group is preferably a C1-C4 alkoxy group, a C1-C2 alkoxy group, and particularly preferably a methoxy group or an ethoxy group.

[0262] Examples of the carbonyl group (—C(═O)—R) include a formyl group (—C(═O)—H), an alkylcarbonyl group, a cycloalkylcarbonyl group, an alkenylcarbonyl group, an alkynylcarbonyl group, an arylcarbonyl group, a heteroarylcarbonyl group, and an aralkylcarbonyl group.

[0263] Examples of the oxycarbonyl group (—C(═O)—OR) include an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group and an aralkyloxycarbonyl group.

[0264] Examples of the carbonyloxy group (—OC(═O)—R) include an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group and an aralkylcarbonyloxy group.

[0265] Examples of the thiocarbonyl group (—C(═O)—SR) include an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group and an aralkylthiocarbonyl group.

[0266] Examples of the carbonylthio group (—SC(═O)—R) include an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group and an aralkylcarbonylthio group.

[0267] Examples of the aminocarbonyl group (—C(═O)—NHR) include an alkylaminocarbonyl group (e.g., a C1-C6 or C1-C4 alkylaminocarbonyl group, particularly an ethylaminocarbonyl group and a methylaminocarbonyl group), a cycloalkylaminocarbonyl group, an alkenylaminocarbonyl group, an alkynylaminocarbonyl group, an arylaminocarbonyl group, a heteroarylaminocarbonyl group and an aralkylaminocarbonyl group. Examples thereof also include a group in which the H atom bonded to the N atom in —C(═O)—NHR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, a aryl group, a heteroaryl group or an aralkyl group.

[0268] Examples of the carbonylamino group (-NH-C(=O)-R) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino and aralkylcarbonylamino. Examples thereof also include groups in which the H atom bonded to the N atom in -NH-C(=O)-R is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or an aralkyl group.

[0269] Examples of the oxycarbonylamino group (-NH-C(=O)-OR) include alkoxycarbonylamino, cycloalkyloxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, and aralkyloxycarbonylamino groups. Examples thereof also include groups in which the H atom bonded to the N atom in -NH-C(=O)-OR is further substituted by an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0270] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino and aralkylsulfonylamino. Examples thereof also include groups in which the H atom bonded to the N atom in -NH-SO2-R is further substituted by an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl group.

[0271] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl and aralkylaminosulfonyl. Examples thereof also include groups in which the H atom bonded to the N atom in -SO2-NHR is further substituted by an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl group.

[0272] Examples of sulfamoylamino (-NH-SO2-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino and aralkylsulfamoylamino. Two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with substituents each independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl, and the two substituents may form a ring.

[0273] Examples of the substituent containing an S atom include groups such as a thiol group (-SH), a sulfenyl group (-SR), a sulfinyl group (-S(=O)-R), a sulfonyl group (-SO2-R) and a sulfo group (-SO3H).

[0274] Examples of the thio group (—SR) which may be selected include an alkylthio group, a cycloalkylthio group, an alkenylthio group, an alkynylthio group, an arylthio group, a heteroarylthio group, an aralkylthio group and the like.

[0275] Examples of the sulfonyl group (-SO2-R) include an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group and an aralkylsulfonyl group.

[0276] Examples of substituents containing a nitrogen atom include groups such as azide (-N3; also known as an "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R; also known as a monosubstituted amino group), tertiary amino (-NR(R'); also known as a disubstituted amino group), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR"')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridinyl, piperidinyl, morpholino and azetidinyl.

[0277] Examples of the secondary amino group (—NH—R: monosubstituted amino group) include an alkylamino group, a cycloalkylamino group, an alkenylamino group, an alkynylamino group, an arylamino group, a heteroarylamino group and an aralkylamino group.

[0278] Examples of tertiary amino groups (-NR(R'): disubstituted amino groups) include amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and aralkyl groups, for example, alkyl (aralkyl) amino groups. These any two substituents may form a ring. Examples thereof specifically include dialkylamino groups, specifically C1-C6 dialkylamino groups, C1-C4 dialkylamino groups, dimethylamino groups and diethylamino groups. In this specification, "C p -C q "Dialkylamino" refers to an amino group separated by two C p -C q Alkyl group substituted group. p -C q The alkyl groups may be the same or different.

[0279] Examples of substituted amidino groups (—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.

[0280] Examples of substituted guanidine groups (-NR-C(=NR'")-NR'R") include groups wherein R, R', R", and R'" are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and groups wherein these substituents form a ring.

[0281] Examples of the aminocarbonylamino group (-NR-CO-NR'R") include groups in which R, R' and R" are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group and an aralkyl group, and groups in which these substituents form a ring.

[0282] In the present specification, the "amino acid residue" constituting a peptide compound may be simply referred to as "amino acid".

[0283] The term "N- substituted amino acid" in this specification means that the main chain amino group in the amino acid defined above is an N- substituted amino acid. Examples of N- substituted amino acids specifically include N- substituted amino acids in which the main chain amino group of the amino acid is an NHR group and R is any group other than hydrogen, such as an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group or a cycloalkyl group, and a cyclic amino acid (such as in proline) in which a carbon atom is bonded to a carbon atom at a N atom and a position α to form a ring. Examples thereof include proline forming a 5-membered ring, azetidine forming a 4-membered ring, and piperidine forming a 6-membered ring. The substituent group of each optionally substituted group is not particularly limited, and examples thereof include a halogen group, an ether group, and a hydroxyl group.

[0284] The "N-alkyl amino acid" in the present invention refers to an amino acid in which the main chain amino acid is substituted by an alkyl group in the N-substituted amino acid defined above. Examples of N-alkyl amino acids specifically include N-C1-C 10 Alkyl amino acids and N-C1-C6 alkyl amino acids. In addition, examples of N-alkyl amino acids specifically include N-methyl amino acids, N-ethyl amino acids, N-(n-propyl) amino acids, and N-(isopropyl) amino acids.

[0285] The "amino acid" in this specification includes all corresponding isotopes. An isotope of an "amino acid" is an isotope in which at least one atom is replaced by an atom having the same atomic number (same number of protons) and a different mass number (a different sum of protons and neutrons). Examples of isotopes included in the "amino acid" herein include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., and they include 2 H. 3 H; 13 C. 14 C; 15 N; 17 O. 18 O; 32 P; 35 S; 18 F; 36 Cl et al.

[0286] The term "proline" in this specification means proline and a group of compounds having one or more arbitrary substituents bonded to any carbon atom forming a 5-membered ring of proline. When there are multiple substituents on the 5-membered ring, the substituents can form a ring together, and the ring can be any aromatic ring. Examples of "proline" include proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-4-fluoro-L-proline, cis-4-fluoro-L-proline and 2-methyl-L-proline. When proline has a hydroxyl group, the hydroxyl group can be protected by an arbitrary protecting group, or an arbitrary substituent can form an ether bond or an ester bond with the oxygen atom of the hydroxyl group. In addition, any of the carbon atoms forming the 5-membered ring can be substituted by an oxygen atom or a sulfur atom. Examples of such compounds specifically include L-thioproline. In addition, proline can have an unsaturated bond in the formed 5-membered ring. Examples of such compounds specifically include 3,4-dehydro-L-proline. They are not necessarily L-type, but can also be D-type.

[0287] In this specification, the term "azetidine" means a cyclic amino acid based on the following compound: one of the nitrogen atom and three carbon atoms forms a 4-membered ring, and the carboxyl group is bonded to the carbon atom adjacent to the nitrogen atom. One or more arbitrary substituents can be bonded to any carbon atom forming a 4-membered ring. When there are multiple substituents on the 4-membered ring, the substituents can form a ring together. The example of "azetidine" specifically includes (S)-azetidine-2-carboxylic acid and (R)-azetidine-2-carboxylic acid. "Azetidine" also includes compounds in which the carboxyl group is bonded to the carbon atom next to the nitrogen atom to form a 4-membered ring. The example of this compound includes azetidine-3-carboxylic acid.

[0288] In this specification, the term "piperidine" means a cyclic amino acid based on the following compound: one nitrogen atom and five carbon atoms form a 6-membered ring, and a carboxyl group is bonded to the carbon atom adjacent to the nitrogen atom. One or more arbitrary substituents may be bonded to any carbon atom forming the 6-membered ring. When there are multiple substituents on the 6-membered ring, the substituents may form a ring together, and the ring may be any aromatic ring. Specific examples of "piperidine" include (R)-piperidine-2-carboxylic acid, (S)-piperidine-2-carboxylic acid, (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0289] In the present specification, examples of the aromatic hydrocarbon solvent include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, and cumene.

[0290] In the present specification, examples of the halogen solvent include dichloromethane, chloroform, 1,2-dichloroethane and carbon tetrachloride.

[0291] In the present specification, examples of the ether solvent include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, tert-butyl methyl ether, 4-methyltetrahydropyran, diglyme, triglyme and tetraglyme.

[0292] In the present specification, examples of the amide solvent include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), M-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP) and formamide.

[0293] In the present specification, examples of the sulfoxide solvent include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide.

[0294] In the present specification, examples of the sulfone solvent include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methylsulfolane, ethyl methyl sulfone and ethyl isopropyl sulfone.

[0295] In the present specification, examples of the urea solvent include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0296] In the present specification, examples of the ester solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, amyl acetate and γ-valerolactone.

[0297] In the present specification, examples of the ketone solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, and diethyl ketone.

[0298] Examples of the carbonate solvent include dimethyl carbonate, diethyl carbonate, and dibutyl carbonate.

[0299] Examples of the phosphoric acid ester solvent include trimethyl phosphate, triethyl phosphate, and tributyl phosphate.

[0300] In the present specification, the term "amidine" means a base represented by the following formula B1:

[0301] [Chem.2]

[0302]

[0303] in

[0304] RB1 and RB4 are each independently a C1-C4 alkyl group, or RB1 and RB4, together with the nitrogen atom to which RB1 is bonded and the carbon atom to which RB4 is bonded, form a 5- to 8-membered ring.

[0305] RB2 and RB3 are each independently a C1-C4 alkyl group, or RB2 and RB3, together with the nitrogen atom to which RB2 is bonded, the nitrogen atom to which RB3 is bonded, and the carbon atom to which the nitrogen atom is bonded, form a 5- to 8-membered ring.

[0306] When RB1 to RB4 are C1-C4 alkyl groups, the C1-C4 alkyl group is preferably a methyl group or an ethyl group.

[0307] When RB1 and RB4 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring or the like.

[0308] When RB2 and RB3 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring or the like.

[0309] Examples of amidines specifically include DBU: 1,8-diazabicyclo[5.4.0]-7-undecene and DBN: 1,5-diazabicyclo[4.3.0]-5-nonene.

[0310] In the present specification, the term "guanidine" means a base represented by the following formula B2:

[0311] [Chem.3]

[0312]

[0313] in

[0314] RB6 is hydrogen or C1-C4 alkyl,

[0315] RB5 and RB7 are each independently a C1-C4 alkyl group, or form a 5- to 8-membered ring together with the nitrogen atom to which RB5 and RB7 are bonded and the carbon atom to which the nitrogen atom is bonded, and

[0316] RB8 is C1-C4 alkyl, or RB9 is hydrogen, C1-C4 alkyl or phenyl, or RB8 and RB9 together with the nitrogen atom to which RB8 and RB9 are bonded and the carbon atom to which the nitrogen atom is bonded form a 5- to 8-membered ring,

[0317] When RB9 is a phenyl group, two benzene rings having a phenyl group in the formula B2 are condensed to form naphthalene.

[0318] When RB5 to RB8 are C1-C4 alkyl groups, the C1-C4 alkyl group is preferably a methyl group, and when RB9 is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably a tert-butyl group.

[0319] When RB5 and RB7 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably an imidazolidine ring, a hexahydropyrimidine ring, 1,3-diazepane, or the like.

[0320] When RB8 and RB9 form a 5- to 8-membered ring, the 5- to 8-membered ring is preferably a 1,4,5,6-tetrahydropyrimidine ring or the like.

[0321] Examples of guanidine specifically include TMG: 1,1,3,3-tetramethylguanidine, TBD: 1,5,7-triazabicyclo[4.4.0]deca-5-ene, and MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene.

[0322] In this specification, the term "phosphazene" means a base represented by the following formula B3 or the following formula B4:

[0323] [Chem.4]

[0324]

[0325] in

[0326] RB 10 is C1-C4 alkyl, or RB 10 and RB 11 and RB 10 and RB 11 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0327] RB 11 is C1-C4 alkyl, unless RB 10 and RB 11 Form 5- to 8-membered rings, or RB 11 and RB 12 and RB 10 and RB 11 The nitrogen atom bonded thereto and the phosphorus atom bonded thereto together form a 5- to 8-membered ring,

[0328] RB 12 is C1-C4 alkyl, unless RB 11 and RB 12 Form 5- to 8-membered rings, or RB 12 and RB 13 and RB 12 and RB 13 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0329] RB 13 is C1-C4 alkyl, unless RB 12 and RB 13 Form 5- to 8-membered rings, or RB 13 and RB 14 and RB 13 and RB 14The nitrogen atom bonded thereto and the phosphorus atom bonded thereto together form a 5- to 8-membered ring,

[0330] RB 14 is C1-C4 alkyl, unless RB 13 and RB 14 Form 5- to 8-membered rings, or RB 14 and RB 15 and RB 14 and RB 15 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0331] RB 15 is C1-C4 alkyl, unless RB 14 and RB 15 Forming a 5- to 8-membered ring, and

[0332] RB 16 is hydrogen, C1-C8 alkyl or C6-C 10 Aryl.

[0333] When RB 10 To RB 15 When RB is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and when RB 16 When it is a C1-C8 alkyl group, the C1-C8 alkyl group is preferably a tert-butyl group or a tert-octyl group.

[0334] When RB 10 and RB 11 , RB 12 and RB 13 , and / or RB 14 and RB 15 When a 5- to 8-membered ring is formed, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring or the like.

[0335] RB 11 and RB 12 , and / or RB 13 and RB 14 Form a 5- to 8-membered ring, preferably the 5- to 8-membered ring does not contain any 11 , RB 12 , RB 13 and RB 14 A 5- to 8-membered saturated ring having a hetero atom other than the nitrogen atom bonded to the phosphorus atom and the nitrogen atom bonded to the phosphorus atom.

[0336] [Chem.5]

[0337]

[0338] in

[0339] RB 17 are independently C1-C4 alkyl, or RB 17 and RB 18 and RB 17 and RB 18 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0340] RB 18 is C1-C4 alkyl, unless RB 17 and RB 18 Form 5- to 8-membered rings, or RB 18 and RB 19 and RB 18 and RB 19 The nitrogen atom bonded thereto and the phosphorus atom bonded thereto together form a 5- to 8-membered ring,

[0341] RB 19 is C1-C4 alkyl, unless RB 18 and RB 19 Form 5- to 8-membered rings, or RB 19 and RB 20 and RB 19 and RB 20 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0342] RB 20 is C1-C4 alkyl, unless RB 19 and RB 20 Forming a 5- to 8-membered ring,

[0343] RB 21 is C1-C4 alkyl, or RB 21 and RB 22 and RB 21 and RB 22 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0344] RB 22 is C1-C4 alkyl, unless RB 21 and RB 22 Form 5- to 8-membered rings, or RB 22 and RB 23 and RB 22 and RB 23 The nitrogen atom bonded thereto and the phosphorus atom bonded thereto together form a 5- to 8-membered ring,

[0345] RB 23 is C1-C4 alkyl, unless RB 22 and RB 23 Form 5- to 8-membered rings, or RB 23 and RB24 and RB 23 and RB 24 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0346] RB 24 is C1-C4 alkyl, unless RB 23 and RB 24 Form 5- to 8-membered rings, or RB 24 and RB 25 and RB 24 and RB 25 The nitrogen atom bonded thereto and the phosphorus atom bonded thereto together form a 5- to 8-membered ring,

[0347] RB 25 is C1-C4 alkyl, unless RB 24 and RB 25 Form 5- to 8-membered rings, or RB 25 and RB 26 and RB 25 and RB 26 The bound nitrogen atoms together form a 5- to 8-membered ring,

[0348] RB 26 is C1-C4 alkyl, unless RB 25 and RB 26 Forming a 5- to 8-membered ring,

[0349] RB 27 is C1-C4 alkyl or C6-C 10 Aryl.

[0350] When RB 17 To RB 26 When RB is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl or ethyl, and when RB 27 When it is a C1-C4 alkyl group, the C1-C4 alkyl group is preferably an ethyl group or a tert-butyl group.

[0351] When RB 17 and RB 18 , RB 19 and RB 20 , RB 21 and RB 22 , RB 23 and RB 24 , and / or RB 25 and RB 26 When a 5- to 8-membered ring is formed, the 5- to 8-membered ring is preferably a pyrrolidine ring, a piperidine ring, an azepane ring or the like.

[0352] When RB 17 and RB 18When all are C1-C4 alkyl, RB is preferred 19 and RB 20 are all C1-C4 alkyl, and preferably RB 17 and RB 18 Forming 5- to 8-membered rings and RB 19 and RB 20 It also forms 5- to 8-membered rings.

[0353] When RB 21 and RB 22 When all are C1-C4 alkyl, RB is preferred 23 , RB 24 , RB 25 and RB 26 All are C1-C4 alkyl, and preferably RB 21 and RB 22 Forming 5- to 8-membered rings and RB 23 and RB 24 and RB 25 and RB 26 They form 5- to 8-membered rings, respectively.

[0354] RB 18 and RB 19 , and / or RB 22 and RB 23 Form a 5- to 8-membered ring, preferably the 5- to 8-membered ring does not contain any 11 , RB 12 , RB 13 and RB 14 A 5- to 8-membered saturated ring having a hetero atom other than the nitrogen atom bonded to the phosphorus atom and the nitrogen atom bonded to the phosphorus atom.

[0355] Specific examples of phosphazenes include P2tBu: 1-tert-butyl-2,2,4,4,4-penta(dimethylamino)-2λ 5 ,4λ 5 -bis(phosphazene), P2Et: tetramethyl(tris(dimethylamino)phosphoranylidene)phosphoric acid triamide-ethylimide, HP1(dma): imino-tris(dimethylamino)phosphine, BTPP: tert-butylimino-tris(pyrrolidine)phosphine, P1tBu: tert-butylimino-tris(dimethylamino)phosphine and BEMP: 2-tert-butylamino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphine.

[0356] In this specification, the term "and / or" is intended to include every combination of the terms "and" and "or" in appropriate combinations. Specifically, for example, the term "A, B and / or C" includes the following seven variations:

[0357] (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0358] (Production Method)

[0359] The present invention relates to a method for producing a peptide by a solid phase method, the method comprising the following steps (1) to (3):

[0360] (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase;

[0361] (2) after step (1), treating the first peptide with one or more bases (including at least a base having a pKa of 23 or greater) as a conjugate acid in acetonitrile in a solvent containing a sulfoxide solvent; and

[0362] (3) After step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

[0363] The term "first peptide" in step (2) means a first peptide having a protecting group containing an Fmoc backbone.

[0364] Step (1)

[0365] Step (1) in the present invention is a step of providing a first peptide having a protecting group containing an Fmoc skeleton and supported on a solid phase.

[0366] The "first peptide having a protective group containing an Fmoc skeleton and supported on a solid phase" in step (1) is usually supported on a solid phase synthetic resin via a functional group (e.g., a carboxyl group) in its C-terminal amino acid residue, but may be supported on a solid phase via a functional group (e.g., a carboxyl group) in an amino acid residue other than the C-terminal. The amino group of the amino acid residue at the N-terminal of the "first peptide having a protective group containing an Fmoc skeleton and supported on a solid phase" is protected by a protective group containing an Fmoc skeleton.

[0367] As a solid phase for loading the first peptide, any solid phase known in the relevant art can be used, and generally, the resin is connected to a predetermined amino acid residue (e.g., an amino acid residue at the C-terminus) of the first peptide. It is particularly preferred that the resin has a resin-bonded group classified as "H (TFA in DCM <5%)" according to the acid sensitivity described in the Handbook of Solid Phase Synthesis (Merck, published on May 1, 2002), and the resin can be appropriately selected according to the functional group on the amino acid side used.

[0368] For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (a phenolic group represented by Tyr) is used as a functional group on the amino acid side, it is preferred to use a trityl chloride resin (Trt resin) or a 2-chlorotrityl chloride resin (Clt resin) as a resin. When an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) is used as a functional group on the amino acid side, it is preferred to use a trityl chloride resin (Trt resin), a 2-chlorotrityl chloride resin (Clt resin) or a 4-methyltrityl chloride resin (Mtt resin) as a resin.

[0369] In the present invention, "a protecting group containing an Fmoc skeleton" means a group in which an arbitrary substituent is introduced at an arbitrary position on the Fmoc group or the skeleton constituting the Fmoc group. Examples of such a protecting group containing an Fmoc skeleton specifically include a protecting group which can be represented by the following formula (1):

[0370] [Chem.6]

[0371]

[0372] in

[0373] R1 to R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl, and trimethylsilyl; and

[0374] R9 to R 10 Each is hydrogen or methyl.

[0375] Examples of the protecting group containing an Fmoc skeleton more specifically include a 9-fluorenylmethoxycarbonyl (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, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, and a 2,7-diisooctyl-Fmoc (dio-Fmoc) group. 7-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc (tdf-Fmoc) group, 2,7-bis(trimethylsilyl)-Fmoc (Fmoc(2TMS)) group, (2-sulfo-9H-fluorene-9-yl)methoxycarbonyl group (Fmoc(2so3h)), [(1S)-1-(9H-fluorene-9-yl)ethoxy]carbonyl group (sm-Fmoc) and [(1R)-1-(9H-fluorene-9-yl)ethoxy]carbonyl group (rm-Fmoc). Such a protecting group containing an Fmoc skeleton is preferably an Fmoc group. Such a protecting group containing an Fmoc skeleton can be introduced by a known method using a commercially available reagent or the like.

[0376] In the present invention, the type and number of amino acid residues constituting the first peptide are not limited, and a peptide having any amino acid sequence including 2 or more amino acid residues, for example, 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues can be used as the first peptide. For amino acids, natural amino acids and / or non-natural amino acids can be used. Preferably, the first peptide contains one or more N-substituted amino acids.

[0377] The first peptide having a protecting group containing an Fmoc backbone provided in step (1) (e.g., a first peptide containing a protecting group containing an Fmoc backbone and loaded on a solid phase) can be obtained from a commercial supplier or produced by applying methods known in the relevant art, such as the method shown in WO 2013 / 100132 or WO 2018 / 225864.

[0378] In some aspects, the first peptide is a dipeptide. In this case, the amino acid residue at the C-terminus of the dipeptide is loaded on a solid phase, for example, via an ester bond, and as shown in the following scheme, the amino acid residue at the N-terminus may react with the carbonyl group in the ester bond to form a poor DKP. By using the method of the present invention, the reduction in the yield of the target peptide due to the elimination of the DKP can be suppressed.

[0379] [Chem.7]

[0380]

[0381] wherein R1 to R4 each represent an arbitrary atom or group, and * represents a position bonding to a solid phase synthetic resin.

[0382] In some aspects, the first peptide is a peptide in which the amino acid at the second residue from its N-terminus is an "N-substituted amino acid". In this case, as shown in the following scheme, the free amino group produced by removing the protecting group of the N-terminal amino acid residue can react with the carbonyl group in the peptide bond between the amino acids of the second residue and the third residue from the N-terminus, so that an undesirable DKP, a DKP elimination compound or an impurity having an amidine skeleton (a 6-membered ring amidine skeleton compound) can be formed. By using the method of the present invention, the formation of DKP, a DKP elimination compound and a 6-membered ring amidine skeleton compound can be inhibited, thereby preventing the yield of the target peptide from being reduced.

[0383] [Chem.8]

[0384]

[0385] Wherein R4 represents any atom or group except hydrogen, R1 to R3 and R5 to R8 each represent any atom or group, and * represents the position bonded to the adjacent amino acid residue or the solid phase, wherein when R6 is hydrogen, both Path A and Path B in the scheme can be used, and when R6 is not hydrogen, only Path A can be used.

[0386] As the "N-substituted amino acid" in the present invention, preferably, there are mentioned N-alkyl amino acids obtained by N-alkylation such as N-methylation, and cyclic amino acids such as proline and azetidine.

[0387] Step (2)

[0388] Step (2) of the present invention is a step of treating the first peptide provided in step (1) with one or more bases (including at least a base having a pKa of 23 or greater) as a conjugate acid in acetonitrile in a solvent containing a sulfoxide solvent.

[0389] In certain aspects, at least a portion of the first peptide obtained in step (2) is in the form of a carbamate. Without being bound by a particular theory, in step (2), dibenzofulvene is formed without decarboxylation to remove the protecting group containing the Fmoc backbone in at least a portion of the first peptide, so that the amino group at the N-terminus is bound to the C(=O)O derived from the protecting group. - Formation of carbamate ions (-NRC(=O)O -, wherein R is hydrogen or any substituent of the amino group), and the carbamate ion forms carbamate with the protonated base in the system (the following is a schematic diagram showing the formation of carbamate when Fmoc is used as a protecting group containing an Fmoc skeleton and DBU is used as a base).

[0390] [Chem.9]

[0391]

[0392] When carbamate is formed, the intramolecular nucleophilic reaction of the terminal amino group can be suppressed, so the formation of the 6-membered ring intermediate can be suppressed even under alkaline deprotection conditions, thereby reducing the formation of DKP elimination compounds and 6-membered ring amidine skeleton compounds as by-products.

[0393] In some aspects, at least a portion of the first peptide obtained in step (2) is in the form of a carbamate. Without being bound by a particular theory, it is believed that when the carbamate is stably present until step (3), the isocyanate formed by the reaction of the condensing agent with the carbamate anion undergoes a nucleophilic attack by the amino group of the adjacent N-terminus, thereby undergoing an intermolecular reaction (herein, the term "intermolecular" is used even if the molecules are supported on the same solid phase) to form a diurea compound.

[0394] [Chem.10]

[0395]

[0396] Therefore, in the step of treating the mixture in step (2) (for example, the step of washing the solid phase synthetic resin), the carbamate can be decomposed and converted into an amine compound to reduce the formation of the dimerurea compound in step (3).

[0397] In the method of the present invention, wherein a base having a pKa of 23 or greater in acetonitrile as a conjugate acid is used in step (2), the first peptide may be present in the form of a carbamate, which can significantly reduce the formation of DKP elimination compounds and 6-membered cyclic amidine skeleton compounds. For example, in the method of the present invention, the carbamate may be formed in an amount such that the molar ratio of the carbamate to the amine form additionally formed as the decarboxylation proceeds (carbamate / amine) is 0.6 or greater, 0.8 or greater, 1.0 or greater, 2.0 or greater, 3.0 or greater, 4.0 or greater, 4.6 or greater, 5.0 or greater, 6.0 or greater, 8.0 or greater, or 10.0 or greater. Such a molar ratio can be obtained by treating a solution of an amino acid having an amide compound (e.g., a dimethylamide compound) containing a protective group for an Fmoc skeleton at the N-terminus of the first peptide with a base. 1 The proton integration ratio in H-NMR was used to determine the

[0398] The carbamate is a salt of any base having a pKa of 23 or more as a conjugate acid in acetonitrile. Examples of such salts specifically include DBU salts, TMG salts, HP1(dma) salts, MTBD salts, P1-tBu salts, and P2-Et salts.

[0399] In certain aspects, the solvent used in step (2) may contain a sulfoxide solvent, and may be a single solvent or a mixed solvent. In the case of a mixed solvent, it is preferred that the solvent contain 50 v / v% or more, 70 v / v% or more, or 90 v / v% or more of a sulfoxide solvent.

[0400] Examples of the sulfoxide solvent used in step (2) specifically include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide and methyl phenyl sulfoxide, among which DMSO is preferred.

[0401] When the solvent used in step (2) is a mixed solvent, in addition to the sulfoxide solvent, the mixed solvent may contain one or more solvents selected from the group consisting of aromatic hydrocarbon solvents, halogen solvents, ether solvents, ester solvents, ketone solvents, carbonate solvents, phosphate solvents, amide solvents, urea solvents and sulfone solvents.

[0402] In certain aspects, when the solvent used in step (2) is a mixed solvent, the mixed solvent may contain one or more solvents selected from the group consisting of an amide solvent, a urea solvent, and a sulfone solvent in addition to the sulfoxide solvent.

[0403] When an aromatic hydrocarbon solvent is used in step (2), examples of the solvent specifically include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene and cumene, among which toluene and cumene are preferred.

[0404] When a halogen solvent is used in step (2), examples of the solvent specifically include dichloromethane, chloroform, 1,2-dichloroethane and carbon tetrachloride, among which dichloromethane is preferred.

[0405] When an ether solvent is used in step (2), examples of the solvent specifically include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxane, 1,2-dimethoxyethane (DME), diisopropyl ether, cyclopentyl methyl ether (CPME), tert-butyl methyl ether, 4-methyltetrahydropyran, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether and tetraethylene glycol dimethyl ether, among which tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane and 1,3-dioxolane are preferred.

[0406] When an ester solvent is used in step (2), examples of the solvent specifically include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate and γ-valerolactone, among which butyl acetate and methyl propionate are preferred.

[0407] When a ketone solvent is used in step (2), examples of the solvent specifically include acetone, methyl ethyl ketone (MEK), methyl isobutyl ketone, cyclohexanone, cyclopentanone and diethyl ketone (3-pentanone), among which methyl ethyl ketone and diethyl ketone are preferred.

[0408] When a carbonate solvent is used in step (2), examples of the solvent specifically include dimethyl carbonate, diethyl carbonate and dibutyl carbonate, among which dimethyl carbonate is preferred.

[0409] When a phosphoric acid ester solvent is used in step (2), examples of the solvent specifically include trimethyl phosphate, triethyl phosphate and tributyl phosphate, among which tributyl phosphate is preferred.

[0410] When an amide solvent is used in step (2), examples of the solvent specifically include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), M-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP) and formamide.

[0411] When a urea solvent is used in step (2), examples of the solvent specifically include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU).

[0412] When a sulfone solvent is used in step (2), examples of the solvent specifically include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methylsulfolane, ethyl methyl sulfone and ethyl isopropyl sulfone.

[0413] In some aspects, the solid phase resin swelling capacity of the solvent used in step (2) is preferably 1.5 mL / g or more, 2.0 mL / g or more, 2.5 mL / g or more, 3.0 mL / g or more, 3.5 mL / g or more, or 4.0 mL / g or more. As an indicator of the solid phase resin swelling capacity of each solvent, the values ​​described in "Tetrahedron Lett., 1998, 39, 8951-8954", "Org. Process Res Dev. 2018, 22, 494-503" or "RSC Adv., 2020, 10, 42457-42492" are used. The solid phase resin swelling capacity (mL / g) is as follows: DMI: 8.5; DMPU: 8.0; NMP: 6.4; THF: 6.0; DMA: 5.8; CHCl3: 5.6; CPME: 5.6; CH2Cl2: 5.4; 2-methyltetrahydrofuran: 5.4; DMF: 5.2; butyl acetate: 5.2; DME: 4.8; MEK: 4.4; toluene: 4.0; AcOEt: 4.2; xylene: 3.0; Et2O: 2.8; dimethyl carbonate: 2.8; MTBE: 2.4; CH3CN: 2.0; tributyl phosphate: 1.6; MeOH: 1.6; and H2O: 1.6. In addition, estimates in the table described in "Green Chem., 2017, 19, 952-962", Table 2 in "Green Chem., 2020, 22, 996-1018", etc. can be adopted.

[0414] In step (2), one or more bases are used, which at least include a base having a pKa of 23 or more as a conjugate acid in acetonitrile. When only one base is used, the base is a base having a pKa of 23 or more as a conjugate acid in acetonitrile. On the other hand, when multiple bases are used, at least one base is a base having a pKa of 23 as a conjugate acid in acetonitrile, and the other bases can be a base having a pKa of 23 or more, or less than 23, as a conjugate acid in acetonitrile.

[0415] The base having a pKa of 23 or more as a conjugate acid in acetonitrile used in step (2) may be a base having a pKa of 34 or less, preferably 30 or less, more preferably 28 or less.

[0416] pKa is an indicator of the degree of basicity, and the larger the pKa, the higher the basicity. As the pKa (pKa (CH3CN)) of the organic base as a conjugate acid in acetonitrile in the present invention, "New J. Chem. 2009, 33, 588" is adopted. 1),"Sigma-Aldrich,Phosphazene Bases:https: / / www.sigmaaldrich.com / chemistry / chemical-synthesis / technology-spotlights / phosphazenes.html" 2) , or the values ​​shown in "Eur.J.Org.Chem.2019,6735-6748" etc.

[0417] Examples of the base having a pKa of 23 or more as a conjugate acid in acetonitrile include amidine, guanidine, phosphazene, and, for example, the bases described below all have a pKa of 23 or more.

[0418] DBU:pKa 1) =24.3

[0419] DBN:pKa 1) =23.8

[0420] TMG:pKa 1) =23.3

[0421] TBD:pKa 1) =26.0

[0422] MTBD:pKa 1) =25.5

[0423] P2tBu:pKa 1) =33.5

[0424] P2Et:pKa 1) =32.9

[0425] HP1(dma):pKa 1) =25.9

[0426] BTPP:pKa 2) =28.4

[0427] P1tBu:pKa 1) =27.0

[0428] BEMP:pKa 2) =27.6

[0429] When a plurality of bases having a pKa of 23 or more as a conjugate acid in acetonitrile are used in this step, for example, two or more, two bases selected from amidines, guanidines and phosphazenes may be used in combination. As a specific combination of such bases, preferably, a combination of DBU and MTBD, DBU and HP1(dma), DBU and P1tBu, DBU and P2Et, etc. are listed.

[0430] When a base having a pKa of 23 or more and a base having a pKa of less than 23 in acetonitrile as a conjugate acid are used in combination in this step, the base having a pKa of 23 or more in acetonitrile as a conjugate acid can be selected from, for example, amidines, guanidines and phosphazenes. The base having a pKa of less than 23 in acetonitrile as a conjugate acid is preferably selected from piperidine, piperazine and morpholine. As the specific combination of this type of base, the combination of DBU and piperidine is preferably enumerated.

[0431] In some aspects, step (2) can be repeated twice or more before step (3). In this case, when repeating step (2), the same solvent and / or the same base can be used, or different solvents and / or different bases can be used. In the present invention, the term "same solvent" means that the type and mixing ratio of the solvent are the same, and the term "different solvents" means that the type and mixing ratio of the solvent are different or both are different. In the present invention, the term "same base" means that the type of base and the concentration of the base in the solvent are the same, and the term "different bases" means that the type of base and the concentration of the base in the solvent are different or both are different.

[0432] In certain aspects, the method of the present invention may include a step of draining the solution between repetitions of step (2) when step (2) is repeated two or more times. This enables the solvent and / or base used in the previous repetition of step (2) to be replaced with the solvent and / or base used in the next repetition of step (2).

[0433] In certain aspects, step (2) is a step of removing the protecting group containing the Fmoc backbone, and / or a step of converting the protecting group containing the Fmoc backbone in the first peptide into a carbamate form.

[0434] In certain aspects, the step of removing the protecting groups containing the Fmoc backbone in the first peptide is substantially performed only in step (2).

[0435] In certain aspects, the method of the present invention does not include a step of treating the first peptide with a single base such as piperidine having a pKa of less than 23 as a conjugate acid in acetonitrile before step (2). Specifically, the present invention may exclude an aspect in which the first peptide having a protecting group containing an Fmoc backbone provided in step (1) is treated with a base having a pKa of less than 23 as a conjugate acid (such as piperidine) in acetonitrile in any solvent (such as in DMF), and then step (2) of the present invention is performed (such as treatment with DBU or a combination of DBU and piperidine in a solvent containing DMSO).

[0436] In some aspects, the alkali used for step (2) can be contained in a solvent at any concentration that enables the reaction of this step to proceed. Specifically, the concentration range is, for example, 0.25 to 20 v / v%, 0.5 to 20 v / v%, 1 to 20 v / v%, 1 to 15 v / v%, 1 to 10 v / v%, 1 to 9 v / v%, 1 to 8 v / v%, 1 to 7 v / v%, 1 to 6 v / v%, 1 to 5 v / v%, 1 to 4 v / v%, 1 to 3 v / v% or 1 to 2 v / v%, preferably 1 to 8 v / v%.

[0437] In some aspects, step (2) may further include a step of contacting the solvent with CO2. In some aspects, the solvent used in step (2) may be a solvent that has been contacted with CO2 prior to this step. The solvent may be contacted with CO2 by, for example, bubbling the solvent with CO2. CO2 may be introduced into the reaction system by contacting the solvent with CO2 during step (2) or before step (2). Without being bound by a particular theory, in step (2), the N-terminal amino group is in at least a portion of the first peptide with a C(=O)O derivative from a protecting group. - The protonated base forms carbamate, and by incorporating CO2 into the reaction system, the amine form in the reaction system can be more easily converted into the carbamate. Therefore, by introducing CO2 into the reaction system, the amount of the amine form during the reaction can be further reduced to further suppress the intramolecular nucleophilic reaction of the terminal amino group, so that even under alkaline deprotection conditions, the formation of the 6-membered ring intermediate can be further suppressed to further reduce the formation of the DKP elimination compound and the 6-membered ring amidine skeleton compound as by-products.

[0438] In certain aspects, the reaction of step (2) can be carried out at a temperature from -100° C. to a temperature close to the boiling point of the solvent, preferably from 0° C. to 50° C. The reaction of step (2) can be carried out for 1 minute to 1 week, preferably from 1 minute to 3 hours, from 3 minutes to 3 hours, or from 5 minutes to 3 hours, more preferably from 1 minute to 20 minutes, from 3 minutes to 20 minutes, or from 5 minutes to 20 minutes.

[0439] Step (3)

[0440] Step (3) of the present invention is a step of condensing the first peptide with carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide after step (2). In this step, the amino group at the N-terminus of the first peptide is condensed with the carboxyl group of the carboxylic acid to synthesize the third peptide.

[0441] In certain aspects, the carboxylic acid used in step (3) can be an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid, or a C6-C8 alkyl carboxylic acid. 10 Aryl carboxylic acids.

[0442] In certain aspects, the carboxylic acid analog used in step (3) can be an active ester of a carboxylic acid or an acyl halide of a carboxylic acid.

[0443] By using an active ester of an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 Aryl carboxylic acid, the condensation reaction of step (3) can be carried out in the absence of a condensing agent. Examples of active esters specifically include: esters in which the carbonyl group in the above-mentioned compound (especially when the compound is a second peptide, the carbonyl group contained in the amino acid residue at the C-terminus of the second peptide) is bonded to a group capable of promoting the reaction with an amine, such as an O-(benzotriazole-1-yl) group (OBt), an O-(7-azabenzotriazole-1-yl) group (OAt), an N-hydroxysuccinimide group (OSu), a pentafluorophenoxy group (OPfp), an SR 11 (where R 11 represents a hydrogen atom, an alkyl group optionally having a substituent, an aryl group optionally having a substituent, an aralkyl group optionally having a substituent, a cycloalkyl group optionally having a substituent, a heteroaryl group optionally having a substituent, an alkenyl group optionally having a substituent, or an alkylene group optionally having a substituent) and the like.

[0444] By using an acyl halide of an amino acid having a protecting group, a second peptide having a protecting group, a C1-C8 alkyl carboxylic acid or a C6-C 10 Aryl carboxylic acid, the condensation reaction of step (3) can be carried out in the absence of a condensing agent. Examples of acyl halides specifically include acyl halides in which the carboxyl group (especially when the compound is a second peptide, the carboxyl group contained in the amino acid residue at the C-terminus of the second peptide) is converted into a chlorocarbonyl group, a fluorocarbonyl group, a bromocarbonyl group, or an iodocarbonyl group.

[0445] When the carboxylic acid or carboxylic acid analogue, such as an amino acid or the second peptide, used in step (3) has a protecting group, the protecting group may be a carbamate type protecting group, a sulfonyl type protecting group or an acyl type protecting group.

[0446] In this specification, the term "carbamate type protecting group" refers to the protecting group constituting the carbamate structure, and includes, for example, the protecting group having the Fmoc skeleton, Alloc, Teoc, Boc, Cbz and 2,2,2-trichloroethoxycarbonyl group (Troc). The term "sulfonyl type protecting group" means the protecting group having the sulfonyl structure, and includes, for example, the methanesulfonyl (Ms) group, the p-toluenesulfonyl (Ts) group, the 2-nitrobenzenesulfonyl (Ns) group, the trifluoromethanesulfonyl (Tf) group and the (2-trimethylsilyl)-ethylsulfonyl (SES) group. The term "acyl type protecting group" means the protecting group having the acyl structure, and includes, for example, the acetyl group, the pivaloyl group, the benzoyl group, the trifluoroacetyl (Tfa) group.

[0447] In certain aspects, when the carboxylic acid or carboxylic acid analog used in step (3) is an amino acid having a protecting group containing an Fmoc backbone, any natural or unnatural amino acid protected with a protecting group containing an Fmoc backbone can be used for the amino acid having a protecting group containing an Fmoc backbone.

[0448] In certain aspects, when the carboxylic acid or carboxylic acid analog used in step (3) is a second peptide having a protecting group containing an Fmoc backbone, the type and number of amino acid residues constituting the second peptide having a protecting group containing an Fmoc backbone are not limited, as long as the N-terminal amino acid of the peptide is protected by a protecting group containing an Fmoc backbone, and a peptide having any amino acid sequence comprising two or more natural and / or non-natural amino acid residues. Preferably, the second peptide contains one or more N-substituted amino acids.

[0449] In certain aspects, when the carboxylic acid or carboxylic acid analog used in step (3) is a C1-C8 alkyl carboxylic acid, a C6-C 10 When the alkyl carboxylic acid or its active ester or acyl halide is substituted, such compound is optionally substituted by one or more substituents independently selected from the group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclic, arylalkyl, heteroarylalkyl, halogen, nitro, dialkylamino, cyano, alkoxycarbonyl and dialkylaminocarbonyl. The C1-C8 alkyl carboxylic acid or its active ester or acyl halide is preferably a C1-C4 alkyl carboxylic acid or its active ester or acyl halide, and examples thereof specifically include acetic acid, propionic acid, butyric acid, valeric acid, and its active ester or acyl halide. C6-C 10 Examples of the arylcarboxylic acid or its active ester or acid halide specifically include benzoic acid, naphthoic acid, and its active ester or acid halide.

[0450] In step (3), 1 to 20 equivalents, preferably 2 to 10 equivalents, of an amino acid having a protecting group containing an Fmoc skeleton, or a second peptide having a protecting group containing an Fmoc skeleton may be used relative to the first peptide.

[0451] In certain aspects, preferably, the first peptide and / or the second peptide having a protecting group containing an Fmoc backbone contains at least one N-substituted amino acid. The amino acid having a protecting group containing an Fmoc backbone is preferably an N-substituted amino acid.

[0452] In certain aspects, the protecting group containing the Fmoc backbone used in the amino acid or the second peptide in step (3) may be the same as or different from the protecting group containing the Fmoc backbone used in the first peptide in step (1).

[0453] In certain aspects, the solvent used in step (3) may contain at least one of an aromatic hydrocarbon solvent, a halogen solvent, an ether solvent, an amide solvent, a sulfoxide solvent, a sulfone solvent, a urea solvent, an ester solvent, a ketone solvent, a carbonate solvent, and a phosphate ester solvent, and may be a single solvent or a mixed solvent. When the solvent used in step (3) is a mixed solvent, it is preferred that the solvent contains at least one of an aromatic hydrocarbon solvent, a halogen solvent, an ether solvent, an amide solvent, a sulfoxide solvent, a sulfone solvent, a urea solvent, an ester solvent, a ketone solvent, a carbonate solvent, and a phosphate ester solvent, and the content is 25 v / v% or more, 50 v / v% or more, or 75 v / v% or more.

[0454] When an aromatic hydrocarbon solvent is used in step (3), examples of the solvent specifically include benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene and cumene, among which toluene and cumene are preferred.

[0455] When a halogen solvent is used in step (3), examples of the solvent specifically include dichloromethane, chloroform, 1,2-dichloroethane and carbon tetrachloride, among which dichloromethane is preferred.

[0456] When an ether solvent is used in step (3), examples of the solvent specifically include diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, 1,3-dioxane, 1,2-dimethoxyethane, diisopropyl ether, cyclopentyl methyl ether, tert-butyl methyl ether, 4-methyltetrahydropyran, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether and tetraethylene glycol dimethyl ether, among which tetrahydrofuran, 2-methyltetrahydrofuran, 1,2-dimethoxyethane and 1,3-dioxolane are preferred.

[0457] When an amide solvent is used in step (3), examples of the solvent specifically include N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), M-ethylpyrrolidone (NEP), N-butylpyrrolidone (NBP) and formamide, among which DMF and NMP are preferred.

[0458] When a sulfoxide solvent is used in step (3), examples of the solvent specifically include dimethyl sulfoxide (DMSO), diethyl sulfoxide, methyl ethyl sulfoxide and methyl phenyl sulfoxide, among which DMSO is preferred.

[0459] When a sulfone solvent is used in step (3), examples of the solvent specifically include diphenyl sulfone, dimethyl sulfone, diethyl sulfone, sulfolane, 3-methylsulfolane, ethyl methyl sulfone and ethyl isopropyl sulfone.

[0460] When a urea solvent is used in step (3), examples of the solvent specifically include 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), among which DMI is preferred.

[0461] When an ester solvent is used in step (3), examples of the solvent specifically include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, butyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, pentyl acetate and γ-valerolactone, among which ethyl acetate, butyl acetate and methyl propionate are preferred.

[0462] When a ketone solvent is used in step (3), examples of the solvent specifically include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone and diethyl ketone, among which methyl ethyl ketone and diethyl ketone are preferred.

[0463] When a carbonate solvent is used in step (3), examples of the solvent specifically include dimethyl carbonate, diethyl carbonate and dibutyl carbonate, among which dimethyl carbonate is preferred.

[0464] When a phosphoric acid ester solvent is used in step (3), examples of the solvent specifically include trimethyl phosphate, triethyl phosphate and tributyl phosphate, among which tributyl phosphate is preferred.

[0465] When the solvent used in step (3) is a mixed solvent, the mixed solvent may include two or more solvents selected from aromatic hydrocarbon solvents, halogen solvents, ether solvents, amide solvents, sulfoxide solvents, sulfone solvents, urea solvents, ester solvents, ketone solvents, carbonate solvents and phosphate solvents, and may contain other solvents.

[0466] When the solvent used in step (3) is a single solvent, the solvent is preferably toluene, isopropylbenzene, 1,2-dichlorobenzene, benzene, anisole, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 4-methyltetrahydropyran, 1,4-dioxane, 1,2-dimethoxyethane, 1,3-dioxane, DMF, NMP, DSMO, DMI, DMPU, ethyl acetate, isopropyl acetate, butyl acetate, methyl propionate, acetone, methyl ethyl ketone, diethyl ketone, dimethyl carbonate, diethyl carbonate, trimethyl phosphate, tributyl phosphate, sulfolane or 3-methylsulfolane.

[0467] In certain aspects, the solvent used in step (3) can be the same as the solvent used in step (2).

[0468] As the solvent used in step (3), it is preferred to use a solvent that can dissolve the condensing agent. In the present invention, the base used in step (2) may remain, and the residual base may cause the formation of by-products of excessive extension of amino acids or peptides. When a solvent that can dissolve the condensing agent is used in step (3), the formation of such excessively extended compounds can be suppressed.

[0469] In certain aspects, the condensing agent used in step (3) is in the form of a salt, and its counter anion is PF6 - or BF4 - Examples of such condensing agents specifically include PyOxim ([ethylcyano(hydroxyimino)acetate-O 2]tris-1-pyrrolidinylphosphonium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), COMU ((1-cyano-2-ethoxy-2-oxyethylideneaminooxy)dimethylaminomorpholinium hexafluorophosphate), HATU (O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate, HCTU (O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), TDBTU (O-(3,4-dihydro-4- The following are the esters of 1-[(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-[((((triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate), 1-[ ...

[0470] In certain aspects, preferably, the condensing agent used in step (3) contains at least one selected from the group consisting of PyOxim, PyAOP, PyBOP, COMU, HATU, HBTU, HCTU, TDBTU, HOTU, TATU, TBTU, TCTU and TOTU, and more preferably any one of these compounds is used.

[0471] In some aspects, the condensation reaction of step (3) can be carried out in the presence of a base. The type of base is not particularly limited as long as it helps the condensation reaction to proceed, and preferably a base with a pKa of 5 to 12 in water as a conjugate acid. Examples of such bases specifically include DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine and pyridine. In step (3), a base with an amount of 1 to 20 equivalents, preferably 2 to 10 equivalents, relative to the first peptide can be used. As the pKa of the base of the conjugate acid in water, a value calculated according to, for example, Advanced Chemistry Development (ACD / Labs) software V11.02 ((c) 1994-2020 ACD / Labs) can be used.

[0472] In certain aspects, the condensing agent used in step (3) is a carbodiimide condensing agent. Examples of condensing agents specifically include diisopropylcarbodiimide (DIC), di-sec-butylcarbodiimide (DsBC), 1-tert-butyl-3-ethylcarbodiimide (tBEC), di-tert-butylcarbodiimide (DtBC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), preferably diisopropylcarbodiimide (DIC) or di-sec-butylcarbodiimide (DsBC).

[0473] When the condensing agent used in step (3) is a carbodiimide condensing agent, the condensation reaction in step (3) can be carried out in the presence of an additive. The type of the additive is not limited as long as it helps the condensation reaction to proceed, and examples of the additive specifically include HOAt, HOBt, HOOBt, Oxyma and Oxyma-B, among which HOAt and Oxyma are preferred.

[0474] In step (3), the condensing agent can be used in an amount of 1 to 20 equivalents, preferably 2 to 10 equivalents, relative to the first peptide.

[0475] In some aspects, the present invention further includes a step (treatment step) of treating the mixture in step (2) between step (2) and step (3). In some aspects, the treatment step is a step of contacting an acid or a salt with the mixture. In the step of contacting an acid or a base with the mixture, the acid or the base can be directly contacted with the mixture, or a solution containing the acid or the base can be added to the mixture. For the solution, any solvent can be used. For example, it is preferred to use the solvent used in step (2), more preferably to use a sulfoxide solvent, and further preferably to use DMSO. The treatment step can be a step of decomposing the carbamate generated in step (2).

[0476] As the acid used in the treatment step, HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid can be mentioned. Among them, HOAt, Oxyma, 4-nitrophenol and HOOBt are preferred. As the salt used in the treatment step, at least one acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid and at least one base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine and pyridine form a salt, and pyridine hydrochloride is preferably exemplified.

[0477] In some aspects, the step of treating the mixture in step (2) can be a step of washing the solid phase synthetic resin. In order to wash the resin, any solvent can be used. For the solution, any solvent can be used. For example, it is preferred to use the solvent used in step (2), more preferably to use a sulfoxide solvent, and further preferably to use DMSO. In some aspects, the solvent for the step of washing the solid phase synthetic resin can be a solution containing an alkali. As the alkali, for example, DIPEA, triethylamine, 2,6-lutidine and 2,4,6-trimethylpyridine can be cited. The resin can be washed twice or more (for example, twice, three times, four times, five times or six times) with the same solvent or different solvents.

[0478] In some aspects, the step of washing the solid phase synthetic resin is a step of washing a sulfoxide solvent solution containing an acid or a salt. As the acid for washing the resin, HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid are preferred. Among them, HOAt, Oxyma, 4-nitrophenol and HOOBt are preferred. As the salt for washing the resin, at least one acid selected from the group consisting of HOAt, Oxyma, 4-nitrophenol, HOOBt and hydrochloric acid and at least one base selected from the group consisting of DIPEA, triethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine and pyridine form a salt, and pyridine hydrochloride is preferably exemplified.

[0479] In certain aspects, in the step of washing the solid phase synthetic resin, a solvent that has been in contact with CO2 before the step (e.g., a solvent bubbled with CO2 gas) may be used, or a solvent that has not been in contact with CO2 may be used. A solvent in contact with CO2 and a solvent that has not been in contact with CO2 may be used in combination. When washing is performed two or more times, it is preferred that the resin be washed one or more times with a solvent that has been in contact with CO2 in advance, and then the resin be washed one or more times with a solvent that has not been in contact with CO2 in advance.

[0480] In certain aspects, the reaction of step (3) can be carried out at a temperature from -100° C. to a temperature close to the boiling point of the solvent, preferably from 0° C. to 60° C. The reaction of step (3) can be carried out for 1 minute to 1 week, preferably 30 minutes to 20 hours.

[0481] In certain aspects, the peptide produced by the method of the present invention contains the third peptide produced by steps (1) to (3). In this case, the peptide produced by the method of the present invention further contains any number of arbitrary amino acids in addition to the third peptide.

[0482] In certain aspects, the peptide produced by the method of the present invention may contain a third peptide produced by steps (1) to (3).

[0483] In certain aspects, the peptide produced by the method of the present invention can contain at least one, or two or more, three or more, four or more, or five or more N-substituted amino acids. The N-substituted amino acids can be N-alkyl amino acids, preferably N-methyl amino acids.

[0484] In certain aspects, the method of the present invention may include a step of separating the peptide from the solid phase synthesis resin after the step of extending the peptide chain. For the separation step, methods known in the art or methods described in the examples can be used.

[0485] In certain aspects, the present invention also relates to a method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton compound impurities formed in peptide production (e.g., peptide production by a solid phase method), the method comprising steps (1) and (2). The method may further comprise step (3): after step (2), in a solvent, in the presence or absence of a condensing agent, condensing the first peptide with a carboxylic acid or a carboxylic acid analogue to obtain a third peptide.

[0486] In certain aspects, the present invention also relates to a method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeleton compound impurities and diurea compound impurities formed in peptide production (e.g., peptide production by a solid phase method), the method comprising steps (1) and (2). The method may further comprise step (3): after step (2), in a solvent, in the presence or absence of a condensing agent, condensing the first peptide with a carboxylic acid or a carboxylic acid analogue to obtain a third peptide.

[0487] All references cited herein are hereby incorporated by reference.

[0488] Examples

[0489] The present invention is described in more detail by examples, comparative examples and reference examples, but the present invention is not limited to the examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0490] In the examples, the following abbreviations are used.

[0491] AA: Ammonium acetate

[0492] Ac: acetyl group

[0493] Alloc: allyloxycarbonyl group

[0494] Boc: tert-butoxycarbonyl group

[0495] Cbz: benzyloxycarbonyl group

[0496] COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholinium hexafluorophosphate

[0497] DBU: 1,8-diazabicyclo[5.4.0]-7-undecene

[0498] DCM: dichloromethane

[0499] DCE: 1,2-dichloroethane

[0500] DMF: N,N-dimethylformamide

[0501] DKP: diketopiperazine

[0502] DIC: N,N'-diisopropylcarbodiimide

[0503] DIPEA: N,N'-diisopropylethylamine

[0504] DMSO: dimethyl sulfoxide

[0505] DsBC: N,N'-di-sec-butylcarbodiimide

[0506] EDCI·HCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0507] EtOAc: ethyl acetate

[0508] FA: Formic acid

[0509] Fmoc: 9-fluorenylmethoxycarbonyl group

[0510] HOAt: 1-Hydroxy-7-azabenzotriazole

[0511] HOBt: 1-Hydroxybenzotriazole

[0512] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

[0513] HATU:O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0514] HBTU: O-(1H-Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0515] HCTU: O-(1H-6-chloro-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0516] HOTU: O-[(Ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0517] HP1(dma): imino-tris(dimethylamino)phosphorane

[0518] MTBD: 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene

[0519] NMP: N-methyl-2-pyrrolidone

[0520] Ns: 2-nitrobenzenesulfonyl group

[0521] Oxyma: Ethyl cyano(imino)acetate

[0522] pip: piperidine

[0523] P1tBu: tert-butylimino-tris(dimethylamino)phosphorane

[0524] P2Et: triamidotetramethyl(tris(dimethylamino)phosphoranylidene)phosphate-ethyleneimine

[0525] PyAOP: (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0526] PyBOP: Benzotriazol-1-yloxy-tris-pyrrolidinyl-phosphonium hexafluorophosphate

[0527] PyOxim: [Ethyl cyano(hydroxyimino)acetate-O2]tris-1-pyrrolidinylphosphonium hexafluorophosphate

[0528] Teoc: 2-trimethylsilylethoxycarbonyl group

[0529] TES: Triethylsilane

[0530] Tfa: trifluoroacetyl group

[0531] TFA: trifluoroacetic acid

[0532] TFE: 2,2,2-trifluoroethanol

[0533] THF: Tetrahydrofuran

[0534] TMG: 1,1,3,3-Tetramethylguanidine

[0535] TATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate

[0536] TBTU: 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate

[0537] TCTU: O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0538] TDBTU: O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0539] TOTU: O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate The LCMS analysis conditions are shown in Table 1.

[0540]

[0541] Example 1: Preparation of amino acids, resin-loaded peptides, etc. used in the examples

[0542] Example 1-1: Fmoc-amino acid is used to synthesize peptides by fully automatic peptide synthesizer

[0543] In the peptide synthesis described in the present specification, the Fmoc-amino acids shown in Tables 2 to 4 were used and the synthesis was carried out by a fully automatic peptide synthesizer.

[0544] The Fmoc-amino acids shown in Table 2 were synthesized according to the methods described in WO 2018 / 225864 and WO 2021 / 090856.

[0545] The Fmoc-amino acids shown in Table 3 were purchased from commercial suppliers.

[0546] The Fmoc-amino acids shown in Table 4 were synthesized according to the scheme shown below.

[0547] [Table 2]

[0548]

[0549] [Table 3]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555] [Table 4]

[0556]

[0557] Synthesis of compound aa3-1: (2S)-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-3-pyridin-3-yl Propionic acid (Fmoc-MeAla(3-Pyr)-OH)

[0558] [Chem.11]

[0559]

[0560] Under nitrogen atmosphere, paraformaldehyde (696 mg, 23.17 mmol) and trifluoroacetic acid (TFA) (5.36 mL, 69.5 mmol) were added to a toluene (10.5 mL) solution of commercially available (2S)-2-[9H-fluorene-9-ylmethoxycarbonylamino]-3-pyridin-3-ylpropionic acid (Fmoc-Ala(3-Pyr)-OH, compound aa3-1-a) (3 g, 7.72 mmol), and the mixture was stirred at 40° C. for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with dichloromethane (DCM), then washed with saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and then filtered. The resulting solution was concentrated under reduced pressure to give compound aa3-1-b (2.95 g, 95%) as a crude product. The compound was used in the next reaction without further purification.

[0561] LCMS (ESI) m / z = 401 (M + H) +

[0562] Retention time: 0.64 minutes (analysis conditions SQDFA05)

[0563] At room temperature, under a nitrogen atmosphere, triethylsilane (TES) (10.59 mL, 66.3 mmol) and trifluoroacetic acid (TFA) (15.32 mL, 199 mmol) were added to a solution of compound aa3-1-b (2.95 g) in 1,2-dichloroethane (DCE) (15.5 mL), and the mixture was stirred at 70 ° C for 5 hours. The reaction solution was cooled to room temperature, the solvent was removed by distillation under reduced pressure, the crude product thus obtained was dissolved in ethyl acetate, tert-butyl methyl ether / n-hexane = 9 / 1 (40 mL) was added, and the mixture was stirred. The mixture was cooled in a refrigerator for 30 minutes, and the obtained solid was then filtered, washed three times with tert-butyl methyl ether / n-hexane = 9 / 1 (30 mL), and then dried under reduced pressure. The obtained crude product was suspended in DCM (50 mL), 3 mol / L sodium dihydrogen phosphate aqueous solution (45 mL, including sodium chloride 1.5 mol / L) was added, the mixture was stirred, the water layer was removed, and then the organic layer was washed twice with 3 mol / L sodium dihydrogen phosphate aqueous solution (45 mL, including sodium chloride 1.5 mol / L). The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure, and the residue thus obtained was purified by reverse phase column chromatography (water / acetonitrile) to obtain compound aa3-1: (2S)-2-[9H-fluorene-9-ylmethoxycarbonyl (methyl) amino]-3-pyridin-3-ylpropionic acid (Fmoc-MeAla (3-Pyr) -OH) (2.67 g, 90%).

[0564] LCMS (ESI) m / z = 403 (M + H) +

[0565] Retention time: 0.53 minutes (analysis conditions SQDFA05)

[0566] Synthesis of compound aa3-2: (2S)-3-[4-trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxy Carbonylamino)propionic acid (Teoc-Phe(4-CF3)-OH)

[0567] [Chem.12]

[0568]

[0569] Under nitrogen atmosphere, commercially available (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propionic acid (H-Phe(4-CF3)-OH, compound aa3-2-a) (2.00 g, 8.58 mmol) was dissolved in water (12.0 mL), and then a mixed solution of triethylamine (2.38 mL, 17.15 mmol) and 1,4-dioxane (10.0 mL) was added, and the mixture was stirred at room temperature for 10 minutes. Then, 2,5-dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl) carbonate (2.45 g, 9.43 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 2-Methyltetrahydrofuran (5 mL) was added to the reaction solution, and the mixture was washed with a 10% aqueous sodium carbonate solution (10 mL). The obtained organic layer was washed with a saturated potassium hydrogen sulfate aqueous solution (10 mL), then washed with a 10% sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, and then filtered. The obtained solution was concentrated under reduced pressure to obtain compound aa3-2: 2S)-3-[4-trifluoromethyl)phenyl]-2-(2-trimethylsilylethoxycarbonylamino)propionic acid (Teoc-Phe(4-CF3)-OH). (2.88 g, 89%)

[0570] LCMS (ESI) m / z = 376 (MH) -

[0571] Retention time: 0.87 minutes (analysis conditions SQDFA05)

[0572] Synthesis of compound aa3-3: 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propionic acid (Ns-Aib-OH)

[0573] [Chem.13]

[0574]

[0575] 2-Nitrobenzenesulfonyl chloride (6.93 g, 31.2 mmol) in DCM (30.0 mL) was added to a solution of commercially available methyl 2-amino-2-methylpropanoate hydrochloride (4.0 g, 26.0 mmol) and DIPEA (10.92 mL, 62.5 mmol) in DCM (22.1 mL), cooled in an ice bath, and the mixture was stirred at 0° C. for 5 minutes, and then at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give the crude product aa3-3-b.

[0576] LCMS (ESI) m / z = 301 (MH) -

[0577] Retention time: 0.64 minutes (analysis conditions SQDFA05)

[0578] Under ice bath cooling, 5N sodium hydroxide aqueous solution (26mL) was added to the obtained crude product aa3-3-b (3.63g) in THF / methanol (1 / 1, 52mL), and the mixture was stirred at 0°C for 5 minutes, and then stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure, the obtained residue was dissolved in 4N hydrochloric acid (30mL), and the solvent was distilled off. The mixture was extracted twice with ethyl acetate, and the organic layer was then dried over anhydrous sodium sulfate, then filtered, and the solution was concentrated under reduced pressure. The obtained crude product was purified twice by reverse phase column chromatography (0.1% acetonitrile containing formic acid / 0.1% distilled water containing formic acid) to obtain compound aa3-3: 2-methyl-2-[(2-nitrophenyl)sulfonylamino]propionic acid (Ns-Aib-OH) (2.46g, 71%).

[0579] LCMS (ESI) m / z = 287 (MH) -

[0580] Retention time: 0.53 minutes (analysis conditions SQDFA05)

[0581] Example 1-2: Preparation of resin-loaded amino acids and peptides used in the examples

[0582] Example 1-2-1: (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid Synthesis of 2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrrole, compound 1-2-1)

[0583] [Chem.14]

[0584]

[0585] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin portion may be drawn as ○. In order to clarify the reaction point of the resin portion, the chemical structure of the reaction portion may be drawn with a compound connected to ○. For example, in the above structure (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrrole (Compound 1-2-1)), the 2-chlorotriethyl group of the resin is bonded to the carboxylic acid on the side chain of Asp via an ester bond. The term "pyrrole" means pyrrolidine, and in the above structure, the carboxylic acid group at the C-terminus forms an amide bond with pyrrolidine.

[0586] [Chem.15]

[0587]

[0588] At 0°C, under a nitrogen atmosphere, EDCI·HCl (67.1 g, 350 mmol), HOBt (43.3 g, 321 mmol) and Fmoc-Asp (OtBu) -OH (120 g, 2929 mmol) were added to DMF (600 mL) in sequence, and the mixture was stirred at 0°C for 1 hour. At 0°C, ethyl acetate (10 v) and 0.5 mol / L aqueous hydrochloric acid solution (2 V) were added to the reaction solution, and the organic layer was separated. The obtained organic layer was washed (in this order) with 0.5 mol / L aqueous hydrochloric acid solution, water, saturated sodium bicarbonate aqueous solution / water (1 / 1 (v / v)), and saturated sodium chloride aqueous solution / water (1 / 1 (v / v)), and dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain compound 1-2-1-a as a crude product. (137.1 g, quantitative)

[0589] LCMS (ESI) m / z = 465 (M + H) +

[0590] Retention time: 1.05 minutes (analysis conditions SQDAA05)

[0591] TFA (271 mL) was slowly added to a DCM (137 mL) solution of compound 1-2-1-a (137 g, 395 mmol) under ice cooling so that the internal temperature did not exceed 10° C. The mixture was stirred at room temperature for 1 hour, then diisopropyl ether (3.4 L) was added in four portions, and the precipitated solid was collected by filtration and dried to give compound 1-2-1-b ((3s)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutanoic acid, Fmoc-Asp-pyrrole). (108.4 g, 90%)

[0592] LCMS (ESI) m / z = 409 (M + H) +

[0593] Retention time: 0.83 minutes (analysis conditions SQDAA05)

[0594] The reaction of loading Fmoc-amino acid on resin was carried out according to the method described in WO 2013 / 100132 or WO 2018 / 225864. 2-Chlorotrityl chloride resin (1.60mmol / g, 100 to 200 mesh, 1% DVB, 48.7g) and dehydrated dichloromethane (500mL) were added to a reaction vessel with a filter, and the mixture was shaken at room temperature for 20 minutes. Nitrogen pressure was applied to remove dichloromethane, and dehydrated methanol (12.63mL) and diisopropylethylamine (DIPEA) (32.6mL) were added to compound 1-2-1-b (15.91g) and dehydrated dichloromethane (350mL) to obtain a mixed solution, which was then added to the reaction vessel, and the mixture was shaken for 60 minutes. Nitrogen pressure was applied to remove the reaction solution, dehydrated methanol (97.3 mL) and diisopropylethylamine (DIPEA) (32.6 mL) were added to anhydrous dichloromethane (350 mL) to obtain a mixed solution, which was then added to a reaction vessel, and the mixture was shaken for 1 hour and 30 minutes. Nitrogen pressure was applied to remove the reaction solution, and dichloromethane (350 mL) was then placed in the reaction vessel, the mixture was shaken for 5 minutes, and nitrogen pressure was then applied to remove the reaction solution. The resin was washed with dichloromethane five times, and the obtained resin was dried under reduced pressure overnight to obtain (3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-pyrrolidin-1-ylbutyric acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrrole, compound 1-2-1, 59.79 g).

[0595] In order to determine the loading rate, the obtained compound 1-2-1 (12.6 mg) was placed in a reaction vessel, DMF (2 mL) was added, and the mixture was shaken at room temperature for 1 hour. Thereafter, DBU (40 μL) was added, and the mixture was shaken at 30 ° C for 30 minutes. Thereafter, DMF (8 mL) was added to the reaction mixture, and 1 mL of the resulting solution was diluted with DMF (11.5 mL). The absorbance (294 nm) of the obtained diluted solution was measured (using Shimadzu, UV-1600PC (cell length: 1.0 cm)), and the loading rate of compound 1-2-1 was calculated to be 0.464 mmol / g.

[0596] Another batch synthesized similarly with a different loading ratio is also used for peptide synthesis, research, etc.

[0597] Example 1-2-2: (S)-3-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl) Synthesis of butyric acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip, compound 1-2-2)

[0598] [Chem.16]

[0599]

[0600] (S)-3-(((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(piperidin-1-yl)butanoic acid-2-chlorotrityl resin (Compound 1-2-2, Fmoc-Asp(O-Trt(2-Cl)-resin)-pip) was synthesized by the method described in the literature (Document: International Publication No. WO 2013 / 100132 A1). The term "pip" refers to piperidine, and in the above structure, the carboxylic acid group at the C-terminus forms an amide bond with piperidine.

[0601] Example 1-2-3: (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butyric acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin, compound 1-2-3)

[0602] [Chem.17]

[0603]

[0604] Using (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butyric acid (Fmoc-D-3-Abu-OH) (7.1 g, 21.82 mmol) and 2-chlorotrityl chloride resin (1.6 mmol / g, 100 to 200 mesh, 1% DVB, 27.25 g, 43.6 mmol) (purchased from a commercial supplier), the same steps as the synthesis of compound 1-2-1 were followed to obtain compound 1-2-3: (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butyric acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl)-resin). (33.44 g, loading rate: 0.598 mmol / g)

[0605] Another batch synthesized similarly but with a different loading ratio was also used for peptide synthesis in this example.

[0606] Example 1-2-4: Preparation of solid-phase supported peptide compounds (compounds 1-2-4 to 1-2-12) used in the examples

[0607] Compounds 1-2-4 to 1-2-12 used in this example were prepared by the Fmoc method using a fully automatic peptide synthesizer (Multipep RS; manufactured by Intavis AG). The detailed operation procedure was consistent with the manual attached to the fully automatic peptide synthesizer.

[0608] The Fmoc-protected amino acid (0.6 mol / L) constituting the target peptide and HOAt, Oxyma or HOOBt (0.375 mol / L) as a carboxylic acid activating agent were dissolved in NMP to prepare solution 1. In the case where the Fmoc-protected amino acid or HOOBt was poorly soluble, DMSO was added to 20% to prepare solution 1. N,N'-diisopropylcarbodiimide (DIC) (10 v / v%) was mixed with N,N-dimethylformamide (DMF) to prepare solution 2.

[0609] 2-Chlorotrityl resin is bonded to the carboxylic acid moiety on the aspartic acid side chain protected by the Fmoc group at the N-terminus or the carboxylic acid moiety on the β amino acid main chain protected by Fmoc at the N-terminus (compound 1-2-1, 1-2-2 or 1-2-3) (100 mg per column), which has been prepared in Example 1-2-1, 1-2-2 or 1-2-3, and is added to a solid phase reaction vessel, and the reaction vessel is set in a fully automatic peptide synthesizer. Dichloromethane (DCM) (1.0 mL) is added to the resin (100 mg), and the mixture is allowed to stand for about 30 minutes to allow the resin to swell. Solution 1 and solution 2 are set in a fully automatic peptide synthesizer, and automatic synthesis by the fully automatic peptide synthesizer is started. The solution is discharged from the glass material, and then the resin is washed twice with DMF (0.7 mL per column).

[0610] Fmoc deprotection step

[0611] A DMF solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the deprotection of the Fmoc group was carried out at room temperature. The mixture was reacted for 4.5 minutes in the deprotection of the first residue and for 10 minutes in the deprotection of the second and subsequent residues, and then the solution was drained from the glass frit. Subsequently, the resin was washed twice with DMF (0.7 mL per column), once with a DMF solution of DIPEA (0.14 mol / L) and HOAt (0.14 mol / L) (0.7 mL per column), and twice with DMF (0.7 mL per column).

[0612] Extension Steps

[0613] Solution 1 (0.3 mL per column) was mixed with solution 2 (0.36 mL per column) by the mixing bottle of the fully automatic peptide synthesizer, and the mixture was added to the resin. In the case of difficulty in extending the sequence, the solid phase reaction vessel was heated to 40°C-60°C, and in the case of difficulty in extending the sequence, the mixture was reacted for 2.5 to 14 hours to carry out a condensation reaction between the amino groups on the resin and the Fmoc-protected amino acids, and then the solution was discharged from the glass material. In the case of low extension efficiency, the condensation reaction of the Fmoc-protected amino acids was further repeated once or twice. Subsequently, the resin was washed three times with DMF (0.7 mL per column).

[0614] The condensation reaction of the Fmoc amino acid after the deprotection reaction of the Fmoc group is set as a cycle, and the cycle is repeated to extend the peptide on the resin surface. After the last amino acid is extended, the Fmoc deprotection step is not carried out, and the resin is washed four times with DCM (1.0 mL per column), dried, and then used for subsequent research. Using this method as standard conditions, the following solid-phase-loaded peptide compounds (compounds 1-2-4 to 1-2-12) were prepared.

[0615] As described above, although 100 mg of each of the compounds 1-2-1, 1-2-2, or 1-2-3 was used per column to prepare the peptide compound, a plurality of columns arranged per sequence were similarly used to prepare the peptide compound.

[0616] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pyrrole (Compound 1-2-4)

[0617] [Chem.18]

[0618]

[0619] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pyrrole (compound 1-2-1, 0.552 mmol / g).

[0620] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(O-Trt(2-Cl)-resin)-pip(Compound 1-2-5)

[0621] [Chem.19]

[0622]

[0623] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0624] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(O-Trt(2-Cl)-resin)-pip(Compound 1-2-6)

[0625] [Chem.20]

[0626]

[0627] This compound was prepared by Fmoc-Asp(O-Trt(2-Cl)-resin)-pip(compound 1-2-2, 0.452 mmol / g) /

[0628] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (Compound 1-2-7)

[0629] [Chem.21]

[0630]

[0631] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0632] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-(O-Trt(2-Cl)-resin)-pip (Compound 1-2-8)

[0633] [Chem.22]

[0634]

[0635] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0636] Fmoc-Ser(nPr)-MeAla-Phe-Asp(O-Trt(2-Cl)-resin)-pip(Compound 1-2-9)

[0637] [Chem.23]

[0638]

[0639] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0640] Fmoc-1-ACPrC-MeAla-Tyr(tBu)-Asp-(O-Trt(2-Cl)-resin)-pip (Compound 1-2-10)

[0641] [Chem.24]

[0642]

[0643] This compound was prepared from Fmoc-Asp(O-Trt(2-Cl)-resin)-pip (compound 1-2-2, 0.452 mmol / g).

[0644] Fmoc-Aib-Pro-D-3-Abu-O-Trt(2-Cl)-resin (Compound 1-2-11)

[0645] [Chem.25]

[0646]

[0647] This compound was prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.558 mmol / g).

[0648] Fmoc-cLeu-MeVal-D-3-Abu-O-Trt(2-Cl)-resin (Compound 1-2-12)

[0649] [Chem.26]

[0650]

[0651] This compound was prepared from Fmoc-D-3-Abu-O-Trt(2-Cl)-resin (compound 1-2-3, 0.558 mmol / g).

[0652] For example, in order to confirm that compound 1-2-4 was obtained, a peptide was separated from a portion of the obtained resin using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS, and the results showed that the target peptide (compound 1-2-4*) was formed. For compounds 1-2-5 to 1-2-12, the formation of the target peptide (compounds 1-2-5* to 1-2-12*) was confirmed by the same method as compound 1-2-4. In this example, the * added to the compound number indicates a compound examined with a peptide separated from the resin to examine the reaction. Compound 1-2-4* represents a peptide compound in which the bond between the carboxylic acid of the peptide contained in compound 1-2-4 and the 2-chlorotrityl group of the resin is broken.

[0653] Fmoc-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrrole (Compound 1-2-4*)

[0654] [Chem.27]

[0655]

[0656] LCMS (ESI) m / z = 928.69 (M+H) +

[0657] Retention time: 0.85 minutes (analysis conditions SQDFA05)

[0658] Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip (Compound 1-2-5*)

[0659] [Chem.28]

[0660]

[0661] LCMS (ESI) m / z = 928.80 (M+H) +

[0662] Retention time: 0.87 minutes (analysis conditions SQDFA05)

[0663] Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip (Compound 1-2-6*)

[0664] [Chem.29]

[0665]

[0666] LCMS (ESI) m / z = 1003.80 (M+H) +

[0667] Retention time: 1.06 minutes (analysis conditions SQDFA05)

[0668] Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip(Compound 1-2-7*)

[0669] [Chem.30]

[0670]

[0671] LCMS (ESI) m / z = 855.73 (M+H) +

[0672] Retention time: 0.76 minutes (analysis conditions SQDFA05)

[0673] Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-pip(Compound 1-2-8*)

[0674] [Chem.31]

[0675]

[0676] LCMS (ESI) m / z = 888.67 (M+H) +

[0677] Retention time: 1.05 minutes (analysis conditions SQDFA05)

[0678] Fmoc-Ser(nPr)-MeAla-Phe-Asp-pip (Compound 1-2-9*)

[0679] [Chem.32]

[0680]

[0681] LCMS (ESI) m / z = 784.66 (M+H) +

[0682] Retention time: 0.86 minutes (analysis conditions SQDFA05)

[0683] Fmoc-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Compound 1-2-10*)

[0684] [Chem.33]

[0685]

[0686] LCMS (ESI) m / z = 810.44 (M+H) +

[0687] Retention time: 2.60 minutes (analysis conditions SQDFA05long)

[0688] Fmoc-Aib-Pro-D-3-Abu-OH (Compound 1-2-11*)

[0689] [Chem.34]

[0690]

[0691] LCMS (ESI) m / z = 508.47 (M+H) +

[0692] Retention time: 0.66 minutes (analysis conditions SQDFA05)

[0693] Fmoc-cLeu-MeVal-D-3-Abu-OH (Compound 1-2-12*)

[0694] [Chem.35]

[0695]

[0696] LCMS (ESI) m / z = 326.41 (MH) -

[0697] MS detection of fragments without Fmoc protection ((MH-Fmoc)-)

[0698] Retention time: 0.79 minutes (analysis conditions SQDFA05)

[0699] Examples 1-3: Preparation of linear peptides used in the examples

[0700] Example 1-3-1: Fmoc-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(OMe)-pip, compound 1-3-1 Synthesis

[0701] [Chem.36]

[0702]

[0703] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-5 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit. Subsequently, TFE / DCM solution (1 / 1 (v / v), 2.0 mL) was added, and the mixture was shaken at room temperature for 2 hours. Thereafter, the solution was discharged from the glass frit into an eggplant-shaped flask. Subsequently, the resin was washed twice with TFE / DCM solution (1 / 1 (v / v), 1.0 mL), and the discharged solution was collected in an eggplant-shaped flask. The same operation was performed on seven solid phase reactors, the discharged solutions were combined, and then the solvent was removed by distillation under reduced pressure. The obtained residue was dissolved in DCE (5 mL), and the solvent was distilled off under reduced pressure twice to obtain compound 1-2-5* (209.2 mg, 71%) as a crude product. This compound was used in the next reaction without further purification.

[0704] LCMS (ESI) m / z = 928.78 (M+H) +

[0705] Retention time: 0.88 minutes (analysis conditions SQDFA05)

[0706] Under a nitrogen atmosphere, a hexane solution of trimethylsilyldiazomethane (0.6M, 0.75mL, 0.45mmol) was added dropwise at room temperature to a solution obtained by dissolving compound 1-2-5* (209.2mg, 0.23mmol) in toluene (0.75mL) and methanol (1.50mL). The mixture was stirred at room temperature for 5 minutes, and then acetic acid (0.013mL, 0.23mmol) was added, the solvent was removed by distillation under reduced pressure, and the residue thus obtained was purified by normal phase column chromatography (DCM / methanol) to obtain compound 1-3-1, Fmoc-Ile-MeGly-Aze (2) -MeCha-MeGly-Asp (OMe) -pip (175.9mg, 83%).

[0707] LCMS (ESI) m / z = 942.63 (M+H) +

[0708] Retention time: 0.94 minutes (analysis conditions SQDFA05)

[0709] Example 1-3-3: Fmoc-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip, compound 1-3-2 Synthesis

[0710] [Chem.37]

[0711]

[0712] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-6 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit. Subsequently, TFE / DCM solution (1 / 1 (v / v), 2.0 mL) was added, and the mixture was shaken at room temperature for 2 hours. Thereafter, the solution was discharged from the glass frit into an eggplant-shaped flask. Subsequently, the resin was washed twice with TFE / DCM solution (1 / 1 (v / v), 1.0 mL), and the discharged solution was collected in an eggplant-shaped flask. The same operation was performed on seven solid phase reactors, the discharged solutions were combined, and the solvent was removed by distillation under reduced pressure. The obtained residue was dissolved in DCE (5 mL), and then the solvent was distilled off under reduced pressure twice to obtain compound 1-2-6* (202.8 mg, 64%) as a crude product. This compound was used in the next reaction without further purification.

[0713] LCMS (ESI) m / z = 1003.70 (M+H) +

[0714] Retention time: 1.06 minutes (analysis conditions SQDFA05)

[0715] Under a nitrogen atmosphere, a hexane solution of trimethylsilyldiazomethane (0.6M, 0.67 mL, 0.40 mmol) was added dropwise at room temperature to a solution obtained by dissolving compound 1-2-6* (202.8 mg, 0.20 mmol) in toluene (0.67 mL) and methanol (1.35 mL). The mixture was stirred at room temperature for 5 minutes, and then acetic acid (0.012 mL, 0.20 mmol) was added, the solvent was removed by distillation under reduced pressure, and the residue thus obtained was purified by normal phase column chromatography (ethyl acetate / hexane) to obtain compound 1-3-2, Fmoc-Thr-(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip (168.1 mg, 82%).

[0716] LCMS (ESI) m / z = 1017.61 (M+H) +

[0717] Retention time: 1.12 minutes (analysis conditions SQDFA05)

[0718] Example 2: Examination of the effects of reagents and solvents on the Fmoc deprotection step, resin washing step, and elongation step in peptide solid phase synthesis Experiments on the effects of step

[0719] In the solid phase synthesis of peptides, amino acid extension is performed after Fmoc deprotection and resin washing. In this example, compound 1-2-4 is used as a peptide-loaded solid phase resin, the reagents and solvents in each step are changed, and the degree of reducing impurities formed in peptide synthesis (diketopiperazine elimination compound, 6-membered ring amidine skeleton compound and over-extension compound) is compared to determine a series of suitable conditions.

[0720] Basic Operation A (Table 5, Runs 1 to 7, 14 to 15, and 29 to 30)

[0721] 100mg of the peptide-loaded solid phase resin (compound 1-2-4) prepared in Example 1-2-4 was placed in a solid phase reaction container, DCM (1.0mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit, and the resin was subsequently washed twice with the solvent (0.7mL per column) used during Fmoc deprotection. Fmoc deprotection solution (0.7mL per column) was added, and the mixture was reacted for 10 minutes at room temperature to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base shown in Table 5 in the Fmoc deprotection solvent at a volume percentage concentration (v / v%) shown in Table 5. The solution was discharged from the glass frit, and then the resin was washed six times with a washing solvent (0.7mL per column).

[0722] To the resin obtained as described above, a solution obtained by mixing a solution of Fmoc-MeLeu-OH (0.6 mol / L) and Oxyma or HOAt (0.375 mol / L) in an extended solvent (0.3 mL per column) and a solution of DIC (10 v / v%) or DsBC (12 v / v%) in an extended solvent (0.36 mL per column) was added, and the mixture was shaken at 40° C. for 2.5 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0723] For example, in Run 5 in Table 5, the following procedure is performed according to Basic Operation A.

[0724] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-4 prepared from Compound 1-2-1 (at 0.552 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0725] To the resin obtained as described above, a solution obtained by mixing a solution of Fmoc-MeLeu-OH (0.6 mol / L) and Oxyma (0.375 mol / L) in DMSO (0.3 mL) and a solution of DIC (10 v / v%) in DMSO (0.36 mL) was added, and the mixture was shaken at 40° C. for 2.5 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMSO (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0726] To check the progress of the reaction, a portion of the obtained resin was taken out, swollen with DCM, and washed twice with DMF, then a DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, the resin was washed four times with DMF, and four times with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDAA05 long) to confirm the formation of the target peptide compound (TM) (2-1*), the diketopiperazine (DKP) elimination compound (compound 2-2*), the 6-membered ring amidine skeleton compound (compound 2-3*), and the MeLeu over-extension compound (compound 2-4*).

[0727] Target peptide compound (TM), H-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrrole (Compound 2-1*)

[0728] [Chem.38]

[0729]

[0730] LCMS (ESI) m / z = 833.58 (M+H) +

[0731] Retention time: 2.72 minutes (analysis conditions SQDAA05 long)

[0732] Diketopiperazine elimination compound, H-MeLeu-Aze(2)-MeCha-MeGly-Asp-pyrrole (Compound 2-2*)

[0733] [Chem.39]

[0734]

[0735] LCMS (ESI) m / z = 635.45 (M+H) +

[0736] Retention time: 2.24 minutes (analysis conditions SQDAA05 long)

[0737] 6-membered cyclic amidine skeleton compound (Compound 2-3*)

[0738] [Chem.40]

[0739]

[0740] LCMS (ESI) m / z = 688.47 (M+H) +

[0741] Retention time: 2.52 minutes (analysis conditions SQDAA05 long)

[0742] MeLeu over-extended compound, H-MeLeu-MeLeu-Ile-MeAla-Aze(2)-MeCha-MeGly-Asp-pyrrole (Compound 2-4*)

[0743] [Chem.41]

[0744]

[0745] LCMS (ESI) m / z = 960.66 (M + H) +

[0746] Retention time: 3.19 minutes (analysis conditions SQDAA05 long)

[0747] Basic Operation B (Table 5, Runs 8 to 13, 16 to 23, and 25 to 28)

[0748] 100mg of the peptide-loaded solid phase resin (compound 1-2-4) prepared in Example 1-2-4 was placed in a solid phase reaction container, DCM (1.0mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit, and the resin was subsequently washed twice with the solvent (0.7mL per column) used during Fmoc deprotection. Fmoc deprotection solution (0.7mL per column) was added, and the mixture was reacted for 10 minutes at room temperature to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base shown in Table 5 in the Fmoc deprotection solvent at a volume percentage concentration (v / v%) shown in Table 5. The solution was discharged from the glass frit, and then the resin was washed six times with a washing solvent (0.7mL per column).

[0749] To the resin obtained as described above, a solution obtained by mixing a solution of Fmoc-MeLeu-OH (4 equivalents) in an extended solvent (0.25 mL), a urea-based condensing agent or a phosphonium-based condensing agent (4 equivalents) in an extended solvent (0.25 mL) and DIPEA (6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0750] For example, in Run 8 in Table 5, the following procedure is performed according to Basic Operation B.

[0751] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-4 prepared from Compound 1-2-1 (at 0.552 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0752] To the resin obtained as described above, a solution obtained by mixing a solution of Fmoc-MeLeu-OH (0.221 mmol, 4 equivalents) in DMSO (0.25 mL), a solution of [ethylcyano(imino)acetate-O2)tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.221 mmol, 4 equivalents) in DMSO (0.25 mL) and DIPEA (0.057 mL, 0.331 mmol, 6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0753] To check the progress of the reaction, a portion of the resulting resin was taken out, swollen with DCM, and washed twice with DMF, then a DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, the resin was washed four times with DMF, and four times with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDAA05 long).

[0754] Basic Operation C (Table 5, Run 24)

[0755] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-4 prepared from Compound 1-2-1 (at 0.552 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0756] To the resin obtained as described above, a mixture of a solution of Fmoc-MeLeu-Cl (described later) (0.221 mmol, 4 equivalents) in NMP (0.7 mL) and 2,4,6-trimethylpyridine (0.55 mmol, 10 equivalents) was added, and the mixture was shaken at 40° C. for 1.5 hours. The solution was discharged from the glass frit, and the resin was then washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried. Fmoc-MeLeu-Cl was prepared as follows. (2S)-2-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]-4-methylpentanoic acid (Fmoc-MeLeu-OH) (0.081 g, 0.22 mmol) was dissolved in dichloromethane (0.44 mL), and DMF (0.017 mL, 0.22 mmol) was added dropwise while the solution was cooled in an ice bath. Next, thionyl chloride (0.024mL, 0.33mmol) was added. The obtained reaction mixture was stirred at room temperature for 30 minutes, and then concentrated under reduced pressure. Toluene (0.81mL, 10v / w) was added to the obtained residue, and the mixture was concentrated under reduced pressure twice. The obtained crude product was dissolved in NMP (0.7mL), and the solution was used for the above reaction.

[0757] To check the progress of the reaction, a portion of the resulting resin was taken out, swollen with DCM, and washed twice with DMF, then a DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, the resin was washed four times with DMF, and four times with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDAA05 long).

[0758] The formation of diketopiperazine elimination compounds is a well-known side reaction in peptide synthesis. The formation of 6-membered ring amidine skeleton compounds has not been reported before, and is a new peptide synthesis problem discovered by the inventors of the present application. Without being bound by a specific theory, when the amino group at the N-terminal nucleophilic attack carbonyl group, a second amide bond is formed from the N-terminal, thereby forming a 6-membered ring, and then by eliminating the original amino group that forms the amide bond, a diketopiperazine elimination compound is formed. On the other hand, when a 6-membered ring is formed similarly, the formation of a 6-membered cyclic amidine skeleton compound is carried out, and the hydroxyl group derived from the carbonyl group formed by the nucleophilic attack is subsequently eliminated. That is, the formation of diketopiperazine elimination compounds and the formation of 6-membered amidine skeleton compounds are common peptide synthesis problems caused by the formation of 6-membered rings.

[0759] The following Table 5 shows the obtained results (the relative ratios of the UV areas of Compound 2-1*, Compound 2-2*, Compound 2-3* and Compound 2-4* in LCMS under the respective conditions are expressed as percentages).

[0760]

[0761]

[0762] Run 1 and Run 2 include an Fmoc deprotection step, a resin washing step, and an extension step, which are performed in a normal peptide synthesis (comparative experiment). For example, under the conditions of Run 2, the base used in the Fmoc deprotection step is DBU, and the solvent used in the Fmoc deprotection step, the resin washing step, and the extension step is DMF. It was confirmed that under this condition, about 2% of a diketopiperazine (DKP) elimination compound lacking two residues was formed, and about 5% of a 6-membered cyclic amidine skeleton compound was formed. On the other hand, it was confirmed that by using DMSO as a solvent in the Fmoc deprotection process, the resin washing step, and the extension process, the formation of the diketopiperazine elimination compound and the 6-membered cyclic amidine skeleton compound can be suppressed, and the formation of a MeLeu over-extension compound as in Run 5 can be suppressed. As shown in the results of Run 3, it was confirmed that under the conditions of using piperidine (pip), piperidine (pip) is the most commonly used base in Fmoc deprotection (pKa 19.35 (in acetonitrile), refer to Eur.J.Org.Chem.2019,6735-6748) and the solvent in the Fmoc deprotection step, the resin washing step and the extension step is DMSO, about 2% of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds are formed respectively. On the other hand, as shown in Runs 4 to 7, it was confirmed that the use of a stronger base with a pKa of 23 or more in the conjugate acid can inhibit the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds.

[0763] As shown in the results of Run 8, Runs 10 to 13, and Runs 16 to 23, it was confirmed that the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds could be suppressed even when various phosphonium / urea condensation agents were used in the extension reaction under the conditions of using DMSO as a solvent in the Fmoc deprotection step, the resin washing step, and the extension step.

[0764] As shown in the results of Run 9, it was confirmed that the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds can be suppressed even when DMSO solvent is used in the Fmoc deprotection step and the resin washing step, and PyOxim is used as a condensation agent and a solvent other than DMSO in the extension step.

[0765] The results of using DMSO solvent in the Fmoc deprotection step and using DMSO solvent in one of the resin washing step and the extension step and using DMF solvent in the other step are shown in Runs 14 and 15. Compared with Run 5 in which DMSO solvent was used in all three steps, when DIC / Oxyma was used as a condensing agent, the use of DMF solvent only in the extension step resulted in a slight increase in the formation of a 6-membered ring amidine skeleton compound. On the other hand, it was confirmed that when DMF solvent was used only in the resin washing step, the formation of a diketopiperazine elimination compound and a 6-membered ring amidine skeleton compound could be suppressed to the same extent as in the case in which DMSO solvent was used in all three steps.

[0766] In the present invention, an acid chloride can be used in the extension step. For example, as shown in Run 24, it was confirmed that even if an acid chloride prepared separately was applied to NMP in the presence of 2,4,6-trimethylpyridine in the extension reaction performed under the condition of using DMSO in the Fmoc deprotection step and the resin washing step, the formation of a diketopiperazine elimination compound and a 6-membered cyclic amidine skeleton compound can be suppressed.

[0767] In the present invention, two or more bases can coexist at the same time in the Fmoc deprotection step. In the case where two bases coexist, for example, even when piperidine, which is generally known to give an adduct with dibenzofulvene formed in the Fmoc deprotection step, coexists, the formation of a diketopiperazine elimination compound and a 6-membered cyclic amidine skeleton compound can be suppressed, as long as the optimal base (here, DBU) is used as one of the bases in Run 25.

[0768] It was confirmed that even when HP1(dma), P1tBu, and P2Et, which are more basic than DBU, coexist, the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds can be suppressed.

[0769] As shown in the results of Runs 29 and 30, it was confirmed that the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds could be suppressed even when DsBC and HOAt were used in the elongation reaction under the conditions of using DMSO in the Fmoc deprotection step, the resin washing step, and the elongation step.

[0770] Therefore, the outline conditions of each step (Fmoc deprotection step, resin washing step and extension step) capable of suppressing the formation of diketopiperazine elimination compound and 6-membered cyclic amidine skeleton compound were determined based on the results of Example 2. When suppressing the formation of diketopiperazine elimination compound and 6-membered cyclic amidine skeleton compound, the combination of reaction conditions is not limited to the conditions shown in Example 2.

[0771] Example 3: Testing the generality of the reduction of diketopiperazine elimination during peptide synthesis under the conditions of the peptide sequence found Universal Experiment

[0772] In this example, for various peptide sequences (peptide sequences) prepared in Example 1-2-4, the amino acids described in Core 1 were extended by a common conventional method (Condition-1) and the method of the present invention (Condition-2) (the peptide sequences of compounds 1-2-5 to 1-2-9 in Table 6 were extended to Core 2), and the amounts of diketopiperazine elimination compounds formed were compared.

[0773] Condition-1 (Traditional method)

[0774] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (compounds 1-2-5 to 1-2-9 (corresponding to runs 1 to 5 in Table 6, respectively)) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and then the resin was washed twice with DMF (0.7 mL per column). A DMF solution of piperidine (20 v / v%, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and then the resin was washed eight times with DMF (0.7 mL per column).

[0775] To the resin obtained as described above, a solution obtained by mixing a solution of an Fmoc-protected amino acid (0.6 mol / L) and HOAt (0.375 mol / L) in DMF (0.3 mL) with a solution of DIC (10 v / v%) in DMF (0.36 mL) was added, and the mixture was shaken for 4 hours at 40° C. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0776] Condition-2 (method of the present invention)

[0777] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (compounds 1-2-5 to 1-2-9 (corresponding to runs 1 to 5 in Table 6, respectively)) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0778] To the resin obtained as described above, add a solution obtained by mixing a DMSO (0.25 mL) solution of an amino acid (4 equivalents) protected by Fmoc, a DMSO (0.25 mL) solution of [ethyl cyano (hydroxyimino) acetate atom-O2] tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (4 equivalents) and DIPEA (6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes, and the mixture is shaken at room temperature for 4 hours. The solution is discharged from the glass frit, and then the resin is washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried. The solution is discharged from the glass frit, and then the resin is washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0779] In order to check the progress of the reaction, a portion of each resin obtained was taken out and swollen with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05 long) to confirm the formation of the target peptide (TM) (compounds 3-1-1* to 3-1-5*) and the diketopiperazine elimination compound (compounds 3-2-1* to 3-2-5*).

[0780] Table 6 shows the amount of diketopiperazine (DKP) elimination compound formed for each sequence and each condition. When the total UV area percentage of the target peptide (TM) and the diketopiperazine (DKP) elimination compound in LCMS is 100%, the amount of diketopiperazine (DKP) elimination compound formed shown in Table 6 is the UV area percentage of the diketopiperazine (DKP) elimination compound.

[0781] Target peptide compound (TM), Fmoc-MeLeu-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp-pip (Run 1) (Compound 3-1-1*)

[0782] [Chem.42]

[0783]

[0784] LCMS (ESI) m / z = 1055.68 (M+H) +

[0785] Retention time: 3.10 minutes (analysis conditions SQDFA05 long)

[0786] Diketopiperazine elimination compound, Fmoc-MeLeu-Aze(2)-MeCha-MeGly-Asp-pip (Run 1) (Compound 3-2-1*)

[0787] [Chem.43]

[0788]

[0789] LCMS (ESI) m / z = 871.61 (M+H) +

[0790] Retention time: 2.97 minutes (analysis conditions SQDA05 long)

[0791] Target peptide compound (TM), Fmoc-Thr(tBu)-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp-pip (Run 2) (Compound 3-1-2*)

[0792] [Chem.44]

[0793]

[0794] LCMS (ESI) m / z = 1160.61 (M+H) +

[0795] Retention time: 3.81 minutes (analysis conditions SQDA05 long)

[0796] Diketopiperazine elimination compound, Fmoc-Thr(tBu)-Cys(StBu)-Phe-Asp-pip (Run 2) (Compound 3-2-2*)

[0797] [Chem.45]

[0798]

[0799] LCMS (ESI) m / z = 918.44 (M+H) +

[0800] Retention time: 3.49 minutes (analysis conditions SQDA05 long)

[0801] Target peptide compound (TM), Fmoc-Nle-MeAla(3-Pyr)-Ser(iPen)-Asp-pip (Run 3) (Compound 3-1-3*)

[0802] [Chem.46]

[0803]

[0804] LCMS (ESI) m / z = 968.59 (M+H) +

[0805] Retention time: 2.50 minutes (analysis conditions SQDA05 long)

[0806] Diketopiperazine elimination compound, Fmoc-Nle-Ser(iPen)-Asp-pip (Run 3) (Compound 3-2-3*)

[0807] [Chem.47]

[0808]

[0809] LCMS (ESI) m / z = 693.72 (M+H) +

[0810] Retention time: 2.88 minutes (analysis conditions SQDA05 long)

[0811] Target peptide compound (TM), Fmoc-Nle-MePhe(3-Cl)-Ser(iPen)-Asp-pip (Run 4) (Compound 3-1-4*)

[0812] [Chem.48]

[0813]

[0814] LCMS (ESI) m / z = 1001.54 (M+H) +

[0815] Retention time: 3.55 minutes (analysis conditions SQDA05 long)

[0816] Diketopiperazine elimination compound, Fmoc-Nle-Ser(iPen)-Asp-pip (Run 4) (Compound 3-2-4*)

[0817] [Chem.49]

[0818]

[0819] LCMS (ESI) m / z = 693.42 (M+H) +

[0820] Retention time: 2.88 minutes (analysis conditions SQDA05 long)

[0821] Target peptide compound (TM), Fmoc-Pro-Ser(nPr)-MeAla-Phe-Asp-pip (Run 5) (Compound 3-1-5*)

[0822] [Chem.50]

[0823]

[0824] LCMS (ESI) m / z = 881.47 (M+H) +

[0825] Retention time: 2.60 minutes (analysis conditions SQDA05 long)

[0826] Diketopiperazine elimination compound, Fmoc-Pro-Phe-Asp-pip (Run 5) (Compound 3-2-5*)

[0827] [Chem.51]

[0828]

[0829] LCMS (ESI) m / z = 667.38 (M+H) +

[0830] Retention time: 2.44 minutes (analysis conditions SQDA05 long)

[0831]

[0832] Therefore, according to the results of Example 3, it was confirmed that in the synthesis of various peptides and specific peptide sequences, when elongation was performed by the method of the present invention, the diketopiperazine elimination compound inhibition effect was exhibited compared with the general conventional method.

[0833] Example 4: In addition to the problem of diketopiperazine elimination, the neutralization step was examined under the conditions of WO2022 / 097540 Experimental study on the effect of acid washing on the order of formation of diurea compounds

[0834] Compound 1-2-10 has a sequence that diketopiperazine is easily eliminated. When the method in WO2022 / 097540 is applied, the elimination of diketopiperazine can be significantly suppressed, but on the other hand, it has been confirmed that diurea compounds derived from carbamates are formed as additional by-products. It is not bound by a specific theory, and it is believed that when carbamates are stably present, the isocyanate formed by the reaction of a condensing agent with a carbamate anion undergoes a nucleophilic attack of an amino group adjacent to the N-terminus, thereby causing an intermolecular reaction to form a diurea compound. In this example, compound 1-2-10 is used as a solid-phase loaded peptide sequence, a neutralization step is added, acid and alkali are added in the resin washing step, and the degree of reduction of impurities (diketopiperazine compounds and diurea compounds) formed in peptide synthesis is determined to be eliminated to determine a series of appropriate conditions that can alleviate these two problems.

[0835] Basic Operation D (Table 7, Runs 1 and 4)

[0836] 100mg of the peptide-loaded solid phase resin (compound 1-2-10) prepared in Example 1-2-4 was placed in a solid phase reaction vessel, DCM (1.0mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit, and the resin was subsequently washed twice with the solvent (0.7mL per column) used in the Fmoc deprotection. Fmoc deprotection solution (0.7mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base shown in Table 7 in the Fmoc deprotection solvent at a volume percentage concentration (v / v%) shown in Table 7. The solution was discharged from the glass frit, and the resin was then washed eight times with DMF in Run 1 and six times with DMSO in Run 4, wherein the amount of solvent was 0.7mL per column.

[0837] To the resin obtained as described above, a mixture of a solution (0.3 mL per column) obtained by dissolving Fmoc-MeAla-OH (0.6 mol / L) and HOAt or Oxyma (0.375 mol / L) in an extended solvent and a solution (0.36 mL per column) obtained by dissolving DIC (10 v / v%) in an extended solvent was added, and the mixture was shaken at 40° C. for 2.5 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0838] For example, in Run 4 in Table 7, the following procedure is performed according to Basic Operation D.

[0839] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-10 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0840] To the resin obtained as described above, a mixture of a solution of Fmoc-MeAla-OH (0.6 mol / L) and Oxyma (0.375 mol / L) in DMSO (0.3 mL) and a solution of DIC (10 v / v%) in DMSO (0.36 mL) was added, and the mixture was shaken for 2.5 hours at 40° C. The solution was drained from the glass frit, and the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0841] To check the progress of the reaction, a portion of the resulting resin was taken out and swollen with DCM, the solution was drained from the glass frit, and then the peptide was separated using a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05long) to confirm the formation of the target peptide compound (TM) (4-1*), diketopiperazine (DKP) elimination compound (compound 4-2*) and diurea (compound 4-3*).

[0842] Target peptide compound (TM), Fmoc-MeAla-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Compound 4-1*)

[0843] [Chem.52]

[0844]

[0845] LCMS (ESI) m / z = 895.61 (M+H) +

[0846] Retention time: 2.66 minutes (analysis conditions SQDFA05long)

[0847] Diketopiperazine elimination compound, Fmoc-MeAla-Tyr(tBu)-Asp-pip (Compound 4-2*)

[0848] [Chem.53]

[0849]

[0850] LCMS (ESI) m / z = 727.52 (M+H) +

[0851] Retention time: 2.81 minutes (analysis conditions SQDFA05long)

[0852] Diurea compound (Compound 4-3*)

[0853] [Chem.54]

[0854]

[0855] LCMS (ESI) m / z = 1201.77 (M+H) +

[0856] Retention time: 2.18 minutes (analysis conditions SQDFA05long)

[0857] Basic Operation E (Table 7, Runs 2, 3, and 6 to 25)

[0858] 100mg of the solid phase resin (compound 1-2-10) loaded with peptide prepared in Example 1-2-4 was placed in a solid phase reaction container, DCM (1.0mL) was added in a glove box purged with nitrogen, and the mixture was left to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass material, and the resin was subsequently washed twice with the solvent (0.7mL per column) used in the Fmoc deprotection. Fmoc deprotection solution (0.7mL per column) was added, and the mixture was reacted for 10 minutes at room temperature to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the alkali shown in Table 7 in the Fmoc deprotection solvent at a volume percentage concentration (v / v%) shown in Table 7. The solution was discharged from the glass material, and the resin was then washed with the washing solvent (0.7mL per column) shown in Table 7. Multiple washing operations were performed using the same solvent or different solvents or in combination with acid washing. Table 7 shows the order and number of washing operations.

[0859] To the resin obtained as described above, a solution obtained by mixing a solution of Fmoc-MeAla-OH (4 equivalents) in an extended solvent (0.25 mL), a solution of a phosphonium condensing agent (4 equivalents) in an extended solvent (0.25 mL) and DIPEA (6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0860] For example, in Run 17 in Table 7, the following procedure is performed according to Basic Operation E. 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-10 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed twice with a DMSO solution of DIPEA (2 equivalents) (0.7 mL per column). Subsequently, the resin was washed once with HOAt (2 eq.) and DIPEA (2 eq.) in DMSO (0.7 mL per column), and then three times with DIPEA (2 eq.) in DMSO (0.7 mL per column).

[0861] To the resin obtained as described above, a solution obtained by mixing a DMSO (0.25 mL) solution of Fmoc-MeAla-OH (0.181 mmol, 4 equivalents), a DMSO (0.25 mL) solution of [ethylcyano (hydroxyamino) acetate ester-O2] tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equivalents) and DIPEA (0.047 mL, 0.271 mmol, 6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was drained from the glass frit, and the resin was then washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0862] To check the progress of the reaction, a portion of the resulting resin was taken out and swollen with DCM, the solution was drained from the glass frit, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05long).

[0863] Basic Operation F (Table 7, Run 5)

[0864] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-10 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction container, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was discharged from the glass frit, and the resin was then washed twice with the solvent used for Fmoc deprotection shown in Table 7 (0.7 mL per column). Fmoc deprotection solution (0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The Fmoc deprotection solution was prepared by dissolving the base shown in Table 7 in the Fmoc deprotection solvent at the volume percentage concentration (v / v%) shown in Table 7. Subsequently, as neutralization step, add the DMSO solution of 0.4mL HOAt (0.25mol / L) (relative to solid phase loaded peptide about 2 equivalents), and mixture was left standstill 5 minutes.Solution is discharged from glass frit, and then with the washing solvent (every post 0.7mL) shown in resin table 7 wash six times.In the resin obtained as mentioned above, add by by Fmoc-MeAla-OH (0.181mmol, 4 equivalents) DMSO (0.3mL) solution, [ethyl cyano (hydroxy amino) acetic acid radical-O ] tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181mmol, 4 equivalents) DMSO (0.3mL) solution and DIPEA (0.047mL, 0.271mmol, 6 equivalents) mixing and mixture was left standstill about 1 to 2 minutes and the solution obtained, and mixture was at room temperature shaken 4 hours. The solution was drained from the frit, and the resin was then washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0865] For example, in Run 5 in Table 7, the following procedure is performed according to Basic Operation F. 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (Compound 1-2-10 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to swell the resin. Thereafter, the solution was discharged from the glass frit. Subsequently, a DMSO solution (2 v / v%, 0.7 mL per column) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added to the resin, and the mixture was allowed to stand for 10 minutes for Fmoc deprotection. Subsequently, as a neutralization step, 0.4 mL of HOAt (0.5 mol / L) (about 4 equivalents relative to the solid phase loaded peptide) of DMSO solution was added, and the mixture was allowed to stand for 5 minutes. The solution was drained from the frit, and the resin was then washed six times with DMSO (0.7 mL per column).

[0866] To the resin obtained as described above, a solution obtained by mixing a DMSO (0.3 mL) solution of Fmoc-MeAla-OH (0.181 mmol, 4 equivalents), a DMSO (0.3 mL) solution of [ethylcyano (hydroxyamino) acetate ester-O2] tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equivalents) and DIPEA (0.047 mL, 0.271 mmol, 6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was discharged from the glass frit, and the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0867] To check the progress of the reaction, a portion of the resulting resin was taken out and swollen with DCM, the solution was drained from the glass frit, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05long).

[0868] The following Table 12 shows the obtained results (the relative ratios of the UV areas of compound 4-1*, compound 4-2* and compound 4-3* in LCMS under the respective conditions are expressed as percentages).

[0869]

[0870]

[0871]

[0872] Run 1 includes an Fmoc deprotection step, a resin washing step, and an extension step, which are typically performed in ordinary peptide synthesis (comparative experiment). Piperidine (pip) is used as a base in the Fmoc deprotection step, and DMF is used as a solvent in the Fmoc deprotection step, the resin washing step, and the extension step. Under this condition, about 75% of diketopiperazine (DKP) elimination compounds lacking two residues are formed, and about 25% of the target product is formed. On the other hand, when the method in WO 2022 / 097540 is used, the formation of diketopiperazine elimination compounds can be suppressed to about 4%, but about 30% of diurea compounds are formed as additional by-products, and the rate of target product formed is about 68%, as shown in the results of Run 2.

[0873] On the other hand, as shown in Run 3, the use of DMSO as a solvent in all Fmoc deprotection steps, resin washing steps, and extension steps reduced the formation of diketopiperazine elimination compounds to about 10%, reduced the formation of diurea compounds to about 7%, and increased the formation of the target product to about 83%.

[0874] To suppress the formation of diketopiperazine elimination compounds and diurea compounds, the condensation was fixed to PyOxim, and the conditions of the resin washing step after the Fmoc deprotection step were investigated.

[0875] First, when the neutralization step of adding 2 equivalents of HOAt is performed after the Fmoc deprotection step, the formation of the diketopiperazine elimination compound can be suppressed to about 8%, and the formation of the diurea compound can be suppressed to about 4%, as shown in Run 5. Without being bound by a particular theory, the present result of being able to reduce the formation of the diurea compound may be due to the decomposition of the carbamate by the neutralization step. When washing with a DMSO solution containing 2 equivalents of HOAt is performed only in the sixth of the six resin washing steps for the same purpose as the neutralization step, the formation of the diketopiperazine elimination compound and the diurea compound can be further suppressed compared to the case of using the neutralization step as shown in Run 6.

[0876] On the other hand, as shown in Runs 6 to 8, the formation of diketopiperazine elimination compounds increased with longer exposure to DMSO solution containing 2 equivalents of HOAt, but still showed excellent results compared to Run 1 (representing the general method).

[0877] Even when the exposure time to the washing solvent was long, the formation of diketopiperazine elimination compounds and diurea compounds could be suppressed to the same extent as in the case of short exposure time by adding DIPEA to the washing solvent containing an acid, as shown in Runs 9 to 12. This demonstrates the applicability to scale-up involving increasing the time required for each step, such as the washing time.

[0878] It was confirmed that when washing with a DMSO solution containing HOAt and DIPEA was performed in the first of six resin washing steps, the formation of diurea compounds was slightly increased compared to the case where the relevant washing was performed in the sixth resin washing step shown in Runs 13 and 14, while when the relevant washing was performed in the third resin washing step, the formation of diketopiperazine elimination compounds and diurea compounds could be suppressed to the same extent as the case where the relevant washing was performed in the sixth resin washing step shown in Runs 15 and 16.

[0879] In addition, as shown in Operations 17 and 18, it was confirmed that among the six resin washings, when washing with a DMSO solution containing HOAt and DIPEA was also performed in the other five resin washing steps except for washing with a DMSO solution containing 2 equivalents of DIPEA, the formation of diketopiperazine elimination compounds could be further suppressed compared with the case where washing was performed only with DMSO in the other five resin washing steps.

[0880] As shown in Runs 19 to 25, it was confirmed that even when Oxyma, 4-nitrophenol, HOOBt, triethylamine, 2,6-lutidine, 2,4,6-collidine and pyridine hydrochloride were used as the acid and base in the washing with the DMSO solution containing the acid and the base and in the washing with the DMSO solution containing the base and in the other five resin washing steps, the production of diketopiperazine elimination compounds and diurea compounds could be suppressed.

[0881] Therefore, according to the results of Example 4, the range of steps (Fmoc deprotection step, resin washing step, and extension step) capable of suppressing the formation of diketopiperazine elimination compounds and diurea compounds was determined, where diketopiperazine elimination compounds in the peptide sequence became a problem, and the formation of diurea compounds under the conditions of WO 2022 / 097540 became another problem. With regard to suppressing the formation of diketopiperazine elimination compounds and diurea compounds, this example is part of the present invention, and the present invention is not limited to the combination of reaction conditions shown in Example 4.

[0882] Example 5: To examine the effect of reducing the elimination of diketopiperazines in peptide synthesis under the conditions for the peptide sequence in Example 4 The universal experiment

[0883] In this example, the various peptide sequences prepared in Example 1-2-4 were extended with the amino acids described in core 1 by a common conventional method (condition-1), the method of the present invention described in Example 3 (condition-2) and the method found in Inventive Example 4 (condition-3) (peptide sequences extended to core 2 in Table 8, compounds 1-2-4 to 1-2-9 and compounds 1-2-11 to 1-2-12), and the amounts of diketopiperazine elimination compounds and carbamate-derived by-products formed were compared.

[0884] In Run 1, the amino acids described in Core 1 were extended by the method described in Example 2, Table 5, Run 1 as a common routine method.

[0885] Condition 3 (method in Example 4)

[0886] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-4 (compounds 1-2-4 to 1-2-9, 1-2-11 and 1-2-12 (corresponding to runs 1 to 8 in Table 6, respectively)) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2% v / v, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and then the resin was washed twice with a DMSO solution of DIPEA (2 equivalents) (0.7 mL per column). Subsequently, the resin was washed once with HOAt (2 eq.) and DIPEA (2 eq.) in DMSO (0.7 mL per column), and then three times with DIPEA (2 eq.) in DMSO (0.7 mL per column).

[0887] To the resin obtained as described above, a solution of DMSO (0.25 mL) solution of an amino acid (4 equivalents) protected by Fmoc, a solution of DMSO (0.25 mL) solution of [ethylcyano (hydroxyamino) acetate atom-O2] tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (4 equivalents) and DIPEA (6 equivalents) were added and the mixture was allowed to stand for about 1 to 2 minutes and the solution obtained, and the mixture was shaken at room temperature for 4 hours. The solution was discharged from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0888] In order to check the progress of the reaction, a portion of each resin obtained was taken out and swollen with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05 long) to confirm the formation of the target peptide (TM) (compounds 3-1-1* to 3-1-5* and compounds 5-1-1* to 5-1-2*) and diketopiperazine elimination compounds (compounds 3-2-1* to 3-2-5* and compounds 5-2-1* to 5-2-2*).

[0889] In Run 1, in order to check the progress of the reaction, a portion of the obtained resin was taken out, swollen with DCM, and washed twice with DMF, then a DMF solution (2 v / v%) of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, the resin was washed four times with DMF, and four times with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDAA05long) to confirm the formation of the target peptide (TM) (Compound 2-1*), the diketopiperazine elimination compound (Compound 2-2*), and the 6-membered ring amidine skeleton compound (Compound 2-3*).

[0890] Table 8 shows the amount of diketopiperazine (DKP) elimination compounds formed for each sequence and each condition. When the total UV area percentage of the target peptide (TM), diketopiperazine (DKP) elimination compounds and carbamate-derived by-products in LCMS is 100%, the amount of diketopiperazine (DKP) elimination compounds formed shown in Table 8 is the UV area percentage of the diketopiperazine (DKP) elimination compounds. The amount of diketopiperazine (DKP) elimination compounds formed in Run 1 shown in Table 8 includes the amount of the 6-membered cyclic amidine skeleton compound formed.

[0891] Target peptide compound (TM), Fmoc-MeIle-Aib-Pro-D-3-Abu-OH (Run 7) (Compound 5-1-1*)

[0892] [Chem.55]

[0893]

[0894] LCMS (ESI) m / z = 633.42 (MH) -

[0895] Retention time: 2.41 minutes (analysis conditions SQDA05 long)

[0896] Diketopiperazine elimination compound, Fmoc-MeIle-D-3-Abu-OH (Run 7) (Compound 5-2-1*)

[0897] [Chem.56]

[0898]

[0899] LCMS (ESI) m / z = 453.55 (M+H) +

[0900] Retention time: 2.46 minutes (analysis conditions SQDFA05 long)

[0901] Carbamate-derived by-product (Run 7) (Compound 5-3-1*)

[0902] [Chem.57]

[0903]

[0904] LCMS (ESI) m / z = 412.23 (M+H) +

[0905] Retention time: 1.03 minutes (analysis conditions SQDFA05 long)

[0906] Target peptide compound (TM), Fmoc-Pro-cLeu-MeVal-D-3-Abu-OH (Run 8) (Compound 5-1-2*)

[0907] [Chem.58]

[0908]

[0909] LCMS (ESI) m / z = 645.41 (MH) -

[0910] Retention time: 2.39 minutes (analysis conditions SQDFA05 long)

[0911] Diketopiperazine elimination compound, Fmoc-Pro-D-3-Abu-OH (Run 8) (Compound 5-2-2*)

[0912] [Chem.59]

[0913]

[0914] LCMS (ESI) m / z = 423.53 (M+H) +

[0915] Retention time: 1.87 minutes (analysis conditions SQDFA05 long)

[0916] Carbamate-derived by-product (Run 8) (Compound 5-3-2*)

[0917] [Chem.60]

[0918]

[0919] LCMS (ESI) m / z = 452.24 (MH) -

[0920] Retention time: 1.58 minutes (analysis conditions SQDFA05 long)

[0921]

[0922]

[0923] From the results of Runs 1 to 6, it was confirmed that in the synthesis of various peptides and the sequence described in Example 4, the diketopiperazine elimination compound inhibition effect was exhibited when elongation was performed by the method found in Example 4 (Condition-3) compared with the common conventional method.

[0924] In Runs 7 and 8, it was confirmed that when the method of Example 3 (Condition 2) was applied to these sequences, the formation of diketopiperazine elimination compounds could be suppressed compared to the conventional method, but carbamate-derived byproducts (Compound 5-3-1* and Compound 5-3-2*) were formed. Without being bound by a particular theory, the formation of byproducts may be because when carbamate is stably present, the isocyanate formed by the reaction of the condensing agent with the carbamate anion is subjected to nucleophilic attack by TFE used in the separation process.

[0925] It was confirmed that when these sequences were extended by the method found in Example 4 (Condition-3) of the present invention, the formation of carbamate-derived by-products and diketopiperazine elimination compounds could be suppressed (Runs 7 and 8). This result of being able to reduce by-products derived from carbamate may be due to the decomposition of carbamate by washing with an acid.

[0926] Therefore, according to the results of Example 5, it was confirmed that in the synthesis of various peptides and specific peptide sequences, when elongation was performed by the method in Example 4, the formation of diketopiperazine elimination compounds and carbamate-derived by-products could be generally suppressed compared with the common conventional method.

[0927] Example 6: Used to test the diketone in the peptide synthesis process for the carboxylic acid substrate to be extended under the conditions in Example 4 Experiments on the universality of the reduction effect of piperazine elimination

[0928] In this example, various carboxylic acids described in Core 1 were extended by the method found in Example 4 (Condition-4) for the peptide sequence prepared in Example 1-2-4 (Compound 1-2-10), and the amounts of diketopiperazine elimination compounds and diurea compounds formed were compared.

[0929] Condition 4 (method in Example 4)

[0930] 100 mg of the peptide-loaded solid phase resin prepared in Example 1-2-2 (Compound 1-2-10 prepared from Compound 1-2-2 (at 0.452 mmol / g) in Example 1-2-4) was placed in a solid phase reaction vessel, DCM (1.0 mL) was added in a glove box purged with nitrogen, and the mixture was allowed to stand for 10 minutes to allow the resin to swell. Thereafter, the solution was drained from the glass frit, and the resin was subsequently washed twice with DMSO (0.7 mL per column). A DMSO solution of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (2 v / v%, 0.7 mL per column) was added, and the mixture was reacted at room temperature for 10 minutes to deprotect the Fmoc group. The solution was drained from the glass frit, and the resin was then washed twice with a DMSO solution of DIPEA (2 equivalents) (0.7 mL per column). Subsequently, the resin was washed once with HOAt (2 eq.) and DIPEA (2 eq.) in DMSO (0.7 mL per column), and then three times with DIPEA (2 eq.) in DMSO (0.7 mL per column).

[0931] To the resin obtained as described above, a solution obtained by mixing a solution of a carboxylic acid to be extended (0.181 mmol, 4 equivalents) (described in Table 9) in DMSO (0.25 mL), a solution of [ethylcyano(hydroxyamino)acetate-O2]tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (0.181 mmol, 4 equivalents) in DMSO (0.25 mL) and DIPEA (0.047 mL, 0.271 mmol, 6 equivalents) and leaving the mixture to stand for about 1 to 2 minutes was added, and the mixture was shaken at room temperature for 4 hours. The solution was drained from the glass frit, and then the resin was washed four times with DMF (0.7 mL per column) and four times with DCM (0.7 mL per column), and dried.

[0932] In order to check the progress of the reaction, a portion of each resin obtained was taken out and swollen with DCM, and then the peptide was separated with a TFE / DCM solution (1 / 1 (v / v)) containing DIPEA (0.045 mol / L). The separated solution was analyzed by LCMS (SQDFA05long or SQDAA05long) to confirm the formation of the target peptide (TM) (compounds 6-1-1* to 3-1-6*), diketopiperazine elimination compounds (compounds 6-2-1* to 6-2-6*) and diurea compounds (compounds 4-3*).

[0933] Table 9 shows the amounts of diketopiperazine elimination compounds (TM-DKP) and diurea compounds formed for each sequence. When the total UV area percentage of the target peptide (TM), diketopiperazine (DKP) elimination compounds and diurea compounds in LCMS is 100%, the amounts of each diketopiperazine (DKP) elimination compound and diurea compound formed shown in Table 9 are the UV area percentages of each diketopiperazine (DKP) elimination compound and diurea compound.

[0934] Target peptide compound (TM), Teoc-Phe(4-CF3)-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 1) (Compound 6-1-1*)

[0935] [Chem.61]

[0936]

[0937] LCMS (ESI) m / z = 947.60 (M+H) +

[0938] Retention time: 2.99 minutes (analysis conditions SQDFA05long)

[0939] Diketopiperazine elimination compound, Teoc-Phe(4-CF3)-Tyr(tBu)-Asp-pip (Run 1) (Compound 6-2-1*)

[0940] [Chem.62]

[0941]

[0942] LCMS (ESI) m / z = 779.53 (M+H) +

[0943] Retention time: 3.11 minutes (analysis conditions SQDFA05long)

[0944] Target peptide compound (TM), Boc-MeAla-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 2) (Compound 6-1-2*)

[0945] [Chem.63]

[0946]

[0947] LCMS (ESI) m / z = 773.62 (M+H) +

[0948] Retention time: 2.18 minutes (analysis conditions SQDFA05long)

[0949] Diketopiperazine elimination compound, Boc-MeAla-Tyr(tBu)-Asp-pip (Run 2) (Compound 6-2-2*)

[0950] [Chem.64]

[0951]

[0952] LCMS (ESI) m / z = 603.58 (MH) -

[0953] Retention time: 2.29 minutes (analysis conditions SQDFA05long)

[0954] Target peptide compound (TN), Cbz-Pro-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 3) (Compound 6-1-3*)

[0955] [Chem.65]

[0956]

[0957] LCMS (ESI) m / z = 819.58 (M+H) +

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

[0959] Diketopiperazine elimination compound, Cbz-Pro-Tyr(tBu)-Asp-pip (Run 3) (Compound 6-2-3*)

[0960] [Chem.66]

[0961]

[0962] LCMS (ESI) m / z = 649.61 (M+H) +

[0963] Retention time: 2.27 minutes (analysis conditions SQDFA05long)

[0964] Target peptide compound (TM), Ac-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 4) (Compound 6-1-4*)

[0965] [Chem.67]

[0966]

[0967] LCMS (ESI) m / z = 630.50 (M+H) +

[0968] Retention time: 2.21 minutes (analysis conditions SQDFA05long)

[0969] Diketopiperazine elimination compound, Ac-Tyr(tBu)-Asp-pip (Run 4) (Compound 6-2-4*)

[0970] [Chem.68]

[0971]

[0972] LCMS (ESI) m / z = 462.42 (M+H) +

[0973] Retention time: 2.17 minutes (analysis conditions SQDFA05long)

[0974] Target peptide compound (TM), Tfa-Aib-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 5) (Compound 6-1-5*)

[0975] [Chem.69]

[0976]

[0977] LCMS (ESI) m / z = 769.56 (M+H) +

[0978] Retention time: 1.90 minutes (analysis conditions SQDFA05long)

[0979] Diketopiperazine elimination compound, Tfa-Aib-Tyr(tBu)-Asp-pip (Run 5) (Compound 6-2-5*)

[0980] [Chem.70]

[0981]

[0982] LCMS (ESI) m / z = 601.54 (M+H) +

[0983] Retention time: 1.98 minutes (analysis conditions SQDFA05long)

[0984] Target peptide compound (TM), Ns-Aib-1-ACPrC-MeAla-Tyr(tBu)-Asp-pip (Run 6) (Compound 6-1-6*)

[0985] [Chem.71]

[0986]

[0987] LCMS (ESI) m / z = 858.54 (M+H) +

[0988] Retention time: 2.09 minutes (analysis conditions SQDFA05long)

[0989] Diketopiperazine elimination compound, Ns-Aib-Tyr(tBu)-Asp-pip (Run 6) (Compound 6-2-6*)

[0990] [Chem.72]

[0991]

[0992] LCMS (ESI) m / z = 690.41 (M+H) +

[0993] Retention time: 2.17 minutes (analysis conditions SQDFA05long)

[0994] [Table 9]

[0995]

[0996] Therefore, the results of Example 6 confirmed that in the elongation of various carboxylic acids and specific Fmoc amino acids, the formation of diketopiperazine elimination compounds and diurea compounds for synthesizing the target peptide during elongation by the method of the present invention can be suppressed.

[0997] Example 7: Examination of the Presence of Carbamate in the Fmoc Deprotection Step and the Effect on the Elimination of Diketopiperazines Inspection

[0998] The following table shows the compounds described in the Examples.

[0999] [Table 10]

[1000]

[1001]

[1002] In the examples, the following abbreviations are used.

[1003] NMR: Nuclear Magnetic Resonance

[1004] ROESY: Rotating-frame Overhauser effect spectroscopy

[1005] NOESY: Nuclear Overhauser Effect Spectroscopy

[1006] HSQC: Heteronuclear Single Quantum Correlation

[1007] HMBC: Heteronuclear Multiple Bond Correlation

[1008] cpd.: compound

[1009] The NMR measurement was performed at 298 K using AVANCE III 600Cryo-TCI or AVANCE III HD 600BBFO manufactured by Bruker.

[1010] When a base containing multiple nitrogen atoms is protonated to form a cation to form a salt with an anion in the system, any cation in which the nitrogen atom is protonated can give a cation source. For example, in the case of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), the three cations shown below can give a cation source, and in this specification, salts formed by any cation and the corresponding anion are not distinguished and are all referred to as DBU salts ( Figure 1 ). The same is true for other bases. For example, salts similarly formed from 1,1,3,3-tetramethylguanidine (TMG) or iminotris(dimethylamino)phosphorane (HP1(dma)) are referred to as TMG salts, HP1(dma) salts, or MTBD salts, respectively.

[1011] [Chem.73]

[1012]

[1013] Synthesis of compound 1-4-1: 9H-fluoren-9-ylmethyl N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl [Fmoc-Phe-NMe 2)

[1014] To a mixed solution of commercially available N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-Phe-OH, CAS: 35661-40-6, compound aa2-12) (2.0 g, 5.16 mmol) in N,N-dimethylformamide (DMF) (14 mL) and dichloromethane (DCM) (4 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (1.19 g, 6.19 mmol) and 1-hydroxybenzotriazole (HOBt) (0.767 g, 5.68 mmol), and then a tetrahydrofuran (THF) solution of dimethylamine (2.0 M, 2.84 mL, 5.68 mmol) was further added, and the mixture was stirred for 15 minutes. Isopropyl acetate (20 mL) was added to the solution, and the organic phase was washed twice with 1M hydrochloric acid (20 mL), once with water (50 mL), twice with half-saturated sodium bicarbonate aqueous solution (20 mL), and twice with half-saturated sodium chloride aqueous solution (20 mL). The obtained organic phase was dried over sodium sulfate, and then filtered, and the solvent of the obtained mother liquor was removed by distillation under reduced pressure. The obtained crude product was azeotropically distilled with toluene, and then cooled at 5 ° C to obtain white crystals (1.88 g, 88%) of compound 1-4-1.

[1015] [Chem.74]

[1016]

[1017] 1 H-NMR (600 MHz, toluene-d8, 298K) δ7.51 (d, 2H, J = 7.2 Hz), 7.43 (d, 2H, J = 7.3 Hz), 7.21-7.16 (m, 2H), 7.14-7.07 (m, 2H), 7.05-7.00 (m, 4H), 7.00-6.95 (m, 1H), 6.00 (d, 1H, J = 8.4 Hz, NH), 4.88 (ddd, 1H, J = 8 .4,8.4,6.0Hz),4.32(dd,1H,J=10.5,7.4Hz),4.23(dd,1H,J=10.5,7.4Hz),4.03(t,1H,J=7 .4Hz), 2.93 (dd, 1H, J = 13.0, 8.4Hz), 2.86 (dd, 1H, J = 13.0, 6.0Hz), 2.49 (s, 3H), 2.02 (s, 3H).

[1018] 13 C-NMR (151 MHz, toluene-d8, 298K) δ 171.1, 155.9, 144.6, 141.8, 129.9, 128.5, 127.8 (overlapping with solvent peak, detected by CH-HSQC), 127.3, 127.0, 125.6, 125.4, 120.2, 67.2, 52.3, 47.7, 40.2, 36.0, 35.0.

[1019] LCMS (ESI) m / z = 415.44 (M+H) +

[1020] Retention time: 0.89 minutes (analysis conditions SQDFA05)

[1021] Example 7-1: Checking the presence of DBU·HOCO-Phe-NMe2 when compound 1-4-1 is treated with DBU

[1022] Example 7-1-1: Compound 1-4-1 was treated with DBU in N,N-dimethylformamide-d7 (DMF-d7) solution to obtain Product of

[1023] The solution of compound 1-4-1 (12.9 mg, 0.031 mmol) in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was measured. 1 H-NMR( Figure 1 -b)). Then, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (5.11 μL, 0.034 mmol) was added to the relevant solution, and the mixture was vigorously stirred, followed by measuring 1 H-NMR( Figure 1-a) and 1D-ROESY(( Figure 2 -a)-2) and ( Figure 2 -a)-3)). As a result, it was confirmed from the NMR spectrum that three compounds were mainly formed. That is, it was confirmed that three compounds: dibenzofulvene, compound 1-4-2 (H-Phe-NMe2) and N-[(1S)-1-benzyl-2-(dimethylamino)-2-oxo-ethyl]carbamic acid DBU salt (compound 1-4-3a and DBU·HOCO-Phe-NMe2) were present in the relevant solution, and compound 1-4-2 and compound 1-4-3a were chemically exchanged. The identification of compound 1-4-2 (H-Phe-NMe2) in the relevant solution and the identification of compound 1-4-3a are described in detail in this example (Example) respectively. 7 -1-1) and examples 7 -1-3.

[1024] Confirm the formation of compound 1-4-2 (H-Phe-NMe2) and compound 1-4-1 in DMF-d7 by applying DBU Chemical exchange and ratio of compound 1-4-3a

[1025] Measure the 1D-ROESY of the relevant solution (selectively excite at 4.84 ppm and 3.95 ppm 1 H signal, Figure 2 -a)-2 and Figure 2 -a)-3), and the results showed that compound 1-4-3a was at 4.84 ppm 1 The H signal and the proton corresponding to the α-methylene of compound 1-4-2 are at 3.95 ppm 1 The H signal undergoes chemical exchange in the system (( Figure 2 -a)-2) and ( Figure 2 -a)-3)). That is, when selectively exciting 4.84 ppm 1 The H signal is at 3.95ppm 1 H signal ( Figure 2 -a)-2) and by selectively exciting 3.95ppm 1 The H signal is at 4.85ppm 1 H signal ( Figure 2 -a)-3), a ROESY signal in phase with the selective excitation signal was observed, and thus it was confirmed that these signals were chemically exchanged ( Figure 2 -a)). In addition, according to 1 In H-NMR 1 The integral ratio of H signals confirmed that the existence ratio of compound 1-4-3a to compound 1-4-2 was 82:18 ( Figure 2 -b)).

[1026] Check the presence of compound 1-4-2 (H-Phe-NMe2) in the relevant solution

[1027] Three (3) μL of the separately provided standards of compound 1-4-2 were mixed with the relevant solution and then measured. 1 H-NMR( Figure 3 -a)-3 and Figure 3 -b)) and 1D-NOSEY( Figure 3 -a)-4 and Figure 3 -a)-5).

[1028] 1) Measure the solution obtained by adding 3 μL of the standard of compound 1-4-2 to the relevant solution. 1 H-NMR and observations showed that the relevant solution 1 H-NMR observed at 3.95 ppm 1 H signal ( Figure 3 -a)-2) and the standard of compound 1-4-2 ( Figure 3 -a)-1) The result is 3.94ppm 1 H signal was observed as triplet at 3.95 ppm 1 H signal, the two signals completely overlap and there is no separation ( Figure 3 -a)-3).

[1029] 2) 1D-NOESY (selective excitation at 4.84 ppm and 3.95 ppm, respectively) of a solution obtained by adding 3 μL of a standard of compound 1-4-2 to a relevant solution 1 H signal), and the results show that: when selectively exciting the 1 The H signal is at 3.95ppm 1 H signal ( Figure 3 -a)-4); and when selectively excited at 3.95 ppm 1 The H signal is at 4.85ppm 1 H signal ( Figure 3 -a)-5), a NOESY signal that was in phase with the selective excitation signal and had the same shape as the original signal (4.85 ppm: double triplet and 3.95 ppm: triplet) was observed.

[1030] 3) Add 3 μL of the standard of compound 1-4-2 to the relevant solution, and add the standard of compound 1-4-3a at 4.84 ppm. 1 The H signal of compound 1-4-2 at 3.95 ppm 1 The integral ratio of H signal was increased from 82:18 (the integral ratio before adding compound 1-4-2 [ Figure 2 -b]) to 61:39( Figure 3 -b)).

[1031] Based on the above three points 1) to 3), the presence of compound 1-4-2 in the relevant solution was confirmed.

[1032] Example 7-1-2: Spraying 13 Identification of compound 1-4-3a formed by applying DBU to compound 1-4-2 using CO2

[1033] The following example 7 -1-2 Experimental operations were all performed in a glove box (under dehydration conditions under a nitrogen atmosphere). MS4A activated by heating to a high temperature with a microwave and then allowing to cool in a vacuum (×2) was used to dry N,N-dimethylformamide-d7 (DMF-d7) for 12 hours or more, which was used as a solvent. The sample was collected in a low pressure / vacuum tube for NMR with a valve in the glove box and completely prevented from contact with the outside air for NMR measurement.

[1034] Preparation of Solution A (SolA)

[1035] To a solution of compound 1-4-2 (4.64 mg, 0.024 mmol) in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was bubbled 13 CO2 gas for 1 minute, then measure 1 H-NMR( Figure 4 -b))、 13 C-NMR( Figure 5 -a)) and 1D-NOESY (selectively exciting 4.77 ppm and 3.96 ppm 1 H signal, ( Figure 4 -c) and ( Figure 4 -d)). Thereafter, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (3.97 μL, 0.027 mmol) was added to prepare a solution A containing compound 1-4-3a (DBU·HOCO-Phe-NMe2), and the 1 H-NMR( Figure 4 -e)) and 13 C-NMR( Figure 5 -b)).

[1036] Identification of compound 1-4-3a in solution A

[1037] The structure of compound 1-4-3a can be represented by 1 H-NMR, 13 The measurement results of C-NMR and 1D-NOESY can be interpreted without any contradiction.

[1038] 4) Measurement of the solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) with 13 The solution obtained by bubbling CO2 gas for 1 minute1 H-NMR, and the results showed the presence of a new 1 H signal, which is present in the raw material compound 1-4-2 1 No ( Figure 4 -a))( Figure 4 -b)) (formation of carbamate anion or carbamic acid, see Figure 5 -a)).

[1039] 5) The solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was measured with 13 The solution obtained by bubbling CO2 gas for 1 minute 1 The H-NMR observed a 1 The H signal at 3.96 ppm corresponding to the α-methylene of compound 1-4-2 1 The integral ratio of the H signal is 52:48 ( Figure 4 -f).

[1040] 6) The solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was measured with 13 1D-NOESY of the solution obtained by CO2 gas treatment for 1 minute, and the results showed that when the 4.76 ppm 1 The H signal is at 3.96ppm 1 H signal ( Figure 4 -c)) and when selectively excited at 3.96 ppm 1 The H signal is 4.76ppm 1 H signal, and the same-phase NOESY signal ( Figure 4 -d)), thus confirming that both signals were chemically exchanged.

[1041] 7) Measure the solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) with 13 The solution obtained by bubbling CO2 for 1 minute and then adding DBU 1 H-NMR, and the results showed that the 4.76 ppm observed before the addition of DBU 1 The H signal shifted to 4.83 ppm under high magnetic field when DBU was added ( Figure 4 -e)) (the formation of compound 1-4-3a was confirmed).

[1042] 8) The solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was measured by 13 The solution obtained by bubbling CO2 gas for 1 minute and then adding DBU 1 H-NMR, and the results showed that the α-methine at 4.83 ppm 1 The H signal is at 161.5ppm 13 C-labeled carbonyl 13 There is a 2.7Hz gap between the C signals. 3 J CH coupling( Figure 5 -b))( Figure 4 -g)).

[1043] 9) Measurement of the solution of compound 1-4-2 in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) with 13 The solution obtained by bubbling CO2 gas for 1 minute 13 C-NMR( Figure 5 -a)), the solution obtained by adding DBU to the above solution was measured. 13 C-NMR( Figure 5 -b)), the former measurement result is compared with the latter measurement result, and the results show that by adding DBU, corresponding to 13 C-labeled carbonyl 13 The C signal shifted from 157.1 ppm to 161.5 ppm under high magnetic field (confirming the formation of compound 1-4-3a).

[1044] According to the six points 4) to 9), it was confirmed that in the preparation of solution A (Sol A), the primary amine of compound 1-4-2 and 13 CO2 gas reacts to form carbamate or carbamic acid, and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) is applied thereto to form compound 1-4-3a (DBU·HOCO-Phe-NMe2). According to the above experiment, it is considered that compound 1-4-3a (DBU·HOCO-Phe-NMe2) is formed without any contradiction. For this structure, in Example 7 A more detailed identification is given in -1-3.

[1045] Example 7-1-3: Structural demonstration of compound 1-4-3a in solution A + solution B (SolA + SolB)

[1046] The experimental operations described in the following Example 7-1-3 were all carried out in a glove box (under dehydration conditions under a nitrogen atmosphere). MS4A activated by heating to a high temperature with a microwave and then allowing cooling in a vacuum (×2) was used to dry N,N-dimethylformamide-d7 (DMF-d7) for 12 hours or more, which was used as a solvent. The sample was collected in a low pressure / vacuum tube for NMR, with a valve in the glove box, and completely prevented from contact with the outside air for measuring NMR.

[1047] Preparation of Solution B (SolB)

[1048] To a solution of compound 1-4-1 (11.5 mg, 0.028 mmol) in N,N-dimethylformamide-d7 (DMF-d7) (0.5 mL) was added 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (4.56 μL, 0.031 mmol) to prepare solution B, and the 1 H-NMR (Solution B, Figure 6 -a)).

[1049] Preparation of Solution A + Solution B (SolA + Sol B)

[1050] Solution B (Sol B) (0.15 mL, Figure 6 -a), 0.0084 mmol) and solution A (Sol A) (0.35 mL, Sol A, Figure 6 -c), 0.0168mmol) were mixed and measured 1 H-NMR( Figure 6 -b))、 13 C-NMR, CH-HMBC ( Figure 7 -c))、CH-HSQC、NH-HMBC( Figure 7 -b)) and NH-HSQC( Figure 7 -a)).

[1051] Check for the presence of compound 1-4-3a in solution B (Sol B)

[1052] It was confirmed that in solution A 1 The α-methine group of compound 1-4-3a at 4.83 ppm observed in H-NMR 1 H signal ( Figure 6 -c)) and in solution B 1 The H-NMR observed a 1 H signal ( Figure 6 -a)) are completely consistent with each other, and each moves to 4.84ppm 1 H signal ( Figure 6-b)). That is, it was confirmed that when solution B was mixed with solution A, 4.85 ppm of 1 The uncoupled low-field side peak in the H signal ( Figure 6 -a) and the peak surrounded by the dotted line in solution A at 4.83 ppm 1 H signal can be identified with 13 C(2.7Hz) coupled low magnetic field side peak ( Figure 6 -b) moves together and transforms into an intermediate peak with a wide shape ( Figure 6 -b)). These results show that compound 1-4-3a ( 1 H signal).

[1053] Structural demonstration of compound 1-4-3a in solution A + solution B (SolA + Sol B)

[1054] By analyzing 1 H-NMR( Figure 6 -b))、 13 C-NMR, CH-HMBC ( Figure 7 -c))、CH-HSQC、NH-HMBC( Figure 7 -b)) and NH-HSQC( Figure 7 -a)), it can be explained without any contradiction that the structure of compound 1-4-3a in solution A + solution B (Sol A + Sol B) is the DBU salt of carbamic acid.

[1055] 10) The CH-HMBC of the mixed solution of solution A (Sol A) + solution B (Sol B) was measured, and it was found that the α-methine at 4.84 ppm 1 H signal and 161.5ppm 13 C-mark 13 C signal (which is higher than the solvent 13 C signal) between the CH-HMBC cross peaks ( Figure 7 -c)). That is, due to the CH-HMBC correlation and 13 C signal value, and due to 13 C is derived from the experimental bubbling 13 CO2 gas, so there will be no contradiction at 161.5ppm 13 The C signal was identified as the carbonyl group of carbamate.

[1056] 11) NH-HSQC of a mixed solution of solution A (Sol A) + solution B (Sol B) was measured, and the results showed that the NH 1 The H signal was identified at 91.4 ppm 15The NH-HSQC correlation between the N signals, thus the 15 The N signal was identified as the nitrogen atom of the carbamate ( Figure 7 -a)). It was also confirmed that the benzyl methylene at 2.91 / 2.83 ppm 1 The H signal is the same as the 91.4 ppm signal corresponding to the nitrogen atom of the carbamate. 15 There is NH-HMBC correlation between N signals ( Figure 7 -b)), and according to 15 The N signal can explain the 91.4ppm 15 The N signal is the nitrogen atom of the carbamate.

[1057] According to 10) and 11), it was confirmed that compound 1-4-3a was present in the system when preparing solution A + solution B (Sol A + Sol B). As described below, the NMR signals of the carbamate part of compound 1-4-3a can be attributed entirely to ( Figure 8 ), and the values ​​of all NMR signals can explain the structure of compound 1-4-3a without any contradiction.

[1058] [Chem.75]

[1059]

[1060] 1 H-NMR (600M Hz, DMF-d7, 298K) δ7.29-7.23 (m, 4H), 7.22-7.11 (m, 1H), 5.30 (brs, 1H, NH), 4.90 (ddd, 1H, J=6.8, 6.2Hz, 3 J CH =2.7Hz), 2.91 (dd, 1H, J = 13.3, 6.8Hz), 2.87 (s, 3H), 2.83 (dd, 1H, J = 13.3, 6.2Hz), 2.78 (s, 3H).

[1061] 13 C-NMR(151M Hz,DMF-d7,298K)δ173.6(qC),161.5(qC),139.4(qC),129.7(CHx2),128.3(CH x2),126.3(CH),53.0(CH),40.0(CH2),36.4(CH3),34.8(CH3).

[1062] 15 N-NMR (61M Hz, DMF-d7, 298K, assigned by NH-HSQC and NH-HMBC) δ 96,91.

[1063] The above results indicate that when the Fmoc-protected amino group is treated with DBU, the amino group is not completely exposed and subsequently decarboxylated to eliminate diphenylfulvene, but most of it exists as DBU·HOCO-Phe-NMe2. It also indicates that it can be 1 Its presence and abundance ratio were examined by H-NMR.

[1064] Example 7-2: Examination of the presence of carbamate when compound 1-4-1 was treated with DBU or piperidine in various solvents exist

[1065] Basic Operation

[1066] A solution of compound 1-4-1 (about 25 mg) in a heavy solvent (1.25 mL, tetrahydrofuran-d8 (THF-d8) [entry 1], toluene-d8 [entry 2], dicyclomethane-d2 (DCM-d2) [entry 3], N,N-dimethylformamide-d7 (DMF-d7) [entry 4]) or dimethyl sulfoxide-d6 (DMSO-d6) [entry 5] was prepared, and then 1.0 mL of the relevant solution was divided into two parts (0.5 mL). 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL) was applied to one part, and piperidine (67.5 μL) was applied to the other part. NMR of each part was measured by 1 The proton integration ratio in H-NMR determined the ratio of compound 1-4-3a to compound 1-4-2 (Table 11). The amount of compound 1-4-1 used in this experiment was: 25.1 mg for entry 1, 24.8 mg for entry 2, 25.0 mg for entry 3, 25.3 mg for entry 4, and 25.2 mg for entry 5.

[1067] [Chem.76]

[1068]

[1069] [Table 11]

[1070]

[1071] From the above results, it was confirmed that when the Fmoc-protected amino group was treated with DBU, the presence ratio of carbamate in any solvent was 50% or more. On the other hand, it was confirmed that when treated with pyridine, carbamate was not detected in any solvent, and even decarboxylation was performed, so that the amino group was completely exposed.

[1072] To discuss the experimental results on the solid surface based on this discovery, without being bound by a specific theory, it can be explained that, as shown in the scheme below, a 6-membered ring is formed, which leads to the reduction of the diketopiperazine elimination which is blocked by the presence of carbamate.

[1073] [Chem.77]

[1074]

[1075] For example, in the solid phase experiment, deprotection with DBU, which has been confirmed to generate carbamate, and deprotection with piperidine, which has not been confirmed to generate carbamate in NMR, were compared, and the effect of carbamate generation was examined. The reaction was carried out by the same method as the basic operation B described in Example 2 using the reagents and solvents shown in Runs 1 and 2 in Table 12. The following Table 12 shows the results obtained (the relative ratio of the UV area in LCMS under the respective conditions of compound 2-1*, compound 2-2*, compound 2-3* and compound 2-4* is expressed as a percentage).

[1076] [Table 12]

[1077]

[1078] Without being bound by a particular theory, from the comparison between Run 1 (2% DBU) and Run 2 (20% piperidine) in Table 12, it can be explained without contradiction why the formation of diketopiperazine elimination compounds and 6-membered cyclic amidine skeleton compounds can be significantly inhibited when treated with DBU, because the presence of carbamate prevents the formation of a 6-membered ring that leads to diketopiperazine elimination.

[1079] Example 7-3: When compound 1-4-1 was treated with various bases in DMSO, the presence of compound 1-4-3a was examined

[1080] Basic Operation

[1081] Five solutions of compound 1-4-1 (about 10 mg, 0.24 mmol) in dimethyl sulfoxide-d6 (DMSO-d6) (0.5 mL) were prepared, and then four bases, piperidine (67.5 μL, entry 1), 1.1.3.3-tetramethylguanidine (TMG) (13.5 μL, entry 2), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (6.8 μL, entry 3), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene (MTBD) (13.5 μL, entry 4) and iminotris(dimethylamino)phosphorane (HP1(dma)) (13.5 μL, entry 5), were applied to the solution, respectively. The NMR of each solution was measured by 1The proton integration ratio in H-NMR determined the ratio of compound 1-4-3a to compound 1-4-2 (Table 13). The amount of compound 1-4-1 used in this experiment was: 10.1 mg for entry 1, 10.0 mg for entry 2, 10.1 mg for entry 3, 10.1 mg for entry 4, and 10.1 mg for entry 5.

[1082] [Chem.78]

[1083]

[1084] [Table 13]

[1085] entry Alkali pKa* Equivalent Compound 1-4-3a: Compound 1-4-2 1 Piperidine 19.35 28.3(20％) 0:100 2 TMG 23.35 4.47(4％) 91:9 3 DBU 24.31 1.88(2％) 92:8 4 MTBD 25.47 3.91(4％) 93:7 5 HP1(dma) 25.85 3.08(4％) 93:7

[1086] From the above results, it was confirmed that when the Fmoc-protected amino group was treated with a base having a pKa of 23 or more in the conjugate acid, the presence ratio of the carbamate was 90% or more.

[1087] In order to discuss the experimental results on the solid surface based on this discovery, without being limited to a specific theory, in Example 2, the reason why the elimination of diketopiperazines, etc. can be reduced under the conditions of Runs 4 to 7 compared to Run 2 is due to the presence of carbamate as described above, which prevents the formation of a 6-membered ring that causes the elimination of diketopiperazines, etc.

[1088] Therefore, without being limited to a particular theory, when Fmoc protection is treated with a base, the presence of the N-terminal amino group as a carbamate can reduce the elimination of diketopiperazine, etc. 7 -3, the range of bases that can exist as carbamates is determined.

[1089] Example 8: Experimental study on the effect of reducing the elimination of diketopiperazine during liquid phase peptide synthesis under known conditions

[1090] In this example, for the linear peptides prepared in Examples 1 to 3, the Fmoc-protected amino acids described in Table 14 were extended by the method of the present invention (Condition-X), and the amounts of diketopiperazine elimination compounds and diurea compounds formed in the liquid phase method were compared.

[1091] Condition-X

[1092] DBU (0.091 mL, 0.061 mmol, 1 eq) was added to a DMSO (0.20 mL) solution of the linear peptide (compounds 1-3-1 and 1-3-2) (0.061 mmol) prepared in Example 1-3, and the mixture was stirred at room temperature for 5 minutes. A DMSO (0.1 mL) solution of HOAt (16.52 mg, 0.121 mmol, 2 eq) and DIPEA (0.021 mL, 0.121 mmol, 2 eq) was added to the reaction solution, and the mixture was stirred at room temperature for 3 minutes to obtain a solution A.

[1093] DIPEA (0.019 mL, 0.109 mmol, 1.8 eq) was added to a DMSO (0.20 mL) solution of the Fmoc-protected amino acid (0.073 mmol, 1.2 eq) and [ethylcyano(hydroxyamino)acetate-O2]tris 1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim) (38.4 mg, 0.073 mmol, 1.2 eq) described in Table 14, the mixture was stirred at room temperature for 3 minutes, and then a separately prepared solution A was added dropwise thereto. The mixture was stirred at room temperature for 1 hour, and then a portion of the reaction solution was analyzed by LCMS (SQDFA05long) to confirm the formation of the target peptide (TM) (compounds 8-1-1 to 8-1-2) and diketopiperazine elimination compounds (compounds 8-2-1 to 8-2-2).

[1094] Table 14 shows the amount of diketopiperazine elimination compound (TM-DKP) for each peptide sequence. When the total UV area percentage of the target peptide (TM) and the diketopiperazine elimination compound (TM-DKP) in LCMS is 100%, the amount of the diketopiperazine elimination compound (TM-DKP) formed shown in Table 14 is the UV area percentage of the diketopiperazine elimination compound (TM-DKP).

[1095] Target peptide compound (TM), Fmoc-MeLeu-Ile-MeGly-Aze(2)-MeCha-MeGly-Asp(OMe)-pip (Run 1) (Compound 8-1-1)

[1096] [Chem.79]

[1097]

[1098] LCMS (ESI) m / z = 1069.97 (M+H) +

[1099] Retention time: 3.35 minutes (analysis conditions SQDFA05long)

[1100] Diketopiperazine elimination compound, Fmoc-MeLeu-Aze(2)-MeCha-MeGly-Asp(OMe)-pip (Run 1) (Compound 8-2-1)

[1101] [Chem.80]

[1102]

[1103] LCMS (ESI) m / z = 885.78 (M+H) +

[1104] Retention time: 3.26 minutes (analysis conditions SQDFA05long)

[1105] Target peptide compound (TM), Fmoc-Thr(tBu)-Thr(tBu)-MeAla-Cys(StBu)-Phe-Asp(OMe)-pip (Run 2) (Compound 8-1-2)

[1106] [Chem.81]

[1107]

[1108] LCMS (ESI) m / z = 1074.90 (M+H) +

[1109] Retention time: 4.05 minutes (analysis conditions SQDFA05long)

[1110] Diketopiperazine elimination compound, Fmoc-Thr(tBu)-Cys(StBu)-Phe-Asp(OMe)-pip (Run 2) (Compound 8-2-2)

[1111] [Chem.82]

[1112]

[1113] LCMS (ESI) m / z = 932.69 (M+H) +

[1114] Retention time: 3.76 minutes (analysis conditions SQDFA05long)

[1115] [Table 14]

[1116]

[1117] Therefore, from the results of Example 8, it was confirmed that the method of the present invention can suppress the formation of a diketopiperazine elimination compound to synthesize a target peptide even in synthesis by a liquid phase method.

[1118] Industrial Applicability

[1119] It was found that according to the present invention, the formation of byproducts such as diketopiperazine and 6-membered cyclic amidine skeleton compounds can be suppressed, thereby effectively extending the peptide chain in peptide production. The present invention is used in the field of peptide synthesis.

Claims

1. A method for producing a peptide, the method comprising the following steps: (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; (2) after step (1), treating the first peptide with one or more bases including at least a base whose conjugate acid has a pKa of 23 or greater in acetonitrile in a solvent containing a sulfoxide solvent; and (3) After step (2), the first peptide is condensed with a carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

2. A method for reducing the amount of diketopiperazine impurities and / or 6-membered cyclic amidine skeleton compound impurities formed in the production of peptides, the method comprising the following steps: (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

3. A method for reducing the amount of at least one impurity selected from the group consisting of diketopiperazine impurities, 6-membered cyclic amidine skeleton compound impurities and dimer urea compound impurities formed in the production of a peptide, the method comprising the following steps: (1) providing a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase; and (2) After step (1), the first peptide is treated with one or more bases including at least a base whose conjugate acid has a pKa of 23 or more in acetonitrile in a solvent containing a sulfoxide solvent.

4. The method according to claim 2 or 3, further comprising the following step: (3) after step (2), condensing the first peptide with carboxylic acid or a carboxylic acid analog in a solvent in the presence or absence of a condensing agent to obtain a third peptide.

5. The method according to any one of claims 1 to 4, which is performed by a solid phase method. The method according to any one of claims 1 to 4, wherein the first peptide is a first peptide having a protecting group containing an Fmoc backbone and supported on a solid phase.

7. The process according to any one of claims 1 to 4, which is carried out by a liquid phase process.

8. The method according to any one of claims 1 to 7, wherein step (2) is a step of removing the protecting group containing the Fmoc backbone in the first peptide, and / or a step of converting the protecting group containing the Fmoc backbone in the first peptide into a carbamate form.

9. The method according to any one of claims 1 to 8, which does not include a step of treating the first peptide with piperidine as a single base before step (2).

10. The method according to any one of claims 1 to 8, which does not include the step of treating the first peptide with a single base whose conjugate acid has a pKa of less than 23 in acetonitrile.

11. The method according to any one of claims 1 to 10, wherein at least a portion of the first peptide obtained in step (2) is in the form of a carbamate.

12. The method according to any one of claims 1 to 11, wherein the solvent in step (2) contains 50 v / v% or more of a sulfoxide solvent. 13 . The method according to claim 1 , wherein the sulfoxide solvent is one or more selected from the group consisting of DMSO, diethyl sulfoxide, methyl ethyl sulfoxide, and methyl phenyl sulfoxide.

14. The method according to any one of claims 1 to 13, wherein the base in step (2) is at least one selected from the group consisting of amidines, guanidines and phosphazenes.

15. The method according to any one of claims 1 to 14, wherein the base in step (2) is at least one selected from the group consisting of DBU, MTBD, TMG, P1tBu, P2Et and HP1(dma).

16. The method of any one of claims 1 to 15, wherein the first peptide contains 2 to 30, 2 to 20, 2 to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues.

17. according to the method described in any one of claims 1 and 4 to 16, wherein the carboxylic acid or carboxylic acid analog is an amino acid or a second peptide with a protecting group; or an active ester of an amino acid or a second peptide with a protecting group; or an acyl halide of an amino acid or a second peptide with a protecting group; wherein the first peptide and / or the second peptide with a protecting group contains one or more N-substituted amino acids, and / or the amino acid with a protecting group is an N-substituted amino acid.

18. The method of any one of claims 1 to 17, wherein the amino acid at the second residue from the N-terminus of the first peptide is an N-substituted amino acid.

19. The method according to any one of claims 1 and 4 to 18, wherein the condensing agent in step (3) is in the form of a salt and its counter anion is PF6 - or BF4 - , or the condensing agent is a carbodiimide condensing agent.

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