JPS6148519B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing N-acylcefamycin C and its organic base salt, and more specifically to a method for producing N-acylcefamycin C, namely, 7β-(5'-carboxy-5'-aminovaleramide). )-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid is acylated to derive N-acylcefamycin C, and then treated with a macroporous nonionic adsorption resin to obtain the resin. N-acylsefamycin C is adsorbed onto the surface of the same material, and then eluted to collect N-acylsefamycin C, which is optionally converted into a salt of an organic base. The present invention relates to a method for producing C or an organic base salt thereof.

Cefamycin C is an antibiotic produced by fermentation of various microorganisms, and is an important substance as a raw material for various useful cephalosporin derivatives.

Conventionally, various methods are known for extracting and purifying cefamycin C from fermentation liquor, but among these, the adsorption/elution method using activated carbon has various operational and efficiency problems and is not economical. isn't it. Furthermore, for cephalosporin C and cephamycin A and B, a method of extraction and purification by adsorption and desorption on a macroporous nonionic adsorption resin is known (see U.S. Pat. No. 3,725,400 and JP-A-46-3286). , cefamycin C cannot be adsorbed onto such resins. Furthermore, an acylating agent is applied to the fermentation broth containing cefamycin C to produce an acyl derivative of cefamycin C, which is extracted with an organic solvent such as ethyl acetate, and then further purified by various methods to obtain cefamycin C. A method for collecting acyl derivatives is known (see JP-A-49-30593), but such a method of directly extracting a large amount of dilute aqueous solution with an organic solvent involves the consumption of a relatively large amount of organic solvent. do not have,
Furthermore, since organic acids and other substances produced by fermentation, as well as excess acylating agents, are extracted at the same time, the degree of purification is not high, and this is not a satisfactory method.

Therefore, the present inventors conducted various studies on new methods for collecting purified cefamycin C, and found that a solution of N-acyl cefamycin C was obtained by treating an acylating agent with an aqueous solution of crude cefamycin C. When treated with a macroporous nonionic adsorption resin, as mentioned above, cefamycin C is not adsorbed to the resin, whereas N-acyl cefamycin C is adsorbed at high density and is also easily adsorbed by the eluent. It was found that N-acylcefamycin C could be obtained with extremely high purity and yield.

The present invention was completed based on this knowledge.

The aqueous solution of crude cefamycin C used as a raw material in the present invention includes cefamycin C
Crude cefamycin C that has been partially purified and concentrated by a known method, such as by treating the culture solution with activated carbon or an anion exchange resin and adsorbing and desorbing cefamycin C thereto.
It may be an aqueous solution or any other solution.

The acylating agent used in the present invention includes:
Examples include various known compounds such as aliphatic carboxylic acids, aromatic carboxylic acids, heterocyclic carboxylic acids, their respective sulfonic acids, carbonic acid esters, carbamic acids, their thio acids, and reactive derivatives of the acids mentioned above. Reactive derivatives include acid chlorides, acid anhydrides, active amides, active esters, isocyanates, thioisocyanates, and the like.

The acylation reaction may be carried out by a known method using these, but in that case, the acyl group introduced into the amino group may be an aliphatic carboxylic acid, an aromatic carboxylic acid, a heterocyclic carboxylic acid, or a similar sulfonic acid. Contains residues obtained by removing OH from acids, carbonic esters, carbamic acids, or other thio acids, and substituents include halogens, nitro groups, alkoxy groups,
Alkanoyl groups such as acetyl, propionyl, butyryl, isobutyryl, etc. with or without alkylthio groups, allyloxy groups, allyl groups, alkoxycarbonyl groups, acyloxy groups, acylamino groups, heterocyclic groups, etc., cycloalkanecarbonyl groups such as cyclohexanecarbonyl , aryloyl groups such as berzoyl, toluoyl, alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, allyloxycarbonyl groups such as phenyloxycarbonyl, benzyloxycarbonyl, etc. Aralkyloxycarbonyl groups, various heterocyclic carbonyl groups, alkylcarbamoyl groups,
Examples include an allylcarbamoyl group, an alkylallylcarbamoyl group, or a similar thiocarbamoyl group. The acylation reaction is preferably carried out under slightly acidic or weakly alkaline conditions, and therefore, the reaction may be carried out while adjusting the pH of the aqueous solution of crude cefamycin C, if necessary.

The thus obtained N-acylcefamycin C
The aqueous solution containing is then treated with a macroporous nonionic adsorption resin, which is preferably one based on a polymer of styrene and divinylbenzene or a polymer of acrylic ester; A specific example is amber light.
XAD-1, XAD-2, XAD-4, XAD-
5 (registered trademark, manufactured by Rohm and Haas) and Diamondion HP-10, Diamondion HP-20, Diamondion HP-
30, HP-40, HP-50 (registered trademark, manufactured by Mitsubishi Kasei Corporation), etc. Specific examples of the latter include Amberlite・Manufactured by Haas Corporation) etc.

To treat a solution containing crude N-acylcefamycin C with a macroporous nonionic adsorption resin, a batch method or a column method may be used, but the column method is generally more efficient. That is, the resin is filled in a column in advance and regenerated by passing, for example, aqueous acetone, aqueous methanol, a solution of these with sodium hydroxide, hydrochloric acid, or sulfuric acid, dilute hydrochloric acid, dilute sulfuric acid, etc., and then thoroughly washed with water. A solution of crude N-acylcefamycin C whose pH has been adjusted to 7.0 or less, preferably 5.0 or less, may be passed through this at a flow rate of SV1 to 5, preferably 2 to 3.

After washing with water, the N-acylcefamycin C adsorbed onto the resin in this manner is eluted using a water-containing solvent such as water-containing methanol or water-containing acetone.
The preferred concentration of methanol or acetone in the aqueous eluent is generally 40-80%.

In addition, the flow rate of the eluent is normally SV 0.5 to 3.

The portion of the eluate containing N-acyl cefamycin C was collected, and N-
Acylcefamycin C can be obtained. For example, the above N-acylcefamycin C-containing portion is concentrated under reduced pressure to remove the organic solvent, and mineral acid is added to adjust the pH to 1.5 to 4.5. After adjustment, a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate,
By extracting with methyl isobutyl ketone or the like and concentrating the extract, N-acylcefamycin C of high purity can be collected in good yield.

N-acylcefamycin C can also be collected as a salt of its organic base. For example, various organic bases such as dicyclohexylamine, trialkylamine, quinoline, N/N-dialkylbenzylamine, diphenylamine, benzylamine, tert-octylamine, etc. The purity of N-acylcefamycin C can be further increased by precipitating the respective salts and separating them.
Moreover, it can be obtained with good yield.

As described above, by the method of the present invention, highly pure N-acylcefamycin C or its organic base salt can be obtained in good yield.

This substance is useful as a raw material for various cefamycin C derivatives.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 800 ml of a culture solution containing cefamycin C (cephamycin C, titer: 3060 μg/ml) was passed through a column filled with 100 ml of anion exchange resin IRA411 (acetic acid type) to adsorb cefamycin C. The resin was washed with water and eluted with a 0.2N pH5.5 acetate buffer to obtain 1250 ml of eluate containing cefamycin C (containing 10.3 g of cefamycin C). This eluate
2N sodium hydroxide solution was added to 250 ml (containing 2.06 g of cefamycin C) to adjust the pH to 8.5, and 2.12 g of well-pulverized paranitrobenzoyl chloride was added while stirring at room temperature. After addition,
The mixture was stirred at room temperature for 2 hours. During this time, 2N sodium hydroxide solution was added at appropriate times to maintain the pH at 8.0 to 8.5. After the reaction was completed, 6N sulfuric acid solution was added with stirring to adjust the pH to 3.5, and the resulting precipitate was removed to give N-paranitrobenzoylcefamycin.
310 ml of solution containing 2.75 g of C was obtained.

This solution was passed through a column packed with 30 ml of Diaion HP-20 at a flow rate of SV2 to adsorb N-paranitrobenzoylcefamycin C. After washing with water, 60% methanol aqueous solution was passed through the column at a flow rate of SV1 for elution.

After concentrating the eluate containing N-paranitrobenzoylcefamycin C and distilling off methanol, the pH was adjusted to 2.0 by adding a 6N sulfuric acid solution.
Extracted three times with 1/2 volume of ethyl acetate. The extracts were combined, washed with saturated saline, dehydrated with anhydrous sodium sulfate, concentrated and dried to give N-paranitrobenzoylcefamycin C, i.e. 7β-(5'-
Carboxy-5'-paranitrobenzoylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid
Obtained 2.20g. The purity as determined by high performance liquid chromatography was 83%, and the yield was 66.4%.

The NMR spectrum of this substance in heavy acetone showed the following characteristics.

1.70 to 2.20 (4H, multiplet) 2.30 to 2.80 (2H, multiplet) 3.50 (3H, singlet) 3.50 (2H, wide singlet) 4.87 and 5.02 (2H, AB-quartet J = 13H2) 5.13 (1H, singlet) 6.06 (2H, wide singlet) 7.85-8.52 (8H) Example 2 Ethyl acetate containing 1.88 g of N-paranitrobenzoylcefamycin C (purity 83%) obtained in Example 1 When 650 mg of dicyclohexylamine was added to 20 ml of the solution with stirring, a white precipitate formed.

After further stirring for 30 minutes, the precipitate was collected, washed with ethyl acetate, and dried to obtain the dicyclohexylamine salt of N-paranitrobenzoylcefamycin C, i.e., 7β-(5'-carboxy-5'-paranitrobenzoylamino). valeramide)-7α-
Methoxy-3-carbamoyloxymethyl-3-
2.13 g of dicyclohexylamine salt of cefem-4-carboxylic acid was obtained. The purity as determined by high performance liquid chromatography was 90% and the yield was 95%.

Example 3 180 ml of treated solution of ion exchange resin Amberlite IRA411 obtained by the method described in Example 1
(containing 1.45 g of cefamycin C) was added with 2N sodium hydroxide solution to adjust the pH to 8.5.
While stirring at room temperature, 1.89 g of parachlorobenzoyl chloride was gradually added over 30 minutes. After addition,
While stirring at room temperature for 2 hours, 2N sodium hydroxide solution was added at appropriate times to maintain the pH between 8.0 and 8.5. After the reaction was completed, 6N sulfuric acid solution was added while stirring to adjust the pH to 3.5, and the resulting precipitate was removed. Add 30ml of this liquid to Diamond Ion HP-
40 at a flow rate of SV2 and N-
Parachlorobenzoylcefamycin C was adsorbed.

Next, wash the resin with water and add 70% methanol aqueous solution.
Elution was performed at a flow rate of SV1. The eluate containing N-parachlorobenzoylcefamycin C was concentrated to remove methanol, and then a 6N sulfuric acid solution was added to adjust the pH to 2.0, followed by extraction with 1/2 volume of ethyl acetate. The extracts were combined, washed with saturated saline, dehydrated with anhydrous sodium sulfate, and then
420 mg of N.N-diethylbenzylamine was added with stirring, resulting in a white precipitate.

After stirring for an additional 30 minutes, the precipitate was collected, washed with ethyl acetate, and dried to form the N-N-diethylbenzylamine salt of N-parachlorobenzoylcefamycin C, i.e., 7β-(5'-carboxy-5 '-parachlorobenzoylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid N・N
-1.69 g of diethylbenzylamine salt was obtained. The purity as determined by high performance liquid chromatography was 90%, and the yield was 62.2%.

Example 4 280 ml of treated solution of ion exchange resin Amberlite IRA411 obtained by the method described in Example 1
(contains cefamycin C 2.34g) and acetone 400%
ml and 2N sodium hydroxide solution was added to adjust the pH to 8.5. A solution of 3.45 g of paratoluenesulfonyl chloride in 20 ml of acetone was added with stirring over a period of 1 hour. After the addition, 2N sodium hydroxide solution was added at appropriate times while stirring at room temperature for 2 hours to maintain the pH between 8.0 and 8.5. Next, the pH was adjusted to 7.0, and acetone was evaporated under reduced pressure at 30°C. A 6N sulfuric acid solution was added to this solution to adjust the pH to 3.5, the resulting precipitate was removed, and the solution was passed through a column filled with 40ml of Diaion HP-20 at a flow rate of SV3 to remove N-paratoluenesulfonyl. Cefamycin C was adsorbed, and after washing with water, elution was performed by passing a 60% methanol aqueous solution through at a flow rate of SV1. After distilling off methanol from the eluate containing N-paratoluenesulfonylcefamycin C, a 6N sulfuric acid solution was added to adjust the pH to 2.0.
and extracted three times with 1/2 volume of ethyl acetate. The extracts were combined, washed with saturated saline, dehydrated with anhydrous sodium sulfate, and 530 mg of quinoline was extracted.
When added with stirring, a precipitate formed. The precipitate was collected, washed with ethyl acetate, and dried to give the quinoline salt of N-paratoluenesulfonylsefamicin C, i.e., 7β-(5'-carboxy-5'-paratoluenesulfonylaminovaleramide)-7α.
-methoxy-3-carbamoyloxymethyl-3
-Quinoline salt of cefem-4-carboxylic acid 2.92g
I got it. The purity as determined by high performance liquid chromatography was 87%, and the yield was 66.1%.

Example 5 100 ml of acetone was added to 290 ml (containing 2.38 g of cefamycin C) of the treatment solution of ion exchange resin Amberlite IRA411 obtained by the method described in Example 1. Add 2N sodium hydroxide solution
The pH was adjusted to 8.5, and a solution of 1.41 g of benzoyl chloride dissolved in 20 ml of acetone was added over 1 hour with stirring.

After addition, 2N sodium hydroxide solution was added at appropriate times while stirring at room temperature for 30 minutes to bring the pH to 8.0~
It was held between 8.5 and 8.5. Next, the pH was adjusted to 7.0, acetone was distilled off under reduced pressure at 30°C, 6N sulfuric acid solution was added to adjust the pH to 3.5, the precipitate formed was removed, and 30ml of
N-benzoylcefamycin C was passed through a column filled with Diaion HP-40 at a flow rate of SV2.
was adsorbed. Next, the resin was washed with water and eluted with a 70% aqueous methanol solution at a flow rate of SV1. After distilling off methanol from the eluate containing N-benzoylcefamycin C, a 6N sulfuric acid solution was added to adjust the pH to 2. 0 and extracted three times with 1/2 volume of ethyl acetate. The extracts were combined, washed with saturated saline, dehydrated with anhydrous sodium sulfate, and then
540 mg of tert-octylamine was added with stirring and a precipitate formed. The precipitate was collected, washed with ethyl acetate, dried and N-benzoylcefamycin C.
tert-octylamine salt, i.e. 7β-
(5'-carboxy-5'-benzoylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid
2.67 g of tert-octylamine salt was obtained. Purity by high performance liquid chromatography method is 86%, yield is
It was 63%.

Example 6 Add 450 ml of acetone to 500 ml of a crude solution containing 4 mg/ml of cefamycin C, adjust the pH to 8.5 by adding 2N sodium hydroxide solution, and maintain the liquid temperature at 5°C while stirring. , 50 ml of acetone solution containing 4 g of isobutoxycarbonyl chloride was added dropwise over 30 minutes, followed by 2 hours at 5°C.
Add normal sodium hydroxide solution to adjust pH to 8.0-8.5
The mixture was stirred for 1 hour while adjusting the temperature. Next, after correcting the pH to 7.0 with 6N sulfuric acid, acetone was distilled off at 30° C. under reduced pressure, and the pH of the resulting aqueous solution was corrected to 3.5 with 6N sulfuric acid, and the resulting precipitate was removed. The resulting aqueous solution was passed through a tower packed with 40 ml of Diaion HP-20 at a flow rate of SV3 to adsorb N-isobutoxycefamycin C, washed with water, and then passed through a column filled with 40 ml of Diaion HP-20 at a flow rate of SV1. Elution was carried out throughout. The eluate containing N-isobutoxycefamycin C was distilled under reduced pressure to remove methanol, the pH was adjusted to 2.0 with 6N sulfuric acid, and the mixture was extracted three times with 1/2 volume of ethyl acetate. . The extracts were combined, washed with saturated brine, dehydrated with anhydrous sodium sulfate, distilled under reduced pressure to obtain a concentrated solution, and a large amount of isopropyl ether was added to it.
The resulting precipitate was collected and dried to give N-isobutoxycefamycin C, i.e. 7β-(5'-carboxy-5'-isobutoxycarbonylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3. -Cefem-4-carboxylic acid powder 1.53g was obtained.

The NMR spectrum of this powder in deuterated acetone was as follows.

0.90ppm (6H, doublet, J=7.0Hz) 2.20~2.70ppm (2H, multiplet) 3.50ppm (2H, wide singlet) 3.50ppm (3H, singlet) 3.83ppm (2H, doublet) , J=7.0Hz) 4.15-4.40ppm (1H, multiplet) 4.87 and 5.03ppm (2H, AB-quartet, J=13
Hz) 5.11ppm (1H, singlet) 6.06ppm (2H, wide singlet) 6.38ppm (1H, doublet, J=8Hz) 8.26ppm (1H, wide singlet) 8.97ppm (2H, wide singlet) singlet) and infrared absorption spectrum (nujiol) is 1770cm
^-1 and showed absorption at 1710 cm ^-1 .

Example 7 Using isopropoxycarbonyl chloride in place of isobutoxycarbonyl chloride in Example 6, and performing the same procedure, N-isopropoxycefamycin C, 7β-(5'-carboxy-5'-iso Propoxycarbonylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid powder
1.4g was obtained. The infrared absorption spectrum (nujiol) showed absorption at 1770 cm ^-1 and 1715 cm ^-1 .

Example 8 500 ml of acetone was added to 560 ml of a solution containing 5 mg/ml of cefamycin C, and the mixture was cooled to 5°C. Next, a 2N sodium hydroxide solution was added to this solution, and a solution of 5 g of ethyl chlorocarbonate dissolved in 50 ml of acetone was added dropwise while maintaining the pH at 8.0 to 8.5.

After completion of the dropwise addition, stirring was continued for 2 hours while adjusting the pH to 8.0 to 8.5 under ice cooling.

After correcting the pH of this reaction solution to 7.0 with 6N sulfuric acid, acetone was distilled off at 30° C. under reduced pressure to obtain an aqueous solution. After correcting the pH of this aqueous solution to 3.5 with 6N sulfuric acid and removing the formed precipitate, 70ml
The flow rate is S.
I passed it with V.2. N-ethoxycarbonylcefamycin C adsorbed on the resin was eluted with a 60% methanol aqueous solution at a flow rate of SV1. Next, the eluate containing N-ethoxycarbonylcefamycin C was distilled under reduced pressure to remove methanol, the pH of the resulting aqueous solution was adjusted to 2.0 with 6N sulfuric acid, and 1/2 volume of ethyl acetate was added. Extracted three times. The extracts were combined, washed with saturated saline, dehydrated with anhydrous sodium sulfate, and then evaporated to dryness under reduced pressure to yield N-ethoxycarbonylcefamycin C, i.e., 7β-
1 g of (5'-carboxy-5'-ethoxycarbonylaminovaleramide)-7α-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid was obtained. The NMR spectrum of this substance in deuterated dimethylformamide showed the following characteristics.

1.20ppm (3H, triplet, J=7Hz) 1,28~1.98ppm (2H, multiplet) 2.08~2.68ppm (2H, multiplet) 3.48ppm (3H, singlet) 3.48ppm (2H, singlet) 4.08ppm (2H, quartet, J=7Hz) 4.08-4.43ppm (1H, multiplet) 4.79 and 4.97ppm (2H, AB-quartet, J=13
Hz) 5.11ppm (1H, singlet) 6.54ppm (2H, wide singlet) 7.00ppm (1H, doublet, J=7Hz) 7.48~7.88ppm (wide singlet) 9.03ppm (1H, wide singlet) Singlet) Also, the infrared absorption spectrum (nujiol) is 1770
cm ^-1 , absorption was observed at 1715 cm ^-1 .

Claims (1)

[Claims] 1 Crude cefamycin C is acylated to induce N-acylcefamycin C, and then treated with a macroporous nonionic adsorption resin to adsorb N-acylcefamycin C to the resin, followed by elution. to collect N-acylcefamycin C,
A method for producing N-acylcefamycin C or an organic base salt thereof, which comprises converting the N-acylcefamycin C into an organic base salt, if necessary.