阅读说明：本技术 柠檬酸衍生物的非对映异构体的制造方法 (process for producing diastereomer of citric acid derivative ) 是由 平石胜也 相马洋之 神间史惠 足立太郎 山冈一平 远藤直之 于 2018-04-23 设计创作，主要内容包括：通过对包含下述式表示的化合物A及柠檬酸的水溶液使用离子交换柱色谱或碳酸钙,从而除去上述水溶液中的柠檬酸,之后经历若干工序而得到结晶性化合物A。另外,高纯度的非结晶性化合物A是通过对包含化合物A及柠檬酸的水溶液使用碳酸钙、硫酸、有机溶剂等来除去溶液中的柠檬酸及结晶性化合物A从而得到的。结晶性化合物A的立体结构按照RS表示法为SS体。非结晶性化合物A的立体结构按照RS表示法为SR体。<Image he="627" wi="514" file="DDA0002242995910000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(An aqueous solution containing a compound a represented by the following formula and citric acid is subjected to ion exchange column chromatography or calcium carbonate to remove citric acid from the aqueous solution, and then subjected to several steps to obtain a crystalline compound a. The highly pure amorphous compound a is obtained by removing citric acid and the crystalline compound a from an aqueous solution containing the compound a and citric acid by using calcium carbonate, sulfuric acid, an organic solvent, or the like. The crystalline compound a has a stereo structure represented by RS as SS. The three-dimensional structure of the amorphous compound a is represented by RS as SR.)

1. A crystal of a compound represented by the following formula (A) (hereinafter referred to as Compound A),

[ chemical formula 1]

2. The crystal of claim 1, having a steric structure according to the RS notation as SS-mer.

3. the crystal according to claim 1, wherein in a powder X-ray diffraction pattern using CuK α rays as an X-ray source, there are peaks at diffraction angles (2 θ) of 11.74 ± 0.20 °, 29.25 ± 0.20 °, 18.36 ± 0.20 °, 21.75 ± 0.20 °, and 15.95 ± 0.20 °.

4. A method for producing a crystal of Compound A, which comprises the following steps (a) to (f),

A step (a) in which an aqueous solution having a pH of 5.0 to 8.5 and containing a compound A and/or a salt thereof and citric acid and/or a salt thereof is passed through a column packed with an anion exchange resin;

A step (b) of introducing an eluent into the column to obtain an aqueous solution containing no citric acid but containing the compound A;

A step (c) of removing the eluent from the aqueous solution obtained in the step (b);

A step (d) of concentrating the aqueous solution from which the eluent has been removed;

A step (e) in which water is added to the concentrated residue to form an aqueous solution, and the aqueous solution is concentrated to precipitate crystals of compound A; and

And (f) obtaining a crystal of the compound A.

5. The production process according to claim 4, wherein the crystal of Compound A has a steric structure represented by SS isomer according to RS representation.

6. The production method according to claim 4 or 5, wherein the eluent is an eluent selected from the group consisting of an aqueous ammonium acetate solution, an aqueous sodium chloride solution, and an aqueous ammonium formate solution.

7. The production process according to any one of claims 4 to 6, wherein the method of removing the eluting solution is a method using a column packed with a cation exchange resin.

8. The production process according to any one of claims 4 to 7, wherein the concentration is carried out by freeze-drying.

9. A method for producing a crystal of Compound A, which comprises the following steps (a) to (f),

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

A step (b) for removing calcium citrate from the aqueous solution;

A step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

A step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A; and

And (f) obtaining a crystal of the compound A.

10. The production process according to claim 9, wherein the crystal of compound A has a steric structure represented by SS isomer according to RS representation.

11. The production process according to claim 9 or 10, wherein the concentration is a concentration under reduced pressure.

12. The production method according to any one of claims 9 to 11, further comprising the following steps (g) to (j) after obtaining the crystal of the compound A,

A step (g) in which an organic solvent is added to the aqueous solution to precipitate calcium citrate;

A step (h) of removing calcium citrate from a mixed solution of an aqueous solution and an organic solvent;

A step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A; and

And (j) obtaining a crystal of the compound A.

13. The production process according to claim 12, wherein the organic solvent is acetone.

14. A process for producing an amorphous diastereomer salt of Compound A, which comprises the following steps (a) to (l),

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

A step (b) for removing calcium citrate from the aqueous solution;

A step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

a step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A;

A step (f) of removing the crystals of the compound A from the aqueous solution;

A step (g) in which an organic solvent is added to the aqueous solution to precipitate crystals of the compound A and calcium citrate;

A step (h) in which the crystals of Compound A and calcium citrate are removed from a mixed solution of an aqueous solution and an organic solvent;

a step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A;

a step (j) of removing the crystals of the compound A from the aqueous solution;

A step (k) of adding a metal salt or an amino acid salt and an alcohol to the aqueous solution to precipitate an amorphous diastereomer salt of the compound A; and

Step (l) is a step of obtaining an amorphous diastereomer salt of Compound A.

15. The process according to claim 14, wherein the amorphous diastereomer salt of Compound A has a stereostructure represented by RS as SR isomer.

16. The method according to claim 14, wherein the organic solvent is acetone.

17. The production method according to any one of claims 14 to 16, wherein the metal salt is a metal salt selected from the group consisting of sodium salt, potassium salt, magnesium salt, and calcium salt.

18. The production method according to any one of claims 14 to 16, wherein the amino acid salt is an amino acid salt selected from the group consisting of an arginine salt, a citrulline salt, an ornithine salt, and a histidine salt.

19. The production method according to any one of claims 14 to 18, wherein the alcohol is ethanol or methanol.

20. A non-crystalline diastereomer of compound a or a salt thereof.

21. The non-crystalline diastereomer of compound a or a salt thereof according to claim 20, wherein the steric structure is SR form according to RS notation.

22. the non-crystalline diastereomer of compound a or a salt thereof according to claim 20 or 21, wherein the salt is a metal salt or an amino acid salt of the non-crystalline diastereomer of compound a.

23. The non-crystalline diastereomer of compound a or a salt thereof according to claim 22, wherein the metal salt is a metal salt selected from the group consisting of a sodium salt, a potassium salt, a magnesium salt, and a calcium salt.

24. compound a, amorphous diastereomer or salt thereof according to claim 22, wherein the amino acid salt is an amino acid salt selected from the group consisting of arginine salt, citrulline salt, ornithine salt, and histidine salt.

Technical Field

the present invention relates to a crystal of a citric acid derivative having an effect of inhibiting liver damage, a highly purified amorphous diastereomer (amorphous diastereomer ) thereof, and a method for producing the same.

Background

Plum (British name: Japanese apricot, scientific name: Prunus mume) belongs to the genus Prunus of the family Rosaceae, the subfamily Pruni, and plum processed products such as dried plum, plum wine, and plum meat extract are being eaten. In addition, the plum extract has antibacterial, fatigue recovery, and stomach protecting effects, and is taken by people for health. Further, the plum extract is known to have an effect of improving blood circulation (see non-patent documents 1 and 2). It is known that the above-mentioned effect of improving blood circulation is brought about by the umefural (Mumefural) contained in the ume meat extract, which is produced by heating an organic acid such as citric acid or malic acid and a sugar, and related compounds thereof (non-patent document 3).

one of health foods containing a plum extract is Misatol (registered trademark) which is commercially available, and Misatol is known to have an autophagy-inducing effect and an effect of inhibiting liver damage in patients with viral hepatitis (patent documents 1 and 2).

The following compounds are known as active substances having a liver injury inhibitory effect: a compound in which two carboxyl groups bonded to the carbon chain of citric acid (IUPAC name: 2-hydroxypropane-1, 2, 3-tricarboxylic acid), i.e., the 1-position carbon (or 3-position carbon) and the 2-position carbon of a propane chain, form an imide bond with the amino group of a specific amino acid; and an amide compound obtained by hydrolyzing the imide compound, wherein the amide compound is formed by a carboxyl group bonded to the carbon at the 1-position (or the carbon at the 3-position) of a citric acid-derived propane chain and an amino acid.

Based on the above findings, the inventors of the present application previously proposed a citric acid derivative containing a compound represented by the following formula and a synthetic method thereof (PCT/JP 2016/004789).

[ chemical formula 1]

(wherein R1 represents a C1-C3 alkyl group which may have a carboxyl group or a hydroxyl group, and R2 represents a hydrogen atom, or R1 and R2 may form a cyclic structure together and represent a C2-C3 alkylene chain.)

However, the citric acid derivative represented by the above chemical formula has two chiral carbons, and thus 4 stereoisomers exist. Among them, in order to obtain these stereoisomers having similar structures by separation, it is necessary to combine a plurality of expensive columns having high separation performance or to repeat the separation a plurality of times, and this method is capable of achieving only a small amount of separation depending on the size of the columns and requires high cost. No technique has been established for separating these stereoisomers in large amounts at low cost and for purifying them.

Disclosure of Invention

problems to be solved by the invention

The invention provides the following method: among the citric acid derivatives represented by the above chemical formula, a crystalline diastereomer compound is isolated in a large amount from a diastereomer mixture of a citric acid derivative represented by the following formula obtained by a reaction of citric acid with L-aspartic acid by an inexpensive method; and purifying the amorphous diastereomer compound in a large amount to a high purity by an inexpensive method.

[ chemical formula 2]

Means for solving the problems

As a result of repeated research and study to solve the above problems, the inventors of the present application have found that crystals of the compound represented by the above formula can be obtained by combining specific steps. The present inventors have also found that by combining specific steps, an amorphous diastereomer salt of the compound can be precipitated. In addition, the inventors of the present application determined the structure of the crystalline diastereomer of the compound represented by the above formula by structural analysis based on single crystal X-ray diffraction.

the present invention has been completed based on the above findings.

namely, the present invention relates to the following aspects.

[1] A crystal of a compound represented by the following formula (A) (hereinafter referred to as Compound A).

[ chemical formula 3]

[2] The crystal according to [1], wherein the crystal has a cubic structure represented by SS according to RS representation.

[3] the crystal according to [1] or [2], wherein, in a powder X-ray diffraction pattern using CuK α rays as an X-ray source, peaks are present at diffraction angles (2 θ) of 11.74 ± 0.20 °, 29.25 ± 0.20 °, 18.36 ± 0.20 °, 21.75 ± 0.20 °, and 15.95 ± 0.20 °.

[4] A method for producing a crystal of Compound A, comprising the following steps (a) to (f).

A step (a) in which an aqueous solution having a pH of 5.0 to 8.5 and containing a compound A and/or a salt thereof and citric acid and/or a salt thereof is passed through a column packed with an anion exchange resin;

A step (b) of introducing an eluent into the column to obtain an aqueous solution containing the compound A and no citric acid;

a step (c) of removing the eluent from the aqueous solution obtained in the step (b);

A step (d) of concentrating the aqueous solution from which the eluent has been removed;

A step (e) in which water is added to the concentrated residue to form an aqueous solution, and the aqueous solution is concentrated to precipitate crystals of compound A; and

And (f) obtaining a crystal of the compound A.

[5] the production process according to [4], wherein the crystal of the compound A has a steric structure represented by SS according to RS representation.

[6] the production method according to [4] or [5], wherein the eluent is an eluent selected from the group consisting of an aqueous ammonium acetate solution, an aqueous sodium chloride solution, and an aqueous ammonium formate solution.

[7] The production method according to any one of [4] to [6], wherein the method of removing the eluent is a method using a column packed with a cation exchange resin.

[8] The production method according to any one of [4] to [7], wherein the concentration is performed by freeze-drying.

[9] a method for producing a crystal of Compound A, comprising the following steps (a) to (f).

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

A step (b) of removing calcium citrate from a 60% methanol-water solution;

A step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

A step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A; and

And (f) obtaining a crystal of the compound A.

[10] The production process according to [9], wherein the crystal of the compound A has a steric structure represented by SS according to RS representation.

[11] the production process according to [9] or [10], wherein the concentration is a concentration under reduced pressure.

[12] The production method according to any one of [9] to [11], further comprising the following steps (g) to (j) after obtaining the crystal of the compound A.

A step (g) in which an organic solvent is added to the aqueous solution to precipitate calcium citrate;

A step (h) of removing calcium citrate from a mixed solution of an aqueous solution and an organic solvent;

A step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A; and

And (j) obtaining a crystal of the compound A.

[13] The production method according to [12], wherein the organic solvent is acetone.

[14] A method for producing an amorphous diastereomer salt of Compound A, which comprises the following steps (a) to (l).

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

A step (b) for removing calcium citrate from the aqueous solution;

A step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

A step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A;

a step (f) of removing the crystals of the compound A from the aqueous solution;

A step (g) in which an organic solvent is added to the aqueous solution to precipitate crystals of the compound A and calcium citrate;

A step (h) in which the crystals of Compound A and calcium citrate are removed from a mixed solution of an aqueous solution and an organic solvent;

A step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A;

A step (j) of removing the crystals of the compound A from the aqueous solution;

A step (k) of adding a metal salt or an amino acid salt and an alcohol to the aqueous solution to precipitate an amorphous diastereomer salt of the compound A; and

step (l) is a step of obtaining an amorphous diastereomer salt of Compound A.

[15] the production process according to [14], wherein the stereostructure of the amorphous diastereomer salt of the compound A is SR by RS representation.

[16] The production method according to [14], wherein the organic solvent is acetone.

[17] The production method according to any one of [14] to [16], wherein the metal salt is a metal salt selected from the group consisting of sodium salt, potassium salt, magnesium salt, and calcium salt.

[18] The production method according to any one of [14] to [16], wherein the amino acid salt is an amino acid salt selected from the group consisting of an arginine salt, a citrulline salt, an ornithine salt, and a histidine salt.

[19] The production method according to any one of [14] to [18], wherein the alcohol is ethanol or methanol.

[20] A non-crystalline diastereomer of compound a or a salt thereof.

[21] the non-crystalline diastereomer of compound A or a salt thereof according to [20], wherein the stereostructure is an SR form according to RS representation.

[22] the non-crystalline diastereomer of Compound A or a salt thereof according to [20] or [21], wherein the salt is a metal salt or an amino acid salt of the non-crystalline diastereomer of Compound A.

[23] The non-crystalline diastereomer of Compound A or a salt thereof according to [22], wherein the metal salt is a metal salt selected from the group consisting of a sodium salt, a potassium salt, a magnesium salt and a calcium salt.

[24] The non-crystalline diastereomer of compound a or a salt thereof according to [22], wherein the amino acid salt is an amino acid salt selected from the group consisting of an arginine salt, a citrulline salt, an ornithine salt, and a histidine salt.

ADVANTAGEOUS EFFECTS OF INVENTION

Obtaining crystals of compound a and highly purified amorphous diastereomers of compound a contributes to the clarification of physiological activities of the respective substances and mechanisms of action on diseases. Further, the crystal of compound a and the highly purified amorphous diastereomer of compound a are easier to handle than a mixture of diastereomers containing them, and therefore are extremely useful for producing a drug containing compound a as an active ingredient, a food or drink containing compound a, or the like. In addition, according to the method described in the present application, it is possible to separate and obtain a crystal and an amorphous diastereomer of compound a in a large amount at low cost in a short time as compared with a purification method based on a high separation performance column or the like (which is used as a general purification means). In the purification method using a column or the like using a carrier having high separation performance (which is used as a general method for separating diastereoisomers), the amount of purification which can be performed in one purification process depends on the size of the column used, but a large-scale purification method using a large-scale column requiring an expensive carrier having high separation performance is not easy from the viewpoint of cost and efficiency. According to the method described in the present application, 20g or more of a high-purity diastereomer can be easily separated from 500mL of a reaction solution in one purification process, and the purification scale can be easily enlarged.

Drawings

FIG. 1 shows an HPLC chromatogram (160-fold dilution) of a reaction solution for synthesizing Compound A.

FIG. 2 shows an HPLC chromatogram of a crystal of Compound A after crystallization in an aqueous solution.

FIG. 3 shows an HPLC chromatogram of the mother liquor solution after isolating crystals of Compound A.

FIG. 4 shows an HPLC chromatogram of Compound A after crystallization in acetone (after recrystallization).

FIG. 5 shows an HPLC chromatogram of the precipitate precipitated at pH3.6 (Ca salt of amorphous Compound A (1)).

FIG. 6 shows an HPLC chromatogram of the precipitate precipitated at pH6.0 (Ca salt of amorphous Compound A (2)). In peak 2 of fig. 6, the amide compound peak obtained by hydrolysis of compound a partially overlaps with the left shoulder of the peak of compound a (the left shoulder of the peak appears to bulge). In addition, peak 3 is a peak considered to be citric acid.

FIG. 7 shows an HPLC chromatogram of a synthetic solution of Compound A after the crude purification.

Fig. 8 shows an HPLC chromatogram of compound a after crystallization in an aqueous solution (analysis result from the same sample as fig. 2).

Fig. 9 shows an HPLC chromatogram of compound a after crystallization in acetone (analysis result from the same sample as fig. 4).

FIG. 10 shows an HPLC chromatogram of the Ca salt of amorphous Compound A precipitated at pH3.6 (the result of analysis from the same sample as in FIG. 5).

FIG. 11 is a flowchart of a method for producing crystals of Compound A by ion exchange chromatography in example 7.

FIG. 12 shows an HPLC chromatogram of a crystal of Compound A.

[ FIG. 13] shows a 1H-NMR spectrum of a crystal of Compound A.

FIG. 14 shows an HPLC chromatogram of a mother liquor obtained by fractionating crystals of Compound A.

FIG. 15 shows a 1H-NMR spectrum of a mother liquor after separation of crystals of Compound A.

Fig. 16 shows a comparison by multiple plotting of powder X-ray diffraction patterns relating to each substance in the order of specimen 1 (crystal obtained from an aqueous solution by column purification), specimen 2 (crystal obtained from an acetone solution), and specimen 3 (Ca salt of an amorphous diastereomer) from above.

Detailed Description

As the crystals of compound a of the present invention, the following crystals may be mentioned: in a powder X-ray diffraction pattern using CuK alpha as an X-ray source, peaks are present at diffraction angles (2 theta) of 11.74 + -0.20 DEG (preferably + -0.10 DEG), 29.25 + -0.20 DEG (preferably + -0.10 DEG), 18.36 + -0.20 DEG (preferably + -0.10 DEG), 21.75 + -0.20 DEG (preferably + -0.10 DEG), and 15.95 + -0.20 DEG (preferably + -0.10 DEG).

As the crystals of compound a of the present invention, the following crystals may be mentioned: in a powder X-ray diffraction pattern using CuK alpha as an X-ray source, in addition to the above, peaks are present at diffraction angles (2 theta) of 24.09 + -0.20 DEG (preferably + -0.10 DEG), 19.32 + -0.20 DEG (preferably + -0.10 DEG), 19.04 + -0.20 DEG (preferably + -0.10 DEG), 26.95 + -0.20 DEG (preferably + -0.10 DEG), and 16.19 + -0.20 DEG (preferably + -0.10 DEG).

As the crystals of compound a of the present invention, the following crystals may be mentioned: in a powder X-ray diffraction pattern using CuK alpha as an X-ray source, in addition to the above, peaks are present at diffraction angles (2 theta) of 26.42 + -0.20 DEG (preferably + -0.10 DEG), 16.68 + -0.20 DEG (preferably + -0.10 DEG), 17.85 + -0.20 DEG (preferably + -0.10 DEG), 21.19 + -0.20 DEG (preferably + -0.10 DEG), and 18.14 + -0.20 DEG (preferably + -0.10 DEG).

A powder X-ray diffraction pattern using CuK α as an X-ray source can be obtained by the method described in example 8 of the present specification.

In compound a, there is a diastereomer with the carbon at position 2 from the propane chain of citric acid being a chiral carbon. In the compound a of the present invention, a chiral carbon is present in the structure derived from aspartic acid, but the stereoconfiguration of the chiral carbon is derived from aspartic acid or asparagine as a raw material to be used. The crystal of compound a of the present invention is derived from L-body aspartic acid.

The crystal of compound a of the present invention is a crystal of either one of the diastereomers of compound a, but it is not possible to distinguish which diastereomer corresponds to in NMR.

As a method for determining which diastereomer the crystal of compound a is, a structural analysis by single crystal X-ray diffraction can be mentioned. Specifically, the method described in example 9 can be given as an example. The analysis result of example 9 shows that crystalline compound a (crystal of compound a) is an enantiomer of the SS isomer or the RR isomer. Further, since L-Asp (S isomer) was used in the synthesis, it was found that the configuration of the crystalline compound A was SS isomer in accordance with the RS representation.

[ chemical formula 4]

The analysis result indicated that the crystalline compound a was an enantiomer of the SS isomer or the RR isomer. Since L-Asp (S isomer) is used in the synthesis, the crystalline compound A is SS isomer. Further, based on this, it is also known that compound a having a property of not crystallizing is an SR form.

the amorphous diastereomer of compound a of the present invention or a salt thereof is a diastereomer (hereinafter, also referred to as an amorphous diastereomer) which is not crystallized by a method of crystallizing compound a described below in one of the diastereoisomers of compound a or a salt thereof.

the amorphous diastereomer of the compound a of the present invention or a salt thereof can be confirmed by qualitative analysis by High Performance Liquid Chromatography (HPLC) as described in example 5, and can be distinguished from the crystalline diastereomer.

As described above, since the crystal of the compound a of the present invention has a stereoconfiguration represented by SS according to RS, the amorphous (amorphous diastereomer) stereoconfiguration of the compound a of the present invention can also be represented by SR according to RS as follows.

[ chemical formula 5]

The amorphous diastereomer of compound a or a salt thereof of the present invention includes an amorphous diastereomer of compound a or a salt thereof, in which the mixing ratio of the crystalline diastereomer is 5% or less, preferably 4% or less, more preferably 3% or less, further preferably 2% or less, and most preferably 1% or less.

examples of the salt of the amorphous diastereomer of compound a of the present invention include a metal salt and an amino acid salt.

The metal salt includes a metal salt selected from the group consisting of sodium salt, potassium salt, magnesium salt, and calcium salt, and most preferably includes calcium salt.

Examples of the amino acid salt include amino acid salts selected from the group consisting of arginine salts, citrulline salts, ornithine salts, and histidine salts. The amino acid salt is preferably an L-form amino acid salt.

The crystal of compound a of the present invention can be obtained by any one of the following methods: crystallizing by ion exchange column chromatography; and crystallizing the crystals using a compound such as calcium carbonate.

(method based on ion exchange column chromatography)

crystals of compound a were produced by the following steps (a) to (f).

A step (a) in which an aqueous solution having a pH of 5.0 to 8.5 and containing a compound A and/or a salt thereof and citric acid and/or a salt thereof is passed through a column packed with an anion exchange resin;

A step (b) of introducing an eluent into the column to obtain an aqueous solution containing the compound A and no citric acid;

A step (c) of removing the eluent from the aqueous solution obtained in the step (b);

A step (d) of concentrating the aqueous solution from which the eluent has been removed;

A step (e) in which water is added to the concentrated residue to form an aqueous solution, and the aqueous solution is concentrated to precipitate crystals of compound A; and

And (f) obtaining a crystal of the compound A.

The salt of compound a and the salt of citric acid in the aqueous solution having a ph of 5.0 to 8.5, which contains compound a and/or a salt thereof and citric acid and/or a salt thereof used in the step (a) are not particularly limited. Examples of the salt of compound a and the salt of citric acid include amino acid salts and metal salts (sodium salt, potassium salt, magnesium salt, calcium salt, etc.). The most preferable examples of the compound a and/or a salt thereof and the citric acid and/or a salt thereof are compound a and citric acid.

The aqueous solution having a ph of 5.0 to 8.5 and containing the compound a and/or a salt thereof and citric acid and/or a salt thereof used in the step (a) is produced, for example, in the following manner. Under heating, citric acid monohydrate is reacted with L-aspartic acid to produce compound a. The compound a may be produced by a method other than a synthetic method such as an enzymatic reaction method or a fermentation method. After the reaction, the reaction solution was left to cool, and methanol was added under acidic conditions. The concentration of the methanol solution may be any concentration as long as it is a concentration at which L-aspartic acid can be precipitated, and may be, for example, a 60V/V% solution. Since the unreacted citric acid and L-aspartic acid among L-aspartic acid contained in the reaction solution precipitate, solid-liquid separation is performed by centrifugal separation or filtration. The aqueous solution after the solid-liquid separation contains compound a and citric acid. When the aqueous solution after the solid-liquid separation is acidic, it is preferable to adjust the pH by adding an aqueous alkali solution such as ammonia. The pH of the aqueous solution is 5.0 to 8.5, preferably 6.0 to 8.0, and most preferably 6.5 to 7.2. The aqueous solution is passed through a column packed with an anion exchange resin.

The anion exchange resin may be any anion exchange resin as long as it can separate the compound A and/or a salt thereof from the citric acid and/or a salt thereof, and examples thereof include TOYOPEARL SuperQ-650M (150 mm. times.500 mm, manufactured by Tosoh Corporation).

The eluent used in the step (b) is not particularly limited, and is selected from the group consisting of an ammonium acetate aqueous solution, a sodium chloride aqueous solution, and an ammonium formate aqueous solution.

The concentration of the eluent is 50 mM-5M, preferably 100 mM-3M, more preferably 150 mM-2M, and most preferably 200 mM-1.5M. The elution may be performed by increasing the concentration of the eluent stepwise.

The flow rate in the elution step is 10 to 500mL/min, preferably 50 to 300mL/min, and most preferably 100 to 200 mL/min.

In the step (b), the "citric acid-free" is preferably completely citric acid-free, but a trace amount of citric acid may be contained to such an extent that crystallization of the compound a is not inhibited.

In the step (c), examples of a method for removing the eluent include a method using a column packed with a cation exchange resin, and electrodialysis, and preferably a method using a column packed with a cation exchange resin. The term "eluent is removed" means that components (solute) in the eluent are removed.

the cation exchange resin may be any cation exchange resin as long as it can separate the compound a from the eluate, and examples thereof include DOWEX50Wx8 (manufactured by Wako Chemical corporation). An aqueous solution such as purified water is sent to a column packed with a cation exchange resin to obtain an aqueous solution containing compound a but no eluent. The "eluent is preferably not contained at all, but a trace amount of the eluent may be contained to such an extent that crystallization of the compound a is not inhibited.

In the steps (d) and (e), examples of the method for concentrating the aqueous solution include heat concentration, concentration under reduced pressure, and freeze drying is most preferable.

in the steps (d) and (e), amorphous and/or crystalline compounds a, a mixture thereof, or a concentrated residue containing them can be obtained by concentrating the aqueous solution. In the case where the compound a is amorphous, the case of a mixture containing amorphous, or the case of a concentrated residue containing them, it is preferable to add water again to form an aqueous mixture and perform the concentration step. By performing this step, crystals of compound a can be precipitated.

In the step (f), examples of a method for obtaining crystals include pressure filtration, suction filtration, and centrifugal separation. Furthermore, the crystals can be washed appropriately in order to reduce the adhesion of the mother liquor and improve the quality of the crystals. The washed crystals can be dried by drying under reduced pressure, fluidized bed drying, aeration drying, etc. to obtain the final product.

In the above method, the crystallization of the compound a may be achieved by repeating the steps (d) to (e) a plurality of times.

(method based on crystallization Using calcium carbonate or the like)

Crystals of compound a were produced in an aqueous solution through the following steps (a) to (f).

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

A step (b) for removing calcium citrate from the aqueous solution;

A step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

A step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A; and

and (f) obtaining a crystal of the compound A.

The aqueous solution containing compound a and citric acid and having a ph of 2.0 or less used in the step (a) is produced, for example, in the following manner. Reacting the citric acid monohydrate with L-aspartic acid under heating to produce Compound A. The compound a may be produced by a method other than a synthetic method such as an enzymatic reaction method or a fermentation method. After the reaction, the reaction solution was left to cool, and methanol was added under acidic conditions. The concentration of the aqueous methanol solution may be any concentration as long as it is a concentration at which L-aspartic acid can be precipitated, and may be, for example, a 60 v/v% solution. Since unreacted citric acid and L-aspartic acid among L-aspartic acid contained in the reaction solution precipitate, solid-liquid separation is performed by centrifugal separation. In this step, it is important to add methanol. Ethanol may not be used. In the subsequent step, an aqueous solution obtained by solid-liquid separation containing compound a and citric acid is used.

When a crystal of compound a is obtained by this method, it is difficult to: citric acid is removed from an aqueous solution comprising compound a and citric acid under acidic conditions. Since compound a is unstable in a neutral to basic aqueous solution (pH5 or higher), it is important to maintain acidic conditions in order to suppress the decomposition of compound a. Therefore, the aqueous solution containing compound a and citric acid and having a pH of 2.0 or less used in step (a) may be an aqueous solution having a pH of preferably 0.6 to 1.8, most preferably 1.0 to 1.6. The inventors of the present application have thought to remove citric acid as a salt, and have repeatedly tried using salts formed from various metal ions, and as a result, have found that citric acid can be removed well by using calcium ions (Ca2 +). The inventors of the present application have found that even in the case of a calcium salt, when calcium chloride (CaCl2) is used, citric acid cannot be removed satisfactorily under acidic conditions, and when calcium carbonate (CaCO3) is used, citric acid can be removed smoothly even under acidic conditions. The reason for this is considered to be that carbonate ions (CO32-) of calcium carbonate form CO2, anions that pair with calcium ions disappear, and therefore citrate ions easily bind to calcium ions. Therefore, in the step (a), calcium carbonate is essential.

The removal of calcium citrate in the step (b) and the removal of calcium sulfate in the step (d) can be performed by, for example, centrifugal separation or filtration.

The reason why sulfuric acid is used in the step (c) is as follows: compound a is not base tolerant; and easy removal of calcium ions (i.e., easy desalination) due to low solubility of calcium sulfate. When the pH is 2.0 or less, calcium ions present as a calcium salt of compound a are dissociated from compound a to form calcium sulfate. The pH at this time is preferably 1.0 to 2.0, more preferably 1.3 to 1.8. When sodium salt (sodium sulfate) or potassium salt (potassium sulfate) is used, desalting is difficult.

As a method of concentrating the aqueous solution in the step (e), specific examples include heating concentration, vacuum concentration and the like, and a method of concentrating under reduced pressure is preferable in order to prevent the property change or decomposition of the mixed component due to heat.

In the step (f), examples of a method for obtaining crystals include pressure filtration, suction filtration, and centrifugal separation. Furthermore, the crystals can be washed appropriately in order to reduce the adhesion of the mother liquor and improve the quality of the crystals. The washed crystals can be dried by drying under reduced pressure, fluidized bed drying, aeration drying, etc. to obtain the final product.

The aqueous solution (crystal mother liquor) after the crystal of the compound a is precipitated in the step (e) contains the crystalline compound a diastereomer. Therefore, the crystals of compound a can be further obtained through the following steps (g) to (j).

a step (g) in which an organic solvent is added to the aqueous solution to precipitate calcium citrate;

A step (h) of removing calcium citrate from a mixed solution of an aqueous solution and an organic solvent;

A step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A; and

And (j) obtaining a crystal of the compound A.

the organic solvent to be added in the step (g) is preferably an organic solvent capable of being mixed with water and dissolving a certain amount of the compound a, and specifically, acetone or the like can be exemplified.

The removal of calcium citrate in the step (h) can be performed by, for example, centrifugation or filtration.

the method for precipitating the crystals of the compound a in the step (i) may be any method as long as the compound is precipitated, and preferred methods include: the aqueous solution was concentrated by removing the solvent by distillation, and the solvent was added again.

In the step (j), examples of a method for obtaining crystals include pressure filtration, suction filtration, and centrifugal separation. Furthermore, the crystals can be washed appropriately in order to reduce the adhesion of the mother liquor and improve the quality of the crystals. The washed crystals can be dried by drying under reduced pressure, fluidized bed drying, aeration drying, etc. to obtain the final product.

The amorphous diastereomer salt of compound a of the present invention can be obtained by a method including the steps (a) to (l) described below.

A step (a) in which calcium carbonate is added to an aqueous solution having a pH of 2.0 or less and containing compound A and citric acid, thereby precipitating calcium citrate;

a step (b) for removing calcium citrate from the aqueous solution;

a step (c) in which sulfuric acid is added to the aqueous solution to a pH of 2.0 or less to precipitate calcium sulfate;

A step (d) for removing calcium sulfate from the aqueous solution;

A step (e) of concentrating the aqueous solution to precipitate crystals of Compound A;

A step (f) of removing the crystals of the compound A from the aqueous solution;

A step (g) in which an organic solvent is added to the aqueous solution to precipitate crystals of the compound A and calcium citrate;

A step (h) in which the crystals of Compound A and calcium citrate are removed from a mixed solution of an aqueous solution and an organic solvent;

A step (i) in which a mixed solution of an aqueous solution and an organic solvent is dehydrated to precipitate a crystal of compound A;

a step (j) of removing the crystals of the compound A from the aqueous solution;

A step (k) of adding a metal salt or an amino acid salt and an alcohol to the aqueous solution to precipitate an amorphous diastereomer salt of the compound A; and

Step (l) is a step of obtaining an amorphous diastereomer salt of Compound A.

The aqueous solution containing compound a and citric acid and having a ph of 2.0 or less used in the step (a) is produced, for example, in the following manner. Under heating, citric acid monohydrate is reacted with L-aspartic acid to produce compound a. The compound a may be produced by a method other than a synthetic method such as an enzymatic reaction method or a fermentation method. After the reaction, the reaction solution was left to cool, and methanol was added under acidic conditions. The concentration of the aqueous methanol solution may be any concentration as long as it is a concentration at which L-aspartic acid can be precipitated, and may be, for example, a 60 v/v% solution. Since unreacted citric acid and L-aspartic acid among L-aspartic acid contained in the reaction solution precipitate, solid-liquid separation is performed by centrifugal separation. In this step, it is important to add methanol. Ethanol may not be used. In the subsequent step, an aqueous solution obtained by solid-liquid separation containing compound a and citric acid is used. The aqueous solution containing compound a and citric acid used in step (a) and having a pH of 2.0 or less may be an aqueous solution having a pH of preferably 0.6 to 1.8, most preferably 1.0 to 1.6.

When a crystal of compound a is obtained by this method, it is difficult to: citric acid was removed from the aqueous solution comprising compound a and citric acid. The inventors of the present application have thought to remove citric acid as a salt, and have repeatedly tried using salts formed from various metal ions, and as a result, have found that citric acid can be removed well by using calcium ions (Ca2 +). The inventors of the present application have found that even in the case of a calcium salt, when calcium chloride (CaCl2) is used, citric acid cannot be removed satisfactorily under acidic conditions, and when calcium carbonate (CaCO3) is used, citric acid can be removed smoothly even under acidic conditions. The reason for this is considered to be that carbonate ions (CO32-) of calcium carbonate form CO2, anions that pair with calcium ions disappear, and therefore citrate ions easily bind to calcium ions. Therefore, in the step (a), calcium carbonate is essential.

the removal of calcium citrate in the step (b) and the removal of calcium sulfate in the step (d) can be performed by, for example, centrifugal separation or filtration.

The reason why sulfuric acid is used in the step (c) is as follows: compound a is not base tolerant; and easy removal of calcium ions (i.e., easy desalination) due to low solubility of calcium sulfate. When the pH is 2.0 or less, calcium ions present as a calcium salt of compound a are dissociated from compound a to form calcium sulfate. The pH at this time is preferably 1.0 to 2.0, more preferably 1.3 to 1.8. When sodium salt (sodium sulfate) or potassium salt (potassium sulfate) is used, desalting is difficult.

As a method of concentrating the aqueous solution in the step (e), specific examples include heating concentration, vacuum concentration and the like, and a method of concentrating under reduced pressure is preferable in order to prevent the property change or decomposition of the mixed component due to heat.

In the step (f), examples of the method for removing crystals include pressure filtration, suction filtration, and centrifugal separation.

the above-mentioned step (g) and step (h) are steps necessary for the purification of the diastereomer salt of compound a. This is because the crystallized compound a remains in the aqueous solution after the crystals of compound a are removed in step (f).

the organic solvent to be added in the step (g) is preferably an organic solvent capable of being mixed with water and dissolving a certain amount of the compound a, and specifically, acetone or the like can be exemplified.

The removal of calcium citrate in the step (h) can be performed by, for example, centrifugation or filtration.

The method for precipitating the crystals of the compound a in the step (i) may be any method as long as the compound is precipitated, and preferred methods include: the aqueous solution was concentrated by removing the solvent by distillation, and the solvent was added again.

In the step (j), examples of the method for removing crystals include centrifugation, pressure filtration, and suction filtration. In the step (j), the crystalline compound a is substantially removed.

examples of the metal salt to be added in the step (k) include sodium salt, potassium salt, magnesium salt, and calcium salt (for example, calcium chloride and calcium acetate) is most preferably used. The pH of the aqueous solution is preferably acidic, and particularly, pH3.6 or less is most preferable.

Examples of the amino acid salt to be added in the step (k) include amino acid salts selected from the group consisting of arginine salt, citrulline salt, ornithine salt and histidine salt, for example, L-arginine hydrochloride, L-citrulline hydrochloride, L-ornithine hydrochloride and L-histidine hydrochloride.

Examples of the alcohol to be added in the step (k) include methanol, ethanol, n-propanol, and isopropanol, and for example, ethanol or methanol can be used. Ethanol is more preferable than methanol in terms of efficiency in salting out the diastereomer of compound A.

The compound A of the present invention can be synthesized using citric acid and L-aspartic acid and/or L-asparagine (most preferably L-aspartic acid) as starting materials. In this case, compound a was synthesized comprising a mixture of 2 diastereomers. The crystallization of compound a and the precipitation of the amorphous diastereomer salt of compound a can be continuously performed by the following steps. In the following steps, the conditions of the respective steps described above can be used.

(1) Under heating, citric acid monohydrate is reacted with L-aspartic acid and/or L-asparagine (most preferably L-aspartic acid) to produce compound A. The heating temperature and the reaction time may be determined in consideration of various conditions (e.g., 121 ℃,8 hours).

(2) After the reaction is completed, the reaction solution is left to cool, and methanol (for example, 60% methanol solution) is added under acidic conditions. Since the unreacted citric acid and L-aspartic acid among L-aspartic acid contained in the reaction solution precipitate, solid-liquid separation is performed by centrifugation or filtration. The isolated L-aspartic acid can be reused. In this step, L-aspartic acid can be removed by adding ethanol or isopropanol in addition to methanol, but methanol is most preferable in terms of solubility of calcium carbonate to be added later.

(3) to the obtained methanol solution containing compound a and citric acid, purified water was added for dilution (for example, 2-fold dilution), followed by addition of calcium carbonate powder (for example, 228mg per 1mL of the reaction solution). Thereby, the reaction solution was foamed, and calcium citrate was precipitated. The precipitated calcium citrate is subjected to solid-liquid separation by centrifugation or filtration. The reaction solution from which calcium citrate was removed was a methanol solution of compound a containing calcium ions.

(4) The reaction solution obtained in (3) was concentrated, methanol was distilled off, and purified water was added to make the volume of the initial reaction solution. When 1M sulfuric acid is added thereto to adjust the pH to 2.0 or less and 1.3 or more, calcium ions form calcium sulfate and precipitate. The precipitated calcium sulfate is subjected to solid-liquid separation by centrifugal separation or filtration. The reaction solution after the solid-liquid separation is a sulfuric acid acidic solution containing the compound a.

(5) When the sulfuric acid acidic solution is concentrated to, for example, about 2/5, crystals of the compound a (referred to as "crystallized compound a") are precipitated, and thus separated, and the remaining portion is used as a mother liquid of crystals.

(6) When the sulfuric acid-acidic crystal mother liquor is concentrated and acetone is added to form an acetone solution (acidic) (e.g., 80% acetone solution) of compound a, a viscous precipitate precipitates and then crystallizes (calcium citrate). The solid-liquid separation is carried out on the calcium citrate by centrifugal separation or filtration.

(7) The crystal mother liquor after solid-liquid separation was concentrated and subjected to dehydration treatment (acetone dehydration). Acetone is added to the crystal mother liquor after the dehydration treatment, and when the acetone in the solvent becomes, for example, 90% or more, the compound a is crystallized and precipitated. The crystallized compound a (referred to as "crystallized compound a") is subjected to solid-liquid separation by centrifugation or filtration.

(8) Purified water was added to the acetone solution of compound a and the acetone was distilled off. When calcium chloride and calcium hydroxide are added to an aqueous solution of compound a from which acetone has been distilled off to adjust the pH to, for example, 3.6 and ethanol (for example, a 70% aqueous ethanol solution) is then added, the calcium salt of the amorphous diastereomer of compound a precipitates. The precipitate (referred to as non-crystalline compound a (Ca salt))) was subjected to solid-liquid separation by centrifugation or filtration.

The crystallized compound a (crystal in aqueous solution) obtained in the step (5) and the crystallized compound a (crystal in acetone) obtained in the step (7) were stored by the following procedure, respectively.

Redissolved in water for recrystallization, purity evaluation by HPLC-based qualitative analysis, and dry storage

The amorphous compound a (Ca salt) obtained in the above step (8) was stored by the following procedure.

Redissolution in water concentration (removal of precipitated calcium citrate) addition of ethanol (e.g., 90 v/v% ethanol in water) to compound a in water solution followed by precipitation for purity evaluation by HPLC qualitative analysis dry storage

The obtained amorphous compound a (Ca salt) can be adjusted to sulfuric acid acidity to remove calcium at the time of use, and the concentration can be evaluated as compound a by qualitative and quantitative analysis based on HPLC.