WO2023166975A1 - Peptide compound production method - Google Patents
Peptide compound production method Download PDFInfo
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- WO2023166975A1 WO2023166975A1 PCT/JP2023/005023 JP2023005023W WO2023166975A1 WO 2023166975 A1 WO2023166975 A1 WO 2023166975A1 JP 2023005023 W JP2023005023 W JP 2023005023W WO 2023166975 A1 WO2023166975 A1 WO 2023166975A1
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- WO
- WIPO (PCT)
- Prior art keywords
- group
- compound
- hydrochloride
- formula
- peptide
- Prior art date
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- 238000004519 manufacturing process Methods 0.000 title claims description 68
- 150000001875 compounds Chemical class 0.000 title claims description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 89
- 229910001510 metal chloride Inorganic materials 0.000 claims abstract description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 19
- 125000000962 organic group Chemical group 0.000 claims description 46
- 125000003277 amino group Chemical group 0.000 claims description 43
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- 125000001424 substituent group Chemical group 0.000 claims description 15
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 14
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 12
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 12
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 11
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- NHGXDBSUJJNIRV-UHFFFAOYSA-M tetrabutylammonium chloride Chemical compound [Cl-].CCCC[N+](CCCC)(CCCC)CCCC NHGXDBSUJJNIRV-UHFFFAOYSA-M 0.000 claims description 10
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- 125000006253 t-butylcarbonyl group Chemical group [H]C([H])([H])C(C(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229940026510 theanine Drugs 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- FTVLMFQEYACZNP-UHFFFAOYSA-N trimethylsilyl trifluoromethanesulfonate Chemical compound C[Si](C)(C)OS(=O)(=O)C(F)(F)F FTVLMFQEYACZNP-UHFFFAOYSA-N 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
Definitions
- the present invention relates to a method for producing a peptide compound.
- the targets for development of drug candidate compounds are changing day by day, from low-molecular-weight drugs to antibody drugs and then to peptide drugs.
- the peptide drug can be obtained relatively easily by solid-phase peptide synthesis (SPPS), and is highly promising in that it can target specific cells.
- SPPS solid-phase peptide synthesis
- ADCs Antibody Drug Conjugates
- ADCs Antibody Drug Conjugates
- the three-dimensional structure of peptides represented by ⁇ -helices, leucine zippers, zinc fingers, coiled coils, etc. is known to be related to the physiological activities of the peptides.
- an ⁇ -helical structure is formed by interactions between side chains of amino acid residues that constitute a peptide, and stapling that bridges the side chains of multiple amino acids strengthens the ⁇ -helical structure. It has been known.
- the nucleophilic thiol group present in the side chain of cysteine is involved in alkylation, Michael reaction, disulfide formation, etc., and serves as a bridge for site-specific modification of peptides and proteins. It is known. It is also known that an intramolecular reaction by a nucleophilic thiol group in a peptide is related to cyclization of the peptide, stapling within the peptide, and the like.
- Non-Patent Document 1 Non-Patent Document 1
- the linker provided between the antibody and the low-molecular-weight compound poses problems in terms of biocompatibility and metabolism, making it necessary to optimize the linker.
- the present inventors have found that the side chain of a cysteine residue having a specific protective group and the side chain of a tryptophan residue are in the presence of hydrochloride or metal chloride. It was found to react below to form cross-links. The present inventors also found that such a cross-linking reaction proceeds both intramolecularly within a compound and between molecules of different compounds.
- the present invention is an invention completed based on these findings, and broadly includes the inventions shown in the following items.
- R 1 represents a monovalent organic group.
- R2 represents a divalent organic group.
- R3 represents a monovalent organic group. Also, R 1 and R 3 may combine with each other to form a ring.
- R 1 , R 2 and R 3 are the same as above.
- R 4 represents 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 the step of cross-linking the compound represented by in the presence of hydrochloride or metal chloride.
- R 1 is a monovalent organic group having a carbonyl group
- R 2 is a divalent organic group having a carbonyl group and an amino group
- R 3 is a monovalent organic group having an amino group. 2. The production method according to item 1 above, wherein the organic group is a divalent organic group.
- Item 3 The production method according to Item 1, wherein each of R 1 , R 2 and R 3 is a peptide residue.
- Item 4 The production method according to Item 1 above, wherein the substituent on the phenyl ring is an electron-donating group.
- 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. 5. The manufacturing method according to item 4 above.
- 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.
- the manufacturing method according to any one of items 1 to 5 above.
- Item 7 Any one of items 1 to 5 above, wherein the metal chloride is at least one selected from the group consisting of magnesium chloride, zinc chloride, lithium chloride, iron chloride (III), calcium chloride and nickel chloride. The manufacturing method described in the item.
- Item 8 The production method according to any one of Items 1 to 7 above, wherein the cross-linking reaction is performed under acidic conditions.
- R 6 , R 7 , R 8 and R 9 each represent a monovalent organic group.
- R 8 and R 9 are the same as above.
- R 10 represents 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 the step of reacting a compound represented by in the presence of a hydrochloride or a metal chloride.
- Item 10 The production method according to Item 9, wherein R 6 and R 8 are monovalent organic groups having a carbonyl group, and R 7 and R 9 are monovalent organic groups having an amino group.
- Item 11 The production method according to Item 9, wherein each of R 6 , R 7 , R 8 and R 9 is a peptide residue.
- Item 12 The production method according to Item 9 above, wherein the substituent on the phenyl ring is an electron-donating group.
- 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. 13.
- 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. , the production method according to any one of items 9 to 13 above.
- Item 15 Any one of items 9 to 13 above, 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. The manufacturing method described in .
- Item 16 The production method according to any one of Items 9 to 15 above, wherein the cross-linking reaction is performed under acidic conditions.
- Item 17 The production method according to any one of Items 9 to 16 above, wherein the cross-linking reaction is further performed in the presence of phenol and/or alkoxybenzene.
- a novel cross-linking reaction between the side chain of a protected cysteine residue and the side chain of a tryptophan residue can be provided.
- Such a cross-linking reaction can form a cross-link at a position different from the main chain bond. Therefore, when the cross-linking reaction is carried out within the molecule of the compound, it is possible to form staples between specific amino acid residues, thus making the conformation such as ⁇ -helix more rigid. can be done.
- the cross-linking reaction is carried out between molecules of different compounds, the backbone of each molecule can be retained, so that while retaining the physiological activity expressed by each compound, direct Each compound can be crosslinked. Therefore, by using the method of the present invention, various peptide drugs can be produced industrially and advantageously.
- FIG. 1-1 is a diagram showing the results of Example 1.
- FIG. 1 to 16 are the results of HPLC analysis of the reactants in each sample. * in the figure is a non-peptidic impurity.
- 1-2 are diagrams showing the results of Example 1.
- FIG. 1 to 16 are the results of HPLC analysis of the reactants in each sample. * in the figure is a non-peptidic impurity.
- FIG. 2 is a diagram showing the results of Example 2.
- FIG. 1 to 5 are the results of HPLC analysis of the reactants in each sample.
- 3 is a diagram showing the results of Example 3.
- FIG. 1 to 5 are the results of HPLC analysis of reactants starting from compounds 6 to 10, respectively. 6 is the result of changing the reaction temperature from 20°C to 37°C in the reaction using compound 10 as a raw material.
- FIG. 1 is the result of HPLC analysis of a reaction product using compound 16 as a raw material.
- 2 is the result of HPLC analysis of the reaction product using compound 17 as a starting material.
- 5 is a diagram showing the results of Example 5.
- FIG. 1 is the result of HPLC analysis of a reaction product using compound 19 as a raw material.
- 6 is a diagram showing the results of Example 6.
- FIG. 7 is a diagram showing the results of Example 7.
- FIG. 1 shows the results of HPLC analysis of compound 24, which is the raw material of compound 25, and 2 shows the results of HPLC analysis of the reaction product of preparing compound 25 using compound 24 as the raw material.
- 3 is the result of measuring the CD spectra of compound 25 (solid line), compound 26 (dotted line), and control (dashed line).
- Control is stERAP. * in the figure is a non-peptidic impurity.
- 8 is a diagram showing the results of Example 8.
- FIG. 1 is a silver-stained image after the reaction.
- 2 is a biotin-stained image after the reaction.
- a in 1 and 2 is a marker and B shows the result of the mixture before the reaction.
- C shows the results of the reactants after the reaction.
- FIG. 9 is a diagram showing the results of Example 9.
- FIG. 1 is the result of HPLC analysis of the reaction product using compound 29 and compound 30 as raw materials.
- 2 is the result of HPLC analysis of the reaction product using compound 29 and compound 32 as raw materials.
- 3 is the result of HPLC analysis of the reaction product using compound 35 and compound 30 as raw materials.
- 4 is the result of HPLC analysis of the reaction product using compound 35 and compound 32 as raw materials.
- the first manufacturing method of the present invention is the following formula (1)
- R 1 represents a monovalent organic group.
- R2 represents a divalent organic group.
- R3 represents a monovalent organic group. Also, R 1 and R 3 may combine with each other to form a ring.
- R 1 , R 2 and R 3 are the same as above.
- R 4 represents R 5 —CH 2 — (R 5 represents an alkylcarbonylamino group) or a benzyl group which may have a substituent on the phenyl ring.
- R 1 represents a monovalent organic group.
- a monovalent organic group having a carbonyl group can be mentioned as a preferred embodiment. More specifically, a monovalent organic group in which a carbonyl group is provided so as to form a peptide bond with an amino group adjacent to R 1 is more preferable.
- Examples of the monovalent organic group having a carbonyl group include a fatty acid group, a monovalent organic group having miniPEG, a peptide residue, and the like.
- the fatty acid group may be linear or branched, but is preferably linear in view of the ease of production. Although the number of carbon atoms in the above fatty acid is not particularly limited, it can usually be about 12 to 18.
- MiniPEG is a registered trademark and is a compound represented by 8-amino-3,6-dioxaoctanoic acid.
- peptide residues are preferred. Peptides constituting such peptide residues may be linear or branched, but are preferably linear in view of ease of production.
- a peptide residue means an atomic group obtained by removing a hydrogen atom from the N-terminus of a peptide and removing a hydroxyl group from the C-terminus of the peptide.
- the amino acids constituting the peptide are not limited to the 20 types of narrowly defined amino acids governed by codons, ⁇ -amino acids such as theanine and ornithine, ⁇ -amino acids such as ⁇ -alanine, All structural isomers of ⁇ -amino acids such as ⁇ -aminobutyric acid (GABA) and the like can be employed, and they may be D-amino acids or L-amino acids.
- GABA ⁇ -aminobutyric acid
- the number of amino acids contained in the peptide residues exemplified for R 1 is not particularly limited.
- the number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10.
- the terminal not bonded to the amino group adjacent to R 1 can be protected with an acetyl group, a benzoyl group, a pivaloyl group, or the like.
- R 2 represents a divalent organic group.
- Such an organic group is not particularly limited as long as the effects of the present invention are exhibited, and a preferred embodiment is a divalent organic group having a carbonyl group and an amino group. More specifically, a divalent organic group containing a carbonyl group so as to form a peptide bond with the amino group adjacent to R 2 and an amino group so as to form a peptide bond with the carbonyl group adjacent to R 2 It is more preferable to
- divalent organic group having a carbonyl group and an amino group examples include a fatty acid group having an amino group, a divalent organic group having miniPEG, a peptide residue, and the like.
- divalent organic groups divalent peptide residues are preferred.
- a peptide constituting such a peptide residue may be linear or branched, but is preferably linear in view of ease of production.
- the number of amino acids contained in the peptide residues exemplified for R2 above is not particularly limited as long as the effects of the present invention are not inhibited.
- the number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10, more preferably about 2 to 5.
- R 3 represents a monovalent organic group.
- a monovalent organic group is not particularly limited, and a monovalent organic group having an amino group can be mentioned. More specifically, it is more preferable to use a monovalent organic group containing an amino group so as to form a peptide bond with the carbonyl group adjacent to R3 .
- the monovalent organic group having an amino group examples include a fatty acid group having an amino group, a monovalent amino group having miniPEG, a peptide residue, and the like.
- peptide residues are preferred.
- a peptide constituting such a peptide residue may be linear or branched, but preferably linear.
- the number of amino acids contained in the peptide residues exemplified for R3 is not particularly limited.
- the number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10.
- the terminal not bonded to the carbonyl group adjacent to R 3 may be protected with a primary amino group, secondary amino group, aromatic amino group, or the like.
- R 1 and R 3 are bonded to each other to form a ring. That is, the compound represented by Formula (1) can mention the aspect which forms a cyclic peptide.
- Other embodiments of the compound represented by formula (1) include, for example, an embodiment containing a disulfide bond via a cysteine residue contained in formula (1), an oxoacid and hydroxyl Examples include an aspect containing an ester bond formed with a group.
- R 4 in the above formula (2) represents R 5 —CH 2 — or a benzyl group which may have a substituent on the phenyl ring.
- R 5 represents an alkylcarbonylamino group.
- the alkylcarbonylamino group is a group represented by the formula below (R 11 represents an alkyl group) and is also called an alkanoylamino group.
- the substituent on the phenyl ring is not particularly limited. Examples of such substituents include electron-donating groups.
- the electron-donating group is not particularly limited as long as it does not inhibit the effects of the present invention. , hydroxyl group, amino group and halogen group.
- alkyl group examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, 1-ethylpropyl group and n-pentyl group. , a neopentyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, and other linear or branched alkyl groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms). Among these alkyl groups, a methyl group is preferred.
- alkoxy group examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, n-pentyloxy, neopentyloxy, and n-hexyloxy groups.
- Linear or branched alkoxy groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) can be mentioned.
- a methoxy group and the like are preferable.
- alkylamino group examples include 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, monoalkylamino groups such as 3-methylpentylamino group; dimethylamino group, diethylamino group, di- Linear or branched alkylamino groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) such as dialkylamino groups such as n-propylamino group can be mentioned.
- alkylcarbonyl group examples include methylcarbonyl group, ethylcarbonyl group, n-propylcarbonyl group, isopropylcarbonyl group, n-butylcarbonyl group, isobutylcarbonyl group, s-butylcarbonyl group, t-butylcarbonyl group, 1 -An alkyl moiety such as an ethylpropylcarbonyl group, n-pentylcarbonyl group, neopentylcarbonyl group, n-hexylcarbonyl group, isohexylcarbonyl group, 3-methylpentylcarbonyl group has 1 to 6 carbon atoms (especially 4), for example, a linear or branched alkylcarbonyl group.
- alkylaminocarbonyl groups include methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, n-butylaminocarbonyl, isobutylaminocarbonyl and s-butylaminocarbonyl groups.
- alkyl moiety such as dialkylaminocarbonyl group such as dimethylaminocarbonyl group, diethylaminocarbonyl group and di-n-propylaminocarbonyl group has 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) Linear or branched alkylaminocarbonyl groups and the like can be mentioned.
- alkylcarbonylamino group examples include methylcarbonylamino group, ethylcarbonylamino group, n-propylcarbonylamino group, isopropylcarbonylamino group, n-butylcarbonylamino group, isobutylcarbonylamino group and s-butylcarbonylamino group.
- t-butylcarbonylamino group 1-ethylpropylcarbonylamino group, n-pentylcarbonylamino group, neopentylcarbonylamino group, n-hexylcarbonylamino group, isohexylcarbonylamino group, 3-methylpentylcarbonylamino group, etc.
- a linear or branched alkylcarbonylamino group having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) in the alkyl moiety of is mentioned.
- halogen group examples include fluorine, chlorine, bromine, and iodine.
- the hydrochloride 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. Specifically, guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine Hydrochloride, tetrabutylammonium chloride (nBu 4 NCl), piperazine hydrochloride, morpholine hydrochloride and the like can be mentioned.
- guanidine hydrochloride dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, piperazine hydrochloride, or morpholine hydrochloride is preferred, and guanidine hydrochloride, diisopropylamine hydrochloride, or piperidine hydrochloride is preferred. Most preferred.
- the amount of hydrochloride used above can be appropriately set within a wide range.
- the hydrochloride can be used in an amount of about 1 to 4 mol, preferably about 2 to 4 mol, relative to the molar amount of the compound represented by formula (2).
- 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 specifically, magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride. , calcium chloride, nickel chloride and the like. Among these metal chlorides, magnesium chloride is preferred.
- the amount of metal chloride used can be set appropriately from a wide range.
- the metal chloride can be used in an amount of about 25 to 40 molar amounts. A molar amount is preferred.
- R 4 in formula (2) represents R 5 —CH 2 — (R 5 is an alkylcarbonylamino group), it is preferred to use a metal chloride.
- the reaction conditions of the first production method of the present invention are not particularly limited as long as the effects of the present invention are exhibited, but for example, it is preferably carried out under acidic conditions.
- the reaction system contains acids such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, trimethylsilyl trifluoromethanesulfonate, 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid, and acetic acid. It is possible to mention that
- the reaction solvent in the first production method of the present invention is not particularly limited as long as it does not inhibit the reaction.
- trifluoroacetic acid and the like can be mentioned.
- trifluoroacetic acid can also be used as an acid.
- the reaction temperature in the first production method of the present invention is not particularly limited.
- the reaction temperature can be about 20 to 70°C, preferably about 30 to 50°C.
- the reaction time of the first production method of the present invention varies depending on the reaction temperature, and although it cannot be generalized, it can be, for example, about 1 to 5 hours.
- the cross-linking 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 appropriately combined with known means such as column chromatography such as HPLC, thin layer chromatography, recrystallization, reprecipitation, distillation, and solvent extraction.
- the second manufacturing method of the present invention is the following formula (3)
- R 6 , R 7 , R 8 and R 9 each represent a monovalent organic group.
- R 8 and R 9 are the same as above.
- R 10 represents 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 in the presence of hydrochloride or metal chloride.
- R 6 , R 7 , R 8 and R 9 in formulas (3), (4) and (5) above are each monovalent organic groups. These monovalent organic groups can be the same as the monovalent organic groups specifically described in the first production method of the present invention.
- R 10 in the above formula (5) may have a substituent on R 5 —CH 2 — or the phenyl ring, like R 4 specifically explained in the first production method of the present invention. It shows a good benzyl group.
- the usage ratio of the compound represented by formula (4) and the compound represented by formula (5) in the second production method of the present invention is not particularly limited.
- the molar ratio of the former to the latter can be about 1:0.5-2, preferably about 1:0.8-1.5, more preferably 1:0.9-1.
- a ratio of about 1 can be used.
- the hydrochloride in the second production method of the present invention can be the same as the hydrochloride specifically explained in the first production method of the present invention.
- the metal chloride in the second production method of the present invention can be the same as the metal chloride specifically explained in the first production method of the present invention.
- R 10 in formula (5) represents R 5 —CH 2 — (R 5 is an alkylcarbonylamino group)
- R 5 is an alkylcarbonylamino group
- reaction conditions in the cross-linking reaction of the second production method of the present invention are also preferably carried out under acidic conditions, similar to the reaction conditions of the first production method of the present invention.
- the means for providing acidic conditions can be as described in detail in the first production method of the present invention.
- the reaction solvent in the second production method of the present invention can be the same as the solvent in the cross-linking reaction in the first production method of the present invention. More specifically, when R 10 is a benzyl group which may have a substituent on the phenyl ring, it is preferred to use methanesulfonic acid in addition to trifluoroacetic acid. When R 10 is R 5 —CH 2 — (R 5 is an alkylcarbonylamino group), the ionic liquid 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid (BMPy ⁇ OTf) can also be used as a solvent.
- BMPy ⁇ OTf 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid
- the cross-linking reaction can also be carried out in the presence of phenol and/or alkoxybenzene.
- the reaction can proceed more efficiently.
- the alkoxybenzene is not particularly limited as long as the effects of the present invention are exhibited, and examples include anisole (methoxybenzene), ethoxybenzene, propoxybenzene, and butoxybenzene. Among these alkoxybenzenes, anisole and ethoxybenzene are preferred, and anisole is particularly preferred. In addition, the said alkoxybenzene can be used individually or in combination of 2 or more types.
- the amount of the phenol and/or alkoxybenzene used can be used in a wide range as long as it does not inhibit the effects of the present invention. can be done.
- the reaction temperature in the cross-linking reaction of the second production method of the present invention can be the same as the reaction temperature of the first production method of the present invention.
- the reaction time in the first production method of the present invention can be the same as the reaction time in the first production method of the present invention.
- R 10 is a benzyl group which may have a substituent on the phenyl ring
- the reaction temperature may be in the range of 0 to 70°C, which is lower than the reaction temperature of the first production method. can.
- the compound obtained by the second production method of the present invention (compound of formula (3)) is subjected to isolation and purification means to obtain a highly pure target product.
- isolation and purification means can be appropriately combined with known means such as column chromatography such as HPLC, thin layer chromatography, recrystallization, reprecipitation, distillation, and solvent extraction.
- MS analysis in the examples described later was performed by Waters MICROMASS (registered trademark) LCT PREMIERTM (ESI-TOF) or LC-MS (Shimadzu, Japan, Prominence-I LC-2030, LCMS-2020). did.
- HPLC analyzes in the examples below were performed using a HITACHI L-7150 L-2400 detector or a Waters Alliance 2695 Separations Module with ELS 2420 System using a Cosmosil 5C18-AR-II analytical column (Nacalai Tesque, 4 .6 ⁇ 250 mm, flow rate 1.0 mL ⁇ min ⁇ 1 ) and Cosmosil 5C18-AR-II semi-preparative columns (Nacalai Tesque, 10 ⁇ 250 mm, flow rate 3.0 mL ⁇ min ⁇ 1 ).
- NMR measurement in the examples described later uses Bruker AV400N (400 MHz) or Bruker AV500 (500 MHz) for 1 H, and Bruker AV400N (100 MHz) or Bruker AV500 (125 MHz) for 13 C. did.
- CD spectra in the examples described later were measured by a conventional method using a JASCO J-1500 CD spectrometer unless otherwise specified.
- Fmoc-SPPS Fmoc SPPS was performed according to the following protocol.
- Cys(Acm)(O)-containing peptide was obtained by subjecting the Cys(Acm)-containing peptide to an oxidation reaction after obtaining the Cys(Acm)-containing peptide by a conventional Fmoc solid-phase synthesis method.
- Example 1 In the following reaction scheme, the following compound 2 was obtained by reacting the following compound 1 as a raw material under various conditions. Table 1 shows specific conditions. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. In this reaction, the solvent used was TFA, which acted as an acid. Compound 1 was produced by the peptide synthesis method described above.
- GAL indicates residues of a peptide consisting of glycine-alanine-leucine in order from the N-terminal side.
- the carbonyl group adjacent to G (glycine) and the amino group of the glycine are peptide-bonded
- the amino group adjacent to L (leucine) and the carbonyl group of leucine are peptide-bonded.
- R indicates an arginine residue.
- TFMSA trifluoromethanesulfonic acid
- MSA methanesulfonic acid
- nBu 4 NCl tetrabutylammonium chloride
- FIG. 1 shows the HPLC chart of the reaction product obtained in this reaction.
- MS data and NMR data of the compound 2 are as follows.
- Example 2 In the reaction scheme below, compound 3 and compound 4 were reacted with compound 5 under various conditions shown in Table 2 below. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction. For compound 3, the carbonyl group of L-tryptophan was converted to a methoxy group by a conventional method in the presence of thionyl chloride. Compound 4 was produced by the peptide synthesis method described above.
- Ac- represents an acetyl group.
- FIG. 2 shows the HPLC chart of the reaction product obtained in this reaction. Moreover, the MS data of the obtained compound 5 are as follows. ⁇ Compound 5> LRMS ( ESI -TOF) m/z: [M+H] + calcd for C25H37N5O6S 534.2 , found 533.9.
- Example 3 In the reaction scheme below, the following compounds 6 to 10 were reacted under the conditions of 1M MSA and 4M guanidine hydrochloride as starting materials. The reaction temperature in this reaction was 20° C., and the reaction time was 3 hours. TFA was used as solvent in this reaction. In addition, the reaction was similarly performed at 37° C. instead of 20° C. using compound 10 as a starting material. Compounds 6-10 were made by the peptide synthesis method described above.
- the MS data of the prepared compounds 6 to 10 are as follows. ⁇ Compound 6> LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C66H102N22O15S 737.4 , found 737.1. ⁇ Compound 7> LRMS (ESI-TOF) m/z: [ M+2H] + calcd for C66H107N21O15S 732.9 , found 732.7. ⁇ Compound 8> LRMS (ESI - TOF) m/z: [M+2H] + calcd for C69H104N20O15S 742.4 , found 742.2.
- Ac- represents an acetyl group
- GAL represents a peptide residue consisting of glycine-alanine-leucine in order from the N-terminal side
- GHRAL represents glycine-histidine-arginine-alanine- in order from the N-terminal side.
- GKRAL indicates a peptide residue consisting of glycine-lysine-arginine-alanine-leucine in order from the N-terminal side
- GFRAL indicates glycine-phenylalanine-arginine-alanine- in order from the N-terminal side
- GMRAL indicates glycine-methionine-arginine-alanine- in order from the N-terminal side
- Peptide residues consisting of leucine are indicated.
- the carbonyl group adjacent to the N-terminal G (glycine) residue and the primary amino group of the glycine residue are peptide-bonded, and the amino acid adjacent to the C-terminal L (leucine) residue A peptide bond is formed between the group and the carbonyl group of the leucine residue.
- RG indicates residues of a peptide consisting of arginine-glycine in order from the N-terminus.
- the carbonyl group adjacent to the R (arginine) residue and the amino group of arginine are peptide-bonded, and the G (glycine) residue has a C-terminal amide structure.
- FIG. 3 shows the HPLC chart of the reaction product obtained in the above reaction.
- the MS data of Compounds 11 to 15 obtained are as follows. ⁇ Compound 11> LRMS ( ESI-TOF) m/z: [M+2H] + calcd for C58H92N22O13S 668.4 , found 668.2 . ⁇ Compound 12> LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C58H97N21O13S 663.9 , found 663.7 .
- cross-linking reaction temperature was 37°C more efficiently than 20°C.
- Example 4 In the reaction scheme below, compounds 16 and 17 below were 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 solvent in this reaction. These compounds 16 and 17 were prepared by the peptide synthesis method described above.
- Ac- represents an acetyl group
- GA represents a peptide residue consisting of glycine-alanine in order from the N-terminus
- GALRA is a peptide consisting of glycine-alanine-leucine-arginine-alanine in order from the N-terminus. Residues are indicated.
- the carbonyl group adjacent to the G (glycine) residue on the N-terminal side and the amino group of the glycine residue are peptide-bonded, and the amino group adjacent to the A (alanine) residue on the C-terminal side A peptide bond is formed between the group and the carbonyl group of the alanine residue.
- R represents an arginine residue.
- FIG. 4 shows the HPLC chart of the reaction product obtained in the above reaction.
- MS data of the obtained compounds 18 and 19 are as follows. ⁇ Compound 18> LRMS ( ESI -TOF) m/z: [ M +H] + calcd for C27H39N10O6S 631.3, found 631.3 . ⁇ Compound 19> LRMS (ESI-TOF ) m /z: [ M+2H] + calcd for C42H68N16O9S 486.3, found 486.3.
- Example 5 In the reaction scheme below, the following compound 20 was used as a starting material and reacted under the conditions of 1M MSA and 4M guanidine hydrochloride. The reaction temperature in this reaction was 25° C. and the reaction time was 30 minutes. TFA was used as solvent in this reaction. This compound 19 was prepared by the peptide synthesis method described above.
- Ac- represents an acetyl group
- GYRAL represents a peptide residue consisting of glycine-tyrosine-arginine-alanine-leucine in order from the N-terminus
- GAL consists of glycine-alanine-leucine in order from the N-terminus.
- Peptide residues are indicated.
- the carbonyl group adjacent to the G (glycine) residue and the amino group of the glycine residue are peptide-bonded, and the primary amino group adjacent to the L (leucine) residue and the leucine residue A peptide bond is formed with the carboxynyl group.
- RG represents a peptide residue consisting of glycine-arginine in order from the N-terminus.
- the carbonyl group adjacent to the R (arginine) residue and the amino group of the arginine residue are peptide-bonded, and the G (glycine) residue has a C-terminal amide structure.
- compound 20 which has a cysteine residue and a tryptophan residue protected by a methylcarbonylamino group contained in the peptide, under acidic conditions in the presence of hydrochloride (guanidine hydrochloride), the cysteine residue and tryptophan residues.
- hydrochloride guanidine hydrochloride
- Example 6 As shown in the scheme below, compound 21 prepared by the peptide synthesis method described above was converted to N-methylpyrrolidone (NMP) containing tripyrrolidinophosphonium hexafluorophosphate (e.g., PyBOP) and N,N-diisopropylethylamine (DIEA). ) at room temperature for 3 hours to obtain a cyclic peptide of compound 22.
- NMP N-methylpyrrolidone
- DIEA N,N-diisopropylethylamine
- this compound 22 was reacted under the conditions of 1M MSA and 4M guanidine hydrochloride.
- the reaction temperature in this reaction was 4° C. and the reaction time was 3 hours.
- TFA was used as solvent in this reaction.
- NPI indicates a peptide residue consisting of asparagine-proline-isoleucine in order from the N-terminus.
- the carbonyl group adjacent to the N (asparagine) residue and the amino group of the asparagine residue are peptide-bonded, and the primary amino group adjacent to the I (isoleucine) residue and the isoleucine residue A peptide bond is formed with the carbonyl group.
- GI indicates a peptide residue consisting of glycine-isoleucine in order from the N-terminus.
- the carbonyl group adjacent to the G (glycine) residue and the primary amino group of the glycine residue are peptide-bonded
- the primary amino group adjacent to the I (isoleucine) residue and the carbonyl of the isoleucine residue are group is peptide-bonded.
- Example 7 In the reaction scheme below, compound 24 was used as a starting material and reacted under the conditions of 1M MSA and 4M guanidine hydrochloride. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction. Compound 24 was made by the peptide synthesis method described above.
- Ac- represents an acetyl group
- SDL represents a peptide residue consisting of serine-aspartic acid-leucine in order from the N-terminus.
- the carbonyl group adjacent to the S (serine) residue and the amino group of the serine residue are peptide-bonded
- the amino group adjacent to the L (leucine) residue and the carbonyl group of the leucine residue is a peptide bond.
- LQLRQR indicates a peptide residue composed of leucine-glutamine-leucine-arginine-glutamine-arginine in order from the N-terminus.
- the carbonyl group adjacent to the L (leucine) residue on the N-terminal side and the amino group of the leucine residue are peptide-bonded, and the R (arginine) on the C-terminal side has a C-terminal amide structure.
- Compound 24 is an analog of the amino acid sequence of stERAP, a breast cancer inhibitory peptide, and the norleucine residue in compound 24 corresponds to the methionine residue of stERAP.
- stERAP a bisamide-mediated cross-linking is performed between a glutamic acid residue provided between a methionine residue and a serine residue and a glutamine residue provided between leucine residues. It is Here, it was assumed that formation of an intramolecular cross-link between the cysteine residue and the tryptophan residue in compound 24 would give a compound having the same three-dimensional structure as stERAP, like compound 25.
- FIG. 7 shows the HPLC chart of the reaction product obtained in the above reaction and the results of the CD spectrum after the reaction.
- Example 8 0.01 mM tratusuzumab (Chugai Pharmaceutical Co., Ltd.) and compound 27 represented by the following formula were mixed and reacted in 0.1% TFA containing 30 mM magnesium chloride and 5% water at 37° C. for 24 hours.
- the reaction system also contained 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid.
- Compound 27 was made by the peptide synthesis method described above.
- a in the above compound 27 is a group shown below, 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 bound to peptides.
- Example 9 In the reaction scheme below, compounds 29, 30, 32, and 35 were reacted under conditions of 1M MSA and 4M diisopropylamine hydrochloride as starting materials. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction.
- H at the N-terminus represents a histidine residue
- EGTFFTSDVSSYLEGQAAKEFIA is glutamic acid-glycine-threonine-phenylalanine-threonine-serine-aspartic acid-valine-serine-serine-tyrosine-leucine in order from the N-terminal side.
- LVRGRG represents a peptide residue consisting of leucine-valine-arginine-glycine-arginine-glycine in order from the N-terminus.
- the carbonyl group adjacent to the N-terminal L (leucine) residue and the amino group of the leucine are peptide-bonded.
- 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 of A group is attached to the peptide.
- A3 is a group shown below. In compound 32, the carbonyl group adjacent to A3 and the amine group of A3 are bonded to the peptide, and the amine group adjacent to A3 and the amine group of A3 are bonded to the peptide . A carbonyl group is attached to the peptide.
- FIG. 9 shows the HPLC chart of the reaction product obtained in the above reaction.
- MS data of the obtained compounds 31, 33, 36 and 37 are as follows.
- compounds 29 and 35 are bioactive peptides, and lipid structures are being actively bound to them.
- Compounds 30 and 32 are expected to increase the in vivo stability of the peptide and suppress renal excretion by introducing a lipid structure.
- Compounds 31, 33, 36 and 37 in which a cysteine sulfoxide protected by a methoxybenzyl group is used to introduce a lipid structure onto tryptophan via intermolecular cross-linking, exhibit more effective physiological activity in vivo. be able to.
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
本発明は、ペプチド化合物の製造方法に関する。
The present invention relates to a method for producing a peptide compound.
医薬品候補化合物の開発対象は、低分子医薬から抗体医薬、そしてペプチド医薬へと日々変遷している。当該ペプチド医薬は、ペプチド固相合成法（ＳＰＰＳ）によって比較的簡便に得ることが可能であり、さらに特定の細胞を標的にすることができる点で極めて有望視されている。抗体やその断片に低分子医薬等を担持させたＡＤＣ（Ａｎｔｉｂｏｄｙ Ｄｒｕｇ Ｃｏｎｊｕｇａｔｅ）の開発も進められている。
The targets for development of drug candidate compounds are changing day by day, from low-molecular-weight drugs to antibody drugs and then to peptide drugs. The peptide drug can be obtained relatively easily by solid-phase peptide synthesis (SPPS), and is highly promising in that it can target specific cells. The development of ADCs (Antibody Drug Conjugates), in which antibodies or fragments thereof carry low-molecular-weight drugs, etc., is also underway.
α－ヘリックス、ロイシンジッパー、ジンクフィンガー、コイルドコイル等に代表されるペプチドの立体構造は、そのペプチドの生理活性に関連することが知られている。例えば、α－ヘリックス構造は、ペプチドを構成するアミノ酸残基の側鎖間の相互作用によって形成され、複数のアミノ酸の側鎖間を架橋するステープリングによって、α－ヘリックス構造をより強固にすることが知られている。
The three-dimensional structure of peptides represented by α-helices, leucine zippers, zinc fingers, coiled coils, etc. is known to be related to the physiological activities of the peptides. For example, an α-helical structure is formed by interactions between side chains of amino acid residues that constitute a peptide, and stapling that bridges the side chains of multiple amino acids strengthens the α-helical structure. It has been known.
天然アミノ酸の一種であるシステインの側鎖に存在する求核性チオール基は、アルキル化、マイケル反応、ジスルフィド形成等に関連し、ペプチドやタンパク質の部位特異的な修飾における橋渡しとして機能する基となることが知られている。また、ペプチドにおける求核性チオール基による分子内反応が当該ペプチドの環化、当該ペプチド内でのステープリング等に関連することも知られている。
The nucleophilic thiol group present in the side chain of cysteine, a type of natural amino acid, is involved in alkylation, Michael reaction, disulfide formation, etc., and serves as a bridge for site-specific modification of peptides and proteins. It is known. It is also known that an intramolecular reaction by a nucleophilic thiol group in a peptide is related to cyclization of the peptide, stapling within the peptide, and the like.
保護されたペプチドを酸性条件下で脱保護する際に、予想外の反応が起こることが知られている。例えば、ペプチド鎖の伸長過程で生成したパラメトキシベンジル保護システインをアニソール存在下で脱保護すると、Ｓ－アリール化システインが合成されることが報告されている（非特許文献１）。
It is known that an unexpected reaction occurs when the protected peptide is deprotected under acidic conditions. For example, it has been reported that deprotection of para-methoxybenzyl-protected cysteine produced in the process of peptide chain elongation in the presence of anisole leads to the synthesis of S-arylated cysteine (Non-Patent Document 1).
また、ＡＤＣの開発において、抗体と低分子化合物との間に設けるリンカーが生体適合性や代謝についての問題点を生じさせることから、リンカーの最適化が必要となっている。
In addition, in the development of ADCs, the linker provided between the antibody and the low-molecular-weight compound poses problems in terms of biocompatibility and metabolism, making it necessary to optimize the linker.
今日まで、ペプチドの生理活性に関連するα－ヘリックス構造をより強固なものとできるステープリングを選択的に実施できる方法が知られているものの、遷移金属を使用する必要がある点においてデメリットがある。さらに付言すると、ステープリングに関連するシステイン残基の側鎖と他のアミノ酸の側鎖を介した架橋反応を選択的に、且つ高効率で行うことができる方法は知られていない。また、システイン残基を含有する化合物とその他の化合物とを、選択的に、且つ効率的に結合させる方法も知られていない。
To date, methods are known that can selectively perform stapling that can strengthen the α-helical structure related to the bioactivity of peptides, but they have the disadvantage of requiring the use of transition metals. . In addition, there is no known method capable of selectively and highly efficiently carrying out a cross-linking reaction via side chains of cysteine residues related to stapling and side chains of other amino acids. Also, a method for selectively and efficiently binding a compound containing a cysteine residue to another compound is not known.
本発明者らは、上記の課題を解決すべく鋭意検討を重ねた結果、特定の保護基を有するシステイン残基の側鎖とトリプトファン残基の側鎖とが、塩酸塩又は金属塩化物の存在下にて反応して、架橋結合を形成することを見出した。また、本発明者らは、このような架橋反応が化合物の分子内であっても、異なる化合物の分子間であっても進行することも見出した。本発明は、これらの知見に基づいて完成された発明であって、下記の各項に示す発明を広く包含する。
As a result of intensive studies to solve the above problems, the present inventors have found that the side chain of a cysteine residue having a specific protective group and the side chain of a tryptophan residue are in the presence of hydrochloride or metal chloride. It was found to react below to form cross-links. The present inventors also found that such a cross-linking reaction proceeds both intramolecularly within a compound and between molecules of different compounds. The present invention is an invention completed based on these findings, and broadly includes the inventions shown in the following items.
項１ 下記式（１）
［式中、Ｒ１は、一価有機基を示す。
Ｒ２は、二価の有機基を示す。
Ｒ３は、一価有機基を示す。
また、Ｒ１とＲ３とが、互いに結合して環を形成していてもよい。］
で表される化合物の製造方法であって、
下記式（２）Item 1 Formula (1) below
[In the formula, R 1 represents a monovalent organic group.
R2 represents a divalent organic group.
R3 represents a monovalent organic group.
Also, R 1 and R 3 may combine with each other to form a ring. ]
A method for producing a compound represented by
Formula (2) below
Ｒ２は、二価の有機基を示す。
Ｒ３は、一価有機基を示す。
また、Ｒ１とＲ３とが、互いに結合して環を形成していてもよい。］
で表される化合物の製造方法であって、
下記式（２）
R2 represents a divalent organic group.
R3 represents a monovalent organic group.
Also, R 1 and R 3 may combine with each other to form a ring. ]
A method for producing a compound represented by
Formula (2) below
で表される化合物を、塩酸塩又は金属塩化物の存在下で架橋反応させる工程を含む、製造方法。
A production method comprising the step of cross-linking the compound represented by in the presence of hydrochloride or metal chloride.
項２ 前記Ｒ１が、カルボニル基を有する一価の有機基であり、前記Ｒ２が、カルボニル基及びアミノ基を有する二価の有機基であり、及び前記Ｒ３が、アミノ基を有する一価の有機基である、上記項１に記載の製造方法。
Item 2 R 1 is a monovalent organic group having a carbonyl group, R 2 is a divalent organic group having a carbonyl group and an amino group, and R 3 is a monovalent organic group having an amino group. 2. The production method according to item 1 above, wherein the organic group is a divalent organic group.
項３ 前記Ｒ１、Ｒ２及びＲ３が、それぞれペプチド残基である、上記項１に記載の製造方法。
Item 3. The production method according to Item 1, wherein each of R 1 , R 2 and R 3 is a peptide residue.
項４ 前記フェニル環上の置換基が、電子供与性基である、上記項１に記載の製造方法。
Item 4 The production method according to Item 1 above, wherein the substituent on the phenyl ring is an electron-donating group.
項５ 前記電子供与性基が、アルキル基、アルコキシ基、アルキルアミノ基、アルキルカルボニル基、アルキルアミノカルボニル基、アルキルカルボニルアミノ基、水酸基、アミノ基及びハロゲン基からなる群より選択される少なくとも一種である、上記項４に記載の製造方法。
Item 5 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. 5. The manufacturing method according to item 4 above.
項６ 前記塩酸塩が、グアニジン塩酸塩、ジメチルアミン塩酸塩、ジイソプロピルアミン塩酸塩、ピペリジン塩酸塩、塩化テトラブチルアンモニウム、ピペラジン塩酸塩及びモルフォリン塩酸塩からなる群より選択される少なくとも一種である、上記項１～項５の何れか一項に記載の製造方法。
Item 6 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. The manufacturing method according to any one of items 1 to 5 above.
項７ 前記金属塩化物が、塩化マグネシウム、塩化亜鉛、塩化リチウム、塩化鉄（III）、塩化カルシウム及び塩化ニッケルからなる群より選択される少なくとも一種である、上記項１～項５の何れか一項に記載の製造方法。
Item 7. Any one of items 1 to 5 above, wherein the metal chloride is at least one selected from the group consisting of magnesium chloride, zinc chloride, lithium chloride, iron chloride (III), calcium chloride and nickel chloride. The manufacturing method described in the item.
項８ 酸性条件下で架橋反応を行う、上記項１～項７の何れか一項に記載の製造方法。
Item 8 The production method according to any one of Items 1 to 7 above, wherein the cross-linking reaction is performed under acidic conditions.
項９ 下記式（３）
Item 9 Formula (3) below
で表される化合物の製造方法であって、
下記式（４）
A method for producing a compound represented by
Formula (4) below
で表される化合物と、
下記式（５）
A compound represented by
Formula (5) below
で表される化合物とを、塩酸塩又は金属塩化物の存在下で反応させる工程を含む、製造方法。
A production method comprising the step of reacting a compound represented by in the presence of a hydrochloride or a metal chloride.
項１０ 前記Ｒ６及びＲ８がカルボニル基を有する一価の有機基であり、前記Ｒ７及びＲ９が、アミノ基を有する一価の有機基である、上記項９に記載の製造方法。
Item 10. The production method according to Item 9, wherein R 6 and R 8 are monovalent organic groups having a carbonyl group, and R 7 and R 9 are monovalent organic groups having an amino group.
項１１ 前記Ｒ６、Ｒ７、Ｒ８及びＲ９が、それぞれペプチド残基である、上記項９に記載の製造方法。
Item 11. The production method according to Item 9, wherein each of R 6 , R 7 , R 8 and R 9 is a peptide residue.
項１２ 前記フェニル環上の置換基が、電子供与性基である、上記項９に記載の製造方法。
Item 12 The production method according to Item 9 above, wherein the substituent on the phenyl ring is an electron-donating group.
項１３ 前記電子供与性基が、アルキル基、アルコキシ基、アルキルアミノ基、アルキルカルボニル基、アルキルアミノカルボニル基、アルキルカルボニルアミノ基、水酸基、アミノ基及びハロゲン基からなる群より選択される少なくとも一種である、上記項１２に記載の製造方法。
Item 13 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. 13. The manufacturing method according to item 12 above.
項１４ 前記塩酸塩が、グアニジン塩酸塩、ジメチルアミン塩酸塩、ジイソプロピルアミン塩酸塩、ピペリジン塩酸塩、塩化テトラブチルアンモニウム、ピペラジン塩酸塩及びモルフォリン塩酸塩からなる群より選択される、少なくとも一種である、上記項９～項１３の何れか一項に記載の製造方法。
Item 14 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. , the production method according to any one of items 9 to 13 above.
項１５ 前記金属塩化物が、塩化マグネシウム、塩化亜鉛、塩化リチウム、塩化鉄（III）、塩化カルシウム及び塩化ニッケルからなる群より選択される少なくとも一種である、上記項９～１３の何れか一項に記載の製造方法。
Item 15 Any one of items 9 to 13 above, 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. The manufacturing method described in .
項１６ 酸性条件下で架橋反応を行う、上記項９～項１５の何れか一項に記載の製造方法。
Item 16 The production method according to any one of Items 9 to 15 above, wherein the cross-linking reaction is performed under acidic conditions.
項１７ 更に、フェノール及び／又はアルコキシベンゼンの存在下で架橋反応させる、上記項９～項１６の何れか一項に記載の製造方法。
Item 17 The production method according to any one of Items 9 to 16 above, wherein the cross-linking reaction is further performed in the presence of phenol and/or alkoxybenzene.
本発明によると、保護されたシステイン残基の側鎖とトリプトファン残基の側鎖との新規な架橋反応を提供することができる。このような架橋反応により、主鎖の結合とは異なる位置での架橋を形成することができる。そのために、当該架橋反応が化合物の分子内で実施される場合、特定のアミノ酸残基間にてステープリングを形成することができ、それ故、α－ヘリックスなどの立体構造をより強固にすることができる。また、当該架橋反応が異なる化合物の分子間で実施される場合、それぞれの分子の主鎖を保持することができるので、それぞれの化合物が発現する生理活性を保持しつつ、リンカーを介することなく直接それぞれの化合物同士を架橋することができる。従って、本発明の方法を利用することにより、様々なペプチド医薬を工業的に有利に製造することができる。
According to the present invention, a novel cross-linking reaction between the side chain of a protected cysteine residue and the side chain of a tryptophan residue can be provided. Such a cross-linking reaction can form a cross-link at a position different from the main chain bond. Therefore, when the cross-linking reaction is carried out within the molecule of the compound, it is possible to form staples between specific amino acid residues, thus making the conformation such as α-helix more rigid. can be done. In addition, when the cross-linking reaction is carried out between molecules of different compounds, the backbone of each molecule can be retained, so that while retaining the physiological activity expressed by each compound, direct Each compound can be crosslinked. Therefore, by using the method of the present invention, various peptide drugs can be produced industrially and advantageously.
以下、本発明について説明する。なお、以下において、特に断らない限り、数値範囲を示す「～」との標記は、「未満」及び「超過」の意味ではなく「以上」及び「以下」を示す。つまり、「Ａ～Ｂ」は「Ａ以上Ｂ以下」を意味し、Ａ及びＢも含まれる。
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" and "exceeding" but "greater than" and "less than". That is, "A to B" means "A or more and B or less", and A and B are also included.
本発明の第一の製造方法は、下記式（１）
The first manufacturing method of the present invention is the following formula (1)
Ｒ２は、二価の有機基を示す。
Ｒ３は、一価有機基を示す。
また、Ｒ１とＲ３とが、互いに結合して環を形成していてもよい。］
で表される化合物の製造方法であって、
下記式（２）
R2 represents a divalent organic group.
R3 represents a monovalent organic group.
Also, R 1 and R 3 may combine with each other to form a ring. ]
A method for producing a compound represented by
Formula (2) below
で表される化合物を、塩酸塩又は金属塩化物の存在下で架橋反応させる工程を含む。
A step of cross-linking the compound represented by in the presence of hydrochloride or metal chloride.
上記式（１）及び（２）において、Ｒ１は、一価有機基を示す。このような一価有機基は、特に限定されないが、カルボニル基を有する一価の有機基を好ましい態様として挙げることができる。より具体的に、Ｒ１に隣接するアミノ基とペプチド結合するようにカルボニル基が設けられた態様の一価の有機基とすることが更に好ましい。
In formulas (1) and (2) above, R 1 represents a monovalent organic group. Although such a monovalent organic group is not particularly limited, a monovalent organic group having a carbonyl group can be mentioned as a preferred embodiment. More specifically, a monovalent organic group in which a carbonyl group is provided so as to form a peptide bond with an amino group adjacent to R 1 is more preferable.
上記のカルボニル基を有する一価の有機基として、例えば脂肪酸基、ｍｉｎｉＰＥＧを有する一価の有機基、ペプチド残基等を挙げることができる。上記脂肪酸基は、直鎖状であっても分枝状であってよいが、簡便に製造できることに鑑みて、直鎖状とすることが好ましい。上記脂肪酸の炭素数は、特に限定されないが、通常１２～１８程度とすることができる。また、ＭｉｎｉＰＥＧは登録商標であり、８－ａｍｉｎｏ－３，６－ｄｉｏｘａｏｃｔａｎｏｉｃ ａｃｉｄで表される化合物である。これらの一価有機基の中でも、ペプチド残基とすることが好ましい。このようなペプチド残基を構成するペプチドは、直鎖状であっても、分枝状であってもよいが、簡便に製造することできることに鑑みて、直鎖状することが好ましい。
Examples of the monovalent organic group having a carbonyl group include a fatty acid group, a monovalent organic group having miniPEG, a peptide residue, and the like. The fatty acid group may be linear or branched, but is preferably linear in view of the ease of production. Although the number of carbon atoms in the above fatty acid is not particularly limited, it can usually be about 12 to 18. Also, MiniPEG is a registered trademark and is a compound represented by 8-amino-3,6-dioxaoctanoic acid. Among these monovalent organic groups, peptide residues are preferred. Peptides constituting such peptide residues may be linear or branched, but are preferably linear in view of ease of production.
なお、本明細書においてペプチド残基とは、ペプチドのＮ末端から水素原子が除かれ、ペプチドのＣ末端から水酸基が除かれた原子団を意味する。ここで、当該ペプチドを構成するアミノ酸は、コドンに支配される２０種類の狭義のアミノ酸に限定されず、テアニンやオルニチン等に代表されるα－アミノ酸、βアラニン等に代表されるβ－アミノ酸、γ－アミノ酪酸（ＧＡＢＡ）等に代表されるγ－アミノ酸等のあらゆる構造異性体を採用することができ、Ｄ－アミノ酸であってもＬ－アミノ酸であってもよい。
As used herein, a peptide residue means an atomic group obtained by removing a hydrogen atom from the N-terminus of a peptide and removing a hydroxyl group from the C-terminus of the peptide. Here, the amino acids constituting the peptide are not limited to the 20 types of narrowly defined amino acids governed by codons, α-amino acids such as theanine and ornithine, β-amino acids such as β-alanine, All structural isomers of γ-amino acids such as γ-aminobutyric acid (GABA) and the like can be employed, and they may be D-amino acids or L-amino acids.
上記Ｒ１の例示として挙げるペプチド残基に含有されるアミノ酸の個数は、特に限定されない。当該アミノ酸の個数として、例えば、通常２～２０個程度を挙げることができ、好ましくは２～１０個程度である。
The number of amino acids contained in the peptide residues exemplified for R 1 is not particularly limited. The number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10.
なお、上記Ｒ１において、Ｒ１に隣接するアミノ基と結合しない末端には、アセチル基、ベンゾイル基、ピバロイル基等で保護されることができる。
In addition, in the above R 1 , the terminal not bonded to the amino group adjacent to R 1 can be protected with an acetyl group, a benzoyl group, a pivaloyl group, or the like.
上記式（１）及び（２）において、Ｒ２は、二価有機基を示す。このような有機基は、本発明の効果を発揮する限りにおいて特に限定されず、カルボニル基及びアミノ基を有する二価の有機基を好ましい態様として挙げることができる。より具体的に、Ｒ２に隣接するアミノ基とペプチド結合するようにカルボニル基が含まれ、Ｒ２に隣接するカルボニル基とペプチド結合するようにアミノ基が含まれる態様の二価の有機基とすることが更に好ましい。
In formulas (1) and (2) above, R 2 represents a divalent organic group. Such an organic group is not particularly limited as long as the effects of the present invention are exhibited, and a preferred embodiment is a divalent organic group having a carbonyl group and an amino group. More specifically, a divalent organic group containing a carbonyl group so as to form a peptide bond with the amino group adjacent to R 2 and an amino group so as to form a peptide bond with the carbonyl group adjacent to R 2 It is more preferable to
上記のカルボニル基及びアミノ基を有する二価の有機基として、例えばアミノ基を有する脂肪酸基、ｍｉｎｉＰＥＧを有する二価の有機基、ペプチド残基等を挙げることができる。これらの二価有機基の中でも、二価のペプチド残基とすることが好ましい。このようなペプチド残基を構成するペプチドは、直鎖状であっても、分枝状であってもよいが、簡便に製造できることなどに鑑みて、直鎖状であることが好ましい。
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 miniPEG, a peptide residue, and the like. Among these divalent organic groups, divalent peptide residues are preferred. A peptide constituting such a peptide residue may be linear or branched, but is preferably linear in view of ease of production.
上記Ｒ２の例示として挙げるペプチド残基に含有されるアミノ酸の個数は、本発明が発揮する効果を阻害しない限りにおいて、特に限定されない。当該アミノ酸の個数として、例えば、通常２～２０個程度を挙げることができ、好ましくは、２～１０個程度、更に好ましくは２～５個程度である。
The number of amino acids contained in the peptide residues exemplified for R2 above is not particularly limited as long as the effects of the present invention are not inhibited. The number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10, more preferably about 2 to 5.
上記式（１）及び（２）において、Ｒ３は、一価有機基を示す。このような一価有機基は、特に限定されず、アミノ基を有する一価の有機基を挙げることができる。より具体的に、Ｒ３に隣接するカルボニル基とペプチド結合するようにアミノ基が含まれる態様の一価の有機基とすることが更に好ましい。
In formulas (1) and (2) above, R 3 represents a monovalent organic group. Such a monovalent organic group is not particularly limited, and a monovalent organic group having an amino group can be mentioned. More specifically, it is more preferable to use a monovalent organic group containing an amino group so as to form a peptide bond with the carbonyl group adjacent to R3 .
上記アミノ基を有する一価有機基として、具体的には、アミノ基を有する脂肪酸基、ｍｉｎｉＰＥＧを有する一価アミノ基、ペプチド残基等を挙げることができる。これらの一価有機基の中でもペプチド残基とすることが好ましい。このようなペプチド残基を構成するペプチドは、直鎖状であっても、分枝状であってもよいが、直鎖状であることが好ましい。
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 miniPEG, a peptide residue, and the like. Among these monovalent organic groups, peptide residues are preferred. A peptide constituting such a peptide residue may be linear or branched, but preferably linear.
上記Ｒ３の例示として挙げるペプチド残基に含有されるアミノ酸の個数は、特に限定されない。当該アミノ酸の個数として、例えば、通常２～２０個程度を挙げることができ、好ましくは、２～１０個程度である。
The number of amino acids contained in the peptide residues exemplified for R3 is not particularly limited. The number of such amino acids is, for example, usually about 2 to 20, preferably about 2 to 10.
なお、上記Ｒ3において、Ｒ3に隣接するカルボニル基と結合しない末端は、一級アミノ基、二級アミノ基、芳香族アミノ基等で保護されることもできる。
In the above R 3 , the terminal not bonded to the carbonyl group adjacent to R 3 may be protected with a primary amino group, secondary amino group, aromatic amino group, or the like.
上記式（１）において、Ｒ１とＲ３とが互いに結合して環を形成する態様を挙げることができる。すなわち、式（１）で表される化合物が、環状ペプチドを形成する態様を挙げることができる。その他の式（１）で表される化合物の態様として、例えば、式（１）に含有されるシステイン残基を介したジスルフィド結合を含有する態様、式（１）に含有されるオキソ酸とヒドロキシ基との間で形成されるエステル結合を含有する態様等を挙げることができる。
In the above formula (1), there is an embodiment in which R 1 and R 3 are bonded to each other to form a ring. That is, the compound represented by Formula (1) can mention the aspect which forms a cyclic peptide. Other embodiments of the compound represented by formula (1) include, for example, an embodiment containing a disulfide bond via a cysteine residue contained in formula (1), an oxoacid and hydroxyl Examples include an aspect containing an ester bond formed with a group.
上記式（２）におけるＲ４は、Ｒ５－ＣＨ２－又はフェニル環上に置換基を有していてもよいベンジル基を示す。ここで、Ｒ５は、アルキルカルボニルアミノ基を示す。アルキルカルボニルアミノ基は、下記式に示す基であり（Ｒ１１は、アルキル基を示す）、アルカノイルアミノ基とも呼ばれることもある基である。
R 4 in the above formula (2) represents R 5 —CH 2 — or a benzyl group which may have a substituent on the phenyl ring. Here, R 5 represents an alkylcarbonylamino group. The alkylcarbonylamino group is a group represented by the formula below (R 11 represents an alkyl group) and is also called an alkanoylamino group.
上記Ｒ４で定義されるベンジル基において、フェニル環上の置換基は、特に限定されない。このような置換基として、例えば電子供与性基を挙げることができる。
In the benzyl group defined by R 4 above, the substituent on the phenyl ring is not particularly limited. Examples of such substituents include electron-donating groups.
上記電子供与性基は、本発明の効果を阻害しないものである限り特に限定されず、具体的に、アルキル基、アルコキシ基、アルキルアミノ基、アルキルカルボニル基、アルキルアミノカルボニル基、アルキルカルボニルアミノ基、水酸基、アミノ基及びハロゲン基等を挙げることができる。
The electron-donating group is not particularly limited as long as it does not inhibit the effects of the present invention. , hydroxyl group, amino group and halogen group.
上記アルキル基として、具体的にメチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｓ－ブチル基、ｔ－ブチル基、１－エチルプロピル基、ｎ－ペンチル基、ネオペンチル基、ｎ－ヘキシル基、イソヘキシル基、３－メチルペンチル基等の炭素数１～６（特に炭素数１～４）の直鎖状又は分岐鎖状アルキル基等を挙げることができる。これらのアルキル基の中でも、メチル基が好ましい。
Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, 1-ethylpropyl group and n-pentyl group. , a neopentyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, and other linear or branched alkyl groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms). Among these alkyl groups, a methyl group is preferred.
上記アルコキシ基として、具体的にメトキシ基、エトキシ基、ｎ－プロポキシ基、イソプロポキシ基、ｎ－ブトキシ基、ｔ－ブトキシ基、ｎ－ペンチルオキシ基、ネオペンチルオキシ基、ｎ－ヘキシルオキシ基の炭素数１～６（特に炭素数１～４）の直鎖状又は分岐鎖状のアルコキシ基等を挙げることができる。これらのアルコキシ基の中でも、メトキシ基等が好ましい。
Specific examples of the alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, n-pentyloxy, neopentyloxy, and n-hexyloxy groups. Linear or branched alkoxy groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) can be mentioned. Among these alkoxy groups, a methoxy group and the like are preferable.
上記アルキルアミノ基として、具体的にメチルアミノ基、エチルアミノ基、ｎ－プロピルアミノ基、イソプロピルアミノ基、ｎ－ブチルアミノ基、イソブチルアミノ基、ｓ－ブチルアミノ基、ｔ－ブチルアミノ基、１－エチルプロピルアミノ基、ｎ－ペンチルアミノ基、ネオペンチルアミノ基、ｎ－ヘキシルアミノ基、イソヘキシルアミノ基、３－メチルペンチルアミノ基等のモノアルキルアミノ基；ジメチルアミノ基、ジエチルアミノ基、ジ－ｎ－プロピルアミノ基等のジアルキルアミノ基等の炭素数１～６（特に炭素数１～４）の直鎖状又は分岐鎖状のアルキルアミノ基等を挙げることができる。
Specific examples of the alkylamino group include 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, monoalkylamino groups such as 3-methylpentylamino group; dimethylamino group, diethylamino group, di- Linear or branched alkylamino groups having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) such as dialkylamino groups such as n-propylamino group can be mentioned.
上記アルキルカルボニル基として、具体的にメチルカルボニル基、エチルカルボニル基、ｎ－プロピルカルボニル基、イソプロピルカルボニル基、ｎ－ブチルカルボニル基、イソブチルカルボニル基、ｓ－ブチルカルボニル基、ｔ－ブチルカルボニル基、１－エチルプロピルカルボニル基、ｎ－ペンチルカルボニル基、ネオペンチルカルボニル基、ｎ－ヘキシルカルボニル基、イソヘキシルカルボニル基、３－メチルペンチルカルボニル基等のアルキル部分が炭素数１～６（特に炭素数１～４）である直鎖状又は分岐鎖状アルキルカルボニル基等を挙げることができる。
Specific examples of the alkylcarbonyl group include methylcarbonyl group, ethylcarbonyl group, n-propylcarbonyl group, isopropylcarbonyl group, n-butylcarbonyl group, isobutylcarbonyl group, s-butylcarbonyl group, t-butylcarbonyl group, 1 -An alkyl moiety such as an ethylpropylcarbonyl group, n-pentylcarbonyl group, neopentylcarbonyl group, n-hexylcarbonyl group, isohexylcarbonyl group, 3-methylpentylcarbonyl group has 1 to 6 carbon atoms (especially 4), for example, a linear or branched alkylcarbonyl group.
上記アルキルアミノカルボニル基として、具体的にメチルアミノカルボニル基、エチルアミノカルボニル基、ｎ－プロピルアミノカルボニル基、イソプロピルアミノカルボニル基、ｎ－ブチルアミノカルボニル基、イソブチルアミノカルボニル基、ｓ－ブチルアミノカルボニル基、ｔ－ブチルアミノカルボニル基、１－エチルプロピルアミノカルボニル基、ｎ－ペンチルアミノカルボニル基、ネオペンチルアミノカルボニル基、ｎ－ヘキシルアミノカルボニル基、イソヘキシルアミノカルボニル基、３－メチルペンチルアミノカルボニル基等のモノアルキルアミノカルボニル基；ジメチルアミノカルボニル基、ジエチルアミノカルボニル基、ジ－ｎ－プロピルアミノカルボニル基等のジアルキルアミノカルボニル基等のアルキル部分が炭素数１～６（特に炭素数１～４）である直鎖状又は分岐鎖状アルキルアミノカルボニル基等を挙げることができる。
Specific examples of the above alkylaminocarbonyl groups include methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, n-butylaminocarbonyl, isobutylaminocarbonyl and s-butylaminocarbonyl groups. , t-butylaminocarbonyl group, 1-ethylpropylaminocarbonyl group, n-pentylaminocarbonyl group, neopentylaminocarbonyl group, n-hexylaminocarbonyl group, isohexylaminocarbonyl group, 3-methylpentylaminocarbonyl group, etc. monoalkylaminocarbonyl group; alkyl moiety such as dialkylaminocarbonyl group such as dimethylaminocarbonyl group, diethylaminocarbonyl group and di-n-propylaminocarbonyl group has 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) Linear or branched alkylaminocarbonyl groups and the like can be mentioned.
上記アルキルカルボニルアミノ基として、具体的にメチルカルボニルアミノ基、エチルカルボニルアミノ基、ｎ－プロピルカルボニルアミノ基、イソプロピルカルボニルアミノ基、ｎ－ブチルカルボニルアミノ基、イソブチルカルボニルアミノ基、ｓ－ブチルカルボニルアミノ基、ｔ－ブチルカルボニルアミノ基、１－エチルプロピルカルボニルアミノ基、ｎ－ペンチルカルボニルアミノ基、ネオペンチルカルボニルアミノ基、ｎ－ヘキシルカルボニルアミノ基、イソヘキシルカルボニルアミノ基、３－メチルペンチルカルボニルアミノ基等のアルキル部分の炭素数が１～６（特に炭素数１～４）である直鎖状又は分岐鎖状アルキルカルボニルアミノ基等を挙げることができる。
Specific examples of the alkylcarbonylamino group include methylcarbonylamino group, ethylcarbonylamino group, n-propylcarbonylamino group, isopropylcarbonylamino group, n-butylcarbonylamino group, isobutylcarbonylamino group and s-butylcarbonylamino group. , t-butylcarbonylamino group, 1-ethylpropylcarbonylamino group, n-pentylcarbonylamino group, neopentylcarbonylamino group, n-hexylcarbonylamino group, isohexylcarbonylamino group, 3-methylpentylcarbonylamino group, etc. A linear or branched alkylcarbonylamino group having 1 to 6 carbon atoms (especially 1 to 4 carbon atoms) in the alkyl moiety of is mentioned.
上記ハロゲン基として、具体的にフッ素、塩素、臭素、ヨウ素等を挙げることができる。
Specific examples of the halogen group include fluorine, chlorine, bromine, and iodine.
本発明の第一の製造方法において使用する塩酸塩は、本発明の効果を発揮できるものである限り特に限定されず、具体的に、グアニジン塩酸塩、ジメチルアミン塩酸塩、ジイソプロピルアミン塩酸塩、ピペリジン塩酸塩、塩化テトラブチルアンモニウム（ｎＢｕ４ＮＣｌ）、ピペラジン塩酸塩、モルフォリン塩酸塩等を挙げることができる。これらの塩酸塩の中でも、グアニジン塩酸塩、ジメチルアミン塩酸塩、ジイソプロピルアミン塩酸塩、ピペリジン塩酸塩、ピペラジン塩酸塩、又はモルフォリン塩酸塩が好ましく、グアニジン塩酸塩、ジイソプロピルアミン塩酸塩又はピペリジン塩酸塩が最も好ましい。
The hydrochloride 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. Specifically, guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine Hydrochloride, tetrabutylammonium chloride (nBu 4 NCl), piperazine hydrochloride, morpholine hydrochloride and the like can be mentioned. Among these hydrochlorides, guanidine hydrochloride, dimethylamine hydrochloride, diisopropylamine hydrochloride, piperidine hydrochloride, piperazine hydrochloride, or morpholine hydrochloride is preferred, and guanidine hydrochloride, diisopropylamine hydrochloride, or piperidine hydrochloride is preferred. Most preferred.
上記の塩酸塩の使用量は、広い範囲から適宜設定することができる。例えば、式（２）で表される化合物のモル量に対して、前記塩酸塩を１～４モル量程度の使用量とすることができ、２～４モル量程度の使用量が好ましい。
The amount of hydrochloride used above can be appropriately set within a wide range. For example, the hydrochloride can be used in an amount of about 1 to 4 mol, preferably about 2 to 4 mol, relative to the molar amount of the compound represented by formula (2).
本発明の第一の製造方法において使用する金属塩化物は、本発明の効果を発揮できるものである限り特に限定されず、具体的に、塩化マグネシウム、塩化亜鉛、塩化リチウム、塩化鉄（III）、塩化カルシウム、塩化ニッケル等を挙げることができる。これらの金属塩化物の中でも、塩化マグネシウムが好ましい。
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 specifically, magnesium chloride, zinc chloride, lithium chloride, iron (III) chloride. , calcium chloride, nickel chloride and the like. Among these metal chlorides, magnesium chloride is preferred.
上記の金属塩化物の使用量は、広い範囲から適宜設定することができる。例えば、式（２）で表される化合物のモル量に対して、前記金属塩化物を２５～４０モル量程度の使用量とすることができ、原料を変性させないことに鑑みて、３０～３５モル量程度の使用量が好ましい。
The amount of metal chloride used can be set appropriately from a wide range. For example, with respect to the molar amount of the compound represented by formula (2), the metal chloride can be used in an amount of about 25 to 40 molar amounts. A molar amount is preferred.
本発明の第一の製造方法において、式（２）のＲ４が、Ｒ５－ＣＨ２－（Ｒ５は、アルキルカルボニルアミノ基）を示す場合、金属塩化物を使用することが好ましい。
In the first production method of the present invention, when R 4 in formula (2) represents R 5 —CH 2 — (R 5 is an alkylcarbonylamino group), it is preferred to use a metal chloride.
本発明の第一の製造方法の反応条件は、本発明の効果を発揮する限りにおいて特に限定されないが、例えば酸性条件下にて実施することが好ましい。具体的には、反応系内に、トリフルオロメタンスルホン酸、メタンスルホン酸、トリフルオロ酢酸、トリフルオロメタンスルホン酸トリメチルシリル、１－ブチル－１－メチルピロリジニウムトリフルオロメタンスルホン酸、酢酸等の酸を含有させることを挙げることができる。
The reaction conditions of the first production method of the present invention are not particularly limited as long as the effects of the present invention are exhibited, but for example, it is preferably carried out under acidic conditions. Specifically, the reaction system contains acids such as trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroacetic acid, trimethylsilyl trifluoromethanesulfonate, 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid, and acetic acid. It is possible to mention that
本発明の第一の製造方法の反応溶媒は、反応を阻害しない限りにおいて特に限定されない。具体的には、トリフルオロ酢酸等を挙げることができる。なお、トリフルオロ酢酸は、酸としても使用することができる。
The reaction solvent in the first production method of the present invention is not particularly limited as long as it does not inhibit the reaction. Specifically, trifluoroacetic acid and the like can be mentioned. Incidentally, trifluoroacetic acid can also be used as an acid.
本発明の第一の製造方法の反応温度は、特に限定されない。例えば、２０～７０℃程度の反応温度とすることができ、３０～５０℃程度が好ましい。
The reaction temperature in the first production method of the present invention is not particularly limited. For example, the reaction temperature can be about 20 to 70°C, preferably about 30 to 50°C.
本発明の第一の製造方法の反応時間は、反応温度により異なり、一概には言えないが、例えば１～５時間程度とすることができる。
The reaction time of the first production method of the present invention varies depending on the reaction temperature, and although it cannot be generalized, it can be, for example, about 1 to 5 hours.
本発明の第一の製造方法で得られる架橋反応物（式（１）の化合物）を単離精製手段に供して、純度の高い目的物を得ることができる。具体的な単離精製手段は、例えばＨＰＬＣ等のカラムクロマトグラフィー、薄層クロマトグラフィー、再結晶、再沈殿、蒸留、溶媒抽出等の公知の手段を適宜組み合わせることができる。
The cross-linking 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 appropriately combined with known means such as column chromatography such as HPLC, thin layer chromatography, recrystallization, reprecipitation, distillation, and solvent extraction.
本発明の第二の製造方法は、下記式（３）
The second manufacturing method of the present invention is the following formula (3)
の製造方法であって、
下記式（４）
A manufacturing method of
Formula (4) below
で表される化合物と、
下記式（５）
A compound represented by
Formula (5) below
で表される化合物とを、塩酸塩又は金属塩化物の存在下で架橋反応させる工程を含む。
and a compound represented by in the presence of hydrochloride or metal chloride.
上記式（３）、（４）及び（５）におけるＲ６、Ｒ７、Ｒ８及びＲ９は、それぞれ一価の有機基である。これらの一価有機基は、本発明の第一の製造方法にて具体的に説明した一価有機基と同様とすることができる。
R 6 , R 7 , R 8 and R 9 in formulas (3), (4) and (5) above are each monovalent organic groups. These monovalent organic groups can be the same as the monovalent organic groups specifically described in the first production method of the present invention.
上記式（５）におけるＲ１０は、本発明の第一の製造方法にて具体的に説明したＲ４と同様に、Ｒ５－ＣＨ２－又はフェニル環上に置換基を有していてもよいベンジル基を示す。
R 10 in the above formula (5) may have a substituent on R 5 —CH 2 — or the phenyl ring, like R 4 specifically explained in the first production method of the present invention. It shows a good benzyl group.
本発明の第二の製造方法における式（４）で表される化合物と、式（５）で表される化合物との使用割合は、特に限定されない。例えば、前者：後者をモル比で、１：０．５～２程度の割合とすることができ、好ましくは１：０．８～１．５程度、更に好ましくは１：０．９～１．１程度の割合とすることができる。
The usage ratio of the compound represented by formula (4) and the compound represented by formula (5) 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 1:0.5-2, preferably about 1:0.8-1.5, more preferably 1:0.9-1. A ratio of about 1 can be used.
本発明の第二の製造方法における塩酸塩は、本発明の第一の製造方法にて具体的に説明した塩酸塩と同様とすることができる。また、本発明の第二の製造方法における金属塩化物も、本発明の第一の製造方法にて具体的に説明した金属塩化物と同様とすることができる。
The hydrochloride in the second production method of the present invention can be the same as the hydrochloride specifically explained in the first production method of the present invention. Also, the metal chloride in the second production method of the present invention can be the same as the metal chloride specifically explained in the first production method of the present invention.
なお、本発明の第二の製造方法において、式（５）のＲ１０が、Ｒ５－ＣＨ２－（Ｒ５は、アルキルカルボニルアミノ基）を示す場合、金属塩化物を使用することが好ましい。
In the second production method of the present invention, when R 10 in formula (5) represents R 5 —CH 2 — (R 5 is an alkylcarbonylamino group), it is preferable to use a metal chloride. .
本発明の第二の製造方法の架橋反応における反応条件も、本発明の第一の製造方法の反応条件と同様に、酸性条件下にて実施することが好ましい。具体的に酸性条件とする手段は、本発明の第一の製造方法にて詳述した通りとすることができる。
The reaction conditions in the cross-linking reaction of the second production method of the present invention are also preferably carried out under acidic conditions, similar to the reaction conditions of the first production method of the present invention. Specifically, the means for providing acidic conditions can be as described in detail in the first production method of the present invention.
本発明の第二の製造方法における反応溶媒は、本発明の第一の製造方法の架橋反応における溶媒と同様とすることができる。より詳細に、Ｒ１０がフェニル環上に置換基を有していてもよいベンジル基であるとき、トリフルオロ酢酸に加えて、メタンスルホン酸を使用することが好ましい。また、Ｒ１０がＲ５－ＣＨ２－（Ｒ５は、アルキルカルボニルアミノ基）であるときは、イオン性液体である１－ブチル－１－メチルピロリジニウムトリフルオロメタンスルホン酸（ＢＭＰｙ・ＯＴｆ）を溶媒として使用することもできる。
The reaction solvent in the second production method of the present invention can be the same as the solvent in the cross-linking reaction in the first production method of the present invention. More specifically, when R 10 is a benzyl group which may have a substituent on the phenyl ring, it is preferred to use methanesulfonic acid in addition to trifluoroacetic acid. When R 10 is R 5 —CH 2 — (R 5 is an alkylcarbonylamino group), the ionic liquid 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid (BMPy·OTf) can also be used as a solvent.
本発明の第二の製造方法では、更にフェノール及び／又はアルコキシベンゼンの存在下で架橋反応させることもできる。このようにフェノール及び／又はアルコキシベンゼンの存在下で架橋反応させることによって、より効率的に反応を進めることができる。
In the second production method of the present invention, the cross-linking reaction can also be carried out in the presence of phenol and/or alkoxybenzene. By carrying out the cross-linking reaction in the presence of phenol and/or alkoxybenzene in this manner, the reaction can proceed more efficiently.
上記アルコキシベンゼンは、本発明の効果を発揮する限りにおいて特に限定されず、例えば、アニソール（メトキシベンゼン）、エトキシベンゼン、プロポキシベンゼン、ブトキシベンゼン等を挙げることができる。これらの上記アルコキシベンゼンの中でも、アニソール、エトキシベンゼンが好ましく、特にアニソールが好ましい。なお、上記アルコキシベンゼンは、単独又は二種以上を併用して使用することができる。
The alkoxybenzene is not particularly limited as long as the effects of the present invention are exhibited, and examples include anisole (methoxybenzene), ethoxybenzene, propoxybenzene, and butoxybenzene. Among these alkoxybenzenes, anisole and ethoxybenzene are preferred, and anisole is particularly preferred. In addition, the said alkoxybenzene can be used individually or in combination of 2 or more types.
上記フェノール及び／又はアルコキシベンゼンの使用量は、本発明の効果を阻害しない限り、広い範囲で使用することができ、例えば、１ミリモルの化合物（３）に対して５～５００ミリモル量とすることができる。
The amount of the phenol and/or alkoxybenzene used can be used in a wide range as long as it does not inhibit the effects of the present invention. can be done.
本発明の第二の製造方法の架橋反応における反応温度は、本発明の第一の製造方法の反応温度と同様とすることができる。また、本発明の第一の製造方法における反応時間も、本発明の第一の製造方法の反応時間と同様とすることができる。なお、Ｒ１０がフェニル環上に置換基を有していてもよいベンジル基であるときの反応温度は、第一の製造方法の反応温度よりも低い０～７０℃の範囲で実施することもできる。
The reaction temperature in the cross-linking reaction of the second production method of the present invention can be the same as the reaction temperature of the first production method of the present invention. Also, the reaction time in the first production method of the present invention can be the same as the reaction time in the first production method of the present invention. In addition, when R 10 is a benzyl group which may have a substituent on the phenyl ring, the reaction temperature may be in the range of 0 to 70°C, which is lower than the reaction temperature of the first production method. can.
本発明の第一の製造方法と同様に、本発明の第二の製造方法で得られる化合物（式（３）の化合物）も単離精製手段に供して、純度の高い目的物を得ることができる。具体的な単離精製手段は、例えばＨＰＬＣ等のカラムクロマトグラフィー、薄層クロマトグラフィー、再結晶、再沈殿、蒸留、溶媒抽出等の公知の手段を適宜組み合わせることができる。
As in the first production method of the present invention, the compound obtained by the second production method of the present invention (compound of formula (3)) is subjected to isolation and purification means to obtain a highly pure target product. can. Specific isolation and purification means can be appropriately combined with known means such as column chromatography such as HPLC, thin layer chromatography, recrystallization, reprecipitation, distillation, and solvent extraction.
以下に、本発明をより詳細に説明するための実施例を示す。本発明が下記に示す実施例に限定されないのは言うまでもない。
Examples for explaining the present invention in more detail are shown below. It goes without saying that the present invention is not limited to the examples shown below.
後記する実施例におけるＭＳ分析は、特に断りのない限り、Ｗａｔｅｒｓ ＭＩＣＲＯＭＡＳＳ（登録商標）ＬＣＴ ＰＲＥＭＩＥＲＴＭ（ＥＳＩ－ＴＯＦ）又はＬＣ－ＭＳ（Ｓｈｉｍａｄｚｕ、Ｊａｐａｎ、Ｐｒｏｍｉｎｅｎｃｅ－Ｉ ＬＣ－２０３０、ＬＣＭＳ－２０２０）により実施した。
Unless otherwise specified, MS analysis in the examples described later was performed by Waters MICROMASS (registered trademark) LCT PREMIERTM (ESI-TOF) or LC-MS (Shimadzu, Japan, Prominence-I LC-2030, LCMS-2020). did.
後記する実施例におけるＨＰＬＣ分析は、特に断りのない限り、ＨＩＴＡＣＨＩ Ｌ－７１５０にＬ－２４００検出器又はＷａｔｅｒｓ Ａｌｌｉａｎｃｅ ２６９５ Ｓｅｐａｒａｔｉｏｎｓ Ｍｏｄｕｌｅ ｗｉｔｈ ＥＬＳ ２４２０ Ｓｙｓｔｅｍで、Ｃｏｓｍｏｓｉｌ ５Ｃ１８－ＡＲ－ＩＩ分析カラム（Ｎａｃａｌａｉ Ｔｅｓｑｕｅ、４．６×２５０ｍｍ、流量１．０ｍＬ・ｍｉｎ－１）及びＣｏｓｍｏｓｉｌ ５Ｃ１８－ＡＲ－ＩＩセミ分取カラム（Ｎａｃａｌａｉ Ｔｅｓｑｕｅ、１０×２５０ｍｍ、流量３．０ｍＬ・ｍｉｎ－１）を使用して実施した。０ｍＬ・ｍｉｎ－１、及びＣｏｓｍｏｓｉｌ ５Ｃ１８－ＡＲ－ＩＩ分取カラム（Ｎａｃａｌａｉ Ｔｅｓｑｕｅ、２０×２５０ｍｍ、流量１０ｍＬ・ｍｉｎ－１）で溶出し、直線勾配系（溶媒Ａ：０．１％ＴＦＡを含有する水、溶媒Ｂ：０．１％ＴＦＡを含有するＭｅＣＮ）で溶出し、２２０ｎｍでＵＶ検出を行った。
Unless otherwise specified, HPLC analyzes in the examples below were performed using a HITACHI L-7150 L-2400 detector or a Waters Alliance 2695 Separations Module with ELS 2420 System using a Cosmosil 5C18-AR-II analytical column (Nacalai Tesque, 4 .6×250 mm, flow rate 1.0 mL·min −1 ) and Cosmosil 5C18-AR-II semi-preparative columns (Nacalai Tesque, 10×250 mm, flow rate 3.0 mL·min −1 ). 0 mL min −1 and eluted with a Cosmosil 5C18-AR-II preparative column (Nacalai Tesque, 20×250 mm, flow rate 10 mL min −1 ), linear gradient system (solvent A: containing 0.1% TFA Water, solvent B: MeCN containing 0.1% TFA) with UV detection at 220 nm.
後記する実施例におけるＮＭＲ測定は、特に言及しない限り、１Ｈは、Ｂｒｕｋｅｒ ＡＶ４００Ｎ（４００ＭＨｚ）又はＢｒｕｋｅｒ ＡＶ５００（５００ＭＨｚ）を使用し、１３Ｃは、Ｂｒｕｋｅｒ ＡＶ４００Ｎ（１００ＭＨｚ）又はＢｒｕｋｅｒ ＡＶ５００（１２５ＭＨｚ）を使用した。
Unless otherwise specified, NMR measurement in the examples described later uses Bruker AV400N (400 MHz) or Bruker AV500 (500 MHz) for 1 H, and Bruker AV400N (100 MHz) or Bruker AV500 (125 MHz) for 13 C. did.
後記する実施例におけるＣＤスペクトルは、特に言及しない限り、ＪＡＳＣＯ Ｊ－１５００ ＣＤスペクトロメーターを用い、常法にて測定した。
The CD spectra in the examples described later were measured by a conventional method using a JASCO J-1500 CD spectrometer unless otherwise specified.
後記する実施例におけるペプチド等は、特に断りのない限りＮｏｖａｓｙｎ（登録商標）ＴＧＲ樹脂（０．２５ｍｍｏｌｇ－１）又はＦｍｏｃ－Ｒｉｎｋ Ａｍｉｄｅ－Ｐｈｅ樹脂（０．２５ｍｍｏｌｇ－１）上でのＦｍｏｃ固相ペプチド合成（Ｆｍｏｃ－ＳＰＰＳ）によって合成した。Ｆｍｏｃ ＳＰＰＳは以下のプロトコルに従って実施した。
Peptides and the like in the examples described later are Fmoc solid-phase peptides on Novasyn (registered trademark) TGR resin (0.25 mmolg −1 ) or Fmoc-Rink Amide-Phe resin (0.25 mmolg −1 ) unless otherwise specified. Synthesized by Synthesis (Fmoc-SPPS). Fmoc SPPS was performed according to the following protocol.
１）Ｆｍｏｃ基の除去を、２０％ｐｉｐｅｒｉｄｉｎｅ／ＤＭＦを用いて室温で１０分間行った。
２）樹脂をＤＭＦで５回洗浄した。
３）標準Ｆｍｏｃ保護アミノ酸（４．０当量）を、Ｎ，Ｎ－ｄｉｉｓｏｐｒｏｐｙｌｃａｒｂｏｄｉｉｍｉｄｅ（ＤＩＰＣＩ）（４．０当量）と１－ｈｙｄｒｏｘｙｂｅｎｚｏｔｒｉａｚｏｌｅ ｍｏｎｏｈｙｄｒａｔｅ（ＨＯＢｔ－Ｈ２Ｏ）（４．０当量）とをＤＭＦ中で１．５時間、室温でカップリングし、カイザーニンヒドリンテストにより反応完了を確認した。カイザーテストが陰性になるまでカップリング反応を繰り返した。
４）樹脂をＤＭＦで三回洗浄した。 1) Removal of the Fmoc group was performed with 20% piperidine/DMF for 10 minutes at room temperature.
2) The resin was washed 5 times with DMF.
3) standard Fmoc-protected amino acids (4.0 eq.), N,N-diisopropylcarbodiimide (DIPCI) (4.0 eq.) and 1-hydroxybenzotriazole monohydrate (HOBt- H2O ) (4.0 eq.) in DMF for 1.5 hours at room temperature and the Kaiser ninhydrin test confirmed reaction completion. The coupling reaction was repeated until the Kaiser test was negative.
4) The resin was washed with DMF three times.
２）樹脂をＤＭＦで５回洗浄した。
３）標準Ｆｍｏｃ保護アミノ酸（４．０当量）を、Ｎ，Ｎ－ｄｉｉｓｏｐｒｏｐｙｌｃａｒｂｏｄｉｉｍｉｄｅ（ＤＩＰＣＩ）（４．０当量）と１－ｈｙｄｒｏｘｙｂｅｎｚｏｔｒｉａｚｏｌｅ ｍｏｎｏｈｙｄｒａｔｅ（ＨＯＢｔ－Ｈ２Ｏ）（４．０当量）とをＤＭＦ中で１．５時間、室温でカップリングし、カイザーニンヒドリンテストにより反応完了を確認した。カイザーテストが陰性になるまでカップリング反応を繰り返した。
４）樹脂をＤＭＦで三回洗浄した。 1) Removal of the Fmoc group was performed with 20% piperidine/DMF for 10 minutes at room temperature.
2) The resin was washed 5 times with DMF.
3) standard Fmoc-protected amino acids (4.0 eq.), N,N-diisopropylcarbodiimide (DIPCI) (4.0 eq.) and 1-hydroxybenzotriazole monohydrate (HOBt- H2O ) (4.0 eq.) in DMF for 1.5 hours at room temperature and the Kaiser ninhydrin test confirmed reaction completion. The coupling reaction was repeated until the Kaiser test was negative.
4) The resin was washed with DMF three times.
上記１）～４）を１サイクル繰り返した。ＴＦＡ－ａｎｉｓｏｌｅ－Ｈ２Ｏ（９０：５：５）のカクテルを用いて、樹脂からのペプチドの遊離を伴う酸－標識保護基の脱保護を行った。樹脂を濾過した後、濾液に冷却したジエチルエーテル（Ｅｔ２Ｏ）を加え、遠心分離して析出物を回収した。得られた沈殿物を０．１％のＴＦＡを含有するＨ２Ｏ－ＭｅＣＮに溶解させた。Ｔｒｐ（Ｂｏｃ）誘導体を用いて合成したペプチドについて、３７℃で１時間インキュベーションを続け、得られた溶液を分取ＨＰＬＣで精製した。
The above 1) to 4) were repeated for one cycle. A cocktail of TFA-anisole-H 2 O (90:5:5) was used to deprotect the acid-labeled protecting groups with release of the peptide from the resin. After filtering the resin, chilled diethyl ether (Et 2 O) was added to the filtrate and centrifuged to collect the precipitate. The resulting precipitate was dissolved in H 2 O-MeCN containing 0.1% TFA. For peptides synthesized with Trp(Boc) derivatives, incubation was continued at 37° C. for 1 hour and the resulting solution was purified by preparative HPLC.
側鎖保護アミノ酸は、特に断りがない限り、以下のものを採用した。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）。なお、Ｃｙｓ（Ａｃｍ）（Ｏ）含有ペプチドは通常のＦｍｏｃ固相合成法でＣｙｓ（Ａｃｍ）含有ペプチドを得た後、これを酸化反応に供することで得た。
The following side chain protected amino acids were used unless otherwise noted. 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 subjecting the Cys(Acm)-containing peptide to an oxidation reaction after obtaining the Cys(Acm)-containing peptide by a conventional Fmoc solid-phase synthesis method.
実施例１
下記の反応スキームにて、下記化合物１を原料に種々の条件で反応を行って下記化合物２を得た。具体的な条件を表１に示す。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応では、溶媒として、酸として働くＴＦＡを使用した。なお、化合物１は、上記ペプチド合成法によって作製した。 Example 1
In the following reaction scheme, the followingcompound 2 was obtained by reacting the following compound 1 as a raw material under various conditions. Table 1 shows specific conditions. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. In this reaction, the solvent used was TFA, which acted as an acid. Compound 1 was produced by the peptide synthesis method described above.
下記の反応スキームにて、下記化合物１を原料に種々の条件で反応を行って下記化合物２を得た。具体的な条件を表１に示す。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応では、溶媒として、酸として働くＴＦＡを使用した。なお、化合物１は、上記ペプチド合成法によって作製した。 Example 1
In the following reaction scheme, the following
作製した化合物１のＭＳデータは、以下の通りである。
＜化合物１＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C41H60N11O9S 882.4, found 882.5. The MS data of the producedcompound 1 are as follows.
<Compound 1>
LRMS (ESI-TOF ) m/z: [M+H] + calcd for C41H60N11O9S 882.4 , found 882.5.
＜化合物１＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C41H60N11O9S 882.4, found 882.5. The MS data of the produced
<
LRMS (ESI-TOF ) m/z: [M+H] + calcd for C41H60N11O9S 882.4 , found 882.5.
上記スキームにおいて、Ａｃ－はアセチル基を示す。ＧＡＬは、Ｎ末端側から順に、グリシン－アラニン－ロイシンからなるペプチドの残基を示す。ここで、Ｇ（グリシン）に隣接するカルボニル基と当該グリシンのアミノ基とがペプチド結合しており、Ｌ（ロイシン）に隣接するアミノ基とロイシンのカルボニル基とがペプチド結合している。Ｒは、アルギニン残基を示す。
In the above scheme, Ac- represents an acetyl group. GAL indicates residues of a peptide consisting of glycine-alanine-leucine in order from the N-terminal side. Here, the carbonyl group adjacent to G (glycine) and the amino group of the glycine are peptide-bonded, and the amino group adjacent to L (leucine) and the carbonyl group of leucine are peptide-bonded. R indicates an arginine residue.
上記表１において、ＴＦＭＳＡは、トリフルオロメタンスルホン酸、ＭＳＡは、メタンスルホン酸、ｎＢｕ４ＮＣｌは、塩化テトラブチルアンモニウムである。
In Table 1 above, TFMSA is trifluoromethanesulfonic acid, MSA is methanesulfonic acid, and nBu 4 NCl is tetrabutylammonium chloride.
この反応にて得られた反応物のＨＰＬＣチャートを図１に示す。また、化合物２のＭＳデータ及びＮＭＲデータは、以下の通りである。
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C33H50N11O7S 744.4, found 744.0.1H-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) FIG. 1 shows the HPLC chart of the reaction product obtained in this reaction. Moreover, MS data and NMR data of thecompound 2 are as follows.
LRMS (ESI-TOF ) m / z: [ M+H] + calcd for C33H50N11O7S 744.4, found 744.0.1H-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 = 7.3Hz, 3H), 0.91-0.81 (m, 6H)
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C33H50N11O7S 744.4, found 744.0.1H-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) FIG. 1 shows the HPLC chart of the reaction product obtained in this reaction. Moreover, MS data and NMR data of the
LRMS (ESI-TOF ) m / z: [ M+H] + calcd for C33H50N11O7S 744.4, found 744.0.1H-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 = 7.3Hz, 3H), 0.91-0.81 (m, 6H)
上記の結果から、メトキシベンジル基によって保護されたシステイン残基を有する化合物１は、種々の塩酸塩の存在する酸性条件下で、当該システイン残基とトリプトファン残基との間で分子内架橋し、化合物２が生成されることが明らかとなった。
From the above results, Compound 1, which has a cysteine residue protected by a methoxybenzyl group, under acidic conditions in the presence of various hydrochlorides, intramolecularly crosslinks between the cysteine residue and the tryptophan residue, It was found that compound 2 was produced.
その一方で、塩酸塩の代わりに硫酸塩を使用したサンプル７では、架橋を確認することができなかった。
On the other hand, cross-linking could not be confirmed in sample 7, which used sulfate instead of hydrochloride.
実施例２
下記の反応スキームにて、化合物３及び化合物４を原料に化合物５を、下記表２に示す種々の条件で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応では、溶媒としてＴＦＡを使用した。なお、化合物３は、塩化チオニルの存在下にて常法により、Ｌ－トリプトファンのカルボニル基をメトキシ基に変換した。なお、化合物４は、上記ペプチド合成法によって作製した。 Example 2
In the reaction scheme below,compound 3 and compound 4 were reacted with compound 5 under various conditions shown in Table 2 below. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction. For compound 3, the carbonyl group of L-tryptophan was converted to a methoxy group by a conventional method in the presence of thionyl chloride. Compound 4 was produced by the peptide synthesis method described above.
下記の反応スキームにて、化合物３及び化合物４を原料に化合物５を、下記表２に示す種々の条件で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応では、溶媒としてＴＦＡを使用した。なお、化合物３は、塩化チオニルの存在下にて常法により、Ｌ－トリプトファンのカルボニル基をメトキシ基に変換した。なお、化合物４は、上記ペプチド合成法によって作製した。 Example 2
In the reaction scheme below,
作製した化合物３及び４のＭＳデータは、以下の通りである。
＜化合物３＞
HRMS (ESI-TOF) m/z: [M+H]+ calcd for C14H16KN2O3 299.0798, found 299.0802
＜化合物４＞
HRMS (ESI-TOF) m/z: [M+Na]+calcd for C19H29N3O5NaS 434.1726, found 434.1724. The MS data of Compounds 3 and 4 prepared are as follows.
<Compound 3>
HRMS (ESI - TOF) m /z: [M+H]+ calcd for C14H16KN2O3 299.0798 , found 299.0802
<Compound 4>
HRMS (ESI -TOF) m/z: [M+Na] + calcd for C19H29N3O5NaS 434.1726 , found 434.1724 .
＜化合物３＞
HRMS (ESI-TOF) m/z: [M+H]+ calcd for C14H16KN2O3 299.0798, found 299.0802
＜化合物４＞
HRMS (ESI-TOF) m/z: [M+Na]+calcd for C19H29N3O5NaS 434.1726, found 434.1724. The MS data of
<
HRMS (ESI - TOF) m /z: [M+H]+ calcd for C14H16KN2O3 299.0798 , found 299.0802
<
HRMS (ESI -TOF) m/z: [M+Na] + calcd for C19H29N3O5NaS 434.1726 , found 434.1724 .
上記、スキームにおいて、Ａｃ－はアセチル基を示す。
In the above scheme, Ac- represents an acetyl group.
この反応にて得られた反応物のＨＰＬＣチャートを図２に示す。また、得られた化合物５のＭＳデータは、以下の通りである。
＜化合物５＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C25H37N5O6S 534.2, found 533.9. FIG. 2 shows the HPLC chart of the reaction product obtained in this reaction. Moreover, the MS data of the obtainedcompound 5 are as follows.
<Compound 5>
LRMS ( ESI -TOF) m/z: [M+H] + calcd for C25H37N5O6S 534.2 , found 533.9.
＜化合物５＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C25H37N5O6S 534.2, found 533.9. FIG. 2 shows the HPLC chart of the reaction product obtained in this reaction. Moreover, the MS data of the obtained
<
LRMS ( ESI -TOF) m/z: [M+H] + calcd for C25H37N5O6S 534.2 , found 533.9.
上記の結果から、メトキシベンジル基によって保護されたシステイン残基を有する化合物３とトリプトファン残基を有する化合物４とが、種々の塩酸塩が存在する酸性条件下で、当該システイン残基とトリプトファン残基との分子間にて架橋を生じさせ、化合物５が生成されることが明らかとなった。
From the above results, compound 3, which has a cysteine residue protected by a methoxybenzyl group, and compound 4, which has a tryptophan residue, under acidic conditions in the presence of various hydrochlorides, the cysteine residue and the tryptophan residue It was clarified that the compound 5 was produced by cross-linking between the molecules.
実施例３
下記の反応スキームにて、下記化合物６～１０を原料に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２０℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。なお、化合物１０を原料として、２０℃ではなく３７℃での反応も同様に行った。化合物６～１０は、上記ペプチド合成法によって作製した。 Example 3
In the reaction scheme below, the followingcompounds 6 to 10 were reacted under the conditions of 1M MSA and 4M guanidine hydrochloride as starting materials. The reaction temperature in this reaction was 20° C., and the reaction time was 3 hours. TFA was used as solvent in this reaction. In addition, the reaction was similarly performed at 37° C. instead of 20° C. using compound 10 as a starting material. Compounds 6-10 were made by the peptide synthesis method described above.
下記の反応スキームにて、下記化合物６～１０を原料に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２０℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。なお、化合物１０を原料として、２０℃ではなく３７℃での反応も同様に行った。化合物６～１０は、上記ペプチド合成法によって作製した。 Example 3
In the reaction scheme below, the following
作製した化合物６～１０のＭＳデータは、以下の通りである。
＜化合物６＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C66H102N22O15S 737.4, found 737.1.
＜化合物７＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C66H107N21O15S 732.9, found 732.7.
＜化合物８＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C69H104N20O15S 742.4, found 742.2.
＜化合物９＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C69H104N20O16S 750.4, found 750.2.
＜化合物１０＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C57H94N20O13S2 734.4, found 734.1. The MS data of theprepared compounds 6 to 10 are as follows.
<Compound 6>
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C66H102N22O15S 737.4 , found 737.1.
<Compound 7>
LRMS (ESI-TOF) m/z: [ M+2H] + calcd for C66H107N21O15S 732.9 , found 732.7.
<Compound 8>
LRMS (ESI - TOF) m/z: [M+2H] + calcd for C69H104N20O15S 742.4 , found 742.2.
<Compound 9>
LRMS (ESI-TOF) m/z: [M+2H] + calcd for C69H104N20O16S 750.4 , found 750.2.
<Compound 10>
LRMS ( ESI -TOF) m/z: [M+2H] + calcd for C57H94N20O13S2 734.4 , found 734.1 .
＜化合物６＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C66H102N22O15S 737.4, found 737.1.
＜化合物７＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C66H107N21O15S 732.9, found 732.7.
＜化合物８＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C69H104N20O15S 742.4, found 742.2.
＜化合物９＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C69H104N20O16S 750.4, found 750.2.
＜化合物１０＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C57H94N20O13S2 734.4, found 734.1. The MS data of the
<
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C66H102N22O15S 737.4 , found 737.1.
<
LRMS (ESI-TOF) m/z: [ M+2H] + calcd for C66H107N21O15S 732.9 , found 732.7.
<
LRMS (ESI - TOF) m/z: [M+2H] + calcd for C69H104N20O15S 742.4 , found 742.2.
<
LRMS (ESI-TOF) m/z: [M+2H] + calcd for C69H104N20O16S 750.4 , found 750.2.
<
LRMS ( ESI -TOF) m/z: [M+2H] + calcd for C57H94N20O13S2 734.4 , found 734.1 .
上記各スキームにおいて、Ａｃ－はアセチル基を示し、ＧＡＬは、Ｎ末端側から順にグリシン－アラニン－ロイシンからなるペプチド残基を示し、ＧＨＲＡＬは、Ｎ末端側から順にグリシン－ヒスチジン－アルギニン－アラニン－ロイシンからなるペプチド残基を示し、ＧＫＲＡＬは、Ｎ末端側から順にグリシン－リジン－アルギニン－アラニン－ロイシンからなるペプチド残基を示し、ＧＦＲＡＬは、Ｎ末端側から順にグリシン－フェニルアラニン－アルギニン－アラニン－ロイシンからなるペプチド残基を示し、ＧＹＲＡＬは、Ｎ末端側から順にグリシン－チロシン－アルギニン－アラニン－ロイシンからなるペプチド残基を示し、ＧＭＲＡＬは、Ｎ末端側から順にグリシン－メチオニン－アルギニン－アラニン－ロイシンからなるペプチド残基を示す。ここで、上記のＮ末端のＧ（グリシン）残基に隣接するカルボニル基と当該グリシン残基の一級アミノ基とがペプチド結合しており、Ｃ末端側のＬ（ロイシン）残基に隣接するアミノ基と当該ロイシン残基のカルボニル基とがペプチド結合している。また、ＲＧは、Ｎ末端から順にアルギニン－グリシンからなるペプチドの残基を示す。ここで、Ｒ（アルギニン）残基に隣接するカルボニル基とアルギニンのアミノ基とがペプチド結合しており、Ｇ（グリシン）残基は、Ｃ末端アミド構造を有する。
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-terminal side, and GHRAL represents glycine-histidine-arginine-alanine- in order from the N-terminal side. Indicates a peptide residue consisting of leucine, GKRAL indicates a peptide residue consisting of glycine-lysine-arginine-alanine-leucine in order from the N-terminal side, GFRAL indicates glycine-phenylalanine-arginine-alanine- in order from the N-terminal side Indicates a peptide residue consisting of leucine, GYRAL indicates a peptide residue consisting of glycine-tyrosine-arginine-alanine-leucine in order from the N-terminal side, GMRAL indicates glycine-methionine-arginine-alanine- in order from the N-terminal side Peptide residues consisting of leucine are indicated. Here, the carbonyl group adjacent to the N-terminal G (glycine) residue and the primary amino group of the glycine residue are peptide-bonded, and the amino acid adjacent to the C-terminal L (leucine) residue A peptide bond is formed between the group and the carbonyl group of the leucine residue. RG indicates residues of a peptide consisting of arginine-glycine in order from the N-terminus. Here, the carbonyl group adjacent to the R (arginine) residue and the amino group of arginine are peptide-bonded, and the G (glycine) residue has a C-terminal amide structure.
上記反応にて得られた反応物のＨＰＬＣチャートを図３に示す。また、得られた化合物１１～１５のＭＳデータは、以下の通りである。
＜化合物１１＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C58H92N22O13S 668.4, found 668.2.
＜化合物１２＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C58H97N21O13S 663.9, found 663.7.
＜化合物１３＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C61H94N20O13S 673.4, found 673.2.
＜化合物１４＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C61H94N20O14S 681.4, found 681.1.
＜化合物１５＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C57H94N20O13S2 665.3, found 665.1. FIG. 3 shows the HPLC chart of the reaction product obtained in the above reaction. The MS data ofCompounds 11 to 15 obtained are as follows.
<Compound 11>
LRMS ( ESI-TOF) m/z: [M+2H] + calcd for C58H92N22O13S 668.4 , found 668.2 .
<Compound 12>
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C58H97N21O13S 663.9 , found 663.7 .
<Compound 13>
LRMS (ESI-TOF) m/z: [ M+2H] + calcd for C61H94N20O13S 673.4 , found 673.2 .
<Compound 14>
LRMS (ESI-TOF ) m/z: [ M+2H] + calcd for C61H94N20O14S 681.4 , found 681.1.
<Compound 15>
LRMS ( ESI-TOF) m/z: [ M+2H] + calcd for C57H94N20O13S2 665.3 , found 665.1.
＜化合物１１＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C58H92N22O13S 668.4, found 668.2.
＜化合物１２＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C58H97N21O13S 663.9, found 663.7.
＜化合物１３＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C61H94N20O13S 673.4, found 673.2.
＜化合物１４＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C61H94N20O14S 681.4, found 681.1.
＜化合物１５＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C57H94N20O13S2 665.3, found 665.1. FIG. 3 shows the HPLC chart of the reaction product obtained in the above reaction. The MS data of
<
LRMS ( ESI-TOF) m/z: [M+2H] + calcd for C58H92N22O13S 668.4 , found 668.2 .
<
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C58H97N21O13S 663.9 , found 663.7 .
<
LRMS (ESI-TOF) m/z: [ M+2H] + calcd for C61H94N20O13S 673.4 , found 673.2 .
<
LRMS (ESI-TOF ) m/z: [ M+2H] + calcd for C61H94N20O14S 681.4 , found 681.1.
<
LRMS ( ESI-TOF) m/z: [ M+2H] + calcd for C57H94N20O13S2 665.3 , found 665.1.
架橋反応の温度は、２０℃よりも３７℃のほうが、より効率よく架橋反応を生じさせることができることも明らかとなった。
It was also found that the cross-linking reaction temperature was 37°C more efficiently than 20°C.
実施例４
下記の反応スキームにて、下記化合物１６及び１７に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２０℃とし、反応時間を３時間とした。この反応における溶媒としてTFAを使用した。これらの化合物１６及び１７は、上記ペプチド合成法によって作製した。 Example 4
In the reaction scheme below, compounds 16 and 17 below were 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 solvent in this reaction. These compounds 16 and 17 were prepared by the peptide synthesis method described above.
下記の反応スキームにて、下記化合物１６及び１７に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２０℃とし、反応時間を３時間とした。この反応における溶媒としてTFAを使用した。これらの化合物１６及び１７は、上記ペプチド合成法によって作製した。 Example 4
In the reaction scheme below, compounds 16 and 17 below were 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 solvent in this reaction. These
作製した化合物１６及び１７のＭＳデータは、以下の通りである。
＜化合物１６＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C35H49N10O8S 769.4, found 769.5.
＜化合物１７＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C50H78N16O11S 555.3, found 555.3. The MS data of Compounds 16 and 17 prepared are as follows.
<Compound 16>
LRMS (ESI-TOF) m/z: [M+H] + calcd for C35H49N10O8S 769.4 , found 769.5 .
<Compound 17>
LRMS (ESI-TOF) m /z: [ M+2H] + calcd for C50H78N16O11S 555.3 , found 555.3.
＜化合物１６＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C35H49N10O8S 769.4, found 769.5.
＜化合物１７＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C50H78N16O11S 555.3, found 555.3. The MS data of
<
LRMS (ESI-TOF) m/z: [M+H] + calcd for C35H49N10O8S 769.4 , found 769.5 .
<
LRMS (ESI-TOF) m /z: [ M+2H] + calcd for C50H78N16O11S 555.3 , found 555.3.
上記各スキームにおいて、Ａｃ－はアセチル基を示し、ＧＡは、Ｎ末端から順にグリシン－アラニンからなるペプチド残基を示し、ＧＡＬＲＡは、Ｎ末端から順にグリシン－アラニン－ロイシン－アルギニン－アラニンからなるペプチド残基を示す。ここで、上記のＮ末端側のＧ（グリシン）残基に隣接するカルボニル基と当該グリシン残基のアミノ基とがペプチド結合しており、Ｃ末端側のＡ（アラニン）残基に隣接するアミノ基とアラニン残基のカルボニル基とがペプチド結合している。また、Ｒは、アルギニン残基を示す。
In each scheme above, Ac- represents an acetyl group, GA represents a peptide residue consisting of glycine-alanine in order from the N-terminus, and GALRA is a peptide consisting of glycine-alanine-leucine-arginine-alanine in order from the N-terminus. Residues are indicated. Here, the carbonyl group adjacent to the G (glycine) residue on the N-terminal side and the amino group of the glycine residue are peptide-bonded, and the amino group adjacent to the A (alanine) residue on the C-terminal side A peptide bond is formed between the group and the carbonyl group of the alanine residue. Also, R represents an arginine residue.
上記反応にて得られた反応物のＨＰＬＣチャートを図４に示す。また、得られた化合物１８及び１９のＭＳデータは、以下の通りである。
＜化合物１８＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C27H39N10O6S 631.3, found 631.3.
＜化合物１９＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C42H68N16O9S 486.3, found 486.3. FIG. 4 shows the HPLC chart of the reaction product obtained in the above reaction. Moreover, the MS data of the obtained compounds 18 and 19 are as follows.
<Compound 18>
LRMS ( ESI -TOF) m/z: [ M +H] + calcd for C27H39N10O6S 631.3, found 631.3 .
<Compound 19>
LRMS (ESI-TOF ) m /z: [ M+2H] + calcd for C42H68N16O9S 486.3, found 486.3.
＜化合物１８＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C27H39N10O6S 631.3, found 631.3.
＜化合物１９＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C42H68N16O9S 486.3, found 486.3. FIG. 4 shows the HPLC chart of the reaction product obtained in the above reaction. Moreover, the MS data of the obtained compounds 18 and 19 are as follows.
<
LRMS ( ESI -TOF) m/z: [ M +H] + calcd for C27H39N10O6S 631.3, found 631.3 .
<
LRMS (ESI-TOF ) m /z: [ M+2H] + calcd for C42H68N16O9S 486.3, found 486.3.
上記の結果から、ペプチドに含有されるメトキシベンジル基によって保護されたシステイン残基とトリプトファン残基との間に２～５個のアミノ酸残基が存在しても、当該ペプチドにて分子内架橋を生じることが明らかとなった。
From the above results, even if there are 2 to 5 amino acid residues between the cysteine residue protected by the methoxybenzyl group and the tryptophan residue contained in the peptide, intramolecular cross-linking can occur in the peptide. was revealed to occur.
実施例５
下記の反応スキームにて、下記化合物２０を原料にして、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２５℃とし、反応時間を３０分とした。この反応における溶媒としてＴＦＡを使用した。この化合物１９は上記ペプチド合成法によって作製した。 Example 5
In the reaction scheme below, the followingcompound 20 was used as a starting material and reacted under the conditions of 1M MSA and 4M guanidine hydrochloride. The reaction temperature in this reaction was 25° C. and the reaction time was 30 minutes. TFA was used as solvent in this reaction. This compound 19 was prepared by the peptide synthesis method described above.
下記の反応スキームにて、下記化合物２０を原料にして、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を２５℃とし、反応時間を３０分とした。この反応における溶媒としてＴＦＡを使用した。この化合物１９は上記ペプチド合成法によって作製した。 Example 5
In the reaction scheme below, the following
作製した化合物２０のＭＳデータは、以下の通りである。
＜化合物２０＞
LRMS (ESI-TOF) m/z: [M+2H]2+ calcd for C64H101N21O16S 725.9, found 725.8. The MS data of the producedcompound 20 are as follows.
<Compound 20>
LRMS (ESI-TOF) m /z: [M+2H] 2+ calcd for C64H101N21O16S 725.9 , found 725.8.
＜化合物２０＞
LRMS (ESI-TOF) m/z: [M+2H]2+ calcd for C64H101N21O16S 725.9, found 725.8. The MS data of the produced
<
LRMS (ESI-TOF) m /z: [M+2H] 2+ calcd for C64H101N21O16S 725.9 , found 725.8.
上記スキームにおいて、Ａｃ－はアセチル基を示し、ＧＹＲＡＬは、Ｎ末端から順にグリシン－チロシン－アルギニン－アラニン－ロイシンからなるペプチド残基を示し、ＧＡＬは、Ｎ末端から順にグリシン－アラニン－ロイシンからなるペプチド残基を示す。ここで、上記のＧ（グリシン）残基に隣接するカルボニル基と当該グリシン残基のアミノ基とがペプチド結合しており、Ｌ（ロイシン）残基に隣接する一級アミノ基と当該ロイシン残基のカルボキニル基とがペプチド結合している。また、ＲＧは、Ｎ末端から順にグリシン－アルギニンからなるペプチド残基を示す。ここで、Ｒ（アルギニン）残基に隣接するカルボニル基と当該アルギニン残基のアミノ基とがペプチド結合しており、Ｇ（グリシン）残基はＣ末端アミド構造を有する。
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 consists of glycine-alanine-leucine in order from the N-terminus. Peptide residues are indicated. Here, the carbonyl group adjacent to the G (glycine) residue and the amino group of the glycine residue are peptide-bonded, and the primary amino group adjacent to the L (leucine) residue and the leucine residue A peptide bond is formed with the carboxynyl group. 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 and the amino group of the arginine residue are peptide-bonded, and the G (glycine) residue has a C-terminal amide structure.
上記反応にて得られた反応物のＨＰＬＣチャートを図５に示す。
The HPLC chart of the reaction product obtained in the above reaction is shown in FIG.
上記の結果から、ペプチドに含有されるメチルカルボニルアミノ基によって保護されたシステイン残基及びトリプトファン残基を有する化合物２０は、塩酸塩（グアニジン塩酸塩）が存在する酸性条件下で、当該システイン残基とトリプトファン残基との分子間にて架橋を生じさせることが明らかとなった。
From the above results, compound 20, which has a cysteine residue and a tryptophan residue protected by a methylcarbonylamino group contained in the peptide, under acidic conditions in the presence of hydrochloride (guanidine hydrochloride), the cysteine residue and tryptophan residues.
実施例６
下記のスキームに示すように、上記ペプチド合成法によって作製した化合物２１をヘキサフルオロリン酸トリピロリジノホスホニウム（例えば、ＰｙＢＯＰ）及びＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＥＡ）を含有するＮ－メチルピロリドン（ＮＭＰ）中で室温、３時間反応させることによって、化合物２２の環状ペプチドとした。 Example 6
As shown in the scheme below,compound 21 prepared by the peptide synthesis method described above was converted to N-methylpyrrolidone (NMP) containing tripyrrolidinophosphonium hexafluorophosphate (e.g., PyBOP) and N,N-diisopropylethylamine (DIEA). ) at room temperature for 3 hours to obtain a cyclic peptide of compound 22.
下記のスキームに示すように、上記ペプチド合成法によって作製した化合物２１をヘキサフルオロリン酸トリピロリジノホスホニウム（例えば、ＰｙＢＯＰ）及びＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＥＡ）を含有するＮ－メチルピロリドン（ＮＭＰ）中で室温、３時間反応させることによって、化合物２２の環状ペプチドとした。 Example 6
As shown in the scheme below,
この化合物２２を原料にして、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。
Using this compound 22 as a raw material, it was reacted under the conditions of 1M MSA and 4M guanidine hydrochloride. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction.
作製した化合物２１のＭＳデータは、以下の通りである。
＜化合物２１＞
LRMS (ESI-TOF) m/z: [M+H]+calcd for C47H67N10O12S 995.5, found 995.0. The MS data of the producedcompound 21 are as follows.
<Compound 21>
LRMS (ESI-TOF) m/z: [M+H] + calcd for C47H67N10O12S 995.5 , found 995.0.
＜化合物２１＞
LRMS (ESI-TOF) m/z: [M+H]+calcd for C47H67N10O12S 995.5, found 995.0. The MS data of the produced
<
LRMS (ESI-TOF) m/z: [M+H] + calcd for C47H67N10O12S 995.5 , found 995.0.
上記スキームにおいて、ＮＰＩは、Ｎ末端から順にアスパラギン－プロリン－イソロイシンからなるペプチド残基を示す。ここで、上記のＮ（アスパラギン）残基に隣接するカルボニル基と当該アスパラギン残基のアミノ基とがペプチド結合しており、Ｉ（イソロイシン）残基に隣接する一級アミノ基と当該イソロイシン残基のカルボニル基とがペプチド結合している。また、ＧＩは、Ｎ末端から順にグリシン－イソロイシンからなるペプチド残基を示す。ここで、Ｇ（グリシン）残基に隣接するカルボニル基と当該グリシン残基の一級アミノ基とがペプチド結合しており、Ｉ（イソロイシン）残基に隣接する一級アミノ基と当該イソロイシン残基のカルボニル基とがペプチド結合している。
In the above scheme, NPI indicates a peptide residue consisting of asparagine-proline-isoleucine in order from the N-terminus. Here, the carbonyl group adjacent to the N (asparagine) residue and the amino group of the asparagine residue are peptide-bonded, and the primary amino group adjacent to the I (isoleucine) residue and the isoleucine residue A peptide bond is formed with the carbonyl group. GI indicates a peptide residue consisting of glycine-isoleucine in order from the N-terminus. Here, the carbonyl group adjacent to the G (glycine) residue and the primary amino group of the glycine residue are peptide-bonded, and the primary amino group adjacent to the I (isoleucine) residue and the carbonyl of the isoleucine residue are group is peptide-bonded.
上記反応にて得られた反応物のＨＰＬＣチャートを図６に示す。
The HPLC chart of the reaction product obtained in the above reaction is shown in FIG.
上記の結果から、環状ペプチドに含有されるメトキシベンジル基によって保護されたシステイン残基とトリプトファン残基との間で、分子内架橋を生じることが明らかとなった。このように環状構造が追加されたペプチドは、その立体構造がより強固になることが想定される。
From the above results, it was clarified that an intramolecular cross-link occurs between the cysteine residue protected by the methoxybenzyl group contained in the cyclic peptide and the tryptophan residue. Peptides to which a cyclic structure has been added in this way are assumed to have a more rigid three-dimensional structure.
実施例７
下記の反応スキームにて、化合物２４を原料に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。化合物２４は、上記ペプチド合成法によって作製した。 Example 7
In the reaction scheme below,compound 24 was used as a starting material and reacted under the conditions of 1M MSA and 4M guanidine hydrochloride. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction. Compound 24 was made by the peptide synthesis method described above.
下記の反応スキームにて、化合物２４を原料に、１ＭのＭＳＡ及び４Ｍのグアニジン塩酸塩の条件下で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。化合物２４は、上記ペプチド合成法によって作製した。 Example 7
In the reaction scheme below,
作製した化合物２４のＭＳデータは、以下の通りである。
＜化合物２４＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C82H132N24O22S 918.5, found 918.6. The MS data of the producedcompound 24 are as follows.
<Compound 24>
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C82H132N24O22S 918.5 , found 918.6.
＜化合物２４＞
LRMS (ESI-TOF) m/z: [M+2H]+ calcd for C82H132N24O22S 918.5, found 918.6. The MS data of the produced
<
LRMS (ESI-TOF ) m/z: [M+2H] + calcd for C82H132N24O22S 918.5 , found 918.6.
上記各スキームにおいて、Ａｃ－はアセチル基を示し、ＳＤＬは、Ｎ末端から順にセリン－アスパラギン酸－ロイシンからなるペプチド残基を示す。ここで、Ｓ（セリン）残基に隣接するカルボニル基と当該セリン残基のアミノ基とがペプチド結合しており、Ｌ（ロイシン）残基に隣接するアミノ基と当該ロイシン残基のカルボニル基とがペプチド結合している。また、ＬＱＬＲＱＲは、Ｎ末端から順にロイシン－グルタミン－ロイシン－アルギニン－グルタミン－アルギニンからなるペプチド残基を示す。ここで、Ｎ末端側のＬ（ロイシン）残基に隣接するカルボニル基と当該ロイシン残基のアミノ基とがペプチド結合しており、Ｃ末端側のＲ（アルギニン）は、Ｃ末端アミド構造を有する。
In each scheme above, Ac- represents an acetyl group, and SDL represents a peptide residue consisting of serine-aspartic acid-leucine in order from the N-terminus. Here, the carbonyl group adjacent to the S (serine) residue and the amino group of the serine residue are peptide-bonded, and the amino group adjacent to the L (leucine) residue and the carbonyl group of the leucine residue is a peptide bond. LQLRQR indicates a peptide residue composed of leucine-glutamine-leucine-arginine-glutamine-arginine in order from the N-terminus. Here, the carbonyl group adjacent to the L (leucine) residue on the N-terminal side and the amino group of the leucine residue are peptide-bonded, and the R (arginine) on the C-terminal side has a C-terminal amide structure. .
化合物２４は、乳ガン抑制ペプチドであるｓｔＥＲＡＰのアミノ酸配列のアナログであり、化合物２４におけるノルロイシン残基は、ｓｔＥＲＡＰのメチオニン残基に相当する。ここで、ｓｔＥＲＡＰでは、メチオニン残基とセリン残基の間に設けられたグルタミン酸残基と、ロイシン残基とロイシン残基との間のグルタミン残基との間で、ビスアミドを介した架橋が施されている。ここで、化合物２４におけるシステイン残基とトリプトファン残基との間で分子内架橋が形成されると化合物２５のように、ｓｔＥＲＡＰと同様の立体構造を有する化合物が得られると想定された。上記反応にて得られた反応物のＨＰＬＣチャート及び反応後のＣＤスペクトルの結果を図７に示す。
Compound 24 is an analog of the amino acid sequence of stERAP, a breast cancer inhibitory peptide, and the norleucine residue in compound 24 corresponds to the methionine residue of stERAP. Here, in stERAP, a bisamide-mediated cross-linking is performed between a glutamic acid residue provided between a methionine residue and a serine residue and a glutamine residue provided between leucine residues. It is Here, it was assumed that formation of an intramolecular cross-link between the cysteine residue and the tryptophan residue in compound 24 would give a compound having the same three-dimensional structure as stERAP, like compound 25. FIG. 7 shows the HPLC chart of the reaction product obtained in the above reaction and the results of the CD spectrum after the reaction.
上記の結果から、化合物２４に含有されるメトキシベンジル基によって保護されたシステイン残基とトリプトファン残基との間で分子間にて架橋を生じ、化合物２５が産生されることが明らかとなった。また、化合物２４のシステイン残基をアラニン残基に代えたペプチドと、架橋反応後の化合物２５が示すＣＤスペクトルの結果は異なっており、化合物２５は、化合物２４よりもヘリックス性が向上したことも明らかとなった。
From the above results, it was clarified that compound 25 was produced by intermolecular cross-linking between the cysteine residue protected by the methoxybenzyl group contained in compound 24 and the tryptophan residue. In addition, the results of the CD spectra of the peptide obtained by replacing the cysteine residue of compound 24 with an alanine residue and the compound 25 after the cross-linking reaction are different, and the helicality of compound 25 is improved more than that of compound 24. It became clear.
実施例８
０．０１ｍＭトラツスズマブ（中外製薬）と下記式に示す化合物２７とを混合し、３０ｍＭの塩化マグネシウム、５％の水を含有する０．１％ＴＦＡ中で、３７℃にて２４時間反応させた。また、この反応系には、１ブチル－１－メチルピロリジニウムトリフルオロメタンスルホン酸を含有させた。化合物２７は、上記ペプチド合成法によって作製した。 Example 8
0.01 mM tratusuzumab (Chugai Pharmaceutical Co., Ltd.) and compound 27 represented by the following formula were mixed and reacted in 0.1% TFA containing 30 mM magnesium chloride and 5% water at 37° C. for 24 hours. The reaction system also contained 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid. Compound 27 was made by the peptide synthesis method described above.
０．０１ｍＭトラツスズマブ（中外製薬）と下記式に示す化合物２７とを混合し、３０ｍＭの塩化マグネシウム、５％の水を含有する０．１％ＴＦＡ中で、３７℃にて２４時間反応させた。また、この反応系には、１ブチル－１－メチルピロリジニウムトリフルオロメタンスルホン酸を含有させた。化合物２７は、上記ペプチド合成法によって作製した。 Example 8
0.01 mM tratusuzumab (Chugai Pharmaceutical Co., Ltd.) and compound 27 represented by the following formula were mixed and reacted in 0.1% TFA containing 30 mM magnesium chloride and 5% water at 37° C. for 24 hours. The reaction system also contained 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonic acid. Compound 27 was made by the peptide synthesis method described above.
上記化合物２７におけるＡは、下記に示す基であり、化合物２７においてＡに隣接するカルボニル基とＡのアミン基とがペプチドと結合しており、Ａに隣接するアミン基とＡのカルボニル基とがペプチドと結合している。
A in the above compound 27 is a group shown below, 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 bound to peptides.
作製した化合物２７のスルホキシドの状態でのＭＳデータは、以下の通りである。
＜化合物２７＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C36H64N9O13S2 894.4, found 894.7. The MS data of the prepared compound 27 in the state of sulfoxide are as follows.
<Compound 27>
LRMS ( ESI -TOF) m/z : [M+H]+ calcd for C36H64N9O13S2 894.4 , found 894.7.
＜化合物２７＞
LRMS (ESI-TOF) m/z: [M+H]+ calcd for C36H64N9O13S2 894.4, found 894.7. The MS data of the prepared compound 27 in the state of sulfoxide are as follows.
<Compound 27>
LRMS ( ESI -TOF) m/z : [M+H]+ calcd for C36H64N9O13S2 894.4 , found 894.7.
上記反応にて得られた反応物と反応前の混合物とを、公知の方法に準じて銀染色及びビオチン染色に供した。その結果を図８に示す。
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. The results are shown in FIG.
図８に示す結果から、反応前のサンプルではビオチン染色においてバンドが検出されなかったのに対して、反応後ではビオチン染色像において、重鎖及び軽鎖に相当する位置に明らかなバンドが検出された。他方、タンパク質全体を検出する銀染色では反応前後のサンプルにおいて、重鎖及び軽鎖に相当する位置に明らかなバンドが検出された。
From the results shown in FIG. 8, no band was detected in the biotin-stained sample before the reaction, whereas in the biotin-stained image after the reaction, clear bands were detected at positions corresponding to heavy and light chains. Ta. On the other hand, silver staining, which detects the whole protein, detected clear bands at positions corresponding to the heavy and light chains in the samples before and after the reaction.
この結果から、上記反応において抗体に含有されるトリプトファン残基と、化合物２７含有されるメチルカルボニルアミノ基によって保護されたシステイン残基との間で分子間架橋を生じていることが想定される。また、この反応は、上記の実験例５とは異なって、塩酸塩ではなく塩化マグネシウムの存在下で架橋反応が進行することも明らかとなった。
From this result, it is assumed that intermolecular cross-linking occurs between the tryptophan residue contained in the antibody and the cysteine residue protected by the methylcarbonylamino group contained in compound 27 in the above reaction. It was also found that this reaction proceeds in the presence of magnesium chloride instead of hydrochloride, unlike Experimental Example 5 above.
また、ＭＳ解析の結果、重鎖の３箇所のトリプトファン及び軽鎖の１箇所のトリプトファンが化合物２７によって修飾されていることが明らかなった。
In addition, as a result of MS analysis, it was revealed that compound 27 modified tryptophan at 3 sites in the heavy chain and tryptophan at 1 site in the light chain.
実施例９
下記の反応スキームにて、化合物２９、３０、３２、３５を原料に、１ＭのＭＳＡ及び４Ｍのジイソプロピルアミン塩酸塩の条件下で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。 Example 9
In the reaction scheme below, compounds 29, 30, 32, and 35 were reacted under conditions of 1M MSA and 4M diisopropylamine hydrochloride as starting materials. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction.
下記の反応スキームにて、化合物２９、３０、３２、３５を原料に、１ＭのＭＳＡ及び４Ｍのジイソプロピルアミン塩酸塩の条件下で反応させた。この反応での反応温度を４℃とし、反応時間を３時間とした。この反応における溶媒としてＴＦＡを使用した。 Example 9
In the reaction scheme below, compounds 29, 30, 32, and 35 were reacted under conditions of 1M MSA and 4M diisopropylamine hydrochloride as starting materials. The reaction temperature in this reaction was 4° C. and the reaction time was 3 hours. TFA was used as solvent in this reaction.
作製した化合物２９、３０、３２、３５のＭＳデータは、以下の通りである。
＜化合物２９＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C151H232N40O47 839.4, found 839.5.
＜化合物３０＞
LRMS (ESI-Q) m/z: [M+H]+ calcd for C33H58N3O7S 640.4, found 640.4.
＜化合物３２＞
LRMS (ESI-Q) m/z: [M+H]+ calcd for C45H80N5O13S 742.4, found 742.2.
＜化合物３５＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C152H234N42O47 849.9, found 850.0. The MS data of the produced compounds 29, 30, 32 and 35 are as follows.
<Compound 29>
LRMS ( ESI -Q) m/z: [M+4H] 4+ calcd for C151H232N40O47 839.4 , found 839.5.
<Compound 30>
LRMS ( ESI -Q ) m/z: [M+H]+ calcd for C33H58N3O7S 640.4, found 640.4.
<Compound 32>
LRMS (ESI-Q) m/z: [M+H] + calcd for C45H80N5O13S 742.4 , found 742.2 .
<Compound 35>
LRMS ( ESI -Q) m/z: [ M+4H] 4+ calcd for C152H234N42O47 849.9 , found 850.0.
＜化合物２９＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C151H232N40O47 839.4, found 839.5.
＜化合物３０＞
LRMS (ESI-Q) m/z: [M+H]+ calcd for C33H58N3O7S 640.4, found 640.4.
＜化合物３２＞
LRMS (ESI-Q) m/z: [M+H]+ calcd for C45H80N5O13S 742.4, found 742.2.
＜化合物３５＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C152H234N42O47 849.9, found 850.0. The MS data of the produced compounds 29, 30, 32 and 35 are as follows.
<Compound 29>
LRMS ( ESI -Q) m/z: [M+4H] 4+ calcd for C151H232N40O47 839.4 , found 839.5.
<
LRMS ( ESI -Q ) m/z: [M+H]+ calcd for C33H58N3O7S 640.4, found 640.4.
<Compound 32>
LRMS (ESI-Q) m/z: [M+H] + calcd for C45H80N5O13S 742.4 , found 742.2 .
<Compound 35>
LRMS ( ESI -Q) m/z: [ M+4H] 4+ calcd for C152H234N42O47 849.9 , found 850.0.
上記各スキームにおいて、Ｎ末端のＨは、ヒスチジン残基を示し、ＥＧＴＦＴＳＤＶＳＳＹＬＥＧＱＡＡＫＥＦＩＡは、Ｎ末端側から順に、グルタミン酸－グリシン－スレオニン－フェニルアラニン－スレオニン－セリン－アスパラギン酸－バリン－セリン－セリン－チロシン－ロイシン－グルタミン酸－グリシン－グルタミン－アラニン－アラニン－リジン－グルタミン酸－フェニルアラニン－イソロイシン－アラニンからなるペプチド残基を示す。ここで、上記のＮ末端側のＥ（グルタミン酸）残基に隣接するカルボニル基と当該グルタミン酸残基のアミノ基とがペプチド結合しており、Ｃ末端側のＡ（アラニン）残基に隣接するアミノ基と当該アラニン残基のカルボニル基とがペプチド結合している。また、ＬＶＲＧＲＧは、Ｎ末端から順にロイシン－バリン－アルギニン－グリシン－アルギニン－グリシンからなるペプチド残基を示す。ここで、Ｎ末端のＬ（ロイシン）残基に隣接するカルボニル基と当該ロイシンのアミノ基とがペプチド結合している。
In each of the above schemes, H at the N-terminus represents a histidine residue, and EGTFFTSDVSSYLEGQAAKEFIA is glutamic acid-glycine-threonine-phenylalanine-threonine-serine-aspartic acid-valine-serine-serine-tyrosine-leucine in order from the N-terminal side. -glutamic acid-glycine-glutamine-alanine-alanine-lysine-glutamic acid-phenylalanine-isoleucine-alanine. Here, the carbonyl group adjacent to the E (glutamic acid) residue on the N-terminal side and the amino group of the glutamic acid residue are peptide-bonded, and the amino group adjacent to the A (alanine) residue on the C-terminal side A peptide bond is formed between the group and the carbonyl group of the alanine residue. LVRGRG represents a peptide residue consisting of leucine-valine-arginine-glycine-arginine-glycine in order from the N-terminus. Here, the carbonyl group adjacent to the N-terminal L (leucine) residue and the amino group of the leucine are peptide-bonded.
上記各スキームにおいて、Ａは、上記Ａと同じであり、化合物３０において、Ａ）に隣接するカルボニル基とＡのアミン基とがペプチドと結合しており、Ａに隣接するアミン基とＡのカルボニル基とがペプチドと結合している。また、Ａ３は、下記に示す基であり、化合物３２において、Ａ３に隣接するカルボニル基とＡ３のアミン基とがペプチドと結合しており、Ａ３に隣接するアミン基とＡ３のカルボニル基とがペプチドと結合している。
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 of A group is attached to the peptide. Further, A3 is a group shown below. In compound 32, the carbonyl group adjacent to A3 and the amine group of A3 are bonded to the peptide, and the amine group adjacent to A3 and the amine group of A3 are bonded to the peptide . A carbonyl group is attached to the peptide.
上記反応にて得られた反応物のＨＰＬＣチャートを図９に示す。また、得られた化合物３１、３３、３６及び３７のＭＳデータは、以下の通りである。
＜化合物３１＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C176H279N43O52S 964.7, found 965.0.
＜化合物３３＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C188H301N45O58S 1037.3, found 1037.5.
＜化合物３６＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C177H281N45O52S 975.3, found 975.5.
＜化合物３７＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C189H303N47O58S 1047.8, found 1048.0. FIG. 9 shows the HPLC chart of the reaction product obtained in the above reaction. Moreover, 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 C176H279N43O52S 964.7 , found 965.0.
<Compound 33>
LRMS ( ESI -Q) m/z: [M+4H] 4+ calcd for C188H301N45O58S 1037.3 , found 1037.5.
<Compound 36>
LRMS (ESI-Q) m/z: [ M+4H] 4+ calcd for C177H281N45O52S 975.3 , found 975.5.
<Compound 37>
LRMS (ESI- Q ) m/z: [M+4H] 4+ calcd for C189H303N47O58S 1047.8 , found 1048.0.
＜化合物３１＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C176H279N43O52S 964.7, found 965.0.
＜化合物３３＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C188H301N45O58S 1037.3, found 1037.5.
＜化合物３６＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C177H281N45O52S 975.3, found 975.5.
＜化合物３７＞
LRMS (ESI-Q) m/z: [M+4H]4+ calcd for C189H303N47O58S 1047.8, found 1048.0. FIG. 9 shows the HPLC chart of the reaction product obtained in the above reaction. Moreover, the MS data of the obtained compounds 31, 33, 36 and 37 are as follows.
<
LRMS ( ESI -Q) m/z: [ M+4H] 4+ calcd for C176H279N43O52S 964.7 , found 965.0.
<
LRMS ( ESI -Q) m/z: [M+4H] 4+ calcd for C188H301N45O58S 1037.3 , found 1037.5.
<
LRMS (ESI-Q) m/z: [ M+4H] 4+ calcd for C177H281N45O52S 975.3 , found 975.5.
<
LRMS (ESI- Q ) m/z: [M+4H] 4+ calcd for C189H303N47O58S 1047.8 , found 1048.0.
上記の結果から、上記化合物２９及び３５は、それぞれ生理活性を有するペプチドであり、これに脂質構造を結合させることが盛んに行われている。上記化合物３０及び３２は、脂質構造の導入によりペプチドの生体内安定性の増加、腎排泄の抑制が期待できる。メトキシベンジル基によって保護されたシステインスルホキシド体を利用してトリプトファン上に分子間架橋を介して脂質構造を導入した化合物３１、３３、３６及び３７は、生体内にて生理活性をより有効に発揮することができる。
From the above results, compounds 29 and 35 are bioactive peptides, and lipid structures are being actively bound to them. Compounds 30 and 32 are expected to increase the in vivo stability of the peptide and suppress renal excretion by introducing a lipid structure. Compounds 31, 33, 36 and 37, in which a cysteine sulfoxide protected by a methoxybenzyl group is used to introduce a lipid structure onto tryptophan via intermolecular cross-linking, exhibit more effective physiological activity in vivo. be able to.
Claims (9)
- 下記式（１）
Ｒ２は、二価の有機基を示す。
Ｒ３は、一価有機基を示す。
また、Ｒ１とＲ３とが、互いに結合して環を形成していてもよい。］
で表される化合物の製造方法であって、
下記式（２）
で表される化合物を、塩酸塩又は金属塩化物の存在下で架橋反応させる工程を含む、製造方法。 Formula (1) below
R2 represents a divalent organic group.
R3 represents a monovalent organic group.
Also, R 1 and R 3 may combine with each other to form a ring. ]
A method for producing a compound represented by
Formula (2) below
A production method comprising the step of cross-linking the compound represented by in the presence of hydrochloride or metal chloride. - 前記Ｒ１が、カルボニル基を有する一価の有機基であり、前記Ｒ２が、カルボニル基及びアミノ基を有する二価の有機基であり、及び前記Ｒ３が、アミノ基を有する一価の有機基である、請求項１に記載の製造方法。 R 1 is a monovalent organic group having a carbonyl group, R 2 is a divalent organic group having a carbonyl group and an amino group, and R 3 is a monovalent organic group having an amino group The production method according to claim 1, which is an organic group.
- 前記Ｒ１、Ｒ２及びＲ３が、それぞれペプチド残基である、請求項１に記載の製造方法。 The production method according to claim 1, wherein said R 1 , R 2 and R 3 are each peptide residues.
- 前記フェニル環上の置換基が、電子供与性基である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the substituent on the phenyl ring is an electron-donating group.
- 前記電子供与性基が、アルキル基、アルコキシ基、アルキルアミノ基、アルキルカルボニル基、アルキルアミノカルボニル基、アルキルカルボニルアミノ基、水酸基、アミノ基及びハロゲン基からなる群より選択される少なくとも一種である、請求項４に記載された製造方法。 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. The manufacturing method according to claim 4.
- 前記塩酸塩が、グアニジン塩酸塩、ジメチルアミン塩酸塩、ジイソプロピルアミン塩酸塩、ピペリジン塩酸塩、塩化テトラブチルアンモニウム、ピペラジン塩酸塩及びモルフォリン塩酸塩からなる群より選択される少なくとも一種である、請求項１～５の何れか一項に記載の製造方法。 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. 6. The production method according to any one of 1 to 5.
- 前記金属塩化物が、塩化マグネシウム、塩化亜鉛、塩化リチウム、塩化鉄（III）、塩化カルシウム及び塩化ニッケルからなる群より選択される少なくとも一種である、請求項１～５の何れか一項に記載の製造方法。 6. The metal chloride 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. manufacturing method.
- 酸性条件下で架橋反応を行う、請求項１～７の何れか一項に記載の製造方法。 The production method according to any one of claims 1 to 7, wherein the cross-linking reaction is performed under acidic conditions.
- 下記式（３）
で表される化合物の製造方法であって、
下記式（４）
で表される化合物と、
下記式（５）
で表される化合物とを、塩酸塩又は金属塩化物の存在下で反応させる工程を含む、製造方法。 Formula (3) below
A method for producing a compound represented by
Formula (4) below
A compound represented by
Formula (5) below
A production method comprising the step of reacting a compound represented by in the presence of a hydrochloride or a metal chloride.
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