Method for producing intermediate for producing endotoxin detection reagent, method for producing endotoxin detection reagent, and endotoxin detection reagent

Abstract

This is a method for producing an intermediate for producing an endotoxin detection reagent wherein the intermediate is a compound represented by formula (3) for producing an endotoxin detection reagent. This method for producing an intermediate for producing an endotoxin detection reagent comprises: a step for obtaining a reaction product that contains a compound represented by formula (3); and a step for reprecipitating the compound represented by formula (3) using a poor solvent for the compound represented by formula (3). (3): R1T1R2X. In formula (3), R1 is a group that contains conjugated multiple bonds, R2 is an alkylene group having 1 to 10 carbon atoms, T1 is a linking group, and X is a leaving group.

Description

Method for producing intermediate for producing endotoxin detection reagent, method for producing endotoxin detection reagent, and endotoxin detection reagent

[0001] Endotoxin is a lipopolysaccharide, a cell wall component of gram-negative bacteria, and is a typical pyrogen ubiquitous in the living environment. When endotoxin enters the bloodstream, it can cause fever, septic shock, multiple organ failure, tachycardia, and other symptoms. Therefore, strict management is required in the manufacture of pharmaceuticals and medical devices, particularly liquids that are directly introduced into the body, as well as pharmaceutical water, syringes, artificial organs, dialysis membranes, and other medical devices. For example, the management standard for water for injection in the "Japanese Pharmacopoeia (JP18) Quality Conformity Test" stipulates that endotoxin levels should be less than 0.25 EU / mL.

[0002] For example, Non-Patent Document 1 describes an endotoxin detection reagent used for detecting endotoxin.

[0003] In order for a detection reagent for detecting endotoxin to function efficiently, it must have high purity. In particular, when detecting endotoxin with high sensitivity or at low concentrations, the detection reagent must have even higher purity. For example, even if the fluorescence to be detected is emitted, if impurities are present, the fluorescence is absorbed, significantly reducing sensitivity. Therefore, the purity of the detection reagent must be, for example, 90% or more, preferably 95% or more.

[0004] Anal. Chem. , 2023, 95(33), 12349-12357

[0005] The endotoxin detection reagent described in Non-Patent Document 1 is produced through two reactions. If the purity of the intermediate obtained by the first reaction is low, a large amount of by-products is produced by the second reaction. As a result, it was found that the yield and purity of the target endotoxin detection reagent are low. This is because the physical properties of the raw materials, intermediates, and impurities are often similar, and separation by column chromatography, a common separation method, tends to result in a significantly low purity of the final product due to poor separation efficiency. Therefore, there has been a need to improve the yield and purity of the endotoxin detection reagent by increasing the purity of the intermediate obtained by the first reaction.

[0006] The problem to be solved by one embodiment of the present disclosure is to provide a method for producing an intermediate for producing an endotoxin detection reagent, which can improve the yield and purity of the endotoxin detection reagent. Another problem to be solved by another embodiment of the present disclosure is to provide a method for producing an endotoxin detection reagent using the method for producing an intermediate for producing an endotoxin detection reagent. Another problem to be solved by another embodiment of the present disclosure is to provide a high-purity endotoxin detection reagent.

[0007] The present disclosure includes the following aspects: <1> A method for producing an intermediate for producing an endotoxin detection reagent, which is a compound represented by the following formula (3), for producing an endotoxin detection reagent, the method comprising the steps of obtaining a reaction product containing the compound represented by formula (3), and reprecipitating the compound represented by formula (3) using a poor solvent for the compound represented by formula (3). １ T １ R ２ X...(3) In formula (3), R １ is a group containing conjugated multiple bonds, R ２ represents an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and T １is a linking group, and X is a leaving group. <2> The method for producing an intermediate for producing an endotoxin detection reagent according to <1>, wherein the compound represented by formula (3) is a compound represented by the following formula (3A), and in the step of obtaining a reaction product, a compound represented by the following formula (1A) is reacted with a compound represented by the following formula (2A). R １ OH...(1A) XR ２ X ... (2A) R １ OR ２ X...(3A) In formulas (1A) to (3A), R １ is a group containing conjugated multiple bonds, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group. <3> The compound represented by formula (3) is a compound represented by the following formula (3B), and in the step of obtaining the reaction product, a compound represented by the following formula (1B) is reacted with a compound represented by the following formula (2B). R １ NH ２ …(1B) XR ２ COOH...(2B) R １ NHCOR ２ X...(3B) In formulas (1B) to (3B), R １ is a group containing conjugated multiple bonds, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group. <4> The compound represented by formula (3) is a compound represented by the following formula (3C), and in the step of obtaining the reaction product, a compound represented by the following formula (1C) is reacted with a compound represented by the following formula (2C). R １ COOH...(1C) XR ２ NH ２ …(2C) R １ CONHR ２X...(3C) In formulas (1B) to (3C), R １ is a group containing conjugated multiple bonds, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group. <5> The poor solvent has an HSP value of 21 MPa １／２ ~30 MPa １／２ <6> A method for producing an intermediate for use in producing an endotoxin detection reagent according to any one of <1> to <5>, wherein the poor solvent is ethanol. <7> R １ <1> is a group containing a naphthalene ring, an anthracene ring, a phenanthrene ring, a pyrene ring, a chrysene ring, a perylene ring, a tetraphenylethylene ring, or an azobenzene ring. <8> A method for producing an endotoxin detection reagent, comprising the steps of: purifying the endotoxin detection reagent production intermediate using the method for producing an endotoxin detection reagent production intermediate according to any one of <1> to <7>; and reacting the endotoxin detection reagent production intermediate with a compound containing a group having a metal coordinating group, an acid group, or a base group in the presence of a base to produce the endotoxin detection reagent. <9> A method for producing the endotoxin detection reagent according to <8>, wherein the compound containing a metal coordinating group, an acid group, or a base group is 2,2'-dipicolylamine or iminodiacetic acid. <10> An endotoxin detection reagent having a purity of 80% or more and represented by the following formula (A): In formula (A), m is an integer of 1 to 10. In formula (A), R １ is a group containing conjugated multiple bonds, R ２ represents an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and T １is a linking group, and Y is a group that interacts with endotoxin. <11> An endotoxin detection reagent having a purity of 80% or more and represented by the following formula (A1): In formula (A1), m is an integer of 1 to 10.

[0008] According to one embodiment of the present disclosure, there is provided a method for producing an intermediate for producing an endotoxin detection reagent, which can improve the yield and purity of the endotoxin detection reagent. According to another embodiment of the present disclosure, there is provided a method for producing an endotoxin detection reagent using the method for producing the intermediate for producing the endotoxin detection reagent. According to another embodiment of the present disclosure, there is provided a highly pure endotoxin detection reagent.

[0009] The contents of the present disclosure are described in detail below. The following description of the constituent elements may be based on representative embodiments of the present disclosure, but the present disclosure is not limited to such embodiments. In the present disclosure, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in the present disclosure, the upper or lower limit value described in a certain numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in the numerical ranges described in the present disclosure, the upper or lower limit value described in a certain numerical range may be replaced with a value shown in the examples. In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0010] In the present disclosure, when a composition contains a plurality of substances corresponding to each component, the amount of each component in the composition refers to the total amount of the plurality of substances present in the composition unless otherwise specified. In the present disclosure, the term "process" includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.

[0011] [Method for producing intermediate for producing endotoxin detection reagent] The method for producing an intermediate for producing an endotoxin detection reagent (hereinafter also simply referred to as "intermediate") according to the present disclosure is a method for producing an intermediate for producing an endotoxin detection reagent, which is a compound represented by formula (3) below, for producing an endotoxin detection reagent, and includes a step of obtaining a reaction product containing the compound represented by formula (3), and a step of reprecipitating the compound represented by formula (3) below using a poor solvent for the compound represented by formula (3). R １ T １ R ２ X...(3) In formula (3), R １ is a group containing conjugated multiple bonds, R ２ represents an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and T １ is a linking group; and X is a leaving group.

[0012] As a result of intensive research, the present inventors have found that the method for producing an intermediate according to the present disclosure can produce an intermediate with higher purity than conventional methods, and that by using the highly pure intermediate, an endotoxin detection reagent with higher yield and higher purity than conventional methods can be obtained.

[0013] An endotoxin detection reagent containing a structure having a response site and a recognition site is usually produced by producing an intermediate containing a structure having the response site, and then reacting the obtained intermediate with a compound containing the recognition site.

[0014] Conventionally, in the production of intermediates, a compound containing a structure having a responsive site is reacted with a compound having a leaving group to obtain an intermediate containing a structure having a responsive site and also having a leaving group, and then a purification process using column chromatography is performed. However, in the purification process using column chromatography, it is difficult to separate the compound containing the responsive site and the compound having the leaving group from the intermediate.

[0015] In contrast, in the method for producing an intermediate according to the present disclosure, after obtaining a reaction product containing the compound represented by formula (3), the compound represented by formula (3) is reprecipitated using a poor solvent for the compound represented by formula (3). Components other than the compound represented by formula (3) contained in the reaction product dissolve in the poor solvent, while the compound represented by formula (3) does not dissolve in the poor solvent, allowing the other components to be separated from the intermediate compound represented by formula (3). This allows for the production of an intermediate with higher purity than conventional methods. Furthermore, by using a highly pure intermediate, an endotoxin detection reagent with higher purity can be obtained in a higher yield than conventional methods.

[0016] Each step in the method for producing an intermediate according to the present disclosure will be described below.

[0017] <Step of Obtaining a Reaction Product Containing a Compound Represented by Formula (3)> The method for producing an intermediate according to the present disclosure includes a step of obtaining a reaction product containing a compound represented by formula (3). The compound represented by formula (3) is an intermediate for producing an endotoxin detection reagent.

[0018] R １ T １ R ２ X...(3) In formula (3), R １ is a group containing conjugated multiple bonds, R ２ represents an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and T １ is a linking group; and X is a leaving group.

[0019] [R １ The group containing a conjugated multiple bond may be linear or may have a ring structure. A heteroatom may be contained at the site of the conjugated multiple bond. Examples of the group containing a conjugated multiple bond include a methine group (—C═C—), an aromatic group, an azo group, and a group formed by combining these groups.

[0020] Also, R １may be a group composed only of conjugated multiple bonds, but may also have a substituent that does not form a conjugated bond, such as an alkyl group, as appropriate. １ When the endotoxin detection reagent is introduced into the membrane, the affinity between the endotoxin detection reagent and the lipid chain of endotoxin is improved, making it possible to measure endotoxin at lower concentrations.

[0021] From the viewpoint of enabling highly sensitive detection of endotoxin, the group containing a conjugated multiple bond is preferably a group containing at least one of an aromatic group and an azo group.

[0022] The aromatic group may be an aromatic hydrocarbon group or an aromatic heterocyclic group. The ring structure may be a monocyclic or polycyclic ring, and the polycyclic ring may be a fused ring. The aromatic group may be unsubstituted or may have a substituent, but is preferably unsubstituted from the viewpoint of enabling high-sensitivity detection of endotoxin.

[0023] The aromatic heterocycle may be a monocycle, a heterocycle having two or more rings, or a fused heterocycle in which a heterocycle and an aromatic ring are fused, but is preferably a fused heterocycle in which a heterocycle and an aromatic ring are fused.

[0024] The number of ring members of the heterocycle is not particularly limited, but is preferably a 2- to 5-membered ring, and more preferably a 2- or 3-membered ring. The heterocycle is preferably a heterocycle having at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom, more preferably having at least one heteroatom selected from a nitrogen atom and an oxygen atom, further preferably containing a nitrogen atom or an oxygen atom, and particularly preferably containing an oxygen atom.

[0025] Examples of heterocyclic rings include a coumarin ring, a pyridine ring, a pyrimidine ring, a thiophene ring, a furan ring, and a pyrrole ring. The heterocyclic ring may be unsubstituted or substituted. The substituent is not particularly limited, and examples thereof include a hydroxy group, a carboxy group, an alkoxy group, an aryloxy group, a halogen atom, an alkyl group, a halogenated alkyl group, an aryl group, an alkyloxycarbonyl group, an alkylcarbonyloxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylthio group, an arylthio group, a dialkylamino group, an alkylarylamino group, a diarylamino group, and a group formed by combining two or more of these. Among these, the substituent is preferably at least one group selected from the group consisting of a hydroxy group and a carboxy group.

[0026] From the viewpoint of enabling high-sensitivity detection of endotoxin, the heterocycle is preferably a heterocycle having a substituent, more preferably a coumarin ring having a substituent, still more preferably a coumarin ring substituted with at least one group selected from the group consisting of a hydroxy group and a carboxy group, and particularly preferably a coumarin ring substituted with a hydroxy group and a carboxy group.

[0027] The aromatic ring may be a single ring or a fused aromatic ring in which two or more aromatic rings are fused, but from the viewpoint of enabling high-sensitivity detection of endotoxin, a fused aromatic ring is preferred. The fused aromatic ring is preferably a fused aromatic ring in which two to eight aromatic rings are fused, more preferably a fused aromatic ring in which two to six aromatic rings are fused, and even more preferably a fused aromatic ring in which two to four aromatic rings are fused. Examples of the fused aromatic ring include a naphthalene ring, an anthracene ring, a phenalene ring, a phenanthrene ring, a pyrene ring, a triphenylene ring, a tetracene ring, a quinoline ring, a chrysene ring, and a picene ring. R １ When is an aromatic group, from the viewpoint of enabling high-sensitivity detection of endotoxin, the aromatic ring is preferably a heterocycle or a fused aromatic ring, more preferably a coumarin ring, a pyrene ring or an anthracene ring, and particularly preferably a pyrene ring.

[0028] Furthermore, when the aromatic ring is monocyclic, the aromatic ring is preferably a benzene ring. When the group containing an aromatic group contains a monocyclic aromatic ring, it may contain one aromatic ring or two or more aromatic rings. Examples of the group containing a monocyclic aromatic ring include a phenyl group, a biphenyl group, a stilbene group, etc., and from the viewpoint of being able to detect endotoxin with high sensitivity, a biphenyl group is preferred.

[0029] From the viewpoint of enabling high-sensitivity detection of endotoxin, the group containing an aromatic group is preferably a group represented by the following formula (A ａｒｏ ) is preferably a group represented by the formula (I).

[0030]

[0031] Formula A aro In the formula, A represents an aromatic ring, and R Ｙ１ each independently represents a monovalent substituent, a represents an integer of 0 to 5, and the wavy line represents T in formula (3). １ Examples of the aromatic ring include the aromatic rings described above, and among these, a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a pyrene ring, a chrysene ring, or a perylene ring is preferred. Ｙ１ are each independently a substituent in the aromatic hydrocarbon group. The substituent may be bonded to ring A via an amide bond or an ester bond. The substituent is preferably an alkyl group, a hydroxy group, or a carboxy group. The alkyl group is preferably an alkyl group having 1 to 15 carbon atoms, more preferably an alkyl group having 2 to 12 carbon atoms, and even more preferably an alkyl group having 5 to 10 carbon atoms. Formula A aro In the formula (I), when a is an integer of 1 to 5, R Ｙ１ is preferably bonded to ring A via an amide bond, and Ｙ１ It is more preferable that at least one of a is an alkyl group having 1 to 15 carbon atoms (preferably having 2 to 12 carbon atoms, more preferably having 5 to 10 carbon atoms) bonded to ring A via an amide bond. a is preferably an integer of 0 to 3, more preferably an integer of 0 to 2.

[0032] When the ring A is a coumarin ring, A is represented by the following formula Aｃｏｕ It is preferable that the formula be represented by the following formula:

[0033]

[0034] Formula A ｃｏｕ Medium, R Ｙ１ each independently represents a monovalent substituent, a represents an integer of 1 or 2, and the wavy line represents T in formula (3). １ represents the binding site with Ｙ１ The substituents in A aro The meanings and preferred embodiments of the substituents are the same as those of the substituents in the above.

[0035] A group containing an azo (-N=N-) structure (hereinafter sometimes referred to as an "azo group") may contain one azo structure or two or more azo structures. From the viewpoint of enabling high-sensitivity detection of endotoxin, a group containing one azo structure is preferred, and a group combining an azo group with an aromatic group is more preferred. In the group combining an azo group with an aromatic group, the aromatic group may be an aromatic hydrocarbon ring or an aromatic heterocycle, but from the viewpoint of excellent color development, an aromatic hydrocarbon group is preferred. The aromatic ring contained in the aromatic hydrocarbon group may be a monocyclic ring or a fused aromatic ring in which two or more aromatic rings are fused. Examples of fused aromatic rings include a naphthalene ring, an anthracene ring, and a pyrene ring. Furthermore, when the aromatic ring is a monocyclic ring, the aromatic ring is preferably a benzene ring. Furthermore, the aromatic hydrocarbon group may be unsubstituted or substituted. The substituent is not particularly limited, and examples thereof include a hydroxy group, a carboxy group, an alkoxy group, an aryloxy group, a halogen atom, an alkyl group, a halogenated alkyl group, an aryl group, an alkyloxycarbonyl group, an alkylcarbonyloxy group, an alkoxycarbonyl group, an aryloxycarbonyl group, an alkylthio group, an arylthio group, a dialkylamino group, an alkylarylamino group, a diarylamino group, and groups formed by combining two or more of these groups.

[0036] From the viewpoint of enabling highly sensitive detection of endotoxin, the group containing an azo (—N═N—) structure is preferably a group in which an aromatic hydrocarbon group is bound to an azo structure, and is represented by the following formula (A ａｚｏ ) is more preferably a group represented by the formula (I).

[0037]

[0038] Formula (A ａｚｏ ) Medium, R Ｚ１ and R Ｚ２ each independently represents a substituent, and the wavy line represents T in formula (3). １ represents the bonding site with the above. Examples of the substituent include the substituents in the aromatic hydrocarbon group. The substituent may be bonded to the azobenzene structure via an amide bond or an ester bond. The substituent is preferably an alkyl group, a hydroxy group, or a carboxy group. The alkyl group is preferably an alkyl group having 1 to 15 carbon atoms, more preferably an alkyl group having 2 to 12 carbon atoms, and even more preferably an alkyl group having 5 to 10 carbon atoms. R Ｚ１ and R Ｚ２ may have the same structure or may be different from each other, but from the viewpoint of micelle formation, R Ｚ１ and R Ｚ２ are preferably different from each other. Ｚ１ and R Ｚ２ may be bonded to the azo group at any of the ortho, meta and para positions. Ｚ１ and R Ｚ２ The substituent in the formula (I) is preferably bonded to the para position relative to the azo group. The bonding site to the oxygen atom may be bonded to the azo group at any of the ortho, meta, and para positions. Among these, the bonding site to the oxygen atom is preferably bonded to the para position relative to the azo group.

[0039] From the viewpoint of excellent separability, R １ is preferably a group containing a naphthalene ring, an anthracene ring, a phenanthrene ring, a pyrene ring, a chrysene ring, a perylene ring, a tetraphenylethylene ring, or an azobenzene ring, and more preferably a group containing a pyrene ring or a tetraphenylethylene ring.

[0040] R １ Specific examples of the compound are shown below. The wavy line represents T in formula (3). １ represents the binding site with

[0041]

[0042] [R ２ 〕 R ２ The alkylene group represented by the formula (I) may be linear, branched, or cyclic. ２ The alkylene group represented by R is preferably linear. ２ The alkylene group represented by the formula (I) may be unsubstituted or may have a substituent. Examples of the substituent include an amide group, a carbonyl group, and an amino group. ２ When the alkylene group represented by the formula (I) has two or more carbon atoms, it may contain a heteroatom between carbon atoms. For example, an ethereal oxygen atom, a sulfur atom, or a nitrogen atom may be located between carbon atoms. ２ The alkylene group represented by the formula (I) preferably has 2 to 8 carbon atoms, and more preferably 2 or 3 carbon atoms.

[0043] [T １ 〕 T １ The linking group represented by the formula (I) is not particularly limited, but is preferably —O—, —NH—, —C(═O)—, or a combination thereof. １ Examples of the group include -O-, -NHC(=O)-, -C(=O)NH-, -C(=O)-O-, and -NHC(=O)O-. １ is preferably —O—, —NHC(═O)—, or —C(═O)NH—.

[0044] [X] Examples of the leaving group represented by X include a halogen atom and sulfonic acid. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among these, from the viewpoint of reactivity, the leaving group is preferably a halogen atom, and more preferably a bromine atom.

[0045] In the step of obtaining a reaction product containing a compound represented by formula (3), the method for obtaining the reaction product is not particularly limited, but from the viewpoint of ease of synthesis, the following method is preferred.

[0046] Method 1: A compound represented by formula (1A) is reacted with a compound represented by the following formula (2A): １ OH...(1A) XR ２ X ... (2A) R １ OR ２ X...(3A) In formulas (1A) to (3A), R １ is a group containing conjugated multiple bonds, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group.

[0047] Method 2: A compound represented by formula (1B) is reacted with a compound represented by the following formula (2B): １ NH ２ …(1B) XR ２ COOH...(2B) R １ NHCOR ２ X...(3B) In formulas (1B) to (3B), R １ is a group containing conjugated multiple bonds, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group.

[0048] Method 3: A compound represented by formula (1C) is reacted with a compound represented by the following formula (2C): １ COOH...(1C) XR ２ NH ２ …(2C) R １ CONHR ２ X...(3C) In formulas (1C) to (3C), R １ is a group containing conjugated multiple bonds, R ２is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and X is a leaving group.

[0049] -Method 1- In Method 1, R in Formula (1A) to Formula (3A) １ , R ２ and the preferred embodiments of X are as described above.

[0050] Examples of the compound represented by formula (2A) include 1,2-dibromoethane and 1,3-dibromopropane. In the reaction between the compound represented by formula (1A) and the compound represented by formula (2A), it is preferable to use a larger amount of the compound represented by formula (2A) than the compound represented by formula (1A). By using an excess amount of the compound represented by formula (2A), it is possible to reduce by-products. The amount of the compound represented by formula (2A) used relative to the amount of the compound represented by formula (1A) is preferably 2 to 10 times, and more preferably 4 to 6 times, the molar equivalent.

[0051] -Method 2- In Method 2, R in Formula (1B) to Formula (3B) １ , R ２ and the preferred embodiments of X are as described above.

[0052] Examples of the compound represented by formula (2B) include bromoacetic acid and 3-bromopropionic acid. In the reaction between the compound represented by formula (1B) and the compound represented by formula (2B), it is preferable to use a larger amount of the compound represented by formula (2B) than the compound represented by formula (1B). By using an excess amount of the compound represented by formula (2B), it is possible to reduce by-products. The amount of the compound represented by formula (2B) used relative to the amount of the compound represented by formula (1B) is preferably 2 to 10 times, and more preferably 4 to 6 times, the molar amount of the compound represented by formula (1B).

[0053] -Method 3- In Method 3, R in Formulas (1C) to (3C) １ , R ２ and the preferred embodiments of X are as described above.

[0054] Examples of the compound represented by formula (2C) include 2-bromoethylamine and 3-bromopropylamine. In the reaction between the compound represented by formula (1C) and the compound represented by formula (2C), it is preferable to use a larger amount of the compound represented by formula (2C) than the compound represented by formula (1B). By using an excess amount of the compound represented by formula (2C), it is possible to reduce by-products. The amount of the compound represented by formula (2C) used relative to the amount of the compound represented by formula (1C) is preferably 2 to 10 times, and more preferably 4 to 6 times, the molar amount.

[0055] The step of obtaining a reaction product containing the compound represented by formula (3) is preferably carried out in the presence of a base.

[0056] The type of base is not particularly limited, and examples thereof include hydrides, hydroxides, carbonates, and quaternary ammonium salts of alkali metals and alkaline earth metals. Among these, from the viewpoints of excellent separability and suppression of side reactions, the base is preferably a carbonate of an alkali metal or alkaline earth metal, and more preferably potassium carbonate or sodium carbonate.

[0057] The amount of the base used is not particularly limited, but is preferably 2 to 10 times, and more preferably 4 to 6 times, the amount of the compound represented by Formula (1A), Formula (1B), or Formula (1C) used, in terms of moles.

[0058] The step of obtaining a reaction product containing a compound represented by formula (3) is preferably carried out in a solution. The reaction solvent is not particularly limited, and a solvent that is inert to and capable of dissolving the raw materials is appropriately selected. The reaction temperature is not particularly limited, and is, for example, 60°C to 100°C. The reaction time is not particularly limited, and is, for example, 8 hours to 42 hours. The pressure during the reaction is not particularly limited, and is usually carried out under atmospheric pressure. The reaction is preferably carried out under an inert gas atmosphere. Examples of inert gases include nitrogen gas and argon gas.

[0059] <Step of Reprecipitating the Compound Represented by Formula (3)> In the method for producing an intermediate according to the present disclosure, a poor solvent for the compound represented by formula (3) is used to reprecipitate the compound represented by formula (3). The poor solvent is appropriately selected from solvents that do not dissolve the compound represented by formula (3).

[0060] From the viewpoint of excellent separation property, the poor solvent has an HSP value of 21 MPa. １／２ ~30 MPa １／２ It is preferable that the solvent is 25 MPa. １／２ ~28 MPa １／２ In the present disclosure, the HSP value refers to the value of the Hansen solubility parameter. The HSP value is calculated based on the energy between molecules.

[0061] The HSP values ​​of each solvent are as follows: Methanol: 29.6 MPa １／２ Dimethyl sulfoxide: 26.7 MPa １／２ Ethanol: 26.1 MPa １／２ 1-propanol: 24.9 MPa １／２ Acetonitrile: 24.8 MPa １／２ Dimethylformamide: 24.7 MPa １／２ Isopropanol: 23.5 MPa １／２ 1-butanol: 23.2 MPa １／２ 2-butanol: 22.2 MPa １／２ Pyridine: 21.8 MPa １／２

[0062] From the viewpoint of achieving superior separation properties, the poor solvent is preferably ethanol or methanol.

[0063] The method for reprecipitating the target product is not particularly limited, and can be carried out by a commonly known method.

[0064] [Method for producing endotoxin detection reagent] The method for producing an endotoxin detection reagent according to the present disclosure includes the steps of producing an intermediate using the method for producing an intermediate according to the present disclosure, and reacting the intermediate with a compound containing a group having a metal coordinating group, an acid group, or a base group in the presence of a base to produce an endotoxin detection reagent.

[0065] <Compound containing a group having a metal coordinating group, an acid group, or a base group> In the method for producing an endotoxin detection reagent according to the present disclosure, an intermediate is reacted with a compound containing a group having a metal coordinating group, an acid group, or a base group.

[0066] The compound containing a group having a metal coordinating group, an acid group or a base group is preferably a compound containing a group having a metal coordinating group or an acid group.

[0067] The metal coordinating group may be a group that directly coordinates with at least one metal or a group that promotes coordination with a metal. Examples of the metal include metal ions (cations) such as alkali metals, alkaline earth metals, and transition metals (Li ＋ , Na ＋ , K. ＋ , Mg ２＋ , Ag ＋ , Ni ２＋ , Co ２＋ , Ni ２＋ , Cu ２＋ , Zn ２＋ Or Cd ２＋ Among these, from the viewpoint of being able to detect endotoxin with high sensitivity, transition metals are preferred, divalent transition metals are more preferred, and zinc ions (Zn ２＋ ), or cadmium ions (Cd ２＋ ) is more preferred, and Zn ２＋ Examples of the metal coordinating group include an aromatic heterocyclic group containing at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom, a group containing a hydroxyl group, a carboxy group, a crown ether group, and the like.

[0068] Examples of aromatic heterocyclic groups include heterocyclic groups containing at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom. From the viewpoint of enabling high-sensitivity detection of endotoxin, the aromatic heterocyclic group is preferably a heterocyclic group containing at least one atom selected from a nitrogen atom and an oxygen atom, more preferably a heterocyclic group containing a nitrogen atom, even more preferably a heterocyclic ring containing a 5- or 6-ring member and a nitrogen atom, and particularly preferably a heterocyclic ring containing a 6-ring member and a nitrogen atom. Examples of heterocyclic rings containing a nitrogen atom include a pyrrole ring, a pyridine ring, a pyrazine ring, and a pyrimidine ring. Furthermore, the heterocyclic ring may be unsubstituted or may have a substituent, but from the viewpoint of enabling high-sensitivity detection of endotoxin, a heterocyclic ring containing a substituent is preferred. Examples of the substituent include an alkyl group, an aryl group, and the like. Among these, the substituent is preferably an alkyl group, and more preferably an alkyl group having 1 to 4 carbon atoms.

[0069] From the viewpoint of enabling high-sensitivity detection of endotoxin, among heterocycles having a substituent, a heterocycle containing a nitrogen atom having an alkyl group is preferred, a heterocycle having an alkyl group with 1 to 4 carbon atoms and containing a nitrogen atom with 5 or 6 ring members is more preferred, a heterocycle having an alkyl group with 1 to 4 carbon atoms and containing a nitrogen atom with 6 ring members is even more preferred, and a pyridyl group having an alkyl group with 1 to 4 carbon atoms is particularly preferred.

[0070] When the metal coordinating group contains an aromatic heterocycle, from the viewpoint of enabling endotoxin to be detected with high sensitivity, the group containing an aromatic heterocycle is preferably a substituted amino group substituted with a heterocyclic group, more preferably a substituted amino group substituted with a heterocycle containing an alkyl group having 1 to 4 carbon atoms (preferably a heterocycle having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom having 5 or 6 ring members, more preferably a heterocycle having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom having 6 ring members, and even more preferably a pyridyl group having an alkyl group having 1 to 4 carbon atoms), and even more preferably a disubstituted amino group substituted with a heterocycle containing an alkyl group having 1 to 4 carbon atoms (preferably a heterocycle having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom having 5 or 6 ring members, more preferably a heterocycle having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom having 6 ring members, and even more preferably a pyridyl group having an alkyl group having 1 to 4 carbon atoms).

[0071] Furthermore, when the metal coordinating group is an aromatic heterocyclic group, it is preferable to add a metal ion capable of forming a complex that physically or chemically interacts with the metal coordinating group and a predetermined site or functional group of endotoxin. Furthermore, since the amount of fluorescent light emitted increases significantly when coordinated to a metal ion, endotoxin may be detected as a decrease in the amount of light emitted. Examples of such metal ions include the metal ions in the above-mentioned metal coordinating group, such as copper ions (Cu ２＋ ), nickel ions (Ni ２＋ ) are listed.

[0072] Examples of the acid group include a group selected from a sulfo group, a carboxy group, a phosphate group, a boronic acid group, a phenol group, a salt thereof, or a desalted structure thereof, and among these, a group selected from a carboxy group, a boronic acid, a salt thereof, or a desalted structure thereof is preferred. Examples of the group having the acid group include an alkyl group having the acid group, an aryl group having the acid group, and an amino group having the acid group. Among these, an aryl group having the acid group or an amino group having the acid group is preferred. Examples of the substituent other than the acid group include an alkyl group and an aryl group, and among these, an alkyl group is preferred, and an alkyl group having 1 to 4 carbon atoms is more preferred.

[0073] From the viewpoint of enabling high-sensitivity detection of endotoxin, the group having an acid group is preferably a phenyl group having an acid group or an amino group having an acid group, preferably an amino group substituted with a phenyl group having an acid group or an alkyl group having 1 to 4 carbon atoms containing an acid group, more preferably a phenyl group containing boronic acid, a pyridyl group containing boronic acid, a fluorophenyl group containing boronic acid, or an amino group substituted with an alkyl group having 1 to 4 carbon atoms containing a carboxy group, even more preferably a disubstituted amino group substituted with a fluorophenyl group containing boronic acid or an alkyl group having 1 to 4 carbon atoms containing a carboxy group, and particularly preferably a disubstituted amino group substituted with a fluorophenyl group containing boronic acid or an alkyl group having 1 or 2 carbon atoms containing a carboxy group. Examples of disubstituted amino groups substituted with an alkyl group having 1 or 2 carbon atoms containing a carboxy group include monovalent groups obtained by removing a hydrogen atom from iminodiacetic acid.

[0074] Examples of the base group include an amino group, a phosphine group, an aromatic ring containing a nitrogen atom, etc. From the viewpoint of enabling highly sensitive detection of endotoxin, the base group is preferably an amino group or a phosphine group.

[0075] From the viewpoint of being able to detect endotoxin with high sensitivity, the compound containing a metal coordinating group, an acid group, or a group having a base group is preferably 2,2'-dipicolylamine, iminodiacetic acid, phenylboronic acid, N-[2-(2-pyridinyl)ethyl]-2-pyridineethanamine, dimethylamine, trimethylamine, trimethylphosphine, diethylphosphine, triethylphosphine, diphenylphosphine, or triphenylphosphine, and more preferably 2,2'-dipicolylamine or iminodiacetic acid.

[0076] In the method for producing an endotoxin detection reagent according to the present disclosure, in the reaction between an intermediate and a compound containing a group having a metal coordinating group, an acid group, or a base group, it is preferable to use a larger amount of the compound containing a group having a metal coordinating group, an acid group, or a base group than the intermediate. By using an excess amount of the compound containing a group having a metal coordinating group, an acid group, or a base group, the intermediate can be eliminated. The amount of the compound containing a group having a metal coordinating group, an acid group, or a base group used relative to the amount of the intermediate used is preferably 2 to 10 times, and more preferably 4 to 6 times, the amount in molar terms.

[0077] The reaction of the intermediate with the compound containing a group having a metal coordinating group, an acid group, or a base group is carried out in the presence of a base. The type of base is not particularly limited, and examples thereof include the same bases as those described above.

[0078] The amount of base used is not particularly limited, but is preferably 2 to 10 times, more preferably 4 to 6 times, the amount of intermediate used in molar terms.

[0079] The reaction between the intermediate and the compound containing a group having a metal coordinating group, an acid group, or a base group is preferably carried out in a solution. The reaction solvent is not particularly limited, and a solvent that is inert to and capable of dissolving the intermediate and the compound containing a group having a metal coordinating group, an acid group, or a base group is appropriately selected. The reaction temperature is not particularly limited, and is, for example, 60°C to 100°C. The reaction time is not particularly limited, and is, for example, 8 hours to 48 hours. The pressure during the reaction is not particularly limited, and is usually carried out under atmospheric pressure. The reaction is preferably carried out under an inert gas atmosphere. Examples of inert gases include nitrogen gas and argon gas.

[0080] In the method for producing an endotoxin detection reagent according to the present disclosure, a purification treatment may be carried out after the reaction is completed. Examples of the purification treatment include purification treatment by size exclusion chromatography.

[0081] Examples of endotoxin detection reagents obtainable by reacting an intermediate with a compound containing a group having a metal coordinating group, an acid group, or a base group include the following compounds: The endotoxin detection reagent may contain a metal ion.

[0082]

[0083] [Endotoxin Detection Reagent] The endotoxin detection reagent according to the present disclosure has a purity of 80% or more and is represented by the following formula (A): In formula (A), m is an integer of 1 to 10. In formula (A), R １ is a group containing conjugated multiple bonds, R ２ represents an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a heteroatom between carbon atoms, and T １ is a linking group, and Y is a group that interacts with endotoxin.

[0084] The purity is preferably 90% or more, more preferably 98% or more.

[0085] R in formula (A) １ , R ２ , and T１ The preferred embodiments are as described above.

[0086] Y is preferably a group having a metal coordinating group, an acid group or a basic group. Preferred embodiments of the group having a metal coordinating group, an acid group or a basic group are as described above.

[0087] The endotoxin detection reagent according to the present disclosure has a purity of 80% or more and is represented by the following formula (A1): In formula (A1), m is an integer of 1 to 10.

[0088]

[0089] By using the method for producing an intermediate according to the present disclosure, the following compound (A0) can be obtained with high purity: In formula (A0), m is an integer of 1 to 10.

[0090]

[0091] In the method for producing an endotoxin detection reagent using the compound (A0), the generation of by-products can be suppressed. As a result, the purity of the compound represented by formula (A1) is obtained at 80% or higher. The purity is preferably 90% or higher, and more preferably 98% or higher. When the method for producing an intermediate according to the present disclosure is used, sensitivity is improved by approximately 10 to 1,000 times compared to existing endotoxin detection reagents, for example, endotoxin detection reagents with a purity of approximately 75%. This makes it possible to detect endotoxin at low concentrations or with reduced amounts of reagent used. Note that when producing the endotoxin detection reagent according to the present disclosure using commercially available conventional reagents, the production is usually carried out via an intermediate for producing the endotoxin detection reagent, which is the compound represented by formula (3). When the method for producing an intermediate according to the present disclosure is used, a highly pure endotoxin reagent can be obtained.

[0092] The present disclosure will be specifically described below with reference to examples, but the present disclosure is not limited to these examples in any way.

[0093] Example 1 1-Hydroxypyrene ("Raw Material 1" in Table 1, 0.545 g, 2.50 mmol), 1,2-dibromoethane ("Raw Material 2" in Table 1, 2.020 g, 10.75 mmol), potassium carbonate (1.750 g, 12.67 mmol), and dehydrated acetonitrile (20 mL) were added to a round-bottom flask under an argon atmosphere. The suspension was heated to reflux at 85°C for 15 hours. After the reaction mixture was cooled to room temperature (25°C), the solvent was removed using a rotary evaporator. Chloroform (30 mL) was added to the residue, and the organic solution was washed with water (4 x 40 mL) using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated using a rotary evaporator. A purification treatment by reprecipitation was performed. Specifically, an excess amount of ethanol (a poor solvent) was added to the concentrate. A white solid precipitated, which was collected by filtration and washed with water and ethanol. The resulting solid was dried under reduced pressure to give the following compound as an intermediate (0.692 g, 2.126 mmol). The yield of this reaction was 85.0%.

[0094]

[0095] Subsequently, the obtained intermediate (0.161 mg, 0.495 mmol), potassium carbonate (0.391 g, 2.828 mmol), potassium iodide (0.377 g, 2.269 mmol), and anhydrous tetrahydrofuran (20 mL) were added to a round-bottom flask under an argon atmosphere. 2,2'-dipicolylamine (0.644 g, 3.230 mmol, designated "raw material 3" in Table 1) dissolved in anhydrous tetrahydrofuran (5 mL) was added dropwise to this mixture with stirring at room temperature (25 °C), and the suspension was refluxed at 70 °C overnight. After cooling to room temperature (25 °C), the reaction mixture was filtered to remove insoluble salts, and the solvent was removed using a rotary evaporator. The residue was dissolved in dichloromethane (30 mL), and the organic solution was subsequently washed with 10% by weight aqueous ammonium chloride solution (2 × 20 mL) and water (2 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated to dryness using a rotary evaporator. The residue was purified by size exclusion chromatography using chloroform as an eluent. The following compound (0.149 g, 0.337 mmol) was obtained as a brown oily substance used as an endotoxin detection reagent. The yield of this reaction was 68.0%. Therefore, the total synthesis yield calculated from the starting materials was 58%. The purity of the final product was 99%.

[0096]

[0097] [Examples 2 to 7] In Examples 2 to 7, raw material 1, raw material 2, and raw material 3 were changed to the compounds shown in Table 1, and the poor solvent used for reprecipitation was changed to the compound shown in Table 1. In all cases, raw material 2 was Br—(CH ２ ) ｎ The compounds represented by —Br were used, and n is shown in Table 1.

[0098] [Comparative Examples 1 and 2] In Comparative Example 1, an endotoxin detection reagent was obtained in the same manner as in Example 1, except that a purification treatment by column chromatography was performed without performing a purification treatment by reprecipitation. In Comparative Example 2, an endotoxin detection reagent was obtained in the same manner as in Example 4, except that a purification treatment by column chromatography was performed without performing a purification treatment by reprecipitation. In Table 1, "Y" is entered in the column column.

[0099] It was confirmed that all of the obtained endotoxin detection reagents were capable of detecting endotoxin with high sensitivity.

[0100]

[0101] As shown in Table 1, in Examples 1 to 7, a purification treatment by reprecipitation was performed in the method for producing the intermediate, and it was found that the endotoxin detection reagent could be obtained in high yield and with high purity.

[0102] The disclosure of Japanese Patent Application No. 2024-002886, filed on January 11, 2024, is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually indicated to be incorporated by reference.

Claims

1. A method for producing an intermediate for manufacturing an endotoxin detection reagent, which is a compound represented by the following formula (3) for producing an endotoxin detection reagent, the method comprising: a step of obtaining a reaction product containing the compound represented by the formula (3); and a step of reprecipitating the compound represented by the following formula (3) using a poor solvent for the compound represented by the formula (3). In the formula (3), R １ T １ R ２ X... (3) In the formula (3), R １ is a group containing a conjugated multiple bond, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms, T １ is a linking group, and X is a leaving group.

2. The compound represented by the formula (3) is a compound represented by the following formula (3A). In the step of obtaining the reaction product, a compound represented by the following formula (1A) is reacted with a compound represented by the following formula (2A). The method for producing an intermediate for producing an endotoxin detection reagent according to claim 1. R １ OH … (1A) XR ２ X … (2A) R １ OR ２ X … (3A) In formulas (1A) to (3A), R １ is a group containing a conjugated multiple bond, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and an alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms. X is a leaving group.

3. The compound represented by the formula (3) is a compound represented by the following formula (3B). In the step of obtaining the reaction product, the compound represented by the following formula (1B) is reacted with the compound represented by the following formula (2B). A method for producing an intermediate for producing an endotoxin detection reagent according to claim 1. R １ NH ２ ... (1B) XR ２ COOH... (2B) R １ NHCOR ２ X... (3B) In the formulas (1B) to (3B), R １ is a group containing a conjugated multiple bond, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms - carbon atoms, X is a leaving group.

4. The compound represented by the formula (3) is a compound represented by the following formula (3C). In the step of obtaining the reaction product, a compound represented by the following formula (1C) is reacted with a compound represented by the following formula (2C). The method for producing an intermediate for producing an endotoxin detection reagent according to claim 1. R １ COOH … (1C) XR ２ NH ２ … (2C) R １ CONHR ２ X … (3C) In formulas (1B) to (3C), R １ is a group containing a conjugated multiple bond, R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms. X is a leaving group.

5. The poor solvent has an HSP value of 21 MPa １／２ to 30 MPa １／２ The method for producing an intermediate for manufacturing an endotoxin detection reagent according to claim 1, wherein the poor solvent is a solvent having an HSP value of 21 MPa to 30 MPa.

6. The method for producing an intermediate for manufacturing an endotoxin detection reagent according to claim 1, wherein the poor solvent is ethanol.

7. The R １ is a group containing a naphthalene ring, an anthracene ring, a phenanthrene ring, a pyrene ring, a chrysene ring, a perylene ring, a tetraphenylethylene ring, or an azobenzene ring, and is a method for producing an intermediate for producing an endotoxin detection reagent according to claim 1.

8. A process for producing an intermediate for manufacturing an endotoxin detection reagent using the method for producing an intermediate for manufacturing an endotoxin detection reagent according to any one of claims 1 to 7, and a step of reacting the intermediate for manufacturing an endotoxin detection reagent with a compound containing a metal-coordinating group, an acid group or a group having a basic group in the presence of a base to produce an endotoxin detection reagent. A method for producing an endotoxin detection reagent, comprising:

9. The method for producing an endotoxin detection reagent according to claim 8, wherein the compound containing a metal-coordinating group, an acid group or a group having a basic group is 2,2'-dipicolylamine or iminodiacetic acid.

10. An endotoxin detection reagent having a purity of 80% or more and represented by the following formula (A). In formula (A), R １ is a group containing a conjugated multiple bond, and R ２ is an alkylene group having 1 to 10 carbon atoms, which may have a substituent, and the alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms, and T １ is a linking group, and Y is a group that interacts with endotoxin.

11. An endotoxin detection reagent having a purity of 80% or more and represented by the following formula (A1). In formula (A1), m is an integer of 1 to 10.

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