KR800000569B1 - Process for preparing cephalosporin c derivatives - Google Patents

Abstract

Cephalosporin C derivatives(I, M =H, ankali metal; X = halogen)were prepd. by reaction of cephalosporin C(II) with phthalic anhydride(III). 16.2 g Tetrabromophthalic anhydride powder contg. acetone 2N NaOH were mixed with a culture broth condensate contg. 1.5 (II) and stirred for 1 hr at room temp. and adjusted pH 9.0-8.5 to give 14.2 g(I) (85%). IR, NMR and UV charts of I were presented.

Description

Preparation of Derivatives of Separosporin C

1 shows the infrared absorption spectrum (KBr purification method) of N- (tetrabromophthaloyl) cephalosporin C,

2 shows the nuclear magnetic resonance spectrum of the above compounds,

3 shows the ultraviolet absorption spectrum (in ethanol) of the above compound,

4 shows the infrared absorption spectrum (KBr purification method) of N- (tetrachlorphthaloyl) cephalosporin C,

5 shows the nuclear magnetic resonance spectrum of the above compound,

FIG. 6 shows the ultraviolet absorption spectrum (in methanol) of the above compound.

The present invention relates to the preparation of novel derivatives of cephalosporin C.

Specifically, the present invention relates to a method for recovering the Separosporin C in an aqueous solution containing Separosporin C in a good yield by converting the Separosporin C to a novel derivative.

In detail, the following Cephalosporin C (II) is represented by the following formula

(Where M represents hydrogen or an alkali metal)

By reacting with phthalic anhydride derivative (III)

(Where X represents a halogen element)

The present invention relates to a method for producing a novel derivative (I) of Sepharoseporin C having the following formula.

(Where X and M have the same meaning as above)

Again, since the derivative of formula (I) has low solubility in water under acidic conditions, the present invention also provides a method for recovering the yield of cephalosporin C in an aqueous solution containing cephalosporin C in a good yield. will be.

In the above formulas (I) and (II), X represents a halogen atom, particularly preferred are chlorine and cancellation.

In formulas (I) and (II), M represents an alkali metal such as sodium, carium and the like.

The above derivative (I) of Sepharoseporin C can be easily converted to 7-aminocephalosporin acid by a known method.

Cephalosporin C is produced by fermentation.

In general, in the culture of cephalosporin C, it is difficult to separate the cephalosporin C and the contaminants, because it is produced as a metabolite of microorganisms, very similar in chemical properties.

Since cephalosporin C is extremely soluble in water, it is extremely difficult to extract it from the culture solution in its organic form as it is.

For this reason, a method of collecting the crystals of cephalosporin C by employing a suitable adsorbent such as activated carbon or an ion exchange resin, adsorbing the cephalosporin C and then eluting and concentrating it with an aqueous solvent or an aqueous solution of dilute salt concentration is employed. (E.g., U.S. Patent No. 3,467,654; West German Patent No. 2,021,696; Japanese Patent Application Laid-Open No. 8-86,890, etc.) Therefore, these methods require many processes to purify, and the yield of each process is not necessarily good. Recovery rate is low.

On the other hand, since the properties of Sepharoserin C are soluble to some extent in an organic solvent, a method of converting Sepharoseporin C into a derivative form and extracting the derivative with an organic agent has been devised. (For example, Belgian Kingdom Patent No. 807,171; US Patent No. 3,573,295; West German Patent No. 2,157,693; Japanese Patent Publication No. 49-24,992, etc.).

Even with these methods, it is inevitable that some of the contaminants are modified in the form of derivatives in the same manner as the cephalosporin C derivatives and extracted with the cephalosporin C derivatives.

For this reason, the purity of the substance obtained tends to be low. The present invention is a method of harvesting a novel derivative of Sepharosporin C with high purity by a series of operations while removing the manufacturing bond as described above.

That is, cephalosporin C is induced to a novel derivative having a solubility in water that is significantly different from that of the contaminant, and is used to separate it from the contaminant impurity using the property, especially as a crystal of free acid without extraction with a solvent. It is characterized by titration.

The gist of this new method is described below and further described as examples.

According to the method of the present invention, the culture solution obtained by filtration of the culture solution of cephalosporin C with a filter aid is made acidic and washed with water and an immiscible organic agent such as ethyl acetate, butyl acetate, or the like. Pass through a mixture of Amberlite IRA-94 and IRC-84 (the solution obtained in this way is called a resin treatment solution), or the resin treatment solution and the ion exchange resin again, such as Amberlite IRA-401 or a nonionic adsorbent resin, For example, it can apply to the solution eluted by adsorb | sucking through the diion HP-20 etc. (the solution obtained in this way is called resin eluent).

량의 친수성유기용제, 예컨데 아세톤, 메타놀, 이소프로파놀 등을 가해 수산화 나트륨 혹은 탄산나트륨 무기염기를 가함으로써 pH를 7.0-10.0, 바람직한 것은 8.0-8.5의 알카리성으로 조정하며, 식(III)으로 표시되는 프탈산 무수물을 세파로스포린 C의 2-5당량 그대로 혹은 위의 친수성 유기용제에 용해하여 교반하면서 서서히 가한다.In an aqueous solution containing such cephalosporin C

By adding an amount of a hydrophilic organic solvent, such as acetone, methanol, isopropanol, and the like to add sodium hydroxide or sodium carbonate inorganic base, the pH is adjusted to an alkalinity of 7.0-10.0, preferably 8.0-8.5, and is represented by the formula (III). Phthalic anhydride is slowly added while stirring with 2-5 equivalents of Separosporin C or dissolved in the above hydrophilic organic solvent.

As the reaction proceeds, the pH changes to acidic side and the pH is maintained at 8.0-8.5 by adding the inorganic base.

The reaction between them is at room temperature.

After the above acid anhydride was added, stirring was continued for another 1-3 hours while maintaining the pH at 8.0-8.5 to complete the reaction.

After completion of the reaction, 1-5 times of water is added to dilute or concentrated under reduced pressure to remove the organic solvent, and then the pH is adjusted to 5.0-6.0 by adding an inorganic acid such as sulfuric acid or hydrochloric acid. Impurities precipitate.

After filtering this precipitate, the pH of 1.5-3.0 is preferably adjusted to 2.0-2.5 again with acid, and the crystals of the derivative of Sepharosporin C represented by the formula (I) are precipitated.

Alternatively, the pH is adjusted to 2.0-2.5, followed by extraction with water and an immiscible organic solvent such as ethyl acetate and butyl acetate, followed by shaking well, followed by adding water to the extract and adding an inorganic base such as sodium hydroxide to adjust the pH to 5.0-8.0. It is adjusted to 6.0-6.5 and converted to water.

When acid is added to the obtained aqueous solution and adjusted to pH 2.0-2.5, the crystal | crystallization of the derivative of cephalosporin C represented by Formula (I) will precipitate.

The crystals are collected, washed with water, dried under reduced pressure, and cephalosporin C derivative (I) is obtained as a crystal of free acid.

The obtained crystals are dissolved in alcohols such as methanol or ethanol, and 0.5-1 equivalents of organic bases, such as triethylene diamine, dissolved in the same solvent are added with stirring to precipitate an organic salt of the Sepharoserin C derivative.

By carrying out this process, impurities can be removed again.

In this way, the cephalosporin C derivative represented by the high purity formula (I) can be obtained in good yield in the form of free acid or organic base salt. As such, the derivative of cephalosporin C can be determined under acidic conditions because the acid anhydride represented by the formula (I) has a halogen substituent, and thus the same kind of phthalic anhydride such as phthalic anhydride, 3- or 4-nitro In phthalic acid and the like, the derivatives of cephalosporin C derived from these acid anhydrides are not determined under acidic conditions.

The method of the present invention is characterized in that the purity is high since the fractionation and precipitation of the derivative can be carried out with good yield because it can be collected as a powder in the form of a corresponding derivative without particularly performing extraction operation with an organic solvent in the reaction solution. It is an extremely industrially useful method.

Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

Example 1

The culture filtrate of cephalosporin C was passed through a mixed resin tower of Amberlite IRA-94 (acetic acid type) and IRC-84 (hydrogen type) to obtain a passage liquid (hereinafter referred to as a resin treatment solution), which was then used as a diamond ion HP-. 200 ml of acetone was added to 400 ml of a solution (hereinafter referred to as a resin eluent) (which contains 4.08 g of cephalosporin C) eluted in a 0.05% sodium hydroxide reaction, and the pH was adjusted to 8.5 in a 2% sodium hydroxide solution. After stirring at room temperature, 9.15 g of finely ground powder of tetrabrophthalic anhydride was added slowly as it was for 30 minutes.

After the addition, the mixture was stirred for 1 hour at room temperature.

In the meantime, 1 N sodium hydroxide solution was added timely and the pH was maintained at 8.0-8.5.

After completion of the reaction, the mixture was diluted twice with water and then stirred to add 6 sulphate sulfuric acid solution to adjust the pH to 5.5 to remove the precipitate. The filtrate was stirred again, and 6 mL of sulfuric acid solution was added to adjust pH to 2.0 to precipitate N- (tetrabromophthaloyl) sephalosporin C crystals. Stirred again at room temperature for 1 hour and then cooled overnight.

선광도

+55°(C 0.5메타놀) 다시 이 결정 2g을 물 200ml에 현탁하여 2규정의 수산화나트륨 용액을 가하여 pH 6.5로 조정하고 용해시켜 활성탄 200mg을 가하여 탈색한 후 활성탄을 제거하고 2규정의 황산용액에서 pH 2.0으로 하여 재결정시켜 1.50g의 결정을 얻었다.The crystals were filtered off, handmade, and dried in vacuo at 40 ° C. to obtain 7.81 g. Purity 92%, Yield 85%, Ultraviolet absorption spectrum by hydroxylamine method:

Optical intensity

+ 55 ° (C 0.5Methanol) 2 g of this crystal was suspended in 200 ml of water, adjusted to pH 6.5 by adding 2 sodium hydroxide solution, dissolved, and decolorized by adding 200 mg of activated carbon. It recrystallized at pH 2.0 and obtained 1.50 g of crystals.

Melting point 121 ° C (decomposition)

Ultraviolet absorption spectrum:

+62°(C 0.5메타놀)Radiance:

+ 62 ° (C 0.5Methanol)

Elemental analysis value (%) (as C ₂₄ H ₁₉ N ₃ O ₁₀ SBr ₄ 2H ₂ O)

Calculated C 31.50 H 2.75 N 4.59 S 3.50 Br 34.92

Found C 31.01 H 2.82 N 4.93 S 3.79 Br 35.01

Silica gel thin layer chromatography using n-butanol-acetic acid-water (3: 1: 1, capacity ratio) as a developing solvent showed a single spot of Rf 0.47 in coloration by iodine vapor.

The infrared absorption spectrum (KBr purification method) of this compound in FIG. 1 is shown, the nuclear magnetic resonance spectrum is shown in FIG. 2, and the ultraviolet absorption spectrum in methanol is shown in FIG.

Example 2

750 ml of acetone was added to 1.5 L of the resin treatment solution obtained in Example 1 (containing 7.275 g of cephalosporin C), and the pH was adjusted to pH 8.5 with two prescribed sodium hydroxide solutions, followed by stirring at room temperature. Bromine phthalic acid was added slowly as it was for 30 minutes as a powder, it stirred for 1 hour, and reacted again.

During this time, the pH was maintained at 8.0-8.5.

After the reaction was completed, 6 mL of sulfuric acid solution was added to about 1.5 L of a solution obtained by removing acetone under reduced pressure, and the resulting precipitate was removed at a pH of 5.5.

Six sulfuric acid solutions were added to the filtrate again to pH 2.0 and cooled overnight.

The crystals were filtered off, washed with water and dried in vacuo at 40 ° C. to obtain 14.2 g of N- (tetrabromophthaloyl) sephalosporin C.

Purity 85%, yield 80% by the hydroxylamine method,

선광도 :

+52°(C 0.5메타놀)Ultraviolet absorption spectrum:

Radiance:

+ 52 ° (C 0.5Methanol)

Example 3

2 g of the crystal of N- (tetrabromophthaloyl) sephalosporin C obtained in Example 2 was dissolved in 40 ml of ethanol and ethanol solution of triethylenediamine (260 mg / 2.6 ml) was added with stirring to generate a white precipitate. After stirring for another 30 minutes, the precipitate was filtered off, washed with ethanol and dried in vacuo to obtain 1.94 g of N- (tetrabromophthaloyl) sephalosporin C triethylenediamine salt.

Purity 91%, yield 95.5% by ultraviolet amine method, ultraviolet absorption spectrum:

Example 4

1,000 ml of the culture filtrate containing 3.89 g of cephalosporin C was adjusted to pH 2.0 using 6 sulfuric acid solution, washed with 500 ml of butyl acetate, and then adjusted to pH 8.5 by adding 2 ml of sodium hydroxide solution. Acetone was added, and 17.36 g of tetrabrophthalic anhydride, which was well ground while stirring at room temperature, was added as it was over about 1 hour as a powder, and then the reaction was continued while stirring again for 3 hours.

In the meantime, 1 N sodium hydroxide solution was added timely to maintain pH 8.0-8.5.

After completion of the reaction, the reaction solution and the equivalent amount of water were added to adjust pH to 6.0 with 6 sulfuric acid solution to remove the precipitate formed. While stirring the filtrate, 6 ml sulfuric acid solution was added to adjust pH to 2.0, extracted 3 times with ½ volume of ethyl acetate, the extracts were combined, ½ amount of water was added, and 1 ml of sodium hydroxide solution was added to make pH 6.0. The obtained aqueous layer was added with 6 prescribed sulfuric acid solutions to pH 2.0 and cooled at 0-5 ° C. overnight.

The resulting crystals were filtered off and dried in vacuo at 40 ° C. to obtain 5.58 g of N- (tetrabromophthaloyl) sephalosporin C.

Purity 75%, yield 52%, ultraviolet absorption spectrum by hydroxylamine method:

Example 5

To 400 ml of the same resin eluate as used in Example 1 (containing 4.08 g of cephalosporin C), 200 ml of acetone was added thereto, followed by the addition of two prescribed sodium hydroxide solutions to adjust the pH to 8.5, and 5.62 g of thin crocrophthalic acid while stirring at room temperature. The finely ground powder of was slowly added and reacted for about 30 minutes.

6.19 g of crystals of N- (tetracrophthaloyl) sephalosporin C were obtained in the same manner as in Example 1 below.

Purity 90%, yield 83%, ultraviolet absorption spectrum by hydroxylamine method:

+65°(C 0.5메타놀) 이 결정 3g을 실시예 1과 동일하게 재결정하여 융점 178℃(분해)의 결정 2.2g을 얻었다.Radiance:

+ 65 ° (C 0.5 ethanol) 3 g of this crystal was recrystallized in the same manner as in Example 1 to obtain 2.2 g of a crystal having a melting point of 178 ° C (decomposition).

Ultraviolet spectrum:

+70°(C 0.5메타놀)Radiance:

+ 70 ° (C 0.5Methanol)

Elemental analysis value (%) (as C ₂₄ H ₁₉ O ₁₀ N ₃ SCL ₄ 3H ₂ O)

Theoretic C 39.09 H 3.42 N 5.70 S 4.34 Cl 19.23

Found C 39.03 H 3.45 N 5.72 S 4.86 Cl 19.62

FIG. 4 shows the infrared absorption spectrum of the compound (KBr purification method), FIG. 5 shows the nuclear magnetic resonance spectrum, and FIG. 6 shows the ultraviolet absorption spectrum in methanol.

Example 6

3.4 g of the crystal of N- (tetrabromophthaloyl) sephalosporin C obtained in Example 2 was suspended in 40 ml of ethylene chloride, followed by dissolving 2.04 g of dimethylaniline and 1.37 g of triethylenediamine.

The mixture was then cooled to -10 ° C, and 3.2 g of chlorotrimethylsilane was slowly added dropwise.

The reaction solution was warmed up to 15 ° C. and stirred at 15-18 ° C. for 1 hour. The reaction solution was cooled to -60 ° C, 30 g of phosphorus pentachloride was added and the mixture was kept at -45 to 50 ° C and stirred for 1 hour. Then, the temperature was gradually raised to -20 ° C and stirred again at -20 to -15 ° C for 30 minutes. .

After quenching to -60 ° C, 14ml of methanol and 0.3g of dimethylaniline were added while maintaining at -55 ° C to -60 ° C, the temperature was gradually raised to -20 ° C, and stirred at -20 ° C to -15 ° C for 30 minutes. The reaction solution was injected with vigorous stirring in a mixture of 40 ml of water and 4 ml of methanol, and ammonium carbonate was added to gradually increase the pH to 3.5.

After cooling overnight, the resulting crystals were filtered, washed with 40 ml of water-methanol mixture (1: 1 volume ratio), 20 ml of methanol, 20 ml of acetone, and dried in vacuo, followed by vacuum drying to obtain 0.70 g of a pale yellow 7-aminocephalosporonic acid (7ACA) crystal. Got.

(263nm, 1%탄산수소나트륨용액), 자외부흡수에 의한 순도 94%, 수율 72%Ultraviolet absorption spectrum:

(263 nm, 1% sodium hydrogen carbonate solution), purity 94% by ultraviolet absorption, 72% yield

Example 7

0.81 g of 7ACA crystals were obtained by the method of Example 6 from 2.4 g of N- (tetrachlorophthaloyl) sephalosporin C crystal obtained in Example 5.

Purity 96.5%, yield 94.8% by ultraviolet absorption

Claims (1)

Separosporin C of formula (I) is characterized by reacting the cephalosporin of formula (II) with phthalic anhydride derivative of formula (II) and crystallizing it with free acid to recover it. Preparation of Derivatives.

Wherein X represents a halogen atom, M represents a hydrogen atom or an alkali metal.

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