JP2020180052A - Novel fluorodinitrophenyl compound and use therefor - Google Patents

Abstract

To provide a novel fluorodinitrophenyl compound, a method of producing the same, and a use therefor.SOLUTION: The present invention provides a compound represented by formula (1) or a salt thereof, a method of producing the same, and an optical resolution labelling agent as a use therefor, (where R1 and R2 are the same or different to represent H, an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. R3 and R4 are the same or different to represent an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, or R3 and R4 may be bound to each other, forming a ring with an adjacent nitrogen atom, and the ring may have a substituent. Alk is an alkylene group.)SELECTED DRAWING: None

Description

The present invention relates to a novel fluorodinitrophenyl compound and its use (optical resolution labeling agent, etc.).

Peptide-based natural products, which have intermediate properties between small molecules and antibodies, are attracting a great deal of attention as next-generation drug discovery seeds that have the advantages of small molecules and antibodies and can overcome the drawbacks of both.

Many peptide-based natural products with specific biological activities that have already been used as drugs or are promising as drug discovery leads contain not only L-amino acids but also D-amino acids as their constituent amino acids. .. Determining the absolute configuration of constituent amino acids is important for determining the structure of peptide-based natural products. To determine the absolute configuration, first acid hydrolyze the peptide compound, derivatize the amino acids of the degradation product with an optically split labeling agent, and then perform high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-). A method (Marfey method) of analyzing by MS) and determining the absolute configuration by comparing with the dissolution time of the labeled amino acid standard is widely used.

Non-Patent Document 1 reports 1-fluoro-2,4-dinitrophenyl-5-L-alanine (L-FDAA), which is an amino acid chiral resolution labeling agent for HPLC developed by Marfey in 1984. There is. Since L-FDAA has a fluorodinitrophenyl skeleton that is susceptible to the aromatic nucleophilic substitution reaction (SNAr reaction), it reacts with the amino groups of various amino acids under mild basic conditions and is labeled with L-FDAA. It is induced by the converted amino acid. Since this labeled amino acid has a characteristic UV absorption near 340 nm, the absolute configuration of the amino acid is arranged by comparing the elution time of the labeled amino acid with the standard by HPLC analysis using this characteristic. Can be determined.

Then, in 1997, Harada et al. Synthesized various FDAA derivatives as optical resolution labeling agents, and reported a method for determining the absolute configuration of amino acids using them (Non-Patent Documents 2 and 3). .. According to this, when LC-MS (Positive Ion Mass Chromatogram) was used as the detection method, 1-fluoro-2,4-dinitrophenyl-5-L-leucine (L-FDLA) became L-FDAA. It has been confirmed that the detection sensitivity is superior in comparison. In addition, by using LC-MS, the mass information of the molecule of the detected peak can be obtained, which enables more accurate analysis.

However, in order to determine the structure of a compound that can be isolated from a rare natural resource in a very small amount, further improvement in detection sensitivity is required.

For example, when determining the absolute configuration of the constituent amino acids of a peptide-based natural product, it is necessary to hydrolyze the sample. In studies targeting rare peptide-based natural products with a very small amount of sample, it is difficult to determine the detailed structure because the amount of constituent amino acids generated after hydrolysis is also extremely small.

In addition, when peptide-based natural products are hydrolyzed under harsh conditions of high temperature, strong acid, and long time, the yield of decomposition products increases, but racemization of constituent amino acids progresses, resulting in erroneous structure determination. (For example, Non-Patent Document 4).

On the other hand, in order to suppress racemization, if hydrolysis is performed under mild conditions of lower temperature, weak acid, and short time, the yield of decomposition products will decrease, and only a very small amount of labeled amino acids will be obtained, and as a result, that It becomes difficult to determine the absolute configuration of labeled amino acids.

Therefore, compared with the conventional optical resolution labeling agent, an optical resolution labeling agent capable of detecting the absolute configuration of even a very small amount of amino acids produced under mild hydrolysis conditions with higher sensitivity was used. A detection method was sought.

Marfey P., Carlsberg Res. Commun., 1984, 49, 591. Harada K. et al, Anal. Chem., 1997, 69, 3346. Harada K. et al, Anal. Chem., 1997, 69, 5146. Kuranaga T. et al. J. Am. Chem. Soc. 2015, 137, 9443.

An object of the present invention is to provide a novel fluorodinitrophenyl compound, a method for producing the same, and an application thereof.

As a result of diligent studies to solve the above problems, the present inventor found that the compound represented by the formula (1) (particularly, the compound represented by the formula (1A)) is a novel compound. It has also been found that it is extremely useful as an optically resolution labeling agent for compounds having an asymmetric center (particularly various amino acids).

Specifically, the compound represented by the formula (1A) is reacted with various amino acids to label the amino acids, which are analyzed by HPLC, LC-MS, etc., and are described in Non-Patent Documents 1 to 3. It has been found that labeled amino acids can be detected with good separability and extremely high sensitivity as compared with the conventional method, and as a result, the absolute configuration of amino acids can be determined accurately and with high sensitivity. Based on such findings, further studies have led to the completion of the present invention.

That is, the present invention provides the following fluorodinitrophenyl compounds, a method for producing the same, and uses thereof.

Item 1. Equation (1):
(In the formula, R ¹ and R ² are the same or different and have a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. R ³ and R ⁴ may be the same or different and have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. It may indicate a heteroaryl group, or R ³ and R ⁴ may be bonded to each other to form a ring with adjacent nitrogen atoms, and the ring may have a substituent. Alk may have an alkylene. Indicates a group.)
A compound represented by or a salt thereof.

Item 2. Equation (1A):
(In the formula, R ^1A and R ^2A are different and may have a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. It is a heteroaryl group, and R ³ , R ⁴ and Alk are the same as described above. The steric chemistry of C * to which R ^1A and R ^2A are bonded shows an absolute configuration.)
Item 2. The compound according to Item 1 or a salt thereof, which is a compound represented by

Item 3. Item 2. The compound or salt thereof according to Item 2, wherein either R ^1A or R ^2A is a hydrogen atom, the other is an alkyl group which may have a substituent, and Alk is a C2-C10 alkylene group. ..

Item 4. Alkyl groups that may have substituents represented by R ^1A and R ^2A are the following groups;
(1) Hydroxyl groups that may be protected,
(2) Alkoxy group,
(3) Carboxyl groups that may be protected,
(4) Amide group,
(5) Amino groups that may be protected,
(6) Guanidino groups that may be protected,
(7) An aryl group that may be substituted with at least one group selected from the group consisting of an alkyl group and a hydroxyl group.
(8) A heteroaryl group that may be substituted with at least one group selected from the group consisting of an alkyl group and a hydroxyl group, and (9) a mercapto group that may be protected.
Item 2. The compound according to Item 2 or 3, or a salt thereof, which is an alkyl group which may be substituted with at least one group selected from the group consisting of.

Item 5. An optically resolution labeling agent containing the compound according to any one of Items 1 to 4 or a salt thereof.

Item 6. A method for producing a compound represented by the formula (1) described in Item 1 or a salt thereof, wherein the formula (5):
^{(Wherein, R 1, R 2, R} 3 and ^{R 4} and Alk are as defined above.)
The compound represented by, or a salt thereof, and the formula (6):
A manufacturing method including a step of reacting a compound represented by.

Item 7. A method for producing a compound represented by the formula (1A) or a salt thereof according to Item 2, wherein the formula (5A):
(In the formula, R ^1A , R ^2B , R ³ and R ⁴ and Alk are the same as described above.)
A production method comprising a step of reacting a compound represented by the above formula or a salt thereof and a compound represented by the formula (6).

Item 8. A method for determining the absolute configuration of a compound having an asymmetric center.
(1) A step of reacting the compound having the asymmetric center with the optical resolution labeling agent according to Item 5 to prepare a labeled compound, and (2) analyzing the labeled compound. Thereby, the step of determining the absolute configuration of the compound having an asymmetric center,
How to include.

Item 9. Item 8. The method according to Item 8, wherein the compound having an asymmetric center is an amino acid or a derivative thereof.

As used herein, the term "optical resolution labeling agent" means a labeling agent for optically resolving a compound (amino acid or the like) having an asymmetric center. For example, this chiral resolution labeling agent is reacted with an amino acid to convert it into a labeled amino acid (eg, diastereomer, etc.), which is then converted using a separable analytical means (HPLC, LC-MS, etc.). By analysis, the absolute configuration of the amino acid can be determined. Specifically, the labeled amino acid and the standard whose absolute configuration is known are analyzed by HPLC, LC-MS, etc., and the elution times (retention times) of the two are compared to obtain the absolute amount of the amino acid. The three-dimensional arrangement can be determined.

The compound represented by the formula (1A) of the present invention is suitable as an optical resolution labeling agent for determining the absolute configuration of a compound (particularly an amino acid or the like) having an asymmetric center (particularly an asymmetric carbon). Specifically, an amino acid and a compound represented by the formula (1A) are reacted in the presence of a weak base to prepare an amino acid labeled with the compound represented by the formula (1A). By analyzing it using HPLC, LC-MS, etc. and measuring the elution time (retention time), isomers of labeled amino acids (diastereomers, etc.) can be detected with high resolution and extremely high sensitivity. Can be done. By comparing the elution time of each isomer with the elution time of labeled amino acids whose absolute configuration is known, the absolute configuration of amino acids can be determined accurately and with high sensitivity.

When determining the structure of a trace amount of rare peptide-based natural product constituent amino acids, the yield of the constituent amino acids may be extremely small if hydrolysis is carried out under milder conditions to suppress racemization. Even in that case, the absolute configuration of amino acids can be determined with higher sensitivity by using the compound represented by the formula (1A) of the present invention.

The results of analysis (LC-MS) of labeled amino acids using the compound represented by the formula (1A) of the present invention were labeled with L-FDAA described in conventional Non-Patent Documents 1 and 2. Compared with the analysis results of amino acids, the labeled leucine, which is a neutral amino acid, is about 10 times, the labeled lysine, which is a basic amino acid, is about 9 times, and the labeled glutamic acid, which is an acidic amino acid, is about 9 times. A 7-fold improvement in sensitivity was observed (see, for example, Tables 1-3, FIGS. 1-1, 1-2, 2 and 3).

In particular, by using the electrospray ionization (ESI) method as the ionization method of the detection device and detecting in the positive ionization mode, more sensitive analysis is possible. The amino acid labeled with the compound represented by the formula (1A) has a tertiary amine structure in the molecule, and the nitrogen atom is easily protonated to improve the cationization efficiency. , It is considered that the detection sensitivity in the positive ionization mode is dramatically improved.
The ability to separate isomers (diastereomers) of amino acids labeled with the optical resolution labeling agent of the present invention is equal to or higher than that labeled with the existing optical resolution labeling agents described in Non-Patent Documents 1 and 2. (See, for example, Table 4, FIGS. 4 and 5, and Non-Patent Document 3).

The compound represented by the formula (1) of the present invention can be produced easily and inexpensively in a short process, and can be easily purified by recrystallization. In addition, it has excellent storage stability and can be stored stably in air, at room temperature, and in light-shielded conditions.

Since the compound represented by the formula (1) of the present invention has the above-mentioned characteristics, it is useful as a reagent for amino acid analysis used in various fields.

The results of analysis of the detection sensitivities of various labels of L-leucine performed in Example 2 (2) by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown. In Example 2 (2), the concentration of various labeled forms of L-leucine was changed, and the detection sensitivities thereof were analyzed by HPLC (UV340 nm) and LC-MS (ESI positive mode). The results of analysis of the detection sensitivities of various labeled L-lysine products performed in Example 2 (3) by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown. The results of analysis of the detection sensitivities of various labeled L-glutamic acid labels performed in Example 2 (4) by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown. The results of analysis of the separation and detection sensitivity of the labeled product by the compound (1a) of various amino acids performed in Example 2 (5) by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown. The results of analysis of the separation and detection sensitivity of the labeled product by the compound (1c) of various amino acids performed in Example 2 (6) by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown. The HPLC results before and after storage of compound (1a) for 3 weeks are shown.

Hereinafter, the present invention will be described in detail.
In the present specification, the expression "including" includes the concepts of "including", "substantially consisting" and "consisting of".

1. 1. Fluorodinitrophenyl compound The fluorodinitrophenyl compound of the present invention is a compound represented by the formula (1) or a salt thereof (particularly, a compound represented by the formula (1A) or a salt thereof), and is used as an optically resolution labeling agent. It is useful.

In the compound represented by the formula (1), examples of the "alkyl group" of the "alkyl group which may have a substituent" represented by R ¹ and R ² include a chain-shaped or branched alkyl group. Be done. It is preferably a C1-C20 alkyl group, more preferably a C1-C10 alkyl group, even more preferably a C1-C6 alkyl group, and particularly preferably a C1-C4 alkyl group. Specific examples thereof include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, n-hexyl group and the like. .. In particular, a methyl group, an isopropyl group, and an isobutyl group are preferable.

Examples of the substituent that the alkyl group may have include, for example.
(1) A hydroxyl group that may be protected (examples of the protecting group of the hydroxyl group include a trialkyl (particularly C1-C4 alkyl) silyl group, a tetrahydropyranyl group, a methoxymethyl group, and the like).
(2) Alkoxy group (particularly, C1 to C4 alkoxy group and the like),
(3) A carboxyl group that may be protected (examples of the protecting group of the carboxyl group include an allyl group, a benzyl group, a methyl group, an ethyl group, a tert-butyl group, etc.),
(4) An amide group (the nitrogen atom of the amide group may have one or two alkyl groups (particularly C1 to C4 alkyl groups)),.
(5) An amino group that may be protected (examples of the protecting group of the amino group include a benzyl group, a benzyloxycarbonyl group, an allyloxycarbonyl group, a tert-butoxycarbonyl group and the like),.
(6) A guanidino group that may be protected (examples of the protecting group of the guanidino group include a tosyl group, a tert-butoxycarbonyl group, etc.),
(7) An aryl group that may be substituted with at least one group (particularly 1 to 3) selected from the group consisting of an alkyl group and a hydroxyl group (the aryl group includes, for example, a phenyl group, a naphthyl group, etc. Examples of the alkyl group include C1 to C4 alkyl groups such as a methyl group.),
(8) A heteroaryl group that may be substituted with at least one group (particularly 1 to 3) selected from the group consisting of an alkyl group and a hydroxyl group (for example, the heteroaryl group includes a pyridyl group and an imidazolyl group). Groups, indrill groups and the like can be mentioned, and examples of the alkyl group include C1 to C4 alkyl groups such as methyl group).
(9) A mercapto group that may be protected (examples of the protecting group of the mercapto group include an alkyl group (particularly a C1 to C4 alkyl group)), and the like.
And so on. The alkyl group may have at least one group (particularly one to three) selected from the group consisting of these substituents.

Examples of the "aryl group" of the "aryl group which may have a substituent" represented by R ¹ and R ² include a phenyl group, a toluyl group, a xsilyl group, a naphthyl group, an anthryl group, a phenanthryl group and the like. Can be mentioned.

Examples of the substituent that the aryl group may have include the substituent that the alkyl group may have. The aryl group may have at least one group (further 1 to 5 types, particularly 1 to 3 types) selected from the group consisting of these substituents.

The "heteroaryl group" of the "heteroaryl group which may have a substituent" represented by R ¹ and R ² includes, for example, a group consisting of a nitrogen atom, an oxygen atom and a sulfur atom as atoms constituting the ring. Examples thereof include heteroaryl groups containing at least one selected from the above. Specific examples thereof include a pyridyl group, an imidazolyl group, an indyl group and the like.

Examples of the substituent that the heteroaryl group may have include the substituent that the alkyl group may have. The heteroaryl group may have at least one group (further 1 to 4 types, particularly 1 to 3 types) selected from the group consisting of these substituents.

In the compound represented by formula (1), "alkyl group which may have a substituent" represented by R ³ and R ^4, the "optionally substituted aryl group" and "substituent As the "heteroaryl group which may have a substituent", "an alkyl group which may have a substituent" and "an aryl group which may have a substituent" represented by R ¹ and R ² above. And, it can be arbitrarily selected from those described in "Heteroaryl group which may have a substituent".

R ³ and R ⁴ may be bonded to each other to form a ring with adjacent nitrogen atoms, and the ring may have a substituent. Examples of the ring in which R ³ and R ⁴ are bonded to each other and formed together with adjacent nitrogen atoms include an aziridine ring, an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and a piperazine ring.

Examples of the substituent that the ring may have include the substituent that the alkyl group may have. The ring may have at least one group (further 1 to 4 types, particularly 1 to 3 types) selected from the group consisting of these substituents.

Examples of the alkylene group represented by ALC include a chain-like or branched alkylene group. It is preferably a C2-C10 alkylene group, more preferably a C2-C6 alkylene group, even more preferably a C2-C5 alkylene group, and particularly preferably a C2-C3 alkylene group. Specifically, for example, a dimethylene group (-CH ₂ CH _2- ), a trimethylene group (-CH ₂ CH ₂ CH _2- ), a tetramethylene group (-CH ₂ CH ₂ CH ₂ CH _2- ), -CH ( i-Pr) CH ₂ -etc.

In formula (1), when R ¹ and R ² are different, the carbon atom to which R ¹ and R ² are bonded can be an asymmetric carbon. In this case, in the compound represented by the formula (1), the stereochemistry of the asymmetric carbon is an arbitrary mixture of R-form, L-form, R-form and L-form (equal mixture of R-form and L-form; racemic). Includes any of the body).

The compound represented by the formula (1) can also form a salt. Since the compound has a characteristic tertiary amine (Alk-NR ³ R ⁴ ) structure, it can form a salt with a predetermined acid (inorganic acid or organic acid). Examples of the inorganic acid include hydrochloric acid, sulfuric acid, nitric acid and the like. Examples of the organic acid include carboxylic acids (for example, formic acid, acetic acid, benzoic acid, etc.) and sulfonic acids (for example, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, etc.).

When the compound represented by the formula (1) has an acidic group (carboxyl group or the like), a salt can be formed in the molecule with the tertiary amine structure of the compound.

A preferred embodiment of the compound represented by the formula (1) or a salt thereof includes a compound represented by the formula (1A) or a salt thereof.

"Alkyl group which may have a substituent", "aryl group which may have a substituent", and "heteroaryl group which may have a substituent" represented by R ^1A and R ^2A. "Has an" alkyl group which may have a substituent "," an aryl group which may have a substituent ", and" a substituent which may have a substituent ", respectively, which are indicated by R ¹ and R ² above. It may be arbitrarily selected from those described in "May be heteroaryl group".

R ^1A or R ^2A is preferably an "alkyl group which may have a substituent", more preferably an alkyl group, and further preferably a chain or branched C1 to C6 alkyl group. Particularly preferably, it is a chain-like or branched C1-C4 alkyl group (for example, a methyl group, an isopropyl group, an isobutyl group, etc.).

One of R ^1A and R ^2A is a hydrogen atom, the other is an alkyl group which may have a substituent (preferably a C1 to C6 alkyl group), and Alk is a C2 to C10 alkylene group (preferably). Compounds that are C2-C5 alkylene groups) are preferred.

Since the stereochemistry of C * to which R ^1A and R ^2A are bound shows an absolute configuration, the compound represented by the formula (1A) is represented by the following formula (1A-1) or (1A-2). Includes any optically active compound.
(In the formula, R ^1A , R ^2A , R ³ , R ⁴ and Alk are the same as above. The stereochemistry of C * shows an absolute configuration.)

2. Production Method The compound represented by the formula (1) or a salt thereof can be produced, for example, as follows.
(In the formula, Y represents a protecting group for the amino group. R ¹ , R ² , R ³ , R ⁴ and Alk are the same as above.)

(2) + (3) → (4):
The compound represented by the formula (2) and the compound represented by the formula (3) are subjected to an amidation reaction (condensation reaction) to obtain a compound represented by the formula (4).

Examples of the protecting group of the amino group represented by Y include a tert-butoxycarbonyl (Boc) group, a benzyloxycarbonyl (Cbz) group, an allyloxycarbonyl (Alloc) group, a benzyl group and the like. Protection and deprotection of the amino groups with these protective groups are all known methods (e.g., Protecting Groups: Foundations of Organic Chemistry , Thieme Publishing Group; 3 rd Edition) can be carried out according or analogously to.

The amidation reaction can be carried out using a known method. For example, a mixed acid anhydride method, an acid chloride method, a method using a condensing agent and the like can be mentioned. As a typical example, a method using the mixed acid anhydride method will be described below.

The compound represented by the formula (2) is reacted with a chloroformic acid ester (for example, isobutyl chloroformate) or a carboxylic acid chloride (for example, acetyl chloride) in the presence of a base (for example, N-methylmorpholin). A compound represented by the formula (4) is obtained by forming a mixed acid anhydride and reacting the compound represented by the formula (3) with the mixed acid anhydride.

Solvents used in the amidation reaction are usually ether solvents (eg, diethyl ether, THF, dioxane, dimethoxyethane, etc.), amide solvents (eg, DMF, DMA, NMP, etc.), aromatic hydrocarbon solvents (eg, benzene, etc.). (Toluene, etc.) and the like.

The amount of the base used is usually 1 to 2 mol (particularly 1 to 1.5 mol) with respect to 1 mol of the compound represented by the formula (2).
The amount of chloroformate or carboxylic acid chloride used is usually 1 to 2 mol (particularly 1 to 1.5 mol) with respect to 1 mol of the compound represented by the formula (2).
The amount of the compound represented by the formula (3) to be used is usually 1 to 2 mol (particularly 1 to 1.5 mol) with respect to 1 mol of the compound represented by the formula (2).

The amidation reaction can usually be carried out at −20 ° C. to 50 ° C. (particularly, from 0 ° C. to room temperature (for example, about 25 ° C.)).

After completion of the reaction, a compound represented by the formula (4) can be obtained by using known isolation or purification means (extraction, washing, concentration, recrystallization, column chromatography, etc.).

(4) → (5):
By removing the protecting group (Y) of the compound represented by the formula (4), the compound represented by the formula (5) is obtained.

Protecting group of the amino group of the compound represented by the formula (4) (Y), depending on the type of protecting groups, known methods (e.g., Protecting Groups: Foundations of Organic Chemistry , Thieme Publishing Group; 3 rd Edition) Can be removed (deprotected) by. For example, when the protecting group of the amino group is a Boc group, it is deprotected by treatment with an acid (for example, trifluoroacetic acid) in the presence or absence of a solvent (for example, dichloromethane or the like), and the formula (5) Obtain the compound represented by.

After the deprotection reaction is completed, excess acid can be removed from the reaction solution and subjected to the next reaction. If necessary, it can also be purified using a known method.

Since the compound represented by the formula (5) has an amino group (-NH ₂ ) and a tertiary amine (Alk-NR ³ R ⁴ ) structure in the molecule, a salt may be formed together with the acid. Examples of the acid include inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid; carboxylic acids (for example, formic acid, acetic acid, benzoic acid and the like) and sulfonic acids (for example, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid and the like). Organic acids can be mentioned.

(5) + (6) → (1):
The compound represented by the formula (1) is obtained by reacting the compound represented by the formula (5) or a salt thereof with the compound represented by the formula (6). The compound represented by the formula (6) is commercially available or can be easily produced by those skilled in the art according to or according to a known method.

This reaction can usually be carried out in the presence of a solvent and in the presence of a base.
As the solvent, water, a ketone solvent (for example, acetone, methyl ethyl ketone, etc.), an ether solvent (for example, diethyl ether, THF, dioxane, dimethoxyethane, etc.), an amide solvent (for example, DMF, DMA, NMP, etc.) and the like are usually used. Can be mentioned. One or more of these can be used.
As the base, an inorganic base (for example, a carbonate of an alkali metal such as sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate; a hydroxide of an alkali metal such as sodium hydroxide and potassium hydroxide, etc.), Alternatively, an organic base (for example, pyridine, triethylamine, etc.) and the like can be mentioned. When an inorganic base is used, it is preferably used in the form of an aqueous solution.

The amount of the base used is usually 1 to 3 mol (particularly 1 to 2.5 mol) with respect to 1 mol of the compound represented by the formula (5).

The amount of the compound represented by the formula (6) to be used is usually 1 to 2 mol (particularly 1 to 1.5 mol) with respect to 1 mol of the compound represented by the formula (5).

The reaction temperature is usually about 0 ° C. to 60 ° C., preferably about 5 ° C. to 50 ° C. The reaction time is usually about 1 minute to 5 hours, preferably about 10 minutes to 3 hours.

After completion of the reaction, a compound represented by the formula (1) can be obtained by using known isolation or purification means (extraction, washing, concentration, recrystallization, column chromatography, etc.).

The compound represented by the formula (1A) is included in the compound represented by the formula (1), and can be produced in the same manner as in the reaction formula 1 as shown in the following reaction formula 2. That is, using the compound represented by the formula (2A) as a raw material, a compound represented by the formula (4A) and then a compound represented by the formula (5A) are prepared and represented by the formula (5A). The compound represented by the formula (1A) can be produced by reacting the compound or a salt thereof with the compound represented by the formula (6).
(In the formula, R ^1A , R ^2A , R ³ , R ⁴ , Alk, Y and C * are the same as above.)

The compound represented by the formula (1) of the present invention can be easily and inexpensively produced in a short process as shown by the above reaction formulas 1 and 2. In addition, the compound represented by the formula (1) can usually be easily purified by recrystallization. In addition, the compound represented by the formula (1) is also excellent in storage stability. In particular, in the case of an alkyl group (particularly an alkyl group) in which R ¹ or R ² may have a substituent, these characteristics are well applicable.

3. 3. Applications The compound represented by the formula (1) of the present invention (particularly, the compound represented by the formula (1A)) is an optically resolution label for determining the absolute configuration of a compound having an asymmetric center (particularly an amino acid). It is useful as an agent.

In particular,
(1) A step of reacting a compound having an asymmetric center with an optical resolution labeling agent containing a compound represented by the formula (1A) to prepare a labeled compound, and (2) the labeling. A step of determining the absolute configuration of a compound having an asymmetric center by analyzing the compound.
By including, the absolute configuration of the compound having an asymmetric center (particularly an amino acid) can be determined.

Examples of the compound having an asymmetric center include a compound having one or more (particularly one) asymmetric carbons and having an amino group in the molecule. Specific examples thereof include amino acids (α-amino acids, β-amino acids, etc.) and derivatives thereof (dipeptides, tripeptides, etc.).

As a typical example, a case where various α-amino acids are labeled with the optical resolution labeling agent of the present invention will be described. The amino acid represented by the formula (7) to be detected is reacted with the compound represented by the formula (1A) to prepare a labeled amino acid represented by the formula (8A). The reaction is represented by the formula (8A) by reacting both in water and / or a water-soluble organic solvent (for example, acetone) in the presence of a base (for example, an alkali metal carbonate such as sodium hydrogen carbonate). Obtain labeled amino acids.
(In the formula, R ⁵ represents the side chain of α-amino acid. C ** represents the asymmetric carbon atom whose absolute configuration is determined. R ^1A , R ^2A , R ³ , R ⁴ , Alk and C * Is the same as above.)

This labeling reaction proceeds under mild conditions (arophilic nucleophilic substitution reaction) to give labeled amino acids (particularly labeled amino acids represented by the formula (8A)) in good yield. By analyzing this labeled amino acid by HPLC, LC-MS or the like, the absolute configuration of the amino acid (particularly the configuration of C ** above) can be determined. Specifically, using HPLC or LC-MS (EIC) analysis, the retention time (elution time) of the labeled amino acid to be inspected and the retention time (elution time) of the labeled amino acid standard whose absolute configuration is known in advance. By comparing with time), the absolute configuration of amino acids (particularly α-amino acids) can be determined.

As the HPLC conditions, general conditions can be applied and there is no particular limitation. Labeled amino acids have characteristic UV absorption near 340 nm and can be detected by UV 340 nm.

As the conditions of LC-MS (EIC), general conditions can be applied and there is no particular limitation. For example, a positive ionization mode using an electrospray ionization (ESI) method can be adopted.

Since the labeled amino acid represented by the formula (8A) has a tertiary amine (-Alk-NR ³ R ⁴ ) structure in the molecule, it was analyzed in a positive ionization mode using an electrospray ionization (ESI) method. In this case, even if the amount is extremely small, the absolute configuration can be detected with good separability and extremely high sensitivity. This is because the nitrogen atom having a tertiary amine structure is easily protonated to improve the efficiency of cationization, and thus the detection sensitivity in the positive ionization mode is dramatically improved. Therefore, when the optical resolution labeling agent of the present invention is used, when the conventional optical resolution labeling agent (L-FDAA, L-FDVA, etc.) reported in Non-Patent Documents 1 to 3 is used. In comparison, labeled amino acids can be detected with high resolution and higher sensitivity (for example, (2) to (5) of Example 2, Tables 1 to 4, FIGS. 1-1 to 5). reference).

When the C * of the optically split labeling agent containing the compound represented by the formula (1A) has an absolute configuration derived from the L-amino acid, the amino acid represented by the formula (7) is labeled using this. , HPLC or LC-MS (EIC) analysis, generally detected in the order of L-amino acid labeled form, followed by D-amino acid labeled form.

The optically resolution labeling agent containing the compound represented by the formula (1) of the present invention (particularly the compound represented by the formula (1A)) is used for amino acid analysis in, for example, foods, cosmetics, pharmaceuticals, pesticides, and chemical fields. It is useful as a reagent.

Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Examples (Synthesis of compounds (1a) to (1d))
Compounds (1a) to (1d) were synthesized according to the following methods.

<Synthesis method>

(1) Synthesis of compound (1a)
N-methylmorpholine (0.33 mL) and chloroforme in a solution of Boc-L-valine (2a) (R ¹ = i-Pr, R ² = H) (585 mg) in THF (13 mL) at 0 ° C. Isobutyl acid (0.39 mL) was added and the mixture was stirred for 10 minutes. Next, the temperature was returned from 0 ° C. to room temperature, N, N-dimethylethylenediamine (0.59 mL) and DMF (10 mL) were added, and the mixture was stirred for 2 hours, and a saturated aqueous sodium hydrogen carbonate solution was added. Subsequently, the mixture was extracted 3 times with ethyl acetate, washed twice with saturated brine, and dried over magnesium sulfate. The ethyl acetate solution of the remaining reaction product was purified by recrystallization from ethyl acetate and hexane to obtain 649 mg (yield 84%) of compound (4a) (R ¹ = i-Pr, R ² = H).

To a solution of the obtained compound (4a) (106 mg) in dichloromethane (6 mL) was added 2 mL of trifluoroacetic acid (TFA), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and azeotropically distilled with toluene three times to completely remove TFA, and then a crude product of compound (5a) was obtained.

Alpine (3 mL) was added to the crude product of the obtained compound (5a) to dissolve it, saturated aqueous sodium hydrogen carbonate solution (2.3 mL) and DFDNB (90.6 mg) were added, and the mixture was stirred under the conditions of 40 ° C. for 1 hour. Water was added to the reaction mixture, filtered through a filter paper, and washed with a water: acetone (19: 1) solution. The residue on the filter paper was dissolved in acetone, concentrated under reduced pressure, and purified by recrystallization from acetone and hexane to obtain 108 mg (yield 79%) of the target compound (1a).

L-FDVDA (1a):
¹ H NMR (500 MHz, CDCl ₃ ) δ 9.11 (d, J = 7.4 Hz, 1H), 8.96 (d, J = 7.4 Hz, 1H), 8.90 (m, 1H), 6.84 (d, J = 13.2 Hz) , 1H), 4.18 (t, J = 6.9 Hz, 1H), 3.90 (m, 1H), 3.58 (m, 1H), 3.22 (t, J = 5.4 Hz, 2H), 2.88 (s, 6H), 2.40 (m, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (d, J = 6.9 Hz, 3H);
¹³ C NMR (125 MHz, CDCl ₃ ) δ 170.29, 160.98, 158.82, 149.01, 128.02, 125.99, 101.90, 63.02, 58.09, 43.39, 34.42, 31.55, 19.30, 18.29;
HRMS (ESI) calcd for C ₁₅ H ₂₃ FN ₅ O ₅ [M + H] ⁺ 372.1678, found 372.1685;
[α] _D ²⁰ = 37.03 (c = 0.79, acetone);

(2) Synthesis of compounds (1b) to (1d) Compounds (1b) to (1d) correspond to the protected amino acids (Boc-D-valine (2b), Boc-L-leucine (2c), Boc, respectively. -D-leucine (2d)) was used as a raw material and synthesized according to the method (1) above.

D-FDVDA (1b):
^{1 1} H NMR and HR-MS (ESI) are the same as L-FDVDA (1a).
[α] _D ²⁰ = -33.54 (c = 0.71, acetone)

L-FDLDA (1c):
¹ H NMR (500 MHz, CDCl ₃ ) δ 9.15 (d, J = 8.0 Hz, 1H), 8.73 (m, 1H), 6.57 (d, J = 13.2 Hz, 1H), 6.53 (m, 1H), 4.00 (dd, J = 13.7, 5.7 Hz, 1H), 3.33 (q, J = 5.5 Hz, 2H), 2.37 (m, 2H), 2.14 (s, 6H), 1.85-1.75 (m, 3H), 1.04 ( d, J = 6.3 Hz 3H), 0.96 (d, J = 6.3 Hz, 3H);
¹³ C NMR (125 MHz, CDCl ₃ ) δ 169.94, 161.03, 158.87, 148.31, 127.76, 126.62, 102.11, 101.89, 57.30, 44.96, 41.87, 36.76, 25.06, 22.80, 21.87;
HRMS (ESI) calcd for C ₁₆ H ₂₅ FN ₅ O ₅ [M + H] ⁺ 386.1834, found 386.1833;
[α] _D ²⁰ = 21.46 (c = 0.46, acetone)

D-FDLDA (1d):
^{1 1} H NMR and HR-MS (ESI) are the same as L-FDLDA (1c).
[α] _D ²⁰ = -30.38 (c = 0.62, acetone)

Comparative example (synthesis of L-FDAA and L-FDLA)
L-FDAA and L-FDLA are the compounds shown below. These compounds were synthesized according to or according to the description of Non-Patent Documents 1 to 3.

Example 2 (Analysis of Labeled Amino Acids)
(1) Labeling of amino acids According to the following formula, various amino acids (7) were reacted with compound (1a) or (1c) to label them to prepare compound (8), and LC-MS analysis was performed.

The case where L-lysine (R ⁵ =-(CH ₂ ) _4- NH ₂ ) is labeled with compound (1a) will be described as a typical example.
A saturated aqueous sodium hydrogen carbonate solution (50 μL) was added to a commercially available aqueous solution of L-lysine (0.05 mg) (50 μL). Further, 25 μL of an acetone solution (10 mg / mL) of compound (1a) was added, and the mixture was stirred at 50 ° C. for 1 hour. Next, 60 μL of 1 M hydrochloric acid was added to the reaction mixture, and the mixture was concentrated to dryness. Methanol (500 μL) was added to the obtained solid, filtered through a membrane filter having a pore size of 0.22 μm, and the obtained methanol solution was analyzed by LC-MS.

According to the above typical example, various amino acids (L-leucine, L-lysine, L-glutamic acid, amino acid mixture) are labeled with each of compound (1a), compound (1c), L-FDAA, and L-FDLA. , LC-MS analyzed.

For LC-MS analysis, LC-MS8040 manufactured by Shimadzu Corporation was used. For ionization, the positive mode of ESI was used, and labeled bodies were detected at a UV wavelength of 340 nm. Imtakt cadenza CD-C18 (150 mm × 3 mm, particle size 3 μm) was used to separate the labeled product. The column oven was set to 40 ° C. and the flow rate was set to 0.2 mL / min. A 0.1% formic acid aqueous solution (A) and a 0.1% formic acid acetonitrile solution (B) were used as eluents.

(2) Comparison of detection sensitivity of various labeled L-leucine L-leucine prepared in (1) above, labeled with L-FDLA, L-FDAA, compound (1a) or compound (1c) The mixture was analyzed under 50% B isocratic conditions. The results are shown in Figure 1-1 and Table 1. When the peak intensity of positive ions of various labeled products was divided by the peak area of various labeled products detected at UV340 nm and compared, L-leucine labeled with compound (1a) or compound (1c) was calculated and compared. Compared with L-leucine labeled with L-FDLA or L-FDAA, a 10-fold increase in detection sensitivity was observed. In addition, compared with the detection by UV340nm, the detection in ESI positive mode was able to detect with 40 times higher sensitivity.

In addition, the results of analyzing the detection sensitivities of various labeled L-leucine by HPLC (UV340 nm) and LC-MS (ESI positive mode) are shown in FIG. 1-2. From this, it was confirmed that when LC-MS (EIC) was used, the labeled product could be detected with higher sensitivity even at a lower concentration than when HPLC was used.

(3) Comparison of detection sensitivity of various labeled products of L-lysine Four types of L-lysine prepared in (1) above labeled with L-FDLA, L-FDAA, compound (1a) or compound (1c). The mixture was analyzed under 55% B isocratic conditions. The results are shown in FIG. 2 and Table 2. When the peak intensity of positive ions of various labeled products was divided by the peak area of various labeled products detected at UV340 nm and compared, L-lysine labeled with compound (1a) or compound (1c) was calculated and compared. Compared with L-lysine labeled with L-FDLA or L-FDAA, a detection sensitivity increase of about 3 to 9 times was observed. In addition, the detection in ESI positive mode was able to detect with about 10 times higher sensitivity than the detection by UV340 nm.

(4) Comparison of detection sensitivity of various labeled L-glutamic acid L-glutamic acid prepared in (1) above is labeled with L-FDLA, L-FDAA, compound (1a) or compound (1c). The mixture was analyzed under 55% B isocratic conditions. The results are shown in FIG. 3 and Table 3. When the peak intensity of positive ions of various labeled products was divided by the peak area of various labeled products detected at UV340 nm and compared, L-glutamic acid labeled with compound (1a) or compound (1c) was compared. Compared with L-glutamic acid labeled with L-FDLA or L-FDAA, a detection sensitivity increase of about 7 to 10 times was observed. In addition, the detection in ESI positive mode was able to detect with about 20 times higher sensitivity than the detection by UV340 nm.

(5) Separation of Labeled Compounds of Compounds of Various Amino Acids (1a) Using 1 μL of a methanol solution containing a mixture of various D and L-amino acids prepared in (1) above labeled with compound (1a). The analysis was performed under a gradient condition of 25% to 55% B over 60 minutes. The results are shown in FIG. 4 and Table 4.

(6) Separation of Labeled Compounds of Compounds of Various Amino Acids (1c) Using 1 μL of a methanol solution containing a mixture of various D and L-amino acids prepared in (1) above labeled with compound (1c). The analysis was performed under a gradient condition of 30% to 60% B over 60 minutes. The results are shown in FIG. 5 and Table 4.

(7) When labeled products of various amino acids are prepared using compound (1b) and compound (1d) and analyzed by HPLC and LC-MS, the labeled products are similarly separated and detected with high sensitivity. be able to.

Example 3 (Stability of Compound (1a))
The compound (1a) was allowed to stand at room temperature for 3 weeks in the dark, and the stability was examined by HPLC (measurement conditions and detection conditions are described in FIG. 6). As a result, it was found that compound (1a) is very stable and can be stored at room temperature.

The compound represented by the formula (1) of the present invention can be used as an optical resolution labeling agent because it can analyze the absolute configuration of amino acids and the like with high sensitivity. It is also useful as an amino acid analysis reagent in various fields.

Claims (9)

Equation (1):
(In the formula, R ¹ and R ² are the same or different and have a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. R ³ and R ⁴ may be the same or different and have an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. It may indicate a heteroaryl group, or R ³ and R ⁴ may be bonded to each other to form a ring with adjacent nitrogen atoms, and the ring may have a substituent. Alk may have an alkylene. Indicates a group.)
A compound represented by or a salt thereof. Equation (1A):
(In the formula, R ^1A and R ^2A are different and may have a hydrogen atom, an alkyl group which may have a substituent, an aryl group which may have a substituent, or a substituent. It is a heteroaryl group, and R ³ , R ⁴ and Alk are the same as described above. The steric chemistry of C * to which R ^1A and R ^2A are bonded shows an absolute configuration.)
The compound according to claim 1 or a salt thereof, which is a compound represented by The compound according to claim 2, wherein either R ^1A or R ^2A is a hydrogen atom, the other is an alkyl group which may have a substituent, and Alk is a C2-C10 alkylene group. salt. Alkyl groups that may have substituents represented by R ^1A and R ^2A are the following groups;
(1) Hydroxyl groups that may be protected,
(2) Alkoxy group,
(3) Carboxyl groups that may be protected,
(4) Amide group,
(5) Amino groups that may be protected,
(6) Guanidino groups that may be protected,
(7) An aryl group that may be substituted with at least one group selected from the group consisting of an alkyl group and a hydroxyl group.
(8) A heteroaryl group that may be substituted with at least one group selected from the group consisting of an alkyl group and a hydroxyl group, and (9) a mercapto group that may be protected.
The compound according to claim 2 or 3, or a salt thereof, which is an alkyl group which may be substituted with at least one group selected from the group consisting of. An optically resolution labeling agent comprising the compound according to any one of claims 1 to 4 or a salt thereof. A method for producing a compound represented by the formula (1) or a salt thereof according to claim 1, wherein the formula (5):
^{(Wherein, R 1, R 2, R} 3 and ^{R 4} and Alk are as defined above.)
The compound represented by, or a salt thereof, and the formula (6):
A manufacturing method including a step of reacting a compound represented by. A method for producing a compound represented by the formula (1A) or a salt thereof according to claim 2, wherein the formula (5A):
(In the formula, R ^1A , R ^2B , R ³ and R ⁴ and Alk are the same as described above.)
A production method comprising a step of reacting a compound represented by the above formula or a salt thereof and a compound represented by the formula (6). A method for determining the absolute configuration of a compound having an asymmetric center.
(1) A step of reacting the compound having the asymmetric center with the optical resolution labeling agent according to claim 5 to prepare a labeled compound, and (2) analyzing the labeled compound. By doing so, the step of determining the absolute configuration of the compound having an asymmetric center,
How to include. The method according to claim 8, wherein the compound having an asymmetric center is an amino acid.