Method for producing ganirelix or salt thereof

Abstract

To provide a novel method for producing ganirelix by a liquid-phase peptide synthesis process.SOLUTION: The present invention pertains to a method for producing ganirelix or a salt thereof by a liquid-phase peptide synthesis process, employing at least one compound in a group represented by formulae below (where R1 and R2 each represent Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde, and R3 represents an amino-protecting group) and a carrier for liquid phase peptide synthesis, as a raw material for a condensation reaction of third and fifth diethyl homoarginine residues from the C terminal.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing the gonadotropin antagonist ganirelix or a salt thereof. [Background technology]

[0002] Ganirelix is ​​a gonadotropin antagonist that is marketed as a drug for preventing premature ovulation under controlled ovarian stimulation. Ganirelix is ​​a decapeptide drug that has diethylhomoarginine residues at the third and fifth positions from the C-terminus, and is produced by solid-phase peptide synthesis (Patent Documents 1 and 2, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] CN102584945A [Patent Document 2] International Publication No. 2015 / 188774 [Patent Document 3] Patent No. 5113118 [Patent Document 4] Patent No. 4500854 [Patent Document 5] Patent No. 5929756 [Patent Document 6] Patent No. 6092513 [Patent Document 7] Patent No. 5768712 [Patent Document 8] Patent No. 5803674 [Patent Document 9] Patent No. 6116782 [Patent Document 10] Patent No. 6201076 [Patent Document 11] Patent No. 6283774 [Patent Document 12] Patent No. 6283775 [Patent Document 13] Patent No. 6322350 [Patent Document 14] Patent No. 6393857 [Patent Document 15] Patent No. 6531235 [Patent Document 16] International Publication No. 2019 / 009317 [Patent Document 17] International Publication No. 2020 / 175472 [Patent Document 18] International Publication No. 2020 / 175473 [Patent Document 19] Special Publication No. 2003-500416 [Patent Document 20] Special Publication No. 2004-516330 [Patent Document 21] Japanese Patent Application Publication No. 4-211096 [Patent Document 22] Japanese Patent Application Publication No. 5-170795 [Non-patent literature]

[0004] [Non-Patent Document 1] Ministry of Health, Labor and Welfare, Pharmaceuticals and Food Safety Bureau, Inspection and Control Division, "Ganilest Subcutaneous Injection 0.25mg Syringe" Deliberation Result Report https: / / www.pmda.go.jp / drugs / 2008 / P200800034 / 170050000_22000AMX01714000_A100_2.pdf Summary of the Invention [Problem to be solved by the invention]

[0005] As a method for producing decapeptides such as ganirelix, solid-phase peptide synthesis is preferable for small amounts of reagents, but it is not suitable for mass production for supply as a pharmaceutical. This is because solid-phase synthesis is a heterogeneous reaction system, has low mixing efficiency, and has a slow reaction rate. Furthermore, since it is not possible to purify the peptide during the reaction, it is necessary to use a large excess of 3 to 4 equivalents of amino acids to be extended and reagents in order to react quantitatively. On the other hand, liquid-phase peptide synthesis is a homogeneous reaction system, and it is possible to reduce the amount of reagent used. However, so-called liquid-phase peptide synthesis has not been performed in recent years due to the decrease in reaction rate caused by the decrease in solubility in organic solvents as the peptide chain elongates, and the need to consider a separation process suitable for the properties of each peptide for each peptide elongation reaction, which is complicated. Thus, the synthesis of ganirelix by liquid-phase peptide synthesis has not been performed.

[0006] In contrast, in recent years, liquid-phase peptide synthesis supports (Tag) have been reported for liquid-phase peptide synthesis (Patent Documents 3-18). Since this support is a highly hydrophobic compound, by binding highly hydrophilic amino acids, peptides, amino acid amides, or peptide amides (hereinafter, sometimes referred to as amino acids, etc.) to this support, the solubility in organic solvents can be greatly improved. Therefore, when peptide elongation reaction is carried out in a state in which amino acids, etc. are bound to this support, the amino acids, etc. bound to the support are dissolved in the organic layer, and unnecessary components, such as excess raw amino acids used in the peptide elongation reaction, their decomposition products, and compounds by-produced when the protecting groups of the raw amino acids are deprotected, are dissolved in the aqueous layer, which has the advantage that the amino acids, etc. bound to the support can be easily purified by liquid-liquid separation. Furthermore, there is also the advantage that crude purification of peptides is possible even in a state in which the support is bound. In this way, by using a liquid-phase peptide synthesis support, it has become possible to mass-produce peptides without the need for complicated separation operations. In this specification, the term "amino acid amide" refers to a structure in which the C-terminal carboxyl group (-COOH) of an amino acid becomes an amide group (-CONH2), and the term "peptide amide" refers to a structure in which the C-terminal carboxyl group of a peptide becomes an amide group. Furthermore, in this specification, the term "liquid-liquid separation" refers to the above-mentioned process, i.e., a process in which amino acids bound to a carrier are dissolved in an organic layer, and unnecessary components, such as surplus raw amino acids used in the peptide elongation reaction, their decomposition products, and compounds produced as by-products when the protecting groups of the raw amino acids are deprotected, are dissolved in an aqueous layer.

[0007] However, in the case of ganirelix, it was found that even when this support for liquid-phase peptide synthesis was used, liquid-liquid separation was difficult during the condensation reaction of the guanidyl group-containing amino acid (diethylhomoarginine) present in the ganirelix structure.

[0008] Therefore, an object of the present invention is to provide a novel method for producing ganirelix by liquid phase peptide synthesis. [Means for solving the problem]

[0009] Therefore, the present inventors have conducted various studies on the means for protecting the guanidyl group of diethylhomoarginine used in the liquid-phase peptide synthesis of ganirelix using a liquid-phase peptide synthesis support. First, they have studied proton protection (Patent Documents 19 to 22), which has been widely used as a means for protecting the guanidyl group of arginines, and found that liquid-liquid separation after the condensation reaction of diethylhomoarginine is difficult. In contrast, they have found that if diethylhomoarginine in which the guanidino group of arginines is protected with a protecting group such as a Boc group is used as a raw material, liquid-liquid separation after the condensation reaction is improved, and ganirelix or a salt thereof can be produced by an industrially advantageous method using a liquid-phase peptide synthesis method, and have completed the present invention.

[0010] That is, the present invention provides the following inventions [1] to

[11] . [1] A method for producing ganirelix or a salt thereof by liquid phase peptide synthesis, comprising the steps of:

[0011] [ka]

[0012] (In the formula, R 1 and R 2 represents Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde; R 3 indicates an amino protecting group) and a support for liquid phase peptide synthesis. [2] The method according to [1], comprising the following steps a to c: a. condensing an amino acid, peptide, amino acid amide, or peptide amide bound to a support for liquid phase peptide synthesis with an amino acid or peptide having a protected amino group in a solvent containing an organic solvent; b. removing the amino-protecting group of the compound having a protected amino group in the reaction solution; c. A process of adding an aqueous solution to the reaction solution, followed by separation to obtain an organic solvent layer containing a condensate of an amino acid, peptide, amino acid amide or peptide amide bound to a support for liquid phase peptide synthesis and an amino acid or peptide from which the amino-protecting group has been removed. [3] The production method according to [1] or [2], further comprising the step of adding a quenching agent for the amino acid activated ester to the reaction solution after the condensation reaction subsequent to the step a. [4] The method for producing the present invention according to any one of [1] to [3], wherein the starting amino acids are, from the C-terminus, D-AlaNH2, Pro, hArg(Et)2, Leu, D-hArg(Et)2, Tyr, Ser, D-3-pyridyl Ala, Dp-chloroPhe, and D-naphthyl Ala, in that order. [5] R 1 and R 2 is Boc, and R 3 The method according to any one of [1] to [4], wherein [6] R 1 and R 2 is Boc, and R 3 The method according to any one of [1] to [5], wherein [7] The method according to any one of [3] to [6], wherein the quenching agent for the amino acid activated ester is a water-soluble amine. [8] The method according to [7], wherein the water-soluble amine is hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, or a primary, secondary or tertiary amine having one or more selected from an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aralkyl group and a heterocyclic group, which may have one or more substituents selected from a hydroxy group, an ether bond, an alkoxy group, a sulfonyl group, a sulfonic acid group, a sulfate group and a phosphate group. [9]R 3 The production method according to any one of [2] to [8], wherein when is Fmoc, the production method further comprises a step of adding a trapping agent for dibenzofulvene subsequent to the step b.

[10] The method according to [9], wherein the trapping agent for dibenzofulvene is a mercapto compound having an alkyl group having 1 to 10 carbon atoms and having one or more substituents selected from carboxylic acid, an alkali metal salt of a carboxylic acid, sulfonic acid, or an alkali metal salt of a sulfonic acid.

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

[10] , wherein the support for liquid phase peptide synthesis is a compound represented by the following formula (I):

[0013] [ka]

[0014] [In the formula, Ring A represents a C4-20 aromatic ring which may contain a heteroatom and may be polycyclic; R 11 is a hydrogen atom, or when ring A is a benzene ring and Rb is a group represented by the following formula (b), R 13 may together with ring A and ring B represent a single bond to form a fluorene ring, or may together with ring A and ring B via an oxygen atom form a xanthene ring; p X's1 each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 15 -(R 15 represents a hydrogen atom, an alkyl group, or an aralkyl group; pR 12 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0015] [ka]

[0016] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR 17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto; p represents an integer of 1 to 4; Ring A is a set of p X 1 R 12 and optionally have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Ra represents a hydrogen atom or an aromatic ring which may be substituted with a halogen atom; Rb is a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by formula (b):

[0017] [ka]

[0018] (In the formula, * indicates a bond position; q represents an integer of 0 to 4; q X's 2each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 18 -(R 18 represents a hydrogen atom, an alkyl group, or an aralkyl group; q R 14 are each independently an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0019] [ka]

[0020] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR 17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto; R 13 indicates a hydrogen atom or R 11 may be taken together with ring A to form a single bond to form a fluorene ring together with ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring; Ring B is a ring of q X 2 R 14 In addition, it may further have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Y is a hydroxyl group, a thiol group, or NHR 20 (R 20 represents a hydrogen atom, an alkyl group or an aralkyl group. ) or a halogen atom. ] Effect of the Invention

[0021] When ganirelix or a salt thereof is produced by the method of the present invention using a protected form of diethylhomoarginine, liquid-liquid separation after the condensation reaction becomes easy, and ganirelix or a salt thereof can be produced industrially with advantage. [Brief description of the drawings]

[0022] [Figure 1] This is a photograph of the liquid surface after shaking the separatory funnel and then leaving it to stand at room temperature for 25 minutes in the liquid-liquid separation in the step of Example (1-d) *1. [Diagram 2] This is a photograph of the liquid surface after shaking the separatory funnel and leaving it at room temperature for 25 minutes during liquid-liquid separation in the process of Reference Example (1-d) *2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Ganirelix, the target compound of the present invention, is a decapeptide having the following structure [N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-L-tyrosyl-N6-(N,N'-diethylcarbamimidoyl)-D-lysyl-L-leucyl-N6-(N,N'-diethylcarbamimidoyl)-L-lysyl-L-prolyl-D-alaninamide)], a gonadotropin antagonist, and is marketed as a drug for preventing premature ovulation under controlled ovarian stimulation.

[0024] [ka]

[0025] In the above structure, the amino acid residues constituting the decapeptide structure may be abbreviated as follows from the C-terminus: D-AlaNH2, Pro, hArg(Et)2, Leu, D-hArg(Et)2, Tyr, Ser, D-3-pyridylAla, Dp-chloroPhe, and D-naphthylAla.

[0026] The method for producing ganirelix or a salt thereof of the present invention is a method for producing ganirelix or a salt thereof by liquid phase peptide synthesis, and comprises using as starting materials for the condensation reaction of the third and fifth diethylhomoarginine residues from the C-terminus, compounds represented by the following formulas (1) to (3):

[0027] [ka]

[0028] (In the formula, R 1 and R 2 represents Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde; R 3 indicates an amino protecting group) and a carrier for liquid phase peptide synthesis.

[0029] First, the compounds represented by the above formulas (1) to (3) used as starting materials for the condensation reaction of diethylhomoarginine residues will be described. R 1 and R 2 represents Boc (tert-butoxycarbonyl), Cbz (benzyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Alloc (allyloxycarbonyl), Trt (trityl), Mmt (4-monomethoxytrityl), Teoc (2-(trimethylsilyl)ethoxycarbonyl), Phth (phthaloyl), SES ((2-trimethylsilyl)-ethanesulfonyl), ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl). Among these, Boc is preferred from the viewpoint of improving the liquid-liquid separation of the condensation reaction product. R 3 represents an amino-protecting group. Examples of the amino-protecting group include Fmoc (9-fluorenylmethyloxycarbonyl), Boc, Cbz, and the like. Among these, Fmoc and Cbz are preferred, and Fmoc, which can be deprotected under basic conditions, is more preferred. In particular, R 1 and R 2is Boc and R 3 More preferred are compounds in which is Fmoc.

[0030] The structures represented by the formulas (1) to (3) are E / Z isomers or imino / amino isomers, and the compound may be a mixture of these isomers.

[0031] The compounds represented by the formulas (1) to (3) can be produced, for example, by reacting α-amino-protected diethylhomoarginine or a conjugate of the same with a support for liquid phase peptide synthesis with an amino-protecting agent such as a Boc agent, e.g., di-tert-butyl dicarbonate, N-tert-butoxycarbonylimidazole, etc. For example, this Boc reaction is preferably carried out in a solvent in the presence of a base. The base may be an organic base such as pyridine, triethylamine, DMAP (4-dimethylaminopyridine), N-methylimidazole, or a mixed organic base thereof, or an inorganic base such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, or sodium hydroxide. The amount of the base added is 0.1 to 30 equivalents, preferably 1 to 20 equivalents, relative to the arginine derivative, but is not limited thereto. The reaction solvent used may be water, THF (tetrahydrofuran), 2-methylTHF, 1,4-dioxane, toluene, DMF (N,N-dimethylformamide), acetonitrile, dichloromethane, chloroform, methanol, ethanol, or a mixture thereof. The reaction is preferably carried out at 0° C. to 40° C. for 1 to 24 hours.

[0032] Next, the support for liquid phase peptide synthesis will be described. Such a support for liquid phase peptide synthesis may be a support that protects the functional groups of amino acids, peptides, amino acid amides, or peptide amides (amino acids, etc.) and solubilizes the protected amino acids, etc. in an organic solvent, and for example, the compounds described in Patent Documents 3 to 18 can be used. Specific examples of such supports for liquid phase peptide synthesis include compounds represented by the following formula (I):

[0033] [ka]

[0034] [In the formula, Ring A represents a C4-20 aromatic ring which may contain a heteroatom and may be polycyclic; R 11 is a hydrogen atom, or when ring A is a benzene ring and Rb is a group represented by the following formula (b), R 13 may together with ring A and ring B represent a single bond to form a fluorene ring, or may together with ring A and ring B via an oxygen atom form a xanthene ring; p X's 1 each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 15 -(R 15 represents a hydrogen atom, an alkyl group, or an aralkyl group; pR 12 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0035] [ka]

[0036] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR 17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto; p represents an integer of 1 to 4; Ring A is a set of p X 1 R 12and optionally have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Ra represents a hydrogen atom or an aromatic ring which may be substituted with a halogen atom; Rb is a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by formula (b):

[0037] [ka]

[0038] (In the formula, * indicates a bond position; q represents an integer of 0 to 4; q X's 2 each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 18 -(R 18 represents a hydrogen atom, an alkyl group, or an aralkyl group; q R 14 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0039] [ka]

[0040] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR 17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto; R 13 indicates a hydrogen atom or R 11may be taken together with ring A to form a single bond to form a fluorene ring together with ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring; Ring B is a ring of q X 2 R 14 In addition, it may further have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Y is a hydroxyl group, a thiol group, or NHR 20 (R 20 represents a hydrogen atom, an alkyl group or an aralkyl group. ) or a halogen atom. ]

[0041] Ring A in formula (I) represents a C4-20 aromatic ring which may contain a heteroatom and may be monocyclic or polycyclic. Examples of the aromatic ring include a C6-20 aromatic hydrocarbon ring and a C4-10 aromatic heterocycle. Specific examples of the C6-20 aromatic hydrocarbon ring include a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a triphenylene ring, a tetracene ring, an indane ring, an indene ring, a fluorene ring, a biphenyl ring, and a 1,1'-binaphthalene ring, etc. Among these, a benzene ring, a naphthalene ring, a phenanthrene ring, and a fluorene ring are more preferable. The C4-10 aromatic heterocycle is preferably a 5- to 10-membered aromatic heterocycle containing 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and specifically includes a pyrrole ring, a furan ring, a thiophene ring, an indole ring, a benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, an indazole ring, an imidazole ring, a pyridine ring, a quinoline ring, an isoquinoline ring, etc. Among these, a 5- to 8-membered aromatic heterocycle containing 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom is preferred, and a pyrrole ring, a furan ring, a thiophene ring, an indole ring, a benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, and an indazole ring are more preferred.

[0042] R11 represents a hydrogen atom, or when ring A is a benzene ring and Rb is a group represented by the above formula (b), R 13 R may be taken together with ring A to form a single bond to form a fluorene ring together with ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring. 11 and R 13 The ring which may be formed by combining with each other is preferably a fluorene ring or a xanthene ring.

[0043] p X's 1 each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 15 -(R 15 represents a hydrogen atom, an alkyl group or an aralkyl group. Here, R 15 is preferably a hydrogen atom, a C1 to 10 alkyl group, or a C7 to 20 aralkyl group. Examples of the alkyl group include linear or branched C1 to 10 alkyl groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group. Examples of the aralkyl group include C7 to C16 aralkyl groups, such as a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, and a 1-naphthylethyl group.

[0044] pR 12 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0045] [ka]

[0046] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3is an oxygen atom or -C(=O)NR 17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto. p represents an integer of 1 to 4.

[0047] In this specification, an organic group having an aliphatic hydrocarbon group is a monovalent organic group having an aliphatic hydrocarbon group in its molecular structure. The position of the aliphatic hydrocarbon group in the organic group having an aliphatic hydrocarbon group is not particularly limited, and may be present at a terminal or at another position. The aliphatic hydrocarbon group present in the organic group is a linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbon group, and from the viewpoint of solubility in an organic solvent, an aliphatic hydrocarbon group of C5 or more is preferable, an aliphatic hydrocarbon group of C5 to 50 is more preferable, and an aliphatic hydrocarbon group of C8 to 30 is even more preferable. Specific examples of the aliphatic hydrocarbon group include an alkyl group, a cycloalkyl group, an alkenyl group, and an alkynyl group, and in particular, an alkyl group, a cycloalkyl group, and an alkenyl group are preferable, and an alkyl group is even more preferable. Furthermore, a linear or branched alkyl group of C5 to 30, a cycloalkyl group of C3 to 8, and a linear or branched alkenyl group of C5 to 30 are preferable, a linear or branched alkyl group of C5 to 30, and a cycloalkyl group of C3 to 8 are more preferable, a linear or branched alkyl group of C5 to 30 is even more preferable, and a linear or branched alkyl group of C8 to 30 is even more preferable.

[0048] Specific examples of the alkyl group include alkyl groups having 1 to 30 carbon atoms, such as monovalent groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, an octyl group, a decyl group, a lauryl group, a tridecyl group, a myristyl group, a cetyl group, a stearyl group, an arachyl group, a behenyl group, a tetracosanyl group, a hexacosanyl group, and an isostearyl group, divalent groups derived therefrom, and groups obtained by excluding hydroxyl groups and the like from various steroid groups. Examples of the branched alkyl group include a 2,3-dihydrophytyl group and a 3,7,11-trimethyldodecyl group. 1 If -NHC(=O)-, then X 1 R 12 Examples of such amides include 2,2,4,8,10,10-hexamethyl-5-dodecanoic acid amide. Examples of the alkenyl group include monovalent groups such as a vinyl group, a 1-propenyl group, an allyl group, an isopropenyl group, a butenyl group, an isobutenyl group, and an oleyl group, and divalent groups derived therefrom. Examples of the alkynyl group include an ethynyl group, a propargyl group, and a 1-propynyl group.

[0049] The above aliphatic hydrocarbon group may be substituted with an aliphatic hydrocarbon group via an oxygen atom. Examples of the aliphatic hydrocarbon group that may be substituted with an aliphatic hydrocarbon group via an oxygen atom include monovalent groups such as linear or branched alkoxy groups having 1 to 20 carbon atoms, alkenyloxy groups having 2 to 20 carbon atoms, and cycloalkyloxy groups having 3 to 6 carbon atoms, and divalent groups derived therefrom. In addition, the aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom may have a repeating structure in which an aliphatic hydrocarbon group is further substituted with an aliphatic hydrocarbon group via an oxygen atom. Specifically, R 12 Examples of the aryloxy group include a 12-docosyloxy-1-dodecyl group, a 3,4,5-tris(octadecyloxy)benzyl group, a 2,2,2-tris(octadecyloxymethyl)ethyl group, and a 3,4,5-tris(octadecyloxy)cyclohexylmethyl group.

[0050] The aliphatic hydrocarbon group includes

[0051] [ka]

[0052] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR17 -(R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and A may be substituted with an organic group which is either a silyl group or an alkyl group having a silyloxy group bonded thereto. The silyl group is preferably a silyl group substituted with three groups selected from a linear or branched alkyl group having 1 to 6 carbon atoms and an aryl group which may have a substituent. Here, examples of the aryl group which may have a substituent include a phenyl group and a naphthyl group. A preferred silyl group is a silyl group substituted with three linear or branched alkyl groups having 1 to 6 carbon atoms, and a more preferred silyl group is a silyl group substituted with three linear or branched alkyl groups having 1 to 4 carbon atoms. The three alkyl or aryl groups substituting the silyl group may be the same or different. The alkyl group bonded to a silyloxy group is preferably a linear or branched alkyl group having 1 to 13 carbon atoms bonded to 1 to 3 silyloxy groups each substituted with 3 groups selected from a linear or branched alkyl group having 1 to 6 carbon atoms and an aryl group which may have a substituent. A preferred silyloxy group is a silyloxy group substituted with 3 linear or branched alkyl groups having 1 to 6 carbon atoms, more preferably a silyloxy group substituted with 3 linear or branched alkyl groups having 1 to 4 carbon atoms. The three alkyl or aryl groups substituted on the silyloxy group may be the same or different. The linear or branched alkyl group having 1 to 13 carbon atoms is preferably a branched alkyl group, and more preferably has a quaternary carbon atom.

[0053] p represents an integer of 1 to 4. Here, p is preferably an integer of 1 to 3, and more preferably 1 or 2.

[0054] Ring A is a set of p X 1 R 12 In addition, it may further have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom. Examples of halogen atoms include chlorine, fluorine, bromine, and iodine atoms. Examples of C1-6 alkyl groups which may be substituted with halogen atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, dichloromethyl, trichloromethyl, and trifluoromethyl groups. Examples of C1-6 alkoxy groups which may be substituted with halogen atoms include methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, trichloromethoxy, and trifluoromethoxy groups.

[0055] Ra represents a hydrogen atom or an aromatic ring which may be substituted with a halogen atom. Here, examples of the aromatic ring include a C6 to C18 aromatic hydrocarbon ring and a C4 to C10 aromatic heterocycle. Specific examples of the C6-18 aromatic hydrocarbon ring include a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a triphenylene ring, a tetracene ring, an indane ring, an indene ring, a fluorene ring, a biphenyl ring, etc. Among these, a benzene ring, a naphthalene ring, a phenanthrene ring, and a fluorene ring are more preferable. The C4-10 aromatic heterocycle is preferably a 5- to 10-membered heterocycle containing 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and specifically includes a pyrrole ring, a furan ring, a thiophene ring, an indole ring, a benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, an indazole ring, an imidazole ring, a pyridine ring, a quinoline ring, an isoquinoline ring, etc. Among these, a 5- to 8-membered heterocycle containing 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom is preferred, and a pyrrole ring, a furan ring, a thiophene ring, an indole ring, a benzofuran ring, a benzothiophene ring, a carbazole ring, a pyrazole ring, and an indazole ring are more preferred. The aromatic ring of Ra may be substituted with 1 to 3 halogen atoms.

[0056] Rb represents a hydrogen atom, an aromatic ring which may be substituted with a halogen atom, or a group represented by the above formula (b). In the formula (b), q represents an integer of 0 to 4. q is preferably 0 to 3, more preferably 1 to 3, and further preferably 1 or 2.

[0057] q X's 2 each independently represents a single bond, -O-, -S-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, or -NR 18 -(R 18 represents a hydrogen atom, an alkyl group or an aralkyl group. Here, R 18 is preferably a hydrogen atom, a C1 to 10 alkyl group, or a C7 to 20 aralkyl group. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group. Examples of the aralkyl group include C7 to C16 aralkyl groups, such as a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, and a 1-naphthylethyl group.

[0058] q R 14 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a);

[0059] [ka]

[0060] However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; 3 is an oxygen atom or -C(=O)NR 17 -(R 17represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and A represents either a silyl group or an alkyl group having a silyloxy group bonded thereto. R 14 The organic group represented by the above R 12 The same as the above R 12 The same is preferably used.

[0061] R 13 indicates a hydrogen atom or R 11 may be taken together with ring A to form a single bond to form a fluorene ring together with ring A and ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring.

[0062] Ring B is a ring of q X 2 R 14 In addition, it may further have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom. Examples of halogen atoms include chlorine, fluorine, bromine, and iodine atoms. Examples of C1-6 alkyl groups which may be substituted with halogen atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, dichloromethyl, trichloromethyl, and trifluoromethyl groups. Examples of C1-6 alkoxy groups which may be substituted with halogen atoms include methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, trichloromethoxy, and trifluoromethoxy groups.

[0063] Y is a hydroxyl group, a thiol group, or NHR 20 (R 20 represents a hydrogen atom, an alkyl group or an aralkyl group. ) or a halogen atom. Here, R 20is preferably a hydrogen atom, a C1 to 10 alkyl group, or a C7 to 20 aralkyl group. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group. Examples of the aralkyl group include C7 to C16 aralkyl groups, such as a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-phenylpropyl group, a naphthylmethyl group, and a 1-naphthylethyl group.

[0064] Among the compounds of formula (I) above, specific examples of preferred supports for liquid phase peptide synthesis include compounds represented by the following formulas (7), (20) and (21). As one of them, a compound represented by formula (7) can be used (Patent Documents 9 and 10).

[0065] [ka]

[0066] (Wherein, Yb is -CHOR 34 (where R 34 represents a hydrogen atom, a halogenocarbonyl group, an active ester carbonyl group, or an active ester sulfonyl group), -CH2NHR 35 (where R 35 represents a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or an aralkyl group, a halogenomethyl group, a formyl group, or an oxime group; R 21 , R 22 , R 23 , R 24 and R 25 At least one of the formulas (8)

[0067] [ka]

[0068] and the remainder represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms; R 26 represents a linear or branched alkylene group having 6 to 16 carbon atoms; X 3 O or CONR 36 (where R 36 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; A is formula (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), or (19).

[0069] [ka]

[0070] (where R 27 , R 28 , R 29 are the same or different and each represents a linear or branched alkyl group having 1 to 6 carbon atoms or an aryl group which may have a substituent; R 30 represents a single bond or a linear or branched alkylene group having 1 to 3 carbon atoms; R 31 , R 32 and R 33 each represents a linear or branched alkylene group having 1 to 3 carbon atoms. (representing a group represented by the formula:

[0071] As a support for liquid phase peptide synthesis, a compound represented by formula (20) can be used (Patent Documents 11, 12, and 15).

[0072] [ka]

[0073] (In the formula, X 4 -OR 51 (where R 51 represents a hydrogen atom, an active ester carbonyl group, or an active ester sulfonyl group), -NHR 35 , azide, halogen, isocyanate, X 5 Together with =N-OH or =O, X 4-OR 51 , -NHR 35 , azide or halogen X 5 represents a hydrogen atom, a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, or a cycloalkyl group; X 4 If isocyanate, X 5 represents a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms, or a cycloalkyl group; R 41 ~R 50 at least one of them represents a group represented by formula (2), and the remainder represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms; X 4 -OR 51 , -NHR 35 , azide or halogen, and X 5 is a hydrogen atom, or X 4 and X 5 When these are combined together =O, R 45 and R 46 may be bonded via an oxygen atom to form a xanthene ring)

[0074] As a support for liquid phase peptide synthesis, a compound represented by formula (21) can be used (Patent Documents 13 and 14).

[0075] [ka]

[0076] (In the formula, X 6 represents a hydroxyl group or a halogen atom, R 61 ~R 75 at least one of the groups represented by formula (2) is a group, and the remainder is a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms; R 70 and R 71 may be bonded via a single bond to form a fluorene ring, or may be bonded via an oxygen atom to form a xanthene ring).

[0077] The support for liquid phase peptide synthesis can also be bound to the carboxyl group of the raw material amino acid, peptide, amino acid amide or peptide amide (amino acid, etc.) via a linker. The linker referred to here is an organic group having two reactive groups, one of which binds to the carboxyl group of the amino acid or the like, and the other of which binds to the support for liquid phase peptide synthesis. A preferred linker is an organic group having a molecular weight of about 2000 or less (preferably about 1500 or less, more preferably about 1000 or less), and is a compound having at least two reactive groups, which may be the same or different, selected from the group consisting of amino groups, carboxyl groups, and halomethyl groups in the molecule. For example, the following compounds can be mentioned.

[0078] [ka]

[0079] [ka]

[0080] (wherein Y is an integer of 1 to 6, preferably 1 to 4).

[0081] [ka]

[0082] where X is a halogen atom, preferably chlorine or bromine.

[0083] [ka]

[0084] (In the formula, Z is an integer of 2 to 40, preferably 2 to 35, and more preferably 2 to 28). (The structural formula of the linker above shows the state before it is bonded to a side chain functional group, etc., and before it is bonded to a support for liquid phase peptide synthesis).

[0085] Next, the peptide elongation reaction will be described. The peptide elongation reaction preferably includes the following steps a, b, and c. The order of steps b and c does not matter, and the order may be step b followed by step c, i.e., the organic solvent layer containing the condensate is obtained after removing the protecting group of the amino group, or the order may be step c followed by step b, i.e., the organic solvent layer containing the condensate is obtained after removing the protecting group of the amino group. a. condensing an amino acid, peptide, amino acid amide, or peptide amide bound to a support for liquid phase peptide synthesis with an amino acid or peptide having a protected amino group in a solvent containing an organic solvent; b. removing the amino-protecting group of the compound having a protected amino group in the reaction solution; c. A process of adding an aqueous solution to the reaction solution, followed by separation to obtain an organic solvent layer containing a condensate of an amino acid, peptide, amino acid amide or peptide amide bound to a support for liquid phase peptide synthesis and an amino acid or peptide from which the amino-protecting group has been removed. In addition, the step a may be followed by a step of adding a quenching agent for amino acid active ester to the reaction solution after the condensation reaction. In addition, when the amino-protecting group is an Fmoc group, the step b may be followed by a step of adding a trapping agent for dibenzofulvene.

[0086] The amino acid, peptide, amino acid amide, or peptide amide bound to the support for liquid phase peptide synthesis described in step a (hereinafter, abbreviated as support-bound peptide for liquid phase peptide synthesis) can be produced as follows. First, the support for liquid phase peptide synthesis is dissolved in an organic solvent such as THF, and, for example, an amino acid or peptide protected with Fmoc and a condensing agent, for example, N,N'-diisopropylcarbodiimide (DIPCI), and a base, for example, DMAP, are added to perform condensation. Then, an N-Fmoc-support-bound peptide for liquid phase synthesis, which is an intermediate in which a support for liquid phase peptide synthesis is bound to the carboxy group of an amino acid or peptide, can be produced. Alternatively, the support for liquid phase peptide synthesis is dissolved in an organic solvent such as toluene, and, for example, an amino acid amide or peptide amide protected with Fmoc and an acid catalyst, for example, mesylic acid, are added to perform condensation. Then, an N-Fmoc-support-bound peptide for liquid phase synthesis, which is an intermediate in which a support for liquid phase peptide synthesis is bound to the amide group of an amino acid amide or peptide amide, can be produced.

[0087] The other raw material, an amino acid or peptide whose amino group is protected with an amino-protecting group (hereinafter, abbreviated as amino-protected amino acid), means an amino acid or peptide whose amino group is protected with an amino-protecting group, while its carboxyl group is not protected and is reactive. When an amino acid or peptide has one or more amino groups, it is sufficient that at least one of the amino groups is protected with an amino-protecting group. Examples of the amino-protecting group include an Fmoc group, a Boc group, and a Cbz group. Among these, the Fmoc group, which can be deprotected under basic conditions, is more preferable. In addition, when the amino-protected amino acid has a highly reactive functional group such as a hydroxyl group, an amino group, a guanidyl group, a carboxyl group, a thiol group, an indole group, or an imidazole group, a general protecting group used in peptide synthesis may be introduced into this functional group, and the target compound can be obtained by removing the protecting group as necessary at any time point after the completion of the reaction. Examples of the protecting group for a hydroxyl group include a tBu group, a Trt group, a Bz (benzoyl) group, an acetyl group, a silyl group, and the like. Examples of the protecting group for a guanidyl group include a Pbf group, a Boc group, a Pmc group, a nitro group, and the like. Examples of the protecting group for a carboxyl group include a tBu group, a methyl group, an ethyl group, a Bz group, and the like. Examples of the protecting group for a thiol group include a Trt group, an Acm (acetamidomethyl) group, a tBu group, a S-tBu (dithio-tert-butyl) group, a Dpm (diphenylmethyl) group, a MBom (4-methoxybenzyloxymethyl) group, and the like. Examples of the protecting group for an indole group include a Boc group, and examples of the protecting group for an imidazole group include a Boc group, a Bom (benzyloxymethyl) group, a Bum (tert-butoxymethyl) group, a Trt group, a Ddm (4,4'-dimethoxydiphenyl) group, a MBom group, and the like.

[0088] An amino-protected amino acid can be produced, for example, by reacting an amino acid or peptide whose amino group is to be protected with an amino-protecting group with Fmoc-OSu or the like in the presence of a base in a mixed solvent such as THF / water.

[0089] Step a of the present invention is a step of condensing the raw materials, and the reaction solvent used in step a is a solvent containing an organic solvent. If an amino acid, peptide, amino acid amide, or peptide amide is protected with the support for liquid phase peptide synthesis used in the present invention, the resulting peptide bound to the support for liquid phase peptide synthesis becomes soluble in an organic solvent, making liquid phase peptide synthesis possible. Examples of such organic solvents include THF, DMF, cyclohexane, CPME, 2-methylTHF, 4-methyltetrahydropyran (4-methylTHP), isopropyl acetate, chloroform, dichloromethane, N-methylpyrrolidone, dimethylacetamide (DMAc), and NFM (N-formylmorpholine), and preferred are THF, DMF, CPME, 2-methylTHF, 4-methylTHP, and N-methylpyrrolidone. Furthermore, a mixed solvent of two or more of the above solvents may be used.

[0090] The condensation reaction can be carried out by mixing the support for liquid phase peptide synthesis or the peptide bound to a support for liquid phase peptide synthesis, the amino-protected amino acid, a condensing agent and a base in a solvent containing the organic solvent.

[0091] The amount of amino-protected amino acid used relative to the carrier-bound peptide for liquid phase peptide synthesis is usually 1.01 to 4 equivalents, preferably 1.03 to 3 equivalents, more preferably 1.05 to 2 equivalents, and even more preferably 1.1 to 1.5 equivalents relative to the carrier-bound peptide for liquid phase peptide synthesis. In the peptide production method of the present invention, following step a, the active ester of the unreacted amino acid can be captured and inactivated with a quenching agent that is added thereafter. Therefore, even if an excess of amino-protected amino acid is used, there is no problem of residues.

[0092] Condensing agents that are generally used in peptide synthesis can also be used in the present invention, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride (DMT-MM), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU( 6-Cl), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl). Preferred are DMT-MM, HBTU, HATU, or COMU. The amount of the condensing agent used is preferably 1 to 4 equivalents, more preferably 1 to 2 equivalents, and even more preferably 1.05 to 1.45 equivalents relative to the peptide bound to the support for liquid phase peptide synthesis. As the base, bases generally used in peptide synthesis can be used in the present invention. For example, DIPEA (N,N-diisopropylethylamine), DMAP, NMM (N-methylmorpholine), and TMP (2,4,6-trimethylpyridine) can be mentioned. DIPEA is preferable.

[0093] In the condensation step, an activator is preferably added to promote the reaction and suppress side reactions such as racemization. Here, the activator is a reagent that, in the presence of a condensation agent, converts an amino acid into a corresponding active ester, symmetrical acid anhydride, or the like, and facilitates the formation of a peptide bond (amide bond). As the activator, an activator generally used in peptide synthesis can be used. For example, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-ethyl triazolecarboxylate (HOCt), 1-hydroxy-7-azabenzotriazole (HOAt), 3-hydroxy-4-ketobenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (HOPht), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), pentafluorophenol, ethyl cyano(hydroxyimino)acetate (Oxyma), and the like can be mentioned. Preferred are HOBt, HOOBt, HOCt, HOAt and Oxyma. The amount of the activator used is preferably 1 to 4 equivalents, more preferably 1 to 2 equivalents, and further preferably 1.05 to 1.45 equivalents relative to the support-bound peptide for liquid phase peptide synthesis.

[0094] The amount of the solvent used is such that the concentration of the carrier-bound peptide for liquid phase peptide synthesis or the like dissolved therein is preferably 0.1 mM to 1 M, and more preferably 1 mM to 0.5 M. The reaction temperature is preferably a temperature generally used in peptide synthesis, for example, −20 to 40° C., more preferably 0 to 30° C. The condensation reaction time is usually 1 minute to 30 hours.

[0095] Step a may be followed by a step of adding an amino acid active ester quenching agent (hereinafter, sometimes referred to as "quenching agent") to the reaction solution after the condensation reaction. In step a, the amino-protected amino acid as the raw material is added in an excess amount relative to the support for liquid phase peptide synthesis or the peptide bound to the support for liquid phase peptide synthesis. Therefore, the amino acid active ester activated by the amino-protected amino acid during the condensation reaction remains in excess after the condensation reaction. This step is to quench this excess amino acid active ester. The amount of the quenching agent added in this step is preferably 1 to 10 equivalents, more preferably 1 to 6 equivalents, and even more preferably 1 to 3 equivalents relative to 1 equivalent of the theoretically remaining active amino acid ester.

[0096] The quenching agent for the amino acid activated ester is a compound having an amino group in the molecule, and compounds described in Japanese Patent No. 6703668, Japanese Patent No. 6713983, International Publication No. 2021 / 132545, Molecules 2021, 26, 3497-3505, etc. can be used. As the quenching agent, hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, alkylamine having a primary or secondary amine, aromatic amine having a primary or secondary amine, or tertiary amine can be used. Furthermore, since the excess quenching agent can be removed into the water layer by liquid-liquid separation, it is preferable that the quenching agent is water-soluble, and amines having hydrophilic substituents such as hydroxyl, sulfo, sulfate, and phosphate groups are preferable. Furthermore, the number of amino groups in the compound may be one (monovalent) or may be divalent or more. Examples of alkylamines that can be used include alkylamines having 1 to 14 carbon atoms, preferably alkylamines having 2 to 10 carbon atoms, more preferably alkylamines having 2 to 8 carbon atoms, and even more preferably alkylamines having 3 to 4 carbon atoms. Examples of aromatic amines that can be used in the present invention include aromatic amines having 1 to 14 carbon atoms, and preferably aromatic amines having 6 to 10 carbon atoms. Specific examples of amines include, but are not limited to, propylamine, methylamine, hexylamine, benzylamine, aniline, toluidine, 2,4,6-trimethylaniline, anisidine, phenetidine, hydroxylamine, 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, piperazine, 2-(2-aminoethoxy)ethanol (AEE), taurine, 2-aminoethyl hydrogen sulfate (2-aminoethyl sulfate, AEHS), etc. In addition, NMI (N-methylimidazole), DMAP, and trimethylamine can be mentioned. Among them, 2-(2-aminoethoxy)ethanol (AEE), taurine, and 2-aminoethyl hydrogen sulfate (2-aminoethyl sulfate, AEHS) are preferred.

[0097] Step b is a step of removing the amino-protecting group from the amino-protected amino acid in the reaction solution. The step of removing the amino-protecting group differs depending on the type of the amino-protecting group. For example, when the amino-protecting group is an Fmoc group, the reaction solution may be placed under basic conditions. When the amino-protecting group is a Boc group, the reaction solution may be placed under acidic conditions. When the amino-protecting group is a Cbz group, catalytic reduction may be performed. Of these, it is more preferable to use an Fmoc group as the amino-protecting group for one-pot liquid-phase synthesis.

[0098] The process for removing the amino-protecting group when the amino-protecting group is an Fmoc group will be described below. In the Fmoc removal step, it is sufficient to make the reaction solution basic, and any amine compound can be used, for example, tertiary amines such as 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), 1,4-diazabicyclo[2.2.2]-octane (DABCO), triethylamine, tributylamine, etc.; or divalent or higher water-soluble amines having at least one primary or secondary amino group, such as 1-methylpiperazine, 4-aminopiperidine, diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, piperidine, piperazine, etc. Preferred are DBU, DBN, piperidine, piperazine, 1-methylpiperazine, 4-aminopiperidine, and diethylenetriamine, and more preferred are DBU, piperidine, piperazine, and 1-methylpiperazine. The amount of the amine compound added in the step b is 1 to 30 equivalents, preferably 4 to 20 equivalents, and more preferably 4 to 10 equivalents relative to the amount of Fmoc groups present in the system.

[0099] In addition to the amine compound, it is preferable to add a trapping agent for DBF (dibenzofulvene) and an adduct of DBF and an amine (DBF-amine adduct) generated by the Fmoc deprotection reaction. Examples of the trapping agent for DBF and DBF-amine adduct used here include mercapto compounds. The mercapto compound that can be used is not particularly limited as long as it has a mercapto group and the compound reacted with DBF is water-soluble, and examples thereof include mercapto compounds having an alkyl group with 1 to 10 carbon atoms and having one or more substituents selected from carboxylic acid, alkali metal salt of carboxylic acid, sulfonic acid, or alkali metal salt of sulfonic acid, and examples thereof include compounds represented by the following general formula (4) or (5).

[0100] [ka]

[0101] (In the formula, L1 and L2 each represent a divalent organic group, and M represents a hydrogen atom or an alkali metal.)

[0102] L1 and L2 in general formula (4) or (5) each represent a divalent organic group. The divalent organic group is preferably a divalent organic group having 1 to 10 carbon atoms, more preferably a linear or branched alkylene group having 1 to 10 carbon atoms which may have a mercapto group, an arylene group having 6 to 10 carbon atoms which may have a mercapto group, or a heteroarylene group having 4 to 9 carbon atoms which may have a mercapto group. Specific examples of the divalent organic group include a methylene group, an ethylene group, a trimethylene group, a propylene group, a mercaptotrimethylene group, a mercaptopropylene group, a tetramethylene group, a butylene group, a pentamethylene group, a phenylene group, a naphthylene group, an indole group, a benzimidazole group, a quinolyl group, and an isoquinoline group. M represents a hydrogen atom or an alkali metal, specifically, a hydrogen atom, sodium, or potassium. Specific examples include 3-mercaptopropionic acid, thiomalic acid, cysteine, sodium mercaptomethanesulfonate, sodium 2-mercaptoethanesulfonate, 2-mercaptoethanesulfonic acid, 3-mercaptopropanesulfonic acid, sodium 3-mercaptopropanesulfonate, 1,3-dimercaptopropanesulfonic acid, and sodium 2-mercaptobenzimidazole-5-sulfonate, with 3-mercaptopropanesulfonic acid being preferred.

[0103] The amount of the mercapto compound added is preferably 1 to 30 equivalents relative to the amount of Fmoc groups, more preferably 1 to 10 equivalents, and even more preferably 1 to 5 equivalents. The amine compound and the mercapto compound may be added simultaneously, or the mercapto compound may be added first and then the amine compound. The Fmoc removal step may be carried out at a temperature of -20 to 40°C for 1 minute to 5 hours.

[0104] Step c is a step of adding an aqueous solution to the reaction solution, followed by separation to obtain an organic solvent layer containing a condensate of an amino acid, peptide, amino acid amide or peptide amide bound to the support for liquid phase peptide synthesis and an amino acid or peptide from which the amino-protecting group has been removed. After adding an aqueous solution to the reaction solution in step b, the aqueous layer and the organic solvent layer are separated. The aqueous layer contains the condensate of the amino acid or peptide from which the amino-protecting group has been removed and the active ester quenching agent, and the DBF-trapping agent adduct. That is, the condensate of the amino acid or peptide from which the amino-protecting group has been removed and the active ester quenching agent is easily extracted into the aqueous layer by simply adding the aqueous solution in step c. On the other hand, the organic solvent layer contains a condensate of an amino acid, peptide, amino acid amide or peptide amide bound to the support for liquid phase peptide synthesis and an amino acid or peptide from which the amino-protecting group has been eliminated. The aqueous solution used here may be water or an aqueous solution having a pH in the vicinity of neutral to basic ranges.Specific examples include water, an aqueous sodium chloride solution, an aqueous calcium chloride solution, an aqueous cesium chloride solution, an aqueous potassium chloride solution, an aqueous lithium chloride solution, an aqueous sodium carbonate solution, an aqueous potassium carbonate solution, an aqueous cesium carbonate solution, an aqueous disodium hydrogen phosphate solution, an aqueous trisodium phosphate solution, an aqueous sodium hydrogen carbonate solution, an aqueous potassium hydrogen carbonate solution, an aqueous dipotassium hydrogen phosphate solution, an aqueous tripotassium phosphate solution, or a mixed solvent of these aqueous solutions with DMF, DMSO, NFM, or NMP.

[0105] The obtained Ganirelix can be converted, if necessary, into an organic acid salt such as acetate, formate, oxalate, succinate or trifluoroacetate, or into a mineral acid salt such as hydrochloride, nitrate, sulfate or phosphate.

[0106] Thus, according to the method of the present invention, by simply adding an aqueous solution and performing liquid separation, poor liquid separation between the amino acid activated ester and the product peptide does not occur. In addition, since solid-liquid separation is not required, one-pot synthesis is possible without the need for complicated separation operations. The above-mentioned series of steps may be carried out using microflow technology. A peptide synthesis technique using microflow technology is described, for example, in Nature Communications 7, Article number: 13491 (2016). EXAMPLES

[0107] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0108] Reference example 1 Synthesis of Fmoc-NH(D2-STag)

[0109] [ka]

[0110] (O=(D2-STag), OH(D2-STag), Fmoc-NH(D2-STag) indicate the structures in the formula.)

[0111] Reference example (a) 41.4g (47.7mmol) of O=(D2-STag) (manufactured by Sekisui Medical Co., Ltd.) was dissolved in a mixed solution of 228mL of toluene and 36.0mL of methanol, 2.16g (57.2mmol) of sodium borohydride was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was washed three times with 83.0mL of water. 20.0g of anhydrous sodium sulfate was added to the obtained organic layer, and the mixture was thoroughly stirred and then filtered to obtain a toluene solution containing OH(D2-STag).

[0112] Reference example (b) 13.7g (57.2mmol) of 9-fluorenylmethyl carbamate and 1.80g (14.3mmol) of oxalic acid dihydrate were added to the toluene solution obtained in the previous step, and the mixture was stirred at 80℃ for 3 hours. The reaction solution was cooled to room temperature, and 414mL of methanol:water = 9:1 and 414mL of heptane were added and separated. The obtained organic layer was washed once with 207mL of 5% aqueous sodium hydrogen carbonate solution (5%Na2CO3aq.) and three times with 414mL of methanol:water = 9:1. The obtained organic layer was concentrated under reduced pressure, and 83.0mL of tetrahydrofuran was added to the residue and concentrated under reduced pressure. The residue was dissolved in 62mL of tetrahydrofuran and added dropwise to 830mL of methanol. The precipitated solid was collected by filtration and dried under reduced pressure to obtain 45.5g of Fmoc-NH(D2-STag). ESIMS (m / z) 1107.9 (M+NH4) +

[0113] Example 1 Synthesis of Ganirelix using Fmoc-hArg(Et)2(Boc)2-OH and Fmoc-D-hArg(Et)2(Boc)2-OH (2-(2-Aminoethoxy)ethanol (AEE) was used as a quenching agent for the Fmoc-amino-active ester.) In the following, any amino acid not specified as being D-form refers to the L-form.

[0114] [ka]

[0115] In the reaction formula, (NH2(D2-STag), HD-Ala-NH(D2-STag), H-Pro-D-Ala-NH(D2-STag), Fmoc-hArg(Et)2(Boc)2-OH, H-hArg(Et)2(Boc)2-Pro- D-Ala-NH(D2-STag), HD-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-A la-NH(D2-STag), Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag), Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser-Tyr-D-hArg(Et)2-Leu-hArg(Et)2-Pro-D-Ala-NH2·nTFA) show the structures in the reaction scheme. Note that Fmoc-hArg(Et)2(Boc)2-OH shows the mixture of the three isomers in the reaction scheme. Therefore, H-hArg(Et)2(Boc)2-Pro-D-Ala-NH (D2-STag) is also presumed to be a mixture of the three isomers. Furthermore, HD-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag) and Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag) are assumed to be three isomers in the reaction scheme for hArg(Et)2(Boc)2 and D-hArg(Et)2(Boc)2, respectively, and may be a mixture of a total of nine isomers.) Also, the * in the structure of R' indicates the attachment point.

[0116] Example (1-a) 2.00 g (1.83 mmol) of Fmoc-NH(D2-STag) was dissolved in 29.3 mL of cyclopentyl methyl ether (CPME), 0.542 g (3.04 mmol) of 3-mercapto-1-propanesulfonic acid sodium salt (MPS) dissolved in 7.33 mL of DMF and 2.55 mL of dimethyl sulfoxide (DMSO) was added, and 0.111 g (0.62 mmol) of MPS was added in the solid state, and 0.548 mL (3.67 mmol) of 2,3,4,6,7,8,9,10-Octahydropyrimidol[1,2-a]azepine(DBU) was added, and the mixture was stirred at room temperature for 1 hour and 35 minutes. The mixture was cooled to 8°C, and 0.319mL (1.83mmol) of N,N-diisopropylethylamine (DIPEA), 3.67mL (3.67mmol) of 1N sulfuric acid, 24.2mL of water, and 1.00mL of CPME were added and separated. 4.55mL of DMF and 6.07mL of 50% aqueous dipotassium hydrogen phosphate (50%K2HPO4aq.) were added to the obtained organic layer, and the mixture was washed and separated to obtain a mixture containing NH2(D2-STag).

[0117] Example (1-b) To the resulting mixture, 1.00 mL of CPME, 8.30 mL of DMF, 0.725 g (2.20 mmol) of Fmoc-D-Ala-OH·HO, 1.28 mL (7.33 mmol) of DIPEA, and 0.942 g (2.20 mmol) of COMU were added and stirred at room temperature for 45 minutes. 44.0 μL (0.444 mmol) of 2-(2-Aminoethoxy)ethanol (AEE) was added and stirred at room temperature for 15 minutes. 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO and 0.284 g (1.59 mmol) of solid MPS were added, cooled to 10°C, and 1.43 mL (9.54 mmol) of DBU was added and stirred for 1 hour and 20 minutes. 11.5 mL (11.5 mmol) of 1N sulfuric acid, 20.2 mL of water, and 0.796 mL of CPME were added and separated. 5.10 mL of DMF and 6.80 mL of 50% K2HPO4aq. were added to the obtained organic layer, and the mixture was separated and washed to obtain a mixture containing HD-Ala-NH(D2-STag).

[0118] Example (1-c) To the resulting mixture, 2.20mL of CPME, 8.30mL of DMF, 0.782g (2.20mmol) of Fmoc-Pro-OH·H2O, 1.28mL (7.33mmol) of DIPEA, and 0.942g (2.20mmol) of COMU were added and stirred at room temperature for 50 minutes. 44.0μL (0.444mmol) of AEE was added and stirred at room temperature for 15 minutes. 0.501g (2.81mmol) of MPS dissolved in 2.35mL of DMSO and 0.284g (1.59mmol) of solid MPS were added and cooled to 8℃. 1.43mL (9.54mmol) of DBU was added and stirred for 1 hour and 20 minutes. 11.5mL (11.5mmol) of 1N sulfuric acid, 20.2mL of water, and 0.861mL of CPME were added and separated. To the obtained organic layer, 5.11 mL of DMF and 6.81 mL of 50% K2HPO4 aq. were added, and the mixture was separated and washed to obtain a mixture containing H-Pro-D-Ala-NH(D2-STag).

[0119] Example (1-d) To the resulting mixture, 2.70 mL of CPME, 8.30 mL of DMF, 1.47 g (2.20 mmol) of Fmoc-hArg(Et)2(Boc)2-OH (a mixture of the three isomers shown in the reaction scheme), 1.28 mL (7.33 mmol), and 0.942 g (2.20 mmol) of COMU were added and stirred at room temperature for 50 minutes. 44.0 μL (0.444 mmol) of AEE was added and stirred at room temperature for 15 minutes. 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO and 0.284 g (1.59 mmol) of solid MPS were added, cooled to 8°C, and 1.43 mL (9.54 mmol) of DBU was added and stirred for 1 hour and 50 minutes. 11.5mL (11.5mmol) of 1N sulfuric acid, 20.3mL of water, and 0.950mL of CPME were added and separated.*1 5.12mL of DMF and 6.83mL of 50% K2HPO4aq. were added to the obtained organic layer, and the mixture was separated and washed to obtain a mixture containing H-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag). ESIMS(m / z) 1463.7 (M+H) + (Mixture of 3 isomers) *1 HPLC purity of the target substance in the organic layer: 76.2% HPLC analysis conditions (1) Column: YMC-Pack Pro C18, S-5μm, 12nm, 250mm×4.6mmI.D. Mobile phase A: 500 mM sodium perchlorate aq. Mobile phase B: THF Flow rate: 1.0mL / min Column temperature: 45℃ Detection wavelength: 280nm Gradient conditions: 75%B (0 min) → 90%B (10 min) → 90%B (20 min) → 75%B (21 min) → 75%B (33 min)

[0120] Example (1-e) As in Example (1-d), the peptide was elongated using Fmoc-Leu-OH, Fmoc-D-hArg(Et)2(Boc)2-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal(3)-OH, Fmoc-D-pClPhe-OH, and Fmoc-D-Nal(2)-OH to obtain a mixture containing HD-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag). Note that Fmoc-D-hArg(Et)2(Boc)2-OH (a mixture of three isomers) has the following structure, with * indicating the binding point.

[0121] [ka]

[0122] Example (1-f) To the resulting mixture, 0.800 mL of CPME, 8.95 mL of DMF, 1.28 mL (7.33 mmol), 0.126 mL (2.20 mmol) of acetic acid, and 0.942 g (2.20 mmol) of COMU were added and stirred at room temperature for 50 minutes. The reaction solution was concentrated under reduced pressure, and the resulting residue was added dropwise to 106 mL of acetonitrile (MeCN). The solid was collected by filtration, and the filter cake was washed with 50.0 mL of MeCN. 106 mL of MeCN was added to the filter cake, and after stirring, the solid was collected by filtration, and the filter cake was washed with 50.0 mL of MeCN. The resulting solid was dried under reduced pressure to give 3.98 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag). ESIMS (m / z) 1467.3 (M+2H) 2+

[0123] Example (1-g) A mixed solution of 13.9 mL (181 mmol) of trifluoroacetic acid, 0.365 mL (1.78 mmol) of triisopropylsilane, and 0.365 mL (20.3 mmol) of water was cooled to 5°C, and 1.50 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag) was added. After 5 minutes, the temperature was raised to room temperature and stirred for 6 hours and 55 minutes. The reaction solution was added dropwise to methyl tert-butyl ether (MTBE) cooled to 10°C, centrifuged at 5°C and 4400 rpm for 1 minute, and the supernatant was removed by decantation to obtain a precipitate. The MTBE washing, centrifugation, and decantation were repeated three more times to obtain a precipitate, which was then dried under reduced pressure to give 0.929 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser-Tyr-D-hArg(Et)2-Leu-hArg(Et)2-Pro-D-Ala-NH2·nTFA. ESIMS (m / z) 785.5 (M+2H) 2+ HPLC purity: 94.3% HPLC analysis conditions (2) Column: Inertstain, S-3μm, 250mm×2.1mmI.D. Mobile phase A: 0.1% TFA aq. Mobile phase B: MeCN containing 0.1% TFA Flow rate: 0.22mL / min Column temperature: 33℃ Detection wavelength: 225 nm Gradient conditions: 33%B (0 min) → 47%B (40 min) → 95%B (60 min) → 95%B (65 min) → 33%B (68 min) → 33%B (85 min)

[0124] Reference example 2 Synthesis of H-hArg(Et)2-Pro-D-Ala-NH (D2-STag) using Fmoc-hArg(Et)2-OH·HCl (Fmoc-amino-active ester quenching agent: AEE)

[0125] [ka]

[0126] (Fmoc-hArg(Et)2-OH·HCl and H-hArg(Et)2-Pro-D-Ala-NH(D2-STag) are shown in the reaction scheme.)

[0127] Reference examples (1-a), (1-b), (1-c) In the same manner as in Examples 1-a, 1-b, and 1-c, a mixed solution containing H-Pro-D-Ala-NH(D2-STag) was obtained from 2.00 g (1.83 mmol) of Fmoc-NH(D2-STag).

[0128] Reference example (1-d) To the resulting mixture, 2.60 mL of CPME, 8.30 mL of DMF, 1.11 g (2.20 mmol) of Fmoc-hArg(Et)2-OH·HCl, 1.28 mL (7.33 mmol) of DIPEA, and 0.942 g (2.20 mmol) of COMU were added and stirred at room temperature for 50 minutes. 44.0 μL (0.444 mmol) of AEE was added and stirred at room temperature for 15 minutes. 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO and 0.284 g (1.59 mmol) of solid MPS were added, cooled to 8 °C, 1.43 mL (9.54 mmol) of DBU was added, and the mixture was stirred for 1 hour and 50 minutes. An attempt was made to separate the solution by adding 11.5 mL (11.5 mmol) of 1N sulfuric acid, 20.3 mL of water, and 0.950 mL of CPME, but an emulsion*2 formed and separation was not possible. ESIMS (m / z) 1263.5 (M+H) + *2 HPLC purity of the target substance in the emulsion: 10.4% HPLC analysis conditions: the same as in Example (1-d)(1).

[0129] Example 2 Synthesis of Ganirelix using Fmoc-hArg(Et)2(Boc)2-OH and Fmoc-D-hArg(Et)2(Boc)2-OH (Quenching agent for Fmoc-amino-active ester: 2-aminoethyl hydrogen sulfate (2-AEHS))

[0130] [ka]

[0131] Example (2-a) A mixed solution containing NH2(D2-STag) was obtained from 0.800 g (0.733 mmol) of Fmoc-NH(D2-STag) in the same manner as in Example (1-a).

[0132] Example (2-b) To the resulting mixture, 0.600 mL of CPME, 3.30 mL of DMF, 0.290 g (0.880 mmol) of Fmoc-D-Ala-OH·HO, 0.511 mL (2.93 mmol) of DIPEA, and 0.377 g (0.880 mmol) of COMU were added and stirred at room temperature for 50 min. 2-AEHS (24.8 mg (0.176 mmol)) dissolved in 0.704 mL of DMSO was added and stirred at room temperature for 15 min. MPS (50.4 mg (0.283 mmol)) dissolved in 0.237 mL of DMSO and solid MPS (0.263 g (1.48 mmol)) were added, cooled to 8 °C, DBU (0.597 mL (3.99 mmol)) was added, and the mixture was stirred for 1 h 50 min. 4.60 mL (4.60 mmol) of 1N sulfuric acid, 8.30 mL of 10% aqueous sodium carbonate solution (10% Na2CO3aq.), and 0.319 mL of CPME were added and separated. 2.04 mL of DMF and 2.72 mL of 50% K2HPO4aq. were added to the obtained organic layer, which was then washed and separated to obtain a mixture containing HD-Ala-NH(D2-STag).

[0133] Example (2-c) To the resulting mixture, 0.800 mL of CPME, 3.30 mL of DMF, 0.313 g (0.880 mmol) of Fmoc-Pro-OH·H2O, 0.511 mL (2.93 mmol) of DIPEA, and 0.377 g (0.880 mmol) of COMU were added and stirred at room temperature for 50 min. 2-AEHS (24.8 mg (0.176 mmol)) dissolved in 0.704 mL of DMSO was added and stirred at room temperature for 15 min. MPS (50.4 mg (0.283 mmol)) dissolved in 0.237 mL of DMSO and solid MPS (0.263 g (1.48 mmol)) were added, cooled to 6°C, DBU (0.597 mL (3.99 mmol)) was added, and the mixture was stirred for 1 h 20 min. 4.60 mL (4.60 mmol) of 1N sulfuric acid, 8.30 mL of 10% Na2CO3 aq., and 0.345 mL of CPME were added and separated. 2.04 mL of DMF and 2.72 mL of 50% K2HPO4 aq. were added to the obtained organic layer, and the mixture was separated and washed to obtain a mixture containing H-Pro-D-Ala-NH (D2-STag).

[0134] Example (2-d) To the resulting mixture, 0.600 mL of CPME, 3.30 mL of DMF, 0.587 g (0.880 mmol) of Fmoc-hArg(Et)2(Boc)2-OH (a mixture of the three isomers shown in the figure), 0.511 mL (2.93 mmol), and 0.377 g (0.880 mmol) of COMU were added and stirred at room temperature for 50 minutes. 24.8 mg (0.176 mmol) of 2-AEHS dissolved in 0.704 mL of DMSO was added and stirred at room temperature for 15 minutes. 50.4 mg (0.283 mmol) of MPS dissolved in 0.237 mL of DMSO and 0.263 g (1.48 mmol) of solid MPS were added, cooled to 7°C, and 0.597 mL (3.99 mmol) of DBU was added and stirred for 1 hour and 50 minutes. 4.60mL (4.60mmol) of 1N sulfuric acid, 8.40mL of 10% Na2CO3aq., and 0.380mL of CPME were added and separated. 3 2.05 mL of DMF and 2.73 mL of 50% K2HPO4 aq. were added to the obtained organic layer, and the mixture was separated and washed to obtain a mixture containing H-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag). ESIMS (m / z) 1463.6 (M+H) + (Mixture of 3 isomers) *3 HPLC purity: 78.0% HPLC analysis conditions: the same as in Example (1-d)(1).

[0135] Example (2-e) As in Example (2-d), the peptide was elongated using Fmoc-Leu-OH, Fmoc-D-hArg(Et)2(Boc)2-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Pal(3)-OH, Fmoc-D-pClPhe-OH, and Fmoc-D-Nal(2)-OH to obtain a mixture containing HD-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH (D2-STag).

[0136] Example (2-f) In the same manner as in Example (1-f), 1.36 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH(D2-STag) was obtained. ESIMS (m / z) 1467.3 (M+2H) 2+

[0137] Example (2-g) In a similar manner to Example (1-g), 0.455 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser-Tyr-D-hArg(Et)2-Leu-hArg(Et)2-Pro-D-Ala-NH2·nTFA was obtained from 0.700 g of Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et)2(Boc)2-Leu-hArg(Et)2(Boc)2-Pro-D-Ala-NH2 (D2-STag). ESIMS (m / z) 785.5 (M+2H) 2+ HPLC purity: 94.4% HPLC analysis conditions: the same as in Example (1-g)(2).

[0138] The liquid separation state of Example (1-d) *1 and Reference Example (1-d) *2 is shown in Figures 1 and 2. In Example (1-d) shown in Figure 1, the interface between the organic layer and the aqueous layer was clear, and liquid-liquid separation was good. On the other hand, in Reference Example (1-d) shown in Figure 2, the entire inside of the separatory funnel became an emulsion, and even after leaving it to stand at room temperature for 25 minutes, the emulsion did not disappear, and separation into the organic layer and the aqueous layer was not possible. Next, the HPLC analysis results of *1 in Example 1, *3 in Example 2, and *2 in Reference Example 1 are shown in Table 1. In Examples 1 and 2, in which Fmoc-hArg(Et)2(Boc)2-OH protected with a Boc group of the present invention was used, separation was good, and H-hArg(Et)2(Boc)2-Pro-D-Ala-NH (D2-STag) was synthesized with high purities of 76.2% and 78.0%. In Examples 1 and 2, the peptide elongation reaction was continued, and the crude product of ganirelix, which was the target product, was obtained with high purities of 94.3% and 94.4%. On the other hand, in Reference Example 1, in which Fmoc-hArg(Et)2-OH·HCl, which is only proton protected instead of a Boc group, was used, separation was not possible when H-hArg(Et)2-Pro-D-Ala-NH (D2-STag) was synthesized. To continue the synthesis of ganirelix, it was necessary to purify H-hArg(Et)2-Pro-D-Ala-NH (D2-STag) by a method other than liquid-liquid separation, which was industrially disadvantageous. Furthermore, the purity of H-hArg(Et)2-Pro-D-Ala-NH (D2-STag) in the emulsion was remarkably low at 10.4%.

[0139] [Table 1]

[0140] From the above results, it was found that by using the Boc-protected Fmoc-hArg(Et)2(Boc)2-OH of the present invention instead of the proton-protected Fmoc-hArg(Et)2-OH·HCl of the conventional technology, the liquid separation properties are improved and ganirelix can be obtained in an industrially advantageous manner.

Claims

1. A method for producing ganirelix or a salt thereof by liquid phase peptide synthesis, comprising: 【Chemistry 1】 (In the formula, R 1 and R 2 represents Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde; R 3 indicates an amino protecting group) and a support for liquid phase peptide synthesis.

2. The method according to claim 1, comprising the following steps a) to c). a. A step of condensing an amino acid, peptide, amino acid amide or peptide amide bound to a support for liquid phase peptide synthesis with an amino acid or peptide having a protected amino group in a solvent containing an organic solvent; b. A step of removing the amino-protecting group of the compound having a protected amino group in the reaction solution; c) A step of adding an aqueous solution to the reaction solution, followed by separation to obtain an organic solvent layer containing a condensate of an amino acid, peptide, amino acid amide or peptide amide bound to the support for liquid phase peptide synthesis and an amino acid or peptide from which the amino-protecting group has been removed.

3. 3. The method according to claim 1, further comprising the step of adding a quenching agent for the amino acid active ester to the reaction solution after the condensation reaction subsequent to the step a.

4. The raw amino acid is D-AlaNH from the C-terminus. 2 ,Pro,hArg(Et) 2 , Leu, D-hArg(Et) 2 4. The method according to claim 1, wherein the order of the aryl groups is Tyr, Ser, D-3-pyridyl Ala, Dp-chloroPhe, and D-naphthyl Ala.

5. The R 1 and R 2 is Boc, and R 3 The method according to any one of claims 1 to 4, wherein is Fmoc or Cbz.

6. The R 1 and R 2 is Boc, and R 3 The method according to any one of claims 1 to 5, wherein is Fmoc.

7. The method according to any one of claims 3 to 6, wherein the quenching agent for the amino acid active ester is a water-soluble amine.

8. The method according to claim 7, wherein the water-soluble amine is hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, or a primary, secondary or tertiary amine having one or more selected from an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aralkyl group and a heterocyclic group, which may have one or more substituents selected from a hydroxy group, an ether bond, an alkoxy group, a sulfonyl group, a sulfonic acid group, a sulfate group and a phosphate group.

9. R 3 The method according to any one of claims 2 to 8, further comprising the step of adding a trapping agent for dibenzofulvene subsequent to step b when is Fmoc.

10. The method according to claim 9, wherein the trapping agent for dibenzofulvene is a mercapto compound having an alkyl group having 1 to 10 carbon atoms and having one or more substituents selected from carboxylic acid, an alkali metal salt of a carboxylic acid, sulfonic acid, or an alkali metal salt of a sulfonic acid.

11. The method according to any one of claims 1 to 10, wherein the support for liquid phase peptide synthesis is a compound represented by the following formula (I): 【Chemistry 2】 [In the formula, Ring A represents a C4-20 aromatic ring which may contain a heteroatom and may be polycyclic; R 11 is a hydrogen atom, or when ring A is a benzene ring and Rb is a group represented by the following formula (b), R 13 may be taken together with a single bond to form a fluorene ring together with ring A and ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring; p X's 1 each independently represents a single bond, —O—, —S—, —C(═O)O—, —C(═O)NH—, —NHC(═O)—, or —NR 15 - (R 15 represents a hydrogen atom, an alkyl group, or an aralkyl group; p R 12 each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a); 【Chemistry 3】 However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; X 3 is an oxygen atom or -C(=O)NR 17 - (R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and A represents a silyl group or an alkyl group having a silyloxy group bonded thereto; p represents an integer of 1 to 4; Ring A is p X 1 R 12 and optionally has a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Ra represents a hydrogen atom or an aromatic ring optionally substituted with a halogen atom; Rb is a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by formula (b): 【Chemistry 4】 (In the formula, * indicates a bond position; q represents an integer of 0 to 4; q X's 2 each independently represents a single bond, —O—, —S—, —C(═O)O—, —C(═O)NH—, —NHC(═O)—, or —NR 18 - (R 18 represents a hydrogen atom, an alkyl group, or an aralkyl group; q R 14 are each independently represents an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic group represented by formula (a); 【Chemistry 5】 However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms; X 3 is an oxygen atom or -C(=O)NR 17 - (R 17 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and A represents a silyl group or an alkyl group having a silyloxy group bonded thereto; R 13 represents a hydrogen atom, or R 11 may be taken together with a single bond to form a fluorene ring together with ring A and ring B, or may be taken together with ring A and ring B via an oxygen atom to form a xanthene ring; Ring B is a ring having q X 2 R 14 In addition, it may further have a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted with a halogen atom, and a C1-6 alkoxy group optionally substituted with a halogen atom; Y is a hydroxyl group, a thiol group, or NHR 20 (R 20 represents a hydrogen atom, an alkyl group or an aralkyl group, or represents a halogen atom.

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