IL32407A - Process for preparing 7-aminocephalosporanic acid - Google Patents

Description

IMPROVED PROCESS FOR PREPARING 7-AMINOCEPHALOSPORANIC ACID JT D01>HSS1 _|·»ΒΚ-7-Π Π301ΠΠ nj3n> j»>D1«D0 The present invention provides an improved process for preparing 7-aroinocephalosporanlc acid (7-ACA) from cephalosporin C and salts thereof.

Cephalosporin C is obtained by fermentation as de- scribed in British patent specification 8lO, 196 published March 11 , 1959 . Cephalosporin C has a low order of biological activity, and it has been found necessary to chemically-convert cephalosporin C into derivatives such as cephalothln ahd.cep- haloglycln in order to obtain active antibiotics. One step in this chemical modification involves cleavage of the amldo group In the 7-Posltion of the cephalosporin nucleus to obtain 7-ACA.

A commercially significant process for the cleavage of the 7-amido group of cephalosporin C is described in -t tirfred-- Israel 1™54 -S-t-afcee Patent ^,ΐ-βδ-,-^Ιϊ · Ir* accordance with that process, cephalosporin C is treated with a reagent such as nitrosyl chloride to prepare a cyclic intermediate which is then subjected to hydrolytlc cleavage to yield 7-ACA. In-tfn-t%ed- Israel 18883 S at-es-Patent -5-,-^6 -953~ there is described an improvement on the above process wherein the cephalosporin C is treated with nitrosyl chloride in a mixed solvent system, the intermediate is then treated with methanol, and the 7-ACA is precipitated from the methanol solution by the addition of ammonium hydroxide .

Both of the above processes result in the production of a 7-ACA containing impurities which Interfere with the subsequent N-acylatlon that is necessary to. obtain biologically active compounds. Therefore, 7-ACA prepared by either of these processes must be subjected to a purification procedure prior to acylation. One such purification procedure Involves carbyl sulfonic acid or nitric acid. Such purification procedures result in losses of 7-ACA and increase the cost of the biologically active derivatives. The advantages to be realized by the elimination of the purification step are ap- parent.

We have now discovered an improved process for the preparation of 7-ACA which yields a product whose purity is such that no further purification is necessary. In accordance with this Improved process cephalosporin C or a salt thereof is treated with a nitrosatlng agent, a carbocyclic arenediazonium salt, or a substance affording a positive halogen, at a temperature of less than about 60°C. in a solvent and the reaction mixture is treated with a lower alkylene oxide containing two to four carbon atoms. This can be done by first treating the reaction mixture with water, a lower alkanol containing one to three carbon atoms, or mixtures thereof, and then adding a lower alkylene oxide to precipitate the 7-ACA. Alternatively, the reaction solvent can be removed by evaporation and the residue treated with the alkylene oxide. The 7-ACA from our process is sufficiently pure that it can be acylated to obtain biologically active compounds of high quality in good yield. Thus, the improved process eliminates the costly purification step.

More particularly, this invention provides a process for the preparation of 7-aminocephalosporanic acid, which comprises treating cephalosporin C or a salt thereof with a reagent comprising a nitrosatlng agent, a carbocyclic arenediazonium salt, or a substance affording positive halogen, at a temperature of less than about 6o°C . in a solvent, and treat-ing the reaction mixture with a lower alkylene oxide contain if halosporanlc acid therefrom.

In a further more specific embodiment of the Invention there Is provided a process for the production of 7-amlno^- cephalosporanlc acid which comprises treating a solution of cephalosporin C sodium salt monohydrate in a mixture of formic acid and acetonitrile with from 2 to 2 .5 moles of nitrosyl chloride at a temperature within the range of -10°C. to 4-5°C, pouring the reaction mixture into methanol, and adding propylene oxide to precipitate the product 7-amlnocephalosporanlc acid.

The first step in the process of this invention comprises treating cephalosporin C or a salt thereof with a nitro- satlng agent, a carbocyclic arenediazonium salt, or a substance affording positive halogen, at a temperature of less than about 6o°C . Cephalosporin C is 7- ( 5' -amino-N' -adipamyl)-cephalosporanic acid. It can be obtained by fermentation as described in British patent specification 810 , 196. Cephalosporin C may be employed in the form of a free acid or as a salt thereof. Suitable salts include metallic salts such as the sodium, potassium, and lithium salts, and ammonium and substituted ammonium salts.

Suitable reagents for reacting with the cephalosporin C Include the following: (l) nitrosatlng agents, such as nitrosyl chloride, nitrosyl bromide, nitrous acid, nitrogen dioxide, nitrogen tetroxlde, nitrogen trioxlde, alkyl nitrites, N-nitroso-5-nitrocarbazole, nitrosylsulfuric acid, nitrosyl fluoborate, nitrosyl hexafluorophosphate , and the like; (2 ) substances affording positive halogen under the reaction conditions, such as N-bromosucclnlmide , N-chlorosuccinimide , N-bromophthalimide , N-chlorophthalimide, N-bromoaeetamlde , and chloride, naphthalenedlazonium chloride, and the like. The preferred reagent for the process is nitrosyl chloride.

The molar ratio of reagent to cephalosporin 0 should be at least 1 and is preferably from about 1.5 to about The use of a larger excess is not recommended since any un- reacted reagent should be destroyed or removed at the completion of the reaction.

The reaction may be run at temperatures below about 60°C. It is preferred to operate at temperatures below about 20°C, and still more preferable to operate at about 0°0. , for example, from about -10° to about -5°C.

Solvents fo 17054 Patents -5,-188 511 and l affect the yield of 7-AOA but will not substantially affect the purity of the product obtained. In order to obtain optimum yields it is preferred to use a mixed solvent system of formic acid with either nitromethane , acetonitrile , or a mixture of nitromethane and 2-nltroproPane.

The reaction proceeds rapidly at the temperatures specified and is complete within a matter of minutes after addition of the treating reagent is complete. Thus, for example, reaction times on the order of 5 to 15 minutes are sufficient. If temperatures of less than about -10°C . are employed, the reaction proceeds more slowly and longer reaction times are needed.

In one embodiment of the process of this invention, the reaction intermediate is treated with water or a lower alkanol containing one to three carbon atoms such as, for example, methanol, ethanol, or propanol. Mixtures may also be used. Methanol is preferred. This treatment may be effected reduced pressure, and adding the water or lower alkanol to the residue. If this procedure Is followed, sufficient water or alcohol is added to dissolve the residue. Alternatively, a large volume of alcohol may "be mixed with the reaction mixture without evaporation of the solvent. In either event there le obtained a solution of 7-ACA in essentially water or an alcohol.

If the solvent is removed prior to treating with water or an alcohol, it is preferred to use an additive to react with excess cleavage reagent prior to solvent evaporation. If the reaction mixture .is mixed with a large volume of alcohol, the alkylene oxide may be present in the alcohol at the time of mixing, or the alkylene pxide may be added later. No essential differences in the results obtained by the two procedures have been observed.

Heretofore, the 7-ACA has been precipitated from solution by the addition of a base such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide to a pH of about 5.5. This procedure results in the precipitation of impur-itles with the 7-ACA so that It is necessary to purif the 7-ACA before it can be used in the preparation of biologically-active derivatives.

In accordance with the process of this invention lower alkylene oxide containing two to four carbon atoms is added to precipitate the 7-^ACA. Examples of suitable alkylene oxides include ethylene oxide, 1,2-propylene oxide, butene-1 oxide, butene-2 oxide and oxirane . In addition, ^ -propio-lactone may also be used in our process. This is not aur- Λ oxetane prising since ^P-propiolactone may be considered to be -ox-i-ran-e— 2-one. Ethylene oxide and 1,2-propylene oxide are preferred. # alkylene oxide has not been definitely established, it appears that the addition of the oxide induces a slow crystallization of 7-AOA. This is in contrast to the direct neutralization of soluble 7-ACA salts and rapid precipitation that occurs upon the addition of a base. When rapid precipitation occurs there is a tendency for dissolved substances to be trapped within the precipitate and to come down with it, thus causing contamination of the precipitate. On the other hand, when slow crystallization and precipitation occur trapped particles have a greater opportunity to escape so there is less chance for contamination.

The amount of alkylene oxide necessary to bring about complete precipitation will depend upon the oxide chosen and the concentration and composition of the solution of 7-AOA. The proper amount of oxide. can be determined on a trial and error basis with a minimum of experimentation. In general, good results are obtained by using from about 5 to about 50 moles of oxide per mole of starting cephalosporin compound. It has been found that it is better to add the oxide rapidly all at once rather than slowly over a period of- time .

It is also advantageous to add a small amount of water a few minutes after the oxide addition in those cases where an alcohol is used to treat the intermediate. Such a procedure results in a purer product. An optimum amount of water appears to be approximately an equal amount, by volume, to the oxide added. More or less than this can also be used.

In a particularly preferred embodiment of the process of this invention, a solution of cephalosporin C sodium salt monohydrate in a mixture of formic acid and acetonitrile temperature of from -10° to *5°C. At the completion of the reaction the mixture is poured Into a large volume of methanpl, and propylene oxide is then added to precipitate the 7-ACA.

The first embodiment of the process of this inven-r tion will be further illustrated by the following examples.

Example 1 To a solution of 168 g. of cephalosporin C sodium salt monohydrate in 20 ml. of formic acid was added 1 1. of a 1 : 1 by volume mixture of nitromethane and 2-nltrppropane .

The solution was cooled to -8°C. and a solution of 39 ml. of nitrosyl chloride in 240 ml. of the nitro«lkane mixture was added over 7 minutes. This mixture was stirred at 0°C. for. an additional 15 minutes and then poured into 5 · 4 1 · of methanol. After stirring the methanol solution for 5 minutes, 250 ml. of propylene oxide was added to precipitate the 7-AOA and the temperature was maintained at 10°C. for 1 hour. The product was collected by filtration, washed with methanol and dried at 42 °0 . in a vacuum oven. The 7~ACA was obtained in 49. 8 percent yield and had a purity of 94.2 percent as de- termlned by ultraviolet spectoscopy.

Examples 2 - 15 A series of cleavage runs was made wherein 28 g. of cephalosporin C sodium salt monohydrate was treated with 6.5 ml. of nitrosyl chloride as in Example 1 in a solvent consisting of formic acid and a diluent. The 7-ACA was recovered by mixing the reaction mixture with a large volume of methanol or ethanol as in Example 1. The results are found in Table 1 . In the Table, the symbol R-NO2 represents the •nitroalkane mixture used in Example 1 . In Examples 2 - 4 and 6 - 11 the alkylene oxide was present in the alcohol at and 12 - 15 the alkylene oxide was added about 5 minutes after the reaction mixture was added to the alcohol. The temperature shown is the temperature at whioh the mixture was held after addition of alkylene oxide and prior to filtration.

Only 14 g. of cephalosporin C was used in Example 14. In Examples 12 - 14 the wate shown in the Table was added after the oxide.

Table 1 2 70 R-N02, 210 CH30H, 900 Propylene oxide, 20 3 70 R-N02, 210 CH^OH, 900 Propylene oxide, 40 70 R-N02, 210 CH^OH, 9,00 Propylene oxide , 100 70 R-N02, 210 CE^OH, 900 Propylene oxide , 40 6 70 R-N02., 210 CH^OH, 5OO Propylene oxide , 100 7 70 R-N02, 210 CH30H, 5OO β -Propiolactone, 100 8 70 R-N02, 210 CH30H, 900 Ethylene oxide , 20 9 70 R-N02, 210 CH OH, 900 Ethylene oxide , 40 70 R-N02, 210 CH30H, 900 Ethylene oxide , 60 11 70 R-N02, 210 CH30H, 600 β-Propiolactone , 60 12 70 R-N02, 210 CH30H, 900 Propylene oxide , 50 H20, 50 13 80 CH3CN, 200 Ci^OH, . 9O.O Propylene oxide , 50 ¾o, 50 14 50 Cft^CN, 100 C2H50H, 5Ο Propylene oxide, 25 ¾o, 25 100 Ci^CN, 200 CH30H, 900 2,2 -Dime t hoxy pr opane , 200 Propylene oxide , 50 Example l6 A 28 g. sample of cephalosporin C sodium salt mono- hydrate was treated with 6.5 ml- °f nitrosyl chloride as described in Example 1 using 100 ml. of formic acid and 200 ml. of acetonitrile as solvent. At the completion of the reaction the solvent was evaporated at reduced pressure and the residue was taken up in 200 ml. of ice water. To this solution was added 50 ml. of propylene oxide and the mixture was allowed to stand at 12°C. The product was recovered by filtration, washed with water, and dried. The purity was 99.0 percent.

In another embodiment of the process of this invention, upon completion of the cleavage reaction the solvent is removed by evaporation, preferably under reduced pressure, to leave a residue which is then treated with a lower alkylene oxide. In a particularly preferred embodiment of the process of this invention an additive which reacts with excess cleavage reagent is employed prior to the evaporation. The use of such an additive results in increased yielcls.

Heretofore, the residue from the evaporation has been dissolved in water or a lower alkanol and the 7-ACA recovered from this solution. In accordance with our process this residue is treated with a lower alkylene oxide containing two to four carbon atoms. No water or alcohol is necessary. Lower alkylene oxides that may be employed include, for example, ethylene oxide, 1,2-propylene oxide, butene-2 oxide, butene-1 oxide, and oxi-ra-n-e. Ethylene oxide and 1,2-propylene oxide are preferred.

Treatment of the residue with an alkylene oxide may be effected by the direct addition of the oxide to the residue. However, it is preferred to use an Inert solvent for this is added to the residue. Alternatively, the residue may first be dissolved or dispersed in the solvent, followed by addition of the oxide to the mixture. In either case there is obtained a slurry of product 7-ACA in the solvent. This product may be collected by any suitable means such as filtration or cen-trifugatlon and dried.

The solvent to be used in this step should be inert, and a poor solvent for 7-ACA. Preferred solvents are diluents Israel for the cleavage reaction as described in -U-.-θ-. Patent 18883 ·3Τ367Γ 53-» Acetonitrile is particularly preferred. Other acceptable solvents Include esters such as ethyl acetate and n-butyl acetate, ketones such as acetone and methyl ethyl ketone, and ethers such as dioxane and tetrahydrofuran . Solvents in which 7-ACA is quite soluble, such as dimethylform-amide and dimethylacetamide , are not well suited for use alone, but may be used advantageously in combination with a non-solvent for 7-ACA. When such combinations are used it is preferred to dissolve the residue in a small amount of the solvent, then add a non-solvent, such as n-butyl acetate, con-tainlng the oxide. The viscous residue is difficult to break up and disperse when a non-solvent is used alone . This problem is overcome by the use of a combination of a good solvent and a non-solvent.

The total function of the alkylene oxide is not clear; therefore, it is. difficult to know how much oxide should be added. The proper amount of oxide can be determined on a trial and error basis with a minimum pf experime tation. In general, good results are achieved using from about 2 to about 50 moles of oxide per mole of starting cephalosporin compound. This is not to imply that the function of the oxide being cleaved; it is merely a convenient way to express the amount of oxide used. Higher yields are obtained if the oxide is added rapidly all at once rather than added slowly over a period of time.

The 7-AC obtained in this manner has a high purity except for certain water-soluble impurities that are present in the precipitate. These water-soluble Impurities may be removed from the 7-ACA by a water wash after the 7-ACA has been thoroughly dried. In this way a high purity 7-ACA is obtained in good yield without the necessity of treating an Intermediate with water or an alcohol or precipitation by the addition of a base. Many acylations of 7-ACA are conducted in an aqueous medium and the water-soluble impurities do not interfere in such case. Therefore, it is not always necessary to water-wash the product .

This embodiment of our process will be further illustrated by the following examples: Example 17 A solution of 56 grams of cephalosporin C sodium salt monohydrate in 200 ml. of formic acid and 280 ml. of acetonltrile was cooled to -8°C. and 100 ml. of acetonitrlle containing 12 . 6 ml. of nitrosyl chloride was added over four minutes. The reaction mixture was stirred at 0°C. for an additional 11 minutes and then 8 g. of ammonium sulfamate was added gradually with cooling to keep the reaction temperature below 4°C The reaction mixture was concentrated to a gum under vacuum. This gum was slurried with 500 ml. of acetonltrile containing 80 ml. of propylene oxide. The slurry was allowed to stand for 15 minutes and the Insoluble product was collected by filtration and washed with 200 ml. of aceto r obtained In 55.2 percent yield. This yield figure is a corrected figure to allow for the purity of 54 · 5 peroent of the 7-ACA obtained. The impurities in the sample were primarily water-soluble materials.

Examples 18 - 26 A series of cleavage reaotions was run in the same manner as in Example 17 using 28 g. of cephalosporin C sodium salt monohydrate. The results of these examples are summarized in Table 2 . In Examples 18 and 19, 6 -5 ml. of nitrosyl chloride was used, while in Examples 20-26, 6.5 ml. of nitrosyl chloride was used. In Examples 20-22 the residue from the evaporation was first dispersed in the inert solvents, then the oxide was added. In the other examples a solution of the oxide in the solvent was added to the residue. In the Table the symbol R-N02 represents a mixture of equal volumes of nitromethane and 2-nitropropane . Ammonium sulfamate was added prior to concentration in all examples. Propylene pxlde was the alkylene oxide used in all but Example 2.6, in which ethylene oxide was used. The product from Examples 18 , 19, and 26 was washed with 200 ml. of acetonitrile before drying. The yield figures are corrected to reflect the yield of pure 7-ACA.

Table 2 HC02H, Diluent, N¾S03Nfi2, Solvent, Example ml. ml. g. ml. 18 100 CI^CN, 200 4 acetonitrile, 19 70 R-N02, 210 . acetonitrile, 100 CH3C , 200 6 acetonitrile, 21 100 CI^CN, 200 6 dioxane, 300 22 100 CH3CN, 200 6 dime thy If ormam n-butyl acetat 23 100 CI^CN, 200 6 R-N02, 250 24 100 CH3CN, 200 6 acetone, 250 100 CH3C , 200 6 ethyl acetate, 26 100 CI^CN, 200 6 acetonitrile, Since the objective of our process is the preparation of 7-ACA sufficiently pure that it can be used in the preparation of biologically active derivatives,, it Is important to determine if the product 7-ACA can be so used.

One such use involves the hydrogenatlon of 7-AOA to 7-anaino- desacetoxycephalosporanic acid (7-ADCA). When the 7-ACA from Example 1 was subjected to this hydrogenatlon 7-ADGA was obtained in 69 percent yield. This yield is comparable to that obtained when the 7-ACA used in the hydrogenatlon has been purified by conversion to the p_-toluenesulfonic acid salt as described In British Patent 1 , 104 , 958 . The hydrogenatlon proceeded smoothly and there were no adverse side reactions.

Many active antibiotic . substances are obtained by the acylation of the 7-amino group of 7-ACA. The introduction of a phenylglycyl group at this position of 7-ACA from Example 1 proceeded in a 6o percent yield. Again, this is comparable to the yield obtained when a purified 7-ACA is used.. The reaction proceeded smoothly. In addition, the 7-ACA from Ex-ample was acylated with thiophene-5-acetyl chloride following the procedure of United Stated Patent 5 , 551 , 597 using acetone and urea. The acylation proceeded smoothly and cephalothln was obtained in good yield and purity.

As a further determination of purity the nuclear magnetic resonance spectra of 7-ACA precipitated with ethylene oxide and propylene oxide were compared with the spectrum of 7-ACA purified by means of the £-toluenesulfonic acid salt. The spectra were superlmposable with no new protons observed. In addition to showing the purity of the 7-ACA, this confirms that there was no reaction of the oxide with the 7-ACA.

Claims (5)

32407/4

1. A process for the preparation of 7-amino solvent is evaporated to leave a residue, and to the residue is added said alkylene oxide to result in the intermediate and to obtain the 7-amlnocephalosporanic acid cherefrom.

2. The process of claim 1, wherein the intermediate is hydrolyzed with water and/or an alkanol containing one to three carbon atoms optionally adding ammonium eulfamate and the 7-aminocephalosporanic acid is precipitated from the resulting solution by adding said alkylene oxide.

3. The process of claim 1 or 2 wherein the alkylene oxide is ethylene oxide or 1,2-propylene £oxide.

4. The process of claims 1, 2 or 3 wherein the intermediate is hydrolyzed wit methanol.

5. The process of any of claims 1 to 4, wherein the reagent is nitrosyl chloride. 0- (j!o&^%) S. HOROWITZ & CO. AGENTS FOR APPLICANTS