WO2023033017A1

WO2023033017A1 - Method for producing ganirelix or salt thereof

Info

Publication number: WO2023033017A1
Application number: PCT/JP2022/032700
Authority: WO
Inventors: 真也矢野; 卓松本
Original assignee: 積水メディカル株式会社
Priority date: 2021-09-01
Filing date: 2022-08-31
Publication date: 2023-03-09
Also published as: TW202328164A

Abstract

Provided is a novel method for producing ganirelix by liquid phase peptide synthesis method.　The present invention pertains to a method that is for producing ganirelix or a salt thereof by a liquid phase peptide synthesis method, and that uses a binding product between a compound represented by formulae (1)-(3) (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 the third and fifth diethyl homoarginine residues from the C terminal.

Description

Method for producing Ganirelix or its salt

The present invention relates to a method for producing a gonadotropin antagonist Ganirelix or a salt thereof.

Ganirelix is a gonadotropin antagonist and is marketed as an anti-premature ovulatory drug under controlled ovarian stimulation. Ganirelix is a decapeptide drug having diethylhomoarginine residues at the 3rd and 5th positions from the C-terminus, and is produced by a solid-phase peptide synthesis method (Patent Documents 1 and 2, Non-Patent Document 1).

CN102584945A WO2015/188774 Japanese Patent No. 5113118 Patent No. 4500854 Japanese Patent No. 5929756 Japanese Patent No. 6092513 Japanese Patent No. 5768712 Japanese Patent No. 5803674 Japanese Patent No. 6116782 Japanese Patent No. 6201076 Japanese Patent No. 6283774 Japanese Patent No. 6283775 Japanese Patent No. 6322350 Japanese Patent No. 6393857 Japanese Patent No. 6531235 WO2019/009317 WO2020/175472 WO2020/175473 Japanese Patent Publication No. 2003-500416 Japanese translation of PCT publication No. 2004-516330 JP-A-4-211096 JP-A-5-170795

As a method for producing decapeptides such as Ganirelix, the solid-phase peptide synthesis method is preferable for small amounts of reagents, but it is not suitable for mass production such as supply as pharmaceuticals. This is because the solid-phase synthesis is a heterogeneous reaction system, the mixing efficiency is low, and the reaction rate is slow. Furthermore, since the peptide cannot be purified during the reaction, it is necessary to use a large excess of 3 to 4 equivalents of amino acids and reagents to be extended for the purpose of quantitative reaction. On the other hand, according to the liquid-phase peptide synthesis method, it is a homogeneous reaction system, and it is possible to reduce the amount of reagents used. However, it is complicated because it is necessary to consider the separation process suitable for the properties of each peptide for each peptide elongation reaction, as well as the decrease in reaction rate due to the decrease in solubility in organic solvents as the peptide chain elongates. Therefore, the so-called liquid-phase peptide synthesis has not been performed in recent years, nor has Ganirelix been synthesized by the liquid-phase peptide synthesis method.

On the other hand, in recent years, liquid-phase peptide synthesis carriers (Tag) have been reported (Patent Documents 3 to 18). Since this carrier 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 carrier, solubility in organic solvents can be improved. can be greatly improved. Therefore, when a peptide elongation reaction is carried out with an amino acid or the like bound to the present carrier, the amino acid or the like bound to the carrier is dissolved in the organic layer, and unnecessary components such as surplus raw material amino acids used in the peptide elongation reaction and its By dissolving in the aqueous layer the decomposition products and compounds produced as by-products when the protective groups of the starting amino acids are deprotected, there is an advantage that the amino acids bound to the carrier can be simply purified by liquid-liquid separation. Furthermore, there is also the advantage that crude purification of the peptide is possible even in a carrier-bound state. Thus, the use of a carrier for liquid-phase peptide synthesis has made it possible to mass-produce peptides without the need for complicated separation procedures.
As used herein, the term "amino acid amide" refers to a structure in which the C-terminal carboxy group (--COOH) of an amino acid is replaced by an amide group (--CONH ₂ ). The term "peptide amide" refers to a structure in which the C-terminal carboxyl group of a peptide is an amide group.
Furthermore, when described as "liquid-liquid separation" in this specification, the above-mentioned step, that is, dissolving the amino acid etc. bound to the carrier in the organic layer, unnecessary components such as surplus raw material amino acids used in the peptide elongation reaction, It is a step of dissolving in the water layer the decomposition product, the compound by-produced when the protecting group of the starting amino acid is deprotected, and the like.

However, in the case of Ganirelix, liquid-liquid separation during the condensation reaction of the guanidyl group-containing amino acid (diethylhomoarginine) present in the structure of Ganirelix is difficult even when this carrier for liquid-phase peptide synthesis is used. found.

Therefore, an object of the present invention is to provide a new method for producing Ganirelix by a liquid-phase peptide synthesis method.

Therefore, the present inventors conducted various studies on means for protecting the guanidyl group of diethylhomoarginine used for liquid-phase peptide synthesis of Ganirelix using a carrier for liquid-phase peptide synthesis. First, when proton protection (Patent Documents 19 to 22), which has been widely used as a means for protecting the guanidyl group of arginines, was investigated, liquid-liquid separation after the condensation reaction of diethylhomoarginine was difficult. On the other hand, if diethylhomoarginine in which the guanidino group of arginines is protected with a protecting group such as a Boc group is used as a starting material, the liquid-liquid separation after the condensation reaction becomes favorable, and the liquid-phase peptide synthesis method is industrially advantageous. The inventors have found that ganirelix or a salt thereof can be produced by a method, and completed the present invention.

That is, the present invention provides the following inventions [1] to [11].
[1] A method for producing Ganirelix or a salt thereof by a liquid-phase peptide synthesis method, wherein the following formulas (1) to (3) are used as starting materials for the condensation reaction of the 3rd and 5th diethylhomoarginine residues from the C-terminus.

(wherein R ¹ and R ² represent Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde, and R ³ represents an amino protecting group)
A method for producing ganirelix or a salt thereof using one or more compounds of the group represented by and a carrier for liquid-phase peptide synthesis.
[2] The production method according to [1], characterized by including the following steps a to c.
a. a step of condensing an amino acid, peptide, amino acid amide or peptide amide bound to a carrier 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 in which the amino group is protected in the reaction solution;
c. After an aqueous solution is added to the reaction solution, the liquids are separated to obtain a condensate of the amino acid, peptide, amino acid amide or peptide amide bound to the carrier for liquid phase peptide synthesis and the amino acid or peptide from which the amino protecting group has been removed. a step of obtaining an organic solvent layer containing;
[3] The amino acid sequence of the Ganirelix or its salt is D-AlaNH ₂ , Pro, hArg(Et) ₂ , Leu, D-hArg(Et) ₂ , Tyr, Ser, D-3-pyridyl Ala from the C-terminal side. , Dp-chloroPhe, and D-naphthyl Ala in that order [1] or [2].
[4] The production method according to any one of [1] to [3], wherein R ¹ and R ² are Boc, and R ³ is Fmoc or Cbz.
[5] The production method according to any one of [1] to [4], wherein R ¹ and R ² are Boc and R ³ is Fmoc.
[6] The production method according to any one of [2] to [5], comprising the step of adding an amino acid active ester quenching agent to the reaction solution after the condensation reaction, following step a.
[7] The production method according to [6], wherein the quenching agent for the amino acid active ester is a water-soluble amine.
[8] the water-soluble amine is hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, or an alkyl group, alkenyl group, cycloalkyl group, cycloalkenyl group, aryl group, aralkyl primary, secondary or tertiary amines having one or more selected from groups and heterocyclic groups, wherein a hydroxy group, an ether bond, an alkoxy group, a sulfonyl group, a sulfonic acid group, a sulfate group, and a phosphoric acid group, the production method according to [7], which is an amine optionally having one or more substituents.
[9] The production method according to any one of [2] to [8], which includes the step of adding a trapping agent for dibenzofulvene subsequent to step b when R ³ is Fmoc.
[10] The dibenzofulvene trapping agent is a mercapto compound having an alkyl group of 1 to 10 carbon atoms and is selected from carboxylic acid, alkali metal salt of carboxylic acid, sulfonic acid, or alkali metal salt of sulfonic acid. The production method according to [9], which is a mercapto compound having one or more substituents.
[11] The production method according to any one of [1] to [10], wherein the carrier for liquid-phase peptide synthesis is a compound represented by the following formula (I).

[In the formula,
Ring A represents a C4-20 aromatic ring which may contain heteroatoms 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), together with R ¹³ represents a single bond, and ring A and may form a fluorene ring together with ring B, or may form a xanthene ring together with ring A and ring B via an oxygen atom;
p X ¹ are each independently 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 are each independently an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic indicating a group;

However, R ¹⁶ represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X ³ is an oxygen atom or —C(=O)NR ¹⁷ — (R ¹⁷ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
p represents an integer of 1 to 4;
Ring A is, in addition to p X ¹ R ¹² , 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 It may have a substituent selected from the group consisting of;
Ra represents a hydrogen atom or an aromatic ring optionally substituted with a halogen atom;
Rb represents a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or formula (b);

(Wherein, * indicates the binding position;
q represents an integer from 0 to 4;
q X ² are each independently 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 through an oxygen atom, or an organic group of formula (a);

However, R ¹⁶ represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X ³ is an oxygen atom or —C(=O)NR ¹⁷ — (R ¹⁷ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
R ¹³ represents a hydrogen atom, represents a single bond together with R ¹¹ to form a fluorene ring together with ring A and ring B, or forms a xanthene ring together with ring A and ring B through an oxygen atom. may form;
Ring B, in addition to q X ² R ¹⁴ , further comprises 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 may have a substituent selected from the group consisting of ) represents a group represented by;
Y represents a hydroxy group, a thiol group, NHR ²⁰ (R ²⁰ represents a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]

If Ganirelix or a salt thereof is produced by the method using the protected form of diethylhomoarginine of the present invention, the liquid-liquid separation after the condensation reaction is facilitated, and Ganirelix or a salt thereof can be industrially produced advantageously.

In the liquid-liquid separation in the step of Example (1-d) *1, it is a photograph of the liquid surface after shaking the separating funnel and allowing it to stand at room temperature for 25 minutes. In the liquid-liquid separation in the step of Comparative Example (d) *2, it is a photograph of the liquid surface after shaking the separating funnel and allowing it to stand at room temperature for 25 minutes.

Ganirelix, the target compound of the present invention, is a decapeptide [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-alanamide)], is a gonadotropin antagonist and is marketed as an anti-premature ovulatory agent under controlled ovarian stimulation.

In the structure above, the amino acid residues constituting the decapeptide structure are, from the C-terminal side, D-AlaNH ₂ , Pro, hArg(Et) ₂ , Leu, D-hArg(Et) ₂ , Tyr, Ser, D- It may be abbreviated as 3-pyridyl Ala, Dp-chloroPhe, and D-naphthyl Ala.

The method for producing Ganirelix or a salt thereof of the present invention is a method for producing Ganirelix or a salt thereof by a liquid-phase peptide synthesis method, wherein the following are used as starting materials for the condensation reaction of the 3rd and 5th diethylhomoarginine residues from the C-terminus: Formulas (1) to (3)

(wherein R ¹ and R ² represent Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde, and R ³ represents an amino protecting group)
A method for producing ganirelix or a salt thereof using one or more of the compounds represented by and a carrier for liquid phase peptide synthesis.

First, the compounds represented by the above formulas (1) to (3) used as starting materials for the condensation reaction of the diethylhomoarginine residue will be described.
R ¹ and R ² are protecting groups, Boc (tert-butoxycarbonyl), Cbz (benzyloxycarbonyl), Troc (2,2,2-trichloroethoxycarbonyl) or 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 preferable from the viewpoint of improving the liquid-liquid separation of the condensation reaction product.
R ³ represents an amino protecting group. The amino-protecting group includes Fmoc (9-fluorenylmethyloxycarbonyl), Boc, Cbz, etc. Among them, Fmoc and Cbz are preferred, and Fmoc, which can be deprotected under basic conditions, is more preferred.
R ¹ , R ² and R ³ are preferably orthogonal protecting groups. That is, preferably the deprotection conditions for R ¹ and R ² do not affect R ³ and the deprotection conditions for R ³ do not affect R ¹ and R ² . Particularly preferred are compounds in which R ¹ and R ² are Boc and R ³ is Fmoc.

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

The compounds represented by the above formulas (1) to (3) are, for example, α-amino-protected diethylhomoarginine, or a conjugate of this with a carrier for liquid-phase peptide synthesis, di-tert-butyl dicarbonate, N- It can be produced by reacting an amino-protecting agent such as a Boc agent such as tert-butoxycarbonylimidazole.
For example, this Boc-forming 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, and an inorganic base such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, sodium hydroxide. A base may be used. The amount of the base to be added is 0.1 to 30 equivalents, preferably 1 to 20 equivalents relative to the arginine derivative, but is not limited thereto.
Reaction solvents include water, THF (tetrahydrofuran), 2-methylTHF, 1,4-dioxane, toluene, DMF (N,N-dimethylformamide), acetonitrile, dichloromethane, chloroform, methanol, ethanol, or mixed solvents thereof. is used. The reaction is preferably carried out at 0° C. to 40° C. for 1 to 24 hours.

Next, the carrier for liquid-phase peptide synthesis will be described. Such a carrier for liquid-phase peptide synthesis may be a carrier that protects 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. , for example, the compounds described in Patent Documents 3 to 18 can be used.
Specific examples of such carriers for liquid-phase peptide synthesis include compounds represented by the following formula (I).

However, R ¹⁶ represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X ³ is an oxygen atom or —C(=O)NR ¹⁷ — (R ¹⁷ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
p represents an integer of 1 to 4;
Ring A is, in addition to p X ¹ R ¹² , 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 It may have a substituent selected from the group consisting of;
Ra represents a hydrogen atom or an aromatic ring optionally substituted with a halogen atom;
Rb represents a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by formula (b);

(Wherein, * indicates the binding position;
q represents an integer from 0 to 4;
q X ² are each independently 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 indicating a group;

However, R ¹⁶ represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X ³ is an oxygen atom or —C(=O)NR ¹⁷ — (R ¹⁷ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
R ¹³ represents a hydrogen atom, represents a single bond together with R ¹¹ to form a fluorene ring together with ring A and ring B, or forms a xanthene ring together with ring A and ring B through an oxygen atom. may form;
Ring B, in addition to q X ² R ¹⁴ , further comprises 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 It may have a substituent selected from the group consisting of;
Y represents a hydroxy group, a thiol group, NHR ²⁰ (R ²⁰ represents a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]

Ring A in formula (I) represents a C4-20 aromatic ring which may contain a heteroatom and may be monocyclic or polycyclic. The aromatic ring includes a C6-20 aromatic hydrocarbon ring and a C4-10 aromatic heterocyclic ring.
Specific C6-20 aromatic hydrocarbon rings include benzene ring, naphthalene ring, anthracene ring, phenanthrene ring, triphenylene ring, tetracene ring, indane ring, indene ring, fluorene ring, biphenyl ring, 1,1′- A binaphthalene ring and the like can be mentioned. 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 nitrogen, oxygen and sulfur atoms, specifically , pyrrole ring, furan ring, thiophene ring, indole ring, benzofuran ring, benzothiophene ring, carbazole ring, pyrazole ring, indazole ring, imidazole ring, pyridine ring, quinoline ring, isoquinoline ring and the like. Among these, a 5- to 8-membered aromatic heterocyclic ring containing 1 to 3 atoms selected from a nitrogen atom, an oxygen atom and a sulfur atom as a heteroatom is preferable, 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.

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

p X ¹ are each independently 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 C1-10 alkyl group or a C7-20 aralkyl group. Alkyl groups include linear or branched C1- Ten alkyl groups are mentioned.
Aralkyl groups include C7-16 aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl and 1-naphthylethyl groups.

p R ¹² are each independently an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic indicating a group;

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

As used herein, an organic group having an aliphatic hydrocarbon group is a monovalent organic group having an aliphatic hydrocarbon group in its molecular structure. The site of the aliphatic hydrocarbon group in the organic group having the aliphatic hydrocarbon group is not particularly limited, and may be present at the terminal or at any other site.
The aliphatic hydrocarbon group present in the organic group is a linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbon group. A hydrogen group is preferred, a C5-50 aliphatic hydrocarbon group is more preferred, and a C8-30 aliphatic hydrocarbon group is even more preferred. Specific examples of the aliphatic hydrocarbon group include an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group and the like, with alkyl groups, cycloalkyl groups and alkenyl groups being particularly preferred, and alkyl groups being more preferred. Further, a C5-30 linear or branched alkyl group, a C3-8 cycloalkyl group, a C5-30 linear or branched alkenyl group are preferred, and a C5-30 linear or branched alkyl group. , a C3-8 cycloalkyl group is more preferred, a C5-30 linear or branched alkyl group is more preferred, and a C8-30 linear or branched alkyl group is even more preferred.

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

The above aliphatic hydrocarbon group may be substituted with an aliphatic hydrocarbon group via an oxygen atom. Examples of the aliphatic hydrocarbon group capable of substituting an oxygen atom on the aliphatic hydrocarbon group include straight-chain or branched-chain alkoxy groups having 1 to 20 carbon atoms, alkenyloxy groups having 2 to 20 carbon atoms, and 3 carbon atoms. monovalent groups such as cycloalkyloxy groups of up to 6, divalent groups derived therefrom, and the like. Further, it may have a repeating structure in which an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group through an oxygen atom is further substituted with an aliphatic hydrocarbon group through an oxygen atom.
Specifically, as R ¹² , 12-docosyloxy-1-dodecyl group, 3,4,5-tris(octadecyloxy)benzyl group, 2,2,2-tris(octadecyloxymethyl)ethyl group, 3,4, 5-tris(octadecyloxy)cyclohexylmethyl group and the like.

The above aliphatic hydrocarbon group may be substituted with an organic group represented by formula (a).

(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 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms) A represents a silyl group or an alkyl group to which a silyloxy group is bonded)
The silyl group is preferably a silyl group substituted by three groups selected from linear or branched alkyl groups having 1 to 6 carbon atoms and aryl groups 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, more preferably three linear or branched alkyl groups having 1 to 4 carbon atoms. It is a substituted silyl group. The three alkyl groups or aryl groups substituting on the silyl group may be the same or different.
In addition, as the alkyl group to which the silyloxy group is bonded, one silyloxy group substituted by three selected from linear or branched alkyl groups having 1 to 6 carbon atoms and aryl groups which may have substituents is used. A linear or branched alkyl group having 1 to 13 carbon atoms with ˜3 bonds is preferred. A preferred silyloxy group is a silyloxy group substituted with three linear or branched alkyl groups having 1 to 6 carbon atoms, more preferably three linear or branched alkyl groups having 1 to 4 carbon atoms. It is a substituted silyloxy group. The three alkyl groups 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 branched, and more preferably has a quaternary carbon atom.

　p represents an integer of 1 to 4. Here, p is preferably 1-3, more preferably 1-2.

Ring A is, in addition to p X ¹ R ¹² , 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 may have a substituent selected from the group consisting of
Halogen atoms include chlorine, fluorine, bromine and iodine atoms. The C1-6 alkyl group optionally substituted with a halogen atom includes 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 and a hexyl group. , a dichloromethyl group, a trichloromethyl group, a trifluoromethyl group, and the like. The C1-6 alkoxy group optionally substituted with a halogen atom includes a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, a butyloxy group, an isobutyloxy group, a sec-butyloxy group, a tert-butyloxy group, and a trichloromethoxy group. groups, trifluoromethoxy groups, and the like.

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

Rb represents a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by the above formula (b).
q in the formula (b) represents an integer of 0-4.
q is preferably 0 to 3, more preferably 1 to 3, even more preferably 1 to 2.

q X ² are each independently 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 C1-10 alkyl group or a C7-20 aralkyl group. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl groups.
Aralkyl groups include C7-16 aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl and 1-naphthylethyl groups.

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 indicates a group.

However, R ¹⁶ represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X ³ is an oxygen atom or —C(=O)NR ¹⁷ — (R ¹⁷ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached.
Examples of the organic group represented by R ¹⁴ include the same groups as those for R ¹² above, and preferably the same groups as those for R ¹² above.

R ¹³ represents a hydrogen atom, represents a single bond together with R ¹¹ to form a fluorene ring together with ring A and ring B, or forms a xanthene ring together with ring A and ring B through an oxygen atom. may be formed.

Ring B, in addition to q X ² R ¹⁴ , further comprises 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 may have a substituent selected from the group consisting of
Halogen atoms include chlorine, fluorine, bromine and iodine atoms. The C1-6 alkyl group optionally substituted with a halogen atom includes 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 and a hexyl group. , a dichloromethyl group, a trichloromethyl group, a trifluoromethyl group, and the like. The C1-6 alkoxy group optionally substituted with a halogen atom includes a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, a butyloxy group, an isobutyloxy group, a sec-butyloxy group, a tert-butyloxy group, and a trichloromethoxy group. groups, trifluoromethoxy groups, and the like.

Y represents a hydroxy group, a thiol group, NHR ²⁰ (R ²⁰ represents a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom.
Here, R ²⁰ is preferably a hydrogen atom, a C1-10 alkyl group or a C7-20 aralkyl group. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl groups.
Aralkyl groups include C7-16 aralkyl groups such as benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl and 1-naphthylethyl groups.

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

(wherein Yb is --CH ₂ OR ³⁴ (wherein R ³⁴ represents a hydrogen atom, a halogenocarbonyl group, an active ester carbonyl group or an active ester sulfonyl group), --CH ₂ NHR ³⁵ (wherein 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, and R ²¹ , R ²² , R ²³ , R ²⁴ and at least one of R ²⁵ represents a group represented by formula (8);

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 represents O or CONR ³⁶ (wherein R ³⁶ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms);
A is represented by formula (9), (10), (11), (12), (13), (14), (15), (16), (17), (18) or (19) indicates a group.

(Here, R ²⁷ , R ²⁸ and R ²⁹ are the same or different and represent a linear or branched alkyl group having 1 to 6 carbon atoms or an aryl group which may have a substituent; ³⁰ represents a single bond or a linear or branched alkylene group having 1 to 3 carbon atoms, and R ³¹ , R ³² and R ³³ each represent a linear or branched alkylene group having 1 to 3 carbon atoms. )

In addition, the compound represented by formula (20) can be used as a carrier for liquid-phase peptide synthesis (Patent Documents 11, 12, 15).

(wherein X ⁴ is —OR ⁵¹ (wherein R ⁵¹ represents a hydrogen atom, an active ester carbonyl group or an active ester sulfonyl group), —NHR ³⁵ , azide, halogen, isocyanate, together with X ⁵ =N-OH or =O, and when X ⁴ is -OR ⁵¹ , -NHR ³⁵ , azide or halogen, X ⁵ is a hydrogen atom or a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms , or represents a cycloalkyl group, and when X ⁴ is isocyanate, X ⁵ represents a linear or branched alkyl group or alkenyl group having 1 to 4 carbon atoms, or a cycloalkyl group;
At least one of R ⁴¹ to R ⁵⁰ 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. indicate;
when X ⁴ is —OR ⁵¹ , —NHR ³⁵ , azide or halogen, and X ⁵ is a hydrogen atom, or when X ⁴ and X ⁵ together are =O, R ⁴⁵ and R ⁴⁶ are oxygen atoms to form a xanthene ring)

In addition, the compound represented by formula (21) can be used as a carrier for liquid-phase peptide synthesis (Patent Documents 13 and 14).

(In the formula, X ⁶ represents a hydroxy group or a halogen atom, at least one of R ⁶¹ to R ⁷⁵ represents a group represented by formula (2), and the remainder are hydrogen atoms, halogen atoms, and 1 carbon atom. 4 alkyl group ^or alkoxy group having 1 to ⁴ carbon atoms; may form)

The carrier for liquid-phase peptide synthesis can also be bound via a linker to the carboxyl groups of amino acids, peptides, amino acid amides or peptide amides (such as amino acids) as raw materials.
The linker as used herein 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 carrier for liquid-phase peptide synthesis. Preferred linkers are organic groups having a molecular weight of about 2000 or less (preferably about 1500 or less, more preferably about 1000 or less) and having reactive groups which may be the same or different, amino, carboxyl, and halomethyl groups. It is a compound having in the molecule at least two groups selected from the group consisting of For example, the following compounds can be mentioned.

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

(wherein X is a halogen atom, preferably chlorine or bromine).

(Wherein, Z is an integer of 2 to 40, preferably 2 to 35, more preferably 2 to 28).
(The structural formula of the above linker shows the state before binding to the side chain functional group or the like and the state before binding to the carrier for liquid-phase peptide synthesis).

Next, the peptide elongation reaction will be explained. The peptide extension reaction preferably has the following steps a, b and c. The order of steps b and c is not critical, and the order of step b and then step c, that is, the organic solvent layer containing the condensate may be obtained after removing the protective group for the amino group, or step c and then step In the order of b, that is, after obtaining the organic solvent layer containing the condensate, the protective group for the amino group may be removed.
a. a step of condensing an amino acid, peptide, amino acid amide or peptide amide bound to a carrier 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 in which the amino group is protected in the reaction solution;
c. After an aqueous solution is added to the reaction solution, the liquids are separated to obtain a condensate of the amino acid, peptide, amino acid amide or peptide amide bound to the carrier for liquid phase peptide synthesis and the amino acid or peptide from which the amino protecting group has been removed. a step of obtaining an organic solvent layer containing;
In addition, following the step a, a step of adding a quenching agent for the amino acid active ester to the reaction solution after the condensation reaction may be carried out. Also, when the amino-protecting group is an Fmoc group, step b may be followed by a step of adding a dibenzofulvene trapping agent.

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

The amino acid or peptide whose amino group is protected with an amino-protecting group (hereinafter abbreviated as amino-protected amino acid), which is the other raw material, is an amino acid or peptide whose amino group is protected with an amino-protecting group, On the other hand, carboxyl groups refer to amino acids or peptides that are unprotected and reactive. When an amino acid or peptide has one or more amino groups, at least one amino group should be protected with an amino-protecting group.
Examples of amino-protecting groups include Fmoc group, Boc group, Cbz group, etc. Among them, Fmoc group, which can be deprotected under basic conditions, is more preferable.
When the amino group-protected amino acid has highly reactive functional groups such as hydroxyl, amino, guanidyl, carboxyl, thiol, indole, and imidazole groups, these functional groups are commonly used in peptide synthesis. A protective group may be introduced, and the target compound can be obtained by removing the protective group as necessary at any point after the completion of the reaction.
Examples of hydroxyl-protecting groups include tBu, Trt, Bz (benzoyl), acetyl, and silyl groups, and examples of guanidyl-protecting groups include Pbf, Boc, Pmc, and nitro groups. Examples of carboxyl-protecting groups include tBu, methyl, ethyl, and Bz groups, and examples of thiol-protecting groups include Trt, Acm (acetamidomethyl), tBu, and S-tBu ( dithio-tert-butyl) group, Dpm (diphenylmethyl) group, MBom (4-methoxybenzyloxymethyl) group and the like, and examples of indole group-protecting groups include Boc group and the like, imidazole group-protecting group Examples include Boc group, Bom (benzyloxymethyl) group, Bum (tert-butoxymethyl) group, Trt group, Ddm (4,4'-dimethoxydiphenyl) group, MBom group and the like.

An amino group-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 a mixed solvent such as THF/water in the presence of a base. can be done.

The step a of the present invention is a step of condensing the raw materials, and the reaction solvent used in the step a is a solvent containing an organic solvent. If amino acids, peptides, amino acid amides or peptide amides are protected with the carrier for liquid-phase peptide synthesis used in the present invention, the obtained carrier-bound peptide for liquid-phase peptide synthesis becomes soluble in an organic solvent. Liquid phase peptide synthesis becomes 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), preferably 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.

The condensation reaction is carried out by mixing the carrier for liquid phase peptide synthesis or the carrier-bound peptide for liquid phase peptide synthesis, the amino group-protected amino acid, the condensing agent and the base in a solvent containing the organic solvent. can be done.

The amount of the amino group-protected amino acid used relative to the carrier-bound peptide for liquid-phase peptide synthesis is generally 1.01-4 equivalents, preferably 1.03-3 equivalents, more preferably 1.03-3 equivalents, relative to the carrier-bound peptide for liquid-phase peptide synthesis. is 1.05 to 2 equivalents, more preferably 1.1 to 1.5 equivalents. In the peptide production method of the present invention, subsequent to step a, the unreacted amino acid active ester can be captured and inactivated by a quenching agent added thereafter. Therefore, even if an excess amino group-protected amino acid is used, the problem of residue does not occur.

Condensing agents commonly 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-azabenzo triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl Uronium hexafluorophosphate (HBTU(6-Cl)), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(6-chloro Benzotriazol-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) can be mentioned. DMT-MM, HBTU, HATU or COMU is preferred. The amount of the condensing agent used is preferably 1 to 4 equivalents, more preferably 1 to 2 equivalents, still more preferably 1.05 to 1.45 equivalents, relative to the carrier-bound peptide for liquid-phase peptide synthesis.
Bases commonly used in peptide synthesis can also be used in the present invention. Examples include DIPEA (N,N-diisopropylethylamine), DMAP, NMM (N-methylmorpholine), TMP (2,4,6-trimethylpyridine). DIPEA is preferred.

In the condensation step, an activator is preferably added in order to promote the reaction and suppress side reactions such as racemization. Here, the activating agent is a reagent that facilitates the formation of a peptide bond (amide bond) by leading an amino acid to a corresponding active ester, symmetrical acid anhydride, or the like in coexistence with a condensing agent. As the activator, an activator commonly used in peptide synthesis can be used. For example, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1,2,3-triazole ethyl carboxylate (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, cyano ( hydroxyimino)ethyl acetate (Oxyma) and the like. 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, still more preferably 1.05 to 1.45 equivalents, relative to the carrier-bound peptide for liquid-phase peptide synthesis.

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

Following step a, 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 may be performed. In step a, an amino group-protected amino acid as a raw material is added in an excessive amount to the carrier for liquid phase peptide synthesis or the carrier-bound peptide for liquid phase peptide synthesis. Therefore, the amino acid active ester produced by activating the amino group-protected amino acid during the condensation reaction remains in excess after the condensation reaction. The step of adding a quenching agent is a step of quenching this excess amino acid active ester.
The amount of the quenching agent to be added in this step is preferably 1 to 10 equivalents, more preferably 1 to 6 equivalents, still more preferably 1 to 3 equivalents, relative to 1 equivalent of the theoretically remaining active amino acid ester.

The amino acid active ester quenching agent is a compound having an amino group in the molecule, and is disclosed in Japanese Patent No. 6703668, Japanese Patent No. 6713983, International Publication No. 2021/132545, Molecules 2021, 26, 3497-3505. . and the like can be used.
Such quenching agents include hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, alkylamines having primary or secondary amines, fragrances having primary or secondary amines. Group amines can be used, and tertiary amines can also be used. Furthermore, since excess quenching agent can be removed to the aqueous layer by liquid-liquid separation, it is preferably water-soluble, and amines having hydrophilic substituents such as hydroxyl group, sulfo group, sulfate group and phosphoric acid group are preferred. Further, the number of amino groups in the compound may be one (monovalent), or may be bivalent 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 still more preferably alkylamines having 2 to 8 carbon atoms. is an alkylamine 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, preferably aromatic amines having 6 to 10 carbon atoms.
Specific amines include, but are not limited to, propylamine, methylamine, hexylamine, benzylamine, aniline, toluidine, 2,4,6-trimethylaniline, anisidine, phenetidine, hydroxylamine, 1-methyl Piperazine, 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) and the like. Also included are NMI (N-methylimidazole), DMAP and trimethylamine. Among them, 2-(2-aminoethoxy)ethanol (AEE), taurine, and 2-aminoethyl hydrogen sulfate (2-aminoethyl sulfate, AEHS) are preferred.

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

The step of removing the amino-protecting group when the amino-protecting group is an Fmoc group will be described.
In the Fmoc elimination step, it is sufficient if the reaction solution can be made basic. .0]-5-nonene (DBN), 1,4-diazabicyclo[2.2.2]-octane (DABCO), triethylamine, tertiary amines such as tributylamine; 1-methylpiperazine, 4-aminopiperidine, Diethylenetriamine, triaminoethylamine, 1-ethylpiperazine, N,N-dimethylethylenediamine, ethylenediamine, piperidine, piperazine, etc. Divalent or higher water-soluble amines having at least one primary or secondary amino group are used. be able to. DBU, DBN, piperidine, piperazine, 1-methylpiperazine, 4-aminopiperidine and diethylenetriamine are preferred, and DBU, piperidine, piperazine and 1-methylpiperazine are more preferred.
The equivalent of the amine compound added in step b is 1-30 equivalents, preferably 4-20 equivalents, more preferably 4-10 equivalents, relative to the amount of Fmoc groups present in the system.

In step b, in addition to the amine compound, a trapping agent for DBF (dibenzofulvene) produced by the de-Fmoc reaction and an adduct of DBF and amine (DBF-amine adduct) (hereinafter referred to as "trapping agent") It is preferable to perform the step of adding Trapping agents for DBF and DBF-amine adducts used herein 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 exhibits water solubility. , carboxylic acids, alkali metal salts of carboxylic acids, sulfonic acids, or mercapto compounds having one or more substituents selected from alkali metal salts of sulfonic acids, and represented by the following general formula (4) or (5) compounds represented.

(Wherein, L1 and L2 each represent a divalent organic group, and M represents a hydrogen atom or an alkali metal)

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 and optionally having a mercapto group. , an arylene group having 6 to 10 carbon atoms which may have a mercapto group, and a heteroarylene group having 4 to 9 carbon atoms which may have a mercapto group. Specifically, methylene group, ethylene group, trimethylene group, propylene group, mercaptotrimethylene group, mercaptopropylene group, tetramethylene group, butylene group, pentamethylene group, phenylene group, naphthylene group, indole group, benzimidazole group, A quinolyl group, an isoquinoline group, and the like can be mentioned.
M represents a hydrogen atom or an alkali metal. Specific examples include a hydrogen atom, sodium, and potassium.
Specifically, 3-mercaptopropionic acid, thiomalic acid, cysteine, sodium mercaptomethanesulfonate, sodium 2-mercaptoethanesulfonate, 2-mercaptoethanesulfonic acid, 3-mercaptopropanesulfonic acid, 3-mercaptopropanesulfonic acid sodium, 1,3-dimercaptopropanesulfonic acid, sodium 2-mercaptobenzimidazole-5-sulfonate, and the like, with 3-mercaptopropanesulfonic acid being preferred.

The amount of the mercapto compound added is preferably 1 to 30 equivalents, more preferably 1 to 10 equivalents, even more preferably 1 to 5 equivalents, relative to the amount of the Fmoc group.
The amine compound and the mercapto compound may be added simultaneously, or the mercapto compound and then the amine compound may be added in this order.
The Fmoc elimination step may be performed at a temperature of −20 to 40° C. for 1 minute to 5 hours.

In step c, an aqueous solution is added to the reaction solution, followed by liquid separation to produce an amino acid, peptide, amino acid amide or peptide amide bound to the carrier for liquid phase peptide synthesis and the amino acid or peptide from which the amino protecting group has been removed. is a step of obtaining an organic solvent layer containing a condensate of
After adding the aqueous solution to the reaction solution in step b, the aqueous layer and the organic solvent layer are separated.
When the step of adding a quenching agent and a trapping agent is performed, the aqueous layer contains a condensate of an amino acid or peptide from which the amino protecting group has been removed and an active ester quenching agent and a 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 only by 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 a carrier for liquid-phase peptide synthesis and an amino acid or peptide from which the amino protecting group has been removed.
The aqueous solution used here includes water or an aqueous solution having a pH in the vicinity of neutral to basic. Specifically, water, sodium chloride aqueous solution, calcium chloride aqueous solution, cesium chloride aqueous solution, potassium chloride aqueous solution, lithium chloride aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution, cesium carbonate aqueous solution, disodium hydrogen phosphate aqueous solution, trisodium phosphate Examples include an aqueous solution, an aqueous sodium hydrogencarbonate solution, an aqueous potassium hydrogencarbonate solution, an aqueous dipotassium hydrogenphosphate solution, an aqueous tripotassium phosphate solution, or a mixed solvent of these aqueous solutions and DMF, DMSO, NFM, or NMP.

The resulting Ganirelix is optionally converted into organic acid salts such as acetates, formates, oxalates, succinates and trifluoroacetates, mineral salts such as hydrochlorides, nitrates, sulfates and phosphates. can be converted to

As described above, according to the method of the present invention, simply adding an aqueous solution and separating liquids does not cause poor liquid separation between the amino acid active ester and the product peptide. Moreover, since solid-liquid separation is not required, one-pot synthesis that does not require complicated separation operations becomes possible. The series of steps described above may be performed using microflow technology. Peptide synthesis technology using microflow technology is described, for example, in Nature Communications 7, Article number: 13491 (2016).

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Synthesis of Reference Example Fmoc-NH(D2-STag)

(O = (D2-STag), OH (D2-STag), Fmoc-NH (D2-STag) represent the structures in the formula.)

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

Reference example (b)
13.7 g (57.2 mmol) of 9-fluorenylmethyl carbamate and 1.80 g (14.3 mmol) of oxalic acid dihydrate are added to the toluene solution obtained in the previous step, and stirred at 80° C. for 3 hours. bottom. The reaction solution was cooled to room temperature, 414 mL of methanol:water=9:1, and 414 mL of heptane were added and separated. The resulting organic layer was washed once with 207 mL of a 5% aqueous sodium hydrogencarbonate solution (5% Na ₂ CO ₃ aq.) and three times with 414 mL of methanol:water=9:1. The obtained organic layer was concentrated under reduced pressure, 83.0 mL of tetrahydrofuran was added to the residue, and the mixture was concentrated under reduced pressure. The residue was dissolved in 62 mL of tetrahydrofuran and added dropwise to 830 mL of methanol. The precipitated solid was collected by filtration and dried under reduced pressure to obtain 45.5 g of Fmoc-NH(D2-STag).
ESIMS (m/z) 1107.9 (M+ _NH4 ) ⁺

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.)
In addition, the amino acid without the notation that it is a D-form shows the L-form hereinafter.

In the reaction formula, (NH ₂ (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-Ala-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-NH ₂ ·nTFA) represents the structure in the reaction scheme. Fmoc-hArg(Et) ₂ (Boc) ₂ -OH represents a mixture of three isomers in the reaction formula. Therefore, H-hArg(Et) ₂ (Boc) ₂ -Pro-D-Ala-NH(D2-STag) is also presumed to be a mixture of 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), 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) is the combination of hArg(Et) ₂ (Boc) ₂ and D-hArg (Et) ₂ (Boc) ₂ is presumed to be each of the three isomers in the reaction formula, possibly resulting in a mixture of nine isomers in total. ) In addition, * in the structure of R' indicates a binding point.

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), 7.33 mL of DMF, and 3-mercapto- dissolved in 2.55 mL of dimethylsulfoxide (DMSO). 0.542 g (3.04 mmol) of sodium 1-propanesulfonate (MPS) and 0.111 g (0.62 mmol) of MPS were added in the solid state to obtain 2,3,4,6,7,8,9,10 -Octahydropyrimidol[1,2-a]azepine (DBU) 0.548 mL (3.67 mmol) was added and stirred at room temperature for 1 hour and 35 minutes. Cool to 8° C., add 0.319 mL (1.83 mmol) of N,N-diisopropylethylamine (DIPEA), 3.67 mL (3.67 mmol) of 1N sulfuric acid, 24.2 mL of water, and 1.00 mL of CPME, and separate the liquids. bottom. 4.55 mL of DMF and 6.07 mL of 50% aqueous solution of dipotassium hydrogen phosphate (50% K ₂ HPO ₄ aq.) were added to the obtained organic layer, washed by separation, and mixed with NH ₂ (D2-STag). I got the liquid.

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.H ₂ O, 1.28 mL (7.33 mmol) of DIPEA, and 0.28 mL of COMU were added. 942 g (2.20 mmol) was 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. Add 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO, 0.284 g (1.59 mmol) of solid MPS, cool to 10° C., add 1.43 mL (9.54 mmol) of DBU, add 1 Stirred for 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. DMF 5.10 mL, 50% K ₂ HPO ₄ aq. 6.80 mL was added, and washing was performed by liquid separation to obtain a mixed solution containing HD-Ala-NH (D2-STag).

Example (1-c)
To the resulting mixture, CPME 2.20 mL, DMF 8.30 mL, Fmoc-Pro-OH.H ₂ O 0.782 g (2.20 mmol), DIPEA 1.28 mL (7.33 mmol), COMU 0.942 g ( 2.20 mmol) was added and stirred at room temperature for 50 minutes. 44.0 μL (0.444 mmol) of AEE was added, and the mixture was stirred at room temperature for 15 minutes. Add 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO, 0.284 g (1.59 mmol) of solid MPS, cool to 8° C., add 1.43 mL (9.54 mmol) of DBU, add 1 Stirred for 20 minutes. 11.5 mL (11.5 mmol) of 1N sulfuric acid, 20.2 mL of water, and 0.861 mL of CPME were added and separated. DMF 5.11 mL, 50% K ₂ HPO ₄ aq. 6.81 mL was added and washing was carried out to obtain a mixed solution containing H-Pro-D-Ala-NH (D2-STag).

Example (1-d)
To the resulting mixture were added 2.70 mL of CPME, 8.30 mL of DMF, and 1.47 g of Fmoc-hArg(Et) ₂ (Boc) ₂ -OH (a mixture of three isomers shown in the reaction formula) (2. 20 mmol), 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 the mixture was stirred at room temperature for 15 minutes. Add 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO, 0.284 g (1.59 mmol) of solid MPS, cool to 8° C., add 1.43 mL (9.54 mmol) of DBU, add 1 Stirred for 50 minutes. 11.5 mL (11.5 mmol) of 1N sulfuric acid, 20.3 mL of water, and 0.950 mL of CPME were added and separated *1. DMF 5.12 mL, 50% K ₂ HPO ₄ aq. 6.83 mL was added, and washing was performed by separation to obtain a mixed solution 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 target in organic layer: 76.2%
HPLC analysis conditions (1)
Column: YMC-Pack Pro C18, S-5 μm, 12 nm, 250 mm×4.6 mm I.D. D.
Mobile phase A: 500 mM sodium perchlorate aq.
Mobile phase B: THF
Flow rate: 1.0 mL/min
Column temperature: 45°C
Detection wavelength: 280 nm
Gradient conditions: 75% B (0 min) → 90% B (10 min) → 90% B (20 min) → 75% B (21 min) → 75% B (33 min)

Example (1-e)
As in Example (1-d), 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, Fmoc-D-Nal(2)-OH and 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( A mixed solution containing D2-STag) was obtained. Fmoc-D-hArg(Et) ₂ (Boc) ₂ -OH (a mixture of 3 isomers) has the following structure, and * indicates a binding point.

Example (1-f)
0.800 mL of CPME, 8.95 mL of DMF, 1.28 mL (7.33 mmol) of DIPEA, 0.126 mL (2.20 mmol) of acetic acid, and 0.942 g (2.20 mmol) of COMU were added to the resulting mixture, and the mixture was cooled to room temperature. and stirred for 50 minutes. The reaction solution was concentrated under reduced pressure, and the resulting residue was added dropwise to 106 mL of acetonitrile (MeCN). Collect the solids by filtration and wash the filter cake with 50.0 mL of MeCN. 106 mL of MeCN was added to the filtrate, and after stirring, the solid was collected by filtration, and the filtrate was washed with 50.0 mL of MeCN. The resulting solid was dried under reduced pressure to give 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) 3.98 g.
ESIMS (m/z) 1467.3 (M+2H) ^<2+>

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 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) 1.50 g was added. After 5 minutes, the temperature was raised to room temperature, and the mixture was 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. This washing with MTBE, centrifugation and decantation were further performed three times to obtain a precipitate. The precipitate is dried under reduced pressure, Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser-Tyr-D-hArg(Et) ₂ -Leu-hArg(Et) ₂ -Pro 0.929 g of -D-Ala-NH ₂ ·nTFA was obtained.
ESIMS (m/z) 785.5 (M+2H) ^<2+>
HPLC Purity: 94.3%
HPLC analysis conditions (2)
Column: Inertsustain, S-3 μm, 250 mm×2.1 mm I.D. D.
Mobile phase A: 0.1% TFA aq.
Mobile phase B: MeCN with 0.1% TFA
Flow rate: 0.22 mL/min
Column temperature: 33°C
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)

Comparative Example Synthesis of H-hArg(Et) ₂ -Pro-D-Ala-NH(D2-STag) Using Fmoc-hArg(Et) 2 -OH.HCl (Quenching Agent _for Fmoc-Amino-Active Ester : AEE)

(Fmoc-hArg(Et) ₂ -OH.HCl and H-hArg(Et) ₂ -Pro-D-Ala-NH(D2-STag) represent structures in the reaction formula.)

Comparative examples (a), (b), (c)
H-Pro-D-Ala-NH(D2-STag) was prepared from 2.00 g (1.83 mmol) of Fmoc-NH(D2-STag) in the same manner as in Examples 1-a, 1-b, and 1-c. A mixture containing

Comparative example (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 0.28 mL of COMU were added. 942 g (2.20 mmol) was added and stirred at room temperature for 50 minutes. 44.0 μL (0.444 mmol) of AEE was added, and the mixture was stirred at room temperature for 15 minutes. Add 0.501 g (2.81 mmol) of MPS dissolved in 2.35 mL of DMSO, 0.284 g (1.59 mmol) of solid MPS, cool to 8° C., add 1.43 mL (9.54 mmol) of DBU, add 1 Stirred for 50 minutes. 11.5 mL (11.5 mmol) of 1N sulfuric acid, 20.3 mL of water, and 0.950 mL of CPME were added to attempt to separate the liquids.
ESIMS (m/z) 1263.5 (M+H) ⁺
*2 HPLC purity of target in emulsion: 10.4%
HPLC analysis conditions: same as (1) in Example (1-d).

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: hydrogen sulfate 2-aminoethyl (AEHS))

Example (2-a)
A mixed solution containing NH ₂ (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).

Example (2-b)
0.600 mL of CPME, 3.30 mL of DMF, 0.290 g (0.880 mmol) of Fmoc-D-Ala-OH.H ₂ O, 0.511 mL (2.93 mmol) of DIPEA, and 0.511 mL of DIPEA were added to the resulting mixture. 377 g (0.880 mmol) was 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, 0.263 g (1.48 mmol) of solid MPS were added, cooled to 8° C., 0.597 mL (3.99 mmol) of DBU was added, and 1 Stirred for 50 minutes. 4.60 mL (4.60 mmol) of 1N sulfuric acid, 8.30 mL of 10% aqueous sodium carbonate solution (10% Na ₂ CO ₃ aq.), and 0.319 mL of CPME were added and separated. 2.04 mL of DMF, 50% K ₂ HPO ₄ aq. 2.72 mL was added, and washing was performed by liquid separation to obtain a mixed solution containing HD-Ala-NH (D2-STag).

Example (2-c)
To the resulting mixture, CPME 0.800 mL, DMF 3.30 mL, Fmoc-Pro-OH.H ₂ O 0.313 g (0.880 mmol), DIPEA 0.511 mL (2.93 mmol), COMU 0.377 g ( 0.880 mmol) was 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, 0.263 g (1.48 mmol) of solid MPS were added, cooled to 6° C., 0.597 mL (3.99 mmol) of DBU was added, and 1 Stirred for 20 minutes. 4.60 mL (4.60 mmol) of 1N sulfuric acid, 10% Na ₂ CO ₃ aq. 8.30 mL and 0.345 mL of CPME were added and separated. 2.04 mL of DMF, 50% K ₂ HPO ₄ aq. 2.72 mL was added and washing was carried out to obtain a mixed solution containing H-Pro-D-Ala-NH (D2-STag).

Example (2-d)
0.600 mL of CPME, 3.30 mL of DMF, and 0.587 g of Fmoc-hArg(Et) ₂ (Boc) ₂ -OH (a mixture of three isomers shown in the figure) (0.587 g) were added to the resulting mixture. 880 mmol), 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 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, 0.263 g (1.48 mmol) of solid MPS were added, cooled to 7° C., 0.597 mL (3.99 mmol) of DBU was added, and 1 Stirred for 50 minutes. 4.60 mL (4.60 mmol) of 1N sulfuric acid, 10% Na ₂ CO ₃ aq. 8.40 mL and 0.380 mL of CPME were added and separated *3. 2.05 mL of DMF, 50% K ₂ HPO ₄ aq. 2.73 mL was added, and washing was performed by separation to obtain a mixed solution 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: same as (1) in Example (1-d).

Example (2-e)
Similar to Example (2-d), 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, Fmoc-D-Nal(2)-OH and 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( A mixed solution containing D2-STag) was obtained.

Example (2-f)
Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et) ₂ 1.36 g of (Boc) ₂ -Leu-hArg(Et) ₂ (Boc) ₂ -Pro-D-Ala-NH(D2-STag) were obtained.
ESIMS (m/z) 1467.3 (M+2H) ^<2+>

Example (2-g)
Ac-D-Nal(2)-D-pClPhe-D-Pal(3)-Ser(tBu)-Tyr(tBu)-D-hArg(Et) ₂ in the same manner as in Example (1-g) (Boc) ₂ -Leu-hArg(Et) ₂ (Boc) ₂ -Pro-D-Ala-NH(D2-STag) from 0.700 g of Ac-D-Nal(2)-D-pClPhe-D-Pal( 3) 0.455 g of -Ser-Tyr-D-hArg(Et) ₂ -Leu-hArg(Et) ₂ -Pro-D-Ala-NH ₂ ·nTFA was obtained.
ESIMS (m/z) 785.5 (M+2H) ^<2+>
HPLC Purity: 94.4%
HPLC analysis conditions: same as (2) in Example (1-g).

1 and 2 show the state of liquid separation in *1 of Example (1-d) and *2 in Comparative Example (d). In Comparative Example (d) shown in FIG. 1, the interface between the organic layer and the aqueous layer was clear, and the liquid-liquid separation was good. On the other hand, in Comparative Example (d) shown in FIG. 2, the entire inside of the separatory funnel became an emulsion, and the emulsion did not dissolve even after standing at room temperature for 25 minutes, and the organic layer and the aqueous layer could not be separated. .
Next, Table 1 shows the HPLC analysis results of *1 of Example 1, *3 of Example 2, and *2 of Reference Example 1. In Examples 1 and 2 using Fmoc-hArg(Et) ₂ (Boc) ₂ -OH protected with a Boc group of the present invention, the liquid separation property was good, and H-hArg(Et) ₂ (Boc) ₂ -Pro-D-Ala-NH (D2-STag) could be synthesized with high purities of 76.2% and 78.0%. In Examples 1 and 2, the peptide elongation reaction was continued, and crude 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 using Fmoc-hArg(Et) ₂ -OH·HCl which is only proton-protected instead of the Boc group, H-hArg(Et) ₂ -Pro-D-Ala-NH(D2-STag ) could not be separated when synthesized. To continue the synthesis of Ganirelix, it is necessary to purify H-hArg(Et) ₂ -Pro-D-Ala-NH(D2-STag) by other methods than liquid-liquid separation, which is industrially disadvantageous. Met. The purity of H-hArg(Et) ₂ -Pro-D-Ala-NH(D2-STag) in the emulsion was extremely low at 10.4%.

Based on the above results, the Boc-protected Fmoc-hArg(Et) 2 ₍ Boc) ₂ -OH of the present invention was used in place of the conventional proton-protected Fmoc-hArg(Et) ₂ -OH·HCl. It was found that by using it, the liquid separation property is improved, and ganirelix can be obtained by an industrially advantageous method.

Claims

In a method for producing Ganirelix or a salt thereof by a liquid-phase peptide synthesis method, the following formulas (1) to (3) are used as starting materials for the condensation reaction of the 3rd and 5th diethylhomoarginine residues from the C-terminus.

(wherein R 1 and R 2 represent Boc, Cbz, Troc, Alloc, Trt, Mmt, Teoc, Phth, SES, or ivDde, and R 3 represents an amino protecting group)
A method for producing ganirelix or a salt thereof using one or more compounds of the group represented by and a carrier for liquid-phase peptide synthesis.
A manufacturing method according to claim 1, characterized in that it comprises the following steps ac.
a. a step of condensing an amino acid, peptide, amino acid amide or peptide amide bound to a carrier 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 in which the amino group is protected in the reaction solution;
c. After an aqueous solution is added to the reaction solution, the liquids are separated to obtain a condensate of the amino acid, peptide, amino acid amide or peptide amide bound to the carrier for liquid phase peptide synthesis and the amino acid or peptide from which the amino protecting group has been removed. a step of obtaining an organic solvent layer containing;
The amino acid sequence of the Ganirelix or its salt is, from the C-terminal side, D-AlaNH 2 , Pro, hArg(Et) 2 , Leu, D-hArg(Et) 2 , Tyr, Ser, D-3-pyridyl Ala, D- The production method according to claim 1, wherein the order is p-chloroPhe and D-naphthylAla.
2. The production method according to claim 1, wherein said R1 and R2 are Boc and said R3 is Fmoc or Cbz.
2. The method according to claim 1, wherein said R1 and R2 are Boc and said R3 is Fmoc.
The production method according to claim 1, comprising the step of adding a quenching agent for the amino acid active ester to the reaction solution after the condensation reaction following step a.
The production method according to claim 6, wherein the quenching agent for the amino acid active ester is a water-soluble amine.
The water-soluble amine is hydroxylamine, amidosulfuric acid, hydroxylamine-O-sulfonic acid, hydroxylamine-O-phosphonic acid, or an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aralkyl group and a hetero Primary, secondary or tertiary amines having one or more selected from cyclic groups, comprising a hydroxy group, an ether bond, an alkoxy group, a sulfonyl group, a sulfonic acid group, a sulfate group, and a phosphoric acid group 8. The production method according to claim 7, wherein the amines optionally have one or more substituents selected from groups.
3. The production method according to claim 2, comprising the step of adding a dibenzofulvene trapping agent subsequent to step b, when R3 is Fmoc.
The dibenzofulvene trapping agent is a mercapto compound having an alkyl group having 1 to 10 carbon atoms, and is at least one selected from carboxylic acid, alkali metal salt of carboxylic acid, sulfonic acid, and alkali metal salt of sulfonic acid. The production method according to claim 9, which is a mercapto compound having a substituent of
The production method according to any one of claims 1 to 10, wherein the carrier for liquid-phase peptide synthesis is a compound represented by the following formula (I).

[In the formula,
Ring A represents a C4-20 aromatic ring which may contain heteroatoms 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), together with R 13 represents a single bond, and ring A and may form a fluorene ring together with ring B, or may form a xanthene ring together with ring A and ring B via an oxygen atom;
p X 1 are each independently 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 are each independently an aliphatic hydrocarbon group, an aliphatic hydrocarbon group substituted with an aliphatic hydrocarbon group via an oxygen atom, or an organic indicating a group;

However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X 3 is an oxygen atom or —C(=O)NR 17 — (R 17 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
p represents an integer of 1 to 4;
Ring A is, in addition to p X 1 R 12 , 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 It may have a substituent selected from the group consisting of;
Ra represents a hydrogen atom or an aromatic ring optionally substituted with a halogen atom;
Rb represents a hydrogen atom, an aromatic ring optionally substituted with a halogen atom, or a group represented by formula (b);

(Wherein, * indicates the binding position;
q represents an integer from 0 to 4;
q X 2 are each independently 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 through an oxygen atom, or an organic group of formula (a);

However, R 16 represents a linear or branched alkylene group having 6 to 16 carbon atoms, and X 3 is an oxygen atom or —C(=O)NR 17 — (R 17 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms). A represents either a silyl group or an alkyl group to which a silyloxy group is attached;
R 13 represents a hydrogen atom, represents a single bond together with R 11 to form a fluorene ring together with ring A and ring B, or forms a xanthene ring together with ring A and ring B via an oxygen atom. may form;
Ring B, in addition to q X 2 R 14 , further comprises 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 may have a substituent selected from the group consisting of )
Y represents a hydroxy group, a thiol group, NHR 20 (R 20 represents a hydrogen atom, an alkyl group or an aralkyl group) or a halogen atom. ]