RU2166505C2 - Method of deacetyl-7-glutarylaminocephalosporinic acid concentrated solution preparing - Google Patents

Abstract

FIELD: chemical technology, antibiotics. SUBSTANCE: invention relates to preparing cephalosporin intermediate compound, in part, to deacetyl-7- glutarylaminocephalosporinic acid. The latter is prepared as a concentrated aqueous solution 10 wt. -%, not less, from an aqueous solution containing deacetyl-7 glutarylcephalosporinic acid in less concentrated solution by extraction with cyclohexanone as a solvent followed by reversed extraction into an aqueous phase. Concentrated solution is in the form that can be used for economical producing 7-aminocephalosporinic acid by the known methods. EFFECT: improved method of preparing. 3 cl, 4 ex

Description

 The invention relates to an improved method for producing concentrated aqueous solutions of 3-hydroxymethylcephalosporin from aqueous fermentation broths. More specifically, it relates to a method for extracting certain intermediates of 3-hydroxymethylcephalosporin cyclohexanone from aqueous fermentation broths, followed by back extraction with an aqueous solution to obtain a concentrated aqueous solution of the target product 3-hydroxymethylcephalosporin.

 7-Aminocephalosporic acid (7-ASA) and its various derivatives are valuable intermediates in the preparation of antibiotics belonging to the class of cephalosporins. 7-ASA and its derivatives having various substituents at position 3 can be obtained from the fermentation product cephalosporin C by known methods. For example, the conversion of cephalosporin C to 7-ACA can be carried out by known chemical and enzymatic methods.

 US Pat. No. 4,533,632 describes a method for producing deacetylcephalosporin C in an aqueous fermentation medium; it is reported that this product has significant advantages over cephalosporin C in the preparation of cephalosporin derivatives.

 Other sources, for example, US Pat. No. 5,424,196, describe enzymatic methods for converting deacetylcephalosporin C obtained in an aqueous fermentation broth into deacetyl-7-glutaryl ACA containing a 3-hydroxymethyl substituent.

 In US patents 4908444 and 5221739, as well as in J. Antibiotics. 47 (1): 64-71, 1994 describes a method for acetylation of deacetyl-7-glutaryl ACA in an aqueous solution with acetic anhydride to obtain 7-glutaryl ACA, which can then be hydrolyzed enzymatically to give 7-ACA in high yield.

 Despite the success in creating an enzymatic method, which involves the use of deacetyl-7-glutaryl ACA, obtained from an aqueous enzymatic broth, this method has not yet become industrial. One serious problem is that deacetyl-7-glutaryl ACA is obtained in a low concentration fermentation broth (≈ 1-3%) and cannot be acetylated at these concentrations in an economical manner known in the art. It is reported, for example, in the aforementioned patents that the acetylation reaction proceeds at high concentrations of deacetyl-7-glutaryl, ACA, preferably above 10%. So far, it has not been possible to obtain a concentrated (≥ 10 wt.% / Vol.) Aqueous solution of deacetyl-7-glutaryl ACA from the enzymatic broths of deacetylcephalosporin C so that the aqueous acetylation step is economical.

 Various organic solvents were used to extract cephalosporins from aqueous fermentation broths, for example, n-butyl acetate, methylene chloride, methyl iso-butyl ketone, chloroform and ethyl acetate. US Pat. No. 3,835,129 mentions that cyclohexanone is considered a good extractant for N-acylcephalosporin C, but in the case of large-scale production, problems have arisen with the formation of an emulsion. US Pat. No. 4,168,375 to the list of possible extractants for sulfonamide derivatives of cephalosporin C mentions cyclohexanone, but says nothing about the extraction of 3-hydroxymethylcephalosporins. US Pat. No. 3,986,644 teaches that cyclohexanone is used to extract certain phosphorus-containing amide derivatives of cephalosporin C, but even 3-hydroxymethylcephalosporins are not mentioned in this case.

 It was found that it is difficult or impossible to extract 3-hydroxymethylcephalosporins, such as deacetyl-7-glutaryl ACA, with common organic solvents used for other cephalosporins, such as N-blocked cephalosporin C, but that the use of cyclohexanone as an extractant according to this The invention allows to obtain from a fermentation broth a sufficiently concentrated aqueous solution of deacetyl-7-glutaryl ACA in order to make the above-described aqueous acetylation step economical and obtain 7-glutaryl ACA, which can then be converted to 7-ACA by known methods.

The present invention provides a method for producing a concentrated (10% w / v) aqueous solution of deacetyl-7-glutaryl ACA from an aqueous solution, preferably from an aqueous enzymatic broth and, most preferably, a processed fermentation broth containing deacetyl-7-glutaryl ACA in lower concentration, which includes the stages:
(a) preparing an aqueous solution, preferably a fermentation broth and, most preferably, a processed fermentation broth containing deacetyl-7-glutaryl ACA;
(b) concentrating the resulting solution with cyclohexanone at a pH of from about 1.5 to about 3 to extract deacetyl-7-glutaryl ACA into the cyclohexanone phase;
(c) separating the cyclohexanone phase from the aqueous phase;
(g) contacting the cyclohexanone phase with water at a pH of from about 5 to about 7.5; and
(e) separating the aqueous phase containing the desired concentrated solution of deacetyl-7-glutaryl ACA.

 As indicated above, the implementation of the enzymatic method for producing 7-ACA from deacetylcephalosporin C on an industrial scale requires sufficient yields at a key stage of acetylation, namely acetylation of deacetyl-7-glutaryl ACA in aqueous medium with acetic anhydride to obtain 7-glutaryl ACA. Sufficient yields at this stage depend on the use of concentrated (10 wt.% / Vol.) Aqueous solutions of the original deacetyl-7-glutaryl ACA. However, when this substance is obtained from the fermentation broth, it is contained in the broth in a lower concentration of the order of ≈ 1-3 wt.% / Vol., Therefore, it is necessary to extract desacetyl-7-glutaryl ACA from the fermentation broth and its concentration so that the acetylation step became economical and could compete with the methods of chemical synthesis of 7-ASA.

 According to a preferred embodiment of the method according to the invention, the treated fermentation broth containing deacetyl-7-glutaryl ACA is used as the starting product. As used herein, the term “processed fermentation broth” means a deacetylcephalosporin C fermentation broth that has been subjected to fermentation processes, for example, as described in US Pat. No. 5,424,196, into a broth containing deacetyl-7-glutaryl ACA. Desacetyl-7-glutaryl ACA should be contained in the treated broth in an amount that can be isolated, usually it is contained in a fairly low concentration of approximately 1-3% (w / v). Treated broth can be used immediately after treatment or after purification, for example, pH adjustment and filtration. The treated broth can also be processed on one or more columns filled with resins for further purification; this latter aqueous medium is sometimes called a “resin eluate”.

An aqueous solution, preferably a treated fermentation broth, is contacted with cyclohexanone as an extractant and mixed or shaken vigorously, two phases of solvents are formed. Before or during extraction with cyclohexanone, the pH of the solution or mixture is adjusted to a value of from about 1.5 to about 3, preferably about 2, with an inorganic acid, for example sulfuric, nitric or hydrochloric. The temperature of the cyclohexanone extraction step is not critical, but it is preferable to carry out the extraction at a temperature in the range of about 0 ^° C. to about room temperature, most preferably in the range of about 5-10 ^° C. After the extraction, the aqueous phase can be treated again with cyclohexanone to obtain additional amounts of deacetyl-7-glutaryl ACA and the cyclohexanone layers are combined. Extraction with cyclohexanone results in a high degree of extraction, usually over 90%, of deacetyl-7-glutaryl ACA from the broth.

 Since the acetylation step to obtain deacetyl-7-glutaryl ACA is carried out in an aqueous medium, it is necessary to back-extract the cyclohexanone layer containing deacetyl-7-glutaryl ACA into water to obtain the desired concentrated aqueous solution. The cyclohexanone phase is separated from the aqueous phase and is contacted with water at a pH of from about 5 to about 7.5 again to obtain two solvent phases, this time the aqueous phase contains deacetyl-7-glutaryl ACA. These solvent phases are separated and the desired aqueous solution is obtained. The basis for establishing pH at this stage is not critical, but it is preferable to use an acetate buffer such as sodium or potassium acetate.

 The temperature at this stage of extraction may be the same as that used for extraction with cyclohexanone. Reverse extraction with water results in high yields, again above 90%, of deacetyl-7-glutaryl ASA released in the aqueous phase.

 The method according to this invention provides for the production of aqueous solutions of deacetyl-7-glutaryl ACA with concentrations of 10%. Concentrations in the range of 10-20 wt.% / Vol. Can be obtained, with a concentration of 15% being easily achieved, and it is preferred in the subsequent acetylation step. The combination of extraction and reverse extraction allows you to remove salts formed during the fermentation process, as well as provide the required concentration of deacetyl-7-glutaryl ACA.

 As indicated above, cyclohexanone is found to be a unique solvent for the extraction of 3-hydroxymethylcephalosporins, such as deacetyl-7-glutaryl ACA. Conventional organic solvents used to extract other intermediate cephalosporin derivatives, such as N-blocked cephalosporin C, from fermentation broths are not suitable for 3-hydroxymethyl-cephalosporins. To illustrate this fact, the degree of extraction of deacetyl-7-glutaryl ACA with various industrial solvents is shown in the table at the end of the description.

 It is clear that cyclohexanone is the best extractant. It was also found that the lactonization of deacetyl-7-glutaryl ACA is significantly reduced when cyclohexanone is used as an extractant. The implementation of the complete extraction / reverse extraction process according to this invention provides a more than 10-fold increase in the concentration of deacetyl-7-glutaryl ACA and the resulting aqueous solution is compatible with the aqueous medium at the stage of acetylation, without requiring further processing.

 The following examples are provided to illustrate the invention.

 Example 1

 The conversion of cephalosporin C to glutaryl-7-ACA under the influence of oxidase D-amino acids. immobilized on Duolith A 365.

Batch conversion
66 g of cephalosporin C sodium salt dihydrate (purity 90.9%) is dissolved in 2 L of 25 mM potassium phosphate buffer containing 0.5 g of sodium metabisulfite.

 A solution of cephalosporin C is fed into a 3-liter reactor with 150 g of crude D-amino acid oxidase immobilized on Duolite A 365.

Thermostating is carried out at 25 ^o C with gentle stirring in a stream of oxygen entering through the lower diffuser at a speed of 1 rpm / min.

 The pH value is maintained by 7.5 by automatically adding 5% ammonia solution.

After 75 minutes, cephalosporin C completely transforms. The percentage composition of cephalosporin transformation products is as follows:
Glutaryl-7-ASA - 90.1%
Ketoadipyl-7-ASA - 6.2%
Glutaryl-7-ASA desacetyl - 1.1%
Glutaryl-7-ASA desacetoxy - 0.9%
Glutaryl-7-ASA fate oxide - 0.8%
Other β-lactams - 0.9%
In order to transform the remaining ketoadipyl-7-ACA into glutaryl-7-ACA, the solution obtained after thermostating is separated from the mass of the immobilized enzyme by filtration. For each liter of filtrate, with gentle stirring, add 10 ml of a 3.5% hydrogen peroxide solution. The mixture was left for 15 minutes at 25 ^° C., after which 0.5 g of sodium pyruvate was added.

The percentage at the end of the processing is as follows:
Glutaryl-7-ASA - 95.5%
Ketoadipyl-7-ASA - 0.1%
Glutaryl-7-ASA desacetyl - 1.1%
Glutaryl-7-ASA desacetoxy - 0.9%
Glutaryl-7-ASA fatefoxide - 1.5%
Other β-lactams - 0.9%
The enzyme load was tested for 100 cycles over 75-120 minutes.

 The total production was 4760 g of glutaryl-7-ACA, which is equivalent to 31.7 g of glutaryl-7-ACA per g of immobilized enzyme.

 Example 2

 A 2.7% aqueous solution (1180 ml) of deacetyl-7-glutaryl ACA and cyclohexanone (1180 ml) are mixed, 6N HCl is added to adjust the pH to 2.0. The mixture is fed to a separation funnel and extraction is carried out. The layers separate, leaving a cyclohexanone layer. The aqueous layer was treated with fresh cyclohexanone (2 x 1000 ml). The combined cyclohexanone layers (3220 ml) are analyzed, and a solution with a concentration of 0.928% is obtained. The degree of extraction of deacetyl-7-glutaryl ACA is 92%. The cyclohexanone extract (3215 ml) containing deacetyl-7-glutaryl ACA (0.928%) was back extracted with a 4.0 M sodium acetate solution (NaOAc) (90.0 ml). The pH of the mixture increases from 2.4 to 5.87. Separation of the layers gives an aqueous layer (151.5 ml) with a concentration of 18.1%. Reverse extraction of deacetyl-7-glutaryl ACA provides a degree of extraction of 92%. The total degree of extraction of deacetyl-7-glutaryl ACA is 84%.

 Example 3

 2.48% aqueous solution (910 ml) of deacetyl-7-glutaryl ACA and cyclohexanone (910 ml) are mixed, 6N HCl is added to bring the pH of the mixture, initially equal to 7.2, to 2.0. The mixture is fed to a separation funnel and subjected to extraction. The layers are separated, leaving a cyclohexanone layer. The aqueous layer was treated with fresh cyclohexanone (2 x 910 ml). The combined cyclohexanone layers (2825 ml) were analyzed; the solution concentration was 0.77%. The degree of extraction of deacetyl-7-glutaryl ACA is 96%. The cyclohexanone extract (2822 ml) containing deacetyl-7-glutaryl ACA (0.77%) was back extracted with 4.8 M potassium acetate (KOAc) (60.0 ml). The pH of the mixture increases from 2.5 to 6.45. The aqueous layer (122.5 ml) (79%) obtained after separation of the layers has a concentration of 14.1%. Reverse extraction with 1.0 M KOAc (2 x 7.0 ml) yields combined aqueous layers (18.2 ml) (7.6%). The degree of extraction of deacetyl-7-glutaryl ACA after reverse extraction is 87.6%. The total degree of extraction of deacetyl-7-glutaryl ACA is 84%.

 Example 4

 Laboratory experience using oxidized UF (Ultrafilter) through which a solution of deacetyl-7-glutaryl ACA was passed. Using a DeLaval separator, filtered fermentation broth (10.19 L) containing deacetyl-7-glutaryl ACA (2.41%) was extracted at pH 2.0 with cyclohexanone (10.19 L). Each extraction is carried out at a volume of 1.9 liters. A fresh portion of the broth passed through the filter is extracted with enriched cyclohexanone from the previous extraction, which simulates a continuous process. A portion of the enriched cyclohexanone (3830 ml) from the final solution after the last stage has a concentration of 1.18% and is treated with 4.0 M NaOAc (150 ml) to obtain an aqueous solution of deacetyl-7-glutaryl ACA (250 ml) (16.3%) . The degree of extraction after reverse extraction is 90%.

Claims (3)

 1. The method of obtaining concentrated not less than 10 wt.% / Vol. an aqueous solution of deacetyl-7-glutarylamino-cephalosporic acid from an aqueous fermentation broth containing desacetyl-7-glutarylamino-cephalosporic acid in a lower concentration, comprising the following steps: (a) preparing an aqueous fermentation broth containing deacetyl-7-glutarylaminosulfonamide-10% by weight. / vol., (b) mixing the resulting broth with cyclohexanone at a pH of about 1.5 - 3 to extract deacetyl-7-glutarylaminocephalosporic acid into cyclohexanone phase: (a) separating the cyclohexanone phase from the aqueous phase, (d) contacting the resulting cyclohexanone phase with water at about pH 5 - 7.5, and (d) separating the aqueous phase containing the concentrated solution of the desired 7-desacetyl-glutarilaminotsefalosporinovoy acid.  2. The method according to claim 1, characterized in that the aqueous fermentation broth in stage (a) is a processed fermentation broth.  3. The method according to claim 1 or 2, characterized in that stage (g) involves the use of acetate buffer.

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