JPS6052752B2 - Method for producing 7α-methoxy-7β-(4-carboxybutanamide)-3-cephem-4-carboxylic acid derivative and salt thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 7α-methoxycephalosporin derivatives and salts thereof.

More specifically, a 7α-methoxycephalosporin compound represented by the general formula (in the formula, x represents a hydrogen atom, a hydroxyl group, an acetoxy group, or a carbamoyloxy group) and a salt thereof, or a phenyl group), an organic base, and a divalent or trivalent metal ion,
7α-methoxy7β-(4-carboxybutanamine)-3-cefemu-4-carboxylic acid derivatives represented by the general formula (wherein X is the same as in general formula 1) characterized by reacting with hydrogen peroxide and its The present invention relates to a method for producing salt.

7α-Methoxy7β-(4-carboxybutanamide)-3-cefemu 4-carboxylic acid derivative (general formula)
is a known compound, and its physicochemical properties have also been clarified (JP-A-51-86187, JP-A-KOKAI-KOKAI-154)
94).

Furthermore, this compound can be used in the production of other more useful synthetic 7α-methoxycephalosporin compounds using the 7α-methoxycephalosporin compound obtained as a fermentation product typified by cefamycin fermentation as a starting material. It is an important compound as an intermediate for The method for producing the general formula according to the prior art is based on a D-amino acid oxidase producing bacterium, Trigonopsis variabilis (TrigOnO
In addition to the disadvantage of having to culture all 7α-methoxycephalosporin compounds one by one, the culture tank is likely to be larger than the equipment required to culture the 7α-methoxycephalosporin compound, which is the starting material, making it extremely economical. Not. In addition, although the action of enzymes is relatively greatly influenced by the environment, certain substances present in the fermentation process of the starting material tend to inhibit the action of D-amino acid oxidase, resulting in a stable reaction rate. The major drawback is that it is difficult to maintain. Furthermore, this method using D-amino acid oxidase is effective when catalase coexists with 7α-
Methoxy-7β-(5-carboxy-5-oxopentanamide)-3-cephemu-4-carboxylic acid derivative (general formula) is symbiotically produced. (In the formula, X is the same as in general formula I) Therefore, in order to selectively obtain the target substance (III), it is necessary to carry out treatment with a Tacarase inhibitor such as sodium azide. An aqueous solution of generates hydrogen azide under acidic conditions.

This hydrogen azide is a tasteless and odorless gas, but it is a highly poisonous gas that is at least as toxic as hydrogen cyanide, and it is also an explosive substance that is dangerous even when handled in small quantities in a chemical laboratory [Wear, J. O. :J. Chem. Ed
uc. ? , A23 (1975)]. However, similar prior art methods (JP-A-51-86187, JP-A-5
3-15494), in order to isolate the reaction product, the pH of the reaction solution is lowered to 1.5-2 and extraction with an organic solvent such as ethyl acetate is performed, but under such conditions, Sodium azide remaining in the reaction solution converts to hydrogen azide, which is dangerous in large quantities. Furthermore, it can be said that it is virtually impossible to implement this method on an industrial scale. D
- A further critical drawback of the method using an amino acid converting enzyme is that the reaction must be carried out in an environment (temperature, pH, etc.) that is unstable for the starting materials and the target compound. Therefore, the yield of the reaction cannot be expected to be high. From this perspective, the inventors of the present invention have conducted extensive research into a completely new method that does not have the above drawbacks.
The 7α-methoxycephalosporin compound represented by the general formula 1 and its salt is prepared by the general formula 5＼ -Vvv↓ product v
By reacting with a compound represented by \18 (in the formula R represents a hydroxyl group, an amino group, or a phenyl group), an organic base, and a divalent or trivalent metal ion, and then reacting with hydrogen peroxide, 7α-Methoxy 7, simply represented by the general formula
They discovered that β-(4-carboxybutanamide)-3-cefemu-4-carboxylic acid derivatives and salts thereof can be produced in high yield, and completed the present invention.

Next, the present invention will be explained in more detail.

The starting materials of the present invention include purified cefamycin A1, cefamycin B1, cefamycin Cl7, crystals of 7α-methoxycephalosporin compound represented by general formula 1 such as α-methoxycephalosporin C (Al6884A), and partially purified fermentation process. It can be used either as a post-treatment liquid or as a fermentation process sterilizing liquid itself.

The production strain used for this fermentation can be obtained from public strain preservation institutions.

For example, Streptomyces lactamyulans (St
reptOmyceslactamdur'Ans)N
RRL38O2, Streptomyces Lipmani (Str
”EptOmycesllpmanii)NRRL35
84, Streptomyces griscens (StreptO
mycesgrisens) NRRL385l, Streptomyces fibriatus (St!'EptOmyc
esfimbriatus) NRRL3954, Streptomyces Halstedei (StreptOmyces
halstedii) NRRL3959, Streptomyces Cinnamonensis (StreptOmycesC
lllrlamOnerlSiS)NRRL3974,
Streptomyces chaatrensin (StreptO
myceschartrensis) NRRL397 maki can be mentioned as a 7α-methoxycephalosporin compound-producing bacterium represented by general formula 1.

The starting material of the present invention can be obtained by culturing these 7α-methoxycephalosporin compound-producing bacteria under normal culture conditions (for example, U.S. Pat. No. 3,719,563, JP-A-4
An example is the method described in No. 6-3287)
. One of the features that makes the present invention advantageous is that it can be carried out in an aqueous medium. Currently, the chemical deacylation methods commonly used normally have to be carried out in anhydrous organic solvents or similar solvent systems, but various improvements have been made to enable them to be carried out in aqueous solvents. However, the method of the present invention not only causes no hindrance to the reaction even when carried out in the presence of water, but also
It gives good results. Another feature that makes the present invention advantageous is its high reaction efficiency.

Generally, cephalosporin compounds have poor temperature stability, and it is desirable to treat them at as low a temperature as possible. However, according to the method of the present invention, the reaction temperature is set at -5 so that the reaction system does not freeze.
If the temperature is above 0.degree. C., the reaction proceeds without any disadvantage, and the reaction rate is better at lower temperatures because of the higher stability. Further, the appropriate pH for the reaction is 2 to 8, but 3 to 7 is most preferable, and a high reaction rate can be obtained. This is generally
This seems to be due to the fact that cephalosporin compounds are most stable in the weakly acidic region. In the present invention, the 7α-methoxycephalosporin compound represented by the general formula 1 is a compound in which the amino group of the α-amino-adipic acid side chain at position 7 is substituted with glyoxylic acid or a derivative thereof represented by the general formula. 7α-methoxy7β shown by formula ()
-(5-carboxy 5-oxopentanamide)-3
-Cefemu 4-carboxylic acid derivative is first formed,
This is easily decarboxylated using an oxidizing agent such as hydrogen peroxide to produce the target compound of the present invention, 7α-methoxy7β-(4-carboxybutanamide)-3-
It is converted to cefhemu 4-carboxylic acid derivative.

For conversion to the 7α-methoxycephalosporin compound represented by the general formula, it is necessary to add a reaction accelerator in addition to glyoxylic acid or its derivative.

These include organic bases (examples include pyridine, picoline, piperazine, imidazole, triethylamine, etc.) and divalent or trivalent metal ions (examples include copper, iron, cobalt, nickel, iron, aluminium, etc.). , manganese, gallium, indium, magnesium, etc.). These metal ions are added in the form of organic acid salts such as acetates and propionates, or inorganic acid salts such as sulfates and hydrochlorides, from the viewpoint of solubility. By appropriately combining these two types of reaction accelerators, the reaction time can be shortened and the reaction rate can be improved.

The amount of organic base used varies depending on other reaction conditions and its type, but for example, in the case of picoline, the starting material 7α
- between equivalent moles and 100 micromoles of methoxycephalosporin compound (1). The best results are obtained when the amount is between 5 and 10 times the mole. The amount of metal ion used also differs depending on other reaction conditions and type, but 0.1 of the starting material
A range of 1 to 1 mole is preferable. When using a cefamycin culture sterilizing solution as a starting material, 7α-
Like the methoxycephalosporin compound, it is deaminated and then decarboxylated, so it is better to give a larger amount of each drug used in the reaction than when using purified 7α methoxycephalosporin compound as a starting material. be.

Additionally, the amount of hydrogen peroxide added when decarboxylating the compound represented by the general formula to produce the target compound represented by the general formula varies greatly depending on other reaction conditions and the type of the starting material. A good range is from 0.01 times mole to 5@mol.

Hydrogen peroxide that remains unconsumed in the reaction may sometimes have a negative effect on the stability of the target compound ().
After the reaction is complete, it is safe to add a reducing agent such as sodium thiosulfate to inactivate the oxidizing power of hydrogen peroxide.
This operation does not have any adverse effect on the purification process of the target compound (). In this case, it is sufficient to add the same mole of sodium thiosulfate as the amount of hydrogen peroxide used in the reaction. After the reaction is completed, the reaction product can be easily separated and purified from the reaction solution by a commonly used method. For example, it can be made acidic to pH 2.5 or lower and extracted with a suitable organic solvent such as ethyl acetate, n-butyl acetate or n-butanol. Good results can also be obtained using a combination of ion exchange resin and organic solvent. Suitable ion exchange resins include liquid amine anion exchange resins. In this case, preferred solvents are ethyl acetate, butyl acetate, n-butanol, and the like. Alternatively, solid ion exchange resins can be used for separation. A suitable eluent in that case should be determined through preliminary experiments. For further purification to obtain a pure substance, conventional methods used for the purification of antibiotics may be used. EXAMPLES In order to further understand the present invention, examples will be described below, but the present invention is not limited thereto.

In addition, high performance liquid chromatography (1°C) in the examples
was carried out under the following conditions. High performance liquid chromatography (...1°C) Carrier: Licrosolve RP-18 (manufactured by Talc) 4 x 15
h Stainless steel column Developing solvent: 3% ammonium acetate-liacetonitrile (100:3) Equipment used: 6 model Hitachi high performance liquid chromatograph Flow rate: 1.5 m1/min Identification and quantification method: External standard method Example 1 7-Methoxycef Allosporin C [7-(5-amino-5-carboxyvaleramide)-3-acetoxymethyl-
Crystals (purity tin%) of JmethoxyΔ3-cephemu-4-carboxylic acid 7.35y and copper sulfate 12.4g were added to 1
Dissolve in water, add 130 ml of γ-picoline, and heat at 0°C.
Stir with.

Glyoxylic acid 14y was added to this mixed solution, and the pH was adjusted to 6. O
After reacting with stirring for 1 hour, 2N sulfuric acid was added to
Set H to 3.0. Further, 17 ml of 35% hydrogen peroxide was added and the mixture was stirred at 0 to 2°C, and then 40 da of sodium thiosulfate was added to terminate the reaction. When this solution was subjected to HPLC, the elution time was 2 minutes. The peak area of the starting material (7-methoxycephalosporin C), which was hot, disappeared by 1%, and the elution time was changed to the target compound 7-(4-carboxybutanamide)-J method in [MethoxyΔ3-cefemu-4-carboxylic acid was produced at 92% conversion.

Example 2 1170 m of the reaction-completed liquid reacted according to Example 1
1 to 1.5 using 2N sulfuric acid.

This mixture was extracted four times with 500 ml of ethyl acetate, the collected ethyl acetate layer was reverse dissolved in 200 ml of a 0.2M aqueous potassium phosphate solution with a pH of 8.5, and the phosphoric acid aqueous solution was diluted with 2N sulfuric acid to remove the phosphoric acid with 200 ml of 2N sulfuric acid. 5 and extracted again four times with 100 ml of ethyl acetate, and the ethyl acetate layers were combined, dehydrated with anhydrous sodium sulfate, and concentrated to dryness to yield the target compound 7-(4-carboxybutanamine)-J. [The white powder of methoxyΔ3-cefemu-4-carboxylic acid is 4.1
f was obtained. Example 3 Streptomyces Lipmanii (S
treptOmycesllpmanll)NRRL3
584 fermentation liquid to pH3. After adjusting to O, 2% of Radiolite NO7OO (manufactured by Showa Kagaku) was added to the fermentation liquid and filtered to obtain 7-methoxycephalosporin Cl5Oγ/m.
A p-liquid 1' containing l was obtained.

Copper sulfate 25' and γ-picoline 250ml are added to this solution 1e.
, glyoxylic acid 28y is added and stirred at 5° C. at pH 6.5. After 1 hour, 30 ml of 35% hydrogen peroxide was added, and after another 5 minutes, 50 y of sodium thiosulfate was added to stop the reaction.

A comparison between before and after the reaction at HH°C revealed that 82% of the starting material was converted to the target compound 7-(4-carboxybutanamide)-JmethoxyΔ3-cefemu-4-carboxylic acid. Example 4 According to Example 3, Streptomyces lipmani (
StreptOmyceslipmanii)NRRL
Culture sterilization of 3584? A liquid 200' was obtained.

This was adsorbed on a column of Mitsubishi Kasei Soku-20 (trade name) 21, and after washing with 2' acidic water of PH2, 0.2 mol of P
Elute with H8 phosphate buffer.

The eluate was fractionated into 100 mL portions and the content of 7-methoxycephaloporin C was measured by HPLC. After the start of elution, 16X. 95% of the 7-methoxycephalosporin C adsorbed from the 4th α line is eluted. Combining this active fraction, 7-methoxycephalosporin Cll4
A solution of 2.51 Oγ/ml was obtained. To this solution, 34.7f1 of copper sulfate, 364ml of γ-picoline, and 39f of glyoxylic acid are added, and the mixture is stirred at pH 6.5 and at -3 to 0°C.
After one powder, 47.6 ml of 35% hydrogen peroxide was added dropwise over 1 hour.

When time-course analysis during the reaction was followed at 1°C, the starting material 7-methoxycephalosporin C was found to be 7-(5-carboxy-5-oxopentanamide)-, which corresponds to the general formula.
It is gradually converted to JmethoxyΔ3-cefemu4-carboxylic acid, which is further converted into the target compound 7-(4-carboxybutanamide)-JmethoxyΔ3-cefemu4- by addition of hydrogen peroxide. It can be seen that it is converted to carboxylic acid. The conversion efficiency of the target compound from the starting material was S%. Examples 5 to 11 The results shown in Table 1 were obtained using a cultured sterilized solution of actinomycetes producing various 7-methoxycephalosporins in exactly the same manner as in Example 3.

Example 12 - Conversion of 7-methoxy heptaphalosporin C to 7-(4-carboxybutanamide)-JmethoxyΔ3-cefemu-4-carboxylic acid with % purity was carried out under various conditions, and the results are shown in Table 2. I got it.

Claims (1)

[Claims] 1. General formula▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (in the formula,
represents a hydrogen atom, a hydroxyl group, an acetoxy group, or a carbamoyloxy group) and its salt;
II) After reacting glyoxylic acid or its derivative represented by (wherein R represents a hydroxyl group, an amino group or a phenyl group) with an organic base and a divalent or trivalent metal ion as a reaction accelerator, Furthermore, there are general formulas ▲mathematical formulas, chemical formulas, tables, etc. that are characterized by reaction with hydrogen peroxide▼(III) (in the formula, X is the same as above) 7α-methoxy-7β-(4-carboxy A method for producing a (butanamido)-3-cephem-4-carboxylic acid derivative and a salt thereof. 2. The 7α-methoxy-7β-(4 -carboxybutanamide)-3-cephem-4-carboxylic acid derivative and its salt. 3. The 7α-methoxy-7β-(4 -carboxybutanamide)-3-cephem-4-carboxylic acid derivative and its salt. 4 The organic base is pyridine, γ-picoline, piperazine,
1 selected from imidazole and triethylamine
7α according to claims 1 to 3 which is a species
-methoxy-7β-(4-carboxybutanamide)-
A method for producing a 3-cephem-4-carboxylic acid derivative and a salt thereof.