JP4659111B2 - Method for producing 3-alkenylcephem compound - Google Patents

Description

  The present invention relates to 7-amino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof (hereinafter collectively referred to as “general name”). In addition, the present invention relates to a method for improving the content of the Z isomer and reducing impurities compared to the E isomer in the production of “alkenyl cephem compound”.

  7-Amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof is a substance useful as an intermediate for the production of cephalosporin antibiotics. is there. In this compound, there are two types of isomers, that is, the steric structure of the alkenyl group at the 3-position is a Z configuration and an E configuration. Among these two types of isomers, it is known that a cephalosporin antibiotic using the same as a raw material expresses an excellent antibacterial action as a pharmaceutical antibacterial agent. Accordingly, when synthesizing a cephalosporin antibiotic from 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof, It is important that only the Z body exists in the system and the E body does not exist as much as possible.

  From this viewpoint, in Patent Document 1, 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid in which a Z form and an E form are mixed or It has been proposed that a high porous polymer or activated carbon is allowed to act on the aqueous solution of the alkali metal salt to increase the content of the Z-form. Examples of the high porous polymer used in this method include acrylic resins, phenolic resins, and styrene resins. On the other hand, as the activated carbon, general activated carbon such as zinc chloride charcoal or steam charcoal is used.

  According to the method described in the above document, 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid having an increased Z-form content or The alkali metal salt is obtained. However, in order to use this compound as an intermediate for producing a cephalosporin antibiotic, it is desired to further improve the content of the Z isomer.

  The salt of 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid is 7-substituted acylamino-3- [2- (4-methyl). It is known to be obtained by subjecting a salt of thiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid to an enzymatic reaction to deprotect the 7-position amide bond. The compound obtained by this deprotection reaction contains phenylacetic acid or a derivative thereof as a by-product of the deprotection reaction as an impurity. When a compound containing these impurities is used as an intermediate for the production of cephalosporin antibiotics, there arises a problem of inhibiting the synthetic reaction of the antibiotics, and therefore it is necessary to reduce these impurities.

  From this point of view, in Patent Document 2, an organic solvent such as methyl isobutyl ketone is used to extract and remove phenylacetic acid or a derivative thereof as an impurity from the aqueous solution after the deprotection reaction, and the content thereof is 100 ppm. It has been proposed that:

JP 2005-343854 A JP 2002-316991 A

  Therefore, an object of the present invention is to provide 7-amino-3- [2- (4-methylthiazol-5-yl) vinyl]-having a high Z-form content and a low content of phenylacetic acid or a derivative thereof as an impurity. The object is to provide a process for producing 3-cephem-4-carboxylic acid or a salt thereof.

The present invention relates to a 7-substituted acylamino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid represented by the following formula (1). 7-amino represented by the following formula (3) containing phenylacetic acid or a derivative thereof, which is a by-product of the deprotection reaction: After obtaining an aqueous solution of -3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof, the deprotection reaction 7-Amino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3 represented by the following formula (3) containing the product phenylacetic acid or a derivative thereof: -For an aqueous solution of cephem-4-carboxylic acid or a salt thereof, using an organic solvent, It performs extraction processing of sulfonyl acid or a derivative thereof, then the following equation after the extraction process (3) represented by 7-amino -3 - [(E / Z) -2- (4- methylthiazole-5- Yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof in contact with activated carbon and then treated with 7-amino-3-[(Z ) -2- (4-Methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof 7-amino-3-[( E / Z) -2- (4-Methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof is provided to achieve the above object .

  According to the present invention, in the preparation of 7-amino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof, Compared to the body, the content of the Z body can be improved as compared with the conventional body, and impurities can be reduced.

  In the present invention, first, a 7-substituted acylamino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4 represented by the above formula (1) is used. -7-amino-3-[(E / Z) -2 represented by the above formula (3) by subjecting a salt of a carboxylic acid to an enzyme reaction to deprotect the amide protecting group at the 7-position. An aqueous solution of a salt of-(4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid is obtained. If this salt is water-soluble, there will be no restriction | limiting in particular in the kind. Examples of water-soluble salts include alkali metal salts and ammonium salts. Therefore, examples of the monovalent cation represented by M in the formula (1) include alkali metal ions such as sodium ions and ammonium ions.

  As the solvent for the enzyme reaction, water is preferably used from the viewpoint of maximizing enzyme activity. The pH of the enzyme reaction is a factor that affects the activity of the enzyme. Although depending on the type of enzyme, it is preferable to maintain the pH at 7.0 to 9.0, particularly 7.2 to 8.8 from this viewpoint. Various alkali aqueous solutions such as sodium hydroxide and potassium hydroxide; alkali metal hydrogen carbonates such as sodium bicarbonate; alkali metal carbonates such as sodium carbonate and potassium carbonate; ammonia An aqueous solution such as water can be used. The temperature of the enzyme reaction is also a factor that affects the activity of the enzyme. Although depending on the type of enzyme, it is preferable to maintain the temperature of the reaction system at 20 to 40 ° C., particularly 22 to 38 ° C. from this viewpoint. The reaction time is not critical in the present invention. In general, the reaction may be carried out until the compound represented by the formula (1) disappears from the reaction system. In general, the reaction time can be 1 to 3 hours, provided that the pH and temperature are within the above ranges.

  The concentration of the compound represented by the formula (1) contained in the solution to be added to the enzyme solution is not critical in the present invention, and may be a low concentration that does not cause crystals to precipitate. It can be in the range of% by weight.

  As the enzyme to be used, a conventionally known penicillin G acylase can be used without particular limitation. For example, penicillin-G amidase PGA-150, PGA-300, PGA-450 manufactured by Boehringer Mannheim; penicillin-G acylase manufactured by Dallas Biotech Limited; penicillin-G amidase manufactured by Roche Molecular Biochemicals; For example, IPA-750 manufactured by Fukurai Biotechnology Co., Ltd .; SynthaCLEC-PA manufactured by Atlas Biologics, Inc. can be used.

  The amount of the enzyme used is preferably 30 to 150 parts by weight, particularly 50 to 100 parts by weight, based on 100 parts by weight of the compound represented by the formula (1), although it depends on the type.

  The compound represented by the formula (1) can be synthesized by a known method. For example, a 7-substituted acylamino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid compound represented by the following formula (4) A compound represented by the formula (1) can be obtained by deprotecting the 4-position carboxylic acid protecting group. As a deprotection reaction, various methods known as a deprotection reaction of a carboxylic acid protecting group in a β-lactam compound can be employed. For example, the deprotection reaction in phenols described in JP-A No. 61-263984 can be employed.

In the formula (4), examples of the carboxylic acid protecting group represented by R ² include a benzyl group optionally substituted with an electron donating group, a diphenylmethyl group optionally substituted with an electron donating group, and the like. Is mentioned. Examples of the electron donating group include an alkyl group having 1 to 6 carbon atoms; a hydroxy group, and an alkoxy group having 1 to 6 carbon atoms.

  In the compound represented by the formula (1) contained in the solution before the enzyme reaction and the compound represented by the formula (3) contained in the solution after the enzyme reaction, There is no particular limitation, and this abundance ratio depends on the production conditions of the compound, etc., but it is generally expressed by the content calculated based on the calculation formula of the E content content used in the examples described later, and generally the E content content. Is 0.3 to 20%, particularly 2 to 12%. In view of the object of the present invention, it is desirable that the abundance ratio of the Z isomer is sufficiently higher than the abundance ratio of the E isomer. However, by using the production method of the present invention, the Z isomer can be obtained easily and with a high yield. It is possible.

  By the above enzymatic reaction, an aqueous solution of a salt of the compound represented by the formula (3) is obtained. In this enzymatic reaction, phenylacetic acid or a derivative thereof (hereinafter collectively referred to as “phenylacetic acids”) is a by-product by deprotection of the amide protecting group at the 7-position in the compound represented by formula (1). Generate as In the aqueous solution of the salt of the compound represented by the formula (3) obtained by the enzymatic reaction, phenylacetic acid is contained at a content calculated based on the formula for phenylacetic acid content used in Examples described later. Representing about 16 to 17%. This phenylacetic acid is an impurity for the alkenylcephem compound having a high content of Z form, which is an object of the present production method, and therefore it is necessary to eliminate its existence as much as possible.

Therefore, in the present invention, in order to reduce the content of phenylacetic acid or a derivative thereof, the following (A 2 ) is used for the aqueous salt solution of the compound represented by the formula (3) obtained by the enzyme reaction. The processing steps are performed. In addition, you may carry out combining the following (A) process and the following (B) process as needed.
(A) 7-amino-3-[(E / Z) -2- (4-methyl) represented by the following formula (3) containing phenylacetic acid or a derivative thereof, which is a by-product of the deprotection reaction. The process of performing the extraction process of the said phenylacetic acid or its derivative using the organic solvent with respect to the aqueous solution of thiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or its salt.
(B) 7-amino-3-[(E / Z) -2- (4-methyl) represented by the following formula (3) containing phenylacetic acid or a derivative thereof, which is a byproduct of the deprotection reaction. From an aqueous solution of thiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof, the 7-amino-3-[(E / Z) -2- (4-methylthiazol-5-yl) Vinyl] -3-cephem-4-carboxylic acid or a step of performing a crystallization treatment for precipitating the salt thereof.

Hereinafter, the processing step (A) will be described.
From the standpoint of reliably extracting and removing phenylacetic acids, the pH of the aqueous solution of the salt of the compound represented by the formula (3) obtained by the enzymatic reaction is set in an acidic range, specifically with a mineral acid prior to the extraction treatment. It is preferable to adjust the salt of the compound represented by the formula (3) in the aqueous solution to the form of the corresponding mineral acid salt by adjusting it to 2 or less, particularly 1 or less. Examples of the mineral acid include hydrochloric acid, nitric acid, sulfuric acid and the like.

Examples of the organic solvent used for the extraction treatment include (a) lower alkyl esters of lower carboxylic acids, (b) ketones, (c) ethers, (d) substituted or unsubstituted aromatic hydrocarbons, ) Halogenated hydrocarbons, (f) aliphatic hydrocarbons, and (g) cycloalkanes. These organic solvents can be used alone or in combination of two or more. Examples of the lower alkyl esters of the lower carboxylic acid (i) include methyl formate, ethyl formate, propyl formate, butyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, methyl propionate, and ethyl propionate. . Examples of (b) ketones include methyl propyl ketone, methyl butyl ketone, methyl isobutyl ketone, and diethyl ketone. Examples of (iii) ethers include diethyl ether, ethyl propyl ether, ethyl butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, methyl cellosolve, dimethoxyethane and the like. Examples of the substituted or unsubstituted aromatic hydrocarbons of (d) include benzene, toluene, xylene, chlorobenzene, anisole and the like. Examples of (e) halogenated hydrocarbons include dichloromethane, chloroform, dichloroethane, trichloroethane, dibromoethane, propylene dichloride, carbon tetrachloride, and the like. Examples of (f) aliphatic hydrocarbons include pentane, hexane, heptane, and octane. Examples of (g) cycloalkanes include cyclopentane, cyclohexane, cycloheptane, cyclooctane and the like.

  Among these organic solvents, those having a solubility in water at 20 ° C. of 1% by weight or less, specifically, toluene, chloroform, chlorobenzene and the like are preferable, and toluene is particularly preferable.

  When an organic solvent having high polarity and high solubility in water is used, the organic solvent is dissolved in the aqueous solution of the alkenyl cephem compound to be extracted. If an aqueous solution of an alkenylcephem compound in which an organic solvent is dissolved is subjected to treatment with activated carbon, which will be described in detail later, the activated carbon will adsorb the organic solvent, so the adsorption removal efficiency of the E-form is reduced and the purity of the Z-form is improved. It becomes difficult to make it. Therefore, when an organic solvent having high solubility in water is used, a concentration step is required to concentrate the aqueous solution after the extraction treatment and remove the organic solvent from the aqueous solution before performing the treatment with activated carbon. Use of an organic solvent having a low solubility of 1% by weight or less at 20 ° C. in water is industrially advantageous because a concentration step is unnecessary.

  These organic solvents are preferably used in an amount of 5 to 50 liters, more preferably 10 to 30 liters per kg of the alkenyl cephem compound in the aqueous solution. Moreover, it is preferable to perform an extraction process at 0-20 degreeC. If it is this preferable ratio, the content rate of phenylacetic acids can be reduced efficiently in an extraction process.

  In the production method of the present invention, after the extraction treatment, phenylacetic acids are represented by the content calculated based on the formula for calculating the phenylacetic acid content used in the examples described later, and reduced to 8% or less. It is preferable to make it. By repeating the extraction process a plurality of times, the content of phenylacetic acid gradually decreases. Therefore, if the content of phenylacetic acid does not become 8% or less in one extraction process, the solvent extraction may be performed a plurality of times. preferable.

Hereinafter, the processing step (B) will be described.
From the viewpoint of precipitating the compound represented by the formula (3) and recovering it with high yield, the pH of the aqueous solution of the salt of the compound represented by the formula (3) obtained by the enzyme reaction is weakly acidic with a mineral acid. The salt of the compound represented by the formula (3) in an aqueous solution as a free acid form by adjusting to a range, specifically 3.5 to 4.8, particularly 3.5 to 4.5, Make it easy to deposit. In addition, pH adjustment can be performed by adding mineral acids, such as hydrochloric acid, nitric acid, and a sulfuric acid, to this aqueous solution.
Subsequently, the compound represented by the formula (3) in the aqueous solution can be precipitated by maintaining the aqueous solution at preferably 20 ° C. or lower, more preferably 1 to 10 ° C. The crystallization treatment may be performed under stirring or standing. The compound represented by formula (3), which is a precipitate, is solid-liquid separated by a conventional method and recovered from a treatment liquid containing phenylacetic acids.

  In the production method of the present invention, after the crystallization treatment, the phenylacetic acids are represented by the content calculated based on the calculation formula for the content of phenylacetic acid used in the examples described later, and are up to 8% or less. It is preferable to reduce. The crystallization treatment has higher separation and removal efficiency of phenylacetic acids than the extraction treatment of the above (A). It is possible to reduce the content of phenylacetic acids to 2% or less by the deposition process. When the content of phenylacetic acid is not 8% or less, preferably 2% or less in one crystallization treatment, it is desirable to perform the crystallization treatment a plurality of times.

  If necessary, the processing step (A) and the processing step (B) may be combined. In that case, which processing step is performed first is not particularly limited.

  Next, 7-amino-3-[(E / Z) -2- (4-methylthiazole) represented by formula (3) after completion of the treatment step (A) or the treatment step (B). -5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof (alkenylcephem compound) is brought into contact with activated carbon to adsorb and remove the E form of the alkenylcephem compound contained in the aqueous solution. Increase the content of Z-form. When the treatment step (A) is performed, the aqueous solution that has been extracted with the organic solvent can be directly brought into contact with activated carbon. When the treatment step (B) is performed, the precipitate (compound represented by formula (3)) obtained in the treatment step is dissolved in water to form an aqueous solution, and the aqueous solution is brought into contact with activated carbon. In any case, the concentration of the alkenyl cephem compound in the aqueous solution brought into contact with the activated carbon is preferably 0.1 to 5% by weight, more preferably 0.5 to 4% by weight from the viewpoint of treatment efficiency.

  The pH of the aqueous solution brought into contact with the activated carbon is set to a weakly alkaline region of 7.1 to 9.0 or an acidic region of pH 2.0 or less, particularly 0.8 to 1.4. It is preferable from the viewpoint of efficient activated carbon treatment. In particular, in the acidic region of pH 2.0 or less, particularly 0.8 to 1.4, phenylacetic acids that could not be removed by the treatment step (A) or (B) are simultaneously adsorbed and removed by the activated carbon treatment. Therefore, it is preferable from the viewpoint of obtaining a target product in which phenylacetic acids are further reduced. For adjusting the pH, aqueous ammonia, amines, the above-mentioned mineral acids, and the like can be used. Examples of amines include trimethylamine and triethylamine.

  There is no restriction | limiting in particular in the method of making activated carbon and the alkenyl cephem compound in aqueous solution contact. For example, a method of adding activated carbon to an aqueous solution of an alkenyl cephem compound, or a method of adding an aqueous solution of an alkenyl cephem compound to the activated carbon can be employed. Alternatively, the activated carbon is packed in a column, and the aqueous solution of the alkenyl cephem compound is fed to the column with a pump, passed through the column, and further circulated through the column a plurality of times, or the molded body such as a filter contains activated carbon. A method in which an aqueous solution of an alkenyl cephem compound is brought into contact with the alkenyl cephem compound may be employed. Although there is no restriction | limiting in particular in the ratio of the quantity of activated carbon and the quantity of an alkenyl cephem compound, For example, activated carbon is 10-200 weight part with respect to 100 weight part of alkenyl cephem compounds contained in aqueous solution, Especially 20-100 weight part. It is preferable to make it contact from the point which can reduce the loss rate of Z body. Moreover, if it is this preferable ratio, the content rate of the phenyl acetic acid which remained after the process process of the above-mentioned (A) or (B) can also be further reduced efficiently in activated carbon treatment.

  When using activated carbon, the activated carbon may be added to an aqueous solution of the alkenyl cephem compound in multiple portions. By doing so, E body content can be reduced further and Z body content can be raised. In addition, when all of the activated carbon to be used is added at once, there is a risk of dust explosion and workability problems, but these problems can be solved by adding them in portions.

  There are no particular restrictions on the conditions for contacting the activated carbon with the alkenyl cephem compound. For example, the temperature at the time of contact can be 0-20 degreeC. By making the temperature at the time of contact within this range, the loss rate of the Z body can be reduced and the E body can be efficiently removed, which is preferable. The contact time is preferably 0.5 to 3 hours, particularly 0.5 to 2 hours, provided that the temperature at the time of contact is in the above range. While the two are in contact with each other, the reaction system may be in a stirred state or may be left in a stationary state.

  The treatment with activated carbon may be performed only once, or may be repeated two or more times for the purpose of increasing the purity of the Z form.

  As the activated carbon, for example, zinc chloride activated activated carbon or water vapor activated activated carbon made of coconut shell, coal, wood material or the like can be used. These can be used alone or in combination of two or more.

  The present inventors diligently investigated activated carbon for selectively adsorbing and removing E form from an alkenyl cephem compound, and it was particularly effective to use activated carbon having a large pore diameter peak and a small pore diameter peak. There was found. Furthermore, as the inventors proceeded with investigations, activated carbon having such a pore size distribution was identified with iodine adsorption performance measured according to JIS K-1474 and methylene blue adsorption performance measured according to JIS K-1474. It was found to be within the range. In the present invention, by using activated carbon having such specific iodine adsorption performance and methylene blue adsorption performance, it is possible to more selectively adsorb and remove E form from the alkenyl cephem compound.

  About the specific iodine adsorption | suction performance mentioned above, it is preferable to use that whose value is 1200 mg / g or more. In addition, since it is extremely difficult to industrially obtain activated carbon having an iodine adsorption performance exceeding 1700 mg / g and having the methylene blue adsorption performance described below, the upper limit of the iodine adsorption performance of the activated carbon used in the present invention is 1700 mg / g. g. Therefore, the range of iodine adsorption performance is preferably 1200 to 1700 mg / g, and more preferably 1400 to 1700 mg / g. However, the higher the value of iodine adsorption performance, the better. Therefore, there is no problem in using activated carbon having an iodine adsorption performance of more than 1700 mg / g.

  About methylene blue adsorption | suction performance, it is preferable to use that whose value is 250 ml / g or more. In addition, since the methylene blue adsorption performance is over 500 ml / g and it is extremely difficult to industrially obtain activated carbon having the iodine adsorption performance described above, the upper limit of the methylene blue adsorption performance of the activated carbon used in the present invention is 500 ml / g. And Therefore, the range of methylene blue adsorption performance is preferably 250 to 500 ml / g, more preferably 260 to 500 ml / g. However, the higher the value of the methylene blue adsorption performance, the better. Therefore, there is no problem in using activated carbon having a methylene blue adsorption performance of more than 500 ml / g.

  Normally, the physical properties of activated carbon used in water treatment have iodine adsorption performance of 1200 mg / g or less and methylene blue adsorption performance of 200 ml / g or less (“Applied technology of activated carbon”, supervised by Hideki Tachimoto, Kunio Abe. , Issued by Techno System Co., Ltd., July 25, 2000, pages 409 and 555), these physical properties of the activated carbon used in the present invention are much higher than those of ordinary activated carbon. It is expensive. This is because large pores and small pores are distributed. In general, iodine adsorption performance is an indicator of small pore distribution (that is, an index of adsorptivity of a compound having a low molecular weight), and methylene blue adsorption performance is an indicator of distribution of a large pore (that is, an adsorptivity of a compound having a large molecular weight). Index).

  Examples of the activated carbon that satisfies the iodine adsorption performance and methylene blue adsorption performance described above include water vapor activated activated carbon using coconut shell, coal, wood material, or the like as a raw material. In this case, the above-described physical property values are satisfied by appropriately controlling the activation conditions and appropriately controlling the granulation conditions. The activated carbon may be in the form of powder, granules or fibers, or may be a molded body. Commercially available products can be used as the activated carbon satisfying the above physical property values. Examples of such commercially available products include unitica activated carbon fiber Adol A-20 (trade name), which is activated carbon available from Unitika Ltd., and liquid phase activated carbon CL-KP (product), which is activated carbon available from Ajinomoto Fine Techno. Name).

  By the above operation, the E form is selectively adsorbed and removed from the alkenyl cephem compound including the Z form and the E form by activated carbon, and the content of the Z form is increased. Thereafter, the activated carbon and the treatment liquid are separated. An alkali such as sodium hydroxide is added to the treatment liquid from which the activated carbon has been separated to adjust the pH of the liquid to a weakly acidic region of 3.8 to 4.8, preferably 0.5 to 3 hours, more preferably 0.5. By aging for ˜1.5 hours, crystals of the compound represented by formula (2) can be precipitated. The obtained crystals are separated by filtration or centrifugation, and washed with water and an organic solvent such as methanol. By adjusting the pH of the treatment liquid to the above range and precipitating the compound represented by the formula (2) in the pH within the range, the target product can be recovered with high purity and high yield.

  As described above in detail, in the present invention, an aqueous solution of an alkenylcephem compound obtained by an enzyme reaction is treated with activated carbon after the treatment step (A) or (B). In the case of carrying out the treatment step (solvent extraction treatment) of (A), in order to achieve the same Z-body content compared with the treatment step of (A) after treating the aqueous solution with activated carbon. Only half the amount of activated carbon is required, which is extremely advantageous from an industrial point of view. This is considered to be because when activated carbon is treated before the solvent extraction treatment, the activated carbon adsorbs not only E-form but also phenylacetic acid, and the E-form adsorption removal efficiency decreases.

  Also in the case of performing the treatment step (crystallization treatment) of (B), phenylacetic acids can be separated and removed by crystallization, as in the case of the treatment step of (A) described above. The amount of activated carbon used for the activated carbon treatment can be reduced. The treatment step (crystallization treatment) of (B) has a higher separation and removal efficiency of phenylacetic acids than the treatment step (solvent extraction treatment) of (A), so the amount of activated carbon used is greatly reduced. can do. When the treatment step (A) is employed, the organic solvent remains in the aqueous solution to be subjected to the activated carbon treatment, but the activated carbon adsorbs the organic solvent. The adsorbing and removing efficiency decreases. On the other hand, when the processing step (B) is employed, such a decrease in the adsorption removal efficiency of the E body does not occur. This point also contributes to a reduction in the amount of activated carbon used when the processing step (B) is adopted.

  Furthermore, although the compound represented by the formula (2) obtained by the conventional method is colored, according to the present invention, since the coloring component is also removed by the extraction treatment or the crystallization treatment, it is finally obtained. The coloring of the object can also be reduced.

  The present invention has been described above based on the preferred embodiments. However, the present invention is not limited to the above-described embodiments, and appropriate modifications within the scope of ordinary creation ability of those skilled in the art belong to the scope of the present invention. It is.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited to such examples. Of the following Examples 1 to 9, Examples 1 to 4 are examples of the present invention, and Examples 5 to 9 are outside the scope of the present invention (reference examples).

Prior to describing the examples and comparative examples, the analysis method used will be described. High performance liquid chromatography (HPLC) was used for the analysis. The details are as follows.
Column: Unison UK-C18, 3 μm, 250 mm × 4.6 mm
-Column temperature: 30 ° C
Mobile phase (volume ratio): acetonitrile 13%, 10 mM sodium heptanesulfonate aqueous solution 87%
・ Flow rate: 0.8ml / min
・ Detection wavelength: 254 nm
・ Injection volume: 10 μl
-Z body retention time: 29.0-30.0 minutes-E body retention time: 31.0-32.0 minutes-E body content (calculation formula): [E body area value / (Z body area value + E Body area value)] x 100 (%)

The method for analyzing the content of phenylacetic acid is as follows.
Column: SUPELCO ODS HYPERSIL 5 μm 250 × 4.6 mm
-Column temperature: 25 ° C
Mobile phase (volume ratio): acetonitrile 20%, 50 mM potassium dihydrogen phosphate aqueous solution 80%
・ Flow rate: 1.0ml / min
・ Detection wavelength: 225 nm
・ Injection volume: 10 μl
-Z-form + E-form retention time: 2.5-3.5 minutes- Phenylacetic acid retention time: 8.5-9.5 minutes- Phenylacetic acid content (calculation formula):
[Phenylacetic acid area value / ((Z + E) body area value + phenylacetic acid area value)] × 100 (%)

[Example 1]
(1) First Step A compound represented by the following formula (5) (content of E-form: 3.5%) is weighed into a 10.0 g four-necked flask, and 240 g of a 6% by weight aqueous sodium hydrogen carbonate solution is added to add sodium. An aqueous salt solution was obtained. To this aqueous solution was added 7.0 g of penicillin-G acylase enzyme (PGA-450, manufactured by Dalas Biotech Limited). The 7-position deprotection reaction of the sodium salt of the compound represented by the formula (5) was performed while adding a 5% by weight sodium carbonate aqueous solution at a liquid temperature of 25 to 35 ° C and controlling the pH to 7.5 to 8.5 It went for 2 hours. After completion of the reaction, the aqueous solution contained 7.0 g of a sodium salt of a compound represented by the following formula (6) containing 3.5% of E form in terms of E form content. Further, phenylacetic acid was contained at 16.6% in terms of phenylacetic acid content.

(2) Second Step The enzyme (PGA-450) is filtered from the aqueous solution obtained in the first step, and concentrated hydrochloric acid is added while keeping the liquid temperature at 0 to 10 ° C. to adjust the pH of the aqueous solution to 0.9. The sodium salt of the compound represented by formula (6) contained in the aqueous solution was used as the hydrochloride of the compound represented by formula (6). The aqueous solution whose pH was adjusted was transferred to a separatory funnel, and 150 ml of toluene was added thereto to extract and remove by-products and impurities while maintaining the liquid temperature at 20 ° C. The phenylacetic acid content after the extraction treatment was 6.1%. The concentration of the alkenyl cephem compound in the aqueous solution after the extraction treatment was 2.2% by weight.

(3) Third Step 3.2 g of activated carbon (Ajinomoto Fine Techno Co., Ltd., trade name SD-2) was added all at once to the aqueous solution after solvent extraction, and the mixture was stirred at 3 ° C. for 1 hour. This activated carbon had an iodine adsorption performance measured in accordance with JIS K-1474 of 1080 mg / g and a methylene blue adsorption performance of 180 ml / g. Thereafter, the activated carbon was filtered off, and a 1N aqueous sodium hydroxide solution was added to the aqueous solution to adjust the pH to 4.3, followed by aging for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. The analysis result of the obtained crystal was as follows. In addition, the Z body yield in the following analysis result is the value computed by the following formulas.
Z body yield (%) = A × B / C
A: Crude yield (%) of crystals obtained after the third step
B: Purity (%) of the compound represented by formula (3) as Z-form
C: Theoretical yield (%) of the Z form of the compound represented by the formula (3) based on the mineral acid salt of the compound represented by the formula (6) in the second step
(result of analysis)
-Z body yield: 90.3%
-E body content: 1.65%
・ Phenylacetic acid content: 0.1%
-Color tone (visually): White- ¹ H-NMR (D ₂ O / DCl) ppm from TSP
2.52 (s, 3H, CH ₃ ),
3.56 to 3.60 (d, 1H, S-CH (H), 18.3 Hz),
3.75 to 3.78 (d, 1H, S-CH (H), 18.6 Hz),
5.25-5.26 (d, 1H, S-CH, 5.2 Hz),
5.44-5.45 (d, 1H, N-CH, 5.2 Hz),
6.78 (s, 2H, CH = CH), 9.78 (s, 1H, S-CH = N)

[Comparative Example 1]
The enzyme reaction was carried out in the same manner as in the first step of Example 1, and the enzyme was filtered off from the obtained aqueous solution containing the sodium salt of the compound represented by formula (6). The total amount of the filtrate was adjusted to pH 0.9 with concentrated hydrochloric acid while keeping the liquid temperature at 20 ° C. This obtained the aqueous solution of hydrochloride of the compound represented by Formula (6). Next, 5.6 g of the same activated carbon as that used in the third step of Example 1 was added to the aqueous solution in a lump and stirred at 3 ° C. for 1 hour. The phenylacetic acid content after the activated carbon treatment was 1.1%. Thereafter, the activated carbon was filtered off, and solvent extraction was performed on the obtained filtrate in the same manner as in the second step of Example 1. A 1N sodium hydroxide aqueous solution was added to the aqueous solution after the solvent extraction to adjust the pH to 4.3, followed by aging for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. The analysis result of the obtained crystal was as follows.
(result of analysis)
-Z body yield: 86.5%
-E body content: 1.46%
・ Phenylacetic acid content: 0.1%
・ Color (visual): pale yellow

[Example 2]
In the third step of Example 1, a compound represented by the formula (3) is obtained in the same manner as in Example 1 except that 2.8 g of CL-KP (trade name) manufactured by Ajinomoto Fine Techno is used as the activated carbon. Crystal was obtained. The activated carbon had an iodine adsorption performance of 1620 mg / g and a methylene blue adsorption performance of 280 ml / g. The analysis results of the obtained crystals are shown in Table 1.

Example 3
In the second step of Example 2, crystals of the compound represented by the formula (3) were obtained in the same manner as in Example 2 except that chloroform was used instead of toluene. The analysis results of the obtained crystals are shown in Table 1.

Example 4
(1) 1st process The enzyme reaction was performed on the same operation and conditions as Example 1. After completion of the reaction, the aqueous solution contained 7.0 g of the sodium salt of the compound represented by the formula (6) containing 3.5% of E form in terms of E form content. Further, phenylacetic acid was contained at 16.6% in terms of phenylacetic acid content.
(2) Second step The enzyme is filtered off from the aqueous solution obtained in the first step, and concentrated hydrochloric acid is added while keeping the filtrate at 10 ° C to adjust the pH of the aqueous solution to 1.1. The sodium salt of the compound represented by formula (6) was used as the hydrochloride of the compound represented by formula (6). The aqueous solution whose pH was adjusted was transferred to a separatory funnel, and 150 ml of toluene was added thereto to extract and remove by-products and impurities while maintaining the liquid temperature at 10 ° C. The phenylacetic acid content after the extraction treatment was 5.9%. The concentration of the alkenyl cephem compound in the aqueous solution after the extraction treatment was 2.3% by weight.
(3) Third Step The aqueous solution after the extraction treatment was adjusted to pH 8.0 with concentrated aqueous ammonia while keeping the aqueous solution at 10 ° C. To this aqueous solution, 1.5 g of CL-KP (trade name) manufactured by Ajinomoto Fine-Techno Co., Ltd. was added as activated carbon in a lump and stirred at 5 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, concentrated hydrochloric acid was added to the aqueous solution to adjust the pH to 4.3, and the mixture was aged for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. The analysis results of the obtained crystals are shown in Table 1.

  In Example 1, the activated carbon treatment was performed after the extraction treatment, while in Comparative Example 1, the extraction treatment was conducted after the activated carbon treatment. In Example 1 and Comparative Example 1, the phenylacetic acid content in the crystals of the finally obtained alkenyl cephem compound is the same at 0.1%. However, Example 1 is compared with Comparative Example 1. The amount of activated carbon required to achieve the same phenylacetic acid content was only about half, and the Z-form yield was improved. Moreover, in Example 1, the obtained alkenyl cephem compound was not colored.

  Furthermore, from the comparison between Example 1 and Example 2, it can be seen that the use of activated carbon having specific iodine adsorption performance and methylene blue adsorption performance can further improve the Z-form yield and E-form content. In addition, it can be seen from the comparison between Example 2 and Example 3 that the use of toluene in the extraction treatment can particularly improve the Z-form yield and the E-form content. Further, from the comparison between Example 2 and Example 4, when the pH of the aqueous solution used for the activated carbon treatment is in the alkaline region, a good Z body yield and E body content can be achieved with a small amount of activated carbon used. It can be seen that the acetic acid content is slightly higher.

Example 5
(1) 1st process The enzyme reaction was performed on the same operation and conditions as Example 1. After completion of the reaction, the aqueous solution contained 7.0 g of the sodium salt of the compound represented by the formula (6) containing 3.5% of E form in terms of E form content. Further, phenylacetic acid was contained at 16.6% in terms of phenylacetic acid content.
(2) Second Step The enzyme (PGA-450) was filtered off from the aqueous solution obtained in the first step, adjusted to pH 4.2 with concentrated hydrochloric acid while keeping the liquid temperature at 10 ° C., and aged for 1 hour as it was. The compound represented by the formula (3) was precipitated by this aging, and then filtered to collect the precipitate. In addition, the phenylacetic acid content rate of the obtained deposit was 0.5%.
(3) Third step 7.1 g (containing 6.5 g of Z body) of the precipitate obtained in the second step was dispersed in 340 g of water, adjusted to pH 1.0 with concentrated sulfuric acid while maintaining at 20 ° C., The precipitate was dissolved. To this aqueous solution, 1.5 g of CL-KP (trade name, iodine adsorption performance 1620 mg / g, methylene blue adsorption performance 280 ml / g) manufactured by Ajinomoto Fine-Techno was added as activated carbon in a lump and stirred at 3 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, and a 1N aqueous sodium hydroxide solution was added to the aqueous solution to adjust the pH to 4.2, followed by aging for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. Table 2 shows the analysis results of the obtained crystals.

Example 6
(1) 1st process The enzyme reaction was performed on the same operation and conditions as Example 1. After completion of the reaction, the aqueous solution contained 7.0 g of the sodium salt of the compound represented by the formula (6) containing 3.5% of E form in terms of E form content. Further, phenylacetic acid was contained at 16.6% in terms of phenylacetic acid content.
(2) Second step The enzyme was filtered off from the aqueous solution obtained in the first step, adjusted to pH 4.3 with 15 wt% sulfuric acid while keeping the filtrate at 10 ° C, and aged for 1 hour as it was. The compound represented by the formula (3) was precipitated by this aging, and then filtered to collect the precipitate. In addition, the phenylacetic acid content rate of the obtained deposit was 0.5%.
(3) Third step 7.1 g (containing 6.5 g of Z-form) obtained in the second step is dispersed in 340 g of water, and adjusted to pH 1.2 with 15% by weight sulfuric acid while maintaining at 10 ° C. The precipitate was dissolved. To this aqueous solution, 1.5 g of CL-KP (trade name) manufactured by Ajinomoto Fine Techno Co. was added as activated carbon in a lump and stirred at 3 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, and a 1N aqueous sodium hydroxide solution was added to the aqueous solution to adjust the pH to 4.3, followed by aging for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. Table 2 shows the analysis results of the obtained crystals.

Example 7
A crystal of the compound represented by formula (3) was obtained in the same manner as in Example 5 except that the third step was as follows.
Disperse 7.1 g (containing 6.5 g of Z-form) of the precipitate obtained in the second step in 340 g of water, and adjust the pH to 8.1 with 5 wt% aqueous ammonia while maintaining the temperature at 10 ° C. Dissolved. To this aqueous solution, 1.5 g of CL-KP (trade name) manufactured by Ajinomoto Fine-Techno Co., Ltd. was added as activated charcoal and stirred at 7 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, concentrated hydrochloric acid was added to the aqueous solution to adjust the pH to 4.3, and the mixture was aged for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. Table 2 shows the analysis results of the obtained crystals.

Example 8
A crystal of the compound represented by formula (3) was obtained in the same manner as in Example 5 except that the third step was as follows.
In 340 g of water, 7.1 g of the precipitate obtained in the second step (containing 6.5 g of Z-form) was dispersed, adjusted to pH 1.1 with concentrated hydrochloric acid while maintaining the temperature at 10 ° C., and the precipitate was dissolved. To this aqueous solution, 1.8 g of Adol A-20 (trade name, iodine adsorption performance 1580 mg / g, methylene blue adsorption performance 310 ml / g) manufactured by Unitika Ltd. was added as activated carbon, and the mixture was allowed to stand at 8 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, 1N aqueous sodium hydroxide solution was added to adjust the pH to 4.3, and the mixture was aged for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. Table 2 shows the analysis results of the obtained crystals.

Example 9
A crystal of the compound represented by formula (3) was obtained in the same manner as in Example 5 except that the third step was as follows.
In 340 g of water, 7.1 g of the precipitate obtained in the second step (containing 6.5 g of Z-form) was dispersed, adjusted to pH 1.1 with concentrated hydrochloric acid while maintaining the temperature at 10 ° C., and the precipitate was dissolved. To this aqueous solution, 1.6 g of SD-2 (trade name) manufactured by Ajinomoto Fine-Techno Co., Ltd. as activated carbon was added all at once and stirred at 4 ° C. for 1 hour. Thereafter, the activated carbon was filtered off, concentrated hydrochloric acid was added to the aqueous solution to adjust the pH to 4.3, and the mixture was aged for 1 hour. By this aging, crystals of the compound represented by the formula (3) were precipitated. The precipitated crystals were collected by filtration, washed with water and methanol, and dried. Table 2 shows the analysis results of the obtained crystals.

As is clear from the comparison between Table 1 and Table 2, the crystallization treatment has a greater reduction in phenylacetic acid content than the extraction treatment, and as a result, when crystallization treatment is performed, a small amount of activated carbon is used. An alkenyl cephem compound having a low E-form content and a low phenylacetic acid content can be obtained with a good Z-form yield.
From the comparison between Examples 5, 6, 8 and Example 9, when using activated carbon having specific iodine adsorption performance and methylene blue adsorption performance in the case of crystallization treatment and extraction treatment, Z-form yield And it turns out that E body content rate can be improved further. Further, from the comparison between Examples 5 and 6 and Example 7, when the pH of the aqueous solution subjected to the activated carbon treatment is in the acidic region, the phenylacetic acid content can be further reduced, and the Z-form yield and the E-form content are also obtained. It turns out that it becomes still more favorable.

Claims (5)

A salt of 7-substituted acylamino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid represented by the following formula (1) is enzyme A 7-amide bond is deprotected by subjecting to the reaction, and 7-amino-3- [7] represented by the following formula (3) containing phenylacetic acid or a derivative thereof as a by-product of the deprotection reaction. After obtaining an aqueous solution of (E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof,
7-amino-3-[(E / Z) -2- (4-methylthiazole-5 represented by the following formula (3) containing phenylacetic acid or a derivative thereof as a by-product of the deprotection reaction. -Yl) vinyl] -3-cephem-4-carboxylic acid or an aqueous solution thereof, an organic solvent is used to extract the phenylacetic acid or a derivative thereof ,
Then, 7-amino-3 represented by the following formula after the extraction process (3) - [(E / Z) -2- (4- methylthiazol-5-yl) vinyl] -3-cephem-4 A 7-amino-3-[(Z) -2- (4-methylthiazole-5) represented by the following formula (2), which is obtained by treating an aqueous solution of a carboxylic acid or a salt thereof with activated carbon. 7-amino-3-[(E / Z) -2- (4-methyl) represented by the formula (3) with an improved content of -yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof Thiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid or a salt thereof .

The production method according to claim 1, wherein the organic solvent in the extraction treatment is toluene.   The method according to claim 1 or 2, wherein the activated carbon has an iodine adsorption performance measured according to JIS K-1474 of 1200 mg / g or more and a methylene blue adsorption performance of 250 ml / g or more. After carrying out the deprotection reaction, the pH is adjusted to the acidic range by addition of mineral acid, and 7 represented by the formula (3) containing phenylacetic acid or a derivative thereof as a by-product of the deprotection reaction. 4. An aqueous solution of a mineral acid salt of -amino-3-[(E / Z) -2- (4-methylthiazol-5-yl) vinyl] -3-cephem-4-carboxylic acid. the process according to one paragraph or. Adjust the pH of the treatment solution after treatment with the activated carbon 3.8 to 4.8, the formula in any one of claims 1 to 4 to precipitate the crystals of the compound represented by formula (2) The manufacturing method as described.