Peptide compound production method

Abstract

The present invention strengthens an α-helix structure involved in a bioactivity of a peptide. The present invention provides causing a reaction between a side chain of a tryptophan residue and a side chain of a cysteine residue provided with a specific protection, in the presence of a hydrochloride or a metal chloride.

Description

Method for producing peptide compounds

[0001] The present invention relates to a method for producing a peptide compound.

[0002] The development targets for drug candidate compounds are constantly shifting from small molecule drugs to antibody drugs and then to peptide drugs. Peptide drugs are considered extremely promising because they can be obtained relatively easily using solid-phase peptide synthesis (SPPS) and can target specific cells. Development of ADCs (Antibody Drug Conjugates), in which small molecule drugs are attached to antibodies or their fragments, is also underway.

[0003] The three-dimensional structure of a peptide, such as α-helix, leucine zipper, zinc finger, coiled coil, etc., is known to be related to the physiological activity of the peptide. For example, it is known that the α-helix structure is formed by interactions between the side chains of the amino acid residues that constitute the peptide, and that the α-helix structure is further strengthened by stapling, which crosslinks the side chains of multiple amino acids.

[0004] The nucleophilic thiol group present in the side chain of cysteine, a natural amino acid, is known to be involved in alkylation, Michael reaction, disulfide formation, etc., and to function as a bridging group in site-specific modification of peptides and proteins. It is also known that intramolecular reactions mediated by the nucleophilic thiol group in peptides are involved in cyclization of the peptide, stapling within the peptide, etc.

[0005] It is known that unexpected reactions occur when a protected peptide is deprotected under acidic conditions. For example, it has been reported that S-arylated cysteine ​​is synthesized when para-methoxybenzyl-protected cysteine, generated during the peptide chain elongation process, is deprotected in the presence of anisole (Non-Patent Document 1).

[0006] Furthermore, in the development of ADCs, the linker placed between the antibody and the low-molecular-weight compound can cause problems with biocompatibility and metabolism, making it necessary to optimize the linker.

[0007] Chem. Pharm. Bull. 1979, 27: 2151-2156

[0008] To date, methods are known that can selectively perform stapling, which can strengthen the α-helical structure associated with the biological activity of peptides. However, these methods have the disadvantage of requiring the use of transition metals. Furthermore, no method is known that can selectively and efficiently perform cross-linking reactions between the side chains of cysteine ​​residues associated with stapling and the side chains of other amino acids. Furthermore, no method is known that can selectively and efficiently bond a compound containing a cysteine ​​residue to another compound.

[0009]

[0006] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that the side chain of a cysteine ​​residue having a specific protecting group reacts with the side chain of a tryptophan residue in the presence of a hydrochloride or a metal chloride to form a crosslink. The present inventors have also found that such a crosslinking reaction occurs both within a compound molecule and between molecules of different compounds. The present invention has been completed based on these findings and broadly encompasses the inventions described in the following sections.

[0010] Item 1: Formula (1) below [In the formula, R １ represents a monovalent organic group. ２ represents a divalent organic group. ３ represents a monovalent organic group. １ and R ３ and may be bonded to each other to form a ring], the method for producing a compound represented by the following formula (2):

[0011] [In the formula, R １ , R ２ and R ３ is the same as above. ４ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group optionally having a substituent on the phenyl ring.] in the presence of hydrochloride or a metal chloride.

[0012] Section 2 Said R １ is a monovalent organic group having a carbonyl group, and the R ２ is a divalent organic group having a carbonyl group and an amino group, and the R ３ is a monovalent organic group having an amino group.

[0013] Section 3 Said R １ , R ２ and R ３ and each represent a peptide residue.

[0014] Item 4. The method according to item 1, wherein the substituent on the phenyl ring is an electron-donating group.

[0015] Item 5. The method according to item 4, wherein the electron-donating group is at least one selected from the group consisting of an alkyl group, an alkoxy group, an alkylamino group, an alkylcarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylamino group, a hydroxyl group, an amino group, and a halogen group.

[0016] Item 6. The method according to any one of Items 1 to 5, wherein the hydrochloride is at least one selected from the group consisting of guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, tetrabutylammonium chloride, piperazine hydrochloride, and morpholine hydrochloride.

[0017] Item 7. The method according to any one of Items 1 to 5, wherein the metal chloride is at least one selected from the group consisting of magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride, calcium chloride, and nickel chloride.

[0018] Item 8. The method according to any one of Items 1 to 7, wherein the crosslinking reaction is carried out under acidic conditions.

[0019] Item 9 The following formula (3)

[0020] [In the formula, R ６、 R ７、 R ８ and R ９and each represent a monovalent organic group.] A method for producing a compound represented by the following formula (4):

[0021] [In the formula, R ６ and R ７ are the same as above.] and a compound represented by the following formula (5):

[0022] [In the formula, R ８ and R ９ are the same as above. １０ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group optionally having a substituent on the phenyl ring.] in the presence of hydrochloride or a metal chloride.

[0023] Item 10 Said R ６ and R ８ is a monovalent organic group having a carbonyl group, and ７ and R ９ is a monovalent organic group having an amino group.

[0024] Item 11 The above R ６ , R ７ , R ８ and R ９ and each represent a peptide residue.

[0025] Item 12. The method according to item 9, wherein the substituent on the phenyl ring is an electron-donating group.

[0026] Item 13. The method according to item 12, wherein the electron-donating group is at least one selected from the group consisting of an alkyl group, an alkoxy group, an alkylamino group, an alkylcarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylamino group, a hydroxyl group, an amino group, and a halogen group.

[0027] Item 14. The method according to any one of Items 9 to 13, wherein the hydrochloride is at least one selected from the group consisting of guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, tetrabutylammonium chloride, piperazine hydrochloride, and morpholine hydrochloride.

[0028] Item 15. The method according to any one of Items 9 to 13, wherein the metal chloride is at least one selected from the group consisting of magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride, calcium chloride, and nickel chloride.

[0029] Item 16. The method according to any one of Items 9 to 15, wherein the crosslinking reaction is carried out under acidic conditions.

[0030] Item 17. The method according to any one of Items 9 to 16, further comprising carrying out the crosslinking reaction in the presence of phenol and / or alkoxybenzene.

[0031] The present invention provides a novel cross-linking reaction between the side chain of a protected cysteine ​​residue and the side chain of a tryptophan residue. This cross-linking reaction can form a cross-link at a position different from the bond of the main chain. Therefore, when the cross-linking reaction is carried out intramolecularly, stapling can be formed between specific amino acid residues, thereby further strengthening a three-dimensional structure such as an α-helix. Furthermore, when the cross-linking reaction is carried out between molecules of different compounds, the main chains of each molecule can be maintained, allowing the compounds to be directly cross-linked without a linker while maintaining the physiological activity expressed by each compound. Therefore, by utilizing the method of the present invention, various peptide drugs can be produced industrially and advantageously.

[0032] Figure 1-1 shows the results of Example 1. 1 to 16 are the results of HPLC analysis of the reaction products of each sample. * in the figure indicates non-peptide impurities. Figure 1-2 shows the results of Example 1. 1 to 16 are the results of HPLC analysis of the reaction products of each sample. * in the figure indicates non-peptide impurities. Figure 2 shows the results of Example 2. 1 to 5 are the results of HPLC analysis of the reaction products of each sample. Figure 3 shows the results of Example 3. 1 to 5 are the results of HPLC analysis of the reaction products using compounds 6 to 10 as raw materials, respectively. 6 is the result of changing the reaction temperature from 20°C to 37°C in the reaction using compound 10 as raw material. * in the figure indicates non-peptide impurities. Figure 4 shows the results of Example 4. 1 is the result of HPLC analysis of the reaction product using compound 16 as raw material. 2 is the result of HPLC analysis of the reaction product using compound 17 as raw material. Figure 5 shows the results of Example 5. Figure 1 shows the results of HPLC analysis of the reaction product using compound 19 as the starting material. Figure 6 shows the results of Example 6. Figure 1 shows the results of HPLC analysis of compound 21, the starting material for compound 23. Figure 2 shows the results of HPLC analysis of compound 22, the starting material for compound 23. Figure 3 shows the results of HPLC analysis of the reaction product using compound 22 as the starting material to produce compound 23. * indicates non-peptide impurities. Figure 7 shows the results of Example 7. Figure 1 shows the results of HPLC analysis of compound 24, the starting material for compound 25. Figure 2 shows the results of HPLC analysis of the reaction product using compound 24 as the starting material to produce compound 25. Figure 3 shows the results of measuring the CD spectra of compound 25 (solid line), compound 26 (dotted line), and a control (dashed line). The control is stERAP. * indicates non-peptide impurities. Figure 8 shows the results of Example 8. Figure 1 shows a silver-stained image after the reaction. Figure 2 shows a biotin-stained image after the reaction. In Figures 1 and 2, A is a marker, and B shows the results of the mixture before the reaction. C shows the results of the reaction product after the reaction. Figure 9 shows the results of Example 9. Figure 1 shows the results of HPLC analysis of the reaction product using compounds 29 and 30 as raw materials. Figure 2 shows the results of HPLC analysis of the reaction product using compounds 29 and 32 as raw materials.3 shows the results of HPLC analysis of the reaction product using Compound 35 and Compound 30 as raw materials. 4 shows the results of HPLC analysis of the reaction product using Compound 35 and Compound 32 as raw materials.

[0033] The present invention will be described below. In the following, unless otherwise specified, the notation "to" indicating a numerical range does not mean "less than" or "exceeding," but rather "equal to or greater than" or "equal to or less than." In other words, "A to B" means "equal to or greater than A and equal to or less than B," and both A and B are included.

[0034] The first production method of the present invention is a method for producing a compound represented by the following formula (1):

[0035] [In the formula, R １ represents a monovalent organic group. ２ represents a divalent organic group. ３ represents a monovalent organic group. １ and R ３ and may be bonded to each other to form a ring], the method for producing a compound represented by the following formula (2):

[0036] [In the formula, R １ , R ２ and R ３ is the same as above. ４ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group optionally having a substituent on the phenyl ring.] in the presence of hydrochloride or a metal chloride.

[0037] In the above formulas (1) and (2), R １ represents a monovalent organic group. Such a monovalent organic group is not particularly limited, but a preferred embodiment is a monovalent organic group having a carbonyl group. More specifically, R １ It is more preferable that the carbonyl group is provided so as to form a peptide bond with the amino group adjacent to the carbonyl group.

[0038] Examples of the monovalent organic group having a carbonyl group include a fatty acid group, a monovalent organic group having miniPEG, and a peptide residue. The fatty acid group may be linear or branched, but linear is preferred in view of ease of production. The number of carbon atoms in the fatty acid is not particularly limited, but can usually be about 12 to 18. MiniPEG is a registered trademark and is a compound represented by 8-amino-3,6-dioxaoctanoic acid. Among these monovalent organic groups, a peptide residue is preferred. The peptide constituting such a peptide residue may be linear or branched, but linear is preferred in view of ease of production.

[0039] As used herein, the term "peptide residue" refers to an atomic group resulting from the removal of a hydrogen atom from the N-terminus of a peptide and a hydroxyl group from the C-terminus of the peptide. The amino acids constituting the peptide are not limited to the 20 types of amino acids in the narrow sense regulated by codons, but may be any structural isomer of α-amino acids such as theanine and ornithine, β-amino acids such as β-alanine, or γ-amino acids such as γ-aminobutyric acid (GABA), and may be either D-amino acids or L-amino acids.

[0040] The above R １ The number of amino acids contained in the peptide residues exemplified above is not particularly limited. The number of amino acids is, for example, usually about 2 to 20, and preferably about 2 to 10.

[0041] In addition, the above R １ In this case, R １ The terminal not bonded to the adjacent amino group can be protected with an acetyl group, a benzoyl group, a pivaloyl group, or the like.

[0042] In the above formulas (1) and (2), R ２ represents a divalent organic group. Such an organic group is not particularly limited as long as it exerts the effects of the present invention, and a divalent organic group having a carbonyl group and an amino group can be given as a preferred embodiment. More specifically, R２ a carbonyl group so as to form a peptide bond with the amino group adjacent to R ２ It is more preferable that the divalent organic group contains an amino group so as to form a peptide bond with the adjacent carbonyl group.

[0043] Examples of the divalent organic group having a carbonyl group and an amino group include a fatty acid group having an amino group, a divalent organic group having a miniPEG, and a peptide residue. Among these divalent organic groups, a divalent peptide residue is preferred. The peptide constituting such a peptide residue may be linear or branched, but linear is preferred in view of ease of production.

[0044] The above R ２ The number of amino acids contained in the peptide residues exemplified above is not particularly limited as long as it does not inhibit the effects of the present invention. The number of amino acids is, for example, usually about 2 to 20, preferably about 2 to 10, and more preferably about 2 to 5.

[0045] In the above formulas (1) and (2), R ３ represents a monovalent organic group. Such a monovalent organic group is not particularly limited, and examples thereof include a monovalent organic group having an amino group. More specifically, R ３ It is more preferable that the monovalent organic group contains an amino group so as to form a peptide bond with the adjacent carbonyl group.

[0046] Specific examples of the monovalent organic group having an amino group include a fatty acid group having an amino group, a monovalent amino group having a miniPEG, and a peptide residue. Among these monovalent organic groups, a peptide residue is preferred. The peptide constituting such a peptide residue may be linear or branched, but is preferably linear.

[0047] The above R ３The number of amino acids contained in the peptide residues exemplified above is not particularly limited. The number of amino acids is, for example, usually about 2 to 20, and preferably about 2 to 10.

[0048] In the above R3, the terminal that is not bonded to the carbonyl group adjacent to R3 can also be protected with a primary amino group, a secondary amino group, an aromatic amino group, or the like.

[0049] In the above formula (1), R １ and R ３ and bond with each other to form a ring. That is, an embodiment in which the compound represented by formula (1) forms a cyclic peptide can be mentioned. Other embodiments of the compound represented by formula (1) include an embodiment containing a disulfide bond via a cysteine ​​residue contained in formula (1), an embodiment containing an ester bond formed between an oxo acid contained in formula (1) and a hydroxy group, etc.

[0050] R in the above formula (2) ４ is R ５ -CH ２ - or a benzyl group which may have a substituent on the phenyl ring. ５ represents an alkylcarbonylamino group. The alkylcarbonylamino group is a group represented by the following formula (R １１ represents an alkyl group), and is a group sometimes called an alkanoylamino group.

[0051]

[0052] The above R ４ In the benzyl group defined as above, the substituent on the phenyl ring is not particularly limited. Examples of such a substituent include an electron-donating group.

[0053] The electron-donating group is not particularly limited as long as it does not inhibit the effects of the present invention, and specific examples thereof include an alkyl group, an alkoxy group, an alkylamino group, an alkylcarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylamino group, a hydroxyl group, an amino group, and a halogen group.

[0054] Specific examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, 1-ethylpropyl, n-pentyl, neopentyl, n-hexyl, isohexyl, and 3-methylpentyl. Of these alkyl groups, a methyl group is preferred.

[0055] Specific examples of the alkoxy group include linear or branched alkoxy groups having 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, n-pentyloxy, neopentyloxy, and n-hexyloxy. Of these alkoxy groups, methoxy is preferred.

[0056] Specific examples of the alkylamino group include monoalkylamino groups such as methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, isobutylamino group, s-butylamino group, t-butylamino group, 1-ethylpropylamino group, n-pentylamino group, neopentylamino group, n-hexylamino group, isohexylamino group, and 3-methylpentylamino group; and linear or branched alkylamino groups having 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as dialkylamino groups such as dimethylamino group, diethylamino group, and di-n-propylamino group.

[0057] Specific examples of the alkylcarbonyl group include linear or branched alkylcarbonyl groups in which the alkyl moiety has 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, s-butylcarbonyl, t-butylcarbonyl, 1-ethylpropylcarbonyl, n-pentylcarbonyl, neopentylcarbonyl, n-hexylcarbonyl, isohexylcarbonyl, and 3-methylpentylcarbonyl.

[0058] Specific examples of the alkylaminocarbonyl group include monoalkylaminocarbonyl groups such as a methylaminocarbonyl group, an ethylaminocarbonyl group, an n-propylaminocarbonyl group, an isopropylaminocarbonyl group, an n-butylaminocarbonyl group, an isobutylaminocarbonyl group, an s-butylaminocarbonyl group, a t-butylaminocarbonyl group, a 1-ethylpropylaminocarbonyl group, an n-pentylaminocarbonyl group, a neopentylaminocarbonyl group, an n-hexylaminocarbonyl group, an isohexylaminocarbonyl group, and a 3-methylpentylaminocarbonyl group; and straight-chain or branched-chain alkylaminocarbonyl groups in which the alkyl moiety has 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as dialkylaminocarbonyl groups such as a dimethylaminocarbonyl group, a diethylaminocarbonyl group, and a di-n-propylaminocarbonyl group.

[0059] Specific examples of the alkylcarbonylamino group include linear or branched alkylcarbonylamino groups having an alkyl moiety of 1 to 6 carbon atoms (particularly 1 to 4 carbon atoms), such as methylcarbonylamino group, ethylcarbonylamino group, n-propylcarbonylamino group, isopropylcarbonylamino group, n-butylcarbonylamino group, isobutylcarbonylamino group, s-butylcarbonylamino group, t-butylcarbonylamino group, 1-ethylpropylcarbonylamino group, n-pentylcarbonylamino group, neopentylcarbonylamino group, n-hexylcarbonylamino group, isohexylcarbonylamino group, and 3-methylpentylcarbonylamino group.

[0060] Specific examples of the halogen group include fluorine, chlorine, bromine, and iodine.

[0061] The hydrochloride used in the first production method of the present invention is not particularly limited as long as it can exert the effects of the present invention, and specifically includes guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, tetrabutylammonium chloride (nBu ４NCl), piperazine hydrochloride, morpholine hydrochloride, etc. Among these hydrochlorides, guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, piperazine hydrochloride, and morpholine hydrochloride are preferred, and guanidine hydrochloride, diisopropylamine hydrochloride, and piperidine hydrochloride are most preferred.

[0062] The amount of the hydrochloride salt used can be appropriately set within a wide range. For example, the amount of the hydrochloride salt used can be about 1 to 4 molar amounts, and preferably about 2 to 4 molar amounts, relative to the molar amount of the compound represented by formula (2).

[0063] The metal chloride used in the first production method of the present invention is not particularly limited as long as it can exhibit the effects of the present invention, and specific examples include magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride, calcium chloride, nickel chloride, etc. Among these metal chlorides, magnesium chloride is preferred.

[0064] The amount of the metal chloride used can be appropriately set within a wide range. For example, the amount of the metal chloride used can be about 25 to 40 molar amounts relative to the molar amount of the compound represented by formula (2), and in order not to denature the raw materials, the amount used is preferably about 30 to 35 molar amounts.

[0065] In the first production method of the present invention, R ４ But, R ５ -CH ２ - (R ５ denotes an alkylcarbonylamino group), it is preferable to use a metal chloride.

[0066] The reaction conditions for the first production method of the present invention are not particularly limited as long as the effects of the present invention are exhibited, but it is preferable to carry out the method under acidic conditions, for example. Specifically, an acid such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, trimethylsilyl trifluoromethanesulfonate, 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate, or acetic acid may be added to the reaction system.

[0067] The reaction solvent for the first production method of the present invention is not particularly limited as long as it does not inhibit the reaction. Specific examples include trifluoroacetic acid. Trifluoroacetic acid can also be used as an acid.

[0068] The reaction temperature in the first production method of the present invention is not particularly limited, and may be, for example, about 20 to 70°C, preferably about 30 to 50°C.

[0069] The reaction time in the first production method of the present invention varies depending on the reaction temperature and cannot be generalized, but can be, for example, about 1 to 5 hours.

[0070] The crosslinking reaction product (compound of formula (1)) obtained by the first production method of the present invention can be subjected to isolation and purification means to obtain a highly pure target product. Specific isolation and purification means can be an appropriate combination of known means such as column chromatography (e.g., HPLC), thin layer chromatography, recrystallization, reprecipitation, distillation, solvent extraction, etc.

[0071] The second production method of the present invention is a method for producing a compound represented by the following formula (3):

[0072] [In the formula, R ６、 R ７、 R ８ and R ９ and each represent a monovalent organic group.], the method for producing a compound represented by the following formula (4):

[0073] [In the formula, R ６ and R ７ are the same as above.] and a compound represented by the following formula (5):

[0074] [In the formula, R ８ and R ９ are the same as above. １０ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group optionally having a substituent on the phenyl ring.] in the presence of a hydrochloride or a metal chloride.

[0075] R in the above formulas (3), (4) and (5) ６ , R ７ , R ８ and R ９ are each a monovalent organic group. These monovalent organic groups can be the same as the monovalent organic groups specifically explained in the first production method of the present invention.

[0076] R in the above formula (5) １０ is R specifically described in the first production method of the present invention. ４ Similarly, R ５ -CH ２ or a benzyl group which may have a substituent on the phenyl ring.

[0077] The ratio of the compound represented by formula (4) to the compound represented by formula (5) used in the second production method of the present invention is not particularly limited. For example, the molar ratio of the former to the latter can be about 1:0.5 to 2, preferably about 1:0.8 to 1.5, and more preferably about 1:0.9 to 1.1.

[0078] The hydrochloride salt in the second production method of the present invention may be the same as the hydrochloride salt specifically described in the first production method of the present invention, and the metal chloride salt in the second production method of the present invention may be the same as the metal chloride salt specifically described in the first production method of the present invention.

[0079] In the second production method of the present invention, R １０ But, R ５ -CH ２ - (R ５ denotes an alkylcarbonylamino group), it is preferable to use a metal chloride.

[0080] The crosslinking reaction in the second production method of the present invention is preferably carried out under acidic conditions, similar to the reaction conditions in the first production method of the present invention. Specific means for creating acidic conditions can be as described in detail in the first production method of the present invention.

[0081] The reaction solvent in the second production method of the present invention may be the same as the solvent in the crosslinking reaction in the first production method of the present invention. １０ When R is a benzyl group which may have a substituent on the phenyl ring, it is preferable to use methanesulfonic acid in addition to trifluoroacetic acid. １０ is R ５ -CH ２ - (R ５ is an alkylcarbonylamino group), an ionic liquid, 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate (BMPy.OTf), can also be used as the solvent.

[0082] In the second production method of the present invention, the crosslinking reaction can be carried out in the presence of phenol and / or alkoxybenzene. By carrying out the crosslinking reaction in the presence of phenol and / or alkoxybenzene, the reaction can be carried out more efficiently.

[0083] The alkoxybenzene is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include anisole (methoxybenzene), ethoxybenzene, propoxybenzene, butoxybenzene, etc. Among these alkoxybenzenes, anisole and ethoxybenzene are preferred, and anisole is particularly preferred. The alkoxybenzenes can be used alone or in combination of two or more.

[0084] The amount of the phenol and / or alkoxybenzene used can be in a wide range as long as it does not impair the effects of the present invention, and can be, for example, 5 to 500 mmol per mmol of compound (3).

[0085] The reaction temperature in the crosslinking reaction in the second production method of the present invention can be the same as the reaction temperature in the first production method of the present invention. In addition, the reaction time in the first production method of the present invention can also be the same as the reaction time in the first production method of the present invention. １０When is a benzyl group which may have a substituent on the phenyl ring, the reaction can be carried out at a temperature in the range of 0 to 70°C, which is lower than the reaction temperature in the first production method.

[0086] As in the first production method of the present invention, the compound (compound of formula (3)) obtained in the second production method of the present invention can also be subjected to isolation and purification procedures to obtain a target product with high purity. Specific isolation and purification procedures can be appropriately combined with known procedures such as column chromatography such as HPLC, thin layer chromatography, recrystallization, reprecipitation, distillation, and solvent extraction.

[0087] The following examples are provided to explain the present invention in more detail, but it goes without saying that the present invention is not limited to the examples shown below.

[0088] Unless otherwise specified, MS analyses in the examples described below were carried out using a Waters MICROMASS (registered trademark) LCT PREMIER™ (ESI-TOF) or LC-MS (Shimadzu, Japan, Prominence-I LC-2030, LCMS-2020).

[0089] Unless otherwise specified, HPLC analyses in the examples described below were performed using a Hitachi L-7150 with an L-2400 detector or a Waters Alliance 2695 Separations Module with an ELS 2420 System on a Cosmosil 5C18-AR-II analytical column (Nacalai Tesque, 4.6 x 250 mm, flow rate 1.0 mL min). －１ ) and a Cosmosil 5C18-AR-II semi-preparative column (Nacalai Tesque, 10 × 250 mm, flow rate 3.0 mL min －１ ) was used. －１ , and a Cosmosil 5C18-AR-II preparative column (Nacalai Tesque, 20 × 250 mm, flow rate 10 mL min －１ ) and a linear gradient system (solvent A: water containing 0.1% TFA, solvent B: MeCN containing 0.1% TFA) with UV detection at 220 nm.

[0090] Unless otherwise specified, NMR measurements in the examples described below were carried out by １ H uses Bruker AV400N (400MHz) or Bruker AV500 (500MHz), １３ C used a Bruker AV400N (100 MHz) or a Bruker AV500 (125 MHz).

[0091] Unless otherwise specified, CD spectra in the examples described below were measured by a conventional method using a JASCO J-1500 CD spectrometer.

[0092] Unless otherwise specified, peptides and the like in the examples described below were prepared using Novasyn (registered trademark) TGR resin (0.25 mmol g －１ ) or Fmoc-Rink Amide-Phe resin (0.25 mmol g －１ The peptide was synthesized by Fmoc solid phase peptide synthesis (Fmoc-SPPS) on a 1000-molecular-weight HPLC column. Fmoc SPPS was performed according to the following protocol.

[0093] 1) The Fmoc group was removed using 20% ​​piperidine / DMF at room temperature for 10 minutes. 2) The resin was washed five times with DMF. 3) A standard Fmoc-protected amino acid (4.0 equivalents) was reacted with N,N-diisopropylcarbodiimide (DIPC1) (4.0 equivalents) and 1-hydroxybenzotriazole monohydrate (HOBt-H ２ 4) The resin was then coupled with 4.0 equiv. of 4-(2-methyl-2-propanol)-2-one (4.0 equiv.) in DMF for 1.5 hours at room temperature, and the reaction was confirmed to be complete by Kaiser ninhydrin test. The coupling reaction was repeated until the Kaiser test was negative. 5) The resin was washed three times with DMF.

[0094] The above steps 1) to 4) were repeated once. ２ Deprotection of the acid-labeled protecting groups was carried out with a cocktail of HCl, HCl, HCl (90:5:5), and HCl (90:5:5), followed by release of the peptide from the resin. After filtering the resin, the filtrate was added with chilled diethyl ether (Et ２ The resulting precipitate was collected by centrifugation. ２For peptides synthesized using Trp(Boc) derivatives, incubation was continued for 1 hour at 37°C, and the resulting solution was purified by preparative HPLC.

[0095] Unless otherwise specified, the following side chain-protected amino acids were used: Arg(Pbf), Asp(OtBu), Cys(MBzl)(O), Cys(ACM), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Trp(Boc), Tyr(tBu). The Cys(Acm)(O)-containing peptide was obtained by first obtaining a Cys(Acm)-containing peptide by conventional Fmoc solid-phase synthesis and then subjecting it to an oxidation reaction.

[0096] Example 1 In accordance with the reaction scheme below, the following compound 1 was used as a starting material under various conditions to obtain the following compound 2. Specific conditions are shown in Table 1. The reaction temperature was 4°C, and the reaction time was 3 hours. TFA, which acts as an acid, was used as the solvent in this reaction. Compound 1 was prepared by the peptide synthesis method described above.

[0097] The MS data of the prepared Compound 1 is as follows: <Compound 1> LRMS (ESI-TOF) m / z: [M+H] + calcd for C 41 H 60 N 11 O9S 882.4, found 882.5.

[0098]

[0099] In the above scheme, Ac- represents an acetyl group. GAL represents a peptide residue consisting of, in order from the N-terminus, glycine-alanine-leucine. Here, the carbonyl group adjacent to G (glycine) is peptide-bonded to the amino group of the glycine, and the amino group adjacent to L (leucine) is peptide-bonded to the carbonyl group of the leucine. R represents an arginine residue.

[0100]

[0101] In Table 1 above, TFMSA represents trifluoromethanesulfonic acid, MSA represents methanesulfonic acid, nBu ４ NCl is tetrabutylammonium chloride.

[0102] The HPLC chart of the reaction product obtained in this reaction is shown in Figure 1. The MS data and NMR data of Compound 2 are as follows: LRMS (ESI-TOF) m / z: [M+H] + calcd for C 33 H 50 N 11 O7S 744.4, found 744.0. 1 H-NMR (400 MHz, D2O): δ= 7.64 (dd, J = 8.0, 0.8 Hz, 1H), 7.44 (dd, J = 8.0, 0.8 Hz, 1H), 7.28 (ddd, J = 8.0, 7.4, 0.8 Hz, 1H), 7.17 (ddd, J = 8.0, 7.4, 0.8 Hz, 1H), 4.96-4.67 (m, 1H), 4.44-4.38 (m, 1H), 4.38-4.33 (m, 1H), 4.22 (q, J = 7.3 Hz, 1H), 4.15-4.09 (m, 1H), 3.96-3.93 (m, 2H), 3.56-3.48 (m, 1H), 3.26-3.16 (m, 3H), 3.13-3.05 (m, 2H), 1.88 (s, 3H), 1.83-1.44 (m, 7H), 1.42 (d, J = 7.3 Hz, 3H), 0.91-0.81 (m, 6H)

[0103] The above results revealed that compound 1, which has a cysteine ​​residue protected by a methoxybenzyl group, undergoes intramolecular cross-linking between the cysteine ​​residue and a tryptophan residue under acidic conditions in the presence of various hydrochlorides, to produce compound 2.

[0104] On the other hand, in Sample 7, in which sulfate was used instead of hydrochloride, no crosslinking was observed.

[0105] Example 2 In the following reaction scheme, compound 5 was reacted using compound 3 and compound 4 as starting materials under various conditions shown in Table 2 below. The reaction temperature was 4°C, and the reaction time was 3 hours. TFA was used as the solvent in this reaction. Compound 3 was prepared by converting the carbonyl group of L-tryptophan to a methoxy group in the presence of thionyl chloride using a standard method. Compound 4 was prepared by the peptide synthesis method described above.

[0106] The MS data of the prepared compounds 3 and 4 are as follows: <Compound 3> HRMS (ESI-TOF) m / z: [M+H]+ calculated for C 14 H 16 KN2O3 299.0798, found 299.0802 <Compound 4> HRMS (ESI-TOF) m / z: [M+Na] + calcd for C 19 H 29 N3O5NaS 434.1726, found 434.1724.

[0107]

[0108] In the above scheme, Ac- represents an acetyl group.

[0109]

[0110] The HPLC chart of the reaction product obtained in this reaction is shown in Figure 2. The MS data of the obtained Compound 5 is as follows: <Compound 5> LRMS (ESI-TOF) m / z: [M+H] + calcd for C 25 H 37 N5O6S 534.2, found 533.9.

[0111] The above results revealed that compound 3 having a cysteine ​​residue protected by a methoxybenzyl group and compound 4 having a tryptophan residue undergo intermolecular crosslinking between the cysteine ​​residue and the tryptophan residue under acidic conditions in the presence of various hydrochlorides, thereby producing compound 5.

[0112] Example 3 In the following reaction scheme, compounds 6 to 10 below were used as starting materials and reacted under the conditions of 1 M MSA and 4 M guanidine hydrochloride. The reaction temperature in this reaction was 20°C, and the reaction time was 3 hours. TFA was used as the solvent in this reaction. Note that the reaction was also carried out using compound 10 as the starting material at 37°C instead of 20°C. Compounds 6 to 10 were produced by the peptide synthesis method described above.

[0113] The MS data of the prepared compounds 6 to 10 are as follows: <Compound 6> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 66 H 102 N 22 O 15 S 737.4, found 737.1. <Compound 7> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 66 H 107 N 21 O 15 S 732.9, found 732.7. <Compound 8> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 69 H 104 N 20 O 15 S 742.4, found 742.2. <Compound 9> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 69 H 104 N 20 O 16 S 750.4, found 750.2. <Compound 10> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 57 H 94 N 20 O 13 S2 734.4, found 734.1.

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] In each of the above schemes, Ac- represents an acetyl group, GAL represents a peptide residue consisting of glycine-alanine-leucine in order from the N-terminus, GHRAL represents a peptide residue consisting of glycine-histidine-arginine-alanine-leucine in order from the N-terminus, GKRAL represents a peptide residue consisting of glycine-lysine-arginine-alanine-leucine in order from the N-terminus, GFRAL represents a peptide residue consisting of glycine-phenylalanine-arginine-alanine-leucine in order from the N-terminus, GYRAL represents a peptide residue consisting of glycine-tyrosine-arginine-alanine-leucine in order from the N-terminus, and GMRAL represents a peptide residue consisting of glycine-methionine-arginine-alanine-leucine in order from the N-terminus. Here, the carbonyl group adjacent to the N-terminal G (glycine) residue is peptide-bonded to the primary amino group of the glycine residue, and the amino group adjacent to the C-terminal L (leucine) residue is peptide-bonded to the carbonyl group of the leucine residue. RG represents a peptide residue consisting of arginine-glycine, in order from the N-terminus. Here, the carbonyl group adjacent to the R (arginine) residue is peptide-bonded to the amino group of arginine, and the G (glycine) residue has a C-terminal amide structure.

[0120] The HPLC chart of the reaction product obtained in the above reaction is shown in Figure 3. The MS data of the obtained compounds 11 to 15 are as follows: <Compound 11> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 58 H 92 N 22 O 13 S 668.4, found 668.2. <Compound 12> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 58 H97 N 21 O 13 S 663.9, found 663.7. <Compound 13> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 61 H 94 N 20 O 13 S 673.4, found 673.2. <Compound 14> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 61 H 94 N 20 O 14 S 681.4, found 681.1. <Compound 15> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 57 H 94 N 20 O 13 S2 665.3, found 665.1.

[0121] It was also revealed that the crosslinking reaction temperature can be more efficiently carried out at 37°C than at 20°C.

[0122] Example 4 In the following reaction scheme, the following compounds 16 and 17 were reacted under the conditions of 1 M MSA and 4 M guanidine hydrochloride. The reaction temperature was 20°C, and the reaction time was 3 hours. TFA was used as the solvent in this reaction. These compounds 16 and 17 were prepared by the above-mentioned peptide synthesis method.

[0123] The MS data of the prepared compounds 16 and 17 are as follows: <Compound 16> LRMS (ESI-TOF) m / z: [M+H] + calcd for C 35 H 49 N 10 O8S 769.4, found 769.5. <Compound 17> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 50 H 78 N16 O 11 S 555.3, found 555.3.

[0124]

[0125]

[0126] In each of the above schemes, Ac- represents an acetyl group, GA represents a peptide residue consisting of glycine-alanine in order from the N-terminus, and GALRA represents a peptide residue consisting of glycine-alanine-leucine-arginine-alanine in order from the N-terminus. Here, the carbonyl group adjacent to the G (glycine) residue on the N-terminus side is peptide-bonded to the amino group of the glycine residue, and the amino group adjacent to the A (alanine) residue on the C-terminus side is peptide-bonded to the carbonyl group of the alanine residue. Furthermore, R represents an arginine residue.

[0127] The HPLC chart of the reaction product obtained in the above reaction is shown in Figure 4. The MS data of the obtained compounds 18 and 19 are as follows: <Compound 18> LRMS (ESI-TOF) m / z: [M+H] + calcd for C 27 H 39 N 10 O6S 631.3, found 631.3. <Compound 19> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 42 H 68 N 16 O9S 486.3, found 486.3.

[0128] The above results revealed that intramolecular cross-linking occurs in a peptide even when there are 2 to 5 amino acid residues between the cysteine ​​residue protected by a methoxybenzyl group and the tryptophan residue contained in the peptide.

[0129] Example 5 In the following reaction scheme, the following compound 20 was used as a starting material and reacted under the conditions of 1 M MSA and 4 M guanidine hydrochloride. The reaction temperature was 25°C and the reaction time was 30 minutes. TFA was used as the solvent in this reaction. Compound 19 was produced by the above-mentioned peptide synthesis method.

[0130] The MS data of the prepared compound 20 is as follows: <Compound 20> LRMS (ESI-TOF) m / z: [M+2H] 2+ calcd for C 64 H 101 N 21 O 16 S 725.9, found 725.8.

[0131]

[0132] In the above scheme, Ac- represents an acetyl group, GYRAL represents a peptide residue consisting of glycine-tyrosine-arginine-alanine-leucine in order from the N-terminus, and GAL represents a peptide residue consisting of glycine-alanine-leucine in order from the N-terminus. Here, the carbonyl group adjacent to the G (glycine) residue is peptide-bonded to the amino group of the glycine residue, and the primary amino group adjacent to the L (leucine) residue is peptide-bonded to the carboxyl group of the leucine residue. Furthermore, RG represents a peptide residue consisting of glycine-arginine in order from the N-terminus. Here, the carbonyl group adjacent to the R (arginine) residue is peptide-bonded to the amino group of the arginine residue, and the G (glycine) residue has a C-terminal amide structure.

[0133] The HPLC chart of the reaction product obtained in the above reaction is shown in FIG.

[0134] The above results revealed that compound 20, which has a cysteine ​​residue and a tryptophan residue protected by a methylcarbonylamino group contained in a peptide, generates intermolecular crosslinks between the cysteine ​​residue and the tryptophan residue under acidic conditions in the presence of hydrochloride (guanidine hydrochloride).

[0135] Example 6 As shown in the following scheme, compound 21, prepared by the above-described peptide synthesis method, was reacted in N-methylpyrrolidone (NMP) containing tripyrrolidinophosphonium hexafluorophosphate (e.g., PyBOP) and N,N-diisopropylethylamine (DIEA) at room temperature for 3 hours to form a cyclic peptide of compound 22.

[0136] Compound 22 was used as a raw material and reacted under the conditions of 1 M MSA and 4 M guanidine hydrochloride. The reaction temperature was 4° C. and the reaction time was 3 hours. TFA was used as a solvent in this reaction.

[0137] The MS data of the prepared compound 21 is as follows: <Compound 21> LRMS (ESI-TOF) m / z: [M+H] + calcd for C 47 H 67 N 10 O 12 S 995.5, found 995.0.

[0138]

[0139] In the above scheme, NPI represents a peptide residue consisting of asparagine-proline-isoleucine in order from the N-terminus. Here, the carbonyl group adjacent to the N (asparagine) residue is peptide-bonded to the amino group of the asparagine residue, and the primary amino group adjacent to the I (isoleucine) residue is peptide-bonded to the carbonyl group of the isoleucine residue. Furthermore, GI represents a peptide residue consisting of glycine-isoleucine in order from the N-terminus. Here, the carbonyl group adjacent to the G (glycine) residue is peptide-bonded to the primary amino group of the glycine residue, and the primary amino group adjacent to the I (isoleucine) residue is peptide-bonded to the carbonyl group of the isoleucine residue.

[0140] The HPLC chart of the reaction product obtained in the above reaction is shown in FIG.

[0141] These results demonstrate that intramolecular cross-linking occurs between the cysteine ​​and tryptophan residues protected by the methoxybenzyl group in the cyclic peptide. It is expected that the 3D structure of the peptide with the added cyclic structure will be more rigid.

[0142] Example 7 In the following reaction scheme, compound 24 was reacted with 1 M MSA and 4 M guanidine hydrochloride. The reaction temperature was 4°C and the reaction time was 3 hours. TFA was used as the solvent in this reaction. Compound 24 was produced by the peptide synthesis method described above.

[0143] The MS data of the prepared compound 24 is as follows: <Compound 24> LRMS (ESI-TOF) m / z: [M+2H] + calcd for C 82 H 132 N 24 O 22 S 918.5, found 918.6.

[0144]

[0145] In each of the above schemes, Ac- represents an acetyl group, and SDL represents a peptide residue consisting of serine-aspartic acid-leucine, in that order from the N-terminus. Here, the carbonyl group adjacent to the S (serine) residue is peptide-bonded to the amino group of the serine residue, and the amino group adjacent to the L (leucine) residue is peptide-bonded to the carbonyl group of the leucine residue. Furthermore, LQLRQR represents a peptide residue consisting of leucine-glutamine-leucine-arginine-glutamine-arginine, in that order from the N-terminus. Here, the carbonyl group adjacent to the N-terminal L (leucine) residue is peptide-bonded to the amino group of the leucine residue, and the R (arginine) on the C-terminal side has a C-terminal amide structure.

[0146] Compound 24 is an analog of the amino acid sequence of the breast cancer inhibitory peptide stERAP, and the norleucine residue in compound 24 corresponds to the methionine residue in stERAP. In stERAP, a bisamide-mediated crosslink is formed between the glutamic acid residue located between the methionine and serine residues and the glutamine residue located between the leucine and leucine residues. It was hypothesized that an intramolecular crosslink formed between the cysteine ​​and tryptophan residues in compound 24 would result in a compound with a three-dimensional structure similar to that of stERAP, such as compound 25. The HPLC chart of the reaction product obtained in the above reaction and the CD spectrum after the reaction are shown in FIG. 7.

[0147] The above results revealed that intermolecular crosslinking occurs between the cysteine ​​residue protected by the methoxybenzyl group contained in compound 24 and the tryptophan residue, producing compound 25. Furthermore, the CD spectrum results of compound 25 after the crosslinking reaction differ from those of a peptide in which the cysteine ​​residue of compound 24 was replaced with an alanine residue, and it was also revealed that compound 25 has improved helicity compared to compound 24.

[0148] Example 8 0.01 mM tratuzumab (Chugai Pharmaceutical) and compound 27 shown in the following formula were mixed and reacted for 24 hours at 37°C in 0.1% TFA containing 30 mM magnesium chloride and 5% water. 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate was also added to this reaction system. Compound 27 was prepared by the peptide synthesis method described above.

[0149]

[0150] In the above compound 27, A is the group shown below, and in compound 27, the carbonyl group adjacent to A and the amine group of A are bonded to the peptide, and the amine group adjacent to A and the carbonyl group of A are bonded to the peptide.

[0151]

[0152] The MS data of the prepared compound 27 in the sulfoxide state is as follows: <Compound 27> LRMS (ESI-TOF) m / z: [M+H]+ calculated for C 36 H 64 N9O 13 S2 894.4, found 894.7.

[0153] The reaction product obtained in the above reaction and the mixture before the reaction were subjected to silver staining and biotin staining according to known methods, and the results are shown in Figure 8.

[0154] 8, no bands were detected by biotin staining in the sample before the reaction, whereas clear bands were detected at positions corresponding to the heavy and light chains in the biotin-stained image after the reaction. On the other hand, silver staining, which detects the entire protein, clearly detected bands at positions corresponding to the heavy and light chains in both the sample before and after the reaction.

[0155] From these results, it is assumed that in the above reaction, intermolecular crosslinking occurs between the tryptophan residue contained in the antibody and the cysteine ​​residue protected by the methylcarbonylamino group contained in compound 27. Furthermore, it was also revealed that, unlike the above Experimental Example 5, this crosslinking reaction proceeds in the presence of magnesium chloride rather than hydrochloride.

[0156] Furthermore, MS analysis revealed that three tryptophans in the heavy chain and one tryptophan in the light chain were modified with Compound 27.

[0157] Example 9 In the following reaction scheme, compounds 29, 30, 32, and 35 were reacted in 1 M MSA and 4 M diisopropylamine hydrochloride. The reaction temperature was 4° C. and the reaction time was 3 hours. TFA was used as the solvent.

[0158] The MS data of the prepared compounds 29, 30, 32, and 35 are as follows: <Compound 29> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 151 H 232N 40 O 47 839.4, found 839.5. <Compound 30> LRMS (ESI-Q) m / z: [M+H]+ calcd for C 33 H 58 N3O7S 640.4, found 640.4. <Compound 32> LRMS (ESI-Q) m / z: [M+H] + calcd for C 45 H 80 N5O 13 S 742.4, found 742.2. <Compound 35> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 152 H 234 N 42 O 47 849.9, found 850.0.

[0159]

[0160]

[0161]

[0162]

[0163] In each of the above schemes, H at the N-terminus represents a histidine residue, and EGTFTSDVSSYLEGQAAKEFIA represents a peptide residue consisting of, in order from the N-terminus, glutamic acid-glycine-threonine-phenylalanine-threonine-serine-aspartic acid-valine-serine-serine-tyrosine-leucine-glutamic acid-glycine-glutamine-alanine-alanine-lysine-glutamic acid-phenylalanine-isoleucine-alanine. Here, the carbonyl group adjacent to the N-terminal E (glutamic acid) residue is peptide-bonded to the amino group of the glutamic acid residue, and the amino group adjacent to the C-terminal A (alanine) residue is peptide-bonded to the carbonyl group of the alanine residue. LVRGRG represents a peptide residue consisting of, in order from the N-terminus, leucine-valine-arginine-glycine-arginine-glycine. Here, the carbonyl group adjacent to the N-terminal L (leucine) residue and the amino group of the leucine are peptide-bonded.

[0164] In each of the above schemes, A is the same as A above, and in compound 30, the carbonyl group adjacent to A) and the amine group of A are bonded to the peptide, and the amine group adjacent to A and the carbonyl group of A are bonded to the peptide. ３ is the group shown below, and in compound 32, A ３ The carbonyl group adjacent to A ３ The amine group of A is bound to the peptide, ３ and the amine group adjacent to A ３ The carbonyl group of the hydroxyl group is bonded to the peptide.

[0165]

[0166] The HPLC chart of the reaction product obtained in the above reaction is shown in Figure 9. The MS data of the obtained compounds 31, 33, 36, and 37 are as follows: <Compound 31> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 176 H 279 N 43 O 52S 964.7, found 965.0. <Compound 33> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 188 H 301 N 45 O 58 S 1037.3, found 1037.5. <Compound 36> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 177 H 281 N 45 O 52 S 975.3, found 975.5. <Compound 37> LRMS (ESI-Q) m / z: [M+4H] 4+ calcd for C 189 H 303 N 47 O 58 S 1047.8, found 1048.0.

[0167] From the above results, compounds 29 and 35 are biologically active peptides, and conjugation of lipid structures to these peptides is currently being widely performed. The introduction of lipid structures into compounds 30 and 32 is expected to increase the in vivo stability of the peptides and inhibit renal excretion. Compounds 31, 33, 36, and 37, in which lipid structures are introduced via intermolecular crosslinking on tryptophan using cysteine ​​sulfoxide protected by a methoxybenzyl group, can more effectively exert their biological activity in vivo.

Claims

1. The following formula (1) 【Chemistry 1】 [In the formula, R １ represents a monovalent organic group. R ２ represents a divalent organic group. R ３ represents a monovalent organic group. Also, R １ and R ３ may be bonded to each other to form a ring. A method for producing a compound represented by the formula: The following formula (2) 【Chemistry 2】 [In the formula, R １ , R ２ and R ３ is the same as above. ４ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group which may have a substituent on the phenyl ring. A production method comprising a step of crosslinking a compound represented by the following formula (1): in the presence of hydrochloride or a metal chloride.

2. The R １ is a monovalent organic group having a carbonyl group, and the R ２ is a divalent organic group having a carbonyl group and an amino group, and the R ３ The method according to claim 1 , wherein is a monovalent organic group having an amino group.

3. The R １ , R ２ and R ３ and each are a peptide residue.

4. The method according to claim 1 , wherein the substituent on the phenyl ring is an electron-donating group.

5. 5. The method according to claim 4, wherein the electron-donating group is at least one selected from the group consisting of an alkyl group, an alkoxy group, an alkylamino group, an alkylcarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylamino group, a hydroxyl group, an amino group, and a halogen group.

6. The method according to any one of claims 1 to 5, wherein the hydrochloride is at least one selected from the group consisting of guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, tetrabutylammonium chloride, piperazine hydrochloride, and morpholine hydrochloride.

7. The method according to any one of claims 1 to 5, wherein the metal chloride is at least one selected from the group consisting of magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride, calcium chloride, and nickel chloride.

8. The method according to any one of claims 1 to 5, wherein the crosslinking reaction is carried out under acidic conditions.

9. The following formula (3) 【Transformation 3】 [In the formula, R ６、 R ７、 R ８ and R ９ Each represents a monovalent organic group. A method for producing a compound represented by the formula: The following formula (4) 【Chemistry 4】 [In the formula, R ６ and R ７ are the same as above.] and a compound represented by The following formula (5) 【Transformation 5】 [In the formula, R ８ and R ９ are the same as above. １０ is R ５ -CH ２ - (R ５ represents an alkylcarbonylamino group) or a benzyl group which may have a substituent on the phenyl ring. and a compound represented by the formula (I) in the presence of a hydrochloride or a metal chloride.

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