JPS5915635B2 - Method for producing 7-(4-carboxybutanamide)-3-cephem-4-carboxylic acid compound - Google Patents

Description

Detailed Description of the Invention The present invention provides 7-(4-carboxybutanamide)-3-cephem- This relates to a method for producing a 4-carboxylic acid compound.

Cephalosporin C (CeC) is converted to 7-aminocephalosporanic acid (7-, ACA), which is then converted into 7-acylamide analogues of CeC with excellent antibacterial activity, such as cephalothin, cephaloridine, cephaloglycin, cefazolin, etc. can be converted to .

However, 7-AC due to N-hypothesis acylation of CeC
Many chemical methods have been disclosed for producing A, but biochemical methods are said to be extremely difficult due to the structure E, in which the side chain is a D-5 amino-5-carboxypentanoyl group. .

Recently, a compound represented by the general formula (wherein X represents the same group as above) or a salt thereof E under aerobic conditions has been developed. - act on bacterial cells having amino acid oxidase activity or processed products thereof,
And when R of the target substance is -COCOOH' group, l
This is a general formula (wherein R is -COOH group or -COCOOH group,
A method for producing a cephalosporin compound represented by (X represents the same group as described above) (Belgium Patent No. 736943, Japanese Patent Application Laid-Open No. 1983-39595) has been disclosed.

According to the above method, since the bacterial cells having the D-amino acid oxidase activity or the treated product E catalase are present, R of formula (1) is determined by the presence or absence of inhibition of the catalase.
is -COOH group, 7-(4-carboxybutanamido)-3-cephem-4-carboxylic acid derivative [I] (hereinafter referred to as compound [I]) or R in formula (1) is -COC
A 7-(5-carboxy-5-oxopentanamido)-3-cephem-4-carboxylic acid derivative, which is an 0OH group, is produced.

However, since the former compound is more stable than the latter compound, it is operationally preferable to aim for the production of the former compound CI).

In that case, catalase is inactivated and the D-amino acid oxidase reaction is carried out.

As a method for inactivating catalase, catalase inhibitors such as ascorbic acid, 3-amino-1,2,3-
) A method using lyazole, an alkali metal azide, especially sodium azide, or a method of deactivation by heat treatment is disclosed.

However, when using sodium azide, when separating and purifying the target compound CI from the reaction solution, the reaction solution is acidified (and then extracted with a non-hydrophilic organic solvent; Sodium azide decomposes and generates highly poisonous hydrogen azide gas, which not only poses a great danger to the human body during work, but also poses a problem that is extremely unsuitable for industrial use, as wastewater is not suitable.

In addition, in the case of the method of inactivating by heat treatment, D-amino acid oxidase is also inactivated under conditions that completely inactivate catalase, and catalase is not inactivated under conditions that do not inactivate D-amino acid oxidase. Compounds CI) and R
G) is not preferable as a method for obtaining only compound CI') at a high rate because it does not produce compound CI) in which is a -COCOOH group.

Therefore, in order to solve the above-mentioned drawbacks, the present inventors searched for various substances that inhibit catalase activity, have no industrial risk, and do not inhibit the D-amino acid oxidase reaction up to compound CI). As a result, it was found that perborate can be used extremely safely and effectively, and that compound (1) can be produced at a high rate (Japanese Patent Application No. 50-14550).

As a result of further research, the present inventors discovered that instead of adding an inhibitor of catalase activity, they added hydrogen peroxide in an amount that would not be decomposed by the action of catalase, thereby increasing the D-amino acid oxidase reaction. It has been found that extremely safe compound (1) can be produced at high efficiency without causing any hindrance.

The present invention was completed based on the above findings,
Compound represented by general formula (II) (hereinafter compound (II)
) or a salt thereof under aerobic conditions to produce a compound CI) by reacting with a D-amino acid oxidase-producing bacterium belonging to the genus Aspergillus, genus Fusarium, genus Cephalosporium, or Trigonopsis parabilis, or a treated product thereof. A method for producing compound CI,l, characterized in that the reaction is carried out in the presence of hydrogen peroxide, the objective of which is a method for producing compound [I] from compound (n). In this method, hydrogen peroxide, which is industrially non-hazardous, can be safely used, and does not inhibit D-amino acid oxidase activity, is used to produce compound [I] with high efficiency.
The method of manufacturing is to sacrifice a corpse.

As the D-amino acid oxidase-producing bacteria used in the present invention E, microorganisms belonging to the genus Aspergillus, Fusarium, Cephalosporium, or Trigonopsis parabilis are appropriately used.

Such microorganisms can be selected from type cultures stored in strain archives or isolated from nature.

In addition, mutant strains obtained from the above-mentioned microorganisms by conventional means to increase the production activity of the target compound [■] can also be advantageously used in the present invention. Ustas IM116045 (Aspe
rgillus ustus ) Aspergillus flavus IM I 52104 ( Aspergillus
s flavus ) Aspergillus flavus oryzae IMI44 ( Aspergillus fla
Vus-oryzae) 242 Aspergillus balasiticus IM115947 (Aspergill
us parasiticus) Fusarium solani M-0009 (Fusarium 5olani
) Fusarium solani M-0718 (FE RM-P
2688) (Mycological properties are described in Japanese Patent Application No. 117205/1982) Cephalosporium potoroni IF
05306 (Cephalosporium pot
ronii) Cephalosporium potoroni IFO5
706 Cephalosporium sp. M-4267
Trichosopsis parabilis CB54095 (Tri
In order to produce the target compound [I] using monoamino acid oxidase-producing bacteria such as S. gonopsis Variabilis), these microorganisms are usually cultured first, and the resulting microorganisms or their processed products are cultured. is preferably allowed to act on compound (n) under appropriate conditions.

As a culture method for obtaining bacterial cells, aerobic culture is usually desirable, and liquid aeration agitation culture is preferably carried out.

The medium composition includes medium commonly used as a mold medium, such as peptone, meat extract, yeast extract, and corn/mold extract.
Nitrogen sources such as steep liquor, soybean protein lysate, amino acids, carbon sources such as molasses, glucose, sucrose, phosphates, magnesium salts, common salt and other inorganic salts, or other growth-promoting substances. A nutrient medium containing an appropriate amount is used after adjusting the pH appropriately.
L-)methionine, D-(or DL-)alanine, D
- (or DL-) When containing valine, etc., excellent D-amino acid oxidase activity can be obtained.

The culture temperature is 23-28°C. The culture time is different depending on the culture conditions (culture equipment, culture composition, culture temperature, etc.)
- It is best to decide to terminate the culture at the time when the amino acid oxidase activity reaches its maximum, and 2 to 10 days is usually appropriate.

The bacterial cells thus obtained or their treated products are compound (II
) is used in the D-amino acid oxidation reaction, but the treated bacterial cells here refer to bacterial cells that have been subjected to appropriate treatment to increase the D-amino acid oxidase activity, which is advantageous for the production of compound [I]. For example, since the D-amino acid oxidase activity in the present invention normally exists within the bacterial body, the D-amino acid oxidase activity in the present invention is collected from a culture of D-amino acid oxidase-producing bacteria. A cell-free extract obtained by applying conventional methods, or partially purified or purified D- obtained by applying a known enzyme separation and purification method from a cell-free extract.
Amino acid oxidase, or partially purified or purified D-amino acid oxidase active form bound to a water-insoluble polymeric substance or inorganic carrier by physical or chemical means, or bacterial cells obtained by activation treatment. This refers to activated bacterial cells, etc.

In the present invention, the preparation and reuse of the soluble enzyme described above has limited practicality (on the other hand, the use of an insoluble enzyme, such as the use of activated bacterial cells, allows recovery and reuse). It is convenient in many ways.

The above-mentioned activation treatment of the bacterial cells can be brought about by subjecting the bacterial cells to conditions that cause some kind of mild damage and do not cause disintegration.

Examples of these activation treatments include freezing the cells at below 10° C. with an acid 1'fEpH, e.g. pH about 3-4, followed by thawing, freezing the cells in one or more organic solvents, e.g. acetone, n. - Processing in an aqueous phase with butanol, 2-phenylethanol, dimethyl ether, dichlorohexane, benzene, toluene, etc., 0°1-10
% surfactants, cationic surfactants such as acetyltrimethylammonium, cetylpyridinium, cetyldimethylbenzylammonium halide, anionic surfactants such as dodecyl sulfate, alkylarylsulfonic acid alkali metal salts, sodium desoxycholate, sorbitan mono Laurate Triton x 1
00, dilute solutions of potassium hydroxide or sulfur hydroxide, suspension in hypertonic solutions such as 2M sucrose solution, and then Examples include a method of rapidly diluting with water.

Since these activation treatments are influenced by various factors such as temperature, treatment time, and concentration of the pH 1 reagent, it is necessary to appropriately confirm the optimal conditions for activation.

The cells of the D-amino acid oxidase-producing bacteria thus obtained or the treated product thereof may be brought into contact with compound (II) in an aqueous medium in the presence of hydrogen peroxide.

Hydrogen peroxide is used in an amount sufficient to remain without being decomposed by the action of catalase, but the amount used depends on the amount of bacterial cells or their processed material, the titer of this enzyme, the action of catalase, and the compound [■] It depends on the amount used and its concentration.

Usually, an amount of 7 to 10 TLl (about 0.07 to 0.1 mol) of 35% hydrogen peroxide solution is used per 10,000 units/TIL of the enzyme titer.

The timing of addition should be determined because if it is added all at once during the reaction, the concentration of hydrogen peroxide in the reaction solution will be extremely high and the D-amino acid oxidase activity may be inactivated, or the sulfur atom at position 1 of compound CI) may not be oxidized. Therefore, it is preferable to add intermittently in small amounts or continuously throughout the reaction.

Therefore, the enzymatic reaction described above is usually carried out under conditions such that hydrogen peroxide is added continuously throughout the reaction.

This enzymatic reaction is usually carried out at a pH of 6 to 8.

The reaction temperature is preferably 30 to 40°C.

The reaction time mainly depends on the enzyme titer, but is usually 1 to 5 hours.

The above enzymatic reaction is carried out under aerobic conditions, usually with stirring or preferably under aeration of air or oxygen.

Although it is difficult to extract CeC from a fermentation process due to its amphoteric structure and hygroscopicity, according to the method of the present invention, CeC can be extracted under appropriate conditions in a CeC fermentation process after removing bacterial cells. The compound CI) (X
-acetoxy group) can be recovered by solvent extraction or ion exchange resin adsorption.

Compound CI, in which X is an acetoxy group, cannot be extracted from the reaction solution by adjusting the pH to 2.5 or lower, for example, and using a suitable organic solvent such as ethyl acetate, n-butanol, etc.

Good results have also been obtained using a combination of ion exchange resin and solvent extraction.

Suitable ion exchange resins are liquid amine anion exchange resins.

Preferred solvents include ethyl acetate, butyl acetate, n-butanol, and the like.

It is advantageous to perform extraction from a reaction solution using CeC fermentation broth by adjusting the pH of the broth to about 3 to 5 in advance.

It is also possible to separate compounds CI) in which X is an acetoxy group using solid ion exchange resins.

A suitable elution solvent in this case can be easily determined through preliminary experiments, but acetone is advantageous.

Compound (I) where X is a hydroxy group or a nucleophilic residue
can be recovered from the aqueous medium in which they were produced in a manner similar to that described above.

In that case, the properties of the X group influence the above-mentioned extraction conditions. These can be easily determined by preliminary experiments.

[■] can be converted into the corresponding 7β-amino compound by converting the corresponding 4-ester compound into an imidohalide, converting this into an iminoether E, and decomposing this.

It is also possible to convert 7β-amino compounds using cultures of microorganisms belonging to the genus Comamonas or Pseudomonas (Japanese Patent Application No. 10471/1989).
No. 49-142761 specification).

The starting material for the method of the present invention in which X is a hydroxy group can be produced by the method described in Japanese Patent Publication No. 39-9894 and Japanese Patent Publication No. 42-7553, and when X is a nucleophilic residue, is, as described in Japanese Patent Publication No. 38-26179, pyridine or other tertiary amine, and as described in Japanese Patent Publication No. 39-17936, a sulfur bond,
Nitrogen bond or inorganic nucleophilic compound and Japanese Patent Publication No. 41-4
No. 714 and JP-B No. 1305-1980, a yellow binding nucleophilic compound and JP-B No. 46-130
Publication No. 23, Special Publication No. 46-14735, Special Publication No. 4
It can be produced by reacting with thiol as described in Japanese Patent No. 6-15951.

The above nucleophilic residues are provided for illustrative purposes only and not as a restrictive list; other nucleophilic residues listed in other specifications relating to cephalosporin compounds may also be used. can.

Next, the method of the present invention will be explained in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference example 1 Trigonopsis parabilis culture 2 parts glucose, 0.3% D-methionine, 0.5 part yeast extract, KH2PO40, 4 parts MgSO4 7H20
0.1%, KCl0.05%, FeSO4” 7
Liquid medium containing 200,0025% H (pH 6,0) 1
Dispense 00d into 1 500wL Erlenmeyer flask, 12
After steam sterilization at 0°C for 15 minutes, Trigonopsis parabilis CB54095 is inoculated and cultured with shaking at 30°C for 48 hours.

After culturing, 34 minutes of the culture solution was collected and centrifuged to collect the bacteria to obtain 68 g of wet bacterial cells (water content about 90 parts).

Reference Example 2 Activation of Trigonopsis palabilis (2a) Wet bacterial cells 209 obtained in Reference Example 1 were suspended in 0.1M phosphate buffer (pH 7.5) to a volume of 100 ml.

Toluene ITLl was added to this, and after stirring at 37°C for 60 minutes, the bacteria were collected to obtain toluene-treated bacterial cells.

(2b) 20 g of the wet bacterial cells obtained in Reference Example 1 are suspended in 0.1 M phosphate buffer (pH 7.5) to a total volume of 100 d.

After adding 11 rLl of toluene and stirring at 50°C for 5 hours, the bacteria were collected to obtain toluene heat-treated bacterial cells.

(2c) 20 g of wet bacterial cells obtained in Reference Example 1 are soaked and shaken four times in 100 TILl of dimethyl ether, and then the dimethyl ether is removed to obtain MOM-treated bacterial cells.

Reference example 3 Fusarium solani culture glucose 2 parts, corn stew liquor 2 parts, DL
-Liquid medium containing 0.2% alanine (pH 5,0) 10
Dispense 0m13 into a 5001d Erlenmeyer flask, 120
After steam sterilization for 15 minutes at °C, Fusarium solani M-
0718 (FE RM-P2688) and 26°
Culture with rotary shaking at C for 2 days.

After culturing, 31 minutes of the culture solution was collected and washed with water to obtain 90 g of wet bacterial cells.

Reference Example 4 Culture of Cephalosporium potoroni In reference example 31, Fusarium solani M-0718
Cephalosporium potoroni IF05306 instead of
to obtain 85 g of wet bacterial cells.

Reference Example 5 Activation of Fusarium solani Fusarium solani M-0718 (FE) obtained in Reference Example 3
RM-P2688) was suspended in 40 TIL of 0.1M phosphate buffer (pH 7.5), and
After adding 0.4 rul of toluene and stirring at 37°C for 1 hour, the bacteria were collected to obtain toluene-treated bacterial cells.

Reference Example 6 Activation of Cephalosporium potoroni Cephalosporium potoroni IF05 obtained in Reference Example 4
306 wet bacterial cells 1 (l) were dissolved in 0.1 M phosphate buffer (p
H7, 5) Suspend 40ml of this and add 0 of toluene.
．． After adding 4 ml and stirring at 37°C for 11 hours, the bacteria were collected to obtain toluene-treated bacterial cells.

The Rf value and the production rate of compound CI) in the Examples were determined by the following method.

(1) Rf value carrier; silica gel Nispot film (manufactured by Tokyo Kasei Co., Ltd.); developing solvent; aln-butanol-acetic acid-water (3:1:1) b:n-
Butanol-acetic acid-water-formaldehyde (3:1:1
:5) It was determined by thin layer chromatography (TLC) using:

(2) Formation rate of compound CI) The sample solution was subjected to high-pressure p paper electrophoresis (3.5 KV, 1 hour, mixed solvent of formic acid-acetic acid-water (22 Nia 5:900) (p
H1, 8) Use] f.

A spot of compound CI) was extracted with 0.1 M IJ phosphate buffer (pH 7,0), and this extract was mixed with O, D, 26
It is calculated by measuring the absorbance at 0 mμ and plotting it from the calibration curve of the standard compound CI) to determine the amount of compound (1).

For example, when CeC is used (this spot is
The remaining CeC moves to the cathode side by 1.3 crfL, and the generated 3-acetoxymethyl-7-(4-carboxybutanamide)-3-cephem-4-carvone (compound CI in which X is an acetoxy group) moves to the anode side. The point shown is 4.1 am.

When 3-acetoxymethyl-7-(5-carboxy-5-oxo-bencunamido)-3-cephem-4-carboxylic acid is generated, the point l moves 5.5 m (m) toward the anode.
This is shown.

Example 1 Production of 3-acetoxymethyl-7-(4-carboxybutanamide)-3-cephem-4-carboxylic acid CeC aqueous solution 51 containing 2336γ/1rLl was
Trigonopsis parabilis CB5409 adjusted to H7.6 and obtained in the same manner as described in Reference Example 2b.
Add 315 TILl of toluene-heat-treated bacterial cells from No. 5 (total monoamino acid oxidase titer 369,000 units) and heat at 37°C.
Under conditions of an aeration rate of 121 revolutions per minute and a stirring speed of 300 revolutions per minute, 23 TL of 35% hydrogen peroxide solution was continuously added over 3 hours.

A solution obtained by removing bacteria from the reaction solution (amount of target substance 1
610γ/TLl, production rate 91.4%, CeC residual rate 9, compound (1) which is Rnee C0C0OH could not be detected) was adjusted to pH 1 with hydrochloric acid and extracted with ethyl acetate.

The ethyl acetate layer was dried over anhydrous sulfuric acid (IJ) and concentrated under reduced pressure.

The residue was treated with ethyl acetate-hexane to obtain 8.25 g of powder of the desired substance.

R, f = 0.48 (a), 0.76 et al.) In the above method, the production rate of the target substance when hydrogen peroxide is not used is 65%, and the remainder is compound (1) where R = -COCOOH The formation of

Example 2 Preparation of 3-acetoxymethyl-7-(4-carboxybutanamido)-3-cephem-4-carboxylic acid In Example II, instead of drying and concentrating the ethyl acetate layer under reduced pressure, E was hydroxylated. Neutralized extraction with an aqueous sodium solution yielded an extract of 1.251 m (target substance content: 5.8 m9/r).
rLl).

(The extract of
It is used in raw form for the production of 7-ACA by the method of No. 142,761.

Example 3 Production of 3-acetoxymethyl-7-(4-carboxybutanamide)-3-cephem-4-carboxylic acid Culture P solution (C
Fusarium solani M-0718 (FERM-P2) obtained in Reference Example 5
Add 6 g of toluene-treated bacterial cells of 688) and stir with a rotary shaker at 37°C for 3 hours.

While stirring, 0.611 Ll of 35% hydrogen peroxide solution was added every 30 minutes in 6 portions.

The production rate of the target substance in the reaction solution was 79.5%, R--CO
No formation of compound (1), which is COOH, was detected.

The production rate of the target substance when hydrogen peroxide solution was not added was 38.

Example 4 Production of 3-acetoxymethyl-7-(4-carboxybutanamide)-3-cephem-4-carboxylic acid In Example 3, Fusarium solani M-0718 (
Using toluene-treated bacterial cells of Cephalosporium potoronii FO5306 instead of the toluene-treated bacterial cells of FERM-P2688), the target substance was obtained at a production rate of 77.6%.

Compound where R=-COCOOH (no formation of 1,1 was detected.

When hydrogen peroxide solution was not added, it was 40 units.

Example 5 Production of 3-acetoxymethyl-7-(4-carboxybutanamide)-3-cephem-4-carboxylic acid In Example 1, reference example 2a was used instead of the toluene heat-treated bacterial cells obtained in reference example 2b. 315 m of toluene-treated bacterial cells obtained
A! Using 40 g of the MOM-treated bacterial cells obtained in Reference Example 2c, the target substances were obtained at production rates of 86% and 72%, respectively.

Example 6 Preparation of 7-(4-carboxybutanamide)-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid 7-(D-amino Adipinamide)-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (100%) in 0.1M phosphate buffer (pH 7.5) To this was added 31rLl of toluene-heat treated bacterial cells obtained in Reference Example 2b, and 3
Stir on a rotary shaker for 3 hours at 7°C.

While stirring, 0.6 yd of 35% hydrogen peroxide solution was added every 30 minutes in 6 portions.

The reaction solution was adjusted to pH 1 with hydrochloric acid and extracted with ethyl acetate.

The ethyl acetate layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the desired substance.

Rf=0.38(a) Example 7 Production of 3-hydroxymethyl-7-(4-carboxybutanamido)-3-cephem-4-carboxylic acid In Example 6, 7-([)-5-amino 3-hydroxymethyl-7-(D=5-aminoadipine)-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid The target substance was obtained using amide)-3-cephem-4-carboxylic acid at a yield of 69.5%.

Rf=0.44 (a) No formation of compound (1) where R=-COCOOH was observed.

Example 8 Preparation of N-(7-(4-carboxybutanamide)-3-cephem-3-ylmethyl]pyridinium-4-carboxylate In Example 6, 7-(D-5-aminoadipinamide)-3- N-(7-(D-5-aminoadipinamido)-3-cephem-3 instead of (5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid -ylmethyl]pyridinium-4
- The target substance was obtained using carboxylate.

Claims (1)

[Scope of Claims] 1. A cephalosporin compound represented by the general formula (wherein X represents a hydroxyl group, an acetoxy group, or a nucleophilic residue) or a salt thereof under aerobic conditions such as Trigonopsis parabilis or Fusarium. D belonging to the genus or Cephalosporium
- 7-(4-carboxybutanamide)-3-cephem-4- represented by the general formula (wherein, 7-(4-
Method for producing (carboxybutanamide)-3-cephem-4-carboxylic acid compound.