FIELD OF THE INVENTION

This invention relates to an improved process for obtaining concentrated aqueous solutions of 3-hydroxymethylcephalosporin compounds from aqueous fermentation broths. More particularly, it relates to the cyclohexanone extraction of certain 3-hydroxymethylcephalosporin intermediates from aqueous fermentation broths followed by back-extraction into an aqueous solution to provide a concentrated aqueous solution of the desired 3-hydroxymethyl-cephalosporin intermediate.

BACKGROUND OF THE INVENTION

7-Aminocephalosporanic acid (7-ACA) and various derivatives thereof are known important intermediates in the production of antibiotics of the cephalosporin class. The 7-ACA and derivatives thereof having various 3- substituents may be obtained from fermentation-derived cephalosporin C by known processes. For example, the conversion of cephalosporin C to 7-ACA can be carried out by chemical process steps, but such chemical processes involve use of environmentally unfriendly reactants (chlorinated solvents, chlorosilanes, dimethylaniline) and severe operating conditions (very low

temperatures such as -50°C).

Many attempts have been made to convert cephalosporin C to 7-ACA by an enzymatic process which would be more favorable to the environment, but to date, none of the proposed processes have been commercially feasible.

U.K. Published Patent Application 2,060,610A describes a process for obtaining desacetyl cephalosporin C in an aqueous fermentation broth and reports that this product has significant advantages over cephalosporin C in the production of cephalosporin derivatives.

Other references such as U.S. Patent 5,424,196 disclose enzymatic processes for converting desacetyl cephalosporin C produced in an aqueous fermentation broth to desacetyl 7-glutaryl ACA containing a 3-hydroxymethyl substituent. As used herein and in the claims, the term "processed fermentation broth" is meant to indicate a desacetylcephalosporin C fermentation broth which has been converted by such enzymatic processes to a broth containing desacetyl 7-glutaryl ACA.

Takeda in U.S. Patent 4,908,444 and Lilly in U.S. Patent 5,221,739 and J, Antibiotics 4Z(1):64-71, 1994, disclose a process for acetylating desacetyl 7- glutaryl ACA in aqueous solution with acetic anhydride to produce 7-glutaryl ACA which can then be enzymatically hydrolyzed to 7-ACA in high yield.

Despite the progress which has been made toward an enzymatic 7-ACA process which would use desacetyl 7-glutaryl ACA produced from an aqueous

fermentation broth, such a process is still not commercially feasible. One serious problem is that the desacetyl 7-glutaryl ACA is obtained in the fermentation broth in low concentrations (-1-3%) and cannot be economically acetylated at these concentrations by the prior art methods. It is reported, for example in the above-mentioned Lilly patent and I. Antibiotics paper, that the

acetylation reaction works best at high concentrations of desacetyl 7-glutaryl ACA, preferably above 10%. So far it has not been possible to obtain an

adequately concentrated (> 10% weight/volume) aqueous solution of desacetyl

7-glutaryl ACA from fermentation broths producing desacetyl cephalosporin C to allow the aqueous acetylation step to be economically feasible.

A variety of organic solvents have been used in the extraction of cephalosporin products from aqueous fermentation broths, e.g. n-butyl acetate, methylene chloride, methylisobutyl ketone, chloroform and ethyl acetate. U.S. Patent 3,835,129 mentions that cyclohexanone was considered to be a good N-acyl cephalosporin C extraction solvent, but that it presented emulsion problems when used in large scale operations. U.S. Patent 4,168,375 discloses cyclohexanone in a list of possible extraction solvents for sulfonamide derivatives of cephalosporin C, but does not mention extraction of 3- hydroxymethylcephalosporins. Cyclohexanone is used to extract certain cephalosporin C phosphorous amide derivatives in U.S. Patent 3,986,644, but again no mention is made of 3-hydroxymethylcephalosporins.

The present inventors have found that 3-hydroxymethylcephalosporins such as desacetyl 7-glutaryl ACA are difficult or impossible to extract with common organic solvents used for other cephalosporin compounds such as

N-blocked cephalosporin C, but that use of cyclohexanone as the extracting solvent according to the process of the present invention allows a sufficiently concentrated aqueous solution of desacetyl 7-glutaryl ACA to be obtained from the fermentation broth to economically practice the aqueous acetylation step to give 7-glutaryl ACA and then 7-ACA.

SUMMARY OF THE INVENTION

The present invention provides a process for preparing concentrated (>

10% weight/volume) aqueous solutions of desacetyl 7-glutaryl ACA from an aqueous fermentation broth of desacetyl cephalosporin C that has been converted to desacetyl 7-glutaryl ACA. The concentrated aqueous solution provided by the invention can be economically converted by the aqueous acetylation route using acetic anhydride under basic conditions.

More particularly, the present invention provides a process for

preparing a concentrated (> 10% weight/volume) aqueous solution of

desacetyl 7-glutaryl ACA from an aqueous solution such as a processed fermentation broth containing the desacetyl 7-glutaryl ACA in lower concentration, which comprises the steps of:

(a) providing an aqueous solution, preferably a processed fermentation

broth, containing desacetyl 7-glutaryl ACA;

(b) contacting the solution with cyclohexanone at a pH of from about 1.5 to about 3 so as to extract the desacetyl 7-glutaryl ACA into the cyclohexanone solvent phase;

(c) separating the cyclohexanone solvent phase from the aqueous phase;

(d) 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 desacetyl 7-glutaryl ACA.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, carrying out the enzymatic process for preparing 7- ACA on a commercial scale requires economical yields in the key acetylation step, i.e. the acetic anhydride aqueous acetylation of desacetyl 7-glutaryl ACA to 7-glutaryl ACA. Economical yields in this step are dependent on using

concentrated (> 10% weight/ volume) aqueous solutions of the desacetyl 7-

glutaryl ACA starting material. When this material is obtained from a fermentation broth, however, it is present in the broth at a much lower

concentration, on the order of ~ 1-3% weight /volume, so it is necessary to extract the desacetyl 7-glutaryl ACA from the fermentation broth and

concentrate it for the acetylation step to be practical and competitive with chemical synthesis methods for preparing 7-ACA.

The process of the present invention uses as the starting material a processed fermentation broth containing desacetyl 7-glutaryl ACA. This compound must be present in the processed broth in a recoverable amount, and is generally present in a fairly low concentration of from about 1-3%. The processed broth can be used directly or after being subjected to purification

steps such as pH adjustment and filtration. The processed broth may be also subjected to treatment by one or more resin columns to further purify it; this latter aqueous medium is sometimes referred to as "resin eluate".

The aqueous processed fermentation broth is contacted with cyclohexanone as the extraction solvent and stirred or otherwise vigorously mixed with the broth to give two solvent phases. Before or during extraction with the cyclohexanone, the solution or mixture is adjusted to a pH of from about 1.5 to about 3, preferably about 2, with an inorganic acid such as sulfuric acid, nitric acid or hydrochloric acid. The temperature for the cyclohexanone extraction step is not critical, but it is preferred to do the extraction at a

temperature in the range of from about 0°C to about room temperature, most

preferably in the range of about 5-10°C. Following the extraction, the aqueous

phase may be extracted again with cyclohexanone to obtain additional

amounts of desacetyl 7-glutaryl ACA and the cyclohexanone layers combined.

The cyclohexanone extraction results in a high percentage, generally over 90%, of the desacetyl 7-glutaryl ACA being extracted from the broth.

Since the acetylation step to produce 7-glutaryl ACA is done under aqueous conditions, it is necessary to back-extract the cyclohexanone layer containing the desacetyl 7-glutaryl ACA with water to get the desired concentrated aqueous solution. The cyclohexanone phase is separated from the aqueous phase and contacted with water at a pH in the range of from about 5 to about 7.5 to again give two solvent phases, this time the aqueous phase containing the desacetyl 7-glutaryl ACA. The solvent phases are separated to give the desired aqueous solution. The base used for pH adjustment in this step is not critical, but it is preferred to use an acetate buffer such as sodium or potassium acetate. The temperature for this extraction step can be the same as that used for the cyclohexanone extraction. The back-extraction with water gives high yields, again over 90% of the desacetyl 7-glutaryl ACA being recovered in the aqueous phase.

The process of the present invention provides aqueous desacetyl 7-

glutaryl ACA solutions in concentrations of > 10%. Concentrations in the

range of 10-20% weight/volume are achievable, with 15% concentrations being easily obtained and preferred for the subsequent acetylation step. The extraction and subsequent back-extraction process in combination allow for

removal of fermentation salts as well as concentration of the desacetyl 7- glutaryl ACA.

As mentioned above, cyclohexanone was found to be a unique solvent for extraction of 3-hydroxymethylcephalosporins such as desacetyl 7-glutaryl ACA. Common organic solvents used to extract other cephalosporin intermediates such as N-blocked cephalosporin C from fermentation broths are not useful for 3-hydroxymethylcephalosporins. To illustrate this, we have found that n-butyl acetate, a common solvent for cephalosporin recovery, allows only 1% of the desacetyl 7-glutaryl ACA to be recovered from the broth compared to 85-95% with cyclohexanone. Similarly, methyl isobutyl ketone, another common cephalosporin solvent, allows only 5% of the desacetyl 7- glutaryl ACA to be recovered, again compared to 85-95% recovery with cyclohexanone. It was also found that lactonization of the desacetyl 7-glutaryl ACA was significantly reduced using cyclohexanone as the extraction solvent. The overall extraction/back-extraction process of the present invention gives a greater than 10-fold concentration of the desacetyl 7-glutaryl ACA and the resulting concentrated aqueous solution is compatible with the aqueous acetylation reaction process without the need for further processing.

The following examples are used to illustrate the invention.

Example 1.

A 2.7% desacetyl 7-glutaryl ACA aqueous solution (1180 ml) and cyclohexanone (1180 ml) were stirred and 6 N HC1 was added to adjust the mixture to pH 2.0. The mixture was transferred to a separatory funnel and extracted. Layers were separated and cyclohexanone layer was retained. The

aqueous layer was extracted with fresh cyclohexanone (2 x 1000 ml). Combined cyclohexanone layers (3220 ml) were evaluated and a 0.928% solution was obtained. The desacetyl 7-glutaryl ACA was extracted with 92% efficiency.

Cyclohexanone extract (3215 ml) containing desacetyl 7-glutaryl ACA (0.928%)

was back extracted with 4.0 M sodium acetate (NaOAc) (90.0 ml). Initial pH of the mixture rose from pH 2.4 to pH 5.87. Separation of the layers afforded an aqueous layer (151.5 ml) at an 18.1% concentration. Back extraction of desacetyl 7-glutaryl ACA was accomplished with 92% efficiency. Overall efficiency of desacetyl 7-glutaryl ACA extraction was 84%.

Example 2

A 2.48% desacetyl 7-glutaryl ACA aqueous solution (910 ml) and cyclohexanone (910 ml) were stirred and 6 N HC1 was added to adjust the mixture from pH 7.2 to pH 2.0. The mixture was transferred to a separatory funnel and extracted. Layers were separated and cyclohexanone layer was retained. The aqueous layer was extracted with fresh cyclohexanone (2 x 910 ml). Combined cyclohexanone layers (2825 ml) were evaluated and a 0.77% solution was obtained. The desacetyl 7-glutaryl ACA was extracted with 96% efficiency. Cyclohexanone extract (2822 ml) containing desacetyl 7-glutaryl

ACA (0.77%) was back extracted with 4.8 M potassium acetate (KOAc) (60.0 ml).

Initial pH of the mixture rose from pH 2.5 to pH 6.45. Separation of the layers afforded an aqueous layer (122.5 ml)(79%) at a 14.1% concentration. Back extraction with 1.0 M KOAc (2 x 7.0 ml) afforded combined aqueous layers (18.2 ml) (7.6%). Back extraction of desacetyl 7-glutaryl ACA was accomplished with

87.6% efficiency. Overall efficiency of desacetyl 7-glutaryl ACA extraction was

84%.

Example 3

Laboratory Scale Using Oxidized UF (Ultrafilter. Permeate Containing Desacetyl 7-Glutaryl ACA Solution: Using a DeLaval separator, filtered fermentation broth (10.19 L) containing desacetyl 7-glutaryl ACA (2.41%) was extracted at pH 2.0 with cyclohexanone (10.19 L). Each extraction was carried out on 1.9 L scale. Fresh permeate was extracted with rich cyclohexanone from the previous extraction to simulate a continuous extraction. A portion of the rich cyclohexanone (3830 ml) from the final solutions (1.18%) was back extracted with 4.0 M NaOAc (150 ml) to afford an aqueous solution of desacetyl 7-glutaryl ACA (250 ml) (16.3%). Back extraction was accomplished in 90% efficiency.