CEPHALOSPORIN C SOLUBILIZATION PROCESS
A method by converting cephalosprin C into a derivative soluble in an organic solvent by acylating the amine moiety in the side chain by a adipoyl alkanoyl group 2 ~ The halo-lower alkanoyl group solubilizer used is a moiety derived from a carboxylic acid in carrying one or two halogen atoms on the carbon atom in a. The preferred halogen atom is chlorine but it is also possible to use other halogens such as bromine and fluorine. Examples of suitable solubilizing groups groups comprise the chloroacetyl, dichloroacetyl, bromoacetyl, [...], a- [...], material is preferred. Is used the procedures of known conventional acylation of 1' skilled in the art by bringing the solubilizing group on the amine group of the side chain of the features | [...] C. Pages may be acylation using a halide or anhydride [...][...] acid. Results were obtained using a particularly good [...] chloride as chloroacetyl chloride. Surprisingly, the use of a mixed anhydride of chloroacetic acid such as acetic anhydride gives almost exclusively chloroacetic N- [...] C cephalosporin. The present invention provides a method for transforming cephalosprin C into a derivative soluble in an organic solvent method comprising acylating the amine moiety in the side chain adipoyl cephalosporin C ion alkanoyl group C2-C4 bone-halogenated or, a-dihalo. The present invention also provides a cephalosporin C soluble in an organic solvent having the formula O S <i \ HO, C-CH (CH,)-MHC-CH-CH Ihas j ii it f* O [...] c h wherein R is an alkanoyl group e2 ~ C4 a- halogenated or bone, oc-dihalo. The principal object in transforming cephalosprin C into a derivative soluble in an organic solvent is to aid to the isolation of cephalosporin C the fermentation broth. To achieve this effect, it is necessary to carry out the acylation reaction in the fermentation broth. Can be easily be done. The preferably the broth before acylation step and may also be concentrated to avoid the handling of large volumes of water. Can then extract [...][...] C the fermentation broth with a water-immiscible organic solvent. For water-immiscible solvent is a solvent whose solubility in water is limited, and not necessarily a totally insoluble solvent. For example, ethyl acetate, which is slightly soluble in water, is an excellent solvent for the extraction. Is extracted preferably at an acidic pH. The organic solvents that can be used in the extraction step include the lower alkyl esters, such as ethyl acetate, n-propyl acetate, isopropyl acetate, and the sec.-butyl acetate. It is sometimes advantageous to mix the ester with a tenth to a volume of a lower [...] as ethyl alcohol or n-butyl alcohol. The other appropriate solvents include nitriles as butyronitrile [...]and 1 1 and ketones, such as cyclohexanone and methyl isobutyl ketone. Preferred solvents are ethyl acetate, butyronitrile and cyclohexanone. May be separated N- [...] C the organic solvent by any method well known. For example, can be [...] the solution to prepare a salt of the cephalosporin derivative C which precipitates the organic solvent by stirring, preferably by cooling the solution. The may then submit the same isolated a cleavage reaction of the side chain to 7-aminocephalosporanic acid. The reaction may also be carried out in the organic solvent used for 1' extracting in that it is not necessary to isolate N- [...] C. The following examples will illustrate the preparation of cephalosporin C soluble and their extraction from the aqueous fermentation broth. In this is used two methods for determining the activity of C. the cephalosporin The first is a method which detects ultraviolet not only the cephalosporin C but also other related substances that will react with the acylating reagent. The second method is to Nicotinamide which is more specific cephalosporin C and utilizing the results of this method for calculating efficiencies. Example 1 It has been found, by analysis by ultraviolet, that a liter of fermentation broth [...] C that was filtered and processed by ion exchange resin contains 89 g of cephalosporin C-like substance and, by analysis to Nicotinamide it has been found that it contains 75.25 g cephalosporin C. At this liter of fermentation broth is added 1 liter of saturated sodium bicarbonate solution containing 25 ml of sodium hydroxide solution to 25 percent and 500 ml of acetone. In fifteen minutes is added 500 ml of a solution of 72 ml of chloroacetyl chloride in acetone. During this addition period is held the pH between 8.0 and 8.7 and is complied with the reaction mixture in a frozen bath to maintain the temperature in the range of 20° to 28 °C. The reaction mixture is stirred for another fifteen minutes after completion of the addition. The volume of the reaction mixture was 3070 ml. A 1520 ml of the original reaction mixture is added 300 ml of benzene and is adjusted pH of the mixture to 3.5 by adding 135 ml of hydrochloric acid 6 N. It has left phase separation to occur and is discharged the benzene thereby maintaining 1220 ml of aqueous solution. These 1220 ml was used in two equal portions that will be designated as A2 Al and solutions. Al A the solution is added an equal volume of acetate cl ' ethyl and is adjusted to pH 2.0 by adding hydrochloric acid N 6. The phases were separated and a second extraction with an equal volume of ethyl acetate. The extracts with ethyl acetate joined have been dried on sodium sulfate solution leaving 1200 ml of acetate [...] shown that the analysis ultraviolet contain 11.5 g activity. A the solution to ethyl acetate is added and 16.6 ml quinoline is' seeded onto the mixture. The mixture is stirred overnight at room temperature and then has cooled before filtering. The solid product was washed with 25 ml of acetone and dried in a vacuum oven to obtain 18.9 g of the quinoline salt N- [...] C. A the solution A2 is added 200 ml of acetone and 810 ml ethyl acetate. The adjusted to pH 2.0 by hydrochloric acid 6 N and left phase separation to occur. It has dried the solution to ethyl acetate on sodium sulfate to obtain 1080 ml of ethyl acetate to extract that the analysis ultraviolet shown contain 12.9 g activity. To this solution is added 18.6 ml quinoline and seeded onto the mixture. The mixture is stirred overnight and cooled before in separating solids by filtration. It has washed the solid product to 1' acetone and dried in a vacuum oven to obtain 17.8 g of the quinoline salt N- [...] C. Been treated another portion of 1520 ml of the original reaction mixture by an equal volume of ethyl acetate and is adjusted to pH 2.0 by adding 235 ml hydrochloric acid 6 N and separated phases. A second extraction with an equal volume of ethyl acetate. Aggregation of the extracted in ethyl acetate had a volume of 3000 ml and ultraviolet analysis showed that it contained 30.9 g activity. Example 2 At a liter of fermentation broth cephalosporin C resin-treated, containing 32.5 g activity ultraviolet analysis, and g 26.15 according to the analysis to Nicotinamide, is added 300 ml of acetone and 100 g of sodium bicarbonate sec while maintaining the mixture in an ice bath to 10 °C. The adjusted to pH 8.5 by adding a solution of sodium hydroxide at 25 percent. While continue to be cooled, is added a solution of 45 ml of dichloroacetyl chloride in [...] original 255 ml of acetone to the reaction mixture within 15 minutes. During the addition, is has maintained the pH to 8.0-8.8 by adding a solution of sodium hydroxide at 25 percent. When the addition has been completed, is removed from the mixture of the ice bath and stirred at 20 °C during 20 minutes. The pH was then adjusted to 3.5 with hydrochloric acid is added 6 [...] and 1700 ml ethyl acetate to extraction while adjusting the pH to 1.9 by hydrochloric acid N 6. It has stirred the mixture for 15 minutes and centrifuged to make the separation. The phase with ethyl acetate had a volume of 1940 ml and ultraviolet analysis highlighted 12.8 g of activity. A an extract with the d * ethyl acetate is added quinoline 41 ml into two approximately equal portions and seeded onto the mixture. After allowing the mixture to stand the refrigerator during the end of the week it is filtered. The solids were washed with acetone and dried at 35 °C in a vacuum oven to obtain 20.6 g of the quinoline salt N- [...] C. Example 3 It has repeated the procedure the Example 2 by replacing the dichloroacetyl chloride by the 2- [...] chloride. The temperature is raised to 40 °C during the addition and it has been difficult to adjust the pH between 7.5 and 9.3 which has been found. The product of the reaction was the quinoline salt N-(2- [...] ) C cephalosporin. Example 4 In according to the operational mode of the Example 2 using 4400 ml fermentation broth containing 145 g activity 1' ultraviolet analysis and 133 ml of chloroacetyl chloride was obtained to extract 11800 ml ethyl acetate containing 123.4 g N- [...] C. Example 5 It has cooled in an ice bath four liters of fermentation broth filtered having a pH of 4.4, cephalosporin C containing 5.77 mg per milliliter in accordance with the analysis to Nicotinamide. Cold At this broth is added 20 g of sodium borate decahydrate and 1200 ml acetone. It has adjusted the pH of the mixture to 8.5 by adding a solution of sodium hydroxide at 25 percent. At cooled mixture is added slowly a solution of chloride [...] 74 ml in 600 ml of acetone while maintaining the pH at 8.5. When the addition has been completed, the mixture is stirred further 30 minutes in maintaining the pH at 8.5. It has lowered the pH to 4.5 by adding sulfuric acid to 25 percent, bone added 6800 ml d * ethyl acetate and been lowered the pH to 2.0 [...] by adding sulfuric acid. After stirring 15 minutes, it was formed an emulsion and the phases do not separate from wanted. A the emulsion is added 200 ml of acetone and 150 ml of a commercial emulsion breaker. The phases were separated and is second extraction of the aqueous phase by ethyl acetate using 1 liter of ethyl acetate. Aggregation of the ethyl acetate phase had a total volume of 9.6 litres. It has concentrated to 4.5 litres and separated in two beakers. Is added in each beaker 70 ml quinoline and the mixture is stirred overnight. In a beaker has formed crystals while in the other it has separated oil. The supernatant liquid has been separated from oil by decantation and seeded. Then is concentrate the solution until solids begin to form. Seal [...][...] It has 1 mixture to [...] half where it had previously formed crystals. It has stirred the mixture and the air has cooled for one hour, and filtered. It has washed the solid salt quinoline N- [...] C with a small amount of acetone and dried in a vacuum oven to. e 400 C to obtain 15.3 g. Example 6 It has adjusted the pH of 10 liters of fermentation broth filtered to 6.2 and is concentrated in a flash [...] to 1.8 liter. The ultraviolet analysis has shown that the broth concentrate contains 46.5 g of cephalosporin C activity per liter. It has cooled the filtered broth concentrated in an ice bath and is added 18 g of sodium borate decahydrate and 540 ml of acetone. The adjusted to pH 8.5 with a sodium hydroxide solution at 25 percent. It has slowly added a solution of 56.5 ml of chloroacetyl chloride in 4085 ml acetone while maintaining the pH at 8.5-9.0 by adding a solution of sodium hydroxide at 25 percent. The mixture is stirred 15 minutes when the addition has been completed. At 1530 ml mixture is added ethyl acetate, is adjusted to pH 2.0 with sulfuric acid to 25 percent. Stirred the It has BAD ORIGINAL mixture 15 minutes and separated phases by centrifugation. Castings a second extraction with 1500 ml of acetate? ethyl and joins the two extracts with ethyl acetate to have a total volume of 3825 ml. It has concentrated extracts and joined to 750 ml is added 1 liter of ethyl acetate saturated with water. Quinoline is added (100 ml), the mixture is cooled in the refrigerator, it is filtered, and the solid is washed with 200 ml of acetone and dried at 40 °C in a vacuum oven overnight to obtain 51.8 g of the quinoline salt N- [...] C. Example 7 It has adjusted the pH of a solution of 4.6 g of sodium salt of cephalosporin C having a purity of 89 percent in 30 ml water, to 9.0 by adding a solution of sodium hydroxide at 20 percent. It has added dropwise a solution of 1.3 ml of chloroacetyl chloride in 8.7 ml acetone while maintaining the pH at 8.0-9.3 by adding 1' sodium hydroxide lo percent. The addition need approximately 15 minutes and is stirred the mixture which is only 5 minutes. The pH was then adjusted to 6.5 with hydrochloric acid 6 N, is added 80 ml ethyl acetate, and been lowered the pH to 2.0 with hydrochloric acid N 6. After stirring 5 minutes, the phases were separated, and is has dried the phase acetate [...] on sodium sulfate. Is added saturated methanolic solution of sodium acetate while the ' pH was at 7.2 which is passed to 7.7, with agitation. It has filtered the white solid that has separated, it is cooled and dried in a desiccator overnight to obtain 2.5 g of the sodium salt of N- [...] C. C The cephalosporin derivatives soluble in an organic solvent have the formula wherein R is an alkanoyl [...] a-halogenated or c ;, a-dihalo. The preferred halogen is chlorine and the array is the preferred [...] chloroacetyl group. In addition to obtain the free acid as shown in the above formula, they can be obtained in the form of salts. These salts can be alkali metal salts, alkaline earth metal, of amines of the carboxylic acids. The typical amines which have been used to form the salts are quinoline, cyclohexylamine, 5-ethyl-2-methylpyridine, 2-picoline, 3-picoline, 4-picoline, N-ethylmorpholine, N-methylmorpholine, 2,6-lutidine, N, N-diethylcyclohexylamine, 1 ' [...], N, N-dimethylbenzylamine, or N, N' -dibenzylethylenediamine. 1324419 N-haloalkanoyl derivatives of cephalosporin C ELI LILLY & CO 12 Oct 1970 [17 Dec 1969] 48320/70 Heading C2A Cephalosporin C is converted to an N-haloalkanoyl derivative of formula wherein R is an α-halo or α,α-dihalo-C 2 -C 4 alkanoyl group, by acylating the amino group in the adipoyl side chain with a corresponding haloalkanoyl halide or anhydride. The cephalosporin C may be treated in aqueous solution, e.g. a productive fermentation broth which preferably has been filtered and if necessary concentrated, to convert it to the said derivative which is easily extracted from the aqueous medium with a water-immiscible solvent such as ethyl acetate. The acylating agent is preferably an α-halo or α,α-dihaloalkanoyl chloride or mixed anhydride (e.g. acetic chloroacetic anhydride). The solvent extraction is best conducted at acid pH and immiscible solvents used include propyl or butyl acetate, nitriles and ketones. Alkyl esters may be mixed with one tenth to one volume of C 2 to C 4 alkanol. The derivative may subsequently be isolated by, e.g. adding an alkali to the solution to precipitate it as a salt. The formation of the derivative provides a convenient means for separating cephalosporin C from its productive fermentation broth. 1. A method of transforming cephalosporin C a derivative soluble in an organic solvent, of acylating the amine moiety in the side chain adipoyl C with a cephalosporin alkanoyl group C C2-4 has"[...] or oi, oc-dihalo. 2. A method as in claim 1, wherein the halogen is chlorine. 3. A method as in claim 1 or 2, wherein the halo-alkanoyl group is the group chloroacetyl. 4. A method as in claim 1,2 or 3, wherein the acylation is carried out in the aqueous fermentation broth. 5. C A cephalosporin soluble in an organic solvent, having the formula O H Ho-c-ch (ch0)-mhc-ch-ch [...] 0=C-N \ CH- O " Ch [...] 2-3 C02 h wherein R is an alkanoyl group C2 ~ C4 a- halogenated or, a-dihalo. 6. A compound as in claim 5, wherein 1' halogen is chlorine. 7. A compound as in claim 5 or 6, wherein R is a chloroacetyl group. 8. A therapeutically active composition comprising as an active ingredient a compound as in claim 5.