DK143030B - PROCEDURE FOR IN-SITU PREPARATION OF A CEPHALOSPORIN C DERIVATE - Google Patents

Description

1 143030

The present invention relates to a process for in situ preparation of a cephalosporin C derivative of the general formula H OH c I · I γ / 0 13 HO C C-C- (CHO) -j-C-N - f

NH .. III

a -CH ~ -0-C-CH, c = ° ^ 2 3

B ° I

co2h where B represents a group of formula -NBR or -OR, where R represents alkyl of 1-10 carbon atoms or a group of formula 2 where n represents an integer from 1 to 6 when B represents OR and from 2 to 3 0 to 6 when B represents NHR and R and R are the same or different and each represents H, Cl, Br, F, NOg, alkyl containing 1-10 carbon atoms or alkoxy containing 1-10 carbon atoms. This derivative can be used to prepare 7-aminocephalosporanoic acid (7-ACA).

Methods for recovering cephalosporin C from a culture medium are disclosed in the disclosures of U.S. Patents Nos. 3,093,638 and 3,094,527, wherein cephalosporin C is adsorbed on an adsorbent instead of solvent extraction. While lower alkoxy-carbonyl or aryloxycarbonyl or (lower) alycarbamoyl or arylcarbamoyl-cephalosporin C derivatives are not described as being formed "in situ" in the cephalosporin C culture medium, or as useful in extracting cephalosporin C from the nutrient medium or as useful as starting materials for the preparation of 7-ACA, some of these compounds are described in the patent literature (British Patent Nos.

1,014,883 and 1,064,495 as well as U.S. Patents Nos. 3,227,709 and 3,227,712 as antibiotics). Several methods of chemically cleaving cephalosporin C or some of its derivatives are described in the patent literature (U.S. Patents Nos. 1,188,311, 3,234,223 and 3,124,576, and British Patent Nos. 1,041,985). None of these processes use N- (lower) alkyloxycarbonyl or N- (lower) alkyl or arylcarbamoylcephalosporin C and a silyl ester thereof as starting material.

7-aminocephalosporanoic acid (7-ACA) having the formula II

0 "_N J_CH9-0 -'C-CH, 2 3

COH 2 II

is an extremely valuable intermediate in the preparation of a large number of semi-synthetic antibacterial cephalosporanoic acid derivatives. Due to the difficulties of total synthesis of 7-ACA on a large scale, this compound is prepared in practice by the chemical degradation of naturally occurring cephalosporanoic acid, ie. cephalosporin C produced by fermentation. Thus, most 7-ACA is made from cephalosporin C (U.S. Patent No. 3,093,638) having the formula 05? S H .S.

h2n - c - (ch2) 3 - C - N X \ COzH o 10 i - 012 '0' C '“3

° X I

CO 2 H 1 15

However, the preparation of 7-ACA by the methods available today is fraught with difficulties ranging from the fermentation to the chemical decomposition of cephalosporin C. Thus, low yields of 7-ACA have made it difficult for cephalosporanoic acids to occupy this additional space. in the field of antibiotic therapy.

Cephalosporin C is characterized by an amino acid function in the side chain. The amino acid exists in the form of a zwitterion in aqueous solution and as such it is highly water soluble. Due to its highly ionic nature, it is extremely difficult to recover by solvent extraction from the culture medium. The recovery method used today involves adsorption of the crude cephalosporin C from the culture medium onto a suitable adsorbent, e.g. charcoal, an ion exchange resin or the like, followed by elution, concentration and precipitation at the isoelectric point or by salt formation (U.S. Patent No. 3,094,527). The low efficiency of the complexity of each step of this process makes it very difficult to prepare 7-ACA.

Further, it is known to prepare 7-ACA by partial degradation of derivatives of cephalosporin C by chemical agents (U.S. Patents Nos. 3,124,576, 3,188,311 and 3,234,223). Again, these methods provide practical yields that are undesirably low. Furthermore, these methods must inevitably use as a starting material a purified form of cephalosporin C which is difficult to obtain.

It has now unexpectedly been found that the in situ process of the cephalosporin C derivative (III) prepared in situ without 05 significant purification steps can be used directly in subsequent cleavage reactions to produce 7-ACA.

The process of the invention for in situ preparation of the cephalosporin C derivative (III) is characterized by the addition of an acylating agent in the form of an isocyanate of the formula 10 R - N = C = O

or a halogen formate of the formula

ISLAND

II

X - C - O - R

Wherein R is as defined above, and X represents chlorine, bromine or iodine, to an acid-incubated culture medium containing cephalosporin C, at a ratio of at least 2 moles of acylating agent per liter. mole of cephalosporin C at a pH above 7 at a temperature below 40 ° C to form said derivative of cephalosporin C.

20 In order to achieve the highest possible yields and the fewest possible by-products, the measures specified in claims 2-6 are preferred.

Cultivation of the mold known cephalosponum or a mutant thereof produces a mixture of compounds, preferably cephalosporin C and smaller amounts of cephalosporin N (now known to be 25 penicillin). Cephalosporin C is acid stable, while cephalosporin N is not. As a result, prior to reacting the culture medium with a halogen formate, it is preferable to destroy in situ possibly co-produced cephalosporin N. This is achieved by acidifying it to a pH of approx. 2 with a mineral acid such as hydrochloric acid, sulfuric acid, phosphoric acid or the like, followed by an incubation period of from 2 to 10 hours. The culture substrate can be filtered before or after acidification and incubation.

After this step, the filtrate culture substrate containing cephalosporin C is adjusted to a pH of 7 to 9, preferably about 8, 35 by the addition of an alkali metal base such as potassium or sodium hydroxide.

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The volume of the substrate produced is increased to approx. 25% by adding a ratio of at least 2, preferably from 2 to 10 moles of acylating agent per minute. mole of cephalosporin C (with the substrate concentration predetermined) and particularly preferred in a ratio of 05 to 8 moles. The pH is kept constant during the slow addition and for at least 30 minutes after the addition. The temperature of the substrate is kept below 40 ° C, preferably at 0-15 ° C during this time. The turnover is usually completed one hour after the addition is complete.

Preferably, half a volume of an immiscible organic solvent, preferably methyl isobutyl ketone (MIBK), is added, when the acylating agent is a halogen formate and n-butanol when the acylating agent is an isocyanate and the pH is adjusted to from 1 to 3. preferably 2. The mixture is stirred and the organic solvent phase containing cephalosporin C derivative III is collected.

The organic solution is concentrated in vacuo to ca. 1/3 of its original volume at a temperature not exceeding 40 ° C. The concentrate is cooled to approx. 20 ° C to 30 ° C and the sodium salt of cephalosporin derivative III is precipitated by the addition of a small excess 20 of sodium 2-ethylhexanoate dissolved in a solvent such as MIBK or n-butanol. The mixture is cooled to 0-5 ° C for 3-4 hours and the solid sodium salt of derivative III is collected by filtration. The filter cake is washed with cold MIBK followed by a washout with n-hexane. The product is dried in vacuo at ca. 25 ° C.

The compounds of formula III or metal or amine salts thereof can be used to prepare 7-ACA without the need for prior purification or chemical conversion to cephalosporin C.

This can be accomplished by a process which essentially consists of the following consecutive steps: mixing a compound of formula H OH -

M0? C-C- (CH9U-C-N --f N

L '0 05 NH

'> -. -CH - 0-C-CH, c = o or L 3

B C02M

Wherein B represents -OR or -NHR, M represents hydrogen, metal or amine cations, and R represents (lower) alkyl, preferably ethyl, n-propyl, isopropyl, n-butyl and isobutyl or an aralkyl group of the formula 15 - ('H2) nf $ 3 2 3 where n represents an integer from 1 to 6, and R and R are the same or 20 different and each represents H, Cl, Br, F, NC (lower) alkoxy, preferably hydrogen, having at least two equivalents of a silyl compound of the formula r * • 25 λ R5 NH NH / NH Λ 6 ·

r4 i / | r4 or R -Si_X

^ Si SC R

, · / Yy.

Wherein R, R and R are hydrogen, halogen, (lower) alkyl, halo,

C C

gene (lower) alkyl or phenyl, with at least one of the groups R, R

7 4 and R are different from halogen or hydrogen, R represents (lower) alkyl, m is an integer p1 1-2 and X represents halogen or the group R8 κ

-N

Wherein R represents hydrogen or (lower) alkyl and R represents hydrogen (lower) alkyl or the group R5.

R 6 - Si - k 7 preferably dimethyl dichlorosilane or trimethyl chlorosilane, under anhydrous conditions in the presence of an acid deactivating tertiary amine which may be triethylamine, dimethylaniline, quinoline, lutidine or pyridine, in an inert solvent which may be methylene chloride, d. , chloroform, tetrachloroethane, nitromethane or diethyl ether, to give the corresponding silyl diester of compound III, (B) mixing the silyl diester with a halogenating agent which may be phosphorus pentachloride, phosphorus pentabromide, phosphorus trichloride phosphorus tribromide, oxalyl chloride, p-toluenesulfonyl halogenide, phosphorus oxychloride, phosgene or the like, in a molar ratio of 20 or more moles of halogenating agent per moles of silyl ester, preferably ca. 2 mol, under anhydrous conditions in an inert solvent such as methylene chloride, dichloroethane, chloroform, tetrachloroethane, nitromethane, diethyl ether or the like in the presence of an acid deactivating tertiary amine in the form of triethylamine, diethylaniline, quinoline, lutidine, pyridine or the like. below 0 ° C, preferably in the range of 40 ° C to -60 ° C, to produce in solution of the corresponding imino halide, (C) mixing said solution of imino halide with an alcohol in the form of aliphatic alcohols containing 1-12 carbon atoms or phenylalkyl alcohols containing 1-7 carbon atoms at a temperature below 0 ° C, preferably -40 ° C to -70 ° C, to produce in solution of the corresponding imino ether and (D) mixing said solution of imino -ether under acidic conditions with water or an alcohol, especially an aliphatic alcohol or a mixture of both to produce 7-aminocephalosporanoic acid.

The process according to the invention is illustrated in the following by way of example: 8

Example 1

Preparation of sodium N-carbisobutoxycephalosporin C 05? S Ϊ / S \ H07C-C- (CH7U-CN - f η Δ, Δ O 0 NH "• JN Λ-CH7-0-C-CH7 c = o 0 ^ 10? C02Na I 6 ch3 ch3 15 2900 liters culture substrate containing cephalosporin C was mixed with 108 kg of filter aid and 300 ml of silicone antifoam and then filtered at a pH of 6.9 at 10 ° C.

To the filtered medium was added sufficient oxalic acid to obtain a pH of 3.1. After this addition, the pH was adjusted to 20 to 2 with the addition of 30% sulfuric acid. 54 kg of fresh filtration aid was added and the mixture was filtered. The filtrate thus obtained was extracted with 1/2 volume of methyl isobutyl ketone (MIBK) at a pH of 2 and then separated. The MIBK phase was discarded.

A quarter volume of acetone was added to the extracted medium and the pH was adjusted to 7.85 with a solution of sodium hydroxide. 2 kg of isobutyl chloroformate per 1000 liters of filtered medium was added slowly with stirring (a molar ratio of 5.9 moles of isobutyl chloroformate per mole of cephalosporin C). The pH was kept constant at 7.8-8.0 by the addition of 15% sodium hydroxide, and the temperature was kept in the range of 0-10 ° C during the addition. Stirring was continued until the pH became constant without the addition of additional sodium hydroxide, for a period of approx. 2 hours.

The pH was adjusted to 2 by the addition of 13% sulfuric acid, 35 and the acylated medium was extracted with 1/2 volume of MIBK.

The MIBK phase was washed with water and then concentrated to ca.

1/3 of the original volume at 36 ° C in vacuo. The water content of the concentrate was 0.2% (Karl Fisher's method).

143030 9

The concentrate was cooled and a solution of sodium ethyl hexanoate in MIBK was added to a pH of 4.8. The sodium salt of a carbisobutoxycephalosporin C precipitated and collected by filtration. The precipitate was washed with MIBK, suspended in petroleum ether, filtered 05 and dried in a vacuum oven at 40 ° C to 60 ° C to yield 7.2 kg of product. Examination of the remaining medium showed the presence of less than 1% cephalosporin C activity. The product collected was estimated to be 42% to 50% pure sodium n-carbisobutoxycephalosporin C. The product was of sufficient purity for the subsequent 7-aminocephalosporanic acid preparation.

Example 2

Preparation of sodium N-carbisobutoxycephalosporin C

The procedure described in Example 1 was repeated, instead of a molar ratio of 5.9: 1 between isobutyl chloroformate and cephalosporin C, a molar ratio of 4.5: 1 was used and 10.4 kg of product was obtained. Examination of the residual culture medium after extraction showed the presence of less than 14% cephalosporin C activity.

20

Example 3

Preparation of N-CN'-butylcarbamoylcephalosporin C 25 H OH / Sv »li» / \ ho9c-c- (Ch7u-c-n - r in 2, v 2'30 NH ", i- -ch2-o-c-ch3

C = 0 S

30. IN

NH07Na I uCIV3 ch3 35 10 143030 2900 liters of culture medium containing cephalosporin C was mixed with 108 kg of filter aid and 300 ml of silicone antifoam and then filtered at a pH of 6.9 at 10 ° C.

To the filtered medium, sufficient oxalic acid was added to obtain 05 a pH of 3.1. After this addition, the pH was adjusted to 2 by the addition of 30% sulfuric acid. 54 kg of fresh filtration aid was added and the mixture was filtered. The filtrate thus obtained was extracted with 1/2 volume of methyl isobutyl ketone (MIBK) at a pH of 2 and then separated. The MIBK phase was removed.

A quarter volume of acetone was added to the extracted medium and the pH was adjusted to 7.85 with a solution of sodium hydroxide. n-Butyl isocyanate (2 kg / 1000 l medium) was added slowly with stirring (a molar ratio of about 6.1 moles of n-butyl isocyanate per mole of cephalosporin C).

The pH was kept constant at 7.8 to 8.0 by the addition of 15% sodium hydroxide, and the temperature was maintained in the range 0-10 ° C during addition. Stirring was continued until the pH remained constant without further addition of sodium hydroxide, for a period of about 20 minutes. 2 hours.

The pH was adjusted to 2 by the addition of 30% sulfuric acid and the acylated medium was extracted with 3/4 volume of n-butanol. The butanol phase was washed with water and then concentrated to ca. half of the output volume in 36 ° C in vacuo. The water content of the concentrate was negligible.

The concentrate was cooled and a solution of sodium ethyl hexanoate in n-butanol was added to a pH of 4.8. The sodium salt of N, N '- butylcarbamoylcephalosporin C precipitated and collected by filtration. The precipitate was washed with butanol, suspended in petroleum ether, washed with acetone, filtered and dried in a vacuum oven at 40-60 ° C to give the product. Examination of the remaining medium showed the presence of less than 15% cephalosporin C activity. The product was sufficiently pure for subsequent preparation of 7-ACA. It appeared as the di-acid.

Analysis calculated for C₂jHHQN4O₂S.H₂O: C, 47.36; H, 6.06; N, 10.54; H 3 O 3.38.

C, 47.85; H, 6.52; N, 10.54; O 3.48.

Claims (4)

Example 4 Preparation of Sodium N-CN'-phenylcarbamoyQ-cephalosporin C The procedure of Example 3 was repeated, using instead of a molar ratio of 6.1: 1 between n-butyl isocyanate and cephalosporin C, a molar ratio of 7: 1 between phenyl isocyanate and cephalosporin C. A product was found which was satisfactory for cleavage to 7-ACA. Examination of the remaining medium after extraction showed the presence of less than 15% cephalo-10 sporin C activity. It was investigated as the diacid. Analysis calculated for C 23 H 26 N 4 O 9 S: C 51.68; H, 4.90; N, 10.48; S, 6.00. C, 51.63; H, 5.27; N, 10.62; S 6.14. 15 Patent claims A process for in situ preparation of a cephalosporin C derivative of the general formula 20 H OH ~, ... ^ S \ H0OC-C- (CHO), -CN - r 1 l 1 £ SO NH "111. V. A-CH - 0-C-CH, C = 0 -N 2 6 B is in CO 2 h where B represents a group of formula -NHR or -OR, where R represents all alkyl of 1-10 carbon atoms or a group of formula -oc: