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

Abstract

To provide a method for producing an intermediate for producing an endotoxin detection reagent, the method being capable of improving both the yield and the purity of the endotoxin detection reagent.SOLUTION: The invention provides a method for producing an intermediate for producing an endotoxin detection reagent, where the intermediate is a compound represented by a formula (3) defined by R1T1R2X for producing an endotoxin detection reagent, where R1 is a group that comprises 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. The 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 the formula (3); and a step for reprecipitating the compound represented by the formula (3) using a poor solvent for the compound represented by the formula (3).SELECTED DRAWING: None

Description

Technical Field

[0001] Endotoxin is lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, and is a typical pyrogen ubiquitously present in the living environment. When endotoxin enters the blood, it causes effects such as fever, septic shock, multiple organ failure, and tachycardia. Therefore, in the manufacture of pharmaceuticals, medical devices, especially liquids directly introduced into the body, pharmaceutical water, syringes, artificial organs, dialysis membranes, and other medical devices, strict management is required. For example, in the control standards for injection water in the "Japanese Pharmacopoeia (JP18) Quality Conformance Test", it is specified to be less than 0.25 EU / mL.

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

[0003] For the detection reagent for detecting endotoxin to function efficiently, high purity is required. In particular, when detecting endotoxin with high sensitivity or at a low concentration, a higher purity of the detection reagent is required. For example, even if the fluorescence to be detected is emitted, if there are impurities, the fluorescence will be absorbed and the sensitivity will be significantly reduced. Therefore, the purity of the detection reagent is required to be, for example, 90% or more, preferably 95% or more.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[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 will be generated by the second reaction. As a result, it has been found that the yield of the target endotoxin detection reagent is low and the purity is also low. This is because the physical properties of the raw material substances, intermediates, and impurities are often similar, and in the separation by column chromatography, which is a common separation method, the separation efficiency is poor, so the purity of the final product tends to be extremely low. Therefore, it has been required 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 that 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.

Means for Solving the Problems

[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, a step of obtaining a reaction product containing the compound represented by formula (3); a step of reprecipitating the compound represented by the following formula (3) using a poor solvent for the compound represented by formula (3), the method for producing an intermediate for producing an endotoxin detection reagent. R 1 T 1 R 2 X …(3) In 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, X is a leaving group. <2> The compound represented by the formula (3) is the compound represented by the following formula (3A), In the step of obtaining the reaction product, the compound represented by the following formula (1A) is reacted with the compound represented by the following formula (2A). The method for producing an intermediate for producing an endotoxin detection reagent according to <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 the 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 the 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). The method for producing an intermediate for producing an endotoxin detection reagent according to <1>. R 1 NH2…(1B) XR 2 COOH …(2B) R 1 NHCOR 2 X …(3B) In formulas (1B) to (3B), R1 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 heteroatom between 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 method for producing an intermediate for producing an endotoxin detection reagent according to <1>, wherein 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 NH2…(2C) R 1 CONHR 2 X …(3C) In the 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 heteroatom between carbon atoms, X is a leaving group. <5> The poor solvent is a solvent having an HSP value of 21 MPa 1 / 2 ~30 MPa 1 / 2 A method for producing an intermediate for producing an endotoxin detection reagent according to any one of <1> to <4>. <6> The poor solvent is ethanol. A method for producing an intermediate for producing an endotoxin detection reagent according to any one of <1> to <5>. <7> 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. A method for producing an intermediate for producing an endotoxin detection reagent according to any one of <1> to <6>. <8> <1> to <7>, using the method for producing an intermediate for producing an endotoxin detection reagent according to any one of <1> to <7>, a step of purifying the intermediate for producing an endotoxin detection reagent, and in the presence of a base, reacting the intermediate for producing an endotoxin detection reagent with a compound containing a group having a metal-coordinating group, an acid group or a base group to produce an endotoxin detection reagent. A method for producing an endotoxin detection reagent, comprising: <9> The compound containing a group having a metal-coordinating group, an acid group or a base group is 2,2'-dipicolylamine or iminodiacetic acid. The method for producing an endotoxin detection reagent according to <8>. <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 from 1 to 10.

Chemical formula

Chemical formula

Advantages of the Invention

[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. Further, according to another embodiment of the present disclosure, there is provided a method for producing an endotoxin detection reagent using the method for producing an intermediate for producing an endotoxin detection reagent. Further, according to another embodiment of the present disclosure, there is provided a high-purity endotoxin detection reagent.

Mode for Carrying Out the Invention

[0009] Hereinafter, the content according to the present disclosure will be described in detail. The description of the constituent elements described below may be based on typical embodiments according to the present disclosure, but the present disclosure is not limited to such embodiments. In the present disclosure, a numerical range indicated by using "~" means a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in the present disclosure, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the upper limit value or the lower limit value of the numerical range described in other stepwise descriptions. Further, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the 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, the amount of each component in the composition means the total amount of the plurality of substances present in the composition when there are a plurality of substances corresponding to each component in the composition, unless otherwise specified. In the present disclosure, the term "step" includes not only an independent step but also a step included in this term if the intended purpose of the step is achieved even when it cannot be clearly distinguished from other steps.

[0011] [Method for Producing Intermediate for Producing Endotoxin Detection Reagent] The method for producing an intermediate for producing an endotoxin detection reagent according to the present disclosure (hereinafter, also simply referred to as "intermediate") is 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, 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) 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 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, X is a leaving group.

[0012] As a result of intensive studies by the present inventors, it has been found that an intermediate having a higher purity can be obtained by the method for producing an intermediate according to the present disclosure as compared with the conventional method. And it has been found that by using an intermediate having a high purity, an endotoxin detection reagent having a higher yield and a higher purity than the conventional one can be obtained.

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

[0014] Conventionally, in the production of an intermediate, after obtaining an intermediate containing a structure having a response site and having a leaving group by reacting a compound containing a structure having a response site with a compound having a leaving group, a purification treatment using column chromatography has been performed. However, in the purification treatment using column chromatography, it has been difficult to separate the compound containing the response site, the compound having the leaving group, and 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). Since other components contained in the reaction product other than the compound represented by formula (3) are dissolved in the poor solvent and the compound represented by formula (3) is not dissolved in the poor solvent, other components and the compound represented by formula (3) which is an intermediate can be separated. Thereby, an intermediate having a higher purity than before can be obtained. Further, by using an intermediate having a high purity, an endotoxin detection reagent having a higher yield and a higher purity than before can be obtained.

[0016] Hereinafter, each step in the method for producing an intermediate according to the present disclosure will be described.

[0017] <Step of obtaining a reaction product containing the 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 the 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 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, T 1 is a linking group, 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 hetero atom 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 combining these groups.

[0020] Also, R 1 may be a group composed only of a conjugated multiple bond, or may have a substituent that does not form a conjugated bond such as an alkyl group as appropriate. As the substituent, for example, when an alkyl group is introduced into R 1 the affinity between the endotoxin detection reagent and the fatty acid chain of endotoxin is improved, so that endotoxin can be measured at a lower concentration.

[0021] From the viewpoint of being able to detect endotoxin with high sensitivity, 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. Further, the above ring structure may be a monocyclic or polycyclic ring, and the polycyclic ring may be a condensed ring. The aromatic group may be unsubstituted or may have a substituent, but from the viewpoint of being able to detect endotoxin with high sensitivity, it is preferably unsubstituted.

[0023] The aromatic heterocyclic ring may be a monocyclic ring, a heterocyclic ring of two or more rings, or a condensed heterocyclic ring in which a heterocyclic ring and an aromatic ring are condensed, but a condensed heterocyclic ring in which a heterocyclic ring and an aromatic ring are condensed is preferable.

[0024] The number of ring members of the heterocyclic ring is not particularly limited, but is preferably a 2-membered ring to a 5-membered ring, and more preferably a 2-membered ring or a 3-membered ring. The heterocyclic ring is preferably a heterocyclic ring 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, still more preferably containing a nitrogen atom or an oxygen atom, and particularly preferably containing an oxygen atom.

[0025] Examples of the heterocyclic ring include a coumarin ring, a pyridine ring, a pyrimidine ring, a thiophene ring, a furan ring, a pyrrole ring, and the like. The above heterocyclic ring may be unsubstituted or may have a substituent. 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 highly sensitive detection of endotoxin, the heterocyclic ring is preferably a heterocyclic ring 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 monocyclic ring or a condensed aromatic ring in which two or more aromatic rings are condensed. From the viewpoint of enabling highly sensitive detection of endotoxin, a condensed aromatic ring is preferred. The condensed aromatic ring is preferably a condensed aromatic ring in which 2 to 8 aromatic rings are condensed, more preferably a condensed aromatic ring in which 2 to 6 aromatic rings are condensed, and still more preferably a condensed aromatic ring in which 2 to 4 aromatic rings are condensed. Examples of the condensed 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, a picene ring, and the like. R 1When it is an aromatic group, from the viewpoint of enabling highly sensitive detection of endotoxin, the aromatic ring is preferably a heterocyclic ring or a condensed aromatic ring, more preferably a coumarin ring, a pyrene ring or an anthracene ring, and particularly preferably a pyrene ring.

[0028] Further, when the aromatic ring is a monocyclic ring, 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. From the viewpoint of enabling highly sensitive detection of endotoxin, a biphenyl group is preferred.

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

[0030]

Chemical formula

[0031] In formula A aro , A represents an aromatic ring, R Y1 each independently represents a monovalent substituent, a represents an integer of 0 to 5, and the wavy line represents the bonding site with T 1 in formula (3). Examples of the aromatic ring include the above aromatic rings. 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. R Y1 each independently represents a substituent in the above 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. As the alkyl group, an alkyl group having 1 to 15 carbon atoms is preferred, an alkyl group having 2 to 12 carbon atoms is more preferred, and an alkyl group having 5 to 10 carbon atoms is still more preferred. Formula A aro In the case where a is an integer from 1 to 5, at least one of R Y1 is preferably bonded to ring A via an amide bond, and at least one of R Y1 is more preferably an alkyl group having 1 to 15 carbon atoms (preferably 2 to 12 carbon atoms, more preferably 5 to 10 carbon atoms) bonded to ring A via an amide bond. a is preferably an integer from 0 to 3, and more preferably an integer from 0 to 2.

[0032] When the above ring A is a coumarin ring, A is preferably represented by the following formula A cou as shown.

[0033]

Chemical formula

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

[0035] A group containing an azo (-N=N-) structure (hereinafter, may also be referred to as an "azo group") may contain one azo structure or two or more azo structures. From the viewpoint of highly sensitive detection of endotoxin, it is preferably a group containing one azo structure, and more preferably a group combining an azo group and an aromatic group. In a group combining an azo group and an aromatic group, the aromatic group may be an aromatic hydrocarbon ring or an aromatic heterocyclic ring, but from the viewpoint of excellent color development property, it is preferably an aromatic hydrocarbon group. The aromatic ring of the aromatic hydrocarbon group may be a monocyclic ring or a condensed aromatic ring in which two or more aromatic rings are condensed. Examples of the condensed aromatic ring include a naphthalene ring, an anthracene ring, and a pyrene ring. When the aromatic ring is a monocyclic ring, the aromatic ring is preferably a benzene ring. The aromatic hydrocarbon group may be unsubstituted or may have a substituent. 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.

[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 bonded to the azo structure, and more preferably a group represented by the following formula (A azo ).

[0037] [Chemical formula]

[0038] In formula (A azo ), R Z1 and R Z2 each independently represent a substituent, and the wavy line represents the bonding site with T 1 in formula (3). Examples of the substituent include the substituents in the above 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 still 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. Also, R Z1 and R Z2 may be bonded to the azo group at any of the ortho, meta, and para positions. Among these, the substituents in R Z1 and R Z2 are preferably bonded to the azo group at the para position. The bonding site with the oxygen atom may be bonded to the azo group at any of the ortho, meta, and para positions. Among these, the bonding site with the oxygen atom is preferably bonded to the azo group at the para position.

[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 R 1 are the compounds shown below. The wavy line represents the bonding site with T

[0041]

Chemical formula

[0042] 〔R 2 〕 R 2 The alkylene group represented by R 2 may be linear, branched, or cyclic. Among these, the alkylene group represented by R R 2The alkylene group represented by may be unsubstituted or may have a substituent. Examples of the substituent include an amide group, a carbonyl group, and an amino group. R 2 When the alkylene group represented by has 2 or more carbon atoms, a hetero atom may be included between carbon atoms. For example, an etheric oxygen atom, a sulfur atom, or a nitrogen atom may be arranged between carbon atoms. R 2 The carbon number of the alkylene group represented by is preferably 2 to 8, more preferably 2 or 3.

[0043] [T 1 T 1 The linking group represented by is not particularly limited, but is preferably -O-, -NH-, -C(=O)-, or a combination thereof. T 1 Examples of include -O-, -NHC(=O)-, -C(=O)NH-, -C(=O)-O-, and -NHC(=O)O-. Among them, T 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 a sulfonic acid. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among them, from the viewpoint of reactivity, the leaving group is preferably a halogen atom, more preferably a bromine atom.

[0045] In the step of obtaining a reaction product containing the 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: React the compound represented by formula (1A) with the 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 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.

[0047] Method 2: React the compound represented by formula (1B) with the compound represented by the following formula (2B). R 1 NH2…(1B) XR 2 COOH …(2B) R 1 NHCOR 2 X …(3B) In 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, X is a leaving group.

[0048] Method 3: React the compound represented by formula (1C) with the compound represented by the following formula (2C). R 1 COOH …(1C) XR 2 NH2…(2C) R 1 CONHR 2 X …(3C) In formulas (1B) to (3C), R 1 is a group containing a conjugated multiple bond, R 2is 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 - carbon atoms, X is a leaving group.

[0049] - Method 1 - In Method 1, the preferred embodiments of R in Formulas (1A) to (3A) 1 , R 2 , and 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 of the compound represented by Formula (1A) and the compound represented by Formula (2A), it is preferable to use more of the compound represented by Formula (2A) than the compound represented by Formula (1A). By using an excess of the compound represented by Formula (2A), by - products can be reduced. 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, more preferably 4 to 6 times in terms of molar conversion.

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

[0052] Examples of the compound represented by Formula (2B) include bromoacetic acid and 3 - bromopropionic acid. In the reaction of the compound represented by Formula (1B) and the compound represented by Formula (2B), it is preferable to use more of the compound represented by Formula (2B) than the compound represented by Formula (1B). By using an excess of the compound represented by Formula (2B), by - products can be reduced. 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, more preferably 4 to 6 times in terms of molar conversion.

[0053] - Method 3 - In Method 3, R in Formula (1C) to Formula (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 of the compound represented by Formula (1C) and the compound represented by Formula (2C), it is preferable to use more of the compound represented by Formula (2C) than the compound represented by Formula (1B). By using an excess of the compound represented by Formula (2C), by-products can be reduced. 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, more preferably 4 to 6 times, in terms of molar conversion.

[0055] The step of obtaining the 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 include hydrides, hydroxides, carbonates of alkali metals and alkaline earth metals, and quaternary ammonium salts. Among them, from the viewpoint of excellent separability and suppression of side reactions, the base is preferably a carbonate of an alkali metal and an alkaline earth metal, 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, more preferably 4 to 6 times, in terms of molar conversion, relative to the amount of the compound represented by Formula (1A), Formula (1B), or Formula (1C).

[0058] The step of obtaining the reaction product containing the 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 the raw materials and can dissolve them is appropriately selected. The reaction temperature is not particularly limited, for example, it is 60°C to 100°C. The reaction time is not particularly limited, for example, it is 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 in an inert gas atmosphere. Examples of the inert gas include nitrogen gas and argon gas.

[0059] <Step of reprecipitating the compound represented by formula (3)> In the method for producing the intermediate according to the present disclosure, a poor solvent of 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 separability, the poor solvent preferably has an HSP value of 21 MPa 1 / 2 ~30 MPa 1 / 2 and more preferably has an HSP value of 25 MPa 1 / 2 ~28 MPa 1 / 2 and is more preferably a solvent of. In the present disclosure, the HSP value means the value of the Hansen solubility parameter. The HSP value is calculated based on the intermolecular energy.

[0061] The HSP values of each solvent are shown below. 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 excellent separability, the lean 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 generally known method.

[0064] [Method for producing endotoxin detection reagent] The method for producing an endotoxin detection reagent according to the present disclosure includes a step of producing an intermediate using the method for producing an intermediate according to the present disclosure, and a step of reacting the intermediate with a compound containing a group having a metal-coordinating group, an acid group, or a basic 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 basic group] In the method for producing an endotoxin detection reagent according to the present disclosure, the intermediate is reacted with a compound containing a group having a metal-coordinating group, an acid group, or a basic group.

[0066] The compound containing a group having a metal-coordinating group, an acid group, or a basic 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 to at least one metal or a group that promotes coordination to the metal. Examples of the above 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+Examples include etc., and 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+ ) are even more preferred, and Zn 2+ is particularly preferred. In addition, examples of the above metal coordinating group include a group containing 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 hydroxyl group, a carboxy group, a crown ether group, and the like.

[0068] Examples of the aromatic heterocyclic group include a heterocyclic group 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 being able to detect endotoxin with high sensitivity, among these, as the aromatic heterocyclic group, it is preferably a heterocyclic group containing at least one kind of atom selected from a nitrogen atom and an oxygen atom, more preferably a heterocyclic group containing a nitrogen atom, still more preferably a heterocyclic ring containing a nitrogen atom with 5 or 6 ring members, and particularly preferably a heterocyclic ring containing a nitrogen atom with 6 ring members. Examples of the heterocyclic ring containing a nitrogen atom include a pyrrole ring, a pyridine ring, a pyrazine ring, a pyrimidine ring, and the like. In addition, the above heterocyclic ring may be unsubstituted or may have a substituent, but from the viewpoint of being able to detect endotoxin with high sensitivity, it is preferably a heterocyclic ring having a substituent. Examples of the substituent include an alkyl group, an aryl group, etc. Among them, the substituent is preferably an alkyl group, and more preferably an alkyl group having 1 to 4 carbon atoms.

[0069] From the viewpoint of being able to detect endotoxin with high sensitivity, among the heterocyclic rings having a substituent, it is preferably a heterocyclic ring containing a nitrogen atom having an alkyl group, more preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with 5 or 6 ring members, still more preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with 6 ring members, and particularly preferably a pyridyl group having an alkyl group having 1 to 4 carbon atoms.

[0070] When the metal-coordinating group contains an aromatic heterocyclic ring, from the viewpoint of enabling highly sensitive detection of endotoxin, the group containing an aromatic heterocyclic ring is preferably a substituted amino group substituted with a heterocyclic group, and more preferably a substituted amino group substituted with a heterocyclic ring containing an alkyl group having 1 to 4 carbon atoms (preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with a ring member number of 5 or 6, more preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with a ring member number of 6, and still 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 heterocyclic ring containing an alkyl group having 1 to 4 carbon atoms (preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with a ring member number of 5 or 6, more preferably a heterocyclic ring having an alkyl group having 1 to 4 carbon atoms and containing a nitrogen atom with a ring member number of 6, and still more preferably a pyridyl group having an alkyl group having 1 to 4 carbon atoms).

[0071] Also, 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 a predetermined site or functional group of endotoxin and the metal-coordinating group. In addition, since the amount of fluorescence emission significantly increases when coordinated with a metal ion, endotoxin may be detectable as a decrease in the amount of emission. Examples of such metal ions include the metal ions in the above-mentioned metal-coordinating groups, for example, copper ion (Cu 2+ ), nickel ion (Ni 2+ ).

[0072] Examples of the acid group include groups selected from a sulfo group, a carboxy group, a phosphoric acid group, a boronic acid group, a phenol group, salts thereof, or de-salted structures thereof. Among these, groups selected from a carboxy group, a boronic acid, salts thereof, or de-salted structures thereof are preferable. 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 preferable. Examples of the substituent other than the acid group include an alkyl group and an aryl group. Among these, an alkyl group is preferable, and an alkyl group having 1 to 4 carbon atoms is more preferable.

[0073] From the viewpoint of enabling highly sensitive detection of endotoxin, the group having the acid group is preferably a phenyl group having the acid group or an amino group having the acid group, more preferably an amino group substituted with a phenyl group having the acid group or an alkyl group having 1 to 4 carbon atoms containing the acid group, still 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 or 2 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 the disubstituted amino group substituted with an alkyl group having 1 or 2 carbon atoms containing a carboxy group include a monovalent group obtained by removing a hydrogen atom from iminodiacetic acid.

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

[0075] From the viewpoint of enabling highly sensitive detection of endotoxin, the compound containing a group having a metal-coordinating group, an acid group or 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 of an intermediate with a compound containing a group having a metal-coordinating group, an acid group or a base group, it is preferable to use more of the compound containing a group having a metal-coordinating group, an acid group or a base group than the intermediate. By using an excessive amount of the compound containing a group having a metal-coordinating group, an acid group or a base group, the intermediate can be made to disappear. 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, more preferably 4 to 6 times, in terms of molar conversion.

[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 the base is not particularly limited, and examples include the same bases as described above.

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

[0079] The reaction of the intermediate with 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 the intermediate and the compound containing a group having a metal-coordinating group, an acid group or a base group and is soluble therein 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, from 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 in an inert gas atmosphere. Examples of the inert gas 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 performed after the reaction. Examples of the purification treatment include purification treatment by size exclusion chromatography.

[0081] Examples of the endotoxin detection reagent obtained 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 metal ions.

[0082] [Chemical formula]

[0083] [Endotoxin detection reagent] The endotoxin detection reagent according to the present disclosure is 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. [Chemical formula] In formula (A), 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, 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 T 1 The preferred embodiments are as described above.

[0086] Y is preferably a group having a metal coordinating group, an acid group or a base group. The preferred embodiments of the group having a metal coordinating group, an acid group or a base 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 from 1 to 10.

[0088]

Chemical formula

[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 from 1 to 10.

[0090]

Chemical formula

[0091] In the method for producing an endotoxin detection reagent using the above compound (A0), the generation of by-products can be suppressed. As a result, the purity of the compound represented by formula (A1) can be obtained at 80% or more. The purity is preferably 90% or more, more preferably 98% or more. When using the method for producing an intermediate according to the present disclosure, the sensitivity is improved by about 10 to 1000 times compared with existing endotoxin detection reagents, for example, an endotoxin detection reagent with a purity of about 75%. Therefore, detection at a low concentration or detection with a reduced amount of the reagent becomes possible. When manufacturing the endotoxin detection reagent of the present disclosure using commercially available ordinary reagents, it is usually manufactured via an intermediate for manufacturing an endotoxin detection reagent, which is a compound represented by the formula (3). By using the method for manufacturing the intermediate according to the present disclosure, a high-purity endotoxin reagent can be obtained.

Example

[0092] Hereinafter, the present disclosure will be specifically described with reference to examples. Note that the present disclosure is not limited by 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 under 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 × 40 mL) using a separatory funnel. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated using a rotary evaporator. Purification by reprecipitation was performed. Specifically, an excessive amount of ethanol (poor solvent) was added to the concentrate. Since a white solid precipitated, the solid was filtered off and washed with water and ethanol. The obtained solid was dried under reduced pressure to obtain the following compound as an intermediate (0.692 g, 2.126 mmol). The yield in this reaction was 85.0%.

[0094]

Chemical formula

[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 dehydrated tetrahydrofuran (20 mL) were added to a round-bottom flask under an argon atmosphere. To this mixture, 2,2'-dipicolylamine (0.644 g, 3.230 mmol, "Raw Material 3" in Table 1) dissolved in dehydrated tetrahydrofuran (5 mL) was added dropwise 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 subsequently, the organic solution was washed with a 10% by mass 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 the eluent. As an endotoxin detection reagent, the following compound, which is a brown oily substance, was obtained (0.149 g, 0.337 mmol). The yield in this reaction was 68.0%. Therefore, the yield in the total synthesis calculated from the starting materials was 58%. Also, the purity of the final product was 99%.

[0096] [Chemical Formula]

[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 compounds shown in Table 1. As Raw Material 2, a compound represented by Br-(CH2) n -Br was used, and n was described in Table 1.

[0098] [Comparative Example 1 and Comparative Example 2] In Comparative Example 1, an endotoxin detection reagent was obtained in the same manner as in Example 1, except that purification by column chromatography was performed without performing the purification treatment using reprecipitation. In Comparative Example 2, an endotoxin detection reagent was obtained in the same manner as in Example 4, except that the purification treatment by column chromatography was performed without performing the purification treatment using reprecipitation. In Table 1, “Y” was described in the column of the column.

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

[0100] [Table 1]

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

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, comprising the 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). A method for producing an intermediate for manufacturing an endotoxin detection reagent. R １ T １ R ２ X …(3) In 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 heteroatom between carbon atoms-carbon atoms. T １ is a linking group, X is a leaving group.

2. The compound represented by the formula (3) is a compound represented by the following formula (3A), and in the step of obtaining the reaction product, the compound represented by the following formula (1A) is reacted with the compound represented by the following formula (2A). The method for producing an intermediate for manufacturing 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 the alkylene group having 2 or more carbon atoms may contain a hetero atom between carbon atoms - carbon atoms, X is a leaving group.

3. The compound represented by the formula (3) is a compound represented by the following formula (3B), and 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). The method for producing an intermediate for manufacturing an endotoxin detection reagent according to Claim 1. 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 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), and in the step of obtaining the reaction product, the compound represented by the following formula (1C) is reacted with the compound represented by the following formula (2C). The method for producing an intermediate for manufacturing 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 - 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 producing an endotoxin detection reagent according to claim 1, wherein the solvent is as described above.

6. The poor solvent is ethanol. The method for producing an intermediate for manufacturing an endotoxin detection reagent according to Claim 1.

7. Said 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 step of 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 producing an endotoxin detection reagent by 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. A method for producing an endotoxin detection reagent.

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

10. An endotoxin detection reagent with a purity of 80% or more and represented by the following formula (A). 【Chemical 1】 In formula (A), 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, T １ is a linking group, Y is a group that interacts with endotoxin.

11. An endotoxin detection reagent with a purity of 80% or more and represented by the following formula (A1). In formula (A1), m is an integer from 1 to 10. [Chemical 2]

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